Some of operating AD performed poor Evaluated by monitoring only the

Chapter 1 Operation General Operation General Introduction Introduction Condition Condition

H H H H H H H H -- Semi Batch Feeding Semi Batch Feeding -- Temp. = 37 Temp. = 37 o o C C

G G G G G G G G S SSSSSSS

Chapter 2

-- Volume = 0.8 L Volume = 0.8 L Functional Functional Bacterial and Archaeal Community Bacterial and Archaeal Community Structures Structures of Major of Major

A A A A A A A A -- pH = 6.5 pH = 6.5 – – 7.5 7.5 Trophic Groups in a Full Trophic Groups in a Full--Scale Anaerobic Sludge Digester Scale Anaerobic Sludge Digester

B B B B B B B B C C C C C C C C -- SRT = 30 d SRT = 30 d -- C load = 1.5 gL C load = 1.5 gL --1 1 Ebetsu City Anaerobic --1 Ebetsu City Anaerobic d d 1

MS MS MS MS MS MS MS MS

Digester Plant Sludge Chapter Digester Plant Sludge p 3 Assessment of Shift Assessment of Shift in Phylogenetic Microbial Diversity in Response in Phylogenetic Microbial Diversity in Response

Digester Digester Digester Digester Digester Digester Digester Digester to a Substrate Perturbation of a Mesophilic Anaerobic Digester to a Substrate Perturbation of a Mesophilic Anaerobic Digester

Milk Added Anaerobic Milk Added Anaerobic Milk Added Anaerobic Milk Added Anaerobic

Digester Sludge Digester Sludge

Performance Performance Performance Performance

Chapter 4 Phylogenetic and Functional Phylogenetic and Functional Diversity of Propionate--Oxidizing Diversity of Propionate Oxidizing Bacteria Bacteria in an Anaerobic Digester Sludge in an Anaerobic Digester Sludge

Phylogenetic Phylogenetic

MAR MAR--DAPI analysis DAPI analysis

Micro-- Micro

manipulation manipulation Chapter 5

Analysis Analysis

MAR MAR--FISH analysis FISH analysis

Phylogeny identity Conclusions Conclusions and Recommendations and Recommendations

Biodiversity and

Functional community

phylogeny affiliation

structure/substrate uptake patterns

of intriguing cell

MAR MAR MAR MAR ‐‐DAPI/FISH technique ‐‐DAPI/FISH technique DAPI/FISH DAPI/FISH technique technique

Confocal Laser

Samples Samples

Scanning Microscopy

CLSM

Incubation [ 14 C] Substrates

3 h, 37 o C DAPI/FISH

MAR

Fluorescence Transmission

Microautoradiography Microautoradiography Washing Fixation

Silver grain

DAPI/FISH DAPI/FISH

by MAR

Making Film Exposure Development

Counting Counting

Functional Functional Functional Functional Microbial Groups of AD Microbial Groups of AD Microbial Groups of AD Microbial Groups of AD

Chapter Chapter Chapter Chapter 2 2 2 2

Lactose Lactose Polym er Polym er

hydrolytic bacteria Functional hydrolytic bacteria Functional Bacterial and Archaeal Community Structures of Bacterial and Archaeal Community Structures of Major Major Trophic Groups in a Full‐‐Scale Anaerobic Sludge Trophic Groups in a Full Scale Anaerobic Sludge

G alactose G alactose G alactose G alactose

G lucose G lucose G lucose G lucose

Digester Digester

ferm entative bacteria ferm entative bacteria

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

Propionate Propionate Propionate Propionate

Discussion Topic: Discussion Topic:

H H 2, C O 2 2, C O 2 B utyrate B utyrate

A cetate A cetate

fatty acids- the community structures (diversity and relative abundance) fatty acids- the community structures (diversity and relative abundance) of of oxidizing bacteria oxidizing bacteria

m ethanogens m ethanogens

major bacterial and archaeal trophic communities groups in major bacterial and archaeal trophic communities groups in

CH CH CH CH 4 4 4 4 CO CO CO CO 2 2 2 2

anaerobic digester sludge anaerobic digester sludge

The The most susceptible members : most susceptible members : propionate propionate ‐‐, butyrate , butyrate‐‐, acetate , acetate‐‐utilizing microbes utilizing microbes

The The The The objectives objectives objectives objectives

Methodology Methodology Methodology Methodology

• mesophilic • mesophilic

phase • • intermittent intermittent (40ºC) two‐‐phase AD (40ºC) two AD

for for 120,000 p.e. 120,000 p.e. substrate of excess sludge from WWTP substrate of excess sludge from WWTP Operation Operation Condition Condition

Their diversity and population sizes Their diversity and population sizes

• pH are • are still unknown still unknown • SRT • pH SRT = ≈≈ 7.5 = 7.5

33 S S S S S S S S d 33 d

H H H H H H H H -- Semi Batch Feeding Semi Batch Feeding

• •C C load = 2.5 kg m load = 2.5 kg m G G G G G G G G ‐3

-- Temp. = 37 Temp. = 37 o day o day C C -- Volume = 0.8 L Volume = 0.8 L

• A • phosphate

production = 1 × production = 1 B B B B B B B B C C C C C C C C ×10 10 6 6 m m 3 3 year year -- pH = 6.5 pH = 6.5 – -- SRT = 30 d SRT = 30 d ‐1 , , with 55% CH with 55% CH – 7.5 7.5 4 4

Information Information is is needed needed to to enhance enhance

Ebetsu City Anaerobic Ebetsu City Anaerobic

MS MS MS MS MS MS MS MS

-- C load = 1.5 gL C load = 1.5 gL --1 1 d d --1 1

the the performance & stability of AD performance & stability of AD p p y y

Digester Plant Sludge Digester Plant Sludge

Milk Added Anaerobic Milk Added Anaerobic Milk Added Anaerobic Milk Added Anaerobic

Digester Digester Digester Digester

Digester Sludge Digester Sludge

Performance Performance

To characterize, quantify : • Functional community structure (diversity & abundance)

Phylogenetic Phylogenetic

MAR--DAPI analysis MAR DAPI analysis

Micro-- Micro

of major trophic groups of propionate-, butyrate-,

Analysis Analysis

MAR MAR--FISH analysis FISH analysis

manipulation manipulation

acetate- and glucose-utilizing microbes

Universal Bacteria and

•Group-specific FISH probes .

Most active MAR-

Archaea primer sets

•[ 14 C]glucose,[ 14 C]propionate,

positive cells

[ 14 C]butyrate,[ 14 C]acetate.

Microbial Microbial Microbial Microbial diversity in AD (clones library) diversity in AD (clones library) diversity in AD (clones library) diversity in AD (clones library)

Microbial Microbial Microbial Microbial diversity in AD (FISH analysis) diversity in AD (FISH analysis) diversity in AD (FISH analysis) diversity in AD (FISH analysis)

Total DAPI = 1.2 × 10 10 cells ‐1 10 g‐VSS

P I (%)

Total Total specific Bacteria probes specific Bacteria probes

Total Total DAPI DAPI

Tot. Tot. specific Archaea probes specific Archaea probes 6 Total Total DAPI DAPI

e /T otal DA e = 13% 4 13%

FISH-positiv

Most Most frequent frequent Bacteria Bacteria clones clones : Bacteroidetes : Bacteroidetes > > Firmicutes Firmicutes > > Chloroflexi Chloroflexi Most Most frequent frequent Archaea Archaea clones clones : Methanosaeta : Methanosaeta > > Methanospirillum Methanospirillum

Bacteria Bacteria ‐‐targeted probes targeted QUANTIFICATION probes QUANTIFICATION Æ Æ FISH FISH using using group‐‐specific probes group specific probes

Name of probe

Archaea Archaea ‐‐targeted probes targeted probes

Cross Cross Cross Cross ‐‐feeding in MAR ‐‐feeding in MAR feeding feeding in MAR‐‐FISH experiment in MAR‐‐FISH experiment FISH FISH experiment experiment Time Time Time Time course analysis at different incubation period course analysis at different incubation period course analysis at different incubation period course analysis at different incubation period

Lactose Lactose Polym er Polym er hydrolytic bacteria hydrolytic bacteria

Samples Samples Samples Samples

Scanning Microscopy Scanning Microscopy Confocal Laser Confocal Laser

Incubation Incubation

CLSM CLSM CLSM CLSM

G alactose G alactose G alactose G alactose G lucose G lucose G lucose G lucose ferm entative bacteria ferm entative bacteria

[ [ 14 14 C] Substrates C] Substrates 3 h, 37 3 h, 37 o o C C

Fluorescence Transmission DAPI/FISH Fluorescence Transmission DAPI/FISH MAR MAR

Propionate Propionate Propionate Propionate

Washing Washing Washing Washing Microautoradiography Microautoradiography Microautoradiography Microautoradiography Fixation Fixation

H H 2 2 ,CO ,CO B utyrate 2 B utyrate 2 A cetate A cetate

Silver grain Silver grain

DAPI/FISH DAPI/FISH DAPI/FISH DAPI/FISH

Making Film Making Film

by MAR by MAR

oxidizing bacteria fatty acids- oxidizing bacteria fatty acids- m ethanogens m ethanogens

Development Development Exposure Exposure

Counting Counting Counting Counting

CH CH CH CH 4 4 4 4 CO CO CO CO 2 2 2 2 Substrate Substrate cross cross‐‐feeding might occur at the feeding might occur at the

All All of the process of the process proceed proceed simultaneously simultaneously

prolonged prolonged incubation time incubation time

9 9 ‐‐h incubation time was appropriate h incubation time was appropriate to to prevent cross‐‐feeding prevent cross feeding

Substrate Substrate Substrate Substrate uptake patterns of microbes uptake patterns of microbes uptake patterns of microbes uptake patterns of microbes Functional Functional Functional Functional community composition (of total cells) community composition (of total cells) community composition (of total cells) community composition (of total cells)

Green Red Green Red : EUB338 : EUB338 : GNSB : GNSB‐‐941 941

Green Green : SmiSR354

Red Red : EUB338 : EUB338 : SmiSR354

Butyrate 3% Acetate

Red Red Green Green : EUB338 : EUB338 : BET42a : BET42a

Red Red Green Green : ARC915 : ARC915 : MX825 : MX825

Others 77%

Glucose Glucose--fermenting bacteria fermenting bacteria

: 10% : 10% Propionate Propionate--utilizing bacteria utilizing bacteria : 4 : 4% % Butyrate Butyrate--utilizing bacteria utilizing bacteria

19 Acetate Acetate--utilizing microbes utilizing microbes

Glucose Glucose Glucose Glucose ‐‐utilizing community composition ‐‐utilizing community composition utilizing utilizing community composition community composition Propionate Propionate Propionate Propionate ‐‐utilizing community composition ‐‐utilizing community composition utilizing utilizing community composition community composition

TM7905 4% Spiro1400

Others 5%

SmiSR354 SmiSR354 : : Synbac824 Synbac824 6% CFB719

GNSB--941 GNSB 941 : : HGC69A HGC69A

Smithella Smithella sp. >> Syntrophobacter sp. >> Syntrophobacter LGC354

GAM42a

Synbac824

GNSB 941 GNSB-941

Hitherto Hitherto unknown function unknown function

Spirochaeta Spirochaeta & &

glucose glucose TM7 candidatus ‐‐utilizers utilizers

TM7 candidatus

were were

BET42a

found found

to be to be

be related with be related with sulfate sulfate concentration concentration

HGC69A

BET42a

BET42a BET42a : : Betaproteobacteria Betaproteobacteria

SmiSR354 SmiSR354 : Smithella : Smithella sp. sp. SR SR

GNSB GNSB ‐‐941 : 941 : Chloroflexi Chloroflexi

Synbac824 Synbac824 : : Syntrophobacter Syntrophobacter sp. sp.

HGC69A HGC69A

: : Actinobacteria Actinobacteria Spiro1400 Spiro1400 : : Spirochaeta Spirochaeta TM7905 TM7905

: : TM7 candidatus TM7 candidatus

Butyrate Butyrate Butyrate Butyrate ‐‐utilizing community composition ‐‐utilizing community composition utilizing utilizing community composition community composition

Acetate Acetate Acetate Acetate ‐‐utilizing community composition ‐‐utilizing community composition utilizing utilizing community composition community composition

Synm700 Synm700 : : LGC354 LGC354

LGC354

Others Bacteria

Bacteria Bacteria : : Archaea Archaea 11%

= = 3 3 : : 1 1 GAM42a

= = 1 1 : : 1 1 LGC354

Hitherto Hitherto found found unknown function unknown function

acetate acetate ‐‐utilizers utilizers Synergistes Synergistes were were

to be to be

Syntrophomonas Syntrophomonas h h group group was was

MX825 MX825

confirmed confirmed

Methanosaeta Methanosaeta sp. sp. Æ most important most

Æ important ‐‐

BET42a

butyrate butyrate ‐‐oxidizer oxidizer to be most important to be most important

acetoclastic acetoclastic

methanogen methanogen

BET42a

Synm700 Synm700 : Syntrophomonas : Syntrophomonas sp. sp.

Others Archaea

LGC354 LGC354

: : Low‐‐G+C bacteria Low G+C bacteria

GAM42a GAM42a : : Gammaproteobacteria Gammaproteobacteria Syn773 Syn773 : Synergistes : Synergistes group group MX825 MX825 : Methanosaeta : Methanosaeta MS821 MS821 : Methanosarcina : Methanosarcina

Identification Identification Identification Identification of active Betaproteobacteria of active Betaproteobacteria of active Betaproteobacteria of active Betaproteobacteria

Betaproteobacteria Betaproteobacteria was was also identified as also identified as fatty fatty ‐‐acids utilizer acids utilizer

This This group group is not affiliated is not affiliated with with syntrophic syntrophic

MAR+ MAR+

association association association association

BET42a BET42a

Micromanipulation, Micromanipulation, FISH FISH using using specific probe, specific probe, & & DAPI DAPI‐‐staining staining

Conclusions Conclusions Conclusions Conclusions

Chapter Chapter Chapter Chapter 3 3 3 3

•• MAR MAR--FISH FISH and and micromanipulation micromanipulation analysis analysis can can be applied be applied successfully for successfully for identification identification and and characterization characterization of of functional functional community structure community structure of of major major trophic trophic groups groups in in AD AD..

Assessment Assessment of Shift in Phylogenetic Microbial Diversity in of Shift in Phylogenetic Microbial Diversity in Response Response to a Substrate Perturbation of a Mesophilic to a Substrate Perturbation of a Mesophilic

•• The fatty The fatty acids acids-- (esp (esp.. propionate propionate-- & & butyrate butyrate--)) utilizing utilizing microorganisms microorganisms had low had low abundance abundance and and were were more specialized more specialized

Anaerobic Anaerobic Digester Digester

to a to few phylogenetic a few phylogenetic groups groups compared compared to to glucose glucose--degrading degrading bacteria.. bacteria

•• Members Members of of Chloroflexi Chloroflexi,, Smithella,, Smithella Syntrophomonas Syntrophomonas and and

Discussion Topic : Discussion Topic :

Methanosaeta Methanosaeta groups groups dominated dominated the glucose the glucose--,, propionate propionate--,, butyrate-- and butyrate and acetate acetate--utilizing utilizing microorganism microorganism community community.

The The comparison comparison of of the the diversity of fatty acids diversity of fatty acids--utilizing utilizing and and •• Despite Despite the the low low abundance, abundance, the the hitherto hitherto unknown unknown metabolic metabolic

a model a model functions functions of of microbes microbes represented represented by by no no or or few few cultivated cultivated

others microbial community others microbial community in a real plant in a real plant and and

laboratory laboratory--scale ADs scale ADs phylogenetically phylogenetically

representatives were representatives were identified identified to to be be members of members of these these groups groups ..

Substrate Substrate Substrate Substrate variation vs microbial diversity variation vs microbial diversity variation vs microbial diversity variation vs microbial diversity

The The The The objectives objectives objectives objectives

COMPLEX

Anaerobic Digester Plant

To compare :

• The phylogenetic diversity of functional groups especially fatty acids-utilizing microbial community in real AD plant sludge and a model laboratory-scale AD sludge sludge

Lab-scale Anaerobic Digester

Ｌ/S Collection Gas Bag

By application of :

SIMPLE

Full Full--length length 16S rRNA cloning analysis

37 C 0.8 L

Magnetic Stirrer

Methodology Methodology Methodology Methodology

Diversity Diversity Diversity Diversity of bacterial and archaeal domain of bacterial and archaeal domain of bacterial and archaeal domain of bacterial and archaeal domain

Operation Operation Condition Condition

H H H H H H H H -- Semi Batch Feeding Semi Batch Feeding o o

MADS MADS = 90 Volume = 0.8 L = 90 = 33 = 33

TM -- Temp. = 37 7 Temp. = 37 C C

G G G G G G G G S SSSSSSS

P1 0 Total O Total OTUs : OTUs :

EADS EADS

-- Volume = 0.8 L

-- pH = 6.5 pH = 6.5 – – 7.5 7.5 O P5 11 Total r Total OTUs : OTUs : EADS EADS = 13 = 13

MADS MADS =4 =4

Ebetsu City Anaerobic MADS Ebetsu City Anaerobic

MS MS MS MS MS MS MS MS

-- C load = 1.5 gL C load = 1.5 gL --1 1 d d --1 1 in ac ob te ae ta

MADS EADS

Digester Plant Sludge Digester Plant Sludge

EADS

(EADS) ia (EADS) Milk Added Anaerobic Milk Added Anaerobic Digester Digester Digester Digester g g g g

Digester Sludge Digester Sludge

Performance Performance Performance Performance

m ma ma

Ga tapr

eob

te ria

(MADS) um (MADS) D el ot ac xi Me rill

th an no

•Gas a

• •FA FA concentration concentration

Be tapr ro te ob Ch lo ro fle

0 5 content (as COD) 10 content (as COD)

•C • C composition composition

OTUs number

M et han

• •Solid Solid content content

OTUs number

Major Major group of group of Bacteria Bacteria ::

Major Major group of group of Archaea Archaea ::

Phylogenetic Phylogenetic MAR--DAPI analysis MAR DAPI analysis

Micro-- Micro

Analysis Analysis MAR MAR--FISH analysis FISH analysis

manipulation manipulation

1. 1. Bacteroidetes Bacteroidetes 2. 2. Firmicutes Firmicutes

1. 1. Methanosaeta Methanosaeta

Universal Bacteria and

3. 3. Chloroflexi Chloroflexi

2. 2. Methanospirillum Methanospirillum

Archaea primer sets

4. 4. Proteobacteria Proteobacteria

3. 3. Methanoculleus Methanoculleus

Diversity Diversity Diversity Diversity of Firmicutes phylum of Firmicutes phylum of Firmicutes phylum of Firmicutes phylum

Pelotomaculumthermopropionicum Pelotomaculumthermopropionicum Desulfotomaculumthermoacetoxidans 57 Desulfotomaculumthermoacetoxidans 57 EADS18 (1/521) EADS18 (1/521)

Nr. Nr.

of clones : of clones :

EADS EADS 54 MADS MADS

54 Thermoanaerobacteriumsp. Thermoanaerobacteriumsp.

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

66 66 Uncultured bacterium SJA Thermoanaerobactermathranii Thermoanaerobactermathranii EADS20 (2/521) EADS20 (2/521) Uncultured bacterium SJA -143 -143

Nr. Nr. of OTUs : of OTUs :

EADS EADS = 25/102 = 25/102

100 100 79 79 Clostridium quercicolum Clostridium quercicolum

MADS8 (1/133) MADS8 (1/133) Uncultured bacterium SJA-112 Uncultured bacterium SJA-112

MADS MADS

100 100 74 74 EADS21 (5/521) EADS21 (5/521) Uncultured bacterium AA01 Uncultured bacterium AA01

59 59 90 EADS22 (4/521) 90 100 100 EADS22 (4/521) MADS9 (2/133) MADS9 (2/133) EADS23 (4/521) EADS23 (4/521)

100 100 EADS25 (7/521) EADS24 (4/521) EADS25 (7/521) EADS24 (4/521) Syntrophomonasflectens Syntrophomonasflectens 54 54 54 54 Syntrophomonassp. MGB-C1 Syntrophomonassp. MGB-C1

Syntrophomonassapovorans Syntrophomonassapovorans

Syntrophomonas Syntrophomonas sp. sp. Æ Æ butyrate butyrate oxidation oxidation

EADS28 (4/521) EADS28 (4/521) EADS27 (3/521) EADS27 (3/521) EADS26 (4/521) EADS26 (4/521) EADS26 (4/521) EADS26 (4/521) 94 94 EADS29 (4/521) 99 EADS29 (4/521) Clostridium lituseburense Clostridium lituseburense MADS10 (1/133) MADS10 (1/133)

‘high ‘high diversity in EADS’ diversity in EADS’

EADS32 (2/521) 100 EADS31 (3/521) EADS32 (2/521) EADS31 (3/521) Clostridium sporosphaeroides Clostridium sporosphaeroides 100 100 100

EADS30 (4/521) EADS30 (4/521)

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

cellulose cellulose & sugar fermentation & sugar fermentation EADS36 (2/521) be responsible in EADS36 (2/521) EADS35 (2/521) EADS35 (2/521) downs to

Clostridia Clostridia group group Æ Æ to be responsible in to

downs to

75 Uncultured bacterium R6b7 Uncultured bacterium R6b7 MADS11 (1/133) MADS11 (1/133)

fatty fatty

75 acids acids

51 51 74 74 86 86 EADS38 (3/521) EADS38 (3/521) EADS37 (2/521) EADS37 (2/521)

Clostridium cellobioparum Uncultured bacterium ZZ12C1 Uncultured bacterium ZZ12C1 MADS12 (2/133) MADS12 (2/133) Clostridium cellobioparum EADS39 (2/521) EADS39 (2/521)

96 96 Clostridium thermocellum EADS41 (5/521) Clostridium thermocellum EADS40 (6/521) EADS40 (6/521)

‘high ‘high diversity in EADS’ diversity in EADS’

82 82 63 63 Clostridium sp. JC3 Clostridium sp. JC3 EADS41 (5/521) EADS42 (5/521) EADS42 (5/521)

Abundances Abundances Abundances Abundances vs specific rates vs specific rates vs specific rates vs specific rates

Conclusions Conclusions Conclusions Conclusions

Glucose Glucose

•• The The phylogeny phylogeny analysis analysis based based on on 16 16S S rRNA rRNA cloning cloning analysis analysis elucidated that elucidated that the the substrate substrate simplification simplification will will simplified simplified the the

0.7 structure structure of of the the anaerobic anaerobic microbial microbial community community in term in term of of species or species or genus genus level level but but not not phyla phyla or or group group level, level, allowed allowed

u u ndance (% ndance (% u u

Gl Gl Glc Glc : : Prop Prop P P : : A Ace A Ace 4% 4% % %

Propionate Propionate Propionate Propionate

more effective more effective utilization utilization of of perturbed perturbed substrate substrate..

Ab Ab = = 12 12 : : 1 1 :: 2 2 •• Due Due to to its its low low abundance abundance and and degradation degradation rate, rate, propionate-- 1.4 propionate

oxidizing bacteria oxidizing bacteria were were expressed expressed to to be a be a limiting limiting factor factor in in AD AD

Acetate Acetate

processes processes..

Specific Rate ( Specific Rate ( x 10 x 10 --4 4 mol g mol g--VSS VSS --1 1 h h --1 1 ))

Propionate oxidation Propionate oxidation is is “bottle neck” “bottle neck” Limiting factor Limiting factor in anaerobic degradation in anaerobic degradation

Introduction Introduction Introduction Introduction

Chapter Chapter Chapter Chapter 4 4 4 4

Under Methanogenic Condition :

Biowastes, Phylogenetic and Functional Diversity of Propionate-- Biowastes, Phylogenetic and Functional Diversity of Propionate

organic sludge organic sludge

Carbohydrate, Carbohydrate,

Acetate Acetate CO CO 2 2 +H +H

2 2 CH CH CO CO 4 4

2 Oxidizing Bacteria (POB) in an Anaerobic 2 Oxidizing Bacteria (POB) in an Anaerobic Propionate Propionate Digester Sludge Digester Sludge

Protein Protein

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

-- Smithella propionica Smithella propionica -- Syntrophobacter fumaroxidans Syntrophobacter fumaroxidans Syntrophobacter fumaroxidans Syntrophobacter fumaroxidans

Discussion Topics : Discussion Topics :

-- Syntrophobacter pfennigii Syntrophobacter pfennigii -- Syntrophobacter wolinii Syntrophobacter wolinii

1. 1. community structure of community structure of active active POB population under different POB population under different

-- Syntrophobacter sulfatireducens Syntrophobacter sulfatireducens

propionate concentrations propionate concentrations

-- Pelotomaculum schinkii Pelotomaculum schinkii

2. 2. comprehensive comprehensive identification of active POB identification of active POB to explore their to explore their phylogeny identity phylogeny identity

Diversity, abundance, and Diversity, abundance, and in situ in situ activity activity

of these bacteria group of these bacteria group have have NOT NOT been demonstrated yet been demonstrated yet

The The The The objectives objectives objectives objectives

Methodology Methodology Methodology Methodology

Operation Operation Condition Condition H H H H H H H H -- Semi Batch Feeding Semi Batch Feeding

To investigate, identify, characterize :

G G G G G G G G S SSSSSSS

-- Temp. = 37 Temp. = 37 o o C C -- Volume = 0.8 L Volume = 0.8 L

-- pH = 6.5 pH = 6.5 – – 7.5 • the community structure ( 7.5 abundance & diversity) of POB A A A A A A A A B B B B B B B B C C C C C C C C

-- SRT = 30 d SRT = 30 d

-- C load = 1.5 gL C load = 1.5 gL --1 1 d d --1 • the 1 effect of propionate concentration on the active Ebetsu City Anaerobic Ebetsu City Anaerobic

MS MS MS MS MS MS MS MS

community composition of POB

Digester Plant Sludge Digester Plant Sludge

Milk Added Anaerobic Milk Added Anaerobic Milk Added Anaerobic Milk Added Anaerobic

Digester Digester Digester Digester Digester Digester Digester Digester

Digester Sludge Digester Sludge

Performance Performance Performance Performance

• •FA FA concentration concentration

• • •Gas •C Gas C composition By application of : composition By application of :

•Solid • Solid content (as COD) content (as COD) content content

rRNA cloning analysis, rRNA cloning analysis, MAR MAR--FISH FISH Æ Æcommunity structure community structure Micromanipulation Micromanipulation Æ Æ phylogeny identification of active POB phylogeny identification of active POB

Phylogenetic Phylogenetic

MAR MAR--DAPI analysis DAPI analysis

Micro Micro--

Analysis Analysis

MAR MAR--FISH analysis FISH analysis

manipulation manipulation

Universal Bacteria

•POB-specific FISH probes .

Active MAR- positive

primer sets

•[ 14 C]propionate.

POB cells

Fate Fate Fate Fate of fatty acids during AD operation of fatty acids during AD operation of fatty acids during AD operation of fatty acids during AD operation Diversity Diversity Diversity Diversity of propionate of propionate‐‐oxidizing bacteria group of propionate‐‐oxidizing bacteria group of propionate oxidizing oxidizing bacteria group bacteria group

Feeding Feeding substrate (milk) substrate (milk)

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

20 20 8 8 Micromanipulated long rod bacterium clone OTU 1 (2 clones) Micromanipulated Micromanipulated long rod bacterium clone landfill leachate clone (AJ853548) landfill leachate clone (AJ853548) long rod bacterium clone

, , ) ) te

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

a te a (m (m 15 15 pH pH

anaerobic trichlorobenzene consortium clone SJA -63 (AJ009471) 7 anaerobic trichlorobenzene consortium clone SJA -63 (AJ009471) 7 contaminated aquifer clone WCHB1 -12 (AF050534) contaminated aquifer clone WCHB1 -12 (AF050534)

Propionate Propionate ‐‐

cet cet a te te

OTU 13 (8 clones) a OTU 13 (8 clones) Acetate Acetate municipal anaerobic digester clone (CR933198) municipal anaerobic digester clone (CR933198) Bacteria Bacteria Oxidizing Oxidizing ,A ,A m m granular sludge clone (AF482441) granular sludge clone (AF482441) Smithella propionica (AF126282) Smithella propionica (AF126282) Smithella propionica (AF126282)

n alkanes -degrading methanogenic consortium clone B3 (AJ133796) , alkanes -degrading methanogenic consortium clone B3 (AJ133796) , For e , For , e

a n a te te

10 10 6 6 pH pH

P P ropio ropio Lac Lac

tat tat

5 5 Propionate Propionate

5 5 terephthalate degrading anaerobic hybrid reactor clone TTA H101 (AY661418) terephthalate degrading anaerobic hybrid reactor clone TTA H101 (AY661418) Syntrophus gentianae (X85132) Syntrophus gentianae (X85132)

OTU 14 (2 clones) OTU 14 (2 clones)

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

OTU 11 (1 clones) Lactate OTU 11 (1 clones) Lactate 0 0 Formate Formate

4 4 Pelotomaculum thermopropionicum (AB035723) Pelotomaculum thermopropionicum (AB035723) Syntrophobacter wolinii (X70906) Syntrophobacter wolinii Syntrophobacter wolinii (X70906) (X70906) Pelotomaculum schinkii (X91169) Pelotomaculum schinkii (X91169)

OTU 18 (1 clones) Syntrophothermus lipocalidus (AB021305) Syntrophobotulus glycolicus (X99706) Syntrophobotulus glycolicus (X99706) Desulfotomaculum thermobenzoicum (Y11574) Desulfotomaculum thermobenzoicum (Y11574) Syntrophothermus lipocalidus (AB021305) OTU 18 (1 clones)

Butyrate ≈≈ 0.01 mM Butyrate ‐‐ ‐‐ H H 2 2 partial partial pressure < 10 pressure < 10 ‐4 atm atm

Time (h) Time (h)

Bacteria Bacteria ‐‐ [CH [CH 4 4 ] ] = 60 = 60‐‐65 mol% 65 mol%

‐‐ [Sulfate] [Sulfate] 0.01 mM

Syntrophomonas sp. TB-6 (AB098336) Syntrophomonas sp. TB-6 (AB098336) Syntrophomonas sapovorans (AF022249) Syntrophomonas sapovorans (AF022249)

Syntrophomonas wolfei (AF022248) Syntrophomonas wolfei (AF022248)

Oxidizing Oxidizing

0.02 0.02 0.02 Syntrophomonas sp. MGB-C1 (AB021306) ‐‐ [CO Syntrophomonas sp. MGB-C1 (AB021306) [CO 2 2 ] ] = 35 = 35‐‐40 mol% 40 mol%

Syntrophospora bryantii (M26491) Syntrophospora bryantii (M26491)

Diversity Diversity Diversity Diversity of propionate of propionate‐‐oxidizing bacteria group of propionate‐‐oxidizing bacteria group of propionate oxidizing oxidizing bacteria group bacteria group

FISH FISH FISH FISH experiment experiment experiment experiment

alkanes -degrading methanogenic consortium clone B1 (AJ133794) landfill leachate clone (AJ853548) landfill leachate clone (AJ853548) alkanes -degrading methanogenic consortium clone B1 (AJ133794)

Application of Genus Specific Probes on Anaerobic Digester Sludge Application of Genus Specific Probes on Anaerobic Digester Sludge

OTU 74 (1 clones) OTU 74 (1 clones) OTU 1 (2 clones) OTU 1 (2 clones) Micromanipulated long rod bacterium clone long rod bacterium clone anaerobic trichlorobenzene consortium clone SJA -63 (AJ009471) anaerobic trichlorobenzene consortium clone SJA -63 (AJ009471) Red : SmiSR354 (Smithella sp. SR)

Micromanipulated long rod bacterium clone Micromanipulated

contaminated aquifer clone WCHB1 -12 (AF050534) contaminated aquifer clone WCHB1 -12 (AF050534) Green : Synbac824 (Syntrophobacter)

Smithella propionica (AF126282) Smithella propionica (AF126282) Smithella propionica municipal anaerobic digester clone (CR933198) OTU 13 (8 clones) OTU 13 (8 clones) municipal anaerobic digester clone (CR933198)

Propionate Propionate ‐‐

Bacteria SmiSR354 SmiSR354 Bacteria

Oxidizing Oxidizing

granular sludge clone (AF482441) granular sludge clone (AF482441) (AF126282)

Total Total cells = 4× cells = 4 ×10 10 10 10 cells cells mg‐‐VSS mg VSS – –1 1

alkanes -degrading methanogenic consortium clone B3 (AJ133796) alkanes -degrading methanogenic consortium clone B3 (AJ133796) OTU 14 (2 clones) OTU 14 (2 clones) alkanes -degrading methanogenic consortium clone B2 (AJ133795) alkanes -degrading methanogenic consortium clone B2 (AJ133795)

Population of POB community : Population Population Population of POB community : of POB community : of POB community :

terephthalate degrading anaerobic hybrid reactor clone TTA H101 (AY661418) terephthalate degrading anaerobic hybrid reactor clone TTA H101 (AY661418) Smithella sp. Smithella sp. SR SR : : 8 8 Syntrophus gentianae (X85132) Syntrophus gentianae (X85132)

Syntrophobacter Syntrophobacter : 6 6× : ×10 10 10 8 8 cells cells mg‐‐VSS VSS – –1 OTU 11 (1 clones) 1 OTU 11 (1 clones)

mg

2× 2 ×10 cells cells

mg‐‐VSS mg VSS –1 – 1

Pelotomaculum thermopropionicum (AB035723) Pelotomaculum thermopropionicum (AB035723) Syntrophobacter wolinii Syntrophobacter wolinii (X70906) Syntrophobacter wolinii (X70906) (X70906) Desulfotomaculum thermobenzoicum (Y11574) Pelotomaculum schinkii (X91169)

Synbac824

Synbac824

Syntrophobotulus glycolicus (X99706) Desulfotomaculum thermobenzoicum (Y11574) Pelotomaculum schinkii (X91169)

Smithella

Smithella >> >>

Syntrophobacter Syntrophobacter

Syntrophobotulus glycolicus (X99706) OTU 18 (1 clones) OTU 18 (1 clones) Syntrophothermus lipocalidus (AB021305) Syntrophothermus lipocalidus (AB021305) Syntrophomonas wolfei (AF022248) Syntrophomonas sp. TB-6 (AB098336) Syntrophomonas sapovorans (AF022249) Syntrophomonas sp. TB-6 (AB098336) Syntrophomonas sapovorans (AF022249)

Butyrate Butyrate

Oxidizing Oxidizing ‐‐

To To

be related with be related with

Syntrophomonas wolfei (AF022248)

Synm700 Synm700

Bacteria Bacteria sulfate sulfate concentration concentration

0.02 0.02 0.02 Syntrophomonas sp. MGB-C1 (AB021306) Syntrophomonas sp. MGB-C1 (AB021306)

Syntrophospora bryantii (M26491) Syntrophospora bryantii (M26491)

Time Time Time Time course analysis at different incubation period course analysis at different incubation period course analysis at different incubation period course analysis at different incubation period Diversity Diversity Diversity Diversity of propionate‐‐oxidizing bacteria group of propionate of propionate‐‐oxidizing bacteria group of propionate oxidizing oxidizing bacteria group bacteria group

100.00 100.00 Smithella sp. SR Smithella sp. SR

Synbac824 Synbac824 unidentified others Smithella sp. LR Syntrophobacter iv Syntrophobacter e iv e Smithella sp. LR unidentified others

10.00 10.00 SmiSR354 EUB338 SmiSR354 EUB338

Methanosaeta Methanosaeta

/t ta lc o o ta tal M tal M o /t o

A R-posit R-posit

lls lls

MAR+ MAR+ A e lc e Smithella SR Smithella SR

MAR+ MAR+

/t /t (%) (%)

Oval Oval L = 1.5 μm p

50.00 50.00 MAR+ MAR+ cells/total cells cells/total cells

5.00 5.00 e lls lls e Short Short rod rod

Syntrophobacter Syntrophobacter

W1 W= 1 μm m iti

ec ec L = 1.5‐3

μm

r robe robe r

v ti v ti

W051 W= 0.5‐1 μm μm

EUB338 EUB338

EUB338 EUB338

MAR MAR

MAR+ Methanosaeta

MAR MAR

Incubation period (hours) Incubation period (hours)

MAR+ MAR+

MAR+ MAR+

Unidentified Unidentified long rod cells long rod cells

Unidentified Unidentified tiny cells tiny cells

Short Short rod rod 3 3 ‐‐h incubation time was appropriate h incubation time was appropriate to to prevent cross‐‐feeding prevent cross feeding

Long Long

L = 10‐20 rod rod μm

L =1 μm

47 W= 0.5‐1 μm

W = 0.5 μm

Identification Identification Identification Identification of long rod POB cells of long rod POB cells of long rod POB cells of long rod POB cells Diversity Diversity Diversity Diversity of propionate‐‐oxidizing bacteria group of propionate of propionate of propionate‐‐oxidizing bacteria group oxidizing oxidizing bacteria group bacteria group

alkanes -degrading methanogenic consortium clone B1 (AJ133794) alkanes -degrading methanogenic consortium clone B1 (AJ133794) Micromanipulated long rod bacterium clone Micromanipulated long rod bacterium clone landfill leachate clone (AJ853548) landfill leachate clone (AJ853548)

SmiLR150 SmiLR150

OTU 74 (1 clones) OTU 1 (2 clones) OTU 1 (2 clones) OTU 74 (1 clones) anaerobic trichlorobenzene consortium clone SJA -63 (AJ009471) anaerobic trichlorobenzene consortium clone SJA -63 (AJ009471)

contaminated aquifer clone WCHB1 -12 (AF050534) contaminated aquifer clone WCHB1 -12 (AF050534) OTU 13 (8 clones) OTU 13 (8 clones)

Propionate Propionate ‐‐

granular sludge clone (AF482441) granular sludge clone (AF482441) Smithella propionica (AF126282) Smithella propionica Smithella propionica (AF126282) municipal anaerobic digester clone (CR933198) municipal anaerobic digester clone (CR933198) (AF126282)

Bacteria Bacteria Oxidizing Oxidizing

SmiSR354 SmiSR354

Micromanipulation Micromanipulation

alkanes -degrading methanogenic consortium clone B3 (AJ133796) alkanes -degrading methanogenic consortium clone B3 (AJ133796) OTU 14 (2 clones) OTU 14 (2 clones) alkanes -degrading methanogenic consortium clone B2 (AJ133795) terephthalate degrading anaerobic hybrid reactor clone TTA H101 (AY661418) alkanes -degrading methanogenic consortium clone B2 (AJ133795) terephthalate degrading anaerobic hybrid reactor clone TTA H101 (AY661418)

16S 16S rRNA rRNA cloning cloning analysis analysis

Syntrophus gentianae (X85132) Syntrophus gentianae (X85132) OTU 11 (1 clones) OTU 11 (1 clones)

Pelotomaculum thermopropionicum (AB035723) Pelotomaculum thermopropionicum (AB035723) Syntrophobacter wolinii (X70906) Syntrophobacter Syntrophobacter wolinii wolinii (X70906) (X70906)

Synbac824 Synbac824

Desulfotomaculum thermobenzoicum (Y11574) Desulfotomaculum thermobenzoicum (Y11574) Pelotomaculum schinkii (X91169) FISH Pelotomaculum schinkii (X91169) FISH probe probe design design

Syntrophothermus lipocalidus (AB021305) Syntrophothermus lipocalidus (AB021305) Syntrophobotulus glycolicus (X99706) Syntrophobotulus glycolicus (X99706) OTU 18 (1 clones) OTU 18 (1 clones)

Syntrophomonas sapovorans (AF022249) Syntrophomonas sapovorans (AF022249)

Butyrate Butyrate ‐‐

Syntrophomonas wolfei (AF022248) Syntrophomonas wolfei (AF022248) Syntrophomonas sp. TB-6 (AB098336) Syntrophomonas sp. TB-6 (AB098336)

Synm700 Synm700

Oxidizing Oxidizing

Syntrophomonas sp. MGB-C1 (AB021306) Syntrophomonas sp. MGB-C1 (AB021306) Syntrophospora bryantii (M26491) Syntrophospora bryantii (M26491)

Bacteria Bacteria

Abundance Abundance Abundance Abundance of propionate‐‐oxidizing bacteria of propionate of propionate‐‐oxidizing bacteria of propionate oxidizing oxidizing bacteria bacteria

Conclusions Conclusions Conclusions Conclusions

) ) ) ) 100 100 100 100 Smithella Smithella sp. SR Smithella sp. SR Smithella

•• The The combination combination of of MAR MAR--FISH, FISH, micromanipulation micromanipulation and and cloning cloning

lls (% lls (% lls (% Smithella sp. LR Smithella sp. LR

analysis analysis can be can applied successfully be applied successfully for for identification identification of of

e e e e lls (% Syntrophobacter Syntrophobacter 80 c c 80 80 80 80 c 80 80 c 80 unidentified others unidentified others

Smithella sp. Smithella sp. SR SR

was major was major MAR+ cells at MAR+ cells at

propionate--oxidizing propionate oxidizing bacteria bacteria which which is is limiting limiting factor factor in in AD AD

A A R ta lM lM ta ta ta lM lM

A R+ + A R + R+

all all concentrations concentrations

microbial microbial community community..

Syntrophobacter Syntrophobacter was was

minor MAR+ cells at minor MAR+ cells at concentration esp. at low conc. concentration esp. at low conc.

•• The The diversity diversity of of propionate propionate--oxidizing oxidizing bacteria bacteria community community covered covered Smithella Smithella sp sp.. SR, SR, Syntrophobacter Syntrophobacter,, Smithella Smithella sp sp.. LR LR

60 60 60 60 60 60 60 60 all all

/ ls / ls / ls /T /T ls / /T /T

Smithella sp. Smithella sp LR Smithella Smithella sp LR sp. LR LR was major MAR+ cells at was was was major major MAR+ cells at major MAR+ cells at MAR+ cells at

and and unidentified unidentified bacterium bacterium..

el c c c el el el c 40 40 40 40 40 40 40 40 lower lower concentrations concentrations

hy hy b b hy b b

•• Members of Members of the the Smithella Smithella sp sp.. SR SR cluster cluster that that hybridized hybridized with with

hy

SmiSR SmiSR354 354 probe probe appeared appeared to to play play a major role a major role in in propionate propionate

ob e ob ob e e ob e 20 20 20 20 20 20 20 20

oxidation, that oxidation, that account account for for 50 50% % of of total total POB POB population population and and the the

most active most active POB POB at all at all concentrations concentrations..

pr A A R + +

pr pr pr R

0 0 0 0 0 0 0 0 5 5 5 5 5 5 5 5 10 10 10 10 10 10 10 10 15 15 15 15 15 15 15 15 •• These These phylogenetically phylogenetically and and functionally functionally diverse diverse and and active active POB POB

communities communities were were dynamically dynamically responding responding to to the the periodical periodical changes changes in in propionate propionate

Propionate concentration (mM) Propionate concentration (mM)

concentration concentration to to ensure ensure stable stable

propionate degradation propionate degradation..

CONCLUDING CONCLUDING CONCLUDING CONCLUDING REMARKS REMARKS REMARKS REMARKS

FUTURE FUTURE FUTURE FUTURE RESEARCH PERSPECTIVES RESEARCH PERSPECTIVES RESEARCH PERSPECTIVES RESEARCH PERSPECTIVES

•• Isolation Isolation and and charaterization charaterization of of active active POB POB to identify to identify their their •• Due Due

to to their their limited limited diversity diversity

and and

abundance abundance

both both

physiology physiology nature nature..

phylogenetically phylogenetically and and physiologically, physiologically,

fatty fatty

acids--utilizing acids utilizing

microorganisms microorganisms (esp (esp.. POB) POB) play play very very important important role role in in •• Population dynamics Population dynamics of of others others fatty fatty acids acids (acetate (acetate-- and and anaerobic anaerobic digestion digestion process process and and are are ‘key ‘key players’ players’ of of carbon carbon

butyrate butyrate--)) utilizing utilizing microorganisms microorganisms at at varied varied concentrations concentrations.. flow flow from from complex complex substances substances downs downs to to methane methane.. •• Phylogeny Phylogeny and and physiology physiology identification identification of of novel novel fatty fatty acids acids-- •• The POB The The The POB POB communities POB communities communities (esp communities (esp (esp (esp.. Smithella Smithella groups) Smithella groups) Smithella groups) groups) might be might might might be be be utilizing utilizing microbial utilizing utilizing microbial microbial groups microbial groups groups groups (e..g (e (e g (e g.. Synergistes Synergistes)) which Synergistes)) which g Synergistes which seemed which seemed seemed to seemed to to be to be be be proposed proposed to to become become a a ‘process ‘process biomarker’ biomarker’ in in a a multi multi--

important in important in completing completing fatty fatty--acids acids degradation degradation.. complexity of complexity of microbial microbial ecosystems ecosystems in in anaerobic anaerobic digestion digestion to to illustrate illustrate and and evaluate evaluate process process stability stability in in full full--scale scale anaerobic anaerobic

microbes microbes with with digesters treating digesters treating domestic domestic wastewater wastewater..

•• Symbiotic Symbiotic

of of

the the

fatty fatty

acids acids--utilizing utilizing

homoacetogenic bacteria homoacetogenic bacteria and and hydrogenotrophic hydrogenotrophic archaea archaea .. •• Auxiliary Auxiliary identification identification of of other other microbial microbial groups groups natured natured in in AD AD

(such (such as as Variovorax Variovorax group) group) which which is is unrelated unrelated with with methane methane generation generation but but seemed seemed to to affect affect its its performance performance and and stability stability..

‘Green’ ‘Green’ ‘Green’ ‘Green’ Anaerobic Anaerobic Anaerobic Anaerobic Digester System ( Digester System ( Digester System ( Digester System ( GR GR GR GR ANDS) ANDS) ANDS) ANDS)

Engine/Generator Engine/Generator

Gas Gas Collection Collection

Storage Lagoon Pump Lagoon Pump Aerator Aerator Storage

Digester heating Digester

Mixer Mixer

heating

Entrance Entrance Sludge Sludge

exit exit Effluent Effluent Sludge Sludge Re To Re To compactor ‐‐use/Land disposal compactor use/Land disposal

Thank you for your attention ! Thank you for your attention ! Thank you for your attention ! Thank you for your attention !

Mix Mix tank tank

ANAEROBIC ANAEROBIC

Gas Gas outlet outlet

Concepts Concepts

DIGESTER DIGESTER

Sludge Sludge inlet inlet

• • ‘Bio ‘Bio biogas biogas ‐‐control’ control’ for for maximum maximum • • ‘zero’ ‘zero’ production production

• Minimum • Minimum loss of energy loss of energy direct direct

controlling controlling monitoring

Digester Digester

• • Low Low monitoring costs of O & M costs of O & M

Insulation Insulation

Sludge Sludge outlet outlet

Heater Heater coil coil

SUSTAINABLE SUSTAINABLE SLUDGE SLUDGE

Gas Gas compressor compressor

TREATMENT TREATMENT

Power Power generator generator

Solids Solids outlet outlet

Introduction Introduction

Process Chemistry Process Chemistry

Strain LYP T cells :

-elongated, slightly sinuous rods -0.5 μm in dia. & 4-5 μm in length -Bodies: poly- β-hydroxyalkanoate -Cells stained Gram-negative -Walls : Gram (-) ultrastructure -Grew axenically on crotonate, but

not on butyrate, propionate, lactate,succinate,malate,fumarate oxalate, isobutyrate, isovalerate, valerate, caproate, pyruvate or

H 2 plus CO 2

Syntrophobacter wolinii :

-Similar dimension with Str.LYP T -Gram-negative -End of cells tapered to blunt point

Pathways of Propionate Degradation Pathways of Propionate Degradation

MAR MAR ‐‐FISH Experiment FISH Experiment Population composition of Population composition of

Propionate--oxidizing bacteria Propionate oxidizing bacteria

Syntrophobacter Syntrophobacter

Smithella Smithella sp. SR sp. SR

Smithella Smithella sp. LR sp. LR

Discussions

Discussions

CO 2

HCO 3 - +H +

2HCO - +H +

Sulfate Sulfate Reducers? (81%)

Acetoclastic reaction

CH 3 ┇ COO - +H 2 O

Æ CH 4 + HCO 3 -

Sekiguchi et al., 1999

Result–16S rDNA cloning analysis 9 Result–16S rDNA cloning analysis n=202 3% 7%

5 N r. of colony 10 15 48% 20

Result–16S rDNA cloning analysis Result–16S rDNA cloning analysis

N r. of colony

f speci

candidate division B R C 1 candidate division T M 7

A cidobacteria

candidate division N K B 19 A ctinobacteria

candidate division O D 1 B acteroidetes

PS-3

candidate division O P 9

C hloroflexi

V errucom icrobia

uncultured bacterium P roteobacteria

S pirochaetes

F irm icutes

M ethanom icrobiaceae M ethanosaetaceae

M ethanospirillaceae uncultured A rchaea

N r. o f colony

0 20 40 N r. of colony 60 80 100

Bacteriodetes

Desulvothiovibrio Spirochaetes TM 7

M ethanobacteriaceae

uncultured Archaea M ethanom icrobiaceae

M ethanosaetaceae

Acidobacteria

Beta-Proteobacteria Delta-Proteobacteria Actinobacteria

PS-2

M ethanospirillaceae

PS-4

Firm icutes Alpha-Proteobacteria

c a n d id a te d iv isio n W S 3 c a n d id a te d iv isio n O P 8 A c id o b a c te ria c a n d id a te d iv isio n O P 9 F irm ic u te s T h e rm o to ga e A c tin o b a c te ria V e rru c o m ic ro b ia

P ro te o b a c te ria u n c u ltu re d b a c te riu m c a n d id a te d iv isio n O D 1 B a c te ro id e te s

M ethanospirillaceae M ethanom icrobiaceae M ethanosaetaceae uncultured A rchaea

OP 11 14 Therm otogae 12 Planctom ycetes

OP 8 OP 5

e c Chloroflexi/GNSB 10 n=25

10 12 8 6 4 2 Nr

. of peci es

N r. o f c olon y

PS-3 = A25f – A1391r

8% 2% Nr .o ies

4 2 0 0 6 8 10 20 N r. of colony 30 f sp 40 50 60

PS-4 = A25f – U1492r PS-5 = A109f – U1492r

PS-1 = B8f – U1492r

PS-2 = B63f – B1387r

PS-5

PS-5

PS-5 = A109f – U1492r

c a n d id a te d iv isio n O P 1 1 C h lo ro fle xi

u n c u ltu re d b a c te riu m c a n d id a te d iv isio n T M 7

M ethanobacteriaceae M ethanospirillaceae

uncultured A rchaea M ethanom icrobiaceae M ethanosaetaceae

Strategy applied Strategy applied

Anaerobic Digestor DNA

16s rDNA

ecosystem

Total DNA

Ebetsu Sludge

Cloning

Overview Sequence ecosystem Of

sequences 16s rDNA

Sequencing

16s rDNA clones

Analysis

Anaerobic Digestor DNA ecosystem

amplificates Extraction 16s rDNA

Total DNA

PCR

SIMPLE

Semi Batch Reactor Sludge

Cloning

Overview Sequence ecosystem Of

sequences 16s rDNA

Sequencing

16s rDNA clones

Analysis