جداسازی و شناسایی باکتری‌های حل‌کننده فسفات مقاوم به دما برای استفاده در کود میکروبی فسفاته

در فرمولاسیون کودهای میکروبی فسفاته که غالباً به فرم گرانوله تهیه می‌شوند از باکتری های حل‌کننده فسفات استفاده می‌شود. از جمله محدودیت‌های تولید این نوع کود، از بین رفتن باکتری ها در فرایند تهیه و خشک نمودن گرانول‌ها می‌باشد. بر این اساس در این تحقیق اقدام به جداسازی باکتری های حل‌کننده فسفات مقاوم به دما شد و تحمل دمایی و توانایی انحلال فسفات (از منابع سنگ فسفات و تری‌کلسیم فسفات) در باکتری‌های بومی جدا شده از خاک، مورد ارزیابی قرار گرفت. از پنج باکتری (جدایه‌های rps4، rps6، rps7، rps8 و rps9) با قابلیت تولید هاله شفاف تنها دو باکتری (rps7 و rps9) قادر به تحمل دمای 55 درجه سلسیوس به مدت 16ساعت بودند. در محیط اسپربر جامد حاوی تری‌‌کلسیم ‌فسفات، باکتری rps9 و rps7 به ترتیب با دارا بودن مقادیر 2/60 و 2/27 برای نسبت (hd/cd) بعد از گذشت 12روز، قدرت بالایی در امر انحلال فسفات معدنی نامحلول داشتند. میزان انحلال فسفات کم‌محلول در روش کمی نیز برای دو باکتری rps9 و rps7 به ترتیب 531/8 و 324/1 میلی‌گرم بر لیتر بدست آمد. نتایج زنده‌مانی باکتری ها نیز نشان داد که زنده‌مانی جمعیت میکروبی در حالت بدون تیمار دمایی تا 6 ماه و در حالت تیمار دمایی به 4 ماه کاهش یافت. شناسایی مولکولی rps7 و rps9 حاکی از آن بود که هر دو جدایه متعلق به گونه pantoea agglomerans هستند.

Isolation and Identification of Temperature Resistant Phosphate Solubilizing Bacteria for Use in Phosphatic Microbial Fertilizer

Introduction: Application of chemical and organic carrier and its integration with useful microorganisms including phosphate solubilizing bacteria (PSB) has facilitatedproduction of phosphate microbial fertilizers (PMFs), which are used in granular or powder form. One of the major limitation of thesebiofertilizers is decline or loss of PSB viable cell in the granule preparation process. Accordingly, in this study, isolation of temperature resistant phosphate solubilizing bacteria was performed and temperature tolerance and ability to dissolve phosphate from rock phosphate (RP) and tricalcium phosphate (TCP) sources were evaluated. Materials and Methods: Firstly, each soil samples incubated for 16 hours at 55 °C, then dilution series were prepared and 100 μl of four final dilutions (106, 107, 108, and 109) were used to spread on Sperber solid medium. After spreading the microbial suspensions from the dilutions in the Sperber solid culture medium and the appearance of colonies, screening based on the resistance to soil temperature treatment and, subsequently, the ability to grow in a solid Sperber solid medium and dissolution of low solubility phosphate (formation of transparent halo), was done to isolate PSB. In order toprepare PMF, each of screened PSB were cultured in NB, andthen 1 ml of overnight culture wasmixed with 9 ml sterile distilled water and added to the basal formulation of rock phosphate (45 g), bagasse (30 g) and sulfur (15 g) with initial temperature of20°C.Temperature treatments (55 °C for 16 hours) of bacteria were performed in three steps: a) on sampled soils, b) pureculture of bacteria and c) bacteria added to the carrier. Microbial population in provided microbial fertilizer was countedin two ways a) half of the microbial fertilizer was kept at normal temperature by maintaining the initial conditions, b) another half after the temperature applied (55 ° C for 16 hours). The semiquantitative and quantitative test of insoluble inorganic phosphate solubility was performed by isolates in solid and liquid Sperber medium. The 16S rRNA molecular method was used to identify the isolated bacteria by general primers 27F and 1492R. Results and Discussion: Five bacteria (RPS4, RPS6, RPS7, RPS8, RPS9) out of nine isolated bacteria were able to solubilize mineral phosphate (TCP and RP) but only two isolates (RPS7 and RPS9) were resistant to temperature (55 °C for 16 h). In tricalcium phosphate medium, the RPS9 and RPS7 bacteria had a high ability to solubilize insoluble inorganic phosphate with average of 2.60 and 2.27 for a ratio of (HD / CD) after 12 days, respectively. There was no halo in Sperber medium containing rock phosphate. The amount of solution in the quantitative methods was also obtained to be 563.8 and 324.1 mg / l for RPS9 and RPS7 bacteria, respectively. The prepared microbial fertilizer was counted in two ways (a): half of the sample fertilizer was kept at normal temperature by maintaining the initial conditions; (b):after the maintaining temperature at 55 °C for 16 hours, the population of other half was determined. During counting the initial microbial population (zero time) at normal temperature, all bacteria used in microbial carrier had an acceptable population. During examining the populationsof microbial in the initial carrier, RPS4, RPS6, RPS7, RPS8 and RPS9 bacteria were 3.6, 3.5, 3.6, 3.5, 3.6 and 3.5 (×106 CFU/g), respectively. After 4 months the populations were 4.6, 6.3, 9.6, 7.4 and 8.6 (×105) and in the 6th month, the populations were 3.9, 3.8, 12.3, 4. 7 and 9.2 (×104) seeming to be favorable for the tested bacteria. It seems that this survival time for the tested bacteria is desirable. During countingactive population of temperature treated microbial fertilizers, the initial population of the microbial carrier (at zero time) decreased 10 times with respect to the nontreated carrier. Active population ofRPS9 and RPS7 (temperatureresistant treatments) in the zero time was 4.5 and 4.3 (×105), respectively. Although the RPS9 and RPS7 microbial populations were able to survive in non temperature treatments for 6 months, it was observed that in the treatment, this viability practically reduced to 4 months. Molecular identification of the isolates RPS7 and RPS9 revealed that they belonged to Pantoeaagglomerans. Conclusions: According to the findings of this research, using phosphate solubilizing bacteria and temperature resistant Pantoeaagglomerans RPS9, recently isolated and identified, can be considered toindustrially produce granular microbial fertilizers. It is worth mentioning thatfurther studies are required to be carried out on the effectiveness of this formulation with these bacteria infield scale.