| Compared with traditional chemical degumming method, the biologic degumming of ramie bast fibers is a kind of method with green and environment-friendly. The biologic degumming shows the advantages of quality improvement of the refined dried-ramie ,without injury fiber and non-pollution. This study involves two complemented research lines, one is microbial culture method, and the other is metagenomics method, which were used to explore the diversity of uncultured microbial communities and obtain novel environmental DNA without any cultivation. The aim of the study is to promote analysis of unknown microorganism from environment and ecological system of ramie natural processing system. Obtaining the effective degumming microbial community was one of key facts to realize energy-saving and pollution-free degumming method. By the traditional microbial culture method, regard the rotten ramie as the research target, 38 microbial strains were obtained, including 21 bacteria, 13 fungi and 4 actinomycetes, all isolated from randomly selected putrid ramie by enrichment with enriched medium. Throngh screening, with a method of transparent zones, obtained 9 microbial strains, which had high and stable degradation ability to gum. The bacteria were indentified as Bacillus, Acinetobacter, Paenibacillus and Burkholderia by morphology, physiological and biochemical reactions, and sequence analysis of 16S rRNA. The degumming effects of the strains indicated that the bacillus subtilis A5 strain isolated from rotten ramie is best, and the residual gum content was only 13.82%.This experiment researched the degumming mechanism of Bacillus subtilis A5 in submerged fermentation by orthogonal experiment methodology and response surface methodology . The study was. undertaken to find optimum conditions of initial pH of the culture medium , cultivation temperature, shaking speed. degumming time and inoculums size for degumming of ramie with Bacillus subtilis A5 by a combined statistical approach of orthogonal design (L16(45)), and response surface methodology. A central composite design was used as an experimental design for the analysis of the allocation of treatment combination. Optimal conditions for minimal residual gum content were determined based on 5 parameters at four different levels. Initially, a screening design methodology was used to evaluate the process variables, which were relevant to residual gum content and the response surfaces applied to find optimal parameters of degumming.Estimated optimum parameters were as follows: pH 8. 5, cultivation temperature 40℃, inoculums size 5%, shaking speed 205rpm and incubation time 96h. under the optimal degumming conditions, As a result, residual gum rate of ramie is 10.68%,decreasing 23.23% than that of non-optimization. Effectively reducing the rate of residual adhesive and improve the degumming effect. The linear density of Bacillus subtilis A5 treated ramie fibers is 6.47dtex, breaking strength is 46.50cN, elongation at break is 5.13%, breaking strength is 7.19cN/dtex, ensuring the high quality of ramie fiber. The bio-degumming treated fibers through dilute alkali treatment, residual gum content reached 1.35%, meet the requirement of the spinning process. The study of Bacillus subtilis A5 Conditions of ramie degumming process provideda theoretical basis for the further study of its biochemical degumming and pure bio-degumming.Evaluation of the diversity of microbial population only rely on microbial incubation system is limited. Metagenomics research method, which extracted directly microbial community genomic DNA from encironmental samples, bypassing the traditional microbiological research methods. Obtained high quality DNA, which is the base of soil microorganisms community structure research. In this experiment, the effectives of six soil DNA extraction methods for isolation of the total microbial DNA from ramie plot were compared. The results showed that the length of DNA was about 23.1kb by all the six methods. The longest length of DNA fragments extracted by IV method as much as 48kb, the highest soil DNA yield was obtained by V method (179.75±0.49μg per gram dry soil), I method is the best one for DNA purity And the III method holded best integrity. The total DNA extracted by all the methods were fit for PCR amplification reaction directly.Ectracted the metagenomic DNA from the ramie garden soil sample, endblunted and ligated with fosmid vector of pCC2FOS. A metagenomic library containing about 5×10~4 clones was constructed. The capacity og foreign DNA cloned in the library was 1.8×10~6 Kb. The library was screened for both xylanase activity and pectinase activity, and 18 positive clones were isolated.
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