Some of operating AD performed poor Evaluated by monitoring only the

(1)

Presented by : Presented by :

HERTO DWI ARIESYADY HERTO DWI ARIESYADY

Ecophysiological

Studies

on

Biodiversity

of

Fatty

Acids

Ecophysiological

Studies

on

Biodiversity

of

Fatty

Acids

‐‐

Utilizing

B

Bacteria

acteria

and

A

Archaea

rchaea

in

Anaerobic

Digester

Treating

Domestic

Wastewater

Analyzed

by

Molecular

Biological

Techniques

Wastewater

Analyzed

by

Molecular

Biological

Techniques

Ecophysiological

Studies

on

Biodiversity

of

Fatty

Acids

Ecophysiological

Studies

on

Biodiversity

of

Fatty

Acids

‐‐

Utilizing

B

Bacteria

acteria

and

A

Archaea

rchaea

in

Anaerobic

Digester

Treating

Domestic

Wastewater

Analyzed

by

Molecular

Biological

Techniques

Wastewater

Analyzed

by

Molecular

Biological

Techniques

Academic Advisor : Prof.

Academic Advisor : Prof. YoshimasaYoshimasa WATANABEWATANABE Research Supervisor : Assoc. Prof. Satoshi OKABE Research Supervisor : Assoc. Prof. Satoshi OKABE

Water Quality Control Engineering Laboratory Water Quality Control Engineering Laboratory Urban Environmental Engineering Division Urban Environmental Engineering Division

Graduate School of Engineering Graduate School of Engineering

•

•ReuseReuse‐‐recyclingrecycling

•

•Land disposalLand disposal

Power consumption for WWTP : 1% Power consumption for WWTP : 1%

30

30‐‐50%50% isis forfor sludgesludge treatmenttreatment

Bio

‐‐

process

sludge

management

(in

Japan)

Bio

‐‐

process

sludge

management

(in

Japan)

Image is adopted from : Image is adopted from : www.veoliawater.com www.veoliawater.com

p p

• •CompostingComposting

•

•AnaerobicAnaerobic‐‐digestiondigestion

40 40‐‐50%50%

to reuse to reuse

<20% to disposal <20% to disposal

2

30 30‐‐40%40% toto ADAD

Wastewater Treatment System Wastewater Treatment System

Wastewater Generation

Wastewater Generation WaterWater DischargeDischarge & Land Disposal & Land Disposal BUILT AND NATURAL ENVIRONMENT

BUILT AND NATURAL ENVIRONMENT

Application

of

anaerobic

digester

(AD)

Application

of

anaerobic

digester

(AD)

Application

of

anaerobic

digester

(AD)

Application

of

anaerobic

digester

(AD)

AD AD

Problems :

Problems : OutcomeOutcome ::

• •SludgeSludge volumevolume

& its characteristics & its characteristics •

•EnergyEnergy consumptionconsumption •

•LimitationLimitation ofof naturalnatural resourcesresources

•

•SmallSmall amountamount disposeddisposed

sludge sludge •

•AlternativeAlternative energyenergy sourcesource •

•ConservationConservation ofof naturalnatural

resources resources

3

Obstacles of AD application : Obstacles of AD application : •

•LowLow growthgrowth raterate ofof microbesmicrobes •

•LongLong startingstarting‐‐up/recoveryup/recovery periodperiod •

•LongLong SolidSolid RetentionRetention TimeTime •

•HighHigh interdependencyinterdependency amongamong microbial groups

microbial groups •

•susceptiblesusceptible toto pHpH depletiondepletion

Problems

of

AD

application

Problems

of

AD

application

Problems

of

AD

application

Problems

of

AD

application

4 Some of operating AD Some of operating AD

performed poor performed poor

Evaluated by monitoring Evaluated by monitoring onlyonly

the

the physicophysico‐‐chemicalchemicalpropertiesproperties

Accumulation

Accumulationofof fattyfatty acidsacids

Propionate Propionate

Significance of propionate Significance of propionate‐‐ degradation : degradation :

•

•SulfateSulfate concentrationconcentration

•

•HH partial pressurepartial pressure

Anaerobic

digestion

processes

Anaerobic

digestion

processes

Anaerobic

digestion

processes

Anaerobic

digestion

processes

CH

CH33CHCH22COOCOO––+ 3+ 3HH22O CHO CH33COOCOO––+ + HCOHCO33––+ 3+ 3HH22+ H+ H++

ΔG

ΔGo o _{= +76 kJ/mol}_{= +76 kJ/mol}

5

We want to optimize We want to optimize

the performance the performance and stability of AD and stability of AD

By monitoring and controlling By monitoring and controlling fattyfatty

acids

acids‐‐oxidizingoxidizing bacteriabacteriainin ADAD •

•HH22partialpartial pressurepressure

•

•SyntrophicSyntrophic associationassociation

1.

1. thethe functionalfunctional communitycommunity structuresstructures ofof fattyfatty‐‐acidsacids (propionate(propionate‐‐,, butyrate

butyrate‐‐andand acetateacetate‐‐)) utilizingutilizing microbialmicrobial communitiescommunitiesasas wellwell asas glucose

glucose‐‐degradingdegrading bacteriabacteria asas majormajor trophictrophic groupsgroups inin aa realreal plantplant

AD AD

Through analyzing and characterizing : Through analyzing and characterizing :

GOAL : Optimizing the performance and stability of AD by

controlling

controlling ‘key‘key‐‐players’players’microbesmicrobes

The

objectives

The

objectives

The

objectives

The

objectives

AD AD,,

2.

2. TheThe comparisoncomparisonofof thethe diversitydiversity ofof fattyfatty acidsacids‐‐utilizingutilizingandand othersothers

microbial community

microbial communityinin aa realreal plantplantandandaa modelmodel laboratorylaboratory‐‐scalescale ADs

ADsphylogenetically,phylogenetically,

3.

3. thetheabundanceabundance andand diversitydiversity ofof syntrophicsyntrophic propionatepropionate‐‐oxidizingoxidizing bacteria (POB) populations

bacteria (POB) populationsandand itsits dynamicsdynamics towardstowards thetheeffecteffect ofof propionate concentration

propionate concentrationonon thosethose populationspopulations,, inin aa modelmodel

laboratory laboratory‐‐scalescale ADAD..

(2)

Chapter 1

General General IntroductionIntroduction

Chapter 2

Functional

Functional Bacterial and Archaeal Community Bacterial and Archaeal Community StructuresStructuresof Majorof Major Trophic Groups in a Full

Trophic Groups in a Full--Scale Anaerobic Sludge DigesterScale Anaerobic Sludge Digester

Chapter 3

The

outline

of

the

thesis

The

outline

of

the

thesis

The

outline

of

the

thesis

The

outline

of

the

thesis

p

Assessment of Shift

Assessment of Shiftin Phylogenetic Microbial Diversity in Responsein Phylogenetic Microbial Diversity in Response to a Substrate Perturbation of a Mesophilic Anaerobic Digester to a Substrate Perturbation of a Mesophilic Anaerobic Digester

Chapter 4

Phylogenetic and Functional

Phylogenetic and Functional Diversity of PropionateDiversity of Propionate--Oxidizing Oxidizing Bacteria

Bacteriain an Anaerobic Digester Sludgein an Anaerobic Digester Sludge

Chapter 5

Conclusions

Conclusionsand Recommendationsand Recommendations

7

Milk Added Anaerobic Milk Added Anaerobic H

A B C

M S G

S H

A B C

M S G

S H

A B C

M S G

S H

A B C

M S G

S H

A B C

M S G

S H

A B C

M S G

S H

A B C

M S G

S H

A B C

M S G

S

Ebetsu City Anaerobic Ebetsu City Anaerobic Digester Plant Sludge Digester Plant Sludge

--Semi Batch Feeding Semi Batch Feeding -- Temp. = 37 Temp. = 37 ooCC

--Volume = 0.8 L Volume = 0.8 L -- pH = 6.5 pH = 6.5 –– 7.57.5

--SRT = 30 d SRT = 30 d

--C load = 1.5 gLC load = 1.5 gL--11_d_d--11

Digester Digester Digester Digester

Operation Condition Operation Condition

Research

design

Research

design

Research

design

Research

design

Phylogenetic Phylogenetic Analysis Analysis

Milk Added Anaerobic Milk Added Anaerobic

Digester Sludge Digester Sludge

MAR

MAR--DAPI analysisDAPI analysis MAR

MAR--FISH analysisFISH analysis

Micro Micro--manipulation manipulation

8

Functional community structure/substrate

uptake patterns

Phylogeny identity of intriguing cell

Digester Digester Performance Performance Digester Digester Performance Performance

Biodiversity and phylogeny affiliation

Samples Samples

Confocal Laser Scanning Microscopy

CLSM

Fluorescence Transmission [14_{C] Substrates}

3 h, 37o_C Incubation

DAPI/FISH

MAR

‐‐

DAPI/FISH

technique

MAR

‐‐

DAPI/FISH

technique

Counting Counting Washing

Fixation

DAPI/FISH

Microautoradiography

Making Film Exposure Development

Silver grain by MAR

9

Functional

Bacterial

and

Archaeal

Community

Structures

of

Functional

Bacterial

and

Archaeal

Community

Structures

of

Major

Trophic

Groups

in

a

Full

Major

Trophic

Groups

in

a

Full

‐‐

Scale

Anaerobic

Sludge

Digester

(Water Research, 2007, doi:10.1016/j.watres.2006.12.036)

Chapter

2

2 Chapter

Chapter

2

Discussion Topic: Discussion Topic:

the community structures (diversity and relative abundance) the community structures (diversity and relative abundance)of of major bacterial and archaeal trophic communities groups in major bacterial and archaeal trophic communities groups in anaerobic digester sludge

anaerobic digester sludge

11

Functional

Microbial

Groups

of

AD

Functional

Microbial

Groups

of

AD

Functional

Microbial

Groups

of

AD

Functional

Microbial

Groups

of

AD

Lactose

G alactose G lucose

Propionate

hydrolytic bacteria

Polym er

G alactose G lucose

Propionate

ferm entative bacteria

Lactose

G alactose G lucose

Propionate

hydrolytic bacteria

Polym er

G alactose G lucose

Propionate

C H₄ C O₂

H₂, C O₂ A cetate

C H₄ C O₂

B utyrate

fatty

acids-oxidizing bacteria m ethanogens

C H₄ C O₂

B utyrate

fatty

The most susceptible members :

The most susceptible members : propionatepropionate‐‐,, butyratebutyrate‐‐,, acetateacetate‐‐utilizingutilizing microbesmicrobes

(3)

Their diversity and population sizes Their diversity and population sizes

are

are still unknownstill unknown

Information is

Information is neededneededtoto enhanceenhance the performance & stability of AD the performance & stability of AD

The

objectives

The

objectives

The

objectives

The

objectives

p y

13

• Functional community structure(diversity & abundance)

of major trophic groups of propionate-, butyrate-,

acetate-andglucose-utilizing microbes To characterize, quantify :

H

--Semi Batch Feeding Semi Batch Feeding

•

• mesophilic (40ºC) twomesophilic (40ºC) two‐‐phase ADphase AD •

• intermittent substrate of excess sludge from WWTP intermittent substrate of excess sludge from WWTP for 120,000 p.e.

• C load = 2.5 kg mC load = 2.5 kg m‐‐33_day_day‐‐11 •

• phosphate phosphate ≈≈4 mM4 mM •

• Gas production = 1 Gas production = 1 ××101066_m_m33_year_year‐‐11_{, with 55% CH}_{, with 55% CH} 4

MAR

•Group-specific FISH probes.

•[14C]glucose,[14C]propionate, [14_C]butyrate,[14_C]acetate.

Most active MAR-positive cells Digester Digester Performance Performance

Universal Bacteriaand

Archaeaprimer sets

14

Microbial

diversity

in

AD

(clones

library)

Microbial

diversity

in

AD

(clones

library)

Microbial

diversity

in

AD

(clones

library)

Microbial

diversity

in

AD

(clones

library)

Most frequent

Most frequentBacteriaBacteriaclones : clones : BacteroidetesBacteroidetes> > FirmicutesFirmicutes> > ChloroflexiChloroflexi Most frequent

Most frequentArchaeaArchaeaclones : clones : MethanosaetaMethanosaeta> > MethanospirillumMethanospirillum

QUANTIFICATION

QUANTIFICATION ÆÆFISHFISHusingusing groupgroup‐‐specificspecific probesprobes

15

otal DA

P

I

(%) TotalTotal specificspecific BacteriaBacteria probesprobes Total DAPI

Total DAPI _Tot._Tot._specific_specific_Archaea_Archaea_probes_probes Total DAPI Total DAPI = 37%37%

= 13%13%

Total DAPI = 1.2 ×1010_{cells g‐VSS}‐1

Microbial

diversity

in

AD

(FISH

analysis)

Microbial

diversity

in

AD

(FISH

analysis)

Microbial

diversity

in

AD

(FISH

analysis)

Microbial

diversity

in

AD

(FISH

analysis)

0 2 4

Name of probe

FISH-positiv

e

Bacteria Bacteria‐‐targetedtargeted probesprobes Archaea Archaea‐‐targetedtargeted probesprobes

16

Lactose

G alactose G lucose

Propionate

hydrolytic bacteria

Polym er

G alactose G lucose

Propionate

Lactose

G alactose G lucose

Propionate

hydrolytic bacteria

Polym er

G alactose G lucose

Propionate

Samples Samples

CLSM

Fluorescence Transmission [14C] Substrates

WashingMicroautoradiographyMicroautoradiography

DAPI/FISH MAR Samples

Samples

CLSM CLSM CLSM

Fluorescence Transmission [14C] Substrates

WashingMicroautoradiographyMicroautoradiography

DAPI/FISH MAR

Cross

‐‐

feeding

in

MAR

‐‐

FISH

experiment

Cross

‐‐

feeding

in

MAR

‐‐

FISH

experiment

C H4 C O2

B utyrate

fatty

C H4 C O2

B utyrate

fatty

acids-oxidizing bacteria m ethanogens CountingCounting

Washing Fixation

DAPI/FISH

DAPI/FISH _{Making Film}

Exposure Development

Silver grain by MAR

DAPI/FISH _{Making Film}

Exposure Development

Silver grain by MAR

All of the process

All of the process proceed simultaneouslyproceed simultaneously

Substrate cross

Substrate cross‐‐feedingfeeding mightmight occuroccur atat thethe prolonged incubation time prolonged incubation time

17

Time

course

analysis

at

different

incubation

period

Time

course

analysis

at

different

incubation

period

Time

course

analysis

at

different

incubation

period

Time

course

analysis

at

different

incubation

period

9

9‐‐hh incubationincubation timetime waswas appropriateappropriateto prevent crossto prevent cross‐‐feedingfeeding

(4)

Green : GNSB Green : GNSB‐‐941941

Red : EUB338 Red : EUB338

Green : SmiSR354

Red : EUB338 Red : EUB338

Substrate

uptake

patterns

of

microbes

Substrate

uptake

patterns

of

microbes

Substrate

uptake

patterns

of

microbes

Substrate

uptake

patterns

of

microbes

Green : BET42a

Red : EUB338 Red : EUB338

Green : MX825

Red : ARC915 Red : ARC915

19

Butyrate 3% Propionate 4%

Acetate 6% Glucose

10%

Functional

community

composition

(of

total

cells)

Functional

community

composition

(of

total

cells)

Functional

community

composition

(of

total

cells)

Functional

community

composition

(of

total

cells)

Others 77%

Glucose

Glucose--fermenting bacteriafermenting bacteria : 10%: 10%

Propionate

Propionate--utilizing bacteria utilizing bacteria : 4: 4%%

Butyrate

Butyrate--utilizing bacteriautilizing bacteria : 3% : 3% Acetate

Acetate--utilizing microbesutilizing microbes : 6%: 6% 20

27% GNSB-941

1%

TM7905 4%

Others

6% GAM42a

5% CFB719 <1% Spiro1400

6%

GNSB

GNSB--941941: : HGC69AHGC69A =

=33::11

Glucose

‐‐

utilizing

community

composition

Glucose

‐‐

utilizing

community

composition

13% HGC69A

GNSB 941

LGC354

39%

BET42a BET42a BET42a : : BetaproteobacteriaBetaproteobacteria

GNSB

GNSB‐‐941 : 941 : ChloroflexiChloroflexi

HGC69A

HGC69A : : ActinobacteriaActinobacteria

Spiro1400 : Spiro1400 : SpirochaetaSpirochaeta

TM7905

TM7905 : TM7 candidatus : TM7 candidatus Hitherto unknown function Hitherto unknown function

Spirochaeta

Spirochaeta&& TM7TM7 candidatuscandidatuswere were found to be

found to be glucoseglucose‐‐utilizersutilizers

21

48% 12%

Synbac824 10% Others

SmiSR354

SmiSR354: : Synbac824Synbac824 =

=33: : 11

Smithella sp. >> Syntrophobacter Smithella sp. >> Syntrophobacter

Propionate

‐‐

utilizing

community

composition

Propionate

‐‐

utilizing

community

composition

48% BET42a

30% SmiSR354

SmiSR354 :

SmiSR354 : SmithellaSmithellasp. SR sp. SR Synbac824 :

Synbac824 : SyntrophobacterSyntrophobactersp.sp. To be related with To be related with

sulfate concentration sulfate concentration

22

11% LGC354

7%

Others Synm700Synm700: : LGC354LGC354

= =33: : 11

h h

Butyrate

‐‐

utilizing

community

composition

Butyrate

‐‐

utilizing

community

composition

48% BET42a

35% Synm700

Synm700 :

Synm700 : SyntrophomonasSyntrophomonassp.sp. LGC354

LGC354 : Low: Low‐‐G+C bacteriaG+C bacteria Syntrophomonas Syntrophomonasgroup was group was confirmed to be most important confirmed to be most important

butyrate butyrate‐‐oxidizeroxidizer

23

5% GAM42a

10% LGC354

1%

Syn773 3%

Others Bacteria

18% MX825

Bacteria Bacteria: : ArchaeaArchaea

= =11: : 11

Hitherto unknown function

Hitherto unknown function SynergistesSynergisteswere were found to be

found to be acetateacetate‐‐utilizersutilizers

Acetate

‐‐

utilizing

community

composition

Acetate

‐‐

utilizing

community

composition

6% Others Archaea 55%

BET42a

MX825

1% MS821

GAM42a :

GAM42a : GammaproteobacteriaGammaproteobacteria

Syn773 :

Syn773 : SynergistesSynergistes groupgroup MX825 :

MX825 : MethanosaetaMethanosaeta

MS821 :

MS821 : MethanosarcinaMethanosarcina Methanosaeta

Methanosaeta sp. sp. ÆÆmost important most important ‐‐

acetoclastic methanogen acetoclastic methanogen

(5)

Betaproteobacteria Betaproteobacteria

was also identified as was also identified as

fatty

fatty‐‐acidsacids utilizerutilizer

This group

This group isis notnot affiliatedaffiliated

with syntrophic

association association

Identification

of

active

Betaproteobacteria

Identification

of

active

Betaproteobacteria

Identification

of

active

Betaproteobacteria

Identification

of

active

Betaproteobacteria

MAR+ MAR+ BET42a BET42a

Micromanipulation, Micromanipulation, FISH

FISH usingusing specificspecific probe,probe,

& DAPI & DAPI‐‐stainingstaining

25

association association

•• MARMAR--FISHFISH andand micromanipulationmicromanipulation analysisanalysis cancan bebe appliedapplied

successfully

successfully forfor identificationidentification andand characterizationcharacterization ofof functionalfunctional community

community structurestructure ofof majormajor trophictrophic groupsgroups inin ADAD..

•• TheThe fattyfatty acidsacids-- (esp(esp.. propionatepropionate-- && butyratebutyrate--)) utilizingutilizing

microorganisms

microorganismshadhad lowlow abundanceabundance andand wereweremoremore specializedspecialized

to

to aa fewfew phylogeneticphylogenetic groupsgroups comparedcompared toto glucoseglucose--degradingdegrading bacteria

bacteria..

Conclusions

•• MembersMembers ofof ChloroflexiChloroflexi,, SmithellaSmithella,, SyntrophomonasSyntrophomonas andand Methanosaeta

Methanosaeta groupsgroups dominateddominated thethe glucoseglucose--,, propionatepropionate--,, butyrate

butyrate-- andand acetateacetate--utilizingutilizing microorganismmicroorganism communitycommunity.

•• DespiteDespite thethe lowlow abundance,abundance, thethe hithertohitherto unknownunknown metabolicmetabolic functions

functions ofof microbesmicrobes representedrepresented byby nono oror fewfew cultivatedcultivated

representatives

representatives werewere identifiedidentified toto bebemembersmembers ofof thesethese groupsgroups..

27

Assessment of Shift in Phylogenetic Microbial Diversity in

Response to a Substrate Perturbation of a Mesophilic

Anaerobic Digester

Chapter

3

3 Chapter

Chapter

3

Discussion Topic : Discussion Topic :

The

The comparisoncomparisonof of the the diversity ofdiversity of fatty acidsfatty acids--utilizingutilizingandand others microbial community

others microbial communityin a real plantin a real plantandanda model a model laboratory

laboratory--scale ADsscale ADsphylogeneticallyphylogenetically

28

COMPLEX Anaerobic Digester Plant

Substrate

variation

vs

microbial

diversity

Substrate

variation

vs

microbial

diversity

Substrate

variation

vs

microbial

diversity

Substrate

variation

vs

microbial

diversity

SIMPLE

Ｌ/ S Gas Collection Bag

Magnetic Stirrer 37 oC 0.8 L

Lab-scale Anaerobic Digester

29

• The phylogenetic diversity of functional groups

especially fatty acids-utilizing microbial community in

real AD plant sludgeand amodel laboratory-scale AD

sludge

To compare :

The

objectives

The

objectives

The

objectives

The

objectives

sludge

By application of :

Full

Full--length

length 16S rRNA

cloning analysis

(6)

H

(EADS)

Digester Digester Digester Digester

Methodology

(MADS) (MADS)

MAR

Micro

Universal Bacteriaand

Archaeaprimer sets

• •FAFA concentrationconcentration

• •GasGas compositioncomposition

•

•CC contentcontent (as(as COD)COD)

• •SolidSolid contentcontent

31

Diversity

of

bacterial

and

archaeal

domain

Diversity

of

bacterial

and

archaeal

domain

Diversity

of

bacterial

and

archaeal

domain

Diversity

of

bacterial

and

archaeal

domain

0 10 20 30

Alphap rote

obac teria

Betapr oteob

OTUs number

Major group of Major group of BacteriaBacteria ::

1.

1. BacteroidetesBacteroidetes 2.

2. FirmicutesFirmicutes 3. 3. ChloroflexiChloroflexi 4.

4. ProteobacteriaProteobacteria

0 5 10 rillum Meth

ano

OTUs number

Major group of Major group of ArchaeaArchaea ::

1.

1. MethanosaetaMethanosaeta 2.

2. MethanospirillumMethanospirillum 3.

3. MethanoculleusMethanoculleus

32

Desulfotomaculumthermoacetoxidans Pelotomaculumthermopropionicum

EADS18 (1/521) EADS19 (1/521)

Thermoanaerobacteriumsp. EADS20 (2/521)

Thermoanaerobactermathranii

Uncultured bacterium SJA-143

Clostridium quercicolum

Uncultured bacterium SJA-112 MADS8 (1/133) Uncultured bacterium AA01 EADS21 (5/521) EADS22 (4/521)

EADS23 (4/521) MADS9 (2/133)

Syntrophomonassapovorans Syntrophomonasflectens

EADS24 (4/521) EADS25 (7/521)

Syntrophomonassp. MGB-C1 EADS26 (4/521) 74

EADS18 (1/521) EADS19 (1/521)

Thermoanaerobacteriumsp. EADS20 (2/521)

Thermoanaerobactermathranii

Clostridium quercicolum

MADS8 (1/133)

Uncultured bacterium AA01 EADS21 (5/521) EADS22 (4/521)

EADS23 (4/521) MADS9 (2/133)

Syntrophomonassapovorans Syntrophomonasflectens

EADS24 (4/521) EADS25 (7/521)

Syntrophomonassp. MGB-C1 EADS26 (4/521) 74

Syntrophomonassp. sp. ÆÆbutyratebutyrate oxidationoxidation

Diversity

of

Firmicutes

phylum

Diversity

of

Firmicutes

phylum

Diversity

of

Firmicutes

phylum

Diversity

of

Firmicutes

phylum

EADS26 (4/521) EADS27 (3/521) EADS28 (4/521)

EADS29 (4/521) MADS10 (1/133)

Clostridium lituseburense

EADS30 (4/521)

Clostridium sporosphaeroides

EADS31 (3/521) EADS32 (2/521)

EADS33 (2/521) EADS34(3/521)

Ruminococcusflavefaciens

EADS35 (2/521) EADS36 (2/521) MADS11 (1/133) Uncultured bacterium R6b7

EADS37 (2/521) EADS38 (3/521)

Uncultured bacterium ZZ12C1 EADS39 (2/521)

Clostridium cellobioparum

MADS12 (2/133) EADS40 (6/521)

Clostridium thermocellum

EADS41 (5/521)

Clostridium sp. JC3 EADS42 (5/521) 100

EADS26 (4/521) EADS27 (3/521) EADS28 (4/521)

EADS29 (4/521) MADS10 (1/133) Clostridium lituseburense

EADS30 (4/521)

Clostridium sporosphaeroides

EADS31 (3/521) EADS32 (2/521)

EADS33 (2/521) EADS34(3/521)

Ruminococcusflavefaciens

EADS35 (2/521) EADS36 (2/521) MADS11 (1/133)

Uncultured bacterium R6b7 EADS37 (2/521) EADS38 (3/521)

Uncultured bacterium ZZ12C1 EADS39 (2/521)

Clostridium cellobioparum MADS12 (2/133) EADS40 (6/521)

Clostridium thermocellum

EADS41 (5/521)

Clostridium sp. JC3 EADS42 (5/521) 100

Clostridiagroup group ÆÆto be responsible in to be responsible in

cellulose & sugar fermentation cellulose & sugar fermentationdowns to downs to fatty acids

ndance (%

)

u

ndance (%

) GlucoseGlucose

Propionate

Abundances

vs

specific

rates

Abundances

vs

specific

rates

Abundances

vs

specific

rates

Abundances

vs

specific

rates

Ab

u

Ab

u PropionatePropionate

Acetate

Specific Rate (

Specific Rate (x 10x 10--44_{mol g}_{mol g--VSS}_VSS--1 1 _h_h--11₎₎

1.4

Propionate oxidation

Propionate oxidation is is “bottle neck”“bottle neck”

Limiting factor

Limiting factorin anaerobic degradationin anaerobic degradation Glc

Glc : : PropProp: : AceAce

= =1212: : 11::22

37

•• TheThe phylogenyphylogeny analysisanalysis basedbased onon 1616SS rRNArRNA cloningcloning analysisanalysis elucidated

elucidated thatthat thethe substratesubstrate simplificationsimplification willwill simplifiedsimplified thethe structure

structure ofof thethe anaerobicanaerobic microbialmicrobial communitycommunity inin termterm ofof species

species oror genusgenus levellevelbutbut notnot phylaphyla oror groupgroup level,level, allowedallowed more

more effectiveeffective utilizationutilizationofof perturbedperturbed substratesubstrate..

Conclusions

•• DueDue toto itsits lowlow abundanceabundance andand degradationdegradation rate,rate, propionate propionate--oxidizing

oxidizing bacteriabacteriawerewere expressedexpressed toto bebe aalimitinglimiting factorfactorinin ADAD processes

processes..

(7)

Phylogenetic and Functional Diversity of Propionate

Phylogenetic and Functional Diversity of

Propionate--Oxidizing Bacteria (POB) in an Anaerobic

Oxidizing Bacteria (POB) in an Anaerobic

Digester Sludge

(Applied Microbiology and Biotechnology, 2007, doi:10.1007/s00253‐007‐0842‐y)

Chapter

4

4 Chapter

Chapter

4

Discussion Topics : Discussion Topics :

1.

1. community structure of community structure of activeactivePOB population under different POB population under different propionate concentrations

propionate concentrations

2.

2. comprehensive comprehensive identification of active POBidentification of active POBto explore their to explore their phylogeny identity

phylogeny identity

39

-- Smithella propionicaSmithella propionica

--Syntrophobacter fumaroxidansSyntrophobacter fumaroxidans

Under Methanogenic Condition :

Acetate organic sludge organic sludge

CH

Syntrophobacter fumaroxidans Syntrophobacter fumaroxidans

--Syntrophobacter pfennigiiSyntrophobacter pfennigii

--Syntrophobacter woliniiSyntrophobacter wolinii

--Syntrophobacter sulfatireducensSyntrophobacter sulfatireducens

--Pelotomaculum schinkiiPelotomaculum schinkii

Diversity, abundance, and

Diversity, abundance, and in situin situactivity activity of these bacteria group of these bacteria group have

haveNOTNOTbeen demonstrated yetbeen demonstrated yet

40

• the effect of propionate concentration on the active

community composition of POB

To investigate, identify, characterize :

• the community structure (abundance&diversity) of POB

The

objectives

The

objectives

The

objectives

The

objectives

By application of : By application of :

rRNA cloning analysis,

rRNA cloning analysis, MARMAR--FISHFISHÆÆcommunity structurecommunity structure Micromanipulation

Micromanipulation ÆÆphylogeny identification of active POBphylogeny identification of active POB

41

Milk Added Anaerobic Milk Added Anaerobic H

--Semi Batch Feeding Semi Batch Feeding

Methodology

MAR

Active MAR- positive POB cells

Digester Digester Performance Performance Digester Digester Performance Performance

Universal Bacteria

primer sets

•POB-specific FISH probes.

•[14_{C]propionate.}

• •FAFA concentrationconcentration

• •GasGas compositioncomposition

•

•CC contentcontent (as(as COD)COD)

• •SolidSolid contentcontent

42

Feeding substrate (milk) Feeding substrate (milk)

Fate

of

fatty

acids

during

AD

operation

Lactate _Formate

0

Lactate _Formate

‐‐[Sulfate] [Sulfate] ≈≈0.01 mM0.01 mM ‐‐HH22partial pressure < 10partial pressure < 10‐‐44atmatm ‐‐[CH[CH44] = 60] = 60‐‐65 mol%65 mol%

‐‐[CO[CO22] = 35] = 35‐‐40 mol%40 mol% ₄₃

alkanes -degrading methanogenic consortium clone B2 (AJ133795) alkanes -degrading methanogenic consortium clone B1 (AJ133794)

OTU 1 (2 clones) OTU 74 (1 clones)

anaerobic trichlorobenzene consortium clone SJA -63 (AJ009471) contaminated aquifer clone WCHB1 -12 (AF050534)

OTU 13 (8 clones)

municipal anaerobic digester clone (CR933198)

Smithella propionica(AF126282) granular sludge clone (AF482441)

alkanes -degrading methanogenic consortium clone B3 (AJ133796) landfill leachate clone (AJ853548)

Micromanipulated long rod bacterium clone

alkanes -degrading methanogenic consortium clone B2 (AJ133795) alkanes -degrading methanogenic consortium clone B1 (AJ133794)

OTU 1 (2 clones) OTU 74 (1 clones)

anaerobic trichlorobenzene consortium clone SJA -63 (AJ009471) contaminated aquifer clone WCHB1 -12 (AF050534)

municipal anaerobic digester clone (CR933198)

Smithella propionica Smithella propionica(AF126282)(AF126282)

granular sludge clone (AF482441)

alkanes -degrading methanogenic consortium clone B3 (AJ133796) landfill leachate clone (AJ853548)

Micromanipulated

Micromanipulated long rod bacterium clonelong rod bacterium clone

Propionate Propionate‐‐

Oxidizing

Bacteria Bacteria

Diversity

of

propionate

Diversity

of

propionate

‐‐

oxidizing

bacteria

group

Diversity

of

propionate

Diversity

of

propionate

‐‐

oxidizing

bacteria

group

0.02

terephthalate degrading anaerobic hybrid reactor clone TTA H101 (AY661418)

Syntrophus gentianae(X85132)

Syntrophobacter wolinii(X70906)

Pelotomaculum thermopropionicum(AB035723)

Pelotomaculum schinkii(X91169)

Desulfotomaculum thermobenzoicum(Y11574)

Syntrophobotulus glycolicus(X99706)

Syntrophothermus lipocalidus(AB021305)

Syntrophomonas sapovorans(AF022249)

Syntrophomonas sp. TB-6 (AB098336)

Syntrophomonas wolfei (AF022248)

Syntrophospora bryantii(M26491)

Syntrophomonas sp. MGB-C1 (AB021306)

0.02 0.02

terephthalate degrading anaerobic hybrid reactor clone TTA H101 (AY661418)

Syntrophus gentianae(X85132)

Syntrophobacter Syntrophobacter woliniiwolinii(X70906)(X70906) Pelotomaculum thermopropionicum(AB035723)

Pelotomaculum schinkii(X91169)

Desulfotomaculum thermobenzoicum(Y11574)

Syntrophobotulus glycolicus(X99706)

Syntrophothermus lipocalidus(AB021305)

Syntrophomonas sapovorans(AF022249)

Syntrophomonas sp. TB-6 (AB098336)

Syntrophomonas wolfei (AF022248)

Syntrophospora bryantii(M26491)

Syntrophomonas sp. MGB-C1 (AB021306)

Butyrate Butyrate‐‐ Oxidizing Oxidizing Bacteria Bacteria