Accepted Manuscript Effect of carbon source on biomass growth and nutrients removal of Scenedesmus obliquus for wastewater advanced treatment and lipid production Qiao-Hui Shen, Jia-Wei Jiang, Li-Ping Chen, Li-Hua Cheng, Xin-Hua Xu, Huan-Lin Chen PII: DOI: Reference:

S0960-8524(15)00560-X http://dx.doi.org/10.1016/j.biortech.2015.04.053 BITE 14894

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

22 February 2015 15 April 2015 16 April 2015

Please cite this article as: Shen, Q-H., Jiang, J-W., Chen, L-P., Cheng, L-H., Xu, X-H., Chen, H-L., Effect of carbon source on biomass growth and nutrients removal of Scenedesmus obliquus for wastewater advanced treatment and lipid production, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.04.053

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Effect of carbon source on biomass growth and nutrients removal of Scenedesmus obliquus for wastewater advanced treatment and lipid production Qiao-Hui Shena, Jia-Wei Jianga, Li-PingChena , Li-Hua Chenga∗ , Xin-Hua Xua, Huan-Lin Chenb a

b

Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, P.R. China Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, P.R. China

Submitted to Bioresource Technology February, 2015

∗

Corresponding author: Tel.(fax): +86-571-88982025 E-mail address: [email protected] (L.-H. Cheng).

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Abstract The combination of tertiary wastewater treatment and microalgal lipid production is considered to be a promising approach to water eutrophication as well as energy crisis. To intensify wastewater treatment and microalgal biofuel production, the effect of organic and inorganic carbon on algal growth and nutrient removal of Scenedesmus obliquus were examined by varying TOC (Total Organic Carbon) concentrations of 20-120 mg L-1 in wastewater and feeding CO2 concentrations in the range of 0.03%-15%, respectively. The results showed that the maximal biomass and average lipid productivity were 577.6 mg L-1 d-1 and 16.7 mg L-1 d-1 with 5% CO2 aeration. The total nitrogen, total phosphorus and TOC removal efficiencies were 97.8%, 95.6% and 59.1% respectively within 6 days when cultured with real secondary municipal wastewater. This work further showed that S. obliquus could be utilized for simultaneous organic pollutants reduction, N, P removal and lipid accumulation.

Keywords: Microalgae; Carbon source; CO2; Wastewater; Lipid.

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1. Introduction The widespread eutrophication aggravates water scarcity. The removal of nitrogen and phosphorus from wastewater is a fundamental way to prevent eutrophication. The energy crisis has also become one of the biggest challenges of the 21st century. Advanced wastewater treatment and exploitation of novel energy forms are the two major approaches to handle the challenges of water scarcity and energy crisis, respectively. Under these scenarios, using urban secondary wastewater that contains nitrogen and phosphorus as a nutrient source to cultivate microalgae and produce lipid simultaneously represents one of the best future measures (Chen et al., 2012). Thus, the coupling of advanced wastewater treatment and biodiesel production based on microalgae is a promising technology. To achieve high efficiency of nitrogen and phosphorus removal and to obtain high lipid productivity are the two purposes of the coupling system. Carbon source availability is the key metabolic factor controlling the growth rate and extent of oil accumulation, and the enhancement of carbon utilization efficiency can intensify the industrial-scale wastewater treatment and lipid production (Fan et al., 2012). The low C/N/P ratio in domestic wastewater, compared to the optimum reported C/N ratio for microalgae growth of 100/18, suggests a potential C limitation during advanced wastewater treatment (Posadas et al., 2013). The C/N ratio about 2/1 of a typical urban secondary effluent is much lower than 100/18. Due to most of the organics are poorly biodegradable within secondary effluent, supplement of carbon source is essential for municipal wastewater treatment and lipid production. 3

Carbon source includes organic carbon and inorganic carbon source. Currently, there are a lot of on-going researches on the wastewater treatment by microalgae culture systems supplemented with carbon source, especially CO2. Besides the inorganic carbon, part of the organic matters in wastewater could also be utilized by some species of microalgae during the mixotrophic cultivation (Wu et al., 2014). As for organic carbon, most researches focus on high concentrations of organic carbon removal (chemical oxygen demand, COD>500 mg L-1), while there is a lack of information on the low concentration of organic carbon (COD500 mg L-1), our study focused on the utilization of low concentrations of organic carbon source (COD 1% CO2 culture (16.1) > 10% CO2 culture (14.3) > ambient air culture (10.9) > 15% CO2 culture (8.1). The differences in lipid productivity among different cultures mainly resulted from the differences in their biomass, since the deviations in lipid contents of S. obliquus within different culture were low in contrast. The enhancement of lipid accumulation triggered by increasing CO2 addition was

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not remarkable as shown in Fig. 3. Lipid accumulation in microalgae under nitrogen starvation was considered to a large extent limited by the carbon supply. Fan et al. (2012) reported that increased lipid accumulation in the starchless mutant of Chlamydomonas was mostly attributed to increased carbon availability in the absence of carbohydrate synthesis. Although the content of carbohydrate increased with the CO2 concentration increasing in Fig.3, carbon availability for carbohydrate synthesis might still be insufficient when supplied with high concentration of CO2 due to the poor solubility of CO2 in water and the low CO2 removal ratio with 15% CO2 mentioned above. Therefore, the increasing supply of CO2 could not attribute to lipid accumulation, and the high lipid productivity with 5% CO2 was mainly due to the high biomass density. Since higher concentration of CO2 was harmful to algal growth, enhancing the mass transfer efficiency would be a feasible pathway to increase the carbon availability for carbohydrate and the following lipid.

3.3. Real wastewater treatment The experiments mentioned above were all conducted with synthetic secondary wastewater. The application of the coupling system to treat real wastewater would meet many difficulties due to the fluctuant components. The wastewater was then prepared using the effluent from a wastewater treatment workshop in Lin’an, and treated in the photobioreactor by S. obliquus. As shown in fig. 4 (a), S. obliquus failed to grow well in the real wastewater, reaching the decline phase within 7 days. The microalgae were observed to decline in

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the 3 rd day and the cells even disrupted after 7 days. The main reason for poor growth of microalgae in the real wastewater might be the inevitable contamination of bacteria which could be observed by the microscope. The cultivation of microalgae within real wastewater was a complicated symbiotic system where the cross infection or contamination by biological pollutants was inevitable, which had become a big constraint in mass cultivation and impede the industrial process (Wang et al., 2013). As shown in Fig. 4 (a), S. obliquus obtained high lipid content after 6 days. However the lipid productivity was only 9 mg L-1 d-1 because of the low biomass density, which was much lower than that within synthetic wastewater. As shown in fig. 4 (b), S. obliquus could remove nitrogen, phosphorus and TOC efficiently. However, the TOC removal efficiency within real wastewater was much lower than that of synthetic secondary effluents due to the poor biodegradability of real secondary effluent. Only the easily biodegradable organic matters could be removed, which made it necessary to adopt chemical oxidation as a pretreatment unit for improvement of the removal efficiency of organic matter. Some proper pretreatments should be planned to improve the TOC removal efficiency within real urban secondary wastewater and enhance the practicability of the coupling system. The metabolism of the microalgae was studied during the entire duration of the real urban wastewater experiment. Analysis of the biomass composition and elemental balance calculations clearly illustrated that nutrients removed from the system were captured within the produced biomass, and microalgae played a major role in removal of these nutrients. Fig. 5 showed the balance of nitrogen and carbon during the

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treatment of real urban wastewater, which exhibited the fractions of various forms of nitrogen and carbon presented in the influent and effluent (the 6th day). The determination of elemental balance was done based on the nutrients concentration and biochemical composition. It was feasible to determine the destination of nutrients by the analysis of biomass composition and nutrient consumption data. As proteins production was the main destination of the nitrogen consumed, nitrogen content could be determined by multiplying protein by the factor 16%. As shown in Fig. 5 (a), the small fraction of TN in the effluent (0.282 mg L-1) indicated that nitrogen removal must have been accomplished by fixation in the biomass, which was also certified by the large fraction 11.72 mg L-1 of biomass-N at the 6th day. About 88% of the nitrogen in the influent was assimilated by microalgae, assuming the biodegradation of heterotrophic bacteria within the system was ignored. Carbon proportion of carbohydrate, protein and lipid could be considered as 40%, 45% and 87%, respectively (Sydney et al. 2010). The carbon balance during the treatment of real urban wastewater in Fig. 5 (b) showed about 72.8% of the initial total carbon source was transformed into algal biomass. The initial total carbon included 19.1 mg L-1 TOC, 19.76 mg L-1 IC (inorganic carbon measured by TOC analyzer) and 376.2 mg L-1 CO2. The mechanism of organic carbon removal by microalgae was still controversial. The organic carbon pollutants removal was attributable to biodegradation of heterotrophic bacteria by Tuantet et al. (2014). In our study, S. obliquus removed TOC efficiently from 120 mg L-1 to 15 mg L-1 within 8 days in Section 3.1 and

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bacteria was hardly observed by microscopy, showing the ability of organic carbon assimilation by microalgae. Nevertheless, the contamination of bacteria was inevitably present in the real urban wastewaters treatment system employing a column bioreactor as shown in Fig. 4(a). Further studies are essential to explore the mechanism of organic carbon removal in the coupling system and the methods to control the pollutions. Many researches have been focused on the interaction between microalgae and bacteria, and the co-existing bacteria are found to be beneficial to algal growth and nutrients removal. More researches should be done to explore the mechanism of organic carbon removal in the coupling system, and study the relations between microalgae and bacteria.

4. Conclusion This study illustrated the remarkable potential of employing S. obliquus for the wastewater advanced treatment and lipid production. Low concentrations of carbon pollutants could be removed efficiently by S. obliquus. The average TOC removal efficiency was 93.3% when cultivated with artificial wastewater. The coupling system could also be used for treatment of real secondary wastewater and reducing nitrogen and phosphorus. After 6 days of cultivation, the TN, TP and TOC of real secondary municipal wastewater had been reduced to low levels, with removal efficiency of 97.8%, 95.6% and 59.1% respectively.

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Acknowledgments This work is based upon research supported by the National Natural Science Foundation of China (No. 21106130, No. 21276221), National High Technology R&D Program of China (2012AA050101) and the Doctoral Discipline Foundation for Young Teachers in the Higher Education Institutions of Ministry of Education (201101011120074).

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Table Captions Table 1. Effect of initial TOC concentration on algal growth and nutrients removal from simulated wastewater effluent Table 2. Effect of inlet CO2 concentration on the biomass growth and CO2 fixation of Scenedesmus obliquus cultured with simulated secondary wastewater effluent

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Table 1 Effect of initial TOC concentration on algal growth and nutrients removal from simulated wastewater effluent

TOC

mg L

C/N

-1

Biomass

Assimilation

Maximum daily

productivity

days

removal rate

-1

-1

-1

Carbo-

Protein

Lipid

hydrate

-1

-1

-1

mg L d

TN

TP

TN mg L d

TP mg L d

%

%

%

0

0

35.9±2.6

14

3

3.89±0.44

2.51±0.17

51.6±1.2

33.8±1.1

14.7±1.0

20

0.8

38.2±3.6

12

2

7.22±0.56

2.82±0.22

53.0±0.9

34.5±0.9

12.5±0.1

40

1.6

36.9±1.7

12

3

7.08±1.03

2.78±0.08

53.9±1.4

33.2±1.2

12.9±0.3

60

2.4

40.5±4.1

11

2

9.94±0.26

2.96±0.06

56.1±1.0

31.5±2.2

12.3±1.3

80

3.2

45.8±3.4

11

2

11.60±1.80

2.93±0.03

57.0±0.6

31.8±2.9

11.2±2.5

120

4.8

52.7±1.6

9

2

14.16±1.61

2.93±0.07

65.4±3.1

22.7±2.1

11.9±1.1

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Table 2 Effect of inlet CO2 concentration on the biomass growth and CO2 fixation of Scenedesmus obliquus cultured with simulated secondary wastewater effluent

microalgae

S.obliquus

CO2 concentration % 0.03 1 5 10 15

biomass productivity mg L-1 d-1 101.08±4.50 215.49±11.59 251.13±7.42 208.67±15.28 126.50±14.19

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Average CO2 fixation rate mg L-1 h-1 1.25±0.54 5.75±0.38 10.69±1.70 16.87±2.09 26.45±1.51

Maximum CO2 fixation ratio % 32.49±2.53 8.45±1.48 4.20±1.02 3.22±1.37 2.73±0.80

Figure Captions Fig. 1 The algal growth (a), TOC (b) and TN removal (c) of S. obliquus obtained during the experiments with six different initial TOC concentrations. Fig. 2 Effect of CO2 concentration on biomass yield of S. obliquus. Time course of Dry weight (a), TN removal (b) and TP removal (c). Fig. 3 Effect of CO2 concentrationon algal components of S. obliquus. Fig. 4 Changes in biomass growth and algal components (a), TN concentration, TP concentration, and TOC concentration (b) of S. obliquus cultured in real urban wastewater. (feed gas, 5.0% CO2; gas rate, 300 mL min-1; light intensity, 40 µmol photons m-2 s-1; temperature: 25 °C) Fig. 5 Nitrogen balance (a) and carbon balance (b) of S. obliquus during the real urban wastewater treatment

25

Fig. 1

0.6

(a)

-1

120

0 mg L -1 20 mg L -1 40 mg L -1 60 mg L -1 80 mg L -1 120 mg L

80 -1

TOC (mg L )

-1

Dry weight (g L )

100 0.4

-1

0 mg L -1 20 mg L -1 40 mg L -1 60 mg L -1 80 mg L -1 120 mg L

0.2

0.0 0

2

4

6

(b)

60

40

20

0

8

0

2

Days of cultivation (d)

4

6

8

Days of cultivation (d)

30

15

2.5

2.0

10

1.0

0.5

0

0.0 2

4

6

8

0

2

4

Days of cultivation (d)

Days of cultivation (d)

26

(d)

1.5

5

0

-1

0 mg L -1 20 mg L -1 40 mg L -1 60 mg L -1 80 mg L -1 120 mg L

1

1 TN (mg L- )

20

3.0

(c)

TP (mg L- )

-1

0 mg L -1 20 mg L -1 40 mg L -1 60 mg L -1 80 mg L -1 120 mg L

25

6

8

Fig. 2

30

2.0

20 -1

1.2

0.8

15

10

5

0.4

0

0.0 0

2

4

6

8

10

0

12

3.0

(c)

air 1% 5% 10% 15%

2.5

2.0 -1

1.5

1.0

0.5

0.0 0

2

4

6

8

2

4

6

8

Days of cultivation (d)

Days of cultivation (d)

TP (mg L )

(b)

air 1% 5% 10% 15%

25

TN (mg L )

-1

Dry weight (g L )

(a)

air 1% 5% 10% 15%

1.6

10

12

Days of cultivation (d)

27

10

12

Fig. 3

Biochemical composition (%)

100

Protein Carbohydrate Lipid

80

60

40

20

0 0.03%

1%

5%

CO2 concentration

28

10%

15%

Fig. 4

1.2

Protein Carbohydrate Lipid

1.0

80

Dry weight

Dry weight (g L )

0.8

-1

Biochemical composition (%)

(a) 100

60 0.6 40 0.4 20

0.2

0

0.0 0

2

4

6

8

Days of cultivation (d)

1.4

(b) 30

14 1.2

25 12

TOC

8 15 6 10 4 5

0 0

2

4

6

Days of cultivation (d)

29

8

-1

TP (mg L )

-1

-1

10

TN (mg L )

20

TOC (mg L )

1.0

TN TP

0.8

0.6

0.4

2

0.2

0

0.0

Fig. 5

20 18

500

Carbon (mg L )

14

-1

-1

Nitrogen (mg L )

16

600

(a)

missing Biomass N Total N

12 10 8 6 4

missing Biomass-C CO2

(b)

IC TOC

400 300 200 100

2 0

0 Initial

Initial

Stable

30

Stable

Highlights:

1. Carbon source supplement enhanced algal growth and nutrients removal.

2. Low concentrations of organic carbon pollutants were removed by S. obliquus.

3. 5% CO2 bubbling accelerated biomass and lipid productivity of S. obliquus.

4.

Pollutants in a real urban wastewater were efficiently utilized by microalgae.