Biological abatement of inhibitors in rice hull hydrolyzate and fermentation to ethanol using conventional and engineered microbes Nancy N. Nichols*, Ronald E. Hector, Badal C. Saha, Sarah E. Frazer, Gregory J. Kennedy Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 N. University St., Peoria, IL 61604, USA 1 article info Article history: Received 12 September 2013 Received in revised form 25 March 2014 Accepted 18 April 2014 Available online Keywords: Rice hulls Fermentation Inhibitors Bioabatement Hemicellulose abstract Microbial inhibitors arise from lignin, hemicellulose, and degraded sugar during pretreat- ment of lignocellulosic biomass. The fungus Coniochaeta ligniaria NRRL30616 has native ability to metabolize a number of these compounds, including furan and aromatic alde- hydes known to act as inhibitors toward relevant fermenting microbes. In this study, C. ligniaria was used to metabolize and remove inhibitory compounds from pretreated rice hulls, which comprise a readily available agricultural residue rich in glucose (0.32e0.33 g glucan/g hulls) and xylose (0.15e0.19 g xylan/g hulls). Samples were dilute-acid pretreated and subjected to bioabatement of inhibitors by C. ligniaria. The bioabated rice hull hemi- cellulose hydrolyzates were then utilized for ethanol fermentations. In bioabated liquors, glucose was converted to 0.58% (w/v) ethanol by Saccharomyces cerevisiae D5a at 100% of theoretical yield, while fermentations of unabated hydrolyzates either failed to exit lag phase or had reduced ethanol yield (80% of theoretical). In fermentations using ethanol- ogens engineered for conversion of pentoses, bioabatement of hydrolyzates similarly improved fermentations. Fermentation of xylose and arabinose by ethanologenic Escher- ichia coli FBR5 yielded 2.25% and 0.05% (w/v) ethanol from bioabated and unabated samples, respectively. Fermentations using S. cerevisiae YRH400 had decreased fermentation lag times in bioabated hydrolyzates. However, xylose metabolism in S. cerevisiae YRH400 was strongly affected by pH and acetate concentration. Published by Elsevier Ltd. 1. Introduction Production of rice, the third most abundant grain crop in the world behind wheat and corn (http://www.irri.org), generates quantities of rice hulls as a by-product. Global production of rice hulls is estimated at 139 million tonnes annually [1]. The hulls, which are harvested with the grain and equal 20% of harvested rice on a dry weight basis, are traditionally considered a low-value or waste residue due to high lignin * Corresponding author. Tel.: þ1 309 681 6271. E-mail address: [email protected](N.N. Nichols). 1 Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. Available online at www.sciencedirect.com ScienceDirect http://www.elsevier.com/locate/biombioe biomass and bioenergy 67 (2014) 79 e88 http://dx.doi.org/10.1016/j.biombioe.2014.04.026 0961-9534/Published by Elsevier Ltd.
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b i om a s s a n d b i o e n e r g y 6 7 ( 2 0 1 4 ) 7 9e8 8
Available online at w
ScienceDirect
http: / /www.elsevier .com/locate/biombioe
Biological abatement of inhibitors in rice hullhydrolyzate and fermentation to ethanol usingconventional and engineered microbes
Nancy N. Nichols*, Ronald E. Hector, Badal C. Saha, Sarah E. Frazer,Gregory J. Kennedy
Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S.
Department of Agriculture, 1815 N. University St., Peoria, IL 61604, USA1
1 Mention of trade names or commercial pnot imply recommendation or endorsemeemployer.http://dx.doi.org/10.1016/j.biombioe.2014.04.0961-9534/Published by Elsevier Ltd.
a b s t r a c t
Microbial inhibitors arise from lignin, hemicellulose, and degraded sugar during pretreat-
ment of lignocellulosic biomass. The fungus Coniochaeta ligniaria NRRL30616 has native
ability to metabolize a number of these compounds, including furan and aromatic alde-
hydes known to act as inhibitors toward relevant fermenting microbes. In this study, C.
ligniaria was used to metabolize and remove inhibitory compounds from pretreated rice
hulls, which comprise a readily available agricultural residue rich in glucose (0.32e0.33 g
glucan/g hulls) and xylose (0.15e0.19 g xylan/g hulls). Samples were dilute-acid pretreated
and subjected to bioabatement of inhibitors by C. ligniaria. The bioabated rice hull hemi-
cellulose hydrolyzates were then utilized for ethanol fermentations. In bioabated liquors,
glucose was converted to 0.58% (w/v) ethanol by Saccharomyces cerevisiae D5a at 100% of
theoretical yield, while fermentations of unabated hydrolyzates either failed to exit lag
phase or had reduced ethanol yield (80% of theoretical). In fermentations using ethanol-
ogens engineered for conversion of pentoses, bioabatement of hydrolyzates similarly
improved fermentations. Fermentation of xylose and arabinose by ethanologenic Escher-
ichia coli FBR5 yielded 2.25% and 0.05% (w/v) ethanol from bioabated and unabated samples,
respectively. Fermentations using S. cerevisiae YRH400 had decreased fermentation lag
times in bioabated hydrolyzates. However, xylose metabolism in S. cerevisiae YRH400 was
strongly affected by pH and acetate concentration.
Published by Elsevier Ltd.
1. Introduction
Production of rice, the third most abundant grain crop in the
world behind wheat and corn (http://www.irri.org), generates
1.gov (N.N. Nichols).roducts in this article is
nt by the U.S. Departme
026
quantities of rice hulls as a by-product. Global production of
rice hulls is estimated at 139 million tonnes annually [1]. The
hulls, which are harvested with the grain and equal 20% of
harvested rice on a dry weight basis, are traditionally
considered a low-value or waste residue due to high lignin
solely for the purpose of providing specific information and doesnt of Agriculture. USDA is an equal opportunity provider and
a 72 h.b Fermentation lag times were calculated as the time to reach 5% of maximum psi in fermentation bottles.c g ethanol produced/g glucose consumed.d Fermentation lag times could be calculated for only the three fermentations (of seven total) that exited lag phase.
Table 4 e Fermentation of bioabated and unabated RHH with S. cerevisiae YRH400.
Bio-abatement pHcontrol
Lag time(h)a
Sugar consumed during fermentation Sugar remaining at the end of fermentation Final xylitol(% w/v)
a Fermentation lag times were calculated as the time to reach 5% of maximum psi in fermentation bottles.b g ethanol produced/g glucose þ xylose consumed.c Fermentations were initiated at pH 4.5, without pH control during fermentations, and monitored for 72 h.d pH was controlled at 5.5 throughout the fermentations, and sampled after 165 h.
hibitor concentrations) when using bioabatement and
YRH400.
5. Conclusion
This work demonstrates that biological abatement facili-
tates fermentation of sugars obtained from dilute acid pre-
treatment of rice hulls. A specially selected microbe, C.
ligniaria NRRL30616, was used to metabolize undesirable
compounds which, if left untreated, can complicate micro-
bial conversion of sugars to the desired end product. In
ethanol fermentations, conversion of glucose in bioabated
hemicellulose hydrolyzates proceeded to completion, while
fermentations in unabated samples either stalled or failed to
completely utilize glucose. Increased utilization of xylose by
ethanologenic E. coli FBR5 and the xylose-utilizing yeast S.
cerevisiae YRH400 was also observed. Bioabatement was
associated with enhanced ethanol production in fermenta-
tions of dilute acid-pretreated rice hulls and may facilitate
utilization of this feedstock, which presently has little
commercial use.
Acknowledgements
The authors thank Patricia O’Bryan for excellent technical
assistance and Rice Hull Specialty Products (Stuttgart, AR) for
the gift of ground rice hulls.
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