Sequential saccharification of corn fiber and ethanol production by the brown rot fungus Gloeophyllum trabeum

Abstract

Degradation of lignocellulosic biomass to sugars through a purely biological process is a key to sustainable biofuel production. Hydrolysis of the corn wet-milling co-product—corn fiber—to simple sugars by the brown rot fungus Gloeophyllum trabeum was studied in suspended-culture and solid-state fermentations. Suspended-culture experiments were not effective in producing harvestable sugars from the corn fiber. The fungus consumed sugars released by fungal extracellular enzymes. Solid-state fermentation demonstrated up to 40% fiber degradation within 9 days. Enzyme activity assays on solid-state fermentation filtrates confirmed the involvement of starch- and cellulose-degrading enzymes. To reduce fungal consumption of sugars and to accelerate enzyme activity, 2- and 3-d solid-state fermentation biomasses (fiber and fungus) were submerged in buffer and incubated at 37 °C without shaking. This anaerobic incubation converted up to almost 11% of the corn fiber into harvestable reducing sugars. Sugars released by G. trabeum were fermented to a maximum yield of 3.3 g ethanol/100 g fiber. This is the first report, to our knowledge, of G. trabeum fermenting sugar to ethanol. The addition of Saccharomyces cerevisiae as a co-culture led to more rapid fermentation to a maximum yield of 4.0 g ethanol/100 g fiber. The findings demonstrate the potential for this simple fungal process, requiring no pretreatment of the corn fiber, to produce more ethanol by hydrolyzing and fermenting carbohydrates in this lignocellulosic co-product.

Introduction

Sustainable biofuel production from lignocellulosic biomass depends on the development of economical methods of feedstock production, transportation, and processing. Corn wet-milling, used for the production of various food products, including starch, oil, and sweetners, and for approximately 20% of total US ethanol production, produces large quantities of the low-value, lignocellulosic co-product corn fiber (Brown, 2003, RFA, 2007). Corn fiber is an abundant and convenient feedstock, which requires no additional harvesting or transportation since it is already at the corn wet-milling plants. Corn steeping at elevated temperatures with sulfur dioxide provides the required lignocellulosic pretreatment, which makes corn wet-milled fiber ready for use. During the wet-milling operation, the hull of the corn kernel (pericarp) is first separated and washed to recover residual starch, and then dried and combined with other by-products including corn gluten, oil cake, steep liquor, and stillage (Butzen and Hobbs, 2002). Corn fiber contains 17% starch, 35% hemicellulose, 18% cellulose, 11% protein, 3% oil, 6% ash, 5% galactan, 1% mannan, and 4% other materials (Abbas et al., 2004). It is dried, pelletized, and sold as the main component in the product corn gluten feed (approximately 21% protein) for dairy cattle.

Wet mills produced 3 million metric tons of corn gluten feed in 2008 (RFA, 2009). The typical yield is 13 lb corn gluten feed per bushel of corn processed (Brown, 2003), and the demand for corn fiber as an animal feed is not expected to increase significantly. Rapidly expanding ethanol production leads to flooding of the market with alternative ruminant feeds, particularly distillers dried grains. Additional uses for the corn fiber will be required to prevent an oversupply of animal feed. One possible approach for value-adding is to utilize the corn fiber as feedstock for production of more bioethanol. Saccharification of the cellulose and residual starch could liberate simple sugars for subsequent yeast fermentation to ethanol. Hemicellulose is another possible source of sugars for ethanol production, provided the strain of yeast or bacteria used is able to ferment five-carbon sugars to ethanol (Ho et al., 1998).

Various approaches, including concentrated and dilute acid hydrolysis and enzymatic hydrolysis, have been investigated to find an economical method of converting lignocellulose to fermentable sugars (US DOE EERE, 2007). Significant obstacles hinder the commercialization of these processes. Concentrated acid hydrolysis is appealing for the near theoretical yields of sugars released; however, the chemical expense, acid recovery and corrosivity, and gypsum waste impede economical, full-scale development. Although dilute acid hydrolysis requires less acid, it is still not considered economically competitive due to the similar expenses of chemicals, corrosion-resistant equipment, and gypsum waste disposal, and the potential production of fermentation inhibitors at the higher temperatures (Brown, 2003).

Enzymatic hydrolysis is considered the most economically promising approach (US DOE EERE, 2007), although it currently still involves biomass pretreatment and the addition of lignocellulolytic enzymes that are produced separately. The direct bioconversion of cellulose and hemicellulose to simple sugars by microorganisms cultured on the feedstock would be advantageous by avoiding the need to produce and recover enzymes separately on more expensive substrates, provided that sufficient quantities of enzymes are produced in situ. White rot and brown rot fungi have great potential for hydrolyzing lignocellulosic biomass, as they are among the most important agents of decay for woody biomass (Dix and Webster, 1995). Brown rot fungi, such as Gloeophyllum trabeum, rapidly metabolize the hemicellulose and cellulose in wood, leaving behind a modified brown lignin residue (Goodell, 2003). The enzymatic and non-enzymatic mechanisms used by G. trabeum to degrade wood could potentially be employed for the bioconversion of other fibrous substrates, such as corn fiber, to fermentable sugars. Under laboratory conditions, G. trabeum has been shown to produce a β-glucosidase (Herr et al., 1978), a thermophilic xylanase (Ritschkoff et al., 1994), and two endoglucanases (Mansfield et al., 1998). G. trabeum releases a processive endoglucanase during the hydrolysis of crystalline cellulose to cleave the cellulose internally and to liberate oligosaccharides (Cohen et al., 2005). The cellulases and hemicellulases produced by G. trabeum are not inhibited by glucose (Varela et al., 2003).

This research used both suspended-culture and solid-state fermentations of corn fiber by G. trabeum to determine the potential for release and subsequent fermentation of simple sugars to ethanol. Saccharomyces cerevisiae was also added under anaerobic conditions to enhance ethanol production from the fermentable sugars.