Appl Microbiol Biotechnol (2006) 71: 783–789 DOI 10.1007/s00253-006-0451-1
MINI-REVIEW
Daniel K. Y. Solaiman . Richard D. Ashby . Thomas A. Foglia . William N. Marmer
Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates) Received: 13 January 2006 / Revised: 29 March 2006 / Accepted: 30 March 2006 / Published online: 18 May 2006 # Springer-Verlag 2006
Abstract Aside from their importance to the survival and general welfare of mankind, agriculture and its related industries produce large quantities of feedstocks and coproducts that can be used as inexpensive substrates for fermentative processes. Successful adoption of these materials into commercial processes could further the realization of a biorefinery industry based on agriculturally derived feedstocks. One potential concept is the production of poly (hydroxyalkanoate) (PHA) polymers, a family of microbial biopolyesters with a myriad of possible monomeric compositions and performance properties. The economics for the fermentative production of PHA could benefit from the use of low-cost agricultural feedstocks and coproducts. This mini-review provides a brief survey of research performed in this area, with specific emphasis on studies describing the utilization of intact triacylglycerols (vegetable oils and animal fats), dairy whey, molasses, and meat-andbone meal as substrates in the microbial synthesis of PHA polymers.
Introduction Poly(hydroxyalkanoates) (PHAs) are microbial polyesters synthesized and sequestered as intracellular granules by microorganisms belonging to the Bacteria and Archaea domains of life. These storage materials are generally Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. D. K. Y. Solaiman (*) . R. D. Ashby . T. A. Foglia . W. N. Marmer Fats, Oils and Animal Coproducts Research Unit, Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA e-mail:
[email protected] Tel.: +1-215-2336476 Fax: +1-215-2336795
considered as the carbon and energy reserves of the producing microorganisms grown under duress. PHAs are commonly grouped into two major categories: the shortchain-length (scl-) and the medium-chain-length (mcl-) PHAs (Fig. 1). The repeat units of scl-PHAs are composed of hydroxy fatty acids (HFA) having three to five carbon atoms, whereas, mcl-PHAs contain HFAs repeat units with six or more carbon atoms. In general, the scl-PHAs are more crystalline than the mcl-PHAs. As such, scl-PHAs usually exhibit thermoplastic-like properties of thermoplastics, while mcl-PHAs behave like elastomers or adhesives. Because of their biodegradable and biocompatible properties, PHAs have been extensively researched as potentially attractive “green” substitutes for petroleumderived polymers for use in medicine, drug-delivery, agriculture and horticulture, the fibers industry, and consumer products (Zinn et al. 2001; Luengo et al. 2003; Steinbüchel and Lütke-Eversloh 2003; Lenz and Marchessault 2005). The genetic loci responsible for PHA biosynthesis have been classified into four classes (see Fig. 2). The type of PHA (i.e., scl- or mcl-) synthesized by a microorganism is largely determined by the class of PHA biosynthesis genes it harbors. Nevertheless, the enzymes responsible for either sclor mcl-PHA polymerization (i.e., PHA synthases) have sufficient flexibility in substrate specificity to also incorporate medium- or short-chain-length HFA monomers, respectively, albeit, at a suboptimal rate. This flexibility has allowed the biosynthesis of numerous PHAs that contain a variety of substituted pendant groups or backbones with different carbon chain lengths and ester linkages. PHAs are, thus, a family of biopolyesters with many structural possibilities and wide-ranging field of applications. There are two routes to biotechnological production of PHA, i.e., microbial fermentation and plant-based production system, each with its own advantages and shortcomings. Currently, commercial efforts to produce PHA employ microbial fermentation processes, whereas, plantbased production systems are still in the research and development stage. For fermentation production of biobased products, the cost of the fermentative substrate
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H
C
C O
O
(CH2)n
m
Fig. 1 Schematic representation of poly(hydroxyalkanoate) (PHA). The stereo drawing depicts the [R–] configuration of the repeat units in PHA isolated from organisms. The majority of naturally isolated PHA have n=1, but may be varied by the feeding of specific substrates. Short-chain-length PHAs contain an R group with one to three carbons. Medium-chain-length PHAs contain R groups with four or more carbons, which may contain double-bond(s)
constitutes a significant proportion of the total production costs. For PHA production specifically, estimates of the contribution of the substrate cost to total production costs were 28–50 % (Lee and Choi 1998; Lynd et al. 1999; Braunegg et al. 2004). Consequently, research efforts abound to exploit inexpensive fermentable raw materials as fermentative substrates for PHA production. Agriculture and its associated industries produce many feedstocks and coproducts that are attractive raw materials for use in microbial production of PHAs. From an ecological point of view, they are renewable and from an economic point-ofview, many of the coproducts being studied are derived from surplus or low-cost processing streams. This minireview provides a quick survey of research in this field, with an emphasis on the utilization of fats and oils, molasses, the glycerol-rich biodiesel coproduct streams, and meat-and-bone meal for PHA production.
Synthesis of short-chain-length PHA The traditional and commonly used substrates for the fermentative production of many microbial products, including the scl-PHA, are glucose and other saccharides. Nevertheless, fatty acids derivable from agricultural triacylglycerols (i.e., vegetable oils, animal fats, and coproducts derived thereof such as recycled grease) have now attracted the attention of researchers because they may serve as a better Fig. 2 Classes of PHA biosynthesis gene clusters. Genes and the enzymes they code for are as follow: phaA β-ketothiolase; phaB acetoacetyl-CoA reductase; phaC, phaC1, and phaC2, PHA synthases; phaE and phaR the second subunit of the class III and class IV PHA synthases, respectively; phaP phasin; phaQ homologous to the helix-turnhelix multiple antibiotic resistance protein; phaZ PHA depolymerase. ORF-1 is an open-reading-frame putatively coding for an unknown protein
fermentative substrate in certain instances for microbial growth and production of bioproducts. From a metabolic point-of-view, fatty acids are energetically advantageous substrates because their complete β-oxidation generates more chemical energy equivalents in the form of ATP molecules than the complete oxidation of a molar equivalent of glucose does. From a natural resource management pointof-view, agricultural triacylglycerols (TAG) are renewable raw materials. The annual global production of agricultural triacylglycerols is estimated at 100 million tons. In the U.S. alone, the annual production of animal fats and vegetable oils is estimated at 16 million metric tons. Nevertheless, additional industrial outlets for agricultural TAGs are constantly sought to ensure the economic health of the farm sector. For fermentation purposes, it is desirable to directly use the TAG as the substrate rather than the fatty acids that must first be obtained through an additional step of saponification. Furthermore, the glycerol generated by microbial hydrolysis of TAG could itself serve as additional carbon and energy source for microbial fermentation. Report of the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HBco-3HHx)) from unsaponified olive oil by Aeromonas caviae first appeared in the early 1990s (Shimamura et al. 1994). (The authors have included the discussion of P(3HB-co-3HHx) in this section of scl-PHA because the mol % of 3HHx is usually less than 20 %.) Subsequently, Fukui and Doi (1998) demonstrated the use of olive oil, corn oil and palm oil to produce P(3HB) and P(3HB-co-3HHx) from Alcaligenes eutrophus (now Ralstonia eutropha) H16 wild type and recombinant R. eutropha PHB−4 (a mutant lacking PHBsynthesizing ability) expressing the PHA synthase gene of A. caviae, respectively. A subsequent scaled-up fermentation study at 10-l capacity by the same laboratory showed that the wild-type R. eutropha H16 synthesized P(3HB) from soybean oil with a volumetric yield of 95.8 g polymer l−1 and the recombinant PHB−4 expressing the A. caviae gene produced P(3HB-co-3HHx) at 102.1 g polymer l−1 (Kahar et al. 2004). In a subsequent work, Loo et al. (2005) studied the suitability of palm kernel oil, palm olein, crude palm oil, and palm acid oil as substrates for scl-PHA synthesis by R. eutropha PHB−4
Class I (Ralstonia eutropha)
phbC
phbA
phbB
Class II (Pseudomonads)
phaC1
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phaC2
Class III (Allochromatium vinosum ) ORF-1 phaC
phaE
phbA
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Class IV (Bacillus megaterium )
phaP
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phaC
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containing the PHA synthase gene of A. caviae. Copolymer P (3HB-co-3HHx) was synthesized at yields ranging from 1.5 to 3.7 g copolymer l−1, and the content of 3HHx in the polymer was 5 mol %. In an attempt to find additional microorganisms capable of utilizing palm-oil products for growth and PHA production, Alias and Tan (2005) isolated a Gram-negative bacterium, FLP1, from palm oil mill effluent by using a culture-enrichment technique. The isolate was tentatively identified as closely related (80 % similarity by BIOLOG determination) to Burkholderia cepacia. FLP1 produced P (3HB) when grown on crude palm oil and palm kernel oil. Supplementation of odd-number fatty acids (i.e., valerate and propionate) resulted in the synthesis of poly(3-hydroxybutyrate- co-3-hydroxyvalerate) P(3HB-co-3HV) by the FLP1 strain. Soybean oil is another agriculturally important vegetable-oil substrate examined for scl-PHA synthesis. As described earlier, Kahar et al. (2004) used these same R. eutropha H16 wild-type and recombinant R. eutropha PHB−4 containing the A. caviea PHA-biosynthesis gene to show the production of P(3HB) and P(3HB-co-3HHx) with soybean oil as substrate. Akiyama et al. (2003) performed a computersimulation study to examine the economics and the environmental impacts of the large-scale production of P (3HB-co-3HHx) from soybean oil by the recombinant R. eutropha strain described above. The calculated cost of production was US$3.5–4.5 kg−1 at a production scale of 5,000 tons per year. In comparison, the production cost of P (3HB) with glucose as feedstock was calculated at US$3.8– 4.2 kg−1. Life cycle inventories of energy consumption and carbon dioxide emissions for the two processes under various presumptions were also calculated. The results indicated that in general, the soybean oil-based production model consumed less total energy and emitted less carbon dioxide than did the glucose-based production model. The dairy industry is an important part of the agricultural sector with a forecast global milk and cheese output of roughly 410 and 14 million metric tons, respectively, in 2005 (USDA/Foreign Agricultural Service, July, 2005). Cheese whey, a large-volume coproduct stream (130–145 million tons per year generated globally) resulting from cheese manufacture, is rich in fermentable nutrients such as lactose (see Fig. 3), lipids and soluble proteins (Athanasiadis et al. 2002). In addition to its surplus status, the environmental concern associated with the direct disposal of cheese whey makes this coproduct stream a prime target for developing new uses such as serving as a feedstock for fermentative processes. Lee et al. (1997) demonstrated that poly(3hydroxybutyrate) (P(3HB)) could be obtained at a yield of 4.5–5.2 g l−1 from the fermentation of whey by recombinant Escherichia coli GCSC4401 and GCSC6576 strains that express the PHB-biosynthesis genes of R. eutropha. Ahn et al. (2000) improved the yield and productivity of P(3HB) to 96.2 g l−1 and 2.57 g l−1 h−1, respectively, by employing a high-density cultivation process with recombinant E. coli CGSC4401 expressing the Alcaligenes latus PHA biosynthesis genes. In this process, the whey was first concentrated to 280 g lactose l−1 before its use as a fermentative feedstock, and the dissolved-oxygen-content (DOC) of the culture was decreased stepwise during the P(3HB)-synthesis stage of the
fermentation process. Ahn et al. (2001) improved the yields and productivity of P(3HB) from whey fermentation by the same recombinant E. coli by employing a cell-recycle fedbatch culture approach. The approach helped overcome the culture volume constraint brought on by continual periodic addition of whey feed stream. With the cell-recycle approach, P(3HB) concentration and productivity of 168 g l−1 and 4.6 g polymer l−1 h−1, respectively, were achieved. The same laboratory subsequently studied pilot plant-scale, fedbatch fermentation production of P(3HB) from whey substrate using the genetically engineered E. coli CGSC 4401 expressing the A. latus PHA biosynthesis genes (Park et al. 2002). A volumetric yield of 35.7 g P(3HB) l−1 at a productivity rate of 1.35 g polymer l−1 h−1 was attained in a 30-l fermenter. In a 300-l fermenter, however, the corresponding values decreased to 20.1 g P(3HB) l−1 and 1.0 g P (3HB) l−1 h−1, respectively. Researchers have also investigated the synthesis of scl-PHA from whey and its major component, lactose, by nonrecombinant organisms. For example, Yellore and Desai (1998) described the isolation of a Methylobacterium sp. ZP24 that grew on cheese whey and produced P(3HB) at a yield of 1.1 g polymer l−1. Supplementation of the whey medium with ammonium sulphate increased the P(3HB) yield to as high as 3 g l−1. Marangoni et al. (2002) investigated the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) by R. eutropha grown on whey-based basal medium supplemented with invert sugar (i.e., an equal mixture of glucose and fructose resulting from the hydrolysis of sucrose) and with periodic feeding of propionic acid. They reported a productivity of only 0.17 g l−1 h−1, but a high content (38 mol %) of 3-hydroxyvalerate comonomer was found in the polymer. Povolo and Casella (2003) reported the ability of Sinorhizobium meliloti 41 and Hydrogenophaga pseudoflava DSM 1034 to use cheese whey for cell growth and P (3HB) production. Their results showed that S. meliloti and H. pseudoflava cultures grew to 0.48 and 0.38 g cell dry weight (CDW) l−1, and produced P(3HB) at 3.5 and 4.4 % of CDW, respectively. They also attempted the isolation of soil bacteria capable of growth on lactose and producing P(3HB). Three strains (Sinorhizobium sp., Bacillus sp., and B. megaterium) were obtained by such screening, but the P (3HB) yields when grown on whey were low. Dhanasekar and Viruthagiri (2005) mathematically modeled and experimentally examined the batch production of P(3HB) by Azotobacter vinelandii using sucrose or cheese whey as a substrate. The study showed that polymer biosynthesis was highly dependent on the initial biomass concentration. For the cheese whey fermentation, a production of 1.8 g l−1 of P (3HB) in 48 h was reported with an initial biomass concentration of 0.3 g l−1. The experimental set up, however, was not described in sufficient detail for further evaluation. Young et al. (1994) devised a two-stage fermentation system to produce P(3HB) from lactose and D-xylose using Pseudomonas cepacia as the producing organism. In this process, cells were grown in a high nitrogen-content medium in the first stage, and in the second stage, production of P (3HB) occurred in a nitrogen-limited medium. They also showed that with lactose as the sole carbon source, they
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could obtain a polymer yield of 2 g P(3HB) l−1 from 1.25-l culture in 120 h. Molasses is a sugar-rich coproduct stream generated by crop refining industries. Sugar beet and sugar cane refining plants are the major sources for sugar molasses that contains high sucrose content. Depending on the grades and sources, sugar molasses cannot be further used in foods or feeds and is, thus, ideal for consideration as an inexpensive carbon source for fermentative processes. According to Tate & Lyle Molasses Germany GMBH (http://www.melasse.de/originsofmolasses.html), the quantities of cane molasses and beet molasses generated worldwide in 2004 were 39 and 12 million tons, respectively. Soy molasses, a by-product from soybean processing to soy proteins, is another coproduct stream that could benefit from new uses to alleviate disposal problems. It contains a high concentration (30 %) of soluble carbohydrates especially sucrose, stachyose, and raffinose (Fig. 4), and is thus, a good carbon source for fermentation by microorganisms that possess the appropriate enzymes (i.e., α-galactosidases and β-furanosidases) and metabolic pathways to utilize these carbohydrates. Although the annual global production numbers for soy molasses are not readily available, the ever-increasing demand for soy protein-based food products is accompanied by increased production of this coproduct stream. Research on the use of molasses for PHA production is, thus, of interest to researchers and of relevance to the industries eager to utilize these materials. Page et al. (1992) reported the production of P(3HB-co-3HV) at 19-22 g polymer l−1 by A. vinelandii UWD grown on beet molasses supplemented with valerate. The 3HV content of the polymer could be varied between 8.5 and 23 mol % by adjusting the substrate feeding pattern and rate of valerate addition. Wu et al. (2001) isolated a Bacillus sp., JMa5, and showed its ability to use cane molasses in conjunction with sucrose to synthesize up to 25 g P(3HB) l−1. Spore formation, however, limited the potential of Bacillus to attain high PHA production. Celik and Beyatli (2005) screened a collection of Pseudomonas spp. isolates and reported the synthesis of P(3HB) from one of these strains, P. cepacia G13. The reported P(3HB) yield from cells grown in a medium containing 3 % sugar molasses was 70 % of cell dry weight. Unfortunately, the total dry cell mass, total amount of the purified polymer, and the composition analysis of the isolated polymer were not reported. Similarly, production of P(3HB) by Rhizobium meliloti (Mercan and Beyatli 2005) and by Bacillus cereus M5 strain (Yilmaz and Beyatli 2005) was demonstrated using
sugar beet molasses as a carbon source. P(3HB) contents of 56 and 74 % of cell dry weigh for R. meliloti and B. cereus, respectively, were reported, but total cell mass and monomer composition were not determined. Biobased fuels, such as ethanol and biodiesel, are important alternative renewable energy sources and are manufactured using agricultural products as raw materials. The production of biodiesel generates significant quantities of a coproduct stream rich in glycerol. New uses for glycerol have been the subject of much research to alleviate a market glut of this commodity and to leverage the economics of biodiesel production. One potential use of glycerol is in industrial fermentation where it can be employed as a substrate for microbial growth and the biosynthesis of microbial bioproducts (see Fig. 5 for glycerol utilization pathway). In the PHA production arena, Borman and Roth (1999) used Methylobacterium rhodesianum to synthesize P(3HB) at a product yield of 10.5 g polymer l−1 culture in a 2-l culture that contained glycerol (50 %) and casein peptone as carbon and nitrogen sources, respectively. A similar experiment with R. eutropha DSM 11348 yielded up to 15 and 17.6 g P (3HB) l−1 of product with glycerol as the carbon source and casein peptone or Casamino acids as the nitrogen source. Ashby et al. (2005) investigated the synthesis of P(3HB) from glycerol in a chemically defined medium by a Pseudomonas oleovorans strain NRRL B-14682 that synthesizes scl-PHA (Solaiman and Ashby 2005). The results showed that P(3HB) was produced at a yield of 0.97 g polymer l−1 in 5 %-glycerol medium in shake-flask cultures. A systematic study further showed that increasing the concentration of glycerol in the medium resulted in the synthesis of lower molecular-weight P(3HB) due to glycerol end-capping, a finding that is certain to have an important implication when using biodiesel-derived glycerol coproduct streams to produce PHA. The actual use of a glycerol-rich biodiesel coproduct stream for scl-PHA production was reported by Koller et al. (2005). These investigators showed that an unidentified osmophilic organism could produce P(3HB-co-3HV) from the glycerol liquid phase from biodiesel production at a yield of 16.2 g Stachyose Raffinose Sucrose
α -D-gal
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Fig. 3 Major fermentable carbohydrate of cheese whey. Lactose is broken down by β-galactosidase activity into galactose and glucose. The galactose and glucose are then metabolized via Leloir and the glycolysis pathways, respectively
Fig. 4 The major soluble carbohydrates of soy molasses. With an appropriate α-galactosidase activity, the galactose units are successively released from stachyose and raffinose to yield sucrose. Sucrose can be broken down into fructose and glucose by βfructosidases. The monosaccharides are subsequently metabolized by the appropriate pathways of the microbial system
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Glucose
Fig. 5 Glycerol as carbon and energy source. Pathways leading from glycerol to glucose or acetyl CoA are shown. The three thermodynamically favorable forward reactions are catalyzed by fructose 1,6bisphosphatase (1), glucose 6-phosphatase (2), and pyruvate kinase (3)
Pi
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OPO32Dihydroxyacetone Phosphate
OPO32Glyceraldehyde 3-Phosphate NAD+ + Pi NADH + H+ ADP ATP
Glycolysis
H2O ADP 3
ATP
Pyruvate
polymer l−1 without the addition of an odd-number fattyacid precursor as typically required in other fermentation systems and that the 3HV content in the polymer was 8– 10 mol %. These authors also described a decrease in molecular weight of the polymer as a result of using glycerol-containing biodiesel coproduct stream as a carbon source. In a companion study to their earlier work that used pure glycerol, Ashby et al. (2004) reported the use of a wellcharacterized biodiesel coproduct stream containing 34 % glycerol as the sole carbon source in a chemically defined basal medium to synthesize P(3HB) by shake-flask cultures of P. oleovorans NRRL B-14682. Again, a decrease in the molecular weight of the polymer was observed with an increase of concentration of the glycerol biodiesel coproduct stream in the culture medium. With the diagnosis of bovine spongiform encephalopathy (BSE) in Britain nearly two decades ago, regulatory agencies and the rendering industry have taken major steps to address this problem. As a result, meat-and-bone meal, which was traditionally used as animal feed, has become a surplus rendering coproduct that urgently needs new uses and/or alternative markets. Although the main focus of meat and bone meal utilization research is on energy generation, researchers have started to investigate its use as an inexpensive source of nitrogen and other nutrients needed in fermentation. In the PHA arena, Koller et al. (2005) described the use of the hydrolysate of meat-andbone meal as a nitrogen source in conjunction with the
Acetyl CoA
glycerol-rich biodiesel coproduct stream as a carbon source for the fermentative production of P(3HB) and obtained a yield of 5.9 g polymer l−1 culture.
Synthesis of medium-chain-length PHA Historically, fatty acids have been the preferred substrates for the microbial synthesis of mcl-PHA. Economy, however, could be achieved by directly using triacylglycerols as feedstocks for fermentation because the costs inherent to the saponification process can be avoided. Cromwick et al. (1996) first demonstrated the use of an intact triacylglycerol (i.e., tallow) for the synthesis of mcl-PHA by Pseudomonas resinovorans. Ashby and Foglia (1998) subsequently demonstrated the synthesis of mcl-PHA by P. resinovorans with other intact oils and fats and showed that the repeat-unit composition of the biopolymer reflected the fatty acyl composition of the oil or fat substrate used for their synthesis. Thus, the PHA derived from the feeding of soybean oil has higher content of unsaturated HFA monomers than the polymer obtained with coconut oil. Pseudomonas stutzeri 1317 was reported also to use soybean oil as a substrate for growth and the synthesis of mcl-PHA at a yield of 1.7 g polymer l−1 culture (He et al. 1998). As the majority of Pseudomonas species that produce mcl-PHA do not utilize triacylglycerols for growth and polymer synthesis, genetic engineering techniques
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have been used to express a lipase gene in these bacteria. A convenient electroporation method has been developed to transform many well-known PHA-producing Pseudomonas such as Pseudomonas putida, P. oleovorans, and Pseudomonas corrugata into triacylglycerol-utilizing strains (Solaiman et al. 2001, 2002). The synthesis of mcl-PHA from glycerol or the glycerolrich biodiesel coproduct stream has also been investigated. Ashby et al. (2004) showed the use of the coproduct stream from soybean oil-based biodiesel production to synthesize mcl-PHA by P. corrugata. Maximum cell yields of 2.1 g CDW of P. corrugata l−1 of culture were achieved at 1 % (v/v) of coproduct stream concentration, and a maximum mcl-PHA content of 42 % CDW was attained at 3 % (v/v) of glycerol coproduct stream level. Unlike the case with scl-PHA synthesis by P. oleovorans NRRL B-14682, the molecular weight of the mcl-PHA produced by P. corrugata grown on a glycerol biodiesel coproduct stream did not decrease with increasing concentration of the glycerol substrate. In a separate study using pure glycerol, Ashby et al. (2005) showed that P. corrugata synthesized mcl-PHA at a maximum yield of 0.67 g polymer l−1 at 2 % (v/v) substrate concentration, and the molecular weight of the polymer decreased 72 % in 5 %-glycerol cultivation medium. These investigators further devised a mixedculture fermentation scheme to use pure glycerol as substrate to produce in situ blends of P(3HB) and mclPHA at varying proportions by controlling the inoculation time and duration of fermentation (Ashby et al. 2005). Molasses had not been extensively studied for use as a substrate in the synthesis of mcl-PHA partly because fatty acids had traditionally been considered as the ideal feedstocks as alluded to earlier in this review. In a recent study, Solaiman et al. (2006) screened several mcl-PHAproducing Pseudomonas species and identified P. corrugata as capable of growing and producing biopolymer when cultivated on soy molasses medium. The biopolymer yields from P. corrugata cells grown in shake-flask cultures in a chemically defined medium supplemented with 5 % (w/v) soy molasses were ≥0.6 g mcl-PHA l−1. Obviously, additional study is needed to increase the polymer productivity to make this inexpensive substrate useful for PHA production.
Perspectives Since the initial attempt by Imperial Chemical Industries (ICI) to commercialize P(3HB-co-3HV) in the early 1980s, subsequent efforts and interests by several global companies to market PHA worldwide have not been sustaining. Currently, Metabolix (USA) and Kaneka (Japan) are among the few companies actively seeking to commercially produce PHA biopolymers. Even though it has been claimed that the current production processes purportedly allow for a cost-effective manufacture of these materials, an even lower total production cost is nevertheless desired to increase profitability. The use of low-cost agricultural feedstock and their coproducts as fermentative substrates
could improve the economics of microbial PHA production. An added attractiveness for using these substrates is that they are renewable resources, making the production process ecologically sound and geopolitically independent. However, the perceived major hurdles for adopting these feedstocks to industrial PHA production are many. The chief among these is the variability of the amount of actual fermentable substrates and the presence of adjunct nonfermentable components in the feedstocks. These factors will invariably affect the productivity of the fermentation and the downstream process. This is particularly imperative because it is estimated that a commercially viable process must have a polymer productivity of well above 100 g polymer l−1 in 2–3 days. It is, thus, desirable that a program or system be devised to monitor the “quality” (such as compositions) and to systematically document the performance of various lots of agricultural feedstocks and coproduct streams used in the fermentative process. Such a database could help define an acceptable range for feedstock variability. It also would be helpful if the bioprocess itself is tolerant to these variable factors, including, for example, the total carbohydrate content of whey and molasses, the glycerol concentrations in biodiesel coproducts and the type, distribution, and total amount of amino acids in meat-and-bone meal preparations. Inherent to the surplus nature of the various coproduct streams is the presence of undefined “ashes” and other adjunct components that almost certainly pose a challenge to further downstream processing of the PHA polymers. Furthermore, any real or perceived difference in the quality and properties of the polymers produced from agriculturally derived feedstocks and coproduct streams need to be ascertained by a thorough characterization of the products. Innovative and better processes may be required to handle these crude product streams. These processes need to also address other technical issues related to the use of crude product streams in large-scale fermentation situations. For example, high cell-density fermentation critical to PHA product-yields is difficult to attain with agricultural feedstocks and coproduct streams containing dilute concentrations of substrates (e.g., sugars and glycerol); and the heat generated in fermentation using fatty acids as substrates necessitates proper cooling mechanism. Once these hurdles have been overcome, then we can realize the utilization of agricultural feedstock and coproduct streams in biorefinery-type industrial production to benefit the producers, consumers and the environment.
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