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Simultaneous Saccharification and Fermentation of Ground Corn Stover for

the Production of Fuel Ethanol Using Phanerochaete chrysosporium,
Gloeophyllum trabeum, Saccharomyces cerev...

Article in Journal of Microbiology and Biotechnology · July 2011

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J. Microbiol. Biotechnol. (2011), 21(7), 703–710
doi: 10.4014/jmb.1010.10044
First published online 11 June 2011

Simultaneous Saccharification and Fermentation of Ground Corn Stover

for the Production of Fuel Ethanol Using Phanerochaete chrysosporium,
Gloeophyllum trabeum, Saccharomyces cerevisiae, and Escherichia coli K011
Vincent, Micky1,2,3, Anthony L. Pometto III4, and J. (Hans) van Leeuwen2,3,5,6*
1
Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan,
Sarawak, Malaysia
2
Department of Civil, Construction, and Environmental Engineering, Iowa State University, Ames, IA 50011, USA
3
Biorenewable Resources and Technology Program, Iowa State University, Ames, IA 50011, USA
4
Department of Food Science and Human Nutrition, Clemson University, Clemson, SC 29634, USA
5
Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA 50011, USA
6
Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA
Received: October 21, 2010 / Revised: April 11, 2011 / Accepted: May 7, 2011

Enzymatic saccharification of corn stover using Phanerochaete The ethanol presently used for transportation purposes is
chrysosporium and Gloeophyllum trabeum and subsequent conventionally produced in large quantities from corn
fermentation of the saccharification products to ethanol grain and sugarcane juice. However, this practice is only a
by Saccharomyces cerevisiae and Escherichia coli K011 temporary solution as it conflicts with the food and feed
were achieved. Prior to simultaneous saccharification and industry [7]. Thus, there is great interest in the development
fermentation (SSF) for ethanol production, solid-state of fuel ethanol from agricultural residues and other
fermentation was performed for four days on ground lignocellulosic feedstocks, which are inexpensive and are
corn stover using either P. chrysosporium or G. trabeum to the most abundant bioresources available in the biosphere
induce in situ cellulase production. During SSF with S. [11]. Currently, corn stover biomass is considered to be
cerevisiae or E. coli, ethanol production was the highest on one of the primary lignocellulosic candidates for use in
day 4 for all samples. For corn stover treated with P. cellulosic bioethanol production because it is an abundant
chrysosporium, the conversion to ethanol was 2.29 g/100 g agricultural by-product in many European countries and in
corn stover with S. cerevisiae as the fermenting organism, the USA, and it can be collected during harvest [46, 51].
whereas for the sample inoculated with E. coli K011, the Although promising, the use of corn stover as a raw material
ethanol production was 4.14 g/100 g corn stover. Corn to produce ethanol presents many challenges; unlike starch
stover treated with G. trabeum showed a conversion 1.90 from corn, the polysaccharides in stovers are cellulose and
and 4.79 g/100 g corn stover with S. cerevisiae and E. coli hemicellulose, which are difficult to degrade [20, 24, 33].
K011 as the fermenting organisms, respectively. Other Thus, hydrolyzing these components into fermentable sugars
fermentation co-products, such as acetic acid and lactic
is essential to the efficient and economical production of
cellulosic ethanol [5].
acid, were also monitored. Acetic acid production ranged
Biohydrolysis of cellulose and hemicellulose is an
between 0.45 and 0.78 g/100 g corn stover, while no lactic
enzymatic process carried out by a family of cellulolytic
acid production was detected throughout the 5 days of
and hemicellulolytic enzymes that are highly specific [24].
SSF. The results of our experiment suggest that it is
These enzyme consortia are usually a mixture of several
possible to perform SSF of corn stover using P. chrysosporium,
enzymes that may include endoglucanases, exoglucanases or
G. trabeum, S. cerevisiae and E. coli K011 for the production cellobiohydrolases, glucosidases or cellobiases, endoxylanases,
of fuel ethanol. xylosidases and galactosidases, among others [1, 31, 50,
Keywords: Phanerochaete chrysosporium, Gloeophyllum 54]. The conventional method for the breakdown of
trabeum, Saccharomyces cerevisiae, Escherichia coli K011, lignocellulosics to fermentable sugars requires the use of
solid subtrate fermentation, simultaneous saccharification and expensive commercial enzymes [12, 26, 53]. However, these
fermentation (SSF) enzymes are not only substrate specific, they are largely
susceptible to inhibition from compounds usually associated
*Corresponding author
Phone: +515 294-5251; Fax: +515 294-8216; with lignin. Thus, prior to enzymatic hydrolysis, pretreatment
E-mail: [email protected] of ground lignocelluloses is required [21].
704 Vincent et al.

Pretreatment of plant biomass is crucial for the production following the method of Crawford and Pometto [9] with slight
of cellulosic ethanol as it greatly improves the enzymatic modification, whereby glass fiber filters (1.6 µm) (Fisherbrand, Fisher
accessibility of the feedstock [13, 19, 25, 41, 42]. In recent Scientific, Pittsburgh, PA, USA) were used instead of Whatman
years, several pretreatment methods have been tested on No.1 filter papers for capturing lignin residues. This assay measures
lignin as the acid-insoluble fraction of lignocellulosic material after
corn stover that involve physical, chemical, or physicochemical
hydrolysis by strong acid (H SO ) and heat. The residue on the filter
procedures or a combination thereof [16, 47, 56]. However, 2 4

paper was thoroughly rinsed with deionized water and dried in an

these technologies are energy intensive, environmentally o
oven at 105 C for 4 days. The Klason lignin content was determined
unfriendly, and may produce many toxic by-products such as the weight of dry residue collected on the filter paper.
as weak acids, phenolic derivatives, and furans that inhibit
alcoholic fermentation [6, 7, 21]. Therefore, it is imperative Microorganisms
to develop alternative means of lignocellulosic saccharification All of the cultures used in this study were obtained from the American
that can overcome these obstacles. Type Culture Collection (ATCC; Rockville, MD, USA) and included
One potential form of pretreatment and hydrolysis of P. chrysosporium (ATCC 24725), G. trabeum (ATCC 11539), S.
lignocellulosic materials relies on biological means [15, cerevisiae (ATCC 24859), and E. coli K011 (ATCC 55124). Fungal
49]. This type of procedure usually involves lignocellulolytic cultures were revived by inoculating them into potato dextrose broth
(PDB) (Difco, Becton Dickinson and Co., Sparks, MD, USA) and
fungal species such as Phanerochaete chrysosporium and
the bacterial culture by inoculating into LB broth (Becton Dickinson),
Gloeophyllum trabeum [38, 40, 43-45]. P. chrysosporium o
followed by incubation with shaking at 24 C [43, 44]. Stock cultures
is a white-rot fungus that has been studied extensively in were stored in yeast malt extract (YM) broth (Becton Dickinson)
the degradation of plant cell wall components including supplemented with 20% (v/v) glycerol at -80 C in an ultralow o

cellulose, hemicellulose, and lignin [23, 54]. P. chrysosporium temperature freezer (So-Low Environmental Equipment Co., Inc.,
performs lignocellulolytic processes using the various Cincinnati, OH, USA) for long-term storage.
ligninolytic peroxidases, cellulases, and hemicellulases it
is known to secrete [30, 50, 54]. G. trabeum is a brown-rot P. chrysosporium and G. trabeum Culture Preparation
basidiomycete. Like a typical brown rot-fungus, G. trabeum P. chrysosporium and G. trabeum seed cultures were prepared from
o

primarily attacks the polysaccharide while leaving the spores in 1 l of YM broth and incubated at 30 C with agitation at
brown pigmented lignin behind [8]. These degradative 150 rpm. After 7 days of growth, fungal mycelia (approximately 2-
3 mm in diameter) were harvested via centrifugation in a sterilized
processes culminate in the rapid loss of wood strength and
1 l polypropylene centrifuge bottle (Nalgene, Nalge Nunc, Rochester,
darkening of the affected substrate [10]. G. trabeum is NY, USA), at 7,277 ×g for 20 min using a Sorvall-RC3B Plus
known to secrete a family of potent cellulolytic enzymes centrifuge (Thermo Fisher Scientific, Wilmington, DE, USA) [38].
consisting of endoglucanases, exoglucanases, beta-glucosidases, The fungal pellets were rinsed with fungal mineral salt solution (pH
and other hemicellulases [8, 22]. In contrast to white-rot 4.5-4.8; 50 mM phosphate buffer + 0.5% (NH ) SO + basal medium).
4 2 4

fungi, G. trabeum rapidly degrades cellulose and hemicellulose Basal medium was prepared according to the formulation of Shrestha
while leaving the undigested lignin to be modified mainly et al. [44], consisting of 0.25 g of KH PO (Fisher Scientific, Pittsburgh,
2 4

through demethoxylation and demethylation mechanisms [4]. PA, USA), 0.063 g of MgSO ·7H O (Fisher Scientific), 0.013 g of
4 2

In this paper, we report the use of in situ cellulases and CaCl ·2H O (Fisher Scientific), and 1.25 ml of trace element solutions
2 2

hemicellulases from P. chrysosporium and G. trabeum in 1 l of deionized water [43].

for the saccharification of corn stover cellulose that is
Solid Substrate Fermentation for Enzyme Induction
subsequently fermented to ethanol by Saccharomyces
All ground corn stover used in this study received no pretreatment
cerevisiae and Escherichia coli K011. We performed our except any weathering that might have occurred in the field prior
work under conditions and with equipment that would to harvest. Prior to the addition of fungal inoculum for enzyme
generate commercially relevant results. induction, 2 g of ground stover and glass marbles with 5 ml of
fungal mineral salt solution were sterilized in 250 ml polypropylene
o
bottles (Nalgene) at 121 C for 1 h followed by rapid exhaust. Two ml
MATERIALS AND METHODS of fungal biomass [1.5% (w/v) P. chrysosporium and 1.0% (w/v) G.
trabeum] in mineral salt solution was then added. The bottles were
Corn Stover Analysis rolled on their sides and the marbles assisted in uniformly dispersing
Corn stover was obtained from the Department of Agronomy, Iowa and coating the corn stover and fungi mixture along the inner
State University, USA. Field-dried corn leaf and corn stalk were ground surface [38, 44]. Solid substrate fermentation was then performed
in a Wiley mill to pass through a 2 mm screen, and then screened using for 4 days at 37 C in a humidified incubator for in situ production
o

o
a 20 mesh sieve and further dried in an oven at 80 C for 4 days of cellulases and hemicellulases prior to the addition of the ethanolic
prior to compositional analysis. The composition of cellulose and microorganism.
hemicellulose was determined by the Department of Agronomy, Iowa
State University, using the ANKOM method (ANKOM Technology Protein Assay
Corp., Fairport, NY, USA) as previously described [52]. The Klason Total protein was analyzed using a NanoDrop 1000 Spectrophotometer
lignin content was determined using a modified Klason lignin assay (Thermo Fisher Scientific). The NanoDrop 1000 module measures
 SIMULTANEOUS SACCHARIFICATION AND FERMENTATION OF CORN STOVER 705

protein absorbance at 280 nm (A280) and calculates the concentration Nelson carbohydrate assay was performed at 500 nm with a glucose
(mg/ml) from a 2 µl sample. Sample aliquots of 1.5 ml were taken standard, whereas total sugars were determined via the phenol-sulfuric
from stover treated with fungal cultures and washed with minimal carbohydrate test at 490 nm with a glucose standard. Absorbance
salt medium on day 4. The supernatant was centrifuged using a was read on a SpectraMax Plus384 spectrophotometer (Molecular
MiniSpin Plus centrifuge (Eppendorf, Hauppauge, NY, USA) at Devices, Inc., Sunnyvale, CA, USA). The absorbance readings were
1,118 ×g for 5 min and filtered through a 0.2 µm nylon syringe filter then converted into equivalent sugar concentrations (g/l) based on a
(VWR International, Batavia, IL, USA). Portions of the filtered standard glucose solution curve. All sugar analyses were performed
solution were also used to perform the enzyme activity assay. in triplicate (n=3).

Enzyme Activity Assay Statistical Analyses

A specific enzyme activity assay was performed using the protocol SSF results were statistically analyzed using JMP 8.0 statistical software
described by the official National Renewable Energy Laboratory (SAS Institute, Inc., Cary, NC, USA). The data on ethanol production
(NREL) procedure [2]. This method is based on the International were fitted to exponential fit models, and a significant difference of
Union of Pure and Applied Chemistry (IUPAC) guidelines to determine p value of 0.05 was employed. Student’s t test analyses were also
cellulase activity in terms of “filter-paper units” (FPU) per milliliter performed on all final data sets to determine multiple comparisons
(FPU/mL) of an original enzyme preparation [18]. of ethanol production. A p-value of less than 0.05 was considered
significantly different.
S. cerevisiae and E. coli K011 Culture Preparation
Culture inocula of S. cerevisiae and E. coli K011 were prepared by
o
growing cultures in 50 ml of sterile YM broth at 32 C with constant RESULTS AND DISCUSSION
agitation at 120 rpm. Cells were harvested via centrifugation in 50 ml
conical centrifuge tubes (BD Falcon, BD, Franklin Lakes, NJ, USA)
Enzyme Induction on Untreated Corn Stover
at 2,852 ×g for 10 min in a Beckman J2-21centrifuge (Beckman
Coulter, Inc., Brea, CA, USA). Prior to use in SSF, cell counts were
In this study, we performed SSF on ground corn stover
set at 10 -10 CFU/ml as determined turbidometrically at 600 nm
7 8 without pretreatment. The main components of the corn
via a standard curve [33]. stover were hemicellulose, cellulose, lignin, and ash (Table 1).
Interestingly, the compositional analysis revealed a high
Simultaneous Saccharification and Fermentation (SSF) ash content. This observation is in agreement with that of a
SSF reactions were carried out in 250 ml polypropylene bottles with previous analysis [36, 48] in which the ash content of corn
batch cultures of 100 ml final volume, consisting of 25 ml of 4× yeast leaf and corn stalk were found to be considerably higher
extract broth [1.8 g of yeast extract (Difco), 0.07 g of CaCl ·2H O 2 2 than that of other biomass. A flow chart of our experimental
(Fisher Scientific), 0.45 g of KH PO (Fisher Scientific), 1.2 g of
2 4 design is shown in Fig. 1. Unlike the SSF process in
(NH ) SO (Fisher Scientific), and 0.3 g of MgSO ·7H O per liter
4 2 4 4 2
previous works, ours does not use pretreated corn stover
(Fisher Scientific)] [44] and 66 ml of basal medium (pH 4.5-4.8;
samples [13, 19, 25, 42] or the addition of expensive
50 mM phosphate buffer + 0.5% (NH ) SO + basal medium). The
4 2 4

bottles were then aseptically inoculated with 10 -10 CFU/ml S.

7 8 commercial enzymes [12, 26, 53]. Instead, cellulases and
cerevisiae and E. coli K011 suspensions. Batch culture fermentations hemicellulases are produced by G. trabeum and P.
o
were incubated at 37 C under static conditions. These samples were chrysosporium in situ upon corn stover enzyme induction
then subjected to SSF under anaerobic conditions for 5 days. The performed via solid substrate fermentation in a pH range of
SSF experiments were performed in triplicate (n=3). 4.5-4.8 at 37oC for 4 days, conditions that are suitable not
only for the growth of the fungi but also for production of
High-Performance Liquid Chromatography (HPLC) Analyses cellulolytic enzymes [38, 43, 44]. As seen in Table 2, our
Sample aliquots of 1.8 ml were taken daily, centrifuged at 1,118 ×g assay using the NanoDrop 1000 spectrophotometer indicated
for 5 min, and filtered through a 0.2 µm nylon syringe filter. Glucose, that protein was produced during the induction stage, and
xylose, and the fermentation products (ethanol, acetic acid, and production was higher in the stover and P. chrysosporium
lactic acid) were analyzed using a Waters High Performance Liquid
combination compared with the stover and G. trabeum
Chromatograph (Millipore Corp., Milford, MA, USA) equipped with a
Waters Model 401 refractive index detector, column heater,
combination, at 14.06 and 11.61 mg/ml, respectively.
autosampler, and computer controller. The separation and analysis of
ethanol and other fermentation constituents were done on a Bio-Rad Table 1. Composition of corn stover (as percentage based on dry
Aminex HPX-8711 column (300.0 × 7.8 mm; Bio-Rad Chemical weight; n=3).
Division, Richmond, CA, USA) using 0.012 N H SO as the mobile
2 4 Composition based on cell mass
phase at a flow rate of 0.6 ml/min, a 20 µl injection volume, and a Main component
(%, w/w)
o
column temperature of 65 C [28, 37].
Cellulose 38.08
Total and Reducing Sugar Assays Hemicellulose 30.72
Filtered supernatants from the fermentation broth were tested for Klason lignin 20.70
free reducing sugar and total reducing sugars via the Somogyi-Nelson Ash 8.77
[3] and phenol-sulfuric [9] methods, respectively. The Somogyi- Others 0.31
706 Vincent et al.

Fig. 2. Time course of reducing sugar production, as determined

by the Somogyi-Nelson method.
Data points represent the average of three independent experiments (n=3).
PC, P. chrysosporium; GT, G. trabeum. Time zero is after 4 days of solid
substrate fermentation with a specific fungus (P. chrysosporium or G.
trabeum).

In Situ Enzymatic Hydrolysis

The efficiency of cellulolytic enzyme hydrolysis of
lignocelluloses was evaluated and validated via assays for
saccharification and fermentation products. Saccharification
of the stover to its free reducing and total sugars was
measured using the Somogyi-Nelson and phenol-sulfuric
methods. After the 4-day enzyme induction period (day 0 of
Fig. 1. Flow chart outlining the steps of solid substrate fermentation SSF), between 2.42 and 2.91 g of reducing sugar per 100 g
by G. trabeum and P. chrysosporium of corn stover without
pretreatment, followed by SSF using S. cerevisiae and E. coli of stover was detected in the broth of the G. trabeum-
K011. treated stover and 0.23-0.29 g in the broth of the P.
chrysosporium-treated stover. Although there was a significant
difference in the amount of reducing sugar, the result was
Next, quantitative enzyme activity was determined quite different for total sugars. The total sugar profile was
according to the IUPAC protocol [18], which interprets the similar for the two treatments on day 0 of SSF and ranged
filter paper unit activity (FPase) based on a value of 2.0 mg from 5.57 to 5.94 g of sugar per 100 g of stover. Both of
of reducing sugar as glucose from 50 mg of filter paper, at these assays support the ability of both fungal strains to
4% conversion, in 1 h (units FPU/ml) [2]. Enzyme assays perform in situ saccharification, and these trends were
to determine the FPase showed that more cellulase was observed throughout the 5-day SSF period (Fig. 2 and 3),
being secreted by the brown-rot fungus, at 1.72 FPU/ml, especially for total sugars.
compared with the white-rot fungus, at 0.65 FPU/ml. This To supplement the carbohydrate assay, the presence of
concentration, however, is not correlated with the amount glucose and xylose were also investigated using HPLC, as
of total protein being produced extracellularly, as mentioned these sugars are the main monomeric end products from
earlier. According to the literature, the white-rot fungus P. cellulosic and hemicellusic polymers [20, 27]. During the
chrysosporium produces additional extracellular enzymes anaerobic fermentation period, no glucose was detected;
including laccases and peroxidases when grown in lignin- this result is a good indication that efficient conversion to
impregnated biomass such as corn stover and other ethanol was achieved. Xylose was detected in all fungi-
lignocellulosic material [50, 54]. treated samples that were inoculated with S. cerevisae, as

Table 2. Enzyme activity and total protein (n=3).

Corn stover + P. chrysosporium Corn stover + G. trabeum
Enzyme assay (FPU/ml)a 0.65 1.72
Protein assay (mg/ml)b 14.06 11.61
a
Filter paper unit activities (FPase) based on a value of 2.0 mg of reducing sugar as glucose from 50 mg of filter paper, at 4% conversion, in 1 h (units FPU/ml).
b
Protein was determined using a NanoDrop 1000 Spectrophotometer.
 SIMULTANEOUS SACCHARIFICATION AND FERMENTATION OF CORN STOVER 707

Fig. 3. Time course of total sugar production, as determined by Fig. 5. Time course of ethanol production.
the phenol-sulfuric method. Data points represent the average of three independent experiments (n=3).
Data points represent the average of three independent experiments (n=3). Note: PC - P. chrysosporium, GT - G. trabeum. Time zero is after 4 days
PC, P. chrysosporium; GT, G. trabeum. Time zero is after 4 days of solid of solid substrate fermentation with a specific fungus (P. chrysosporium or
substrate fermentation with a specific fungus (P. chrysosporium or G. G. trabeum).
trabeum).

shown in Fig. 4. This observation was expected since S. during saccharification were readily converted to ethanol.
cerevisae cannot utilize pentoses such as xylose [14, 29]. Ethanol production was highest on day 4 in all samples
inoculated with either P. chrysosporium or G. trabeum. For
Simultaneous Saccharification and Fermentation of stover treated with P. chrysosporium, the conversion to ethanol
Corn Stover was 2.29 g/100 g of stover for the sample inoculated with
The fermentability of the saccharification products was S. cerevisiae, whereas for the sample inoculated with E.
evaluated using S. cerevisiae and E. coli K011 as the coli K011, the ethanol concentration was 4.14 g/100 g
fermenting organisms. Both of these microorganisms were stover. For stover treated with G. trabeum, the conversion
chosen because they are efficient ethanolic fermenters, to ethanol was 1.90 and 4.79 g/100 g stover for the samples
with the former capable of fermenting glucose from the inoculated with S. cerevisiae and E. coli K011, respectively.
breakdown of cellulose, and the latter capable of fermenting In general, fungi-treated samples inoculated with E. coli
both glucose and other fermentable sugars such as xylose, K011 had a greater ethanol yield. This result is due to the
arabinose, and galactose from the enzymatic hydrolysis of ability of E. coli K011 to ferment both hexoses (C6 sugars,
hemicelluloses [29]. i.e., glucose) and pentoses (C5 sugars, i.e., xylose) [29].
From the graph in Fig. 5, it can be seen that ethanol The results shown in Fig. 4 further support this observation;
production started on day 1 and increased steadily in all stover that was not inoculated with E. coli K011 still
corn stover samples, indicating that the sugars released contained xylose even after day 5 of SSF.

Fig. 4. Time course of xylose production. Fig. 6. Time course of acetic acid production.
Data points represent the average of three independent experiments (n=3). Data points represent the average of three independent experiments (n=3).
PC, P. chrysosporium; GT, G. trabeum. Time zero is after 4 days of solid PC, P. chrysosporium; GT, G. trabeum. Time zero is after 4 days of solid
substrate fermentation with a specific fungus (P. chrysosporium or G. substrate fermentation with a specific fungus (P. chrysosporium or G.
trabeum). trabeum).
708 Vincent et al.

Table 3. Summary of nonlinear (exponential) fit models of ethanol production (n=3).

R2 F-value
Corn stover + E. coli K011 0.911 0.0265
Corn stover + P. chrysosporium + S. cerevisiae 0.925 0.0022
Corn stover + P. chrysosporium + E. coli K011 0.839 0.0103
Corn stover + G. trabeum + S. cerevisiae 0.893 0.0044
Corn stover + G. trabeum + E. coli K011 0.937 0.0015

One interesting observation regarding the ethanol profile treatments, whereas no lactic acid production was detected
is the production of a trace amount of ethanol (1.45 g/ throughout the 5 days of SSF.
100 g stover on day 4) in the sample inoculated only with Statistical analysis via nonlinear regression using exponential
E. coli K011. This, however, is not a new finding, as E. model fits, as summarized in Table 3, strongly endorses the
coli has been documented to secrete cellulases and several reliability of the ethanol production, with all p-values being
hemicellulases, such as xylanases, mannosidase, and galactases, < 0.05. Further analysis performed using the Student’s t
that may have liberated xylose from hemicellulose polymers test showed statistically significant ethanol yield (Fig. 7)
[17, 34, 35]. On day 5, no ethanol was detected in the among the different treatments. This result reinforces the
broth. One possible explanation is that E. coli consumed interrelationship between fungal species treatments and the
the minute amount of ethanol present earlier in the broth. fermenting organisms used.
This is a normal observation when an alternative carbon To realize large-scale applications for cellulosic feedstocks
source is needed for E. coli growth [39]. such as corn stovers, low conversion costs are essential.
Throughout the experiment, other fermentation co-products The use of commercial enzymes makes the production of
such as acetic acid and lactic acid were also recorded (Fig. 6). fuel ethanol neither economically feasible nor profitable.
Acetic acid production ranged between 0.45 and 0.78 g/ Furthermore, these enzymes are highly susceptible to inhibition
100 g stover in the samples subjected to different fungal and are very substrate specific. An ideal lignocellulolytic
biocatalyst should degrade the three main components of
stovers; namely, cellulose, hemicellulose, and lignin. Thus,
using P. chrysosporium and G. trabeum to provide in situ
enzymes for the degradation of the lignocellulosic components
of stovers offers a promising solution. In the production of
fuel ethanol from corn stovers, the optimization of this process
can lead to reduced costs, as ethanol plants can produce their
own enzymes to supplement the use of commercial enzymes.
Another advantage in using this process is that it is an
environmentally friendlier approach that eliminates the
need to perform potentially detrimental pretreatments.

Acknowledgments
This work was financially supported by Universiti Malaysia
Sarawak (UNIMAS) and the Malaysian Ministry of Higher
Education. We thank Patrick Murphy and Dr. Kenneth
Moore (Department of Agronomy, Iowa State University,
Ames, IA, USA) for help with the corn stover compositional
analysis and Carol Ziel and Dr. John Strohl for technical
assistance.

Fig. 7. Maximum ethanol production using different fungal

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