Next-generation biomass feedstocks for biofuel production

A review of the potential for biofuel production in the United States from timber and non-grain crops

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency.

Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate - an intermediate of the shikimate pathway - into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass

Potential for genetic improvement of sugarcane as a source of biomass for biofuels

Sugarcane (Saccharum spp. hybrids) has great potential as a major feedstock for biofuel production worldwide. It is considered among the best options for producing biofuels today due to an exceptional biomass production capacity, high carbohydrate (sugar + fiber) content, and a favorable energy input/output ratio. To maximize the conversion of sugarcane biomass into biofuels, it is imperative to generate improved sugarcane varieties with better biomass degradability. However, unlike many diploid plants, where genetic tools are well developed, biotechnological improvement is hindered in sugarcane by our current limited understanding of the large and complex genome. Therefore, understanding the genetics of the key biofuel traits in sugarcane and optimization of sugarcane biomass composition will advance efficient conversion of sugarcane biomass into fermentable sugars for biofuel production. The large existing phenotypic variation in Saccharum germplasm and the availability of the current genomics technologies will allow biofuel traits to be characterized, the genetic basis of critical differences in biomass composition to be determined, and targets for improvement of sugarcane for biofuels to be established. Emerging options for genetic improvement of sugarcane for the use as a bioenergy crop are reviewed. This will better define the targets for potential genetic manipulation of sugarcane biomass composition for biofuels

How Alkyl Chain Length of Alcohols Affects Lignin Fractionation and Ionic Liquid Recycle During Lignocellulose Pretreatment

Abstract Alcohols of increasing alkyl chain length were investigated as precipitants in an ionic liquid (IL) pretreatment system. Switchgrass samples pretreated by 1-ethyl-3-methylimidazolium acetate were characterized after the use of different alkyl chain lengths of alcohols as antisolvents. The resulting IL-pretreated switchgrass (PSG) samples were characterized by enzymatic hydrolysis, cross polarization/magic angle spinning (CP/MAS) 13C nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and 2D NMR spectroscopy. Glucan digestibilities of PSG samples were ∼80 % after 72 h at 5 mg protein g−1 glucan regardless of the antisolvent used. The use of 1-octanol as an antisolvent, with 10 % water to allow for use of wet biomass, enabled a partial lignin fractionation and multiphase separation for the IL recycle without compromising the chemical structure of the carbohydrates and lignin from the PSG. Lignin fragments were observed in the IL after pretreatment by gel permeation chromatography (GPC). After separation, both the IL and the octanol antisolvent were reused for switchgrass pretreatment and precipitation for an additional 3 cycles. The PSG samples derived from recycled IL were rapidly hydrolyzed, and a high glucan digestibility of 80 % was obtained even at a low enzyme loading of 5 mg protein g−1 glucan. 2D NMR analysis of residual solids of PSG post-enzymatic hydrolysis revealed that lignin in these residual solids was depolymerized. This strategy enables an ease in separation of pretreated lignocellulosic solids, reduced water use, and recycle of both IL and the antisolvent.United States. Dept. of Energy. Office of Biological and Environmental Research (Joint BioEnergy Institute (JBEI) (Contract number DE-AC02- 05CH11231)

Efficient eucalypt cell wall deconstruction and conversion for sustainable lignocellulosic biofuels

In order to meet the world’s growing energy demand and reduce the impact of greenhouse gas emissions resulting from fossil fuel combustion, renewable plant-based feedstocks for biofuel production must be considered. The first-generation biofuels, derived from starches of edible feedstocks, such as corn, create competition between food and fuel resources, both for the crop itself and the land on which it is grown. As such, biofuel synthesized from non-edible plant biomass (lignocellulose) generated on marginal agricultural land will help to alleviate this competition. Eucalypts, the broadly defined taxa encompassing over 900 species of Eucalyptus, Corymbia, and Angophora are the most widely planted hardwood tree in the world, harvested mainly for timber, pulp and paper, and biomaterial products. More recently, due to their exceptional growth rate and amenability to grow under a wide range of environmental conditions, eucalypts are a leading option for the development of a sustainable lignocellulosic biofuels. However, efficient conversion of woody biomass into fermentable monomeric sugars is largely dependent on pretreatment of the cell wall, whose formation and complexity lend itself toward natural recalcitrance against its efficient deconstruction. A greater understanding of this complexity within the context of various pretreatments will allow the design of new and effective deconstruction processes for bioenergy production. In this review, we present the various pretreatment options for eucalypts, including research into understanding structure and formation of the eucalypt cell wall

Evidence supporting dissimilatory and assimilatory lignin degradation in Enterobacter lignolyticus SCF1

Lignocellulosic biofuels are promising as sustainable alternative fuels, but lignin inhibits access of enzymes to cellulose, and by-products of lignin degradation can be toxic to cells. The fast growth, high efficiency and specificity of enzymes employed in the anaerobic litter deconstruction carried out by tropical soil bacteria make these organisms useful templates for improving biofuel production. The facultative anaerobe Enterobacter lignolyticus SCF1 was initially cultivated from Cloud Forest soils in the Luquillo Experimental Forest in Puerto Rico, based on anaerobic growth on lignin as sole carbon source. The source of the isolate was tropical forest soils that decompose litter rapidly with low and fluctuating redox potentials, where bacteria using oxygen-independent enzymes likely play an important role in decomposition. We have used transcriptomics and proteomics to examine the observed increased growth of SCF1 grown on media amended with lignin compared to unamended growth. Proteomics suggested accelerated xylose uptake and metabolism under lignin-amended growth, with up-regulation of proteins involved in lignin degradation via the 4-hydroxyphenylacetate degradation pathway, catalase/peroxidase enzymes, and the glutathione biosynthesis and glutathione S-transferase (GST) proteins. We also observed increased production of NADH-quinone oxidoreductase, other electron transport chain proteins, and ATP synthase and ATP-binding cassette (ABC) transporters. This suggested the use of lignin as terminal electron acceptor. We detected significant lignin degradation over time by absorbance, and also used metabolomics to demonstrate moderately significant decreased xylose concentrations as well as increased metabolic products acetate and formate in stationary phase in lignin-amended compared to unamended growth conditions. Our data show the advantages of a multi-omics approach toward providing insights as to how lignin may be used in nature by microorganisms coping with poor carbon availability

Attributions for Relatives' Behavior and Perceived Criticism: Studies With Community Participants and Patients With Anxiety Disorders

The relationship between perceived criticism from one’s relative and attributions about that relative’s behavior was examined in two studies. In Study 1, 50 community couples volunteered to participate in a study of marital interaction. Participants rated their interaction-specific perceived criticism after a 10-min problem-solving interaction and their attributions for their spouses’ behavior during a review of the videotaped interaction. In Study 2, 70 outpatients with obsessive-compulsive disorder (n = 41) or panic disorder with agoraphobia (n = 29) completed a measure of global perceived criticism in their relationship with their spouse or other family member and on another occasion participated in a 10-min problem-solving interaction with that relative. Using interaction transcripts, coders extracted and coded attributions from patients’ speech and, using the videotapes themselves, rated relatives’ observable criticism. In both studies higher scores on negative attributions were related to higher perceived criticism ratings. In Study 2, negative attributions contributed to the prediction of perceived criticism above and beyond the contribution of observed criticism. These findings suggest that targeting attributions about perceived criticism may be fruitful in reducing the negative impact of perceived criticism on treatment outcome for a variety of psychiatric disorders

Draft Genome Sequence of the Lignin-Degrading Burkholderia sp. Strain LIG30, Isolated from Wet Tropical Forest Soil

Burkholderia species are common soil Betaproteobacteria capable of degrading recalcitrant aromatic compounds and xenobiotics. Burkholderia sp. strain LIG30 was isolated from wet tropical forest soil and is capable of utilizing lignin as a sole carbon source. Here we report the draft genome sequence of Burkholderia sp. strain LIG30

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 themajor 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 formost 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 contentwas 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 lignincarbohydrate complexes, also impacts the rate of cellulose hydrolysis in addition to cellulose crystallinity