Exploiting the inter-strain divergence of Fusarium oxysporum for microbial bioprocessing of lignocellulose to bioethanol

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Abstract

Microbial bioprocessing of lignocellulose to bioethanol still poses challenges in terms of substrate catabolism. A targeted evolution-based study was undertaken to determine if inter-strain microbial variability could be exploited for bioprocessing of lignocellulose to bioethanol. The microorganism studied was Fusarium oxysporum because of its capacity to both saccharify and ferment lignocellulose. Strains of F. oxysporum were isolated and assessed for their genetic variability. Using optimised solid-state straw culture conditions, experiments were conducted that compared fungal strains in terms of their growth, enzyme activities (cellulases, xylanase and alcohol dehydrogenase) and yield of bioethanol and the undesirable by-products acetic acid and xylitol. Significant interstrain divergence was recorded in regards to the capacity of studied F. oxysporum strains to produce alcohol from untreated straw. No correlation was observed between bioethanol synthesis and either the biomass production or microbial enzyme activity. A strong correlation was observed between both acetic acid and xylitol production and bioethanol yield. The level of diversity recorded in the alcohol production capacity among closely-related microorganism means that a targeted screening of populations of selected microbial species could greatly improve bioprocessing yields, in terms of providing both new host strains and candidate genes for the bioethanol industry. © 2012 Ali et al.

Figures

  • Figure 1 Genetic diversity among eight strains of Fusarium oxysporum, based on DNA sequence data from fragments of the (A) internal transcribed spacer (ITS) region of nrDNA (B) elongation factor-1 alpha (EF-1a) gene. Sequences analysed were derivedfrom F. oxysporum strains 4E, 7E, 12A, 13C, 11C, 13B, 21D, 27E or F. oxysporum sequences within the NCBI Nucleotide database (http://www.ncbi.nlm.nih.gov/nuccore): F-X.1.7-030520-03 (GenBank No. EU364857.1), FO-10 (GenBank No. AY928417.1), FoChz10c (GenBank No. EU313533.1) and NRRL 38291 (GenBank No. FJ985370.1). The ITS was amplified using PCR primers ITS4 and ITS5 as described by White et al. (White et al. 1990). The 5’ portion of EF1a was amplified using the primers EF-1 and EF-2 as described by O’Donnell et al. (1998). DNA sequences were aligned using European Bioinformatics Institutes’s ClustalW2 tool (www.ebi.ac.uk) (Larkin et al. 2007) and phylogenetic trees were generated using the Neighbor-joining method (Saitou & Nei 1987)
  • Figure 2 Growth and ethanol production by strains of Fusarium oxysporum cultivated on a straw/bran lignocellulosic substrate (10:1 ratio of straw to bran). Fungal isolates were grown in solid-state culture on minimal medium, pH 7 (Mishra et al. 1984) supplemented with 1 g milled straw/bran (initial moisture content was 91% vw-1). Cultures were incubated at 35°C for 4 days of aerobic followed by 4 days of oxygen-limited conditions. Ethanol produced in the culture was estimated using QuantiChrom™ Ethanol Assay Kit (DIET-500) (BioAssay Systems, USA) according to manufacturer’s instruction. Fungal biomass was estimated as described earlier by Scotti et al. (Scotti et al. 2001). Bars indicate SEM (LSD 0.05 bioethanol = 8.93; LSD 0.05 biomass = 0.023).
  • Figure 3 Acetic acid (A) and xylitol (B) produced as a byproduct during bioethanol production by different strains of Fusarium oxysporum cultivated on a straw/bran lignocellulosic substrate (10:1 ratio of straw to bran). Fungal isolates were grown in solid-state culture on 1 g straw/bran supplemented with 5 ml-1 minimal medium, pH 7 (Mishra et al. 1984) (initial moisture content was 91% vw-1). Cultures were incubated at 35°C for 4 days of aerobic followed by 4 days of oxygen-limited conditions. Acetic acid and xylitol content of the culture supernatant were determined enzymatically using the Megazyme™ acetic acid and Xylitol assay kit respectively (Megazyme, Ireland) according to manufacturer’s instruction. Bars indicate SEM (LSD0.05 acetic acid = 0.45478; LSD0.05 xylitol = 0.3004)
  • Figure 4 Specific activity of the major cellulases, xylanase and alcohol dehydrogenase produced by strains of Fusarium oxysporum during growth on wheat straw/bran. Specific activity of (A) endoglucanase (EC 3.2.1.4), (B) exoglucanase (EC 3.2.1.91), (C) b-glucosidase (EC 3.2.1.21), (D) endoxylanase (EC 3.2.1.8), (E) b-xylosidase (EC 3.2.1.37) and (F) alcohol dehydrogenase (ADH) (EC 1.1.1.1) as determined for F. oxysporum strains 11C, 12A, 13C, 27E, 4E and 7E and commercial enzyme preparations, i.e. Celluclast®, Novozyme 188, Commercial mix. (83% vv-1 Celluclast®, 17% vv-1 Novozyme 188) and ADH (Sigma Chemical, St. Louis, USA). Fungal isolates were grown in solid-state culture on minimal medium, pH 7 (Mishra et al. 1984) supplemented with 1 g milled straw/bran (initial moisture content was 91% vw-1). Cultures were incubated at 30°C. Activities of cellulases enzymes in the water extract of the fermented straw (after 4 days of aerobic growth) were determined as described earlier by Wood & Bhat (1986) and Thygesen et al. (Thygesen et al. 2003). ADH activity in the cellular extract of the fermented straw (after 4 days of aerobic and 4 days oxygen-limited growth) were determined as described earlier by Kayali et al. (Kayali et al. 2005) and Ke et al. (Ke et al. 1995). Total protein in the extracts were measured by Bradford assay (Bradford 1976) and specific activity of the enzymes expressed as nkat μg1of crude protein. Bars indicate SEM (LSD 0.05 for parts A - F = 89.92, 6.85, 0.52, 962.96, 0.48 and 60.41, respectively)

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Ali, S. S., Khan, M., Fagan, B., Mullins, E., & Doohan, F. M. (2012). Exploiting the inter-strain divergence of Fusarium oxysporum for microbial bioprocessing of lignocellulose to bioethanol. AMB Express, 2(1), 1–9. https://doi.org/10.1186/2191-0855-2-16

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