Microcomputers in biochemistry

The development of the microcomputer has meant that for the first time individual departments of biochemistry or even laboratories can have their own computing facilities. For less than the cost of a single-beam spectrophotometer it is possible to purchase a complete microcomputer system that is relatively easily programmed and operated. However, a considerable investment of labour is needed tom ake the microcomputer effective in biochemistry and it is a challenge to pare away the mystique from small-scale computing so that it becomes as common a tool as spectrophotometry. In the belief that there are few biochemists who could not benefit from the use of the microcomputer this brief article will explore some of the current and future applications of the microcomputer in biochemistry.     

发酵过程中细胞浓度在线检测系统*

利用微机软、硬件技术,设计了一套比浊法细胞浓度在线检测系统,并应用于5L发酵罐中木糖醇发酵过程细胞浓度在线检测。该系统能及时、较准确地反映发酵过程中菌体浓度随时间的变化情况,在规模化生产中具有较强的适用性、通用性,可为实现其它分析仪器的计算机在线检测系统提供值得借鉴的设计思路和实现方法。     

Production of ethanol,organic acids and hydrogen: an opportunity for mixed culture biotechnology?

Anaerobic fermentation of biodegradable organic materials is usually carried out to obtain the final product, methane, a valuable energy source. However, it is also well known that various intermediates are produced in this process, e.g. ethanol, volatile organic acids and hydrogen. All these species have applications and value as fuels or chemicals. This paper shows a critical analysis of the potential of using anaerobic fermentation by mixed cultures to produce intermediates, e.g. ethanol, acetic, lactic and butyric acid and hydrogen, rather than methane. This paper discusses the current processes to produce these chemicals and compares them with the alternative approach of using open mixed cultures to produce them simultaneously via fermentation from renewable resources. None of these chemicals is currently produced via mixed culture fermentation: ethanol and lactic acid are usually produced in pure culture fermentation using food crops, e.g. corn or sugar cane, as starting materials; hydrogen, acetic and butyric acids are mainly produced via chemical synthesis from fossil fuel derived starting materials. A possible flow-sheet for the production of these chemicals from organic waste using mixed culture fermentation is proposed and the advantages and disadvantages of this process compared to current processes are critically discussed. The paper also discusses the research challenges which need to be addressed to make this process feasible.     

A robust soft sensor based on artificial neural network for monitoring microbial lipid fermentation processes using Yarrowia lipolytica

Microbial oils produced by Yarrowia lipolytica offer an environmentally friendly and sustainable alternative to petroleum as well as traditional lipids from animals and plants. The accurate measurement of fermentation parameters, including the substrate concentration, dry cell weight, and lipid accumulation, is the foundation of process control, which is indispensable for industrial lipid production. However, it remains a great challenge to measure the complex parameters online during the lipid fermentation process, which is nonlinear, multivariate, and characterized by strong coupling. As a type of AI technology, the artificial neural network model is a powerful tool for handling extremely complex problems, and it can be employed to develop a soft sensor to monitor the microbial lipid fermentation process of Y. lipolytica. In this study, we first analyzed and emphasized the volume of sodium hydroxide and dissolved oxygen concentration as central parameters of the fermentation process. Then, a soft sensor based on a four-input artificial neural network model was developed, in which the input variables were fermentation time, dissolved oxygen concentration, initial glucose concentration, and additional volume of sodium hydroxide. This provides the possibility of online monitoring of dry cell weight, glucose concentration, and lipid production with high accuracy, which can be extended to similar fermentation processes characterized by the addition of bases or acids, as well as changes of the dissolved oxygen concentration.     

Adaptive on-line simulation of bioreactors: Fermentation monitoring and modeling system

Summary In order to study and control fermentation processes, indirect on-line measurements and mathematical models can be used. Here an on-line model for fermentation processes is presented. The model is based on atom and partial mass balances as well as on stability equations for the protolytes. The model is given an adaptive form by including transport equations for mass transfer and expressions for the fermentation kinetics. The state of the process can be estimated on-line using the balance component of the model completed with measurement equations for the input and the output flows of the process. Adaptivity is realized by means of on-line estimation of the parameters in the transport and kinetic expressions using recursive regression analysis. On-line estimation of the kinetic and mass transfer parameters makes model-based predictions possible and enables intelligent process control while facilitating testing of the validity of the measurement variables. A practical MS-Windows 3.1 model implementation called FMMS—Fermentation Monitoring and Modeling System is shown. The system makes it easy to configure the operating conditions for a run. It uses Windows dialogs for all set-ups, model configuration parameters, elemental compositions, on-line measurement devices and signal conditioning. Advanced on-line data analysis makes it possible to plot variables against each other for easy comparison. FMMS keeps track of over 100 variables per run. These variables are either measured or estimated by the model. Assay results can also be entered and plotted during fermentation. Thus the model can be verified almost instantly. Historical fermentation runs can be re-analyzed in simulation mode. This makes it possible to examine different signal conditining filters as well as the sensitivity of the model. Combined, the data analysis and the simulation mode make it easy to test and develop model theories and new ideas.     

Generic plasmid DNA production platform incorporating low metabolic burden seed‐stock and fed‐batch fermentation processes

DNA vaccines have tremendous potential for rapid deployment in pandemic applications, wherein a new antigen is “plugged” into a validated vector, and rapidly produced in a validated, fermentation—purification process. For this application, it is essential that the vector and fermentation process function with a variety of different antigen genes. However, many antigen genes are unpredictably “toxic” or otherwise low yielding in standard fermentation processes. We report cell bank and fermentation process unit operation innovations that reduce plasmid‐mediated metabolic burden, enabling successful production of previously known toxic influenza hemagglutinin antigen genes. These processes, combined with vector backbone modifications, doubled fermentation productivity compared to existing high copy vectors, such as pVAX1 and gWiz, resulting in high plasmid yields (up to 2,220 mg/L, 5% of total dry cell weight) even with previously identified toxic or poor producing inserts. Biotechnol. Bioeng. 2009;103: 1129–1143. © 2009 Wiley Periodicals, Inc.     

Detection of phase shifts in batch fermentation via statistical analysis of the online measurements: a case study with rifamycin B fermentation

Industrial production of antibiotics, biopharmaceuticals and enzymes is typically carried out via a batch or fed-batch fermentation process. These processes go through various phases based on sequential substrate uptake, growth and product formation, which require monitoring due to the potential batch-to-batch variability. The phase shifts can be identified directly by measuring the concentrations of substrates and products or by morphological examinations under microscope. However, such measurements are cumbersome to obtain. We present a method to identify phase transitions in batch fermentation using readily available online measurements. Our approach is based on Dynamic Principal Component Analysis (DPCA), a multivariate statistical approach that can model the dynamics of non-stationary processes. Phase-transitions in fermentation produce distinct patterns in the DPCA scores, which can be identified as singular points. We illustrate the application of the method to detect transitions such as the onset of exponential growth phase, substrate exhaustion and substrate switching for rifamycin B fermentation batches. Further, we analyze the loading vectors of DPCA model to illustrate the mechanism by which the statistical model accounts for process dynamics. The approach can be readily applied to other industrially important processes and may have implications in online monitoring of fermentation batches in a production facility.     

Improved performances and control of beer fermentation using encapsulated alpha-acetolactate decarboxylase and modeling

The use of the enzyme alpha-acetolactate decarboxylase allows the acceleration of beer fermentation/maturation because it shunts diacetyl formation, whose elimination is the rate-limiting step of the process. To obtain a cost reduction by using this exogenous enzyme, we propose a new process involving recoverable encapsulated alpha-acetolactate decarboxylase. The performance of traditional and new processes was investigated by a modeling approach. A simple model, focused on alpha-acetolactate and diacetyl profiles during beer fermentation, was set up. The simulated profiles are consistent with literature data. This study shows also that encapsulated alpha-acetolactate decarboxylase allows the acceleration of beer fermentation as efficiently as free alpha-acetolactate decarboxylase. The advantage of immobilized alpha-acetolactate decarboxylase versus free enzyme is that it is recoverable and reusable, which means a process cost reduction.     

Anaerobic fixed film wastewater treatment

The anaerobic methane fermentation process has long been used in the field of wastewater engineering in sludge processing, mainly for waste stabilization and solids reduction. Recently, major advances in the fundamental understanding of the process microbiology and biochemistry, along with the development of new reactor configurations have promoted a resurgence of interest in the use of this technology for the processing of liquid industrial and municipal wastewaters. Three of these new processes, the anaerobic filter, expanded/fluidized bed, and upflow anaerobic sludge blanket, are discussed.Each of these processes is a fixed film process, which enables the attainment of high solids retention times for good system efficiency and stability, with low hydraulic retention times for system economy. Fixed film anaerobic processes are able to realize many of the benefits of anaerobic processes while overcoming many of the problems historically associated with anaerobic processes.Each of the processes is described, and examples are presented for industrial and municipal applications. Finally, the processes are qualitatively compared. At present, it is not possible to say which reactor configuration is best. In fact, the selection is often dependent on wastewater characteristics, local factors, and several other factors. More full-scale data and operating experience along with basic research needs are needed to clarify further this situation, and to design these systems optimally.     

Recent advances in production of succinic acid from lignocellulosic biomass

Production of succinic acid via separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) are alternatives and are environmentally friendly processes. These processes have attained considerable positions in the industry with their own share of challenges and problems. The high-value succinic acid is extensively used in chemical, food, pharmaceutical, leather and textile industries and can be efficiently produced via several methods. Previously, succinic acid production via chemical synthesis from petrochemical or refined sugar has been the focus of interest of most reviewers. However, these expensive substrates have been recently replaced by alternative sustainable raw materials such as lignocellulosic biomass, which is cheap and abundantly available. Thus, this review focuses on succinic acid production utilizing lignocellulosic material as a potential substrate for SSF and SHF. SSF is an economical single-step process which can be a substitute for SHF — a two-step process where biomass is hydrolyzed in the first step and fermented in the second step. SSF of lignocellulosic biomass under optimum temperature and pH conditions results in the controlled release of sugar and simultaneous conversion into succinic acid by specific microorganisms, reducing reaction time and costs and increasing productivity. In addition, main process parameters which influence SHF and SSF processes such as batch and fed-batch fermentation conditions using different microbial strains are discussed in detail.     

Yeasts in sustainable bioethanol production: A review

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.     

Comparison of prediction performances between models obtained by the group method of data handling and neural networks for the alcoholic fermentation rate in enology

The modelling of winemaking processes, to predict as far ahead as possible the fermentation performance, is necessary for enhanced supervision and to enable appropriate corrective action to be taken to remedy incorrect fermentation before it is too late. In this paper, we briefly present two heuristic modelling methods—the Group Method of Data Handling (GMDH) and Neural Networks (NN)—which can be used to obtain unstructured models. The identification and prediction performances of the models obtained with these two methods are compared with respect to the alcoholic fermentation rate (dCO₂/dt) at five prediction horizons and for four fermentations. It is shown that predictive models obtained with neural network methodology are more accurate than those obtained with GMDH. On the other hand, GMDH models are more versatile when used for the prediction of the fermentation rate of a different fermentation than the one used in the learning process.     

Importance of bacteriophages in fermentation processes

Any bacterial strain can be infected by virulent phages or harbour one or more prophages. Therefore, bacteria-phage interactions are to be regarded as fundamental properties of bacteria. In current industrial fermentation processes phages can be advantageously employed for the identification of bacterial production strains (phage typing). In some cases phages are involved in the production of enzymes and special substances. The fundamental importance of phages in any technical fermentation process, however, is based on the peculiarities of their obligately parasitic life cycle. The propagation of phages in fermentation processes can cause complete (or at least partial) lysis of the production strains and, consequently, serious disturbances in the production process and considerable economic losses. The phage problem in the fermentation industry has not yet been completely solved. For the protection of technical processes against virulent phages five measures are discussed: phage-protected sterile fermentation, employment of alternative cultures, employment of phage-resistant mutants, employment of phage inhibitors, and employment of immobilized bacterial cells. The problem of the protection of bacterial production strains from prophage induction is more difficult and practically unsolved. Two possibilities to minimize the process risk due to temperate phages, the elimination of inducing factors during the fermentation process, and the selection of production strains which are difficult to induce, are discussed.     

Adaptive on-line model for aerobic Saccharomyces cerevisiae fermentation

In order to study and control fermentation processes, indirect on-tine measurements and mathematical models can be used. In this article we present a mathematical on-line model for fermentation processes. The model is based on atom and partial mass balances as well as on equations describing the acid-base system. The model is brought into an adaptive form by including transport equations for mass transfer and unstructured expressions for the fermentation kinetics. The state of the process, i.e., the concentrations of biomass, substrate, and products, can be estimated on-line using the balance part of the model completed with measurement equations for the input and output flows of the process. Adaptivity is realized by means of on-line estimation of parameters in the transport and kinetic expressions using recursive regression analysis. These expressions can thus be used in the model as valid equations enabling prediction of the process. This makes model-based automation of the process and testing of the validity of the measurement variables possible. The model and the on-line principles are applied to a 3.5-L laboratory tormentor in which Saccharomyces cerevisiae is cultivated. The experimental results show that the model-based estimation of the state and the predictions of the process correlate closely with high-performance liquid chromatography (HPLC) analyses. (c) 1995 John Wiley & Sons, Inc.     

Modelling of simultaneous hydrolysis and fermentation of maltodextrines to ethanol

In this study the possibilities for simultaneous carrying out of ethanol fermentation process and maltodextrines hydrolysis by immobilized glucoamylase were investigated. By this way it is possible to maintain concentration of glucose (strong inhibitor of both processes) at low levels. This results in a considerable enhancement of the ethanol production as compared to the conventional process. Mathematical model of simultaneous processes is constructed and solved. Good agreement with experimental results is observed.     

Fermentative hydrogen production from low-value substrates

Hydrogen is a promising energy source that is believed to replace the conventional energy sources e.g. fossil fuels over years. Hydrogen production methods can be divided into conventional production methods which depend mainly on fossil fuels and alternative production methods including electrolysis of water, biophotolysis and fermentation hydrogen production from organic waste materials. Compared to the conventional methods, the alternative hydrogen production methods are less energy intensive and negative-value substrates i.e. waste materials can be used to produce hydrogen. Among the alternative methods, fermentation process including dark and photo-fermentation has gained more attention because these processes are simple, waste materials can be utilized, and high hydrogen yields can be achieved. The fermentation process is affected by several parameters such as type of inoculum, pH, temperature, substrate type and concentration, hydraulic retention time, etc. In order to achieve optimum hydrogen yields and maximum substrate degradation, the operating conditions of the fermentation process must be optimized. In this review, two routes for biohydrogen production as dark and photo-fermentation are discussed. Dark/photo-fermentation technology is a new approach that can be used to increase the hydrogen yield and improve the energy recovery from organic wastes.     

Current challenges facing one-step production of l-ascorbic acid

l-ascorbic acid (L-AA, vitamin C) is an essential vitamin that is widely used as a nutrient or medicine in the pharmaceuticals, cosmetics, food, beverage and feed additive industries, and accounts for the largest share of the global vitamins market. L-AA is mainly produced by a classic two-step fermentation process that suffers from the use of a multi-step mixed culture system and two rounds of sterilisation, which significantly increases the cost of the final product. One-step fermentation has been attempted, but a method rivalling the efficiency of the two-step process has not yet been achieved on an industrial scale. In this review, based on the current classical two-step fermentation processes and other potential routes for L-AA production, the challenges and pitfalls of a one-step fermentation process are summarised. The prospects for one-step fermentation production of L-AA and how this might be achieved are also discussed.     

Chemometrics and visible-near infrared spectroscopic monitoring of red wine fermentation in a pilot scale

The modern wine industry needs tools for process control and quality assessment in order to better manage fermentation or bottling processes. During wine fermentation it is important to measure both substrate and product concentrations (e.g. sugars, phenolic compounds), however, the analysis of these compounds by traditional means requires sample preparation and in some cases several steps of purification are needed. The combination of visible/near-infrared (Vis/NIR) spectroscopy and chemometrics potentially provides an ideal solution to accurately and rapidly monitor physical or chemical changes in wine during processing without the need for chemical analysis. The aim of this study was to assess the possibility of combining spectral and multivariate techniques, such as principal component analysis (PCA), discriminant partial least squares (DPLS), or linear discriminant analysis (LDA), to monitor time-related changes that occur during red wine fermentation. Samples (n = 652) were collected at various times from several pilot scale fermentations with grapes from either Cabernet Sauvignon or Shiraz varieties, over three vintages (2001-2003) and scanned using a monochromator instrument (Foss-NIRSystems 6500, Silver Spring, MD) in transmission mode (400-2,500 nm). PCA was used to demonstrate consistent progressive spectral changes that occur through the time course of the fermentation. LDA using PCA scores showed that regardless of variety or vintage, samples belonging to a particular time point in fermentation could be correctly classified. This study demonstrates the potential of Vis/NIR spectroscopy combined with chemometrics, as a tool for the rapid monitoring of red wine fermentation.     

Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery

Clostridial acetone–butanol fermentation from renewable carbohydrates used to be the largest biotechnological process second only to yeast ethanol fermentation and the largest process ever run under sterile conditions. With the rising prices for mineral oil, it has now the economical and technological potential to replace petrochemistry for the production of fuels from renewable resources. Various methods for using non-food biomass such as cellulose and hemicellulose in agricultural products and wastes have been developed at laboratory scale. To our knowledge, the AB plants in Russia were the only full-scale industrial plants which used hydrolyzates of lignocellosic waste for butanol fermentation. These plants were further developed into the 1980s, and the process was finally run in a continual mode different from plants in Western countries. A biorefinery concept for the use of all by-products has been elaborated and was partially put into practice. The experience gained in the Soviet Union forms a promising basis for the development of modern large-scale processes to replace a considerable fraction of the current chemical production of fuel for our future needs on a sustainable basis.     

Traditional food fermentations as industrial resources

Asia is rich in resources which can contribute widely to food processing and production over the next 207–50 years as world population reaches 6 billion. The world needs low cost methods of providing nutritious proteinrich meat analogues for its thousands of millions of consumers. The Indonesian tempe fermentation will serve as a model. A bacterium present in commercial tempe can be used to add vitamin B-12 to other vegetarian foods. Fuel requirements for cooking can be decreased by applying a fungal fermentation of the tempe/ontjom type to legume substrates. The world needs high quality meat-flavors derived from vegetable protein. The soy sauce (kecap)/miso (tauco) processes and the fish/shrimp sauce and paste processes can be modified to yield a wide variety of meat-like flavors for use in formulating new foods. The protein content of high starch substrates can be increased by applying the Indonesian tape fermentation. Leavened sour-dough bread-like products can be produced without the use of wheat or ryeflours using the Indian idli/dosai fermentation. Coconut protein, if extracted without denaturing, can serve as a valuable base for a new type of puddings and related foods. Finally Asia is an almost endless source of cultures of edible microorganisms that, with further study of their synthetic abilities, particularly regarding amino acids and vitamins, could lead to new industries.