Mixed culture biotechnology

for production of bioplastics

Katja Johnson
Robbert Kleerebezem
Mark van Loosdrecht

D lft U
Delft i it off Technology,
University T h l D t t off Biotechnology
Department Bi t h l
The Netherlands
 Outline

¾ Plastics / bioplastics
¾ Mixed culture biotechnology
¾ How to produce bioplastic with mixed culture
biotechnology
¾ Comparison of pure and mixed culture
bi t h l
biotechnology ffor bi
bioplastic
l ti production
d ti
 Plastics
245 million tons per year (2006)*
CO2

• Light
• Versatile
• Cheap
• Durable

* Source: PlasticsEurope
 Plastics in the environment
• “Marine trash, mainly plastic, is killing more than a million
seabirds and 100 000 mammals and sea turtles each
year” (U.N. Secretary-General Kofi Annan, 2004)
• Microscopic plastic fragments and fibers are present in
oceans and sediments; Amount of plastic in plankton
samples tripled from 1960s to 1990s (Thompson et al. Science
304 (5672):838, 2004)
 Bioplastics
0.3 million tons per year (2005)*
CO2

• Light
• Versatile
• Less cheap
• Less durable
• Sustainable

* Source: European Bioplastics

 Types of bioplastics

(Petrochemical based)
• (Petrochemical-based)

• Starch-based

• Cellulose-based

• Polylactic acid (PLA)

• Polyhydroxyalkanoate (PHA)
 Polyhydroxyalkanoate (PHA)

¾ Monomers and chain length

determine physical properties

(Lee et al. 1996)

 Polyhydroxyalkanoate (PHA)
• Common microbial storage polymer (C, energy)
• Stored inside cell in granules
• Often produced under imbalanced /
dynamic growth conditions
• Up
U tto > 80 wt%
t% off cellll d
dry weight
i ht
• Most common form is PHB, but
also copolymers e.g. P(HB-HV) (Jendrossek et al. 2002)
 PHA production
• Pure bacterial cultures
– Natural PHA producers
– Mutants / engineered natural producers
– Recombinant bacteria (e
(e.g.
g E.coli)
E coli)

• Transgenic plants
– E.g. switchgrass

• Mixed bacterial cultures

 Mixed culture biotechnology

Open,
p undefined mixed cultures

• Waste water treatment

• Biogas production

• Composting

• Traditional fermented foods

 Mixed culture biotechnology
“Ecosystem Engineering”:

Explore the microbial diversity available in

nature
t by
b iimposing
i a selective
l ti pressure on a
natural ecosystem for the production of
chemicals and energy

= engineering
g g the environment rather than the
organism to favour a certain metabolism
 How does it work?
• Inoculum: e.g. sludge from wastewater
treatment plant
• Open (non-sterile) reactor
• Application of selective pressure for desired
metabolism via process parameters, e.g.
– Feeding regime (dynamic or continuous)
– Aerobic or anaerobic conditions
– Temperature, pH
– Culture residence time
– Substrate
 Selection for PHA producers
• Dynamic supply of substrate (feast/famine)
Substrate

PHA storing
i b bacteria
i

not PHA storing

bacteria
 Selection for PHA producers
¾ PHA storing bacteria prevail in feast/famine
system
¾ Competitive advantage:
– Higher specific substrate uptake rate under
dynamic conditions
– More balanced growth
g
 PHA production strategy

1 Cultivation: selection and growth

of PHA producing mixed culture 2 PHA production: maximizing
the cellular PHA content

Fatty acids Fatty acids

(acetate) (acetate)

Sequencing Fed batch Biomass

batch reactor reactor + PHA
feast/famine

Biomass
 1. Cultivation

Reaction phase
Start phase
feast / famine

Filling phase

Sludge
Effluent phase withdrawal
ithd l

Settling phase
Sequencing batch reactor
Operational
p cycle
y e.g.
g 4h
 1. Cultivation
Feast Famine
100 10

SBR p
phases: 90 9

N/l],PHB [%]
0-10 Start 80 8

10-13 Filling 70
DO PHB Acetate Ammonium
7

ol/l] ,NH4 [mg N

13 216
13-216 Reaction 60 6
DO [%]

216-218 Sludge 50 5

218-233 Settling 40 4

Ac [mmo
30 3
233-240 Effluent
20 2

10 1

0 0
0 20 40 60 80 100 120 140 160 180 200 220 240
Time [min]
 2. PHA production
Feast
100

90 DO Acetate PHB
DO [%] , Ac [mmol/l] , PHB [%] 80

70

60

50

40

30

20

10

0
0 120 240 360 480 600 720 840 960 1080 1200 1320 1440
Time [min]
 Advantages mixed cultures
+ Cheap substrates: (fermented) waste streams
 Advantages mixed cultures
+ Cheap substrates: (fermented) waste streams
+ Cheap equipment (open process)
 Advantages mixed cultures
+ Cheap substrates: (fermented) waste streams
+ Cheap equipment (open process)
+ Low energy costs (no sterilisation)
 Advantages mixed cultures
+ Cheap substrates: (fermented) waste streams
+ Cheap equipment (open process)
+ Low energy costs (no sterilisation)
+ Continuous process scheme possible

PFR

CSTR Batch
 Advantages mixed cultures
+ Cheap substrates: (fermented) waste streams
+ Cheap equipment (open process)
+ Low energy costs (no sterilisation)
+ Continuous process scheme possible
+ PHA contents comparable to pure cultures
 Advantages mixed cultures
+ Cheap substrates: (fermented) waste streams
+ Cheap equipment (open process)
+ Low energy costs (no sterilisation)
+ Continuous process scheme possible
+ PHA contents comparable to pure cultures
+ Higher rates of PHA production
 Advantages mixed cultures
+ Cheap substrates: (fermented) waste streams
+ Cheap equipment (open process)
+ Low energy costs (no sterilisation)
+ Continuous process scheme possible
+ PHA contents comparable to pure cultures
+ Higher rates of PHA production
+ Non-GMO
 Disadvantages mixed cultures

- Only common / natural PHAs

- More difficult to influence polymer composition
- Possibly higher downstream processing costs
 Conclusions

• Mixed culture biotechnology might be a cost

efficient and sustainable alternative for

bioplastic production

• Products from waste

• Optimisation of process has only just begun…