И. А. Грецкий, Е. Н. Громозова, С. К. Воцелко
Институт микробиологии и вирусологии имени Д. К. Заболотного НАН Украины
ИСПОЛЬЗОВАНИЕ КОМПОЗИЦИЙ ПОЛИСАХАРИДОВ МИКРОБНОГО ПРОИСХОЖДЕНИЯ ДЛЯ КУЛЬТИВИРОВАНИЯ ЛЮМИНЕСЦЕНТНЫХ БАКТЕРИЙ PHOTOBACTERIUM PHOSPHOREUM ІМВ В-7071
Разработана гелевая композиция на основе природного экзополисахарида ксантана и экзополисахаридполиакриламида для удешевления питательной среды для культивирования люминесцентных бактерий. По показателям интенсивности люминесценции и сохранения жизнеспособности оптимальной для культивирования P. рhosphoreum является композиция ЭПАА и ЭПС в пропорции (70%+30%) с концентрацией NaCl 3%.
Ключевые слова: биолюминесценция, Photobacterium phosphoreum, ксантан, экзополисахаридполиакриламид
I. A. Gretsky, E. N. Gromozova, S. K. Votselko
D. K. Zabolotny Institute of Microbiology and Virology of the NASU, Ukraine
USE OF THE POLYSACCHARIDES COMPOSITION OF MICROBIAL ORIGIN FOR CULTIVATION LUMINOUS BACTERIA PHOTOBACTERIUM PHOSPHOREUM ІMV B-7071
Nowadays pollution is biggest problems of people that caused by development of technology. Many analytical methods for monitoring of pollutants are costly because need an expensive equipment and cumbersome pretreatment of samples of environment. The most interested that classical methods show biosensor based on cell of microorganisms. From all types of biosensor devices are more interest Biotest of luminescent bacteria, based on the exceptional sensitivity of these microorganisms to various substances. The intensity of light emission fluorescent bacteria is an integral factor of their metabolism, which causes high sensitivity and speed of response to various influences, simplicity and efficiency of bioassays. Bioelement is the key of devices and consists of luminescent bacteria. The physiological state of one is the top characteristics of the biosensor. The main of necessary is quality requirements luminescent bacterial sensors and their ability to maintain a high intensity luminescence for a long time. The success of the receipt of receptor depends on choice acceptable carrier and method of immobilization of bacteria. Biomass of luminescent bacteria gets from complex nutrient medium, which contents components like as peptones, yeast extract, fish and meat hoods and others. The defect of medium is difficulty their production and relatively a high cost components. In this case, development and price redaction is the importance for increase of duration and stability of highest activity of luminescent microorganisms. It is known the method uses of exopolysaccharides (EPS) of microbial origin in the preparation of medium. There is expedience and effectiveness of this method. The EPS is exogenous macromolecular by biosynthesis of microorganisms that have several advantages to chemical analogues: resistance to mechanical degradation, temperature and low pH, non-toxic and biodegradability. The aim of this work was development and price reduction of liquid medium, based on EPS. EPS may be provide duration and high luminescence of Photobacterium phosphoreum. The IMV B-7071 strain of the luminous marine bacterium P. phosphoreum from the culture collection of the Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine was used as the object of the study. The species identification of the bacteria was confirmed by the sequencing of 16S rRNA gene region. The nucleotide sequence was submitted to the GenBank nucleotide sequence database (http://www.ncbi.nlm.nih.gov/genbank) under accession number KF656787. To create a composition for culturing fluorescent bacteria used polysaccharide compounds based on the following components: EPS xanthan by Xantomonas campestris pv. campestis IMV B - 8158; EPAA – exopolysaccharide acrylamide, copolymer synthesized by polymerization of acrylamide (AA) and xanthan. EPAA obtained by polymerization of acrylamide in an aqueous solution of bacterial polysaccharides (xanthan) in the ratio of these components 7: 3 in the presence of redox initiators. The first step was creation of composition that consist of EPAA and EPS xanthan that provides high vitality and glow luminescent bacteria during storage for a month. The best result among the options using the EPAA obtained using a composition of 30% + 70%. We studied the viable cells of bacteria P. phosphoreum IMV B-7071 at different variants compositions of inoculum that storage at different temperatures. After 30 days of storage when cultured at + 4°C higher than the control and amounted to more than 1010 cells / ml. When cultured at + 21°C titer of viable cells decreased significantly over the period of storage. Found that sticky composition as the substrate improves the duration of luminescence bacteria and provides intensive development of microorganisms in relation to other media. Microorganisms can be used exopolysaccharides as energy substrates and electron donors in dehydrogenation reactions, which is a mandatory step phase redox processes in the cell of microorganism, as well as sources of carbon metabolism, including the destruction of components in the overall exchange. Data of luminescence intensity and persistence viability has shown optimal environment for culturing P. phosphoreum. It is composition of EPAA and EPS at proportion (70% + 30%) with concentration of 3% NaCl. Summarize, a gel composition based on natural gum and xanthan EPS EPAA are promising components for improving the properties of microbial preparations with prolonged shelf life and stable properties, and increase the intensity of luminescence bacteria.
Keywords: bioluminescence, Photobacterium phosphoreum, xanthan, exopolyacrylamide