Effects of Phosphorus Removal and pH Changes in the Culture Medium of Spirulina sp. on the Production Rate of Polyhydroxybutyrate
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Abstract
The aim of this study was to investigate the production of polyhydroxybutyrate (PHB) by Spirulina sp., a type of cyanobacterium recognized for its capacity to produce this biopolymer. PHB accumulation takes place in Spirulina when it undergoes nitrogen and phosphorus limitation, and carbon abundance, acting as a vital reserve material for the microorganism. The study was conducted under autotrophic conditions, phosphorous deficiency, and varying acidity levels to confirm how the interplay between phosphorus deficiency and pH can affect PHB production in Spirulina without reducing microalgal biomass. Microalgal cultures in the phosphorus-free treatments at pH 8, 10, and 12 were performed, and their growth quality including levels of photosynthetic pigments, malondialdehyde, anthocyanin, phenol, and flavonoid were measured. PHB was extracted and analyzed using Fourier Transform Infrared (FTIR) spectroscopy for qualitative analysis and gas chromatography (GC) for quantitative evaluation. The amounts of anthocyanins, phenols, and flavonoids, which are important constituents of the antioxidant defense system, were highest in the treatment with a pH of 10, which also had the lowest levels of the stress indicator malondialdehyde. The extracted polyhydroxybutyrate amounts in the control treatment and treatments with phosphorus deficiency at pH levels of 8, 10, and 12 were 6.82%, 3.54%, 7.04%, and 4.23% of cell dry weight, respectively, according to GC. Based on the results, altering the acidity of the Spirulina culture medium had a limited effect on increasing in polyhydroxybutyrate accumulation compared to optimal acidity conditions and simultaneous phosphorus removal, which was partly due to the consumption of some polyhydroxybutyrate produced under phosphorus deficiency stress.
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References
Mukherjee, C., Varghese, D., Krishna, J.S., Boominathan, T., Rakeshkumar, R., Dineshkumar, S., Rao, C.V.S.B. and Sivaramakrishna, A., 2023. Recent advances in biodegradable polymers–properties, applications and future prospects. European Polymer Journal, 192(10), https://doi.org/10.1016/j.eurpolymj.2023.112068.
Bugnicourt, E., Cinelli, P., Lazzeri, A. and Alvarez, V.A., 2014. Polyhydroxyalkanoate (PHA): Review of synthesis, characteristics, processing and potential applications in packaging. Express Polymer Letters, 8(11), 791-808, https://doi.org/10.3144/expresspolymlett.2014.82.
Bhati, R. and Mallick, N., 2015. Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production by the diazotrophic cyanobacterium Nostoc muscorum Agardh: Process optimization and polymer characterization. Algal Research, 7, 78-85, https://doi.org/10.1016/j.algal.2014.12.003.
Taepucharoen, K., Tarawat, S., Puangcharoen, M., Incharoensakdi, A. and Monshupanee, T., 2017. Production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) under photoautotrophy and heterotrophy by non-heterocystous N2-fixing cyanobacterium. Bioresource Technology, 239, 523-527, https://doi.org/10.1016/j.biortech.2017.05.029.
Singh, M.K., Rai, P.K., Rai, A., Singh, S. and Singh, J.S., 2019. Poly-β-hydroxybutyrate production by the cyanobacterium Scytonema geitleri Bharadwaja under varying environmental conditions. Biomolecules, 9(5), https://doi.org/10.3390/biom9050198.
Krasaesueb, N., Incharoensakdi, A. and Khetkorn, W., 2019. Utilization of shrimp wastewater for poly-β-hydroxybutyrate production by Synechocystis sp. PCC 6803 strain ΔSphU cultivated in photobioreactor. Biotechnology Reports, 23, https://doi.org/10.1016/j.btre.2019.e00345.
Costa, S.S., Miranda, A.L., de Morais, M.G., Costa, J.A.V. and Druzian, J.I., 2019. Microalgae as source of polyhydroxyalkanoates (PHAs)—A review. International Journal of Biological Macromolecules, 131, 536-547,https://doi.org/10.1016/j.ijbiomac.2019.03.099.
Duangsri, C., Mudtham, N.-A., Incharoensakdi, A. and Raksajit, W., 2020. Enhanced polyhydroxybutyrate (PHB) accumulation in heterotrophically grown Arthrospira platensis under nitrogen deprivation. Journal of Applied Phycology, 32, 3645-3654.
Bottomley, P. and Stewart, W.D.P., 1976. ATP pools and transients in the blue-green alga, Anabaena cylindrica. Archives of Microbiology, 108, 249-258, https://doi.org/10.1007/BF00454849.
García, G., Sosa-Hernández, J.E., Rodas-Zuluaga, L.I., Castillo-Zacarías, C., Iqbal, H. and Parra-Saldívar, R., 2020. Accumulation of PHA in the microalgae Scenedesmus sp. under nutrient-deficient conditions. Polymers, 13(1), https://doi.org/10.1371/journal.pone.0158168.
de Morais, E.G., Druzian, J.I., Nunes, I.L., de Morais, M.G. and Costa, J.A.V., 2019. Glycerol increases growth, protein production and alters the fatty acids profile of Spirulina (Arthrospira) sp. LEB 18. Process Biochemistry, 76, 40-45, https://doi.org/10.1016/j.procbio.2018.09.024.
Jaeschke, D.P., Mercali, G.D., Marczak, L.D.F., Müller, G., Frey, W. and Gusbeth, C., 2019. Extraction of valuable compounds from Arthrospira platensis using pulsed electric field treatment. Bioresource Technology, 283, 207-212, https://doi.org/10.1016/j.biortech.2019.03.035.
Park, W.S., Kim, H.-J., Li, M., Lim, D.H., Kim, J., Kwak, S.-S., Kang, C.-M., Ferruzzi, M.G. and Ahn, M.-J., 2018. Two classes of pigments, carotenoids and c-phycocyanin, in Spirulina powder and their antioxidant activities. Molecules, 23(8), https://doi.org/10.3390/molecules23082065.
Matos, J., Cardoso, C.L., Falé, P., Afonso, C.M. and Bandarra, N.M., 2020. Investigation of nutraceutical potential of the microalgae Chlorella vulgaris and Arthrospira platensis. International Journal of Food Science and Technology, 55(1), 303-312, https://doi.org/10.1111/ijfs.14278.
Troschl, C., Meixner, K. and Drosg, B., 2017. Cyanobacterial PHA production—review of recent advances and a summary of three years’ working experience running a pilot plant. Bioengineering, 4(2), https://doi.org/10.3390/bioengineering4020026.
Chu, F.-F., Chu, P.-N., Cai, P.-J., Li, W.-W., Lam, P.K.S. and Zeng, R.J., 2013. Phosphorus plays an important role in enhancing biodiesel productivity of Chlorella vulgaris under nitrogen deficiency. Bioresource Technology, 134, 341-346.
Shen, X.-F., Chu, F.-F., Lam, P.K.S. and Zeng, R.J., 2015. Biosynthesis of high yield fatty acids from Chlorella vulgaris NIES-227 under nitrogen starvation stress during heterotrophic cultivation. Water Research, 81, 294-300, https://doi.org/10.1016/j.watres.2015.06.003.
Xin, L., Hong-Ying, H. and Yu-Ping, Z., 2011. Growth and lipid accumulation properties of a freshwater microalga Scenedesmus sp. under different cultivation temperature. Bioresource Technology, 102(3), 3098-3102, https://doi.org/10.1016/j.biortech.2010.10.055.
Madadi, R., Maljaee, H., Serafim, L.S. and Ventura, S.P.M., 2021. Microalgae as contributors to produce biopolymers. Marine Drugs, 19(8), https://doi.org/10.3390/md19080466.
Zarrouk, C., 1966. Contribution to the study of a cyanophycea: influence of various physical and chemical factors on the growth and photosynthesis of Spirulina maxima', Ph.D. University of Paris, France.
Wegmann, K., 1971. Osmotic regulation of photosynthetic glycerol production in Dunaliella. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 234(3), 317-323.
Markou, G., Chatzipavlidis, I. and Georgakakis, D., 2012. Effects of phosphorus concentration and light intensity on the biomass composition of Arthrospira (Spirulina) platensis. World Journal of Microbiology and Biotechnology, 28(8), 2661-2670, https://doi.org/10.1007/s11274-012-1076-4.
Eijckelhoff, C. and Dekker, J.P., 1997. A routine method to determine the chlorophyll a, pheophytin a and β-carotene contents of isolated photosystem II reaction center complexes. Photosynthesis Research, 52, 69-73, https://doi.org/10.1023/A:1005834006985.
Ismaiel, M.M.S., El-Ayouty, Y.M. and Piercey-Normore, M., 2016. Role of pH on antioxidants production by Spirulina (Arthrospira) platensis. Brazilian Journal of Microbiology, 47, 298-304, https://doi.org/10.1016/j.bjm.2016.01.003.
Yap, P., Jain, A. and Trau, D., 2018. Determination of biomass in Spirulina cultures by photopette. Life Science Application Note 050, Version 1, 1-3.
Leganés, F., Sánchez-Maeso, E. and Fernández-Valiente, E., 1987. Effect of indoleacetic acid on growth and dinitrogen fixation in cyanobacteria. Plant and Cell Physiology, 28(3), 529-533,https://doi.org/10.1093/oxfordjournals.pcp.a077324.
Morales, M. and Munné-Bosch, S., 2019. Malondialdehyde: Facts and artifacts. Plant Physiology, 180(3), 1246-1250, https://doi.org/10.1104/pp.19.00405.
Hara, M., Oki, K., Hoshino, K. and Kuboi, T., 2003. Enhancement of anthocyanin biosynthesis by sugar in radish (Raphanus sativus) hypocotyl. Plant Science 164(2), 259-265, https://doi.org/10.1016/S0168-9452(02)00408-9.
Slinkard, K. and Singleton, V.L., 1977. Total phenol analysis: automation and comparison with manual methods. American Journal of Enology and Viticulture 28(1), 49-55, https://doi.org/10.5344/ajev.1977.28.1.49.
Miliauskas, G., Venskutonis, P.R. and Van Beek, T.A., 2004. Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chemistry, 85(2), 231-237, https://doi.org/10.1016/j.foodchem.2003.05.007.
Heath, R.L. and Packer, L., 1968. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics, 125(1), 189-198, https://doi.org/10.1016/0003-9861(68)90654-1.
Hondo, S., Takahashi, M., Osanai, T., Matsuda, M., Hasunuma, T., Tazuke, A., Nakahira, Y., Chohnan, S., Hasegawa, M. and Asayama, M., 2015. Genetic engineering and metabolite profiling for overproduction of polyhydroxybutyrate in cyanobacteria. Journal of Bioscience and Bioengineering, 120(5), 510-517, https://doi.org/10.1016/j.jbiosc.2015.03.004.
Shrivastav, A., Mishra, S.K., Shethia, B., Pancha, I., Jain, D. and Mishra, S., 2010. Isolation of promising bacterial strains from soil and marine environment for polyhydroxyalkanoates (PHAs) production utilizing Jatropha biodiesel byproduct. International Journal of Biological Macromolecules, 47(2), 283-287, https://doi.org/10.1016/j.ijbiomac.2010.04.007.
Leung, D.Y., Wu, X. and Leung, M.K.H., 2010. A review on biodiesel production using catalyzed transesterification. Applied Energy, 87(4), 1083-1095.
Troschl, C., Meixner, K. and Drosg, B., 2017. Cyanobacterial PHA production—Review of recent advances and a summary of three years’ working experience running a pilot plant. Bioengineering, 4(2), https://doi.org/10.3390/bioengineering4020026.
Li, M., Shi, X., Guo, C. and Lin, S., 2016. Phosphorus deficiency inhibits cell division but not growth in the dinoflagellate Amphidinium carterae. Frontiers in Microbiology, 7, https://doi.org/10.3389/fmicb.2016.00826.
Çelekli, A. and Yavuzatmaca, M., 2009. Predictive modeling of biomass production by Spirulina platensis as function of nitrate and NaCl concentrations. Bioresource Technology, 100(5), 1847-1851, https://doi.org/10.1016/j.biortech.2008.09.042.
Chowdury, K.H., Nahar, N. and Deb, U.K., 2020. The growth factors involved in microalgae cultivation for biofuel production: a review. Water, Energy and Environmental Engineering, 9(4), 185-215, https://doi.org/10.4236/cweee.2020.94012.
Solovchenko, A., Gorelova, O., Karpova, O., Selyakh, I., Semenova, L., Chivkunova, O., Baulina, O., Vinogradova, E., Pugacheva, T., Scherbakov, P., Vasilieva, S., Lukyanov, A. and Lobakova, E., 2020. Phosphorus feast and famine in cyanobacteria: is luxury uptake of the nutrient just a consequence of acclimation to its shortage?. Cells, 9(9), https://doi.org/10.3390/cells9091933.
Jentzsch, L., Grossart, H.-P., Plewe, S., Schulze-Makuch, D. and Goldhammer, T., 2023. Response of cyanobacterial mats to ambient phosphate fluctuations: phosphorus cycling, polyphosphate accumulation and stoichiometric flexibility. ISME Communications, 3(1), https://doi.org/10.1038/s43705-023-00215-x.
Assunção, J., Amaro, H.M., Tavares, T., Malcata, F.X. and Guedes, A.C., 2023. Effects of temperature, pH, and NaCl concentration on biomass and bioactive compound production by Synechocystis salina. Life, 13(1), https://doi.org/10.3390/life13010187.
Soltani, N., Khavari-Nejad, R.A., Tabatabaei Yazdi, M., Shokravi, S. and Fernández-Valiente, E., 2005. Screening of soil cyanobacteria for antifungal and antibacterial activity. Pharmaceutical Biology, 43(5), 455-459, https://doi.org/10.1080/13880200590963871.
Leong, S.Y., Burritt, D.J., Hocquel, A., Penberthy, A. and Oey, I., 2017. The relationship between the anthocyanin and vitamin C contents of red-fleshed sweet cherries and the ability of fruit digests to reduce hydrogen peroxide-induced oxidative stress in Caco-2 cells. Food Chemistry, 227, 404-412, https://doi.org/10.1016/j.foodchem.2017.01.110.
Reyes, L.F. and Cisneros-Zevallos, L., 2007. Degradation kinetics and colour of anthocyanins in aqueous extracts of purple-and red-flesh potatoes (Solanum tuberosum L.). Food Chemistry, 100, 885-894.
Dedaldechamp, F., Uhel, C. and Macheix, J.-J., 1995. Enhancement of anthocyanin synthesis and dihydroflavonol reductase (DFR) activity in response to phosphate deprivation in grape cell suspensions. Phytochemistry, 40, 1357-1360.
Obouayeba, A., Djyh, B.N., Diabate, S., Djaman, J., N'guessan, J.D., Koné, M. and Kouakou, T.H., 2014. Phytochemical and antioxidant activity of roselle (Hibiscus sabdariffa L.) petal extracts. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 5(2), 1453-1465.
Kaurinovic, B. and Vastag, D., 2019. Flavonoids and Phenolic Acids as Potential Natural Antioxidants. London: IntechOpen.
Khajepour, F., Hosseini, S.A., Nasrabadi, R.G. and Markou, G., 2015. Effect of light intensity and photoperiod on growth and biochemical composition of a local isolate of Nostoc calcicol. Applied Biochemistry and Biotechnology, 176, 2279-2289, https://doi.org/10.1007/s12010-015-1717-9.
Le Roux, J.H., 1969. Fischer–tropsch waxes. III. Indications of molecular order in molten wax. Journal of Applied Chemistry, 19(7), 209-212, https://doi:.org/10.1002/jctb.5010190708.
Ansari, S. and Fatma, T., 2016. Cyanobacterial polyhydroxybutyrate (PHB): screening, optimization and characterization. PLoS ONE, 11(6), https://doi.org/10.1371/journal.pone.0158168.
Obruca, S., Sedlacek, P. and Koller, M., 2021. The underexplored role of diverse stress factors in microbial biopolymer synthesis. Bioresource Technology, 326, https://doi.org/10.1016/j.biortech.2021.124767.
Suzuki, T., Tsygankov, A.A., Miyake, J., Tokiwa, Y. and Asada, Y. 1995. Accumulation of poly-(hydroxybutyrate) by a non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides RV at different pH. Biotechnology Letters, 17, 395-400, https://doi.org/10.1007/BF00130796.
Sharma, L. and Mallick, N. 2005. Accumulation of poly-β-hydroxybutyrate in Nostoc muscorum: regulation by pH, light–dark cycles, N and P status and carbon sources. Bioresource Technology, 96(11), 1304-1310, https://doi.org/10.1016/j.biortech.2004.10.009.
Negi, S., Barry, A.N., Friedland, N., Sudasinghe, N., Subramanian, S., Pieris, S., Holguin, F.O., Dungan, B., Schaub, T. and Sayre, R., 2016. Impact of nitrogen limitation on biomass, photosynthesis, and lipid accumulation in Chlorella sorokiniana. Journal of Applied Phycology, 28, 803-812, https://doi.org/10.1007/s10811-015-0652-z.
Singh, A.K., Sharma, L., Mallick, N. and Mala, J., 2017. Progress and challenges in producing polyhydroxyalkanoate biopolymers from cyanobacteria. Journal of Applied Phycology, 29, 1213-1232, https://doi.org/10.1007/s10811-016-1006-1.
Markou, G., 2012. Alteration of the biomass composition of Arthrospira (Spirulina) platensis under various amounts of limited phosphorus. Bioresource Technology, 116, 533-535, https://doi.org/10.1016/j.biortech.2012.04.022.
Satchasataporn, K., Duangsri, C., Charunchaipipat, W., Laloknam, S., Burut-Archanai, S., Powtongsook, S., Akrimajirachoote, N. and Raksajit, W., 2022. Enhanced production of poly-3-hydroxybutyrate and carotenoids by Arthrospira platensis under combined glycerol and phosphorus supplementation. Science Asia, 48, https://doi:10.2306/scienceasia1513-1874.2022.072.
Dadashi, D., Norastehnia, A. and Moradi, F., 2018. Comparative study of Spirulina sp. growth under conditions of different concentrations of nitrogen and phosphorus. 20th National and 8th International Congress on Biology of Iran. Maragheh, Iran, August 22-24, 2018, p. 70.
Haase, S.M., Huchzermeyer, B. and Rath, T., 2012. PHB accumulation in Nostoc muscorum under different carbon stress situations. Journal of Applied Phycology, 24, 157-162, https://doi.org/10.1007/s10811-011-9663-6.
Panda, B., Jain, P., Sharma, L. and Mallick, N., 2006. Optimization of cultural and nutritional conditions for accumulation of poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803. Bioresource Technology, 97(11), 1296-1301, https://doi.org/10.1016/j.biortech.2005.05.013.
Arias, D.M., Uggetti, E., García-Galán, M.J. and García, J., 2018. Production of polyhydroxybutyrates and carbohydrates in a mixed cyanobacterial culture: Effect of nutrients limitation and photoperiods. New Biotechnology, 42, 1-11, https://doi.org/10.1016/j.nbt.2018.01.001.
Hosseini, S.A., Khajepour, F. and Mousavian, Z., 2017. Effects on phosphorus and pH change of Nostoc calcicola medium on production of poly-β-hydroxybutyrate', Shil 5(1), 25-31.
Montiel-Corona, V. and Buitrón, G., 2022. Polyhydroxybutyrate production in one-stage by purple phototrophic bacteria: Influence of alkaline pH, ethanol, and C/N ratios. Biochemical Engineering Journal, 189, https://doi.org/10.1016/j.bej.2022.108715.