Bioresource Technology 181 (2015) 54–61

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Nutrients removal and lipids production by Chlorella pyrenoidosa cultivation using anaerobic digested starch wastewater and alcohol wastewater Libin Yang, Xiaobo Tan, Deyi Li, Huaqiang Chu, Xuefei Zhou, Yalei Zhang ⇑, Hong Yu State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China Key Laboratory of Yangtze Water Environment for Ministry of Education, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China

h i g h l i g h t s  Microalgae culture used anaerobic starch wastewater and alcohol wastewater.  Addition of alcohol wastewater obviously improved biomass and lipids production.  Pollutants in wastewater were efficiently removed.

a r t i c l e

i n f o

Article history: Received 12 November 2014 Received in revised form 6 January 2015 Accepted 9 January 2015 Available online 17 January 2015 Keywords: Chlorella pyrenoidosa (C. pyrenoidosa) Anaerobic digested starch wastewater (ADSW) Alcohol wastewater (AW) Lipids production Pollutants removal

a b s t r a c t The cultivation of microalgae Chlorella pyrenoidosa (C. pyrenoidosa) using anaerobic digested starch wastewater (ADSW) and alcohol wastewater (AW) was evaluated in this study. Different proportions of mixed wastewater (AW/ADSW = 0.176:1, 0.053:1, 0.026:1, v/v) and pure ADSW, AW were used for C. pyrenoidosa cultivation. The different proportions between ADSW and AW significantly influenced biomass growth, lipids production and pollutants removal. The best performance was achieved using mixed wastewater (AW/ADSW = 0.053:1, v/v), leading to a maximal total biomass of 3.01 ± 0.15 g/L (dry weight), lipids productivity of 127.71 ± 6.31 mg/L/d and pollutants removal of COD = 75.78 ± 3.76%, TN = 91.64 ± 4.58% and TP = 90.74 ± 4.62%. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction In China, annual starch production has increased to more than 10 million tons, generating about 60 million cubic meters of starch wastewater (SW) by approximately 600 starch production plants (Lu et al., 2009; Xue et al., 2010). SW is generated from extraction processes, and contains abundant nutrients, including organic matters, nitrogen (N) and phosphorus (P). SW treatments generally focus on simple sedimentation for suspended solids recovery to produce alcohol, whereas supernatant treatments use traditional

⇑ Corresponding author at: State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China. Tel.: +86 21 65983803; fax: +86 21 65988885. E-mail addresses: [email protected] (X. Zhou), [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.biortech.2015.01.043 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

anaerobic–aerobic (A/O) processes, such as an upflow anaerobic sludge bed (UASB), an expanded granular sludge bed (EGSB) and the sequencing contact oxidation process or sequencing batch reactor (SBR) (Chan et al., 2009). Anaerobic processes are appropriate for the treatment of high-strength SW to recover most organic matters using methane. However, it is controversial to achieve nitrogen and phosphorus removal goals using aerobic processes, especially for anaerobic effluents containing large amounts of nutrients (Yang et al., 2011). Aerobic processes for nitrogen and phosphorus removal require high energy costs and generate abundant greenhouse gases and sludge into the environment (Fricke et al., 2005). Thus, an optimal alternative treatment process should be more energy-saving and environmentally friendly to utilize the production of useful organisms and remove pollutants from wastewater. Integration of wastewater treatment and microalgae cultivation may be an optimal alternative treatment process. Microalgae have

L. Yang et al. / Bioresource Technology 181 (2015) 54–61

recently attracted considerable attention as a next generation energy feedstock (Brennan and Owende, 2010; Fenton and Ó hUallacháin, 2012). Compared to other oil crops, microalgae have a number of compelling characteristics to support their development, including high per-acre productivity, short growth cycle, and utilization of non-arable land and a wide variety of water sources (fresh, saline, and wastewater) (Wu et al., 2012). However, the costs of microalgae-based energy are much higher than traditional fossil fuels, in which the supplements of water resources and nutrient elements (i.e., nitrogen, phosphorus and potassium) represent a major obstacle to cost reduction (Behzadi and Farid, 2007; Komolafe et al., 2014). Both stable water resources and nutrient elements in wastewater are considered to be good resources for microalgae cultivation, and simultaneously, microalgae growth can effectively remove pollutants from wastewater associated with little sludge production and carbon emission. Anaerobic digested wastewater generally contains abundant dissolved nutrients that can be directly utilized for microalgae growth. Several researchers have explored the cultivation of microalgae using anaerobic digestion liquids: Marques et al. (2013) tested the potential combination of the anaerobic process and Chlorella vulgaris cultivation for vinasse treatment, Park et al. (2010) cultured green algal Scenedesmus sp. in an anaerobic digestion effluent of livestock waste for ammonia removal and Bahr et al. (2013) used an alkaliphilic microalgal-bacterial consortium for nutrients removal from anaerobic effluents and flue gas capture. These reports demonstrated that the effluents from anaerobic digesters are good mediums for microalgae culture. Anaerobic digested starch wastewater (ADSW) is rich in dissolved nitrogen phosphorus and other trace elements, which might also be a good medium for microalgae culture. Microalgae, by means of mixotrophic or heterotrophic growth utilizing organic carbon as the major carbon source, can promote biomass production and lipids content. Reports have demonstrated that simple dissolved organics (i.e., glucose, short-chain fatty acid) were beneficial to microalgae mixotrophic growth (Zhang et al., 2014). However, the content of organic carbon in ADSW was limited; even part of the organic matters were inert after the anaerobic processes (Cai et al., 2013; Levine et al., 2011). Usually pure artificial organics (i.e., glucose) are preferred, but are costly, and their utilization may cause an adverse effect on food production. Therefore cheaper alternatives need to be explored. Alcohol processing is usually associated with starch production (recovering suspended solids from starch processing wastewater), and alcohol wastewater (AW) which contains abundant simple dissolved organics as an appropriate carbon source for microalgae mixotrophic growth. This suggests that combining ADSW and AW with an appropriate ratio may have potential advantages to optimize both biomass production and lipids content. In this study, Chlorella pyrenoidosa (C. pyrenoidosa) was chosen as the inoculation candidate to generate complex biomolecules mixotrophically utilizing organic carbon or CO2 as a carbon source. To investigate the optimal ratio of ADSW and AW for microalgae growth and nutrients removal, three different proportions of mixed wastewater (AW/ADSW = 0.176:1, 0.053:1, 0.026:1, v/v) and single pure ADSW, AW were evaluated for C. pyrenoidosa cultivation as well as for biomass production, lipids content and pollutants removal.

55

produced from the starch processing plant was first settled in the tank. Then the supernatant was treated using a UASB reactor, followed by two-stage aerobic processing (anoxia-biological contact oxidation process); the suspended solids (SS) recovered by sedimentation were utilized for alcohol production through fermentation. Alcohol wastewater (AW) was first settled for three or four days by a rapid acidification process to obtain abundant dissolved organics, especially volatile fatty acids. In addition, both anaerobic digested starch wastewater (ADSW) and pretreated AW still contained high suspended solids that could hinder the growth of microalgae photosynthesis. Thus, all of the wastewater samples were allowed to settle for several hours and were filtered using a 0.45 lm polyester filter. The wastewater after sterilization was stored at 4 °C prior to the experiments. 2.2. Microalgae strain and culture medium C. pyrenoidosa (C. pyrenoidosa, FACHB-9) was obtained from the Institute of Hydrobiology (the Chinese Academy of Sciences, Wuhan, China). Prior to being cultured in wastewater, C. pyrenoidosa was cultured under sterile conditions in sterilized SE medium, which consists of NaNO3 (0.25 g/L), K2HPO4 (0.075 g/L), MgSO4 7H2O (0.075 g/L), CaCl22H2O (0.025 g/L), KH2PO4 (0.175 g/L), NaCl (0.025 g/L), FeCl36H2O (0.005 g/L), H3BO3 (2.86 mg/L), MnCl24H2O (1.86 mg/L), ZnSO47H2O (0.22 mg/L), Na2MoO42H2O (0.39 mg/L), CuSO45H2O (0.08 mg/L) and Co (NO3)26H2O (0.05 mg/L). The pH value of the SE medium is approximately 7. The cultivation conditions were as follows: light intensity = 127 lmol m2 s1, light/ dark ratio = 12:12, temperature = 25 ± 1 °C and artificial intermittent shaking four times in a day for 6–8 days. To ensure normal microalgae growth in ADSW, a two-phase cultivation strategy was adopted for microalgae adaption according to the reports by Tan et al. (2014): (I) C. pyrenoidosa was cultured in wastewater at low temperature (15 °C) and low light intensity (60 lmol m2 s1) for 3–4 days, and (II) the culture was subjected to high temperature (35 °C) and high light intensity (220 lmol m2 s1) for 6–8 days. Finally, C. pyrenoidosa adapted successfully and exhibited a good growth performance in ADSW. 2.3. Experiments design The samples of AW and ADSW after pretreatment were used for C. pyrenoidosa cultivation. To investigate the optimal ratio of AW and ADSW for microalgae growth and nutrients removal, three experimental groups (total volume of 1000 ml) were evaluated, including G1 (AW/ADSW = 0.176:1, v/v), G2 (AW/ADSW = 0.053:1, v/v) and G3 (AW/ADSW = 0.026:1, v/v). Additionally, comparative experiments were also conducted using single pure ADSW (G4) and AW (G5) for C. pyrenoidosa cultivation. All of the experiments were performed in triplicate. The initial inoculation concentration of C. pyrenoidosa was approximately 0.50 g/L (dry weight). The initial pH was controlled at 6–7 by adding 1 mol/L NaOH solution. C. pyrenoidosa were cultivated in 2 L glass conical flasks placed in an illumination incubator (GZX-300BS-III, CIMO Medical Instrument, Shanghai, China). The cultivation conditions were as follows: light intensity = 127 lmol m2 s1, light/dark ratio = 12: 12, temperature = 25 ± 1 °C and artificial intermittent shaking four times per day for 9 days. 2.4. Analytical procedure

2. Methods 2.1. Wastewater used for microalgae cultivation All of the wastewater samples were collected from a starch processing plant in Shandong Province, China. Starch wastewater

2.4.1. Microalgae biomass The total biomass was determined based on the relationship between the dry cell weight and Chlorophyll a. To determine the chlorophyll a concentration, a 5 ml solution was collected each day and centrifuged at 4500 rpm for 10 min. The cell pellets were

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re-suspended in 5 ml of 95% methanol, incubated at 60 °C for 5 min, and then centrifuged again for 10 min. The absorbance of the supernatant at 665 and 652 nm was measured and the chlorophyll a concentration was calculated with Eq. (1) from Porra et al. (1989):

Chlorophyll a ðmg=LÞ ¼ 16:29  A665  8:54  A652

ð1Þ

1

The specific growth rate l (day ) in the exponential phase of algal growth was measured using Eq. (2) from Zhu et al. (2013):

l ¼ Ln ðN2 =N1 Þ=ðt2  t1 Þ

ð2Þ

where N1 and N2 are defined as the dry biomass (g/L) at times t1 and t2, respectively. A calculation of the organics producing algae coefficient is shown in Eq. (3) from Hongyang et al. (2011):

Pn

Y B=COD

SBMt ¼ Pn t¼1 ðS t¼1 ðt1Þ  St Þ

ð3Þ

where YB/COD (g algal/g COD) is the yield coefficient, SBMt (mg/L) is the algae concentration at time t, and St (mg/L) is the total organic matters (COD) in the culture medium at time t. 2.4.2. Microalgae elemental analysis, total lipids and fatty acid methyl ester (FAME) contents The algae samples were dried in a vacuum freeze dryer and ground to powder for analysis. C. pyrenoidosa was analyzed for carbon (C), hydrogen (H) and nitrogen (N) contents using an elemental analyzer (Vario EL III, Elementar Analysen Systeme, USA). The total lipids in biomass were determined using methods described by Yoo et al. (2010). Lipids were extracted in batch with a chloroform–methanol (2:1, v/v) solution for 8 h; methanol and water were then added producing a final solvent ratio of chloroform: methanol: water = 1:1:0.9. The solvent was separated into two layers (chloroform and aqueous methanol layers), and the chloroform layer was washed with 20 ml of a 5% NaCl solution and were evaporated to dryness. The remaining mass was weighed and represented the total lipids. The lipids productivity (Lp, mg/L/d) of the microalgae is calculated in Eq. (4):

Lp ¼ Lc  Bm =T

ð4Þ

where Lp (mg/L/d) is the lipids productivity, Lc (%) is the lipids content in the biomass, Bm (mg) is the weight of accumulated biomass, and T (d) is the rapid growth time of microalgae. At the end of the batch experiments, the composition and content of FAME were determined using a GC–MS (Agilent) equipped with a flame ionization detector and a Supelco DB-FFAP capillary column (30.0 m  0.25 mm  0.25 lm). The chromatographic conditions were as follows: injection volume = 1 ll; split ratio = 1:10; air = 450 ml/min; H2 = 40 ml/min; gas carrier (N2) = 45 ml/min; injector temperature = 250 °C; detector temperature = 300 °C; and oven temperatures starting at 140 °C for 2 min and raised to 240 °C at a rate of 10 °C/min, and then held at 240 °C for 2 min. The FAME contents were determined by comparing their peak areas with an internal standard (methyl benzoate). 2.4.3. Chemical analysis The concentrations of chemical oxygen demand (COD), total + phosphorus (TP), orthophosphate (PO3 4 ), ammonium (NH4-N), and total nitrogen (TN) were measured according to the Chinese State Environmental Protection Agency Standard Methods (Wei et al., 2002). The values of pH were measured by a pH meter (Rex Electric Chemical, China). Trace elements and heavy metals in wastewater were measured by ICP-AES (Optima 2100DV, UK) after digestion using a microwave digestion instrument.

To analyze the volatile fatty acids (VFAs) in mediums, samples were first filtered using a 0.45 lm polyester filter. The filtration was placed in a 1.5 ml gas chromatography (GC) vial, and 3% H3PO4 was added to adjust the pH to approximately 4.0. A gas chromatograph (HP6890II, USA) equipped with a flame ionization detector (FID) and an analytical column of CPWAX52CB (30 m  0.53 mm  1 lm) was used to determine the concentrations of VFAs (C2–C5). The sample injection volume was 1.0 ll. The temperatures of the injector and FID were set at 200 and 220 °C, respectively. Nitrogen was used as the carrier gas at a flow rate of 50 ml/min. The GC oven was first set to 110 °C for 2 min, and then, the temperature increased to 220 °C at a rate of 10 °C min1 and was held at 220 °C for 2 min.

3. Results and discussion 3.1. Wastewater for microalgae cultivation Both the characteristics of anaerobic digested starch wastewater (ADSW) and alcohol wastewater (AW) after pretreatment are summarized in Table 1. As shown in Table 1, ADSW contains abundant nitrogen (total nitrogen of 265.1 ± 19.12 mg/L) and phosphorus (total phosphorus of 28.34 ± 1.20 mg/L). More importantly, dissolved ammonium and orthophosphate account for more than 90% of the total nitrogen (TN) and phosphorus (TP). These dissolved nutrients can be directly utilized for microalgae growth. In addition, other nutrients and trace elements that are necessary for microalgae growth, including potassium, magnesium, iron, copper, zinc, manganese and boron, were all detected in wastewater, whereas few or no hazardous heavy metal ions (e.g., chrome, cadmium, lead and arsenic) were observed. These characteristics suggest that ADSW is a good medium for cultivating microalgae autotrophically. However, the organic matters content (COD) in ADSW was limited, as most of the organic matters were recovered by methane. The organic matters content in wastewater was much less than the demand of the carbon source (50% of the total biomass weight) for microalgae, which cannot maintain continuous heterotrophic or mixotrophic growth. Thus, a supplement of the carbon source was necessary for microalgae mixotrophic growth to obtain higher biomass and lipids production. Alcohol wastewater (AW) after acidification pretreatment may represent a good alternative carbon source for microalgae mixotrophic growth. In this study, the AW was collected in the same factory in which the solids recovered from starch wastewater were used for alcohol production. From Table 1, AW after the

Table 1 Characteristics of wastewaters used for C. pyrenoidosa cultivation (mean ± standard deviation). Characteristics

AW (alcohol wastewater)

ADSW (anaerobic digested starch wastewater)

pH Chemical oxygen demand (COD) (mg/L) TN (mg/L) NH+4-N (mg/L) TP (mg/L) Al (mg/L) B (mg/L) Ca (mg/L) Fe (mg/L) K (mg/L) Mg (mg/L) Mn (mg/L) Na (mg/L) Zn (mg/L)

3.7–4.2 65,000 ± 1208

7.1–7.3 926.3 ± 65.2

618.68 ± 48.31 279.72 ± 20.41 47.16 ± 1.02 0.21 ± 0.01 2.45 ± 0.12 96.14 ± 5.68 1.47 ± 0.08 157.75 ± 10.65 152.20 ± 8.11 0.57 ± 0.01 787.74 ± 44.56 0.10 ± 0.01

265.10 ± 19.12 240.88 ± 18.89 28.34 ± 1.20 0.12 ± 0.01 4.01 ± 0.23 98.40 ± 5.10 32.86 ± 1.54 174.47 ± 10.71 181.16 ± 9.34 0.13 ± 0.01 719.40 ± 45.32 0.86 ± 0.01

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acidification process contains a large amount of organic matters (COD of 65,000 mg/L). Additionally, the wastewater is rich in volatile fatty acids (VFAs), in which the contents of acetic acid, propionic acid, iso-butyric acid, n-butyric acid, iso-valeric acid and nvaleric were up to 2912.4 ± 241.5 mg/L, 2852.1 ± 250.1 mg/L, 315.9 ± 31.2 mg/L, 4288.7 ± 401.9 mg/L, 375.5 ± 34.6 mg/L and 214.3 ± 19.8 mg/L, respectively. These dissolved short-chain fatty acids have been proven to be good carbon sources for microalgae mixotrophic growth (Bhatnagar et al., 2011). Thus, a proper addition of AW into ADSW will significantly improve biomass production and lipids content. 3.2. Microalgae growth The addition of AW into ADSW significantly influenced C. pyrenoidosa (C. pyrenoidosa) growth (Fig. 1). Compared to pure ADSW for microalgae culture, a moderate addition of AW into ADSW obviously advanced the biomass growth. From Fig. 1, the maximal biomass concentration was only 1.45 ± 0.12 g/L (dry weight) using pure ADSW (G4) without AW addition. In contrast, with the addition of AW in G2 (AW/ADSW = 0.053:1, v/v) and G3 (AW/ ADSW = 0.026:1), the maximal biomass concentrations increased to 3.01 ± 0.15 g/L and 2.21 ± 0.10 g/L, respectively. Enhancement of microalgae growth from the addition of a carbon-based resource into wastewater was also observed in previous studies. Hongyang et al. (2011) used raw soybean processing wastewater and wastewater with a glucose addition (10 g/L) for C. pyrenoidosa growth and found that the maximal biomass concentration increased from 2.09 g/L to 6.20 g/L with the glucose addition. However, an excessive addition of AW may impose a negative influence on C. pyrenoidosa growth. As shown in Fig. 1, the maximal biomass in G1 (AW/ADSW = 0.176:1) declined to only 0.89 ± 0.13 g/L, and in pure AW medium (G5), microalgae cannot even survive. This is mainly because C. pyrenoidosa has difficulty surviving in wastewater containing excessive high-strength organics (Wang et al., 2012; Zhu et al., 2013). To describe the biomass growth evolution in every group, the mean productivity of biomass was introduced and calculated according to Eq. (5) instead of the classic equation used to calculate productivity by Eq. (6) (Ruiz et al., 2013). Eq. (5) excludes the lag phase and the stationary phase of batch growth and is more suitable for calculating productivity in the exponential growth phase.

Mean productivity ¼ ð0:9X max  1:1X 0 Þ=ðt 0:9  t1:1 Þ

ð5Þ

Classic productivity ¼ ðX max  X 0 Þ=ðt max  t 0 Þ

ð6Þ

3.0

G1

Biomass (dry weigt, g/L)

G2 2.5

G3 G4

2.0

G5

1.5 1.0 0.5 0.0 0

1

2

3

4

5

6

7

8

9

Time (d) Fig. 1. Growth of C. pyrenoidosa in a batch culture using different wastewaters.

Table 2 Parameters of C. pyrenoidosa growth in different wastewaters. Group

Maximal biomass (g/L)

Mean productivity (g/L/d)

Maximal specific growth rate l (d1)

1 2 3 4

0.89 ± 0.13 3.01 ± 0.15 2.21 ± 0.10 1.45 ± 0.12

0.06 ± 0.01 0.58 ± 0.03 0.30 ± 0.02 0.15 ± 0.02

0.18 ± 0.02 0.56 ± 0.01 0.44 ± 0.02 0.28 ± 0.02

where Xmax (g/L) is the maximal algal cell density; X0 is the initial algal cell density; t0.9 and tm are the time at which algal cell density reaches 0.9Xmax and Xmax, respectively; and t1.1 is the time at which algal cell density reaches 1.1 X0. Similar to the maximal biomass concentration, the mean productivity of biomass also significantly increased from the addition of AW into ADSW (Table 2). The results show that the mean biomass productivity during the exponential growth phase in pure ADSW medium (G4) was only 0.15 ± 0.02 g/L/d. With the addition of AW in G2 and G3, the mean biomass productivities increased by 2.87 and 1 times. By contrast, the value declined to only 0.06 ± 0.01 g/L/d in G1 due to microalgae growth inhibition by excessive AW addition. The specific growth rate (l) is an another important parameter to assess the microalgae growth curve and determine a proper hydraulic retention time (HRT) in continuous culture (Ruiz et al., 2013). In continuous mode, the maximal biomass productivity was theoretically achieved at an HRT of 2 l1; when HRT > 2 l1, the biomass productivity will decline with longer HRT, although the biomass concentration in the reactor always increased to the maximal with increasing HRT. If the objective was solely to obtain high biomass productivity in a continuous culture, HRT should been controlled at 2 l1. A higher value of l leads to a shorter HRT in continuous culture. Different specific growth rates were obtained in different groups (Table 2). The maximal specific growth rate (0.56 ± 0.01 d1) was achieved in G2 with a moderate AW addition, consistent with the maximal biomass and productivity in G2. Compared to the pure ADSW (G4) medium, the maximal value of l approximately doubled in G2. This indicates that with the addition of AW (AW/ADSW = 0.053:1), the HRT to obtain the highest biomass productivity would reduce by a half compared to the pure ADSW culture. 3.3. Nutrients removal 3.3.1. Organic matters removal Carbon (C) is a fundamental element in microalgae cells, accounting for approximately 50% of microalgae biomass (Singh and Das, 2014). Some species of microalgae have the ability to utilize organic carbon for heterotrophic or mixotrophic growth. C. pyrenoidosa can change their metabolic pathway according to the supply of organic substrates, such as organic acids or glucose, suggesting that they can sustain heterotrophic growth in addition to common autotrophic growth using CO2 as the sole carbon source (Tan et al., 2014). Thus, C. pyrenoidosa growth in wastewater was considered to be an effective approach to remove organic matters from wastewater. The removal of organic matters in wastewater was also observed in this study. As shown in Fig. 2, the organic matters concentrations (COD) decreased at the end of the culture in every group (except in G5, where C. pyrenoidosa had difficulty surviving). However, the removal efficiencies and COD rates varied greatly among different groups. The highest removal efficiency and COD rate were obtained in G2 with the best performance of biomass growth, reaching 75.78 ± 3.76% and 629.01 ± 39.7 mg/L/d, respectively. Too little or too much addition of AW into ADSW caused a decline in the COD removal efficiency associated with

L. Yang et al. / Bioresource Technology 181 (2015) 54–61

7.12% removal

CODinitial CODfinal

10000 8000 6000

75.78% removal

68.74% removal

4000

61.54% removal

2000 0

1

2

3

4

Group Fig. 2. Organic matters (COD) removal during the microalgae culture periods in every group.

low biomass production in G1 and G3. The removal efficiency and COD rate declined to only 7.12 ± 0.76%, 149.76 ± 12.73 mg/L/d in G1 with an excessive addition of AW and 68.74 ± 2.35%, 292.13 ± 25.71 mg/L/d in G3 with little addition of AW. The organic matters removal rate was only 95.00 ± 12.13 mg/L/d in G4 using pure ADSW due to the carbon source shortage causing a slow biomass growth rate. To evaluate organic matters removal by C. pyrenoidosa growth, a parameter of YB/COD (organics producing algae coefficient, the ratio between biomass production and COD removal, g algae/g COD) was introduced. The values of YB/COD obviously increased with

VFAs cencentrations (mg/L)

700

acetic n-butyric

600

piopionic iso-valeric

iso-butyric n-valeric

500 400 300 200 100 0

more addition of AW. This indicates that the metabolic pathway of C. pyrenoidosa changed with the addition of the organic carbon source. In the pure ADSW medium, the value of YB/COD reached1.67 ± 0.13 g algae/g COD, which is much higher than the values in other groups (G1of 0.52 ± 0.04, G2 of 0.80 ± 0.07 and G3 of 0.98 ± 0.02 g algae/g COD). The highest value of YB/COD in pure ADSW indicates that the growth of C. pyrenoidosa was dominated by autotrophic growth utilizing CO2 as the major carbon source. In contrast, the lowest value of YB/COD (0.52 ± 0.04 g algae/g COD) was obtained in G1 with the highest concentration of organic matters, similar to the results (YB/COD = 0.50 g/g) reported by Yang et al. (2008) using cassava ethanol fermentation for a C. pyrenoidosa heterotrophic culture. This suggests that the growth of C. pyrenoidosa in G1 was dominated by heterotrophic growth utilizing organic matters as the major carbon source. Volatile fatty acids (VFAs) are abundant in pre-treated AW, in which the total amount of acetic acid, propionic acid and n-butyric acid reached 10,000 mg/L, accounting for 90% of the total VFAs. These VFAs in AW may be considered to be good carbon sources for C. pyrenoidosa growth. Several reports have demonstrated that Na-acetate was the best carbon source for microalgae heterotrophic growth (Bhatnagar et al., 2010). The changes of six different VFAs (acetic acid, propionic acid, iso-butyric acid, n-butyric acid, iso-valeric acid and n-valeric) during culture periods in G1–G4 were analyzed (Fig. 3). Along with the microalgae growth in G2–G4, all types of VFAs exhibited a continuous decline. As shown in Fig. 3, the concentrations of VFAs in G2–G4 dramatically decreased during the initial three days; however, the microalgae biomass had no obvious increase in this period. This might be ascribed to direct absorption or assimilation for subsequent utilization by microalgae. During the next exponential growth periods, the concentrations of VFAs showed rapid declines in G2–G4. At the end of the culture, more than 90% of all types of VFAs were

250

acetic n-butyric

150 100 50

1

2

3

4

5

6

7

8

9

0

1

2

150

piopionic iso-valeric

iso-butyri n-valeric

100 75 50 25

0

1

2

3

4

5

6

7

8

9

acetic n-butyric

60

6

8

7

9

piopionic iso-valeric

iso-butyric n-valeric

50 40 30 20 10 0

0

1

2

3

Time (d)

G3

5

G2

acetic n-butyric

125

4

3

Time (d)

G1

VFAs concentrations (mg/L)

iso-butyric n-valeric

200

Time (d)

0

piopionic iso-valeric

0

0

VFAs concentrations (mg/L)

COD concentrations (mg/L)

12000

VFAs cencentrations (mg/L)

58

4

5

Time (d)

G4

Fig. 3. Changes of VFAs during the culture periods in every group.

6

7

8

9

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TN concentrations (mg/L)

19.34% removal 300

TNfinal

91.64% removal

66.40% removal

45.24% removal

200

100

0

TP concentrations (mg/L)

TNinitial

400

50 40

2

3

4

38.98% removal

90.74% removal

1

2

96.95% removal

98.87% removal

30 20 10 0

1

TPinitial TPfinal

3

Group

Group

(a)

(b)

4

Fig. 4. Removal of total nitrogen (a) and phosphorus (b) during the microalgae culture periods in every group.

removed from wastewater in G2–G4. These data indicates that VFAs including acetic acid, propionic acid and n-butyric, are good carbon sources for microalgae heterotrophic growth. 3.3.2. Nitrogen and phosphorous removal Microalgae have large potential storage capacities for nitrogen (i.e., protein) and phosphorus (i.e., phospholipid, nucleic acid synthesis) (Markou and Georgakakis, 2011; Pittman et al., 2011). Thus, microalgae have been widely applied for nutrients removal in wastewater treatment, such as high rate algal ponds (HRAPs). With the continuous growth of microalgae, dissolved nitrogen and phosphorus in wastewater could be removed effectively. As shown in Fig. 4, the concentrations of total nitrogen and phosphorus declined at the end of experiments in G1–G4. However, the nitrogen and phosphorus efficiencies varied greatly among different groups due to the different microalgae growth. In G2, approximately 257.61 mg of nitrogen was removed after nine days of cultivation; in contrast, in G1 and G4, with the relative low biomass production, the nitrogen removal was only 58.40 ± 3.21 mg and 119.92 ± 6.14 mg, respectively. According to elemental analysis of the microalgae biomass in this study, the nitrogen content in biomass ranged from 8.24% to 10.17% (mean value of 9.29 ± 0.66%). This suggests that generating 1 g of microalgae biomass can at least remove 82.4–101.7 mg of nitrogen from wastewater. However, nitrogen removal for generating 1 g of microalgae biomass was higher than the nitrogen content in the biomass. For example, nitrogen removal per generating 1 g of microalgae in G1, G2, G3 and G4 was 149.74 ± 9.51, 102.63 ± 7.74, 106.36 ± 7.14 and 126.23 ± 8.86 mg, respectively. These data suggest that other non-microalgae activities may play important roles in nitrogen removal, such as ammonia gasification. During the rapid growth period, the pH values in the medium often rose above 8.5–9.5 due to the consumption of CO2 in the later periods of the algae culture. Ammonium ion–ammonia equilibrium in the aqueous phase was governed by solution pH and temperature, and free ammonia dominated above a pH of 9.25. Nitrogen in the form of free ammonia can be stripped out of wastewater and lead to a higher efficiency of nitrogen removal. C. pyrenoidosa cultivation in wastewater achieved high removal efficiencies (more than 90%) of phosphorous in every group, except in G1 (Fig. 4b). Similar to nitrogen removal, the amounts of phosphorus removal in G2–G4 were much higher than the theoretical phosphorus uptake by the microalgae growth. The amounts of phosphorus removal ranged between 10.58 and 29.43 mg per 1 g biomass, which was higher than the 5.9–7.1 mg uptake by biomass growth. At the end of the experiments, the residual phosphorus was very little (generally below 0.5 mg/L). The excessive removal

of phosphorus was primarily due to phosphorus precipitation with some metal ions. Many researchers have demonstrated that Ca2+, 2+ + PO3 in the aqueous phase can produce insoluble 4 , NH4, and Mg precipitates, such as hydroxyapatite and struvite, at pH values between 8 and 10 (Hao et al., 2008; Tan et al., 2014). Hydroxyapatite is preferentially formed when sufficient Ca2+ coexists. The presence of Ca2+ and Mg2+ in the wastewater and the alkaline conditions caused by microalgae growth promote phosphorus precipitation. Indeed, these formed deposits were helpful for phosphorus removal from wastewater; however, deposits in the reactor and pipeline, especially struvite and hydroxyapatite formation, were fatal to the long-term operation of large-scale cultivation. 3.4. Lipids productivity and FAME compositions A high lipids content and productivity are crucial to the commercial success of microalgae biofuels production. However, most wild microalgae have difficulty obtaining a high lipids content and productivity simultaneously in natural conditions. These microalgae that are rich in lipids are generally associated with low growth rates; in contrast, microalgae with high growth rates generally have a low lipids content. These characteristics of microalgae cause a low lipids productivity, which represents a major barrier for large-scale microalgae biofuels production. Methods to improve lipid synthesis and productivity in microalgae biomass have been widely investigated. Previous studies have demonstrated that the addition of organic compounds used as a carbon source for microalgae growth could simultaneously trigger biomass growth and lipids accumulation (Qiao and Wang, 2009). However, the use of artificial carbon nutrients (e.g., glucose, NaAc) could impose heavy loads in operational costs. Thus, the recycling of these low-cost carbon nutrients in wastewater may be a feasible and economical approach to improve microalgae lipids productivity instead of artificial carbon sources. In this study, the improvement of the lipids content and productivity in biomass from the addition of carbon nutrients in AW was achieved (Table 3). The lipids content in biomass was only 270.86 ± 27.99 mg in G4 using pure ADSW; with the additions of AW into ADSW, the lipids contents increased by 182.7% and 98.0% in G2 and G3; respectively. The lipids contents in biomass had no obvious differences among G1, G2 and G3; however, the lipids productivities varied greatly associated with different biomass productions. As shown in Table 3, the mean lipids productivity in G2 reached 127.71 ± 6.31 mg/L/d, whereas the mean lipids productivities in G1 and G3 with low biomass production were only 18.21 ± 1.16 and 69.17 ± 4.26 mg/L/d, respectively. Compared to the lipids productivity in G4 using pure ADSW, the lipids productivity in G2 improved more than 3.3-fold by adding

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Table 3 Elements content in the biomass and lipids productivity of C. pyrenoidosa cultured in different wastewaters. Group

Maximal biomass (g/L)

Elements (%) C

N

P

1 2 3 4

0.89 ± 0.13 3.01 ± 0.15 2.21 ± 0.10 1.45 ± 0.12

49.94 ± 2.01 49.73 ± 2.34 49.65 ± 1.89 48.84 ± 2.21

10.17 ± 0.83 9.25 ± 0.66 9.41 ± 0.69 8.24 ± 0.56

0.71 ± 0.02 0.63 ± 0.04 0.62 ± 0.04 0.59 ± 0.05

Table 4 FAME compositions (%) of C. pyrenoidosa cultured in different wastewaters. FAME compositions

2

3

4

Saturated fatty acids (SUFAs) C16:0 30.83 ± 2.15

28.85 ± 1.78

29.70 ± 3.04

29.16 ± 2.01

Monoenoic fatty acids (MUFAs) C16:1 0.59 ± 0.07 C18:1 1.61 ± 0.09 Total of MUFAs 2.20 ± 0.05

1.18 ± 0.08 2.09 ± 0.12 3.27 ± 0.09

1.19 ± 0.02 2.31 ± 0.06 3.50 ± 0.02

1.12 ± 0.09 2.24 ± 0.31 3.36 ± 0.15

11.91 ± 1.89 18.14 ± 1.83 37.84 ± 2.01 67.79 ± 1.91

11.25 ± 2.41 19.31 ± 1.29 36.25 ± 1.54 66.81 ± 1.75

12.15 ± 2.30 17.94 ± 1.88 37.39 ± 1.63 67.48 ± 1.94

Polyenoic fatty acids C16:2 C18:2 C18:3 Total of PUFAs

1

(PUFAs) 10.44 ± 2.31 21.32 ± 2.01 35.21 ± 1.88 66.97 ± 1.73

a moderate amount of AW into ADSW. The results suggest that a moderate addition of AW into ADSW can not only improve the lipids content in biomass, but more importantly can improve the mean lipids productivity. Table 4 presents the fatty acid methyl ester (FAME) compositions of the C. pyrenoidosa biomass in every group. Six fatty acids were identified by lipids analysis, including saturated fatty acids (SUFAs) of tetradecanoic acid (C16:0), monosaturated fatty acids (MUFAs) of hexadecanoic acid (C16:1) and oleic acid (C18: 1), and polyunsaturated fatty acids (PUFAs) of hexadecatrienoic acid (C16:2), linoleic acid (C18:2) and linolenic acid (C18:3). According to the FAME composition analysis, there were no obvious differences in FAME compositions among different groups. Tetradecanoic acid (C16:0), hexadecatrienoic acid (C16:2), linoleic acid (C18:2) and oleic acid (C18:3) were the main species of fatty acids accounting for 97% of the total lipids, similar to the results obtained by Cho et al. (2011). 4. Conclusions A moderate addition of alcohol wastewater (AW) into anaerobic digested starch wastewater (ADSW) significantly improved the microalgae biomass production and lipids content. The highest biomass and lipid productivity was achieved with an optimal addition of AW into ADSW (AW/ADSW = 0.053:1, v/v). Compared to the culture using pure ADSW, the maximal biomass concentrations and lipids productivity increased by 107.6% and 331.7%, respectively. Acknowledgements This work was partially supported by the fundamental research project of the Science and Technology Commission of Shanghai (11JC1412600), New Century Excellent Talents in University (NCET-11-0391), National Natural Science Foundation of China (21246001, 51138009) and by the National Key Technology R&D Program of China (No. 2012BAJ25B02).

Lipids content (% dry biomass)

Lipids productivity (mg/L/d)

23.35 ± 2.34 25.44 ± 1.78 24.27 ± 2.11 18.68 ± 1.93

18.21 ± 1.16 127.71 ± 6.31 69.17 ± 4.26 29.58 ± 1.83

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