Biolms 5 - Multispecies biolms

Examples of multispecies biolms Microbial competition in chemostats Microbial competition in biolms 2D/3D modelling of multispecies biolms

Most biolms are multispecies

Any biolm from an industrial system will contain a very wide range of microorganims able to grow under an extreme range of environmental conditions of: - temperature - pH - salinity - nutrients - toxicity - water activity

Examples:

phototrophic biolms oral biolms industrial biofouling mixed species biolms for wastewater treatment (e.g. nitrifying biolms)

Phototrophic biolms

Commonly in

submerged surfaces natural aquatic systems driven by sun light

The community is
freshwater biolm on sedimentation tank (wasterwater treatment) seawater biolm on polypropilene slides

Multispecies phototrophic community

photosynthetic microbes produce organic material and oxygen that sustains the growth of other (heterotrophic) organisms As a result, a layered organization is observed
Cyanobacteria (CYA); Colorless Sulfur Bacteria (CSB), Purple Sulfur Bacteria (PSB), Sulfate reducing bacteria (SRB), and Methanogens (MET)

Phototrophic biolms such as algal mats are extremely important for nutrient cycle in ecosystems

Layered structure in microbial mats

Algal mats are suitably modelled with 1D models

Oral biolms

Dental plaque develops naturally in our teeth The composition of the community is stable (microbial homeostasis) Homeostasis may break down due to the repeated intake of fermentable sugars. In periodontal diseases, there is a shift in the composition of the plaque microora to a more anaerobic community, which can induce damage to tissues.

Arrangement of species

Kolenbrander, P.E. and J. London, Adhere Today, Here Tomorrow - Oral Bacterial Adherence. Journal Of Bacteriology, 1993. 175(11): p. 3247-3252.

Mixed-species biolms in waste water treatment

Waste treatment processes are not in aseptic conditions. All these biolms are multispecies consortia

Trickling lters Biolm airlift reactors Activated sludge ocs Aerobic and anaerobic granular sludge

Equations describing microbial competition

QV, Sin
Influent

Equation for monospecies growth

QV, S, X

V X,S

efluent

dX S = max X DX dt S + KS dS 1 S = max X D Sin S dt Y S + KS

biomass

substrate (nutrients) - The steady state D S = KS max D X = Y Sin S

QV, Sin
Influent

QV, S, X1, X2 V X1, X2, S

efluent

dX1 S = max,1 X1 DX1 dt S + K S,1 dX 2 S = max,2 X 2 DX 2 dt S + KS dS 1 S 1 S = max,1 X1 max,2 X 2 D Sin S dt Y1 S + K S,1 Y2 S + K S,2

With the same: max KS Y

S X1 X2

X1 S X2 (X2 wash-out) and the same: K S, Y

with:

max,1 > max,2

Multispecies modelling in biolms

Multidimensional (2D or 3D) modeling of biofilms - spreading the biomass liquid

x

X1

X2

Spreading of the biomass

biofilm
carrier

z y

The problem with a single species:

How does the biofilm front advance?

The problem with more species:

How do species advance within each other?

Based on a discretization of the biolm along a vertical direction. Each layer is modelled as "compartment" in which local composition in multi-species are modelled:

solid surface

X2 X1
Liquid Biofilm

fi 1 g i = ifi t i z
fi is the fraction of biomass i in that compartment gi is the ux of biomass i, resulting from biomass production or consumption

Orthogonal grid discretization. Each grid node belongs to one of the species and grows following species specic kinetics
S X1 t + t = X1 t + t max,1 X1 t S + K S,1

(
(

()

()

S X 2 t + t = X 2 t + t max,2 X2 t S + K S,2

()

()

When biomass divides it is placed at the end of a random walk This is counter-intuitive and produces unrealistic mixing of species

cell dividing placement of daughter cell

X1

X2

Round biomass particles moving in continuous coordinates. Like in CA: Each particle node belongs to one of the species and grows following species specic kinetics

solid surface

Biofilm

(A) before division

When a particle devides the daughter is placed locally And the neighbors are shoved

This produces clusters of species

(B) after division

X1 X2

The new model of Erik Alpkvist uses biomass fraction fi as in the Dockery and Klapper 1D model Each position in space contains all species but in different fractions

solid surface

f1

f2

NO3 O2
Aerobic nitrite oxidizers

NO2 O2
Aerobic ammonium oxidizers

Inert biomass

NH4

Picioreanu, C., J.U. Kreft, and M.C.M. Van Loosdrecht, Particle-based multidimensional multispecies model. Appl Environ Microbiol, 2004. 70(5): p. 3024-3040.

Xavier, J.B., C. Picioreanu, and M.C. van Loosdrecht, A framework for multidimensional modelling of activity and structure of multispecies biolms. Environ Microbiol, 2005. 7(8): p. 1085-103.

Aerobic granular sludge

Aerobic granule reactors:

Kluyver Laboratory for Biotechnology

! Well settling sludge; ! Small surplus sludge production; ! Small area requirement; ! Use instead of present activated sludge systems;

de Kreuk et al 2004

Introduction Granulation Conversions Full-scale?Conclusions

Example of a present wastewater treatment system

Activated sludge system with presettling, bio-P and sludge digestion

Kluyver Laboratory for Biotechnology

Pre-treatment

Selector

Influent

Aerobic tank

Denitrification

Anaerobic tank

Effluent
Sludge Digester

CH4
Sludge Dewatering

Sludge

de Kreuk et al 2004

Introduction Granulation Conversions Full-scale?Conclusions

Example of a wastewater treatment system with the granule SBR

Granular reactor system with presettling and sludge digestion

Kluyver Laboratory for Biotechnology

Pre-treatment

Effluent

Influent

SBR

CH4
Sludge Digester Sludge Dewatering
Sludge

de Kreuk et al 2004

Introduction Granulation Conversions Full-scale?Conclusions

Goals of the research:

Kluyver Laboratory for Biotechnology

! Understanding of aerobic granulation without carrier material; ! Optimisation of simultaneous N, P and COD removal; ! Full-scale design and feasibility of the granular reactor process in practice

de Kreuk et al 2004

IntroductionGranulation Conversions Full-scale?Conclusions

FILL REACT
Kluyver Laboratory for Biotechnology

IDLE
influent 60 min. 1 min. Cycle 5 min. 3 min. effluent 121 min. ai r

DRAIN
de Kreuk et al 2004

SETTLE

IntroductionGranulation Conversions Full-scale?Conclusions

Criteria for granular growth

Kluyver Laboratory for Biotechnology

! Shear ! Selection

de Kreuk et al 2004

IntroductionGranulation Conversions Full-scale?Conclusions

Kluyver Laboratory for Biotechnology

Filaments and small particles (low density)

Mixture of filaments and granules

Effluent

Granules

Aeration
de Kreuk et al 2004

Settling
IntroductionGranulation Conversions Full-scale?Conclusions

Criteria for granular growth

Kluyver Laboratory for Biotechnology

! Shear ! Selection ! G-number (Picioreanu et.al, 1998 ): Ratio between the biomass growth rate and the substrate conversion rate !feeding phase

de Kreuk et al 2004

IntroductionGranulation Conversions Full-scale?Conclusions

Kluyver Laboratory for Biotechnology

Long anaerobic feed: ! Acetate conversion to PHB; ! Selection of PAO (low maximum growth rate);

Acetate concentrat io

low G-number: smooth and dense granule

Inuent de Kreuk et al 2004

IntroductionGranulation Conversions Full-scale?Conclusions

Development of the granules

Kluyver Laboratory for Biotechnology

Day 4

Day 13

Day 87 de Kreuk et al 2004

Day 346

IntroductionGranulation Conversions Full-scale?Conclusions

Conversion processes in the granule

Layered structure
Kluyver Laboratory for Biotechnology

Concentration

Heterotrophs: Aerobe Anaerobe

surfa ce

O
2 PH

NO
x

Autotrophs de Kreuk et al 2004

Penetration depth
IntroductionGranulation Conversions Full-scale?Conclusions

Experimental set-up
mfc air in

Kluyver Laboratory for Biotechnology

CO2-analyser air out Offgas recirculation Air mixing vessel air in

mfc Membrane pump mfc

Settling height

N2 gas in

downcomer effluent

riser
pH-electrode + pH control

DO-electrode

influent (acetate)

SBAR 2 SBBR1 & SBAR R 2 ecycle off-gas

de Kreuk et al 2004

IntroductionGranulation Conversions Full-scale?Conclusions

Conversions in the reactor

Kluyver Laboratory for Biotechnology

Full COD removal

7.0 Acetate (C-mmol) 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0 50 100

Acetate PHB

2.0 1.5 %HB 1.0 0.5 0.0

150

time (minutes)

IntroductionGranulation Conversions Full-scale?Conclusions

Conversions in the reactor

Kluyver Laboratory for Biotechnology

! Full COD removal ! Full phosphate removal

4 4 3 3 2 2 1 1 0 0 50 100 Time (min.) 150

PO4-P (mmol/l)

IntroductionGranulation Conversions Full-scale?Conclusions

Conversions in the reactor

Kluyver Laboratory for Biotechnology

! Full COD removal ! Full phosphate removal ! 61% - 98 % N-removal

N-removal (85.3%), 40% DO 1.5 NH4/NOx (mmol/l) 1.0 0.5 0.0 0 50 100 Time (min.) 150 NH4 NO2 NO3

IntroductionGranulation Conversions Full-scale?Conclusions

Multiscale model of the reactor

Kluyver Laboratory for Biotechnology

de Kreuk et al 2004