Identification of Carbohydrates Metabolic Related Enzymes for Lipid Production in Botryococcus sp., a Microalgae Isolated from Taman Negara Endau Rompin

Carbohydrates metabolic enzymes for lipid production

Authors

  • ZUBAINATU ABBA Department of Technology and Natural Resources, Faculty of Applied Science and Technology, UTHM, High Education Hub Pagoh, KM 1, 84600 Panchor, Johor, Malaysia
  • SITI FATIMAH ZAHARAH MOHAMAD FUZI Department of Technology and Natural Resources, Faculty of Applied Sciences and Technology, University Tun Hussein Onn Malaysia, Johor, 84600, Pagoh Education Hub, Muar, Johor, Malaysia
  • HAZEL MONICA MATIAS-PERALTA College of Fisheries-Freshwater Aquaculture Center, Central Luzon State University, 3120 Science City of Munoz, Nueva Ecija, Philippines

DOI:

https://doi.org/10.33736/bjrst.5080.2023

Keywords:

Botryococcus sp., carbohydrate metabolism, enolase, lipid, phosphoenolpyruvate

Abstract

Botryococcus is a microalgal genus known for its ability to generate and accumulate substantial amounts of lipids via carbohydrate metabolism. This work determined the metabolic pathways and enzymes involved in carbohydrate metabolism leading to increased synthesis of fat in Botryococcus sp. Relevant intracellular and extracellular metabolites were extracted and quantified using chromatographic analysis. Enzymes involved in carbohydrate metabolism leading to lipid formation in Botryococcus sp. under natural conditions were also discovered by one-dimensional gel electrophoresis followed by proteomic mass spectrometry (LC-MS/MS) and database searching. Proximate analysis demonstrated 23.0% total carbohydrate, 16.0% protein and 61.0% lipid per milligram biomass dry weight of Botryococcus sp. The extracellular metabolites constitute mostly of cyclohydrocarbons, nitrogenated hydrocarbons, siloxanes, phenols, and phenol derivatives. A glycolytic enzyme “enolase,” which can create phosphoenolpyruvate and subsequently convert it into pyruvate, was found in this study. This study revealed that enolase provided an alternate pathway to export fixed carbon to the cytoplasm, hence providing a shorter route to lipid production than the normal process via the plastid leading to the manufacture of more lipids in the cells of Botryococcus sp. than other microalgae of the same group.

 

References

Ashokkumar, V. & Rengasamy, R. (2012). Mass culture of Botryococcus braunii Kutz. under open raceway pond for biofuel production. Bioresource Technology, 104: 394-399.

Baroukh, C., Muñoz-Tamayo, R., Steyer, J.P. & Bernard, O. (2015). A state of the art of metabolic networks of unicellular microalgae and cyanobacteria for biofuel production. Metabolic Engineering, 30: 49-60.

Bayona, K.C.D. & Garcés, L.A. (2014). Effect of different media on exopolysaccharide and biomass production by the green microalga Botryococcus braunii. Journal of Applied Phycology, 26(5): 2087-2095.

Bi, Z. & He, B.B. (2013). Characterization of microalgae for the purpose of biofuel production. Transactions of the ASABE, 56(4): 1529-1539.

Blifernez-Klassen, O., Chaudhari, S., Klassen, V., Wördenweber, R., Steffens, T., Cholewa, D. & Kruse, O. (2018). Metabolic survey of Botryococcus braunii: Impact of the physiological state on product formation. PloS One, 13(6): e0198976.

Boyd, C. (1973). Amino acid composition of freshwater algae. Arch Hydrobiology, 72: 1-9.

Chatsungnoen, T. & Chisti, Y. (2016). Harvesting microalgae by flocculation–sedimentation. Algal Research, 13: 271-283.

Chen, C.Y., Zhao, X.Q., Yen, H.W., Ho, S.H., Cheng, C.L., Lee, D.J. & Chang, J.S. (2013). Microalgae-based carbohydrates for biofuel production. Biochemical Engineering Journal, 78: 1-10.

Cheng, Y.S., Labavitch, J. & VanderGheynst, J. (2015). Elevated CO2 concentration impacts cell wall polysaccharide composition of green microalgae of the genus Chlorella. Letters in Applied Microbiology, 60(1): 1-7.

Cheng, P., Zhou, C., Wang, Y., Xu, Z., Xu, J., Zhou, D. & Liu, T. (2018). Comparative transcriptome analyses of oleaginous Botryococcus braunii race: A reveal significant differences in gene expression upon cobalt enrichment. Biotechnology for Biofuels, 11(1): 333.

Chisti, Y. (2007). Biodiesel from microalgae. Biotechnology Advances, 25(3): 294-306.

Contreras, L., Ritter, A., Dennett, G., Boehmwald, F., Guitton, N., Pineau, C. & Correa, J.A. (2008). Two‐dimensional gel electrophoresis analysis of brown algal protein extracts. Journal of Phycology, 44(5): 1315-1321.

Dayan, C., Kumudha, A., Sarada, R. & Ravishankar, G. (2010). Isolation, characterization, and outdoor cultivation of green microalgae Botryococcus sp. Scientific Research Essays, 5(17): 2497-2505.

Dayananda, C., Sarada, R., Rani, M. U., Shamala, T. & Ravishankar, G. (2007). Autotrophic cultivation of Botryococcus braunii for the production of hydrocarbons and exopolysaccharides in various media. Biomass and Bioenergy, 31(1): 87-93.

Dzieciatkowska, M., Hill, R. & Hansen, K.C. (2014). GeLC-MS/MS analysis of complex protein mixtures. In: Martins-de-Souza, D. (eds) Shotgun Proteomics. Methods in Molecular Biology. Humana Press, New York. 1156

Environmental Protection Agency Method 9071B. (1998). n-Hexane Extractable Material (HEM) for Sludge, Sediment, and Solid Samples. United States. 1-13.

Gani, P., Sunar, N.M., Matias-Peralta, H. & Jamaian, S.S. (2016). Effects of different culture conditions on the phycoremediation efficiency of domestic wastewater. Journal of Environmental Chemical Engineering, 4(4): 4744-4753.

Garibay-Hernández, A., Barkla, B.J., Vera-Estrella, R., Martinez, A. & Pantoja, O. (2017). Membrane proteomic insights into the physiology and taxonomy of an oleaginous green microalga. Plant Physiology, 173(1): 390-416.

Ghaffour, N., Bundschuh, J., Mahmoudi, H. & Goosen, M.F. (2015). Renewable energy-driven desalination technologies: A comprehensive review on challenges and potential applications of integrated systems. Desalination, 356: 94-114.

Gouveia, J.D., Ruiz, J., van den Broek, L.A., Hesselink, T., Peters, S., Kleinegris, D.M. & Wijffels, R.H. (2017). Botryococcus braunii strains compared for biomass productivity, hydrocarbon, and carbohydrate content. Journal of Biotechnology, 248: 77-86.

Grima, E.M., Belarbi, E.H., Fernández, F.A., Medina, A.R. & Chisti, Y. (2003). Recovery of microalgal biomass and metabolites: process options and economics. Biotechnolology Advances, 20(7-8): 491-515.

Gundry, R.L., White, M.Y., Murray, C.I., Kane, L. A., Fu, Q., Stanley, B.A. & Van Eyk, J.E. (2010). Preparation of proteins and peptides for mass spectrometry analysis in a bottom‐up proteomics workflow. Current Protocol in Molecular Biology, 90(1):1-23.

Hershey, J.C., Hautmann, M., Thompson, M.M., Rothblum, L.I., Haystead, T.A. & Owens, G.K. (1995). Angiotensin II-induced hypertrophy of rat vascular smooth muscle is associated with increased 18S rRNA synthesis and phosphorylation of the rRNA transcription factor, upstream binding factor. Journal of Biological Chemistry, 270(42): 25096-25101.

Ho, S.H., Chen, C.Y., Lee, D.J. & Chang, J.S. (2011). Perspectives on microalgal CO2-emission mitigation systems a review. Biotechnology Advance, 29(2): 189-198.

Khan, M.I., Shin, J.H. & Kim, J.D. (2018). The promising future of microalgae: current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microbial Cell Factory, 17(1): 36.

Knothe, G. (2005). Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Processing Technology, 86(10): 1059-1070.

Knothe, G. (2008). “Designer” biodiesel: optimizing fatty ester composition to improve fuel properties. Energy Fuel, 22(2): 1358-1364.

Kurinjimalar, C., Kavitha, G., Shamshath-Begum, S., Rajaram, G., Nagaraj, S., Senthilkumar, N. & Rengasamy, R. (2017). Optimization and production of Botryococcus braunii biomass using commercial nutrients by Response Surface Methodology. Iranica Journal of Energy and Environment, 8(1): 18-25.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193(1): 265-275.

Mata, T.M., Martins, A.A. & Caetano, N.S. (2010). Microalgae for biodiesel production and other applications: A review. Renewable and Sustainable Energy Reviews, 14(1): 217-232.

Melis, A. (2013). Carbon partitioning in photosynthesis. Current Opinion in Chemical Biology, 17(3): 453-456.

Molnár, I., Lopez, D., Wisecaver, J. H., Devarenne, T.P., Weiss, T.L., Pellegrini, M. & Hackett, J.D. (2012). Bio-crude transcriptomics: gene discovery and metabolic network reconstruction for the biosynthesis of the terpenome of the hydrocarbon oil-producing green alga, Botryococcus braunii race B (Showa). BMC Genomics, 13(1): 576.

Mori, N., Moriyama, T. & Sato, N. (2019). Uncommon properties of lipid biosynthesis of isolated plastids in the unicellular red alga Cyanidioschyzon merolae. FEBS Open Bio, 9(1): 114-128.

Nascimento, I.A., Marques, S.S.I., Cabanelas, I.T. D., Pereira, S.A., Druzian, J.I., de Souza, C.O. & Nascimento, M.A. (2013). Screening microalgae strains for biodiesel production: lipid productivity and estimation of fuel quality based on fatty acids profiles as selective criteria. Bioenergy Research, 6(1): 1-13.

Nichols, H.W. (1973). Growth media-freshwater. In: Handbook of phycological methods: culture methods and growth measurements, pp 7–24. Ed. by J. R. Stein. Cambridge: Cambridge University Press.

Polle, J.E., Neofotis, P., Huang, A., Chang, W., Sury, K. & Wiech, E.M. (2014). Carbon partitioning in green algae (Chlorophyta) and the enolase enzyme. Metabolites, 4(3): 612-628.

Powell, E. & Hill, G. (2009). Economic assessment of an integrated bioethanol–biodiesel–microbial fuel cell facility utilizing yeast and photosynthetic algae. Chemical Engineering Research and Design, 87(9): 1340-1348.

Prabhakar, V., Löttgert, T., Gigolashvili, T., Bell, K., Flügge, U.I. & Häusler, R.E. (2009). Molecular and functional characterization of the plastid-localized phosphoenolpyruvate enolase (ENO1) from Arabidopsis thaliana. FEBS Letters, 583(6): 983-991.

Ruangsomboon, S. (2015). Effects of different media and nitrogen sources and levels on growth and lipid of green microalga Botryococcus braunii KMITL and its biodiesel properties based on fatty acid composition. Bioresource Technology, 191: 377-384.

Ruangsomboon, S., Dimak, J., Jongput, B., Wiwatanaratanabutr, I. & Kanyawongha, P. (2020). Outdoor open pond batch production of green microalga Botryococcus braunii for high hydrocarbon production: enhanced production with salinity. Scientific Reports, 10(1): 1-12.

Ruangsomboon, S., Ganmanee, M. & Choochote, S. (2013). Effects of different nitrogen, phosphorus, and iron concentrations and salinity on lipid production in newly isolated strain of the tropical green microalga, Scenedesmus dimorphus KMITL. Journal of Applied Phycology, 25(3): 867-874.

Schwenzfeier, A., Wierenga, P. A. & Gruppen, H. (2011). Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp. Bioresource Technology, 102(19): 9121-9127.

Shi, Q., Chen, C., Zhang, W., Wu, P., Sun, M., Wu, H., Wu, H., Fu, P. & Fan, J. (2021). Transgenic eukaryotic microalgae as green factories: providing new ideas for the production of biologically active substances. Journal of Applied Phycology, 33: 705-728.

Stansell, G.R., Gray, V.M. & Sym, S.D. (2012). Microalgal fatty acid composition: implications for biodiesel quality. Journal of Applied Phycology, 24(4): 791-801.

Troncoso-Ponce, M.A., Garcés, R. & Martínez-Force, E. (2010). Glycolytic enzymatic activities in developing seeds involved in the differences between standard and low oil content sunflowers (Helianthus annuus L.). Plant Physiology and Biochemistry, 48(12): 961-965.

Tsarenko, P.M. (2011). Trebouxiophyceae. In: Algae of Ukraine: diversity, nomenclature, taxonomy, ecology and geography. Volume 3: Chlorophyta. (Tsarenko, P.M., Wasser, S.P. & Nevo, E. Eds), pp. 61-108. Ruggell: A.R.A. Gantner Verlag K.-G.

Van Wychen, S. & Laurens, L.M.L. Determination of total carbohydrates in algal biomass: Laboratory analytical procedure (LAP), report, December 1, 2013; Golden, Colorado. (https://digital.library.unt.edu/ark:/67531/metadc871393/: accessed February 18, 2022).

Voll, L.M., Hajirezaei, M.R., Czogalla-Peter, C., Lein, W., Stitt, M., Sonnewald, U. & Börnke, F. (2009). Antisense inhibition of enolase strongly limits the metabolism of aromatic amino acids but has only minor effects on respiration in leaves of transgenic tobacco plants. New Phytologist, 184(3): 607-618.

Wang, W., Vignani, R., Scali, M. & Cresti, M. (2006). A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. Electrophoresis, 27(13): 2782-2786.

Wang Y., Tibbetts S.M. & McGinn P.J. (2021) Microalgae as sources of high-quality protein for human food and protein supplements. Foods, 10(12): 1-18.

Zhou, W., Min, M., Hu, B., Ma, X., Liu, Y., Wang, Q. & Ruan, R. (2013). Filamentous fungi assisted bio-flocculation: A novel alternative technique for harvesting heterotrophic and autotrophic microalgal cells. Separation and Purification Technology, 107: 158-165.

Published

2023-06-30

How to Cite

ZUBAINATU ABBA, SITI FATIMAH ZAHARAH MOHAMAD FUZI, & HAZEL MONICA MATIAS-PERALTA. (2023). Identification of Carbohydrates Metabolic Related Enzymes for Lipid Production in Botryococcus sp., a Microalgae Isolated from Taman Negara Endau Rompin: Carbohydrates metabolic enzymes for lipid production. Borneo Journal of Resource Science and Technology, 13(1), 41–53. https://doi.org/10.33736/bjrst.5080.2023