Bioresource Technology 173 (2014) 52–58

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Effects of various organic carbon sources on the growth and biochemical composition of Chlorella pyrenoidosa Weiguo Zhang, Peiliang Zhang, Hao Sun, Maozhen Chen, Shan Lu, Pengfu Li ⇑ State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China

h i g h l i g h t s  Addition of sugars and starch increased the growth of C. pyrenoidosa.  Monosaccharides showed stronger stimulative effects on the algal growth.  Addition of monosaccharides significantly affected the biochemical composition.  Disaccharides and starch did not significantly affect the biochemical composition.

a r t i c l e

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Article history: Received 20 July 2014 Received in revised form 12 September 2014 Accepted 17 September 2014 Available online 28 September 2014 Keywords: Microalgae Mixotrophic growth Sugar Starch Biochemical composition

a b s t r a c t The aim of this study was to investigate the effects of various organic carbon sources (glucose, galactose, fructose, sucrose, maltose, lactose and starch) on the growth and biochemical composition of Chlorella pyrenoidosa. Monosaccharides were found to exert stronger stimulative effects on the algal growth than disaccharides and starch. After 10-day culture, addition of 0.5–5.0 g L1 glucose and galactose significantly reduced the cellular protein contents by 27.7–63.7% and 22.6–60.5%, respectively, and significantly increased the carbohydrate contents by 103.2–266.5% and 91.9–240.0%, respectively. However, addition of 0.5–5.0 g L1 disaccharides and starch did not significantly affect the contents of lipid, protein and carbohydrate. Similar to the normal nitrogen condition, the cellular biochemical composition was not significantly affected by addition of 3.0 g L1 disaccharides and starch under the low nitrogen condition. Finally, the significance of this work in the biotechnological application of mixotrophic cultivation of C. pyrenoidosa was further discussed. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae have been widely used as high-value animal feed or as healthy food for humans (Spolaore et al., 2006; Das et al., 2011). Moreover, microalgal biomass has great potential for biofuel feedstock production (Chisti, 2008; Posten and Schaub, 2009; Carioca, 2010). Currently, photoautotrophic culture is commonly used to produce microalgal biomass, but it has several disadvantages, including low biomass concentration and long cultivation period. Some reports have shown that microalgal culture under mixotrophic conditions can increase the biomass concentration and productivity because it overcomes the light limitation of pure photoautotrophic culture (Ceron Garcia et al., 2006; Garci et al., 2000; Sanchez et al., 2001; Ip et al., 2004).

⇑ Corresponding author. Tel.: +86 25 83686755; fax: +86 25 83592705. E-mail address: [email protected] (P. Li). http://dx.doi.org/10.1016/j.biortech.2014.09.084 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

Although mixotrophic cultivation of microalgae provides higher biomass than photoautotrophic conditions, the organic carbon source is essential for mixotrophic cultivation. Glucose is used as the organic carbon source in some studies (Cheirsilp and Torpee, 2012; Yeh and Chang, 2012). Moreover, integration of algal biomass production and wastewater treatment has been proposed to reduce the cost of organic carbon substrate in mixotrophic cultivation. Some wastewaters have been considered to be candidate organic carbon substrates for mixotrophic cultivation of microalgae, such as piggery wastewater (Kumar et al., 2010; Wang et al., 2012), industrial dairy waste (Abreu et al., 2012), fish processing wastewater (Riaño et al., 2011), olive mill wastewater (Sanchez et al., 2001) and soybean processing wastewater (Su et al., 2011). Therefore, it is necessary to investigate the effects of various organic nutrients on the growth and biochemical composition of microalgae in order to efficiently produce microalgal biomass by mixotrophic cultivation. However, only a little information is known on this topic in the literature.

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Due to its high protein content, Chlorella has been commercially cultivated at a large scale to produce healthy food for humans and animal nutritional supplements (Metting, 1996; Spolaore et al., 2006). Because some species of Chlorella have a high carbohydrate or lipid content, the biomass of these algae represents a promising feedstock for producing bioethanol or biodiesel (Yeh and Chang, 2012; Ho et al., 2013). Furthermore, Chlorella sp. are common microalgae used to treat wastewater because of their high tolerance to soluble organic compounds (de la Noue and Basseres, 1989; Metting, 1996). Addition of glucose to medium has been reported to affect the lipid contents of Chlorella sp., Chlorella sorokiniana and Chlorella vulgaris ESP-31 compared with autotrophically grown cells (Wan et al., 2011; Cheirsilp and Torpee, 2012; Yeh and Chang, 2012). However, it is urgent to study the effects of more organic nutrients on the biochemical composition of more Chlorella strains in order to deeply evaluate the biotechnological potential of mixotrophic cultivation. The aim of this study was to investigate the effects of different organic carbon source (glucose, galactose, fructose, sucrose, maltose, lactose, and starch) on the growth and biochemical composition of Chlorella pyrenoidosa, and to explore the significance of this work in the biotechnological application of mixotrophic cultivation. 2. Methods

Thermo Nicolet, USA). The data were processed with OMNIC 6.0 software. The spectrum baseline was corrected by a rubber-band method using 64 baseline points with exclusion of CO2 bands. The characteristic peak areas of lipid (AL), protein (AP) and carbohydrate (AC) were calculated by integration. The weights (mg) of lipid (WL), protein (WP) and carbohydrate (WC) were calculated according to the formulas (1)–(3) (Pistorius et al., 2009):

AL ¼ 2:30 þ 78:96  W L

ð1Þ

AP ¼ 0:27 þ 12:72  W P

ð2Þ

AC ¼ 0:07 þ 2:05  W C

ð3Þ

Assuming that the algal cells consisted of only lipid, protein and carbohydrate, the contents (%) of lipid (CL), protein (CP) and carbohydrate (CC) were calculated with the following formulas (Feng et al., 2013):

CL ¼

WL  100 WL þ WP þ WC

ð4Þ

CP ¼

WP  100 WL þ WP þ WC

ð5Þ

CC ¼

WC  100 WL þ WP þ WC

ð6Þ

2.1. Algal strain, medium and culture conditions The green alga C. pyrenoidosa was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. The algal cells were axenically grown at 28 ± 0.5 °C under a 16/8-h light/dark cycle with exposure to 45 lE m2 s1 provided by cool-white fluorescent lights. The exponentially growing cells were harvested and inoculated into the BBM medium containing compositions as follows (per liter): 0.25 g NaNO3, 0.075 g K2HPO4, 0.075 g MgSO47H2O, 0.025 g CaCl22H2O, 0.175 g KH2PO4, 0.025 g NaCl, 0.75 mg Na2EDTA, 0.097 mg FeCl36H2O, 1 mg vitamin B1, 0.25 lg biotin, 0.15 lg vitamin B12, 0.041 mg MnCl24H2O, 0.005 mg ZnCl27H2O, 0.004 mg Na2MoO42H2O and 0.002 mg CoCl26H2O. For mixotrophic growth, 0.5–12 g L1 sugar (glucose, galactose, fructose, sucrose, maltose or lactose) or starch was added to the culture medium. For the low nitrogen condition, the NaNO3 concentration in the medium was reduced to 5% of the normal concentration. For nitrogen deficiency, the NaNO3 concentration in the medium was zero. The initial inoculation density was 2  106 cells mL1. The flasks were manually shaken three times daily. All the experiments were conducted in triplicate.

2.4. Statistical analysis Data were presented as means ± standard error of the mean, and statistical significances were assessed by analysis of variance (ANOVA) followed by Fisher’s post hoc test using the IBM SPSS Statistics 21.0 program (IBM, Armonk, New York, USA). A P value of less than 0.05 was considered as statistically significant. 3. Results and discussion 3.1. Effects of sugars and starch on the growth of C. pyrenoidosa Addition of glucose is known to increase the growth of green algae C. vulgaris (Yeh and Chang, 2012), Chlorella sp. (Cheirsilp and Torpee, 2012) and Botryococcus braunii (Zhang et al., 2011) compared with autotrophic growth. Addition of maltose, sucrose, lactose and starch can also increase the growth of B. braunii (Zhang et al., 2011). Similarly, addition of sugars and starch increased the growth of C. pyrenoidosa compared with control

2.2. Determination of growth

2.3. Determination of lipid, protein and carbohydrate contents The biochemical composition of algae was determined by Fourier transform infrared (FTIR) spectrometer (Pistorius et al., 2009). The FTIR analysis was performed as previously described by Feng et al. (2013). Briefly, the algal culture was centrifuged at 8000g for 10 min. The cell pellets were washed twice with deionized water and then resuspended in deionized water at a concentration of approximately 1.0 mg mL1 (dry weight). A total of 200 lL suspension was dropped on a KRS-5 window (30  5 mm) and dried at 40 °C in the vacuum drying oven. The transmittance spectra were collected between 400 and 4000 cm1 at a resolution of 4 cm1 with 32 scans on a FTIR spectrometer (NEXUS 870,

Glucose Galactose Fructose Sucrose Maltose Lactose Starch

10 Cell density (×107 cells mL-1)

The algal growth was determined by monitoring the cell density with haemocytometer under a microscope.

8 6 4 2 0 0

2

4

6

8

10

12

Sugar and starch concentration (g L-1) Fig. 1. Effects of sugars and starch on cell densities of C. pyrenoidosa after 10-day culture.

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respectively, while 0.5–1.0 and 5.0 g L1 glucose and galactose, and 0.5–5.0 g L1 fructose, disaccharide and starch did not significantly affect the lipid content. Several reports have shown the effect of glucose on lipid content in Chlorella. The lipid content of mixotrophic cells in C. sorokiniana with addition of 5 g L1 glucose is higher than that of autotrophic cells (Wan et al., 2011). Addition of 10 g L1 glucose increases the lipid content of C. vulgaris ESP-31 (Yeh and Chang, 2012), while the lipid content in Chlorella sp. is not affected by addition of 2 g L1 glucose (Cheirsilp and Torpee, 2012). These data suggested that the effect of glucose on lipid content in Chlorella might be strain specific and in a glucose concentrationdependant manner. More obvious change of the cellular protein content induced by addition of monosaccharides was observed compared with the cellular lipid content (Fig. 2). Addition of 0.5–5.0 g L1 glucose and galactose significantly reduced the cellular protein contents by 27.7–63.7% and 22.6–60.5%, respectively; and the inhibitory effects of these two sugars on the protein content were likely to be in a sugar concentration-dependant manner. Moreover, addition of 3.0 and 5.0 g L1 fructose resulted in a significant decrease of protein content by 18.0% and 20.8%, respectively, although it was not significantly affected by addition of 0.5 or 1.0 g L1 fructose. Consistent with the lipid content, the protein content was not significantly affected by addition of 0.5–5.0 g L1 disaccharide and starch. Corresponded to the decrease of protein content, addition of 0.5–5.0 g L1 glucose and galactose significantly increased the cellular carbohydrate contents by 103.2–266.5% and 91.9–240.0%, respectively; and addition of 3.0 and 5.0 g L1 fructose also led to a significant increase of carbohydrate content by 85.8% and 83.8%, respectively (Fig. 2). Similar to lipid and protein contents, the carbohydrate content was not significantly affected by addition of 0.5–5.0 g L1 disaccharides and starch. With the increase of monosaccharide concentration from 0 to 5.0 g L1, the cell density and the carbohydrate content increased, and the protein content

(without addition of sugars or starch) in this study (Figs. 1 and S1–S3). Glucose is the optimal carbon source for the mixotrophic growth of B. braunii among the six tested carbon sources (glucose, maltose, sucrose, lactose, starch and glycerol) (Zhang et al., 2011). Similarly, the results of this study showed that monosaccharides exerted stronger stimulative effects on the growth of C. pyrenoidosa than disaccharides and starch (Fig. 1). Among the tested monosaccharides, glucose was the best carbon source for the mixotrophic growth of C. pyrenoidosa, followed by galactose and fructose. Among the tested disaccharides, maltose was the best carbon source for the mixotrophic growth, followed by sucrose and lactose. These data showed different stimulative effects on the growth of C. pyrenoidosa among starch and different sugars. The monosaccharide transporter, which transports monosaccharides into the cell, has been identified in Chlorella (Stadler et al., 1995). However, the mechanism of disaccharide utilization in Chlorella and other green algae remains unknown. It might be hypothesized that the monosaccharides were generated through hydrolysis of disaccharides by a specific enzyme, or the disaccharides were directly transported across the plasmalemma by disaccharide carrier. Because the activity of extracellular amylase has been found in some Chlorella species (Kessler, 1978), the monosaccharides or oligosaccharides could be produced through hydrolysis of starch. Therefore, different mechanisms of utilizing different organic carbon sources (monosaccharide, disaccharide and starch) could result in a faster growth of C. pyrenoidosa with addition of monosaccharides. 3.2. Effects of sugars and starch on the biochemical composition of C. pyrenoidosa

Lipid content (%)

Fig. 2 shows the effects of sugars and starch on the cellular lipid content of C. pyrenoidosa. Addition of 3.0 g L1 glucose and galactose significantly reduced the lipid content by 27.5% and 27.9%,

Control Glucose Galactose Fructose Sucrose Maltose Lactose Starch

15 * * 10 5

Carbohydrate content (%) Protein content (%)

0 60 *

*

*

*

* *

40

* *

*

*

20 0

* *

60

*

*

*

* * *

40

*

*

20 0

0.0

0.5

1.0

3.0

5.0

Sugar and starch concentration (g L-1) Fig. 2. Effects of sugars and starch on lipid, protein and carbohydrate contents of C. pyrenoidosa after 10-day culture. Control, without addition of sugars and starch. The asterisks (*) at the top of bars indicate P < 0.05 compared to the control.

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Carbohydrate content (%) Protein content (%)

Lipid content (%)

decreased (Figs. 1 and 2). This suggested that utilization of monosaccharides resulted in the fast growth of the alga. However, with the higher concentration of monosaccharides transported from the medium into algal cells, the faster synthesis of cellular carbohydrate occurred, thereby reducing the cellular protein content. To further investigate the effects of various organic carbon sources on biochemical composition during algal growth, the lipid, protein and carbohydrate contents of C. pyrenoidosa with addition of 3.0 g L1 sugars and starch were analyzed after incubation of 3, 6 and 10 days (Fig. 3). The variation patterns of biochemical composition affected by addition of different sugars and starch were similar on day 3, day 6 and day 10. Addition of glucose and galactose significantly reduced the lipid and protein contents during incubation of from day 3 to day 10. Furthermore, addition of monosaccharides significantly increased the carbohydrate content. The effects of disaccharides and starch on the biochemical composition were not as remarkable as three monosaccharides during growth. The above data of this study indicated that the biochemical compositions of mixotrophic cells with addition of three disaccharides and starch were similar to those of autotrophic cells, while addition of three tested monosaccharides led to more remarkable changes in the biochemical compositions. Addition of the monosaccharides resulted in a higher increase in the growth of C. pyrenoidosa, but its protein content was significantly decreased. Addition of sucrose, maltose and lactose at 0.5–5.0 g L1 increased the cell densities by 65.3–98.9%, 70.6–135.7% and 55.3–59.6%, respectively, and addition of starch at 1.0–5.0 g L1 increased the cell densities by 43.8–160.9% without the change in the biochemical composition (Figs. 1 and 2). The high protein content is important for Chlorella biomass that is used as healthy food for humans and feed for fish and other livestock (Metting, 1996). Therefore, disaccharides and starch may have a biotechnological potential to increase the productivity of C. pyrenoidosa biomass while maintaining a high protein content in its biomass.

15 10

* *

*

3.3. Effects of sugars and starch on the biochemical composition of C. pyrenoidosa under the low nitrogen condition and nitrogen deficiency Fig. 4 shows that the growth of C. pyrenoidosa was inhibited under the low nitrogen condition and nitrogen deficiency. Addition of organic carbon source had stronger effects on the algal growth under the normal nitrogen condition compared with nitrogen stress conditions. Under the normal nitrogen condition and the low nitrogen condition, addition of three monosaccharides significantly increased the cell densities by 192.2–471.5% and 64.0– 116.9%, respectively, and addition of glucose significantly increased the cell density by 86.8% under nitrogen deficiency. Addition of disaccharides significantly increased the cell densities by 59.6–126.9% under the normal nitrogen condition, whereas had no significant effect on the algal growth under nitrogen stress conditions. Under the normal nitrogen condition, the low nitrogen condition and nitrogen deficiency, addition of starch significantly increased the cell densities by 122.0%, 52.1% and 52.6%, respectively. It has been reported that nitrogen concentration in the medium can affect the biochemical composition of algae. When autotrophically grown in low-nitrogen medium, the lipid content is increased and the protein content is decreased in five Chlorella strains, and the carbohydrate content is increased in C. vulgaris and decreased in the other four strains, including C. emersonii, C. protothecoides, C. sorokiniana and C. minutissima (Illman et al., 2000). In this study, low nitrogen condition led to similar biochemical change patterns in autotrophic cells and in mixotrophic cells grown in the presence of three disaccharides and starch (Fig. 5). The protein content of these cells significantly decreased from 64.6–66.8% to 33.3–43.9%, and the carbohydrate content significantly increased from 18.3–20.2% to 42.6–52.8% (P < 0.05). However, the biochemical change induced by low nitrogen condition in mixotrophic cells in the presence of monosaccharides was different from that in autotrophic cells. In mixotrophic cells with

Control Glucose Galactose Fructose Sucrose Maltose Lactose Starch

* *

*

* *

* *

5 0 75

* * * *

*

60

*

*

45 *

30 15 0 75 60

*

*

*

* *

*

*

* *

*

45 30

*

*

*

*

15 0

*

3

6

10

Culture time (day) Fig. 3. Lipid, protein and carbohydrate contents of C. pyrenoidosa during growth with addition of 3.0 g L1 sugars and starch. Control, without addition of sugars and starch. The asterisks (*) at the top of the bars on day 3, day 6 and day 10 indicate P < 0.05 compared to their respective controls.

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*

10

Control Glucose Galactose Fructose Sucrose Maltose Lactose Starch

Cell density (×107 cells mL-1)

8

* 6

* *

4

*

* * * *

2

*

0

Low-N

Normal medium

*

*

*

N-deficiency

Nitrogen nutrient condition

Lipid content (%)

Fig. 4. Cell densities of C. pyrenoidosa after 10-day culture under the normal nitrogen condition, the low nitrogen condition and nitrogen deficiency with addition of 3.0 g L1 sugars and starch. Control, without addition of sugars and starch. Low-N, 5% nitrogen concentration. N-deficiency, no nitrogen. The asterisks (*) at the top of the bars in the normal medium, under the low nitrogen condition and under nitrogen deficiency indicate P < 0.05 compared to their respective controls.

Control Glucose Galactose Fructose Sucrose Maltose Lactose Starch

15 *

*

10 5

Carbohydrate content (%)

Protein content (%)

0 *

60 40 *

* *

20 0 80

*

*

*

*

*

*

* *

*

60 40

*

20 0 Normal medium

Low-N

N-deficiency

Nitrogen nutrient condition Fig. 5. Lipid, protein and carbohydrate contents of C. pyrenoidosa after 10-day culture under the normal nitrogen condition, the low nitrogen condition and nitrogen deficiency with addition of 3.0 g L1 sugars and starch. Control, without addition of sugars and starch. Low-N, 5% nitrogen concentration. N-deficiency, no nitrogen. The asterisks (*) at the top of the bars in the normal medium, under the low nitrogen condition and under nitrogen deficiency indicate P < 0.05 compared to their respective controls.

glucose, the protein content significantly decreased from 25.3% to 9.4%, and the lipid content significantly increased from 11.5% to 14.7% (P < 0.05). In mixotrophic cells with galactose, the protein content significantly decreased from 25.9% to 9.9%, while the carbohydrate content significantly increased from 62.9% to 76.3%

(P < 0.05). In mixotrophic cells with fructose, the protein content significantly decreased from 53.8% to 16.5%, and the carbohydrate content significantly increased from 34.4% to 69.7% (P < 0.05). Consistent with normal nitrogen condition, no significant difference was observed in terms of lipid, protein and carbohydrate contents

W. Zhang et al. / Bioresource Technology 173 (2014) 52–58

in mixotrophic cells with addition of three disaccharides and starch compared with autotrophic cells under the low nitrogen condition. Addition of monosaccharides significantly decreased the cellular protein content by 50.4–71.8%, and significantly increased the carbohydrate content by 32.2–44.5% under the low nitrogen condition, but induced no significant change in the lipid content. The effects of various organic carbon sources on biochemical composition were further assessed under nitrogen deficiency (Fig. 5). Nitrogen deficiency resulted in a significant decrease of lipid content in autotrophic cells of C. pyrenoidosa (P < 0.05). On the contrary, the lipid content in autotrophic cells of Chlorella zofingiensis is increased under the nitrogen deficiency (Zhu et al., 2014), suggesting that the effect of nitrogen deficiency on the lipid content was species-specific. In the mixotrophic cells with addition of glucose, the lipid content was significantly increased under the nitrogen deficiency compared with the normal nitrogen condition (P < 0.05) (Fig. 5). Nitrogen deficiency did not lead to any significant change of lipid content in the mixotrophic cells with sucrose and starch (P > 0.05). The protein content was all significantly decreased in mixotrophic cells with addition of glucose, sucrose and starch as well as in autotrophic cells under the nitrogen deficiency (P < 0.05). Because nitrogen is required for protein biosynthesis, the protein content was decreased under nitrogen deficiency as well as under the low nitrogen condition. Nitrogen deficiency triggers carbohydrate accumulation in C. vulgaris FSP-E (Ho et al., 2013). In this study, a significant increase of carbohydrate content was induced by nitrogen deficiency in mixotrophic cells with addition of sucrose and starch as well as in autotrophic cells (P < 0.05), while no significant change of carbohydrate content was observed in mixotrophic cells with addition of glucose (P > 0.05) (Fig. 5). Moreover, there was no significant difference in lipid, protein and carbohydrate contents between autotrophic cells and mixotrophic cells with addition of sucrose and starch under the nitrogen deficiency, which was consistent with the results under the normal nitrogen condition and low nitrogen condition. The results under different nitrogen conditions demonstrated that the biochemical composition of C. pyrenoidosa was significantly affected by addition of monosaccharides, while it was not significantly affected by addition of disaccharides and starch. Thus, the effects of monosaccharides on cellular biochemical composition could be different from those of disaccharides and starch when this alga was mixotrophically cultivated for biotechnological application. For example, when the alga was used to treat wastewater, various sugars and starch in the wastewater would have different effects on the cellular biochemical composition. The nitrogen stress strategies are commonly used to stimulate the accumulation of lipid or carbohydrate in Chlorella and other microalgae for biofuel production (Illman et al., 2000; Ho et al., 2013). Both nitrogen stress under autotrophic condition and addition of monosaccharides sharply increased the carbohydrate content of C. pyrenoidosa, and combination of nitrogen stress and addition of monosaccharides further enhanced the carbohydrate accumulation (Fig. 5). Since lipid contents are sharply increased by nitrogen stress under autotrophic condition in some other Chlorella strains, such as C. zofingiensis, C. emersonii and C. minutissima (Illman et al., 2000; Zhu et al., 2014), it would be interesting to investigate the combined effects of different organic carbon sources and nitrogen stresses on their biochemical compositions (especially lipid contents). 4. Conclusions In this study, monosaccharides were found to impose greater stimulative effects on the growth of C. pyrenoidosa than disaccha-

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rides or starch. Under both normal nitrogen condition and nitrogen stress condition, the lipid, protein and carbohydrate contents were significantly affected by addition of monosaccharides, but they were not significantly affected by addition of disaccharides and starch. Thus, when this alga was mixotrophically cultivated for biotechnological applications, these various organic carbon sources could have different impacts on biochemical composition. Disaccharides and starch may have a potential to increase the algal productivity while maintaining a high protein content in the biomass. Acknowledgements The authors thank the anonymous reviewers for their valuable comments. This study was financially supported by Science and Technology Support Project of Jiangsu Province (BE2013669).

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Effects of various organic carbon sources on the growth and biochemical composition of Chlorella pyrenoidosa.

The aim of this study was to investigate the effects of various organic carbon sources (glucose, galactose, fructose, sucrose, maltose, lactose and st...
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