UNIVERSITY OF CALIFORNIA Los Ange es Comp ete Bio … · UNIVERSITY OF CALIFORNIA Los Ange es Comp...
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UNIVERSITYOFCALIFORNIA LosAngees CompeteBioogicaDechorination ofChorinatedEthyenestoNon-toxicEthyene underMethanogenicConditions Adissertationsubmittedinpartiasatisfactionofthe requirementsforthedegreeDoctorofPhiosophy inCiviEngineering by Chia-JiTeng 1994
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Transcript of UNIVERSITY OF CALIFORNIA Los Ange es Comp ete Bio … · UNIVERSITY OF CALIFORNIA Los Ange es Comp...
UNIVERSITY OF CALIFORNIA
Los Ange�es
Comp�ete Bio�ogica� Dech�orination
of Ch�orinated Ethy�enes to Non-toxic Ethy�ene
under Methanogenic Conditions
A dissertation submitted in partia� satisfaction of the
requirements for the degree Doctor of Phi�osophy
in Civi� Engineering
by
Chia-Ji Teng
1994
The dissertation of Chia-Ji Teng is approved .
O&ajw~4~xt"Menachem E�ime�ech
Robert A. Mah
Michae� K. Stenstrom, Committee Chair
University of Ca�ifornia, Los Ange�es
1994
11
TABLE OF CONTENTS
in
LIST OF FIGURES
LIST OF TABLES
ACKNOWLEDGMENTS
VITA
ABSTRACT
page
vi
x
xu
xm
xiv
1 . INTRODUCTION 1
2 . LITERATURE REVIEW 5
2 .1 . Xenobiotics and reca�citrance 5
2 .2 . Adaptation 6
2 .3 . Cometabo�ism 7
2.4 . PCE-degradative abi�ity 8
2.4 .1 . Carbon source 9
2.4.2 .
Sequentia� transformation 10
2.4.3 .
Cu�tures 11
2.5 . Degradabi�ity of TCE and �ess ch�orinated compounds 12
2.6 . Microbio�ogica� aspects of anaerobic digested s�udge 13
3 . MATERIALS and METHODS 16
3.1 . Chemica�s 16
3.2 . Fresh s�udge and pond sediment 16
3.3 . Methano�-enrichment cu�ture 17
3.4 . Anaerobic nutrient medium 18
iv
3.5 . Ana�ysis 20
3.5 .1 . Purgeab�e ch�orinated compounds 20
3 .5 .2 . Methane and ethy�ene 22
3.6. Experiment 22
3.6.1 . PCE-dech�orinating abi�ity test 22
3.6.2 . Treatabi�ity of ch�orinated compounds 24
3 .6 .3 . Dech�orinated products and reductive dech�orination
progression test 25
3.6.4 . Toxicity and highest to�erab�e concentration 26
3.6.5 . Semi-continuous operation 27
3.6.6 . Effect of Methano� (e�ectron donor) on PCE
dech�orination rate 28
3 .6 .7 . Effect of mixing on PCE dech�orination test 28
3 .6 .8 . Effect of temperature on PCE dech�orination test 30
3 .6 .9 . PCE-dech�orinating abi�ity in o�d s�udges test 30
3 .6.10. Bio�ogica� activated carbon process (BAC) test 31
3 .6.11 . Off�ine bioregeneration (OBR) test 32
4. RESULTS 36
4.1 . Ch�orinated chemica� �oss contro� 36
4.2 . Mass ba�ance 40
4.3 . PCE-dech�orinating abi�ity and a genera� characteristic of
s�udges 46
4.4 . Treatabi�ity of ch�orinated compounds 67
4.5 . Reductive dech�orination progression 69
4.6 . Toxicity effect 81
v
4.7 . Highest to�erab�e concentration and ethy�ene production 84
4.8 . Semi-continuous operation 92
4.9 . Effect of methano� 101
4.10 . Effect of mixing on PCE dech�orination 110
4.11 . Effect of temperature on PCE dech�orination 119
4.12 . Retardation of PCE dech�orination in o�d digested s�udges 119
4.13 . Effect of activated carbon on PCE dech�orination 121
4 .14 . Comp�ete PCE dech�orination in bio�ogica� activated carbon
system 125
4 .15 . Bio�ogica� regeneration of PCE �aden carbon 128
5 . DISCUSSION 134
5.1 . PCE dech�orination - A common characteristic of s�udges 134
5.2 . Dech�orination progression and comp�ete dech�orination . 137
5.3 . Dech�orination rate 141
5.4 . Nutrient requirements 145
5.5 . E�ectron donor 147
5 .6 . Competition and inhibition 149
5.7 . Carbon addition and dech�orination substrate avai�abi�ity 155
5 .8 . Bio�ogica� activated carbon process 157
5 .9 . Off�ine bio�ogica� regeneration 161
6 . CONCLUSIONS 169
Appendix A. Experimenta� data 176
References 235
LIST OF FIGURES
Figure
Page
1 . F�ow digram of the research .
4
2.
The mu�tistep nature of anaerobic digestion.
15
3 . Schematic of bio�ogica� activated carbon process (BAC) .
33
4 . Schematic of off�ine bio�ogica� regeneration process (OBR) .
34
5.
The �oss of vo�ati�e ch�orinated chemica�s in D .I.water contro�samp�e without foi� .
37
6.
The �oss of vo�ati�e ch�orinated chemica�s in D .I.water contro�samp�e with foi� .
38
7.
PCE �oss and its abiotic products in an autoc�aved s�udge contro�samp�e.
39
8. The measured concentration of ch�orinated compounds in �iquidphase without consideration of biosorption and vo�ati�ity.
45
9. Mass ba�ance of ch�orinated compounds in the Hyperion s�udgesystem when biosorption and vo�ati�ity are ref�ected .
47
10. PCE-dech�orinating abi�ity and formation of intermediates in abatch cu�ture of fresh anaerobic Hyperion s�udge after sucessiveadditions of PCE and 123 µmo� . of methano� .
49
11 . PCE-dech�orinating abi�ity and formation of intermediates inanaerobic pond sediment after sucessive additions of PCE and 123p.mo�. of methano� .
50
12 . PCE-dech�orinating abi�ity and formation of intermediates in amixture (1 :1) of fresh anaerobic Hyperion s�udge and pondsediment after sucessive additions of PCE and 123 µmo� . ofmethano� .
51
13 . PCE-dech�orinating abi�ity and formation of intermediates in abatch cu�ture of fresh anaerobic Chino Basin (RP2) s�udge I withhigh initia� methano� and PCE additions .
53
14 . PCE-dech�orinating abi�ity and formation of intermediates inmethano�-enrichment cu�ture incubated with methano� as the carbonand energy source .
55
vi
15 . PCE-dech�orinating abi�ity and formation of intermediates inmethano�-enrichment cu�ture incubated with methano� andhydrogen as the carbon and energy source .
55
16 . VC-dech�orinating abi�ity and formation of intermediates inmethano�-enrichment cu�ture incubated with methano� andhydrogen as the carbon and energy source.
56
17 . PCE-dech�orinating abi�ity and formation of intermediates in theconcentrated methano�-enrichment cu�tures incubated withmethano� as the carbon and energy source .
57
18. The comp�ete dech�orination of PCE in a batch cu�ture of freshanaerobic s�udge obtained from Termina� Is�and WastewaterTreatment P�ant.
59
19 . The partia� dech�orination of PCE in a batch cu�ture of freshanaerobic s�udge obtained from Chino Basin (RP1) WastewaterTreatment P�ant.
61
20. The comp�ete dech�orination of PCE in a batch cu�ture of freshanaerobic s�udge obtained from A�varado Wastewater TreatmentP�ant .
63
21 . The comp�ete dech�orination of PCE in a batch cu�ture of freshanaerobic s�udge obtained from Va�encia Wastewater TreatmentP�ant .
65
22. The comp�ete dech�orination of PCE in a batch cu�ture . of freshanaerobic s�udge obtained from JWPC Wastewater TreatmentP�ant .
66
23 . Reductive dech�orination progression of PCE at a high PCEconcentration in fresh anaerobic Hyperion s�udge .
71
24. Reductive dech�orination progression of TCE at a high TCEconcentration in fresh anaerobic Hyperion s�udge .
73
25 . Reductive dech�orination progression of cis-DCE at a high cis-DCEconcentration in fresh anaerobic Hyperion s�udge .
74
26. Reductive dech�orination progression of trans-DCE at a high trans-DCE concentration in fresh anaerobic Hyperion s�udge .
75
27 . Reductive dech�orination progression of 1 .1-DCE at a high 1 .1-DCE concentration in fresh anaerobic Hyperion s�udge .
76
28 . Reductive dech�orination progression of VC at a high VCconcentration in fresh anaerobic Hyperion s�udge .
77
vu
vin
29 . The effect of viny� ch�oride on PCE dech�orination in a freshanaerobic Hyperion s�udge cu�tures without methano� addition . 82
30 . The effect of the presence of TCE and VC on PCE dech�orinationin a fresh anaerobic Hyperion s�udge cu�tures without methano�addition . 83
31 . Toxico�ogica� effect of PCE on methanogenic acyivity in freshHyperion s�udge . 85
32. Toxico�ogica� effect of TCE on methanogenic acyivity in freshHyperion s�udge . 85
33 . Toxico�ogica� effect of cis-DCE on methanogenic acyivity in freshHyperion s�udge . 86
34. Toxico�ogica� effect of trans-DCE on methanogenic acyivity infresh Hyperion s�udge . 86
35 . Toxico�ogica� effect of � .1-DCE on methanogenic acyivity in freshHyperion s�udge. 87
36. Toxico�ogica� effect of VC on methanogenic acyivity in freshHyperion s�udge. 87
37 . Long term dech�orination of PCE and formation of intermediates ina semi-continuous operation serum bott�e . 97
38 . Long term dech�orination of VC and formation of intermediates in asemi-continuous operation serum bott�e . 98
39 . Long term dech�orination of PCE and formation of intermediates ina semi-continuous operation serum bott�e in which 60 m� ofmixture of acc�imated anaerobic digested s�udge was used as thedech�orinating organisms . 100
40. The effect of methano� and fresh medium on PCE dech�orination . 108
41 . The effect of mixing on PCE dech�orination and methaneproduction . 112
42. PCE dech�orination in a batch cu�ture of fresh anaerobic Hyperiondigested s�udge under �aboratory conditions without temperatureregu�ation. 120
43 . The effect of activated carbon on the acc�imation of fresh anaerobicHyperion s�udge to PCE dech�orination . 122
44. Comparison of methane production and ethy�ene formation duringPCE dech�orination when two different amounts of GAC areintroduced to acc�imated s�udge .
124
45. Effect of varying PCE concentrations on dech�orinating efficiencyin BAC system .
127
46. Ethy�ene production and accumu�ation of PCE dech�orinationproducts in BAC system with various amounts of PCE added (t =60 days).
129
47. Effect of varying PCE concentrations on dech�orinating efficiencyin OBR system .
131
48. Ethy�ene production and accumu�ation of PCE dech�orinationproducts in OBR system with various amounts of PCE added (t =60 days).
132
49. Schematic for the common observed dech�orination pattern forconversion ch�orinated compounds to ethy�ene .
139
ix
LIST OF TABLES
Tab�e
Page
1 . Composition of feed so�ution for methano�-enrichment cu�tures.
19
2.
Retention times and detection �imits .
21
3 . Summary of Henry's �aw coefficient .
42
4. Summary of Freund�ich characteristic constants of biosorption .
44
5 .
Abi�ity of anaerobic Hyperion s�udge to degrade variousch�orinated compounds to ethy�ene .
68
6 . Summary of ethy�ene formation from dech�orination of variousch�orinated ethy�enes .
79
7. Summary of dech�orination pattern for each ch�orinatedcompound .
80
8. Dech�orination of PCE and formation of dech�orinated products atvarious initia� dosages during anaerobic incubation in freshHyperion s�udge .
89
9. Dech�orination of TCE and formation of dech�orinated products atvarious initia� dosages during anaerobic incubation in freshHyperion s�udge.
90
10. Dech�orination of cis-DCE and formation of dech�orinated productsat various initia� dosages during anaerobic incubation in freshHyperion s�udge.
91
11 . Dech�orination of trans-DCE and formation of dech�orinatedproducts at various initia� dosages during anaerobic incubation infresh Hyperion s�udge .
93
12 . Dech�orination of 1 .1-DCE and formation of dech�orinatedproducts at various initia� dosages during anaerobic incubation infresh Hyperion s�udge .
94
13 . Dech�orination of VC and formation of dech�orinated products atvarious initia� dosages during anaerobic incubation in freshHyperion s�udge .
95
x
xi
14 . Data showing the inhibitory effect of MeOH on ethy�eneproduction . 103
15 . The effect of mixing on PCE dech�orination - 1st Run . 113
16 . The effect of mixing and reduced medium rep�acement on PCEdech�orination - 2nd Run . 114
17. The effect of mixing and CO2 on PCE dech�orination - 3rd Run . 116
18. The effect of mixing and CO2 on PCE dech�orination - 4rd Run . 117
19 . The effect of mixing and CO2 on PCE dech�orination - 5rd Run . 118
20. Bioregeneration efficiency of PCE �aden carbon . 133
21 . Summary of PCE dech�orination and formation of intermediates ina�� test anaerobic cu�tures . 135
22 . The increasing rate of PCE dech�orination by fresh Hyperions�udge at initia� �ag period. 143
ACKNOWLEDGMENTS
I fee� most gratefu� to my wife, Li-Huei, and my sons, Kevin and Wi��iam, for
accompanying me to overcome many difficu�ties and prob�ems during the past four
years. I wou�d �ike to give a�� my g�ory to them.
I thank very sincere�y my advisor Dr . Michae� Stenstrom for his encouragement
and academic guidance during the who�e period of this research . I a�so thank Dr. Robert
Mah for generous�y offering his �ab faci�ities to me. Most of this research work was
conducted in Dr. Mah's �aboratory . I wou�d a�so �ike to thank Dr . Larry Baresi and Dr.
Jean-Louis Garcia for their time�y guidance and encouragement .
I am gratefu� to Fe�ipe A�atriste M ., who a�ways gave his assistance and
direction whenever I needed. Ryan Miya took a �ot of time in correcting my writing .
Yih-Shian Tsu, Chi-Yuh Huang and Chia-Jung Tsay a�ways provided a joke when the
�aboratory work tired me. I specia��y thank Dr. Roger Wi��iam Babcock for his he�p at
the beginning stage of this research . Many students from the UCLA Water Qua�ity
Laboratory are a�so the constant inspiration to me . I a�so thank the members of my
committee; Menachem E�ime�ech and Thomas Harmon
xii
May 24, 1956
1978
1980
1980-1982
1982-1983
1983-1984
1984-1986
1986-1990
1991
1991-1994
VITA
Born, Taiwan, R.O.C .
B .S . Civi� EngineeringNationa� Cheng Kung UniversityTainan, Taiwan
M.S. Civi� EngineeringNationa� Cheng Kung UniversityTainan, Taiwan
Instructor of MathematicsFirst NCO Schoo� of Chinese ArmyTaoyan, Taiwan
Instructor of Sanitary EngineeringFu-Shing Institute of Techno�ogyYi�an, Taiwan
Environmenta� EngineerSuper Max Engineering Enterprise Co ., Ltd .Taipei, Taiwan
Senior Environmenta� EngineerEnvironmenta� Protection DepartmentTaipei, Taiwan
First Section ChiefEnvironmenta� Protection DepartmentTaipei, Taiwan
M.S. Civi� and Environmenta� EngineeringUniversity of Ca�ifornia, Los Ange�es
Research and Teaching AssistantCivi� Engineering DepartmentUniversity of Ca�ifornia, Los Ange�es
ABSTRACT OF THE DISSERTATION
Comp�ete Bio�ogica� Dech�orination
of Ch�orinated Ethy�enes to Non-toxic Ethy�ene
under Methanogenic Conditions
by
Chia-Ji Teng
Doctor of Phi�osophy in Civi� Engineering
University of Ca�ifornia, Los Ange�es, 1994
Professor Michae� K . Stenstrom, Chair
Perch�oroethy�ene-dech�orinating abi�ity was examined first in seven different s�udges,
pond sediment, methano�-enrichment cu�ture, and a mixture of Hyperion s�udge and
sediment. Hyperion s�udge was then chosen to conduct the treatabi�ity, dech�orination
progression, toxico�ogica� effect and semi-continuous operation tests for
perch�oroethy�ene (PCE), trich�oroethy�ene (TCE), 3 isomers of dich�oroethy�ene (DCE)
and viny� ch�oride (VC) . The effect of methano�, mixing and activated carbon addition
on PCE dech�orination was a�so eva�uated with Hyperion s�udge . Reductive
dech�orination of PCE through TCE to cis-DCE was observed in a�� anaerobic test
cu�tures . However, comp�ete dech�orination of PCE to ethy�ene (ETH) was on�y
observed in 6 s�udges and the concentrated methano�-enrichment cu�tures. This indicates
that PCE dech�orination may be a genera� characteristic of s�udges and different s�udges
xiv
may vary in their potentia� to comp�ete�y dech�orinate PCE to ethy�ene . Comp�ete
dech�orination of a�� six ch�orinated ethy�enes to ethy�ene was a�so demonstrated in
Hyperion s�udge. Among them, PCE, TCE and cis-DCE were more �ike�y to produce
ethy�ene. From dech�orination progression test, it was further confirmed that a�� six
compounds may be dech�orinated to ethy�ene through an identica� co-metabo�ic route
(i .e . PCE -> TCE -> DCE -> VC -> ETH). The �ess ch�orinated ethy�enes seemed on�y
to uti�ize part of the route, whi�e the fu��y ch�orinated ethy�ene (PCE) uti�ized the who�e
route during the reductive dech�orination . For reductive dech�orination of each
ch�orinated compound, the tota� ethy�ene production increased with their initia�
concentration up to an upper �imit. However, 100% comp�ete dech�orination to ethy�ene
was never observed for a�� test ch�orinated compounds. On average, on�y about 30 to 40
percent of the cumu�ative�y added ch�orinated ethy�enes were recovered as ethy�ene in
�ong term semi-continuous operation. Excess added methano� or mixing conditions were
further demonstrated to be more favorab�e for methanogenesis and thereby inverse�y
affect the rate and extent of PCE dech�orination in acc�imated s�udge. Furthermore,
comp�ete dech�orination of PCE was a�so achieved in the bio�ogica� activated carbon
process (BAC) and the off�ine bio�ogica� regeneration process (OBR) .
xv
1 . INTRODUCTION
Perch�oroethy�ene (PCE) and trich�oroethy�ene (TCE) are two of the most
frequent�y identified contaminants in groundwaters . Since both are suspected human
carcinogens, thousands of sma�� drinking water systems which re�y on groundwater
sources may be jeopardized. Consequent�y, a feasib�e means of contro��ing or treating
ch�orinated ethy�enes in groundwaters is essentia� .
Perch�oroethy�ene and TCE are ha�ogenated a�iphatic hydrocarbons, a particu�ar
c�ass of chemica�s representing one of the most important categories of industria�
chemica�s with respect to production vo�umes, dispersion in the environment,
toxico�ogica� effects and popu�ation exposure . Both of them are wide�y used in
industria� degreasing so�vents, dry-c�eaning f�uids, and fumigants . The annua� wor�d
production vo�umes of PCE and TCE ( 1 .1 and 0.6 mi��ion tons, respective�y) are
among the 10 �eading compounds of the ha�ogenated a�iphatic hydrocarbons (Leisinger,
1983 ) . However, intensive wor�dwide app�ication and spi��s from industry have
resu�ted in extensive po��ution of the environment . Contamination by PCE and/or TCE
may occur in groundwater, surface water, soi�, and air . The majority of such
contamination is widespread in groundwaters because a significant fraction of these
chemica�s was discarded in waste dumps and then infi�trated into the groundwater
Westrick, Me��o and Thomas, 1984 ; Travis and Doty, 1990 ) . Concern about
groundwater contamination with PCE and/or TCE is growing because it is potentia��y
dangerous to human hea�th even in very �ow concentration .
Groundwater represents more than 95 percent of a�� avai�ab�e freshwater in the
United States. Approximate�y 80 percent of a�� pub�ic water supp�iers in this country
1
re�y on groundwater for potab�e water sources, and about 96 percent of a�� water used
for rura� domestic purposes is obtained from groundwater . The continued va�ue of
groundwater as a future source of potab�e water wi�� depend on contro��ing
contaminants in groundwater, either through reduction of the source of the compounds
or through the rec�amation or treatment of groundwater supp�ies a�ready affected .
However, the effectiveness and widespread use of PCE and TCE in various app�ications
makes a major reduction in their use un�ike�y in the near future . Techniques most
frequent�y used for the treatment of contaminated groundwater inc�ude physica� and
chemica� processes (e.g., air stripping and adsorption). However, there is considerab�e
interest in bio�ogica� remediation processes, especia��y in anaerobic environments,
because they are capab�e of converting the heavi�y ch�orinated compounds to harm�ess
metabo�ites, rather than transferring them from one part of the environment to another .
Bio�ogica� techniques that comp�ete�y degrade contaminants without generating
toxic end products may be best suited for treating �arge vo�umes of contaminated
groundwaters and industria� wastewaters . For the ha�ogenated a�iphatic compounds,
there are three genera� kinds of initia� microbia� transformation they may undergo :
nuc�eophi�ic substitution, oxidation, and reductive deha�ogenation (Leisinger, 1983) .
Whi�e the former two transformations have been demonstrated under aerobic conditions,
reductive deha�ogenation is probab�y responsib�e for the anaerobic transformation of
ha�ogenated a�iphatic compounds . PCE and TCE, because of their vo�ati�ity, are difficu�t
to hand�e in aerobic systems . However, the anaerobic system is ab�e to prevent
vo�ati�ization of the added compounds . PCE dech�orination has been wide�y reported
under anaerobic conditions, but it is converted on�y to tri- and dich�orinated compounds
in most cases . These �ess ch�orinated compounds tend to persist �onger in anaerobic
2
environments than high�y ch�orinated compounds . However, if the environment
becomes aerobic, the �ess ch�orinated chemica�s may be degraded by aerobic bacteria .
Recent�y, a two-stage anaerobic-aerobic process was used to achieve comp�ete
destruction of ch�orinated a�iphatic hydrocarbons (Fathepure and Voge�, 1991); however
this process suffered from the high vo�ati�ization of chemica�s in the aerobic process . A
�ot of work must be done to prevent vo�ati�e �oss of such chemica�s from the treatment
system. The present study concentrates on the feasibi�ity of a comp�ete�y anaerobic
biotransformation of PCE and TCE to ethy�ene (ETH), a non-toxic and environmenta��y
acceptab�e product, by a sing�e-stage treatment process using anaerobic digested s�udge .
The conditions required for transformation by the digested s�udge are a�so reported . The
f�ow diagram for this research is depicted in Fig . 1 .
Because PCE and TCE are detected a�most everywhere in the environment, it
wou�d be a promising strategy to use existing wastewater treatment p�ants for the
rec�amation or treatment of groundwater contaminated with PCE and TCE. This cou�d
be accomp�ished in anaerobic s�udge digestors if degradabi�ity can be demonstrated .
3
Fig.1 F�ow diagram of the research
Bioavai�abi�ity of PCE andits dech�orinated products1 . so�ubi�ity2. vo�ati�ity3. biosorption
iCompetition betweenMeOH & ch�orinatedcompounds
Bioremediation of PCE and�ess ch�orinate ethv�enes
Literature review
1PCE-dech�orinating abi�ity1 . Hyperion s�udge2. Chino s�udge3. MeOH enrichment4. pond sediments5. mixture of 1 & 4
etc .
Scope of this research
Experiments
V
i(Semi-continuous treatment
Studyof
enhancement of treatment
GAC adsorption ofch�orinated compounds
IDeve�opment of theadvance treatmentprocesses
Adsorption + Desorption + Biotreatment
Eva�uation of feasibi�ityand
Summary
Treatabi�ity &dech�orinating progression1 . PCE2 . TCE3 . cis-DCE4 . trans-DCE5 . 1 .1-DCE6 . V .C.
1Toxicity & highestto�erab�e concentration .1 . PCE2 . TCE3 . cis-DCE4. trans-DCE5 . 1 .1-DCE6. V.C .
Desorption of ch�orinatedcompounds
2. LITERATURE REVIEW
Groundwater contamination with the organic so�vents PCE and TCE is a serious
prob�em, and concern about it is growing . Among various treatment techno�ogies
avai�ab�e to contro� groundwater contaminants, bio�ogica� treatment is a promising
method for comp�ete�y degrading these reca�citrant compounds . Environmenta�
contamination by PCE and TCE is of industria� origin and not due to natura� occurrence
of these compounds . These nove� chemica�s, to which microorganisms have not been
exposed in evo�utionary history, are not attacked by microbes and accumu�ate in the
environment. Such compounds are ca��ed xenobiotics since they are foreign to the
norma� environment . To se�ect bacteria capab�e of degrading previous�y nondegradab�e
xenobiotics, successfu� adaptation of a mixed microbia� popu�ation to metabo�ize the
reca�citrant xenobiotics is a prerequisite . Furthermore, reductive dech�orination of
ch�orinated a�iphatic compounds by microorganisms may not be sustainab�e un�ess an
additiona� carbon substrate is provided because it is a co-metabo�ic process (Fathepure
and Boyd, 1988a ) . Therefore, this review wi�� cover definitions of " xenobiotics and
reca�citrance ", " adaptation ", and " co-metabo�ism " first, then a summary of previous
research on biotransformation of PCE and TCE under both anaerobic and aerobic
conditions . Fina��y, the microbio�ogica� aspects of anaerobic digested s�udge wi�� be
presented .
2.1 . Xenobiotics and reca�citrance
A xenobiotic compound is a chemica� to which microorganisms have not been
exposed in evo�utionary history (Leisinger, 1983) . Hence, most of the anthropogenic
5
compounds (e.g . PCE, TCE, etc .) which do not occur natura��y are categorized as
xenobiotics. Most xenobiotics are not degraded under circumstances apparent�y
adequate for microbia� growth . This �eads to their accumu�ation in the environment, i .e .
to persistence or reca�citrance of the chemica� . However, the reca�citrance of a given
compound may a�so be due to inso�ubi�ity, or �imited adsorption onto biomass, or
inhibition to microorganisms at the high concentration . At the enzymatic �eve�, the
compound may not be degraded because it is unab�e to enter the ce�� or the organism
does not possess the appropriate enzyme (Painter and King, 1985) . But it is be�ieved
that a�� organic compounds can be eventua��y biodegraded . A process, i.e. adaptation by
which a mixed popu�ation of microorganisms deve�ops the abi�ity to degrade a substrate
hitherto not biodegradab�e, must be performed .
2.2 . Adaptation
Adaptation is a process to se�ect bacteria capab�e of degrading previous�y
nondegradab�e xenobiotics (Leisinger, 1983). It a�so covers the situation in which the
popu�ations deve�op to�erance to inhibitory substances (Painter and King, 1985) .
Therefore, determining if popu�ations in anaerobic digested s�udge can adapt to degrade
PCE and TCE (nove� compounds) is a prerequisite for this study . There are two genera�
mechanisms by which acquisition of new degradative abi�ities can be achieved ; first, the
adaptation of existing catabo�ic enzymes and, second, the evo�ution of comp�ete
metabo�ic progressions (Painter and King, 1985) . Usua��y, mixed microbia� popu�ations
of many different species have a much greater probabi�ity of adapting to the degradation
of new substrates than a sing�e species . In most instances, the adaptation is the resu�t of
co��ective activity of a metabo�ica��y structured community . Except for this, nothing has
6
been reported as to why the various periods of adaptation were adopted and no agreed
ways of adaptation exist . Litt�e is known about the effects of factors such as time and
pattern of exposure, concentration of the test substance, presence of other substrates, on
the adaptive processes (Painter and King, 1985) . After acc�imation is achieved, the
acc�imated cu�tures can be further enriched to increase rates in degrading previous�y
nondegradab�e xenobiotics .
2.3. Co-metabo�ism
Co-metabo�ism is the transformation of a non-growth substrate in the ob�igate
presence of a growth substrate or another transformab�e compound (Da�ton and Stir�ing,
1982). Compared to the traditiona� biodegradation, it is a new phenomenon in which an
organism grows on one substrate, but a�so has the abi�ity to transform one or more other
compounds, perhaps through on�y a few steps, without being ab�e to derive energy or
growth from the process. A�though co-metabo�izing organisms do not derive benefits
from the metabo�ism of non-growth substrates, co-metabo�ism is thought to occur
wide�y in nature and is probab�y more significant in the degradation of xenobiotics
(Leisinger,1983). Co-metabo�ism can resu�t from a simu�taneous attack on the growth
and non-growth substrates by the same enzyme or sequence of enzymes. It may a�so
occur through the activity of enzymes not direct�y associated with the catabo�ism of the
growth substrate (Painter and King, 1985) . Genera��y speaking, co-metabo�ic
transformation in the environment does not necessari�y resu�t in the comp�ete oxidation
of xenobiotics but may �ead to the accumu�ation of transformation products with
increased or decreased toxicity as compared to the origina� compound (A�exander,
1981). However, if the co-metabo�ic intermediate product from the xenobiotic by one
7
species can be comp�ete�y oxidized for growth by another species, comp�ete
minera�ization may be achieved .
For this reason, biodegradation of PCE and TCE can be considered comp�ete if
the carbon ske�eton is converted to non-toxic metabo�ites and the ch�orine is returned to
the minera� state . Another issue re�ated to co-metabo�ism is that co-metabo�ism of
xenobiotics (e.g . PCE and TCE) usua��y occurs at a much s�ower rate than metabo�ism
of growth substrates. When readi�y uti�izab�e carbon sources are offered to these
microorganisms together with xenobiotics, no se�ective pressure exists for growth on
the xenobiotic compounds .
2.4. PCE-degradative abi�ity
PCE is persistent in aerobic environments but degraded in anaerobic
environments. Reductive deha�ogenation is an important biodegradation mechanism for
ha�ogenated compounds under anaerobic conditions. Reductive dech�orination (i.e. the
rep�acement of ch�orine with hydrogen) of PCE is wide�y reported under a variety of
anaerobic environments inc�uding methanogenic fixed-fi�m reactors (Bouwer and
McCarty, 1983 ; Voge� and McCarty, 1985; Fathepure and Voge�, 1991), anaerobic soi�s
(Doo�ey-Dana, Foge� and Find�ay, 1989), muck (Parsons et a� ., 1984), mixed
methanogenic enrichments (Fathepure et a� ., 1987 ; Freedman and Gossett, 1989), and
10% anaerobic sewage s�udge (Fathepure and Boyd, 1988b) . In most cases, PCE was
on�y partia��y dech�orinated to TCE or cis-DCE . The observed characteristics of
reductive dech�orination of PCE are: (1) it typica��y occurs in a fashion of sequentia�
remova� of ch�orine substituents from PCE ; (2) the dech�orination rate decreases as the
8
number of ch�orine atoms per mo�ecu�e decreases, and (3) reductive dech�orination is
not sustainab�e un�ess a carbon substrate is provided because PCE dech�orination by
anaerobic bacteria is a co-metabo�ic process (Fathepure and Boyd, 1988a) .
2.4.1. Carbon source
During methanogenic co-metabo�ism of PCE, e�ectrons generated during the
formation of methane may be diverted to PCE for reductive dech�orination . Hence, the
primary carbon substrate is very �ike�y to be the source of reducing equiva�ents for both
methane formation and reductive dech�orination . Fathepure and Boyd (1988a) showed
that the reductive dech�orination of PCE is direct�y proportiona� to the concentration of
the primary carbon substrate and the number of methy� moieties associated with the
primary substrate. This indicates dependence of PCE degradation on the methane-
yie�ding capacity of a particu�ar primary substrate . From a study on the dependence of
PCE dech�orination on methanogenic substrate consumption, Fathepure and Boyd
showed that adequate quantities of a carbon source and CH4 biosynthesis were
necessary for reductive dech�orination to occur . No additiona� dech�orination (TCE
formation ) was noted in experimenta� bott�es after the substrate, methano�, was
exhausted and CH4 production ceased .
Fathepure and Boyd a�so found that different substrates (i.e ., acetate, methano�,
methy�amine, and trimethy�amine) affect the extent of PCE dech�orination due to
different growth rates of methanogens . The appropriate substrate shou�d provide
conditions under which the most extensive reductive dech�orination can occur .
Freedman and Gossett (1989) demonstrated that methano� provides the greatest ethy�ene
9
production in dech�orination of PCE . Acetate, formate, g�ucose, or hydrogen were not
as effective. The higher dech�orination rate may be re�ated to the greater reducing power
of methano� metabo�ism (six reducing equiva�ents) than acetate metabo�ism (two
reducing equiva�ents) .
Furthermore, the extent of PCE dech�orination a�so depends on the concentration
of ce�� mass (Fathepure and Boyd, 1988b) . No change in the remova� efficiencies of the
ha�ogenated hydrocarbons was observed whi�e the feed substrate (acetate) was
decreased (Bouwer and McCarty, 1983) . They suggested that the remova�s were more a
function of organism concentration than of the quantity of the primary substrate .
2.4 .2 . Sequentia� transformation
The principa� intermediate in the anaerobic biotransformation of PCE is TCE . If
continued, TCE can be further transformed to dich�oroethy�ene (DCE) and then to viny�
ch�oride (VC) by sequentia� reductive dech�orination . Each ch�orine is rep�aced by
hydrogen. Fathepure and Voge� (1991) have shown that a significant amount of PCE
underwent reductive dech�orination to �ess ch�orinated products within a 37 .5-h
hydrau�ic residence time . Freedman and Gossett (1989) a�so indicated that it took on�y
2-3 days to convert PCE to VC, but, to further degrade VC to ethy�ene or CO 2 required
a much �onger retention time . The dech�orination of VC to ethy�ene was s�ow and on�y
partia��y comp�eted . In a fo��ow-up study (DiStefano, Gossett, and Zinder, 1991),
dech�orinating high concentrations (330 µM) of PCE to ethy�ene and sma�� amounts of
VC was achieved in a mixed anaerobic methano�-PCE enrichment cu�ture . In genera�,
the re�ative rate of dech�orination decreases as ch�orine atoms are sequentia��y removed .
1 0
Thus, the transformation products of PCE, such as TCE, DCE, and VC with �ess
ch�orine atoms per mo�ecu�e, tend to persist �onger in anaerobic environments than PCE .
A bio�ogica� process for remediation of groundwater contaminated with PCE and
TCE can on�y be app�ied if the transformation products are environmenta��y acceptab�e
(Bouwer and McCarty, 1983) . Conversion of PCE to TCE or �ess ch�orinated a�kenes
is of �itt�e or no benefit . Trich�oroethy�ene, cis-1,2-DCE, trans-�,2-DCE and VC are
a�so regu�ated under the 1989 Safe Drinking Water Act Amendments . In order to
achieve the comp�ete dech�orination of PCE, many researchers have attempted to coup�e
anaerobic processes fo��owed by aerobic processes, because the �ess ch�orinated
compounds are more �ike�y to be degraded by aerobic bacteria . However, PCE and its
biotransformation products are very vo�ati�e and hence wou�d be difficu�t to hand�e in
aerobic systems where aeration and stripping are possib�e . For anaerobic bio�ogica�
treatment to be usefu� and feasib�e, a�� of the possib�e intermediates from the reductive
dech�orination of PCE have to be further degraded . Recent�y, DiStefano et a� ., (1991)
showed that viny� ch�oride, the most resistant intermediate of reductive dech�orination of
PCE, can be further degraded to ethy�ene, a nonch�orinated and environmenta��y
acceptab�e product under anaerobic conditions . This resu�t suggests that reductive
dech�orination under anaerobic conditions may be best suited for treating PCE and TCE
contamination in groundwaters and industria� eff�uents .
2.4.3. Cu�tures
Methanogens / methanogenesis p�ay an important ro�e in the anaerobic
dech�orination of PCE . Fathepure and coworkers (1987 ; 1988a,b) demonstrated that
1 1
pure cu�tures of two methanogenic bacteria (Methanosarcina strains) were ab�e to
convert PCE to TCE . Various other methanogenic environments were used to achieve
the biotransformation of PCE . The reductive dech�orination of PCE may be caused by a
member(s) of a methanogenic consortium, such as strain DCB-1, a ch�orobenzoate-
dech�orinating organism iso�ated from a methanogenic consortium that was ab�e to
degrade 3-ch�orobenzoate (Fathepure and Boyd, 1987), and/or by methanogens
themse�ves . PCE dech�orination occurred in both pure cu�ture and in a methanogenic
consortium. In pure cu�ture, on�y a re�ative�y sma�� fraction of PCE underwent reductive
dech�orination. This is consistent with the genera� observation that anaerobes are �ess
active against xenobiotics in pure cu�ture. In addition, the different strains of
methanogens and ce�� mass cou�d a�so significant�y affect the extent of dech�orination .
Genera��y, even though both the methanogens be�ong to the same genus, they differ in
their abi�ity to dech�orinate PCE . Severa� studies (Bouwer and McCarty, 1983a,b ; Voge�
and McCarty, 1985) a�so found more rapid dech�orination under methanogenic
conditions than under su�fate-reducing environments and denitrifying conditions .
2.5. Degradabi�ity of TCE and �ess ch�orinated compounds
As stated ear�ier, TCE and �ower ch�orinated compounds may sti�� undergo
reductive dech�orination but at much s�ower rates . Under aerobic conditions these
compounds are a�so genera��y persistent in natura� environments. However, certain
aerobic microorganisms (e.g . methanotrophs) may degrade these �ess ch�orinated
compounds via oxidative mechanisms which are ineffective for heavi�y ch�orinated
compounds such as PCE. Aerobic environments have �itt�e chance to remediate waters
contaminated with a mixture of ch�orinated ethy�enes .
1 2
Under aerobic conditions in the presence of methane, TCE was bio�ogica��y
oxdized to CO2 (Wi�son et a� ., 1985) . The mechanism is TCE oxidation to
trich�oroethene epoxide by methane monooxygenase and then rapid hydro�ysis (Foge� et
a�., 1986). This biodegradation process did not produce other vo�ati�e ch�orinated
compounds, and the degradation rates decreased for more-ch�orinated compounds .
Viny� ch�oride was degraded more rapid�y than TCE, and PCE was not degraded at a�� .
2.6. Microbio�ogica� aspects of anaerobic digested s�udge
Methanogens / methanogenesis may be invo�ved in reductive dech�orination in
anaerobic communities . Anaerobic s�udge digestors are important methanogenic sites
where favorab�e growth conditions exist . Many anaerobic organisms are invo�ved in
anaerobic digestion. A common feature of anaerobic digestion is the formation of
methane. This gas formation in anaerobic digestors is a syntrophic process depending
upon the action of severa� types of anaerobic bacteria . The mechanism invo�ved in this
process inc�udes four steps : (1) hydro�ysis, (2) acidogenesis, (3) acetification, and (4)
methanogenesis. Biodegradab�e organic compounds are first hydro�yzed and degraded
to simp�er compounds by a variety of facu�tative and anaerobic organisms . These
simp�er compounds are subsequent�y transformed to short-chain fatty acids, carbon
dioxide and hydrogen gas by other non-methanogenic acidogens . Fatty acids are then
metabo�ized by hydrogen-producing acetogenic bacteria to acetate and hydrogen . These
organisms are unab�e to grow at partia� pressures of hydrogen >10 -3 atm., therefore
their maintenance within the methanogenic consortium depends on the continued
remova� of hydrogen by methanogens. Some hydrogen may be converted to acetate by
hydrogen-consuming acetogens in this step. In the fina� step, methane is generated
1 3
most�y from acetate by the acetic�astic methanogens. Part of the methane production can
be derived from hydrogen and carbon dioxide by hydrogenotrophic methanogens
(Levett, 1990) .
Genera��y, the methanogenic bacteria are very substrate specific and are
dependent on the non-methanogenic bacteria for their supp�y of substrate (Grady and
Lim, 1980). Within digestors, methanogenesis occurs at an optimum rate in the range of
pH 6-8. The optimum temperature for mesophi�ic digestion is 40°C . The mu�tistep
nature of anaerobic digestion is depicted in Fig. 2. In anaerobic s�udge, many kinds of
non-methanogenic bacteria may form hydrogen . Genera��y, this hydrogen serves as a
source of the reductant required for reductive dech�orination of PCE and its partia��y
dech�orinated intermediates if suitab�e enzyme(s) exist to divert hydrogen to
dech�orinating processes .
Many dech�orinating bacteria potentia��y exist in anaerobic digestors .
Methanogenic bacteria are an important group of anaerobic bacteria with dech�orinating
capacity . Because of their extreme habitat diversity, these bacteria may be present in
s�udge digestors . However, sewage s�udges from different sources vary in their
potentia� to dech�orinate various substrates (She�ton and Tiedje, 1984)
14
Fig. 2 Mu�tistep nature of anaerobic digestion of organic matter(Levett,1990)
Comp�ex organic matter
Proteins
Carbohydrate
Lipids
Hydro�ysis(c�ostridia)
Amino acids
Sugars
Fatty acids
Acidification(acetogens)
(H2 producing acetogens &H2-consuming acetogens)
Acetification
(acetic�astic methanogens)
Methanogenesis
/
H2O
C02
Acetate
Intermediate products
Acetate, propionate, butyrate, H2. C02
1 5
CH4
H2/C02
3. MATERIALS and METHODS
3.1. Chemica�s
The ha�ogenated organic compounds used were reagent-grade
tetrach�oroethy�ene (PCE), tich�oroethy�ene (TCE), cis-dich�oroethy�ene (cis-DCE),
trans-dich�oroethy�ene (trans-DCE), 1 .1-dich�oroethy�ene (1 .1-DCE), and viny� ch�oride
(V.C .) . A�� of them were purchased from A�drich Chemica� Co ., Mi�waukee,
Wisconsin, at >99% purity except V.C., which was obtained from F�uka Company,
Ronkon Koma, New York. The fo��owing non-ch�orinated chemica�s were used :
methane (A�drich Co .), ethane (A�drich Co.), and ethy�ene (ETH ; A�drich Co.), and
methano� (Fisher Scientific Co ., Pittsburgh, Pa.) .
3.2. Fresh s�udge and pond sediment
Fresh anaerobic digested s�udge for PCE-dech�orinating abi�ity test at the outset
was obtained from the anaerobic digestor at the Hyperion Wastewater Treatment P�ant,
LA., CA., and Chino Basin Wastewater Treatment P�ant (RP2), Chino, CA . Both of
the p�ants receive more than 70% of their wastewater from residentia� sources. The
retention times of the anaerobic digestor are about 15 and 30 days for Hyperion and
Chino Basin, respective�y . The anaerobic s�udge typica��y contains about 20,000 mg of
so�ids per �iter of mixed �iquid . Un�ess otherwise stated, the s�udge samp�es used in
various tests throughout the course of this study were prepared as fo��ows . After
transport to the �aboratory, fresh s�udge (100 mL) was dispensed into 120 mL capacity
serum bott�es in an anaerobic hood. The serum bott�e was then sea�ed with a Tef�on-
1 6
�ined rubber septum (Supe�co, Inc ., Be�fonte, PA) and an a�uminum crimp cap . These
s�udge samp�es contained no detectab�e �eve�s of PCE . A�� experiments were conducted
at 35°C under quiescent conditions. Fresh anaerobic pond sediment was obtained from
the botanica� garden at UCLA and transfered into serum bott�es as described above .
After PCE-dech�orinating abi�ity was demonstrated in the above cu�tures, five
more s�udges obtained from different wastewater treatment p�ants in Ca�ifornia were
examined to determine if PCE dech�orination was a genera� characteristic of anaerobic
s�udges. These five wastewater treatment p�ants are :
1 . Termina� Is�and Wastewater Treatment P�ant, San Pedro, CA .,
2. Chino Basin Wastewater Treatment P�ant (RP1), Rancho Cucamonga, CA.,
3. A�varado Wastewater Treatment P�ant, Union, CA .,
4. Va�encia Wastewater Treatment P�ant, Va�encia, CA ., and
5. JWPCP Wastewater Treatment P�ant, Carson, CA .
3.3. Methano�-enrichment cu�ture
An anaerobic methaogenic methano�-enrichment cu�ture was deve�oped in two
2.1-�iter Er�enmeyer f�asks on stir p�ates . Initia��y, 630 m� of digested s�udge obtained
from an anaerobic digester (Hyperion Wastewater Treatment P�ant, Los Ange�es, CA)
was used to seed each f�ask . Temperature was contro��ed at 35 °C. Each f�ask was fed a
non-steri�e so�ution (Tab�e 1) containing 100 mM methano� as the carbon source. Both
f�asks were comp�ete�y mixed and operated semi-continuous�y (refed dai�y) . The dai�y
feed vo�ume was increased gradua��y from 200 m� to 2000 m� . The mixing in both
1 7
f�asks was discontinued for 3 hours prior to dai�y feeding to a��ow the biomass to sett�e
and �imit biomass wash-out.
After 6 months of operation, the MLSS in both f�asks had increased to
approximate�y 5000 mg/L and the MLVSS was 65-75 % of that. The methane
production was about 65-70% of tota� gas production .
3.4. Anaerobic nutrient medium
To maintain the activity of the anaerobic s�udge used in various dech�orination
tests, an anaerobic minera� medium (modified from Freedman and Gossett, 1989) p�us
50% of supernatant of fresh Hyperion anaerobic digestor s�udge was prepared and used
whenever the nutrients in the s�udge treatment system needed rep�enishing . The
anaerobic medium was made up of 50% deionized water and 50% fresh s�udge
supernatant. The supernatant was obtained by centrifuging the s�udge �iquid at 3500 rpm
for 20 minutes. The minera� materia�s in the medium consisted of ( per �iter of �iquid
medium) : NH4C1, 0 .20 g; K2HP04 . 3H20, 0.01 g; KH2PO4, 0.055 g; MgC12 .6H2O,
0.20 g; trace meta� so�ution (per �iter, 0 .1 g of MnC12 .4H2O; 0.17 g of CoC12 .6H2O;
0.10 g of ZnC12; 0.20 g of CaC12 ; 0.019 g of H3BO4 ; 0.05 g of NiC12 .6H2O; and
0.020 g of Na2Mo04 . 2H2O, adjusted to pH 7 with NaOH or HCI), 10 m� ; resazurin,
0.001 g; Na2S •9H2O, 0.50 g; FeC12 .4H20, 0.10 g; NaHCO3, 5 .0 g; and yeast extract,
0.50 g. The first six components were boi�ed with deionized water and s�udge
supernatant to remove oxygen, and then coo�ed under an N2 purge. After coo�ing, the
remaining components except sodium su�fide were added and the purge gas was
1 8
switched to C02/N2 (20%/80%). The medium was autoc�aved and stored in 120 m�-
serum bott�es unti� they were needed. Sodium su�fide was then added before use .
Tab�e 1 : Composition of Feed So�ution (Voge� and McCarty, 1985)
Organics
Methano�
3200
Note: 6N HC1 was used to adjust pH to 7 .2 - 7.4
1 9
Compound Concentration (mg/�iter)
Inorganics
NaHCO3 3360
NH4C1 215
MgS04.7H2O 150
K2HPO4 60
CaC12 25
KC� 25
FeC12 5 .0
CoC12 0 .5
NiC12 0.25
3.5 . Ana�ysis
3.5.1 . Purgeab�e ch�orinated compounds
Parent compounds (PCE/TCE) and dech�orinated products (TCE, DCE and VC)
in the experimenta� serum bott�es were identified and quantified using a 5890A Hew�ett-
Parkard gas chromatograph equipped with a f�ame ionization detector (FID) and a
purge-and-trap device (Tekmar Mode� LSC-2, Cincinnati, Ohio) . The bott�es were
vigorous�y shaken by hand and then centrifuged 5 min. at 3500rpm. before each time
samp�ing. Water samp�es (5 mL) were prepared by adding 100 µL of supernatant to 4 .9
mL of deionized water . 100 µL of supernatant was taken from each bott�e with a 100-
µL syringe and then transferred into a 5-mL gas-tight syringe in which 4 .9 mL of
deionized water has been fi��ed . Samp�es (5 mL) were purged onto a Tenax TA
absorbent trap (Supe�co Co ., Cat. No. 2-0294M) with he�ium (40 mL/min for 8 min)
and desorbed (180°C for 4 min) onto a DB-624 capi��ary co�umn (30 m by 0 .53-mm
inner diameter, J&W Scientific Co., Fo�som, CA). The carrier gas was he�ium (8 to 10
m�/min). The temperature program was as fo��ows : the initia� temperature was 35°C
with a 5 min ho�d, then increased to 70 °C at 5 °C/min, with a fina� ho�d of 1 min at
70°C. The detector temperature was 200°C. The air and hydrogen gas f�ow rates to the
FID were 310 and 31 m�/min, respective�y . Identification and quantification of
chemica�s was accomp�ished by comparison to carefu��y prepared externa� standards .
The output signa� was recorded using a Hew�ett-Packard Mode� 3392 integrator .
Retention times and detection �imits for each compound of interest in this research under
these GC conditions are �isted in Tab�e 2 . Lower detection �imits are possib�e with the
20
ana�ytica� system used, but the va�ues given in Tab�e 2 are adequate for the purposes of
these experiments .
In order to eva�uate the accuracy of the ana�ytica� method, contro� bott�es were
prepared with 50 µcoo� of each tested chemica� per 100 mL and incubated for at �east 12
hours. Both �iquid and gas samp�es were ana�yzed as described above, and the tota�
mass was compared to the added mass to ca�cu�ate the percent recovery for each
chemica�. The ana�ytica� method yie�ded resu�ts that were within +1-5% of the correct
amount of tested chemica� added in each bott�e .
Tab�e 2 : Retention Times and Detection Limits
21
Parameter Formu�a Retention time(min) Detection �imit(p.g/L)
Pech�oroethy�ene C12C=CC12 9.99 0.7
Trich�oroethy�ene C12C=CHCI 5 .80 0.5
Cis- 1,2-dich�oroethy�ene C1HC=CHCI 3 .35 0.5
Trans-�,2- C1HC=CHCI 2 .35 0.3
dich�oroethy�ene
1 . 1 -dich�oroethy�ene C12C=CH2 1 .87 0.5
Viny� ch�oride CIHC=CH2 1 .56 0.3
3.5.2. Methane and ethy�ene
The methane and ethy�ene concentrations in the headspace of the serum bott�es
were measured by a Varian 3760 gas chromatograph equipped with a f�ame ionization
detector and a g�ass co�umn packed with 80/100 Porapak Q. The carrier gas was he�ium
at a f�ow rate of 30 mL/min . A 100-µL gas-tight syringe was used to remove 100-µL
gas samp�e for ana�ysis .
3.6. Experiments
Un�ess otherwise indicated, bio�ogica� experiments in this research were
conducted with 120 mL serum bott�es fi��ed with 100 mL of fresh digested s�udge that
were sea�ed with Tef�on-�ined rubber septa and a�uminum crimp caps .
3.6.1 . PCE-dech�orinating abi�ity test
At first, PCE-dech�orinating abi�ity of severa� cu�tures obtained from different
anaerobic habitats were tested. The tested cu�tures inc�uded:
1 . Hyperion digested s�udge .
2. Pond sediment.
3 . Mixture (1 :1) of Hyperion digested s�udge and pond sediment .
4. Chino Basin (RP2) digested s�udge .
5. Methano�-enrichment cu�ture .
22
The experiments were carried out in 120 m� serum bott�es containing 100 m� of
tested cu�ture �iquid. Methano� was used as the carbon source and the e�ectron donor .
The tests of Hyperion s�udge, pond sediment and their mixture were started with a �ow
PCE concentration (2.5 µmo� / L in �iquid phase) . The PCE concentration was achieved
by adding 0.3 µmo�es of PCE to each bott�e. Once the added PCE was consumed, fresh
PCE was added. After 28 days of operation, the amount of PCE added to each bott�e
was increased to test the dech�orinating abi�ity under higher PCE concentrations . In
testing of each of these three cu�tures, three different amounts (2.5, 25, and 123 µmo�)
of methano� were used in separate bott�es to observe differences in PCE dech�orination .
For the Chino Basin s�udge test, 369 and 4920 µmo�es of methano� (in tota�)
were app�ied to two separate bott�es containing a high initia� PCE concentration (49
µmo�es per bott�e). In the third bott�e containing a �ow initia� PCE concentration (0 .6
µmo�es per bott�e), 369 µmo�es of methano� (in tota�) was used . Two s�udge samp�es
obtained from two different digestors at the Chino Basin Treatment P�ant (RP2) were
tested. Identica� experiments for each of these s�udge samp�es were conducted.
The dech�orinating abi�ity of methano�-enrichment cu�tures was investigated in
the presence of methano� and hydrogen as the e�ectron donor. Three bott�es were
prepared for the methano�-enrichment cu�ture test . The first bott�e contained 49 µmo�es
of PCE and 123 µmo�es of methano� . The same amount of PCE and methano� was used
in the second bott�e but a�so under a H2/C02 (80 :20) atmosphere. In the third bott�e, 49
µmo�es of VC gas was incubated with 123 µmo�es of methano� .
23
To eva�uate if PCE dech�orination was a genera� characteristic of anaerobic
s�udges, five more fresh anaerobic digested s�udges obtained from different wastewater
treatment p�ants (Termina� Is�and, Chino Basin (RP1), A�varado, Va�encia and JWPCP
P�ant ) were tested. S�udge samp�es used in this test were prepared as decribed before .
For each s�udge, the PCE dech�orination test was started with a high PCE concentration
( about 67 mg/� in the �iquid phase without accounting for biosorption) and performed
in trip�icate. Forty nine µmo�es of PCE was added to each samp�e without adding any
carbon source . For Va�encia and JWPCP s�udges, an additiona� bott�e was prepared to
test the dech�orinating abi�ity under an even higher PCE concentration (about 134 mg/1) .
The PCE concentration was achieved by adding 98 µmo�es of PCE to each bott�e . A��
experiments were conducted at 35 °C under quiescent conditions .
3.6.2 . Treatabi�ity of ch�orinated compounds
Hyperion digested s�udge was chosen to conduct the treatabi�ity test for PCE,
TCE, cis-DCE, trans-DCE, 1 .1-DCE, and VC . The initia� dose for a�� of the ch�orinated
compounds was 0.3 µmo� per bott�e. After the first chemica� addition was consumed,
the dose for each test chemica� was doub�ed. Each test chemica� was tested in separate
bott�es to avoid interference with each other . 5-10 µmo�es of methano� was added to the
bott�es at the same time that PCE was added . For the PCE treatabi�ity test, two identica�
bott�es, with and without methano�, were prepared . The purpose was to determine if the
fresh digested s�udge was capab�e of degrading PCE without adding methano� (e�ectron
donor). After 42 days of semi-continuous operation, the dose of each chemica� was
increased to 49 µmo�es, and a higher amount of methano� was added (123 mo�es in
24
each bott�e). This was done to determine if acc�imation to a �ow concentration wou�d
faci�itate the degradation under high concentrations .
The treatabi�ity of a high initia� concentration of PCE and TCE was a�so
eva�uated. 49 µmo�es of PCE and TCE were initia��y added into two separate bott�es .
Once the initia� chemica�s were consumed, the same amount of PCE or TCE was added
again to observe the dech�orination in �ong-term operation . Each time, 246 µmo�es of
methano� was added with the PCE or TCE . An additiona� bott�e was used to test
treatabi�ity of high PCE concentration without methano� addition . These experiments
were accompanied by a contro� samp�e of water and an autoc�aved s�udge samp�e with
the same �iquid phase vo�ume (deionized water and s�udge, respective�y) and the
addition of the ch�orinated compounds .
3.6.3. Dech�orinated products and reductive dech�orination progression
test
The sequentia� reductive dech�orination of ch�orinated compounds (inc�uding
PCE, TCE, 3 isomers of DCE, and V.C.) in fresh anaerobic Hyperion s�udge was
examined. Six identica� fresh s�udge samp�es were used to assess the dech�orination fate
for each tested chemica� under anaerobic conditions without any addition of methano� .
For PCE dech�orination, one additiona� bott�e (methano� bott�e) received 5 µ� of
methano� (about 123 µmo�es) as the added carbon source and e�ectron donor. About 49
µmo�es of each test chemica� were added to separate bott�es containing 100 m� of fresh
s�udge. These serum bott�es were incubated in the dark without shaking at 35°C .
Routine GC ana�ysis of the supernatant was performed on a�� of the bott�es to observe
25
the change in the concentration of tested compounds and formation of intermediates over
time. A water contro� samp�e (100 m� of deionized water p�us the same amount of each
ch�orinated compounds) was incubated under the same conditions in order to examine
chemica� �osses from the serum-bott�e system .
The effect of TCE and VC presence on PCE dech�orination was a�so studied at
this stage. Dech�orination of a mixture of 49 µmo�es of PCE and 49 Rmo�es of VC was
conducted in a serum bott�e with the same amount of fresh s�udge, whi�e the mixture
containing 49 µmo�es of each of PCE, TCE, and VC was incubated in an additiona�
bott�e. Both of them contained 246 µmo�es of methano� as the e�ectron donor .
3.6.4. Toxicity and highest to�erab�e concentration
A�� of the six ch�orinated compounds were tested for their toxicity to
methanogenesis in Hyperion s�udge. For each test compound, 6 - 8 different dosages
(approximate�y 0 .55 to 250 µmo�es per bott�e) were injected to each of a series of
bott�es. After 3 weeks of incubation, 100 µ� of headspace gas samp�e was removed
from each bott�e for methane production ana�ysis . The methane production from each
bott�e was then compared with that of the b�ank contro� in each set of experiments
(containing the same amount of identica� s�udge cu�tures without the addition of
ch�orinated compounds) to determine the re�ative inhibition of methane production at
various chemica� concentrations .
In this experiment, supernatant �iquid samp�es from a�� of the bott�es were a�so
periodica��y ana�yzed to observe the extent of dech�orination of each ch�orinated
26
compound at various initia� concentrations and the possib�e ethy�ene production from
dech�orination of each ch�orinated compound .
3.6.5 . Semi-continuous operation
The six s�udge bott�es created in the dech�orination progression test for PCE,
TCE, 3 isomers of DCE, and VC respective�y, were periodica��y spiked with the same
chemica� to eva�uate semi-continuous operation . When the added ch�orinated compound
in each bott�e was degraded, the same amount of test chemica� (49 µmo�es) was
repetitive�y added to the bott�e accompanied with the addition of 123 µmo�es of
methano� . From the 59th day on, 5 .0 m� of supernatant was a�so removed and rep�aced
with fresh anaerobic nutrient medium (containing 50% of supernatant of fresh digested
s�udge and 500 mg of yeast extract per �iter) in each addition of ch�orinated compounds .
In addition to these cu�tures, two identica� mixed-cu�tures were created. 10 mL
samp�es from each of the six bott�es (mentioned above) were used to create a mixture to
determine if it cou�d dech�orinate PCE better and faster than a sing�e acc�imated cu�ture .
The mixture bott�es were operated semicontinuous�y . Whenever PCE was degraded to
VC, 5 mL of mixed �iquid was removed and rep�aced with fresh medium (containing 5
tL of MeOH) p�us PCE. After 16 days, semi-continuous operation was fo��owed by an
incubation period (40 days) when PCE additions were stopped to see if a�� the VC can
be further degraded to ethy�ene . During this period, rep�acement of mixed �iquid with
fresh medium and addition of methano� was continued . At the end of the incubation
period, the pressure in the headspace of each bott�e was measured and used to determine
the tota� mass of ethy�ene and methane production .
27
3.6.6. Effect of Methano� (e�ectron donor) on PCE dech�orination rate
The effect of methano� on PCE reductive dech�orination was examined by
incubating the acc�imated s�udge with various amounts of methano� and PCE at 49
µmo�es per bott�e . The methano� concentrations app�ied to each bott�e were 0, 123, 123,
1230, 2460, and 4920 µmo�es per bott�e (123 µmo�es was chosen in two bott�es) . At
the beginning, six bott�es, each containing 100 m� of acc�imated Hyperion s�udge and 49
µmo�es of PCE, were incubated with 123 µmo�es of methano� . After repetitive PCE
degradation in these six bott�es was demonstrated, various amounts of methano� were
added to each bott�e with the same amount of PCE dose (49 µmo�es). Whenever PCE
was degraded, 5 m� of �iquid was removed and rep�aced with the same amount of fresh
medium p�us PCE and methano�, except for one 123 µmo�es-methano� bott�e from
which 20 m� of �iquid was removed . The addition of PCE and methano� was repeated
severa� times to a��ow cu�tures in each bott�e reach stab�e condition. These resu�ts were
used to eva�uate the effect of methano� on the extent of PCE dech�orination and reaction
rate .
3.6.7. Effect of mixing on PCE dech�orination test
Five identica� bott�es containing 100 m� of fresh Hyperion digested s�udge were
prepared. The fresh s�udge in each bott�e was incubated with 49 µmo�es of PCE to
acc�imate to degrade PCE first . Once the added PCE was consumed, fresh PCE was
added. At the same time, 5 m� of supernatant was a�so removed and rep�aced with fresh
anaerobic nutrient medium p�us 123 µmo�es of methano� . After repetitive PCE
28
degradation in these bott�es was demonstrated, a�� s�udge �iquid was mixed together with
25 m� of nutrient medium and then dispensed into 5 bott�es again to create the
homogeneity of mixed cu�ture in each bott�e . These five bott�es designated as S 1 - S5
were used for this series of experiments .
At first, whi�e other samp�es were sti�� incubated under quiescent conditions, S5
samp�e was p�aced in a 35 °C shaker to observe differences in PCE dech�orination . After
10 days of incubation, the first run was ended . S 1 - S4 samp�es and 10 m� of nutrient
medium were mixed together and redispensed into origina� bott�es . At the same time,
2.5 m� of s�udge �iquid was removed and rep�aced with nutrient medium for S5 samp�e .
During the second run, S5 p�us S4 were incubated in the 35 °C shaker to test if the
sma��er amount of medium cou�d improve PCE dech�orination because the partia�
dech�orination of PCE accompanying a much higher methane production occurred in S5
which was p�aced in the shaker during the �ast run .
After 6 days, S 1- S3 were mixed with 7 .5 m� of medium as decribed above and
2.5 m� of s�udge from S4 and S5 was rep�aced with medium. In addition, nitrogen was
used to rep�ace the gas in the headspace of S2 and S3 whi�e other samp�es were sti��
under a N2/C02 (about 70 :30) atmosphere. This was done for preventing the methane
formation from carbon dioxide. The third run was conducted with 3 samp�es (S3 - S5)
incubated in the shaker. After this run (6 days), 2 .5 m� of s�udge �iquid was removed
from each bott�e and rep�aced with the same amount of medium . Nitrogen was sti�� used
for S2 and S3 samp�es . During the fourth run, S5 was removed from the shaker to test
if the comp�ete dech�orination abi�ity cou�d be recovered under quiescent conditions . The
fourth run proceeded 6 days .
29
The fifth run was started with the same conditions as those done for the �ast run
except no PCE being added to S5 . In this experiment, 49 µmo�es of PCE p�us 123
µmo�es of methano� were added to each bott�e before each run was started .
3.6.8. Effect of temperature on PCE dech�orination test
Temperature is an important factor which can inf�uence the microbia� activity .
The effect of temperature on the dech�orination process was tested by incubating the
fresh digested s�udge cu�tures with 49 µmo�es of PCE under �aboratory conditions
without temperature regu�ation .
3.6.9. PCE-dech�orinating abi�ity in o�d s�udges test
To examine the reductive dech�orination abi�ity in o�d s�udge, eight fresh
Hyperion s�udge samp�es prepared as described previous�y were incubated for 1, 2, 3,
4, 12, 14, 20 and 21 days, respective�y before PCE injection . For purposes of
comparison, these experiments were accompanied by a contro� samp�e of fresh
Hyperion s�udge with the same �iquid phase vo�ume and the addition of PCE (49
µmo�es) .
30
3.6.10. Bio�ogica� activated carbon process (BAC) test
Activated carbon is an amorphous form of carbon . It has been wide�y used for
its exce��ent adsorptive capabi�ity for a variety of substances because of the
characteristics of high porosity and high surface area. Nuchar granu�ar activated carbon
(grade WV-5, 10x25 mesh size), produced by Westvaco Corporation (Covington, VA)
was used throughout this study .
Granu�ar activated carbon (GAC) and/or powdered activated carbon (PAC) have
been increasing�y used for the treatment of water and wastewater through most of the
twentieth century. At the beginning, inf�uent water is app�ied to GAC co�umn . In this
case, bio�ogica� growth a�ways �ed to p�ugging prob�ems . Nonethe�ess, these operation
prob�ems �ed to the idea of adding PAC to the aeration tank of an activated s�udge p�ant .
However, both of them were referred to as bio�ogica� activated carbon. The bio�ogica�
activated carbon process (BAC) integrates bio�ogica� treatment and adsorption into a
sing�e reactor. In comp�ete mixing reactor, such system overcomes the p�ugging
prob�ems and �owers the adsorptive remova�s because the carbon tends toward
equi�ibrium with the treated eff�uent (Benedek, 1980) . The organic remova�s are a�so
higher than those of separate bio�ogica� and adsorption treatment .
This experiment concentrated on the effect of carbon addition on the PCE
dech�orination in a bio�ogica� treatment system . Six bott�es designated as U1 -U6 were
prepared for this series of experiments . Two grams of virgin activated carbon and 100
m� of anaerobic Hyperion digested s�udge preacc�imated with PCE degradation were
used in each bott�e . After bott�es were sea�ed, 49, 98, 196, 294, 392, and 490 µmo� of
31
pure PCE were injected into bott�e U1 to U6, respective�y . The above dosages resu�ted
in PCE concentrations ranging from 490 to 4900 gmo�/L (81 .3 to 813 mg/L) in �iquid
phase without considering the headspace-�iquid partitioning and adsorption on s�udge
and carbon. Both �iquid and gas samp�es were ana�yzed over the �ength of the
experiment .
3.6.11. Off�ine bioregeneration (OBR) test
The another way to combine bio�ogica� treatment with carbon adsorption is
off�ine bio�ogica� regeneration (OBR) . The process was proposed by Sigurdson and
Robinson in 1978 . In such a process, exhausted carbon is regenerated through
biodegradation. Bacteria are supposed to degrade adsorbed organics . Bio�ogica�
regeneration of the spent carbon and subsequent recovery and recyc�e of the carbon to
the adsorption process may be practiced because PCE has been demonstrated to be
biodegradab�e in anaerobic digested s�udge. Usua��y, regeneration of the spent carbon is
more economica� than rep�acement with virgin carbon . The conceptua� difference
between BAC and OBR is i��ustrated schematica��y in Figures 3 and 4 .
To examine the feasibi�ity of OBR for PCE �aden carbon, activated carbon was
initia��y saturated with PCE in adsorption bott�es containing deionized water, then was
regenerated in bioregeneration bott�es by the acc�imated anaerobic Hyperion digested
s�udge.
32
Grou
ndwa
ter
Nutr
ient
s
Bio�
ogica� treatment unit
Recyc�e of s�udge containing carbon
C�arification
unit
Figure 3
. Sc
hema
tic
of B
io�o
gica
� Ac
tiva
ted
Carb
on P
roce
ss (
BAC)
Bio�ogica� regeneration unit
C�arification unit
Granu�ar activated carbon adsorption unit
Figure 4
. Schematic of off�ine bio�ogica� regeneration p
roce
ss (
OBR)
Six of two grams of exhausted granu�ar activated carbon (preequi�ibrated
separate�y with 49, 98, 196, 294, 392, and 490 µmo� of PCE in 100 m� deionized
water) were taken out of those PCE-preadsorption bott�es and p�aced in six new bott�es
designated as S 1 - S6. These amounts of PCE were chosen based on pre�iminary
adsorption test resu�ts which provided an acceptab�e range of PCE �iquid concentration
for biodegradation . Each bott�e contained 100 m� of acc�imated s�udge �iquid and
approximate�y 20 m� of headspace . At the beginning a�� PCE was adsorbed on the added
carbon and no PCE was free in s�udge �iquid . Due to the non-equi�ibrium of PCE
distribution between the �iquid and carbon, desorption of previous�y adsorbed PCE
from carbon to the �iquid was supposed to occur to approach the equi�ibrium . This
c�osed bott�e system was designed to emp�oy desorption and microbia� activity for the
regeneration of spent, PCE-bearing carbon . This was a�so used for the investigation of
the concept of off�ine bioregeneration. Bott�es were operated under batch condition and
incubated at 35 °C quiescent�y. Both �iquid and gas samp�es (100µ�) were withdrawn
periodica��y to determine PCE and its dech�orinated product concentration in the
bioregeneration bott�e .
35
4. RESULTS
4.1. Ch�orinated chemica� �oss contro�
First, the integrity of the serum bott�e system sea�ed with Tef�on-�ined rubber
septa and a�uminum crimp caps to prevent sorption and vo�ati�e �osses of ch�orinated
compounds was demonstrated . Over a period of 60 days, the decreases in tota� masses
of PCE, TCE, cis-DCE, trans-DCE, 1 .1-DCE, and VC in a water contro� ( shown in
respective�y, in the water contro� bott�e wrapped with foi� (shown in Fig . 6). The
reduced �oss of ch�orinated compounds observed in the bott�e wrapped with foi� is
consistent with the more stab�e chemica� characteristics of ch�orinated a�iphatic
compounds without exposure to �ight . By and �arge, ch�orinated compounds are re�ative
unstab�e under �ight .
PCE �oss and abio�ogica� reductive dech�orination of PCE were a�so examined in
an autoc�aved-s�udge contro� . Data are shown in Fig . 7. Over a period of 135 days,
about 81% of 245 µmo�es of cumu�ative PCE dose sti�� remained . During the same
period, 6 .63 µmo�es of TCE and trace amounts of cis-DCE (0 .41 µmo�es) were
produced. This experiment a�so demonstrated that the integrity of the serum-bott�e
system is acceptab�e and the abio�ogica��y-mediated reductive dech�orination of PCE is
insignificant in anaerobic digested s�udge .
36
Fig. 5 ) which contained 49 µmo�es of each compound were 42, 37, 19, 36, 25, 25%,
respective�y . Over the same period, the �osses were on�y 28, 26, 13, 28, 10, 15%,
Fig.
5Th
e�o
ssof
vo�
ati�
e ch
�ori
nate
d ch
emic
a�s
in D
.Lwa
ter
cont
ro�
samp
�e w
itho
ut a
�umi
nium
foi�
.10
0 80
CI U U U40 20 0
010
2030
Time (days)
4050
60
Fig.
6The
�oss
of
vo�a
ti�e
ch�
orin
ated
chemi
ca�s
in
D .Lw
ater
contro� samp�e with a�uminium foi� c
over
to
prev
ent
degradation from �ight
.10
0 80 60 40 20-
00
10
20
30
40
50
60
Time
(da
ys.)
70
-
PCE
tran
s,DC
E
-TCE
1.1,
DCE
cis,
DCE
Viny
� ch
�ori
deQ
Fig .
7PC
E �o
ss a
nd i
ts a
biot
ic p
rodu
cts
in a
n au
toc�
aved
s�ud
ge c
ontr
o� s
amp�
e.
0.
.
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PCE
•
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..
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- cumu�ative addition
............
.... ..... i,*-,*
-*...... .... .
. .. ...... ......
............
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I
....
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50
100
Reac
tion
Tim
e (d
ays)
•
150
400
350
0 E30
0
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020
0
150
100 50
LU P-4
0
4.2. Mass ba�ance
A methodo�ogy for performing mass ba�ances of ch�orinated compounds in the
120 m�-serum-bott�e system containing 100 m� of digested s�udge was deve�oped by
considering the headspace-�iquid partitioning and biosorption within the bott�e .
Genera��y, the tota� mass of ch�orinated compounds in an experimenta� bott�e can be
expressed as :
TM =E((C�,iV 1 +Cg,iVg+KiC �,i(1/ni)M)/MW .)
(1)1
where
TM= tota� mass (µmo�e),C �,i= concentration of ch�orinated compound (i) in �iquid phase (µg/1),
V1 = vo�ume of �iquid phase (�iter),
Cg,i = concentration of ch�orinated compound (i) in gas phase (µg/1),
Vg= vo�ume of gas phase (�iter),
Ki = equi�ibrium constant indicative of adsorptive capacity,ni = constant indicative of adsorption intensity,M= mass of bio-adsorbent (gram), andMWi= mo�ecu�ar weight of ch�orinated compound (i) (gram/mo�e) .
However, for di�ute rea� so�utions, a chemica� concentration in gas phase is
proportiona� to the concentration of that component in the �iquid phase based on the
Henry's �aw, expressed as :
Cg,i = HciC1j
(2)
where Hci = Henry's �aw coefficient of ch�orinated compound (i)
40
By substituting equation (2) into equation (1), tota� mass can be expressed as:
TM=)((C1,iV1 +HCiC�,iVg+KiC�,i(11ni)M)/MW1.) (3)
To obtain the tota� mass of ch�orinated compounds, vo�ati�ity and biosorption of
each ch�orinated compound must be determined .
Henry's �aw coefficient
The modified EPICS (Equi�ibrium Partitioning in C�osed Systems) procedure
(Gossett, 1987) was app�ied to measure Henry's �aw coefficient for a�� chemica�s at
35°C. For each chemica�, Henry's coefficients were measured in six 60-m� serum
bott�es. Three contained 50-m1 �iquid (D .I.water) vo�umes; the other three, 5 m�. Bott�es
were prepared as fo��ows: D .I. water (5 or 50 m�) was pipetted to each serum bott�e, the
bott�es were sea�ed with tef�on-�ined rubber septa and a�uminum crimp caps ; the
appropriate chemica� was injected into each bott�e with a gas-tight syringe . The �iquid
vo�ume and the quantity of injected chemica� were determined by gravimetric means .
The six EPICS serum bott�es were then incubated for 24 hours at 35°C . The �iquid
phase concentration in each bott�e was ana�yzed for the determination of Henry's �aw
coefficient . For each chemica�, nine possib�e pairings of trip�icate high and �ow �iquid
vo�ume EPICS bott�es provided nine possib�e estimates of Henry's �aw coefficient
based on the fo��owing equation :
4 1
where
r = M2C1,1M1C1,1,2
H - V1,2-NI'�
(4)
c rVg, �-Vg,2
M = tota� mass of a vo�ati�e so�ute added to a serum bott�e (µg),
V = vo�ume (�iter),
C = concentration (µg/1),
1, g = �iquid and gas, respective�y, and
1, 2 = bott�e 1 and bott�e 2, respective�y .
Henry's �aw coefficients for a�� ch�orinated ethy�enes are �isted in Tab�e 3 .
Furthermore, Henry's �aw coefficients were a�so determined in the s�udge system by
direct measurement of gas and �iquid equi�ibrium concentrations in a 120-m� serum
bott�e containing 100 m� anaerobic digested s�udge .
Tab�e 3 . Summ of He 's �aw coefficient (unit�ess)
( ) : standard deviation .
42
Henry'scoeff.
PCE TCE cis-DCE trans-DCE 1 .1-DCE VC
in watersystem
1 .06 (0.07) 0.61 (0.02) 0.26 (0.01) 0.65 (0.06) 1 .40 (0.08) 1 .45 (0.18)
in s�udgesystem
0.81 (0.05) 0.52 (0.02) 0.22 (0.01) 0.53 (0.01) 1 .63 (0 .09) 1.39 (0.07)
Biosorption
The characteristic constants of biosorption for each test ch�orinated compound in
the s�udge system at 35°C were determined by using the Freund�ich Isotherm . It has the
fo��owing form
x/M = KCe 1/n
where
x = mass of adsorbate adsorbed on bio-adsorbent (µg),
M = mass of bio-adsorbent (gram),
K = equi�ibrium constant indicative of adsorptive capacity,
n = constant indicative of adsorption intensity, and
Ce = so�ution concentration at equi�ibrium after adsorption (µg/1) .
The constants used in equation (5) were determined by p�otting the mass
adsorbed on per unit mass of bio-adsorbent versus the equi�ibrium concentration on �og-
�og paper. The intercept is K, whi�e 1/n is the s�ope . A�� of the six ch�orinated
compounds were tested for their biosorption in the 120 m�-serum-bott�e system
containing 100 m� of Hyperion digested s�udge . For each test compound, 6 to 8
different masses (approximate�y 0.55 to 250 µmo�es per bott�e) were injected to each of
a series of bott�es . After 10 to 24 hours of incubation, both headspace gas and aqueous
concentrations in each bott�e were measured . The amount of chemica� adsorbed on the
s�udge was obtained by subtracting the masses of the chemica� in both gas and aqueous
(5)
43
phases from the tota� added chemica� in each bott�e . The experimenta� resu�ts are
summarized in Tab�e 4 .
Tab�e 4. Summ of Freund�ich characteristic constants of bioso tion
Based on the Freund�ich mode�, x /M = K Ce ^ ( 1/n), (Units : Ce = µg/1 ; x = µg ; M = g) .
Figure 8 shows typica� resu�ts of the serum bott�e experiment when sequentia�
dech�orination of PCE to VC occurs. The measured concentration do not ba�ance the
added PCE concentration due to vo�ati�ization into the bott�e's headspace and adsorption
to the bioso�ids .
To account for vo�ati�ization and biosorption, the respective fractions were
estimated using Henry's �aw and an adsorption isotherm . The amounts vo�ati�ized and
adsorbed are significant, as the fo��owing examp�e shows . If 49 µmo�es PCE were
added, it wou�d correspond to 490 µmo�es per �iter (81 .3 mg/1) in the �iquid phase .
44
Constant PCE TCE cis-DCE trans-DCE 1 .1-DCE VCK 0.2042 0.2907 0.1050 0.0185 0.0748 0.0910n 1 .0040 1 .2309 1 .3993 1 .0821 1 .1946 1 .4186Experimenta�range (Min .)
(µ�a)
182.50 213.95 381 .41 9993 .42 273 .83 2013 .87
Experimenta�range (Max .)
(µms)
60569 135161 180147 160443 113174 111741
Corre�ationcoefficient (r)
0.9998 0.9928 0.9690 0.9874 0.9766 0.9234
Fig.
8Th
e measured concentrations of ch�orinated compounds
in �
iqui
d ph
ase
with
out
cons
ider
atio
n of
bio
sorp
tion
and vo
�ati
�ity
.600
500
400
H30
0
200
100 0
0
PCE
14
TCE-
......
.......
......
......
.......
...-
cis.
DCF,
.
trans,DCE
0
:
;DCE
......
.......
.....
.......
.....
......
....
.......
......
.....
...'
---*
!-VC
'Nomina� conc
.--••••
... .----••••••
..v;
;> .
..........
.;-----
--....
5010
015
0
200
250
Reac
tion
Tim
e (h
rs.)
300
350
Note
:When biosorption and vo�ati�ity are not taken into consideration, the measured
conc
entr
ations for each compound do not add up to the tota� no
mina
� co
ncen
trat
ion
.
Once these
fac
tors
are
tak
en i
nto
acco
unt,
the
sum
of
meas
ured chemica�s
at e
ach
time
poi
nt d
o ad
d up
to
the
cumu
�ati
ve a
ddit
ions
int
o the system
(com
pare
Fig
. 8 to Fig
. 9).
400
Using Henry's �aw, this concentration wou�d be reduced to on�y 421 µmo�es/1 (69 .9
mg/1). The actua� measured concentration was on�y 107 µmo�es/1(17 .8 mg/1). Using the
isotherm shown in Tab�e 4, to account for biosorption and vo�ati�ization, the ca�cu�ated
concentration in �iquid phase is 104 µmo�es/�, which is very c�ose to the measured
concentration of 107 µmo�es/� . This indicates that the methodo�ogy is �ogica��y
reasonab�e for performing mass ba�ances of ch�orinated compounds in the serum bott�e
system.
Figure 9 shows a ca�cu�ated mass ba�ance using the above procedures . It is
observed that the tota� app�ied and measured p�us ca�cu�ated concentrations very c�ose�y
ba�ance. The sma�� departure at the end of the test is probab�y due to ethy�ene
production, which was not measured in this test .
4.3. PCE-dech�orinating abi�ity and a genera� characteristic of s�udges
PCE-dech�orinating abi�ity :
PCE-dech�orinating abi�ity was demonstrated in a�� of the first five anaerobic
cu�tures tested. Viny� ch�oride (VC) was the major accumu�ated dech�orination product
in a�� of these cu�tures, exc�uding pond sediment (ethy�ene production was not measured
at this stage because it was expected to be be�ow the detection �imit if there is any
produced). PCE dech�orination on�y proceeded to cis-DCE in pond sediment cu�ture ;
however, after mixing with Hyperion digested s�udge, cis-DCE cou�d be further
dech�orinated to VC. This resu�t suggests that the Hyperion s�udge cu�ture is main�y
responsib�e for PCE dech�orination in the mixture.
46
Fig
.9
Mass
ba�ance of ch�orinated compound
s in
the
Hyp
erio
ns�
udge system when biosorption and vo�ati�ity are inc�uded
.
0
E
1.1,DCE
TEE
Viny
� ch
�ori
de
:..
.---•--
.. -
.cis,DCE----- Sum
trans,DCE
~ PC
E do
sage
:
:... .
........
........
........
........
........
.....49 µmot of PCE
was added
100
200
300
400
reac
tion
tim
e(hr.
)50
060
070
0
I U O
120
a A O
100 80
0
o W~-
60
b w
40
U20 0
Hyperion s�udge cu�ture was observed to have the greatest abi�ity to
dech�orinate PCE among a�� the tested cu�tures . The first addition of 0 .3 µmo�es of PCE
was tota��y degraded to VC in 3 days without any significant �ag period. Under the
condition of �ow initia� PCE concentration, a tota� (3 .45 µmo�es) of 8 successive PCE
additions were degraded to VC in 29 days . After demonstrating PCE dech�orination at
�ow concentrations, higher concentrations were tested . The s�udge cu�ture sti��
transformed PCE to VC, but , it took �onger. The resu�ts are shown in Fig. 10 .
Different amounts of methano� addition (123, 25, and 2 .5 µmo�es) in a series of bott�es
did not significant�y change PCE dech�orination rates . The resu�ts for different amount
of methano� addition are provided in Appendix A (Fig . A-1 and A-2) .
Pond sediment ( un�ike Hyperion s�udge ) demonstrated on�y partia� PCE-
dech�orinating abi�ity (see Fig . 11). In a�� three test bott�es (with different methano�
additions), cis-DCE was the major end product . A significant �ag period (from 3 to 17
days) was observed before PCE transformation was observed . In the bott�e with the
�east methano� added (2.5 µmo�es, each time), the �ag period is shortest but PCE-
dech�orinating abi�ity was decreased when PCE dose was increased, which was
be�ieved to be a resu�t of TCE accumu�ation . The who�e data are provided in Appendix
A (Fig. A-3 and A-4) .
Mixture (1 :1) of fresh Hyperion s�udge and pond sediment showed a simi�ar
abi�ity of PCE dech�orination to that observed in 100% Hyperion s�udge (see Fig . 12).
PCE dech�orination resu�ted in VC formation and accumu�ation . However in the samp�e
with 25 µmo�es of methano� added each time, about 50% of PCE was degraded on�y to
cis-DCE. After �onger incubation, it was converted to VC . It appears that the Hyperion
48
0
Fig. 10 PCE-dech�orinating abi�ity and formation of intermediatesin a batch cu�ture of fresh anaerobic Hyperion s�udge aftersuccessive additions of PCE and 123 µmo�es of methano� .
350 PCE
0
300
-X - TCE- -6 - cis,DCE
0 trans,DCE
i y
250 0 I.I,DCE-~- Vc
a
200
;-,0 ~
150
r) 100UNA
50 == see rig. 8aI rI00
Fig. 10a PCE-dech�orinating abi�ity and formation of intermediatesin a batch cu�ture of fresh anaerobic Hyperion s�udge aftersuccessive additions of PCE and 123 µmo�es of methano� .
20
0 5
20 40
60Reaction Time (days)
10
15
20Reaction Time (days)
80
25
Note :The major product from PCE dech�orination was VC .
49
100
30
A
PCE-dech�orinating abi�ity and formation of intermediates inanaerobic pond sediment after successive additions of PCEand 123 µmo�es of methano� .
=,
3 0v
aN+ ~oa
25bob
.~
20o.a~~
15M
0
10
5
0
0
.00W*.. .-
0
20
5
50
10
15
20Reaction Time (days)
40
60
80
100Reaction Time (days)
Fig . 11a PCE-dech�orinating abi�ity and formation of intermediates inanaerobic pond sediment after successive additions of PCEand 123 µmo�es of methano�.
40
35
25 30
Fig. 11
60
050
y wU IV
bO 'b
40
30.~ a
o.,
20
A 10'
Fig . 12 PCE-dech�orinating abi�ity and formation of intermediates ina mixture cu�ture (1:1) of fresh anaerobic Hyperion s�udgeand anaerobic pond sediment after successive additions of PCEand 123 µmo�es of methano� .
15
10
5
00
0
20
Fig . 12a PCE-dech�orinating abi�ity and formation of intermediatess ina mixture cu�ture (1 :1) of fresh anaerobic Hyperion s�udgeand anaerobic pond sediment after successive additions of PCEand 123 µmo�es of methano� .
25
5
40
60Reaction Time (days)
10
15
20Reaction Time (days)
5 1
80
25
100
30
s�udge cu�ture has contributed to the further dech�orination of cis-DCE. The
dech�orination resu�ts for different amounts of methano� addition are provided in
Appendix A (Fig . A-5 and A-6) .
Chino Basin (RP2) s�udge a�so showed partia� PCE-dech�orinating abi�ity
in most cases . During the first 90 days, on�y one PCE addition was added to each
bott�e, and cis-DCE was found as the major dech�orination product in most of the bott�es
with various amounts of PCE and methano� added (Shown in Appendix A (Fig . A-7
through A-11)) . However, from one of Chino Basin (RP2) s�udge with 49 µmo�es of
initia� PCE dose and a tota� methano� addition of 4920 µmo�es, comp�ete PCE
dech�orination to non-toxic ethy�ene was observed at the end of the 90 day of
incubation. About 22 .6 µmo�es of ethy�ene were produced from the dech�orination of 49
µmo�es of PCE initia��y added. After that, PCE was comp�ete�y dech�orinated to
ethy�ene at even faster rates (51 µmo�es of ethy�ene produced in 29 days) . The resu�ts
are shown in Fig. 13. To try to improve the PCE-dech�orinating abi�ity after 90 days of
operation, the supernatant in each bott�e was rep�aced with the same vo�ume of fresh
anaerobic medium . The medium was prepared with 50% of fresh Hyperion s�udge
supernatant and 50% of D .I. water containing known concentrations of sa�ts . Methano�
was a�so used and the initia� dose of PCE was 49 µmo�es for each bott�e . After two
months of incubation, significant ethy�ene productions (50.9 and 44.3 µmo�es,
respective�y) were observed from two more bott�es, whi�e cis-DCE was sti�� the major
end product of PCE dech�orination in three other bott�es . The improvement of the
dech�orinating-abi�ity was achieved in part of these bott�es . However, the factor(s) that
has contributed to the improvement in comp�ete PCE dech�orination abi�ity is not c�ear .
52
Fig.
13PCE-dech
�ori
nati
on a
bi�i
ty a
nd f
orma
tion
of intermediates in a
batch cu
�tur
e of
fre
sh a
naer
obic
Chi
no B
asin (RP2) s�udge I
.100
A20 0
JAJ
0
MeOH
add
itio
n=49
20 µ
coo�
.PC
E ad
diti
on=4
9 µc
oo�
.
50
100
Reaction Time (days)
Note
:1 . 4
9 µm
o�es of PCE was added at the beginning of both sta
ges
(A &
B).
2 . Data ob
tain
ed a
fter
90
days
ind
icat
es P
CE d
ech�
orin
atio
n and formation
of i
ntermediates with initia� addition of 49 µmo�es of PCE
, de
crea
sed
concentration of methano�, and fresh medium rep�acemen
t.
150
PCE
1.1,DCE
TCE
--~-- VC
MeOH
add
itio
n=24
60 µ
mot
.PC
E ad
diti
on=4
9 It
mo�
.ci
s,DC
EEt
hy�e
netr
ans,
DCE
Methano�-enrichment cu�ture was deve�oped with the inocu�um of
Hyperion digested s�udge. After one-year exposure of methano� (enriched by methano�
(100 mM) as the on�y carbon and energy source), the enrichment cu�ture was found not
to degrade PCE any better than the origina� fresh Hyperion s�udge ; on the contrary, its
abi�ity to dech�orinate PCE is even worse . Over 79 days of incubation, PCE was main�y
dech�orinated to TCE in methano�-enrichment cu�ture with methano� (615 µmo�es tota�)
as the major carbon source (see Fig . 14). In the second bott�e with the same amount of
methano� under a H2/CO2 (80/20%) atmosphere (1 .34 Kg/cm2), a �onger �ag period
(about 30 days) was observed before PCE transformation to TCE significant�y
occurred. TCE was the on�y observed end-product suggesting that the hydrogen can not
induce �ater transformation steps .(see Fig. 15) .
In the third methano�-enrichment cu�ture bott�e, VC was incubated with the same
amount of methano� and hydrogen to test VC dech�orination . No detectab�e VC
dech�orination was observed (Fig . 16) .
Improved PCE-dech�orinating abi�ity was found in concentrated mixture. After
80 days of incubation, the ce�� masses in the three methano�-enrichment cu�ture bott�es
were centrifuged and transferred into one bott�e, and then taken up to 100 m� by adding
fresh anaerobic medium. 49 µmo�es of PCE was comp�ete�y dech�orinated to ethy�ene in
24 days. The same amount of PCE that was added thereafter was stoichiometrica��y
transformed to ethy�ene at an even faster dech�orination rate within 9 days .(Fig. 17) .
The improvement of PCE-dech�orination abi�ity was possib�y due to the concentrated
ce�� mass, rep�acement of nutrient medium, or induction .
54
Fig. 14 PCE-dech�orination abi�ity and formation of intermediatesin a batch system of MeOH-enrichment cu�tures .(methano� was used as a so�e carbon and energy source .)
60
50
40
30
20
10
0
A
20
0
0
0
10 20
PCE-dech�orination abi�ity and formation of intermediatesin a batch system of MeOH-enrichment cu�tures.(methano� & H2 were used as carbon and energy source.)
e °
\
ee
10 20
30
40
50Reaction Time (days)
55
PCETCE
6
cis,DCE0
trans,TCE9
1.1,DCE-I- Viny� ch�oride
F - Sum
60 70
6030
40
50Reaction Time (days)
Note: These resu�ts indicate that H2 does not appear to have an effect on PCEdech�orination in the MeOH-enrich cu�tures .
70
80
80
Fig.
16Viny� ch�oride-dech�orination abi�ity and formation of
interm
edia
te i
n a
batc
h sy
stem
of
MeOH
-enr
ichm
ent
cu�tur
es. (methano� & H2
were
use
d as
car
bon
and energy source.
)100 0
Low ce�� mass
0
Viny
� ch
�ori
de&
�ow MeOH,
H2
Ethy�ene
S -
Sum
-,,
1
1
1
1
1
1
1
1
1
1
1
1I
1
1
1
11
1
1
1
1
1
1
1
1
1
1
0
10
20
30
40
50
60
70
80
Reac
tion
Tim
e (d
ays)
0
Fig
.17
Improved PCE-dech�orination abi�ity and formation of intermediates
in a
bat
ch system of concentrated MeOH-enrichment cu�tures
.(methano� was used as a so�e carbon and energy source
.)
100
=.
80
U Q0
PCE
TCE
cis,DCE
tran
s,TC
EO
1.1,
DCE
0
Viny� ch�oride
Ethy�ene
Low ce�� mass
& �ow MeOH
High
ce�
� ma
ss*
& high MeOH
020
40
60
80Re
acti
on T
ime
(day
s)100
* : The high ce��
mas
s is
the
con
cent
rate
d bi
omas
s of
the
3 M
eOH-
enrichment
cu�t
ures
exposed to PCE+MeOH, PCE+MeOH+H
2,and VC+MeO
H+H
2.Th
e im
prov
ed P
CE-dech�orination abi�ity was possib�y due to the
conc
entr
ated
ce�� mass, rep�a
ceme
nt o
f nu
trie
nt m
ediu
m, o
r in
duct
ion
.
120
PCE dech�orination - A genera� characteristic of s�udges :
The dech�orination of PCE in fresh anaerobic digested s�udges obtained from
five more different wastewater treatment p�ants (Termina� Is�and, Chino Basin (RP1),
A�varado, Va�encia, and JWPCP P�ant ) was tested to eva�uate if it is a genera�
characteristic of s�udges to metabo�ize PCE . Throughout the course of this study, the
trip�icate samp�es for each s�udge performed simi�ar�y . The sequentia� reductive
dech�orinations of PCE were observed in a�� s�udges . Comp�ete dech�orination of PCE
to non-ch�orinated ethy�ene was demonstrated in a�� test s�udges, except for the Chino
Basin (RP 1) s�udge cu�ture in which PCE was on�y dech�orinated to cis-DCE after 4
months of incubation. The comp�ete�y dech�orinating abi�ity under a higher PCE
concentration (162 .6 mg/1) was a�so demonstrated in the cu�tures obtained from Va�encia
and JWPCP Treatment P�ants . Without the addition of extra carbon source, PCE
dech�orination was achieved in a�� s�udge samp�es . This indicated that a�� fresh anaerobic
digested s�udges contain sufficient and adequate nutrients for PCE dech�orination .
Termina� Is�and s�udge degraded PCE via sequentia� reductive
dech�orinations . PCE was step-by-step dech�orinated to ethy�ene through TCE, cis-DCE
and viny� ch�oride. About 25 µmo�es of ethy�ene was formed from the dech�orination of
49 µmo�es of PCE after 20 days of incubation, whi�e the rest of PCE was ending up as
viny� ch�oride . Over 30 days of incubation, at �east 40 µmo�es of ethy�ene was observed
in a�� trip�icate samp�es . More ethy�ene production was observed after repetitive PCE
addition to these bott�es. At the end of this experiment, 82, 88, and 142 µmo�es of
ethy�ene were accumu�ated in the bott�es which received 98, 98, and 147 µmo�es of
PCE, respective�y. The data are shown in Fig. 18 .
58
yV7•
•d
0a Oo -
O0 ET
.0
A
V1VV0.b•
CIY .~
A
'C7 n•
0Y .naw0
0a
oou0A
Fig . 18
The comp�ete dech�orination of PCE in a batch cu�tureof fresh anaerobic s�udge obtained from Termina� Is�andWastewater Treatment P�ant.
100
80
60
40
20
80
60
40
140
100
80
60
40
20
0 40
60
80
100Reaction Time (day)
Note: figures a-c represent ana�yses of 3 Termina� Is�and wastewater s�udge samp�es .
20
59
120
Chino Basin (RP1) s�udge on�y dech�orinated PCE partia��y to cis-DCE . In
trip�icate samp�es, cis-DCE was the major end product. The comp�ete dech�orination
product, ethy�ene, was never detected . However, no any significant �ag period was
observed for PCE dech�orination even though it was dech�orinated to TCE very s�ow�y
during the first week (see Fig. 19). It took much �onger (about 50 days observed from
two of trip�icate samp�es) to tota��y degrade PCE to TCE. The transformation of TCE to
cis-DCE spent another 40 days . After that, no additiona� dech�orination ( VC formation )
was noted. A�though cis-DCE was continuous�y dec�ined from day 20 to 119 ( see Fig .
19a), it was most �ike�y due to �eakage �osses because no V .C. accumu�ation was
observed. For Chino Basin (RP 1) s�udge, much more methane production was
observed. After 20 days of incubation, a tota� of about 3000 µmo�es of methane was
yie�ded from Chino Basin s�udge, whi�e on�y 750 µmo�es of methane production was
observed from Termina� Is�and s�udge samp�es. If it were the reason for the s�ow and
partia� dech�orination of PCE is not c�ear . The another factor which may cause the worse
dech�orinating abi�ity is that Chino Basin (RP1) P�ant on�y receives �ess than 5% of their
wastewater from industria� sources. The exposure to a �ow percentage of industria�
wastewater may resu�t in a dramatic reduction in the comp�exity of microorganisms
invo�ved in the s�udge . Consequent�y, many dech�orinating bacteria may be exc�uded .
A�varado s�udge performed PCE dech�orination simi�ar�y to that in Termina�
Is�and s�udge . However, for an unknown reason, it a�so took �onger ( about 30 - 35
days) to comp�ete�y dech�orinate 49 µmo�es of PCE to VC . In addition, cis-DCE was no
�onger the on�y major product of TCE dech�orination . A significant amount of trans-
DCE (15 µmo�es ; about 30% of TCE) was observed to accumu�ate in the s�udge system
during the transformation of TCE to DCE . Fortunate�y, it was not so persistent as
60
V1YVa'0 ..w -~a,
0
oah
.C1
T =L
uq
10
0
500
o^
0
hvab r.
0a .a
+~ o
'aHa
T ::L
A
Fig. 19 The comp�ete dech�orination of PCE in a batch cu�tureof fresh anaerobic s�udge obtained from Chino Basin (RP1)Wastewater Treatment P�ant .
60
50
40
30
20
40
30
40
30
20
10
61
0
20
40
60
80Reaction Time (day)
Note: The time needed for each samp�e to dech�orinate PCE and TCE isdifferent . It may be due to the variation within the origina� s�udge .
100 120
expected. Trans-DCE and cis-DCE were comp�ete�y degraded to VC in 10 days . Over
83 days of incubation, 19 - 30 µmo�es of ethy�ene were formed in the trip�icate samp�es .
To investigate the dech�orination abi�ity in the o�d s�udge, an additiona� s�udge samp�e
was incubated 3 days under the same conditions without PCE addition . After that, PCE
was injected into the bott�e . PCE sti�� can be comp�ete�y dech�orinated to ethy�ene but it
took even �onger in each step of PCE dech�orination . More trans-DCE (24 µmo�es)
accumu�ated during the transformation of TCE to DCE than that observed in fresh
s�udge samp�es . At the end of the experiment (119 days of incubation), a tota� of 22 .5
µmo�es of ethy�ene was measured. The data are shown in Fig . 20. During the period of
experiment, an average of 2500 µmo�es of methane production was observed with the
fresh s�udge samp�es, whi�e the o�d s�udge samp�e produced 3000 µmo�es of methane in
tota� .
Va�encia s�udge took on�y 7 days to dech�orinate 49 µmo�es of PCE to cis-
DCE, without any significant formation of the other two isomers (trans-DCE and 1 .1-
DCE) . A�� of the cis-DCE was further dech�orinated to viny� ch�oride in the next 14
days. About 4.5 to 6 µmo�es of ethy�ene were produced from the trip�icate samp�es on
day 28 . After that, ethy�ene was formed at an even s�ower rate . The average methane
production from trip�icate samp�es was around 2500 µmo�es . The dech�orination abi�ity
under a higher initia� PCE concentration (98 µmo�es of PCE per bott�e) was investigated
in an additiona� s�udge samp�e . Interesting�y, the doub�ed amount of PCE did not take
any �onger for comp�ete dech�orination . Within 7 days, PCE was tota��y degraded to cis-
DCE. A�� of the cis-DCE was found to end up as viny� ch�oride on day 21 . However,
much more ethy�ene (22 .2 µmo�es) was produced on day 28, compared to the ethy�ene
production from the samp�e with a �ower initia� PCE concentration . This indicated that a
62
y
4 ..d.
O
oAyo4 =LOauvA
A
Mu7O y
w~O
00-in oe6.w -0-AC,
A
u
''C ..Od
QI YO
OAY Or.T :L.S -.CuinA
Fig. 20
The comp�ete dech�orination of PCE in a batch cu�tureof fresh anaerobic s�udge obtained from A�varadoWastewater Treatment P�ant .
60
50
40
30
20
10
0
50
40
30
20
10
0
50
40
30
20
10
0
50
40
30
20
10
00 40
60
80Reaction Time (day)
Note: 1 . a-c represent ana�yses of 3 A�varado wastewater s�udge samp�es .2 . d represents the resu�ts from an o�der s�udge samp�e (incubated3 days before PCE inocu�ation) .
20
63
100 120
PCE--o-- FM-0
Usns-DCE
- D-0
I .1-DCETCE-X-ds-DCE -~-- v.C .
_ ,
PCE ET11O
trans-DCE
•°
1.1-DCE-Q-
TCE--I-°
V .C.ds-DCE--6 -
0
PCE0° ET0 - none-DCE
•-°
1 .1-DCEWE-ds-DCE -°- V.C .---e--
1 , o
1
1
PCE EM-0-- asps-DCE
--IB --9-- 1 .1-DCE
-0-- if
TCE
0--6
cis-DCE --
4
V.C .
O /, '
higher concentration of viny� ch�oride may not inhibit the formation of ethy�ene in the
s�udge system . However, during the same period, on�y a sma��er amount of methane
(about 1900 µmo�es) was produced from the high PCE concentration samp�e . Whether
it was due to the competition between methanogenesis and reductive dech�orination for
e�ectrons avai�ab�e in the system, or the toxicity of ch�orinated compounds to
methanogens is not known current�y . The resu�ts are shown in Fig. 21 .
JWPCP s�udge took the shortest period of time (�ess than 13 days) to degrade
49 µmo�es of PCE to VC and make ethy�ene (1 .8 - 2.3 µmo�es) emerge ear�iest from the
comp�ete dech�orination of PCE among a�� tested s�udge . However, the ethy�ene
production rates observed from JWPCP s�udge were s�ower than that from Termina�
Is�and s�udge. Within 29 days, JWPCP s�udge on�y yie�ded 18 - 19 µmo�es of ethy�ene
from the dech�orination of 49 µmo�es of PCE, whi�e about 43 - 45 µmo�es of ethy�ene
had been produced in Termina� Is�and s�udge . At the end of the experiment (49 days),
34 - 40 µmo�es of ethy�ene and an average of 2500 µmo�es of methane were
accumu�ated in trip�icate samp�es . For the samp�e with a higher initia� PCE concentration
(98 µmo�es per bott�e), the comp�ete PCE dech�orination proceeded simi�ar�y to that in
Va�encia s�udge, but with a higher fina� ethy�ene production (83 µmo�es) . Again, the
fina� methane production (1700 µmo�es) was much �ower than that produced from the
samp�es with a �ower initia� PCE concentration . The data are shown in Fig . 22 .
Based on the above resu�ts, reductive PCE dech�orination was observed with a��
tested anaerobic digested s�udges . This indicates that the dech�orination of PCE may be
a genera� characteristic of anaerobic digested s�udges. However, due to an unknown
64
A
dA
dA
H
°
40v ..°
°da.
30
10W
0
40
20
50
40
0
0
The comp�ete dech�orination of PCE in a batch cu�tureof fresh anaerobic s�udge obtained from Va�enciaWastewater Treatment P�ant.
°
30
°
30
65
W
_ PCE --~- 1.1-DCE- TCE
cis-DCE ETH-~-O
traps-DCE
1
_--0.- PCETCE
$ 1.1-DCEV.C .-~
----E- E[Hx -e-- cis-DCEO-
trans-DCE
\
----0--M
PCE
9
1 .1-DCETCE
--°- V .C .Na-DCE
•
ETHtram-DCE
--6---0
t
-0-- PCE A
1 .1-DCE°
V.C .X TCEcis-DCE
-[-- ECHtrans-DCE
A0
0
Note :
10 20
30Reaction Time (day)
40 50
1 . a-c represent ana�yses of 3 Va�encia wastewater s�udge samp�es .2. d represents the resu�ts of PCE dech�orination under a higherinitia� PCE concentration.
A
yuC000Y .r
0ƒoff
M ..2-AVd
yV
0 0
G
Fig . 22
The comp�ete dech�orination of PCE in a batch cu�tureof fresh anaerobic s�udge obtained from JWPCPWastewater Treatment P�ant .
50
40
10
0
40
30
10
0
40
20
10
0
80
40
20
00 20
30
40Reaction Time (day)
Note: 1 . a-c represent ana�yses of 3 JWPCP wastewater s�udge samp�es .2. d represents the resu�ts of PCE dech�orination under a higherinitia� PCE concentration .
66
10
°
30
°
30
°
60
50
PCE 1 .1-DCE- p-0
v.C .i
---~(-- TCE
6 - cis-DCEv iram-DCE
--•- E-m
t--0--' PCE -- $ - 1 .1-DCE1
TCE-h- cts-DCC
-°-- V.C .'-W ETH
-0-- tsars-DCE ..
PCE--0-- L1-DCE-$--~- V.C .- TCE--
-~- cis-DCE -a EM- 0 =a-DCE
---..0-
PCE 9 1 .1-DCE
f!)--*-- TCE--6-- cis-DCE
aa
V.C.ETH
. .- 0- tnns-DCE
reason, digested s�udges from different sources varied in their potentia� to comp�ete�y
dech�orinate PCE to ethy�ene .
4.4 . Treatabi�ity of ch�orinated compounds
The biodegradabi�ity (defined as the capacity of a substance to undergo microbia�
attack, Painter and King, 1985) of each ch�orinated compound tested was significant�y
different from each other in fresh Hyperion digested s�udge . However, when the period
of incubation was �ong enough, a�� of the six tested ch�orinated compounds were
anaerobica��y biodegradab�e in Hyperion s�udge . The observed �ag periods for trans-
DCE, 1 .1-DCE, and VC were 12, 4, and 10 days, respective�y under a �ow initia� dose
(0.3 µmo�es of each tested compound p�us 5 µmo�es of methano� per bott�e) . No �ag
was observed in the degradation of PCE, TCE, and cis-DCE . After 6 additions into each
bott�e, PCE ,TCE, cis-DCE tended to accumu�ate . In order to determine if the
dech�orination capacity in each bott�e has been consumed, 49 µmo�es (much higher than
the dose (0.3-0.6 µmo�es) used before) of each ch�orinated compound p�us 123 µmo�es
of methano� were added to each corresponding bott�e and the remaining dech�orination
capacity of the Hyperion s�udge was tested in each bott�e . After another 58 days of
incubation, a�� of added compounds in each bott�e were either partia��y or comp�ete�y
dech�orinated and ethy�ene was detected in a�� the test bott�es . A summary of the resu�ts
is given in Tab�e 5 . This suggested that a�� six ch�orinated compounds can be degraded
and the higher concentrations of each ch�orinated compound may have contributed to the
faster dech�orination rates observed in each bott�e . Among a�� of the ch�orinated
compounds tested, dech�orination of PCE, TCE, and cis-DCE was more �ike�y to resu�t
67
Tab�
e 5 .
Abi
�ity
of
anae
robi
c Hy
peri
on s
�udg
e to
deg
rade
var
ious
ch�
orin
ated
eth
y�en
es t
o et
hy�e
ne.
ON 00
Note
: th
e me
than
o� a
ddit
ion
in e
ach
samp
�e w
as 1
23 µ
mo�e
s.
Dech�orinated products
Samp�e
Time
(days)
Init
ia�
dose
(µmo
t.)
PCE
()1coo
�.)TCE
(µco
o�.)
cis-DCE
(µco
o�.)
tran
s-DC
E(i
t-01
.)1.1-
DCE
(µmo�.
)V.
C.(µmo�.
)ET
H(µcoo�
.)PCE
049
.00
49.0
06
45.9
90.
730.
380.
000.
002.
1320
0.49
0.07
0.00
0.36
0.00
37.8
834
0.00
0.00
0.00
0.00
0.00
8.01
37.1
258
0.00
0.00
0.00
0.00
0.00
3.05
41.4
8PC
E wi
th0
49.0
049
.00
higher conc.
648
.80
0.37
0.04
0.00
0.00
3.61
of M
eOH
2042
.65
0.88
0.12
0.01
0.00
1.17
(123
0µmo
�es)
3415
.44
1.01
0.11
0.00
0.00
18.9
50.
3058
0.06
0.00
0.00
0.00
0.00
15.5
217
.33
TCE
049
.00
49.0
06
34.3
30 .
160.
130.
001.
9220
17.0
62.
610.
460.
006.
3834
0.00
0.15
0.00
0.00
10.9
111
.90
580.
000.
000.
000.
002.
9422
.66
cis-DCE
049
.00
49.0
06
48.4
40.
110.
002.
2120
0.00
0.00
0.00
29.6
534
0.00
0.00
0.00
6.00
44.5
858
0.00
0.00
0.00
4.71
53.1
3trans-DCE
049
.00
49.0
06
47.8
10.
000.
0620
35.6
10.
000.
9534
24.5
00.
003.
19N.
D.58
18.8
50.
005.
590.
231.1-DCE
049
.00
49.0
06
43.2
91.
5020
0.00
31.6
434
0.00
22.1
66.
4158
0.00
19.0
19.
02V.
C.0
49.0
049
.00
644
.62
2041
.70
3441
.41
N.D.
5833
.28
5.38
in ethy�ene production . The detai�ed data for the treatabi�ity test of each compound are
provided in Appendix A (Fig . A-12 through A-17) .
Figure A- 18 (in Appendix A) shows the resu�t of reduced ethy�ene production
from PCE (49 µmo�es) dech�orination with higher methano� addition (1230 µmo�es) .
With the higher added amount of methano�, PCE dech�orination was s�ower than that
with �ower methano� dosage (Fig . A-12 shown in Appendix A) .
The PCE-dech�orinating capacity of the fresh Hyperion digested s�udge was
tested by successive�y adding PCE (49 µmo�es each time) into each of two separate
s�udge bott�es incubated with and without methano� addition, respective�y . Simi�ar
resu�ts (as shown in Fig . A-19, Appendix A) were observed from both cases . After 245
µmo�es of PCE were added, the s�udge system appeared to �ose the dech�orinating
abi�ity and the major end products accumu�ated were TCE and a �ower than expected
amount of VC. The accumu�ation of TCE may be due to nutrient deprivation even
though 246 µmo�es of methano� have been added accompanying with each PCE
addition .
4.5. Reductive dech�orination progression
Viny� ch�oride has been observed as a major intermediate in reductive
dech�orination. The mechanism and path of PCE and other ch�orinated compound
dech�orination through VC to ethy�ene is sti�� not c�ear . To further understand the
mechanism of reductive dech�orination in the anaerobic digested s�udge system, a�� of
their possib�e dech�orinated products were investigated in fresh Hyperion s�udge . The
69
resu�ts indicate that a�� of the tested ch�orinated ethy�enes may be dech�orinated to
ethy�ene through an identica� co-metabo�ic route (i.e . PCE -> TCE -> DCE -> VC ->
ETH). The �ess ch�orinated ethy�enes seemed on�y to uti�ize part of the route, whi�e the
fu��y ch�orinated ethy�ene (PCE) uti�ized the who�e route during the reductive
dech�orination .
Reductive dech�orination progression of PCE at a high PCE
concentration in fresh Hyperion digested s�udge is shown in Fig . 23 . After a �ag period
of 4 days, PCE started to dech�orinate to TCE . After on�y one more day, 49 µmo�es of
PCE comp�ete�y disappeared and 47 µmo�es of TCE were created . TCE thereupon was
transformed to cis-DCE (49 µmo�es) in the fo��owing 3 days of incubation without any
significant formation of the other two isomers (trans-DCE and 1 .1-DCE) . For an
unknown reason, it took �onger (8 days) to comp�ete�y dech�orinate cis-DCE to VC .
On�y 45 µmo�es of VC, �ess than expected, was measured . The difference between PCE
consumption and VC production was most �ike�y due to �eakage �osses and
undetermined ethy�ene production. This high recovery (around 92% ) suggests that PCE
was stoichiometrica��y dech�orinated, through TCE and cis-DCE, to VC . It was
sequentia��y reduced. In each step, one ch�orine atom in the ch�orinated compound was
rep�aced with a hydrogen coming from an e�ectron donor. A simi�ar resu�t (data not
shown) was observed from the same experiment conducted under conditions without
methano�. This suggested that sufficient nutrients contained in fresh digested s�udge are
adequate for PCE degradation .
The step-by-step dech�orination of PCE was a�so monitored from the samp�e
with a �ow initia� PCE concentration (see Fig . A-20 in Appendix A) . There was no �ag
70
Fig.
23Re
ductive
dech
�ori
nati
on p
rogr
essi
onof PCE at a high PCE
concentration in a fresh anaerobic
Hyperion s�udge
.
0
2
4
6
8
10Re
acti
on T
ime
(day
s)Note
: The steps of PCE dech�orination appear to more defined at higher
PCE concentration.
1214
16
100 80
U Oa)
;_a
a~
600
.o o ~.
40
U N A20 0
period observed and the characteristic step-by-step dech�orination was not as c�ear as in
the high concentration PCE experiments . The steps of PCE dech�orination were more
defined at a higher PCE concentration .
Reductive dech�orination progression of TCE a�so went through cis-
DCE to VC. The resu�t is shown in Fig . 24. Compared to PCE dech�orination, a �onger
�ag period (about 12 days) was required . After the �ag period, it on�y took 7 days to
entire�y dech�orinate TCE to VC . But, in PCE bott�e, the same amount of TCE produced
from the first step of PCE dech�orination took 11 days to terminate at VC .
Reductive dech�orination progression of three isomers of DCE is
shown in Fig . 25 to 27. Interesting�y, the transformations of a�� three isomers proceeded
via the simi�ar reductive dech�orination, a�� resu�ting in the formation of �ower
ch�orinated ethy�ene, VC . Among them, trans-DCE was observed to be most resistant to
reductive dech�orination . Trans-DCE took 17 days before its dech�orination was
observed significant�y, whi�e cis-DCE and � .1-DCE on�y took 6 and 5 days,
respective�y. After that, trans-DCE was dech�orinated at a much s�ower rate than that
observed in cis-DCE and 1 .1-DCE samp�es. The VC produced from dech�orination of
trans-DCE was not accumu�ated as much as that from other two isomers . Most of it
ended up as ethy�ene . This is possib�e due to the s�ower re�ease of VC, a �onger
incubation period or different organisms and mechanisms invo�ved in the formation of
ethy�ene .
Reductive dech�orination progression of VC to ethy�ene was observed
after 30 days of incubation . The data shown in Fig . 28 were taken after 50 days of
72
Reductive
dech�orination
progression
ofTCE
ata
high
TCE
conc
entr
atio
nin
afr
esh
anaerobic
Hyperion
s�ud
ge.
0
)(TCE
0
1.1,DCE
p
cis,
DCE-0
Viny
�ch�oride
trans,DCE
TCE
510
Reac
tion
Tim
e (d
ays)
15
V.C
.
20
Fig.
24
100
rA80
0
0 o.60
= o
t E
040
0 A
20
U °
0°
. °
°o 03
U 4) A
Fig
.25
100 80 60 40 20 0
Reductive dech�orination
progression
ofcis-DCE
ata
high
cis-DCE
concentration
ina
fresh
anaerobic Hyperion
s�ud
ge.
05
10
15
20Reaction Time (days)
2530
Fig
.26
Reductive
dech�orination
progression
oftrans-DCE
ata
high
trans-DCE
concentration in
afresh
anae
robi
c Hy
peri
ons�
udge
.
100 80 0
0 Note
:
50
100
Reac
tion
Tim
e (d
ays)
The
majo
r en
d pr
oduc
t of
tra
ns-D
CE d
ech�
orin
atio
n is ethy�ene, and not VC.
Thi
sma
y be
due
to
a �o
nger
inc
ubat
ion
peri
od t
hat
was
observed with this cu�ture
.
150
Fig.
27
150
A
0
0
Reductive
dech
�ori
nati
on p
rogr
essi
onof 1
.1-DCE
ata
high
1.1-DCE
concentration
ina
fresh
anae
robic
Hyperion
s�udge
.
48
12Re
acti
on T
ime
(day
s)16
20
030 20
A
Fig.
28
60 50 10 0
Reductive dech�orination
prog
ress
ion
ofVC
ata
high
VC
concentration
ina
fresh
anaerobic
Hyperion
s�udge
.
u
0
II
I~
1I
II
iI
I1
11
11
I~
11
Ii
~I
II
I~
II
5
10
15
20
25
30
35Re
acti
on T
ime
(day
s)No
te:
these data points were taken after 50 days initia� incub
ation with 49 µmo�es of VC
.
semi-continuous operation fed with VC and methano� . With 49 µmo�es of initia� VC
dose, 40 µmo�es of VC was consumed and 36 µmo�es of ethy�ene was produced in 35
days. This indicates that VC has undergone a reductive dech�orination ending up as
ethy�ene.
Ethy�ene formation was detected from the comp�ete dech�orination of a�� six
ch�orinated ethy�enes in fresh anaerobic Hyperion s�udge . To investigate the possib�e
ethy�ene production from a�� six ch�orinated ethy�enes, a new series of six 120-m1 serum
bott�es containing 100 m� of fresh Hyperion s�udge were prepared again as decribed
previous�y . Each bott�e contained one kind of test compound and was incubated under
the same conditions as described before . The initia� dose was 49 µmo�es for each
chemica� except VC. In order to enhance the ethy�ene production from VC
dech�orination, a higher initia� VC dose (122 µmo�es) was app�ied to the �ast bott�e .
After 15 days, PCE, TCE, cis-DCE, and 1.1-DCE were observed to entire�y end up as
VC. Trans-DCE and VC showed a more persistent characteristic to reductive
dech�orination as expected . Most of them continue to exist . From the 15th day on,
ethy�ene ana�ysis was conducted for each samp�e . Ethy�ene production (summarized in
Tab�e 6) was demonstrated from a�� test ch�orinated compounds after 54 days of
incubation. From Tab�e 6, during the second run, much more ethy�ene production was
observed from the dech�orination of trans-DCE and VC ( the two most persistent
compounds) after 12 days of incubation . This suggested that if the right environmenta�
conditions were provided, even the most resistant compound can easi�y undergo
comp�ete dech�orination in the anaerobic s�udge . The nutrient conditions (inc�uding the
amount, type of e�ectron donors and e�ectron acceptors) and the concentration of target
78
Tab�
e 6.
Summary of ethy�ene fo
rmat
ion
from
dec
h�or
inat
ion
of v
ario
us c
h�orinated ethy�enes
Time
PCE
TCE
cis-DCE
trans-DCE
1.1-DCE
V.C
.(d
ay)
Accumu�ation ofV.C
.(µmo�)
1st
Run
1543
.330
.78
46.47
1.28
44.7
104.
02TD=43.35 µcoo� (residue)
Ethy�ene production (µmo�)
1st
Run
150.
920.
7trace
N.D
.2 .
930.
7417
2.28
4 .03
trace
trace
8.14
0.81
204.
25 .
67trace
trace
8.56
1.75
245.
327.
380.
6trace
9.06
2.13
469.
3910
.52
0.6
1.13
8.42
14.8
554
12.46
11.36
2.4
5.55
8.21
20.62
2nd
Run
40.
616.
682.
213
.38
127.
9122
.35
1.91
27.96
ch�orinated compound may be two of the most important environmenta� factors for the
reductive dech�orination in the anaerobic digested s�udge .
The dech�orination patterns for each ch�orinated compound tested in the fresh
Hyperion s�udge are summarized in Tab�e 7 .Tab�e 7. Summary of dech�orination pattern for each ch�orinated
compound in anaerobic digested s�udge
The effect of the presence of TCE and VC on PCE dech�orination .
When PCE was incubated with VC in the fresh Hyperion s�udge, the dech�orination
pattern for PCE was the same as observed with PCE a�one . The dech�orination of PCE
went through TCE, cis-DCE, VC and terminated at ethy�ene . Interesting�y, the
dech�orination of the initia��y added VC did not occur unti� a�� PCE was transformed to
VC. In addition, a faster VC dech�orination rate was observed, compared to the resu�t
shown in Fig. 28 and Tab�e 6 . After the initia� added PCE was entire�y transformed to
VC, 79 µmo�es of VC, from a tota� of 99 µmo�es of VC, were dech�orinated to 74
80
Ch�orinated
compounds
Major products in sequentia� reductive dech�orination
PCE
-~ TCE-4cis-DCE-->V .C .--ETH
TCE
- cis-DCE-*V .C .--ETH
cis-DCE
-* V .C . -*ETH
trans-DCE -4 V . C . -ETH
1.1-DCE -4 V . C .-SETH
V.C.
-~ ETH
µmo�es of ethy�ene in on�y 10 days . The resu�t is shown in Fig . 29. A simi�ar
phenomenon a�so occurred in the s�udge bott�e which was incubated with PCE, TCE,
and VC simu�taneous�y . TCE dech�orination occurred after the comp�etion of PCE
dech�orination. When a�� of the TCE was transformed to VC, VC started to be degraded
and ethy�ene emerged ( shown in Fig . 30) .
The resu�ts shown in Fig. 29 and 30 suggested that the dech�orination of �ess
ch�orinated ethy�enes may be inhibited by the presence of higher ch�orinated ethy�enes .
This was a�so common�y suggested in previous �iterature and �ead peop�e to be�ieve that
PCE is the most susceptib�e to the reductive dech�orination among six ch�orinated
ethy�enes. However, if PCE can actua��y inhibit the dech�orination of �ess ch�orinated
ethy�enes in the s�udge ? We wi�� discuss it �ater.
4 .6. Toxicity effect
Methane production was used as an indicator to estimate the toxic effect of each
ch�orinated compound on the methanogenic activity of fresh Hyperion s�udge . A variety
of effects on methane production was observed with different ch�orinated compounds .
The term, LC50 was used to describe the concentration of a test ch�orinated compound
which reduced methanogenic activity by 50% . It is be�ieved that the decrease in methane
production may be partia��y or comp�ete�y due to the competition for reduction between
the e�ectron acceptors used by methanogens in the s�udge system and the test ch�orinated
compound. In some situations, it is possib�e that e�ectrons are more easi�y diverted to
ch�orinated compounds than to the precursor materia�s of methane formation .
81
y
A
Fig . 29
The effect of viny� ch�oride on PCE dech�orination ina fresh anaerobic Hyperion s�udge cu�tures withoutmethano� addition .
0 20 40
60Reaction Time (days)
80 100
Fig. 29a The effect of viny� ch�oride on PCE dech�orination ina fresh anaerobic Hyperion s�udge cu�tures withoutmethano� addition .
0 5 10 15
20
25Reaction Time (days)
82
30 35 40
200
150
100
50
0
Fig. 30 The effect of the presence of TCE and VC on PCEdech�orination in a fresh anaerobic Hyperion s�udgecu�tures without methano� addition .
0
0 5
20
40
10
60Reaction Time (days)
15
20
25reaction time (days)
83
80 100
Fig . 30a The effect of the presence of TCE and V.C. on PCEdech�orination in a fresh anaerobic digested s�udgecu�tures without methano� addition .
30 35
From Fig. 31 to 36, we found that the ch�orinated compounds (inc�uding PCE,
TCE, and cis-DCE) which were more rapid�y dech�orinated, have �ower LC50 va�ues .
Compounds with �ower rates of dech�orination (such as trans-DCE, 1 .1-DCE and VC)
exhibited higher LC50 va�ues . The LC50 va�ues obtained from each chemica� corre�ate
with their treatabi�ity (the amenabi�ity of compounds to dech�orination during bio�ogica�
treatment ) .
4.7. Highest to�erab�e concentration and ethy�ene production
Tab�e 8 to 13 show the change in tota� mass of each tested compound and their
dech�orinated products over a 20-55 day period of incubation in the fresh Hyperion
digested s�udge. PCE showed the highest potentia� to produce ethy�ene, then TCE and
cis-DCE. Trans-DCE, 1 .1-DCE and VC were more resistant to degradation and ethy�ene
productions. In these experiments, we did not observe 100% comp�ete dech�orination at
any initia� dosage for each tested compound. The maximum �eve� of ethy�ene production
was observed from the samp�e with an initia� dose of 97 .86 µmo�es of PCE . About 74%
of the PCE was recovered as ethy�ene (72 .4 µmo� .) after 32 days . No instances of
100% transformation were observed during the incubation period, even for the samp�e
with the �owest initia� dosage of PCE . Viny� ch�oride and trans-DCE were dech�orinated
most s�ow�y. The recovery for most of samp�es were �ess than 100% . This is due to the
�osses of chemica�s from the re�eased gas (for re�easing pressure resu�ting from gas
production in serum bott�es containing fresh digested s�udge) .
PCE. After one month incubation, the highest dose of PCE that was most
readi�y degraded to ethy�ene was 97 .86 µmo�es per bott�e . A�though degradation of PCE
84
Fig. 31 Toxico�ogica� Effect of PCE on Anaerobic Digested S�udge
LC50 : �etha� concentration fifty100
1
1
.80
---;; . .----:-;;..--- . ;~:. . - - ------------ -
,
. .. .;{°,iirjj;i;i;j;i_
. . . . .. .... . .40
`-
.--~-- .
, ;
.
;
.
.
20
+---.. :'
;--- . . ;. . .. ~ ..---- LC so180 gmo�JL- :... --=- ° °. .
.-
-
- ° ° -
---;° . ----..; . .--- . ;;; . . . . - --
-- '
.
> .
;
;
. ._
0
500
1000
1500
2000
PCE Concentration(tmo� ./L)
40
20
0
0
Fig. 32 Toxico�ogica� Effect of TCE on Anaerobic Digested S�udgeLC 50 : �etha� concentration fifty
100
°
1
i
,
!
i
!
!
!
I
!
!
!
i
.
..
.
.
.
-}pii;i, . .--- ..},i. . . . . . . }i. . .i_
500
1000
1500
2000TCE Concentration(µmo� ./L)
LC5070 Mo� .R,---'--
--
--
..-
i
85
0
Fig. 33 Toxico�ogica� Effect of cis-DCE on Anaerobic Digested S�udge
LC50: �etha� concentration fifty
I
y
+
+
+
+
+
+
i
L -J
+
+
+
-
-
--
100
. ....=-°--- . . .
_
-
o
.
-- --+v-
.-b
80 > -
CIO _j;~- . ...Y ..---+'°`i- .-- .}-..-.{::j;j . . . .-:.;j .---
.+{..-.-
.
.
.
.
~
.
.
~
---------
4 --° . --- --
-
--
y
;
°
20 -..y1ii+;iy+-..- yj... .- ;.i+- .-..j
. . . . . ... .LCSO=200µmo�/L .- ..---
. .;..-- - °. . . ; . . .
0 iiIiiiIiiiIiiiiii
0
400
800
1200
1600
2000
2400
Cis-DCE Concentration(gmo� ./L)
Fig. 34 Toxico�ogica� Effect of trans-DCE on Anaerobic Digested S�udge
LC50 : �etha� concentration fifty
100
60
20
0
120
0 400 800
1200
1600
2000
Trans-DCE Concentration(µmo� ./L)
86
°
80
°
40
2400
Fig. 35 Toxico�ogica� Effect of 1.1,DCE on Anaerobic Digested S�udge
LC50 : �etha� concentration fifty
Fig. 36 Toxico�ogica� Effect of Viny� Ch�oride on Anaerobic Digested S�udge
LC50 : �etha� concentration fifty
I
I
I
..
.
------------- }+°i-°- - °-
100
80
60
40
20
0
0
0
200 400
600
800
1000
12001 .1,DCE concentration(gmo� ./L)
1400
500
1000
1500Viny� Ch�oride Concentration(gmo�./L)
87
1600
2000
120
100
oub0 80
b
N 60
A0 40
UQ 20
0
was sti�� carried out at higher concentrations (up to 195 .72 µmo�es per bott�e), the
dech�orination that occurred within this period was not as comp�ete. The major product
of PCE dech�orination at 195 µmo�es per bott�e was viny� ch�oride, instead of ethy�ene .
The tota� ethy�ene production increased with the initia� PCE concentration up to an upper
�imit. It seemed that the ethy�ene formation from the reductive dech�orination may be
a�so inf�uenced by the concentration of viny� ch�oride . There was no any detectab�e
ethy�ene produced from the samp�es with a �ow initia� PCE concentration which resu�ted
in a corresponding �ow VC concentration . Resu�ts of this experiment are shown in Tab�e
8 .
TCE. The resu�ts for TCE dech�orination were simi�ar to those observed from
PCE dech�orination. With up to 196.21 µmo�es of TCE per bott�e, dech�orination of
TCE sti�� occurred; however 91 % of TCE was recovered as VC and on�y 9% of TCE
was comp�ete�y dech�orinated to ethy�ene . The maximum ethy�ene production (48.8
µmo�es) was observed from the samp�e incubated with 98.11 µmo�es of TCE, whi�e the
samp�e with 73 .58 µmo�es of TCE showed the highest percentage (about 56%) of
transformation from TCE to ethy�ene (see Tab�e. 9) .
cis-DCE. The production of ethy�ene from cis-DCE was much more than those
observed with the other two isomers. The highest to�erab�e cis-DCE concentration
appeared to be 105 .62 µmo�es per bott�e producing the maximum amount of ethy�ene
(see Tab�e 10) . For the highest cis-DCE dosage (211 .24 µmo�es per bott�e), on�y 112
µmo�es of cis-DCE was degraded to VC after 39 days of incubation with no ethy�ene
produced .
88
N.D
.:not detected; Sub-tota�=PCE+TCE+cis-DCE+trans-DCE+
1.1-DCE+V.C
. ; R
ecov
ery=
(3)/(2).
Tab�e 8.
Dech
�ori
nati
on o
f PC
E an
d th
e fo
rmat
ion
of d
ech�
orin
ated
pro
duct
s at
var
ious initia� dosages
duri
ng a
naerobic incubation in fresh Hyperion s�udge
.
Major dech�orinated products
Samp�e
Time
PCE
TCE
cis-
DCE
tran
s-DC
E 1.1
-DCE
V.C
.Su
b-to
ta�
PCE added
(1)/
(2)
ETH
Tota
�-(3
)(3)/(2)
No.
(days)
(gmo�.
)(gmo�.
) (.
tmo�
.)(tmo�.
)(pmo�.
)(µmot.
)(tmo�.
)-(�
)(gmo
�.)-(2)
(%)
(µcoo�
.)(µ
mo�.
)(%
)1
120 .
030.
680.
720.
5612
80.
72128
190.
500.
500.
5690
N.D
.0.
5090
320.
500.
500.
5689
N.D
.0.
5089
212
0 .03
1.02
1.05
1.40
751.
0575
190.
021.
071.09
1.40
78N
.D.
1.09
7832
0.96
0.96
1.40
69N
.D.
0.96
69
312
1.35
0.09
6.86
8.29
9.79
858.
2985
190.
050.
068.
348.
449.
7986
N.D
.8.
4486
327.
587.
589.
7977
N.D
.7.
5877
412
0.20
10.6
50.6
611
.15
22.6
624
.46
9322
.66
9319
0.66
18.2
218
.88
24.46
771.
8220
.70
8532
15.5
015
.50
24.46
634.
2019
.70
81
512
3.36
22.1
32.
0310
.91
38.4
348
.93
7938
.43
7919
18.5
918
.59
48.93
3826
.40
44.9
992
3212
.44
12.4
448
.93
2535
.63
48.07
98
612
31.4
120
.11
3.37
7.38
62.2
873
.39
8562
.28
8519
37.9
037
.90
73.3
952
43.1
481
.04
110
3223
.47
23.4
773
.39
3259
.12
82.5
9113
712
61.3
210
.15
4.57
7.61
83.6
597
.86
8583
.65
8519
0.11
10.2
956
.06
66.4
697
.86
6844
.49
110.
95113
3226
.78
26.7
897
.86
2772
.40
99.18
101
812
0.77
154.
823.
411.09
1.01
0.66
161.
76195.7
283
161.
7683
190.
0614
2 .22
6.91
2.00
0.03
1.41
152.
6419
5.72
78N.D
.152.
6478
2889
.33
17.3
92.
000.
9621
.78
131.
45195.
7267
131.
4567
3249
.76
15.1
32.
291.
1865
.80
134.
16195.
7269
N.D
.134.
1669
3511
.57
2.06
2.54
126.
9514
3.13
195.
7273
143.
1373
N.D
.:no
t de
tect
ed;
Sub-
tota
�=PC
E+TC
E+ci
s-DC
E+tr
ans-
DCE+
1.
1 -D
CE+V
.C.; Recovery=
(3)/
(2).
Ta�e
9.
Dech�orination
of
TCE
and
the
form
atio
n of
dec
h�or
inat
ed p
rodu
cts
at v
ario
us i
niti
a� d
osag
esduring anaerobic incubation in fresh Hyperion s�udge
.
Major dech�orinated products
Samp
�eTime
PCE
TCE
cis-DCE trans-DCE 1.1-DCE
V.C
.Su
b-to
ta�
TCE
adde
d(1)/(2)
ETH
Tota�-(3)
(3)/(2)
No.
(day
s)(4
mo�.
)(p.mo
�.)
(9mo�.
)(µ
mo� .
)(�
imo�
.)(µ
mo�.
)(µmo�.
)-(�
)(gmo�.
)-(2
)(%)
(gmo�.
)(µ
mot.
)(%)
124
0.51
0.51
0.55
92N.D
.0.
5192
380.
420.
420.
5576
N.D
.0.
4276
224
0.01
0.76
0.77
1.40
55N.D
.0.
7755
380.
570.
571.
4041
N.D
.0.
5741
324
0.53
5.46
5.99
11.1
554
0.00
5.99
5438
4.13
4.13
11.1
537
N.D
.4.
1337
424
0.23
12.9
813
.21
24.5
354
0.00
13.2
154
380.
5010
.85
11.3
524
.53
46N.D
.11
.35
46
524
0.86
11.4
612
.32
49.0
525
23.44
35.7
573
385.
855.
8549
.05
1218
.35
24.2
049
624
1.72
26.0
227
.74
73.58
3841
.22
68.9
694
3821
.98
21.9
873
.58
3036
.49
58.4
779
724
2.30
37.5
139
.81
98.11
4148
.82
88.6
390
3832
.55
32.5
598
.11
3347
.93
80.4
882
824
1.22
47.7
92 .
00143.
89194.
91196.
2199
N.D
.194.
9199
382.
23176 .
81179.
0419
6.21
9118
.80
197.
84101
N.D
.: n
ot d
etec
ted;
Sub
-tot
a�=P
CE+TCE+cis-DCE+trans-DCE+1
.1-D
CE+V
.C.; Recovery= (3)/(2)
;CD=cis-DCE.
Tab�
e 10
.De
ch�o
rina
tion of cis-DCE and the formation of dech�orinated products at var
ious
ini
tia�
dos
ages
during anaerobic incubation in fresh Hyperion s�udge
.
Major dech�orinated products
Samp�e
Time
PCE
TCE
cis-
DCE
tran
s-DC
E 1.
1-DC
EV.C
.Su
b-to
ta�
CD added
(1)/
(2)
ETH
Tota�-(3)
(3)/
(2)
No.
(days)
(Rmo
�.)
(µmo
�.)
(µmo
�.)
(µmo
�.)
(µmo
�.)
(µmo
t.)
(gmo
�.)-(
1)(gmo�.
)-(2)
(%)
(4mo�.
)(t
mo�.
)(%)
125
0.45
0.45
0.56
80N.D
.0.
4580
390.
300.
300.
5653
N.D
.0.
3053
225
0.59
.0.
591.
4142
N.D
.0.
5942
390.
350.
351.
4125
N.D
.0.
3525
325
12.61
12.61
13.2
096
N.D
.12
.6196
3911
.60
11.6
013
.20
88N.D
.11
.60
88
425
11.48
11.48
26.4
143
0.32
11.8
045
398.
008.
0026
.41
300.
598.
5933
525
13.37
13.3
752
.81
2531
.23
44.6
084
3913
.58
13.5
852
.81
2624
.99
38.5
773
625
30.33
30.3
379
.22
3835
.81
66.1
483
3927
.25
27.2
579
.22
3429
.80
57.0
572
725
60.13
60.13
105.
6257
31.7
291
.85
8739
38.6
838
.68
105.
6237
49.6
988
.37
84
825
160.
890.
3828
.61
189.
88211.
2490
N.D
.189.
8890
3976
.76
0.38
112.
03189.
17211.
2490
N.D
.189.
1790
trans-DCE . Trans-DCE was high�y resistant to dech�orination in the fresh
digested s�udge. After 54 days of incubation, no significant amount of trans-DCE was
transformed in any samp�e tested (Tab�e 11) . This is consistent with ear�ier observations
for the trans-DCE progression test . No ethy�ene production was observed even after 70
days of incubation (data not shown here) .
1 .1-DCE . At a dose of 50 µmo�es of 1 .1-DCE, the maximum amount of
ethy�ene production was observed (16 .7 µmo�es) . At higher concentrations of 1 .1-DCE
incomp�ete dech�orination was observed in a�� samp�es after 55 days of incubation
period (see Tab�e 12) .
VC. Genera��y, VC is regarded as the most resistant compound to reductive
dech�orination (Freedman and Gossett, 1989) . In spite of this, we sti�� found an
optimum concentration at which the dech�orination of VC to ethy�ene was maximized,
whi�e VC was a�most tota��y resistant at higher initia� doses. VC was maxima��y
dech�orinated at the initia� dose of 73 .57 µmo�es per bott�e. After 51 days, about 35
µmo�es of ethy�ene was recovered from VC (refer to Tab�e 13) . This indicates that the
concentration of VC inf�uences its rate of dech�orination to ethy�ene .
4.8. Semi-continuous operation
In order to eva�uate �ong-term dech�orination efficiency for each ch�orinated
ethy�ene, the six s�udge bott�es created in the dech�orination progression test and two
mixed-cu�ture bott�es were maintained under semi-continuous feeding . In PCE bott�e,
49 µmo�es of PCE was tota��y degraded to VC in 15 days at the beginning . After 5 runs,
92
N.D
.:not detected
; Su
b-to
ta�=
PCE+
TCE+
cis-
DCE+
tran
s-DC
E+1.1-DCE+V
.C.; R
ecov
ery=
(3)/(2);TD
=tra
ns-D
CE.
Tab�e 1.
Dech
�ori
nati
on o
f tr
ans-
DCE
and
the
form
atio
n of
dec
h�or
inat
ed p
rodu
cts
at various initia� dosages
duri
ng a
naer
obic incubation in fresh Hyperion s�udge
.
Major dech�onnated products
Samp�e
Time
PCE
TCE
cis-DCE trans-DCE 1.1
-DCE
V.C
.Su
b-to
ta�
TD added
(1)/(2)
ETH
Tota
�-(3
)(3)/(2)
No.
(days)
(�imo�
.)(µmot.
)(gmo�.
)(µmo�.
)(µmot.
)(µmot.
)(µmot.
)-(1)
(µmo
t.)-
(2)
(%)
(gmo
�.)
(gmo
�.)
(%)
130
10.3
00.
0510
.35
13.20
78N.D
.10
.35
7854
7.16
0.19
7.35
13.20
56N.D
.7.
3556
230
20.8
80.
8621
.74
26.4
182
N.D
.21
.74
8254
14.7
50.
2014
.95
26.4
157
N.D
.14
.95
57
330
45.0
50.
7345
.78
52.8
187
N.D
.45
.78
8754
38.1
30.
1138
.24
52.8
172
N.D
.38
.24
72
430
64.5
02.
4166
.91
79.22
84N.D
.66
.91
8454
56.3
40.
5156
.85
79.22
72N.D
.56
.85
72
530
87.5
42.
6090
.14
105.
6285
N.D
.90
.14
8554
72.6
12.
8675
.47
105.
6271
N.D
.75
.47
71
630
168.
653.
0517
1.70
211.24
81N.D
.171.
7081
5413
9.66
0.00
139.
66211.24
66N.D
.13
9.66
66
Tab�e 12
.
Dech�orination
of
1.1-DCE and the formation of dech�orinated products at v
ario
us i
niti
a�do
sage
sduring anaerobic incubation in fresh Hyperion s�udge
.
N.D.
: not detected;
Sub-
tota
�=PC
E+TCE+cis-DCE+trans-DCE+1.1
-DCE
+V.C
.; R
ecov
ery=
(3)/
(2);VD
=1.1-DCE
.
Major dech�orinated products
Samp
�eTime
PCE
TCE
cis-DCE trans-DCE 1.1
-DCE
V.C
.Su
b-to
ta�
VD a
dded
(1)/(2)
ETH
Tota�-(3)
(3)/(2)
No.
(day
s)(µ
mo�.
)(g
mo�.
)(gmo�.
)(p.mo
�.)
(.tmo�
.)(gmo�.
)(g
mo�.
)-(1
)(µmo�.
)-(2
)(%)
(µcoo�
.)(mot
.)
(%)
130
9.18
9.18
12.5
173
N.D
.9.
1873
556.
746.
7412
.51
542.
859.
5977
230
13.4
913
.49
25.02
54N.D
.13
.49
5455
7.45
7.45
25.02
30N.D
.7.
4530
330
39.4
539
.45
50.0
479
6.46
45.9
192
5517
.22
17.2
250
.04
3416
.67
33.89
68
430
60.5
35.
8766
.40
75.06
88N.D
.66
.40
8855
30.6
528
.50
59.1
575
.06
79N.D
.59
.15
79
530
85.8
77.
5693
.43
100.
0893
N.D
.93
.43
9355
46.4
040
.97
87.3
7100.
0887
4.77
92.1
492
630
158.
334.
31162.
64200.1
781
N.D
.162.
6481
55134.
025.
92139.
94200.
1770
N.D
.139.
9470
N.D
.:no
t de
tect
ed; Sub-tota�=PCE+TC
E+ci
s-DC
E+tr
ans-
DCE+
1.1
-DCE
+V.C
.; R
ecov
ery=
(3)/
(2).
Tab�
e 13
.De
ch�o
rina
tion of viny� ch�oride and the formation of dech�orinated products
at
vari
ous
init
ia�
dosa
ges
during anaerobic incubation in fresh Hyperion s�udge
.
Majo
r de
ch�o
nnat
ed p
rodu
cts
Samp
�eTime
PCE
TCE
cis-DCE trans-DCE 1.1-DCE
V.C
.Su
b-to
ta�
V.C
. ad
ded
(1)/(2)
ETH
Tota�-(3)
(3)/(2)
No.
(day
s)(R
mo�.
)(µ
mo�.
)(gmo�.
)(µ
mo�.
)(gmo�.
)(µ
mo� .
)(gmo�.
)-(1
)(tmo�.
)-(2
)(%)
(Rmo�.
)(µmo�.
)(%)
120
3.68
3.68
4.09
90N.D
.3.
6890
513.
003.
004.
0973
N.D
.3.
0073
220
8.71
8.71
9.81
89N.D
.8.
7189
516.
936 .
939.
8171
N.D
.6.
9371
320
18.2
918
.29
24.5
275
N.D
.18
.29
7551
16.2
016
.20
24.5
266
N.D
.16
.20
66
420
40.2
940
.29
49.0
582
0.50
40.79
8351
35.5
135
.51
49.0
572
N.D
.35
.51
72
520
63.9
763
.97
73.5
787
8.79
72.76
9951
26.2
726
.27
73.5
736
34.87_
61.14
83
620
90.3
590
.35
98.0
992
N.D
.90
.35
9251
68.4
268
.42
98.0
970
7.40
75.82
77
720
183.
08183.
08196.
1993
N.D
.18
3.08
9351
168.
13168.
1319
6.19
86N.D
.16
8.13
86
820
221.0
9221.
09245.
2490
N.D
.221.
0990
51210.
06210.
06245.
2486
N.D
.210.
0686
on�y 3 days were required to degrade the same amount of PCE . A tota� of 686 µmo�es
of PCE was degraded over a period of 145 days . At the end of the experiment, a tota�
of 33% of PCE was recovered as ethy�ene. VC was the major product accumu�ated in
the serum bott�e from PCE dech�orination . The resu�ts are shown in Fig . 37. Simi�ar
resu�ts (shown in Appendix, Fig. A-21) were obtained under conditions in which no
methano� was added for the first 60 days . 275 µmo�es of ethy�ene were produced from
735 µmo�es of PCE cumu�ative�y added in 145 days . Furthermore, �ong term semi-
continuous tests carried out using TCE, cis-DCE, and 1 .1-DCE showed simi�ar resu�ts
with �arge amounts of VC accumu�ation . The data are provided in Appendix A (Fig . A-
22 to A-24) .
In the trans-DCE bott�e, 61 µmo�es of ethy�ene were produced in 130 days from
a tota� of 98 µmo�es of trans-DCE cumu�ative addition . There were on�y 10 µmo�es of
VC accumu�ated in the bott�e at the end of the experiment (see Fig . 26) .
VC was a�so very s�ow�y dech�orinated . During the period of 135 days, a tota�
of 196 µmo�es of VC was added and 141 µmo�es of ethy�ene were recovered. At the
end of the test, VC was sti�� not comp�ete�y dech�orinated . A�though this resu�t indicated
that dech�orination of VC under anaerobic condition is possib�e, comp�ete
transformation of VC from the treatment system is more difficu�t and may take �onger .
The importance of methano� to VC dech�orination was a�so tested in this experiment by
ending methano� addition on 85th day . Dech�orination of VC and production of ethy�ene
were inhibited significant�y unti� the 114th day, at which time methano� was added again
( see Fig. 38). In this case methano� appears to be necessary for VC dech�orination to
take p�ace.
96
Fig.
37Long t
erm
dech
�ori
nati
on o
f PC
E an
d formation of
inte
rmed
iate
s in
a s
emi-
cont
inuo
us o
pera
tion
ser
um b
ott�
e
700
600
300
U A
200
100 0
iO/ o
„o
^~
0 0
00 0 0
F.FA
i
11
::
2PC
ETCE
cis,
DCE
tran
s,DC
E1.
1,DCE
V.C
.et
hy�e
nePCE consumption
050
100
Reac
tion
Tim
e (d
ays)
150
Fg
.38
Lg
erde
h�r
V.C
.d
ds
-u
usb
.
200
O=L
U 0 Q50
0
0
--0
.C.
y
v
.C.
. :1 11
50
100
150
Reac
tion
Tim
F x - , b 1 (B 1) x -
99
F . 39 L PCE-
b w 60 L x (*)b w
.
500
400
300
0
500
400
~ 300
0A
~ 200
100
0
0
--0--- PCE
O
LLDCE-x
CE
6
.C.- .
.DCE
-ES- y
---0- .DCE
-}- PCE
B2
0
20
20
40
60
( y )
40
60( y )
80
80
(*) : x 10 x (60 ).
1 00
100
100
PCE 1 .1,DCEICE .C .µ B1,DCE,DCE 0
yPCE
4.9. E .
I b b w
( y b ) x v PCE,
w b w v PCE 49 µ
100 L q . w
x PCE. A w ,
PCE . A q ,
y .
. x w
w PCE v x
. , b
k . B , PCE
.
b w .
b w 230 23 v .
k 4 , w 30 w
( ). G , 6 w b
b b v w 6 . A
b , , PCE (49 ) .
b z b A-1 (A A).
D w , w PCE
b w b b w v
10 1
. H w v , v
b . F 4 ,
w b v w v
. D 4 , b 8 - 19 w
w 1230 , w 42.9
57 .6 w w 0 123
, v . w b 14 .
w b v b
PCE . 20 CE 3 -DCE w
w (4920 )
4 b 3 , w PCE (49 )w
C . b w
b v w . A 6
b , 49 PCE w 32.7
CE 15.4 -DCE . A , PCE
w -DCE . v
v q PCE w .
PCE b z w .
0 M OH b . PCE w w
b . A b , 5 w
v w 49 PCE.
A ' , b w v
. 42.8 w
49 PCE C ( b 50 b v
102
b14
.D
wb
MOH
(*):A
b,
, PC
EMOH
.M
OH b
.
D#
(*)
Ib
0 MOH
5 MOH
5 20MOH+
M50 M
OH
100
MOH
200MOH
222
EP
()
442
.76
45.2
634
.17
48.96
43.66
47.86
324
642
.87
57.6
226
.69
7.91
18.94
0.91
49 52 53 54 55
8 8 8 8 _8
0 3 4 5 6
0.0
014
.40
23.2
131
.04
42.4
0
0.0
022
.26
33.1
741
.50
57.0
4
0.0
03.7
99.7
914
.89
22.9
4
0.00
17.31
21.86
27.29
34.19
0.00
0.00
0.00
0.00
0100
0.00
0.00
0.00
0.00
0.00
I
) 4 . D , 58 w
. A , v w 83 (1918
). Ov 230 - , 1127
PCE w 644 w w
451 C. b 57% .
F , w v b
b w .
w b w w k
w 6 ( 41), z
. w w b 35 °C
k PCE
11 12 . ( )
. 104 .
b w 55 °C b 4 . A , k 10
49 PCE C b
. b b w
v 30 - 60 .
5 M OH b . 5 (123 ) 5
w PCE . D 2 ,
b w b b v 45
49 PCE 4 .
w 65 . A , 775 w
1127 PCE, w PCE C.
104
68 .8% . v w 96
(2210 v ) .
5 M OH+20 M b . 5 1 20
w PCE .
. A
b , 85% PCE w v 5 .
42 ( b )
b 20 . H w v , w b v . O
v w 114 . I bv
b PCE
. Ov , 1005 PCE w
449 .
44.7%.
50 M OH b . A (50 , q v 1230
) 5 w PCE . A
b , b w b
. I w b b b
w b v w . A
, b 350
b ( b 10 -20 ). H w v , PCE
b C w
. I v
, b w k 67 80. , PCE
105
b -DCE . b w
v v w b . D w
, PCE w 15 14 ,
v . H w v , q PCE
w b w
v -DCE. A 686 PCE w
229 w v 230 . A
, 5248 w -DCE
C 156 308 , v .
33 .4% .
100 M OH b . 100 (2460 ) 5
w b w PCE w . PCE
b b w v . A
b , PCE b w
b v b . H w v , 4 , CE (32.7 ) -DCE
(15.4 ) . Ev b w ,
. A , PCE
w -DCE .
, 392 PCE 19926 w . A
, 116 4436 , v w
. 29.6%. H w v ,
b b ,
w v z .
106
200 M OH b . 200 (4920 ) 5
w b PCE . A 39606
w .
(7405 ) b .
H w v , w v PCE- b w
b v w . A 3 , PCE (49 )
CE (20 ) -DCE (3.4 ), w PCE w
C b . A ,
b w b v 132 ( ) 449
w 48 14 . H
w v w
v 2300 . C -DCE w j PCE
PCE b v . ,
k b PCE .
, 392 PCE 114
142 96 -DCE C,
v . 29.1 % . w
F . 40 b.
G k , b PCE w
b v b w
. b v b w
v .
PCE b b . A
, v PCE, b , w
107
1200
1000
800
600
400
200
0
0 35000
v
30000
25000
E 20000E
15000
1000005000
0
7000
6000
5000
E
40000v
30000
2000
1000
0
700
600
500
E
400$v
300
w
200100
0
F . 40( )PCE .
108
F-
F . 40(b)PCE .
600
100 -
50 -
---- 0 L M OH- - 5 L M OH-- -- 5 L M OH + 20 L M
0- 50 L M OH100 L M OH
-- 200 L M OH
0
50
1 09
250
b v . I k
b w
b b v . I , b
w b v b PCE . B
v - b . H w v , -
w k w b 50%
b . k b
w .
4.10. E PCE .
P v
w w . F b ,
b v . H w v , b PCE
b H w b v b b .
F , PCE w .
, b w w
b q .
v . B b b v
PCE .
M PCE w 1
. A 10 b , PCE -DCE (S5) w
, w PCE w C w
. D , w
1 10
b v . 302.2 w 5 (S5) v
241 w .
b v .
w F . 41 b 15. F F . 41, w b PCE w
-DCE, w
b w w w . A , -DCE w
w b w
. ( ) v
b PCE CE .
I b , v
w 2 b w b v
w v . A 2 , PCE w
C ( b 35 ) (14.6 )
4 (S4) w w v k b . I
, ,
b v . D w b 16.
b w b v w
. F 5 w , PCE w -DCE .
I , b w v
2 3 b 3 . A 6 b ,
b v 3 (w b w
C02) w b b v 4 (w w C02).
b v .
1 1 1
OE
00F
F . 41350
300
250
200
150
100
50
0
6
5
4
3
2
1
0
500
40
30
020
H10
0
10
0
500
40
30
0
20F
10
00
PCE. D 1 .
2
1 12
4
6( )
8 10
M
--- ,- W-
M
W
EE H w/E H---X
w/
.C .
.C .--- -
w/.C .---)E- w/
-DCE
-9- -DCE w/- - -DCE w/
PCE
PCE w/I PCE w
b 15.PCE - 1
N : 1 . PCE 49 .2 . .C . b b10 - 15 .
1 1 3
( )PCE
( )CE
(I. )CD
( )C
( )E H
(. )CH4
( )S 1 (w )
2 0.00 127.343 0.00 140.627 0.00 199.6210 0.00 0.00 0.00 56.79 4.61 249.63
S 2 (w )2 108.993 130.167 0.00 204.1810 0.00 0.00 0.00 54.47 3.71 241 .36
S 3 (w )1 52.30 0.65 0.08 10.312 0.36 0.15 45 .11 13.94 121 .583 0.23 0.00 44.73 11 .78 146.284 0.16 0.00 43.56 13.265 0.23 0.00 42.21 16.366 0.00 0.00 38.42 22.277 0.92 0.00 30.14 26.49 0.00 214.078 0.18 0.00 15.14 43.829 0.11 0.00 0.00 52.8710 0.00 0.00 0.00 54.57 6.76 246.08
S 4 (w )2 106.473 126.807 1.18 187.9310 0.00 0.00 0.00 57.04 5.56 225.78
S 5 (w )1 54.06 0.69 0.09 15.18 0.002 0.29 0.00 47.93 18.61 0.07 143.193 0.09 0.00 45.91 15.71 0.01 164.184 0.09 0.00 45.28 15.915 0.12 0.00 46.31 17.526 0.00 0.00 45.72 19.307 0.74 0.00 42.90 18.06 263.278 0.00 0.18 42.19 21 .519 0.30 0.00 41 .32 21 .7710 0.00 0.00 39.93 22.15 0.00 302.20
b 16.
N :
PCE - 2
1 . v b w5 , 2.5 L .
2 . PCE .C . bw b PCE .C . .
1 14
( )PCE
( )CE
( )CD
( )C
( )E H
( )CH4(4 )
S 1 (w )0 49.00 21.00 0.00 0.004 7.77 136.116 0.00 0.00 0.00 54.44 16.11 177.82
S 2 (w )0 49.00 25.00 0.00 0.004 8.71 126.656 0.00 0.00 0.00 51 .99 22.34 183 .61
S 3 (w )0 49.00 30.00 0.00 0.004 0.00 0.00 0.61 64.09 12.78 136.196 0.00 0.00 0.00 48.65 31.25 168.40
S 4 (w )0 49.00 20.00 0.00 0.004 0.00 0.00 0.42 65.06 5.22 143.826 0.00 0.00 0.00 55.38 14.58 212.46
S 5 (w ),0 49.00 32.00 18.00 0.00 0.004 0.16 0.00 81 .14 17.85 0.00 163.956 0.00 0.00 77.33 18.02 0.00 207.67
w b 17. A , PCE w -DCE
5 v b w b w
.
b w v b
, 5 w v k q b
b 4 . A 6 b , b 21 -DCE w
b C. D , -DCE w b v
3 4 ( ). H w v , v b w
b 3 w CO2 w . z
b 18 .
F , v b , b w
PCE w 5 5 . A
b , b w v b
b (64 ) w 5 .
b w v b
. M w b v 1 3. A
, PCE w -DCE C w
4 w CO2. C 3,
b b v
. D w b 19.
1 1 5
b 17.
N :
bPCE - 3
1 . v b w 2.5 L.2 . S 2 3 w w , w
w w 30% b 70% .3 . PCE .C . bw b PCE .C . .
1 1 6
( )PCE
( )CE
( )CD
( )C
( )E H
( )CH4
(. )
S 1 (w : w b )0 49.00 34.00 0.00 0.002 5.82 80.836 0.00 0.00 0.00 57.73 25.72 129.49
S 2 (w : w b )0 49.00 23.00 0.00 0.002 2.25 24.626 0.00 0.00 0.00 56.02 16.07 61 .76
S 3 (w : w b )0 49.00 21.00 0.00 0.002 3.53 48.126 0.11 0.00 0.00 60.65 10.06 102.75
$ 4 (w : w b )0 49.00 31.00 0.00 0.002 7.15 96.276 0.08 0.00 0.00 75.95 4.92 143.62
S 5 (w : w b )0 49.00 65.00 16.00 0.00 0.002 0.00 99.356 0.08 0.00 114.27 16.61 0.00 173.72
b 18.
N :
bPCE - 4
1 . v b w 2.5 L.2 . S 2 3 w w , w
w w 30% b 70% .3. S #5 w v b k b w
-DCE b .4. PCE .C . bw b PCE .C . .
1 1 7
( )PCE
(. )CE
( !)CD
(. )C
( !)E H
( !)CH4
( )
S I (w . w b )06
49.000.00
28.0068.76
0.008.92
0.0039.150.00
0.02
S 2 (w : w b )0 49.00 32.00 0.00 0.006 0.00 0.00
0.00 64.48 17.08 37.47
S 3 (w : w b )0 49.00 30.00 0.00 0.006 0.00 0.00
19.75 59.28 0.14 94.39
S 4 (w 2: w b )0 49.00 45.00 0.00 0.006 0.00 0.00
40.44 54.24 0.00 77.47
S 5 (w : w b )0 49.00 103.00 16.00 0.00 0.006 0.00 0.00 131 .60 37.24 0.00 144.27
b 19.
N :
bPCE - 5
1 . v b w 2.5 L.2 . S 2 3 w w , w
w w 30% b 70% .3 . PCE w #5 -DCE
b .4 . PCE .C . bw b PCE .C. .
1 1 8
PCE
CE
CD( ) ( ) ( ) (M01)
C( )
E H( )
CH4( )
S I (w : w b )0
49.00 44.00 0.00 0.006
0.00
0.00
9.36 84.629
0.00
0.00
0.00 87.3911
0.00
0.00
0.00 84.38 8 .17 103.5321
0.00
0.00
0.00 77.36 13.11 126.8032 47.96 68.24
S 2 (w . w b )0
49.00 42.00 0.00 0.006
0.00
0.06
15.02 69.0511
0.00
0.00
0.83 83.43 8.81 113.8521
0.00
0.00
0.83 83.43 17.18 140.9032 49.65 85 .05
S 3 (w : w b )0
49.00
15.00 38.00 0.00 0.006
0.07
0.00
53.94 48.6111
0.00
0.00
40.35 63.6516
0.00
0.00
19.71 81 .5921
0.00
0.00
1 .81 98.15 0.00 190.5432 36.00 100.32
S 4 (w ; w b )0
49.00
35.00 34.00 0.00 0.006
0.00
0.00
70.41 47.9811
0.00
0.00
57.13 59.9916
0.00
0.00
41.07 71 .1721
0.00
0.00
28.18 83 .19 0.00 196.6632 0.00 103.96
S 5 (w : w b )0
0.00
124.00 23.00 0.00 0.006
0.10
0.05
53.50 94.4211
0.00
0.00
41.69 105.98- 0.00 204.2114
0.00
0.00
6.37 140.0621
0.00
0.00
0.00 99.38 44.55 209.9732 64.33 84.18
4.11. E PCE .
v . z
b ,
30 38°C. F , b w . I
, w PCE w
w . A w b , w
.
PCE w H
b w . I k 6
PCE. I w 18 , PCE w
-DCE. A , -DCE w v w . I k 3
-DCE C. E w 146. A
(182 ), 21 w v
49 PCE ( F . 42). H w v , b 35°C
b , k 23 ( F . A-25,
A A).
4.12. PCE .
I w v v
. I , PCE
CE w v w . I . E
(1, 2, 3, 4, 12, 14, 20, 21- , v ) w . A
1 19
F.42
PCE
bb
Hb
w.
.50
100
150
()
200
v . C v , w
w b v w , w
w . v A A (F . A-25 A-33) .
I , CE -DCE w
. F . A-28, 18 .4 PCE w
w 12.0 C 8.6 38.
bv v w k. I
b w
. A b b
v w w
.
4.13. E v b PCE .
A b v b ,
w v b b PCE
b . A ,
PCE b w w w b
w . A , q w
w b b . F
, w b w b v
( F . 43) . v v
q . H w v , v
PCE, w b v w
1 21
F . 43
v bb H - PCE .
( )
Q7
3
Q0
3
Q
3
L3
2000
1500
1000
500
.C. q ( /1)
_-
• 60
=
=' 40
-3
0-DCE q ( / )
-8
6
4
2
80
20
3
0
1 .5
0 .5
0 .0
15
5
0
CE q ( /1)
PCE q ( A)
122
2000
1500
01000
500
1 .0
10
0
0
b . A , PCE w k b w -DCE C
w b k w w PCE
w b ( F . A-34,
A A) . I w b b w b
b b , v b b
b b .
I w- , b w b
. W 2 b w
w 2 b , (w b )
b w 2
b . w F . 44 F .
A-35 ( A A). b
v b w b . w w
. , b v b .
w b v w w 8
b 196 PCE w . A 85 b ,
w . H w v , 75
w w (4 )
b PCE ( F . A-36 A-37, A A).
b w v
b PCE b - .
1 23
0 40 W
F.
44C
PCE
ww
GAC
.40 35 30 25 20 15 10
0
E
H/4AC
v
/4AC
E
H/2AC
/2
AC
510
15(
)20
700
-60
0
500
400
CD b
300
'20
0
100
0
4.14. C PCE b v b
.
v q v
b PCE w b v BAC . A 66 b ,
b w 49, 98, 196, 294, 392,
490 PCE, w 34.5, 44 .2, 85 .2, 108.2, 142.4,
188 .9 , v . A , PCE b
C. PCE w
PCE. M PCE w
b PCE b v .
v v b v
PCE b PCE.
I , v b w
PCE w b v . PCE w , w v
PCE v .
F b , -DCE w b v
b . O 3, -DCE b
w PCE . PCE
b w b w b
PCE. H w v , PCE
C w b b PCE b
w . k PCE C w : 26
1 b w 49 PCE, 36 2 4 b w 98 294
1 25
PCE, 56 5 6 b w 392 490 PCE. I
w PCE .
A w b v PCE b
( 2 6) . E w b
C b . H w v , b ( 1) w PCE ,
w PCE C.
b C . F b , b
w C w b 90
/ (5.6 /1) q . I ,
, . . PCE -DCE, b
b w 6 b
w C 50 / q . A (
26), PCE, CE, -DCE q 6 b
w 41, 106, 1550 / , v . H w v ,
b w PCE CE .
PCE b v A A (F . A-38
A-43) . v PCE BAC
w F . 45 .
G k , w
PCE b w . I ,
PCE b w C .
C b w w PCE . H w v ,
1 26
W
W
A
0.
F . 45
E v PCE .(B v b )
1500
1000
500
0
150
100
50
0
200
150
100
50
100
80
60
40
20
00 10 20
127
30
40( )
50 60 70
M ( )
PCE- 49 PCE
98
PCE
196
PCE294
1 PCE-- -392
PCE-- ----F- 490 PCE
E ( )
49
PCE
98
PCE-
196
PCE294
PCE
392
PCE6
- - 490 PCE
-DCE q( )
49
PCE- --98
PCE-19
PCE- II294
PCE-- -392
PCE-- --490
PCE-*-
PCE q ( )
49
PCE
98
PCE-196
PCE-294
PCE- -490
PCE-
w b v . w F .
46.
4.15. B PCE b .
PCE b
b . k b v
w w PCE. q w ,
w b v w .
I v b k
w w . H w v , v b
b v w b . I q
, b
w , b
b w k w
, v PCE w . P
.
F PCE b b , ,
, w b v BAC .
OB w BAC w b v
b . b PCE b w
PCE BAC PCE
w F . A-50 A-55 ( A A).
1 28
F.
46E
PCE
BAC
wv
PCE
. (
60b
)1247
.
500
400
300
200
E]
M
49
98
196
294
392
FPC
E D
()
490
PCE b b b b
w .
v A A (F . A-44 A-49). A
, b w w
PCE. H w v , PCE
C . v PCE
w F . 47. F k w , PCE
w v w w 392 PCE
30 . A , b w v .
PCE z F . 48 .
j b
b (OB ) PCE v
b . , PCE-b v b b b
w v . k ,
PCE b b w v b PCE v
q . H w v , PCE
b b . b v
b . b ,
w v v b v b b
b v . , b b
b ,
q w b v . A
1 30
GZN
W
F . 47
E v PCE .(B PCE b )
1600
1400
1200
1000
800
600
400
200
0
120
100
80
60
40
20
0
200
150
100
50
60
50
20
10
0
1 .5
0 .5
0 .010 20
1 3 1
30
40( )
50 60 70
b PCEM (
- 0-49 ICE?CF
490 ,
E ( )
49
PCE
98
P
*
29964IL PCE
392
PCE- --- 490 PCE
-DCE q(
49
PCE-
98 PCE196
PCE-+- 01294
PCE-- -392
PCE- -- 490 PCEW
PCE q ( )
49
PCE-98 PCE-
196
PCE
294
PCE- --392
PCE-A- -- 490 W PCE
F.
48E
PCE
OB
wv
PCE
bb
. (
60b
)
700
600
100
1330
49
EL]
M
98
196
294
392
Ab
PCE D
()
490
, PCE b w C.
b z b 20.
b 20. M PCE b .
* : C q b .
= (2 + 3) / 1 % .
F b 20, b PCE b w
v . B ,
b PCE. O , b b
. b v
b b b w
w PCE CE.
133
A b PCE .C.* E H E H / PCE
( ) - 1 ( ) - 2 ( ) - 3 (%) (%)
49 15 .6 35.3 72 .0 103 .9
98 37 .3 45.2 46 .1 84.2
196 80.3 80.5 41 .1 82.0
294 127.7 116.8 39 .7 83 .2
392 170.6 123.8 31 .6 75.1
490 222.3 110.4 22 .5 67.9
5. DISC SSION
5.1. PCE - A .
PCE- b b .
F , PCE , -
v b , w v
, b C B ( P1) W w
P ( b 21). PCE
M OH .
PCE b b ,
b . v v PCE b
w b b . w b v PCE
w b w
q PCE v v . H w v , k w
w b PCE. W
k w w ( ) PCE
. b v
PCE w b
. G k , v PCE k
. S k
b v k b b
b . , v w
v
v . B ,
1 34
b q PCE b k b
v b b . I ,
b w
.
b 21. S PCEb .
N : 1. * 3 b .2. ** .3 . -DCE b
.4. *** b v
CE .5 . ( ) E H b b w
.
1 35
M j
A b CE -
DCE
-
DCE
1 .1-
DCE
C F
H ' E H
C - P I 'J '1 -DCE
C - P2 * E H*
I . E H
A v , E H
* * * E H
JWPCP *** E H
P -DCE
M C/E H ( )
M OH * * E H* *
There is no attempt to identify which organism(s) is responsib�e for the reductive
dech�orination in this study . In the �iterature, the occurrence of reductive dech�orination
has been demonstrated in both pure cu�ture and mixed microbia� popu�ation . However,
on�y a re�ative�y s�ow rate and partia� dech�orination was achieved in most cases
studying with pure cu�tures (e.g . 0.25 - 0.45 mo�es of PCE per �iter per day observed
in Fathepure and Boyd's work, 1988b) . In the present study, we observed that PCE
dech�orination proceeded at a much faster rate in s�udges . On average, 49 µmo�es of
PCE can be entire�y dech�orinated to VC with a significant amount of ethy�ene
accumu�ated in 15 days for most test s�udges . The dech�orination rate corresponding to
these reactions is about 32 .7 µmo�es per �iter per day . Furthermore, PCE was
dech�orinated not on�y to TCE but a�so further to VC and ethy�ene in this study .
Therefore, both the rate and the extent of PCE dech�orination have been improved .
Usua��y, pure cu�tures are more un�ike�y to degrade a reca�citrant compound so
comp�ete�y. The comp�ete dech�orination of PCE in digested s�udges is more �ike�y to be
the co-metabo�ic resu�t of a methanogenic consortium .
In anaerobic digested s�udge, many kinds of non-methanogenic bacteria may
form hydrogen . Genera��y, this hydrogen serves as a source of the reductant required
for reductive dech�orination of PCE and its partia��y dech�orinated intermediates . The
bio�ogica� reductive dech�orination of PCE may be a nonspecific reaction and mediated
nonspecifica��y by coenzymes invo�ved in methanogenesis . In addition, because PCE is
dech�orinated sequentia��y, the conditions (nutritiona� and process requirements) for
each step of the comp�ete dech�orination of PCE may be different . To achieve optimum
dech�orinating efficiency, the environmenta� conditions for each step shou�d be
optimized separate�y. However, it wou�d be comp�ex and difficu�t to e�ucidate .
136
5.2 . Dech�orination progression and comp�ete dech�orination .
The fresh anaerobic Hyperion s�udge incubated with PCE exhibited a typica�
resu�t for the reductive dech�orination pattern (Fig .23). The comp�ete reduction of 49
µmo�es of PCE per 100 mL occurred within 5 days . The transient accumu�ation of TCE
reached its maximum �eve� on day 5 . After this time, the tota� mass of TCE started to
decrease and cis-DCE started to increase. Then cis-DCE decreased and V.C. began to
accumu�ate in the system. The digested s�udge incubated with PCE contained TCE, cis-
DCE, and V.C. after 4, 6, and 9 days, respective�y, suggesting that TCE, cis-DCE, and
V.C. were sequentia� intermediates of PCE dech�orination (PCE was dech�orinated
stepwise via TCE, cis-DCE, and VC to ethy�ene) .
Ethane (a possib�e reduced product of ethy�ene) has never been detected in any
significant amount throughout the course of this study . Apparent�y, the methanogenic
consortium in the anaerobic digested s�udge is not on�y capab�e of degrading PCE but
a�so capab�e of degrading a�� of its possib�e biotransformation products (i.e . TCE, 3
isomers of DCE, and V .C.) . However, it is sti�� not known whether the same
microorganisms are invo�ved in the dech�orination of different compounds or different
species are needed for each step of dech�orination . With the �ag periods observed in each
step of PCE dech�orination, it was very possib�e that the different dech�orination steps
might be effected by different microorganisms . Severa� different microorganisms are
needed to achieve comp�ete dech�orination of PCE. To identify the species that posses
dech�orinating activity is most important . The dech�orinated intermediates for each
parent target compound �isted in Tab�e 7 and their sequence of appearance and
1 37
disappearance of the dech�orinated products shown in Fig . 24 to 28 appeared to indicate
that a�� the tested ch�orinated compounds have undergone a dech�orination progression
that is consistent with a simi�ar mechanism which �eads to the same intermediates and
end products. The common observed dech�orination pattern is summarized in Fig . 49 .
The dech�orination pattern is simi�ar to that observed previous�y in other methanogenic
environment (Voge� and McCarty, 1985 ; Barr�o-Lage et a� ., 1986) .
The findings of comp�ete reductive dech�orination of PCE to ethy�ene in
anaerobic digested s�udges obtained from six different wastewater treatment p�ants and
in methano�-enrichment cu�tures enriched from Hyperion s�udge are of great importance
for bioremediation app�ications . This is the first report of such a comp�ete dech�orination
of PCE occurring in fresh anaerobic digested s�udge at such a high rate. These findings
make reductive dech�orination in anaerobic digested s�udge an attractive method for
remova� of PCE in bioremediation processes .
In this study, not on�y PCE but a�so TCE, 3 isomers of DCE, and VC were
demonstrated to readi�y undergo the comp�ete reductive dech�orination in anaerobic
digested s�udge. It is apparent from these studies that reductive dech�orination is the
primary mechanism in the degradative sequence of ch�orinated compounds in anaerobic
digested s�udge. A�� of tested ch�orinated compounds were dech�orinated in a stepwise
fashion and a�� of them can be dech�orinated to ethy�ene . These resu�ts are consistent
with recent�y observed dech�orinations under anaerobic conditions (Freedman and
Gossett, 1989, 1991 ; Bruin and Zehnder, 1992). The evidence of these dech�orination
are based on the disappearance of parent compounds (i.e . PCE, TCE, DCE and V.C.)
and the formation of dech�orinated products under strict anaerobic conditions . Whether
13 8
Fig. 49 Schematic for the common observed dech�orinationpattern for conversion of ch�orinated compounds toethy�ene.
C� \
/ C�C C
C1 /
\ C1
H\C C/H
cis-1.2-DCE
139
H2
HC�
PCE
TCE
C1
H\C
C /H /
\ C�
trans-1,2-DCE
VC
Ethy�ene
biooxidation mechanisms were a�so in part invo�ved in the transformations of
ch�orinated compounds was not determined because CO2 was not measured in this
study .
In this study, we did not conduct radioisotope studies to determine whether the
ethy�ene formed was a consequence of PCE degradation . Indirect evidences suggest that
the formation of ethy�ene is direct�y re�ated to the dech�orination of PCE . These
evidences are : (1) ethy�ene was detected on�y from the time when VC started to
disappear, (2) in most of cases, the amount of ethy�ene formed was near�y
stoichiometrica��y equa� to the responding decrease in VC, and (3) there was no any
detectab�e ethy�ene observed in the s�udge samp�e which was incubated without PCE
under the same conditions . A�though the transformation of PCE to ethy�ene, and
eventua��y to ethane was monitored in Bruin et a� .'s report (1992), there was no
significant amount of ethane being found throughout this study . Furthermore, we have
not tried to determine whether products not detectab�e by the purge-and-trap method
were a�so being formed .
PCE-dech�orinating abi�ity may vary with the microorganisms present and
physicochemica� environment. According�y, reductive dech�orination may degrade PCE
to various extents under different conditions . In most previous studies of bio�ogica�
dech�orination of PCE, on�y partia� dech�orination was reported . The partia� PCE-
dech�orinating abi�ity was a�so found in the pond sediment obtained from UCLA's
botanica� garden. In that case, PCE was initia��y dech�orinated to TCE and cis-DCE .
After one month of semi-continuous operation cis-DCE was the on�y end product . The
cis-DCE did not further dech�orinated even after 6 months of extended incubation . The
140
�ess ch�orinated products may not be further degraded under the same conditions . Not
surprising�y, in contaminated anaerobic environments, such as waste dumping sites,
subsoi�, and groundwater, partia� dech�orinated products of PCE are often found . For
examp�e, cis-DCE is frequent�y the major environmenta� contaminant when PCE or TCE
has been re�eased in a dump. Therefore, in assessing the biodegradation of PCE, one
must a�so assess the partia� dech�orination products formed and their possibi�ity to
further biodegradation, not simp�y the disappearance of PCE .
5.3. Dech�orination rate
In this study, PCE dech�orination commenced most�y after a short initia� �ag .
The insignificant �ag time might be due to the sufficient, appropriate primary carbon
source, e�ectron acceptor, or �arger ce�� popu�ation in the anaerobic digested s�udge . The
initia� �ag time wi�� a�so depend upon the ch�orinated compound . For examp�e, V.C.
and trans-DCE needed much �onger period of time to start being degraded . If PCE
dech�orination were bio�ogica��y cata�yzed, the re�ative�y short acc�imation time is a�so
probab�y due to the continua� presence of dech�orination cata�yst, that may be a non-
specific cata�yst, in anaerobic digested s�udge . The possib�e exp�anations for the periods
of acc�imation are : (i) genetic change, (ii) induction, (iii) exhaustion of preferred
substrates, or (iv) growth of the active popu�ation from a very �ow initia� density (Mohn
and Tiedje, 1992). Exhaustion of preferred substrates may be responsib�e for the �ag
period observed in the reductive dech�orination because PCE dech�orination occurred at
the initia� period, but on�y at a very s�ow rate (a typica� data from PCE dech�orination
was shown in Tab�e 22). This appeared to indicate that microorganisms prefer to
degrade non-PCE materia�s other than PCE at initia� stage . For both cases of trans-DCE
1 41
and VC with �onger acc�imation periods, their methane productions observed at initia�
stage were re�ative higher than those observed in the s�udge acc�imated to other
compounds causing shorter acc�imation periods .
Induction may not be used to exp�ain the reductive dech�orination observed in
the digested s�udge because there was a detectab�e dech�orination occurred at the initia�
period and hence the PCE-dech�orination activity in the s�udge cu�tures shou�d be
expected initia��y. Genetic change, which might invo�ve mutation or genetic exchange,
was a�so un�ike�y because of the reproducibi�ity of the acc�imation periods . The �ast
exp�anation may a�so be app�ied to exp�ain the case of dech�orination because the
dech�orination was initia��y present at a �ow rate . If it is true, the �ag period in PCE
dech�orination is not true acc�imation . The short acc�imation period required for the
reductive dech�orination of PCE a�so suggests that popu�ations capab�e of dech�orinating
PCE are popu�ar in anaerobic digested s�udge .
In this study, PCE was very quick�y dech�orinated to V.C., which was
dech�orinated to ethy�ene at a much s�ower rate than previous steps . On average, after
the �ag time 49 µmo�es of added PCE cou�d be degraded to VC in 4 days in the 100 m�-
s�udge system (that is, the ca�cu�ated maximum dech�orination rate was about 122.5
µmo�es of PCE per �iter per day), whi�e on�y about 30% of PCE was recovered as
ethy�ene. It takes much �onger to produce more ethy�ene . Over the 40 days of extended
incubation observed from the mixed-cu�ture bott�e (B2), 119 more µmo�es of ethy�ene
was produced from dech�orinating VC . The VC dech�orination rate corresponding to
this reaction is on�y about 3 .0 µmo�es per �iter per day . Therefore, the dech�orination of
VC to ethy�ene wou�d be the rate-�imiting step in comp�ete dech�orination of PCE .
142
Optimizing the dech�orination conditions to enhance the dech�orination in this step is
important in using reductive dech�orination in bioremediation processes .
Tab�e 22 The increasing rate of PCE dech�orination by freshHyperion s�udge at initia� �ag period .
* PCE = 49 - (TCE + cis-DCE + VC) ; ** Initia� PCE dosage .
When PCE was dech�orinated in digested s�udge, a series of �ess ch�orinated
intermediates have been observed to accumu�ate transient�y . Such sequentia�
dech�orination not on�y revea�ed the anaerobic biodegradation progression of PCE, but
a�so provided the information that the more ch�orinated congeners (such as PCE and
TCE) are thermodynamica��y more favorab�e to be dech�orinated . After a short �ag time,
PCE was tota��y degraded to TCE in 0 .8 days in the Hyperion s�udge, then two more
days were required to convert to cis-DCE, and then cis-DCE took 7 days to become VC .
An even �onger period is genera��y expected to carry out the dech�orination of VC to
ethy�ene. The dech�orination of pure VC to ETH was observed to be much more
143
reaction time
(days)
PCE*
(µmo�es)
TCE
(µmo�es)
cis-DCE
(µmo�es)
VC
(µmo�es)
CH4
(µmo�es)
0 49.0** 0.00 0.00 0.00 0.00
0.67 48 .66 0.34 0.00 0.00 1046
1 .63 48 .30 0.68 0.02 0 .00 1438
2 .63 47 .03 0.94 0.03 0 .00 1621
3 .96 43 .82 5 .10 0 .08 0.00 1776
4.79 1 .78 46 .6 0.62 0.00 1831
resistant than that for any other tested compounds . During the process of sequentia�
reductive dech�orination of a�� tested compounds, the conversion of VC to ETH was
a�so s�ow, but it was notab�y faster than the pure VC conversion to ETH . The possib�e
reason has been suggested in DiStefano's dissertation(1992) that the presence of PCE
and/or its �esser ch�orinated products (TCE, DCEs) may faci�itate transformation of VC
to ETH .
However, as suggested previous�y in this study, the required environmenta�
conditions may be different for each compound's dech�orination . Therefore, perhaps,
the resistance of VC to reductive dech�orination has resu�ted from the presence of
inappropriate environmenta� conditions . After the dech�orination of more ch�orinated
compounds the environment may undergo changes to fit the required conditions for
dech�orinating �ess ch�orinated compounds . If so, it shou�d not be the presence of higher
ch�orinated compounds to inhibit the dech�orination of �ess ch�orinated compounds .
A�though this inhibition was indeed observed in the fresh Hyperion s�udge cu�tures to
which PCE, TCE and VC were simu�taneous�y added at the beginning (see Fig . 29 and
30), the concurrent dech�orination of PCE, TCE, cis-DCE and VC was a�so observed in
many o�d s�udge samp�es (see Fig . A-27, 28 and 29 in Appendix A) . It indicated that
before PCE is comp�ete�y dech�orinated to TCE, the dech�orination of TCE is possib�e to
occur. Dech�orination of VC is a�so achievab�e with the presence of TCE and cis-DCE .
It is not necessary to a�ways expect the s�owest rate for VC dech�orination if the right
environmenta� conditions are provided . Based on the observations of this study, the
factor most possib�y responsib�e for the tardy VC dech�orination is methane-yie�ding
capacity of the s�udge used . For the o�d s�udge samp�es, after most methane-yie�ding
capacity has been consumed (i.e . there is no excess e�ectron acceptor existing for
144
methanogenesis), VC wou�d become more competitive for reductive reaction and
thereby resu�t in a faster dech�orination .
5.4. Nutrient requirements
The presence of a suitab�e e�ectron donor is necessary to prevent the
accumu�ation of dech�orinated intermediates . The dech�orination of PCE has to be
stimu�ated by e�ectron donors �ike �actate, formate, g�ucose and methano� . But in most
previous studies, a comp�ete dech�orination was not achieved . This is possib�y because
reducing equiva�ents were not avai�ab�e for reductive dech�orination resu�ted from
competitions for e�ectrons by other species in the same system . For examp�e, PCE
dech�orination was partia��y inhibited in Hyperion s�udge cu�tures incubated with
methano� more than 1230 µmo�es (comparing Fig . A-12 and Fig. A-�d). Experiments
conducted with s�udge obtained from Chino Basin Wastewater Treatment P�ant showed
s�ower dech�orination . This was probab�y due to carbon source deficiency in this s�udge
because much �ess methane production was observed in these samp�es than that in the
bott�es containing the fresh s�udge obtained from Hyperion Wastewater Treatment P�ant .
The fresh Hyperion s�udge was a�so found to gradua��y �ose their dech�orinating abi�ity
from the bott�e without rep�enishing nutrients . The apparent inabi�ity of digested s�udge
to dech�orinate PCE in the absence of carbon substrate suggests that PCE dech�orination
by digested s�udge is a co-metabo�ic process .
However, autoc�aved Hyperion s�udge (possessing p�enty of nutrients) amended
with PCE, TCE, DCE, V.C . showed no significant transformation, suggesting that
dech�orination is bio�ogica��y dependent and no significant�y abiotic dech�orination have
145
occurred. In digested s�udge cu�tures reductive dech�orination of PCE is bio�ogica��y
dependent, but it is not c�ear that the activity is bio�ogica��y cata�yzed because we found
6.5 µmo�es of TCE and 0 .4 µmo�es of cis-DCE productions in the autoc�aved digested
s�udge incubated with PCE after 4 months . Mohn and Tiedje (1992) have pointed out
that the inhibition of reductive dech�orination by steri�ization on�y imp�ies either
bio�ogica� or bio�ogica��y dependent cata�ysis . Steri�ization may abo�ish the cata�yst of
reductive dech�orination or the source of reductants, which are products of bio�ogica�
activity and needed for the reductive dech�orination . If PCE dech�orination were not
bio�ogica��y cata�yzed, it is possib�e to occur in autoc�aved s�udge containing the
reductant(s) .
In dech�orination of PCE, on�y the ch�orine substituents were removed and
degradation of the remaining hydrocarbon compound (i.e . ETH) did not occur .
Therefore, we can conc�ude that such substrate did not support growth. In order to
maintain the activity of microorganisms, nutrients a�so have to be rep�enished . In the
study of dech�orination of ch�orinated ethy�enes by fresh digested s�udge, major
required nutrients were presumab�y supp�ied initia��y by the source s�udges . When PCE
and other tested compounds were s�ow�y degraded, these existing nutrient materia�s
were a�so metabo�ized to provide e�ectrons and carbon . Later on, an organic substrate
(methano�) p�us autoc�aved anaerobic medium so�ution (containing 50% of supernatant
of fresh digested s�udge and 500 mg per �iter of yeast extract) was provided as the
potentia� e�ectron donor and carbon source. The purposes of the addition of substrate are
to serve as an e�ectron donor for reductive dech�orination and to support the growth of
the dech�orinating organisms . Throughout this study, yeast extract was used to provide
essentia� amino acids, vitamins, and trace e�ements . But the nutritiona� requirements of
146
reductive�y dech�orinating communities are sti�� not understood yet . For their use in
bioremediation, the know�edge of nutritiona� requirements is essentia� .
5.5. E�ectron donor
As mentioned above, the presence of the suitab�e e�ectron donor is necessary to
reductive dech�orination . The avai�abi�ity of reducing equiva�ents wi�� possib�y affect the
extent of reductive dech�orination. H2 is the most possib�e source of reducing
equiva�ents for the reductive dech�orination of ch�orinated compounds by an anaerobic
consortium (Do�fing, J., and Tiedje, J.M., 1986) . H2 can be obtained from the
oxidation of organic substrates, e.g. from the acetogenic oxidation . But very �itt�e
information about the origin of the reducing equiva�ents needed for the dech�orination in
monocu�ture has been deduced .
Differences in the nature of reducing equiva�ents used for reductive
dech�orination may account for the enhanced rates of PCE dech�orination in the
consortium (Fathepure and Boyd, 1987) . Under methanogenesis, it is proposed that the
reducing equiva�ents for PCE dech�orination are derived from methane biosynthesis and
the e�ectrons can be diverted to PCE by a reduced e�ectron carrier invo�ved in methane
formation (Fathepure and Boyd, 1988a) . If e�ectrons from the primary carbon source
are being shared between methanogenesis and reductive dech�orination processes, a
competition for grabbing the e�ectrons between these two reductive reaction processes is
expected. According to our resu�ts, that too much methano� added to the s�udge did not
resu�t in more or faster dech�orination reaction in the s�udge treatment system, may be
due to the predomination of methanogenesis . Most of e�ectrons generated from methano�
147
have f�owed to the methane formation . This is confirmed by the observation (from the
s�udge bott�e containing 49 µmo�es of PCE and 4920 µmo�es of methano� in MeOH
effect test) that the ca�cu�ated rate of methane formation was 73 times higher ( 600
µmo�es per day per 100 mL) than the rate of PCE dech�orination (8 .2 µmo�es per day
per 100 mL) . Furthermore, in this bott�e, reductive dech�orination was obvious�y
inhibited and PCE was partia��y dech�orinated to cis-DCE on�y .
However, based on the simi�ar resu�ts, Fathepure and Boyd (1988a) deduced
that the reductive dech�orination of PCE is direct�y proportiona� to the concentration of
the primary carbon substrate because it is the source of reducing equiva�ents for both
methane biosynthesis and reductive dech�orination . However, when the amount of TCE
formed, shown in the Tab�e 1 of Fathepure and Boyd's report (1988a), is norma�ized on
the basis of methano� consumed, we wi�� get TCE production rate per mi��imo�e of
methano� consumed from 52 to 31 nmo� per mi��imo�e of methano�, for the tota� added
mass of methano� from 0.25 to 5 .0 mmo�es, respective�y. It is apparent that the
dech�orination of PCE per unit of methano� is inverse�y proportiona� to the concentration
of methano�. Besides, the incubation period (2 weeks) is too �ong to observe the inverse
effect of high methano� concentration on the PCE dech�orination.
In fact PCE dech�orination is indeed �inked to the methano� consumption during
the methanogenesis. But too much methano� added wi�� inhibit the ch�orinated
compounds to undergo reductive dech�orination . In reductive dech�orination, a��
ch�orinated compounds are e�ectron acceptors. The presence of e�ectron acceptors wi��
affect the composition of species present in the digested s�udge . Therefore, the presence
of reductive�y dech�orinating organisms in the digested s�udge may be affected by
148
e�ectron acceptors. E�ectron acceptors do affect the dech�orination activity in anaerobic
digested s�udge . A�so it is possib�e to have a competition for e�ectron donors between
different e�ectron acceptors . So, the f�ow of e�ectrons may be affected by the avai�abi�ity
of e�ectron acceptors . This effect might occur via intrace��u�ar channe�ing of e�ectrons or
via interspecific competition for e�ectron donors (Mohn and Tiedje, 1992) . Methano�-
uti�izing methanogens might compete with PCE-dech�orinating popu�ations for avai�ab�e
e�ectron donors. This may be a direct inhibition of PCE dech�orination by the e�ectron
acceptor because methano� might have higher potentia� being reduced to produce
methane gas .
5.6. Competition and inhibition
Competition between dech�orination and methanogenesis .
Reductive dech�orination of PCE is main�y known under anaerobic conditions .
Reductive dech�orination invo�ves the rep�acement of ch�orine substituent with
hydrogen. This process requires a reductant. The reductant may be derived from
numerous microbio�ogica� activities in anaerobic s�udge systems . However, in such
systems the reductant is a�so strong�y required by methanogenic reactions and it may be
eco�ogica��y or thermodynamica��y more favorab�e for methanogenesis . Therefore, a
competition for reductants between dech�orination and methanogenesis wou�d be
expected to occur in the systems . Furthermore, it is possib�e that the avai�abi�ity of
various e�ectron acceptors wi�� determine the f�ow of e�ectrons . Many evidences
149
observed in this study indeed showed that e�ectron acceptors used by methanogens do
affect dech�orination activity in digested s�udge .
Reductive dech�orination rates were found to be inverse�y corre�ated to methane
productions in the test of methano� effect on PCE dech�orination . The higher amount of
added methano� resu�ting a more methane production did not stimu�ate a faster
dech�orination. Instead, PCE dech�orination was on�y s�ower and partia� under higher
initia� methano� concentrations whi�e it was sti�� comp�ete for other samp�es with �ower
methano� concentrations . The high methane production was the evidence of e�ectrons
having most�y f�owed to methanogenic process, rather than reductive dech�orination .
A�though it has been wide�y suggested in the �iterature that
methanogens/methanogenesis are required for dech�orination of PCE under
methanogenic conditions, a high methanogenic activity sti�� need to be avoided .
In order to enhance the dech�orination of PCE, mixing was app�ied to some test
s�udge samp�es to provide sufficient interaction between the treated compound and
microorganisms. However, it seemed that mixing was more favorab�e for
methanogenesis. As simi�ar�y observed in methano� test, reductive dech�orination was
significant�y inhibited by the higher methane production under mixing conditions . In
addition, the presence of CO2, a methanogenic e�ectron acceptor (used by
methanogens), a�so showed a significant inf�uence on PCE dech�orination under mixing
conditions . The presence of CO2 resu�ted in a higher methane production but a sma��er
ethy�ene formation during PCE dech�orination under mixing conditions . However, there
1 50
was no significant difference observed between the samp�es with and without CO2
incubated under quiescent conditions . This may be due to the much s�ower gas transfer
efficiency for CO2 under quiescent conditions .
In the test of o�d s�udges, a favorab�e environmenta� conditions may have been
created through exhaustion of substrates and reduction of redox potentia� because a�� test
s�udges have been stored anaerobica��y in a 35 °C incubator for a few days to a��ow
methane evo�ution before their uses . However, an apparent �onger �ag time was found in
o�d s�udges. This is �ike�y due to the decrease in viabi�ity during storage. After the �ag
time, dech�orination of PCE, TCE, cis-DCE and even VC (the most resistant one) was
observed to occur concurrent�y under the �ess active�y methanogenic conditions . That
the higher ch�orinated ethy�enes inhibit dech�orination of �ess ch�orinated ones was no
�onger observed in o�d s�udges . The simi�ar resu�ts were a�so common�y observed in
acc�imated Hyperion s�udge. The step-by-step dech�orination in acc�imated s�udge was
not as c�ear as in the fresh anaerobic digested s�udge samp�es . This phenomenon might
be brought about by various possib�e reasons ; e.g., (i) different ch�orinated substrates
might have different abi�ity to compete with methanogenic e�ectron acceptors for
e�ectrons and (ii) the same organism(s) cata�yze the different dech�orination steps but
different environmenta� conditions are required for each step . Based on these resu�ts,
reductive dech�orination of each ch�orinated ethy�ene appeared to have variab�e
responses to the concentration of e�ectron acceptors used by methanogens. For fresh
digested s�udges, a p�enty of e�ectron acceptors is avai�ab�e for methanogenesis .
Methanogenic reactions might outcompete reductive dech�orinations of �ess ch�orinated
ethy�enes by virtue of a higher potentia� for e�ectron consumption . However, at this
1 5 1
time, PCE and/or TCE might have a higher competing abi�ity for e�ectrons and sti�� can
undergo reductive dech�orination. For o�d or acc�imated s�udges, in contrast, on�y a few
methanogenic e�ectron acceptors are avai�ab�e . The �ower concentration of methanogenic
e�ectron acceptors might reduce the redox potentia� of the system and make �ess
ch�orinated ethy�enes (e.g . cis-DCE and VC with a �ower competing abi�ity for
e�ectrons) more readi�y to undergo dech�orination . Therefore, the to�erab�e concentration
of methanogenic substrate may be different for dech�orination of each compound . To
achieve comp�ete dech�orination of PCE, the required environmenta� conditions for each
step may have to be created separate�y .
The different abi�ity of competition for e�ectrons with methanogenesis for each
ch�orinated ethy�ene was demonstrated more obvious�y in the toxicity test . The abi�ity of
competition for e�ectrons for different ch�orinated ethy�enes had the fo��owing order :
TCE > PCE > cis-DCE > 1 .1-DCE > trans-DCE > VC . The s�udge samp�e incubated
with more readi�y degradab�e ch�orinated compounds produced �ess methane ; on the
contrary, more methane production was observed from the samp�es with more persistent
ch�orinated compounds .
However, for the same test compound, the competing abi�ity and dech�orination
rate were observed to be proportiona� to the concentration of ch�orinated compound
present, up to some maximum va�ue (see Tab�e 8 to 13). This suggested that the
concentration of each ch�orinated compound is a�so an important factor which may affect
the competition between dech�orination and methanogenesis .
152
Methanogenesis has been wide�y imp�ied to p�ay an important ro�e in reductive
dech�orination of PCE under methanogenic conditions . However, methanogens may not
necessari�y be invo�ved direct�y in the transformation of ch�orinated ethy�enes if they can
support conditions where the avai�ab�e e�ectrons may be diverted to PCE dech�orination
process (Baker and Herson, 1994) . In the s�udge system, redox conditions may
determine the type of bio�ogica� community that deve�ops . However, the concentrations
of bio�ogica��y produced reductants and methanogenic e�ectron acceptors wou�d affect
the redox potentia� of the system . Therefore, the concentrations of reductants and
methanogenic e�ectron acceptors are two of the most important factors to be contro��ed .
For comp�ete dech�orination of PCE, the concentration of methanogenic e�ectron
acceptors must be decreased be�ow a certain �eve� before the use of PCE or other �ess
ch�orinated compounds as an e�ectron acceptor starts .
Toxicity and inhibition .
PCE and other �ess ch�orinated ethy�enes are usua��y mentioned in regard to their
environmenta� persistence and toxicity . However, it is a difficu�t question to answer
proper�y if PCE or other ch�orinated ethy�enes are toxic materia�s . Many factors may
inf�uence their toxicity to s�udge cu�tures ; e .g ., characteristics of these compounds,
concentration, abi�ity of microorganisms to adapt to their presence, and time of exposure
(Baker and Herson, 1994) . Usua��y, for a non-inhibitory substrate, the biodegradation
rate increases with the substrate concentration, up to an upper �imit . Beyond this �imit,
the rate remains constant . However, for an inhibitory substrate, the degradation rate wi��
1 53
dec�ine when the substrate concentration is beyond the maximum va�ue . Based on this
criterion, we may categorize a�� these compounds as toxic materia�s . In the highest
to�erab�e concentration test, dech�orination rates for each test compound were observed
to increase with their concentrations, up to an upper va�ue. Beyond that �imit,
dech�orination rate was decreased significant�y for a�� test compounds . In addition, the
methane productions for each compound were a�so decreased . It suggested that the high
concentrations of ch�orinated compounds have a�so inhibited methanogenic activity in
the s�udges .
The toxicity of PCE was a�so demonstrated in the s�udge samp�e with activated
carbon added . After the system has reached a stab�e condition, a faster dech�orination
accompanying a higher methane production was observed . This may be due to the �ower
toxicity because most PCE was adsorbed on carbon and PCE concentration in �iquid
phase has been dramatica��y decreased . Due to the toxicity, the concentration of each
compound app�ied to the bio�ogica� treatment process must be �arge�y restricted .
However, the highest to�erab�e concentration for a�� ch�orinated ethy�enes can be further
enhanced in the s�udge system with activated carbon addition because a�� of these
compounds are adsorbab�e on activated carbon .
154
5.7. Carbon addition and dech�orination substrate avai�abi�ity
The avai�abi�ity of dech�orination substrates to microorganisms has been
demonstrated to affect their dech�orination rates in the highest to�erab�e concentration test
(see Tab�e 8 through 13) . The dech�orination rate for each test compound was observed
to be proportiona� to their initia� concentration, up to an upper �imit . To increase
substrate avai�abi�ity, activated carbon is often added to �iquid treatment systems because
it can concentrate substrates on the carbon surface and thereby enhance the contact of
microorganisms and substrates. The concentration enhancement may occur when a
significant fraction of the bacteria can �ive in the pores and these bacteria can direct�y
metabo�ize adsorbed chemica�s on the carbon . However, most bacteria are severa�
orders of magnitude �arger than the average pore size and can not enter the pores .
Therefore, it is sti�� questionab�e if bacteria can direct�y attack the chemica� adsorbed on
carbon. In this experiment, no direct evidence showed that the addition of activated
carbon has improved the dech�orination rate in the acc�imated s�udge system.
Neverthe�ess, the carbon addition indeed offered significant protection against inhibitory
factors to comp�ete dech�orination .
For an unknown reason, partia� dech�orination of PCE to cis-DCE fo��owed a
retardation of further dech�orination was observed in severa� para��e� test s�udge bott�es
without any carbon addition . However, in the bott�es with carbon added, PCE was
dech�orinated to ethy�ene with no exception. The introduction of activated carbon into
1 55
the acc�imated s�udge system may have adsorbed inhibitory or toxic substances, thereby
prevent bacteria from toxicity .
In addition, the overa�� dech�orinating abi�ity observed in both BAC and OBR
systems exceeded the abi�ity of the pure s�udge system a�one . With on�y one time of
nutrient rep�acement, the s�udge cu�ture with carbon added degraded PCE as much as
490 µmo�es. There are two things here which are not achievab�e in the pure s�udge
system. First�y, the reductive dech�orination wou�d have been tota��y inhibited if 490
µmo�es of PCE had been added to the pure s�udge system . However, it is not so
surprising because the adsorptive capacity of activated carbon is expected to reduce the
concentration of adsorbab�e species in the �iquid phase . Second�y, it is impossib�e for
the s�udge to entire�y dech�orinate 490 µmo�es of PCE with such a sma�� amount of
supp�ementary nutrient (e�ectron donors) . Compared to the methane production (about
1300 µmo�es) from the samp�es (U 1 and S 1) with 49 µmo�es of PCE addition, a much
sma��er amount (about 200 µmo�es) of methane was produced from the highest initia�
PCE dosage samp�es (U6 and S6) . This indicated that most e�ectrons avai�ab�e in the
system have been diverted to the reductive dech�orination . The synergism of the
presence of activated carbon and biodegradation may have created an environmenta�
condition which is more favorab�e for reductive dech�orination . The toxicity of PCE was
reduced through carbon adsorption . Consequent�y, bio�ogica� activity was enhanced . In
addition, the biodegradation of PCE in �iquid phase disturbed the equi�ibrium of PCE
between carbon and aqueous phases and resu�ted in the desorption of PCE from carbon .
This continuous supp�ement of PCE may a�so resu�t in dech�orination occurred more
competitive�y than methanogenesis .
1 56
Genera��y speaking, the added activated carbon is ab�e to adsorb a substantia�
part of PCE and, thus, �ower its inf�uence on the biomass and reduce its inhibitory effect
on the bioprocess .
5.8. Bio�ogica� activated carbon process
Bio�ogica� activated carbon process is a combined adsorption and biodegradation
process. Adsorption and biodegradation are two of the most important remova�
mechanisms of the process. However, the interaction between these two remova�
mechanisms has not been fu��y understood . Co��ective�y, biodegradation and adsorption
occur simu�taneous�y and competitive�y . In addition, these two mechanisms may be
functioning synergistica��y . The preferentia� adsorption of toxic or inhibitory substances
can enhance bio�ogica� activity. Simi�ar�y, bio�ogica� activity may regenerate the
adsorption capabi�ity of the carbon and great�y extend the period of performance .
Remova� mechanisms :
In this study, adsorption was initia��y a faster process than dech�orination . The
major added PCE, which may cause biotoxicity, was preferentia��y adsorbed onto
carbon before significant dech�orination occurred . Carbon adsorption has obvious�y
caused a �ower PCE concentration in the �iquid phase . However, the sequentia�
157
reductive dech�orination of PCE was sti�� observed . About 36 to 70% of initia� added
PCE was recovered as ethy�ene at the end of the test, whi�e a�� remaining PCE was
transformed to V .C. in each different initia� PCE dose samp�e . During the period of
experiment, the sequentia� adsorption, desorption and biodegradation seemed to be the
major mechanisms for PCE dech�orination occurred in BAC process . Prior to
degradation by the s�udge cu�tures, the major added PCE in BAC experimenta� bott�es
was adsorbed on carbon . This may be due to a much faster adsorption rate on carbon .
As degradation occurred, desorption of adsorbed PCE from carbon may be caused by
the reversed concentration gradient . The decrease in PCE and appearance of �ess
ch�orinated ethy�enes is indicative of microbia� transformation .
Comp�ete dech�orination :
Feasibi�ity of the BAC process for comp�ete dech�orination of PCE was
successfu��y demonstrated at a�� initia� PCE doses . The initia� concentration of PCE
achieved for comp�ete reductive dech�orination is much higher than reported previous�y
in the �iterature . At the highest PCE concentration of 813 mg/L, PCE sti�� cou�d be
tota��y dech�orinated and a significant amount (189 µmo�es) of PCE was recovered as
ethy�ene. Most of PCE in each bott�e was dech�orinated and terminated at V.C. This is
very possib�y due to that the right environmenta� conditions were not maintained
continuous�y . A�though we sti�� do not know the exact nature of the right environment
required for comp�ete dech�orination, the possib�e factors might inc�ude temperature,
inorganic trace e�ements, carbon sources (e�ectron donors), and e�ectron acceptors . To
achieve optimum performance in BAC and bioregeneration systems, these
environmenta� factors shou�d be further studied and optimized .
1 58
Carbon performance :
During the operation of BAC, difficu�t-to-degrade or nondegradab�e organics
shou�d accumu�ate on the carbon. This wou�d be the major factor responsib�e for the
decrease in adsorption capacity gradua��y . The adsorbed organics which are degradab�e
wi�� be desorbed into �iquid phase and then degraded when their concentrations are
dropped be�ow equi�ibrium . In bio�ogica� activated carbon process, part of the added
virgin PCE may undergo biodegradation before it is adsorbed on carbon, whi�e most of
added PCE is expected to be adsorbed first and then desorbed to be degraded .
Typica��y, there has no attempt to distinguish between these two in this study .
Activated carbon adsorption is prominent in its attenuation of high transient peak
inf�uent PCE concentrations . The distinctive feature of the addition of activated carbon
to the bio�ogica� treatment observed in the BAC processes is, at �east, to improve
stabi�ity to shock �oads and toxic upsets . However, it is not c�ear if biodegradation rates
in the combined bio�ogica� degradation and carbon adsorption system have been
improved. A major disadvantage of the use of activated carbon is the costs associated
with the rep�acement or regeneration of the spent carbon . Common�y, regeneration of
spent carbon can be more economica� than rep�acement with virgin carbon . Among
many regeneration methods (inc�uding so�vent regeneration, steam stripping, therma�
regeneration, etc .), microbia� regeneration of spent carbon might be the most
advantageous if the adsorbate is biodegradab�e (Sigurdson and Robinson, 1978) .
1 59
However, very few work has been concentrated on the deve�opment of bio�ogica�
regeneration as an a�ternative to traditiona� methods of carbon regeneration .
Effectiveness of BAC process :
This study focused on the effectiveness of granu�ar activated carbon on the
improvement of microbia� to�erance of high initia� PCE dosage during the dech�orination
of PCE. In bio�ogica� activated carbon process, carbon rapid�y reduced PCE
concentration in the �iquid phase and increased methane production compared to the
amount produced from the system without carbon addition . The high adsorption
efficiency and probab�y �ow desorption and subsequent biodegradation of PCE and its
dech�orinated products have enab�ed the improvement of bio�ogica� system in the
to�erance of high PCE �oading during the dech�orination processes .
The success of PCE dech�orination in the BAC process is significant for uti�izing
existing anaerobic digestors for the treatment of PCE contamination . It is a �ess
expensive way because adding on carbon adsorption co�umns to existing bio�ogica�
treatment faci�ities needs �arge capita� expenditures and increased operation costs
(Sub�ette, et a� ., 1982)
1 60
5.9 . Off�ine bio�ogica� regeneration
Off�ine bio�ogica� regeneration is another way to combine bio�ogica� treatment
with carbon adsorption . In such a process, sorbed substrate is supposed to desorb from
carbon and undergo biodegradation in bu�k �iquid . Bio�ogica� activity is uti�ized to
regenerate activated carbon. The major purpose of this study is to examine the feasibi�ity
of the off�ine bio�ogica� regeneration process for PCE �aden carbon . The regeneration of
spent, PCE-bearing granu�ar activated carbon by anaerobic digested s�udge cu�tures was
achieved in a�� test samp�es . The PCE adsorbed on carbon cou�d be comp�ete�y
detoxified to non-toxic ethy�ene in acc�imated s�udge cu�tures. The nature of
dech�orination products is exact�y same as that observed in the s�udge system without
carbon .
Regeneration mechanisms:
Numerous studies have shown that adsorbed substrates on carbon can be
degraded bio�ogica��y . It is feasib�e to regenerate the spent carbon with a bio�ogica�
cu�ture. However, if microorganisms can direct�y uti�ize an adsorbed substrate is sti��
not exact�y understood yet . In this study, PCE dech�orination in OBR system seemed
more �ike�y to be a two-step reaction, that is, adsorbed PCE was desorbed from carbon
first and then degraded by s�udge cu�tures in the �iquid phase. Desorption continuous�y
occurred unti� a�� of the PCE, adsorbed and unadsorbed, was degraded .
1 61
Bioregeneration efficiency :
Usua��y, bioregeneration is defined as the renewa� of the adsorptive capacity of
activated carbon by the microbia� activity that is capab�e of metabo�izing the adsorbed
compounds subsequent to their desorption from the carbon (Sub�ette, et a� ., 1982) and
is recognized by the increase in adsorptive capacity . However, many other mechanisms
can a�so achieve the remova� of materia� adsorbed on carbon and hence increase
adsorptive capacity. To recognize the occurrence of bioregeneration in this study, PCE-
dech�orinated products were used as the indicator . Once these compounds appear in the
�iquid phase, a certain amount of adsorbed PCE wou�d have been removed from the
carbon and bio�ogica��y degraded . In addition, ethy�ene, the end product of PCE
dech�orination, was found to be the best one for eva�uating bioregeneration efficiency
because it adsorbs neither on carbon nor on s�udge materia�s . Tota� ethy�ene production
based on the ana�ysis of gas samp�es wou�d be re�ative�y more accurate when it is used
for the eva�uation of the regenerated portion of the carbon's adsorptive capacity .
At the end of the experiment, the percentages of PCE recovered as ethy�ene
production for each initia� dose ranged from 22 .5 to 72.0% (see Tab�e 20). However, to
correct�y estimate the bioregeneration efficiency of carbon, any dech�orination product
which has not been adsorbed on the carbon and stay in the �iquid and s�udge so�id
phases shou�d be accounted. Therefore, the ethy�ene production p�us the tota� mass of
VC found in both �iquid and s�udge phases is used as an indicator for the eva�uation of
bioregeneration efficiency because on�y VC and ethy�ene were found at the end of the
test. Based on the experimenta� resu�ts, a high efficiency of bioregeneration of PCE
�aden carbon was achieved in a�� test samp�es . The tota� regeneration efficiency for each
162
PCE dose ranges from 68 to 100% . It obvious�y depends upon the initia� amount of
adsorbed PCE. In this study, the effect of various PCE �oading on bioregeneration
efficiency at different time points was a�so investigated . The resu�ts showed that
bioregeneration efficiency is a�so dependent of regeneration time. Besides, a sma�� part
of work was conducted as a function of the carbon dosage . The amount of activated
carbon added was a�so demonstrated to inverse�y affect bioregeneration . This may be
due to a s�ower desorption of chemica�s resu�ting from the introduction of too much
carbon.
Genera��y speaking, bioregeneration efficiency wi�� be high�y inf�uenced by the
environmenta� conditions . Such environmenta� factors as temperature, trace nutrients,
e�ectron donors, e�ectron acceptors, spent carbon adsorbate �oading, carbon type, ratio
of carbon mass to biomass, tota� biomass, and retention time can be modified to
optimize the environment for bioregeneration (Baker and Herson, 1994 ; Sigurdson and
Robinson, 1978; Goeddertz, Matsumoto, and Weber, 1988) .
Reproducibi�ity :
Bioregeneration observed from another regeneration tria� using Hyperion s�udge
obtained on different date is quite simi�ar . The reproducib�e bioregeneration may be due
to the reproducibi�ity of PCE reductive dech�orination which has been previous�y
observed in anaerobic digested s�udge .
163
1 64
Equi�ibrium between carbon and bu�k �iquid :
During bioregeneration, it was found that the isotherm constants for virgin
carbon in deionized water were not app�icab�e to the spent carbon being regenerated in
the s�udge system. A change in carbon adsorption characteristics may have occurred
during the regeneration process because the possib�e bacteria� growth around the carbon
partic�e and the re�ative thick so�id materia�s of digested s�udge were expected to
decrease the carbon adsorption and desorption abi�ity .
Biodegradabi�ity :
From the ana�ysis of �iquid samp�es, it was observed that TCE, cis-DCE, V .C .
and ethy�ene appeared sequentia��y and then vanished in the same order except ethy�ene
which was gradua��y accumu�ated during the bioregeneration process . However, 100%
of recovery of ethy�ene from adsorbed PCE was never achieved among test samp�es .
V.C. was the major accumu�ated dech�orination product . This indicated that each
compound's biodegradabi�ity wi�� high�y affect the efficiency of bioregeneration . For
more biodegradab�e compound, the �oss of effective adsorption site may be �ess during
each cyc�e of a mu�tip�e adsorption and bioregeneration .
Loss of adsorptive capacity :
Because of the difficu�ty of remova� of the adherent s�udge materia�s to the
carbon, the equi�ibrium adsorptive capacity of the regenerated carbon was not
experimenta��y tested . However, after three runs, the initia� equi�ibrium PCE
concentration in the �iquid phase of the samp�e used for pre�iminary BAC test was sti��
c�ose to the va�ue of the first run . This suggested that there is no significant �oss
occurred for the PCE equi�ibrium adsorption capacity of carbons that were regenerated
bio�ogica��y three times . In Sigurdson and Robinson's study, it was a�so observed that
no appreciab�e change in the bio�ogica��y regenerated carbon adsorption capacity after
the first regeneration. The �oss (about 53% of the fresh carbon adsorption capacity
observed in their experiment) occurred during the first regeneration is probab�y due to
the �imit of the physica� desorption process, i .e. adsorbate might be no �onger desorbed
from carbon once the amount of adsorbed compound on carbon is be�ow a certain �eve� .
Based on a study of adsorbate distribution in the pores of carbon (Jiang and
Huang,1982 ; cited in Zhang's paper, 1991), the strength of adsorption was observed to
be part�y dependent upon the ratio of the pore diameter to the adsorbate diameter. When
the ratio is �ess than three, it is very difficu�t for so�vent regeneration because the
adsorbate is tight�y adsorbed in pores . The another reason for the incomp�ete desorption
of adsorbate may be due to the existence of a thresho�d concentration of the adsorbate in
the �iquid for the microbia� degradation . Liquid concentration �ower than the thresho�d
va�ue, wi�� not be achievab�e during bioregeneration . In addition, reduction in the
adsorptive capacity of regenerated carbon may resu�t from microbia� fou�ing of the pore
openings or irreversib�e adsorption of metabo�ites- during the bioregeneration
(Hutchinson and Robinson, 1990b) .
If any intermediate metabo�ite produced from the metabo�ism of treated
compounds were irreversib�y adsorbab�e or reversib�y adsorbab�e but
165
nonbiodegradab�e, it wou�d eventua��y cause a significant �oss in adsorption capacity of
bio�ogica��y regenerated carbon . In this study, PCE was sequentia��y converted to TCE,
cis-DCE, V .C. and eventua��y ended at ethy�ene without any other intermediate
metabo�ite . Therefore, such kind of regeneration �oss may not be encountered in PCE-
bearing carbon bioregeneration process .
Rate-�imiting step :
For free suspended microorganisms in the �iquid phase, the avai�ab�e substrate is
those disso�ved in the bu�k aqueous phase. The biodegradation of PCE in �iquid phase
wi�� disturb the equi�ibrium of PCE between carbon and aqueous phases and resu�t in
the desorption of PCE from carbon . In such a case, if the biodegradation rate is much
greater than the rate of PCE desorption, bioregeneration of carbon wi�� be governed by
the rate of PCE desorption and the equi�ibrium of PCE between carbon and the bu�k
aqueous so�ution wi�� never be reached. Otherwise, adsorbed-PCE on carbon is a�ways
equi�ibrating with the PCE concentration in the �iquid phase and biodegradation wi��
�imit the bioregeneration rate . In bioregeneration test, we found that PCE desorption
from carbon is more �ike�y to be the �imiting reaction when compared to biodegradation
of PCE in pure s�udge . Actua��y, these two reactions are a�� possib�e to be the rate-
�imiting step during the bioregeneration . For examp�e, in Goeddertz's study (1988)
desorption was observed to �imit regeneration rate, whi�e biodegradation was reported to
be the �imiting step in Sigurdson's research (1978) . It can be a�tered by many factors,
such as the nature of adsorbate and carbon, biodegradabi�ity of adsorbate and
characteristic of microorganisms, etc .
1 66
Bioregenerabi�ity :
In bioregeneration process, PCE wi�� desorb from the carbon unti� a specified
fina� equi�ibrium is reached . The specified equi�ibrium has to be determined based on the
bio�ogica� regenerabi�ity of carbon . The microorganisms used in the regeneration
process wi�� high�y inf�uence the regenerabi�ity . Genera��y speaking, bio�ogica�
regeneration in anaerobic digested s�udge can produce a significant degree of carbon
regeneration . It makes bioregeneration a promising method to regenerate the spent,
PCE-bearing carbon . However, to obtain a conc�usive resu�t, a �ong-term cyc�ica�
adsorption-bioregeneration study is essentia� .
The high bioregenerabi�ity achieved in a�� OBR test bott�es demonstrates not on�y
the feasibi�ity of the off�ine bio�ogica� regeneration process for PCE �aden carbon, but
a�so the concept of a three-stage treatment process consisting of adsorption of PCE by
activated carbon fo��owed by desorption and anaerobic dech�orination for the
rec�amation or treatment of groundwater contaminated with PCE and/or other �ess
ch�orinated ethy�enes. The process wi�� use activated carbon to adsorb PCE from �iquid
phase (contaminated groundwater), instead of a��owing it to direct�y enter the bio�ogica�
treatment system. Then, the activated carbon wi�� be regenerated in an off�ine bio�ogica�
regeneration system. PCE wi�� be detoxified comp�ete�y in the bioregeneration system .
Using this scheme, deoxygenation of the PCE contaminated groundwater wou�d be un-
necessary and 100% of the remova� of PCE from the contaminated groundwater can be
achieved without any secondary po��ution . A�so, higher concentration of PCE can be
obtained which wi�� probab�y resu�t in higher treatment efficiencies in the bio�ogica�
167
system. The use of activated carbon co�umn shou�d be more re�iab�e than direct
bio�ogica� treatment for the remediation of po��uted groundwater .
1 68
6. CONCLUSIONS
The widespread contamination of groundwater and surface water with PCE and
TCE is a serious prob�em facing the industria�ized wor�d today . A�though many
regu�atory strategies have been adopted in an attempt to reduce the use of PCE and TCE,
this contamination is sti�� �ike�y to occur in the future because of their versati�ity as
industria� organic so�vents . To remediate such environmenta� contamination and keep
the continued va�ue of PCE and TCE as industria� organic so�vents, a techno�ogy
capab�e of detoxifying these ch�orinated compounds is needed .
Among severa� techniques used previous�y, bio�ogica� remediation processes are
most potentia��y suited for comp�ete�y treating �arge vo�umes of contaminated
groundwaters and industria� wastewaters without generating toxic end products . Many
previous studies have demonstrated that PCE and TCE are susceptib�e to reductive
biodegradation under a variety of anaerobic environments . However, �itt�e is known
about the feasibi�ity of the exp�oitation of microorganisms in anaerobic digested s�udge
(the most reducing environment) to treat such contamination . In view of this
consideration, the present research concentrates on the study of comp�ete detoxification
of ch�orinated ethy�enes in anaerobic s�udge and the feasibi�ity of using existing
wastewater treatment p�ants to the rec�ame or treat of groundwater contaminated with
PCE and TCE .
Reductive dech�orination was demonstrated to be the primary mechanism
invo�ved in the bio�ogica� transformation of six ch�orinated ethy�enes under
169
methanogenic conditions in a�� test anaerobic s�udges . In this process, ch�orine atoms
were sequentia��y removed and rep�aced with hydrogen .
Comp�ete dech�orination of six ch�orinated ethy�enes (inc�uding PCE, TCE, 3
isomers of DCE and VC) to non-toxic ethy�ene was demonstrated in fresh anaerobic
Hyperion digested s�udge . Reductive dech�orination of PCE was even proved to be a
genera� characteristic of anaerobic digested s�udges obtained from different wastewater
treatment p�ants. A�� of these compounds were transformed under methanogenic
conditions. PCE and TCE, the higher ch�orinated ethy�enes, more readi�y undergo
reductive dech�orination . The �ower ch�orinated ethy�enes are �ess reactive to
dech�orination under methanogenic conditions . In fresh digested s�udge, dech�orination
of TCE and VC can not occur unti� PCE is entire�y dech�orinated . However, in "o�d"
s�udge, a�� these compounds can be dech�orinated simu�taneous�y . This indicates that it
is not necessari�y the presence of higher ch�orinated compounds that inhibits the
dech�orination of �ower ch�orinated compounds . Furthermore, it may be due to
decreasing methanogenic activity a��owing the �ower ch�orinated compounds to
successfu��y compete for e�ectrons in the "o�d" s�udge .
The competition for e�ectrons between methanogenesis and reductive
dech�orination may exist in the digested s�udge incubated with ch�orinated compounds .
A s�ower and partia� dech�orination of PCE is observed from the s�udge samp�e with
higher methanogenic activity resu�ting from a higher amount of methano� added . The
partia� inhibition and retardation of PCE dech�orination is a�so observed with the s�udge
samp�e under mixing condition which causes a higher amount of methane production .
This suggests that reductive dech�orination is re�ated to methanogenesis in digested
170
s�udge and, for each ch�orinated ethy�ene, the reductive dech�orination requires different
methanogenic conditions .
Comp�ete dech�orination of PCE in a bio�ogica� activated carbon process can be
achieved at much higher initia� PCE concentration (813 mg/� without accounting for
vo�ati�ization and sorption) . The added activated carbon protects dech�orinating bacteria
from inhibitory substances and improves the bio�ogica� system's to�erance of high PCE
�oading.
The feasibi�ity of bioregeneration of PCE-�aden carbon is demonstrated in
digested s�udge . The tota� regeneration efficiency is inverse�y proportiona� to the amount
of PCE initia��y added under the experimenta� conditions . The high bioregenerabi�ity
a�so demonstrates that the concept of a three-stage treatment process consisting of
adsorption of PCE by activated carbon fo��owed by desorption and anaerobic
dech�orination is a promising method for the rec�amation or treatment of groundwater
contaminated with PCE and/or other �ess ch�orinated ethy�enes .
A summary of the most important findings in this research is as fo��ows :
1 . Reductive dech�orination of PCE is a genera� characteristic of anaerobic digested
s�udges. PCE-dech�orination abi�ity was demonstrated in a�� the tested anaerobic
cu�tures, inc�uding seven fresh digested s�udges (obtained from Hyperion,
Termina� Is�and, A�varado, Va�encia, JWPCP, Chino Basin (RP�) and (RP2)
Wastewater Treatment P�ants), pond sediment, a mixture of pond sediment and
Hyperion s�udge, and methano�-enrichment cu�tures . The dech�orination abi�ity
varied in the extent of dech�orination of PCE depending upon the cu�ture tested .
17 1
Whi�e comp�ete dech�orination of PCE to ethy�ene was found in most of s�udge
cu�ture samp�es and concentrated methano�-enrichment cu�tures, partia�
dech�orination of PCE to cis-DCE was observed in Chino Basin (RP1) digested
s�udge and pond sediment.
2. An improved PCE-dech�orination abi�ity was achieved in both methano�-
enrichment cu�ture and Chino Basin (RP2) s�udge cu�ture by the renewa� of
nutrient medium and concentrated ce�� mass. Without the rep�acement of nutrient
medium, PCE-dech�orination abi�ity of the fresh Hyperion s�udge cu�tures cou�d
not be sustained .
3 . An apparent �ack of specificity for the reductive dech�orination of ch�orinated
ethy�enes was observed in the fresh anaerobic Hyperion s�udge . A�� six
ch�orinated ethy�enes (inc�uding PCE, TCE, 3 isomers of DCE, and VC) were
biodegraded via reductive dech�orination. Among these six ch�orinated ethy�enes,
PCE is most readi�y dech�orinated comp�ete�y to ethy�ene, whi�e trans-DCE and
VC showed most resistance to reductive dech�orination .
4 . Comp�ete dech�orination, resu�ting in the formation of ethy�ene (an
environmenta��y harm�ess metabo�ite), was demonstrated under methanogenic
conditions for a�� ch�orinated ethy�enes . The same dech�orination pattern was
observed from dech�orinations of a�� six ch�orinated ethy�enes. The observed
dech�orination pattern is summarized in Fig . 49. In the comp�ete dech�orination
process, the dech�orination of VC to ethy�ene was observed as a rate �imiting step .
VC was the major end product of reductive dech�orination of ch�orinated
ethy�enes.
5 . The presence of higher ch�orinated compounds wi�� not inhibit dech�orination of
the �ess ch�orinated compounds . The inhibition of dech�orination of the �ess
172
ch�orinated ethy�enes may be due to the increasing methanogenic activity in the
fresh digested s�udge . In o�d s�udge, dech�orination of PCE, TCE, cis-DCE and
VC occurred concurrent�y .
6 . Of the three possib�e isomers of DCE, cis-DCE is the most significant
intermediate from dech�orination of PCE and TCE . The other two isomers are on�y
produced insignificant�y in most cases .
7 . The �ower LC50 va�ues obtained from PCE, TCE, and cis-DCE corre�ate with the
chemica�s that appear to be dech�orinated easier by the Hyperion s�udge cu�tures . It
appears to indicate that the reductive dech�orination and methanogenesis are in
competition with each other for �imited e�ectrons.
8 . For most ch�orinated ethy�enes (except trans-DCE which showed no significant
dech�orination at a�� concentrations), the dech�orination rate of each compound was
observed to be proportiona� to their concentration, up to an upper �imit .
9 . In semi-continuous operation, a tota� of 30 to 40% of PCE cou�d be recovered as
ethy�ene . Simi�ar resu�ts were a�so obtained from dech�orinations of other
ch�orinated ethy�enes . With the rep�acement of nutrient medium and addition of
methano�, 49 µmo�es per bott�e (81 mg/L) repetitive doses of PCE were
dech�orinated within 4 days, for 5 months of semi-continuous operation .
10 . There seems to be an inverse re�ationship between reductive dech�orination and
methanogenic reaction . Over the concentration of 24.6 mM of methano�, both of
the extent and the rate of PCE dech�orination were significant�y inhibited by
methanogenesis. Under such a condition, PCE was on�y partia��y dech�orinated to
cis-DCE, at a s�ower rate. A competition for e�ectrons between reductive
dech�orination and methanogenesis may exist .
173
11 . PCE dech�orination was retarded by mixing . Mixing is more favorab�e for
methanogenesis . The use of CO2 as an e�ectron acceptor by methanogens a�so
significant�y inhibited reductive dech�orination under mixing conditions .
12 . The comp�ete dech�orination of PCE to ethy�ene at high PCE concentration makes
reductive dech�orination in anaerobic digested s�udge an attractive method for
remova� of PCE in bioremediation processes .
13 . The amount of added activated carbon wi�� inf�uence the effectiveness of carbon
addition on the improvement of comp�ete PCE dech�orination in the carbon-s�udge
system. Too much carbon added wou�d retard comp�ete dech�orination .
14 . In the bio�ogica� activated carbon process, the carbon adsorption provided
significant protection against inhibitory factors to comp�ete dech�orination . The
overa�� comp�ete dech�orinating capacity observed in both BAC and OBR systems
exceeded the abi�ity of the pure s�udge system a�one .
15 . In the bio�ogica� activated carbon process, the tota� ethy�ene production increased
with the amount of PCE added if the incubation time was �ong enough . However,
an inverse re�ationship between methane production and initia� PCE dose was
demonstrated. The more PCE added, the more e�ectrons were diverted to PCE for
reductive dech�orination .
16 . Bioregeneration of PCE-�aden carbon was demonstrated in batch serum bott�es
with acc�imated digested s�udge . A high bioregeneration efficiency of the
adsorptive capacity of activated carbon was achieved in this study . The concept of
the treatment process, consisting of activated carbon adsorption of PCE fo��owed
by desorption and bio�ogica� treatment system for the remediation of groundwater
contaminated with PCE and/or other �ess ch�orinated ethy�enes, was proved. The
off�ine bio�ogica� regeneration process is appropriate for the use in this case .
174
17 . Further research is needed for comp�ete�y understanding the bioregeneration
process .
175
Appendix A
Experimenta� Data
176
Appendix A
List of figures
page
A-� . PCE-dech�orinating abi�ity and formation of intermediates in a
batch cu�ture of fresh anaerobic Hyperion s�udge after successive
additions of PCE and 25 µmo� . of methano� .
184
A-2. PCE-dech�orinating abi�ity and formation of intermediates in a
batch cu�ture of fresh anaerobic Hyperion s�udge after successive
additions of PCE and 2.5 µmo�. of methano� .
185
A-3. PCE-dech�orinating abi�ity and formation of intermediates in
anaerobic pond sediment after successive additions of PCE and
25 .tmo�. of methano� .
186
A-4. PCE-dech�orinating abi�ity and formation of intermediates in
anaerobic pond sediment after successive additions of PCE and
2.5 µmo�. of methano� .
187
A-5. PCE-dech�orinating abi�ity and formation of intermediates in a
mixture (1 :1) of fresh anaerobic Hyperion s�udge and pond
sediment after successive additions of PCE and 25 pmo�. of
methano� .
188
A-6. PCE-dech�orinating abi�ity and formation of intermediates in a
mixture (1 :1) of fresh anaerobic Hyperion s�udge and pond
sediment after successive additions of PCE and 2 .5 µmo� . of
methano� .
189
177
178
A-7. PCE-dech�orinating abi�ity and formation of intermediates in a
batch cu�ture of fresh anaerobic Chino Basin (RP2) s�udge I with
�ow initia� PCE and methano� additions . 190
A-8 . PCE-dech�orinating abi�ity and formation of intermediates in a
batch cu�ture of fresh anaerobic Chino Basin (RP2) s�udge I with
�ow initia� methano� addition but high initia� PCE addition . 191
A-9 . PCE-dech�orinating abi�ity and formation of intermediates in a
batch cu�ture of fresh anaerobic Chino Basin (RP2) s�udge II
with �ow initia� PCE and methano� additions . 192
A-10. PCE-dech�orinating abi�ity and formation of intermediates in a
batch cu�ture of fresh anaerobic Chino Basin (RP2) s�udge II
with �ow initia� methano� addition but high initia� PCE addition . 193
A-11 . PCE-dech�orinating abi�ity and formation of intermediates in a
batch cu�ture of fresh anaerobic Chino Basin (RP2) s�udge II
with high initia� methano� and PCE additions . 194
A-12. Anaerobic Hyperion s�udge abi�ity to degrade PCE . 195
A- 13. Anaerobic Hyperion s�udge abi�ity to degrade TCE. 196
A- 14. Anaerobic Hyperion s�udge abi�ity to degrade cis-DCE . 197
A-15 . Anaerobic Hyperion s�udge abi�ity to degrade trans-DCE . 198
A-16. Anaerobic Hyperion s�udge abi�ity to degrade 1 .1-DCE. 199
A-17 . Anaerobic Hyperion s�udge abi�ity to degrade VC . 200
A-18. Anaerobic Hyperion s�udge abi�ity to degrade PCE at high
methano� concentration . 201
A-19. Decrease in PCE-dech�orinating abi�ity without rep�acement of
nutrient media . 202
A-20. Reductive dech�orination progression of PCE at a �ow PCE
179
concentration . 203
A-2 1 . Long term dech�orination of PCE and formation of intermediates
in a semi-continuous operation serum bott�e (There was no
methano� added from day 0 to 60) . 204
A-22. Long term dech�orination of TCE and formation of intermediates
in a semi-continuous operation serum bott�e . 205
A-23. Long term dech�orination of cis-DCE and formation of
intermediates in a semi-continuous operation serum bott�e . 206
A-24. Long term dech�orination of 1 .1-DCE and formation of
intermediates in a semi-continuous operation serum bott�e . 207
A-25. Dech�orination of PCE and formation of intermediates in a batch
cu�ture of fresh anaerobic Hyperion s�udge . 208
A-26. Dech�orination of PCE and formation of intermediates in a batch
cu�ture of 1-day o�d anaerobic Hyperion s�udge . 209
A-27. Dech�orination of PCE and formation of intermediates in a batch
cu�ture of 2-day o�d anaerobic Hyperion s�udge. 210
A-28 . Dech�orination of PCE and formation of intermediates in a batch
cu�ture of 3-day o�d anaerobic Hyperion s�udge. 211
A-29. Dech�orination of PCE and formation of intermediates in a batch
cu�ture of 4-day o�d anaerobic Hyperion s�udge . 212
A-30. Dech�orination of PCE and formation of intermediates in a batch
cu�ture of 12-day o�d anaerobic Hyperion s�udge . 213
A-3 1 . Dech�orination of PCE and formation of intermediates in a batch
cu�ture of 14-day o�d anaerobic Hyperion s�udge . 214
180
A-32 . Dech�orination of PCE and formation of intermediates in a batch
cu�ture of 20-day o�d anaerobic Hyperion s�udge . 215
A-33. Dech�orination of PCE and formation of intermediates in a batch
cu�ture of 21-day o�d anaerobic Hyperion s�udge . 216
A-34. Initia� PCE dech�orination in the s�udge cu�tures with and without
GAC addition . 217
A-35 . The effect of different amounts of carbon addition on PCE
dech�orination and methane formation (1) . 218
A-36. The effect of activated carbon on PCE dech�orination . 219
A-37. The effect of different amounts of carbon addition on PCE
dech�orination (2) . 220
A-38 . PCE dech�orination in a bio�ogica� activated carbon system with
acc�imated anaerobic digested s�udge (initia� free PCE dose = 49
µmo�) . 221
A-39. PCE dech�orination in a bio�ogica� activated carbon system with
acc�imated anaerobic digested s�udge (initia� free PCE dose = 98
µmo�) . 222
A-40. PCE dech�orination in a bio�ogica� activated carbon system with
acc�imated anaerobic digested s�udge (initia� free PCE dose = 196
p mo�) . 223
A-41 . PCE dech�orination in a bio�ogica� activated carbon system with
acc�imated anaerobic digested s�udge (initia� free PCE dose = 294
p.mo�) . 224
A-42 . PCE dech�orination in a bio�ogica� activated carbon system with
181
acc�imated anaerobic digested s�udge (initia� free PCE dose = 392
µmo�) . 225
A-43. PCE dech�orination in a bio�ogica� activated carbon system with
acc�imated anaerobic digested s�udge (initia� free PCE dose = 490
4mo�) . 226
A-44. Bio�ogica� regeneration of PCE �aden carbon (adsorbed PCE =
49 µmo�) . 227
A-45 . Bio�ogica� regeneration of PCE �aden carbon (adsorbed PCE =
98 µmo�) . 222
A-46. Bio�ogica� regeneration of PCE �aden carbon (adsorbed PCE =
196 µmo�) . 223
A-47. Bio�ogica� regeneration of PCE �aden carbon (adsorbed PCE =
294 µmo�). 224
A-48 . Bio�ogica� regeneration of PCE �aden carbon (adsorbed PCE =
392 µmo�) . 225
A-49. Bio�ogica� regeneration of PCE �aden carbon (adsorbed PCE =
490 µmo�). 226
A-50. A comparison of anaerobic dech�orinating systems with adsorbed
or free PCE dose (PCE dose = 49 µmo�) . 227
A-51 . A comparison of anaerobic dech�orinating systems with adsorbed
or free PCE dose (PCE dose = 98 tmo�) . 228
A-52. A comparison of anaerobic dech�orinating systems with adsorbed
or free PCE dose (PCE dose = 196 µmo�) . 229
A-53 . A comparison of anaerobic dech�orinating systems with adsorbed
or free PCE dose (PCE dose = 294 µmo�) .
230
A-54. A comparison of anaerobic dech�orinating systems with adsorbed
or free PCE dose (PCE dose = 392 µmo�) .
231
A-55. A comparison of anaerobic dech�orinating systems with adsorbed
or free PCE dose (PCE dose = 490 µmo�) .
232
182
Appendix A
List of Tab�es
page
A-1 . Feeding summary for the experiment of the MeOH and media
effect on PCE dech�orination .
233
A-2. Comp�ete dech�orinating abi�ity of acc�imated anaerobic Hyperion
s�udge .
234
183
A
Fig. A-1 PCE-dech�orinating abi�ity and formation of intermediatesin a batch cu�ture of fresh anaerobic Hyperion s�udge aftersuccessive additions of PCE and 25 µmo�es of methano� .
0 20 40
60Rreaction Time (days)
80 100
Fig . A-1a PCE-dech�orinating abi�ity and formation of intermediatesin a batch cu�ture of fresh anaerobic Hyperion s�udge aftersuccessive additions of PCE and 25 µmo�es of methano� .
25
20
00 5 10
15
20Reaction Time (days)
184
25 30
50
40
0
baa
30
r
0 ;~20 I
0r
Fig. A-2 PCE-dech�orinating abi�ity and formation of intermediatesin a batch cu�ture of fresh anaerobic Hyperion s�udge aftersuccessive additions of PCE and 2.5 µmo�es of methano� .
25
E
20
o a+
15obfs. 'acZoa19S
10C00UA 5
00
------------0
20
Fig. A-2a PCE-dech�orinating abi�ity and formation of intermediatesin a batch cu�ture of fresh anaerobic Hyperion s�udge aftersuccessive additions of PCE and 2 .5 µmo�es of methano� .
5
40
60Reaction Time (days)
10
15
20Reaction Time (days)
185
80
25
100
30
r.
0Ev
UA
Fig. A-3 PCE-dech�orinating abi�ity and formation of intermediates inanaerobic pond sediment after successive additions of PCEand 25 µmo�es of methano� .
60
50
40
30
20
10
Fig. A-3a PCE-dech�orinating abi�ity and formation of intermediates inanaerobic pond sediment after successive additions of PCEand 25 µmo�es of methano� .
35
25
20
15
10
5
0
0
0 5
20 40
60Reaction Time (days)
80
100
10
15
20Reaction Time (days)
186
25 30
Fig. A-4 PCE-dech�orinating abi�ity and formation of intermediates inanaerobic pond sediment after successive additions of PCEand 2.5 µmo�es of methano� .
30
25
20
5
0
° 0
0
Fig A-4a PCE-dech�orinating abi�ity and formation of intermediates inanaerobic pond sediment after successive additions of PCEand 2.5 µmo�es of methano� .
40
35
0 5
20 40
60Reaction Time (days)
10
15
20Reaction Time (days)
187
80
25
100
30
Fig. A-5 PCE-dech�orinating abi�ity and formation of intermediates ina mixture cu�ture (1:1) of fresh anaerobic Hyperion s�udgeand anaerobic pond sediment after successive additions of PCEand 25 µmo�es of methano� .
50
a
'
o
00_
Fig . A-5a PCE-dech�orinating abi�ity and formation of intermediates ina mixture cu�ture (1 :1) of fresh anaerobic Hyperion s�udgeand anaerobic pond sediment after successive additions of PCEand 25 µ.mo�es of methano� .
20
16
12
8
4
00 5 10
15
20Reaction Time (days)
188
25 30
Fig. A-6 PCE-dech�orinating abi�ity and formation of intermediatesin a mixture cu�ture (1:1) of fresh anaerobic Hyperions�udge and anaerobic pond sediment after successiveadditions of PCE and 2.5 µmo�es of methano�.
Fig. A-6a PCE-dech�orinating abi�ity and formation ofintermediates in a mixture cu�ture (1 :1) offresh anaerobic Hyperion s�udge and anaerobicpond sediment after successive additions of PCEand 2.5 µmo�es of methano� .
I
I
30
20
15
10
5
0
0
see Fig. 16a
PCE
TCE
cis,DCE
Irans.DCE
1 .1,DCE
VC
0
20
5
.ft.-00w'~`~
189
40
60Reaction Time (days)
80
10
15
20Reaction Time (days)
Note : The major product from PCE dech�orination was VC . These resu�tsappear to indicate that the Hyperion s�udge cu�ture is predominate�yresponsib�e for PCE dech�orination when both are present.
25
100
30
1500
OE
1000z,a
o :a500
rI
O
VA
I
I
1
I
I
Fig. A-7 PCE-dech�orination abi�ity and formation of intermediatesin a batch cu�ture of fresh anaerobic Chino Basin (RP2)s�udge I .
400
300
250
150
100
50
0
Fig. A-7a PCE-dech�orination abi�ity and formation ofintermediates in a batch cu�ture of fresh anaerobicChino Basin (RP2) s�udge I .
Tota� MeOH addition=369 µmo� .•
- - PCE
-~~ TCE
•
--~- cis,DCE
-0-- trans,DCE
-0-- I .I .DCE
--~- VC
rsee Fig . 17a
f1
1 dtoi I b' Is,
'6 '''------------------------0
50
100Reaction Time (days)
ota� MeOH addition=369 gmo�.
150
40
60Reaction Time (days)
Note: 1 . On�y initia� 0 .6 µmo�es of PCE was added in the first 90 days .
2. Data obtained after 90 days indicates PCE dech�orination and formationof intermediates with initia� addition of 49 µmo�es of PCE, increasedconcentration of methano�, and fresh medium rep�acement .
0 20
190
80
•
350
•
200
100
Fig.
A-8
PCE-
dech
�ori
nati
on a
bi�i
ty a
nd f
orma
tion of intermediates in a
batc
h cu
�tur
e of
fre
sh a
naer
obic
Chi
no Basin (RP2) s�udge I.
100
U80 40 20 0I 0
MeOH addition=369 µmot
.PC
E addition--49 µcoo�
.
50
100
Reac
tion
Tim
e (d
ays)
Note
: 1.
49 µmo�es of PCE was added at the beginning of both s
tage
s (A
& B
).2.
Dat
a ob
tain
ed a
fter
90
days
ind
icat
es P
CE d
ech�
orin
ation and formation
of int
erme
diat
es w
ith
init
ia�
addi
tion
of
49 µ
mo�e
s of
PCE, increased
conc
entr
atio
n of
met
hano
�, a
nd f
resh
med
ium
rep�
acemen
t.
150
PCE
1.1,
DCE
XTCE
0VC
MeOH addition=3075 µmo�.
cis,
DCE
Ethy�ene
PCE addition=49 µcoo�.
otr
ans,
DCE
Fig. A-9 PCE-dech�orination abi�ity and formation of intermediatesin a batch cu�ture of fresh anaerobic Chino Basin (RP2)s�udge II .
400
1~ 350OP=L 300y•
250Oa 200a0
C• 150O•
100NA
50
0
V- PCE~~TCE-e- cis,DCE
i, McOH addition=369 µmo�.k PCE addidon-0 .6 µmot .
r
r
II
see Fig. 20a
-----------------0
50
trans,DCE1 .1,DCEvc
Reaction Time (days)
Fig. A-9a PCE-dech�orination abi�ity and formation of intermediatesin a batch cu�ture of fresh anaerobic Chino Basin (RP2)s�udge II.
MeOH addition=2460 µmot .PCE addition-49 µmo� .
100
150
A
0
0 PCE
MeOH addition=369 pmo� .TCE
PCE addition=0.6 µmot.
j
cis,DCEtrans,DCE
-o- 1 .1,DCE
20 40
60Reaction Time (days)
80 100
Note: 1 . On�y initia� 0.6 µmo�es of PCE was added in the first 90 days .2. Data obtained after 90 days indicates PCE dech�orination and formation
of intermediates with initia� addition of 49 µmo�es of PCE, increasedconcentration of methano�, and fresh medium rep�acement .
192
Fig.
A-10
PCE-de
ch�o
rina
tion
abi
�ity
and
for
mati
on of intermediates in a
batch
cu�t
ure
of f
resh
ana
erob
ic C
hino
Basin (RP2) s�udge II
.10
0 60
0
MeOH a
ddit
ion=
369
µcoo
�.
PCE ad
diti
on=4
9 µc
oo�
.
IAI
50
100
Reaction Time (days)
B
Note
: 1.
49
µmo�es of PCE was added at the beginning of both sta
ges
(A &
B).
2. Data
obta
ined
aft
er 9
0 da
ys i
ndic
ates
PCE
dec
h�or
inat
ion and formation
of intermediates with initia� addition of 49 µmo�es of PCE, increased
concen
trat
ion
of m
etha
no�,
and
fre
sh m
ediu
m re
p�ac
ement
.
150
01.
1,DCE
PPCOE
cis,
DCE
0-
Ethy
�ene
MeOH addition=2460 µcoo�
.tr
ans,
DCE
PCE
addi
tion
=49
µcoo
�.
Fig.
A-11 PCE-dech�orination abi�ity and formation of
int
erme
diat
es i
n a
batch cu
�tur
e of
fre
sh a
naer
obic
Chi
no B
asin (RP2) s�udge II
.11-1 O E
500
400
C) Q
0
MeOH
add
itio
n=36
90 µ
mo�
PCE addition=49 µmot
.
MeOH addition=2460 µmot.
PCE addition=49 µmo�
.
B
50
100
150
Reaction Time (days)
Note
: 1.
49
µmo�es of PCE was added at the beginning of both sta
ges
(A &
B).
2 . Data ob
tain
ed a
fter
90
days
ind
icat
es P
CE d
ech�
orin
atio
n and formation
of i
ntermediates with initia� addition of 49 µmo�es of PCE
, de
crea
sed
conc
entr
atio
n of
met
hano
�, a
nd f
resh
med
ium
rep�
acem
ent.
PCE
tran
s,DC
E)(
TCE
O1.1,DCE
pci
s,DC
E0-
VC
w----o- PCE
_)( TCE- II cis,DCE
W
40 - O
trans,DCEU
LLDCE
2 o VC
m Ethy�ene
ji
30
20
A
Fig. A-12 Anaerobic Hyperion s�udge abi�ity to degrade PCE .
50
10 -II -1I
0 1
0
Fig. A-12a Anaerobic Hyperion s�udge abi�ity to degrade PCE.5
1
0
0
it
Tota� McOH addition=323 µmo�.Tota� PCE addition=53 µmo� .
I
I I
I
I
see Fig . A-12a
20
10
IoII
40
60
80Reaction Time (days)
195
40
6100
20
30Reaction Time (days)
Note: 1 . At the first 45 days, �ow PCE concentration was tested .2. After 45 days, high PCE concentration was tested .
120
50
A
A
Fig. A-13 Anaerobic Hyperion s�udge abi�ity to degrade TCE.
50
2
1
0
TCE
cis,DCE
trans.DCE
I .1,DCE
VC
w
Ethy�ene
20
Fig . A-13a Anaerobic Hyperion s�udge abi�ity to degrade TCE .5
)(TCE
-g- cis,DCE
--0--- trans,DCE
-O- t.I,DCE
40
60
80Reaction Time (days)
I d
196
Tota� McOH addition=323 µmo� .Tota� PCE addition=53 µmo� .
100
120
Tota� McOH addition=200 µmo� .Tota� PCE addition=3.9 µmo� .
'7C
0
10
20
30
40
50Reaction Time (days)
Note: 1 . At the first 45 days, �ow TCE concentration was tested .2. After 45 days, high TCE concentration was tested .
HU
Fig. A-14 Anaerobic Hyperion s�udge abi�ity to degrade cis-DCE.
60
50
ba y 40[ O
R 0 30Eov
20A
10 1 ,=-
see Fig . A-14aII
2
1
0
0
cis,DCE
0
trans,DCE
1 .1,DCE
VC
Ethy�ene
20
Fig . A-14a Anaerobic Hyperion s�udge abi�ity to degrade cis-DCE .5
0
Note:
10
40 60Reaction Time (days)
Tota� McOH addition=323 µmo� .Tota� PCE addition=53 grant .
80
100
120
20
30
40
50Reaction Time (days)
1. At the first 45 days, �ow cis,DCE concentration was tested .2. After 45 days, high cis,DCE concentration was tested .
197
cis,DCE Tota� MeOH addition=200 µmo� .Tota� PCE addition=3.9 µmo� .
-6-
p trans,DCE
1.1,DCE_ -0
-~ -- VC
NQ
A
Fig. A-15 Anaerobic Hyperion s�udge abi�ity to degrade trans-DCE .
50
20
3
2
I
0
10
20
30
40
50Reaction Time (days)
Note: 1. At the first 45 days, �ow trans,DCE concentration was tested .2. After 45 days, high trans,DCE concentration was tested .
198
0
O trans,DCE
o I .�.DCE
VC
ƒ
Ethy�ene
20
0 - trans,DCE
1 .1,D-CE
=-~-- VC
40
60
80Reaction Time (days)
Fig . A-15a Anaerobic Hyperion s�udge abi�ity to degrade trans-DCE .5
Tota� MeOH addition=200Itmo� .Tota� PCE addition=3 .9 tmo�.
0-0
100
0
120
Fig . A-16 Anaerobic Hyperion s�udge abi�ity to degrade 1.1-DCE.
Q
Ub
O iaoa ,ao-.° Eo =LeUN'L1
Fig. A-16a Anaerobic Hyperion s�udge abi�ity to degrade 1 .1-DCE .5
4
3
2
1
0
0
0
-
.
vc
20
Tota� McOH addition=323 µmo� .Tota� PCE addition=53 µmo� .
40
60
80Reaction Time (days)
p t.I,DCE
Tota� McOH addition=200 µmot .Tota� PCE addition=3.9 µcoo�.
100
10 20
30reaction time (days)
199
40
Note : 1 . At the first 45 days, �ow 1 .1,DCE concentration was tested .2. After 45 days, high 1 .1,DCE concentration was tested .
120
50
UNA
Fig. A-17 Anaerobic Hyperion s�udge abi�ity to degrade VC.
50
1
0
0
vcE
Ethy�ene
10 � _
.1 -
see Fig. A-17a
L~ - >_ - - . N -
20
--. VC
Tota� MeOH addition=323 µmo� .Tota� PCE addition=53 µma� .
40
60
80Reaction Time (days)
Tota� MeOH addition=200 µcoo�.Tota� PCE addition--3 .9 µcoo� .
100
Fig . A-17a Anaerobic Hyperion s�udge abi�ity to degrade VC .
5
IIIIt,,tI�,ttItt,tItt,t
200
120
0
10
20
30
40
50Reaction Time (days)
Note: 1 . At the first 45 days, �ow VC concentration was tested .2. After 45 days, high VC concentration was tested .
0.0 cd
O20
oO A
10
Anaero
bic
Hype
rion
s�udge
abi�
ity
todegradePCE
athi
ghme
thano� concentration
.
0
PCE
TCE
cis,DCE
tran
s,DC
E1.1,DCE
vc Ethy
�ene
2040
60
80Re
acti
on T
ime
(day
s)Tota
� Me
OH addition=1230 µcoo�
.Tota� PCE
addi
tion
=53
µcoo
�.
100
Note
: Co
mpar
ed to the resu�ts shown in Fig
. A-
12,
the
high
er m
ethano� concentration
does
not improve the PCE dech�orination and ethy�ene pro
duct
ion.
120
Fig
.A-
18
50
I U b ~
40 30
Fig.
A-19
Decrease in PCE-dech�orination abi
�ity
wit
hout
rep�
acem
ent
of n
utri
ent
medi
um.
020
40 R
eact
ionTi
me(d
ays)
80
100
Notice that when nutrient media is not rep�enished, major end products accumu�ated are
TCE
and �ower than expected amounts of VC. Accumu�ation of TCE that is usua��y
conv
erte
d co
mp�e
te�y
to
VC d
emon
stra
tes
that
som
e in
hibi
tion
s may be due to
nutrient
dep
riva
tion
.
120
300
I250
U o200
0o'
-:150
0
°o
=.100
U N A50 0
Fig.
A-20
Red
ucti
ve d
ech�
orin
atio
n pr
ogre
ssio
n of
PCE
at
a �o
w PC
Eco
ncen
trat
ion
in a
fre
sh a
naer
obic
Hyp
erio
n s�
udge
.
1.2
1 .0
0.4
Q
0.2
0.0
41
0
0
0
I1-4.0.1ma
1st
]PCI
Einjection
PCE
TCE
2nd
PCEiniection_~
PCE
TCE
cis,
DCE
0
tran
s,DC
E
1.1,
DCE
-+--
vc
04
8
12Re
acti
on T
ime
(day
s)16
20
U C) A
Fig.
A-21
Long
ter
m de
ch�o
rina
tion
of
PCE
and formation of
inte
rmed
iate
s in
a s
emi-
cont
inuo
us o
pera
tion
ser
um b
ott�
e.
(The
re w
as n
o me
than
o� a
dded
fro
m da
y 0
to 6
0.)
800
700
600
U
N
a
500
o400
oG •
o ~.
300
200
100 0
0
PCE
)(
TCE
cis,
DCE
trans,DCE
1 .1,
DCE
•
V.C
.et
hy�e
nePCE consumption
vId
r.
50
100
Reac
tion
Tim
e (d
ays)
150
4-1 A
Fig.
A-22
Long
ter
m de
ch�o
rina
tion
of
TCE
and
form
atio
n of
intermediates in a semi-continuous operation serum bott�e
.
500
400
300
200
100 0
0
TCE
cis,
DCE
tran
s,DC
E
1.1,
DCE
-f- Viny� ch�oride
o
consumption of TCE
2040
60
80Re
acti
on T
ime
(day
s)100
120
Fig.
A-23
Long term dech�orination of cis-DCE and
form
atio
n of
intermediates in a semi-continuous operation serum bott�e.
050
100
Reaction Time (days)
150
700
600
500
U O O N
a40
00
o- 0
030
0o U A
200
100 0
(1) AFig.
A-24
Long term dech�orination of 1.1-DCE and formation of
intermediates in a semi-continuous o
pera
tion
ser
um b
ott�
e.
800
700
500
400
300
200
100 0
050
100
Reaction Time (days)
150
Fig.
A-25
Dech�orination of PCE and formation of int
erme
diat
esin
a b
atch
cu�
ture
of
fres
h an
aero
bic
Hype
rion
s�u
dge .
100 80 60 40 20
CD
'~ COD
Fig.
A-26
Dech�orination of PCE and formation of intermediates in a
batch cu�ture of one-day o�d* anaerobic Hyperion s�udge
. 4000
050
10
20
30
40Re
acti
on T
ime
(day
)No
te:
One-day o�d s�udge means that the s�udge cu�ture was
incubated for
one
day
befo
re P
CE i
njec
tion
.
3500
3000
2500
2000
1500
1000
500
4 CD w o 0
100 80
-v Z b 26 a
600 0
0 o~
40-
U N A20
-
Fig.
A-27
Dech
�ori
nati
onof PCE and formation of inte
rmed
iate
s in
abatch cu�ture of two-day o�d anaerobic Hyperion s�udge
o ~
00
0 O=L
0 G) A
100
020
40
60
80Reaction Time (day)
100
opa� oe
re
-49A
::
120
5000
4000
3000
1000
rA U O^ a5O
0 IdO
QE
0 U N A
Fig.
A-28
Dech
�ori
nati
on o
f PC
E an
d fo
rmat
ion
of i
nter
medi
ates
in
aba
tch
cu�t
ure
of t
hree
-day
o�d
ana
erob
ic H
yper
ion
s�ud
ge.
100
o
e
020
40
60
80Reaction Time (day)
100
120
5000
4000
CD
3000
ii
2000
1000
0
Fig.
A-29
Dech�orination of PCE and formation of intermediates in a
batc
h cu
�tur
e of
fou
r-da
y o�
d an
aero
bic
Hype
rion
s�u
dge
.
A
100 80 40
.2E~
U N
20 0
o ;jy0 0
0
.MMA
....
Aer
010
2030
40
50
60Re
acti
on T
ime
(day
)70
80
5000
4000
3000
2000
1000
0
Methane
PCE
trans-DCE
ETH
XTCE
1.1-DCE
cis-
DCE
µV
.C.
Fig.
A-30
Dech�orination of PCE and formation of int
erme
diat
es i
na batch cu�ture of 12-day o�d anaerobic Hyperion s�udge
.
100 40 20
020
40
60Re
acti
on T
ime
(day
)80
100
6000
5000
4000
3000
0
2000
1000
Fig.
A-31
100 80
-
60-
40 20
Dech�orination of PCE and formation of intermediates in
a ba
tch
cu�t
ure
of 1
4-da
y o�
d an
aero
bic
Hype
rion
s�u
dge
.
0
Meth
ane
0
PCE
0
tran
s-DC
E
m
ETH
TCE
0
1.1-DCE
cis-DCE
---0
V.C
.
2040
60Reaction Time (day)
80100
6000
5000
4000
3000
2000
1000
a CD o o' 0
Fig .
A-32
Dech�orination of PCE and formation of intermediates in
a ba
tch
cu�t
ure
of 2
0-da
y o�
d an
aero
bic
Hype
rion
s�u
dge
.
-20
020
40
60Reaction Time (day)
8010
0
Fig.
A-33 Dech�or
inat
ion
of P
CE a
nd f
orma
tion
of
intermediates in
a ba
tch
cu�t
ure
of 2
1-da
y o�
d an
aero
bic
Hype
rion
s�u
dge
.
100 80 20
-20
020
40
60Reaction Time (day)
80100
aba_y
C)G9o .?N
ob 3vWUa
Fig . A-34 Initia� PCE dech�orination in the s�udge cu�turewith GAC addition.
300
250
200
150
100
50
0 20 40
60
80Reaction Time (day)
100
Initia� PCE dech�orination in the s�udge cu�turewithout GAC addition .
new PCE addition
2 17
°
Ethy�ene -
120
25
5O
700
600 =
PCEoTCEx
-.6 cis-DCEa trans-DCE
500
o
o 1.1-DCE0 - V.C .
Fig . A-35 The effect of different amounts of carbon additionon PCEdech�orination and methane production .
300
700
on:a Z
C . .1OOPav
n
�.N f3
o O
V[:a Za ~..a
06a
2 1 8
0
00
10
20
30
40
50Reaction Time (day)
Note : a). Cu�ture with 2 grams of GAC initia��y, and add 2 more grams of GAC after 30 days .b). Cu�ture with 2 grams of GAC throughout the experiment .
Fig. A-36 The effect of activated carbon on PCE dech�orination.
0 2 4 6
8
10Reaction Time (day)
2 1 9
12 14
03F
00
~aoB
U
Fig. A-37
The effect of different amounts of carbonn i
0
80
60
40
20
0
0 .10
0 .08
0 .06
0 .04
0 .02
0 .00
0 .5
0 .4
0 .3
0 .2
0 .1
0 .00 20 40
60Reaction Time (day)
220
80 100
0 .80
0.60
gi gn"=
0.40
~+ aso $ oµ
0.20
0.0
0.10
0.080
0 .060
0.040
0.020
0 .0
Methane formation
-e-0g-GAC8g-GAC
- -4g-GAC
Ethy�ene formation
V .C . concentration in �iquid phase (•mo�I�)
0
cis-DCE concentration in �iquid phase (pmo�/�)
TCE concentration in �iquid phase (pmo�I�)
Init PCE dose-49 pmo� .
PCE concentration in�iquid phase (•mo�/�)
Initia� PCE dose=196
(free) .gmo�Initia� PCE dose=196 �inte� (adsorbed).
Fig . A-38
PCE dech�orination in a bio�ogica� activatedcarbon system with acc�imated anaerobic digesteds�udge. (Initia� free PCE dose = 49•mo�)
P
Fig. A-44
50
40
30
20
10
0 10
Bio�ogica� regeneration of PCE �aden carbon.(Adsorbed PCE = 49•mo�)
20 30
40
50Reaction Time (day)
22 1
60 70
1600
1400
1200
1000
800
600
400
200
Fig. A-39 PCE dech�orination in a bio�ogica� activatedcarbon system with acc�imated anaerobic digesteds�udge. (Initia� free PCE dose = 98•mo�)
.d a.0a an
y09:abA µ,ƒ"
40
v
U o
20a
=Lv
Ov
100
80
60
0
Fig. A-45
0 10 20 30
40
50Reaction Time (day)
60
Bio�ogica� regeneration of PCE �aden carbon .(Adsorbed PCE = 98•mo�)
70
0 10 20 30
40
50Reaction Time (day)
222
60 70
500
400
100
0
Fig . A-40 PCE dech�orination in a bio�ogica� activatedcarbon system with acc�imated anaerobic digesteds�udge (Initia� free PCE dose = 196gmo�)
200
150
100
0
Fig. A-46
Bio�ogica� regeneration of PCE �aden carbon.(Adsorbed PCE = 196gmo�)
0 10 20 30
40
50Reaction Time (day)
223
60 70
Fig . A-41 PCE dech�orination in a bio�ogica� activatedcarbon system with acc�imated anaerobic digesteds�udge. (Initia� free PCE dose = 294•mo�)
300
50
Fig. A-47
0 10 20
Bio�ogica� regeneration of PCE �aden carbon .(Adsorbed PCE = 294•mo�)
0 10 20
30
40
50Reaction Time (day)
224
60 70
30
40
50
60
70Reaction Time (day)
300
yay+ ~g 250O
WAD200
d 150
b _g100
Wo 50
Fig . A-42
PCE dech�orination in a bio�ogica� activatedcarbon system with acc�imated anaerobic digesteds�udge. (Initia� free PCE dose = 392gmo�)
200
100
0
0 10 20 30
40Reaction Time (day)
Fig. A-48
Bio�ogica� regeneration of PCE �aden carbon .(Adsorbed PCE = 392gmo�)
0 10 20 30
40
50Reaction Time (day)
225
50
60
60
070
70
500
400
100
5 00
400
300
200
100
v
2.
Fig . A-43 PCE dech�orination in a bio�ogica� activatedcarbon system with acc�imated anaerobic digesteds�udge. (Initia� free PCE dose = 490•mo�)
250
200
0
Fig. A-49
0 10 20 30
40
50Reaction Time (day)
Bio�ogica� regeneration of PCE �aden carbon .(Adsorbed PCE = 490•mo�)
0 10 20 30
40
50Reaction Time (day)
226
60
60
70
70
300
250
150
100
50
0
eY0.
V
u
Va.
Fig. A-50
1600
1400
1200
1000
800
600
400
200
0
35
30
25
20
15
10
5
0
15
10
5
0
1 .5
VO
1 .0
0 .5
0
A comparison of anaerobic PCE dech�orinating systemswith adsorbed or free PCE doses. (PCE dose = 49 •mo�)
10 20 30
40Reaction time (day)
227
50 60 70
-o-- Free PCEAdsorbed PCE
Tota� methane production (•mo�)-ss-
-o-- Free ICEAdwrbed PCE
Tota� ethy�ene production (•mod)
-a--
- -~ Free PCE- V.C. in �iquid and s�udge phases (•moo_ ~- Ad-bed PCE
- -Free PCE_ Ads bed PCE
cis-DCE in �iquid and s�udge phases (•mop--a-
-Free PCEAd-bed PcE
PCE in �iquid and s�udge phases (pmo0-
Vaa
V
W
U
mUd
400
300
200
100
0
50
40YCY.
30A
20
10
0
35
30
25
20
15
10
5
0
10 .0
8.0tatUO
6.0
4.0
2.0
0
A comparison of anaerobic PCE dech�orinating systemswith adsorbed or free PCE doses. (PCE dose = 98 •mot)
10 20 30
40Reaction time (day)
228
50 60 70
Fan PCE
Ad.ubed PCE
Tota� methane production (•mop
'Fme PCE'-'~- Tota� ethy�ene production (•not)
K
e
V .C. in �iquid and s�udge phases (•mop
Adwabed PCs- a-
~- Fae PCE
Advabed PCE
Fax PCEcis-DCE in �iquid and s�udge phases (•m01)
~-
Adwsbed PCE-a--
~- Fwe FCEAd-bedFCE
PCE in �iquid and s�udge phases (•mop~.-
aa
U
400
300
200
100
20
0
80
60
40
20
0
20
U
15
0
10
5
0
10
8
roU
6a
4
2
00
A comparison of anaerobic PCE dech�orinating systemswith adsorbed or free PCE doses. (PCE dose = 196 •mot)
10 20 30
40Reaction time (day)
229
50 60 70
Free PCE _-_ - Adsorbed PC E
Tota� methane production Quno�)
-
_
F�ee PCE Tota� ethy�ene production (•nap
I
-a-
Adorbed PCE-a--
1
_
F�ee PCE V.C. in �iquid and s�udge phases (•nap--a-
Adsorbed PCE-a-
Free PCEcis-DCE in �iquid and s�udge phases (�inte�)
'
-b--
Adsorbed PCE-r--
P- PCE
d ICEAd-bPCE in �iquid and s�udge phases (•map
uCaa
uC
Aa
U
Fig . A-53
400
350
300
250
200
150
100
50
0
100
50
60
40
20
0
120
100
so
60
40
20
0
30
25
W
20UA
15
10
5
0
40
30
20
10
00
A comparison of anaerobic PCE dech�orinating systemswith adsorbed or free PCE doses. (PCE dose = 294 •mo�)
10 20 30
40Reaction time (day)
230
50 60 70
'~- Fax PCEAdaobcd PCE
Tota� methane oduction (•0n1)-a--
„
e
- Pox PCE
Ad-bed PCE-a-PCE In �iquid and s�adge p�anes (•map
YCaY
YC
Tam
ci
Fig . A-54
400
350
300
250
200
150
100
50
0
100
50
0
150
100
50
0
50
40WUO
30
u20
10
0
30
25
20cia
15
10
5
0
A comparison of anaerobic PCE dech�orinating systemswith adsorbed or free PCE doses . (PCE dose = 392 •mo�)
10 20 30
40Reaction time (day)
23 1
50 60 70
-~ Fr… PCEAd-bed FCE
Tota� methane production (•mo��
-.~
0
rf
---o-- FRe �CE
Adnwbad PCETota� ethy�ene production (It=))
-u--
---o-- Free PCE
Adwrbod PCE
and and s�udge phases (•mot)
-.r-
Free PCE-o-
Ad-bed PCE-w-
cis-DCE In �iquid and s�udge phases (•mop
'--o-- F- PCE
- Aduabed PCEPCE In �iquid and s�udge phases (•mop
YCdCY
U
200
150
100
50
150
YCY>n
100
W
50
0
200
150
100
50
0
40
W
30UO
20
10
0
100
80
W5.
60d
40
20
0
00
A comparison of anaerobic PCE dech�orinating systemswith adsorbed or free PCE doses . (PCE dose = 490 •mot)
10 20 30
40Reaction time (day)
232
50 60 70
Am PCE Tota� methane production (•coo�)-r~
Ad`ahed FCC--w-
- Free FCC
Adsorbed FCCTota� ethy�ene production (•mop
-w-
Fan FCE-
A6arbed FCC-a--
cis-DCE in �iquid and s�udge phases (•no�)
---v- Fme FCCAdmbOd Fm---w-
FCC in �iquid and s�udge phases (•mob
Tab�e
A-1.
Feed
in S
umma
for
the
ex e
rime
nt o
f th
e Me
OH a
nd m
edi
ff0 MeOH
5 McCH
20 Medium
50 M
eOH
100
MeOH
200
MeOH
Time
NM
PN
MP
NM
PN
MP
NM
PN
MP
(day)
(m�-)
(•L)
(•L)
(m�-
)(•
L)(•L)
(m�-
)(•
L)(•
L)(m
�-)
(•L)
(•L)
(m�-
)(•
L)(•
L)(mL)
(•L)
(•L)
135
05
55
520
55
550
55
100
55
200
518
50
55
55
205
55
505
510
05
520
05
225
05
55
205
55
505
510
05
520
05
265
05
55
520
55
550
55
100
55
200
532
50
55
55
205
55
505
510
05
520
038
50
55
55
205
55
505
55
445
05
55
520
55
550
55
100
55
200
549
50
55
55
205
55
505
510
05
200
555
05
55
520
55
550
55
561
50
55
205
55
505
55
675
05
55
520
55
550
55
573
50
55
55
205
55
505
55
55
55
802.5
05
2.5
55
2.5
55
2.5
505
872.5
05
2.5
55
105
2.5
505
55
932.5
05
2.5
05
992.5
05
2.5
05
2.5
505
2.5
100
52.
520
05
105
50
55
05
200
5115
50
55
05
200
5122
05
50
510
05
133
50
55
05
100
5147
50
55
05
100
5161
50
55
05
100
7.5
201
50
55
55
55
5
Tota
�105
011
510
575
115
338
7510
367
.575
070
67.5
810
4067
.516
1040
Tota
�(•
mot)
011
2718
4511
2718
4510
0518
450
686
1992
639
239
606
392
Note
:N =
Nutr
ient
med
ium
;M
=Me
than
o�;P
=PCE
.
Tab�e A-2 .Comp�ete dech�orinating abi�ity of acc�imatedanaerobic Hyperion s�udge
234
Time(day)
PCE(•mot)
V.C.(•coo�)
ETH(•mo�)
Methane(•mot)
Samp�e 10 49.00 27.0114 0.00 22.09 56.69 33.11
Samp�e 20 49.00 28.2414 0.00 20.54 55.46 29.33
Samp�e 30 49.00 27.9614 0.00 21 .76 56.02 34.52
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