Xenorhabdus nematophila发酵动力学研究

在分批发酵中,研究了Xenorhabdus nematophila YL001的生长、基质消耗及抗菌物质产生的特性.基于Logistic方程和Luedeking-Piret方程,得到了描述分批发酵过程的动力学模型及模型参数,同时对实验数据与模型进行了验证比较.模型计算值与实验数据拟合良好,模型基本反映了Xenorhabdus nematophila YL001分批发酵过程的动力学特征.分批发酵中细胞生长与产物合成属于偶联型.     

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.     

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。     

Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus

Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimer's disease. Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-l bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-l fermentor by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.     

Fed-batch fermentation dealing with nitrogen limitation in microbial transglutaminase production by Streptoverticillium mobaraense

In the later stages of a batch fermentation for microbial transglutaminase production by Streptoverticillium mobaraense the availability of a nitrogen source accessible to the microorganism becomes critical. Fed-batch fermentation is investigated with the aim of avoiding this substrate limitation. When peptone is used as a nitrogen source in the feed, no significant improvement of growth and transglutaminase production is observed. This is probably due to crosslinking of the nitrogen source by the transglutaminase produced. Using an inorganic nitrogen source alone does not give satisfactory growth and production. A fed-batch fermentation method has thus been developed to deal with this problem. In the batch phase of the fermentation, an initial medium containing peptone, designed on the basis of the stoichiometric requirements of the microorganism, is used to ensure optimal growth. In the feeding phase, ammonium sulphate is used instead to avoid the crosslinking effect. The feed composition, mainly the amount of nitrogen and carbon source, is also based on the stoichiometric requirements of the organism, taking into account the replacement of peptone by ammonium sulphate. By using this fed-batch fermentation technique, cell-mass dry weight and transglutaminase production could be increased by 33% and 80% respectively, compared to those in a batch fermentation. Received: 10 July 1997 / Received revision: 24 October 1997 / Accepted: 24 October 1997     

Role of ultrasound in assisted fermentation technologies for process enhancements

Abstract

Biological molecules are widely produced by fermentation technology using bacteria, fungi or yeast. Fermentation is a biochemical process wherein the rate of bioconversion is governed by the organisms involved. The growth of the organism is mainly limited by mass transfer rates of nutrients and gases that directly affect the product formation in fermentation. Attempts have been made to enhance the growth rate and yield using mutational, recombinant strain development approach at microbial level as well as fed batch and continuous processing approach at bioprocess level in the past. The growth rate of microbes can be accelerated by increased mass transfer rates and cell wall permeability with the use of controlled low frequency ultrasound irradiation. The present review provides insights into the application of acoustic cavitation in process intensification of fermentation approaches and the role of various factors involved are highlighted with typical examples.     

无花果曲霉发酵生产β-葡萄糖苷酶的动力学分析

在7L生物反应器的分批发酵中,通过对无花果曲霉UV-29液态发酵茵丝体的生长、基质消耗（以总糖计）及β-葡萄糖苷酶产生的特性研究,发现总糖是无花果曲霉生长的限制性基质;β-葡萄糖苷酶的增长趋势明显滞后于细胞生长的增长趋势,其发酵过程属于部分相关模型,即Ga—den提出的Ⅱ型发酵;基于logistic方程,建立了发酵动力学模型,同时对实验数据与模型进行了验证比较,模型计算值与实验数据拟合良好。在7L生物反应器的最大茵体生物量（干重）达到1．17g／100mL,β-葡萄糖苷酶最高酶活达到22．25IU／mL。     

A study of producing ethanol from cellulose using Clostridium thermocellum

The feasibility of producing ethanol in a continuous system from cellulose using Clostridirrrn thermocellum was investigated. This anaerobic and therniophilic bacterium was able to degrade cellulose directly into ethanol with acetic acid, hydrogen. and carbon dioxide as by-products of this fermentation. The fermentation was first carried out in a batch mode to study the effects of buffers, temperature, and agitation on microbial growth and ethanol production. From the compounds used to control pH. sodium bicarbonate had the most preferred effects on generation time and ethanol production. As expected, there was a positive relationship between temperature and growth rate. On the other hand, agitation did not benefit from ethanol production or microbial growth. The possibility of noncompetitive inhibition within such a system was deduced from the calculation of the kinetic constants K(m) and V(max). Continuous fermentations were carried out at 60 degrees C and pH 7.0 using 1.5 and 3% pure cellulose as a limiting substrate. The maximum ethanol concentration reached during the 1.5% cellulose fermentation was 0.3%. and 0.9% during the 3% cellulose fermentation. The yield of ethanol was about 0.3 grams per gram of consumed cellulose. The overall yield in both schemes was around 0.45 and 0.75 grams per gram of cellulose degraded. It was concluded that cellulose could be degraded continuously in a system with C. thermocellum for production of ethanol. While the continuous system like the batch method is feasible, it may not be promising as yet because of the slow generation time of this microorganism.     

Study of fermentation behavior and formulation of a semidefined nutrient medium based on acid-production measurements in Z. mobilis cultures

Batch fermentations of glucose to ethanol by Z. Mobilis.(ATCC 10988) were examined in several semidefined nutrient media. The measurement of acid produced by the microorganism was used to study its transient fermentation characteristics. Limitation of nitrogen source in the semidefined medium of Rogers and coworkers(2) was found to limit the growth of this microorganism in the late stages of batch fermentations, when the initial glucose concentration was 75 g/L and higher. The microorganism exhibits a preference for inorganic nitrogen over preformed organic nitrogen provided by yeast extract. The microbial growth occurs exponentially in the presence of ammonium sulfate and yeast extract. However, in the absence of ammonium sulfate, the growth occurs in a linear fashion. The "linear" growth phase is characterized by poor cell-mass yields, and during this phase, growth and ethanol production are decoupled. An improved semi-defined growth medium is established which supports better growth rate and cellular yield, without affecting the ethanol yield.     

Large-scale production of diesel-like biofuels – process design as an inherent part of microorganism development

Industrial biotechnology is playing an important role in the transition to a bio-based economy. Currently, however, industrial implementation is still modest, despite the advances made in microorganism development. Given that the fuels and commodity chemicals sectors are characterized by tight economic margins, we propose to address overall process design and efficiency at the start of bioprocess development. While current microorganism development is targeted at product formation and product yield, addressing process design at the start of bioprocess development means that microorganism selection can also be extended to other critical targets for process technology and process scale implementation, such as enhancing cell separation or increasing cell robustness at operating conditions that favor the overall process. In this paper we follow this approach for the microbial production of diesel-like biofuels. We review current microbial routes with both oleaginous and engineered microorganisms. For the routes leading to extracellular production, we identify the process conditions for large scale operation. The process conditions identified are finally translated to microorganism development targets. We show that microorganism development should be directed at anaerobic production, increasing robustness at extreme process conditions and tailoring cell surface properties. All the same time, novel process configurations integrating fermentation and product recovery, cell reuse and low-cost technologies for product separation are mandatory. This review provides a state-of-the-art summary of the latest challenges in large-scale production of diesel-like biofuels.     

Detection of phase shifts in batch fermentation via statistical analysis of the online measurements: a case study with rifamycin B fermentation

Industrial production of antibiotics, biopharmaceuticals and enzymes is typically carried out via a batch or fed-batch fermentation process. These processes go through various phases based on sequential substrate uptake, growth and product formation, which require monitoring due to the potential batch-to-batch variability. The phase shifts can be identified directly by measuring the concentrations of substrates and products or by morphological examinations under microscope. However, such measurements are cumbersome to obtain. We present a method to identify phase transitions in batch fermentation using readily available online measurements. Our approach is based on Dynamic Principal Component Analysis (DPCA), a multivariate statistical approach that can model the dynamics of non-stationary processes. Phase-transitions in fermentation produce distinct patterns in the DPCA scores, which can be identified as singular points. We illustrate the application of the method to detect transitions such as the onset of exponential growth phase, substrate exhaustion and substrate switching for rifamycin B fermentation batches. Further, we analyze the loading vectors of DPCA model to illustrate the mechanism by which the statistical model accounts for process dynamics. The approach can be readily applied to other industrially important processes and may have implications in online monitoring of fermentation batches in a production facility.     

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.     

Enantiomeric effect of paclobutrazol on the microorganism composition during wine fermentation

Pesticide residues in food can bring potential risks to human health and has been widely concerned in recent years. In the current study, the influence of paclobutrazol, which resided in raw material (grape) on wine fermentation process, were investigated. The degradation kinetic results indicated that the enantiomers of paclobutrazol not be degraded during 30 days of fermentation process. In order to achieve the fermented microorganism information of diversity, community composition, and function, the analysis of 16S rRNA and ITS sequencing were performed. Results demonstrated that the dominant microorganisms multiplied and the microbial diversity in the samples decreased as the fermentation process progresses. Furthermore, the paclobutrazol stimulated the growth of Pichia, which was observed during wine fermentation and which may have an underlying impact on the quality of the wine. The above results inferred that paclobutrazol residue could disturb the microbial community stability during wine fermentation, and the stable existence of paclobutrazol will cause potential risks to food safety and human health. In this work, we have successfully devised a method to investigate the influences of pesticide residues in raw materials during food processing and conclusions from this study could provide basis for dietary risk assessment.     

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.     

The behavior of kinetic parameters in production of pectinase and xylanase by solid-state fermentation

Solid-state fermentation (SSF) is defined as the growth of microbes without a free-flowing aqueous phase. The feasibility of using a citrus peel for producing pectinase and xylanase via the SSF process by Aspergillus niger F3 was evaluated in a 2 kg bioreactor. Different aeration conditions were tested to optimize the pectinase and xylanase production. The best air flow intensity was 1 V kg M (volumetric air flow per kilogram of medium), which allowed a sufficient amount of O2 for the microorganism growth producing 265 U/g and 65 U/g pectinases and xylanases, respectively. A mathematical model was applied to determine the different kinetic parameters related to SSF. The specific growth rate and biomass oxygen yield decreased during fermentation, whereas an increase in the maintenance coefficient for the different employed carbon sources was concurrently observed.     

The application of a novel heat flux calorimeter for studying growth of Escherichia coli W in aerobic batch culture

The modification and principle of a novel heat flux calorimeter for the in situ, on-line measurement of the heat generated during microbial growth is described. Data concerning the physical characterization of the calorimeter as a fermentor, including stability and sensitivity of the heat signal, are presented. The calorimeter has been successfully applied to the study of the aerobic batch culture of Escherichia coli W on glucose under carbon and nitrogen limitation. A direct correlation between growth and heat evolution was obtained. Quantitative analysis of the data suggests that the new calorimetric technique could be used for monitoring growth and specific metabolic events, for convenient medium optimization, and as a basis for a novel fermentation process control system.     

Physiological state control of fermentation processes

In this article a novel approach to the control of fermentation processes is introduced. A "physiological state control approach" has been developed using the concept of representing fermentation processes through the current physiological state of the cell culture. No conventional mathematical model is required for the synthesis of such a control system.The main idea is based on the fact that during batch, feed-batch, or even continuous cultivation the physiological characteristics of the cell population, jointly expressed by the term "physiological state", are not constant but rather variable, which is reflected in expected or unexpected changes in the behavior of the control plant, and which requires flexible alteration of the current control strategy. The proposed approach involves decomposition of the physiological state space into several subspaces called "physiological situations." In every physiological situation the cell population expresses stable characteristics, and therefore an invariant control strategy can be effectively applied. The on-line functions of the physiological state control system consist of the calculation of physiological state variables, determination of the current physiological situation as an element of a previously defined set of known physiological situations, switching of the relevant control strategy, and calculation of the control action. Attention is focused on the synthesis of the novel and nonstandard part of the control system - the algorithm for online recognition of the current physiological state. To this end an effective approach, based on artificial intelligence methods, particularly fuzzy sets theory and pattern recognition theory, was developed. Its practical realization is demonstrated using data from a continuous fermentation process for single cell protein production.     

Degradation of pectins with different degrees of esterification by Bacteroides thetaiotaomicron isolated from human gut flora

A complete human fecal flora and cultures of defined species obtained from fecal flora were investigated in vitro to determine their ability to ferment the dietary fiber pectin. Bacteroides thetaiotaomicron was tested as a pectin-degrading microorganism alone and in coculture with Escherichia coli. Macromolecular pectins with different degrees of esterification were used as substrates in microbial degradation studies. The levels of oligogalacturonic acids formed in batch cultures were estimated during a 24- or 48-h incubation period by using high-performance thin-layer chromatography and high-performance anion-exchange chromatography. The spectrum and the amount of unsaturated oligogalacturonic acids formed as intermediate products of pectin fermentation changed permanently in the culture media during incubation with the complete fecal flora. After 24 h, no oligogalacturonic acids were detected. The pectin-degrading activities of pure cultures of B. thetaiotaomicron were lower than the pectin-degrading activity of a complete fecal flora. Cocultures of B. thetaiotaomicron and E. coli exhibited intermediate levels of degradation activity. In pure cultures of E. coli no pectin-degrading activity was found. Additionally, the rate of pectin degradation was affected by the degree of esterification of the substrate. Saturated oligogalacturonic acids were not found during pectin fermentation. The disappearance of oligogalacturonic acids in the later stages of fermentation with both the complete fecal flora and B. thetaiotaomicron was accompanied by increased formation of short-chain fatty acids.     

红茶菌群中菌株相互作用影响菌体生长和代谢

[背景]红茶菌是一种由细菌和酵母菌共生发酵而成的传统茶饮料.该饮料中含有多种有益人体健康的营养物质,具有促进消化、消炎、抗菌、抗糖尿病等生理作用.这些有益的代谢物是以醋酸菌和酵母菌为主的微生物相互作用而产生的.因此,红茶菌是一个优良的研究微生物相互作用的体系.[目的]分析不同菌株单独培养和混合培养对菌体生长和代谢产物的...