A recombinant E. coli XL1-Blue (pKSSE5.3) habouring PHA sythase gene (phaC) of Ralstonia eu-tropha H16 and 4-hydroxybutyric acid: CoA transferase gene (orfZ) of Clostridium kluyveri was used to produce poly (4-hydroxybutyric acid), P (4HB), homopoiyester in M9 salts medium with glucose and 4-hydroxybutyric acid as carbon sources. Optimization of medium type and cultivation conditions and fed-batch culture were carried out. The results showed that the final cell dry weight, P (4HB) concentration and P (4HB) content were 13 g/L, 5g/L, and 36%, respectively in a 27L stirred and aerated fermentor after 68 hours of fed-batch culture. A large amount of P (4HB) was isolated by extraction of lyophilized cells with chloroform and make it possible to further examine the material properties and applications of P (4HB) homopoiyester from recombinant E. coli.

Continuous planting of muskmelon and excessive application of chemical fertilizers have caused a series of problems, such as imbalance of the soil micro-ecological environment, serious soil-borne diseases and yield loss. Application of Bacillus subtilis agent is an important way to improve soil micro-ecological environment, prevent soil-borne diseases, and promote plant growth. In this study, B. subtilis was used as experimental agent to analyze the effects of different application methods on the soil microbial diversity and growth of muskmelon in greenhouse. The number of culturable microorganisms in soil was measured by dilution-plate method. The diversity of soil uncultivated microorganisms was determined by Illumina Miseq sequencing technology. The yield of muskmelon was measured by weighing method. The number of culturable bacteria in the root irrigation, hole application and dipping root application groups was higher than that of the control in different muskmelon growth stages, but there was no significant difference among the three different application methods. The number of soil fungi from B. subtilis agent treatment groups in flowering stage was significantly lower in comparison to the control group. However, B. subtilis agent treatment did not cause significant difference on soil fungi number at the fruiting and pulling stage. Diversity analysis of uncultured microorganisms showed that the Shannon index values of bacteria were higher and Simpson index values were lower respectively in the three B. subtilis treatment groups than that in the control. Moreover, the dipping root treatment produced the lowest Shannon index value and the highest Simpson index value of fungi. NMDS and cluster analysis showed that B. subtilis agents dipping root treatment significantly affected the bacterial and fungal flora, both of which were clustered into one independent branch. The application of B. subtilis agents, especially dipping root treatment, significantly decreased the abundance of Bacteroidetes, increased the abundance of Actinobacteria and Acidobacteria. The B. subtilis agent treatment didn't produce significant effect on the diversity of fungal flora except Chytridiomycota. The height, stem diameter and leaf area of muskmelon increased by applying B. subtilis agents, and dipping root treatment produced the most significant effect. As a new type of environmental protection fertilizer, B. subtilis agent can increase the number of soil culturable microorganisms, improve soil microbial diversity, and promote growth and yield. This study would provide a scientific basis for the rational application of B. subtilis.
Bacillus subtilis/genetics* ; Fertilizers ; Fungi ; Soil ; Soil Microbiology