Influence of pH on butyrate uptake and solvent fermentation by a mutant strain of Clostridiumacetobutylicum

The effect of pH (between 4.4 and 6.6) on butyrate uptake by the mutant strain of Clostridium acetobutylicum was studied using the fermentation broth from fermentor-2 (solventogenic stage) of a two-fermentor continuous system. Low pH (?1 butyrate, under batch incubation at 30?°C, was not inhibited at pH?>?5.2, however, at pH??1. Batch incubation at relatively higher temperatures (35° and 37?°C) indicated a similar trend i.e., a pH of >5.5 was required for uptake of >8?g l^?1 butyrate. Optimization studies for butyrate uptake by C. acetobutylicum suggested a direct correlation between minimum pH and butyrate concentration or temperature. The role of undissociated butyric acid appears to be critical in regulation of butyrate uptake.     

Improvement of l-lactic acid production by osmotic-tolerant mutant of Lactobacillus casei at high temperature

l-Lactic acid production by Lactobacillus casei was used as a model to study the mechanism of substrate inhibition and the strategy for enhancing l-lactic acid production. It was found that the concentration of cell growth and l-lactate decreased with the increase of glucose concentration and fermentation temperature. To enhance the osmotic stress resistance of the strain at high temperature, a mutant G-03 was screened and selected with 360?g/L glucose at 45°C as the selective criterion. To further increase the cell growth for lactic acid production, 3?g/L of biotin was supplemented to the medium. As a result, l-lactate concentration by the mutant G-03 reached 198.2?g/L (productivity of 5.5?g?L^?1?h^?1) at 41°C in a 7-L fermentor with 210?g/L glucose as carbon source. l-Lactate concentration and productivity of mutant G-03 were 115.2% and 97.8% higher than those of the parent strain, respectively. The strategy for enhancing l-lactic acid production by increasing osmotic stress resistance at high temperature may provide an alternative approach to enhance organic acid production with other strains.     

Xylitol production from a mutant strain of Candida tropicalis

Aims: To characterize the kinetics of growth, sugar uptake and xylitol production in batch and fed‐batch cultures for a xylitol assimilation‐deficient strain of Candida tropicalis isolated via chemical mutagenesis. Methods and Results: Chemical mutagenesis using nitrosoguanidine led to the isolation of the xylitol‐assimilation deficient strain C. tropicalis SS2. Shake‐flask fermentations with this mutant showed a sixfold higher xylitol yield than the parent strain in medium containing 25 g l^?1 glucose and 25 g l^?1 xylose. With 20 g l^?1 glycerol, replacing glucose for cell growth, and various concentrations of xylose, the studies indicated that the mutant strain resulted in xylitol yields from xylose close to theoretical. Under fully aerobic conditions, fed‐batch fermentation with repeated addition of glycerol and xylose resulted in 3·3 g l^?1 h^?1 xylitol volumetric productivity with the final concentration of 220 g l^?1 and overall yield of 0·93 g g^?1 xylitol. Conclusions: The xylitol assimilation‐deficient mutant isolated in this study showed the potential for high xylitol yield and volumetric productivity under aerobic conditions. In the evaluation of glycerol as an alternative low‐cost nonfermentable carbon source, high biomass and xylitol yields under aerobic conditions were achieved; however, the increase in initial xylose concentrations resulted in a reduction in biomass yield based on glycerol consumption. This may be a consequence of the role of an active transport system in the yeast requiring increasing energy for xylose uptake and possible xylitol secretion, with little or no energy available from xylose metabolism. Significance and Impact of the Study: The study confirms the advantage of using a xylitol assimilation‐deficient yeast under aerobic conditions for xylitol production with glycerol as a primary carbon source. It illustrates the potential of using the xylose stream in a biomass‐based bio‐refinery for the production of xylitol with further cost reductions resulting from using glycerol for yeast growth and energy production.     

Enhanced thermotolerance and ethanol tolerance in Saccharomyces cerevisiae mutated by high-energy pulse electron beam and protoplast fusion

To increase thermotolerance and ethanol tolerance in Saccharomyces cerevisiae strain YZ1, the strategies of high-energy pulse electron beam (HEPE) and three rounds of protoplast fusion were explored. The YF31 strain had the characteristics of resistant to high-temperature, high-ethanol tolerance, rapid growth and high yield. The YF31 could grow on plate cultures up to 47?°C, containing 237.5?g?L^?1 of ethanol. In particular, the mutant strain YF31 generated 94.2?±?4.8?g?L^?1 ethanol from 200?g glucose L^?1 at 42?°C, which was 2.48 times the production of the wild strain YZ1. Results demonstrated that the variant phenotypes from the strains screening by HEPE irradiation could be used as parent stock for yeast regeneration and the protoplast fusion technology is sufficiently powerful in combining suitable characteristics in a single strain for ethanol fermentation.     

α-Ketoglutaric acid production from rapeseed oil by Yarrowia lipolytica yeast

The possibility of using rapeseed oil as a carbon source for microbiological production of α-ketoglutaric acid (KGA) has been studied. Acid formation on the selective media has been tested in 26 strains of Yarrowia lipolytica yeast, and the strain Y. lipolytica VKM Y-2412 was selected as a prospective producer of KGA from rapeseed oil. KGA production by the selected strain was studied in dependence on thiamine concentration, medium pH, temperature, aeration, and concentration of oil. Under optimal conditions (thiamine concentration of 0.063 μg?g cells^?1, pH?3.5, 30 °C, high dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and oil concentration in a range from 20 to 60 g?l^?1), Y. lipolytica VKM Y-2412 produced up to 102.5 g?l^?1 of KGA with the mass yield coefficient of 0.95 g?g^?1 and the volumetric KGA productivity (Q _KGA) of 0.8 g?l^?1?h^?1.     

Metabolic,physiological and kinetic aspects of the alcoholic fermentation of whey permeate by Kluyveromyces fragilis NRRL 665 and Kluyveromyces lactis NCYC 571

Optimum growth conditions for the fermentation of non-concentrated whey permeate by Kluyveromyces fragilis NRRL 665 have been defined. Use of 3.75 g yeast extract l^?1, a growth temperature of 38°C and a pH of 4.0 allowed a maximum productivity of 5.23 g ethanol l^?1 h^?1 in continuous culture with a yield 91% of theoretical. Complete batch fermentation of permeate with 100 g lactose l^?1 was possible with a maximum specific growth rate of 0.276 h^?1 without any change in ethanol yield. Fermentation of concentrated permeate resulted, however, in a general decrease of specific substrate consumption rate, demonstrated by the inability to completely convert an initial 90 or 150 g lactose l^?1 in continuous culture, even at dilution rates as low as 0.05 and 0.08 h^?1, respectively. The decrease could be related to substrate inhibition, to an increase in osmotic pressure caused by lactose and salts, and to ethanol inhibition of both alcohol and biomass yield. The decrease in specific productivity could be counterbalanced by use of high cell density cultures, obtained by cell recycle of K. fragilis. Fermentation of a non-concentrated permeáte at a dilution rate of 1 h^?1 resulted in a productivity of 22 g l^?1 h^?1 at 22 g ethanol l^?1. Cell recycle using flocculating Kluyveromyces lactis NCYC 571 was also tested. With this strain a productivity of 9.3 g l^?1 h^?1 at 45 g product l^?1 was attained at a dilution rate of 0.2 h^?1, with an initial lactose concentration of 95 g l^?1.     

Production of l-rhamnulose,a rare sugar,from l-rhamnose using commercial immobilized glucose isomerase

Abstract

A commercial immobilized d-glucose isomerase from Streptomyces murines (Sweetzyme) was used to produce l-rhamnulose from l-rhamnose in a packed-bed reactor. The optimal conditions for l-rhamnulose production from l-rhamnose were determined as pH 8.0, 60?°C, 300?g L^?1 l-rhamnose as a substrate, and 0.6?h^?1 dilution rate. The half-life of the immobilized enzyme at 60?°C was 809?h. Under the optimal conditions, the immobilized enzyme produced an average of 135?g L^?1 l-rhamnulose from 300?g L^?1 l-rhamnose after 16 days at pH 8.0, 60?°C, and 0.6?h^?1 dilution rate, with a productivity of 81?g/L/h and a conversion yield of 45% in a packed-bed reactor.     

Improvement of the multiple-stress tolerance of an ethanologenic <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain by freeze-thaw treatment

An effective, simple, and convenient method to improve yeast’s multiple-stress tolerance, and ethanol production was developed. After an ethanologenic Saccharomyces cerevisiae strain SC521 was treated by nine cycles of freeze-thaw, a mutant FT9-11 strain with higher multiple-stress tolerance was isolated, whose viabilities under acetic acid, ethanol, freeze-thaw, H₂O₂, and heat-shock stresses were, respectively, 23-, 26-, 10- and 7-fold more than the parent strain at an initial value 2 × 10⁷ c.f.u. per ml. Ethanol production of FT9-11 was similar (91.5 g ethanol l⁻¹) to SC521 at 30°C with 200 g glucose l⁻¹, and was better than the parent strain at 37°C (72.5 g ethanol l⁻¹), with 300 (111 g ethanol l⁻¹) or with 400 (85 g ethanol l⁻¹) g glucose l⁻¹.     

Construction of lactose-consuming Saccharomyces cerevisiae for lactose fermentation into ethanol fuel

Two lactose-consuming diploid Saccharomyces cerevisiae strains, AY-51024A and AY-51024M, were constructed by expressing the LAC4 and LAC12 genes of Kluyveromyces marxianus in the host strain AY-5. In AY-51024A, both genes were targeted to the ATH1 and NTH1 gene-encoding regions to abolish the activity of acid/neutral trehalase. In AY-51024M, both genes were respectively integrated into the MIG1 and NTH1 gene-encoding regions to relieve glucose repression. Physiologic studies of the two transformants under anaerobic cultivations in glucose and galactose media indicated that the expression of both LAC genes did not physiologically burden the cells, except for AY-51024A in glucose medium. Galactose consumption was initiated at higher glucose concentrations in the MIG1 deletion strain AY-51024M than in the corresponding wild-type strain and AY-51024A, wherein galactose was consumed until glucose was completely depleted in the mixture. In lactose medium, the Sp. growth rates of AY-51024A and AY-51024M under anaerobic shake-flasks were 0.025 and 0.067 h^?1, respectively. The specific lactose uptake rate and ethanol production of AY-51024M were 2.50 g lactose g CDW^?1 h^?1 and 23.4 g l^?1, respectively, whereas those of AY-51024A were 0.98 g lactose g CDW^?1 h^?1 and 24.3 g lactose g CDW^?1 h^?1, respectively. In concentrated cheese whey powder solutions, AY-51024M produced 63.3 g l^?1 ethanol from approximately 150 g l^?1 initial lactose in 120 h, conversely, AY-51024A consumed 63.7 % of the initial lactose and produced 35.9 g l^?1 ethanol. Therefore, relieving glucose repression is an effective strategy for constructing lactose-consuming S. cerevisiae.     

Microbial production of 2,3-butanediol by a newly-isolated strain of Serratia marcescens

A newly-isolated strain of Serratia marcescens, G12, was characterized for 2,3-butanediol (2,3-BD) production. In shake-flask and batch fermentations, 2,3-BD reached 48.5 and 51 g l^?1, respectively. Low amounts of (~8 g l^?1) of acetoin were also formed. In fed-batch fermentations, strain G12 produced 72.8 g 2,3-BD l^?1 with glucose initially at 130 g l^?1. When aeration rate was increased to 2.5 vvm for the fermentation process, 2,3-BD reached 87.8 g l^?1 and the highest productivity was 1.6 g l^?1 h^?1. Acetoin was at 6.2 g l^?1. G12 therefore may be a suitable candidate strain for large-scale production of 2,3-BD.     

Bacterial dynamics in two high-arctic lakes

The heterotrophic planktonic bacteria in two high-arctic lakes were studied by direct microscope count and the enzymatic uptake of ¹⁴C labelled glucose which generally conformed to Michaelis-Menten kinetics. Bacterial numbers and activity in oligo-trophic Char Lake ranged from 0.1 to 2.0×10^?3 bacteria/l and a maximum uptake velocity (V_max) of 1.8 × 10^?3μg glucose l^? h^?1. Nearby Meretta Lake received waste water from the Department of Transport Base at Resolute and this eutrophication was reflected in higher bacterial numbers of 2-80 × 10⁸/1 and Kmax of 0.1 × 10^?1-7.5 × 10^?1 fig glucose l^?1 h^?1 The Kmax per cell in Char Lake was 3 × 10^?11μg glucose l^?1 h^?1 and changed little between the period of solid ice cover in May and ice-free conditions in August. Bacterial cycles could not be related to phytoplankton cycles in either lake. Comparison of kinetic data from several lakes suggests a relationship between the bacterial uptake rate of glucose and phytoplankton production. Both bacterial numbers and activity in Char Lake may be very close to the minima to be expected in undisturbed freshwater environments.     

Inorganic carbon uptake by phytoplankton in Vineyard Sound,Massachusetts. II. Comparative primary productivity and nutritional status of winter and summer assemblages

Using time-course, natural-light incubations, we assessed the rate of carbon uptake at a range of light intensities, the effect of supplemental additions of nitrogen (as NH₄⁺ or urea) on light and dark carbon uptake, and the rates of uptake of NH₄⁺ and urea by phytoplankton from Vineyard Sound, Massachusetts from February through August 1982. During the winter, photoinhibition was severe, becoming manifested shortly after the start of an incubation, whereas during the summer, there was little to no evidence of photoinhibition during the first several hours after the start of an incubation. At light levels which were neither photoinhibiting nor light limiting, rates of carbon uptake normalized per liter were high and approximately equal during winter and summer (22–23 μg C·l^?1 · h^?1), and low during spring (<10 μgC·l^?1· h^?1). In contrast, on a chlorophyll a basis, rates of carbon fixation were as high during spring (15–20μg C·μg Chl a^?1·h^?1), when concentrations of chlorophyll a were at the yearly minimum (<0.5 μg · l^?1) as during the summer, when chlorophyll a concentrations were substantially higher (0.8–1.3 μg · l^?1). Highest rates of NH₄⁺ and urea uptake were observed during summer, and at no time of the year was there evidence for severe nitrogen deficiency, although moderate nitrogen nutritional stress was apparent during the summer months.     

Production of a novel compound, 10,12-dihydroxystearic acid from ricinoleic acid by an oleate hydratase from Lysinibacillus fusiformis

A recombinant oleate hydratase from Lysinibacillus fusiformis converted ricinoleic acid to a product, whose chemical structure was identified as the novel compound 10,12-dihydroxystearic acid by gas chromatograph/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance analysis. The reaction conditions for the production of 10,12-dihydroxystearic acid were optimized as follows: pH?6.5, 30 °C, 15 g?l^?1 ricinoleic acid, 9 mg?ml^?1 of enzyme, and 4 % (v/v) methanol. Under the optimized conditions, the enzyme produced 13.5 g?l^?1 10,12-dihydroxystearic acid without detectable byproducts in 3 h, with a conversion of substrate to product of 90 % (w/w) and a productivity of 4.5 g?l^?1?h^?1. The emulsifying activity of 10,12-dihydroxystearic acid was higher than that of oleic acid, ricinoleic acid, stearic acid, and 10-hydroxystearic acid, indicating that 10,12-dihydroxystearic acid can be used as a biosurfactant.     

Poly(β-hydroxybutyric acid) thermoplastic production by Alcaligenes latus: Behavior of fed-batch cultures

Fed-batch culture of Alcaligenes latus, ATCC 29713, was investigated for producing the intracellular bioplastic poly(β–hydroxybutyric acid), PHB. Constant rate feeding, exponentially increasing feeding rate, and pH-stat fed batch methods were evaluated. pH-stat fed batch culture reduced or delayed accumulation of the substrate in the broth and led to significantly enhanced PHB productivity relative to the other modes of feeding. Presence of excessive substrate appeared to inhibit PHB synthesis, but not the production of cells. In fed-batch culture, the maximum specific growth rate (0.265?h^?1) greatly exceeded the value (0.075?h^?1) previously observed in batch culture of the same strain. Similarly, the maximum PHB production rate (up to 1.15?g?·?l^?1?·?h^?1) was nearly 8-fold greater than values observed in batch operations. Fed-batch operation was clearly superior to batch fermentation for producing PHB. A low growth rate was not a prerequisite for PHB accumulation, but a reduced or delayed accumulation of substrate appeared to enhance PHB accumulation. Under the best conditions, PHB constituted up to 63% of dry cell mass after 12?h of culture. The average biomass yield coefficient on sucrose was about 0.35, or a little less than in batch fermentations. The highest PHB concentrations attained were about 18?g?·?l^?1.     

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH₂PO₄ in 0.2 mM CaSCO₄ at the average rate of 34.3 ±3.2 μg P_i g^?1 (fr. m.) h^?1,i.e. 0.35± 0.02 μmol P_i g^?1 (fr. m.) h^?1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO₄. This increase is completely blocked by the presence of 10 μg ml^?1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following K_m and V_max values were found: K_m[mM] : 0.37 - 3.82 - 27.67 V_max[μg P_i g^?1 (fr. m.) h^?1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH₂PO₄, while uptake from 5 and 10 mM KH₂PO₄ was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10^?3 M NEM by 81.6%, 10⁴ M NEM by 42% and 10^?4 M PCMB by 42%.     

Influence of the nutritional conditions on haloalcohol dehalogenase HheC production by recombinant Escherichia coli P84A/MC1061

In this research, we first determined the three most significant nutrient factors affecting haloalcohol dehalogenase HheC production by Escherichia coli P84A/MC1061. These were glycerol, yeast extract, and ammonium sulfate. The steepest ascent method was then applied to obtain the optimal design intervals of the three factors. An application of center composite design was used, and the ingredients of the optimized medium were 1.8 g l^?1 glycerol, 48 g l^?1 yeast extract, 2.2 g l^?1 ammonium sulfate, 5 g l^?1 compound phosphate, 1 g l^?1 magnesium sulfate, and 1.19?×?10^?5?g l^?1 ferric sulfate. The enzyme activity reached 109,365 U ml^?1 under the most favorable conditions, which is a 277.7 % increase compared with the control group. Our study of cellular respiration parameters (oxygen uptake rate and carbon dioxide emission rate) revealed that the metabolic activity of the strain was strongly promoted under these optimal nutrient conditions and that yeast extract had a positive effect on respiratory intensity and the expression levels of HheC.     

Large-scale bioreactor production of the herbicide-degrading Aminobacter sp. strain MSH1

The Aminobacter sp. strain MSH1 has potential for pesticide bioremediation because it degrades the herbicide metabolite 2,6-dichlorobenzamide (BAM). Production of the BAM-degrading bacterium using aerobic bioreactor fermentation was investigated. A mineral salt medium limited for carbon and with an element composition similar to the strain was generated. The optimal pH and temperature for strain growth were determined using shaker flasks and verified in bioreactors. Glucose, fructose, and glycerol were suitable carbon sources for MSH1 (μ?=?0.1 h^?1); slower growth was observed on succinate and acetic acid (μ?=?0.01 h^?1). Standard conditions for growth of the MSH1 strain were defined at pH 7 and 25 °C, with glucose as the carbon source. In bioreactors (1 and 5 L), the specific growth rate of MSH1 increased from μ?=?0.1 h^?1 on traditional mineral salt medium to μ?=?0.18 h^?1 on the optimized mineral salt medium. The biomass yield under standard conditions was 0.47 g dry weight biomass/g glucose consumed. An investigation of the catabolic capacity of MSH1 cells harvested in exponential and stationary growth phases showed a degradation activity per cell of about 3?×?10^?9 μg BAM h^?1. Thus, fast, efficient, large-scale production of herbicide-degrading Aminobacter was possible, bringing the use of this bacterium in bioaugmentation field remediation closer to reality.     

Characterization of a recombinant l-rhamnose isomerase from Dictyoglomus turgidum and its application for l-rhamnulose production

A putative recombinant enzyme from Dictyoglomus turgidum was characterized and immobilized on Duolite A568 beads. The native enzyme was a 46 kDa tetramer. Its activity was highest for l-rhamnose, indicating that it is an l-rhamnose isomerase. The maximum activities of both the free and immobilized enzymes for l-rhamnose isomerization were at pH 8.0 and 75 °C in the presence of Mn²⁺. Under these conditions, the half-lives of the free and immobilized enzymes were 28 and 112 h, respectively. In a packed-bed bioreactor, the immobilized enzyme produced an average of 130 g l-rhamnulose l^?1 from 300 g l-rhamnose l^?1 after 240 h at pH 8.0, 70 °C, and 0.6 h^?1, with a productivity of 78 g l^?1 h^?1 and a conversion yield of 43 %. To the best of our knowledge, this is the first report describing the enzymatic production of l-rhamnulose.     

Improved ethanol production of a newly isolated thermotolerant Saccharomyces cerevisiae strain after high-energy-pulse-electron beam

Aims: To isolate thermotolerant Saccharomyces cerevisiae with high‐energy‐pulse‐electron (HEPE) beam, to optimize the mutation strain fermentation conditions for ethanol production and to conduct a preliminary investigation into the thermotolerant mechanisms. Methods and Results: After HEPE beam radiation, the thermotolerant S. cerevisiae strain Y43 was obtained at 45°C. Moreover, the fermentation conditions of mutant Y43 were optimized by L3³ orthogonal experiment. The optimal glucose content and initial pH for fermentation were 20% g l^?1 and 4·5, respectively; peptone content was the most neglected important factor. Under this condition, ethanol production of Y43 was 83·1 g l^?1 after fermentation for 48 h at 43°C, and ethanol yield was 0·42 g g^?1, which was about 81·5% of the theoretical yield. The results also showed that the trehalose content and the expression of the genes MSN2, SSA3 and TPS1 in Y43 were higher than those in the original strain (YE0) under the same stress conditions. Conclusions: A genetically stable mutant strain with high ethanol yield under heat stress was obtained using HEPE. This mutant may be a suitable candidate for the industrial‐scale ethanol production. Significance and Impact of the Study: High‐energy‐pulse‐electron radiation is a new efficient technology in breeding micro‐organisms. The mutant obtained in this work has the advantages in industrial ethanol production under thermostress.     

Expression of exoinulinase genes in Saccharomyces cerevisiae to improve ethanol production from inulin sources

To improve inulin utilization and ethanol fermentation, exoinulinase genes from the yeast Kluyveromyces marxianus and the recently identified yeast, Candida kutaonensis, were expressed in Saccharomyces cerevisiae. S. cerevisiae harboring the exoinulinase gene from C. kutaonensis gave higher ethanol yield and productivity from both inulin (0.38 vs. 0.34 g/g and 1.35 vs. 1.22 g l^?1 h^?1) and Jerusalem artichoke tuber flour (0.47 vs. 0.46 g/g and 1.62 vs. 1.54 g l^?1 h^?1) compared with the strain expressing the exoinulinase gene from K. marxianus. Thus, the exoinulinase gene from C. kutaonensis is advantageous for engineering S. cerevisiae to improve ethanol fermentation from inulin sources.