Ethanol produced from biomass, such as corn grain or sugar cane, is a renewable fuel that helps lessen dependence on petroleum-based fossil fuels. Currently, the majority of ethanol production in the United States utilizes corn grain, which supplied about 14 billion gallons of ethanol to the market in 2012. In the near future, corn supplies for ethanol are expected to be limited due to concerns that corn grown for fuel limits or competes with corn grown for feed and food. However, lignocellulosic biomass is abundant in the United States, and has the potential to produce fuel ethanol to meet 30% of current gasoline consumption; nevertheless, this technology faces significant challenges. Corn stover is the biomass remaining after harvesting the grain and is the most abundant agricultural residue in the United States, with an estimated 170–256 million tons available annually. Unlike corn grain, the carbohydrates in lignocellulosic biomass are not readily available for ethanol production, so pretreatment is required. Pretreatment processes that use harsh chemicals or steam can be effective, but currently are not economically feasible and/or environmentally sustainable. This fact sheet discusses research on the effectiveness of using a white-rot fungus to pretreat three different corn stover fractions: leaves, stalks and cobs.
The basic process for ethanol production from plant material is shown in figure 1. For corn grain, the first step is liquefaction, during which ground corn is mixed with water to form a slurry, and hydrolytic enzymes are added to convert corn starch to sugars. Yeast is then added to the slurry and fermentation occurs, generating ethanol and water. During distillation, the slurry is heated to separate the ethanol from the water and solids. In the dehydration step, the small amount of water remaining in the ethanol is removed using molecular sieves.
Although lignocellulosic biomass (ex. corn stover) has a high carbohydrate content, conversion of this material to ethanol requires pretreatment due to its complex structure. Corn stover is comprised of 35–40% cellulose, 20–25% hemicellulose, and 15–20% lignin. It is recalcitrant, or resistant, to hydrolytic enzyme attack due to the crystalline structure of the cellulose, the chemical bonds formed between the hemicellulose and lignin, and the limited surface area. Thermochemical pretreatments that use harsh chemicals and steam can be effective in reducing this recalcitrance but can be expensive and generate waste streams. Fungal pretreatment of corn stover has the potential to be a cost-effective and environmentally friendly method for reducing recalcitrance of biomass for enzymatic hydrolysis.
Figure 1. Corn ethanol vs. lignocellulosic ethanol production processes.
Fungal Pretreatment of Corn Stover
The structure and composition of corn stover varies considerably throughout the growing season and between various fractions of the plant. These differences affect pretreatment conditions and the fermentable sugar potential. In general, less recalcitrant plant fractions require less severe pretreatment. In this study, three corn stover fractions (leaves, stalks and cobs) were pretreated separately with a white-rot fungus, Ceriporiopsis subvermispora. For each fraction, the effectiveness of the pretreatment over a period of 35 days was evaluated based on the degree of degradation of the lignocellulosic components during pretreatment as well as sugar yields of enzymatic hydrolysis.
The degradation caused by the white-rot fungus was determined by measuring the initial composition of each fraction and comparing it to the composition every 3–4 days during the 35-day pretreatment. As shown in figure 2, the lignin in leaves tended to be degraded more quickly by C. subvermispora than in stalks and cobs, although the rate of degradation in leaves slowed after day 17. For the stalks and cobs, the degradation was slow initially but increased after day 17. Lignin degradation after 35 days reached 30–45% for all fractions. In contrast, the cellulose loss was much lower than the lignin degradation (figure 3). The cellulose loss of leaves was about 15–20% after two weeks of treatment, which was much higher than that of the stalk (5–10%) and cobs (<3%).
Figure 2. Degradation of lignin in corn stover fractions.
Figure 3. Degradation of cellulose in corn stover fractions.
Enzyme activity of fungi
To understand how C. subvermispora acted on different corn stover fractions, the activity levels of three different lignin-degrading enzymes (lignin peroxidase, manganese peroxidase, and laccase) that are typically secreted by white-rot fungi were measured. It was found that lignin peroxidase activity was absent with C. subvermispora grown on corn stover fractions across all time points. The effects of the other two groups of lignin degradation enzymes varied depending on the fraction being degraded. Manganese peroxidase activity peaked on day 3 for cobs and on day 10 for leaves, with activity decreasing sharply afterwards. For the fungi grown on stalks, the overall manganese peroxidase activity was much lower, but by day 17 the activity on stalks was greater than on cobs and leaves due to decreased manganese peroxidase activity on these two fractions at that time point. The enzyme laccase showed similar activity for leaves and stalks, peaking on day 10, with the highest activity associated with leaves. Laccase activity for cobs initially peaked on day 3 then decreased until day 13, after which it gradually increased, reaching the highest level at the end of pretreatment.
Although enzyme production profiles varied among the corn stover fractions or changed during the pretreatment process of a single fraction, there was no direct relationship between levels of enzyme activity and the extent of lignin degradation. This result was similar to other studies and may reflect the degraded biomass to glucose (sugar). Although low lignin content is usually associated with increased cellulose digestibility, it was not true in this study.
One challenge associated with fungal pretreatment is preservation of cellulose, as some fungi secrete enzymes that convert this polymer into simple sugars that are used by the fungus for growth and metabolism. C. subvermispora is a selective fungus that typically degrades hemicellulose and lignin as the main carbon sources. However, in this study, increased cellulase activity was observed in leaves and stalks, which corresponded to significant cellulose loss.
Following pretreatment, hydrolytic enzymes were added to each corn stover fraction to convert the degraded biomass to glucose (sugar). Although low lignin content is usually associated with increased cellulose digestibility, it was not true in this study. For example, although leaves and cobs have similar lignin contents, the untreated leaves yielded 37% glucose while the untreated cobs yielded less than 20% (Figure 4). Similarly, untreated cobs and stalks have different lignin contents but had similar glucose yields.
Fungal pretreatment did substantially improve the enzymatic hydrolysis of all corn stover factions. Glucose yield of leaves increased with pretreatment time, leveling off after 30 days. For stalks and cobs, glucose yields increased steadily throughout the 35-day study. For each fraction, cellulose digestibility was closely related to lignin degradation, presumably because the pretreated materials were much less resistant to enzymatic hydrolysis due to lignin removal.
Figure 4. Glucose yields of corn stover fractions pretreated with fungi.
Although fungal pretreatment of corn stover fractions did not yield more total glucose than that from the entire plant, variations in degradation and glucose yields indicate that selectively processing the corn stover fractions with fungi could be beneficial. For example, leaves were the least resistant to the fungus, requiring 25 days to reach maximum glucose yield while cobs required 35 days. Thus, the biorefining process could be optimized by pretreating fractions separately, reducing operational costs by reducing treatment time and maximizing sugar yield.
However, while leaves were the least recalcitrant to the fungal pretreatment, they had the lowest total glucose yield; whereas the cobs required longer pretreatment but had the highest glucose yield. If soil sustainability and sugar conversion efficiency are considered, it may be desirable to harvest only cobs and stalks, with the leaves remaining in the field where they can be readily broken down for improved soil organic matter and erosion control. Choosing the best harvesting scenario of corn stover for fungal pretreatment will depend on the goals of the biorefinery and will require thorough economic analysis.
¹This fact sheet is based on a publication by Z. Cui, C. Wan, S. Jian, R. Sykes, and Y. Li titled, "Enzymatic digestibility of corn stover fractions in response to fungal pretreatment" published in Industrial & Engineering Chemistry (2012) 51:7153–7159.