Sources and methods of manufacturing xanthan by fermentation of various carbon sources

Xanthan gum, an anionic polysaccharide with an exceptionally high molecular weight, is produced by the bacterium Xanthomonas sp. It is a versatile compound that has been utilized in various industries for decades. Xanthan gum was the second exopolysaccharide to be commercially produced, following dextran. In 1969, the US Food and Drug Administration (FDA) approved xanthan gum for use in the food and pharmaceutical industries. The food industry values xanthan gum for its exceptional rheological properties, which make it a popular thickening agent in many products. Meanwhile, the cosmetics industry capitalizes on xanthan gum's ability to form stable emulsions. The industrial production process of xanthan gum involves fermenting Xanthomonas in a medium that contains glucose, sucrose, starch, etc. as a substrate and other necessary nutrients to facilitate growth. This is achieved through batch fermentation under optimal conditions. However, the increasing costs of glucose in recent years have made the production of xanthan economically unviable. Therefore, many researchers have investigated alternative, cost-effective substrates for xanthan production, using various modified and unmodified raw materials. The objective of this analysis is to investigate how utilizing different raw materials can improve the cost-efficient production of xanthan gum.     

Influence of pH and malate-glucose ratio on the growth kinetics of Leuconostoc oenos

Summary Growth, substrate utilization and product formation were studied in batch cultures of a Leuconostoc oenos strain. The effect of various culture conditions, i.e. pH-control at different values and various initial concentrations of malate and glucose, on growth and metabolism were investigated. Addition of malate resulted in a marked stimulation of growth, with only a slight increase in final biomass but a high conversion yield of glucose. Under pH control this stimulation was much greater than could be accounted for from changes in pH profile resulting from malate utilization. The specific rate of malate utilization was maximal at pH 4.0 whereas the specific rate of glucose consumption was highest at pH 5.5. During co-metabolism of malic acid and glucose, substrate utilization and product formation agreed with the stoichiometric relationships of the malo-lactic reaction and the heterolactic fermentation of glucose. Offsprint requests to: A. Pareilleux     

Rapid ethanol fermentation of cellulose hydrolysate. II. Product and substrate inhibition and optimization of fermentor design

High concentrations of both ethanol and sugar in the fermentation broth inhibit the growth of yeast cells and the rate of product formation. Inhibitory effects of ethanol on the yeast strain Saccharomyces cerevisiae NRRL-Y-132 were studied in batch and continuous chemostat cultures. Growth was limited by either glucose or ethanol. Feed medium was supplemented with different ethanol concentrations. Ethanol was found to inhibit growth and the activity of yeast to produce ethanol in a noncompetitive manner. A linear kinetic pattern for growth and product formation was observed according to μ = μ_m (1 – P/P_m) and v = v_m (1 – P/P_m′), where μ_m is the maximum specific growth rate at P = 0 (hr^?1); P_m is the maximum specific product formation rate at P = 0 (hr^?1); P_m is the maximum ethanol concentration above which cells do not grow (g/liter); P_m′ is the maximum ethanol concentration above which cells do not produce ethanol (g/liter). Substrate inhibition studies were carried out using short-time experimental techniques under aerobic and anaerobic condition. The degree of substrate inhibition was found to be higher than that has been reported for ethanol fermentation of pure sugar. The kinetic relationships thus obtained were used to compute growth, substrate utilization, and alcohol production patterns and have been discussed with reference to batch and continuous fermentation of enzymatically produced bagasse hydrolysate.     

Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

Kinetics of polysaccharide B-1459 fermentation

Polysaccharide gum was made by fermentation with Xanthomonas campestris NRRL B-1459 in a medium of glucose, minerals, and distillers' solubles. The effect of distillers' solubles on growth rate can be described by the familiar saturation equation. Although a quasistoichiometric relationship was observed between nitrogen utilization and growth, total nitrogen supply was not growth limiting, nor was polymer formation growth associated. Cell growth primarily took place in the early part of the fermentation; polysaccharide biosynthesis occurred throughout the fermentation. Glucose was converted to polysaccharide at a fairly constant yield, which was 70–80% of glucose consumed, under optimum conditions. The kinetic patterns observed indicate that multistage continuous fermentation will be suitable for polysaccharide production.     

Cultivation of hybridoma cells in continuous cultures: kinetics of growth and product formation

Mouse hybridoma cells were grown in suspension in continuous stirred bioreactors. Cell growth, substrate utilization, and monoclonal antibody (MAb) production were studied using serum-free medium. Steady-state data were obtained at different dilution rates, between 0.012 and 0.039 h(-1) Viability was profoundly affected by dilution rate, particularly near the lower end of the dilution-rate range investigated. MAb concentration and productivity went through a maximum with respect to dilution rate. Lactate yield on glucose declined with in creasing dilution rate. Experiments were carried out to study the effects of medium glucose concentration on cell growth, product formation, and lactate yield on glucose. Reduction of glucose concentration in the feed medium did not considerably affect cell density and MAb concentration in the culture, but lactate levels dropped sharply; lactate yield on glucose declined substantially, indicating alterations in cell metabolic path ways for energy metabolism. Optimization strategy for continuous cell culture is discussed.     

Diagnosing lactic acid fermentation based on specific rates of growth,substrate consumption and product formation

The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.List of Symbols 1/h specific rate of cell growth - 1/h specific rate of substrate consumption - 1/h specific rate of product formation - ^* dimensionless specific rate of cell growth - ^* dimensionless specific rate of substrate consumption - ^* dimensionless specific rate of product formation - _max 1/h maximum specific rate of cell growth - _max 1/h maximum specific rate of substrate consumption - _max 1/h maximum specific rate of product formation - X g/l cell mass concentration - S g/l substrate concentration - S ^* dimensionless substrate concentration - S ₀ g/l initial substrate concentration - P g/l product concentration     

Production of α-galactosidase by a novel actinomycete <Emphasis Type="Italic">Streptomyces griseoloalbus</Emphasis> and its application in soymilk hydrolysis

α-Galactosidase production by a newly isolated actinomycete Streptomyces griseoloalbus under submerged fermentation was investigated. The influence of initial pH of medium, incubation temperature, inoculum age and inoculum size on α-galactosidase formation was studied. Various carbon sources were supplemented in the medium to study their effect on enzyme production. The influence of the concentration of locust bean gum on enzyme production also was optimized. Optimization of process parameters resulted in a highest α-galactosidase activity of 20.4 U/ml. The highest α-galactosidase activity was obtained when the fermentation medium with initial pH 6.0 and containing 1% locust bean gum as growth substrate was inoculated with 10% (v/v) of 72 h grown inoculum and incubated at 30°C. The hydrolysis of flatulence-causing oligosaccharides in soymilk by the enzyme was also investigated. Thin layer chromatographic analysis of enzyme-treated soymilk samples showed the complete hydrolysis of soy oligosaccharides liberating galactose, the final product.     

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.     

Substrate and energy costs of the production of exocellular enzymes by Bacillus licheniformis

Substrate and energy costs of the production of exocellular enzymes from glucose and citrate by B. Iicheniformis S1684 as well as molar growth yields corrected for these costs of product formation were calculated using data from chemostat experiments. The calculations showed that 1.46-1.73 mol glucose and 2.31-2.77 mol citrate are needed for formation and excretion of 1 mol protein. Consequently, the values of the maximal product yield from substrate (Y(psm') g/mol) are 80 < Y(psm) < 95 when product is formed from glucose and 50 < Y(psm) < 60 when product is formed from citrate. The higher substrate costs for product formation from citrate are due to a higher level of CO(2) production during protein formation and a higher substrate requirement for the energy supply of product formation and excretion than when product is formed from glucose. The theoretical ATP requirement for protein synthesis could be determined reasonably well, but the energy costs of protein excretion could not be determined exactly. The energy costs of protein formation are higher than those of biomass formation or protein excretion. Molar growth yields corrected for the substrate costs of product formation were high, indicating a high efficiency of growth.Growth and production parameters were determined as well from experimental data of recycling fermentor experiments using a parameter optimization procedure based on a mathematical model describing biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of biomass growth rate. The fitting procedure yielded two growth and production domains during glucose limitation. In the first domain the values for the maximal growth yield and maintenance coefficient were in agreement with those found in chemostat experiments at corresponding values of Y(spm). Domain 2 could be described best with linear growth and product formation. In domain 2 the rate of product formation decreased and more substrate became available for biomass formation. As a consequence the specific growth rate increased in the shift from domain 1 to 2. Domain 2 behavior most probably is caused by the rel-status of B. Iicheniformis S1684.     

Enzyme production in a cell recycle fermentation system evaluated by computer simulations

Enzyme production in a cell recycle fermentation system was studied by computer simulations, using a mathematical model of -amylase production by Bacillus amyloliquefaciens. The model was modified so as to enable simulation of enzyme production by hypothetical organisms having different production kinetics at different fermentation conditions important for growth and production. The simulations were designed as a two-level factorial assay, the factor studied being fermentation with or without cell recycling, repression of product synthesis by glucose, kinetic production constants, product degradation by a protease, mode of fermentation, and starch versus glucose as the substrate carbon source.The main factor of importance for ensuring high enzyme production was cell recycling. Product formation kinetics related to the stationary growth phase combined with continuous fermentation with cell recycling also had a positive impact. The effect was greatest when two or more of these three factors were present in combinations, none of them alone guaranteeing a good result. Product degradation by a protease decreased the amount of product obtained; however, when combined with cell recycling, the protease effect was overshadowed by the increased production. Simulation of this type should prove a useful tool for analyzing troublesome fermentations and for identifying production organisms for further study in integrated fermentation systems.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - c starch concentration (g/l) - c ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - e intrinsic intracellular amylase concentration (g product/g cell mass) - E extracellular amylase concentration (g/l) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA/cell - K ₁ intracellular repression constant - K ₂ intracellular repression constant - K _s Monod saturation constant (g/l) - k ₃ product excretion rate constant (h^–1) - k _I translation constant (g product/g mRNA/h) - k _d first order decay constant (h^–1) - k _dw first order decay constant (h^–1) - k _gl rate constant for glucose production (g/l/h) - k _{m, dgr} saturation constant for product degradation (g/l) - k _st rate constant for starch hydrolysis (g/l/h) - k _t1 proportionality constant for amylase production (g mRNA/g substrate) - k _t2 proportionality constant for amylase production (g mRNA *h/g substrate) - k _w protease excretion rate constant (h^–1) - k _wt1 proportionality constant for protease production (g mRNA/g substrate) - k _wt2 proportionality constant for protease production (g mRNA *h/g substrate) - k _wI translation constant (g protease/g mRNA/h) - m maintenance coefficient (g substrate/g cell mass/h) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function, K₁/K₂ less than or equal to 1.0 - Q _w repression function, K₁/K₂ less than or equal to 1.0 - r intrinsic amylase mRNA concentration (g mRNA/g cell mass) - r _m intrinsic protease mRNA concentration (g mRNA/g cell mass) - R _ex retention by the filter of the compounds x=: C starch, E amylase, or S glucose - R _t amylase transport rate (g product/g cell mass/h) - R _wt protease transport rate (g protease/g cell mass/h) - R _s rate of glucose production (g/l/h) - R _c rate of starch hydrolysis (g/l/h) - S ₀ feed concentration of free reducing sugar (g/l) - s extracellular concentration of reducing sugar (g/l) - t time (h) - V volume (1) - w intracellular protease concentration (g/l) - W extracellular protease concentration (g/l) - X cell mass concentration (dry weight) (g/l) - Y yield coefficient (g cell mass/g substrate) - substrate uptake (g substrate/g cell mass/h) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) - _m,dgr maximum specific rate of amylase degradation (h^–1) This study was supported by the Nordic Industrial Foundation Bioprocess Engineering Programme and the Center for Process Biotechnology, The Technical University of Denmark.     

Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium brockii.

Specific changes in the chemical and microbial composition of Thermoanaerobium brockii fermentations were compared and related to alterations of process rates, end product yields, and growth parameters. Fermentation of starch as compared with glucose was associated with significant decreases in growth rate and intracellular fructose-1,6-bisphosphate concentration and with a dramatic increase in the ethanol/lactate product ratio. Glucose or pyruvate fermentation in the presence of acetone was correlated with increased substrate consumption, growth (both rate and yield), acetate yield, and quantitative reduction of acetone to isopropanol in lieu of normal reduced fermentation products (i.e., H2, ethanol, lactate). Acetone altered pyruvate phosphoroclastic activity of cell extracts in that H2, lactate, and ethanol levels decreased, whereas the acetate concentration increased. Glucose fermentation in the presence of exogenous hydrogen was associated with inhibition of endogenous H2 production and either increased ethanol/acetate product ratios and decreased growth at less than 0.5 atm (51 kPa) of H2 or total growth inhibition at 1.0 atm (102 kPA). The effects of exogenous hydrogen on glucose fermentation were totally reversed by the addition of acetone. Glucose fermentation in coculture with Methanobacterium thermoautotrophicum correlated with increased growth (both rate and yield), acetate yield, and the formation of methane in lieu of monoculture reduced products. In coculture, but not monoculture, T. brockii grew on ethanol as the energy source, and acetate and methane were the end products as a direct consequence of hydrogen consumption by the methanogen.     

Monensin has no effect on growth and metabolism ofMegasphaera elsdenii

A rumen strain ofMegasphaera elsdenii was grown on glucose and lactate in monensin-free and monensin-supplemented medium (10 mg/L). Monensin had no effect on growth rate, growth yields, metabolic pattern and composition of cells. Growth yields of dry matter and protein were higher in cultures supplied with glucose than in cultures supplied with lactate. The bacterium compensated the lower gain of energy from fermentation of lactate by rapid utilization of this substrate. Cells grown on glucose contained more saccharide and less protein than lactate-grown cells.     

Kinetics of soluble microbial product formation in substrate-sufficient batch culture of activated sludge

The kinetics of soluble microbial product (SMP) formation under substrate-sufficient conditions appear to exhibit different patterns from substrate-limited cultures. However, energy spilling-associated SMP formation is not taken into account in the existing kinetic models and classification of SMP. Based on the concepts of growth yield and energy uncoupling, a kinetic model describing energy spilling-associated SMP formation in relation to the ratio of initial substrate concentration to initial biomass concentration (S ₀/X ₀) was developed for substrate-sufficient batch culture of activated sludge, and was verified by experimental data. The specific rate of energy spilling-associated SMP formation showed an increasing trend with the S ₀/X ₀ ratio up to its maximum value. The SMP productivity coefficient (α _p/e) was defined from the model on the basis of energy spilling-associated substrate consumption. Results revealed that less than 5% of energy spilling-associated substrate consumption was converted into SMP. Electronic Publication     

Inhibition of alcoholic fermentation by substrate and ethanol

The effect of ethanol and sugars on rates of fermentation was studied. We used a strain of Canadida pseudotropicalis. The specific rate of fermentation was determined by using the Warburg manometer. The effect of ethanol was formulated as an exponential function of ethanol concentration, but the empirical constant was different when glucose or lactose was used as a substrate. The effects of both ethanol and substrate were formulated. It was demonstrate that when lactose and glucose were present in the medium with a small amount of alcohol, a synergistic effect on the rate of fermentation appeard. This phenomenon considerably limits the rate of fermentation.     

Influence of sugar source (lactose,glucose, galactose) on 2,3-butanediol production by Klebsiella oxytoca NRRL-B199

The ability of Klebsiella oxytoca NRRL-B199 to use either lactose or the mixture of glucose and galactose as substrate for the production of 2,3-butanediol was studied in batch fermentations with different conditions of aeration and pH. 2,3-butanediol was undetected, or present in minute concentration in the fermentation broths with lactose, while it was the main product from glucose+galactose with final concentrations of up to 18.8 g/l in media at pH 6.0. Under conditions optimal for 2,3-butanediol synthesis, when aeration limited growth, the rate of biomass growth was more tightly related to the aeration rate in lactose medium than in glucose+galactose medium. These relations suggest that the growth rate is very low on lactose but still considerable on glucose+galactose when aeration rate tends toward zero. Correspondingly, the metabolism is more oxidative in the former medium, yielding mainly acetate as product.Abbreviations CDW cell dry weight     

Regulatory aspects of bakers' yeast metabolism in aerobic fed-batch cultures

A highly instrumented computer-coupled bioreactor is used to investigate metabolic changes of Saccharomyces cerevisiae in aerobic fed-batch systems which are generally applied in bankers' yeast manufacture. The four types of metabolism (oxidation of glucose, aerobic fermentation, oxidation of glucose and ethanol, and oxidation of ethanol) appearing in such systems are characterized by four significant fermentation parameters: Respiratory quotient (RQ), glucose uptake rate (Q_g), ethanol turnover rate (Q_EtOH), and growth yield on glucose (Y_g). Below the critical glucose concentration glucose and ethanol are utilized simultaneously. The shift from aerobic fermentation to nondiauxic growth on glucose and ethanol is not only dependent on glucose concentration. but also on the precultivation on cells. The uptake of ethanol is controlled by the glucose supply except in the case when ethanol is limiting; the oxygen uptake rate (Q_o2), however, is unaffected by the ratio of Q_g and Q_EtOH. Critical glucose concentration is not a constant value for a particular strain, but varies corresponding to the nutritional state of the cells.     

Xanthan gum: an economical substitute for agar in plant tissue culture media

Xanthan gum, a microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used as a solidifying agent for plant tissue culture media. Its suitability as a substitute to agar was demonstrated for in vitro seed germination, caulogenesis and rhizogenesis of Albizzia lebbeck, androgenesis in anther cultures of Datura innoxia, and somatic embryogenesis in callus cultures of Calliandra tweedii. Culture media used for eliciting these morphogenic responses were gelled with either 1% xanthan gum or 0.9% agar. Xanthan gum, like agar, supported all these responses.     

Software sensors for fermentation processes

Four software sensors based on standard on-line data from fermentation processes and simple mathematical models were used to monitor a number of state variables in Escherichia coli fed-batch processes: the biomass concentration, the specific growth rate, the oxygen transfer capacity of the bioreactor, and the new R _O/S sensor which is the ratio between oxygen and energy substrate consumption. The R _O/S variable grows continuously in a fed-batch culture with constant glucose feed, which reflects the increasing maintenance demand at declining specific growth rate. The R _O/S sensor also responded to rapid pH shift-downs reflecting the increasing demand for maintenance energy. It is suggested that this sensor may be used to monitor the extent of physiological stress that demands energy for survival.     

Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed-batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO₂ evolution rate, O₂ uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (δ_h) with the fraction of available electrons transferred to oxygen (?_O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ?_O2 than δ_h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.