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Performance of photoperiod and light intensity on biogas upgrade and biogas effluent nutrient reduction by the microalgae Chlorella sp.
Accepted Manuscript Performance of photoperiod and light intensity on biogas upgrade and biogas effluent nutrient reduction by the microalgae Chlorella sp Cheng Yan, Zheng Zheng PII: DOI: Reference:
S0960-8524(13)00661-5 http://dx.doi.org/10.1016/j.biortech.2013.04.054 BITE 11714
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
22 March 2013 12 April 2013 13 April 2013
Please cite this article as: Yan, C., Zheng, Z., Performance of photoperiod and light intensity on biogas upgrade and biogas effluent nutrient reduction by the microalgae Chlorella sp, Bioresource Technology (2013), doi: http:// dx.doi.org/10.1016/j.biortech.2013.04.054
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Performance of photoperiod and light intensity on biogas upgrade and biogas effluent nutrient reduction by the microalgae Chlorella sp.
Cheng Yan, Zheng Zheng* Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, P.R. China *
Corresponding author
Prof. Zheng Zheng Department of Environmental Science and Engineering, Fudan University, 220 Handan Road, Shanghai 200433, P.R. China Tel.: +86-21-65642948 Fax: +86-21-65642948 E-mail: [email protected]
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Abstract Biogas is an environment-friendly fuel but that must be upgraded before being utilized. The method about removing CO2 from biogas by microalgal culturing using biogas effluent as nutrient medium in this study could effectively upgrade biogas and simultaneously reduce the biogas effluent nutrient. Results showed that the optimum parameters for microalgal growth and biogas effluent nutrient reduction was moderate light intensity with middle photoperiod. While low light intensity with long photoperiod and moderate light intensity with middle photoperiod obtained the best biogas CO2 removal and biogas upgrade effects. Therefore, the optimal parameters were moderate light intensity 350 mol m-2 s-1 with middle photoperiod 14 h light:10 h dark. Under this condition, the microalgal dry weight, CH4 concentration, reduction efficiency of chemical oxygen demand, total nitrogen, and total phosphorus was 615.84 ± 33.07 mg L-1, 92.16% ± 2.83% (v/v), 88.74% ± 3.45%, 83.94% ± 3.51%, and 80.43% ± 4.17%, respectively.
Keywords: CO2 removal; Chemical oxygen demand removal; Total nitrogen removal; Total phosphorus removal; Dry weight
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1. Introduction Biogas is an environment-friendly, low-cost, versatile fuel that can well substitute for conventional fossil fuel that is currently causing environmental problems (Santosh et al., 2004; Demirbas et al., 2011). Crude biogas is mainly composed of methane (CH4; 50% to 70%, v/v) and carbon dioxide (CO2; 30% to 45%, v/v). Crude biogas also has trace components such as water (H2O), oxygen (O2), and hydrogen sulfide (H2S) (Starr et al., 2012). Among such components, CH4 is the most important particularly for the combustion process in engines. However, the presence of relatively high concentrations of incombustible components such as CO2, H2S, and H2O reduces the calorific value of crude biogas (Demirbas et al., 2011), which must thus be upgraded to meet the requirement of efficient combustion (i.e., CH4 concentration >90%, v/v) (Ryckebosch et al., 2011). The most commonly used biogas upgrade technologies are chemical absorption, water scrubbing, membrane separation, pressure swing adsorption, and cryogenic separation (Nordberg et al., 2012). The operational costs of and investments on these upgrade technologies allow economical utilization only on large-scale biogas plants with crude biogas outputs >500 m3 h-1 (Raab et al., 2012). Therefore, such technologies are unsuitable for decentralized energy supply with a large number of relatively small biogas upgrade plants (Nordberg et al., 2012; Raab et al., 2012). High amounts of thermal energy and auxiliary materials are needed, and wastes that require treatment are generated (Ryckebosch et al., 2011). The removed CO2 is also usually released directly into the atmosphere (Baciocchi et al., 2012). However, microalgal photosynthesis is one of the most cost-effective ways to sequester CO2, and approximately half of microalgal dry weight (DW) biomass is carbon from CO2 (Jeong et al., 2013). Given that this CO2 in biogas is of biogenic 3
origin, its emissions should be regarded as climate neutral. Thus, by storing the removed CO2 during microalgal photosynthesis, emissions can be substantially decreased (Baciocchi et al., 2012). Furthermore, culturing microalgae in biogas effluent provides a perfect freely obtained nutrient medium and reduces environmental pollution (Singh et al., 2011). Continuous cultivation of some specific microalgal species (e.g., Chlorella sp.) can produce value-added products from their biomass (Ryckebosch et al., 2011). Therefore, removing CO2 from biogas by microalgal culturing using biogas effluent is a highly feasible method of biogas upgrade. Light is the basic energy source in microalgal photosynthesis. Thus, the light intensity and photoperiod (light–dark cycle) are crucial in determining the growth rate of microalgal cultivation (Parmar et al., 2011). However, varying the illumination conditions of natural sunlight can inhibit microalgal growth because of a shortage in light energy supply ensues (e.g., very low light intensities during rainy days) or of photoinhibition caused by excessive lighting (e.g., very high light intensities at noontimes during summer) (Ugwu et al., 2007). Using an artificial light source is a solution to this concern because both the light intensity and photoperiod can be controlled (Carvalho et al., 2006). However, studies on biogas upgrade and biogas effluent nutrient reduction using microalgae, especially regarding the effects of different light intensities and photoperiods, are limited. Thus, this research aimed to determine the influence of various photoperiods and light intensities on microalgal growth, biogas upgrade, biogas CO2 removal, and biogas effluent nutrient reduction by the microalgae Chlorella sp. These factors were optimized by analyzing intrinsic connections among them.
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2. Method and materials 2.1. Materials The artificial light source used was cool-white fluorescent light. Light intensity was controlled by varying the number of fluorescent lights used. The microalgal-based photo-bioreactor was a transparent polyethylene bag (6 dm × 4 dm × 1 dm) pretreated by washing with distilled water twice and sterilized with an ultraviolet sterilizer, for 2 min (SKW- UV-U01, SKYUV Water Treatment Co., Ltd., P.R. China). The microalgae Chlorella sp. used was from stock cultures and were confirmed to be of high CO2-fixation rate based on Li (2012) and Li et al. (2013). The stock culture was on standard BG-11 medium (Tansakul et al. 2005), and the culture conditions were as follows: cool-white light-emitting diode (LED) light with an internal surface light intensity of 250 mol m−2 s−1; 25 ± 0.5 °C; 12 h light:12 h dark photoperiod; and artificial intermittent shaking four times a day (08:00, 14:00, 07:00, and 23:00 h). The microalgal DW of the stock cultures was 64.52± 13.06 mg L-1. The crude biogas and biogas effluent were both derived from an anaerobic digester in Hongmao Hacienda, Kunshan City, Jiangsu Province, P.R. China. The biogas was pretreated by a chemical absorption process to decrease the H2S concentration to <0.005% (v/v) (Chung et al., 2006). The components of crude biogas were 70.65% ± 4.38% (v/v) CH4, 26.14% ± 2.57% (v/v) CO2, 0.23% ± 0.02% (v/v) O2, and 3.11% ± 0.35% (v/v) H2O. To prevent potential interference from sediment and other microorganisms, the biogas effluent was pretreated with a glass microfiber filter (GF/C, Whatman, USA) and an ultraviolet sterilizer (SKW- UV-U01, SKYUV Water Treatment Co., Ltd., P.R. China) for 2 min. The pH was 6.82 ± 0.05, and the dissolved inorganic carbon (DIC), dissolved oxygen (DO), chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) of the biogas effluent before 5
pretreatment were 1005.73 ± 32.46, 5.41 ± 0.36, 998.27 ± 67.51, 364.82 ± 19.71, and 40.78 ± 5.32 mg L-1, respectively. After pretreatment, the pH was 6.77 ± 0.03, and the DIC, DO, COD, TN, and TP were 996.51 ± 47.85, 5.49 ± 0.47, 986.05 ± 73.16, 357.41 ± 36.09, and 37.24 ± 6.48 mg L-1, respectively. The COD, TN, and TP only slightly decreased after pretreatment, whereas the pH, DIC, and DO almost remained unchanged. Therefore, the pretreatment process did not significantly affect the characteristics of biogas effluent, and its characteristics can be represented by the filtrate. 2.2. Experimental procedures The photo-bioreactor bag contained 21.0 L of biogas and 3.0 L of biogas effluent culture (initial Chlorella sp. DW was 267.51 ± 24.03 mg L-1). A 3 L biogas effluent culture was prepared by the following procedure: (a) approximately 12.50 L of microalgal stock culture was harvested by centrifugation at 3000 × g for 5 min to obtain approximately 806.50 mg of microalgal DW; (b) the pelleted microalgal cells in the centrifugal tubes were rinsed with the pretreated biogas effluent, and the washing fluid was placed in a 5 L measuring cylinder; and (c) rinsing was stopped when the volume of the washing fluid was about 2.5 L, and then the pretreated biogas effluent was added to reach 3.0 L. The optimum light intensity and photoperiod for microalgal growth, biogas upgrade, and biogas effluent nutrient reduction were determined by exposing the photo-bioreactor bag treatments to low (300 mol m-2 s-1), moderate (350 mol m-2 s-1), and high (400 mol m-2 s-1) light intensities under short (12 h light:12 h dark), middle (14 h light:10 h dark), and long (16 h light:8 h dark) photoperiod. All treatments were performed in quadruplicates. The photo-bioreactor bag treatments were placed in an illuminating incubator (SPX-400I-G, Boxun Industry & Commerce 6
Co., Ltd., P.R. China) equipped with cool-white LEDs as a light source and a timer for photoperiod control. During the 6 days of experimentation, the culture temperature was maintained at 25.0 ± 0.5 °C, and 3 min of artificial intermittent shaking was performed four times a day. 2.3. Analysis The photo-bioreactor bag treatments were sampled and analyzed daily at 10:00 h to 11:00 h. The concentrations (v/v) of CH4, CO2, O2, and H2O in the biogas were measured with a circulating gas analyzer (GA94, ONUEE Co., Ltd., P.R. China). The biogas effluent culture was sampled through the sampling hole of the photo-bioreactor using a 50 mL syringe. Then, the sampled biogas effluent culture was treated as follows: (a) 30 mL of culture was passed through a glass microfiber filter (GF/C, Whatman, USA); (b) the filter with the attached microalgae was dried at 120 °C for 24 h and then cooled to room temperature in a desiccator; (c) the difference between the filter weights before and after filtration was calculated as microalgal DW; and (d) the filtrates were analyzed for COD, TN, and TP according to a standard method (APHA-AWWA-WPCF, 1995). The pH, DIC, and DO were measured using a pH meter (Orion 250 Aplus ORP Field Kit, USA), DIC analyzer (Shimazu TOC 5000A, Japan), and DO meter (TP350, Beijing Timepower Measurement and Control Equipment Co., Ltd., P.R. China), respectively. Light intensity was measured with a waterproof light meter (CEM, DT-1308, Shenzhen Everbest Machinery Industry Co., Ltd., P.R. China) on the inner surface of the culture. The efficiency of biogas CO2 removal or biogas effluent nutrient reduction was calculated as follows:
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C R = 1 − i ×100 C0
(1)
where R is the efficiency of biogas CO2 removal or the biogas effluent nutrient reduction (%), C0 is the initial CO2 concentration of the biogas (%, v/v) or the nutrient concentration of the biogas effluent culture (mg L-1), and Ci is the CO2 concentration of the biogas (%, v/v) or the nutrient concentration of the biogas effluent culture (mg L-1) at time i. [Table 1 inserts here]
3. Results
3.1. Physical and chemical parameters Table 1 showed the variations in pH, DIC, and DO in the biogas effluent culture throughout the experiment. Each parameter similarly changed under different light intensities (300, 350, and 400 mol m-2 s-1) and photoperiod (12 h light:12 h dark, 14 h light:10 h dark, and 16 h light:8 h dark). The pH and DIC only slightly changed from 6.85 ± 0.62 to 6.67 ± 0.58 and from 1009.43 ± 74.05 mg L-1 to 990.46 ± 49.86 mg L-1, respectively. The DO values mildly increased from 5.49 ± 0.47 mg L-1 to 5.73 ± 0.35, 5.77 ± 0.49, and 5.69 ± 0.48 mg L-1 for the light intensity treatment of 300, 350, and 400 mol m-2 s-1, respectively. 3.2. Microalgal DW Figure 1 demonstrated that the microalgal DW constantly increased throughout the experiment and reached the highest values of 553.67 ± 26.82, 615.84 ± 33.07, and 436.86 ± 25.32 mg L-1 under low light intensity–long photoperiod, moderate light intensity–middle photoperiod, and high light intensity–short photoperiod, respectively. Therefore, the optimum parameters for microalgal growth was moderate light 8
intensity 350 mol m-2 s-1 with middle photoperiod 14 h light:10 h dark. [Table 2 inserts here] [Figure 1 inserts here] 3.3. Biogas upgrade effect Throughout the experiment, the biogas CO2 removal efficiency (Fig. 2) increased with increased biogas CH4 concentration (v/v) (Fig. 3). The highest biogas CO2 removal efficiency was 81.68% ± 3.28%, 86.15% ± 3.94%, and 53.25% ± 3.21% at low light intensity–long photoperiod, moderate light intensity–middle photoperiod, and high light intensity–short photoperiod, respectively. The biogas CH4 concentration (v/v) had similar variation trends and reached the highest values of 90.1% ± 3.54%, 92.16% ± 2.83%, and 83.91% ± 3.42% at low light intensity–long photoperiod, moderate light intensity–middle photoperiod, and high light intensity–short photoperiod, respectively. In this research, the biogas CH4 concentration (v/v) represented the biogas upgrade effect. In addition, the concentration (v/v) of O2 and H2O in the biogas remained almost unchanged throughout the experiment (Table 2). The O2 concentration (v/v) slightly varied from 0.23% ± 0.02% to 0.94% ± 0.05%, whereas the H2O concentration (v/v) remained between 3.05% ± 0.24% and 4.18% ± 0.36%. Consequently, the optimum parameters for biogas CO2 removal and biogas upgrade were low light intensity 300 mol m-2 s-1 with long photoperiod 16 h light:8 h dark and moderate light intensity 350 mol m-2 s-1 with middle photoperiod 14 h light:10 h dark. [Figure 2 inserts here] [Figure 3 inserts here] 3.4. Biogas effluent nutrient reduction Figures 4-6 illustrates the biogas effluent nutrient reduction efficiencies under 9
various photoperiods and light intensity treatments. The variation trends of COD, TN, and TP reduction efficiencies similarly increased throughout the experiment. For low light intensity treatments, the highest nutrient reduction efficiencies were all obtained at long photoperiod with values of 84.07% ± 3.17%, 83.26% ± 4.02%, and 76.18% ± 3.51% for COD, TN, and TP removal (Figs. 4a, 5a, and 6a), respectively. For moderate intensity treatments, the highest nutrient reduction efficiencies were all reached at middle photoperiod with values of 88.74% ± 3.45%, 83.94% ± 3.51%, and 80.43% ± 4.17% for COD, TN, and TP removal (Figs. 4b, 5b, and 6b), respectively. For high light intensity treatments, the highest nutrient reduction efficiencies were reached at short photoperiod with values of 70.34% ± 3.06%, 68.43% ± 4.16%, and 65.32% ± 3.67% for COD, TN, and TP removal (Figs. 4c, 5c, and 6c), respectively. Therefore, moderate light intensity 350 mol m-2 s-1 with middle photoperiod 14 h light:10 h dark was the optimum parameters for biogas effluent nutrient reduction. [Figure 4 inserts here] [Figure 5 inserts here] [Figure 6 inserts here]
4. Discussion
Throughout the experiment, both pH and DIC values in the biogas effluent culture only slightly changed because the CO2 concentration almost remained unchanged (Table 1). A large amount of CO2 had already dissolved in the biogas effluent during the anaerobic digestion process. Thus, the biogas effluent culture at a relatively higher biogas CO2 concentration did not achieve a lower pH value. Meanwhile, the biogas effluent culture had an initially high concentrated DIC because CO2 was its main component (Lei et al., 2007). Furthermore, the biogas CO2 always met the level 10
required for microalgal growth because sufficient CO2 was supplied to dissolve in the biogas effluent culture (Carvalho et al., 2006). Although the dissolved CO2 in the biogas effluent culture was used by microalgal cells for growth, the consumed CO2 in the culture was immediately replaced by CO2 in the biogas, thereby maintaining the DIC and pH (Papazi et al., 2008). The variation ranges of DO of the biogas effluent culture were always below the excessive DO level (35 mg L-1), which enabled the avoidance of microalgal growth inhibition (De Godos et al., 2010). Carvalho et al. (2006) reported that in a closed photo-bioreactor (same as the photo-bioreactor bag used in this study), O2 in the initial biogas or that produced during microalgal photosynthesis may inhibit microalgal growth, because the accumulation of O2 in the microalgal culture caused photorespiration. This phenomenon did not occur in this research because of the existence of a relatively higher initial CO2 concentration in the photo-bioreactor bag and the organic carbon in the biogas effluent culture (Douskova et al., 2009). Van Den Hende et al. (2011) suggested that the increase in the CO2:O2 ratio by the addition of more inorganic carbon to the photo-bioreactor or the addition of organic carbon to the microalgal culture were both able to overcome the potential microalgal photosynthesis inhibition. Light was found to be the most important factor affecting microalgal growth because it was the fundamental energy source of the synthesis of microalgal cell protoplasm. When the light intensity was insufficient (Fig. 1a), the microalgal DW was lower than that under the optimum moderate light treatment (Fig. 1b) because the microalgae consumed carbohydrates during photorespiration (Jeong et al., 2013). In this state, microalgal growth always occurred under a photo-limitated condition. Thus, an increase in the photoperiod (i.e., longer lighting time) realized better microalgal growth (Xue et al., 2011). By contrast, when microalgae were exposed to light levels 11
above the light saturation limit (Fig. 1c), the microalgae were unable to use light efficiently because the excessive light intensity caused overloaded photosystems, bleached pigments, and ultimately destroyed photosystems (Jeong et al., 2013). In this state, microalgal growth occurred under conditions that were always beyond the saturation light intensity. Thus, an decrease in the photoperiod (i.e., shorter lighting time) realized higher microalgae growth (Xue et al., 2011). Under the moderate light intensity treatment (Fig 1b), only the middle photoperiod treatment realized the highest microalgal DW; longer or shorter photoperiod treatments obtained even lower microalgal DW values than those under the insufficient light intensity treatments (Fig. 1a). These findings can be explained by the theory that microalgal growth is affected by the light intensity and the duration of lighting in each photoperiod, i.e., the amount of light energy offered in each photoperiod light-dark cycle (Toro, 1989). Thus, the moderate light intensity treatments with photoperiod 16 h light:8 h dark or 12 h light:12 h dark provided relatively excess (20.16 mol m-2) or inadequate (15.12 mol m-2) light energy, respectively. The light energy of photoperiod 14 h light:10 h dark (17.64 mol m-2) was the most appropriate. Furthermore, Kumar et al. (2010) suggested that the availability of lighting was important for microalgal growth, and the maximum photosynthetic efficiencies can be achieved when photoperiod approached the microalgal photosynthetic unit turnover time. However, the treatments under different light intensities with equal light energy (i.e., 17.28 mol m-2 for photoperiod 16 h light:8 h dark under 300 mol m-2 s-1 in Fig. 1a and photoperiod 12 h light:12 h dark under 400 mol m-2 s-1 in Fig. 1c; 20.16 mol m-2 for photoperiod 16 h light:8 h dark under 350 mol m-2 s-1 in Fig. 1b and photoperiod 14 h light:10 h dark under 400 mol m-2 s-1 in Fig. 1c) did not achieve the same microalgal DW. In fact, the microalgal DW in the 12
higher light intensity treatment (Fig. 1c ) always led to much lower values. This finding was due to the fact that the high illumination intensities damaged the photosynthetic receptor system of the microalgae within a few minutes, and this phenomenon is known as photoinhibition (Anderson, 2005). Only treatments under moderate light intensity with middle photoperiod and low light intensity with long photoperiod efficiently removed CO2 from biogas and achieve the standard for efficient combustion (i.e., CH4 concentration >90%, v/v) (Ryckebosch et al., 2011). Furthermore, their O2 concentration (v/v) was outside the explosive range (O2 concentration = 0%–4%, v/v) (Ryckebosch et al., 2011). These results agreed with the variation trends of microalgal DW (Fig. 1) because approximately half of the microalgal DW was carbon derived from CO2 (Chisti, 2007). The microalgal photosynthesis consisted of two steps: light reactions that occurred only when the cells were illuminated, and dark reactions (i.e., carbon-fixation reactions) that occurred both in the presence and absence of light. In the light reactions, light energy was used to transfer electrons from water to coenzyme II (i.e., nicotinamide-adenine dinucleotide phosphate, NADP +) forming nicotinamide adenine dinucleotide phosphate (NADPH) and generate the highly energetic compound adenine triphosphate (ATP). Then, light energy transformed into the chemical energy ATP, which was stored for later use in the CO2-fixation reactions (Iverson, 2006; Baba and Shiraiwa, 2012). The microalgae developed the CO2-concentrating mechanism for adapting to changes in CO2 concentration. The chloroplast envelope and carbonic anhydrases of microalgae could facilitate the diffusion by stimulating the indirect supply of CO2 from outside the cells to Rubisco. Meanwhile, the inorganic carbon transporters were able to facilitate the DIC membrane transport of CO2 or bicarbonate through plasmalemma (Baba and Shiraiwa, 2012). In addition, the existence of O2 and 13
H2O (Table 2) in upgraded biogas were not expected to exert any harmful effect on microalgal growth because crude biogas was always saturated with water. Given that microalgae were cultured in liquid (biogas effluent) in this research water and O2 were the compounds consumed during microalgal photosynthesis (Kubler et al., 1999). The nutrient in the biogas effluent was efficiently reduced during the biogas upgrade process. The variation trends of reduced efficiencies of COD, TN, and TP (Figs. 4–6) agreed with that of microalgal growth (Fig. 1) and CO2 removal (Fig. 2). This finding can be attributed to the fact that microalgal photosynthesis and microalgal-CO2 fixation were environmentally sustainable when combined with wastewater treatment (Ahrens and Sander, 2010). Nutrient reduction was mainly achieved by the assimilation of microalgal photosynthesis because the microalgal reproduction requires abundant carbon, nitrogen, and phosphorous sources for synthesizing nucleic acids, phospholipids, and proteins (Munoz and Guieysse, 2006; Kumar et al., 2010). Moreover, the metabolic process of biogas effluent nutrient involved its accumulation of the chemical energy source ATP (the reduced form of nicotinamide-adenine dinucleotide) and the components of microalgal backbones. These phenomena were able to promote the autotrophic metabolism process of the microalgae (Rittenberg, 1969). Particularly, the accumulated ATP can be used as an enzyme activator during the autotrophic metabolism process (i.e., the abovementioned dark reactions and carbon-fixation reactions) (Moore and Cohen, 1967).
5. Conclusion
In this research, method about removing CO2 from biogas by microalgal culturing using biogas effluent as nutrient medium could effectively upgrade biogas and simultaneously reduce biogas effluent nutrient. Microalgal growth achieved the 14
highest DW value at moderate light intensity with middle photoperiod. Whereas, the low light intensity with long photoperiod and moderate light intensity with middle photoperiod both obtained the best biogas upgrade effects. Meanwhile, the moderate light intensity with middle photoperiod also had the highest biogas effluent nutrient reduction. Therefore, the optimal parameters were moderate light intensity 350 mol m-2 s-1 with middle photoperiod 14 h light:10 h dark.
Acknowledgments
This study was sponsored by the Beijing Green Future Environment Foundation, the National Key Special Project for Water Pollution Control and Treatment (Grant no. 2012ZX07102-004), and the National Science Funds of China (Grant no. 51102136/E021301).
Reference
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[23] Parmar, A., Singh, N.K., Pandey, A., Gnansounou, E., Madamwar, D., 2011. Cyanobacteria and microalgae: a positive prospect for biofuels. Bioresour. Technol. 102, 10163-10172. [24] Raab, K., Lamprecht, M., Brechtel, K., Scheffknecht, G., 2012. Innovative CO2 separation of biogas by polymer resins: operation of a continuous lab-scale plant. Eng. Life Sci. 12, 327-335. [25] Regeneration of a spent alkaline solution from a biogas upgrading unit by carbonation of APC residues. Chem. Eng. J. 179, 63-71. [26] Rittenberg, S.C., 1969. The roles of exogenous organic matter in the physiology of chemolithotrophic bacteria. Adv. Microb. Physiol. 3, 159-196. [27] Ryckebosch, E., Drouillon, M.H., Vervaeren, H., 2011. Techniques for transformation of biogas to biomethane. Biomass. Bioenerg. 35, 1633-1645. [28] Santosh, Y., Sreekrishnan, T.R., Kohli, S., Rana, V., 2004. Enhancement of biogas production from solid substrates using different techniques-a review. Bioresour. Technol. 95, 1-10. [29] Singh, A., Poonam, S.N., Jerry, D.M., 2011. Mechanism and challenges in commercialisation of algal biofuels. Bioresour. Technol. 102, 26-34. [30] Starr, K., Gabarrell, X., Villalba, G., Talens, L., Lombardi, L., 2012. Life cycle assessment of biogas upgrading technologies. Waste Manag. 32, 991-999. [31] Tansakul, H.P., Savaddiraksa, Y., Prasertsan, P., Tongurai, C., 2006. Cultivation of the hydrocarbon-rich alga, Botyococcus braunii in secondary treated effluent from a sea food processing plant. Thai. J. Agric. Sci. 38, 71-76. [32] Toro, J.E., 1989. The growth rate of two species of microalgae used in shellfish hatcheries cultured under two light regimes. Aqua. Fish. Manage. 20, 249-254. [33] Ugwu, C.U., Aoyagi, H., Uchiyama, H., 2007. Influence of irradiance, dissolved 18
oxygen concentration, and temperature on the growth of Chlorella sorokiniana. Photosynthetica 45, 309-311. [34] Xue, C., Qianru, Y.G., Weifeng, T., Iqbal, H., Wei, N.C., Raymond, L., 2011. Lumostatic strategy for microalgae cultivation utilizing image analysis and chlorophyll a content as design parameters. Bioresour. Technol. 102, 6005-6012.
19
Figure Captions Fig. 1 Microalgae dry weight under various photoperiods and light intensities: (a) 300,
(b) 350, and (c) 400 mol m-2 s-1. Fig. 2 Biogas CO2 removal efficiency under various photoperiods and light intensities:
(a) 300, (b) 350, and (c) 400 mol m-2 s-1. Fig. 3 Biogas CH4 concentration under various photoperiods and light intensities: (a)
300, (b) 350, and (c) 400 mol m-2 s-1. Fig. 4 Biogas effluent chemical oxygen demand removal efficiency under various
photoperiods and light intensities: (a) 300, (b) 350, and (c) 400 mol m-2 s-1. Fig. 5 Biogas effluent total nitrogen removal efficiency under various photoperiods
and light intensities: (a) 300, (b) 350, and (c) 400 mol m-2 s-1. Fig. 6 Biogas effluent total phosphorus removal efficiency under various photoperiods
and light intensities: (a) 300, (b) 350, and (c) 400 mol m-2 s-1.
20
700
-1
Microalgae dry weight (mg L )
650
550 500 450 400 350 300 250
-1
Microalgae dry weight (mg L )
650
b
600 550 500 450 400 350 300 250 200 700
650 -1
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
600
200 700
Microalgae dry weight (mg L )
a
c
600 550 500 450 400 350 300 250 200 0
1
2
3
4
Experimental time (d)
Fig. 1 21
5
6
100
Biogas CO2 removal efficiency (%)
90
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
80 70 60 50 40 30 20 10 0 100
90
Biogas CO2 removal efficiency (%)
a
b
80 70 60 50 40 30 20 10 0 100
Biogas CO2 removal efficiency (%)
90
c
80 70 60 50 40 30 20 10 0 1
2
3
4
Experimental time (d)
Fig. 2 22
5
6
Biogas CH4 concentration (%)
100
a
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
95
90
85
80
75
70
Biogas CH4 concentration (%)
100
95
90
85
80
75
70 100
Biogas CH4 concentration (%)
b
c
95
90
85
80
75
70 0
1
2
3
4
Experimental time (d)
Fig. 3 23
5
6
100
a
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
COD removal efficiency (%)
90 80 70 60 50 40 30 100
b
COD removal efficiency (%)
90 80 70 60 50 40 30
100
c
COD removal efficiency (%)
90 80 70 60 50 40 30 1
2
3
4
Experimental time (d)
Fig. 4 24
5
6
100
a
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
TN removal efficiency (%)
90 80 70 60 50 40 30 100
b
TN removal efficiency (%)
90 80 70 60 50 40 30 100
c
TN removal efficiency (%)
90 80 70 60 50 40 30 1
2
3
4
Experimental time (d)
Fig. 5 25
5
6
100
a
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
TP removal efficiency (%)
90 80 70 60 50 40 30 100
b
TP removal efficiency (%)
90 80 70 60 50 40 30 100
c
TP removal efficiency (%)
90 80 70 60 50 40 30 1
2
3
4
Experimental time (d)
Fig. 6
26
5
6
Table 1 Variations of physical and chemical parameters in biogas effluent culture Treatments Light intensity (μmol m-2 s-1)
Photoperiod (light : dark) 12 h :12 h
300
14 h : 10 h
16 h : 8 h
12 h :12 h
350
14 h : 10 h
16 h : 8 h
400
12 h :12 h
Experimental time (day) Parameters
0
1
2
3
4
5
6
pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1)
6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47
6.72±0.51 997.34±52.91 5.52±0.41 6.81±0.53 998.34±56.26 5.51±0.38 6.71±0.35 1007.23±64.37 5.56±0.38 6.73±0.52 999.34±48.38 5.51±0.28 6.73±0.45 1001.23±58.47 5.56±0.52 6.82±0.41 1003.23±67.24 5.52±0.36 6.75±0.58 994.30±57.93 5.53±0.50
6.81±0.42 999.73±68.03 5.56±0.53 6.82±0.42 993.23±36.36 5.54±0.52 6.78±0.30 995.48±59.30 5.60±0.51 6.79±0.41 1004.33±57.43 5.56±0.32 6.82±0.52 999.54±38.65 5.58±0.37 6.69±0.53 998.35±63.93 5.56±0.50 6.73±0.69 992.47±40.93 5.56±0.61
6.74±0.52 985.32±71.36 5.61±0.42 6.75±0.65 1003.34±52.34 5.58±0.31 6.82±0.54 1003.73±85.39 5.64±0.42 6.72±0.64 996.45±53.20 5.60±0.48 6.83±0.71 994.52±59.60 5.61±0.42 6.74±0.49 999.54±47.39 5.59±0.61 6.82±0.49 1006.35±73.94 5.58±0.49
6.70±0.48 1004.32±32.50 5.63±0.50 6.70±0.43 1009.43±74.05 5.61±0.40 6.84±0.63 997.42±69.43 5.67±0.61 6.83±0.43 999.03±73.21 5.63±0.41 6.80±0.36 1007.45±86.43 5.67±0.58 6.78±0.56 1003.24±34.23 5.63±0.48 6.80±0.72 1004.34±54.02 5.63±0.40
6.68±0.35 1008.79±46.25 5.66±0.36 6.69±0.64 993.45±65.38 5.67±0.38 6.83±0.53 999.52±64.94 5.70±0.22 6.67±0.58 1005.32±57.48 5.64±0.30 6.75±0.59 994.36±62.95 5.72±0.63 6.82±0.64 993.23±56.39 5.67±0.51 6.74±0.51 999.40±58.46 5.67±0.52
6.82±0.64 998.31±51.47 5.68±0.42 6.74±0.31 997.36±56.89 5.68±0.61 6.73±0.54 994.52±76.40 5.73±0.35 6.75±0.58 1001.35±64.35 5.69±0.42 6.79±0.53 1000.36±69.25 5.77±0.49 6.85±0.62 990.46±49.86 5.75±0.34 6.79±0.60 1001.24±62.38 5.69±0.48
1
14 h : 10 h
16 h : 8 h
pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1)
6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47
6.81±0.46 1005.32±57.24 5.53±0.35 6.75±0.53 1005.39±84.32 5.51±0.38
6.75±0.39 1002.94±73.29 5.56±0.52 6.74±0.62 999.83±57.32 5.53±0.44
2
6.71±0.52 998.39±73.23 5.61±0.64 6.79±0.48 1006.34±52.60 5.56±0.47
6.83±0.61 999.05±39.23 5.63±0.47 6.68±0.42 993.42±83.23 5.58±0.59
6.72±0.49 1007.32±49.32 5.65±0.60 6.70±0.61 995.34±89.57 5.61±0.65
6.80±0.65 1006.75±73.21 5.67±0.32 6.82±0.53 994.34±37.43 5.63±0.47
Table 2 Concentrations of O2 and H2O in the biogas Treatments Light intensity (μmol m-2 s-1)
Photoperiod (light : dark) 12 h :12 h
300
14 h : 10 h 16 h : 8 h 12 h :12 h
350
14 h : 10 h 16 h : 8 h 12 h :12 h
400
14 h : 10 h 16 h : 8 h
Experimental time (day) Concentration (%, v/v)
0
1
2
3
4
5
6
O2 H2O O2 H2O O2 H2O O2 H2O O2 H2O O2 H2O O2 H2O O2 H2O O2 H2O
0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35
0.24±0.02 3.16±0.31 0.26±0.04 3.25±0.36 0.26±0.03 3.19±0.32 0.25±0.03 3.37±0.41 0.27±0.04 3.31±0.25 0.25±0.03 3.26±0.25 0.25±0.04 3.14±0.23 0.25±0.03 3.10±0.32 0.25±0.03 3.28±0.34
0.30±0.04 3.28±0.27 0.33±0.04 3.28±0.36 0.34±0.03 3.10±0.54 0.34±0.04 3.25±0.37 0.39±0.03 3.28±0.24 0.33±0.04 3.29±0.21 0.31±0.03 3.26±0.42 0.32±0.03 3.15±0.31 0.31±0.03 3.28±0.27
0.41±0.04 3.14±0.32 0.45±0.05 3.19±0.28 0.47±0.04 3.27±0.35 0.44±0.03 3.36±0.21 0.50±0.03 3.18±0.27 0.43±0.03 3.20±0.31 0.37±0.04 3.29±0.37 0.36±0.05 3.34±0.21 0.37±0.04 3.19±0.47
0.60±0.05 4.18±0.36 0.63±0.06 3.26±0.31 0.64±0.05 3.29±0.31 0.62±0.05 3.17±0.22 0.68±0.04 3.21±0.35 0.61±0.04 3.17±0.21 0.56±0.04 3.25±0.36 0.47±0.04 3.15±3.11 0.43±0.04 3.28±0.31
0.68±0.06 3.05±0.34 0.71±0.05 3.17±0.38 0.73±0.06 3.16±0.24 0.71±0.06 3.25±0.26 0.79±0.06 3.36±0.24 0.71±0.05 3.42±0.32 0.61±0.06 3.29±0.24 0.56±0.05 3.24±0.26 0.49±0.05 3.15±0.31
0.79±0.08 3.17±0.30 0.87±0.07 3.14±0.32 0.89±0.07 3.19±0.28 0.83±0.06 3.14±0.23 0.94±0.05 3.25±0.25 0.82±0.06 3.05±0.24 0.67±0.06 3.17±0.28 0.64±0.06 3.35±0.31 0.54±0.05 3.17±0.26
3
Highlights > Moderate light intensity with middle photoperiod was optimum for microalgal
growth. > Moderate light intensity with middle photoperiod was optimum for nutrient reduction. > Low light intensity with long photoperiod obtained the best biogas upgrade effects. > Moderate light intensity with middle photoperiod was also optimum for biogas upgrade. > The upgraded CH4 concentration was 92.16% ± 2.83% (v/v).
29
S0960-8524(13)00661-5 http://dx.doi.org/10.1016/j.biortech.2013.04.054 BITE 11714
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
22 March 2013 12 April 2013 13 April 2013
Please cite this article as: Yan, C., Zheng, Z., Performance of photoperiod and light intensity on biogas upgrade and biogas effluent nutrient reduction by the microalgae Chlorella sp, Bioresource Technology (2013), doi: http:// dx.doi.org/10.1016/j.biortech.2013.04.054
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Performance of photoperiod and light intensity on biogas upgrade and biogas effluent nutrient reduction by the microalgae Chlorella sp.
Cheng Yan, Zheng Zheng* Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, P.R. China *
Corresponding author
Prof. Zheng Zheng Department of Environmental Science and Engineering, Fudan University, 220 Handan Road, Shanghai 200433, P.R. China Tel.: +86-21-65642948 Fax: +86-21-65642948 E-mail: [email protected]
1
Abstract Biogas is an environment-friendly fuel but that must be upgraded before being utilized. The method about removing CO2 from biogas by microalgal culturing using biogas effluent as nutrient medium in this study could effectively upgrade biogas and simultaneously reduce the biogas effluent nutrient. Results showed that the optimum parameters for microalgal growth and biogas effluent nutrient reduction was moderate light intensity with middle photoperiod. While low light intensity with long photoperiod and moderate light intensity with middle photoperiod obtained the best biogas CO2 removal and biogas upgrade effects. Therefore, the optimal parameters were moderate light intensity 350 mol m-2 s-1 with middle photoperiod 14 h light:10 h dark. Under this condition, the microalgal dry weight, CH4 concentration, reduction efficiency of chemical oxygen demand, total nitrogen, and total phosphorus was 615.84 ± 33.07 mg L-1, 92.16% ± 2.83% (v/v), 88.74% ± 3.45%, 83.94% ± 3.51%, and 80.43% ± 4.17%, respectively.
Keywords: CO2 removal; Chemical oxygen demand removal; Total nitrogen removal; Total phosphorus removal; Dry weight
2
1. Introduction Biogas is an environment-friendly, low-cost, versatile fuel that can well substitute for conventional fossil fuel that is currently causing environmental problems (Santosh et al., 2004; Demirbas et al., 2011). Crude biogas is mainly composed of methane (CH4; 50% to 70%, v/v) and carbon dioxide (CO2; 30% to 45%, v/v). Crude biogas also has trace components such as water (H2O), oxygen (O2), and hydrogen sulfide (H2S) (Starr et al., 2012). Among such components, CH4 is the most important particularly for the combustion process in engines. However, the presence of relatively high concentrations of incombustible components such as CO2, H2S, and H2O reduces the calorific value of crude biogas (Demirbas et al., 2011), which must thus be upgraded to meet the requirement of efficient combustion (i.e., CH4 concentration >90%, v/v) (Ryckebosch et al., 2011). The most commonly used biogas upgrade technologies are chemical absorption, water scrubbing, membrane separation, pressure swing adsorption, and cryogenic separation (Nordberg et al., 2012). The operational costs of and investments on these upgrade technologies allow economical utilization only on large-scale biogas plants with crude biogas outputs >500 m3 h-1 (Raab et al., 2012). Therefore, such technologies are unsuitable for decentralized energy supply with a large number of relatively small biogas upgrade plants (Nordberg et al., 2012; Raab et al., 2012). High amounts of thermal energy and auxiliary materials are needed, and wastes that require treatment are generated (Ryckebosch et al., 2011). The removed CO2 is also usually released directly into the atmosphere (Baciocchi et al., 2012). However, microalgal photosynthesis is one of the most cost-effective ways to sequester CO2, and approximately half of microalgal dry weight (DW) biomass is carbon from CO2 (Jeong et al., 2013). Given that this CO2 in biogas is of biogenic 3
origin, its emissions should be regarded as climate neutral. Thus, by storing the removed CO2 during microalgal photosynthesis, emissions can be substantially decreased (Baciocchi et al., 2012). Furthermore, culturing microalgae in biogas effluent provides a perfect freely obtained nutrient medium and reduces environmental pollution (Singh et al., 2011). Continuous cultivation of some specific microalgal species (e.g., Chlorella sp.) can produce value-added products from their biomass (Ryckebosch et al., 2011). Therefore, removing CO2 from biogas by microalgal culturing using biogas effluent is a highly feasible method of biogas upgrade. Light is the basic energy source in microalgal photosynthesis. Thus, the light intensity and photoperiod (light–dark cycle) are crucial in determining the growth rate of microalgal cultivation (Parmar et al., 2011). However, varying the illumination conditions of natural sunlight can inhibit microalgal growth because of a shortage in light energy supply ensues (e.g., very low light intensities during rainy days) or of photoinhibition caused by excessive lighting (e.g., very high light intensities at noontimes during summer) (Ugwu et al., 2007). Using an artificial light source is a solution to this concern because both the light intensity and photoperiod can be controlled (Carvalho et al., 2006). However, studies on biogas upgrade and biogas effluent nutrient reduction using microalgae, especially regarding the effects of different light intensities and photoperiods, are limited. Thus, this research aimed to determine the influence of various photoperiods and light intensities on microalgal growth, biogas upgrade, biogas CO2 removal, and biogas effluent nutrient reduction by the microalgae Chlorella sp. These factors were optimized by analyzing intrinsic connections among them.
4
2. Method and materials 2.1. Materials The artificial light source used was cool-white fluorescent light. Light intensity was controlled by varying the number of fluorescent lights used. The microalgal-based photo-bioreactor was a transparent polyethylene bag (6 dm × 4 dm × 1 dm) pretreated by washing with distilled water twice and sterilized with an ultraviolet sterilizer, for 2 min (SKW- UV-U01, SKYUV Water Treatment Co., Ltd., P.R. China). The microalgae Chlorella sp. used was from stock cultures and were confirmed to be of high CO2-fixation rate based on Li (2012) and Li et al. (2013). The stock culture was on standard BG-11 medium (Tansakul et al. 2005), and the culture conditions were as follows: cool-white light-emitting diode (LED) light with an internal surface light intensity of 250 mol m−2 s−1; 25 ± 0.5 °C; 12 h light:12 h dark photoperiod; and artificial intermittent shaking four times a day (08:00, 14:00, 07:00, and 23:00 h). The microalgal DW of the stock cultures was 64.52± 13.06 mg L-1. The crude biogas and biogas effluent were both derived from an anaerobic digester in Hongmao Hacienda, Kunshan City, Jiangsu Province, P.R. China. The biogas was pretreated by a chemical absorption process to decrease the H2S concentration to <0.005% (v/v) (Chung et al., 2006). The components of crude biogas were 70.65% ± 4.38% (v/v) CH4, 26.14% ± 2.57% (v/v) CO2, 0.23% ± 0.02% (v/v) O2, and 3.11% ± 0.35% (v/v) H2O. To prevent potential interference from sediment and other microorganisms, the biogas effluent was pretreated with a glass microfiber filter (GF/C, Whatman, USA) and an ultraviolet sterilizer (SKW- UV-U01, SKYUV Water Treatment Co., Ltd., P.R. China) for 2 min. The pH was 6.82 ± 0.05, and the dissolved inorganic carbon (DIC), dissolved oxygen (DO), chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) of the biogas effluent before 5
pretreatment were 1005.73 ± 32.46, 5.41 ± 0.36, 998.27 ± 67.51, 364.82 ± 19.71, and 40.78 ± 5.32 mg L-1, respectively. After pretreatment, the pH was 6.77 ± 0.03, and the DIC, DO, COD, TN, and TP were 996.51 ± 47.85, 5.49 ± 0.47, 986.05 ± 73.16, 357.41 ± 36.09, and 37.24 ± 6.48 mg L-1, respectively. The COD, TN, and TP only slightly decreased after pretreatment, whereas the pH, DIC, and DO almost remained unchanged. Therefore, the pretreatment process did not significantly affect the characteristics of biogas effluent, and its characteristics can be represented by the filtrate. 2.2. Experimental procedures The photo-bioreactor bag contained 21.0 L of biogas and 3.0 L of biogas effluent culture (initial Chlorella sp. DW was 267.51 ± 24.03 mg L-1). A 3 L biogas effluent culture was prepared by the following procedure: (a) approximately 12.50 L of microalgal stock culture was harvested by centrifugation at 3000 × g for 5 min to obtain approximately 806.50 mg of microalgal DW; (b) the pelleted microalgal cells in the centrifugal tubes were rinsed with the pretreated biogas effluent, and the washing fluid was placed in a 5 L measuring cylinder; and (c) rinsing was stopped when the volume of the washing fluid was about 2.5 L, and then the pretreated biogas effluent was added to reach 3.0 L. The optimum light intensity and photoperiod for microalgal growth, biogas upgrade, and biogas effluent nutrient reduction were determined by exposing the photo-bioreactor bag treatments to low (300 mol m-2 s-1), moderate (350 mol m-2 s-1), and high (400 mol m-2 s-1) light intensities under short (12 h light:12 h dark), middle (14 h light:10 h dark), and long (16 h light:8 h dark) photoperiod. All treatments were performed in quadruplicates. The photo-bioreactor bag treatments were placed in an illuminating incubator (SPX-400I-G, Boxun Industry & Commerce 6
Co., Ltd., P.R. China) equipped with cool-white LEDs as a light source and a timer for photoperiod control. During the 6 days of experimentation, the culture temperature was maintained at 25.0 ± 0.5 °C, and 3 min of artificial intermittent shaking was performed four times a day. 2.3. Analysis The photo-bioreactor bag treatments were sampled and analyzed daily at 10:00 h to 11:00 h. The concentrations (v/v) of CH4, CO2, O2, and H2O in the biogas were measured with a circulating gas analyzer (GA94, ONUEE Co., Ltd., P.R. China). The biogas effluent culture was sampled through the sampling hole of the photo-bioreactor using a 50 mL syringe. Then, the sampled biogas effluent culture was treated as follows: (a) 30 mL of culture was passed through a glass microfiber filter (GF/C, Whatman, USA); (b) the filter with the attached microalgae was dried at 120 °C for 24 h and then cooled to room temperature in a desiccator; (c) the difference between the filter weights before and after filtration was calculated as microalgal DW; and (d) the filtrates were analyzed for COD, TN, and TP according to a standard method (APHA-AWWA-WPCF, 1995). The pH, DIC, and DO were measured using a pH meter (Orion 250 Aplus ORP Field Kit, USA), DIC analyzer (Shimazu TOC 5000A, Japan), and DO meter (TP350, Beijing Timepower Measurement and Control Equipment Co., Ltd., P.R. China), respectively. Light intensity was measured with a waterproof light meter (CEM, DT-1308, Shenzhen Everbest Machinery Industry Co., Ltd., P.R. China) on the inner surface of the culture. The efficiency of biogas CO2 removal or biogas effluent nutrient reduction was calculated as follows:
7
C R = 1 − i ×100 C0
(1)
where R is the efficiency of biogas CO2 removal or the biogas effluent nutrient reduction (%), C0 is the initial CO2 concentration of the biogas (%, v/v) or the nutrient concentration of the biogas effluent culture (mg L-1), and Ci is the CO2 concentration of the biogas (%, v/v) or the nutrient concentration of the biogas effluent culture (mg L-1) at time i. [Table 1 inserts here]
3. Results
3.1. Physical and chemical parameters Table 1 showed the variations in pH, DIC, and DO in the biogas effluent culture throughout the experiment. Each parameter similarly changed under different light intensities (300, 350, and 400 mol m-2 s-1) and photoperiod (12 h light:12 h dark, 14 h light:10 h dark, and 16 h light:8 h dark). The pH and DIC only slightly changed from 6.85 ± 0.62 to 6.67 ± 0.58 and from 1009.43 ± 74.05 mg L-1 to 990.46 ± 49.86 mg L-1, respectively. The DO values mildly increased from 5.49 ± 0.47 mg L-1 to 5.73 ± 0.35, 5.77 ± 0.49, and 5.69 ± 0.48 mg L-1 for the light intensity treatment of 300, 350, and 400 mol m-2 s-1, respectively. 3.2. Microalgal DW Figure 1 demonstrated that the microalgal DW constantly increased throughout the experiment and reached the highest values of 553.67 ± 26.82, 615.84 ± 33.07, and 436.86 ± 25.32 mg L-1 under low light intensity–long photoperiod, moderate light intensity–middle photoperiod, and high light intensity–short photoperiod, respectively. Therefore, the optimum parameters for microalgal growth was moderate light 8
intensity 350 mol m-2 s-1 with middle photoperiod 14 h light:10 h dark. [Table 2 inserts here] [Figure 1 inserts here] 3.3. Biogas upgrade effect Throughout the experiment, the biogas CO2 removal efficiency (Fig. 2) increased with increased biogas CH4 concentration (v/v) (Fig. 3). The highest biogas CO2 removal efficiency was 81.68% ± 3.28%, 86.15% ± 3.94%, and 53.25% ± 3.21% at low light intensity–long photoperiod, moderate light intensity–middle photoperiod, and high light intensity–short photoperiod, respectively. The biogas CH4 concentration (v/v) had similar variation trends and reached the highest values of 90.1% ± 3.54%, 92.16% ± 2.83%, and 83.91% ± 3.42% at low light intensity–long photoperiod, moderate light intensity–middle photoperiod, and high light intensity–short photoperiod, respectively. In this research, the biogas CH4 concentration (v/v) represented the biogas upgrade effect. In addition, the concentration (v/v) of O2 and H2O in the biogas remained almost unchanged throughout the experiment (Table 2). The O2 concentration (v/v) slightly varied from 0.23% ± 0.02% to 0.94% ± 0.05%, whereas the H2O concentration (v/v) remained between 3.05% ± 0.24% and 4.18% ± 0.36%. Consequently, the optimum parameters for biogas CO2 removal and biogas upgrade were low light intensity 300 mol m-2 s-1 with long photoperiod 16 h light:8 h dark and moderate light intensity 350 mol m-2 s-1 with middle photoperiod 14 h light:10 h dark. [Figure 2 inserts here] [Figure 3 inserts here] 3.4. Biogas effluent nutrient reduction Figures 4-6 illustrates the biogas effluent nutrient reduction efficiencies under 9
various photoperiods and light intensity treatments. The variation trends of COD, TN, and TP reduction efficiencies similarly increased throughout the experiment. For low light intensity treatments, the highest nutrient reduction efficiencies were all obtained at long photoperiod with values of 84.07% ± 3.17%, 83.26% ± 4.02%, and 76.18% ± 3.51% for COD, TN, and TP removal (Figs. 4a, 5a, and 6a), respectively. For moderate intensity treatments, the highest nutrient reduction efficiencies were all reached at middle photoperiod with values of 88.74% ± 3.45%, 83.94% ± 3.51%, and 80.43% ± 4.17% for COD, TN, and TP removal (Figs. 4b, 5b, and 6b), respectively. For high light intensity treatments, the highest nutrient reduction efficiencies were reached at short photoperiod with values of 70.34% ± 3.06%, 68.43% ± 4.16%, and 65.32% ± 3.67% for COD, TN, and TP removal (Figs. 4c, 5c, and 6c), respectively. Therefore, moderate light intensity 350 mol m-2 s-1 with middle photoperiod 14 h light:10 h dark was the optimum parameters for biogas effluent nutrient reduction. [Figure 4 inserts here] [Figure 5 inserts here] [Figure 6 inserts here]
4. Discussion
Throughout the experiment, both pH and DIC values in the biogas effluent culture only slightly changed because the CO2 concentration almost remained unchanged (Table 1). A large amount of CO2 had already dissolved in the biogas effluent during the anaerobic digestion process. Thus, the biogas effluent culture at a relatively higher biogas CO2 concentration did not achieve a lower pH value. Meanwhile, the biogas effluent culture had an initially high concentrated DIC because CO2 was its main component (Lei et al., 2007). Furthermore, the biogas CO2 always met the level 10
required for microalgal growth because sufficient CO2 was supplied to dissolve in the biogas effluent culture (Carvalho et al., 2006). Although the dissolved CO2 in the biogas effluent culture was used by microalgal cells for growth, the consumed CO2 in the culture was immediately replaced by CO2 in the biogas, thereby maintaining the DIC and pH (Papazi et al., 2008). The variation ranges of DO of the biogas effluent culture were always below the excessive DO level (35 mg L-1), which enabled the avoidance of microalgal growth inhibition (De Godos et al., 2010). Carvalho et al. (2006) reported that in a closed photo-bioreactor (same as the photo-bioreactor bag used in this study), O2 in the initial biogas or that produced during microalgal photosynthesis may inhibit microalgal growth, because the accumulation of O2 in the microalgal culture caused photorespiration. This phenomenon did not occur in this research because of the existence of a relatively higher initial CO2 concentration in the photo-bioreactor bag and the organic carbon in the biogas effluent culture (Douskova et al., 2009). Van Den Hende et al. (2011) suggested that the increase in the CO2:O2 ratio by the addition of more inorganic carbon to the photo-bioreactor or the addition of organic carbon to the microalgal culture were both able to overcome the potential microalgal photosynthesis inhibition. Light was found to be the most important factor affecting microalgal growth because it was the fundamental energy source of the synthesis of microalgal cell protoplasm. When the light intensity was insufficient (Fig. 1a), the microalgal DW was lower than that under the optimum moderate light treatment (Fig. 1b) because the microalgae consumed carbohydrates during photorespiration (Jeong et al., 2013). In this state, microalgal growth always occurred under a photo-limitated condition. Thus, an increase in the photoperiod (i.e., longer lighting time) realized better microalgal growth (Xue et al., 2011). By contrast, when microalgae were exposed to light levels 11
above the light saturation limit (Fig. 1c), the microalgae were unable to use light efficiently because the excessive light intensity caused overloaded photosystems, bleached pigments, and ultimately destroyed photosystems (Jeong et al., 2013). In this state, microalgal growth occurred under conditions that were always beyond the saturation light intensity. Thus, an decrease in the photoperiod (i.e., shorter lighting time) realized higher microalgae growth (Xue et al., 2011). Under the moderate light intensity treatment (Fig 1b), only the middle photoperiod treatment realized the highest microalgal DW; longer or shorter photoperiod treatments obtained even lower microalgal DW values than those under the insufficient light intensity treatments (Fig. 1a). These findings can be explained by the theory that microalgal growth is affected by the light intensity and the duration of lighting in each photoperiod, i.e., the amount of light energy offered in each photoperiod light-dark cycle (Toro, 1989). Thus, the moderate light intensity treatments with photoperiod 16 h light:8 h dark or 12 h light:12 h dark provided relatively excess (20.16 mol m-2) or inadequate (15.12 mol m-2) light energy, respectively. The light energy of photoperiod 14 h light:10 h dark (17.64 mol m-2) was the most appropriate. Furthermore, Kumar et al. (2010) suggested that the availability of lighting was important for microalgal growth, and the maximum photosynthetic efficiencies can be achieved when photoperiod approached the microalgal photosynthetic unit turnover time. However, the treatments under different light intensities with equal light energy (i.e., 17.28 mol m-2 for photoperiod 16 h light:8 h dark under 300 mol m-2 s-1 in Fig. 1a and photoperiod 12 h light:12 h dark under 400 mol m-2 s-1 in Fig. 1c; 20.16 mol m-2 for photoperiod 16 h light:8 h dark under 350 mol m-2 s-1 in Fig. 1b and photoperiod 14 h light:10 h dark under 400 mol m-2 s-1 in Fig. 1c) did not achieve the same microalgal DW. In fact, the microalgal DW in the 12
higher light intensity treatment (Fig. 1c ) always led to much lower values. This finding was due to the fact that the high illumination intensities damaged the photosynthetic receptor system of the microalgae within a few minutes, and this phenomenon is known as photoinhibition (Anderson, 2005). Only treatments under moderate light intensity with middle photoperiod and low light intensity with long photoperiod efficiently removed CO2 from biogas and achieve the standard for efficient combustion (i.e., CH4 concentration >90%, v/v) (Ryckebosch et al., 2011). Furthermore, their O2 concentration (v/v) was outside the explosive range (O2 concentration = 0%–4%, v/v) (Ryckebosch et al., 2011). These results agreed with the variation trends of microalgal DW (Fig. 1) because approximately half of the microalgal DW was carbon derived from CO2 (Chisti, 2007). The microalgal photosynthesis consisted of two steps: light reactions that occurred only when the cells were illuminated, and dark reactions (i.e., carbon-fixation reactions) that occurred both in the presence and absence of light. In the light reactions, light energy was used to transfer electrons from water to coenzyme II (i.e., nicotinamide-adenine dinucleotide phosphate, NADP +) forming nicotinamide adenine dinucleotide phosphate (NADPH) and generate the highly energetic compound adenine triphosphate (ATP). Then, light energy transformed into the chemical energy ATP, which was stored for later use in the CO2-fixation reactions (Iverson, 2006; Baba and Shiraiwa, 2012). The microalgae developed the CO2-concentrating mechanism for adapting to changes in CO2 concentration. The chloroplast envelope and carbonic anhydrases of microalgae could facilitate the diffusion by stimulating the indirect supply of CO2 from outside the cells to Rubisco. Meanwhile, the inorganic carbon transporters were able to facilitate the DIC membrane transport of CO2 or bicarbonate through plasmalemma (Baba and Shiraiwa, 2012). In addition, the existence of O2 and 13
H2O (Table 2) in upgraded biogas were not expected to exert any harmful effect on microalgal growth because crude biogas was always saturated with water. Given that microalgae were cultured in liquid (biogas effluent) in this research water and O2 were the compounds consumed during microalgal photosynthesis (Kubler et al., 1999). The nutrient in the biogas effluent was efficiently reduced during the biogas upgrade process. The variation trends of reduced efficiencies of COD, TN, and TP (Figs. 4–6) agreed with that of microalgal growth (Fig. 1) and CO2 removal (Fig. 2). This finding can be attributed to the fact that microalgal photosynthesis and microalgal-CO2 fixation were environmentally sustainable when combined with wastewater treatment (Ahrens and Sander, 2010). Nutrient reduction was mainly achieved by the assimilation of microalgal photosynthesis because the microalgal reproduction requires abundant carbon, nitrogen, and phosphorous sources for synthesizing nucleic acids, phospholipids, and proteins (Munoz and Guieysse, 2006; Kumar et al., 2010). Moreover, the metabolic process of biogas effluent nutrient involved its accumulation of the chemical energy source ATP (the reduced form of nicotinamide-adenine dinucleotide) and the components of microalgal backbones. These phenomena were able to promote the autotrophic metabolism process of the microalgae (Rittenberg, 1969). Particularly, the accumulated ATP can be used as an enzyme activator during the autotrophic metabolism process (i.e., the abovementioned dark reactions and carbon-fixation reactions) (Moore and Cohen, 1967).
5. Conclusion
In this research, method about removing CO2 from biogas by microalgal culturing using biogas effluent as nutrient medium could effectively upgrade biogas and simultaneously reduce biogas effluent nutrient. Microalgal growth achieved the 14
highest DW value at moderate light intensity with middle photoperiod. Whereas, the low light intensity with long photoperiod and moderate light intensity with middle photoperiod both obtained the best biogas upgrade effects. Meanwhile, the moderate light intensity with middle photoperiod also had the highest biogas effluent nutrient reduction. Therefore, the optimal parameters were moderate light intensity 350 mol m-2 s-1 with middle photoperiod 14 h light:10 h dark.
Acknowledgments
This study was sponsored by the Beijing Green Future Environment Foundation, the National Key Special Project for Water Pollution Control and Treatment (Grant no. 2012ZX07102-004), and the National Science Funds of China (Grant no. 51102136/E021301).
Reference
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19
Figure Captions Fig. 1 Microalgae dry weight under various photoperiods and light intensities: (a) 300,
(b) 350, and (c) 400 mol m-2 s-1. Fig. 2 Biogas CO2 removal efficiency under various photoperiods and light intensities:
(a) 300, (b) 350, and (c) 400 mol m-2 s-1. Fig. 3 Biogas CH4 concentration under various photoperiods and light intensities: (a)
300, (b) 350, and (c) 400 mol m-2 s-1. Fig. 4 Biogas effluent chemical oxygen demand removal efficiency under various
photoperiods and light intensities: (a) 300, (b) 350, and (c) 400 mol m-2 s-1. Fig. 5 Biogas effluent total nitrogen removal efficiency under various photoperiods
and light intensities: (a) 300, (b) 350, and (c) 400 mol m-2 s-1. Fig. 6 Biogas effluent total phosphorus removal efficiency under various photoperiods
and light intensities: (a) 300, (b) 350, and (c) 400 mol m-2 s-1.
20
700
-1
Microalgae dry weight (mg L )
650
550 500 450 400 350 300 250
-1
Microalgae dry weight (mg L )
650
b
600 550 500 450 400 350 300 250 200 700
650 -1
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
600
200 700
Microalgae dry weight (mg L )
a
c
600 550 500 450 400 350 300 250 200 0
1
2
3
4
Experimental time (d)
Fig. 1 21
5
6
100
Biogas CO2 removal efficiency (%)
90
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
80 70 60 50 40 30 20 10 0 100
90
Biogas CO2 removal efficiency (%)
a
b
80 70 60 50 40 30 20 10 0 100
Biogas CO2 removal efficiency (%)
90
c
80 70 60 50 40 30 20 10 0 1
2
3
4
Experimental time (d)
Fig. 2 22
5
6
Biogas CH4 concentration (%)
100
a
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
95
90
85
80
75
70
Biogas CH4 concentration (%)
100
95
90
85
80
75
70 100
Biogas CH4 concentration (%)
b
c
95
90
85
80
75
70 0
1
2
3
4
Experimental time (d)
Fig. 3 23
5
6
100
a
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
COD removal efficiency (%)
90 80 70 60 50 40 30 100
b
COD removal efficiency (%)
90 80 70 60 50 40 30
100
c
COD removal efficiency (%)
90 80 70 60 50 40 30 1
2
3
4
Experimental time (d)
Fig. 4 24
5
6
100
a
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
TN removal efficiency (%)
90 80 70 60 50 40 30 100
b
TN removal efficiency (%)
90 80 70 60 50 40 30 100
c
TN removal efficiency (%)
90 80 70 60 50 40 30 1
2
3
4
Experimental time (d)
Fig. 5 25
5
6
100
a
12 h light : 12 h dark 14 h light : 10 h dark 16 h light : 8 h dark
TP removal efficiency (%)
90 80 70 60 50 40 30 100
b
TP removal efficiency (%)
90 80 70 60 50 40 30 100
c
TP removal efficiency (%)
90 80 70 60 50 40 30 1
2
3
4
Experimental time (d)
Fig. 6
26
5
6
Table 1 Variations of physical and chemical parameters in biogas effluent culture Treatments Light intensity (μmol m-2 s-1)
Photoperiod (light : dark) 12 h :12 h
300
14 h : 10 h
16 h : 8 h
12 h :12 h
350
14 h : 10 h
16 h : 8 h
400
12 h :12 h
Experimental time (day) Parameters
0
1
2
3
4
5
6
pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1)
6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47
6.72±0.51 997.34±52.91 5.52±0.41 6.81±0.53 998.34±56.26 5.51±0.38 6.71±0.35 1007.23±64.37 5.56±0.38 6.73±0.52 999.34±48.38 5.51±0.28 6.73±0.45 1001.23±58.47 5.56±0.52 6.82±0.41 1003.23±67.24 5.52±0.36 6.75±0.58 994.30±57.93 5.53±0.50
6.81±0.42 999.73±68.03 5.56±0.53 6.82±0.42 993.23±36.36 5.54±0.52 6.78±0.30 995.48±59.30 5.60±0.51 6.79±0.41 1004.33±57.43 5.56±0.32 6.82±0.52 999.54±38.65 5.58±0.37 6.69±0.53 998.35±63.93 5.56±0.50 6.73±0.69 992.47±40.93 5.56±0.61
6.74±0.52 985.32±71.36 5.61±0.42 6.75±0.65 1003.34±52.34 5.58±0.31 6.82±0.54 1003.73±85.39 5.64±0.42 6.72±0.64 996.45±53.20 5.60±0.48 6.83±0.71 994.52±59.60 5.61±0.42 6.74±0.49 999.54±47.39 5.59±0.61 6.82±0.49 1006.35±73.94 5.58±0.49
6.70±0.48 1004.32±32.50 5.63±0.50 6.70±0.43 1009.43±74.05 5.61±0.40 6.84±0.63 997.42±69.43 5.67±0.61 6.83±0.43 999.03±73.21 5.63±0.41 6.80±0.36 1007.45±86.43 5.67±0.58 6.78±0.56 1003.24±34.23 5.63±0.48 6.80±0.72 1004.34±54.02 5.63±0.40
6.68±0.35 1008.79±46.25 5.66±0.36 6.69±0.64 993.45±65.38 5.67±0.38 6.83±0.53 999.52±64.94 5.70±0.22 6.67±0.58 1005.32±57.48 5.64±0.30 6.75±0.59 994.36±62.95 5.72±0.63 6.82±0.64 993.23±56.39 5.67±0.51 6.74±0.51 999.40±58.46 5.67±0.52
6.82±0.64 998.31±51.47 5.68±0.42 6.74±0.31 997.36±56.89 5.68±0.61 6.73±0.54 994.52±76.40 5.73±0.35 6.75±0.58 1001.35±64.35 5.69±0.42 6.79±0.53 1000.36±69.25 5.77±0.49 6.85±0.62 990.46±49.86 5.75±0.34 6.79±0.60 1001.24±62.38 5.69±0.48
1
14 h : 10 h
16 h : 8 h
pH DIC (mg L-1) DO (mg L-1) pH DIC (mg L-1) DO (mg L-1)
6.77±0.63 996.51±47.85 5.49±0.47 6.77±0.63 996.51±47.85 5.49±0.47
6.81±0.46 1005.32±57.24 5.53±0.35 6.75±0.53 1005.39±84.32 5.51±0.38
6.75±0.39 1002.94±73.29 5.56±0.52 6.74±0.62 999.83±57.32 5.53±0.44
2
6.71±0.52 998.39±73.23 5.61±0.64 6.79±0.48 1006.34±52.60 5.56±0.47
6.83±0.61 999.05±39.23 5.63±0.47 6.68±0.42 993.42±83.23 5.58±0.59
6.72±0.49 1007.32±49.32 5.65±0.60 6.70±0.61 995.34±89.57 5.61±0.65
6.80±0.65 1006.75±73.21 5.67±0.32 6.82±0.53 994.34±37.43 5.63±0.47
Table 2 Concentrations of O2 and H2O in the biogas Treatments Light intensity (μmol m-2 s-1)
Photoperiod (light : dark) 12 h :12 h
300
14 h : 10 h 16 h : 8 h 12 h :12 h
350
14 h : 10 h 16 h : 8 h 12 h :12 h
400
14 h : 10 h 16 h : 8 h
Experimental time (day) Concentration (%, v/v)
0
1
2
3
4
5
6
O2 H2O O2 H2O O2 H2O O2 H2O O2 H2O O2 H2O O2 H2O O2 H2O O2 H2O
0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35 0.23±0.02 3.11±0.35
0.24±0.02 3.16±0.31 0.26±0.04 3.25±0.36 0.26±0.03 3.19±0.32 0.25±0.03 3.37±0.41 0.27±0.04 3.31±0.25 0.25±0.03 3.26±0.25 0.25±0.04 3.14±0.23 0.25±0.03 3.10±0.32 0.25±0.03 3.28±0.34
0.30±0.04 3.28±0.27 0.33±0.04 3.28±0.36 0.34±0.03 3.10±0.54 0.34±0.04 3.25±0.37 0.39±0.03 3.28±0.24 0.33±0.04 3.29±0.21 0.31±0.03 3.26±0.42 0.32±0.03 3.15±0.31 0.31±0.03 3.28±0.27
0.41±0.04 3.14±0.32 0.45±0.05 3.19±0.28 0.47±0.04 3.27±0.35 0.44±0.03 3.36±0.21 0.50±0.03 3.18±0.27 0.43±0.03 3.20±0.31 0.37±0.04 3.29±0.37 0.36±0.05 3.34±0.21 0.37±0.04 3.19±0.47
0.60±0.05 4.18±0.36 0.63±0.06 3.26±0.31 0.64±0.05 3.29±0.31 0.62±0.05 3.17±0.22 0.68±0.04 3.21±0.35 0.61±0.04 3.17±0.21 0.56±0.04 3.25±0.36 0.47±0.04 3.15±3.11 0.43±0.04 3.28±0.31
0.68±0.06 3.05±0.34 0.71±0.05 3.17±0.38 0.73±0.06 3.16±0.24 0.71±0.06 3.25±0.26 0.79±0.06 3.36±0.24 0.71±0.05 3.42±0.32 0.61±0.06 3.29±0.24 0.56±0.05 3.24±0.26 0.49±0.05 3.15±0.31
0.79±0.08 3.17±0.30 0.87±0.07 3.14±0.32 0.89±0.07 3.19±0.28 0.83±0.06 3.14±0.23 0.94±0.05 3.25±0.25 0.82±0.06 3.05±0.24 0.67±0.06 3.17±0.28 0.64±0.06 3.35±0.31 0.54±0.05 3.17±0.26
3
Highlights > Moderate light intensity with middle photoperiod was optimum for microalgal
growth. > Moderate light intensity with middle photoperiod was optimum for nutrient reduction. > Low light intensity with long photoperiod obtained the best biogas upgrade effects. > Moderate light intensity with middle photoperiod was also optimum for biogas upgrade. > The upgraded CH4 concentration was 92.16% ± 2.83% (v/v).
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