Bioethanol production from lignocellulosic materials is hindered by the high costs of pretreatment and the enzymes. The present study aimed to evaluate whether co-cultivation of four selected cellulolytic fungi yields higher cellulase and xylanase activities compared to the monocultures and to investigate whether the enzymes from the co-cultures yield higher saccharification on selected plant materials without thermo-chemical pretreatment. The fungal isolates, Trichoderma reesei F118, Penicillium javanicum FS7, Talaromyces sp. F113, and Talaromyces pinophilus FM9, were grown as monocultures and binary co-cultures under submerged conditions for 7 days. The cellulase and xylanase activities of the culture filtrates were measured, and the culture filtrates were employed for the saccharification of sugarcane leaves, Guinea grass leaves, and water hyacinth stems and leaves. Total reducing sugars and individual sugars released from each plant material were quantified. The co-culture of Talaromyces sp. F113 with Penicillium javanicum FS7 and of T. reesei F118 with T. pinophilus FM9 produced significantly higher cellulase activities compared to the corresponding monocultures whereas no effect was observed on xylanase activities. Overall, the highest amounts of total reducing sugars and individual sugars were obtained from Guinea grass leaves saccharified with the co-culture of T. reesei F118 with T. pinophilus FM9, yielding 63.5% saccharification. Guinea grass leaves were found to be the most susceptible to enzymatic saccharification without pre-treatment, while water hyacinth stems and leaves were the least. Accordingly, the study suggests that fungal co-cultivation could be a promising approach for the saccharification of lignocellulosic materials for bioethanol production.

Department of Botany Faculty of Science University of Peradeniya Kandy Sri Lanka

National Institute of Fundamental Studies Hantana Road Kandy Sri Lanka

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Ahamed A, Vermette P (2008) Enhanced enzyme production from mixed cultures of Trichoderma reesei RUT-C30 and Aspergillus niger LMA grown as fed batch in a stirred tank bioreactor. Biochem Eng J 42(1):41–46. https://doi.org/10.1016/j.bej.2008.05.007 DOI

Ahmed S, Riaz S, Jamil A (2009) Molecular cloning of fungal xylanases: an overview. Appl Microbiol Biotechnol 84:19–35. https://doi.org/10.1007/s00253-009-2079-4 PubMed DOI

Amarasinghe SR (2021) Use of invasive water hyacinth for composting of ordinary leaf litter. Sri Lankan J Agric Ecosyst 3(1):5–16 DOI

Amore A, Giacobbe S, Faraco V (2013) Regulation of cellulase and hemicellulase gene expression in fungi. Curr Genom 14(4):230–249 DOI

Bajaj P, Mahajan R (2019) Cellulase and xylanase synergism in industrial biotechnology. Appl Microbiol Biotechnol 103:8711–8724 PubMed DOI

Ball S, Bullock S, Lloyd L, Mapp KP, Ewen A (2011) Analysis of carbohydrates, alcohols, and organic acids by ion-exchange chromatography. Agilent Hi-plex Columns Applications Compendium. Agilent Technologies Inc., Santa Clara, California. https://www.agilent.com/cs/library/applications/5990-8801EN%20Hi-Plex%20Compendium.pdf

Burke SV, Wysocki WP, Zuloaga FO, Craine JM, Pires JC, Edger PP, Mayfield-Jones D, Clark LG, Kelchner SA, Duvall MR (2016) Evolutionary relationships in panicoid grasses based on plastome phylogenomics (Panicoideae; Poaceae). BMC Plant Biol 16(1):1–11. https://doi.org/10.1186/s12870-016-0823-3 DOI

Carpita NC, Gibeaut DM (1993) Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J 3(1):1–30 PubMed DOI

Deshpande SK, Bhotmange MG, Chakrabarti T, Shastri PN (2008) Production of cellulase and xylanase by Trichoderma reesei (QM 9414 mutant), Aspergillus niger and mixed culture by solid state fermentation (SSF) of water hyacinth (Eichhornia crassipes). Ind J Chem Tech 15:449–456

Fatma S, Saleem A, Tabassum R (2021) Wheat straw hydrolysis by using co-cultures of Trichoderma reesei and Monascus purpureus toward enhanced biodegradation of the lignocellulosic biomass in bioethanol biorefinery. Biomass Convers Biorefin 11:743–754 DOI

Foster CE, Martin TM, Pauly M (2010) Comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part II: carbohydrates. J vis Exp 37:1837. https://doi.org/10.3791/1837 DOI

Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59(2):257–268 DOI

Global biodiversity information facility (2019) GBIF. Gbif.org. https://www.gbif.org/ . Accessed 24 February 2024

Gottschalk LMF, Oliveira RA, Bon EPS (2010) Cellulases, xylanases, β-glucosidase and ferulic acid esterase produced by Trichoderma and Aspergillus act synergistically in the hydrolysis of sugarcane bagasse. Biochem Eng J 51:72–78. https://doi.org/10.1016/j.bej.2010.05.003 DOI

Guna V, Ilangovan M, Anantha Prasad MG, Reddy N (2017) Water hyacinth: a unique source for sustainable materials and products. ACS Sustain Chem Eng 5(6):4478–4490. https://doi.org/10.1021/acssuschemeng.7b00051 DOI

Gupta MN, Bisaria VS (2018) Stable cellulolytic enzymes and their application in hydrolysis of lignocellulosic biomass. Biotechnol J. https://doi.org/10.1002/biot.201700633 PubMed DOI

Gusakov AV, Sinitsyn AP (2012) Cellulases from Penicillium species for producing fuels from biomass. Biofuels 3(4):463–477 DOI

Hodgson-Kratky K, Papa G, Rodriguez A, Stavila V, Simmons B, Botha F, Furtado A, Henry R (2019) Relationship between sugarcane culm and leaf biomass composition and saccharification efficiency. Biotechnol Biofuels. https://doi.org/10.1186/s13068-019-1588-3 PubMed DOI PMC

Kariyawasam CS, Kumar L, Ratnayake SS (2021) Potential risks of invasive alien plant species on agriculture under climate change scenarios in Sri Lanka. Curr Res Environ Sustain 3:100051 DOI

Kurasawa T, Yachi M, Suto M, Kamagata Y, Takao S, Tomita F (1992) Induction of cellulase by gentiobiose and its sulfur-containing analog in Penicillium purpurogenum. Appl Environ Microbiol 58(1):106–110 PubMed DOI PMC

Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66(3):506–577. https://doi.org/10.1128/MMBR.66.3.506-577.2002 PubMed DOI PMC

Mandels M (1975) Microbial sources of cellulase. Biotechnol Bioeng Symp 5:81–105

Mandels M, Weber J (1969) The production of cellulases. Adv Chem 95:391–414

Mandels M, Andreotti R, Roche C (1976) Measurement of saccharifying cellulase. Biotechnol Bioeng Symp 6:21–33

Margeot A, Hahn-Hagerdal B, Edlund M, Slade R, Monot F (2009) New improvements for lignocellulosic ethanol. Curr Opin Biotechnol 20(3):372–380. https://doi.org/10.1016/j.copbio.2009.05.009 PubMed DOI

Messner R, Hagspiel K, Kubicek CP (1990) Isolation of a β-glucosidase binding and activating polysaccharide from cell walls of Trichoderma reesei. Arch Microbiol 154(2):150–155. https://doi.org/10.1007/BF00423325 DOI

Miller GL (1959) Use of Dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428. https://doi.org/10.1021/ac60147a030 DOI

Moodley P, Kana EBG (2017) Microwave-assisted inorganic salt pretreatment of sugarcane leaf waste: Effect on physiochemical structure and enzymatic saccharification. Bioresour Technol 235:35–42. https://doi.org/10.1016/j.biortech.2017.03.031 PubMed DOI

Morilla EA, Taddia A, Sortino M, Tubio G (2023) Mixed cultures of Aspergillus niger and Rhizopus oryzae using lignocellulosic substrates to improve hydrolytic enzyme production. BioEnergy Res 16:2285–2296 DOI

O’ Neill MA, York WS, (2003) The composition and structure of plant primary cell walls. Annual plant reviews, 8th edn. Rose JKC Blackwell publishing Ltd, Oxford, pp 1–54

Odorico FH, Morandim-Giannetti ADA, Lucarini AC, Torres RB (2018) Pretreatment of guinea grass (Panicum maximum) with the ionic liquid 1-ethyl-3-methyl imidazolium acetate for efficient hydrolysis and bioethanol production. Cellulose 25:2997–3009. https://doi.org/10.1007/s10570-018-1753-z DOI

Peláez RDR, Wischral D, Cunha JRB, Mendes TD, Pacheco TF, Siqueira FGD, Almeida JRMD (2022) Production of enzymatic extract with high cellulolytic and oxidative activities by co-culture of Trichoderma reesei and Panus lecomtei. Fermentation 8(10):522 DOI

Peterson R, Nevalainen H (2012) Trichoderma reesei RUT-C30 – thirty years of strain improvement. Microbiol 158:58–68. https://doi.org/10.1099/mic.0.054031-0 DOI

Premaratne S, Ibrahim MNM, Perera HGD (1993) Effect of stage of growth and additives on digestibility and palatability of Guinea (Panicum maximum, Jacq) grass silage. J Natl Sci Found Sri Lanka 21(2):175–182 DOI

Santana JC, Souza Abud AK, Wisniewski A, Navickiene S, Romão LPC (2020) Optimization of an organosolv method using glycerol with iron catalysts for the pretreatment of water hyacinth. Biomass Bioenergy 133:105454. https://doi.org/10.1016/j.biombioe.2019.105454 DOI

Shallom D, Shoham Y (2003) Microbial hemicellulases. Curr Opin Microbiol 6(3):219–228. https://doi.org/10.1016/S1369-5274(03)00056-0 PubMed DOI

Silva JCR, Salgado JCS, Vici AC et al (2020) A novel Trichoderma reesei mutant RP698 with enhanced cellulase production. Braz J Microbiol 51:537–545. https://doi.org/10.1007/s42770-019-00167-2 PubMed DOI

Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D (2005) Determination of ash in biomass. National Renewable Energy Laboratory, USA. http://www.nrel.gov/docs/gen/fy08/42622.pdf

Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2008) Determination of structural carbohydrates and lignin in biomass. National Renewable Energy Laboratory, USA. http://www.nrel.gov/docs/gen/fy13/42618.pdf

Soeder DJ (2021) Fossil fuels and climate change. In: Fracking and the Environment. Springer, Cham. https://doi.org/10.1007/978-3-030-59121-2_9

Trollope K, Nel DW, Volschenk H (2018) The heterologous expression potential of an acid-tolerant Talaromyces pinophilus β-glucosidase in Saccharomyces cerevisiae. Folia Microbiol 63:725–734 DOI

Wilson DB (2011) Microbial diversity of cellulose hydrolysis. Curr Opin Microbiol 14(3):259–263 PubMed DOI

Yilmaz N, Visagie CM, Houbraken J, Frisvad JC, Samson RA (2014) Polyphasic taxonomy of the genus Talaromyces. Stud Mycol 78:175–341 PubMed DOI PMC

Zhang YHP, Ding SY, Mielenz JR, Cui JB, Elander RT, Laser M, Himmel ME, McMillan JR, Lynd LR (2007) Fractionating recalcitrant lignocellulose at modest reaction conditions. Biotechnol Bioeng 97(2):214–223. https://doi.org/10.1002/bit.21386 PubMed DOI

Zhang C (2019) Lignocellulosic ethanol: technology and economics. In: Yun Y (ed) Alcohol fuels-current technologies and future prospect, IntechOpen