补料发酵工艺的应用及其研究进展

综述了补料工艺在发酵工业中应用和研究。介绍了补料发酵工艺及其优点,着重讨论了补料发酵动力学和控制理论研究,以期为补料发酵的应用提供充分的参考依据。     

Optimization of exopolysaccharide production by Tremella mesenterica NRRL Y-6158 through implementation of fed-batch fermentation

In liquid culture conditions, the yeast-like fungus Tremella mesenterica occurs in the yeast state and synthesizes an exopolysaccharide (EPS) capsule, which is eventually released into the culture fluid. It is composed of an α-1,3-D-mannan backbone, to which β-1,2 side chains are attached, consisting of D-xylose and D-glucuronic acid. Potato dextrose broth (PDB) seemed to be an excellent medium for both growth of the yeast cells and synthesis of the EPS. This medium is composed solely of an extract of potatoes to which glucose was added. Yet an important disadvantage of this production medium is the presence of starch in the potato extract, since Tremella cells are not capable of metabolizing this component; furthermore, it coprecipitates upon isolation of the polymer [3]. In this respect, it was essential to remove the starch in order to achieve high polysaccharide production and recovery. A good method was the removal of starch through ultrafiltration of the PDB medium before inoculation of the strain. This resulted in an excellent starch-free medium in which other components essential for polysaccharide production were still present [3]. Through implementation of single and cyclic fed-batch fermentations with glucose feed, 1.6- and 2.2-fold increases in EPS yield were obtained, respectively. Lowering the carbon source level by using a cyclic fed-batch technique might decrease the osmotic effect of glucose or any catabolite regulation possibly exerted by this sugar on enzymes involved in EPS synthesis. Journal of Industrial Microbiology & Biotechnology (2002) 29, 181–184 doi:10.1038/sj.jim.7000276 Received 18 March 2002/ Accepted in revised form 20 May 2002     

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l⁻¹) and less than 200 g l⁻¹ total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l⁻¹ xylitol concentration, 0.75 g xylitol g xylose⁻¹ xylitol yield and 3.9 g xylitol l⁻¹ h⁻¹ volumetric productivity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 16–19 doi:10.1038/sj.jim.7000257 Received 15 October 2001/ Accepted in revised form 30 March 2002     

腈水解酶psn基因工程菌的发酵及产酶条件优化

来自恶臭假单胞菌的腈水解酶具有高效催化3-氰基吡啶产烟酸的能力,对表达该酶的基因psn进行发酵和产酶条件优化,通过对C源、N源、磷酸盐、金属离子、温度、诱导剂浓度和诱导时间进行单因素考察,获得最适培养基条件(g/L):葡萄糖5、蛋白胨15、酵母粉5、(NH4)2SO45、K2HPO424.5、KH2PO45.76、MgSO40.48;最佳诱导条件:培养2.5 h后添加IPTG诱导,浓度0.2 mmol/L,诱导温度30℃。在该条件下培养,重组大肠杆菌的腈水解酶比酶活可达到45.67 U/mL,比优化前提高了2.26倍。在此基础上,于5 L发酵罐上进行C、N源的补料研究,获得最适分批补料策略,发现其腈水解酶活力可达到75.40 U/mL,是优化前的3.74倍。     

Optimal nonsingular control of fed-batch fermentation

Presented is a new simple method for multidimensional optimization of fed-batch fermentations based on the use of the orthogonal collocation technique. Considered is the problem of determination of optimal programs for fermentor temperature, substrate concentration in feed, feeding profile, and process duration. By reformulation of the state and control variables is obtained a nonsingular form of the optimization problem which has considerable advantage over the singular case since a complicated procedure for determination of switching times for feeding is avoided. The approximation of the state variables by Lagrange polynomials enables simple incorporation of split boundary conditions in the approximation, and the use of orthogonal collocations provides stability for integration of state and costate variables. The interpolation points are selected to obtain highest accuracy for approximation of the objective functional by the Radau-Lobatto formula. The control variables are determined by optimization of the Hamiltonian at the collocation points with the DFP method. Constraints are imposed on state and control variables.The method is applied for a homogeneous model of fermentation with volume, substrate, biomass, and product concentrations as the state variables. Computer study shows considerable simplicity of the method, its high accuracy for low order of approximation, and efficient convergence.     

Ethanol yields during the feeding phase in fed-batch fermentation of sugar-cane blackstrap molasses

The efficiency of ethanol yield increased from 61% to 88% of the theoretical value as the filling-up time was approached in fed-batch fermentation with Saccharomyces cerevisiae. Temporary accumulation of ethanol within the yeast cells may explain the above variation.The author is with the Instituto Mauá de Tecnologia, Estrada das Lágrimas 2035, 09580-900. Sao Caetano Do Sul, SP, Brazil     

甘蔗渣和糖蜜混合发酵制备燃料丁醇

以抗逆突变株Clostridium beijerinckii IB4为研究对象,葡萄糖为C源,对其进行补料分批发酵过程的优化,同时将该优化工艺应用于甘蔗渣和糖蜜混合发酵制备燃料丁醇。结果表明:在5 L发酵罐中,先加入作为还原糖的甘蔗渣酸解糖液10 g/L,16 h后补加甘蔗糖蜜30 g/L,于35℃、100 r/min发酵50 h,丁醇和总溶剂产量分别达到11.1和15.3 g/L,丁醇比例高达72.5%。     

基因工程菌高密度发酵工艺研究进展

阐述了基因工程菌高密度发酵工艺的几个主要影响因素,包括重组菌构建、培养条件、生长抑制因子以及它们的控制技术。通过高密度发酵可以提高细胞生长密度、目的蛋白的表达含量。在高密度发酵过程中,会产生一些有害抑制代谢副产物,但通过分批补料可以降低影响。     

Enhanced phenylpyruvic acid production with Proteus vulgaris in fed-batch and continuous fermentation

Phenylpyruvic acid is a deaminated form of phenylalanine and is used in various areas such as development of cheese and wine flavors, diagnosis of phenylketonuria, and to decrease excessive nitrogen accumulation in the manure of farm animals. However, reported phenylpyruvic acid fermentation studies in the literature have been usually performed at shake-flask scale with low production. In this study, phenylpyruvic acid production was evaluated in bench-top bioreactors by conducting fed-batch and continuous fermentation for the first time. As a result, maximum phenylpyruvic acid concentrations increased from 1350 mg/L (batch fermentation) to 2958 mg/L utilizing fed-batch fermentation. Furthermore, phenylpyruvic acid productivity was increased from 48 mg/L/hr (batch fermentation) to 104 and 259 mg/L/hr by conducting fed-batch and continuous fermentation, respectively. Overall, this study demonstrated that fed-batch and continuous fermentation significantly improved phenylpyruvic acid production in bench-scale bioreactor production.     

Heterotrophic production of ascorbic acid by microalgae

An aerobic fermentation process has been developed for the production of L-ascorbic acid (vitamin C). After an extensive screening program for microorganisms capable of heterotrophically synthesizing L-ascorbic acid, a unicellular green microalga,Chlorella pyrenoidosa, was selected. This organism has a number of characteristics that recommend it as an industrial organism: (1) it can double every 3.5 h when growing aerobically in the dark on a glucose-minimal salts medium; (2) its small size and tough cell wall make it very insensitive to shear, allowing very high impeller velocities; (3) it can be grown to 100 g L^–1 cell dry weight; (4) it is readily mutable by classical mutagenesis techniques; and (5) it has efficient growth kinetics with respect to yield of cell mass on glucose and oxygen. Fermentation process development and classical strain improvement have resulted in a greater than 70-fold increase in intracellular ascorbic acid concentration compared to the parent strainC. pyrenoidosa UTEX 1663. The process is compatible with existing industrial fermentation technology and equipment and is described in U.S. Patent 5,001,059. Patents have been submitted for a process in which the ascorbic acid accumulates extracellularly.     

Comparative proteome analysis of robust <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> insights into industrial continuous and batch fermentation

A robust Saccharomyces cerevisiae strain has been widely applied in continuous and batch/fed-batch industrial fermentation. However, little is known about the molecular basis of fermentative behavior of this strain in the two realistic fermentation processes. In this paper, we presented comparative proteomic profiling of the industrial yeast in the industrial fermentation processes. The expression levels of most identified protein were closely interrelated with the different stages of fermentation processes. Our results indicate that, among the 47 identified protein spots, 17 of them belonging to 12 enzymes were involved in pentose phosphate, glycolysis, and gluconeogenesis pathways and glycerol biosynthetic process, indicating that a number of pathways will need to be inactivated to improve ethanol production. The differential expressions of eight oxidative response and heat-shock proteins were also identified, suggesting that it is necessary to keep the correct cellular redox or osmotic state in the two industrial fermentation processes. Moreover, there are significant differences in changes of protein levels between the two industrial fermentation processes, especially these proteins associated with the glycolysis and gluconeogenesis pathways. These findings provide a molecular understanding of physiological adaptation of industrial strain for optimizing the performance of industrial bioethanol fermentation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Software sensing for glucose concentration in industrial antibiotic fedbatch culture using fuzzy neural network

In order to control glucose concentration during fed-batch culture for antibiotic production, we applied so called “software sensor” which estimates unmeasured variable of interest from measured process variables using software. All data for analysis were collected from industrial scale cultures in a pharmaceutical company. First, we constructed an estimation model for glucose feed rate to keep glucose concentration at target value. In actual fed-batch culture, glucose concentration, was kept at relatively high and measured once a day, and the glucose feed rate until the next measurement time was determined by an expert worker based on the actual consumption rate. Fuzzy neural network (FNN) was applied to construct the estimation model. From the simulation results using this model, the average error for glucose concentration was 0.88 g/L. The FNN model was also applied for a special culture to keep glucose concentration at low level. Selecting the optimal input variables, it was possible to simulate the culture with a low glucose concentration from the data sets of relatively high glucose concentration. Next, a simulation model to estimate time course of glucose concentration during one day was constructed using the on-line measurable process variables, since glucose concentration was only measured off-line once a day. Here, the recursive fuzzy neural network (RFNN) was applied for the simulation model. As the result of the simulation, average error of RFNN model was 0.91 g/L and this model was found to be useful to supervise the fed-batch culture.     

蛋白质组学技术在工业发酵梭菌研究中的应用

介绍了目前应用较广泛的蛋白质组学技术的原理、应用及优缺点;总结了发酵工业中常用的梭菌属细菌;重点阐述了蛋白质组学技术在工业发酵梭状芽孢杆菌研究中的应用,为工业发酵菌种的改良和发酵工艺的优化提供理论依据。最后讨论了今后工业发酵菌种蛋白质组学研究的重点和方向。     

补料分批发酵过程中两阶段发酵生产1,3-丙二醇

在补料分批发酵过程中提高比生长速率不仅减少乙醇、甲酸的生成,而且提高1,3-丙二醇的得率和比生产速率.发酵后期甘油的浓度在15～26 g/L时有利于提高1,3-丙二醇的生产.采取在发酵前期控制菌体较高比生长速率和发酵后期控制适宜甘油浓度相结合的策略,有效地提高了1,3-丙二醇的生产,降低副产物乳酸和乙醇的生成.     

Biodegradation of phenols by microalgae

Two green microalgae, Ankistrodesmus braunii and Scenedesmus quadricauda, degraded phenols (each tested at 400 mg ml^–1) selected from olive-oil mill wastewaters, within 5 days, with a removal greater than 70%. Green algae may, therefore, represent an alternative to other biological treatment used for the biodegradation of phenol-containing wastewaters.     

Statistical medium optimization and DO-STAT fed-batch fermentation for enhanced production of tyrosine phenol lyase in recombinant Escherichia coli

Abstract

Tyrosine phenol lyase (TPL) is a robust biocatalyst for the production of L-dihydroxyphenylalanine (L-DOPA). The improvement of TPL production is conducive to the industrial potential. In this study, the optimization of culture medium of recombinant Escherichia coli harboring TPL from Fusobacterium nucleatum (Fn-TPL) was carried out. Sucrose and combination of yeast extract and peptone were selected as carbon and nitrogen source, respectively. Their optimal concentrations were determined by Box-Behnken design and the synergistic effect between yeast extract and peptone was found to be significant, with p-value < 0.05. The DO-STAT fed-batch fermentation under optimized culture condition was established and the oxygen level was fixed at 20%. Both the biomass and Fn-TPL activity were significantly increased, which were 35.6 g dcw/L and 12292 U/L, respectively. The results obtained significantly promote the industrial production of L-DOPA production.     

Cell retention culture with an internal filter module: continuous ethanol fermentation

A new internal filter feedback system with a stainless steel filter was introduced and its application for continuous ethanol fermentation was investigated. The filter performance was highly influenced by agitation speed and yeast concentration. Retention coefficient with a filter of 2 mum pore size was found more than 97.5%, and the filter was suitable for yeast separation. Maximum yeast concentration was 157 g/L and the best operable cell concentration was between 90 and 150 g/L. Which was similar to that obtained in the external membrane cell recycle culture. The cell concentration in the fermentor was maintained by manipulation of dilution rate and bleed ratio with the growth rate. The internal filter feedback system was successfully operated for more than 10 days. This study shows that the internal filter feedback system with a stainless steel filter can be used high-density cell culture and ethanol fermentation. Furthermore, it can be scaled up more easily than the external cell recycle system. (c) 1993 John Wiley & Sons, Inc.     

高生物量富硒酵母的选育及发酵条件的研究

采用硒浓度耐性培养,从8株酿酒酵母中筛选驯化得到一株高生物量富硒酵母菌株NH-7,并对其发酵条件进了行优化。采取流加培养,在菌株稳定期,即18 h开始分批添加亚硒酸钠至总硒浓度为30μg/mL,控制乙醇浓度为0.4%和0.7%,分别得到20.14 g的高生物量(每升培养液中获得的酵母菌体干重)和82.52%的硒转化率。在60μg/mL总硒浓度的流加培养中,得到高达2 411μg/g的有机硒含量,每升发酵液菌体干重为18.83 g。     

Interactions between planktonic microalgae and protozoan grazers

For an algal bloom to develop, the growth rate of the bloom-forming species must exceed the sum of all loss processes. Among these loss processes, grazing is generally believed to be one of the more important factors. Based on numerous field studies, it is now recognized that microzooplankton are dominant consumers of phytoplankton in both open ocean and coastal waters. Heterotrophic protists, a major component of microzooplankton communities, constitute a vast complex of diverse feeding strategies and behavior which allow them access to even the larger phytoplankton species. A number of laboratory studies have shown the capability of different protistan species to feed and grow on bloom-forming algal species. Because of short generation times, their ability for fast reaction to short-term variation in food conditions enables phagotrophic protists to fulfill the function of a heterotrophic buffer, which might balance the flow of matter in case of phytoplankton blooms. The importance of grazing as a control of microalgae becomes most apparent by its failure; if community grazing controls initial stages of bloom development, there simply is no bloom. However, if a certain algal species is difficult to graze, e.g. due to specific defense mechanisms, reduced grazing pressure will certainly favor bloom development. The present contribution will provide a general overview on the interactions between planktonic microalgae and protozoan grazers with special emphasis on species-specific interactions and algal defense strategies against protozoan grazers.     

在林可霉素生物合成过程中铵离子调控作用的酶学研究

在FUS-50L发酵罐内,用林可链霉菌发酵生产林可霉素。研究发现,NH4^＋对林可霉素发酵过程具有显著的调控效应：补入硫酸铵前,发酵液中的NH4^＋浓度由3．0mmol／L消耗至1．0mmol／L以下的控制过程非常关键,这样可能使林可霉素合成酶大量合成,同时,解除了NH4^＋对谷氨酰合成酶（GS）的阻遏效应;20～24h,尽可能提高硫酸铵的平均补入速率,但最高NH4^＋的瞬时浓度应控制在19．0mmol／L以下,一方面可缩短生物量的积累时间,另一方面可避免过高浓度的NH4^＋对GS的抑制作用;24h后逐渐降低NH4^＋的平均补入速率,使主代谢流转入次级代谢;在发酵中期和后期,应将最低NH4^＋浓度控制在3．0～4．0mmol／L的范围内,避免铵离子对GS的阻遏效应,GS比活力与林可霉素的产量呈正相关关系。最终建立了动态的硫酸铵补加工艺。