Studies On Process And Technology Of The Production Of Poly-γ-Glutamic Acid By Bacillus Amyloliquefaciens C1-6in Solid-State Fermentation

Poly-y-glutamic acid (y-PGA), a naturally occurring anionic high molecular weight biopolymer, was connected by y-amide bonds between y-carboxyl and a-amino groups of D-and/or L-glutamic acid by certain microbial species. y-PGA is water-soluble, biodegradable, edible and nontoxic to human and environment. In addition, it has a good affinity to nutrients and a strong ability to preserve moisture and retain water because there are large amounts of free carboxyl in its intramolecules. In recent years, y-PGA has been used for various applications in foods, cosmetics, medicine, waste water treatments and agriculture. The objective of this dissertation is to develop a new type of bio-organic fertilizer by solid-state fermentation (SSF). The present work consists of:(1) isolation and identification of the strain producing y-PGA;(2) mutation breeding of the y-PGA productive strain;(3) optimization of the parameters and medium of SSF for producing y-PGA;(4) the effects of y-PGA on germination of cucumber seeds and the effects of the products of SSF on growth of maize seedlings repectively;(5) the dynamics shifts of microbial communities during SSF by denaturing gradient gel electrophoresis (DGGE);(6) quantification of the total amount of bacteria and B. amyloliquefaciens in the solid-state fermentation by real-time PCR assay;(7) cloning and heterologous expression of y-PGA degradation gene, ywtD. The main results obtained in this study were summarized as below:1.39strains with viscous colonies on plates were isolated from vegetable soil, among which a strain that can produce y-PGA with a yield of18.4g/L was screened. The product of C1produced by liquid fermentation had a maximum absorption wavelength at209nm and its molecular weight was more than130kDa. With the analysis of colony morphology, physiological and biochemical characteristics, as well as the phylogenetic analysis of16S rRNA gene sequence, Cl was identified as a Bacillus amyloliquefaciens, which was named as Bacillus amyloliquefaciens C1.2. B. amyloliquefaciens C1was treated with an ultraviolet-nitrosoguanidine composite mutation. The resulted mutant strain C1-6was screened with a y-PGA productivity of24.2g/L, which was increased by31.52%compared with the wild strain and had a good genetic stability after8streaks. Soaking cucumber seeds with y-PGA could promote the growth of germinative cucumber seeds under water shortage condition. In contrast with the treatment of full-strength nutrient solution, remarkable increase of root and shoot biomass, height and accumulation of N, P and K of the maize seedlings were obtained under low-strength nutrient solution treatments. In addition, all y-PGA treatments increased the relative chlorophyll content (SPAD) of maize seedlings.3. The parameters and medium components of SSF for the production of y-PGA by B. amyloliquefaciens C1-6were optimized by single-factor experiments and response surface methodology (RSM) using agro-industrial organic wastes as basic substrate respectively. The optimal SSF medium (20g substrates with50%initial moisture and initial pH7.0) was determined to contain5.51g dairy manure compost,1.91g soybean cake,0.57g corn flour,2.15g monosodium glutamate production residues (MGPR),1.5g wheat bran,0.5g rapeseed cake,0.1g citric acid,0.05g MgSO4·7H2O and0.03g MnSO4·H2O, in which C1-6produced y-PGA up to4.37%when fermented for48h at37℃.SDS-PAGE showed that the molecular weight of the y-PGA produced by SSF was more than130kDa. The SSF product was applied as bio-organic fertilizer (CBIOF) in pot experiments, which showed that the biomass and height of maize seedlings, length and width of leaf, and stem thickness were significantly increased. Besides, the contents of soluble protein, soluble sugar, and root activity were all increased in the CBIOF treatment. At equal fertilization level, the promotion effects on maize seedlings by applied with CBIOF was much better than applied with regular organic fertilizer (OF).4. Scale-up SSF experiments based on the result of optimized medium in flask were carried out outdoor. The whole SSF process could be divided into three phases:an initial short mesophilic period (1～2days), followed by a long thermophilic period (3-29days) and a curing period with a decrease in temperature (30-33days). The production of y-PGA reached a maximum of0.6%after20days fermentation, and then it decreased a little to0.57%at the end of the fermentation. PCR-DGGE profile showed that the microbes in the substrate of SSF could be classed into6orders:Firmicutes Bacillales; Firmicutes Lactobacillales; Actinobacteria Actinobacteridae; Proteobacteria Alphaprobacteria; Proteobacteria Deltaprobacteria; Proteobacteria Gammaprobacteria. A remarkable reduction of microbial diversity was detected during the fermentation process, while the inoculum, B. amyloliquefaciens C1-6, was detected as the dominant organism through the whole process. In the mesophilic phase of SSF, Proteobacteria was the dominant microbe, which was replaced by Firmicutes and Actinobacteria in the thermophilic phase. In the mature stage of fermentation, the microbial community gradually tended to be stable. The molecular analysis of the bacterial diversity has significant potential for instructing the maturing process of SSF to produce y-PGA at a large-scale level, which could be a benefit in the production of high quality and stable SSF products.5. The total number of bacteria and the starting functional strain played an important role in controlling the process of solid-state fermentation and characterize of the fermentation products. SYBR Green real-time PCR and TaqMan real-time PCR were employed to quantify the total number of bacteria and the starting functional strain B. amyloliquefaciens respectively in solid-state fermentation that produce y-PGA. For the development of the methodology based on TaqMan probe, the primer pair pgsB726-f/pgsB791-r and the pgsB-probe were designed from one of the y-PGA synthetase gene (pgsB) of B. amyloliquefaciens. SYBR Green real-time PCR showed the amount of total bacteria reached3.95×10916S rRNA gene copies/g sample after30days, which was about only twice compared with that at the beginning. TaqMan real-time PCR revealed that the number of B. amyloliquefaciens was2.62×106pgsB gene copies/g sample at the beginning of the fermentation, while it increased to2.482×108pgsB gene copies/g sample at the end of the fermentation. B. amyloliquefaciens growed well and became the dominant strain in the fermentation product thus increased the y-PGA production.6. A key property of y-PGA required for practical applications is molecular weight. The ywtD gene, which expresses an enzyme that degrades y-PGA, was cloned from B. amyloliquefaciens Cl-6and ligated to an expression vector pET29a(+) to obtain pET29a-ywtD). The recombinant plasmid was then transformed into the competence cell of E. coli BL21. Histidine-tagged YwtD was induced by1mM IPTG and purified by Ni-chelating affinity chromatography from sonicated cells of the positive transformant. YwtD, which had a molecular weight of46.6kDa detected by SDS-PAGE, was proved to be an endo-hydrolase enzyme and exhibited a remarkable activity in y-PGA degradation at a wide range of temperature (25～45℃) and pH (pH4.0～8.0). The optimal condition for YwtD was at30℃and pH4.0, under which y-PGA with a molecular weight of1,800kDa was degraded to375.2kDa. The activity of YwtD enzyme was found to be inhibited by Zn2+, while Ca2+, Fe3+, Mn2+, Ni2+and Mg2+showed no effects on the activity of YwtD.