PVA共固定化双菌种发酵海藻酒的研究*

采用PVA为载体共固定化酿酒酵母和产香酵母发酵海藻,酿造海藻酒。对游离细胞与固定化细胞的分批发酵和连续发酵的动力学进行了研究并建立了相应的发酵动力学方程。实验结果表明：酿酒酵母和产香酵母二种菌种菌量的最佳配比为4：l,发酵温度20℃．共固定化细胞分批发酵和连续发酵凝胶粒的充填系数分别为0．25和0．5,游离混合细胞的发酵时间为7d。共固定化细胞连续发酵稀释速率0．12／h,其发酵时间为0.5d左右。经160d连续发酵实验,PVA固定化细胞粒子的机械强度良好。     

PVA共固定化双菌种发酵海藻酒的研究

采用ＰＶＡ为载体共固定化酿酒酵母和产香酵母发酵海藻,酿造海藻酒。对游离细胞与固定化细胞的分批发酵和连续发酵的动力学进行了研究并建立了相应的发酵动力学方程。实验结果表明：酿酒酵母和产香酵母二种菌种菌量的最佳配比为４∶１,发酵温度２０℃,共固定化细胞分批发酵和连续发酵凝胶粒的充填系数分别为０２５和０５,游离混合细胞的发酵时间为７ｄ,共固定化细胞连续发酵稀释速率０１２／ｈ,其发酵时间为０５ｄ左右。经１６０ｄ连续发酵实验,ＰＶＡ固定化细胞粒子的机械强度良好。     

固定化酵母酒精生成动力学及其数学模型

本文采用解析法对海藻酸钙为载体,以葡萄糖为底物进行酒精连续发酵的管式反应器内所呈现的固定化K字酵母酒精发酵动力学进行了较为系统的研究,建立了由6个方程组成的酒精生成动力学数学模型,并对此模型进行了应用方面的研究。结果表明：此动力学呈现葡萄糖的反竞争性和酒精非竞争性联合抑制的不可逆特征,其数学模型在所涉及的参数拟合及分别在不同反应器和浓度扩展时的发酵过程所进行的实际校验中,最大的算术平均百分误差为13．8l％,在薯干糖化液的发酵动力学应用中,具有平均差为8．28％之良好预测精度,同时,通过模型中参数式计算得葡萄糖抑制的浓度范围为130—450g／L,而酒精抑制的浓度范围是3．07—16．45％(v／v)。     

固定化酵母酒精发酵动力学性质的初步研究

用固定化细胞连续发酵生产酒精是酒精发酵工艺的重大改革,有关这方面的研究已有许多报道,部分已完成了中试。不过、到目前为止,固定化酵母酒精发酵动力学性质方面的研究只有少数报道。本文用海藻酸钠固定化酵     

微球载体固定化纤维素酶的反应动力学模型研究

建立了固定化纤维素酶的反应动力学模型,该模型以米氏方程为基础并考虑了产物竞争性抑制的影响。在此模型的基础上进行模拟,系统研究了产物竞争性抑制、内扩散限制、溶液中的宏观底物浓度、载体大小等因素对球形载体内部的底物、产物浓度分布和效率因子的影响。产物竞争性抑制的存在将增加载体颗粒内的底物浓度,对效率因子的影响较小。底物内扩散系数或者反应体系中底物浓度增大时,载体颗粒内的底物浓度和效率因子都将增大。载体粒径增大,载体颗粒内的底物浓度和效率因子均减小。     

膜生物反应器连续发酵法制取甲醇及其发酵动力学初探

采用膜生物反应器系统连续发酵制取甲醇能够及时分离产物,有效抑制甲醇对细胞的毒副作用,因此延长稳定期产甲醇的时间以提高产量。本文对比间歇发酵,研究了不同稀释率0．05h^-1～0．13h^-1下连续发酵的甲醇生成和甲烷氧化菌株Methylomonas．QJ16生长状况,并且初步探讨了该菌株的生长、甲醇形成的动力学特性。结果表明,稀释率为0．1h^-1时,菌体积累和甲醇的体积产率均较高,最长连续发酵持续时间为300h左右;描述连续发酵过程的动力学模型,菌体生长和产物合成的曲线拟合优度分别为0．991、0．994,基本反映了该甲烷氧化菌株连续发酵过程的动力学特征。     

利用固定化细胞连续发酵生产酸牛奶

本文报道了利用固定化技术连续发酵生产酸牛奶的方法。对单菌种与双菌种固定化、最适发酵温度和pH、发酵时间、固定化方式等进行了研究,得出了在实验室条件下,连续发酵生产酸牛奶的最佳技术条件。与传统的间歇生产工艺相比,可简化菌种制备过程,反复利用乳酸菌种,充分利用发酵酸化设备、便于自动化控制等优点。作者尚未见国内外利用固定化技术连续生产酸牛奶的报道。     

固定化根霉发酵生产脂肪酶

以聚氨酯为少根根霉固定化载体,对固定化后的细胞连续重复批次发酵进行了研究。优化了重复批次发酵培养基组成。在取代发酵液40mL,取代培养基组成为全脂豆粉3%,花生油0.5％条件下,固定化菌体摇瓶实验可连续使用140h,重复9批次。酶的时空产率提高6倍。5L发酵罐小试固定化菌体可连续发酵6批次。固定化细胞连续发酵,大大缩短了发酵的时间,酶的时空产率获得大幅提高。     

无载体固定化细胞的研究进展

以无载体固定化酵母细胞酒精连续发酵的成功工业化应用为实例,并与通常的载体固定化细胞技术比较,阐述了无载体固定化细胞技术的优缺点,系统提出了无载体固定化细胞技术的概念,进而对无载体固定化细胞技术在其它微生物发酵和动植物细胞培养过程的应用前景进行了展望。     

聚乙烯醇固定化酵母细胞制备CTP研究

以聚乙烯醇为固定化载体,固定化冷冻处理过的啤酒酵母细胞从CMP制备CTP;分别从胶的型号、浓度和固定化方法的优化等方面摸索了最适的固定化条件,固定化细胞在试验条件下连续发酵8次,转化率维持在85%～95%。同时,还对固定化细胞的稳定性进行试验研究,并用HPLC对产品进行了分离鉴定。     

Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.     

Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose

Escherichia coli FBR5 containing recombinant genes for ethanol production on plasmids that are also required for anaerobic growth was cultivated continuously on 50 g/l xylose or glucose in the absence of antibiotics and without the use of special measures to limit the entry of oxygen into the fermenter. Under chemostat conditions, stable ethanol yields of ca. 80–85% of the theoretical were obtained on both sugars over 26 days at dilution rates of 0.045/h (xylose) and 0.075/h (glucose), with average plasmid retention rates of 96% (xylose) and 97% (glucose). In a continuous fluidized bed fermenter, with the cells immobilized on porous glass beads, the extent of plasmid retention by the free cells fell rapidly, while that of the immobilized cells remained constant. This was shown to be due to diffusion of oxygen through the tubing used to recirculate the medium and free cells. A change to oxygen-impermeable tubing led to a stable high rate of plasmid retention (more than 96% of both the free and immobilized cells) with ethanol yields of ca. 80% on a 50 g/l xylose feed. The maximum permissible level of oxygen availability consistent with high plasmid retention by the strain appears to be of the order of 0.1 mmol per hour per gram dry biomass, based on measurements of the rate of oxygen penetration into the fermenters. Revertant colonies lacking the ethanologenic plasmid were easily detectable by their morphology which correlated well with their lack of ampicillin resistance upon transfer plating.     

A general solution for the steady-state kinetics of immobilized enzyme systems

A power series solution is presented which describes the steady-state concentration profiles for substrate and product molecules in immobilized enzyme systems. Diffusional effects and product inhibition are incorporated into this model. The kinetic consequences of diffusion limitation and product inhibition for immobilized enzymes are discussed and are compared to kinetic behavior characteristic of other types of effects, such as substrate inhibition and substrate activation.     

Simultaneous uptake and utilisation of glucose and xylose by Clostridium thermohydrosulfuricum

Abstract Growth studies of Clostridium thermohydrosulfuricum Rt8.B1 demonstrated that glucose and xylose were used simultaneously when supplied together at nonlimiting concentrations in pH-controlled batch culture. Under conditions of hyperbolic growth, both catabolite repression and inducer exclusion were absent. Glucose did not repress xylose metabolism (i.e. xylose permease and xylose isomerase genes were expressed in the presence of glucose and were not subject to catabolite inhibition when glucose was added to cultures growing on high concentrations of xylose). The kinetics of glucose and xylose utilisation indicated that separate systems were present for the uptake of these substrates when supplied together. Glucose utilisation was biphasic, indicating high- and low-affinity systems for glucose uptake. Xylose utilisation was directly proportional to the xylose concentration, suggesting a facilitated diffusion mechanism was operative for uptake.     

A simple kinetic model for the hydrolysis of α-d-glucans using glucoamylase immobilized on titanium (IV) activated porous silica

A simple kinetic model which describes the hydrolysis of α-d-glucans by immobilized glucoamylase (exo-1,4-d-glucosidase, EC 3.2.1.3) is reported. The hydrolysis of starch, amylose, amylopectin, maltose and 40DE starch hydrolysates using glucoamylase immobilized on alkylamine derivatives of titanium(IV) activated porous silica are described by a kinetic model based on Langmuir-Hinshelwood kinetics. This model involves enzyme kinetics with or without product inhibition and reverse reactions as well as mass transfer and diffusion effects in immobilized enzyme reactors. The results of other authors are also interpreted by the model developed in this article.     

Application of a compatible xylose isomerase in simultaneous bioconversion of glucose and xylose to ethanol

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose mixture was carried out by the yeastSaccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose andS. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and 30°C. This compatible xylose isomerase fromCandida boidinii, having an optimum pH and temperature range of 4.5–5.0 and 30–50°C respectively, was partially purified and immobilized on an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol byS. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed) and xylose conversion efficiency of 42.8%.     

Sugar cane bagasse as a possible source of fermentable carbohydrates. II. Optimization of the xylose isomerase reaction for isomerization of xylose as well as sugar cane bagasse hydrolyzate to xylulose in laboratory-scale units

Both the forward and backward reactions of xylose isomerase (Sweetzyme Q) with xylose and glucose as substrates have been studied in terms of kinetics and thermodynamics. The relationship between the two reactions can thus be determined. Much attention has been given to the reaction with xylose as substrate. The optimal conditions of the xylose reaction in terms of pH, buffer, metal ions, substrate concentration, temperature, and ionic strength have been determined. These findings did not differ much from those reported for the glucose reaction. Equilibrium constants for the aldose to ketose conversion were more favorable in the case of glucose. The results obtained with continuous isomerization of xylose in columns packed with either Sweetzyme Q or Taka-Sweet were very similar to those obtained from batch isomerization processes. Particle size had a definite effect on reaction rate, which indicates that diffusion limitations do occur with the immobilized enzyme particles. Heat stability of Sweetzyme Q was good with t(1/2) of 118, 248, and 1200 h at 70, 55, and 40 degrees C, respectively. A novel method for the separation of xylose-xylulose mixtures with water as eluant on a specially prepared Dowex 1 x 8 column was developed. This technique has the capability of producing pure xylulose for industrial or research applications. A writ for a patent regarding this technique is at present prepared.     

Phosphorus-31 and carbon-13 nuclear magnetic resonance study of glucose and xylose metabolism in agarose-immobilized Candida tropicalis.

Candida tropicalis can ferment both hexose and pentose sugars. Here, we have used 31P and 13C nuclear magnetic resonance spectroscopy to study the capacity of this yeast species to metabolize glucose or xylose when immobilized in small (< 1-mm-diameter) agarose beads. Immobilized C. tropicalis metabolizing glucose showed rapid initial growth within the beads. A corresponding drop in the intracellular pH (from 7.8 to 7.25) and hydrolysis of intracellular polyphosphate stores were observed. Although the initial rate of glucose metabolism with immobilized C. tropicalis was similar to the rate observed previously in cell suspensions, a decrease by a factor of 2.5 occurred over 24 h. In addition to ethanol, a significant amount of glycerol was also produced. When immobilized C. tropicalis consumed xylose, cell growth within the beads was minimal. The intracellular pH dropped rapidly by 1.05 pH units to 6.4. Intracellular ATP levels were lower and intracellular Pi levels were higher than observed with glucose-perfused cells. Consumption of xylose by immobilized C. tropicalis was slower than was previously observed for oxygen-limited cell suspensions, and xylitol was the only fermentation product.     

Mathematical modelling of ethanol production from glucose/xylose mixtures by recombinant Zymomonas mobilis

A model has been developed for the fermentation of mixtures of glucose and xylose by recombinant Zymomonas mobilis strain ZM4(pZB5), containing additional genes for xylose assimilation and metabolism. A two-substrate model based on substrate limitation, substrate inhibition, and product (ethanol) inhibition was evaluated, and experimental data was compared with model simulations using a Microsoft EXCEL based program and methods of statistical analysis for error minimization. From the results it was established that the model provides good predictions of experimental batch culture data for 25/25, 50/50, and 65/65 g l^–1 glucose/xylose media.     

Steady state model for evaluation of external and internal mass transfer effects in an immobilized biofilm

A steady model for the evaluation of external liquid film diffusion and internal pore diffusion effects in an immobilized biofilm system under continuous mode of operation was developed. The model takes into account, substrate diffusion through external liquid film and biofilm. Average rate of substrate consumption in the biofilm was considered. The overall efficiency of the biofilm was mathematically represented by considering the combined effects of substrate penetration and substrate utilization in the biofilm. The model was illustrated using a case study of pyridine biodegradation in a rotating biological contactor immobilized with pyridine degrading microbial film. The model is able to effectively predict both internal and external mass transfer effects in an immobilized biofilm system.