Microbial Poly-3-Hydroxybutyrate and Related Copolymers

CHAPTER 17

Microbial Poly-3-Hydroxybutyrate and Related Copolymers Raveendran Sindhu, Parameswaran Binod, Ashok Pandey Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India

Contents 1. Introduction 2. PHB-Producing Microbes 2.1 Screening of Microorganisms 2.2 PHB Production Using Wild-Type Bacteria 2.3 PHB Production Using Recombinant Bacteria 2.4 PHB Production Using Mixed or Cocultures 2.5 PHB Production by Cyanobacteria 3. Fermentation Strategies 3.1 Submerged Fermentation

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3.1.1 Batch 3.1.2 Fed-Batch 3.1.3 Continuous

3.2 Solid-State Fermentation 4. Downstream Operations 4.1 Chemical Methods 4.2 Biological Methods 5. Characterization Techniques 5.1 Fourier Transform Infrared Spectroscopy 5.2 Nuclear Magnetic Resonance 5.3 Thermogravimetric Analysis 5.4 Differential Scanning Calorimetry 5.5 Gel Permeation Chromatography 5.6 Atomic Force Microscopy 6. Strain Improvement, Mutation, and Metabolic Engineering 7. Substrate Manipulation for the Production of Various Classes of PHB 7.1 Importance of External Substrate Addition 7.2 Manipulation of Carbon Sources

7.2.1 Properties of PHB due to Carbon Manipulation

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7.3 Effect of Nitrogen and Phosphorus 7.4 Inhibitor Addition 8. Applications 8.1 Medical

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8.1.1 Targeted Drug Delivery 8.1.2 Nanofibrous Matrices as Cell Supporting Materials

Industrial Biorefineries and White Biotechnology http://dx.doi.org/10.1016/B978-0-444-63453-5.00019-7

Copyright © 2015 Elsevier B.V. All rights reserved.

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8.1.3 Biomedical Application of PHB Sutures 8.1.4 Organic-Soluble Chitosan/PHB Ultrafine Fibers for Skin Regeneration 8.1.5 PHB Composite Materials for Bone Tissue Regeneration

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8.2 Industrial

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8.2.1 Packaging Applications 8.2.2 Recombinant Protein Purification

9. Conclusion and Perspectives 601 References601

1. INTRODUCTION Pollution by plastics is one of the major concerns all over the world. The major factors that attract these synthetic polymers are that they are cheap, can easily produce from petrochemicals, and are very durable and flexible. These synthetic plastics are inevitable for day-to-day human life.The main disadvantages of these polymers are its nonbiodegradable characteristics and these are produced from nonrenewable natural resource. So there is a need for alternative sources of plastics that can be produced from renewable resources and it should be biodegradable. Several researchers are constantly involved in the search for alternative sources of plastics, and this has resulted the discovery of biodegradable polymers in several life forms such as plants, microbes, and animals. Among these, microbes are the preferred source because the culture condition and other bioprocess can be easily controlled and monitored. Several microorganisms such as bacteria can synthesize polymers like poly-3-hydroxybutyrate (PHB) that are biodegradable, and utilization of these biopolymers will help to reduce the dependence of petroleum derived plastics. These bacterial PHB can be produced from renewable resource that offers ecological advantages as compared to thermoplastics and elastomers produced from fossil carbon sources.1 PHB is a short chain length (SCL) polymer with several properties similar to polypropylene and it can be easily degraded aerobically or anaerobically. It is a crystalline material with high melting temperature and high degree of crystallinity. The physical and mechanical properties are similar to polypropylene, but PHB is stiff and brittle and the degree of brittleness depends on the degree of crystallinity, glass transition temperature, and microstructure. One of the main drawbacks of this polymer is that it is thermally unstable which will lead to decrease in viscosity and molar mass. The structure of PHB is given in Figure 17.1. The properties of PHB can be improved by blending with other polymers or by addition of lubricants or plasticizers or by suppression of crystallization and by lowering the glass transition temperature. The blending of PHB with other polymers allows a variety of copolymers to be produced with flexibility and tensile strength. Poly (hydroxybutyrate–valerate) (PHBV) is such a copolymer produced by certain bacteria when grown under specific carbon source. It is less stiff and tougher than PHB and can be used as packaging material (Figure 17.2).

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

Figure 17.1  Structure of poly-3-hydroxybutyate.

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2

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2 &+&

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Figure 17.2  Structure of PHBV.

Several microorganisms are known to accumulate PHB within the cells as an intracellular storage material for carbon and energy source. In microbes PHB synthesis takes place by a sequence of reactions catalyzed by three enzymes—3-ketothiolase, acetoacetylcoA reductase and PHB synthase.The first step is catalyzed by the enzyme 3-ketothiolase (E.C. 2.3.1.16) which condenses acetyl-CoA to acetoacetyl-CoA. This intermediate is reduced to D (-)-β-hydroxybutyryl-CoA by an Nicotinamide Adenine Dinucleotide Phosphate (NADPH)-dependent acetoacetyl-CoA reductase (E.C.1.1.1.36) and the enzyme PHB synthase catalyzes the head to tail polymerization of the monomer to PHB (Figure 17.3). PHB is naturally accumulating in a wide variety of microorganisms. Ralstonia eutropha is the well-known bacterium which accumulates PHB up to 80% of the cell dry weight utilizing various carbon sources. The PHB exists as water insoluble inclusions in bacteria and it shows mechanical properties similar to that of polypropylene.2 One of the main limitations for the bulk production of PHB is its high production costs as well as recovery costs. However with the development of genetic and metabolic engineering techniques allowed PHB biosynthesis in several recombinant bacteria and yeasts by improving the yields and thereby reducing the overall production costs.3 By implementing several metabolic engineering strategies like external substrate manipulation, inhibitor addition, recombinant gene expression, host cell genome manipulation, and protein engineering, it is possible to construct microbial plastic factories to produce biopolymers with desirable structure as well as properties.4 Utilization of inexpensive renewable carbon sources like plant oils, waste materials, and carbon dioxide is essential to reduce the production cost further. PHB finds applications in the development of biomedical devices and related products, as scaffolds in tissue engineering, development of vascular grafts and artificial heart valves and as a packaging material. One of the major limitations of using PHB is due to

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Acetyl-CoA β-ketothiolase Acetoacetyl-CoA Acetoacetyl CoA reductase (R)-3-Hydroxyacyl-CoA PHB synthase

PHB granule

Figure 17.3  Mechanism of poly-3-hydroxybutyrate synthesis within bacterial cell.

weak mechanical properties. Hence these polymers need to be blended with synthetic polymers for improved applications. Cost of production is one of the major limiting factors for large-scale production of PHB. Recent developments in gene manipulation, metabolic engineering, and utilization of cheaper sources as well as new fermentation strategies for production lead to the reduction of production cost. This chapter discusses about PHB and the related copolymers, its production, characterization, factors affecting production as well as applications in various sectors.

2. PHB-PRODUCING MICROBES Several species of bacteria, recombinant strains as well as cyanobacteria are known to produce PHB. Table 17.1 shows list of microbes producing PHB.

2.1 Screening of Microorganisms Screening and selection of microorganisms producing PHB is the first step in the bioprocess development of PHB. Several screening protocols have been developed to detect PHB in microbial cells. PHB confers some opacity to the bacterial cells thus allows a primary screening of PHB positive and negative colonies. This effect is only observed when the

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

Table 17.1  Microbes producing poly-3-hydroxybutyrate Microorganism PHB% References

Bacillus megaterium Bacillus mycoides Bacillus sphaericus NII 0838 Bacillus firmus NII 0830 Corynebacterium glutamicum Azotobacter vinelandii Chlorococcus Oscillatoria Anabaena Pseudoanabaena Synchocystis

45% 69.4% 31% 89% 36% – – – – – –

Chaijamrus and Udpuay8 Borah et al.10 Sindhu et al.11 Sindhu et al.12 Jo et al.19 Zanzig and Scholz17 Beyatli et al.37 Beyatli et al.37 Beyatli et al.37 Beyatli et al.37 Beyatli et al.37

difference is relatively high. The lipophilic dyes such as Sudan black, Nile blue, and Nile red have been used to distinguish between PHB accumulating and nonaccumulating strains. Nile red produces a strong orange fluorescence (emission maximum, 598 nm) with an excitation wavelength of 543 nm (maximum) upon binding to PHB granules.5 The oxazone form (Nile pink or Nile red) is probably formed from the basic oxazine dye Nile blue A by spontaneous oxidation in aqueous solution, thereby producing fluorescence.6 The lipophilic dyes must be dissolved in organic solvents such as ethanol (for Sudan black B) or acetone (for Nile red) and then are poured onto the agar plates. Since the cells are killed during the staining of the colonies, master plates have to be prepared. Spiekermann et al.7 developed a protocol for a viable colony staining method based on direct inclusion of the Nile red or Nile blue A in the agar medium. In such case the growth of the cells is not affected and the occurrence of PHB in the colonies can be directly monitored. This viable-colony staining method can be done with Nile red as well as Nile blue A. However, with Nile blue A, the intensity of the fluorescence was generally weaker and this can be enhanced by increasing the concentration of Nile blue A in the medium. The fluorescent microscopic image of PHB positive strain is shown in Figure 17.4.

2.2 PHB Production Using Wild-Type Bacteria Several wild-type bacterial strains were reported to produce PHB. Bacteria of the genera Bacillus, Ralstonia, Pseudomonas, Alcaligenes, Azotobacter, Halomonas, Corynebacteria, Lactobacillus, and Vibrio species were reported to produce PHB as a storage compound in response to nutrient imbalance caused by growth under conditions of excess carbon source and limitation of other nutrients like nitrogen or phosphorous. PHB is reported to be produced by several Bacillus species like Bacillus megaterium,8,9 Bacillus mycoides,10 Bacillus sphaericus NII 0838,11 Bacillus firmus NII 0830,12 Bacillus thuringiensis,13 Bacillus cereus,14 Azotobacter beijerinckii,15 Azotobacter chroococcum,16 Azotobacter vinelandii,17 Actinobacillus,18 Corynebacterium glutamicum19 etc.

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Figure 17.4  Primary screening profile of bacteria based on Nile blue staining.

2.3 PHB Production Using Recombinant Bacteria Recombinant microorganisms offer several characteristics for the production of PHB in a cost-effective manner. The costs can be reduced by including the use of cheap substrates or by enhancement of the product yield.20 Escherichia coli is a suitable host as a heterologous expression background for foreign genes that can be easily manipulated and improved by means of recombinant DNA methodologies. High-cell-density cultivation strategies for numerous E. coli strains are well established.21 Escherichia coli cells that accumulate large amounts of PHB become fragile, facilitating the isolation and purification of the biopolymer, and the bacterium does not express PHA (polyhydroxyalkanoate)degrading enzymes.22 Genes responsible for PHB biosynthesis (pha or phb genes) from a number of microorganisms, such as Cupriavidus necator, formerly called Alcaligenes eutrophus23; Pseudomonas aeruginosa24; Alcaligenes latus25; Thiocapsa pfennigii26; and Streptomyces aureofaciens27 have been introduced into E. coli. In most cases, the biosynthetic genes were expressed under the control of their native promoters, and the resulting recombinants were able to accumulate PHA from different carbon sources.22 de Almeida et al.28 conducted several studies where PHB production has been evaluated in E. coli and metabolic engineering has been carried out to improve productivity. Nikel et al.22 reported a mutation in arcA encoding, a protein that regulates aerobic respiration under microaerophilic conditions, resulted in accumulation of higher amounts of PHB in the cell. Low agitation has a positive effect on PHB synthesis in E. coli carrying phaCAB operon and phaP encoding phasin, a granule-associated protein. Several studies have been carried out by Kocharin et al.29 to transfer the PHB biosynthetic pathways to alternative hosts which lack enzymes involved in depolymerization. With the help of genetic and protein engineering techniques, it is possible to enhance polymer productivity. Recombinant E. coli are capable of producing ultrahigh molecular

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

weight PHB with superior properties after stretching of the material. Metabolically engineered Pseudomonas sp. 61-3 produced PHB copolymers which are similar to low density polyethylene (LDPE). Hydrogenase 3 and acetyl-CoA synthetase enzymes are very important in PHB synthesis. Wang et al.30 enhanced coproduction of hydrogen and PHB by recombinant E. coli by over expressing hydrogenase 3 and acetyl-CoA synthetase. Anaerobic metabolic pathways dedicated to coproduction of hydrogen and PHB were established due to the advantages of directing fluxes away from toxic compounds like acetate and formate to useful products. Overexpression of hydrogenase 3 resulted in an increase in PHB yield from 0.55 to 5.34 mg PHB/g glucose in MS medium with glucose and acetate as carbon source.

2.4 PHB Production Using Mixed or Cocultures One of the main disadvantages of using pure culture for the production of PHB includes the high cost of the pure substrates utilized and the cost of sterile precultivation of the bacteria utilized as well as the sterile operation of the final production process. Mixed or coculture systems are widely used in several fermentation processes. There are many fermentation systems, where microorganisms assimilate one substrate and convert it to an intermediate metabolite which is converted from other microorganisms to metabolitetarget product. Mixed or coculture systems will serve as an attractive addition to traditional pure culture-based technology for the production of PHB. To reduce their production cost, several efforts have been made for developing better bacterial strains and more efficient fermentation as well as recovery processes.The use of mixed cultures and cheap substrates can reduce the production cost of PHB. Accumulation of PHB by mixed cultures occurs under transient conditions caused mainly by intermittent feeding and variation in the electron donor/acceptor presence. The maximum capacity for PHB storage and the PHB production rate are dependent on the substrate and the operating conditions used.31 Ganduri et al.32 reported better PHB productivity through coculturing Lactobacillus delbrueckii and R. eutropha. PHB concentrations of 12 g/L and 40 g/L were reported for laboratory experiments and fed-batch fermentation, respectively.The study revealed that equally high yield is possible in simple batch fermentations by controlling the mixing intensity. Shalin et al.33 reported utilization of mixed microbial cultures for PHB production. Bacillus firmus NII 0830 was used for the production of PHB since it accumulates a large amount of PHB and a second organism L. delbrueckii NII 0925 was used to provide lactic acid. Enhanced economy, simple process control, nonrequirement of mono-septic processing are some of the advantages of using mixed culture system. Tohayama et al.34 reported the effect of controlling lactate concentration and periodic change in dissolved oxygen concentration (DOC) affects PHB production using a mixed culture of L. delbrueckii and R. eutropha. Here, the glucose was converted to lactate by L. delbrueckii and the lactate was converted to PHB by R. eutropha.

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The results indicate that periodic fermentation resulted in superior PHB yield with high PHB productivity when DOCs were controlled to be constant at less than 1 ppm or 3 ppm. Short- and long-term temperature effects on aerobic PHB producing mixed cultures were evaluated by Johnson et al.35 Temperature coefficient related to PHB metabolism was higher than short-term temperature changes. The specific growth rate on acetate decreased with increase in temperature. At higher temperature (30 °C), the culture produced 84% PHB. At lower temperature (15 °C), the production was decreased and the feast phase was longer and growth occurred predominantly on acetate rather than on stored PHB.

2.5 PHB Production by Cyanobacteria Several phototrophic bacteria produce PHB as an intracellular reserve for reducing power.These microorganisms accumulate more glycogen than PHB. Its synthesis is stimulated when reducing equivalents are excess or quickly interrupted when balanced growth is permitted; here PHB acts as a buffer system for regulating the intracellular redox balance.36 Several cyanobacteria were reported to produce PHB. This includes Chlorococcus, Oscillatoria, Anabaena, Pseudoanaebaena, and Synechocystis.37 Sharma and Mallick38 explored the potential of cyanobacteria for the production of PHB. Due to their minimal nutrient requirement and ability to grow in waste waters in presence of sunlight and CO2 serves as an alternative source for PHB production since the biomass is converted to PHB by solar energy.The study revealed that by media engineering, PHB production was improved fourfold. Phosphorous limitations as well as exogenous supply of carbon sources like acetate, glucose, maltose, fructose, and ethanol were found to have a positive effect on PHB accumulation.

3. FERMENTATION STRATEGIES 3.1 Submerged Fermentation 3.1.1 Batch Several reports were available on utilizing batch cultures for production of PHB. A nitrogen limitation condition is not ideal for batch fermentation employing microorganisms which cannot grow under nitrogen limiting conditions. Jiang et al.39 investigated the use of lactate and a lactate and acetate mixture for enrichment of PHB production by mixed cultures in sequencing batch reactors. The mixed cultures enriched on lactate can accumulate over 90% PHB within 6 h, which is the best result reported for a bacterial culture in terms of final PHB content and the biomass specific PHB production rate. The second mixed culture enriched on lactate and acetate produced 84% PHB after 8 h. The study revealed that the use of different substrates has no significant effect on the functionality of PHB production process.

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

PHB production using R. eutropha ATCC 17697 and A. latus ATCC 29712 was evaluated using a bioreactor as batch by Azhar et al.40 Maximum sugar consumption and sugar utilization efficiency were attained after 100 h; ammonia was completely assimilated after 80 h. The cell dry weight after 100 and 80 h were 10.18 and 8.73 g/L, respectively. PHB synthesis from a mutant strain A. vinelandii using glucose in a batch reactor was optimized by Dhanasekar et al.41 The initial medium pH significantly affects the productivity. Incubation temperature does not have a significant role in PHB production. Substrate concentration significantly affects the productivity as well as the PHB yield. It was observed that maximum productivity was observed with short fermentation period and lag phase were also minimized. 3.1.2 Fed-Batch Fed-batch cultivation is one of the best methods to produce high cell density with high PHB content. The important strategy for fed-batch fermentation is to feed the growth limiting substrates at the same rate as the rate of substrate is utilized by the organism.This helps in preventing the formation of by-products that are produced when the substrate is excess and leads to production of product of interest. PHB production from glycerol by Zobella denitrificans MW1 using high cell density fed-batch fermentation was reported by Ibrahim and Steinbuchel.42 The strain showed an increased PHB content at a low concentration of ammonium chloride. In this method, a much higher concentration of PHB (54.3 g/L) and highest cell density (81.2 g/L) were obtained in the presence of 20 g/L NaCl with optimized feeding of glycerol and ammonia to support both cell growth and polymer accumulation over a period of 50 h. PHB production by Saccharophagus degradans using raw starch as carbon source in a fed-batch culture was evaluated by González-Garcia et al.43 The strain accumulated 21.35% and 17.46% of PHB using glucose and starch as sole carbon source.The physical properties of these polymers were similar. Patnaik44 reported fed-batch optimization of PHB synthesis through mechanistic, cybernetic, and neural approaches. Enhancement of PHB productivity was investigated by applying two artificial neural networks to a bioreactor with finite dispersion and noise in feed streams. One network filtered the noise and other controlled the filtered feed rates of carbon and nitrogen sources. The study revealed that neural optimization doubled the maximum PHB concentration in fed-batch fermentation with R. eutropha by optimizing the time dependent feed rates. High cell density fed-batch fermentation of A. eutrophus was carried out for the production of PHB in a 60 L fermentor by Ryu et al.45 The PHB production was carried out by maintaining constant pH using NH4OH solution and PHB accumulation was induced by phosphate limitation. The fed-batch fermentation resulted in final cell concentration of 281 g/L and PHB concentration of 232 g/L and a productivity of 3.14 g/L/h.

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Yeo et al.46 reported enhancement of PHB production by a two stage supplementation of carbon sources and continuous feeding of NH4Cl. The study revealed that gluconate was used to maximize specific growth rate during initial stage of growth while glucose was used to maximize PHB synthesis. The sequential feeding of gluconate and glucose resulted in 50% enhancement in PHB production when compared to glucose alone.The glucose-grown culture showed a higher level of NADPH during the NH4Clexausted PHB accumulation stage than was observed in the gluconate-grown culture, which reflects that the reason of higher PHB production observed when glucose was used as a carbon source. NH4Cl feeding following the depletion of initial NH4Cl resulted in elevated levels of both ATP and NADPH, which increased the PHB biosynthesis rate, and also in a decrease in the level of NADH, which reflected the alleviation of the inhibitory effects on the cells caused by nitrogen depletion. A recombinant E. coli K24K strain was constructed and evaluated for PHB production from whey and corn steep liquor as main carbon and nitrogen source by Nikel et al.22 PHB was efficiently produced by the recombinant bacteria grown aerobically in fed-batch cultures in a laboratory-scale bioreactor in semisynthetic medium supplemented with agro-industrial by-products. The cells accumulated 72.9% of PHB with a productivity of 2.13 g/L/h. Quillaguiaman et al.47 achieved high PHB content and volumetric productivity by fed-batch culture of Halomonas boliviensis. Shake flask cultivation was carried out in minimal medium and growth was supported with supplementation of aspartic acid, glycine, or glutamine. Addition of glutamine resulted in high cell weight and when it was replaced with monosodium glutamate there was no change in cell density. 3.1.3 Continuous Continuous fermentation is a fermentation strategy having the possibility of achieving high productivity with strains having high maximum specific growth rate, which can be operated as a single stage or multistage process. An early nutrient limitation would result in low biomass with high PHB content while late induction of nutrient limitation would result in high biomass but with less PHB content. Hughes and Richardson48 patented a fermentation process for the production of PHB from Alcaligenes sp. continuously in a fermentor. A medium containing nutrient salts, carbon and energy source, and a water-soluble compound that is assimilated by microbes was supplied continuously removing an equal amount of medium containing bacterial cells from the medium thereby maintaining the amount of aqueous medium in the vessel at constant level. PHB is produced in R. eutropha under unbalanced growth conditions, hence a twostage continuous culture system to be adopted. In the first stage maximum cell biomass were produced and in the second stage PHB was produced. The PHB content and productivity were 47.6% and 1.43 g/L/h, respectively.

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

Henderson and Jones49 reported growth of A. eutrophus in a continuous system under various conditions of dilution rate, nutrient limitation, and carbon substrate. The study revealed that maximum PHB content, productivity, and carbon substrate utilization rate when grown on glucose medium were maximal at low dilution rate under ammonia limitation. The PHB content decreased in a linear manner as a function of dilution rate. The strain showed highest growth rate when grown on media containing lactate. The study revealed that A. eutrophus cannot regulate the rate at which it takes up excess carbon substrate which is solely required for growth especially during growth on lactate at low dilution rate thereby producing PHB as a means of avoiding harmful effects of high concentration of intracellular metabolites. A two-stage continuous culture system where a phase-wise optimization was carried out which maximizes the residual biomass growth rate in the first stage followed by a PHB production rate in the second stage was reported by Lee et al.50 Maximum PHB productivity of 2.86 g/L/h was obtained in this process. Khanna and Srivastava51 reported members of the genus Wautersia uptake excessive carbon from the medium and accumulate PHB. Continuous cultivation of Wautersia eutropha was carried out in a 7 L reactor. Reactor was operated in a batch mode for initial 15 h followed by a fed-batch mode for sufficient biomass and PHB production, followed by a continuous mode so that PHB production was continuously maintained and released from the reactor.

3.2 Solid-State Fermentation Few reports are available on PHB production by solid-state fermentation (SSF). Ramadas et al.52 have developed a novel SSF bioprocess in which polyurethane foam (PUF) was used as a physical inert support for the production of PHB by B. sphaericus NII 0838. Media engineering for optimal PHB production was carried out using response surface methodology (RSM) adopting a Box–Behnken design. The factors optimized by RSM were inoculum size, pH, and (NH4)2SO4 concentration. Under optimized conditions—6.5% inoculums size, 1.7% (w/v) (NH4)2SO4, and pH 9.0, PHB production and biomass were 0.169 and 0.4 g/g PUF, respectively. This is the first report on PHB production by SSF using PUF as an inert support. The results demonstrate that SSF can be used as an alternative strategy for the production of PHB. The major inherent problem associated with PHB production in SSF systems is the biomass retrieval of bacterial cells. This limitation can, however, be overcome by using PUF as an inert support. Oleivera et al.53 reported PHB production by R. eutropha using agro-industrial residues. The PHB productivity and content were 4.9 mg/g medium in 60 h and 39%, respectively. Production was carried out using soy cake alone or supplemented with sugarcane molasses. The results obtained showed that the biopolymer obtained by SSF has similar properties as commercial PHB, except for the higher molar mass and the

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lower degree of crystallinity. Thus, the present data indicate that SSF is an interesting alternative for the production of PHB, allowing the production of biopolymers with adequate properties from low-cost, renewable resources. SSF process provides a biopolymer that is identical to a commercial PHB produced by submerged fermentation, as well as to other PHB data reported in literature. The only differences noted for the polymers produced by SSF were a higher molar mass and a lower degree of crystallinity, which both represent advantages for the SSF process, since these properties enable a broader range of applications for the PHB produced by this method.

4. DOWNSTREAM OPERATIONS The extraction as well as purification of the PHB is one of the key steps in the bioprocess. An ideal purification method is that which leads to high purity and recovery level at a low production cost.

4.1 Chemical Methods The common chemical methods for extraction of PHB are solvent extraction, chemical digestion using sodium hypochlorite, sodium hypochlorite and chloroform, surfactants, surfactant hypochlorite digestion, surfactant chelate digestion, chelate hydrogen peroxide treatment, selective dissolution of nonpolymer cell mass by proton etc. Solvent extraction method is one of the oldest techniques adopted for extraction of PHB. The mechanism of action of solvent is that it modifies the cell membrane permeability and then solubilize PHB. This process was first adopted by Lemoigne and Baptist for the extraction of PHB from B. megaterium and Rhodospirillum rubrum.54 Several solvent mixtures were tried for the separation of PHB from the solvent by solvent evaporation or by precipitation in a nonsolvent. Vanlautem and Gilain55 used liquid halogenated solvents like chloroethanes and chloropropanes for the extraction of PHB. This method was found to be superior when compared to extraction using other solvents like tetrahydrofuran methyl cyanide, tetrahydrofuran ethyl cyanide, and acetic anhydride. Noda56 developed a method with a mixture of PHB solvent and nonsolvent, the insoluble biomass is separated leaving behind a suspension of precipitated PHB in the nonsolvent. Another method for extraction of PHB is by using sodium hypochlorite digestion method.57 With this method, 86% purity was observed with R. eutropha and 93% purity with E. coli. One of the major drawbacks of this method is that it causes degradation of PHB resulting in 50% reduction in molecular weight. Hahn et al.58 developed a novel strategy for PHB extraction by combining the advantage of both differential digestions by hypochlorite and solvent extraction. Adopting this method for extraction of PHB from R. eutropha, three separate phases were obtained, an upper phase of hypochlorite solution, a middle phase of non-PHB cell materials and

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

undisrupted cells, and the chloroform phase contains the PHB. The polymer is recovered by precipitation in a nonsolvent and filtration and by adopting this method, the degradation of polymer is significantly reduced. Dong and Sun59 adopted a combination of surfactant and hypochlorite process for extraction of PHB. By this method, polymer can be extracted to a purity of 98%. In this method, a freezing pretreatment was also used for cell lysis. The main advantages of this method are its low operating cost and limited degradation of polymer. Chen et al.60 reported that by adding a chelate to the surfactant increase the release of polymer. Addition of chelate destabilizes the outer membrane forming complexes with divalent cations. This in turn cause changes in inner membrane and improves cell lysis and gives a high purity polymer. The advantages of this method are high purity of the product and low environmental pollution. One of the main drawbacks of this method is generation of large volume of waste water during the product recovery step. Liddell and Locke61 developed a combination of chelate treatment with hydrogen peroxide for the extraction of PHB produced by R. eutropha. For the recovery of polymer, first a heat pretreatment was carried out followed by treatment with chelating agent like diethylene triamine pentamethylene phosphonic acid and hydrogen peroxide. By adopting this method polymer can be recovered with a purity of 99.5%. A novel strategy for selective dissolution of nonpolymer cell mass by protons in aqueous solutions and crystallization of the polymer was developed by Yu and Chen.62 Polymer extracted by this method showed high purity and high yield. Studies revealed that this method is much cheaper than conventional methods used for recovering polymer and reduces the overall cost of polymer recovery by 90%. Fiorese et al.63 developed a novel method for recovery of PHB from C. necator biomass by solvent extraction with 1, 2-propylene carbonate. Process parameters like temperature, contact time, precipitation period, and pH affects the extraction efficiency and polymer properties. The highest yield of 90% and purity of 84% were obtained at temperature of 130 °C, contact time of 30 min, and precipitation period of 48 h. Under these conditions, high molecular weight PHB was obtained and physical properties like glass transition temperature, melting temperature, melting enthalpy, and crystallinity were not affected. The polymer yield did not improve with the heat/pH treatment; this treatment increased the PHB molecular weight and purity.

4.2 Biological Methods Holmes and Lim64 developed the enzymatic extraction process for the extraction of PHB. Proteolytic enzymes were used for this process.The process involves a heat treatment followed by enzymatic hydrolysis, surfactant treatment, and decolorization with hydrogen peroxide. The advantage of this pretreatment is that the recovery rate was higher and the major drawback of this technology is the high cost of the enzyme.

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Harrison et al.65 extracted polymer from R. eutropha upon treatment with lytic enzymes of Cytophaga sp. without any mechanical processing and 90% purity was achieved by this method. Lu et al., 200666 developed a combined method involving enzyme and sodium hypochlorite for extraction of polymer from Burkholderia sp. PTU9. A recovery of 89% was achieved by this process. De Koning and Witholt67 reported a combined process involving sequential treatment with heat, alcalase, and SDS assisted by Ethylene Diamine Tetraacetic Acid (EDTA) treatment for the recovery of the polymer from Pseudomonas. A recovery rate of 95% was achieved by this process. Kapritchkoff et al.68 observed that for enzymatic recovery and purification of PHB from R. eutropha, proteases were the most suitable enzymes for non-PHB biomass solubilization and purification. Due to high efficiency and low-cost, pancreatin makes an ideal candidate for large-scale applications.

5. CHARACTERIZATION TECHNIQUES Characterization techniques were used to determine molecular mass, molecular structure, morphology, thermal as well as mechanical properties. Various techniques commonly used for characterization of PHB include FTIR, NMR, DSC, TGA, GPC, and AFM.

5.1 Fourier Transform Infrared Spectroscopy Fourier transform infrared spectroscopy (FTIR) is a technique which is used to obtain infrared spectrum of absorption, emission, and photoconductivity of solid, liquid, and gas. It is used to detect different functional groups in PHB. FTIR spectrum is recorded between 4000 and 400 cm−1. For FTIR analysis, the polymer was dissolved in chloroform and layered on a NaCl crystal and after evaporation of chloroform, the polymer film was subjected to FTIR. The spectrum of PHB shows peaks at 1724 cm−1 and 1279 cm−1, which corresponds to specific rotations around carbon atoms. The peak at 1724 cm−1 corresponds to C–O stretch of the ester group present in the molecular chain of highly ordered structure and the adsorption band at 1279 cm−1 corresponds to ester bonding.69 Figure 17.5 shows FTIR spectrum of PHB.

5.2 Nuclear Magnetic Resonance Nuclear magnetic resonance (NMR) is a physical process in which nuclei in a magnetic field absorb and reemit electromagnetic radiation. Analysis of NMR spectra allows the determination of polymer composition, and the distribution of monomer units can be deduced from the diad and triad sequences by NMR spectral analysis. For characterization of polymer, the extracted polymer will be dissolved in CDCl3 followed by NMR analysis. The NMR spectrum for PHB shows three characteristic signals. A doublet at 1.53 ppm represented the methyl group (CH3) coupled to one proton while a doublet of

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

quadruplet at 2.75 ppm resulted from methylene group (CH2) adjacent to an asymmetric carbon atom bearing a single proton.The third signal was a multiplet at 5.52 ppm, which was attributed to a methyne group (CH). Figure 17.6 shows NMR spectrum of PHB.

5.3 Thermogravimetric Analysis Thermal degradation of the polymer was studied using thermogravimetric analysis (TGA). Here, the change in physical and chemical properties of the polymer was

95 %T 90 85

127881

80 75

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70 65 60 3750

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3250

3000

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2250

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1500

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Figure 17.5  FTIR spectrum of poly-3-hydroxybutyrate.

4

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4000 std

Figure 17.6  NMR spectrum of poly-3-hydroxybutyrate.

0

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30.05C 498.04C -2.483mg -98.532%

195.36C 279.19C -2.429mg -96.389%

29.34C 195.36C -0.007mg -0. 278%

279.19C 497.33C -0.045mg -1.786%

Figure 17.7  TGA curve of poly-3-hydroxybutyrate.

measured as a function of increasing temperature. PHB is very brittle and has low melting temperature. Several studies revealed that blending of PHB with other polymers is advantageous in terms of cost reduction with improved properties when compared to PHB alone.70 Figure 17.7 shows the thermal degradation profile of PHB.

5.4 Differential Scanning Calorimetry Differential scanning calorimetry (DSC) is a thermoanalytical technique used to study the thermal properties of the polymer using a differential scanning calorimeter. In this process, the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature. Sample and reference will be maintained at same temperature throughout the experiment. DSC curves were plotted based on heat flux versus temperature or time. Thermal transitions of polymer can be determined by this technique. DSC is widely used for the decomposition behavior determination of the polymer. Figure 17.8 shows the DSC curves of PHB.

5.5 Gel Permeation Chromatography Gel permeation chromatography (GPC) is a type of size exclusion chromatography technique used for determination of molecular weight of polymers, since molecular mass is an important factor determining the physical properties of polymers. Tetrahydrofuran is used

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

Figure 17.8  DSC curve of poly-3-hydroxybutyrate.

to dissolve the polymers prior to determination of molecular mass using GPC. For analysis, two columns HT3 and HT5 and a refractive index detector with chloroform as the elution solvent were used. Several factors affect the molecular weight of PHB. Effect of substrate and culture conditions affecting the molecular weight of PHB was reported by Chen and Page.71 The protocol adopted for extraction of the polymer can also lead to loss of molecular mass of polymer.72

5.6 Atomic Force Microscopy Atomic force microscopy (AFM) is a novel method for imaging the surface architecture of cells and cellular components. It provides a real-time three-dimensional images under natural conditions and gives high resolution images of surface topography. A study conducted by Sudesh et al.73 revealed that AFM can be used to observe directly and characterize proteins associated with native polymer granules. The study revealed three-dimensional images of proteins associated with native polymer granules as well as PHB single crystals. One of the main advantages of using AFM as imaging tool is that minimum sample pretreatment is required and it will not damage the sample. Immunogold labeling will help to understand the molecular mechanism of PHB granule formation, mobilization, and regulatory machinery involved. Figure 17.9 shows AFM image of PHB matrix.

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800 600 400 μm 200 0 10 8 6 4 2 1

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μm

Figure 17.9  Atomic force microscopic image of poly-3-hydroxybutyrate matrix.

6. STRAIN IMPROVEMENT, MUTATION, AND METABOLIC ENGINEERING Recombinant E. coli serves as a good candidate for PHB production.The main drawback is the cost associated with Luria-Bertani medium, ampicillin, and requirement of pure oxygen. Hence, large-scale production is not practically feasible. Utilization of cheaper carbon source makes the process economically viable. Liu et al.74 reported PHB production from molasses by recombinant E. coli. The fermentation with molasses was economically viable when compared to that of glucose. The PHB content and productivity were 80% w/w and 1 g/L/h, respectively. The study revealed that molasses concentration had a significant effect on PHB synthesis and cell growth. A fed-batch strategy was adopted to overcome substrate inhibition as well as to improve cell growth and PHB production. PHB synthesis was evaluated using a recombinant E. coli arcA mutant using glycerol as sole carbon source by Nikel et al.75 Casein amino acids showed a significant effect on PHB production. The study revealed that microaerophilic fed-batch cultivation leads to a 2.57-fold increase in volumetric productivity when compared to batch cultivation. Lee76 reported PHB production from several recombinant E. coli strains harboring A. eutrophus PHA biosynthesis genes utilizing xylose from cotton seed hydrolyzate or soy bean hydrolyzate as sole carbon source.The study revealed that there is a twofold increase in PHB production when the medium is supplemented with a small amount of cotton seed hydrolyzate or soy bean hydrolyzate.The PHB concentration and PHB content were

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

4.4 g/L and 73.9%, respectively. Among the various recombinant E. coli strains, tested E. coli TG1 (pSYL107) showed highest productivity using cheap hemicelluloses hydrolyzate. A new fermentation strategy using cell recycle membrane system was developed by Ahn et al.77 for the efficient production of PHB from whey by recombinant E. coli strain CGSC 4401 harboring the A. latus PHA biosynthesis genes. The working volume of fermentation was constantly maintained by cell recycle and by fed-batch cultivation employing an external membrane module. The PHB concentration and PHB content were 168 g/L and 87%, respectively. Slater et al.78 reported cloning and expression of A. eutrophus H16 PHB pathway in E. coli. Library was constructed in cosmid pVK 102. The study revealed the relation between PHB transcriptional control and transcriptional control in other regulations, and PHB biosynthetic pathway is controlled by nitrogen and oxygen limitation. Cosmid clone was subcloned and the PHB biosynthetic pathway gene with a fragment size of 5.2 kb was cloned into multicopy vectors, which can direct the PHB synthesis in E. coli to 80% dry weight. To endow the superior PHB biosynthetic machinery to E. coli, Choi et al.25 cloned the PHA biosynthetic genes from A. latus. Three PHA biosynthetic genes that form an operon consisting of PHA synthase, β-ketothiolase, and reductase genes were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains accumulated more amount of PHB when compared to R. eutropha. The PHB productivity was 4.36 g/L/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness. This study provided a strategy of enhancing PHB productivity by developing recombinant E. coli strains harboring the more efficient PHA biosynthesis machinery of A. latus. The effect of different amino acids supplements on the synthesis of PHB by recombinant E. coli was evaluated by Mahishi and Rawal.79 The study revealed that when the basal medium is supplemented with amino acids, except glycine and valine, all other amino acid supplements enhanced PHB accumulation in recombinant E. coli harboring PHB synthesizing genes from S. aureofaciens. Cysteine, isoleucine, or methionine supplementation increased PHB accumulation by 60, 45, and 61%, respectively. Amino acid biosynthetic enzyme activities in several pathways are repressed by end product supplementation. End product inhibition in the cysteine biosynthetic pathway controls the carbon flow due to sensitivity of serine transacetylase to cysteine. Hence, supplementation of cysteine favors a change in carbon flux that eliminates the requirement of acetyl-CoA for serine transacetylation which in turn provides more carbon source and acetyl-CoA for PHB synthesis. Degradation of methionine and isoleucine yields succinyl CoA, an intermediate of tricarboxylic acid cycle and allows more acetyl-CoA to enter the PHB biosynthetic pathway. Effect of anaerobic promoters on the microaerobic production of PHB in recombinant E. coli was reported by Wei et al.80 Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from R. eutropha for the microaerobic production of PHB in recombinant E. coli. Among the various promoters,

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alcohol dehydrogenase was found to be most effective. Recombinant E. coli strain showed a PHB accumulation of 48% after 48 h of static culture while with native promoter produced only 30% PHB. The molecular weights of PHB produced under microaerophilic conditions were found to be higher. The study conducted by de Almeida et al28 revealed that aeration affects PHB synthesis from glucose and glycerol in recombinant E. coli carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol at low agitation accumulated more PHB and ethanol than at high agitation. When glucose is used low agitation led to a decrease in PHB formation. Small variations in available oxygen can lead to significant changes in metabolism of E. coli cultures, which reflect the metabolic adjustments that take place to optimize cell growth in facultative aerobe, vary when using glycerol or glucose affecting the synthesis of products. Heterologous expression of the bacterial PHB biosynthesis pathway in Saccharomyces cerevisiae involves the utilization of acetyl-CoA, an intermediate of the central carbon metabolism, as precursor and NADPH, a redox cofactor used during anabolic metabolism, as a required cofactor for the catalyzing enzymes in the PHB biosynthesis pathway. Provision of acetyl-CoA and NADPH by alteration of the endogenous pathways and/ or implementation of a heterologous gene/pathway was investigated with the aim to improve PHB production in S. cerevisiae. Engineering of the central carbon and redox metabolism substantially improve PHB production. By adopting metabolic engineering, microorganisms can be engineered to produce new products with higher yield and productivities. Kocharin et al.29 expressed the bacterial PHB pathway in S. cerevisiae and evaluated the effect of engineering the formation of acetyl-CoA, which is an intermediate of the central carbon metabolism and precursor of PHB biosynthetic pathway for heterologous PHB production by yeast. Kocharin et al.29 engineered the acetyl-CoA metabolism by cotransformation of a plasmid containing genes for native S. cerevisiae alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA acetyltransferase (ERG10), and a Salmonella enterica acetyl-CoA synthetase variant (acsL641P), resulting in acetoacetyl-CoA overproduction, together with a plasmid containing the PHB pathway genes coding for acetyl-CoA acetyltransferase (phaA), NADPH-linked acetoacetyl-CoA reductase (phaB), and PHB polymerase (phaC) from R. eutropha H16. Enhancement of acetyl-CoA production by coexpression of genes on the acetyl-CoA boost plasmid improved the productivity of PHB during growth on glucose and further enhanced the productivity of PHB approximately 16.5 times bioreactor cultivations and reduce the flux from acetyl-CoA to lipids. Li et al.81 over expressed Nicotinamide Adenine Dinucleotide (NAD) kinase to enhance the accumulation of PHB in recombinant E. coli harboring PHB synthesis pathway by an accelerated supply of NADPH, one of the important factors affecting PHB production. The study revealed that NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35% weight compared

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

to controls. The availability of NADPH is an important factor for improving PHB productivity. The NAD kinase overexpression enhanced PHB production in recombinant E. coli harboring the PHB synthesis operon due to effective supply of NADPH. Mutation can be carried out by chemical methods, UV radiation, or by genetic elements such as transposons. UV mutagenesis seems to be one of the promising methods for strain improvement. The study conducted by Ugwu et al.82 revealed that UV mutagenesis can be successfully used in generating mutants for extracellular production of PHB. This is the first report on the use of mutants for extracellular production of (R)3-HB. Production of (R)-3-HB using acetoacetate seems to be an interesting biosynthetic route in C. necator. Mutational effects of PHB polymerase on PHB accumulation was reported by Taguchi et al.83 The assay system consists of a PCR-mediated random mutagenesis and two assay procedures based on plate assay and High Performance Liquid Chromatography (HPLC) assay based on the conversion of PHB to crotonic acid.The level of PHB accumulation, an activity estimation of the R. eutropha polymerase would be efficiently achieved by monitoring the level of PHB accumulation using this in vivo assay system.

7. SUBSTRATE MANIPULATION FOR THE PRODUCTION OF VARIOUS CLASSES OF PHB PHB production by wild as well as recombinant strains consists of a cell growth phase and a PHB production phase. A nutrient-rich medium will be used to obtain high cell mass in the early cell growth phase followed by a PHB production phase, where the cell growth is limited by depletion of some nutrients like nitrogen, phosphorous, magnesium, or oxygen which favor the metabolic shift for biosynthesis of PHB. These biopolymers have been drawing much attention as promising substitutes for chemically synthesized polymers due to their similar mechanical properties to petroleum-derived plastics and complete biodegradability. There are several reports about PHAs consisted of both SCL and MCL (medium chain length) monomer units (SCL-MCL-PHA). The physical properties of PHAs are highly dependent upon their monomer units, and therefore, biodegradable polymers having a wide range of properties can be made by incorporating different monomer units. The monomeric composition of PHB can be engineered using various metabolic engineering and substrate manipulation approaches. The composition of the monomer in a copolymer depends on the hosts PHB synthase as well as on the hydroxyacyl-CoA thioester precursors supplied to the enzyme, which in turn depend on the metabolic pathways operating in the cell and on the external carbon source.The primary objective of metabolic engineering strategy is to include various controlling factors that determine polymer material properties like monomeric composition, chain length, and copolymer microstructure for optimizing yield. Pathway engineering for PHB production

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offers the opportunity to synthesize novel polymers with desirable properties in lowcost, high-productivity fermentations.

7.1 Importance of External Substrate Addition The simplest metabolic engineering approach for the production of PHB copolymers is to manipulate the carbon sources in the culture medium.This strategy has been exploited to modify polymer composition by varying the feed ratio of different substrate precursors. Currently, there have been novel applications of this strategy to generate unusual sulfur-containing polymers. Ewering et al.84 reported a recombinant R. eutropha producing poly (3-hydroxy-S-propyl-o-thioalkanoate) copolymers containing thioether linkages in their side chains when fed with alkylthioalkanoates (thio fatty acids). The intermittent addition of valerate to wild R. eutropha leads to formation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (P (3HB-co-3HV)).

7.2 Manipulation of Carbon Sources Carbon sources play a major role in PHB copolymer biosynthesis. The addition of various carbon sources in different concentration will enable to engineer the polymer with different physical properties. Various microorganisms like R. eutropha, A. latus, and Pseudomonas sp. were reported to produce PHA by the condensation or modification of acetyl-CoAs generated from sugars. Ralstonia eutropha as well as other bacteria are capable of accumulating PHB from sugars except for R. rubrum, where two acetyl-CoA moieties are condensed to form acetoacetyl-CoA by β-ketothiolase (phaA). Acetoacetyl-CoA is reduced to (R)-3hydroxybutyryl-CoA by an NADPH-dependent reductase (phaB). PHA synthase (phaC) finally links (R)-3-hydroxybutyryl-CoA to the growing chain of PHB. In R. rubrum, NADH-linked acetoacetyl-CoA reductase reduces acetoacetyl-CoA to (S)-hydroxybutyrylCoA. Two crotonases convert (S)-hydroxybutyryl-CoA to (R)-hydroxybutyryl-CoA, and finally polymerized by PHA synthase to PHA. Many Pseudomonas belonging to the rRNA homology group I except Pseudomonas oleovorans, use 3-hydroxyacyl-ACPs generated from fatty acid biosynthesis pathway as precursors for PHA production. The enzyme 3-hydroxydecanoyl-ACP CoA transacylase coded by phaG gene was cloned from Pseudomonas putida will serve as intermediate for fatty acid biosynthesis pathway for PHA production from sugars. The phaG gene present in P. oleovorans cannot synthesize MCL-PHAs since the expression of this gene is suppressed at transcriptional level resulting in no MCL-PHAs synthesis from glucose or gluconate. A schematic representation of bacterial PHB synthesis from glycerol is shown in Figure 17.10. Pseudomonas oleovorans and most pseudomonads belonging to the rRNA homology group I can accumulate MCL-PHAs using 3-hydroxyacyl-CoA intermediates of β-oxidation pathway when grown on various alkanes, alkanols, or fatty acids. Aeromonas sp. utilize β-oxidation pathway to supply PHA precursors, especially 3HB and

Microbial Poly-3-Hydroxybutyrate and Related Copolymers Bacterial cell

glycerol NAD+ NADH2 Dihydroxyacetone ATP ADP Dihydroxyacetone phosphate ATP ADP

NAD+ NADH2

Phosphoenolpyruvate ADP ATP Pyruvate CO2 Acetyl-CoA

PHB granule (R)-3-Hydroxyacyl-CoA

Acetoacetyl CoA reductase Acetoacetyl-CoA β-ketothiolase

PHB synthase

Figure 17.10  Schematic representation of bacterial poly-3-hydroxybutyrate synthesis from glycerol.

3HHx monomers from various fatty acids. The composition of the PHA formed by pseudomonads is highly dependent on the structure of the carbon substrate especially when the PHA precursors are supplied from β-oxidation pathway. Generally, the chain length of monomers in PHAs is the same as that of carbon sources or shortened by 2, 4, or 6 carbon atoms. The three putative enzymes involved in providing hydroxyacylCoA from β-oxidation pathway include hydratase, epimerase and 3-ketoacyl-CoA reductases. Tsuge et al.85 reported cloning of the (R)-specific enoyl-CoA hydratase genes from Aeromonas caviae and P. aeruginosa, revealed that hydratase will supply 3-hydroxyacylCoAs from β-oxidation pathway. Two expression plasmids for phaJ1 (Pa) and phaJ2 (Pa) were constructed and introduced into E. coli DH5α strain. The recombinants harboring phaJ1 (Pa) or phaJ2 (Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities for SCL enoyl-CoA or MCL enoyl-CoA. The coexpression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14–29 wt% of cell dry weight from dodecanoate as a sole carbon source. This study revealed that phaJ1 (Pa) and phaJ2 (Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle. The recombinant E. coli harboring R. eutropha PHA biosynthesis genes could accumulate PHB from glucose; E. coli has been metabolically engineered to produce various PHAs. The general metabolic pathway for the production of PHB from fatty acids is shown in Figure 17.11.

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HS—CoA H2O

RCH2CH2CO—SCoA (Acyl-CoA) acyl-CoA dehydrogenase

FAD FADH2

RCH:CHCO—SCoA (trans-2,3-dehyroacyl-CoA) H 2O

enoyl-CoA hydratase R H

OH

CO—SCoA (L-3-hydroxyacyl-CoA)

3-hydroxyacyl-CoA epimerase R H

CO—SCoA (D-3-hydroxyacyl-CoA)

OH

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Figure 17.11  Poly-3-hydroxybutyrate synthetic pathway from fatty acids.

7.2.1 Properties of PHB due to Carbon Manipulation The material property of PHB is similar to polypropylene but its high melting temperature of 170 °C makes the processing of PHB difficult. P (3HB-co-3HV) copolymer is less stiff and tougher and shows higher elongation to break and reduced melting point ranging from 160 °C to 100 °C based on polymer composition (0–25 mol% 3HV). MCL-PHAs can be used as a biodegradable rubber and coating material due to a much lower crystallinity and higher elasticity. PHAs consisting of both SCL- and MCL-monomer units have been produced by several bacteria. Among those, poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (P (3HB-co-3HHx)) and poly (3-hydroxybutyrate-co-3-hydroxyalkanoate) (P (3HB-co3HA)) have the superior mechanical properties similar to LDPE depending on the monomer composition. The superior properties of SCL-MCL-PHA copolymers are attracting various industrial applications.

7.3 Effect of Nitrogen and Phosphorus Wang and Lee86 reported a nutrient limitation strategy to increase PHB content for the development of economically attractive process. Among the various nutrient limitation

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

condition including N, P, Mg, and S, nitrogen limitation was found to be the best strategy since it allowed the greatest enhancement of PHB production. Nitrogen-limited conditions lead to an increase in residual cell concentration. During fed-batch culture of A. latus, two feeding strategies during nitrogen limitation were applied.87 Since the DOC response was more sensitive than the pH response on the carbon depletion, the DO-stat was used during actively growing stage. After nitrogen limitation, feeding strategy was changed from the DO-stat method to the optimally determined feeding profile due to the no apparent DO increase upon carbon depletion under nitrogen-limited condition.The feeding profile was determined experimentally by calculating the sucrose consumption rate during the nitrogen-­ limited period. Nitrogen limitation was applied at a cell concentration of 76 g/L, which has been suggested to be optimal for both high cell and PHA concentration from fedbatch cultures of R. eutropha. Sucrose concentration was maintained within 5–20 g/L. After 8 h of incubation under nitrogen limitation, cell concentration, PHB concentration, and PHB content reached were 111.7 g/L, 98.7 g/L, and 88 wt%, respectively, resulting in the productivity of 4.94 g/L/h.The highest productivity of 5.13 g/L/h was obtained at 16 h.86

7.4 Inhibitor Addition PHA synthesis can be altered by the addition of inhibitors. These compounds have particularly useful for incorporating MCL monomers derived from β-oxidation into PHAs. Acrylate was used to inhibit β-oxidation in wild-type R. eutropha during growth on octanoate so that the bacteria accumulated a copolymer of 3-hydroxypropionate, 3HB, 3-hydroxyhexanoate, and 3-hydroxyoctanoate (P (3HP-co-3HB-co-3HHX-co-3HO)) containing both SCL and MCL monomers.

8. APPLICATIONS 8.1 Medical 8.1.1 Targeted Drug Delivery Arsenic trioxide loaded biocompatible PHB–PVA1 nanoparticles (<100 nm in size) with folate-functionalized surface were synthesized using PHB produced by B. firmus NII 0830. Folate functionalization was carried out by using dicyclohexyl carbodiimide as catalyst and 10-bromodecanol as linker molecule to conjugate glutamic acid terminal of folate with the hydroxylate groups present on the surface of PHBA–PVA2 nanotrojans.The effect of fabrication parameters on shape, size distribution, and PDI of the PHB nanoparticles were evaluated by Althuri et al.88 It was observed that increase in sonication time and polyvinyl alcohol (PVA) concentration greatly reduced the size of nanoparticles. The drug release studies on arsenic trioxide incorporated PHB–PVA nanoparticles were conducted at physiological pH and temperature. FOL–PHBA–PVA3 nanoparticles showed greater extent of cytotoxicity toward murine fibrosarcoma L929

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cells than PHBA–PVA nanoparticles alone without conjugated folate, indicating the significance of folate as ligand for specific targeting of FR+ cancer cells. The study concludes that folate functionalized PHB nanoparticles are ideal biocompatible polymer matrix to carry toxic drug compounds to the targeted sites for the treatment of lifethreatening diseases such as cancer.88 8.1.2 Nanofibrous Matrices as Cell Supporting Materials Li et al.89 evaluated the potential of nanofiber matrices of polymer mimicking the real microenvironment of extracellular matrix for cell growth.The study revealed that nanofiber matrices favor better support cell attachment and cell viability compared to solid-walled matrices.Three-dimensional structures showed similarity to the extracellular matrices component collagen.These nanomatrices combined the advantages of biodegradation, mechanical strength, and nanostructure of natural extracellular matrix, which leads to better cell compatibility thereby indicating its application as future implant biomaterial development. 8.1.3 Biomedical Application of PHB Sutures Volova et al.90 reported that PHB sutures implanted intramuscularly do not produce any adverse effect on physiological, biochemical, and functional parameters of the animal. Monofilament sutures made of high purity PHB provide necessary strength of suture throughout the period of wound healing. These sutures do not cause any acute vascular reaction at the site of implantation. 8.1.4 Organic-Soluble Chitosan/PHB Ultrafine Fibers for Skin Regeneration Ma et al.91 studied the effect of organic-soluble chitosan/PHB fibers for skin regeneration that was prepared by electrospinning.The cytotoxic study was evaluated with mouse fibroblast cells (L929) and the cell culture results revealed that it benefits promoting the cell attachment and proliferation and can be used as tissue engineering for skin regeneration. 8.1.5 PHB Composite Materials for Bone Tissue Regeneration The efficacy of poly-3-hydroxybutyrate-co-3-valerate (PHBV) was evaluated by Cool et al.92 for bone tissue engineering.The in vitro osteogenic and inflammatory properties of PHBV were evaluated. The study revealed that the addition of nanosized reinforcing phase to PHBV improves osteogenic properties as well as reduces the proinflammatory response. This provides a new strategy for improving the suitability of PHBV-based materials for bone tissue regeneration.

8.2 Industrial 8.2.1 Packaging Applications PHB finds numerous applications in packaging. It can be used to prepare films or molded objects. They are compatible with various food products like beverages, dairy, meat etc. So far, the large-scale application of PHB as packaging material is hampered by their high cost.

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

8.2.2 Recombinant Protein Purification Banki et al.93 developed a novel and economic method for purification of recombinant proteins by combining two well-established technologies to generate a breakthrough in protein purification. First, PHB was produced in recombinant E. coli, this was combined with a self-cleaving affinity tags based on protein splicing elements. By combining these techniques with a PHB-specific binding protein, a self-contained protein expression and purification system has been developed where the PHB-binding proteins act as an affinity tag for desired product proteins. Expression of these tagged proteins in E. coli produces intracellular PHB where it binds to PHB by PHB-binding tag. By allowing the bacterial cells to produce both the affinity resin and tagged target protein, the cost associated with purification of recombinant proteins can be greatly reduced.

9. CONCLUSION AND PERSPECTIVES The most important aspects addressed while using biodegradable polymers include the limited use of petroleum-based plastics, environmental protection as well as reduction in CO2 emissions. Currently, the major limitation in usage of PHB is the cost associated with its production. This can be overcome to certain extent by developing biorefinery concept as well as development of high value product applications of PHB. Surfacebinding proteins can be developed for protein purification or targeted drug delivery. Several new applications of PHB are to be developed to make the process economically feasible. Integrated strategies for the development of more efficient metabolically engineered strains that can utilize cheap carbon source with increased PHB production are to be developed in near future. With these strategies, PHB will become the polymer of the next decade.

REFERENCES 1. Savenkova L, Gercberga Z, Muter O, Nikolaeva V, Dzene A, Tupureina V. Over-expression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly (3-hydroxybutyrate) production. Process Biochem 2002;37:719–22. 2. Tsuge T. Metabolic improvements and use of inexpensive carbon sources in microbial production of polyhydroxyalkanoates. J Biosci Bioeng 2002;94:579–84. 3. Luengo JM, Garcia B, Sandoval A, Naharro G, Oliver ER. Bioplastics from microorganisms. Curr Opin Biotechnol 2003;6:251–60. 4. Aldor IS, Keasling JD. Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates. Curr Opin Biotechnol 2003;14:475–83. 5. Degelau A, Scheper T, Bailey JE, Guske C. Fluorometric measurement of poly-β-hydroxybutyrate in Alcaligenes eutrophus by flow cytometry and spectrofluorometry. Appl Microbiol Biotechnol 1995;42:653–7. 6. Ostle AG, Holt JG. Nile blue A as a fluorescent stain for poly-β-hydroxybutyrate. Appl Environ Microbiol 1982;44:238–41. 7. Spiekermann P, Bernd HA, Kalscheuer RR, Baumeister D, Steinbuchel A. A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 1999;171:73–80.

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