Degradation of Toluene and Trichloroethylene by Burkholderia cepacia G4 in Growth-Limited Fed-Batch Culture

Burkholderia (Pseudomonas) cepacia G4 was cultivated in a fed-batch bioreactor on either toluene or toluene plus trichloroethylene (TCE). The culture was allowed to reach a constant cell density under conditions in which the amount of toluene supplied equals the maintenance energy demand of the culture. Compared with toluene only, the presence of TCE at a toluene/TCE ratio of 2.3 caused a fourfold increase in the specific maintenance requirement for toluene from 22 to 94 nmol mg of cells (dry weight)-1 h-1. During a period of 3 weeks, approximately 65% of the incoming TCE was stably converted to unidentified products from which all three chlorine atoms were liberated. When toluene was subsequently omitted from the culture feed while TCE addition continued, mutants which were no longer able to grow on toluene or to degrade TCE appeared. These mutants were also unable to grow on phenol or m- or o-cresol but were still able to grow on catechol and benzoate. Plasmid analysis showed that the mutants had lost the plasmid involved in toluene monooxygenase formation (pTOM). Thus, although strain G4 is much less sensitive to TCE toxicity than methanotrophs, deleterious effects may still occur, namely, an increased maintenance energy demand in the presence of toluene and plasmid loss when no toluene is added.

Comparison of quenching and extraction methodologies for metabolome analysis of Lactobacillus plantarum

<p>Abstract</p> <p>Background</p> <p>A reliable quenching and metabolite extraction method has been developed for <it>Lactobacillus plantarum</it>. The energy charge value was used as a critical indicator for fixation of metabolism.</p> <p>Results</p> <p>Four different aqueous quenching solutions, all containing 60% of methanol, were compared for their efficiency. Only the solutions containing either 70 mM HEPES or 0.85% (w/v) ammonium carbonate (pH 5.5) caused less than 10% cell leakage and the energy charge of the quenched cells was high, indicating rapid inactivation of the metabolism.</p> <p>The efficiency of extraction of intracellular metabolites from cell cultures depends on the extraction methods, and is expected to vary between micro-organisms. For <it>L. plantarum</it>, we have compared five different extraction methodologies based on (i) cold methanol, (ii) perchloric acid, (iii) boiling ethanol, (iv) chloroform/methanol (1:1) and (v) chloroform/water (1:1). Quantification of representative intracellular metabolites showed that the best extraction efficiencies were achieved with cold methanol, boiling ethanol and perchloric acid.</p> <p>Conclusion</p> <p>The ammonium carbonate solution was selected as the most suitable quenching buffer for metabolomics studies in <it>L. plantarum </it>because (i) leakage is minimal, (ii) the energy charge indicates good fixation of metabolism, and (iii) all components are easily removed during freeze-drying. A modified procedure based on cold methanol extraction combined good extractability with mild extraction conditions and high enzymatic inactivation. These features make the combination of these quenching and extraction protocols very suitable for metabolomics studies with <it>L. plantarum</it>.</p

Degradation of Chloroaromatics: Purification and Characterization of a Novel Type of Chlorocatechol 2,3-Dioxygenase of Pseudomonas putida GJ31

A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native Mr value under nondenaturating conditions by gel filtration gave a molecular mass of 135 ± 10 kDa, indicating a homotetrameric enzyme structure (4 × 33.4 kDa). The pI of the enzyme was estimated to be 7.1 ± 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40°C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.

Efficient hydrogen production from the lignocellulosic energy crop Miscanthus by the extreme thermophilic bacteria Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana

<p>Abstract</p> <p>Background</p> <p>The production of hydrogen from biomass by fermentation is one of the routes that can contribute to a future sustainable hydrogen economy. Lignocellulosic biomass is an attractive feedstock because of its abundance, low production costs and high polysaccharide content.</p> <p>Results</p> <p>Batch cultures of <it>Caldicellulosiruptor saccharolyticus </it>and <it>Thermotoga neapolitana </it>produced hydrogen, carbon dioxide and acetic acid as the main products from soluble saccharides in <it>Miscanthus </it>hydrolysate. The presence of fermentation inhibitors, such as furfural and 5-hydroxylmethyl furfural, in this lignocellulosic hydrolysate was avoided by the mild alkaline-pretreatment conditions at a low temperature of 75°C. Both microorganisms simultaneously and completely utilized all pentoses, hexoses and oligomeric saccharides up to a total concentration of 17 g l^-1in pH-controlled batch cultures. <it>T. neapolitana </it>showed a preference for glucose over xylose, which are the main sugars in the hydrolysate. Hydrogen yields of 2.9 to 3.4 mol H₂per mol of hexose, corresponding to 74 to 85% of the theoretical yield, were obtained in these batch fermentations. The yields were higher with cultures of <it>C</it>. <it>saccharolyticus </it>compared to <it>T. neapolitana</it>. In contrast, the rate of substrate consumption and hydrogen production was higher with <it>T. neapolitana</it>. At substrate concentrations exceeding 30 g l^-1, sugar consumption was incomplete, and lower hydrogen yields of 2.0 to 2.4 mol per mol of consumed hexose were obtained.</p> <p>Conclusion</p> <p>Efficient hydrogen production in combination with simultaneous and complete utilization of all saccharides has been obtained during the growth of thermophilic bacteria on hydrolysate of the lignocellulosic feedstock <it>Miscanthus</it>. The use of thermophilic bacteria will therefore significantly contribute to the energy efficiency of a bioprocess for hydrogen production from biomass.</p