FACULTY OF APPLIED SCIENCES

DEPARTMENT OF CHEMISTRY

ANALYTICAL CHEMISTRY IV
MODULE 2 (ANCH 421)

CHEMISTRY PROJECT -
PRE-TREATMENT OF SUGARCANE BAGASSE
FOLLOWED BY ENZYMATIC HYDROLYSIS TO
SUGARS
NAME: S. RAMKISSON
STUDENT NUMBER: 21351652
LECTURER: PROF. N. DEENADAYALU

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 CONTENTS

1. Abstract……………………………………………………………………..……………………………………… Page 3

2. Introduction …………………………………………………………………………………..…………………. Page 4

2. History and current research……………………………………………………………………………… Page 7-8

3. Materials and Methodology………………………………………………………………………………. ….. Page 9

3.1. Materials……………………………………………………………………………………………………………Page 9

3.2. Instruments…………………………………………………………………………………………………. ….. Page 9

3.3. Instrument Parameters for HPLC………………………………………………………………………Page 9

3.4. Analysis……………………………………………………………………………………………………….…. Page 9-10

3.5. Safety Precautions……………………………………………………………………………………………………………. Page 12

4. Results and Discussion…………………………………………………………………………………………….……………. Page 12

5. Conclusion…………………………………………………………………………………………………………………………Page 13

8. References………………………………………………………………………...…………………………………………. Page 14-15

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 DILUTE AMMONIA PRE-TREATMENT OF SUGARCANE BAGASSE
FOLLOWED BY ENZYMATIC HYDROLYSIS TO SUGARS

1.ABSTRACT
In the present study, the evaluation of the effects of dilute ammonia pre-treatment on the solid
yields, delignification and the recoveries of glucose and xylose at different pre-treatment times
(0-300min) had been investigated. Dilute ammonia pre-treatment had been expected to break
apart the lignin and hemicellulose of Sugarcane Bagasse which resists accessibility of enzymes
to cellulose. The pretreatment performed at 1700, 50min and 25%(w/w) had produced xylose
18.75%w/w and 80.76% w/w glucose yields from enzymatic hydrolysis. The trend of reaction
times tended to gradually decrease from 50-300minutes in glucose and xylose yields which
indicates that dilute ammonia pre-treatment does not require long reaction time conditions. The
improved digestibility of dilute ammonia pre-treated sugarcane bagasse had been due to
delignification of sugarcane bagasse confirmed by Fourier Transform Infrared Spectroscopy.

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2. INTRODUCTION
Fossil fuels is a general term for buried combustible geologic deposits of organic materials,
formed from decayed plant and animal matter that had been converted into several types of fossil
fuels depending on the combination of organic material that had been present, length of time of
burial and the temperature and pressure conditions that existed. It comprises of mainly coal, oil
and natural gas. Fossil fuels are sources of energy which is used in various applications such as
transportation, production of plastics, medicine, cosmetics, lubricants, synthetic fabrics and a
power source in electricity plants. [1]

Although fossil fuels are a valuable resource, there are many disadvantages of utilizing this
energy source because of the negative impacts that it has on the environment. Pollution is a main
concern when using fossil fuels because of the releasing of harmful gases such as carbon dioxide,
carbon monoxide, nitrogen oxides and sulphur oxides during combustion. Carbon dioxide
emissions causes an entrapment of heat in the earth’s atmosphere which contributes to global
warming. Sulphur oxides react readily with water in atmosphere to produce sulphuric acid which
causes acid rain. Environmentally, the mining of fossil fuels leads to the destruction of wide
ranges of land. [1]

Growing worldwide research has been taken into finding an alternative method to the utilization
of fossil fuels due to the inevitable depletion and negative impact that fossil fuels has on the
environment. Biomass is an alternate energy source that can be defined as “any material,
excluding fossil fuels which was a living organism that can be used as a fuel either directly or
after a conversion process,” [2-3]. Biomass energy is abundant, secure, sustainable,
environmentally friendly and can overcome the limitations of fossil fuels. Biomass can be
converted to organic fuels for transportation such as bioethanol, bio-methanol, biodiesel, and
additives for reformulated gasoline.

Bioethanol is considered as an important renewable fuel to partly replace fossil-derived fuels.

Bioethanol is a liquid alcohol that can be produced from biomass by fermentation by bacteria to
metabolize sugars in oxygen-lean conditions to produce ethanol and carbon dioxide. [4]
Bioethanol has a high-octane rating, which increases the energy efficiency of an engine and
reduces emissions because engine can completely combust fuel. Bioethanol production from
biomass consists of four main steps – pre-treatment of biomass, enzymatic hydrolysis of
cellulose and hemicellulose, fermentation and distillation.

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Sugarcane Bagasse is a fibrous material which is an abundant agricultural solid residue after the
extraction of juice from sugarcane. The South African Sugar Industry has sugarcane growers
extending from Northern Poundland in the Eastern Cape to the Mpumalanga Lowveld. There are
14 operational sugar mills in South Africa. [3]

South Africa’s sugar cane growers annually produce on average 19.9 million tons of cane,
farmed more than 371 662 hectares under sugar cane and make a substantial contribution to the
economy. Sugarcane bagasse consists of 30% of crushed cane which ranges from 5-6 million
tons of sugarcane bagasse in sugar production. Approximately, 4.5 million tons of sugarcane
bagasse is used as in-house fuel for power generation or as a raw material for producing low
value products such as mulch or ceiling tiles. The remaining 1.5 million tons of sugarcane
bagasse is considered waste and can be used as a raw material for cellulosic bioethanol
production. [5] Sugarcane bagasse will provide an economic, abundant, renewable and a
sustainable feedstock that will reduce the waste from sugarcane plants [4]

Figure 2.1.1 Raw Sugar Cane Bagasse

Sugarcane bagasse, as well as other types of plant biomass, consists mainly of biopolymers of
cellulose, hemicellulose, lignin and trace amounts of extractives and mineral salts. These
polymers are associated with each other in a hetero-matrix to different degrees and varying
relative composition depending on the species, type and sources of the plant biomass. [5] The
relative abundance of cellulose, hemicellulose and lignin is a major factor in determining the
optimum energy conversion route for diverse types of plant biomass. [6]

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2.2. Cellulose

Cellulose is a sustainable, abundant biopolymer that can be found in various sources such as
plants, animals, bacteria and amoebas. It can be used as an energy source, in textiles, building
material and clothing. [7]

Cellulose is the most significant structural component of a plant cell wall and is a polysaccharide
compound composed of repeated unbranched β-(1,4)-glucosidic linked glucose monomers. In
plants, cellulose chains are bunched together by hydrogen bonding into semi-crystalline
microfibrils containing crystalline allomorphs and cellulose (β and α). [8] In the crystalline
regions, cellulose chains are closely packed together by a strong ad highly intricate intra- and
intermolecular hydrogen bonding network. [9] As a naturally occurring material, cellulose may
contain by-products leading to application issues and difficulty in chemical modifications
reactions. [10]

2.2 Hemicellulose

Hemicellulose is the heterogenous polysaccharide composed of D-xylose, D-glucose, D-

mannose, D-galactose, L-arabinose, D- glucuronic acid and 4-O-methyl-D-glucuronic acid. The
specific combination varies in different plants. Classification is according to the main sugar in
the main backbone. Sugarcane bagasse is composed of a backbone of xylose, branched with
glucose and arabinose units [11]. Xylan can be extracted easily in an acidic or alkali
environment. Removing hemicellulose from by these methods greatly facilitates cellulose
digestion.

2.3. Lignin

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Lignin is the third most abundant polymer compound that is imbedded in the cellulose and
hemicellulose in a plant cell. Lignin imparts rigidity and offers impermeable resistance to
microbial and chemical attack. Lignin is an amorphous, heteropolymer network of phenyl
propane units (p-coumaroyl, coniferyl and sinapyl alcohol) held together at different linkages.
[10] Lignin prevents the saccharification of cellulose and hemicellulose acting as a physical
barrier between cellulolytic enzyme and substrate.

Consequently, the rate of saccharification of cellulose is inversely proportional to the lignin

content, with maximum recovery of sugars occurring only after bulk of lignin is removed. Pre-
treatment for ethanol production is the most favourable way to synthesize sugars, overcoming
limitations of cellulose crystallinity and physical and chemical associations of lignin with
producing potentially fermentable monosaccharides for ethanol production

The objective of a pre-treatment step is to alter the structure of plant cell to remove lignin and
hemicellulose, and de-crystallization of cellulose which would lead to an increase in the rate of
enzymatic hydrolysis and greater sugar yield for fermentation. [12]

Figure 1.1.2. Structure of lignin, hemicellulose and cellulose present in plant material (adapted
from Wu-Jinn Liu, Hong Jiang, Han-Qing Yu (2015) Thermochemical conversion of lignin to
functional materials; a review and future directions)

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2.4. Pretreatment

Many pre-treatment processes of enzymolysis of lignocellulosic biomass has been investigated.

The purpose of the pretreatment is to remove lignin and hemicellulose, reduce cellulose
crystallinity, and increase the porosity of the materials. Pretreatment must meet the following
requirements: (1) improve the formation of sugars or the ability to form sugars by enzymatic
hydrolysis; (2) avoid the loss or bio-degradation of carbohydrates; (3) avoid the formation of by-
products inhibitory to the subsequent enzymatic hydrolysis and fermentation processes; and (4)
be economical. Physical (e.g. Milling and grinding), physio-chemical (e.g. Autohydrolysis,
liquid hot water, steam, supercritical fluids) chemical (e.g. acid, alkaline, organic solvents,
oxidizing agents) and biological processes (e.g. fungi) or combinations have been used for
pretreatment of lignocellulosic materials. [15 -18]. These pre-treatment processes have
limitations regarding sugar yields, reaction conditions and great economic costs. Therefore,
research has been ventured into finding alternative pre-treatment processes that is able to
produce greater sugar yields, alter lignin and hemicellulose structures and de-crystallize cellulose
effectively within economical means.

Dilute ammonia (NH4OH) pre-treatment of sugarcane bagasse has been investigated as it is a

promising method for the release of carbohydrates from sugarcane bagasse. Dilute ammonia has
been selected as a reagent because it is non-corrosive, inexpensive and recycling considerations.
Dilute ammonia pre-treatment has indicated remarkable success in delignification of
lignocellulosic biomass. [14] Ammonia tends to have a selectivity towards reactions with lignin
than that of carbohydrates.

The treatment of lignocellulosic biomass with alkali hydrolyses the intermolecular ester bonds
between lignin, hemicellulose and cellulose to remove lignin leaving behind hemicellulose and
cellulose. Ammonia can remove lignin by cleaving C-O-C bonds in lignin and other ether and
ester bonds in the biomass resulting in the release of cellulose and hemicellulose from the
lignocellulosic biomass. [19] The mechanism of alkaline hydrolysis is believed to be
saponification of intermolecular ester bonds crosslinking xylan hemicelluloses and other
components, for example, lignin and other hemicellulose. The porosity of the lignocellulosic
materials increases with the removal of the crosslinks. [21]

2.4. Chlorite Bleaching

Bleaching is an additional process that has been used to eliminate all residual hemicellulose and
lignin present. Sodium Chlorite under acidic conditions has been prominently used for bleaching
of various pulp materials. Growing research has been taken into the use of sodium chlorite as
oxidant replacing chlorine-based reagents. [32]

8
Sodium chlorite undergoes an oxidation reaction with non-cellulosic compounds to obtain highly
purified cellulose. The use of sodium chlorite has been expected to increase thermal stability,
crystallinity content, and flattened morphology when compared to untreated cellulose fibres. [33]

2.5 Cellulase

Cellulase belongs to a family of enzymes which can be subdivided into three groups which
provide a collaborative effort in cellulose degradation in nature. They are endoglucanases (1,4-β-
D- glucan glucanohydrolases), exoglucanases (1,4-β-D- glucan cellobiohydrolases) and β-
glucosidases (β-D-glucoside glucohydrolases). [12-13]

Cellulase is synthesised from fungi, bacteria and plants but most research is focused on microbial
cellulases. Microbial cellulases, such as Trichoderma reesei, is used as a catalyst because it is
most resistant chemical inhibitors, and exhibits better stability at 500C than other fungal
cellulases. The factors that affect the enzymatic hydrolysis of cellulose include substrates,
cellulase activity, and reaction conditions (temperature, pH, as well as other parameters). To
improve the yield and rate of the enzymatic hydrolysis, research has focused on optimizing the
hydrolysis process and enhancing cellulase activity (Cantwell et al., 1988; Durand et al., 1988;
Orpin, 1988). The mechanism of a basic enzymatic hydrolysis consists of three different steps:
adsorption of biocatalyst to the cellulose surface, biodegradation of cellulose and ending with
enzyme desorption. [14] The conversion of hemicellulose and cellulose can be expressed as a
reaction of glucan (for hexoses) and xylan (for pentose) with water:

(C6H10O5)n + nH2O  n C6 H12 O6

(C5H8O4)n + nH2O  n C5H10O5

Many factors affect the enzymatic hydrolysis efficiency of cellulose in lignocellulosic biomass,
such as the cellulose crystallinity, specific surface area, particle size, the degree of cellulose
polymerization, the lignin barrier, hemicellulose content and porosity. The experimental
conditions of temperature, pH substrate and enzyme loadings influences the overall yield of
glucose and kinetics. [12]

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Figure 1.1 indicates the basic hydrolysis reaction of cellulose and hemicellulose. The catalyst
refers to cellulytic enzymes that reacts with cellulose and hemicellulose to break down structure
to glucose and xylose respectively. (adapted from Hydrolysis of woody biomass-derived reusable
heterogenous catalyst, Hirokazu Kobayashi, 2015)

The aim of the present study is the evaluation of the effects of dilute ammonia pre-treatment on
the solid yields, delignification and the recoveries of glucan and xylan at different pre-treatment
reaction times - 0-300min.

3. HISTORICAL AND CURRENT RESEARCH

Dilute ammonia pretreatment of sugarcane bagasse followed by enzymatic hydrolysis to sugars
(2014) Hongdan Zhang, Shubin Wu evaluated the dilute pre-treatment to break the intricate
structure of sugarcane bagasse which resists the accessibility to cellulose. The effects of
temperature, times, and ammonia dosage on the recoveries of glucan, xylan and acid in soluble
lignin were evaluated. The pretreatment performed at 1700C, 60 minutes and an ammonia %
(w/w) of 15% (w/w) will provided delignification of 36.9% and retaining 94.7% of glucan and
63.1% of xylan in the raw material.

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Dilute acid and enzymatic hydrolysis of sugarcane bagasse for biogas production -Rui Nuno
Leitato de Carvalho (2009) which evaluated a study on dilute sulphuric acid pretreatment
followed by enzymatic hydrolysis. The obtained saccharifications were low due to low
concentration of acid hydrolysis. However, this hydrolysate possessed a good biochemical
methane potential which was promising for biogas production.

Enzymatic hydrolysis of cellulosic biomass (2011) Yang B, Dal Z, Ding S-Y, Wyman C.E,
evaluated the diversity, stability and activity of individual enzymes and their synergistic effects
in deconstructing complex biomass. The review highlights the impact of key substrates and
enzyme features that influence performance, to better understand enzymatic hydrolysis of
cellulosic biomass for biological conversion of fuels and chemicals.

Pretreatment technologies for an efficient bioethanol production on enzymatic hydrolysis.

Alvira, E, Tomas-Pejo, Ballesteros M, Negro MJ, (2010) reviewed different types of pre-
treatments for ethanol production for lignocellulose and several key properties that should be
targeted for low-cost and advanced pretreatment processes. Alkaline treatment depends on the
effect of the bases on lignocellulosic biomass which are effective dependant on the lignin content
of the biomass. Alkaline treatments increase cellulose digestibility and are more effective for
lignin solubilisation, exhibiting minor cellulose and hemicellulose than acid and hydrothermal
processes.

Cellulosic Ethanol production from sugarcane bagasse without enzymatic saccharification

(2008) Dawson L, Boopathy R, investigated a chemical pretreatment of alkaline peroxide and
acid hydrolysis without enzymatic hydrolysis the results indicated that ethanol can be produced
from the sugarcane bagasse.

Alkali and enzymatic delignification of sugarcane bagasse to expose cellulose polymers for
saccharification and bio-ethanol production (2013) Asghar M evaluated that the waste biomass
would be of particular interest, since it seems an eco-friendly approach for bio-ethanol
production. Alkali pre-treatment had provided better ethanol production than ligininolytic
application.

Biomass Pre-treatment: Fundamentals toward application (2011) Agbhor V, Cicek N, Sparling

R, Berlin A, presents a survey of pre-treatment technologies with particular interest on concepts,
mechanism of reaction and practicality. Pretreatment of alkali such as KOH, Ca(OH)2 and
ammonia causes swelling of the biomass, which increases the surface area of the biomass and
decreases the degree of polymerization and cellulose crystallinity. Alkaline pre-treatment
disrupts the lignin structure and breaks the linkage between lignin and other carbohydrates.
Alkali pretreatment tended to show best results in biomass with low concentrations of lignin
present.

Pre-treatments to enhance the digestibility of lignocellulosic biomass (2009) Zeeman G ,

A.T.W.M Hendriks were steam, lime, hot-water and ammonia based pre-treatments had been

11
reviewed. Alkaline pretreatment tended to have a major effect on increasing accessible surface
area of cellulose and solubility of lignin and hemicellulose.

4.MATERIALS AND METHOD

4.1 Materials

The sugarcane bagasse used in this study will be provided from Sugar Milling Research Institute
(SMRI).

Ammonia

Enzymes – Cellulase - 20FPU/g

Acetic acid /Sodium acetate buffer (pH = 4.8)

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Deionized water

D-Xylose (1 - 4mg/mL)

D-Glucose (1 – 4 mg/mL)

4.2. Apparatus

Analytical Balance

Heating Mantle

Round bottom flasks

Vacuum Filtration system

Centrifugation

4.3. Instrumentation

4.3.1 Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy had been recorded using a FT-IR spectrometer to
determine the presence of main organic groups that constitutes the lignocellulosic structure of
sugarcane bagasse. Fourier Transform Infrared Spectroscopy is a simple, non-destructive
analytical technique that can provide qualitative analysis based on determining the presence of
various functional groups in a sample.

Figure 4.4 A schematic diagram of a Fourier Transform Infra-

red Spectrophotometer (Adapted from Wikipedia 2017)

Figure 4.4 A schematic diagram of a Fourier Transform Infra-red Spectrophotometer (Adapted

from Wikipedia 2017)

Attenuated Total Reflectance has become a widely used FT-IR sampling tool. Speed of analysis
is increased by the decrease in sample preparation times. An advantage of ATR sampling comes
from a thin sample path length or depth of penetration of IR beam exposed to sample. In

13
comparison to FT-IR sampling by transmission, where sample had to be diluted with a IR
transparent salt and pressed into a disc prior to analysis to prevent totally absorbing bands in
infrared spectra. [36]

Using Attenuated Total Reflector Accessory, the infrared beam is directed towards a crystal with
a relatively higher refractive index than that of the sample. Infrared beam is reflected from
internal surface of the crystal and creates the evanescent wave which is projected into the sample
in contact with attenuated total reflector crystal. Some of the energy of the wave is absorbed by
the sample and the reflected radiation is returned to detector. An Attenuated Total Reflector
Accessory operating on principle is listed below:

A Perkin Elmer 100 FTIR spectrometer equipped with Attenuated Total Reflection Accessor had
been used. Scans were performed in a region of 4000-600cm-1.

4.3.2 High-pressure Liquid chromatography

Liquid chromatography is a quantitative and qualitative technique that is used for the separation,
purification and determination of thermochemical constant of volatile compounds. In liquid
chromatography, the different components are separated based on their polarity. The analytical
technique’s simplicity, sensitivity, and effectiveness in separating components of mixtures makes
determination of the monomers of cellulose and hemicellulose possible.

The method consists of introducing the standards or sample into the mobile phase by being
injected into the injector. The liquid is passed, by the pressure introduced to the column, through
the packed column known as the stationary phase, through which the components of the sample
move at velocities that are influenced by the partitioning of each constituent with the stationary
phase. The substances having the greater interaction with the stationary phase are kept in the
column and consequently separate from those with smaller interaction. As the components elute
from the column, it can be quantified by a detector.

Column
The column is a Biorad Aminex HPHX 87-P equipped with an appropriate guard column.

Injection volume: 10 – 50 μL, dependent on concentration

Mobile phase: HPLC grade water, 0.22 μm filtered and degassed

Flow rate: 0.5mL / minute

Column temperature: 80 - 85°C

pH range : 5 - 9

Detector

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The ELSD-LTII utilizes a high-efficiency LED and enhanced digital signal treatment to
minimize noise and deliver optimum sensitivity for trace analysis. An innovative cell design also
reduces band broadening and allows sensitivity levels below 200 picograms. The high-
performance optical detection system can also be used for combinatorial chemistry and high-
throughput screening applications.
The Low Temperature Evaporative Light Scattering Detector (ELSD-LTII) is an easy-to-operate,
Universal detector that identifies analytes with uniform sensitivity regardless of spectroscopic
properties. It is not a spectroscopic detector. Instead, it removes the mobile phase through
evaporation and then makes a light scattering measurement of the dried analyte particles
Pressure: 2.5 bar

Temperature of Detector: 450C

Flow diagram 3.4.2. indicating methodology of dilute ammonia pre-treatment and enzymatic
hydrolysis of sugarcane bagasse

Sugarcane Ammonia Pre-

Washing
Bagasse treament

Vaccuum Filtering
Bleaching Washing
and drying

Vaccuum Filtering Monomers of

and drying Centrifugation Cellulose and
Hemicellulose

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5.4 Analysis

Chemical composition of Sugarcane bagasse raw sample had been determined using Liquid
Chromatography and Fourier transform infrared spectroscopy.

Sugarcane bagasse sample of 5g and deionized water (200mL) had been added to a heating
mantle with an agitation of 500 revolutions per minute.

Reactor will be heated using an electrical heating jacket at 1700C and held at a certain
temperature from 0-300 minutes at an initial ammonia dosage from 25% weight according to the
dry raw material.

The pre-treated solution had been separated by filtration. Solid fraction requires washing with
water until a neutral pH is obtained. Solid fraction had been dried in an oven at 80-100oC and
stored in a refrigerator for further use

Solid fraction had undergone bleaching process by using 5% sodium hypochlorite solution,
sodium chlorite solid and acetic acid for four hours. Subsequently, solid fraction had been
washed with water until neutral pH was obtained. Solid fraction had been dried in an oven at 80-
100oC and stored in a refrigerator for further use.

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Enzymatic hydrolysis had been performed in 50mL bottles using 0.05mol/L acetic acid and
sodium acetate buffer (pH : 4.8) and 2% dry matter at 500C on a shaker at 150 revolutions per
minute for 24 hours. 50mg of sample had been dissolved in 24.5mL sodium acetate and enzyme
solution. Cellulase of a filter paper activity of 20FPU/mL had been used for reaction. Enzymes
had been added to dry substrate, along with acetic ether to inhibit microbial contamination or
growth.

Samples had undergone centrifugation and liquid hydrolysate had been separated from solid
residue. Liquid hydrolyzate had been neutralized to pH of 5-6 using solid calcium carbonate and
settling of solid. The pH of the liquid after settling will be approximately 7.

The sugars in the enzyme hydrolysate had been analysed by High-Pressure Liquid
Chromatography equipped with an Evaporative Light Scattering Detector.

A series of mixed calibration standards ranging from 1 – 4mg/mL of xylose and glucose had
been prepared by serial dilution for quantification of xylose and glucose using High Pressure
Liquid chromatography.

Analysis of the calibration standards and samples by HPLC had been performed using a Biorad
Aminex HPX-87P column equipped with the appropriate guard column which was in accordance
to NREL Laboratory Guidelines.

The Glucan and Xylan conversion (%) was calculated by :

𝑚𝑔
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 ( ) 𝑥 𝑉𝑜𝑙𝑢𝑚𝑒 𝑥 𝐻𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝐹𝑎𝑐𝑡𝑜𝑟
𝑚𝐿
% Conversion of Glucan = x 100%
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑟𝑎𝑤 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝑥 𝑔 𝑝𝑜𝑙𝑦𝑠𝑎𝑐𝑐ℎ𝑖𝑟𝑖𝑑𝑒
𝑔 𝑏𝑖𝑜𝑚𝑎𝑠𝑠

𝑚𝑔
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑋𝑦𝑙𝑜𝑠𝑒( ) 𝑥 𝑉𝑜𝑙𝑢𝑚𝑒 𝑥 𝐻𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝐹𝑎𝑐𝑡𝑜𝑟
𝑚𝐿
% Conversion of Xylan = x 100%
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑟𝑎𝑤 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙

‘The resulting glucose, cellobiose, and xylose concentrations (mg/mL) calculated for each
digestion mixture should be converted to anhydro-glucose, anhydrocellobiose, and anhydro-
xylose concentrations, respectively, by subtracting out the proportional weight added to each
molecule by the water of hydrolysis (i.e., multiplying by 0.95 for cellobiose, by 0.90 for glucose,
and by 0.88 for xylose.)’ [30]

Fourier Transform Infrared Spectroscopy had been recorded from AT-IR spectrometer.

17
4. SAFETY PRECAUTIONS
Ammonia is a severe irritant of the eye, skin and mucous membranes of humans. Precaution
must be taken to ensure that ammonia is not inhaled or ingested. Gloves and googles must be
worn when encountering ammonia.

Acetic acid is a flammable, corrosive liquid that must kept away from sources of ignition. Acetic
acid is very hazardous in case of contact with skin and eyes. Inhalation of Acetic acid may
produce severe irritation of respiratory tract, characterized by coughing, shortness of breath and
choking.

18
5. RESULTS AND DISCUSSION
5.1 Compositional Analysis of Sugarcane Bagasse

The composition analysis of untreated depith, mill run sugarcane bagasse is given in the pie chart
below. The compositional analysis of untreated depith and mill-run sugarcane bagasse consisted
of 55% Cellulose, 28.2% Hemicellulose, 7.2% Lignin and 6.2% of Ash. It can be observed that
no monomers of cellulose and hemicellulose had been present. [34]

The homogeneity of sugarcane bagasse had not been consistent in particle size. Particle size had
been <5mm but consistency tended to vary in sample. Some sections of the waste material
tended to be powdered and finely ground while other sections tended to be greater than 5mm
particle size. This inconsistency in homogeneity may have affected the reactivity and
reproducibility of results.

Particle size is an crucial factor for surface area and exposure to chemical modification with
regards to pre-treatments and enzymatic hydrolysis. It can be noted that a pre-treatment is
required to increase the accessible surface area of substrate for the enzymatic attack. Pre-
treatment can be enhanced if the particle size of raw material decreases. [37]

Compostional Analysis of Untreated Sugarcane Bagasse

6.1
7.2

28.2
55

Cellulose Hemicellulose Lignin Ash

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5.2. Characterization of Untreated and Pre-treated Sugarcane Bagasse

5.2.1. Chemical Composition

The ammonia pre-treatment required a pressure reactor to elevate pressure of system with
aqueous ammonia solutions and sugarcane bagasse raw material. The releasing of pressure
allows the expansion of ammonia gas that causes swelling and physical disruption of sugarcane
bagasse raw material and partial de-crystallization of cellulose. [28,37] Since the reaction had
been prepared in a heating mantle at atmospheric pressure, the swelling and physical disruption
and de-crystallization had not been very effective for enzymatic hydrolysis. This had been noted
by the unchanged physical appearance of samples after treatment. The texture of samples tended
to be light to dark brownish texture that appeared to be bonded to each other of sugarcane
bagasse fibres.

From Table 5.2.1 shows that the solid yields of pre-treated samples mostly ranged from 69.5 to
75.6 %, which indicated that some constituents (mainly lignin and hemicellulose) were removed
because of dilute ammonia conditions. As the pretreatment conditions became more severe, the
solid yields decreased gradually, and more mass was lost. This mass loss can also be attributed to
sample being lost during washing and filtering of samples using a vacuum filtration system.

Table 5.2.1 indicates the Ammonia Pre-treatment Reaction Time against Solid Yield Recovery of
Material

Reaction Time of Ammonia Solid Yield

Treatment (minutes) Recovery After
Ammonia Treatment
(%)

50 71.0

100 75.6

150 69.5

200 63.9

300 64.5

20
A further bleaching step had been required to increase digestibility, porosity and surface area of
cellulose of ammonia treated material. To account for this lack of de-crystallisation of cellulose,
a bleaching step had been introduced involving sodium chlorite and acetic acid at 60-700C for 4
hours using an oil bath. The bleached samples appeared bright white and contained fine fibres of
cellulose. Precaution had been taken to ensure that the bleached step was followed consistently
and each sample had received the same bleaching conditions.

The solid yield recoveries after bleaching treatment mostly ranged around 79.8-85.7% as can be
seen in table 5.2.2 below. This mass loss can also be attributed to sample being lost during
washing and filtering of samples using a vacuum filtration system.

Table 5.2.2. the Ammonia Pre-treatment Reaction Time against Solid Yield Recovery of
Bleached Material

Reaction Time of Solid Yield Recovery

Ammonia after Bleaching
Treatment Treatment (%)
(minutes)

50 83.4

100 85.6

150 80.5

200 85.7

300 79.8

Washing of samples after each pre-treatment and bleaching step should be thorough to ensure
that traces of ammonia or bleaches does not interfere with enzymes used during enzymatic
hydrolysis process.

5.2.1. Fourier Transform Infrared Spectroscopy

To facilitate fast and effective hydrolysis of polysaccharides, the sugarcane bagasse requires a re-
treatment. Lignin acts an inhibiting barrier to enzymes attack and should be removed before
enzymatic hydrolysis.

21
 FT-IR is a qualitative technique that has been used evaluate structural analysis of the material
before and after chemical treatments. Figure 5.2.1 indicates the comparison AT-IR spectra of
sugarcane bagasse raw material, dilute ammonia pre-treated solid, bleached sample and
enzymolysis residue. Comparisons of intensities of carbonyl bond of acetyl groups in
hemicellulose, xylan and lignin bonds can be made between raw material, pre-treated and
bleached solids.

The bands at 3400-3200cm-1 for all spectra indicates the O-H stretching of hydroxyl groups
present in cellulose, hemicellulose and lignin. [31] The decreasing of the band from raw material
to bleached pre-treated solid indicates that hemicellulose and lignin had been removed from raw
material.

The characteristic band at around 2970-2850cm-1 in spectra corresponds to C-H stretching of

vibrations of alkyl groups in aliphatic bonds of cellulose, lignin and hemicellulose. [35] The
spectral band had remained unaffected by ammonia treatment but had been significantly lowered
in bleached pre-treated solid.

Spectral bands at 1730-1740cm-1 corresponds to C=O of acetyl groups of hemicellulose. [36] the
spectral band at dilute ammonia pre-treatment had remained unchanged. The bleached pre-
treated solid appears to be weaker in comparison.

Aromatic ring of lignin at 1510cm-1 [21] tended to decrease from sugarcane bagasse raw material
to bleached pre-treated solid. The band at 1510cm-1 assigned to the aromatic C=C vibration in
lignin significantly decreased from raw material to bleached indicating that delignification of
sample had occurred.

The bands at 1430-1420cm-1 is evaluated as CH2 scissoring motion in cellulose. Spectral bands
at 1430-1420cm-1 and 900-893cm-1are used to explain crystal structure cellulosic material. [32-
33] The equations indicate the Lateral Orientation Index (LOI) and Total Crystallinity
Index(TCI) which can be calculated using Infrared ratios.

The band at 1200-1300cm-1 in the FT-IR spectrum is reduced drastically from raw material to
bleached sample indicating the binding components of biomass had been removed by the
chemical pretreatment. This phenomenon can be explained by most of hemicellulose(xylan) and
cellulose(glucan) being removed during pre-treatment, bleaching and/or enzymolysis.

A peak at 895cm-1 indicates the peaks are the characteristic peak of β-D glycosidase Cellulose
visible in both the raw material and pre-treated solids.

Figure 5.2.1 indicates the comparison AT-IR spectra of sugarcane bagasse raw material, dilute
ammonia pre-treated solid, bleached pre-treated solid and enzymolysis residue

22
 Key

Dark Blue: Enzymatic Residue

Yellow: Raw sugarcane bagasse

Orange: Alkali Pre-treated Sample

Light Blue: Bleached Sample

Table 5.2 indicates the frequency and corresponding functional groups

Frequency (cm-1) Corresponding to Functional

Groups

3400-3200 O-H stretching of Hydroxyl

groups of Cellulose,
Hemicellulose and Lignin

23
 2891 C-H stretching of alkyl groups

1730 C=O acetyl groups of

hemicellulose

1510 Aromatic ring of lignin

1430-1420 C-H stretching of Cellulose

900-895 β-D glycosidase of Cellulose

5.3 Characterization after Enzymatic Hydrolysis

5.3.1 High Pressure Liquid Chromatography

Chemical composition of enzymatic hydrolysis liquid had been determined using Liquid
Chromatography. The analysis had been performed for the quantitative and qualitative analysis
of the hydrolysate produced from enzymatic hydrolysis of the cellulose extracted from The
concentration of xylose and glucose in enzymolysis residue and individual components from
recovered solid after pre-treatment was determined using calibration standard graphs.

Samples and standards were analysed by HPLC. Two peaks had been present with The
calibration curves were drawn from peak height and concentration of Xylose and Glucose
standards ranging from 1-4mg/mL which was used to determine the concentrations present in
samples. The calibration curves are below at Figure 5.3.1 and 5.3.2. From the R2 value, glucose
and xylose calibration curve’s R2 =0.9842 and 0.9998 respectively. R2 is a statistical measure of
how close data are to a fitted linear regression. Linear regression calculates an equation that
minimizes the distance between fitted and the data points. A value close to 1 indicates the model
explains all the variability of the response data around the mean.

The chromatograms attached in appendixes for the HPLC analysis of glucose and xylose
concentrations had retention times of 13.5 and 14.6 respectively. First four appendices included
chromatograms for standards ran. Results had been detected three times and can be found in
Table 5.3.3. which indicates the Concentrations of Glucose and %Conversion of Glucan to
Glucose and Table 5.3.4 indicates the Concentrations of Xylose and %Conversion of Xylan to
Xylose.

The concentration of glucose and xylose had been calculated with equations from linear
regression equation below:

Concentration of Glucose =Peak Height = 7.257 x (concentration of Glucose) - 3.412

24
Concentration of Xylose = Peak Height = 11.891x (concentration of Xylose) - 6.0325

The Glucan and Xylan conversion (%) was calculated by :

𝑚𝑔
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 ( ) 𝑥 𝑉𝑜𝑙𝑢𝑚𝑒 𝑥 𝐻𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝐹𝑎𝑐𝑡𝑜𝑟
𝑚𝐿
% Conversion of Glucan = 𝑔 𝑝𝑜𝑙𝑦𝑠𝑎𝑐𝑐ℎ𝑖𝑟𝑖𝑑𝑒 x 100%
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑟𝑎𝑤 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝑥 𝑔 𝑏𝑖𝑜𝑚𝑎𝑠𝑠

𝑚𝑔
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓𝑋𝑦𝑙𝑜𝑠𝑒 ( ) 𝑥 𝑉𝑜𝑙𝑢𝑚𝑒 𝑥 𝐻𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝐹𝑎𝑐𝑡𝑜𝑟
𝑚𝐿
% Conversion of Xylan = 𝑔 𝑝𝑜𝑙𝑦𝑠𝑎𝑐𝑐ℎ𝑖𝑟𝑖𝑑𝑒 x 100%
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑟𝑎𝑤 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙 𝑥 𝑔 𝑏𝑖𝑜𝑚𝑎𝑠𝑠

∑𝑋
Mean = 𝑛

(∑ 𝑋−𝑋𝑚)2
Standard Deviation : √ 𝑛−1

Variance : (standard deviation)2

Figure 5.3.1 indicates Calibration Graph of Concentration of Glucose against Peak Height

25
 Graph of Peak Height and Concentration of
Glucose
30
25
Peak Height(mV)

20
15
10
5 y = 7.2572x - 3.412
0 R² = 0.9844
0 1 2 3 4 5
Concentration of Glucose (mg/mL)

Figure 5.3.2 indicates calibration graph of Concentration of Xylose against Peak Height

Graph of Peak Height against Concentration of

Xylose
45
40
35
Peak Height(mV)

30
25
20
15
10 y = 11.891x - 6.035
5 R² = 0.9998
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Concentration of Xylose (mg/mL)

Table 5.3 indicates the Concentrations of Glucose ,Conversion of Glucan to Glucose

26
 Peak Concentration Concentration Mass of % Relative
Height of Glucose (x10 of Bleached Convers Standard
(mV.s) dilution) Glucose(mg/mL) Sample( ion Deviation
(mg/mL) mg)

50 9.814 1.822 18.225 501.3 80.16

50 9.986 1.846 18.46 501.3 81.20

50 9.913 1.836 18.36 501.3 80.76

80.76 0.52

100 9.828 1.824 18.24 506.3 79.44

100 9.947 1.842 18.42 506.3 80.12

100 9.415 1.767 17.67 506.3 79.96

78.87 1.38

150 8.627 1.609 16.09 507.8 69.87

150 8.364 1.622 16.22 507.8 70.43

150 8.395 1.622 16.22 507.8 70.43

70.24 0.26

Diluted
2.5 *

200 20.960 3.358 8.396 501.3 36.95

200 20.977 3.360 8.401 501.3 36.97

200 21.955 3.496 8.739 501.3 38.46

37.46 0.8

No
Dilution

300 16.148 2.690 2.690 586.7 25.46

27
 Peak Concentration of Concentration of Mass of % Relative
Height Glucose(Diluted) Glucose(mg/mL) Bleached Conversi Standard
(mV.) (mg/mL) Sample(m on Deviation
g)

300 16.182 2.700 2.700 586.7 25.50

25.48 0.02

Diluted
2.5x

50 24.654 2.58 6.43 501.8 27.65

23.352 2.47 6.17 501.8 26.51

24.780 2.59 6.45 501.8 27.65

27.27 0.46

100 28.526 2.88 7.2 506.3 30.66

24.693 2.58 5.16 506.3 21.97

22.47 2.39 5.99 506.3 25.51

26.04 1.5

150 20.53 2.23 5.58 507.8 23.69

20.78 2.25 5.62 507.8 23.86

21.25 2.29 5.74 507.8 24.37

23.97 0.25

200 12.83 1.59 3.96 501.3 17.01

200 13.00 1.60 4 501.3 17.20

200 13.84 1.67 4.17 501.3 17.90

17.37 0.33

28
300 10.886 1.423 1.423 586.7 5.25

300 9.247 1.285 1.285 586.7 4.75

5.00 0.35

Reaction Mean % Relative Variance

Times Conversion standard
minutes (w/w) (n=3) deviation

50 80,76 0,5 0,25

100 78,87 1,3 1,69
150 70,24 0,2 0,04
200 37,46 0,8 0,64
300 10,13 0,02 0,0004

Table 5.1 indicates the pre-treatment conditions

The percentage conversion of monomers correlates to delignification, removal of hemicellulose

and increasing of crystallinity of cellulose. [33] From results obtained with high yields of
monomers achieved, it can be noted that the combination of ammonia pre-treatment and
bleaching process had successfully removed lignin and traces of hemicellulose while retaining
cellulose from sugarcane bagasse material.

29
Table 5. indicates the variation of xylose and glucose yields from enzymatic hydrolysis with
different pre-treatment conditions. It can be noted that glucose had been the main carbohydrate
contributor in the liquid enzymatic hydrolyzate.

As the pretreatment retention times increased, the glucose yield gradually decreased from
80.76% in 50 minutes to 10.13% in 300 minutes. It is observed that the optimum time for
ammonia pre-treatment is 50 minutes at 1700C and 25% w/w ammonia had removed the most
amount of lignin and hemicellulose.

As the pretreatment retention times increased, the xylose yields gradually decreased from 18.75
in 50 minutes to 5% at 300 minutes. The removal of lignin resulted in pore formation and
swelling of biomass, thus increasing the surface area and subsequently improving enzyme
accessibility.

In accordance with

The standard deviation

The variance

5.3.2. Factors Affecting Enzymatic Hydrolysis

Enzymatic hydrolysis depends of optimization of conditions for maximal efficiency and suffers
from product inhibition and biomass structural restraints.[36] Factors that affect enzyme
saccharification include hydrolysis temperature, time, pH, enzyme loading and substrate
conditions.

pH

Different physical factors affect the conversion of cellulose and pH is a key factor affecting
cellulase production. The effect of pH on cellulase

30
 Graph of Reaction Times against
%Conversion of Glucan to Glucose
90
%Conversion of Glucan to Glucose

80
80.76 78.87
70
70.24
60
50
40
30 37.46
20
10
10.13
0
50 100 150 200 300
Reaction Times(minutes)

Chart of % Conversion of Xylan to

Xylose against Reaction Times
20
18
% Conversion of Xylan to Xylose

18.75
16 17.44
14 15.92

12 13.45
10
8
6
4 5
2
0
50 100 150 200 300
Reaction Times

31
 5.2 FT-IR Analysis of Enzymatic Residue

Key

Dark Blue: Enzymatic Residue

Yellow : Ammonia Treated Sample

Orange : Raw untreated Sample

Light Blue : Bleached sample

Figure above shows the ATR FTIR spectra of the sugarcane bagasse raw material, dilute
ammonia pre-treated solid, bleached solid and enzymatic hydrolysis residue. The spectra were
scanned from 4000cm-1 to 650cm-1.

32
The bands at 3400-3200cm-1 for all spectra indicates the O-H stretching of hydroxyl groups
present in cellulose, hemicellulose and lignin. [31] These bands had been extremely prominent in
enzymatic residue compared to raw untreated sugarcane bagasse.

Compared to the raw material, dilute ammonia pre-treated solid and bleached solid, the lignin
bond at 1510cm-1(aromatic ring of lignin) in enzymolysis residue was significantly enhanced.
This phenomenon can be explained by most of hemicellulose(xylan) and cellulose(glucan) being
removed during pre-treatment, bleaching and/or enzymolysis.

In Fig. 5.3.2. as can be seen, the intensity of carbonyl bond at 1,740 cm-1, which has been
ascribed to the C=O of acetyl groups in hemicellulose, compared to raw untreated material which
has a prominent peak at 1740cm-1 which had disappeared gradually in the enzymolysis residue.

The enzymatic residue tended to have a greater peak in 1200-1300cm-1 region which is a
distinctive characteristic of hemicellulose and lignin components.

Table 5.4. indicates the frequency and functional groups

Frequency (cm-1) Corresponding to Functional

Groups

3400-3200 O-H stretching of Hydroxyl

groups of Cellulose,
Hemicellulose and Lignin

2891 C-H stretching of alkyl groups

1730 C=O of acetyl groups of

hemicellulose

1510 Aromatic ring of lignin

1430-1420 C-H stretching of Cellulose

33
7.CONCLUSION AND RECOMMENDATIONS
The aim of the study had been to determine the viability of the preparation of monomers of
hemicellulose and cellulose from sugarcane bagasse using dilute ammonia treatment, bleaching
and enzymatic hydrolysis and to determine optimum conditions of ammonia treatment. It is
hoped that further study can be driven into ammonia treatment conditions with regards to
preparation of monomers of cellulose and hemicellulose from biomass.

Biomass pretreatment remains a key aspect in the bioprocessing of lignocellulosic material for
biofuels production. The investigation into finding a sustainable, profitable and environmentally
method has been extensively researched.

Sugarcane Bagasse can be utilized as a source of cellulose and hemicellulose for producing
monomers of polysaccharides mentioned. The preparation of cellulose from sugarcane bagasse
was produced by two steps involving ammonia treatment and acidic sodium chlorite which
produced white coloured purified cellulose and hemicellulose solid. This solid was further
enzymatically hydrolysed to extract monomers of cellulose and hemicellulose.

The aim of the study was to is evaluate the effects of dilute ammonia pre-treatment on the solid
yields, delignification and the conversion of glucan and xylan to xylose and glucose at different
pre-treatment reaction times ranging from 0-300min.

The experiments

X-Ray Diffraction,Thermal Gravimetric Analysis and Scanning Electron Microscopy can be

used to determine properties of sample about composition, morphological, structure and thermal
stability information. Therefore, dilute ammonia pretreatment can be successfully applied to
sugarcane bagasse to improve the total sugar yields during enzymatic hydrolysis.

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38
 Peak Concentration of Concentration of Mass of Conversion Standard
Height Glucose (x10 Xylose(mg/mL) Raw Percentage Deviation
(mV) dilution) (mg/mL) sample

50 4.414 0.4264 4.264 501.3 18.34

50 4.584 0.4584 4.584 501.3 19.047

50 4.536 0.4382 4.382 501.3 18.85

Mean 18.75 0.36

100 3.830 0.3700 3.700 506.3 15.74

100 3.602 0.3479 3.479 506.3 14.81

100 5.298 0.5118 5.11 506.3 21.78

Mean 17.44 3.78

150 3.740 0.361 3.613 507.8 15.33

150 4.040 0.390 3.902 507.8 16.56

150 3.868 0.373 3.735 507.8 15.86

Mean 15.92 0.62

Diluted 2.5
*

200 12.283 1.186 2.965 501.3 12.76

200 13.00 1.255 3.138 501.3 13.50

200 13.561 1.309 3.274 501.3 14.09

Mean 13.45 0.66

No Dilution

300 10.886 1.423 1.423 586.7 5.257

300 9.247 1.285 1.285 586.7 4.75

Mean 5.00 0.35

39