Molecular Microbial Ecology – Application in EEWS (Energy, Environment, Water, and Sustainability)
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Transcript of Molecular Microbial Ecology – Application in EEWS (Energy, Environment, Water, and Sustainability)
Molecular Microbial Ecology – Application in EEWS (Energy,
Environment, Water, and Sustainability)
Quan, Zhe-Xue ( 全 哲 学 )
School of Life Sciences, Fudan University, Shanghai, China E-mail: [email protected]
History of Molecular Microbial Ecology
Carl Woese –
Analysis of 16S rRNA
1) represent a new kingdom “Archaebacteria”
2) A universal and quantitative phylogeny
is possible
The “Woesian” Revolution
Alignment of a highly conserved region
of the 16S/18S rRNA
Homo sapiens ...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAG...S. cereviceae ...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAG...Zea maize ...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAG... Escherichia coli ...GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCG...Anacystis nidulans ...GTGCCAGCAGCCGCGGTAATACGGGAGAGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCG...Thermotoga maritima ...GTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTACCCGGATTTACTGGGCGTAAAGGG...Methanococcus vannielii ...GTGCCAGCAGCCGCGGTAATACCGACGGCCCGAGTGGTAGCCACTCTTATTGGGCCTAAAGCG... Thermococcus celer ...GTGGCAGCCGCCGCGGTAATACCGGCGGCCCGAGTGGTGGCCGCTATTATTGGGCCTAAAGCG... Sulfolobus sulfotaricus ...GTGTCAGCCGCCGCGGTAATACCAGCTCCGCGAGTGGTCGGGGTGATTACTGGGCCTAAAGCG...
E. coli
Human
Yeast
Corn
Green algae
Thermophile
Prokaryotes Eukaryotes
Macroorganisms
Bacteria Archaea Eukarya
Not include virus
Three domain theory
Now: uncultured: ~800,000 cultured: ~200,000
Total number of Archaeal 16S rRNA gene sequences retrived from EMBL sequence database and introduced into ARB database over the last 11 years
0
2000
4000
6000
8000
10000
12000
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005
Time
Num
ber
of s
eque
nces
Uncultivated
Cultivated
“Domain” of Bacteria
1994 - 13 divisions (all cultured)
1997 - 36 divisions 24/12
2003 - 53 divisions 26/27
2004 - 80 divisions 26/54
2008: 30/70
Research Field
Taxonomy Ecology
Bioinformatics
Cultured <5% Uncultured >95%
Molecular Microbial Ecology
MetagenomeMicrobial Diversity
Cultivation Classification
Molecular analysis
Nitrogen Cycle
Environmental Technology
Removal of organic carbon
Removal ofnitrogen and phosphate
EcologyCarbon cycle
(Greenhouse gas) Coupling of
Carbon and Nitrogen cycles
Ammonium-Oxidizing Microorganisms
Aerobic Ammonium-Oxidizing Bacteria
Aerobic Ammonium-Oxidizing Archaea
Anaerobic Ammonium-Oxidizing (ANAMMOX) Bacteria
Diversity of Ammonium-Oxidizing Bacteria and Archaea
in Changjiang (Yangtze River) Estuary
(PNAS 2005, 102, 14683-14688)
Diversity of ammonium-oxidizing archaea
(Nature, 2005, 437, 543-546)
Chongming Island • At the estuary of Yangtze river• The 3rd largest island in China • Area: >1000 square kilometers,
increasing >10 square kilometers per year
Diversity of Ammonium-Oxidizing Bacteria in a Granular Sludge Anaerobic ammonium-
Oxidizing (ANAMMOX) Reactor
ANAMMOX (anaerobic ammonium oxidation)
In 1977, the existence of chemolithoautotrophic anammox bacteria was predicted:
NH4+ + NO2
- → N2 + 2H2O (ΔG= -357 kJ/mol)
(Z Allg Mikrobiol, 1977, 17, 491-493)
In 1995, it was scientifically confirmed thatANAMMOX is biologically mediated process
15NH4+ + 14NO3
- → 14,15 N2(98%) 5NH4
+ + 3NO3- → 4N2 + 9H2O + 2H+
NH4+ + NO2
- → N2 + 2H2O(AEM, 1995, 61,1246-1251)
ANAMMOX in marine ecological system
30-50% of fixed-nitrogen in marine environment would be removed through ANAMMOX process. Black Sea and Golfo Dulce, Costa Rica (Nature, 2002,422, 608-611; 606-608) Benguela upwelling system (PNAS, 2005, 102,6478-6483)
ANAMMOX application in wastewater treatment
The first full-scale ANAMMOX reactor (2002) at the Dokhaven wastewater treatment plant, Rotterdam, the Netherlands.(http://www.anammox.com/research.html)
•Normal nitrogen –removal process: NH4
+ + 2O2 → NO3- + H2O + 2H+
NO3- + CH2O → N2 + CO2
•ANAMMOX Process: ( NH4
+ + 1.5O2 → NO2- + H2O + 2H+)
NH4+ + NO2
- → N2 + 2H2O
Reactor operation
Influent
Recycle
Gas
Effluent
Water b
ath
Artificial Wastewater:NaNO2 + NH4HCO3 (1:1), KH2PO4 10 mg/l, yeast extract 5 mg/l, and TE.
Sludge: river sediment (1400 mg VSS/l)
Loading rate: 1-130 days: at 0.3 kg NH4
+-N/(m3·d) Up to 250 days: 0.4-0.8 kg NH4
+-N/(m3·d) (80% removal) 351 days: stable removal 82-86%
Sludge sampling: day 377.
ANAMMOX reactor
Phylogenetic tree based on 16S rRNA gene sequences amplified from the anammox reactor sludge using Planctomycetales-specific primers.
The relationships of the different families of anammox bacteria among the Planctomycetes. (Nature Reviews Microbiology 2008, 6, 320-326 )
Defined as the fifth ANAMMOX genus
Anammox bacteria
Matched contigs 3042
Number of assembled reads 269,212 (31.7%)
Sum of contig length 561.25Kb
Matched ORFs in Kuenenia 1346
Best match with Kuenenia 3023
Best match with KSU-1 19
Best match with others 145
Metagenomic analysis
- Microbial population in ANAMMOX reactor
- Isolation of novel species from ANAMMOX reactor
- Anaerobic ammonium oxidation with sulfate reduction
Cowork with environmental engineers :
Biological treatment of metal containing wastewater
Free heavy metalsCyanide-complexed
heavy metals
High concentration of heavy metals
High concentration of cyanide
Heavy metal wastewater
Biological treatment ofheavy metal containing wastewater
Treatment of heavy metals with sulfate reduction
Sulfide productionSO4
2- + 2CH2O + 2H+ H2S + 2H2O + 2CO2
Metal sulfide precipitation
H2S + Me2+ MeS(s) +2H+
Bacteria
Infl ue nt
Off - ga s
20 c m10 c m
12 c m
( IV )
( II )
( Se c t io n I )
( III )Dir e c t io n o f fl o w
Gr ave l
So lid s ubs t r a te s
20 c m
20 c m
15 c m
5 c m
5 c m
10 c m
Solid substrates: UASB granule
Cow manure
Method for recovering heavy metals
from the drainage containing heavy metals,
10-0414891, Korea.
Acid mine drainage
Production
(a) FeS2 + 7/2 O2 + H2O
→ Fe2+ + 2 SO42- + 2 H+
(b) Fe2+ + 1/4O2 + H+
→ Fe3+ + 1/2 H2O
(c) FeS2 + 14 Fe3+ + 8 H2O
→ 15 Fe2+ + 2 SO42-+16 H+
The rate of (b) increase million times by bacteria.
SO42- + 2CH2O + 2H+
→ H2S + 2H2O + 2CO2
H2S + Me2+ → MeS(s) + 2H+
Pilot-scale treatment
Anaerobic treatment of cyanide- and metal- containing wastewater
Cyanide- and metal-containing wastewater
Me2+, CN-, [Me(CN) 4]
2-
CN-, [Me(CN) 4]2-
MeS CO2, NH3
Cyanide degrading SRBGranular sludge
(SRB)
Time (day)
0 2 4 6 8 10 12 14
Su
lfat
e (m
M)
0
1
2
3
4
5
6
Free cyanideZinc-complexed cyanideCopper-complexed cyanideNickel-complexed cyanide
12
13
14
15
16
17
18
19
20
0 2 4 6 8 10 12
Time (d)
Sulfate
(m
M)
Time (d)
0 1 2 3 4 5 6 7
Su
lfat
e (m
M)
0
1
2
3
4
5
6
0 mM0.5 mM 1 mM 2 mM 5 mM
[ Ni(CN)4]2- -> Ni2+ + CO2 + NH3
Ni2+ + S2- -> NiS
Aerobic treatment of metal-complexed cyanide
Time (h)
0 5 10 15 20 25
Free
cya
nide
(mM
)
0.0
0.5
1.0
1.5
2.0
Time (h)
0 5 10 15 20 25
Zinc
com
plex
ed c
yani
de (m
M)
0.0
0.5
1.0
1.5
2.0
Time (h)
0 5 10 15 20 25
Nic
kel c
ompl
exed
cya
nide
(mM
)
0.0
0.5
1.0
1.5
2.0
ControlRe-FC sludgeRe-ZC sludgeRe-NC sludge
Operation time (days)
0 10 20 30 40 50
Cya
nid
e re
mo
val
per
cen
t
0
20
40
60
80
100
Re-FCRe-ZCRe-NC
24 h 12 h48 h 6 h
Analysis with DGGE
Free heavy metalsCyanide-complexed
heavy metals
High concentration of heavy metals
High concentration of cyanide
Heavy metal wastewater
Degrade different types cyanide in aerobic condition
Precipitate heavy metal with sulfate reduction
Degrade cyanide in sulfate reducing
condition
Biological treatment of metal containing wastewater
Monitoring of microorganisms in water
Microbial Monitoring for Drinking Water
Evaluation of drinking water
- Previous: Plate counting of total bacteria and enterobacteria
- New: Pathogenic protozoa Cryptosporidium, Giardia
- Future: Detection of viruses
Detection of viruses:
using fecal bacteriophage
(host: E. coli, Bacteroides fragilis)
using real-time PCR quantification
cultivating viruses in cells and
detecting with quantum-dot nanocomplexes
Change of microbial populations in swimming pools treated
with non-chlorine disinfectant
Requirement of the company
the identity of the organisms found in each sample
an idea of the proportions of each (i.e. which are the dominant bacteria/and fungi in each sample)
how this dynamic changes over the sampling time during the pool summer
if there are distinct differences in the ecology of the two groups of pools that were sampled.
Samples
12 swimming pools (6 pools were treated with original chemical and the others
were treated with new chemicals)
Three sites: pool water, sand filter, pipe line
8 sampling times: (June-Sept. two weeks interval)
DNA extraction Liquid nitrogen grinding Enzyme extraction
o Lysozyme, Lyticase
PCR amplification Reconditioning PCR
o (18S, most sand_16S, some water_16S (7 sample) )
Nested PCRo (ITS, all water_16S, some
sand_16S)
PCR primers 16S
o 27F+1390Ro 27F+1512R, 519F+1390R
18S
o 18S-nu0817+18S-nu1536
ITS
o NSA3+NLC2, NSI1 +NLB4
Construction of clone libraries
Software for sequence analysis
Library typeTotal number
of clonessequenced
OTU uncertainty value_0(%)
OTU uncertainty value_1(%)
OTU uncertainty value_2(%)
OTU uncertainty value_3(%)
OTU uncertainty value_4(%)
OTU uncertainty
value_10(%)
16S rRNA gene
11009 79.0 1.2 1.7 0.5 3.9 13.7
ITS 9860 49.8 17.7 7.6 7.1 7.5 10.3
18S rRNA gene
3540 8.8 11.2 29.4 13.1 27.2 10.3
Percent of uncertainty level during microbial identification
from different type clone libraries (Sample sources: Swimming Pool)
Average contents of major types bacteria in different group sand samples. (‘Sand-new-good-743(13)’ means the data is analyzed from the 743 clones from 13 ‘good’ samples treated with new chemical.) The number at X-axis are matching to the order of major bacterial type.
0.0
5.0
10.0
15.0
20.0
25.0
30.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Major bacterial type
Ave
rage
con
tent
in e
ach
sam
ple
Sand-new-good-743(13) Sand-new-bad-652(12)Sand-old-good-620(11) Sand-old-bad-1161(21)
Methylobacterium (甲基杆菌属)
Chemical Library Sand (%) Water(%)
Old
GO 1.8 3.2
GR 1.8 3.3
MA 2.6 1.1
MU 5.2 7.5
SC 18.7 1.9
WI 3.3 4.6
old average 5.6 3.6
New
BR 0.8 6.6
BT 12.8 11.6
RI 29.0 10.4
ST 32.3 7.8
ZJ 16.8 5.8
ZP 9.4 4.8
new average 16.8 7.8
Pipe 30.3
Content of Methylobacterium in each swimming pool
Average contents of major types bacteria in different group water samples. (‘Water-new-good-855(14)’ means the data is analyzed from the 855 clones from 14 ‘good’ samples treated with new chemical.) The number at X-axis are matching to the order of major bacterial type.
0.0
5.0
10.0
15.0
20.0
25.0
30.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Major bacterial tpye
Ave
rage
con
tent
in e
ach
sam
ple
Water-new-good-855(14) Water-new-bad-700(12)Water-old-good-662(11) Water-old-bad-1288(22)
Sphingomonas (鞘氨醇单胞菌)
Relationships between turbidity and content of Sphingomonas in the water samples.
Content of Shingomonas (%)
0 20 40 60
Tu
rbid
ity
0
1
2
3
4
Y=0.033*X+0.44R2=0.39
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19Major fungal type
Ave
rage
con
tent
in
each
sam
ple
Sand-new-good-835(14) Sand-new-bad-713(13)Sand-old-good-633(11) Sand-old-bad-1286(22)
Average contents of major types fungi identified by ITS gene in different group sand samples. The number at X-axis are matching to the order of major fungal type with ITS analysis.
Candida (念珠菌)
Alternaria (链格孢)Epicoccum (附球菌)
Cladosporium (枝孢霉)
Average contents of major types fungi identified by ITS gene in different group water samples. The number at X-axis are
matching to the order of major fungal type with ITS gene analysis.
0. 0
5. 0
10. 0
15. 0
20. 0
25. 0
30. 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Maj or f ungal type
Aver
age
cont
ent
in e
ach
samp
le (
%)
Water- new- good- 710(15) Water- new- bad- 548(11)Water- ol d- good- 458(10) Water- ol d- bad- 1195(23)
Candida
BioEnergy
Gro
wth
rate
of
en
erg
y
con
su
mp
tion
per
year(
%)
Growth rate of energy consumption in future 20 years
2004 2005 2006 2007 2008
United States 223 261 335 457 656
Europe 52 80 130 154 214
Russia 0 0 0 0 0
India 3 4 4 5 5
Indonesia 0.0 0.2 1.5 2.6 3.5
Vietnam 0 0 0 0 0
South Korea 0.1 0.2 0.9 1.8 3.2
China 17 21 28 35 38
Total Biofuels Production (Thousand Barrels Per Day)
USAKorea (South)
India Indonesia Vietnam China
Urban SO2 concentration (mg/m3)
15.43 (114/141)
52.41 (63) 27.55 (93) 51.05 (65) 64.07 (51) 97.07 (28)
SO2 emissions per populated area (thousand metric tons/squ)
1680 (38/141) 19430 (2) 1150 (47) 360 (84) 260 (92) 2680 (22)
Urban N2O concentration (mg/m3)
60.57 (45/141)
52.86 (65) 29.7 (122) 34.6 (111) 65.5 (30) 71.7 (15)
NOx emissions per populated area (thousand metric tons/squ)
1.29 (13/141) 1.24 (14) 0.52 (33) 0.18 (94) 0.56 (32) 0.75 (27)
CO2 Emissions (kt)5788181 (1/195)
455878 (9)
1273175 (4)
295033 (21)76095 (41)
4143494(2)
CO2 Emissions (kt/1000 people) 19.9 (11/196) 9.5 (34) 1.2 (119) 1.4 (114) 0.9 (129) 3.2 (89)
CO2 from fossil fuels 2000 (per $ GDP) (per $100 million)
0.0133 (9/25)
0.0124 (11)
0.0076 (22)
0.0084 (20) -0.0107
(14)
CO2 from fossil fuels 2000 (per capita) (per 1 million people)
5.31 (1/25) 2.36 (12) 0.23 (25) 0.29 (24) - 0.59 (22)
Forest area (sq. km)3030890 (4/195)
62650 (68)
677010 (10)
884950 (8)129310
(41)1972900
(5)
Forest area > (% of land area) 33 (84/195) 63 (20) 23 (115) 49 (42) 41.7 (59) 21 (122)
Forest area (sq. km/1000 people)10225
(44/195) 1297 (135)
619 (162) 4012 (87) 1556(126) 1512 (130)
Fertiliser consumption (hundred grams/hectare)
1117 (48/141) 5117 (8) 1040 (52) 1546 (34) 3416(15) 2825 (21)
Air Pollution Reated data (ranking)
USASouth Korea
India Indonesia China
Organic water pollutant (BOD) emissions (Kg/d)
1805861 315177 1519842 732965 6088663
Organic water pollutant (BOD) emissions (Kg/d/worker)
0.13 (47/115) 0.12 (49) 0.2 (14) 0.18 (15) 0.14 (60)
Water pollution source (% of total BOD emissions)
chemical industry 14 (9/114) 13 (11) 9.24 (27) 9.17 (13) 14.8 (8)
food industry 42 (31/114) 26 (45) 53.7 (14) 53.7 (12) 28.1 (60)
metal industry 9.6 (13/94) 11.3 (9) 12.2 (7) 2.5 (19) 20.4 (5)
paper &pulp industry 10.6 (35/111) 18.9 (16) 7.6 (46) 8.2 (25) 10.9 (43)
textile industry 5.4 (40/114) 13.6 (16) 12.8(19) 19.4 (8) 15.47 (16)
Water Polution Related Data (Ranking)
Source: www.nationmaster.com
Energy
Petroleum Biosurfactant Application of in-situ microorganisms
Coal Desulfuration
Bioleaching Biofuel
Microalgae Anerobic digestion Hydrogen-gas production MFC (microbial Fuel Cells) Plant: Cellulase
Production Imports Exports Consumption
United States 19089 4608 822 23047
Europe 10716 15305 6115 20106
Russia 23064 2059 8377 16746
India 1108 352 0 1460
Indonesia 2422 0 1199 1224
Japan 190 3377 0 3738
South Korea 14 1179 0 1231
China 2446 138 95 2490
Natural Gas Overview 2007 (Billion Cubic Feet)
Metagenomics (Environmental genomics)
Example:
Ethanol from starch and lignocellulose
Metagenomic screening of
applicable cellulase
Source: rumen (IM), termite (SIBS),
biogas fermentation reactor
with rice straw (SIBS)
Enzyme screening for new source of energy
The U.S. Department of Energy (DOE) Office of Science:
support sequencing- 485 microbial genomes - 30 microbial communities (metagenomes)
Objectives: seek solutions to difficult DOE mission challenges: - alternative sources of energy
- cleaning up environmental wastes- understanding biological carbon cycling as it relates to global climate change (sustainability)
Identification of novel bacteria
Novel Bacteria
Genus novel : Henriciella marina Joostella marina
Species novel : Altererithrobacter dongtanensis Flavobacterium dongtanense Pseudomonas caeni Chryseobacterium caeni Azonexus caeni Rhizobium daejeonense
Novel bacteria list accepted by ICSB (International committee on systematic bacteriology)
( as first or corresponding author )
Class novel
Im WT, Kim KY, Rhee SK, Jung HM, Meng H, Lee ST, & ZX Quan* Description of Fimbriimonadia class nov. of the phylum Armatimonadetes and the diversity and abundance of this class in various environments. Appl Environ Microbiol (submitted)
Full genome sequencing
Published SCI Papers -First author
No. Titles Time Journal Citation
1 Henriciella marina gen. nov., sp. nov., a
novel member of the family Hyphomonadaceae isolated from the East Sea
2009.4 J Microbiol
(IF 1.5)
2Diversity of ammonium-oxidizing bacteria in
a granular sludge anaerobic ammonium-oxidizing (anammox) reactor
2008.11Environ Microbiol
( IF 4.9 )14
3Joostella marina gen. nov., sp. nov., a novel
member of the family Flavobacteriaceae isolated from the East Sea.
2008.6.Int J Syst Evol Microbiol
( IF 2.1 )1
4Chryseobacterium caeni sp. nov., isolated
from bioreactor sludge.2007.1
Int J Syst Evol Microbiol (IF 2.1)
13
5Azonexus caeni sp. nov., a denitrifying bacterium isolated from the sludge of
wastewater treatment plant2006. 5
Int J Syst Evol Microbiol ( IF 2.1 ) 3
6Rhizobium daejeonense sp. nov., nickel-
complexed cyanide-degrading bacterium2005. 11
Int J Syst Evol Microbiol ( IF 2.1 ) 12
7Hydrolyzed molasses as an external carbon
source in biological nitrogen removal2005. 10
Bioresource Technol( IF 4.3 ) 19
Published SCI Papers -Corresponding author
No. Titles Time Journal Citation
1Flavobacterium dongtanense sp. nov., isolated from the rhizosphere of reed in wetland
2010.3Int J Syst Evol Microbiol
(IF 2.1)
2 Bacterial diversity of water and sediment in the Changjiang estuary and coastal area of the East China Sea
2009.11 FEMS Microbiol Ecol
(IF 3.6)1
3 Pseudomonas caeni sp. nov., denitrifying bacteria isolated from sludge of an anaerobic ammonium-oxidizing bioreactor
2009.10Int J Syst Evol Microbiol
(IF 2.1)
4Could nested-PCR be applicable for the study of microbial diversity?
2009.8World J Microbiol
Biotechnol
(IF 1.1)
5The bacterial diversity in an anaerobic ammonium-oxidizing (anammox) reactor community
2009.7Syst Appl Microbiol
(IF 2.6)2
6Analyses of Microbial Consortia in the Starter of Fen Liquor
2009.4Lett Appl Microbiol
(IF 1.6)
Recent Projects –Project manager
2011.1-2013.12 “Study of carbon- and nitrogen- cycle related active microbial population in soil of tidal flat”, Supported by National Natural Foundation of China.
2010.7-2012.6 “Investigation of pollutant contamination and bioremediation potential on the seashores neighboring on the Yellow Sea in Korea and China” , Supported by the NSFC-NRF Scientific Cooperation Program
2008.3-2009.4 “Population of microbiology in fermentation of Fen-liquor” Supported as the Project of Scientific and Technological Innovation in Shanxi province, China.
2007.1-2009.12 “Diversity of anaerobic ammonium-oxidizing bacteria and metagenomic research”, Supported by National Natural Foundation of China.
2006.6-2006.12 “Microbial diversity in swimming pools” Supported by one of Chemical Compony in USA
2005.7-2008.3 “Metagenomics of anaerobic nitrogen removal bacteria and isolation of related microorganisms”, Supported by Korea Advanced Institute of Science and Technology
Lab members