TERTIARY WASTEWATER TREATMENT THROUGH MICROALGAE AND SEPARATION OF ITS VALUE-ADDED PRODUCTS

Abstract

Algae-based wastewater treatment (ABWWT) has been experimented for a few decades as a green technology that procures sustainable renewable energy, lowers greenhouse gas (GHG) emissions, remediates wastewater by reducing nutrients and pathogens and also produces valuable biomass. A few studies have reported the potential of sustainable bioremediation of wastewater (WW) through microalgae and the production of its value-added products on a large scale. In wastewater treatment (WWT), algae can be used at the secondary as well as tertiary treatments for the removal of organic matter, nutrients, pathogens, as well as the production of valuable biomass. Hence, developing ABWWT strategies provides a new opportunity for achieving a profitable wastewater treatment facility. In the present study, microalgal strains were isolated, characterized, and used for carrying out the WWT by varying different factors which affect the treatment efficiency. Therefore, the first objective of the study was to isolate, identify, and characterize the microalgal strains from the wastewater sample procured from locally available waste stabilization pond (WSP), Lakkarghat (Rishikesh). The isolated strains were characterized by using the 18S rDNA sequencing method, and two strains were found to be closely related to Chlorella sorokiniana. The second objective was to optimize the inoculum dose (ID) of isolated microalgal strains (C.sorokiniana, A&B) grown optimally at pH 9 (A) and 7 (B). These strains were used to check their efficacy for nutrients, and organic carbon removal from the tertiary synthetic wastewater using two different photoperiod regimes (PR) [Continuous illumination (CI) and Light/dark (L/D) cycles (12/12h)]. For optimization of ID, four different IDs of strain A (50 mg/L, 100 mg/L, 400 mg/L, and 700 mg/L) were used to check their effect on WWT under CI. Results demonstrated that the increase in the IDs, lead to decrease in hydraulic retention time (HRT) from 6 days to 1 day and highest specific growth rate (d-1) of 0.19 ± 0.007 d-1 was observed in 24 hours for strain A. Experiments revealed variation in the percentage removal i.e. 12-100% of total kjeldahl nitrogen (TKN), 53-96% NO3-N, 59-92% PO4-P, 17-47 % of chemical oxygen demand (COD). Despite the same growth environment, strain A with the ID of 700 mg/L showed the best performance in terms of biomass production, overall organic carbon, and nutrient removal. Both the strains showed luxury phosphorus uptake and were found suitable for advanced WWT. Conventional WWT systems have been designed for reducing BOD, COD, and to some extent, pathogens from the treated wastewater. The results obtained from the first and second objective showed that the isolated algal strains couldn’t reduce COD and BOD as per the required regulatory limits. To further the WWT efficiency of the isolated algal strains, the third objective of this study was designed to improve the COD, BOD removal efficiency of microalgae (strain B) by cultivating it in real tertiary II WW. Two reactors, one with an inoculum of Algae+sludge (A+S) and the other with Algae (A) in WW only, were set up and were subjected to various L/D cycles of 12/12 h, 16/8 h and 24/0 h to understand the effect of various PR on biomass production and nutrient removal in both systems. Control reactors, one with only sludge (S) and other WW blank, were also set up. Results showed that in the A+S reactor due to the presence of sludge, COD removal efficiency was 50-55% more, but nitrogen removal efficiency was 88 % less as compared to the algae system. This is because nitrifiers were found predominant in the A+S reactor and converted ammonia to nitrate, and due to low light intensity, the present algal strain could not uptake nitrate during the study period. Contrary to this, the addition of sludge didn't have any a significant effect on phosphorus removal, as it remained approximately the same in both the reactors (~80%). In the algae reactor, an increase of light duration had no significant effect on nitrogen (N) and phosphorus (P) removal, whereas COD removal was increased by 35%. In the A+S reactor, variation in L/D cycles improved the removal efficiency of COD, BOD, and TKN, but it had no effect on P removal. In terms of algal biomass, the highest biomass productivity (461 mg/L/d) as well as the growth rate (0.49 d-1) were observed in the algae reactor with 24/0 h PR. A chlorophyll based method was developed to differentiate between algae and sludge biomass. The last two objectives of the thesis were to investigate economic benefits in terms of value-added product recovery and pathogen removal from ABWWT. These experiments were parallelly performed with the above experiments. In the fourth objective, strain B was further investigated for its feasibility of removing pathogens present in real tertiary WW. For this, microbial counts of total heterotrophic bacteria, E.coli, Salmonella, Shigella, and total fungus were determined by using colony forming units/mL (CFU/mL) in A reactor and A+S reactor with same culture conditions as mentioned above in third objective. Overall, the population of all the pathogens decreased with time. Algae reactor showed approximately complete removal of E.coli from an initial count of 2.9*106 to 980 CFU/mL after completion of 24/0 h PR and also 5 log unit reduction in heterotrophic plate count (HPC), whereas addition of sludge improved removal of Salmonella, Shigella and Total fungus by 1-1.5 log unit. By comparing all the reactors, the removal rate of HPC and E.coli was solely improved by algae as in the sludge reactor, there is very little change in the removal of these pathogenic strains. Highest removal was observed in elevated photoperiod in both cases. Hence PR made a significant difference in pathogen removal. The fifth and last objective of this study was to investigate the value-added product recovery through algae biomass obtained from ABWWT. For this, the accumulation of proteins and carbohydrates was simultaneously estimated at the end of each experiment. Both the products were estimated through their respective standard methods. Results from the study with synthetic WW demonstrated that III variation of ID had a significant effect on value-added products. By increasing ID (from 50-700 mg/L), the content of proteins and carbohydrates increased from 5% to 18.60% and 9.4 to 38%, respectively. In synthetic WW, Strain A showed the best performance in terms of increase in carbohydrate and protein content by 42% and 13% in L/D, respectively. Light/dark cycle improved the carbohydrate accumulation. Furthermore, a real WW study with strain B showed that the light period had a significant effect on the accumulation of carbohydrates and proteins in both the systems (A and A+S). The highest % content of protein (14.45) was observed in the algae system cultivated in 24/0 h PR, and carbohydrates (6.67) were observed in the A+S system with PR of 16/8 h. Overall, strain B showed better accumulation of proteins cultivated in real WW. In a comparative study, in-spite of having similar culture conditions such as inoculum dose, photoperiod regimes etc., microalgae strain and composition of WW made a significant effect in the variation of accumulation of value-added products. Hence, the research carried out on above-mentioned factors such as different inoculum doses of microalgae, photoperiod regimes and addition of particular sludge ratio emphasizes their significant effect in terms of biomass production, nutrient removal, pathogen removal and value-added product recovery in ABWWT.