Synchronous Use of Hindakia Sp. For Electricity Generation and Dairy Wastewater Treatment

This paper illustrates the potential of microalgae in electricity production in concurrence with wastewater treatment. In order to explore the potential biogenic algal strains, our study focused on the isolation of microalgal strains from various aquatic sources. Cyclic voltammetry was performed to detect the electrogenic activity and out of 18 algal isolates, six algal strains were screened. The cyclic voltammograms of Hindakia sp from the culture collection revealed the well-dened redox peak in contrast to the other algal strains. The electrogenic Hindakia sp. was also analyzed for their potential to remove nutrients in wastewater treatment. A fteen days trial under lab scale race way pond was conducted to evaluate the performance of electrogenic algae. A signicant decrease in N, P, BOD, COD, and TOC was observed. The removal eciency of NH 4 -N, NO 3 -N, P, BOD, COD and TOC were 90.38, 90.24, 66.75, 67.15, 69.44, and 83.51 respectively. Hindakia sp was able to produce 13.79 mg/ml of EPS which paves way to a hydrated biolm matrix that aids in better electrogenicity. To the best of our knowledge, EPS production, electrogenic activity and their utility in waste water treatment are reported for the rst time in Hindakia sp. The results of our study demonstrated the combined benecial traits of microalgae towards electricity production and waste water treatment.


Introduction
Hitherto, the global energy demand is coped up by fossil fuels which are estimated to be exhausted in the ensuing years (1). There is a pressing need for alternative energy sources and the reliability of renewable energy extends their demand around the globe in the ever-expanding energy crisis scenario. Various renewable energy viz., solar, wind, and geothermal are well-rounded and ready to be offered at the commercial level (2). However, the energy generation from biomas products with the growth of plants and crops, algae, organic waste would be economically sound and environmentally safe. In a bio-based economy both macro and micro algae have gained worthwhile interest as a potential feedstock (3). Microalgae are e cient converters of solar energy, therby holds a wide application in mass production for high value products. The photosynthetic productivity of algae on earth is well acknowledged contributing more than 50%. The promising technology for the production of electricity viz algae has been evolved in microbial fuel cells. The metabolic activity of the algae with the potential to transfer electrons extracellularly to interact with conductive material results in the electric current (4). They are effectively used in biological photovoltaics to generate sustainable long-term energy. Various green algae are reported in the exploitation of bio-electrochemical fuel cells such as Chlorella vulgaris, Dunaliella tertiolecta (5), Chlamydomonas, Geobacter(6), Synechocystis (7) etc. The different electron transfer strategies reported in various organisms namely direct electron transfer and mediated electron transfer have been an object of inquisitiveness in microbial bioelectrochemical systems.
Energy production employing wastewater as a resource further assists in ameliorating the burden on other technologies. The electron-producing catalytic process in photosynthetic algae to generate electricity is a promising technology(8). The use of bio-electrochemical systems has set steps to treat wastewater with the support of electro-active microorganisms (9). The photosynthetic machinery endowed with the algae serves as a potential alternate for diverse forms of energy including biohydrogen, biodiesel, bioethanol etc., Bioelectrochemical systems assisting electro-active microbes are gaining interest in the recent days due to their ability to afford the power and to treat wastewater. The recruitment of microalgae in BES offers simultaneous consumption of CO 2 and organic matter production. The environmental challenge has posed numerous threats besides increasing global population and one amongst is wastewater production. The conventional tertiary treatment can be altered with the growth of microalgae for the removal of nitrogen and phosphorus (10). Several studies have described the potential of several species of microalgae including Chlorella, Chlamydomonas, Botryococcus, Chlamydomonas, Phormidum in wastewater treatment (11,12). Various pollution including nitrogen, phosphorus, and other nutrients in the sewage causes concern in the environmental safety (13). The untreated wastewater contains nutrients like nitrogen and phosphorus, whose discharge poses threat to various forms of life.
The treatment of industrial wastewater is crucial in protection against health risk, freshwater demand, eutrophication of lakes etc., Waste water treatment (WWT) assisted with microalgae ensures a key solution for nutrient removal due to their uptake and desolating ammonia (14). The technology of employing algae in WWT is utterly natural and the biomass produced can be reused for extracting products to clinch a circular economy. Many studies have employed dairy waste wastewater for cultivating algae as they could e ciently remove complex organics and high contents of COD, which are distinct characteristics of dairy wastewater .
The main aim of the study is to i) demonstrate the electrogenic potential of microalgae ; ii) investigate the potential of electrogenic microalgae in WWT by nutrient removal.

Sample collection
The algal samples were collected from aquatic regions of various districts. The stock algal strains from the culture collection of the Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore were also used.

Isolation of microalgal cultures
The collected samples were serially diluted from 10 1 to 10 5 times and plated on sterile plates containing BG11 medium. The plates were incubated at an algal growth chamber supplying 16h: 8h of light: dark (1400 l), with a temperature of (28 ± 2ºC) for 7-10 days. Cold uorescent lamps were used for illuminating the growth chamber. The colonies grown were picked and continuously sub-cultured in BG11 agar plate, supplemented with ampicillin (100 µg/ml) and kanamycin (100 µg/ml), cychloheximide (100 µg/ml) ensuring the absence of bacterial and fungal contamination respectively. The puri ed colonies were cultured in BG11 broth and used for further analysis.
Screening for electrogenic activity Cyclic voltammetry (CV) method, under electrochemical work station (AMETEK, scienti c instruments, USA) was performed using three-electrode system, namely glassy carbon electrode (3mm diameter) as a working electrode, a platinum wire and Ag/AgCl (3M KCl) as a counter and reference electrodes respectively. The electron transfer mechanism was analyzed using a working electrode covered with a concentrated microalgal coat. The coated electrode was shade dried and incubated at room temperature (28 ± 2ºC) for two days to induce the interaction between electrode and algae. CV traces were determined in the potential range of -0.8 to + 0.8 V at a scan rate of 10 mVs − 1 . Deoxygenated, deionized water was used as an electrolyte to ensure the absence of oxidation reduction.

Authentication of electrogenic activity
Hindakia sp. was grown in BG 11broth and waste water separately along with stainless steel mesh (anode) and incubated in above-mentioned speci cations of the light chamber. One end of the anode was let outside the rubber cork to measure the electron ow. They were connected to the cathode of the multimeter (MASTECH, MAS830L) and the circuit is closed by connecting its anode to a graphite plate (air cathode) as depicted in Fig. 1. The power density was recorded on alternate days up to the 25th day. The study level current and voltage were calculated using the formula I = V/R, where I is the current, V is the voltage measured and R is external resistance. The power density P was calculated from the measured voltage (P = V 2 /RA). The cathode area in our study used is 64 cm 2 and the resistance provided is 500 Ω .

Estimation of Extracellular polymeric substances (EPS)
The algae at the log phase were harvested on the 20th day and the cells were heated at 45ºC for 20 min. It was then centrifuged at 8000 rpm for 20 min at room temperature. The supernatant was collected and added with three volumes of acetone and stored at − 20ºC for 24h. After 24h, it was spun down at 4 ºC for 20 min. Along the sides of the tube, the precipitated polysaccharides were dried to measure the EPS yield gravimetrically (15).

Microscopic analysis
The screened algal cultures were observed in a light microscope and their morphological characteristics were studied. Further, to analyze the live and dead cells, algal strains were stained with trypan blue(16).

E uent collection
The raw industrial e uents from the processing plant were collected from Aavin Dairy Plant, Coimbatore District Co-operative Milk Producers Union Ltd., Pachapalayam, Perur, Coimbatore (10.9755° N, 76.9153°E ).

Experimental setup
The screened algal strain was inoculated into wastewater at lab-scale reactor. The preliminary trials were carried out with lab scale raceway pond fabricated for the growth of microalgae of 190 L capacity made up of acrylic sheet. It is a transparent plastic material of 5mm thickness. The laboratory setup was fabricated with a length of 1.2m, width 0.41 m and height 0.3 m. The pond was assembled over a steel structure to the height of 0.62 m from the ground level. A paddle wheel of acrylic sheet and xed to the axle. The axle is supported by two bearings in the structure. The gearbox is used for regulating the speed of an electric motor, which in turn maintains the speed of the paddle wheel. The operating speed is maintained in the range of 10 to 40 rpm throughout the study. The movement is transmitted from the motor to the axle by a drive chain. The operational view of constructed lab-scale raceway pond is given in Fig. 2.
The quantity of wastewater was maintained up to 20 cm height throughout the experiment. The experimental setup was maintained under sunlight (360 x 10 2 l). The trial was conducted for 15 days.

E uent treatment with algae
The screened algal isolate was treated with wastewater. The raw wastewater was fed into raceway pond solely without dilution. To 100 l of wastewater, 2% of Hindakia cultures at the optical density of 0.5 at A 680 nm were inoculated.

Statistical analysis
The dataset generated from our study were subjected to one-way ANOVA, in SPSS (Version 20, IBM, N, USA). Statistically signi cant differences between algal treatments were analyzed using Tukey HSD. The graphs were plotted using Origin, 2018.

Light microscopic observation of electrogenic microalgae
The brief notes on the algal isolates are presented in Table 1. The morphology of algal isolates screened with electrogenic activity was observed under the light microscope (Fig. 3). Among the isolates analyzed, Hindakia displayed higher EPS production (13.79 mg/ml) followed by Anabena azollae (9.83 mg/ml) and Chlorella sp. (8.21 mg/ml) (Fig. 4). EPS production of Chlorococcum sp, ULU and MDU were on par ranging from 3.12 to 5.34 mg/ml Electrogenic activity of the algal isolates To assess the electrogenic activity of the algal isolates, CV measurements of the microalgae were carried out on the glassy carbon. Among the 21 isolates, six were found to have electrogenic activity. It could be observed the supernatant of the culture (control) displayed only background current elucidating their electro inactivity of the broth. However, the electrogenic activity was observed in Chlorella sp. Chlorococcum sp. Anabena azollae and unidenti ed isolates of our study (Fig. 5), but distinct redox peak was obtained only in Hindakia sp. over a potential range of -0.8 to 0.4V versus Ag/Ag Cl 2 underpinning their redox activity. Among the screened strains Hindakia displayed the oxidation peak at the potential range of + 100 to + 200 mV under anaerobic conditions, while no distinct peaks were observed in other strains. Cyclic voltammograms studies on Hindakia supernatant, recorded nearly negotiable electrochemical response and in contrast, signi cant response was observed in pellets. Hence following electrochemical analysis, the Hindakia strain was chosen for further investigation.

Proof of electrogenic activity
The stainless steel (SS) mesh in the conical ask holds dimensions of radius and area of 3.5 cm and 38.48 cm 2 respectively. The microscopic view of algae in SS (Fig. 6A) and without algae is given in the Wastewater treatment by electrogenic algae The physical and chemical properties of wastewater taken for the study are illustrated in Table 2. The algal strains were inoculated into the wastewater and routine measurements taken are provided in supplementary Fig. 1. The results of nutrient removal displayed no signi cant difference upon various days. The multiple comparisons between the parameters were performed in Tukey HSD and the results are furnished in Table 3. Algae inoculated with wastewater exhibited a signi cant reduction of nutrients. Hindakia sp removed 90.38, 90.24, 66.75 of NH4-N, NO 3 -N, P during 15 days (Fig. 7). A removal percentage of 67.15, 69.44, and 83.51 were observed for BOD, COD and total organic carbon respectively.  Electrochemical techniques are the key tools for the analysis and interpretation of the electrode reactions occurring in the microalgal fuel cells. Since the constructions of fuel cells are cumbersome, electrochemical analysis at a lab scale is the reliable and bene cial method for preliminary screening (15). The redox activity of the isolates and the stains from the culture collections were evaluated by CV measurements on glassy carbon (GC) electrode, as it is highly conductive (23). The distinct redox peak in Hindakia sp. were observed and might be attributed to the EPS production of cells indicating the viable cells for active transfer of electrons, as depicted in Fig. 8. Similar results were obtained by (15) in Scenedesmus sp. whose enhanced EPS production displayed electrogenic activity witnessing their plausible use in photosynthetic algal microbial fuel cells. (24) demonstrated cathodic reduction for generation of electricity in Scenedesmus obliqus, in which the quantity of available oxygen was claimed to be essential for the reduction peak. In analogy, the redox peak observed might be due to the oxygen produced from the algal strains for electrogenecity.
The photosynthetic activity of microalgae generates electrical current due to the release of electrons from them, which could be employed in biophotovoltaic devices. Studies by (25) reported NADPH oxidase activity in Phaeadactylum tricomutum and Thalassiosira pseudonona responsible for long-term output power. The mediated electron transfer has been reported in Shewanellaceae which exploits melanin, menaquinone, ribo avin and their derivatives as redox shuttles (26, 27). (28) have interpreted the importance of EPS matrix in contact of microorganisms with the electrode. The result of our study correlates the EPS production with electrogenecity, which beholds a corresponding relation. Among the strains in our study, Hindakia sp expressed higher EPS production (Fig. 4), which is selfsame for electrogenecity. The reason could be attributed to the mediator role of EPS for electron transfer, rendering more electrogenic activity to Hindakia sp. Similarly, (29) have demonstrated EPS as a crucial site for binding of c-type cytochromes which are indeed essential for electron transfer to the electrode.

Current density analysis
The assessment of the current density of Hindakia sp. in multimeter con rms their electricity production potential. (30) have investigated the electricity production of Chloricystis sp. using a sensitive current collector in which the translated photochemical signal is obtained. The polarization curves of the anode under the in uence of Hindakia grown in BG11 broth and wastewater are represented in Fig. 3B. The experiment was carried out at 1400 lux and the maximum current recorded grown on BG11 broth and wastewater was 26.4 mW m − 2 and 31.4 mW m − 2 respectively which is signi cantly higher than that of the control. The photosynthetic algal MFC produced a maximum current density of 539 mA/m 2 with at power production of 110 mW/m 2 (31). (32) have revealed the correlation between MFC grown on cathode and their COD degradation potential. In analogy, the culture used in our study had potential to grow on the anode and also COD removal. The results were in analogy to the study conducted by (33) using photo microbial fuel cells of Desmodesmus sp. whose recorded power intensity was 99.09 mW m − 2 at 3000 lux. It could be interpreted as with the increase in light intensity, the power density could be enhanced. The electron ow from algae passes through the anode, gets neutralized on the graphite plate where oxygen from air serves as electron acceptor. The e cient current production of algal isolate on wastewater over BG11 broth con rms its potential in photovoltaics.

Removal of nutrients in wastewater
The results of algal inoculation on wastewater show that Hindakia sp. could able to grow on milk processing waste water. The effect of microalgal treatment nutrients by removal of wastewater with respect to days was measured. Microalgae can assimilate nutrients for its growth from wastewater and convert into biomass constituents. pH value is crucial criterion for the growth of microalgae (34,35). pH increased from 8.6 to 9.5 at the 5th day which gradually decreased and was found signi cant with COD and TOC (Tukey HSD, P < 0.05). Studies by (36) on C. vulgaris treatment in piggery wastewater showed the negative impact upon alkaline pH. Similarly, in our study the initial pH was slightly alkaline, which became neutral upon algal growth. The crucial necessitate of waste water treatment is the removal of BOD as it deteriorates the dissolved oxygen.  (39) in the treatment of Cassava processing wastewater using electrogenic and biomass production potential of Spirulina platensis for COD reduction. Among all the observed measurements, TOC displayed signi cant differences (Tukey HSD, P < 0.05) between all parameters except COD. On the other hand COD is found signi cant except DO and TOC. From Table 3, it can be interpreted as COD and TOC are effectively reduced followed by BOD. As depicted in supplementary Fig. 1, the initial concentration of total nitrogen in the wastewater was 103.6 mg L − 1 and it was decreased upon algal inoculation. The decline in amount of total nitrogen indicates that Hindakia used wastewater as a source of nitrogen for growth. Studies have reported the NH 4 -N removal e ciency of Chlorella sp.in municipal wastewater was 81%. (40). In analogy, Hindakia sp have also utilized NH 3 from 39.3 to 3.75 mg l − 1 contributing to the removal rate of 90.38%. During the test period, phosphates in the wastewater reduced gradually. Fate of P in the bioreactors followed the same trend as that of N with the removal from 8.0 mg L − 1 to 2.66 mg L − 1 . The result was comparable to the removal rate of phosphorus with the study conducted by (41) by Galidieria sulphuraria in primary e uent. The C, N and P removal were higher in waste water upon algal inoculation and the same in uninoculated were lower as depicted in Fig. 7 This in turn can be correlated with higher electrogenecity in wastewater. The study conducted by (42) in dairy wastewater with cultivation of Chlorella vulgaris yielded 0.45 g − 1 l − 1 day − 1 at 4th day of cultivation.. Correspondingly, in our study the biomass productivity gradually increased upon time and reached maximum at 15th day recording 4.61g − 1 l − 1 day − 1 witnessing their probable use in large scae cultivation.

Conclusion
Hindakia sp. displayed a reduction peak in the potential range of -0.4V to -0.2V and oxidation peak in 5-10 in 10 − 6 mA. The result of our study depicts the feasibility of using Hindakia sp. in wastewater sampled from a dairy products processing plant, which effectively removes nutrients and roughly produces 314 mW m − 2 per liter of wastewater. Elimination of nutrients and BOD, COD of the wastewater was evaluated in our study with subsequent electricity production. Their power density production can further be enhanced by optimizing several parameters like light intensity, cell load, selection of anode, etc, in future studies. Additionally, progress in design and operation for algal photovoltaic cell can still augment the electro genesis, which could be exploited in wastewater treatment plants.
Declarations Figure 1 Experimental setup for electrogenic activity con rmation    Removal percentage of nutrients upon inoculation of algae vs uninoculated Figure 8