Using C. vulgaris assisted microbial desalination cell as a green technology in land ﬁ ll leachate pre-treatment: a factor-performance relation study

Algae biocathodes have become one of the most sustainable replacements for abiotic cathodes which were expensive and had toxic chemical oxidant by-products. In this study, a pure culture of Chlorella vulgaris from a photobioreactor was pumped into a photosynthetic microbial desalination cell to treat real land ﬁ ll leachate (had undergone physical treatment) under varying ‘ factor-conditions (FC) ’ to embark on a factor-performance relation (FPR) study. This aimed at determining the relationship between operating factors and to depict the most favourable conditions (and range) in order to boost the overall performance of the reactor/cell. Three groups of FC (A, B and C) were adapted, in that, under FC A external resistance was varied, FC B varied pumping rate and FC C varied temperature, light intensity and dissolved oxygen under conditions ﬂ ow and recirculation mode. Results showed 95% COD removal, a maximum power density of 121.57 mWm (cid:1) 2 (anodic volume) and an average desalination rate of 3.93 mg/L/h. The varying results at different FC showed the signi ﬁ cant impact of operating conditions on performance. Algae biocathodes also proved to be an essential bene ﬁ t in boosting the sustainable application of MDC in wastewater and land ﬁ ll pre-treatment as well as the generation of bioenergy.


INTRODUCTION
A microbial desalination cell is a bio-based technology that has received attention due to its sustainable potential applications such as bioenergy generation, wastewater treatment, water desalination (Luo et al. ), remediation of groundwater (Tong & He ), water softening (Arugula et al. Albeit, the impact some operating factors have on the reactor's performance is more significant than others, it is essential to understand that the presence or absence of one factor (or condition) can influence the impact another factor (or condition) has on the reactor's performance and efficiency ( Jingyu et al. ). In spite of this, remarkable improvement in finding solutions to some of the limitations in this technology, understanding of the relationship between the operating factors and conditions has been mildly explored. In as much many studies have discovered the potential of this technology, it is equally important to comprehend the mechanism, operating conditions and performance in detail. In this research, we investigate pre-treatment of real landfill leachate using a C. vulgaris aided 'photosynthetic' microbial desalination cell (PMDC) under varying factor-conditions (operations conditions) to determine the influence these conditions have on the general performance (power density, desalination rate and COD removal) of the reactor. Our results discuss the influence of inter-membrane distance, membrane surface area and other prominent factors on performance. To the best of our knowledge, this is the first paper to report the use of C. vulgaris biocathode MDC in pre-treatment of real landfill leachate (anolyte) under varying operating condition as a factor-performance relation (FPR) study.  The temperature was kept at an average of 25 C. The culture was then transferred into a 2 L glass bottle after a period of time as the catholyte (photobioreactor). The culture medium was prepared at algae to medium ratio of 1:1 mL (both medium and algae were in liquid form).

Real landfill leachate was sampled from Changchun
Sandao MSW Landfill in Sandao, Erdao District of Changchun City and pre-treated at Key Laboratory of Songliao Aquatic Environment, Changchun, China. The leachate was poured into a 5 L reservoir where it was stirred continuously for 2 h with an automated stirrer. 3 L of the mixture was sieved twice, to remove the solids and particles, into a clear reservoir and allowed to settle for 48 h. The decantate was skimmed to remove the oil and grease floating on the surface. Finally, 1 L of the physically pre-treated leachate was used as the anolyte.

PMDC set-up
The reactor was manufactured with cast acrylic cylindrical tube stock with a 4 cm and 5 cm inner and outer diameter, respectively, in our laboratory. The reactor had a working volume of 150 mL (anode chamber), 50 mL (desalination chamber) and 100 mL (cathode chamber), giving the reactor a volumetric ratio of 6:1:4 (V anode :V saline :V cathode ). Each chamber had two 8 mm diameter holes connected to a 2.5 cm long cylindrical acrylic tube protruding outside the chambers as inlet and outlets. The reactor was thoroughly washed with hot water (about 60 C) and allowed to dry in an incubator at 30 C prior to use. Since the reactor was operated in a continuous mode (with recirculation of electrolytes), there was no need for stoppers at the openings.
The anode and cathode electrodes in the MDC reactor were made from Toray carbon fibre brushes twisted in titanium metalcore (Shenzhen Kang Preston Technology Co. Ltd, China). Anode brushes were 10 cm in length and 4 cm in diameter while the cathode brushes were 8 cm by 4 cm. All electrodes were pretreated as previously described (Wang & Logan 2017). After pretreatment, the electrodes were inserted into the electrode hole drilled in the reactor and sealed with neutral silicone sealant and allowed to harden. All adhesives were completely dried in 24 h.
Between each chamber where two flat gaskets were placed were also membranes: anion exchange membrane (AEM, AMI-7001) (between the anode and the desalination chamber) and cation exchange membrane (CEM, CMI-7000) (between the desalination and cathode chamber).
Both were purchased from Membranes International Inc., USA. Prior to use, both ion exchange membranes were immersed in 5% sodium chloride solution for 24 h and then allowed to dry before installing into the reactor.
Start-up protocol, inoculation, and operation of MDC The whole system was fed by three 2 L reservoir tanks containing anolyte, saline and catholyte (simple photobioreactor) solutions. Leachate used as the anolyte had an initial COD of 2,769 mg/L, conductivity of 432 μs/cm, salinity of 216 mg/L and pH of 4.75, which was adjusted to 8.35 using NaHCO 3 . The cathode chamber was also fed continuously from a 2 L algal-photobioreactor containing 1 L of concentrated algae water and BG 11 media, with a constant light supply at 5,000 Lm intensity (PL-X300D series xenon lamp, China). 500 mL of 15 g/L NaCl was introduced into the desalination chamber. Saline water was recirculated from a 2 L clear glass bottle as a reservoir.
An efficient start-up protocol, as proposed by Borjas et al. (), was adopted in that, prior to inoculation N 2 gas was passed through the anolyte and the anode chamber for 2 h to ensure an anoxic environment. After the reactor was fully assembled, the electrolytes and saline water in the reservoirs were connected to the cell with 8 mm inner diameter tubes. PTFE tape was used at the connecting ends to prevent leakages. Three one-channel peristaltic pumps were used for recirculation. At a flow rate of 70 mL/min, the electrolytes were recirculated to test for leaks and to ensure there were no stagnant zones, and this lasted overnight. Recirculation was stopped as the enriched inoculum of anaerobic microbes was inoculated onto the anode brush and remained overnight without recirculation to enable the microbes to adhere to the anode brush surface.
The recirculation of solutions (at an initial flow rate of 50 mL/min) was resumed for a period of time until a more stable current density was attained. Meanwhile, air was constantly supplied to the photobioreactor at 3 L/min. Then the parameter reading devices were connected placed to commence data recording in an open circuit (no external power supply was connected to increase the cell potential since this was to study MDC's self-sustenance). After 24 h of operation when a stable voltage was attained, the circuit was closed with an external load of 500 Ω using a 0.5 mm diameter titanium wire connected to the electrodes as electron collectors across a resistance box. PMDC with algae biocathode was operated in a continuous mode with varying operation conditions as summarized in Table 1. Operating conditions influencing performance were varied periodically to enhance the monitoring and interpretation of results, also shown in Table 1. Operating conditions were divided into three (A, B, C) groups according to their relations and altered at: factor study A, day 2; B, day 7; and C, day 14. Figure 1 shows the details of the experimental set-up together with the data collection point and flow directions.

Monitoring and analysis of cell performance
The voltage (V, mV) produced across the potential ends of the electrodes was recorded every minute with a VersaSTAT 4 data acquisition system running on a computer software program, Versa studio. The current density (I, mAcm À2 ) and volumetric power density (P, W/m 3 ) were calculated as previously (Logan 2011). Electrical conductivity, salinity, total dissolved solids (TDS), pH and dissolved oxygen measurements were carried out using DZS-706 Multi-Parameter (and pH/Oxi 340i) also running on computer software, REX Instrument Analysis software. The desalination rate (Q D , mg/L/h) was calculated using Equation (1): where C 0 and C t are the initial and the final TDS of saltwater in the desalination chamber and t is the time period (h).
Step test was conducted to determine the performance of the cell by analysing a polarization curve from the data obtained. Using the resistance box, voltage rate of change was recorded over 20 min of each step from 9,000 Ω reducing to 10 Ω, repeatedly. These data helped to determine where voltage or power losses may have occurred and also served as a reference for how much current the cell can generate at a given voltage. By analysing these data, the maximum power possibly generated by the cell was determined as well as the corresponding current at which it was attained. We could also approximately determine the internal resistance of the MDC cell (since maximum power occurs when the external resistance is approximately equal to the internal resistance in a simple resistive circuit). Power density and current density were plotted against current to enhance the above-mentioned analysis. The COD removal efficiency was calculated by the COD difference between the influent and effluent divided by HRT; where HRT is the volume of the reactor (anode chamber) divided by fed rate in a continuous operation.

RESULTS AND DISCUSSION
Performance of the PMDC was analysed in terms of electricity generation (current density, mAcm À2 ; power density, mWm À2 ), desalination or salt removal (desalination rate, mg/h) and COD removal (COD removal and rate). Operating conditions were varied, as shown in Table 1, to enhance the analysis of the relations between factors affecting the performance of the cell in a continuous mode of operation.
FCs influence and changes in electricity generation (voltage) The first couple of data produced by the cell prove the efficiency of the start-up protocol adapted. After the microbes inoculated on the anode were allowed to adhere overnight, the peristaltic pumps were activated allowing monitoring

Polarization characteristics
Polarization curve was measured on the 13th day of continuous operation. Figure 5 shows the electrochemical parameters obtained from the polarization curves, such as the maximum power density (mWm À2 ), the current density obtained at maximum power density (mAm À2 ) and the internal resistance (kΩ). Current density increased sharply from 10 to 100 Ω and then steadily decreased until 700 Ω.
It increased again reaching its maximum value and steadily decrease between 1 kΩ and 9 kΩ. The maximum power den-

FCs influence on desalination or salt removal
The results showed a low desalination rate and efficiency as well as conductivity readings, especially at the initial stage of the experiment (data not shown). This low performance was mainly attributed to back diffusion from the anolyte chamber into the desalination stream. Apparently, the migration of ion through the AEM is mainly driven by concentration gradient which is caused by water transport, molecule transport and Donnan effect (Milner et al. ; Santoro et al. a, b). Nevertheless, in spite the increase in conductivity at the initial days of operation, the results showed a drastic reduction in conductivity under FC B (see Table 1 and

Relationship between operating factors
From the study, a significant relationship can be deduced between the operating factors (and conditions) that were varied during the studies, some of which have been confirmed by other studies. Understanding this relationship between operating factors such as pumping rate, external resistance, temperature, reactor configuration (dimension), etc. and performance (COD removal, desalination and bioenergy generation) will aid significantly in the advancement and application of this technology. Table 2    The external load determines the electric current density (mAcm À2 ) of the system, which is related to the COD removal rate, as well as the anodic and cathodic potential. In addition, desalination rate is related to current density Flow rate Pumping rate HRT. Current density. COD removal Depending on the working volume of the reactor, the flow rate determines how long (in hours) a volume of wastewater and/or saline water will stay in the chamber (reactor) to be treated thus hydraulic retention time (HRT). The higher the HRT (low flow rate), the more effective the reactor and its processes are. Both continuous and batch flow modes have a distinct influence on the performance on the cell but the most significant factor is to increase HRT in order to enhance better mass transport inside the cell With the introduction of biocathodes in MDC, passive algae biocathodes for in situ oxygen generation has become very prominent. The availability of light has significant effects on the growth and performance of the photosynthetic microalgae (biocathode) as well as the concentration of oxygen (by-product).
In the presence of passive algae biocathodes, the source is light is vital for good performance Oxygen Dissolved oxygen DO concentration. Energy generation Among the numerous electron acceptors applied in MDC, oxygen has proved to be a practical terminal electron acceptor due to its high reduction potential, oxygen reduction reaction (ORR) in the presence of transferred electrons and hydrogen ions at the surface of the cathode. The power density generated by the cell as well as desalination process is highly dependent on this phenomenon. Therefore, the higher the concentration of DO, the higher the reactor's performance We also propose some issues and areas that need further investigation in this area of PMDC application: i. The use of saline-tolerant anaerobic microorganisms in the anode chamber.
ii. The significance of pure and mixed cultures both in the bio-anodes and cathodes and their feasibility in real-life application.
iii. The influence of algae on ion exchange membranes.
iv. Standards for MDC reactor configuration with respect to inter-membrane distance, membrane surface area and vertical and horizontal dimensions.
v. Storage of the electricity produced by the cell to monitor its capabilities over time. As the biological driving force of this technology, its tolerance and adaptation to environmental conditions are very crucial. Apart from pH imbalance and temperature fluctuation, which have been solved by recirculation and temperature control measures, the anolyte composition is one of the major factors impeding the growth and activities of microbes and performance in the end. Adapting skilful start-up protocol is one of the most effective solutions. From culturing stage to the start-up process, microbial species can adapt resistance to extreme conditions in order to enhance their performance in the reactor Cell or reactor Reactor configuration Inter membrane distance. The vertical or horizontal length. Membrane surface area. Desalination rate. Power generation It is always a priority to reduce internal resistance in the cell to boost general performance. With reactor configuration, it is important to consider: (1) Intermembrane distance (the distance between the IEMs)can be used to decrease ohmic losses by reducing this distance (depending on the total volume of the reactor).
(2) Vertical versus horizontal length (determines the surface area available for membrane exposure)preferably, reactors should be longer vertically than horizontally. This phenomenon helps to increase the surface area of the membrane as it reduces the intermembrane distance to enhance general performance.
(3) Membrane surface area (surface of the IEMs exposed in the desalination chamber)generally, the desalination process is significantly enhanced when a larger surface of the membrane is exposed to the saline water improving the manuscript. We also appreciate Dr Yuehong Wang for her assistance in SEM analysis. The authors declare that there no conflict of interest.