Strategies for Large-Scale Production of Polyhydroxyalkanoates, Chem

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Strategies for Large-Scale Production of Polyhydroxyalkanoates, Chem G. KAUR and I. ROY, Strategies for Large-scale Production of Polyhydroxyalkanoates, Chem. Biochem. Eng. Q., 29 (2) 157–172 (2015) 157 Strategies for Large-scale Production of Polyhydroxyalkanoates G. Kaur and I. Roy* doi: 10.15255/CABEQ.2014.2255 Faculty of Science and Technology, University of Westminster, Review 115 New Cavendish Street, London W1W6UW, United Kingdom Received: August 23, 2014 Accepted: June 9 ,2015 Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible intracellular polyesters that are accumulated as energy and carbon reserves by bacterial species, under nutrient limiting conditions. Successful large-scale production of PHAs is dependent on three crucial factors, which include the cost of substrate, downstream processing cost, and process development. In this respect, design and implementation of bioprocess strat- egies for efficient PHA bioconversions enable high PHA concentrations, yields and pro- ductivities. Additionally, development of PHA fermentation processes using inexpensive substrates, such as agro-industrial wastes, facilitates further cost reduction, thus benefit- ting large-scale PHA production. Thus, the aim of this review is to highlight various bi- oprocess strategies for high production of PHAs and their novel copolymers in relatively large quantities. This review also discusses the application of kinetic analysis and math- ematical modelling as important tools for process optimization and thus improvement of the overall process economics for large-scale production of PHAs. Key words: polyhydroxyalkanoates, PHA copolymers, large-scale production, PHA optimization, bi- oprocess strategies Introduction products as compared to the toxicity resulting from a huge accumulation of lactic acid generated by Polyhroxyalkanoates (PHAs) are macromolec- PLLA degradation. The latter leads to complica- ular storage polyesters produced by metabolic trans- tions in medical applications.6 The monomeric com- formation of carbon sources by microorganisms, position also affects the degradation rate of PHAs. under nutrient limiting conditions. The latter in- Thus, the possibility of a tailored composition of volves excess of carbon source and limiting concen- PHAs provides a means to control their degradation trations of at least one other essential nutrient, such at a desired rate (short or extended period of time) as nitrogen, phosphorous, and/or sulphur.1 PHA ac- depending upon the application.7 Both in-vitro and cumulation as intracellular carbon and energy re- in-vivo tests have proved PHAs to be biocompatible serves can be rationalised due to their oxidized state with bone, cartilage, blood and various other cell and consequent high calorific value of >20 KJ g–1.2 lines. Their biocompatibility has been reviewed in 8 The ever increasing popularity of PHAs is based on detail by Valappil et al. Other polymers, such as their biodegradable and biocompatible nature along starch, exhibit unsatisfactory mechanical properties 9 with the possibility of tailored molecular structure and difficult processing. Thus, the lack of variabil- and/or composition, which makes them highly ver- ity in structure, extensive material properties, and satile candidate materials for a range of different controlled degradation for PLLA or starch-based applications, including bulk and medical.3,4 This has polymers makes them less attractive than PHAs. led to the development of more than 150 different Several microorganisms, including both native types of biotechnologically produced PHAs to date.5 and recombinant strains, have been employed for PHAs offer several distinct advantages over other PHA production. Industrial production processes popular biopolymers such as poly-L-lactic acid for PHAs have generally been developed using (PLLA) or starch-based polymers e.g., starch-poly- Gram negative strains, such as Cupriavidus necator ethylene. PHA degradation mechanism involves and Alcaligenes latus, mainly due to the relatively surface erosion rather than bulk degradation report- high PHA yields offered by them and the ability of some strains to synthesize PHAs under nutrient ed for PLLA. This allows a more predictable degra- 10–11 dation profile of PHA-based medical products than non-limiting conditions. However, huge efforts PLLA. In addition, PHAs have reduced immune have also been directed towards process develop- response due to lower acidity of their degradation ment based on Gram positive strains such as Bacil- lus sp. and Corynebacterium glutamicum. Their * Corresponding author: [email protected] primary advantage is the absence of the lipopoly- 158 G. KAUR and I. ROY, Strategies for Large-scale Production of Polyhydroxyalkanoates, Chem. Biochem. Eng. Q., 29 (2) 157–172 (2015) saccharide (LPS) layer in the Gram positive strains, ties. The latter leads to higher capital and operating which makes the PHAs derived from them to be costs, especially for large bioreactors. ideal for medical applications.12 Improvement in process economics is possible The PHAs can be divided into three main cate- by design and implementation of efficient biopro- gories based on the number of carbon atoms in their cess strategies for improving the overall process ki- monomeric units i.e., short-chain length (scl-PHAs) netics, and thereby resulting in higher PHA concen- containing 3–5 carbon atoms, medium-chain length trations and productivities.22 Besides natural strains, (mcl-PHAs) containing 6–14 carbon atoms, and tremendous efforts in metabolic engineering in the long-chain length (lcl-PHAs) containing more than recent years have led to the development of high 14 carbon atoms.13–14 Another type of classification product-yielding strains, which are now being in- divides PHAs into homopolymers i.e., containing creasingly used in process development. However, only one type of monomer unit, and heteropolymers while dealing with recombinant microorganisms, (copolymers), which are composed of more than stability of the plasmids is an important aspect that one type of monomer unit.13 Copolymers could needs to be clearly established and ensured before be a combination of only scl-/mcl-PHA monomers the engineered strain could be used for large-scale or consist of both scl- and mcl-PHA units. Of all production. Otherwise, there is a risk that the plas- the PHAs, the scl-PHA, poly-3-hydroxybutyrate, mid might lose its function over several runs of fer- P(3HB), has been most widely explored.4 However, mentation (production cycles).23–24 Additionally, one of the major challenges with P(3HB) is its brit- maintenance of plasmid stability usually requires tle and stiff nature, which limits the applications of addition of expensive antibiotics in the medium, this polymer. On the other hand, mcl-PHAs are which is not very suitable for economic large-scale flexible and elastomeric polymers.14–15 Their struc- production. In such a case, other plasmid mainte- tural diversity allows more flexibility with respect nance strategies, such as creation of an auxotrophic to tailoring their properties to suit specific applica- mutant with a deletion and then complementing that tions.16 Additional advantages are offered by PHA mutation with a plasmid containing the deleted scl-mcl copolymers, which interestingly combine gene, could be used.24 specific properties of their constituent monomers In addition, the use of thermophilic or thermo- and facilitate in catering to more diverse applica- tolerant strains, especially for industrial production tions.4,16 of PHAs, offers several cost advantages. These in- While research efforts towards large-scale PHA clude operation at medium and elevated tempera- production have been continued for more than three ture, which in turn accelerates the reaction rates, decades, the final quantity and quality of the bio- reduces the cost for cooling/heating, and decreases polymer are the two major factors that dictate their the chances of cross-contamination from other mi- market applications.17 The PHA production process croorganisms.25 Another method to reduce the fer- involves a series of complex steps beginning from mentation cost is the development of production biomass growth, polymer accumulation, cell har- protocols for the bioconversion of inexpensive and vesting to polymer extraction and purification which renewable carbon (and/or nitrogen) substrates, in- affect the final polymer quality. The nature of mi- cluding waste and by-products from agriculture and croorganism, inherited metabolic pathway, medium industrial sources, instead of pure and refined sub- constituents and bioprocess strategy are additional strates.20 In fact, recent years have witnessed an in- important factors controlling the polymer yield and creasing trend towards exploration of unconven- quality.18 In fact, the major challenge facing bio- tional substrates for cost efficient production of technological polymer production has been the de- several types of PHAs. velopment of economic PHA production competi- Thus, the present review highlights the impor- tive with the petrochemical synthesis. Major factors tance of process development towards efficient leading to relatively higher cost of bio-based PHA PHA fermentations enabling high PHA concentra- production include high cost of substrate, low poly- tions, yields and productivities. Results of various mer concentration, yield, and productivity of the bioprocess strategies including batch, fed-batch production processes.19–20 Cost of the substrate is with different types of feeding regimes, continuous
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