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

Enzymatic saccharification of IL-pretreated switchgrass by JTherm at 70°C pH 5.5.

<p>The supernatant from the thermophilic community at fixed concentration of 0.6× was augmented with various amounts of CBH and BG, and liberated glucose (♦) and cellobiose (▪) from IL-pretreated switchgrass were measured after 72 h incubation. Enzyme combinations were as follows: (A) supernatant, CBH, and BG; (B) CBH and BG without supernatant. The reaction was in a 1 ml volume with 25 mg of IL-pretreated switchgrass.</p

Cellulase and xylanase from the thermophilic community identified by proteomics.

<p>Predicted function of the proteins identified by proteomics is based on comparisons of the genes in the metagenome to the pfam, Clusters of Orthologous Groups (COGs), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The IMG gene oid is the gene identifier for The Joint Genome Institute's Integrated Microbial Genomes database <a href="http://img.jgi.doe.gov/" target="_blank">http://img.jgi.doe.gov/</a>. The pfam assignment of the metagenome gene is indicated.</p

Growth of an <i>E. coli</i> strain engineered to produce biodiesel on CTec2 hydrolysate

<p>(A) and control sample containing 2% glucose and 1% xylose (B). Oxygen transfer rate (OTR), and cell density (OD₆₀₀) were monitored during the fermentation to determine the impacts of the hydrolysate on growth and respiration.</p

Glycoside hydrolase activities produced by the thermophilic community.

<p>Endoglucanase and endoxylanase activities were determined using the DNS assay on carboxymethyl cellulose or birtchwood xylan. Other activities were assessed using <i>p</i>-nitrophenyl substrates. U = µmol/min and is reported as the mean and standard deviation of triplicate experiments.</p

A flow diagram of two potential biomass-to-biofuel bioprocessing configurations that utilize IL-pretreatment.

<p>A) Diagrams a configuration based on methods currently established in the literature and lists some potential barriers to commercialization (Problems). B) This configuration combines IL-pretreatment and saccharification into a single pot and may overcome barriers outlined in A (as listed in the solutions section), but requires an IL-tolerant cellulase cocktail, such as JTherm.</p

Effects of ionic liquids on biodiesel production by an engineered <i>E. coli</i> strain.

<p>The strain was fed either 2% glucose or a CTec2 hydrolysate of IL- pretreated switchgrass containing 0–1% (w/v) [C2mim][OAc] [(A). The percentage of glucose remaining after fermentation was measured (B). Glucose levels were adjusted to 2% for all hydrolysates and controls. The CTec2 control contained equivalent amounts of purified glucose and xylose as the hydrolysate. No xylose was consumed during the fermentation (data not shown). Error bars indicate the standard deviation of triplicate experiments.</p

A pie chart showing the percent relative abundance of each taxon in the McCel-adapted thermophilic bacterial consortia.

<p>SSU pyrosequencing was conducted to identify community members. Only members with a relative abundance greater than 1% are reported. Relative abundance is calculated as a percentage of the total number of SSU reads for the community. The closest taxon to each organism in the community is reported in the legend. The percent identity between the consortial and closest taxon SSU sequence is in parentheses.</p

Activity of the JTherm and CTec2 cellulase cocktails on ionic-liquid pretreated switchgrass at various temperatures and in the presence of the ionic liquid [C2mim][OAc].

<p>Samples were run at 2.5% w/v biomass loadings in 1 ml and incubated at pH 5.5 for 72 h with shaking.</p