1.

0 OBJECTIVES
To convert the lignocellulose to fermentable sugars
To determine the glucose concentration using Samogyi Nelsons method
To compare the effectiveness of converting the lignocellulose to fermentable
sugars between steam treatment and acid hydrolysis.

2.0 INTRODUCTION
Lignocellulosic materials serve as a cheap and abundant feedstock for production of bio
energy. In this experiment, agriculture residue which is sugarcane bagasse is used as the raw
material to convert lignocellulosic material into fermentable sugar. The conversion of
sugarcane bagasse into fermentable sugars required two steps which are pre-treatment and
hydrolysis. Cellulose is the major targeted polysaccharide as it consisted of D-glucose units,
which is a fermentable sugar for most microorganisms. However, pentoses released from
hemicelluloses are not fermented by most of the yeasts used in ethanol production
(Hernndez-Salas et al. 2009).
In this experiment, steam and acid hydrolysis are used as a pre-treatment .The aim of
the pretreatment is to separate the lignin and break the structure of lignocellulose and it is one
of the most critical steps in the process of converting biomass to fermentable sugars
Samongyi Nelsons method is used for the determination of glucose concentration. The sugar
concentration is expressed by using the following formula

Y = (CV/W)*100

Where C is the concentration of reducing sugars (g/L), V is total volume of the liquid
phase (L) and W is dry weight of the corresponding lignocellulosic material (g). Y is the yield
of sugars expressed as percent of reducing sugars in dry weight.
Agro-industrial wastes can acts as suitable substrates for fermentative processes since
they are abundant in source, rich in carbon, and also reduce environmental problem of
disposal. The conversion of this biomass to fermentable sugars is a process that involves a
combination of pre-treatment (chemical or mechanical) and hydrolysis (chemical or
enzymatic). Pre-treatment is important to increase the accessibility of lignocellulosic
materials for hydrolysis. Usually, dilute-acid pre-treatment with high-pressure steam
explosion is the common pre-treatment being used. During this pre-treatment, hemicellulose
is hydrolyzed into monomeric sugars and a complex mixture of compounds that can inhibit
the fermentation of sugars while cellulose is essentially inert. In the other hand, lignin does
not contribute to ethanol production, yet it can be separated after pre-treatment and can be
used as a solid fuel for the generation of electricity (Gassara et al. 2010).
An effective pre-treatment as shown in figure 1.1 is characterized by several criteria
included preserving hemicelluloses fraction to yield maximum fermentable sugar contents,
limiting the loss of carbohydrate to minimize the formation of inhibitors due to degradation
products, minimize energy input and the process is economically efficient as well as cost
effective (Anwar et al.,2104).

Figure 1.1: Schematic presentation of effects of pre-treatment on lignocellulosic biomass

(Hsu et al, 1980)

Anwar, Z., Gulfraz, M., & Irshad, M. (2014). Agro-industrial lignocellulosic biomass a key to
 unlock the future bio-energy: A brief review. Journal of Radiation Research and Applied
Sciences, 7(2), 163173. https://doi.org/http://dx.doi.org/10.1016/j.jrras.2014.02.003

Binod P, Satyanagalakshmi K, Sindhu R, Janu KU, Sukumaran RK, Pandey A. Short duration
microwave assisted pretreatment enhances the enzymatic saccharification and
fermentable sugar yield from sugarcane bagasse. Renew Energ. 2012; 37: 109-116.

Gassara F, Brar SK, Tyagi RD, Verma M, Surampalli RY. Screening of agro-industrial wastes
to produce ligninolytic enzymes by Phanerochaete chrysosporium. Biochem Eng J.
2010; 49: 388-394.

Hsu, T.A., M.R. Ladisch, et al. (1980): Alcohol from Cellulose. Chemtech. 10: 315-319

Hernndez-Salas JM, Villa-Ramrez MS, Veloz-Rendn JS, Rivera-Hernndez KN,

Gonzlez-Csar RA, Plascencia-Espinosa MA, et al. Comparative hydrolysis and
fermentation of sugarcane and agave bagasse. Bioresour Technol. 2009; 100: 1238-1245.

3.0 PROCEDURES

Part A: Preparation of lignocelluloses

1. Sugarcane bagasse is dried in the oven to reduce its moisture content
2. Then, it is cut into smaller pieces to increase its surface area.
Part B Pre-treatment

I. Steam Pre-treatment
1. 2g of sugarcane bagasse is mixed with 60 ml of distilled water
2. The mixture is then autoclaved at 121C and 1.1 kg/cm2 for 4 hr.
3. Next, the mixture is filtered using Whatmann filter paper and the filtrate is kept to
analyze total reducing sugar using Samogyi Nelsons Method.

II. Acid Hydrolysis

1. (1.2%) of HCl is added to 2g of bagasse sample in a ratio of 5:1, 10:1 or 15:1 (mL of
solution/g of bagasse by weight)
2. The mixture is autoclaved at 121C and 1.1 kg/cm2 for 4 hr.
3. After hydrolysis, 0.1 N NaOH is added and the pH is adjusted to 5.0
4. The mixture is diluted with distilled water to a final dilution of 30:1 (30 ml of liquid
phase/g of bagasse)
5. The mixture is filtered and the filtrate is kept for analysis
6. The reducing sugar is determined by using Samogyi Nelsons method.
7. The sugar concentration is calculated by using the following formula

Y = (CV/W)*100

Where C is the concentration of reducing sugars (g/L), V is total volume of the liquid
phase (L) and W is dry weight of the corresponding lignocellulosic material (g). Y is
the yield of sugars expressed as percent of reducing sugars in dry weight.

Appendix

Determination of glucose by using Samogyi Nelsons method

1. The copper reagent is prepared by adding 12g of sodium tartarate and 24g of sodium
carbonate in 250ml of distilled water. Then, 4g of copper sulphate and 16g of sodium
hydrogen carbonate are added. Gently mixed the mixture until all the salts are
dissolved. Next, 180g of sodium sulphate is dissolved in 250ml distilled water. The 2
solution are mixed and distilled water is top up to 1000ml.
2. The Nelsons reagent is prepared by adding 25g of ammonium molydate in 450ml of
distilled water and 21ml of concentrated sulphuric acid is slowly added into the
solution. At the same time, 3g of disodium hydrogen arseno sulphate is added in 25ml
of distilled water. Next, the two solutions are mixed together and distilled water is top
up to the total volume of of 500ml.
3. 1.0 ml of the sample is added into 1.0ml of the copper reagent and incubated in the
water bath at the temperature of 60C for 10 minutes.
4. The reaction mixture is cooled down to room temperature and 1.0 ml of the Nelsons
reagent is added, followed by 10ml of distilled water. The solution is allowed to stand
for 15 minutes.
5. The optical density of the reaction mixture is determined at 660nm.
6. A standard curve for glucose is prepared by using the concentration of pure glucose in
the range of 0-1000mg/L. (Noted: The concentration of pure glucose stock solution is
1.00g/L). Then continued with steps 3-5.
7. The standard curve is used to determine the unkonown glucose concentration.