Abstract
A critical factor in implementing microalgal biofuels for mass production is the nutrient requirements. The current study investigated the fate of macro- and micronutrients and their availability in a sequential phototrophic-heterotrophic production process for the lipid rich microalga Auxenochlorella protothecoides. More than 99 % (by weight) of overall process nutrients were supplied during the initial photoautotrophic stage reflecting its significantly larger volume. Under photoautotrophic growth conditions only 9–35 % of supplied Mn, S, Fe, N, Mg, and Cu and less than 5 % of P, Mo, Co, B, Zn, and Ca were consumed by the algae. The rest of these nutrients remain in the spent growth media during the culture concentration-down from an 800 L phototrophic pond to a 5 L heterotrophic fermenter. In contrast, Zn, Mo, Mn, Mg, Ca, and N were exhausted (90–99 % removal) during the first 25 h of the heterotrophic growth stage. The depletion of these key nutrients may have ultimately limited the final biomass density and/or lipid productivity achieved. Approximately 10–20 % of the total supplied S, Mn, Fe, N, and Cu and 5 % of Ca and Zn were assimilated into algal biomass. Several elements including N, P, Mn, B, Cu, Ca, Mg, S, and Fe were released back into the liquid phase by anaerobic digestion (AD) of the residual biomass after lipid extraction. The nutrients recovered from the AD effluent and remaining in the spent medium should be recycled or their initial concentration to the phototrophic stage decreased to enhance process economics and sustainability for future commercialization of algal-derived biofuels.
Similar content being viewed by others
References
Aksu Z (2002) Determination of the equilibrium, kinetic and thermodynamic parameters of the batch biosorption of nickel(II) ions onto Chlorella vulgaris. Process Biochem 38(1):89–99, doi:10.1016/s0032-9592(02)00051-1
Ajanovic A (2011) Biofuels versus food production: does biofuels production increase food prices? Energy 36(4):2070–2076, doi:10.1016/j.energy.2010.05.019
Bligh EG, Dyer WJ (1959) A rapid method for total lipid extraction and purification. Can J Biochem Physiol 37:911–917
Borowitzka MA, Moheimani NR (2010) Sustainable biofuels from algae. Mitig Adapt Strat Gl 18(1):13–25, doi:10.1007/s11027-010-9271-9
Brown MR, Jeffrey SW, Volkman JK, Dunstan GA (1997) Nutritional properties of microalgae for mariculture. Aquaculture 151(1–4):315–331, doi:10.1016/s0044-8486(96)01501-3
Chen F (1996) High cell density culture of microalgae in heterotrophic growth. Trends Biotechnol 14(11):421–426, doi:10.1016/0167-7799(96)10060-3
Chen M, Tang H, Ma H, Holland TC, Ng KYS, Salley SO (2010) Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta. Bioresource Technol 102(2):1649–1655, doi:10.1016/j.biortech.2010.09.062
Chen R, Li R, Deitz L, Liu Y, Stevenson RJ, Liao W (2012) Freshwater algal cultivation with animal waste for nutrient removal and biomass production. Biomass Bioenergy 39:128–138, doi:10.1016/j.biombioe.2011.12.045
Davis R, Aden A, Pienkos PT (2011) Techno-economic analysis of autotrophic microalgae for fuel production. Appl Energ 88(10):3524–3531, doi:10.1016/j.apenergy.2011.04.018
Doi M (2001) Chlorophyll degradation in a Chlamydomonas reinhardtii mutant: an accumulation of pyropheophorbide a by anaerobiosis. Plant cell physiol 42(5):469–474, doi:10.1093/pcp/pce057
Eaton AD, Franson MAH (2005) Standard methods for the examination of water & wastewater, 21st edn. APHA, AWWA, and the WEF, New York
Feng Y, Li C, Zhang D (2011) Lipid production of Chlorella vulgaris cultured in artificial wastewater medium. Bioresource Technol 102(1):101–105, doi:10.1016/j.biortech.2010.06.016
Fernandes B, Teixeira J, Dragone G, Vicente AA, Kawano S, Bišová K, Přibyl P, Zachleder V, Vítová M (2013) Relationship between starch and lipid accumulation induced by nutrient depletion and replenishment in the microalga Parachlorella kessleri. Bioresource Technol 144:268–274, doi:10.1016/j.biortech.2013.06.096
Fernandez E, Galvan A (2007) Inorganic nitrogen assimilation in Chlamydomonas. J Exp Bot 58(9):2279–2287, doi:10.1093/jxb/erm106
Gadd GM (1990) Heavy metal accumulation by bacteria and other microorganisms. Experientia 46(8):834–840. doi:10.1007/bf01935534
Garnham G, Codd G, Gadd G (1992) Kinetics of uptake and intracellular location of cobalt, manganese and zinc in the estuarine green alga Chlorella salina. Appl Microbiol Biot 37(2) doi:10.1007/bf00178183
Goldman CR (1966) Micronutrient limiting factors and their detection in natural phytoplankton populations. In: Goldman CR (ed) Primary productivity in aquatic environments. University of California, Berkeley, pp 123–125
Golueke CG, Oswald WJ (1959) Biological conversion of light energy to the chemical energy of methane. Appl Microbiol 7(4):219–227
Haehnel W (1984) Photosynthetic electron transport in higher plants. Annu Rev of Plant Phys 35(1):659–693, doi:10.1146/annurev.pp. 35.060184.003303
Hamdy AA (2000) Biosorption of heavy metals by marine algae. Curr Microbiol 41(4):232–238. doi:10.1007/s002840010126
Hänsch R, Mendel RR (2009) Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl). Curr opin plant biol 12(3):259–266. doi:10.1016/j.pbi.2009.05.006
Harvey M, Pilgrim S (2011) The new competition for land: food, energy, and climate change. Food Policy 36:S40–S51. doi:10.1016/j.foodpol.2010.11.009
Hortensteiner S (1999) Chlorophyll breakdown in higher plants and algae. Cell Mol Life Sci 56(3–4):330–347. doi:10.1007/s000180050434
Hortensteiner S, Chinner J, Matile P, Thomas H, Donnison IS (2000) Chlorophyll breakdown in Chlorella protothecoides: characterization of degreening and cloning of degreening-related genes. Plant Mol Biol 42(3):439–450. doi:10.1023/a:1006380125438
Hu B, Min M, Zhou W, Du Z, Mohr M, Chen P, Zhu J, Cheng Y, Liu Y, Ruan R (2012) Enhanced mixotrophic growth of microalga Chlorella sp. on pretreated swine manure for simultaneous biofuel feedstock production and nutrient removal. Bioresource Technol 126:71–79. doi:10.1016/j.biortech.2012.09.031
Huss VAR, Frank C, Hartmann EC, Hirmer M, Kloboucek A, Seidel BM, Wenzeler P, Kessler E (1999) Biochemical taxonomy and molecular phylogeny of the genus Chlorella sensu lato (Chlorophyta). J Phycol 35(3):587–598, doi:10.1046/j.1529-8817.1999.3530587.x
Khummongkol D, Canterford GS, Fryer C (1982) Accumulation of heavy metals in unicellular algae. Biotechnol Bioeng 24(12):2643–2660. doi:10.1002/bit.260241204
Lardon L, Hélias A, Sialve B, Steyer J-P, Bernard O (2009) Life-cycle assessment of biodiesel production from microalgae. Environ Sci Technol 43(17):6475–6481. doi:10.1021/es900705j
Leonardo T, Farhi E, Boisson AM, Vial J, Cloetens P, Bohic S, Rivasseau C (2014) Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques—subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis. Metallomics 6(2):316. doi:10.1039/c3mt00281k
Leone DE (1963) Growth of Chlorella pyrenoidosa in recycled medium. Appl Microbiol 11:427–429
Leusch A, Holan ZR, Volesky B (1995) Biosorption of heavy metals (Cd, Cu, Ni, Pb, Zn) by chemically-reinforced biomass of marine algae. J Chem Technol Biot 62(3):279–288. doi:10.1002/jctb.280620311
Li Y, Horsman M, Wang B, Wu N, Lan CQ (2008) Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl Microbiol Biot 81(4):629–636. doi:10.1007/s00253-008-1681-1
Liang Y, Sarkany N, Cui Y (2009) Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnol Lett 31(7):1043–1049, doi:10.1007/s10529-009-9975-7
Link DD, Walter PJ, Kingston HM (1998) Development and validation of the New EPA microwave-assisted leach method 3051A. Environ Sci Technol 32(22):3628–3632. doi:10.1021/es980559n
Lundquist TJ, Woertz IC, Quinn N, Benemann JR (2010) A realistic technology and engineering assessment of algae biofuel production. Energy Biosciences Institute University of California, Berkeley, p 178
Mandalam RK, Palsson BO (1998) Elemental balancing of biomass and medium composition enhances growth capacity in high-density Chlorella vulgaris cultures. Biotechnol Bioeng 59(5):605–611
Miao X, Wu Q (2006) Biodiesel production from heterotrophic microalgal oil. Bioresource Technol 97(6):841–846. doi:10.1016/j.biortech.2005.04.008
Mizuno Y, Sato A, Watanabe K, Hirata A, Takeshita T, Ota S, Sato N, Zachleder V, Tsuzuki M, Kawano S (2013) Sequential accumulation of starch and lipid induced by sulfur deficiency in Chlorella and Parachlorella species. Bioresource Technol 129:150–155. doi:10.1016/j.biortech.2012.11.030
Morel FMM, Hudson RJM, Price NM (1991) Limitation of productivity by trace metals in the sea. Limnol Oceanogr 36(8):1742–1755. doi:10.4319/lo.1991.36.8.1742
Morel FMM, Reinfelder JR, Roberts SB, Chamberlain CP, Lee JG, Yee D (1994) Zinc and carbon co-limitation of marine phytoplankton. Nature 369(6483):740–742. doi:10.1038/369740a0
Morrison GMP (1989) Trace element speciation and its relationship to bioavailability and toxicity in natural waters. In: Batley GE (ed) Trace element speciation: analytical methods and problems. CRC Press, Boca Raton, pp 25–41
NRC (2012) Sustainable Development of Algal Biofuels in the United States. The National Academies Press
Ogden KL, Herraras VC (2013) Understanding the relationships between productivity and water recycle in outdoor algal cultivation systems. Oral presentation. In: The 3rd international conference on algal biomass. Biofuels and Bioproducts 16–19 June, Toronto
Oshio Y, Hase E (1969) Studies on red pigments excreted by cells of Chlorella protothecoides during the process of bleaching induced by glucose or acetate I. Chemical properties of the red pigments. Plant Cell Physiol 10(1):41–49
Owen WF, Stuckey DC, Healy JB, Young LY, McCarty PL (1979) Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res 13(6):485–492. doi:10.1016/0043-1354(79)90043-5
Pate R, Klise G, Wu B (2011) Resource demand implications for US algae biofuels production scale-up. Appl Energ 88(10):3377–3388. doi:10.1016/j.apenergy.2011.04.023
Pilon M, Abdel-Ghany SE, Cohu CM, Gogolin KA, Ye H (2006) Copper cofactor delivery in plant cells. Curr opin plant biol 9(3):256–263. doi:10.1016/j.pbi.2006.03.007
Pittman JK, Dean AP, Osundeko O (2011) The potential of sustainable algal biofuel production using wastewater resources. Bioresource Technol 102(1):17–25. doi:10.1016/j.biortech.2010.06.035
Raalte CD, Valiela I, Teal JM (1976) Production of epibenthic salt marsh algae: light and nutrient limitation. Limnol Oceanogr 21(6):862–872. doi:10.2307/2835051
Ras M, Lardon L, Bruno S, Bernet N, Steyer J-P (2011) Experimental study on a coupled process of production and anaerobic digestion of Chlorella vulgaris. Bioresource Technol 102(1):200–206
Raven JA, Evans MCW, Korb RE (1999) The role of trace metals in photosynthetic electron transport in O2-evolving organisms. Photosynth Res 60(2/3):111–150. doi:10.1023/a:1006282714942
Rhee G-Y (1978) Effects of N:P atomic ratios and nitrate limitation on algal growth, cell composition, and nitrate uptake. Limnol Oceanogr 23(1):10–25
Rodolfi L, Chini Zittelli G, Bassi N, Padovani G, Biondi N, Bonini G, Tredici MR (2009) Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol Bioeng 102(1):100–112. doi:10.1002/bit.22033
Schlee J, Komor E (1986) Ammonium uptake by Chlorella. Planta 168(2):232–238. doi:10.1007/BF00402968
Schumacher T, Davis K, Kassai S, Harroff L, Reardon KF Medium recycling in Neochloris oleoabundans cultivation. In: Algae Biomass Summit, 2013.
Sheehan J, Dunahay T, Benemann J, Roessler P (1998) A look back at the U.S. Department of Energy’s aquatic species program: biodiesel from algae. NREL, Golden CO, p 323
Shigeji A, Mitsuo M, Eiji H (1965) De- and re-generation of chloroplasts in the cells of Chlorella protothecoides: V. Degeneration of chloroplasts induced by different carbon sources, and effects of some antimetabolites upon the process induced by glucose. Plant Cell Physiol 6(3):487–498
Shihira-Ishikawa I, Hase E (1964) Nutritional control of cell pigmentation in Chlorella protothecoides with special reference to the degeneration of chloroplast induced by glucose. Plant Cell Physiol 5(2):227–240
Sialve B, Bernet N, Bernard O (2009) Anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable. Biotechnol Adv 27(4):409–416
Singh A, Nigam PS, Murphy JD (2011) Renewable fuels from algae: an answer to debatable land based fuels. Bioresource Technol 102(1):10–16. doi:10.1016/j.biortech.2010.06.032
Skrupski B, Wilson KE, Goff KL, Zou J (2012) Effect of pH on neutral lipid and biomass accumulation in microalgal strains native to the Canadian prairies and the Athabasca oil sands. J Appl Phycol 25(4):937–949. doi:10.1007/s10811-012-9930-1
Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101(2):87–96. doi:10.1263/jbb.101.87
Stoddard JL (1987) Micronutrient and phosphorus limitation of phytoplankton abundance in Gem Lake, Sierra Nevada, California. Hydrobiologia 154(1):103–111. doi:10.1007/bf00026834
Su C-H, Chien L-J, Gomes J, Lin Y-S, Yu Y-K, Liou J-S, Syu R-J (2010) Factors affecting lipid accumulation by Nannochloropsis oculata in a two-stage cultivation process. J Appl Phycol 23(5):903–908, doi:10.1007/s10811-010-9609-4
Suzuki T, Shioi Y (2002) Re-examination of Mg-dechelation reaction in the degradation of chlorophylls using chlorophyllin a as a substrate. Photosynth Res 74(2):217–223, doi:10.1023/A:1020915812770
Twiss MR, Auclair J-C, Charlton MN (2000) An investigation into iron-stimulated phytoplankton productivity in epipelagic Lake Erie during thermal stratification using trace metal clean techniques. Can J Fish Aquat Sci 57(1):86–95, doi:10.1139/f99-189
Tyrrell T (1999) The relative influences of nitrogen and phosphorus on oceanic primary production. Nature 400(6744):525–531. doi:10.1038/22941
Volesky B, Holan ZR (1995) Biosorption of heavy metals. Biotechnol Progr 11(3):235–250. doi:10.1021/bp00033a001
Wan M, Liu P, Xia J, Rosenberg J, Oyler G, Betenbaugh M, Nie Z, Qiu G (2011) The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana. Appl Microbiol Biot 91(3):835–844, doi:10.1007/s00253-011-3399-8
Wan M-X, Wang R-M, Xia J-L, Rosenberg JN, Nie Z-Y, Kobayashi N, Oyler GA, Betenbaugh MJ (2012) Physiological evaluation of a new Chlorella sorokiniana isolate for its biomass production and lipid accumulation in photoautotrophic and heterotrophic cultures. Biotechnol Bioeng 109(8):1958–1964. doi:10.1002/bit.24477
Wang L, Li Y, Chen P, Min M, Chen Y, Zhu J, Ruan RR (2010a) Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp. Bioresource Technol 101(8):2623–2628, doi:10.1016/j.biortech.2009.10.062
Wang L, Wang Y, Chen P, Ruan R (2010b) Semi-continuous cultivation of Chlorella vulgaris for treating undigested and digested dairy manures. Appl Biochem Biotech 162(8):2324–2332. doi:10.1007/s12010-010-9005-1
Xia L, Ge H, Zhou X, Zhang D, Hu C (2013) Photoautotrophic outdoor two-stage cultivation for oleaginous microalgae Scenedesmus obtusus XJ-15. Bioresource Technol 144:261–267, doi:10.1016/j.biortech.2013.06.112
Xin L, Hong-ying H, Ke G, Ying-xue S (2010) Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp. Bioresource Technol 101(14):5494–5500. doi:10.1016/j.biortech.2010.02.016
Xiong W, Li X, Xiang J, Wu Q (2007) High-density fermentation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production. Appl Microbiol Biot 78(1):29–36. doi:10.1007/s00253-007-1285-1
Xu H, Miao X, Wu Q (2006) High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters. J Biotechnol 126(4):499–507. doi:10.1016/j.jbiotec.2006.05.002
Yan D, Dai J, Wu Q (2013) Characterization of an ammonium transporter in the oleaginous alga Chlorella protothecoides. Appl Microbiol Biot 97(2):919–928. doi:10.1007/s00253-012-4534-x
Yang J, Xu M, Zhang X, Hu Q, Sommerfeld M, Chen Y (2011) Life-cycle analysis on biodiesel production from microalgae: water footprint and nutrients balance. Bioresource Technol 102(1):159–165, doi:10.1016/j.biortech.2010.07.017
Yeh K-L, Chang J-S (2011) Nitrogen starvation strategies and photobioreactor design for enhancing lipid content and lipid production of a newly isolated microalga Chlorella vulgaris ESP-31: implications for biofuels. Biotechnol J 6(11):1358–1366, doi:10.1002/biot.201000433
Acknowledgments
The authors gratefully acknowledge financial support from U.S. DOE CCS Program (Grant No. DE-FE0001888 to Phycal), U.S. NSF CBET Program (Grant No.1236691 to JHU), and the Bureau of Education and Cultural Affairs of U.S. Department of State through an International Fulbright Science and Technology Award to Pavlo Bohutskyi.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 228 kb)
Rights and permissions
About this article
Cite this article
Bohutskyi, P., Liu, K., Kessler, B.A. et al. Mineral and non-carbon nutrient utilization and recovery during sequential phototrophic-heterotrophic growth of lipid-rich algae. Appl Microbiol Biotechnol 98, 5261–5273 (2014). https://doi.org/10.1007/s00253-014-5655-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-014-5655-1