Enhancement of Ethanol Production from Napiergrass (<i>Pennisetum purpureum</i> Schumach) by a Low-Moisture Anhydrous Ammonia Pretreatment

doi:10.4236/jsbs.2013.33025

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Journal of Sustainable Bioenergy Systems, 2013, 3, 179-185

http://dx.doi.org/10.4236/jsbs.2013.33025 Published Online September 2013 (http://www.scirp.org/journal/jsbs)

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: *[email protected]

Received July 18, 2013; revised August 18, 2013; accepted August 25, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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.

M. YASUDA ET AL.

180

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

napiergrass.

Components/wt%

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.

M. YASUDA ET AL.

181

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

(Shimadzu).

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

Napiergrass

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 L−1e)

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 L−1. 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

M. YASUDA ET AL.

182

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

100

120

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.

M. YASUDA ET AL. 183

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·L−1f)

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·L−1 of xylose

and 4.4 g·L−1 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·L−1.

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

Evaporation

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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