The potential of fungal co-cultures as biological inducers for increased ligninolytic enzymes on agricultural residues

  • G. N. IjomaEmail author
  • R. Selvarajan
  • M. Tekere
Original Paper


Dual cultivation or co-cultivation of fungi and the exploitation of interspecific interaction, even antagonism, show considerable promise as a strategy in enhancing enzyme production. The aim of this work was to investigate the phenomenon of antagonistic invasion as a strategy to possibly accelerate secondary metabolism in ligninolytic fungi and thereby to increase enzyme activity. Ten different fungal strains were cultivated on various substrates like corn cob, wheat straw and sugar cane bagasse and evaluated for the effects of invasion antagonistic interaction on enzyme production. Monoculture enzyme activities were compared with co-culture enzyme activities. However, strains such as Trichoderma sp. KN10, Rhizopus microsporus KN2, Fomitopsis sp. KN1 and Coriolopsis sp. KN6 demonstrated strong tendencies of invasion and replacement in co-cultures. Also, significant changes in morphology and concomitant increase in enzyme activity were demonstrated in a majority of these interactions with remarkable results observed with invasions involving Trichoderma sp. KN10. Analysis of mean values of enzyme activity showed dual culture interactions involving KN10 with values for manganese peroxidase production approximately at 1.46 U/mL compared to monoculture of 0.06 U/mL. Furthermore, dual culture laccase values were approximately 0.09 U/mL compared to monocultures of 0.05 U/mL. Overall, the highest enzyme activity was observed using wheat straw reflecting similar patterns of invasion interactions. This study demonstrates that dual cultivation of fungi with the appropriate substrates results in improved production of biotechnologically relevant enzymes.


Co-cultures Antagonism Invasion Ligninolytic enzymes Fungi Agro-residues 



The authors wish to thank Mr Oliver Wanjau for his tremendous support during the course of the laboratory work, Miss Annie Monanga for her critical evaluation of this work during the writing and Mr Taboga Mathiba for his contribution to the statistical analysis. They would like to acknowledge the Pearson Institute of Higher Education for the provision of their laboratory facilities. This work was financially supported by the National Student Financial Aid Scheme (NSFAS) project (Grant Number 54003/74721) managed by the University of South Africa (UNISA).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13762_2018_1672_MOESM1_ESM.docx (18 kb)
Supplementary material 1 (DOCX 18 kb)


  1. Aiken L, Appelbaum M, Boo-doo G et al (1999) Statistical methods in psychology journals. Am Psychol 54:594–604CrossRefGoogle Scholar
  2. Altschul SF, Madden TL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefGoogle Scholar
  3. Aryantha INP, Guest DI (2006) Mycoparasitic and antagonistic inhibition on Phytophthora cinnamomi rands by microbial agents isolated from manure composts. Plant Pathol J 5:291–298CrossRefGoogle Scholar
  4. Bader J, Mast-Gerlach E, Popovic M et al (2010) Relevance of microbial coculture fermentations in biotechnology. J Appl Microbiol 109:371–387. CrossRefGoogle Scholar
  5. Bagal UR, Leebens-Mack JH, Lorenz WW, Dean JFD (2012) The phenylalanine ammonia lyase (PAL) gene family shows a gymnosperm-specific lineage. BMC Genom 13:S1. Google Scholar
  6. Balan V (2014) Current challenges in commercially producing biofuels from lignocellulosic biomass. ISRN Biotechnol 2014:1–31CrossRefGoogle Scholar
  7. Balan V, da Costa Sousa L, Chundawat SPS et al (2008) Mushroom spent straw: a potential substrate for an ethanol-based biorefinery Mushroom spent straw—a potential substrate for an ethanol-based biorefinery. J Ind Microbiol Biotechnol 35:293–301. CrossRefGoogle Scholar
  8. Baldrian P (2004) Increase of laccase activity during interspecific interactions of white-rot fungi. FEMS Microbiol Ecol 50:245–253. CrossRefGoogle Scholar
  9. Basu S, Bose C, Ojha N et al (2015) Evolution of bacterial and fungal growth media. Bioinformation 11:182–184CrossRefGoogle Scholar
  10. Boddy L (2000) Interspecific combative interactions between wood-decaying basidiomycetes. FEMS Microbiol Ecol 31:185–194CrossRefGoogle Scholar
  11. Bonugli-Santos RC, Durrant LR, Sette LD (2010) Laccase activity and putative laccase genes in marine-derived basidiomycetes. Fungal Biol 114:863–872. CrossRefGoogle Scholar
  12. Coll PM, Fernandez-abalos JM, Villanueva JR et al (1993) Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971). Appl Environ Microbiol 59:2607–2613Google Scholar
  13. Collins PJ, Dobson ADW (1997) Regulation of laccase gene transcription in trametes versicolor. Appl Environ Micro Biol 63:3444–3450Google Scholar
  14. Cooke RC, Rayner AD (1984) Ecology of saprophytic fungi. Longman, New YorkGoogle Scholar
  15. Crowe JD, Olsson S (2001) Induction of laccase activity in Rhizoctonia solani by antagonistic Pseudomonas fluorescens strains and a range of chemical treatments. Appl Environ Microbiol 67:2088–2094. CrossRefGoogle Scholar
  16. Cupul CW, Abarca GH, Carrera DM, Vazquez RR (2014) Enhancement of ligninolytic enzyme activities in a Trametes maxima: Paecilomyces carneus co-culture—key factors revealed after screening using a Plackett–Burman experimental design. Electron J Biotechnol 17:114–121. CrossRefGoogle Scholar
  17. Dashtban M, Schraft H, Syed TA, Qin W (2010) Fungal biodegradation and enzymatic modification of lignin. Int J Biochem Mol Biol 1:36–50Google Scholar
  18. Daugas E, Susin SA, Zamzami N et al (2000) Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J 14:729–739CrossRefGoogle Scholar
  19. de Souza WR (2013) microbial degradation of lignocellulosic biomass. In: Chandel A (ed) Sustainable degradation of lignocellulosic biomass-techniques, applications and commercialization. InTech, Rijeka, pp 207–245Google Scholar
  20. Dhouib A, Hamza M, Zouari H et al (2005) Screening for ligninolytic enzyme production by diverse fungi from Tunisia. World J Microbiol Biotechnol 21:1415–1423. CrossRefGoogle Scholar
  21. Eggert C, Temp U, Eriksson K-EL (1996) The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl Environ Microbiol 62:1151–1158Google Scholar
  22. Elisashvili V, Kachlishvili E (2009) Physiological regulation of laccase and manganese peroxidase production by white-rot Basidiomycetes. J Biotechnol 144:37–42. CrossRefGoogle Scholar
  23. Emri T, Molnár Z, Pócsi I (2005) The appearances of autolytic and apoptotic markers are concomitant but differently regulated in carbon-starving Aspergillus nidulans cultures. FEMS Microbiol Lett 251:297–303. CrossRefGoogle Scholar
  24. Faget M, Nagel KA, Walter A et al (2013) Root–root interactions: extending our perspective to be more inclusive of the range of theories in ecology and agriculture using in vivo analyses. Ann Bot 112:253–266. CrossRefGoogle Scholar
  25. Falconer RE, Bown JL, White NA, Crawford JW (2008) Modelling interactions in fungi. J R Soc Interface 5:603–615. CrossRefGoogle Scholar
  26. Fargione J, Hill J, Tilman D et al (2008) Land clearing and the biofuel carbon debt. Science 319:1235–1238. CrossRefGoogle Scholar
  27. Flores C, Casasanero R, Trejo-Hernandez MR et al (2010) Production of laccases by Pleurotus ostreatus in submerged fermentation in co-culture with Trichoderma viride. J Appl Microbiol 108:810–817. CrossRefGoogle Scholar
  28. Gomaa OM, Momtaz OA (2015) Copper induction and differential expression of laccase in Aspergillus flavus. Braz J Microbiol 46:285–292CrossRefGoogle Scholar
  29. Griffith GW, Easton GL, Detheridge A et al (2007) Copper deficiency in potato dextrose agar causes reduced pigmentation in cultures of various fungi. FEMS Microbiol Lett 276:165–171. CrossRefGoogle Scholar
  30. Halliwell B, Aruoma OI (1991) DNA damage by oxygen-derived species: its mechanism and measurement in mammalian systems. FEBS Lett 281:9–19CrossRefGoogle Scholar
  31. Haruta S, Cui Z, Huang Z et al (2002) Construction of a stable microbial community with high cellulose-degradation ability. Appl Microbiol Biotechnol 59:529–534. CrossRefGoogle Scholar
  32. Hatakka A (1994) Lignin-modifying enzymes from selected white-rot fungi: production and role in lignin degradation. FEMS Microbiol Rev 13:125–135CrossRefGoogle Scholar
  33. Heilmann-Clausen J, Boddy L (2005) Inhibition and stimulation effects in communities of wood decay fungi: exudates from colonized wood influence growth by other species. Microb Ecol 49:399–406. CrossRefGoogle Scholar
  34. Hiscox J, Baldrian P, Rogers H, Boddy L (2010) Changes in oxidative enzyme activity during interspecific mycelial interactions involving the white-rot fungus Trametes versicolor. Fungal Genet Biol 47:562–571. CrossRefGoogle Scholar
  35. Hiscox J, Savoury M, Vaughan IP et al (2015) Antagonistic fungal interactions influence carbon dioxide evolution from decomposing wood. Fungal Ecol 14:24–32. CrossRefGoogle Scholar
  36. Hiscox J, Clarkson G, Savoury M et al (2016) Effects of pre-colonisation and temperature on interspecific fungal interactions in wood. Fungal Ecol 21:32–42. CrossRefGoogle Scholar
  37. Howell CR (2003) Mechanisms employed by trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis 87:4–10CrossRefGoogle Scholar
  38. Hu HL, Van Den Brink J, Gruben BS et al (2011) Improved enzyme production by co-cultivation of Aspergillus niger and Aspergillus oryzae and with other fungi. Int Biodeterior Biodegrad 65:248–252. CrossRefGoogle Scholar
  39. Hyun MW, Yun YH, Kim JY, Kim SH (2011) Fungal and plant phenylalanine ammonia-lyase. Mycobiology 39:257–265. CrossRefGoogle Scholar
  40. Iakovlev A, Olson A, Elfstrand M, Stenlid J (2004) Differential gene expression during interactions between Heterobasidion annosum and Physisporinus sanguinolentus. FEMS Microbiol Lett 241:79–85. CrossRefGoogle Scholar
  41. Jiang X, Wang X (2004) Cytochrome C-mediated apoptosis. Annu Rev Biochem 73:87–106. CrossRefGoogle Scholar
  42. Keller L, Surette MG (2006) Communication in bacteria: an ecological and evolutionary perspective. Nat Rev Microbiol 4:249–258. CrossRefGoogle Scholar
  43. Kirk TK, Croan S, Tien M (1986) Production of multiple ligninases by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain. Enzyme Microb Technol 8:27–32. CrossRefGoogle Scholar
  44. Kumar P, Barrett DM, Delwiche MJ, Stroeve P (2009) Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind Eng Chem Res 48:3713–3729. CrossRefGoogle Scholar
  45. Leiter E, Szappanos H, Oberparleiter C, Kaiserer L (2005) Antifungal protein PAF severely affects the integrity of the plasma membrane of Aspergillus nidulans and induces an apoptosis-like phenotype. Antimicrob Agents Chemother 49:2445–2453. CrossRefGoogle Scholar
  46. Leonowicz A, Matuszewska A, Luterek J et al (1999) Biodegradation of lignin by white rot fungi biodegradation of lignin by white rot fungi. Fungal Genet Biol 27:175–185. CrossRefGoogle Scholar
  47. Levin L, Forchiassin F, Ramos A (2002) Copper induction of lignin-modifying enzymes in the white-rot fungus Trametes trogii. Mycologia 94:377–383. CrossRefGoogle Scholar
  48. Li LY, Luo X, Wang X (2001) Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 412:95–99. CrossRefGoogle Scholar
  49. Lu Z, Tombolini R, Woo S et al (2004) In vivo study of Trichoderma-pathogen-plant interactions, using constitutive and inducible green fluorescent protein reporter systems. Appl Environ Microbiol 70:3073–3081. CrossRefGoogle Scholar
  50. Mahajan S (2011) Characterization of the white-rot fungus, Phanerochaete carnosa, through proteomic methods and compositional analysis of decayed wood fibre characterization of the white-rot fungus. University of Toronto, TorontoGoogle Scholar
  51. Marra R, Ambrosino P, Carbone V et al (2006) Study of the three-way interaction between Trichoderma atroviride, plant and fungal pathogens by using a proteomic approach. Curr Genet 50:307–321. CrossRefGoogle Scholar
  52. Moore-Landecker E (2002) Fundamentals of the Fungi, 4th edn. Prentice Hall, New JerseyGoogle Scholar
  53. Morris SJ, Friese CF, Allen MF (2007) Disturbance in natural ecosystems: scaling from fungal diversity to ecosystem functioning. In: Esser K, Kubicek CP, Druzhinina IS (eds) The Mycota, 2nd edn. Springer, Bochum, pp 31–42Google Scholar
  54. Pinan-Lucarré B, Balguerie A, Clave C (2005) Accelerated cell death in Podospora autophagy mutants †. Eukaryot Cell 4:1765–1774. CrossRefGoogle Scholar
  55. Prasher I, Chauhan R (2015) Effect of carbon and nitrogen sources on the growth, reproduction and ligninolytic enzymes activity of Dictyoarthrinium synnematicum somrith. Adv Zool Bot 3:24–30. Google Scholar
  56. Qi-He C, Krügener S, Hirth T et al (2011) Co-cultured production of lignin-modifying enzymes with white-rot fungi. Appl Biochem Biotechnol 165:700–718. CrossRefGoogle Scholar
  57. Searchinger T, Heimlich R (2015) Avoiding bioenergy competition for food crops and land. Washington, DCGoogle Scholar
  58. Searchinger T, Heimlich R, Houghton RA et al (2008) Use of U.S. croplands for biofuels increases greenhouse gases through emissions from land-use change. Science 423:1238–1240CrossRefGoogle Scholar
  59. Selvarajan R, Felföldi T, Tauber T et al (2015) Screening and evaluation of some green algal strains (Chlorophyceae) isolated from freshwater and soda lakes for biofuel production. Energies 8:7502–7521. CrossRefGoogle Scholar
  60. Sharma G, Pandey RR (2010) Influence of culture media on growth, colony character and sporulation of fungi isolated from decaying vegetable wastes. J Yeast Fungal Res 1:157–164Google Scholar
  61. Sharon A, Finkelstein A, Shlezinger N, Hatam I (2009) Fungal apoptosis: function, genes and gene function. FEMS Microbiol Rev 33:833–854. CrossRefGoogle Scholar
  62. Sims R, Taylor M, Saddler J, Mabee W (2008) From 1st to 2nd generation biofuel technologies: an overview of current and RD&D activities. ParisGoogle Scholar
  63. Slifkin M, Cumbie R (1988) Congo Red as a fluorochrome for the rapid detection of fungi. J Clin Microbiol 26:827–830Google Scholar
  64. Sorek N, Yeats TH, Szemenyei H et al (2014) The implications of lignocellulosic biomass chemical composition for the production of advanced biofuels. Bioscience 64:192–201. CrossRefGoogle Scholar
  65. Tamura K, Stecher G, Peterson D et al (2013) MEGA6: molecular evolutionary genetics analysis version 6. 0. Mol Biol Evol 30:2725–2729. CrossRefGoogle Scholar
  66. Ujor VC (2010) The physiological response of the white-rot fungus, Schizophyllum commune to Trichoderma viride, during interspecific mycelial combat. University of Westminster, WestminsterGoogle Scholar
  67. Vrsanska M, Buresova A, Damborsky P, Adam V (2015) Influence of different inducers on ligninolytic enzyme activities. J Met Nanotechnol 3:64–70Google Scholar
  68. Wang W, Yuan T, Cui B (2014) Biological pretreatment with white rot fungi and their co-culture to overcome lignocellulosic recalcitrance for improved enzymatic digestion. BioResources 9:3968–3976Google Scholar
  69. Wongwilaiwalin S, Rattanachomsri U, Laothanachareon T et al (2010) Analysis of a thermophilic lignocellulose degrading microbial consortium and multi- species lignocellulolytic enzyme system. Enzyme Microb Technol 47:283–290. CrossRefGoogle Scholar
  70. Koley S, Mahapatra SS (2015) Evaluation of culture media for growth characteristics of Alternaria solani, causing early blight of tomato. Plant Pathol Microbiol. Google Scholar
  71. Yang S-J, Kataeva I, Hamilton-Brehm SD et al (2009) Efficient degradation of lignocellulosic plant biomass, without pretreatment, by the thermophilic anaerobe “Anaerocellum thermophilum” DSM 6725. Appl Environ Microbiol 75:4762–4769. CrossRefGoogle Scholar
  72. Yorimitsu T, Klionsky DJ (2005) Autophagy: molecular machinery for self-eating. Cell Death Differ 12:1542–1552CrossRefGoogle Scholar
  73. Zhang X, Liu C (2015) Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids. Mol Plant 8:17–27. CrossRefGoogle Scholar

Copyright information

© Islamic Azad University (IAU) 2018

Authors and Affiliations

  1. 1.Department of Environmental SciencesUniversity of South AfricaFloridaSouth Africa
  2. 2.Department of Applied SciencesPearson Institute of Higher EducationMidrandSouth Africa

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