Determination of the intrinsic fermentation kinetics of Saccharomyces cerevisiae cells immobilized in Ca-alginate beads and observations on their growth

Continuous fermentation experiments in a well-stirred fermentor with Saccharomyces cerevisiae cells immobilized in Ca-alginate beads of small diameter (approx. 1 mm) have been performed in order to discover their intrinsic fermentation kinetics, and compare them to the fermentation kinetics for free cells, by fitting both sets of results to the same model. The results show similar kinetic parameters for free and immobilized cells. The changes in cell concentration inside the beads and microscopical observations of transverse sections throughout the experiments, allowed discernment of two different scenarios of cell growth inside the beads: low cell density and fully developed growth. Correspondence to: F. Gòdia     

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.     

A phenomenological model to represent the kinetics of growth by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for lysine production

Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.     

Batch kinetics of microbial polysaccharide biosynthesis

A modified form of logistic equation has been proposed to quantity the batch kinetics of microbial growth during the biosynthesis of extra- and intracellular polymers. Based on the experimental data developed in this study, the proposed model appeared to provide adequate growth and fermentation kinetics of Aureobasidium pullulans. The model was also applicable for representing the reported data on pullulan, xanthan, and poly-beta-hydroxybutyricacid. In comparison to the logistic and Monod kinetics, this model fitted the data better and more accurately described the overall fermentation, both concentrations and fermentation time.     

An error estimation of Michaelis-Menten (Monod)-type kinetics

Due to research on biochemistry and genetic engineering, mathematical models of microbial growth have become more complicated but Michaelis-Menten or Monod type expressions have still been used for conversion rates, uptake rates, etc. It is worth examining the error that can be caused by these quasi-steady-state-hypotheses. This paper presents a simple but very effective rationale function that describes the error of the quasi-steady-state hypothesis in enzyme kinetics. A simplified fermentation kinetic model was used for comparison of microbial growth but no analytical error function has been found for batch cultivation. In the case of continuous fermentation the error can be given in an analytical form. Many simulations, based on real SCP experiments, show a significant effect of the quasi-steady-state hypothesis. Since the rate constants of intracellular events are not really known, we have to be very careful when taking into account Michaelis-Menten type expressions in the building of complicated models. Correspondence to: L. Szigeti     

Fermentation kinetics of spent sulfite liquor by Saccharomyces cerevisiae

The growth kinetics of the yeast Saccharomyces cerevisiae and the production rate of ethanol have been studied in batch fermentation under anaerobic conditions in a 20-L fermentor. Two substrates were used in fermentation trials: a synthetic mixture of three fermentable sugars, D-glucose, D-mannose, and D-galactose, and a low-yield liquor originating from a bisulfite cooking process. The Monod model adequately described the system in relation to the specific growth rate mu(x) and the specific product formation rate mu(P). Different fermentation parameters (growth rate, substrate utilization, and product formation) were determined for the synthetic mixture and the bisulfite liquor. It was observed that the specific growth rate is much lower in spent sulfite liquor than in a synthetic medium. However, the specific product formation rate remains the same in both media.     

赖氨酸流加发酵最优控制的研究

在赖氨酸发酵动力学模型和庞特里金明小值原理的基础上,得出流加发酵的最优化底物流加方式。并进一步对流加发酵的全过程进行了分析,得出了在实际控制中较为可行的流加发酵全过程的总控制策略,实际控制表明在小型反应器中赖氨酸产生菌FB42的发酵水平为81．6g／l。、转化率为0．418％、生产强度为1．16g／h·L,和分批发酵相比分别提高了45．4％、9．7％和28．4％。     

Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry

AIMS: To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. METHODS AND RESULTS: Saccharomyces cerevisiae was grown in batch in a defined medium with glucose (200 g l(-1)) as the only carbon and energy source, and nitrogen supplied as ammonium sulphate or phosphate forms under different concentrations. The initial nitrogen concentration in the media had no effect on specific growth rates of the yeast strain PYCC 4072. However, fermentation rate and the time required for completion of the alcoholic fermentation were strongly dependent on nitrogen availability. At the stationary phase, the addition of ammonium was effective in increasing cell population, fermentation rate and ethanol. CONCLUSIONS: The yeast strain required a minimum of 267 mg N l(-1) to attain complete dryness of media, within the time considered for the experiments. Lower levels were enough to support growth, although leading to sluggish or stuck fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to elucidate the role of nitrogen on growth and fermentation performance of wine yeast. This information might be useful to the wine industry where excessive addition of nitrogen to prevent sluggish or stuck fermentation might have a negative impact on wine stability and quality.     

Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their growth-associated biosynthesis

Four Streptococcus thermophilus strains ( Strep. thermophilus BTC, Strep. thermophilus LY03, Strep. thermophilus 480 and Strep. thermophilus Sfi20) have been examined for their exopolysaccharide production capacity. All strains produced a polymer composed of the neutral sugars glucose and galactose, but in different ratios. It was clearly shown that the biosynthesis of exopolysaccharides from Strep. thermophilus LY03 is growth-associated and hence displays primary metabolite kinetics. The monomer ratio of the exopolysaccharide synthesized did not vary throughout the fermentation cycle. The production kinetics and exopolysaccharide yields were strongly dependent on the fermentation conditions. Physical factors such as temperature, pH and oxygen tension as well as chemical factors (medium composition, initial lactose concentration, carbon/nitrogen levels) were of utmost importance.     

A kinetic study of the lactic acid fermentation. Batch process at controlled pH. Reprinted from Journal of Biochemical and Microbiological Technology Engineering Vol. I, No. 4. Pages 393-412 (1959)

Kinetic data are needed to develop basic understanding of fermentation processes and to permit rational design of continuous fermentation processes. The kinetics of the fermentation of glucose to lactic acid have been studied at six constant pH levels between 4. 5 and 6.0 by measuring the instantaneous rates of bacterial growth and of lactic acid formation throughout each fermentation. It was found that the instantaneous rate of acid formation dP/dt, should be related to the instantaneous rate of bacterial growth dN/dt, and to the bacterial density N, throughout a fermentation at a given pH, by the expression when the constants alpha and beta are determined by the pH of the fermentation.     

产人表皮生长因子的重组大肠杆菌的发酵动力学

对产人表皮生长因子的重组大肠杆菌Escherichia coli(E.Coli)的发酵动力学进行了研究,采用了发酵体系中含质粒的工程菌与不含质粒的非工程菌的共处模型,分析了细胞生长、底物消耗、基因工程产物生成的过程,由模型计算的结果与实验结果基本吻合。     

山梨糖发酵产生2—酮基—L—古龙酸氮源代谢规律

对山梨糖发酵产生2-酮基-L-古龙酸氮源代谢规律进行了初步研究。通过对这一混合发酵体系蛋白和尿素代谢的研究表明,氮源代谢与单一菌体发酵相比有其特殊性,主要表现在尿素的加入有两个作用,即作为生理碱性物质调节体系pH和为菌体代谢提供部分氮源,而体系的蛋白含量随发酵时间的延续不断增加,其增加的原因是巨大芽孢杆菌由营养体转变成芽孢所致,这是该发酵体系的特点。本文还对该发酵体系各种氨基酸变化规律进行了讨论,将一共17种氨基酸按其变化规律分成了三类,较好地解释了各种氨基酸的变化情况,为进一步深入研究该体系的动力学特性提供了数据基础。     

Methanol bioconversion by Butyribacterium methylotrophicum--batch fermentation yield and kinetics

Butyribacterium methylotrophicum is an anaerobic bacterium that can convert methanol to butyrate. This ability to produce longer-chain carbon compounds from C(1) substrates could be of commercial significance. The fermentation rates and product formation depend on the methanol/bicarbonate ratios during fermentation. The kinetics of batch fermentation fit the Luedeking-Piret model with growth and maintenance associated product formation. Butyrate yield of 0.256 mol/mol methanol (ca. 85% of theoretical yield) has been obtained in batch fermentation.     

Ethanol inhibition of yeast growth and fermentation: Differences in the magnitude and complexity of the effect

Summary The effect of ethanol on yeast growth and fermentation has been studied in two strains, NCYC479 (a commercial saké yeast) and 5D-cyc (a laboratory haploid strain). The effect of ethanol on growth was similar in the two strains. It showed complex kinetics which resulted from both the inhibition of the growth rate itself and also a reduction in cell viability. The growth and viability effects had different inhibition constants. Ethanol was less inhibitory toward fermentation than toward growth. Fermentation in the saké yeast was more ethanol tolerant than in the laboratory strain. The inhibition kinetics for fermentation were less complex than those for growth and followed the classical noncompetitive pattern.     

Effects of glycerol on alcohol fermentation. Inhibition mechanism and diffusion limitations

Batch alcohol fermentations have been carried out varying the starting level of glycerol in the broth and keeping constant all the other fermentation parameters, in order to study the effect of the accumulation of this metabolite on the fermentation kinetics. A linear slow decrease of the maximum specific ethanol productivity with increasing glycerol level has been followed by a sharp fall of this parameter over a glycerol concentration threshold. A kinetic study through unstructured integrated models demonstrates that, at low concentrations, glycerol behaves as a non competitive inhibitor of fermentation, while, over a concentration threshold (105 kg/m³), an additional effect takes place, likely ascribable to diffusion limitations provoked by excess viscosity of the broth.     

Model aided dynamic process analysis and optimization for the nourseothricin fermentation

The relation between product formation and growth kinetics could be characterized by two facts: the specific product formation rate depends on the ageing of the population and on the specific growth rate. These relation was formulated and quantified by a mathematical model, which was fitted to experimental data of a representative fermentation run und used to predict an optimal fermentation mode. In the result of this discussion cyclic fed batch fermentation was found to be optimal.     

Investigation on the biodegradability of mycotoxins nivalenol (NIV) and deoxynivalenol (DON) in a rusitec fermentor and their monitoring by HPLC/MS

Biodegradation of two B-trichothecenes nivalenol (NIV) and deoxynivalenol (DON) have been studied in a RUSITEC (rumen simulation technique) system. The fermentation studies were carried out in vessels containing the rumen fluid. To reach steady state an adaptation period of one week was carried out. The mycotoxin standards were then added into the fermentor in a concentration of 1ppm NIV and 2ppm DON. The kinetics of NIV and DON biodegradability during the fermentation process were monitored by using a rapid HPLC method combined with a mass spectrometer and an atmospheric pressure chemical ionisation (APCI^-) interface. Also the effect of mycotoxin addition on different parameters such as ammonia production, pH, gas production and volatile fatty acids have been studied.     

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.     

药用昆虫蜣螂对灵芝发酵和抗小鼠肝癌活性的影响

采用液体深层发酵方式,研究了药用昆虫蜣螂对灵芝细胞生长、关键活性产物发酵动力学和抗小鼠肝癌活性的影响。结果表明,药用昆虫蜣螂在各添加浓度下对灵芝的细胞生长均无显著促进作用;但在添加量为5g/L时,对灵芝多糖和灵芝三萜的发酵动力学有显著影响(P<0.05),在发酵第7天时,灵芝总多糖和总三萜的产量分别达到2.81g/L和539.0mg/L(对照组分别为2.25g/L和428.2mg/L)。小鼠体内抗肝癌结果表明,灵芝对照发酵物的抑癌率为41.61%,灵芝-蜣螂配合物的抑癌率为42.24%;而补加蜣螂发酵后的灵芝加蜣发酵物的抑癌率高达57.21%,与灵芝对照发酵物的抑癌率相比,提高了37.49%。研究表明,采用昆虫蜣螂补料-分批发酵后,灵芝发酵物抗小鼠肝癌的活性得到显著增强。