Factors that affect the removal of nitrogen and phosphorous from piggery wastewater using microalgae –bacteria consortium - Ngo Van Chien

Vietnam Journal of Science and Technology 55 (4C) (2017) 198-203 FACTORS THAT AFFECT THE REMOVAL OF NITROGEN AND PHOSPHOROUS FROM PIGGERY WASTEWATER USING MICROALGAE –BACTERIA CONSORTIUM Ngo Van Chien 1 , Vu Ngoc Thuy 1,* , Miyashita Hideaki 2 , Doan Thi Thai Yen 1 1 Department of Environmental Engineering; School of Environmental Science and Technology (INEST) - Ha Noi University of Science and Technology,1 Dai Co Viet, Ha Noi, Viet Nam 2 Graduate School of Global and Environmental Studies (GSGES) and Graduate School of Human and Environmental Studies (GSGHS) at Kyoto University, Kyoto 606-8501, JAPAN * Email: thuy.vungoc@hust.edu.vn Received: 30 June 2017, Accepted for publication: 15 October 2017 ABSTRACT Currently, most piggery farms in Viet Nam use biogas tank as a primary wastewater treatment system. However, even after the anaerobic treatment process, the concentration of nutrients was still high and exceeded the national effluent standard. If the wastewater was directly discharged into the water bodies, it was certainly hazardous to the environment and causes eutrophication. To remove the residual nutrients, several processes have been used, mostly chemical processes. The disadvantages of those chemical processes are high cost and the increase in amount of sludge. In this study, the biological method using microalgae-bacteria consortium was applied for the removal of nitrogen and phosphorous from wastewater from biogas treatment process. This study focused on factors which affect nitrogen and phosphorous removal efficiency in a batch reactor using microalgae-bacterial consortium. Studying on Chlorella sp A8. Activated sludge co-cultivation at different ratio (Chl/AS) gave good results in hydraulic retention time of 20 to 24 hours when C/N in wastewater was 4/1(m/m). In the fully mixing condition, DO range from 0.3 - 0.5 mg O2/L, the highest removal efficiencies of COD was 71 - 76 %; NH4 + -N was 40 - 47 % and PO4 3- -P was 64 - 77 % at Chl /As was 1.5:1, 2.5:1 and 1:1( g/g). Keywords: microalgae-bacterial consortium, nitrogen and phosphorous removal, Piggery wastewater treatment. 1. INTRODUCTION In Vietnam, the dumping of untreated or unproperly treated wastewater have been caused serious environmental problems. This is due to the high expenses for construction and operation costs of wastewater treatment systems. Furthermore, some conventional treatment methods still can not meet the discharge standard due to the complex composition of the raw wastewater. Wastes from pig farm are usually treated by conventional anearobic digestion, namely “biogas tank”. The effluent from biogas tanks still contain large amount of nitrogen and phosphorus Development of nitrogen and phosphorous removal from piggery wastewater 199 compounds, which must be further removed with natural treatments, such as stalibization ponds or long oxidation ditches, that required large area. In the other methods, the activated sludge process can be applied for the effluents from anaerobic treatment, but has raised high aeration costs and resulted in the production of large amounts of activated sludge [1]. In recent years, microalgae-bacteria-based biotechnology has received more and more attention as an alternative method of conventional multi-step wastewater treatment processes, especially for wastewater containing high concentration of nitrogen and phosphorous compounds [2, 3]. Microalgae are eukaryotic microscopic aquatic plants that carry out photosynthesis that is the same process and mechanism with higher plants in converting sunlight, H2O and CO2 into biomass and O2. Algae provide an efficient way to consume nutrients and provide oxygen needed for the growth of aerobic bacteria. By using microalgae-bacteria- based biotechnology, the treatment cost can be reduced. Oxygen generated by microalgae can be an alternative source of oxygen instead of mechanical aeration. It might eliminate the costs of aerobic treatment in daytime. Moreover, microalgal biomass instead of activated sludge is produced, which is valuable as a renewable resource for a wide range of applications (e.g. biofuel, agricultural fertilizers or animal feeds) [4 - 6]. Main purpose of this study is to clarify the factors which affect the nitrogen and phosphorous removal efficiency by microalgae– bacteria consortium growing in piggery wastewater after anaerobic treatment. 2. MATERIALS AND METHODS 2.1. Microorganisms and cultivation Microalgae strain used in this study was Chlorella sp. A8, which was isolated from a pond in a Son Tay pig farm and characterized in the previous publication [7]. The strain was maintained in Bold’s Basal medium (BBM) under sterile conditions [8]. Cells of Chlorella sp. A8 acclimated in wastewater for several generations before used for experiments, starting with cell concentration of 400 mg/L. Activated sludge was initially cultured in an artificial medium with the nutrient ingredients: KNO3 250 g/L, KH2PO4 30 g/L, K2HPO4 60 g/L, MgSO4 100 g/L, FeC6H5O7 0.25 g/L. Sucrose was used as the carbon source in the artificial effluent. When the biomass concentration of the activated sludge was about 3000 mg/L, the activated sludge was adapted with the piggery wastewater for several weeks before mixing with the microalgae with different ratios. 2.2. Wastewater and experimental design Wastewater samples were taken at the outlet of biogas system at Thanh Hung farm, Thanh Tri, Hanoi in different stages during the period from August 2016 to May 2017, following Vietnamese standards, such as TCVN 6663-1:2011, TCVN 6663-3:2008, TCVN 5999: 1995. Then, samples were filtrated via filter papers to reduce suspension particles. The effluent stream was characterized and the results were: pH = 6.9-7.2; COD = 396 - 893 mg/L; BOD5 = 110 - 150 mg/L; TN = 565 - 585 mg/L; NH4 + -N = 88 - 134 mg/L; TP = 45 - 64 mg/L; PO4 3- -P = 18 - 55. mg/L; SS = 300 - 402 mg/L. The data showed that all parameters of effluent stream were higher than national standard for discharging, extremely total nitrogen contents were almost 4 times higher. Organic carbons after anaerobic treatment (i.e. COD and BOD5) could be removed further by bacteria, as well as N and P could be consumed by microalgae. Experiment was established in batch mode in a transparent tank made of polyethylene plastic, Ngo Van Chien, Vu Ngoc Thuy, Miyashita Hideaki, Doan Thi Thai Yen 200 completely stirred at 150 rpm, reaction volume was 2 liters. The light irradiation was four T8- fluorescent bulbs for illumination of 1200 lumens, placed in one side. The temperature in the reaction tank was 27 - 30 o C. Dissolved oxygen (DO) in containers was measured by DO meter. Effect of hydraulic retention time (HRT) to the treatment efficiency: The ratio of algae/ bacteria biomass was 1/1 (w/w); total biomass concentration was maintained about 1000 mg/L; COD/NH4 + -N(C/N) = 4/1( m/m); DO from 5 to 7 mgO2/L; initial pH = 6.9. The reaction time lasted from 10 h to 52 h. Samples were taken every 8 hours. Effect of dissolved oxygen (DO) to treatment efficiency: Experiment was carried out with two values of DO content: from 5 - 7 mg O2/L (aeration mode) and 0.3 - 0.5 mg O2/L (stirring mode). The initial conditions of experiment: C/N was 4/1; NH4 + -N was 115 mg/L, ratio of algae/ bacteria biomass was 1/1(w/w), initial pH was 7.0 and observation time in 24 h. Effect of C/N ratio: C/N ratios of 2 and C/N = 4, were conducted. In each experiment of each C/N ratio, the Chlorella/activated sludge biomass was 1:1 (w/w) and total amount of all biomass was 1000 mg/L, using stirring mode to generate DO = 0.3 - 0.5 mgO2/L, initial pH was 7. Effect of inoculum ratios of Chlorella to suspended Activated sludge (Chl/AS ratio): Different inoculum ratios (Chl/AS) of both Chlorella A8 and suspended activated sludge were 1; 1.5; and 2. Control the total biomass concentration of Chlorella and AS was 1000 mg/L for all experiments of inoculum ratio. Input parameters of wastewater were kept constant with C/N was 4, initial pH = 7.0, DO = 0.3 - 0.5 mg O2/L. 2.3. Analytical methods Biomass, consisted of algae and bacteria biomass, was collected by centrifugation at 6500 rpm for 5 minutes and its pellet was washed two times with distilled water. The pellet of total biomass was dried at 105 °C for 24 hours, then weighed to determine dry biomass. COD was determined by Dichromate Method (TNT822, HACH); BOD5 was determined using Oxitop methods; NH4 + _N: using TNT (Salicylate, product#:2606945, HACH); PO4 3- _P: using Molybdovanadate Test (product#:2767245, HACH). Chlorophyll- a: following TCVN 6662- 2000, using ethanol extraction (ethanol absolute for analysis, Merck). Figure 1. Overview of experiment procedures. Microalge biomass Activated sludge biomass Chlorella A8 Activated sludge In Basal Bold Medium culturing Adapt with WW for acclimation Synthetic WW culturing Adapt with WW for acclimation Chlorella sp.A8 and activated sludge consortium Wastewater(WW) Filtration process Effect of HRT Effect of DO Effect of COD/NH4+ Effect of initial conc. of consortium Development of nitrogen and phosphorous removal from piggery wastewater 201 3. RESULTS AND DISCUSSION 3.1. Effect of hydraulic retention time (HRT) As shown in Fig. 2, the removal efficiency increased from 10 to 24 hours and reached maximum 73 % for COD, 57 % for ammonium after 24 hours. However, PO4 3- _P removal efficiency increased reach maximum 63 % after 52 hours. The COD removal efficiency reduced dramatically after 40 hours, which could be due to the substrate content declined, that caused to decrease in the food/microorganism (F/M) ratio. After 24 h, a rapid self-oxidation process kept the F/M ratio in balance. Besides, due to Chlorella sp. A8 utilized inorganic carbon as the main source of carbon (resulted from previous investigation), the organic matters in the wastewater were removed negligibly by microalgae. Ammonium and phosphorus were removed from 40 - 50 % and 25 - 35 %, respectively. The HRT was chosen about 24 hours for the next experiments to keep bacteria in active condition. This HRT was similar to that in the case of microalgae–activated sludge flocs that treated salicylate in industrial wastewater (1 day), and it was lower than that of dairy processing of industry wastewater, about 48 hours [9, 10]. 3.2. Effect of dissolved oxygen (DO) to treatment efficiency In this study, the ammonia treatment capacity under agitation was not significantly different compared to that of aeration (Fig. 3). This may be due to the amount of oxygen in both regimes was not enough for ammonium oxidation. Besides, the growth of microorganisms was inhibited when pH > 8. Growth of Chlorella sp. A8 was almost the same under two DO conditions and pH fluctuations (Chlorophyll-a contents was 9.8 mg/L in aeration and 12 mg/L in stirring mode). Treatment efficiency of phosphorus under stirring conditions tended to be better than that under aeration condition, i.e. 78.1 % and 62.6 % at 24 hours, respectively. In aeration mode, COD treatment efficiency of the microalgae- bacteria consortium was slightly poorer than that under stirring mode (i.e. 56 % and 66 %, respectively). Generally, the stirring condition mode showed better efficiency for phosphorus and COD removal and saved energy to operate the system, similar to the results of published studies [11]. COD-Cr NH4+-N PO43--P DO = 3 - 7 mgO2/L 55.9 47 62.6 DO = 0.3 - 0.6 mgO2/L 66.3 45 77.7 0 10 20 30 40 50 60 70 80 90 Re m ov al E ff ic ie nc y ,% Figure 3. Effect of DO on removal efficiency at 24 h. 0 10 20 30 40 50 60 70 80 90 9 18 24 41 52 R e m o v a l e ff ic ie n c y , % HRT, h COD ammonium octophosphate figure 3. 1 Figure 2. Effect of HRT on removal efficiency (Chl : AS = 1:1). Ngo Van Chien, Vu Ngoc Thuy, Miyashita Hideaki, Doan Thi Thai Yen 202 3.3. Effect of COD / NH4 +-N (C/N) ratios to treatment efficiency In this experiment, two different C/N ratios, C/N = 2 and C/N = 4, were conducted. The results in Fig. 4 showed that the removal efficiency of COD, NH4 + -N and PO4 3- -P when C/N = 4 was higher than those when C/N = 2. With C/N = 4, the highest removal efficiency of COD and PO4 3- -P was 64 % at 20 h and 78 % at 24 h, respectively. At the same time, the efficiency of COD, PO4 3- -P when C/N =2 was lower than 15 %. The removal efficiency of NH4 + -N with C/N = 2 was increasing during early stage and reached a maximum of 42 % at 20 hours, while when C/N = 4, the ammonium treatment efficiency was highest at 46 %, which was not significantly different. This result displayed the role in ammonium consumption of microalgae in wastewater, leading to the increase of treatment efficiency in the first 20 hours. The reduction of the treatment efficiency after 20 hours at the ratio of C/N = 2 was due to the self-degradation of bacterial sludge by lacking substrate for their growth (Fig. 4). 3.4. Effect of the inoculum ratio of Chlorella sp.A8 and activated sludge (Chl/AS) The system of Chlorella sp.A8 and activated sludge exhibited different removal performances depending upon their inoculum ratio, 1/1, 1.5/1 and 2.5/1 (g/g). The results in Fig. 5 showed that the increase of inoculum ratio of Chlorella seemed to be increase the removal efficiency. However, the removal efficiency of 1.5 and 2.5 inoculum ratio was not increased significantly. This result was similar to the results in research [3] with the optimum ratio of Chl/AS was 2/1. This could be explained that the increase of inoculum ratio of algae concomitantly leaded to the decrease of the amount of activated sludge. That might be resulted to reduce the amount of CO2 produced by bacteria in activated sludge, then caused a shortage for the photosynthesis of microalgae. In addition, under CO2-limited condition, microalgae grew slowly, that resulted to low removal efficiency of NH4 + -N. Exceptionally, the PO4 3- -P was removed in Chl : As = 1:1 Chl : As =1.5:1 Chl : As =2.5:1 COD 62.9 70.8 73.5 NH4+ 39.4 45.7 46.8 PO43- 62.8 76.5 77.6 0 10 20 30 40 50 60 70 80 90 R e m o va l E ff ic ie n cy , % Figure 5. Effect of Chl:As ratios on removal efficiency at 20 hours. Figure 4. Effect of the COD/NH4 + ratio on removal efficiency (Chl/As = 1:1). 0 10 20 30 40 50 0 10 20 30 R e m o va l E ff ic ie n cy , % HRT, h C/N = 4 COD NH4+-N PO43--P 0 10 20 30 40 50 0 10 20 30 R e m o va l E ff ic ie n cy , % HRT, h C/N = 2 COD NH4+-N PO43--P Development of nitrogen and phosphorous removal from piggery wastewater 203 high efficiency (76 %), which due to being synthesized for microorganism biomass of consortium and/or absorbed capability on activated sludge flocs [11]. 4. CONCLUSION In this study, microalgae-bacterial consortium could be applied for wastewater treatment from piggery farm after biogas treatment process. After 20 hours of HRTs, removal efficiency of COD, NH4+_N and PO43-_P reached maximum at 71 %, 46 % and 77 % , respectively, under complete stirring condition, C/N was 4/1 and inoculum ratio of Chlorella sp. A8 to activated sludge was 1.5. 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