Effects of laccase on lignin depolymerization and enzymatic hydrolysis of ensiled corn stover
Highlights
► TMAH–GC–MS can effectively elucidate molecular changes in plant cell wall lignin. ► Laccase directly contributed to lignin decomposition at a molecular level. ► Laccase increased cellulose digestibility in ensiled stover.
Introduction
Ensilage amended with cellulases and/or hemicellulases has been demonstrated as an effective and stable long-term storage strategy for lignocellulosic biomass, as well as a beneficial platform for downstream pretreatment (Ren et al., 2007, Richard et al., 2002, Shinners et al., 2007, Vervaeren et al., 2010). However, saccharification of cellulose during and after ensilage is limited due to the presence of lignin in biomass. Lignin is a semi-random three-dimensional aromatic polymer composed of phenylpropanoid subunits linked together by a variety of ether and carbon–carbon bonds. Lignin is always intimately interlaced with hemicellulose in the plant cell wall, forming a matrix to envelop the crystalline cellulose microfibrils (Kirk and Farrell, 1987). Its complex structure and high molecular weight make lignin degradation very difficult (Call and Mücke, 1997, Ke et al., 2011, Shi et al., 2009). To overcome the recalcitrance of lignocelluloses associated with lignin, delignification strategies such as supercritical fluid extraction and hydrogenolysis have attracted increasing attention (Gosselink et al., 2012, Moilanen et al., 2011, Torr et al., 2011). Chemical and biological delignification are two major methods of lignin depolymerization. The latter is considered superior to the former due to its friendly environmental characteristics and lower energy demand. However, within the category of biological delignification strategies, microbial treatment is usually slower than enzymatic treatment, and is often achieved at the expense of considerable dry matter loss (Lechner and Papinutti, 2006, Pinto et al., 2012, Shi et al., 2009). Although enzymatic treatment is challenging due to the high molecular mass of lignin degrading enzymes, their high cost, and the requirement of enzyme co-factors, it has advantages over microbial treatment because it targets specifically selected reactions and minimizes the interference of side reactions (Roberts et al., 1995).
Among the limited number of enzymes known to participate in delignification, the laccase system has been studied most extensively. Laccase (EC 1.10.3.2) is a class of copper containing enzymes produced by fungi, bacteria and plants (Call and Mücke, 1997, Palonen, 2004). These rather nonspecific enzymes degrade polymeric products by reacting with low molecular mass mediators (Call and Mücke, 1997, Moilanen et al., 2011, Palonen, 2004). A common characteristic of most effective mediators is the prevalence of N-heterocylic bearing N–OH groups. The most widely used low-molecular mass mediator of laccases is HBT (1-hydroxybenzotriazole) (Call and Mücke, 1997).
In recent years the application of laccases to industrial lignin degradation has gained considerable attention (Kirk and Jeffries, 1996, Kolb et al., 2012, Tengerdy and Szakacs, 2003). The laccase system has been shown to be effective in degrading the lignin of pulped fibers from the paper and pulping industry, as well as in laboratory studies of degradation of synthesized or purified lignin or soluble phenolic monomers (Kirk and Jeffries, 1996, Kolb et al., 2012, Tengerdy and Szakacs, 2003). Previously reported laccase loading rates have varied widely, from 20 U to 80,000 U per gram of substrate, where one activity unit refers to the amount of enzyme that oxidizes 1 μmol of substrates per minute (Call and Mücke, 1997, Camarero et al., 2004, Sealey and Ragauskas, 1998). For example, Sealey and Ragauskas (1998) found a laccase loading rate of around 40,000 U per gram of kraft pulp was optimal for lignin degradation during 24 h experiments. However, successful application of laccase or other lignin-degrading enzymes for delignification has previously been limited to separated, isolated, or synthesized lignin. For example, lignin is partially separated from paper fibers during normal heat treatment in the pulp and paper industry, which has been shown to also facilitate access and surface contact of lignin-degrading enzymes (Camarero et al., 2004, Kirk and Jeffries, 1996, Tengerdy and Szakacs, 2003). The large macromolecular size of lignin-degrading enzymes limits access to the lignin components in an intact plant cell wall, so without separating lignin from the plant cell walls by chemical or physical pretreatment, the efficacy of these lignin-degrading enzymes is constrained (Kirk and Farrell, 1987). The overall goal of this study is to better understand the effect of the laccase system on the lignin in plant cell walls, especially in biomass stored using ensilage.
In order to determine the fate of lignin before and after treatment, we applied TMAH–GC–MS technology to the corn stover biomass. TMAH–GC–MS has previous been used to analyze lignin model compounds, synthetic lignins, wood and wheat straw lignin and to monitor the molecular change of lignin by fungal and insect decay (Geib et al., 2008, Ke et al., 2011, Vane et al., 2001). This method has not previously been used to analyze corn stover lignin, nor has TMAH–GC–MS been used to monitor the chemical change of plant cell wall lignin as affected by the laccase system or other lignin degrading enzymes.
To our knowledge, there have been no prior studies on the use of ensilage or other biological pretreatment strategies to increase the accessibility of lignin degrading enzymes to the plant cell wall. Similarly, synergies among hemicellulase, cellulase and lignin-degrading enzymes during the ensilage process have not previously been reported. Furthermore, in spite of increasing interest in a variety of hydrolysis treatments (Moilanen et al., 2011, Pinto et al., 2012, Velmurugan and Muthukumar, 2012), little is known about the influence of delignification by the laccase system on the downstream digestibility of ensiled corn stover.
To expand the current state of knowledge of these lignin degrading mechanisms, the specific objectives of this research are:
- 1.
To explore whether corn stover lignin, not previously isolated from plant cell walls by physical or chemical processes, can be degraded by the laccase system after ensilage (an anaerobic, acidogenic solid-state fermentation).
- 2.
To investigate whether the TMAH–GC–MS method can be used to characterize corn stover lignin and identify any molecular changes in that lignin associated with activity of the laccase system.
- 3.
To examine whether lignin degradation by the laccase system after ensilage can increase the digestibility of ensiled stover, and how the digestibility of ensiled stover was influenced by varying the laccase loading rate.
Section snippets
Overview of methodology
In order to achieve the stated goals and objectives, a two part series of experiments were designed to investigate effects of the laccase system on lignin depolymerization and enzymatic hydrolysis of ensiled corn stover (Fig. 1). In part I, focused on lignin analysis, gas chromatography–mass spectroscopy (GC–MS) was followed by tetramethylammonium hydroxide thermochemolysis technology to monitor the molecular change of corn stover lignin (objectives 1 and 2). Part II focused on identifying the
Results and discussion
This study examined how laccase and mediators influenced lignin decomposition and enzyme digestibility of ensiled corn stover. Detailed changes in the chemical structure of stover lignin were measured by TMAH–GC–MS analysis, while saccharification assays indicated how digestibility of ensiled stover changed at different laccase loading rates.
Conclusions
Given the short time that the ensiled stover was treated by the laccase-mediator system, it was surprising to observe such a significant change in the lignin structure and cellulose digestibility. It is possible that ensilage enhanced the effects of biological pretreatment by providing channels for laccase to enter the complex biomass, and because cellulose and hemicellulose were partially hydrolyzed into soluble sugars by cellulase, hemicellulase and organic acid during the ensilage process.
Acknowledgements
The authors thank Dr. Bob Minard sincerely for assisting with TMAH–GC–MS analysis; Professors Ali Demirci and Dawn Luthe for lab support; and Rosa Jarvis for laboratory assistance. Funding was provided by the USDA-DOE Biomass Research and Development Initiative (contract # 68-3A75-4-137) and the Pennsylvania Agricultural Experiment Station.
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