Investigation and isolation of some diatoms (bacillariophyceae) in the red river in ha noi

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - 🙞🙞🙞🙞🙞🙞 - GRADUATION THESIS INVESTIGATION AND ISOLATION OF SOME DIATOMS (BACILLARIOPHYCEAE) IN THE RED RIVER IN HANOI HANOI, 12/2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - 🙞🙞🙞🙞🙞🙞 - GRADUATION THESIS INVESTIGATION AND ISOLATION OF SOME DIATOMS (BACILLARIOPHYCEAE) IN THE RED RIVER IN HANOI Student name : BUI THI TUOI Student code : 637436 Class : K63-CNSHE Department : Supervisor : Assoc Prof Dr Nguyen Duc Bach BIOTECHNOLOGY HANOI, 12/2022 COMMITMENT I hereby declare that I have written the thesis independently under the guidance of Assoc Prof Dr Nguyen Duc Bach, Faculty of Biotechnology, Faculty of Agronomy in Vietnam National University of Agriculture (VNUA) The data and results written in the thesis are completely honest I have not used any external support except for the cited documents and other sources mentioned at the end of this thesis i ACKNOWLEDGEMENTS First of all, I would like to thank the teachers of the Department of Biotechnology for teaching, imparting knowledge and sharing experiences during my study at the Faculty of Biotechnology I would like to express my deep gratitude to Assoc.Prof.Dr Nguyen Duc Bach, Faculty of Biotechnology, Vietnam National University of Agriculture (VNUA) enthusiastically guided, instructed and helped and also gave ideas and inspirations during the implementation of the thesis topic I would also like to thank the research staff of the Institute of Microalgae Research and Cosmeceutical Medicine for helping me in the research process and completing my graduation thesis Finally, I want to give deep affection and love to my parents for giving me all their enthusiasm, encouragement and motivation throughout my life Hanoi,Deceber 2022 Students Bui Thi Tuoi ii LIST OF CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii LIST OF CONTENTS iii LIST OF ABBREVIATIONS v LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT viii CHAPTER I INTRODUCTION 1.1 Introduction 1.2 Objectives and requirements 1.2.1 Objectives 1.2.2 Requirements 1.3 Scientific significance and practical significance 1.3.1 Scientific meaning 1.3.2 Practical significance CHAPTER II LITERATURE OVERVIEW 2.1 General introduction to microalgae 2.1.1 Classification of microalgae 2.1.2 Biotechnological potential of microalgae 2.2 Introduction to the class of diatoms (Bacillariophyceae) 14 2.2.2 Morphological and biological characteristics of diatoms 14 2.2.3 Applications of diatoms 16 CHAPTER III MATERIALS AND METHODS 17 3.1 Research area 17 3.2 Identification and quantification of microalgae 18 3.2.1 Microalgae collection 18 3.2.2 Identification of microalgae 19 iii 3.3 Diatoms isolation 20 3.3.1 Incubation increases the density of microalgae 20 3.3.2 Isolation on agar medium/solid medium 22 3.3.3 Growing microalgae in liquid medium 23 3.3.4 Morphological description methods 24 3.3.5 Retention of microalgae species 24 3.4 Microalgae biomass culture 26 3.4.1 Installation of a biomass multiplication system 26 3.4.2 Preparation of the culture medium 27 3.4.3 Preparation of microalgae species for growing biomass 30 3.4.4 The effect of the culture environment on algae growth 30 3.4.5 The effect of temperature on algae growth 31 3.4.6 Cell density monitoring 31 PART IV RESULTS AND DISCUSSION 32 4.1 Sampling results 32 4.2 Microalgae isolation results 34 4.2.1 Isolation results 34 4.2.2 Identification of isolated microalgae by morphological characteristics 35 4.2.2.1 Morphology of Nitzschia sp 35 4.2.2.2 Morphology of Navicula sp 36 4.2.2.3 Morphology of Gomphonema sp 36 4.3 Physiological and biochemical characteristics of two species of algae Navicula sp and Nitzschia sp 37 CHAPTER V CONCLUSIONS AND RECOMMENDATIONS 43 5.1 Conclusion 43 5.2 Recommendation 43 REFERENCES 44 iv LIST OF ABBREVIATIONS BBM BG-11 F/2 Strep Peni cell/ml UV PUFA EPA DHA SEDD CBZ DA NMO ASP :Bold's Basal Medium :BG-11 Medium :F/2 Medium : streptomycin : penicillin G :number of cells per ml : Ultraviolet : polyunsaturated fatty acids : axit eicosapentaenoic : Docosa Hexaenoic Acid : self-emulsifying drug delivery system : self-emulsifying phospholipid suspension of carbamazepine : domoic acid : Nylon monofilament : amnesic shellfish poisoning v LIST OF TABLES Table Composition of BG-11 medium with added salt Na SiO 27 Table Composition of BBM medium with added salt Na SiO 28 Table Composition of F/2 medium with added salt Na SiO 29 Table Location and physicochemical parameters during two sampling at sites 32 Table Diatoms density during two sampling at sites 33 Table Growth rate of Navicula sp in different nutrient environments 38 Table Growth rate of Nitzschia sp in different nutrient environments 39 vi LIST OF FIGURES Figure 1: Some typical diatoms 15 Figure 2: Map of water sample sites in the Red River in Hanoi 18 Figure 3: Collecting water samples 19 Figure 4: Quantification in collected water samples 20 Figure 5: Samples after incubation increased algae density 22 Figure 6: Steps of microalgae culture in solid medium 23 Figure 7: Microalgae culture in liquid medium 24 Figure 8: Diatoms isolation process 25 Figure 9: Microalgae culture system 27 Figure 10: Process of preparing culture 30 Figure 11 Nitzschia sp observed under the microscope 40X 35 Figure 12: Navicula sp observed under the microscope 40X 36 Figure 13: Gomphonema sp observed under the microscope 40X 37 Figure 14: Growth curve of Nitzschia sp and Nacuvila sp obtained at different temperatures 41 vii ABSTRACT The study "Investigation and isolation of some diatoms (Bacillariophyceae) in the Red River in Hanoi" was conducted in the laboratory of the Institute for Microalgae and Pharmacosmetics, Vietnam National University of Agriculture (VNUA) from July 2022 to December 2022 The objective of the study was to isolate and culture microalgae belonging to the class Bacillariophyceae collected in the Red River area to enrich microalgae genetic resources, preserve the genetic resources of indigenous microalgae species Water was collected in the Red River basin by a microalgae filter to identify microalgae species present in the river basin The microalgae obtained were evaluated and isolated by a combination of different methods including: sequential dilution, single colony selection and agar culture The experiments were arranged to demonstrate the ratio of physical and chemical variables to the abundance and growth rate of diatoms Although macronutrients are often selected and quantified into conventional culture media to meet the general requirements of many diatoms, the availability and optimization of micronutrients is not a problem are easily affected by the specific physiological conditions of each species The purpose of this study is to characterize the growth characteristics of two species of diatoms are Navicula sp Nitzschia sp was isolated from the Red River in Hanoi in culture experiments under different conditions of culture medium (BG-11, F/2, BBM) with simultaneous addition of silicate salts (Na SiO ) into the culture medium to increase cell density, culture temperature (10℃, 15℃ and 20℃) In the medium BG-11 supplemented with salt Na SiO , under constant lighting conditions, both Navicula sp and Nitzschia viii dilutions were made in 50 ml of BG-11 nutrient medium supplemented with Na SiO , inoculating each microalgae for three days, confirming the presence of a single species Inoculated by scraping a liquid sample in a test tube containing 10 ml of agar With this method, we obtained pure microalgae samples of two species of Nitzschia sp and Navicula sp 4.2.2 Identification of isolated microalgae by morphological characteristics 4.2.2.1 Morphology of Nitzschia sp Nitzschia is a marine pennate diatom known to be responsible for Amnesic shellfish poisoning (ASP) due to domoic acid (DA) production, a type of neurotoxin The cell size is approximately 40-50 µm in length and µm in width The surface of the cell cortex is long and rod-shaped, with two spiky cell poles There is no central valve, but valves running along one side of the body are difficult to observe under a microscope Chromatophores are present in abundance Based on the morphological characteristics of the standard samples of the algae, the observed species is likely to be Nitzschia sp ` Figure 11 Nitzschia sp observed under the microscope 40X 35 4.2.2.2 Morphology of Navicula sp Navicula is a genus of diatoms Navicula belongs to the family Naviculaceae Individuals with relatively large cells and thick shells live solitarily The surface of the cell shell is long and rhombus-shaped, with rounded ends The two ends in the middle of the shell surface are clearly enlarged and far apart, and the two shell faces have the same structure Cell length is 25-30 μm, width is about μm, and this strain has characteristics of the genus Navicula, species Navicula sp Figure 12: Navicula sp observed under the microscope 40X 4.2.2.3 Morphology of Gomphonema sp This strain has characteristics of the genus Gomphonemataceae, species Gomphonema sp It is 30-40 µm in length and 10-14 µm in width, with asymmetrical markings along the axis, a wide and truncated head, and a fissure extending from the main cell of the body to the two poles Additionally, the cells have only transverse striations, may be parallel pigmented, and contain two cytoskeletons Therefore, preliminarily identify the strain as Gomphonema sp 36 Figure 13: Gomphonema sp observed under the microscope 40X 4.3 Physiological and biochemical characteristics of two species of algae Navicula sp and Nitzschia sp a) Effect of nutrition on the growth of microalgae Nutrition is an essential factor in microalgae culture The nutrient environment affects the growth and cell density of microalgae The biomass multiplication ability of two species of diatoms was investigated on media BG-11, BBM and F/2, all medias were supplemented with Na SiO salt (10mg/l) ) at a temperature of 25℃, pH 8.5 After 20 days of monitoring and counting cell density every days The assessment of the growth rate is recorded every days and is summarized in the table below 37 Table Growth rate of Navicula sp in different nutrient environments Culture time Density of microalgae Navicula sp (x 105 cells) (days) BG-11 BBM F/2 1,35 ± 0,01 1,35 ± 0,01 1,35 ± 0,01 1,3 ± 0,02 1,35 ± 0,1 1,25 ± 0,02 2,05 ± 0,01 1,75 ± 0,01 1,95 ± 0,01 3,35 ± 0,01 2,85 ± 0,01 3,1 ± 0,01 4,95 ± 0,01 3,40 ± 0,01 3,75 ± 0,01 10 6,3 ± 0,02 4,2 ± 0,02 5,25 ± 0,2 12 8,2 ± 0,02 5,0 ± 0,01 6,35 ± 0,01 14 10,5 ± 0,01 7,4 ± 0,03 9,0 ± 0,02 16 15,45 ± 0,1 14,80 ± 0,01 13,15 ± 0,01 18 21,15 ± 0,02 13,50 ± 0,03 17,45 ± 0,01 20 20,15 ± 0,01 12,05 ± 0,01 16,65 ± 0,01 38 Table Growth rate of Nitzschia sp in different nutrient environments Culture time Density of microalgae Nitzschia sp (x 105 cells) (days) BG-11 BBM F/2 1,00 ± 0,01 1,00 ± 0,01 1,00 ± 0,01 1,05 ± 0,02 1,00 ± 0,1 1,05 ± 0,02 2,00 ± 0,1 1,15 ± 0,02 1,85 ± 0,01 3,15 ± 0,01 2,75 ± 0,01 3,00 ± 0,01 4,95 ± 0,01 3,40 ± 0,01 3,75 ± 0,01 10 6,13 ± 0,2 4,03 ± 0,02 5,05 ± 0,2 12 8,2 ± 0,02 5,0 ± 0,01 6,1 ± 0,01 14 8,0 ± 0,1 4,8 ± 0,01 5,9 ± 0,01 16 7,12 ± 0,2 4,03 ± 0,02 5,25 ±0 ,2 18 6,15 ± 0,25 4,00 ± 0,002 5,05 ± 0,2 20 5,15 ± 0,2 3,5 ± 0,01 4,2 ± 0,1 Through the above data table, it was shown that both diatoms (Bacillariophyta) had high cell density in BG-11 medium added salt Na SiO However, in BBM medium, both species showed low cell density Thus, BG11 medium was used to culture and collect the biomass of microalgae Navicula sp., Nitzschia sp for 20 days for further experiments The maximum cell density in Navicula sp appeared on day nineteen with 2115000 cells/ml with a 39 final density at day 20 of 201500 cells/ml in BG-11 medium supplemented with Na2SiO3 salt, under culture conditions at an average temperature of 20-25℃ and pH is 8.5 While for Nitzschia sp cultured for maximum density on day 12 with 82000 cells/ml ending with 51500 cells/ml at 1ml/L dose of BG-11 supplemented with Na2SiO3 salt and cultured at 20- 25℃ b) Effect of temperature on the growth of diatoms (Bacillariophyta) Temperature is a particularly important influencing factor on the growth of algae as well as living organisms in the world Each species of algae has a suitable temperature range for growth; most diatoms can withstand temperatures between 16°C and 27°C, with an optimal range of 20°C to 25°C Temperatures below 10°C and above 35°C can lead to death (Fulks and Main, 1991) Temperature also affects the determination of species that will thrive in outdoor conditions, as well as the growth of each species under certain environmental temperature conditions Investigate the effect of temperature on the growth rate of microalgae from the temperature thresholds of 10℃, 15℃ and 20℃, pH 8.5 on BG-11 medium supplemented with Na SiO salt with cell density onthefirstday First is 1x105 cells/ml After 20 days of culture, the growth rate of microalgae is shown in the following chart: 40 Figure 14: Growth curve of Nitzschia sp and Nacuvila sp obtained at different temperatures A: Nitzschia sp B: Nacuvila sp Through the chart, we can see that both diatoms grow well at the temperature range of 15℃-20℃ At a temperature of 10℃, Nacuvila sp growth is slower, there is a clear stratification in the growth rate of algae Nitzschia sp There was no big difference in cell density when cultured at temperatures from 10℃ to 20℃ Thus, we can see that when storing algae in the same nutrient environment but with different storage temperatures, the growth of algae when growing biomass is also different It is the storage temperature that affects the cleavage of algae cells, so the number of cells in each batch and at each time is also different At the temperature threshold of 20℃ microalgae grow to the highest density of 820,100 cells/ml, but at the threshold of 10℃ microalgae reach the highest density of 5000000 cells/ml Clearly show the difference in these two temperature thresholds.When the number of algal cells participating in cleavage is high, the density of algae increases faster When the algae reaches the maximum density, the nutrient content is gradually depleted, the 41 ability of each cell to receive light decreases due to the self-obstruction, so the algae gradually decrease in density and die in the following days 42 CHAPTER V CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusion Isolation of Navicula sp and Nitzschia sp was carried out during the removal of microalgae present in the collected water samples, using BG-11 medium supplemented with Na SiO and solid agar Collected water samples were incubated at an incubation temperature of 25℃ to 27℃ under continuous lighting conditions with a light intensity of 2500-3000 lux at the Institute for Microalgae and Pharmacosmetic, Vietnam National University of Agriculture (VNUA) Isolation was performed Navicula sp lasted for 22 days and Nitzschia sp isolated for 27 days using solid culture 1% agar and by serial dilution with BG-11 nutrient medium supplemented with Na SiO A suitable environment for raising Navicula sp and Nitzschia sp is BG11 medium with added salt Na SiO at a temperature of 20℃-25℃, a pH of 8.5, and a light intensity of 2500-3000 lux The maximum cell density of Navicula sp was observed on day 18, with 2115000 cells/ml, and a final density of 201500 cells/ml on day 20 For Nitzschia sp., the maximum density was observed on day 12, with 82000 cells/ml, and a final density of 51500 cells/ml 5.2 Recommendation It is necessary to collect and isolate more varieties of class Bacillariophyceae in the Red River in Hanoi Continue to study in more intensive conditions to classify the remaining algae species Conduct experiments to further analyze the suitable environmental conditions for suitable growth of native gene-enriched algae 43 REFERENCES A.Shirora 1966 The plankton of south Viet Nam Fresh Water and Marine Plankton Overseas Technical Cooperation Agency Japan Abdelhamid FM, Elshopakey GE, Aziza AE (2020) Ameliorative effects of dietary Chlorella vulgaris and β-glucan against diazinon-induced toxicity in Nile tilapia (Oreochromis niloticus) Fish & shellfish immunology, 96, 213222 https://doi.org/10.1016/j.fsi.2019.12.009 Acquah C, Tibbetts SM, Pan S, Udenigwe C (2020) Chapter 19 - Nutritional quality and bioactive properties of proteins and peptides from microalgae In: Jacob-Lopes E, Manzoni Maroneze M, Queiroz MI, Queiroz Zepka L (eds) Handbook of Microalgae-Based Processes and Products, Academic Press, Pages 493-531, https://doi.org/10.1016/B978-0-12-8185360.00019-1 Al-Jabri H, Das P, Khan S, Thaher M, Abdul Quadir M (2021) Treatment of wastewaters by microalgae and the potential applications of the produced biomass A - review Water 13(1):27 https://doi.org/10.3390/w13010027 Amorim ML, Soares J, Coimbra JS dos R, Leite M de O, Albino LFT, Martins MA (2020) Microalgae proteins: production, separation, isolation, quantification, and application in food and feed https://doi.org/10.1080/10408398.2020 Azimi A, Azari A, Rezakazemi M, Ansarpour M (2017) Removal of heavy metals from industrial wastewaters: a review ChemBioEng Reviews, 4: 3759 https://doi.org/10.1002/cben.201600010 Barsanti L, Gualtieri P (2014) Algae: anatomy, biochemestry and biotechnology 2nd edn CRC Press Beardall J, Berman T, Heraud P, Kadiri MO, Light BR, Patterson G, Roberts S, Sulzberger B, Sahan E, Uehlinger U, Wood B (2001 a) A comparison of methods for detection of phosphate limitation in microalgae Aquat Sci 63(1):107-121 https://doi.org/10.1007/Pl00001342 Bastidas, O, 2008 Neubauer chamber formula, chamber calculations CELEROMICS http://www.celeromics.com/es/resources/Technical%20Notes/neubauercha mber-cell-concentration/formula-chamber-neubauer-concentration Date of access: 3/08/15 44 Blanco AM, Moreno J, Del Campo JA, Rivas J, Guerrero MG (2017) Outdoor cultivation of lutein-rich cells of Muriellopsis sp in open ponds Appl Microbiol Biotechnol 73, 1259–1266 https://doi.org/10.1007/s00253006-0598-9 10.Borowitzka MA (2018, b) Chapter - Microalgae in Medicine and Human Health: A Historical Perspective In: Levine IA, Fleurence J (eds) Microalgae in Health and Disease Prevention, Academic Press, Pages 195210 https://doi.org/10.1016/B9780-12-811405-6.00009-8 11.Borowitzka,MA (2018, a) Chapter - Biology of Microalgae Levine IA, Fleurence J (eds) Microalgae in Health and Disease Prevention, Academic Press, Pages 23-72 https://doi.org/10.1016/B978-0-12-811405-6.00003-7 12.Camacho F, Macedo A, Malcata F “Potential industrial applications and commercialization of microalgae in the functional food and feed industries: A short review” (2019) 13.Caporgno MP, Mathys A (2018) Trends in microalgae incorporation into innovative food products with potential health benefits Frontiers in Nutrition, Volume https://doi.org/10.3389/fnut.2018.00058 14.Carvalho M, Montero D, Rosenlund G, Fontanillas R, Ginés R, Izquierdo M (2020) Effective complete replacement of fish oil by combining poultry and microalgae oils in practical diets for gilthead sea bream (Sparus aurata) fingerlings Aquaculture, Volume 529 https://doi.org/10.1016/j.aquaculture.2020.735696 15.Chatsungnoen T, Chisti Y (2019) Chapter 11 - Flocculation and electroflocculation for algal biomass recovery In: A Pandey, J-S Chang, CR Soccol, D-J Lee, Y Chisti (eds) Biomass, Biofuels, Biochemicals, Biofuels from Algae (Second Edition), Elsevier, Pages 257-286, ISBN 9780444641922 https://doi.org/10.1016/B978-0-444-64192-2.00011-1 16.Chatsungnoen T, Chisti Y (2019) Chapter 11 - Flocculation and electroflocculation for algal biomass recovery In: A Pandey, J-S Chang, CR Soccol, D-J Lee, Y Chisti (eds) Biomass, Biofuels, Biochemicals, Biofuels from Algae (Second Edition), Elsevier, Pages 257-286, ISBN 9780444641922 https://doi.org/10.1016/B978-0-444-64192-2.00011-1 17.Cole, G M., Ma, Q.-L., and Frautschy, S A (2010) Dietary fatty acids and the aging brain Nutr Rev 68, (Suppl 2), S102–S111 18.Colla, E , et al "Microalgae: A new and promising source of food." (2020) 19.Connelly, R "Second-Generation Biofuel from High-Efficiency AlgalDerived Biocrude." Bioenergy Research Advances & Applications (2014) 45 20.Da Silva Vaz, Bds , et al "Microalgae as a new source of bioactive compounds in food supplements." Current Opinion in Food Science 7(2016) 21.Dineshbabu G, Goswami G, Kumar R, Sinha A, Das D (2019) Microalgae– nutritious, sustainable aqua- and animal feed source, Journal of Functional Foods, Volume 62, 103545 https://doi.org/10.1016/j.jff.2019.103545 22.Dolatabadi, J E N., Mashinchian, O., Ayoubi, B., Jamali, A A., Mobed, A., Losic, D., et al (2011) Optical and electrochemical DNA nanobiosensors TrAC Trends Anal Chem 30, 459–472 doi: 10.1016/j.trac.2010.11.010 23.Dyall, S C (2015) Long-chain omega-3 fatty acids and the brain: a review of the independent and shared effects of EPA, DPA and DHA Front Aging Neurosci 7:52 doi: 10.3389/fnagi.2015.00052 24.Ferreira AS, Ferreira SS, Correia A, Vilanova M, Silva TH, Coimbra MA, Nunes C (2020) Reserve, structural and extracellular polysaccharides of Chlorella vulgaris: A holistic approach Algal Research, 45, 101757 https://doi.org/10.1016/j.algal.2019.101757 25.Fulks W And Main K L (Eds.) (l991) Rotifer and Microalgae Culture Systems Proceedings of a U.S - Asia Workshop, Honolulu, Hawaii, 28-31 January 1991,3-l2,The Oceanic Institute, Honolulu, 364 pp 26.Halarry B Erica S (1982) Culturing algae A guide for schools and colleges 27.Henchion M, Hayes M, Mullen AM, Fenelon M, Tiwari B Future Protein Supply and Demand: Strategies and Factors Influencing a Sustainable Equilibrium Foods 2017; 6(7):53 https://doi.org/10.3390/foods6070053 28.Hunter MC, Smith RG, Schipanski ME, Atwood LW, Mortensen DA (2017) Agriculture in 2050: Recalibrating Targets for Sustainable Intensification BioScience, Volume 67, Issue 4, Pages 386–391 https://doi.org/10.1093/biosci/bix010 29.Jin, P., and Agustí, S (2018) Fast adaptation of tropical diatoms to increased warming with trade-offs Sci Rep 8:17771 30.Koreivienė J, Valčiukas R, Karosienė J, Baltrėnas P (2014) Testing of Chlorella/Scenedesmus microalgae consortia for remediation of wastewater, CO2 mitigation and algae biomass feasibility for lipid production J Environ Eng Landsc Manag., Volume 22, Pages 105-114 https://doi.org/10.3846/16486897.2013.911182 31.Madeira MS, Cardoso C, Lopes PA, Coelho D, Afonso C, Bandarra NM, Prates JAM (2017) Microalgae as feed ingredients for livestock production 46 and meat quality: A review Livestock Science, Volume 205, Pages 111-121 https://doi.org/10.1016/j.livsci.2017.09.020 32.Martínez-Roldán A de J, Cizares-Villanueva RO (2020) Chapter Wastewater treatment based in microalgae In: JacobLopes E, Manzoni Maroneze M, Queiroz MI, Queiroz Zepka L (eds) Handbook of MicroalgaeBased Processes and Products Academic Press 33.Mata TM, Martins AA, Caetano NS (2010) Microalgae for biodiesel production and other applications: A review Renewable and Sustainable Energy Reviews 14: 217-232 https://doi.org/10.1016/j.rser.2009.07.020 34.Matos J, Cardoso C, Bandarra NM, Afonso C (2017) Microalgae as healthy ingredients for functional food: a review Food Funct Aug 1;8(8):26722685 https://doi.org/10.1039/C7FO00409E 35.Mishra, M., Arukha, A P., Bashir, T., Yadav, D., and Prasad, G B K S (2017) All new faces of diatoms: potential source of nanomaterials and beyond Front Microbiol 8:1239 doi: 10.3389/fmicb.2017.01239 36.Murphy CD “The microbial cell factory”.Org Biomol Chem., 10, 1949 (2012) 37.Pereira, I , Rangel, A , Chagas, B , Moura, B D , & Castro, C (2021) Microalgae Growth under Mixotrophic Condition Using Agro-Industrial Waste: A Review 38.Posadas E, García-Encina P-A, Soltau A, Domínguez A, Díaz I, Moz R (2013) Carbon and nutrient removal from centrates and domestic wastewater using algal–bacterial biofilm bioreactors Bioresource Technology, Volume 139, Page 50-58 39.Rizwan, M , Mujtaba, G , Memon, S A , Lee, K , & Rashid, N (2018) Exploring the potential of microalgae for new biotechnology applications and beyond: a review Renewable and Sustainable Energy Reviews, 92(SEP.), 394-404 40.Salama E-S, Kurade MB, Abou-Shanab RAI, El-Dalatony MM, Yang I-S, Min B, Jeon B-H (2017) Recent progress in microalgal biomass production coupled with wastewater treatment for biofuel generation Renewable and Sustainable Energy Reviews, Volume 79, Pages 1189-1211, https://doi.org/10.1016/j.rser.2017.05.091 41.Sharma, N , Simon, D P , Diaz-Garza, A M , Fantino, E , & I DesgagnéPenix (2021) Diatoms biotechnology: various industrial applications for a greener tomorrow Frontiers in Marine Science, 47 42.Singh, A K., Sharma, N., Farooqi, H., Abdin, M Z., Mock, T., and Kumar, S (2017) Phycoremediation of municipal wastewater by microalgae to produce biofuel Int J Phytoremediation 19, 805–812 doi: 10.1080/15226514.2017.1284758 43.Tréguer, P., Nelson, D M., Van Bennekom, A J., DeMaster, D J., Leynaert, A., and Quéguiner, B (1995) The silica balance in the world ocean: a reestimate Science 268, 375–379 doi: 10.1126/science.268.5209.375 44.Uyeh DD, Mallipeddi R, Pamulapati T, Park T, Kim J, Woo S, Ha Y (2018) Interactive livestock feed ration optimization using evolutionary algorithms Computers and Electronics in Agriculture, Volume 155, Pages 1-11 https://doi.org/10.1016/j.compag.2018.08.031 45.Vossen, E, Raes K, Van Mullem D, De Smet S (2017) Production of docosahexaenoic acid (DHA) enriched loin and dry cured ham from pigs fed algae: Nutritional and sensory quality Eur J Lipid Sci Technol., 119: 1600144 https://doi.org/10.1002/ejlt.201600144 46.Wang, H., and Daggy, B P (2017) The role of fish oil in inflammatory eye diseases Biomed Hub 2, 1–12 doi: 10.1159/000455818 47.Wang, X C., and Zhao, H M (2007) Uptake and biodegradation of polycyclic aromatic hydrocarbons by marine seaweed J Coastal Res SI50, 1056–1061 48.Watson JE, Venter O, Lee J, Jones KR, Robinson JG, Possingham HP et al (2018) Protect the last of the wild Nature 563, 27–30 49.Wen, Z Y., and Chen, F (2001a) A perfusion–cell bleeding culture strategy for enhancing the productivity of eicosapentaenoic acid by Nitzschia laevis Appl Microbiol Biotechnol 57, 316–322 doi: 10.1007/s002530100786 50.Xia, S., Wang, K., Wan, L., Li, A., Hu, Q., and Zhang, C (2013) Production, characterization, and antioxidant activity of fucoxanthin from the marine diatom Odontella aurita Mar Drugs 11, 2667–2681 doi: 10.3390/md11072667 51.Yaakob Z, Ali E, Zainal A, Mohamad M, Takriff MS (2014) An overview: Biomolecules from microalgae for animal feed and aquaculture J of Biol Res-Thessaloniki 21, https://doi.org/10.1186/2241-5793-21-6 52.Zhang, et al.(2013) "Expression of fatty acid desaturase genes and fatty acid accumulation in Chlamydomonas sp ICE-L under salt stress." 53.Zhang, Z , and S Mitmesser (2018)."Dietary Intakes of EPA and DHA Omega-3 Fatty Acids among U.S Pregnant Women." 54.http://diatoms.org/species 48 55.http://www.unifr.ch/biol/ecology/ebert/lab/counting/ 56.https://diatoms.org/species 57.https://www.credenceresearch.com/report/algae-products-market 58.https://www.fao.org/documents/card/en/c/cb4474en 49