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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
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
23 February 2008 17
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
23 February 2008 22
Time (h)
Granulation at low SAVGranulation at low SAVSuperficial up-flow airflow velocity: 0.5 cm/sec
GranulesFlocs
23 February 2008 23
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
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
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
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