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Investigation of initial pH effects on growth of an oleaginous microalgae Chlorella sp. HQ for lipid production and nutrient uptake Qiao Zhang, Ting Wang and Yu Hong

ABSTRACT Using microalgae for synchronous biodiesel production and wastewater treatment is a promising technology. The growth, lipid accumulation and nutrient uptake characteristics of an oleaginous microalga Chlorella sp. HQ were evaluated at different initial pH from 5.0 to 11.0. The pH values changed towards neutrality and ended in the range 6.0–9.0 without artificial control. The alkalinity change before 8 days was in accordance with pH changing. The alkalinity increase after 8 days might

Qiao Zhang Ting Wang Yu Hong (corresponding author) College of Environmental Science and Engineering, Beijing Forestry University, Beijing 100083, China E-mail: [email protected]

be due to the nitrate consumption, CO2 absorption and the algal release at stationary phases. The algal maximal cell density and population growth rate increased with initial pH values while the specific growth rate kept high without significant difference. After 30 days, the maximal algal lipid yield reaching 167.5 mg · L1 occurred at initial pH of 7.0 and the triacylglycerols content was significantly enhanced to 63.0% at initial pH of 5.0 but with a peak of 54.4 mg · L1 at initial pH of 9.0. Furthermore, nutrients were taken up by the alga obviously at all initial pH values. The total nitrogen (TN) and total phosphorus (TP) uptake efficiencies in neutral/alkalic circumstances were larger than that in acid circumstance. The TN and TP were removed by 87.77% and 92.05%, respectively, at initial pH of 7.0. Key words

| alkalinity, Chlorella sp., lipid accumulation, microalgae, nutrient uptake, pH

INTRODUCTION The exploitation of renewable and environmentally friendly energy has become a hotspot worldwide due to the continuous increasing consumption of fossil fuels as well as the increasing emission of greenhouse gas CO2. Biodiesel offers a viable alternative due to its renewable and non-toxic nature. Recently, microalgae are considered as the most promising feedstock for biodiesel production because microalgae have advantages over traditional feedstock which competes for freshwater resource and arable land and is limited in the ability for high-level biodiesel production (Sharma et al. ). As the energy storage in microalgae, triacylglycerols (TAGs) can be transesterified to produce biodiesel and can be induced under adverse circumstances, especially nitrogen deficiency (Fuentes-Grunewald et al. ). In addition, microalgae can be cultivated for biofuel production and wastewater treatment synchronously to save huge resources, which led to the decrease in the cost input and in the potential for eutrophication in waters (Hu et al. ). At present, a large number of studies have been conducted doi: 10.2166/wst.2014.285

utilizing microalgae to produce lipid and remove nutrients in different kinds of wastewater. Huo et al. () found that the Chlorella zofingiensis in bench-scale outdoor ponds could accumulate lipid content up to 31.8% and remove 97.5% PO3 4 and 51.7% total nitrogen (TN) from the dairy wastewater. Zhou et al. () studied the properties of a growing wastewater-borne microalga Auxenochlorella protothecoides UMN280, and the result showed that the TN and total phosphorus (TP) were removed by more than 59% and 81%, respectively, and significantly high biomass (269 mg · L1 · d1) and lipid productivity (78 mg · L1 · d1) were obtained synchronously. Unfortunately, a strong negative correlation between growth rate and lipid content was observed for all species (Roleda et al. ). In order to obtain both high algal biomass and lipid content, a two-stage cultivation strategy was proposed whereby sufficient nutrient was supplied in the first stage to produce large biomass and nutrient deprivation occurred in the second stage to boost lipid

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synthesis. As a result of adopting this strategy the high lipid productivity would be attained as expected. To further optimize the algal growth conditions, some factors were investigated; for example under N deprivation (2.5 mg · L1) and P deprivation (0.1 mg · L1), the lipid contents of Scenedesmus sp. LX1 were boosted up to 30% and 53%, respectively (Li et al. a). Also, with nitrate or urea as N source, the alga grew well and removed both TN (90%) and TP (100%) efficiently (Li et al. b); the highest eicosapentaenoic acid (EPA) percentages of total fatty acids in Pinguiococcus pyrenoidosus 2078 were obtained at 20 C (20.83%) and at 100 μmol photons · m2 · s1 (31.37%), respectively (Sang et al. ). pH is an important factor regulating the algal abundance and distribution in freshwater systems. However, only a few reports have been conducted about the effect of initial pH on algal properties in batch culture for coupled system of lipid production and wastewater treatment; for example Li et al. (b) reported that the proper pH for Scenedesmus sp. LX1’s growth and nutrient uptake was 7.0 when ammonium was the main N source in wastewater; Sang et al. () found that the highest percentages of total polyunsaturated fatty acids and EPA in Pinguiococcus pyrenoidosus 2078 of total fatty acids were reached at an initial pH of 6.0. The pH change of the culture medium was also accompanied by changing alkalinity. The main components of alkalinity include hydroxide ion, carbonates and bicarbonates. Since alkalinity affects algal inorganic carbon source, changes in the latter in culture system can be understood and adjusted with the determination of alkalinity. Lee et al. () reported that one of the possible limitations for the use of sulfur-utilizing denitrification was the alkalinity requirement for the neutralization of protons produced with sulfate. Accordingly, this paper aims at studying the effects of different initial pH values on the growth, lipid accumulation and nutrient uptake properties of Chlorella sp. HQ as well as alkalinity changes in culture system. Based on the results, optimal pH values could be provided for the coupled system of synchronous lipid accumulation and nutrient uptake in wastewater. W

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Collection Center) used in this study was isolated in our previous study (Zhang & Hong ). Experimental set-up Chlorella sp. HQ was incubated in 300 mL sterile modified 50% BG11 medium (Zhang & Hong ) in 500-mL conical flasks with an initial density of 2 × 105 cells · mL1. To determine the effect of initial pH value, the temperature, illumination and cyclic illumination period were set at 25 C, 60 μmol photons · m2 · s1 and 14 (light):10 (dark) hours. The initial pH was adjusted to 5.0, 7.0, 9.0, 10.0 or 11.0 by using either 0.1 mol · L1 HCl or 0.1 mol · L1 NaOH solution. All the experiments were conducted in triplicate and the conical flasks were hand shaken two to three times per day. The algal density, the pH value, and the concentrations of TN and TP in each growth medium were tested every 2 days. The alkalinity of each medium was measured every 8 days and at the end of the 30-day cultivation. Also, the dry weight of algal biomass, lipid content (%, dry weight), and TAGs content (%, dry lipid) were assayed after 30 days of cultivation. W

Analytical methods The algal density was determined by counting cell numbers using a hemocytometer under an optical microscope (XSZHS3, COIC, China) (Zhang & Hong ). The relationship between algal growth and density was described by a logistic model as shown in Equations (1)–(3) (Zhang & Hong ). Equation (2) was transformed from Equation (1), the values of a and r were obtained by a regression line between N and t. When N reached half of K, the population growth rate (R) was at its maximal value (Rmax), which was calculated in Equation (3) N¼  ln

K 1 þ eart

 K  1 ¼ a  rt N

Rmax ¼ rK=4

(1)

(2)

(3)

MATERIALS AND METHODS Microalga and culture medium The microalga Chlorella sp. HQ (Collection No. GCMCC7601 in the China General Microbiological Culture

N is the algal cell density at time t (d), K (cells · mL1) is the maximal algal cell density in the culture medium, a is a constant, and r (d1) is the intrinsic specific growth rate, and Rmax (cells · mL1 · d1) is the maximal population growth rate.

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The microalgal biomass, total lipid content (dry weight), TAGs content (dry lipid) and the concentrations of TN and TP were measured as described by Zhang & Hong (). The pH value in the algal culture medium was tested by a pH meter with the cultivation time. The alkalinity was determined by potentiometric titration using the combination of an automatic titrator with a pH meter. The samples of each medium, which were all filtered with a 0.45 μm filter membrane, were titrated with 0.1 mol · L1 HCl standard solution and the potentiometric titration ended up with pH ¼ 3.7 (State Environmental Protection Administration ). All analyses were conducted for three independent cultures. The significance analysis was conducted by an independent-samples t-test using SPSS (version 18.0) statistical software (a P-value 0.05). As reported, the r of Scenedesmus sp. LX1 dropped to 0.2 d1 as initial pH became lower than 6.0 (Li et al. b). It indicates that Chlorella sp. HQ could maintain relatively high growth rate in a wide range of initial pH values. However, the K and Rmax were notably affected by initial pH (P < 0.05). For instance, at initial pH 5.0, the values of K and

Figure 1

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Microalgal growth curves in culture media with varied initial pH values.

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Table 1

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The K, r and Rmax of Chlorella sp. HQ at different values of initial pH (SD: standard deviation)

Initial pH

5.0

7.0

9.0

10.0

11.0

K ± SD 1.9 ± 0.0 2.7 ± 0.3 3.1 ± 0.2 3.0 ± 0.5 3.2 ± 0.2 (107 cells · mL1) r ± SD (d1)

0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.1 0.5 ± 0.0 0.5 ± 0.0

Rmax ± SD (107 0.2 ± 0.0 0.4 ± 0.0 0.4 ± 0.1 0.4 ± 0.1 0.4 ± 0.0 cells · mL1 · d1)

Rmax were obviously lower than that at other pH values. Hence, the neutral or alkaline condition should be adopted for the growth of Chlorella sp. HQ. Generally, different algae might have different growth responses to the changes of pH values. As reported, the cells of Chlorella protothecoides grew very fast at the pH value of 5.0, and the profile of specific cell growth rate was higher than at other pH values (4.0, 4.5, 5.5, 6.0, 6.5, 7.0). Concisely, its proper pH was suggested to be controlled at 5.0 (Liang et al. ). Also, alkaline pH was reported to inhibit the growth of Chlorella CHLOR1 based on morphological observations (Guckert & Cooksey ). In contrast, the r of Scenedesmus sp. LX1 was distinctly higher with initial pH in a range of 6.0–10.3 than that with pH less than 6.0 (Li ). Therefore, to disclose the growth changes under different pH values for specific algae with high-lipid-yield abilities is of great importance for its application during the coupling technique for microalgal lipid production and wastewater treatment.

pH change tendency in algal cultures during the cultivation The changes of pH value in algal culture during 30-day batch experiments are shown in Figure 2. It was observed that the pH value in the medium was slightly increased with cultivation and finally maintained at around 6.0 at initial pH value of 5.0. When the initial pH was in a range of 7.0–11.0, the pH values declined obviously in the third day, lifted slightly in the fourth day and was maintained between 7.0 and 8.0 from the fourth day to thirteenth day, afterwards followed by a slow decrease during the residual culture time. It has been a common conclusion that photosynthesis could cause the pH to rise with cultivation time increase. Hu et al. () investigated the changes of pH value of algal cultures during a 5-day batch experiment of Chlorella

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Figure 3 Figure 2

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Changes in pH values during the 30-day batch experiment in culture media with varied initial pH values.

sp. on 20-fold diluted raw and sterilized, traditionally digested liquid swine manure, and raw and sterilized and acidogenically fermented liquid swine manure, and the result showed that the pH values increased from 7.0 to 8.0–9.0. Similar results reported by Su et al. () also showed that the pH values of algal reactors (including Phormidium sp., Chlamydomonas reinhardtii, Chlorella vulgaris and Scenedesmus rubescens) changed from initial 7.0 to around 10.0 after 7 days of cultivation for the four algae species. However, in this study, the pH values showed a change tendency towards neutrality and ended in a range of 6.0–9.0. The results above indicated that Chlorella sp. HQ had a good ability to adjust the pH values to be suitable for cell growth in either acidic or alkaline culture medium. Algal biomass, lipid and TAGs accumulation at different initial pH After 30 days of cultivation, the algal biomass levels obtained at different initial pH values are presented in Figure 3(a). As represented, the dry weights of algal biomass were in a range of 0.38–0.46 g · L1, and the minimal value was achieved at the initial pH of 5.0. When the initial pH was above 7.0, the biomass varied in a small range of 0.46–0.48 g · L1. Generally, the common algal biomass levels in typical cultivation were between 0.3 g · L1 and 0.5 g · L1. Li () reported that the algal biomass of Scenedesmus sp. LX1 increased from 0.04 to 0.37 g · L1 as the initial pH value rose from 5.3 to 10.9. The results imply that Chlorella sp. HQ could accumulate a significantly high level of algal biomass in a wide range

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Algal biomass, lipid yield, TAGs yield (a), Lipid contents per algal biomass (%, dry weight) and TAGs contents per lipid (%, dry weight) (b) of Chlorella sp. HQ after 30 days of cultivation at different initial pH values.

of initial pH changes, which is beneficial to wastewater purification and lipid production based on microalgae. The lipid contents per algal biomass and lipid yields of Chlorella sp. HQ after 30 days of cultivation at varied initial pH values are shown in Figures 3(a) and 3(b). During the cultivation, both lipid content and yield increased to the maxima at the initial pH of 7.0 firstly, and then followed a linear decrease as initial pH value rose to 10.0; afterwards the lipid content was maintained at this level, whereas the lipid yield continued increasing. It was observed that the change tendencies in the above parameters were consistent with the changes in pH values. After 30 days of cultivation, the algal lipid contents ranged between 14.7 and 32.8%, and the lipid yields were in a range of 52.5–167.5 mg · L1. Notably, the lipid content was found to be at a relatively high level (18.3%) at the initial pH value of 5.0, which was higher than at initial pH of 10.0 and 11.0 (around 14.7%). It can be reasonably inferred that the initial pH-limited (pH ¼ 5.0) condition could enhance the lipid content to a certain degree. So far, large numbers of researchers have examined the effect of nutrient concentrations on lipid accumulation of microalgae, and a common conclusion was obtained, which was that nutrient deficiency was an efficient approach to enhance the lipid content (Rodolfi et al. ; Roleda et al. ). However, the studies conducted on the effect of initial pH values on algal lipid accumulation property were limited. Liang et al. () reported that the optimal pH value for lipid accumulation of Chlorella protothecoides during batch culture was 6.5. Li () found that Scenedesmus sp. LX1 attained distinctly higher lipid content at initial pH value of 3.9 (above 30%), as compared to that at initial pH of 5.3–10.9 (the minimum value: about 15%).

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Hence, through comprehensive analysis, the results in this study verified the conclusion that adjusting the initial pH values of cultures provided another way to optimize the lipid accumulation of microalgae, and the optimal initial pH value for lipid accumulation of Chlorella sp. HQ should be controlled between 7.0 and 9.0. The TAGs in microalgae, as the main feedstock for biodiesel production, were assayed at varied values of initial pH. After 30 days of cultivation, the TAGs contents per dry lipid and total TAGs yields of Chlorella sp. HQ were obtained, as shown in Figures 3(a) and 3(b). The TAGs contents at different initial pH of 5.0–11.0 remained at a significantly high level, ranging from 24.5 to 63.0%. The peak value of TAGs content was achieved at initial pH value of 5.0, and with initial pH increasing to 7.0, it declined dramatically to the minimum. When the initial pH was above 9.0, the TAGs contents decreased slightly and varied in a small range of 38.3–44.4%. Generally, the TAGs yield was decided by the algal biomass associated with TAGs content. Chlorella sp. HQ achieved maximal TAGs yield of 54.4 mg · L1 at initial pH 9.0 and a second largest value of 43.7 mg · L1 at initial pH 5.0. Factors such as nutrient concentrations, irradiance, and ethyl-2methyl acetoacetate (EMA) have been shown to affect TAGs accumulation in many algae. As reported, the TAGs of Chlamydomonas reinhardtii increased rapidly in response to nutrient starvation, especially sulfur deprivation (Cakmak et al. ), and generally the algae cells channeled the excess carbon and energy into lipid storage (mainly TAGs) under N deprivation (Rodolfi et al. ). Remarkably, the TAGs content per lipid (about 20%) and TAGs productivity (about 23 mg · L1) of Scenedesmus sp. LX1 were increased by 79 and 40%, respectively, under EMA concentrations of 1.0– 2.0 mg · L1, comparing with the one without EMA treatment (Li et al. c). However, quite a few studies have

Figure 4

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investigated the effect of initial pH values on TAGs accumulation of microalgae. The TAG accumulation of Chlorella CHLOR1 was induced by alkaline pH stress (Guckert & Cooksey ), whereas the TAGs content of Scenedesmus sp. LX1 was boosted slightly under pH-limited condition (pH ¼ 3.9), in contrast, the TAGs yield related to the initial pH value positively (Li ). Accordingly, the pH limitation (pH ¼ 5) provides a viable and efficient approach to enhance both TAGs content and yield of Chlorella sp. HQ in this study, and the optimal initial pH value should be set in the range of 5.0–9.0. Nitrogen and phosphorus uptake efficiencies at different initial pH The changes in the concentrations of TN and TP during the cultivation of Chlorella sp. HQ at different initial pH values are shown in Figure 4, and after the cultivation, the uptake efficiencies were obtained, as shown in Table 2. It is clear that the uptake rate of TP by Chlorella sp. HQ was higher than that of TN. At the 10th day of cultivation, the concentrations of TP dramatically decreased with initial pH value ranging from 7.0 to 11.0, while the concentrations of TN still remained at a relatively high level until the 18th day with pH values between 5.0 and 11.0. After the cultivation, the uptake efficiencies of TP at different initial pH values varied in a small range from 92.05 to 93.80%. However, the TN uptake efficiency in acid circumstance was relatively lower than other experimental groups, reaching 79.54%. At pH value of 7.0, the largest amount of TN was removed, and the uptake efficiency was obtained as 87.77%. Based on the property that microalgae can take up a large amount of nutrients for cells growth, a new concept was proposed that the excess TN and TP could be extensively removed by microalgae, implying that almost all the available ammonia N appeared in the form of algal cell

The concentrations of TN and TP versus cultivation time in culture media with different initial pH values.

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Table 2

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TN and TP uptake efficiencies of Chlorella sp. HQ after 30 days of cultivation at different initial pH values

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Alkalinity change tendency of culture media during the cultivation

pH value 5.0

7.0

9.0

10.0

11.0

TN uptake efficiency (%)

79.54

87.77

85.99

84.55

84.69

TP uptake efficiency (%)

92.64

92.05

92.79

93.45

93.80

material under optimal operating conditions. This novel technology has been developed rapidly and used extensively in the past half a century. Shi et al. () reported that Chlorella vulgaris and Scenedesmus rubescens removed over 90% TN and 90% TP in secondary wastewater within 9 days using a twin-layer system. To further obtain a substantial saving in the overall cost, using oleaginous microalgae to uptake nutrients from wastewater is an efficient approach, which has drawn significant attention in recent years. Farooq et al. () found that Chlorella vulgaris (UTEX-265) could remove more than 90% of both TN and TP from brewery wastewater, and synchronously the algal lipid productivity could reach up to 108.0 mg · L1d1. In addition, Chlorella ellipsoidea YJ1 was found to remove more than 99% TN and 90% TP from domestic secondary effluent and achieve lipid content per algal biomass as high as 43% (Yang et al. ). Indeed, the nutrient uptake efficiency based on microalgae can be influenced by some factors, such as N/P ratio, salinity, illumination and pH. However, the studies about the effect of initial pH on nutrient uptake of microalgae were quite few. Generally, the pH values rise with the algal photosynthesis (Hoffman ; Garcia et al. ). Garcia et al. () reported that the diurnal rhythm of the algal photosynthetic activity in the mixed liquor of high rate ponds causes variations of dissolved oxygen and pH during the day. It was reported that the algal growth was inhibited with significantly high pH values (8.5–9.0), which was not conducive to the nutrient uptake (Li et al. ). For Scenedesmus sp. LX1, the proper pH to remove TN (81.3–95.7%) and TP (nearly 100.0%) was in the range of 7.3–10.3 (Li ). In this study, the value of initial pH should be controlled between 7.0 and 11.0 to remove N and P efficiently by Chlorella sp. HQ. Through comprehensive analysis, Chlorella sp. HQ mainly produced biofuels in the wastewater with initial pH of 5.0–9.0, and mainly removed TN and TP from wastewater with initial pH of 7.0–11.0. Hence, the optimal initial pH values to produce biofuels as well as remove nutrients were suggested to be in the range of 7.0–9.0.

The changes of alkalinity in algal culture during 30-day batch experiments are shown in Figure 5. Since the initial pH was adjusted to different levels, initial alkalinities of all cultures differed from each other. And it is clear that there exists a positive correlation between the initial alkalinity of different cultures and the initial pH values, which hints that the concentration of inorganic carbon source including carbonate and bicarbonate increased with the initial pH values. It is worth mentioning that there were large gaps for initial alkalinities among the cultures with different initial pH values, although the gaps were reduced with increasing cultivation time. Especially at the eighth day, the alkalinities in the cultures with the initial pH of 7.0 and 9.0 increased but the alkalinities with the initial pH of 10.0 and 11.0 decreased obviously. Starting from the eighth day, alkalinities of all cultures showed an increasing tendency. Interestingly, the alkalinities in the cultures with the initial pH of 11.0 and 5.0 still kept the maximum and minimum level during the whole cultivation time, respectively. Carbonates and bicarbonates are the most common and most important components of alkalinity (Wurts & Durborow ), and are a carbon source in algae culture medium. The relative concentrations of CO2, HCO 3 , and CO2 of the carbonate system and the pH of the water 3 system are closely linked. As pH increases, carbonate increases and bicarbonate and molecular CO2 decrease (Chen & Durbin ). Uusitalo () conducted a pHdrift experiment on three marine algae and found that Ascophyllum nodosum was able to take up carbon, while

Figure 5

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Changes of alkalinity during the 30-day batch experiment in culture medium.

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remaining at the same pH, due to lowered alkalinity caused by proton excretion; Ulva lactuca did not change alkalinity while taking up carbon and consequently showed the expected pH increase, an effect of the carbon uptake; Polysiphonia nigrescens released carbon while remaining at the same pH, the latter being a consequence of increased alkalinity. And Brewer & Goldman () found that uptake of NO 3 by algae in culture medium caused an increase in alkalinity, whereas uptake of NHþ 4 produced a decrease. Generally photosynthesis could cause pH to rise with cultivation time; hence it is speculated that alkalinity of the culture medium could be changed at the same time. In this study, the pH values showed a change tendency towards neutrality and ended in a range of 6.0–9.0, which was in accordance with the change tendency of alkalinity of the first 8 days. In actual conditions, pH increase may occur during algal blooms which form hydroxide as algae consumes all carbonate alkalinity (Gao et al. ). And it can be observed that alkalinity of all cultures tends to increase after 8 days. More precisely, the alkalinity increased significantly after 8 days of cultivation but approached a plateau between 16 and 24 days, which agreed with the uptake tendency of TN, reaching nearly the largest uptake efficiencies at 16 days, with sodium nitrate as the main nitrogen source, and demonstrated that uptake of NO 3 by algae in culture medium caused an increase in alkalinity (Brewer & Goldman ). During the last 6 days of cultivation, pH values tended to be almost steady but the alkalinity still increased, which may be due to the increase of carbonates and bicarbonates in culture media, absorbed from the atmosphere, or the release of algae cells at stationary phases of growth.

CONCLUSIONS Chlorella sp. HQ maintained high growth rate with initial pH between 5.0 and 11.0. Both the lipid content and yield in microalgae reached the maxima (32.8%, 168 mg · L1) at initial pH of 7.0. Promisingly, the pH limitation (initial pH ¼ 5) provides a viable and efficient approach to enhance both algal TAGs content (up to 63.0%) and yield (up to 43.7 mg · L1), and the optimal initial pH value to produce TAGs should be set in the range of 5.0–9.0. In addition, to synchronously remove the TN (>84.55%) and TP (>92.05%) efficiently, the initial pH should be controlled between 7.0 and 11.0. The pH values showed a change tendency towards neutrality, which was in accordance with the change tendency of alkalinity during the first 8 days. And the increasing tendency of alkalinity could be observed for all

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cultures after 8 days with the consumption of nitrate, which was followed by maintenance of the increasing trend possibly due to CO2 absorption from the atmosphere and the release of algae cells at stationary phases of growth. It was suggested that the optimal initial pH values for Chlorella sp. HQ to produce biofuels, combined with purification of wastewater, should be controlled between 7.0 and 9.0, and the characteristics of this alga should be considered for application in the coupled system.

ACKNOWLEDGEMENTS We are grateful for the support of Professor Hu Hong-Ying for the dedicated technical assistance and the assistance of Prof. Hou Yang-Long, Prof. Qiang Zhi-Min, Dr Wu YinHu and Dr Yu Yin for paper revision. This study was supported by Beijing Nova Program (grant no. 2010B019).

REFERENCES Brewer, P. G. & Goldman, J. C.  Alkalinity changes generated by phytoplankton growth. Limnology and Oceanography 21 (1), 108–117. Cakmak, T., Angun, P., Demiray, Y. E., Ozkan, A. D., Elibol, Z. & Tekinay, T.  Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii. Biotechnology and Bioengineering 109 (8), 1947–1957. Chen, C. Y. & Durbin, E. G.  Effects of pH on the growth and carbon uptake of marine phytoplankton. Marine Ecology Progress Series 109 (1), 83–94. Farooq, W., Lee, Y. C., Ryu, B. G., Kim, B. H., Kim, H. S., Choi, Y. E. & Yang, J. W.  Two-stage cultivation of two Chlorella sp. strains by simultaneous treatment of brewery wastewater and maximizing lipid productivity. Bioresource Technology 132, 230–238. Fuentes-Grunewald, C., Garcés, E., Alacid, E., Sampedro, N., Rossi, S. & Camp, J.  Improvement of lipid production in the marine strains Alexandrium minutum and Heterosigma akashiwo by utilizing abiotic parameters. Journal of Industrial Microbiology & Biotechnology 39 (1), 207–216. Gao, Y., Cornwell, J. C., Stoecker, D. K. & Owens, M. S.  Effects of cyanobacterial-driven pH increases on sediment nutrient fluxes and coupled nitrification-denitrification in a shallow fresh water estuary. Biogeosciences 9, 2697–2710. Garcia, J., Green, B. & Oswald, W.  Long term diurnal variations in contaminant removal in high rate ponds treating urban wastewater. Bioresource Technology 97 (14), 1709–1715. Guckert, J. B. & Cooksey, K. E.  Triglyceride accumulation and fatty acid profile changes in Chlorella (chlorophyta)

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First received 19 December 2013; accepted in revised form 12 June 2014. Available online 25 June 2014

Investigation of initial pH effects on growth of an oleaginous microalgae Chlorella sp. HQ for lipid production and nutrient uptake.

Using microalgae for synchronous biodiesel production and wastewater treatment is a promising technology. The growth, lipid accumulation and nutrient ...
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