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Conditions for the cultivation of new Bacillus bacteria being micro bioproduct producers.

Most cultivars of agricultural crops in the average implement only 20-25 % of the genetic potential of productivity. With protection from diseases, pests and weeds they can produce a much greater yield. The average world level of losses due to infection of agricultural plants with phytopathogenic microorganisms is estimated at 12 % (1). To protect plants from diseases chemical fungicides are widely used. Their application efficiency can be as high as 100%, but there arise a number of problems, the main of them being environmental pollution and toxicity of the products (2). In this regard, it is most important to use environmentally safe methods of plant protection against pathogenes. One of the ways of solving this problems of plant protection is the shift towards creation and application of microbial agents.

Among aerobic spore-forming bacteria it is Bacillus genus that is most widely used as the basic substance for biologics against plant diseases (3,4). Bacteria of Bacillus subtilis species are highly productive and promising representatives of Bacillus genus in microbiological industry (5-7).

Bacterial fungicides based on Bacillus genus strains possess a number of positive features: a high antifungal activity (8-10); a rapid antagonistic effect (11-12); the bacilli's ability of colonizing various parts of the plant and formation of biofilms in the rhizosphere and on the leaf surface (13); the absence of pathogen resistance to bacilli and compounds formed by them (14); the possibility of their use at different stages of plant development, as well as (including) for seed and soil treatment; their compatibility with chemicals (15).

Nowadays there are many Bacillus genus bacteria-based biologics used worldwide: Avogreen (B. subtilis), Ballad (B. pumilus), Bio safe (B. subtilis), Biosubtilin (B. subtilis), Botrybel (B. velezensis), Cease (B. subtilis), Companion (B. subtilis), Kodiak (B. subtilis), Ecoshot (B. subtilis), EcoGuard TM (B. licheniformis), FZB24WG (B. subtilis), Rhizo Plus (B. subtilis), HiStick (B. subtilis), RhizoVital42 (B. amyloliquefaciens), Subtilex (B. subtilis), Pro-Mix (B. subtilis), Rhapsody (B. subtilis), Serenade (B. subtilis), Sonata (B. pumilus), Sublic (Bacillus sp.), Yield Shield (B. pumilus) (the USA, Canada, China, India, etc.) (16).

Despite the above advantages and numerous research works in the field, the quantity of ready-made biopreparations in the Russian market is quite insufficient. In 2013 the following Bacillus-oased biopreparations were allowed to be used at the Russian Federation territory: Bactofit (B. subtilis), Phytosporin-M (B. subtilis), Alirin B (B. subtilis), Gamair (B. subtilis), Vitaplan (B. subtilis), Bisolbisan (B. subtilis) (17). Such a poor choice is, among other things, due to the absence of up-to-date standards and biotechnologies for obtaining biopesticides (18).

It should be born in mind that the active element of biologics are living cells of microorganisms. That is why they have a number of special characteristics:

* titer and biological activity decrease in the process of long storage (19);

* sensitivity to environmental factors (20);

* loss of activity by biologics producer strains (21);

* critical or limited time of application (22,23);

* short residual time of activity (5);

* pollution by foreign micro biota (24).

At the biologics production stage the bioagent's sensitivity to environmental factors (components of nutrient media) is of utmost significance. Thus, temperature influences the velocity of cell reactions, the nature of metabolism, nutrition demands and the biomass composition. The culture pH level influences the end-products of anaerobic transformation of carbon and energy sources, affects the medium composition and the nature of surface of the microorganisms, tells on acids and bases dissociation. The organic matter carbon is used by bacteria for construction of their cells and as a source of energy for cell material biosynthesis, for the cell's growth and motility. The nitrogen source metabolism ensures mainly synthesis of proteins, nucleic acids and cell membrane polymers (25-21).

Therefore, it is a very important (actual) task to ensure the vitality, biological activity and purity of the producing strains cell culture, to develop and improve the production technologies of biopreparations based on them, as well as to improve production technologies taking into account trophic demands of microorganisms, and their resistance to all kinds of effects. With optimal combination of all components in qualitative and quantitative relation the medium provides sufficiently quick growth and development of microorganism population and is considered to be well-balanced (28,29).

Thus, one of the essential branches of biotechnology today is the search of optimal conditions for cultivating producer strains of biologics: choosing new substrates and nutrition sources, improvement of cultivation methods, studying vitality and activity of microorganisms under different cultivation conditions.

Creating effective biological plant protection involves the development of laboratory regulations of mass production of biologics based on the study of physiological features of promising strains-producer. Therefore, the purpose of the present work was to study the quantitative patterns of promising bacterial strains population growth in batch cultivation conditions, and evaluation of their antifungal activity, which subsequently will solve the problem of optimization of processing methods of use of biologics.


Bacillus subtilis strains

The object under study was new producer strains of biopreparations test species for protecting winter wheat against Fusarium root rot pathogen from the ARRIBPP microorganism operation collection: B. subtilis BZR 336 g, B. subtilis BZR 511 (30-34), the phytopathogenic fungus Fusarium graminearum Schwabe test-culture.

Determination of optimal conditions of cultivation

The incubation was carried out in thermostatized systems of cell cultivation (180 rot./min) New Brunswick Scientific Excella E25 (USA) for 48 hours. The batch cultivation was performed in conic flasks (350 ml) with the culture medium volume of 100 ml and pretreatment with the sown (stock) culture (2.0 % of the culture medium volume). The stock culture was obtained by means of introducing agar blocks with the strains under study into the conic flasks and their successive cultivation (35).

To determine the optimal temperature of cultivation the strains were grown at temperatures 20, 25, 30 and 35[degrees]C. To determine the optimal culture medium pH the strains were grown on liquid potato glucose substrate (PGS) (500.0 ml potato broth, 20.0 g glucose per litre, Russia) (36). By adding lactic acid or alkaline (4 N solution of NaOH, Russia) the substrate reaction was kept within the 3.0, 6.0, 8.0 and 10.0 pH measured by a pH meter Sartorius PB11 (Germany).

Determination of the optimal nutrition sources

As the carbon sources sucrose, glucose, molasses and glycerol were added in the test media. In the study of carbon sources nitrogen sodium nitrate served as the unchanged nutrition component. In determining the optimal sources of nitrogen nutrition peptone, NaN[O.sub.3], yeast and corn extracts were tested with glucose as the only (constant) carbon source. Chapek medium for bacteria was used as basic (0.5 g KCl, 0.5 g MgS[O.sub.4], 1.0 g [K.sub.2]HP[O.sub.4], 3.0 g CaC[O.sub.3], 1.0 ml of 1% solution FeSO4, 2.0 g NaN[O.sub.3], 20.0 g glucose per litre, all the components of the Russian manufacture) (37). The concentrations of the introduced carbon and nitrogen sources, as well as the composition of the (original) culture medium (orig.), as elements of the production know-how for new biopreparations, are the objects protected as a trade secret All-Russian Research Institute of Biological Plant Protection (order number 42-p of 28 November 2012). As a control, the King's B liquid medium (KB) (20.0 g peptone, 15.0 g glycerol, 1.5 g MgSO4, 1.5 g [K.sub.2]HP[O.sub.4] per litre, all the components of the Russian manufacture) was used (38) and the PGS.

Determination of the number of colony forming units

Upon completion of the cultivation in all the experiments the number of bacterial cells was determined. To study the quantitative patterns of population growth of the strains under different conditions the Koch method was used (39). The determination of number of cells by this method consists of three stages: preparation of dilutions, sowing on the nutrition medium in PE. Calculation of the grown colonies was performed using a system for automatic counting of colonies Color Qcount, Spiral Biotech (USA).

Determination of the antagonistic activity

Determination of the antagonistic activity of the strains under study was performed by double (counter) cultures method (40-41) on potato-glucose agar (PGA), agarized KB medium and the optimized medium (orig.). An agar block with the mycelium of a pathogen was inoculated into a Petri dish, with the bacterial strain deposited by the stroke method at the distance of 6 cm from the pathogen block. The cultures were incubated for 20 days at + 28[degrees]C. Pure cultures of bacteria and fungus pathogen sown separately were used as control options. The recordings were taken at the 5-th, 10-th, 15-th and 20-th days. The type of relationship of the fungus and bacteria was marked: the presence or absence of zones, their size, changes in color, density, thickness and direction of the pathogen mycelium growth. The degree of inhibition of the pathogen mycelium growth was determined according to the formula (42):

I = (1 - (A/B)) x 100,

with I being the inhibition of the pathogen mycelium growth, %;

A-fungus growth in the option, mm;

B-fungus growth in the control, mm.


Temperature is an important factor for the growth of microorganisms. The investigation showed that a high density (concentration) of cells in the replication with the strain B. subtilis BZR 336g was marked at the cultivation temperature of 30[degrees]C making 4.1 x [10.sup.10] CFU/ml. The highest LA titer of strain B. subtilis BZR 517 was marked at the temperature of 35[degrees]C: 5.1 x [10.sup.10] CFU/ml. Based on the data obtained it is safe to conclude that the most optimal temperatures for bacillary strains are these: 30.0 and 35.0[degrees]C (Fig. 1).

The pH is of critical significance for cultivation of microorganisms. When it changes to the worse, the microorganisms cease to grow, even with all other conditions being optimal.

The optimal pH for growth of the strain B. subtilis BZR 336g is established to be 6.0 and 8.0: 1.6 x [10.sup.8] CFU/ml and 1.7 x [10.sup.8] CFU/ml, correspondingly. The pH values of 3.0 and 10.0 proved to be limiting. The highest titer for strains B. subtilis BZR 517 was marked at pH 10.0: 1.0 x [10.sup.9] CFU/ml. The number of colony-forming units in LA based on this strain was growing proportionally to the increase of the medium pH level (Fig. 2).

The maximum cell concentration in prototype LA based on the strain B. subtilis BZR 336g was observed on the medium with molasses as the carbon source: 1.1 x 109 CFU/ml. In options with the addition of glucose, sucrose and glycerol the LA titer was lower by two orders . For strain B. subtilis BZR 517 medium supplemented with molasses was also optimal for growth, the LA titer was 3.5 x [10.sup.11] CFU/ml (Fig. 3) . These results are explained by the fact that molasses being a byproduct of sugar production contains not only sucrose, but other organic nitrogen-free substances and minerals.

High LA titer in the option of the strain B. subtilis BZR 336g was observed on the nutrient medium with peptone, yeast extract and corn used as the nitrogen source: 1.5-5.7 x [10.sup.8] CFU/ml. These components were also optimal for strain B. subtilis BZR 517-2.8-5.5 x [10.sup.8] CFU/ml (Fig. 4). In all likelihood, this is due to the fact that peptone is a product obtained by the action of proteolytic enzymes, making it more available for digestion, while in the composition of the yeast extract and corn there are vitamins, organic acids, macro -and micronutrients (39).

Based on the data obtained, we selected the first samples of the original optimized culture media. As a control the KB culture medium was used, on which the biological agents under study produced from natural sources were actively growing and exhibited a high antifungal activity. The studies found that the number of colonyforming units of LA based on the strain B. subtilis BZR 336g on the optimized medium was significantly higher than in the KB and PGS media and made 8.7 x [10.sup.10] CFU/ml. For strain B., subtilis BZR 517 the optimized nutrient medium was also preferable (effective) by this criterion compared to standard media: the LA titer was 7.2 x [10.sup.10] CFU/ml (Table 1).

It is well-known that the development of biotechnologies to obtain complex action biofungicides demands LA with optimum density of microbial cells in combination with high concentrations of antifungal agents. Therefore, at the next stage we studied antifungal activity of producing strains of biological products on various nutrient media.

For strain B. subtilis BZR 336g maximum antifungal activity was noted on the optimized medium, and made 90.0% on the fifth day, 83.6 % on the tenth day and 78.2% on the fifteenth day. On PGS and KB medium the degree of inhibition of the pathogen was increased to the tenth day of incubation, but remained significantly lower than in the option with the optimized medium. By the fifteenth day the antifungal activity in these media began to decline (Fig. 5). It is important to note for that matter, that only on the optimized medium, this strain has a high mobility: as early as on the fifth day of co-incubation the biological agent occupied the full area of the nutrient medium blocking the pathogen growth (Fig. 7).

The degree of inhibition of F. graminearum in the option with strain B. subtilis BZR 517 increased by the tenth day in all nutrient media, but no significant difference in antifungal activity was observed, as is in the case with strain of B. subtilis BZR 336g (Fig. 6). However, as in the case of the strain B. subtilis BZR 336g, B. subtilis BZR 517 on the optimized medium had a higher mobility (Fig. 7).

Furthermore, among the impact characteristics of metabolites of the active bacteria strains on F. graminearum the following should be noted: in the antagonistic action area of bacteria in some options the lysis of an already formed mycelium growth was observed, with inhibition and change of colour of the pathogen mycelium.

Thus, as a result of the research, the optimal conditions for cultivation of strains B. subtilis BZR 336g and B. subtilis BZR 517 were found, original optimized culture media samples were obtained, providing the LA with the required number of colony forming units (no less than 1.0 x 1010 CFU/ml) in combination with a high antifungal activity and mobility.


This work was financially supported by the Eurasec Interstate Task Program "Innovative Biotechnologies" GC No. 16.M04.11. 0026 of 15.07.2011 in 2011-2015 Ministry of Education and Science of the Russian Federation, the Russian Foundation for Basic Research and Krasnodar region administration grant (1) 13-08-96533 r_yug_a.


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A.M. Asaturova, A.I. Homyak, N.S. Tomashevich, M.D. Pavlova, N.A. Zhevnova, V.M. Dubyaga, A.Ye. Kozitsin, T.M. Sidorova, V.D. Nadykta, V.Ya. Ismailov

All-Russian Research Institute of Biological Plant Protection.

(Received: 16 October 2015; accepted: 17 November 2015)

* To whom all correspondence should be addressed. Tel.: +7(861) 228-17-74; E-mail:

Caption: Fig. 5. Antifungal activity of strain B. subtilis BZR 336g against F. graminearum

Caption: Fig. 6. Antifungal activity of strain B. subtilis BZR 517 against F. graminearum

Caption: Fig. 7. Antifungal activity of strains B. subtilis BZR 336g and B. subtilis BZR 517 against F. graminearum on different nutrient media
Table 1. Growth of strains B. subtilis BZR 336g and B. subtilis BZR
517 on different nutrient media in batch cultivation process

Strain               Nutrient medium, LA titer, CFU/ml

              KB                    PGS

B. subtilis   (5,7 [+ or -] 0,06)   (1,3 [+ or -] 0,14)
  BZR 336g      x [10.sup.7]          x [10.sup.8]
B. subtilis   (3,7 [+ or -] 0,14)   (1,8 [+ or -] 0,07)
  BZR 517       x [10.sup.8]          x [10.sup.8]

Strain        Nutrient medium, LA
                 titer, CFU/ml
                Optimized medium

B. subtilis   (8,7 [+ or -] 0,66)
  BZR 336g      x [10.sup.10]
B. subtilis   (7,2 [+ or -] 0,42)
  BZR 517       x [10.sup.10]

* --the table shows the average values and standard deviation

Fig. 1. Temperature effect on the growth of strains B. subtilis BZR
336g and B. subtilis BZR 517 in the process of batch cultivating

Temperature   B. subtilis   B. subtilis
[degrees]C      BZR 336g      BZR 517

20                671           3660
25               1870            183
30              40700             87
35               1840          56300

Note: Table made from bar graph.

Fig. 2. pH effect on the growth of strains B. subtilis BZR 336g
and B. subtilis BZR 517 in the process of batch cultivating

pH   B. subtilis   B. subtilis
       BZR 336g      BZR 517

3          3             4
6        162            39
8        169           122
10        60          1050

Note: Table made from bar graph.

Fig. 3. The effect of carbon nutrition sources on the growth of
strains B. subtilis BZR 336g and B. subtilis BZR 517 in batch
cultivation process

Carbon     B. subtilis   B. subtilis
source       BZR 336g      BZR 517

sucrose         18             21
glucose         23             23
molasses      1110         350000
glycerol        24             18

Note: Table made from bar graph.

Fig. 4. The effect of nitrogen nutrition sources on the growth
of strains B. subtilis BZR 336g and B. subtilis BZR 517 in
batch cultivation process

Nitrogen         BZR 336g B.   BZR 517 B.
source            subtilis      subtilis

peptone              152          270
NaNO3                 38           24
yeast extracts       220          210
corn extracts        570          550

Note: Table made from bar graph.
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Author:Asaturova, A.M.; Homyak, A.I.; Tomashevich, N.S.; Pavlova, M.D.; Zhevnova, N.A.; Dubyaga, V.M.; Kozi
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Dec 1, 2015
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