MANUSCRIPT

Fermentation production of poly(3-hydroxyalkanoats)

Ronny Purwadi School of Engineering Hgskolan i Bors Allgatan 1 501 90 Bors, Sweden Tel. +46 33 435 4429 Fax. +46 33 435 40 08 Email : ronny.purwadi@hb.se

Abstract Polyhydroxyalkanoates (PHAs) are polyesters synthesized by numerous bacteria as an intracellular carbon and energy storage compound stored in the cytoplasm of cells. Bacteria including R. eutropha, A. latus, Pseudomonas sp. and recombinant E. coli have been used for the production of PHAs with high productivities. Many kind of polymer are produced by cultivating these bacteria using different carbon sources to obtain different kind of polymer. In fact, numerous enzymes control metabolic pathways of PHA synthesis. Many researches have been carried out to clone and express these enzymes into other micro-organisms which are more productive in term of ability to taken up wide range of cheap carbon sources with high yield and productivity. E. coli has been successfully cloned and become the most powerful bio-factory of PHA production. Additionally, the techniques of fermentation are also the key of success in PHA production. Fed-batch and multi-stages continuous cultivations are chosen as the best fermentation mode for PHA fermentation because they can perform two fermentation phase: growing phase and accumulation phase. In particular, the condition and technique of fermentation are very specific to individual micro-organism. Lastly, product separation is also important since this step has been one of major components of PHA production cost. Solvent extraction and cellular component removal are two methods usually employed for PHA separation. Separation cost is largely caused by the use of removal agents such as solvent and enzyme/detergent, therefore a new low cost separation method is a great interest for making PHA as economically competitive bioplastic.

Keywords: Polyhydroxyalkanoate; Polyhydroxybutyrate; Bioplastics; Fermentation; biopolymers

Contents Abstract ......................................................................................................................................2 Contents .....................................................................................................................................3 1. Introduction............................................................................................................................4 2. Polyhydroxyalkanoates (PHA) ..............................................................................................5 2.1 History..............................................................................................................................5 2.2 Family of polyhydroxyalkanoate .....................................................................................6 2.3 Biosynthesis .....................................................................................................................7 2.3 Properties .........................................................................................................................9 3. Production of PHA by Fermentation ...................................................................................10 3.1 Micro-organisms ............................................................................................................10 3.2 Substrates .......................................................................................................................12 3.4 Fermentation Techniques...............................................................................................12 3.4.1 Fermentation modes................................................................................................13 3.4.1.1 Fed-batch Cultivation.......................................................................................13 3.4.1.2 Continuous Cultivation ....................................................................................14 3.4.2 PHA production in R. eutropha ..............................................................................15 3.4.3 PHA production in A. latus.....................................................................................16 3.4.4 PHA production in Pseudomonas species...............................................................16 3.4.5 PHA production by recombinant E. coli.................................................................17 3.4.6 Anaerobic digestion of biological wastes ...............................................................18 3.5 Product Separation .........................................................................................................18 3.5.1 Solvent extraction ...................................................................................................19 3.5.2 Cellular component removal...................................................................................19 4. Application and commercialization .....................................................................................20 5. Conclusion ...........................................................................................................................21 References................................................................................................................................23

1. Introduction Plastic materials have become an integral part of contemporary life because of many desirable properties including durability and resistance to degradation. There are used in almost every aspect of human lives ranging from household to manufacturing industry. Plastics are synthetic polymers that can be manipulated chemically to have a wide range of strength and shapes. They have molecular weight ranging from 50,000 to 1,000,000 Da [1] and can be easily molded into almost any desire shape. They have high chemical resistance and are more or less elastic, hence popular in many durable, disposal goods and as packaging materials. The disadvantage of plastics is the difficulty in their disposal. In the recent years, there has been increasing public concern over the harmful effect of petrochemical-derived plastic materials in the environment. This has prompted many countries to start developing biodegradable plastic. According to an estimate, more than 100 million tonnes of plastic are produced every year. Incinerating plastic has been one option in dealing with non-degradable plastics, but in addition to its high cost, it is dangerous since harmful chemicals like hydrogen chloride and hydrogen cyanide are released during incineration. Recently, the problems concerning the global environment and solid waste management have created much interest in the development of biodegradable plastics, which must still retain the desired physical and chemical properties of conventional synthetic plastics. Some of the biodegradable plastic materials under development include polyhydroxyalkanoate (PHA), polylactide, aliphatic polyesters, polysaccharides, and the copolymers and/or blends of these. However, one of the problems facing the development of biodegradable polymers as substitutes for conventional plastics is their high production cost compared with petroleum-derived plastics.

Polyhydroxyalkanoates are 100% biodegradable polymers. They are polyester of various hydroxyalkanoates (HAs) which are synthesized by numerous micro-organisms as energy reserve materials under imbalance condition of substrates. They have similar properties to various synthetic thermoplastic like polypropylene and hence can be used in their place. They are also totally degraded to water and carbon dioxide under aerobic conditions and to methane under anerobic conditions by micro-organisms in soil, lake water, sewages and sea water. To date, much effort has been devoted to develop a process for the economical production of PHAs. A cost estimation for PHA production has been reported [2]. The particular components of the cost are mainly the raw material costs, the running/operational cost, and labour cost. The first term is dependent on cultivation yield, while the second term is dependent on cultivation productivity. It is obvious that both yield and productivity are closely related to choosing a good strain and applying appropriate cultivation methods. This paper is an attempt to summarize recent advance of PHA production, by discussing the basic knowledge, biosynthesis and the production aspects of PHA including micro-organism candidates, raw materials and fermentation techniques.

2. Polyhydroxyalkanoates (PHA) 2.1 History PHAs have been observed as a granules in bacterial cells under microscope since at least back to Beijerinck in 1888 [3]. The composition of PHA was discovered by Lemoigne (1927) who identified the exertion of 3-hydroxybutyric acid by Bacillus megaterium. A proposal of functional role for poly(3-hydroxybutyric acid) or P(3HB) came from Macrae and Wlkinson in 1958. They observed that B. megaterium stored the homopolymer when the glucose-to-nitrogen source ration of the medium was high. They concluded that P(3HB) was

a carbon- and energy-reserve material. In the following fifteen years, the research was developed in various aspects from micro-organisms to the properties of the polymer produced. The potential usefulness of PHAs were initiated in the 1960s, where many patents related to P(3HB) were published. However, petroleum-based plastic which were cheap and ease hinder the idea to use natural plastics. The fact that fossil fuels would not remain low priced and a public concern for environmental issues gave little incentive to push for the development of a natural plastics industry. Only after the oil crisis of 1973, the doubts on the future of petroleum-based polymer industry lead the search for alternate types of plastic materials. In 1976, Imperial Chemical Industries (ICI) started to investigate if P(3HB) is profitable enough to be commercialized by bacterial fermentation. The research was diverse to the properties of P(3HB) such as biodegradability and biocompatibility, piezoelectric properties and source of optically active molecules. The first report of the hetero-polymer of PHA was from Wallen and Davis (1972) who found the presence of 3-hydroxyvalerate (3HV) unit in the P(3HB) chains. This finding pushed the research to find another possibility in regard of polymer properties. Today, PHAs is produced by various companies with different trademarks. Many strains of microorganism have been employed and engineered to produce a highly content of polymers with a tremendous cells growth. This attempt is likely to beat the comparable price with the synthetic plastics. 2.2 Family of polyhydroxyalkanoate PHAs are polyester compromising hydroxycarboxylate monomers. These monomers have carbon chains ranging from 3 to 18 carbons. The structure of PHAs including homoand hetero-polymer of PHA is shown in Fig. 1. Ninety-one the most possible hydroxyalkanoate as constituent of biosynthetic PHAs has been reported [4]. In nature, PHAs occur as water-insoluble inclusion bodies within the cytoplasm of a variety of bacteria.

Biological PHA is composed of 3-hydroxy-carboxylate units, all in the (R) configuration, i.e., biological PHAs are isotactic. Depending on the number of carbon atom in the chain PHAs can be divided into two groups: short-chain length (SCL) which consist 3-5 carbon atoms, and medium-chain length (MCL) which consist 6-14 carbon atoms [5]. This difference is mainly due to the substrate specificity of the PHA synthases that can accept 3HAs of a certain range of carbon length. It means that different combinations of substrates and micro-organism lead to various type of polymer. The difference of those groups or its elements are regarding to the properties of the polymers which will be discussed later. The other well-known PHASCL is the copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV), which comprise of fourand five-carbon monomeric units (Fig. 1c). The proportion of these monomeric units can vary, and this affects the physical properties of the polymer, i.e. less brittle with increasing proportion of 3HV unit. 2.3 Biosynthesis PHAs are obtained from three ways: biosynthesis by micro-organisms, photosynthesis by transgenic plants, and in vitro biosynthesis using appropriate enzymes. The last two are beyond of our discussion since we are focusing to the fermentation production of PHA. In most bacteria, cells synthesize PHA under growth-limiting substrates other than carbon source such as nitrogen, phosphorus or oxygen. Accumulated PHA serves as both carbon and energy source during starvation. PHA also serves as a sink for reducing power and could therefore be regarded as a redox regulator within the cell. The biosynthesis pathways of PHA production are varied regarding to carbon sources applied as well as PHA products wanted. At least there are four major pathways in producing PHA found to date [3, 5-9]. The first pathway is a typical PHB synthesis from glucose which is usually found in R. eutropha: two acetyl-CoA are condensed by a -ketothiolase (PhaA) to

acetoacetyl-CoA. An NADPH-dependent reductase (PhaB) then carries out its conversion to (R)-3-hydroxybutyril-CoA. The final step is the polymerisation reaction catalyzed by PHA synthase (PhaC). The second pathway is commonly found in Rhodopsuedomonas rubrum (not shown in Fig. 1): acetoacetyl-CoA formed by -ketothiolase is reduced by a NADH dependent reductase to (S)-3-hydroxybutyril-CoA which is then converted further to (R)-3hydroxybutyril-CoA by two enoyl-CoA hydratases. When propionic acid present in the medium, -ketothiolase condenses one propionic-CoA with one acetyl-CoA to form ketovaleryl-CoA which is polymerized after reduction to 3-hydroxyvalerate monomer by PHA synthase. This pathway is one example to produce PHA copolymer from other substrates. Another pathway is related to the production of medium length PHA polymer, which usually found in Psuedomonas species including -oxidation of fatty acid biosynthesis. Through these paths, the bacteria can accumulate PHA consisting 3-hydroxyalkanoic acid when the cells are cultivated on alkanes, alkanols or alkanoic acids. If fatty acids are used as substrates, the -oxidation of fatty acid take place as chain elongation reaction with 2ketoacetyl-CoA, enoyl-CoA and (S)-3-OH-hydroxyacyl-CoA as intermediate precursors to PHA synthesis. At the absence of fatty acid, cells can assemble their own fatty acid from carbohydrate substrates through de novo fatty acid synthesis where (R)-3-hydroxyacyl-ACP is an intermediate precursor for PHA synthesis. Various factors could affect the accumulation of PHA of cells, hence also the final product properties such as molecular weight and size. The pH of culture medium can greatly affect the molecular weight of the PHA produced [6] while concentration of the carbon sources supplied can also affect the molecular weight. The number of granule in cytoplasm remains constant at eight to twelve in the nitrogen limitation condition. Furthermore, the accommodation of additional polymer was done through increases in the diameter of the granules, gradually forcing the cells to change their shape from cylindrical to spherical. The

cells stopped increase at around 80% of the polymer. Therefore, the physical constrains could be one of limiting factor for polymer accumulation. 2.3 Properties The PHAs are non-toxic, biocompatible, biodegradable thermoplastics that can be produced from renewable sources. They have high degree of polymerization, are highly crystalline, optical active and isotactic (stereochemical regularity in repeating units), piezoelectric and insoluble in water. These features make them highly competitive with polypropylene, the petrochemical-derived plastic [1, 8]. The copolymer of PHA has more useful material properties such as lower crystallinity, melting temperature, stiffness, and tensile strength while toughness and oxygen permeability are increased [8]. Moreover, addition of plastisizer and nucleation agent lead to lower glass temperature and lower crystallinity [10]. The physicochemical properties comparison of homo-polymer, co-polymer and polypropylene is shown in Table 1. One unique property of biological PHA is its biodegradability in environments. Many reports have been shown the biodegradability of PHA materials in natural environments such as soil, sea water and lake water, which was influenced by a number of factor such as microbial population, temperature, moisture level, pH, nutrient supply as well as composition, crystallinity, additives and surface area of PHA itself. Various micro-organisms in nature excrete extracellular PHA depolymerases that hydrolyse PHA into water soluble oligomers and monomers and subsequently utilize it as nutrients within the cells. Many such micro-organisms including bacteria and fungi have been isolated from various environments as shown in Table 2.

3. Production of PHA by Fermentation 3.1 Micro-organisms Bacteria used for the production of PHAs can be divided to two major groups based on the culture conditions required for PHA synthesis [5]. First group of bacteria requires the limitation of an essential nutrient such as nitrogen, phosphorus, magnesium, or sulphur for the synthesis of PHA from an excess carbon source. The bacteria included in this group are R. eutropha, Protomonas extorquens, and Protomonas oleovorans. The second group of bacteria do not require nutrient limitation for PHA synthesis and the polymer is accumulated during growth phase. It is included Alcaligenes latus, a mutant strain of Azotobacter vinelandii, and recombinant E. coli. These characteristics are important to be considered while production of PHA. About 300 different bacteria have been identified having ability to synthesize PHA [11]. Many works have been carried out to discover a new PHA synthesis species as well as to optimize the existing bacteria. These works reported the use of various bacteria including cyanobacteria [12], Paracoccus denitrificans [13], P. stutzeri [14], P. aeruginosa [15], recombinant Bacillus subtilis [16], Delftia acidovorans [17], Azotobacter chroococcum [18], Azobacter vinelandii [19], Comamonas sp [20], Streptomyces aureofaciens [21], and Aeromonas hydrophila [22]. However, only few bacteria are used for industrial production of PHA including R. eutropha which is largely used for PHB biosynthesis, Psuedomonas oleovorans and Psuedomonas putida for production of PHAMCL, while all fuctional genes for PHA production are successfully cloned and expressed in E. coli [8]. E. coli has been chosen as a better commercial producer of PHA because it can use a wider range of cheap carbon sources, and also it is relative easy to extract and purify the polymer from this bacteria. Moreover, E. coli do not have an intracellular PHA depolymerase since this bacteria is not a

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PHA producer, thus the synthesized PHA will not directly degraded. This is probably one of the reason that had been enabled the production of very high molecular weight PHA using recombinant E. coli [6]. The current knowledge of their biosynthetic pathways and regulation has allowed the construction of recombinant organisms (other microbes, yeasts and plants) able to synthesize PHA from inexpensive carbon sources (e.g. molasses, sucrose, lactose, glycerol, oils and methane) [7]. The important enzyme in PHA synthesis is PHA synthase which is bound to the surface of PHA granule together with other proteins. The biosynthesis of PHA through this enzyme is independent from a template and with the high activity of the enzyme; various products with relative high molecular weight could be formed. More than 60 PHA synthase genes from eurobacteria have been cloned and sequenced. The knowledge on PHA synthases and efficient screening system are now utilized to obtain modified PHA synthases with new properties or enhanced in vivo or in vitro PHA synthase activity by in vivo random mutagenesis, in vitro site-directed mutagenesis and gene shuffling [4]. The genes of the PHA synthesis has also been successfully established in eucaryote such as yeast [23]. Even though the PHA has been produced, the yield was still low enough compare with bacteria. As an alternative of single culture, mixed culture could also be used to synthesize PHA. The mixed cultures are usually used in wastewater treatment. Activated sludge, a wellknown mixed culture, is able to store PHA as carbon and energy storage material under unsteady condition. The micro-organisms involved experience rapidly changing conditions of availability of nutrients and can adapt continuously to change in substrate since biological wastewater treatment usually occurs under dynamic conditions. Activated sludge accumulates PHA to around 20% of dry weight under unaerobic conditions compare with pure culture which can reach more than 88% of cell dry weight [11]. The PHA content of activated sludge can be increase to 62% in a microaerophilic-aerobic sludge process. The use of mixed culture

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would be very interesting since has an enhanced economy, simpler process control, requirement of monoseptic processing, and an improved use of wastes. 3.2 Substrates PHAs are very attractive as a substitute for petroleum-derived polymers regarding to its possibility to produce from renewable resources and harmless to the environment due to its biodegradability. However, the major hurdle facing commercial production and application of PHA in consumer products is the high cost of bacterial fermentation. It makes bacterial PHA production 5-10 times more expensive than the petroleum-derived polymers such polyethylene and polypropylene [5]. The significant factor of the production cost of PHA is the cost of substrate (mainly carbon source). In order to decrease this cost, the use of cheap carbon sources as substrates are developed. The researches have been carried out to develop recombinant strains utilising a cheap carbon source, while corresponding fermentation strategies have been developed and optimized. The uses of alternative carbon sources and corresponding strain developments have been reported including glycerol as co-product of many industrial processes using P. oleovorans [24], wastewater using activated sludge [25-27], glutamic acid in the wastewater [28], olive oil mill effluents [18, 29], palm oil mill effluents [30], soyabean oil [31], and agricultural waste [32]. However, the polymer concentration and content obtained were considerably lower than those obtained using purified carbon substrates. Therefore, there is a need for development of more efficient fermentation strategies for production of these polymers from a cheap carbon source. 3.4 Fermentation Techniques In general, the fermentation condition should be design in order to direct the cells to accumulate the carbon source inside their body in a form of PHA polymer rather than using it

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for reproduction. However, the production of PHA by fermentation is very dependent to the nature of micro-organism used for this purpose. Hence, the discussion of general fermentation modes which are usually employed in the production of PHA is discussed in advance, followed by the discussion of fermentation using particular type of PHA synthesis micro-organisms. 3.4.1 Fermentation modes In general, the goal of fermentation of PHA production is to let micro-organism to accumulate available carbon source in medium as a carbon- and energy-source in form of PHA polymer inside their body rather than to use it for their reproduction. For this purpose, imbalance nutrients should be provided to prevent the cells to carry out their reproduction. However, initial cells concentration should be high enough in order to achieve high productivity. Hence, the strategies of fermentation are to increase the density of cells by providing enough nutrients in medium and followed by introducing a condition where growth nutrients except carbon source are limited. These strategies have been achieved by fed-batch or multistage continuous fermentation. 3.4.1.1 Fed-batch Cultivation In fed-batch fermentation, an initial batch step is carried out to achieve high cell density and followed by a continuous step which limits growth nutrients except carbon source. These cultivation strategies usually rely on open-loop control and indirect monitoring and control system using dissolved oxygen concentration in the bioreactor as a measure of carbon source uptake rate. Other method use a pH-stat system which couple pH control to nutrient or substrate feeding. Although these control systems are useful for single substrate feeding condition, they are not suitable for fermentation with two or more carbon sources, i.e. for production of co-polymer. In these cases, on-line closed-loop control for maintaining each

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substrate concentration is necessary. A number of direct concentration analyses have been used such as on-line high-performance liquid chromatography (HPLC) and flow injection analysis (FIA). Use of a computer based automated on-line close-loop control for high-celldensity fermentation system for the production of PHAMCL has been reported [33]. At discontinuous regime of fed-batch cultures, a high carbon-to-nitrogen ratio (i.e. 40 mol/mol) is usually employed as a substrate. Decreasing the C/N ratio led to a gradually inhibition of polymer synthesis. It is obvious because a limitation of essential nutrients not only stops cell growth, but also affects cell physiology and performance [9]. 3.4.1.2 Continuous Cultivation Generally, growth step and accumulation step can be performed in multi-stages (at least two stages) of continuous cultivation. The first-stage is cultivation on glucose and excess nitrogen to produce a high density of cells, and then followed with second-stage which is cultivation on non-carbon-growth-nutrients limited condition to accumulate polymers inside the cells. The rate of growth and polymer accumulation are usually different and cause a difference in retention times of first- and second-stage fermenter thus dilution rates. As an example, production of PHAMCL by P. oleovorans has been carried out in two-stage continuous cultivation with dilution rate of 0.21 and 0.16 h1 for first- and second-stage respectively [34]. The capability of P. oleovorans to accumulate PHA is dependent on its specific growth rate, and the accumulation reach the maximum point when the strain grows at 0.2 h1, which is lower than the maximum specific growth rate of 0.48 h1. Even though continuous cultivation has higher productivity due to its flow rate, incomplete utilization of substrates because of high carbon-to-nitrogen ratio in feed is often encountered. These unutilized substrates can be recovered easily and cheaply and recycled back in to the process. However, the use of multi-stage cultivation can advantageously increase the time of exposure for the bacteria to conditions favourable for polymer 14

accumulation, leading to higher yield and productivity [3]. Therefore, the use of multi-stage cultivation could be better than substrate recycle to achieve a high yield and profitable production of PHAs. The use of other type of bio-reactor such as continuous plug flow bio-reactor as a second-stage fermenter has also been reported in case of PHA production from carbohydrate using R. eutropha and A. latus [3] 3.4.2 PHA production in R. eutropha R. eutropha is a typical bacterium which accumulates PHA in a nutrient-limited condition except carbon source. This bacterium has been studied most extensively due to its ability to accumulate large amount of P(3HB), around 80% (w/w) of dry cell mass, from simple carbon source. In fed-batch cultivation, R. eutropha is grown in a glucose-salts medium containing only the calculated amount of phosphate to support a desire amount of cell growth. The phosphate limitation has an advantage due to independency of a use of nitrogen source (NH4OH) which usually used as a pH control solution [35]. Cells encounter phosphate limitation after 60 h and accumulate P(3HB) during the next 40-60 h from supplied glucose. In example, by controlling the glucose concentration at 10 to 20 g/L during the fedbatch culture, the final cell mass reach 164 g/L with P(3HB) concentration of 121 g/L and P(3HB) content of 76% [5, 36]; resulting in the highest productivity of 2.42 g/L.h. In a production of P(3HB-co-3HV), both glucose and propionic acid are fed during the polymer accumulation phase. Typically, P(3HB-co-3HV) copolymer containing 0 to 30 mol% of 3-hydroxyvalerate are produced, and that fraction can be controlled by varying the ratio of glucose to propionic acid in the feed [36]. Continuous production of P(3HB) by R. eutropha in two-stage culture system has also been demonstrated [5, 37]. The first-stage cultivation on glucose produced maximum cell dry weight of 27.1 g/L at dilution rate of 0.21 h1 while in the second-stage cultivation; 47.1 g/L

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PHB polymer was accumulated at a dilution rate of 0.14 h1. PHB productivity was 1.23 g/L.h, yield PHB to glucose was 0.36 g/g and PHB content was 72.1%. 3.4.3 PHA production in A. latus A. latus is considered as a good P(3HB) synthesizer since it grows fast and accumulates P(3HB) during the growth phase [5, 36]. It is also reported that the bacteria can utilize cheap carbon sources such as sucrose, beet or cane molasses for P(3HB) production. A. latus DSM 1123 grow and produce P(3HB) at 35C, thus lowering cooling demands. In addition, this strain only need 5 h to reach an intracellular P(3HB) concentration of 80% of dry cell mass and synthesis of PHB is growth-associated. The P(3HB) concentration in this strain has been increase from 50 to 87% in fed-batch cultivation by introducing of nitrogen limitation. The use of cheap carbon sources such as molasses or sugar syrup is also interesting. Batch culture of A. latus ATCC 29713 was reported to produce P(3HB) up to 63% of dry cell mass after 93 h of culture when sucrose used as a carbon source. The cultivation achieves a yield of 0.4 g/g. Fed-batch culture with the same strain reaches maximum specific growth rate of 0.265 h1 compared with 0.075 h1 achieved in batch culture. The maximum P(3HB) productivity was reported as 1.15 g/L.h [5]. 3.4.4 PHA production in Pseudomonas species Production of PHAMCL by fed-batch and continuous culture of P. oleovorans has been investigated to achieve high concentration of PHA and high productivity. With optimization of continuous culture conditions the steady state cell concentration has been achieved as 11.6 g/L with productivity of 0.58 g/L.h [36]. P. stutzeri 1317 was found to synthesize a variety of PHAs when grown in glucose and/or fatty acid. It was found that the monomeric structure of the PHAs was dependent on the structure of related fatty acids taken as carbon sources.

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Monomer content in PHA was adjusted by regulating ratios of fatty acids in growth substrates. Thus, the PHA structures can be tailor made by using structure-related fatty acids and varying ratios of fatty acids [5]. In the production of PHAMCL by native PHAMCL-producing strains, once PHAMCL granules are formed, the growth rate decreases [9]. It may be because the intercellular granules interfere with cell division. Hence, the production of a desire high cell density in advance with sufficient nutrients supply is one of preferred strategy, while then continued with cultivation in limited nutrient condition to accumulate the polymers. In using PHAMCLproducing recombinant strains, polymer synthesis can be induced by desired, without nutrient starvation. This may be important because a limitation fro essential nutrient not only stops cell growth, but also affects cell physiology and performance. 3.4.5 PHA production by recombinant E. coli Escherichia coli have been used for production of various proteins because it has been studied most extensively. The advantages of PHA production by recombinant E. coli are (1) fast growth to a high cell density resulting in high productivity, (2) accumulation of a large amount of polymers, (3) ability to utilize several inexpensive carbon sources, (4) easy purification of PHA, and (5) the lact of a depolymerase system active against the synthesized polymers. PHA biosynthesis genes of R. eutropha were cloned into E. coli, and it was found that these genes were constitutively expressed and a large amount of P(3HB) could be accumulated. The accumulation of P(3HB) in recombinant E. coli could reach 80-90% of dry cell mass at the end of cultivation of a fed-batch culture with the P(3HB) concentration of higher than 80 g/L and productivity of greater than 2 g P(3HB)/L.h. The use of stable highcopy-number plasmid was necessary to allow efficient synthesis of P(3HB) in recombinant E. coli.

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The P(3HB-co-3HV) copolymer could also be produced employing mutan strain E. coli using glucose and propionic acid as substrates. Copolymer consisting up to 40 mol % of 3-hydroxyvalerate could be obtained. E. coli bacteria do not need a limitation of special nutrient, but is dependent on the amount of acetyl-CoA available. The production of P(3HB) was significantly enhanced by the addition of various complex nitrogen sources, amino acid or oleic acid, which seemed to be due the fact that in these conditions more acetyl-CoA and/or NADPH, a cofactor of the reductase in the PHA synthetic pathway, were available for PHA synthesis. However, the problem of the high oxygen demand during the high cell density culture of recombinant E. coli needs to be solved to allow this process to be an economical alternative to the ones currently in use. 3.4.6 Anaerobic digestion of biological wastes Production of PHA using mixed culture is usually carried out in a wastewater treatment. An anaerobic digestion is used to carry out this process. Anaerobic digestion is a multi-step process. First, complex bio-molecules are hydrolyzed into soluble, biodegradable organic materials. Then acidogenic and acetogenic bacteria metabolize the hydrolytic product to lower volatile fatty acid (VFAs) which are the precursor to PHA synthesis. Sources of VFAs include municipal wastes, banana, damaged food grains, pea shells, apple pomace and palm oil mill effluent. Various bacteria like R. eutropha, Bacillus megaterium, P. oleovorans, Azotobacter beijerincki, Rhizobium, Nocardia utilize lower VFAs as substrate for PHA production [1]. 3.5 Product Separation After the fermentation, cells containing PHA polymer are separated from the medium by common solid separation such as centrifugation, filtration or flocculation-centrifugation.

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Separation of PHA from inclusion body can be carried out by two different categories of methods. The first is to extract the PHA polymer from cells while the second is to remove the cellular component other than PHA. Since the PHA is water-insoluble, the first category uses PHA soluble solvent to extract PHA and leave the cellular component in the water solution. On the contrary, the second category breaks inclusion body and washes the insoluble PHA from the cell debits. 3.5.1 Solvent extraction Solvents such as chloroform, methylene chloride, propylene chloride, and dichlororethane are usually employed for extracting PHA polymer from cell body. High amount of solvent is used for extracting PHA polymer due to the high viscosity of even dilute PHA solution. About 20 of solvent are required to extract 1 of polymer. The large amount of solvent required makes this method economically unattractive, even after the recycle of the solvent. Hypochlorite salt solution can also be employed for PHA extraction. This method is effective in the digestion of non-PHA cellular materials. However, hypochlorite causes severe degradation of P(3HB) resulting in a 50% reduction in the molecular weight [36]. The mixture of hypochlorite and chloroform significantly reduce degradation of PHA due to immediate PHA dissolution by chloroform after isolated by hypochlorite and thus protects polymer from degradation. Normally, purity of greater than 95% is obtained by hypochlorite treatment. 3.5.2 Cellular component removal Enzymes such as lysozyme, proteinases, DNAses, etc. can be used to lyse the cells thus separate PHA out from the cells. By this method, most of cell materials are solubilized by the enzymes but not the PHA. After digestion, the polymer is washed with anionic surfactant to separate non-PHA cellular materials. Treatment with sodium hydroxide or

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ammonium hydroxide is suitable for the cells with high accumulation of PHA because the cell wall become fragile at high polymer content. A. vinelandii and recombinant E. coli are the example of this type of bacteria. It also has been report that such method decreases the production cost of P(3HB) by 25% [38]. The recovery of PHA contributes significantly to the overall PHA production cost. Development of a process that allows the simple and efficient extraction of polymers will be well rewarded. 4. Application and commercialization PHAs have been considerably industrial interest as biodegradable and/or biocompatible plastic for wide range of application. Initially, they were used in packaging films mainly in bags containers and paper coatings. Similar applications as conventional commodity plastics include the disposable items, such as razors, utensils, diapers, feminine hygiene products and cosmetic containers (shampoo bottles and cups). PHAs are also useful as stereo regular compounds which can serve as chiral precursors for the chemical synthetic of optically active compounds. Such compounds are particularly used as biodegradable carriers for long-term dosage of drugs, medicines, hormones, insecticides and herbicides [1]. They are also used as osteosynthetic materials in the stimulation of bone growth owing to their piezoelectric properties, in bone plates, surgical sutures and blood vessel replacements [5]. PHAMCL have interesting potential applications in coating, and in medical temporary implants, such as scaffolding for the regeneration of arteries and nerves axons. Recent examples include the use of PHAMCL in water-based latex paints, developed by ATO in the Netherlands and the use of PHAMCL in vein scaffolding for vascular-deficient patients, developed by Metabolix [9].

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Commercial application and wide use of PHA is hampered due to its price. The cost of PHA using the natural producer R. eutropha is US$16 per Kg which is 18 times more expensive than polyethylene [1]. With recombinant E. coli as producer of PHA, price can be reduced to US$4 per Kg, which is close to other biodegradable plastic materials such as PLA and aliphatic polyester. The commercially viable price should come to US$3-5 per Kg. A number of polymer companies have been commercially producing PHA polymer, and some of them are listed in Table III. Metabolix has developed many kinds of PHA polymer including its copolymers and terpolymers, using high-productivity E. coli K12. They claim that PHA is accumulated with concentration of 100 g/L in 40 hours and reaches up to 90% of dry cell mass at the end of cultivation [39]. Their commercial scale trials have validated production cost of US% 1.00 per pound [40]. ZENECA Bio Products produces Biopol at commercial scale from R. eutropha (H16) with the capacity of 1000 tonnes per annum.

5. Conclusion The prices of PHAs are the most interesting issue to date. Although PHAs have been produced in commercial scales, the uses of these materials are still limited due to their high prices compare to petroleum-derived plastic such as polyethylene and polypropylene. However, the production of bioplastics such as PHAs are still considered as sustainable processes where use renewable resources and also produce no net additional CO2 to the atmosphere. The biodegradability of these bioplastics gives additional positive value in regard to the environmental issues. Current advances in fermentation and purification technology as well as development of superior bacterial strains by recombinant DNA technology are likely to lower the price of PHA. The isolation and development of bacterial strains that can utilize cheap carbon

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substrate should be pursued intensively. Even though several bacteria strains will be employed for the production of PHAs in the next few years, eventually metabolically engineered bacterial strains may likely surpass the wild-type bacteria currently in use. In addition, unusual PHAs, such as those containing various functional side groups, will remain expensive due to the high substrate cost, and await the development of suitable application areas. Furthermore, inexpensive but effective recovery process needs to be developed for overall cost reduction. Multidisciplinary research effort by microbiologists, geneticists, chemists, polymer scientists, and government officials is required for the successful commercialization of PHAs. With all these effort, it is to be expected that PHAs will become a major biodegradable plastic material in a wide range of application in the near future.

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References [1] [2] [3] Reddy CSK, Ghai R, Kalia RVC. Polyhydroxyalkanoates: an overview. Bioresource Technology 2003; 87:137-146. Yamane T. Cultivation engineering of microbial bioplastics production. FEMS Microbiology Letters 1992; 103(2-4):257. Braunegg G, Lefebvre G, Genser KF. Polyhydroxyalkanoates, biopolyesters from renewable resources: Physiological and engineering aspects. Journal of Biotechnology 1998; 64:127-161. Steinbuchel A, Valentin HE. Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiology Letters 1995; 128(3):219. Khanna S, Srivastava AK. Recent advances in microbial polyhydroxyalkanoates. Process Biochemistry 2005; 40(2):607-619. Sudesh K, Abe H, Doi Y. Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Progress in Polymer Science 2000; 25:1503-1555. Luengo JM, Garcia B, Sandoval A, Naharro G, Olivera ER. Bioplastics from microorganisms. Current Opinion in Microbiology 2003; 6(3):251. Tan IKP. Polyhydroxyalkanoates. In: editor^editors. Kirk-Othmer Encyclopedia of Chemcial Technology. John Wiley & Sons, Inc, 2003. p. Witholt B, Kessler B. Perspectives of medium chain length poly(hydroxyalkanoates), a versatile set of bacterial bioplastics. Current Opinion in Biotechnology 1999; 10(3):279. El-Hadi A, Schnabel R, Straube E, Muller G, Henning S. Correlation between degree of crystallinity, morphology, glass temperature, mechanical properties and biodegradation of poly (3-hydroxyalkanoate) PHAs and their blends. Polymer Testing 2002; 21(6):665. Salehizadeh H, Loosdrecht MCMV. Production of polyhydroxyalkanolates by mixed culture: recent trends and biotechnological importance. Biotechnology Advances 2004; 22:261-279. Asada Y, Miyake M, Miyake J, Kurane R, Tokiwa Y. Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria--the metabolism and potential for CO2 recycling. International Journal of Biological Macromolecules 1999; 25(1-3):37. Gao D, Maehara A, Yamane T, Ueda S. Identification of the intracellular polyhydroxyalkanoate depolymerase gene of Paracoccus denitrificans and some properties of the gene product. FEMS Microbiology Letters 2001; 196(2):159. Guo-Qiang C, Jun X, Qiong W, Zengming Z, Kwok-Ping H. Synthesis of copolyesters consisting of medium-chain-length [beta]-hydroxyalkanoates by Pseudomonas stutzeri 1317. Reactive and Functional Polymers 2001; 48(1-3):107. Hori K, Marsudi S, Unno H. Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa. Biotechnology and Bioengineering 2002; 78(6):699-707. Law KH, Cheng YC, Leung YC, Lo WH, Chua H, Yu HF. Construction of recombinant Bacillus subtilis strains for polyhydroxyalkanoates synthesis. Biochemical Engineering Journal 2003; 16(2):203. Mothes G, Ackermann JU. Synthesis of Poly(3-hydroxybutyrate-co-4-hydrobutyrate) with a Target Mole Fraction of 4-Hydroxybutyric Acid Units by Two-Stage Continuous Cultivation of Delftia acidovorans P4a. Engineering in Life Sciences 2005; 5(1):58-62.

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Figures:
CH3 (CH2)n O CH O CH2 (a) CH3 CH3 CH O CH2 O C O x (c) CH2 CH CH2 y O C C O x CH3 CH CH2 (b) O C x

Figure 1. Structures of PHAs: (a) General structure of P(3HA), (b) homopolymer of P(3HB), (c) copolymer of P(3HB-co-3HV).

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Figure 2. The different pathways involved in the biosysthesis and catabolism of PHAs. The enzymes involved in: alkane oxidation (1) Alkane 1-momoxygenase, (2) alcohol dehydrogenase, (3) aldehyde dehydrogenase; fatty-acid -oxidation (4) acyl-CoA ligase, (5) acyl-CoA dehydrogenase, (6) enoyl-CoA hydratase, (7) 3-hydroxiacyl-CoA dehydrogenase, (8) 3-ketothiolase, (9) (R)-enoyl-CoA hydratase, (10) 3-ketoacyl-CoA reductase; biosynthesis from carbohydrate (11) -ketohiolase, (15) 3-ketoacyl-ACP synthase (16) 3-ketoacyl-ACP reductase, (17) 3-hydroxyacyl-ACP reductase (18) enoyl-ACP reductase, (19) 3-hydroxyacyl-ACP-CoA transacylase.

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Tables:
Polymer Molecular weight (Mw, 105) Melting temperature (Tm, C) Glass transition temperature (Tg, C) 5 6 Tensile strength (MPa) Young's modulus (GPa) Elongation to break (%) 8 10 300450 400

P(3HB) 2 180 40 4.0 P(3HB-3HV) 3 137 30 0.7 25 mol% 3HV P(3HO)a 2 68 35 610 2.59 polypropylene 2 180 10 38 1.7 a Polyhydroxyoctanoate is P(3HO), a PHAMCL with predominant hydroxyoctanoate monomers.

Table 1. Physicochemical properties comparison of PHA and polypropylene. (adopted from [8])

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Source from which isolated Soil

Micro-organism Aspergillus fumigatus Acidovorax faecalis Comamonas sp. Pseudomonas lemoignei Variovorax paradoxus A. faecalis Pseudomonas fluorescens Comamonas tertosteroni P. stutzeri Ilyobacter delafieldii

Activated sludge Sea water Lake water Anaeorbic sludge

Table 2. PHA degrading micro-organism isolated from various environments (adopted from [5])

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Company ZENECA Bio Products (UK) ZENECA Seeds (UK) Metabolix, Inc. (USA) Monsanto (USA) Polyferm, Inc. (Canada) BioVentures Alberta Inc. (Canada) Bioscience Ltd (Finland) Berlin Packaging Corp. (USA)

Area of interest Production of P(3HB) by fed-batch culture of R. eutropha Production of PHA by transgenic plants (rapeseed) Production of PHA by transgenic plants. Production of PHA by transgenic plants (rapeseed and soybean) Production of PHA from cheap substrate (hemicellulose) Production of PHA by recombinant E. coli Medical application of PHAs Marketing, sales, and distribution of ZENECAs BIOPOL

Table 3. Industrial production of PHAs and applications

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