V. A. Alferov

Yeast-based self-organized hybrid bio-silica sol-gels for the design of biosensors.

The methylotrophic Pichia angusta VKM Y-2559 and the oleaginous Cryptococcus curvatus VKM Y-3288 yeast cells were immobilized in a bimodal silica-organic sol-gel matrix comprised of tetraethoxysilane (TEOS), the hydrophobic additive methyltriethoxysilane (MTES) and the porogen polyethylene glycol (PEG). Under carefully optimized experimental conditions, employing basic catalysts, yeast cells have become the nucleation centers for a silica-organic capsule assembled around the cells. The dynamic process involved in the formation of the sol-gel matrix has been investigated using optical and scanning electron microscopic techniques. The results demonstrated the influence of the MTES composition on the nature of the encapsulation of the yeast cells, together with the architecture of the three-dimensional (3D) sol-gel biomatrix that forms during the encapsulation process. A silica capsule was found to form around each yeast cell when using 85 vol% MTES. This capsule was found to protect the microorganisms from the harmful effects that result from exposure to heavy metal ions and UV radiation. The encapsulated P. angusta BKM Y-2559 cells were then employed as a biosensing element for the detection of methanol. The P. angusta-based biosensor is characterized by high reproducibility (Sr, 1%) and operational stability, where the biosensor remains viable for up to 28 days.

Bioanode for a Microbial Fuel Cell Based on Gluconobacter oxydans Immobilized into a Polymer Matrix

Acetic acid bacteria Gluconobacter oxydans subsp. industrius RKM V-1280 were immobilized into a synthetic matrix based on polyvinyl alcohol modified with N-vinylpyrrolidone and used as biocatalysts for the development of bioanodes for microbial fuel cells. The immobilization method did not significantly affect bacterial substrate specificity. Bioanodes based on immobilized bacteria functioned stably for 7 days. The maximum voltage (fuel cell signal) was reached when 100–130 μM of an electron transport mediator, 2,6-dichlorophenolindophenol, was added into the anode compartment. The fuel cell signals reached a maximum at a glucose concentration higher than 6 mM. The power output of the laboratory model of a fuel cell based on the developed bioanode reached 7 mW/m2 with the use of fermentation industry wastes as fuel.

Biosensors based on modified screen-printed enzyme electrodes for monitoring of fermentation processes

Biosensor array based on modified screen-printed electrodes containing enzymes (glucose oxidase, alcohol oxidase, lactate oxidase, mixture of glucose oxidase and γ-amylase) immobilized in a membrane of cross-linked bovine serum albumin was developed for the determination of the content of glucose, ethanol, lactic acid, and starch in fermentation media. The analytical and metrological characteristics of the developed biosensor system were determined. Samples of the fermentation mass and of alcoholic and fermented milk products were analyzed. Statistical analysis of the results obtained showed that the results of determining the content of glucose, ethanol, lactic acid, and starch with the developed biosensor system and by reference methods differed insignificantly. The developed biosensor system is considered as a base for fabricating a prototype of an analyzer for use in fermentation biotechnology.

Efficiency of bioelectrocatalytic oxidation of ethanol by whole cells and membrane fractions of Gluconobacter Oxydans bacteria in the presence of mediators of ferrocene series

Bioelectrocatalytic oxidation of ethanol by whole cells and membrane fraction of Gluconobacteroxydans bacteria is studied on modified graphite-paste electrodes in mediator biosensors. Ferrocene derivatives are used as electron transport mediators for effective coupling of enzymatic and electrochemical processes on graphite electrodes. Electrochemical kinetics of the processes are studied; the obtained data are interpreted in the terms of the mechanism of two-substrate enzymatic reaction. It is shown that mediators of ferrocene series are promising compounds for development of mediator biosensors based both on whole cells of Gluconobacter oxydans bacteria and on membrane fractions of these bacteria. Bioelectrocatalytic processes of ethanol oxidation on graphite paste electrodes occur more efficiently when the bacterial membrane fraction is used as a biocatalyst and ferrocenemonocarboxylic acid is used as a mediator.

Three-Dimensional Organization of Self-Encapsulating Gluconobacter oxydans Bacterial Cells

Self-organized bacteria have been the subject of interest for a number of applications, including the construction of microbial fuel cells. In this paper, we describe the formation of a self-organized, three-dimensional network that is constructed using Gluconobacter oxydans B-1280 cells in a hydrogel consisting of poly(vinyl alcohol) (PVA) with N-vinyl pyrrolidone (VP) as a cross-linker, in which the bacterial cells are organized in a particular side-by-side alignment. We demonstrated that nonmotile G. oxydans cells are able to reorganize themselves, transforming and utilizing PVA–VP polymeric networks through the molecular interactions of bacterial extracellular polysaccharide (EPS) components such as acetan, cellulose, dextran, and levan. Molecular dynamics simulations of the G. oxydans EPS components interacting with the hydrogel polymeric network showed that the solvent-exposed loops of PVA–VP extended and engaged in bacterial self-encapsulation.

Effect of polyethylene glycol additives on structure, stability, and biocatalytic activity of ormosil sol–gel encapsulated yeast cells

Biohybrid materials based on ormosil encapsulated yeast cells were synthesized through a one-step sol–gel route with base-catalyst (NaF) using tetraethoxysilane (TEOS), methyltriethoxysilane (MTES) and polyethylene glycol (PEG) with different molar weights as a structure-controlling agent. Phase contrast microscopy and scanning electron microscopy were employed to evidence possible structures of the materials. The addition of PEG during cell encapsulation has induced structural changes within the biohybrids, which depend on PEG molecular weights. The biocatalytic activity of the living hybrids has been investigated by a biosensor which was based on the Clark-type oxygen electrode.Graphical Abstract

Competition between redox mediator and oxygen in the microbial fuel cell

The maximal rates and effective constants of 2,6-dichlorphenolindophenol and oxygen reduction by bacterim Gluconobacter oxydans in bacterial fuel cells under different conditions were evaluated. In an open-circuit mode, the rate of 2,6-dichlorphenolindophenol reduction coupled with ethanol oxidation under oxygen and nirogen atmospheres were 1.0 and 1.1 μM s–1 g–1, respectively. In closed-circuit mode, these values were 0.4 and 0.44 μM s–1 g–1, respectively. The initial rate of mediator reduction with the use of membrane fractions of bacteria in oxygen and nitrogen atmospheres in open-circuit mode were 6.3 and 6.9 μM s–1 g–1, whereas these values in closed-circuit mode comprised 2.2 and 2.4 μM s–1 g–1, respectively. The oxygen reduction rates in the presence and absence of 2,6-dichlorphenolindophenol were 0.31 and 0.32 μM s–1 g–1, respectively. The data obtained in this work demonstrated independent electron transfer from bacterial redox centers to the mediator and the absence of competition between the redox mediator and oxygen. The results can make it possible to reduce costs of microbial fuel cells based on activity of acetic acid bacteria G. oxydans.