(275b) Switchgrass Solubilization By Mixed Methanogenic Enrichments with Comparison to Pure Cultures of Clostridium Thermocellum

To meet anticipated global demand for low carbon liquid transportation fuels, second generation biofuels will likely be produced from lignocellulose, a low cost and abundant plant biomass resource. However, the lignocellulose in plant cell walls is difficult to convert. Increasing lignocellulose conversion will significantly enhance biofuel production and lower capital & operational cost. Microbially-mediated fermentation of lignocellulose is a promising approach. Diverse, mixed microbial enrichment cultures provide an indication of the achievable extent of biomass solubilization. Yet there have been few fundamental studies of grass fermentation under the conditions anticipated for a high-rate, low cost industrial process, including very high solids concentration, short residence times and high temperatures. Motivated by this perspective, we have initiated study of thermophilic switchgrass fermentation using mixed culture anaerobic enrichments at decreasing residence times & increasing grass concentrations.

Inoculum was collected from a two-stage anaerobic digester (Vermont Technical College) fed with manure and food waste. Triplicate semi-continuous, anaerobic digesters were operated at 55^oC and 30g/L mid-season switchgrass concentration for 214 days, during which two of them were allowed to come to steady-state sequentially at residence times (RT) of 20 days, 10 days, 5 days and 3.3 days, with the other one running as control at RT = 20 days. This feed rate was maintained by regular replacement of 1/10 of reactor contents to achieve the nominal residence times. Whereas we expected a shift from methanogenesis to acidogenesis as RT decreased to 5 days, surprisingly, even at RT=3.3 days stable methane production was still seen and acids inhibition was not observed, which indicates that the digesters were still healthy and balanced. Total carbohydrate solubilization at RT = 20, 10, 5 and 3.3 days was 69.1%, 62.8%, 54.9% and 50.0% respectively. In addition to these continuous trials, batch studies compared the performance of mixed enrichment cultures to pure cultures of Clostridium thermocellum, where solubilization was similar despite the more limited enzyme diversity of the pure culture species.

16S rRNA analysis was used to characterize microbial populations dynamics. It was found that Firmicutes dominated each of the reactors, representing between 54-96% of the sequence reads, and these populations increased with reduced residence time. In addition, Synergistetes, Euryarchaeota etc. taxa were also well represented and relatively stable. By using bioinformatics tools, species present in the consortia were also matched to closely related cultured species including Clostridium clariflavum that have cellulolytic activities. Plate culture isolation further revealed that Clostridium clariflavum was one of the major cellulolytic species existing in the mixed culture consortia. Data at feed concentrations � 75 g/L, approaching industrial conditions, will also be presented.