Origin and maintenance of fungi
F. oxysporum strains were isolated from agricultural soil, peat and plant samples collected from different sites in Ireland between October 2007 to February 2008 (Additiona file 1: Table S1) using Komada's selective medium (Komada 1975) (see Additiona file 1 information for more details). Prior to use, fungal isolates were sub-cultured onto PDA plates and incubated at 25°C for 5 days.
DNA extraction, F. oxysporum-specific PCR analysis and DNA sequencing
Genomic DNA was isolated using the rapid mini-preparation procedure described by Edel et al. (Edel et al. 2000). F. oxysporum-specific polymerase chain reaction (PCR) analysis was conducted using the primer pair PFO2/PFO3 and the conditions described by Edel et al. (2000) in order to amplify a 70 bp DNA fragment of the 28S rDNA D2 domain. PCR products (10 μl) were electrophoresed through 1% (wv-1) agarose gels containing 0.5 μgml−1 ethidium bromide. Products were visualised using Imagemaster VDS and Liscap software (Pharmacia Biotech, San Francisco, CA).
Fragments of both the ITS region of nuclear ribosomal DNA (nrDNA) and EF-1α gene amplified using primer pairs ITS4/ITS5 and EF-1/EF-2 and the conditions described by White et al. (1990) and O'Donnell et al. (1998), respectively. PCR products were electrophoresed through 1% (wv-1) agarose gels containing 0.5 μgml−1 ethidium bromide; bands were visualised by UV transillumination and were excised. PCR products were cleaned up using the mi-Gel Extraction Kit (Metabion, Germany) and were sequenced by Macrogen (Seoul, Korea). Consensus sequences (derived from forward and reverse sequences) were subjected to BLAST analysis (http://www.ncbi.nlm.nih.gov) (Altschul et al. 1997). Sequences were aligned using European Bioinformatics Institutes's ClustalW2 tool (http://www.ebi.ac.uk) (Larkin et al. 2007) and phylogenetic trees were generated using the Neighbor-joining method (Saitou & Nei 1987).
Solid-state cultivation (SSC) on straw/bran
The carbon substrate used for most SSC experiments was based on non-alkali-treated wheat straw blended with wheat bran. Dry wheat straw (cultivar Einstein) was ground in a coffee grinder (Model 203C, KRUPS, Poland, Mexico), passed through a sieve (2 mm pore size) and blended with unprocessed wheat bran (particle size ≤ 3 mm) (10:1 ratio of straw to bran). The straw composition of cultivar Einstein was determined as follows: cellulose 38.46%, hemicellulose 27.50% and lignin 13.18% (Ali et al. unpubl data). One gram of the straw/bran blend was mixed with 5 ml minimal medium (see below; excluding a C-source) and autoclaved (121°C for 15 min) in a 100 ml Erlenmeyer flask. Except where otherwise stated below, the cultures were grown in a minimal medium earlier described by Mishra et al. (1984) (pH 5); they contained 91% initial moisture (vw-1) and were maintained at 25°C. Fungal conidial inoculums was produced in Mung bean broth as described by Brennan et al. (2005) and were re-suspended in the minimal medium at a concentration of 106 ml-1. Flasks were supplemented with either 4 ml of conidial suspension or minimal medium (negative controls). For the aerobic growth periods, Erlenmeyer flasks were plugged with non-absorbent cotton and covered with aluminium foil. For the oxygen-limited growth period, Erlenmeyer flasks were plugged with cork and sealed with parafilm.
SSC experiments were conducted in order to determine the influence of the following factors on the bioethanol production capacity of F. oxysporum: (a) the duration of SSC - the length of aerobic/oxygen-limited growth phases were varied as follows: 2/4, 3/4, 3/7, 4/2, 4/4 and 4/7 days; (b) minimal medium composition - SSC was conducted using the fungal media and associated pH earlier described by Uchida et al. (2003), Crawford (1987), Christakopoulos et al. (1991b), Mishra et al. (1984), Bollok and Reczey (2000) and Panagiotou et al. (2003); (c) temperature - SSC cultures were incubated at either 20, 25, 30, 32 or 35°C; (d) pH - SSC cultures were adjusted to initial pH 4, 5, 6, 7 or 8 and cultures were incubated at 35°C; (e) moisture content - adjusted to either 80, 85 and 91% initial moisture (vw-1) by adjusting the amount of minimal media added to the flask before autoclaving and cultures were incubated at 35°C. For each treatment, three replicate flasks were used and each experiment was conducted twice.
Shake flask cultivation of delignified straw were also conducted in order to determine the efficacy of F. oxysporum in producing ethanol from it. The culture conditions were not as above; those described by Christakopoulos et al. (1991a) were used in order to compare yields with those previously reported for other strains of F. oxysporum. In brief, fungal inoculum was grown aerobically in a 250 ml Büchner flask containing 50 ml minimal medium and 1% (wv-1) alkali treated cellulose and 0.15% (wv-1) wheat bran as carbon sources. After 3 days, cultures were amended with 1.5 g of dry-sterilised alkali-treated wheat straw and grown under oxygen-limiting condition for 4-6 days. For switching from aerobic to oxygen-limiting condition inside the flask, the cotton plugs were replaced with a rubber bung and sealed with paraflim while the hose barb was fitted with a silicon tube and the end was dipped in water filled beaker.
Estimation of bioethanol
Following SSC, flasks were incubated at 4°C for 1 h in order to condense any synthesised alcohol. Then flasks were supplemented with 10 ml of sterile cold water, plugged with cork and incubated at 150 rpm, 25°C for 1 h. All ethanol extraction procedures thereafter were conducted in a cold room (4°C). Flasks were incubated for 1 h and two sub-samples (2 ml) of liquid were removed to sterile tubes, and centrifuged at 10,000 rpm at 4°C for 20 min. The supernatant was decanted and stored at −70°C until further use. Bioethanol estimation (mg-1 wheat straw/bran) was performed using the QuantiChrom™ Bioethanol Assay Kit (DIET-500) (BioAssay Systems, CA, USA) according to manufacturer's instruction. Results were based on duplicate analyses conducted for each sub-sample.
Estimation of acetic acid and xylitol
Acetic acid and xylitol content of the culture supernatants were determined enzymatically using the Megazyme™ acetic acid and Xylitol assay kits (Megazyme, Co. Wicklow, Ireland) according to manufacturer's instruction. Results were based on duplicate analyses conducted for each sub-sample.
Estimation of fungal biomass
Fungal biomass was estimated based on the glucosamine content of cell wall chitin. Chitin was hydrolysed into N-acetyl glucosamine as previously described by Scotti et al. (2001), which was assayed by the modified colorimetric method described by Ride and Drysdale (1972).
For enzyme assays, SSC was conducted using the medium described by Mishra et al. (1984) (pH 7). Fungal cultures contained 91% initial moisture and were incubated at 30°C. For cellulase assays, flasks were harvested after 4 days of aerobic growth because an initial screen identified this as the time point when cellulase activity reached maximal levels for all the F. oxysporum strains (data not shown). Following SSC, cultures were supplemented with 10 ml of distilled water and were incubated at 25°C, 150 rpm for 1 h before being transferred to sterile tubes and centrifuged at 10,000 rpm for 20 min. Supernatants were harvested, flash-frozen in liquid nitrogen and stored at −70°C until further use. Total protein content in the supernatant was determined using the Bradford assay and bovine serum albumin (BSA) as a standard (Bradford 1976). Exoglucanase (EC 126.96.36.199) activity was measured using 1% Avicel (Sigma Chemical, St. Louis, USA) in 100 mM sodium acetate buffer (pH 4.8), as described by (Wood & Bhat 1988) and activity was expressed in nkat μg-1 of crude protein. The activity of β-glucosidase (EC 188.8.131.52), β-xylosidase (EC 184.108.40.206), endoglucanase (EC 220.127.116.11) and endoxylanase (EC 18.104.22.168) in the supernatant was determined as described by Thygesen et al. (2003) and was expressed in nkat μg-1 of crude protein. In each enzyme assay, the specific activity of two commercially available cellulase enzyme mixes, Celluclast® from Trichoderma reesei (Cat. no. 9012-54-8, Sigma Chemical, St. Louis, USA) and Novozyme 188 from Aspergillus niger (Cat. no. C6105, Sigma Chemical, St. Louis, USA) were also estimated, as was that of a mixture of Celluclast® (83% v v-1) and Novozyme 188 (17% vv-1), as described earlier by Thygesen et al. (2003).
When assaying alcohol dehydrogenase (ADH) (EC 22.214.171.124) activity, SSC was conducted as described for cellulase enzyme assays, except that incubation conditions were 4 days aerobic followed by 4 days oxygen-limited growth. Following SSC, solid residue (including fungal mycelium) was washed twice with sterile distilled water. Samples were flash-frozen in liquid nitrogen and stored at −70°C until use. Cell extract was obtained as described by Kayali et al. (2005). The total protein in the extract was determined as above and ADH activity was assayed and expressed in nkat μg-1 of protein in the cell free extract as described by Ke et al. (1995). Commercially available ADH from Saccharomyces cerevisiae (Cat. No. A3263, Sigma Chemical, St. Louis, USA) was also included in these assays.
See supplementary information for information regarding data distribution, transformation and pooling. The significance of treatment effects was analysed within the Statistical Package for the Social Sciences (SPSS 11.0, SPSS Inc.) by either (i) normally distributed data - one-way ANOVA with Post Hoc pair wise Least Significance Difference (LSD) comparisons (P = 0.050), or (ii) non-normally-distributed data - the Kruskal-Wallis H test. Correlation analysis between specific enzyme activities and either bioethanol yield or fungal biomass data was performed by one-tailed correlation analysis conducted using mean data values (non-normal data: Spearman Rank; normal data: Pearson product moment) within SPSS. Correlation analysis between bioethanol yield and fungal biomass data (transformed) was performed using one-tailed Pearson product moment correlation analyses within SPSS.