Evaluation of various Bacillus biopreparations as microbial fertilizers for optimum production conditions and performance

In our studies, 440 Bacillus sp. obtained from "EU Bioengineering Department Bacterial Culture Collection" were tested for their inorganic phosphate solubilizing abilities by in vitro pre-screening on solid medium. After that, solubilized phosphate quantities were measured in liquid medium for 51 isolates evaluated as positive for phosphate solubility and indole-3-acetic acid production capabilities were determined. In the study, Bacillus microorganisms which can fix atmospheric nitrogen through non-symbiotic way were also isolated from various sources and the nitrogen fixing abilities of 25 isolated strains were demonstrated by the acetylene reduction assay. Six different Bacillus isolates were selected according to the quantitative data obtained after in vitro experiments (based on the amount of inorganic phosphate (ml-1), the ability to nitrogen fixation and the amount of IAA (μg ml-1) produced by isolates in the nutrient medium). In order to reveal the effects of the six selected isolates on the one year plants seedlings (tomato, pepper, eggplant) growth and development parameters and to compare them with each other in the open field, performance tests were carried out in pots. The effects of selected isolates on in vitro germination of plant seeds were also investigated. As a result of the performance tests carried out with the plants, one of the isolates having the characteristics of being designated as microbial fertilizer (Bacillus subtilis EGE-B-24.4i) was specifically studied to determine the optimum medium and conditions for the production of a high number of endospores in the flasks in shaker. In this study, response surface method (RSM) was used to obtain a production medium which is economic and suitable to use in organic agriculture. With the optimized production medium obtained as a result of the experiments, the production of spores in Bacillus subtilis EGE-B-24.4i was determined as 3.12x1011 endospore/ml in flasks. With the aim of to get maximum endospore count optimized medium was used in a 2 L stirred bioreactor using batch production at 400 rpm agitation speed and 2 vvm aeration rate and 8.5x1010 endospore/mL were obtained. In order to increase the endospore concentration, 3.5x1011 endospore/ml spore count was reached by feeding glucose solution in optimized medium in fed batch production. In the scale up studies, with optimized medium and a working volume of 20 litres in a 30 litre capacity stirred bioreactor at 30°C and 400 rpm, 2.7x1011 endospore/ml spore count was obtained. 993 grams of biomass separated from the production fluid by centrifugation was dried by lyophilization and ground into powder. 5x1011 endospore/g were determined in one gram of the powder biopreparate. At the end of the study, on the basis of their in vitro microbial fertilizer properties and performance tests carried out with plants, 8 indigenous Bacillus isolates selected to become microbial fertilizers were molecularly identified. With this study an important step has been taken for the development of a biopreparation consisting of the original strain isolated from the soil of our country which is also suitable for large-scale production and determined as efficient microbial fertilizer.