Brian H. Davison

Lignin Valorization: Improving Lignin Processing in the Biorefinery

Background Lignin, nature’s dominant aromatic polymer, is found in most terrestrial plants in the approximate range of 15 to 40% dry weight and provides structural integrity. Traditionally, most large-scale industrial processes that use plant polysaccharides have burned lignin to generate the power needed to productively transform biomass. The advent of biorefineries that convert cellulosic biomass into liquid transportation fuels will generate substantially more lignin than necessary to power the operation, and therefore efforts are underway to transform it to value-added products. Production of biofuels from cellulosic biomass requires separation of large quantities of the aromatic polymer lignin. In planta genetic engineering, enhanced extraction methods, and a deeper understanding of the structure of lignin are yielding promising opportunities for efficient conversion of this renewable resource to carbon fibers, polymers, commodity chemicals, and fuels. [Credit: Oak Ridge National Laboratory, U.S. Department of Energy] Advances Bioengineering to modify lignin structure and/or incorporate atypical components has shown promise toward facilitating recovery and chemical transformation of lignin under biorefinery conditions. The flexibility in lignin monomer composition has proven useful for enhancing extraction efficiency. Both the mining of genetic variants in native populations of bioenergy crops and direct genetic manipulation of biosynthesis pathways have produced lignin feedstocks with unique properties for coproduct development. Advances in analytical chemistry and computational modeling detail the structure of the modified lignin and direct bioengineering strategies for targeted properties. Refinement of biomass pretreatment technologies has further facilitated lignin recovery and enables catalytic modifications for desired chemical and physical properties. Outlook Potential high-value products from isolated lignin include low-cost carbon fiber, engineering plastics and thermoplastic elastomers, polymeric foams and membranes, and a variety of fuels and chemicals all currently sourced from petroleum. These lignin coproducts must be low cost and perform as well as petroleum-derived counterparts. Each product stream has its own distinct challenges. Development of renewable lignin-based polymers requires improved processing technologies coupled to tailored bioenergy crops incorporating lignin with the desired chemical and physical properties. For fuels and chemicals, multiple strategies have emerged for lignin depolymerization and upgrading, including thermochemical treatments and homogeneous and heterogeneous catalysis. The multifunctional nature of lignin has historically yielded multiple product streams, which require extensive separation and purification procedures, but engineering plant feedstocks for greater structural homogeneity and tailored functionality reduces this challenge. The Lignin Landscape Lignin is a chemically complex polymer that lends woody plants and trees their rigidity. Humans have traditionally either left it intact to lend rigidity to their own wooden constructs, or burned it to generate heat and sometimes power. With the advent of major biorefining operations to convert cellulosic biomass into ethanol and other liquid fuels, researchers are now exploring how to transform the associated leftover lignin into more diverse and valuable products. Ragauskas et al. (10.1126/science.1246843) review recent developments in this area, ranging from genetic engineering approaches that tune lignin properties at the source, to chemical processing techniques directed toward extracting lignin in the biorefinery and transforming it into high-performance plastics and a variety of bulk and fine chemicals. Research and development activities directed toward commercial production of cellulosic ethanol have created the opportunity to dramatically increase the transformation of lignin to value-added products. Here, we highlight recent advances in this lignin valorization effort. Discovery of genetic variants in native populations of bioenergy crops and direct manipulation of biosynthesis pathways have produced lignin feedstocks with favorable properties for recovery and downstream conversion. Advances in analytical chemistry and computational modeling detail the structure of the modified lignin and direct bioengineering strategies for future targeted properties. Refinement of biomass pretreatment technologies has further facilitated lignin recovery, and this coupled with genetic engineering will enable new uses for this biopolymer, including low-cost carbon fibers, engineered plastics and thermoplastic elastomers, polymeric foams, fungible fuels, and commodity chemicals.

How biotech can transform biofuels

For cellulosic ethanol to become a reality, biotechnological solutions should focus on optimizing the conversion of biomass to sugars.

Lignin content in natural Populus variants affects sugar release

The primary obstacle to producing renewable fuels from lignocellulosic biomass is a plants recalcitrance to releasing sugars bound in the cell wall. From a sample set of wood cores representing 1,100 individual undomesticated Populus trichocarpa trees, 47 extreme phenotypes were selected across measured lignin content and ratio of syringyl and guaiacyl units (S/G ratio). This subset was tested for total sugar release through enzymatic hydrolysis alone as well as through combined hot-water pretreatment and enzymatic hydrolysis using a high-throughput screening method. The total amount of glucan and xylan released varied widely among samples, with total sugar yields of up to 92% of the theoretical maximum. A strong negative correlation between sugar release and lignin content was only found for pretreated samples with an S/G ratio < 2.0. For higher S/G ratios, sugar release was generally higher, and the negative influence of lignin was less pronounced. When examined separately, only glucose release was correlated with lignin content and S/G ratio in this manner, whereas xylose release depended on the S/G ratio alone. For enzymatic hydrolysis without pretreatment, sugar release increased significantly with decreasing lignin content below 20%, irrespective of the S/G ratio. Furthermore, certain samples featuring average lignin content and S/G ratios exhibited exceptional sugar release. These facts suggest that factors beyond lignin and S/G ratio influence recalcitrance to sugar release and point to a critical need for deeper understanding of cell-wall structure before plants can be rationally engineered for reduced recalcitrance and efficient biofuels production.

Assessing the molecular structure basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments

The production of cellulosic ethanol from biomass is considered a promising alternative to reliance on diminishing supplies of fossil fuels, providing a sustainable option for fuels production in an environmentally compatible manner. The conversion of lignocellulosic biomass to biofuels through a biological route usually suffers from the intrinsic recalcitrance of biomass owing to the complicated structure of plant cell walls. Currently, a pretreatment step that can effectively reduce biomass recalcitrance is generally required to make the polysaccharide fractions locked in the intricacy of plant cell walls to become more accessible and amenable to enzymatic hydrolysis. Dilute acid and hydrothermal pretreatments are attractive and among the most promising pretreatment technologies that enhance sugar release performance. This review highlights our recent understanding on molecular structure basis for recalcitrance, with emphasis on structural transformation of major biomass biopolymers (i.e., cellulose, hemicellulose, and lignin) related to the reduction of recalcitrance during dilute acid and hydrothermal pretreatments. The effects of these two pretreatments on biomass porosity as well as its contribution on reduced recalcitrance are also discussed.

Variation of S/G ratio and lignin content in a Populus family influences the release of xylose by dilute acid hydrolysis.

Wood samples from second generation Populus cross were shown to have different lignin contents and S/G ratios (S: syringyl-like lignin structures; G: guaiacyl-like lignin structures). The lignin contents varied from 22.7% to 25.8% and the S/G ratio from 1.8 to 2.3. Selected samples spanning these ranges were hydrolyzed with dilute (1%) sulfuric acid to release fermentable sugars. The conditions were chosen for partial hydrolysis of the hemicellulosic fraction to maximize the expression of variation among samples. The results indicated that both lignin contents and S/G ratio significantly affected the yield of xylose. For example, the xylose yield of the 25.8% lignin and 2.3 S/G (hihg lignin, high S/G) sample produced 30% of the theoretical yield, whereas the xylose yield of the 22.7% lignin and 1.8 S/G (low lignin, low S/G) was 55% of the theoretical value. These results indicate that lignin content and composition among genetic variants within a single species can influence the hydrolyzability of the biomass.

Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations

BackgroundZymomonas mobilis ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. Z. mobilis performs best under anaerobic conditions, but is an aerotolerant organism. However, the genetic and physiological basis of ZM4s response to various stresses is understood poorly.ResultsIn this study, transcriptomic and metabolomic profiles for ZM4 aerobic and anaerobic fermentations were elucidated by microarray analysis and by high-performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Greater amounts of other end-products such as acetate, lactate, and acetoin were detected under aerobic conditions and at 26 h there was only 1.7% of the amount of ethanol present aerobically as there was anaerobically. In the early exponential growth phase, significant differences in gene expression were not observed between aerobic and anaerobic conditions via microarray analysis. HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point.Differences in extracellular metabolite profiles between conditions became greater as the fermentations progressed. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 contained lower levels of amino acids such as alanine, valine and lysine, and other metabolites like lactate, ribitol, and 4-hydroxybutanoate under anaerobic conditions relative to aerobic conditions. Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold. Transcripts for Entner-Doudoroff (ED) pathway genes (glk, zwf, pgl, pgk, and eno) and gene pdc, encoding a key enzyme leading to ethanol production, were at least 30-fold more abundant under anaerobic conditions in the stationary phase based on quantitative-PCR results. We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation.ConclusionHigh oxygen concentrations present during Z. mobilis fermentations negatively influence fermentation performance. The maximum specific growth rates were not dramatically different between aerobic and anaerobic conditions, yet oxygen did affect the physiology of the cells leading to the buildup of metabolic byproducts that ultimately led to greater differences in transcriptomic profiles in stationary phase.

Common processes drive the thermochemical pretreatment of lignocellulosic biomass

Lignocellulosic biomass, a potentially important renewable organic source of energy and chemical feedstock, resists degradation to glucose in industrial hydrolysis processes and thus requires expensive thermochemical pretreatments. Understanding the mechanism of biomass breakdown during these pretreatments will lead to more efficient use of biomass. By combining multiple probes of structure, sensitive to different length scales, with molecular dynamics simulations, we reveal two fundamental processes responsible for the morphological changes in biomass during steam explosion pretreatment: cellulose dehydration and lignin-hemicellulose phase separation. We further show that the basic driving forces are the same in other leading thermochemical pretreatments, such as dilute acid pretreatment and ammonia fiber expansion.

Continuous direct solvent extraction of butanol in a fermenting fluidized-bed bioreactor with immobilizedClostridium acetobutylicum

ImmobilizedClostridium acetobutylicum was used to ferment glucose into acetone and butanol in a fluidized-bed bioreactor. A nontoxic immiscible solvent, oleyl alcohol, was added to, and removed directly from, the fermenting columnar reactor and extracted the majority of the inhibitory butanol from the aqueous broth. The extracting solvent had a distribution coefficient of near 3 for butanol. Nonfermenting system tests indicated that equilibrium between the phases could be reached in one pass through the column. Steady-state results are presented for the fermentation with and without the extractive solvent addition. One run, with a continuous aqueous feedstream containing 40 g/L glucose, was operated for 23 d. A steady state was established with just the aqueous feedstream. Approximately half of the glucose was consumed, and the pH fell to 4.5 from 6.5. Then, during multiple intervals, the flow of organic extractive solvent (oleyl alcohol) was begun into the fermenting columnar reactor. A new apparent steady state was reached in about 4 h. The final aqueous butanol concentration was lowered by more than half. The total butanol production rate increased by 50–90% during the solvent extraction, as the organic-to-aqueous ratio increased from 1 to 4, respectively. There was an observed maximum volumetric productivity of 1.8 g butanol h−1L−1 in this nonoptimized system. The butanol yield apparently improved because of the removal of the inhibition. More substrate is going to the desired product, butanol, and less to maintenance or acid production, resulting in 10–20% increases in the ratio of butanol relative to all products.

Production of Succinic Acid by Anaerobiospirillum succiniciproducens

The effect of an external supply of carbon dioxide and pH on the production of succinic acid byAnaerobiospirillum succiniciproducens was studied. In a rich medium containing yeast extract and peptone, when the external carbon dioxide supply was provided by a 1.5M Na2CO3 solution that also was used to maintain the pH at 6.0, no additional carbon dioxide supply was needed. In fact, sparging CO2 gas into the fermenter at 0.025 L/L-min or higher rates resulted in significant decreases in both production rate and yield of succinate. Under the same conditions, the production of the main by-product acetate was not affected by sparging CO2 gas into the fermenter. The optimum pH (pH 6.0) for the production of succinic acid was found to be in agreement with results previously reported in the literature. Succinic acid production also was studied in an industrial-type inexpensive medium in which light steep water was the only source of organic nutrients. At pH 6.0 and with a CO2 gas sparge rate of 0.08 L/L-min, succinate concentration reached a maximum of 32 g/L in 27 h with a yield of 0.99 g succinate/g glucose consumed.

Economic analysis of fuel ethanol production from corn starch using fluidized-bed bioreactors.

Abstract The economics of fuel ethanol production from dry-milled corn starch were studied in fluidized-bed bioreactors (FBRs) using immobilized biocatalysts. Glucoamylase immobilized on porous diatomaceous earth was used for hydrolysis of the starch to glucose in a packed-bed reactor. The fermentation of glucose to ethanol was carried out in FBRs using Zymomonas mobilis immobilized in κ -carageenan beads. Volumetric ethanol productivities of up to 24 g/l h were achieved in non-optimized laboratory-scale systems. For a 15 million gal/yr ethanol plant, an economic analysis of this process was performed with Aspen Plus (Aspen Technology, Cambridge, MA) process simulation software. The analysis shows that an operating cost savings in the range of 1.1–3.1 cents/gal can be realized by using the FBR technology.