12th Nordic-Baltic IHSS Symposium, 2009

Dark fermentation of biomass and organic waste for production of

renewables

T. Vaalu, M. Michelis, A. Mets, V. Lepane,

M. Kaljurand, J. Suurväli, A. Menert

Dark fermentation

The efficiency of dark fermentation (yield of biogas, etc) depends on a number of process parameters (kind of substrate, loading rate, hydraulic retention time, etc) of which the hydrolysis rate of organic material has the precedence.

Pretreatment of material is applied for increasing it, resulting in converting the substrate more accessible to anaerobic

2

converting the substrate more accessible to anaerobic microorganisms, accompanied by acceleration of digestion process, increase of methane yield, decrease of the amount of digested sludge and improvement of the process energy balance.

Anaerobic digestion of organic material needs additional heat however this can be compensated by the methane evolved in the same process and its energetic value.

14-17 June 2009 T. Vaalu, M. Michelis, A. Mets, V. Lepane,


12th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology

The main goal of sludge treatment

so far has been caused by environmental issues (hygienization and stabilization) in order to produce biogas (methane) and to use residual sludge in agriculture.

Anaerobic digestion is most commonly used for sludge treatment whereas part of organic matter of sludge is decayed by bacteria into CH4 and CO2.

3

A novel challenge for use of residual sludge would be production of H2

in addition to CH4. Moreover, it is advantageous to steer the fermenting process towards production of H2 instead of methane because combustion of methane causes CO2-release. Methane itself is a dangerous greenhouse gas having a 21 times higher global warming potential as compared to CO2. Production of bioH2 from sludge would thus effect positively on climate change mitigation.




The main aims and working hypothesis of the study

Thermal pre-treatment of organic material partly or entirely increases its biodegradability. Although this procedure needs additional heat this can be compensated by the energetic value of methane evolved.

Humic substances well known for their ability to facilitate thebioremediation of pollutants can be possibly applied in the

4

bioremediation of pollutants can be possibly applied in thetreatment of wastewaters.

Although members of biogas producing microbial community are engaged in metabolic symbiosis, it is possible to characterize this community and find out key species necessary for normaloperation of the digester.




Scheme of Tallinn Wastewater Treatmentplant

Outlet

Waste activated sludge

(residual sludge)

InletMethane

reactors

Storage of

digested sludge

Storage of

sludge




Influence of various pretreatment techniques on thebiodegradability of residual sludge

Temperature(T)

Ratio (R)

Minimum +70 °C 1:1:8

Average +85 °C 1:4.5:4.5

Maximum +95 °C 1:8:1

A two-factor three-level full factorial design was usedfor planning the experiment and

calculating the data produced

Bottle # Temperature Level Ratio Level

1 +70 °C -1 1:1:8 -1

2 +70 °C -1 1:4.5:4.5 0

7

3 +70 °C -1 1:8:1 1

4 +85 °C 0 1:1:8 -1

5 +85 °C 0 1:4.5:4.5 0

6 +85 °C 0 1:8:1 1

7 +95 °C 1 1:1:8 -1

8 +95 °C 1 1:4.5:4.5 0

9 +95 °C 1 1:8:1 1

Experimental factors - pre-treatment

temperature and ratio of individual

components of the mixture (inoculum,

treated sludge, raw sludge), were varied

at three levels.

Response factors - cumulative biogas

production, cumulative VFAs production,

ratio of propionate and acetate

14-17 June 2009T. Vaalu, M. Michelis, A. Mets, V. Lepane,



Monitoring of anaerobic digestion process

Gas sample

Liquid sample

8

Oxitop® Control AN 6, WTW Germany

Respirometric Oxitop® method, normally exploited for biodegradability tests and BOD5 measurement can be used for monitoring the digestion process




Change of VFAs composition during anaerobic mesophilic digestion (t=37°°°°)

inoculum : treated sludge (70 Co) : raw sludge; 1 : 1 : 8

0

5

10

15

20

25

Acid

co

ncentr

atio

n (

mM

)

0

1

2

3

4

5

Gas p

rodu

ctio

n (

mM

d-1)

Gas production rateAceticPropionicButyricValeric

a inoculum : treated sludge (70 Co) : raw sludge; 1 : 1 : 8

0

5

10

15

20

25

Acid

co

ncentr

atio

n (

mM

)

0

1

2

3

4

5

Gas p

rodu

ctio

n (

mM

d-1)


a inoculum : treated sludge (95 Co) : raw sludge; 1 : 4.5 : 4.5

0

5

10

15

20

25

30

35

0 5 10 15 20 25 30 35 40

Acid

co

ncen

trati

on

(m

M)

0

1

2

3

4

5

Gas p

rod

ucti

on

(m

M d

-1)


c inoculum : treated sludge (95 Co) : raw sludge; 1 : 4.5 : 4.5

0

5

10

15

20

25

30

35

0 5 10 15 20 25 30 35 40

Acid

co

ncen

trati

on

(m

M)

0

1

2

3

4

5

Gas p

rod

ucti

on

(m

M d

-1)


c

0 5 10 15 20 25 30 35 40 45

Time (d)

0 5 10 15 20 25 30 35 40 45

Time (d)

inoculum : treated sludge (85 Co) : raw sludge; 1 : 4.5 : 4.5

0

5

10

15

20

25

0 5 10 15 20 25 30 35 40 45

Time (d)

Acid

concentr

ation

(m

M)

0

1

2

3

4

5

Gas p

roductio

n (

mM

d-1

)


b inoculum : treated sludge (85 Co) : raw sludge; 1 : 4.5 : 4.5

0

5

10

15

20

25

0 5 10 15 20 25 30 35 40 45

Time (d)

Acid

concentr

ation

(m

M)

0

1

2

3

4

5

Gas p

roductio

n (

mM

d-1

)


b0 5 10 15 20 25 30 35 40

Time (d)

0 5 10 15 20 25 30 35 40

Time (d)

Depending on treatment temperature and sludge make-up

(ratio of inoculum, treated and untreated sludge) three

different VFAs formation profiles were observed:

acetate was formed in two stages with maximums up to 15

mM on the day 10 and day 20, with abundant biogas and

methane generation (balanced growth);

moderate VFAs formation, poor consumption of VFAs and

biogas evolution (overproduction of propionate);

large amounts of acetate were formed only at the beginning of

digestion with moderate biogas and methane formation

(overproduction of acetate);




Global optimisation with desirability function

0,50

0,60

0,70

0,80

0,90

1,00

global optimisation with desirability function

(gas, cum acids, max prp/ac)

0,90-1,00

0,80-0,90

0,70-0,80

0,60-0,70

0,50-0,601 : 5.4 : 2.6

1 : 6.3 : 1.7

1 : 7.1 : 0.9

1 : 8.0 : 1.0

Mix

ture

, ra

tio

Global optimisation with desirability function

(gas, cum acids, max prp/ac)

0,80-1,00

From the 32 design for each response, model of second-degree polynomial function was used. To find the optimum point for all the responses simultaneously, graphical study of the desirability function was performed. By plotting the overall desirability values against the two factors, temperature and ratio of components, it was possible to find optimum conditions for all responses together.

10

70 74 78 81 85 88 90 93 95

1 : 1

.0 : 8

.0

1 : 2

.3 : 5

.7

1 : 4

.5 : 4

.5

1 : 6

.3 : 1

.7

1 : 8

.0 : 1

.0

0,00

0,10

0,20

0,30

0,40

0,50

Tempmix

0,50-0,60

0,40-0,50

0,30-0,40

0,20-0,30

0,10-0,20

0,00-0,10

70 74 78 81 85 88 90 93 95

1 : 1.0 : 8.0

1 : 1.1 : 7.9

1 : 2.3 : 5.7

1 : 3.4 :4.6

1 : 4.5 : 4.5

Temperature, °C

Mix

ture

, ra

tio

0,80-1,00

0,60-0,80

0,40-0,60

0,20-0,40

0,00-0,20

Optimum conditions regarding biogas production, cumulative VFAs production

and propionate acetate ratio were t=70°C and ratio of inoculum : treated

sludge and raw sludge 1 : 8 : 1.




Biogaasi tootlikuse sõltuvus osalisest termilisest eeltöötlusest (70 oC)

40

60

80

100B

iog

aasi h

ulk

(m

mo

l)

10% inokulum + 80% toormuda + 10% 70C

10% inokulum + 90% toormuda

Rate of biogas production

11

0

20

0 5 10 15 20 25 30 35 40

Aeg (päeva)

Bio

gaasi h

ulk

(m

mo

l)

Later on it was found that even partial thermal pre-treatment (10% of raw sludge) increased the production of biogas up to 20%. Considering products of metabolism during anaerobic digestion the acetoclasticmetanogenesis dominated.




Production of VFAs in residual sludgewith low proportion of pre-treated sludge (1:1:8)

Inokulum: töödeldud sete (70oC): toorsete 1:1:8

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35 40 45 50 55Aeg, päev

Org

.üh

end

id,

mM

0

10

20

30

40

50

60

70

Met

aan

i,

mm

ol

Propionaat Atsetaat Võihape Laktaat etanool Metaan

70°°°°


40

50

60

70

Org

. ü

hen

did

,

40

50

60

70

Meta

an

i,

mm

ol

95°°°°

Methane yields

Metaani saagised

0,44

0,30 0,280,30

0,40

0,50

0,60

m3 m

etaa

ni/

kg K

HT

Metaani erisaagis: m3 CH4/kg ärastatud KHT

Metaani saagis: m3 CH4/kg KHT

Specific yield of methane m3 CH4/kg COD removed

Methane yield m3 CH4/kg COD

12



0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30 35 40 45 50 55

Aeg, päev

Org

. ü

hen

did

,

mM

0

10

20

30

40

50

60

70

80

Meta

an

i,

mm

ol


85°°°°

0

10

20

30

0 5 10 15 20 25 30 35 40 45 50 55Aeg, päev

Org

. ü

hen

did

,

mM

0

10

20

30

Meta

an

i,

mm

ol

Propionaat Atsetaat Võihape Laktaat Etanool Metaan

0,18 0,16 0,14

0,00

0,10

0,20

0,30

inokulum: töödeldud

(+70C): toorsete; 1:1:8


(+85C): toorsete; 1:1:8


(+95C): toorsete; 1:1:8

Katsesegu koostis

m3 m

etaa

ni/

kg K

HT




Humic substances

Humic substances as potential electron

acceptors facilitate degradation of recalcitrant

compounds. One of the objects under study

was residual sludge from WWTP that can be

modified by thermal pre-treatment. Humic

substances were extracted from various

environments - ecologically clean environment

(peat) and polluted environment (wastewater

sediments) and fractionated to humic and fulvic

13T. Vaalu, M. Michelis, A. Mets, V. Lepane,


sediments) and fractionated to humic and fulvic

acids.

The fractions obtained were characterized by

size-exclusion chromatrography (HPLC-SEC),

calculating average molecular masses of humic

substances and determining molecular mass

distribution. By these parameters it was

possible to make conclusions on the behaviour

of humic substances in these environments.

14-17 June 200912th Nordic-Baltic IHSS Symposium on Natural Organic Matter in Environment and Technology

Molecular masses of humic and fulvic acids and polydispersity in peat samples

Weight average

molecular mass, Mw

Numeral average

molecular mass, Mn

Polydispersity

Sample 1

Fulvic acid 8360 583 14

Humic acid 84300 332 254

Sample 2


Humic acid 140300 171 610

Sample 3


Fulvohape, turvas

0 10 20 30

Retentsiooni aeg, min

UV

, 254 n

m

Fulvic acid, peat

Retention time, minHumic acid 228300 579 394

Sample 4


Humic acid 170800 318 537

ii

i

i

ii

nMh

h

N

MNM

/∑

∑=

∑

∑=

i

ii

ii

ii

wh

Mh

MN

MNM

∑

∑=

∑

∑=

2

wM nM

/

Retentsiooni aeg, minRetention time, min

Humiinhape, turvas

0 10 20 30

Retentsiooni aeg, min

UV

, 254 n

m

Humic acid, peat

Retention time, min

Co-digestion

Co-digestion - simultaneous digestion of homogeneous mixture oftwo or more substrates improves the processing qualities of differentwastes and increases the production of biogas. Usually a major amount of basic substrate (e.g. manure or sewage sludge) is mixedand digested together with minor amounts of a single or a variety ofadditional substrates (garbage waste, paper mill residues, slaughterhouse waste, animal manures, saw dust, energy crops, food industrywaste, pharmaceutical wastes). Co-digestion offers several

15

waste, pharmaceutical wastes). Co-digestion offers severalecological, technological and economical benefits:

� digester operational advantages, � improved overall process economics (higher biogas yield - 40-200%)� most of chemical energy of the substrate is turned into biogas, less

spent solids are to be processed.




Dependence of biogas yield on thermal pre-treatment (+70°C) and addition of humic substances (peat)

Thermal pre-treatment ofresidual sludgeappeared to be aneffective pre-treatmenttechnique, the best was 20

30

40

50

60

70

Meth

an

e (

mm

ol)

16

technique, the best wascombination of thermalpre-treatment with thesimultaneous use ofhumic substances.

0

10

20

0 10 20 30 40 50 60 70

Time (days)10% inokulum + 80% raw sludge + 10% 70Cpre-treated sludge10% inoculum + 80% raw sludge + 10% 70C pre-treated sludge + 10 ml peat10% inoculum + 80% raw sludge + 10% 70C pre-treated sludge + 30ml peat10% inoculum + 80% raw sludge + 10% 70C pre-treated sludge + 1,6g AQDS10% inoculum + 90% raw sludge10% inoculum + 90% raw sludge + 10ml peat10% inoculum + 90% raw sludge + 30ml peat




Buffering capacity ofhumic substances prevents accumulation of organic acids

Inoculum 10%, raw sludge 90%

20 60

Acetic acid Propionic acidButyric acid Lactic acidMethane

Inoculum 10%, raw sludge 90%

+ 10ml peat

10

15

20

Org

an

ic c

om

po

un

ds (

mm

ol/L

)

30

45

60

Meth

an

e (

mm

ol)

Lactic acid Acetic acidPropionic acid Butyric acidMethane

17

0

5

10

15

0 10 20 30 40 50 60

Time (days)

Org

an

ic c

om

po

un

ds

(m

mo

l/L

)

0

15

30

45

Me

tha

ne

(m

mo

l)

0

5

10

0 10 20 30 40 50 60

Time (days)O

rgan

ic c

om

po

un

ds (

mm

ol/L

)

0

15

30

Meth

an

e (

mm

ol)




Some conclusions on use of humic substances

� Peat as asource of humic substances accelerates the process of anaerobic

digestion, increases the yield of biogas and makes the process more effective

and stabile.

� One reason might be the buffering capacity of humic substances that does

not allow organic acids to accumulate and leads the process towards more

oxidized products. Humic substances act as electron carriers and mediators

on decaying the organic substance.

� The combination of thermal pre-treatment with addition of humic substances

18

� The combination of thermal pre-treatment with addition of humic substances

(peat) accelerated the process most. Use of only 10% of pre-treated sludge in

the mixture is sufficient, 80% of pre-treated sludge in the mixture resulted in

less biogas yield.

� Antraquinone-2,6-disulphonate(AQDS) is not a good model for humic

substances. Quinone respiration was preferred as to methane respiration – in

the experiments with AQDS, methane yield was 2-4 times less as compared

to peat. AQDS performed as terminal electron acceptor.




Humic substances as co-digestion substrates

� Humic substances well known for their ability to facilitate the bioremediation of pollutants can be possibly applied in the treatment of wastewaters. Experiments have clarified by now the effect humic substances (one of co-substrates) on the production of biogas.

� Thermal pre-treatment of residual sludge appeared to be an effective pre-treatment alternative, the best was combination of thermal pre-treatment with the simultaneous use of humic substances.

19

� Humic substances can increase the yield of biogas as electron donors and additional carbon sources. 50 or 150 mmol of extra carbon was added as humic substances, however, in the experiments only 10-30 or 40-60 mmol methane was formed and twice less CO2. Evidently, the rest of carbon was bound into refractory inert polymeric compounds.The resultant digestate is more stabile and has higher dry solids content.




Substrate conversion patterns associated with anaerobic treatment of

wastewaters

1. Hydrolysis of organic polymers;

2. Fermentation of organic monomers;

3. Oxidation of propionic and butyric acids and alcohols by OHPA;

4. Acetogenic respiration of bicarbonate;

5. Oxidation of propionic and butyric acids and alcohols by SRB and NRB;

6. Oxidation of acetic acid by SRB and NRB;

7. Oxidation of hydrogen by SRB and NRB;

8. Aceticlastic methane fermentation;

9. Methanogenic respiration of bicarbonate

OHPA – obligatory hydrogen producing anaerobes

SRB – sulfate reducing bacteria

NRB – nitrate reducing bacteria

(Harper and Pohland, 1987)

Some redox half-reactions responsible for anaerobic microbial conversion of selected substrates

Reactions

Oxidations (electron donating reactions) ∆G0, KJ

Propionate→Acetate: CH3CH2COO- + 3H2O → CH3COO- + H+ + HCO3- + 3H2 +76.1

Butyrate →Acetate: CH3CH2CH2COO- + 2H2O → 2CH3COO- + H+ + 2H2 +48.1

Ethanol →Acetate: CH3CH2OH + H2O → CH3COO- + H+ + 2H2 +9.6

Lactate →Acetate: CH3CHOHCOO- + 2H2O → CH3COO- + HCO3- + H+ + 2H2 -4.2

Acetate →Methane: CH3COO- + H2O → HCO3- + CH4 -31.0

21

Acetate →Methane: CH3COO- + H2O → HCO3- + CH4 -31.0

Respirative (electron accepting reactions)

HCO3- →Acetate: 2HCO3

- + 4H2 + H+ → CH3COO- + 4H2O -104.6

HCO3- →Methane: HCO3

- + 4H2 + H+ → CH4 + 3H2O -135.6

Sulfate→ Sulfide: SO42- + 4H2 + H+ → HS- + 4H2O - 151.9

CH3COO- + SO42- + H+ → 2HCO3

- + H2S -59.9

Nitrate →Ammonia: NO3- + 4H2 + 2H+ → NH4

+ + 3H2O -599.6

CH3COO- + NO3- + H+ + H2O → 2HCO3

- + NH4+ -511.4

Nitrate → Nitrogen gas: 2NO3- + 5H2 + 2H+ → N2 + 6H2O -1120.5

Adapted from Harper and Pohland, 1986)




Selected substrates and methaneproducing reactions

Reactions ∆G’o (kJ/mol) T (°C)

Hydrogenotrophic reactions:

CO2 + 4H2 = CH4 + 2H2O -131 35

4CHOO- + 4H+ = CH4 + 3CO2 + 2H2O -144,5

4 (2-propanol) + CO2 = CH4 + 4 acetone + 2H2O

Aceticlastic reaction:

CH3COO- + H+ = CO2 + CH4 -31,0 25

22

Disproportionation reactions:

4CH3OH + 2H2O = 3CH4 + CO2 + 4H2O -319,5 35

4CH3OH + CH3COO- = 4 CH4 + 2 HCO3- + H+ -346

CH3OH + H2 = CH4 + H2O -113

4CH3NH3+ + 3H2O = 3CH4 + HCO3

- + 4NH4 + + H+ -225

2 (CH3)2NH2+ + 3H2O = 3CH4 + HCO3

- + 2NH4+ + H+ -220

4(CH3)3NH+ + 9 H2O = 9 CH4 + 3HCO3- + 4NH4

+ + 3H+ -670

2Dimethyl sulfide + 2H2 = 3CH4 + CO2 + H2S

Jones, 1991; Thauer, 1977; Zinder, 1993; Lovley et al., 1983




Conversion of acetate to methane

23

Corr – corrinoid containing protein;

CODH – carbon monooxide dehydrogenase

CH3COO- + H+ = CO2 + CH4 ∆G°’ = - 31 kJ

Madigan, M. T. & Martinko, J. M. 2006. Brock Biology of

Microorganisms“ 11th Edition, Southern Illinois University,

Carbondale




The methanogenic pathway of CO2 reduction with H2

MF – metanofurane, coenzyme participatingin C1 transfer from CO2 to CH4;

MP – metanopterine, conenzyme, C1 carrier in intermediate stages;

CoM – coenzyme M, participates inconversion of methyl group (CH3) to CH4

F – reduced coenzyme F ;

24

CO2 + 4H2 → CH4 + 2H2O ∆G°’ = - 135,6 kJ

F420red – reduced coenzyme F420;

F430 – coenzyme F430

CoB – coenzyme B

Yellow – C-atom to be reduced;

Brown – electron donor



Carbondale




Methanogenic archea

Genus Suitable substrates for

methanogenesis

Methanobacteriale

Methanobacterium

Methanobrevibacter

Methanosphaera

Methanothermus

Methanothermobacter

H2 + CO2, formic acid


methanol + H2 (both necessary)

H2 + CO2, (S0); hyper thermophilic

H2 + CO2, formic acid; thermophilic

Methanococcale

Methanococcus

Methanothermococcus

H2 + CO2, CO2 + pyruvate, formic acid


Methanosarcinale

Methanosarcina

Methanolobus

Methanohalobium

Methanococcoides

H2 + CO2, methanol, methylamines, acetate

methanol, methylamines

methanol, methylamines; halophilic

methanol, methylamines

25

Methanocaldococcus

Methanotorris

H2 + CO2

H2 + CO2

Methanomicrobiale

Methanomicrobium

Methanogenium

Methanospirillum

Methanoplanus

Methnocorposculum

Methanoculleus

Methanofollis

Methanolacinia





H2 + CO2, formic acid, alcohols

H2 + CO2, alcohols, formic acid


H2 + CO2, alcohols

Methanohalophilus

Methanosaeta

Methanosalsum

methanol, methylamines, methylsulfides; halophilic

acetate

methanol, methylamines, dimethylsulfide

Methanopyrales

Methanopyrus H2 + CO2; hyper thermophilic (110°C)



Carbondale




DNA-based methods for direct process monitoring of the consortia used

In industrial applications the use of mixed cultures from organic wastes for CH4 and H2 production is more advantageous as pure cultures can easily become contaminated with H2 consumers. Many members of these microbial communities are non-culturable bacteria. Thus for characterization of these potent CH4 and H2 producing

26

characterization of these potent CH4 and H2 producing microorganisms analyzing microbial communities, using culture independent techniques is inevitable.




Identifying on nonculturable bacteria by denaturating gradient gel electrophoresis (DGGE)

DNA extraction

Community DNA

Amplification of a fragment of 16S

rRNA gene (rDNA) using PCR

DGGE-gel. Different

bands in the gel

represent different

microbial speciesDenaturating

Checking PCR-

products using

agarose gel

elektrophoresis

Cutting out bands from

DGGE gel and

reamplifying DNA from

the bands

microbial species

CTGAATCGTA

Sequencing of

purified DNA

originated from

DGGE bands

Identifying

microorganisms by

comparing 16S rDNA

sequences to DNA

databases and

constructing a

phylogenetic tree

Denaturating

gradient gel

elecrrophoresis

(DGGE)

Phylogenetic tree

elektrophoresis




Denaturating gradient gel electrophoresis (DGGE) of bacterial16S rRNA gene fragments

8

9

10

21

23

24

43

44

45

46

47

51

53

54 55

35

36

37

38 39

40

5261

60

62

6364

69

70

717280

77

78

79

81

84

85

8687

90

91

9293

97

98

99100

104

105

106

110

111

112

116

117

121

122

3

1

2

4

15

16

17

31

28

29

32

30 2211

1B 1B 4 4 4B 7A 7B

T=70°C T=85°C T=95°C

12

13

14

25

26

27

48

49

50

56

57

58

59

41

42

65

66

67

68

73

74

75

76

82

83

88

89

94

95

96

101

102

103

107

108

109

113

114

115

118

119

120

123

124

5

6

7

18

19

20

33

34

from samples ofthermally pre-treated residualsludge of WWTP

DGGEwas used in order to determine the impact of different pretreatment temperatures on the microbial community structure.

Characterization of microbial concortia

Most of the bacteria identified were representatives of the phyla Chloroflexi and Bacteroidetes;

All the archaeal strains identified were shown to represent the genus Methanosarcina – anaerobic methanogens, the main biogas

29

producers occurring in landfills, WWTPs, in sea sediments and mammal guts. These archaea are able to produce methane by all three known methanogenic pathways – the hydrogenothrophic, acetoclastic and methylotrophic pathway.




Phylogenetic analyses of bacteria, coloured bacterial strains –obtained from Tallinn WWTP

Most of the bacteria

identified were

representatives of the

phyla Chloroflexi and

Bacteroidetes




The most numerous bacteria were representatives from the phylum Chloroflexi.

The closiest species to the sequences determined was Levilinea saccharolytica

(B). Levilinea saccharolytica has been previously isolated as pure culture from

sugar industry wastewater (containing sucrose and easily degradable VFAs)

sludge granules (Yamada et al., 2005)

A – Anaerolinea thermolimosa; B – Levilinea saccharolytica; C – Leptolinea tardivitalis. (Yamada et al., 2005)




Archea from Tallinn WWTP determined with DGGE

1

3

4 8 10

11

14

70ºC 85ºC 95ºC 70ºC 85ºC 95ºC

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5

7

9

13

12

1516

1718

19

2

0

21

2223

Genus Methanosarcina, coloured archeal strains –coloured archeal strains –obtained from Tallinn WWTP




Archae from the genus Methanosarcina

Genus Methanosarcina, sequences determined

closiest to species Methanosarcina mazei and

Methanosarcina barkeri.

Genus Methanosarcina,

family Methanosarcinaceae,

order Methanosarcinales,

class Methanomicrobia,

phylum Euryarchaeota.

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phylum Euryarchaeota. Multicell form of Methanosarcina acetivorans

(http://www-

genome.wi.mit.edu/annotation/microbes/methano

sarcina/background.html)

Methanosarcinae have the largest genome among

archea – the genome of M. acetivorans has

5,751,492 nucleotides (Galagan et al., 2002).

22nd amino acid

aminohape –

pyrrolysine from

Methanosarcina

barkeri




Methanosarcinae – anaerobic methanogens

Methanosarcinae have specific pathway for methane production – methylotrophic methanogenesis using methanol, methylamines and methyltiols for methane production (Galagan et al., 2002).

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Three pathways ofmethanogenesis)




Acknowledgement

The financial support from

the Estonian Science Foundation (Grant No 5889), from Nordic Energy Research (Grant No 06-Hydr-C13)and from Enterprise Estonia (Grant No EU27358) aregratefully acknowledged.

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gratefully acknowledged.




Thank you for your attention!

12th Nordic-Baltic IHSS Symposium, 2009

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Transcript of 12th Nordic-Baltic IHSS Symposium, 2009