Influence of the parameters processes of cultivation of yeast Saccharomyces cerevisiae in simple periodic culture on the yield and biosynthesis of some cellular components

Для цитирования For citation Меледина Т.В., Иванова В.А., Харба Разан, Головинская О.В., Новикова И.В., Коростелев А.В. Влияние параметров процесса культивирования дрожжей Saccharomyces cerevisiae в простой периодической культуре на выход биомассы и биосинтез некоторых клеточных компонентов //Вестник ВГУИТ. 2018. Т. 80. No 2. С. 175–181. doi:10.20914/2310-1202-2018-2-175-181 Meledina T.V., Ivanova V.A., Harbah Razan, Golovinskaya O.V., Novikova I.V., Korostelev A.V. Influence of the parameters processes of cultivation of yeast Saccharomyces cerevisiae in simple periodic culture on the yield and biosynthesis of some cellular components. Vestnik VGUIT [Proceedings of VSUET]. 2018. vol. 80. no. 2. pp. 175–181. (in Russian). doi:10.20914/2310-1202-2018-2-175-181 175 Оригинальная статья/Original article УДК 663.12 DOI: http://doi.org/10.20914/2310-1202-2018-2-175-181


Introduction
In order to achieve the maximum efficiency of the biomass upstream process, i.e. to achieve the maximum intensity of multiplication with a biomass high yield, it is necessary to provide oxidative yeast metabolism and optimal physico-chemical growth environment.The influence of physical and chemical factors is quite thoroughly studied, while the matter of the proper yeast nutrition is still being solved.
In this regard it is necessary to study the interrelation of energy exchange with physiology and constructive yeast exchange because the intensity of growth and biomass yield will be influenced by reproductive activity of the seed material and by the balance of all components of the nutrient medium, especially the growth factors of nitrogen, macroelements (P, K, Mg) and microelements (Cu, Fe, Mn, Zn).
Nitrogen-containing substances are the main structural components of cells.These are amines, amides, amino acids, proteins and nucleic acids.These compounds account for 37 to 60% of the cell dry solids [1,2].
Most of them are represented by proteins (40.6-58%) and nucleic acids (15-26%); free amino acids and peptides account for 6.5 to 9.3% [3], i.e. most of them are represented by substances that are associated with both energy metabolism and constructive exchange in the cell.
The nitrogen metabolism is closely related to the phosphorus metabolism, which content expressed in terms of Р 2О5 in yeast varies from 1.9 to 5.5% of dry ingredients in baker's yeast [4] and from 1.4 to 2.0% in brewers' yeast [1,2].
Phosphorus is a part of nucleic acids, phospholipids, polymers of a cell wall.It can be accumulated in the form of polymethophosphates or volutin.The greatest part of phosphorus is found in ATP.
Other important components of the nutrient medium are growth factors, in particular, it is biotin, the need for which is characteristic of all Saccharomyces yeasts.The lack of biotin in the culture medium entails an imbalance of all types of metabolism: protein, fat, carbohydrate, and nucleic acid synthesis metabolism [5,6].Typically, to compensate for the lack of biotin it is added into complex nutrient media for example, to molasses or malt wort on the basis of 0.1 to 0.25 mg per 100 g of growth expressed in terms of absolutely dry biomass of yeast [2,4].
The amount of associated biotin in enzymes is always constant [7,8].Having said so it should be borne in mind that yeast has the ability to accumulate intracellular biotin reserves [9].The excess of biotin is also undesirable, because in this case there is slowing-down of its transport to the cell, so when calculating the biotin sources flowrate for the cultivation process, the content of biotin in the medium and in the yeast itself should also be taken into account [9].
The relationship of the amount of pitching and the biotin content in the medium, as well as the interrelation of the biotin metabolism with energy and constructive exchange has been poorly studied so far.Meanwhile, this really matters both in science terms and on practical grounds.
The amount of nitrogen and phosphorus salts in the medium determines their physiological state [3].
It is found that the resistance of cells to stress depends on the content of reserve carbohydrates, in particular trehalose and glycogen [10].The protective effect of trehalose is based on the ability of yeast to maintain the osmotic pressure in the cells, while glycogen is the source of endogenous glucose for initiating the onset of glycolysis.It is shown that the synthesis of trehalose and glycogen in a simple discontinuous culture begins in the phase of cell growth retardation.First, glycogen is synthesized, and then trehalose does [11].The source of carbon for the endogenous synthesis of trehalose can be the free amino acids of yeast, which content is inversely related to the amount of trehalose in yeast [12,13].
Directed synthesis of certain cellular components can be achieved by changing the culture conditions.It is known that a shift-down of population growth is accompanied by the synthesis of reserve carbohydrates, by the same token, the growth rate can be regulated by the process limiting with various nutritional components.Synthesis of reserve carbohydrates can be accelerated even when the culture is limited by carbon.
The reproductive activity of yeast during the lag phase of growth probably depends on the inoculum dose, the quality of which is determined by the culture conditions and by the content of reserve carbohydrates in them.
The purpose of these studies is to determine the effect of inoculum dosing on the yield of biomass and the relationship of this index with the composition of the nutrient medium (nitrogen, phosphorus and biotin content) and the chemical composition of the yeast derived.

Microorganism and cultivation conditions.
The Saccharomyces cerevisiae yeast strain RCAM 02150 (Russian National Collection of Industrial Microorganisms -VKPM) was cultivated in a simple discontinuous culture with aeration using a laboratory fermenter (Biostat A, Sartorius) with a working volume of 2L.The temperature of cultivation was 30 °C.The рН value was 4.4 ± 0.2.
The ammonium sulphate was used as the source of nitrogen, the orthophosphoric acid -as the phosphorus source.
The biomass (dry weight) accumulation was determined by gravimetric way after drying of the washed yeast suspension to constant weight at 105 °C.
The economic coefficient.The economic coefficient or biomass yield was calculated by dividing the gather of cells containing 25% of dry matter by the amount of used substrate (glucose) and it was expressed in %.
Determination of protein content in cells.The total amount of protein substances in the yeast cells was determined using the Lowry method [15,16].
Determination of the yeast amino acid pool.In this paper, a method of extraction of amino acid pool by boiling the cells in distilled water for 15 minutes was applied.Quantification of the pool was carried out using the Swenson & Befts method [17], who modified the Moore & Stein method [18,19,20].This method has the same sensitivity to all amino acids and gives an idea of the total content of free amino acids in cells.
Determination of reserve carbohydrates.The method of step fractionation of carbohydrates [21, 22, 23, 24] was used, followed by the determination of individual carbohydrates in each fraction [22].
Complete factorial experiment [25].The main advantage of this approach is the possibility of studying the simultaneous impact of a large number of factors on the process efficiency.In addition, this method makes possible establishing the availability of interfactor interactions in the system along with a quantitative consideration of each individual factor and allows to estimate the effect of those.
For the function у = f (х1, х2, х3…, хn) the regression equation is used, which is the expansion of this function in a power series (Eq.1): whereхij are variable factors, in coded units; bi, bjregression coefficients at the corresponding variables.
The number of variants of the experiments that must be put in the initial series depends on the number of initial factors.In total, on two levels for n factors, the number of variants of the experiments will be: 2 n .In the first part of this paper, the number of factors studied was equal 4, respectively, the number of experiments was 16.In the second part of this paper, there were 3 variability factors and 8 experiments.
Regression coefficients calculation formulas (Eq.2,3) can be presented in a general form: (2) (3) whereхiu is the value of the variable in the corresponding column of the experiment plan; u y -the result of the u-th experiment, the arithmetic mean value; N is the total number of experiments; u is the number of the variant of the experiment; i is the factor's number.
To confirm the validity of the regression equation obtained, the following actions were carried out for the observed process: ─ statistical analysis of the significance of regression coefficients bj; ─ validity check of the regression equation.
Along the statistical analysis of the significance of the regression coefficients, the distribution error bi was calculated.The regression coefficient was assumed to be different from zero, i.e. significant, if the following inequality was satisfied (Eq.4): where 2 i b S -is the error in bi determining in the experiment; t -Student's criterion for 5% significance level, which allowed us to predicate the significance of bi values at the 95% credible level.
In order to convince ourselves that the obtained regression equations sufficiently (adequately) describe the process under investigation, in each case the Fisher's criterion F (Eq.5) was determined: where 2 ad S -is the adequacy variance (the magnitude of error of the reproducible results); 2 y S -is the variance of the y mean value.
The calculated Fаd criterion was compared to the tabulated value of F (for 5% significance level).
then there is the equation.If , ad F F < then this indicates that the process proximity to the optimal region, i.e. in this case it is not possible to be limited to the linear approximation, it is necessary to take into account the quadratic terms (Eq.1).

Results and discussion
In this study, the following values were taken as factors influencing the main parameters: Х1 -yeast pitching value with 25% dry matter content, the% of fermentable carbohydrates; Х2 -content of nitrogen in the culture medium, g dm -3 ; Х3 -phosphorus content expressed in terms of Р2 05 in the culture medium, g dm -3 ; Х4 -the biotin content in the culture medium, mg dm -3 .
The yield was calculated by the ratio of biomass increment with a content of 25% solids to the amount of glucose to be disposed and it was expressed in %.
During calculation of influence of factors on the biomass yield, it is found that the lower level of factor score Х3 (phosphorus content) is in the optimal region and does not limit the reproduction of yeast.
The relationship between the inoculum value and the economic coefficient at different concentrations of nitrogen and biotin in the medium was found.In that case when both these components, as well as the inoculum amount value, are at the lower level (No. 1, 5, 7, 11, 13 of Table 1), the yield is 44… 48%; when these factors move to the upper level (No. 3 and 15 of Table 1), the yield is increased up to 64… 69%, i.e. up to the value that is achieved in experiments with a large seed material flow (No. 8, 12, 16 in Table 1).
In view of the great importance of the Х1, Х2 и Х4 factors, the effect of seeding, nitrogen and biotin content in the environment on the biochemical composition of cells was studied, upon that the variation interval of nitrogen concentration in the medium made 0.45-0.75g dm -3 .The phosphorus concentration, expressed in terms of Р2 05made 0.4 g dm -3 .The experiments were also carried out according to the full factorial experiment plan.After data processing, multiple regression equations were obtained in which the role of each of the factors was demonstrated and, what is especially important for biological objects, the interaction of these factors in the synthesis of important cellular components was demonstrated too.The equations take into account only those factors and their interaction, whose influence on the process exceeds 10%.
The economic coefficient reflects the biosynthetic activity of yeast and in the studied area of factor change the following is established: ─ the biomass synthesis is influenced most of all by the biotin content in the medium.Reduction of the dose of inoculum increases the cells biotin requirement; ─ the protein synthesis in cells is greatly influenced by the nitrogen concentration in the culture medium; ─ the reserve carbohydrates content depends little on the amount of seed material; ─ the free amino acids content increases at decreasing in seed material flowrate, at increasing in the concentration of nitrogen content and at decreasing in the biotin level; ─ in the biotin-deficient medium, as well as in the medium rich with this growth factor, the value of the free amino acids pool is inversely related to the consumption of the seed material: with an increase in the initial population intensity, the amount of amino acids in the cells decreases.
The increase in the pool value with the increase in the nitrogen level is explained quite simply, because in this case, the amount of exogenous nitrogen significantly exceeds the need for yeast, so the resulting amino acids do not have time to be disposed of in biosynthetic processes and accumulate in cells (Table 2).
In the case when the biotin content is insufficient in the medium, the average content of amino acids in yeast is 14.7%.Increase in the amount of free amino acids with a low seeding value (even in the absence of a limit on nitrogen nutrition) indicates a low activity of biosynthetic processes, as a result of which there is a low yield of biomass (No. 1 and 3 in Table 2).In the biotin-rich medium, the average amino acid content in cells is 8.7%, and the maximum amount of amino acids is contained in yeast obtained in a medium with a high nitrogen content and a low seed material flowrate.In this case, one cannot speak of low yeast activity, because the yields in No. 6 and 7 (Table 2) are almost the same; on the contrary, it follows from these data that when the inoculum content is low in a medium that does not limit cell growth, the yeast reproductive activity will be higher.
Conclusions The composition of the nutrient medium should be adjusted in accordance with the dose of the seed material.In case of the decrease in the amount of inoculum, the cells biotin and sources of nitrogen nutrition needs are increased.The content of reserve carbohydrates in yeast at the end of the cultivation process doesn't practically depend on the pitching value.The increase in pitching decreases the protein content in cells and reduces the free amino acids pool in them.

Table 2 .
Influence of cultivation conditions on the economic coefficient and biochemical biomass composition