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Biobutanolproductionfromricebranandde-oiledricebranbyClostridiumsaccharoperbutylacetonicumN1-4

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Bioprocess and BiosystemsEngineering ISSN 1615-7591 Bioprocess Biosyst EngDOI 10.1007/s00449-011-0664-2

Biobutanol production from rice branand de-oiled rice bran by Clostridiumsaccharoperbutylacetonicum N1-4

Najeeb Kaid Nasser Al-Shorgani, MohdSahaid Kalil & Wan Mohtar Wan Yusoff

1 23

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ORIGINAL PAPER

Biobutanol production from rice bran and de-oiled rice branby Clostridium saccharoperbutylacetonicum N1-4

Najeeb Kaid Nasser Al-Shorgani • Mohd Sahaid Kalil •

Wan Mohtar Wan Yusoff

Received: 22 August 2011 / Accepted: 28 November 2011

� Springer-Verlag 2011

Abstract Rice bran (RB) and de-oiled rice bran (DRB)

have been treated and used as the carbon source in ace-

tone–butanol–ethanol (ABE) production using Clostridium

saccharoperbutylacetonicum N1-4. The results showed that

pretreated DRB produced more ABE than pretreated RB.

Dilute sulfuric acid was the most suitable treatment method

among the various pretreatment methods that were applied.

The highest ABE obtained was 12.13 g/L, including

7.72 g/L of biobutanol, from sulfuric acid. The enzymatic

hydrolysate of DRB (ESADRB), when treated with XAD-4

resin, resulted in an ABE productivity and yield of 0.1 g/

L h and 0.44 g/g, respectively. The results also showed that

the choice of pretreatment method for RB and DRB is an

important factor in butanol production.

Keywords Biobutanol � Clostridium

saccharoperbutylacetonicum N1-4 � Rice bran �De-oiled rice bran � Pretreatment

Introduction

The growing demand of biobutanol as an alternative form

of fuel for transportation has created considerable interest

due to the occurrence of global warming caused by the

increase of carbon dioxide emissions into the atmosphere

from the combustion of fossil fuels.

As a biofuel, butanol has many advantages over ethanol.

The vapor pressure of butanol is lower than that of ethanol,

while its energy content is higher; these attributes make

butanol to be safer for use in blending gasoline for sub-

sequent use as fuel, because the blend has a better fuel

economy as a fuel than blends obtained from the mixture of

ethanol and gasoline. The ability of butanol to tolerate

contamination from water makes its blends with gasoline

averagely stable and this greatly increases the efficiency of

the existing gasoline supply and distribution channels. In

addition, butanol can be used in conventional engines

without modification [1].

The fermentation process through the use of solvent-

producing Clostridium for the production of butanol has

some problems ranging from toxicity and low productivity

to high recovery cost. However, the results of various

researches have yielded new methods for reducing the

difficulty encountered in the process previously. These new

techniques include the procurement of higher butanol-tol-

erant strains and enhancement of yield through mutagen-

esis or the periodic collection of solvent to prevent their

toxicity [2].

To reduce the cost associated with recovery, many

efforts have been made to develop in situ separation pro-

cesses. These include pervaporation, adsorption, liquid–

liquid extraction, gas stripping and reverse osmosis [2].

Also, to increase the butanol concentration and butanol

selectivity as the major product in the total solvent, com-

plementation of the adhE1 and ctfAB genes in a mega-

plasmid-less C. acetobutylicum M5 strain has been

proposed [3]. The new approach is focused on developing a

more efficient process in the production of butanol through

the use of system-level techniques. Some of these

N. K. N. Al-Shorgani (&) � W. M. W. Yusoff

School of Bioscience and Biotechnology, Faculty of Science

and Technology, Universiti Kebangsaan Malaysia,

43600 UKM Bangi, Selangor, Malaysia

e-mail: nag_nas2001@yahoo.com

M. S. Kalil

Department of Chemical and Process Engineering,

Faculty of Engineering, Universiti Kebangsaan Malaysia,

43600 UKM Bangi, Selangor, Malaysia

123

Bioprocess Biosyst Eng

DOI 10.1007/s00449-011-0664-2

Author's personal copy

techniques, for instance genome sequencing and engi-

neering, construction of genome-scale metabolic net-

works of clostridia, omics studies, transcription machinery

engineering and construction of synthetic pathways, have

been utilized in developing more efficient butanol pro-

ducers [4]. To overcome poor solvent resistance and

low productivity in butanol production, a new strategy

involving the use of in situ recovery techniques alongside

strain design has been proposed [5]. One of the most

important factors in butanol fermentation is the avail-

ability and price of the substrate. Butanol can be produced

from various raw materials or renewable agricultural crops

including sago starch [6, 7], corn [8], molasses [9] and

whey permeate [10]. Rice is the staple food for a large

portion of the world’s population and the annual world

rice production is more than 500 million metric tons. The

primary waste product of the rice industry is rice bran,

of which approximately 40 million metric tons are pro-

duced every year and are discarded as unfit for human

consumption.

Rice bran (RB), the residue of brown rice that is

created during the production of white rice, is abun-

dantly available in Southeast Asia. This agricultural by-

product contains a number of carbohydrates and other

nutrients such as proteins, lipids, fiber, Ca2?, Mg2?,

phosphate, silica, Zn2?, thiamin and niacin [11]; but its

industrial applications are limited to being an animal

feed additive or for the production of rice bran oil. This

residue, after extracting rice bran oil, is called de-oiled

rice bran (DRB). Therefore, there are two different

kinds of rice bran available from the rice-processing

industry: RB and DRB. DRB still contains many car-

bohydrates and cellulosic polysaccharides [12], and the

presence of a high amount of carbohydrate (39% cel-

lulose and 31% hemicellulose) and less lignin (4%)

make it an attractive feedstock for conversion into a

variety of value-added products, such as single cell

proteins and ethanol [13].

Dilute acid hydrolysis is a simple and easy method to

perform and is regularly used for the depolymerization of

biomass into fermentable sugars [13]. However, it has

some limitations. For example, if higher temperatures (or

longer residence time) are applied, the hemicellulosic-

derived monosaccharides will degrade and give rise to

fermentation inhibitors such as furan compounds, weak

carboxylic acids and phenolic compounds. These fermen-

tation inhibitors are known to affect the performance of

bioethanol and biobutanol production in fermenting

microorganisms [14–16].

In the present study, Clostridium saccharoperbutyl-

acetonicum N1-4 was used to produce butanol from RB

and DRB hydrolysates, because this bacterium is known to

be a hyper-butanol-producing strain [17, 18].

Materials and methods

Microorganism and inoculum preparation

Clostridium saccharoperbutylacetonicum N1-4 was pro-

vided by the Biotechnology Laboratory, Chemical and

Process Engineering Department of UKM. It was kept at

4 �C as a suspension of spores in potato glucose medium

(PG medium) as a stock culture. The inoculum was pre-

pared by transferring the suspension of spores (1 mL) into

10 mL of PG medium with subsequent heat shock for

1 min in boiling water, cooling in ice water and incubating

for 1–2 days at 30 �C under anaerobic conditions. The

colony morphology and Gram-staining behavior of the

inoculum were verified to ensure that the culture was pure

[19]. The culture was then transferred to tryptone–yeast

extract–acetate medium (TYA), incubated for 18 h and

used as the inoculum.

RB and DRB pretreatment

RB was collected from Kilang Beras BERNAS, Tanjung

Karang, Selangor, Malaysia; and DRB was obtained by

extracting the oil using hexane. The oil was extracted by

solvent extraction using a Soxhlet apparatus according to

the method of Aryee and Simpson [20]. Briefly, one part of

the rice bran sample of a known weight was injected into a

33 9 94 mm cellulose extraction thimble, covered with

silanized glass wool and extracted with 20 parts of hexane

(1:20 w/v) for 10 h at the boiling point of hexane (70 �C)

[20].

The RB and DRB were treated by acidic and enzymatic

methods prior to fermentation. For acid pretreatment,

100 g of RB or DRB was soaked in 1 L of 1% (v/v) HCl or

1% (v/v) H2SO4 and autoclaved at 121 �C for 1 h (dilute

sulfuric acid pretreatment) or at 80 �C for 3 h (dilute

hydrochloric pretreatment), to generate hydrochloric acid

rice bran hydrolysate (HARB), hydrochloric acid de-oiled

rice bran hydrolysate (HADRB), sulfuric acid rice bran

hydrolysate (SARB) and sulfuric acid de-oiled rice bran

hydrolysate (SADRB), respectively. A combined enzy-

matic and acid treatment was carried out after the acid

treatment to generate EHARB, EHADRB, ESARB and

ESADRB. The pH was then adjusted to 4.5 with 10 M

NaOH, and a mixture of two enzyme solutions (6 mL/

100 g RB or DRB of each enzyme) [Celluclast 1.5 L

(cellulase; Novo, Malaysia) and Novozyme 188 (b-gluco-

sidase; Novo, Malaysia)] was added and the solution was

incubated at 45 �C for 72 h with agitation at 80 rpm. After

72 h, the hydrolysate was centrifuged at 7,0009g for

10 min to remove the sediments, and the supernatant was

supplemented with P2 solution and autoclaved at 121 �C

for 15 min and used as a fermentation medium.

Bioprocess Biosyst Eng

123

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The DRB was also treated enzymatically using amylase

enzymes. For this, the pH of the DRB solution was

adjusted to 6.2 by 10 M NaOH and calcium was added,

using calcium chloride (Ca?2 C40 ppm), to stabilize the

enzyme. A heat-stable a-amylase (Termamyl 120 L; Novo,

Malaysia) (0.05 mL/100 g DRB) was added to the DRB

mixture, heated to 100 �C and held at this temperature for

10 min. The mixture was then cooled to 90 �C and held at

this temperature for an additional 2 h to increase hydro-

lysis. After liquefaction, the pH was reduced to 4.5 by 5 M

HCl, and the solution was cooled to 60 �C. Saccharification

was carried out by adding glucoamylase enzyme (AMG

300 L; Novo, Malaysia) (0.1 mL/100 g DRB) at pH 4.5

and incubating at 60 �C for 24 h.

Inhibitor removal by overliming and XAD-4 resin

Chemical treatment methods result in the production of

undesirable secondary products that can inhibit microbial

growth. One of the ways to reduce or remove these

inhibitors is through the overliming method, where calcium

hydroxide is added under fast stirring at 80 �C until the pH

rises to 10, followed by the addition of 1 g/L Na2SO3

(overliming). Sedimentation of the inhibitors was allowed

for 1 h with intermittent mixing followed by cooling to

room temperature. The mixture was then filtered using

vacuum filtration to remove precipitates. The pH of the

clear filtrate was adjusted to 7 using concentrated H2SO4

and was again vacuum filtered to remove any remaining

traces of salt residue.

The hydrolysate was centrifuged again at 5,0009g for

10 min, and the fermentation inhibitors were removed by

passing the clear supernatant through a nonionic polymeric

adsorbent resin [Amberlite XAD-4 (Sigma Chemicals)]

column (510 9 15 mm) at a flow rate of 8 mL/min.

Approximately, 500 mL of hydrolysate was passed through

the column, which was then packed using 75 g of XAD-4

resin. Previous studies have shown that 60–80% of furfural,

hydroxymethyl furfural (HMF) and ferulic compounds can

be removed using XAD-4 resin [21].

Analytical methods

Samples were taken periodically for the analysis of sol-

vents, acids, pH and sugars. After centrifugation at

7,0009g for 10 min, the supernatants were filtered through

a 0.2-lm cellulose acetate filter. The solvent (acetone,

butanol and ethanol) concentrations were analyzed by gas

chromatography (7890A GC-System, Agilent Technolo-

gies, Palo Alto, CA, USA) equipped with a flame ioniza-

tion detector and a 30-m capillary column (Equity1;

30 m 9 0.32 mm 9 1.0-lm film thickness; Supelco Co,

Bellefonate, PA, USA). The oven temperature was

programmed to increase from 40 to 130 �C at a rate of

8 �C/min. The injector and detector temperatures were set

at 250 and 280 �C, respectively. Helium, as the carrier gas,

was set at a flow rate of 1.5 mL/min.

Sugars and organic acids (acetic and butyric acid) were

measured by high-performance liquid chromatography

(HPLC 12000 Series, Agilent technologies, Palo Alto, CA,

USA). Acids were analyzed using a Genesis C18 120A

column (25 cm 9 4.6 mm; Jones Chromatography,

Tempe, AZ, USA) at a column temperature of 40 �C, with

20 mM H2SO4 as a mobile phase, at a flow rate of 0.6 mL/

min. Detection was accomplished with a UV detector at a

wavelength of 220 nm. Sugars were analyzed using a

Shodex Asahipak NH2P-50 4E column (4.6 mm

ID 9 250 mm; Shodex, Japan), and the concentrations

were detected with a refractive index detector (RID 1200,

Agilent Technologies, Palo Alto, CA, USA) at 30 �C and a

flow rate 1 mL/min, using a mixture of acetonitrile and

water (H2O/CH3CN = 40/60) as a mobile phase.

The glucose concentration was detected with a Bio-

chemistry Analyzer (YSI 2700D, YSI Inc., Life Sciences,

Yellow Springs, OH, USA). Cell density was measured

using a Genesys-10 Scanning UV–VIS Spectrophotometer

(Thermo Spectronic, Rochester, USA) at 660 nm. ABE

yield was defined as the amount of ABE produced divided

by the total amount of sugars, and productivity was defined

as the ABE concentration divided by the fermentation time.

Media preparation

PG medium was used to prepare the inoculum and consisted of

150 g/L potato, 10 g/L glucose, 0.5 g/L (NH4)2SO4 and 3 g/L

CaCO3.

TYA medium was also used to prepare the inoculum and

consisted of the following components: 20 g/L glucose,

6 g/L tryptone, 2 g/L yeast extract, 3 g/L ammonium

acetate, 0.5 g/L KH2PO4, 0.3 g/L MgSO4�7H2O, and

0.01 g/L FeSO4�7H2O.

P2 medium was used as a fermentation medium and

consisted of the following: 0.5 g/L KH2PO4, 0.5 g/L

K2HPO4, 0.4 g/L MgSO4�7H2O, 0.01 g/L MnSO4�4H2O,

0.01 g/L FeSO4�5H2O, 1.0 g/L yeast extract and 0.5 g/L

cysteine. A final concentration of 80 lg/L biotin and 1 mL

of a solution containing 1 mg/L 4-aminobenzoic acid were

added to 1 L of P2 medium.

Batch fermentation

The anaerobic batch fermentation was conducted in

250-mL Schott (Duran) bottles with a working volume of

150 mL. P2 medium was used as a fermentation medium,

and 10% (w/v) of either RB or DRB hydrolysates were

used as the sole carbon.

Bioprocess Biosyst Eng

123

Author's personal copy

The initial pH of the medium was adjusted to 6.5. The

medium was then sterilized by autoclaving at 121 �C for

15 min. To generate an anaerobic condition, the medium

was sparged with oxygen-free nitrogen, and the vitamin

solution was filter-sterilized and added aseptically into the

sterilized medium. The batch culture was initiated by

inoculation of the medium with 10% fresh inoculum of

C. saccharoperbutylacetonicum N1-4 that had been previ-

ously grown in TYA medium for 18 h. The Schott bottles

were incubated at 30 �C without shaking under anaerobic

conditions. All fermentations were performed in duplicate,

and measurements are mean values.

Results and discussion

In this study, RB and DRB were pretreated prior to fer-

mentation by various methods. This pretreatment included

using diluted sulfuric and hydrochloric acid (1% v/v) fol-

lowed by a combination of acidic and enzymatic hydroly-

sis. Treatment with H2SO4 and HCl generates SARB,

HARB, SADRB and HADRB. Subsequent treatments of

RB and DRB using the combination of dilute acid followed

by enzymatic hydrolysis generated ESARB, EHARB, ES-

ADRB and EHADRB, respectively.

RB as a by-product of rice production and DRB as a by-

product of rice oil production are cheap and renewable

agricultural products available around the world. Hydro-

lysis of 100 g/L RB and DRB using dilute sulfuric acid

produced sufficient amounts of reducing sugars for butanol

fermentation by C. saccharoperbutylacetonicum N1-4 at

31.43 and 33.35 g/L, respectively. The solvent produced

from the DRB hydrolysate, after the XAD-4 resin treat-

ment, showed concentrations as high as 12.13 g/L of ABE.

ABE fermentation using untreated RB and DRB

as media

RB and DRB without hydrolysis/treatment were used as a

control and supplemented in P2 medium. The reduced

sugar content in untreated rice bran was found to be 6.74 g/

L. The presence of this small amount of sugar may result

from the sterilization effect, which causes the thermal

degradation of polysaccharides in RB. DRB showed lesser

amounts of reducing sugars (2.7 g/L), possibly due to the

oil extraction process.

During the fermentation of untreated RB using C. sac-

charoperbutylacetonicum N1-4, the sugar concentrations of

8.43 and 9.88 g/L were detected after 12 and 24 h,

respectively, demonstrating the ability of C. sacchar-

operbutylacetonicum N1-4 to secrete the respective

enzymes for the hydrolysis of different RB constituents

(Fig. 1). A similar phenomenon was observed with the

fermentation of the untreated DRB (Fig. 2).

ABE production by C. saccharoperbutylacetonicum

N1-4 was carried out using untreated RB and DRB as a

carbon source in P2 medium. During the 96 h of RB fer-

mentation, 2.75 g/L total ABE was produced, containing

0.23 g/L acetone, 2.31 g/L butanol and 0.21 g/L ethanol

(Fig. 1). The total concentration of acids produced was

3.14 g/L (1.90 g/L butyric acid and 1.24 g/L acetic acid).

The ABE productivity was 0.03 g/L h when untreated RB

was used. This culture favored the production of acids

(3.14 g/L total acids), and the lower solvent production can

be attributed to the lack of sugars in the culture. A previous

study reported that the presence of less than 10 g/L glucose

in batch cultures and 4 g/L per day in fed-batch cultures of

C. acetobutylicum can only produce acids, and no shift to

ABE production occurs [22]. However, the initial con-

centration of reducing sugars found in DRB was less than

that found in RB, yet the total ABE obtained from 120 h of

fermentation of DRB was 5.16 g/L, containing 1.70 g/L

acetone, 3.31 g/L butanol and 0.15 g/L ethanol (Fig. 2),

with an ABE productivity of 0.043 g/L h.

Fig. 1 Production of ABE from RB in a batch culture using

C. saccharoperbutylacetonicum N1-4. a Solvent versus fermentation

time; b acids, sugars and pH versus fermentation time

Bioprocess Biosyst Eng

123

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From these results, it can be suggested that the treatment

of RB and DRB or other agricultural residues prior to

fermentation is an important factor in improving the ABE

yield and productivity. Similar results were obtained from a

study conducted by Lee et al. [11], where only 2.22 g/L

butanol was produced from untreated RB; however, when

it was supplemented with P2 medium and fermented using

C. beijerinckii NCIMB 8052 in a batch reactor, 5.03 g/L of

ABE was obtained versus 5.16 g/L of ABE obtained from

DRB in our study. Our results are in agreement with those

reported by Lee et al. [11].

ABE fermentation using glucose-based medium

The control batch culture was carried out using P2 medium

containing 30 g/L glucose, as a comparison to pretreated

RB containing the same amount of sugar. After 72 h,

6.27 g/L total ABE was obtained from this fermentation

with the following composition: 4.86 g/L butanol, 0.56 g/L

acetone and 0.85 g/L ethanol. An ABE productivity and

yield of 0.087 g/L h and 0.28 g/g, respectively. Organic

acids (acetic and butyric acids) were also measured; the

total concentration of acids was 2.71 g/L, which was

composed of 1.61 g/L butyric acid and 1.1 g/L acetic acid.

ABE fermentation using HARB- and HADRB-based

medium

RB and DRB were pretreated with dilute hydrochloric acid

(1%) to release more sugars. The sugar content of the HARB-

and HADRB-based medium was 9.7 and 27.78 g/L, respec-

tively. The hydrolysates were fermented into ABE using

C. saccharoperbutylacetonicum N1-4. The HARB hydrolysate

produced 5.56 g/L total ABE, with a yield of 0.57 g/g, based

upon the sugar utilized. The ABE productivity was 0.06 g/L h.

The concentrations of acetone, butanol and ethanol were

0.4, 4.93 and 0.23 g/L, respectively. Compared to untreated

RB, HARB produced twice the concentration of ABE and the

total acids were 5.55 g/L, compared to 3.14 g/L in the

untreated RB. The accumulation of acids (5.55 g/L) can be

attributed to the lack of sugar, thus halting the production of

additional solvent; the bacteria could not convert the acids for

the same reason (Table 1). In addition, 6.38 g/L total ABE

resulted from HADRB hydrolysate, with a yield and produc-

tivity of 0.32 g/g and 0.053 g/L h, respectively. At the end of

HADRB fermentation, 7.84 g/L sugars and 3.89 g/L organic

acids were observed as residuals.

Fig. 2 Production of ABE from DRB in a batch culture using

C. saccharoperbutylacetonicum N1-4. a Solvent production, b acids,

sugars and pH

Table 1 ABE fermentation of pretreated rice bran (RB) by C. saccharoperbutylacetonicum N1-4

Medium Sugars (g/L) Production (g/L) Acetic

acid

(g/L)

Butyric

acid

(g/L)

Total

acids

(g/L)

Time

(h)

ABE

productivity

(g/L h)

YABE/S

(g/g)Initial Consumed Acetone Butanol Ethanol ABE

RB 6.74 – 0.23 2.31 0.21 2.75 1.24 1.9 3.14 96 0.03 NA

HARB 9.7 9.7 0.40 4.93 0.23 5.56 3.17 2.38 5.55 96 0.06 0.57

EHARB 19.3 19.3 1.76 5.59 0.24 7.59 0.95 1.25 2.2 120 0.06 0.39

SARB 31.43 29.37 2.04 6.32 0.25 8.61 1.57 2.06 3.63 96 0.09 0.29

ESARB 32.1 31.3 2.03 6.57 0.28 8.88 0.92 1.33 2.25 120 0.07 0.28

ESARB ? XAD-4 28.0 28.0 2.12 7.22 0.58 9.92 0.82 0.93 1.75 120 0.08 0.35

RB rice bran, HARB RB hydrolyzed by 1% HCl, EHARB HARB enzymatic hydrolysate using cellulase enzymes, SARB RB hydrolyzed by 1%

H2SO4, ESARB SARB enzymatic hydrolysate using cellulase enzymes, ESARB ? XAD4 enzymatic hydrolysate of SARB treated by XAD-4

resin, YABE/S yield of ABE to sugars consumed, NA not applicable

Bioprocess Biosyst Eng

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ABE fermentation using EHARB- and EHADRB-based

medium

The combined pretreatment using dilute hydrochloric acid

(1%) followed by cellulolytic enzymes was performed on

RB and DRB to generate more sugars. This treatment

generates EHARB and EHADRB hydrolysates, and it

resulted in an increased production of up to 19.3 g/L

reducing sugars in EHARB, having 18 g/L glucose and

1.3 g/L xylose. The fermentation of EHARB resulted in

7.59 g/L ABE, which contained 1.76 g/L acetone, 5.56 g/L

butanol and 0.24 g/L ethanol. ABE productivity and yield

were 0.06 g/L h and 0.39 g/g, respectively. Although the

ABE production from EHARB improved compared to

HARB, the productivity remained unchanged (0.06 g/L h);

this is likely due to the fermentation time. The prolonged

fermentation time may be due to the existence of microbial

inhibitors resulting from the hydrolysis of lignocellulosic

materials, although we avoided the use of high tempera-

tures in this treatment (only heating at 80 �C for 3 h).

The total sugar concentration resulting from the pretreat-

ment of HADRB with cellulolytic enzymes was 29.51 g/L,

and the fermentation of this hydrolysate produced 6.56 g/L

total ABE, containing 1 g/L acetone, 4.71 g/L butanol and

0.84 g/L ethanol. In this study, EHARB produced more ABE

and butanol compared to EHADRB; the productivity was also

increased, although the reducing sugar concentration was

higher in EHADRB.

ABE fermentation using SARB- and SADRB-based

medium

The SARB- and SADRB-based medium contained 30.79

and 33.35 g/L reducing sugars, respectively. The concen-

trations of glucose, xylose and cellobiose in SARB were

23.46, 5.64 and 1.69 g/L, respectively; and in SADRB, the

concentrations were 19.96, 8.21 and 5.18 g/L, respectively.

The glucose percentage was 76.2% in SARB and 60% in

SADRB, indicating the efficiency of using dilute sulfuric

acid as a pretreatment method to generate simple sugars

from agricultural residues.

The fermentation profiles of the SARB and SADRB

hydrolysates by C. saccharoperbutylacetonicum N1-4 in

batch culture utilized 29.37 and 28 g/L of reducing sugar,

respectively. During the first 96 h of SARB hydrolysate

fermentation, 93% of the reducing sugar was consumed,

resulting in a total ABE production of 8.61 g/L, containing

6.23 g/L butanol, 2.04 g/L acetone and 0.25 g/L ethanol.

An ABE yield of 0.29 g/g of sugar was consumed, and a

productivity of 0.09 g/L h was achieved. A regular

increase in solvent production was observed until 96 h of

incubation and became constant thereafter. The maximum

solvent production (9.66 g/L) was measured after 120 h

from SADRB medium, with an ABE yield of 0.35 g/g and

a productivity of 0.081 g/L h (Table 2).

A maximum value of 8.61 g/L ABE resulting from the

SARB fermentation was lower than that reported by Lee

et al. [11]. This may be due to the ability of the C. beijerinckii

NCIMB 8052 strain to more efficiently convert the SARB to

butanol compared to C. saccharoperbutylacetonicum N1-4.

It may also be due to the presence of inhibitors in the SARB.

The sulfuric acid showed a superior effect for the hydrolysis

of hemicelluloses, but generated inhibitors for microbial

fermentation.

ABE fermentation using ESARB- and ESADRB-based

medium

SARB and SADRB were treated with cellulase enzymes

(cellulase and cellobiose) as mentioned in ‘‘Materials and

methods’’. The hydrolysates of ESARB ESADRB were

detoxified by overliming and used for solvent production.

Table 2 ABE fermentation of pretreated de-oiled rice bran (DRB) by C. saccharoperbutylacetonicum N1-4

Medium Sugars (g/L) Production (g/L) Acetic

acid

(g/L)

Butyric

acid

(g/L)

Total

acids

(g/L)

Time

(h)

ABE

productivity

(g/L h)

YABE/S

(g/g)Initial Consumed Acetone Butanol Ethanol ABE

DRB 2.70 – 1.70 3.31 0.15 5.16 1.35 2.47 3.83 120 0.043 NA

HADRB 27.78 19.94 1.83 4.39 0.16 6.38 2.00 1.89 3.89 120 0.053 0.32

EHADRB 29.51 24.30 1.00 4.71 0.84 6.56 0.78 1.38 2.16 120 0.055 0.27

SADRB 33.35 28.00 2.55 6.75 0.36 9.66 2.16 0.74 2.90 120 0.081 0.35

ESADRB 32.65 26.28 2.12 7.10 1.29 10.51 0.87 1.04 1.91 120 0.088 0.40

ESDRB ? XAD-4 30.00 28.57 3.18 7.72 1.23 12.13 1.63 0.50 2.13 120 0.101 0.44

EDRB 29.00 24.54 1.22 4.96 0.19 6.38 2.73 1.21 3.94 120 0.053 0.26

DRB de-oiled rice bran, HADRB DRB hydrolyzed by 1% HCl, EHADRB HARB enzymatic hydrolysate using cellulase enzymes, SADRB DRB

hydrolyzed by 1% H2SO4, ESADRB SADRB enzymatic hydrolysate using cellulase enzymes, ESDRB ? XAD4 enzymatic hydrolysate of DRB

treated by XAD-4 resin, EDRB DRB treated by amylase enzymes, YABE/S yield of ABE to sugars, NA not applicable

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ESARB did not enhance the production of reducing

sugars and the sugar content in ESARB was 32.1 g/L

compared to the 31.43 g/L found in SARB; a similar effect

was observed with ESADRB (Tables 1, 2). These results

demonstrate that a dilute sulfuric acid treatment has the

ability to convert the lignocellulosic materials to ferment-

able sugars while saving the cost associated with enzymatic

hydrolysis. During the fermentation of the ESARB

hydrolysate, only 8.88 g/L total ABE was produced,

resulting in a productivity of 0.07 g/L h and a yield of

0.28 g/g, while ESADRB resulted in 10.51 g/L total ABE,

with a productivity of 0.088 g/L h and an ABE yield of

0.4 g/g. Moreover, enzymatic hydrolysis of DRB using

amylolytic enzymes resulted in 29 g/L reducing sugars and

only 6.38 g/L of ABE with a productivity of 0.053 g/L h,

which is similar to that seen in HADRB.

Previously, it was reported that butanol fermentation

from renewable agricultural residues requires pretreatment

to increase the amounts of fermentable sugars [15, 23, 24].

The most popular and effective hydrolysis method for these

renewable lignocellulosic materials is acid treatment using

dilute sulfuric acid; yet, this type of treatment suffers from

microbial inhibitor generation, such as furans, aliphatic

acids and phenolic compounds [25, 26], thus reducing the

yield and productivity of butanol. In addition, the inhibitors

that affect microbial growth and ABE fermentation are

required to be reduced or removed. However, this acid

treatment has been confirmed to be a cost-effective pre-

treatment method [11]. In this study, the presence of more

than 30 g/L reducing sugars was enough for solvent pro-

duction by C. saccharoperbutylacetonicum N1-4.

In an attempt to resolve the inhibition problem, XAD-4

resin was used to remove the fermentation inhibitors from

the ESARB and ESADRB hydrolysates prior to fermen-

tation. It has been reported that XAD-4 resin has the ability

to reduce furfural and hydroxymethyl furfural (HMF),

which are the common inhibitors resulting from biomass

treatment [21]. Fermentation of XAD-4 treated ESARB

produced 9.92 g/L total ABE, 2.12 g/L acetone, 7.22 g/L

butanol and 0.58 g/L ethanol (Fig. 3). The ABE produc-

tivity and yield were 0.08 g/L h and 0.35, respectively.

Furthermore, XAD-4 treatment of ESADRB improved

solvent production, productivity and yield. In this study, a

maximum ABE production of 12.13 g/L was obtained from

ESADRB treated with XAD-4 resin, which gave a pro-

ductivity of 0.101 g/L h (Fig. 4). These results showed that

XAD-4 resin markedly enhanced the ABE concentration,

yield and productivity compared to that obtained from non-

treated ESARB and ESADRB (Tables 1, 2).

In a similar study, Qureshi et al. [15] enhanced the

performance of corn fiber hydrolysate significantly when

the hydrolysate was treated with XAD-4 resin. In their

study, only 1.7 g/L ABE was produced from untreated corn

fiber hydrolysate, but the treatment with XAD-4 improved

the production to 9.3 g/L ABE.

Various agricultural residues, including corn fiber [15],

wheat bran [27], wheat straw [28], rice bran [11] and rice

straw [29], have been reported to have ABE production

when using different species of solvent-producing clos-

tridia (Table 3). In our study, a maximum ABE concen-

tration of 12.13 g/L was identified in the XAD-4-treated

ESADRB fermentation, which is higher than that reported

by Qureshi et al. [15] and Liu et al. [27]. However, it is

lower than that reported by Qureshi et al. [23], Lee et al.

[11] and Soni et al. [29], as summarized in Table 3. The

differences in ABE production may be due to the differing

nature of the agricultural substrates, the sugar content, the

different Clostridium strains and the existence of the fer-

mentation inhibitors still present in the hydrolysate even

after XAD-4 treatment. Both may have contributed to the

inefficient utilization of the sugars present in the ESADRB

treated with XAD-4 resin for solvent production.

Comparison of corn fiber [15], RB and DRB (our study)

using the same pretreatment method (enzymatic hydrolysis

followed by XAD-4 resin to remove fermentation inhibi-

tors) was found to produce different amounts of reducing

Fig. 3 ABE fermentation from ESARB treated by XAD-4 using C.saccharoperbutylacetonicum N1-4. a Solvent production, b acids,

sugars and pH

Bioprocess Biosyst Eng

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sugars with values of 46.3, 28 and 30 g/L, respectively.

The total solvents (ABE) produced from the hydrolysates

of corn fiber, RB and DRB were found to be 9.3, 9.92 and

12.13 g/L, respectively (Table 3). These results show the

efficiency of our fermentation system compared to that

reported by Qureshi et al. [10].

In all the fermentations in this study, it was observed

that the hydrolysates of DRB showed higher solvent

production, one explanation being that the oil content of

RB may have a negative effect on ABE production when

using C. saccharoperbutylacetonicum N1-4. A similar

phenomenon was observed when crude palm oil (CPO)

was added to palm oil mill effluent medium to investigate

the effect of CPO on ABE fermentation using C. sac-

charoperbutylacetonicum N1-4 (data not shown). An

earlier study reported that the effect of oil on ABE fer-

mentation by C. saccharoperbutylacetonicum N1-4 can

be attributed to interference with sugar uptake and sol-

vent production during fermentation [30]. Extraction of

the oil from RB can facilitate greater solvent production

at a lesser cost and the extracted oil can be used in other

applications, such as biodiesel production, food or med-

ical uses.

Fig. 4 ABE fermentation from ESADRB treated by XAD-4 resin

using C. saccharoperbutylacetonicum N1-4. a Solvent production,

b acids, sugars and pH

Ta

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Bioprocess Biosyst Eng

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Conclusion

This study suggested that rice bran (RB) and de-oiled rice

bran (DRB) should be hydrolyzed to increase the fer-

mentable sugars and consequent butanol productivity. The

use of dilute sulfuric acid and enzymatic hydrolysis by

cellulolytic enzymes as pretreatment methods for RB and

DRB demonstrated similar amounts of reducing sugars,

whereas hydrolysis of DRB by amylase enzymes produced

comparable concentrations of total ABE and reducing

sugars when compared with dilute hydrochloric acid

hydrolysis of DRB. Among all the hydrolysates, the highest

total solvent and butanol production (12.13 and 7.72 g/L,

respectively) was obtained when ESADRB was treated

with XAD-4 resin, with a productivity of 0.1 g/L h and a

yield of 0.44 g/g.

It was found that the poor butanol fermentation perfor-

mance in RB hydrolysates compared to DRB hydrolysates

was likely due to the presence of oil in the rice bran. This

study showed that the extraction of oil from rice bran could

improve the butanol production by 88%. The oil produced

can then be used in other applications such as with bio-

diesel. An enhancement of butanol production was found

when the hydrolysates of RB or DRB were treated with

XAD-4 resin, thus demonstrating the presence of fermen-

tation inhibitors accompanying the hydrolysis of cellulosic

biomass and the ability of XAD-4 resin to remove some

inhibitors.

Acknowledgments We would like to thank Prof. Dr Yoshino

Sadazo, Kyushu University, Japan, who provided us with C. sacchar-operbutylacetonicum N1-4. This research was supported by the

UKM-GUP-KPB -08-32-128 grant.

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