Research Article | Volume: 4, Issue: 4, July-August, 2016

Fungal pellets as potential tools to control water pollution: Strategic approach for the pelletization and subsequent microcystin-LR uptake by Mucor hiemalis

Evelyn Balsanoa Maranda Esterhuizen-Londta Enamul Hoquec Stephan Pflugmachera b   

Open Access   

Published:  Aug 26, 2016

DOI: 10.7324/JABB.2016.40403

Microcystin-LR is one of the most prevalent and toxic secondary metabolites produced by cyanobacteria worldwide, causing global concerns because of its hazardousness to ecosystems and human health. Green Liver Systems® have been developed to purify contaminated water, however, system capacities need to be extended to allow season- and location independent applications. Therefore, mycoremediation using temperature resistant Mucor hiemalis in pellet morphology was considered. In submerged liquid cultures, fungal morphology is species specific and strongly depends on the cultivation environment. One main focus of the present study was the investigation of diverse factors influencing pelletization. Moreover, we translated the pellet product into an immediate application and studied its biosorption ability towards microcystin-LR. Our results showed that pH was a key factor stimulating pellet formation of M. hiemalis and that inoculum size played an essential role as well. Final pellet size was limited by the available space in the flask and is therefore directly related to inoculum size. Microcystin-LR was found to be taken up by pelletized M. hiemalis as quantified via LC-MS/MS measurements. Our results report for the first time optimized pelletization of M. hiemalis and cyanotoxin uptake by these fungal pellets in liquid cultures.

Keyword:     Mucor hiemalis pelletization mycoremediation microcystin-LR uptake.


Balsano E, Esterhuizen-Londt M, Hoque E, Pflugmacher S. Fungal pellets as potential tools to control water pollution: Strategic approach for the pelletization and subsequent microcystin-LR uptake by Mucor hiemalis. J App Biol Biotech. 2016; 4 (04): 031-041. DOI: 10.7324/JABB.2016.40403

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

HTML Full Text

1. Imanishi S, Kato H, Mizuno M, Tsuji K, Harada KI. Bacterial degradation of microcystins and nodularin. Chemical Research in Toxicology. 2005; 18(3):591-598.

2. Namikoshi M, Sivonen K, Evans WR, Sun F, Carmichael WW, Rinehart KL. Isolation and structures of microcystins from a cyanobacterial water bloom (Finland). Toxicon. 1992; 30(11):1473-1479.

3. Luukkainen R, Namikoshi M, Sivonen K, Rinehart KL, Niemelä SI. Isolation and identification of 12 microcystins from four strains and two bloom samples of Microcystis spp.: Structure of a new hepatotoxin. Toxicon. 1994; 32(1):133-139.

4. Sivonen K, Namikoshi M, Evans WR, Färdig M, Carmichael WW, Rinehart KL. Three new microcystins, cyclic heptapeptide hepatotoxins, from Nostoc sp. strain 152. Chemical Research in Toxicology. 1992; 5(4):464-469.

5. Lanaras T, Cook CM. Toxin extraction from an Anabaenopsis milleri - dominated bloom. Science of The Total Environment. 1994; 142(3):163-169.

6. Azevedo SMFO, Carmichael WW, Jochimsen EM, Rinehart KL, Lau S, Shaw GR, Eaglesham GK. Human intoxication by microcystins during renal dialysis treatment in Caruaru-Brazil. Toxicology. 2002; 181–182(0):441-446.

7. Jochimsen EM, Carmichael WW, An J, Cardo DM, Cookson ST, Holmes CEM, Antunes MB, de Melo Filho DA, Lyra TM, Barreto VST, Azevedo SMFO, Jarvis WR. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. New England Journal of Medicine. 1998; 338(13):873-878.

8. Falconer IR, Humpage AR. Tumour promotion by cyanobacterial toxins. Phycologia. 1996; 35(6S):74-79.

9. Falconer IR. Tumor promotion and liver injury caused by oral consumption of cyanobacteria. Environmental Toxicology and Water Quality: An International Journal. 1991; 6(2):177-184.

10. Nishiwaki-Matsushima R, Ohta T, Nishiwaki S, Suganuma M, Kohyama K, Ishikawa T, Carmichael WW, Fujiki H. Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR. Journal of Cancer Research and Clinical Oncology. 1992; 118(6):420-424.

11. Yoshida T, Makita Y, Nagata S, Tsutsumi T, Yoshida F, Sekijma M, Tamura S, Ueno Y. Acute oral toxicity of microcystin-LR, a cyanobacterial hepatotoxin, in mice. Natural Toxins. 1997; 5(3): 91-95.

12. DeMott WR, Zhang QX, Carmichael WW. Effects of toxic cyanobacteria and purified toxins on the survival and feeding of a copepod and three species of Daphnia. Limnology and Oceanography. 1991; 36(7):1346-1357.

13. Ghazali IE, Saqrane S, Carvalho AP, Ouahid Y, Oudra B, del Campo FF, Vasconcelos V. Compensatory growth induced in zebrafish larvae after pre-exposure to a Microcystis aeruginosa natural bloom extract containing microcystins. International Journal of Molecular Sciences. 2009; 10(1):133-146.

14. Saqrane S, Ghazali IE, Ouahid Y, Hassni ME, Hadrami IE, Bouarab L, del Campo FF, Oudra B, Vasconcelos V. Phytotoxic effects of cyanobacteria extract on the aquatic plant Lemna gibba: Microcystin accumulation, detoxication and oxidative stress induction. Aquatic Toxicology. 2007; 83(4):284-294.

15. LeBlanc S, Pick FR, Aranda-Rodriguez R. Allelopathic effects of the toxic cyanobacterium Microcystis aeruginosa on duckweed, Lemna gibba L. Environmental Toxicology. 2005; 20(1):67-73.

16. Pflugmacher S, Codd GA, Steinberg CEW. Effects of the cyanobacterial toxin microcystin-LR on detoxication enzymes inaquatic plants. Environmental Toxicology. 1999; 14(1):111-115.

17. Pflugmacher S. Possible allelopathic effects of cyanotoxins, with reference to microcystin-LR, in aquatic ecosystems. Environmental Toxicology. 2002; 17(4):407-413.

18. Kormas KA, Lymperopoulou DS. Cyanobacterial toxin degrading bacteria: Who are they? BioMed Research International. 2013; 2013(0):1-12.

19. Pflugmacher S, Kühn S, Lee SH, Choi JW, Baik S, Kwon KS, Contardo-Jara V. Green liver systems® for water purification – using the phytoremediation potential of aquatic macrophytes for the removal of different cyanobacterial toxins from water. American Journal of Plant Sciences. 2015; 6(9):1607-1618.

20. Nimptsch J, Wiegand C, Pflugmacher S. Cyanobacterial toxin elimination via bioaccumulation of MC-LR in aquatic macrophytes: An application of the “Green Liver Concept”. Environmental Science & Technology. 2008; 42(22):8552-8557.

21. Tien M. Properties of ligninase from Phanerochaete chrysosporium and their possible applications. Critical Reviews in Microbiology. 1987; 15(2):141-168.

22. Ulmer D, Leisola M, Puhakka J, Fiechter A. Phanerochaete chrysosporium: Growth pattern and lignin degradation. European Journal of Applied Microbiology and Biotechnology. 1983; 18(3):153-157.

23. Novotný ÄŒ, Svobodová K, Erbanová P, Cajthaml T, Kasinath A, Lang E, Šašek V. Ligninolytic fungi in bioremediation: Extracellular enzyme production and degradation rate. Soil Biology and Biochemistry. 2004; 36(10):1545-1551.

24. Reddy CA. The potential for white-rot fungi in the treatment of pollutants. Current Opinion in Biotechnology. 1995; 6(3):320-328.

25. Arjmand M, Sandermann Jr H. N-(Chlorophenyl)-succinimides: A novel metabolite class isolated from Phanerochaete chrysosporium. Pesticide Biochemistry and Physiology. 1987; 27(2):173-181.

26. Arjmand M, Sandermann Jr H. Mineralization of chloraniline/lignin conjugates and of free chloranilines by the white-rot fungus Phanerochaete chrysosporium. Journal of Agricultural and Food Chemistry. 1985; 33(6):1055-1060.

27. Hickey WJ, Fuster DJ, Lamar RT. Transformation of atrazine in soil by Phanerochaete chrysosporium. Soil Biology and Biochemistry. 1994; 26(12):1665-1671.

28. Mougin C, Laugero C, Asther M, Dubroca J, Frasse P, Asther M. Biotransformation of the herbicide atrazine by the white-rot fungus Phanerochaete chrysosporium. Applied and Environmental Microbiology. 1994; 60(2):705-708.

29. Bumpus JA. Biodegradation of polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium. Applied and Environmental Microbiology. 1989; 55(1):154-158.

30. Eaton DC. Mineralization of polychlorinated biphenyls by Phanerochaete chrysosporium: A ligninolytic fungus. Enzyme and Microbial Technology. 1985; 7(5):194-196.

31. Fernando T, Bumpus JA, Aust SD. Biodegradation of TNT (2,4,6-trinitrotoluene) by Phanerochaete chrysosporium. Applied and Environmental Microbiology. 1990; 56(6):1666-1671.

32. Mileski G, Bumpus JA, JurekMA, Aust SD. Biodegradation of pentachlorophenol by the white-rot fungus Phanerochaete chrysosporium. Applied and Environmental Microbiology. 1988; 54(12):2885-2889.

33. Joshi DK, Gold MH. Degradation of 2,4,5-trichlorophenol by the lignin-degrading basidiomycete Phanerochaete chrysosporium. Applied and Environmental Microbiology. 1993; 59(6):1779-1785.

34. Cammarota MC, Sant'Anna Jr GL. Decolorization of kraft bleach plant E1 stage effluent in a fungal bioreactor. Environmental Technology. 1992; 13(1):65-71.

35. Lu Y, Yan L, Wang Y, Zhou S, Fu J, Zhang J. Biodegradation of phenolic compounds from coking wastewater by immobilized white rot fungus Phanerochaete chrysosporium. Journal of Hazardous Materials. 2009; 165(1–3):1091-1097.

36. Miranda Rde C, Gomes Ede B, Pereira Jr N, Marin-Morales MA, Machado KM, Gusmão NB. Biotreatment of textile effluent in static bioreactor by Curvularia lunata URM 6179 and Phanerochaete chrysosporium URM 6181. Bioresource Technology. 2013; 142(0):361-367.

37. Mielgo I, Moreira MT, Feijoo G, Lema JM. Biodegradation of a polymeric dye in a pulsed bed bioreactor by immobilized Phanerochaete chrysosporium. Water Research. 2002; 36(7):1896-1901.

38. Lamar RT, Larsen MJ, Kirk TK, Glaser JA. Growth of the white-rot fungus Phanerochaete chrysosporium in soil. In: Barkley NP, Martin JF, editors. Land disposal, remedial action, incineration and treatment of hazardous waste: Proceedings of the 13th annual research symposium, U.S. EPA, Cincinnati, OH: Hazardous waste and engineering research laboratory, Office of research and development; 1987, p. 419-424.

39. Hoque E, inventor; Patent De, assignee. Verfahren zum Abbau von Xenobiotika durch Pilzarten mit Monooxygenase-/ Dioxygenase- Aktivität in Gegenwart von Pilzen mit Glutathion-S-Transferase-Aktivität. Deutsches Patentdokument DE10125365C2. 2003 June 5.

40. Rønhede S, Jensen B, Rosendahl S, Kragelund BB, Juhler RK, Aamand J. Hydroxylation of the herbicide isoproturon by fungi isolated from agricultural soil. Applied and Environmental Microbiology. 2005; 71(12):7927-7932.

41. Shroff KA, Vaidya VK. Kinetics and equilibrium studies on biosorption of nickel from aqueous solution by dead fungal biomass of Mucor hiemalis. Chemical Engineering Journal. 2011; 171(3):1234-1245.

42. Tewari N, Vasudevan P, Guha BK. Study on biosorption of Cr(VI) by Mucor hiemalis. Biochemical Engineering Journal. 2005; 23(2):185-192.

43. Fritscher J, Hoque E, Stöckl M, inventor; PatentDe, assignee. Fungus Mucor hiemalis, useful for the removal of heavy metals e.g. mercury, chromium, uranium and aluminum in ground- and surface water, purification plants, waste water and industrial water is new. Deutsches Patentdokument DE102004020837B4. 2006 Feb 23.

44. Balsano E, Esterhuizen-Londt M, Hoque E, Pflugmacher S. Toxin resistance in aquatic fungi poses environmentally friendly remediation possibilities: A study on the growth responses and biosorption potential of Mucor hiemalis EH5 against cyanobacterial toxins. International Journal of Water and Wastewater Treatment. 2015; 1(1):1-9.

45. Gibbs PA, Seviour RJ, Schmid F. Growth of filamentous fungi in submerged culture: Problems and possible solutions. Critical Reviews in Biotechnology. 2000; 20(1):17-48.

46. Kim JH, Lebeault JM, Reuss M. Comparative study on rheological properties of mycelial broth in filamentous and pelleted forms. European Journal of Applied Microbiology and Biotechnology. 1983; 18(1):11-16.

47. Olsvik E, Kristiansen B. Rheology of filamentous fermentations. Biotechnology Advances. 1994; 12(1):1-39.

48. Liao W, Liu Y, Chen S. Studying pellet formation of a filamentous fungus Rhizopus oryzae to enhance organic acid production. In: Mielenz JR, Klasson KT, Adney WS, McMillan JD, editors. Applied Biochemistry and Biotechnology, Biotechnology for Fuels and Chemicals, The Twenty-Eight Symposium, New York: Humana Press; 2007, p. 689-701.

49. Liao W, Liu Y, Frear C, Chen S. A new approach of pellet formation of a filamentous fungus – Rhizopus oryzae. Bioresource Technology. 2007; 98(18):3415-3423.

50. Nyman J, Lacintra MG, Westman JO, Berglin M, Lundin M, Lennartsson PR, Taherzadeh MJ. Pellet formation of zygomycetes and immobilization of yeast. New Biotechnology. 2013; 30(5):516-522.

51. Žnidaršič P, Komel R, Pavko A. Studies of a pelleted growth form of Rhizopus nigricans as a biocatalyst for progesterone 11α-hydroxylation. Journal of Biotechnology. 1998; 60(3):207-216.

52. Papagianni M, Mattey M. Morphological development of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Application of neural network and cluster analysis for characterization of mycelial morphology. Microbial Cell Factories. 2006; 5(3):1-12.

53. Dynesen J, Nielsen J. Surface hydrophobicity of Aspergillus nidulans conidiospores and its role in pellet formation. Biotechnology Progress. 2003; 19(3):1049-1052.

54. Carlsen M, Spohr AB, Nielsen J, Villadsen J. Morphology and physiology of an α-amylase producing strain of Aspergillus oryzae during batch cultivations. Biotechnology and Bioengineering. 1996; 49(3):266-276.

55. Xia C, Zhang J, Zhang W, Hu B. A new cultivation method for microbial oil production: Cell pelletization and lipid accumulation by Mucor circinelloides. Biotechnology for Biofuels. 2011; 4(15):1-10.

56. Hoque E, Pflugmacher S, Fritscher J, Wolf M. Induction of glutathione S-transferase in biofilms and germinating spores of Mucor hiemalisstrain EH5 from cold sulfidic spring waters. Applied and Environmental Microbiology. 2007; 73(8):2697-2707.

57. Kirk TK, Schultz E, Connors WJ, Lorenz LF, Zeikus JG. Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Archives of Microbiology. 1978; 117(3):277-285.

58. Sambrook J, Russel T. Preparation of reagents and buffers used in molecular cloning, phosphate-buffered saline (PBS). In: Argentine J, Irwin N, Janssen KA, Curtis S, Zierler M, McInerny N, Brown D, Schaefer S, editors. Molecular cloning: a laboratory manual, New York: Cold Spring Harbor Laboratory Press; 2001, p. A1.7.

59. Tucker KG, Thomas CR. Mycelial morphology: The effect of spore inoculum level. Biotechnology Letters. 1992; 14(11):1071-1074.

60. Zhou Y, Du J, Tsao GT. Mycelial pellet formation by Rhizopus oryzae ATCC 20344. Applied Biochemistry and Biotechnology. 2000; 84(1):779-789.

61. Sladdin M, Lynch JM. Antimicrobial properties of calcium peroxide in relation to its potential use as a seed dressing. Journal of General Microbiology. 1983; 129(7):2307-2314.

62. Douglas HW, Collins AE, Parkinson D. Electric charge and other surface properties of some fungal spores. Biochimica et Biophysica Acta. 1959; 33(2):535-538.

63. Takahashi J, Yamada H. Studies on the effect of some physical conditions on the submerged mold culture. Part II. On the two types of pellet formation in the shaking culture. Journal of Agricultural Chemical Society. 1959; 33(8):707-710.

64. Van Suijdam JC, Kossen NWF, Paul PG. An inoculum technique for the production of fungal pellets. European Journal of Applied Microbiology and Biotechnology. 1980; 10(3):211-221.

65. Gerin PA, Dufrene Y, Bellon-Fontaine MN, Asther M, Rouxhet PG. Surface properties of the conidiospores of Phanerochaete chrysosporium and their relevance to pellet formation. Journal of Bacteriology. 1993; 175(16):5135-5144.

66. Wu J, Li QB. Biosorption of lead by Phanerochaete chrysosporium in the form of pellets. Journal of Environmental Sciences. 2002; 14(1):108-114.

67. Liu Y, Liao W, Chen S. Study of pellet formation of filamentous fungi Rhizopus oryzae using a multiple logistic regression model. Biotechnology and Bioengineering. 2008; 99(1):117-128.

68. Henzler HJ, Schedel M. Suitability of the shaking flask for oxygen supply to microbiological cultures. Bioprocess Engineering. 1991; 7(3):123-131.

69. Driouch H, Hänsch R, Wucherpfennig T, Krull R, Wittmann C. Improved enzyme production by bio-pellets of Aspergillus niger: Targeted morphology engineering using titanate microparticles. Biotechnology and Bioengineering. 2012; 109(2):462-471.

70. Liu Y, Liao W, Chen S. Co-production of lactic acid and chitin using a pelletized filamentous fungus Rhizopus oryzae cultured on cull potatoes and glucose. Journal of Applied Microbiology. 2008; 105(5):1521-1528.

71. Pera LM, Callieri DA. Influence of calcium on fungal growth, hyphal morphology and citric acid production in Aspergillus niger. Folia Microbiologica. 1997; 42(6):551-556.

72. Pflugmacher S, Wiegand C, Beattie KA, Codd GA, Steinberg C. Uptake of the cyanobacterial hepatotoxin microcystin-LR by aquatic macrophytes. Journal of Applied Botany. 1998; 72(5-6):228-232.

73. Mitrovic SM, Allis O, Furey A, James KJ. Bioaccumulation and harmful effects of microcystin-LR in the aquatic plants Lemna minor and Wolffia arrhiza and the filamentous alga Chladophora fracta. Ecotoxicology and Environmental Safety. 2005; 61(3):345-352.

Article Metrics

219 Absract views 185 PDF Downloads 404 Total views

Related Search

By author names

Citiaion Alert By Google Scholar