| Microalgae,a group of microscopic single-celled algae in the form of individuals,chains or colonies that contain chlorophyll a and perform photosynthesis.Microalgae can proliferate by absorbing and using inorganic C,N and P in the water column under photosynthesis.Therefore,the treatment of aquaculture wastewater by means of microalgae bio-purification has become a hot research topic in recent years.Industrial culture of microalgae can be divided into photoautotrophic culture and heterotrophic culture according to its nutrient supply mode.In most cases,heterotrophic culture of microalgae has the advantages of shorter growth cycle,higher biomass productivity and lower economic cost than photoautotrophic culture.So it has greater application potential and broad industrialization development prospect.In this thesis,we investigated the effect of heterotrophic culture of Chlorella pyrenoidosa as inoculum on the purification of shrimp culture tailwater and the regulation of water quality and bacterial phase in shrimp pond culture.The relevant results could provide guidance for the application of heterotrophic cultivated microalgae in the marine aquaculture.1.Experiment on purification of tail water of shrimp pond culture with heterotrophic cultivated Chlorella pyrenoidosa.By photoautotrophic conversion and salinity acclimation,various initial densities of heterotrophic cultivated Chlorella pyrenoidosa,were inoculated into the tail water.The indexes of phosphate,ammonia nitrogen,nitrate nitrogen and nitrite nitrogen were measured regularly to analyze the absorption and utilization of nutrients.The results showed that under the conditions of light intensity of 6600 Ix,light-dark cycle of 16:8,and temperature of 25±0.5℃,the growth rate of Chlorella pyrenoidosa with three inoculation densities(1.0 × 105 cell/mL,5.0× 105 cell/mL and 1.0 × 106 cell/mL,respectively)increased rapidly within 10 days and slowed down thereafter.At the 20th day,the biomass of algae cells increased 39.25,7.98 and 4.07 times,respectively.Chlorella pyrenoidosa could significantly reduce the concentration of nitrogen and phosphate in the tail water.The phosphate removal rate increased with the growth of algae cells,and the removal rates were 58.82±0.72%,72.86±1.70%and 81.40 ±9.86%at the 21st days,respectively.The utilization of nitrogen salts was followed by ammonia nitrogen,nitric nitrogen and nitrite nitrogen.The removal rates of ammonia nitrogen at the 6th day were 81.88±6.00%,96.24± 1.16%,95.44 ± 1.24%,nitric nitrogen at the 21st day were 82.01 ± 1.35%,93.32 ±4.41%,91.78 ± 2.77%,nitrite nitrogen at the 21st day were 84.33± 3.52%,23.51 ±2.5%,3.36 ± 2.16%,respectively.2.Effects of heterotrophic cultivated Chlorella pyrenoidosa on water quality factors and microflora structure of Litopenaeus vannamei pond culture.Aiming to validate the application of heterotrophic microalgae as an alternative to photoautotrophic microalgae in shrimp culture ponds,the experiment was carried out in two Litopenaeus vannamei outdoor ponds by inoculating heterotrophic cultivated Chlorella pyrenoidosa and photoautotrophic concentrated Chlorella vulgaris.The initiation densities were adjusted of 7.0×105 cell/mL,respectively.The algal solution was replenished once after 15 d of culture with a supplemental algal cell density of 2.0×105 cell/mL.The indexes of algal cell density,pH,nutrient concentration,BOD and COD were measured regularly,and the dynamic change of microflora was tracked by 16S rDNA high-throughput sequencing.As a result,the proliferation rates of Chlorella pyrenoidosa and Chlorella vulgaris exhibited similar patterns of change during the 30 experimental days,both showing a decreasing trend followed by an increasing trend.No significant difference was dected in algal cell densities between the two groups at the end of the experiment.Heterotrophic cultivated Chlorella pyrenoidosa as an inoculum exhibited advantages in maintaining pH,phosphate,ammonia,nitrate and COD of the pond water.In terms of regulating the microbial phase in the inoculating heterotrophic cultivated Chlorella pyrenoidosa pond,the proportion of Proteobacteria and Actinobacteria increased significantly from 28.21%and 10.03%to 40.1%and 33.32%,respectively,and the proportion of Dependentiaea decreased.In the inoculating phototrophic Chlorella vulgaris pond,the proportion of Verrucomicrobiota increased from 6.63%to 28.22%,while the Actinobacteria decreased from 39.51%to 16.23%.Chlorella pyrenoidosa promotes the increase of beneficial bacteria,improves the microbial diversity and richness of the culture pond water body,and better maintains the bacterial community of the pond.The application of heterotrophic cultivated Chlorella pyrenoidosa as a substitute for phototrophic Chlorella vulgaris can achieve the purpose of regulating the water quality index and bacterial phase in mariculture by utilizing its advantage of high yield and low cost.This study provided technical support for the application of heterotrophic microalgae in shrimp pond culture.3.Effects of heterotrophic microalgae replacing photoautotrophic microalgae on growth,digestive enzymes,nonspecific immunoenzyme activities and intestinal flora of Litopenaeus vannamei.A comparative study of the growth,digestive enzymes activitiy,and non-specific immune enzyme activities of Litopenaeus vannamei was conducted by inoculating heterotrophic cultivated Chlorella pyrenoidosa and photoautotrophic concentrated Chlorella vulgaris in two separate shrimp culture pond.The diversity,composition and dynamic changes of intestinal microbial of Litopenaeus vannmei were also analyzed.The results showed that the final body mass of shrimp in the groups inoculating with heterotrophic cultivated Chlorella pyrenoidosa and photoautotrophic Chlorella vulgaris were 9.44 ± 0.30 g and 9.66± 0.47 g,weight gain rate was 157.32± 0.43%and 159.53±0.36%,and specific growth rate was 3.11± 0.01%and 3.15 ± 0.01%,respectively,exhibiting no significant differences.The lipase activity of the shrimp in the Chlorella pyrenoidosa added.group was significantly higher than that of the Chlorella vulgaris added group,while the activity of pepsin was significantly lower.The lysozyme activity was slightly higher than that of the Chlorella vulgaris added group,and there was no significant difference in alkaline phosphatase activity between the two groups.The results of the analysis of the intestinal bacterial composition of shrimp showed that the dominant intestinal bacterial species in both groups were relatively consistent at the phylum,order and genus levels.However,the abundance of the dominant microbial and its variation pattern were slightly different.Therefore,the relevant results exhibited the feasibility of heterotrophic microalgae replacing phototrophic microalgae in shimp pond culture.In summary,this study demonstrates that the application of heterotrophic cultivated Chlorella pyrenoidosa as inoculating algal fluid can achieve efficient nitrogen and phosphorus removal and bacteria phase regulation in shrimp aquaculture.The pilot application provided a guidance for establishing the application process of heterotrophic microalgae products in land-based mariculture ponds and exploring efficient,feasible and low-cost mariculture wastewater treatment technology in the future.Further studies are needed to select suitable microalgae species,establish heterotrophic cultivated methods,optimize delivery strategies,and reveal the mechanism of water quality regulation,so as to provide scientific guidance for better utilization of heterotrophic microalgae in aquaculture to achieve high efficiency and low cost water quality and microbial phase regulation technology. |
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