Effects of temperature and reaction time of liquid hot water pretreatment for sugar production from cassava pulp

THAI NGUYEN UNIVERSITY
UNIVERSITY OF AGRICULTURE AND FORESTRY

TO HONG ANH
THESIS TITLE:
EFFECTS OF TEMPERATURE AND REACTION TIME
OF LIQUID HOT WATER PRETREATMENT FOR SUGAR
PRODUCTION FROM CASSAVA PULP

BACHELOR THESIS

Study Mode : Full - time
Major

: Food Technology

Faculty

: Biotechnology and Food Technology

Batch

: 2014 – 2018

Thai Nguyen, 2018


THAI NGUYEN UNIVERSITY
UNIVERSITY OF AGRICULTURE AND FORESTRY

TO HONG ANH

THESIS TITLE:
EFFECTS OF TEMPERATURE AND REACTION TIME
OF LIQUID HOT WATER PRETREATMENT FOR SUGAR PRODUCTION
FROM CASSAVA PULP

BACHELOR THESIS
Study Mode : Full - time
Major

: Food Technology

Faculty

: Biotechnology and Food Technology

Batch

: 2014 – 2018

Supervisors : Assoc. Prof. Dr. Pawinee Chaiprasert
Mr. Suppanut Varongchayakul
Msc. Trinh Thi Chung

Thai Nguyen, 2018
i


DOCUMENTATION PAGE WITH ABSTRACT
Thai Nguyen University of Agriculture and Forestry
Major

Food Technology

Student name

To Hong Anh

Student ID

DTN 1453170065
Effects of temperature and reaction time of liquid

Thesis Title

hot water pretreatment for sugar production from
cassava pulp
Assoc. Prof. Dr. Pawinee Chaiprasert.

Supervisors

Mr. Suppanut Varongchayakul
Msc. Trinh Thi Chung

Abstract:
Cassava is one of the important food crops in the world. Most of cassava was
cultivated in the tropical or subtropical area [1]. Thailand was the major country that
cultivated cassava. Cassava root was rich in starch which was mostly used as raw
material in cassava starch production.
Cassava pulp was the waste residue by product from cassava starch manufacture [2].
Cassava pulp contains about 50–70% starch and 20–30% of lignocellulose (LCMs)

on a dry weight basis [3]. However, the direct hydrolysis of cassava pulp had low
efficiency due to the recalcitrant properties of LCM and starch was trapped in cell
wall of LCMs [4]. To fully utilize, pretreatment step was applied before hydrolysis
step which impact on cell wall properties of cassava pulp to improve the hydrolysis
efficiency next step [5].
Pretreatment used physical, chemical, physico-chemical or biological agent [6].
Liquid hot water (LHW) pretreatment

used the combination of physical and

chemical process .The pressure in this method was used to keep water in the liquid
state under high temperature [7] .During LHW, water is penetrated into the cell wall
structure and caused the hydrating of starch and cellulose, solubilization of
hemicellulose and partial remove lignin of the structure. These effects caused
increasing the surface area which lead to improve the efficiency in hydrolysis step
ii


and enhance the sugar recovery [8]. Moreover, the effectiveness of LHW depends on
many factors such as time reaction, temperature, pressure and solid:liquid ratio [9].
In this work, the effective of process variables (temperature and reaction time) in
LHW pretreatment of cassava pulp for sugar production was addressed by means of
design of experiments. The result show that condition at 180°C for 15 minutes,
obtained the highest amount of total sugar at 703 mg/g CP.

Keywords:

Cassava

pulp,

liquid

hot

water

pretreatment,

temperature reaction time, sugar production

Number of pages:

23

Date of Submission:

08/06/2018

iii


ACKNOWLEDGMENTS
To complete this bachelor thesis, in addition to my own efforts, I have received
great encouragement and supports from many individuals and groups during the
internship since December 18th, 2017 to May 25th, 2018.
Firstly, I would like to express my gratitude to my supervisors Assoc. Prof. Dr.
Pawinee Chaiprasert and my mentor Mr. Suppanut Varongchayakul from School of
Bioresources and Technology, King Mongkut’s University of Technology Thonburi
(KMUTT), Bangkok, Thailand and Msc. Trinh Thi Chung from Department of Food

Technology, Faculty of Biotechnology and Food Technology, Thai Nguyen University
of Agriculture and Forestry (TUAF), Thai Nguyen City, Vietnam, who help and give
me the best conditions to complete the bachelor thesis.
A special thanks to the Phytobioactive and Eco-Waste Lab members for
motivating and teaching me about lab research works. Moreover, I would like to
thanks my family, my friends for supporting me as an internship at KMUTT.
I sincerely appreciate all supports from lecturers at Faculty of Biotechnology
and Food Technology, TUAF, I also thank the Vietnamese students and my friends in
Thailand for helping me during this internship.

iv


TABLE OF CONTENTS
PAGE
DOCUMENTATION PAGE WITH ABSTRACT .........................................................i
ACKNOWLEDGMENTS .............................................................................................iv
TABLE OF CONTENTS ............................................................................................... v
LIST OF FIGURES .......................................................................................................vi
LIST OF TABLES ...................................................................................................... vii
LIST OF ABBREVIATIONS .................................................................................... viii
PART I. INTRODUCTION ........................................................................................... 1
1.1. Rationale ...................................................................................................................1
1.2. Problem statement and justification .........................................................................3
1.3. Objectives .................................................................................................................6
1.4 Hypothesis .................................................................................................................6
1.5. Expect benefits .........................................................................................................6
PART II. METHODOLOGY ......................................................................................... 7
2.1. Materials ...................................................................................................................7
2.2. Methods ....................................................................................................................7

2.2.1. Sample preparation .........................................................................................7
2.2.2. Analytical methods .........................................................................................8
2.2.3. Liquid hot water (LHW) pretreatment ..........................................................14
PART III. RESULTS AND DISCUSSION ................................................................. 16
3.1. Characterization of substrate ..................................................................................16
3.2 Effect of temperature and reaction time on solid remaining ...................................17
3.3. Effect of temperature and reaction time on total sugar and reducing sugar of
cassava pulp. ..................................................................................................................18
3.3.1. Reducing sugar .............................................................................................20
3.3.2. Total sugar ....................................................................................................21
PART IV. CONCLUSION AND RECOMMENDATION .......................................... 23
5.1 Conclusion ...............................................................................................................23
5.2 Recommendation .....................................................................................................23
REFERENCES ............................................................................................................. 24
APPENDICES A .......................................................................................................... 30

v


LIST OF FIGURES
Fig 1.1 Top 10 country production of cassava in the world (FAO, 2016) ......................1
Fig 1.2 Lignocellulose materials structure .....................................................................2
Fig 2.1 Cassava pulp after dry .........................................................................................8
Fig 3.2 The result of characterization of cassava pulp in % dry weigh basis ...............16
Fig 3.2 The content of reducing sugar in hydrolysate after LHW pretreatment. ..........20
Fig 3.3 The content of total sugar in hydrolysate after LHW pretreatment. .................21

vi

LIST OF TABLES
Table 2.2 Following the LHW pretreatment ...............................................................15
Table 3.1 Chemical composition of cassava pulp in % dry ........................................16
Table 3.2 Results of substrate after LHW pretreatment .............................................18
Table 3.3 Results of reducing sugar and total sugar ...................................................18
Table A1. The temperature and reaction time value of LHW were range of 140°C 180°C and 0 - 30 min for total sugar and reducing sugar ............................30
Table A2. Effect of liquid hot water pretreatment at 140oC with various reaction time
conditions for total sugar and reducing sugar ..............................................31
Table A3. Effect of liquid hot water pretreatment at 160oC with various reaction time
conditions for total sugar and reducing sugar ..............................................32
Table A4. Effect of liquid hot water pretreatment at 180oC with various reaction time
conditions for total sugar and reducing sugar ..............................................33

vii


LIST OF ABBREVIATIONS
µg

Microgram

µL

Microlitre

ADF

Acid detergent fiber

ADL

Acid detergent lignin

Conc.

Concentration

CP

Cassava pulp

Fig

Figure

g

Gram

hr

Hour

L

Litre

LCMs

Lignocellulose material

LHW

Liquid hot water

mg

Milligram

min

Minute

mL

Millilitre

mm

Milli mol

NDF

Neutral detergent fiber

nm

Nano mettre

rpm

Revolutions per minute

RS

Reducing sugar

sec

Seconds

TS

Total sugar

viii


PART I. INTRODUCTION
1.1. Rationale
Cassava is the third-largest source of food carbohydrates in the world [10].
Cassava is a major staple food in the developing world, providing a basic diet for over
half a billion people [11]. It is mainly use for processing chips, pellets and starch- by
processing. The following statistics of FAOSTAT (2013) reported that Thailand was
mainly used for food, feed, and fuel. Thailand is the first world exporter of cassava
chips and cassava starch, as well as the second largest producer after Nigeria in
cassava production in 2016 [12]. According the data showed in the chart below.
70

Production (Mtonnes)

57.14

50
31.16

30
21.1

20.75

17.8

10

14.7

11.06 10.21

9.98

9.1

-10

Country

Fig 1.1 Top 10 country production of cassava in the world (FAO, 2016)
Cassava roots are very rich in starch that was used main material of cassava
starch industry. When dried to a powdery (or pearly) extract, is called cassava

starch (Tapioca). During cassava starch processing, the range of using cassava roots to
production 1 ton of cassava starch were about 3.5- 4.8 tons. For agriculture residues
from cassava starch production included cassava peel was 50- 160 kg and cassava pulp
was 1.0- 2.8 tons [12]. By this conclusion, the mount of cassava pulp from cassava
starch processing have a huge solid residue - by product. This is available and cheap
material but the utilization of cassava pulp did not have high efficiency. Normally
cassava pulp was sale for animal feed at low price. Therefore the enhancement of
1


value products of cassava pulp is necessary, to convert into high value products. There
are many considerable numbers of scientific literature about the characteristics and
composition of cassava pulp that utilization of cassava pulp for value added product,
such as production of ethanol [13], hydrogen [14], biogas [15], and organic fertilizer..,
However, little discussion on sugar production from cassava pulp was analyzed.
Cassava pulp contains about 50–70% starch, 20–30% lignocellulose and
content small amount of protein and fat [3]. Due to the low amount of protein in
cassava pulp, it was not suitable for animal feedstock [16]. Although it has still high
organic but utilization did not have high efficiency on direct hydrolysis cassava pulp.
Due to the recalcitrant properties of LCMs and starch was trapped in cell wall of
LCMs which was difficult to break down [17, 18]. Hemicellulose and lignin provide
the protective sheath around the cellulose. In addition, the high crystalline structure of
cellulose is obstacle to hydrolysis, this is like a barrier to prevent the degradation from
surrounding environment (Fig.1.2). [19] Thus, the pretreatment become important step
that applied before hydrolysis step to improve the hydrolysis efficiency.

Fig 1.2 Lignocellulose materials structure [1]
The propose of pretreatment is breakdown cell wall of LCMs. Increase of
accessible surface area lead to improve recrystallization of cellulose, solubilization of
hemicellulose and remove of lignin were the effects of pretreatment on LCMs [20, 21].

When cell wall of LCMs was breakdown, starch in cassava pulp could be easy of
approach during hydrolysis step. From there improve the hydrolysis of cassava pulp
for next step.
There are many previous researches that use pretreatment methods on LCMs.
There were four types of pretreatment: (1) physical pretreatment, (2) chemical
2


pretreatment, (3) physico-chemical pretreatment and (4) biological pretreatment [22].
Each of these methods has different mechanism, advantages and disadvantages.
Depend on substance and objective that choose different methods. Liquid hot water
(LHW) pretreatment was used the combination of physical and chemical process that
does not employ any catalyst, particularly promising [6]. This method has been
reported to have the potential to remove most hemicellulose while increasing cellulose
hydrolysis and minimizing sugar degradation reactions from inhibitors [7, 23]. These
effects suitable for the aim product sugar from cassava pulp [24]. The optimization of
each factors in LHW pretreatment were considered to avoid the unpleasant effects
such as the generation of inhibitors and high recovery sugar depends on many factors
such as reaction time , temperature, pressure and ratio [25].
This study aims to look at the effects of temperature and reaction time of liquid
hot water pretreatment for sugar production from cassava pulp.
1.2. Problem statement and justification
Cassava was mainly cultivated in tropical areas and used as food and feedstock.
In Thailand, approximately 10 million tons of fresh cassava tubers are consumed
annually as a starch staple [12]. When starch is extracted from cassava tubers during
manufacturing, grated cassava tubers are separated into starch granules and fibrous
residual materials by water extraction followed by centrifugation. The fibrous residual
material, called cassava pulp, accounts for approximately 10–30% by weight (wet) of
the original tubers. Therefore, the tapioca starch industry. In Thailand is estimated to
generate at least one million ton of cassava pulp annually from 10 million tons of fresh

tubers [12]. According to reports [26] and processing practices in Thailand, a large
amount of starch (up to 60%, on a dry weight basis) together with cellulosic fiber is
contained in the cassava pulp [20]. It shows that the large amount of cassava pulp was
generated together with high content in organic compounds. This is available and
cheap material that were gain attention to convert into high value products such as
production of ethanol, sugar, hydrogen, biogas and organic fertilizer [27].
Recently, there has been more interest on using pretreatment step before apply
hydrolysis step. Because it is easily applied, cheaper and more efficient direct
3


hydrolysis [28]. The aim of pretreatment method is breakdown cell wall of LCMs of
cassava pulp. Increase of accessible surface area lead to starch in cassava pulp could
be easy of approach during hydrolysis step. From there improve the hydrolysis [29] of
cassava pulp for next step. The selection of pretreatment methods to apply in industrial
scale was the great concern due to the economical point of view to reach the
commercial scale. There were several choice and factors that have to be considered
such as capital and operational cost, the loading of LCMs, high recovery of
carbohydrate [23] and low degradation of sugar into inhibitors [30]. Since several
pretreatments have been studied but only a few of pretreatment methods were reached
the cost effective and applied in industrial scale [31]. These pretreatment methods
were steam explosion, liquid hot water, concentrated and diluted acid pretreatment and
biological pretreatment.
The using of acid in lignocellulose pretreatment was to solubilization of
hemicellulose structure in order to make cellulose more accessibility. In addition,
amorphous regions of cellulose structure could hydrolyze and partial of lignin was
removed but the solubility of lignin was low. For acid concentration, concentrated (3070%) or diluted (below 4%) were used. However, the using of diluted acid
concentration was more favorable than concentrated acid [32]. Due to it was very
dangerous, highly toxic, corrosive and the high cost of equipment and maintenance.
The using of diluted acid pretreatment was integrated with temperature either low

(below 120℃) and high (above 180℃) to reach the same reaction rate of concentrated
acid. Longer reaction time was performed under low temperature (30- 90 min). For
short reaction time, the reaction was conducted under high temperature (1- 5 min).
Hydrochloric acid, nitric acid and phosphoric acid were used but sulfuric acid was the
most commonly used due to inexpensive and had high efficiency [33]. Although, the
advantages were obtained high efficiency in hydrolysis of hemicellulose and increase
the accessibility of cellulose structure, the main drawbacks were the sugar
decomposition into inhibitors, furfural, hydroxyl methyl furfural and acetic acid, and
the cost of equipment, maintenance, and neutralization process after pretreatment [34]
Steam explosion or auto hydrolysis, this pretreatment method was one of the
most commonly used for LCMs. During the pretreatment, LCMs are subjected to the
4


steam under high pressure (0. 69- 4. 83 MPa) and high temperature (160- 260℃) for
particular reaction time [35]. Then, the pressure is dramatically reduced which cause
the disruption of the lignocellulose structure. Moreover, acetyl group of hemicellulose
structure is hydrolyzed during the pretreatment, which consequently make the acid
condition [36]. In acid condition, the hydrolysis of ether bonds are occurred, which
results in hemicellulose hydrolysis, partial hydrolyzed lignin and increase surface area
[37]. This pretreatment method was gained promising in commercial scale due to the
low energy consumption, no chemicals and low cost for neutralization in downstream
process. However, steam explosion could generate inhibitors by the degradation of
sugars. To reduce the concentration of inhibitors, water was used to remove and this
causes the major loss of sugar [8]. The optimizations of steam explosion conditions
were studied to reduce the formation of inhibitors.
Liquid

hot

water

pretreatment

sometimes

also

called

hydrothermal

pretreatment. The pressure in this method was used to keep water in the liquid state
under different temperature (140- 240℃). The main objective of using LHW was to
solubilize hemicellulose which increases accessible to cellulose structure. The
mechanism of LHW was similar to steam explosion. The hydronium ion was
generated from water and catalyzed glycoside bonds. During LHW, water is
penetrated into the cell wall structure and caused the hydrating of cellulose,
solubilization of hemicellulose and partial remove lignin of the structure. These effects
were lead to increase surface area of the structure. After the pretreatment, hydrolysate
could be directly subjected to hydrolysis or fermentation step because low amount of
inhibitors were generated.
The different between steam explosion and LHW, the sugar recovery was
higher and the formation of inhibitors was lower. These were advantages of this
method. However, due to the large amount of water was used, causing low sugar
concentration, and large amount of energy consumption. By this reasons, hydrolysis
step need to apply after LHW pretreatment step to fully utilization of sugar from
cassava pulp [38]. Application of LHW on cassava pulp have been done on the
utilization for value added product such as the combination of pretreatment and
fermentation to was made to convert its components to ethanol [39]; or to enhance

5


sugar recovery from wheat straw by way using combination of LHW and emzyme
hydrolysis [9]. Several study of LHW on LCMs were observed such as wheat straw
[40] , sugarcane bagasse [41], soybean straw [42], corn fiber [43], rice straw [44] and
soy hulls [45].
The optimization of each factors in LHW pretreatment were considered to get
maximum yield of sugar [25]. In this study, the effects of temperature and reaction
time of LHW for sugar production.
1.3. Objectives
 To assess temperature and reaction time of LHW on Cassava pulp
 To prepare the substrate for hydrolysis step on sugar production
1.4 Hypothesis
 Liquid hot water pretreatment could be hydrolysis of cassava pulp for sugar
production
1.5. Expect benefits

 To extend the knowledge regarding effect of the temperature and reaction time
of LHW for the high sugar production
 To improve the substrate is easily access in further step hydrolysis

6


PART II. METHODOLOGY
2.1. Materials
1. Cassava pulp from Choncharoen company, Bangkok, Thailand
2. Water bath (Julabo TW20)
3. Zip bags, Nylon bags, Fiber bags

4. Hot air oven (Memmert, UFE600, Germany)
5. Vortex mixture (vortex genie, Scientific Industries, USA)
6. UV-Visible spectrophotometer (UV - Pharo 100, Spectroquant, Germany)
7. Fume hood (Well model 2000, Thailand)
8. Analytical scales
9. Pressure Reactor (Parr Reactor 4848, America)
10. Automatic fiber (Fibretherm ft12, Gerhardt)
11. Furnace (Thermolyne 48000, Model F48025-80)
12. Centrifuge (PLC - 05, Germany Industrial crop, Taiwan
13. Laboratory instruments such as a micropipette, cuvette, pipette tip, crucible,
beaker, petri dish, hycon plastic, dispensing bottle, Erlenmeyer flask,
desiccator.
14. The chemical used in the experiment: Sulfuric acid 98 %, Phenol, Sodium
hydroxide (Sigma Chemicals, NJ, USA), Red Metheny, mixed catalyst
digestion tablets (Potassium sulfate and Selenium), Boric acid were used for
protein analysis. Petroleum ether was used for fat content analysis. Sulfuric
acid, sodium hydroxide (Sigma Chemicals, NJ, USA), Sodium hydroxide,
Sodium postassium tartrate, Dinitrosalicylic acid , Sodium pyrosulfite
2.2. Methods
2.2.1. Sample preparation
Cassava pulp (CP), the solid residues left after cassava starch processing, were
supplied by the Choncharoen, Bangkok, Thailand. It was milled and screened to 8 -18
mesh and dried in a hot air oven at 55oC to constant weigh (~10h). After that, it was
kept in zip bags and stored at the room temperature [46] ( Fig. 2.1)

7


Fig 2.1 Cassava pulp after dry
2.2.2. Analytical methods

* Proximate Composition
 Characterization as: Moisture, Ash, Total solid, Volatile solid, Protein,
Lipid were determined by AOAC 2000.
 Chemical compositions as:
1. Total sugar by Phenol-Sulfuric acid method [47] (Dubois, 1956 )
2. Reducing sugar by DNS method [48] (Miller, 1959)
3. Starch content by enzymatic method [49] (Ref )
4. Lignocellulose (NDF, ADF, ADL) by Detergent method [50] (Van
Soest, 1991)
2.2.2.1. Analyze moisture
* Method
1. Place the crucible in the furnace at 550°C overnight to ensure that impurities
on the surface of the crucible are burned off.
2. Cool the crucible in the desiccator (30 min).
8


3. Weigh the crucible to 4 decimal places.
4. Weigh about 1- 2 g sample into the crucible. Dry for 3 hr at 105 °C.
5. Cool down in the desiccator (30 min)
6. Re-Weigh and calculation moisture content
* Calculation moisture:
Moisture (%) =

𝑾𝟏−𝑾𝟐
𝑾𝟏

x 100%

Where: W1: Weigh (g) of sample before dry

W2: Weigh (g) of sample after dry
2.2.2.2. Analyze Ash
* Method:
1. Place the crucible in the furnace at 550°C overnight to ensure that impurities
on the surface of the crucible are burned off.
2. Cool the crucible in the desiccator (30 min).
3. Weigh the crucible to 4 decimal places.
4. Weigh about 1- 2 g sample into the crucible. Dry for 3 hr at 105°C.
5. Heat at 550°C overnight
6. Cool down in the desiccator for 30 min
7. Weigh the ash with crucible when the sample turns to gray and calculation
of ash content
* Calculation Ash:
Ash (%) =

Where

𝐰𝟐−𝐰𝟏
𝐰𝟑−𝐰𝟏

x 100%

w1: weight of crucible;
w2: weight of crucible and sample;
w3: weight of sample

2.2.2.3. Analyze protein by the Kjeldahl method
* Prepare material:

9

Weigh approximately 1 g of sample (to a precision of 2 decimal places) using a
weighing paper. Transfer the sample into Kjeldahl flask tube and add one small spoon
of mixed catalyst digestion tablets.
* Method:
Protein analysis has 3 steps: Homogeneous sample, distillation and titration.
In the first step, additional 20 mL 98% Sulfuric acid solution to each Kjeldahl
flask contain the sample and mixed catalyst. Digest the sample at 360 °C for about 180
minutes. Then, place a titration flask containing 20 ml sulfuric acid (C = 0.1 mol/L)
and a few drops of Tashiro indicator into the unit, raise the platform and began to the
distillation process. After the distillation process has been done, we can then do titrate
the contents of the receiver flask with Sulfuric acid (N=0.1 mol/L) to the neutral
endpoint. Record the volume of Sulfuric acid required. Blank titration is carried out to
test the color change of Tashiro indicator.
*Calculation Protein:
Protein (%) =

(𝐀−𝐁)× 𝐍 ×𝟏.𝟒𝟎𝟎𝟕 ×𝐄
𝐖

Where
A: volume (ml) of 0.1 N H2SO4 used sample titration
B: volume (ml) of 0.1 H2SO4 used blank titration
N: normality of H2SO4
14.007: atomic weight of nitrogen
E: Empirical factor
W: weight (g) of sample
2.2.2.4. Analyze lipid by Soxhlet method
* Method

1. Place the beakers and lid in the incubator at 105°C about 5 hr and weight of
beakers.
2. Weigh about 1 g of the sample to paper filter and wrap.
3. Take the sample into extraction thimble and transfer into soxhlet.
4. Fill petroleum ether about 250 mL into the round bottom flask and take it on
the heating mantle.
5. Connect the soxhlet apparatus and turn on the water to cool them and then
switch on the heating mantle.
10


6. Heat the sample about 14 hr (heat rate of 150 drops/min).
7. Evaporate the solvent by hot air oven at 105°C until the solvent is completely
evaporated and bottles are completely dry.
8. After drying, transfer the bottle with a partially covered lid to the desiccator
to cool. Re-weighs the bottles and it dried content.
* Calculation Lipid:
% Fat =

𝑾𝟐−𝑾𝟏
𝑾𝟑

x 100

Where
w1: weight of beaker initial, gram;
w2: weight of fat and beaker, gram;
w3: weight of sample initial, gram
2.2.2.5. Analyze Starch content by hydrolysis method
Procedure:

1.

Accurately weigh 75- 100 mg sample in duplicate into 100 mL beaker.
Samples should contain up to 100 mg starch. Alternatively, filter 80% Et
OH extracted 0.2 g sample through Whatman and transfer entire filter
paper to beaker. Beside samples, include an empty beaker to which all
reagents will be added that will act as your reagent blank

2.

Add 20 mL of dH2O to the sample and stir with a magnetic stir bar

3.

Add 0.1 mL heat-stable -α- amylase to sample and water and stir with a
magnetic stir bar.

4.

Cover beaker with Aluminum foil and place in 90C in water bath for 1
hour. Remove beaker from water bath and cool on bench for 15 minutes

5.

Filter samples through glass wool plugs in funnels into 100 mL volumetric
flasks. Rinse the beaker, the funnel and glass wool thoroughly with d H2O.
Adjust filtered solutions to volume with d H2O. Mix solutions thoroughly
through repeated inversion and shaking of capped or stoppered flasks

6.

Pipette a 1 mL aliquot of each sample into individual 50 mL volumetric
flasks

7.

Add 8 mL of 0.1 M sodium acetate buffer (pH ~ 4.5) to each flasks
11


8.

Add 50 𝜇L of amyloglucosidase. Gently swirl flask to mix

9.

Incubate flasks in 60°C water bath for 30 minutes

10. Bring sample to volume with d H2O
11. Assay the hydrolysed sample for glucose to determine starch content.
2.2.2.6. Analyze Lignocellulose (NDF, ADF, ADL) by Detergent method
* Analyze NDF:
Procedure:
1. Weigh exactly 1g sample
2 Add 100 mL Neutral detergent and 0.2 mL heat- Stable -α amylase
3. Reflux for 1 hour
4 After 1 hr measured from the time boiling began, filter sample
5. Thoroughly rinse the beaker into the filter with boiling dH20
6. Rinse the residue 2x with boiling dH20
7. Rinse the residue 2x with acetone

8. Dry of residue after filter sample (AOAC)
9. Determine ash of residue after filter sample (AOAC)
Calculation:
% NDF=

𝑾 𝒂𝒔𝒉 −𝑾 𝒄𝒓𝒖𝒄𝒊𝒃𝒍𝒆
𝑾 𝒔𝒂𝒎𝒑𝒍𝒆

x 100%

12


* Analyze ADF:
Procedure:
1. Dry the sample at 55C (85% dry matter)
2. Weigh 1 g (W1) sample into Berzelius beaker.
3. Add 100 mL acid-detergent solution at room temperature.
4. Place beaker on heater under the cold water condenser. Heat to boiling in 510 min; reduce heat to avoid foaming as boiling begins.
5. Reflux 60 min from onset of boil
6. After about 30 min, Remove beaker, swirl, and filter through tare fritted
glass crucible, using minimal vacuum.
7. Rinse twice with boiling (95-100C) water.
8. Rinse twice with 30-40 mL acetone
9. Dry 3 hr or overnight in forced-air oven (105C) and weight
Calculation:

% ADF =

𝑾𝒅𝒓𝒚 −𝑾𝒄𝒓𝒖𝒄𝒊𝒃𝒍𝒆

𝑾𝒔𝒂𝒎𝒑𝒍𝒆∗𝟖𝟓%

∗ 𝟏𝟎𝟎%

* Analyze ADL :
From ADF step, take fiber bags contain sample put in 100 mL beaker. The
solution Conc.H2SO4 72% added in beaker. Every 10 min stir fiber bags as mix well
until 3 hours. Rinse the sample with water. The filter bags from the Acid Hydrolysis
Step are placed in an oven overnight and dried at 105°C. After that, heat 3 hours in
Furnace at 550 C.
Calculation:

% ADL =

𝑾𝒂𝒔𝒉 −𝑾𝒄𝒓𝒖𝒄𝒊𝒃𝒍𝒆
𝑾 𝒔𝒂𝒎𝒑𝒍𝒆

∗ 𝟏𝟎𝟎%

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2.2.2.7. Analyze Total Sugar by Phenol- Sulfuric acid method
The setup we used for determined Total sugar can be found is reported from
Dubois, 1956 with some modifications. The 1 mL of appropriately diluted sample
(hydrolysis after LHW) and 1 mL of 2% Phenol and add 5ml Conc.H2SO4 after that
mixed well using a Vortex. The solution was incubated at room temperature (25oC) for
30 min. The absorbance was measured at 490 nm using a spectrophotometer and the
results were compared glucose standard curve.
Additional dilution was done if the absorbance value measured was over the

linear range of the standard curve.
2.2.2.8. Analyze Reducing Sugar by DNS method
The setup we used for determined Reducing sugar by DNS method from Miller,
1959. The 1 mL of appropriately diluted sample (hydrolysis after LHW) added with 1
mL of DNS reagent and then boiling water in bath water at 80oC for 15 minutes. After
that, put it in ice bath for 15 minutes. Then add 5 mL distilled water and mixed well
using a Vortex. The solution was incubated at room temperature (25oC) for 10
minutes. The absorbance was measured at 570nm using a spectrophotometer and the
results were compared to glucose standard curve.
Additional dilution was done if the absorbance value measured was over the
linear range of the standard curve.
2.2.3. Liquid hot water (LHW) pretreatment
2.2.3.1. Pretreatment
Details of the LHW pretreatment are described elsewhere (Yu et al., 2012,
2010). About 5 g of the CP (5% w/v in water) was put into the reactor, which was
sealed and heated to the reaction temperature with the magnetic agitator operating at
500 rpm. The reaction pressure was controlled by the addition of nitrogen [51, 52].
At the end of reaction, the hydrolysate was collected and the reactors were cooled
rapidly to terminate the reaction. Then the solution was separated by filtration to
separate hydrolysate and solid residue. Solid residue was recovered from the reactor
14


using for the measurement of weight loss. The hydrolysate after LHW was analyzed
for reducing sugar and total sugar as describe in 2.2.2.7 and 2.2.2.8.
2.2.3.2. Statistical design of experiments of LHW pretreatment.
The experiments conditions of design are show in table 2.2. In this case,
temperature and reaction time value of LHW were delimited within a range of 140°C 180°C and 0 - 30 minutes under saturated vapor pressure with room temperature as
control and autoclave condition as widely method used to pretreat CP.
Hydrolysate obtained after LHW pretreatment were analyzed to determine total

sugar and reducing sugar.

Table 2.2 Following the LHW pretreatment
Run

Process conditions
Temp

Time

Pressure

( C )

(minutes)

(bar)

Control

Room temp

30

-

1

121

15

1

2

140

0

3.6

140

15

3.6

140

30

3.6

160

0

6.2

160

15

6.2

160

30

6.2

180

0

10.5

180

15

10.5

180

30

10.5

3

4

15


PART III. RESULTS AND DISCUSSION
3.1. Characterization of substrate
Chemical composition of Cassava Pulp used the analytical and calculation. The
results were showed in table 3.1. (Each experiment were replicated at least 3 times)
Table 3.1 Chemical composition of cassava pulp in % dry
Component

% Dry basis

Starch content

47.95

Cellulose

18.95

Hemicellulose

10.05

Lignin

3.98

Protein content

2.54

Crude Fat

0.55

Moisture

7.07

Ash content

2.03

50
40
Starch
30

Cellulose
Hemicellulose

20

Lignin
Moisture

10

Ash
Protein

0

Crude Fat

Fig 3.2 The result of characterization of cassava pulp in % dry weigh basis

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