Sensitive determination of L-lysine with a new amperometric microbial biosensor based on Saccharomyces cerevisiae yeast cells

A new amperometric microbial biosensor based on Saccharomyces cerevisiae NRRL-12632 cells, which had been induced for lysine oxidase enzyme and immobilized in gelatin by a cross-linking agent was developed for the sensitive determination of L-lysine amino acid. To construct the microbial biosensor S. cerevisiae cells were activated and cultured in a suitable culture medium. By using gelatine (8.43 mg cm(-2)) and glutaraldehyde (0.25%), cells obtained in the logarithmic phase of the growth curve at the end of a 14 h period were immobilized and fixed on a pretreated oxygen sensitive Teflon membrane of a dissolved oxygen probe. The assay procedure of the microbial biosensor is based on the determination of the differences of the respiration activity of the cells on the oxygenmeter in the absence and the presence of L-lysine. According to the end point measurement technique used in the experiments it was determined that the microbial biosensor response depended linearly on L-lysine concentrations between 1.0 and 10.0 microM with a 1 min response time. In optimization studies of the microbial biosensor, the most suitable microorganism quantities were found to be 0.97x10(5)CFU cm(-2). In addition phosphate buffer (pH 7.5; 50 mM) and 30 degrees C were obtained as the optimum working conditions. In characterization studies of the microbial biosensor some parameters such as substrate specificity, interference effects of some substances on the microbial biosensor responses, reproducibility of the biosensor and operational and storage stability were investigated.     

Amperometric determination of total assimilable sugars in fermentation broths with use of immobilized whole cells

A microbial sensor consisting of immobilized living whole cells of Brevibacterium lactofermentum and an oxygen electrode was prepared for continuous determination of total assimilable sugars (glucose, fructose and sucrose) in a fermentation broth for glutamic acid production. Total assimilable sugars were evaluated from oxygen consumption by the immobilized microorganisms. When a sample solution containing glucose was applied to the sensor system, increased consumption of oxygen by the microorganisms caused a decrease in the dissolved oxygen around the Teflon membrane of the oxygen electrode and the current of the electrode decreased markedly with time until steady state was reached. The response time was ≈ 10 min by the steady state method and 1 min by the pulse method. A linear relationship was found between the decrease in current and the concentration of glucose (<1 mM), fructose (<1 mM) and sucrose (<0.8 mM). The ratio of the sensitivity of the microbial sensor to glucose, fructose and sucrose was 1.00:0.80:0.92. The decrease in current was reproducible to within 2% of the relative standard deviation when a sample solution containing glucose (0.8 mM) was employed for experiments. The selectivity of the microbial sensor for assimilable sugars was satisfactory for use in the fermentation process. The additivity of the response of the microbial sensor for glucose, fructose and sucrose was examined. The difference between the observed and calculated values was within 8%. The microbial sensor was applied to a fermentation broth for glutamic acid production. Total assimilable sugars can be determined by the microbial sensor which can be used for more than 10 days and 960 assays.     

An amperometric microbial biosensor development based on Candida tropicalis yeast cells for sensitive determination of ethanol

Different branchs of industry need to use ethanol in their production and some progress and not only the industry also to determine ethanol sensitively, accurately, fast and economical is very important. For the sensitive determination of ethanol a new amperometric biosensor based on Candida tropicalis cells, which contains alcohol oxidase enzyme, immobilized in gelatin by using glutaraldehyde was developed. In the study, before the microbial biosensor construction C. tropicalis cells were activated and cultured in a culture medium. By using gelatine and glutaraldehyde (0.1%) C. tropicalis cells obtained in logarithmic phase were immobilized and fixed on a pretreated teflon membrane of a dissolved oxygen probe. Ethanol determination is based on the assay of the differences on the respiration activity of the cells on the oxygenmeter in the absence and the presence of ethanol. The microbial biosensor response was depend linearly on ethanol concentration between 0.5 and 7.5 mM with 2 min response time. In the optimization studies of the microbial biosensor the most suitable microorganism amount were found as 4.42 mg cm(-2) and also phosphate buffer (pH:7.5; 50 mM) and 30 degrees C were obtained as the optimum working conditions. In the characterization studies of the microbial biosensor some parameters such as substrate specificity, interference effects of some substances on the biosensor response, operational and storage stability were carried out.     

Development of a flow-cell system with dual fuel-cell electrodes for continuous monitoring of microbial populations

Summary Two electrode pairs in a flow-through dual fuel-cell system were used to determine the electrochemical potential generated by microbial populations. The potential difference between the counting and the reference electrode pairs was reciprocally proportional to the cell density of microorganisms in the medium, and the response time varied from 20 to 40 minutes at 30°C. Presently, the system is suitable for continuous electrochemical determination of microbial cell populations with a slow growth rate.     

Microbial biosensor array with transport mutants of Escherichia coli K12 for the simultaneous determination of mono-and disaccharides

An automated flow-injection system with an integrated biosensor array using bacterial cells for the selective and simultaneous determination various mono- and disaccharides is described. The selectivity of the individually addressable sensors of the array was achieved by the combination of the metabolic response, measured as the O(2) consumption, of bacterial mutants of Escherichia coli K12 lacking different transport systems for individual carbohydrates. Kappa-carrageenan was used as immobilization matrix for entrapment of the bacterial cells in front of 6 individually addressable working electrodes of a screen-printed sensor array. The local consumption of molecular oxygen caused by the metabolic activity of the immobilized cells was amperometrically determined at the underlying screen-printed gold electrodes at a working potential of -600 mV vs. Ag/AgCl. Addition of mono- or disaccharides for which functional transport systems exist in the used transport mutant strains of E. coli K12 leads to an enhanced metabolic activity of the immobilized bacterial cells and to a concomitant depletion of oxygen at the electrode. Parallel determination of fructose, glucose, and sucrose was performed demonstrating the high selectivity of the proposed analytical system.     

A novel biosensor based on Lactobacillus acidophilus for determination of phenolic compounds in milk products and wastewater

Different branches of industry need to use phenolic compounds (PCs) in their production, so determination of PCs sensitively, accurately, rapidly, and economically is very important. For the sensitive determination of PCs, some biosensors based on pure polyphenol oxidase, plant tissue and microorganisms were developed before. But there has been no study to develop a microbial phenolic compounds biosensor based on Lactobacillus species, which contain polyphenol oxidase enzyme. In this study, we used different forms of Lactobacillus species as enzyme sources of biosensor and compared biosensor performances of these forms for determination of PCs. For this purpose, we used lyophilized Lactobacillus cells (containing L. bulgaricus, L. acidophilus, Streptococcus thermophilus), pure L. acidophilus, pure L. bulgaricus, and L. acidophilus- and L. bulgaricus adapted to catechol in Lactobacilli MRS Broth. The most suitable form was determined and optimization studies of the biosensor were carried out by using this form. For preparing the bioactive layer of the biosensor, the Lactobacillus cells were immobilized in gelatin by using glutaraldehyde. In the study, we used catechol as a substrate. Phenolic compound determination is based on the assay of the differences on the respiration activity of the cells on the oxygen meter in the absence and the presence of catechol. The microbial biosensor response depends directly on catechol concentration between 0.5 and 5.0 mM with 18 min response time. In the optimization studies of the microbial biosensor the most suitable microorganism amount was found to be 10 mg, and also phosphate buffer (pH 8.0; 50 mM) and 37.5 °C were obtained as the optimum working conditions. In the characterization studies of the microbial biosensor some parameters such as substrate specificity on the biosensor response and operational and storage stability were examine. Furthermore, the determination of PC levels in synthetic wastewater, industrial wastewater, and milk products was investigated by using the developed biosensor under optimum conditions.     

Determination of ascorbic acid with immobilized green zucchini ascorbate oxidase

Ascorbate oxidase from zucchini squash was immobilized onto CH-Sepharose via carbodiimide. The properties of the immobilized enzyme were found to be similar to those of the free ascorbate oxidase. The immobilized enzyme was utilized in a flow-through system equipped with a polarographic detector which monitors the oxygen depletion due to the reaction ascorbic acid + 1/2 O2----dehydroascorbic acid + H2O. This method, the response of which is linear between 3 X 10(-7) and 5 X 10(-4) M ascorbate, was utilized to measure the ascorbic acid in biological samples such as human plasma and fruit juices at a rate of about 60 determinations every hour with a standard deviation lower than 5%.     

Oxygen supply to immobilized cells: 5. Theoretical calculations and experimental data for the oxidation of glycerol by immobilized Gluconobacter oxydans cells with oxygen or p-benzoquinone as electron acceptor

Theoretical calculations of reaction kinetics were done for one-step reactions catalyzed by cells immobilized in spherical beads. The reactions catalyzed by free cells were assumed to obey Michaelis-Menten kinetics for a one-substrate reaction. Both external (outside the beads) and internal (inside the beads) mass transfer of the substrate were considered for the immobilized preparations. The theoretical calculations were compared with experimental data for the oxidation of glycerol to dihydroxyacetone by Gluconobacter oxydans cells immobilized in calcium alginate gel. Glycerol was present in excess so that the reaction rate was limited by oxygen. The correlation between experimental data and theoretical calculations was quite good. The calculations showed how the overall effectiveness factor was influenced by, for example, the particle size and the cell density in the beads. In most cases the reaction rate was mainly limited by internal mass transfer of the substrate (oxygen). As shown previously, p-benzoquinone can replace oxygen as the electron acceptor in this reaction. The same equations for reaction kinetics and mass transfer were used with p-benzoquinone as the rate-limiting substrate. Parameters such as diffusivity, maximal reaction rate, and K were, of course, different. In this case also, the correlation between the model and the experimental results was quite good. Much higher production rates were obtained with p-benzoquinone as the electron acceptor compared to when oxygen was used. The reasons for this fact were that p-benzoquinone gave a higher maximal reaction rate for free cells and the solubility of p-benzoquinone was higher than for oxygen. Different methods of increasing the rate of microbial oxidation reactions are discussed.     

Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column.

A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow-injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol.     

Detection of halogenated hydrocarbons by a microbial sensorsystem using a stop-flow-technique

To improve the applicability and efficiency of the microbial assay developed by W. Hutter, J. Peter, H. Swoboda, W. Hampel, E. Rosenberg, D. Krämer, and R. Kellner (1995) a stop-flow-technique was developed for the determination of halogenated hydrocarbons in water samples. Cells of Rhodococcus sp. DSM 6344 were immobilized in alginate beads and placed in a stirred flow-through reactor. The time of incubation, the bacterial cell density and the amount of alginate beads in the reactor on the response of the system as determined by the drop in EMF was investigated. Optimal conditions were achieved with 2 g beads containing a bacterial cell concentration of 0,1 g cells wet wt/g matrix and an incubation time of 20 min. Calibrations with chlorinated and brominated substrates like ethyl bromide, 1,2-dibromopropane, isobutyl bromide, 1-chlorobutane and 1,5-dichloropentane showed a non-linear dependence at low substrate concentrations. The detection limits for ethyl bromide and 1-chlorobutane were estimated as 0.02 mg/l and 0.45 mg/l, respectively, the relative standard deviation was below 10 %. The great advantage of the stop-flow-technique compared to discontinuous measurements can be seen in a simplified handling and an increase of sample capacity.     

On-line determination of glucose and lactate concentrations in animal cell culture based on fibre optic detection of oxygen in flow-injection analysis.

A flow-injection analysis (FIA) system based on fibre optic detection of oxygen consumption using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is described for the on-line monitoring of glucose and lactate concentrations in animal cell cultures. The consumption of oxygen was determined via dynamic quenching by molecular oxygen of the fluorescence of an indicator. GOD and LOD were immobilized on controlled pore glass (CPG) in enzyme reactors which were directly linked to a specially designed fibre optic flow-through cell covering the oxygen optrode. The system is linear for 0-30 mM glucose, with an r.s.d. of 5% at 30 mM (five measurements) and for 0-30 mM lactate, with an r.s.d. of 5% at 30 mM (five measurements). The enzyme reactors used were stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per hour. The system has been successfully applied to the on-line monitoring of glucose and lactate concentrations of an animal cell culture designed for the production of recombinant human antithrombine III (AT-III). Results of the on-line measurement obtained by the FIA system were compared with the off-line results obtained by a glucose and lactate analyser from Yellow Springs Instrument Company (YSI).     

Development and characterization of a novel immobilized microbial membrane for rapid determination of biochemical oxygen demand load in industrial waste-waters

The rapid determination of waste-water quality of waste-water treatment plants in terms of pollutional strength, i.e. biochemical oxygen demand (BOD) is difficult or even impossible using the chemical determination method. The present study reports the determination of BOD within minutes using microbial BOD sensors, as compared to the 5-day determination using the conventional method. Multiple criteria establish the basis for the development of a BOD biosensor useful for rapid and reliable BOD estimation in industrial waste-waters. Of these, preparation of a suitable novel immobilized microbial membrane used in conjunction with an apt transducer is discussed. As a result, a microbial biosensor based on a formulated, synergistic, pre-tested microbial consortium has been developed for the measurement of BOD load of various industrial waste-waters. The sensor showed maximum response in terms of current difference, when a cell concentration of 2.25 x 10(10) CFU, harvested in their log phase of growth were utilized for microbial membrane construction. The sensor showed a stability of 180 days when the prepared membranes were stored at a temperature of 4 degrees C in 50 mM phosphate buffer of pH 6.8. The reusability of the immobilized membranes was up to 200 cycles without appreciable loss of their response characteristics. A linear relationship between the current change and a glucose-glutamic acid (GAA) concentration up to 60 mg l(-1) was observed (r=0.999). The lower detection limit was 1.0 mg l(-1) BOD. The sensor response was reproducible within +/-5% of the mean in a series of ten samples having 44 mg l(-1) BOD using standard a GGA solution. When used for the BOD estimation of industrial waste-waters, a relatively good agreement was found between the two methods, i.e. 5-day BOD and that measured by the developed microbial sensor.     

Simultaneous enzymatic/electrochemical determination of glucose and L-glutamine in hybridoma media by flow-injection analysis

A split-stream flow-injection analysis system is described for simultaneous determination of glucose and L-glutamine in serum-free hybridoma bioprocesses media. Amperometric measurement of glucose is based on anodic oxidation of hydrogen peroxide produced by immobilized glucose oxidase within a triple layer membrane of an integrated flow-through glucose-selective biosensor. Determination of L-glutamine is based on quantitating ammonium ions produced in a flow-through enzymes reactor containing immobilized glutaminase enzyme, and subsequent downstream potentiometric detection of these ions by a nonacting-based ion-selective polymer membrane electrode. Endogenous potassium and ammonium ion interference in the L-glutamine determination are eliminated by using a novel in-line tubular cation-exchange membrane unit to exchange these interferent species for cations undetectable by the membrane electrode. The first generation split-steam flow-injections system can assay 12 samples/h using direct injections of 50 muL of media samples, with linear responses to glucose in the range of 0.03 to 30mM, and log-linear response to L-glutamine from 0.1 to 10 mM. (c) 1993 Wiley & Sons, Inc.     

Microbial electrode sensor for alcohols

A microbial electrode consisting of immobilized microorganisms, a gas permeable Teflon membrane, and an oxygen electrode was prepared for the continuous determination of methyl and ethyl alcohols. Immobilized Trichosporon brassicae was employed for a microbial electrode sensor for ethyl alcohol. When a sample solution containing ethyl alcohol was injected into a microbial electrode system, the current of the electrode decreased markedly with time until a steady state was reached. The response time was within 10 min by the steady state method and within 6 min by the pulse method. A linear relationship was observed between the current decrease and the concentration of ethyl alcohol below 22.5 mg/liter. The current was reproducible within ± 6% of the relative error when a sample solution containing 16.5 mg/liter ethyl alcohol. The standard deviation was 0.5 mg/liter in 40 experiments. The selectivity of the microbial electrode sensor for ethyl alcohol was satisfactory. The microbial electrode sensor was applied to a fermentation broth of yeasts and satisfactory comparative results were obtained (correlation coefficient 0.98). The current output of the microbial electrode sensor was almost constant for more than three weeks and 2100 assays. A microbial electrode sensor using immobilized bacteria for methyl alcohol was also described.     

Biotransformation and Dissolution of Petroleum Hydrocarbons in Natural Flowing Seawater at Low Temperature

The objective of this study was to establish methods for controlled studies of hydrocarbon depletion from thin oil films in cold natural seawater, and to determine biotransformation in relation to other essential depletion processes. Mineral oil was immobilized on the surface of hydrophobic Fluortex fabrics and used for studies of microbial biodegradation in an experimental seawater flow-through system at low temperatures (5.9-7.4 degrees C) during a test period of 42 days. The seawater was collected from a depth of 90 m, and microbial characterization by epifluorescence microscopy, fluorescence in situ hybridization, and most-probable number analysis showed relatively larger fractions of archaea and oil-degrading microbes than in the corresponding surface water. Chemical analysis of hydrocarbons attached to the fabrics during the test period showed that n-alkanes (C10-C36) were decreased by 98% after 21 days, while naphthalenes were depleted by 99-100% during the same period. At the end of the period 4-5 ring polyaromatic hydrocarbon (PAH) compounds were removed by 82% from the fabrics. Analysis of the recalcitrant pentacyclic triterpane C30 17alpha(H),21beta(H)-hopane showed that the oil remained adsorbed to the fabrics during the test period. Comparison of depletion analysis with calculation of hydrocarbon dissolution in a flow-through system indicated that naphthalenes and smaller PAH compounds (alkylated 2-ring and 3-ring compounds) were removed from the fabrics by dissolution. The data further implied that depletion of n-alkanes and 4-5 ring PAH hydrocarbons were the result of biotransformation processes. PCR amplification of bacterial 16S rRNA genes from microbes adhering on the immobilized oil surfaces showed the dominance of a few bands when analysed in denaturing gradient gel electrophoresis (DGGE). Sequence analysis of DGGE bands revealed phylogenetic affiliation to the alpha- and gamma-subdivisions of proteobacteria and to the Chloroflexus-Flavobacterium-Bacteroides group.     

Enhanced bioconversion of colchicine to regiospecific 3-demethylated colchicine (3-DMC) by whole cell immobilization of recombinant E. coli harboring P450 BM-3 gene

Biotransformation of colchicine into regiospecific 3-demethylated colchicine (3-DMC) which is pharmacologically active and a potent anti-cancer drug, mediated by immobilization of recombinant microbial monooxygenases is a novel and promising strategy for its production. In the present study, recombinant Escherichia coli expressing P450 BM-3 was immobilized in calcium-alginate beads and its ability to catalyze colchicine into 3-DMC was investigated. Characteristics of immobilized system showed that optimum conditions for activity of microbial cells were not affected due to immobilization. The optimum pH and temperature for both free and immobilized cells were found to be 7.5 and 37.5 °C, respectively. Experimental variables under consideration such as Ca²⁺ concentration, alginate concentration, P450 BM-3 enzyme activity and colchicine concentration were optimized using response surface methodology. The immobilized cells exhibited a markedly improved thermal stability as compared to free cells. The yield of 3-DMC with immobilized microbial cells was found to be an average of 69%, with 82, 73 and 52% across three independent batches in succession as against bioconversion by free cells, which indicated improved operational stability and reusability of immobilized cells in batch processes. Additionally, a packed bed reactor has been proposed for the immobilized biocatalytic system for bioconversion of colchicine and other biochemicals.     

Effect of oxygen supply on metabolism of immobilized and suspended Escherichia coli

The effect of reduced oxygen supply on the production of a recombinant protein (plasmid-encoded beta-galactosidase) was investigated in Escherichia coli. A novel modified bubble tank reactor was used to provide a direct comparison between immobilized and suspended cells in identical environments except for the immobilization matrix. Decreased oxygen supply led to increased beta-galactosidase synthesis by both immobilized and suspended cells. Immobilized cells produced similar amounts of beta-galactosidase as the suspended cells. Lactose consumption and acetate production, on a per cell basis, were significantly higher in immobilized cells, suggesting that immobilized cells utilized fermentative metabolism. However, a transport analysis of the immobilized cell system showed that immobilized cells were not subject to either external or internal mass transfer gradients.     

Whole cell immobilized amperometric biosensor based on Saccharomyces cerevisiae for selective determination of vitamin B1 (thiamine)

A new amperometric whole cell biosensor based on Saccharomyces cerevisiae immobilized in gelatin was developed for selective determination of vitamin B1 (thiamine). The biosensor was constructed by using gelatin and crosslinking agent glutaraldehyde to immobilize S. cerevisiae cells on the Teflon membrane of dissolved oxygen (DO) probe used as the basic electrode system combined with a digital oxygen meter. The cells were induced by vitamin B1 in the culture medium, and the cells used it as a carbon source in the absence of glucose. So, when the vitamin B1 solution is injected into the whole cell biosensor system, an increase in respiration activity of the cells results from the metabolic activity and causes a decrease in the DO concentration of interval surface of DO probe related to vitamin B1 concentration. The response time of the biosensor is 3 min, and the optimal working conditions of the biosensor were carried out as pH 7.0, 50mM Tris-HCl, and 30 degrees C. A linear relationship was obtained between the DO concentration decrease and vitamin B1 concentration between 5.0 x 10(-3) and 10(-1) microM. In the application studies of the biosensor, sensitive determination of vitamin B1 in the vitamin tablets was investigated.     

Diffusion coefficients of metabolites in active biofilms

Diffusion coefficients of actual metabolites in completely active biofilms can be determined by applying a new concept that is based on a constant local activity in the entire biofilm. In that case, a concentration step will be transmitted unattenuated. Subsequently, the diffusion coefficient can be calculated from the response monitored with a microelectrode positioned in the biofilm without quantitative knowledge of the local microbial kinetics. The conditions required for such a constant microbial biofilm activity were formulated in terms of the Thiele modulus and the substrate concentration in the bulk liquid. This proposed method was successfully applied to determine diffusion coefficients of oxygen and glucose in agar gels containing various fractions of active immobilized microorganisms. The values obtained were compared to experimental results from well-defined inert systems. The transient response of oxygen was far more affected by the presence of the immobilized cells than glucose. This can be explained by partition of the diffusing solute between the microbial cells and the aqueous phase.     

Continuous production of L-arginine using immobilized growing Serratia marcescens cells: Effectiveness of supply of oxygen gas

Summary The immobilized growing cell system using Serratia marcescens was applied to continuous L-arginine production. From the determination of oxygen uptake rate, it was shown that the cells entrapped in carrageenan gel were in an oxygen-limited state due to the diffusion barrier to oxygen transport created by the gel layer. This limited state in gel was relieved by supply of oxygen-enriched gas instead of air into the medium. The maximum population of immobilized cells increased to five times that of free cells with the supply of pure oxygen gas. The L-arginine-producing activity of the immobilized growing cells was proportional to the concentration of oxygen gas supplied and was 6 mg/h per millilitre in gel supplied with pure oxyges gas. The continuous L-arginine containing production was constantly maintained by controlling the medium penicillin G at pH 6.5 and more than 10 mg/ml of L-arginine were obtained at 10h of residence time for at least 12 days.