Lipid production by a mixed culture of oleaginous yeast and microalga from distillery and domestic mixed wastewater

Bioresource Technology 173 (2014) 132–139

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Lipid production by a mixed culture of oleaginous yeast and microalga from distillery and domestic mixed wastewater Jiayin Ling, Saiwa Nip, Wai Leong Cheok, Renata Alves de Toledo, Hojae Shim ⇑ Department of Civil and Environmental Engineering, Faculty of Science and Technology, University of Macau, Taipa, Macau SAR, China

h i g h l i g h t s  Mixed culture improved lipid production and nutrients removal from real wastewater.  Mixed culture saved cost for chemicals, compared to pure microalgal culture.  Harvesting part of biomass in the middle of the process increased lipid production.  Process suitable for real wastewater, without sterilization and pH adjustment.

a r t i c l e

i n f o

Article history: Received 5 July 2014 Received in revised form 10 September 2014 Accepted 11 September 2014 Available online 22 September 2014 Keywords: Lipid production Microalga Mixed culture Mixed wastewater Oleaginous yeast

a b s t r a c t Lipid productivity by mixed culture of Rhodosporidium toruloides and Chlorella pyrenoidosa was studied using 1:1 mixed real wastewater from distillery and local municipal wastewater treatment plant with initial soluble chemical oxygen demand (SCOD) around 25,000 mg/L, initial cell density of 2  107 cells/mL (yeast) and 5  106 cells/mL (microalga), at 30 °C and 2.93 W/m2 (2000 lux, 12:12 h light and dark cycles). Lipid content and lipid yield achieved were 63.45 ± 2.58% and 4.60 ± 0.36 g/L with the associated removal efficiencies for SCOD, total nitrogen (TN), and total phosphorus (TP) at 95.34 ± 0.07%, 51.18 ± 2.17%, and 89.29 ± 4.91%, respectively, after 5 days of cultivation without the pH adjustment. Inoculation of microalgae at 40 h of the initial yeast cultivation and harvesting part of inactive biomass at 72 h by sedimentation could improve both lipid production and wastewater treatment efficiency under non-sterile conditions. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Biodiesel, as one kind of green and renewable energy, has been gaining an increasing attention worldwide. Although the biodiesel generated from food crops is an industrial reality, its production is a land and water intensive technology, which takes land away from its two other primary uses, food production and environmental preservation (Rajagopal et al., 2007). The utilization of biodiesel produced from crops as the substitute of fossil fuel has not been efficient in the reduction of greenhouse gas emission and consequently to relieve the global warming (Noorden, 2013), resulting in only 10% less carbon dioxide emission generated by biodiesel compared to diesel (Atadashi et al., 2010). In addition, more green land was turned to farmland and consequently less green mantle of vegetation would be available to absorb carbon dioxide (Noorden, 2013). Therefore, more attention has been focused on biodiesel ⇑ Corresponding author. Tel.: +853 8397 4374. E-mail address: [email protected] (H. Shim). http://dx.doi.org/10.1016/j.biortech.2014.09.047 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

produced from lipid generated by such oleaginous microorganisms as microalgae and yeasts, especially from wastewater. There are some merits and drawbacks for the microbial lipid production from wastewater by oleaginous microalgae or yeasts. Microalgae are usually effective in nutrients (nitrogen and phosphorus) removal, uptake of carbon dioxide, and oxygen production. However, they are not very effective in organic matter removal from wastewater with the initial chemical oxygen demand (COD) over 5 g/L and their growth is relatively slow, requiring the cultivation time of 6–30 days to reach a notable biomass and lipid production (Ji et al., 2013; Su et al., 2011). In comparison, oleaginous yeasts are effective in organic matter removal (COD removal could be as high as 68–86%) and have shown a remarkable lipid production in high strength wastewater with the initial COD at 15–50 g/L in a relatively short incubation time (30–144 h) (Peng et al., 2013; Xue et al., 2010b; Zhou et al., 2013). However, yeasts are less effective in nutrients removal and most studies were performed under sterile conditions (Peng et al., 2013; Xue et al., 2010a; Zhou et al., 2013).

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Utilizing a mixed culture of microalgae and yeasts could be a strategy to improve both lipid production and removal of organic matters and nutrients from wastewater. Microalgae could produce oxygen for yeasts while yeasts could provide carbon dioxide for microalgae. Yeasts mainly uptake organic matters and microalgae essentially require nitrogen and phosphorus from wastewater. Previous study suggested that biomass production and lipid content for mixed culture of Rhodotorula glutinis and Chlorella vulgaris in seafood processing effluent was 4.63 g/L and 62.20% while the results for pure cultures of yeast and microalga were 2.10 g/L and 61.9% and 1.10 g/L and 45.45%, respectively (Cheirsilp et al., 2011). However, very few studies have been done to produce microbial lipid from high strength real non-sterile wastewater without the addition of other nutrients for the mixed culture of yeast and microalga. Our previous study (Ling et al., 2013) showed that the increase of yeast initial cell density could make the remarkable lipid production possible even in real non-sterile wastewater. Rhodosporidium toruloides is a promising oleaginous yeast for biodiesel production. Its composition of lipid produced from the medium containing glucose as sole carbon source and distillery wastewater was reported to be mainly palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linoleic acid (Lin et al., 2010; Wu et al., 2010, 2011). Chlorella pyrenoidosa, on the other hand, is a green alga used in wastewater treatment and biofuel production (Perez-Garcia et al., 2011; Su et al., 2011). This study was to explore the potential of further improvement in lipid productivity of oleaginous yeast in a mixed culture with microalga in real mixed wastewater obtained from distillery and local domestic wastewater treatment plants as well as to investigate the associated removal efficiencies for organic matters (COD) and nutrients (nitrogen and phosphorus) present in wastewater under non-sterile conditions. 2. Methods 2.1. Strain, medium, and wastewater Yeast strain R. toruloides AS 2.1389 was purchased from the China General Microbiological Culture Collection Center, subcultured on distillery wastewater agar plates, and maintained on distillery wastewater agar slants at 4 °C. The distillery wastewater agar medium was made of rice wine distillery wastewater and 20 g/L of agar with pH adjusted to 5.5. The YPD medium used for the seed culture contained (per liter) glucose 20 g, yeast extract 10 g, and peptone 20 g at pH 6.0 (Lin et al., 2010). Both media were sterilized at 121 °C for 20 min before use. Microalgal strain C. pyrenoidosa FACHB-9 was purchased from the Institute of Hydrobiology (Chinese Academy of Sciences, Wuhan, China) and maintained on SE (soil extract) medium agar plate (Song et al., 1999) at room temperature with the continuous light supply of 2.93 W/m2. The rice wine distillery wastewater was obtained from the S1 distillery in Foshan, China, with the initial soluble COD (SCOD) at 39,800–59,950 mg/L, total nitrogen (TN) at 1570–2680 mg/L, total

phosphorus (TP) at 300–873 mg/L, and pH 3.6–3.7. The domestic wastewater was obtained from a local wastewater treatment plant in Macau Special Administrative Region (SAR), China. The wastewater samples were filtered through the filter paper (47 mm diameter, 0.7 lm pore size glass-fiber) and then stored at 4 °C before use. 2.2. Experimental setup The oleaginous yeast R. toruloides grown on the distillery wastewater medium slant or plate was transferred to 150-mL flask containing 30 mL YPD medium, cultivated at 30 °C and 200 rpm for 36 h, and used as seed culture. The seed culture was then centrifuged at 4000 rpm for 10 min to obtain the high cell density of 1.2  109 cells/mL. The cell density was measured and calculated using a cell chamber. C. pyrenoidosa was taken from the SE agar plate, cultivated in 150-mL conical flasks containing 30 mL of sterile mixed (distillery and domestic, 1:1) wastewater (pH adjusted to 6.5) under the light intensity of 2.93 W/m2 (2000 lux, with continuous light) at 30 °C and 80 rpm for 6–7 days, and used as seed culture. The cell density of microalgal seed culture was around 3  107 cells/mL. Distillery wastewater was mixed with domestic wastewater at the 1:1 ratio (v/v) (Table 1). It should be noted that the main practical reason for this mixed wastewater used in current study was due to the fact that the wastewater generated from distillery would eventually be combined with the domestic wastewater and to further investigate the competitiveness of the mixed culture of yeast and microalga against indigenous organisms while maintaining high lipid production simultaneously with high removal of organics and nutrients from the mixed wastewater. Thirty milliliters (for the yeast pure culture experiment) and 25 mL (for the mixed culture experiment) of real mixed wastewater were first added to 150-mL flasks. Then, yeast cells with the initial cell density of 2  107 cells/mL were inoculated and cultivated at 30 °C and 200 rpm for 7 days for the pure culture experiment. For the microalgal experiment, after 25 mL of real mixed wastewater with the pH adjusted to 6.5 was added to 150-mL flasks, the seed culture with the cell density of 3  107 cells/mL was inoculated and cultivated at 30 °C and 140 rpm for 7 days with the light supply of 2.93 W/m2 (2000 lux, 12:12 h light and dark cycles). In case of the mixed culture experiment I (Table 2), 5 mL of microalgal seed culture at the cell density of 3.0  107 cells/mL were added after 40 h of the initial yeast cultivation and incubated at 30 °C and 200 rpm with the light supply of 2.93 W/m2 (2000 lux, 12:12 h light and dark cycles), without the pH adjustment. For the ‘Mix 1’, part of biomass was harvested at 72 h by sedimentation for 80 min using a split funnel. The production of lipid and biomass for sample cultivation from days 4 to 7 was calculated as the sum of biomass harvested on the 3rd day plus the accumulated sum of biomass harvested on each day. For the ‘Mix 2’, the production of lipid and biomass for sample cultivation from days 4 to 7 was calculated as the amount of biomass harvested on each day. In the mixed culture experiment II (Table 2), 5 mL of microalgal seed

Table 1 Composition of wastewater samples.

a b c

Wastewater

SCODa (mg/L)

TNb (mg/L)

TPc (mg/L)

NH3–N (mg/L)

pH

Distillery wastewater Domestic wastewater Mix wastewater (1:1, experiment I) Mix wastewater (1:1, experiment II)

39,800–59,950 82–184 30,500 17,150

1570–2680 39–51 1315 720

300–873 4–6 181 349

140–218 26–32 95 91

3.6–3.7 7.5–7.8 3.7 3.8

Soluble chemical oxygen demand. Total nitrogen. Total phosphorus.

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J. Ling et al. / Bioresource Technology 173 (2014) 132–139

Table 2 Summary of experimental setups. Experiment

Test No.

Culture type

Initial cell density of yeast (cells/mL)

Initial cell density of microalga seed culture (cells/mL)

Initial cell density of microalga (cells/mL)

Microalgal inoculation at 40 h (Y/N)

Part of biomass harvested on 3rd day (Y/N)

pH adjustment

Mixed culture experiment I

T1

Yeast pure culture Microalgal pure culture Mix 1 Mix 2

2  107

0

0

Mixed culture Mixed culture Mixed culture Yeast pure culture

T2

T3 T4 Mixed culture experiment II

T5 T6 T7 T8

7

N

N

N

6

N

N

pH adjusted to 6.5

0

3.0  10

5.0  10

2  107 2  107

3.0  107 3.0  107

5.0  106 5.0  106

Y Y

Y N

N N

2  107

2.5  107

4.2  106

Y

Y

7

7

6

Y

Y

pH adjusted to 6.5–7.0 at 40 and 72 h

2  10

1.5  10

2.5  10

2  107

1.0  107

1.7  106

Y

Y

2  107

0

0

N

N

N

culture at different cell densities (2.5  107, 1.5  107, and 1.0  107 cells/mL) were inoculated after 40 h of the initial yeast cultivation. Light was supplied from 40 h at 2.93 W/m2 (2000 lux) with 12:12 h light and dark cycles. The pH of wastewater was adjusted to 6.5–7.0 with the addition of 0.1 mL HCl (4 mol/ L) at 40 and 72 h. Part of biomass was harvested at 72 h by sedimentation for 80 min using a split funnel. The production of lipid and biomass during cultivation from days 4 to 7 was calculated as the sum of biomass harvested on the 3rd day plus the accumulated sum of biomass harvested on each day (from days 4 to 7). Samples were collected in every 24 h for the analyses of COD, TN, TP, NH3–N, and pH in supernatants and biomass and lipid yield in pellets. All the experiments were performed in replicates. 2.3. Analytical methods The concentrations of COD, TN, and TP were measured using Hach’s reagents with Hach reactor DRB200 and spectrophotometer DR2800, following the HACH methods (HACH DR/2800, Hach Company, Loveland, Colorado) and the Standard Methods (APHA/ AWWA/WEF, 1995). The pH was measured using HACH ONE laboratory pH meter. After the culture was centrifuged at 4000 rpm for 10 min at ambient temperature, cell pellets were washed with distilled water twice and then dried at 65 °C until the weight remained constant. The dry weight of biomass was calculated as biomass production. The total lipids were analyzed according to Bligh and Dyer (1959) with modifications described by Li et al. (2001) and Ling et al. (2013). The software SPSS was applied in statistical analysis. Data for the lipid production were statistically analyzed by one-way analysis of variance (ANOVA, at 99% confidence) with different types of culture (pure cultures of yeast and microalga or mixed culture of yeast and microalga) as the source of variance and lipid production as the dependant variable. The T-test (paired samples test) was applied to compare the lipid production data for mixed culture samples with and without harvesting part of biomass at 72 h of cultivation. 3. Results and discussion 3.1. Performance comparison between pure and mixed cultures Yeast (3.4 g/L) and microalga (3.0 g/L) as pure cultures showed the highest lipid yield on 3rd and 7th day, respectively (Fig. 1). The difference in optimal cultivation period was due to the faster growth of yeast compared to microalga. When the mixed culture

Fig. 1. Lipid production by pure and mixed cultures of yeast and microalga from the mixed wastewater. (The initial cell density of yeast and microalga was 2  107 and 0.5  107 cells/mL, respectively.)

was used, an increase in lipid yield was obtained (4.0–4.6 g/L) on 5th day of cultivation, suggesting the potential use of mixed culture as a strategy for the improved lipid production. The utilization of mixed culture to boost lipid production was also mentioned by Cheirsilp et al. (2011, 2012) and Xue et al. (2010a). In order to know the significant difference between different treatments (pure or mixed culture), the lipid production data were statically analyzed by ANOVA. The factor culture type (pure or mixed culture) was highly significant as evidenced by the very low probability value (P = 0.010 < 0.05) for yeast pure culture and mixed culture, as well as for the lipid production data for mixed culture and microalgal pure culture with P = 0.000 < 0.05. Two different organisms may have a symbiotic relationship in mixed culture, further providing higher lipid production and more biomass compared to

135

1.88

1.90 1.45 1.51 8.95 0.25 54.48 ± 7.10 20.14 ± 1.96 87.87 ± 0.18 2.53 ± 0.72 5.67 ± 0.14 44.77 ± 13.83

1.86 2.40

2.21 1.43

1.41 12.83

11.69 0.32

0.29 59.86 ± 6.08

58.42 ± 15.71 33.33 ± 9.82

37.50 ± 0.98 92.61 ± 0.87

90.51 ± 1.20 3.50 ± 1.24

3.21 ± 0.92 5.04 ± 1.65

5.95 ± 2.20 58.44 ± 2.69

64.14 ± 6.00

1.29

1.58 1.57 1.30 13.05 0.29 54.66 ± 8.87 26.39 ± 5.89 91.72 ± 2.02 3.46 ± 0.89 5.27 ± 0.76

0 2 N T8

N

2.5

1.7 2

2

Y T7

Y

Y T6

Y

2 Y

5.0

5.0 2

2 N

N

4.2

17,150

49:2:1

65.24 ± 9.41

1.31 5.45

5.38 2.33

2.31 11.38

12.69 0.37

0.33 90.57 ± 1.07

89.29 ± 4.91 51.18 ± 2.17

51.84 ± 6.30 94.54 ± 0.36

95.34 ± 0.07 4.60 ± 0.36

4.12 ± 0.11 6.68 ± 1.09

7.25 ± 0.77 63.45 ± 2.58

62.71 ± 9.54

5.44

1.56 6.09

15.40 7.53

2.79 9.70

12.54 0.94

0.30 86.21 ± 1.59

75.14 ± 11.48 29.28 ± 17.74

46.31 ± 4.57 91.54 ± 0.66

61.75 ± 14.48 2.35 ± 0.28

3.02 ± 1.01 6.19 ± 0.31

4.19 ± 1.03 56.92 ± 7.38

48.48 ± 13.97 169:7:1 30,500

5.0

0 2

0 Y

N

Specific TN removal rate (10 9 mg/ L cell day) Specific COD removal rate (10 7 mg/ L cell day) Specific lipid content (lipid (g/L)/(g/L) biomass day) Specific lipid yield (10 10 g/ L cell day) TP removal efficiency (%) TN removal efficiency (%) COD removal efficiency (%) Lipid yield (g/L) Biomass (g/L) Lipid content (%, g lipid/g biomass) SCOD: TN:TP Initial SCOD (mg/L) Microalgal initial cell density (106 cells/ mL)

Y

Fig. 2. Biomass production by pure and mixed cultures of yeast and microalga from the mixed wastewater. (The initial cell density of yeast and microalga was 2  107 and 0.5  107 cells/mL, respectively.)

T5

7

Y

6

N

3 4 5 Time (day)

T4

2

T3

1

N

0

N

Microalga

T2

Mix 2 (without part of biomass harvested on 3rd day)

2

T1

Mix 1 (with part of biomass harvested on 3rd day)

Yeast initial cell density (107 cells/ mL)

4

pH adjustment (Yes/No)

Yeast

Part of biomass harvested on 3rd day (Yes/No)

6

Test No.

Biomass (g/L)

8

Table 3 Lipid production and removal of organics and nutrients on the 5th day by pure or mixed culture of yeast and microalga in distillery and domestic mixed wastewater.

the pure cultures of each organism. Cheirsilp et al. (2011) also reported that the symbiotic relationship can exist between yeast and microalga and it is beneficial in terms of increasing lipid yield. The mixed culture may mitigate or even eliminate the stresses caused by CO2 for yeast and O2 for microalga. Zhang et al. (2014) further indicated this synergistic effect not only on gas exchange between two different organisms but also on dissolved oxygen, pH adjustment, and substance exchange. As shown in Figs 1 and 2, the highest lipid yield (4.60 ± 0.36 g/L) and biomass (7.25 ± 0.77 g/L) were achieved under the conditions of initial cell density of yeast at 2  107 cells/mL, mix cultured with microalga at the initial cell density of 5  106 cells/mL, without pH adjustment, after 5 days of cultivation, and with part of biomass harvested on 3rd day by sedimentation, further resulting in the associated removal efficiencies for SCOD, TN, and TP at 95.34 ± 0.07%, 51.18 ± 2.17%, and 89.29 ± 4.91%, respectively (Table 3; Test 4). When both organisms were added to the cultivation medium, no considerable increase of biomass due to the depletion of nutrients was observed. According to Cheirsilp et al. (2012), a nutrient imbalance in the culture medium is known to cause lipid accumulation in oleaginous microorganisms. At a high C/N ratio (low nitrogen levels), microorganisms cannot multiply and the excess of carbon is assimilated continuously to produce storage lipid. Another possible reason for the low increase of biomass may be related to the poor growth of microalga in mixed culture. The light might have hardly penetrated through the high concentration of yeast cells in the mixed culture, further contributing to the final low biomass of mixed culture (Cheirsilp et al., 2011). The effect of mixed culture on removal efficiencies for nutrients in mixed wastewater (1:1, v/v) was evaluated using different microalgal initial cell densities, and the results were compared with the pure cultures of yeast and microalga (Table 3). The removal efficiencies for COD, TN, and TP for the mixed culture were generally higher than those for the pure cultures, regardless of the initial cell density of microalga in the mixed culture. Especially the TN and TP removal efficiencies were increased by 5–10% when yeast was mixed with microalga. Table 3 also shows the specific lipid yield and lipid content together with the specific removal rates for COD, TN, and TP. In general, the specific lipid yield of mixed culture samples (Tests 3–4 and 5–7) was similar to each other and slightly higher than those of yeast pure culture (Tests 1 and 8) when grown in same wastewater samples with higher total initial cell densities. The specific lipid yield and specific removal rates of COD, TN, and TP for mixed culture samples (Tests 3–7) were lower compared to the microalgal pure culture (Test 2) due to the higher microalgal initial cell density, but the associated lipid yield and removal efficiencies for COD, TN, and TP were higher compared to the pure culture samples of yeast or microalga.

Specific TP removal rate (10 9 mg/ L cell day)

J. Ling et al. / Bioresource Technology 173 (2014) 132–139

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J. Ling et al. / Bioresource Technology 173 (2014) 132–139

As shown in Fig. 3, the pH of mixed (distillery and domestic at 1:1 ratio, v/v) wastewater increased from about 3.7 to around 7.5 after 48 h of incubation in the yeast pure culture, also reported in previous study (Ling et al., 2013). The brewery or distillery wastewater contains several kinds of organic acids including malic acid, citric acid, lactic acid, acetolactic acid, and tartaric acid, which are produced by the metabolism of wine yeast during fermentation (Saayman and Viljoen-Bloom, 2006; Whiting, 1976). It has also been reported that yeasts could use such organic acids as malic acid, citric acid, and lactic acid as single carbon source to produce single cell protein (Gao et al., 2008) and lipid (Zhou et al., 2011). Therefore, one possible reason for the pH increase after 48 h could be attributed to the uptake of organic acids by yeasts during cultivation. On the other hand, this pH increase would be even more beneficial to the growth of microalga, together with the savings of chemicals to be used to adjust pH from 3.7 to 6.5 to grow microalga in the mixed culture otherwise. The influence of pH adjustment on pH of the mixed wastewater and on the growth of mixed culture in current study was negligible, as shown in Fig. 3. After the pH was adjusted to 6.5–7.0 at 40 and 72 h by adding 0.1 mL HCl (4 mol/L), pH further increased to 7.3–7.7 at 48 h and to 7.8–8.4 at 96 h. The pH adjustment did not substantially improve lipid production (Tests 5–7) compared to without pH adjustment (Tests 3–4) (Table 3). To maintain the pH of wastewater at 6.5–7.0 would require much more chemicals and frequent pH adjustment, which may improve the lipid production to some extent but also increase the process cost.

Harvesting part of yeast biomass during cultivation of mixed culture proved to be effective in increasing lipid yield because of the decrease in yeast biomass when its growth entered the late stationary phase or death phase, as shown in Figs 1 and 2. The

harvesting was done by simple sedimentation on 3rd day of cultivation. Since yeast cells are larger than microalgae and bacteria, the yeast biomass is easier to sediment and handle. As shown in Fig. 2, as the biomass of yeast pure culture started to decrease, the microalgal pure culture biomass showed the increasing trend after 4th day due to the different growth behaviors between yeast and microalga, although with some fluctuations maybe caused by the growth of indigenous microorganisms. The doubling or generation time of yeast was reported about 90 min (Sherman, 2002) with a cell cycle time of 140–160 min (Abe et al., 1984; Lovrics et al., 2006), while for microalgae, the doubling time is 3.5–72 h (Chisti, 2007; Sheehan et al., 1998), further suggesting the best harvest time for yeast is earlier than the one for microalga. The biomass left at the bottom after sedimentation may be the least active yeast cells that might have accumulated a large amount of lipids from the mid stationary or early death phase. As suggested by our previous study (Ling et al., 2013), the optimal harvest time for the yeast pure culture in this kind of mixed wastewater was 72 h. In current study, there was no significant further increase in biomass after the inoculation of different microalgal cell densities (1.7–4.2  106 cells/mL), which might be attributed to the suppression of yeast growth when the mixed culture reached an alkaline condition with the pH up to 8.0 (data not shown) (Huang et al., 2013). The removal of part of the least active yeast biomass may also spare more resources for the growth of active yeast and microalgal cells in mixed culture and further improve the overall performance. Su et al. (2011) also reported that C. pyrenoidosa could be harvested by self-sedimentation, suggesting the possibility for the simultaneous harvest of yeast and microalgal biomass in mixed culture by sedimentation. It is well known that the process of biodiesel production from microorganisms is costly and has been the biggest obstacle for its industrialization (Wang et al., 2012). Therefore, any methodology capable of even slightly increasing the lipid production without

Fig. 3. Lipid production by yeast pure culture (initial cell density, 2  107 cells/mL) and mixed culture of yeast (initial cell density, 2  107 cells/mL) and microalga (three different initial cell densities) from the mixed wastewater.

Fig. 4. Changes in pH of mixed wastewater with pure and mixed cultures.

3.2. Influence of harvesting part of biomass in mixed culture

Table 4 Lipid production by pure or mixed culture of oleaginous yeasts and microalgae under different conditions. Substrate

Inoculum size/ initial cell density (cells/mL)

Sterile condition (Yes/No)

Time (day)

Biomass (g/L)

Lipid (% w/ w)

Lipid (g/L)

COD removal (%)

Nutrient removal (%)

Spirulina platensis and Rhodotorula glutinis

Monosodium glutamate wastewater (COD, 43,210 mg/ L; reducing sugar, 2.9 ± 0.1 g/L)

10% (v/v) yeast, 20% (v/v) microalga

Y

5

1.6

13.75

0.22

100 (glutamic acid), 35 (NH+4–N)

Rhodotorula glutinis and Chlorella vulgaris

Seafood processing effluent (pH, 5.3; COD, 34,680 mg/L; nitrate, 32 mg/L) with 1% molasses (62% total sugar contained) 3% pure glycerol and urea with molar C/N = 32, pH = 6.0

2.8  105 yeast, 2.6  105 microalga

Y

5

4.63

62.2

2.88

73 (reducing sugar removal, 94 ± 1%) 79

1.0  106 yeast, 3.0  105 microalga

Y

5

3.2

34.38

1.1

5% (v/v)

Y

5

3.8

20.79

5% (v/v)

Y

5

3.8

10% (v/v)

Y

5

N

5

Rhodotorula glutinis and Chlorella vulgaris Lipomyces starkeyi

Rhodosporidium toruloides Y2 Scenedesmus sp. Rhodosporidium toruloides and Chlorella pyrenoidosa a

Fishmeal wastewater (COD, 78.4 g/L + 21.3 g/L (glucose) = 99.7 g/L; TN = 4.3 g/L; TP = 0.95 g/L) Bioethanol wastewater (COD, 34 g/L) with glucose, 1.2 g/ (L days) added from 48 h Synthetic dark fermentation effluent (COD = 11.04 g/L) Distillery wastewater (COD = 30.5 g/L; TN = 1315 mg/L; TP = 181 mg/L)

7

2  10 (yeast), 0.5  107 (microalga)

Specific lipid yield (10 10 g/ L cell day)

Specific lipid content (lipid (g/L)/ (g/L) biomass d)

Specific COD removal rate (10 7 mg/ L cell day)

References

0.04

2.75

5.13

Xue et al. (2010a)

N/Aa

10.67

12.68

70.64

Cheirsilp et al. (2011)

76.7 (glycerol removal)

N/A

1.69

6.88

0.79

35.83

N/A

0.50

4.16

22.69

34.9

1.33

72.3

N/A

0.84

6.98

7.56

Zhou et al. (2013)

1.8

37

0.67

36.9

N/A

0.21

7.40

1.29

7.25 ± 0.77

63.45 ± 2.58

4.60 ± 0.36

95.34 ± 0.07

51.18 (TN), 89.29 (TP)

0.37

12.69

2.33

Ren et al. (2014) Current work

N/A

Cheirsilp et al. (2012) Huang et al. (2011)

J. Ling et al. / Bioresource Technology 173 (2014) 132–139

Organism

Not applicable/available.

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the additional cost could be promising for the microbial biodiesel production. Table 3 shows that the lipid production (4.60 ± 0.36 g/L) for the ‘Mix 1’ samples with part of biomass harvested on 3rd day, with the specific lipid yield of 0.37  10 10 g/L cell days and the lipid content of 63.45 ± 2.58%, was slightly higher compared to the ‘Mix 2’ samples, with the lipid production of 4.12 ± 0.11 g/L, the specific lipid yield of 0.33  10 10 g/L cell days, and the lipid content of 62.71 ± 9.54% when part of biomass (lipid) was not harvested on 3rd day. The T-test (paired samples test) applied to the lipid production data for mixed culture samples showed that the factor with or without part of biomass harvested on 3rd day had a significant difference in lipid production with a very low probability value, P (2-tailed) = 0.025 < 0.05 (detailed data not shown). 3.3. Influence of initial SCOD, TN, and TP concentrations As shown in Table 3, a higher production of biomass and lipids was obtained with the higher initial SCOD value. This behavior was expected since increasing the initial SCOD available in wastewater increases biomass and lipid produced by microorganisms. However, in case of the lipid content of dry biomass, the ratio of SCOD:TN:TP may play a more important role than the initial SCOD value, especially in the yeast pure culture (Figs. 1 and 4). The lipid content of yeast on the 3rd day was around 35% when the SCOD:TN:TP was 49:2:1, while over 40% was obtained when the ratio was 169:7:1. However, this effect was much smaller in the mixed culture samples, resulting in a similar lipid content of dry biomass at different SCOD:TN:TP ratios. Table 3 also shows that the removals of SCOD, TN, and TP were slightly higher when the initial concentrations of SCOD, TN, and TP were higher, suggesting that the microorganisms used in this study were not very good at the organics and nutrients removal when the initial concentrations of SCOD, TN, and TP were too low. In addition, microorganisms did not grow very well and the removal of organics and nutrients occurred more slowly in the later stage of cultivation. 3.4. Influence of microalgal initial cell density in mixed culture Cheirsilp et al. (2011) suggested the optimal ratio of yeast and microalgal cells in mixed culture for the lipid production in seafood processing wastewater at 1:1. Following our previous study (Ling et al., 2013), which showed that the optimal initial cell density of 2  107 cells/mL enabled R. toruloides to survive in the mixed (distillery and domestic) wastewater in the presence of indigenous microorganisms, current study mainly investigated the effect of initial cell density of microalga in mixed culture on lipid production and treatment efficiency in mixed wastewater. The initial microalgal cell densities studied were 1.7  106, 2.5  106, 4.2  106, and 5.0  106 cells/mL, with the initial yeast cell density fixed at 2.0  107 cells/mL, corresponding to the ratio of yeast to microalga at 12:1, 8:1, 5:1 and 4:1, respectively. As shown in Fig. 4 and Table 4, the mixed culture of yeast and microalga, regardless of the initial microalgal cell density, generally showed higher lipid yields compared to the yeast pure culture. However, the increased microalgal initial cell density from 1.7  106 to 4.2  106 cells/mL did not show any significant improvement in both lipid yield and wastewater treatment efficiencies. Samples with the microalgal initial cell density of 5.0  106 cells/mL showed a slight improvement in both parameters and this result could be partially attributed to the higher initial COD concentration of the mixed wastewater. These results further suggests that a higher initial cell density of microalga with the initial cell density of yeast fixed at 2.0  107 cells/mL and the mixed ratio of microalga to yeast in mixed culture in the range of 1:1–3:1 may result in a better overall

performance, as also reported by others (Cheirsilp et al., 2011; Xue et al., 2010a). The increase in the initial cell density may reduce the time that microalga required to exert its positive effect in mixed culture since microalga grow much more slowly than yeast. Therefore, to obtain the higher microalgal cell density in seed culture would take a longer time (possibly 2–3 weeks), which may not be that much practical for the batch culture experiment for 5– 7 days, even though it could be more applicable to the continuous process. Table 4 summarizes the performance comparison between current study and other previously published ones, especially in terms of lipid production and removal of organics and nutrients by pure or mixed cultures. When the mixed cultures were used (Cheirsilp et al., 2011, 2012; Xue et al., 2010a), both lipid yield and lipid content from current study were higher. Even though specific lipid yield, specific lipid content, and specific COD removal rate obtained from current study were lower compared to others, due to the higher initial cell number used, the cost of using a high initial cell density under non-sterile condition in current study would not be significant, considering even a higher cost for maintaining the sterile condition in most other studies, especially for the continuous process. In addition, current study showed such other advantages as saving for chemical cost because no external nutrients were added to the cultivation medium and good performance under non-sterile condition. Both lipid content and lipid yield using the mixed culture of yeast and microalga were also higher than the ones obtained when pure cultures of yeast (Huang et al., 2011; Zhou et al., 2013) and microalga (Ren et al., 2014) were used. These results further imply this mixed culture of yeast and microalga as a promising strategy for the lipid production simultaneously with the treatment of such wastewater as food industry wastewater. The growth of mixed culture of yeast and microalga may take a longer time compared to the yeast pure culture to achieve the highest lipid production, as shown in Table 4. On the other hand, the development of continuous process could be a feasible way to take full advantages of this mixed culture. In addition, a relatively high initial cell density is another way to achieve a remarkable lipid production under non-sterile conditions (Lin et al., 2010; Ling et al., 2013), as well as at low temperature and low pH (Santamauro et al., 2014). The cost for seed culture or pre-culture preparation may increase if a high cell density is used, while it could be reduced if the spent seed culture medium could be recycled or reused. Furthermore, this cost would become much less important if operated as a continuous process since seed culture or pre-culture would only need to be prepared at the beginning.

4. Conclusion As the growth of yeast R. toruloides increased the pH of mixed wastewater to the level suitable for microalga C. pyrenoidosa, the inoculation of microalga after 40 h of the initial yeast cultivation could save chemicals for the pH adjustment, compared to the microalgal pure culture. The addition of microalga to the yeast culture and harvesting part of biomass at 72 h further increased lipid yield and lipid content as well as removal of nutrients, compared to the yeast and microalgal pure cultures. The approach of using mixed culture is feasible to improve the lipid production from real mixed wastewater under non-sterile conditions.

Acknowledgements This research was supported by the University of Macau MultiYear Research Grant and by grant from the Macau Science and Technology Development Fund (FDCT).

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