R. Clinton Fuller

Ability of the phototrophic bacterium Rhodospirillum rubrum to produce various poly (β-hydroxyalkanoates): Potential sources for biodegradable polyesters

Studies have been carried out in order to optimize growth and culture conditions for the intracellular formation of poly(beta-hydroxyalkanoates) (PHA) in the phototrophic, purple, non-sulphur bacterium Rhodospirilum rubrum. Its potential to produce novel copolymers was investigated. Recently, it has become of industrial interest to evaluate these polyesters as potentially biodegradable plastics for a wide range of possible applications. On an industrial scale, the use of photosynthetic bacteria could harness sunlight as an energy source for the production of these materials. R. rubrum was grown anaerobically in the light on different linear and branched beta-hydroxycarboxylic acids and various n-alkanoic acids. Under nitrogen-limiting conditions a PHA content of up to 45% of cellular dry weight was detected. When R. rubrum was grown on different concentrations of various n-alkanoic acids, intracellular PHA production was detected on all acids used. In most of the cases, the storage polymer contained beta-hydroxybutyrate (HB) and beta-hydroxyvalerate (HV) monomer units. Grown on n-alkanoic acids with a chain length of four carbon atoms and more, R. rubrum produced a copolymer containing the beta-hydroxyhexanoate (HC) repeating unit in addition to the HB and HV monomer. Using beta-hydroxyheptanoic acid as the carbon source, a polyester which contained HB, HV, HC, and beta-hydroxyheptanoate was formed. These copolyesters represent a novel class of biodegradable thermoplastics. The results demonstrate the metabolic flexibility of R. rubrum to form many different types of polyesters which might substitute plastics synthesized from petrochemicals.

Formation of poly(3-hydroxyalkanoates) by phototrophic and chemolithotrophic bacteria

The formation of poly(3-hydroxyalkanoic acid), PHA, by various strains of chemolithotrophic and phototrophic bacteria has been examined. Chemolithotrophic bacteria were grown aerobically under nitrogen-limiting conditions on various aliphatic organic acids. Phototrophic bacteria were grown anaerobically in the light on a nitrogen-rich medium and were subsequently transferred to a nitrogen-free medium containing acetate, propionate, valerate, heptanoate or octanoate as carbon source. All 41 strains investigated in this study were able to synthesize and accumulate PHA. All 11 strains of chemolithotrophic bacteria and all 15 strains belonging to the non-sulfur purple bacteria synthesized a polymer, which contained 3-hydroxy-valerate (3HV) beside 3-hydroxybutyrate (3HB), if the cells were cultivated in the presence of propionate, valerate or heptanoate. Many non-sulfur purple bacteria synthesized copolyesters of 3HB and 3HV even with acetate as carbon source. In contrast, most sulfur purple bacteria did not incorporate 3HV at all. Among 15 strains tested, only Chromatium vinosum strain 1611, C. purpuratum strain BN5500 and Lamprocystis roseopersicina strain 3112 were able to synthesize polyesters containing 3HV with propionate, valerate or heptanoate as carbon source.

Production of unsaturated polyesters by Pseudomonas oleovorans

Pseudomonas oleovorans was grown separately on 3-hydroxy-6-octenoic acid and 3-hydroxy-7-octenoic acid as the only carbon source and under ammonium nutrient-limiting conditions to produce storage polyesters. The polyesters produced contained mainly unsaturated C8 units. Small amounts of both the saturated and the unsaturated C6 units were also present, but only about 1% of the saturated 3-hydroxyoctanoate units was detected. The polyester obtained from 3-hydroxy-6-octenoic acid, which was a mixture of the cis and trans isomers, also contained units with cis and trans double bonds. The weight average molecular weights of the polymers produced were in the range of 339,000-383,000 as determined by g.p.c. relative to polystyrene, with Mw/Mn ratios of 1.8-2.1. The mechanism of PHA formation from n-octene previously reported is discussed in relation to the present results, and the two were found to be in good agreement.

Bacterial polyesters containing branched poly(β-hydroxyalkanoate) units☆

Pseudomonas oleovorans was grown on mixtures of methyloctanoates with n-octanoate. Polymers were also obtained from organisms grown on pure 7-methyloctanoate, but not from pure 5- or 6-methyloctanoate. The polyesters obtained from 7-methyloctanoate and from its mixtures with n-octanoate contained units with the methyl branches in the pendant group, as did the copolymers from the mixtures of 5- and 6-methyloctanoate with n-octanoate. The methyl branched repeating units contained two diastereomers, and the 13C-n.m.r. spectra of these polymers indicated that the 5-methyloctanoate units had a higher content of one of the two isomers, but not in the 6-methyloctanoate units. The weight average molecular weights of the copolyesters produced were in the range of 220,000 to 410,000, with Mw/Mn ratios of 1.7 to 1.9.

A unique photosynthetic reaction center from Heliobacterium chlorum

A previously unknown type of photosynthetic reaction center in the brownish‐green bacterium Heliobacterium chlorum is described. The reaction center is tightly bound in a highly proteinaceous undifferentiated plasma membrane and contains bacteriochlorophyll g, which has major in vivo absorbancies at 788, 576 and 370 nm. The purified membrane shows a reversible photobleaching at 798 nm; the reaction center bacteriochlorophyll g is designated P798. A reversible photobleaching at 553 nm is assigned to photooxidation of a membrane‐bound c‐type cytochrome. The membrane structure, pigment composition and photochemical properties suggest that H. chlorum may represent a fifth family of anoxygenic photosynthetic procaryotes.

Menaquinone is the sole quinone in the facultatively aerobic green photosynthetic bacterium Chloroflexus aurantiacus

Abstract The role of quinones was investigated in Chloroflexus aurantiacus, a thermophilic green bacterium capable of photosynthetic or respiratory growth. Thin-layer chromatography, ultraviolet difference spectroscopy and high-pressure liquid chromatography showed that menaquinone is the only quinone present in both photosynthetic and respiratory Chloroflexus cultures. Menaquinone-10 and menaquinone-8 are the predominant homologues in both cultures. For comparative purposes the quinone compositions in photoheterotrophic cultures of Chromatium vinosum and Chlorobium limicola were also analyzed. Chloroflexus is the only facultatively aerobic photosynthetic bacterium that does not possess ubiquinone. Menaquinone appears to be the only quinone involved in the photosynthetic and oxidative electron transport in this organism.

The accumulation of poly(3-hydroxyalkanoates) in Rhodobacter sphaeroides

In recent years industrial interest has been focussed on the evaluation of poly(3-hydroxyalkanoates) (PHA) as potentially biodegradable plastics for a wide range of technical applications. Studies have been carried out in order to optimize growth and culture conditions for the intracellular formation of PHA in the phototrophic, purple, non-sulfur bacterium Rhodobacter sphaeroides. Its potential to produce polyesters other than poly(3-hydroxybutyrate) (PHB) was investigated. On an industrial scale, the use of photosynthetic bacteria could harness sunlight as an energy source for the production of these materials. R. sphaeroides was grown anaerobically in the light on different carbon sources. Under nitrogenlimiting conditions a PHA content of up to 60 to 70% of the cellular dry weight was detected. In all of the cases studied, the storage polymer contained approximately 98 mol% of 3-hydroxybutyrate (HB) and 2 mol% 3-hydroxyvalerate (HV) monomer units. Decreasing light intensities did not stimulate PHA formation. Compared to Rhodospirillum rubrum (another member of the family of Rhodospirillaceae), R. sphaeroides showed a limited flexibility in its ability to form PHA with varying monomer unit compositions.

The fate of ‘biodegradable’ plastics in municipal leaf compost

SummaryBlends of starch with polypropylene, starch with polyethylene, polycaprolactone with polyethylene, and a copolymer of β-hydroxybutyrate and β-hydroxyvalerate (PHB/V) were exposed to degrading leaves in a municipal leaf composting operation. Every month for 6 months, duplicate samples were analyzed for changes in weight and tensile properties, and many of these samples were further analyzed for changes in molecular weight and surface morphology. All results were compared to controls which were incubated for 6 months in moist, sterile leaves at a leaf compost temperature. Very little change was noted for any of the polyolefin blends over the 6-month period. In contrast, PHB/V samples showed massive deterioration with substantial weight loss. Although there was a decrease in molecular weight and a loss of tensile properties in leaf-exposed PHB/V films, the sterile control films also showed similar changes, but without weight loss. Of the microbial isolates from film surfaces, only fungi possessed PHB/V depolymerase activity.

The complete amino acid sequence of a bacteriochlorophyll a binding polypeptide isolated from the cytoplasmic membrane of the green photosynthetic bacterium Chloroflexus aurantiacus

A polypeptide soluble in organic solvents was isolated from whole membrane fractions of the green thermophilic bacterium Chloroflexus aurantiacus by chromatography on Sephadex LH‐60, Whatman DE‐32 and Bio Gel P‐10. The complete amino acid sequence of this 4.9 kDa polypeptide (44 amino acid residues) was determined. The polypeptide shows a 3‐domain structure, similar to the domain structure of the antenna BChI polypeptides of purple photosynthetic bacteria, and sequence homologies (27–39%) to the light‐harvesting α‐polypeptides of the B870 (890) antenna complexes from purple bacteria. Therefore, the 4.9 kDa polypeptide is designated B(808‐866)‐α. The typical His residue (conserved His residue identified in all antenna polypeptides of purple bacteria as possible BChI binding site) is found within the hydrophobic domain, which extends from Asn 10 to Leu 30.

Bacterial production of poly-3-hydroxyalkanoates containing arylalkyl substituent groups

6-Phenylhexanoic acid (6PHxA), 7-phenylheptanoic acid (7PHpA), 9-phenylnonanoic acid (9PNA), 11-phenylundecanoic acid (11PUA), 9-p-tolylnonanoic acid (9TNA) and 9-p-styrylnonanoic acid (9SNA) were prepared and evaluated as substrates for cell growth and polyester production by Pseudomonas oleovorans and Pseudomonas putida. P. putida was more effective than P. oleovorans for producing polyesters from these aromatic substrates. Poly-3-hydroxyalkanoates, PHAs, were obtained from 6PHxA, 7PHpA, 9PNA and 11PUA. The PHAs produced from all of these substrates contained mostly 3-hydroxy-5-phenylvalerate (H5PV) and 3-hydroxy-6-phenylhexanoate (H6PHx) units. Polymer yields ranging from 3 to 47% of cell dry weight were obtained with molecular weights ranging from 156 000 to 37 000 and polydispersities from 2.3 to 2.9. Cofeeding of most of these substrates with nonanoic acid produced mixtures of two different PHAs with different glass transitions, one in the region of −8 to 12°C for the PHA with arylalkyl substituent groups, and one in the region of −14 to −35°C for the PHA from nonanoic acid. The PHA from 9TNA also had a crystalline melting transition.