Probiotic Role of Salt Pan Bacteria in Enhancing the Growth of Whiteleg Shrimp, Litopenaeus vannamei

  • Samantha Fernandes
  • Savita Kerkar
  • Joella Leitao
  • Abhishek Mishra


Development of probiotics to improve the growth of cultured species is a key to sustainable aquaculture. The present study investigates the potential of salt pan bacteria as probiotics for Litopenaeus vannamei. Halotolerant bacteria (100) were screened for enzyme production and mucus adhesion in vitro. The bacteria (SK07, SK27, ABSK55, FSK444, TSK17, TSK71) exhibiting promising enzyme activity and adhesive property in vitro were selected to study their effect on the growth and metabolism of L. vannamei in vivo. When administered to shrimps individually as a water additive in experiment I, SK07, SK27 and TSK71 significantly (p < 0.05) increased shrimp weight as compared to the control. In experiment II, a lyophilized bacterial consortium (test) prepared with the four best isolates (SK07, SK27, ABSK55, TSK71), exhibited significantly higher weight gain of shrimps, better feed efficiency and final yield as compared to control. Total enzyme activity (amylase, protease, lipase) in the shrimp gut was significantly higher in the test than the control. The four isolates showed 99% nBLAST similarity with Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Pseudomonas sp. Presence of these bacteria in the shrimp gut was confirmed by using specific PCR-based molecular probes and 16S rDNA sequencing. Safety evaluation by antibiotic susceptibility test and hemolytic activity test indicated that the bacteria are safe as bioinoculants. The increased enzyme activity by colonisation of the isolates in the shrimp gut, along with improved growth and feed utilisation efficiency, strongly confirms that these salt pan bacteria are prospective probiotics in shrimp aquaculture.


Probiotic Halotolerant Salt pan Growth enhancement Shrimp aquaculture 



The authors are thankful to the Head of the Department of Biotechnology, Goa University, for the facilities. The authors also wish to thank the Head of Department of Zoology, Goa University, and National Institute of Oceanography (NIO) for providing the aquaculture facilities.


Samantha Fernandes is thankful to Department of Science, Technology and Environment, Goa for the Research fellowship (No. 8-210-2013/STE-DIR/Acct.//714).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Research Involving Human Participants and/or Animals

Not applicable.

Supplementary material

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ESM 1 (DOCX 1455 kb)


  1. 1.
    Sahu MK, Swarnakumar NS, Sivakumar K, Thangaradjou T, Kannan L (2008) Probiotics in aquaculture: importance and future perspectives. Indian J Microbiol 48:299–308CrossRefGoogle Scholar
  2. 2.
    Hoseinifar SH, Zare P, Merrifield DL (2010) The effects of inulin on growth factors and survival of the Indian white shrimp larvae and postlarvae (Fenneropenaeus indicus). Aquac Res:1365–2109Google Scholar
  3. 3.
    Zhang D, Wang F, Dong S, Lu Y (2016) De novo assembly and transcriptome analysis of osmoregulation in Litopenaeus vannamei under three cultivated conditions with different salinities. Gene 578(2):185–193CrossRefGoogle Scholar
  4. 4.
    Akiyama DM, Dominy WG, Lawrence AL (1992) Penaeid shrimp nutrition. In: marine shrimp culture: principles and practices. Developments in aquaculture and fisheries science, vol 23. Elsevier Science Publisher B.V., The Netherlands, pp 535–568Google Scholar
  5. 5.
    Abd El-Rhman AM, Khattab YA, Shalaby AM (2009) Micrococcus luteus and Pseudomonas species as probiotics for promoting the growth performance and health of Nile tilapia, Oreochromis niloticus. Fish Shellfish Immunol 27:175–180CrossRefGoogle Scholar
  6. 6.
    Zokaeifar H, Balcazar JL, Saad CR, Kamarudin MS, Sijam K, Arshad A et al (2012) Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol 33:683–689CrossRefGoogle Scholar
  7. 7.
    Nimrat S, Suksawat S, Boonthai T, Vuthiphandchai V (2012) Potential Bacillus probiotics enhance bacterial numbers, water quality and growth during early development of white shrimp (Litopenaeus vannamei). Vet Microbiol 159:443–450CrossRefGoogle Scholar
  8. 8.
    Tzuc JT, Escalante DR, Herrera RR, Cortes GG, Ortiz ML (2014) Microbiota from Litopenaeus vannamei: digestive tract microbial community of Pacific white shrimp (Litopenaeus vannamei). SpringerPlus 3:1–10CrossRefGoogle Scholar
  9. 9.
    El-Haroun ER, Goda AMAS, Chowdhury MAK (2006) Effect of dietary probiotic biogens supplementation as a growth promoter on growth performance and feed utilization of Nile tilapia Oreochromis niloticus (L.). Aquac Res 37:1473–1480CrossRefGoogle Scholar
  10. 10.
    Mohapatra S, Chakraborty T, Prusty AK, Das P, Pani prasad K, Mohanta N (2012) Use of probiotics in the diet of rohu, Labeo rohita fingerlings: effects on growth nutrient digestibility, retention digestive enzyme activities and intestinal microflora. Aquac Nutr 18:1–11CrossRefGoogle Scholar
  11. 11.
    Balcazar JL, Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Muzquiz JL (2006) The role of probiotics in aquaculture. Vet Microbiol 114:173–186CrossRefGoogle Scholar
  12. 12.
    Vine NG, Leukes SD, Kaiser H (2006) Probiotics in marine larviculture. FEMS Micriobiol Rev 30:404–427CrossRefGoogle Scholar
  13. 13.
    Hoseinifar SH, Dadar M, Ringø E (2017) Modulation of nutrient digestibility and digestive enzyme activities in aquatic animals: the functional feed additives scenario. Aquac Res 48:3987–4000CrossRefGoogle Scholar
  14. 14.
    Zorriehzahra MJ, Delshad ST, Adel M, Tiwari R, Karthik K, Dhama K, Lazado CC (2016) Probiotics as beneficial microbes in aquaculture: an update on their multiple modes of action: a review. Vet Q 36:228–241CrossRefGoogle Scholar
  15. 15.
    NavinChandran M, Iyapparaj P, Moovendhan S, Ramasubburayan R, Prakash S, Immanuel G, Palavesam A (2014) Influence of probiotic bacterium Bacillus cereus isolated from the gut of wild shrimp Penaeus monodon in turn as a potent growth promoter and immune enhancer in P. monodon. Fish Shellfish Immunol 36:38–45CrossRefGoogle Scholar
  16. 16.
    Kongnum K, Hongpattarakere T (2011) Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi. Fish Shellfish Immunol 32:170–177CrossRefGoogle Scholar
  17. 17.
    Zhou X, Wang Y, Li W (2009) Effect of probiotic on larvae shrimp (Penaeus vannamei) based on water quality, survival rate and digestive enzyme activities. Aquaculture 287:349–353CrossRefGoogle Scholar
  18. 18.
    Wang YB (2007) Effect of probiotics on growth performance and digestive enzyme activity of the shrimp Penaeus vannamei. Aquaculture 269:259–264CrossRefGoogle Scholar
  19. 19.
    Vieira FDN, Pedrotti FS, Neto CCB, Mourino JLP, Beltrame E, Martins ML et al (2007) Lactic–acid bacteria increase the survival of marine shrimp, Litopenaeus vannamei, after infection with Vibrio harveyi. Braz J Oceanogr 55:251–255CrossRefGoogle Scholar
  20. 20.
    Li J, Tan B, Mai K, Ai Q, Zhang W, Xu W, Liufu Z, Ma H (2006) Comparative study between probiotic bacterium Arthrobacter XE–7 and chloramphenicol on protection of Penaeus chinensis post–larvae from pathogenic vibrios. Aquaculture 253:140–147CrossRefGoogle Scholar
  21. 21.
    Solano JLO, Soto JO (2006) The functional property of Bacillus for shrimp feeds. Food Microbiol 23:519–525CrossRefGoogle Scholar
  22. 22.
    Preetha R, Jayaprakash NS, Singh IS (2007) Synechocystis MCCB 114 and 115 as putative probionts for Penaeus monodon post–larvae. Dis Aquat Org 74:243–247CrossRefGoogle Scholar
  23. 23.
    Ansari ZA, Pandiyarajan RS, Ayajuddin M (2012) Khazan land of Goa and its fishery potential. Fishcoop. NIO Goa, pp 3–6Google Scholar
  24. 24.
    Rodrigues CM, Bio A, Amat F, Vieira (2011) Artisanal salt production in Aveiro/Portugal—an ecofriendly process. Saline Syst 7(3):1–14Google Scholar
  25. 25.
    Kerkar S (2004) Ecology of hypersaline microorganisms. In: Ramaiah N (ed) Marine microbiology facets and opportunities. NIO Goa, India, pp 53–67Google Scholar
  26. 26.
    Alariya SS, Sethi S, Gupta S, Lal B, Lal GB (2013) Amylase activity of a starch degrading bacteria isolated from soil. Arch Appl Sci Res 5:15–24Google Scholar
  27. 27.
    Shivakumar S (2012) Production and characterisation of an acid protease from a local Aspergillus sp. by solid substrate fermentation. rch Appl Sci Res 4:188–199Google Scholar
  28. 28.
    Kumar S, Karan R, Kapoor S, Singh SP, Khare SK (2012) Screening and isolation of halophilic bacteria producing industrially important enzymes. Braz J Microbiol 43:1595–1603CrossRefGoogle Scholar
  29. 29.
    Liang YL, Zhang Z, Wu M, Wu Y, Feng JX (2014) Isolation, screening, and identification of cellulolytic bacteria from natural reserves in the subtropical region of China and optimization of cellulase production by Paenibacillus terrae ME27-1. Biomed Res Int 2014:1–13Google Scholar
  30. 30.
    Worthington V (1993) Worthington enzyme manual. Enzymes and related biochemicals. Worthington Chemical, ColumbusGoogle Scholar
  31. 31.
    Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268CrossRefGoogle Scholar
  32. 32.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275Google Scholar
  33. 33.
    Kanwar SS, Verma HK, Kaushal RK, Gupta R, Chimni SS, Kumar Y, Chauhan GS (2005) Effect of solvents and kinetic parameters on synthesis of ethyl propionate catalysed by poly (AAc-co-HPMAcl-MBAm)-matrix-immobilized lipase of Pseudomonas aeruginosa BTS-2. World J Microbiol Biotechnol 21:1037–1044CrossRefGoogle Scholar
  34. 34.
    Balakrishna A (2013) In vitro evaluation of adhesion and aggregation abilities of four potential probiotic strains isolated from guppy (Poecilia reticulata). Braz Arch Biol Technol 56:793–800CrossRefGoogle Scholar
  35. 35.
    Hasan B, Guha B, Datta S (2012) Optimization of feeding efficiency for cost effective production of Penaeus monodon Fabricius in semi-intensive pond culture system. J Aquac Res Dev 3:1–7Google Scholar
  36. 36.
    Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM (2009) The ribosomal database project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37:D141–D145CrossRefGoogle Scholar
  37. 37.
    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real time PCR experiments. Clin Chem 55:611–622CrossRefGoogle Scholar
  38. 38.
    Bauer AW, Kirby MM, Sherris JC, Tuurck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45:493–496CrossRefGoogle Scholar
  39. 39.
    Luis-Villasenor E, Macias-Rodriguez ME, Gomez-Gil B, Ascencio-Valle F, Campa-Cordova A (2011) Beneficial effects of four Bacillus strains on the larval cultivation of Litopenaeus vannamei. Aquaculture 321:136–144CrossRefGoogle Scholar
  40. 40.
    Gómez RG, Shen MA (2008) Influence of probiotics on the growth and digestive enzyme activity of white Pacific shrimp (Litopenaeus vannamei). J Ocean Univ China 7:215–218CrossRefGoogle Scholar
  41. 41.
    Ringo E, Olsen RE, Gifstad TO, Dalmo RA, Amlund H, Hemre GI et al (2010) Prebiotics in aquaculture: a review. Aquac Nutr 16:117–136CrossRefGoogle Scholar
  42. 42.
    Llewellyn MS, Boutin S, Hoseinifar SH, Derome N (2014) Teleost microbiomes: the state of the art in their characterization, manipulation and importance in aquaculture and fisheries. Front Microbiol 5:207CrossRefGoogle Scholar
  43. 43.
    Verschuere L, Rombaut G, Sorgeloos P, Verstraete W (2000) Probiotic bacteria as biological control agents in aquaculture. Microbiol Mol Biol Rev 64:655–671CrossRefGoogle Scholar
  44. 44.
    Gesheva V, Vasileva-Tonkova E (2012) Production of enzymes and antimicrobial compounds by halophilic Antarctic Nocardioides sp. grown on different carbon sources. World J Microbiol Biotechnol 28:2069–2076CrossRefGoogle Scholar
  45. 45.
    Das S, Ward LR, Burke C (2010) Screening of marine Streptomyces spp. for potential use as probiotics in aquaculture. Aquaculture 305:32–41CrossRefGoogle Scholar
  46. 46.
    Venkat HK, Sahu NP, Jain KK (2004) Effect of feeding Lactobacillus-based probiotics on the gut microflora, growth and survival of postlarvae of Macrobrachium rosenbergii (de man). Aquac Res 35:501–507CrossRefGoogle Scholar
  47. 47.
    Fernandes R, Sridhar M, Sridhar N (2011) Effect of lactic acid bacteria administered orally on growth performance of Penaeus indicus (H.Milne Edwards) juveniles. Res J Microbiol 6(5):466–479CrossRefGoogle Scholar
  48. 48.
    Henriques AO, Moran CP (2000) Structure and assembly of the bacterial endospore coat. Methods 20:95–110CrossRefGoogle Scholar
  49. 49.
    Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64:548–572CrossRefGoogle Scholar
  50. 50.
    Ziaei-Nejad S, Rezaei MH, Takami GA, Lovett DL, Mirvaghefi AR, Shakouri M (2006) The effect of Bacillus spp. bacteria used as probiotics on digestive enzyme activity, survival and growth in the Indian white shrimp Fenneropenaeus indicus. Aquaculture 252:516–524CrossRefGoogle Scholar
  51. 51.
    Yanbo W, Zirong X (2006) Effect of probiotics for common carp (Cyprinus carpio) based on growth performance and digestive enzyme activities. Anim Feed Sci Technol 127:283–292CrossRefGoogle Scholar
  52. 52.
    Li K, Zheng T, Tian Y, Xi F, Yuan J, Zhang G, Hong H (2007) Beneficial effects of Bacillus licheniformis on the intestinal microflora and immunity of the white shrimp, Litopenaeus vannamei. Biotechnol Lett 29:525–530CrossRefGoogle Scholar
  53. 53.
    Gullian M, Thompson F, Rodriguez J (2004) Selection of probiotic bacteria and study of their immunostimulatory effect in Penaeus vannamei. Aquaculture 233:1–14CrossRefGoogle Scholar
  54. 54.
    Vaseeharan B, Ramasamy P (2003) Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Lett Appl Microbiol 36:83–87CrossRefGoogle Scholar
  55. 55.
    Zhang Q, Tan B, Mai K, Zhang W, Ma H, Ai Q, Wang X, Liufu Z (2011) Dietary administration of Bacillus (B. licheniformis and B. subtilis) and isomaltooligosaccharide influences the intestinal microflora, immunological parameters and resistance against Vibrio alginolyticus in shrimp, Penaeus japonicus (Decapoda: Penaeidae). Aquac Res 42:943–952CrossRefGoogle Scholar
  56. 56.
    Dong H, Su Y, Mao Y, You X, Ding S, Wang J (2013) Dietary supplementation with Bacillus can improve the growth and survival of the kuruma shrimp Marsupenaeus japonicus in high-temperature environments. Aquac Int 22:607–617CrossRefGoogle Scholar
  57. 57.
    Gamboa-Delgado J, Molina-Poveda C, Cahu C (2003) Digestive enzyme activity and food ingesta in juvenile shrimp Litopenaeus vannamei (Boone, 1931) as a function of body weight. Aquac Res 34:1403–1411CrossRefGoogle Scholar
  58. 58.
    Kaktcham PM, Temgoua J, Zambou FM, Diaz-Ruiz G, Wacher C, Perez-Chabela M (2018) In vitro evaluation of the probiotic and safety properties of bacteriocinogenic and non bacteriocinogenic lactic acid bacteria from the intestines of Nile tilapia and common carp for their use as probiotics in aquaculture. Probiotics Antimicrob Proteins 10:98–109CrossRefGoogle Scholar
  59. 59.
    Delcour AH (2009) Outer membrane permeability and antibiotic resistance. Biochim Biophys Acta 1794(5):808–816CrossRefGoogle Scholar
  60. 60.
    Abebe E, Tegegne B, Tibebu S (2016) A review on molecular mechanisms of bacterial resistance to antibiotics. EJAS 8(5):301–310Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Samantha Fernandes
    • 1
  • Savita Kerkar
    • 1
  • Joella Leitao
    • 1
  • Abhishek Mishra
    • 1
  1. 1.Department of BiotechnologyGoa UniversityTaleigao PlateauIndia

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