Influence of agitation and aeration on growth and antibiotic production by <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K _L a). With increase in K _L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml⁻¹ and 19.58 g l⁻¹, respectively, productivity in cell and antibiotic was up more than 30% when K _L a increased from 115.9 h⁻¹ to 185.7 h⁻¹. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l⁻¹ and 249 U ml⁻¹, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min⁻¹, 2.5 l min⁻¹).     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

Growth of <Emphasis Type="Italic">Trametes versicolor</Emphasis> on phenol

Trametes versicolor 1 was shown to grow on phenol as its sole carbon and energy source. The culture growth and degradation ability dependence on culture medium pH value was observed. The optimal pH value of a liquid Czapek salt medium was 6.5. The investigated strain utilized completely 0.5 g/l phenol in 6 days. The dynamics of the phenol degradation process was investigated. The process was characterized by specific growth rate μ_max 0.33 h⁻¹, metabolic coefficient k = 4.4, yield coefficient Y _x/s  = 0.23 and rate of degradation Q = 0.506 h⁻¹. The intracellular activities of phenol hydroxylase (0.333 U/mg protein) and cis,cis-muconate lactonizing enzyme (0.41 U/mg protein) were demonstrated for the first time in this fungus. In an attempt to estimate the occurrence of gene sequences in T. versicolor 1 related to phenol degradation pathway a dot blot analysis with total DNA isolated from this strain was performed. Two synthetic oligonucleotides were used as hybridizing probes. One of the probes was homologous to the 5′end of phyA gene coding for phenol hydroxylase in Trichosporon cutaneum ATCC 46490. The other probe was created on the basis of cis,cis-muconate lactonizing enzyme coding gene in T. cutaneum ATCC 58094. The results of these investigations showed that T. versicolor 1 may carry genes similar to those of Trichosporon cutaneum capable to degrade phenol.     

Isolation and Characterization of a Pseudomonas Oleovorans Degrading the Chloroacetamide Herbicide Acetochlor

To date, no pure bacterial cultures that could degrade acetochlor have been described. In this study, one strain of microorganism capable of degrading acetochlor, designated as LCa2, was isolated from acetochlor-contaminated soil. The strain LCa2 is Pseudomonas oleovorans according to the criteria of Bergey’s manual of determinative bacteriology and sequence analysis of the partial 16S rRNA gene. Optimum growth temperature and pH were 35 °C and 8.0, respectively. The strain could degrade 98.03% of acetochlor treated at a concentration of 7.6 mg l⁻¹ after 7 days of incubation and could tolerate 200 mg l⁻¹ of acetochlor. When the acetochlor concentration became higher, the degradation cycle became longer. The acetochlor biodegradation products were identified by GC–MS based on mass spectral data and fragmentation patterns. The main plausible degradative pathways involved dechlorination, hydroxylation, N-dealkylation, C-dealkylation and dehydrogenation.     

Evaluation of different culture conditions ofClostridium bifermentans DPH-1 for cost effective PCE degradation

Clostridium bifermentans strain DPH-1 has already been found to dechlorinate perchloroethylene (PCE) tocis-dichloroethylene (cis-DCE)via trichloroethylene (TCE). In this study, our investigation on different culture conditions of this DPH-1 strain was extended to find a more efficient and cost effective growth medium composition for this DPH-1 strain in bioremediation practices. Temperature dependency of strain DPH-1 showed that the growth starting time and PCE degradation at 15°C was very slow compared to that of 30°C, but complete PCE degradation occurred in both cases. For the proper utilization of strain DPH-1 in more cost effective bioremediation practices, a simpler composition of an effective media was studied. One component of the culture medium, yeast extract, had been substituted by molasses, which served as a good source of electron donor. The DPH-1 strain in the medium containing molasses, in the presence of K₂HPO₄ and KH₂PO₄, showed identical bacterial multiplication (0.135 mg protein mL⁻¹h⁻¹) and PCE degradation rates (0.38 μM/h) to those of the yeast extract containing medium.     

Response of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> F1 cultures to fluctuating toluene loads and operational failures in suspended growth bioreactors

The response of Pseudomonas putida F1 to process fluctuations and operational failures during toluene biodegradation was evaluated in a chemostat suspended growth bioreactor. The ability of P. putida F1 to rapidly increase its specific toluene degradation capacity resulted in no significant variation in process removal efficiency when toluene load was increased from 188 to 341 g m⁻³ h⁻¹. Likewise, bacterial activity rapidly reached steady state performance (in less than 1.5 h after the restoration of steady state operational conditions) following an 8 h process shutdown, or after episodes of toluene or mineral nutrients deprivation. Process performance was however highly sensitive to pH, as pH levels below 4.5 dramatically inhibited bacterial activity, decreasing severely process robustness and inducing a cycle of periodic process collapses and recoveries. This pH mediated deterioration of bacterial activity was confirmed by further respirometric tests, which revealed a 50–60% reduction in the O₂ consumption rate during the degradation of both toluene and 3-methyl catechol when pH decreased from 5.05 to 4.55. Finally, process robustness was quantified according to methods previously described in literature.     

Optimization and modeling of phenanthrene degradation by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. 6PY1 in a biphasic medium using response-surface methodology

In the present paper, the degradation of phenanthrene, a model polycyclic aromatic hydrocarbon compound, by the Mycobacterium strain 6PY1 was optimized in a biphasic culture medium. The optimization and modeling were performed using the design of experiments methodology. The temperature, the silicone oil/mineral salts medium volume ratio, and the initial cell concentration, were used as the central composite design parameters. In all experiments, the phenanthrene was degraded to undetectable levels. Response surface methodology was successfully employed to derive an empirical model describing the rate and time of degradation and to deduce the optimal degradation conditions. As a result of the optimization processes, the optimal responses for the degradation rate, the volumetric degradation rate, and the 90% degradation time were estimated to be 0.172 mg h⁻¹, 22 mg l⁻¹ h⁻¹, and 18 h, respectively.     

Biodegradation of nicotine from tobacco waste extract by <Emphasis Type="Italic">Ochrobactrum intermedium</Emphasis> DN2

Ochrobactrum intermedium DN2 was used to degrade nicotine in tobacco waste extracts. The optimal temperature and pH of nicotine degradation by strain DN2 was 30–37 °C and 7.0, respectively. Under these optimal conditions, the average degradation rate of nicotine in a 30L fed-batch culture was 140.5 mg l⁻¹ h⁻¹. The results of this study indicate that strain DN2 may be useful for reducing the nicotine content of reconstituted tobacco.     

Purification and characterization of pectin lyase secreted by <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K _m and k _cat values were found to be 1 mg/ml and 76 sec⁻¹, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992.     

Degradation of diphenyl phthalate by <Emphasis Type="Italic">Sphingomonas chungbukensis</Emphasis>

More than 80% of diphenyl phthalate (DPP) at 100 mg l⁻¹ was degraded by Sphingomonas chungbukensis KCTC 2955 in a mineral salts medium at pH 7.0 and 30°C within 48 h. The maximum specific degradation rate was 5 mg DPP l⁻¹ h⁻¹. It was rapidly converted to monophenyl phthalate and phthalic acid which were further degraded.     

Bacterial Decolorization of Azo Dyes by Rhodopseudomonas palustris

Summary The ability of Rhodopseudomonas palustris AS1.2352 possessing azoreductase activity to decolorize azo dyes was investigated. It was demonstrated that anaerobic conditions were necessary for bacterial decolorization, and the optimal pH and temperature were pH 8 and 30–35 °C, respectively. Decolorization of dyes with different molecular structures was performed to compare their degradability. The strain could decolorize azo dye up to 1250 mg l⁻¹, and the correlation between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Long-term repeated operations showed that the strain was stable and efficient during five runs. Cell extracts from the strain demonstrated oxygen-insensitive azoreductase activity in vitro.     

Aerobic batch degradation of phenol using immobilized Pseudomonas putida

Alginate concentrations between 2 and 4% had little effect on the degradation rate of phenol by alginate-immobilized Pseudomonas putida. Ten-degree shifts from 25°C resulted in approximately 30% slower degradation. Maximal degradation rates were favored at pH 5.5–6.0. The response of degradation rate to increased air flow in the bubble column used was almost linear and an optimal higher than 16 vol vol⁻¹ was indicated, although free cells appeared in the reaction medium above 12 vol vol⁻¹. When the initial phenol concentration was raised, degradation rate was not significantly affected until levels higher than 1200 mg ml⁻¹ where performance was markedly reduced. Increasing the ratio of total bead volume to medium volume gave progressively smaller increases in degradation rate. At a medium volume to total bead volume ratio of 5:1, the maximum degradation rate was 250 mg L⁻¹ h⁻¹. Received 24 November 1998/ Accepted in revised form 27 January 1999     

Rapid biodegradation of metsulfuron-methyl by a soil fungus in pure cultures and soil

Summary Four strains of bacteria, 9 strains of fungi and 20 strains of actinomycetes capable of utilizing metsulfuron-methyl as sole carbon and energy source were isolated from a metsulfuron-methyl-treated soil by the enrichment culture method. A fungus named DS11F was selected as the most highly effective one according to the maximum tolerance concentration of 1,200 mg l⁻¹ and metsulfuron-methyl-degrading rate of 0.0716 g g⁻¹ cells h⁻¹, and was identified as an unknown strain of Penicillium sp. on the basis of colony growth, morphology and biochemical characteristics.␣Through liquid pure culture, the optimal metsulfuron-methyl-degrading conditions of DS11F were determined to be metsulfuron-methyl concentration 22.6 mg l⁻¹, inoculum concentration 12.25 mg l⁻¹, pH 7.0 and temperature 30°C. As additional C sources, supernatant of soaked compost could increase metsulfuron-methyl degradation by 8%, but glucose was ineffective. DS11F inoculation was found to significantly enhance the degradation of metsulfuron-methyl in soil, with the reduction of the concentration reaching 50% in 6 days. Admixture of compost could promote metsulfuron-methyl degradation to some extent. The growth of the inocula in the soils remained dominant and degradation resumed immediately when metsulfuron-methyl was applied again. The results show that addition of the isolated Penicillium sp. enhances the degradation of metsulfuron-methyl in water and soil.     

Single-culture aerobic granules with <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

Aerobic granules are cultivated by a single bacterial strain, Acinetobacter calcoaceticus, in a sequencing batch reactor (SBR). This strain presents as a good phenol reducer and an efficient auto coagulator in the presence of phenol, mediated by heat-sensitive adhesins proteins. Stable 2.3-mm granules were formed in the SBR following a 7-week cultivation. These granules exhibit excellent settling attributes and degrade phenol efficiently at concentrations of 250–2,000 mg l⁻¹. The corresponding phenol degradation rate reached 993.6 mg phenol g⁻¹ volatile suspended solids (VSS) day⁻¹ at 250 mg l⁻¹ phenol and 519.3 mg phenol g⁻¹ VSS day⁻¹ at 2,000 mg l⁻¹ phenol concentration. Meanwhile, free A. calcoaceticus cells were fully inhibited at phenol >1,500 mg l⁻¹. Denaturing gradient gel electrophoresis fingerprint profile demonstrated no genetic modification in the strain during aerobic granulation. The present single-strain granules showed long-term structural stability and performed high phenol degrading capacity and high phenol tolerance. The confocal laser scanning microscopic test revealed that live A. calcoaceticus cells principally distributed at 200–250 μm beneath the outer surface, with an extracellular polymeric substance layer covering them to defend phenol toxicity. Autoaggregation assay tests demonstrated the possibly significant role of secreted proteins on the formation of single-culture A. calcoaceticus granules.     

Mechanism of cyanide and thiocyanate decomposition by an association of Pseudomonas putida and Pseudomonas stutzeri strains

The intermediate and terminal products of cyanide and thiocyanate decomposition by individual strains of the genus Pseudomonas, P. putida strain 21 and P. stutzeri strain 18, and by their association were analyzed. The activity of the enzymes of nitrogen and sulfur metabolism in these strains was compared with that of the collection strains P. putida VKM B-2187^T and P. stutzeri VKM B-975^T. Upon the introduction of CN⁻ and SCN⁻ into cell suspensions of strains 18 and 21 in phosphate buffer (pH 8.8), the production of NH ₄ ⁺ was observed. Due to the high rate of their utilization, NH₃, NH ₄ ⁺ , and CNO⁻ were absent from the culture liquids of P. putida strain 21 and P. stutzeri strain 18 grown with CN⁻ or SCN⁻. Both Pseudomonas strains decomposed SCN⁻ via cyanate production. The cyanase activity was 0.75 μmol/(min mg protein) for P. putida strain 21 and 1.26 μmol/(min mg protein) for P. stutzeri strain 18. The cyanase activity was present in the cells grown with SCN⁻ but absent in cells grown with NH ₄ ⁺ . Strain 21 of P. putida was a more active CN⁻ decomposer than strain 18 of P. stutzeri. Ammonium and CO₂ were the terminal nitrogen and carbon products of CN⁻ and SCN⁻ decomposition. The terminal sulfur products of SCN⁻ decomposition by P. stutzeri strain 18 and P. putida strain 21 were thiosulfate and tetrathionate, respectively. The strains utilized the toxic compounds in the anabolism only, as sources of nitrogen (CN⁻ and SCN⁻) and sulfur (SCN⁻). The pathway of thiocyanate decomposition by the association of bacteria of the genus Pseudomonas is proposed based on the results obtained. Original Russian Text ? N.V. Grigor’eva, T.F. Kondrat’eva, E.N. Krasil’nikova, G.I. Karavaiko, 2006, published in Mikrobiologiya, 2006, Vol. 75, No. 3, pp. 320–328.     

Evidence for the role of zinc on the performance of dibenzothiophene desulfurization by <Emphasis Type="Italic">Gordonia alkanivorans</Emphasis> strain 1B

Gordonia alkanivorans strain 1B is able to desulfurize dibenzothiophene (DBT) to 2-hydroxybiphenyl (2-HBP), the final product of the 4S pathway. However, both the cell growth and the rate of desulfurization can be largely affected by the nutrient composition of the growth medium due to cofactor requirements of many enzymes involved in the biochemical pathways. In this work, the effect of several metal ions on the growth and DBT desulfurization by G. alkanivorans was studied. From all the metal ions tested, only the absence of zinc significantly affected the cell growth and the desulfurization rate. By increasing the concentration of Zn from 1 to 10 mg L⁻¹, 2-HBP productivity was improved by 26%. The absence of Zn²⁺, when sulfate was also used as the only sulfur source, did not cause any difference in the bacterial growth. Resting cells grown in the presence of Zn²⁺ exhibited a 2-HBP specific productivity of 2.29 μmol g⁻¹ (DCW) h⁻¹, 7.6-fold higher than the specific productivity obtained by resting cells grown in the absence of Zn²⁺ (0.30 μmol g⁻¹ (DCW) h⁻¹). These data suggests that zinc might have a key physiological role in the metabolism of DBT desulfurization.     

Effects of Rhizobium inoculation on growth and nodulation ofAlbizia falcataria (L.) Fosh. andAcacia mangium Willd. in the nursery

Three separate experiments were conducted in the nursery using grassland soil as a growing medium. The first experiment was conducted to assess the nodulation of the two legume trees grown in unamended soil, the second was done to determine the effects of N-fertilizer application on the interaction of the five Rhizobium isolates withA. falcataria and the third experiment was conducted to determine the effects of liming on theRhizobium × A. mangium interaction. Three local Rhizobium isolates, A₁₆, A₁₈, and A₁₄ were effective forA. falcataria with A₁₆ as the most promising strain under the conditions described. In general, application of combined nitrogen suppressed nodulation ofA. falcataria. Nodulation in the absence of combined N exceeded those fertilized with 30 kg N ha⁻¹ by 114.0%, 60 kg N ha⁻¹ by 209.6% and 100 kg N ha⁻¹ by 237.1%. Two local isolates, Am₁₀ and Am₂, and an introduced strain, NA 1533 from Australia were promising forA. mangium. Unlike inA. falcataria, the application of combined nitrogen at the rate of 100 kg N ha⁻¹ did not suppress nodulation inA. mangium. Liming the soil to pH 6.5 regardless of nitrogen fertilizer application improved the performance of the Rhizobium—A. mangium symbiosis.     

Biodegradation of 8-anilino-1-naphthalenesulfonic acid by Pseudomonas aeruginosa

Pseudomonas aeruginosa, isolated from soil near tannery effluent was able to degrade 8-anilino-1-naphthalenesulfonic acid (ANSA), a sulfonated aromatic amine. The organism degraded this amine up to a concentration of 1,200 mg l⁻¹ using glucose and ammonium nitrate as carbon and nitrogen sources respectively. The degradation started when the organism reached its late exponential growth phase. Salicylic acid and β-ketoadipic acid were identified as intermediate compounds using HPLC and GC–MS and provide evidence for ortho pathway reactions. Further proof for the pathway is obtained from the dioxygenase activity of the strain growing exponentially in medium with ANSA and glucose.     

Biosynthesis and properties of an extracellular thermostable serine alkaline protease from <Emphasis Type="Italic">Virgibacillus pantothenticus</Emphasis>

In this communication, we report the presence of a newly identified serine alkaline protease producing bacteria, Virgibacillus pantothenticus (MTCC 6729) in the fresh chicken meat samples and the factors affecting biosynthesis as well as characterization of protease. The strain produced only 14.3 U ml⁻¹ protease in the standard medium after 72 h of incubation, while in optimized culture conditions the production of protease was increased up to 18.2 U ml⁻¹. The strain was able to produce protease at 40°C at pH 9.0. The addition of dextrose and casein improved protease production. The protease was partially purified and characterized in terms of pH and temperature stability, effect of metal ions and inhibitors. The protease was found to be thermostable alkaline by retaining its 100% and 85% stability at pH 10.0 and at 50°C respectively. The protease was compatible with some of the commercial detergents tested, and was effective in removing protein stains from cotton fabrics. The V. pantothenticus, MTCC 6729 protease appears to be potentially useful as an additive in detergents as a stain remover and other bio-formulations.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.