中国科学院海洋研究所机构知识库

随着集约化海水养殖业的迅速发展，大量的海水养殖废水直接排入临近海域。海水养殖废水中含有大量的有机物、营养盐、悬浮固体、病原微生物、抗生素、重金属等污染物，加剧了周围水环境的污染负荷，严重影响海水养殖业的可持续发展。因此对海水养殖废水的资源化与无害化处理十分必要。由于海水养殖废水具有高盐度、处理量大，潜在污染物浓度相对较低的特点，增加了其处理难度。因此，寻求低成本、高效能、运行简便、多功能生态化的海水养殖废水处理系统具有重要的经济价值和社会价值。 

本研究通过前期调研不同海水养殖厂不同养殖品种的养殖废水排放情况，了解海水养殖废水的水质特点，并根据海水养殖废水的特点构建复合人工湿地系统（沉淀池—牡蛎过滤池—毕氏海蓬子人工湿地）。微生物是海水养殖废水处理系统的关键部分，包括病原性微生物和功能性微生物。病原性微生物的种类和数量直接影响养殖生物的存活率。

功能性微生物在水处理过程中执行着重要功能，包括有机物降解、氮磷转化、病原菌的拮抗等。因此本研究以微生物为研究对象，包括复合湿地系统对典型病原性微生物—杀鲑气单胞菌的去除效能，及毕氏海蓬子人工湿地的微生物菌群，并进一步探讨复合湿地系统净化污染物的机制，以期为复合湿地系统处理海水养殖废水的推广应用提供理论依据和技术支持。 

本研究的主要成果如下： 

1.  本研究选取天津某水产养殖集中养殖区作为水产养殖污染调查点。调研了半滑舌鳎（Cynoglossus semilaevis）、大菱鲆（Scophthal musmaximus）、欧鲈（Dicentrarchus labrax）、凡纳滨对虾（Penaeus vannamei）、日本对虾（Marsupenaeus Japonicus）养殖水质情况及 6 个海水养殖厂排放水质情况。监测表明，在调研时的养殖情况下，海水养殖废水中总氨氮浓度（TAN）0.27-2.12 mg/L，亚硝酸盐氮（NO₂^–-N）浓度 0.10-0.65 mg/L，化学需氧量（COD）浓度 1.44-10.08 mg/L，总磷酸盐（TP）浓度 0.19-0.90 mg/L，悬浮固体（SS）含量较高，达 12-75 mg/L。不同养殖品种的水质情况差别较大，当单一品种养殖时，海水养殖废水的 TAN 浓度范围为 0.13-9.65 mg/L，NO₂^–-N 浓度范围为 0.01-2.51 mg/L，COD 浓度 0.64 -1.04 mg/L，TP 浓度范围为 0.09-2.36 mg/L；SS 浓度为 4.20-161.00 mg/L。综上，海水养殖废水具有排放量大，单位体积的营养盐、有机物等含量较低，悬浮固体浓度含量高等特点。本调研结果以期为海水养殖废水处理技术提供基础参数，以更好地设计合适的海水养殖废水处理技术，达到良好的处理效果。

2.  本研究构建了由沉淀池—牡蛎过滤池—毕氏海蓬子人工湿地池串联组成的复合湿地系统海水养殖废水处理模式，并在生产条件下，评估复合湿地系统对大西洋鲑典型病原菌—杀鲑气单胞菌（C4）的去除效能，研究结果表明，复合湿地系统可有效去除杀鲑气单胞菌，总去除率可达 64%-99%，其中，牡蛎过滤单元去除率达22%-94%，人工湿地单元去除率达17%-99%。  

3.  为了进一步探讨牡蛎对杀鲑气单胞菌 C4 的净化机制，本研究在实验室条件下，验证牡蛎（牡蛎幼虫和成体）和绿色荧光菌（C4-GFP）的去除机制，并在实验室条件下评估了牡蛎幼虫和成体牡蛎的去除效率和摄食速率。实验结果表明，牡蛎能够滤除水体中的 C4-GFP，其滤除作用与牡蛎的滤食作用有关，C4-GFP 被牡蛎滤食后有两种可能：（1）通过牡蛎的肠道后进入胃等内脏团，在内脏团中被分解、消化；（2）通过牡蛎的肠道后未进入内脏团而是从肛门排出，排出的菌体已失活。牡蛎幼虫对 C4-GFP 的去除率达 88%-95%，摄食速率 6.4×10³-6.2× 10⁵ CFU/h·ind。牡蛎成体对 C4-GFP 的去除率达 79%-92%，摄食速率 2.1 × 10⁴-3.1×10⁶ CFU/h·ind。以上结果为牡蛎在复合人工湿地中的应用，为净化海水养殖废水提供理论基础。 

4.  为了进一步从微生物的角度探讨人工湿地净化污染物的机制，本实验揭示了处理不同总氨氮浓度海水养殖废水的毕氏海蓬子人工湿地中根际和基质微生物菌群信息。实验结果表明，处理海水养殖废水的毕氏海蓬子人工湿地微生物菌群的丰度和多样性较高；在门水平上，共有12个优势菌门（11个细菌门，1个古菌门）：变形菌门（Proteobacteria），厚壁菌门（Firmicutes），蓝藻菌门（Cyanobacteria），拟杆菌门（Bacteroidetes），浮霉菌门（Planctomycetes），酸杆菌门（Acidobacteria），放线菌门（Actinobacteria），疣微菌门（Verrucomicrobia），绿弯菌门（Chloroflexi），WS3，绿菌门（Chlorobi），奇古菌门（Thaumarchaeota）。其中，变形菌门（Proteobacteria）是根际最优势菌门，而蓝藻菌门（Cyanobacteria）是基质最优势菌门。属水平上，湿地系统中存在一些丰度较高的功能菌属：假交替单胞菌属（Pseudoalteromonas），不动杆菌属（Acinetobacter），芽孢杆菌属（Bacillus），假单胞菌属（Pseudomonas），弧菌属（Vibrio），寡养单胞菌属（Stenotrophomonas），丛毛单胞菌属（Comamonas），Nisaea，硝化刺菌属（Nitrospina），亚硝化单胞菌属（genus  of  Nitrosomonadaceae），亚硝化侏儒菌属（Nitrosopumilus），浮霉菌属（Planctomyces）等功能菌属。其中，对 NO₃⁻-N 去除影响最大的是亚硝化侏儒菌属（Nitrosopumilus），对 NO₂⁻-N 去除影响最大的是假交替单胞菌属（Pseudoalteromonas），浮霉菌属（Planctomyces）影响总氨氮的去除和磷酸盐的去除。硝化刺菌属（Nitrospina），不动杆菌属（Acinetobacter），假单胞菌属（Pseudomonas）和弧菌属（Vibrio）能够促进植物的生长。另外，实验中检测到能够拮抗 C4 的芽孢杆菌属（Bacillus）。微生物菌群多样性指数、组成及通过 UPGMA 聚类和 PCoA 进行的群落结构组间差异分析表明：人工湿地中微生物菌群具有空间差异性，根际和基质上的微生物菌群具有明显的差异，水体中的总氨氮浓度对微生物菌群具有一定的影响。 

 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 tank and 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 fom 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 0.27-2.12 mg/L TAN, 0.10-0.65 mg/L NO₂^–-N, 1.44-10.08 mg/L COD, 0.19-0.90 TP, 12-75 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 0.09 to 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 88-95% of C4-GFP was removed by oyster larvae at an ingestion rate of 6.4 × 10³-6.2 × 10⁵ CFU/h·ind, while 79%-92% of C4-GFP was removed by adult oysters at an ingestion rate of 2.1×10⁴-3.1×10⁶ 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.