Screening for microorganisms producing D-malate from maleate.

More than 300 microorganisms were screened for their ability to convert maleate into D-malate as a result of the action of maleate hydratase. Accumulation of fumarate during incubation of permeabilized cells with maleate was shown to be indicative of one of the two enzymes known to transform maleate. The ratio in which fumarate and malate accumulated could be used to estimate the enantiomeric composition of the malate formed. Many strains (n = 128) were found to be capable of converting maleate to D-malate with an enantiomeric purity of more than 97%. Pseudomonas pseudoalcaligenes NCIMB 9867 was selected for more detailed studies. Although this strain was not able to grow on maleate, permeabilized cells were able to degrade maleate to undetectable levels, with a concomitant formation of D-malate. The D-malate was formed with an enantiomeric purity of more than 99.97%.     

Screening for microorganisms producing D-malate from maleate.

More than 300 microorganisms were screened for their ability to convert maleate into D-malate as a result of the action of maleate hydratase. Accumulation of fumarate during incubation of permeabilized cells with maleate was shown to be indicative of one of the two enzymes known to transform maleate. The ratio in which fumarate and malate accumulated could be used to estimate the enantiomeric composition of the malate formed. Many strains (n = 128) were found to be capable of converting maleate to D-malate with an enantiomeric purity of more than 97%. Pseudomonas pseudoalcaligenes NCIMB 9867 was selected for more detailed studies. Although this strain was not able to grow on maleate, permeabilized cells were able to degrade maleate to undetectable levels, with a concomitant formation of D-malate. The D-malate was formed with an enantiomeric purity of more than 99.97%.     

利用Pimelobacter simplex DM18从马来酸转化制备D-苹果酸

利用PimelobactersimplexDM18从马来酸高效制备D 苹果酸 ,优化了菌株DM18的发酵条件 ,最佳发酵培养基组成为 (g/L) :柠康酸 ,2 5 ;葡萄糖 ,3;磷酸二氢钾 ,10 5 ;硝酸铵 ,2 0 ;硫酸镁 ,0 2 5 ;七水硫酸亚铁 ,0 0 15 ;酵母膏 ,2 8;玉米浆 5 ;pH值 7 2。在最适培养基中 30℃培养 30h ,菌体OD660 可达到 4～ 4 5 ,酶活达到 14g/ (L·h) (反应菌液OD660 为 10 )。采用间歇添加马来酸钠反应 32h ,D 苹果酸质量浓度为 15 3 9g/L ,D 苹果酸的收率达到 96 6 7%。,经离子配位体色谱分析 ,产物D 苹果酸光学纯度为 96 98%。     

Microbial production of isoquinoline from indene

A purified microbial isolate, identified as a strain of Rhodococcus sp., metabolized indene primarily to iso quinoline and lesser amounts of indandiol and indanone. Isoquinoline production was dependent on the presence of microbial culture, indene, and ammonium ions as the source of nitrogen in the molecule. The ability to produce isoquinoline was induced by growth on benzene or naphthalene and by the presence of indene itself. The culture produced compounds tentatively identified as 3-methylisoquinoline and 3-ethylisoquinoline from 2-methylindene and from 2-ethylindene, respectively. Deuterated indene was converted to deuterated isoquinoline, deuterated indanone, and deuterated indandiol. Experiments with [15N]ammonium nitrate and ammonium [15N]nitrate confirmed ammonium as the source of nitrogen in the isoquinoline products.     

Microbial production of ethanol from carbon monoxide

Production of ethanol from fermentation of CO has received much attention in the last few years with several companies proposing to use CO fermentation in their ethanol production processes. The genomes of two CO fermenters, Clostridium ljungdahlii and Clostridium carboxidivorans, have recently been sequenced. The genetic information obtained from this sequencing is aiding molecular biologists who are enhancing ethanol and butanol production by genetic manipulation. Several studies have optimized media for CO fermentation, which has resulted in enhanced ethanol production. Also, new reactor designs involving the use of hollow fiber membranes have reduced mass transfer barriers that have hampered previous CO fermentation efforts.     

Microbial production of methane from municipal refuse

A heat and caustic pretreatment process has been tested to determine the increase in gas yield that can be obtained from fermentation of organic municipal refuse. A treatment temperature of 130°C and a NaOH concentration of 3 g/100 g of dry solids resulted in the highest gas yield for the conditions tested. The probable increase in gas yield was 20% for the best conditions tested. The treatment procedure also substantially increased the rates of gas production. A high conversion efficiency is possible at much shorter reactor retention times with pretreatment than without pretreatment.     

D-malate production by permeabilized Pseudomonas pseudoalcaligenes; optimization of conversion and biocatalyst productivity

For the development of a continuous process for the production of solid D-malate from a Ca-maleate suspension by permeabilized Pseudomonas pseudoalcaligenes, it is important to understand the effect of appropriate process parameters on the stability and activity of the biocatalyst. Previously, we quantified the effect of product (D-malate2 -) concentration on both the first-order biocatalyst inactivation rate and on the biocatalytic conversion rate. The effects of the remaining process parameters (ionic strength, and substrate and Ca2 + concentration) on biocatalyst activity are reported here. At (common) ionic strengths below 2 M, biocatalyst activity was unaffected. At high substrate concentrations, inhibition occurred. Ca2+ concentration did not affect biocatalyst activity. The kinetic parameters (both for conversion and inactivation) were determined as a function of temperature by fitting the complete kinetic model, featuring substrate inhibition, competitive product inhibition and first-order irreversible biocatalyst inactivation, at different temperatures simultaneously through three extended data sets of substrate concentration versus time. Temperature affected both the conversion and inactivation parameters. The final model was used to calculate the substrate and biocatalyst costs per mmol of product in a continuous system with biocatalyst replenishment and biocatalyst recycling. Despite the effect of temperature on each kinetic parameter separately, the overall effect of temperature on the costs was found to be negligible (between 293 and 308 K). Within pertinent ranges, the sum of the substrate and biocatalyst costs per mmol of product was calculated to decrease with the influent substrate concentration and the residence time. The sum of the costs showed a minimum as a function of the influent biocatalyst concentration.     

Microbial production of spider silk proteins.

The remarkable properties of spider dragline silk and related protein polymers will find many applications if the materials can be produced economically. We have demonstrated the production of high molecular weight spider dragline silk analog proteins encoded by synthetic genes in several microbial systems, including Escherichia coli and Pichia pastoris. In E. coli, proteins of up to 1000 amino acids in length could be produced efficiently, but the yield and homogeneity of higher molecular weight silk proteins were found to be limited by truncated synthesis, probably as a result of ribosome termination errors. No such phenomenon was observed in the yeast P. pastoris, where higher molecular weight silk proteins could be produced without heterogeneity due to truncated synthesis. Spider dragline silk analog proteins could be secreted by P. pastoris when fused to both the signal sequence and N-terminal pro-sequence of the Saccharomyces cerevisiae alpha-mating factor gene.     

Microbial production of D-mannitol and D-fructose from glycerol

In view of the recent development that some petrochemical products are efficiently available as substrates for the fermentation industry, glycerol manufactured from propylene by chemical synthesis would also be hoped for the purpose. This paper describes some of the factors influencing mannitol production from glycerol by Torulopsis yeasts and a microbial conversion of glycerol to D -fructose via mannitol, in which two sequential steps of yeast and Acetobacter fermentation are involved. Torulopsis mannitofaciens CBS 5981 and Torulopsis vcrsatilis CBS 1752, exceptionally good mannitol producers, were selected for the study. High concentrations of nitrogen sources and KH₂PO₄ in the medium markedly decreased mannitol yield in spite of good utilization of the substrate. T. mannitofaciens produced mannitol in yield of 31% of the glycerol consumed at optimal condition. The fermentation by washed yeast cells gave much higher mannitol yield of more than 50%. A sequential fermentation process was carried out without isolation and purification of the intermediate and yielded.51.7%. D -fructose from the glycerol.     

Microbial production of 2,3-butanediol from whey permeate

Summary Of four organisms tested in semi-synthetic medium for the production of 2,3-butanediol from lactose, Klebsiella pneumoniae N.C.I.B. 8017 proved to be the most promising. When tested using rennet whey permeate as substrate, a butanediol concentration of 7.5 g/l, representing a yield of 0.46 g/g lactose utilized, was observed after 96 h incubation. In whey permeate where the lactose had been hydrolysed enzymatically prior to the fermentation, a butanediol concentration of 13.7 g/l, representing a yield of 0.39 g/g sugar utilized was obtained. These results indicate that lactose utilization may be a limiting step in the fermentation process.     

Microbial community engineering for biopolymer production from glycerol

In this work, the potential of using microbial community engineering for production of polyhydroxyalkanoates (PHA) from glycerol was explored. Crude glycerol is a by-product of the biofuel (biodiesel and bioethanol) industry and potentially a good substrate for bioplastic production. A PHA-producing microbial community was enriched based on cultivation in a feast–famine regime as successfully applied before for fatty acids-based biopolymer production. A glycerol-fed sequencing batch reactor operated at a 2-day liquid and biomass residence time and with feast–famine cycles of 24 h was used to enrich a mixed community of PHA producers. In a subsequent fed-batch PHA production step under growth-limiting conditions, the enriched mixed community produced PHA up to a dry weight content of 80 wt.%. The conversion efficiency of substrate to PHA on electron basis was 53%. Since glycerol is entering the metabolic pathways of the cell in the glycolytic pathway, it was anticipated that besides PHA, polyglucose could be formed as storage polymer as well. Indeed, polyglucose was produced in low amounts (∼10 wt.%). The results indicated that the feast–famine-based enrichment strategy was comparably successful to obtain a microbial community compared to fatty acids-based enrichment described before.     

Microbial production of single-cell protein from deproteinized whey concentrates

Deproteinized sweet and sour cheese whey concentrates were investigated for their suitability as substrates for the production of single-cell protein with Kluyveromyces marxianus CBS 6556 up to a 100-l scale. An important factor for gaining high cell concentrations was the use of the Crabtree-negative strain K. marxianus CBS 6556. Supplements such as trace elements, ammonium and calcium were required for the complete conversion of sweet whey concentrates into biomass, whereas sour whey concentrates had to be supplemented with ammonium, trace elements and vitamins. After improvement, biomass dry concentrations of up to 50 g l⁻¹ could be reached with Yx/s values of 0.52 for sweet whey and of up to 65 g l⁻¹ with Yx/s values of 0.48 for sour whey concentrates. The chemical oxygen demand of the whey concentrates were reduced by 80%. The cells were used for the analysis of amino acid and ash composition, showing a distinct increase of eight out of ten essential amino acids compared to sweet and sour whey protein and exceeding the World Health Organisation guidelines for valine, leucine, isoleucine, threonine, phenylalanine and tyrosine.     

Microbial d-xylonate production

D-Xylonic acid is a versatile platform chemical with reported applications as complexing agent or chelator, in dispersal of concrete, and as a precursor for compounds such as co-polyamides, polyesters, hydrogels and 1,2,4-butanetriol. With increasing glucose prices, D-xylonic acid may provide a cheap, non-food derived alternative for gluconic acid, which is widely used (about 80?kton/year) in pharmaceuticals, food products, solvents, adhesives, dyes, paints and polishes. Large-scale production has not been developed, reflecting the current limited market for D-xylonate. D-Xylonic acid occurs naturally, being formed in the first step of oxidative metabolism of D-xylose by some archaea and bacteria via the action of D-xylose or D-glucose dehydrogenases. High extracellular concentrations of D-xylonate have been reported for various bacteria, in particular Gluconobacter oxydans and Pseudomonas putida. High yields of D-xylonate from D-xylose make G. oxydans an attractive choice for biotechnical production. G. oxydans is able to produce D-xylonate directly from plant biomass hydrolysates, but rates and yields are reduced because of sensitivity to hydrolysate inhibitors. Recently, D-xylonate has been produced by the genetically modified bacterium Escherichia coli and yeast Saccharomyces cerevisiae and Kluyveromyces lactis. Expression of NAD(+)-dependent D-xylose dehydrogenase of Caulobacter crescentus in either E. coli or in a robust, hydrolysate-tolerant, industrial Saccharomyces cerevisiae strain has resulted in D-xylonate titres, which are comparable to those seen with G. oxydans, at a volumetric rate approximately 30?% of that observed with G. oxydans. With further development, genetically modified microbes may soon provide an alternative for production of D-xylonate at industrial scale.     

Microbial production of xylitol from D-xylose using Candida tropicalis

Candida tropicalis DSM 7524 was used to produce xylitol from d-xylose. The fermentation conditions were optimized during continuous cultivation. The strain employed showed no great dependence upon temperature in a range between 30° C and 37° C. It achieved its best yield of xylitol from d-xylose at a pH value of 2.5. Such low pH values allow non sterile cultivation, which is a major economic factor. With an oxygen uptake rate of 0.8–1 ml oxygen per litre culture medium, the C. tropicalis produce xylitol at a yield of between 77% and 80% of the theoretical value. Higher yeast extract concentrations prevent the conversion of d-xylose into xylitol. d-xylose acts as a growth inhibitor in higher concentrations. The maximum xylitol yield was reached at a d-xylose concentration of around 100 g/l. In a non sterile batch culture with substrate shift 220 g/l xylitol were produced from 300 g/l d-xylose at a xylitol productivity rate of 0.37 g/(lh). In order to increase the specific yield, C. tropicalis was immobilised on porous glass and cultivated in a fluidized bed reactor. In a continuous non sterile cultivation with immobilised cells 155 g/l d-xylose produced 90–95% g/l xylitol with a productivity of 1.35 g/(lh).Mr. S. S. da Silva was a visiting scientist to the GBF. He was supported by a scholarship from the National Council of Scientific and Technological Development, Brasilia, Brazil (CNPq).We also would like to gratefully acknowledge the support of Prof. Dr. Michele Vitolo of the University of Sao Paulo, and the Centre for Biotechnology and Chemistry, Lorena, S. P. Brazil, in particular the Department of Fermentative Process.We are grateful to Prof. Rainer Jonas, head of the International Cooperation between Germany/Brazil for the helpful discussions and Dr. Heinrich Lönsdorf (GBF) for the Scanning electron micrographs.Dedicated to the 65th birthday of Prof. Dr. Fritz Wagner.     

Microbial production of poly-D-3-hydroxybutyrate from CO2

This short review covers the biotechnological aspects of the production of poly-D-3-hydroxybutyric acid, P(3HB), from H2, O2 and CO2 by autotrophic culture of the hydrogen-oxidizing bacterium, Ralstonia eutropha. Considering the efficiency of utilization of a gas mixture as substrate, a practical fermentation process using R. eutropha for the mass production of P(3HB) from CO2 should be designed on the basis of a recycled-gas, closed-circuit culture system. Also, maintaining the O2 concentration in a gas phase lower than 6.9% (v/v) is essential to prevent the gas mixture from exploding. Our study, using an explosion-proof fermentation bench plant and a two-stage culture system with a newly designed air-lift fermenter, demonstrated that very high P(3HB) yield and productivity could be obtained while the O2 concentration was maintained below 6.9%. However, a study on the continuous production of P(3HB) from CO2 by chemostat culture of R. eutropha revealed that the productivity and content of P(3HB) in the cells was considerably lower than by fed-batch culture. It is deduced that the use of the hydrogen-oxidizing bacterium, Alcaligenes latus, which accumulates P(3HB) even in the exponential growth phase, will be useful for the effective production of P(3HB) from CO2.     

Microbial production of dihydroxyacetone

Dihydroxyacetone is extensively used in cosmetic industry as an artificial suntan besides having clinical and biological applications. Thus, it is important to meet the commercial demand of dihydroxyacetone at an economical and qualitative level. Microbial route of production is found to be more favorable for dihydroxyacetone as compared to chemical methods. This review gives detailed information about the microbial route of dihydroxyacetone production. Till date the microorganism which is most utilized for dihydroxyacetone production is Gluconobacter oxydans. Some limitations associated with dihydroxyacetone production by G. oxydans like substrate inhibition, product inhibition and oxygen limitation are discussed here. Various fermentation modes and culture conditions have been tried for their ability to overcome these limitations. It has been found that fed-batch mode of fermentation provides a better yield as compared to batch mode for dihydroxyacetone production. Two-stage repeated fed-batch mode of fermentation has been found to be the most optimized mode. Immobilization has also been recognized as a much better alternative for fermentation since it avoids the problem of substrate and product inhibition to a greater extent. Although these methods have increased the dihydroxyacetone production to a prominent level yet the production has not reached the level required to meet the commercial demand. One looks for future prospects of developing recombinant microbial method for dihydoxyacetone production.