Cultivation of Chlorella sp. using raw dairy wastewater for nutrient removal and biodiesel production: Characteristics comparison of indoor bench-scale and outdoor pilot-scale cultures

Accepted Manuscript Cultivation of Chlorella sp. Using Raw Diary Wastewater for Nutrient Removal and Biodiesel Production: Characteristics Comparison of Indoor Bench-scale and Outdoor Pilot-scale Cultures Weidong Lu, Zhongming Wang, Xuewei Wang, Zhenhong Yuan PII: DOI: Reference:

S0960-8524(15)00767-1 http://dx.doi.org/10.1016/j.biortech.2015.05.094 BITE 15065

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

17 April 2015 24 May 2015 25 May 2015

Please cite this article as: Lu, W., Wang, Z., Wang, X., Yuan, Z., Cultivation of Chlorella sp. Using Raw Diary Wastewater for Nutrient Removal and Biodiesel Production: Characteristics Comparison of Indoor Bench-scale and Outdoor Pilot-scale Cultures, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech. 2015.05.094

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Cultivation of Chlorella sp. Using Raw Diary Wastewater for Nutrient Removal and Biodiesel Production: Characteristics Comparison of Indoor Bench-scale and Outdoor Pilot-scale Cultures Weidong Lua,b,c, Zhongming Wanga*, Xuewei Wangd, Zhenhong Yuana a

Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion,

Chinese Academy of Sciences, Guangzhou 510640, China b

School of Chemistry and Environmental Engineering, Shaoguan University, Shaoguan

512005, China c

Universtity of Chinese Academy of Sciences, Beijing 100049, China

d

Foshan Sanshui Energy Environmental Technology Innovation and Incubation Center,

Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Foshan 528137, China

*

Corresponding author. Tel:+86-20-3702-9685; E-mail address: [email protected] (Z. Wang)

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Abstract: The biomass productivity and nutrient removal capacity of simultaneous Chlorella sp. cultivation for biodiesel production and nutrient removal in raw dairy wastewater (RDW) in indoor bench-scale and outdoor pilot-scale photobioreactors were compared. Results from the current work show that maximum biomass productivity in indoor bench-scale cultures can reach 260 mg·L-1·day-1, compared to that of 110 mg·L-1·day-1 in outdoor pilot-scale cultures. Maximum chemical oxygen demand (COD), total nitrogen (TN), and total phosphorous (TP) removal rate obtained in indoor conditions was 88.38, 37.28, and 2.03 mg·L-1·day-1, respectively, this compared to 41.31, 6.58, and 2.74 mg·L-1·day-1, respectively, for outdoor conditions. Finally, dominant fatty acids determined to be C16/C18 in outdoor pilot-scale cultures indicated great potential for scale up of Chlorella sp. cultivation in RDW for high quality biodiesel production coupling with RDW treatment. Keywords:

Microalgae; Dairy wastewater; Nutrient removal; Biodiesel production

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1. Introduction Inappropriate discharge of livestock effluent with high concentration of nitrogen and phosphorous as well as high turbidity not only cause environment pollution but also waste lots of valuable nutrients which have substantially positive impacts on agricultural development. Biological treatment, such as anaerobic digestion is conventionally employed for aquaculture wastewater treatment (Rico et al., 2011; Deng et al., 2012). However, COD, TN and TP concentration in the effluents from anaerobic digestion facilities are still higher than the allowable discharge limits in China. Therefore, further treatment process is required to reduce COD, TN and TP to an acceptable level. In general, aerobic treatment processes, such as active sludge systems, involve an oxygen supply with an energy demand and extra sludge generation. High consumption of fossil fuels in industry and transportation sectors accelerate energy depletion and emit substantial quantities of greenhouse gases (GHG) into the atmosphere. Under these scenarios, the development of renewable energy is regarded as a promising approach to avoid further aggravation of the energy crisis and global climate change (Lam et al., 2012). Microalgae based biofuels, such as biodiesel, have great potential to be employed as a supplement to conventional fuel. Furthermore, oleaginous microalgae has further advantage as a biofuel feedstock compared to food crops, since microalgae biomass has a much shorter growth circle time, higher lipid content and can be cultivated in abandon land and wastewater. Therefore, using microalgae as algal based biodiesel feedstock has great potential in raw material assurance for biodiesel production and can avoid competition of limited arable land and fresh water. Finally, high culture medium cost and complicated production processes are still the most prominent bottlenecks that hinder commercialization of algal based

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biodiesel production. Thus, cultivation of microalgae in nutrient rich wastewater, such as aquaculture wastewater, makes it possible recover nutrients through microalgae assimilation and produce microalgae biomass economically. Currently, there is a great deal of research looking to integrate microalgae cultivation and wastewater treatment (including municipal sewage, brewery wastewater, and dairy industry wastewater, etc.) (Van et al., 2014a; Kothari et al., 2012; Ramos et al., 2014; Farooqet al., 2013). Cultivation of microalgae in dairy wastewater (DW) has also received increased attention. Huo et al. (2012) evaluated the feasibility of cultivation Chlorella zofingiensis in DW in bench-scale outdoor ponds with pH regulation by CO2 and acetic acid, and achieved a maximum TP and TN removal percentage of 97.5% and 51.7% in five days, respectively. Chen et al. (2012) concluded that non-filamentous green algae (Chlorella and Scenedesmus) were able to tolerate high nutrient loading of chemically pre-treated anaerobic digestion during long term continuous cultivation in outdoor raceway ponds. Van et al. (2014b) found that aquaculture wastewater could be remedied by microalgal bacteria flocs in sequencing reactors through photosynthetic aeration with a COD, TN and TP removal rate of 65, 9.8 and 1.56 mg·L-1·day-1, respectively. Ding et al. (2014) cultivated microalgae in DW without sterilization and obtained a maximum biomass concentration of 0.86 g·L-1 in 20% DW, NH3-N, TP and COD removal ratio of 83.20-99.26%, 89.92-91.97% and 84.18%-89.70% in 5%, 10%, and 20% RDW, respectively, in an 8-day indoor lab-scale experiment. The literature available on the combination of nutrient removal and microalgae cultivation in RDW (without sterilization, disinfection or chemical pre-treatment) in outdoor pilot-scale photobioreactors is limited. In addition, reports on microalgae cultivation process without aeration pure CO2, addition of other carbon source or pH

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adjustment are also rare. This is especially important, since the development of economically favorable production processes is imperative in facilitating the industrial-scale production of microalgae biomass for algal based biodiesel (Samori et al., 2013). Finally, it is necessary to ascertain the growth characteristic and nutrient removal capacity difference between indoor bench-scale and outdoor pilot-scale cultures for guidance of cost-effective microalgae based biodiesel commercialization, however, information from the literature is currently lacking. The objectives of this study are to: (1) evaluate the feasibility of simultaneous treatment of RDW and production microalgae based biodiesel at an outdoor pilot-scale site without disinfection, chemical pre-treatment, extra carbon source addition or pH control; (2) elucidate the distinction between indoor bench-scale and outdoor pilot-scale cultivation in microalgae growth characteristic and pollutant removal efficiency; (3) compare the fatty acid composition of indoor bench-scale and outdoor pilot-scale cultures and assess the scale-up potential of Chlorella sp. cultivation in RDW in outdoor photobioreactors. 2. Materials and Methods 2.1. Materials The Chlorella sp. strain was obtained from Guangzhou Institute of Energy Conversion (GIEC), Chinese Academic of Sciences (CAS). The RDW was collected from the floor flushing effluent discharge point of a local dairy farm breeding about one thousand dairy cattle, close to the Demonstration Base of Energy Microalgae Cultivation, GIEC, CAS, in Foshan, China. The RDW was settled by gravity overnight and subsequently filtered through gauze before use. The physico-chemical parameters of RDW were determined and presented in Table 1.

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2.2. Cultivation of microalgae in RDW The microalgae were firstly subjected to pre-culture in Erlenmeyer flasks in the lab in BG-11 medium (Rippka et al., 1979), then inoculated to four indoor bench-scale photobioreators (iPBRs, 1.3 L glass photobioreactors, Φ6×70 cm) containing 500 mL 25% RDW to achieve an initial algal density of 0.17 g·L-1 for two iPBRs and 0.34 g·L-1 for the other two iPBRs. A control experiment was done without inoculation. After inoculation, the cultures were sparged with air through the bottom of the iPBRs, and maintained at temperature of 25 ± 1 °C and illumination intensity of 3000 ± 10 lux at the single side all day round. As a comparison, the seeds were transferred to outdoor pilot-scale photobioreactors (oPBRs, 40L, Ø272×H1720) in Foshan, China (23°30′29.63″N, 112°51′13.13″E) in Autumn. The oPBRs contained 30 L RDW, the same as used in the indoor experiments. The initial microalgae biomass was kept at 0.34 g·L-1 for all outdoor treatments. All the experiments were run in duplicate. 2.3. Water quality analysis Approximately 10 mL microalgae suspension was taken out from each photobioreactor for COD, ammonium, total nitrogen (TN) and total phosphorous (TP) concentration analysis according to experimental design. The samples were first centrifuged at 5000 rpm for 5min. Then, the supernatants were filtered using a 0.22 µm nylon membrane filter, after which the filtrates were appropriately diluted. Finally, COD, ammonium, TN and TP concentration of the filtrates were measured following the Hach DR2700 Spectrophotometer Manual (Hach, 2008). The removal percentage (RP) and removal rate (RR) were calculated by the following formulas: RP (%) = (C0-Ct) / C0, RR (mg·L-1·day-1) = (C0-Ct)/(t-t0), where Ct and C0 are the nutrients concentration at time t and the beginning, respectively.

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2.4. Biomass concentration and productivity determination 1 mL wastewater containing biomass sampled from photobioreactors every day was filtered with a pre-dried and weighed (w1) 0.45 µm nominal pore size glass microfiber filter (Whatman, GF/F) and washed twice with distilled water under 0.1 MPa vacuum pressure, and subsequently dried at 70.0 ℃ until the weight was constant (w2) . The biomass concentration (as total suspend solids, TSS) was calculated by subtracting w1 from w2. The productivity and specific growth rate during the culture period was obtained by equations below: Biomass productivity (mg·L-1·day-1) = ( Xt - X0) / (t - t0); Specific growth rate (day-1) = Ln (Xt / X0) / (t - t0), Where Xt and X0 are biomass density at time t and t0 (at the beginning), respectively. After the eight-day experiment, culture broths were firstly harvested by gravity sedimentation and then centrifuged at 5000 rpm, for 5min. Supernatants were collected for water quality measurement and biomass pellets were washed with distilled water and freeze-dried at -80 °C. The dried biomass was milled and collected in screw capped containers for fatty acid composition analysis. 2.5. Preparation and analysis of fatty acid methyl esters (FAMEs) Lipids are conversed to FAMEs following an in-situ transesterification approach as described in the literature (Indarti et al., 2005), with slight modification. Specifically, 20 mg of freeze-dried biomass samples, 2.5 mL of methanol with 2 wt.% of concentrated sulfuric acid as the catalyst were added in a glass tube with a magnetic bar. The reaction mixture was sealed and incubated at 80 °C in a water bath for 2.5 h. On completion of the reaction, the mixtures were cooled down to room temperature. 1 mL of saturated NaCl aqueous solution and 2 mL of hexane were added into the mixtures to fully dissolve the FAMEs. Then the mixture was subjected to vortex oscillators for 30 s to

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ensure thorough mixing. Finally, the mixture was centrifuged (3000 rpm, 17 min) to form two phases, and the upper phase containing FAMEs was filtered through a 0.22 µm nylon membrane filter and transferred to a clean bottle and dried under N2 stream. Until now, the sample was ready for the FAME composition and content analysis, which was carried out by gas chromatography equipped with an FID detector (Shimadzu GC-2010, Japan). The injector and detector temperature was 300 and 250 °C, respectively. The temperature was initially programmed at 190 °C for 5 min, and then elevated by 10 °C/min to 250 °C for 7 min. Methyl hexadecanoate (C17:0) was used as internal standard and Ar as carrier gas. FAMEs were identified and quantified by comparison with the retention time of standards and the internal standard concentration. All the experiments were carried out in duplicate and average values as well as standard deviations were reported. Results were performed with EXCEL (Microsoft Office Enterprise, 2003) and origin pro 8.6 (Origin Lab, USA) and analysis of variance (ANOVA) was determined wherever applicable. 3. Results and Discussion 3.1. Biomass growth and productivity The variations of biomass concentration throughout the culture period are illustrated in Fig. 1. It is easily to see that the Chlorella sp. is able to quickly adapt to the RDW in the indoor bench-scale conditions with the two tested initial inoculation concentration. Dramatic increase of biomass concentration was observed after one day growth, and sustained buildup of this value could also be observed in the remaining culture time. Finally, the maximum biomass concentration reached 2.25 g·L-1 and 3.05 g·L-1 for 0.17 g·L-1 and 0.34 g·L-1 initial inoculation concentration, respectively. No static and decay phase were noticed since the experiment lasted only eight days. The biomass

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productivity and specific growth rate of 0.17 g·L-1 and 0.34 g·L-1 initial inoculation concentration was 260.0 mg·L-1·day-1, 0.3229 day-1 and 338.8 mg·L-1·day-1, 0.2742 day-1, respectively (Table 1). The biomass concentration and productivity in this study is much higher than that in the research carried out by Qin et al. (2014) who cultivated Chlorella vulgaris in untreated, UV pretreated and NaClO pre-treated DW and obtained biomass concentration ranged from 0.861 to 1.870 g·L-1, which could possibly be due to the different species and initial inoculated concentration and varied characteristics of DW. The remarkable inhibitory effect of RDW on the microalgae was observed in the first two days for all treatments under outdoor pilot-scale cultivation conditions (Fig. 1). This can be attributed to the undulation of temperature and illumination intensity outdoors during cultivation, as well as the possibly more intense competition from the indigenous microorganisms in the RDW of higher temperature outdoors. After that, a steady increase of biomass concentration was found in 25% RDW. For the treatments of 5% RDW and 10% RDW, however, no significant biomass accumulation could be observed before the fourth day of the experiment. This can be attributed to lack of nutrient in 5% RDW and 10% RDW treatments. The maximum biomass concentration achieved for 5% RDW, 10% RDW and 25% RDW in outdoor pilot-scale culture reached 0.70, 1.60, and 1.20 g·L-1, respectively, in the eight-day incubation. The higher biomass productivity obtained in indoor bench-scale cultures can be attributed to the controlled environment indoors, while temperature and irradiation intensity fluctuated significantly outdoors as illustrated in Fig. 2 (Juneja et al., 2013). During the experiment, the outdoor ambient temperature varied from 14.0 to 32.0 °C, and the irradiation intensity ranged from 123 to 1418 µmol·m-2·s-1 (Fig. 2). Considering the cost-effectiveness of commercial production, however, outdoor cultivation can cut down

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energy input and thus reduce the algal biomass production cost. The biomass productivity and specific growth rate of 5% RDW, 10% RDW, and 25% RDW in outdoor pilot-scale were 47.5 mg·L-1·day-1, 0.0978 day-1, 160.0 mg·L-1·day-1, 0.2012 day-1 and 110.0 mg·L-1·day-1, 0.1652 day-1, respectively, which was lower than that reported by Wang et al. (2010) who cultivated wildly isolated Chlorella sp. in anaerobic digested dairy manure with dilution multiples of 10, 15, 20 and 25 and achieved average specific growth rate of 0.282, 0.350, 0.407 and 0.409 day-1, respectively, in the first 7 days. This could possibly be due to the different composition of the raw and digested dairy manure as well as distinct cultivation surroundings with the present study (Nogueira et al., 2015). The lowest biomass concentration obtained in the 5% RDW treatment was caused by nutrient depletion as illustrated in Fig. 3, Fig. 4, Fig. 5 and Fig. 6, which was in accordance with the point stated by Chen et al. (2012). The lower biomass growth rate of with 25% RDW than 10% RDW was possibly caused by higher turbidity as more solid colloidal particles and organic matter which decreased the opacity of the culture medium (Chen et al., 2012). 3.2. Nutrient removal Nutrients such as carbon, nitrogen and phosphorous are the main ingredients in the eukaryotic algae culture medium. RDW contains high concentration of carbon, nitrogen and phosphorous, which has great potential to be used as an alternative to the conventional culture medium in microalgae cultivation. The changes of TN, ammonium, TP, and COD concentration in RDW during cultivation in outdoor pilot-scale are exhibited in Fig. 3, Fig. 4, Fig. 5 and Fig. 6, respectively. From Fig. 3, it is evident to see that there is a continuous reduction of TN within the first two days for all treatments. Nitrogen can be reduced in two approaches, one is assimilated by algae (Razzak et al.,

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2013), another is air stripping due to fierce aeration under high alkalinity of RDW (Li et al., 2011). Afterwards, TN concentration decreased very slowly in the 5% RDW and 10% RDW compared to continuing significant decrease in 25% RDW experiments, which could be attributed to a much higher ammonium concentration which remained in 25% RDW (Fig. 4). There is no obvious difference in the final TN removal percentage between these treatments, all of which ranged from 83.38% to 85.17% (Table 3). There was no notable difference between the TN removal efficiency in this study and our previous test, in which a TN removal percentage of 82.70% was achieved by cultivating Chlorella zonfigiensis in the autoclaved piggery wastewater (Zhu, et al., 2013). However, much higher TN removal rate in 25% RDW (6.58 mg·L-1day-1) was achieved compared to only 2.05 mg·L-1day-1 and 2.62 mg·L-1day-1 for 5% RDW and 10% RDW, respectively. The reason was that no more nitrogen was available for microalgae uptake after a quick decline within two days in 5% RDW and 10% RDW. The characteristic of TN variation suggests that nitrogen supplement at appropriate interval (in continuous or semi-continuous cultivation mode) is necessary to maximize nitrogen removal rate. The variation of ammonium concentration during incubation is plotted in Fig. 4. For 25% RDW, ammonium concentration was reduced from 43.20 mg·L-1 to 11.80 mg·L-1 in the first four days, with a removal ratio of 72.70%. As a comparison, it took only one day for microalgae grown in 5% RDW and 10% RDW to reduced ammonium concentration from 8.80 and 16.70 mg·L-1 to 0.00 and 5.85 mg·L-1, respectively. By the end of the cultivation, ammonium could not be detected in all treatments, which was consistent with the results reported by Wang et al. (2010). Fig.5 shows the changes of TP concentration during the experiment, similar to the removal of TN, a sharp decrease can also be observed in 5% RDW and 10% RDW in

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the initial two days although the biomass concentration was still in lag phase. It had been reported that there were two kinds of phosphorous removal mechanisms, including biomass assimilation and chemical precipitation induced by alkalinity of culture medium (Sara et al., 2014). In the present study, the culture pH remained above 8.5 during the experiments (data not shown). Therefore, it is reasonable to deduce that the removal of phosphorous in the initial two days is attributed to chemical precipitation, after which, TP concentration nearly levelled off and was reduced to only 0.1 mg·L-1 at the end of the cultivation. For treatment of 25% RDW, similar patterns of TP concentration variation was recorded as that of the other two treatments. Phosphorous was still removed slowly, however, after four days and decreased to 7.35 mg·L-1 at the end of the experiment, which was lower than the discharge limits of China (8.0 mg·L-1). The TP removal percentage was only 65.33% in 25% RDW, compared to 66.68 % and 88.47% of 5% RDW and 10% RDW, respectively (Table 3). Much higher TP removal rate was observed for 25% RDW, however, with 2.74 compared to 1.04 and 0.63 mg·L-1day-1 for 5% RDW and 10% RDW, respectively. Therefore, higher TP concentration in the substrate would help to accelerate TP removal efficiency. A few species of microalgae can use dissolved organic carbon as carbon sources (Wang et al., 2010). The COD data are depicted in Fig. 6. Microalgae was not so highly efficient in removing organic substance compared to nitrogen and phosphorous. The maximum COD removal percentage and removal rate of 54.82% and 41.31 mg·L-1day-1, respectively, are achieved in 25% RDW, which is higher than that reported by Wang et al. (2010), in which COD removal percentage is only 27.4% to 38.4%. The better performance of COD removal percentage in the present research can be attributed to the degradation effect by the indigenous bacteria in RDW as indicated in table 3, where

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COD removal ratio of 84.33% was achieved in the control of indoor experiments. The COD removal ratio in outdoor test of the current study, however, is lower than that obtained by Qin et al. (2014) who reported the maximum COD removal cultivating Chlorella vulgaris in NaClO and UV pre-treated DW ranged from 73.7 to 74.8% and 51.5 to 65.8%, respectively. The possible reasons are the drastic varation of iridiance and ambient temperature which affect the microalgae and indigenous bacteria growth. In addition, lower utility of the organic substances in RDW for microalgae growth could also lead to worse COD removal performance (Passero et al., 2014). After the second day, the steadily rise of COD in 5% RDW and 10% RDW might be attributed to soluble organic compounds released by algae (Fig. 4). As a comparison, Chlorella sp. was also cultivated in RDW in indoor bench-scale in terms of nutrient removal percentage and removal rate under different initial inoculation concentration simultaneously. It can be seen from Table 3 that, no significant difference in nutrient removal percentage and removal rate was observed among different treatments in indoor cultures. However, cultures in indoor conditions showed much higher nutrient removal capacity than that in outdoor conditions. Specifically, COD removal rate could be achieved ranging from 88.88 to 91.75 mg·L-1day-1, compared to only 3.88 to 41.31mg·L-1day-1 in outdoor pilot scale cultures. Higher nutrient removal efficiency in indoor bench-scale cultures can be attributed to the controlled conditions, which is beneficial to microalgae growth. 3.3. Fatty acid profiles and biodiesel productivity FAMEs are the principal component of biodiesel and the chemical structure of FAMEs play a critical role in the properties of biodiesel. Previous studies have indicated that, by alternating the environmental conditions the microalgae is exposed to during growth, it is possible to change the biosynthesis of fatty acids significantly (Los et al.,

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2004). Thus it is crucial to uncover the distinct profiles of fatty acids between indoor and outdoor cultures. Table 4 reveals the composition and distribution of fatty acids derived from the biomass cultivated indoor (0.17 g·L-1 initial concentration indoor) and outdoor (25% RDW outdoor) in the current study. As illustrated in Table 4, hexadecanoic acid (C16:0), palmitoleic acid (C16:1), octadecenoic acid (C18:1) and linoleic acid (C18:2) were the abundant fatty acids in indoor cultures, while C16:0, stearic acid (C18:0), C18:1 and linolenic acid (C18:3) were more prominent in outdoor biomass. Therefore, the FAMEs obtained by cultivating Chlorella sp. in RDW is an ideal alternative for biodiesel according to the point stated by Schenk et al. (2008). However, C16:1 and C18:2 in outdoor biomass are lower than that in indoor cultures, while there is a greater distribution of C18:0 and C18:3 distributed more in outdoor cultivation conditions. Tridecanecarboxylic acid (C14:0), Myristoleic acid (C14:1), Eicosanoic acid (C20:0), Eicosadenoic acid (C20:2), Arachidonic Acid (C20:4), Docosanoic acid (C22:0) and Lignoceric acid (C24:0) could also be detected in both indoor and outdoor culture samples. With regard to proportion of unsaturated fatty acids, indoor bench-scale had a significant higher value than that in outdoor pilot-scale (62.63% vs 51.03%), which might partially be attributed to higher temperature and light intensity in outdoor (Fig. 2) (Feng et al., 2014 and Subhash et al., 2014). The increased unsaturated fatty acids facilitate the biodiesel to have lower pour point in cold areas. However, augment in saturated fatty acid can result in superior oxidative stability of biodiesel. As indicated in Table 4, FAMEs productivity of indoor bench-scale are superior to that in outdoor pilot-scale culture, which was caused by the higher biomass productivity and FAME yield in indoor cultures (Fig. 1 and Table 4). The FAME content in biomass obtained was 55.54 and 34.90 mg·(g-1 dry weight) in indoor and

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outdoor cultures, which were both lower than that obtained by previous research carried out in autoclaved and NaClO-pretreated piggery wastewater both indoors and outdoors (Zhu et al., 2013). 4. Conclusions This study has clearly demonstrated the differences of integrating nutrient removal from RDW and biodiesel production in both indoor bench-scale and outdoor pilot-scale conditions. The fatty acid profiles of biomass suggesting that simultaneously remediation of RDW and production Chlorella sp. biomass in outdoor pilot-scale facilities is a feasible process for biodiesel production. The optimal culture conditions still required further investigation, however, for maximum FAMEs productivity in outdoor cultivation. Finally, evaluation of the potential challenge of loss of productivity of the system due to biofouling and growth inhibition in long term outdoor cultivation should be carried out in further study.

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Acknowledgments The authors gratefully thank Dr. William Nock of University of Southampton for a proof-reading of English. This research was supported by grants from the National Natural Science Foundation of China (Grant No. 51476177) and the CAS-Foshan cooperation project (2012HY100415), providing partial financial support, are also greatly acknowledged. The authors would also like to thank the two anonymous reviewers for their helpful comments and suggestions that greatly improved the manuscript.

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References 1. Chen, R., Li, R., Deitz, L., Liu, Y., Stevenson, R.J., Liao, W., 2012. Freshwater algal cultivation with animal waste for nutrient removal and biomass production. Biomass Bioenerg., 39, 128-138. 2. Deng, L.W., Chen, Z.A., Yang, H. , Zhu, J.Y. , Liu, Y., Long, Y. , Zheng, D., 2012. Biogas fermentation of swine slurry based on the separation of concentrated liquid and low content liquid. Biomass Bioenerg., 45, 187-194. 3. Ding, J.F., Zhao, F.M., Cao, Y.F., Xing, L., Liu, W., Mei, S., Li, S.J., 2014. Cultivation of microalgae in dairy wastewater without sterilization. Int. J. Phytoremediat., 17, 222-227. 4. Farooq, W ., Lee, Y.C., Ryu, B.G., Kim, B.H., Kim, H.S., Choi, Y.E., Yang, J.W., 2013. Two-stage cultivation of two Chlorella sp. Strains by simultaneous treatment of brewery wastewater and maximizing lipid productivity. Bioresour. Technol., 132, 230-238. 5. Feng, P.Z., Deng, Z.Y., Hu, Z.Y., Wang, Z.M., Fan, L., 2014. Characterization of Chlorococcum pamirum as a potential biodiesel feedstock. Bioresour. Technol., 162,115-122. 6. Hach. 2008. Procedure Manual. Hach, Loveland, CO. 7. Huo S. H., Wang Z. M., Zhu S.N., Zhou W. Z., Dong R.J., Yuan Z.H., 2012. Cultivation of Chlorella zofingiensis in bench-scale outdoor ponds by regulation of pH using dairy wastewater in winter, South China. Bioresour. Technol., 121, 76-82. 8. Indarti, E., Majid, M.I.A., Hashim, R., Chong, A., 2005. Direct FAME synthesis for rapid total lipid analysis from fish oil and cod liver oil. J. Food Compos. Anal., 18, 161-170.

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9. Juneja, A., Ceballos, R.M., Murthy, G.S., 2013. Effects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production: a review. Energies, 6, 4607-4638. 10. Kothari R., Pathak V.V. , Kumar V., Kumar V., Singh D.P., 2012. Experimental study for growth potential of unicellular alga Chlorella pyrenoidosa on dairy wastewater: An integrated approach for treatment and biofuel production. Bioresour. Technol., 116, 466-470. 11. Lam M.K., Lee K.T., Mohamed A.R., 2012. Current status and challenges on microalgae-based carbon capture. Int. J. Greenh. Gas Control, 10, 456-469. 12. Li Y. C., Chen Y. F., Chen P., Min M., Zhou W.G., Martinez, B., Zhu J., Ruan R., 2011. Characterization of a microalga Chlorella sp. well adapted to highly concentrated municipal wastewater for nutrient removal and biodiesel production. Bioresour. Technol., 102, 5138-5144. 13. Los D. A., Murata N., 2004. Membrane fluidity and its roles in the perception of environmental signals. BBA-Biomembr., 1666, 142-157. 14. Nogueira D.P.K., Silva A.F., Araujo O.Q.F., Chaloub, R.M., 2015. Impact of temperature and light intensity on triacylglycerol accumulation in marine microalgae. Biomass Bioenerg., 72, 280-287. 15. Passero M.L., Cragin B., Hall A.R., Staley, N., Coats, E.R., McDonald, A.G., Feris, K., 2014. Ultraviolet radiation pre-treatment modifies dairy wastewater, improving its utility as a medium for algal cultivation. Algal Res., 6, 98-110. 16. Qin L., Shu Q., Wang Z. M., Shang C.H, Zhu S.N., Xu J.L., Li R.Q., Zhu L.D., Yuan Z.H., 2014. Cultivation of Chlorella vulgaris in dairy wastewater pretreated by UV irradiation and sodium hypochlorite. Appl. Biochem. Biotechnol., 172, 1121-1130.

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17. Razzak,S.A., Hossain, M.M., Lucky, R.A., Bassi, A.S., de Lasa, H., 2013. Integrated CO2 capture, wastewater treatment and biofuel production by microalgae culturing-A review. Renew. Sust. Energ. Rev., 27, 622-653. 18. Rico, C., Rico, J.L., Tejero, I., Munoz, N., Gomez, B., 2011. Anaerobic digestion of the liquid fraction of dairy manure in pilot plant for biogas production: Residual methane yield of digestate. Waste Manage., 31, 2167-2173. 19. Rippka, R., Deruelles, J., Waterbury, J., Herdman, M., Stanier, R.Y., 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol., 111, 1-61. 20. Samori, G., Samori, C., Guerrini, F., Pistocchi, R., 2013. Growth and nitrogen removal capacity of Desmodesmus communis and of a natural microalgae consortium in a batch culture system in view of urban wastewater treatment: part I. Water Res., 47, 791-801. 21. Sara, R. A., Nima, M. N., Saeedeh, S., Azam, S., Aboozar, K., Pegah, M., Mohammad, A.M., Younes, G., 2014. Removal of nitrogen and phosphorus from wastewater using microalgae free cells in batch culture system. Biocatal. Agric. Biotechnol., 3, 126-131. 22. Schenk, P. M., Thomas-Hall, S. R., Stephens, E., Marx, U.C., Mussgnug, J.H., Posten, C., Kruse, O., Hankamer, B., 2008. Second generation biofuels: high-efficiency microalgae for biodiesel production. BioEnergy Res., 1, 20-43. 23. Subhash G. V., Rohit M.V. , Devi P. M., Swamy, Y.V., Venkata Mohan, S., 2014. Temperature induced stress influence on biodiesel productivity during mixotrophic microalgae cultivation with wastewater. Bioresour. Technol., 169, 789-793. 24. Ramos Tercero, E. A., Sforza, E., Morandini, M., Bertucco, A., 2014. Cultivation of

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Chlorella protothecoides with urban wastewater in continuous photobioreactor: Biomass productivity and nutrient removal. Appl. Biochem. Biotechnol., 172, 1470-1485. 25. Van den Hende S., Beelen, V., Bore, G., Boon, N., Vervaeren, H., 2014a. Up-scaling aquaculture wastewater treatment by microalgal bacterial flocs: From lab reactors to an outdoor raceway pond. Bioresour. Technol., 159, 342-354. 26. Van den Hende S., Carre, E., Cocaud E., Beelen, V., Boon, N., Vervaeren, H., 2014b. Treatment of industrial wastewaters by microalgal bacterial flocs in sequencing batch reactors. Bioresour. Technol., 161, 245-254. 27. Wang L., Li Y. C., Chen P., Min, M., Chen Y.F., Zhu J., Ruan R.R., 2010. Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp. Bioresour. Technol., 101, 2623-2628. 28. Zhu L. D., Wang Z. M., Takala J., Hiltunen, E., Qin, L., Xu, Z.B., Qin, X.X., Yuan Z.H., 2013. Scale-up potential of cultivating Chlorella zofigiensis in piggery wastewater for biodiesel production. Bioresour. Technol., 137, 318-325.

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Fig.1. Variation of biomass concentration during cultivation.

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Fig.2. The variation of ambient temperature and irradiance of each day (8:00 am, 12:00 and 16:00) under outdoor conditions (means ± SD)

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Fig.3. TN concentration variation during cultivation.

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Fig.4. Variation of ammonium concentration during cultivation.

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Fig.5. Variation of TP concentration during cultivation.

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Fig.6. Variation of COD during cultivation.

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Table 1 Physico-chemical characteristics of DW(means ± SD) Parameters pH Suspended solid (mg· L-1) TN (mg· L-1) Ammonium (mg· L-1) TP (mg· L-1) COD (mg· L)

Value 8.18±0.03 1.74±0.09 283.00±12.73 181.50±9.19 115.90±7.50 2593.00±15.56

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Table 2 Specific growth rate and biomass productivity of indoor and outdoor culture Treatments

Specific growth rate (day-1)

Biomass productivity (mg· L-1·day-1)

0.17 g·L-1 initial con. indoor

0.3229±0.0039

260.0±0.0088

0.2742±0.0029

338.8±0.0088

-1

0.34 g·L initial con. indoor 5% RDW outdoor

0.0978±0.0000

47.50±0.0000

10% RDW outdoor

0.2012±0.0000

160.0±0.0000

25% RDW outdoor

0.1652±0.0000

110.0±0.0000

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Table 3 Nutrient removal rate and removal percentage in indoor and outdoor cultures

Treatments 0.00 g·L-1 initial con. indoor 0.17 g·L-1 initial con. indoor 0.34 g·L-1 initial con. indoor 5% RDW outdoor 10% RDW outdoor 25% RDW outdoor

COD Removal Removal Rate percentage (mg· L-1d-1) (%)

Removal rate (mg· L-1d-1)

TN Removal percentage (%)

Removal rate (mg· L-1d-1)

Removal percentage (%)

91.75±0.35

84.33±1.22

27.15±1.43

57.75±2.18

1.63±0.40

82.97±0.38

88.38±1.59

81.21±0.6

37.28±2.02

79.29±3.09

2.03±0.40

100.0±0.00

88.88±0.35

81.68±0.54

38.34±1.65

81.57±2.29

2.03±0.41

99.73±0.38

3.88±0.00

21.99±0.66

2.05±0.44

85.12±3.37

0.63±0.04

88.47±3.90

11.88±1.59

38.88±4.09

2.62±0.08

85.17±0.35

1.04±0.22

66.68±1.29

41.31±0.09

54.82±0.91

6.58±0.02

83.83±1.19

2.74±0.09

65.33±2.05

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TP

Table 4 Fatty acid profiles of indoor and outdoor cultures Distribution (%, of total FAMEs) Constituents

Indoor bench-scale

Outdoor pilot-scale

C14:0 6.36 ± 0.52 6.69 ± 0.18 C14:1 2.86 ± 0.08 2.08 ± 0.02 C16:0 24.87 ± 1.37 26.51 ± 1.14 C16:1 13.22 ± 0.84 7.66 ± 0.99 C18:0 6.44 ± 0.07 12.10 ± 0.48 C18:1 10.31 ± 1.54 15.62 ± 0.12 C18:2 28.18 ± 3.17 9.90 ± 0.01 C18:3 7.08 ± 0.51 13.94 ± 0.79 C20:0 0.28 ± 0.11 0.47 ± 0.05 C20:2 0.19 ± 0.03 0.16 ± 0.05 C20:4 1.67 ± 0.89 2.07 ± 0.07 C22:0 0.60 ± 0.01 1.15 ± 0.09 C24:0 0.12 ± 0.04 1.66 ± 0.40 Percentage of saturated fatty acids 37.37± 2.03 48.97 ± 1.70 (% of total FAME) Percentage of unsaturated fatty acids 62.63 ± 2.03 51.03 ± 1.70 (% of total FAME) FAME yield (mg· g-1 dry 55.54 ± 5.04 34.90 ± 3.21 weight)** FAME productivity 15.60 ± 0.93 5.23 ± 0.48 (mg· L-1·d-1 )*** ** -1 FAME yield (mg· g dry weight)=total FAME/dry weight of biomass *** FAME productivity(mg· L-1·d-1)=total FAME/(maximum biomass concentration×cultivation time)

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Highlights




Chlorella sp. was successfully cultivated in raw dairy wastewater outdoors.




Nutrients were efficiently removed in outdoor pilot-scale cultures.




110 mg·L-1·d-1 of biomass was achieved in outdoor pilot-scale cultures.




Scale up of Chlorella sp. production using raw dairy wastewater is possible.

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