Selective utilization of fructose to glucose by Candida magnoliae, an erythritol producer

Candida magnoliae isolated from honeycomb is an industrially important yeast with high erythritol-producing ability. Erythritol has been used as functional sugar substitute for various foods. In order to analyze the physiological properties of C. magnoliae, a study on sugar utilization pattern was carried out. The fermentation kinetics of glucose and fructose revealed that C. magnoliae has the discrepancy in glucose and fructose utilization when it produces erythritol. In contrast to most yeasts, C. magnoliae showed preference for fructose to glucose as a carbon source, deserving the designation of fructophilic yeast. Such a peculiar pattern of sugar utilization in C. magnoliae seems to be related to the evolutionary environment.     

Evaluation of Cashew Apple Juice for the Production of Fuel Ethanol

A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentrations. Maximal ethanol, cell, and glycerol concentrations were obtained when 103.1 g L⁻¹ of initial sugar concentration was used. Cell yield (Y _X/S) was calculated as 0.24 (g microorganism)/(g glucose + fructose) using cashew apple juice medium with 41.3 g L⁻¹ of initial sugar concentration. Glucose was exhausted first, followed by fructose. Furthermore, the initial concentration of sugars did not influence ethanol selectivity. These results indicate that cashew apple juice is a suitable substrate for yeast growth and ethanol production.     

Optimum conditions for transformed Panax ginseng hairy roots in flask culture

Panax ginseng hairy roots were transformed by Agrobacterium rhizogenes KTCT 2744. They showed an active branching pattern and fast growth in hormone-free medium, and good growth at 23°C, pH 5.8, 1/2 MS medium, and 3% sucrose. Sucrose provided the highest growth among seven carbon sources tested. Six complex media were also tested. In the combined sugar study, hairy roots grew better on sucrose without glucose or fructose than with glucose or fructose. In the 1/2 MS basal medium, 30 mM in nitrogen and 0.62 mM phosphate salt concentration was the optimum. The growth ratio was maximal at an inoculum size of 0.4% (w/v). Crude saponin and polysaccharide levels were also measured.     

Continuous fermentation studies with xylose-utilizing recombinant Zymomonas mobilis

This study examined the continuous cofermentation performance characteristics of a dilute-acid “prehydrolysate-adapted” recombinant Zymomonas 39676:pZB4L and builds on the pH-stat batch fermentations with this recombinant that we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the coferm entation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5c/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because of competition for a common sugar transporter that exhibits a higher affinity for glucose, utilization of glucose was complete whereas xylose was always present in the chemostat effluent. The ethanol yield, based on sugar used, was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilization for determining the maximum dilution rate (D _max), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted D _max from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), D _max was 0.07/h. By comparison to a similar investigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose, we observed that at pH 5.0, the D _max was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium with 3% xylose and 1.8% glucose at pH 5.75, the D _max for the adapted recombinant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L·h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid did not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sugar/(g of cell·h).     

GpdA-promoter-controlled production of glucose oxidase by recombinant aspergillus niger using nonglucose carbon sources

The gpdA-promoter-controlled exocellular production of glucose oxidase (GOD) by recombinant Aspergillus niger NRRL-3 (GOD3-18) during growth on glucose and nonglucose carbon sources was investigated. Screening of various carbon substrates in shake-flask cultures revealed that exocellular GOD activities were not only obtained on glucose but also during growth on mannose, fructose, and xylose. The performance of A. niger NRRL-3 (GOD3-18) using glucose, fructose, or xylose as carbon substrate was compared in more detail in bioreactor cultures. These studies revealed that gpdA-promoter-controlled GOD synthesis was strictly coupled to cell growth. The gpdA-promoter was most active during growth on glucose. However, the unfavorable rapid GOD-catalyzed transformation of glucose into gluconic acid, a carbon source not supporting further cell growth and GOD production, resulted in low biomass yields and, therefore, reduced the advantageous properties of glucose. The total (endo- and exocellular) specific GOD activities were lowest when growth occurred on fructose (only a third of the activity that was obtained on glucose), whereas utilization of xylose resulted in total specific GOD activities nearly as high as reached during growth on glucose. Also, the portion of GOD excreted into the culture fluid reached similar high levels (≅ 90%) by using either glucose or xylose as substrate, whereas growth on fructose resulted in a more pelleted morphology with more than half the total GOD activity retained in the fungal biomass. Finally, growth on xylose resulted in the highest biomass yield and, consequently, the highest total volumetric GOD activity. These results show that xylose is the most favorable carbon substrate for gpdA-promoter-controlled production of exocellular GOD.     

Response Surface Optimization of the Critical Medium Components for Pullulan Production by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> FB-1

Culture conditions for pullulan production by Aureobasidium pullulans were optimized using response surface methodology at shake flask level without pH control. In the present investigation, a five-level with five-factor central composite rotatable design of experiments was employed to optimize the levels of five factors significantly affecting the pullulan production, biomass production, and sugar utilization in submerged cultivation. The selected factors included concentration of sucrose, ammonium sulphate, yeast extract, dipotassium hydrogen phosphate, and sodium chloride. Using this methodology, the optimal values for concentration of sucrose, ammonium sulphate, yeast extract, dipotassium hydrogen phosphate, and sodium chloride were 5.31%, 0.11%, 0.07%, 0.05%, and 0.15% (w/v), respectively. This optimized medium has projected a theoretically production of pullulan of 4.44%, biomass yield of 1.03%, and sugar utilization of 97.12%. The multiple correlation coefficient ‘R’ was 0.9976, 0.9761 and 0.9919 for pullulan production, biomass production, and sugar utilization, respectively. The value of R being very close to one justifies an excellent correlation between the predicted and the experimental data.     

Fermentation of xylose into acetic acid by Clostridium thermoaceticum

For optimum fermentation, fermenting xylose into acetic acid by Clostridium thermoaceticum (ATCC 49707) requires adaptation of the strain to xylose medium. Exposed to a mixture of glucose and xylose, it preferentially consumesxylose over glucose. The initial concentration of xylose in the medium affects the final concentration and the yield of acetic acid. Batch fermentation of 20 g/L of xylose with 5g/L of yeast extract as the nitrogen source results in a maximum acetate concentration of 15.2 g/L and yield of 0.76 g of acid/g of xylose. Corn steep liquor (CLS) is a good substitute for yeast extract and results in similar fermentation profiles. The organism consumes fructose, xylose, and glucose from a mixture of sugars in batch fermentation. Arabinose, mannose, and galactose are consumed only slightly. This organism loses viability on fed-batch operation, even with supplementation of all the required nutrients. In fed-batch fermentation with CSL supplementation, d-xylulose (an intermediate in the xylose metabolic pathway) accumulates in large quantities.     

Production of ethanol from cellulosic biomass hydrolysates using genetically engineered saccharomyces yeast capable of cofermenting glucose and xylose

Recent studies have proven ethanol to be the idael liquid fuel for transportation, and renewable ligno cellulosic materials to be the attractive feed stocks for ethanol fuel production by fermentation. The major fermentable sugars from hydrolysis of most cellulosic biomass are D-glucose and D-xylose. The naturally occurring Saccharomyces yeasts that are used by industry to produce ethanol from starches and cane sugar cannot metabolize xylose. Our group at Purdue University succeded in developing genetically engineered Saccharomyces yeasts capable of effectively cofermenting glucose and xylose to ethanol, which was accomplished by cloning three xylose-metabolizing genes into the yeast. In this study, we demonstrated that our stable recombinant Sacharomyces yeast, 424A (LNH-ST), which contains the cloned xylose-metabolizing genes stably integrated into the yeast chromosome in high copy numbers, can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.     

Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis

The commercial production of mannitol involves high-pressure hydrogenation of fructose using a nickel catalyst, a costly process. Mannitol can be produced through fermentation by microorganisms. Currently, a few Lactobacillus strains are used to develop an efficient process for mannitol bioproduction; most of the strains produce mannitol from fructose with other products. An approach toward improving this process would be to genetically engineer Lactobacillus strains to increase fructose-to-mannitol conversion with decreased production of other products. We cloned the gene mtlK encoding mannitol-2-dehydrogenase (EC 1.1.1.67) that catalyzes the conversion of fructose into mannitol from Lactobacillus brevis using genomic polymerase chain reaction. The mtlK clone contains 1328 bp of DNA sequence including a 1002-bp open reading frame that consisted of 333 amino acids with a predicted molecular mass of about 36 kDa. The functional mannitol-2-dehydrogenase was produced by overexpressing mtlK via pRSETa vector in Escherichia coli BL21pLysS on isopropyl-β-d-thiogalactopyranoside induction. The fusion protein is able to catalyze the reduction of fructose to mannitol at pH 5.35. Similar rates of catalytic reduction were observed using either the NADH or NADPH as cofactor under in vitro assay conditions. Genetically engineered Lactobacillus plantarum TF103 carrying the mtlK gene of L. brevis indicated increased mannitol production from glucose. The evaluation of mixed sugar fermentation and mannitol production by this strain is in progress. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the names by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Hexavalent Molybdenum Reduction to Molybdenum Blue by <Emphasis Type="Italic">S. Marcescens</Emphasis> Strain Dr. Y6

A molybdate-reducing bacterium has been locally isolated. The bacterium reduces molybdate or Mo⁶⁺ to molybdenum blue (molybdate oxidation states of between 5+ and 6+). Different carbon sources such as acetate, formate, glycerol, citric acid, lactose, fructose, glucose, mannitol, tartarate, maltose, sucrose, and starch were used at an initial concentration of 0.2% (w/v) in low phosphate media to study their effect on the molybdate reduction efficiency of bacterium. All of the carbon sources supported cellular growth, but only sucrose, maltose, glucose, and glycerol (in decreasing order) supported molybdate reduction after 24 h of incubation. Optimum concentration of sucrose for molybdate reduction is 1.0% (w/v) after 24 h of static incubation. Ammonium sulfate, ammonium chloride, valine, OH-proline, glutamic acid, and alanine (in the order of decreasing efficiency) supported molybdate reduction with ammonium sulfate giving the highest amount of molybdenum blue after 24 h of incubation at 0.3% (w/v). The optimum molybdate concentration that supports molybdate reduction is between 15 and 25 mM. Molybdate reduction is optimum at 35 °C. Phosphate at concentrations higher than 5 mM strongly inhibits molybdate reduction. The molybdenum blue produced from cellular reduction exhibits a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. The isolate was tentatively identified as Serratia marcescens Strain Dr.Y6 based on carbon utilization profiles using Biolog GN plates and partial 16s rDNA molecular phylogeny.     

Preparation and properties of glucose isomerase immobilized on Indion 48-R

Partially purified glucose isomerase fromStreptomyces thermonitrificans when coupled to glutaraldehyde-activated Indion 48-R, retained 30–40% activity of the soluble enzyme. However, an approximately twofold increase in the activity could be achieved by binding the enzyme in the presence of glucose. Binding the enzyme to matrices presaturated with either glucose or fructose and influence of lysine modification on the activity of the soluble enzyme revealed that the comparatively low activity observed in case of the enzyme bound in the absence of substrate is the result of the nonspecific binding of either substrate or product to the matrix. Immobilization did not affect the pH and temperature optima of the enzyme, but it lowered the temperature stability. Immobilization resulted in a marginal increase in theK _m and a threefold decrease in theV _max . Substrate concentrations as high as 36% glucose could be converted to 18.5% fructose in 5 h, at pH 7.0 and 70‡C. The bound enzyme, however, showed inferior stability to repeated use and lost approx 40% of its initial activity after five cycles of use. Indion 48-R bound glucose isomerase could be stored, in wet state, for 30 d without any apparent loss in its initial activity.     

Butanol production by Clostridium beijerinckii BA101 in an immobilized cell biofilm reactor

Acetone butanol ethanol was produced in a continuous immobilized cell (biofilm) plug-flow reactor inoculated with Clostridium beijerinckii BA101. To achieve high reactor productivity, C. beijerinckii BA101 cells were immobilized by adsorption onto clay brick. The continuous plug-flow reactor offers high productivities owing to reduced butanol inhibition and increased cell concentration. Although high productivity was achieved, it was at the expense of low sugar utilization (30.3%). To increase sugar utilization, the reactor effluent was recycled. However, this approach is complicated by butanol toxicity. The effluent was recycled after removal of butanol by pervaporation to reduce butanol toxicity in the reactor. Recycling of butanolfree effluent resulted in a sugar utilization of 100.7% in addition to high productivity of 10.2g/(L·h) at a dilution rate of 1.5 h⁻¹. A dilution rate of 2.0h⁻¹ resulted in a reactor productivity of 16.2g/(L·h) and sugar utilization of 101.4%. It is anticipated that this reactor-recovery system would be economical for butanol production when using C. beijerinckii BA101.     

Xanthan gum production from cassava bagasse hydrolysate with Xanthomonas campestris using alternative sources of nitrogen

Cassava bagasse was hydrolyzed using HCl and the hydrolysate was used for the production of xanthan gum using a bacterial culture of Xanthomonas campestris. Cassava bagasse hydrolysate with an initial concentration of approx 20 g of glucose/L proved to be the best substrate concentration for xanthan gum production. Among the organic and inorganic nitrogen sources tested to supplement the medium—urea, yeast extract, peptone, potassium nitrate, and ammonium sulfate—potassium nitrate was most suitable. Ammonium sulfate was the least effective for xanthan gum production, and it affected sugar utilization by the bacterial culture. In media with an initial sugar concentration of 48.6 and 40.4 g/L, at the end of fermentation about 30 g/L of sugars was unused. Maximum xanthan gum (about 14 g/L) was produced when fermentation was carried out with a medium containing 19.8 g/L of initial reducing sugars supplemented with potassium nitrate and fermented for 72 h, and it remained almost the same until the end of fermentation (i.e., 96 h).     

Characterization of a high-productivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/xylose mixtures

The fermentation characteristics of a recombinant strain of Zymomonas mobilis ZM4(pZB5) capable of converting both glucose and xylose to ethanol have been further investigated. Previous studies have shown that the strain ZM4(pZB5) was capable of converting a mixture o 65 g/L of glucose and 65 g/L of xylose to 62 g/L of ethanol in 48 h with an overall yield of 0.46 g/g. Higher sugar concentrations (e.g., 75/75 g/L) resulted in incomplete xylose utilization (80 h). In the present study, further kinetic evaluations at high sugar levels are reported. Acetate inhibition studies and evaluation of temperature and pH effects indicated increased maximum specific uptake rates of glucose and xylose under stressed conditions with increased metabolic uncoupling. A high-productivity system was developed that involved a membrane bioreactor with cell recycling. At sugar concentrations of approx 50/50 g/L of glucose/xylose, an ethanol concentration of 50 g/L, an ethanol productivity of approx 5 g/(L·h), and a yield (Y _p/s) of 0.50 g/g were achieved. Decreases in cell viability were found in this system after attainment of an initial steady state (40–60 h); a slow bleed of concentrated cells may be required to overcome this problem.     

Carbon stable isotope ratio of phloem sugars in mature pine trees throughout the growing season: comparison of two extraction methods

The study presents a comparison of two phloem sugar extraction methods. The amount of phloem sugar extracted and the carbon isotope composition (δ¹³C) of the total extracts and of the main phloem compounds separated by high‐performance liquid chromatography (sucrose, glucose, fructose and pinitol) are compared. These two phloem sap extraction methods are exudation in distilled water and a new method using centrifugation, which avoids the addition of any solvent. We applied both extraction methods on phloem discs sampled from 38‐year‐old Pinus pinaster trees in south‐western France throughout the period from June 2007 to December 2008 on different time‐scales: hourly, daily and monthly. We found that the centrifugation method systematically extracted ca. 50% less compounds from the phloem discs than the exudation method. In addition, the two extraction methods provided similar δ¹³C values of the total extracts, but the values obtained by the exudation method were 0.6‰ more negative than those calculated from the mass balance using the individual constituents. Over the growing season, both extraction methods exhibited lower total sugar content and more ¹³C‐enriched phloem sap in summer compared with winter values. These findings suggest that both extraction methods can be applied to study the carbon isotope composition of phloem sap, and the centrifugation method has the advantage that no solvent has to be added. The exudation method, however, is more appropriate for the quantification of the amounts of phloem sugars. Copyright © 2009 John Wiley & Sons, Ltd.     

Growth ofschizosaccharomyces pombe on glucose-malte mixtures in continuous cell-recycle cultures kinetics of substrate utilization

The aerobic growth ofSchizosaccharomyces pombe on mixtures of glucose and malate was investigated during continuous high cell density cultures with partial cell-recycle using a membrane bioreactor. Determination of the specific metabolic rates relative to substrates and products allowed the capacity of the yeast to metabolize malic acid under both oxidative metabolism (carbon limited cultures) and oxidofermentative metabolism (carbon sufficient cultures) situations to be characterized. Under carbon limiting conditions, the specific rate of malate utilization was dependent on the residual concentration and a limit for a purely oxidative breakdown without ethanol formation was observed for a characteristic ratio between the rates of substrate consumption qm/qg of 1.63 g.g^-1. In addition, the mass balance analysis revealed the incorporation of malic acid into biomass. In carbon excess environments, the specific rate of malate utilization was dependent on both the residual malate and the specific rate of glucose consumption indicating that in addition to its conversion into ethanol malate can be respiratively metabolized for qm/qg ratios higher than 0.4 g.g^-1.     

Fermentation kinetics of ethanol production from glucose and xylose by recombinant Saccharomyces 1400(pLNH33)

Fermentation kinetics of ethanol production from glucose, xylose, and their mixtures using a recombinant Saccharomyces 1400 (pLNH33) are reported. Single-substrate kinetics indicate that the specific growth rate of the yeast and the specific ethanol productivity on glucose as the substrate was greater than on xylose as a substrate. Ethanol yields from glucose and xylose fermentation were typically 95 and 80% of the theoretical yield, respectively. The effect of ethanol inhibition is more pronounced for xylose fermentation than for glucose fermentation. Studies on glucose-xylose mixtures indicate that the recombinant yeast co-ferments glucose and xylose. Fermentation of a 52.8 g/L glucose and 56.3 g/L xylose mixture gave an ethanol concentration of 47.9 g/L after 36 h. Based on a theoretical yield of 0.51 g ethanol/g sugars, the ethanol yield from this experiment (for data up to 24 h) was calculated to be 0.46 g ethanol/g sugar or 90% of the theoretical yield. The specific growth rate of the yeast on glucose-xylose mixtures was found to lie between the specific growth rate on glucose and the specific growth rate on xylose. Kinetic studies were used to develop a fermentation model incorporating the effects of substrate inhibition, product inhibition, and inoculum size. Good agreements were obtained between model predictions and experimental data from batch fermentation of glucose, xylose, and their mixtures.     

Continuous ethanol production from nonsterilized carob pod extract by immobilizedSaccharomyces cerevisiae on mineral kissiris using a two-reactor system

The continuous production of ethanol from nonsterilized carob pod extract by immobilizedSaccharomyces cerevisiae on mineral kissiris using one- and two-reactor systems has been investigated. A maximum ethanol productivity of 9.6 g/L/h was obtained at an initial sugar concentration of 200 g/L and D = 0.4 h^-1 with 68% of theoretical yield and 34% of sugar utilization using the one-reactor system. AtS ₀ = 200 g/L, D = 0.05 h^-1, 83% of theoretical yield, and 64% of sugar utilization, an ethanol productivity of 2.6 g/L/h was achieved. In the tworeactor system, a maximum ethanol productivity of 11.4 g/L/h was obtained at S₀ = 200 g/L and D = 0.4 h^-1 with 68.5% of theoretical yield and 41.5% of sugar utilization. The two-reactor system was operated at a constant dilution rate of 0.3 h^-1 for 60 d without loss of the original immobilized yeast activity. In this case, the average ethanol productivity, ethanol yield (% of theoretical), and sugar utilization were 10.7 g/L/h, 71.5%, and 48%, respectively.     

Bioemulsifier production in batch culture using glucose as carbon source by Candida lipolytica

The yeast Candida lipolytica IA 1055 produced an inducible extracellular emulsification activity while utilizing glucose at different concentrations as carbon source during batch fermentation at 27°C. In all glucose concentrations studied, maximum production of emulsification activity was detected in the stationary phase of growth, after pH reached minimal values. The bioemulsifier isolated was a complex biopolymer constituting proteins, carbohydrates, and lipids. The results obtained in this work show that the biosynthesis of a bioemulsifier is not simply a prerequisite for the degradation of extracellular hydrocarbon.