Aeromonas Salmonicida Reduction And Microbial Community Characterization In Integrated Constructed Wetlands For Treatment Of Marine Aquaculture Effluents

With the rapid growth of the intensive mariculture industry,one of the main challenges for its sustainable development is to minimize the impact on the environment.Significant amounts of suspended solids,organic matter,inorganic nutrients?nitrogen,phosphorus?,trace elements and pathogenic microorganisms from uneaten feed,faeces and excretory products contained in aquaculture wastewater can damage the adjacent ecosystems.So it is quite necessary to purify the wastewater ecologically.Due to large volumes of wastewater with high salinity and low level in contaminants,it remains a challenge for treatment of effluents from land-based marine aquaculture.Development of low costs,simple operation,small energy requirements,environmental friendliness and efficient aquaculture effluents treatment system is crucial for environmental protection and economic development.This study try to understand the water quality characteristics of aquaculture wastewater through a preliminary investigation.Based on that,we build an integrated constructed wetlands?CWs?consisting of settling pond,oyster?Crassostrea gigas?filter tankand Salicornia Bigelovii CWs to purify mariculture wastewater.Microorganisms including pathogenic microorganisms and functional microorganisms,are key part in the mariculture wastewater treatment system.Pathogenic microorganisms can infect the culture species and influence the survival rate,resulting in disastrous economic losses to the fish farming industry.Functional microorganisms as the key player in the degradation of multiple pollutants i.e.organics fermentation,nutrients?N,P?transformation and pathogen disinfect.Therefore,we conducted the present study from a microorganism perspective and tried to uncover the removal mechanism of contaminants in CWs.The removal ability of an important pathogenic bacteria—Aeromonas salmonicida?C4?were evaluated and the microbial community in the CWs were characterized.The findings of this study could serve as a basis for the potential use of integrated CWs system for treatment of effluents from land-based marine aquaculture and broaden the knowledge of the removal mechanism of contaminants.The main results are summarized as follows:1.In order to understand the water quality characteristics of mariculture wastewater,we made an investigation on a mariculture zone of various species in Tianjin Province.The wastewater quality of Cynoglossus semilaevis,Scophthal musmaximus,Dicentrarchus labrax,Penaeus vannamei,Marsupenaeus Japonicus and effluents from 6 land-based marine aquaculture farm were monitored.Results showed that aquaculture effluents were complicated with 0.27².12 mg/L TAN,0.10⁰.65 mg/L NO₂^–-N,1.44¹0.08 mg/L COD,0.19⁰.90 TP,12⁷5 mg/L SS under current investigation.However,there are a big difference among the wastewater when culture different species,with TAN varying from 0.13 to 9.65 mg/L,NO₂^–-N varying from 0.01 to 2.51 mg/L,COD varying from 0.64 to 11.04 mg/L,TP varying from 0.09to 2.36 mg/L,SS varying from 4.20 to 161.00 mg/L.In conclusion,the mariculture wastewater are low level in contaminants as organic matter and inorganic nutrients?N,P?,but high level in SS.2.Based on the mariculture wastewater quality characteristics,we build an integrated CWs consisting of settling pond,oyster filter tank and Salicornia Bigelovii CWs to purify mariculture wastewater.And we evaluated the removal ability of an important pathogenic bacteria—Aeromonas salmonicida?C4?by the integrated CWs system.As the result showed,the integrated CWs system has the ability to remove C4 from wastewater with a high removal efficiency?64%⁹9%?.The oyster filter tank constributed 22%⁹4%to the removal efficiency and the Salicornia Bigelovii CWs unit contributed 17%⁹9%to the removal efficiency.3.To discuss the possible C4 removal mechanism by oysters,we carried a series of a series of laboratory experiment.Both larvae and adult oysters?C.gigas?were used to remove C4 strain of A.salmonicida with a plasmid containing the green fluorescent protein?GFP?gene?C4-GFP?.Based on current findings,we concluded that one of the mechanisms was largely attributed to the feeding behavior and the ingestion process.There are two possible fates of C4-GFP by oysters:?1?after entering into the gut,part of C4-GFP subsequently reached the visceral mass,and were digested there;?2?after entering into the gut,part of C4-GFP were rejected for entry into the visceral mass and discarded through the anus,exhibiting zoogleal masses inactivated.The removal efficiency and the ingestion rate were also evaluated.Approximately 88⁹5%of C4-GFP was removed by oyster larvae at an ingestion rate of 6.4×10³⁶.2×10⁵ CFU/h·ind,while 79%⁹2%of C4-GFP was removed by adult oysters at an ingestion rate of 2.1×10⁴³.1×10⁶ CFU/h·ind.This research not only provides a theoretical basis,but also a baseline data for the practical application of oysters as a biofilter of aquaculture wastewater.4.Microorganisms play an essential role in the performance of CWs for wastewater treatment.This study aimed to characterize the microbial communities of pilot-scale CWs with Salicornia bigelovii for treatment of mariculture wastewater.Illumina high-throughput sequencing was employed to identify the profile of microbial communities of three CWs receiving wastewater under different total ammonia nitrogen?TAN?concentrations.Results of this study showed remarkable spatial variations in diversity and composition of microbial communities between roots and substrates in the three CWs,with distinct response to different TAN concentrations.A variety of microbial phyla?11 bacteria phyla and 1 archaea phylum?were found in wetland microbial communities,including Proteobacteria,Firmicutes,Cyanobacteria,Bacteroidetes,Planctomycetes,Thaumarchaeota,Acidobacteria,Actinobacteria,Verrucomicrobia,Chloroflexi,WS3,Chlorobi,while Proteobacteria were the most dominated phyla in all the substrate and Cyanobacteria was the most abundant phyla in all root samples.Moreover,redundancy analysis indicated that specific functional genera,such as Acinetobacter,Nisaea,Nitrosopumilus,Comamonas,Bacillus,Pseudomonas,Vibrio,Stenotrophomonas,Pseudoalteromonas and genera of Nitrosomonadaceae.Among them,Nitrosopumilus,Pseudoalteromonas and Planctomyces contributed most to the nitrogen transformation.Planctomyces had an impact on the phosphate removal.Nitrospina,Acinetobacter,Pseudomonas and Vibrio could promote the growth of Salicornia.It is worth noting that Bacillus,a currently known bacterial strain with anti-A.salmonicida properties,was detected abundant in the CWs.From a microorganism perspective,the findings of this study could contribute to better understanding of contaminants removal mechanism and improved management of CWs for treatment of effluents from marine aquaculture.