Biosynthesis and incorporation of side-chain-truncated lignin monomers to reduce lignin polymerization and enhance saccharification

Lignocellulosic biomass is utilized as a renewable feedstock in various agro‐industrial activities. Lignin is an aromatic, hydrophobic and mildly branched polymer integrally associated with polysaccharides within the biomass, which negatively affects their extraction and hydrolysis during industrial processing. Engineering the monomer composition of lignins offers an attractive option towards new lignins with reduced recalcitrance. The presented work describes a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers to reduce lignin polymerization degree (DP). Biosynthesis of these ‘DP reducers’ is achieved by expressing a bacterial hydroxycinnamoyl‐CoA hydratase‐lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side‐chain of hydroxycinnamoyl‐CoA lignin precursors to produce the corresponding hydroxybenzaldehydes so that plant stems expressing HCHL accumulate in their cell wall higher amounts of hydroxybenzaldehyde and hydroxybenzoate derivatives. Engineered plants with intermediate HCHL activity levels show no reduction in total lignin, sugar content or biomass yield compared with wild‐type plants. However, cell wall characterization of extract‐free stems by thioacidolysis and by 2D‐NMR revealed an increased amount of unusual C₆C₁ lignin monomers most likely linked with lignin as end‐groups. Moreover the analysis of lignin isolated from these plants using size‐exclusion chromatography revealed a reduced molecular weight. Furthermore, these engineered lines show saccharification improvement of pretreated stem cell walls. Therefore, we conclude that enhancing the biosynthesis and incorporation of C₆C₁ monomers (‘DP reducers’) into lignin polymers represents a promising strategy to reduce lignin DP and to decrease cell wall recalcitrance to enzymatic hydrolysis.     

Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging

Enzymatic hydrolysis of biomass is an established method for producing biofuels. Lignocellulosic biomass such as corn stover is very inhomogeneous material with big variation on conversion rates between individual particles therefore leading to variable recalcitrance results. In this study, we used noninvasive optical microscopy techniques, such as two-photon microscopy and fluorescence lifetime imaging microscopy, to visualize and analyze morphological and chemical changes of individual corn stover particles pretreated with sulfuric acid during hydrolysis. Morphochemical changes were interpreted based on the fluorescence properties of isolated building blocks of plant cell wall, such as cellulose, hemicellulose, and lignin. Enzymatic hydrolysis resulted in particle size reduction, side wall collapse, decrease of second harmonic signal from cellulose, redshifting of autofluorescence emission, and lifetime decrease attributed to the relative increase of lignin. Based on these observations, tracking compositional change after hydrolysis of individual particles was accomplished. The methodologies developed offer a paradigm for imaging and analyzing enzymatic hydrolysis in vitro and in situ, which could be used for screening enzymes cocktails targeting specific recalcitrant structures or investigating locally enzyme anti-inhibitory agents.     

Lignin Structural Alterations in Thermochemical Pretreatments with Limited Delignification

Lignocellulosic biomass has a complex and rigid cell wall structure that makes biomass recalcitrant to biological and chemical degradation. Among the three major structural biopolymers (i.e., cellulose, hemicellulose, and lignin) in plant cell walls, lignin is considered the most recalcitrant component and generally plays a negative role in the biochemical conversion of biomass to biofuels. The conversion of biomass to biofuels through a biochemical platform usually requires a pretreatment stage to reduce the recalcitrance. Pretreatment renders compositional and structural changes of biomass with these changes ultimately governing the efficiency of the subsequent enzymatic hydrolysis. Dilute acid, hot water, steam explosion, and ammonia fiber expansion pretreatments are among the leading thermochemical pretreatments with a limited delignification that can reduce biomass recalcitrance. Practical applications of these pretreatment are rapidly developing as illustrated by recent commercial scale cellulosic ethanol plants. While these thermochemical pretreatments generally lead to only a limited delignification and no significant change of lignin content in the pretreated biomass, the lignin transformations that occur during these pretreatments and the roles they play in recalcitrance reduction are important research aspects. This review highlights recent advances in our understanding of lignin alterations during these limited delignification thermochemical pretreatments, with emphasis on lignin chemical structures, molecular weights, and redistributions in the pretreated biomass.     

Low temperature alkali pretreatment for improving enzymatic digestibility of sweet sorghum bagasse for ethanol production

A low temperature alkali pretreatment method was proposed for improving the enzymatic hydrolysis efficiency of lignocellulosic biomass for ethanol production. The effects of the pretreatment on the composition, structure and enzymatic digestibility of sweet sorghum bagasse were investigated. The mechanisms involved in the digestibility improvement were discussed with regard to the major factors contributing to the biomass recalcitrance. The pretreatment caused slight glucan loss but significantly reduced the lignin and xylan contents of the bagasse. Changes in cellulose crystal structure occurred under certain treatment conditions. The pretreated bagasse exhibited greatly improved enzymatic digestibility, with 24-h glucan saccharification yield reaching as high as 98% using commercially available cellulase and β-glucosidase. The digestibility improvement was largely attributed to the disruption of the lignin-carbohydrate matrix. The bagasse from a brown midrib (BMR) mutant was more susceptible to the pretreatment than a non-BMR variety tested, and consequently gave higher efficiency of enzymatic hydrolysis.     

Biomass Enzymatic Saccharification Is Determined by the Non-KOH-Extractable Wall Polymer Features That Predominately Affect Cellulose Crystallinity in Corn

Corn is a major food crop with enormous biomass residues for biofuel production. Due to cell wall recalcitrance, it becomes essential to identify the key factors of lignocellulose on biomass saccharification. In this study, we examined total 40 corn accessions that displayed a diverse cell wall composition. Correlation analysis showed that cellulose and lignin levels negatively affected biomass digestibility after NaOH pretreatments at p<0.05 & 0.01, but hemicelluloses did not show any significant impact on hexoses yields. Comparative analysis of five standard pairs of corn samples indicated that cellulose and lignin should not be the major factors on biomass saccharification after pretreatments with NaOH and H₂SO₄ at three concentrations. Notably, despite that the non-KOH-extractable residues covered 12%–23% hemicelluloses and lignin of total biomass, their wall polymer features exhibited the predominant effects on biomass enzymatic hydrolysis including Ara substitution degree of xylan (reverse Xyl/Ara) and S/G ratio of lignin. Furthermore, the non-KOH-extractable polymer features could significantly affect lignocellulose crystallinity at p<0.05, leading to a high biomass digestibility. Hence, this study could suggest an optimal approach for genetic modification of plant cell walls in bioenergy corn.     

Analysis of Crystallinity Index and Hydrolysis Rates in the Bioenergy Crop Sorghum bicolor

Maximum yield from any cellulosic bioenergy crop is largely dependent upon total dry weight at harvest and process-specific bioconversion rates. Using enzymatic hydrolysis rate as a bioconversion metric, we have investigated the relationship between the biomass crystallinity index (CI) and hydrolysis yield potential (HYP) among ??20 Sorghum bicolor varieties grown in two environments. The comparison of HYP to CI revealed a significant negative correlation in both environments indicating that high cellulose crystallinity in sorghum can have an impact on conversion yield. Interestingly, no correlation was seen between CI and HYP after pretreatment. Compositional analysis revealed a significant positive correlation between lignin content and CI, as well as a significant negative correlation between lignin content and HYP. Additionally, CI and HYP were found to be significantly correlated only after 24?h of hydrolysis. These results suggest that when a sorghum cultivar is being considered for industrial scale production, the inclusion of cellulose crystallinity should be factored into the decision along with total biomass yield and lignin composition.     

The Minor Wall-Networks between Monolignols and Interlinked-Phenolics Predominantly Affect Biomass Enzymatic Digestibility in Miscanthus

Plant lignin is one of the major wall components that greatly contribute to biomass recalcitrance for biofuel production. In this study, total 79 representative Miscanthus germplasms were determined with wide biomass digestibility and diverse monolignol composition. Integrative analyses indicated that three major monolignols (S, G, H) and S/G ratio could account for lignin negative influence on biomass digestibility upon NaOH and H₂SO₄ pretreatments. Notably, the biomass enzymatic digestions were predominately affected by the non-KOH-extractable lignin and interlinked-phenolics, other than the KOH-extractable ones that cover 80% of total lignin. Furthermore, a positive correlation was found between the monolignols and phenolics at p<0.05 level in the non-KOH-extractable only, suggesting their tight association to form the minor wall-networks against cellulases accessibility. The results indicated that the non-KOH-extractable lignin-complex should be the target either for cost-effective biomass pretreatments or for relatively simply genetic modification of plant cell walls in Miscanthus.     

Inhibition of enzymatic cellulolysis by phenolic compounds

Phenolics derived from lignin and other plant components can pose significant inhibition on enzymatic conversion of cellulosic biomass materials to useful chemicals. Understanding the mechanism of such inhibition is of importance for the development of viable biomass conversion technologies. In native plant cell wall, most of the phenolics and derivatives are found in polymeric lignin. When biomass feedstocks are pretreated (prior to enzymatic hydrolysis), simple or oligomeric phenolics and derivatives are often generated from lignin modification/degradation, which can inhibit biomass-converting enzymes. To further understand how such phenolic substances may affect cellulase reaction, we carried out a comparative study on a series of simple and oligomeric phenolics representing or mimicking the composition of lignin or its degradation products. Consistent to previous studies, we observed that oligomeric phenolics could exert more inhibition on enzymatic cellulolysis than simple phenolics. Oligomeric phenolics could inactivate cellulases by reversibly complexing them. Simple and oligomeric phenolics could also inhibit enzymatic cellulolysis by adsorbing onto cellulose. Individual cellulases showed different susceptibility toward these inhibitions. Polyethylene glycol and tannase could respectively bind and degrade the studied oligomeric phenolics, and by doing so mitigate the oligomeric phenolic's inhibition on cellulolysis.     

Lignin monomer composition affects Arabidopsis cell-wall degradability after liquid hot water pretreatment

Background

Lignin is embedded in the plant cell wall matrix, and impedes the enzymatic saccharification of lignocellulosic feedstocks. To investigate whether enzymatic digestibility of cell wall materials can be improved by altering the relative abundance of the two major lignin monomers, guaiacyl (G) and syringyl (S) subunits, we compared the degradability of cell wall material from wild-type Arabidopsis thaliana with a mutant line and a genetically modified line, the lignins of which are enriched in G and S subunits, respectively.

Results

Arabidopsis tissue containing G- and S-rich lignins had the same saccharification performance as the wild type when subjected to enzyme hydrolysis without pretreatment. After a 24-hour incubation period, less than 30% of the total glucan was hydrolyzed. By contrast, when liquid hot water (LHW) pretreatment was included before enzyme hydrolysis, the S-lignin-rich tissue gave a much higher glucose yield than either the wild-type or G-lignin-rich tissue. Applying a hot-water washing step after the pretreatment did not lead to a further increase in final glucose yield, but the initial hydrolytic rate was doubled.

Conclusions

Our analyses using the model plant A. thaliana revealed that lignin composition affects the enzymatic digestibility of LHW pretreated plant material. Pretreatment is more effective in enhancing the saccharification of A. thaliana cell walls that contain S-rich lignin. Increasing lignin S monomer content through genetic engineering may be a promising approach to increase the efficiency and reduce the cost of biomass to biofuel conversion.     

Understanding biomass recalcitrance in grasses for their efficient utilization as biorefinery feedstock

One of the main challenges for the deployment of lignocellulosic biorefineries in future years is to find renewable and secured biomass sources in order to obtain bio-sourced products, as an alternative to petroleum-based commodities. Grass biomass, considering its characteristics (availability, composition, productivity, possibility of being harvested from both arable (post-harvest residues) and non-agricultural lands), can be considered as a biomass source for the future. Nevertheless, because of its complex structure and composition, which need deconstructive pre-treatments to render possible further biological conversions, grasses utilisation in biorefinery is today not widespread. Indeed, recalcitrance to polymers degradation in grasses concerns structural and compositional characteristics and can result in costly and complicated biorefinery processes. Grass recalcitrance is due to various natural factors strongly related and difficult to dissociate: rind and vascular structures; composition (lignin content is a key factor for cellulose hydrolysis acting like a physical barrier while hemicelluloses seem to play a more significant role in woody biomass than in grass plants); physical structures (crystalline nature and insoluble surface of cellulose, specific surface area, particle size), etc. Physico-chemical pretreatments are efficient solutions to overcome recalcitrance, while phenotypic selections are interesting but not efficient enough to obtain an optimal enzymatic hydrolysis. In some cases, the structural elements of grass biomass can be negatively affected by physico-chemical pretreatments, causing pre-treatment-induced recalcitrance, like cellulose hornification (irreversible alteration of cellulose microfibers), vascular structure collapsed and reduced cellulose bioaccesibility to enzymes due to cellulose covering by lignin, following lignin solubilisation.     

Designed for deconstruction--poplar trees altered in cell wall lignification improve the efficacy of bioethanol production

? There is a pressing global need to reduce the increasing societal reliance on petroleum and to develop a bio-based economy. At the forefront is the need to establish a sustainable, renewable, alternative energy sector. This includes liquid transportation fuel derived from lignocellulosic plant materials. However, one of the current limiting factors restricting the effective and efficient conversion of lignocellulosic residues is the recalcitrance of the substrate to enzymatic conversion. ? In an attempt to assess the impact of cell wall lignin on recalcitrance, we subjected poplar trees engineered with altered lignin content and composition to two potential industrial pretreatment regimes, and evaluated the overall efficacy of the bioconversion to ethanol process. ? It was apparent that total lignin content has a greater impact than monomer ratio (syringyl : guaiacyl) on both pretreatments. More importantly, low lignin plants showed as much as a 15% improvement in the efficiency of conversion, with near complete hydrolysis of the cellulosic polymer. ? Using genomic tools to breed or select for modifications in key cell wall chemical and/or ultrastructural traits can have a profound effect on bioenergy processing. These techniques may therefore offer means to overcome the current obstacles that underpin the recalcitrance of lignocellulosic substrates to bioconversion.     

Statistical correlation of spectroscopic analysis and enzymatic hydrolysis of poplar samples

Spectroscopic characterization of poplar wood samples with different crystallinity indices, lignin contents, and acetyl contents was performed to determine changes in the biomass spectra and the effects of these changes on the hydrolysis yield. The spectroscopic methods used were X-ray diffraction for determining cellulose crystallinity (CrI), diffuse reflectance infrared (DRIFT) for changes in C-C and C-O bonds, and fluorescence to determine lignin content. Raman spectroscopy was also used to determine its effectiveness in the determination of crystallinity and C-C and C-O bond changes in the biomass as a complement to better-known methods. Changes in spectral characteristics and crystallinity were statistically correlated with enzymatic hydrolysis results to identify and better understand the fundamental features of biomass that influence enzymatic conversion to monomeric sugars. In addition, the different spectroscopic methods were evaluated separately to determine the minimum amount of spectroscopic data needed to obtain accurate predictions. The principal component regression (PCR) model with only the DRIFT data gives the best correlation and prediction for both initial rate of hydrolysis and also the 72-h hydrolysis yield. The factor that most affects both the initial rate and the 72-h conversion is the O-H bond content of the sample, which directly relates to the breakage of structural carbohydrates into smaller molecules.     

RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass

Sugarcane is a prime bioethanol feedstock. Currently, sugarcane ethanol is produced through fermentation of the sucrose, which can easily be extracted from stem internodes. Processes for production of biofuels from the abundant lignocellulosic sugarcane residues will boost the ethanol output from sugarcane per land area. However, unlocking the vast amount of chemical energy stored in plant cell walls remains expensive primarily because of the intrinsic recalcitrance of lignocellulosic biomass. We report here the successful reduction in lignification in sugarcane by RNA interference, despite the complex and highly polyploid genome of this interspecific hybrid. Down‐regulation of the sugarcane caffeic acid O‐methyltransferase (COMT) gene by 67% to 97% reduced the lignin content by 3.9% to 13.7%, respectively. The syringyl/guaiacyl ratio in the lignin was reduced from 1.47 in the wild type to values ranging between 1.27 and 0.79. The yields of directly fermentable glucose from lignocellulosic biomass increased up to 29% without pretreatment. After dilute acid pretreatment, the fermentable glucose yield increased up to 34%. These observations demonstrate that a moderate reduction in lignin (3.9% to 8.4%) can reduce the recalcitrance of sugarcane biomass without compromising plant performance under controlled environmental conditions.     

High-Throughput Profiling of the Fiber and Sugar Composition of Sugarcane Biomass

Lignocellulosic biomass from sugarcane (Saccharum spp. hybrids) could potentially be a major feedstock for second-generation biofuel production. Consequently, selecting sugarcane varieties with favorable biomass characteristics, typically less enzymatic recalcitrance and better saccharification yield without sugar-yield penalty, will be important in sugarcane breeding. Economical and high-throughput techniques for profiling the major biomass components of this complex system will facilitate selection of clones with ideal lignocellulosic composition from large numbers of genotypes in breeding programs. We used a combined high-throughput profiling approach to evaluate the biomass composition of samples from a sugarcane germplasm collection. This employed near-infrared (NIR) spectroscopy for fiber characterization and high-performance liquid chromatography (HPLC) for determining the sugar content in juice. The results for 331 samples, from a diverse sugarcane population of 186 genotypes, derived from 143 parents of different genetic backgrounds, showed that high-quality NIR spectroscopic predictions were feasible for cellulose, hemicellulose, lignin, and extractives values in fiber, and sugars in juice were suitably analyzed by HPLC. The analysis of total biomass indicated that this NIR- and HPLC-based high-throughput method allowed a robust phenotypic assessment of a large number of samples for the key biomass traits in the sugarcane system, including total dry biomass, fiber, sugar content, and theoretical ethanol yields, and could potentially become the method of choice for sugarcane germplasm screening in breeding programs targeting the support of biofuel production.     

Substrate-Specific Development of Thermophilic Bacterial Consortia by Using Chemically Pretreated Switchgrass

Microbial communities that deconstruct plant biomass have broad relevance in biofuel production and global carbon cycling. Biomass pretreatments reduce plant biomass recalcitrance for increased efficiency of enzymatic hydrolysis. We exploited these chemical pretreatments to study how thermophilic bacterial consortia adapt to deconstruct switchgrass (SG) biomass of various compositions. Microbial communities were adapted to untreated, ammonium fiber expansion (AFEX)-pretreated, and ionic-liquid (IL)-pretreated SG under aerobic, thermophilic conditions using green waste compost as the inoculum to study biomass deconstruction by microbial consortia. After microbial cultivation, gravimetric analysis of the residual biomass demonstrated that both AFEX and IL pretreatment enhanced the deconstruction of the SG biomass approximately 2-fold. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments and acetyl bromide-reactive-lignin analysis indicated that polysaccharide hydrolysis was the dominant process occurring during microbial biomass deconstruction, and lignin remaining in the residual biomass was largely unmodified. Small-subunit (SSU) rRNA gene amplicon libraries revealed that although the dominant taxa across these chemical pretreatments were consistently represented by members of the Firmicutes, the Bacteroidetes, and Deinococcus-Thermus, the abundance of selected operational taxonomic units (OTUs) varied, suggesting adaptations to the different substrates. Combining the observations of differences in the community structure and the chemical and physical structure of the biomass, we hypothesize specific roles for individual community members in biomass deconstruction.     

Reaction wood – a key cause of variation in cell wall recalcitrance in willow

Background

The recalcitrance of lignocellulosic cell wall biomass to deconstruction varies greatly in angiosperms, yet the source of this variation remains unclear. Here, in eight genotypes of short rotation coppice willow (Salix sp.) variability of the reaction wood (RW) response and the impact of this variation on cell wall recalcitrance to enzymatic saccharification was considered.

Results

A pot trial was designed to test if the ‘RW response’ varies between willow genotypes and contributes to the differences observed in cell wall recalcitrance to enzymatic saccharification in field-grown trees. Biomass composition was measured via wet chemistry and used with glucose release yields from enzymatic saccharification to determine cell wall recalcitrance. The levels of glucose release found for pot-grown control trees showed no significant correlation with glucose release from mature field-grown trees. However, when a RW phenotype was induced in pot-grown trees, glucose release was strongly correlated with that for mature field-grown trees. Field studies revealed a 5-fold increase in glucose release from a genotype grown at a site exposed to high wind speeds (a potentially high RW inducing environment) when compared with the same genotype grown at a more sheltered site.

Conclusions

Our findings provide evidence for a new concept concerning variation in the recalcitrance to enzymatic hydrolysis of the stem biomass of different, field-grown willow genotypes (and potentially other angiosperms). Specifically, that genotypic differences in the ability to produce a response to RW inducing conditions (a ‘RW response’) indicate that this RW response is a primary determinant of the variation observed in cell wall glucan accessibility. The identification of the importance of this RW response trait in willows, is likely to be valuable in selective breeding strategies in willow (and other angiosperm) biofuel crops and, with further work to dissect the nature of RW variation, could provide novel targets for genetic modification for improved biofuel feedstocks.

     

Genetic Parameters of Factors Affecting the Biomass Recalcitrance of Douglas Fir Trees

Although forestry residuals provide a potentially abundant source of biomass, Douglas fir is a particularly challenging feedstock to utilize in the biochemical conversion to fuels and value-added chemicals due to its high levels of biomass recalcitrance. A greater understanding of the underlying factors behind highly recalcitrant biomass is critical in overcoming this problem. Building on earlier efforts to establish a protocol and a “recalcitrance factor” for screening the recalcitrance of trees, this study attempts to provide the first look into the genetic factors behind recalcitrance in Douglas fir with the possibility of using selective breeding and tree improvement practices to reduce recalcitrance in future generations of our forest products. Samples from over 250 Douglas fir trees in a second-cycle progeny test were collected and subjected to screening. Samples were subjected to a dilute-acid pretreatment and a subsequent enzymatic hydrolysis procedure, ultimately measuring the raw wood density, pretreatment yield, the holocellulose content of pretreated samples, hydrolyzability, and recalcitrance factor. From these data, the heritability, genetic gains, and genetic correlations were estimated. Based on these results, we predict that modifying recalcitrance in tree improvement may be feasible, but would likely require some additional understanding and improved screening techniques.     

Pretreatment and Lignocellulosic Chemistry

Lignocellulosic materials such as wood, grass, and agricultural and forest residues are promising alternative energy resources that can be utilized to produce ethanol. The yield of ethanol production from native lignocellulosic material is relatively low due to its native recalcitrance, which is attributed to, in part, lignin content/structure, hemicelluloses, cellulose crystallinity, and other factors. Pretreatment of lignocellulosic materials is required to overcome this recalcitrance. The goal of pretreatment is to alter the physical features and chemical composition/structure of lignocellulosic materials, thus making cellulose more accessible to enzymatic hydrolysis for sugar conversion. Various pretreatment technologies to reduce recalcitrance and to increase sugar yield have been developed during the past two decades. This review examines the changes in lignocellulosic structure primarily in cellulose and hemicellulose during the most commonly applied pretreatment technologies including dilute acid pretreatment, hydrothermal pretreatment, and alkaline pretreatment.