载菌纳米级硅酸盐微粒的构建及其净化养殖水质的效果和机理探讨
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摘要
随着高密度人工水产养殖快速发展，池塘水质不断恶化，直接或间接导致了水体的富营养化、水产病害的频繁发生以及水产品失去固有的鲜味等，这给水产养殖业造成了巨大损失，严重影响了水产养殖业的可持续发展。与此同时，未经处理的养殖废水的任意排放，不仅带来二次污染，而且还威胁到人类自身的健康和生存。鉴于此，本课题针对水产养殖中这一特殊池塘水体的理化特性，以纳米科技为平台，结合微生态理论，开展集吸附、降解和转化为一体的载菌纳米级硅酸盐微粒(PSPN)的构建及其净化养殖水质的机理探讨。
主要包括以下几个方面的内容：(1)采用AODC法定量分析了精养池塘悬浮颗粒附着细菌以及与悬浮颗粒、游离细菌和颗粒有机碳之间的关系，并计数了悬浮颗粒上的附着细菌数，为进一步构建PSPN提供了现实依据和模型；(2)结合国内外净化养殖水质所采用的益生微生物，分离、筛选和鉴定了包括沼泽红假单胞菌(Rp．palustris，编号为HZ0201)、凝结芽孢杆菌(B．coagulans，编号为HZ0202)和啤酒酵母(S．cerevisiae，编号为HZ0203)在内的益生菌株，并对它们的培养和生长特性进行了研究；(3)对筛选的益生菌株进行了复合优化组合和安全性试验研究；(4)制备纳米级硅酸盐附载材料(SCN)，并且以未处理的层状硅酸盐(OLS)为对照对SCN进行了表征，比较研究了SCN的晶体结构、表面特性和吸附性能；(5)针对养殖池塘水体的特殊理化性质，利用制备的SCN和分离筛选的有益微生物，构建了PSPN，并对构建条件进行了优化研究，此外，探讨了SCN与复合有益微生物的作用机理；(6)分离和原代培养了尼罗罗非鱼(Oreochromis nilotica)鳃和肠道上皮细胞，并以其为细胞模型，研究了一定浓度的PSPN对上皮细胞增殖、存活率、通透性、胞浆游离Ca~(2+)和磷脂酶A_2活性的影响；(7)对构建的PSPN净化室内和池塘养殖水质的效果和机理进行了探讨。主要研究结果如下：
1．池塘悬浮颗粒附着细菌的研究结果显示，附着细菌与附菌颗粒、未附菌颗粒和悬浮颗粒之间呈显著正相关，表明了在南美白对虾池塘养殖水体中，附着细菌的数目主要取决于水体中悬浮颗粒的数目，而与颗粒有机碳的含量并没有显著的相关性(P>0.05)。被细菌附着的悬浮颗粒数量占总悬浮颗粒数量的比例约50％，而附着4个细菌以上的悬浮颗粒占总附菌颗粒的比例却少于10％，说明颗粒物质的表面积并非附着细菌的限制因素。南美白对虾养殖池塘水体附着细菌的数量均值为6.33×10~4个/ml，仅占细菌总数的1.76％。但相对于游离细菌，附着细菌具有更大的代谢活力，主要表现在附着细菌对营养物质吸收速率快，而且具有较大的细胞体积。
2．分别分离和筛选了三种益生菌株，通过形态学、理化特性以及核酸特征测定，鉴定为沼泽红假单胞菌菌株HZ0201、凝结芽孢杆菌菌株HZ0202和酵母菌菌株HZ0203。并分别研究了各自的生长和培养特性，结果表明它们对生长条件的要求不苛刻，具有广泛的适应性，适合于大量培养与在水产养殖中应用。
3．菌株的优化组合试验结果表明，HZ0201、HZ0202和HZ0203三种微生物之间均不存在相互拮抗，可以在复合培养基中共同培养且生长具有一定的共性。复合菌株的溶血性试验结果发现血平板上均没有透明圈形成，说明复合菌株没有溶血性。复合菌株对仔虾的安全性试验结果显示，与添加无菌海水的组比较差异并不显著，但与添加致病副溶血弧菌的组比较差异显著(P<0.01)，说明筛选得到的三种菌株的复合培养物对于南美白对虾没有致病性。
4．基于层状硅酸盐特殊的结构特征和纳米属性制备了SCN，X射线衍射谱线表明为典型的钙质硅酸盐。进一步表征结果显示，SCN无论从离子交换容量、BET比表面积、微孔表面积、外表面积还是孔体积、微孔体积上都要优于OLS，具备纳米材料的特性。对复合菌体胞内酶活性的影响结果表明，与对照组相比，SCN的添加对复合菌体胞内酶乳酸脱氢酶、丙氨酸氨基转移酶和天冬氨酸氨基转移酶活性均没有显著的影响。这说明SCN不影响复合菌体细胞壁或细胞膜的完整性，从一定程度上表明SCN对于复合菌体没有毒害作用。
5．针对养殖池塘水体的特殊理化性质，构建了PSPN，并分别研究了SCN附载菌数与SCN重量、复合菌液起始菌数和浓度以及菌液体积的关系，优化了构建条件。此外，通过测定不同粒径的硅酸盐材料，不同的pH值、离子强度和温度条件下对复合菌体悬液的吸附率，探讨SCN与有益复合菌体的作用机理。结果表明，随着硅酸盐粒径的减少，对复合菌体的吸附能力明显增加，吸附率从OLS的22.70％上升到SCN的90.20％，差异显著(P<0.01)。SCN对复合菌体的吸附能力随着pH值的升高而逐渐降低，不同离子强度条件下，呈现相同趋势。不同温度条件下，SCN对复合菌体的吸附率呈现出先升高后降低的趋势，当温度为30℃时吸附率最高为89.74％。这说明复合菌体的吸附不仅依赖于生物体的生物化学属性和表面属性，也受到环境温度等条件的影响。
6．PSPN对尼罗罗非鱼上皮细胞的影响结果显示，与对照组相比，添加适当浓度的PSPN、复合菌株和SCN对细胞增殖、存活率、细胞通透性、胞浆游离Ca~(2+)和磷脂酶A_2活性均有一定影响，但是差异不显著。此外，观察PSPN对尼罗罗非鱼鳃和肠道上皮细胞生长形态的影响，结果没有出现任何异常形态。说明PSPN在一定时间内对尼罗罗非鱼鳃和肠道上皮细胞没有毒害作用。
7．模拟室外池塘养殖试验，对构建的PSPN进行了应用效果和机理研究，结果表明PSPN不仅可以改善鲫鱼养殖水质，还能够提高其生长性能。与对照组相比，添加0.01g/L的PSPN可以显著(P<0.01)降低氨氮、亚硝酸盐氮、COD和硫化物的含量，净化了养殖水质。同时，PSPN添加显著提高了鲫鱼的日增重和相对增重率(P<0.01)。这主要得益于组成其的附载材料和复合有益菌株的共同作用。
8．净化甲鱼养殖池塘水质的研究结果表明，添加PSPN可以显著降低水体中氨氮、亚硝酸盐氮、硝酸盐、硫化物和COD的浓度(P<0.05)，但是对pH值的影响不大(P>0.05)。净化南美白对虾养殖池塘水质的研究结果显示，添加PSPN不仅显著改善了池塘水体的透明度和COD浓度(P<0.05)，可溶性无机氮和可溶性正磷酸磷浓度上升的幅度也显著小于对照组。池塘菌群含量也发生了变化，结果表明添加PSPN的试验组水体中芽孢杆菌、氨化细菌和蛋白质矿化细菌的数量明显高于对照组(P<0.05)。试验组水体中总菌的数量与对照组差异不显著(P>0.05)，但是弧菌的数量(4.37×10~2cfu/ml)要明显低于对照组(2.09×10~3cfu/ml)。此外，添加PSPN的试验组，南美白对虾具有明显的生长优势。净化南美白对虾养殖池塘底质的研究结果表明，PSPN的添加减缓了底质中总氮、总磷、有机碳和硫化物的沉积，缓解了底质恶化的趋势，延缓了底质的老化。此外，与对照组相比，试验组池塘底质中拥有较高数量的芽孢杆菌、氨化细菌、硫氧化细菌和硫还原细菌等益生菌群。而养殖末期弧菌的数量却明显低于对照组(P<0.05)。这些与构建PSPN的SCN和复合菌株在养殖池塘水体中的共同作用是分不开的。
综上所述，PSPN有效地结合了纳米硅酸盐材料和复合微生物的优势，当添加到养殖水体中后，硅酸盐材料和微生物对水体中的污染物质都发生作用，导致水相中污染物质的浓度下降。在这个混合体系中不仅存在着硅酸盐材料对污染物质的吸附和解吸过程，还存在着微生物的降解作用。PSPN的添加净化了养殖水质和提高了养殖动物的生长性能，为水产养殖业的进一步可持续发展提供了新的思路。
Intensive farming of aquatic animals such as fish and shrimp is widely practiced in China. However, intensification results in stressful conditions of ponds water that can cause problems for aquaculture. As a result the ponds eutrophication and aquatic disease often break out and aquatic food products lose intrinsic characters. All of these problems induce enormous economy loss in Chinese aquaculture and make a full impact on the environment-friendly and sustaining aquaculture. Furthermore, unsettled contaminative ponds water could cause the second environmental pollution. However with the increasing concern over the potential harm of aquaculture effluents on receiving water bodies, worries over the contamination of aquatic food products with bioaccumulative and potentially harmful chemicals and antibiotics, and human health risks associated with contaminative aquatic products, the research and application of probiotics and other purificants are progressively increasing in aquaculture of the world. Thus the research of preparation of probiotics-loaded silicate particulates based on nano-technology(PSPN) and its application mechanism for pond water quality was conducted according to the physical and chemical characters of particular ponds water in aquaculture.
In the present study, the numbers of suspended particles, particles attached by bacteria, epibacteria and total plate count were counted by the acridine orange direct count method(AODC method) and the concentration of particulate organic carbon(POC) was measured. Furthermore, the correlation among the particles, epibacteria and POC was analyzed. Thus it provided the true model and basis for the further research of designing PSPN. Three strains including Rp. palustris(No. HZO201), B. coagulans(No. HZ0202) and S. cerevisiae(No. HZ0203) were isolated and characterized as a potential component for the purpose of probiotics. The taxonomic identification and other physiological and biochemical tests and culture characters were also made. These probiotics were optimized for the sake of obtaining the mixture microorganisms and proved to be safe to the aquatic animals. Then the silicate carrier nano-material(SCN) was prepared by original layer silicate(OLS) and measured. The crystal structure, surface characters and absorption capability were also studied comparing with the OLS. PSPN was prepared by SCN and mixture microorganisms and optimized the condition of preparation. Furthermore, the mechanism between SCN and mixture microorganisms was discussed in this study. Gill and intestinal epithelial cells of tilapias, Oreochromis nilotica, were isolated and cultivated by primary culture of cells as the cells model to research the effect of PSPN on the epithelia viability, epithelia livability, epithelia permeability, cytosolic free calcium and membranous phospholipase A_2(PLA_2) activity of tilapias. At last, the application effect and mechanism of PSPN for pond water quality was conducted and discussed in aquaculture. The results were as follows.
1. The bacteria attached to suspended particles, epibacteria, in northern white shrimp, Penaeus vannamei, ponds were studied. In this study, the numbers of suspended particles(SP), particles attached by bacteria(BP), epibacteria(EB) and total plate count(TPC) were counted and the concentration of particulate organic carbon(POC) was measured. Furthermore, the correlation among the particles, epibacteria and POC was analyzed. There were highly significantly lower(P<0.01) counts of BP, EB and TPC of the first culture year ponds in all the phase. However, there was not significantly different(P>0.01) for POC between the four shrimp ponds. In this study, the number of particles which attached by 1, 2, 3 and 4 bacteria was 46%, 22%, 14% and 9% of the total particles attached by bacteria. However, the percent of the number of particles attach by more than 4 bacteria was less than 10%. There was a remarkable correlation between the number of epibacteria and that of particles attached by bacteria(P<0.01). The number of epibacteria was also correlated with the number of suspended particles(P<0.01) and the total number of bacteria(TPC)(P<0.01), but was not correlated with POC(P>0.05). The EB and TPC of four ponds were 6.33×10~4 per ml and 3.59×10~6 per ml, respectively, which the percent of EB was only 1.76% to TPC. However, the epibacteria had higher metabolic activity comparing with the unattached bacteria and showed higher incorporation rates and bigger cells cubage.
2. Three probiotics strains were isolated and identified as Rp. palustris(No. HZ0201), B. coagulans(No. HZ0202) and S. cerevisiae(No. HZ0203) by taxonomic identification, the Gram staining test and other physiological and biochemical tests, respectively. Furthermore, their growth characters and culture optimization were also studied and the results showed that these strains possessed widely accommodation ability and could culture at poor substrate to use in aquaculture.
3. The results of experiment compounding and optimizing the probiotics strains indicated that there was no antagonism among three strains including HZ0201, HZ0202 and HZ0203 using cross-streaking method. The mixture microorganisms could be cultured in the same mixture substrate and grow well. Haemolytic test of mixture microorganisms proved that the strains had no haemolytic character. The trials treated with the mixture microorganisms showed higher survival percent than that treated with vibrio pathogen, V. parahaemolyticus,(ATCC 27519)(P<0.01) in the security experiment of larvae shrimp, Penaeus vannamei. However, there was no significant between the trail treated with probiotics and the control treated with sterile seawater. The results showed that both the probiotics strains and the culture substrates had no pathogenicity to shrimp.
4. SCN was prepared based on the special structure characters of original layer silicate and nanoattribute and proved to be typical Ca-silicate through using the X-ray diffraction(XRD). The further research results indicated that SCN was better than OLS in cation exchange capacity, BET surface area, micropore surface area, external surface area, pore volume and micropore volume and was provided with nanoattribute. The effect of SCN on endoenzyme activity of mixture strains was also studied and there was no significant(P>0.01) in the activity of lactate dehydrogenase(LDH), alanine aminotransferase(ALT) and aspartate aminotransferase(AST) comparing with the control treated without adding SCN. These results showed that SCN did not affect the integrality of cells membrane and cells wall and was safe to the mixture strains from a certain extent.
5. PSPN was prepared according to the special physical and chemical characters of ponds water in aquaculture. The relationship between the amount of adsorption microorganisms and weight of SCN, the initial concentration of microorganisms and the volume of mixture strains was researched to optimize the preparation condition. Furthermore, the application mechanism between SCN and mixture strains was discussed through measuring the absorption percent of SCN to mixture strains during the different conditions including the various size of silicate, pH values, concentration of ionic and temperature. The adsorption ability increased significantly(P<0.01) with the decrease of silicate size. The rate of OLS was only 22.70%, but that of SCN was up to 90.20%. The adsorption ability decreased with the increasing pH values and presented the same trend in different ionic concentration. At different temperature, the adsorption rate of SCN to mixture strains increased firstly and then decreased and the highest adsorption percent was up to 89.74% at 30℃. This result indicated that the strains adsorption depended on not only the biochemical and surface characters, but also the environmental condition such as temperature.
6. The gill and intestinal epithelial cells of tilapias, Oreochromis nilotica, were cultured primarily as model to study the effect of different concentration PSPN, mixture strains and SCN on the epithelia viability, epithelia livability, epithelia permeability, cytosolic free calcium and membranous PLA_2 activity of tilapias, but there was no significance comparing with the control. Furthermore, the effect of PSPN on growth morphological variety of gill and intestinal epithelial cells of tilapias was also observed and the result did not showed any morphological abnormity. This indicated that PSPN was harmless to the growth of epithelial cells of tilapias for a period of determinate time.
7. The effect of prepared PSPN on the application and mechanism was studied in indoor aquaria simulated as the ponds culture and showed that PSPN improved the water quality of crucian carp culture and increased the growth characters. The trial treated with PSPN(0.01g/L) could significantly(P<0.01) decrease the concentration of ammonia nitrogen, nitrite, COD and sulfide comparing with the control and resulted in improving the water quality. At the same time, daily weight gain(DWG) and relative gain rate(RGR) were also increased significantly(P<0.01) through adding the PSPN. The remarkable results were profited from the combined action between the carrier material and mixture strains of probiotics in this study.
8. The concentration of ammonia nitrogen, nitrite, nitrate, COD and sulfide was decreased significantly(P<0.05) in turtle ponds treated with PSPN comparing with the control. However, there was no significance(P>0.05) in pH values. The effectiveness on water quality, population density of bacteria and shrimp production in ponds treated with commercial probiotics was tested in Penaeus vannamei ponds. Results showed that the PSPN could improve the population density of beneficial bacterial flora, reduce concentrations of nitrogen and phosphorus and increase yields of shrimp. The use of PSPN significantly increased(P<0.05) transparency and reduced(P<0.05) dissolved reactive phosphorus, total inorganic nitrogen and COD. The average counts of Bacillus sp., ammonifying bacteria and protein mineralizing bacteria were found to be significantly higher in treated ponds compared to control(P<0.05). In control ponds, an increase in presumptive vibrios was observed and the average density was up to 2.09×10~3 cfu mL~(-1), whereas that was only 4.37×10~2 cfu mL~(-1) in treated ponds(P<0.05). This indicates that the addition of PSPN had a noticeable influence on water quality of shrimp ponds and shrimp production. Commercial shrimp, Penaeus vannamei ponds receiving PSPN were monitored for changes in total nitrogen, total phosphate, organic carbon, sulfide and bacterial populations in ponds sediment. Results showed that PSPN could improve the bottom quality, decrease the nutrients(nitrogen, phosphate, carbon and sulphur) accumulation and mend the composing of bacteria populations in ponds sediment. In our study, final average presumptive vibrios of sediment in treated ponds adding PSPN was significantly(P<0.05) lower than the control ponds, perhaps indicating the inhibiting effects of PSPN on pathogenic bacteria. The average number of Bacillus sp., ammonifying bacteria and presumptive sulphur oxidizing bacteria in treated ponds sediment was also generally higher(P<0.05) than that of the control ponds. These results attributed to the synergism exhibited by the mixture strains associated with the SCN in the ponds water.
In a word, PSPN combined effectively the predominance of nano-silicate material and mixture microorganisms and showed preferable results in ponds water and growth of aquatic animals when it was used in aquaculture. Both SCN and mixture strains adsorbed and assimilated the pollution substances to improve the water quality and decrease the concentration of contamination in the ponds water. The use of PSPN in aquaculture provided a new developing road for the environment-friendly and sustaining aquaculture.
主要包括以下几个方面的内容：(1)采用AODC法定量分析了精养池塘悬浮颗粒附着细菌以及与悬浮颗粒、游离细菌和颗粒有机碳之间的关系，并计数了悬浮颗粒上的附着细菌数，为进一步构建PSPN提供了现实依据和模型；(2)结合国内外净化养殖水质所采用的益生微生物，分离、筛选和鉴定了包括沼泽红假单胞菌(Rp．palustris，编号为HZ0201)、凝结芽孢杆菌(B．coagulans，编号为HZ0202)和啤酒酵母(S．cerevisiae，编号为HZ0203)在内的益生菌株，并对它们的培养和生长特性进行了研究；(3)对筛选的益生菌株进行了复合优化组合和安全性试验研究；(4)制备纳米级硅酸盐附载材料(SCN)，并且以未处理的层状硅酸盐(OLS)为对照对SCN进行了表征，比较研究了SCN的晶体结构、表面特性和吸附性能；(5)针对养殖池塘水体的特殊理化性质，利用制备的SCN和分离筛选的有益微生物，构建了PSPN，并对构建条件进行了优化研究，此外，探讨了SCN与复合有益微生物的作用机理；(6)分离和原代培养了尼罗罗非鱼(Oreochromis nilotica)鳃和肠道上皮细胞，并以其为细胞模型，研究了一定浓度的PSPN对上皮细胞增殖、存活率、通透性、胞浆游离Ca~(2+)和磷脂酶A_2活性的影响；(7)对构建的PSPN净化室内和池塘养殖水质的效果和机理进行了探讨。主要研究结果如下：
1．池塘悬浮颗粒附着细菌的研究结果显示，附着细菌与附菌颗粒、未附菌颗粒和悬浮颗粒之间呈显著正相关，表明了在南美白对虾池塘养殖水体中，附着细菌的数目主要取决于水体中悬浮颗粒的数目，而与颗粒有机碳的含量并没有显著的相关性(P>0.05)。被细菌附着的悬浮颗粒数量占总悬浮颗粒数量的比例约50％，而附着4个细菌以上的悬浮颗粒占总附菌颗粒的比例却少于10％，说明颗粒物质的表面积并非附着细菌的限制因素。南美白对虾养殖池塘水体附着细菌的数量均值为6.33×10~4个/ml，仅占细菌总数的1.76％。但相对于游离细菌，附着细菌具有更大的代谢活力，主要表现在附着细菌对营养物质吸收速率快，而且具有较大的细胞体积。
2．分别分离和筛选了三种益生菌株，通过形态学、理化特性以及核酸特征测定，鉴定为沼泽红假单胞菌菌株HZ0201、凝结芽孢杆菌菌株HZ0202和酵母菌菌株HZ0203。并分别研究了各自的生长和培养特性，结果表明它们对生长条件的要求不苛刻，具有广泛的适应性，适合于大量培养与在水产养殖中应用。
3．菌株的优化组合试验结果表明，HZ0201、HZ0202和HZ0203三种微生物之间均不存在相互拮抗，可以在复合培养基中共同培养且生长具有一定的共性。复合菌株的溶血性试验结果发现血平板上均没有透明圈形成，说明复合菌株没有溶血性。复合菌株对仔虾的安全性试验结果显示，与添加无菌海水的组比较差异并不显著，但与添加致病副溶血弧菌的组比较差异显著(P<0.01)，说明筛选得到的三种菌株的复合培养物对于南美白对虾没有致病性。
4．基于层状硅酸盐特殊的结构特征和纳米属性制备了SCN，X射线衍射谱线表明为典型的钙质硅酸盐。进一步表征结果显示，SCN无论从离子交换容量、BET比表面积、微孔表面积、外表面积还是孔体积、微孔体积上都要优于OLS，具备纳米材料的特性。对复合菌体胞内酶活性的影响结果表明，与对照组相比，SCN的添加对复合菌体胞内酶乳酸脱氢酶、丙氨酸氨基转移酶和天冬氨酸氨基转移酶活性均没有显著的影响。这说明SCN不影响复合菌体细胞壁或细胞膜的完整性，从一定程度上表明SCN对于复合菌体没有毒害作用。
5．针对养殖池塘水体的特殊理化性质，构建了PSPN，并分别研究了SCN附载菌数与SCN重量、复合菌液起始菌数和浓度以及菌液体积的关系，优化了构建条件。此外，通过测定不同粒径的硅酸盐材料，不同的pH值、离子强度和温度条件下对复合菌体悬液的吸附率，探讨SCN与有益复合菌体的作用机理。结果表明，随着硅酸盐粒径的减少，对复合菌体的吸附能力明显增加，吸附率从OLS的22.70％上升到SCN的90.20％，差异显著(P<0.01)。SCN对复合菌体的吸附能力随着pH值的升高而逐渐降低，不同离子强度条件下，呈现相同趋势。不同温度条件下，SCN对复合菌体的吸附率呈现出先升高后降低的趋势，当温度为30℃时吸附率最高为89.74％。这说明复合菌体的吸附不仅依赖于生物体的生物化学属性和表面属性，也受到环境温度等条件的影响。
6．PSPN对尼罗罗非鱼上皮细胞的影响结果显示，与对照组相比，添加适当浓度的PSPN、复合菌株和SCN对细胞增殖、存活率、细胞通透性、胞浆游离Ca~(2+)和磷脂酶A_2活性均有一定影响，但是差异不显著。此外，观察PSPN对尼罗罗非鱼鳃和肠道上皮细胞生长形态的影响，结果没有出现任何异常形态。说明PSPN在一定时间内对尼罗罗非鱼鳃和肠道上皮细胞没有毒害作用。
7．模拟室外池塘养殖试验，对构建的PSPN进行了应用效果和机理研究，结果表明PSPN不仅可以改善鲫鱼养殖水质，还能够提高其生长性能。与对照组相比，添加0.01g/L的PSPN可以显著(P<0.01)降低氨氮、亚硝酸盐氮、COD和硫化物的含量，净化了养殖水质。同时，PSPN添加显著提高了鲫鱼的日增重和相对增重率(P<0.01)。这主要得益于组成其的附载材料和复合有益菌株的共同作用。
8．净化甲鱼养殖池塘水质的研究结果表明，添加PSPN可以显著降低水体中氨氮、亚硝酸盐氮、硝酸盐、硫化物和COD的浓度(P<0.05)，但是对pH值的影响不大(P>0.05)。净化南美白对虾养殖池塘水质的研究结果显示，添加PSPN不仅显著改善了池塘水体的透明度和COD浓度(P<0.05)，可溶性无机氮和可溶性正磷酸磷浓度上升的幅度也显著小于对照组。池塘菌群含量也发生了变化，结果表明添加PSPN的试验组水体中芽孢杆菌、氨化细菌和蛋白质矿化细菌的数量明显高于对照组(P<0.05)。试验组水体中总菌的数量与对照组差异不显著(P>0.05)，但是弧菌的数量(4.37×10~2cfu/ml)要明显低于对照组(2.09×10~3cfu/ml)。此外，添加PSPN的试验组，南美白对虾具有明显的生长优势。净化南美白对虾养殖池塘底质的研究结果表明，PSPN的添加减缓了底质中总氮、总磷、有机碳和硫化物的沉积，缓解了底质恶化的趋势，延缓了底质的老化。此外，与对照组相比，试验组池塘底质中拥有较高数量的芽孢杆菌、氨化细菌、硫氧化细菌和硫还原细菌等益生菌群。而养殖末期弧菌的数量却明显低于对照组(P<0.05)。这些与构建PSPN的SCN和复合菌株在养殖池塘水体中的共同作用是分不开的。
综上所述，PSPN有效地结合了纳米硅酸盐材料和复合微生物的优势，当添加到养殖水体中后，硅酸盐材料和微生物对水体中的污染物质都发生作用，导致水相中污染物质的浓度下降。在这个混合体系中不仅存在着硅酸盐材料对污染物质的吸附和解吸过程，还存在着微生物的降解作用。PSPN的添加净化了养殖水质和提高了养殖动物的生长性能，为水产养殖业的进一步可持续发展提供了新的思路。
Intensive farming of aquatic animals such as fish and shrimp is widely practiced in China. However, intensification results in stressful conditions of ponds water that can cause problems for aquaculture. As a result the ponds eutrophication and aquatic disease often break out and aquatic food products lose intrinsic characters. All of these problems induce enormous economy loss in Chinese aquaculture and make a full impact on the environment-friendly and sustaining aquaculture. Furthermore, unsettled contaminative ponds water could cause the second environmental pollution. However with the increasing concern over the potential harm of aquaculture effluents on receiving water bodies, worries over the contamination of aquatic food products with bioaccumulative and potentially harmful chemicals and antibiotics, and human health risks associated with contaminative aquatic products, the research and application of probiotics and other purificants are progressively increasing in aquaculture of the world. Thus the research of preparation of probiotics-loaded silicate particulates based on nano-technology(PSPN) and its application mechanism for pond water quality was conducted according to the physical and chemical characters of particular ponds water in aquaculture.
In the present study, the numbers of suspended particles, particles attached by bacteria, epibacteria and total plate count were counted by the acridine orange direct count method(AODC method) and the concentration of particulate organic carbon(POC) was measured. Furthermore, the correlation among the particles, epibacteria and POC was analyzed. Thus it provided the true model and basis for the further research of designing PSPN. Three strains including Rp. palustris(No. HZO201), B. coagulans(No. HZ0202) and S. cerevisiae(No. HZ0203) were isolated and characterized as a potential component for the purpose of probiotics. The taxonomic identification and other physiological and biochemical tests and culture characters were also made. These probiotics were optimized for the sake of obtaining the mixture microorganisms and proved to be safe to the aquatic animals. Then the silicate carrier nano-material(SCN) was prepared by original layer silicate(OLS) and measured. The crystal structure, surface characters and absorption capability were also studied comparing with the OLS. PSPN was prepared by SCN and mixture microorganisms and optimized the condition of preparation. Furthermore, the mechanism between SCN and mixture microorganisms was discussed in this study. Gill and intestinal epithelial cells of tilapias, Oreochromis nilotica, were isolated and cultivated by primary culture of cells as the cells model to research the effect of PSPN on the epithelia viability, epithelia livability, epithelia permeability, cytosolic free calcium and membranous phospholipase A_2(PLA_2) activity of tilapias. At last, the application effect and mechanism of PSPN for pond water quality was conducted and discussed in aquaculture. The results were as follows.
1. The bacteria attached to suspended particles, epibacteria, in northern white shrimp, Penaeus vannamei, ponds were studied. In this study, the numbers of suspended particles(SP), particles attached by bacteria(BP), epibacteria(EB) and total plate count(TPC) were counted and the concentration of particulate organic carbon(POC) was measured. Furthermore, the correlation among the particles, epibacteria and POC was analyzed. There were highly significantly lower(P<0.01) counts of BP, EB and TPC of the first culture year ponds in all the phase. However, there was not significantly different(P>0.01) for POC between the four shrimp ponds. In this study, the number of particles which attached by 1, 2, 3 and 4 bacteria was 46%, 22%, 14% and 9% of the total particles attached by bacteria. However, the percent of the number of particles attach by more than 4 bacteria was less than 10%. There was a remarkable correlation between the number of epibacteria and that of particles attached by bacteria(P<0.01). The number of epibacteria was also correlated with the number of suspended particles(P<0.01) and the total number of bacteria(TPC)(P<0.01), but was not correlated with POC(P>0.05). The EB and TPC of four ponds were 6.33×10~4 per ml and 3.59×10~6 per ml, respectively, which the percent of EB was only 1.76% to TPC. However, the epibacteria had higher metabolic activity comparing with the unattached bacteria and showed higher incorporation rates and bigger cells cubage.
2. Three probiotics strains were isolated and identified as Rp. palustris(No. HZ0201), B. coagulans(No. HZ0202) and S. cerevisiae(No. HZ0203) by taxonomic identification, the Gram staining test and other physiological and biochemical tests, respectively. Furthermore, their growth characters and culture optimization were also studied and the results showed that these strains possessed widely accommodation ability and could culture at poor substrate to use in aquaculture.
3. The results of experiment compounding and optimizing the probiotics strains indicated that there was no antagonism among three strains including HZ0201, HZ0202 and HZ0203 using cross-streaking method. The mixture microorganisms could be cultured in the same mixture substrate and grow well. Haemolytic test of mixture microorganisms proved that the strains had no haemolytic character. The trials treated with the mixture microorganisms showed higher survival percent than that treated with vibrio pathogen, V. parahaemolyticus,(ATCC 27519)(P<0.01) in the security experiment of larvae shrimp, Penaeus vannamei. However, there was no significant between the trail treated with probiotics and the control treated with sterile seawater. The results showed that both the probiotics strains and the culture substrates had no pathogenicity to shrimp.
4. SCN was prepared based on the special structure characters of original layer silicate and nanoattribute and proved to be typical Ca-silicate through using the X-ray diffraction(XRD). The further research results indicated that SCN was better than OLS in cation exchange capacity, BET surface area, micropore surface area, external surface area, pore volume and micropore volume and was provided with nanoattribute. The effect of SCN on endoenzyme activity of mixture strains was also studied and there was no significant(P>0.01) in the activity of lactate dehydrogenase(LDH), alanine aminotransferase(ALT) and aspartate aminotransferase(AST) comparing with the control treated without adding SCN. These results showed that SCN did not affect the integrality of cells membrane and cells wall and was safe to the mixture strains from a certain extent.
5. PSPN was prepared according to the special physical and chemical characters of ponds water in aquaculture. The relationship between the amount of adsorption microorganisms and weight of SCN, the initial concentration of microorganisms and the volume of mixture strains was researched to optimize the preparation condition. Furthermore, the application mechanism between SCN and mixture strains was discussed through measuring the absorption percent of SCN to mixture strains during the different conditions including the various size of silicate, pH values, concentration of ionic and temperature. The adsorption ability increased significantly(P<0.01) with the decrease of silicate size. The rate of OLS was only 22.70%, but that of SCN was up to 90.20%. The adsorption ability decreased with the increasing pH values and presented the same trend in different ionic concentration. At different temperature, the adsorption rate of SCN to mixture strains increased firstly and then decreased and the highest adsorption percent was up to 89.74% at 30℃. This result indicated that the strains adsorption depended on not only the biochemical and surface characters, but also the environmental condition such as temperature.
6. The gill and intestinal epithelial cells of tilapias, Oreochromis nilotica, were cultured primarily as model to study the effect of different concentration PSPN, mixture strains and SCN on the epithelia viability, epithelia livability, epithelia permeability, cytosolic free calcium and membranous PLA_2 activity of tilapias, but there was no significance comparing with the control. Furthermore, the effect of PSPN on growth morphological variety of gill and intestinal epithelial cells of tilapias was also observed and the result did not showed any morphological abnormity. This indicated that PSPN was harmless to the growth of epithelial cells of tilapias for a period of determinate time.
7. The effect of prepared PSPN on the application and mechanism was studied in indoor aquaria simulated as the ponds culture and showed that PSPN improved the water quality of crucian carp culture and increased the growth characters. The trial treated with PSPN(0.01g/L) could significantly(P<0.01) decrease the concentration of ammonia nitrogen, nitrite, COD and sulfide comparing with the control and resulted in improving the water quality. At the same time, daily weight gain(DWG) and relative gain rate(RGR) were also increased significantly(P<0.01) through adding the PSPN. The remarkable results were profited from the combined action between the carrier material and mixture strains of probiotics in this study.
8. The concentration of ammonia nitrogen, nitrite, nitrate, COD and sulfide was decreased significantly(P<0.05) in turtle ponds treated with PSPN comparing with the control. However, there was no significance(P>0.05) in pH values. The effectiveness on water quality, population density of bacteria and shrimp production in ponds treated with commercial probiotics was tested in Penaeus vannamei ponds. Results showed that the PSPN could improve the population density of beneficial bacterial flora, reduce concentrations of nitrogen and phosphorus and increase yields of shrimp. The use of PSPN significantly increased(P<0.05) transparency and reduced(P<0.05) dissolved reactive phosphorus, total inorganic nitrogen and COD. The average counts of Bacillus sp., ammonifying bacteria and protein mineralizing bacteria were found to be significantly higher in treated ponds compared to control(P<0.05). In control ponds, an increase in presumptive vibrios was observed and the average density was up to 2.09×10~3 cfu mL~(-1), whereas that was only 4.37×10~2 cfu mL~(-1) in treated ponds(P<0.05). This indicates that the addition of PSPN had a noticeable influence on water quality of shrimp ponds and shrimp production. Commercial shrimp, Penaeus vannamei ponds receiving PSPN were monitored for changes in total nitrogen, total phosphate, organic carbon, sulfide and bacterial populations in ponds sediment. Results showed that PSPN could improve the bottom quality, decrease the nutrients(nitrogen, phosphate, carbon and sulphur) accumulation and mend the composing of bacteria populations in ponds sediment. In our study, final average presumptive vibrios of sediment in treated ponds adding PSPN was significantly(P<0.05) lower than the control ponds, perhaps indicating the inhibiting effects of PSPN on pathogenic bacteria. The average number of Bacillus sp., ammonifying bacteria and presumptive sulphur oxidizing bacteria in treated ponds sediment was also generally higher(P<0.05) than that of the control ponds. These results attributed to the synergism exhibited by the mixture strains associated with the SCN in the ponds water.
In a word, PSPN combined effectively the predominance of nano-silicate material and mixture microorganisms and showed preferable results in ponds water and growth of aquatic animals when it was used in aquaculture. Both SCN and mixture strains adsorbed and assimilated the pollution substances to improve the water quality and decrease the concentration of contamination in the ponds water. The use of PSPN in aquaculture provided a new developing road for the environment-friendly and sustaining aquaculture.
引文
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