Granular biofilms: their generation and application in environmental biotechnology

Venugopalan VP, Nancharaiah YVVenugopalan VP, Nancharaiah YV

Biofouling and Biofilm Processes SectionBiofouling and Biofilm Processes SectionggWater and Steam Chemistry DivisionWater and Steam Chemistry DivisionBARC Facilities, Kalpakkam 603 102BARC Facilities, Kalpakkam 603 102

23 February 2008 1

Structure of the talkG l bi filG l bi filGranular biofilmsGranular biofilmsCultivationCultivationSequencing Batch ReactorsSequencing Batch ReactorsDevelopmentDevelopmentDevelopmentDevelopmentStructureStructureP t ti l li tiP t ti l li tiPotential applicationsPotential applications

23 February 2008 2

Biofilms

SubstratumSubstratum--associated microbial associated microbial communities, bound by EPS matrixcommunities, bound by EPS matrix

Granular biofilmsGranular biofilms

Dense, spherical, selfDense, spherical, self--immobilised immobilised i bi l ti h ld t th bi bi l ti h ld t th bmicrobial consortia, held together by microbial consortia, held together by

EPS, without any carrier substratumEPS, without any carrier substratum

23 February 2008 3

Aerobic microbial granules ...

.... Granular sludge g

.... Particulate biofilms.... Particulate biofilms

23 February 2008 4

Aerobic microbial granules:Aerobic microbial granules: characteristics

First reported by Mishima and First reported by Mishima and Nakamura (1991)Nakamura (1991)Nakamura (1991)Nakamura (1991)Smooth and round morphologySmooth and round morphology

High settling velocity (30High settling velocity (30--70 m/h)70 m/h)

High specific gravity (1 004 to 1 065)High specific gravity (1 004 to 1 065)High specific gravity (1.004 to 1.065)High specific gravity (1.004 to 1.065)

23 February 2008 5

Granular biofilms:-advantages in wastewater treatment

High biomass retentionHigh biomass retentionExcellent settleability (high settling velocity)Excellent settleability (high settling velocity)Stable rate of metabolism (due to Stable rate of metabolism (due to syntrophic associations)syntrophic associations)Resilience to shocks (due to protection by Resilience to shocks (due to protection by matrix)matrix)Good storage stabilityGood storage stabilityPossibility for bioaugmentationPossibility for bioaugmentation

23 February 2008 6

Possibility for bioaugmentationPossibility for bioaugmentation

Granular biofilm: development(Weber et al, AEM 2007)

Activated sludge (seed material) Activated sludge (seed material)

St lk d t fl & fSt lk d t fl & fStalked protozoans grow on flocs & from Stalked protozoans grow on flocs & from treetree--like colonieslike colonies

Ciliates are overgrown by bacteriaCiliates are overgrown by bacteria

Smooth compact granules are formed onSmooth compact granules are formed onSmooth compact granules are formed on Smooth compact granules are formed on the frameworkthe framework

23 February 2008 7

Transformation of activated sludge to granulesTransformation of activated sludge to granules

Fl l t biFlocculent biomass

Highly filamentous

GranulationGranulation

Sequencing Batch Reactor

Granules

Inoculum (activated sludge flocs)

23 February 2008 8

SVI: ~180 ml/g SVI: 40 ml/gSettling velocity: ~70 m/h

Cultivation of aerobic granules

hydrodynamic shear force and

t

Selecting fast settling granules Vs. slow settling flocs

oxygen stressg

formation of suspendedRepetitive “feast and formation of suspended biofilm aggregates from

flocculent sludgefamine” condition

23 February 2008 9

Reactor with completely granular biomass

Microbial granules cultivated in lab scale SBRMicrobial granules cultivated in lab scale SBR

23 February 2008 10

23 February 2008 11

Activated sludge process – continuous flow system

influenteffluent

O2effluent

excessinternal recirculation

excesssludgeReturn sludge

separate vessels for separate processes

23 February 2008 12

Sequencing Batch Reactor (SBR)modified activated sludge process- modified activated sludge process

influentSBR-cycle

influent

effluent

O2

effluent

excessl d

aerate settlemixfill decantsludge

• sequence of processes separated along the time axis

• one vessel for all processes

23 February 2008 13

one vessel for all processes

Aerobic granulation in SBRAerobic granulation in SBRgg

In an SBR, microbes are In an SBR, microbes are a S , c obes a ea S , c obes a esubjected to a periodic subjected to a periodic operational cycleoperational cycle Aeration &Feeding ReactFeeding

Decant Settling

23 February 2008 14

Typical SBR Cycle

PeriodPeriod Duration (min)Duration (min)

Fill (no mix, anaerobic)Fill (no mix, anaerobic) 6060

Aeration Aeration 282282

Settling Settling 33Settling Settling 33

Decant Decant 1010

IdleIdle 55

Total cycleTotal cycle 360360

23 February 2008 15

“Feast-and-famine“ cycle

25 25

20 20

DO

temperature

10

15

2] m

g /l

10

15

T (°

C)

ANAEROBIC AEROBIC SETTLEDRAW

FILL

0

5

[O2

0

5

T

-5

00 50 100 150 200 250 300

t (min)

-5

0

23 February 2008 16

t (min)

Granular sludge settling characteristicsGranular sludge settling characteristics

5 sec 30 sec 60 sec 180 sec

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SBR bank used forSBR bank used for culturing microbial granules

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granules

25 litre SBR for bi l tibiogranulation experiments

23 February 2008 19

Factors influencing biogranulationg g

Reactor configuration (column type, Reactor configuration (column type, upflowupflow))Substrate composition (carbon source)Substrate composition (carbon source)Organic loading rate (2Organic loading rate (2--15 kg/m15 kg/m33 COD)COD)Hydrodynamic shear force (mnm. SAV of Hydrodynamic shear force (mnm. SAV of y y (y y (1.2 cm/s reported)1.2 cm/s reported)Settling time (a major selection pressure)Settling time (a major selection pressure)Sett g t e (a ajo se ect o p essu e)Sett g t e (a ajo se ect o p essu e)Hydraulic retention timeHydraulic retention timeAerobic starvation (feastAerobic starvation (feast andand famine cycle)famine cycle)

23 February 2008 20

Aerobic starvation (feastAerobic starvation (feast--andand--famine cycle)famine cycle)

Aggregation in bacteriagg gAutoAuto--aggregationaggregation

CoCo--aggregationaggregation

23 February 2008 21

Simple aggregation assaySimple aggregation assay

Decrease in OD as an indication of aggregationDecrease in OD as an indication of aggregation

1.1

0.8

0.9

1.0

600 nm

0 5

0.6

0.7

sorb

ance

at

0.3

0.4

0.5

Res

idua

l abs

0 cm sec-1

0.5 cm sec-1

1.0 cm sec-1

1

0 1 2 3 4 180.1

0.2

Time (h)

1.5 cm sec-1

2.0 cm sec-1

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Time (h)

Granulation at low SAVGranulation at low SAVSuperficial up-flow airflow velocity: 0.5 cm/sec

GranulesFlocs

23 February 2008 23

A B

0.4 cm/s

DC

0.8 cm/s

23 February 2008 24

after 5 days after 90 days

D i l b i l i l f l ?Do single bacterial strains also form granules?

YES

Upflow reactor, 0.5 l volumeAfter 7 da s

Pure culture (AG08)

23 February 2008 25

After 7 days

Role of AHL (QS molecules)

1 More granules per reactor

in aerobic granule formation

1.More granules per reactor2.Smaller granules3.Better circularity

10 nM BHL 1µM BHLy

No BHL

23 February 2008 26

StructureStructureDense core zone

Loose fringe zoneLoose fringe zone

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Internal architecture of granules

xy-projection xy-slice

23 February 2008 28Mostly rod or cocci shaped bacteria

Granule: optical sectioning usingGranule: optical sectioning usingconfocal microscope

23 February 2008 29

Aerobic granular sludge: potential applications

Biodegradation of nitrilotriacetic acidg

Biodegradation of tributyl phosphate

Biosorption of heavy metals

23 February 2008 30

Free NTA degradation

0.8

0.9 Cycle 1 Cycle 2Cycle 3

0.5

0.6

0.7

(mM

)

y Cycle 4

0 2

0.3

0.4NT

A

0 2 4 6 8 10 12 14 160.1

0.2

Time (Hours)

23 February 2008 31

Free and Fe(II)-NTA biodegradation

1 6

1.8

2.0

2.2

Test Control

1 6

1.8

2.0

2.2

Test Control

1.0

1.2

1.4

1.6

NTA

(mM

)

0 8

1.0

1.2

1.4

1.6

NTA

(mM

)0.2

0.4

0.6

0.8N

0.2

0.4

0.6

0.8

0 2 4 6 8 10 12 14 16 18

Time (Hours)

0 10 20 30 40 50 60 70 80

Time (Hours)

23 February 2008 32

TBP biodegradation by microbial granules

3.0

TBP (mM)

350

2 0

2.5( )

Phosphate (µg/L)

mM

250

300

1.5

2.0

TBP

in m

150

200

Phosphat

1.0

Res

idua

rl

100

te (ppb)

0.5

R

0

50

23 February 2008 33

0 10 20 30 40 500.0

Time (hours)

Cr (VI) removal by granules

0.2 mM

Cr (VI) removal by granules

2.5

3.0

(mM

)

0.4 mM 0.6 mM 0.8 mM 1 mM

1.5

2.0in

g C

r (V

I) ( 1.5 mM

2 mM 3 mM

0.5

1.0

Rem

ain

0 2 4 6 8 10 12 140.0

Time (d)

23 February 2008 34

Cr (VI) removal by granulesCr (VI) removal by granules

0.15

0.20

) (m

M) 5 g

10 g 15 g20 g

0.10m

aini

ng C

r (V

I 20 g Blank MM

0 1 2 3 4 5 60.00

0.05Re

Time (d)

23 February 2008 35

Biosorption of uraniumBiosorption of uranium

120

130

140

150

6 ppm 10 ppm 50 ppm100 ppm

7080

90

100

110

um (m

g/l)

100 ppm 140 ppm

20

30

40

50

60

Ura

niu

0 1 2 3 4 240

10

Time(h)

23 February 2008 36

BioaugmentationBioaugmentation- advantages -

Enhancement of degradative abilityEnhancement of degradative abilityReduction in reactor startReduction in reactor start--up timeup timepp

23 February 2008 37

Bioaugmentation of granulesBioaugmentation of granules

Do cultured strains colonize and integrate Do cultured strains colonize and integrate with mixed species consortia?with mixed species consortia?

Can they effectively transfer their plasmid to Can they effectively transfer their plasmid to granules?granules?gg

Can improved degradation pathway be Can improved degradation pathway be established through horizontal gene transferestablished through horizontal gene transferestablished through horizontal gene transfer established through horizontal gene transfer to enhance biodegradation?to enhance biodegradation?

23 February 2008 38

BIOAUGMENTATION:In situ monitoring of gene transferIn situ monitoring of gene transfer

Live cell marker genes Excitation λ Emission λ

Green fluorescent protein (Gfp)

488 nm BP 515-545 nm

Red fluorescent protein (D R d)

543 nm LP 570 nm

NalR×

(DsRed)

DsRedTcR

GFP,KmR

NalR

R D

ChromosomePlasmid (pWWO) R T D

23 February 2008 39P. putida KT2442::dsRed NalR TcR pWWO::gfpmut3b KmR

Gene transfer in biofilms throughGene transfer in biofilms through conjugation: in situ monitoring by CLSM

DsRed

GFP

23 February 2008 40

Bioaugmentation using TOL plasmid….. … for more efficient biodegradation

25 µm25 µm

23 February 2008 41

Blue = syto 60; pink = donor Yellow = donor; green = transconjugant

Bioaugmentation in lab reactorsEffect on benzyl alcohol degradationEffect on benzyl alcohol degradation

4.0

4.5

5.0

)

4.0

4.5

5.0

2.0

2.5

3.0

3.5

yl a

lcoh

ol (m

M)

2.0

2.5

3.0

3.5

al a

lcoh

ol (m

M)

0 0

0.5

1.0

1.5

Benz

y

Cycle 1 Cycle 3 Cycle 5 Cycle 7 Cycle 9 0.5

1.0

1.5

Ben

zy

Cycle 1 Cycle 3 Cycle 5 Cycle 7Cycle 9

0 1 2 3 4 5 6 7 80.0

Cycle time (h)0 1 2 3 4 5 6 7 8

0.0

Cycle time (h)

Cycle 9

23 February 2008 42

Areas of interest / future work

Bioaugmentation of granules using GMOsBioaugmentation of granules using GMOsMi bi l it t t f lMi bi l it t t f lMicrobial community structure of granules Microbial community structure of granules (molecular methods)(molecular methods)Role of cellRole of cell--cell interactions in biogranulation cell interactions in biogranulation Role of ROS and PCD in granule architecture Role of ROS and PCD in granule architecture developmentdevelopment

23 February 2008 43

Conclusions

Granular biofilms are easy to cultivate Granular biofilms are easy to cultivate using SBRsusing SBRsusing SBRsusing SBRsThey have useful characteristicsThey have useful characteristicsAmenable to bioaugmentationAmenable to bioaugmentationPromising biotechnological applicationsPromising biotechnological applicationsg g ppg g pp

23 February 2008 44

Thank YouThank YouThank YouThank You

23 February 2008 45

CONFOCAL LASER SCANNING MICROSCOPE

23 February 2008 46

45 Days

Aerobic granules formation by Aerobic granules formation by marine bacteriamarine bacteriamarine bacteriamarine bacteria

23 February 2008 47

SBR – operating strategies

tcycle

Vreactor

Vfill

fill time ratior = tfill / t l

tfill

volumetric exchange ratiorfill = tfill / tcycle volumetric exchange ratio VER = Vfill / Vreactor

C l d hi h VERCS low rfill and high VER

high rfill and high VER

23 February 2008 48

tcycle

SBR – operational limitations

20 cm

Vfill, maxClarified effluent

20 cm

Sludge volume gafter sedimentation

Maximum Vfill is determined byfill y

• sludge settling characteristics

• maximum conversion capacity

23 February 2008 49

maximum conversion capacity