The methylotrophic yeast, immobilized in sol-gel matrix, as a basis for stable heterogeneous biocatalysts

The methylotrophic yeast, immobilized in sol-gel matrix, as a basis for stable heterogeneous biocatalysts

Kamanina O.A. Burmistrova T.V., Rogova T.V.

Tula State University

At present we have acute problem of development of rapid methods of ethanol determination in fermentation mass for quality control at biotech industries. There are several methods of determining of alcohol concentration: bottle method, aerometric method and gas chromatography. Thus, the development of analytical method, which would simplify and reduce cost of analysis is very important. The biosensor method me propose do not require highly qualified personnel and helps to increase the economic returns of facilities.

Immobilization of microbial cells is very efficient in intensification and increasing of economic attractiveness of advanced biotechnological processes, because it allows us to use the same cells for a long time. It greatly simplifies the process and reduces the cost of analysis comparing to processes that require using of free cells. In these latter days method of biomaterial immobilization by including it into bimodal silicon sol-gel matrix attracted particular interest. Because of its properties the matrix can be applied for the immobilization of whole cells in creating heterogeneous biocatalysts.

The purpose was to create a stable heterogeneous biocatalysts by encapsulating the methylotrophic yeast Pichia angusta in sol-gel matrix based alkoksiproizvodnyh silica and polyethylene glycol. The influence of hydrophobic additives alkilalkoksisilana on the properties of heterogeneous biocatalysts that depend on the structure and properties of crosslinked polymer, physiological and biochemical behavior of immobilized microorganisms. As an analytical signal representative of the biocatalyst used the methylotrophic yeast respiratory activity in the presence of ethanol. A convenient tool for studying the properties of the immobilized biomaterial are biosensors that biocomponent is in direct contact with the physical and chemical transmitter (oxygen electrode). In this work we received five receptor elements, which are immobilized in bimodal silicon sol-gel matrix methylotrophic yeast Pichia angusta. We have determined the main characteristics of the developed receptor elements: sensitivity, detection range, operational and long-term stability. Comparative analysis of parameters of heterogeneous biocatalysts showed that the increase in the share of alkiltrialkoksisilana matrix up to 83% increase in the hydrophobicity of the matrix, which results in an increase in the coefficient of sensitivity and decrease the detection limit (Fig. 1).

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Fig. 1 Detection limit and sensitivity coefficient for different heterogeneous biocatalysts

However, a further increase in the content of hydrophobic additives not recommended, as it results in a decrease of analytical characteristics obtained heterogeneous biocatalysts.

As a result of the work, received receptor elements were tested on commercial samples of vodka and model samples of fermentation mass (table 1).

Table 1

Vodka sample	Concentration C₂H₅OH , %
	Gas chromatography		The biosensor method
Zhuravli	40,1±0,5		40,1±0,4
Slavyanskaya	40,0±0,3		39,9±0,2
Zelenaya marka	40,3±0,8		40,1±0,3
Belenkaya	39,9±0,2		39,7±0,3

The fermentation, h	Concentration C₂H₅OH, mM
0	20±3	20±4
24	43±1	46±5
48	168±1	173±2
72	205±3	209±5

We carried out a statistical analysis of the data (Fisher criterion and the modified t-test). These two methods (gas chromatography and biosensor determination of ethanol content in the samples) are homogeneous in reproducibility and differ insignificantly.

The work was supported by the Federal Program "Scientific and scientific-pedagogical personnel of innovative Russia" (GK № 14.B37.21.1231 and № 16.740.11.0766)