Jonsson et al. (1998) studied the detoxification effect of laccase, phenol oxidase, and lignin peroxidase on hydrolysates. S. cerevisiae was used for subsequent ethanol fermentation. The results showed more rapid consumption of glucose and a higher ethanol productivity for samples treated with laccase than for untreated samples. Treatment of hydrolysates with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involved removal of monoaromatic phenolic compounds present in the hydrolysate (Jonsson et al. 1998).

Chandel et al. (2007) conducted laccase detoxification tests using sugarcane bagasse hydro­lysates produced by 2.5% (v/v) HCl, which contained 30.39 g/L of total reducing sugars along with various fermentation inhibitors such as furans, phenolics, and acetic acid. The laccase reduced total phenolics by 77.5% without affecting furans and acetic acid content in the hydrolysate. In comparison, the anion-exchange resin brought about a maximum reduction of 63.4% in furans and 75.8% in total phenolics, while the treatment with activated charcoal caused 38.7% and 57.5% reduction in furans and total phenolics, respectively. Fermentation of these hydrolysates with C. shehatae NCIM 3501 showed maximum ethanol yield (0.48 g/g) from ion exchange-treated hydrolysates, followed by activated charcoal (0.42 g/g), laccase (0.37g/g), overliming (0.30g/g), and neutralized hydrolysates (0.22g/g).

Adaptation of Microorganisms

Microorganisms have the ability to adapt to perturbations of the surrounding environment to grow (Dinh et al. 2008). Utilizing the microorganism of a previous experiment as the inocu­lum of the next one, the adaptation of a microorganism to the hydrolysate is another biological method for improving the fermentation of lignocellulosic hydrolysate media (Mussatto and Roberto 2004). To analyze the adaptation process of S. cerevisiae to a high ethanol concen­tration, Dinh et al. (2008) performed repetitive cultivations with a stepwise increase in the ethanol concentration in the culture medium. They found that the mother cells of the adapted yeast were significantly larger than those of the non-adapted strains and that the content of palmitic acid in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol.

Martm et al. (2007) adapted a xylose-utilizing genetically engineered strain of S. cerevisiae with sugarcane bagasse hydrolysates by 353-hour cultivation using a medium with increasing concentrations of phenols (from 1.5 to 2.3 g/L), furfural (from 0.7 to 3.4 g/L), and aliphatic acids (from 2.5 to 8.7 g/L). The performance of the adapted strain was compared with the parental strain: the ethanol yield after 24 h of fermentation of the bagasse hydrolysate with inhibitors (phenols: 1.4 g/L, furfural: 2.2 g/L, apliphatic acids: 5.0 g/L) increased from 0.18 g/g of total sugar with the non-adapted strain to 0.38 g/g with the adapted strain. The specific ethanol productivity increased from 1.15 g ethanol per gram initial biomass per hour with the non-adapted strain to 2.55g/g/h with the adapted strain.

Agbogbo et al. (2008) investigated the effect of adaptation of P. stipitis in acid-pretreated CSH without detoxification for ethanol fermentation. Fermentation results showed that the solid agar adaptation improved both the sugar consumption rate and the rate of ethanol pro­duction. Liquid and solid agar adaptation increased the sugar consumption from 64 to 72% after 96 hours of fermentation at 100 rpm. The ethanol concentration (g/L) was increased from unadapted 16.3 ± 0.51 to 18.4 ± 0.20 (liquid adapted) and 19.4 ± 0.12 (solid adapted). The solid agar-adapted stains started using xylose after 96 hours of fermentation while wild strains did not consume xylose. However, when rotation speed in the flask was increased to 150 rpm, 92% of the total sugar was consumed within 72 hours of fermentation.

Conclusion

The presence of inhibitors in lignocellulosic hydrolysates directly influences biofuel fermen­tation. Due to a lack of understanding about the synergistic interactions among inhibitors and the mechanisms of these interactions, highly inhibitor-resistant microorganisms might not be expected in the short term. Problems associated with biomass hydrolysates, however, may be resolved by the development of inhibitor-tolerant strains using genetic modification and metabolic engineering. From an economic standpoint, the ultimate goal is to develop a decon­struction process without detoxification. The main features of a number of detoxification methods are summarized in this chapter. Some of them are relatively new, while others have existed for decades but need some improvements for optimal performance. Among the methods, the biological detoxification methods are promising. With the isolation and develop­ment of some inhibitors degrading microorganisms and mutants, there exist some prospects of SSF of biomass to biofuel or combining a biological detoxification step with the SSF process.

Acknowledgem ent

This work was financially supported by Energy Biosciences Institute.