Journal of Sustainable Bioenergy Systems, 2013, 3, 179-185 Published Online September 2013 (
Enhancement of Ethanol Production from Napiergrass
(Pennisetum purpureum Schumach) by a Low-Moisture
Anhydrous Ammonia Pretreatment
Masahide Yasuda1*, Keisuke Takeo1, Hayato Nagai1, Takuya Uto1, Toshifumi Yui1,
Tomoko Matsumoto2, Yasuyuki Ishii3, Kazuyoshi Ohta4
1Department of Applied Chemistry, Faculty of Engineering,
University of Miyazaki, Miyazaki, Japan
2Center for Collaborative Research and Community Cooperation,
University of Miyazaki, Miyazaki, Japan
3Department of Animal and Grassland Sciences, Faculty of Agriculture,
University of Miyazaki, Miyazaki, Japan
4Department of Biochemistry and Applied Biosciences, Faculty of Agriculture,
University of Miyazaki, Miyazaki, Japan
Email: *
Received July 18, 2013; revised August 18, 2013; accepted August 25, 2013
Copyright © 2013 Masahide Yasuda et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Napiegrass (Pennisetum purpureum Schumach) was treated with a low-moisture anhydrous ammonia (LMAA) pre-
treatment by adding an equal weight of water and keeping it under atmospheric ammonia gas at room temperature for
four weeks. After the removal of ammonia and washing with water, a simultaneous saccharification and fermentation
(SSF) was conducted for the LMAA-pretreated napiergrass (1.33 g) in a buffer solution (8 mL) using a mixture of a
cellulase (80 mg) and a xylanase (53 mg) as well as the cell suspension (0.16 mL) of Saccharomyces cerevisiae. Etha-
nol and xylose resulted in 91.2% and 62.9% yields, respectively. The SSF process was scaled up using LMAA-pre-
treated napiergrass (100.0 g) to give ethanol (77.2%) and xylose (52.8%). After the removal of ethanol, the pentose
fermentation of the SSF solution (40 mL), which contained 1.00 g of xylose, using cell suspension of Escherichia coli
KO11 (70 mL) gave 86.3% yield of ethanol. Total ethanol yield reached 68.9% based on xylan (21.4 wt%) and glucan
(39.7 wt%) of the LMAA-pretreated napiergrass.
Keywords: LMAA; SSF; Cellulase; Xylanase; Saccharomyces cerevisiae; Escherichia coli KO11
1. Introduction
Biomass has gained much attention as a new sustainable
energy source alternative to petroleum-based fuels [1].
The second-generation bioethanol from lignocellulosic
materials became a promising approach since the lingo-
celluloses are not directly in competition with food
sources. However, a variety of pretreatment methods to
remove the lignin components and/or to promote an en-
zymatic digestibility of the cellulosic components have
been required for efficient bio-ethanol production from
lignocellulose [2].
It has been known that cellulose chains are incorpo-
rated into a number of distinct crystal phases each dif-
fering in chemical reactivity and material characteristics.
The naturally occurring crystal phases, cellulose I [3] can
be transformed into cellulose IIII by treating them with
liquid ammonia [4] or various amines [5-9]. Therefore,
the ammonia fiber explosion (AFEX) pretreatment was
performed by heating the biomass with ammonia gas at
90˚C under 21 atm [10,11]. This was interpreted as an
increase in the cellulose IIII phase which has a high reac-
tivity toward enzymatic degradation [12]. Also, soaking
in aqueous ammonia (SAA) at 40˚C - 80˚C in a room
atmosphere was used as the pretreatment of lignocellu-
lose [13,14]. Recently, Kim et al. [15] developed a pre-
treatment using gaseous ammonia, low-moisture anhy-
drous ammonia (LMAA) pretreatment, where the lingo-
cellulose was kept in a flask filled with ammonia gas at
80˚C for 84 h.
*Corresponding author.
opyright © 2013 SciRes. JSBS
We were interested in ethanol production from herba-
ceous lignocellulosic napiergrass (Pennisetum purpureum
Schumach) because of its low lignin content and high
harvest amount per year and per area [16,17]. Ethanol
was produced from non-pretreated napiergrass through
simultaneous saccharification and fermentation (SSF) as
well as pentose fermentation (PF) with recombinant Es-
cherichia coli KO11 [18]. However, the total ethanol
yield was still low (44.2%). Moreover, usual alkali-pre-
treatment did not operate effectively to enhance the
ethanol yield [19]. Therefore, we applied a LMAA pre-
treatment to enhance the ethanol yield from napiergrass.
2. Materials and Methods
2.1. LMAA Pretreatment
The LMAA pretreatment was performed by modifying
the Kim method [15]. Water (100 g) was added dropwise
to the dry powdered napiergrass (100 g, volume 320 mL)
in the flask (1 L). The resulting moist powdered napier-
grass in the flask was evacuated with a pump under 20
mm Hg and then gaseous ammonia was introduced into
the flask. This operation was performed three times until
the atmosphere inside the flask was entirely replaced
with ammonia. The amount of ammonia presented in the
flask was 1.1 g. The moist powdered napiergrass was
kept under ammonia gas atmosphere at room temperature
for four weeks. After the treatment, the ammonia was
removed with an evaporator. The treated napiergrass was
washed with water (2 L) three times to separate the brow-
nish aqueous solution of the lignin. The treated napiergrass
was dried at 60˚C to weigh out the precise amount of
napiergrass in the following biological treatment.
2.2. Chemical Components of Napiergrass
The powdered napiergrass (30 g) was treated with a 1%
aqueous solution of NaOH (400 mL) at 95˚C for 1 h. The
holocellulose was isolated as a pale yellow precipitate
from the treated mixture by centrifugation and filtration.
The supernatant solution was neutralized to pH 5.0 by a
dilute HCl solution. The resulting dark brown precipitate
identified as lignin was collected by centrifugation at
10,000 rpm for 10 min. Sugars in holocellolose were
determined according to the methods published by the
National Renewable Energy Laboratory (NREL) [20] as
follows. Sulfuric acid (72 wt%, 3.0 mL) was added
slowly to holocellulose (300 mg) and kept at 30˚C for 1 h.
The resulting solution was diluted by water (84 mL) until
the concentration of sulfuric acid was 4 wt%. Acid hy-
drolysis was performed by autoclaving at 121˚C for 1 h
in an autoclave. After the neutralization by CaCO3, the
solution was subjected to the centrifugation to give the
supernatant solution (ca. 87 mL), which was concen-
trated to 30 mL by evaporation. The solution was ana-
lyzed by HPLC. The peaks of glucose and xylose ap-
peared whereas the peaks of galactose and arabinose
were very weak because of their low contents. The
amounts of glucan and xylan were determined from the
amounts of glucose and xylose determined by HPLC. It
was confirmed that the sum amounts of glucan and xylan
were equaled to the amounts of hollocellolose. The ash
component in lignocellulose was obtained by the burning
of the lignocellulose (2.0 g) in an electric furnace
(KBF784N1, Koyo, Nara, Japan) for 2 h at 850˚C. Thus,
the chemical components of napiergrass were determined,
as shown in Table 1.
2.3. Hydrolytic Enzyme
A cellulase from Acremonium cellulolyticus (Acremo-
zyme KM, Kyowa Kasei, Osaka, Japan) was selected by
comparing its activity with other cellulases such as
Meycellase (Meiji Seika), a cellulase from Trichoderma
viride (Wako Chemicals, Osaka, Japan) and a cellulase
from Aspergillus niger (Fluka Japan, Tokyo) [18,21].
The cellulase activity of Acremozyme was determined to
be 1320 units/mg by the method of breaking down filter
paper [18]. A xylanases from Trichoderma longi-
brachiatum (reesei) (Sumizyme X, Shin Nihon Chemi-
cals, Anjyo, Japan, 5000 u/g) was selected from com-
mercially available hemicellulase.
2.4. Preparation of the Inoculum Culture of
Saccharomyces cerevisiae and Escherichia
coli KO11
Saccharomyces cerevisiae NBRC 2044 was grown at
30˚C for 24 h in a basal medium (initial pH 5.5) consist-
ing of glucose (20.0 g/L), polypeptone (1.0 g/L), yeast
extract (1.0 g/L), KH2PO4 (1.0 g/L), and MgSO4 (3.0
g/L). After incubating for 24 h, the cell suspension of S.
cerevisiae whose grown culture of S. cerevisiae showed a
cell density of 7.7 × 107 cells/mL, was obtained [18]. E.
coli KO11 was grown in the LB medium (200 mL) con-
sisting of tryptone (2.0 g/L, Difco), yeast extract (1.0
g/L), and NaCl (2.0 g/L) under shaking at 150 rpm at
37˚C for 24 h. The KO11 cell suspension contained a dry
weight of 0.52 mg/mL of E. coli KO11.
Table 1. Components of the NO- and LMAA-pretreated
PTa) Holocellulose
(glucan : xylan) Lignin Ash Others
NOb) 48.2 (31.3:16.9) 12.6 13.9 25.3
LMAA61.1 (39.7:21.4) 7.1 7.1 24.7
a) Pretreatment (PT): NO: non-treatment. LMAA: low-moisture anhydrous
ammonia pretreatment at room temperature for four weeks. b) The compo-
nents of the LMAA-pretreatment napiergrass without washing with water
was considered to be same as those of non-treated napiergrass.
Copyright © 2013 SciRes. JSBS
Copyright © 2013 SciRes. JSBS
2.5. Simultaneous Saccharification and
Fermentation (SSF)
The LMAA-pretreated napiergrass (1.33 g) was sus-
pended in the acetate buffer (5 mL, pH 5.0) and then
autoclaved at 121˚C for 20 min. After cooling to room
temperature under UV-irradiation, the cell suspension
(0.16 mL) of S. cerevisiae and the hydrolytic enzyme
(133 mg) in an acetate buffer solution (3.0 mL, pH 5.0)
were added to the suspension of the LMAA-treated
napiergrass [18]. After air was purged with N2, the SSF
was initiated by stirring the solution vigorously with a
magnetic stirrer at an optimal temperature of 34˚C. The
evolved CO2 was collected over water by a messcylinder,
and the reaction was monitored by the volume of CO2.
The SSF reaction was continued for 48 h until CO2 evo-
lution was ceased. Table 2 lists the data expressed as
averages of the experiments in three times.
2.6. Analysis
Saccharides were analyzed on a high-performance liquid
chromatography system (LC-20AD, Shimadzu, Kyoto,
Japan) equipped with RI detector (RID-10A) using an
anion exchange column (NH2P-50 4E; Shodex Asahipak,
250 mm in length and 4.6 mm in ID, Yokohama, Japan).
Acetonitrile-water (8:2 v/v) was flowed at 1.0 mL/min as
mobile phase. Ethanol was analyzed by gas-liquid chro-
matography using 2-propanol as an internal standard on a
Shimadzu gas chromatograph (model GC-2014) equipped
with a glass column of 5% Thermon 1000 on Sunpak-A
3. Results
3.1. Napiergrass as Lignocellulosic Materials
For the herbaceous lignocellulose, we selected a dwarf
type of napiergrass (Pennisetum purpureum Schumach),
which is a digestible tropical grass [16,17]. Napiergrass
was cultivated in the Kibana Agricultural Science Station,
at the University of Miyazaki. Leaf blades of the napier-
grass were separated from the stem and then cut by a
cutter and dried at 70˚C for 72 h. The dried matter was
ground until the 70% of the particles were in a range of
32 - 150 μm in length.
3.2. Strategy for Ethanol Production from
In the case of lignocelluloses with high xylan contents,
PF is an unavoidable process. In many cases, the PF us-
ing recombinant E. coli [22-24] or recombinant S. cere-
visiae [25] has been incorporated with the process as
cofermentation with hexose fermentation [15,26]. How-
ever, we performed the PF step separately, since HF us-
ing S. cerevisiae was well-established technique. Our
process outline proceeded through SSF using a hydro-
lytic enzyme and S. cerevisiae followed by PF using E.
coli KO11.
3.3. SSF of the LMAA-Pretreated Napiergrass
The results of the SSF process were summarized in Ta-
ble 2. Without the pretreatment, the SSF using cellulase
(Acremozyme KM) produced ethanol and xylose in
Table 2. The product yields in the SSF of napier grassa).
Product/mg (Yield/%) b)
Run PTc) FXd)
Glucose Xylose Ethanol
Ethanol/g L1e)
1 f) NO 0.0 13 ± 2 (2.8) 77 ± 29 (30.8) 122 ± 15 (51.6) 15.3
2 LMAA 0.0 32 ± 4 (5.5) 150 ± 14 (47.6) 179 ± 3 (59.7) 22.4
3 LMAA 0.1 25 ± 4 (4.2) 172 ± 5 (53.5) 182 ± 4 (60.5) 22.8
4 LMAA 0.2 20 ± 2 (3.5) 155 ± 14 (48.9) 197 ± 1 (65.7) 24.6
5 LMAA 0.3 22 ± 2 (3.7) 160 ± 3 (50.5) 219 ± 22 (72.9) 27.3
6 LMAA 0.4 18 ± 10 (3.1) 199 ± 18 (62.9) 273 ± 7 (91.2) 34.2
7 LMAA 0.5 14 ± 1 (2.3) 173 ± 2 (54.8) 260 ± 10 (86.5) 32.4
8 LMAA 0.6 23 ± 22 (4.0) 146 ± 5 (46.3) 197 ± 8 (65.5) 24.0
9 LMAA 0.7 26 ± 10 (4.4) 150 ± 9 (47.6) 198 ± 12 (66.0) 24.7
10f) NO 0.4 142 ± 35 (30.7) 120 ± 23 (48.3) 98 ± 1 (41.3) 12.2
11f) LMAA g) 0.4 34 ± 16 (7.4) 133 ± 6 (45.2) 178 ± 4 (75.1) 22.3
a) The SSF was performed for the pretreated napiergrass (1.33 g) in buffer solution (8 mL) containing hydrolytic enzyme (133 mg) and the cell suspension of S.
cerevisiae (0.16 mL) at 34˚C for 48 h. The data were expressed as averages of three experiments. b) In the case of LMAA, the yields of glucose, xylose, and
ethanol were calculated based on the theoretical amounts, 587 mg, 316 mg, and 300 mg, respectively; c) Pretreatment (PT). NO: non-treatment. LMAA:
low-moisture anhydrous ammonia pretreatment at room temperature for four weeks. d) The FX was the fraction of xylanase in the mixture (133 mg) of cellulase
and xylanase. e) Concentration of ethanol in g L1. f) The yields of glucose, xylose, and ethanol were calculated based on the theoretical amounts, 463 mg, 249
g, and 237 mg, respectively. g)In the case of the LMAA-pretreatment without washing with water. m
51.6% and 30.8% yields, respectively (run 1). In the
LMAA-pretreated napiergrass case, the yields of ethanol
and xylose increased to 59.7% and 47.6% yields, respec-
tively (run 2). However, the yields were still low. There-
fore, we used a xylanase (Sumizyme X) in addition to the
cellulase. The fraction of xylanase (FX) in the mixture
(133 mg) of cellulase and xylanase was defined. The
maximum yields were observed at 0.4 of FX among 0.1 -
0.7 of FX (runs 3-9). The yields of ethanol and xylose
reached 91.2% and 62.9%, respectively under the opti-
mal condition of the SSF where the LMAA-pretreated
napiergrass (1.33 g) was treated in a buffer solution (8
mL) using cellulase (80 mg), xylanase (53 mg), and the
cell suspension (0.16 mL) of S. cerevisiae (run 6). On the
other hand, in the non-treated napiergrass case, the etha-
nol yield was still low even at 0.4 of FX (run 10). In the
case of LMAA-pretreatment that was not washed with
water (run 11), the ethanol yield was 75.1%. This
showed that washing with water was needed to enhance
the ethanol yield.
3.4. Pentose Fermentation with E. coli KO11
The SSF process was scaled up under optimized condi-
tions of an acetate buffer (600 mL) containing cellulase
(6.0 g), xylanase (4.0 g), the suspension of S. cerevisiae
(12.0 mL), and the LMAA-pretreated napiergrass (100 g)
was stirred at 34˚C. After the SSF for 18 h, 17.4 g
(77.2%) of ethanol and 12.6 g (52.8%) of xylose were
formed whereas 2.2 g (5.0%) of glucose remained un-
changed. Ethanol was removed from the SSF-treated
solution (600 mL) by distillation under a reduced pres-
sure until the volume of the residual solution reached 500
mL. The condensed SSF solution contained 25.2 g/L of
xylose and 4.4 g/L of glucose.
A portion (50-80 mL) of the inoculum culture E. coli
KO11 was poured into the condensed SSF solution (40 -
50 mL). After pH was adjusted to 6.6, the PF was per-
formed by shaking the solution at 150 rpm at 37˚C for 96
h. The yields of ethanol are summarized in Table 3.
Ethanol (531 mg) was obtained under optimal conditions
where the cell suspension of E. coli KO11 (70 mL) and
SSF solution (40 mL) were used (run 4). Since the glu-
cose disappeared after being fermented by E. coli KO11,
it was assumed that the glucose was entirely turned to
ethanol. Therefore, the ethanol amount produced from
xylose was calculated to be 441 mg by subtracting 90 mg
from 531 mg. Thus, the ethanol yield from xylose was
determined to be 86.3%.
4. Discussion
Usual pretreatment of lignocelluloses has been per-
formed by the removal of the lignin-component using an
alkali-pretreatment. However, it was found that the al-
kali-pretreatment was not a useful method for the bio-
logical process of napiergrass with its low lignin-content,
because of the retardation of the fermentation rate by the
inhibitory materials derived from the alkali-pretreatment
and the loss of nutrients for the fermentation [19]. The
LMAA-pretreatment did not take place in the retardation
of fermentation, as shown in Figure 1 where the time
profile of CO2 evolution per 1 g of hollocellulose were
compared among LMAA-pretreated, non-treated, and
alkali-pretreated napiergrass. Moreover, it is possible to
easily recycle gaseous ammonia with low energy in
LMAA-pretreatment compared with the AFEX and SAA
pretreatments which demanded energy to strip and re-
cover ammonia from aqueous ammonia. Thus, the
LMAA-pretreatment will be one of the useful pretreat-
ment method that can be achieved with low energy to
enhance the SSF processes of lignocelluloses.
5. Conclusion
A successfully efficient bio-ethanol production from
napiergrass was achieved by the combination of the
LMAA-pretreatment, the SSF used a mixture of cellu-
lase and xylanase, and the pentose was fermented by E.
coli KO11. The total mass balance was constructed in
Figure 2. With 100 g of the LMAA-pretreated napier-
grass, 24.1 g of ethanol can be totally produced through
SSF and PF. The total ethanol yield reached 68.9% based
on the amounts of xylan (21.4 g) and glucan (39.7 g) in
SSF time/h
Evolved amount of CO2/mL
02040 6080
Figure 1. The time-conversion of CO2 evolved from the SSF
reaction of LMAA-treated (), non-treated (), and al-
kali-treated () napiergrass. The amounts of CO2 were
presented as the evolved CO2 from 1.0 g of holocellolose.
Copyright © 2013 SciRes. JSBS
Table 3. Pentose fermentation with E. coli KO11.
Run KO11/mLa) SSF/mLb) Xylose/mg (Yield/%)c) Ethanol/mg (Yield/%)d) [Yield/%]e) Ethanol/g·L1f)
1 50 50 807 ± 9 (64.0) 352 ± 28 (46.5) [37.2] 3.52
2 60 50 457 ± 122 (36.3) 440 ± 75 (58.1) [50.8] 4.00
3 70 30 0 (0.0) 309 ± 3 (72.0) [68.6] 3.09
4 70 40 0 (0.0) 531 ± 4 (88.4) [86.3] 4.82
5 70 50 60 ± 10 (4.7) 594 ± 77 (77.5) [73.6] 4.95
6 80 50 86 ± 38 (6.7) 540 ± 59 (70.4) [65.4] 4.15
a) The volume of the cell suspension of E. coli KO11 in ml. b)Pentose fermentation was performed for the SSF solution which contained 25.2 g·L1 of xylose
and 4.4 g·L1 of glucose. c) Recovered xylose. d) The values in parenthesis are ethanol yields from both xylose and glucose. e) The values in blanket are the etha-
nol yields from xylose. f) Concentration of ethanol in g·L1.
LMAA (ro o m temp , 4 w e e k s )
Water 127 mL
NH31.4 g
Buffer 588 mL
Cellulase6.0 g
Xylanase4.0 g
Cell suspension of
S. cere vi s ia e12 mL
Cell suspension
of E. c oli.KO11
882 mL
Dry powdered napiergrass(127 g)
Glucan 39.7 g, Xylan21.4 g, Lignin 16.1 g
Glucose 2.2 g
Xylose 12.6 g
Ethanol 17.4 g
PF (37 ºC, 94 h)
Glucose 2.2 g
Xylose 12.6 g
Water 500 mL
SSF (34 ºC, 48 h)
Et ha no l 6.7 g
Glucan 7.1 g
Xylan 10.1 g
Lignin 7.1 g
Water 1.38 L
Et ha no l 17 .4 g
Water 100 mL
Remov al of NH 3
Was h in g with water
Dr ynes s
Glucan7.1 g
Xylan 10.1 g
Lignin 7.1 g
Water 7.6 L
NH31.4 g
Lignin 9.0 g
Water 7.7 L
Glucan7.1 g
Xylan 10.1 g
Lignin 7.1 g
LMAA-treated napiergrass (100 g)
Glucan 39.7 g, Xylan21.4 g, Lignin 7.1 g
Figure 2. Process outline and total mass balance from LMAA-pretreated napiergrass (100 g).
100 g of LMAA-pretreated napiergrass. If the PF process
was combined with the SSF process run 6 of Table 2
which was run under optimal conditions of the small
scale using 1.33 g of LMAA-pretreated napiergrass, the
total yield will become 80.5%.
6. Acknowledgements
We sincerely thank Lonnie O. Ingram from the Univer-
sity of Florida for providing the E. coli KO11 strain. This
study was done as a part of the project entitled “Research
and Development of Catalytic Process for Efficient Con-
version of Cellulosic Biomass into Biofuels and Chemi-
cals (2009-2013)” through Special Funds for Education
and Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.
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