Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source.

TL;DR: Mixotrophic cultivation of C. vulgaris using the main dairy industry by-product could be considered a feasible alternative to reduce the costs of microalgal biomass production, since it does not require the addition of expensive carbohydrates to the culture medium.

About: This article is published in Bioresource Technology.The article was published on 2012-08-01 and is currently open access. It has received 320 citations till now. The article focuses on the topics: Chlorella vulgaris & Biomass.

Summary

1. Introduction

• Microalgae cultivation has been carried out throughout the world in order to produce animal feed or high-value added products, such as cosmetics, pharmaceuticals and health supplements (Das et al., 2011) .
• Nonetheless, this culture mode, known as photoautotrophic, presents several disadvantages including low cell densities and long cultivation periods.
• The cost of the organic carbon substrate is estimated to be about 80% of the total cost of the cultivation medium (Bhatnagar et al., 2011) .
• Cheese whey (CW), the liquid by-product remaining from the cheese manufacturing process constitutes a serious environmental problem of dairy industries due to its high organic matter content (Dragone et al., 2009) .

2.1. Microalgal strain and inoculum preparation

• The freshwater microalga C. vulgaris (strain P12) was used in all experiments.
• The microalgal inoculum was prepared according to previous studies (Fernandes et al., 2010) and conducted at 30 °C in 0.5-L glass photobioreactors under photoautotrophic conditions.

2.2. Media and culture conditions

• Four different cultivation conditions were carried out in duplicate (Table 1 ).
• Sweet CW powder was supplied by Lactogal (Porto, Portugal).
• After reaching the stationary growth phase, cells were collected and centrifuged at 8750Âg for 10 min, washed with distilled water and then freeze-dried for further biochemical characterization.
• The supernatant was also collected and frozen for subsequent sugar analyses.

2.3. Determination of microalgal cell concentration

• Cell concentration in the photobioreactor cultures was measured regularly by using an improved Neubauer hemocytometer.
• Biomass concentration was estimated by cell dry weight after centrifugation of the sample (8750Âg for 10 min), washing with distilled water and drying at 105 °C until constant weight.

2.6. Measurement of lipid and protein contents in microalgae

• Total lipids were determined by the classic Folch chloroformbased lipid extraction protocol.
• The protein content of microalgae was quantified according to the method of Lowry.

2.7. Measurement of chlorophylls and total carotenoids concentration

• Chlorophylls and carotenoids in C. vulgaris were extracted with methanol and spectrophotometrically determined as described by Dere et al. (1998) .
• Total pigment content was obtained by summing chlorophylls and carotenoids contents.

3.1. Growth parameters of microalgae cultivated under photoautotrophic and mixotrophic conditions

• Specific growth rate, final biomass concentration and biomass productivity of C. vulgaris cultivated under photoautotrophic and mixotrophic conditions were compared and summarized in Table 2 .
• These values were almost 3.5 times higher than those obtained when cells were grown in inorganic medium supplemented with non-hydrolyzed CW powder solution, and under photoautotrophic mode of nutrition.
• It can be observed in Table 2 that the highest values of X max (3.58 g/L) and P max (0.75 g/L d) achieved in the mixotrophic culture using hydrolyzed CW powder solution resulted in 2.9-and 7.5-fold increase respectively, when compared to the values obtained in the photoautotrophic culture.
• These results are in agreement with a previous study, which reported that mixotrophic C. vulgaris growth in glucose yielded higher biomass content and productivity than cells grown under photoautotrophic conditions (Kong et al., 2011) .
• It is worth mentioning that the organic substrate played an important role in promoting biomass accumulation of C. vulgaris during microalgae cultivation.

3.2. Consumption of glucose and galactose by C. vulgaris

• The above presented results demonstrated that the microalgal species used in this study was able to grow mixotrophically in the presence of glucose and galactose.
• It was found that glucose and galactose were consumed in larger quantities during microalgal growth in the presence of the hydrolyzed CW powder solution, in comparison to the culture supplemented with a mixture of pure sugars.
• Means in the same column followed by different letters represent significant differences (p < 0.05).
• * l max = Specific growth rate during exponential growth phase.
• Regardless of the media used, glucose was more efficiently assimilated than galactose by C. vulgaris cells grown under mixotrophic conditions.

3.3. Influence of nutritional modes on biochemical composition of C. vulgaris

• The lipid content and lipid productivity of C. vulgaris under different growth conditions were compared and depicted in Fig. 1 .
• On the other hand, the highest lipid productivity (253 mg/L d) was achieved when cells were grown in culture medium supplemented with hydrolyzed CW powder solution, due to the highest growth rate and cell density.
• Therefore, since the starch productivity was calculated by multiplying the biomass productivity by the starch content (w/w) in microalgae, no differences on the values of this parameter were observed for the cells cultivated under mixotrophic conditions using hydrolyzed CW powder solution, and a mixture of glucose and galactose as organic carbon sources.
• Cultivation of C. vulgaris P12 using hydrolyzed CW powder solution as organic carbon source led to the highest protein content (63.5%) and protein productivity (474 mg/L d).
• The higher content of chlorophylls obtained in the photoautotrophic culture when compared to mixotrophic cultures confirms such observation.

4. Conclusions

• When compared with the photoautotrophic control culture, mixotrophic microalgae grew faster, providing higher productivities of biomass, lipids, starch and proteins.
• Furthermore, microalgal biomass production and carbohydrate consumption were enhanced by supplementing the inorganic culture medium with hydrolyzed CW powder solution, than supplementing with a mixture of pure glucose and galactose, as a consequence of stimulatory effects arising from growth-promoting nutrients in CW.

Figures

Table 2 Growth parameters of C. vulgaris cultivated under photoautotrophic and mixotrophic conditions at 30 C.

Table 4 Total pigment (chlorophylls + carotenoids) content of C. vulgaris cultivated under photoautotrophic and mixotrophic conditions at 30 C.

Table 1 Different cultivation conditions of C. vulgaris and respective carbon sources.

Fig. 3. Effect of nutritional mode on protein content and protein productivity of C. vulgaris.

Fig. 2. Comparison of starch content and starch productivity of C. vulgaris grown under photoautotrophic and mixotrophic conditions.

Frequently asked questions

Q1. What are the contributions in "Mixotrophic cultivation of chlorella vulgaris using industrial dairy waste as organic carbon source" ?

In this paper, the growth parameters and biochemical composition of the green microalga Chlorella vulgaris cultivated under different mixotrophic conditions were determined and compared to those obtained from a photoautotrophic control culture. 

Q2. What is the effect of CW powder on the growth of C. vulgaris?

The stimulatory effect of hydrolyzed CW powder solution on biomass production is probably related to the presence of some nutrients in CW powder composition, such as phosphorous and calcium. 

Q3. What was the effect of the hydrolyzed CW powder on the growth of C.?

In particular, glucose was completely consumed and only 4% of the initial galactose concentration remained in the growth medium at the end of cultivation when hydrolyzed CW powder solution was used as carbon source. 

Q4. What is the effect of the supplementation of pure glucose and galactose on micro?

microalgal biomass production and carbohydrate consumption were enhanced by supplementing the inorganic culture medium with hydrolyzed CW powder solution, than supplementing with a mixture of pure glucose and galactose, as a consequence of stimulatory effects arising from growth-promoting nutrients in CW. 

Q5. What was the source of the mixotrophic growth of C. vulgaris?

The organic carbon sources used for mixotrophic cell growth were: a non-hydrolyzed CW powder solution, a mixture of pure glucose and galactose, and a hydrolyzed CW powder solution. 

Q6. What is the cost of the organic carbon substrate?

Despite mixotrophic cultivation of microalgae provides higher biomass and lipid productivities than cultivation under photoautotrophic conditions, the cost of the organic carbon substrate is estimated to be about 80% of the total cost of the cultivation medium (Bhatnagar et al., 2011). 

Q7. What was the effect of the hydrolyzed CW powder on the microalga?

It was found that glucose and galactose were consumed in larger quantities during microalgal growth in the presence of the hydrolyzed CW powder solution, in comparison to the culture supplemented with a mixture of pure sugars. 

Q8. What is the effect of mixotrophy on microalgae?

since the amount of energy dissipated is minimal, mixotrophy provides higher energetic efficiency than other cultivation modes (Lalucat et al., 1984). 

Q9. What is the effect of the photoautotrophic culture on microalgae?

When compared with the photoautotrophic control culture, mixotrophic microalgae grew faster, providing higher productivities of biomass, lipids, starch and proteins. 

Q10. What is the effect of CW powder on microalgal growth?

a higher microalgal biomass concentration than that found in their study could have been obtained by using acid CW powder due to the higher concentrations of calcium and phosphorous presented in that type of whey (Mavropoulou and Kosikowski, 1973). 

Q11. What is the effect of the CW powder on the lipid content of C. vulgaris?

Other authors (Liang et al., 2009) have also shown that the amount of lipids accumulated in C. vulgaris under photoautotrophic growth conditions may surpass that from mixotrophic growth. 

Q12. What is the effect of CW on microalgae?

As shown in Table 2, supplementation of the inorganic culture medium with hydrolyzed CW powder solution led to higher biomass concentration than supplementation with a mixture of glucose and galactose. 

Q13. What is the effect of low chlorophylls in mixotrophic cells?

On the other hand, Yan et al. (in press) reported that low chlorophyll content in mixotrophic cells decreases the dependence on light. 

Q14. What was the specific growth rate of the cells at the end of the exponential growth phase?

The specific growth rate (l, day 1) was calculated from the Eq. (1), where N1 and N2 were the concentration of cells at the beginning (t1) and at the end (t2) of the exponential growth phase, respectively.l ¼ ðln N2 ln N1Þ=ðt2 t1Þ ð1ÞBiomass productivity (Pmax, g/L d) during the culture period was calculated from the Eq. (2), where Xt was the biomass concentration (g/L) at the end of the exponential growth phase (tx) and X0 the initial biomass concentration (g/L) at t0 (day):Pmax ¼ ðXt X0Þ=ðtx t0Þ ð2ÞProductivity of starch, lipids and proteins at the end of cultivation were calculated from the Eq. (3), where Pcomponent was the productivity of starch, lipids or proteins, Pmax was the biomass productivity and Fcomponent was the mass fraction (w/w) of each component. 

Q15. How much lipid content was obtained in C. vulgaris cultivated under mixotrophic?

1. When compared with mixotrophic cultures, higher lipid content (42%) was obtained in photoautotrophic mode at the beginning of the stationary growth phase (approximately 190 h). 

Q16. How much glucose was consumed by C. vulgaris?

On the other hand, after nearly 90 h of cultivation, initial contents of glucose and galactose dropped 80.5% and 49.5%, respectively, in the culture supplemented with both sugars.