| In this study, four kinds of bacteria, normal temperature feed-degrading bacteria, low temperature feed-degrading bacteria, normal temperature bacillus bacteria, low temperature bacillus bacteria, were isolated and selected with a series of laboratory experiment methods, respectively, after enriching and culturing the bacterial samples taken from sea cucumber (Apostichopus japonicus Selenka) outdoor ponds. Then we studied optimizing and compounding of high effective strains, specific degrading characteristics of some special strains, all of which were screened from four kinds of bacteria above. The main results are detailed as follows:1. Both the normal and low temperature feed-degrading bacteria were isolated and screened from sea cucumber culturing ponds after the process of enrichment and domestication under the normal temperature of (28±1℃) and low temperature of (15±1℃), respectively. And five normal temperature strains and seven low temperature strains were selected eventually, named CR1, CR5, CR18, CR19, CR24 and DR3, DR4, DR7, DR8, DR11, DR12, DR13 respectively. All of these strains can highly decompose some components of sea cucumber feed, such as organic compounds, ammonia nitrogen, feed amylum and proteins. The studies showed that degrading capability of strains vary from each other in terms of each component of sea cucumber feed. In normal temperature bacteria, CR1 and CR24 are the best degrading strains in terms of organic compounds and feed proteins with high degradation rate of 41.0%-44.5% and 44.3%-54.5% respectively, in five days. As to feed amylum, CR18 and CR19 are the best ones with degradation rate of 49.4%-57.2% in five days. While in terms of ammonia nitrogen, all of the degrading strains behaved badly. In low temperature bacteria, DR7 and DR8 are the best degrading strains in terms of organic compounds with high degradation rate of 37.7%-50.0% in five days. And DR7, DR8 and DR11 are the best ones in terms of ammonia nitrogen with degradation rate of 95.0%-99.8% in five days. All the strains can decompose feed amylum and proteins, but degradation rates are not very high. Based on the physiological and biochemical tests and the 16S rDNA sequence similarity analysis, strain CR1 was identified as Bacillus pumilus, CR5 as Staphylococcus sp., CR18 as Bacillus sp., CR19 and CR24 as Bacillus subtilis, strains DR3, DR7, DR11, DR12 and DR13 were identified as Pseudomonas spp., strain DR4 as Plantibacter sp., and DR8 as Paracoccus sp..2. Both the normal and low temperature bacillus bacteria were isolated and screened from sea cucumber culturing ponds after the process of enrichment and domestication under the normal temperature of (28±1℃) and low temperature of (15±1℃), respectively. And five normal temperature strains and four low temperature strains were selected eventually, named Yll, Y20, Y22, Y24, Y26 and YD11, YD13, YD19, YD20 respectively. All of these strains can highly decompose some components of sea cucumber feed, such as organic compounds, ammonia nitrogen, feed amylum and proteins. The studies showed that degrading capability of strains varies from each other in terms of each component of sea cucumber feed. In normal temperature bacteria, Y20, Y24 and Y26 are the best degrading strains in terms of organic compounds with high degradation rate of 32.9%-42.3% in five days. As to the degrading of feed amylum and proteins, all the strains behaved well, and Y22 and Y26 are the best ones with degradation rate of 66.9%-68.7%,34.4%-49.1% respectively, in five days. While in terms of ammonia nitrogen, all of the degrading strains behaved badly. Comparatively, strains Y24 and Y26 are the best. In low temperature bacteria, YD11 are the best degrading strain in terms of organic compounds and ammonia nitrogen with high degradation rate of 50.0% and 98.0% respectively, in five days. And YD13, YD19 and YD20 are the best ones in terms of feed amylum and proteins with degradation rate of 40.6%-53.2%,29.8%-63.9% respectively, in five days. Based on the physiological and biochemical tests and the 16S rDNA sequence similarity analysis, strains Y11 and Y20 were identified as Bacillus spp., Y22 as Bacillus cereus, Y24 as Bacillus pumilus, Y26 as Bacillus amyloliquefaciens, and strains YD11, YD13, YD19 and YD20 were identified as Bacillus spp..3. Comparative study was investigated about three high effective low temperature feed-degrading bacteria, DR7, DR8 and DR11, by examing their degrading capability of COD and NH3-N in sea cucumber feed. This study contains the growth characteristics of three strains, dynamic process of feed degrading and the different effects of strains optimization on the degrading of the feed. On this basis, further studies were done for the best feed-degrading strain DR8 about its specific degrading characteristics, including its optimal temperature, optimal pH and optimal feed mass concentration for feed degrading. The results showed that these three strains are all very effective in feed degrading of COD and NH3-N, and the degrading dynamic process is significant. Among them, DR8 is the best in feed degrading. When inoculated into the sea cucumber-feed-enrichment-medium by less than 5%o, strain DR8 was able to utilize 54.4% of COD and 99.0% of NH3-N within five days at 15℃. Further degrading studies showed that the appropriate range of temperature and pH value for growth of strain DR8 was 15-30℃and pH 7-10, respectively, while the optimal temperature and pH value conditions for feed degrading was 15-20℃and pH 8.0-8.5, respectively. It could decompose feed mass concentration of 10-20g/L with 56.9%-65.7% of COD degradation rate, and mass concentration of 1-20g/L with 91.7%-99.9% of NH3-N degradation rate, respectively, within 3 days after inoculated by 5%o. Based on the physiological and biochemical tests and the 16S rDNA sequence similarity analysis, strain DR8 was similar to Paracoccus marcusii, with 99.7% sequence similarity.4. This study is about the optimizing and compounding of those four kinds of high effective strains, including normal temperature feed-degrading bacteria, low temperature feed-degrading bacteria, normal temperature bacillus bacteria and low temperature bacillus bacteria. Acording to the effect of bacterial combinations on the degradation of organic compounds and ammonia nitrogen in sea cucumber feed, the best one is selected. The results showed that some combinations were better than the control of the best single bacterium in degrading capability of COD and NH3-N, others were worse. By the process of screening, CR5-CR19 is the best bacterial combination of normal temperature feed-degradng bacteria, with COD degradation rate of 45.0% and NH3-N degradation rate of 70.3% in five days, respectively. For low temperature feed-degradng bacteria, the best bacterial combination is DR4-DR7-DR11, of which COD degradation rate is 49.4% and NH3-N degradation rate is 98.4% respectively, in five days. At the same time, the combination of DR7-DR11 is much effective in degrading of NH3-N as well, with the rate of 96.7%. These both combinations are much better than the control of the single bacterium in degrading ability of COD and NH3-N. In normal temperature bacillus bacteria, Y11-Y26 is the best combination in terms of degradation of COD and NH3-N, while Y11-Y24 is the best one among COD degrading combinations with the rate of 44.0%, which is better than the control. And in low temperature bacillus bacteria, YD11-YD13-YD19 is the best combination in terms of COD degrading with the rate of 75.9% in five days, which is much better than the control. And YD11-YD20 is the best combination of NH3-N degrading with the rate of 91.6% in five days. |
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