Fermentation Production of Polyhydroxyalkanoates

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Fermentation Production of Polyhydroxyalkanoates

• Ronny Purwadihttp//www.adm.hb.se/rpu
• E-mail ronny.purwadi_at_hb.se

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Introduction
Biodegradable plastics
No net additional CO2 to atmosphere
Non-biodegradable plastics
Sustainable system
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History of Polyhydroxyalkanoates
1900
1950
2000
Lemoigne (1927)Found 3-hydroxybutyric acid as an
exertion of Bacillus megaterium
Macrae and Wilkinson (1958) Function of
P(3HB) Observed B. megaterium stored a
homopolymer when glucose-to-nitrogen source ratio
was high. ? PHA was a carbon- and energy-reserve
material
Imperial Chemical Industries (ICI)
(1976) Investigation of the commercialization of
PHA by bacterial fermentation
Oil crisis (1973) Challenge the search of
alternative type of plastic materials which is
independent to fossil fuel as a raw material.
To date About 91 most possible PHA
structures. About 300 different bacteria
producing PHA. Has been commercially produced by
Monsanto, ZENECA, Chimie Linz etc.
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Polyhydroxyalkanoate (PHA)
Poly(3-hydroxybutyrate) or P(3HB)
Poly(3-hydroxyalkanoate) with Ralkyl or
functional groups
Poly(3-hydroxypropionate)
Poly(3-hydroxyvalerate) or P(3HV)
PHASCL short-chain length PHA 3-5 carbon
atoms PHAMCL medium-chain length PHA 6-14
carbon atoms
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Polyhydroxyalkanoate (PHA)
-co-
3-hydroxybutyrate
3-hydroxyvalerate
The copolymer has enhancement in physico-chemical
properties
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Biosynthesis of PHA

• Function of PHA production
• Carbon- and energy storage
• Sink for reducing power

• Conditions for production
• Excess carbon source
• Limited growth nutrient except carbon source

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Biosynthesis of PHA
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Properties of PHA
Adopted from http//www.metabolix.com
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Micro-organisms
Two types of PHA producing micro-organisms
R. eutropha, Protomonas oleovorans
A. latus, Azobacter vinelandii, recombinant E.
coli
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Recombinant micro-organism
Native bacteria
PhaC
Glucose
Acetyl-CoA
3-hydroxybutyril-CoA
PHA
PhaA PhaB
PhaZ
PhaA PhaB
PhaC
No degradation of PHA results higher yield
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Substrates
Corn, sugar cane, wheat, potato, tapioca etc
Glucose
Carbohydrate
Agriculture, Industrial and Municipal waste
Alkanoates (propionic acid, butyric acid,
valeric acid etc.)
Vegetable oil,and fats
Fatty acids
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Fermentation modes
Fed-batch
1. Introduce an aliquot part of nutrients for
cell cultivation. With appropriate nitrogen and
carbon source, adequate cell density will be
obtained.
N
C
2. Controlled addition of limited growth nutrient
except carbon.The cells convert carbon source
into PHA
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Fermentation modes
Multi-stages Continuous Cultivation

• First fermenter
• biomass production
• aliquot part of nutrients.
• Second fermenter
• biosynthesis of PHA
• limited nutrients except carbon source.
• Non-assimilated nutrients are sended back to the
  second fermenter.

Using additional multi-stages fermenter following
second fermenter increase the time of exposure
for bacteria to conditions favorable for polymer
accumulation
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PHA production in specific bacteria

• R. Eutropha
• Content 76, cell conc. 121 g/L, prod. 2.42 g/L.h
  (fed-batch)
• Content 72.1, yield 0.36 g/g, prod. 1.23 g/L.h
  (cont.cult.)
• A. latus ATTC 29713
• Content 63, yield 0.4 g/g, prod. 1.15 g/L.h
  (fed-batch)
• Pseudomonas sp.
• cell conc. 11.6 g/L, prod. 0.58g/L.h.
  (cont.cult.)
• Recombinant E. coli
• Content 80-90, cell. conc.gt 80 g/L, prod. gt 2
  g/L.h (fed-batch)
• Applicable for cheap substrates
• Need acetyl-CoA sources, more oxygen, stable
  plasmid

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Anaerobic digestion of biological wastes

• Run in dynamic condition
• Change in concentration and kind of substrates
• Mixed culture
• Contain various bacteria which work symbiotically
  (i.e. activated sludge)
• Biomolecules ? (small) fatty acid ? PHA
• Content 20 (62 in microaerophilic-aerobic
  cond.)
• Advantages
• Simple process control, improve the use of waste,
  enhance economy

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Product Separation

• Solvent Extraction
• Use solvents chloroform, methylene chloride,
  propylene chloride, dichloroethane.
• High amount of solvent (201) ? high cost
• Hypochlorite salt cheaper but cause degradation
• Hypochloritechloroform purity 95
• Cellular component removal
• Use enzymes lisozyme, proteinases, DNAses
• Cell derbits is solubilized but not PHA
• Sodium hydroxide for cells with fragile cell
  walls (recombinant E. coli and A. vinelandii)

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Application Commercialization

• Applications
• Household, packaging film, coating, etc.
• Precursor of optically active compound synthesis
• Biodegradable carrier in drug delivery processes
• Osteosynthetic materials
• Commercialization
• ZENECA production capacity of 1000 tones/annum.
• Metabolix 100 g/L in 40 hours with content of
  90 at the end of cultivation. Production cost
  US1.00 per pound

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Conclusion

• PHAs are a wide range biopolymers
• Properties, feedstock, applications
• Produced by various bacteria as well as its
  recombinant
• Genetic engineering contributions
• Future outlook in PHA production lower price
• Larger production scale
• More efficient bacteria as bio-factory of PHA
• Use of cheap carbon sources
• Better optimization and control of PHA production
  system

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Thank you