Получение бактериальной целлюлозы при совместном культивировании Komagataeibacter sucrofermentans с продуцентами декстрана Leuconostoc mesenteroides и ксантана Xanthomonas campestris

Abstract

Bacterial cellulose (BC) is an extracellular product of bacterial metabolism. Like plant cellulose, BC has the same molecular formula but its structure is significantly different. Due to its unique properties (high degree of crystallinity, purity, good water-holding capacity), BC is widely used in many areas of human life. However, despite all the advantages of BC over plant polymers, its production is a relatively expensive process. Thus, one of the ways to increase the polymer yield can be to jointly cultivate a BC producer strain with other polysaccharide producers. The positive effect of some water-soluble polysaccharides on the BC output is known from the literature data. In addition, many biosynthetic genes remain silent and not expressed in vitro, thereby severely limiting the chemical diversity of microbial compounds that can be obtained by fermentation. In contrast, the co-cultivation of two or more different microorganisms mimics a real "situation" where microorganisms coexist in complex microbial communities. It has been proven that competition or antagonism occurring within co-cultivation leads to a significant increase in the existing compounds and / or accumulation of new compounds which are not found in axial cultures of the producer strain. The purpose of this study is to investigate cocultivation as a way to increase the yield of BC during the cultivation of BC producers with other polysaccharide-forming strains. The strain of Komagataeibacter sucrofermentans B-11267 was used as a BC producer, Xanthomonas campestris was used as a xanthan producer, and Leuconostoc mesenteroides was used as a dextran producer. The cultivation was carried out under dynamic conditions on a medium with molasses. The polysaccharide yield was expressed as the absolute dry weight of the polymers per unit volume of the culture medium. We have studied the BC morphology using atomic force microscopy (AFM) and FTIR spectroscopy. Crystallinity was checked by X-ray diffraction analysis. The interest in BC makes it necessary to synthesize it in large quantities on an industrial scale. The problem of increasing productivity was solved by co-cultivating the BC producer Komagataeibacter sucrofermentans with the producers of dextran Leuconostoc mesenteroides and xanthan Xanthomonas campestris, since the addition of water-soluble polysaccharides is known to increase the viscosity of the medium and facilitate the dispersion of bacterial cellulose granules. Thereby increasing the number of free cells, which can accelerate sugar consumption and polymer formation. At the first stage of the study, the most optimal conditions for co-cultivation of the BC producer with the producers of xanthan and dextran were selected, namely, the optimal pH value of the medium. Monoculture of bacteria X. campestris, L. mesenteroides, and K. sucrofermentans was carried out at different pH values (See Fig. 1-3). Based on the data obtained, we can say that the most optimal pH value for co-cultivation of microorganisms is pH 5.0. In this regard, at the second stage of this study, we carried out joint cultivation of the BC producer strain K. sucrofermentans with the xanthan and dextran producers X. campestris and L. mesenteroides, respectively, on molasses medium. From the data presented (See Fig. 4), it can be seen that the largest amount of polysaccharide is formed on the third day during cocultivation of the BC producer and the dextran producer. The amount of BC was 5.99 ± 0.02 g/l, which is two and a half times higher than the amount of polymer formed during monocultivation of a BC producer (2.25 ± 0.05 g/l). Co-cultivation of the BC producer strain with the xanthan producer did not lead to an increase in the polysaccharide yield. Therefore, no further study of co-cultivation of these microorganisms was carried out. To assess the success of the joint cultivation of BC and dextran producer strains and investigate the properties of the obtained polysaccharide, studies using AFM, FTIR spectroscopy, and X-ray structural analysis were carried out. The surface relief of the obtained BC was studied using the AFM method (See Fig. 7). Analysis of the AFM images showed the presence of an association of K. sucrofermentans and L. mesenteroides cells in the BC. Also, the obtained BC was investigated using the method of FTIR spectroscopy (See Fig. 8). The obtained IR spectra show similarity of the detected peaks with the literature data of peaks corresponding to BC. To determine the degree of crystallinity, the structure of cellulose was studied by X-ray structural analysis (See Fig. 9). The degree of crystallinity of the studied cellulose samples is 64% and 32% with monocultivation of K. sucrofermentans and co-cultivation of K. sucrofermentans and L. mesenteroides, respectively. The article contains 9 Figures, 1 Table, 37 References.