Glucose uptake and phosphorylation in Pseudomonas fluorescens

Pseudomonas fluorescens ATCC 13525 and a particulate glucose oxidase (d-glucose:oxygen oxidoreductase, EC 1.1.3.4) mutant of this organism, gox-7, were examined to determine if glucose oxidation via particulate glucose oxidase is a required first step for glucose uptake. Initial [(14)C]glucose-uptake rates in parent and gox-7 cells were qualitatively similar. Initial [(14)C]glucose-uptake product analysis revealed that glucose was accumulated via active transport and was rapidly metabolized to glucose-6-phosphate and gluconate-6-phosphate in both parent and gox-7 cells. Cell extracts contained soluble adenosine 5'-triphosphate specific kinase activity for phosphorylation of glucose. Glucose uptake was induced by glucose and not gluconate, thus, establishing independent regulation of glucose transport and glucose catabolism in p. fluorescens. The results prove that glucose oxidase was not an obligatory reaction for glucose carbon permeation in P. fluorescens. A general unifying scheme for glucose utilization in the aerobic fluorescent pseudomonads is suggested for the purpose of clarifying glucose uptake in these bacteria.     

Enrichment of polyunsaturated fatty acids from tuna oil using immobilized Pseudomonas fluorescens lipase

Immobilized Pseudomonas fluorescens lipase enzyme was used to enrich the important polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from tuna oil. Hydrolysis, esterification, and transesterification reactions were studied in detail to find out the fractionation pattern of DHA and EPA during these processes due to preferential selectivity for or against these PUFA. Hydrolysis with P. fluorescens biotype I lipase with stoichiometric amount of water content gave more than 80% of DHA and EPA in the free fatty acid (FFA) form after around 60% of hydrolysis. After some preferential specificity during the early stages of hydrolysis, P. fluorescens lipase exhibits nonselective characteristics on extended hydrolysis. Esterification of FFA extracted from the completely hydrolyzed mixture of tuna oil was found to be better with long chain fatty alcohol like octanol which lead to good enrichment (44.5% for DHA and 11.3% for EPA) and yields of the PUFA in the FFA form. Transesterification (ethanolysis) with immobilized P. fluorescens lipase enzyme resulted in good enrichment and recovery of DHA and EPA in the glyceride mixture. After around 60% of ester synthesis, 74% of (DHA + EPA) enrichment was achieved with yields of more than 90% in the glyceride mixture.     

Gluconate Regulation of Glucose Catabolism in Pseudomonas fluorescens

Induction of Entner-Doudoroff pathway enzymes in Pseudomonas fluorescens was investigated to study the role of gluconate as a possible inducer. Glucose oxidase-deficient mutants were isolated and characterized. One of these mutants, gox-7, was deficient in particulate glucose oxidase; another mutant, gox-17, was deficient in particulate glucose and gluconate oxidase activities. Gluconate, but not glucose, induced synthesis of gluconokinase and 6-phosphogluconate dehydratase in both mutants. High constitutive levels of 2-keto-3-deoxy-6-phosphogluconate aldolase were found when both mutants were grown on glucose. Growth of parent and both mutant strains on glycerol also resulted in high levels of Entner-Doudoroff pathway enzymes. It was concluded that glucose cannot serve as an inducer molecule for derepression of Entner-Doudoroff pathway enzymes in P. fluorescens. Evidence presented provides good support for gluconate being the true inducer of this pathway in P. fluorescens. A relationship is presented for explaining distribution of the Entner-Doudoroff pathway in certain groups of bacteria.     

Influence of Temperature on Glucose Utilization by Pseudomonas fluorescens

The influence of temperature on the conversion of glucose into cell material and into energy for maintenance was determined for Pseudomonas fluorescens by a steady-state turbidity method and by a substrate utilization method. Conversion of glucose into cell material was measured as yield; conversion of glucose into energy for maintenance was measured as specific maintenance, the minimum dilution rate in continuous culture below which a steady state is not possible. The values obtained by the two methods were nearly identical; with both, the yield and specific maintenance decreased with decreasing temperature. The specific maintenance consumption rate (milligrams of glucose taken up per milligram of cell dry weight per hour at zero growth) was also calculated by the substrate utilization method and found to decrease with decreasing temperature. However, the amount of glucose consumed per generation for maintenance increased with decreasing temperature. This increased glucose consumption for maintenance may provide a partial explanation for the decrease in yield at low temperatures. Small amounts of glucose were also converted into pigment at all temperatures tested, with the greatest amount formed at 20 C.     

Mass transfer effects in fermentations using immobilized whole cells

The immobilization of whole cells for fermentation processes has many potential advantages over fermentation with free cells, including higher cell concentrations, higher productivites and a higher level of operational stability. Most of the research reported in the literature has been directed towards demonstrating the feasibility of using these systems for various fermentations. The ultimate goal of research in this area is to bring the understanding of immobilized whole cells to the level of heterogeneous catalysis. Immobilized whole cell systems are examined from a mass transfer perspective. Evidence for external and internal mass transfer limitations is presented. Procedures for quantifying these effects in terms of effectiveness factors and determining the reaction kinetics in their presence are reviewed. Development of the reactor design equations and the reactor performance results for fermentations with immobilized cells are also discussed.     

Biochemical energy conversion using immobilized whole cells of Clostridium butyricum

Hydrogen producing bacteria, Clostridium butyricum, were immobilized in agar gel (2 per cent). The immobilized whole cells were employed for continuous production of hydrogen from alcohol factory waste waters. The hydrogen production rate became constant above BOD 1500 ppm when hydrogen production was performed with a batch system. The immobilized whole cells continuously produced hydrogen over a 20-day period. The amount of hydrogen produced was about 6 ml/min/kg wet gels. Hydrogen produced was supplied to the hydrogen-oxygen (air) fuel cells. The maximum cell voltage of cell I and II was about 0.55 and 0.66 V respectively when the flow rate of hydrogen was 6 ml/min. The limiting current density changed from 0.4 to 40 mA/cm2 as the resistance between the electrodes changed from 1 to 100 ohmz. The fuel cell was left on for 7 days and the current from 550 to 500 mA was obtained continuously over a 7 day period.     

Survival ofalginate-ecapsulated Pseudomonas fluorescens cells in soil

Alginate-encapsulated and unencapsulated cells of Pseudomonas fluorescens Rsf were introduced into soil microcosms with and without wheat plants to evaluate bacterial survival and colonization of the rhizoplane and rhizosphere. Encapsualtion of cells in alginate amended with skim milk or with skim milk plus bentonite clay significantly enchanced long-term survival of the cells. There was a negligible effect on long-term bacterial survival when cells were encapsulated in alginate amended with TY medium or soil extract, as compared to water. Drying of beads resulted in a significant reduction in bacterial viability. After addition to soil, cells in dried beads increased in numbers and exhibited stable population densities, whereas cells added in moist beads showed stable dynamics at a higher level. Cells encapsulated in dried beads or fresh beads survived better than unencapsulated cells added to soil. Both cells in moist and dried alginate beads also survide a dry/wet cycle in soil, whereas unencapsulated cells were sensitive to these moisture fluctuations. Shortly after inoculation and 63 days after this, cells from moist beads colonized wheat roots at significantly higher levels than unencapsulated cells, whereas cells in dried beads did so at levels similat to unencapsulated cells. Cells in beads initially placed at different distance from developing root mat were able to move towards and colonize the rhizosphere, at levels of roughly 10⁴ to 10⁶ colony-forming units fo P. fluorescens R2f per gram of dry soil. Correspondence to: J. T. Trevors or J. D. van Elsas     

Glucose oxidase immobilized polyethylene-g-acrylic acid membrane for glucose oxidase sensor

A polyethylene-g-acrylic acid (PE-g-AA) graft copolymer was prepared via gamma-ray-irradiation-induced postirradiation procedures, and was used as support material for the immobilization of glucose oxidase. Soluble carbodiimides were used as the coupling agent. Reasonable yields were obtained with CMC but not with EDAC, EEDQ, or WRK. A number of factors were studied. (1) The use of water-soluble carbodiimides as condensing agent was attempted and the optimum condition for coupling glucose oxidase to PE-g-AA was established; (2) the effect of pH and temperature on the reactivity of native and immobilized glucose oxidase was studied. When exposed to temperatures in excess of 60 degrees C, the immobilized glucose oxidase was less sensitive to thermal inactivation than the native enzyme. The optimum pH value for the performance of the enzyme-immobilized membrane was 5. 6. For 200 tests, the response error of glucose sensor was less than 4% and its linear detected range was 0-1000 ppm. The obtained glucose oxidase-immobilized PE-g-AA membranes were kept in pH 5. 6 acetate buffer solution at 4 degrees C. The glucose oxidase activity of the membrane was determined at sevenday intervals. The membranes still have 92% glucose oxidase activity even after eight weeks of storage.     

Biodegradation of benzidine based azodyes Direct red and Direct blue by the immobilized cells of Pseudomonas fluorescens D41

Benzidine based azodyes are proven carcinogens, mutagens and have been linked to bladder cancer of human beings and laboratory animals. The textile and dyestuff manufacturing industry are the two major sources that released azodyes in their effluents. The dye, Direct blue contains two carcinogenic compounds namely benzidine (BZ), 4-amino biphenyl (4-ABP), while the dye Direct red has benzidine (BZ). Among 40 isolates of Pseudomonas fluorescens screened, one isolate designated as D41 was found to be capable of extensively degrading the dyes Direct blue and Direct red. Immobilized cells of P. fluorescens D41 efficiently degraded Direct red (82%) and Direct blue (71%) in the presence of glucose.     

Glucose determination using immobilized polyphosphate glucokinase.

Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate:glucose phosphotransferase) was covalently coupled to collagen-coated silica gel beads. The immobilized enzyme, as a packed-bed reactor, was used to determine glucose in serum and other samples. The method was based on a spectrophotometric measurement of NADPH produced by two consecutive reactions, similar to the hexokinase method. The described approach takes advantage of the greater stability of polyphosphate compared to that of ATP, the greater specificity of polyphosphate glucokinase versus that of hexokinase, and the reusability of the immobilized enzyme. Linearity, precision, and accuracy of the method were tested and found to be very good. The results were linear between 10 and 50 nmol of glucose in a 50-microliter sample and the coefficient of variation was less than 4% in five successive determinations. The recovery of glucose was about 100% after calibration of the method. The results of the measurements correlated well with those obtained with soluble polyphosphate glucokinase (r = 0.997, y = 1.036x - 0.016). The immobilized-enzyme reactor showed good operational stability during a month of use, losing about 12% of its initial activity.     

Urocanic acid production using whole cells immobilized in a hollow fiber reactor

The feasibility of using hollow fiber membrane dialyzers (C-DAK) for immobilization of microbial whole cells was investigated. The cells are located on the shell side of the dialyzer, while substrates and products are free to diffuse across the hollow fiber membranes. The biochemical reaction studied was the conversion of L -histidine to urocanic acid and catalyzed by L -histidine ammonia-lyase. C-DAK dialyzers containing a heat-treated suspension of Pseudomonas fluorescens ATCC 11299b (with L -histidine ammonia–lyase activity) were incorporated into constant volume recycle reactor systems for continuous product formation. A simple model successfully correlated the data and predicted performance. It was found that the reaction was not likely to be diffusion limited, and such a cell immobilization scheme is convenient and workable for continuous production of biochemicals.     

Biological control of soil-pathogenic nematodes infecting mungbean using Pseudomonas fluorescens

The addition of organic matters to soil has been explored as an alternative means of nematode control under field conditions. Several oil-seed cakes of neem (Azadirachta indica), castor (Ricinus communis), groundnut (Arachis hypogeae), linseed (Linum usitatissimum) and sunflower (Helianthus annuus) were found to be highly effective in reducing the multiplication of soil-pathogenic nematodes Meloidogyne incognita, Rotylenchulus reniformis, Tylenchorhynchus brassicae, etc. The plant growth parameters such as plant weight, per cent pollen fertility, number of pods per plant, root-nodulation and chlorophyll content of mungbean increased significantly. The multiplication rate of nematodes and number of root-galls were less in the presence of Pseudomonas fluorescens as compared to its absence. Damage caused by the nematodes was further reduced when P. fluorescens was added along with the oil-seed cakes. Neem cake was found most effective in combination with P. fluorescens.     

Respiratory activity of alginate-encapsulated Pseudomonas fluorescens cells introduced into soil

Summary Alginate-entrapped cells of Pseudomonas fluorescens were introduced into soil microcosms to evaluate their respiratory activity (O₂ consumption and CO₂ evolution) and survival during a 14-day incubation period at 20°C. Alginate-entrapped cells and cells resuspended in sterile distilled water and introduced into sterile soil exhibited relatively similar O₂ consumption/CO₂ evolution and survival over the 14-day period. The same treatments in non-sterile soil exhibited lower respiratory activity and a population density decrease of about 2.0 Log. cfu/g after 14 days. Alginate-entrapped bacterial cells may be a useful method for introducing genetically-engineered and non-engineered bacterial strains into the soil environment.     

Hydantoinase activity of immobilized non-growing Pseudomonas putida cells

Summary Hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) activity of Pseudomonas putida DSM 84 was evaluated using cells immobilized in alginate beads and in a microporous hollow fibre bioreactor. Conversion of dihydrouracil into N-carbamyl--alanine was most efficient with alginate-immobilized cells. A 40 to 45% conversion was obtained in shake flasks and in continuous mode with packed bed columns. The highest volumetric productivity was obtained with a packed bed column operated at a dilution rate of 0.5 h^-1 (99 g of product. 100 l^-1 per hour). After 96 h the alginate beads began to swell and break apart; no free cells were detected however. Despite some initial loss of cells from the microporous hollow fibre bioreactor, a steady state was later established and maintained for 400 h at dilution rates of 0.1 and 0.25 h^-1.     

Glucose oxidase biosynthesis using immobilized mycelium of Aspergillus niger

A simple method is described for the immobilization of Aspergillus niger GIV-10 which produces an extracellular glucose oxidase. A. niger conidia were immobilized on sintered glass Raschig rings, pumice stones or polyurethane foam. Mycella growing out from the spores produced extracellular glucose oxidase: the highest production was with the pumice stone carrlers. This technique facilitates the growth of the filamentous cultures in the spongy structure of a support with continuous accumulation of biomass. After 24 to 36 h, a culture liquid with 2.7 to 3.1 U of glucose oxidase/ml was obtained. This procedure also made possible repeated batch enzyme production and as many as 25 subsequent 24-h batches could be fermented by using the same carrier with only a small loss of glucose oxidase activity.The authors are with the Institute of Microbiology, M. Curie-Sklodowska University, Akademicka 19, 20-003 Lublin, Poland.