Studies On Characteristics Of Sporulation And Germination Of Paenibacillus Polymyxa SQR-21

Paenibacillus polymyxa (P. polymyxa) strains, that can produce two types of peptide antibiotics and can form endospores and survive efficiently in soil make them very popular in biocontrol. P. polymyxa SQR-21 is a spore-forming Gram-positive bacterium, which was isolated from plant rhizosphere soil by the Plant-Microbial Interaction Laboratory, Nanjing Agricultural University. It has been identified as a potential biological control agent and classified as a plant growth-promoting rhizobacterium (PGPR). SQR-21 strain has also been introduced into matured composts to make bioorganic fertilizer. These fertilizers have been shown to exhibit strong biological control of Fusarium wilt disease in cucumber and watermelon when applied to Fusarium oxysporum infested soils. In this thesis, we studies the factors influencing spore formation and germination of SQR-21, and the application of SQR-21 spores in amino acid organic fertilizer to produce bioorganic fertilizer (BIO). The main results were as follows:1. The cell membrane of strain SQR-21 contains 12 species of PLFA. The content of anteriso-C15 fatty acid was much higher than other PLFA. The unsaturated fatty acids including 16:1w7c alcohol,16:1 w11c, ISO 17:1 w10c and 17:1 ANTEISO B/i I were about 9.8% of total PLFA. The strain was identified as P. polymyxa by the Sherlock Microbial Identification System (Sherlock MIS). The spore and sporulation process of P. polymyxa SQR-21 were examined by electron microscopy. The dimensions of the oval-shaped spore, formed at terminal portion of cell, were 0.5-0.8μm x 1.5-1.8μm. The spore coat of SQR-21 consisted of three morphologically distinguishable layers, the inner, the middle and the outer spore coat. Eight to eleven ridges were found on a cross section image, which was different from other Bacillus. spp with star-shape. The process of sporulation was essentially the same as that of other Bacillus. spp. During the period of prespore and the mother cell membrane, the substances to form cortex began to accumulate. In the later stage of the sporulation, the electron dense materials were synthesized and became coat structure. At the end, the mature spore released from the mother cell. 2. In order to enhance cell growth and spore formation of P. polymyxa SQR-21, the impact of medium composition and culture conditions were examined. The optimal medium composition was determined as follows:glucose 1g/L; beef extract 16g/L; Mn2+ 0.1 mmol/L; Mg2+0.5g/L; Ca2+1 mmol/L and Fe2+0.5μmol/L. The optimal pH and temperature for growth and sporulation were 7.0-8.0 and 37℃respectively. The bottle filling capacity 100ml/250ml was best. The maximum total cell number (2.22 x1010 cfu/ml) and spore yields (2.21 x 1010 cfu/ml) were obtained at 37℃,170r/min for 48h.3. To optimize the role of Paenibacillus polymyxa SQR-21 in bioorganic fertilizer, a study of spore germination under various conditions was conducted. The abilities to induce spore germination of Paenibacillus polymyxa ACCC10252 and SQR-21 by a mixture of L-asparagine, glucose, fructose and K+(AGFK), and single sugar (glucose, fructose, sucrose and lactose) plus L-alanine were evaluated. Spore germination was measured as a decrease in optical density at 600 nm. The effects of heat activation and germination temperature were important for germination of spores of both strains on AGFK in Tris-HCl. L-alanine alone showed a weak ability to induce spore germination. However, fructose plus L-alanine significantly induced spore germination and the maximum spore germination rate was observed with 10 mmol l-1 L-alanine in the presence of 1 mmol l-1 fructose in PBS. In contrast, fructose plus L-alanine hardly induced spore germination in Tris-HCl. However, addition of 10 mmol l-1 NaCl into Tris-HCl, the percentages of OD600 fall increased by 19.6 and 24.3%for ACCC10252 and SQR-21, respectively. AGFK-induced spore germination needed much more strict germination temperature than fructose plus L-alanine. For both strains, fructose plus L-alanine-induced spore germination was not sensitive to pH.4. Effect of spore-forming temperatures on the spore characteristics including dipicolinic acid (DPA) content, heat resistance, fatty acids composition, germination, and spore coat proteins was studied with Paenibacillus polymyxa SQR-21. Spores were formed rapidly at 37℃and showed higher heat resistance than those formed at 25℃and 30℃. However, the germination rate was negatively correlated to the sporulation temperature. The maximum germination rate of spores formed at 25℃was 1.27 times higher than that formed at 30℃, which may be due to the higher permeability of the spores’inner membrane. The pellets of spores formed at higher temperature showed much darker color than those formed at lower temperature and the number of spore coat proteins were increased with the increase of sporulation temperature.5. The influences of BIO storage temperature, water content and SQR-21 physiologically inoculated status on BIO were studied by plating method and FISH method. The spore and vegetative cell numbers detected by FISH were 10-100 times higher than that by plate counting, suggesting that FISH might be a better method to detect bacterial numbers. When inoculated with 100% spores of SQR-21 into the amino acid fertilizer with 20%-30% water content, the maximum spore forming rate and total cell number were obtained at 20℃. When incubated at 30℃, the optimal conditions for BIO storage were 40% water content with 100% spores. When the BIO was placed at 40℃, lower water content and inoculation with 100% spores favored the BIO storage. In all words, inoculation with 100% spores and maintaining of water moisture at the range of 20%-30% was favourable toward BIO storage. These results were useful in producing high quality of bio-organic fertilizers and keeping them in a good way.