Isolation of cellulolytic bacteria from cultivated soil samples

. Cellulose decomposition is widespread in all types of soils in the world, which plays an important role in the carbon cycle and the decomposition of plant residues, which is especially important for agriculture. Cellulolytic bacteria can be actively used in the processes of increasing soil fertility, especially in those areas where the natural content of such microorganisms is not high. Within the framework of this work, cellulolytic bacteria were isolated from soil actively used for growing crops. 39 strains of microorganisms were selected, their lytic activity in relation to cellulose compounds was determined. It was observed that 5 strains showed high cellulose activity already within 24 hours. The study of some biological properties of isolated microorganisms showed their strong difference from each other. The unifying feature of all isolated strains was the absence of tryptophan fermentation. The obtained data allow us to continue creating a collection of cellulolytic microorganisms with their further identification.


Introduction
Cellulose, the most common component of plant biomass, is found in nature almost exclusively in the cell walls of plants, although it is produced by some animals and several bacteria [3][4][5].Despite the large differences in the composition and anatomical structure of cell walls of different plant taxa, high cellulose content -usually in the range of about 35 to 50% of the dry weight of the plant -is a unifying feature [12].In some cases, cellulose is in an almost pure state.However, in most cases, cellulose fibers are embedded in a matrix of other structural biopolymers, primarily hemicellulose and lignin, which make up from 20 to 35 and from 5 to 30% of the dry weight of plants [1;7;13].
It is known that microorganisms, such as fungi and bacteria, play an important role in the decomposition of cellulose and starch components [11].The splitting of these components leads to the formation of simple sugars [14].Although a large number of microorganisms can decompose cellulose, only a few of them produce a significant amount of free enzymes capable of completely hydrolyzing crystalline cellulose [2].Microorganisms play a significant role in the decomposition of plant residues in the environment and therefore the selection of suitable strain is important for the individual process [8][9].The use of biological preparations based on cellulose-decomposing bacteria is a promising direction in the intensification of agriculture.A significant amount of plant residues in the fields can be effectively used as fertilizers for subsequent plant crops [10].Natural processes taking place in the soil take a considerable period of time to convert cellulose into compounds acceptable for plants.In this regard, the search and isolation of prospective production strains of cellulose-decomposing bacteria is an urgent problem.
The conducted monitoring of cellulose-destroying bacteria will make it possible to form, characterize and systematize a collection of these microorganisms reflecting their diversity and geographical prevalence within the region.
The selected bacteria with the best enzymatic properties will be used in the future when designing an experimental biological preparation for further testing on plant biomass.
The purpose of this work was to isolate cellulose-destroying bacteria from soil samples.
Research aim: -Study of selected soil samples for the presence of cellulose-destroying bacteria.
-Determination of cellulolytic activity of isolated strains.
-Study of some biological properties of isolated strains.

Materials and methods
Collection and preparation of samples.
As an object of research, 17 soil samples obtained from grain growing sites in the Ulyanovsk region were used.Sampling was carried out from places where the decomposition of plant residues was noted.
A number of different cellulose-decomposing bacteria has been isolated.Both grampositive (18 strains) and gram-negative species (21 strains) were found.
Nutrient media.
Isolation of microorganisms.Isolation of microorganisms from soil samples was carried out according to the scheme shown in the fig. 1.
Isolation of bacteria was carried out in the following way.A sample of soil weighing 10 g was suspended in 100 ml of saline solution for 24 hours, constantly stirring.After that, the suspensions were allowed to settle, after which the add-ons were selected in a volume of 5 ml and a series of successive dilutions of 1/10 were made.Experimentally, it was found that the optimal number of dilutions is 3, as a result of which a 1000-fold dilution of the initial suspension was obtained.Then the suspension was seeded from the third dilution on a Getchinson medium containing 1% CMC.The crops were cultivated for 48 hours at a temperature of 28°С.
Evaluation of cellulolytic activity.
The decomposition of cellulose was studied in LB medium with the addition of 1% cellulose.Seeding of studied microorganisms was carried out in several dilutions to assess the decrease in cellulolytic activity with decrease in the number of bacterial cells.Storage of isolated strains.The isolated bacteria were permanently stored at a temperature of 2-4oC on a Getchinson medium containing 1% myeliokariocyte.

Results and Discussion
In the process of isolation of cellulose-destroying microorganisms, it was found that on the selected nutrient medium, despite the absence of other carbohydrates besides cellulose, a sufficiently large number of colonies of microorganisms with clarification zones grew when Lugol solution was added to the medium.Lugol solution reacting with cellulose colors the latter in a dark blue color.The presence of clarification zones around the grown colonies indicates the fermentation of cellulose compounds by grown microorganisms.Note that the sizes of the clarification zones were different in each case.First of all, the selection was carried out of those colonies that had the largest clarification zones.At this stage, it was decided to select only 3-4 colonies with the largest degradation zone of CMC.The passage of the selected colonies was carried out on a Getchinson liquid medium containing melyokariocytes, after which the crops were incubated for 24-48 hours at a temperature of 28oC until the appearance of visual signs of bacterial growth in the test tube.Then the grown bacterial cultures were re-sown on Petri dishes with LB medium to control their purity.In the case of a positive result and the absence of extraneous growth, we proceeded to the identification of isolated microorganisms and the study of some of their properties.
According to the results of the conducted studies, 39 strains of cellulose-destroying bacteria were isolated.All isolated strains showed a different degree of degradation of CMC in the nutrient medium, which was reflected in the size of clarification zones after the introduction of the corresponding reagent into the medium (fig.2).
Further, the isolated strains were cultured on a Getchinson medium with MKC for 48 hours.According to the results of cultivation, both the formation of films on the surface of medium and bottom growth were noted.The Gram staining of isolated strains showed that at this stage 2 groups of bacteria were obtained, distributed almost equally.
Gram-positive rods -the character of stained cells was as similar as possible between individual strains, there were both single and paired or in short chains of cells.
Cellulolytic activity of isolated strains.
The experiment showed that all the studied strains had different levels of cellulolytic activity and showed it differently when the concentration of bacterial cells decreased (figure 3).As it can be seen from picture 2, part of the strains visually showed practically no cellulolytic activity, although when removing the bacterial mass and coloring with the use of congo red on the surface of medium at the colony growth site, it was possible to detect zones of clarification. 5 strains with the largest zones of clarification on the used medium were noted from the isolated ones.In some cases, the radius of the zone of enzymatic activity around the grown colony was up to 15 mm, while the average radius did not exceed 3-4 mm.The summarized data is presented in the table 1.
Further the influence of the beginning concentration of bacteria on the degree of cellulose decomposition was studied.During the experiment, up to 6 dilutions of the daily studied bacterial culture were sown per cup.After cultivation, clarification zones were detected for 24 hours using congo red dye or Lugol solution.It is worth noting that the use of Lugol's solution made it possible in some cases to identify areas of carification much better than congo red, even after applying the latter.
The obtained results showed that the cellulolytic activity was displayed in different ways.In some strains, it practically did not change with the change in number of bacterial cells (P 7.3).Others had a decrease in activity with a decrease in the initial concentration of bacterial cells (P 9.3).In the third case, on the contrary, we observed an increase in bacterial activity with a decrease in titer (P 17.42).The difference in the zone of lytic activity between individual strains may be due to the more discharged growth of individual colonies on the nutrient medium, as a result of which the zone of access to nutrients also increased.
Study of some biological properties.
The study of individual biological properties of isolated bacteria was carried out to find similarities between individual isolated strains.It should be noted that in the studied collection, all strains showed a lack of ability to convert tryptophan into indole.In addition, it was found that over 80% of studied cultures were catalase-positive, showed enzymatic activity against starch and gelatin.In relation to various sugars, we have obtained diverse results.Only about half of the studied cultures fermented individual sugars, while the other part was not able to do this.At the same time, it was practically impossible to find relationship between individual strains that would allow separating some cultures of microorganisms into separate groups with respect to their biological properties.In the future, it is planned to continue working with a large number of "field" strains to detect the relationships between them.

Conclusion
Within the framework of this work, the creation of a collection of cellulose-destroying microorganisms was initiated.The conducted studies made it possible to select 39 strains from 14 soil samples, of which 18 were gram-positive bacteria, 21 strains were classified as gram-negative.In the process of studying their cellulolytic properties, it was found that the level of activity between individual strains is very different.5 strains (P 5.1, P 7.3, P 9.3, P17.41, P17.42) with the largest size of the cellulolytic activity zone were selected.The study of some biological properties of isolated microorganisms showed their noticeable difference between each other.However, some similarities have also been noted, such as a lack of activity against tryptophan.

Fig. 1 .
Fig. 1.Scheme of isolation of cellulolytic microorganisms.Study of biological properties.The research was carried out on the basis of available and tested methods of the staff of the Department of microbiology, virology, epizootology and veterinary-sanitary expertise of the Ulyanovsk state agrarian university named after P.A. Stolypin.Storage of isolated strains.The isolated bacteria were permanently stored at a temperature of 2-4oC on a Getchinson medium containing 1% myeliokariocyte.

Table 1 .
Zone size of cellulolytic activity of isolated strains.