Community dynamics of cellulose-adapted thermophilic bacterial consortia.

Enzymatic hydrolysis of cellulose is a key process in the global carbon cycle and the industrial conversion of biomass to biofuels. In natural environments, cellulose hydrolysis is predominately performed by microbial communities. However, detailed understanding of bacterial cellulose hydrolysis is primarily confined to a few model isolates. Developing models for cellulose hydrolysis by mixed microbial consortia will complement these isolate studies and may reveal new mechanisms for cellulose deconstruction. Microbial communities were adapted to microcrystalline cellulose under aerobic, thermophilic conditions using green waste compost as the inoculum to study cellulose hydrolysis in a microbial consortium. This adaptation selected for three dominant taxa--the Firmicutes, Bacteroidetes and Thermus. A high-resolution profile of community development during the enrichment demonstrated a community transition from Firmicutes to a novel Bacteroidetes population that clusters in the Chitinophagaceae family. A representative strain of this population, strain NYFB, was successfully isolated, and sequencing of a nearly full-length 16S rRNA gene demonstrated that it was only 86% identical compared with other validated strains in the phylum Bacteroidetes. Strain NYFB grew well on soluble polysaccharide substrates, but grew poorly on insoluble polysaccharide substrates. Similar communities were observed in companion thermophilic enrichments on insoluble wheat arabinoxylan, a hemicellulosic substrate, suggesting a common model for deconstruction of plant polysaccharides. Combining observations of community dynamics and the physiology of strain NYFB, a cooperative successional model for polysaccharide hydrolysis by the Firmicutes and Bacteroidetes in the thermophilic cellulolytic consortia is proposed.

Three Novel Rice Genes Closely Related to the Arabidopsis IRX9, IRX9L, and IRX14 Genes and Their Roles in Xylan Biosynthesis.

Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx) mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9), Os01g48440 (OsIRX9L), and Os06g47340 (OsIRX14), from glycosyltransferase family 43 as putative orthologs to the putative ß-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the over-expression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx) mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase (XylT) activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in XylT activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength.

Impact of high biomass loading on ionic liquid pretreatment.

BACKGROUND: Ionic liquid (IL) pretreatment has shown great potential as a novel pretreatment technology with high sugar yields. To improve process economics of pretreatment, higher biomass loading is desirable. The goal of this work is to establish, the impact of high biomass loading of switchgrass on IL pretreatment in terms of viscosity, cellulose crystallinity, chemical composition, saccharification kinetics, and sugar yield. RESULTS: The pretreated switchgrass/IL slurries show frequency dependent shear thinning behavior. The switchgrass/IL slurries show a crossover from viscous behavior at 3 wt% to elastic behavior at 10 wt%. The relative glucan content of the recovered solid samples is observed to decrease with increasing levels of lignin and hemicelluloses with increased biomass loading. The IL pretreatment led to a transformation of cellulose crystalline structure from I to II for 3, 10, 20 and 30 wt% samples, while a mostly amorphous structure was found for 40 and 50 wt% samples. CONCLUSIONS: IL pretreatment effectively reduced the biomass recalcitrance at loadings as high as 50 wt%. Increased shear viscosity and a transition from 'fluid' like to 'solid' like behavior was observed with increased biomass loading. At high biomass loadings shear stress produced shear thinning behavior and a reduction in viscosity by two orders of magnitude, thereby reducing the complex viscosity to values similar to lower loadings. The rheological properties and sugar yields indicate that 10 to 50 wt% may be a reasonable and desirable target for IL pretreatment under certain operating conditions.

Survey of renewable chemicals produced from lignocellulosic biomass during ionic liquid pretreatment.

BACKGROUND: Lignin is often overlooked in the valorization of lignocellulosic biomass, but lignin-based materials and chemicals represent potential value-added products for biorefineries that could significantly improve the economics of a biorefinery. Fluctuating crude oil prices and changing fuel specifications are some of the driving factors to develop new technologies that could be used to convert polymeric lignin into low molecular weight lignin and or monomeric aromatic feedstocks to assist in the displacement of the current products associated with the conversion of a whole barrel of oil. We present an approach to produce these chemicals based on the selective breakdown of lignin during ionic liquid pretreatment. RESULTS: The lignin breakdown products generated are found to be dependent on the starting biomass, and significant levels were generated on dissolution at 160°C for 6 hrs. Guaiacol was produced on dissolution of biomass and technical lignins. Vanillin was produced on dissolution of kraft lignin and eucalytpus. Syringol and allyl guaiacol were the major products observed on dissolution of switchgrass and pine, respectively, whereas syringol and allyl syringol were obtained by dissolution of eucalyptus. Furthermore, it was observed that different lignin-derived products could be generated by tuning the process conditions. CONCLUSIONS: We have developed an ionic liquid based process that depolymerizes lignin and converts the low molecular weight lignin fractions into a variety of renewable chemicals from biomass. The generated chemicals (phenols, guaiacols, syringols, eugenol, catechols), their oxidized products (vanillin, vanillic acid, syringaldehyde) and their easily derivatized hydrocarbons (benzene, toluene, xylene, styrene, biphenyls and cyclohexane) already have relatively high market value as commodity and specialty chemicals, green building materials, nylons, and resins.

Comparison of the impact of ionic liquid pretreatment on recalcitrance of agave bagasse and switchgrass.

Lignocellulose represents a sustainable source of carbon for transformation into biofuels. Effective biomass to sugar conversion strategies are needed to lower processing cost without degradation of polysaccharides. Since ionic liquids (ILs) are excellent solvents for pretreatment/dissolution of biomass, IL pretreatment was carried out on agave bagasse (AGB-byproduct of tequila industry) and digestibility and sugar yield was compared with that obtained with switchgrass (SWG). The IL pretreatment was conducted using ([C2mim][OAc]) at 120 and 160 °C for 3h and 15% biomass loading. While pretreatment using [C2mim][OAc] was very effective in improving the digestibility of both feedstocks, IL pretreatment at 160 °C resulted in higher delignification for AGB (45.5%) than for SWG (38.4%) when compared to 120 °C (AGB-16.6%, SWG-8.2%), formation of a highly amorphous cellulose structure and a significant enhancement of enzyme kinetics. These results highlight the potential of AGB as a biofuel feedstock that can produce high sugar yields with IL pretreatment.

Engineering of plants with improved properties as biofuels feedstocks by vessel-specific complementation of xylan biosynthesis mutants.

BACKGROUND: Cost-efficient generation of second-generation biofuels requires plant biomass that can easily be degraded into sugars and further fermented into fuels. However, lignocellulosic biomass is inherently recalcitrant toward deconstruction technologies due to the abundant lignin and cross-linked hemicelluloses. Furthermore, lignocellulosic biomass has a high content of pentoses, which are more difficult to ferment into fuels than hexoses. Engineered plants with decreased amounts of xylan in their secondary walls have the potential to render plant biomass a more desirable feedstock for biofuel production. RESULTS: Xylan is the major non-cellulosic polysaccharide in secondary cell walls, and the xylan deficient irregular xylem (irx) mutants irx7, irx8 and irx9 exhibit severe dwarf growth phenotypes. The main reason for the growth phenotype appears to be xylem vessel collapse and the resulting impaired transport of water and nutrients. We developed a xylan-engineering approach to reintroduce xylan biosynthesis specifically into the xylem vessels in the Arabidopsis irx7, irx8 and irx9 mutant backgrounds by driving the expression of the respective glycosyltransferases with the vessel-specific promoters of the VND6 and VND7 transcription factor genes. The growth phenotype, stem breaking strength, and irx morphology was recovered to varying degrees. Some of the plants even exhibited increased stem strength compared to the wild type. We obtained Arabidopsis plants with up to 23% reduction in xylose levels and 18% reduction in lignin content compared to wild-type plants, while exhibiting wild-type growth patterns and morphology, as well as normal xylem vessels. These plants showed a 42% increase in saccharification yield after hot water pretreatment. The VND7 promoter yielded a more complete complementation of the irx phenotype than the VND6 promoter. CONCLUSIONS: Spatial and temporal deposition of xylan in the secondary cell wall of Arabidopsis can be manipulated by using the promoter regions of vessel-specific genes to express xylan biosynthetic genes. The expression of xylan specifically in the xylem vessels is sufficient to complement the irx phenotype of xylan deficient mutants, while maintaining low overall amounts of xylan and lignin in the cell wall. This engineering approach has the potential to yield bioenergy crop plants that are more easily deconstructed and fermented into biofuels.

Understanding changes in lignin of Panicum virgatum and Eucalyptus globulus as a function of ionic liquid pretreatment.

Ionic liquids (ILs) have shown great potential for the reduction of lignin in biomass after pretreatment. Although dilute acid and base pretreatments have been shown to result in pretreated biomass with substantially different lignin composition, there is scarce information on the composition of lignin of IL pretreated biomass. In this work, temperature dependent compositional changes in lignin after IL pretreatment were studied to develop a mechanistic understanding of the process. Panicum virgatum and Eucalyptus globulus were pretreated with 1-ethyl-3-methylimidazolium acetate ([C(2)mim][OAc]). Measurement of syringyl and guaiacyl ratio using pyrolysis-GC/MS and Kamlet-Taft properties of [C(2)mim][OAc] at 120 °C and 160 °C strongly suggest two different modes of IL pretreatment. Preferential breakdown of S-lignin in both eucalyptus and switchgrass at high pretreatment temperature (160 °C) and breakdown of G-lignin for eucalyptus and no preferential break down of either S- or G-lignin in switchgrass was observed at lower pretreatment temperatures (120 °C).

Impact of ionic liquid pretreatment conditions on cellulose crystalline structure using 1-ethyl-3-methylimidazolium acetate.

Ionic liquids (ILs) have been shown to affect cellulose crystalline structure in lignocellulosic biomass during pretreatment. A systematic investigation of the swelling and dissolution processes associated with IL pretreatment is needed to better understand cellulose structural transformation. In this work, 3-20 wt % microcrystalline cellulose (Avicel) solutions were treated with 1-ethyl-3-methylimidazolium acetate ([C(2)mim][OAc]) and a mixture of [C(2)mim][OAc] with the nonsolvent dimethyl sulfoxide (DMSO) at different temperatures. The dissolution process was slowed by decreasing the temperature and increasing cellulose loading, and was further retarded by addition of DMSO, enabling in-depth examination of the intermediate stages of dissolution. Results show that the cellulose I lattice expands and distorts prior to full dissolution in [C(2)mim][OAc] and that upon precipitation the former structure leads to a less ordered intermediate structure, whereas fully dissolved cellulose leads to a mixture of cellulose II and amorphous cellulose. Enzymatic hydrolysis was more rapid for the intermediate structure (crystallinity = 0.34) than for cellulose II (crystallinity = 0.54).

Effect of ionic liquid treatment on the structures of lignins in solutions: molecular subunits released from lignin.

The solution structures of three types of isolated lignin--organosolv (OS), Kraft (K), and low sulfonate (LS)--before and after treatment with 1-ethyl-3-methylimidazolium acetate were studied using small-angle neutron scattering (SANS) and dynamic light scattering (DLS) over a concentration range of 0.3-2.4 wt %. The results indicate that each of these lignins is comprised of aggregates of well-defined basal subunits, the shapes and sizes of which, in D(2)O and DMSO-d(6), are revealed using these techniques. LS lignin contains a substantial amount of nanometer-scale individual subunits. In aqueous solution these subunits have a well-defined elongated shape described well by ellipsoidal and cylindrical models. At low concentration the subunits are highly expanded in alkaline solution, and the effect is screened with increasing concentration. OS lignin dissolved in DMSO was found to consist of a narrow distribution of aggregates with average radius 200 ± 30 nm. K lignin in DMSO consists of aggregates with a very broad size distribution. After ionic liquid (IL) treatment, LS lignin subunits in alkaline solution maintained the elongated shape but were reduced in size. IL treatment of OS and K lignins led to the release of nanometer-scale subunits with well-defined size and shape.

Loss of Cellulose synthase-like F6 function affects mixed-linkage glucan deposition, cell wall mechanical properties, and defense responses in vegetative tissues of rice.

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked ß-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles and leaves retain trace amounts of MLG only in specific cell types such as sclerenchyma fibers. These results correlate with the absence of endogenous MLG synthase activity in mutant seedlings and 4-week-old sheaths. Mutant cell walls are weaker in mature stems but not seedlings, and more brittle in both stems and seedlings, compared to wild type. Mutants also display lesion mimic phenotypes in leaves, which correlates with enhanced defense-related gene expression and enhanced disease resistance. Taken together, our results underline a weaker role of MLG in cell expansion than previously thought, and highlight a structural role for MLG in nonexpanding, mature stem tissues in rice.

Rapid determination of syringyl: guaiacyl ratios using FT-Raman spectroscopy.

Lignin composition in relation to its basic phenylpropanoid units, particularly the syringyl to guaiacyl (S/G) ratio, is an important property for biomass characterization and varies greatly as a function of species, genotype and environment. A rapid screening method is highly desirable to assess lignin composition in a large number of samples. We have developed a nondestructive and label-free Fourier transform Raman (FT-Raman) spectroscopic method that is capable of rapidly and reliably measuring the S/G ratio with minimal sample preparation. A variety of feedstocks, including hardwood (Eucalyptus globulus), softwood (Pinus radiata), herbaceous plants (Zea mays, Panicum virgatum, and Sorghum bicolor), and a model dicot (Arabidopsis thaliana) were measured using this technique and the corresponding S/G ratio was calculated after spectral deconvolution based on the S and G bands identified using a known library of model compounds. The results obtained using this technique were successfully validated by pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS). This technique holds significant promise in the rapid screening of engineered feedstocks as part of a comprehensive screening methodology that is correlated with biomass recalcitrance.

Transition of cellulose crystalline structure and surface morphology of biomass as a function of ionic liquid pretreatment and its relation to enzymatic hydrolysis.

Cellulose is inherently resistant to breakdown, and the native crystalline structure (cellulose I) of cellulose is considered to be one of the major factors limiting its potential in terms of cost-competitive lignocellulosic biofuel production. Here we report the impact of ionic liquid pretreatment on the cellulose crystalline structure in different feedstocks, including microcrystalline cellulose (Avicel), switchgrass (Panicum virgatum), pine ( Pinus radiata ), and eucalyptus ( Eucalyptus globulus ), and its influence on cellulose hydrolysis kinetics of the resultant biomass. These feedstocks were pretreated using 1-ethyl-3-methyl imidazolium acetate ([C2mim][OAc]) at 120 and 160 °C for 1, 3, 6, and 12 h. The influence of the pretreatment conditions on the cellulose crystalline structure was analyzed by X-ray diffraction (XRD). On a larger length scale, the impact of ionic liquid pretreatment on the surface roughness of the biomass was determined by small-angle neutron scattering (SANS). Pretreatment resulted in a loss of native cellulose crystalline structure. However, the transformation processes were distinctly different for Avicel and for the biomass samples. For Avicel, a transformation to cellulose II occurred for all processing conditions. For the biomass samples, the data suggest that pretreatment for most conditions resulted in an expanded cellulose I lattice. For switchgrass, first evidence of cellulose II only occurred after 12 h of pretreatment at 120 °C. For eucalyptus, first evidence of cellulose II required more intense pretreatment (3 h at 160 °C). For pine, no clear evidence of cellulose II content was detected for the most intense pretreatment conditions of this study (12 h at 160 °C). Interestingly, the rate of enzymatic hydrolysis of Avicel was slightly lower for pretreatment at 160 °C compared with pretreatment at 120 °C. For the biomass samples, the hydrolysis rate was much greater for pretreatment at 160 °C compared with pretreatment at 120 °C. The result for Avicel can be explained by more complete conversion to cellulose II upon precipitation after pretreatment at 160 °C. By comparison, the result for the biomass samples suggests that another factor, likely lignin-carbohydrate complexes, also impacts the rate of cellulose hydrolysis in addition to cellulose crystallinity.

Remediation of PCB contaminated soils using iron nano-particles.

In this study, iron nano-particles were used to remediate PCB contaminated soil and an attempt was made to maximize PCB destruction in each treatment step. The results show that nano-particles do aid in the dechlorination process and high PCB destruction efficiencies can be achieved. The destruction efficiency during the preliminary treatment (mixing of soil and iron nano-particles in water) can be increased by increasing the water temperature. The maximum thermal destruction (pyrolysis/combustion of soil after preliminary treatment) of soil-bound PCBs occurs at 300 degrees C in air. A minimum total PCB destruction efficiency of 95% can be achieved by this process. The effect of changing treatment parameters such as type of mixing, time of mixing and mixing conditions and application of other catalysts like iron oxide and V(2)O(5)/TiO(2) was also investigated. It was found that at 300 degrees C in air, iron oxide and V(2)O(5)/TiO(2) are also good catalysts for remediating PCB contaminated soils.