CHAPTER THREE

3.0 MATERIAL AND METHODOLOGY

3.1 Materials

Materials used in these researches include reagents and apparatus

3.1.1 Reagent/Chemicals

- Boric acid

- Diethyl-ether

- Sulphuric acid

- DNS reagent

- Absolute ethanol

- Ammonium Sulphate

- Distilled water

3.1.2 Apparatus

- Sugarcane Bagasse

- Drying oven

- Muffle furnace

- Desiccator

- Digestion flask

- Filter paper

- Volumetric flask

- Aluminum foil

- UV-Spectrophotometer

- pH-meter

1
 - Shaking incubator

- Crucibles

3.2 Methods

3.2.1 Sample Collection

Sugarcane bagasse was collected from a local sugarcane mild at Tudun wada Kaduna

State Nigeria

3.2.2 Sample Preparation

The sample collected was air dried completely to remove all the moisture present in it

before drying in an oven at 105°C. The dried sample was grounded to fine powder and

sieved to obtained higher surface area for reaction. The powder was stored in a

polyethylene bag to prevent any possible degradation or spoilage at low temperature until

required.

3.2.3 Determination of Moisture Content

A crucible was weighed, and its mass was recorded as M 1, 2g of the sample was

transferred to the weighed crucibles, placed in an oven at temperature of 105 oC and

heated for 24hours. The process was repeated at one-hour interval until a constant weight

was attained. Therefore, the crucible was removed from the oven, cooled in a desiccator

containing silica gel and weighed as M2.

3.2.4 Determination of Ash Content

The ash of the sample was determined by weighing a crucible and charged with 2g of the

sample. The crucible and its content were put into a muffle furnace and heated at 600oc

for 3hours, cooled in a desiccator containing silica gel and weighed.

2
3.2.5 Determination of Nitrogen Content

[Link] Procedure:

2g of each dried sample was weighed into digestion flask and 0.5g of Kjeldahl table was

added followed by addition of 10cm3 of concentrated tetraoxosulphates (VI) acid. The

content was then heated in Kjeldahl digestion unit until the digest became clear in 2hours.

After the digestion had been completed, the flask was cooled, diluted with 10cm3

distilled water and filtered with a whatman No.1 filter paper into a 100cm3 volumetric

flask and made up to the mark with distilled water. 10cm3 of homogeneous aliquot

solution was pipetted into distillation flask and 20cm3 of 45% NaOH solution was added.

The content was diluted to about 200cm3 with distilled water using distillation apparatus.

The distillate was collected in receiving flask containing 10cm3 boric acid indicator

solutions. After the distillation, the distillate was titrated with standardized 0.01M HCL

to the end point. Blank was determined using all the reagent in the same quantities as

described above. The process was carried out in triplicate and the crude protein (CP)

3.2.6 Pretreatment of Sugarcane Bagasse

The pre-treatment was done on the sample as described below.

Three 1000 cm3 different volumetric flasks, fifty gram (50g) of the powdered sample

were transferred, 250cm3 of diethyl-ether was then added into each flasks (to obtain ratio

of 1:5w/v), covered with aluminum foil for 24hours in order to remove fat extractives,

and the residue left was washed with distilled water.

3.2.7 Testing for Glucose In the Hydrolyzed Bagasse

The reducing sugars were estimated by using DNS reagent. The glucose standard was

prepared by dissolving 0.1g of glucose into 100cm3 of distilled water, 10cm3 portion of

3
first standard was pipetted and transferred into another 100cm3 volumetric flask and

made to the mark using distilled water. To each tube 3cm3 of DNS reagent was added.

The content of each tube was placed in boiling water bath for 10minutes to develop red

brown colour. Then 1cm3 of 40% potassium sodium titrate solution was added to

stabilize the colour while hot then cooled at room temperature. The absorbance was

measured at 508nm with a UV-visible spectrophotometer.

4
 CHAPTER FOUR

4.0 RESULT AND DISCUSSION

4.1 RESULT

4.1.1 Determination of Moisture Content

Weight of sample =1g

Weight loss =0.9089g

%moisture content = weight of sample – weight loss (g) x 100%

Weight of sample

%moisture content = 1 – 0.9089 (g) x 100% = 0.0911 x 100% = 9.11%

%moisture content = 9.11%

4.1.2 Determination of Ash Content

Weight of sample =1g

Amount of Ash =0.2837g

%Ash content = Amount of Ash x 100%

Weight of sample

%Ash content = 0.2837g x 100% = 0.2837 x 100% = 28.37%

%Ash content = 28.37%

4.1.3 Determination of Nitrogen Content

Titre value for digest=a = 15cm3

Titre value for the blank =b =14.936 cm3

Volume to which the digest was made up=c = 14cm3

5
 Volume of aliquot used in distillation=d = 1.0cm3

Weight of dried sample=e =10g

%Nitrogen = (a-b) x 0.01M HCl x 14 x c x 100

dxe

%nitrogen = (15 – 14.936) x 0.01 x 14 x10 x100 = 8.96 = 0.896%

1.0 x 10 10

%nitrogen = 0.896%

4.1.4 Crude Protein

Crude protein = %nitrogen x 6.25

0.896% x 6.25 = 5.6%

4.1.5 Determination of Organic Matter

Organic matter = 100% - (%ash + %moisture)

Organic matter = 100% - (28.37% + 9.11%) = 100% - 37.48 % = 62.52%

Organic matter = 62.52%

4.1.6 Determination of Percentage Carbon

% Carbon = 0.58 x organic matter = 0.58 x 62.52 = 36.2616%

4.1.7 Determination of Carbon – Nitrogen Ratio

Carbon: Nitrogen = %organic carbon + %nitrogen

C:N = 36.2616% + 0.896%

C:N = 37.1576

6
4.1.8 Determination of pH

pH = 4.2

4.1.9 Quantification of Reducing Sugars

Glucose level = 13.2%

4.1.10 Quantitative Estimation of Ethanol

% Yield of Ethanol = Mass of Ethanol Yield x 100

Total Mass of Substrate

Mass of ethanol = density of ethanol x volume

Mass of ethanol = 0.8033g/ml x 99 ml

1000
Mass of ethanol = 0.0795g

Total mass of substrate = 0.7098g

% yield of ethanol = Mass of Ethanol Yield x 100

Total Mass of Substrate

% yield of ethanol = 0.0795g x 100

0.7098g

% yield of ethanol = 11.2%

7
Table 4.1.1: Result obtained from analysis of sugar cane

S/N PARAMETERS VALUE

1 Moisture content 9.11%
2 Ash content 28.37%
3 Nitrogen content 0.89%
4 Crude protein 5.56%
5 Organic matter 62.52%
6 Percentage carbon 36.26%
7 Carbon – Nitrogen ratio 37.16
8 Ph 4.2
9 Glucose level 13.2%
10 Yield of ethanol 11.2%

4.2 DISCUSSION

Lignocellulosis biomass cannot be saccharified by enzymes to yield high sugar with

pretreatment, mainly because the lignin in the plant biomass forms a barrier against
enzymes attack (Sewait et al., 1997) from the methodology the weight of sample is 2g.
At the end of the drying period an equilibrium weight was achieved at 0.9089g from the
initial weight of 1/2g. The weight loss which is the moisture content of the sample was
obtained to be 0.0911g. From these percentage moisture content of the sample was
calculated to be 9.11% which is within acceptable level of moisture content.

Ash content is the residue of the sample after completely burnt, or in the combustible
component remaining after the sample is completely burnt. Percentage Ash Content
obtained is 28.37% which represents amount of inorganic materials in the sample.

Kjeldahl method is a universally accepted method for determination of total nitrogen.

Nitrogen content corresponds to protein in content in a sample though as one of the
limitations of Kjeldahl method, the result is affected by non-protein nitrogen nitrogen.
From the procedure the percentage nitrogen content obtained is 0.896%. this signifies that
there is a relatively low amount of nitrogen in the sample.

8
It is a general assumption that pure protein should contain 16% Nitrogen. It is based on
Assumption that a Nitrogen –to-the-protein conversion factor of 6.25 (100/16) was
adopted. This general conversion factor is used because the non-protein nitrogen content
of a sample is calculated from the determined Nitrogen content by multiplying the
Nitrogen-to-the Protein conversion factor. Hence, the amount of crude protein present in
the sample is 5.6%.

The total composition of the sample include organic matter, inorganic matter and
moisture. The total percentage composition of the sample less the sum of percentage Ash
content and moisture content reveals the percentage organic matter of 62.52%. the high
organic matter content implies that the materials could also mineralized quickly and
benefit short lived (Bhadha et al., 2017).

Organic matter are carbon based compounds with about 58% of organic matter existing
as carbon. 36261% is the calculated percentage carbon content in the sample.
37.6% carbon Nitrogen Ratio, although a high C/N ration is usually required for lignin
degradation by fungi, nitrogen sources greatly affect this activity (Lopez et al., 2006).

4.2% pH also investigated by the influence of pH and continous product of Xylose from
sugar cane bagasse hemicellulosic hydrolysate by C. guilliermondii (Martinez et al.,
2003).

13.2 % Glucose level, the yield of conversion lignocellulosic materials into glucoseis
usually very low if the enzymatic hydrolysis is carried out without applying a
pretreatment (Luo et al., 2010).

Table 4.1.11 above shows the composition of sugar cane bagasse used for this study.
Carbon content of sugar cane biomass which accounts for about 35.26% of dry mass,
makes it as interesting feedstock for ethanol production . the powdered sample were first
treated with diethyl- ether before hydrolysis to remove fat extractive. Cellulose
molecules are composed of long chains of glucose molecule in the acid hydrolysis
process, this chains are broken down to free the sugar before it is fermented for alcohol

9
production. Acid hydrolysis was done with sulphuric acid to promote hemicellulose break
down in xylose and other sugar in the biomass.

Fermentation is a metabolic process that converts sugar to acid, gases and alcohol.
Microorganisms such as bacteria, yeast or fungi ferments carbohydrates to ethanol under
oxygen free conditions.

C6H12O16 + Yeast 2C2H5 + 2CO2

According to the reaction above, theoretical maximum yield is 0.5kg Ethanol and 0.49kg
carbondioxide per Kg sugar. For this study the preferred microorganisms for the
fermentation process is Saccharomyces cerevisiae .

11.2% Ethanol yield was shown in table 4.1.1 was obtained and the previous studies
suggested that sugar cane bagasses is the best substrate for ethanol production among
several tested subscripts under simultaneous saccharification and fermentation
(Shrivastava et al., 2014).

10
 CHAPTER FIVE

CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION

From the result obtained, the hydrolyzed sugarcane bagasse contains glucose in various

concentrations. Ethanol was produced from the hydrolyzed sugarcane bagasse and it can

be seen from result that 10cm3 of concentrated acid gave the optimum yield of ethanol at

a pH of 4.2 which is slightly acidic. The result obtained from analysis of sugarcane are

obtained as follows, 9.11%, 28.37%, 0.89%, 5.56%, 62.52%, 62.52%, 36.26%, and

37.16% respectively. A similar result was obtained by (Ballesteros et al., 2004) in SSF

experiments using acid hydrolysis.

5.2 RECOMMENDATION

This research have successfully proved that production of ethanol from sugarcane waste

is a good venture that could reduce unemployment in Nigeria where high sugarcane is

generated annually. There is the need for further research to improve the production of

high quality ethanol from sugarcane waste. The pretreatment of sugarcane waste or other

non-chemical treatment methods can be explored and used as an alternative to chemical

methods of treatment to save the environment from the dangers of these chemicals.

Finally, use of sugarcane waste for fermentation reduces environmental pollution.

11
References

Abril, D. and Abril, A. (2009). Ethanol from lignoce-llulosic biomass. Ciencia e

Investigación Agraria. vol. 36, 177-190.
Ajao A. T., Abdullahi H.J., Atere T. G. & Kolawole O. M. (2009). Studies on the
biodegradation and utilisation of selected tuber wastes by Penicillium expansum,
Bioresource Research Communication, Vol.21, No 5.
Alessandra Verardi, Isabella De Bari, Emanuele Ricca and Vincenza Calabrò (2012).
Hydrolysis of Lignocellulosic Biomass: Current Status of Processes and
Technologies and Future Perspectives, Bioethanol,Prof. Marco Aurelio Pinheiro
Lima (Ed.), Available
from:[Link]
biomass current-status-of-processes-and technologies-and-future-perspect.
Alkasrawi, M., Galbe, M., and Zacchi, G. (2002). “Recirculation of process streams in
fuel ethanol production from softwood based on simultaneous saccharification
and fermentation,” App. Biochem. and Biotech. 98-100, 849-861
Alonso Pippo W, Luengo CA, Alonsoamador Morales Alberteris L, Garzone P,
Cornacchia G. Energy recovery from sugarcane-trash in the light of 2nd
generation biofuels. Part 1: current situation and environmental aspects. Waste
Biomass Valoriz. 2011;2:1–16.
Arora A., Nain L. & Gupta J. K. (2005). Solid-state fermentation of wood residues by
Streptomyces griseus B1, a soil isolate, and solubilization of lignins. World
Journal of Microbiology and Biotechnology, 21(3): 303-308.
Bailey, B.K. (2005). Performance of ethanol as a transportation fuel. In: Wyman, C.E. ed.
Handbook on bioethanol: production and utilization. Taylor and Francis, Bristol,
p. 37-60.
Ballesteros, I.; Negro, J.M.; Oliva, J.M.; Cabañas, A.; Manzanares, P. and Ballesteros, M.
(2006). Etha-nol production from steam-explosion pretreated wheat straw.
Applied Biochemistry and Biotech-nology. vol. 129-132, 496-508.
Ballesteros, I.; Oliva, J.M.; Negro, M.J.; Manzanares, P. and Ballesteros, M. (2002).
Proceso de sacari-cación y fermentación simultánea para la conversión de la
fracción celulósica del residuo de laextracción del aceite de oliva en etanol.
Grasas y Aceites. vol. 53, 282-288.
Ballesteros, M., Oliva, J. M., Negro, M. J., Manzanares, P., and Ballesteros, I. (2004).
“Ethanol from lignocellulosic materials by a simultaneous saccharification and
fermentation process (SFS) with Kluyveromyces marxianus CECT 10875,”
Process Biochem. 39(12), 1843-1848
Barl, B., Biliaderis, C. G., Murray, E. D., and Macgregor, A. W. (1991). “Combined
chemical and enzymatic treatments of corn husk lignocellulosics,” J. Sci. Food
Agric. 56(2), 195-214.

12
Brooks A. A. (2008). Ethanol production potential of local yeast strains isolated from ripe
banana [Link] Journal of Biotechnology,7(20):3749-3752110.
Cara, C.; Ruiz, E.; Oliva, J.M.; Sáez, F. and Castro, E. (2008). Conversion of olive tree
biomass into fermentable sugars by dilute acid pretreatment and enzymatic
sacchariffication. Bioresource Techno-logy. vol. 99, 1869–1876.
Chen, Y.; Sharma-Shirappa, R.R.; Keshwani, D. and Chen, C. (2007). Potential of
agricultural residues for bioethanol production. Applied Biochemistry and
Biotechnology. vol. 142, 276-290.
Churdawat S.P., Venkatesh B. & Dale B. E. (.2007). Effect of particle size based
separation of milled corn stover on AFEX pretreatment and enzymatic
digestibility. Biotechnology and Bioengineering 96(2): 219-31.
Current Opinion in Biotechnology, 16:407– 415
Damisa D., Ameh J. & Umoh V. J. (2008). Effect of chemical pretreatment of some
lignocellulosic wastes on the recovery of cellulase from Aspergillus niger AH3
mutant. African Journal of Biotechnology, 7 (14): 2444-2450.
Das M. & Hossain S. K. M. (2000). Studies on lignin biodegration of non- conventional
lignocellulosic agro-waste material by using P. chrysosporium - A pollution
control biopulping process. J. Ind. Pollut. Control 16(2): 195-200.
Das M. & Hossain S. K. M. (2000). Studies on lignin biodegration of non- conventional
lignocellulosic agro-waste material by using P. chrysosporium - A pollution
control biopulping process. J. Ind. Pollut. Control 16.
Dawson L., Boopathy R. (2007). Use of post-harvest sugarcane residue for ethanol
production. Bioresource Technology, 98:1695 – 1699
Dhillon G. S., Bansal S. & Oberoi H. S. (2007). Cauliflower waste incorporation into
cane molasses improves ethanol production using Saccharomyces cerevisiae
MTCC 178. Indian Journal of Microbiology, 47:353– 357.
Elena Patrascu, Gabriela Rapeanu, Traian Hopulele (2009). Current Approaches to
Efficient Biotechnological Production of Ethanol. Innovative Romanian Food
Biotechnology, 4: 1-11.
Elijah I.O. (2010). Emerging Bio-ethanol Projects in Nigeria: Their Opportunities and
Challenges. Energy Policy Reviews. 38(11) 7161-7168.
Elijah I.O. (2010). Emerging Bio-ethanol Projects in Nigeria: Their Opportunities and
Challenges. Energy Policy Reviews. 38(11) 7161-7168.
Ferreira-Leitão, V.; Perrone, C.C.; Rodrigues, J.; Franke, [Link].; Macrelli, S. and
Zacchi, G. (2010). An approach to the utilisation of CO2 as impregnating agent in
steam pretreatment of sugar cane bagasse and leaves for ethanol production.
Biote-chnology for Biofuels. vol. 3, 7. doi:10.1186/1754-6834-3-7.
Geddes et al., 2011 C.C. Geddes, I.U. Nieves, L.O. Ingram Advances in ethanol
production Curr. Opin. Biotechnol., 22 (3) (2011), pp. 312-319
Geddes, C.C.; Mullinnix, M.T.; Nieves, I.U.; Peter-son, J.J.; Homan, R.W.; York, S.W.;
Yomano, L.P.; Miller, E.N.; Shanmugam, K.T. and Ingram, L.O. (2011).
13
 Simplified process for ethanol production from sugarcane bagasse using
hydrolysate resistant Escherichia coli strain MM160. Bioresource Technology.
vol. 102, 2702-11. doi:10.1016/[Link]-tech.2010.10.14.
Graeme M. Walker (2010). Bioet h an ol: Science an d t ech n ology of fuel
alcohol ,Ventus Publishing company Scotland. Pp 8–14.
Hendriks A. T. W. M. & Zeeman G. (2009). Pretreatments to enhance the digestibility of
lignocelluloses biomass. Bio resource Technology, 100(1)10-18.
Hendriks, A.T. and Zeeman, G. (2009) Pretreatments to Enhance the Digestibility of
Lignocellulosic Biomass. Bioresource Technology, 100, 10-18.
Inoue H, Yano S, Endo T, Sakaki T and Sawayama S (2008), Combining hot-compressed
water and ball milling pretreatments to improve the efficiency of the enzymatic
hydrolysis of eucalyptus, Biotechnology for biofuels 1(1): 2.
DOI:org/10.1186/1754-6834-1-2
Ivanova V., Petrova P. & Hristov J. (2011). Application in the ethanol fermentation of
immobilized yeast cells in matrix of alginate/magnetic nanoparticles, on chitosan-
magnetite microparticles and cellulose-coated magnetic nanoparticles.
International Review of Chemical Engineering , 3:289 –299.
Jorgensen H., Kristensen J. B., & Felby C. (2007). Enzymatic conversion of
lignocellulose into fermentable sugars: Challenges and opportunities. Biofuels,
Bio-production and Biorefining (1)119-134
Jørgensen, H., Vibe-Pedersen, J.; Larsen, J. and Felby, C. (2007). Liquefaction of
lignocellulose at high solids concentrations. Biotechnology and Bioen-ginnering.
vol. 96, 862-870
K. Olofsson, M. Bertilsson and G. Lidén, (2008)“A Short Review on SSF—An
Interesting Process Option for Ethanol Production from Lignocellulosic
Feedstocks
Kaar, W.E.; Gutierrez, C.V. and Kinoshita, C.M. (1998). Steam explosion of sugarcane
bagasse as a pretreatment for conversion to ethanol. Biomass and Bioenergy. vol.
14, 277-287.
Koon Ong L. (2004).Conversion of Lignocellulosic Biomass to Fuel Ethanol- A brief
review. Since cellulases are inhibited. The Planter, Kuala Lumpur , 80 (941)pp.
517-524
Laurence B Davin and Norman G Lewis (2005). Lignin primary structures and dirigent
sites,
Laurence B Davin and Norman G Lewis (2005). Lignin primary structures and dirigent
sites, Current Opinion in Biotechnology, 16:407 – 41.
Lebo Stuart E. Jr., Gargulak Jerry D., & McNally Timothy J. (2001). "Lignin".Kirk-
Othmer Encyclopedia of Chemical Technology. John Wiley & Sons, [Link]
104 – 109

14
Lee, Y.L.; Chung, C.H. and Day, D.F. (2009). Sugarcane bagasse oxidation using a
combination of hypochlorite and peroxide. Bioresource Technology. vol. 100,
935-941
Lin, Y. and Tanaka, S. (2006). Ethanol fermentation from biomass resources: current
state and prospects. Applied Microbiology and Biotechnololy. vol. 69, 627-642
Lin, Y. and Tanaka, S. (2013). Ethanol fermentation from biomass resources: current
state and pros-pects. Applied Microbiology and Biotechnololy. vol. 69, 627-642
Macrelli S, Galbe M, Wallberg O. Effects of production and market factors on ethanol
profitability for an integrated first and second generation ethanol plant using the
whole sugarcane as feedstock. Biotechnol Biofuels. 2014;7:26–41.
Martín, C.; Galbe, M.; Nilvebrant, N.O. and Jönsson, L.J. (2010). Comparison of the
fermentability of enzymatic hydrolyzates of sugarcane bagasse pre-treated by
steam explosion using different impregnating agents. Applied Biochemistry and
Biotechnology. vol. 98-100, 699-716
Martín, C.; Marcet, M. and Thomsen, A.B. (2008).
McMillan J. D., Himmel M. E., Baker J. O. & Overend R. P. (1994). Pretreatment
of lignocellulosic biomass. In Enzymatic Conversion of Biomass for Fuels
Production; American Chemical Society: Washington. DC, Pp 292-324
McMillan, (2010). "Pretreatment of lignocellulosic biomass," in ACS Symposium Series
(Enzymatic Conversion of Biomass for Fuels Production), Himmel, M. E., Baker,
J. O., and Overend, R. P. (eds.), 292-324.
Mohammed M. N. (2011). Determination of the chemical constituents of selected food
stuffs in Niger State, Nigeria, for the development of animal protein supplement,
PhD Thesis in Analytical chemistry Federal University of Technology, Minna, ,
Pp 195– 198
Mtui G. & Nakamura Y. (2005). Bioconversion of lignocellulosic waste from selected
dumping sites in Dares Salaam, Tanzania Biodegradation, 16(6): 493-499.
Mtui G. & Nakamura Y. (2008). Lignocellulosic enzymes from Flavodon flavus, a
fungus isolated from Western Indian Ocean off the Coast of Dar es Salaam,
[Link] Journal of Biotechnology, 7(17): 3066-3072.
Nasidi M., Blackwood D., Akunna J. & Walker G. M. (2010) Bioethanol in Nigeria:
comparison of sugar cane and sweet sorghum feedstocks. Energy and
Environmental Science DOI:10.1039/C0EE00084A.
Nasidi M., Blackwood D., Akunna J. & Walker G. M. (2010). Bioethanol in Nigeria:
comparison of sugar cane and sweet sorghum feedstocks. Energy and
Environmental Science DOI:10.1039/C0EE00084A.
Nathan Mosier, Charles Wyman, Bruce Dale, Richard Elander, Y.Y. Lee, Mark
Holtzapple, & Michael Ladisch (2005). Features of promising technologies for
pretreatment of lignocellulosic biomass. Bioresource Technology (96) 673– 686.

15
Negro M. J., Manzanares P. & Ballesteros I. (2003). Hydrothermal pretreatment
conditions to enhance ethanol production from poplar biomass. Applied
Biochemistry and Biotechnology, 105: 87－100.
Saha, B.C. (2003). Hemilcellulose bioconversion. Journal Industrial Microbiology and
Biotechno-logy. vol. 30, 279-291. doi: 10.1007/s 10295-003-0049.
Saratale, G.D. and Oh, S. (2012). Lignocellulosics to ethanol: The future of the chemical
and energy industry. African Journal of Biotechnology. vol. 11 No 5, 1002-1013.
doi: 10.5897/AJB10.897.
Sewalt VJH, Glasser WG, Beauchemin KA. 1997. Lignin impact on fiber degradation,
Reversal of inhibition on enzymatic hydrolysis by chemical modification of lignin
and by additive. J Agri Food Sci. 45:1823–1828. doi:
10.1021/jf9608074 [Crossref], [Web of Science ®], [Google Scholar].
Shi J., Chinn M. S. & Sharma-Shivappa R. R. (2008). Microbial pretreatment of cotton
stalks by solid state cultivation of Phanerochaete chrysosporium. Bio-resource
Technology, 99(14) 6556-6564.
Shrivastava S, Tekriwal KG, Kharkwal AC, Varma A. 2014. Bio-ethanol production by
simultaneous saccharification and fermentation using microbial consortium. Int J
Curr Microbiol App. Sci. 3:505–511. [Google Scholar].
Stuart E. Jr., Gargulak Jerry D., & McNally Timothy J. (2001). "Lignin". Kirk-Othmer
Encyclopedia of Chemical Technology. John Wiley & Sons, Inc. Pp 104– 109.
Sun Y, Cheng J. (2002) Hydrolysis of lignocellulosic materials for ethanol production: a
review. Bioresour Technol. 2002;83:1–11.
Sun Y. & Cheng J.(2002). Hydrolysis of lignocellulosic materials for ethanol production:
A review. Bioresource Technology,(3) 1— 11.
Taherzadeh and Karimi, (2011). M.J. Taherzadeh, K. Karimi Pretreatment of
lignocellulosic wastes to improve ethanol and biogas production: a review Int. J.
Mol. Sci., 9 (9) (2008), pp. 1621-1651 CrossRefView Record in ScopusGoogle
Scholar
Xu F, Ding H, Osborn D, Tejirian A, Brown K, Albano W, Sheehy N, Langston J.
Partition of enzymes between the solvent and insoluble substrate during the
hydrolysis of lignocellulose by cellulases. Journal of Molecular Catalysis B:
Enzymatic. 2008;51:42–48.
Yi Zheng, Zhongli Pan & Ruihong Zhang (2009). Overview of biomass pretreatment for
cellulosic ethanol production. International Journal of Agriculture &
Biological Engineering, 2 (3) 51 –60.
Zhao Y., Wang Y., Zhu J. Y., Ragauskas A. & Deng Y. (2007). Enhanced enzymatic
hydrolysis of spruce by alkaline pretreatment at low temperature. Biotechnology
and Bioengineering, 99(6). 1320-1328.

16