A Thermophilic Ionic Liquid-Tolerant Cellulase Cocktail for the Production of Cellulosic Biofuels

Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels

Perspectives on the use of transcriptomics to advance biofuels

As a field within the energy research sector, bioenergy is continuously expanding. Although much has been achieved and the yields of both ethanol and butanol have been improved, many avenues of research to further increase these yields still remain. This review covers current research related with transcriptomics and the application of this high-throughput analytical tool to engineer both microbes and plants with the penultimate goal being better biofuel production and yields. The initial focus is given to the responses of fermentative microbes during the fermentative production of acids, such as butyric acid, and solvents, including ethanol and butanol. As plants offer the greatest natural renewable source of fermentable sugars within the form of lignocellulose, the second focus area is the transcriptional responses of microbes when exposed to plant hydrolysates and lignin-related compounds. This is of particular importance as the acid/base hydrolysis methods commonly employed to make the plant-based cellulose available for enzymatic hydrolysis to sugars also generates significant amounts of lignin-derivatives that are inhibitory to fermentative bacteria and microbes. The article then transitions to transcriptional analyses of lignin-degrading organisms, such as Phanerochaete chrysosporium, as an alternative to acid/base hydrolysis. The final portion of this article will discuss recent transcriptome analyses of plants and, in particular, the genes involved in lignin production. The rationale behind these studies is to eventually reduce the lignin content present within these plants and, consequently, the amount of inhibitors generated during the acid/base hydrolysis of the lignocelluloses. All four of these topics represent key areas where transcriptomic research is currently being conducted to identify microbial genes and their responses to products and inhibitors as well as those related with lignin degradation/formation.clos

Identification of bacterial cultures from archaeological wood using molecular biological techniques

Anaerobic bacteria were isolated from a 1700-year-old wooden spear shaft, excavated from an archaeological site that dates from the iron age, in the southern part of Jutland, Denmark. The bacteria were cultivated in glucose- and xylose-supplemented media at 14°C and 20°C. A gene library with 21 clones was constructed by extracting and amplifying 16S rDNA sequences from the individual cultures. One clone was phylogenetically affiliated to the Spirochaeta. Eleven clones affiliated to an unidentified member of the α-Proteobacteria were present in all culture samples. Three clones were affiliated to the β-Proteobacteria. Four clones were clustered among the Geobacteriaceae, in the δ-Proteobacteria. A single clone was clustered with gram-positives. All the identified bacterial families are commonly found in soil or bog environments and many are able to utilize cellulose as their carbon or energy source. © 2003 Elsevier Ltd. All rights reserved

Identification of bacterial cultures from archaeological wood using molecular biological techniques

Anaerobic bacteria were isolated from a 1700-year-old wooden spear shaft, excavated from an archaeological site that dates from the iron age, in the southern part of Jutland, Denmark. The bacteria were cultivated in glucose- and xylose-supplemented media at 14°C and 20°C. A gene library with 21 clones was constructed by extracting and amplifying 16S rDNA sequences from the individual cultures. One clone was phylogenetically affiliated to the Spirochaeta. Eleven clones affiliated to an unidentified member of the α-Proteobacteria were present in all culture samples. Three clones were affiliated to the β-Proteobacteria. Four clones were clustered among the Geobacteriaceae, in the δ-Proteobacteria. A single clone was clustered with gram-positives. All the identified bacterial families are commonly found in soil or bog environments and many are able to utilize cellulose as their carbon or energy source. © 2003 Elsevier Ltd. All rights reserved