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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
The “Woesian” Revolution
Carl Woese –
Analysis of 16S rRNA
1) represent a new kingdom “Archaebacteria”
2) A universal and quantitative phylogeny
is possible
Alignment of a highly conserved region
of the 16S/18S rRNA
Human
Yeast
Corn
E. coli
Green algae
Thermophile
Homo sapiens
S. cereviceae
Zea maize
Escherichia coli
Anacystis nidulans
Thermotoga maritima
Methanococcus vannielii
Thermococcus celer
Sulfolobus sulfotaricus
...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAG...
...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAG...
...GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAG...
...GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCG...
...GTGCCAGCAGCCGCGGTAATACGGGAGAGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCG...
...GTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTACCCGGATTTACTGGGCGTAAAGGG...
...GTGCCAGCAGCCGCGGTAATACCGACGGCCCGAGTGGTAGCCACTCTTATTGGGCCTAAAGCG...
...GTGGCAGCCGCCGCGGTAATACCGGCGGCCCGAGTGGTGGCCGCTATTATTGGGCCTAAAGCG...
...GTGTCAGCCGCCGCGGTAATACCAGCTCCGCGAGTGGTCGGGGTGATTACTGGGCCTAAAGCG...
Three domain theory
Prokaryotes
Bacteria
Eukaryotes
Archaea
Eukarya
Macroorganisms
Not include virus
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
12000
Uncultivated
10000
Number of sequences
Cultivated
8000
6000
4000
2000
0
1993
1994
1995
1996
1997
1998
1999
Time
2000
2001
2002
2003
2004
2005
“Domain” of Bacteria
1994 - 13 divisions
(all cultured)
1997 - 36 divisions
24/12
2008: 30/70
2003 - 53 divisions
26/27
2004 - 80 divisions
26/54
Research Field
Bioinformatics
Uncultured >95%
Cultured <5%
Molecular
Microbial Ecology
Taxonomy
Cultivation
Classification
Ecology
Metagenome
Microbial
Diversity
Molecular analysis
Nitrogen Cycle
Environmental
Technology
Ecology
Removal of
organic carbon
Carbon cycle
(Greenhouse gas)
Removal of
nitrogen and phosphate
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 ammoniumOxidizing (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 that
ANAMMOX is biologically mediated process
15NH +
4
+ 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
•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
The first full-scale ANAMMOX reactor
(2002) at the Dokhaven wastewater
treatment plant, Rotterdam, the Netherlands.
(http://www.anammox.com/research.html)
Reactor operation
Artificial Wastewater:
NaNO2 + NH4HCO3 (1:1), KH2PO4 10 mg/l,
yeast extract 5 mg/l, and TE.
Gas
Sludge: river sediment (1400 mg VSS/l)
Water bath
Recycle
Effluent
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.
Influent
ANAMMOX reactor
Phylogenetic tree based on 16S rRNA gene sequences amplified from
the anammox reactor sludge using Planctomycetales-specific primers.
Defined as the fifth
ANAMMOX genus
The relationships of the different families of anammox bacter
ia among the Planctomycetes.
(Nature Reviews Microbiology 2008, 6, 320-326 )
Metagenomic analysis
Anammox
bacteria
Matched contigs
Number of assembled reads
Sum of contig length
3042
269,212 (31.7%)
561.25Kb
Matched ORFs in Kuenenia
1346
Best match with Kuenenia
3023
Best match with KSU-1
19
Best match with others
145
 Cowork with environmental engineers：
- Microbial population
in ANAMMOX reactor
- Isolation of novel species
from ANAMMOX reactor
- Anaerobic ammonium oxidation with sulfate reduction
Biological treatment of metal
containing wastewater
Biological treatment of
heavy metal containing wastewater
Heavy metal wastewater
Free heavy metals
Cyanide-complexed
heavy metals
High concentration
of heavy metals
High concentration
of cyanide
Treatment of heavy metals with sulfate reduction
O ff- g a s
10 cm
Sulfide production
SO42- + 2CH2O + 2H+
H2S + 2H2O + 2CO2
20 cm
10 cm
( IV )
Bacteria
Metal sulfide precipitation
H2S + Me2+
MeS(s) +2H+
Solid substrates:
UASB granule
Cow manure
20 cm
( III )
D i re c ti o n o f fl o w
S o l i d s u b s tra te s
20 cm
( II )
15 cm
( S e c ti o n I )
5 cm
5 cm
G ra v e l
12 cm
I n fl u e n t
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.
Pilot-scale treatment
 SO42- ＋ 2CH2O ＋ 2H+
→ H2S ＋ 2H2O ＋2CO2
 H2S ＋ Me2+ → MeS(s) ＋ 2H+
Anaerobic treatment of
cyanide- and metal- containing wastewater
Cyanide- and metalcontaining wastewater
CN-, [Me(CN) 4]2-
Me2+, CN-,
Granular sludge
2[Me(CN) 4]
(SRB)
Cyanide degrading SRB
MeS
CO2, NH3
6
Sulfate (mM)
5
4
0 mM
0.5 mM
1 mM
2 mM
5 mM
3
2
1
0
0
1
2
3
4
5
6
7
Time (d)
20
6
Sulfate (mM)
4
Sulfate (mM)
Free cyanide
Zinc-complexed cyanide
Copper-complexed cyanide
Nickel-complexed cyanide
5
3
2
19
18
17
16
15
14
13
12
1
0
0
2
4
6
8
Time (day)
10
12
14
0
2
4
6
8
10
12
Time (d)
[ Ni(CN)4]2- -> Ni2+ + CO2 + NH3
Ni2+ + S2- -> NiS
Cyanide removal percent
Aerobic treatment of metal-complexed cyanide
100
80
60
Re-FC
Re-ZC
Re-NC
40
20
6h
12 h
24 h
48 h
0
0
10
20
30
40
50
Free cyanide (mM)
2.0
1.5
1.0
0.5
0.0
0
5
10
15
Time (h)
20
25
Nickel complexed cyanide (mM)
Zinc complexed cyanide (mM)
Operation time (days)
2.0
1.5
1.0
0.5
0.0
0
5
10
15
Time (h)
20
25
2.0
1.5
1.0
Control
Re-FC sludge
Re-ZC sludge
Re-NC sludge
0.5
0.0
0
5
10
15
Time (h)
20
25
Analysis with DGGE
Biological treatment of
metal containing wastewater
Heavy metal wastewater
Free heavy metals
Cyanide-complexed
heavy metals
High concentration
of heavy metals
Precipitate
heavy metal with
sulfate reduction
Degrade cyanide
in sulfate reducing
condition
High concentration
of cyanide
Degrade different
types cyanide in
aerobic condition
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
treated with new chemicals)
Three sites:
pool water, sand filter, pipe line
8 sampling times:
(June-Sept. two weeks interval)
were
Construction of clone libraries
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 PCR
o
(ITS, all water_16S, some
sand_16S)
PCR primers
 16S
o 27F+1390R
o 27F+1512R, 519F+1390R
 18S
o 18S-nu0817+18S-nu1536
 ITS
o NSA3+NLC2, NSI1 +NLB4
Software for sequence analysis
Percent of uncertainty level during microbial identification
from different type clone libraries (Sample sources: Swimming Pool)
Library type
Total number
of clones
sequenced
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
Average content in each sample
30.0
Methylobacterium（甲基杆菌属）
25.0
20.0
15.0
10.0
5.0
0.0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Major bacterial type
Sand-new-good-743(13)
Sand-old-good-620(11)
Sand-new-bad-652(12)
Sand-old-bad-1161(21)
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.
Content of Methylobacterium in each swimming pool
Chemical
Library
Sand (%)
Water(%)
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
5.6
3.6
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
16.8
7.8
Old
old average
New
new average
Pipe
30.3
Average content in each sample
30.0
Sphingomonas （鞘氨醇单胞菌）
25.0
20.0
15.0
10.0
5.0
0.0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Major bacterial tpye
Water-new-good-855(14)
Water-old-good-662(11)
Water-new-bad-700(12)
Water-old-bad-1288(22)
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.
Turbidity
4
Y=0.033*X+0.44
R2=0.39
3
2
1
0
0
20
40
60
Content of Shingomonas (%)
Relationships between turbidity and content of Sphingomonas
in the water samples.
Alternaria （链格孢）
Epicoccum（附球菌）
20.0
Average content in each sample
18.0
Cladosporium （枝孢霉）
16.0
14.0
Candida（念珠菌）
12.0
10.0
8.0
6.0
4.0
2.0
0.0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Major fungal type
Sand-new-good-835(14)
Sand-old-good-633(11)
Sand-new-bad-713(13)
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.
18
19
Average content in each sample (%)
30.0
Candida
25.0
20.0
15.0
10.0
5.0
0.0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Major fungal type
Water-new-good-710(15)
Water-old-good-458(10)
Water-new-bad-548(11)
Water-old-bad-1195(23)
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.
19
BioEnergy
Growth rate of energy
consumption per year(%)
Growth rate of energy consumption in future 20 years
Total Biofuels Production (Thousand Barrels Per Day)
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
0.0
0.2
1.5
2.6
3.5
0
0
0
0
0
South Korea
0.1
0.2
0.9
1.8
3.2
China
17
21
28
35
38
Indonesia
Vietnam
Air Pollution Reated data (ranking)
USA
Korea
(South)
India
Indonesia
Vietnam
China
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)
5788181
(1/195)
455878
(9)
1273175
(4)
295033 (21)
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)
5.31 (1/25)
2.36 (12)
0.23 (25)
0.29 (24)
-
0.59 (22)
3030890
(4/195)
62650
(68)
677010
(10)
884950 (8)
129310
(41)
1972900 (5)
33 (84/195)
63 (20)
23 (115)
49 (42)
41.7 (59)
21 (122)
Urban SO2 concentration
(mg/m3)
CO2 Emissions (kt)
CO2 from fossil fuels 2000
(per capita) (per 1 million people)
Forest area (sq. km)
Forest area > (% of land area)
76095 (41) 4143494(2)
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)
1040 (52)
1546 (34)
3416(15)
2825 (21)
5117 (8)
Water Polution Related Data (Ranking)
USA
South
Korea
India
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)
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)
Indonesia
China
Water pollution source
(% of total BOD emissions)
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
Natural Gas Overview 2007 (Billion Cubic Feet)
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
2446
138
95
2490
China
Enzyme screening for new source of energy
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)
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
Novel bacteria list accepted by ICSB
(International committee on systematic bacteriology)
（as first or corresponding author）
Genus novel：
 Henriciella marina
 Joostella marina
Species novel：
 Altererithrobacter dongtanensis
 Flavobacterium dongtanense
 Pseudomonas caeni
 Chryseobacterium caeni
 Azonexus caeni
 Rhizobium daejeonense
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
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)
2
Diversity of ammonium-oxidizing bacteria in
a granular sludge anaerobic ammoniumoxidizing (anammox) reactor
2008.11
Environ Microbiol
（IF 4.9）
14
Joostella 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
4
Chryseobacterium caeni sp. nov., isolated
from bioreactor sludge.
2007.1
Int J Syst Evol Microbiol
(IF 2.1)
13
5
Azonexus caeni sp. nov., a denitrifying
bacterium isolated from the sludge of
wastewater treatment plant
2006. 5
Int J Syst Evol Microbiol
（IF 2.1）
3
6
Rhizobium daejeonense sp. nov., nickelcomplexed cyanide-degrading bacterium
2005. 11
Int J Syst Evol Microbiol
（IF 2.1）
12
7
Hydrolyzed molasses as an external carbon
source in biological nitrogen removal
2005. 10
Bioresource Technol
（IF 4.3）
19
3
Citation
Published SCI Papers -Corresponding author
No.
Time
Journal
1
Flavobacterium dongtanense sp. nov.,
isolated from the rhizosphere of reed in
wetland
2010.3
Int 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)
3
Pseudomonas caeni sp. nov., denitrifying
bacteria isolated from sludge of an
anaerobic ammonium-oxidizing bioreactor
2009.10
Int J Syst Evol Microbiol
(IF 2.1)
4
Could nested-PCR be applicable for the
study of microbial diversity?
2009.8
World J Microbiol
Biotechnol
(IF 1.1)
The bacterial diversity in an anaerobic
ammonium-oxidizing (anammox) reactor
community
2009.7
Syst Appl Microbiol
(IF 2.6)
Analyses of Microbial Consortia in the
Starter of Fen Liquor
2009.4
Lett Appl Microbiol
(IF 1.6)
5
6
Titles
Citation
1
2
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