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Heterotrophic culture of Chlorella pyrenoidosa using sucrose as the sole carbon source by co-culture with immobilized yeast
Accepted Manuscript Heterotrophic Culture of Chlorella pyrenoidosa Using Sucrose as the Sole Carbon Source by Co-culture with Immobilized Yeast Shi-Kai Wang, Xu Wang, Hui-Hui Tao, Xiang-Sheng Sun, Yong-Ting Tian PII: DOI: Reference:
S0960-8524(17)31878-3 https://doi.org/10.1016/j.biortech.2017.10.049 BITE 19090
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
24 August 2017 8 October 2017 11 October 2017
Please cite this article as: Wang, S-K., Wang, X., Tao, H-H., Sun, X-S., Tian, Y-T., Heterotrophic Culture of Chlorella pyrenoidosa Using Sucrose as the Sole Carbon Source by Co-culture with Immobilized Yeast, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.10.049
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Research Article for Bioresource Technology
Heterotrophic Culture of Chlorella pyrenoidosa Using Sucrose as the Sole Carbon Source by Co-culture with Immobilized Yeast Shi-Kai Wang*, Xu Wang, Hui-Hui Tao, Xiang-Sheng Sun, Yong-Ting Tian Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Yangzhou 225009, P. R. China
*Corresponding author Dr. Shi-Kai Wang Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Yangzhou 225009, P. R. China E-mail address: [email protected] 1
Abstract Glucose is normally used as the carbon source for heterotrophic cultivation of algal cells, whereas sucrose is difficult to be heterotrophicly utilized by them. In this study, a new co-culture system was developed through mixed culture of Chlorella pyrenoidosa with immobilized Saccharomyces cerevisiae in the dark to effectively obtain pure algal suspension using sucrose as only carbon source. In this system, a pure algal suspension with a concentration of 2.08 g/L was obtained. The lipid content reached 29%, which was higher than that obtained in glucose contained system. In addition, the immobilized yeast beads were repeatedly used for at least three times. Through immobilization, the choice for the yeast strains that are able to hydrolyze sucrose was not limited by its product and pure algal suspension was efficiently obtained. This strategy may effectively decrease the cost of carbon source in the heterotrophic cultivation of microalgae.
Key words: Chlorella pyrenoidosa; Immobilized yeast; Co-culture; Sucrose; Biomass
2
1. Introduction In recent years, heterotrophic cultivation is regarded as a promising approach for the efficient culture of algae to obtain abundant algal biomass (Perez-Garcia et al., 2011; Morales-Sánchez et al., 2015; Feng et al., 2014). In heterotrophic cultivation, the light requirement is eliminated. Thus, the design and operation of bioreactor are significantly simplified (Morales-Sánchez et al., 2015). In addition, by assimilating exogenous carbon source, the growth of algal cells is greatly enhanced, high biomass density and short culture period are achieved, and the accumulation of some specific products can also be remarkably enhanced (Lin and Wu, 2015). Furthermore, the high cell density gained in the heterotrophic culture effectively decreases the biomass harvesting cost. Exogenous carbon source is a necessary element in the heterotrophic culture. Due to the excellent culture performance compared to any other substrates, glucose is the most commonly used organic carbon source (Perez-Garcia et al., 2011). However, the high cost of glucose greatly increases the cost of medium and therefore, makes the heterotrophic culture of microalgae economically unfeasible (Menetrez, 2012). Recently, many efforts have been made to develop more economical substrates instead of glucose for the heterotrophic culture of algae, such as corn powder hydrolysate, sugarcane bagasse hydrolysate, cassava starch hydrolysate, and sorghum bagasse hydrolysate (Xu et al., 2006; Mu et al., 2015; Wei et al., 2009; Liang et al., 2012). However, pre-treatment, such as hydrolysis into monosaccharide or low molecular carbohydrate using acid, alkali or enzyme, is usually necessary for these raw substrates before use in heterotrophic culture, because of that algal cells are usually unable to directly utilize the components. This pre-treatment process complicates the culture process 3
and increases the cost. Some sugar-rich wastes or by-products have enriched carbohydrate content (as high as more than 50% w/w). Therefore, they are regarded as promising alternative carbon sources in fermentation industry (Andrade and Costa, 2007; Girard et al., 2014). Among these substrates, the sucrose-rich molasses accumulated in sugar production from sugarcane or sugar beets are efficient and economical alternative carbon sources and have been widely used in the fermentation of ethanol, succinic acid, isomaltulose, etc. (Castaneda-Ayarza and Cortez, 2017; Ma et al., 2014; Wu et al., 2017). In addition, sucrose is also an efficient substrate in the mixotrophic cultivation of Chlorella sp. Y8-1 (Lin and Wu, 2015). However, under heterotrophic cultivation, most of the investigated algal species were difficult to utilize sucrose as the growth of microalgae was poor or even cannot survive (Zhang et al., 2014a; Moon et al., 2013). So far, it was hypothesized that the poor utilization efficiency of sucrose in heterotrophic cultivation may be due to that the algal cell either lack specific sucrose transporter or are unable to extracellularly hydrolyze sucrose into monosaccharide (Zhang et al., 2014a). In our previous study, by mixing cultured green alga, Chlorella pyrenoidosa, with Rhodotorula glutinis, a co-culture system was developed to solve the issue that the algal cells cannot heterotrophicly utilize sucrose. R. glutinis is a yeast strain that is able to secrete extracellular β-fructofuranosidase (invertase, EC 3.2.1.26) and can extracellularly hydrolyze sucrose into glucose and fructose. In this co-culture system, C. pyrenoidosa grew well by utilizing the accumulated glucose and fructose hydrolyzed from sucrose by R. glutinis. Finally, not only a high cell density was achieved, but also the lipid content was enhanced compared 4
with pure culture (Wang et al., 2016). However, in this co-culture system, algal and yeast cells were difficult to be separated from each other. Therefore, the co-culture system is only feasible when both alga and yeast cells accumulate same products. This hinders the further broad application of this approach. Immobilization technology is an effective technique to restrict the freedom movement of cell in cell culture (Lam and Lee, 2012). After being immobilized, the viable cells keep its metabolic activity and it has many advantages over the conventional free cell culture system, such as the improved biological stability, the accelerated reaction rates, and easy separation, etc. (Moriwaki et al., 2014). This technique has been widely applied in various cell culture and fermentation process (Bleve et al., 2016). In this study, the most commonly used yeast, Saccharomyces cerevisiae, which can be easily obtained from brewery, was immobilized in calcium alginate beads and then was heterotrophicly co-cultured with C. pyrenoidosa using sucrose as the sole carbon source to obtain pure algal suspension at last. In this system, C. pyrenoidosa grew well under the condition of accumulated glucose and fructose hydrolyzed from sucrose by yeast cells as previous research investigated. Furthermore, the yeast cells immobilized in the beads were easily separated by simple filtration method. The strategy provided in this study can achieve the pure culture of algal biomass and can be widely used in the heterotrophic culture of microalgae using sucrose or sucrose-rich by-product as the sole carbon source.
2. Material and methods 2.1 Microorganism strains and inoculum culture 5
C. pyrenoidosa FACHB-9 was purchased from Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB), Chinese Academy of Sciences. For inoculum culture, the stored cells were activated in liquid BG-11 medium and incubated at 25°C in an orbital shaker at 120 rpm under a 16 h light period per day with 35 µmol·m-2·s-1. After cultured for two weeks, the cells were used as inoculum for the following experiments. S. cerevisiae, kindly provided by Prof. Agen Huang (College of Food Science and Engineering, Yangzhou University), was stored on Yeast Extract Peptone Dextrose Medium (YPD) medium containing 1% glucose, 1% peptone, 0.5% yeast extract, and 2% agar. For inoculum culture, the cells were maintained at liquid medium as the same components without agar and kept at 25°C in an orbital shaker at 150 rpm for two days culture. The liquid cultured cells were used for immobilization and other experiments. 2.2 Preparation of immobilized yeast beads (IYB) Yeast cell immobilization was performed as described by El-Dalatony et al. (2016). Briefly, 50 mL Na-alginate solution (2%, w/v) was prepared by dissolving Na-alginate powder in distilled water. After counting the cell density using a hemacytometer and a microscope (Olympus BX53, Japan), the yeast culture was concentrated through centrifugation at 4000 rpm, 10 min and then mixed with above mentioned Na-alginate solution at room temperature. After being thoroughly mixed, the mixture was extruded dropwise through a 10 mL disposable plastic syringe to 100 mL CaCl2 (2%, w/v) solution to prepare uniform spherical beads. After hardening at 4°C for 2 h, the beads were washed with sterile distilled water for three times to remove unbound impurities, and stored at 4°C for further use. The diameters of the IYB were 2~3 mm as measured using a digital vernier caliper. 6
2.3 Comparison of the culture of free yeast and immobilized yeast Free and immobilized yeast cells of same cell density were inoculated in liquid BG-11 medium with 1% sucrose. The culture was kept at 25°C in an orbital shaker at 150 rpm. Samples were taken at 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h to measure the cell concentration, pH, and extracellular invertase activity. 2.4 Co-culture of microalgae and IYB 100 mL of Liquid BG-11 medium with 1% sucrose was added into Erlenmeyer flasks (250 mL) for the following experiments. Various numbers of IYB (30, 60, 90, and 120) were added to each culture. The cultures were incubated at 25°C in an orbital shaker at 150 rpm under a dark environment. 2.5 Reusability of IYB At the end of the co-culture process, the IYB was collected and washed three times using sterile distilled water. The IYB was reused in the following co-culture process as described previously and its culture performance was evaluated. 2.6 Analytic methods 2.6.1 Determination of cell growth Biomass concentration of microalgae and free culture yeast was determined gravimetrically. Briefly, after centrifuging at 4°C, 8000 rpm for 10 min, the cell pellets were collected and washed by distilled water twice, and then dried at 80°C for 24 h. For the determination of immobilized yeast cells, the beads were firstly solubilized using 2 % (w/v) sodium carbonate solution and then dried at 80°C for 24 h (Lam and Lee, 2012). The dry cell weight (DCW, g/L) of the cells was determined gravimetrically. 7
The growth kinetic parameters of the algal cells in the co-culture process were calculated as described by Wang et al. (2014). 2.6.2 Measurement of extracellular invertase activity Extracellular invertase activity in the culture of free and immobilized yeast was measured according to the method modified from Arez et al. (2014). In detail, the culture was centrifuged at 9400 rpm, 4 °C for 10 min and the supernatant was taken as the crude enzymatic extract. The enzymatic reaction mixture containing 100 µL crude enzymatic extract and 900 µL 1% (w/v) sucrose in 50 mM sodium citrate buffer, pH 5.5. After being incubated at 45°C for 10 min, the reaction was immediately inactivated by putting the reaction mixtures on ice. 2 mL dinitrosalicylic acid (DNS) reagent was added into the mixture and then put the mixture in boiling water for 5 min. After cooling on ice to room temperature, 9 mL distilled water was added into the mixture. The absorbance was determined at 540 nm in a 722N visible spectrophotometer (Shanghai P&S instrument CO., LTD, China). Blank controls were prepared with inactivated enzyme crude extracts treated by boiling for 5 min. One unit of invertase activity (U) was defined as the amount of enzyme responsible for the production of 1 µmol of reducing sugar per minute under the above conditions. 2.6.3 Measurement of lipid content Lipids were extracted and gravimetrically quantified as reported by Morales-Sánchez et al. (2013). Briefly, cell pellet was collected by centrifuging (11,700 rpm, 10 min, 4°C). After washing
twice
with
deionized
water,
the
cell
pellet
was
resuspended
in
methanol/dichloromethane (2:1, v/v) containing 0.5 mg of butylated hydroxytoluene and stored at 4°C overnight. After centrifugation at 4°C, 11,700 rpm for 10 min, the supernatant 8
was transferred to another tube, and the residue was extracted twice with 4 mL of methanol/dichloromethane (1:1; v/v) in an ultrasonic cleaner for 10 min. The solvents in the combined organic phase were removed using an evaporator at 40°C and atmospheric pressure. After drying under nitrogen atmosphere, the lipids were gravimetrically quantified. 2.7 Statistical analysis Data were presented as means ± standard error of the mean based on three parallel experiments. The statistical significances were analyzed by one-way analysis of variance (ANOVA) (P＜0.05) using Origin 9 (OriginLab, USA). 3. Results and discussion 3.1 Comparison of the culture of immobilized yeast and free yeast As shown in Fig. 1A, S. cerevisiae could grow in both free and immobilized cultures in BG-11 medium with sucrose, although the growth rate was much lower than that in YPD medium (data not shown) due to the scanty nitrogen source and other nutrients in BG-11 medium. Compared to the rapid growth of yeast, a relatively slow growth was necessary for the long culture of IYB as that abundant yeast cells easily escaped from the beads. The growth of free and immobilized cells was similar at initial 48 h. However, after 48 h, the growth rate of free culture was much faster than that in immobilized culture. Meanwhile, the pH fluctuation in free yeast culture was also much more violent than that in immobilized yeast culture. As shown in Fig. 1B, the pH decreased to 3.62 in free culture from the initial 7.77 after 96 h culture while the final pH kept at 4.48 in the immobilized culture. It was reported that in the growth of yeast, it produces various organic acids, such as lactic acid, acetic acid, malic acid, succinic acid, etc., resulting in the continuous decrease of pH along 9
with culture (Li and Borodina, 2015). Combined the results of cell growth and pH variation, it was concluded that the immobilization of yeast can partly inhibit the metabolic activity of yeast cells. Furthermore, the decreased competitiveness of yeast cells after been immobilized is benefit for the growth of other microorganism in the co-culture system. After being immobilized, the secretion of sucrase of the yeast cells was also been reduced as shown in Fig. 1C. The sucrase activity was undetectable at initial 12 h in immobilized culture while it reached 2.8×103 U/mL at 12 h in free yeast culture. In addition, the highest activity in free culture was also greatly higher than that in immobilized culture. In immobilized culture, the sucrase activity was relatively stable in the whole culture period after 12 h. This confirmed that immobilization can effectively improve the biological stability of cells (Moriwaki et al., 2014). The stable and relatively low sucrase activity resulted in a slower hydrolysis of sucrose, which may be helpful for algae growth in co-culture as the utilization rate of monosaccharide was much lower than the hydrolysis rate of sucrose as previous study stated (Wang et al., 2016). Similar with the previous study (Wang et al., 2016), obvious monosaccharides accumulation was detected in this study, which can supply as the available carbon source for algal cells in the mixed culture process. 3.2 Growth of C. pyrenoidosa and S. cerevisiae in the co-culture of microalgae and IYB The growth curves of algal cells co-cultured with different numbers of IYB were displayed in Fig. 2A. At the beginning of culture, the growth of algal cells exhibited a short lag phase. This was mainly due to that in this phase, sucrose was not yet hydrolyzed by the immobilized yeast. Therefore, the metabolic activity of algae cells was weak. However, in this lag phase, the metabolic activity of yeast kept increasing, resulting in the drastic decrease of pH in all 10
culture system as shown in Fig. 2B. After 2 days, the hydrolysis of sucrose led to abundant accumulation
of
monosaccharide
and
C.
pyrenoidosa
effectively
utilized
these
monosaccharides for its growth, resulting in the algal cells entered the exponential growth stage as shown in Fig. 2A. With the increasing growth of microalgae, the pH presented a significant increase. It has been detected that pH was kept increasing in the culture of microalgae and even developed more than 10 under both autotrophic and heterotrophic conditions without CO2 supplementation based on our previous study. Similar result was also detected in the culture of Haematococcus pluvialis, in which the pH reached 10.5 in the culture process (Zhang et al., 2016). This indicated that the metabolism of C. pyrenoidosa was vigorous in this co-culture system. As shown in Fig. 3, the growth of immobilized yeast in the co-culture system was significantly restricted. At the beginning of the culture, the yeast concentration was slightly increased in all cultures. However, after 2 days culture, the growth of yeast cells was completely stagnated and slightly declined, accompanied by that the growth of C. pyrenoidosa entered the exponential phase. This was might due to that the composition of the medium used in this study, BG-11, was more suitable for the growth of microalgae. Furthermore, after being immobilized, the transfer of nutrition and dissolved oxygen for the immobilized yeast was greatly limited. It has been investigated that the dissolved oxygen has significant influence on yeast growth (Li et al., 2017). In our previous study in which C. pyrenoidosa was co-cultured with free yeast, the final percentage of yeast was more than 50% (based on cell number), which was unfavourable for microalgae growth (Wang et al., 2016). Through immobilization, the growth of yeast could be greatly limited and this was benefit for the 11
accumulation of algal biomass. The growth kinetic parameters of C. pyrenoidosa in the co-culture process with different numbers of IYB was calculated in Table 1. It could be concluded that the more IYB, the higher maximum specific growth rate. The highest final biomass concentration and biomass productivity were 2.08 g/L and 0.34 g/L/day, respectively, in the condition of 150 IYB (100 mL culture). However, the biomass concentration and biomass productivity had no significant difference from cultures with 150 IYB and 120 IYB except that the maximum specific growth rate was slightly higher in the cultures with 150 IYB. Compared with the results obtained from the co-culture system with free yeast, the biomass accumulation and biomass productivity of C. pyrenoidosa was greatly enhanced in the co-culture system with immobilized yeast (Wang et al., 2016). These results indicated that the immobilization of yeast significantly enhance the accumulation of algal biomass, which is benefit for the production of alga-based products. Till now, no algal species have been successfully cultured using sucrose as the sole carbon source at heterotrophic conditions. The investigated algal species, including Chlorella, Chlamydomonas reinhardtii, and Botryococcus braunii, grew poor and even could not survive in the condition of sucrose as the sole carbon source (Lin and Wu, 2015; Moon et al., 2013; Zhang et al., 2011). This study solved the issue that alga are unable to heterotrphicly utilize sucrose and furthermore, a good culture performance of alga was achieved. 3.3 The reusability of IYB As shown in Fig. 4, both of the final biomass concentration of algal cells co-cultured with re-used IYB at second round and third round reached about 1.80 g/L, as high as that obtained 12
from the first round. However, the culture period was gradually shortened from the first round to third round. Only 4 days were taken to reach the final biomass concentration at the third round compared to that of 5 days at the first round. As the result, the biomass productivity was increased from 0.31 g/L/day to 0.44 g/L/day. This indicated that the immobilized yeast cells can maintain its metabolic activity for long culture and they exhibited a better and better reusability for alga growth. However, along with the culture, the mechanical strength of the IYB was obviously weakened and some debris of IYB was detected at the third culture round in the culture suspension, whereas no free yeast was detected in the culture broth. Further optimization of the synthesis condition of IYB is necessary to enhance its mechanical strength to fulfill the multiple cycles of the IYB. 3.4 Lipid accumulation of C. pyrenoidosa in the co-culture of microalgae and IYB A lipid content of 25.2% was obtained by using glucose as the sole carbon source. In contrast, the lipid content of C. pyrenoidosa co-cultured with different numbers of IYB was ranged from 28.24% to 29.70%, which has no significant difference (p＞0.05) between themselves, but was significantly higher (p＜0.05) than that obtained in the control of glucose as sole carbon source. This result was consistent with the data obtained in the co-culture system with free yeast (Wang et al., 2016). After being immobilized, the yeast cells still maintained its metabolism and the calcium alginate could permit the substance exchange with the environment (Moriwaki et al., 2014). Therefore, the immobilized yeast cells also had a synergistic effect on lipid accumulation of algal cells as previously detected (Wang et al., 2016; Xue et al., 2010; Zhang et al., 2014b; Yen et al., 2015). Besides, in the mixotrophic culture of Chlorella sp. Y8-1 using sucrose, it has been found that the lipid content of algal 13
cells was much higher than that of glucose, which indicated that sucrose was a promising carbon source for lipid accumulation in algal culture (Lin and Wu, 2015). This study provides a novel way for the heterotrophic culture of microalgae using sucrose or sucrose-riched substrates. This effectively solves the issue that alga are unable to heterotrphicly utilize sucrose. More importantly, pure algal suspension was efficiently obtained in this system as that the IYB can be easily separated through simple filtration method compared with our previous study (Wang et al., 2016). Meanwhile, the choice for the yeast strains that are able to hydrolyze sucrose was not limited by its product and the utilization of the most commonly used yeast, S. cerevisiae, can further decrease the cost as it can be easily obtained from brewery.
4. Conclusion A new co-culture system was developed through mixed culture of C. pyrenoidosa and immobilized yeast. By immobilization, the growth of yeast was partly inhibited and a higher algal concentration was obtained compared to that of co-culture with free yeast. A pure algal suspension with a concentration of 2.08 g/L was obtained in this system. In addition, the immobilized yeast beads were repeatedly used for at least three times. This strategy can be widely used in the heterotrophic culture of microalgae using sucrose-riched wastes and may effectively decrease the cost of carbon source in the heterotrophic cultivation of microalgae.
Acknowledgments This work was financially supported by the Natural Science Foundation of Jiangsu Province 14
(No. BK20170495), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (17KJB240003), and
Postdoctoral Science Foundation of China (No.
2016M591933).
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18
Wang et al., Table 1
Table 1 Growth kinetic parameters of the algal cells in the co-culture process with different number of IYB. Number
of Final biomass
IYB (/100 mL)
(g/L)
Biomass
Maximum
Doubling time
productivity
specific growth (days)
(g/L/day)
rate µmax (day-1)
30
0.90 ± 0.15, a
0.15 ± 0.03, a
0.73 ± 0.03, a
0.95 ± 0.03, a
60
1.46 ± 0.04, b
0.24 ± 0.01, b
0.84 ± 0.02, b
0.83 ± 0.02, b
90
1.88 ± 0.04, c
0.31 ± 0.01, c
0.93 ± 0.02, c
0.75 ± 0.03, c
120
1.96 ± 0.07, c, d
0.32 ± 0.01, c, d
0.94 ± 0.02, c
0.74 ± 0.01, c
150
2.08 ± 0.03, d
0.34 ± 0.01, d
1.01 ± 0.03, d
0.69 ± 0.03, d
Values in a column with different letters are significantly different (P < 0.05).
19
Wang et al., Figure captions
Fig. 1. Comparison of biomass (A), pH (B), and sucrase activity (C) in the culture of immobilized and free S. cerevisiae. (I: Immobilized culture; F: Free culture)
Fig. 2. The growth curves of C. pyrenoidosa (A) and the variation of pH (B) in the co-culture process with different numbers of IYB (in 100 mL culture).
Fig. 3. The growth curves of S. cerevisiae in the co-culture process with different numbers of IYB (in 100 mL culture).
Fig. 4. The growth curve of C. pyrenoidosa co-cultured with the re-used IYB (90 beads in 100 mL culture).
20
Wang et al., Fig. 1
21
22
Wang et al., Fig. 2
A
B
23
Wang et al., Fig. 3
24
Wang et al., Fig. 4
25
Research Highlights A new co-culture system was developed by co-culture of C. pyrenoidosa and immobilized yeast. Alga could effectively utilize sucrose via the hydrolysis of sucrose by immobilized yeast.
Pure algal suspension with a concentration of 2.08 g/L was obtained as the IYB can be easily separated by filtration.
26
S0960-8524(17)31878-3 https://doi.org/10.1016/j.biortech.2017.10.049 BITE 19090
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
24 August 2017 8 October 2017 11 October 2017
Please cite this article as: Wang, S-K., Wang, X., Tao, H-H., Sun, X-S., Tian, Y-T., Heterotrophic Culture of Chlorella pyrenoidosa Using Sucrose as the Sole Carbon Source by Co-culture with Immobilized Yeast, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.10.049
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Research Article for Bioresource Technology
Heterotrophic Culture of Chlorella pyrenoidosa Using Sucrose as the Sole Carbon Source by Co-culture with Immobilized Yeast Shi-Kai Wang*, Xu Wang, Hui-Hui Tao, Xiang-Sheng Sun, Yong-Ting Tian Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Yangzhou 225009, P. R. China
*Corresponding author Dr. Shi-Kai Wang Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Yangzhou 225009, P. R. China E-mail address: [email protected] 1
Abstract Glucose is normally used as the carbon source for heterotrophic cultivation of algal cells, whereas sucrose is difficult to be heterotrophicly utilized by them. In this study, a new co-culture system was developed through mixed culture of Chlorella pyrenoidosa with immobilized Saccharomyces cerevisiae in the dark to effectively obtain pure algal suspension using sucrose as only carbon source. In this system, a pure algal suspension with a concentration of 2.08 g/L was obtained. The lipid content reached 29%, which was higher than that obtained in glucose contained system. In addition, the immobilized yeast beads were repeatedly used for at least three times. Through immobilization, the choice for the yeast strains that are able to hydrolyze sucrose was not limited by its product and pure algal suspension was efficiently obtained. This strategy may effectively decrease the cost of carbon source in the heterotrophic cultivation of microalgae.
Key words: Chlorella pyrenoidosa; Immobilized yeast; Co-culture; Sucrose; Biomass
2
1. Introduction In recent years, heterotrophic cultivation is regarded as a promising approach for the efficient culture of algae to obtain abundant algal biomass (Perez-Garcia et al., 2011; Morales-Sánchez et al., 2015; Feng et al., 2014). In heterotrophic cultivation, the light requirement is eliminated. Thus, the design and operation of bioreactor are significantly simplified (Morales-Sánchez et al., 2015). In addition, by assimilating exogenous carbon source, the growth of algal cells is greatly enhanced, high biomass density and short culture period are achieved, and the accumulation of some specific products can also be remarkably enhanced (Lin and Wu, 2015). Furthermore, the high cell density gained in the heterotrophic culture effectively decreases the biomass harvesting cost. Exogenous carbon source is a necessary element in the heterotrophic culture. Due to the excellent culture performance compared to any other substrates, glucose is the most commonly used organic carbon source (Perez-Garcia et al., 2011). However, the high cost of glucose greatly increases the cost of medium and therefore, makes the heterotrophic culture of microalgae economically unfeasible (Menetrez, 2012). Recently, many efforts have been made to develop more economical substrates instead of glucose for the heterotrophic culture of algae, such as corn powder hydrolysate, sugarcane bagasse hydrolysate, cassava starch hydrolysate, and sorghum bagasse hydrolysate (Xu et al., 2006; Mu et al., 2015; Wei et al., 2009; Liang et al., 2012). However, pre-treatment, such as hydrolysis into monosaccharide or low molecular carbohydrate using acid, alkali or enzyme, is usually necessary for these raw substrates before use in heterotrophic culture, because of that algal cells are usually unable to directly utilize the components. This pre-treatment process complicates the culture process 3
and increases the cost. Some sugar-rich wastes or by-products have enriched carbohydrate content (as high as more than 50% w/w). Therefore, they are regarded as promising alternative carbon sources in fermentation industry (Andrade and Costa, 2007; Girard et al., 2014). Among these substrates, the sucrose-rich molasses accumulated in sugar production from sugarcane or sugar beets are efficient and economical alternative carbon sources and have been widely used in the fermentation of ethanol, succinic acid, isomaltulose, etc. (Castaneda-Ayarza and Cortez, 2017; Ma et al., 2014; Wu et al., 2017). In addition, sucrose is also an efficient substrate in the mixotrophic cultivation of Chlorella sp. Y8-1 (Lin and Wu, 2015). However, under heterotrophic cultivation, most of the investigated algal species were difficult to utilize sucrose as the growth of microalgae was poor or even cannot survive (Zhang et al., 2014a; Moon et al., 2013). So far, it was hypothesized that the poor utilization efficiency of sucrose in heterotrophic cultivation may be due to that the algal cell either lack specific sucrose transporter or are unable to extracellularly hydrolyze sucrose into monosaccharide (Zhang et al., 2014a). In our previous study, by mixing cultured green alga, Chlorella pyrenoidosa, with Rhodotorula glutinis, a co-culture system was developed to solve the issue that the algal cells cannot heterotrophicly utilize sucrose. R. glutinis is a yeast strain that is able to secrete extracellular β-fructofuranosidase (invertase, EC 3.2.1.26) and can extracellularly hydrolyze sucrose into glucose and fructose. In this co-culture system, C. pyrenoidosa grew well by utilizing the accumulated glucose and fructose hydrolyzed from sucrose by R. glutinis. Finally, not only a high cell density was achieved, but also the lipid content was enhanced compared 4
with pure culture (Wang et al., 2016). However, in this co-culture system, algal and yeast cells were difficult to be separated from each other. Therefore, the co-culture system is only feasible when both alga and yeast cells accumulate same products. This hinders the further broad application of this approach. Immobilization technology is an effective technique to restrict the freedom movement of cell in cell culture (Lam and Lee, 2012). After being immobilized, the viable cells keep its metabolic activity and it has many advantages over the conventional free cell culture system, such as the improved biological stability, the accelerated reaction rates, and easy separation, etc. (Moriwaki et al., 2014). This technique has been widely applied in various cell culture and fermentation process (Bleve et al., 2016). In this study, the most commonly used yeast, Saccharomyces cerevisiae, which can be easily obtained from brewery, was immobilized in calcium alginate beads and then was heterotrophicly co-cultured with C. pyrenoidosa using sucrose as the sole carbon source to obtain pure algal suspension at last. In this system, C. pyrenoidosa grew well under the condition of accumulated glucose and fructose hydrolyzed from sucrose by yeast cells as previous research investigated. Furthermore, the yeast cells immobilized in the beads were easily separated by simple filtration method. The strategy provided in this study can achieve the pure culture of algal biomass and can be widely used in the heterotrophic culture of microalgae using sucrose or sucrose-rich by-product as the sole carbon source.
2. Material and methods 2.1 Microorganism strains and inoculum culture 5
C. pyrenoidosa FACHB-9 was purchased from Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB), Chinese Academy of Sciences. For inoculum culture, the stored cells were activated in liquid BG-11 medium and incubated at 25°C in an orbital shaker at 120 rpm under a 16 h light period per day with 35 µmol·m-2·s-1. After cultured for two weeks, the cells were used as inoculum for the following experiments. S. cerevisiae, kindly provided by Prof. Agen Huang (College of Food Science and Engineering, Yangzhou University), was stored on Yeast Extract Peptone Dextrose Medium (YPD) medium containing 1% glucose, 1% peptone, 0.5% yeast extract, and 2% agar. For inoculum culture, the cells were maintained at liquid medium as the same components without agar and kept at 25°C in an orbital shaker at 150 rpm for two days culture. The liquid cultured cells were used for immobilization and other experiments. 2.2 Preparation of immobilized yeast beads (IYB) Yeast cell immobilization was performed as described by El-Dalatony et al. (2016). Briefly, 50 mL Na-alginate solution (2%, w/v) was prepared by dissolving Na-alginate powder in distilled water. After counting the cell density using a hemacytometer and a microscope (Olympus BX53, Japan), the yeast culture was concentrated through centrifugation at 4000 rpm, 10 min and then mixed with above mentioned Na-alginate solution at room temperature. After being thoroughly mixed, the mixture was extruded dropwise through a 10 mL disposable plastic syringe to 100 mL CaCl2 (2%, w/v) solution to prepare uniform spherical beads. After hardening at 4°C for 2 h, the beads were washed with sterile distilled water for three times to remove unbound impurities, and stored at 4°C for further use. The diameters of the IYB were 2~3 mm as measured using a digital vernier caliper. 6
2.3 Comparison of the culture of free yeast and immobilized yeast Free and immobilized yeast cells of same cell density were inoculated in liquid BG-11 medium with 1% sucrose. The culture was kept at 25°C in an orbital shaker at 150 rpm. Samples were taken at 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h to measure the cell concentration, pH, and extracellular invertase activity. 2.4 Co-culture of microalgae and IYB 100 mL of Liquid BG-11 medium with 1% sucrose was added into Erlenmeyer flasks (250 mL) for the following experiments. Various numbers of IYB (30, 60, 90, and 120) were added to each culture. The cultures were incubated at 25°C in an orbital shaker at 150 rpm under a dark environment. 2.5 Reusability of IYB At the end of the co-culture process, the IYB was collected and washed three times using sterile distilled water. The IYB was reused in the following co-culture process as described previously and its culture performance was evaluated. 2.6 Analytic methods 2.6.1 Determination of cell growth Biomass concentration of microalgae and free culture yeast was determined gravimetrically. Briefly, after centrifuging at 4°C, 8000 rpm for 10 min, the cell pellets were collected and washed by distilled water twice, and then dried at 80°C for 24 h. For the determination of immobilized yeast cells, the beads were firstly solubilized using 2 % (w/v) sodium carbonate solution and then dried at 80°C for 24 h (Lam and Lee, 2012). The dry cell weight (DCW, g/L) of the cells was determined gravimetrically. 7
The growth kinetic parameters of the algal cells in the co-culture process were calculated as described by Wang et al. (2014). 2.6.2 Measurement of extracellular invertase activity Extracellular invertase activity in the culture of free and immobilized yeast was measured according to the method modified from Arez et al. (2014). In detail, the culture was centrifuged at 9400 rpm, 4 °C for 10 min and the supernatant was taken as the crude enzymatic extract. The enzymatic reaction mixture containing 100 µL crude enzymatic extract and 900 µL 1% (w/v) sucrose in 50 mM sodium citrate buffer, pH 5.5. After being incubated at 45°C for 10 min, the reaction was immediately inactivated by putting the reaction mixtures on ice. 2 mL dinitrosalicylic acid (DNS) reagent was added into the mixture and then put the mixture in boiling water for 5 min. After cooling on ice to room temperature, 9 mL distilled water was added into the mixture. The absorbance was determined at 540 nm in a 722N visible spectrophotometer (Shanghai P&S instrument CO., LTD, China). Blank controls were prepared with inactivated enzyme crude extracts treated by boiling for 5 min. One unit of invertase activity (U) was defined as the amount of enzyme responsible for the production of 1 µmol of reducing sugar per minute under the above conditions. 2.6.3 Measurement of lipid content Lipids were extracted and gravimetrically quantified as reported by Morales-Sánchez et al. (2013). Briefly, cell pellet was collected by centrifuging (11,700 rpm, 10 min, 4°C). After washing
twice
with
deionized
water,
the
cell
pellet
was
resuspended
in
methanol/dichloromethane (2:1, v/v) containing 0.5 mg of butylated hydroxytoluene and stored at 4°C overnight. After centrifugation at 4°C, 11,700 rpm for 10 min, the supernatant 8
was transferred to another tube, and the residue was extracted twice with 4 mL of methanol/dichloromethane (1:1; v/v) in an ultrasonic cleaner for 10 min. The solvents in the combined organic phase were removed using an evaporator at 40°C and atmospheric pressure. After drying under nitrogen atmosphere, the lipids were gravimetrically quantified. 2.7 Statistical analysis Data were presented as means ± standard error of the mean based on three parallel experiments. The statistical significances were analyzed by one-way analysis of variance (ANOVA) (P＜0.05) using Origin 9 (OriginLab, USA). 3. Results and discussion 3.1 Comparison of the culture of immobilized yeast and free yeast As shown in Fig. 1A, S. cerevisiae could grow in both free and immobilized cultures in BG-11 medium with sucrose, although the growth rate was much lower than that in YPD medium (data not shown) due to the scanty nitrogen source and other nutrients in BG-11 medium. Compared to the rapid growth of yeast, a relatively slow growth was necessary for the long culture of IYB as that abundant yeast cells easily escaped from the beads. The growth of free and immobilized cells was similar at initial 48 h. However, after 48 h, the growth rate of free culture was much faster than that in immobilized culture. Meanwhile, the pH fluctuation in free yeast culture was also much more violent than that in immobilized yeast culture. As shown in Fig. 1B, the pH decreased to 3.62 in free culture from the initial 7.77 after 96 h culture while the final pH kept at 4.48 in the immobilized culture. It was reported that in the growth of yeast, it produces various organic acids, such as lactic acid, acetic acid, malic acid, succinic acid, etc., resulting in the continuous decrease of pH along 9
with culture (Li and Borodina, 2015). Combined the results of cell growth and pH variation, it was concluded that the immobilization of yeast can partly inhibit the metabolic activity of yeast cells. Furthermore, the decreased competitiveness of yeast cells after been immobilized is benefit for the growth of other microorganism in the co-culture system. After being immobilized, the secretion of sucrase of the yeast cells was also been reduced as shown in Fig. 1C. The sucrase activity was undetectable at initial 12 h in immobilized culture while it reached 2.8×103 U/mL at 12 h in free yeast culture. In addition, the highest activity in free culture was also greatly higher than that in immobilized culture. In immobilized culture, the sucrase activity was relatively stable in the whole culture period after 12 h. This confirmed that immobilization can effectively improve the biological stability of cells (Moriwaki et al., 2014). The stable and relatively low sucrase activity resulted in a slower hydrolysis of sucrose, which may be helpful for algae growth in co-culture as the utilization rate of monosaccharide was much lower than the hydrolysis rate of sucrose as previous study stated (Wang et al., 2016). Similar with the previous study (Wang et al., 2016), obvious monosaccharides accumulation was detected in this study, which can supply as the available carbon source for algal cells in the mixed culture process. 3.2 Growth of C. pyrenoidosa and S. cerevisiae in the co-culture of microalgae and IYB The growth curves of algal cells co-cultured with different numbers of IYB were displayed in Fig. 2A. At the beginning of culture, the growth of algal cells exhibited a short lag phase. This was mainly due to that in this phase, sucrose was not yet hydrolyzed by the immobilized yeast. Therefore, the metabolic activity of algae cells was weak. However, in this lag phase, the metabolic activity of yeast kept increasing, resulting in the drastic decrease of pH in all 10
culture system as shown in Fig. 2B. After 2 days, the hydrolysis of sucrose led to abundant accumulation
of
monosaccharide
and
C.
pyrenoidosa
effectively
utilized
these
monosaccharides for its growth, resulting in the algal cells entered the exponential growth stage as shown in Fig. 2A. With the increasing growth of microalgae, the pH presented a significant increase. It has been detected that pH was kept increasing in the culture of microalgae and even developed more than 10 under both autotrophic and heterotrophic conditions without CO2 supplementation based on our previous study. Similar result was also detected in the culture of Haematococcus pluvialis, in which the pH reached 10.5 in the culture process (Zhang et al., 2016). This indicated that the metabolism of C. pyrenoidosa was vigorous in this co-culture system. As shown in Fig. 3, the growth of immobilized yeast in the co-culture system was significantly restricted. At the beginning of the culture, the yeast concentration was slightly increased in all cultures. However, after 2 days culture, the growth of yeast cells was completely stagnated and slightly declined, accompanied by that the growth of C. pyrenoidosa entered the exponential phase. This was might due to that the composition of the medium used in this study, BG-11, was more suitable for the growth of microalgae. Furthermore, after being immobilized, the transfer of nutrition and dissolved oxygen for the immobilized yeast was greatly limited. It has been investigated that the dissolved oxygen has significant influence on yeast growth (Li et al., 2017). In our previous study in which C. pyrenoidosa was co-cultured with free yeast, the final percentage of yeast was more than 50% (based on cell number), which was unfavourable for microalgae growth (Wang et al., 2016). Through immobilization, the growth of yeast could be greatly limited and this was benefit for the 11
accumulation of algal biomass. The growth kinetic parameters of C. pyrenoidosa in the co-culture process with different numbers of IYB was calculated in Table 1. It could be concluded that the more IYB, the higher maximum specific growth rate. The highest final biomass concentration and biomass productivity were 2.08 g/L and 0.34 g/L/day, respectively, in the condition of 150 IYB (100 mL culture). However, the biomass concentration and biomass productivity had no significant difference from cultures with 150 IYB and 120 IYB except that the maximum specific growth rate was slightly higher in the cultures with 150 IYB. Compared with the results obtained from the co-culture system with free yeast, the biomass accumulation and biomass productivity of C. pyrenoidosa was greatly enhanced in the co-culture system with immobilized yeast (Wang et al., 2016). These results indicated that the immobilization of yeast significantly enhance the accumulation of algal biomass, which is benefit for the production of alga-based products. Till now, no algal species have been successfully cultured using sucrose as the sole carbon source at heterotrophic conditions. The investigated algal species, including Chlorella, Chlamydomonas reinhardtii, and Botryococcus braunii, grew poor and even could not survive in the condition of sucrose as the sole carbon source (Lin and Wu, 2015; Moon et al., 2013; Zhang et al., 2011). This study solved the issue that alga are unable to heterotrphicly utilize sucrose and furthermore, a good culture performance of alga was achieved. 3.3 The reusability of IYB As shown in Fig. 4, both of the final biomass concentration of algal cells co-cultured with re-used IYB at second round and third round reached about 1.80 g/L, as high as that obtained 12
from the first round. However, the culture period was gradually shortened from the first round to third round. Only 4 days were taken to reach the final biomass concentration at the third round compared to that of 5 days at the first round. As the result, the biomass productivity was increased from 0.31 g/L/day to 0.44 g/L/day. This indicated that the immobilized yeast cells can maintain its metabolic activity for long culture and they exhibited a better and better reusability for alga growth. However, along with the culture, the mechanical strength of the IYB was obviously weakened and some debris of IYB was detected at the third culture round in the culture suspension, whereas no free yeast was detected in the culture broth. Further optimization of the synthesis condition of IYB is necessary to enhance its mechanical strength to fulfill the multiple cycles of the IYB. 3.4 Lipid accumulation of C. pyrenoidosa in the co-culture of microalgae and IYB A lipid content of 25.2% was obtained by using glucose as the sole carbon source. In contrast, the lipid content of C. pyrenoidosa co-cultured with different numbers of IYB was ranged from 28.24% to 29.70%, which has no significant difference (p＞0.05) between themselves, but was significantly higher (p＜0.05) than that obtained in the control of glucose as sole carbon source. This result was consistent with the data obtained in the co-culture system with free yeast (Wang et al., 2016). After being immobilized, the yeast cells still maintained its metabolism and the calcium alginate could permit the substance exchange with the environment (Moriwaki et al., 2014). Therefore, the immobilized yeast cells also had a synergistic effect on lipid accumulation of algal cells as previously detected (Wang et al., 2016; Xue et al., 2010; Zhang et al., 2014b; Yen et al., 2015). Besides, in the mixotrophic culture of Chlorella sp. Y8-1 using sucrose, it has been found that the lipid content of algal 13
cells was much higher than that of glucose, which indicated that sucrose was a promising carbon source for lipid accumulation in algal culture (Lin and Wu, 2015). This study provides a novel way for the heterotrophic culture of microalgae using sucrose or sucrose-riched substrates. This effectively solves the issue that alga are unable to heterotrphicly utilize sucrose. More importantly, pure algal suspension was efficiently obtained in this system as that the IYB can be easily separated through simple filtration method compared with our previous study (Wang et al., 2016). Meanwhile, the choice for the yeast strains that are able to hydrolyze sucrose was not limited by its product and the utilization of the most commonly used yeast, S. cerevisiae, can further decrease the cost as it can be easily obtained from brewery.
4. Conclusion A new co-culture system was developed through mixed culture of C. pyrenoidosa and immobilized yeast. By immobilization, the growth of yeast was partly inhibited and a higher algal concentration was obtained compared to that of co-culture with free yeast. A pure algal suspension with a concentration of 2.08 g/L was obtained in this system. In addition, the immobilized yeast beads were repeatedly used for at least three times. This strategy can be widely used in the heterotrophic culture of microalgae using sucrose-riched wastes and may effectively decrease the cost of carbon source in the heterotrophic cultivation of microalgae.
Acknowledgments This work was financially supported by the Natural Science Foundation of Jiangsu Province 14
(No. BK20170495), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (17KJB240003), and
Postdoctoral Science Foundation of China (No.
2016M591933).
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18
Wang et al., Table 1
Table 1 Growth kinetic parameters of the algal cells in the co-culture process with different number of IYB. Number
of Final biomass
IYB (/100 mL)
(g/L)
Biomass
Maximum
Doubling time
productivity
specific growth (days)
(g/L/day)
rate µmax (day-1)
30
0.90 ± 0.15, a
0.15 ± 0.03, a
0.73 ± 0.03, a
0.95 ± 0.03, a
60
1.46 ± 0.04, b
0.24 ± 0.01, b
0.84 ± 0.02, b
0.83 ± 0.02, b
90
1.88 ± 0.04, c
0.31 ± 0.01, c
0.93 ± 0.02, c
0.75 ± 0.03, c
120
1.96 ± 0.07, c, d
0.32 ± 0.01, c, d
0.94 ± 0.02, c
0.74 ± 0.01, c
150
2.08 ± 0.03, d
0.34 ± 0.01, d
1.01 ± 0.03, d
0.69 ± 0.03, d
Values in a column with different letters are significantly different (P < 0.05).
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Wang et al., Figure captions
Fig. 1. Comparison of biomass (A), pH (B), and sucrase activity (C) in the culture of immobilized and free S. cerevisiae. (I: Immobilized culture; F: Free culture)
Fig. 2. The growth curves of C. pyrenoidosa (A) and the variation of pH (B) in the co-culture process with different numbers of IYB (in 100 mL culture).
Fig. 3. The growth curves of S. cerevisiae in the co-culture process with different numbers of IYB (in 100 mL culture).
Fig. 4. The growth curve of C. pyrenoidosa co-cultured with the re-used IYB (90 beads in 100 mL culture).
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Wang et al., Fig. 1
21
22
Wang et al., Fig. 2
A
B
23
Wang et al., Fig. 3
24
Wang et al., Fig. 4
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Research Highlights A new co-culture system was developed by co-culture of C. pyrenoidosa and immobilized yeast. Alga could effectively utilize sucrose via the hydrolysis of sucrose by immobilized yeast.
Pure algal suspension with a concentration of 2.08 g/L was obtained as the IYB can be easily separated by filtration.
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