Materials
The adult mussels (Hyriopsis cumingii) were provided by Zhejiang Qinghu Agricultural Science and Technology Co., Ltd. As shown in Fig. 1a, mussels (400 ± 50 g in wet weight, 14 ± 2.2 cm in shell length, 9 ± 0.5 cm in shell width, 4.3 ± 0.2 cm in shell height) with strong water spray were selected. In order to reduce the external influence on the wastewater treatment experiment, the selected mussels were placed into tap water for 2–3 day after impurities on shell surface was cleaned first.
(a) Adult mussel used in the study, (b) large-scale cultivation of microalgae in a tank.
Chlorella vulgaris which had high photosynthetic efficiency, short breeding cycle, and strong environmental adaptability was chosen in this study. They were obtained from the Institute of Hydrobiology at the Chinese Academy of Sciences, and precultured in bottles containing liquid BG-11 medium. To maintain their growth, Chlorella vulgaris were put in an illumination incubator with 12:12 light: dark cycles. The microalgae cells reached exponential phase on day 7. As shown in Fig. 1b, large-scale Chlorella vulgaris were cultivated in a tank where microalgae biomass concentration was about 10 mg/L.
Bacillus subtilis strain YFFJ-2 and Bacillus licheniformis strain NJ-6 were isolated and screened in our laboratory. Their physiological and biochemical characteristics are shown in Table 1. Bacillus subtilis strain YFFJ-2 were cultivated in sealed glass bottles which contained the following components: 4.0 g/L beef extract, 3.8 g/L peptone, 3.3 g/L NaCl, 4.0 g/L glucose, 3.3 g/L starch, 3.4 g/L KH2PO4, 0.2 g/L MnSO4·7H2O. After 16 h of cultivation, the bacteria of exponential phase were harvested and used in the following experiment. Bacillus licheniformis strain NJ-6 were cultivated in medium which contained 2.0 g/L beef extract, 5.0 g/L peptone, 3.0 g/L yeast powder, 0.005 g/L MnSO4, 2.0 g/L NaCl, 3.0 g/L K2HPO4, 0.02 g/L MgSO4. The biomass was harvested after 48 h of cultivation.
Investigation of mussel breeding density
The influence of mussel breeding density on wastewater treatment performance was investigated. Aquaculture wastewater was collected from a fish-farming area in Hefei city of China. The concentration of total nitrogen (TN), total phosphorus (TP), suspended solid (SS), ammonia nitrogen (NH3-N) and dissolved oxygen (DO) in tested wastewater samples was 3.0, 0.3, 95.0, 2.0, 6.2 mg/L, respectively. The experiments were conducted in several rectangular plastic tanks (2 m in length, 0.5 m in width). 5 cm-thick sediment from fish farming zone was first laid on the bottom of tanks, and 0, 2, 4, and 8 mussels were put in each tank containing 1000 L aquaculture wastewater, separately. Batch experiment was then conducted in outdoor under the natural environment. 50 mL of water sample was taken from each tank per day, and TP and NH3-N concentration were analyzed. At the same time, pH and DO were recorded during the whole process.
Influence of bacteria and microalgae dose on the water quality
After the optional density of mussel was settled, an orthogonal experiment was carried out to explore the effect of bacteria and microalgae dose on wastewater treatment efficiency. Our previous experiments suggested that when the microalgae amount was more than 3 mL, filter-feeding ability of mussel would be inhibited by rapid growth of microalgae, resulting in increase of turbidity and chromaticity in water. So, the volume of microalgae was set around 1–3 mL. When too much Bacillus subtilis and Bacillus licheniformis biomass was added to wastewater, their massive reproduction could consume most of oxygen in water, leading to significant decrease of DO concentration which was bad for the growth of mussels. So, 0.5–2 mL was chosen as the dose for bacteria. Thus, inoculation quantity of bacteria and microalgae on NH3-N removal ability was investigated in this study. Three levels parameters of orthogonal experimental are shown in Table 2.
Wastewater treatment by mussel/microalgae/bacteria in batch experiment
Batch experiment was undertaken to investigate pollutant removal ability by mussels in the presence of bacteria and microalgae, which was named as mussel/microalgae/bacteria ecosystem. The procedure was described as follows. First, several mussels (Hyriopsis cumingii) were put in rectangular plastic tanks containing 1000 L aquaculture wastewater, then certain volume of bacteria (Bacillus subtilis YFFJ-2 and Bacillus licheniformis NJ-6) and microalgae (Chlorella vulgaris) with optimal inoculation quantity were added. The whole experiment was conducted in outdoor under natural environment for 6 days. 20 mL of supernate was taken from the tank at 12:00 PM every day, and residual COD, TP and NH3-N in wastewater were analyzed. In addition, aquaculture wastewater treatment by Hyriopsis cumingii alone (named as mussel system) was conducted as described above. Aquaculture wastewater treatment performance by Hyriopsis cumingii with adding Bacillus subtilis YFFJ-2 and Bacillus licheniformis NJ-6 (named as mussel/bacteria system) was also studied. Moreover, COD, TP and NH3-N removal ability by Hyriopsis cumingii along with Chlorella vulgaris (named as mussel/algae system) was investigated.
After the treatment, three mussels were sampled from each system for measuring the activities of digestive enzymes in their digestive gland and stomach. First, these mussels were opened by a special shell opener, and stomachs were cut out without damage. Then, they were flushed with ice-cold sterilized saline water to remove body fluids and some impurities, and excess saline water was wiped out with filter paper. About 0.3 g of digestive gland tissue samples were centrifuged (12,000 rpm) for 10 min at 4 °C, and the supernatant containing crude enzyme extract was obtained and frozen at − 80 °C. Amylase and pepsin activity were further studied using standard kits according to the manufacturer’s instructions (Jiancheng Bioengineering Institute, Nanjing, China), respectively. The digestive enzyme activities were expressed in standard units (U/mg prot).
Field experiment
Chaohu lake was one of five major lakes in the middle and lower reaches of Yangtze River. There were 35 rivers along the lake, and our study site was Kulv river (2–3 m deep) which was located in Feixi city. The increase of pond-farming and overuse of fertilizers at the upper reaches of Kulv river resulted in nitrogen and phosphorus pollution. Conventional aquaculture model was made of cylindrical nets in which the mussels were placed and then suspended for culture30. Different from this model, an automation pipe network supply feeding system was built in our study. It mainly included central controller, compounding tank, lifting pump, agitation equipment, feeding pipe network and multiple foster boxes (Fig. S1). The central controller was connected with compounding tank, lifting pump and mixing feed device, respectively. Mussels were placed inside the foster boxes (Fig. 2a) that were specifically designed to secure and maintain shellfish for aquaculture grow-out. The foster boxes were 25 m long, 0.15 m wide, and 0.3 m high. They were often set 0.5 m under the water surface through automatic jacking system (Fig. 2b). Under the management of central controller, bacteria solution was added into large-scale microalgae in the compounding tank, and then the formed mixture was transported to the mixing feed station through lifting pump. After mixing the microalgae and bacteria, the prepared ingredients were transported into pipe network. While, the small pipe in the foster boxes was connected with pipe network through a drilled hole on mussel (as shown in Fig. 1a). So, the formed nutrient mixture was directly fed to hyriopsis cumingii in the foster boxes through this automatic pipe network regularly (Fig. 2c), which can also prevent the discharge of nutrient into water. There were about 90 thousand mussels per hectare of water surface, and 120 thousand mussels were farmed in total. The filtering capacity of one mature mussel was about 50 L/day. So, mussels in one hectare could treat about 4500 m3 of aquaculture wastewater every day. The field experiment had run for several months. NH3-N, TN, COD and TP concentration of water samples were monitored every 7 days according to standard methods31. pH and DO were detected at 0.5 m below the water surface using a portable Hach (HQ40D) portable multi meter.
(a) Photos of foster boxes designed for mussels’ cultivation, (b) automatic jacking system connected with foster box, (c) pipe network of feeding for mussels.
Statistical analysis
All the batch and field experiments were carried out in triplicate, and collected data was expressed as mean ± SD. One-way ANOVA was performed with SPSS 17.0 software to determine the significant difference of digestive enzyme activities between different treatment systems. P < 0.05 was set as a significant difference.
Ethical approval
This manuscript is an original work and never has been published elsewhere in any form or language.
