Steve Goodwin

Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other metals

The gram-negative metal-reducing microorganism, previously known as strain GS-15, was further characterized. This strict anaerobe oxidizes several short-chain fatty acids, alcohols, and monoaromatic compounds with Fe(III) as the sole electron acceptor. Furthermore, acetate is also oxidized with the reduction of Mn (IV), U (VI), and nitrate. In whole cell suspensions, the c-type cytochrome(s) of this organism was oxidized by physiological electron acceptors and also by gold, silver, mercury, and chromate. Menaquinone was recovered in concentrations comparable to those previously found in gram-negative sulfate reducers. Profiles of the phospholipid ester-linked fatty acids indicated that both the anaerobic desaturase and the branched pathways for fatty acid biosynthesis were operative. The organism contained three lipopolysaccharide hydroxy fatty acids which have not been previously reported in microorganisms, but have been observed in anaerobic freshwater sediments. The 16S rRNA sequence indicated that this organism belongs in the delta proteobacteria. Its closest known relative is Desulfuromonas acetoxidans. The name Geobacter metallireducens is proposed.

Hydrogen concentrations as an indicator of the predominant terminal electron-accepting reactions in aquatic sediments

Abstract Factors controlling the concentration of dissolved hydrogen gas in anaerobic sedimentary environments were investigated. Results, presented here or previously, demonstrated that, in sediments, only microorganisms catalyze the oxidation of H 2 coupled to the reduction of nitrate, Mn(IV), Fe(III), sulfate, or carbon dioxide. Theoretical considerations suggested that, at steady-state conditions, H 2 concentrations are primarily dependent upon the physiological characteristics of the microorganism(s) consuming the H 2 and that organisms catalyzing H 2 oxidation, with the reduction of a more electrochemically positive electron acceptor, can maintain lower H 2 concentrations than organisms using electron acceptors which yield less energy from H 2 oxidation. The H 2 concentrations associated with the specified predominant terminal electron-accepting reactions in bottom sediments of a variety of surface water environments were: methanogenesis, 7–10 nM; sulfate reduction, 1–1.5 nM; Fe(III) reduction, 0.2 nM; Mn(IV) or nitrate reduction, less than 0.05 nM. Sediments with the same terminal electron acceptor for organic matter oxidation had comparable H 2 concentrations, despite variations in the rate of organic matter decomposition, pH, and salinity. Thus, each terminal electron-accepting reaction had a unique range of steady-state H 2 concentrations associated with it. Preliminary studies in a coastal plain aquifer indicated that H 2 concentrations also vary in response to changes in the predominant terminal electron-accepting process in deep subsurface environments. These studies suggest that H 2 measurements may aid in determining which terminal electron-accepting reactions are taking place in surface and subsurface sedimentary environments.

Bacterial poly-3-hydroxyalkenoates with epoxy groups in the side chains

Pseudomonas oleovorans was grown on 10-undecenoic acid and on a mixture of sodium octanoate and 10-undecenoic acid. With either or both of these substrates, cells produced polyesters containing unsaturated side chains in the repeating units. In the case of 10-undecenoic acid as the sole nutrient, the microorganism produced a polymer containing only repeating units with unsaturated side chains. A second bacterium, Rhodospirillum rubrum was grown on 4-pentanoic acid alone and it produced polyesters with shorter pendant groups in the repeating units, including both saturated and unsaturated side chains. Epoxidation of these different bacterial polyesters with m-chloroperbenzoic acid, as 4 chemical reagent, yielded to quantitative conversions of the unsaturated groups into epoxy groups as determined by 1H- and 13C-NMR, no side reaction was observed on the macromolecular chain by molecular weights measurements. More important, it has been possible to produce new functional bacterial polyesters containing terminal epoxy groups in the side chains, in variable proportions up to 37% by growing P. oleovorans on a 10-epoxyundecanoic acid and sodium octanoate culture feed mixture.

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.

Hydrolase activity of an extracellular depolymerase from Aspergillus fumigatus with bacterial and synthetic polyesters

An extracellular depolymerase produced by the fungus Aspergillus fumigatus was found to have hydrolytic activity towards both bacterial and synthetic polyesters. The enzyme catalysed the hydrolysis of the bacterial polyesters: poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB/4HB) over the entire range of compositions. In addition, this fungal depolymerase also catalysed the hydrolysis of synthetic, aliphatic polyesters including poly(ethylene adipate) (PEA), poly(ethylene succinate) (PES), poly(1,4-tetramethylene adipate) (PTMA) and several commercial poly(ester urethanes) with the trade name “Bionolle.” However, the enzyme was inactive for other synthetic polyesters, including poly([R]-lactide), (P[R]-LA), poly(5-valerolactone) (PVL) and poly(6-caprolactone) (PCL). The results on enzyme specificity and activity, and the types of degradation products obtained, were analyzed by molecular modelling. The analysis indicated that there is a relationship between the activity of the depolymerase, as indicated by the relative rates of hydrolysis of the different polyesters, and the molecular dimensions of their repeating units.

Comparative study of the relationship between monomer structure and reactivity for two polyhydroxyalkanoate synthases.

Abstract. Using organically synthesized hydroxyalkanoate coenzyme A thioesters, the activities of two short-chain polyhydroxalkanoate (PHA) synthases were investigated – Ralstonia eutropha PHA synthase (a type I PHA synthase) and Ectothiorhodospira shaposhnikovii PHA synthase (a type III synthase). The results indicate that the two synthases have similar activities towards most of the monomers tested. 3-Hydroxybutyryl CoA was found to be the most efficient substrate for both synthases. Changes in the side-chain length of the monomers affect monomer reactivity, with shortening of the side-chain length having the more severe effect. Hydrophobicity in the side chain appears to play an important role in the catalytic reaction. The configuration and the position of the hydroxyl group also affect the reactivity of a monomer. Monomers with the [S] configuration can not be recognized by either synthase. Moving the hydroxyl group from the β carbon to the α carbon has a much more severe effect on the reactivity of the monomer than moving the hydroxyl group to the γ carbon. The results demonstrate that the in vitro system can be used to prepare entirely novel polymers that may not be obtainable from living cells because of metabolic restrictions.

Characterization, Seasonal Occurrence, and Diel Fluctuation of Poly(hydroxyalkanoate) in Photosynthetic Microbial Mats

ABSTRACT In situ poly(hydroxyalkanoate) (PHA) levels and repeating-unit compositions were examined in stratified photosynthetic microbial mats from Great Sippewissett Salt Marsh, Mass., and Ebro Delta, Spain. Unlike what has been observed in pure cultures of phototrophic bacteria, the prevalence of hydroxyvalerate (HV) repeating units relative to hydroxybutyrate (HB) repeating units was striking. In the cyanobacteria-dominated green material of Sippewissett mats, the mole percent ratio of repeating units was generally 1HB:1HV. In the purple sulfur bacteria-dominated pink material the relationship was typically 1HB:2HV. In Sippewissett mats, PHA contributed about 0.5 to 1% of the organic carbon in the green layer and up to 6% in the pink layer. In Ebro Delta mats, PHA of approximately 1HB:2HV-repeating-unit distribution contributed about 2% of the organic carbon of the composite photosynthetic layers (the green and pink layers were not separated). Great Sippewissett Salt Marsh mats were utilized for more extensive investigation of seasonal, diel, and exogenous carbon effects. When the total PHA content was normalized to organic carbon, there was little seasonal variation in PHA levels. However, routine daily variation was evident at all sites and seasons. In every case, PHA levels increased during the night and decreased during the day. This phenomenon was conspicuous in the pink layer, where PHA levels doubled overnight. The daytime declines could be inhibited by artificial shading. Addition of exogenous acetate, lactate, and propionate induced two- to fivefold increases in the total PHA levels when applied in the daylight but had no effect when applied at night. The distinct diel pattern of in situ PHA accumulation at night appears to be related, in some phototrophs, to routine dark energy metabolism and is not influenced by the availability of organic nutrients.

Epoxidation of bacterial polyesters with unsaturated side chains. II. Rate of epoxidation and polymer properties

Poly(3-hydroxyoctanoate-co-3-hydroxy-10-undecenoate)s (PHOUs) with controlled amounts of unsaturated repeating units were epoxidized to various extents with m-chloroperbenzoic acid (MCPBA) in homogeneous solution. The epoxidation reaction was second order, with an initial rate constant of 1.1 × 10−3Lmol−1.s−1 at 20°C, regardless of the unsaturated unit content in PHOU. No substantial change in either molecular weight or molecular weight distribution occurred as a result of epoxidation, but the melt transition temperature and enthalpy of melting both decreased as the unsaturated groups were increasingly converted into epoxide groups. In contrast, the glass transition temperature (Tg) increased by approximately 0.25°C for each 1 mol % of epoxidation, irrespective of the composition of the PHOU.

Epoxidation of bacterial polyesters with unsaturated side chains : IV. Thermal degradation of initial and epoxidized polymers

The thermal decomposition of poly(3-hydroxyoctanoate-co-3-hydroxy-10-undecenoate), PHOU, and the completely epoxidized form of this polymer, poly(3-hydroxyoctanoate-co-3-hydroxy-10,11-epoxyundecanoate), PHOE, have been studied by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The thermograms of seven different samples of PHOE, which differed in the contents of epoxy, E, units, contained a three-step degradation process, while those of the initial PHOU samples exhibited only one-step degradation process. This degradation behavior of the PHOEs, which have a higher thermal stability as measured by weight loss, is probably controlled by crosslinking reactions of the pendant epoxide groups in the polymer, which occur during the degradation process, and the occurrence of such reactions can be assigned to the exothermic peaks in their DSC thermograms. An isothermal study of these polymers at 250°C for 1 h indicated that the residual weight correlated directly with the amount of epoxide groups in the PHOE samples.

Extracellular degradation of medium chain length poly(beta-hydroxyalkanoates) by Comamonas sp.

The PHA-degrading isolate, strain P37C, was enriched from residential compost for its ability to hydrolyze the medium chain length PHA, poly(beta-hydroxyoctanoate) (PHO). It was subsequently found to grow on a wide range of PHAs, including both short chain length and medium chain length PHAs. The isolate was identified as belonging to the genus Comamonas. Strain P37C formed clear zones on poly(beta-hydroxybutyrate) (PHB), (PHO) and poly(beta-hydroxyphenylvalerate) (PHPV) overlay plates. PHA clear zone tubes were prepared using seven different kinds of PHAs, ranging from PHB with four-carbon repeating units, to poly(beta-hydroxyoctanoate-co-beta-hydroxyundecanoate) (PHOU) with 8- and 11-carbon repeating units. There was a direct correlation between PHA side chain length and rate of hydrolysis of the PHAs. A series of PHOUs containing varying percentages of unsaturated bonds were used to make a series of epoxidized PHOUs (PHOEs) with varying percentages of epoxy functions. Results of clear zone tube assays showed that these functionalized PHAs were all biodegradable by strain P37C, and there was no apparent correlation between rate of biodegradation and the proportion of functional groups in the PHAs. Biodegradability of these PHAs was verified using respirometry and enzyme assays. Cell-free supernatants containing activity toward PHAs were prepared, and strain P37C was shown to synthesize at least two distinct PHA depolymerases for the hydrolysis of SCL and MCL PHAs.