Mass transfer effects in fermentations using immobilized whole cells

The immobilization of whole cells for fermentation processes has many potential advantages over fermentation with free cells, including higher cell concentrations, higher productivites and a higher level of operational stability. Most of the research reported in the literature has been directed towards demonstrating the feasibility of using these systems for various fermentations. The ultimate goal of research in this area is to bring the understanding of immobilized whole cells to the level of heterogeneous catalysis. Immobilized whole cell systems are examined from a mass transfer perspective. Evidence for external and internal mass transfer limitations is presented. Procedures for quantifying these effects in terms of effectiveness factors and determining the reaction kinetics in their presence are reviewed. Development of the reactor design equations and the reactor performance results for fermentations with immobilized cells are also discussed.     

Theme 4: Immobilized Cell Technology in Wine Production

Abstract

In spite of its traditional nature, wine making is largely concerned with the progress of biotechnology. High cell density reactors have potential for enology: improved performance of alcoholic and malolactic fermentations, smaller scale fermentation facilities, adaptation to continuous processes. Among the immobilization techniques, cell entrapment in alginate beads seems to be an impressive one. Alcoholic fermentation of wine, malolactic fermentation, bottle fermentation known as “Methode champenoise” and sparkling wine are among the industrial applications. Knowledge of kinetics and physiology in microorganisms in heterogeneous media has expanded in the last few years. The use of immobilized yeast cells for the champagne method would greatly simplify “remuage”. The compared metabolism of entrapped and free cells during the bottle fermentation shows differences, but the final product does not reveal significant sensory disparity. New products can be obtained with more thoroughly controlled conditions.     

Immobilized Cells in Meat Fermentation

Abstract

The immobilization of microbial cells can contribute to fermented meat technology at two basic levels. First, the solid/semisolid nature (low available water) of the substrate restricts the mobility of cells and results in spatial organizations based on “natural immobilization” within the fermentation matrix. The microniches formed influence the fermentation biochemistry through mass transfer limitations and the subsequent development and activity of the microflora. This form of immobilization controls the nature of competition between subpopu-lations within the microflora and ultimately exerts an effect on the ecological competence (ability to survive and compete) of the various cultures present. Second, immobilized cell technology (ICT) can be used to enhance the ecological competence of starter cultures added to initiate the fermentation. Immobilization matrices such as alginate can provide microniches or microenvironments that protect the culture during freezing or lyophilization, during subsequent rehydration, and when in competition with indigenous microflora. The regulated release of cells from the microenvironments can also contribute to competitive ability. The regulation of both immobilization processes can result in enhanced fermentation activity.     

Food Bioconversions and Metabolite Production Using Immobilized Cell Technology

Abstract

This review explores recent advances in the use of immobilized cells for the production of metabolites used in the food industry, such as enzymes, amino acids, organic acids, alcohols, aroma compounds, polysaccharides, and pigments. Some food bioconversions such as fermentation of soy sauce and various hydrolysis are also considered. Special emphasis was placed on existing or potential industrial processes. This article also reports the effects of the reactor (configuration and working conditions), the immobilized cell physiological status (growing, nongrowing, or permeabilized), and of the carrier type, configuration, and size on the performance of immobilized cell systems. Compared with free cell fermentation, the main advantage of using immobilized cells is an increase in productivity, particularly in the case of continuous fermentation. For monoenzymatic reactions, nongrowing immobilized cells are often reported to exhibit a higher stability than free or immobilized enzymes.     

Production and Mass Transfer Characteristics of Non-Newtonian Biopolymers for Biomedical Applications

ABSTRACT:?

The market for microbial biopolymers is currently expanding to include several emerging biomedical applications. Specifically, these applications are drug delivery and wound healing. A fundamental understanding of the key fermentation parameters is necessary in order to optimize the production of these biopolymers. Considering that most microbial biopolymer systems exhibit non-Newtonian rheology, oxygen mass transfer can be an important parameter to optimize and control. In this article, we present a critical review of recent advances in rheological and mass transfer characteristics of selected biopolymers of commercial interest in biomedical applications.     

Immobilized cell microchannel bioreactor for evaluating fermentation characteristics of mixed substrate consumption and product formation

An immobilized cell microchannel bioreactor was designed to test continuous fermentation. The fermentation set-up included a bottom hydrophilic quartz channel to immobilize cells using 0.4 wt% polyethyleneimine and a top channel designed to continuously remove metabolically generated carbon dioxide using hydrophobic polypropylene. To evaluate fermentation characteristics of immobilized cells, ethanol fermentation was carried out using Saccharomyces cerevisiae and Pichia stipitis. The immobilized cell microchannel bioreactor was used to identify long-term activity of immobilized S. cerevisiae cells. The continuous flow microchannel bioreactor was operated stably over a period of 1 month. The immobilized cell microchannel bioreactor was used to examine the characteristics cells that consumed mixed substrates. The concentration ratio of glucose to xylose for simultaneous utilization of hemicellulosic sugars was evaluated using the microchannel bioreactor and the results were compared with those obtained by using conventional batch fermentation with P. stipitis.     

Peptide antibiotic production through immobilized biocatalyst technology

The use of immobilized biocatalysts for producing known or new antibiotics is presented. An evaluation of the applicability of this concept in the fascinating field of peptide antibiotic bioconversions and fermentations is also given.The use of immobilized enzymes, organelles and cells to synthesize antibiotics as an alternative method to conventional fermentation is discussed. In vitro total enzymatic antibiotic synthesis is illustrated with the ‘multienzyme thiotemplate mechanism’ of Bacillus brevis, the producer of gramicidin S. Total synthesis of peptide antibiotics, based on immobilized living cells, has recently been demonstrated with penicillin, bacitracin, nisin and a few other antibiotics.As an industrial example of the use of enzymes or cells to convert peptide antibiotics into therapeutically useful derivatives, free and immobilized penicillin acylases, producing the penicillin nucleus 6-aminopenicillanic acid (6-APA), are reviewed as well as their potential to synthesize semisynthetic β-lactams (penicillins, cephalosporins).Acylases, acetylesterases and α-amino acid ester hydrolases acting on cephalosporin-compounds and yielding valuable intermediary or end products have also gained wide interest. Stereospecific enzymic side-chain preparations for semisynthetic penicillin and cephalosporin production have recently reached the industrial stage. Bioconversion possibilities with the novel β-lactam compounds are suggested.These examples of simple single-step, as well as complex multi-step, enzyme reactions point to the vast potential of immobilized biocatalyst technology in fermentation science, in organic synthesis and in biotechnological processes in general.     

Adhesion of yeast cells to different porous supports, stability of cell-carrier systems and formation of volatile by-products

The aim of our research was to study how the conditions of immobilization influence cell attachment to two different ceramic surfaces: hydroxylapatite and chamotte tablets. Three fermentative yeast strains, namely brewery TT, B4 (ale, lager) and distillery Bc15a strains belonging to Saccharomyces spp., and one strain of Debaryomyces occidentalis Y500/5 of weak fermentative nature, but with high amylolytic activity due to extracellular ??-amylase and glucoamylase, were used in this study. Different media, including cell starvation, were applied for immobilization of yeast strains as well as different phases of cell growth. Immobilization of selected yeasts on a hydroxylapatite carrier was rather weak. However, when incubation of starved yeast cells was conducted in the minimal medium supplemented by calcium carbonate, the scale of immobilization after 24?h was higher, especially for the D. occidentalis strain. Adhesion to hydroxylapatite carriers in wort broth was of reversible character and better results of adhesion were observed in the case of another ceramic carrier-chamotte. The number of immobilized cells was about 10⁶?C10⁷ per tablet and cell adhesion was stable during the whole fermentation process. The comparison of the volatile products that were formed during fermentation did not show any significant qualitative and quantitative differences between the free and the immobilized cells. This is the first time when a cheap, porous chamotte surface has been applied to yeast adhesion and fermentation processes.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Modeling of immobilized cell columns for bioconversion and wastewater treatment

Immobilized cells are widely used in bioconversions to produce biological products as well as in wastewater treatment such as solvent removal from wastewater streams. In this work, a rate model is proposed to simulate this kind of process in an axial-flow fixed-bed column packed with porous particles containing immobilized cells. The transient model considered various mass transfer mechanisms including axial dispersion, interfacial film mass transfer, and intraparticle diffusion. Cell death in the immobilized cell system was also considered. Effects of various parameters such as kinetic constants and mass transfer parameters were studied. Operational situations such as feed fluctuation flow rate increase and two columns in series were also investigated. The model can be used to study the behavior and characteristics of immobilized cell columns in order to perform scale-up predictions of effluent profiles and for the purpose of process optimization.     

Influence of the oxygenation level on d-xylose fermentation by free and agar-entrapped cultures of Candida shehatae

This paper investigates the effects of the oxygenation level on the performance of d-xylose alcoholic fermentation by free- and immobilized-cell batch cultures of Candida shehatae (ATCC 22984). Yeast cells were immobilized in composite agar layer/microporous membrane structures. Fermentations were performed under varying oxygenation levels corresponding to different O₂ flow rates (OFRs). Low OFRs enhanced the fermentation performance of free and immobilized yeasts. The best ethanol yield coefficient, obtained at an OFR of 5 mmol O₂ h^–1 dm^–3 for both culture modes, was slightly higher (0.425 g g^–1) for immobilized cultures than for their free counterparts (0.39 g g^–1). More sustained aeration inhibited ethanol production by free and immobilized organisms. However, this inhibition was more pronounced for agar-entrapped cultures. Xylitol production of free cultures normally decreased as the OFR increased. At high OFR, however, immobilized organisms surprisingly produced more xylitol than at lower OFR or in anaerobiosis. This effect is discussed by referring to the mass transfer limitations that occur inside the immobilized-cell structures. Gel-entrapped cultures displayed higher specific and volumetric production rates of ethanol and xylitol than free-cell cultures.     

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.     

Syngas fermentation of Clostridium carboxidivoran P7 in a hollow fiber membrane biofilm reactor: Evaluating the mass transfer coefficient and ethanol production performance

Gasification followed by syngas fermentation is a unique hybrid process for converting lignocellulosic biomass into fuels and chemicals. Current syngas fermentation faces several challenges with low gas–liquid mass transfer being one of the major bottlenecks. The aim of this work is to evaluate the performance of hollow fiber membrane biofilm reactor (HFM-BR) as a reactor configuration for syngas fermentation. The volumetric mass transfer coefficient (K_La) of the HFM-BR was determined at abiotic conditions within a wide range of gas velocity/flowrate passing through the hollow fiber lumen and liquid velocity/flowrate passing through the membrane module shell. The K_La values of the HFM-BR were higher than most reactor configurations such as stir tank reactors and bubble columns. A continuous syngas fermentation of Clostridium carboxidivorans P7 was implemented in the HFM-BR system at different operational conditions, including the syngas flow rate, liquid recirculation between the module and reservoir, and the dilution rate. It was found that the syngas fermentation performance such as syngas utilization efficiency, ethanol concentration and productivity, and ratio of ethanol to acetic acid depended not only on the mass transfer efficiency but also the characteristics of biofilm attached on the membrane module (biofouling or abrading of the biofilm). The HFM-BR results in a highest ethanol concentration of 23.93 g/L with an ethanol to acetic acid ratio of 4.79. Collectively, the research shows the HFM-BR is an efficient reactor system for syngas fermentation with high mass transfer.     

拉曼光谱分析技术在资源微生物发酵性能评价中的应用

微生物发酵过程是细胞新陈代谢进行物质转化的过程,为了提高目标产物的转化率,需要对微生物发酵动态特性进行实时分析,以便实时优化发酵过程。拉曼光谱(Raman spectroscopy)量化测试作为一种有应用前景的在线过程分析技术,可以在避免微生物污染的条件下,实现精准监测,进而用于优化控制微生物发酵过程。【目的】以运动发酵单胞菌(Zymomonas mobilis)为例,建立微生物发酵过程中葡萄糖、木糖、乙醇和乳酸浓度拉曼光谱预测模型,并进行准确性验证。【方法】采用浸入式在线拉曼探头,收集运动发酵单胞菌发酵过程中多个组分的拉曼光谱,采用偏最小二乘法对光谱信号进行预处理和多元数据分析,结合离线色谱分析数据,对拉曼光谱进行建模分析和浓度预测。【结果】针对运动发酵单胞菌,首先实现拉曼分析仪对单一产品乙醇发酵过程的精准检测,其次基于多元变量分析,建立葡萄糖、乙醇和乳酸浓度变化的预测模型,实现对发酵过程中各成分浓度变化的准确有效分析。【结论】成功建立了一种评价资源微生物尤其是工业菌株发酵液多种组分的拉曼光谱分析方法。该方法为运动发酵单胞菌等工业菌株利用多组分底物工业化生产不同产物的实时检测,以及其他微生物尤其工业菌株的选育和过程优化提供了新方法。     

Hydrodynamics, mass transfer and rheological studies of gibberellic acid production in an airlift bioreactor

Although a lot of research has been done into modelling microbial processes, the applicability of these concepts to problems specific for bioreactor design and optimization of process conditions is limited. This is partly due to the tendency to separate the two essential factors of bioreactor modelling, i.e. physical transport processes and microbial kinetics. The deficiencies of these models become especially evident in industrial production processes where O₂ supply is likely to become the limiting factor, e.g. production of gibberellic acid and other organic acids. Hydrodynamics, mass transfer and rheology of gibberellic acid production by Gibberella fujikuroi in an airlift bioreactor is presented in this work. Important hydrodynamic parameters such as gas holdup, liquid velocity in the riser and in the downcomer, and mixing time were determined and correlated with superficial gas velocity in the riser. Mass transfer was studied evaluating the volumetric mass transfer coefficient, which was determined as a function of superficial gas velocity in the riser and as a function of fermentation time. Culture medium rheology was studied through fermentation time and allowed to explain the volumetric mass transfer coefficient behaviour. Rheological behaviour was explained in terms of changes in the morphology of the fungus. Finally, rheological studies let us obtain correlations for gas holdup and volumetric mass transfer coefficient estimation using the superficial gas velocity in the riser and the culture medium apparent viscosity.     

粘细菌Corallococcus sp. strain EGB及其培养发酵液的安全性评估

【目的】研究粘细菌Corallococcus sp. strain EGB及其细胞培养发酵液的毒理安全性,为菌株EGB作为新型生防微生物菌剂的开发和环境施用安全性提供一定的科学基础。【方法】通过Ames试验、小鼠骨髓嗜多染红细胞微核试验和小鼠睾丸染色体畸变试验测定粘细菌EGB菌体及其细胞培养发酵液的遗传毒性;通过经口灌胃的方式测定粘细菌EGB菌体及其细胞培养发酵液对ICR小鼠的急性毒性和28d亚急性毒性。【结果】Ames试验、微核试验和精母细胞染色体畸变试验结果表明,与对照组相比,EGB菌体及其细胞培养发酵液无基因突变能力,对ICR小鼠无明显的遗传毒性。EGB菌体及其细胞培养发酵液对ICR小鼠的急性经口半致死剂量(LD50)>10g/kg BW (body weight);连续灌胃28 d后,处理组ICR小鼠的体重变化、采食饮水、血液生化指标、血常规、主要脏器指数和主要器官病理切片与对照组相比无显著差异(P<0.05)。【结论】粘细菌EGB菌体及其细胞培养发酵液的毒理安全性属于无毒类别,粘细菌的生物安全性使其在工农业领域的植物病害控制和生物转化等方面具有潜在的应用价值。     

Lignocellulose Biotechnology: Current and Future Prospects

Abstract

Lignocellulose is the most abundant biomass available on Earth. It has attracted considerable attention as an alternate feed stock and energy resource because of the large quantities available and its renewable nature. The potential uses of lignocelluloses are in pulp and paper industries, production of fuel alcohol and chemicals, protein for food, and feed using biotechnological means. The current industrial activity of lignocellulosic biomass fermentation is limited mainly because of the difficulty in economic bioconversion of these materials to value-added products. Considerable improvement in many processes related to lignocellulose biotechnology appeared during the last decade. Current uses of lignocellulosic biomass, process constraints, and areas of future research are discussed here.     

海绵来源链霉菌S52-B中氨酰胺天然产物的分离与鉴定

【背景】海洋微生物是复杂海洋生态环境中重要的生物资源之一。海洋微生物所产生的活性天然产物极为丰富,是药物或药物先导化合物的重要来源。【目的】探索海洋中海绵来源链霉菌Streptomycessp.S52-B的优势生长条件,挖掘其次级代谢产物,以期分离具有良好生物活性的天然产物。【方法】根据"One Strain Many Compounds"(OSMAC)策略,寻找利于Streptomyces sp. S52-B生长和次级代谢产物产生的优势培养基,结合质谱及特征性的紫外吸收谱图,选择培养基进行大量发酵。利用正相硅胶柱色谱、葡聚糖凝胶柱色谱和制备型高效液相色谱等进行分离纯化,并应用高分辨质谱和核磁共振光谱进行化合物结构解析。【结果】确定培养基A–D为海洋链霉菌S52-B的优势培养基,基于紫外吸收光谱与质谱分析,从培养基A的大量发酵物中分离鉴定3个具有吡咯并[4,3,2-de]喹啉核心结构的含氯化合物,属于氨酰胺类天然产物,其中Ammosalic acid为新结构化合物。【结论】已知含有吡咯并喹啉母核的氨酰胺类家族化合物具有优良的抗癌活性。本研究从海绵来源链霉菌S52-B中分离鉴定了3个氨酰胺类化合物,其中一个是新结构化合物,不仅丰富了此类化合物家族的结构类型,也为研究其生物合成途径中的未知机理奠定了基础,还有利于结合培养条件和基因组信息从这株海绵来源链霉菌中挖掘新结构的活性天然产物。     

Cell-recycle batch fermentation using immobilized cells of flocculent Saccharomyces cerevisiae wine strains

Five, highly flocculeng strains of Saccharomyces cerevisiae, isolated from wine, were immobilized in calcium alginate beads to optimize primary must fermentation. Three cell-recycle batch fermentations (CRBF) of grape musts were performed with the biocatalyst and the results compared with those obtained with free cells. During the CRBF process, the entrapped strains showed some variability in the formation of secondary products of fermentation, particularly acetic acid and acetaldehyde. Recycling beads of immobilized flocculent cells is a good approach in the development and application of the CRBF system in the wine industry.     

Immobilization of Champagne Yeasts by Inclusion into Cryogels of Polyvinyl Alcohol: Means of Preventing Cell Release from the Carrier Matrix

Wine champagnizing, a process involving the use of champagne yeasts immobilized by inclusion into cryogels of polyvinyl alcohol, has been studied. Treatment of yeast cells with the autoregulatory factor d ₁ was proposed as a means of preventing the cell release from the carrier matrix. Such a treatment inhibited growth and proliferation processes in yeast cells, without affecting the activity of fermentation; the resulting champagne had the same organoleptic and chemical characteristics as its counterparts obtained using conventional techniques.