Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016     

智舌快速鉴定水产品多种致病性弧菌

【目的】探索研究能很好显示被检测液态食品样品综合信息的电子舌是否也能很好的显示致病性弧菌液体培养物的综合信息,籍此探讨一类致病性弧菌的快速鉴定鉴别新技术。【方法】基于多频脉冲伏安法的智舌,结合主成分分析,对源自水产品的11种致病性弧菌进行区分鉴定研究,得到最佳电极阵列和频率段组合。【结果】结果显示,区分致病性弧菌效果好的电极和频率段分别是:钛电极的100Hz、银电极的100Hz和钨电极的1Hz、10Hz。钨电极的1Hz频率段能够独立的把11种致病性弧菌在同一张主成分得分图上区分开;钛电极的100Hz、银电极的100Hz和钨电极的10Hz两两组合也能把11种致病性弧菌主成分得分图上区分开。【结论】研究结果表明,智舌伏安法结合主成分分析法区分致病性弧菌是可行的,具有很大的持续研究价值,有望发展成一类很有发展优势的快速鉴定致病性弧菌乃至延伸至其他致病菌的新技术。     

Multiplication and Fermentation of Saccharomyces cerevisiae under Carbon Dioxide Pressure in Wine

Conditions for rapid fermentation of sugar in wine under pressure were sought for use in continuous production of naturally fermented sparkling wine. Wine yeast growth and fermentation were measured under CO(2) pressure. The medium was white wine with added glucose. Pressure was very inhibitory to growth, especially at low pH or high alcohol concentration. Use of various strains of wine yeast, cultures of various ages, or cells adapted to wine did not give more rapid growth. Addition of nutrients increased growth, but under no conditions was growth rapid enough to bring about sufficiently rapid fermentation rates. Conditions for rapid fermentation were sought by use of high levels of cells as inocula. Fermentation rates in wine also were inhibited by pressure, and were dependent on pH and alcohol levels. Addition of nutrients did not increase the fermentation rate, but rapid fermentation rates were obtained, under pressure, by inoculation with high levels of cells adapted several weeks to the base wine. Thus, continuous sparkling-wine production might be practical with proper amounts of adapted cells used as inocula, or perhaps with reuse of the fermentation culture.     

电子嗅在线反馈控制毕赤酵母糖化酶发酵过程中甲醇浓度新方法的应用

探索了电子嗅传感仪直接通过发酵尾气进行发酵液中甲醇浓度在线检测的方法,建立了毕赤酵母表达糖化酶过程中甲醇浓度的自动化反馈补料控制模型,可准确实现发酵过程中甲醇浓度的精确控制;研究表明,当利用电子嗅将培养液中甲醇浓度稳定控制在(890±35)ppm水平下,发酵诱导培养到128h时目的蛋白糖化酶酶活达到了8 153U/ml,与甲醇浓度控制在(350±26)ppm时的发酵水平相比提升了48.8%。该方法具有无需前处理、与发酵液非接触、快速和准确性的优点,为提升工程酵母在工业发酵培养过程工艺的优化控制具有重要的指导作用。     

多频大幅脉冲传感系统监测3种食源性致病菌的生长趋势

【目的】探究智能电子舌伏安法结合特定的统计分析系统是否适用于示踪食品致病性细菌生长情况的快速监测。【方法】利用智能电子舌――多频大幅脉冲传感系统的伏安法,监测液体培养基中3种食源性致病菌的16个时段生长情况所致液体基质变化过程的综合信息;结合主成分分析法对获得的复杂综合信息数据进行统计学分析,按获得的主成分得分图分析检测样品。【结果】监测能力强的电极、频率段分别是：金黄色葡萄球菌的为钨电极的100 Hz、铂电极的1 Hz、银电极的10 Hz和钛电极的10 Hz频率段;大肠杆菌O157:H7的为金电极的100 Hz、铂电极的1 Hz、钛电极的1 Hz和钨电极的100 Hz频率段;枯草芽孢杆菌的为钯电极的1、10和100 Hz 3个频率段。【结论】本试验首次用多频大幅脉冲传感系统伏安法结合主成分分析法,能够有效的监测样品细菌的生长情况,有望成为一种具有多种优点的新型检测细菌生长情况的快速监测系统。     

Microwave-Assisted Development of Orally Disintegrating Tablets by Direct Compression

Orally disintegrating tablets (ODTs) are challenged by the need for simple technology to ensure good mechanical strength coupled with rapid disintegration. The objective of this work was to evaluate microwave-assisted development of ODTs based on simple direct compression tableting technology. Placebo ODTs comprising directly compressible mannitol and lactose as diluents, super disintegrants, and lubricants were prepared by direct compression followed by exposure to >97% relative humidity and then microwave irradiation for 5 min at 490 W. Placebo ODTs with hardness (>5 kg/cm²) and disintegration time (<60 s) were optimized. Palatable ODTs of Lamotrigine (LMG), which exhibited rapid dissolution of LMG, were then developed. The stability of LMG to microwave irradiation (MWI) was confirmed. Solubilization was achieved by complexation with beta-cyclodextrin (β-CD). LMG ODTs with optimal hardness and disintegration time (DT) were optimized by a 2³ factorial design using Design Expert software. Taste masking using sweeteners and flavors was confirmed using a potentiometric multisensor-based electronic tongue, coupled with principal component analysis. Placebo ODTs with crospovidone as a superdisintegrant revealed a significant increase in hardness from ～3 to ～5 kg/cm² and a decrease in disintegration time (<60 s) following microwave irradiation. LMG ODTs had hardness >5 kg/cm², DT?<?30s, and rapid dissolution of LMG, and good stability was optimized by DOE and the design space derived. While β-CD complexation enabled rapid dissolution and moderate taste masking, palatability, which was achieved including flavors, was confirmed using an electronic tongue. A simple step of humidification enabled MWI-facilitated development of ODTs by direct compression presenting a practical and scalable advancement in ODT technology.     

Physiologically Motivated Monitoring of Fermentation Processes by Means of an Electronic Nose

An on‐line approach of non‐invasive monitoring of the physiological changes in fermentation processes is presented. In yeast batch and bacterial fed‐batch fermentations it is shown that metabolic state changes can be revealed using an electronic nose. The transient responses of the gas sensors to the changes in the composition of the volatiles emitted from the cell cultures during fermentation are used to retrieve a semi‐quantitative representation of the physiological state of the cultures. With the sensor responses of the electronic nose it is shown that physiological variables such as rates of growth, substrate uptake and product formation can be depicted. The non‐invasive method thus seems as a pertinent alternative to conventional bioreactor monitoring methods.     

Monitoring the aroma production during wine-must fermentation with an electronic nose.

This work discusses the feasibility of using the electronic nose for the on-line and real-time monitoring of the production of a complex aroma profile during a bioconversion process. As a case study, the formation of the muscatel aroma during the wine-must fermentation was selected. During wine-must fermentation, aroma compounds responsible for the organoleptic character are produced in the ppm range, while simultaneously one of the main metabolic products, ethanol, is produced in much higher quantities (up to 10% wt). Because the sensors of the electronic nose array are cross-selective to different volatile compounds, it was investigated in detail how far the electronic nose was able to evaluate the aroma profile along the fermentation. This article discusses and evaluates subsequently the integration of a membrane separation process-organophilic pervaporation-for selectively enriching aroma compounds relative to ethanol, to improve sample discrimination.     

Monitoring bacterial and archaeal community shifts in a mesophilic anaerobic batch reactor treating a high-strength organic wastewater

Shifts in bacterial and archaeal communities, associated with changes in chemical profiles, were investigated in an anaerobic batch reactor treating dairy-processing wastewater prepared with whey permeate powder. The dynamics of bacterial and archaeal populations were monitored by quantitative real-time PCR and showed good agreement with the process data. A rapid increase in bacterial populations and a high rate of substrate fermentation were observed during the initial period. Growth and regrowth of archaeal populations occurred with biphasic production of methane, corresponding to the diauxic consumption of acetate and propionate. Bacterial community structure was examined by denaturing gel gradient electrophoresis (DGGE) targeting 16S rRNA genes. An Aeromonas -like organism was suggested to be mainly responsible for the rapid fermentation of carbohydrate during the initial period. Several band sequences closely related to the Clostridium species, capable of carbohydrate fermentation, lactate or ethanol fermentation, and/or homoacetogenesis, were also detected. Statistical analyses of the DGGE profiles showed that the bacterial community structure, as well as the process performance, varied with the incubation time. Our results demonstrated that the bacterial community shifted, reflecting the performance changes and, particularly, that a significant community shift corresponded to a considerable process event. This suggested that the diagnosis of an anaerobic digestion process could be possible by monitoring bacterial community shifts.     

Measurement of oxygen solubility in fermentation media: a colorimetric method

Methods of measuring oxygen solubility in culture media are scarce, and those available are tedious to apply. A simple colorimetric assay was developed and applied to the analysis of oxygen solubility during alcoholic fermentation. The method was based on the consumption of oxygen by glucose oxidase activity and the production of the pink quinone of syringaldazine by coupled peroxidase activity. Color formation at 526 nm progressed through an optimum that was a linear function of the oxygen added to the assay. Sensitivity was maximized by operating at pH 7 and limiting the medium sample volume added. Each assay took 10-15 min to prepare and react. Reaction time was minimized by using abundant glucose and enzyme concentrations. Data obtained by the assay developed showed good agreement with published oxygen solubilities in water and selected media at various temperatures. Subsequent analyses of fermentation broths indicated falling sugar concentration to be primarily responsible for increases in oxygen solubility during fermentation. For example, during fermentations started with 230 g/L xylose or glucose, oxygen solubility could increase by 41% due to sugar consumption alone. This procedure can provide the solubility data needed to accurately calibrate in-line electronic probes for monitoring dissolved oxygen concentration during fermentation processes.     

Inline noninvasive Raman monitoring and feedback control of glucose concentration during ethanol fermentation

Raman spectroscopy as a process analytical technology tool was implemented for the monitoring and control of ethanol fermentation carried out with Saccharomyces cerevisiae. The need for the optimization of bioprocesses such as ethanol production, to increase product yield, enhanced the development of control strategies. The control system developed by the authors utilized noninvasive Raman measurements to avoid possible sterilization problems. Real-time data analysis was applied using partial least squares regression (PLS) method. With the aid of spectral pretreatment and multivariate data analysis, the monitoring of glucose and ethanol concentration was successful during yeast fermentation with the prediction error of 4.42 g/L for glucose and 2.40 g/L for ethanol. By Raman spectroscopy-based feedback control, the glucose concentration was maintained at 100 g/L by the automatic feeding of concentrated glucose solution. The control of glucose concentration during fed-batch fermentation resulted in increased ethanol production. Ethanol yield of 86% was achieved compared to the batch fermentation when 75 % yield was obtained. The results show that the use of Raman spectroscopy for the monitoring and control of yeast fermentation is a promising way to enhance process understanding and achieve consistently high production yield.     

α-Amylase,Limit Dextrinase,and α-Glucosidase Enzymes in Barley and Malt

Abstract

A parallel discussion is presented of recent developments in fermentation monitoring and control in both research institutes and in industry. The discussion is based around the areas of measurement (on-line and off-line), data conditioning and analysis, modeling, fault analysis, and control. Recent progress in industrial fermentation monitoring and control is used as a guide to identify potential areas of research that might have a most rapid and direct impact on industrial fermentation operation.     

Rapid quantitation and monitoring of plasmid DNA using an ultrasensitive DNA-binding dye

A sensitive fluorescence-based method for monitoring plasmid DNA during production was investigated. This simple method of assaying for plasmid DNA allows rapid monitoring of plasmid yields from a recombinant Escherichia coli fed-batch fermentation. The assay has several advantages over traditional methods of plasmid DNA measurement. The fluorescent dye is highly specific and can measure total plasmid DNA concentration in about 5 min. The assay is sensitive over a wide range of plasmid concentrations of between 15 and 280 ng/mL, even in the presence of impurities that occur within alkaline lysate preparations. The technique can also be applied to monitoring fermentation and downstream purification steps.     

Increase of xylitol production rate by controlling redox potential in Candida parapsilosis

The effect of redox potential on xylitol production by Candida parapsilosis was investigated. The redox potential was found to be useful for monitoring the dissolved oxygen (DO) level in culture media, especially when the DO level was low. An increase in the agitation speed in a 5 L fermentor resulted in an increased culture redox potential as well as enhanced cell growth. Production of xylitol was maximized at a redox potential of 100 mV. As the initial cell concentration increased from 8 g/L to 30 g/L, the volumetric productivity of xylitol increased from 1.38 g/L. h to 4.62 g/L. h. A two-stage xylitol production strategy was devised, with stage 1 involving rapid production of cells under well-aerated conditions, and stage 2 involving cultivation with reduced aeration such that the culture redox potential was 100 mV. Using this technique, a final xylitol concentration of 180 g/L was obtained from a culture medium totally containing 254.5 g/L xylose in a 3,000 L pilot scale fermentor after 77 h fermentation. The volumetric productivity of xylitol during the fermentation was 2.34 g/L. h.     

Rapid estimation of glucose and amylase in fermentation culture broth with test strips by reflectance photometry

Summary The applicability of clinical multilayer test strips based on dry reagent chemistries for the quantitative analysis of fermentation media constituents was evaluated. For glucose and amylase determinations a significant correlation to conventional wet chemical methods was obtained. Bacterial biomass in the range of 0.5–5 g/l has no interfering effect. It is a rapid and cost-effective method for fermentation process monitoring and control.     

Using a Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Chicken Carcass

Chicken is one of the most popular meat products in the world. Salmonella Typhimurium is a common foodbome pathogens associated with the processing of poultry. An optical Surface Plasmon Resonance （SPR） biosensor was sensitive to the presence of Salmonella Typhimurium in chicken carcass. The Spreeta biosensor kits were used to detect Salmonella Typhimurium on chicken carcass successfully. A taste sensor like electronic tongue or biosensors was used to basically ＂taste＂ the object and differentiated one object from the other with different taste sensor signatures. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with dif- ferent antisera or other molecular species. The selectivity of the SPR biosensor was assayed using a series of antibody con- centrations and dilution series of the organism. The SPR biosensor showed promising to detect the existence of Salmonella Typhimurium at 1 x 106 CFU/ml. Initial results show that the SPR biosensor has the potential for its application in pathogenic bacteria monitoring. However, more tests need to be done to confirm the detection limitation.     

Image analysis for monitoring the barley tempeh fermentation process

AIMS: To develop a fast, accurate, objective and nondestructive method for monitoring barley tempeh fermentation. METHODS AND RESULTS: Barley tempeh is a food made from pearled barley grains fermented with Rhizopus oligosporus. Rhizopus oligosporus growth is important for tempeh quality, but quantifying its growth is difficult and laborious. A system was developed for analysing digital images of fermentation stages using two image processing methods. The first employed statistical measures sensitive to image colour and surface structure, and these statistical measures were highly correlated (r=0.92, n=75, P<0.001) with ergosterol content of tempeh fermented with R. oligosporus and lactic acid bacteria (LAB). In the second method, an image-processing algorithm optimized to changes in images of final tempeh products was developed to measure number of visible barley grains. A threshold of 5 visible grains per Petri dish indicated complete tempeh fermentation. When images of tempeh cakes fermented with different inoculation levels of R. oligosporus were analysed the results from the two image processing methods were in good agreement. CONCLUSION: Image processing proved suitable for monitoring barley tempeh fermentation. The method avoids sampling, is nonintrusive, and only requires a digital camera with good resolution and image analysis software. SIGNIFICANCE AND IMPACT OF THE STUDY: The system provides a rapid visualization of tempeh product maturation and qualities during fermentation. Automated online monitoring of tempeh fermentation by coupling automated image acquisition with image processing software could be further developed for process control.     

Sensor systems,electronic tongues and electronic noses,for the monitoring of biotechnological processes

Production of biofuel is based on the conversion by microorganisms of complex organic substrates into the methane or ethanol, which are consequently used as energy sources. Real time monitoring of the fermented media composition is of paramount for the effectiveness of the whole process. However, despite the fact that products worth billions of dollars are produced through fermentation processes annually, analytical instruments used for these processes’ monitoring remain relatively primitive. Established laboratory techniques produce exhaustive information about media composition but analysis is often quite time-consuming, expensive, requires skilled personnel and hardly can be automated. Lack of on-line sensors for the fermentation monitoring is commonly stressed in the literature. One of the techniques particularly suitable for this purpose is chemical sensors. Such features as low prices, relatively simple instrumentation, minimal sample preparation and easy automation of measurements make chemical sensors an attractive tool for industrial process control. However, practical use of chemical sensors in complex media is often hindered by their insufficient selectivity. For example, only pH and oxygen probes are routinely used in bio-reactors. One of the emerging approaches permitting to overcome the selectivity problems is the use of systems instead of discrete sensors. Such systems for liquid and gas analysis were named electronic tongues and electronic noses correspondingly. They are capable to perform both quantitative analysis (components’ concentrations) and classification or recognition of multicomponent media. This review presents recent achievements in the R&D and applications of electronic tongues and noses to the monitoring of biotechnological processes. JIMB-2008: BioEnergy—Special issue.     

Development of an On-line Glucose Sensor for Fermentation Monitoring

A dimethylferrocene-mediated enzyme electrode for glucose (Cass et al., 1984) was evaluated for application to microbial fermentation monitoring and control. The deficiencies revealed - insufficient stability, progressive increase in response time and progressive decrease in linear range - were investigated using electrochemical and radiochemical techniques. The enzyme immobilization was improved with a novel procedure using periodate-oxidized glucose oxidase and an alky lamine electrode coating. An in-situ probe and a computer-controlled analytical system were developed for fermentation monitoring. The glucose concentration of an Escherichia coli batch culture was successfully monitored on line using this analytical system.     

Evaluation of protein hydrolysates using the fermentation activity of immobilized yeast cells

A rapid, easy, and reproducible method for evaluation of protein hydrolysates has been developed using the ethanol fermentation of immobilized yeast cells as a monitoring system. The method is sensitive to the origin, concentration, and degree of hydrolysis of the hydrolysates.