Poly-β-hydroxybutyrate metabolism in Azospirillum brasilense and the ecological role of PHB in the rhizosphere

Abstract Azospirillum brasilense is a rhizosphere microorganism which has potential use for promoting plant growth in economically important crops. Its ability to survive the adverse conditions imposed by nutrient starvation and competition in the rhizosphere is of great importance. A. brasilense accumulates up to 70% of its cell dry weight with poly-β-hydroxybutyrate (PHB). In the presence of stress factors such as ultraviolet radiation, desiccation and osmotic stress, PHB-rich cells survived better than PHB-poor cells. Polymer-rich cells of Azospirillum fixed N₂ in the absence of exogenous carbon and combined nitrogen. The enzymes of the PHB cycle in both the synthesis and degradation processes as well as during starvation were more active in PHB-rich cells. After 24 h of starvation there was a peak of activity of d (−)β-hydroxybutyrate dehydrogenase, β-ketothiolase and thiophorase due to PHB degradation. Additionally, acetoacetyl-CoA reductase dropped to a minimum level because PHB could not be synthesized. The possible utilization of PHB as a sole carbon and energy source by A. brasilense and other bacteria during establishment, proliferation and survival in the rhizosphere will be discussed.     

Microbial Conversion of Methane into poly-β-hydroxybutyrate (PHB): Growth and intracellular product accumulation in a type II methanotroph

The biochemical pathways of methane to poly-β-hydroxybutyrate (PHB) in type II methanotrophs have been analyzed and used to propose stoichiometric equations for cell biomass and PHB. Conditions necessary for PHB accumulation in a batch culture of Methylocystis parvus OBBP were studied. In nitrogen limited cultures PHB started accumulating in the declining growth phase and maximum rate of PHB formation occurred during the late growth and early stationary phases. Inoculum age had an effect on maximum level of intracellular PHB, which increased with inoculum age up to ca. 70% of the cell dry weight using a 70 h inoculum. It has been shown that oxygen and methane requirements are high and pose a great stress on the mass transfer in the system; this limits the obtainment of high cell concentrations. When conditions for increased mass transfer of both CH₄ and O₂ were used, 5 g/l of cells could be produced. Finally an enrichment strategy was investigated so that cells would start accumulating PHB earlier during the growth phase. Alternatives to increase cell mass concentrations are discussed.     

Kinetic analysis on formation of poly(3-hydroxybutyrate) from acetic acid by Ralstonia eutropha under chemically defined conditions

Batch cultures of Ralstonia eutropha in chemically defined media with acetic acid (HAc) as the sole carbon source were conducted to investigate acetate utilization, formation of poly(3-hydroxybutyrate) (PHB) and growth of active biomass (ABM) under different carbon to nitrogen (C/N) weight ratios. The specific acetate utilization rate based on ABM approached 0.16 g/g ABM h⁻¹, which was not affected very much by the extracellular HAc concentration from 1 to 5 g/l, but was affected by the C/N weight ratio. A low C/N ratio or high nitrogen supply sped up the specific acetate utilization rate to produce more ABM and less PHB. A high HAc concentration (>6 g/l), however, depressed acetate utilization as well as the ABM growth and PHB formation. A high cell mass concentration enhanced the tolerance of R. eutropha to the toxicity of HAc at pH 7 to 8.5. The viscosity-average molecular size of PHB generally increased first and then declined in batch cultures. Larger PHB molecules and less PHB per ABM were produced at a low C/N ratio with enough nutrient nitrogen than those under a high C/N ratio with less nutrient nitrogen available. Journal of Industrial Microbiology & Biotechnology (2001) 26, 121–126. Received 06 June 2000/ Accepted in revised form 21 October 2000     

The effect of dissolved oxygen on PHB accumulation in activated sludge cultures

Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio. In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-beta-hydroxybutyrate (PHB). The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB. Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates. However, at the lower DO supply rates (k(L)a 6 to 16 h(-1), 0 mg L(-1) DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (k(L)a 30, 51 h(-1), DO >0.9 mg L(-1)). Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (Y(PHB/Ac) 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (Y(PHB/Ac) 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth. It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L(-1) PHB) accounted for an additional denitrification potential of up to 18 mg L(-1) nitrate-nitrogen. The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments. Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell. In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB yields. The results suggest that oxygen management is crucial to conserving reducing power during the feast phase of SBR operation, as excessive aeration rates decrease the PHB yield and allow higher biomass growth.     

Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production.

Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H2-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (-1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A. succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO2. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed.     

Polyhydroxybutyrate production by Saccharophagus degradans using raw starch as carbon source

Biosynthesis of poly(3‐hydroxybutyrate) (PHB) from raw starch as the carbon source by the polysaccharide‐digesting bacteria Saccharophagus degradans was investigated in a fed‐batch culture. The production and properties of the PHB synthesized from starch were compared to those obtained using glucose as carbon source. In fed‐batch cultures, S. degradans accumulated 21.35 and 17.46% of PHB, using glucose or starch as carbon source, respectively. The physical properties of the biopolymer produced from each carbon source were similar between them. Molecular mass, melting temperature and heat of fusion were 54.23 kDa, 165.61°C and 59.59 J/g, respectively, using glucose; and 57.07 kDa, 174.31°C and 67.66 J/g, respectively, using starch. This is the first work describing the capability of S. degradans to utilize raw starch as the sole carbon source for the production of PHB.     

Microbial Utilization of Electrically Reduced Neutral Red as the Sole Electron Donor for Growth and Metabolite Production

Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H₂-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (−1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A. succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO₂. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed.     

Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: effect of various carbon and nitrogen sources

Recombinant Escherichia coli (ATCC:PTA-1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB. Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E. coli were studied. Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation. No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source. Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources. The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37 degrees C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources. Scanning electron microscopy of the PHB granules isolated from recombinant E. coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 pm with the mean value of 0.23 +/- 0.06 pm.     

Growth and Polysaccharide Production by Methylocystis parvus OBBP on Methanol

Methylocystis parvus OBBP, an obligate methylotroph originally isolated as a methane-utilizing bacterium, was cultivated on methanol as a sole source of carbon. After adaptation to high methanol levels, this organism grew on methanol with a maximum specific growth rate of 0.65 h. The pH optimum for growth was between 7 and 9, and the temperature optimum was between 30 and 37 degrees C. Methanol concentrations higher than 5% (by weight) were toxic. Formaldehyde, at a concentration greater than 1 mM, inhibited growth. Formate was neither a substrate nor an inhibitor. An extracellular viscous heteropolysaccharide was produced during growth. The maximum production of the total biomass was 14.5 g (dry weight) per liter of broth. The dried biomass contained 22% (wt/wt) crude protein and 62% (wt/wt) polysaccharide. The main components of the polysaccharide were d-glucose (82%) and l-rhamnose (14%).     

Electron Partitioning During Light- and Nutrient-Powered Hydrogen Production by Rhodobacter sphaeroides

The partitioning of reducing power into different electron-accepting pathways was evaluated during growth and stationary phases of H₂-producing Rhodobacter sphaeroides cultures. For this, an electron balance method was developed using the chemical oxygen demand concept to quantitatively analyze the partitioning of nutrient electrons into H₂, cell biomass, polyhydroxybutyrate (PHB), and soluble microbial products (SMP). Overall, these four electron sinks were accounted for greater than 85% of the electrons provided by the nutrients. Glucose, lactate, succinate, fumarate, and pyruvate were individually provided as the main carbon source, and in all cases, glutamate was provided as a nitrogen source in order to enhance H₂ production. About 25–35% of the electrons ended up in H₂ during growth, while up to 60% of the electrons partitioned into H₂ in some stationary phase cultures. The other two major electron sinks in the growth phase were cell mass and PHB, while in stationary phase, SMP were accounted for >30% of the substrate electrons utilized. In general, the largest portion of SMP comprised low-molecular weight (<3 kDa) compounds mostly produced during stationary phase, although larger-size molecules were also detected in both phases. Overall, the fractions of electrons that partitioned into H₂ (0.21 to 0.35) and PHB (0.06 to 0.21) were highly correlated with the standard free energy change of the substrate oxidation half-reaction equation, normalized per electron equivalent. In a PHB(?) mutant, electron redistribution increased H₂ production, the extent of which depended on the carbon source provided.     

Preparation,statistical optimization and characterization of poly(3-hydroxybutyrate) fermented by Cupriavidus necator utilizing various hydrolysates of alligator weed (Alternanthera philoxeroides) as a sole carbon source

Alligator weed (Alternanthera philoxeroides) is a stoloniferous, amphibious and perennial herb which has invaded many parts of the world and led to serious environmental and ecological problems. In order to exploit cheap carbon source for poly(3-hydroxybutyrate) (PHB) production, alligator weed hydrolysates were prepared by acid and enzyme treatment and used for PHB production via Cupriavidus necator. The bacterium utilized alligator weed enzymatic hydrolysate and produced the PHB concentration of 3.8 ± 0.2 g/L at the conditions of pH 7.0, 27.5°C, 1.5 g/L of nitrogen source, and 25 g/L of carbon source, this exceeded the value of 2.1 ± 0.1 g/L from acid hydrolysate media at the same conditions. In order to obtain the optimum conditions of PHB production, response surface methodology was employed which improved PHB content. The optimum conditions for PHB production are as follows: carbon source, 34 g/L; nitrogen source, 2 g/L; pH, 7; temperature, 28°C. After 72 hr of incubation, the bacterium produced 8.5 g/L of dry cell weight and 4.8 g/L of PHB. The PHB was subjected to Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and Molecular weight analysis and found the melting temperature, number average molecular mass, and polydispersity were 168.20°C, 185 kDa, and 2.1, respectively.     

Occurrence of Poly-β-Hydroxybutyrate in the Azotobacteriaceae

Poly-beta-hydroxybutyrate (PHB) from various representative strains of the genera Azotobacter, Beijerinckia, and Derxia was isolated and characterized. During growth in shake culture, with glucose as a carbon and energy source, and molecular nitrogen as a nitrogen source, increase in dry weight appeared linear, and PHB formed a constant percentage of the dry weight. In a medium containing 1% (w/v) glucose, PHB declined with the onset of the stationary phase of growth; with 2% (w/v) glucose, an increase in PHB content during stationary phase was noted in the case of some strains, before a subsequent decline. The decrease in PHB as a percentage of dry cellular weight (not of total amount present in the culture) during growth of some strains with 2% as opposed to 1% (w/v) glucose may be ascribed to a greater production of capsular polysaccharide. PHB content could not be used as a taxonomic criterion. Strain differences were as great as or greater than species differences. The only strain of Beijerinckia fluminensis obtained contained PHB, but it could not be grown on the nitrogen-free medium used. Two species of the genus Azotomonas, reported to be aerobic, nonsymbiotic nitrogen-fixers, did not grow on the nitrogen-free medium used and did not produce PHB during growth with a combined nitrogen source.     

Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low cost agricultural waste material

Background

Polyhydroxybutyrates (PHBs) are macromolecules synthesized by bacteria. They are inclusion bodies accumulated as reserve materials when the bacteria grow under different stress conditions. Because of their fast degradability under natural environmental conditions, PHBs are selected as alternatives for production of biodegradable plastics. The aim of this work was to isolate potential PHB producing bacteria, evaluate PHB production using agro-residues as carbon sources.

Result

Among fifty bacterial strains isolated from different localities, ten PHB accumulating strains were selected and compared for their ability to accumulate PHB granules inside their cells. Isolate Arba Minch Waste Water (AWW) identified as Bacillus spp was found to be the best producer. The optimum pH, temperature, and incubation period for best PHB production by the isolate were 7, 37 °C, and 48 h respectively at 150 rpm. PHB production was best with glucose as carbon source and peptone as nitrogen source. The strain was able to accumulate 55.6, 51.6, 37.4 and 25% PHB when pretreated sugar cane bagasse, corn cob, teff straw (Eragrostis tef) and banana peel were used as carbon sources respectively. Fourier transform-infrared authentication results of the extracted and purified PHB identified its functional units as C–H, CH₂, C=O and C–O groups. UV–Vis spectrophotometric analysis and biodegradability test confirmed the similarity of the extract with standard PHB and its suitability for bioplastic production.

Conclusion

The isolated Bacillus sp can be used for feasible production of PHB using agro-residues especially sugarcane bagasse which can reduce the production cost in addition to reducing the disposal problem of these substrates. The yield of PHB can further be boosted by optimization of production parameters as substrates.

     

Nuclear Magnetic Resonance Studies of Poly(3-Hydroxybutyrate) and Polyphosphate Metabolism in Alcaligenes eutrophus

The metabolic pathways of poly(3-hydroxybutyrate) (PHB) and polyphosphate in the microorganism Alcaligenes eutrophus H16 were studied by ¹H, ¹³C, and ³¹P nuclear magnetic resonance (NMR) spectroscopy and by conventional analytical techniques. A. eutrophus cells accumulated two storage polymers of PHB and polyphosphate in the presence of carbon and phosphate sources under aerobic conditions after exhaustion of nitrogen sources. The solid-state cross-polarization/magic-angle spinning ¹³C NMR spectroscopy was used to study the biosynthetic pathways of PHB and other cellular biomass components from ¹³C-labeled acetate. The solid-state ¹³C NMR analysis of lyophilized intact cells grown on [1-¹³C]acetate indicated that the carbonyl carbon of acetate was selectively incorporated both into the carbonyl and methine carbons of PHB and into the carbonyl carbons of proteins. The ³¹P NMR analysis of A. eutrophus cells in suspension showed that the synthesis of intracellular polyphosphate was closely related to the synthesis of PHB. The roles of PHB and polyphosphate in the cells were studied under conditions of carbon, phosphorus, and nitrogen source starvation. Under both aerobic and anaerobic conditions PHB was degraded, whereas little polyphosphate was degraded. The rate of PHB degradation under anaerobic conditions was faster than that under aerobic conditions. Under anaerobic conditions, acetate and 3-hydroxybutyrate were produced as the major extracellular metabolites. The implications of this observation are discussed in connection with the regulation of PHB and polyphosphate metabolism in A. eutrophus.     

Dynamics of activity of the key enzymes of polyhydroxyalkanoate metabolism in Ralstonia eutropha

The dynamics of accumulation of polyhydroxybutyrate (PHB) and the activities of the key enzymes of PHB metabolism (beta-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, D-hydroxybutyrate dehydrogenase, and PHA depolymerase) in the hydrogen bacterium Ralstonia eutropha B5786 were studied under various conditions of carbon nutrition and substrate availability. The highest activities of beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase were recorded at the stage of acceleration of PHB synthesis. The activities of enzymes catalyzing PHB depolymerization (PHB depolymerase and D-hydroxybutyrate dehydrogenase) were low, being expressed only at stimulated endogenous PHB degradation. The change of carbon source (CO2 or fructose) did not cause any marked changes in the time course of enzyme activity.     

Atypical Polyphosphate Accumulation by the Denitrifying Bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-β-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.