| Coal will emit sulfur dioxide harmful substances in the combustion process, which have a negative impact on human health, vegetation, metals, construction and ecological environment. Biological desulfurization technology was limited for currently screened strain applying a single desulfurization capacity. L1 could selectively remove inorganic sulfur in coal and D4 could selectively remove organic sulfur. In this paper, L1 and D4 was applied as the starting strain, and genetic technology was used to improve the desulfurization capacity of wild strains. An engineering bacteria was constructed which can remove both organic sulfur and inorganic sulfur in coal. It provided a meaningful reference for the industrial application of biological desulfurization. The main research contents and results include the following aspects:Combined with the ability of the original sulfur removing by strain L1 and inorganic sulfur removing by strain D4, effects on biological leaching of sulfide in raw coal were compared between single strain and multi strains: Total sulfur of coal sample was determined with Elemental analyzer, pyrite sulfur content and sulphate sulphur content in coal were determined by National Standard of The People’s Republic of China(GB/T 215-2003). Scanning electron microscopy(SEM) analysis, Fourier transform infrared(FTIR) analysis, X-ray diffraction(XRD) analysis and thermal gravimetric analysis(TG) analysis were alao used for further analysis. The results showed that desulfurization rate of total sulfur reached 51%, pyrite sulfur desulphurization rate was about 52.1%, and organic sulfur desulphurization rate reached 39.5% after two-step bioleaching. In addition, the two-step microbial desulfurization technology did not reduce the calorific value of coal in the desulfurization process, and coal surface, chemical bonding and crystal showed differences. That is, two-step bioleaching could be one of desirable methods of microbial desulfurization.Using the total DNA of the L1 as a template for PCR amplification, the target gene CysH with the size of 783 bp was cloned. The total DNA of the D4 was used as the template for PCR amplification, target gene of DszA, DszB and DszC were obtained. The sizes of which were 1374 bp, 1110 bp, and 1266 bp, respectively. OE-PCR amplification was used to connect four target genes and gained desulfurization operon DszA-DszB-DszC-CysH. pET-28a(+) as expression vector to construct a recombinant desulfurization operon of pET-DszA-DszB-DszC-CysH(for short pET-Dsz-CysH). The recombinant expression vector was transformed into Escherichia coli BL21(DE3) by electroporation. One complex desulfurization genetic engineering bacteria E. Coli BL21(DE3-pET-Dsz-CysH L-D(for short L-D) was obtained suceessfully.Induced by IPTG, recombinant enzyme was detected by SDS-PAGE. The results showed that obvious specificity protein bands appeared in the area of 49.5KDa, 44.8 KDa, 39 KDa and 29.9KDa, respectively, which consistent with DszA, DszC, DszB and CysH protein size. The enzyme activities of APS reductase enzyme of the recombinant strain were detected, and the optimum temperature and pH were 30 ℃ and 6. In addition, removal activity of DBT by engineering bacteria L-D was cetified. Results showed that the correct expression of the complex desulfurases.Impact factors of reaction temperature, speed, pH, reaction time, particle size, coal slurry concentration and inoculum size of coal were examined to investigat influence on coal desulfurization of engineering bacteria L-D. Considering the actual desulfurization rate and economic benefits, the optimum desulfurization conditions of engineering bacteria L-D was determined as follow, shaking speed of 170 r/min, the culture temperature of 30 ℃, initial medium pH of 6, concentration of coal slurry of 6.0%, desulfurization time of 6 d, inoculation amount of 10%. Under optimum conditions, the total sulfur removal rate of engineering bacteria L-D reached 46%, the organic desulfurization rate reached 38%, the inorganic desulfurization rate reached 62%.One gene engineering desulfurization bacteria of L-D was successfully constructed in this work, desulfurization characteristics, the desulfurization rate and growth characteristics of L-D on raw coal desulfurization was examined, the optimal growth conditions and the desulfurization conditions were obtained. This word provides a useful reference for the industrial production of coal bio desulfurization technology. |
