Environmental Science and Pollution Research

, Volume 24, Issue 34, pp 26763–26777 | Cite as

Optimisation of critical medium components and culture conditions for enhanced biomass and lipid production in the oleaginous diatom Navicula phyllepta: a statistical approach

  • Sanyo Sabu
  • Isaac Sarojini Bright Singh
  • Valsamma Joseph
Research Article


Diatoms hold great promise as potential sources of biofuel production. In the present study, the biomass and lipid production in the marine diatom Navicula phyllepta, isolated from Cochin estuary, India and identified as a potential biodiesel feedstock, were optimized using Plackett-Burman (PB) statistical experimental design followed by central composite design (CCD) and response surface methodology (RSM). The growth analyses of the isolate in different nitrogen sources, salinities and five different enriched sea water media showed the best growth in the cheapest medium with minimum components using urea as nitrogen source at salinity between 25 and 40 g kg−1. Plackett-Burman experimental analyses for screening urea, sodium metasilicate, sodium dihydrogen phosphate, ferric chloride, salinity, temperature, pH and agitation influencing lipid and biomass production showed that silicate and temperature had a positive coefficient on biomass production, and temperature had a significant positive coefficient, while urea and phosphate showed a negative coefficient on lipid content. A 24 factorial central composite design (FCCD) was used to optimize the concentration of the factors selected. The optimized media resulted in 1.62-fold increase (64%) in biomass (1.2 ± 0.08 g L−1) and 1.2-fold increase (22%) in estimated total lipid production (0.11 ± 0.003 g L−1) compared to original media within 12 days of culturing. A significantly higher biomass and lipid production in the optimized medium demands further development of a two-stage strategy of biomass production followed by induction of high lipid production under nutrient limitation or varying culture conditions for large-scale production of biodiesel from the marine diatom.


Diatom Navicula phyllepta Biodiesel Growth medium Plackett-Burman design Response surface methodology 



The authors acknowledge Dr. Sunitha Poulose and Dr. Sareen Sarah John for supporting analyses using Design Expert Software.

Funding information

The authors acknowledge the University Grants Commission, Government of India for the financial support under the major research grant (File No. 41-568/2012 (SR)),

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Acıkel U, Erşan M, Açıke YS (2010) Optimization of critical medium components using response surface methodology for lipase production by Rhizopus delemar. Food Bioprod Process 88:31–39Google Scholar
  2. Adenan NR, Yusoff FMD, Sheriff M (2013) Effect of salinity and temperature on growth of diatoms and green algae. J Fish Aqua Sci 8:397–404CrossRefGoogle Scholar
  3. Alverson AJ (2007) Strong purifying selection in the silicon transporters of marine and freshwater diatoms. Limnol Oceanogr 52:1420–1429CrossRefGoogle Scholar
  4. Amin SA, Parker MS, Armbrust EV (2012) Interactions between diatoms and bacteria. Microbiol Mol Biol Rev 76:667–684CrossRefGoogle Scholar
  5. Andersen RA, Berges JA, Harrison PJ, Watanabe MM (2005) Recipes for freshwater and seawater media. In: Anderson RA (ed) Algal culturing techniques. Elsevier, Amsterdam, pp 429–538Google Scholar
  6. Araujo GS, Matos LJBL, Gonçalves LRB, Fernandes FAN, Farias WRL (2011) Bioprospecting for oil producing microalgal strains: evaluation of oil and biomass production for 10 microalgal strains. Bioresour Technol 102:5248–5250CrossRefGoogle Scholar
  7. Arora N, Patel A, Pruthi PA, Pruthi V (2016) Synergistic dynamics of nitrogen and phosphorous influences lipid productivity in Chlorella minutissima for biodiesel production. Bioresour Technol 213:79–87CrossRefGoogle Scholar
  8. Azma M, Mohamed MS, Mohamad R, Rahim RA, Ariff AB (2011) Improvement of medium composition for heterotrophic cultivation of green microalgae, Tetraselmis suecica, using response surface methodology. Biochem Eng J 53:187–195CrossRefGoogle Scholar
  9. Bartleya ML, Boeinga WJ, Corcorana AA, Holguinb FO, Schaubb T (2013) Effects of salinity on growth and lipid accumulation of biofuel microalga Nannochloropsis salina and invading organisms. Biomass Bioenergy 54:83–88CrossRefGoogle Scholar
  10. Becker EW (1994) Microalgae: biotechnology and microbiology. Cambridge University Press, CambridgeGoogle Scholar
  11. Bellinger EG, Sigee DC (2015) Freshwater algae: identification, enumeration and use as bioindicators, 2nd edn. Wiley Blackwell, UKGoogle Scholar
  12. Bilad MR, Arafat HA, Vankelecom FJ (2014) Membrane technology in microalgae cultivation and harvesting: a review. Biotechnol Adv 32:1283–1300CrossRefGoogle Scholar
  13. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Phys 37:911–917CrossRefGoogle Scholar
  14. Buhmann M, Schleheck D, Windler M, Kroth PG (2011) Bacteria influence diatom biofilm formation. Eur J Phycol 46:80–80Google Scholar
  15. Chen JJ, Li YR, Lai WL (2014) Application of experimental design methodology for optimization of biofuel production from microalgae. Biomass Bioenergy 64:11–19CrossRefGoogle Scholar
  16. Chen JJ, Li YR, Xie MZ, Chiu CY, Liao SW, Lai WL (2012) Factorial design of experiment for biofuel production by Isochrysis galbana. Int Proc Chem Biol Environ Eng 33:91–95Google Scholar
  17. Cheng KC, Ren M, Ogden KL (2013) Statistical optimization of culture media for growth and lipid production of Chlorella protothecoides UTEX 250. Bioresour Technol 128:44–48CrossRefGoogle Scholar
  18. Clavero E, Hernandez-Marine M, Grimalt JO, Garcia-Pichel F (2000) Salinity tolerance of diatoms from thalassic hypersaline environments. J Phycol 36:1021–1034CrossRefGoogle Scholar
  19. Coull BC (1999) Role of meiofauna in estuarine soft-bottom habitats. Aust J Ecol 24:327–343CrossRefGoogle Scholar
  20. Crofcheck C, Xinyi E, Shea A, Montross M, Crocker M, Andrews R (2012) Influence of media composition on the growth rate of Chlorella vulgaris and Scenedesmus acutus utilized for CO2 mitigation. J Biochem Tech 4:589–594Google Scholar
  21. Dawes CJ (1998) Microalgae and their communities, 2nd edn. Marine botany, John Wiley and Sons, NY, pp 168–204Google Scholar
  22. D’Ippolito G, Sardo A, Paris D, Vella FM, Adelfi MG, Botte P, Gallo C, Fontana A (2015) Potential of lipid metabolism in marine diatoms for biofuel production. Biotechnol Biofuels 8:1–28CrossRefGoogle Scholar
  23. Doghri I, Lavaud J, Dufour A, Bazire A, Lannelu I, Sablé S (2016) Cell-bound exopolysaccharides from an axenic culture of the intertidal mudflat Navicula phyllepta diatom affect biofilm formation by benthic bacteria. J Appl Phycol:1–13Google Scholar
  24. Duong VT, Thomas-Hall SR, Schenk PM (2015) Growth and lipid accumulation of microalgae from fluctuating brackish and sea water locations in South East Queensland-Australia. Front Plant Sci 6:359CrossRefGoogle Scholar
  25. Eustance E, Gardner RD, Moll KM, Menicucci J, Gerlach R, Peyton BM (2013) Growth, nitrogen utilization and biodiesel potential for two chlorophytes grown on ammonium, nitrate or urea. J Appl Phycol 25:1663–1677CrossRefGoogle Scholar
  26. Fakhry EM, El Maghraby DM (2015) Lipid accumulation in response to nitrogen limitation and variation of temperature in Nannochloropsis salina. Bot Stud 56:6CrossRefGoogle Scholar
  27. Fawzy MA (2017) Fatty acid characterization and biodiesel production by the marine microalga Asteromonas gracilis: statistical optimization of medium for biomass and lipid enhancement. Mar Biotechnol:1–13Google Scholar
  28. Fields MW, Hise LEJ, Bell T, Gardner RD, Corredor L, Moll K, Peyton BM, Characklis GW, Gerlach R (2014) Sources and resources: importance of nutrients, resource allocation, and ecology in microalgal cultivation for lipid accumulation. Appl Microbiol Biotechnol 98:4805–4816CrossRefGoogle Scholar
  29. Gagneux-Moreaux S, Moreau C, Cosson RP (2007) Diatom artificial medium (DAM): a new artificial medium for the diatom Haslea ostrearia and other marine microalgae. J Appl Phycol 19:549–556CrossRefGoogle Scholar
  30. García N, López-Elías JA, Miranda A, Martínez-Porchas M, Huert N, García A (2012) Effect of salinity on growth and chemical composition of the diatom Thalassiosira weissflogii at three culture phases. Lat Am J Aquat Res 40:435–440CrossRefGoogle Scholar
  31. Ghadge SV, Raheman H (2006) Process optimization for biodiesel production from mahua (Madhuca indica) oil using response surface methodology. Bioresour Technol 97:379–384CrossRefGoogle Scholar
  32. Glass JB, Wolfe-Simon F, Anbar AD (2009) Coevolution of metal availability and nitrogen assimilation in cyanobacteria and algae. Geobiology 7:100–123CrossRefGoogle Scholar
  33. Guillard RRL (1973) Division rates. In: Stein (ed) Handbook of phycological methods. Vol. 1. Cambridge University Press, Cambridge, pp 289–312Google Scholar
  34. Guillard RRL (1975) Culturing of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH (eds) Culture of marine invertebrates animals. Plenum Press, NY, pp 26–60Google Scholar
  35. Guillard RRL, Hargraves PE (1993) Stichochrysis immobilis is a diatom, not a chrysophyte. Phycologia 32:234–236CrossRefGoogle Scholar
  36. Guillard RR, Sieracki MS (2005) Counting cells in cultures with the light microscope. In: Anderson RA (ed) Algal culturing techniques, 1st edn. Academic Press, Elsevier, pp 239–252Google Scholar
  37. Hildebrand M, Dahlin K (2000) Nitrate transporter genes from the diatom Cylindrotheca fusiformis (Bacillariophyceae): mRNA levels controlled by nitrogen source and by the cell cycle. J Phycol 36:702–713CrossRefGoogle Scholar
  38. Hildebrand M, Davis AK, Smith SR, Traller JC, Abbriano R (2012) The place of diatoms in the biofuels industry. Biofuels 3:221–240CrossRefGoogle Scholar
  39. Huang X, Huang Z, Wen W, Yan J (2013) Effects of nitrogen supplementation of the culture medium on the growth, total lipid content and fatty acid profiles of three microalgae (Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis). J Appl Phycol 25:129–137CrossRefGoogle Scholar
  40. Imamura S, Terashita M, Ohnuma M, Maruyama S, Minoda A, Weber AP, Inouye T, Sekine Y, Fujita Y, Omata T, Tanaka K (2010) Nitrate assimilatory genes and their transcriptional regulation in a unicellular red alga Cyanidioschyzon merolae: genetic evidence for nitrite reduction by a sulfite reductase-like enzyme. Plant Cell Physiol 51(5):707–717CrossRefGoogle Scholar
  41. Ji F, Hao R, Liu Y, Li G, Zhou Y, Dong R (2013) Isolation of a novel microalgae strain Desmodesmus sp. and optimization of environmental factors for its biomass production. Bioresour Technol 148:249–254CrossRefGoogle Scholar
  42. Jia Z, Liu Y, Daroch M, Geng S, Cheng JJ (2014) Screening, growth medium optimisation and heterotrophic cultivation of microalgae for biodiesel production. Appl Biochem Biotechnol 173:1667–1679CrossRefGoogle Scholar
  43. Joseph MM, Renjith KR, John G, Nair SM, Chandramohanakumar N (2016) Biodiesel prospective of five diatom strains using growth parameters and fatty acid profiles. Biofuels:1–9Google Scholar
  44. Juneja A, Ceballos RM, Murthy GS (2013) Effects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production: a review. Energies 6:4607–4638CrossRefGoogle Scholar
  45. Kamalanathan M, Pierangelini M, Shearman LA, Gleadow R, Beardall J (2015) Impacts of nitrogen and phosphorus starvation on the physiology of Chlamydomonas reinhardtii. J Appl Phycol 28:1509–1520CrossRefGoogle Scholar
  46. Karthikeyan P, Jayasudha S, Sampathkumar P, Manimaran K, Santhoshkumar C, Ashokkumar S, Ashokprabu V (2010) Effect of industrial effluent on the growth of marine diatom, Chaetoceros simplex (Ostenfeld, 1901). J Appl Sci Environ Manag 14:35–37Google Scholar
  47. Kim G, Mujtaba G, Lee K (2016) Effects of nitrogen sources on cell growth and biochemical composition of marine chlorophyte Tetraselmis sp. for lipid production. Algae 31:257–266CrossRefGoogle Scholar
  48. Krammer K, Lange-Bertalot H (1986) Bacillariophyceae. 1. Teil: Naviculaceae. In: Ettl H, Gerloff J,Heynig H, Mollenhaurer D (eds) Süsswasserflora von Mitteleuropa, Band 2/1. VEB G. Fischer, Jena, pp 876Google Scholar
  49. Kuczynska P, Jemiola-Rzeminska M, Strzalka K (2015) Photosynthetic pigments in diatoms. Mar Drugs 13:5847–5881CrossRefGoogle Scholar
  50. Katarzyna L, Sai G, Singh OA (2015) Non-enclosure methods for non-suspended microalgae cultivation: literature review and research needs. Renew Sust Energ Rev 42:1418–1427CrossRefGoogle Scholar
  51. Lee ME, van der Vegt NF (2006) Does urea denature hydrophobic interactions? J Am Chem Soc 128:4948–4949CrossRefGoogle Scholar
  52. Levitan O, Dinamarca J, Hochman G, Paul G. Falkowski (2014) Diatoms: a fossil fuel of the future. Trends Biotechnol 32:117–124Google Scholar
  53. Lewis PR, Knight DP (1977) Staining methods for sectioned material. In: Glauert AM (ed) Practical methods in electron microscopy: Vol. 5. Elsevier, North HollandGoogle Scholar
  54. Liu T, Wanga J, Hub Q, Chenga P, Jia B, Liua J, Chena Y, Zhanga W, Chena X, Chena L, Gaoa L, Jia C, Wanga H (2013) Attached cultivation technology for microalgae for efficient biomass feedstock production. Bioresour Technol 127:216–222CrossRefGoogle Scholar
  55. Mandalam RK, Palsson B (1998) Elemental balancing of biomass and medium composition enhances growth capacity in high density Chlorella vulgaris cultures. Biotechnol Bioeng 59:605–611CrossRefGoogle Scholar
  56. Mansour MP, Frampton DMF, Nichols PD, Volkman JK, Blackburn SI (2005) Lipid and fatty acid yield of nine stationary-phase microalgae: applications and unusual C24–C28 polyunsaturated fatty acids. J Appl Phycol 17:287–300CrossRefGoogle Scholar
  57. Martin-Jézéquel V, Hildebrand M, Brzezinski MA (2000) Silicon metabolism in diatoms: implications for growth. J Phycol 36:821–840CrossRefGoogle Scholar
  58. Matsumoto M, Sugiyama H, Maeda Y, Sato R, Tanaka T, Matsunaga T (2010) Marine diatom, Navicula sp. strain JPCC DA0580 and marine green alga, Chlorella sp. strain NKG400014 as potential sources for biodiesel production. Appl Biochem Biotechnol 161:483–490CrossRefGoogle Scholar
  59. McDonald SM, Plant JN, Worden AZ (2010) The mixed lineage nature of nitrogen transport and assimilation in marine eukaryotic phytoplankton: a case study of Micromonas. Mol Biol Evol 27:2268–2283CrossRefGoogle Scholar
  60. Moll KM, Gardner RD, Eustance EO, Gerlach R, Peyton BM (2014) Combining multiple nutrient stresses and bicarbonate addition to promote lipid accumulation in diatoms. Algal Res 5:7–15CrossRefGoogle Scholar
  61. Muthu A, Agarwal A, Arya MC, Ahmed Z (2013) Influence of nitrogen sources on biomass productivity of microalgae Scenedesmus bijugatus. Bioresour Technol 131:246–249CrossRefGoogle Scholar
  62. Nguyen RT, Harvey HR (2001) Preservation of protein in marine systems: hydrophobic and other non-covalent associations as major stabilizing forces. Geochim Cosmochim Acta 65:1467–1480CrossRefGoogle Scholar
  63. Nurachman Z, Panggabean LMG, Anita S (2010) Screening of local marine microalgae for biodiesel production. SEAMEO-SEARCA, SEARCA Agriculture & Development Paper Series 3Google Scholar
  64. Qin JZ, Song FF, Qiu YF, Li XX, Guan X (2013) Optimization of the medium composition of a biphasic production system for mycelial growth and spore production of Aschersonia placenta using response surface methodology. J Invertebr Pathol 112:108–115CrossRefGoogle Scholar
  65. Renaud SM, Thinh LV, Lambrinidis G, Parry DL (2002) Effect of temperature on growth, chemical composition and fatty acid composition of tropical Australian microalgae grown in batch cultures. Aquaculture 211:195–214CrossRefGoogle Scholar
  66. Sabbe K, Verleyen E, Hodgson DA, Vanhoutte K, Vyverman W (2003) Benthic diatom flora of freshwater and saline lakes in the Larsemann Hills and Rauer Islands, East Antarctica. Antarct Sci 15:227–248CrossRefGoogle Scholar
  67. Sabu S, Singh ISB, Joseph V (2017) Molecular identification and comparative evaluation of tropical marine microalgae for biodiesel production. Mar Biotechnol.
  68. Said KA, Amin MA (2016) Overview on the response surface methodology (RSM) in extraction processes. J Appl Sci Process Eng 2:8–17Google Scholar
  69. Sandnes JM, Kallqvist T, Wenner D, Gislerod HR (2005) Combined influence of light and temperature on growth rates of Nannochloropsis oceanica: linking cellular responses to large-scale biomass production. J Appl Phycol 17:515–525CrossRefGoogle Scholar
  70. Sanjay KR, Nagendra PMN, Anupama S, Yashaswi BR, Deepak B (2013) Isolation of diatom Navicula cryptocephala and characterization of oil extracted for biodiesel production. Afr J Environ Sci Technol 7:41–48Google Scholar
  71. Saumya D, Kannan DC, Dhawan V (2016) Understanding urea assimilation and its effect on lipid production and fatty acid composition of Scenedesmus sp. SOJ Biochem 2:7Google Scholar
  72. Shenbaga Devi A, Santhanam P, Rekha V, Ananth S, Prasath BB, Nandakumar R, Jeyanthi S, Kumar SD (2012) Culture and biofuel producing efficacy of marine microalgae Dunaliella salina and Nannochloropsis sp. J Algal Biomass Utln 3:38–44Google Scholar
  73. Singh P, Guldhe A, Kumari S, Rawat I, Bux F (2015) Investigation of combined effect of nitrogen, phosphorus and iron on lipid productivity of microalgae Ankistrodesmus falcatus KJ671624 using response surface methodology. Biochem Eng J 94:22–29CrossRefGoogle Scholar
  74. Smol JP, Stoermer EF (2010) The diatoms: applications for the environmental and earth sciences, 2nd edn. Cambridge University Press, CambridgeGoogle Scholar
  75. Song L, Qin JG, Su S, Xu J, Clarke S, Shan Y (2012) Micronutrient requirements for growth and hydrocarbon production in the oil producing green alga Botryococcus braunii (Chlorophyta). PLoS ONE 7:e41459. CrossRefGoogle Scholar
  76. Spilling K, Ylostalo P, Simis S, Seppala J (2015) Interaction effects of light, temperature and nutrient limitations (n, p and si) on growth, stoichiometry and photosynthetic parameters of the cold-water diatom Chaetoceros wighamii. PLoS ONE 10:e0126308. CrossRefGoogle Scholar
  77. Suman KK, Devi T, Sarma UK, Nittala S (2012) Culture medium optimization and lipid profiling of Cylindrotheca, a lipid- and polyunsaturated fatty acid-rich pennate diatom and potential source of eicosapentaenoic acid. Bot Mar 55:1–11CrossRefGoogle Scholar
  78. Valenzuela J, Carlson RP, Gerlach R, Cooksey KE, Peyton BM, Bothner B, Fields MW (2013) Nutrient re-supplementation arrests bio-oil accumulation in Phaeodactylum tricornutum. Appl Microbiol Biotechnol 97:7049–7059CrossRefGoogle Scholar
  79. Valenzuela J, Mazurie A, Carlson RP, Gerlach R, Cooksey KE, Peyton BM, Fields MW (2012) Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum. Biotechnol Biofuels 5:1–17CrossRefGoogle Scholar
  80. Vanelslander B, Créach V, Vanormelingen P, Ernst A, Chepurnov VA, Sahan E, Muyzer G, Stal LJ, Vyverman W, Sabbe K (2009) Ecological differentiation between sympatric pseudocryptic species in the estuarine benthic diatom Navicula phyllepta (Bacillariophyceae). J Phycol 45:1278–1289CrossRefGoogle Scholar
  81. Wagenen JV, Miller TW, Hobbs S, Hook P, Crowe B, Huesemann M (2012) Effects of light and temperature on fatty acid production in Nannochloropsis salina. Energies 5:731–740CrossRefGoogle Scholar
  82. Wah NB, Ahmad AL, Chieh DC, Hwai AT (2015) Changes in lipid profiles of a tropical benthic diatom in different cultivation temperature. Asian J Appl Sci Eng 4:91–101Google Scholar
  83. Wijanarko A (2011) Effect of the presence of substituted urea and also ammonia as nitrogen source in cultivation medium on Chlorella’s lipid content. In: Shaukat SS (ed) Progress in biomass and bioenergy production. InTech, pp 273–282Google Scholar
  84. Windler M, Leinweber K, Bartulos CR, Philipp B, Kroth PG (2015) Biofilm and capsule formation of the diatom Achnanthidium minutissimum are affected by a bacterium. J Phycol 51:343–355CrossRefGoogle Scholar
  85. Willis A, Chiovitti A, Dugdale TM, Wetherbee R (2013) Characterization of the extracellular matrix of Phaeodactylum tricornutum (Bacillariophyceae): structure, composition, and adhesive characteristics. J Phycol 49:937–949Google Scholar
  86. Witkowski A, Lange-Bertalot H, Metzeltin D (2000) Diatom flora of marine coasts I. Iconogr Diatomol 7:1–925Google Scholar
  87. Wu LF, Chen PC, Lee CM (2013) The effects of nitrogen sources and temperature on cell growth and lipid accumulation of microalgae. Int Biodeter Biodegr 85:506–510CrossRefGoogle Scholar
  88. Yadavalli R, Rao R (2013) Response surface methodological approach to optimize process parameters for the biomass production of Chlorella pyrenoidosa. Int J Biotechnol Res 1:37–48Google Scholar
  89. Yang ZK, Niu YF, Ma YH, Xue J, Zhang MH, Yang WD, Liu JS, Lu SH, Guan Y, Li HY (2013) Molecular and cellular mechanisms of neutral lipid accumulation in diatom following nitrogen deprivation. Biotechnol Biofuels 6:67CrossRefGoogle Scholar
  90. Yang F, Long L, Sun X, Wu H, Li T, Xiang W (2014a) Optimization of medium using response surface methodology for lipid production by Scenedesmus sp. Mar Drugs 12:1245–1257CrossRefGoogle Scholar
  91. Yang M, Zhao W, Xie X (2014b) Effects of nitrogen, phosphorus, iron and silicon on growth of five species of marine benthic diatoms. Acta Ecol Sin 34:311–319CrossRefGoogle Scholar
  92. Yongmanitchai W, Ward OP (1991) Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions. Appl Environ Microbiol 57:419–425Google Scholar
  93. Zhang J, Fu D, Xu Y, Liu C (2012) Optimization of parameters on photocatalytic degradation of chloramphenicol using TiO2 as photocatalyst by response surface methodology. J Environ Sci 22:1281–1289CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Sanyo Sabu
    • 1
  • Isaac Sarojini Bright Singh
    • 1
  • Valsamma Joseph
    • 1
  1. 1.National Centre for Aquatic Animal HealthCochin University of Science and TechnologyKochiIndia

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