Richard D. Ashby

Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates).

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-and-bone meal as substrates in the microbial synthesis of PHA polymers.

Rapid and specific identification of medium-chain-length polyhydroxyalkanoate synthase gene by polymerase chain reaction

Abstract A polymerase chain reaction (PCR) protocol was developed for the specific detection of genes coding for type II polyhydroxyalkanoate (PHA) synthases. The primer-pair, I-179L and I-179R, was based on the highly conserved sequences found in the coding regions of Pseudomonas phaC1 and phaC2 genes. Purified genomic DNA or lysate of colony suspension can serve equally well as the target sample for the PCR, thus affording a simple and rapid screening of phaC1/C2-containing microorganisms. Positive samples yield a specific 540-bp PCR product representing partial coding sequences of the phaC1/C2 genes. Using the PCR method, P. corrugata 388 was identified for the first time as a medium-chain-length (mcl)-PHA producer. Electron microscopic study and PHA isolation confirmed the production of mcl-PHA in P. corrugata 388. The mcl-PHA of this organism has a higher molecular weight than that of similar polymers produced by other pseudomonads.

Poly(hydroxyalkanoate) biosynthesis from triglyceride substrates

Abstract The biosynthesis of poly(hydroxyalkanoates) (PHA) by Pseudomonas resinovorans from triglyceride substrates was investigated. Each triglyceride, whether animal fat or vegetable oil, supported cellular growth to relatively high average cell yields (3.3 ± 0.2 g/l). PHA yields ranged from 1.1 g/l to 2.1 g/l, representing approximately 45% of the bacterial cell dry weight. The repeat-unit composition of the polymers was determined by gas chromatography (GC) and GC/mass spectrometry of the β-hydroxyalkanoate methyl esters from the hydrolyzed polymers. With the exception of PHA from soybean oil (PHA-soy), each polyester was composed of β-hydroxyacyl moieties with chain lengths ranging from C4 to C14, with C8 and C10 being the predominant species. PHA-soy contained an additional fraction (2%) of C16 monomers. The alkyl side-chains of the PHA contained varying degrees of unsaturation. PHA from coconut oil was composed entirely of saturated side-chains, whereas PHA-soy contained 4.2 mol% olefinic groups in its side-chains. The increase in the degree of side-chain unsaturation caused decreased melting temperatures, enthalpies of fusion, and glass transition temperatures. The molar masses of the polymers were relatively constant and ranged from 6.5 × 104 to 10.1 × 104 g/mol.

Analysis and Characterization of Sophorolipids by Liquid Chromatography with Atmospheric Pressure Chemical Ionization

SummaryA reversed phase high performance liquid chromatographic method combined with atmospheric pressure chemical ionization mass detection (LC/APCI-MS) has been developed for the separation and analysis of sophorolipids produced byC. bombicola when grown on fatty acid mixtures. Using this method it was found that the incorporation of palmitic, linoleic, and linolenic acids into the sophorolipid structure was dependent on the initial fatty acid, content of these acids, whereas the incorporation of oleic acid, was independent of its initial content in the mixture. Also observed was the incorporation of esterified glycerol into the sophorolipid structure, which has not been reported previously.

Viscoelastic properties of linseed oil-based medium chain length poly(hydroxyalkanoate) films: effects of epoxidation and curing

Medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers derived from linseed oil (PHA-L) have a relatively small molar mass and contain a high concentration of unsaturated side-chains. As such, these polymers are amorphous and take on the consistency of a viscous liquid at room temperature. In order to increase the application potential of this material, the side-chain olefinic groups of PHA-L were converted to epoxy derivatives (PHA-LE) using m-chloroperoxybenzoic acid (m-CPBA). Epoxidation resulted in a 37% conversion of olefinic to epoxy groups. The epoxy groups enhanced the PHA-LE film susceptibility to crosslinking upon exposure to air. PHA-LE films began to crosslink and stiffen in less than 25 days, whereas PHA-L films began to crosslink between days 50 and 75. The PHA-LE films showed an increase in tensile strength (TS, from 4.8 to 20.7 MPa) and Youngs modulus (YM, from 12.9 to 510.6 MPa) between 25 and 100 days. In contrast, PHA-L had a TS of 25.0 MPa and YM of 767.8 MPa after 100 days. Epoxidation helped induce crosslink formation; however, aging for 100 days ultimately resulted in crosslinked films from both PHA-L and PHA-LE with higher strength and durability than the original materials.

LC/MS analysis and lipase modification of the sophorolipids produced by Rhodotorula bogoriensis**

The extracellular glycolipids produced by the yeast, Rhodotorula bogoriensis (formerly Candida bogoriensis), were analyzed using an LC/API-MS method. The analysis confirmed that the predominant form the sophorolipid structure contained a C22 hydroxy carboxylic acid. A minor amount (<10%) of a C24 hydroxy carboxylic acid in the sophorolipid was also found, which had not been reported previously. The sophorolipid product, which contained varying degrees of acetylation at the primary hydroxy groups of the sophorose sugar, was deacetylated with sodium methoxide. The des-acetylated sophorolipid was esterified using an immobilized lipase as catalyst in tetrahydrofuran and the product analyzed by mass spectrometric techniques. The product was screened for dimer or polymer formation but only a monomeric lactonized sophorolipid structure was detected.

The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown Pseudomonas oleovorans

Pseudomonas oleovorans NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV). Acetone fractionation verified the presence of PHB/mcl-PHA mixtures. The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms. In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (Tm) between 177 and 179°C, enthalpies of fusion (ΔHf) of 20 cal/g and glass transition temperatures (Tg) of 3–4°C. This was due to the large PHB content in the polymer mixtures. On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature. In both cases, the Tm values were 161°C, ΔHf values were 7cal/g and Tg values were −3°C. Journal of Industrial Microbiology & Biotechnology (2002) 28, 147–153 DOI: 10.1038/sj/jim/7000231

Physiological Characterization and Genetic Engineering of Pseudomonas corrugata for Medium-Chain-Length Polyhydroxyalkanoates Synthesis from Triacylglycerols

Pseudomonas belonging to the rRNA-DNA homology group I produce medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). We show that P. corrugata, a member of this group, accumulates 0.5–1.0 g of mcl-PHA/L of culture when grown on glucose (Gl) or oleic acid (Ol). The predominant monomers of Gl-PHA and Ol-PHA are β-hydroxydecanoate and β-hydroxyoctanoate, respectively. The molecular masses and polydispersity of P. corrugata PHAs are higher than those typically found with other Pseudomonas. We electrotransformed P. corrugata with a plasmid pCN51lip-1 carrying Pseudomonas lipase genes to generate strain III111-1. The recombinant strain grew on intact triacylglycerols (TAGs) to 1.9–2.7 g of cell-dry-weight/L of culture. The yields and the predominant repeat-units of PHAs obtained from the lard- and tallow-grown III111-1 were similar to those of Ol-PHA from wild-type cells. In contrast to other Pseudomonas species, P. corrugata III111-1 grown on TAGs at temperatures up to 36°C was not significantly affected with regard to cell yields, amounts of PHA produced, and the repeat unit compositions of the polymer.

Glucose/lipid mixed substrates as a means of controlling the properties of medium chain length poly(hydroxyalkanoates).

Glucose-triacylglycerol (TAG) mixed substrates were used to modulate the physical and mechanical properties of medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs). Pseudomonas resinovorans NRRL B-2649 grew and produced mcl-PHAs on glucose and TAGs (coconut oil, C; soybean oil, S) after 24 h in a shake flask culture. However, with the exception of coconut oil, maximum cell productivity was not reached in any of the cultures until 72 h post-inoculation. Here, 50:50 mixtures of glucose and coconut oil (glc/C) or glucose and soybean oil (glc/S) resulted in intermediate cell productivities with a maximum of 57% and 48% of the CDW at 72 h, respectively. In addition, mixed substrates resulted in mcl-PHAs with compositions that varied slightly over time. PHA-glc/C and PHA-glc/S were composed of 7 mol % and 8 mol % 3-hydroxydodecenoic acid (C(12:1)), respectively at 72 h. These concentrations were intermediate to the C(12:1) concentration of PHA-glc and respective PHA-TAG. Also, significant amounts of 3-hydroxytetradecanoic acid (C(14:0)), 3-hydroxytetradecenoic acid (C(14:1)), and 3-hydroxytetradecadienoic acid (C(14:2)) were present in PHA-glc/C and PHA-glc/S, which were derived from the respective TAG, as glucose resulted in almost no C(14:)(X) monomers. The molar masses of each of the polymers remained relatively constant between 24 and 96 h. At 72 h, the number-average molar masses (M(n)) of PHA-glc/C and PHA-glc/S were 178,000 and 163,000 g/mol, respectively, which were also intermediate to the M(n) of PHA-glc (225,000 g/mol) and the respective PHA-TAG (PHA-C = 153,000 g/mol; PHA-S = 75,000 g/mol). These physical differences caused variations in the mechanical properties of mcl-PHA films, thus providing a new and effective method of modifying their properties.

Glycerine and levulinic acid: renewable co-substrates for the fermentative synthesis of short-chain poly(hydroxyalkanoate) biopolymers.

Glycerine (a biodiesel co-product) and levulinic acid (a pulp and paper co-product) were used as co-substrates for the fermentative synthesis of short-chain polyhydroxyalkanoate (sc-PHA) biopolymers with tunable monomer and molecular weight characteristics. Pseudomonas oleovorans NRRL B-14682 utilized glycerine alone to produce poly(3-hydroxybutyrate) (PHB). When levulinic acid was added to the media at shake-flask scale in concentrations ≤0.6 wt.%, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/V) copolymers were produced with 3-HV contents ranging from 37 to 97 mol%; a glycerine:levulinic acid ratio of 0.2%:0.8% (w/v) resulted in poly(3-hydroxyvalerate) (PHV). Ten-liter batch fermentations using glycerine:levulinic acid ratios of 1%:0, 0.75%:0.25%, 0.5%:0.5% and 0.25%:0.75% (w/v) resulted in PHB, P(73%-3HB-co-27%-3HV), P(30%-3HB-co-70%-3HV) and PHV with increasing number average molecular weights (×10(3) g/mol) of 328, 511, 728 and 1330, respectively, owing to glycerine-based chain termination. These results provide a novel means by which glycerine and levulinic acid can be used collectively to produce an array of distinct sc-PHA biopolymers.