Pentose degradation in archaea: Halorhabdus species degrade D-xylose,L-arabinose and D-ribose via bacterial-type pathways

Extremophiles - The degradation of the pentoses d-xylose, l-arabinose and d-ribose in the domain of archaea, in Haloferax volcanii and in Haloarcula and Sulfolobus species, has been shown to...     

Ethanol production by extractive fermentation

The ideal method to produce a terminal metabolite inhibitor of cell growth and production is to remove and recover it from the fermenting broth as it formed. Extractive fermentation is achieved in the case of ethanol production by coupling both fermentation and liquid-liquid extraction, The solvent of extraction is 1-dodecanol (or a mixture 1-dedecanol, 1-tetradecanol); study of the inhibitory effect of primary aliphatic alcohols of different chain lengths shows that no growth is observed in the presence of alcohols which have between 2 and 12 carbons. This effect is suppressed when the carbon number is 12 or higher. A new reactor has been used-1 pulsed packed column. Pulsation is performed pneumatically. Porous material used as a package adsorbs the cells. The fermentation broth is pulsed in order to (1) increase the interfacial area between the aqueous phase and the dodecanol, (2) decrease gas holdup. Alcoholic fermentation, performed at 35 degrees C on glucose syrup, permits the total utilization of glucose solution of 409 g/L with a yeast which cannot-in classical process- completely use solutions with 200 g/L of glucose. The feasibility of a new method of fermentation coupling both liquid-liquid extraction and fermentation is demonstrated. Extension of this method is possible to any microbial production inhibited by its metabolite excretion.     

Ethanol production from D-xylose and other sugars by the yeastPachysolen tannophilus

Summary D-Xylose was fermented to ethanol by a strain ofPachysolen tannophilus in yields greater than 0.3g ethanol per g xylose consumed. Ethanol production was influenced by xylose concentration and was at a maximum at 10%, w/v. Ethanol formation occurred at pH 2.75-2.50 but the yeast would not grow at this pH when the initial pH of the medium was less than 3.0. Ethanol was consumed by the yeast when the xylose concentration became limiting. L-Arabinose, D-glucose, D-fructose, cellobiose, D-glucuronic acid, but not sucrose,were also fermented to ethanol byPachysolen tannophilus. Kinetic studies on xylose fermentation established various parameters involved in growth, substrate utilization and ethanol formation when the yeast was fermenter grown.     

发酵法生产D-核糖

Ｄ－核糖是一种重要的生理物质,可用于合成维生素Ｂ２,风味提高剂以及多种核酸药物等,具有广泛的应用前景,由于化学合成法存在污染公害,故近年来各国相继研究微生物发酵法生产Ｄ－核糖,并致力于工业化生产。本文对国内外Ｄ－核糖的研究进展作了综述,并对Ｄ－核糖的提取方法进行了探讨。     

Induction of NADPH-linked D-xylose reductase and NAD-linked xylitol dehydrogenase activities in Pachysolen tannophilus by D-xylose, L-arabinose, or D-galactose

Considerable interest in the D-xylose catabolic pathway of Pachysolen tannophilus has arisen from the discovery that this yeast is capable of fermenting D-xylose to ethanol. In this organism D-xylose appears to be catabolized through xylitol to D-xylulose. NADPH-linked D-xylose reductase is primarily responsible for the conversion of D-xylose to xylitol, while NAD-linked xylitol dehydrogenase is primarily responsible for the subsequent conversion of xylitol to D-xylulose. Both enzyme activities are readily detectable in cell-free extracts of P. tannophilus grown in medium containing D-xylose, L-arabinose, or D-galactose and appear to be inducible since extracts prepared from cells growth in media containing other carbon sources have only negligible activities, if any. Like D-xylose, L-arabinose and D-galactose were found to serve as substrates for NADPH-linked reactions in extracts of cells grown in medium containing D-xylose, L-arabinose, or D-galactose. These L-arabinose and D-galactose NADPH-linked activities also appear to be inducible, since only minor activity with L-arabinose and no activity with D-galactose is detected in extracts of cells grown in D-glucose medium. The NADPH-linked activities obtained with these three sugars may result from the actions of distinctly different enzymes or from a single aldose reductase acting on different substrates. High-performance liquid chromatography and gas-liquid chromatography of in vitro D-xylose, L-arabinose, and D-galactose NADPH-linked reactions confirmed xylitol, L-arabitol, and galactitol as the respective conversion products of these sugars. Unlike xylitol, however, neither L-arabitol nor galactitol would support comparable NAD-linked reaction(s) in cellfree extracts of induced P. tannophilus. Thus, the metabolic pathway of D-xylose diverges from those of L-arabinose or D-galactose following formation of the pentitol.     

Ethanol production from lactose by extractive fermentation

Summary The suitability of extractive fermentation as a technique for the production of ethanol from lactose by Candida pseudotropicalis was examined as a potential improvement over conventional methods. A biocompatible solvent was selected through determination of the critical log P (octanol-water distribution coefficient) of the fermentation organism. Using Adol 85 NF, the selected solvent, extractive fed-batch and conventional fed-batch systems were operated for 160 h. The extractive system showed a 60% improvement in lactose consumption and ethanol production, as well as a 75% higher volumetric productivity.     

Effect of nitrogen sources on oxidoreductive enzymes and ethanol production during D-xylose fermentation by Candida shehatae.

The effect on D-xylose utilization and the corresponding xylitol and ethanol production by Candida shehatae (ATCC 22984) were examined with different nitrogen sources. These included organic (urea, asparagine, and peptone) and inorganic (ammonium chloride, ammonium nitrate, ammonium sulphate, and potassium nitrate) sources. Candida shehatae did not grow on potassium nitrate. Improved ethanol production (Y(p/s), yield coefficient (grams product/grams substrate), 0.34) was observed when organic nitrogen sources were used. Correspondingly, the xylitol production was also higher with organic sources. Ammonium sulphate showed the highest ethanol:xylitol ratio (11.0) among all the nitrogen sources tested. The ratio of NADH- to NADPH-linked D-xylose reductase (EC 1.1.1.21) appeared to be rate limiting during ethanologenesis of D-xylose. The levels of xylitol dehydrogenase (EC 1.1.1.9) were also elevated in the presence of organic nitrogen sources. These results may be useful in the optimization of alcohol production by C. shehatae during continuous fermentation of D-xylose.     

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

Background  

Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials.     

Optimization of culture medium and growth conditions for production of L-arabinose isomerase and D-xylose isomerase by Lactobacillus bifermentans

Lactobacillus bifermentans was used to produce the intracellular enzymes L-arabinose isomerase and D-xylose isomerase. Various factors of cultivation (temperature, pH, or incubation period) and culture medium composition (mineral salts, carbon and nitrogen source) were studied to select the conditions that maximize production of these enzymes. Arabinose isomerase and xylose isomerase activities were 9.4 and 7.24 U/ml, respectively. They were highest at 9 h of cultivation in the optimized medium, 1.6 times higher than that in the basic MRS broth. The optimal medium composition and cultivation conditions were determined. On the other hand, the strain required for growth Tween 80 (1 g/l) and a source of inorganic nitrogen (e.g. ammonium citrate). The bacterium had no requirement for sodium acetate for both growth and production of isomerases. The production rate of enzymes was increased when metal ions were added and mainly manganese (2.5 mM).     

Fermentation of L-arabinose,D-xylose and D-glucose by ethanologenic recombinant Klebsiella oxytoca strain P2

Summary Recombinant Klebsiella oxytoca strain P2 carrying genes for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis was evaluated for its ability to ferment arabinose, xylose and glucose alone and in mixtures in pH-controlled batch fermentations. This organism produced 0.34–0.43 g ethanol/g sugar at pH 6.0 and 30°C on 8% sugar substrate and demonstrated a preference for glucose. Sugar utilization was glucose > arabinose > xylose and ethanol production was xylose > glucose > arabinose.     

木糖发酵生产乙醇的研究

选育出一株优良的木糖发酵菌株树干毕赤酵母菌7124,并利用纯木糖优化了木糖发酵条件,利用海藻酸钠固定化树干毕赤酵母菌增殖细胞,不仅能较好满足限氧发酵条件,而且能耐较高糖浓度,使乙醇发酵浓度提高到20g/L。利用半纤维素水解液进行了乙醇发酵的初步研究,基本达到了纯木糖发酵的效果。     

Ethanol production from alfalfa fiber fractions by saccharification and fermentation

This work describes ethanol production from alfalfa fiber using separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) with and without liquid hot water (LHW) pretreatment. Candida shehatae FPL-702 produced 5 and 6.4 g/l ethanol with a yield of 0.25 and 0.16 g ethanol/g sugar respectively by SHF and SSF from alfalfa fiber without pretreatment. With LHW pretreatment using SSF, C. shehatae FPL-702 produced 18.0 g/l ethanol, a yield of 0.45 g ethanol/g sugar from cellulosic solids or ‘raffinate’. Using SHF, it produced 9.6 g/l ethanol, a yield of 0.47 g ethanol/g sugar from raffinate. However, the soluble extract fraction containing hemicelluloses was poorly fermented in both SHF and SSF due to the presence of inhibitors. Addition of dilute acid during LHW pretreatment of alfalfa fiber resulted in fractions that were poorly saccharified and fermented. These results show that unpretreated alfalfa fiber produced a lower ethanol yield. Although LHW pretreatment can increase ethanol production from raffinate fiber fractions, it does not increase production from the hemicellulosic and pectin fractions.     

Ethanol extraction by supported liquid membrane during fermentation

A supported liquid membrane system was developed for the extraction of ethanol during semicontinuous fermentation of Saccharomyces bayanus. it consisted of a porous Teflon sheet as support, soaked with isotridecanol. This assembly permitted combining biocompatibility, permeation efficiency, and stability. The removal of ethanol from the cultures led to decreased inhibition and, thus, to a gain in conversion of 452 g/L glucose versus 293 g/L glucose without extraction. At the same time, the ethanol volumetric productivity was enhanced 2.5 times, due to an improvement of yeast viability, while the substrate conversion yield was maintained above 95% of its theoretical value. Besides these improvements in fermentation performances, the process resulted in ethanol purification, since the separation was selective towards microbial cells and carbon substrate, and likely selective to mineral ions present in the fermentation broth. For pervaporation, a concentration of ethanol four times greater was obtained in the collected permeate.     

Ethanol productions from D-xylose and cellobiose by Kluyveromyces cellobiovorus

Yeasts capable of fermenting both D-xylose and cellobiose to ethanol were screened. Of 213 species of yeasts surveyed, Kluyveromyces cellobiovorus sp. nov., a new species belonging to genus of Kluyveromyces, was selected as the sole strain. This strain accumulated 32, 22, and 19 g/L of ethanol from 8% glucose, D-xylose, and cellobiose, respectively. It was also shown that this strain produced ethanol from the enzymatic bagasse hydrolysate containing hexoses and pentoses more efficiently than Saccharomyces cerevisiae.     

Role of D-ribose as a cometabolite in D-xylose metabolism by Saccharomyces cerevisiae.

The influence of D-ribose as a cosubstrate on the uptake and metabolism of the non-growth substrate D-xylose by Saccharomyces cerevisiae ATCC 26602 was investigated. Xylose was taken up by means of low- and high-affinity glucose transport systems. In cells exposed for 2 days to a mixture of xylose and ribose, only the high-affinity system could be detected. Glucose strongly inhibited the transport of xylose by both systems. Starvation or exposure to either xylose or ribose resulted in inactivation of xylose transport, which did not occur in the presence of a mixture of ribose and xylose. A constitutive non-glucose-repressible NADPH2-dependent xylose reductase with a specific activity of ca. 5 mU/mg of protein that converted xylose to xylitol was present in a glucose-grown culture. No activity converting xylitol to xylulose or vice versa was found in crude extracts. Both xylose and ribose were converted to their corresponding polyols, xylitol and ribitol, as indicated by 13C nuclear magnetic resonance spectroscopy. Furthermore, ethanol was detected, and this implied that pathways for the complete catabolism of xylose and ribose exist. However, the NADPH2 required for the conversion of xylose to xylitol is apparently not supplied by the pentose phosphate pathway since the ethanol produced from D-[1-13C]xylose was labelled only in the C-2 position. Acetic acid was produced from ribose and may assist in the conversion of xylose to xylitol by cycling NADPH2.     

Alcoholic fermentation of D-xylose by immobilizedPichia stipitis yeast

Summary Pichia stipitis NRRL Y-7124 yeast cells were for the first time immobilized both in agar gel beads and on fine nylon net for ethanol fermentation on D-xylose, in order to investigate the possibility of using the biocatalyst for improved utilization of the biomass pentose fraction. With free cells the initial xylose level affected little ethanol production, with a maximum of 22 g/l ethanol obtained in 5 days on 5% and of 40 g/l in 8 days on 10% xylose, and an average volumetric productivity of about 0.22 g/lh. The maximum ethanol concentration of 19.5% on 5% xylose with the nylon net attached cells in a continuous packed-bed column reactor was obtained with 35 h residence time. The volumetric productivities of 0.56 g/lh at 19.5 g/l ethanol and 1.0 g/lh at 15.0 g/l ethanol were markedly higher than those obtained with free cells. The stability of the immobilized biocatalyst was excellent. The same reactor could be used for at least 80 days without significant activity loss.     

Engineering Pseudomonas putida S12 for efficient utilization of D-xylose and L-arabinose

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to utilize xylose as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli. The initial yield on xylose was low (9% [g CDW g substrate(-1)], where CDW is cell dry weight), and the growth rate was poor (0.01 h(-1)). The main cause of the low yield was the oxidation of xylose into the dead-end product xylonate by endogenous glucose dehydrogenase (Gcd). Subjecting the XylAB-expressing P. putida S12 to laboratory evolution yielded a strain that efficiently utilized xylose (yield, 52% [g CDW g xylose(-1)]) at a considerably improved growth rate (0.35 h(-1)). The high yield could be attributed in part to Gcd inactivity, whereas the improved growth rate may be connected to alterations in the primary metabolism. Surprisingly, without any further engineering, the evolved D-xylose-utilizing strain metabolized l-arabinose as efficiently as D-xylose. Furthermore, despite the loss of Gcd activity, the ability to utilize glucose was not affected. Thus, a P. putida S12-derived strain was obtained that efficiently utilizes the three main sugars present in lignocellulosic hydrolysate: glucose, xylose, and arabinose. This strain will form the basis for a platform host for the efficient production of biochemicals from renewable feedstock.