Preparation Of Probiotics-loaded Silicate Particulates Based On Nano-technology And Its Application Mechanism For Pond Water Quality

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 A₂(PLA₂) 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 6.33×10⁴ per ml and 3.59×10⁶ 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 PLA₂ 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 2.09×10³ cfu mL^-1, whereas that was only 4.37×10² 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.