Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.     

表面活性剂对出芽短梗霉多糖生产影响的研究

研究了表面活性剂对出芽短梗霉细胞培养过程中多糖释放的影响。在摇瓶中,比较添加0.05%(w/v)的Tween 80、Tween 60、Tween 40,结果显示几种表面活性剂均能促进细胞释放多糖,其中以Tween 80的效果最佳。在5L发酵罐中,以100g/L玉米粉水解液做碳源的出芽短梗霉细胞培养液中分别添加了表面活性剂Tween 80 0.01%、0.05%、0.1%,其中以添加Tween 800.05%时的效果最好,与不添加表面活性剂相比多糖产量提高25%左右,发酵周期缩短了将近2d。     

Degradation of benzo[a]pyrene by the litter-decomposing basidiomycete Stropharia coronilla: role of manganese peroxidase

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn(2+)) supplied at a concentration of 200 micro M stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn(2+) than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn(2+). Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter(-1) was converted in an in vitro reaction solution containing 1 U of MnP ml(-1) within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter(-1)) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).     

Toxicity of phenanthrene dissolved in nonionic surfactant solutions to Pseudomonas putida P2

The fraction in which direct contact occurs between micellar-phase phenanthrene and the bacterial cell surface was estimated by measuring the toxicity of nonionic surfactant (Tween 80 and Triton X-100) solutions to the phenanthrene-degrading bacterium, Pseudomonas putida P2. Cell viability of completely dissolved phenanthrene decreased by 30% at concentrations greater than 0.3 mg L(-1), which is equal to approximately one third of its solubility. Both nonionic surfactants had no effect on cell viability up to 5 g L(-1). Cell viability increased with increasing surfactant concentration at a fixed phenanthrene concentration, due to the decreased concentration of aqueous-pseudophase phenanthrene and the reduced fraction of direct contact. The fraction of direct contact was c. 20% or more below 3 g L(-1) of Triton X-100. The fraction of direct contact for Tween 80 was estimated to be lower than Triton X-100.     

Utilization of Molasses Sugar for Lactic Acid Production by Lactobacillus delbrueckii subsp. delbrueckii Mutant Uc-3 in Batch Fermentation

Efficient lactic acid production from cane sugar molasses by Lactobacillus delbrueckii mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid fermentation using molasses was not significantly affected by yeast extract concentrations. The final lactic acid concentration increased with increases of molasses sugar concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter was obtained at a molasses sugar concentration of 190 g/liter with a productivity of 4.15 g/liter/h. Such a high concentration of lactic acid with high productivity from molasses has not been reported previously, and hence mutant Uc-3 could be a potential candidate for economical production of lactic acid from molasses at a commercial scale.     

Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium.

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

Estimation of direct-contact fraction for phenanthrene in surfactant solutions by toxicity measurement

The toxicity of solutions containing nonionic surfactants Tween 80, Brij 35 and/or phenanthrene to Pseudomonas putida ATCC 17484 was investigated. The fraction of direct contact between micellar-phase phenanthrene and bacterial cell surface was estimated by using the toxicity data and a mathematical model. The mathematical model was used to calculate phenanthrene concentration in the micellar phase and aqueous pseudophase separately. The first-order death rate constant increased from 0.088+/-0.016 to 0.25+/-0.067 h(-1) when the phenanthrene concentration was increased from 0 to 5.17 x 10(-6)M (equals water solubility). The intrinsic toxicity of surfactant was higher in Brij 35 than in Tween 80. When phenanthrene concentration was increased to 9.7 x 10(-5)M in surfactant solutions, the death rate constant increased to 1.8 +/- 0.024 and 0.41 +/- 0.088 h(-1) for 8.4 x 10(-4)M Brij 35 and 7.6 x 10(-4)M Tween 80. The direct-contact fraction was 0.083 and 0.044 for Brij 35 and Tween 80, respectively, under these conditions using exponential model. The toxicity increased with increasing phenanthrene concentration at a fixed surfactant concentration. The toxicity decreased with increasing the surfactant concentration at a fixed phenanthrene concentration due to decreased contact of bacteria with phenanthrene present in the interior of surfactant micelles.     

Growth characteristics of Candida utilis on volatile substrate in a multistage tower fermentor.

The influence of increasing ethanol concentration in the feed on growth and physiological activity of the yeast Candida utlis was studied. The measurements were made at steady states of continuous culture under constant values of dilution rate, temperature, and pH in all stages of the fermentor; Synthetic ethanol was used as the sole source of carbon and energy in the concentration range 10-100 g/liter. The maximum biomass concentration in the effluent and maximum productivity was achieved at 75 g ethanol/liter in the feed. In respect to ethanol losses in the outlet and biomass yield, the optimum ethanol concentration in the input of the growth medium was found to be about 50 g/liter using a four-stage system.     

Bacteroides species: maintenance of laboratory strains.

A medium composed of blood agar base (40 g/liter), yeast extract (5 g/liter), and cysteine hydrochloride (0.05 g/liter), completely filling screw-cap tubes (13 by 100 mm), can keep Bacteroides species alive for at least 10 months without refrigeration.     

Erucic acid production using porcine pancreas lipase: Enhancement by mixed surfactants

Application of mixed surfactants coupled with statistical optimization in lipase catalyzed oil hydrolysis is presented for the first time in this study. Selective hydrolysis of brown mustard oil to erucic acid by porcine pancreas lipase was enhanced by mixed surfactants comprising of an oil-soluble nonionic surfactant (Span 80) and a watersoluble nonionic surfactant (Tween 80). The production of erucic acid was maximized using statistically designed experiments and subsequent analysis of their result by response surface methodology. The most significant variables were enzyme concentration and concentration of Tween 80. Small changes in pH and concentration of Span 80 also produced a significant change in the production of erucic acid. Temperature and speed of agitation were insignificant variables and were fixed at 35oC and 900 rpm, respectively. Under these conditions, the optimal combination of other variables were pH 9.65, 2.13 mg/g enzyme in oil, 9.8 × 10⁻³ M Span 80 (in oil), and 4 × 10⁻³ M Tween 80 (in buffer). These conditions led to formation of 99.69% of the total erucic acid in 1.25 h. Interaction of enzyme concentration with pH significantly affected erucic acid production.     

Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli.

Recombinant Escherichia coli XL1-Blue harboring a high-copy-number plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate synthesis genes could efficiently synthesize poly(3-hydroxybutyrate) (PHB) in a complex medium containing yeast extract and tryptone but not in a defined medium. One of the reasons for the reduced PHB production in a defined medium was thought to be severe filamentation of cells in this medium. By overexpressing an essential cell division protein, FtsZ, in recombinant E. coli producing PHB, filamentation could be suppressed and PHB could be efficiently produced in a defined medium. A high PHB concentration of 149 g/liter, with high productivity of 3.4 g of PHB/liter/h, could be obtained by the pH-stat fed-batch culture of the filamentation-suppressed recombinant E. coli in a defined medium. It was also found that insufficient oxygen supply at a dissolved oxygen concentration (DOC) of 1 to 3% of air saturation during active PHB synthesis phase did not negatively affect PHB production. By growing cells to the concentration of 110 g/liter and then controlling the DOC in the range of 1 to 3% of air saturation, a PHB concentration of 157 g/liter and PHB productivity of 3.2 g of PHB/liter/h were obtained. For the scale-up studies, fed-batch culture was carried out in a 50-liter stirred tank fermentor, in which the DOC decreased to zero when cell concentration reached 50 g/liter. However, a relatively high PHB concentration of 101 g/liter and PHB productivity of 2.8 g of PHB/liter/h could still be obtained, which demonstrated the possibility of industrial production of PHB in a defined medium by employing the filamentation-suppressed recombinant E. coli.     

Fed-batch approach to production of 2,3-butanediol by Klebsiella pneumoniae grown on high substrate concentrations

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol by Klebsiella pneumoniae grown on high sugar concentrations was investigated. When K. pneumoniae was grown under finite air conditions in the presence of added acetic acid, 50 g of D-glucose and D-xylose per liter could be converted to 25 and 27 g of butanediol per liter, respectively. The efficiency of bioconversion decreased with increasing sugar substrate concentrations (up to 200 g/liter). Butanediol production at low sugar substrate concentrations was less efficient when the organism was grown under aerobic conditions; however, final butanediol values were higher for cultures grown on an initial sugar concentration of 150 g/liter, particularly when the inoculum was first acclimatized to high sugar levels. When a double fed-batch approach (daily additions of sugars together with yeast extract) was used under aerobic conditions, up to 88 and 113 g of combined butanediol and acetyl methyl carbinol per liter could be obtained from the utilization of 190 g of D-xylose and 226 g of D-glucose per liter, respectively.     

Fed-batch approach to production of 2,3-butanediol by Klebsiella pneumoniae grown on high substrate concentrations.

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol by Klebsiella pneumoniae grown on high sugar concentrations was investigated. When K. pneumoniae was grown under finite air conditions in the presence of added acetic acid, 50 g of D-glucose and D-xylose per liter could be converted to 25 and 27 g of butanediol per liter, respectively. The efficiency of bioconversion decreased with increasing sugar substrate concentrations (up to 200 g/liter). Butanediol production at low sugar substrate concentrations was less efficient when the organism was grown under aerobic conditions; however, final butanediol values were higher for cultures grown on an initial sugar concentration of 150 g/liter, particularly when the inoculum was first acclimatized to high sugar levels. When a double fed-batch approach (daily additions of sugars together with yeast extract) was used under aerobic conditions, up to 88 and 113 g of combined butanediol and acetyl methyl carbinol per liter could be obtained from the utilization of 190 g of D-xylose and 226 g of D-glucose per liter, respectively.     

Direct ethanol fermentation from lignocellulosic biomass by Antarctic basidiomycetous yeast Mrakia blollopis under a low temperature condition

Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10 °C. Maximally, 48.2 g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2 g/l, 12.5 g/l and 7.2 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1–1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in cold environments.     

Enhancement of enzymatic hydrolysis of cellulose by surfactant

Effects of surfactants on enzymatic saccharification of cellulose have been studied. Nonionic, amphoteric, and cationic surfactants enhanced the saccharification, while anionic surfactant did not. Cationic and anionic surfactants denatured cellulase in their relatively low concentrations, namely, more than 0.008 and 0.001%, respectively. Using nonionic surfactant Tween 20, which is most effective to the enhancement (e.g., the fractional conversion attained by 72 h saccharification of 5 wt % Avicel in the presence of 0.05 wt % Tween 20 is increased by 35%), actions of surfactant have been examined. As the results, it was suggested that Tween 20 plays an important role in the hydrolysis of crystalline cellulose and that Tween 20 disturbs the adsorption of endoglucanase on cellulose, i.e., varies the adsorption balance of endo- and exoglucanase, resulting in enhancing the reaction. The influence of Tween 20 to the saccharification was found to remain in simultaneous saccharification and fermentation of Avicel.     

Rapid ethanol fermentation of cellulose hydrolysate. I. Batch versus continuous systems

Rapid fermentation of bagasse hydrolysate to ethanol under anaerobic conditions by a strain of Saccharomyces cerevisiae has been studied in batch and continuous cultures at pH 4.0 and 30°C temperature with cell recycle. By using a 23.6 g/liter cell concentration, a concentation of 9.7% (w/v)ethanol was developed in a period of 6 hr. The rate of fermentation was found to increase with supplementation of yeast vitamins in the hydrolysate. In continuous culture employing cell recycle and a 0.127 v/v/m air flow rate, a cell mass concentration of 48.5 g/liter has been achieved. The maximum fermentor productivity of ethanol obtained under these conditions was 32.0 g/liter/hr, which is nearly 7.5 times higher than the normal continuous process without cell recycle and air sparging. The ethanol productivity was found to decrease linearly with ethanol concentration. Conversion of glucose in the hydrolysate to ethanol was achieved with a yield of 95 to 97% of theoretical.     

Tween 80 effect on bacteriocin synthesis by Lactococcus lactis subsp. cremoris J46

E. HUOT. C. BARRENA-GONZALEZ AND H. PETITDEMANGE. 1996. Sorbitan polyoxyethylene monooleate (Tween 80) suppressed bacteriocin cell adhesion. Within the range 0–1% (v/v), there was an increase in bacteriocin production in regulated (pH 5.5 or 6.0) batch cultures with increasing Tween 80 concentration. For example, at pH 5.5 and in the presence of 1% Tween 80, bacteriocin production was about fourfold higher than in its absence. However, further increase in Tween 80 concentration did not result in a significant modification of the bacteriocin titre. It was shown that the increase was not linked to an activating effect of the surfactant on preformed enzyme, to an increase of bacteriocin availability or to a sensitization of the target cell, demonstrating that Tween 80 promoted bacteriocin production.     

Flocculant production from kerosene

Cultivation of Corynebacterium hydrocarboclastus, which is capable of synthesizing an extracellular polymer and utilized hydrocarbons, has been reported. Growth studies in shake flasks and fermenters were made to obtain maximum polymer production. Polymer formation was found to be growth associated. The highest level of polymer accumulation was attained after 50–60 hr cultivation in the fermenter and it amounted to approximately 5.5–6 g/liter of fermentation broth. The medium contained initially 2% (v/v) kerosene as a carbon source. The maximum yield obtained corresponds to 37–40% (w/w) of kerosene supplied. At the same time the cell concentration was 10–13 g/liter which represents the yield of 67–87% (w/w). The rate of polymer production in the exponential phase was 0.25 g/liter hr and cell production rate was 0.27 g/liter hr. Sodium nitrate, 0.5%, and yeast extract, 0.3%, (w/w) were the best nigrogen sources for polymer formation. The highest level of polymer produced in broth was 6 g/liter.     

Bio-ethanol Production from Green Onion by Yeast in Repeated Batch

Considered to be the cleanest liquid fuel, bio-ethanol can be a reliable alternative to fossil fuels. It is produced by fermentation of sugar components of plant materials. The common onions are considered to be a favorable source of fermentation products as they have high sugar contents as well as contain various nutrients. This study focused on the effective production of ethanol from Green onion (Allium fistulosum L.) by the yeast “Saccharomyces cerevisiae” in repeated batch. The results showed that the total sugar concentration of onion juice was 68.4 g/l. The maximum rate of productivity, ethanol yield and final bio-ethanol percentage was 7 g/l/h (g ethanol per liter of onion juice per hour), 35 g/l (g ethanol per liter of onion juice) and 90 %, respectively.