Alistair J. Anderson

Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126☆

A number of taxonomically-related bacteria have been identified which accumulate poly(hydroxyalkanoate) (PHA) copolymers containing primarily 3-hydroxyvalerate (3HV) monomer units from a range of unrelated single carbon sources. One of these, Rhodococcus sp. NCIMB 40126, was further investigated and shown to produce a copolymer containing 75 mol% 3HV and 25 mol% 3-hydroxybutyrate (3HB) from glucose as sole carbon source. Polyesters containing both 3HV and 3HB monomer units, together with 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV) or 3-hydroxyhexanoate (3HHx), were also produced by this organism from certain accumulation substrates. With valeric acid as substrate, almost pure (99 mol% 3HV) poly(3-hydroxyvalerate) was produced. N.m.r. analysis confirmed the composition of these polyesters. The thermal properties and molecular weight of the copolymer produced from glucose were comparable to those of PHB produced by Alcaligenes eutrophus.

A survey of the accumulation of novel polyhydroxyalkanoates by bacteria

SummaryA wide range of bacterial strains were examined for their ability to accumulate polyhydroxyalkanoates (PHA) from various carbon sources. Strains were selected from those reported to accumulate poly-3-hydroxybutyrate (PHB), related organisms and laboratory stocks. Other strains known to utilize n-alkanes, n-alcohols or n-acids were chosen to investigate their ability to produce long-chain PHAs. Five strains accumulated only PHB, 13 accumulated PHAs containing only C4 and C5 units and 7 accumulated PHAs containing 3-hydroxyacid units in the range C5 to C10.

CHAIN TERMINATION IN POLYHYDROXYALKANOATE SYNTHESIS: INVOLVEMENT OF EXOGENOUS HYDROXY-COMPOUNDS AS CHAIN TRANSFER AGENTS

We have identified a range of compounds which, when present during poly(3-hydroxybutyrate) [P(3HB)] accumulation by Ralstonia eutropha (reclassified from Alcaligenes eutrophus), can act as chain transfer agents in the chain termination step of polymerization. End-group analysis by 31P NMR of polymer derivatized with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane revealed that all these compounds were covalently linked to P(3HB) at the carboxyl terminus. All chain transfer agents possessed one or more hydroxyl groups, and glycerol was selected for further investigation. The number-average molecular mass (Mn) of P(3HB) produced by R. eutropha from glycerol was substantially lower than for polymer produced from glucose, and we identified two new end-group structures. These were attributed to a glycerol molecule bound to the P(3HB) chain via the primary or secondary hydroxyl groups. When a primary hydroxyl group of glycerol is involved in chain transfer, the end-group structure is in both [R] and [S] configurations, implying that chain transfer to glycerol is a random transesterification and that PHA synthase does not catalyse chain transfer. 3-Hydroxybutyric acid is the most probable chain transfer agent in vivo, with propagation and termination reactions involving transfer of the P(3HB) chain to enzyme-bound and free 3-hydroxybutyrate, respectively. Only carboxyl end-groups were detected in P(3HB) extracted from exponentially growing bacteria. It is proposed that a compound other than 3-hydroxybutyryl-CoA acts as a primer in the initiation of polymer synthesis.

Biosynthesis and composition of bacterial poly(hydroxyalkanoates)

It is well established that Alcaligenes eutrophus can accumulate a copolymer containing 3-hydroxybutyrate and 3-hydroxyvalerate, but longer 3-hydroxyacid monomers have not been reported to occur in this organism. The properties of the enzymes of poly(hydroxyalkanoate) (PHA) biosynthesis are discussed and it is proposed that the substrate specificity of the polymerizing enzyme restricts the range of monomer units incorporated into PHA. Various other bacteria produce similar copolymers from propionic acid and/or valeric acid. A number of Pseudomonas species accumulate PHAs containing longer-chain monomer units from linear alkanoic acids, alkanes and alcohols.

Effect of carbon source and concentration on the molecular mass of poly(3-hydroxybutyrate) produced by Methylobacterium extorquens and Alcaligenes eutrophus

In shake-flask culture, Methylobacterium extorquens accumulated poly(3-hydroxybutyrate) (PHB) possessing a substantially higher weight-average molecular mass (MW) than previously reported for this organism. The MW of PHB produced by M. extorquens was dependent on the initial concentration of methanol or sodium succinate, used as sole carbon sources. The highest MW values (0.6 and 1.7 × 106) were obtained with low initial concentrations of methanol or sodium succinate (4.0 and 3.0 g l−1, respectively) and the latter substrate always yielded PHB of higher MW than that produced from methanol. Thus PHB with an MW in the range 0.2–1.7 × 106 could be produced by selection of the carbon source and its concentration. In contrast to the findings with M. extorquens, the MW of PHB produced by Alcaligenes eutrophus was high (1.1–1.6 × 106) and generally unaffected by the choice or concentration of the carbon source. The use of glycerol as sole carbon source did, however, result in the accumulation of PHB with a markedly lower MW (5.5–8.5 × 105) than that produced from other sole carbon sources by this organism under similar conditions.

Production of docosahexaenoic acid by Crypthecodinium cohnii grown in a pH-auxostat culture with acetic acid as principal carbon source

Crypthecodinium cohnii, a marine alga used for the commercial production of docosahexaenoic acid (DHA), was cultivated in medium containing sodium acetate as principal carbon source; the pH was maintained at a constant value by addition of acetic acid, which also provided an additional carbon source in a controlled manner. The accumulation of lipid by C. cohnii in this pH-auxostat culture was significantly greater than previously reported for batch cultures using glucose as principal carbon source. Of six strains tested in pH-auxostat cultures, C. cohnii ATCC 30772 was the best, with the cells reaching 20 to 30 g dry weight per liter after 98 to 144 h and containing in excess of 40% (w/w) total lipid, with DHA representing approximately half of the total fatty acids in the triacylglycerol fraction. A productivity of 36 mg DHAL−1 h−1 was achieved during cultivation for 98 h using a 5% (vol/vol) inoculum, and DHA production was in excess of 3 g per liter of culture. Most of the DHA was present in neutral lipids.

Phenotypic variation of lipid composition in Burkholderia cepacia : a response to increased growth temperature is a greater content of 2-hydroxy acids in phosphatidylethanolamine and ornithine amide lipid

Burkholderia cepacia produces an unusual range of polar lipids, which includes two forms each of phosphatidylethanolamine (PE) and ornithine amide lipid (OL), differing in the presence or absence of 2-hydroxy fatty acids. By using chemostat cultures in chemically defined media, variations in the lipid content and the proportions of individual lipids have been studied as a function of (a) growth temperature, (b) growth rate and (c) growth-limiting nutrient (carbon, magnesium, phosphorus or oxygen). Total cellular lipid in carbon-limited cultures was lowest at high growth temperatures and low growth rates. Increases in growth temperature over the range 25-40 degrees C led to increases in the proportions of molecular species of PE and OL containing 2-hydroxy acids, without changing the PE:OL ratio. Growth temperature did not alter the balance between neutral and acidic lipids, but the contribution of phosphatidylglycerol to the latter increased with rising growth temperature and growth rate. Pigmentation of cells and the presence of flagella were also temperature-dependent. Change in growth rate also affected the PE:OL ratio and the extent to which monoenoic acids were replaced by their cyclopropane derivatives. Whereas similar lipid profiles were found for carbon-, magnesium- and oxygen-limited cultures, ornithine amides were the only polar lipids detected in phosphorus-limited cells.

The Production of Polyhydroxyalkanoates from Unrelated Carbon Sources

Several bacteria have been found to accumulate polyhydroxyalkanoates (PHAs) containing 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) monomers from glucose and other carbon sources. These bacteria are members of the taxonomically related genera Rhodococcus, Nocardia and Corynebacterium. The proportion of 3HV and 3HB monomers present in PHA is dependent on the carbon source but 3HV is generally the major 3-hydroxyacid.

Production of a co-polyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from succinic acid by Rhodococcus ruber: biosynthetic considerations

The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of polyhydroxyalkanoate by Rhodococcus ruber from succinic acid was investigated using nuclear magnetic resonance analysis. Polymer produced from [2,3-13C]- and [1,4-13C]succinate showed that the C-1-C-2 and C-4-C-5 fragments of 3HV were derived from carbons 2 and 3 of succinate, essentially without bond cleavage, and carbon 3 of 3HV was derived from a carboxyl carbon of succinate. Using [1,2-13C]succinate it was demonstrated that the C-1-C-2 bond of succinate was cleaved during polymer biosynthesis. Methylmalonyl-coenzyme A (CoA) mutase activity was detected in cell-free extracts of R. ruber by enzyme assay and HPLC analysis of reaction products. A pathway, involving the known methylmalonyl-CoA pathway for propionate formation in Propionibacteria, followed by the established pathway for PHA biosynthesis from propionyl-CoA and acetyl-CoA, is proposed for the biosynthesis of 3HV from succinate by R. ruber.

Alternative Carbon Sources for Heterotrophic Production of Docosahexaenoic Acid by the Marine Alga Crypthecodinium cohnii

Publisher Summary In recent years, interest in Polyunsaturated Fatty Acids (PUFA) has increased considerably due to their various physiological functions in the human body and their beneficial effects on human health. Of key importance is the heterotrophic marine dinoflagellate, Crypthecodinium cohnii , that has been studied intensively, and that, together with Schizochytrium and related genera, represents the major commercial source of Docosahexaenoic Acid (DHA). Important parameters for optimal DHA productivity include growth rate, final biomass concentration, the total lipid content, and the DHA proportion of the lipid. In most of the documented commercial cultivation processes, glucose is used as the carbon and energy source. However, it is not the only possible substrate. This chapter discusses the use of alternative carbon sources like acetic acid and ethanol on growth, lipid accumulation, and DHA productivity of C. cohnii in fed-batch cultures. A comment is also made regarding the possible uses of glycerol as a feedstock material.