Development of an automated microbial sensor system

An automated whole cell biosensor system was developed by integration of immobilized microbial cells in a flow-through system with screen-printed flow-through electrodes as detectors. The detectors used were thick-film Pt-electrodes in a 3-electrode configuration constructed as sandwich flow-through cells with a volume of about 36 microliters polarized at -900 mV. The measuring principle was the determination of oxygen consumption due to the microbial metabolism. Fructose was used as model analyte. The microorganisms were immobilized on cellulose-acetate membranes and integrated into a newly created reaction chamber (membrane reactor). The microbial cells used were Rhodococcus erythropolis and Issatchenkia orientalis known to be suitable for the determination of biological oxygen demand.     

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.     

Microbial sensor for selective determination of sulphide

A microbial sensor consisting of immobilized Thiobacillus thiooxidans, a gas-permeable membrane, and an O₂ electrode was prepared for the determination of sulphide. When a sample solution containing sulphide was passed into the flow cell, the output of the microbial sensor decreased markedly with time until a steady state was reached. The total time required for an assay was 20–30 min by the steady-state method. In the pulse method, the total time required for an assay was about 5 min. A linear relationship was obtained between the sensor output and the concentration of sodium sulphide below 0.40 mm. The minimum detectable concentration of sodium sulphide was 0.02 mm. Selectivity of the sensor was satisfactory. The microbial sensor was applied to the determination of sulphide in spring water. A good agreement was obtained between the microbial sensor and the methylene blue method. The regression coefficient was 0.97 for five experiments. The activity of the microbial membrane was stable for more than 25 days. The response was reproducible with 2.5% of the relative standard deviation when a sample solution containing 0.2 mm sodium sulphide was employed. *** DIRECT SUPPORT *** AG903053 00005     

应用FIA型微生物传感器测定谷氨酸含量的研究

目前应用酶或微生物细胞作为分子识别元件构建生物传感器测定谷氨酸含量的研究引起了广泛的兴趣,并陆续有实例报道。在这些报道中人们依据不同的酶催反应选择相应的离子选择性电极,如CO₂电极、NH₄⁺电极等,而测量对象有直接的测定谷氨酸,也有测定谷氨酸单钠的间接测定法。本文选用了可固定大量细胞的固定化细胞柱与流动注入法相结合的测量方法,并根据细胞柱的动力学模型分析动态响应曲线,计算测量结果,提高了测量精度,扩大了测量范围。     

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.     

A new method for immobilization of microbial cells by cross-linking

A method for immobilization of microbial cells was designed. The method uses generation of reactive aldehyde groups on the cell wall surface under conditions of periodate oxidation. The linking of aldehyde groups by various bifuctional aromatic diamines and then by glutaraldehyde produced immobilized cells, which are promising for use in biocatalysis with high-molecular-weight substrates.     

Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry

As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors. The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line. This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary. Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture.     

Protein determination of cells immobilized in cross-linked synthetic gels

Summary An assay for the determination of the protein content of whole cells immobilized in cross-linked synthetic gels was developed. The assay is based on a three step procedure: a) methanol dehydration, b) protein extraction by 1.0 M alkali at 125°C c) colorimetric assay of the extracted protein according to Bradford's procedure (Bradford M. M. (1976), Anal. Biochem. 72:248–254). The procedure worked out was found adequate for the determination of the protein content of microbial cells immobilized in synthetic and native polymer-gel-systems.     

A simplification of the protein assay method of Ramsay et al. for the quantification of Thiobacillus ferrooxidans in the presence of ferric precipitates

A variation on Ramsay's method for microbial protein determination has been developed in order to quantify Thiobacillus ferrooxidans attached to ferric precipitates or in aqueous suspensions containing such precipitates. Some modifications have been introduced to provide a method that is more sensitive, simple and rapid. A linear standard curve is presented to permit a direct correlation between the protein concentration (mg/l) and the cell concentration (10⁶ cells/ml). An application of this method has been demonstrated in the quantification of biomass immobilized on the surface of polyurethane foam particles in a packed bed reactor, several experiments having been conducted to establish the best conditions for the quantification studies. Received: 12 August 1999 / Received revision: 21 December 1999 / Accepted: 29 December 1999     

On-line determination of animal cell concentration.

A new approach for the indirect determination of cell concentration in the case of nonconstant metabolic rates has been developed. The specific glucose-uptake rate was shown to be nonconstant in batch cultures of free suspended and immobilized CHO SSF3 cells. Time-independent models correlating the specific rate to the limiting substrate concentration were established, thus providing a continuous determination of the specific rate through on-line measurement of the limiting substrate. The method could be applied to determine on-line cell concentration in both free suspended and immobilized cell cultures. Results were verified off-line by crystal violet nuclei counting. The predicted cell concentration was in very good agreement with the off-line reference during the whole exponential-growth phase, until the specific glucose-uptake rate tended to zero.     

A method for the quantification of bacterial protein in the presence of Jarosite

A variation on Peterson's modification of the Lowry method for microbial protein determination was developed in which 10% (w/v) oxalic acid was used to remove jarosite. This allowed the quantification of Thiobacillus ferrooxidans entrapped in solid jarosite or in aqueous suspensions containing jarosite. The quantity of protein measured was not affected by the amount of jarosite in the culture, the concentration of oxalic acid, or the time of exposure (up to 72 h) of the sample to oxalic acid. An application of this method was demonstrated in the quantification of biomass immobilized in jarosite on the surface of polystyrene beads in an inverse fluidized bed bioreactor used for the rapid microbial oxidation of ferrous iron.     

Analysis of Distributed Growth of Saccharomyces cerevisiae Cells Immobilized in Polyacrylamide Gel

A technique is described for the quantitative determination of the distributed growth of Saccharomyces cerevisiae immobilized in polyacrylamide gel. Gel specimens were embedded in paraffin or gelatin and paraffin before sectioning and staining. Photomicrographs of specimen sections were enlarged, and cell microcolony volumes were determined as a function of position in the gel by grid transparency analysis. Overall cell densities within the gel were calculated for a quantitative comparison with values measured by a second spectrophotometric method. The results show good agreement and demonstrate the sigmoidal growth of the immobilized cells, reaching a maximum steady-state value. The technique shows promise as a general method for following the transient growth of organisms immobilized within gel particles.     

Biosensor design: microbial loading capacity of acetylcellulose membranes

Summary In the construction of biosensors incorporating probe oxygen electrodes as the biochemical signal transducer, acetylcellulose membranes have proved to be a popular but poorly characterized matrix for microbial cell immobilization. In this report the relationship between immobilized whole microbial cell concentration/mm³ of acetylcellulose membrane and the reduction in oxygen diffusion across the microbial electrode has been determined using enteric bacteria. Total inhibition of oxygen diffusion occurred when the cell concentration was raised from 1×10⁶ to 1×10⁷ cells mm³: equivalent to a microbial cell volume of 1.5% compared to the maximum theoretical matrix volume. To facilitate future biosensor design a biosensor immobilized cell concentration Bicc value and a zero oxygen diffusion ZO₂D value are suggested, and the parameters of immobilized cell concentration, reduction in oxygen diffusion by immobilized cells and microbial cell size are discussed.     

Preservation of immobilized bacterial cell-matrix by drying for direct use in microbial sensors

A simple and effective method for the drying of immobilized bacterial cells to be used directly in a microbial biosensor for measurement of activity is reported. As a case example, plasmid-bearing cells of Alcaligenes eutrophus JMP 134, DSM 4058 were immobilized on various carriers and liquid-dried. The dried cell-matrix was used directly after rehydration/reactivation as the biological component of a biosensor for determining the concentration of xenobiotic compounds in the environment. Good viability results were obtained after long-term storage and cells exhibited no loss of plasmids responsible for the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The activity of the cells for 2,4-D was proved using a respiration electrode. No time-consuming, repeated cell cultivation and harvesting was required, as the cells preserved from a single batch served as a continuous source for activity measurements. Many other microbial cultures can be preserved by this method and the cells preserved in the form of immobilized dried cell-matrix can be used directly to perform enzymatic tests, complex biochemical conversions and for production in the reactors. The dried cell-matrix can serve as a stable interchangeable component for a multipurpose biosensor.     

A new method for immobilizing microbial cells by crosslinking

A method for immobilization of microbial cells was developed designed. The method uses based on generation of reactive aldehyde groups on the cell wall surface under conditions of periodate oxidation. The linking of aldehyde groups by various bifunctional aromatic diamines and then by glutaraldehyde produced immobilized cells, which are promising for the use in biocatalysis with high-molecular-weight substrates of high molecular weight.     

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.     

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.     

Enhancement of coulombic efficiency and salt tolerance in microbial fuel cells by graphite/alginate granules immobilization of Shewanella oneidensis MR-1

Coulombic efficiency and stability of electricity output are crucial for practical applications of microbial fuel cells (MFCs). In this study, a cell immobilization method for electrogenic microorganism in MFCs using graphite/alginate granules is developed. The MFC with immobilized cell granules delivered a much more stable electricity output than that with suspension cells, and resulted in a ～0.8 to 1.7 times improvement on coulombic efficiency compared to the suspension mode. Impressively, with the conductive graphite/alginate/cells granules, the internal resistance of the MFC decreased dramatically. Moreover, the cell immobilized MFC showed a much higher tolerance to the shock of high salt concentration than the MFC with suspension cells. The results substantiated that immobilization of electrogenic microorganism for MFCs could be achieved by the method developed here, and it is promising for practical application in energy harvesting from wastewater by MFCs.     

Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.     

Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.