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Nitrate Removal from Groundwater Using Immobilized Heterotrophic Algae

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The treatment efficiency of Chlorella sorokiniana and Scenedesmus species, immobilized in sodium alginate, was evaluated for removing nitrate from groundwater. The experiments were performed initially in batch mode and the best-performing conditions were replicated in sequencing batch reactor mode. S. sp. showed a higher nitrate uptake in short term than C. sorokiniana. Immobilized S. sp. and C. sorokiniana cells showed 90% nitrate removal in 9 and 12 days, respectively. The optimal ratio of algal beads/water was found to be 12.5% (v:v). Comparatively, suspended S. sp. cells were able to remove only up to 35% of nitrate in 8 days. Alginate immobilized S. sp. beads were capable of uptaking nitrate for 100 consecutive days in sequencing batch reactor mode. When tested in actual groundwater, 90% of nitrate was eliminated in 2 days without need for any additional carbon source. Immobilized algal beads can be a low-cost alternative technique to remove nitrate from groundwater as they are water-insoluble, non-toxic, easy to harvest, and offer high removal efficiency.

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  • Abreu, A. P., Fernandes, B., Vicente, A. A., Teixeira, J., & Dragone, G. (2012). Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source. Bioresource Technology, 118, 61–66.

  • Agency, U. E. P. (2018). 2018 edition of the drinking water standards and health advisories tables. US EPA.

    Google Scholar 

  • AlMomani, F. A., & & Örmeci, B. (2016). Performance of Chlorella vulgaris, Neochloris Oleoabundans, and mixed indigenous microalgae for treatment of primary effluent, secondary effluent and centrateFares. Ecological Engineering, 280–289.

  • Berdalet, E., Latasa, M., & Estrada, M. (1994). Effects of nitrogen and phosphorus starvation on nucleic acid and protein content of Heterocapsa sp. Journal of Plankton Research, 16(4), 303–316.

  • Breuer, G., Lamers, P. P., Martens, D. E., Draaisma, R. B., & Wijffels, R. H. (2012). The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresource Technology, 217–226.

  • Breuer, G., Lamers, P. P., Martens, D. E., Draaisma, R. B., & Wijffels, R. H. (2013). Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus. Bioresource technology, 1–9.

  • City of Hastings (2016). Aquifer storage and restoration project. Accessed 17 July 2019.

  • Di Caprio, F., Altimari, P., & Pagnanelli, F. (2018). Effect of Ca2+ concentration on Scenedesmus sp. growth in heterotrophic and photoautotrophic cultivation. New biotechnology, 228–235.

  • Eroglu, E., Agarwal, V., Bradshaw, M., Chen, X., Smith, S. M., Raston, C. L., & Iyer, K. S. (2012). Nitrate removal from liquid effluents using microalgae immobilized on chitosan nanofiber mats. Green Chemistry, 14(10), 2682–2685.

  • Fernandez, E., & Galvan, A. (2007). Inorganic nitrogen assimilation in Chlamydomonas. Journal of Experimental Botany, 58(9), 2279–2287.

    Article  CAS  Google Scholar 

  • Figueroa-Martinez, F., Nedelcu, A. M., Smith, D. R., & Reyes-Prieto, A. (2015). When the lights go out: the evolutionary fate of free-living colorless green algae. New Phytologist, 206(3), 972–982.

  • Karlander, E. P., & Krauss, R. W. (1966). Responses of heterotrophic cultures of Chlorella vulgaris Beyerinck to darkness and light. I. Pigment and pH changes. Plant physiology, 41(1), 1–6.

  • Kim, S., Park, J. E., Cho, Y. B., & Hwang, S. J. (2013). Growth rate, organic carbon and nutrient removal rates of Chlorella sorokiniana in autotrophic, heterotrophic and mixotrophic conditions. Bioresource Technology, 8–13.

  • Kleinübing, S. J., Gai, F., Bertagnolli, C., & Silva, M. G. C. D. (2013). Kleinübing, S. J., Gai, F., Bertagnolli, C., & Si Extraction of alginate biopolymer present in marine alga Sargassum filipendula and bioadsorption of metallic ions. Kleinübing, S. J., Gai, F., Bertagnolli, C., & Silva, M. G. C. D. (2013). Extraction of alginate biopolymer presenMaterials Research, 2, 481–488.

  • Kodama, Y. (2016). Time gating of chloroplast autofluorescence allows clearer fluorescence imaging in Planta. Plos One, 11(3), ARTN e0152484.

    Article  CAS  Google Scholar 

  • Lawniczak, A. E., Zbierska, J., Nowak, B., Achtenberg, K., Grzeskowiak, A., & Kanas, K. (2016). Impact of agriculture and land use on nitrate contamination in groundwater and running waters in central-west Poland. Environmental Monitoring and Assessment, 188, (3), ARTN 172, 10.1007/s10661-016-5167-9.

  • Liang, Y., Sarkany, N., & Cui, Y. (2009). Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnology Letters, 7, 1043–1049.

  • Liu, M., Liu, F., Bawa, M. L., & Chen, H. (2012). Nitrate in drinking water: a major polluting component of groundwater in gulf region aquifers, south of Togo. International Journal of Physical Sciences, 7(1), 144–152.

  • Moreno-Garrido, I. (2008). Microalgae immobilization: current techniques and uses. Bioresource Technology, 99(10), 3949–3964.

    Article  CAS  Google Scholar 

  • Moreno-Garrido, I. (2013). Microalgal immobilization methods. In Immobilization of Enzymes and Cells (pp. 327, Totowa–347). Humana Press.

  • Perez-Garcia, O., & Bashan, Y. (2015). Microalgal heterotrophic and mixotrophic culturing for bio-refining: from metabolic routes to techno-economics. In Algal biorefineries (pp. 61-131): Springer.

  • Perez-Garcia, O., Escalante, F. M., de Bashan, L. E., & Bashan, Y. (2011). Heterotrophic cultures of microalgae: metabolism and potential products. Water Research, 45(1), 11–36.

  • Rai, M. P., & Gupta, S. (2016). Growth and lipid production from Scenedesmus sp. under mixotrophic condition for bioenergy application. In Proceedings of the First International Conference on Recent Advances in Bioenergy Research, New Delhi (pp. 159–167): Springer.

  • Rhoades, M. G., Meza, J. L., Beseler, C. L., Shea, P. J., Kahle, A., Vose, J. M., et al. (2013). Atrazine and nitrate in public drinking water supplies and non-Hodgkin lymphoma in Nebraska, USA. USA. Environmental health insights.

  • Ruiz-Marin, A., Mendoza-Espinosa, L. G., & Stephenson, T. (2010). Growth and nutrient removal in free and immobilized green algae in batch and semi-continuous cultures treating real wastewater. Bioresource Technology, 101(1), 58–64.

  • Samejima, H., & Myers, J. (1958). On the heterotrophic growth of Chlorella pyrenoidosa. Microbiology, 18(1), 107–117.

  • Selimoglu, S. M., & Elibol, M. (2010). Alginate as an immobilization material for MAb production via encapsulated hybridoma cells. Critical Reviews in Biotechnology, 30(2), 145–159.

  • Shaker, S., Nemati, A., Montazeri-Najafabady, N., Mobasher, M. A., Morowvat, M. H., & Ghasemi, Y. (2015). Treating urban wastewater: Nutrient removal by using immobilized green algae in batch cultures. International Journal of Phytoremediation, 17(12), 1177–1182.

  • Shi, X.-M., Liu, H.-J., Zhang, X.-W., & Chen, F. (1999). Production of biomass and lutein by Chlorella protothecoides at various glucose concentrations in heterotrophic cultures. Process Biochemistry, 34(4), 341–347.

  • Sorial, G. A., Smith, F. L., Suidan, M. T., Pandit, A., Biswas, P., & Brenner, R. C. (1997). Evaluation of trickle bed air biofilter performance for BTEX removal. Journal of Environmental Engineering-Asce, 123(6), 530–537.

    Article  CAS  Google Scholar 

  • Survey, U. G. (2010). Groundwater use in the United States. USGS.

  • Wang, J., Yang, H., & Wang, F. (2014). Mixotrophic cultivation of microalgae for biodiesel production: status and prospects. Applied Biochemistry and Biotechnology, 7, 3307–3329.

  • Wang, J. F., Liu, J. L., & Liu, T. Z. (2015). The difference in effective light penetration may explain the superiority in photosynthetic efficiency of attached cultivation over the conventional open pond for microalgae. Biotechnology for Biofuels, 8, ARTN 49.

    Article  CAS  Google Scholar 

  • Wheeler, D. C., Nolan, B. T., Flory, A. R., DellaValle, C. T., & Ward, M. H. (2015). Modeling groundwater nitrate concentrations in private wells in Iowa. Science of the Total Environment, 536, 481–488.

    Article  CAS  Google Scholar 

  • Xu, X., Shen, Y., & Chen, J. (2015). Cultivation of Scenedesmus dimorphus for C/N/P removal and lipid production. Electronic Journal of Biotechnology, 18(1), 46–50.

  • Zhang, E., Wang, B., Ning, S., Sun, H., Yang, B., Jin, M., et al. (2012a). Ammonia-nitrogen and orthophosphate removal by immobilized Chlorella sp. isolated from municipal wastewater for potential use in tertiary treatment. African Journal of Biotechnology, 11(24), 6529–6534.

  • Zhang, Y., Ghyselbrecht, K., Vanherpe, R., Meesschaert, B., Pinoy, L., & Van der Bruggen, B. (2012b). RO concentrate minimization by electrodialysis: techno-economic analysis and environmental concerns. Journal of Environmental Management, 107, 28–36.

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The authors received the financial support of the University of Nebraska-Lincoln under Research Council Seed Grant. The National Water Center and United Arab Emirates University (UAEU) also partly financed this project under grant no. G00003297.

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Correspondence to Ashraf Aly Hassan.

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• Utilization of immobilized algae is a practical method for nitrate treatment

• Cultivation of heterotrophic algae reduces retention time needed for treatment

• Actual groundwater is more suitable for nitrate removal than simulated lab water

• In natural water, additional carbon source is not required for successful removal

• Same algae beads could be reused for treatment for repeated cycles up to 100 days

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Mollamohammada, S., Aly Hassan, A. & Dahab, M. Nitrate Removal from Groundwater Using Immobilized Heterotrophic Algae. Water Air Soil Pollut 231, 26 (2020).

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