Aquaculture International

, Volume 25, Issue 2, pp 743–753 | Cite as

Evaluation of pyocyanin toxicity in various life stages of Penaeus monodon and in nitrifying bacterial consortia for their safe application in recirculating aquaculture systems (RAS) to abrogate pathogenic vibrios

  • P. Priyaja
  • P. Jayesh
  • M. Haseeb
  • Blessy Jose
  • Rosamma Philip
  • I. S. Bright SinghEmail author


Pyocyanin has recently been recognized as antimicrobial agent in aquaculture industry, an alternative to antibiotics, as it accorded higher level inhibitory effect on aquaculture pathogens. As pyocyanin-induced toxicity is a major concern, this study has been undertaken to evaluate the toxicity of purified pyocyanin and to determine the LC50 value for its safe application in aquaculture. The dose-dependent pyocyanin-induced toxicity was tested on various life stages of P. monodon such as nauplius, zoea, mysis and post-larva as well as on nitrifying bacterial consortia used in aquaculture in order to fix the concentration limits to the animal rearing systems. The study revealed that pyocyanin was a potential inhibitory compound to V. harveyi, bacteriostatic at 5 mg L−1 and bactericidal at 10 mg L−1. The LC50 values of pyocyanin at various life stages of P. monodon were higher than the concentration required to abrogate pathogenic V. harveyi. Moreover, pyocyanin did not inhibit nitrification, specifically of nitrifying bacterial consortia used for activating nitrifying bioreactors, at different salinities, suggesting its potential application in recirculating aquaculture systems to control pathogenic vibrios.


Pyocyanin Pseudomonas Toxicity Penaeus monodon RAS 



This work was supported by Department of Biotechnology, Government of India under Program Support in Marine Biotechnology (Order No: BT/PR4012/AAQ/03/204/2003). The first author thanks DBT for fellowship.


  1. Achuthan C, Kumar VJR, Manju NJ, Philip R, Singh ISB (2006) Development of nitrifying bacterial consortia for immobilizing nitrifying bioreactors designed for penaeid and non-penaeid larval rearing systems in the tropics. Indian J Mar Sci 35:240–248Google Scholar
  2. Anjaiah V, Cornelis P, Koedam N (2003) Effect of genotype and root colonization in biological control of Fusarium wilts in pigeon pea and chickpea by Pseudomonas aeruginosa PNA1. Can J Microbiol 49:85–91CrossRefPubMedGoogle Scholar
  3. Arunkumar G, Rao SG, Shivananda PG (1997) Anti-staphylococcal activity of Pseudomonas aeruginosa. Curr Sci 72:580–582Google Scholar
  4. Balcazar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Muzquiz JL (2006) The role of probiotics in aquaculture. Vet Microbiol 114:173–186CrossRefPubMedGoogle Scholar
  5. Bano N, Musarrat J (2003) Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent. Curr Microbiol 46:324–328CrossRefPubMedGoogle Scholar
  6. Bendschneider K, Robinson RJ (1952) A new spectrophotometric method for the determination of nitrite in sea water. J Mar Sci 11:87–96Google Scholar
  7. Cabello FC (2006) Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol 8:1137–1144CrossRefPubMedGoogle Scholar
  8. Chaythanya R, Karunasagar I, Karunasagar I (2002) Inhibition of shrimp pathogenic vibrios by a marine Pseudomonas I-2 strain. Aquaculture 208:1–10CrossRefGoogle Scholar
  9. Chen JC, Cheng SY (1995) Changes of oxy-hemocyanin and protein levels in the hemolymph of Penaeus japonicus exposed to ambient nitrite. Aquat Toxicol 33:215–226CrossRefGoogle Scholar
  10. Chen K, Hu H, Wang W, Zhang X, Xu Y (2008) Metabolic degradation of phenazine-1-carboxylic acid by the strain Sphingomonas sp DP58: the identification of two metabolites. Biodegradation 19:659–667CrossRefPubMedGoogle Scholar
  11. Costa AL, Cusmano V (1975) Anti-mycotic activity of pyocyanin in vitro and in vivo on a pathogenic strain of Candida albicans. Gen Bacteriol Virol Immunol 66:297–308Google Scholar
  12. Dive D (1973) Action of exocellular pigments secreted by Pseudomonas aeruginosa on the growth and division of Colpidium campylum. Protistologica 9:315–318Google Scholar
  13. Gloyne LS, Grant GD, Perkins AV, Powell KL, McDermott CM, Johnson PV, Anderson GJ, Kiefel M, Dukie SA (2011) Pyocyanin-induced toxicity in A549 respiratory cells is causally linked to oxidative stress. Toxicol In Vitro 25:1353–1358CrossRefPubMedGoogle Scholar
  14. Hai NV, Buller N, Fotedar R (2009) Effects of probiotics (Pseudomonas synxantha and Pseudomonas aeruginosa) on the growth, survival and immune parameters of juvenile western king prawns (Penaeus latisulcatus Kishinouye, 1896). Aquac Res 40:590–602CrossRefGoogle Scholar
  15. Harwing J, Scott PM (1971) Brine shrimp (Artemia salina) larvae as a screening system for fungal toxins. Appl Microbiol 21:1011–1016Google Scholar
  16. Hasanuzzaman M, Umadhay-Briones KM, Zsiros SM, Morita N, Nodasaka Y, Yumoto I, Okuyama H (2004) Isolation, identification and characterization of a novel, oil-degrading bacterium, Pseudomonas aeruginosa T1. Curr Microbiol 49:108–114CrossRefPubMedGoogle Scholar
  17. Hill J, Johnson G (1969) Microbial transformation of phenazines by Aspergillus sclerotiorum. Mycologia 61:452–467CrossRefPubMedGoogle Scholar
  18. Kerr JR, Taylor GW, Rutman A, Hoiby N, Cole PJ, Wilson R (1999) Pseudomonas aeruoginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth. J Clin Pathol 52:385–387CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kesarcodi-Watson A, Kaspar H, Lategan MJ, Gibson L (2008) Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes. Aquaculture 274:1–14CrossRefGoogle Scholar
  20. Kumar RS, Ayyadurai N, Pandiaraja P, Reddy AV, Venkateswarlu Y, Prakash O, Sakthivel N (2005) Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits. Appl Microbiol 98:145–154CrossRefGoogle Scholar
  21. Kumar VJR, Achuthan C, Manju NJ, Philip R, Singh ISB (2009a) Activated packed bed bioreactors (PBBR) for the rapid nitrification in brackish water hatchery systems. J Ind Microbiol Biotechnol 36:355–365CrossRefPubMedGoogle Scholar
  22. Kumar VJR, Achuthan C, Manju NJ, Philip R, Singh ISB (2009b) A stringed bed suspended bioreactor (SBSBR) for in situ nitrification in penaeid and non-penaeid hatchery systems. Aquacult Int 17:479–489CrossRefGoogle Scholar
  23. Kumar VJR, Achuthan C, Manju NJ, Philip R, Singh ISB (2009c) Mass production of nitrifying bacterial consortia for establishing instantaneous nitrification in saline recirculating aquaculture systems. World J Microbiol Biotechnol 25:407–414CrossRefGoogle Scholar
  24. Kumar VJR, Joseph V, Philip R, Singh ISB (2010) Nitrification in brackishwater recirculating aquaculture system integrated with activated packed bed bioreactor. Water Sci Technol 613:797–805CrossRefGoogle Scholar
  25. Lavilla-Pitogo CR, Leano EM, Paner MG (1996) Mortalities of pond-cultured juvenile shrimp, Penaeus monodon, associated with dominance of luminescent bacteria, Vibrio harveyi in the rearing environment. SICCPPS book of abstracts, SEAFDEC, Iloilo City, p 40Google Scholar
  26. Lavilla-Pitogo CR, Leano EM, Paner MG (1998) Mortalities of pond-cultured juvenile shrimp Penaeus monodon associated with dominance of luminescent vibrios in the rearing environment. Aquaculture 164:337–349CrossRefGoogle Scholar
  27. Lightner DV, Lewis DH (1975) A septicemic bacterial disease syndrome of penaeid shrimp. Mar Fish Rev 37(5–6):25–28Google Scholar
  28. Lightner DV, Bell TA, Redman RM, Mohney LL, Natividad JM, Rukyani A, Poernomo A (1992) A review of some major diseases of economic significance in penaeid shrimps/shrimps of the Americas and Indo-Pacific. In: Shariff M, Subasinghe R, Arthur JR (eds) Proceeding of 1st symposium on diseases in Asian aquaculture. Fish Health Section, Asian Fisheries Society, Manila, pp 57–80Google Scholar
  29. Muller M, Li Z, Maitz PK (2009) Pseudomonas pyocyanin inhibits wound repair by inducing premature cellular senescence: role for p38 mitogen-activated protein kinase. Burns 35:500–508CrossRefPubMedGoogle Scholar
  30. Ohfuji K, Sato N, Hamada-Sato N, Kobayashi T (2004) Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa. Biosens Bioelectron 19:1237–1244CrossRefPubMedGoogle Scholar
  31. Pai SS, Anas A, Jayaprakash NS, Priyaja P, Sreelakshmi B, Preetha R, Singh ISB (2010) Penaeus monodon larvae can be protected from Vibrio harveyi infection by pre-emptive treatment of a rearing system with antagonistic or non-antagonistic bacterial probiotics. Aquac Res 41:847–860CrossRefGoogle Scholar
  32. Pham TH, Boon N, Maeyer K, Hofte M, Rabaey K, Verstraete W (2008) Use of Pseudomonas species producing phenazine-based metabolites in the anodes of microbial fuel cells to improve electricity generation. Appl Microbiol Biotechnol 80:985–993CrossRefPubMedGoogle Scholar
  33. Preetha R, Jose S, Prathapan S, Vijayan KK, Jayaprakash NS, Philip R, Singh ISB (2010) An inhibitory compound produced by Pseudomonas with effectiveness on Vibrio harveyi. Aquac Res 41:1452–1461Google Scholar
  34. Priyaja P, Jayesh P, Correya NS, Sreelakshmi B, Sudheer NS, Philip R, Singh ISB (2014) Antagonistic effect of Pseudomonas aeruginosa isolates from various ecological niches on Vibrio species pathogenic to crustaceans. J Coast Life Med 2:76–84Google Scholar
  35. Priyaja P, Jayesh P, Philip R, Singh ISB (2016) Pyocyanin induced in vitro oxidative damage and its toxicity level in human, fish and insect cell lines for its selective biological applications. Cytotechnology 68:143–155CrossRefPubMedGoogle Scholar
  36. Rahman S, Khan N, Naser MN, Karim MM (2011) Safety issues of isolated probiotic natured bacteria from Bangladesh coastal waters for controlling shrimp diseases. J Sci Res 3:659–668Google Scholar
  37. Reszka KJ, O’Malley Y, McCormick ML, Denning GM, Britigan BE (2004) Oxidation of pyocyanin, a cytotoxic product from Pseudomonas aeruginosa, by microperoxidase 11 and hydrogen peroxide. Free Radical Bio Med 36:1448–1459CrossRefGoogle Scholar
  38. Solorzano L (1969) Determination of ammonia in natural waters by the phenol hypochlorite method. Limnol Oceanogr 14:799–801CrossRefGoogle Scholar
  39. Tang X, Zhu Y, Meng Q (2007) Enhanced crude oil biodegradability of Pseudomonas aeruginosa ZJU after preservation in crude-oil containing media. World J Microbiol Biotechnol 23:7–14CrossRefGoogle Scholar
  40. Tinh NTN, Yen VHN, Dierckens K, Sorgeloos P, Bossier P (2008) An acyl homoserine lactone-degrading microbial community improves the survival of first-feeding turbot larvae (Scophthalmus maximus). Aquaculture 285:56–62CrossRefGoogle Scholar
  41. Verschuere L, Rombaut G, Sorgeloos P, Verstraete W (2000) Probiotics bacteria as biological control agents in aquaculture. Microbiol Mol Biol Rev 64:655–671CrossRefPubMedPubMedCentralGoogle Scholar
  42. Vijayan KK, Singh ISB, Jayaprakash NS, Alavandi SV, Pai SS, Preetha R, Rajan JJS, Santiago TC (2006) A brackish water isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaied and non-penaied rearing systems. Aquaculture 251:192–200CrossRefGoogle Scholar
  43. Vinayak RC, Sabu AS, Chatterji A (2011) Bio-prospecting of a few brown seaweeds for their cytotoxic and antioxidant activities. Evid Based Complement Alternat Med. doi: 10.1093/ecam/neq024 PubMedPubMedCentralGoogle Scholar
  44. Warren JB, Loi R, Rendell NB, Taylor GW (1990) Nitric oxide is inactivated by the bacterial pigment pyocyanin. Biochem J 266:921–923PubMedPubMedCentralGoogle Scholar
  45. Watson SW (1965) Characteristics of a marine nitrifying bacterium, Nitrosocystis oceanus sp. Limnol Oceanogr 10:274–289CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • P. Priyaja
    • 1
  • P. Jayesh
    • 1
  • M. Haseeb
    • 1
  • Blessy Jose
    • 1
  • Rosamma Philip
    • 2
  • I. S. Bright Singh
    • 1
    Email author
  1. 1.National Centre for Aquatic Animal HealthCochin University of Science and TechnologyKochiIndia
  2. 2.Department of Marine Biology, Microbiology and BiochemistryCochin University of Science and TechnologyKochiIndia

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