Biotechnol Lett DOI 10.1007/s10529-014-1655-6

ORIGINAL RESEARCH PAPER

An auto-flocculation strategy for Chlorella vulgaris Y. Shen • Z. Fan • C. Chen • X. Xu

Received: 24 July 2014 / Accepted: 27 August 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Extracellular polymeric substances (EPS) excreted by microalgae are effective for microalgal flocculation. An auto-flocculation strategy was conducted by adding adequate glycine into the medium, stimulating EPS secretion to achieve auto-flocculation, and recycling the supernatant medium for further cultivation. Bound EPS positively corresponded with the solid concentration achieved. Increasing the mixing time enhanced the secretion of bound EPS but the influence of glycine was affected by light intensity. Increasing the glycine dose decreased the production of bound EPS with light intensity of 250 lmol m-2 s-1, but increased the production of bound EPS with light intensity of 125 lmol m-2 s-1. Maximum solid concentration of 21.2 g l-1 with biomass recovery rate of 71 % was achieved under light intensity of 250 lmol m-2 s-1, mixing time of 3 days and glycine at 0.1 g l-1. Keywords Auto-flocculation  Biomass recovery rate  Extracellular polymeric substances  Glycine  Microalgae

Y. Shen (&)  Z. Fan  C. Chen  X. Xu College of Mechanical Engineering and Automation, Fuzhou University, Fuzhou 350108, Fujian, China e-mail: [email protected]

Introduction Microalgae are a source of many highly valuable products such as polyunsaturated fatty acids, astaxanthin and various bioactive compounds. However, due to their small size (5–20 lm), negative surface charge (about -7.5 to -40 mV) and low biomass concentration (0.5–1 g l-1), harvesting microalgal biomass from growth medium is a challenge, that accounts for 20–30 % of the total production cost from algae to biodiesel (Liu et al. 2013). Conventional harvesting processes, such as centrifugation and filtration, require large capital and operational costs (Shen et al. 2013). Flocculation conducted by adding cationic salts into growth medium to neutralize the negative charge on cell surface was a cost-effective process to harvest biomass. However, it is difficult to recover the metal ions from medium, which will ultimately contaminate the water and final products. Auto-flocculation usually occurs in the stationary period of the microalgal culture, under which the extracellular polymeric substances (EPS) production reached maximum. Extracellular polymeric substances play an important role in controlling the flocculation and floc properties, including settling and dewatering (Bura et al. 1998). The mechanism of the auto-flocculation is interpreted as a result of the interaction of those polymers that have sufficiently accumulated at the microbial surface during endogenous growth. The EPSs present a dominant bridging mechanism between the floc components, namely

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cellular, bio-organic, and inorganic compounds (Hoa et al. 2003). By controlling EPS production, the settling and dewatering of biomass can be improved. Various operational conditions can affect EPS production, such as light intensity, nutrient concentration, pH and so on (Sheng et al. 2010). As an organic carbon and nitrogen source, glycine promotes biofilm formation (Shen et al. 2014a) and is possibly related to the secretion of EPS from microalgae. To our best knowledge, this is the first paper that revealed the effect of light intensity, glycine dosage and mixing time on EPS production and its influence on solid concentration and biomass recovery of auto-flocculation of Chlorella vulgaris.

Materials and methods Algae culture and flocculation The freshwater alga Chlorella vulgaris (FACHB-31) was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology (Wuhan, China). Modified basal medium was applied in inoculation of FACHB-31 (Shen et al. 2014b). Algae were grown in Erlenmeyer flasks and then transferred to a 35 l airlift raceway pond (Weng et al. 2014). Algae were continuously cultured during March 2014 (Fuzhou, Fujian). The average sunlight intensity was *812 ± 250 lmol m-2 s-1, the average temperature was *20 ± 10 °C. Auto-flocculation experiments were conducted 14 days after inoculation, when the cell growth reached stationary period. The initial biomass concentration was 1.16 ± 0.08 g l-1. The influence of three concentrations of glycine (0.1, 0.5 and 1 g l-1) and mixing time (1, 2 and 3 day) on flocculation efficiencies were compared under both high (250 lmol m-2 s-1) and low (125 lmol m-2 s-1) light intensity. The following procedures were followed in all flocculation experiments. First, the correct amount of glycine was added into each sample (150 ml) and the sample was completely mixed and cultivated in a rotary shaker (125 rpm min-1) with high/low light intensity. Secondly, after 1–3 days mixing, 100 ml was transferred to a 100 ml cylinder. Four hours settling time was determined from previous tests (settling time ranged from 30 min to 24 h) based

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on the concern of both flocculation efficiency and capital cost (data not presented). At the end of the 4 h, the volume of the upper clear supernatant was read, and a fraction of the supernatant was carefully removed using a fixed volume pipette without disturbing the bottom concentrated algae. Biomass dry weight (in the supernatant) was measured gravimetrically. Algae were washed with de-ionized water to remove the attached salt, and then filtered to pre-weighed 0.45 lm GF/C filter membrane. The filter was dried at 105 °C for 5 h. Algal biomass dry weight was determined by the difference of the two weights. Thirdly, another 50 ml sample was also washed with de-ionized water, and then applied for biomass (before flocculation) and EPS measurement. Three parts of EPS, including soluble EPS, loosely bound EPS and tightly bound EPS were isolated in turn based on the methods introduced by Liang et al. (2010). Extracellular polymeric substance composition, in terms of polysaccharide and protein, were then measured by anthrone colorimetric method (Yuan and Wang 2012) and Coomassie Brilliant Blue method (Zheng et al. 2012). Finally, to test whether the supernatant medium can be recycled as inoculum, 20 ml supernatant was mixed with 100 ml modified Basal medium for further cultivation. All the experiments were duplicated. Data are means ± standard deviations of two replicates. Data analysis Biomass recovery rate (BRR), dewatering rate (DR), and solid concentration achieved (SCA) are defined by Eq. 1, 2 and 3. BC1  0:1  BC2  V BC1  0:1  BC2  V ¼ 1  100 % BC1  0:1

BRR ¼

DR ¼

V  100% 0:1

SCA ¼

BC1  0:1  BC2  V BC1  BRR ¼ 0:1  V 1  DR

ð1Þ

ð2Þ ð3Þ

where BC1 is the initial algal biomass concentration before flocculation (g l-1); BC2 and V are the biomass concentration (g l-1) and volume (l) of the supernatant.

Biotechnol Lett

60

1-day

A

2-days

60

3-days

2-days

3-days

50

Bound EPS (mg g-1)

50

Bound EPS (mg g-1)

1-day

B

40 30 20

40 30 20 10

10

0

0 0.1

0.5

Glycine dose (g

0.1

1

l-1)

0.5

Glycine dose (g

1

l-1)

Fig. 1 Influence of glycine dose and mixing time on production of bound EPS with light intensity of 250 lmol m-2 s-1 (a) and 125 lmol m-2 s-1 (b)

25

A

1-day

2-days

25

3-days

2-days

3-days

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SCA (g l-1)

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SCA (g l-1)

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B

15

10

15 10 5

5

0

0 0.1

0.5

1

Glycine dose (g l-1)

0.1

0.5

Glycine dose (g

1

l-1)

Fig. 2 Influence of glycine dose and mixing time on solid concentration achieved (SCA) with light intensity of 250 lmol m-2 s-1 (a) and 125 lmol m-2 s-1 (b)

Results Figure 1 shows the production of bound EPS under various conditions. Increasing the mixing time enhanced the secretion of bound EPS. However, the influence of glycine on the production of bound EPS was affected by light intensity. Increasing glycine dosage decreased the production of bound EPS with light intensity of 250 lmol m-2 s-1 (Fig. 1a) but increased the production of bound EPS at a light intensity of 125 lmol m-2 s-1 (Fig. 1b). The maximum bound EPS produced in high and low light

intensity were 47.3 ± 5.2 mg g-1 (glycine dose of 0.1 g l-1, mixing time of 3 days) and 44.2 ± 1.6 mg g-1 (glycine dose of 1 g l-1, mixing time of 3 days), respectively. As shown in Figs. 1 and 2, bound EPS positively corresponded with SCA. Samples obtained the maximum bound EPS in high and low light intensity, were resulted with maximum SCA of 21.2 ± 1.35 and 17.2 ± 1.83 g l-1. The trend of the variation of BRR was slightly different. As shown in Fig. 3, either increasing mixing time or decreasing glycine dose increased the BRR. The maximum BRR

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A

1-day

2-days

80

3-days

70

70

60

60

50

50

BRR (%)

BRR (%)

80

40

1-day

B

2-days

3-days

40

30

30

20

20

10

10 0

0 0.1

0.5

Glycine dose (g

0.1

1

l-1)

0.5

Glycine dose (g

1

l-1)

Fig. 3 Influence of glycine dose and mixing time on biomass recovery rate (BRR) with light intensity of 250 lmol m-2 s-1 (a) and 125 lmol m-2 s-1 (b)

1-day

A

2-days

1.8

3-days

1.7

Biomass concentration (g l-1)

Biomass concentration (g l-1)

1.8

1.6 1.5 1.4 1.3 1.2 1.1

1-day

B

2-days

3-days

1.7 1.6 1.5 1.4 1.3 1.2 1.1 1

1 0.1

0.5

1

0.1

Glycine dose (g l-1)

0.5

1

Glycine dose (g l-1)

Fig. 4 Influence of glycine dose and mixing time on biomass concentration with light intensity of 250 lmol m-2 s-1 (a) and 125 lmol m-2 s-1 (b)

achieved in high and low light intensity were 71 ± 1.8 % (glycine dose of 0.1 g l-1, 3 days mixing) and 69.6 ± 0.6 % (glycine dose of 0.1 g l-1, 3 days mixing), respectively. As shown in Fig. 4, the biomass concentration varied from 1.2 ± 0.04 to 1.75 ± 0.03 g l-1 under various conditions. The data proved that glycine was consumed by FACHB-31 to stimulate the biomass and EPS growth. The supernatant medium was successfully recycled as inoculum.

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Discussion Biomass recovery rate has been considered in most studies of algae flocculation. Biomass recovery rate represents the percentage of algae retained in the concentrated broth; however, it cannot tell the DR of harvesting, which is critical in downstream biomass processing, such as cell disruption, lipid extraction, and product separation, especially when drying of biomass is needed. Solid concentration achieved is a

Biotechnol Lett

factor that relates to both BRR and the DR. It reflects the dry weight concentration of algae in the final harvested broth as a result of biomass recovery and dewatering. Therefore, it is very important to achieve the highest possible SCA in algae harvesting to reduce energy consumption in drying while keeping BRR in consideration (Shen et al. 2013). Extracellular polymeric substances produced by algae biofilms in a trickling filter enhanced solids flocculation in a later clarifier operation (Shipin et al. 1999). Extracellular polymeric substance often divide into two major fractions: soluble EPS and bound EPS (Li and Yang 2007). The adhesion of soluble EPS to cells is weak and, as a result, soluble EPS are often in solution. The inner layer of bound EPS consists of tightly-bound EPS, whereas the outer layer consists of loosely-bound EPS (Sheng et al. 2010). Sedimentation of phytoplankton blooms has been positively correlated with an increase in bound EPS concentrations (Bhaskar and Bhosle 2005). Thus, the bound EPS can be considered as the flocculant in some cases. The results were in consistent with this study. Increasing bound EPS enhanced SCA in auto-flocculation of FACHB-31. The secretion of bound EPS was affected by light, mixing time and glycine dose. Generally, EPS production was maximum at the end of the growth phase (Bhaskar and Bhosle 2005), although light and temperature conditions also affect bioflocculation (Wolfstein and Stal 2002). Therefore, increasing mixing time was effective to enhance the SCA, but also increased the operational cost. Hoa et al. (2003) reported that both nitrogen deficiency (COD: N \ 100:2) and nitrogen excess (COD: N [ 100:10) increased the EPS production. This result was close to that of this study. The nutrient and light source were provided not only for biomass growth but also for EPS production. It is possible to increase SCA when there are sufficient EPS surround microalgal cells. In this case, when light intensity was 250 lmol m-2 s-1, the biomass growth rate was increased in nitrogen excess (glycine dose of 1 g l-1) situation, and thus, the EPS surrounded each single cell was reduced, leading to the decrease of SCA. In contrary, when light intensity was 125 lmol m-2 s-1, the cell growth was relatively slow, and more EPS was secreted in nitrogen excess (glycine dose of 1 g l-1) situation, and therefore, increased the solid concentration.

Conclusions Glycine stimulated the secretion of bound EPS of FACHB-31, leading to auto-flocculation. Increasing the mixing time was effective on increasing both solid concentration and BRR. The influence of glycine dose was affected by light intensity. The optimal auto-flocculation condition was: light intensity of 250 lmol m-2 s-1, glycine dose of 0.1 g l-1, mixing time of 3 days, under which the solid concentration of 21.2 g l-1 with BRR of 71 % was achieved. The supernatant medium was effectively recycled as the inoculum for further cultivation. Acknowledgments This research was financially supported by the Natural Science Foundation of China (Award 51108085), the Natural Science Foundation of Fujian Province (Award No. 2013J01129) and the Program of the Education Department of Fujian Province (Award JA12018).

References Bhaskar PV, Bhosle NB (2005) Microbial extracellular polymeric substances in marine biogeochemical processes. Curr Sci 88:45–53 Bura R, Cheung M, Liao B, Finlayson J, Lee BC, Droppo IG, Leppard GG, Liss SN (1998) Composition of extracellular polymeric substance in the activated sludge floc matrix. Water Sci Technol 37:325–333 Hoa PT, Nair L, Visvanathan C (2003) The effect of nutrients on extracellular polymeric substance production and its influence on sludge properties. Water SA 29:437–442 Li XY, Yang SF (2007) Influence of loosely bound extracellular polymeric substances (EPS) onthe flocculation, sedimentation and dewaterability of activated sludge. Water Res 41:1022–1030 Liang ZW, Li WH, Yang SY, Du P (2010) Extraction and structural characteristics of extracellular polymeric substances (EPS), pellets in autotrophic nitrifying biofilm and activated sludge. Chemosphere 81:626–632 Liu JX, Zhu Y, Tao YJ, Zhang YM, Li AF, Li T, Sang M, Zhang CW (2013) Freshwater microalgae harvested via flocculation induced by pH decrease. Biotechnol Biofuel 6:98–108 Shen Y, Cui Y, Yuan W (2013) Flocculation Optimization of Microalga Nannochloropsis oculata. Appl Biochem Biotechnol 169:2049–2063 Shen Y, Chen C, Chen W, Xu X (2014a) Attached culture of Nannochloropsis oculata for lipid production. Bioprocess Biosyst Eng. doi:10.1007/s00449-014-1147-z Shen Y, Xin X, Zhao Y, Lin X (2014b) Influence of algae species, substrata and culture conditions on attached microalgal culture. Bioprocess Biosyst Eng 37:441–450 Sheng GP, Yu HQ, Li XY (2010) Extracellular polymeric substances (EPS) of microbial aggregates in biological

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Biotechnol Lett wastewater treatment systems: a review. Biotechnol Adv 28:882–884 Shipin OV, Rose PD, Meiring PGJ (1999) Microbial processes underlying the PETRO concept (trickling filter variant). Water Res 33:1645–1651 Weng B, Shen Y, Chen JC, Li HB, Liu LM, Deng BC, Huang GB (2014) CFD Simulation of an airlift-driven raceway bioreactor for microalgae cultivation. Adv Mater Res 955:289–292 Wolfstein K, Stal LJ (2002) Production of extracellular polymeric substances (EPS) by benthic diatoms: effect of irradiance and temperature. Mar Ecol Prog Ser 236:13–22

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Yuan DQ, Wang YL (2012) Study on the stratification components of extracellular polymeric substances (EPS) in activated sludge and their variation characteristics in physicochemical properties. Environ Sci 33:3522–3528 Zheng NI, Tao YG, Wang WQ, Qin CY, Xu YQ (2012) Optimization of extraction conditions for extracellular polymeric substances from activated sludge response surface methology. J Anhui Polytech Univ 27:17–24

An auto-flocculation strategy for Chlorella vulgaris.

Extracellular polymeric substances (EPS) excreted by microalgae are effective for microalgal flocculation. An auto-flocculation strategy was conducted...
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