LYOPHILIZATION EFFECT ON PRODUCTIVITY OF BUTANOL-PRODUCING STRAINS

Investigation of lyophilization effect on the productivity of butanol-producing strains was the aim of our research. For this purpose we used butanol-producing strains; technical glycerol; biomass of switchgrass Panicum virgatum L. Lyophilization was performed using a lyophilization-drying. The effect of the protective medium on residual moisture of freezedrying cultures suspensions depending on the concentration of glucose and sucrose was studed. It was shown that the lowest residual moisture was attained by using glucose and sucrose in amount of 10% and if the samples of freeze-drying bacteria had been saved for one month at 4 ο C the productivity did not decrease. As temperature preservation was increased the productivity of the cultures was gradually decreased and it was greatly reduced at 30 ο C. So the protective medium composition was optimized for lyophilization of butanol-producing strains as follows: sucrose 10.0%; gelatin 10.0%; agar 0.02%. It was shown that the preservation of samples of freeze-drying bacteria for six months at a temperature of 4 ο C did not affect the productivity of strains. It was found that cultures could use glycerol as a carbon source for butanol accumulation before lyophilization.

The main problem of preservation of microorganisms in collections (museums) is the variability of biological material.During storage microorganisms on artificial media, there is a danger of a significant reduction or even loss of their physiological and enzymatic properties.For the "conservation" materials of biological origin the method of lyophilization has received prevalence.In the process of lyophilization of biological materials free water contained therein is removed by sublimation -evaporation directly from the solid state (ice).The temperature of the material that undergoes dehydration during the whole period of free water removal remains below freezing point resulting proteins do not denature at elevated concentrations of electrolytes [1][2][3].
The structure of the material after lyophilization drying is equally important.It should be dry, porous, without free water mass that almost kept the volume and structure of the original substance.Freezing followed by drying does not guarantee the full preservation of all properties of biological material but this method significantly increases time of storage of cultures of microorganisms, live vaccine, serum and plasma [4][5][6].
When freezing any material that contains water and dissolved salts, there is an eutectic separation of solution and elevated concentrations of salt which can cause denaturation of proteins or cell destruction.The degree of damage depends primarily on the nature of dissolved salts, cooling rate and initial concentration of the suspension material.If the cooling is quicker the effect of concentrated salt solutions is lower.Through the selection and use of so-called "protective" medium of different composition during freezing and lyophilization drying it can prevent negative effects of eutectic concentration on microorganisms [7][8][9][10][11].
The aim of this work was to study the effect of lyophilization drying on productivity of butanol-producing microorganisms' strains.

Materials and Methods
For the study their were used butanolproducing strains Clostridium acetobutylicum ІМВ В-7407 (IFBG C6H) and its mutant Clostridium sp.ІМВ В-7570 (IFBG C6H 5M) from "Collection microorganism's stains and plants line for food and agriculture biotechnology" of the Institute of Food Biotechnology and Genomics of the National Academy of Sciences of Ukraine; technical glycerol; biomass of switchgrass Panicum virgatum L. (Gryshko National Botanic Garden).
To prepare mashes of switchgrass, samples of 50.0 g of the substance were taken per 1 l of water, and sterilized for 2 h at a pressure 0.2 MPa.Biomass of switchgrass was dried at 30 ± 1 С during 168 h.The dried biomass was ground using mill Cyclone MSh 1 (Ukraine) to particles sized 200 mesh.The moisture was determined by moisture analyzer RADWAG MA 50/C/1 (Poland).
Culture of microorganisms at solid medium was performed following [12] in anaerobic culture apparatus AE 01 (RF) under a nitrogen atmosphere.The apparatus was kept in thermostat at 35 ± 1 С.In five days, the fermentation was stopped and the cells were precipitated using ultracentrifuge Labofuge 400R (Germany), then the supernatant was distilled and fermentation products defined.
To confirm the effect of protection medium on cell viability after lyophilization drying, medium of the following composition was used (%): glucose or sucrose at appropriate concentrations -1.0; 10.0; 30.0; gelatin -10.0; agar -0.2.
Winogradsky medium at first was used for the accumulation of cultures of butanol producing stains [12].Bacteria carried in a protective medium at the rate of cell concentration 410 9 cell/ml, then in the amount of 5 ml contributed carry in to penicillin bottles.
Samples were frozen in low-temperature refrigerator LAB 11/EL19LT (Elcold, Denmark) at -80 С.Frozen samples were transferred into special cassettes in the precooled camera (temperature condenser -50 С) freeze drying CRUODOS-50 (TELSTAR, Spain).Lyophilization was performed at constant temperature -50 С (drying temperature is not regulated) to stable residual pressure in the chamber 0.0018 Pa.Drying time was 72 h.The residual moisture was determined by moisture analyzer.
Preparing freeze-drying material for the study of productivity was necessary to fulfill two conditions: as accurately as possible restore first (to dry) volume of material and to derive living cells from a state of anabiosis.To fulfill these conditions freeze-drying material was adjusted with distilled water to a volume of 5 ml and kept at room temperature for 30 min.
Statistical data analysis was performed using Microsoft Excel program.All experiments were done in triplicate.The difference between two averages was considered  2 probable at P < 0.05 (significant results marked with *) [13].Aseptic environment was taken as control.

Results and Discussion
For the preparation of Clostridium acetobutylicum IMV B-7407 (IFBG C6H) and Clostridium sp.IMB B-7570 (IFBG C6H 5M) befor lyophilization the dependence on residual moisture of freeze-drying cultures was first investigated, after lyophilization, on the concentration of glucose or sucrose in a protective medium (Table 1).
As follows from the data, under the same conditions of lyophilization the residual moisture of freeze-drying cultures after lyophilization depends on the type and concentration of carbohydrates.Lowest rates of residual moisture were obtained using glucose or sucrose at a concentration of 10%.The protective medium according to the research composition was optimized for lyophilization butanol-producing strains (10.0%sucrose; 10.0%gelatin; 0.02% agar).
Temperature conditions were particular important in the process of storage microorganisms after lyophilization drying.With increasing of the temperature of storage the number of viable cells of microorganisms and their productivity were decreased.Strains were cultivated on mashes of switchgrass to determine their productivity.The productivity (accumulation butanol) of freeze-drying microorganisms dependence by storage temperature after one month are shown in Fig. 1.
Productivity of the cultures did not decreased if the samples of freeze-drying bacteria preserve for one month at 4 C.With increasing temperature of preservation the productivity of bacteria was gradually decreased, and at a temperature of 30 C it was greatly reduced.
Further storage freeze-drying cultures was carried out at a temperature of 4 С for 6 months, exploring their productivity through every month on mashes of switchgrass (Table 2).
It is seen from the table 2 that even after storage at a temperature of 4 С for 6 months, the renewed freeze-drying culture (strain IFBG C6H 5M) was viable and the accumulation of butanol in cultural liquid after cultivation was hardly changed compared to accumulation before lyophilization 2.7 g/l [14].Similar results were obtained for strain IFBG C6H.
One of the main factors that effect on lyophilization was a stage of culture development.As a source of energy the different carbohydrates and carbohydrate containing compounds for the development of microorganisms were used.Technical glycerol could be one of these sources of energy.To optimize the nutrient medium before lyophilization we researched alcohols accumulation by strain IFBG C6N using technical glycerol as a carbon source (Fig. 2).
The study results show that the greatest accumulation of butanol (10.0 g/l) in cultural liquid was at the glycerol concentration 100.0 g/l.Most bioconversion of substrate was observed at concentrations of glycerol 50.0 g/l with the accumulation of butanol 8.0 g/l.
Fig. 3 showes alcohols accumulation (productivity) strain IFBG C6H 5M at different concentrations of glycerol.Mutant strain IFBG C6H 5M had the changed properties concerning glycerol fermentation, compared with the original strain.Like with the original strain the most accumulation of butanol by strain IFBG C6H 5M was observed at concentrations of glycerol 100 g/l and exactly such concentration of glycerol was optimal for the development of culture before lyophilization.However, the accumulation of butanol by mutant strain IFBG C6H 5M was decreased to 8 g/l in comparison with the original strain IFBG C6H 10 g/l.From the analysis of the data and the results of [14], we can conclude that the strain IFBG C6H 5M was characterized by higher cellulose activity and reduced bioconversion of glycerol.So, composition of the protective medium (sucrose 10.0%; gelatin 10.0%; agar 0.02%) for lyophilization butanolproducing strains was optimized.The preservation of samples of freeze-drying cultures for 6 months at 4 C does not effect on the productivity of strains.It is found that glycerol can be used as a carbon source for accumulation of butanol by cultures before lyophilization.

Fig. 1 .
Fig. 1.Effect of storage temperature on the accumulation of butanol