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。     

Process monitoring of an industrial fed-batch fermentation

Market demand places great emphasis in industry on product quality. Consequently, process monitoring and control have become important aspects of systems engineering. In this article we detail the results of a 2-year study focusing on the development of a condition monitoring system for a fed-batch fermentation system operated by Biochemie Gmbh in Austria. We also demonstrate the suitability and limitations of current state of the art technologies in this field and suggest novel modifications and configurations to improve their suitability for application to a fed-batch fermentation system.     

固态间歇补料乙醇生料发酵新工艺

浓醪发酵是酒精生产的发展方向。与现行酒精厂普遍采用的热蒸煮工艺相比, 生料发酵技术的发展使得浓醪发酵更容易进行。本研究首次在生料发酵中直接采用固态原料间歇补料, 比较了STARGENTM生淀粉水解酶间歇补料工艺和传统无补料工艺, 并对不同补料方式进行了研究。结果表明: 与传统无补料生料发酵工艺相比, 在相同的干基配料浓度30%、相同的生料酶添加量0.22%(W/W)的条件下, 采用15%的起始配料浓度、发酵15~25 h进行间歇补料的新工艺, 酒精产量从17.06%提高到18.50%。该间歇补料优化工艺的建立, 丰富了生料发酵技术的应用。     

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.     

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％。     

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%。     

Optimization of fermentation conditions for the production of ethylene-diamine-disuccinic acid by Amycolatopsis orientalis

The production of EDDS (ethylene-diamine-disuccinic acid), a potential substitute for EDTA, has been optimized up to a product concentration of 20 grams per litre in fermentations of Amycolatopsis orientalis. Decisive steps for the increase in productivity were variation of the synthetic medium composition, investigation of the influence of metal ions on product formation, controlled feeding of carbon and nitrogen sources in fed-batch fermentations and improvement of the downstream processing steps. Received 05 May 1997/ Accepted in revised form 13 August 1997     

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.     

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。     

冬虫夏草菌液体培养补料对菌丝体和腺苷产量的影响

为提高冬虫夏草菌菌丝体及腺苷产量,探索了液体培养中进行营养物质补料的方法。通过在培养10d时进行碳、氮源联合补料,将浓度分别补充至47g/L和0.76g/L,则冬虫夏草菌丝体干重和腺苷产量分别达到27.94g/L和1.15mg/g（20d培养周期）,为对照组的2.43倍和19.2倍。这项研究对冬虫夏草菌的大规模工业应用有参考意义。     

微藻规模化培养研究进展

微藻作为地球上最古老的物种之一,其诞生可追溯到35亿多年前.微藻的种类十分丰富,形态也多种多样.微藻一般都含有叶绿体,因此可进行光合作用,有研究表明微藻固定CO2的能力是陆地植物的10倍.微藻以其丰富的代谢产物及独特的生理特性在可再生能源、生物医药、食品工业和环境监测等方面有着广泛的应用.然而如何在控制成本的前提下对微...     

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.     

Manipulating fatty-acid profile at unit chain-length resolution in the model industrial oleaginous microalgae Nannochloropsis

The chain length (CL) of fatty acids (FAs) is pivotal to oil property, yet to what extent it can be customized in industrial oleaginous microalgae is unknown. In Nannochloropsis oceanica, to modulate long-chain FAs (LCFAs), we first discovered a fungi/bacteria-originated polyketide synthase (PKS) system which involves a cytoplasmic acyl-ACP thioesterase (NoTE1). NoTE1 hydrolyzes C16:0-, C16:1- and C18:1-ACP in vitro and thus intercepts the specific acyl-ACPs elongated by PKS for polyunsaturated FA biosynthesis, resulting in elevation of C16/C18 monounsaturated FAs when overproduced and increase of C20 when knocked out. For medium-chain FAs (MCFAs; C8-C14), C8:0 and C10:0 FAs are boosted by introducing a Cuphea palustris acyl-ACP TE (CpTE), whereas C12:0 elevated by rationally engineering CpTE enzyme's substrate-binding pocket to shift its CL preference towards C12:0. A mechanistic model exploiting both native and engineered PKS and type II FAS pathways was thus proposed for manipulation of carbon distribution among FAs of various CL. The ability to tailor FA profile at the unit CL resolution from C8 to C20 in Nannochloropsis spp. lays the foundation for scalable production of designer lipids via industrial oleaginous microalgae.     

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。