Sequential saccharification and fermentation of corn stover for the production of fuel ethanol using wood-rot fungi, Saccharomyces cerevisiae and Escherichia coli K011
Anthony L. Pometto Iii
World oil consumption for energy and transportation applications has increased tremendously over the past decades as the world population grew, and more countries becoming industrialized. Even domestic products like plastics, chemicals, toiletries, clothes, food packaging, automobile parts and building materials are made from petrochemicals. In the United States, world's number one oil consumer, approximately 70% of crude oil goes to the transportation sector. To supplement these fossil based fuels, several ethanol-gasoline blends are currently in the market, and since 2006, a massive increase in the utilization of ethanol is reported in the United States, and this trend is also observed globally. While the present first generation fuel ethanol are produced mainly from sugary and starchy feedstock, numerous efforts are underway in the research, development and production of second generation bioethanol that are derived from lignocellulosic biomass. The latter platform has not fully matured due to the various process and economic challenges in efficiently producing market friendly ethanol from lignocellulosic biomass. Therefore, it is imperative to develop means of bioprocesses that may reduce cost associated with lignocellulosic ethanol production.
In our study, we aim to develop a sequential biological process that converts cellulosic materials into fermentable sugars and ultimately ethanol as a transportation fuel. We performed solid state fermentation at ambient conditions to induce lignocellulolytic activities from three fungal species, namely Phanerochaete chrysosporium, Gloeophyllum trabeum and Trichoderma reesei. We cultivated each of the fungal species on pure cellulose and corn stover to induce the secretion of cellulases, hemicellulase and lignolytic enzymes via solid state fermentation for several days. Corn stover was chosen as the main material as it is one of the most abundant agricultural residues. The mold mediated processes liberate simple carbohydrates, suitable substrates for downstream microbial utilization. Next, we performed simultaneous saccharification and fermentation (SSF) of the cellulosic materials to produce more sugars that are converted to ethanol.
Prior to the SSF studies on the corn stover, we initially performed enzymatic studies of these fungal species on pure cellulose to evaluate their in situ enzyme production and hydrolytic abilities. Filter paper was used in the screening in accordance to the recommendations of several previously reported studies. The efficiency of the fungal species in saccharifying the filter was compared against a low dose (25 FPU/g cellulose) of a commercial cellulase. Fermentation was achieved by using the yeast Saccharomyces cerevisiae. Total sugar, cellobiose and glucose concentrations were monitored during the fermentation period, along with three main fermentation products, namely ethanol, acetic acid and lactic acid. Results indicated that the most efficient fungal species in saccharifying the filter paper was T. reesei with 5.13 g/100 g filter paper of ethanol being produced at days 5, followed by P. chrysosporium at 1.79 g/100 g filter paper. No ethanol was produced from the filter paper treated with G. trabeum throughout the five day fermentation stage. Acetic acid was only produced in the sample treated with T. reesei and the commercial enzyme, with concentration 0.95 g and 2.57 g/100 g filter paper, respectively at day 5.
Next, we performed enzymatic saccharification of corn stover using P. chrysosporium and G. trabeum. Subsequent fermentation of the saccharification products to ethanol was achieved via the use of Saccharomyces cerevisiae and Escherichia coli K011. During the SSF period with S. cerevisiae or E. coli, ethanol production was highest on day 4 for all samples inoculated with either P. chrysosporium or G. trabeum. For the corn stover treated with P. chrysosporium, the conversion of corn stover to ethanol was 2.29 g/100 g corn stover for the sample inoculated with S. cerevisiae, whereas for the sample inoculated with E. coli K011, the ethanol concentration was 4.14 g/100 g corn stover. While for the corn stover treated with G. trabeum, the conversion of corn stover to ethanol was 1.90 g and 4.79 g/100 g corn stover for the sample inoculated with S. cerevisiae and E. coli K011, respectively. Other fermentation co-products, such as, acetic acid and lactic acid were also recorded. Acetic acid production ranged between 0.45 g and 0.78 g/100 g corn stover for the samples under different fungal treatments, while no lactic acid production was detected throughout the 5 days of SSF.
In the later stages of our study, we further explore the coupling of mild chemical (dilute NaOH) and biological pretreatment and saccharification on the corn stover. Ethanol production was achieved via the sequential saccharification and fermentation of dilute sodium hydroxide (2% w/w NaOH in corn stover) treated corn stover using P. chrysosporium and G. trabeum. Ethanol production peaked on day 3 and day 4 for the samples inoculated with either P. chrysosporium or G. trabeum, slightly plateauing or decreasing thereafter. Ethanol production was highest for the combination of G. trabeum and E. coli K011 at 6.68 g/100 g corn stover, followed by the combination of P. chrysosporium and E. coli K011 at 5.00 g/100 g corn stover. Combination of both the fungi with S. cerevisiae generally had lower ethanol yields, ranging between 2.88 g (P. chrysosporium treated corn stover) and 3.09 g/100 g corn stover (G. trabeum treated corn stover). Acetic acid production ranged between 0.53 g and 2.03 g/100 g corn stover for the samples under different fungal treatments, while lactic acid production was only detected in samples treated with G. trabeum, throughout the 5 days of SSF.
The results of our study indicated that mild alkaline pretreatment coupled with fungal saccharification offer a promising bioprocess for ethanol production from corn stover without the addition of commercial enzymes. We believe these sequential procedures are potentially applicable to various other lignocellulosic materials (i.e. switchgrass, poplar, corn cobs) and may assist in environmentally, economical and technological friendlier ethanol production processes.