11
Degradation and applications of polyhydroxyalkanoatesl Helmut Brandl, Reinhard Bachofen, Jorg Mayer, and Erich Wintermantel Abstract: A series of tests is available to study the biodegradation of plastic materials under either laboratory or field conditions. Most of the standard methods have been published by the American Society for Testing and Materials. All of them describe techniques to investigate the biodegradation of plastics under laboratory conditions. Microbially formed polyhydroxyalkanoates (PHAs) have been marketed recently as biodegrad- able plastics. However, currently only a few articles made from PHAs (e.g., bottles) are commercially available. A series of microorganisms (prokaryotes as well as eukaryotes) has been characterized as being able to degrade PHAs. With one exception (Ilyobacter delafieldii), all of them were isolated from aerobic environments. So far, over 10 different extracellular PHA depolymerases have been purified and charac- terized. Depolymerases that preferentially attack PHAs with monomer units other than 3-hydroxybutyrate have been found only in Pseudomonasjluorescens and Pseudomonas lemoignei. Key words: poly(3-hydroxybutyrate), polyhydroxyalkanoates, biodegradation, industrial applications. Resume : Une skrie de tests est disponible pour Ctudier la biodkgradation de matibres plastiques sous des conditions soit de laboratoire soit de terrain. La plupart des mkthodes standards ont Ctk publikes par la American Society for Testing and Materials. Elles dkcrivent toutes des techniques pour Ctudier la biodkgradation de plastiques sous des conditions de laboratoire. Des polyhydroxyalcanoates (PHAs) d'origine microbienne ont Ctk mis en march6 rkcemment comme plastiques biodkgradables. Cependant, jusqu'h aujourd'hui seulement quelques objets fabriquks h partir de PHAs (p. ex., des bouteilles) sont disponibles commercialement. Une sQie de microorganismes (aussi bien procaryotes qu'eucaryotes) a kt6 caractkriske c ome Ctant capable de dkgrader les PHAs. A part une exception (Ilyobacter delafieldii), tous ont Cte isolks d'environnements akrobies. Jusqu'h prCsent, plus de 10 PHA dkpolymCrases extracellulaires diffkrentes ont CtC purifikes et caracterisCes. Des dkpolymkrases qui attaquent prkfkrentiellement les PHAs avec des unitks monomkriques autres que le 3-hydroxybutyrate sont connues seulement chez les Pseudomonasfluorescens et Pseudomonas lemoignei. Mots cle's : poly(3-hydroxybutyrate), polyhydroxyalcanoates, biodkgradation, applications industrielles. [Traduit par la RCdaction] Introduction High molecular weight polymers play an important role in nature as structural or storage components (e.g., cellulose and Received August 15, 1994. Revision received November 10, 1994. Accepted December 15,1994. H. Brand12 and R. Bachofen. Universitat Zurich, Institut fiir Pflanzenbiologie, Zollikerstrasse 107, CH-8088 Ziirich, Switzerland. J. Mayer and E. Wintermantel. Eidgenossische Technische Hochschule, Professur fiir biokompatible Werkstoffe und Bauweisen, Wagistrasse 6, CH-8952 Schlieren, Switzerland. 1 H. Brand1 dedicates this paper to his close friend Dr. Hubert E. Arter, who died tragically on December 28, 1994. 2 Author to whom all correspondence should be addressed. starch). Other polymers are of immense importance for techni- cal applications (e.g., plastics such as polyethylene or polystyrene). These plastic materials have, in contrast to bio- polymers (e.g., nucleic acids, proteins, and polysaccharides), almost unlimited life-spans. Since the first development of plastics at the beginning of this century it has been the goal of industry and science to improve the resistance of these materials to microbial attack. Recently, science and industry have focussed on the develop- ment and application of biodegradable plastic materials. Bio- degradable plastics are defined by the American Society for Testing and Materials as degradable plastics in which the degradation results from the action of naturally occurring microorganisms such as bacteria, fungi, and algae (American Society for Testing and Materials 1992). Our increased percep- tion of today's ecological problems as well as legislative Can. J. Microbiol. 41(Suppl. 1): 143-153 (1995). Printed in Canada I Imprime au Canada Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Dalian Nationalities University on 06/06/13 For personal use only.

Degradation and applications of polyhydroxyalkanoates

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Degradation and applications of polyhydroxyalkanoatesl

Helmut Brandl, Reinhard Bachofen, Jorg Mayer, and Erich Wintermantel

Abstract: A series of tests is available to study the biodegradation of plastic materials under either laboratory or field conditions. Most of the standard methods have been published by the American Society for Testing and Materials. All of them describe techniques to investigate the biodegradation of plastics under laboratory conditions. Microbially formed polyhydroxyalkanoates (PHAs) have been marketed recently as biodegrad- able plastics. However, currently only a few articles made from PHAs (e.g., bottles) are commercially available. A series of microorganisms (prokaryotes as well as eukaryotes) has been characterized as being able to degrade PHAs. With one exception (Ilyobacter delafieldii), all of them were isolated from aerobic environments. So far, over 10 different extracellular PHA depolymerases have been purified and charac- terized. Depolymerases that preferentially attack PHAs with monomer units other than 3-hydroxybutyrate have been found only in Pseudomonasjluorescens and Pseudomonas lemoignei.

Key words: poly(3-hydroxybutyrate), polyhydroxyalkanoates, biodegradation, industrial applications.

Resume : Une skrie de tests est disponible pour Ctudier la biodkgradation de matibres plastiques sous des conditions soit de laboratoire soit de terrain. La plupart des mkthodes standards ont Ctk publikes par la American Society for Testing and Materials. Elles dkcrivent toutes des techniques pour Ctudier la biodkgradation de plastiques sous des conditions de laboratoire. Des polyhydroxyalcanoates (PHAs) d'origine microbienne ont Ctk mis en march6 rkcemment comme plastiques biodkgradables. Cependant, jusqu'h aujourd'hui seulement quelques objets fabriquks h partir de PHAs (p. ex., des bouteilles) sont disponibles commercialement. Une sQie de microorganismes (aussi bien procaryotes qu'eucaryotes) a kt6 caractkriske c o m e Ctant capable de dkgrader les PHAs. A part une exception (Ilyobacter delafieldii), tous ont Cte isolks d'environnements akrobies. Jusqu'h prCsent, plus de 10 PHA dkpolymCrases extracellulaires diffkrentes ont CtC purifikes et caracterisCes. Des dkpolymkrases qui attaquent prkfkrentiellement les PHAs avec des unitks monomkriques autres que le 3-hydroxybutyrate sont connues seulement chez les Pseudomonas fluorescens et Pseudomonas lemoignei.

Mots cle's : poly(3-hydroxybutyrate), polyhydroxyalcanoates, biodkgradation, applications industrielles. [Traduit par la RCdaction]

Introduction

High molecular weight polymers play an important role in nature as structural or storage components (e.g., cellulose and

Received August 15, 1994. Revision received November 10, 1994. Accepted December 15,1994.

H. Brand12 and R. Bachofen. Universitat Zurich, Institut fiir Pflanzenbiologie, Zollikerstrasse 107, CH-8088 Ziirich, Switzerland. J. Mayer and E. Wintermantel. Eidgenossische Technische Hochschule, Professur fiir biokompatible Werkstoffe und Bauweisen, Wagistrasse 6, CH-8952 Schlieren, Switzerland.

1 H. Brand1 dedicates this paper to his close friend Dr. Hubert E. Arter, who died tragically on December 28, 1994. 2 Author to whom all correspondence should be addressed.

starch). Other polymers are of immense importance for techni- cal applications (e.g., plastics such as polyethylene or polystyrene). These plastic materials have, in contrast to bio- polymers (e.g., nucleic acids, proteins, and polysaccharides), almost unlimited life-spans. Since the first development of plastics at the beginning of this century it has been the goal of industry and science to improve the resistance of these materials to microbial attack.

Recently, science and industry have focussed on the develop- ment and application of biodegradable plastic materials. Bio- degradable plastics are defined by the American Society for Testing and Materials as degradable plastics in which the degradation results from the action of naturally occurring microorganisms such as bacteria, fungi, and algae (American Society for Testing and Materials 1992). Our increased percep- tion of today's ecological problems as well as legislative

Can. J. Microbiol. 41(Suppl. 1): 143-153 (1995). Printed in Canada I Imprime au Canada

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Table 1. Factors influencing environmental degradability of plastics (Brand1 et. al. 1990; Palmisano and Pettigrew 1992; In't Veld 1992).

Parameter Factor

Physicochemical parameters of an ecosystem Temperature

pH Water content Oxygen content Redox potential Nutrient supply Presence of inhibitors

Microbiological parameters of an ecosystem Population density

Microbial diversity Microbial activity Spatial distribution of

microorganisms Ability to adapt

Primary material properties Polymer composition Molecular weight Molecular weight distribution Crystallinity Glass transition temperature Porosity Hydrophobicity Steric configuration Bond type between monomers

Material processing Type of processing Surface characteristics Material thickness Additives Fillers Coatings

pressure have led to the introduction of these materials. According to a survey, two thirds of the American population agree with the assertion that plastics pose the greatest threat to the environment because of their nonbiodegradability (Rogers 1990).

In industrialized countries, waste reduction has been the focus of much debate. In certain waste incineration plants the amount of plastics in the waste stream is almost 20%, because of the separation of the waste before incineration (i.e., composting of the organic parts and recycling of glass, aluminum, paper, and cardboard). It is assumed that the utilization of degradable plastics would help to reduce the percentage of plastics in the waste. In addition, the use of these materials is based on renewable resources and contributes to a material cycling analogous to the natural biogeochemical cycles in nature.

In nature, polymers are degraded preferentially by hydrolytic reactions. The presence of ester, ether, or amide bonds facilitates biological degradation. Biodegradation is influenced by a series of different environmental parameters (Table 1). The physicochemical and microbiological parameters of the ecosystem, primary material properties, and material processing affect the extent of biodegradation.

Classification of degradable plastics Degradable plastics can be classified into several categories (Fig. 1). The type of degradation (chemical, physical, biological) is the first criterion. Biodegradation can take place under either septic or aseptic conditions. Aseptic conditions (sterile conditions without any microbial activity) are required for medical applications. Polymers are degraded by hydrolytic reactions and (or) bioresorbed under those conditions. In contrast, biodegradable polymers for industrial applications are degraded under septic conditions (systems with microbial activities such as soil, compost, and lake water) by the catalysis of microorganisms. There are two types of microbially degradable plastic materials, namely completely degradable polymers and polymers consisting of a degradable additive and a nondegradable matrix.

Polyhydroxyalkanoates as biodegradable plastics Microbially formed polyhydroxyalkanoates (PHAs) have been used recently as biodegradable plastics (Brand1 et al. 1990; Steinbuchel 1992). PHAs are bacterial biopolymers and are formed as naturally occurring storage polyesters by a wide range of microorganisms (Brand1 et al. 1990; Steinbuchel 1991). Poly(3-hydroxybutyrate) (P(3-HB)) is the best known representative of the PHA family. Microorganisms are able to incorporate up to 60 different monomer types into their storage polymer (Steinbuchel 1991). A series of PHAs with different monomeric compositions (i.e., different physical and chemical properties) can be produced because of either the metabolic flexibility of a particular organism to incorporate different monomer units or the wide diversity of microorganisms that can form PHAs. On a fairly large scale, PHA is produced industrially by Zeneca Bio Products (Great Britain) and is commercialized as biodegradable plastic under the trade name BiopolB. This product is a copolymer consisting of 3-HB and 3-hydroxyvalerate (3-HV) repeating units with various ratios.

PHA can be completely mineralized to H20 and C02 in aerobic systems (Krupp and Jewel1 1992). H20, C02, and CH4 are the final end products when PHA is degraded under anaerobic conditions (Budwill et al. 1992).

Techniques for the determination of biodegradability

Test methods There are several different methods and techniques to determine the degradability of plastic materials (Table 2). Methods are based on visual observation of plastic materials, quantitative determination of microbial growth when plastic materials are used as carbon source, polymer utilization by microorganisms, determination of changes in polymer characteristics during incubation, and determination of microbial activities such as oxygen consumption or gas production. Most of the tests are laboratory methods. Only a very limited number can be applied as field or in situ tests. Results from laboratory tests can rarely be transferred to conditions in natural ecosystems because of the complexity of these systems. Therefore, field or in situ experiments are an unalterable prerequisite for the determination of biodegradability.

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Fig. 1. Schematic classification of degradable plastics.

Solubilization - Solublepolymers

C Photosensitive additives

Chemical

i f Photochemical Photosensitive copolymers

Oxidation - Oxidizable polymers

Degradable Polymers

Aseptic - Resorbable polymers \-< Polymers Completely degradable

Microbial

Degradable additives

Mechanical -b All polymers

Physical

I Thermal -b All polymers

Field degradation tests As an example, biodegradation of dogbone-shaped PHA test pieces (homopolymers of 3-HB as well as copolymers of 3-HB and 3-HV) has been studied under natural conditions by measuring the mass loss after incubation of the polymeric material in ecosystems such as soil, freshwater, seawater, and compost (Mergaert et al. 1992~).

Bottles made from Biopolm were utilized to study the degradation of PHA under in situ conditions in a freshwater lake and sediment (Brand1 and Puchner 1992). Bottles made from polyethylene-starch blends were also used in this work.

Laboratory degradation tests Biodegradation under aerobic conditions can be studied using a modified turbidometric assay (clear zone test) under different conditions (Augusta et al. 1993; Brand1 et al. 1995). An agar plate (1.5% (wlw) in tap water) was poured and a template was used to create a cross- or star-shaped pattern of polymer granules on the agar surface. Wells were formed in the center of the plate using a sterile cork borer. The bottom of the well was sealed with two drops of agar. Details of the methods are published elsewhere (Brand1 et al. 1995). The modification of the clear zone test (use of a template to strew powdered polymers onto agar surfaces) allowed biodegradation studies under aerobic conditions with minimal amounts of polymeric material. Inoculated plates were incubated and tho size of the

clear zone along the polymer lines was periodically determined.

Samples from natural ecosystems can be taken to study the degradation capabilities of different systems. Wells in the agar plates were filled with either soil, freshwater lake sediments, sewage sludge, or compost. Pure cultures of Acidovorax delafieldii (ATCC 17505) as well as crude extracts of extracellular P(3-HB) depolymerase from this organism were also used to catalyze biodegradation (Schwegler 1992). All of the samples used had the potential to degrade PHAs.

Degradation of polyhydroxyalkanoates

Polyhydroxyalkanoate-degrading microorganisms and their enzymes

In contrast to studies on pathways of PHA biosynthesis only a few data are available on the extracellular degradation of these polymers (Steinbuchel 1992). Although the first investigations on PHA degradation by Pseudomonas spp. were performed in the early 1960s, the list of P(3-HB)-degrading microorganisms has been drastically expanded only recently (Table 3). A large number of PHA degraders were isolated from soil. Information on PHA-degrading microorganisms from lacustrine as well as marine systems is scarce. To our knowledge there are only a few known PHA degraders from freshwater systems, although PHA degradation in lakes has been described (Brand1 and

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Table 2. Test methods for the determination of biodegradability of plastics materials.

Method Reference Indicator

Surface growth ASTM 1986a Surface growth

Surface growth ASTM 1986h Surface growth Microbial growth Arninabhavi et al. 1990 Biomass

Polymer utilization Brandl and Piichner 1992 Weight loss Mergaen el al. 1 992a

Polymer utilization GGilmore et al. 1990 nubidi ty

Polymer utilization Gilmore et al. 1990 Turbidity Change in polymer

characteristics Sawada 1994 Tensile strength Kaplan et al. 1994

Change in polymer characteristics ASTM t994c Molecular weight

Microbial activities ASTM 1994a Gas production Microbial activities ASTM 1994b Gas production Microbial activities MITI 1983 Nr. 117 Oxygen utilization

Microbial activities ASTM 1994d Oxygen utili7stion Microbial activities ASTM 1994e Gas production Microbial activities Tilstra and Johnsonbaugh 1993 Oxygen utilization

Microbial activities Albertsson 1989 Gas production

Observation or measurement

Visual

Nwa1 Turhidomctric Protein content Phospholipid content Gravirnetric

Turbidometric

Different material tests

Chromatographic

Titrirnetric Gas chromatographic Electrorhemical

Electrochemical Ktrimetric Manometric

Radiometric

Laboratory Organism or system test Field test

Aspe~:qil/zts niger ( ATCC 9642) x

Prnicillhrmfinftulnsum (ATCC 9644) Cl~aeromiurn glnbos~rm (ATCC 6205) GIiocIadium virens (ATCC 9645) Aw-eobosidirrm pullulans (ATCC 9348) Pse~rifnmonns aeruginosa (ATCC 13388') x Pure or mixed culture x

Pure or mixed culture or natural ecosystem x

Pure or mixed cuIture or sampIes from natural x ecosystems

Cdl-free enzyme extract x

Pure or mixed culture or natural ecosystem x

Streptonlyces hadilts 252 (ATCC 391 17) Sn-eptornyces setonii 75Vi2 ( ATCC 39 I 16) Streptonryces virirluspororus T7A (ATCC 391 1 5 ) Phantrorhaer~ rhryxo.tporium (ATCC 3454 1) Sewage sludge Sewage sludge Pure or mixed culture or samples from natural

ecosystems Sewage sludge Compost AspergiI!~.~ niger ( ATCC 9642) Penirr lliumfMnicrrlos~im (ATCC 9644) Chaetolriiuni glol~nsum (ATCC 6205) Cliocladi~mt t~ i r~n~v (ATCC 9645) Aur~obosidirun pul~uulans (ATCC 9348) Pure or mixed culture or samples from natural

ecosystems

NOTE: ATCC, American Type Culture Collection; ASTM, American Society for Testing and Materials; MITI, Agency of Indushial Science and Technology, Japan.

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Table 3. Microorganisms able to degrade polyhydroxyalkanoates.

Organism Ecosystem Group Domain Reference

Acidovorax facilis Acidovorax delafieldii Acrernonium sp. Acidovorax facilis Alcaligenes faecalis Arthrobacter viscosus Aspergillus sp. Aspergillus fumigatus Aspergillus penicilloides Bacillus megaterium Bacillus polymyxa Cephalosporium sp. Cladosporium sp. Comarnonas testosteroni Comamonas acidovorans Cytophaga johnsonae Eupenicillium sp. Ilyobacter delafieldiia Mucor sp. Paecilomyces marquandii Penicillium adametzii Penicillium chermisinum Penicillium daleae Penicillium funiculosum Penicillium ochrochloron Penicillium restrictum Penicillium simplicissimum Polyporus circinatus Pseudomonas sp. Pseudomonas cepacia Pseudomonas fluorescens Pseudomonas lemoignei Pseudomonas pickettii Pseudomonas stutzeri Pseudomonas syringae Pseudomonas vesicularis Streptomyces sp. Variovorax paradoxus Verticillium leptobactrum Xanthomonas maltophilia Zoogloea ramigera

Soil, compost Soil Soil Soil Sewage sludge Soil Soil, compost Soil Soil, compost Soil, compost Soil Soil Soil Sea water Lake water Soil Soil Estuarine sediment Soil Soil Soil Soil Soil Soil Soil Soil Soil, compost Soil Soil, compost Lake water Sewage sludge Soil Laboratory Lake water Soil Lake water Soil, compost Soil, compost Compost Soil Soil

Gram negative Gram negative Deuteromycetes Gram negative Gram negative Gram positive Ascomycetes Ascomycetes Ascomycetes Gram positive Gram positive Deuteromycetes Deuteromycetes Gram negative Gram negative Gram negative Deuteromycetes Gram negative Zygomycetes Deuteromycetes Ascomycetes Ascomycetes Ascomycetes Ascomycetes Ascomycetes Ascomycetes Ascomycetes Basiodiomycetes Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Gram negative Actinomycetes Gram negative Ascomycetes Gram negative Gram negative

Prokaryote Prokaryote Eukaryote Prokaryote Prokaryote Prokaryote Eukaryote Eukaryote Eukaryote Prokaryote Prokaryote Eukaryote Eukaryote Prokaryote Prokaryote Prokaryote Eukaryote Prokaryote Eukaryote Eukaryote Eukary ote Eukaryote Eukaryote Eukaryote Eukaryote Eukaryote Eukaryote Eukaryote Prokaryote Prokaryote Prokaryote Prokaryote Prokaryote Prokaryote Prokaryote Prokaryote Prokaryote Prokaryote Eukaryote Prokaryote Prokaryote

Mergaert et al. 1992a Chowdhury 1963 Mergaert et al. 1992a Jendrossek et al. 1993 Tanio et al. 1982 Mergaert et al. 1992b Mergaert et al. 1992a Mergaert et al. 1992b Mergaert et al. 1992a Mergaert et al. 1993 Mergaert et al. 1992a Matavulj and Molitoris 1992 Matavulj and Molitoris 1992 Mukai et al. 1993 Mukai et al. 1994 Mergaert et al. 1993 McLellan and Halling 1988 Janssen and Harfoot 1990 Matavulj and Molitoris 1992 Mergaert et al. 1992a Mergaert et al. 1992a Mergaert et al. 1992a Mergaert et al. 1992a Brucato and Wong 199 1 Mergaert et al. 1992a Mergaert et al. 1992a McLellan and Halling 1988 Matavulj and Molitoris 1992. Mergaert et al. 1992 Mukai et al. 1994 Schirmer et al. 1993 Delafield et al. 1965 Mukai et al. 1994 Mukai et al. 1994 Mergaert et al. 1993 Mukai et al. 1994 Mergaert et al. 1992a Mergaert et al. 1992a Mergaert et al. 1992b Mergaert et al. 1992b Jendrossek et al. 1993

allyobacter dela4eldii is the only representative of strictly anaerobic microorganisms known to degrade poly(3-hydroxybutyrate).

Piichner 1992; Mukai et al. 1994). Gram-positive as well as Gram-negative aerobic bacteria are known for their ability to degrade PHA. The extracellular degradation by archaea is unknown. Recently, the fermentative degradation by I. delafieldii under strictly anaerobic conditions was described (Janssen and Harfoot 1990). There seems to exist a wide distribution of PHA-degrading abilities among fungi (Mergaert et al. 19926; Matavulj and Molitoris 1992).

Only the P(3-HB) depolymerase of Alcaligenes faecalis T1 has been characterized at the molecular level (Saito et al. 1989; Steinbuchel 1992). Table 4 shows a list of known extracellular depolymerases. Although PHA degradation is widespread in nature, depolymerizing enzymes from only a limited-number of organisms have been described. PHA depolymerases from anaerobic microorganisms are not yet characterized. In most cases P(3-HB) was used as substrate. Depolymerases that preferentially attack PHAs with monomer-units other than

3-HB have been found only in two organisms (Muller and Jendrossek 1993; Schirmer et al. 1993). Besides P(3-HB), depolymerases from Alcaligenes faecalis, Comamonas testosteroni, Pseudomonas lemoignei, Pseudomonas pickettii, and Pseudomonas stutzeri are able to use homopolymers of 4-hydroxybutyrate as substrate (Mukai et al. 1994).

Field studies Considering the structural diversity of PHAs and the related material properties, information on the biodegradation of different PHAs is very scarce. Detailed knowledge of PHA-degrading organisms, their distribution in a specific ecosystem, and the enzymatic degradation mechanisms is of fundamental importance for the use of PHAs as biodegradable plastics.

A series of field experiments was performed to study PHA (Biopolm bottles; molecular composition: 92% 3-HB,

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Table 4. Extracellular enzymes responsible for the primary degradation of polyhydroxyalkanoates.

Molecular Organism Enzyme weight Structure

Substrate specificity

Alcaligenes faecalis (TI) P(3-HB) nd

depolymerase

Alcaligenes faecalis (Tl) P(3-HB) 48 OOOb

depolymerase 50 OOOc

Alcaligenes faecalis (TI) Oligomer 68 OOOb

hydrolase 74 OOOC (EC 3.1.1.22)

Cornonas sp. ( DSM 678 1) PQ-HB) 44 OOOC

depolymerase 45 OOod

Commonas testostmni (YM 1004) P(3-HB) 50 OOOC

dep l ymerase

fi~niccllos~m (ATCC 9644) P(3-HB) 36 O0OC

depolymerase 38 0 0 0 ~

Pseudomonas jluorescens (GK13) P(3-HO) 25 OOOC

depolymerase 48 OOod

Monomer

Monomer

Monomer

Monomer

nd

Monomer

Dimer

P(3-HB) 3-HR-trirner Tetramer Pentamer Hexamer Heptamer Octamer Dodecamer Hexadecamcr

P(3-HB) 3-HB-[rimer Tetnuner Pentamer

3-HB-dimer Trimer Tetramer Pentamer Octamer Dodecamer

Products pH,

o~timal IEP

Optimal temperature

("C)

nd

nd

nd

29-35

nd

nd

30-32

DTT

Deoxycholat, DFP, PMSF, Triton X- 100

DFP

DTT

DFP, DIT, PMSF, Tween 20

DAN, DFP, DTT, EPW, HgC12, PMSE Triton X-100. Tween 80

DIT. EDTA. HgCI2, KCN. MIA. NaN3, PMSF

Reference

Shirakura et al. 1986

Tanio et al. 1982

Shirakura et al. 1983

Jendrossek et al. 1993

Mukai et al. 1993

Brucato and Wong 1991

Schirmer et al. 1993

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Table 4 (concluded).

Organism

Pseudomonas lemoignei (LMG 2207)

Pseudomonas lemoignei (LMG 2207)

Pseudomonas lemoignei (ATCC 17989)

Pseudomonas lemoignei (ATCC 17989)

Pseudomonas pickettii (YM-b)

Pseudomonas stutzeri (YM1414)

Pseudomonas (PI)

Enzyme

P(3-HB) depolymerase

P(3-HV) depolymerase

P(3-HB) depolymerase

Isoenzymes A1 A2 B1 B2

P(3-HB) depolymerase

P(3-HB) depolymerase

P(3-HB) depolymerase

P(3-HB) depolymerase

7 . Optimal

Molecular Substrate pH, temperature 2. 5

weight Structure specificity KIT, (PM) Products optimal IEP ("C) Inhibitorsa Reference ffl Y s 0

nd nd P(3-HB) 100 p,g.m~-l 3-HB 8.0 nd 65 nd Muller and (D

P(3-HV) Jendrossek 1993 P(3-HB-CO-3-HV)

53 0 0 0 ~ Monomer P(3-HB) 65 p,g-mI-l 3-HV 8.0 nd 55 DTT, PMSF Muller and 54 OOOC P(3-HV) 77 , . ~ g . d - l Jendrossek 1993

P(3-HB-CO-3-HV)

Monomer P(3-HB) 3-HB-trimer Tetramer Pentamer

45 O O O ~ 54 OOOC 49 O O O ~ 58 OWC

73 - 131 p,g.m~-' (3-HB) 8.0 nd DFP, DIT, Nakayarna et al. 1.7-5.0 Dimer PMSF 1985 0.22-0.26 Trimer 0.091 -0.099

9.7 10.0 10.0 10.6

3-HB 8.0 >9.5 ncl CETAB, Delafield et al. 1965 Dimer ED'I'A. SDS, (Trimer) Triton X- 100,

Tween 80

40 OOOC nd P(3-HB) nd 3-HB 5.5 7.7 40 DFP. DTT, Yamada et al. 1993 P(3-HB-co-3-HV) (Dimer) PMSF P(3-HB-CO-4-HV)

48 OOOC nd P(3-HB) nd 3-HB 9.5 9.2 55 DFP. DTT, Mukai et al. 1994 Dimer PMSF, Trimer Tween 20

nd nd P(3-HB) nd 3-HB 9.8 >8.6 45 Dm, DTT, Chowdhury 1963 Propyl-HB PMSF

NOTE nd, nor determined 'DAN, diazmorleucinmethy1ester. DFP, dii.wpropylflum~phwphate: MT. dithiothreitol; CETAB, cetyltrimethylammoniumbromide; EDTA, ethyldiaminetetraacetate; EPNP, 1,2-epoxy-3-(p-

niuophe~xy)propane: MIA. rnono~odoacetate: PMSF. phenylmethylsulfonylfluoride. hGel filtration. SDSPAGE (sodium cladecyl sulfate - polyacrylamide gcl elecfrophmsis).

"AGE (native).

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Fig. 2. In situ degradation of bottles made from BiopolB or polyethylene-starch (PE+S) blends (the percentage of starch is in parentheses) in Lake Lugano, Switzerland. The BiopolB bottles were made available by Wella AG (Darmstadt, Germany) and had a molecular composition of 92% 3-HB and 8% 3-HV. The average mass of a bottle was 31.90 g (n = 13). The inner and outer surface areas were 336 and 356 cm2, respectively. Bottles were incubated precisely at the sediment-water interface at a depth of 85 m (Brand1 and Piichner 1992).

90 1 I I I I I I 0 50 100 150 200 250 300

Time (d)

8% 3-HV) biodegradation under in situ conditions in an aquatic ecosystem (Fig. 2). The inner and outer surface areas of the bottles were 336 and 356 cm2, respectively. The average mass was 31.90 g (n = 13). Degradation studies were carried out in Lake Lugano, Switzerland. The plastic bottles were attached to a buoy for incubation at different water depths. For the precise placement of the bottles directly on the sediment surface at a depth of 85 m a manned research submarine was used. Detailed descriptions of the sampling site and the techniques used are published elsewhere (Brand1 and Piichner 1992). Degradation rates of 10-20 mgld can be measured, giving an average lifetime of 5- 10 years for the specific bottle type (Brand1 and Piichner 1992). The degradation rates vary with the water depth, that is, with the spatial heterogeneity of the ecosystem.

Bottles made from polyethylene-starch (PE+S; 9% and 13% starch, respectively) blends showed no degradation at all. For reasons of comparison they had the same form and size as the Biopolm bottles. The dry mass even increased during incubation, suggesting the incorporation of water into the polymer that could not be removed be drying (24 h at 105°C). The results demonstrate that PHAs can be degraded in a freshwater lake ecosystem even under relatively extreme conditions, such as constantly low temperatures, no exposure to sunlight, seasonal variation in the oxygen concentration, and increased hydrostatic pressure. PE+S blends, materials that are

supposed to biodegrade in natural ecosystems (Maddever and Campbell 1990), do not degrade under these conditions. This phenomenon was also observed by other investigators (Krupp and Jewel1 1992).

Applications of polyhydroxyalkanoates

General PHAs can be used for a multitude of industrial and medical applications in which biodegradability offers a selective advantage over traditional, petrochernically based plastics. However, currently only a very limited range of items made from PHAs are commercially available. Most of the possible applications are still under development.

Articles made from PHAs are advertised as being completely biodegradable in natural ecosystems and are therefore environmentally friendly. However, only a few studies that report PHA degradation in natural ecosystems such as soil, compost, freshwater, or seawater have been published so far (e.g., Brand1 and Piichner 1992; Gilmore et al. 1992; Mergaert 1992a; Mergaert et al. 1993; Kimura et al. 1994). For all applications it is important to determine the life-spans of the materials in specific environments, to characterize the degradation mechanisms, and to describe the microorganisms involved in biodegradation. An important goal of further research is the ecotoxicological evaluation (final fate, accumu- lation and toxicity of intermediates, and biocompatibility) of the biodegradable materials used. In addition, life-cycle analyses of the different materials have to be conducted.

Bottles Industrial containers (e.g., bottles) are one possible technical application for PHAs. In 1990, Wella, a German hair-care company, released shampoo bottles made from BiopolB. During the pilot phase, material properties were improved so that the bottles are now definitively on the market. In Japan and the U.S.A., Biopolm bottles have been commercially available since 1991 and 1992, respectively.

Fiber-reinforced, biodegradable bicycle helmet Reinforcing PHAs with biodegradable fibers opens the field of industrial applications to anisotropic engineering materials (strength up to 400 MPa, Youngs modulus up to 15 GPa). The bicycle helmet shown in Fig. 3 illustrates an application wherein knitted cellulosic high-perfomance fibers had been used to reinforce BiopolB (9% 3-HV) to optimize the strength and energy absorbance behavior (Koch 1993). It has been shown that the composite retains its mechanical properties in a normal outdoor environment, whereas in soil, complete disintegration and degradation were observed after 40 days, as illustrated in Fig. 4.

Biodegradable, autoseparative filter Biodegradable air filters offer the opportunity to separate filter matrices from filtered particles by the degradation of the filter matrix. This filter behavior is called autoseparative and considerably reduces the volume of filtrate, which has to be treated as critical waste (Wintermantel et al. 1992). The investigated depth filter membranes were made out of a composite of 90 vol% NaCl granules and 10 vol% BiopolB (molecular composition: 81% 3-HB and 19% 3-HV). After

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Fig. 3. Bicycle helmet made from ~ i o ~ o l @ (molecular composition: 91% 3-HB, 9% 3-HV) reinforced with knitted high-performance cellulosic fibers (35 ~01%) that showed crash resistance comparable to that of commercial bicycle helmets (Koch 1993).

Fig. 4. Disintegration and degradation of ~ i o ~ o l @ (molecular Fig. 5. Autoseparative biodegradable air filter made out of a composition: 81% 3-HB, 19% 3-HV) reinforced with cellulosic composite of 90 vol% salt granules and 10 vol% Biopoln fibers, after 20 days in soil. The fiber-matrix interface is (molecular composition: 81% 3-HB, 19% 3-HV). After thermal considered to trigger the degradation process, wherein Biopoln consolidation of the filter composite, the salt granules were degrades slightly faster than the crystalline fiber (Koch 1993). removed by dissolution in water (Wintermantel et al. 1992).

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thermal consolidation of the filter composite, the salt granules were removed by dissolution in water, resulting in an open porous filter matrix. The interconnecting pore structure of the filter is illustrated in Fig. 5.

References

Albertsson, A.C. 1989. The synergism between biodegradation of polyethylene and environmental factors. In Advances in the stabilization and controlled degradation of polymers. Vol. 1. Edited by A.V. Patsis. Technomic Publishing, Lancaster, Basel. pp. 115-122.

American Society for Testing and Materials. 1986a. Standard practice for determining resistance of polymeric materials to fungi (G21-70). In Annual book of ASTM standards. Vol. 8.03. American Society for Testing and Materials, Philadelphia, Pa. pp. 8 17-821.

American Society for Testing and Materials. 1986b. Standard practice for determining resistance of plastics to bacteria (G22-76). In Annual book of ASTM standards. Vol. 8.03. American Society for Testing and Materials, Philadelphia, Pa. pp. 822-825.

American Society for Testing and Materials. 1992. Standard terminology relating to plastics (D833-91a). In Annual book of ASTM standards. Vol. 1 1.01. American Society for Testing and Materials, Philadelphia, Pa. pp. 322-335.

American Society for Testing and Materials. 1994a. Standard test method for determining the aerobic biodegradation of plastic materials in the presence of municipal sludge (D5209-92). In Annual book of ASTM standards. Vol. 8.03. American Society for Testing and Materials, Philadelphia, Pa. pp. 372-375.

American Society for Testing and Materials. 1994b. Standard test method for determining the anaerobic biodegradation of plastic materials in the presence of municipal sludge (D5210-92). In Annual book of ASTM standards. Vol. 8.03. American Society for Testing and Materials, Philadelphia, Pa. pp. 376-379.

American Society for Testing and Materials. 1994c. Standard test method for determining the aerobic biodegradability of degradable plastics by specific microorganisms (D5247-92). In Annual book of ASTM standards. Vol. 8.03. American Society for Testing and Materials, Philadelphia, Pa. pp. 396-399.

American Society for Testing and Materials. 1994d. Standard test method for determining the aerobic biodegradation of plastic materials in an activated-sludge-wastewater-treatment system (D527 1-93). In Annual book of ASTM standards. Vol. 8.03. American Society for Testing and Materials, Philadelphia, Pa. pp. 406-41 1.

American Society for Testing and Materials. 1994e. Standard test method for determining the aerobic biodegradation of plastic materials under controlled composting conditions (D5338-92). In Annual book of ASTM standards. Vol. 8.03. American Society for Testing and Materials, Philadelphia, Pa. pp. 439-443.

Aminabhavi, T.M., Balundgi, R.H., and Cassidy, P.E. 1990. A review on biodegradable plastics. Polym. Plast. Technol. Eng. 29: 235-262.

Augusta, J., Muller, R.J., and Widdecke, H. 1993. A rapid evaluation plate-test for the biodegradability of plastics. Appl. Microbiol. Biotechnol. 39: 673-678.

Brandl, H., and Puchner, P. 1992. Biodegradation of plastic bottles made from "Biopol" in an aquatic ecosystem under in situ conditions. Biodegradation, 2: 237-243.

Brandl, H., Gross, R.A, Lenz, R.W., and Fuller, R.C. 1990. Plastics from bacteria and for bacteria: poly(P- hydroxyalkanoates) as natural, biocompatible, and biodegradable polyesters. Adv. Biochem. Eng. Biotechnol. 41: 77-93.

Brandl, H., Aeberli, B., Bachofen, R., Schwegler, I., Muller, H.-M., Biirger, H.M., Hoffmann, T., Lengweiler, V.D., and Seebach, D. 1995. Biodegradation of cyclic and substituted linear oligomers of poly(3-hydroxybutyrate). Can. J. Microbiol. 41(Suppl. 1): 180- 186.

Brucato, C.L., and Wong, S.S. 1991. Extracellular poly(3-hydroxybutyrate) depolymerase from Penicillium funiculosum: general characteristics and active site studies. Arch. Biochem. Biophys. 290: 497-502.

Budwill, K., Fedorak, P.M., and Page, W.J. 1992. Methanogenic degradation of poly(3-hydroxyalkanoates). Appl. Environ. Microbiol. 58: 1398-1401.

Chowdhury, A.A. 1963. Poly-P-hydroxybuttersaure abbauende Bakterien und Exoenzym. Arch. Mikrobiol. 47: 167-200.

Delafield, F.P., Doudoroff, M., Palleroni, N.J., Lusty, C.J., and Contopoulos, R. 1965. Decomposition of poly-P-hydroxybutyrate by pseudomonads. J. Bacteriol. 90: 1455-1466.

Gilmore, D.F., Fuller, R.C., and Lenz, R.W. 1990. Degradation of poly(beta-hydroxyalkanoates). In Degradable materials. Edited by S.A. Barenberg, J.L. Brash, R. Narayan, and A.E. Redpath. CRC Press, Boca Raton, Fla. pp. 481-507.

Gilmore, D.F., Antoun, S, Lenz, R.W., Goodwin, S., Austin, R., and Fuller, R.C., 1992. The fate of "biodegradable" plastics in municipal leaf compost. J. Ind. Microbiol. 10: 199-206.

In't Veld, P.J.A. 1992. Biodegradable polyesteramides. Ph.D. dissertation, University of Twente, The Netherlands.

Janssen, P.H., and Harfoot C.G. 1990. Ilyobacter delafieldii , sp.nov., a metabolically restricted anaerobic bacterium fermenting PHB. Arch. Microbiol. 154: 253-259.

Jendrossek, D., Knoke, I., Habibian, R.B., Steinbuchel, A., and Schlegel, H.G. 1993. Degradation of poly(3-hydroxybutyrate), PHB, by bacteria and purification of a novel PHB depolymerase from Comamonas sp. J. Environ. Polym. Degrad. 1: 53-63.

Kaplan, D.L., Mayer, J.M., Greenberger, M., Gross, R.A., and McCarthy, S. 1994. Degradation methods and degradation kinetics of polymer films. Polym. Degrad. Stab. 45: 165-172.

Kimura, M., Toyota, K., Iwatsuki, M., and Sawada, H. 1994. Effects of soil conditions on biodegradation of plastics and responsible microorganisms. In Biodegradable plastics and polymers. Edited by Y. Doi and K. Fukada. Elsevier, Amsterdam, London, New York, Tokyo. pp. 92-106.

Koch, B. 1993. D6veloppement d'un nouveau composite biodkgradable : exemples concernant son procCd6 de fabrication, son comportement lors de sa dkgradation et

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Dal

ian

Nat

iona

litie

s U

nive

rsity

on

06/0

6/13

For

pers

onal

use

onl

y.

Minireview / Minisynthese

ses utilisations. Ph.D. thesis, Department of Material Science, Eidgenossische Technische Hochschule, Zurich.

Krupp, L.R., and Jewell, W.J. 1992. Biodegradability of modified plastic films in controlled biological environments. Environ. Sci. Technol. 26: 193-198.

Maddever, J., and Campbell, P.D. 1990. Modified starch based environmentally degradable plastics. In Degradable materials. Edited by S.A. Barenberg, J.L. Brash, R. Narayan, and A.E. Redpath. CRC Press, Boca Raton, Fla. pp. 237-255.

Matavulj, M., and Molitoris, H.P. 1992. Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi. FEMS Microbiol. Rev. 103: 323-33 1.

McLellan, D.W., and Halling, P.J. 1988. Acid tolerant poly(3-hydroxybutyrate) hydrolases from moulds. FEMS Microbiol. Lett. 52: 215-218.

Mergaert, J., Anderson, C., Wouters, A., Swings, J., and Kersters, K. 1992a. Biodegradation of polyhydroxy- alkanoates. FEMS Microbiol. Rev. 103: 317-322.

Mergaert, J., Wouters, A., Swings, J., and Kersters, K. 19923. Microbial flora involved in the biodegradation of polyhydroxyalkanoates. In Biodegradable polymers and plastics. Edited by M. Vert, J. Feijen, A. Albertsson, G. Scott, and E. Chiellini. Royal Society of Chemistry, Cambridge. pp. 207-210.

Mergaert, J., Webb, A., Anderson, C., Wouters, A., and Swings, J. 1993. Microbial degradation of poly(3- hydroxybutyrate) and poly(3-hydroxybutyrate-co-3- hydroxyvalerate) in soils. Appl. Environ. Microbiol. 59: 3233-3238.

Mukai, K., Yamada, K., and Doi, Y. 1993. Enzymatic degradation of poly(hydroxyalkanoates) by a marine bacterium. Polym. Degrad. Stab. 41: 85-91.

Mukai, K., Yamada, K., and Doi, Y. 1994. Efficient hydrolysis of polyhydroxyalkanoates by Pseudomonas stutzeri YM1414 isolated from lake water. Polym. Degrad. Stab. 43: 319-327.

Muller, B., and Jendrossek, D. 1993. Purification and properties of poly(3-hydroxyvaleric acid) depolymerase from Pseudomonas lemoignei. Appl. Microbiol. Biotechnol. 38: 487-492.

Nakayama, K., Saito, T., Fukui, T., Shirakura, Y., and Tomita, K. 1985. Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerase from Pseudomonas lemoignei. Biochim. Biophys. Acta, 827: 63-72.

Palmisano, A.C., and Pettigrew, C.A. 1992. Biodegradability of plastic. Bioscience, 42: 680-685.

Rogers, J.K. 1990. Dow calls for media blitz to stem anti-plastics tide. Mod. Plast. Vol. 67. 1990(March): 10-12.

153

Saito, T., Suzuki, K., Yamamoto, J., Fukui, T., Miwa, K., Kenichi, T., Nakanishi, S., Odani, S., Suzuki, J.I., and Ishikawa, K. 1989. Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. J. Bacteriol. 171: 184-189.

Sawada, H. 1994. Field testing of biodegradable plastics. In Biodegradable plastics and polymers. Edited by Y. Doi and K. Fukada. Elsevier, Amsterdam, London, New York, Tokyo. pp. 298-312.

Schirmer, A., Jendrossek, D., and Schlegel, H.G. 1993. Degradation of poly(3-hydroxyoctanoic acid) [P(3H0)] by bacteria: purification and properties of a P(3HO) depolymerase from Pseudomonas Juorescens GK13. Appl. Environ. Microbiol. 59: 1220-1227.

Schwegler, I. 1992. Untersuchungen des extrazellularen Abbaus von unterschiedlich geschutzten, offenkettigen und zyklischen (R)-3-Hydroxybuttersaure-Oligomeren mit dem Mikroorganismus Pseudomonas delafieldii. Diploma thesis, Department of Chemistry, Eidgenossische Technische Hochschule, Zurich.

Shirakura, Y., Fukui, T., Tanio, T., Nakayama, K., Matsuno, R., and Tomita, K. 1983. An extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Alcaligenes faecalis. Biochim. Biophys. Acta, 748: 331-339.

Shirakura, Y., Fukui, T., Saito, T., Okamoto, Y., Narikawa, T., Koide, K., Tomita, K., Takemasa, T., and Masamune, S. 1986. Degradation of poly(3- hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Biochim. Biophys. Acta, 880: 46-53.

Steinbuchel, A. 1991. Polyhydroxyalkanoic acids. In Biomaterials. Edited by D. Byrom. Stockton Press, New York. pp. 123-213.

Steinbuchel, A. 1992. Biodegradable plastics. Curr. Opin. Biotechnol. 3: 291-297.

Tanio, T., Fukui, T., Saito, Y., Tanid, T., and Tomita, K. 1982. An extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Eur. J. Biochem. 124: 71-77.

Tilstra, L., and Johnsonbaugh, D. 1993. A test method to determine rapidly if polymers are biodegradable. J. Environ. Polym. Degrad. 1: 247-255.

Wintermantel, E., Teichrnann, M., Koch, B., and Mayer, J. 1992. Russ weg - Filter weg, Herstellung eines biologisch abbaubaren Filters. Umweltprojekte 92. Austellung uber Umwelttechnik and Umweltforschung an der Eidgenossischen Technischen Hochschule, Zurich.

Yamada, K., Mukai, K., and Doi, Y. 1993. Enzymatic degradation of poly(hydroxya1kanoates) by Pseudomonas pickettii. Int. J. Biol. Macromol. 15: 215-220.

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