Main Article Content
The effect of three nitrogen (N) sources in the nutrient medium – sodium nitrate (NaNO3), urea (CO(NH2)2), and ammonium chloride (NH4Cl) – on the morphological and physiological characteristics of the green microalga Chromochloris (Chlorella) zofingiensis, a potential commercial producer of lipids and of a ketocarotenoid astaxanthin, was studied. The alga was batch-cultivated in glass conical flasks from starting cell density (n) around 2.3·106 per mL and dry weight (DW) content of 0.06 g·L−1 in all variants at 120 μmol·m−2·s−1 PAR, +20…+21 °C, and air bubbling at a rate of 0.3 L·min−1·L−1. The concentration of nitrogen sources (as elemental N) in the modified BBM nutrient was 8.83 mmol·L−1, the cultivation duration was 17 days. The dynamics of n and cell volumes, DW content, chlorophylls a and b (Chla and Chlb), total carotenoids (Car), and lipids (Lip) in the cultures, concentration of N sources in the nutrient medium and its pH were recorded. It was shown that the growth rate, size distribution of the cell populations and the biomass chemical composition depended significantly on the nitrogen source in the nutrient medium. Using NH4Cl as N source caused on the second day growth inhibition, cell swelling, aggregation, and discoloration and, by the seventh day, culture crash. The cells of C. zofingiensis took up NaNO3 and CO(NH2)2 from the medium at a similar rate (0.626 and 0.631 mmol N·L−1·day−1, respectively), but the growth of the culture fed with CO(NH2)2 lagged, its cell volume and Chla, Chlb, and total Car contents declined profoundly. The average dry matter productivity (PDW) in the culture grown on CO(NH2)2 [(0.086 ± 0.004) g·L−1·day−1] was 32.6 % lower than in the culture grown on NaNO3 [(0.114 ± 0.005) g·L−1·day−1]. At the same time, lipid productivity (PLip) of the urea-fed culture was comparable with that of the nitrate-fed culture (PLip = 28 and 26 mg·L−1·day−1, respectively). The lipid DW percentage of the former exceeded significantly that of the nitrate-fed culture (31.6 % vs 23.1 %, respectively). From the standpoint of profitability, the lag in biomass accumulation recorded in the urea-fed culture on PDW is not critical since it is compensated by lowering the cost of nitrogen source for the nutrient medium (approximately by 230 %) and a higher biomass lipid content. C. zofingiensis grown in media with urea as the only N source deserves further investigation.
Minyuk G. S., Chelebieva E. S., Chubchikova I. N., Dantsyuk N. V., Drobetskaya I. V., Sakhon E. G., Chivkunova O. B., Chekanov K. A., Lobakova E. S., Sidorov R. A., Solovchenko A. E. pH and CO2 effects on Coelastrella (Scotiellopsis) rubescens growth and metabolism. Russian Journal of Plant Physiology, 2016, vol. 63, no. 4, pp. 566–574. https://doi.org/10.1134/S1021443716040105
Sapozhnikov V. V. Metody gidrokhimicheskikh issledovanii osnovnykh biogennykh elementov. Moscow: VNIRO, 1988, 119 p. (in Russ.)
Terent'yeva N. V., Drobetskaya I. V., Chubchikova I. N., Minyuk G. S. Effect of light illumination on physiological and biochemical characteristics of green microalga Haematococcus pluvialis Flotow (Chlamydomonadales). Ekologiya morya, 2008, iss. 75, pp. 82–88. (in Russ.)
Arumugam M., Agarwal A., Arya M. C., Ahmed Z. Influence of nitrogen sources on biomass productivity of microalgae Scenedesmus bijugatus. Bioresource Technology, 2013, vol. 131, pp. 246–249. https://doi.org/10.1016/j.biortech.2012.12.159
Becker E. W. Microalgae: Biotechnology and Microbiology. Cambridge: Cambridge University Press, 1994, pp. 196–250. (Cambridge Studies in Biotechnology ; vol. 10).
Bekheet I. A., Syrett P. J. Urea-degrading enzymes in algae. British Phycological Journal, 1977, vol. 12, iss. 2, pp. 137–143. https://doi.org/10.1080/00071617700650151
Bligh E. G., Dyer W. J. A rapid method for total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 1959, vol. 37, no. 8, pp. 911–917. https://doi.org/10.1139/o59-099
Bold H. C. The cultivation of algae. Botanical Review, 1942, vol. 8, iss. 2, pp. 69–138. https://doi.org/10.1007/BF02879474
Chelebieva E. S., Dantsyuk N. V., Chekanov K. A., Chubchikova I. N., Drobetskaya I. V., Minyuk G. S., Lobakova E. S., Solovchenko A. E. Identification and morphological-physiological characterization of astaxanthin producer strains of Haematococcus pluvialis from the Black Sea region. Applied Biochemistry and Microbiology, 2018, vol. 54, no. 6, pp. 639–648. https://doi.org/10.1134/s0003683818060078
Chen J., Liu L., Wei D. Enhanced production of astaxanthin by Chromochloris zofingiensis in a microplate-based culture system under high light irradiation. Bioresource Technology, 2017, vol. 245, pp. 518–529. https://doi.org/10.1016/j.biortech.2017.08.102
El-Sayed A. B., Abdel-Maguide A. A. Growth response of Chlorella vulgaris to acetate carbon and nitrogen forms. Nature and Science, 2011, vol. 9, no. 9, pp. 53–58.
Eustance E., Gardner R. D., Moll K. M., Menicucci J., Gerlach R., Peyton B. M. Growth, nitrogen utilization and biodiesel potential for two chlorophytes grown on ammonium, nitrate or urea. Journal of Applied Phycology, 2013, vol. 25, no. 6, pp. 1663–1677. https://doi.org/10.1007/s10811-013-0008-5
Feng P., Deng Z., Fan L., Hu Z. Lipid accumulation and growth characteristics of Chlorella zofingiensis under different nitrate and phosphate concentrations. Journal of Bioscience and Bioengineering, 2012, vol. 114, no. 4, pp. 405–410. https://doi.org/10.1016/j.jbiosc.2012.05.007
Fučíková K., Lewis L. A. Intersection of Chlorella, Muriella and Bracteacoccus: Resurrecting the genus Chromochloris Kol et Chodat (Chlorophyceae, Chlorophyta). Fottea, 2012, vol. 12, iss. 1, pp. 83–93. https://doi.org/10.5507/fot.2012.007
Hsieh C.-H., Wu W.-T. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresource Technology, 2009, vol. 100, iss. 17, pp. 3921–3926. https://doi.org/10.1016/j.biortech.2009.03.019
Huo S., Wang Z., Zhu S., Shu Q., Zhu L., Qin L., Zhou W., Feng P., Zhu F., Yuan Z., Dong R. Biomass accumulation of Chlorella zofingiensis G1 cultures grown outdoors in photobioreactors. Frontiers in Energy Research, 2018, vol. 6, art. 49. (8 p.). https://doi.org/10.3389/fenrg.2018.00049
Ip P. F., Chen F. Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark. Process Biochemistry, 2005, vol. 40, pp. 733–738. https://doi.org/10.1016/j.procbio.2004.01.039
Kim D-Y., Vijayan D., Praveenkumar R., Han J.-I., Lee K., Park J.-Y., Chang W.-S., Lee J.-S., Oh Y.-K. Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus. Bioresource Technology, 2016, vol. 199, pp. 300–310. https://doi.org/10.1016/j.biortech.2015.08.107
Lemoine Y., Schoefs B. Secondary ketocarotenoid astaxanthin biosynthesis in algae: A multifunctional response to stress. Photosynthesis Research, 2010, vol. 106, pp. 55–177. https://doi.org/10.1007/s11120-010-9583-3
Li Y., Horsman M., Wang B., Wu N., Lan C. Q. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Applied Microbiology and Biotechnology, 2008, vol. 81, pp. 629–636. https://doi.org/10.1007/s0253-008-1681-1
Li Y., Huang J., Sandmann G., Chen F. High-light and sodium chloride stress differentially regulate the biosynthesis of astaxanthin in Chlorella zofingiensis (Chlorophyceae). Journal of Phycology, 2009, vol. 5, pp. 635–641. https://doi.org/10.1111/j.1529-8817.2009.00689.x
Li T., Zheng Y., Yu L., Chen S. High productivity cultivation of a heat-resistant microalga Chlorella sorokiniana for biofuel production. Bioresource Technology, 2013, vol. 131, pp. 60–67. https://doi.org/10.1016/j.biortech.2012.11.121
Liu J., Mao X., Zhou W., Guarnieri M. T. Simultaneous production of triacylglycerol and high-value carotenoids by the astaxanthin-producing oleaginous green microalga Chlorella zofingiensis. Bioresource Technology, 2016, vol. 214, pp. 319–327. https://doi.org/10.1016/j.biortech.2016.04.112
Liu J., Sun Z., Gerken H., Liu Z., Jiang Y., Chen F. Chlorella zofingiensis as an alternative microalgal producer of astaxanthin: Biology and industrial potential. Marine Drugs, 2014, vol. 12, pp. 3487–3515. https://doi.org/10.3390/md12063487
Markou G., Monlau F. Nutrient recycling for sustainable production of algal biofuels. In: Biomass, Biofuels and Biochemicals: Biofuels from Algae. 2nd ed. / Pandey A., Chang J.-S., Soccol C. R., Lee D.-J., Chisti Y. (Eds). Elsevier, 2019, chapt. 6, pp. 109–133. https://doi.org/10.1016/B978-0-444-64192-2.00006-8
Park J.-J., Wang H., Gargouri M., Deshpande R. R., Skepper J. N., Holguin F. O., Juergens M., Shachar-Hill Y., Hicks L. M., Gang D. R. The response of Chlamydomonas reinhardtii to nitrogen deprivation: A systems biology analysis. The Plant Journal, 2015, vol. 81, iss. 4, pp. 611–624. https://doi.org/10.1111/tpj.12747
Podevin M., De Francisci D., Holdt S. L., Angelidaki I. Effect of nitrogen source and acclimatization on specific growth rates of microalgae determined by a high-throughput in vivo microplate autofluorescence method. Journal of Applied Phycology, 2015, vol. 27, iss. 4, pp. 1415–1423. https://doi.org/10.1007/s10811-014-0468-2
Ramanna L., Guldhe A., Rawat I., Bux F. The optimization of biomass and lipid yields of Chlorella sorokiniana when using wastewater supplemented with different nitrogen sources. Bioresource Technology, 2014, vol. 168, pp. 127–135. https://doi.org/10.1016/j.biortech.2014.03.064
Richardson J. W., Johnson M. D., Outlaw J. L. Economic comparison of open pond raceways to photo bio-reactors for profitable production of algae for transportation fuels in the Southwest. Algal Research, 2012, vol. 1, pp. 93–100. https://doi.org/10.1016/j.algal.2012.04.001
Sanz-Luque E., Chamizo-Ampudia A., Lamas A., Galvan A., Fernandez E. Understanding nitrate assimilation and its regulation in microalgae. Frontiers in Plant Science, 2015, vol. 6, pp. 899–916. https://doi.org/10.3389/fpls.2015.00899
Dhup S., Kannan D. C., Dhawan V. Understanding urea assimilation and its effect on lipid production and fatty acid composition of Scenedesmus sp. SOJ Biochemistry, 2016, vol. 2, no. 1, pp. 1–7. http://dx.doi.org/10.15226/2376-4589/2/1/00112
Shah M. M. R., Liang Y., Cheng J. J., Daroch M. Astaxanthin-producing green microalga Haematococcus pluvialis: from single cell to high value commercial products. Frontiers in Plant Science, 2016, vol. 7, art. 531. (28 p.) https://doi.org/10.3389/fpls.2016.00531
Shi X.-M., Zhang X.-W., Chen F. Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources. Enzyme and Microbial Technology, 2000, vol. 27, iss. 3–5, pp. 312–318. https://doi.org/10.1016/s0141-0229(00)00208-8
Smith R. V., Foy R. H. Improved hydrogen ion buffering of media for the culture of freshwater algae. British Phycological Journal, 1974, vol. 9, no. 3, pp. 239–245. https://doi.org/10.1080/00071617400650271
Sun N., Wang Y., Li Y. T., Huang J. C., Chen F. Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta). Process Biochemistry, 2008, vol. 43, no. 11, pp. 1288–1292. https://doi.org/10.1016/j.procbio.2008.07.014
Vonshak A. Laboratory techniques for cultivation of microalga. In: Handbook of Microalgal Mass Culture / A. Richmond (Ed.). Boca Raton ; London ; New York: CRC Press ; Taylor & Francis Group, 1986, pp. 117–145.
Wellburn A. R. The spectral determination of chlorophyll a and b, as well as total carotenoids, using various solvents with spectro-photometers of different resolution. Journal of Plant Physiology, 1994, vol. 144, pp. 307–313. https://doi.org/10.1016/S0176-1617(11)81192-2
Wijanarko A. Effect of the presence of substituted urea and also ammonia as nitrogen source in cultivied medium on Chlorella's lipid content. In: Progress in Biomass and Bioenergy Production / S. Shaukat (Ed.). IntechOpen, 2011, pp. 273–282. https://doi.org/10.5772/19358
Wood A. M., Everroad R. C., Wingard L. M. Measuring growth rates in microalgal cultures. In: Algal Culturing Techniques / R. A. Anderson (Ed.). Burlington: Elsevier Academic Press, 2005, pp. 269–288.