Recirculating Aquaculture System Strategy

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Chapter 3 – Materials and Methods

MFC Setup

The designed MFC was constructed as a tubular reactor (30 cm long and 5 cm in diameter). The anodic chamber was built by a rolled-up anion exchange membrane (AEM) with a volume of 220 mL. Carbon brush was inserted in the anodic chamber as the electrode. The carbon brush was soaked in acetone solution overnight, and then heated at 450 ℃ for 30-min before putting into the anode chamber (Wang et al. 2009, Li et al. 2015). The AEM was also acted as a separator between anode and cathode. The carbon cloth was used as cathode electrode, which was coated with carbon powder with loading rate of 0.2 mg of C per cm2. The cathode cloth was wrapped and covered the anode membrane tube. The tube was put in a cylindrical container, which was the cathode component, and left a volume of 0.8 L for the cathode chamber. The air was pumped into the cathode chamber at the bottom of container. One gas bag was installed at the top of anode for collecting the gas that generated from denitrification process. Titanium wire was used to connect anode and cathode to the external resistor.

Operating Conditions

The anode was inoculated with anaerobic sludge from Christiansburg Wastewater Treatment Plant (Christiansburg, VA, USA), and was operated under room temperature at 20 °C. A synthetic solution was feed into the anode to mimic wastewater, which contains: 1g glucose, NH4Cl 0.15 g; NaCl 0.5 g; MgSO4 0.015 g; CaCl2 0.02 g; NaHCO3 1g, and 1 mL trace elements per liter of tap water. The anolyte was operated as the continuous flow mode with a hydraulic retention time (HRT) of 24-hour or 0.15 mL per minute with a pump. Aquaculture water tested in this study was obtained from recirculating aquaculture systems at the Virginia Tech Department of Food Science and Technology, and was applied under the batch mode in the cathode chamber. In order to have a relatively constant water quality, the culture water sample was collected for the study at one time.
To better mimic the RAS system with a constant nitrate accumulation rate of 50 mg L-1 day-1 NO3-N, 10 ml concentrated NaNO3 solution (24.28 g L-1) was added to the cathode chamber with a syringe. Meanwhile, 10 ml water sample was taking out of the system for analyzing each day to maintain a constant cathodic volume (0.8 L).
For the situation that nitrification tank is replaced by MFC, with a constant ammonium generation rate of 50mg L-1 day-1 NH4+-N, 10 ml concentrated NH4HCO3 solution (22.57 g L-1) was added to the cathode chamber with a syringe. Meanwhile, 10 ml effluent sample was taking out of the system for analyzing each day to maintain a constant cathodic volume. The samples from both chambers were collected periodically with 24-hour interval.

Data Measurements and Analysis

A voltage across an external resistor was recorded every 5-minutes by a digital multimeter for the MFC reactor. The current was calculated based on Ohm’s Law. The pH value for the cathode was monitored by a pH controller. The pH for the anode and the conductivity for both chambers were measured by a benchtop pH meter and a benchtop conductivity meter, respectively. The concentration of chemical oxygen demand (COD) was tested by Hach COD Test’n’Tube. The ammonium, nitrate, and nitrite was measured by corresponding Hach powder methods. The Ion Chromatography was also used to test whether other anions or cations were generated from the system. The monitored anions including chloride, sulfate, Phosphate; whereas the cations including sodium, calcium, potassium, and magnesium (Appendix C, and Appendix D).
The coulombic efficiency was calculated based on the equation below (Yang et al. 2016):
The estimated energy consumption was analyzed based on the pumping system. The pumping system for the continuous flow of anolyte and the aeration for the cathode were calculated based on the equation below (Li et al. 2015, Liu et al 2016):
Where P is the power requirement in kW, Q is the flowrate (m3 s-1), γ is 9800 (N m-3), and E is the head loss per m of H2O.

Results and Discussion

MFC for Denitrification in RAS

Feasibility Test

The BES has a potential to enhance nitrate removal for the RAS. To test this hypothesis, the triplicate tests of different resistors was conducted. Based on Ohm’s Law, the current is inverse proportional to the resistor under a constant voltage. The voltage value was recorded across 3.3 M Ohm (very little current generation) and 1 Ohm resistor (relatively high current generation) by the digital multimeter. Both cathode and anode were operated under batch mode. The cathode was injected with 0.8 L aquaculture water with the composition in Table 1.
Due to the RAS has a relatively mature technique for COD removal which resulted a low COD concentration in the aquaculture recirculating water, the COD removal for aquaculture water was not considered in this study.
With a same 24-hour operation, the nitrate was first transferred from cathode chamber to anode chamber by concentration gradient. However, it was showed from Figure 6 that with 1-Ohm resistor, a higher electricity was produced which drove more nitrate to the anode for denitrification. With 1-Ohm resistor, the residual nitrate was 4.00 ±0.88 mg L-1 NO3-N, whereas for 3.3-M Ohm resistor, the residual nitrate was 22 ±1.13 mg L-1 NO3-N. This indicated that more nitrate was removed under a higher electricity generation during a same period, and gave a better nitrate removal efficiency in the system.
To enable a better understanding of the removal performance and potential of the MFC under RAS, the different nitrate concentrations were also tested. Although fish does not have the sensitive response for nitrate concentration in the aquaculture system, the nitrate concentration was kept around 50 mg L-1 NO3-N in most of the time. However, due to the massive fish production, the aquaculture water was also found to have the nitrate concentration up to 200 mg L-1 NO3-N (Sandu et al 2013). Two kinds of aquaculture water with different nitrate concentration were obtained from VT Department of Food Science, one with the 51.8 ±0.2 mg L-1 NO3-N, the other was 210.50 ±16.48 mg L-1 NO3-N. With the knowledge of electricity generation can drive more nitrate from cathode to anode, 1-Ohm external resistor was applied for MFC. Both cathode and anode were operated under batch mode with 3 cycles testing. To provide an enough carbon source (3000 mg L-1 COD) and recirculating for the system, the anode was built for 220 mL internal volume plus a 200 mL reservoir outside of the reactor. The cathode was injected with 0.8 L aquaculture water with different nitrate concentration.
From Figure 7, although the MFC remove more amount of nitrate aquaculture water with 200 mg L-1 NO3-N under a same period of operation than that of 50 mg L-1 NO3-N, the residual of nitrate was still a concern. The 50 mg L-1 NO3-N brought a lower nitrate residual and a higher removal efficiency.

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Electricity Generation with MFC for Denitrification

With the feasibility study, the MFC exhibited a better performance under 1-Ohm resistor and 50 mg L-1 NO3-N. The anode chamber with 220 ml volume was fed with synthetic wastewater (960 mg L-1 COD) under continuous mode. 0.8 L aquaculture water was pumped into and remained in the cathode chamber for 15 days. 10 ml NaNO3 was injected into the system for supporting a 50 mg L-1 NO3-N in cathodic aquaculture water with a syringe every day. The electricity was generated in the system as showed in Figure 8 below, which brought a daily average current density of 19.87 ± 7.26 mA m-3. The current kept decreasing over the operational time with the accumulation of nitrate in the system. After 15 days of adding dose, the current returned to its initial value from day-16 also indicated denitrification gradually dominated the activity in the anode chamber with the limited amount of carbon source.

Removal of Nitrates and Organics with MFC for Denitrification

The removal efficiency for both nitrate and organics are the key elements for testing the system’s performance. With 24-hour HRT of the anode or an anodic recirculation rate of 0.15 mL/min, the anode removed 876.06 ±7.59 mg L-1 COD, resulting in 91.26% COD removal efficiency. The immigration of nitrate in cathode was continuously moving to the anode to achieve a simultaneous nitrate removal through heterotrophic denitrification. With the daily nitrate accumulation rate of 50 mg L-1 NO3-N, the water should be replaced in every day. However, with the MFC installed in the system, it would consume much less fresh water due to the less accumulation of the nitrate via the denitrification process. This indicated the MFC can slow down the fresh water replacement frequency (Figure 9), plus the generated energy can further shorten the energy consumption. The other ions and cations concentrations which tested by Ion Chromatography were showed in Appendix C and Appendix D.

MFC for Nitrification and Denitrification in RAS

The BES can improve nitrate removal based on the installed nitrification process in RAS. However, MFC may also has a potential to achieve ammonia removal for the RAS with combined processes of nitrification and denitrification. To test this hypothesis, the biofilm was cultured on the carbon cloth for the nitrification. Aquaculture water with a same water quality and chemical composition showed in Table 1 was applied in this section.
The anode chamber with 220 ml volume was fed with synthetic wastewater (960 mg L-1 COD) under continuous mode. 0.8 L aquaculture water was pumped into and remained in the cathode chamber for 20 days. 10 ml concentrated NH4HCO3 was injected into the system for supporting a 50 mg L-1 NH4+-N generation in cathodic aquaculture water with a syringe every day. With a same 24-hour operation, the ammonia was first converted to nitrate in cathode chamber, then the nitrate moved to the anode for the denitrification. The pH would drop down during the nitrification process in cathode chamber. In order to best mimic the RAS system, NH4HCO3 was chose to provide the ammonia while adjusting the pH and supporting an appropriate alkalinity level in the water. pH and conductivity were monitored on a daily basis, and were presented in Figure 11 and Figure 12, respectively. The cathode maintained a pH of 7.78 ± 0.11, and anode of 6.78 ± 0.29. The conductivity was gradually increasing in the cathode due to the ammonia accumulation, whereas the conductivity of anode was more depending on the nitrogen removal performance of the system.

Electricity Generation and Organics Removal with MFC for Nitrification and Denitrification

The daily averaged current density generated from the system were presented in Figure 13. With the combination of nitrification and denitrification processes, the current density was jumping between 49.36 and 88.70 A m-3, which gave a higher electricity production than that of only denitrification system. A possible reason for this may be the biofilm attached on the carbon cloth enhanced electron transfer. Figure 14 also indicated that electricity generation had a same pattern with coulombic efficiency. During 20 days of operation, the MFC removed 826.43 mg LCOD in average, a COD removal of 86.09% ± 9.83, and a coulombic efficiency of 58.86 ± 8.76%. Some of the carbon source were oxidized for denitrification without involving in the electricity generation would be an appropriate reason to explain the high COD consumption and a low Coulombic efficiency in the system.
The distribution of COD consumption was illustrated in Figure 15. The denitrification and electricity generation were considered as the major parts to consume carbon source, whereas the other factors were assumed as system loss. From the analysis, the electricity generation used almost 59% of the COD, denitrification consumed 23% of the COD, and the system loss took 18% of the consumed COD in average.
The consumed COD was calculated based on the equation below (Strohm et al 2007):
5 C6H12O6 + 24 NO3 + 24 H+ à 30 CO2 + 12 N2 + 42 H2O

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Nitrogen Compounds removal with MFC for Nitrification and Denitrification

The total inorganic nitrogen balance was conducted as showed in Figure 16 under the steady state of the system with the assumption of 0 organic nitrogen compounds generation or decay. Due to the system complexity, the analysis simply tested and calculated the chemicals in and out of the MFC on a daily basis.
As illustrated in Figure 17, The ammonium was the only nitrogen source entered into the system or the cathode chamber. However, the effluent had the combination of ammonium, nitrate, and nitrite due to the system limitations on nitrification and denitrification. From Figure 18, the total nitrogen removal efficiency in the system was achieved at 38.72 ±4.99%, with a system removal rates of 0.02 kg N m-3 d-1. The ammonia residual in cathode chamber was 5.10 ± 0.6 mg L-1 NH4+-N and would cause health issue to the aquaculture products. This concern can be further investigated by changing the MFC configurations, scales, HRT, and through the fresh water replacement.

Energy Consumption with MFC for Nitrification and Denitrification

Energy consumption is estimated with the consideration of feeding anode chamber continuously, and pumping air for nitrification aerating in cathode chamber. The aerating took 8.82×10-4 kWh m-3, whereas feeding anode used 2.67×10-6 kWh m-3 and brought a total energy consumption of 8.85 ×10-4 kWh m-3. Further energy analysis is needed to conduct based on the treated water volume and the possible fish production in the system.


The developed MFC presented a potential to be applied in RAS with a much lower energy consumption than the conventional nitrification and denitrification processes. Further energy generation and consumption are needed to investigate with the consideration of fish production.
Although there are several advantages to insert MFC into RAS, the biofilms attaching on the MFC may also bring a concern of health issues for the fish or other aquaculture product. Hence, the water quality under inserting MFC are needed to further investigate with the real RAS system.
The ammonium level was not as low as expected, thus replacing the system with certain amount of water is an alternative for applying MFC under RAS. Manipulating he biofilm coverage, the pH, and the aeration of oxygen can be tested for further improving the nitrification performance. Looking further for the accurate DO and pH control can also be conducted in the future.
The ways to increase capacity of BES system are also encouraged. MFC is one of the form of bio-electrochemical, the other configurations of BES may provide an even better performance for RAS.
In this study, a synthetic wastewater was supplied as the carbon source for the denitrification. Although the system showed a desirable COD removal performance, other anions in wastewater may also migrate across the membrane into the RAS system (i.e. cathode chamber), which may bring a further health concern for the living fish. Inserting a digester for digesting the organic compounds and use the digested wastes to feed MFC from the system is worth to consider.
Further mathematical modelling can be performed to better evaluate the performance of MFC based on different nitrogen-compounds level, the thickness and coverage area of biofilm, and the configurations.
Finally, scaling-up is always the challenge for applying MFC in the real industry. An appropriate strategy for system scaling-up is needed to investigate from laboratory scale into a practical technology in the future study.


In conclusion, the study illustrated the MFC with an anion exchange membrane has the potential to apply under RAS facility, which can achieve nitrogen removal for the system. The current design approached the ammonium and nitrate removal from RAS, and organic carbon removal from synthetic wastewater. With the nitrification process integrated in MFC, a prompt energy generation with a low energy consumption was found, which also indicated a potential for bio-cathode applied into the RAS MFC study. Further study regarding the health concerns, improving the water quality, alternating carbon sources, are needed for applying and scaling-up MFC under RAS into the real practice in the future.

Table of Contents
General Audience Abstract 
Table of Contents 
List of Figures
List of Tables 
Chapter 1 – Introduction 
Chapter 2 – Literature Review
2.1 Introduction to Recirculating Aquaculture System Strategy
2.2 Introduction to Bioelectrochemical Systems (BES) and Microbial Fuel Cells (MFC)
2.3 BES and MFC Applied in Aquaculture Systems
Chapter 3 – Materials and Methods
3.1 MFC Setup
3.2 Operating Conditions
3.3 Data Measurements and Analysis
Chapter 4 – Results and Discussion 
4.1 MFC for Denitrification in RAS
4.2 MFC for Nitrification and Denitrification in RAS
Chapter 5 – Perspectives
Chapter 6 – Conclusions 
Nitrogen Removal from Closed Aquaculture System by Bio-electrochemical System

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