Development of a thermophilic coculture for corn fiber conversion to ethanol

Abstract

The fiber in corn kernels, currently unutilized in the corn to ethanol process, represents an opportunity for introduction of cellulose conversion technology. We report here that Clostridium thermocellum can solubilize over 90% of the carbohydrate in autoclaved corn fiber, including its hemicellulose component glucuronoarabinoxylan (GAX). However, Thermoanaerobacterium thermosaccharolyticum or several other described hemicellulose-fermenting thermophilic bacteria can only partially utilize this GAX. We describe the isolation of a previously undescribed organism, Herbinix spp. strain LL1355, from a thermophilic microbiome that can consume 85% of the recalcitrant GAX. We sequence its genome, and based on structural analysis of the GAX, identify six enzymes that hydrolyze GAX linkages. Combinations of up to four enzymes are successfully expressed in T. thermosaccharolyticum. Supplementation with these enzymes allows T. thermosaccharolyticum to consume 78% of the GAX compared to 53% by the parent strain and increases ethanol yield from corn fiber by 24%.

Coculture with Hemicellulose-Fermenting Microbes Reverses Inhibition of Corn Fiber Solubilization by C. Thermocellum at Elevated Solids Loadings

“…Corn fiber hemicellulose released by C. thermocellum is structurally heterogeneous [24], and the sum of total sugars we measured may not reveal the level of the particular molecular species that are inhibitory. In addition, there may be inhibitors other than hemicellulose that accumulate during corn fiber solubilization that are removed by the coculture partners.…”

“…Strains, media and fermentation conditions Fermentations were carried out with C. thermocellum strain LL1043, also known as M1570 [33]; the M1442 strain of T. saccharolyticum [34] and various derivatives of T. thermosaccharolyticum strain LL1244, also known as ATCC 31960 and HG-8. LL1355 was a novel isolate as reported elsewhere [24].…”

“…Destarched corn bran and growth media was prepared as previously described [24]. The fermentations were carried out as described previously [35] in 3L (2-L working volume) Biostat A-plus bioreactors (Sartorius Stedim, Bohemia, NY) with the temperature maintained at 55°C using a resistive heat blanket and stirred at 200 rpm.…”

“…Growth rate measurements were performed in a Powerwave XS plate reader (BioTek Corporation, Winooski, VT) in an anaerobic chamber at 55°C as previously described [44]. The media used for growth tests was CC6 [24] with 5-10 g/L cellobiose and 10 g/L MOPS. A 2% inoculum grown on the same medium was used.…”

“…Corn fiber, the fibrous portion of corn kernels, has almost no lignin and a high hemicellulose to cellulose ratio [23], and is expected to release a substantial amount of hemicellulose oligomers in fermentation, even at moderate solids loading. We recently reported structural characterization of corn fiber hemicellulose released by C. thermocellum [24] . While the glucuronoxylan carbohydrate was recalcitrant to most bacteria we tested, we identified a novel isolate Herbinix spp.…”