272 Appl Biochem Biotechnol (2011) 165:270–278 Fig. 1 Summary of the method used to determine the EPF index of WIAS polysaccharides The bottles were capped with 1-cm-thick butyl rubber stoppers, sealed with aluminum caps, and incubated at 30 °C. Atmospheric gases were sampled over the course of the MC phase of the experiment. The pressure in the gas space was measured using a Thermo Fisher Scientific Traceable pressure meter. Earlier observations indicated that the MC microbial communities were dominated by Gram-positive clostridia. To verify this dominance, we isolated organotrophs by plating serial diluted suspensions of compost tea on glucose–cellulose–agar plates (30 g/L tryptic soy broth; 10 g/L D-glucose; 15 g/L agar; and 2 g/L cellulose (Sigma, C6288); adjusted to pH 6.5). Inoculated media were incubated at 30 °C under aerobic conditions (~21% O2) and also under microaerophilic conditions (~1.6% O2 in N2). Fifty clones were picked up randomly and saved in a library. The isolated clones were grown for 2 days in the same culture medium (minus agar and cellulose), cells were separated by centrifugation (13,000 rpm, 2 min at 2 °C), and the genomic DNA (gDNA) was extracted by direct boiling for 5 min in 1× TE [19, 20]. After centrifugation (13,000 rpm, 10 min at 2 °C), ~70% of the supernatant was recovered, and the dsDNA content was quantified using a NanoDrop 1000 instrument. The PCR amplification mixtures contained each primer at 5 μM concentration, ~3.6 ng/μl gDNA, and 1% DMSO to minimize inhibition by melanin. All clones were verified with the 16S rDNA universal primers 8F 5′-AGAGTTTGATCCTGGCTCAG and 1492R 5′-GGTTACCTTGTTACGACTT [20], and with the 16S rDNA Clostridium-specific primers Chis150F 5′-AAAGGRAGATTAATACCGCATAA and ClostIR 5′-TTCTTCCTA ATCTCTACGCA [21, 22]. For PCR amplification, we used an Applied Biosystems GeneAmp 2700 instrument and the following conditions: denaturing at 95 °C for 2 min; 40 cycles of 95 °C for 30 s, 46 °C for 30 s, and 72 °C for 2 min; and final extension at 72 °C for 5 min. Amplicon sizes were verified by electrophoresis in 0.7% agarose gels stained with ethidium bromide. The strains showing amplification with the Chis150F–ClostIR primer pair were considered to be phylogenetically related to Clostridium. Microbial activity was monitored by following changes in O2 and CO2 content in the headspaces and formation of VOAs, alcohols, and ketones. For the analysis of atmospheric