ARTICLE IN PRESS

Available at wwwsciencedirectcom

B I O M A S S A N D B I O E N E R G Y 3 2 ( 2 0 0 8 ) 9 7 1 ndash 9 7 7

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Lime pretreatment enzymatic saccharification andfermentation of rice hulls to ethanol

Badal C Saha Michael A Cotta

US Department of Agriculture1 Fermentation Biotechnology Research Unit National Center for Agricultural Utilization Research

Agricultural Research Service 1815 N University Street Peoria IL 61604 USA

a r t i c l e i n f o

Article history

Received 6 December 2007

Received in revised form

15 January 2008

Accepted 18 January 2008

Available online 5 March 2008

Keywords

Rice hulls

Ethanol

Lime pretreatment

Enzymatic saccharification

Separate hydrolysis and

fermentation

Simultaneous saccharification and

fermentation

nt matter Published by Eioe200801014

thor Tel +1 309 681 6276BadalSahaarsusdagovde names or commercialndation or endorsement

a b s t r a c t

Rice hulls used in this study contained 356701 cellulose and 120707 hemicellulose

The maximum yield of monomeric sugars from rice hulls (150 wv) by lime pretreatment

(100 mg g1 hulls 121 1C 1 h) and enzymatic saccharification (45 1C pH 50 72 h) using a

cocktail of three commercial enzyme preparations (cellulase b-glucosidase and hemi-

cellulase) at the dose level of 015 ml of each enzyme preparation g1 hulls was

15471 mg g1 (32 yield) The lime pretreatment did not generate any detectable furfural

and hydroxymethyl furfural in the hydrolyzate The concentration of ethanol from lime-

pretreated enzyme-saccharified rice hull (138 g) hydrolyzate by recombinant Escherichia coli

strain FBR5 at pH 65 and 35 1C in 19 h was 98705 g l1 with a yield of 049 g g1 available

sugars The ethanol concentration was 110710 g l1 in the case of simultaneous

saccharification and fermentation by the E coli strain at pH 60 and 35 1C in 53 h

Published by Elsevier Ltd

1 Introduction

In the USA the production of fuel ethanol from corn starch

reached 184106 m3 (486 billion US gallons) in 2006 [1]

Developing ethanol as fuel beyond its current role as a fuel

oxygenate will require developing lignocellulosic biomass as

a feedstock because of its relative abundance and low cost

Rice hulls which represent 20 dry weight of the harvested

rice can serve as a low-cost abundant feedstock for produc-

tion of fuel alcohol in the USA and other rice-producing

countries In 2005 world rice production was about 61844

million tonnes (Mt) of which about 1012 Mt was produced in

the USA [2] Arkansas (493 Mt) is the leading rice-producing

state in the nation followed by California (176 Mt) Louisiana

lsevier Ltd

fax +1 309 681 6427(BC Saha)products in this article isby the US Department o

(140 Mt) Mississippi (076 Mt) Missouri (064 Mt) and Texas

(061 Mt) [3] However most of the rice is grown in localized

regions of each state The total available rice hulls are 202 Mt

and 12369 Mt in the US and globally respectively Rice hulls

are considered waste materials because of their low value as

animal feed due to low digestibility peculiar size low bulk

density high ashsilica contents and abrasive characteristics

They can be easily collected from rice-processing sites and

contain about 36 cellulose and 12 hemicellulose The

theoretical ethanol production from rice hulls is 62105 m3

(164 million US gallons) in the US while global production

level is 36106 m3 (95 billion US gallons) [4] However rice

hulls also contain high quantities of lignin (16) and ash

(20) which complicates their use as lignocellulosic feedstock

solely for the purpose of providing specific information and doesf Agriculture

ARTICLE IN PRESS

B I O M A S S A N D B I O E N E R G Y 3 2 ( 2 0 0 8 ) 9 7 1 ndash 9 7 7972

for conversion to ethanol Gasification of rice hulls has been

considered as a potential waste disposal and energy recovery

method and very little literature is available on the pretreat-

ment enzymatic saccharification and fermentation of rice

hulls [5ndash10]

The objective of this work was to develop methods for cost-

effective pretreatment and enzymatic saccharification of rice

hull cellulose and hemicellulose into fermentable sugars and

fermentation of the hydrolyzate to ethanol In our previous

study we have evaluated dilute acid pretreatment enzymatic

saccharification and fermentation of rice hulls to ethanol [11]

Rice hulls can be enzymatically saccharified to sugars with a

maximum yield of 60 after dilute acid pretreatment under

optimized conditions More than 50 of the cellulose in the

dilute acid-pretreated rice hulls remained unhydrolyzed by

enzymes Moreover inhibitors were also produced during

dilute acid pretreatment which needed to be removed by

overliming before fermentation The hemicellulose in rice

hulls contains both arabinoxylan and xyloglucan [1213]

Recently we have shown that alkaline peroxide pretreatment

under optimized conditions released about 90 of the sugars

present in rice hulls [14] However alkaline peroxide pre-

treatment of rice hulls under the conditions used is not cost-

effective Lime pretreatment has been studied on various

biomass substrates such as switchgrass corn stover wood

and municipal wastes [15ndash18] Lime offers certain advantages

such as inexpensive (006 $ kg1) safe to handle and can be

recovered easily In the present study the efficacies of lime as

pretreatment option enzymatic saccharification and fermen-

tation of lime-pretreated rice hulls were examined

The production of ethanol from any lignocellulosic biomass

generally involves four process stepsmdashfeedstock pretreat-

ment enzymatic saccharification fermentation and ethanol

recovery [19] In order to reduce the cost of ethanol produc-

tion from lignocellulosic biomass integration of these process

steps is essential Moreover during enzymatic saccahrifica-

tion the cellulases and hemicellulases are severely inhibited

by their own products (sugars) [20] In order to relieve the

product inhibition simultaneous saccharification and fer-

mentation (SSF) of the pretreated hydrolyzate is preferred

where the fermentative microorganism would convert the

sugars into ethanol as soon as they are formed However the

optimal conditions (mainly pH and temperature) for enzy-

matic saccharification and fermentation are different [21] In

this paper the ethanol production by a recombinant Escher-

ichia coli strain from lime-pretreated and enzyme-hydrolyzed

rice hulls (separate hydrolysis and fermentation SHF) and

also from SSF of lime-pretreated rice hulls has been

presented

2 Materials and methods

21 Materials

Rice hulls (rough grade) were purchased from Rice Hull

Specialty Products Inc Stuttgart AR They were dried in a

forced-air oven at 55 1C for 24 h and milled in a hammer mill

to pass through a 127-mm screen The milled rice hulls were

stored at room temperature Celluclast 15 L and Novozym 188

were purchased from Brenntag Great Lakes Milwaukee WI

Glucose xylose arabinose Tween 20 furfural hydroxymethyl

furfural (HMF) and lime (calcium hydroxide) were purchased

from Sigma Chemical Co St Louis MO Viscostar 150 L was

supplied by Dyadic Corp Jupiter FL Aminex HPX 87P column

(30078 mm2) Aminex HPX 87 H column (30078 mm2)

De-ashing cartridge (3046 mm2) Carbo-P micro-guard car-

tridge (3046 mm2) and Cation H micro-guard cartridge

(3046 mm2) were purchased from Bio-Rad Laboratories

Inc Hercules CA All other chemicals used were of analytical

grades

22 Pretreatment

Unless otherwise stated milled rice hulls (150 wv) and

lime (15 wv) were slurried in water mixed and autoclaved

at 121 1C for 1 h The pH of the lime-pretreated rice hulls was

adjusted to 50 using concentrated HCl before performing

enzymatic saccharification

23 Enzyme assays

Carboxymethyl cellulase (CMCase) b-glucosidase xylanase

b-xylosidase a-L-arabinofuranosidase and ferulic acid ester-

ase activities were assayed using 1 (wv) carboxymethyl

cellulose 4 mM p-nitrophenyl b-D-glucoside 1 (wv) oat

spelt xylan 2 mM p-nitrophenyl b-D-xyloside 1 mM p-nitro-

phenyl a-L-arabinofuranoside and 09 mM methyl ferulate

respectively as substrate by the procedures described pre-

viously [11] One unit (U) of each enzyme activity is defined as

the amount of enzyme which produces 1 mmole product in the

reaction mixture per min under the assay conditioned used

24 Enzymatic saccharification

The enzymatic saccharification of the lime-pretreated rice

hulls was performed by shaking gently (100 rpm) at 45 1C after

adjusting the pH to 50 with HCl and adding a cocktail of three

commercial enzyme preparations at each enzyme dose of

005 ml g1 of rice hulls for 72 h unless otherwise stated

Samples (1 ml) were withdrawn and kept at 20 1C until

analyzed

25 Bacterial strain and preparation of seed culture

Recombinant E coli strain FBR5 maintained in glycerol vials

at 20 1C was used as a working stock [22] It was plated on

Luria broth (LB 10 g tryptone 5 g yeast extract and 5 g NaCl)

containing 40 g xylose and 20 mg tetracycline solidified with

15 g agar l1 Plates were incubated at 35 1C Cells from a single

well-isolated colony were inoculated into a 125 ml flask

containing 100 ml of LB supplemented with 2 g xylose and

2 mg tetracycline and grown at 35 1C and 100 rpm for 24 h to

use as seed culture

26 Fermentation

For SHF the fermentation was performed in a pH-controlled

500 ml fleaker with a working volume of 350 ml under

semianaerobic condition at pH 65 35 1C and 130 rpm

ARTICLE IN PRESS

Table 1 ndash Activity level of three commercial enzymepreparations used in lime pretreated rice hulls hydrolysis

Enzymea Activity (U ml1)

Celluclast

(cellulase)

Novozym 188

(b-glucosidase)

Viscostar 150L

(hemicellulase)

B I O M A S S A N D B I O E N E R G Y 3 2 ( 2 0 0 8 ) 9 7 1 ndash 9 7 7 973

essentially as described previously [23] The liquid portion of

the lime-pretreated enzymatically saccharified (at pH 50 and

45 1C) rice hull hydrolyzate after separating it from the solids

by filtration over glass fiber filter (10ndash15mm pore size 75 mm

diameter Nalgene Rochester NY) was used as substrate The

medium was prepared by dissolving 10 g tryptone and 5 g

yeast extract in 1 l hydrolyzate and autoclaving at 121 1C for

15 min The pH was controlled at 65 using 4 M KOH Samples

were withdrawn periodically to determine cell density

ethanol organic acids and residual sugars and stored at

20 1C prior to analysis Base consumption and pH were also

recorded For SSF 2 l fermenter (Biostat B B Braun Biotech-

nology International Allentown PA) with a working volume

of 15 l was used at pH 60 and 35 1C at the agitation rate of

150 rpm The lime-pretreated whole rice hull slurry was

added to the fermenter as substrate after adjusting the pH

to 60 with concentrated HCl and autoclaved for 15 min at

121 1C before adding the filter-sterilized enzyme cocktail and

seed culture Inoculum size was 5 (vv) in both cases

27 Analytical procedures

Cell growth of the recombinant E coli was monitored by

measuring the optical density of the appropriately diluted

culture broth at 660 nm Sugars ethanol acetic acid succinic

acid furfural and HMF were analyzed by using high-pressure

liquid chromatography (HPLC) The separation system

consisted of a solvent delivery system (P2000 pump Spectra-

Physics San Jose CA) equipped with an autosampler

(Model 717 Waters Chromatography Division Millipore Corp

Milford MA) a refractive index detector (Model 410 differ-

ential refractometer Waters) a dual l absorbance detector

(Model 2487 Waters) and a computer software-based integra-

tion system (Chromquest 40 Spectra-Physics) Two ion-

moderated partition chromatography columns (Aminex

HPX-87P with De-ashing and Carbo-P micro-guard cartridges

Aminex HPX 87H with Cation H micro-guard cartridge) were

used The Aminex HPX-87P column was maintained at 85 1C

and the sugars were eluted with filtered (Milli-Q Millipore

Corp Bedford MA) deionized water at a flow rate of

06 ml min1 The Aminex HPX-87 H column was maintained

at 65 1C and the sugars organic acids furfural HMF and

ethanol were eluted with 10 mM HNO3 prepared using filtered

deionized water at a flow rate of 06 ml min1 Peaks were

detected by refractive index (glucose xylose galactose

arabinose ethanol acetic acid succinic acid and ethanol) or

UV absorption at 277 nm (furfural and HMF) and were

identified and quantified by comparison to retention times

of authentic standards

Carboxymethyl

cellulase

941742 770 698763

b-Glucosidase 3870 68677 8170

Xylanase 103972 6777 119497639

b-Xylosidase 4671 770 3070

a-L-arabino-

furanosidase

870 1470 1670

Ferulic acid

esterase

1370 8273 170

a At pH 50 and 50 1C

3 Results and discussion

31 Effect of lime dose and duration of pretreatment onenzymatic saccharification of rice hulls

Rice hulls used in this study contained 356701 cellulose

and 120707 hemicellulose (total carbohydrate content

476708) 154702 lignin 187700 ash and 62700

moisture [9] Three commercial enzyme preparations

(Celluclast 15 L cellulase preparation Novozym 188

b-glucosidase preparation Viscostar 150 L hemicellulase

preparation) were used The activity levels of CMCase

b-glucosidase xylanase b-xylosidase a-L-arabinofuranosi-

dase and ferulic acid esterase in each of these enzyme

preparations are presented in Table 1 Initially the effects of

lime doses (25 50 and 100 mg g1 rice hulls) on the pretreat-

ment of rice hulls (150 wv) for 6 min 30 min and 1 h at

121 1C were evaluated The resultant yield of glucose and total

sugars in terms of mg g1 rice hulls after enzymatic sacchar-

ification using the cocktail of three commercial enzyme

preparations at a dose level of 005 ml of each enzyme

preparation g1 substrate at 45 1C and pH 50 for 72 h is shown

in Fig 1A and B respectively The yields of glucose as well as

total sugars increased with increasing lime concentration for

pretreatment There was not much difference between 30 min

and 1 h pretreatments The yield of total sugars (g1) was

increased from 8571 to 12671 mg (47 increase) with the

increase of lime dose from 25 mg to 100 mg g1 of hulls for 1 h

pretreatment Using the same lime dose (100 mg g1 rice

hulls) the yield of total sugars increased from 9271 to

12671 mg (36 increase) by increasing the pretreatment time

from 6 min to 1 h The maximum yield of total sugars

(12671 mg g1 hulls glucose 9371 mg xylose 2770 mg

arabinose 670 mg 32 conversion) was achieved at

100 mg lime g1 hulls and 1 h pretreatment time Thus it

was decided to use 100 mg lime g1 of hulls and pretreatment

time of 1 h for subsequent studies No galactose (detectable

limit 25mg ml1 by HPLC) was detected in any of the lime-

pretreated hydrolyzates even though acid pretreatment

released 16 mg galactose g1 of rice hulls [11] No furfural or

HMF (detectable limit 1 mg ml1) was detected in any of the

lime-pretreated rice hull hydrolyzates The enzymatic sac-

charification (45 1C pH 50 72 h) of pretreated rice hulls

without lime (1 h 121 1C control using water instead of lime)

using the same enzyme cocktail generated 4772 mg glucose

570 mg xylose 070 mg arabinose and total sugars

5272 mg g1 hulls (11 conversion) Thus lime pretreatment

aided in 24-fold increase in the saccharification of rice hulls

by enzymes over the control (without lime)

ARTICLE IN PRESS

Fig 1 ndash Effect of lime dose (25 50 100 mg g1) and duration

(6 30 60 min) of pretreatment at 121 1C on the enzymatic

saccharification of pretreated rice hulls (150 wv) using a

cocktail of three commercial enzyme preparations

(cellulase b-glucosidase and hemicellulase) at each enzyme

preparation dose level of 005 ml g1 hulls at pH 50 and 45 1C

for 72 h The data presented are averages of two individual

experiments (A) glucose (B) total sugars

pH35 40 45 50 55 60 65 70 75

Suga

r (m

g g-1

)

0

20

40

60

80

100

120

140

Fig 2 ndash Effect of pH on the enzymatic saccharification of

lime-pretreated (100 mg g1 121 1C 1 h) rice hulls (150

wv) using a cocktail of three commercial enzyme

preparations (cellulase b-glucosidase and hemicellulase) at

each enzyme dose level of 005 ml g1 hulls at 45 1C for 72 h

The data presented are averages of two individual

experiments Symbols glucose (K) xylose () and total

sugars (rsquo)

Temperature (degC)20 25 30 35 40 45 50 55 60 65 70

Suga

r (m

g g-1

)

0

20

40

60

80

100

120

140

Fig 3 ndash Effect of temperature on the enzymatic

saccharification of lime-pretreated (100 mg g1 121 1C 1 h)

rice hulls (15 wv) at pH 50 for 72 h using a cocktail

of three commercial enzyme preparations (cellulase

b-glucosidase and hemicellulase) at each enzyme dose level

of 005 ml g1 hulls The data presented are averages of two

individual experiments Symbols glucose (K) xylose ()

and total sugars (rsquo)

B I O M A S S A N D B I O E N E R G Y 3 2 ( 2 0 0 8 ) 9 7 1 ndash 9 7 7974

32 Effect of pH and temperature on enzymaticsaccharification of lime-pretreated rice hulls

The effects of pH (35ndash65) and temperature (25ndash70 1C) on the

enzymatic hydrolysis of lime-pretreated (100 mg g1 sub-

strate 121 1C 1 h) rice hulls (150 wv) using the three-

enzyme cocktails at each enzyme preparation dose level of

005 ml g1 substrate were investigated Figs 2 and 3 show the

release of glucose xylose arabinose and total sugars at 72 h

for various pH and temperatures respectively The combined

enzyme cocktail worked well over a pH range of 40ndash55 with

an optimum pH of 50 for the release of all sugars (Fig 2) The

relative sugar yields at pH 60 and 65 were 83 and 56 of the

maximum level observed at pH 50 and the enzyme cocktail

worked better at the lower pH side of optimum pH 50 than at

the higher pH side With regard to temperature the three-

enzyme combination worked optimally at 45 1C (Fig 3) The

yields of total sugars at 37 50 and 60 1C were 96 87 and

57 of that at 45 1C respectively Thus pH 50 and 45 1C are

optimal for saccharification of lime-pretreated rice hulls by

the enzyme cocktail

33 Effect of enzyme combinations on saccharification oflime-pretreated rice hulls

The effect of eight different combinations of the three

commercial enzyme preparations on the saccharification of

lime-pretreated rice hulls at 45 1C and pH 50 for 72 h is

ARTICLE IN PRESS

Table 2 ndash Effect of three-enzyme combination (cellulase b-glucosidase and hemicellulase preparations) on enzymatichydrolysis of lime-pretreated rice hulls at 45 1C and pH 50 for 72 h

Celluclast(ml g1)

Novozym(ml g1)

Viscostar(ml g1)

Glucose(mg g1)

Xylose(mg g1)

Arabinose(mg g1)

Total sugars(mg g1)

15 15 15 5973 1971 570 8374

150 15 15 7773 2770 670 11073

15 150 15 7772 2571 770 10971

15 15 150 9675 3173 770 13478

150 150 15 8174 2771 870 11676

15 150 150 10573 3271 870 14574

150 15 150 9573 2971 870 13276

150 150 150 10972 3671 970 15471

Rice hulls (150 wv) were pretreated with lime (15 wv) at 121 1C for 1 h The pH was adjusted to 50 and enzyme combinations were added

The data presented are averages of two individual experiments

Time (h)0 12 24 36 48 60 72 84 96 108 120

Suga

r (m

g g-1

)

0

20

40

60

80

100

120

140

160

Fig 4 ndash Time course of enzymatic saccharification of lime-

pretreated (100 mg g1 121 1C 1 h) rice hulls (150 wv)

using a cocktail of three commercial enzyme preparations

(cellulase b-glucosidase and hemicellulase) at each enzyme

preparation dose level of 015 ml g1 hulls at 45 1C and pH

50 The data presented are averages of two individual

experiments Symbols glucose (K) xylose () arabinose

(E) and total sugars (rsquo)

Time (h)0 6 12 18 24 30 36 42 48

Suga

r or E

than

ol (g

l-1)

0

4

8

12

16

20

24

Cel

l Den

sity

(A66

0nm

)

0

1

2

3

4

5

6

7

8

Fig 5 ndash Time course of ethanol production by recombinant

Escherichia coli FBR5 from lime-pretreated (100 mg g1

121 1C 1 h) enzymatically saccharified (45 1C pH 50 72 h)

rice hull (150 wv) hydrolyzate at pH 65 and 35 1C The

data presented are averages of two individual experiments

Symbols glucose (K) xylose () arabinose (E) total sugars

(rsquo) ethanol (m) and cell density (amp)

B I O M A S S A N D B I O E N E R G Y 3 2 ( 2 0 0 8 ) 9 7 1 ndash 9 7 7 975

presented in Table 2 It is evident that for maximum release of

sugars the three-enzyme combination performed better than

any two enzyme combinations The time course of the release

of each sugar (glucose xylose and arabinose) as well as total

sugars g1 of rice hulls by the three-enzyme cocktail using

each enzyme preparation at a dose level of 015 ml g1

substrate at 45 1C and pH 50 is presented in Fig 4 The yield

of total sugars at 72 h was 15471 mg (glucose 10972 mg

xylose 3671 mg arabinose 970 mg) g1 hulls which is about

32 of the total carbohydrates present in the hulls The data

indicate that only 31 cellulose and 37 hemicellulose of

lime-pretreated rice hulls were saccharified The total sugar

yields were 12173 mg (glucose 9473 mg xylose 2070 mg

arabinose 670 mg) g1 hulls (25 conversion) after 6 h and

14073 mg (glucose 10273 mg xylose 3070 mg arabinose

870 mg) g1 rice hulls (29 conversion) after 24 h These data

clearly indicate that both cellulose and hemicellulose sac-

charification rates were much higher during initial reaction

times After longer time incubation (120 h) the yield of total

sugars increased only negligibly to 15672 mg g1 hulls This

indicates that enzymes are not effective in hydrolyzing

completely both cellulose and hemicellulose of lime-pre-

treated rice hulls under the conditions used

34 Effect of surfactant on enzymatic saccharification oflime-pretreated rice hulls

Tween 20 is known to enhance the enzymatic saccharification

of cellulose [2425] The effect of Tween 20 (0 125 and 25 g l1)

on the enzymatic action at two enzyme dose levels (005 and

015 ml of each enzyme preparation g1 substrate) was tested

Addition of Tween 20 did not have any effect on the release of

sugars from lime-pretreated rice hulls (data not shown) The

ARTICLE IN PRESS

Table 2 ndash Effect of three-enzyme combination (cellulase b-glucosidase and hemicellulase preparations) on enzymatichydrolysis of lime-pretreated rice hulls at 45 1C and pH 50 for 72 h

Celluclast(ml g1)

Novozym(ml g1)

Viscostar(ml g1)

Glucose(mg g1)

Xylose(mg g1)

Arabinose(mg g1)

Total sugars(mg g1)

15 15 15 5973 1971 570 8374

150 15 15 7773 2770 670 11073

15 150 15 7772 2571 770 10971

15 15 150 9675 3173 770 13478

150 150 15 8174 2771 870 11676

15 150 150 10573 3271 870 14574

150 15 150 9573 2971 870 13276

150 150 150 10972 3671 970 15471

Rice hulls (150 wv) were pretreated with lime (15 wv) at 121 1C for 1 h The pH was adjusted to 50 and enzyme combinations were added

The data presented are averages of two individual experiments

Time (h)0 12 24 36 48 60 72 84 96 108 120

Suga

r (m

g g-1

)

0

20

40

60

80

100

120

140

160

Fig 4 ndash Time course of enzymatic saccharification of lime-

pretreated (100 mg g1 121 1C 1 h) rice hulls (150 wv)

using a cocktail of three commercial enzyme preparations

(cellulase b-glucosidase and hemicellulase) at each enzyme

preparation dose level of 015 ml g1 hulls at 45 1C and pH

50 The data presented are averages of two individual

experiments Symbols glucose (K) xylose () arabinose

(E) and total sugars (rsquo)

Time (h)0 6 12 18 24 30 36 42 48

Suga

r or E

than

ol (g

l-1)

0

4

8

12

16

20

24

Cel

l Den

sity

(A66

0nm

)

0

1

2

3

4

5

6

7

8

Fig 5 ndash Time course of ethanol production by recombinant

Escherichia coli FBR5 from lime-pretreated (100 mg g1

121 1C 1 h) enzymatically saccharified (45 1C pH 50 72 h)

rice hull (150 wv) hydrolyzate at pH 65 and 35 1C The

data presented are averages of two individual experiments

Symbols glucose (K) xylose () arabinose (E) total sugars

(rsquo) ethanol (m) and cell density (amp)

B I O M A S S A N D B I O E N E R G Y 3 2 ( 2 0 0 8 ) 9 7 1 ndash 9 7 7 975

presented in Table 2 It is evident that for maximum release of

sugars the three-enzyme combination performed better than

any two enzyme combinations The time course of the release

of each sugar (glucose xylose and arabinose) as well as total

sugars g1 of rice hulls by the three-enzyme cocktail using

each enzyme preparation at a dose level of 015 ml g1

substrate at 45 1C and pH 50 is presented in Fig 4 The yield

of total sugars at 72 h was 15471 mg (glucose 10972 mg

xylose 3671 mg arabinose 970 mg) g1 hulls which is about

32 of the total carbohydrates present in the hulls The data

indicate that only 31 cellulose and 37 hemicellulose of

lime-pretreated rice hulls were saccharified The total sugar

yields were 12173 mg (glucose 9473 mg xylose 2070 mg

arabinose 670 mg) g1 hulls (25 conversion) after 6 h and

14073 mg (glucose 10273 mg xylose 3070 mg arabinose

870 mg) g1 rice hulls (29 conversion) after 24 h These data

clearly indicate that both cellulose and hemicellulose sac-

charification rates were much higher during initial reaction

times After longer time incubation (120 h) the yield of total

sugars increased only negligibly to 15672 mg g1 hulls This

indicates that enzymes are not effective in hydrolyzing

completely both cellulose and hemicellulose of lime-pre-

treated rice hulls under the conditions used

34 Effect of surfactant on enzymatic saccharification oflime-pretreated rice hulls

Tween 20 is known to enhance the enzymatic saccharification

of cellulose [2425] The effect of Tween 20 (0 125 and 25 g l1)

on the enzymatic action at two enzyme dose levels (005 and

015 ml of each enzyme preparation g1 substrate) was tested

Addition of Tween 20 did not have any effect on the release of

sugars from lime-pretreated rice hulls (data not shown) The

ARTICLE IN PRESS

Table 3 ndash Ethanol production from lime-pretreated rice hull hydrolyzate by recombinant Escherichia coli strain FBR5 at 35 1C

Hydrolyzate Fermentation time(h)

Total sugars(g l1)

Ethanol(g l1)

Ethanol(g g1 sugar)

Separate hydrolysis and fermentation (SHF) 19 198706 98705 049

Simultaneous saccharification and fermentation

(SSF)

53 ndash 110710 ndash

The medium contained hydrolyzates from 138 g rice hulls l1 Rice hulls (150 wv) were pretreated with lime (15 ww) at 121 1C for 1 h

Enzymatic hydrolysis was performed using cellulase (Celluclast 15 L) b-glucosidase (Novozym 188) and hemicellulase (Viscostar 150 L) at 45 1C

and pH 50 for 72 h Each enzyme preparation used 015 ml g1 rice hulls Fermentation experiments were performed at pH 65 for SHF and pH

60 for SSF The data presented are averages of two individual experiments

B I O M A S S A N D B I O E N E R G Y 3 2 ( 2 0 0 8 ) 9 7 1 ndash 9 7 7976

reason is not clear but the exact mechanism of enhancement

of enzymatic hydrolysis of cellulose by Tween 20 is also not

clear It plays an important role in preventing the non-specific

binding of cellulases to lignin residues allowing more

enzymes to be available for the conversion of cellulose

resulting in a higher conversion rate [25] In comparison

Tween 20 enhanced the enzymatic hydrolysis of lime-

pretreated wheat straw by 10 using the same enzyme

cocktail at the dose level of 005 ml of each enzyme g1

substrate [26] This result emphasizes the importance of the

effectiveness of feedstock pretreatment prior to enzymatic

hydrolysis

35 Separate saccharification and fermentationof lime-pretreated rice hulls

The time course of ethanol production by the recombinant

E coli strain FBR5 from lime-pretreated enzymatically hydro-

lyzed rice hulls is shown in Fig 5 The concentration of

ethanol from rice hull (138 g) hydrolyzate by the bacterium

was 98705 g l1 with a yield of 049 g g1 of available sugars

(007 g g1 rice hulls) in 19 h by SHF The bacterium produced

small amounts of succinic and acetic acids as by-products in

addition to ethanol The cell density (A660 nm) reached a

maximum of 65705 in 19 h after which it declined slightly to

62705 in 27 h There is little growth (A660 nm 0447002) of

the recombinant E coli strain in the control medium where

water was substituted for the hydrolyzate No detectable

ethanol succinic acid or acetic acid was found to be produced

in the control medium by the strain Regarding the mixed

sugar utilization by the bacterium glucose was utilized first

(Fig 5) Xylose utilization only began after almost all the

glucose was consumed Arabinose was slowly utilized from

the beginning but finished before xylose Similar patterns of

mixed sugar utilization were also observed in the cases of

fermentation of both dilute acid and alkaline peroxide-

pretreated enzymatically saccharified rice hulls hydrolyzates

by the recombinant bacterium [1114]

36 SSF of lime-pretreated rice hulls

For SSF the concentration of ethanol was 110710 g l1 in 53 h

(008 g g1 rice hulls) There was no accumulation of

glucose during the time period However there was slight

accumulation of xylose and arabinose during the initial phase

of the SSF These sugars disappeared slowly (data not shown)

Considering the time required for separate enzymatic hydro-

lysis (72 h) and fermentation (19 h) the total time of conver-

sion of pretreated hulls to ethanol by SHF was 99 (72+19) h

whereas the total time was 53 h in the case of SSF In this

respect SSF worked much better than SHF

The summary of the results of both SHF and SSF of lime-

pretreated rice hulls by the recombinant E coli strain FBR5 is

presented in Table 3 The SHF approach worked better than

the SSF method with respect to fermentation time

The fermentative microorganism is generally unable to

withstand the inhibitory compounds formed during typical

dilute acid and autohydrolysis pretreatments of any ligno-

cellulosic biomass at a high temperature and a detoxification

step can be used to improve fermentability of the hydrolyzate

[19] This was also true with our previous studies on the

fermentation of the dilute acid hydrolyzates of rice hulls and

wheat straw [1127] The inhibitor problem does not appear to

be an issue for fermentation of lime-pretreated rice hull

hydrolyzates since none were detected and fermentation

proceeded rapidly However the lime pretreatment is not

an option for converting rice hulls to sugars due to low

conversion yield Attempts will be made to study the

feasibility of using other inexpensive methods for pretreat-

ment of rice hulls

Acknowledgments

The authors thank Gregory J Kennedy for technical assis-

tance and Bruce S Dien for providing recombinant E coli

strain FBR5

R E F E R E N C E S

[1] Renewable Fuels Association httpwwwethanolrfaorgindustrystatisticsS December 2007

[2] International Rice Research Institute httpwwwrrriorgSDecember 2007

[3] US Department of Agriculture National Agricultural Statisticsservice httpnassusdagovQuickStatsPullData_USjspSDecember 2007

ARTICLE IN PRESS

B I O M A S S A N D B I O E N E R G Y 3 2 ( 2 0 0 8 ) 9 7 1 ndash 9 7 7 977

[4] US Department of Energy Energy Efficiency and RenewableEnergy httpwww1eereenergygovbiopmassethanol_yield_calculatorhtmlS December 2007

[5] Natarajan E Nordini A Rao AN Overview of combustion andgasification of rice husk in fluidized bed reactors Biomassand Bioenergy 199814533ndash46

[6] Sharma A Khare SK Gupta MN Hydrolysis of rice hull bycross-linked Aspergillus niger cellulase Bioresource Techno-logy 200178281ndash4

[7] Moniruzzaman M Ingram LO Ethanol production from diluteacid hydrolyzate of rice hulls using genetically engineeredEscherichia coli Biotechnology Letters 199820943ndash7

[8] Kumagai S Hayashi N Sasaki T Nakada M Shibata MFractionation and saccharification of cellulose and hemi-cellulose in rice hull by hot-compressed-water treatmentwith two-step heating Journal of Japan Institute of Energy200483776ndash81

[9] Vegas R Alonso JL Dominguez H Parajo JC Processing of ricehusk autohydrolysis liquors for obtaining food ingredientsJournal of Agricultural and Food Chemistry 2004527311ndash7

[10] Yanez R Alonso JL Parajo JC Enzymatic saccharification ofhydrogen peroxide-treated solids from hydrothermal pro-cessing of rice husks Process Biochemistry 2006411244ndash52

[11] Saha BC Iten LB Cotta MA Wu YV Dilute acid pretreatmentenzymatic saccharification and fermentation of rice hulls toethanol Biotechnology Progress 200521816ndash22

[12] Watanabe T Shida M Furuyama Y Tsukamoto K NakajimaT Matsuda K Structure of arabinoxylan of rice hullCarbohydrate Research 198312383ndash95

[13] Watanabe T Shida M Murayama T Furuyama Y Nakajima TMatsuda K Xyloglucans in cell walls of rice hull Carbohy-drate Research 1984129229ndash42

[14] Saha BC Cotta MA Enzymatic saccharification and fermen-tation of alkaline peroxide pretreated rice hulls to ethanolEnzyme and Microbial Technology 200741528ndash32

[15] Chang VS Burr B Holtzapple MT Lime pretreatment ofswitchgrass Applied Biochemistry and Biotechnology199763ndash653ndash19

[16] Chang VS Nagwani M Kim C-H Holtzapple MT Limepretreatment of crop residues bagasse and wheat strawApplied Biochemistry and Biotechnology 199874135ndash59

[17] Karr WE Holtzapple MT Using lime pretreatment to facil-itate the enzyme hydrolysis of corn stover Biomass andBioenergy 200018189ndash99

[18] Kim S Holtzapple MT Lime pretreatment and enzymatichydrolysis of corn stover Bioresource Technology 2005961994ndash2006

[19] Saha BC Lignocellulose biodegradation and applications inbiotechnology In Saha BC Hayashi K editors Lignocellulosebiodegradation Washington DC American Chemical So-ciety 2004 p 2ndash34

[20] Saha BC Enzymes in lignocellulosic biomass conversion InSaha BC Woodward J editors Fuels and chemicals frombiomass Washington DC American Chemical Society 1996p 46ndash56

[21] Wyman CE Spindler DD Grohman K Simultaneous sacchar-ification and fermentation of several lignocellulosic feed-stocks to fuel ethanol Biomass and Bioenergy 19923301ndash7

[22] Dien BS Nichols NN OrsquoBryan PJ Bothast RJ Development ofnew ethanologenic Escherichia coli strains for fermentation oflignocellulosic biomass Applied Biochemistry and Biotech-nology 200084ndash86181ndash6

[23] Bothast RJ Saha BC Flosenzier AV Ingram LO Fermentationof L-arabinose D-xylose and D-glucose by ethanologenicEscherichia coli Biotechnology Letters 199416401ndash6

[24] Kaar WE Holtzapple MT Benefits from tween duringenzymic hydrolysis of corn stover Biotechnology andBioengineering 19989419ndash27

[25] Eriksson T Borjesson J Tjerneld F Mechanism of surfactanteffect in enzymatic hydrolysis of lignocellulose Enzyme andMicrobial Technology 200231353ndash64

[26] Saha BC Cotta MA Enzymatic hydrolysis and fermentationof lime pretreated wheat straw to ethanol Journal ofChemical Technology and Biotechnology 200782913ndash9

[27] Saha BC Iten LB Cotta MA Wu YV Dilute acid pretreatmentenzymatic saccharification and fermentation of wheat strawto ethanol Process Biochemistry 2005403693ndash700