The substrate specificity of the membrane fraction of bacteria G. oxydans in conditions of the biofuel cell

Minaycheva P.R., Kirillov I.M., Alferov S.V.

Tula State University

Bacteria of Gluconobacter sp. as well as Acetobacter sp. are long known in biotechnology for their unique oxidative metabolism. Gluconobacter cells contain multiple membrane-bound dehydrogenase.

Oxidation of monosaccharide and alcohol is carried out either in the pentose phosphate cycle in the cytosol by cytoplasmic, preferably NADH-dependent enzymes, or partial oxidation reactions surfactant localized dehydrogenase. It is known that the acetic acid bacteria contain membrane-bond glucose and alcohol dehydrogenase. Individual enzymes and whole cells of microorganisms are competing types of biocatalysts for use in the biofuel cell (BFC). Membrane localization of Gluconobacter oxydans enzyme allows use a membrane fraction of the biocatalyst that can be an alternative to the use of enzymes. Firstly, it is more economically advantageous since the process of obtaining a membrane fraction is an intermediate step in isolation of individual enzymes, and therefore less expensive. Second, the is the priority use of a wide range of substrates that can be achieved using a membrane fraction containing various membrane-bond dehydrogenase.

Enzyme preparations containing membrane-bond dehydrogenase G. oxydans, prepared by ultrasonically disrupted of the bacterial biomass by means of sequential centrifugation. From the literature about using membrane fractions of bacteria G. oxydans mediator in biosensors. Given the similarity of structures mediator graphite paste electrode with the electrodes used in the BFC, we can talk about the possibility of using membrane fractions of bacteria G. oxydans as a biocatalyst in the layout of the biofuel cell.

A typical plot of the potential generated from the BFC model based on membrane fraction of G. oxydans is shown in figure 1. Potential measurements have been carried out in the sodium phosphate buffer solution using galvanicpotentiostatic analyzer IPC Micro. Graphite rods have been dipped into electrochemical cell. The registration of the potential generation while substrate was added to the anode chamber has been carried out after stationary potential range achievement. The measurable parameter during the biocatalytic substrate oxidation was the potential signal amplitude. Evaluation of the catalytic activity of the bacterial enzyme systems were performed in the closed external circuit mode.

Fig.1 Typical plot of the potential generated from the BFC model based on membrane fraction of G. oxydans

It is known that bacteria Gluconobacter typical substrates are carbohydrates and alcohols, most of these compounds are oxidized by oxygen and membrane-bond dehydrogenase associated with bacterial respiratory chain. This process is also the basis for recording the oxidative activity of enzyme systems using bacterial synthetic electron acceptor (2,6-dichlorophenol indophenol).

The absolute values of the potentials generated in the layout of the BFC using the membrane fraction of G. oxydans are shown in figure 2.

Fig. 2 Absolute values of the potentials generated in the layout of the BFC using the membrane fraction of G. oxydans

It was found that the maximum potential generated observed with glucose (200 ± 10 mV), galactose (160 ± 10 mV), and butanol-2 (170 ± 10 mV) as substrates.

Thus, the relative magnitude of the potential difference in response to galactose was made 83% other monosaccharides - from 22 to 70%. In response to the relative values of the generated alcohols capacity lie within the range from 35 to 84% compared to glucose.

Previously, the layout of the biofuel cell based on a suspension of G. oxydans values were obtained power characteristics. Comparative evaluation of the power characteristics for BFC models based on a variety of biocatalysts is shown in table 1. Glucose was used as substrate (concentration of 10 mM).

Тable 1

Power characteristics of the BFC models with biocatalysts based on G. oxydans

Type of biocatalyst	Voltage, mV	External resistance, kΩ	Internal resistance, kΩ	Power, ŋW	Power density, µW·m^-2
Supension of bacterial cells	120±10	240	300	52±3	7
Membrane fraction	200±10	100	120	160±10	20

The use of biocatalysts based on the membrane fraction can double the voltage compare with the suspension of bacterial cells, and the 2,5 times lower internal resistance and to increase power density three times. Thus, membrane fraction of bacteria G. oxydans as a biocatalyst is promising to significantly increase the energy characteristics of the layout biofuel cell.

The work was supported by government assignment № 4412 and President Grant for young Ph.D. № 16.120.11.4341-MK.