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

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Cane and trash

Biofertilizers

EthanoldistilleryEthanol

Can

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Mol

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Sugarfactory

Suga

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Cogenplants

Bagasse and trash

Steam & electricity

Foodproducts

Leaves & trash

SolventsBioplastic

factory

Stea

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Bioplastics

Bio-refinery concept