Elsevier

Enzyme and Microbial Technology

Volume 39, Issue 5, 4 September 2006, Pages 1137-1144
Enzyme and Microbial Technology

Enzymatic saccharification of hot-water pretreated corn fiber for production of monosaccharides

https://doi.org/10.1016/j.enzmictec.2006.02.022Get rights and content

Abstract

Corn fiber, currently produced at wet milling facilities, is readily available as a potential feedstock for production of fermentable sugars. Destarched corn fiber (DSCF) can be conveniently prepared for enzymatic saccharification by treating with liquid hot-water. Treating DSCF with hot-water (HW-DSCF) at 160 °C for 20 min dissolved 58% of the solids and 75% of the xylan. Preparations of hydrolytic enzymes were next used to saccharify the cellulose and xylan. The needed enzymes were prepared from culture supernatants of Trichoderma reesei Rut C30 and Aspergillus niger NRRL 2001, each grown on HW-DSCF. The harvested cultures were found to have a broad range of carbohydrase activities. The enzyme profiles varied considerably from one another and the preparations were determined to be most effective for saccharifying HW-DSCF when used in combination. Monosaccharide sugar yields obtained using the blended preparations were 74 and 54% of the available arabinose and xylose, respectively. Arabinose and xylose yields were both further increased to 80% by increasing the hot-water pretreatment time to 30 min and adding a commercial preparation of β-glucosidase, which also contained β-xylosidase side-activity.

Introduction

The U.S. annual production of ethanol is 3.4 billion gallons of ethanol (Renewable Fuel Association, 2005), which consumes 1.4 billion bushels of corn (National Corn Growers Association, 2005). Ethanol is manufactured from corn by either the dry grind or wet mill process. Corn fiber is a residue of wet milling that is currently incorporated into corn gluten feed, a low-protein animal feed product. Redirecting this fiber for conversion to ethanol would simultaneously increase the ethanol yield from wet milled corn (by as much as 10%) and increase the value of the corn gluten feed by reducing its fiber content. Enough corn fiber is produced each year in the U.S. to generate an additional 385 million gallons of ethanol per year [1].

Corn fiber contains approximately 70% carbohydrates including cellulose, xylan, and residual starch. The first step for converting corn fiber to ethanol is saccharifying these carbohydrates to fermentable monosaccharide sugars. Most research on corn fiber conversion to ethanol has focused on pretreating corn fiber with dilute sulfuric acid. Typically this involves lowering the pH to 1.0–1.3 with sulfuric acid and treating at a temperature of 150–180 °C for anywhere from 2 to 20 min [2], [3], [4], [5]. The sugar yields from these studies are generally very high [4], [5], [6]. However, from a processing view-point, dilute-acid pretreatment has several shortcomings, including the need for expensive reactors capable of withstanding the combination of low-pH and high-temperature, formation of pretreatment-associated side-products that often stall the subsequent fermentation, and generation of acid-related waste streams (e.g., gypsum) that adds to the cost of waste treatment and complicates downstream processing.

Hot-water pretreatment is an effective alternative to dilute-acid for pretreating herbaceous biomass [7], [8], [9], [10], including corn fiber [11], [12], [13]. The major advantages of hot-water pretreatment compared to dilute-acid are avoiding the use of mineral acid (with its myriad of disadvantages) and reducing sugar degradation products. The technology is also conveniently integrated into a wet milling operation as demonstrated by Ladisch and co-workers [14]. However, corn fiber treated with hot-water requires further processing than that treated with dilute-acid because the former is not severe enough to saccharify xylan sugars, as needed for their fermentation. Fermentation of xylan is critical because it represents approximately 50 wt% of the corn fiber carbohydrates [5]. Recovering corn fiber xylan as monosaccharide sugars is difficult because its xylan is exceedingly complex in structure and resilient to treatment with most commercially available xylanases.

This paper examines the feasibility of using custom hydrolytic enzyme blends for recovering carbohydrates from hot-water treated corn fiber as monosaccharide sugars. Enzymes were prepared by growing Trichoderma reesei Rut C30 and Aspergillus niger NRRL 2001 on hot-water treated corn fiber. The recovered cultures were profiled for carbohydrase activities and evaluated on hot-water treated corn fiber for release of monosaccharide sugars. The effect of adding additional enzyme activities and modifying the pretreatment conditions was also examined for the possibility of increasing final sugar yields.

Section snippets

Materials

Microbial strains were obtained from the ARS Culture Collection (NCAUR, Peoria, IL). Corn fiber was received from Aventine Renewable Fuels (Perkin, IL) and stored at −20 °C. Feruloyl esterase is a recombinant purified enzyme originating from Clostridium thermocellum that has been over expressed in E. coli [15]. A purified xylosidase/arabinosidase, isolated from Selenomonas ruminantium, was expressed in and purified from E. coli [16]. The following commercial enzymes were used: glucoamylase

Pretreating destarched corn fiber with hot-water

The corn fiber used in this study was partially destarched to minimize background glucose originating from the starch; corn fiber contains up to 20% (w/w) residual starch. The corn fiber was destarched by treating with a purified glucoamylase (Megazyme) and repeatedly washing with distilled water. The partially destarched corn fiber (DSCF) was determined to have the following carbohydrate content (per gram DSCF, db): 145 mg arabinose, 286 mg xylose and galactose, and 276 mg glucose of which 76 mg

Discussion

Hot-water treatment of corn fiber is an efficient method for dissolving intact xylan. However, these dissolved carbohydrates still need to be saccharified to monosaccharides before they are fermentable by most ethanol producing microorganisms. This is problematic because commercially available xylanases that have been evaluated are largely ineffective for saccharifying corn fiber (pericarp). For example, Hespell et al. [17] was only able to release 18% of the sugars from ammonia fiber explosion

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