Ethanethiol degradation by Ralstonia eutropha

In the present study, a pure culture of Ralstonia eutropha was used to degrade gaseous ethanethiol. Ethane thiol at various initial concentrations ranging from 115 to 320 mg/m³ was degraded almost completely within 120 ～ 168 h, while at higher concentrations up to 452 mg/m³, removal efficiency declined. It was likely that ethanethiol was used as the source of energy by R. eutropha, since no clear increase in the biomass concentration was observed. Kinetic data of ethanethiol bidegradation could be fitted using the Monod model. The kinetic parameters were q _m = 0.23 (mg ethanethiol/g biomass/h), and K _s = 1.379 (mg/L). The mineralization pathway of ethanethiol through sulphate, as the detected product, and the energy production were discussed in some detail.     

Importance of different tfd genes for degradation of chloroaromatics by Ralstonia eutropha JMP134

The tfdC(I)D(I)E(I)F(I,) and tfdD(II)C(II)E(II)F(II) gene modules of plasmid pJP4 of Ralstonia eutropha JMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D). However, activity of tfd(I)-encoded enzymes was usually higher than that of tfd(II)-encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only one tfd gene module. The tfdD(II)-encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors of R. eutropha strains containing different tfd gene modules. The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro- and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to form trans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far.     

Enzymatic recovery and purification of polyhydroxybutyrate produced by Ralstonia eutropha

Polyhydroxybutyrate (PHB) is the most studied among a wide variety of polyhydroxyalkanoates, bacterial biodegradable polymers known as potential substitutes for conventional plastics. This work aimed at evaluating the use of enzymes to recover and purify the PHB produced by Ralstonia eutropha DSM545. Screening experiments allowed the selection of trypsin, bromelain and lysozyme among six enzymes, based on their efficiency in lysing cells of a non-PHB producing R. eutropha strain. Then, process conditions for high efficiency in PHB purification from the DSM545 cells were searched for the enzymes previously selected. The best result was achieved with 2.0% of bromelain (enzyme mass per biomass), equivalent to 14.1 U ml(-1), at 50 degrees C and pH 9.0, resulting in 88.8% PHB purity. Aiming at improving the process efficiency and reducing the enzyme cost, experiments were carried out with pancreatin, leading to 90.0% polymer purity and an enzyme cost three times lower than the one obtained with bromelain. The molecular mass analysis of PHB showed no polymer degradation. Therefore, this work demonstrates the potential of using enzymes in order to recover and purify PHB and bacterial biopolymers in general.     

Catabolism of 3-Nitrophenol by Ralstonia eutropha JMP 134

Ralstonia eutropha JMP 134 utilizes 3-nitrophenol as the sole source of nitrogen, carbon, and energy. The entire catabolic pathway of 3-nitrophenol is chromosomally encoded. An initial NADPH-dependent reduction of 3-nitrophenol was found in cell extracts of strain JMP 134. By use of a partially purified 3-nitrophenol nitroreductase from 3-nitrophenol-grown cells, 3-hydroxylaminophenol was identified as the initial reduction product. Resting cells of R. eutropha JMP 134 metabolized 3-nitrophenol to N-acetylaminohydroquinone under anaerobic conditions. With cell extracts, 3-hydroxylaminophenol was converted into aminohydroquinone. This enzyme-mediated transformation corresponds to the acid-catalyzed Bamberger rearrangement. Enzymatic conversion of the analogous hydroxylaminobenzene yields a mixture of 2- and 4-aminophenol.     

Production of polyhydroxybutyrate by Ralstonia eutropha from protein hydrolysates

Polyhydroxybutyrate (PHB) was produced by Ralstonia eutropha DSM 11348 (formerly Alicaligenes eutrophus) in media containing 20–30 g l⁻¹ casein peptone or casamino acids as sole sources of nitrogen. In fermentations using media based on casein peptone, permanent growth up to a cell dry mass of 65 g l⁻¹ was observed. PHB accumulated in cells up to 60%–80% of dry weight. The lowest yields were found in media without any trace elements or with casamino acids added only. The residual cell dry masses were limited to 10–15 g l⁻¹ and did not contain PHB. The highest productivity amounted to 1.2 g PHB l⁻¹ h⁻¹. The mean molecular mass of the biopolymer was determined as 750 kDa. The proportion of polyhydroxyvalerate was less than 0.2% in PHB. The bioprocess was scaled up to a 300-l plant. During a fermentation time of 39 h the cells accumulated PHB to 78% w/w. The productivity was 0.98 g PHB l⁻¹ h¹. Received: 8 July 1998 / Accepted: 26 August 1998     

Genetic and biochemical characterization of a 2,4,6-trichlorophenol degradation pathway in Ralstonia eutropha JMP134

Ralstonia eutropha JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH2)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (tcpABC) from JMP134 by using primers designed from conserved regions of FADH2-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH2-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The tcpA mutant failed to degrade 2,4,6-TCP, while both tcpB and tcpC mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of tcpB may have led to a polar effect on downstream tcpC, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH2 was supplied by an Escherichia coli flavin reductase. TcpC produced in E. coli oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that tcpB may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.     

真氧产碱杆菌在双营养（碳、氮）限制区内合成聚羟基烷酸酯

研究了真氧产碱杆菌以混合有机酸为碳源,硫酸铵为氮源,在双营养（碳、氮）限制区内聚羟基烷酸酯的生物合成。结果表明：双营养限制区的长度与聚羟基烷酸酯的产量呈正相关。同时,在对两种不同的双营养限制区实现方式进行比较后发现,首先限制碳源的双营养限制方式比首先限制氮源的双营养限制方式更有利于聚羟基烷酸酯的合成;在这两种不同营养限制方式下,PHAs的最高产量分别为3.72 g/L和2.55 g/L。     

Degradation of formaldehyde in packed-bed bioreactor by kissiris-immobilized Ralstonia eutropha

The ability of the Ralstonia eutropha cells to utilize formaldehyde (FA) as the only source of carbon and energy was studied in the kissiris-immobilized cell bioreactor (KICB) in batch-recirculation and continuous modes of operation. In batch-recirculation experiments, the test bacterium could tolerate concentrations of FA up to 1,400 mg/L at 30°C and aeration rate equal to 0.75 vvm (r _S = 7.25 mg/L/h, q S = 0.019 g_FA/g_cell/h). However, further increase of initial FA concentration resulted in degradation reaction of FA to stop at 1,600 mg/L. Results of continuous mode experiments showed that the biodegradation performance of the KICB was dependent on both feed flow rate and inlet FA concentration parameters. The optimum feed flow rate which corresponded to the highest biodegradation rate (r _S = 240.3 mg/L/h) was observed at Q = 18 mL/min when KICB did not operate under the external mass transfer limiting regime. Substrate inhibition kinetics (Edwards and Luong equations) were used to describe the experimental specific degradation rates data. According to the Luong model, the values of the maximum specific degradation rate (q _max), half-saturation coefficient (K _S), the maximum allowable FA concentration (S _m), and the shape factor (n) were 0.178 g_FA/g_cell/h, 250.9 mg/L, 1,600 mg/L, and 1.86, respectively.     

Biosynthesis of short-chain-length-polyhydroxyalkanoates during the dual-nutrient-limited zone by Ralstonia eutropha

Biosynthesis of PHAs by Raltonia eutropha during the dual nutrient-limitation-zone was investigated with mixed organic acids as carbon sources and (NH₄)₂SO₄ as nitrogen source. Two different methods of maintaining the dual-nutrient-limitation zone were adopted by feeding mixed acids and (NH₄)₂SO₄ at determined rates into the fermentation cultures which were initially free of carbon sources (method A) or nitrogen sources (method B). The results indicate that, firstly, with the increase of the width of the dual-nutrient-limitation zone, the yield of short-chain-length-polyhydroxyalkanoates also increases and it suggests that most of the short-chain-length-polyhydroxyalkanoates were biosynthesized during the dual-nutrient-limitation zone. Secondly, in contrast with the dual-nutrient-limitation method of limiting the nitrogen source first (method B), the dual-nutrient-limitation method of limiting the carbon source first (method A) was more favourable for the production of short-chain-length-polyhydroxyalkanoates, and the maximum production of short-chain-length-polyhydroxyalkanoates of these two methods are 3.72 and 2.55 g/l, respectively.     

Conversion of volatile fatty acids into polyhydroxyalkanoate by Ralstonia eutropha

Aims:  The aims of this study were to optimize condensed corn solubles (CCS) as a medium for growth of Ralstonia eutropha and to determine the effects of individual volatile fatty acids (VFAs) on polyhydroxyalkanoate (PHA) production .
Methods and Results:  A CCS medium of concentration 240 g l⁻¹ with a carbon : nitrogen ratio of 50 : 1 was developed as the optimal medium. Cultures were grown in 1-l aerated flasks at 250 rev min⁻¹ at 30°C for 120 h. Comparable growth rates were observed in CCS vs a defined medium. At 48 h, VFAs were fed individually at different levels. Optimal levels of all the acids were determined to maximize PHA production. An overall comparison of the VFAs indicated that butyric and propionic acids provided the best results.
Conclusion:  An optimized CCS medium supported growth of R. eutropha . Butyric and propionic acids were the most efficient carbon sources to maximize PHA production when added at the 5 g l⁻¹ level.
Significance and Impact of the Study:  The study shows that a byproduct of ethanol industry can be effectively used as a low cost medium for PHA production, thus partly reducing the cost of commercialization of biopolymers.     

Bacterial cometabolic degradation of chlorinated paraffins

Summary Cometabolic dechlorination of chlorinated paraffins was demonstrated in the presence of n-hexadecane by bacterial strains (HK-3, HK-6, HK-8, and HK-10) isolated from soil samples.Eleven per cent of chlorine of chlorinated paraffin-150 (CP-150) was released by strain HK-3. The mixed culture of strain HK-3, catalyzing the dechlorination of terminal chlorine of chloroalkane, and strain H15-4, capable of releasing the chlorine from 2-chlorinated fatty acids, dechlorinated CP-150 up to 13%. The mixed culture of the four strains (HK-3, HK-6, HK-8, and HK-10) performed the dechlorination of CP-150 by cometabolism in a jar fermentor pH at 7.0. The amount of chloride released from the chlorinated paraffins tested was in the range of 15–57%.The activated sludge acclimatized to n-hexadecane for 60 days showed a little dechlorination activity to CP-150.     

The synthesis of hydroxybutyrate and hydroxyvalerate copolymers by the bacterium Ralstonia eutropha

The paper deals with the study of the synthesis of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymers by the bacterium Ralstonia eutropha B-5786 grown under different carbon nutrition conditions (growth on carbon dioxide, fructose, and CO2-valerate and fructose-valerate mixtures). The parameters to be analyzed included the yield of biomass, the yield, synthesis rate, and composition of copolymers, the activity of the key enzymes of polyhydroxyalkanoate (PHA) synthesis (beta-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase), the maximum tolerable concentration of valerate to the bacterium, and the conditions that govern the incorporation of hydroxyvalerate to copolymers. This allowed the relationship between cultivation conditions and the proportion of monomers in the copolymers to be deduced. We were able to synthesize a range of 3HB/3HV copolymers and found that the thermal characteristics and the degree of crystallinity of these copolymers depend on the molar fraction of 3HV.     

The synthesis of hydroxybutyrate and hydroxyvalerate copolymers by the bacterium Ralstonia eutropha

The paper deals with the study of the synthesis of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) copolymers by the bacterium Ralstonia eutropha B-5786 grown under different carbon nutrition conditions (growth on carbon dioxide, fructose, and CO₂-valerate and fructose-valerate mixtures). The parameters to be analyzed included the yield of biomass; the yield, synthesis rate, and composition of copolymers; the activity of the key enzymes of polyhydroxyalkanoate (PHA) synthesis -ketothiolase, acetoacetyl-CoA reductase, and PHA synthase); the maximum tolerable concentration of valerate to the bacterium; and the conditions that govern the incorporation of hydroxyvalerate into copolymers. This allowed the relationship between cultivation conditions and the proportion of monomers in the copolymers to be deduced. We were able to synthesize a range of 3HB/3HV copolymers and found that the thermal characteristics and the degree of crystallinity of such copolymers depend on the molar fraction of 3HV.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 63–69.Original Russian Text Copyright © 2005 by Volova, Kalacheva.     

Chemoselective nitro group reduction and reductive dechlorination initiate degradation of 2-chloro-5-nitrophenol by Ralstonia eutropha JMP134.

Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134. 2-Chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene.     

Growth and localization of polyhydroxybutyrate granules in Ralstonia eutropha

The bacterium Ralstonia eutropha forms cytoplasmic granules of polyhydroxybutyrate that are a source of biodegradable thermoplastic. While much is known about the biochemistry of polyhydroxybutyrate production, the cell biology of granule formation and growth remains unclear. Previous studies have suggested that granules form either in the inner membrane, on a central scaffold, or in the cytoplasm. Here we used electron cryotomography to monitor granule genesis and development in 3 dimensions (3-D) in a near-native, "frozen-hydrated" state in intact Ralstonia eutropha cells. Neither nascent granules within the cell membrane nor scaffolds were seen. Instead, granules of all sizes resided toward the center of the cytoplasm along the length of the cell and exhibited a discontinuous surface layer more consistent with a partial protein coating than either a lipid mono- or bilayer. Putatively fusing granules were also seen, suggesting that small granules are continually generated and then grow and merge. Together, these observations support a model of biogenesis wherein granules form in the cytoplasm coated not by phospholipid but by protein. Previous thin-section electron microscopy (EM), fluorescence microscopy, and atomic force microscopy (AFM) results to the contrary may reflect both differences in nucleoid condensation and specimen preparation-induced artifacts.     

Production and purification of self-assembling peptides in Ralstonia eutropha

Self-assembling peptides have emerged as an attractive scaffold material for tissue engineering, yet the expense associated with solid phase chemical synthesis has limited their broad use. In addition, the fidelity of chemical synthesis constrains the length of polypeptides that can be produced homogeneously by this method. Template-derived biosynthesis by recombinant DNA technology may overcome both of these problems. However, recovery of polypeptides from recombinant protein expression systems typically involves multi-step purification schemes. In this study, we report an integrated approach to recombinantly produce and purify self-assembling peptides from the recently developed expression host Ralstonia eutropha. The purification is based on the specific affinity of carbohydrate binding modules (CBMs) to cellulose. In a first step, we identified CBMs that express well in R. eutropha by assembling a fusion library of green fluorescent protein (GFP) and CBMs and determining the fluorescence of cell-free extracts. Three GFP::CBM fusions were found to express at levels similar to GFP alone, of which two CBMs were able to mediate cellulose binding of the GFP::CBM fusion. These two CBMs were then fused to multiple repeats of the self-assembling peptide RAD16-I::E (N-RADARADARADARADAE-C). The fusion protein CBM::E::(RAD16-I::E)4 was expressed in R. eutropha and purified using the CBM's affinity for cellulose. Subsequent proteolytic cleavage with endoproteinase GluC liberated RAD16-I::E peptide monomers with similar properties to the chemically synthesized counterpart RAD16-I.     

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     

Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.