The structure of Gluconacetobacter hansenii GH 1/2008 population cultivated in static conditions on various sources of carbon

Bacterial cellulose (BC) is a natural polymer that has a number of unique properties that determine the need to synthesize large amounts of it and to search the ways to increase the productivity of strains and to optimize the nutritive media. It is known that the choice of the producer for BC synthesis has an impact on its final properties and on the productivity of this polymer production. Under static liquid phase cultivation conditions, all cellulose-producing bacteria form a uniform film on the medium surface that serves as a scaffold for cells immobilization, thus providing them with the access to the air/liquid interface, where the access to oxygen is not limited. Meanwhile, when cultivation goes under agitating conditions, most of Gluconacetobacter xylinus strains produce less cellulose in form of globules of various sizes, despite the better oxygen access. Several authors explain the lower cellulose outcome when cultivated under agitated conditions by the appearance of spontaneous mutants that do not produce cellulose in the population. It was revealed that when grown on agarized media, cellulose-non-producing mutants form colonies of a specific mucoid type, while non-mucoid phenotype cells form smooth colonies of non-mucoid type. To our knowledge, there is no published research on the impact of cultivation conditions and nutritive medium composition on the appearance of spontaneous phenotype mutations in the population of Gluconacetobacter hansenii representatives. The aim of the present research is to elucidate the impact of the carbon source on the productivity of G. hansenii strain and the appearance of cellulose-negative mutants under static cultivation conditions. We studied the strain G. hansenii GH 1/2008 (VKPM B-10547) as a BC source. Liquid phase static cultivation of G. hansenii GH 1/2008 was carried out using the modified Hestrin-Schramm (HS) medium, containing 4% of monosaccharides (glucose, fructose and galactose) or disaccharides (sucrose, maltose, lactose) as carbon sources. The occurrence of mutants was calculated considering phenotypes of colonies obtained by seeding the samples of cultural liquid and wash-offs of cells from films produced by the cultivation of the producer on modified agarized HS media. The polymer outcome was expressed as the film absolute dry weight (a.d.w.) per cultivation medium volume unit. We studied the morphology of the producer’s wild type and mutant cells by means of atomic force microscopy (AFM) (See Fig. 8). The structural organization of the produced films and gel was revealed by means of scanning electron microscopy (SEM) performed after freeze-drying. The composition of the fibers was checked acquiring FTIR Spectroscopy. We established that G. hansenii GH 1/2008 produces a dense film on media containing fructose, glucose and sucrose, while the polymer has gel consistence when grown on maltose, galactose and lactose (See Fig. 1). The maximal quantity (a.d.w.) of polymer was produced on fructose- and sucrose-containing media. The overall number of immobilized producer cells was considerably higher when grown on media with glucose, fructose and sucrose than on gels grown on those containing maltose, galactose and lactose (See Table 1). SEM imaging revealed considerable difference in the microscale organization of films and gels produced by G. hansenii GH 1/2008 on various carbon sources (See Fig. 2). Fructose-containing medium yields the densest structure with dense layers separated by 2μm thick areas filled with non-ordered BC fibrils. The microscale organization of sucrose- and glucose-based films were very similar and had a cell-like structure. In cases where the synthesized polymer had squeezable gel consistence, its microstructure was not layered but close to isotropic. The studies of gels by means of FTIR spectroscopy showed that the gels are also formed of BC molecules; the spectra were almost identical (See Fig. 4). The only difference, i.e. the intensity of the 1638 cm^-1 peak, can be explained by the presence of a higher amount of bound water in the latter. It is known that some strains of this species may produce glucuronic acid oligomers under unfavorable conditions, but peaks corresponding to carboxyl or carbonyl groups were not revealed in the spectra. This is the evidence that no detectable amounts of glucuronic acid were produced under conditions studied. The analysis of colonies of G. hansenii GH 1/2008 cultivated under static conditions on media containing various carbon sources revealed colonies with two dominating phenotypes: non-mucoid smooth convex colonies and mucoid flat ones (See Fig. 5). The number of cells forming smooth non-mucoid colonies on agarized media was maximal in the inoculations of cultural liquids after the cultivation on media containing fructose and sucrose, i.e. those carbon sources that demonstrated high productivity per 1L of cultural liquid (See Fig. 6). In the inoculations of the cultural liquid and wash-offs of cells immobilized on gels obtained by the cultivation on media containing galactose and lactose, the number of mucoid colonies was considerably higher (See Table 2). The clones forming mucoid type colonies did not produce BC films when reinoculated in liquid media, while those forming colonies of mucoid (smooth) type produce films on the 3rd day of cultivation (See Fig. 7). The analysis of cells shape and sizes by means of AFM did not reveal any statistically valid difference between the mutants and the wild type. The present research shows that the source of carbon is a selective factor in the formation of the inner composition of the population of clones of the bacterial cellulose producer Gluconacetobacter hansenii GH 1/2008. The proliferation of cellulosenegative cells arouses competition for the substrate with cellulose-positive cells of G. hansenii GH 1/2008 that reduces the number of the latter and the production of the exopolymer. The paper contains 8 Figures, 2 Tables and 38 References. Acknowledgments: The authors thank the Department of Structural Studies, ND Zelinsky Institute of Organic Chemistry, Moscow for carrying out SEM imaging. The Authors declare no conflict of interest. The paper contains 8 Figures, 2 Tables and 38 References. The Authors declare no conflict of interest.

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