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: quanzx@fudan.edu.cn

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