Genetic modification of lignin biosynthesis for improved biofuel production

The energy in cellulosic biomass largely resides in plant cell walls. Cellulosic biomass is more difficult than starch to break down into sugars because of the presence of lignin and the complex structure of cell walls. Transgenic down-regulation of major lignin genes led to reduced lignin content, increased dry matter degradability, and improved accessibility of cellulases for cellulose degradation. This review provides background information on lignin biosynthesis and focuses on genetic manipulation of lignin genes in important monocot species as well as the dicot potential biofuel crop alfalfa. Reduction of lignin in biofuel crops by genetic engineering is likely one of the most effective ways of reducing costs associated with pretreatment and hydrolysis of cellulosic feedstocks, although some potential fitness issues should also be addressed.     

Ethanol production from lignocellulosic biomass of energy cane

Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum based liquid fuels. The release of many new sugarcane varieties by the United States Department of Agriculture to be used as energy crops is a promising feedstock alternative. Energy cane produces large amounts of biomass that can be easily transported, and production does not compete with food supply and prices because energy cane can be grown on marginal land instead of land for food crops. The purpose of this study was to evaluate energy cane for lignocellulosic ethanol production. Energy cane variety L 79-1002 was pretreated with weak sulfuric acid to remove lignin. In this study, 1.4 M sulfuric acid pretreated type II energy cane had a higher ethanol yield after fermentation by Klebsiella oxytoca without enzymatic saccharification than 0.8 M and 1.6 M sulfuric acid pretreated type II energy cane. Pretreated biomass was inoculated with K. oxytoca for cellulose fermentation and Pichia stipitis for hemicellulose fermentation under simultaneous saccahrification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) conditions. For enzymatic saccharification of cellulose, the cellulase and ??-glucanase cocktail significantly increased ethanol production compared to the ethanol production of fermented acid pretreated energy cane without enzymatic saccharification. The results revealed that energy cane variety L 79-1002 produced maximum cellulosic ethanol under SHF (6995 mg/L) and produced 3624 mg/L ethanol from fermentation of hemicellulosic sugars.     

Genetic improvement of C4 grasses as cellulosic biofuel feedstocks

C4 grasses are among the most productive plants and most promising cellulosic biofuel feedstocks. Successful implementation of cellulosic biofuel feedstocks will depend on the improvement of critical crop characteristics and subsequent conversion technologies. The content and composition of lignin, cellulose, and hemicellulose, their biomass yields, and their biotic and abiotic stress tolerances are critical factors which can be enhanced by molecular breeding methods, including marker-assisted selection and transgenic approaches. To maximize biomass yield, no flowering or late flowering and no grain set would be ideal for cellulosic biofuel crops. Reducing fecundity also reduces the risk of undesired gene transfer and invasiveness, thus accelerating deregulation processes and permitting faster implementation of highly improved genotypes in cellulosic feedstock production.     

Natural lignin modulators improve bagasse saccharification of sugarcane and energy cane in field trials

The burgeoning cellulosic ethanol industry necessitates advancements in enzymatic saccharification, effective pretreatments for lignin removal, and the cultivation of crops more amenable to saccharification. Studies have demonstrated that natural inhibitors of lignin biosynthesis can enhance the saccharification of lignocellulose, even in tissues generated several months post-treatment. In this study, we applied daidzin (a competitive inhibitor of coniferaldehyde dehydrogenase), piperonylic acid (a quasi-irreversible inhibitor of cinnamate 4-hydroxylase), and methylenedioxy cinnamic acid (a competitive inhibitor of 4-coenzyme A ligase) to 60-day-old crops of two conventional Brazilian sugarcane cultivars and two energy cane clones, bred specifically for enhanced biomass production. The resultant biomasses were evaluated for lignin content and enzymatic saccharification efficiency without additional lignin-removal pretreatments. The treatments amplified the production of fermentable sugars in both the sugarcane cultivars and energy cane clones. The most successful results softened the most recalcitrant lignocellulose to the level of the least recalcitrant of the biomasses tested. Interestingly, the softest material became even more susceptible to saccharification.     

Improvement of biomass through lignin modification

Lignin, a major component of the cell wall of vascular plants, has long been recognized for its negative impact on forage quality, paper manufacturing, and, more recently, cellulosic biofuel production. Over the last two decades, genetic and biochemical analyses of brown midrib mutants of maize, sorghum and related grasses have advanced our understanding of the relationship between lignification and forage digestibility. This work has also inspired genetic engineering efforts aimed at generating crops with altered lignin, with the expectation that these strategies would enhance forage digestibility and/or pulping efficiency. The knowledge gained from these bioengineering efforts has greatly improved our understanding of the optimal lignin characteristics required for various applications of lignocellulosic materials while also contributing to our understanding of the lignin biosynthetic pathway. The recent upswing of interest in cellulosic biofuel production has become the new focus of lignin engineering. Populus trichocarpa and Brachypodium distachyon are emerging as model systems for energy crops. Lignin research on these systems, as well as on a variety of proposed energy crop species, is expected to shed new light on lignin biosynthesis and its regulation in energy crops, and lead to rational genetic engineering approaches to modify lignin for improved biofuel production.     

A comparison of canopy evapotranspiration for maize and two perennial grasses identified as potential bioenergy crops

In the Midwestern US, perennial rhizomatous grasses (PRGs) are considered one of the most promising vegetation types to be used as a cellulosic feedstock for renewable energy production. The potential widespread use of biomass crops for renewable energy production has sparked numerous environmental concerns, including the impacts of land‐use change on the hydrologic cycle. We predicted that total seasonal evapotranspiration (ET) would be higher for PRGs relative to maize resulting from higher leaf area and a prolonged growing season. We further predicted that, compared with maize, higher aboveground biomass associated with PRGs would offset the higher ET and increase water‐use efficiency (WUE) in the context of biomass harvests for liquid biofuel production. To test these predictions, ET was estimated during the 2007 growing season for replicated plots of Miscanthus×giganteus (miscanthus), Panicum virgatum (switchgrass), and Zea mays (maize) using a residual energy balance approach. The combination of a 25% higher mean latent heat flux (λET) and a longer growing season resulted in miscanthus having ca. 55% higher cumulative ET over the growing season compared with maize. Cumulative ET for switchgrass was also higher than maize despite similar seasonal‐mean λET. Based on total harvested aboveground biomass, WUE was ca. 50% higher for maize relative to miscanthus; however, when WUE calculated from only maize grain biomass was compared with WUE calculated from miscanthus harvested aboveground biomass, this difference disappeared. Although WUE between maize and miscanthus differed postsenescence, there were no differences in incremental WUE throughout the growing season. Despite initial predictions, aboveground biomass for switchgrass was less than maize; thus WUE was substantially lower for switchgrass than for either maize scenario. These results indicate that changes in ET due to large‐scale implementation of PRGs in the Midwestern US would likely influence local and regional hydrologic cycles differently than traditional row crops.     

Eastern gamagrass as an alternative cellulosic feedstock for bioethanol production

Eastern gamagrass (Trypsacum dactyloides) is a C₄ perennial grass, native to the USA with desirable characteristics that warrants further investigation as a new lignocellulosic crop for bioethanol production. Chemical composition assays showed that eastern gamagrass had comparable cellulose, hemicellulose and lignin compositions to those of switchgrass (Panicum virgatum). With the cellulose solvent-based lignocellulose fractionation (CSLF) pretreatment and subsequent enzymatic saccharification, 80.5–99.8% of cellulosic glucose was released from the gamagrass biomass, which was 10–17% greater than the glucose release efficiency from switchgrass (73.5–87.1%). Furthermore, the hydrolysate of gamagrass supported greater ethanol fermentation yield (up to 0.496 g/g glucose) than the hydrolysates of switchgrass. As such, in the whole process of biomass-to-ethanol conversion, gamagrass could yield 13–35% more ethanol per gram of biomass than switchgrass, indicating that gamagrass has high potential as an alternative energy feedstock for lignocellulosic ethanol production.     

Hemicelluloses negatively affect lignocellulose crystallinity for high biomass digestibility under NaOH and H2SO4 pretreatments in Miscanthus

ABSTRACT: BACKGROUND: Lignocellulose is the most abundant biomass on earth. However, biomass recalcitrance has become a major factor affecting biofuel production. Although cellulose crystallinity significantly influences biomass saccharification, little is known about the impact of three major wall polymers on cellulose crystallization. In this study, we selected six typical pairs of Miscanthus samples that presented different cell wall compositions, and then compared their cellulose crystallinity and biomass digestibility after various chemical pretreatments. RESULTS: A Miscanthus sample with a high hemicelluloses level was determined to have a relatively low cellulose crystallinity index (CrI) and enhanced biomass digestibility at similar rates after pretreatments of NaOH and H2SO4 with three concentrations. By contrast, a Miscanthus sample with a high cellulose or lignin level showed increased CrI and low biomass saccharification, particularly after H2SO4 pretreatment. Correlation analysis revealed that the cellulose CrI negatively affected biomass digestion. Increased hemicelluloses level by 25% or decreased cellulose and lignin contents by 31% and 37% were also found to result in increased hexose yields by 1.3-times to 2.2-times released from enzymatic hydrolysis after NaOH or H2SO4 pretreatments. The findings indicated that hemicelluloses were the dominant and positive factor, whereas cellulose and lignin had synergistic and negative effects on biomass digestibility. CONCLUSIONS: Using six pairs of Miscanthus samples with different cell wall compositions, hemicelluloses were revealed to be the dominant factor that positively determined biomass digestibility after pretreatments with NaOH or H2SO4 by negatively affecting cellulose crystallinity. The results suggested potential approaches to the genetic modifications of bioenergy crops.     

Stochastic model of lignocellulosic material saccharification

The processing of agricultural wastes towards extraction of renewable resources is recently being considered as a promising alternative to conventional biofuel production. The degradation of agricultural residues is a complex chemical process that is currently time intensive and costly. Various pre-treatment methods are being investigated to determine the subsequent modification of the material and the main obstacles in increasing the enzymatic saccharification. In this study, we present a computational model that complements the experimental approaches. We decipher how the three-dimensional structure of the substrate impacts the saccharification dynamics. We model a cell wall microfibril composed of cellulose and surrounded by hemicellulose and lignin, with various relative abundances and arrangements. This substrate is subjected to digestion by different cocktails of well characterized enzymes. The saccharification dynamics is simulated in silico using a stochastic procedure based on a Gillespie algorithm. As we additionally implement a fitting procedure that optimizes the parameters of the simulation runs, we are able to reproduce experimental saccharification time courses for corn stover. Our model highlights the synergistic action of enzymes, and confirms the linear decrease of sugar conversion when either lignin content or crystallinity of the substrate increases. Importantly, we show that considering the crystallinity of cellulose in addition to the substrate composition is essential to interpret experimental saccharification data. Finally, our findings support the hypothesis of xylan being partially crystalline.     

Developmental Control of Lignification in Stems of Lowland Switchgrass Variety Alamo and the Effects on Saccharification Efficiency

The switchgrass variety Alamo has been chosen for genome sequencing, genetic breeding, and genetic engineering by the US Department of Energy Joint Genome Institute (JGI) and the US Department of Energy BioEnergy Science Center. Lignin has been considered as a major obstacle for cellulosic biofuel production from switchgrass biomass. The purpose of this study was to provide baseline information on cell wall development in different parts of developing internodes of tillers of switchgrass cultivar Alamo and evaluate the effect of cell wall properties on biomass saccharification. Cell wall structure, soluble and wall-bound phenolics, and lignin content were analyzed from the top, middle, and bottom parts of internodes at different developmental stages using ultraviolet autofluorescence microscopy, histological staining methods, and high-performance liquid chromatography (HPLC). The examination of different parts of the developing internodes revealed differences in the stem structure during development, in the levels of free and well-bound phenolic compounds and lignin content, and in lignin pathway-related gene expression, indicating that the monolignol biosynthetic pathway in switchgrass is under complex spatial and temporal control. Our data clearly show that there was a strong negative correlation between overall lignin content and biomass saccharification efficiency. The ester-linked p-CA/FA ratio showed a positive correlation with lignin content and a negative correlation with sugar release. Our data provide baseline information to facilitate genetic modification of switchgrass recalcitrance traits for biofuel production.     

Substrate-Related Factors Affecting Enzymatic Saccharification of Lignocelluloses: Our Recent Understanding

Enzymatic saccharification of cellulose is a key step in conversion of plant biomass to advanced biofuel and chemicals. Many substrate-related factors affect saccharification. Rather than examining the role of each individual factor on overall saccharification efficiency, this study examined how each factor affects the three basic processes of a heterogeneous biochemistry reaction: (1) substrate accessibility to cellulose—the roles of component removal and size reduction by pretreatments, (2) substrate and cellulase reactivity limited by component inhibition, and (3) reaction conditions—substrate-specific optimization. Our in-depth analysis of published literature work, especially those published in the last 5 years, explained and reconciled some of the conflicting results in literature, especially the relative importance of hemicellulose vs. lignin removal and substrate size reduction on enzymatic saccharification of lignocelluloses. We concluded that hemicellulose removal is more important than lignin removal for creating cellulase accessible pores. Lignin removal is important when alkaline-based pretreatment is used with limited hemicellulose removal. Partial delignification is needed to achieve satisfactory saccharification of lignocelluloses with high lignin content, such as softwood species. Rather than using passive approaches, such as washing and additives, controlling pretreatment or hydrolysis conditions, such as pH, to modify lignin surface properties can be more efficient for reducing or eliminating lignin inhibition to cellulase, leading to improved lignocellulose saccharification.     

Modifying plants for biofuel and biomaterial production

The productivity of plants as biofuel or biomaterial crops is established by both the yield of plant biomass per unit area of land and the efficiency of conversion of the biomass to biofuel. Higher yielding biofuel crops with increased conversion efficiencies allow production on a smaller land footprint minimizing competition with agriculture for food production and biodiversity conservation. Plants have traditionally been domesticated for food, fibre and feed applications. However, utilization for biofuels may require the breeding of novel phenotypes, or new species entirely. Genomics approaches support genetic selection strategies to deliver significant genetic improvement of plants as sources of biomass for biofuel manufacture. Genetic modification of plants provides a further range of options for improving the composition of biomass and for plant modifications to assist the fabrication of biofuels. The relative carbohydrate and lignin content influences the deconstruction of plant cell walls to biofuels. Key options for facilitating the deconstruction leading to higher monomeric sugar release from plants include increasing cellulose content, reducing cellulose crystallinity, and/or altering the amount or composition of noncellulosic polysaccharides or lignin. Modification of chemical linkages within and between these biomass components may improve the ease of deconstruction. Expression of enzymes in the plant may provide a cost‐effective option for biochemical conversion to biofuel.     

Using satellite vegetation and compound topographic indices to map highly erodible cropland buffers for cellulosic biofuel crop developments in eastern Nebraska,USA

Cultivating annual row crops in high topographic relief waterway buffers has negative environmental effects and can be environmentally unsustainable. Growing perennial grasses such as switchgrass (Panicum virgatum L.) for biomass (e.g., cellulosic biofuel feedstocks) instead of annual row crops in these high relief waterway buffers can improve local environmental conditions (e.g., reduce soil erosion and improve water quality through lower use of fertilizers and pesticides) and ecosystem services (e.g., minimize drought and flood impacts on production; improve wildlife habitat, plant vigor, and nitrogen retention due to post-senescence harvest for cellulosic biofuels; and serve as carbon sinks). The main objectives of this study are to: (1) identify cropland areas with high topographic relief (high runoff potentials) and high switchgrass productivity potential in eastern Nebraska that may be suitable for growing switchgrass, and (2) estimate the total switchgrass production gain from the potential biofuel areas. Results indicate that about 140,000 hectares of waterway buffers in eastern Nebraska are suitable for switchgrass development and the total annual estimated switchgrass biomass production for these suitable areas is approximately 1.2 million metric tons. The resulting map delineates high topographic relief croplands and provides useful information to land managers and biofuel plant investors to make optimal land use decisions regarding biofuel crop development and ecosystem service optimization in eastern Nebraska.     

Advances in modifying lignin structures for largely enhancing high-lignin biomass saccharification

Traditional chemical treatments require high energy and concentrated agents to decrease high-lignin biomass recalcitrance during lignin removal. A low-cost and environment-friendly method via modifying the lignin structure without lignin degradation overcomes lignin barrier in wood-feeding termites. In this study, thermal hydroxyl radicals in the biomimetic system performed in high-lignin cassava stillage residues did not reduce total lignin level but altered the lignin monostructure. The major β-O-4′ remained conserved, whereas hydroxylation and modification of selective intermonomer side-chain linkages occurred in the treated lignin. The treatment can enhance enzymatic saccharification with cellulose conversion of cassava stillage residues at 97%, twice higher than that of the control. In addition, the modified lignin exhibited low invalid adsorption capacity into commercial cellulases, thereby improving the performance of cellulosic feedstock. Modifying the lignin structure, instead of removing lignin, provides new insights for development of novel treatments for high-lignin biomass.     

Chromatographic detection of lignin-carbohydrate complexes in annual plants by derivatization in ionic liquid

The opportunity for detecting the presence and the amount of lignin-carbohydrate complexes (LCCs) in renewable feedstocks is a major issue for the complete utilization of biomass. Indeed, LCCs are known to shield cellulose from enzymatic hydrolysis, reducing the efficiency of the digestion processes needed for the production of biobased products. This study is focused on the chromatographic characterization of lignocellulose from agricultural residues (rice husk, wheat straw) and herbaceous energy crops ( Arundo donax , Miscanthus sinesis ) and their fractionation products (hemicellulose, cellulose, and lignin). Exploiting alternative chemical derivatizations on the aforementioned samples, it was possible to discern the connectivity among the various lignocellulosic components. The complete acetylation and benzoylation of the milled native substrates in ionic liquid media, and the systematic comparison between their GPC-UV chromatograms collected at different wavelengths has revealed itself as a straightforward technique in the detection of LCCs. This novel approach proved an extensive connectivity between the lignin and the hemicellulosic for all the analyzed specimens, whereas the cellulosic fraction was conceived as a substantially unbound moiety, accounting for the sample composition at higher molecular weights. Moreover, the collected lignin fractions were extensively characterized by means of (31)P NMR and 2D-HSQC techniques.     

Evapotranspiration of annual and perennial biofuel crops in a variable climate

Eddy covariance measurements were made in seven fields in the Midwest USA over 4 years (including the 2012 drought year) to estimate evapotranspiration (ET) of newly established rain‐fed cellulosic and grain biofuel crops. Four of the converted fields had been managed as grasslands under the USDA's Conservation Reserve Program (CRP) for 22 years, and three had been in conventional agriculture (AGR) soybean/corn rotation prior to conversion. In 2009, all sites were planted to no‐till soybean except one CRP grassland that was left unchanged as a reference site; in 2010, three of the former CRP sites and the three former AGR sites were planted to annual (corn) and perennial (switchgrass and mixed‐prairie) grasslands. The annual ET over the 4 years ranged from 45% to 77% (mean = 60%) of the annual precipitation (848–1063 mm; November–October), with the unconverted CRP grassland having the highest ET (622–706 mm). In the fields converted to annual and perennial crops, the annual ET ranged between 480 and 639 mm despite the large variations in growing‐season precipitation and in soil water contents, which had strong effects on regional crop yields. Results suggest that in this humid temperate climate, which represents the US Corn Belt, water use by annual and perennial crops is not greatly different across years with highly variable precipitation and soil water availability. Therefore, large‐scale conversion of row crops to perennial biofuel cropping systems may not strongly alter terrestrial water balances.     

Development and status of dedicated energy crops in the United States

The biofuel industry is rapidly growing because of increasing energy demand and diminishing petroleum reserves on a global scale. A multitude of biomass resources have been investigated, with high-yielding, perennial feedstocks showing the greatest potential for utilization as advanced biofuels. Government policy and economic drivers have promoted the development and commercialization of biofuel feedstocks, conversion technologies, and supply chain logistics. Research and regulations have focused on the environmental consequences of biofuels, greatly promoting systems that reduce greenhouse gas emissions and life-cycle impacts. Numerous biofuel refineries using lignocellulosic feedstocks and biomass-based triglycerides are either in production or pre-commercial development phases. Leading candidate energy crops have been identified, yet require additional efforts to realize their full potential. Advanced biofuels, complementing conventional biofuels and other renewable energy sources such as wind and solar, provide the means to substantially displace humanity’s reliance on petroleum-based energy.     

Candidate perennial bioenergy grasses have a higher albedo than annual row crops

The production of perennial cellulosic feedstocks for bioenergy presents the potential to diversify regional economies and the national energy supply, while also serving as climate ‘regulators’ due to a number of biogeochemical and biogeophysical differences relative to row crops. Numerous observational and model‐based approaches have investigated biogeochemical trade‐offs, such as increased carbon sequestration and increased water use, associated with growing cellulosic feedstocks. A less understood aspect is the biogeophysical changes associated with the difference in albedo (α), which could alter the local energy balance and cause local to regional cooling several times larger than that associated with offsetting carbon. Here, we established paired fields of Miscanthus × giganteus (miscanthus) and Panicum virgatum (switchgrass), two of the leading perennial cellulosic feedstock candidates, and traditional annual row crops in the highly productive ‘Corn‐belt’. Our results show that miscanthus did and switchgrass did not have an overall higher α than current row crops, but a strong seasonal pattern existed. Both perennials had consistently higher growing season α than row crops and winter α did not differ. The lack of observed differences in winter α, however, masked an interaction between snow cover and species differences, with the perennial species, compared with the row crops, having a higher α when snow was absent and a much lower α when snow was present. Overall, these changes resulted in an average net reduction in annual absorbed energy of about 5 W m^?2 for switchgrass and about 8 W m^?2 for miscanthus relative to annual crops. Therefore, the conversion from annual row to perennial crops alters the radiative balance of the surface via changes in α and could lead to regional cooling.     

Enhancement of enzymatic hydrolysis by simultaneous attrition of cellulosic substrates

It has been shown that simultaneous attrition of cellulose in an attritor containing stainlesssteel beads results in a substantial enhancement of the enzymatic hydrolysis. The attrition exerts two opposing effects, continuous delamination and comminution of the substrate with formation of new reactive sites and a gradual denaturation and inactivation of the enzyme. Consequently, the hydrolysis proceeds very rapidly at first and levels off at about 70% saccharification of the substrate. Accumulation of hydrolysis products is also responsible for inhibition of the enzyme. The attrition method is effective for the saccharification of cottonwood in which the cellulosic microfibrils are embedded in a matrix of lignin and hemicelluloses. A comparison between the saccharification of wood, lignocellulose, holocellulose, and cellulose with simultaneous attrition showed that the lignin component provided more hindrance toward the saccharification process than hemicelluloses, which are themselves subject to enzymatic hydrolysis.