- Top of page
- Materials and Methods
Globally there is a pressing need to establish a bio-based, renewable energy sector. Increasing population growth, coupled with increased industrialization, is exponentially amplifying societal reliance on petroleum-derived energy and consequently resulting in elevated amounts of carbon release into the atmosphere in the form of the glasshouse gas CO2. These anthropogenic activities are therefore a major contributor to global climate change. In an attempt to offset the negative impacts of the existing petroleum-dominated economy, mitigation strategies have been prioritized, including activities aimed at reducing consumption and establishing alternative energy sources. Fuel derived from fermentable sugars, such as bioethanol, to supplement the liquid fuel transportation sector offers one such opportunity, and has the potential to be produced with a positive energy balance, capitalizing on the capture of solar energy by biomass crops (Rubin, 2008).
Along with what is derived from sugarcane, a significant amount of ethanol is generated from grain-derived starch, such as maize, throughout North America. Although established, such production is not likely the best long-term strategy, since the current agricultural-derived capacity is not sufficient to sustainably produce the substantial projected requirements. Equally important, there is an inherent competition for land use for food production (Karp & Shields, 2008). In contrast, a promising source of ethanol is the abundant lignocellulosic feedstocks, including wood and fiber-derived biomass, produced from forested lands and marginal agricultural lands (Li et al., 2008; Mansfield, 2009). Lignocellulosic biomass, which is available in a number of forms, represents an abundant, inexpensive, and generally locally available feedstock. Despite the promising economic and environmental (carbon-neutral) benefits associated with lignocellulosic biomass bioenergy production, key biological, technical, and socioeconomic challenges must be overcome (Gregg et al., 1998; Huang et al., 2009). Lignocellulosic feedstocks are chemically and structurally more complex than the currently employed substrates such as soluble sugars derived from sugar cane or starch in corn-derived ethanol. Lignocellulosic feedstocks consist of plant cell walls composed of chemically linked polymeric macromolecules composed of cellulose, lignin, and hemicelluloses. The structure and chemistry of woody feedstocks inherently make these substrates recalcitrant to breakdown into fermentable sugars, owing to the compact structure of crystalline cellulose microfibrils, the lack of substrate porosity and the presence of higher lignin concentrations (Mansfield et al., 1999; Chang & Holtzapple, 2000). Therefore, the innate plant cell wall structure and biochemistry are key determinants of the utility of lignocellulosic feedstocks for biofuel applications. Similarly, maximizing woody biomass productivity is also critical for minimizing feedstock costs. Thus, technological advances improving growth rates and/or the design of plant cell wall biochemistries that are more amenable to conversion to fermentable sugars for bioethanol production could have a substantial impact on the overall efficacy of the bioenergy process.
Lignin, inherent in the plant secondary cell wall, significantly impedes enzymatic accessibility (Mooney et al., 1998; Chang & Holtzapple, 2000) and competitively binds cellulolytic enzymes (Cleresci et al., 1985; Eklund et al., 1990; Berlin et al., 2005). This underpins the innate recalcitrance of wood-derived lignocellulosic feedstocks for production of liquid biofuels. Lignin formation in higher plants has been studied extensively, and most of the enzymes directly involved in its biosynthesis have now been identified (Boerjan et al., 2003; Weng & Chapple, 2010). Furthermore, several different approaches have been employed to modify lignin quantity or subunit composition by directed genetic modification (Vanholme et al., 2008; Mansfield, 2009). In recent years, genetically controlling/restricting monolignol flux to lignin polymer biosynthesis has been shown to lead to overall reductions in lignin quantity, in several potential bioenergy crops, including poplar (Hu et al., 1999; Halpin et al., 2007; Coleman et al., 2008a; Vanholme et al., 2008), switchgrass (Fu et al., 2011), and alfalfa (Reddy et al., 2005; Chen & Dixon, 2007; Shadle et al., 2007). Using a variety of genetic tools, perturbations in many of these genes have resulted in varying effects, from extreme reductions in lignification to more mild reductions with concurrent alterations in monolignol composition. For example, caffeoyl CoA 3-O-methyltransferase (CCoAOMT) suppression in poplar (Meyermans et al., 2000) showed that there was a decrease in the syringyl lignin content and a concomitant appearance of 5-hydroxyguaiacyl residues, while antisense regulation of cinnamoyl CoA reductase (CCR) (Lepléet al., 2007) resulted in a 20% reduction in total lignin content. Using RNAi-mediated suppression of p-coumarate 3′-hydroxylase (C3′H), Coleman et al. (2008a) showed up to a 50% reduction in total cell wall lignin content. Similar results were observed by Hu et al. (1999), who used antisense to misregulate 4-(hydroxy)cinnamoyl CoA ligase (4CL) in poplar and observed 45% reductions in lignification. Recent studies have shown that in genetically engineered energy crops, such as alfalfa (Chen & Dixon, 2007) and poplar (Voelker et al., 2011), as well as native stands of poplar (Studer et al., 2011), lignin content impacts sugar release during biochemical bioconversion processes.
Alternatively, overexpressing a cytochrome P450-dependent monooxygenase gene which encodes ferulate 5-hydroxylase (F5H) has been shown to impact the quality of lignin, resulting in a dramatic shift in the ratio of monomers, in favour of syringyl subunits, without impacting the total lignin content (Franke et al., 2000). Earlier, these transgenic poplar trees carrying a C4H::F5H fusion were shown to substantially improve the ease of chemical pulping (Huntley et al., 2003). This wood could be pulped in half the time normally required and yielded higher-quality cellulose, indicating that high syringyl lignins are much more readily degraded during the chemical pulping process. In this paper, we extend this work and clearly demonstrate the influence of lignin quantity and quality on production of liquid biofuels. We fundamentally demonstrate that targeted transgenics, which augment the biosynthesis of key cell wall constituents, can positively impact the production of ethanol from feedstocks that have traditionally been regarded as highly recalcitrant.
- Top of page
- Materials and Methods
Cellulosic polymers can be degraded by a number of different means; however, primary degradation results from either chemical or enzymatic hydrolysis of the polymeric substrate into oligomeric and monomeric soluble sugars. During enzymatic hydrolysis, the structural differences in cellulose allomorphs, and the intricate association with other biomolecules in lignocellulosic substrates are very important factors in controlling their susceptibility to degradation (Mansfield et al., 1999). Furthermore, as a result of the inherent insolubility and physical complexity, several different enzymes are needed for solubilization, and in most cases, complete solubilization is never achieved. The current understanding of enzyme-mediated hydrolysis of native cellulose, by extracellular microbial enzyme systems, results from the synergistic and complementary activities of several monocomponent enzymes that lead to an enhancement of the activity over the added activity of the individual enzymes (Mansfield & Meder, 2003). It has often been suggested that the initial rapid rate of hydrolysis followed by a slowing and sometimes incomplete solubilization of cellulose is related to the initial catalysis of the more ‘amorphous’ moieties of the cellulosic substrate. However, ‘recalcitrance’ of the residual material may be ascribed, along with crystallinity (Fan et al., 1980, 1981), to several other key substrate factors, including, irreversible binding of the enzyme to the substrate (Cleresci et al., 1985; Eklund et al., 1990; Berlin et al., 2005), and the lignin itself acting as a barrier and limiting the accessibility of the cellulases to the cellulosic residue (Converse et al., 1990; Mooney et al., 1998). Herein, we examine the impact of lignin quantity and quality (monomer distribution) on hydrolysis, and the response to pretreatment followed by conversion efficiencies.
It is apparent that the chemical composition of lignin in poplar wood has no impact on the efficacy of hydrolysis of the wood substrate by cellulase preparations. Wood derived from C4H::F5H transgenic poplar, which has similar total amounts of lignin with varying concentrations of subunit monomers, did not impact the rate or extent of hydrolysis. These findings are consistent with those of Chen & Dixon (2007) who showed that transgenic alfalfa lines with misregulated F5H or CCoAOMT had comparable enzymatic conversion efficiencies to wildtype plants. In contrast, using trees with perturbed lignification, via down-regulation of C3′H, show a clear relationship between cell wall lignin content and the efficiency of hydrolysis. Again, these results concur with Chen & Dixon (2007), who showed that plants with lower lignin contents were more amenable to hydrolysis. More importantly, these authors showed that improved hydrolysis can be achieved by several of the genes in the lignin biosynthetic pathway, including hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT), cinnamate 4-hydroxylase (C4H), p-coumarate 3′-hydroxylase (C3′H) and caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) (Chen & Dixon, 2007). Equally important is the recent work by Studer et al. (2011) that demonstrates that in a screen of 1100 naturally occurring unrelated Poplar genotypes, below 20% total cell wall lignin content wood hydrolyzability was directly related to lignin content.
Biological degradation of lignocellulosics, while environmentally benign, is not effective at solubilizing the structural carbohydrate moieties away from the other associated wall constitutes, as depicted in Fig. 1, which clearly demonstrates the traditionally observed wood hydrolysis profile. This limited hydrolysis has often been linked to the inherent heterogeneous nature of the supramolecular structures of the naturally occurring lignocellulosic matrices. Furthermore, it is apparent that the efficacy of enzymatic complexes to hydrolyze these substrates is inextricably linked to substrate modifications that occur to the substrate as saccharification proceeds. Therefore, as a means to improve the biological conversion of woody biomass, and improve the effective enzymatic liberation of the carbohydrate polymer, there is a heavy reliance on some form of physicochemical pretreatment (i.e. dilute acid, steam explosion, ammonia fibre expansion (AFEX) or organosolv). Herein, we evaluated two different pretreatment regimes to assess the impact of altered S : G ratio and total cell wall lignin content on conversion efficiencies.
Previous work (Brownell & Saddler, 1987) has shown that steam pretreatment of hardwoods can improve both the rate and yield of enzymatic hydrolysis, while at the same time recovering a substantial component of the lignin (90%). We show that steam explosion pretreatment and subsequent hydrolysis and fermentation efficiencies of poplar wood substrates can be substantially improved by manipulating plant cell wall lignification. First, it is apparent that lignin monomer composition can improve the overall steam explosion process by as much as c. 15%, which is associated with a significant increase in the monomeric carbohydrate concentrations in the saccharification stream that ultimately improves the amount of ethanol derived from fermentation. These results are consistent with Li et al. (2010), who showed that when a liquid hot water (LHW) pretreatment was included before enzyme hydrolysis, the S-lignin-rich tissue gave a much higher glucose yield than either the wildtype or G-lignin-rich tissue. However, it should be noted that the fermentation of the prehydrolyzate is negatively affected. This is likely a consequence of elevated concentrations of syringyl-derived lignin degradation products that have previously been shown to inhibit yeast fermentation (Keating et al., 2006) and/or process inhibitors derived from the addition of acid as a catalyst to the steam explosion operation (Haemelinck et al., 2005).
The extent of total cell wall lignin content also positively impacted the efficiency of the steam explosion process, as shown in other potential energy crops, such as grain (Mussatto et al., 2008), corn stover (Öhgren et al., 2007), sugarcane (Martín et al., 2007) in which lower lignin content improved the subsequent hydrolysis of the wood residue and fermentation processes. These findings are consistent with work evaluating a dilute acid pretreatment on control and transgenic alfalfa (Chen & Dixon, 2007) and more recently with naturally occurring poplar genotypes (Studer et al., 2011). In all cases, there is a clear relationship between decreasing lignin content and the ability for cellulolytic enzymes to access the remaining wall carbohydrate matrices. And, more importantly in extreme cases, there are indications that significant reductions in lignification can obviate the need for pretreatment, as demonstrated here with poplar and previously with alfalfa (Chen & Dixon, 2007). However, despite the improved hydrolyzability observed with RNAi-C3′H poplar, substantial reductions in lignin come with a yield penalty (Table 1). Similar yield penalties have also been observed in field-grown antisense down-regulation of 4CL poplar. Coleman et al. (2008b) suggest that the severe inhibition of secondary cell wall lignification produced trees with a collapsed xylem phenotype, resulting in compromised vascular integrity and reduced hydraulic conductivity. This resulted in a greater susceptibility to wall failure and cavitation. Interestingly, they also demonstrated that leaf starch and soluble sugars were accumulating to high degrees, suggesting that the trees with substantially reduced cell wall lignin were not carbon-limited and that reductions in sink strength were, instead, limiting photosynthesis (Coleman et al., 2008b).
During steam explosion, the residual lignin undergoes substantial chemical modifications (Shevchenko et al., 1999) as a result of the high pressures and temperatures associated with the pretreatment process. A proportion of the ß-O-4 bonds present in lignin are cleaved, resulting in lignin moving into the elastic state (Kallavus & Gravitis, 1995), and agglomerates forming 5-5 biphenyl bonds (Michalowicz et al., 1991). These ensuing 5-5 linkages formed (Shevchenko et al., 1999) are much more recalcitrant to chemical cleavage than other interunit linkages inherent to native lignin (although there are small amounts of native 5-5 linkages). Therefore, the recovered steam explosion lignin is generally only useful for its calorific value, and as such is burned to provide energy in the form of heat. This limited use for lignin restricts the utility of this pretreatment process. The employment of high-mol % S-lignin transgenic trees, which contain methoxyl units bound to both the 3- and 5-positions of the benzene ring, could negate this limiting factor and improve the utility of the recoverable lignin for co-product development.
It has been shown, via the results of techno-economic modelling (Gregg et al., 1998), that if the wood-to-ethanol process is to be viable, it must generate high-quality, lignin-derived co-products other than heat energy in addition to the ethanol derived from the carbohydrates to drive this otherwise marginally cost-effective biorefinery process. Thus, a second pretreatment process, organosolv pulping, was evaluated. This process uses the product as a key reactant, and has been shown to facilitate the recovery lignin that can be employed in the manufacture of several industrial co-products such as adhesives or biodegradable polymers (Kubo & Kadla, 2004).
Employing a series of conditions previously shown to work well with hybrid poplar (Pan et al., 2006a), we evaluated the impact of cell wall lignin on its solubilization and cellulose recovery by acid-catalyzed ethanolysis. The results clearly show that the incorporation of higher than normal (c. 65–70%) syringyl moieties into lignin by genetic engineering improves the solubilization of the lignin. This might be expected since it has been shown that the transgenic C4H::F5H lignin is more linear and of much smaller molecular mass (Stewart et al., 2009), and as such should be more amenable to cleavage and subsequent extraction, as has been shown in alkaline pulping reactions (Huntley et al., 2003). Of the conditions examined, 65% ethanol supplemented with 0.76% acid catalyst appears to be the most effective at solubilizing the lignin, in both wildtype wood and transgenic wood, and are similar to previous observations (Pan et al., 2006a). More importantly, the high S-lignin improves cellulose recovery by c. 10%, and offers a substrate that is readily hydrolyzed by enzymes and contains few deleterious inhibitors to fermentation, thereby improving the overall ethanol yield and bioconversion process.
Again, the ethanolysis significantly improved cellulose recovery and substantially improved the efficiency of hydrolysis and ethanol yield. And, more importantly, it is clear that lignin content has a greater impact then monomer distribution on pretreatment and ethanol productions. These general findings are consistent with those of Chen & Dixon (2007) and Studer et al. (2011). Although it was apparent that reducing cell wall lignification could obviate the need for pretreatment (in the extreme case), the severe misregulation comes with a yield penalty. As such, there is a pressing need to investigate the impact of plants with only modestly down-regulated lignin deposition, where improvements in biofuel processing can be obtained and plant form and function are not compromised. Plants of this type would more realistically represent trees that could withstand field conditions. It is known, however, that these transgenic events are stably integrated into the host genome and continue to confer the observed trait, year after year (Table 3).
Table 3. Annual determination of wood chemical characteristics in wildtype and transgenic C4H::F5H poplar trees after 8 yr of growth in the glasshouse, as determined by annual growth ring evaluation (standard error of the mean in brackets)
|Line||Mol S-lignin (%) (years 1–2)||Mol S-lignin (%) (years 3–5)||Mol S-lignin (%) (years 7–8)|
|Wildtype||65.2 (0.31)||68.3 (0.66)||69.2 (0.32)|
|C4H::F5H 82||87.2 (1.47)||85.5 (1.17)||84.5 (0.48)|
|C4H::F5H 64||94.9 (0.18)||94.3 (0.21)||93.48 (0.41)|