Bioplastics Produced by Microorganisms
Iman Rusmana
Department of BiologyBogor Agricultural University
1869 Celluloid John Wesley Hyatt
1912 (1838 erstmals
erzeugt)
PVC (Polyvinylchlorid) Fritz Klatte
1920-28 Theorien über den Aufbau von Kunststoffen (Nobelpreis 1953)
Hermann Staudinger
1930 Neopren DuPont / USA
1930 Polystyrol (Styropor) BASF, IG Farben
1931 Polyethylen ICI / UK
1935 Polyamid (Nylon) DuPont / USA
1937 Polyamid (Perlon) BASF, IG Farben
1937 Polyurethan BASF, IG Farben
1938 Polytetrafluorethylen (Teflon) DuPont / USA
1941 Polyethylenterephthalat (PET) Calico Printers / UK
1951 Polypropylen Philipps Petroleum / USA
1953 Polycarbonat Bayer
Importance2003- North America
107 billion pounds of synthetic plastics produced from petroleumTake >50 years to degradeImproper disposal and failure to recycle overflowing landfills
Degradable polymers that are naturally degraded by the action of microorganisms such as bacteria, fungi and algae
What are Bioplastics?
Benefits Include:100 % biodegradableProduced from natural, renewable resourcesAble to be recycled, composted or burned without producing toxic byproducts
Carbon Cycle of BioplasticsCO2
H2O Biodegradation
CarbohydratesPlastic Products
Plants
Fermentation PHA Polymer
Photosynthesis
Recycle
Polyhydroxyalkanoates (PHAs)
Polyesters accumulated inside microbial cells as carbon & energy source storage
Ojumu et al., 2004
Polyhydroxyalkanoates, or PHAs, are homo- or heteropolyesters synthesized and intracellularly stored bynumerous prokaryotes.
Polyhydroxyalkanoates (PHAs)
Produced under conditions of:Low limiting nutrients (P, S, N, O)Excess carbon
2 different types:Short-chain-length 3-5 CarbonsMedium-chain-length 6-14 Carbons
Bacillus megaterium,BacillusPseudomonasAlcaligenes Azotobacter HydrogenomonasChromatium Cyanobacteria,and many others
~250 different bacteria have been found to produce some form of PHAs
"Bacterial Polyester"
• Poly-3-Hydroxybutyrat (PHB) in Bacillus megateri1926 Maurice Lemoigne (Institut Pasteur)
PHA - Polyhydroxyalkanoate
Polyhydroxybutyrate (PHB)
Example of short-chain-length PHAProduced in activated sludgeFound in Alcaligenes eutrophusAccumulated intracellularly as granules (>80% cell dry weight)
Lee et al., 1996
PHA Biosynthesis
Ojumu et al., 2004
phbC-A-B Operon in A. eutrophus
Structural genes encoded in single operonPHA synthaseβ-ketothiolaseNADPH-dependent acetoacetyl-CoA reductase
Lee et al 1996
Ralstonia eutropha (Alcaligenes eutrophus)
PHB biosynthesis
PHB > 70% cell dry weight
DepolymerasePhasin
Ralstonia eutropha (Alcaligenes eutrophus)
PHB-Cycle
Production of PHA in BacteriaPhaA: β-KetothiolasePhaB: Acetoacetyl-CoA-ReduktasePhaC: PHA-Synthase
PhaJ: (R)-specific Enoyl-CoA-HydrPhaG: Hydroxyacyl-ACP:CoA Tran
PHAs
Pseudomonas putida
Maximum: 85-90% dry weight
Prieto (2007) J. Bacteriol. 189:289-29Alcanivorax borkumensis
• Synthesis Propionyl-CoA PHBV Synthesis
PHA-Synthesis in Bakteri vs Transgenic Plants
Production PHA PurificationOf PHA
Productionof starch
Isolation of starch
Hydrolysis Fermentation Purification of PHA
90% 90% 90%30%
90%
Plant
TrangenicPlants
Bacteria
Accumulation of PHA in transgenic A. thaliana
Chloroplast Nukleus Peroxisom
• 3-Ketothiolase endogen (Cytoplasma)Reduktase (phaB) & Synthase (phaC) transgen (R. eutropha)
NOS-PolyAphaB35S
NOS-PolyAphaC35S
• Expression in Plastides (phaA, phaB, phaC):14% TG (Multigenvektor: bis 40%)
NOS-PolyAphaB35S
NOS-PolyAphaA35S
NOS-PolyAphaC35S TPSS
TPSS
TPSS
Slater et al. 1999 Nature Biotechnol. 17:1011-1016.
Synthesis of PHBV-Copolymers in PlantsArabidopsis thaliana, Brassica napus
• 4 Transgenes
(Monsanto)
• Expression: A. thaliana: 35S-PromotorRuBisCo-small-subunit Transit-Peptid (Chloroplasten2 Vektor
B. napus: Promotor (P-Lh; Hydroxylase; Lesquerella)RuBisCo-small-subunit Transit-Peptid (Leukoplasten)1 Vektor
• Substrates: Acetyl-CoA & Propionyl-CoA
BioplasticsICI (UK, 1975) 1982: BIOPOL™ A. eutrophus - Fermentatio
C-Quelle: Glukose/Saccharose (PHB)+ Propionates (PHV)
Recovery of PHAs from Cells
PHA producing microorganisms stained with Sudan black or Nile blueCells separated out by centrifugation or filtrationPHA is recovered using solvents (chloroform) to break cell wall & extract polymerPurification of polymer
Bioplastic PropertiesSome are stiff and brittle
Crystalline structure rigiditySome are rubbery and moldableProperties may be manipulated by blending polymers or genetic modificationsDegrades at 185°CMoisture resistant, water insoluble, optically pure, impermeable to oxygenMust maintain stability during manufacture and use but degrade rapidly when disposed of or recycled
BiodegradationFastest in anaerobic sewage and slowest in seawaterDepends on temperature, light, moisture, exposed surface area, pH and microbial activityDegrading microbes colonize polymer surface & secrete PHA depolymerasesPHA CO2 + H2O (aerobically)PHA CO2 + H2O + CH4 (anaerobically)
Biodegradation by PHA depolymerases
Controlled Degradation
PET PHBV
Canebiomass
Biotechnologytools
Bio
fert
ilize
rsco
2
Cane and trash
Biofertilizers
EthanoldistilleryEthanol
Can
e ju
ice
Mol
asse
s
Sugarfactory
Suga
r exp
orts
Cogenplants
Bagasse and trash
Steam & electricity
Foodproducts
Leaves & trash
SolventsBioplastic
factory
Stea
m a
ndel
ectr
icity
Bioplastics
Bio-refinery concept