Energy-Based Dynamic Model for Variable Temperature Batch Fermentation by Lactococcus lactis

We developed a mechanistic mathematical model for predicting the progression of batch fermentation of cucumber juice by Lactococcus lactis under variable environmental conditions. In order to overcome the deficiencies of presently available models, we use a dynamic energy budget approach to model the dependence of growth on present as well as past environmental conditions. When parameter estimates from independent experimental data are used, our model is able to predict the outcomes of three different temperature shift scenarios. Sensitivity analyses elucidate how temperature affects the metabolism and growth of cells through all four stages of fermentation and reveal that there is a qualitative reversal in the factors limiting growth between low and high temperatures. Our model has an applied use as a predictive tool in batch culture growth. It has the added advantage of being able to suggest plausible and testable mechanistic assumptions about the interplay between cellular energetics and the modes of inhibition by temperature and end product accumulation.     

Modeling of yeast metabolism and process dynamics in batch fermentation

Much is known about yeast metabolism and the kinetics of industrial batch fermentation processes. In this study, however, we provide the first tool to evaluate the dynamic interaction that exists between them. A stoichiometric model, using wine fermentation as a case study, was constructed to simulate batch cultures of Saccharomyces cerevisiae. Five differential equations describe the evolution of the main metabolites and biomass in the fermentation tank, while a set of underdetermined linear algebraic equations models the pseudo-steady-state microbial metabolism. Specific links between process variables and the reaction rates of metabolic pathways represent microorganism adaptation to environmental changes in the culture. Adaptation requirements to changes in the environment, optimal growth, and homeostasis were set as the physiological objectives. A linear programming routine was used to define optimal metabolic mass flux distribution at each instant throughout the process. The kinetics of the process arise from the dynamic interaction between the environment and metabolic flux distribution. The model assessed the effect of nitrogen starvation and ethanol toxicity in wine fermentation and it was able to simulate fermentation profiles qualitatively, while experimental fermentation yields were reproduced successfully as well.     

Modeling and experimental studies on intermittent starch feeding and citrate addition in simultaneous saccharification and fermentation of starch to flavor compounds

Simultaneous saccharification and fermentation (SSF) is a combined process of saccharification of a renewable bioresource and fermentation process to produce products, such as lactic acid and ethanol. Recently, SSF has been extensively used to convert various sources of cellulose and starch into fermentative products. Here, we present a study on production of buttery flavors, namely diacetyl and acetoin, by growing Lactobacillus rhamnosus on a starch medium containing the enzyme glucoamylase. We further develop a structured kinetics for the SSF process, which includes enzyme and growth kinetics. The model was used to simulate the effect of pH and temperature on the SSF process so as to obtain optimum operating conditions. The model was experimentally verified by conducting SSF using an initial starch concentration of 100 g/L. The study demonstrated that the developed kinetic was able to suggest strategies for improved productivities. The developed model was able to accurately predict the enhanced productivity of flavors in a three stage process with intermittent addition of starch. Experimental and simulations demonstrated that citrate addition can also lead to enhanced productivity of flavors. The developed optimal model for SSF was able to capture the dynamics of SSF in batch mode as well as in a three stage process. The structured kinetics was also able to quantify the effect of multiple substrates present in the medium. The study demonstrated that structured kinetic models can be used in the future for design and optimization of SSF as a batch or a fed-batch process. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A maximum production strategy of lysine based on a simplified model derived from a metabolic reaction network

The aim of this study is to develop a strategy for maximum production of a target product with a simplified model derived from a metabolic reaction network through an example of lysine production. Based on the model, a search for the optimal specific growth rate profile was conducted among the available conditions of batch fermentation based on the derived model, when the total fermentation time was fixed. The optimal specific growth rate was obtained as a boundary control: initially, the specific growth rate was maintained at a maximum value and was subsequently switched to a critical value giving the maximum specific production rate. To make the specific growth rate follow this optimal profile as accurately as possible in batch mode, first, an appropriate initial concentration of leucine was employed in the experiment. Second, the feeding strategy of leucine was further studied. The specific growth rate profile with feeding was closer to the optimal one and the amount of lysine produced at the final stage of fermentation was increased about twofold, compared to that in the batch fermentation. Finally, the strategy was summarized as an algorithm for general use of this method.     

Cell proliferation-cephamycin biosynthesis relationship in the culture of Streptomyces lactamdurans L 2/6: phosphate regulation

During batch cultivation of Streptomyces lactamdurans a diauxic growth has been shown taking into consideration dry cell mass, protein, and DNA determination. This phenomenon was connected with substantial changes in environmental conditions such as concentrations of ammonia and amino nitrogen, soluble inorganic phosphate and pH. The antibiotic production was preceded by the preparatory phase occurring at the beginning of the second growth phase. A hypothetical pattern of cell physiology in the curse of cephamycin fermentation is discussed. An essential part in the biphasic cell growth and the induction of antibiotic synthesis in this model is played by phosphate depletion in the medium.     

温度对谷胱甘肽分批发酵的影响及动力学模型

研究了24～32℃范围内产朊假丝酵母生产谷胱甘肽的分批发酵过程,发现较高温度对细胞生长有促进作用,而较低温度则更有利于谷胱甘肽产量的提高。应用改进的Logistic和LuedekingPiret方程分别对细胞生长动力学和谷胱甘肽合成动力学进行了模拟,得到不同温度下各种动力学参数。在此基础上,进一步研究了温度同细胞生长动力学参数之间的内在联系,得到谷胱甘肽分批发酵过程中细胞浓度的变化同温度以及底物浓度之间的一般关系式：dX-dt＝［0.0224(T+1.7)］2X(1-X/X_max)1+S｛8.26×10.6×exp［-31477/R/(T+273)］｝。验证实验结果表明,该模型具有很好的适用性。     

模拟青霉素发酵过程中菌体生长动态的细胞自动机模型

在青霉素发酵生产机理及其动力学微分方程模型的基础上,建立了模拟青霉素分批发酵过程中菌体生长动态的细胞自动机模型(CABGM)。CABGM采用三维细胞自动机作为菌体生长空间,采用Moore型邻域作为细胞邻域,其演化规则根据青霉素分批发酵过程中菌体生长机理和动力学微分方程模型设计。CABGM中的每一个细胞既可代表单个的青霉素产生菌,又可代表特定数量的青霉素产生菌,它具有不同的状态。对CABGM进行了统计特性的理论分析和仿真实验,理论分析和仿真实验结果均证明了CABGM能一致地复现动力学微分方程模型所描述的青霉素分批发酵菌体生长过程。最后,对所建模型在实际生产过程中的应用问题进行了分析,指出了需要进一步研究的问题。     

Global changes in gene expression observed at the transition from growth to stationary phase in Listeria monocytogenes ScottA batch culture

Listeria monocytogenes is a food-borne Gram-positive bacterium that is responsible for a variety of infections (worldwide) annually. The organism is able to survive a variety of environmental conditions and stresses, however, the mechanisms by which L. monocytogenes adapts to environmental change are yet to be fully elucidated. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. We have utilized a proteomic approach to investigate the response of L. monocytogenes batch cultures to the transition from exponential to stationary growth phase. Proteomic analysis showed that batch cultures of L. monocytogenes perceived stress and began preparations for stationary phase much earlier (approximately A(600) = 0.75, mid-exponential) than predicted by growth characteristics alone. Global analysis of the proteome revealed that the expression levels of more than 50% of all proteins observed changed significantly over a 7-9 h period during this transition phase. We have highlighted ten proteins in particular whose expression levels appear to be important in the early onset of the stationary phase. The significance of these findings in terms of functionality and the mechanistic picture are discussed.     

Pullulan from peat hydrolyzate fermentation kinetics

The Luedeking-Piret equation was used to fit the kinetic data of pullulan fermentations from peat hydrolyzate substrate. In batch mode, the kinetic parameters m, n, alpha, and beta varied as a function of fermentation conditions: aeration rate, agitation speed, and temperature. In constant-feed fed-batch mode, the parameters Varied according to the feed rates. In peat hydrolyzate medium, the polysaccharide synthesis was strongly growth associated in batch and continuous fermentations but entirely growth associated in fedbatch fermentations. The fed-batch mode of fermentation with an appropriate feed rate is more advantageous with respect to batch and continuous fermentations. Therefore, if the fermentation is started batchwise and then followed by fed-batch mode at a constant feed rate, the overall polysaccharide productivity (g pullulan/L h) is significantly higher than those obtained with batch or continuous fermentations using the same total medium volume.     

Are responses of herbivores to environmental variability spatially consistent in alpine ecosystems?

Animal responses to global climate variation might be spatially inconsistent. This may arise from spatial variation in factors limiting populations' growth or from differences in the links between global climate patterns and ecologically relevant local climate variation. For example, the North Atlantic Oscillation (NAO) has a spatially consistent relation to temperature, but inconsistent spatial relation to snow depth in Scandinavia. Furthermore, there are multiple mechanistic ways by which climate may limit animal populations, involving both direct effects through thermoregulation and indirect pathways through trophic interactions. It is conceptually appealing to directly model the predicted mechanistic links. This includes the use of climate variables mimicking such interactions, for example, to use growing degree days (GDD) as a proxy for plant growth rather than average monthly temperature. Using a unique database of autumn body mass of 83331 domestic lambs from the period 1992–2007 in four alpine ranges in Norway, we demonstrate the utility of hierarchical, mechanistic path models fitted using a Bayesian approach to analyse explicitly predicted relationships among environmental variables and between lamb body mass and the environmental variables. We found large spatial variation in strength of responses of autumn lamb body mass to the NAO, to a proxy for plant growth in spring (the Normalized Difference Vegetation Index, NDVI) and effects even differed in direction to local summer climate. Average local temperature outperformed GDD as a predictor of the NDVI, whereas the NAO index in two areas outperformed local weather variables as a predictor of lamb body mass, despite the weaker mechanistic link. Our study highlights that spatial variation in strength of herbivore responses may arise from several processes. Furthermore, mechanistically more appealing measures do not always increase predictive power due to scale of measurement and since global measures may provide more relevant “weather packages” for larger scales.     

Fermentation Kinetics and Continuous Process of L-Asparaginase Production

For the purpose of obtaining L-asparaginase in quantities from Erwinia aroideae, cell growth and enzyme formation were investigated in both batch and continuous fermentation. Using yeast extract as a growth-limiting substrate, the relationship between specific growth rate and substrate concentration was found to fit the Monod equation. The optimum temperature for enzyme production was 24 C, although cell growth was higher at 28 C. The enzyme yield reached its maximum of 4 IU/ml during the negative acceleration growth phase which occurs just prior to stationary growth. Compared to batch fermentations, the continuous fermentation process gave a lower enzyme yield except when the fermentation was conducted at a dilution rate of 0.1 hr(-1). The graphical method frequently used for prediction of continuous fermentation does not apply to L-asparaginase production by E. aroideae. The optimum temperature for enzyme production in continuous process was 24 C, which was the same as in batch process. Increasing the temperature from 24 to 28 C resulted in a 20% loss of enzyme yield.     

Evaluation of Various Unstructured Kinetic Models for the Production of Protease by Bacillus sphaericus MTTC511

Bacillus sphaericus MTCC511 was used for the production of protease in submerged batch fermentation. Maximum protease activity of 1010 U/L was obtained during a fermentation period of 24 h under optimized conditions of 30 °C in a medium with an initial pH of 7 and at a shaking rate of 120 rpm. The maximum biomass obtained in the batch fermentation was 2.55 g/L after 16 h. Various unstructured models were analyzed to simulate the experimental values of microbial growth, protease activity and substrate concentration. The unstructured models, i.e. the Monod model for microbial growth, the Monod incorporated Luedeking‐Piret model for the production of protease and the Monod‐incorporated modified Luedeking‐Piret model for the utilization of substrate were capable of predicting the fermentation profile with high coefficient of determination (R²) values of 0.9967, 0.9402 and 0.9729, respectively. The results indicated that the unstructured models were able to describe the fermentation kinetics more effectively.     

Cybernetic modeling of iron-limited growth and siderophore production

The cybernetic modeling framework developed by Ramkrishna and co-workers has been applied to a case of bacterial metabolite production, namely the production of siderophores (iron-chelating agents) associated with iron-limiting fermentation conditions. Experimental growth data showed that, even though final biomass levels were controlled by exhaustion of the carbon source, iron-limiting conditions also affected the biomass yield. A structured model which includes the process of an iron-limiting energy resource production was able to quantitatively account for this apparent dual-substrate limitation over a wide range of batch and continuous operating conditions. The experiments data also showed quite large difference in iron uptake over the wide range of operating condition and iron levels investigated. The inclusion in the model of the processes of low and high (siderophore-mediated) affinity iron transport, and siderophore production led to simulation results that were in good quantitative agreement with the siderophore, medium and cell iron levels, in both batch and steady-state continuous culture operating conditions.     

Fermentation technologies for the production of probiotics with high viability and functionality

There is growing scientific evidence supported by mechanistic and clinical studies that probiotics can provide health benefits. As probiotics are highly sensitive to many environmental factors, and because the propagation of many strains of intestinal origin is not straightforward, most commercial strains are selected on the basis of their technological properties - ruling out some strains with promising health properties. To date, probiotic production has almost exclusively been carried out using conventional batch fermentation and suspended cultures, in some cases combined with the use of sublethal stresses to enhance cell viability, the addition of protectants or microencapsulation to provide cell protection. However, other less conventional fermentation technologies, such as continuous culture and immobilized cell systems, could have potential for enhancing the performance of these fastidious organisms. These technologies might be employed to develop strains with improved physiology and functionality in the gut and to enlarge the range of commercially available probiotics, as well as expanding product applications.     

Environmental variables influencing the carbon balance at the alpine treeline: a modeling approach

Abstract. Growth of coniferous trees at alpine treeline locations has traditionally been viewed as primarily temperature dependent. In this study, we use a physiologically mechanistic process model, ATE-BGC (Alpine Treeline Ecotone - BioGeochemical Cycles), to study the effects of a suite of environmental variables on the carbon balance of krummholz growth forms of subalpine Abies lasiocarpa under environmental conditions representative of treeline locations in Glacier National Park, Montana, USA. We perform a sensitivity analysis of ATE-BGC to determine the relative effects of temperature, insolation, snow, rain, soil depth, leaf area index, winter injury and elevation on krummholz carbon balance. Our results indicate that ATE-BGC carbon balance estimates are most highly influenced by temperature and winter injury. Leaf area index, soil depth, rainfall, insolation and snowfall follow temperature and winter injury in their effect on carbon balance. Due to the sensitivity of the model to moisture related variables, we propose that local irregularities in the location of the alpine treeline ecotone are the result of localized xeric conditions.     

Stochastic gene expression in fluctuating environments

Stochastic mechanisms can cause a group of isogenic bacteria, each subject to identical environmental conditions, to nevertheless exhibit diverse patterns of gene expression. The resulting phenotypic subpopulations will typically have distinct growth rates. This behavior has been observed in several contexts, including sugar metabolism and pili phase variation. Under fixed environmental conditions, the net growth rate of the population is maximized when all cells are of the fastest growing phenotype, so it is unclear what fitness advantage is conferred by population heterogeneity. However, unlike ideal laboratory conditions, natural environments tend to fluctuate, either periodically or randomly. Here we use a stochastic population model to show that, during growth in such fluctuating environments, a dynamically heterogenous bacterial population can sometimes achieve a higher net growth rate than a homogenous one. By using stochastic mechanisms to sample several distinct phenotypes, the bacteria are able to anticipate and take advantage of sudden changes in their environment. However, this heterogeneity is beneficial only if the bacterial response rate is sufficiently low. Our results could be useful in the design of artificial evolution experiments and in the optimization of fermentation processes.     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

Genetic structure of a novel biofuel-producing microorganism community

Biofuels are an important alternative, renewable source of energy in the face of the ongoing depletion of fossil fuels. Cheese whey is a dairy industry waste characterized by high lactose concentration, which represents a significant environmental problem. Bio-ethanol production by cheese whey could be an effective nonvegetable source for renewable energy production. Here, we report the isolation of a mixed microbial population, able to produce ethanol as main fermentation product from fermenting whey. The microbial consortium has been used to perform a batch fermentation of crude whey in both anoxic and hypoxic conditions. Maximum ethanol concentrations achieved in this study was obtained using the mixed culture in hypoxic conditions, grown at pH 4 and 30°C, with ethanol production yield of 60 g/L. Our research has pointed out an alternative way to both dispose and valorize cheese whey, a dairy by-product that could cause water pollution and harm to the environment if not properly treated.     

Kinetics and modeling of temperature effects on batch xanthan gum fermentation

Batch fermentation kinetics of xanthan gum production from glucose by Xanthomonas campestris at temperatures between 22 degrees C and 35 degrees C were studied to evaluate temperature effects on cell growth and xanthan formation. These batch xanthan fermentations were modeled by the logistic equation for cell growth, the Luedeking-Piret equation for xanthan production, and a modified Luedeking-Piret equation for glucose consumption. Temperature dependence of the parameters in this model was evaluated. Growth-associated rate constants increased to a maximum at approximately 30 degrees C and then decreased to zero at approximately 35 degrees C. This temperature effect can be modeled using a square-root model. On the contrary, non-growth-associated rate constants increased with increasing temperature, following the Arrhenius relationship, in the entire temperature range studied. The model developed in this work fits the experimental data very well and can be used in a simulation study. However, due to the empirical nature of the model, the parameter values need to be reevaluated if the model is to be applied to different growth conditions.