Bioresource Technology 205 (2016) 111–117
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A comparative study on flocculating ability and growth potential of two microalgae in simulated secondary effluent Junping Lv, Junyan Guo, Jia Feng, Qi Liu, Shulian Xie ⇑ School of Life Science, Shanxi University, Taiyuan 030006, China
h i g h l i g h t s The self-flocculating property of Chlorococcum sp. GD was firstly reported. Chlorococcum sp. GD had excellent flocculating ability. The excellent flocculating ability of Chlorococcum sp. GD was related to EPS. Pollutants in secondary effluent were effectively removed by Chlorococcum sp. GD. The lipid content of Chlorococcum sp. GD was high.
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Article history: Received 21 November 2015 Received in revised form 10 January 2016 Accepted 11 January 2016 Available online 23 January 2016 Keywords: Microalgae Flocculating ability Pollutants removal Biomass and lipid accumulation Secondary effluent
a b s t r a c t The flocculating ability was an important property to microalgal harvesting, especially in secondary effluent. In this study, the flocculating ability of two microalgae, Chlorococcum sp. GD and Parachlorella kessleri TY, was evaluated after 10 d of cultivation in secondary effluent. After 180 min of settling, the flocculating ability of Chlorococcum sp. GD and P. kessleri TY was 84.43% and 16.23%, respectively. It was suggested that Chlorococcum sp. GD was an excellent self-flocculating microalgae. The mechanism on selfflocculating of Chlorococcum sp. GD was probably related to hydrophobic extracellular polymeric substances (EPS). Besides, compared to P. kessleri TY, the nitrogen and phosphorus removal efficiency of Chlorococcum sp. GD was high, which was up to 66.51% and 74.19%, respectively. Chlorococcum sp. GD also had high lipid content and biomass concentration. Therefore, Chlorococcum sp. GD could be regarded as a promising candidate for microalgal cultivation and harvesting in secondary effluent. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction The secondary effluent from wastewater treatment plants (WWTPs) is commonly rich in nitrogen and phosphorus owing to the defect of process itself or inadequate operation. As described in literatures, the total nitrogen and total phosphorus concentration of secondary effluent range from 5 to 30 mg/L, and from 0.2 to 3 mg/L respectively (Wu et al., 2014), which are much higher than the threshold of nitrogen and phosphorus causing the eutrophication in streams (Chambers et al., 2012). Therefore, it is essential to develop some technologies for advanced treatment of secondary effluent. Microalgae as the largest photoautotrophic group of plant taxa have high photosynthetic efficiency, rapid growth rate, and strong adaptability. Many microalgae are rich in oil, which can be further
⇑ Corresponding author. Tel./fax: +86 351 7018121. E-mail address: [email protected] (S. Xie). http://dx.doi.org/10.1016/j.biortech.2016.01.047 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
processed into biodiesel (Maity et al., 2014). Also, microalgae can metabolize nitrogen and phosphorus via different metabolic pathways (Cai et al., 2013). Therefore, microalgal-based biotechnology has been gradually developed for biodiesel production and wastewater treatment over the past few years (Rawat et al., 2013; Zeng et al., 2015). As an interesting topic, the integration of microalgae-based advanced treatment of secondary effluent and lipid production has been widely concerned in recent years (Arbib et al., 2014; Ji et al., 2013; Li et al., 2010; Sydney et al., 2011; Yang et al., 2011). However, there is a difficulty on microalgal harvesting from secondary effluents due to the relatively low cell density (Ji et al., 2013; Li et al., 2010). Currently, microalgal harvesting methods include centrifugation, chemical coagulation, gravity sedimentation, filtration, flotation, electrophoresis technique, and immobilized technique (Barros et al., 2015). Although these techniques can effectively harvest microalgal biomass from wastewaters, the huge consumption of chemicals and energies is a major challenge.
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Also, some chemicals show a certain degree of biomass toxicity (Rawat et al., 2013). Consequently, all drawbacks above limit the use of these technologies for large-scale harvesting of microalgae, especially in secondary effluent. Self-flocculating microalgae is a kind of microalgae, which can aggregate together to form large particles by themselves. Then, the aggregate can be easily harvested by sedimentation. Owing to environmental friendliness and no additional economic costs, screening and cultivating self-flocculating microalgae has been regarded as an alternative way to harvest microalgae. Till now, a few investigations have reported that some microalgae, such as Ankistrodesmus falcatus, Chlorella vulgaris, Ettlia texensis, Scenedesmus obliquus and Tetraselmis suecica, belong to self-flocculating microalgae (Alam et al., 2014; Guo et al., 2013; Salim et al., 2012, 2013, 2014). Especially, the flocculation efficiency of E. texensis after 3 h of sedimentation was around 90% (Salim et al., 2013). Nevertheless, the diversity of self-flocculating microalgae is low. Also, the flocculation performance of self-flocculating microalgae in secondary effluent has not been reported. Thus, it is important to screen more self-flocculating microalgae and evaluate their flocculating ability after cultivation of them in secondary effluent. Compared to a large number of microalgae successfully cultivated in municipal wastewater, industry wastewater, agricultural wastewater and anaerobic digestion wastewater (Chen et al., 2015; Morales-Amaral et al., 2015), the number of microalgae adapting to secondary effluent is limited. The main groups focus on Botryococcus, Chlorella and Scenedesmus, and have low biomass production without any supplement of exogenous carbon sources (Li et al., 2010; Sawayama et al., 1992; Yang et al., 2011). Thus, it is essential to screen more microalgae suitable for growth in secondary effluent, and evaluate the potential of lipid accumulation and biomass production. In the present study, two microalgae isolated from Shanxi Province are evaluated on flocculating ability, growth potential and pollutants removal efficiency in simulated secondary effluent. It is expected that the finding of this investigation can provide some bases for microalgal cultivation and efficient biomass recovery in secondary effluent.
The simulated secondary effluent in this study was mainly composed by C6H12O6, NaNO3, KH2PO4, NaHCO3, NaCl, MgSO4, FeSO4, CaCl2, H3BO3, MnCl2, ZnSO4, Na2MoO4, CuSO4 and Co(NO3)2, and the chemical oxygen demand (COD), nitrate and total phosphorus concentrations were around 40 mg/L, 6.5 mg/L and 0.7 mg/L, respectively. 2.2. Flocculating ability test The flocculating ability test was carried out according to Alam et al. (2014) with some modification. After 10 d of cultivation, the culture of the two microalgae was harvested. 25 mL culture was distributed in 25 mL cylindrical glass tubes, and followed by gently mixing for 1 min at room temperature. An aliquot of the culture was withdrawn at a height of two-thirds from the bottle when the culture was settled for 30, 60, 120 and 180 min, respectively. After that, the optical density of above aliquots was measured at 680 nm. The flocculating ability was calculated according to the equation as following:
Flocculating ability ¼ ðA BÞ=A 100% where A and B were the optical density (OD680) of the aliquot before and after flocculation. 2.3. Extraction and analysis of EPS The EPS were extracted according to the procedure from Yang and Li (2009) with some modifications. After 10 d of cultivation, microalgae suspension was dewatered by centrifugation at 5000 rpm for 5 min. The pellet was then washed with deionized water and centrifuged at 5000 rpm for 5 min. The washed pellet was diluted with deionized water and was heated to 80 °C for 30 min. Subsequently, the mixture was centrifuged at 10,000 rpm for 10 min. After that, the supernatant was filtered with 0.45 lm acetate cellulose membranes and the filtrate was regarded as the EPS fraction. Proteins were measured by the coomassic brilliant blue method (Bradford, 1976) using BSA as the standard. Carbohydrates were measured by the Anthrone method (Gaudy, 1962) with glucose as the standard. 2.4. Excitation-emission matrix (EEM) fluorescence spectroscopy
2. Methods 2.1. Microalgal strains and cultivation Chlorococcum sp. GD and Parachlorella kessleri TY were isolated from the moss Entodon obtusatus and soil of Shanxi Province, respectively. The details were in accordance with methods described by Rasoul-Amini et al. (2009) and Zhang et al. (2014). The two microalgae were cultivated in BG11 medium with the following compositions: 1500 mg/L NaNO3, 40 mg/L K2HPO43H2O, 75 mg/L MgSO47H2O, 20 mg/L Na2CO3, 27 mg/L CaCl2, 6 mg/L citric acid monohydrate, 6 mg/L ammonium ferric citrate, 1 mg/L Na2EDTA, and 1 mL trace metal solution (2.86 mg/L H3BO3, 1.81 mg/L MnCl24H2O, 0.222 mg/L ZnSO47H2O, 0.079 mg/L CuSO45H2O, 0.050 mg/L CoCl26H2O, 0.39 mg/L Na2MoO42H2O). At the end of cultivation period, the culture was centrifuged at 5000 rpm for 5 min. Then, the pellet was washed with deionized water and centrifuged at 5000 rpm for 5 min. After that, the pellet was suspended in the simulated secondary effluent for inoculation, and the initial microalgal biomass concentration was about 80 mg/ L. The two microalgae were cultivated in 2 L conical flasks with batch mode, and conical flasks were stirred at 160 rpm. The temperature was set at 25 °C. The fluorescent lamps were used to provide incident light intensity with 3000 lux and light/dark period was 14 h/10 h. All these experiments were performed in triplicate.
EEM spectra of microalgal EPS were measured with F-280 fluorescence spectrophotometer (Gangdong, China). Spectra were collected with subsequent scanning of emission spectra from 250 to 550 nm at 1 nm increments by varying the excitation wavelength from 200 to 450 nm at 10 nm increments. Excitation and emission slits were maintained at 5 nm and the scanning speed was set at 1200 nm/min. The voltage of the photomultiplier tube (PMT) was set to 700 V. The software Origin 8.0 was employed for handling EEM data. 2.5. Relative hydrophobicity (RH) of microalgae The RH of microalgae was measured by following the protocol from Zhang et al. (2007) with some modifications. After 10 d of cultivation, 10 mL of hexadecane was mixed with 20 mL of microalgal suspension and the mixture was inverted for 10 min at room temperature. Then, the mixture was settled for 30 min, and the two phases separated completely. After that, the aqueous phase was transferred. The relative hydrophobicity was expressed as the ratio of the optical density (OD680) in the aqueous phase after emulsification (OD680-E) to the optical density (OD680) in the aqueous phase before emulsification (OD680-O). The RH was evaluated as
RH ¼ ð1 OD680E =OD680O Þ 100%
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90
After 10 d of cultivation, a suspension of microalgae (240 lL) was stained with 0.5 mg/mL Nile red (9-diethylamino-5H-benzo( a)phenoxazine-5-one, dissolved in DMSO, 1 lL), and the mixture was incubated for 10 min at 37 °C. Then, the fluorescence of the mixture was determined using a microplate reader Tecan Infinite 200 Pro (Tecan, Switzerland) with a 96-well plate. The fluorescence of microalgae alone was measured. In addition, the fluorescence of Nile red alone was also measured. The fluorescence intensity of microalgal lipid was obtained when the autofluorescence of microalgae and Nile red was subtracted. The excitation and emission wavelengths were 543 and 598 nm, respectively. Each experiment was performed in triplicate. It the present study, specific Nile Red fluorescence intensity was calculated as described by Gardner et al. (2013) with some modification. The equation was as following:
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sFI ¼ 10 000 FI N1 c where sFI was the specific fluorescence intensity (a.u./cell), FI was the total Nile Red fluorescence intensity of the microalgal suspension (a.u./240 lL), and Nc was cellular number in 240 lL microalgal suspension. An arbitrary scaling factor of 10,000 was used to shift results to 0–100. 2.7. The analysis of microalgal biomass concentration and water quality The microalgal concentration was determined by the method of mixed liquid suspended solids (MLSS) according to standard methods (APHA-AWWA-WEF, 2005) Nitrate and total phosphorus were analyzed by ultraviolet spectrophotometric screening method and ascorbic acid method, respectively, according to APHA-AWWA-WEF (2005). 2.8. Statistical analysis In this study, the measured value was expressed as the mean ± standard deviation. The flocculating ability, the intensity of EPS analyzed by EEM, relative hydrophobicity and sFI between Chlorococcum sp. GD and P. kessleri TY was analyzed by T-test conducting by SPSS software (version 19.0). There was a statistically significant difference when p < 0.05.
Flocculating ability (%)
2.6. Nile red fluorescence determination of microalgal lipid
Chlorococcum sp. GD Parachlorella kessleri TY
70 60 50 40 30 20 10 0
30
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Time (min) Fig. 1. The flocculating ability of Chlorococcum sp. GD and P. kessleri TY after different time of setting.
3.2. EPS analysis of the two microalgae EPS were mainly composed by extracellular proteins and extracellular carbohydrates. Fig. 2 showed the respective content of extracellular proteins and carbohydrates in Chlorococcum sp. GD and P. kessleri TY. The proteins content of Chlorococcum sp. GD was 6.21 mg/g dry weight. Whereas the proteins content of P. kessleri TY was 11.00 mg/g dry weight. The extracellular carbohydrates of Chlorococcum sp. GD were only 8.33 mg/g dry weight, which was far less than that of P. kessleri TY (82.04 mg/g dry weight). Generally speaking, both extracellular proteins and extracellular carbohydrates of Chlorococcum sp. GD were lower than those of P. kessleri TY. Nevertheless, it was found that the ratio of proteins and carbohydrates of EPS in the Chlorococcum sp. GD was 0.75, while the corresponding value in the P. kessleri TY was only 0.13. It meant that Chlorococcum sp. GD had a relatively large ratio of extracellular proteins in EPS, compared to P. kessleri TY. Fig. 3 summarized the EEM results of EPS from Chlorococcum sp. GD and P. kessleri TY. Only one main peak B (Ex/Em: 290/355 nm; tryptophan protein-like substances) emerged in the EPS of Chlorococcum sp. GD and P. kessleri TY. Although the component of EPS was similar between Chlorococcum sp. GD and P. kessleri TY, the intensity of Peak B (675.88 a.u./0.05 g dry weight biomass) in the EPS of Chlorococcum sp. GD was significantly higher than that
3. Results
After 10 d of cultivation in secondary effluent, the flocculating ability of Chlorococcum sp. GD and P. kessleri TY was evaluated. As shown in Fig. 1, there was a significant difference on flocculating ability between Chlorococcum sp. GD and P. kessleri TY (p < 0.05), and Chlorococcum sp. GD showed excellent flocculating ability. Concretely, the flocculating ability of Chlorococcum sp. GD was 47.73% after 30 min of settling. The corresponding flocculating activity was increased to 66.97%, 82.23% and 84.43% after 60 min, 120 min and 180 min of settling. The P. kessleri TY showed a poor flocculating activity. After 30 min of settling, the flocculating activity of P. kessleri TY was only 7.61%. With the increase of settling time to 60, 120 and 180 min, the corresponding flocculating activity was only slightly increased to 11.09%, 14.71% and 16.23%, respectively. Therefore, it was suggested that Chlorococcum sp. GD was an excellent self-flocculating microalgae. The cultivation of Chlorococcum sp. GD was beneficial to effectively separate microalgal biomass from secondary effluent.
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Concentartion (mg/g dry weight)
3.1. The flocculating ability of the two microalgae
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Chlorococcum sp. GD Parachlorella kessleri TY
70 60 50 40 30 20 10 0 Proteins
Carbohydrates
Fig. 2. The EPS concentration of Chlorococcum sp. GD and P. kessleri TY after 10 d of cultivation.
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peak B Maximum fluorescence intensity = 608.10 a.u./0.05 g dry weight biomass 300
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Fig. 3. The EEM spectra of Chlorococcum sp. GD and P. kessleri TY EPS after 10 d of cultivation.
(608.10 a.u./0.05 g dry weight biomass) of P. kessleri TY (p < 0.05). The result indicated that more tryptophan protein-like substances existed in EPS of Chlorococcum sp. GD. Besides, it was also determined the hydrophobicity of the two microalgae. As shown in Fig. 4, there was a significant difference on the hydrophobicity between Chlorococcum sp. GD and P. kessleri TY (p < 0.05). The hydrophobicity of Chlorococcum sp. GD was 46.89%, whereas the hydrophobicity of P. kessleri TY was 38.52%. The Chlorococcum sp. GD exhibited higher hydrophobic property than P. kessleri TY.
3.3. The pollutants removal efficiency, biomass productivity and lipid content of the two microalgae The pollutants removal of Chlorococcum sp. GD and P. kessleri TY was presented in Fig. 5. For Chlorococcum sp. GD, the initial concentration of nitrate in secondary effluent was 6.42 mg/L. After 10 d of cultivation, nitrate concentration was reduced to 2.15 mg/L, and the removal efficiency of nitrate was 66.51%. The concentration of phosphorus was decreased from 0.62 mg/L to 0.16 mg/L, and the removal efficiency was up to 74.19%. For P. kessleri TY, the concentration of nitrate was decreased from 6.30 mg/L to 3.28 mg/L and the removal efficiency was 47.94%. The concentration of phosphorus was 0.75 mg/L in initial cultivation. After 10 d of cultiva-
Relative Hydrophobicity (%)
60 50 40 30 20 10 0 Chlorococcum sp. GD
Parachlorella kessleri TY
Fig. 4. The relative hydrophobicity of Chlorococcum sp. GD and P. kessleri TY after 10 d of cultivation.
tion, the phosphorus concentration was reduced to 0.31 mg/L, and the removal efficiency was 58.67%. These results indicated that Chlorococcum sp. GD had a better pollutants removal capacity, compared with P. kessleri TY. The microalgal biomass of Chlorococcum sp. GD and P. kessleri TY was illustrated in Fig. 6a. The initial inoculation concentration of Chlorococcum sp. GD and P. kessleri TY was 85.19 and 72.23 mg/L, respectively. After 10 d of cultivation, the microalgal biomass was increased to 244.44 and 215.56 mg/L, respectively. Besides, the biomass productivity of Chlorococcum sp. GD and P. kessleri TY was 15.93 and 14.33 mg/L/d, respectively. Both the two microalgae could well grow in secondary effluent. By the end of culture, the lipid content of the two microalgae was evaluated by sFI. sFI represented the Nile Red fluorescence intensity of each microalgal cell, and the high value represented high lipid content. As shown in Fig. 6b, there was a significant difference on the sFI between Chlorococcum sp. GD and P. kessleri TY (p < 0.05). The sFI for Chlorococcum sp. GD was 233.25 a.u./cell after 10 d of cultivation in secondary effluent. The sFI was only 11.42 a. u./cell for P. kessleri TY. Obviously, Chlorococcum sp. GD had better performance for lipid production than P. kessleri TY.
4. Discussion Currently, high cost of microalgal biomass harvesting was one of the bottlenecks for commercialization of microalgae-based industrial processes. In order to solve the problem, many chemical and physical flocculation methods were developed and showed a high harvesting efficiency (Wan et al., 2015). However, the huge consumption of chemicals and energy were major challenges. Also, some chemicals showed a certain degree of biomass toxicity (Rawat et al., 2013). Self-flocculating microalgae were a kind of microalgae, which could aggregate together by themselves. Then, the aggregate could be easily harvested by sedimentation. The self-flocculating process of microalgae was a spontaneous behavior, and didn’t have high consumption of energy and potential biological toxicity, compared to physical and chemical flocculation. Therefore, self-flocculation was regarded as an environmental friendly and low-cost method for microalgal biomass harvesting. Unfortunately, only small number of microalgae, such as A. falcatus, C. vulgaris, E. texensis, S. obliquus and T. suecica, had been regarded as self-flocculating microalgae so far (Alam et al., 2014; Guo et al., 2013; Salim et al., 2012, 2013, 2014). In this study, the Chlorococcum sp. GD isolated from the moss E. obtusatus of Shanxi Province, China, was investigated to enrich the diversity of self-flocculating
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Nitrate Concentration (mg/L)
8 initial inoculation after harvestation
7 6 5 4 3 2 1 0 Chlorococcum sp. GD
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a
240 220 200 180 160 140 120 100 80 60 40 20 0
Chlorococcum sp. GD
Parachlorella kessleri TY
Specific Fluorescence Intensity (a.u./cell)
Fig. 5. The pollutants removal of Chlorococcum sp. GD and P. kessleri TY after 10 d of cultivation.
300 275
b
250 225 200 175 150 125 100 75 50 25 0
Chlorococcum sp. GD
Parachlorella kessleri TY
Fig. 6. The microalgal biomass (a) and specific fluorescence intensity (b) of Chlorococcum sp. GD and P. kessleri TY after 10 d of cultivation.
microalgae and also provide some bases for microalgal cultivation and efficient biomass recovery in wastewaters. For WWTPs, the defect of process itself or inadequate operation might influence pollutants removal efficiency, which led to the concentration of nitrogen and phosphorus in secondary effluent was higher than the perfect state. Therefore, it was necessary to strengthen tertiary treatment to reduce the risk of the eutrophication. Currently, although secondary effluent could be used as medium for microalgal growth, the biomass production of microalgae was far less than that in municipal wastewater, industry wastewater, agricultural wastewater and anaerobic digestion wastewater. Obviously, low biomass concentration from secondary effluent caused some difficulties for microalgal harvesting. At the present study, it was found that Chlorococcum sp. GD showed excellent flocculating ability when cultivating in secondary effluent for 10 d. The flocculating activity could be up to 84.43% after 180 min of settling. Salim et al. (2013) cultivated E. texensis in freshwater medium with high concentration of nitrate and phosphorus. When the growth of E. texensis went into the stationary phase, 90% recovery of E. texensis was achieved after 3 h settling. As described by Guo et al. (2013), S. obliquus cultivated in Detmer’s Medium could flocculate together and form flocs large enough to be seen by the naked-eye. After 13 d of culture, the flocculation efficiency was up to around 80% when the culture was stayed for about 30 min. Alam et al. (2014) reported that C. vulgaris JSC-7 cultivated in modified Bold’s Basal Medium with 3-fold nitrogen sup-
plementation for 14 d exhibited high flocculation efficiency of 76%, after 30 min settling. Besides, some other microalgae, such as A. falcatus and T. suecica, also showed excellent self-flocculating property (Salim et al., 2012). In short, the flocculating ability of Chlorococcum sp. GD was comparable to excellent selfflocculating microalgae above. The self-flocculating property of Chlorococcum sp. GD was firstly reported in the study, which enriched the self-flocculating microalgae pool. Also, it was the first attempt to harvest microalgal biomass from secondary effluent by utilizing the self-flocculating property of microalgae. Of course, the energy recovery amount expected by using this biomass was not elucidated in the study and above literatures. Nevertheless, it was considered that it had low energy recovery amount compared to physical and chemical flocculation. The detailed comparison would be done in the next investigation. EPS composed by extracellular proteins and extracellular carbohydrates were regarded as an important factor influencing the selfflocculating of microalgae (Salim et al., 2013, 2014). Owing to the different flocculating ability between Chlorococcum sp. GD and P. kessleri TY, the property of EPS was also analyzed. Unlike the high amount of extracellular proteins and extracellular carbohydrates (proteins: 233 mg/g dry weight; carbohydrates: 96 mg/g dry weight) for the self-flocculating microalgae E. texensis (Salim et al., 2014), the concentration of extracellular proteins and carbohydrates of Chlorococcum sp. GD was low. Commonly, EPS were metabolites which were secreted out of cells. As reviewed by
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Sheng et al. (2010), EPS production and composition of microorganisms were significantly influenced by substrate type, nutrient content and growth phase. For the cultivation of Chlorococcum sp. GD, the nitrogen and phosphorous content in secondary effluent was far less than those in medium cultivating the selfflocculating microalgae reported in literatures (Alam et al., 2014; Guo et al., 2013; Salim et al., 2013). Hence, it was reasonable to believe that low nutrient content in secondary effluent affected the EPS production of Chlorococcum sp. GD. In this study, it was also found that the EPS concentration of Chlorococcum sp. GD was lower than that of non-flocculating microalgae P. kessleri TY. Although all of existed literatures on microalgal EPS analysis showed that EPS content of self-flocculating microalgae was higher than that of non-flocculating microalgae (Salim et al., 2014), there were also some cases demonstrating that the amount of total EPS was not related to microbial settleability (Liao et al., 2001; Lv et al., 2014). Further, EEM results illustrated that Chlorococcum sp. GD had more tryptophan protein-like substances than P. kessleri TY. As described by some literatures (Dignac et al., 1998), most of the amino acid composition of proteins was hydrophobic, whereas carbohydrates contained a high proportion of hydrophilic group. Therefore, compared to P. kessleri TY, it was speculated that more hydrophobic protein components in EPS of Chlorococcum sp. GD was beneficial for its flocculation, which was also supported by the result that Chlorococcum sp. GD had stronger hydrophobicity than P. kessleri TY. Besides excellent flocculating ability, high biomass productivity was also important to microalgal cultivation in secondary effluent. In the present study, after 10 d of cultivation, the microalgal biomass of Chlorococcum sp. GD and P. kessleri TY was increased to 244.44 and 215.56 mg/L, respectively. As described by Li et al. (2010), the microalgal biomass of Scenedesmus sp. LX1 was 110 mg/L after 15 d of cultivation in secondary effluent, which was lower than that in this study. Several other microalgae, such as Botryococcus braunii and Chlorella ellipsoidea YJ1, were also cultivated in secondary effluent, and the microalgal biomass was up to 350 and 425 mg/L, respectively (Sawayama et al., 1992; Yang et al., 2011). Although microalgae above had higher biomass concentration than those in this study, the biomass concentration was at the same order of magnitude. More importantly, it was demonstrated that both the two microalgae could be regarded as excellent microalgae growing in secondary effluent, by comparison with existing literatures. Additionally, pollutants removal efficiency was also evaluated. For Chlorococcum sp. GD, it was showed that the removal efficiency of nitrate and phosphorus was 66.51% and 74.19%, respectively. According to existing investigations, microalgae could remove more than 80% of nitrogen and phosphorus in secondary effluent (Ji et al., 2013; Li et al., 2010; Sawayama et al., 1992; Yang et al., 2011). The possible reason resulting in the difference on pollutants removal efficiency was different cultivation conditions, such as initial inoculation concentration, microalgal immobilization, and CO2 supplementation, etc. Also, there were some differences on pollutants removal efficiency between Chlorococcum sp. GD and P. kessleri TY, although cultivation conditions were consistent. It was speculated to be species-dependent. The lipid was an important metabolite for microalgal cultivation, which could be used as the resource for biodiesel production. At the present study, it was showed that Chlorococcum sp. GD had better performance for lipid production than P. kessleri TY. Also, the lipid content of Chlorococcum sp. GD was comparable to that of Scenedesmus sp. strain WC-1 via optimized cultivation (Gardner et al., 2011, 2012). Therefore, it was believed that Chlorococcum sp. GD was a potential microalga for lipid production in the future. Based on the above analysis, Chlorococcum sp. GD exhibited excellent flocculating ability, pollutants removal efficiency, and growth potential in simulated secondary effluent. The investiga-
tion enriched the self-flocculating microalgal pool and provided a cost-effective way to cultivate and harvest microalgae in secondary effluent. Also, it was reasonable to believe that the Chlorococcum sp. GD could be cultivated in pilot or industrial scale and on real effluent for self-flocculating harvesting. Related investigations would be carried out in future steps.
5. Conclusions In the present study, a comparative study on flocculating ability and growth potential of two microalgae, Chlorococcum sp. GD and P. kessleri TY, was conducted. Compared to P. kessleri TY, Chlorococcum sp. GD possessed excellent flocculating ability, which was likely to be related to hydrophobic EPS based on the EEM analysis. Besides, the nitrogen and phosphorus removal efficiency, lipid content and biomass concentration of Chlorococcum sp. GD was also high after 10 d of cultivation in secondary effluent. It was suggested that Chlorococcum sp. GD was a promising candidate for microalgal cultivation and harvesting in secondary effluent.
Acknowledgements This research was financed by the Key Scientific Development Project of Shanxi Province, China (No. FT-2014-01), and the Natural Science Foundation of Shanxi Province, China (No. 2015021159).
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
A comparative study on flocculating ability and growth potential of two microalgae in simulated secondary effluent Junping Lv, Junyan Guo, Jia Feng, Qi Liu, Shulian Xie ⇑ School of Life Science, Shanxi University, Taiyuan 030006, China
h i g h l i g h t s The self-flocculating property of Chlorococcum sp. GD was firstly reported. Chlorococcum sp. GD had excellent flocculating ability. The excellent flocculating ability of Chlorococcum sp. GD was related to EPS. Pollutants in secondary effluent were effectively removed by Chlorococcum sp. GD. The lipid content of Chlorococcum sp. GD was high.
a r t i c l e
i n f o
Article history: Received 21 November 2015 Received in revised form 10 January 2016 Accepted 11 January 2016 Available online 23 January 2016 Keywords: Microalgae Flocculating ability Pollutants removal Biomass and lipid accumulation Secondary effluent
a b s t r a c t The flocculating ability was an important property to microalgal harvesting, especially in secondary effluent. In this study, the flocculating ability of two microalgae, Chlorococcum sp. GD and Parachlorella kessleri TY, was evaluated after 10 d of cultivation in secondary effluent. After 180 min of settling, the flocculating ability of Chlorococcum sp. GD and P. kessleri TY was 84.43% and 16.23%, respectively. It was suggested that Chlorococcum sp. GD was an excellent self-flocculating microalgae. The mechanism on selfflocculating of Chlorococcum sp. GD was probably related to hydrophobic extracellular polymeric substances (EPS). Besides, compared to P. kessleri TY, the nitrogen and phosphorus removal efficiency of Chlorococcum sp. GD was high, which was up to 66.51% and 74.19%, respectively. Chlorococcum sp. GD also had high lipid content and biomass concentration. Therefore, Chlorococcum sp. GD could be regarded as a promising candidate for microalgal cultivation and harvesting in secondary effluent. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction The secondary effluent from wastewater treatment plants (WWTPs) is commonly rich in nitrogen and phosphorus owing to the defect of process itself or inadequate operation. As described in literatures, the total nitrogen and total phosphorus concentration of secondary effluent range from 5 to 30 mg/L, and from 0.2 to 3 mg/L respectively (Wu et al., 2014), which are much higher than the threshold of nitrogen and phosphorus causing the eutrophication in streams (Chambers et al., 2012). Therefore, it is essential to develop some technologies for advanced treatment of secondary effluent. Microalgae as the largest photoautotrophic group of plant taxa have high photosynthetic efficiency, rapid growth rate, and strong adaptability. Many microalgae are rich in oil, which can be further
⇑ Corresponding author. Tel./fax: +86 351 7018121. E-mail address: [email protected] (S. Xie). http://dx.doi.org/10.1016/j.biortech.2016.01.047 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
processed into biodiesel (Maity et al., 2014). Also, microalgae can metabolize nitrogen and phosphorus via different metabolic pathways (Cai et al., 2013). Therefore, microalgal-based biotechnology has been gradually developed for biodiesel production and wastewater treatment over the past few years (Rawat et al., 2013; Zeng et al., 2015). As an interesting topic, the integration of microalgae-based advanced treatment of secondary effluent and lipid production has been widely concerned in recent years (Arbib et al., 2014; Ji et al., 2013; Li et al., 2010; Sydney et al., 2011; Yang et al., 2011). However, there is a difficulty on microalgal harvesting from secondary effluents due to the relatively low cell density (Ji et al., 2013; Li et al., 2010). Currently, microalgal harvesting methods include centrifugation, chemical coagulation, gravity sedimentation, filtration, flotation, electrophoresis technique, and immobilized technique (Barros et al., 2015). Although these techniques can effectively harvest microalgal biomass from wastewaters, the huge consumption of chemicals and energies is a major challenge.
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Also, some chemicals show a certain degree of biomass toxicity (Rawat et al., 2013). Consequently, all drawbacks above limit the use of these technologies for large-scale harvesting of microalgae, especially in secondary effluent. Self-flocculating microalgae is a kind of microalgae, which can aggregate together to form large particles by themselves. Then, the aggregate can be easily harvested by sedimentation. Owing to environmental friendliness and no additional economic costs, screening and cultivating self-flocculating microalgae has been regarded as an alternative way to harvest microalgae. Till now, a few investigations have reported that some microalgae, such as Ankistrodesmus falcatus, Chlorella vulgaris, Ettlia texensis, Scenedesmus obliquus and Tetraselmis suecica, belong to self-flocculating microalgae (Alam et al., 2014; Guo et al., 2013; Salim et al., 2012, 2013, 2014). Especially, the flocculation efficiency of E. texensis after 3 h of sedimentation was around 90% (Salim et al., 2013). Nevertheless, the diversity of self-flocculating microalgae is low. Also, the flocculation performance of self-flocculating microalgae in secondary effluent has not been reported. Thus, it is important to screen more self-flocculating microalgae and evaluate their flocculating ability after cultivation of them in secondary effluent. Compared to a large number of microalgae successfully cultivated in municipal wastewater, industry wastewater, agricultural wastewater and anaerobic digestion wastewater (Chen et al., 2015; Morales-Amaral et al., 2015), the number of microalgae adapting to secondary effluent is limited. The main groups focus on Botryococcus, Chlorella and Scenedesmus, and have low biomass production without any supplement of exogenous carbon sources (Li et al., 2010; Sawayama et al., 1992; Yang et al., 2011). Thus, it is essential to screen more microalgae suitable for growth in secondary effluent, and evaluate the potential of lipid accumulation and biomass production. In the present study, two microalgae isolated from Shanxi Province are evaluated on flocculating ability, growth potential and pollutants removal efficiency in simulated secondary effluent. It is expected that the finding of this investigation can provide some bases for microalgal cultivation and efficient biomass recovery in secondary effluent.
The simulated secondary effluent in this study was mainly composed by C6H12O6, NaNO3, KH2PO4, NaHCO3, NaCl, MgSO4, FeSO4, CaCl2, H3BO3, MnCl2, ZnSO4, Na2MoO4, CuSO4 and Co(NO3)2, and the chemical oxygen demand (COD), nitrate and total phosphorus concentrations were around 40 mg/L, 6.5 mg/L and 0.7 mg/L, respectively. 2.2. Flocculating ability test The flocculating ability test was carried out according to Alam et al. (2014) with some modification. After 10 d of cultivation, the culture of the two microalgae was harvested. 25 mL culture was distributed in 25 mL cylindrical glass tubes, and followed by gently mixing for 1 min at room temperature. An aliquot of the culture was withdrawn at a height of two-thirds from the bottle when the culture was settled for 30, 60, 120 and 180 min, respectively. After that, the optical density of above aliquots was measured at 680 nm. The flocculating ability was calculated according to the equation as following:
Flocculating ability ¼ ðA BÞ=A 100% where A and B were the optical density (OD680) of the aliquot before and after flocculation. 2.3. Extraction and analysis of EPS The EPS were extracted according to the procedure from Yang and Li (2009) with some modifications. After 10 d of cultivation, microalgae suspension was dewatered by centrifugation at 5000 rpm for 5 min. The pellet was then washed with deionized water and centrifuged at 5000 rpm for 5 min. The washed pellet was diluted with deionized water and was heated to 80 °C for 30 min. Subsequently, the mixture was centrifuged at 10,000 rpm for 10 min. After that, the supernatant was filtered with 0.45 lm acetate cellulose membranes and the filtrate was regarded as the EPS fraction. Proteins were measured by the coomassic brilliant blue method (Bradford, 1976) using BSA as the standard. Carbohydrates were measured by the Anthrone method (Gaudy, 1962) with glucose as the standard. 2.4. Excitation-emission matrix (EEM) fluorescence spectroscopy
2. Methods 2.1. Microalgal strains and cultivation Chlorococcum sp. GD and Parachlorella kessleri TY were isolated from the moss Entodon obtusatus and soil of Shanxi Province, respectively. The details were in accordance with methods described by Rasoul-Amini et al. (2009) and Zhang et al. (2014). The two microalgae were cultivated in BG11 medium with the following compositions: 1500 mg/L NaNO3, 40 mg/L K2HPO43H2O, 75 mg/L MgSO47H2O, 20 mg/L Na2CO3, 27 mg/L CaCl2, 6 mg/L citric acid monohydrate, 6 mg/L ammonium ferric citrate, 1 mg/L Na2EDTA, and 1 mL trace metal solution (2.86 mg/L H3BO3, 1.81 mg/L MnCl24H2O, 0.222 mg/L ZnSO47H2O, 0.079 mg/L CuSO45H2O, 0.050 mg/L CoCl26H2O, 0.39 mg/L Na2MoO42H2O). At the end of cultivation period, the culture was centrifuged at 5000 rpm for 5 min. Then, the pellet was washed with deionized water and centrifuged at 5000 rpm for 5 min. After that, the pellet was suspended in the simulated secondary effluent for inoculation, and the initial microalgal biomass concentration was about 80 mg/ L. The two microalgae were cultivated in 2 L conical flasks with batch mode, and conical flasks were stirred at 160 rpm. The temperature was set at 25 °C. The fluorescent lamps were used to provide incident light intensity with 3000 lux and light/dark period was 14 h/10 h. All these experiments were performed in triplicate.
EEM spectra of microalgal EPS were measured with F-280 fluorescence spectrophotometer (Gangdong, China). Spectra were collected with subsequent scanning of emission spectra from 250 to 550 nm at 1 nm increments by varying the excitation wavelength from 200 to 450 nm at 10 nm increments. Excitation and emission slits were maintained at 5 nm and the scanning speed was set at 1200 nm/min. The voltage of the photomultiplier tube (PMT) was set to 700 V. The software Origin 8.0 was employed for handling EEM data. 2.5. Relative hydrophobicity (RH) of microalgae The RH of microalgae was measured by following the protocol from Zhang et al. (2007) with some modifications. After 10 d of cultivation, 10 mL of hexadecane was mixed with 20 mL of microalgal suspension and the mixture was inverted for 10 min at room temperature. Then, the mixture was settled for 30 min, and the two phases separated completely. After that, the aqueous phase was transferred. The relative hydrophobicity was expressed as the ratio of the optical density (OD680) in the aqueous phase after emulsification (OD680-E) to the optical density (OD680) in the aqueous phase before emulsification (OD680-O). The RH was evaluated as
RH ¼ ð1 OD680E =OD680O Þ 100%
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After 10 d of cultivation, a suspension of microalgae (240 lL) was stained with 0.5 mg/mL Nile red (9-diethylamino-5H-benzo( a)phenoxazine-5-one, dissolved in DMSO, 1 lL), and the mixture was incubated for 10 min at 37 °C. Then, the fluorescence of the mixture was determined using a microplate reader Tecan Infinite 200 Pro (Tecan, Switzerland) with a 96-well plate. The fluorescence of microalgae alone was measured. In addition, the fluorescence of Nile red alone was also measured. The fluorescence intensity of microalgal lipid was obtained when the autofluorescence of microalgae and Nile red was subtracted. The excitation and emission wavelengths were 543 and 598 nm, respectively. Each experiment was performed in triplicate. It the present study, specific Nile Red fluorescence intensity was calculated as described by Gardner et al. (2013) with some modification. The equation was as following:
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sFI ¼ 10 000 FI N1 c where sFI was the specific fluorescence intensity (a.u./cell), FI was the total Nile Red fluorescence intensity of the microalgal suspension (a.u./240 lL), and Nc was cellular number in 240 lL microalgal suspension. An arbitrary scaling factor of 10,000 was used to shift results to 0–100. 2.7. The analysis of microalgal biomass concentration and water quality The microalgal concentration was determined by the method of mixed liquid suspended solids (MLSS) according to standard methods (APHA-AWWA-WEF, 2005) Nitrate and total phosphorus were analyzed by ultraviolet spectrophotometric screening method and ascorbic acid method, respectively, according to APHA-AWWA-WEF (2005). 2.8. Statistical analysis In this study, the measured value was expressed as the mean ± standard deviation. The flocculating ability, the intensity of EPS analyzed by EEM, relative hydrophobicity and sFI between Chlorococcum sp. GD and P. kessleri TY was analyzed by T-test conducting by SPSS software (version 19.0). There was a statistically significant difference when p < 0.05.
Flocculating ability (%)
2.6. Nile red fluorescence determination of microalgal lipid
Chlorococcum sp. GD Parachlorella kessleri TY
70 60 50 40 30 20 10 0
30
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Time (min) Fig. 1. The flocculating ability of Chlorococcum sp. GD and P. kessleri TY after different time of setting.
3.2. EPS analysis of the two microalgae EPS were mainly composed by extracellular proteins and extracellular carbohydrates. Fig. 2 showed the respective content of extracellular proteins and carbohydrates in Chlorococcum sp. GD and P. kessleri TY. The proteins content of Chlorococcum sp. GD was 6.21 mg/g dry weight. Whereas the proteins content of P. kessleri TY was 11.00 mg/g dry weight. The extracellular carbohydrates of Chlorococcum sp. GD were only 8.33 mg/g dry weight, which was far less than that of P. kessleri TY (82.04 mg/g dry weight). Generally speaking, both extracellular proteins and extracellular carbohydrates of Chlorococcum sp. GD were lower than those of P. kessleri TY. Nevertheless, it was found that the ratio of proteins and carbohydrates of EPS in the Chlorococcum sp. GD was 0.75, while the corresponding value in the P. kessleri TY was only 0.13. It meant that Chlorococcum sp. GD had a relatively large ratio of extracellular proteins in EPS, compared to P. kessleri TY. Fig. 3 summarized the EEM results of EPS from Chlorococcum sp. GD and P. kessleri TY. Only one main peak B (Ex/Em: 290/355 nm; tryptophan protein-like substances) emerged in the EPS of Chlorococcum sp. GD and P. kessleri TY. Although the component of EPS was similar between Chlorococcum sp. GD and P. kessleri TY, the intensity of Peak B (675.88 a.u./0.05 g dry weight biomass) in the EPS of Chlorococcum sp. GD was significantly higher than that
3. Results
After 10 d of cultivation in secondary effluent, the flocculating ability of Chlorococcum sp. GD and P. kessleri TY was evaluated. As shown in Fig. 1, there was a significant difference on flocculating ability between Chlorococcum sp. GD and P. kessleri TY (p < 0.05), and Chlorococcum sp. GD showed excellent flocculating ability. Concretely, the flocculating ability of Chlorococcum sp. GD was 47.73% after 30 min of settling. The corresponding flocculating activity was increased to 66.97%, 82.23% and 84.43% after 60 min, 120 min and 180 min of settling. The P. kessleri TY showed a poor flocculating activity. After 30 min of settling, the flocculating activity of P. kessleri TY was only 7.61%. With the increase of settling time to 60, 120 and 180 min, the corresponding flocculating activity was only slightly increased to 11.09%, 14.71% and 16.23%, respectively. Therefore, it was suggested that Chlorococcum sp. GD was an excellent self-flocculating microalgae. The cultivation of Chlorococcum sp. GD was beneficial to effectively separate microalgal biomass from secondary effluent.
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Concentartion (mg/g dry weight)
3.1. The flocculating ability of the two microalgae
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Chlorococcum sp. GD Parachlorella kessleri TY
70 60 50 40 30 20 10 0 Proteins
Carbohydrates
Fig. 2. The EPS concentration of Chlorococcum sp. GD and P. kessleri TY after 10 d of cultivation.
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peak B Maximum fluorescence intensity = 675.88 a.u./0.05 g dry weight biomass 300
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peak B Maximum fluorescence intensity = 608.10 a.u./0.05 g dry weight biomass 300
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Fig. 3. The EEM spectra of Chlorococcum sp. GD and P. kessleri TY EPS after 10 d of cultivation.
(608.10 a.u./0.05 g dry weight biomass) of P. kessleri TY (p < 0.05). The result indicated that more tryptophan protein-like substances existed in EPS of Chlorococcum sp. GD. Besides, it was also determined the hydrophobicity of the two microalgae. As shown in Fig. 4, there was a significant difference on the hydrophobicity between Chlorococcum sp. GD and P. kessleri TY (p < 0.05). The hydrophobicity of Chlorococcum sp. GD was 46.89%, whereas the hydrophobicity of P. kessleri TY was 38.52%. The Chlorococcum sp. GD exhibited higher hydrophobic property than P. kessleri TY.
3.3. The pollutants removal efficiency, biomass productivity and lipid content of the two microalgae The pollutants removal of Chlorococcum sp. GD and P. kessleri TY was presented in Fig. 5. For Chlorococcum sp. GD, the initial concentration of nitrate in secondary effluent was 6.42 mg/L. After 10 d of cultivation, nitrate concentration was reduced to 2.15 mg/L, and the removal efficiency of nitrate was 66.51%. The concentration of phosphorus was decreased from 0.62 mg/L to 0.16 mg/L, and the removal efficiency was up to 74.19%. For P. kessleri TY, the concentration of nitrate was decreased from 6.30 mg/L to 3.28 mg/L and the removal efficiency was 47.94%. The concentration of phosphorus was 0.75 mg/L in initial cultivation. After 10 d of cultiva-
Relative Hydrophobicity (%)
60 50 40 30 20 10 0 Chlorococcum sp. GD
Parachlorella kessleri TY
Fig. 4. The relative hydrophobicity of Chlorococcum sp. GD and P. kessleri TY after 10 d of cultivation.
tion, the phosphorus concentration was reduced to 0.31 mg/L, and the removal efficiency was 58.67%. These results indicated that Chlorococcum sp. GD had a better pollutants removal capacity, compared with P. kessleri TY. The microalgal biomass of Chlorococcum sp. GD and P. kessleri TY was illustrated in Fig. 6a. The initial inoculation concentration of Chlorococcum sp. GD and P. kessleri TY was 85.19 and 72.23 mg/L, respectively. After 10 d of cultivation, the microalgal biomass was increased to 244.44 and 215.56 mg/L, respectively. Besides, the biomass productivity of Chlorococcum sp. GD and P. kessleri TY was 15.93 and 14.33 mg/L/d, respectively. Both the two microalgae could well grow in secondary effluent. By the end of culture, the lipid content of the two microalgae was evaluated by sFI. sFI represented the Nile Red fluorescence intensity of each microalgal cell, and the high value represented high lipid content. As shown in Fig. 6b, there was a significant difference on the sFI between Chlorococcum sp. GD and P. kessleri TY (p < 0.05). The sFI for Chlorococcum sp. GD was 233.25 a.u./cell after 10 d of cultivation in secondary effluent. The sFI was only 11.42 a. u./cell for P. kessleri TY. Obviously, Chlorococcum sp. GD had better performance for lipid production than P. kessleri TY.
4. Discussion Currently, high cost of microalgal biomass harvesting was one of the bottlenecks for commercialization of microalgae-based industrial processes. In order to solve the problem, many chemical and physical flocculation methods were developed and showed a high harvesting efficiency (Wan et al., 2015). However, the huge consumption of chemicals and energy were major challenges. Also, some chemicals showed a certain degree of biomass toxicity (Rawat et al., 2013). Self-flocculating microalgae were a kind of microalgae, which could aggregate together by themselves. Then, the aggregate could be easily harvested by sedimentation. The self-flocculating process of microalgae was a spontaneous behavior, and didn’t have high consumption of energy and potential biological toxicity, compared to physical and chemical flocculation. Therefore, self-flocculation was regarded as an environmental friendly and low-cost method for microalgal biomass harvesting. Unfortunately, only small number of microalgae, such as A. falcatus, C. vulgaris, E. texensis, S. obliquus and T. suecica, had been regarded as self-flocculating microalgae so far (Alam et al., 2014; Guo et al., 2013; Salim et al., 2012, 2013, 2014). In this study, the Chlorococcum sp. GD isolated from the moss E. obtusatus of Shanxi Province, China, was investigated to enrich the diversity of self-flocculating
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Fig. 5. The pollutants removal of Chlorococcum sp. GD and P. kessleri TY after 10 d of cultivation.
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Fig. 6. The microalgal biomass (a) and specific fluorescence intensity (b) of Chlorococcum sp. GD and P. kessleri TY after 10 d of cultivation.
microalgae and also provide some bases for microalgal cultivation and efficient biomass recovery in wastewaters. For WWTPs, the defect of process itself or inadequate operation might influence pollutants removal efficiency, which led to the concentration of nitrogen and phosphorus in secondary effluent was higher than the perfect state. Therefore, it was necessary to strengthen tertiary treatment to reduce the risk of the eutrophication. Currently, although secondary effluent could be used as medium for microalgal growth, the biomass production of microalgae was far less than that in municipal wastewater, industry wastewater, agricultural wastewater and anaerobic digestion wastewater. Obviously, low biomass concentration from secondary effluent caused some difficulties for microalgal harvesting. At the present study, it was found that Chlorococcum sp. GD showed excellent flocculating ability when cultivating in secondary effluent for 10 d. The flocculating activity could be up to 84.43% after 180 min of settling. Salim et al. (2013) cultivated E. texensis in freshwater medium with high concentration of nitrate and phosphorus. When the growth of E. texensis went into the stationary phase, 90% recovery of E. texensis was achieved after 3 h settling. As described by Guo et al. (2013), S. obliquus cultivated in Detmer’s Medium could flocculate together and form flocs large enough to be seen by the naked-eye. After 13 d of culture, the flocculation efficiency was up to around 80% when the culture was stayed for about 30 min. Alam et al. (2014) reported that C. vulgaris JSC-7 cultivated in modified Bold’s Basal Medium with 3-fold nitrogen sup-
plementation for 14 d exhibited high flocculation efficiency of 76%, after 30 min settling. Besides, some other microalgae, such as A. falcatus and T. suecica, also showed excellent self-flocculating property (Salim et al., 2012). In short, the flocculating ability of Chlorococcum sp. GD was comparable to excellent selfflocculating microalgae above. The self-flocculating property of Chlorococcum sp. GD was firstly reported in the study, which enriched the self-flocculating microalgae pool. Also, it was the first attempt to harvest microalgal biomass from secondary effluent by utilizing the self-flocculating property of microalgae. Of course, the energy recovery amount expected by using this biomass was not elucidated in the study and above literatures. Nevertheless, it was considered that it had low energy recovery amount compared to physical and chemical flocculation. The detailed comparison would be done in the next investigation. EPS composed by extracellular proteins and extracellular carbohydrates were regarded as an important factor influencing the selfflocculating of microalgae (Salim et al., 2013, 2014). Owing to the different flocculating ability between Chlorococcum sp. GD and P. kessleri TY, the property of EPS was also analyzed. Unlike the high amount of extracellular proteins and extracellular carbohydrates (proteins: 233 mg/g dry weight; carbohydrates: 96 mg/g dry weight) for the self-flocculating microalgae E. texensis (Salim et al., 2014), the concentration of extracellular proteins and carbohydrates of Chlorococcum sp. GD was low. Commonly, EPS were metabolites which were secreted out of cells. As reviewed by
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Sheng et al. (2010), EPS production and composition of microorganisms were significantly influenced by substrate type, nutrient content and growth phase. For the cultivation of Chlorococcum sp. GD, the nitrogen and phosphorous content in secondary effluent was far less than those in medium cultivating the selfflocculating microalgae reported in literatures (Alam et al., 2014; Guo et al., 2013; Salim et al., 2013). Hence, it was reasonable to believe that low nutrient content in secondary effluent affected the EPS production of Chlorococcum sp. GD. In this study, it was also found that the EPS concentration of Chlorococcum sp. GD was lower than that of non-flocculating microalgae P. kessleri TY. Although all of existed literatures on microalgal EPS analysis showed that EPS content of self-flocculating microalgae was higher than that of non-flocculating microalgae (Salim et al., 2014), there were also some cases demonstrating that the amount of total EPS was not related to microbial settleability (Liao et al., 2001; Lv et al., 2014). Further, EEM results illustrated that Chlorococcum sp. GD had more tryptophan protein-like substances than P. kessleri TY. As described by some literatures (Dignac et al., 1998), most of the amino acid composition of proteins was hydrophobic, whereas carbohydrates contained a high proportion of hydrophilic group. Therefore, compared to P. kessleri TY, it was speculated that more hydrophobic protein components in EPS of Chlorococcum sp. GD was beneficial for its flocculation, which was also supported by the result that Chlorococcum sp. GD had stronger hydrophobicity than P. kessleri TY. Besides excellent flocculating ability, high biomass productivity was also important to microalgal cultivation in secondary effluent. In the present study, after 10 d of cultivation, the microalgal biomass of Chlorococcum sp. GD and P. kessleri TY was increased to 244.44 and 215.56 mg/L, respectively. As described by Li et al. (2010), the microalgal biomass of Scenedesmus sp. LX1 was 110 mg/L after 15 d of cultivation in secondary effluent, which was lower than that in this study. Several other microalgae, such as Botryococcus braunii and Chlorella ellipsoidea YJ1, were also cultivated in secondary effluent, and the microalgal biomass was up to 350 and 425 mg/L, respectively (Sawayama et al., 1992; Yang et al., 2011). Although microalgae above had higher biomass concentration than those in this study, the biomass concentration was at the same order of magnitude. More importantly, it was demonstrated that both the two microalgae could be regarded as excellent microalgae growing in secondary effluent, by comparison with existing literatures. Additionally, pollutants removal efficiency was also evaluated. For Chlorococcum sp. GD, it was showed that the removal efficiency of nitrate and phosphorus was 66.51% and 74.19%, respectively. According to existing investigations, microalgae could remove more than 80% of nitrogen and phosphorus in secondary effluent (Ji et al., 2013; Li et al., 2010; Sawayama et al., 1992; Yang et al., 2011). The possible reason resulting in the difference on pollutants removal efficiency was different cultivation conditions, such as initial inoculation concentration, microalgal immobilization, and CO2 supplementation, etc. Also, there were some differences on pollutants removal efficiency between Chlorococcum sp. GD and P. kessleri TY, although cultivation conditions were consistent. It was speculated to be species-dependent. The lipid was an important metabolite for microalgal cultivation, which could be used as the resource for biodiesel production. At the present study, it was showed that Chlorococcum sp. GD had better performance for lipid production than P. kessleri TY. Also, the lipid content of Chlorococcum sp. GD was comparable to that of Scenedesmus sp. strain WC-1 via optimized cultivation (Gardner et al., 2011, 2012). Therefore, it was believed that Chlorococcum sp. GD was a potential microalga for lipid production in the future. Based on the above analysis, Chlorococcum sp. GD exhibited excellent flocculating ability, pollutants removal efficiency, and growth potential in simulated secondary effluent. The investiga-
tion enriched the self-flocculating microalgal pool and provided a cost-effective way to cultivate and harvest microalgae in secondary effluent. Also, it was reasonable to believe that the Chlorococcum sp. GD could be cultivated in pilot or industrial scale and on real effluent for self-flocculating harvesting. Related investigations would be carried out in future steps.
5. Conclusions In the present study, a comparative study on flocculating ability and growth potential of two microalgae, Chlorococcum sp. GD and P. kessleri TY, was conducted. Compared to P. kessleri TY, Chlorococcum sp. GD possessed excellent flocculating ability, which was likely to be related to hydrophobic EPS based on the EEM analysis. Besides, the nitrogen and phosphorus removal efficiency, lipid content and biomass concentration of Chlorococcum sp. GD was also high after 10 d of cultivation in secondary effluent. It was suggested that Chlorococcum sp. GD was a promising candidate for microalgal cultivation and harvesting in secondary effluent.
Acknowledgements This research was financed by the Key Scientific Development Project of Shanxi Province, China (No. FT-2014-01), and the Natural Science Foundation of Shanxi Province, China (No. 2015021159).
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