The combination of a feedstock with increased enzymatic digestibility in combination with the CBP approach, which will eliminate the need for exogenous hydrolytic enzymes, has the potential to further reduce the cost of biofuels. Therefore we examined the fermentation performance of both wild-type and transgenic switchgrass lines using Clostridium thermocellum
, Caldicellulosiruptor obsidiansis
, and Caldicellulosiruptor bescii.
Using three lines of switchgrass down-regulated in the COMT gene
], we have shown that a milder pretreatment process does not impact the improved product yield generated by fermentation of the COMT down-regulated switchgrass biomass during yeast-based SSF. However, when a CBP-capable bacterium is tested, a significant differential of fermentation inhibition is detected, as judged by product yield on carbohydrate. In the case of C. thermocellum
fermentations of dilute acid pretreated feedstock, the cellulosome and/or free carbohydrolases appear functional, as indicated by high levels of liberated unfermented glucose and cellobiose in the fermentation broth. At the same time, COMT transgenic feedstock lines clearly generate greater inhibition compared to the wild-type switchgrass, in the case of C. thermocellum
fermentation. The inhibition of fermentation was shown to be removed after hot water extraction was applied to the dilute acid pretreated feedstock lines, suggesting that the inhibition is caused by water-soluble constituents.
The picture is quite different for the Caldicellulosiruptor sp. strains tested. Fermentation of dilute acid pretreated and hot water extracted biomass that was readily fermented by C. thermocellum caused significant reduction in fermentation yield for T1-2-TG and T1-3-TG substrates with both Caldicellulosiruptor sp. strains. In addition, there were only low levels of unconsumed sugar remaining in the broth at the end of fermentation, indicating that both fermentation and hydrolysis were negatively impacted for the two highly down-regulated COMT feedstock lines. Moreover, the apparent differential of fermentation inhibition between the three CBP microorganisms, measured by unconsumed carbohydrates or low product yields, was readily detected when a less severe hot water pretreatment was used to prepare the feedstock lines.
The apparent differential of inhibition between bacterial fermentations was particularly interesting because it was not seen in yeast-based SSF, and was an unexpected result. We hypothesize that the reduction in fermentation yield could be a biomass, microbe, or a biomass-microbe combined effect. A result that supports the hypothesis of a biomass effect contributing to the apparent inhibition is the significant reduction in yield of the Caldicellulosiruptor sp. strains’ fermentation of dilute acid pretreated, highly down-regulated COMT T1-2 and T1-3 lines, which is not present in the moderately down-regulated T1-12 transgenic line or the wild-type lines. Another possible reason for the apparent differential of inhibition is the various modes of interaction and hydrolysis employed by the hydrolytic system used by the microorganisms. As a result, they may release different or varying concentrations of inhibitory aromatic constituents, including mono-phenolic acids and sugar-aromatic conjugates. It is also not unreasonable to expect that the three microorganisms have different levels of tolerance for various inhibitory compounds.
We analyzed the fermentation broth and appropriate controls with GC-MS based metabolite profiling in an attempt to determine if mono-phenolic acids or other aromatic constituents were causing the observed inhibition. We showed temperature, media components, and fungal enzymes alone did not generate aromatic constituents or mono-phenolics, which are components of plant cell walls and known to inhibit bacterial fermentation
]. The aromatic constituents, including mono-phenolic acids found in the fermentation broth for hot water versus dilute acid pretreatment are different. The variation in lignin derived constituents may be explained by the difference in pretreatment severity affecting the lignin structure and content
In the case of hot water pretreatment, there was a mild biomass effect. Of specific interest was the increased relative abundance of aromatic constituents in the Caldicellulosiruptor sp. strains in comparison to C. thermocellum. This indicates that C. thermocellum’s hydrolytic system (cellulosome and free enzymes) might be producing a cleaner (less aromatic constituents) carbohydrate hydrolysate from the hot water pretreated switchgrass feedstocks than the Caldicellulosiruptor sp. strains. In contrast to the hot water pretreated feedstock results, dilute acid pretreated feedstocks did not show a notable difference in aromatic or mono-phenolic acid content between the different types of biomass or microorganisms. However, results showed that a tentatively identified compound, coumaroyl-benzaldehyde, was present in statistically different levels for both the biomass and microbe effect. The minimal biomass effect for either pretreatment was surprising, because our original hypothesis was based on the premise that the modification of the lignin pathway altered the lignin composition and content of the transgenic feedstock lines, and therefore, the concentration or composition of lignans generated and or released during pretreatment and bacterial hydrolysis and fermentation would appear quite different in comparison to the wild-type feedstock.
The differential of bacterial fermentation inhibition may, in part, be explained by the aromatic constituents in the fermentation broth. Additionally, it may also be explained by the microorganisms having varying degrees of tolerance to these compounds. In general, the reduction in recalcitrance drastically improved the susceptibility to bioconversion for yeast-based SSF and, after the inhibition was removed; high levels of fermentation products were produced by C. thermocellum. As a result, biomass sources with reduced recalcitrance resulting from lignin pathway modification are a valuable resource for producing economical biofuels, but the impact of the lignin modification on the three bacteria’s fermentation performance needs to be further studied to determine the cause of reduction in fermentation yield.