3 Biotech (2018) 8:15

https://doi.org/10.1007/s13205-017-1033-x

ORIGINAL ARTICLE

Bioethanol production from rice husk using different pretreatments

and fermentation conditions
Joshua Osuigwe Madu1 · Bolade Oyeyinka Agboola1

Received: 3 April 2017 / Accepted: 4 December 2017 / Published online: 11 December 2017
© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract
Bioethanol is an environmentally friendly alternative to petroleum energy sources. This study evaluated the effects of ­H2O,
HCl, NaOH and ­FeCl3 pretreated rice husk feedstocks on the production of bioethanol. The pretreatments were carried out
using water, 0.1 M HCl, NaOH and ­FeCl3 at 121 °C for 15 min, followed by simultaneous saccharification and fermentation
(SSF) as well as separate hydrolysis and fermentation (SHF). The raw and pretreated lignocellulosic feedstocks were analyzed
using Fourier transform infrared spectroscopy. Saccharification and fermentation were accomplished using Trichoderma ree-
sei cellulase and Saccharomyces cerevisiae, respectively. The products obtained after saccharification and fermentation were
collected and analyzed for reducing sugars and ethanol contents using 3,5-dinitrosalicylic acid and high-performance liquid
chromatography, respectively. Enzyme hydrolysis of the ­FeCl3 and HCl treated samples resulted in hydrolysates containing
3.845 and 3.402 mg/ml glucose equivalent, respectively. In all pretreatments, SSF for each pretreatment produced more
ethanol than the SHF method; the F ­ eCl3 pretreatment gave the highest ethanol yield of 3.011 ± 0.034 and 3.802 ± 0.041%
in the SHF and SSF methods, respectively. Utilization of ­FeCl3 pretreatment of rice husk is a potential option for bioethanol
production in the future.

Keywords Energy · Bioethanol · Pretreatment · Lignocellulosic biomass · Food and environment

Introduction that these food crops are modified to improve yield at very
short periods, in the future, utilization of these food sources
Globally, there is a rising demand for food and energy due for ethanol production will further complicate the already
to increasing human population; and this has stimulated existing food and energy crisis as human population con-
research into the development of alternative energy sources tinues to increase (de Fraiture et al. 2008; Koh and Ghazoul
that are sustainable, renewable, economically competitive, 2008). Therefore, utilization of lignocellulosic biomass is
environmentally friendly and do not compete with human seen as an attractive feedstock for future supplies of ethanol
food sources. Alternatives to petroleum-derived fuels such considering its great availability, low cost and non-competi-
as bioethanol are sought for to reduce the world’s depend- tion with human food sources (Kim and Dale 2004; Tilman
ence on non-renewable resources (Gray et al. 2006; Hahn- et al. 2006; Balat et al. 2009).
Hagerdal et al. 2007; Balat and Balat 2008). Significant Economic and environmental concerns are the major pro-
efforts are being made to enhance the production of ethanol pelling factors that have made the search for alternatives to
from lignocellulose feedstock and make this alternative, eco- fossil fuels a subject of intensive research; demands for these
nomically feasible and competitive with gasoline. alternative sources continue to increase and lignocellulosic
Currently, commercial bioethanol is derived from corn plant biomass have been identified as promising renewable
grains (starch) and sugarcane (sucrose) and these sources feedstock for biofuel production (Hannon et al. 2010; Kumar
compete for fertile land as well as food production. Except and Sahu 2013). Lignocellulosic biomass from plants is
cheap and readily available. However, its components are
* Bolade Oyeyinka Agboola difficult to ferment or degrade biologically depending on the
[email protected] nature and composition of a particular plant; but with care-
fully selected pretreatment methods, efficient enzymes for
1
Department of Petroleum Chemistry, American University the saccharification and optimized fermentation conditions;
of Nigeria, Yola, Nigeria

13
Vol.:(0123456789)
15 Page 2 of 6 3 Biotech (2018) 8:15

the cellulose and hemicellulose components of lignocellu- Organism used for fermentation and inoculum
losic biomass can be efficiently converted to ethanol con- preparation
sidering the large amounts of glucose monomers present in
them (Ibeto et al. 2011; Bhagwat et al. 2015; Welker et al. Commercial yeast (S. cerevisiae) was used for the bioetha-
2015). nol production. The yeast was maintained according to
In Nigeria, rice is widely cultivated principally for its the method described by Ishola et al. (2013). Prior to use,
grains and its husks are abundantly available as waste and a pre-culture of the S. cerevisiae used was grown on 1%
in few areas used as feeds for ruminant. Hence, rice husk are (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glu-
of little economic use presently; therefore, could be used as cose for 60 min and was washed four times at 8500 rpm
suitable lignocellulosic substrate for bioethanol production. using sterile normal saline.
The present study focuses on the potentials of rice husk
to produce bioethanol under different pretreatment and fer-
mentation methods. Simultaneous saccharification and fermentation
of pre‑treated rice husk feedstock

Materials and methods The fermentation medium described by Sun and Tao
(2010) consisting of 3 g/l of yeast extract; 0.25 g/l of urea;
Materials 0.25 g/l of calcium chloride; 0.25 g/l of magnesium sul-
fate and 2.5 g/l of potassium phosphate monobasic was
Trichoderma reesei ATCC 26921 Cellulase was from Sigma adapted for this study. The samples were loaded at 10%
Chemical Co., USA; bacteriological yeast extract powder w/v and where adjusted to pH 5, followed by sterilization
and peptone were from Fisher Scientific UK. Other reagents at 121 °C for 15 min and were allowed to cool to room
were of high purity and analytical grade from reputable temperature. For enzymatic hydrolysis, Sigma aqueous
vendors. cellulase enzyme solution from T. reesei ATCC 26921
with activity of ≥ 700 units/ml at a loading of 30 units/g
Raw materials of substrate was used for the hydrolysis and 2 ml of the S.
cerevisiae with optical density 0.5 was simultaneously and
Rice husk was collected from a rice mill located in Yola, aseptically transferred from the pre-culture to the fermen-
Adamawa State, Nigeria. The husks obtained were dried and tation media at 35 °C and 50 rpm in a shaker. Flasks were
of uniform particle size, it was stored in plastic container and maintained in a shaker for 48 h and samples were collected
kept at room temperature (27 ± 2 °C) until use. to determine the amount of sugars and ethanol produced.

Pretreatment of lignocellulosic samples

Separate hydrolysis and fermentation to ethanol
Rice husk samples were subjected to different pretreatments
at 10% w/v loading with water and 0.1 M of ­FeCl3, HCl, The samples were loaded at 10% w/v and adjusted to pH 5
and NaOH in triplicates at 121 °C for 15 min, the samples for enzymatic hydrolysis. Cellulase from T. reesei ATCC
were washed with tap water until a neutral pH was obtained 26921 aqueous enzyme solution of ≥ 700 units/ml at a load-
for all treatment. The pretreated-washed samples were oven ing of 30 units/g of substrate was used for the hydrolysis at
dried at 90 °C, overnight (12–18 h) and were used for further temperature of 50 °C and 50 rpm in a shaker for 48 h. After
studies. the hydrolysis, samples were centrifuged at 8500 rpm for
20 min and supernatants were incorporated fermentation
FTIR analysis of pre‑treated and raw samples media components described by Sun and Tao (2010), steri-
lized at 121 °C for 15 min and were allowed to cool to room
Evaluation of the chemico-structural changes that occurred temperature. The samples were then subjected to fermen-
with the different pretreatments were carried out on a BUCK tation using pre-cultured commercial active S. cerevisiae.
Scientific FTIR model 530. Two milligram of dried samples Two milliliters of the S. cerevisiae with optical density 0.5
were mixed with 250 mg of dried KBr, pressed to pellets and were used for each flask; conditions of fermentation were
scanned over the range 4000–600 cm−1 wavenumber, with a pH 5, temperature 35 °C, agitation 50 rpm for 48 h. The
4 cm−1 spectral resolution. The spectra (from the KBr pel- fermentation for each treatment was carried out in a 250-ml
lets) were used to evaluate the chemico-structural changes Erlenmeyer flask in triplicates. Samples were collected to
that occurred with the different pretreatments (Himmelsbach determine the residual sugars and ethanol content.
et al. 2002; Pavia et al. 2005).

13
3 Biotech (2018) 8:15 Page 3 of 6 15

Estimation and analysis of reducing sugars production (Vermaas et al. 2015). The treatments of the rice
and ethanol produced husk using water, 0.1 M of ­FeCl3, HCl, and NaOH resulted
in visible altered physical appearance and increased surface
The sugars produced during enzyme hydrolysis were esti- area compared to the untreated sample. In this form, when
mated using the 3,5-dinitrosalicylic acid (DNSA) method the biomass are exposed to the cellulase, they can be more
described by Miller (1959). For ethanol analysis, samples easily degraded to produce sugar, as most of the pretreat-
obtained after fermentation were centrifuged to separate the ment methods used are known to make the biomass more
biomass from the product and filtered through a 0.22-μm porous with increased surface area and easier for degrada-
membrane filter and then analyzed using the Agilent HPLC tion. Figure 1 shows the physical appearance of the rice
1200 Series equipped with an auto injector and isocratic husks before and after various pretreatments.
pump. HPLC analyses were carried out using the following Qualitative analysis of the chemico-structural changes
conditions: column, HyperSil BDS C18 RP; 10 µl of sample in lignocellulosic biomass using FTIR is well known. The
was injected into the HPLC system. The mobile phase was FTIR spectra of samples have been utilized in the investi-
0.01 M sulphuric acid pH 2; at a flow rate of 1.0 ml/min and gation of the relative loss or changes in the key chemical
the detection was set at a wavelength of 254 nm. components of samples in association with visible changes
in structure (Himmelsbach et al. 2002). The structural/com-
Statistical analysis positional changes which occurred as a result of the acid,
base, water and ­FeCl3 pretreatment of the rice husk indicates
The results obtained are expressed as mean ± standard error that all the treatment resulted to varying forms of structural
of mean (SEM) of three replicate experiments. Statistical alterations of the lignocelluloses biomass compared to the
analysis was performed by one way analysis of variance raw sample (Fig. 2a). The FTIR analysis of the raw and pre-
(ANOVA) followed by Tukey’s multiple comparison tests treated samples confirmed the cleavage of lignin-carbohy-
using graph pad prism 5. p values < 0.05 were considered drate complex. Previous studies on lignocellulose biomass
significant. showed that the structure of lignin–carbohydrate complex
can be identified by the FTIR spectrum with the frequencies
of around 1600, 1509, 1464 and 1422 (Chen et al. 2011;
Results and discussion Dai et al. 2015; Sun et al. 2003). Even though some slight
differences were observed in the peak values attributed to
Appearance of the samples lignin compared to values published in literature, prominent
before and after pretreatment bands occurred at 1600–1410 cm−1 for the raw sample but
were of lower intensity and slightly shifted positions in the
Production of bioethanol from plant lignocellulosic/cellu- treated samples. The raw (untreated) sample had a sharp and
losic feedstock is a practicable venture. However, there is intense peak at 1541 and 1650 cm−1 (Fig. 2b, c), however,
a major challenge with the structural/compositional vari- these were either lower or absent in the treated samples and
ability of different lignocellulose components of available can be related to the different experimental conditions to
feedstocks, as plant biomass contain lignin–carbohydrate which the husk were treated.
complex that gives rise to complex intractable crystalline The critical role played by pretreatment on the release of
structure (Pu et al. 2013; Sorek et al. 2014). This crystalline sugars and eventually improved yield and reduced cost have
structure of lignocellulose prevents cellulases from bind- resulted into investigation of novel pretreatment materials
ing onto the cellulose surfaces to liberate sugars for biofuel such as ­FeCl3 (Chen et al. 2015), and NaOH/Urea (Dai et al.

Fig. 1  Appearance of rice husk Untreated H2 O HCl NaOH FeCl3 Pretreated

before and after various treat- Sample Pretreated Pretreated Pretreated
ments

13
15 Page 4 of 6 3 Biotech (2018) 8:15

Fig. 2  a Complete spectra of Complete FTIR spectra for raw and treated rice husk
the raw and treated rice husk b a 150
and c are excerpt of overlapping 140
finger print region of the FTIR
130 FeCl3
spectra of raw rice husk, NaOH,
HCl, ­H2O and F ­ eCl3 pretreated 120 NaOH T reament
rick husk separated for ease of 110 HCl

Transmittance
comparison and clarity 100 H2O

90
Raw

80

70

60

50

40

30

3959.2 3319.2 2679.2 2039.2 1399.2 759.2

Wavenumber cm-1
b 140

120
Transmittance

100

-1
1524 cm
1744 cm -1

80 FeCl3
NaOH Treament -1
1651 cm
HCl
H2O
60
Raw

40
1889.6 1809.6 1729.6 1649.6 1569.6 1489.6 1409.6 1329.6
-1
Wavenumber cm

c 120

FeCl3
110 NaOH Treament
HCl
H2O
Transmittance

100
Raw

90

80

70
1554.4 1474.4 1394.4 1314.4
-1
Wavenumber cm

13
3 Biotech (2018) 8:15 Page 5 of 6 15

2015); these pretreatments are needed to render the cellu- and independent saccharification and fermentation is shown
lose and other carbohydrates components in plant biomass in Table 2. The rice husk being non-edible and of low eco-
accessible for enzymatic hydrolysis and ethanol fermenta- nomic profile makes the utilization of the plant part of
tion, thereby improving ethanol yield in fermentation (Jöns- advantage in ethanol production.
son and Martín 2016). The yields of ethanol from F ­ eCl3 and NaOH pretreat-
ments methods showed that these pretreatment methods on
Reducing sugars obtained from pretreatment rice husk did not produce substances which were capable
and enzymatic hydrolysis of inhibiting the yeast cells from converting the reducing
sugars to ethanol; as ­FeCl3 treated sample were observed
The choice of pretreatment takes into account the sugar- to have yielded the highest amount of reducing sugars
release pattern and the compatibility/suitability of these compared to other treatments. Furthermore, analysis of the
sugars in the overall process of ethanol production. Any pre- fermentation broth after fermentation indicates that most
treatment with much yield of inhibitors capable of inhibit- of the initially present reducing sugars were converted to
ing cellulase activity or hindering the fermenting organism ethanol via fermentation unlike the water treated samples
from growth is usually not considered suitable (Larissa et al. which still have much reducing sugars left (0.270 ± 0.018
2012). Table 1 shows the sugar content obtained from the and 0.232 ± 0.048 mg/ml for SHF and SSF, respectively)
rice husk biomass under SSF and SHF. HCl and ­FeCl3 pre- after fermentation. The inhibition of enzymatic activities
treatments gave high reducing sugars on treatment with the in the water treated fermenting media may be as a result of
­ eCl3 treat-
T. reesei cellulase, but the yield obtained for the F direct inhibition of catabolic enzymes, generation of reactive
ment was significantly (p < 0.05) higher compared to other oxygen species, decreased intracellular pH, ATP depletion,
treatments (Table 1). The difference between the amounts toxic anion accumulation (Westman et al. 2014). The ethanol
of sugar produced after the different pretreatments can be produced from water pretreatment was the lowest. However,
attributed to pretreatment and other by-products these pre- its yield was comparable to that of the HCl treatment. The
treatments produce. result of this water treatment could be advantageous to the
industry as no additional chemicals are required and may
Ethanol obtained from fermentation media also not require costly materials that are resistant to corro-
sion for the scale-up.
Yeast (S. cerevisiae) has been historically used for second- The SSF of samples resulted higher ethanol yield com-
generation bioethanol production because of its fermenta- pared to the SHF for the same treatment (Table 2). The SSF
tive capacity and ethanol tolerance. The yield of ethanol process combines the saccharification of cellulose and fer-
obtained from different treatments using both simultaneous mentation of glucose which diminishes end-product sugar

Table 1  Results of reducing sugar content of the sample for SHF and SSF
Treatments Reducing sugar contents of the samples (mg/ml)

Before fermentation After fermentation

HCl NaOH H2O FeCl3 HCl NaOH H2O FeCl3

SHF 3.402 ± 0.059ab 3.200 ± 0.116cd 2.624 ± 0.080ace 3.845 ± 0.083bde 0.126 ± 0.027 0.167 ± 0.054 0.270 ± 0.018h 0.089 ± 0.011h
f fg
SSF ND ND ND ND 0.158 ± 0.015 0.082 ± 0.004 0.232 ± 0.048 0.095 ± 0.019g

The sugar contents were determined after enzyme hydrolysis for SHF and after fermentation for SHF and SSF
ND not determined
Results are presented as mean ± SEM (n = 3). Values with the same superscripts on the same row are significantly different from each other
(p < 0.05)

Table 2  Ethanol contents of the Ethanol contents of the samples after fermentation (%)
samples after fermentation
Treatments HCl NaOH Water FeCl3

SHF 2.716 ± 0.079c 2.848 ± 0.010c 2.421 ± 0.011 3.011 ± 0.034

SSF 3.330 ± 0.017a 3.664 ± 0.058b 3.327 ± 0.017a 3.802 ± 0.041b

Results are presented as mean ± SEM (n = 3). Values with the same superscripts on the same row are not
significantly different from each other (p < 0.05)

13
15 Page 6 of 6 3 Biotech (2018) 8:15

inhibition and yields. In all fermentation methods, extension Hannon M, Gimpel J, Tran M, Rasala B, Mayfield S (2010) Biofuels
of fermentation periods up to 72 h resulted to the production from algae: challenges and potential. Biofuels 1(5):763–784
Himmelsbach DS, Khalili S, Akin DE (2002) The use of FT-IR micro-
of acetic acid for ­FeCl3 and NaOH pretreatments. spectroscopic mapping to study the effects of enzymatic retting of
flax (Linum usitatissimum L) stems. J Sci Food Agric 82:685–696.
https://doi.org/10.1002/jsfa.1090
Conclusion Ibeto CN, Ofoefule AU, Agbo KE (2011) A global overview of biomass
potentials for bioethanol production: a renewable alternative fuel.
Trends Appl Sci Res 6:410–425
In this study, four different thermochemical pretreatments Ishola MM, Jahandideh A, Haidarian B (2013) Simultaneous sacchari-
were performed on rice husk with the aim of evaluating the fication, filtration and fermentation (SSFF): a novel method for
potentials of each treatment for producing bioethanol and bioethanol production from lignocellulosic biomass. Bioresour
Technol 133:68–73
meeting the increasing demand of alternative sources of Jönsson LJ, Martín C (2016) Pretreatment of lignocellulose: forma-
energy with and noncompetitive with human food sources. tion of inhibitory by-products and strategies for minimizing their
Significant differences in the sugar-release patterns and etha- effects. Bioresour Technol 199:103–112
nol produced were observed, with ­FeCl3 and NaOH pro- Kim S, Dale BE (2004) Global potential bioethanol production from
wasted crops and crop residue. Biomass Bioenergy 26:361–375
ducing high quantities of ethanol. Optimization of the pre- Koh LP, Ghazoul J (2008) Biofuels, biodiversity, and people: under-
treatment, saccharification and fermentation condition will standing the conflicts and finding opportunities. Biol Conserv
prove its potential and feasibility to use as a good cellulosic 141:2450–2460
material for second-generation bioethanol production but Kumar K, Sahu O (2013) Hydrogen energy as advance renewable
resource. Sustain Energy 1(2):32–37
evaluation of the costs for these options are also required. Larissa C, Anuj KC, dos Santos Suzane, Milessi T, Felipe Antônio
FA, Wagner L, Costa F, das Gracas Almeida Felipe M, da Silva
Acknowledgements We are grateful to the departments of Petro- S (2012) Bioconversion of sugarcane biomass into ethanol: an
leum Chemistry, and Natural and Environmental Sciences, American overview about composition, pretreatment methods, detoxification
University of Nigeria Yola for providing all reagents and facilities used of hydrolysates, enzymatic saccharification, and ethanol fermenta-
for this work. tion. J Biomed Biotechnol. https://doi.org/10.1155/2012/989572
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination
Author contributions BOA, conceived, designed the study and criti- of reducing sugar. Anal Chem 31(3):426–428
cally revised the manuscript. JOM, performed analysis on all sam- Pavia DL, Lampman GM, Ktiz GS, Engel GR (2005) Introduction to
ples, interpreted the results and drafted the manuscript. Both authors organic laboratory techniques, a small scale approach, 2nd edn.
approved the final version of the manuscript. Thomson Brooks/Cole, Belmont, CA
Pu Y, Hu F, Huang F, Davison BH, Ragauskas AJ (2013) Assessing
the molecular structure basis for biomass recalcitrance during
dilute acid and hydrothermal pretreatments. Biotechnol Biofuels
References 6(15):13. https://doi.org/10.1186/1754-6834-6-15
Sorek N, Yeats TH, Szemenyei H, Youngs H, Somerville CR (2014)
Balat M, Balat H (2008) Recent trends in global production and utiliza- The implications of lignocellulosic biomass chemical composition
tion of bio-ethanol fuel. Appl Energy 86:2273–2282 for the production of advanced biofuels. Bioscience 64:192–201
Balat M, Balat H, Oz C (2009) Progress in bioethanol processing. Progr Sun WL, Tao WY (2010) Comparison of cell growth and ethanol pro-
Energy Combust Sci 34:551–573 ductivity on different pretreatment of rice straw hemicellulose
Bhagwat S, Ratnaparkhe S, Kumar A (2015) Biomass pre-treatment hydrolysate by using Candida shehatae CICC 1766. Afr J Micro-
methods and their economic viability for efficient production of biol 4(11):1105–1109
biofuel. Br Biotechnol J 8(2):1–17 Sun JX, Sun XF, Sun RC, Paul F, Mark SB (2003) Inhomogeneities in
Chen WH, Tu YJ, Sheen HK (2011) Disruption of sugarcane bagasse the chemical structure of sugarcane bagasse lignin. J Agric Food
lignocellulosic structure by means of dilute sulfuric acid pre- Chem 51:6719–6725
treatment with microwave-assisted heating. Appl Energy Tilman D, Hill J, Lehman C (2006) Carbon-negative biofuels from low-
88:2726–2734 input high-diversity grassland biomass. Science 314:1598–1600
Chen L, Chen R, Fu S (2015) ­FeCl3 pretreatment of three lignocel- Vermaas JV, Petridis L, Qi X, Schulz R, Lindner B, Smith JC (2015)
lulosic biomass for ethanol production. ACS Sustain Chem Eng Mechanism of lignin inhibition of enzymatic biomass decon-
3:1794–1800. https://doi.org/10.1021/acssuschemeng.5b00377 struction. Biotechnol Biofuels 8:217. https://doi.org/10.1186/
Dai Y, Si M, Chen Y, Zhang N, Zhou M, Liao Q, Shi D, Liu Y (2015) s13068-015-0379-8
Combination of biological pretreatment with NaOH/Urea pretreat- Welker CM, Balasubramanian VK, Petti C, Rai KM, DeBolt S, Mendu
ment at cold temperature to enhance enzymatic hydrolysis of rice V (2015) Engineering plant biomass lignin content and composi-
straw. Bioresour Technol 198:725–731 tion for biofuels and bioproducts. Energies 8:7654–7676
de Fraiture C, Giordano M, Liao Y (2008) Biofuels and implications Westman JO, Mapelli V, Taherzadeha MJ, Franzénb CJ (2014) Floccu-
for agricultural water use: blue impacts of green energy. Water lation causes inhibitor tolerance in Saccharomyces cerevisiae for
Policy 10(Supplement 1):67–81 second-generation bioethanol production. Appl Environ Microbiol
Gray KA, Zhao L, Emptage M (2006) Bioethanol. Curr Opin Chem 80(22):6908–6918. https://doi.org/10.1128/AEM.01906-14
Biol 10:141–146
Hahn-Hagerdal B, Galbe M, Gorwa-Grauslund MF, Liden G, Zacchi
G (2007) Bio-ethanol–the fuel of tomorrow from the residues of
today. Trends Biotechnol 24(12):549–556

13