Screening And Compounding Of Indigenous Bacillus Of Hainan And Study On The Ability Of Sea-water Purification

Hainan is a major province exporting with aquatic products in China. The coastal water quality is good. And the aquatic products are not only rich in quantity, but also in species. In recent years, the rapidly expanding of the intensive aquiculture has greatly increased organic materials discharging into water. The water quality in aquacultural ponds thus declining, causes an increasing frequency of the diseases and huge economic loss. Currently, the safe, highly efficient and environment-friendly microbial preparation is considered as a significant measure to improve aquacultural environment and to reduce diseases. Bacillus, exhibiting effective water purification, is recognized as a good type of microecological bacterium.. However, due to the special geographical environment of Hainan, the effects of the probiotics from other areas are not always desirable. In this article, two strains with high protease activity and four strains with high lipase activity were isolated and identified from the maricultural systerms of Huiwen town, Wenchang City. These strains are all indigenous Bacillus of Hainan. The optimal formula and the ability of compound strains which constructed with two strains with high protease activity and one strain with high lipase activity to purify the water quality were studied. The results were as follows:1. Water and sediment samples used in this study were collected from maricultural systems of Huiwen town, Wenchang city, and247strains were initially separated from the samples with Bacillus selective medium (50℃,70%oNaCl). Taking two strains separated from a frequently-used microbial preparation as control strains, two strains (WC11011and WC11148) with high protease activity were isolated by skim milk plate method. Their protease activities were significantly higher than those of the control strains (P<0.01). Both strains were identified as Bacillus licheniformis by means of colony morphologies analysis, biochemical identification and16S rDNA molecular identification. Keep the Litopenaeus vannamei in seawater for48hours with a bacteria concentration of1×107CFU/mL. Results of acute attack test showed that both B. licheniformis strains were safe to aquaculture animals. With a inoculum concentration of2×105CFU/mL, both strains could degrade the proteins (5%) in sterilized aquacultural wastewater completely in12hours. As for proteins (5%) in unsterilized aquacultural wastewater, WC11148could degrade most of it in12hours, while complete degradation of the proteins in the same wastewater for WC11148only needed8hours.2. Four strains with high lipase activity were screened from the water and sediment samples by means of enrichment culture, primary screening on lipidic plate, separation of Bacillus, fermenting in shake flask and measurements of lipase activity. The lipase activities of these four strains were14.60U/mL for WC11225,9.75U/mL for WC11023,10.24U/mL for WC11021, and7.66U/mL for WC11041. These strains were identified as Bacillus subtilis by means of colony morphologies analysis, biochemical identification and16S rDNA sequencing Keeping the Litopenaeus vannamei in seawater for48hours with a bacteria concentration of1×107CFU/mL for acute attack test, all the tested strains were safe to the animals. In24hours, with an inoculum concentration of1.8×105CFU/mL, all the strains could completely degrade the olive oil (1.96%) in sterilized aquacultural waste water. As for the olive oil (1.96%) in unsterilized aquacultural wastewater, WC11023and WC11225could degrade it completely, while strains WC11021and WC11041only degrade most of it. As for the olive oil (1.96%) in sterilized aquacultural wastewater with common seawater medium added, WC11021and WC11225could degrade a significant part of it, while WC11023and WC11041just degrade a small percentage of it. As for the olive oil (1.96%) in sterilized aquacultural wastewater, the degradation effects of the four strains were not desirable. Only the supernatants of WC11021and WC11225could just degrade a small percentage of it.3. Using strains WC11011, WC11148and WC11225, the optimal formula of the compound strains was studied by means of single factor experiment and orthogonal experiment. And the degrading rate of COD in artificial formulated wastewater (30%oNaCl) was used for assayed parameter.(1) Results of single factor experiment:In24hours, the three strains showed the largest degradation of COD at16hours when the inoculum concentrations were4×104CFU/mL for WC11011,4×104CFU/mL for WC11148and1.2×104CFU/mL for WC11225. Their degradation rates were87.4%,92.5%and65.8%, respectively.(2) Results of orthogonal experiment of three factors (strains) at three different levels (inoculum concentrations):The optimal formula of the compound strains was3×104CFU/mL (WC11011),4×104CFU/mL (WC11148) and1.2×104CFU/mL (WC11225). And the degradation rate of COD16h in artificial formulated wastewater was96.4%, which was significantly higher than that of strains WC11011(P<0.01), WC11225(P<0.01) and WC11148(P<0.05). Meanwhile, the abilities of excluding NO2-N and NH4-N in artificial formulated wastewater within24hours were studied. The results showed that strains WC11011, WC11148and WC11225could degrade NO2-N and NH4-N significantly, with the degradation rate of32.6%,67.5%and30.3%to NO2-N and the degradation rate of0%,29.1%and35%NH4-N for the three strains in24hours. Degradation rates of N02-N and NH4-N of compound strains were81.8%and62.9%, respectively, which were significantly higher than those of single strain (P<0.01).