An on-line advisory control system for the lactic acid fermentation process

A fuzzy expert system was developed for online diagnosing and controlling of bioprocesses. The system was constructed in object-oriented Smalltalk/V for diagnosing and controlling of bioprocesses. Lactic acid fermentation with an industrial strain ofLactobacillus casei was chosen as the model system. The performance of the fuzzy expert system and the knowledge base utilizing experts' knowledge and several facts obtained from the experiments were successfully validated with on-line fermentations. The fuzzy expert system could diagnose a fault on-line and give reasonable advice to the process operator. In order to achieve the diagnosing faculty, a database, a knowledge base, and both backward and forward chaining procedures were implemented employing the object-oriented programming environment. A defuzzifier was implemented in the system to achieve on-line control. In order to realize a decision-making system with a human operator and a fuzzy expert system, a new control strategy namedAdvice was also introduced. Several cultivations were carried out in order to collect knowledge on the effects concerned with inoculum properties to the process and to construct a database including standard time-course profiles. The performance of the fuzzy expert control system was successfully tested with on-line experiments.Visiting scientist from HUT at RIKEN     

A fuzzy expert system for the optimization of glutamic acid production

A fuzzy expert system was developed to allow the effective use of semi-quantitative or imprecise information in the determination of the optimal operating conditions of a fermentation process. The system incorporated fuzzy relations representing both the trends of experimental data and semi-quantitative information obtained from the literature. The inference method used consisted primarily of search and reasoning based on fuzzy set theory. The resulting expert system incorporated a generalized inference engine designed to deal with various types of fermentation processes, it only being necessary to alter the knowledge database in order to adapt the system to process modifications. The construction of the knowledge database from experimental data or semi-quantitative information was designed to be carried out semi-automatically using a graphic computer tool.The expert system was applied to the optimization of glutamic acid production by fermentation. The optimal conditions predicted by the expert system were found experimentally to give maximum production.List of Symbols A, B names of objects - A _i , B _i linguistic level of A and B, respectively - E₁, E₂ ordinary sets - I depth of search for inference - I _M maximum of I - M, X, Y names of sets - R ₁, R ₂ fuzzy relations - P_total g amount of glutamic acid production - Pen⁺ h penicillin addition time - S_vp1 h^–1 specific rate of glutamic acid production in 1st production phase - S_vp2 h^–1 specific rate of glutamic acid production in 2nd production phase - T _growth h growth period - T _lag h lag time - T _plag h lag time of production defined in Fig. 6 - T _p1 h period of 1st production - T _p2 h period of 2nd production - T _total h total production time - X _p g average cell mass of production phase - x, y, z real numbers - max(x, y) or x y maximum of x and y - min(x, y) or x y minimum of x and y     

A knowledge based system for diagnosing microbial activities during a fermentation process

A knowledge based system, LAexpert, was developed to diagnose microbial activities during a fermentation process on the basis of specific rates determined on-line. The LAexpert is a supervisor for a process control system and assists operators in fault diagnosis. The LAexpert was implemented using a fuzzy expert system shell based on the object oriented programming tool Smalltalk V/Mac running in a Macintosh II computer. The shell can handle uncertainties both in the measurements and knowledge by fuzzy reasoning.List of Symbols X g/l biomass dry weight (g/l) - S g/l substrate concentration (g/l) - P g/l product concentration (g/l) - _c, _c, _c 1/h specific rates calculated from on-line measured data of X, S and P (1/h) - _d, _d, _d 1/h specific rates read from database of BIOACS (1/h)     

Fuzzy reasoning system for fault diagnosis of physiological activities in a cultivating process.

Aiming at development of a system which supports cultivating operations, a method to diagnose physiological activities in a cultivating process is presented, and a fuzzy expert system for diagnosing Lactobacillus casei cultivating process is implemented in this paper. This system can calculate specific rates of cell growth, substrate consumption, and product formation with measuring cell mass concentration, substrate concentration, and product concentration by using a turbidity sensor and HPLC. A database is implemented, where standard curves on specific rates representing characteristics of microorganisms are stored according to normalized substrate consumption. Comparing the calculated specific rates with standard values derived from the database, the system diagnoses physiological activities of the microorganisms. As a case study, a knowledge base for diagnosing lactic acid production process is implemented. The use of fault diagnosis on pH malfunctions by the expert system proves its reasonable performance.     

Knowledge based diagnosis of inoculum properties and sterilization time in lactic acid fermentation

Summary A knowledge based system has been shown to be a powerful tool for diagnosing microbial activities during a fermentation process. Knowledge about lactic acid fermentation was collected by an experimental study ofLactobacillus casei. The effects of the inoculum properties and sterilization time on the cultivation were expressed in a form of a fuzzy rule-based knowledge network. The system was able to detect abnormal inoculum or sterilization conditions which caused malfunctions in the cultivations.     

Impact of balanced substrate flux on the metabolic process employing fuzzy logic during the cultivation of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var.<Emphasis Type="Italic"> Galleriae</Emphasis>

Summary The insecticidal crystal protein (ICP) synthesized at the onset of sporulation by Bacillus thuringiensis var. galleriae (Btg) is lethal against specific pests. Attempts were made to enhance the synthesis of biomass and ICP by Btg employing process optimization strategies. The process optimization was carried out with residual glucose concentration control in a bench scale bioreactor. A fuzzy logic-based feedback control system for maintaining the residual glucose concentration at a constant level during cultivation was developed in LabVIEW. This control system indicated the possibilities in providing a balanced substrate flux during cultivation. The identified optimum level of 2.72 g/l in residual glucose concentration was maintained by fed-batch cultivation with glucose and yeast extract fed at equal concentration with the above control system. High cell density of 16.0 g/l with specific growth rate of 0.69 h^-1 was obtained during the cultivation. The balanced flux of substrate during cultivation has resulted in the enhanced synthesis of biomass and ICP. This optimized process could be commercially exploited by comparing the fluxes of basal compounds in different media sources used in fermentation.     

Generation of rules for expert systems by statistical methods of fermentation data analysis

Five new methods for determining the relations between kinetic data of fermentations are described and applied to an industrial antibotic fermentation process. The input data for these method are the elements of the distance matrix d_ij, which quantify the sum of the deviation squares between the time dependent kinetics x (t) of the fermentation runs i and j. For each measurable or calculable kinetic state variable, one n x n distance matrix must be calculated where n is the number of fermentation runs. All methods compare these distance matrices by statistical or graph-theoretical approaches. The algorithms obtained are universally applicable if enough kinetic data are available, especially from more than 10 comparable fermentation runs. The algorithms were developed for the use in knowledge acquisition modules of expert systems.     

Design and simulation of a fuzzy controller for fed-batch yeast fermentation

In baker's yeast fermentation, the process is non-linear and the response of the system to changes in glucose feeding has a very long delay time. Therefore, a conventional system can not give satisfactory results. In this paper, a fuzzy controller designed to control a fed-batch fermenter is presented. The fuzzy controller uses Respiratory Quotient (RQ) as a controller input and produces glucose feeding rate as control variable. The controller has been tested on a simulated fed-batch fermenter. The results show that the maximum yeast production is possible by keeping the specific growth rate (μ) and the glucose concentration (C _s) at preset values (μ _Cand C _s,c) and minimizing the ethanol production.     

A pathway finding system for the cell signaling networks database

We report on a knowledge-based pathway-finding system that builds on the cell-signaling networks database, CSNDB, which we developed previously. This new system, PaF-CSNDB, uses a general inference engine to apply rules for finding and coupling pathways between or around specific biomolecules from the CSNDB database. We show how PaF-CSNDB finds relationships in a large but fragmented collection of cell-signaling knowledge by filtering out and composing together those sections of pathways specified from an extensive and complex set of binary or pair-wise cell-signaling reactions.     

An expert system applied to the physiological analysis of early stage of beer fermentation

A fuzzy expert system was applied to the knowledge analysis of yeast physiology in the early stage of beer fermentation, when the wort was aerated. We used ergosterol and glycogen concentration in the wort as a suitable marker of physiological state of the cell population. The amount of both compounds influences the rate of fermentation, cell growth and the final taste of beer. The concentrations of ergosterol and glycogen including the number of cells can not be measured immediately during the relatively short aeration period, and incomplete experimental data are therefore found in laboratory logbooks. We therefore suggested that the fuzzy relation between the directly measurable dissolved oxygen concentration and the rate of ergosterol or glycogen formation should be identified and a fuzzy expert system should be used to analyze the behavior of the yeast.     

Fermentation analysis by clustering

To supervise, stabilize and optimize antibiotic fermentations in the industrial scale expert systems are presently worked out. For the knowledge acquisition various classifiers are tested using a set of 27 nourseothricin fermentation runs. Two methods are applied: optimal clustering by help of minimum variance criterion and hierarchical clustering by help of dendrograms. The fermentations are classified with respect to the specific material costs as well as the product formation kinetics.List of Symbols a kg/m³ initial value of linearized product kinetics - b kg/(m³ · h) slope of linearized product kinetics - B binary variable (value 0 or 1) - C DM/kg specific costs - d distance - m number of samples - p kg/m³ product concentration - pO₂ % dissolved oxygen concentration - t h fermentation time - T h initial time of linearized product kinetics - n number of fermentation runs     

Lactic acid production with Lactobacillus casei ssp. rhamnosus NBIMCC 1013: Modeling and optimization of the nutrient medium

The medium needed to perform a fermentation process with viable cells of Lactobacillus casei ssp. rhamnosus NBIMCC 1013 for the production of lactic acid was modeled and optimized. On the basis of single‐factor experiments and statistical analysis, the significant factors affecting the fermentation process, i.e. the concentration of carbon source, concentrations of both yeast and meat extracts, and the range of variability of these components were determined. Modeling and optimization of the medium contents were performed using central composite design. The composition of the medium used for the production of lactic acid (g/L) was as follows: glucose 69.8, meat extract 17.07, yeast extract 10.9, CH₃COONa 10, K₂HPO₄ 0.25, KH₂PO₄ 0.25, MgSO₄·7H₂O 0.05, and FeSO₄ 0.05. The maximum specific growth rate of the lactic acid bacteria (μ=0.51 h⁻¹) and other kinetic parameters were determined during cultivation in a laboratory bioreactor using the logistic equation and the Luedeking–Piret model. The obtained medium allows the production of lactic acid under optimum conditions, at high specific sugar assimilation rates and high lactic acid accumulation rates. The positive results of the paper are the new nutrient medium for lactic acid production and the process kinetic model, enabling scaling up and switching to a continuous process.     

Production of hirudin by recombinant Saccharomyces cerevisiae in a membrane-recycle fermentor

Summary Recombinant Saccharomyces cerevisiae was employed to continuously produce hirudin in a membrane cell recycle fermentor. The gene cooing for the anticoagulant protein was combined with the GAL10 promoter for controlled expression and the MF 1 signal sequence for secretion to the fermentation broth. A dilution rate of 0.1h^–1 yielded a maximum hirudin concentration of 59mg / l with a specific hirudin concentration of 2.4 mg /g cell mass among dilution rates studied ranging from 0.05h^–1 to 0.3h^–1. Cell bleeding gave the same fermentation results as cell recycle fermentation without cell bleeding. The productivity of the cell recycle fermentation process was 6.0mg hirudin/l · hr, corresponding to a 1.7-fold increase compared with a conventional continuous culture.     

Studies on Oxygen Transfer in Submerged Fermentations

In conventional shaken culture system, control of oxygen supply is performed by changing liquid volume in flasks and it necessarily introduces variation in the effectiveness of agitation and in the partial pressure of carbon dioxide. In jar or tank culture system, also, the changes in mechanical agitation and in the flow rate of air for control of aeration induce similar problems. It is impossible, therefore, to isolate the effects of oxygen on microbial metabolism from these accompanying ones. Hence, there is a basic requirement of making clear distinction among them, and in this paper the effects of agitation and carbon dioxide on product formation are presented in glutamic acid fermentation using the apparatus of controlling the level of dissolved oxygen throughout the fermentation.

To obtain fundamental knowledge required for attaining adequate aeration, the rate of oxygen demand in glutamic acid fermentation was discussed in connection with its fermentation rates. On the basis of specific rates, rates of change per unit mass of cells, glutamic acid fermentation was found to fall in the process pattern of Gaden’s type II, in which a constant rate of oxygen demand was sustained for a considerable time. On the basis of volumetric rates, rates of change per unit volume of broths, oxygen demand was recognized to be correlated with growth, sugar utilization and product formation, and it was pointed out particularly that the oxygen demand was closedly related with sugar utilization. In the particular cases where rapid utilization of sugar occurred, therefore, oxygen deficiency was liable to be evoked being unable to fill the growing oxygen demand. This finding might be useful for scale-up studies or process design.     

Expert systems in histopathology. II. Knowledge representation and rule-based systems

Two aspects of expert systems for use in diagnostic histopathology and cytopathology are examined: knowledge representation and the structure and operation of rule-based systems. Knowledge may be represented, e.g., in semantic networks, frames, multiple contexts and model-based structures; the choice of structure should be matched to the type of information to create an efficient and logically adequate expert system. In a rule-based system, knowledge is represented as "rules," often in the form of "IF (condition)-THEN (conclusion)" rules. The anatomy of such rules and their operation is explored via the use of examples. Uncertainty in rules is briefly addressed, and their processing by the symbolic reasoning of the "inference engine" of the expert system is described, including both "forward-chaining" ("data-driven") operations and "backward-chaining" ("goal-driven") operations.     

A fuzzy logic controller.

This paper describes a fuzzy sets method which is very useful for handling uncertainties and essential for knowledge acquisition of a human expert. Kinetics of a reactor is often complex and not trivial to describe by mathematical equations. Reactor control by traditional control technology is therefore difficult. A novel technology is presented. In the following a fuzzy inference (approximate reasoning) is used for decision making in analogy to human thinking, facilitating a more sophisticated control. Readers of this paper do not need any advanced mathematics beyond the four basic operations in arithmetic (+, -, x, divided by) and using the maximum and minimum values. This fuzzy inference is introduced to construct a fuzzy logic controller which is suitable for a nonlinear, multivariable and time variant system applied to a bioreactor.     

Scale-up fermentation of recombinant <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using the GAP promoter

The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation. The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells, controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate high-level product. A stable lipase activity of approximately 14,000 IU ml⁻¹ and a cell wet weight of ca. 500 g l⁻¹ at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further scale-up for industrial lipase production.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain     

Braindex--an expert system for the diagnosis of brain death

An IBM PC-based system was developed as a decision-supporting system for the diagnosis of brain death. Braindex has been realized with the expert system Tool PC-Plus, and includes a knowledge base containing 300 rules organized in 8 frames. Both forward chaining and backward chaining are used in the inference engine. Braindex also includes a text archive and a lexicon. The data of 42 brain death patients are stored in the data bank (300 parameter per patient). The system is presently in the first stage of clinical application.     

Multistrain probiotic production by co‐culture fermentation in a lab‐scale bioreactor

Most commercial probiotic products intended for pharmaceutical applications consist of combinations of probiotic strains and are available in various forms. The development of co‐culture fermentation conditions to produce probiotics with the correct proportion of viable microorganisms would reduce multiple operations and the associated costs. The aim of this study was to develop a fermentation medium and process to achieve biomass comprising the desired proportion of two probiotic strains in co‐culture. Initially, a quantification medium was developed, and the method was optimized to allow the quantification of each strain's biomass in a mixture. The specific growth rates of Lactobacillus delbrueckii spp. bulgaricus and Lactobacillus plantarum were determined in media with different carbon sources. The inoculum volume was optimized to achieve equal proportion of biomass in co‐culture fermentation in test tubes. Next, fermentation was carried out in a 3‐L bioreactor. A biomass concentration of 2.06 g/L, with L. delbrueckii spp. bulgaricus and L. plantarum in the ratio of 47%:53% (by weight), was achieved with concomitant production of 12.69 g/L of lactic acid in 14 h. The results show that with careful manipulation of process conditions, it is possible to achieve the desired proportion of individual strains in the final biomass produced by co‐culture fermentation. This process may serve as a model to produce multistrain probiotic drugs at industrial scale.