BAHIR DAR UNIVERSITY INSTITUTE OF TECHNOLOGY

SCHOOL OF RESEARCH AND POST GRADUATE STUDIES

FACULTY OF CHEMICAL AND FOOD ENGINEERING

THESIS REPORT
TITLE:-Lactic Acid Production from Water Hyacinth by Solid-acid Graphene
oxide catalyst

BY:- Birhanie Getachew

Main Advisor:

Prof. Nigus Gabbiye Habtu (PhD)

October : 2023

Bahir Dar, Ethiopia

 BAHIR DAR INSTITUTE OF TECHNOLOGY BAHIR DAR UNIVERSITY
SCHOOL OF RESEARCH AND POST GRADUATE STUDIES
FACULTY OF CHEMICAL AND FOOD ENGINEERING
THESIS REPORT

Student Name: Signature: Date:

Birhanie Getachew …………………… ………………

The following graduate faculty members certify that this student has successfully presented the
necessarily written thesis proposal and oral presentation of this proposal for partial fulfillment of
the thesis-option requirements for the Degree of Master of Science in Process Engineering

Approved:

Advisor Name: Signature: Date:

Prof. Nigus Gabbiye (PhD) …………… …………

Chair Holder Name: Signature: Date:

……………………………. …………… ……........

Faculty Dean Name: Signature: Date:

……………………………. …………… …………

I
 DECLARATION
I, the undersigned, that this thesis is my original work and has not been presented for the award
of a degree in any university and all the sources of the materials used for this thesis have been
duly acknowledged.

Birhanie Getachew Alemayehu ……….. …………

Name of student signature date

Professor Nigus Gabbiye Habtu (PhD) ………… …………

Main advisor name signature date

II
ACKNOWLEDGEMENT
Above all, I would like to thank the Almighty God for always being with me during my work. I
would like to acknowledge my advisor, Professor Nigus Gabbiye Habtu, for his useful
comments guidance willingness to supervise my research, support as well as professional advice
from the starting to completion of my thesis. I would also like to express my gratitude to the
laboratory technician and assistance's of FCFE in BIT and Mr. Gashaw and Mr. Alebel in
particular for their time and help during my experimental work.

III
 Abstract
Water hyacinth biomass (WHB)was used as a substrate for lactic acid production in the present
study. Lactic acid (LaA) is one of the top fifteen rostrum chemicals derived from lignocellulosic
biomass. For the increase of the required product quality, pretreatment of the substrate (WHB)
was needed. Among the pretreatment method, the Organosoly pretreatment process was used in
this study due to its simplicity, low cost, ease of separation that reduces energy consumption, and
less toxicity as compared to other chemical pretreatment processes. This pretreatment process
was applied with EtOH/water in different mixing ratios. The raw and pretreated WHB were
characterized for their composition using NREL and ASTM protocols. It is found that the raw
WHB has a composition of 44.8% cellulose, 25.12% hemicellulose, and 6.58% lignin with a
balance of extractive and ash. The EtOH/water pretreatment was performed by CCD for
delignification and enhancing sugar content in order to upgrade the quality of lactic acid and
simplify the hydrolysis step. The hydrolysis process was done by using the heterogeneous
catalyst for strengthening of environmental regulation, enhancing an adsorption and an
isomerization of reduced sugar on the catalyst surface. It is an attractiveness strategy for the
replacement of corrosive and harmful traditional mineral acid catalyst due to the presence of
oxygenated and carboxylic groups together with Lewis acid site caused by the electron inductive
effect of sulfonic groups In this study, the effect of pretreatment conditions on the hemicellulose
recovery and delignification was investigated in order to optimize the hydrolysis process
conditions for lactic acid production. The optimization was done by using BBD taking account
of temperature, time, and catalyst concentration. It is found that temperature of 140 ℃, contact
time of 90 min, and EtOH/Water ratios of 12.5L/g were the optimal conditions for the
pretreatment process. At this optimal condition, 60.07% of lignin removal, 84.2% of
holocellulose recovery, and the reduced sugar (32.13g/L or 64.26%) was measured by the DNS
method. The recovered hollocellulose was hydrolyzed by the solid-acid Graphene oxide catalyst
synthesized from pure graphite powder through the improved hammers method and
characterized by FTIR and XRD. Lastly, 0.3432g of lactic acid per gram of biomass or 34.2%
yield at 45 minute) was quantified by different analytical methods like LA standard method and
FTIR spectrum.

Keywords:- Water Hyacinth, Organosoly Pretreatment, Acid Hydrolysis, graphene oxide

catalyst, Lactic Acid.

IV
 ABBREVIATION
AC Ash Content
AFEX Ammonium Fiber Explosion
(CF)n Poly( Carbon Mono-Fluoride)
CL Catalyst load
D-LaA D-Lctic acid
DI De-ionized Water
DNS Di-Nitrosalicylic Acid Method
GRAS Generally Regarded As Safe
EC Eichhornia Crassipes
FC Fixed Carbon
FCED Food and Chemical Engineering Department
MC Moisture Content
FT-IR Fourier Transform-Infrared Ray
GO Graphene Oxide
HR Holocellulose Recovery
HT Hydrolysis temperature
Ht Hydrolysis time
L-LaA L-Lactic Acid
LB Lignocellulosic Biomass
LR Lignin Removal
MAS Magic-Angle Spinning
PLA Polylactic Acid
NMR Nuclear Magnetic Resonance
Rpm Revolution Per Minute
SE Steam Explosion
TGA Thermo-gravimetric Analyzer
WH Water Hyacinth
WHB Water Hyacinth Biomass
USFDA The United States Food And Drug Administration
VM Volatile matter

V
Table of Contents
DECLARATION ............................................................................................................................ II

ACKNOWLEDGEMENT ............................................................................................................ III

Abstract ......................................................................................................................................... IV

ABBREVIATION.......................................................................................................................... V

Table of Contents .......................................................................................................................... VI

List of Table ................................................................................................................................... X

List of Figure................................................................................................................................ XII

CHAPTER ONE ............................................................................................................................. 1

INTRODUCTION .......................................................................................................................... 1

1.1 Background ........................................................................................................................... 1

1.2 Statement of Problems .......................................................................................................... 4

1.3 Objectives .............................................................................................................................. 5

1.3.1 General Objectives ......................................................................................................... 5

1.3.2 Specific Objectives ......................................................................................................... 5

1.4 Scope of the study ................................................................................................................. 5

1.5 Significance of the study ....................................................................................................... 5

CHAPTER TWO ............................................................................................................................ 6

LITERATURE REVIEW ............................................................................................................... 6

2.1 Background ........................................................................................................................... 6

2.2 Water hyacinth in Ethiopia .................................................................................................... 7

2.2.1 Composition of water hyacinth....................................................................................... 8

2.3 Pre-treatment of water hyacinth .......................................................................................... 10

2.4 Potential utilization of water hyacinth ................................................................................ 12

2.4.1 For Power alcohol production ..................................................................................... 13

VI
 2.4.2 In Biogas production as Raw Biomass ......................................................................... 13

2.4.3 Used as a Compost ....................................................................................................... 14

2.4.4 Other Utilization ........................................................................................................... 15

2.5 Lactic Acid Production Technology ................................................................................... 15

2.5.1 Chemical synthesis of Lactic Acid ............................................................................... 16

2.5.2 Enzymatic Hydrolysis................................................................................................... 18

2.6 Characterization of Lactic Acid .......................................................................................... 19

2.7 Properties of Lactic Acid..................................................................................................... 19

2.8 Application of Lactic Acid (C3H6O3) .................................................................................. 20

CHAPTER THREE ...................................................................................................................... 21

MATERIALS AND METHODS .................................................................................................. 21

3.1 Sample Collection ............................................................................................................... 21

3.2 Chemicals and Materials required ....................................................................................... 21

3.2.1 Chemicals ..................................................................................................................... 21

3.2.2 Equipment ..................................................................................................................... 21

3.3 Process flow diagram and Framework of the study ............................................................ 23

3.4 Sample preparation .............................................................................................................. 23

3.4.1 Characterization and Composition Determination ....................................................... 24

3.5 Pretreatment experiments .................................................................................................... 28

3.5.1 Hydrolysis experiments ................................................................................................ 29

3.5.2 Total reduced sugar determination ............................................................................... 29

3.6 Synthesis of Graphene Oxide by Improved Hammers Method .......................................... 31

3.7 Fourier Transform Infrared Spectroscopy Analysis ............................................................ 31

3.8 Thermogravimetric analysis ................................................................................................ 31

3.8.1 TGA analysis of WHB sample ..................................................................................... 31

VII
 3.8.2 TGA analysis of GO sample ......................................................................................... 32

3.8.3 XRD Pattern analysis of GO samples........................................................................... 32

3.9 Lactic Acid Production (solid-acid-catalyzed or chemical hydrolysis) ............................. 32

3.9.1 Product Quantity Determination ................................................................................... 33

3.10 Data Analysis .................................................................................................................... 34

3.10.1 Experimental design for CCD, BBD, and Response Surface Method ....................... 34

CHAPTER FOUR ......................................................................................................................... 36

RESULTS AND DISCUSSION ................................................................................................... 36

4.1 Proximate and Compositional Analysis of WHB ............................................................... 36

4.2 FT IR Spectroscopy, TGA and XRD analysis .................................................................... 36

4.2.1 FTIR analysis of water Hyacinth .................................................................................. 36

4.2.2 Thermogravimetric analysis Results of water Hyacinth............................................... 38

4.2.3 FTIR analysis of Graphene oxide ................................................................................. 39

4.2.4 TGA analysis of Graphene oxide ................................................................................. 40

4.2.5 XRD analysis Results of Graphene oxide .................................................................... 41

4.3 Pretreatment Results ............................................................................................................ 42

4.4 Optimiztion of Pretreatmen Process via RSM Analysis ..................................................... 44

4.5 Statistical Analysis of the Experimental Results................................................................. 45

4.5.1 ANOVA( analysis of variance) for pretreatment process ........................................... 45

4.6 Effects of Parameters on Lignin Removal, and Holocellulose Recovery ........................... 59

4.6.1 Interaction effect of pretreatment factors ..................................................................... 60

4.6.2 Effects of pretreatment parameters on reduced sugar yield ......................................... 63

4.7 Effects of hydrolysis Operating Conditions on total amount of lactic acid ........................ 65

4.7.1 Individual effect of Operating Conditions on lactic acid yield .................................... 65

4.7.2 Interaction Effect of Hyadrolysis Operating Conditions on Lactic Acid Yield ........... 67

VIII
 4.8 Countor and 3-D Response Surface for Yield with Parameter Interaction ......................... 70

4.9 FTIR Analysis for Produced Lactic Acid ............................................................................ 71

4.10 Design expert for acid hydrolysis process......................................................................... 72

4.10.1 Graphical analysis (diagnostic plot) for lactic acid yield ........................................... 77

4.11 Optimization of Acid Hydrolysis Production of LA from Water Hyacinth Plant ............ 85

CHAPTER FIVE .......................................................................................................................... 87

CONCLUSION AND RECOMMENDATION ............................................................................ 87

5.1 Conclusion........................................................................................................................... 87

5.2 Recommendation ................................................................................................................. 88

References ..................................................................................................................................... 89

Appendeces ................................................................................................................................... 98

Appendex 1 Design Combination of Pretreatment Process ...................................................... 98

Appendex 2 Organosoly pretreatment process of raw water hyacinth biomass sample ........... 99

Appendex 3 improved hammers method synthesis of graphene oxide from fine graphite
powder ..................................................................................................................................... 100

Appendex 4 Standard curve of lactic acid ............................................................................... 100

Appendex 5 The BOX bohnken design for the lactic acid hydrolysis .................................... 101

Appendex 6 The Interaction effect of factors on lignin content and reduced sugar yield using
countor surface plot ................................................................................................................. 101

Appendix 7 The Interaction effect of factors on lignin content and reduced sugar yield using 3-
D Surface ................................................................................................................................. 105

Appendix 8 the main components of reactors used both in pretreatment and hydrolysis process
................................................................................................................................................. 108

IX
List of Table
Table 2.1 Summary of the pretreatment methods of lignocellulosic material 11
Table 3.1 Equipment and their purposes during the study 22
Table 3.2 Glucose standard solution data and calibration curve 30
Table 3. 3 Experimental design for Pretreatment process 35
Table 3. 4 Experimental design for hydrolysis process for lactic acid production 35
Table 4. 1The proximate analysis and Compositional analysis of raw water hyacinth biomass 36
Table 4. 2 Result of hollocellulose, lignin content, and reduced sugar from pretreated biomass 43
Table 4. 3 Sequential Model Sum of Squares for lignin removal 45
Table 4. 4 Sequential model sum of squares for holo-cellulose recovery 45
Table 4. 5 Sequential model sum of squares for reduced sugar 46
Table 4. 6 Results of Lack of Fit test for lignin removal 46
Table 4. 7 Results of Lack of Fit test For holocellulose recovery 47
Table 4. 8 Results of Lack of Fit test For reduced sugar 47
Table 4. 9 Results Model Summary Statistics Test for lignin removal 47
Table 4. 10 Results of model summary statistics test for holocellulose recovery 48
Table 4. 11 Results of model summary statistics test for reduced sugar 48
Table 4.12 The ANOVA result for the significance of main and interaction effects on lignin
removal 49
Table 4.13 The ANOVA result for the significance of main and interaction effects on
holocellulose recovery 50
Table 4.14 The ANOVA result for the significance of main and interaction effects on reduced
sugar 51
Table 4. 15 Results of R- squared test for lignin removal 52
Table 4. 16 Results of R- squared test For holocellulose recovery 52
Table 4. 17 Results of R- squared test for reduced sugar 52
Table 4. 18 Build Information of experimental analysis of hydrolysis process 72
Table 4. 19 Fit Summary test for lactic acid yield 72
Table 4. 20 Sequential Model Sum of Squares 72
Table 4. 21 Model Summary Statistics 73
Table 4. 22 Lack of Fit Tests 73

X
Table 4. 23 ANOVA for Quadratic model of Lactic Acid 74
Table 4. 24 Fit statstics test table 75
Table 4. 25 Coefficients in Terms of Coded Factors 76
Table 4. 26 Diagnostics report of Lactic Acid yield 84
Table 4. 27 Optimization for The Maximum Lactic Acid Production in a Given Constraints and
its solutions 85

XI
List of Figure
Figure 2. 1 Structures of water hyacinth with stolons 7
Figure 2. 2 Chemical structure of Celluloses 9
Figure 2. 3 P-coumaryl-,coniferyl-, and sinaplyl- alcohol 10
Figure 2. 4 Chemical structure of Hemicellulose 10
Figure 2.5 Proposed pathway for the conversion of actual lignocelluloses to LaA 17
Figure 2.6 Chemical reaction pathway of lactic acid production from LB 18
Figure 2.7 Fermentation pathway of lactic acid production 19
Figure 3. 1 Framework and Flow diagram of the study 23
Figure 3. 2 The process of sample preparation 24
Figure 3. 3 Experimental setup for pretreatment process 29
Figure 3. 4 Experimental set up for acid catalyzed hydrolysis process 33
Figure 4. 1 FT-IR analysis of untreated and pretreated WHB samples 37
Figure 4. 2 Thermogavimetric curve of water hyacinth sample 39
Figure 4. 3 FTIR analysis for GO 39
Figure 4. 4 TGA curve for graphene oxide 41
Figure 4. 5 XRD pattern of GO 42
Figure 4. 6 a) normal plot of residuals, b) predicted vs actual lignin response of the experiment
55
Figure 4. 7 a) normal plot of residuals, b) predicted vs actual plot for holocellulose response of
the experiment. 56
Figure 4. 8a) normal plot of residuals, b) predicted vs actual plot for reduced sugar yield 57
Figure 4. 9 Residual vs predicted and residual vs run plots for pretreatment process 59
Figure 4. 10 The Interaction Effect of pretreatment parameters on lignin removal 62
Figure 4.11 Interaction effect of pretreatment parameters on reduced sugar yield 64
Figure 4. 12 Graphical represention fot the individual effect of hydrolysis condition on LA yield
66
Figure 4. 13 Temperature -Time interaction effect on total lactic acid yield 68
Figure 4.14 Catalyst load -Time interaction effect on total lactic acid yield 69
Figure 4. 15 Catalyst load -Temperature interaction effect on total lactic acid yield 70
Figure 4. 16 FTIR analysis for lactic acid 71

XII
Figure 4. 17 (a) Normal plots of residuals and (b) predicted versus actual plots for lactic acid
yield 79
Figure 4. 18 (a) Residual versus predicted and (b) Residual versus run number plots for lactic
acid yield. 80
Figure 4.19 Countor plot response surface plot for (a) HT-Ht,(b) CL-Ht, and (c) CL-HT
interaction effect on lactic acid yield 82
Figure 4. 20 The interaction effect of hydrolysis parameters on lactic acid yield by 3-D surface
83

XIII
 CHAPTER ONE
INTRODUCTION

1.1 Background
Lactic acid (lactate) is a degradation product of glucose metabolism. Lactic acid is a substance
made from sugars in milk, by the action of certain enzymes. It is used in skincare products to
reduce wrinkles and soften the skin. It has the chemical formula C3H6O3 and is an organic acid.
It's also known as milk acid. Normally, Lactic acid is acquired or created when milk sugar
(lactose) is fermented. It can be found in cottage cheese, leban, sour milk, yogurt, and Koumiss..

The US Energy Agency publish a list of compounds stated to be future building blocks in 2010,
and Lactic acid is one of them (Komesu et al., 2017). Lactic acid (LA) is an attractive alternative
to petroleum plastics due to the necessity for environmental suitability and biomass utilization
(Komesu et al., 2017). Polylactic acid (PLA) is applicable for the production of surgical sutures,
prostheses, orthopedic fixation devices, and drug delivery systems in the medicinal sector
(Antonetti et al., 2016; Komesu et al., 2017;). Lactic acid has a very high global demand due to
its wide range of industrial applications. It will rise from 1220 kilo ton in 2016 to 1960 kilo ton
in 2025(Zhang et al., 2018). An extremely low-cost Lactic acid is required by an industrial users.
The cost of food-grade LA is 1.38 $kg-1 (50 percent purity) and 1.54 $kg-1 (88 percent purity),
whereas the cost of technical grade LA is 1.59 $kg-1 (88 percent purity) (Antonetti et al., 2016;
Komesu et al., 2017; Yankov, 2022; Zhang et al., 2018).

Searching an alternate and tenable resources to supplement and replace fossil fuels is critical for
the economy and human society's progress (Antonetti et al., 2016; Komesu et al., 2017; Zhang
et al., 2018, Yankov, 2022 ). Lignocellulosic biomass is a good potential alternative since it is a
renewable and abundantly available resource processed into a number of value-added
compounds(Sudarsanam et al., 2018). Lactic acid (LaA) is one of the top fifteen rostrum
chemicals derived from lignocellulosic biomass, with two enantiomers (D-LaA and L-LaA)(
Kebede, 2007; Castillo Martinez et al., 2013; Ganguly et al., 2013, Antonetti et al., 2016,
Komesu et al., 2017; Sudarsanam et al., 2018, Zhang et al., 2018; Yankov, 2022;). Because of its
good-looking and profitable poly-functional qualities, it is also contemplating an essential
industrial product with a big and constantly developing market, particularly in the polylactic acid

1
(PLA) industry (Mohan et al., 2020). In comparison to edible crops and pure cellulose/xylan,
genuine lignocellulosic biomass is core promising as a starting material for producing LaA, due
to its low cost and abundance, as well as the coexistence of cellulose and hemicellulose, both of
which can provide LaA (Ghaffar et al., 2014). Due to fact that the cellulose and hemicellulose
components in lignocelluloses are not directly available for bio-conversion to LaA due to their
close association with lignin and the lack of hydrolytic enzymes, actual lignocelluloses are still
rarely used as the feedstock for LaA production by fermentation (Idrees et al., 2013). In addition,
in fermentation, simultaneous conversion of the sugar mixture (containing C6 and C5 sugar) is
still problematic. Chemo-catalysis has recently been reported as a possible technique for cost-
effective LaA synthesis when authentic or real lignocelluloses are used as a primary commodity
(Liu et al., 2020). More importantly, when employing genuine lignocellulose as raw material, the
expensive pre-treatment for lignocellulose degradation to separated cellulose/xylan are restricted
by chemo-catalysis, and the LaA manufacturing cost lowering greatly. Furthermore, unlike in the
case of fermentation, it has been demonstrated that lignin contained in actual lignocellulose does
not interfere with chemo-catalysts for glucose conversion. Regrettably, the yield of LaA straight
from genuine lignocelluloses (45%) without any pretreatment is significantly lower than the
yield of pure cellulose, even under extreme circumstances (Wu et al., 2021).

Commercial (Antonetti et al., 2016) LA production is now made using an enzymatic hydrolysis
process (over 90%), which has several advantages, including low production temperature, low
energy usage, and high purity with a suitable strain (Van Der Pol et al., 2016). Pretreatment of
feed-stocks, anaerobic fermentation, acidification, and separation and purification of LA are the
four basic processes in the production of LA by conventional fermentation starting from
cellulosic biomass. However, it has several disadvantages, such as limited productivity, the
necessity for expensive enzymes (tight pH and temperature), and difficult separation and
purification requirements, which are always present. As a result, difficulties of sustainability in
the up-scaling of the current fermentation process must be addressed by replacing it with a
promising alternative method, chemo-catalysis, which might be considered a research hot spot in
the current state of lactic acid research.

Chemical catalysis (homogeneous or heterogeneous) is becoming increasingly popular as a

powerful technique for converting cellulosic biomass into value-added compounds with adequate

2
selectivity(Yuan and Wang, 2021). Chemo-catalysts are particularly important in the
fundamental and new production path of lactic acid (or lactates), which use sugars or genuine
lignocellulosic biomass (such as water hyacinth or cornstalk) in a rostrum approach. Using
homogeneous or heterogeneous acid catalysts and hydrolysis to fractionate lignocellulosic
biomass into reduced sugars, the chemical catalysis process creates lactic acid directly from
lignocellulosic biomass.

Graphene oxide (GO) is a compound that contains carbon, oxygen, and hydrogen. It is an
oxidized analogy of graphene which is recognized as the only intermediate for obtaining the
latter on large scale, (Feicht and Eigler, 2018). GO is a sheet of graphene functionalized with
different oxygenated groups. In general, the GO contains carboxyl, lactone, phenol, carboxyl,
anhydride, ether, Quinone, and epoxy groups (Gado, 2018, Sun, 2019, Samsul et al., 2020). Due
to the presence of H2SO4 impurities during synthesis, GO have also sulfur-containing functional
groups (Yu, 2020, Elazab, 2021) that provides acidic property. Graphene oxide can be
synthesized by the oxidation of graphite to form acidic functional groups such as –SO3H and
carboxylic acid on the surface of the material. Due to these functional groups, graphene oxide is
used in organic syntheses, such as in the cellulose hydrolysis reaction (Garg et al., 2014; Zhu et
al., 2014; Yang, 2015; Said, 2017). It is a potentially acidic heterogeneous catalyst with good
solubility in aqueous solutions (or polar solvents) due to the presence of many oxygen-rich
functional groups (-OH, -COOH, -CHO, etc.), which are highly hydrophilic (Nguyen, 2019).

Water hyacinth (Eichhornia crassipes) is an aquatic weed native to the world and anxious beauty
due to its beautiful purple flowers. It continues to spread aggressively throughout temperate,
tropical, and sub-tropical climates (Mujere, 2017). It has become a problem in all continents
apart from Europe as well as in Ethiopia. Water hyacinth grows over a wide variety of wetland
types such as lakes, streams, ponds, waterways, ditches, and backwater areas (Williams, 2017).
According to (Mitan, 2019), the high growth rate of the water hyacinth (17.5 tons/hectare/day)
under favorable conditions, and its daily average productivity of 0.26 ton hectare-1 in all seasons.
This makes it a promising lignocellulosic feedstock for bio-fuel and value-added chemical
production. It reaches productivity up to 100-140 tons/year per hectare depending on the time of
the year and the location.

3
1.2 Statement of Problems
The catalytic conversion of LCB such as water hyacinth plant into value added chemicals like
lactic acid as a building block for useful chemical production has attracted significant attention
worldwide. On the other hand, enzymatic conversion of water hyacinths to lactic acid becomes
difficult due to cost of enzyme as well as its downstream process as compared to its yield.
Therefore, the study focuses on exploring the effect of chemical hydrolysis, pretreatment
conditions on the yield of lactic acid production from water hyacinth, and optimizing the
hydrolysis process. With the increasing demand for higher efficiency, lower environmental
impacts, and better economics, chemical catalysis serves as an option for industrial chemical
manufacturing processes. These factor alerts the country to find alternative catalyst type for the
demand of Lactic Acid as a building block material.

Lactic acid could be processed from water hyacinth biomass in the presence of chemical catalyst
with acid hydrolysis. The most ancient method for the conversion of biomass to lactic acid use
homogeneous mineral acid catalysts due to their high catalytic activity, easy availability and their
efficiency towards high LA yield. But the homogeneous catalyst has an effect on the material
used as well as on environment harshment due to their toxicity and corrosiveness behavior. As a
result of ever strengthening environmental regulation, enhancing an adsorption and an
isomerization of reduced sugar on the catalyst surface, the solid acid heterogeneous catalyst like
metal oxide and their sulfonated derivatives like graphene oxide are the most attractive for the
replacement of corrosive and harmful traditional mineral acid catalyst. Their activity is due to the
presence of oxygenated and carboxylic groups together with Lewis acid site caused by the
electron inductive effect of sulfonic groups. Thus, the chemical synthesis or heterogeneous solid-
acid hydrolysis of lactic acid as a building block material may improve the catalyst life time and
ultimately enhance the productivity of the conversion process. It will also contribute for effective
management of invasive weed infestation by utilization method. To increase cellulosic material
recovery or reduced sugar generating and lactic acid quality, water hyacinth plants was treated
carefully by using Organosoly pretreatment methods with an ethanol as organic solvent.

So, in this thesis, the effect of pretreatment conditions and chemical hydrolysis on reduced sugar
production and on lactic acid yield and ability of catalyst have been investigated.

4
1.3 Objectives
1.3.1 General Objectives
The objective of this research is to optimize the chemical hydrolysis of lactic acid production
from water hyacinth using Graphene oxide solid-acid catalyst.

1.3.2 Specific Objectives

 To determine the proximate and chemical composition of raw and treated water hyacinth
biomass
 Investigating the effect of pretreatment and chemical hydrolysis parameters on lignin
removal, holocellulose recovery as well as on reduced sugar and lactic acid yield
 Synthesizing the heterogeneous solid-acid GO catalyst using improved hammers method.
 To produce and optimize lactic acid from water hyacinth using solid acid GO catalyst
 Characterizing and quantifying the yield of lactic acid through different analytical
methods

1.4 Scope of the study

Pretreatment of water hyacinth, proximate and composition analysis of WH, effect of process
conditions on the response, catalyst synthesis and chemical hydrolysis of water hyacinth biomass
through Graphene oxide catalyst for the synthesis of lactic acid were all covered in this thesis
study.

1.5 Significance of the study

This study contributes to the effective use of an abundantly available and renewable
lignocellulosic feedstock for the extraction and conversion of these materials to value-added
chemicals such as lactic acid. The present practice of water hyacinth control is focused on trying
to clear or discard the weed from water bodies with harvesting manually, mechanically, and
burning or dispose which leads to ineffective utilization of bio-resource. Therefore, this study
adds up to the research activities to include lactic acid production in applying “waste to value-
added chemicals” concept as part or class of integrated management strategies in controlling and
halting the invasive water hyacinth and its disturbing effect on water bodies to a non-problematic
level. This allows both academics and investors to investigate the possibility of replacing an
expensive commercial lactic acid with a local made lactic acid.

5
 CHAPTER TWO

LITERATURE REVIEW

2.1 Background

Lactic acid is a valuable platform chemical with both traditional and newer applications. It was
widely used as a neutralizer, preservative, or acidulant in food and beverage, cosmotics,
pharceutical, and othet industries.in moment, lactic acid is applied as a building block for various
biodegradable polymers or precursor for environmentally friendly solvents. The yearly
international market for lactic acid in 2020 is valued at USD 1.1 billion with an increasing
tendency to double till 2025 (Yankov, 2022). The global market for lactic acid was 750.00
kilotons in 2017 and is proposed to reach 1,845.00 kilotons by 2022 (Yankov, 2022). Lactonitrile
is made by allowing acetaldehyde to react with hydrogen cyanide in the presence of a base
catalyst in the liquid phase and under high pressure to form lactic acid. Base-catalyzed sugar
degradation, oxidation of propylene glycol, carbon monoxide, and water at high temperature and
pressure, hydrolysis of chloro-propionic acid, and nitric acid oxidation of propylene are some of
the additional chemical methods for lactic acid synthesis (John et al., 2008).

Because of several properties, lactic acid is an important industrial product used as a predecessor
of tiny (propylene glycol) or huge (acrylic polymers) compounds. Their polymers are compos-
table and bio-compatible (Van Der Pol et al., 2016). Poly-lactic acid, for example, provides a
number of advantages in the textile, medicinal, and pharmaceutical industries. It is also used in
the cosmetic industry to make hygiene and esthetic products, as well as oral hygiene products. It
is utilized in the pharmaceutical industry as a supplement in the production of dermatologic
medications and as an anti-osteoporosis agent. In 2007, the global demand for lactic acid is
expected to be between 130,000 and 150,000 metric tons per year, with commercial pricing for
food-grade lactic acid ranging between 1.38 US$ kg1 (50 %) and 1.54 US$ kg1 (100 %) (88 %of
purity)( John et al., 2007). According to Mujtaba, its production should increase significantly
over the coming years which is expected to reach 259,000 metric tons in 2012 for satisfying the
polylactic acid manufacturing industries (Mujtaba et al., 2012).

The most pernicious aquatic weed, water hyacinth (Eichhornia crassipes), continues to spread at
random throughout temperate, tropical, and sub-tropical areas (Williams, 2017). The weed is

6
used for different applications. It serves as a potential lignocellulosic feedstock for bio-ethanol
and different value-added chemical production. It has higher growth rate of 17.5 tons/hectare/day
(Ruan et al., 2016) and its daily average productivity of 0.26 tons of dry biomass per hectare
(Kumar and Singh, 1984) in all seasons under favorable condition. The production exceeds up to
100-140 tons per year per hectare based on the time of the year and destination or location. As it
can be seen in figure 2.1 below, it is a floating biomass with roots, long spongy stems, and
leaves. Roots, rhizomes, stolons, leaves, inflorescence, and fruit clusters make up the mature WH
(Saning et al., 2019).

Source: (Saning et al., 2019).

Figure 2. 1 Structures of water hyacinth with stolons

Where ar; adventitious root, dp; daughter plant, in; inflorescence, lb; leaf blade, li; leaf litmus,
pt; petiole, pf; peduncle of the flower spike, rt; root, and st; stolon

2.2 Water hyacinth in Ethiopia

In the early 1900s, the water hyacinth brought into Africa from South America (Villamagna &
Murphy, 2010) and it become a serious weed throughout Southern Africa, the Congo Basin, and
the Upper Nile since the 1950s. Water hyacinth is without any doubt the major aquatic weed
problem all over the tropical and sub-tropical regions of the world. Its incidence in water bodies
causes enormous problems to the economies and the environment of the countries. Water
hyacinth is introduced into the Rift Valley's water bodies in the 1950s as a decorative plant, and
occasional visits and cleanup attempts are done in 1959, 1968, 1979, and 1988 on the different

7
water bodies of Ethiopia (Firehun et al., 2014). The weed began to proliferate on reservoirs,
irrigation, and drainage structures at Wonji-Shoa Sugar state, in 1996 when the plantation is
flooded by the overflow of Awash River. The lack of natural enemies together with nutrient-
enriched water bodies facilitate the spread of water hyacinth in temperate, tropical and sub-
tropical waters. The weed causes a variety of socioeconomic and environmental problems when
its rapid mat-like proliferation covers areas of fresh water. Water hyacinth is considered invasive
throughout the world because it grows rapidly and can form thick layers over the water. These
mats shade out the other aquatic plants. Eventually these shaded plants die and decay. The
decaying process depletes the amount of dissolved oxygen in the water. In 2005, the persistence
of the crisis is recognized soon, and it is decided to launch a nationwide weed management
strategy. Manual, mechanical, biological, and chemical controls are all part of an action-oriented
for control program (Yirefu et al., 2007). This weed has also infested to Lake Ellen and Lake
Tana which entails more research into the application of effective management master-plan both
environmentally sound and practical, as well as cost-efficient (Tessema, 2012). When water
hyacinth quickly spreads to fill freshwater environments, it can create a number of issues. The
issues including; hindrance to water transport, clogging of intakes of irrigation, hydro-power and
water supply systems, blockage of canals and rivers causing flooding, micro-habitat for a variety
of disease vectors, increased evapo-transpiration, problems related to fishing, and reduction of
biodiversity are the most common. The utilization of this weed after it has been removed from
water bodies to be processed into a useful product such as bio-ethanol, value-added chemical,
and others are examples of these types of measures.

2.2.1 Composition of water hyacinth

The composition of WH is characterized by low DM concentration, high CP and ash content.
Plant leaves and petioles that are yet growing have a lighter green color and a higher protein
content than mature plant leaves and petioles. The protein level of the leaves is higher than that
of sweet potato leaves and the whole plant’s higher protein content makes it a protein increment
in low-quality diets. The amino acids glutamine, asparagine, and leucine are especially abundant
in leaf protein (Auma et al., 2019). The chemical composition of WH varies with season,
habitats, and harvesting time (Carlini et al., 2018). Water hyacinth comprises cellulose,
hemicellulose, and lignin. Lignocellulosic fibers are made up of helically wound cellulose micro-
fibrils that are bonded together by lignin and hemicellulose (Tham, 2012).

8
2.2.1.1 Cellulose
Cellulose, which is made up of ß-D glucose units, is the most common natural polymer. Straight
chains are formed by joining the monomer units together by ß (1-4) glycosidic linkages. Three
hydroxyl groups are present in each monomer. Because the hydroxyl groups of the glucose units
on one chain can create hydrogen bonds with an oxygen atom on the other chain linked by
hydrogen bonds. Micro-fibrils are formed when cellulose molecules are grouped, having highly
ordered regions and less ordered parts (Hany et al., 2021). Cellulose is insoluble in water and
most organic solvents (Masykuri et al., n.d.).

Source: (Masykuri et al., n.d.)

Figure 2. 2 Chemical structure of Celluloses

2.2.1.2 Lignin
Lignin is a racemic, cross-linked macro-molecule that is hydrophobic and aromatic. It covers the
cellulose skeleton and forms the lignocellulosic matrix by filling the gap between the
hemicellulose structures (Paudel et al., 2017). It's a sugar-free three-dimensional highly branched
polyphenolic polymer. In most acids, it is insoluble, but not in alkali's. It consists of p-coumary
alcohol, coniferyl alcohol, and sinapyl alcohols found as phenylpropane units in lignin
(Mukaratirwa-muchanyereyi et al., 2016). Lignin differs in terms of the degree of carbon-carbon
cross-linking between phenyl groups (Alzagameem et al., 2018). The chemical and physical
properties of lignin are determined by the lignin isolation process. The most prevalent functional
groups are aliphatic and phenolic hydroxyl groups (9-11%), methoxy groups (13-26%), and
carbonyl groups (Mukaratirwa-Muchanyereyi, 2016).

9
Sources: (Mukaratirwa-Muchasnyereyi, 2016)

Figure 2. 3 P-coumaryl-,coniferyl-, and sinaplyl- alcohol

2.2.1.3 Hemicellulose
Hemicellulose is a heterogeneous polysaccharides with a low strength random and amorphous
structure found between the lignin and cellulose fibers. Hemicellulose is classified into four
categories based on the structure of the cell wall. Xylan, mannan, glucan with mixed bonds, and
xylo-glucan are examples (Liu et al., 2019). Among them, Xylan is the major component
substituted with sugar units. Hemicellulose is a polymer of a variety of monosaccharides unit.
The hemicellulose and cellulose are bound to lignin with hydrogen bonds (Mukaratirwa-
Muchanyereyi, 2016).

Figure 2. 4 Chemical structure of Hemicellulose

2.3 Pre-treatment of water hyacinth

Pretreatment is a process used to change the individual component content or its structure to
achieve an efficient separation. Conventional pretreatment methods can be used widely but with
different challenges. Among the methods, Chemical pretreatment, Thermal Pretreatment,
Physical Pretreatment, Biological Pretreatment, and Physio-chemical pretreatment are the most
common processes and summarized in the table 2.1. In this study, the chemical pretreatment
method which is Organosoly pretreatment process was used for the treating water hyacinth

10
biomass used for lactic acid production. It is the most selective method as compared to another
chemical pretreatment methods. Because it avoids an adverse downstream effects like cell wall
swelling and component solubility due to contradict the efficient product separation(Sidars,
2022).

Organosoly by itself defined as an initial pretreatment process owing to its fundamental

advantages such as, easy recapture of the solvents by distillation, recycles the solvents back to
pretreatment and utilization of high-quality lignin remote from this process as value-added
byproducts for industrial applications. However, there also deceptions a few major drawbacks to
the organosolv pretreatment. Most of the organic solvents are too expensive and need to be
recovered as much as possible which is an energy-demanding process. In addition, high
flammability and volatility of the organic solvents make the pretreatment to be carried out under
especially controlled conditions (Baruah et al., 2018).

Table 2. 1 Summary of the pretreatment methods of lignocellulosic material

Pretreatment process Advantage Disadvantage

Physical Mechanical Reduces cellulose power consumption is
pretreatment comminution crystallinity usually higher than
inherent biomass energy
Pyrolysis produces gas and liquid high temperature; ash
products production
Physio- Steam causes hemicellulose destruction of a portion
chemical explosion degradation and lignin of the Xylan fraction;
pretreatment transformation; incomplete disruption
cost-effective of the lignin-
carbohydrate matrix;
generation of
compounds inhibitory to
microorganisms
Ammonia increases accessible not efficient for biomass
fiber surface area removes with high lignin content
explosion lignin and hemicellulose

11
 to an extent; does not
produce inhibitors for
downstream processes
CO2 It increases accessible It does not modify lignin
explosion surface area; cost- or hemicellulose
effective, and it does not
cause the formation of
inhibitory compounds
Chemical Ozonolysis It reduces lignin content A large amount of ozone
pretreatment and does not form toxic is required and it is
residues expensive
Acid It hydrolyzes It is costly and causes
hydrolysis hemicellulose to xylose equipment corrosion as
and other sugars and well as the formation of
alters the lignin structure toxic substances
Alkaline removes hemicellulose It requires long
hydrolysis and lignin; residence times,
forms irrecoverable
increases accessible salts, and is incorporated
surface area into biomass
Organosoly hydrolyzes lignin and solvents need to be
hemicellulose drained from the reactor
evaporated, condensed,
and recycled; high cost
Biological Biological degrades lignin and rate of hydrolysis is very
pretreatment hemicellulose; low
low energy requirements
Source:Harmsen et al., 2010

2.4 Potential utilization of water hyacinth

Although, water hyacinth is frequently considered a weed that causes many of the issues
mentioned above, there are different schools of thought that support the plant's practical uses.
12
Despite having a water content of more than 95%, water hyacinth can be used for a number of
purposes due to its fibrous tissue, high protein, and energy content, and high water content
(Rezania et al., 2015). The following discussion covers a variety of potential applications for the
plant, some of which have already been discovered and others that are just beginning.

2.4.1 For Power alcohol production

The hunt for efficient alternative renewable sources to meet future energy demands has been
sparked by awareness of the non-renewable nature of fossil fuels. The lignocellulosic biomass'
hemicellulose component is regarded as a desirable raw material for the manufacturing of fuel
ethanol. The water hyacinth has a rather high hemicellulose content (30–55 % of dry weight),
which suggests that it might be a viable source of hemicellulose for bio-conversion.
Additionally, the water hyacinth has significant levels of crude protein, which could serve as the
process' nitrogen supply. However, pre-hydrolysis, hydrolysis, fermentation, and distillation are
all steps in the multistage process of producing fuel ethanol from biomass. Many of the
byproducts of hydrolysis may also act as inhibitors on the microorganisms used in later
fermentation processes. However, studies into the viability of producing methanol from water
hyacinth have been documented. It has been investigated to saccharify water hyacinth and use
Saccharomyces cerevisiae to ferment the reducing sugars into alcohol. The study improved a
number of fermentation parameters, and the highest output of 0.38g alcohol/g sugar
concentration was reached at 29°C, pH 4, and 3d of incubation. The cellulosic wastes' acid
hydrolysis was shown to be far inferior to enzymatic saccharification. Using Pichia stipitis
NRRL Y- 7124, also looked into the manufacture of ethanol from untreated and treated water
hyacinth hemicellulose acid hydrolysate. For the treated material, the author recorded a
maximum output of 0.35g ethanol/g substrate concentration, which is comparable to the results
of baggasse fermentation from sugar cane. However, the hydrolysates acetic acid content
significantly reduced productivity and ethanol generation. Compared to ethanol or acetone
butanol fermentation, where the end products are far more hazardous, butanediol fermentation
provides a competitive advantage. A plan has been developed for the microbial generation of 2,
3-butanediol from water hyacinth.
2.4.2 In Biogas production as Raw Biomass
Biogas production from organic matter (often human or animal waste) is a well-known small-
and medium-scale technique. A long-standing topic of intense interest is the potential for turning

13
water hyacinth into biogas. Volatile matter biogas yields of up to 0.67l gg-1 have been reported.
A combination of animal waste and water hyacinth produces superior biogas yields (Sindhu et
al., 2017), and the mixed feed sludge, which has a higher nitrogen, phosphorus, and potassium
content, can be used as excellent manure. However, there are some issues with using the water
hyacinth for digestion in a conventional digester, including the size of the digester, the lower
biogas conversion efficiency (due to the extremely high water content), and the requirement for
pre-treatment prior to digestion (to remove air entrapped in the tissue). The biodegradability of
water hyacinth can be increased through pretreatment with fungi or chemicals to produce enough
biogas (Ferdeș et al., 2020). All of these issues suggest that using a traditional, one stage reactor
for water hyacinth-based biogas plants is not practical. Due to this, multi-phasic reactors that can
solve a number of issues, such as feeding, foaming, clogging, and low reactor efficiency, have
been thoroughly studied over the past 20 years. For the creation of sensible and economical two-
and three-stage methane recovery systems from the water hyacinth, the ideas of feed
pretreatment, phase separation, and whole-cell immobilization technology have been combined.
According to (Sawyerr et al., 2019) an unique feed batch digestion system might be used to
successfully digest water hyacinth and provide a consistent flow of biogas. The digestion and gas
production of the water hyacinth are influenced by the composition of the meal, according to the
authors. Also described is the solid-phase fermentation of water hyacinth caused by the daily
sprinkling of an aqueous suspension of biodegradative bacteria on the water hyacinth biomass
substrate. One of these possibilities could be the production of biogas from water hyacinth.
2.4.3 Used as a Compost
The water hyacinth can be utilized as compost or as surface mulch on the ground. In comparison
to the control (no mulching) treatment, it was discovered that water hyacinth mulching field
crops increased the yield of lady's finger (67%), potato (14%) and tomato (90%). Therefore, care
must be taken to ensure that water hyacinth is not sprayed with pesticides before being used as
mulch or compost. Traditional composting can be accomplished by combining dried plants with
ash, soil, some animal manure, and organic municipal garbage. This method is appropriate for
labor-intensive, low-capital manufacturing. Because the water hyacinth loses its ability to
reproduce vegetatively after passing through the earthworm gut, vermicomposting it is more
favorable. Additionally, vermi-casts made during vermicomposting are thought to have
hormones and enzymes that promote plant growth while discouraging infections. In India,

14
significant work has been done on vermicomposting water hyacinths, including developing high-
rate vermi-reactors, optimizing earthworm species and worm density, and pretreating the water
hyacinth before composting. The use of the water hyacinth vermicompost has also been shown to
have no negative effects on the development and flowering of vegetable crops.
2.4.4 Other Utilization
The stems of the water hyacinth plant can be used to manufacture rope, baskets, or even high-
quality paper when combined with waste paper or jute. Small-scale cottage industry paper-
making initiatives have proven effective in a number of nations, including the Philippines,
Indonesia, and India. It has also been successfully examined how the water hyacinth might be
used as a pulp material to make grease-proof paper. Production of fiber boards for a number of
end purposes, including bituminized board for inexpensive roofing material and interior partition
walls, is another way that water hyacinth is used. The suggested technology for briquetting
charcoal dust produced from the pyrolysis of the water hyacinth can also be used to extensively
utilize water hyacinth. The water hyacinth is widely used as a medicinal plant in India, mostly to
cure the goiter condition. A lot of cytosine-based proteins, notably metallothioneins, are thought
to be present in the water hyacinth. It is widely known that the amino acid cytosine acts as an
antioxidant against O2- and OH free radicals and is a key component of glutathione. An estimate
of 40 ± 2nmol eg/gdp for the water hyacinth leaves' antioxidant activity (equivalent glutathione
per gram of dry plant material). Vitamins and minerals are abundant in these leaves. Therefore, it
is desirable to maximize the process of extracting vitamins from this plant. According to the
findings, the fermentation medium including 50% hydrolysate and 50% basal medium made up
of glucose, asparagine, etc. contained the maximum concentration of riboflavin. These studies
imply that the water hyacinth could be used as a substrate for cellular growth. Recent research
has shown that the water hyacinth biomass can also be used to successfully culture the oyster
mushroom (Pleurotus sajorcaju) (Patel, 2012).

2.5 Lactic Acid Production Technology

Lactic acid can be produced industrially in one of two ways: chemically or through microbial
fermentation. However, the least expensive option (fermentation by microbes) has some
potential advantages, such as the ability to obtain pure lactic acid, whereas chemical lactic acid
synthesis always results in a raceme mixture (Chowdhary et al., 2012). Poly-lactic acids have a

15
high crystallinity and melting point due to the presence of L- lactic acid, which has a high optical
purity.

2.5.1 Chemical synthesis of Lactic Acid

Lactonitrile is used in the commercial process of chemically synthesizing lactic acid. Hydrogen
cyanide is added to the acetaldehyde in presence of a base to generate Lactonitrile. The reaction
takes place in a liquid phase at high atmospheric pressures. A racemic combination of DL-lactic
acid results from the chemical production method. Following reactions are involved in this
process (Ghaffar et al., 2014). Unlike enzymatic processes, chemo-catalytic processes use less
expensive inorganic molecules that may work under a wider variety of reaction conditions,
making vast amounts of glucose treatment industrially possible. As a result, much effort has gone
into improving fructose selectivity and yield across a variety of homogeneous or heterogeneous
catalytic systems. Heterogeneous catalysts are preferred because they are easy to separate from
the post-reaction mixture and can be reused in subsequent reaction cycles using a straightforward
technique. Lewis acid and, more recently, Lewis / Bronsted tandem catalysts have been used to
treat the sites in question. As a result, the most recent advancements are focused on the tandem
system, with the primary goal of simplifying biomass Valorization processes.

16
Sources: (Ghaffar et al., 2014).

Figure 2.5 Proposed pathway for the conversion of actual lignocelluloses to LaA

17
Sources: (Ghaffar et al., 2014)

Figure 2.6 Chemical reaction pathway of lactic acid production from LB

2.5.2 Enzymatic Hydrolysis

Lactic acid is produced through the fermentation of various sugars (glucose, lactose), starchy or
lignocellulosic hydrolysates by various bacteria, primarily lactobacilli. pH, temperature,
nutrition, substrate, product concentrations, and other factors can all influence lactic acid
fermentation. Lactic acid fermentation can be carried out in this case by employing water
hyacinth. It is well recognized that pretreatment and enzyme hydrolysis maximize the water
hyacinth's potential. Lactic acid bacteria are used to make lactic acid. All cells were grown at 37
℃ with an initial pH of 5.5. HPLC is used to measure lactic acid production. From pretreatment
of water hyacinth, all Lactobacillus strains could create lactic acid. Lactic acid fermentation by

18
L. delbrueckii for D-form lactic acid synthesis and L. helveticus for L-form lactic acid
production yields the maximum lactic acid. L. delbrueckii had a lactic acid content of 10.70 g/L
and converted glucose in the medium to lactic acid practically perfectly. When fermentation was
carried out at a pH of 5.5, lactic acid production increased.

Sources: (Komesu et al., 2017)

Figure 2.7 Fermentation pathway of lactic acid production

2.6 Characterization of Lactic Acid

HPLC can be used for the characterization of lactic acid product obtained from lignocellulosic
biomass using various synthesis methods. For the detection of Lactic acid levels in a variety of
biological samples, High-Performance Liquid Chromatography employs a differential refractive
index detector (RID). This Methodology gives accurate, dependable, and repeatable lactic acid
measurement data, allowing us to examine lactic acid levels in vitro and in vivo.

2.7 Properties of Lactic Acid

Lactic acid, also known as 2-hydroxypropanoic acid, is a naturally occurring organic acid. It is
the most basic chiral 2-hydroxycarboxylic acid, possessing two enantiomeric forms. Lactic acid
has the chemical formula C3H6O3. Lactic acid is formed when milk sugar (lactose) is fermented.

19
Lactic acid has two optical isomers. In its solid state, it is white, water soluble and, while in its
liquid state, it is colourless. 1 mM lactic acid has a PH of around 3.51.

2.8 Application of Lactic Acid (C3H6O3)

Lactic acid is an essential industrial product that is utilized as a precursor for tiny (propylene
glycol) and large (acrylic polymers) chemicals due to a variety of characteristics (Abdel-Rahman
et al., 2011). Their polymers are suitable for packaging and labeling, for prosthetic devices,
sutures, and internal drug administration (Martinez et al., 2013, Thomas et al., 2018). Among
them, poly-lactic acid (Martinez et al., 2013) has several applications in the textile, medical and
pharmaceutical industries (Singh et al., 2010). Lactic acid is used in the cosmetic industry to
make hygiene and esthetic products, as well as oral hygiene products. Lactic acid derivatives,
such as lactate esters, are widely used in a variety of applications (Gao et al., 2011). It is utilized
in the pharmaceutical sector as a supplement for the production of dermatologic medicines and
for the treatment of osteoporosis (Li et al., 2017). Approximately 70% of lactic acid generated is
used in the food business. Lactic acid forms spontaneously in the field of grain production.
Controlled lactic fermentation improves the shelf life, palatability, and nutritional value of silage
in terms of animal nutrition. Many processed goods, such as bakery, dairy products, and soft
drinks, contain it as an acidulant, preservative, flavoring, or buffering agent. The US FDA and
other regulatory organizations have designated it as GRAS (generally regarded as safe) for food
additives.

20
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Sample Collection

The water hyacinth sample was collected from Lake Tana which is geographically located in the
North- Western part of Ethiopia between latitudes of 10o 58'-12o 47'N and longitudes 36o 45'-38o
14'E with variations brought on by rising silation levels. The fresh water hyacinth plant was
harvested and transported to Bahir Dar university and was kept in the chemical and food
engineering back side of process control Laboratory.

3.2 Chemicals and Materials required

3.2.1 Chemicals

During this study, NaOH(99.89%), H2SO4(98%), Peroxide(30%), 3,5-dinitro salicylic acid,

ethanol (96%), ferric chloride (FeCl3), graphite powder, H3PO4(85%), KMnO4, and lactic
acid(88%) were purchased from market suppliers while distilled water and De-ionized water
were obtained in the chemistry lab, BDIT.

Ethanol: used as a solvent in both pretreatment and hydrolysis process

Ferric chloride: used for the preparation of lactic acid standard solution

KMnO4, H3PO4, H2SO4 : for the synthesis of Graphene oxide catalyst as oxidizing agent

DNS: used for the determination sugar content in the biomass

Peroxide: used to eliminate the excess amount of KMnO4 during GO synthesis

3.2.2 Equipment
Various equipment is used in the pretreatment and Hydrolysis of WHB, as well as the generation
and analysis of lactic acid.

21
 Table 3. 1 Equipment and their purposes during the study

Equipment Application

Digital balance For measuring samples

Soxhlet To do extractive

Oven For drying of the samples for characterization

Crucible Used as sample holder to do ash and VM

hammer mill For size reduction

Reactors(GLASS/SS316, 4609) Used for the hydrolysis of biomass

whatman paper Used as a filter media

plastic bags For sample container

Furnance(Bahnhofstr.20, B180) For burning samples to determine ash and VM

vaccum pump filter To separate hydrolysate from biomass residue

UV spectrum (lambda 35) Used to determine sugar content and concentration of product

FTIR( FT/IR 6600) To determine structural and functional groups

Pressure vessel(SS316, 4570) For biomass pretreatment and hydrolysis

TG analyzer (HTC-1) To determine the degradation temperature of the sample

22
3.3 Process flow diagram and Framework of the study

Figure 3. 1 Framework and Flow diagram of the study

3.4 Sample preparation

The harvested plant roots, stems, and leaves were used in the experimentation process. The roots,
stems, and leaves were washed and submerged carefully in tap water to remove dirts and
contaminants. Surface moisture and the moisture that was entrapped in the water hyacinth are
removed by drying the sample using sunlight. It took two weeks to dry the whole plant, but it
only took five days when the root, stem, and leaves were separated and cut into small sized
sample. The samples were then dried in an oven at 105℃ until constant weight is recorded.
Since the size of the sample was very large to directly send to the next process, size reduction
took place to bring them to the available size range by using the centrifugal mill between 0.5mm

23
and 1mm sieve in the mechanical unit operation laboratory. Afterward, the samples were washed
with tap water and filter in vaccum pump to remove very fine particles and different contaminant
materials. The residue were dried in drying cabinet until its moisture is fully removed. The dried
material was packed using plastic bag until used for the next process at room temperature. The
process is described in figure 3.2 below from left to right direction.

Figure 3. 2 The process of sample preparation

3.4.1 Characterization and Composition Determination

3.4.1.1 Characterization of Water Hyacinth
The structural features of treated and untreated water hyacinth can provide a piece of information
that is used to describe the hydrolysis performance. The WHB was characterized by the
compositional analysis using UV and functional properties by FTIR spectroscopy. The proximate
analysis of the biomass sample were analyzed by standard ASTM method. The contents of
cellulose, hemicellulose and lignin were also determined by standard ASTM and NREL method.
The hydrolyzed reduced sugar were determined by the DNS method and standard solution
method.

3.4.1.1.1 Proximate Analysis of Water Hyacinth Biomass

Proximate analysis is the determination by prescribed methods of moisture, volatile matter, fixed
carbon, and ash.

24
Determination of moisture contents
The moisture contents of the biomass samples were determined using the standard ASTM E-871
method. The 2g weight of the sample was measured and the measured sample were dried in an
oven at 105℃ and weighed at regular interval until constant weight was observed. The sample
was cooled, after constant weight recorded, to room temperature in a desiccators and the
moisture content was determined using the formula:-

( ) [ ] 0 ......................................eqn.3.1

Determination of Ash content

The Ash content of water hyacinth biomass was determined using the ASTM D 482 standard
procedure. A biomass sample of 2g size from the oven-dried sample was taken in the crucible
and placed in a maffle furnace at 600℃ for a period of 4 hours. Then it was cooled to room
temperature in a desiccators and its weight was recorded. The percentage of the ash in the sample
was determined using the equation:-

( ) [ ] …………. .......................…….eqn.3.2

Determination of Volatile Matter

Volatile matter was determined in maffle furnace using (ASTM, E-872). To measure the volatile
content, 2g of dried sample was taken to a crucible and kept inside the maffle furnace at 900℃
for 7 minutes. After incineration the sample was cooled in standard desiccators at room
temperature and recorded the weight. Volatile matter was calculated by the method below.

( ) [ ] …..................……….eqn.3.3

Determination of Fixed Carbon

Fixed carbon content is calculated from 100% reduced by moisture content, ash content and
volatile matter (ASTM D-3173). The reduced moisture content means that the fixed carbon is
higher.
FC = 100% – (moisture content % - Ash content % – volatile matter %) ......................eqn 3.4

25
3.4.1.1.2 Compositional Analysis of WHB
Determination of extractives
A cellulose thimble of soxhlet extractor was loaded with a dried biomass sample of 2.5g. With
the soxhlet extractor set up, 160ml ethanol was used. This served as a solvent for extraction with
the sample left on the heating mantle for 5-h run, the stage retention time for boiling and rising
were adjusted to 80℃ and 20 min, respectively with care. After extraction , the sample was air
dried at room temperature for 5 min and then oven dried at 105℃ until the constant weight of
extracted sample was recorded. The ratio of the difference in weight of sample before and weight
of sample after extraction to the weight before multiplied by 100 is considered as extractives(%).

Extractive(%)= ((W before ext - W after ext)/ W before extraction)* 100 ....…………..eqn 3.5

Hemicellulose Determination
For the determination of hemicellulose present in the biomass, 0.5 g sample from dried extractive
free was taken into a test tube and 5ml of of 0.5M of NaOH solution was added to it. Then the
solution was kept in boiling water bath for three and half hour at 80℃. After reach its reaction
time, the sample was washed with distilled water until the pH was neutralized. Then after, the
residue was dried in an oven at 105℃ till getting constant weight was recorded. The
hemicellulose content was calculated by ASTM method as follows below.

( ) [ ] ...…………………………..eqn 3.6

Lignin determination
The standard ASTM D 1106-96 method was adopted in the determination of lignin in WHB
samples. WHB sample of 0.3g was added to 3ml of 72% H2SO4, the solution was well mixed and
incubated for one hour at 30℃. After the initial hydrolysis, 84ml of distilled water was added to
the solution until 4% concentration reach, thus making it slurry. The succeeding hydrolysis step
was initiated using an autoclave for one hour at 121℃. Then the slurry was allowed to cool at
room temperature. The hydrolysate was then filtered via vaccum filtration with the application of
filtering crucible. Lignin (acid insoluble) was estimated through drying of the residue at 105℃
and were incinerated at 575±25℃ in a maffle furnace to ash for one hour. The liquid filtrate
obtained was the acid soluble lignin and the acid hydrolyzed sample absorbance was measured
and recorded at 320nm by UV/visible spectra-photometer.

26
The ash obtained was weighed and the lignin was calculated using the following expressions.The
wt(%) acid insoluble residue (AIR) was calculated using the following expression,

%AIR = ([(Wt of(Crucible+AIR)) – Wt of (crucible)] /ODWsample) x 100 ...............eqn 3.7

The oven dried sample initially measured for lignin determination in the acid hydrolysis process
was considered as an oven dried sample (ODW).
Weight of the acid soluble lignin (AIL) on an extractives free basis was derived from the
formula,
%AIL = %AIR - (([Wt (Crucible+ash)– Wt of (crucible)] – [Wt of Protein])/ODW sample )
x 100 .....................................................................................................................................eqn 3.8
Where, weight of protein was the amount of protein present in the acid insoluble residue and
assuming zero because it is very low in amount. The amount of acid soluble lignin (ASL) on an
extractive free basis,
%ASL = {[UV-abs x volume of filtrate x dilution]/ [(ε) x ODW (sample) x Path length]} x
100 …………………………………………………………………………………….eqn 3.9

Where, UV-abs reading was the absorption reading displayed in the spectrometer, the average
ultra violet visual absorbance for the sample at appropriate wave length of 320nm. ε was the
absorptivity of the biomass at specific wavelength, was 25nm and the path length was the path
length of ultra violet visual cell in cm, which was 1cm. The dilution was derived as,

Dilution = [(V of sample) + (V of diluting sample)]/ (V of sample) ........................…..eqn 3.10

In this case, the value of dilution factor D is unity due to no need of diluting sample.

Total amount of lignin on an extractives free basis was calculated as,

%Lignin (Extractives free) = %AIL + % ASL ........................................................…..eqn 3.11

The total lignin obtained or received from the analysis was estimated using the formula,
%Lignin(as received) = %Lignin (Ext. free) x [(100 - %extractives)/ 100]............….eqn 3.12

27
Cellulose determination
The cellulose in the biomass was calculated by using the values obtained for their corresponding
extractives, lignin, hemicellulose and ash contents. The cellulose was calculated using the
expression,

Cellulose = 100 – (Lignin + Hemicellulose + Ash + extractives)..........................……eqn 3.13

3.5 Pretreatment experiments

This step is crucial to make the cellulose of the material easily available for the acid hydrolysis
process. It reduces the amount of lignin present in the material and decreases the crystallinity of
the cellulose. In this study the pretreatment process was conducted with ethanol and distilled
water in a 1:1 ratio. The samples that were dried and ground (which are between 0.5mm and 1
mm) were taken to the reactor where the delignification process took place. A solution was
prepared which comprised 96% ethanol and distilled water in 1:1 ratio in a beaker. The ratio of
the dried water hyacinth to the above mentioned solution was at 1:10, 1:15, and 1:12.5 with low,
high and medium level respectively (1 gram dried water hyacinth : 10, 15, 12.5 ml of solution).
The solution was added to the dried sample in the reactor. The rpm of the mixer was fixed at 400
rpm at a pressure of 4 bar and the sample stayed in the reactor for different temperature and time
interval based on the design expert v.11 display. Nextly, the pretreated sample was taken out of
the reactor and filtered off. Then the sample was washed by distilled water on a funnel vaccum
filter until it become neutral. The final procedure in the pretreatment process was drying the
washed sample. Washed sample was placed in drying cabinet to vaporize all the moisture from
the sample. The dried sample was packed by using plastic bag at room temperature until it was
characterized and used for the next step or hydrolysis step. The pretreatment step was assigned
in the figure from Appendix 2. The experimental set up for the pretreatment process is described
as figure 3.3 below the number representation was in appendex 8.

28
Figure 3. 3 Experimental setup for pretreatment process

3.5.1 Hydrolysis experiments

The Holocellulose materials which are composed of long chain simple sugars are broken down
into their corresponding simple sugars like glucose, before it is acid catalyzed for lactic acid
production. The residue from the pretreatment was oven dried and weighed for pre- hydrolysis in
order to determine the total reducing sugar present in the water hyacinth biomass or sample. The
hydrolysis process was carried out in vertical autoclave using 2% sulfuric acid at a temperature
of 121℃ in 15 psi for each individual pretreated sample for 90 minutes in 1:20(w/v) biomass to
sulfuric acid ratio. After the hydrolysis reach its residence time, the reduced sugar was analyzed
using UV spectrometer by DNS method and the concentration of reduced sugar (RS) was
determined based on the standard curve linear equation proved from the glucose standard
solution concentration and its absorbance from UV/visible spectroscopy.

3.5.2 Total reduced sugar determination

The total reducing sugar present in the water hyacinth biomass was determined using 3,5 Di-
Nitrosalicylic acid (DNS) method. For this, 1000 ppm standard glucose solution was prepared in
100ml volumetric flask (0.1g of glucose in 100ml distilled water) and it's series assay were
prepared in different volume of the stock solution in six 10 ml test tube. Another six test tubes
were prepared and 1ml of each solution were added into each test tube. 2ml of DNS reagents and
1ml of distilled water were added into prepared glucose containing solution. The test tube were
covered by aluminum foil and inserted into water bath at 80 ℃ for 10 minute. After 10 minute,

29
the test tube were left and cool down to room temperature and 1 ml of sodium potassium tertrate
solution was added in order to adjust the color of the sample and subjected to UV analysis. The
absorbance of the sample was recorded at 540nm against the reagent blank. Using the glucose
standard solution, the linear regression equation was obtained with 95% confidence level for
concentration range from 0.1-0.6 mg/ml. 80µl of the test sample( hydrolysate) were taken and 2
ml of DNS reagents and 1.92 ml of distilled water were added into a test tube. The mixture was
well mixed and kept in a boiling water bath for 10 minute. 1ml of 40% potassium sodium tertrate
was added to stabilize the red brown color formed. After cooling to room temperature, the
absorbance of the sample were recorded at 540nm against reagent blank. The concentration of
unknown sugar samples were derived from the standard curve generated from the relation
between glucose concentration and the absorbance values measured at 540nm using unknown
sample absorbance value.

3.5.2.1 Preparation of DNS reagent

DNS reagent solution was prepared by dissolving 2 g Di-Nitrosalicylic acid, 0.4g phenol, 0.1g
sodium sulphite and 2g sodium hydroxide in 200ml volumetric flask with distilled water. Then
after, the solution was heated for 30 minute in a hot plate. After 30 minute, the volumetric flask
was cooled and covered with an aluminum foil. A 40% potassium sodium tertrate aqueous
solution was prepared in another 50ml volumetric flask. The concentration to absorbance data
were arranged as follows below the table 3.
Table 3.2 Glucose standard solution data and calibration curve

30
3.6 Synthesis of Graphene Oxide by Improved Hammers Method
GO was synthesized from pure and fine graphite powder through improved Hammers method
using different oxidizing agents. In this study, 1:9 mixture of 85% phosphoric acid (160ml) and
98% sulfuric acid (1440ml) were mixed and 12g of graphite powder was added to the solution.
After 15 minute stirring, 72g of potassium permanganate was slowly added to the mixture as an
oxidizing agent and stirred continuously until the solution become dark green using magnetic
stirrer. The reaction undergoes an exothermic process at 50⁰C for 24 hrs. Before it reaches final
residence time, the solution changes its color from dark green to light purple and after some
times later it turns back to the dark green color. After 24 hr heating the solution was cooled at
room temperature for 30 minutes before proceed with washing process. 4000 ml of ice was
added to the solution followed by 16 ml of 30% concentrated hydrogen peroxide (H2O2) dropped
slowly into the solution and stirred for 10 minutes and the solution undergone centrifugation with
the spin speed of 180 rpm for 15 minutes. 40 ml of 10% hydrochloric acid (HCl) was added and
ultrasonicate for 10 minutes by mechanical shaker before second centrifugation process. Then
after the HCl was discarded and 120ml of distilled water was added and further ultrasonication
process was undergoing for another 10 minutes before last centrifugation process. This process
was repeated two times followed by centrifuge until the pH of the solution was neutralized.
After washing the product it was dried at 90 ⁰C using convection oven for 24 hrs to obtain
Graphene oxide paste. After drying the GO, it was grounded through mortar and pestle into small
sized particle or powder form. This powder form (<0.5mm) of Graphene oxide was packed in
polyethylene plastic bag until it was used as solid acid catalyst for the catalytic synthesis of lactic
acid. The synthesis process of Graphene oxide from graphite powder is shown in Appendix 3.
3.7 Fourier Transform Infrared Spectroscopy Analysis
FT-IR was used for studying the changes of functional groups of materials before and after
pretreatment process. Dried powder of water hyacinth biomass samples before and after
treatment, Graphene oxide, and lactic acid were tested and the spectra of the samples were
recorded in the range of 4000 to 400 cm-1 wave number with 4 resolutions.

3.8 Thermogravimetric analysis

3.8.1 TGA analysis of WHB sample
The thermal degradation behavior of the dried and pretreated water hyacinth sample was
investigated using a Perkin Elmer Thermal Analyzer Lambda 35 under air nitrogen environment.

31
Sample weights of 10mg were used. The water hyacinth sample was heated with gas flowrate of
20 ml/min to maintain air N2 for the thermal degradation. The temperature range for the
pyrolysis investigation was from room temperature to 900℃ with a heating rate of 20℃/min. the
average dried biomass particle size of 0.8mm was used.

3.8.2 TGA analysis of GO sample

The thermal degradation behavior of the synthesized GO sample was investigated using a Perkin
Elmer Thermal Analyzer Lambda 35 under air N2 environment. Sample weights of 10mg were
used. The GO sample was heated with gas flowrate of 20 ml/min to maintain inert atmosphere
for the thermal degradation. The temperature range for the pyrolysis investigation was room
temperature to 900℃ with a heating rate of 20℃/min. the average dried biomass particle size of
0.5mm was used.

3.8.3 XRD Pattern analysis of GO samples

The structural properties of graphene oxide were determined by the XRD method. The XRD
analysis observes the crystalline and amorphous structure of the synthesized GO sample. The
XRD diffractograms were collected at room temperature within a 2θ range from 5 to 60° using a
diffractometer (Krystalloflex D500, Siemens, Germany), Kα (Cu) radiation (40 kV and 30 mA),
and a wavelength of 1.5406 Å reported by Díaz et al (Wauton & William-Ebi, 2019). The
crystallinity index (CrI) was determined using the Segal method, as shown in (1):

–
( ) [ ] …………………………………………eqn 3.14

Where CrI represents the relative degree of crystallinity, I002 is the intensity of the 002-lattice
diffraction of total area and Iam is the intensity of diffraction of amorphous region at 2θ . I002
represents both crystalline and amorphous regions, while Iam represents only the amorphous
region discussed by Flauzino Neto et al (Wauton & William-Ebi, 2019).

3.9 Lactic Acid Production (solid-acid-catalyzed or chemical hydrolysis)

Lactic acid can be made through the chemo-catalytic synthesis process which is cost-effective,
although the product quality is lower than that of enzymatic hydrolysis or fermentation due to the
presence of binding of different chemical precipitates which are used during the process. The
samples were chemically hydrolyzed with an acid catalyst called Graphene oxide with catalyst to
sample ratio range of 1/4 to 3/4 to fractionate the cellulose and hemicellulose into reduced sugar

32
units and finally converted to lactic acid in the presence of ethanol as a solvent in addition to
distilled water. The reaction was conducted through a vertical pressure vessel at a different
combination of process parameters at a pressure of 1bar. After the reaction reaches the process
temperature and time, the hydrolysate was filtered-off and subjected to UV/ vis for absorbance
measurement. The concentration of the produced lactic acid was calculated from the calibration
curve of standard solution using absorbance of unknown sample and calibration equation. The
sludge was washed out many times with tap water to neutralize the pH followed by a final rinse
in distilled water. After that, the sludge was dried at 90℃ in a hot air oven for 24h and subjected
for checking catalyst recyclability as well as separability. The experimental set up for hydrolysis
is shown below figure 3.4 and numbering representation was assigned at appendex 8.

Figure 3. 4 Experimental set up for acid catalyzed hydrolysis process

3.9.1 Product Quantity Determination

Lactic acid's chemical behavior is determined by its physio-chemical properties, which include:
a) acidic character in an aqueous medium; b) bi-functional reactivity associated with the
presence of a carboxyl and hydroxyl group, which provides it with great reaction versatility; and
c) asymmetric optical activity of C2. The LaA was analyzed qualitatively and quantitatively
using UV/visible spectroscopy with wavelength detector. By comparing the concentrations of
racemic lactic acid to the standard calibration curve, the concentrations of unknown sample can
be determined. The yield of LaA is given as follows below.

33
Yield (%) = ( )* 100 ....................................................................................eqn 3.15

3.9.1.1 Lactic acid standard preparation

1.2 g of 88% lactic acid was added into 10ml volumetric flask and diluted with distilled water. A
stock solution with x concentration of lactic acid was obtained and its assay were prepared in
different volume using two fold dilutions. A solution of 0.2% Iron (III) Chloride (0.3g) in 100ml
volumetric flask was prepared by diluting with distilled water and stirred until complete
dissolution of salt was happened. 50µL of the prepared lactic acid stock solution was mixed with
2ml of 0.2% Iron (III) Chloride and stirred until color formation of the solution was appeared.
The absorbance of the colored solution was measured at wavelength of 390nm with the reference
solution containing 2ml of 0.2% FeCl3. The linear regression equation was developed from the
relation between the concentration of stock solution and the absorbance give the calibration
curve used to determine the concentration of the unknown test sample generated from the solid-
acid hydrolysis of pretreated water hyacinth biomass. The standard curve is presented at
appendix 4.

3.10 Data Analysis

The experimental method was designed to optimize pretreatment and acid hydrolysis of water
hyacinth using randomized central composite design and response surface methodology with no
transformation. The effects of solvent concentration, acid concentration, reaction temperature
and reaction time on the yield of lactic acid were evaluated. Regression analysis and analysis of
variance (ANOVA) were performed using response surface methodology and data were
interpreted from ANOVA. The concentration and quality of lactic acid yield was analyzed either
by polynomial or quadratic equation significancy and by its reactivity in ethyl lactate production.

3.10.1 Experimental design for CCD, BBD, and Response Surface Method
Using a Central Composite Design and response surface technique, an experimental method was
created to optimize acid hydrolysis step. The experiments were planed and conducted according
to CCD type response surface design using three levels for each pretreatment parameters and for
the hydrolysis process, the BBD model was used with the three levels of respective hydrolysis
parameters. The process temperature, time, liquid-solid ratio(solvent concentration) (for WHB
Pretreatment) and solid acid catalyst concentration(for Hydrolysis of LCW) were selected as
process parameters and to be optimized in order to ensure their effect on the yield. Each factor
34
consisted of three levels of temperature(100, 120, 140)⁰C, time (60, 90, 120) minute, ratio (10,
12.5, 15) l/g were for the pretreatment process and temperature of (90, 105, 120)⁰C , time (30,
45, 60) minute and catalyst load of (0.25, 0.5, 0.75) gram were consisting for hydrolysis process.
Based on the number of factors and levels required, 33 total runs of WHB pretreatment with their
replication were conducted for randomized CCD response surface and 15 runs of biomass
hydrolysis were conducted by BBD response surface. The effects of solvent concentration, acid
catalyst concentration, temperature, and reaction duration on the yield of lactic acid were
investigated. To analyze the effect of process variables, factorial experimental design was
performed. In the pretreatment process batch reactor, the factors to be considered were the liquid
to solid ratio, temperature, solid acid GO load, and time that determines the amount of
hollocellulose, reduced sugar and hydrolysis yield of lactic acid in BBD model shown in
Appendix 5.

Table 3. 3 Experimental design for Pretreatment process

Variable Name Unit Factor Levels

minimum middle maximum
Temperature ℃ A 100 120 140

Pretreatment time min B 60 90 120

Liquid-solid ratio L/g C 10 12.5 15

Table 3. 4 Experimental design for hydrolysis process for lactic acid production

Variable Name Unit Factor Levels

minimum middle maximum
Catalyst load gram A 0.25 0.5 0.75

Hydrolysis temperature ℃ B 90 105 120

Hydrolysis time min C 30 45 60

35
 CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 Proximate and Compositional Analysis of WHB
From the experimental work, the proximate and composition of whole water hyacinth plant were
determined by using the standard ASTM method and the results were presented in the table 4.1
below. The proximate analysis of moisture , ash , volatile matter , fixed carbon content and the
compositional analysis of lignin content (6.58%), hemicellulose content (25.12%) and cellulose
content (44.8%) recorded from the laboratory analysis were almost similar to the previous study.
The ash content (2.04%) and average moisture content (89%) for wet basis and (5.77%) for dry
basis of water hyacinth in this study were recorded and in close agreement with the study of
Reales-Alfaro (Reales-Alfaro et al., 2013).

Table 4. 1The proximate analysis and Compositional analysis of raw water hyacinth biomass

Authors Parameters (%)

Cellulose Hemicellulose Lignin Ash Moisture Volatile Fixed

content content matter carbon
R. Kumar, 2009 18.4 49.2 3.55 - - - -
Reales –Alfaro, 32.84 24.7 8.10 1.614 93.1 - -
2013
A.S.Kalamdhad, 31.67 27.33 3.93 - - - -
2017
Nigam, 2002 40-65 48 3.5-4.6 - - - -
This study 44.8 25.12 6.58 2.04 89 (wet) 6.06 2.9
5.77(dry)

4.2 FT IR Spectroscopy, TGA and XRD analysis

4.2.1 FTIR analysis of water Hyacinth
The FT-IR analysis of untreated water hyacinth and pretreated WHB samples are shown in figure
below. The FT-IR analysis of spectrum for the biomass, the entanglement degree of the bond
between each group involved in the biomass was observed and the regions that the functional
group bounded were investigated based on the transmittance versus wave number graph.

36
Figure 4. 1 FT-IR analysis of untreated and pretreated WHB samples

The peaks observed at around 3378.86 and 3832.84 cm-1, at 1038 cm -1, 1591.67 cm -1, and
2343.88 cm -1 are due to the presence of the O-H, C-O, N-O and O=C=O bond stretching respect
to their functional group respectively for raw water hyacinth sample. For treated WH, the
intensity of peaks at 1776.14 cm-1 was deacreased that revealed the deformation of C=O
(stretching) in hemicellulose and the O-H bond present in untreated biomass was stretching in
treated biomass as compared from FTIR analysis. Also, the peak happens around the wave length
of 2839.38 cm-1 was observed in treated biomass due to the C-H stretching vibration band of
cellulose component as almost the same to the previous report that the peaks were appeared at
around 3734 and 3614 cm-1 , at 1032 cm -1
, at 1527.86 cm -1
, and 2346.92 cm -1
due to the
presence of free O-H stretching sharp peak vibration, C-O stretch to inform about alcohol
structure in raw sample, N-H stretching vibration for band of protein amide structure as well as
N-O stretching of nitro compounds and O=C=O stretching of carbon dioxide respectively
(Boontum et al., 2019). This change of the structure resulted from the pretreatment process is
due to the influence of process parameters. Changes in structural characteristics such as porosity
and crystallinity are always followed by changes in DP (Energy & Technologies, 2022).

37
4.2.2 Thermogravimetric analysis Results of water Hyacinth
Figure 4.2 below is the TGA profile showing the thermal decomposition characteristics of water
hyacinth at a heating rate of 20 ℃/min. The profile indicates that most of the loss in weight
happened in the temperature range of about 245 to 550 ℃. This establishes the temperature
range for pyrolysis reactions. The TGA profile of WH sample involves three lumped consecutive
reactions: vaporization, devolatilization and secondary cracking reactions in harmony with
literatures (Wauton & William-Ebi, 2019). The first weight loss is attributed to the removal of
moisture and light volatiles in the biomass which is followed by the decomposition of volatile
matter and removal of volitiles (i.e devolatilization) at about 245 to 550 ℃, indicated by a sharp
endotherm in the TGA. In this phase, most of the weight loss was takes palced and the pyrolysis
products of tar or bio-oil, non-condensible gases and char were happened due to Devolatilization
reaction. The hemicellulose and cellulose decomposition were takes placed at 230-380℃ and
lignin degradation happened between 420℃-550℃. The loss of lignin typically occurs at a
slower rate over a much wide temperature range of 175-900℃ and it is almost in line with the
previous study (Wauton & William-Ebi, 2019) and reports (Girisuta et al., 2008) on the
characterization of ligninocellulosic biomass using TGA analysis. As compared to the second
stage, the lower rate of weight loss was observed in the third region or stage i.e due to the
degradation of heavier volatiles or tar, breaking of C-C bonds and formation of char decomposes
the tar to gas that leads gasification reaction at temperature range from 550℃ to 900℃ and the
lignin decomposition continues to this temperatue region as reported by Mostafa,et. al (Wauton
& William-Ebi, 2019). In order to control the formation of char for the maximization of bio-oil
production, the secondary reaction shoul be minimized in the design of pyrolysis system as
reported by Swart et al ((Wauton & William-Ebi, 2019).

38
Figure 4. 2 Thermogavimetric curve of water hyacinth sample

4.2.3 FTIR analysis of Graphene oxide

100 GO
O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n

98

O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n
% (Transmitance)

96

O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n

94

O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n

92

O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n
90

O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n
88

O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n
86

0 500 1000 1500 2000 2500 3000 3500 4000 4500

wave number (1/cm)

Figure 4. 3 FTIR analysis for GO

The FTIR spectroscopy analysis was done in order to investigate the structural change and
functional groups in Graphene oxide. The FTIR spectrum of GO in the above figure shows a

39
broad peak between 3002.665 and 3713.349 cm-1 in the higher- frequency area corresponding to
the stretching and bending variation of OH groups of water molecule adsorbed on GO.
Therefore, it can be concluded as the sample has a strong hydrophilic property. In this study, the
GO sheet showed apparrent adsorption peak bands for the carboxyl C=O (1762.216 cm-1),
aromatic C=C (1580.29765 cm-1), epoxy C–O (1200.4665 cm-1), alkoxy C–O (1051.53266 cm-1)
and hydroxy –OH (3399.489 cm-1) groups and it is almost similar to the previous study as
reported that peak occur at the wave length of 1723 cm-1 , 1621 cm-1, 1220 cm-1, 1043 cm-1, and
3391 cm-1 for carboxyl, aromatic, epoxy, alkoxy, and hydroxyl groups respectively. There are
also valence vibrations of S=O bonding included in the –SO3H group at 1040, 1059, and 1108
cm-1 (Nguyen, 2019). The presentation of oxygen-containing functional groups, such as C=O and
C–O, further confirmed that the graphite indeed was oxidized into GO and was cosistent with the
literature's (Yadav and Bhaduri, 2023).

4.2.4 TGA analysis of Graphene oxide

Fig. 4.4 displays the TGA curves for GO samples synthesized via the improved Hammers
method measured between room temperature and 900℃ in air nitrogen environment. The TGA
curve exhibits three weight losses: from room temperature to 793℃. The weight loss of the GO
samples was due to the decarboxylation of the carboxylic groups and the loss of CO2 ( Tohamy et
al., 2020) . The initial weight loss from 110-227℃ was likely due to the removal of moisture
contents from the sample. The medium weight loss from 227-570℃ was the result of several
concident processes like dehydroxylation, combined with pyrolytic fragmentation leading to the
development of aromtized units and volatile products i.e pyrolysis of the most oxygenated
functional groups such as hydroxyl group had occurred, which then released CO, CO2 and steam
in the second decomposition step. The third and the final decomposition from 570-793℃ was
ascribed/ certified to the decomposition of the carbonaceous residue to form low molecular
weight gaseous products i.e the thermal decomposition was likely related to the combustion of
the cross-linked aromatized units formed in the second step. Generally, the lower rate of weight
loss over temperature in the final region may be due to the burning of most stable oxygen
functionalities i.e the double bond between carbon and oxygen of the carboxyl groups (Tohamy
et al., 2020). Further decomposition take place up to 800 ℃ . This curve is in agreement with
the TGA curve recorded for GO produced by the Hummer process (Abdelkader et al., 2014).

40
 Figure 4. 4 TGA curve for graphene oxide

4.2.5 XRD analysis Results of Graphene oxide

The X-Ray Diffraction (XRD) patterns of graphene oxide (GO) is shown in figure 4.5. The XRD
analysis allows or describes the crystalline structure of the GO sample at the peaks to be formed.
The GO sample shows a sharp peak at 2θ = 26.8° in the XRD pattern corresponds to the plane
which may be attributable to an increase in the interlayer spacing due to oxygen containing
functional groups on the graphene oxide surface(Sahila Grace & Littis Malar, 2020) and due to
the incorporation of oxygen functionalized group like epoxy, carboxyl, hydroxyl and carbonyl in
graphene layers, which confirms the successful preparation of GO (Kamel et al., 2019). The
interlayer distances, calculated using the Braggs equation is 0.332nm which is relatively similar
to 0.35 to 0.765 nm calculated in the previous study (Kamel et al., 2019) after graphene
oxidation owing to interlayer spacing of oxygen containing functional groups. The average
interlayer distances or spacing of the graphene oxide calculated is approximately 0.446 nm
which is in range of 0.335nm to 0.822nm reported by the the previous researcher(Abdelkader et
al., 2014). Based on segal method the crystalinity index of GO is 62.79%.

41
Figure 4. 5 XRD pattern of GO

4.3 Pretreatment Results

In this study, chemical pretreatment using Organosoly method with ethanol as organic solvent
with distilled water in 1:1 ratio is used. The Organosoly pretreatment process of the biomass was
done in 10:1, 12.5:1, and 15:1 ratio of ethanol solution to biomass sample at different
pretreatment conditions. The holocellulose, lignin content, and reduced sugar present in water
hyacinth at different pretreatment conditions is presented in the table 4.2. The analysis of
variance (ANOVA) was used to check the adequacy of the model for the responses in the
experimentation. Based on the result from the experiment, the significancy of the pretreatment
factors was observed and investigated. This investigation was done by interpreting the data from
ANOVA analysis through F-value, P- value, sum of squares, and mean squares. Those
parameters helps to optimize the pretreatment process of water hyacinth biomass for
holocellulose recovery, reduced sugar and lignin removal. The significancy of the factors from
the observation indicates that the effect of pretreatment conditions on the delignification rate and
hollocellulose content or recovery in water hyacinth biomass as well as on reducing sugar yield.
During biomass pretreatment process, mass loss and reactivity of biomass in the solvent/water
solution were observed. The influence of pretreatment conditions on the delignification rate or
lignin content, reduced sugar, and holocellulose content existed in water hyacinth biomass were

42
analyzed through residual, predicted and actual product graph, 3D- surface analysis and their
interaction effect.

Table 4.2 Result of hollocellulose, lignin content, and reduced sugar from pretreated biomass

Factor 1 Factor 2 Factor 3 Response 1 Response 2 Response 3

Run A:T(℃) B:t (min) C:LSR(L/g) L.C (%) H.R (%) RS yield(mg/ml)
1 100 60 10 7.25 68.39 19.94
2 120 120 12.5 7.91 75.51 26.96
3 120 90 10 9.13 77.79 25.67
4 140 60 10 7.94 71.29 27
5 120 90 12.5 8.81 82.43 26.89
6 140 60 10 7.95 71.28 26.23
7 140 120 10 7.48 66 24.9
8 140 90 12.5 8.5 77.21 26.85
9 140 120 10 7.49 66.05 24.89
10 120 60 12.5 7.93 79.17 26.38
11 120 120 12.5 7.86 75.49 27.02
12 100 120 10 7.57 64.46 25.67
13 100 120 15 7.15 71.15 25.52
14 100 60 10 7.23 68.37 19.93
15 120 90 15 8.87 84.18 28.22
16 100 120 15 7.19 71 25.8
17 140 60 15 7.93 76.5 32.07
18 140 60 15 7.92 76.51 32.13
19 120 90 15 8.85 84.2 28.56
20 120 90 12.5 8.79 82 26.42
21 120 90 10 9.12 77.76 26.42
22 140 120 15 7.53 73.91 25.98
23 100 60 15 6.65 73.78 21.67
24 100 90 12.5 7.84 73.94 23.79
25 120 90 12.5 8.87 81.89 27.73

43
 26 100 60 15 6.62 73.76 21.42
27 140 90 12.5 8.43 75.5 28.03
28 100 120 10 7.55 64.48 26.42
29 120 90 12.5 8.85 81.95 27.84
30 100 90 12.5 7.86 74.42 23.82
31 120 60 12.5 7.8 79.12 26.73
32 120 90 12.5 8.82 81.45 26.73
33 140 120 15 7.52 73.89 25.57

4.4 Optimiztion of Pretreatmen Process via RSM Analysis

For this study, a second-order RSM model is utilized to find a suitable approximation for the
functional relationship between independent variables and the response surface. Thirty three
experimental runs were performed according to the CCD model. Regression analysis was done to
fit the response function as per the equation. From several possible models of Design expert v 11
software, a quadratic model was found to be adequate for the prediction of the given yield as
shown by the following equations for the three responses.

( ) = 8.81 + 0.2890A + 0.0015B – 0.124C – 0.2144AB +0.1269AC + 0.0344BC –

0.6436A2 – 0.9261B2 +0.1914C2……………………………………………………………………… eqn.4.1

( ) = 81.7 + 1.22 A – 1.81 B + 3.15C –0.1575AB + 0.1375AC + 0.4838BC –6.28 A2 –

4.22 B2 – 0.5617C2 …… ……………………………………………………………………………………… eqn.4.2

R.S (g/L) = 24.28 + 1.11A + 0.6265B + 1.12C –1.89AB + 0.775AC + 1.63BC – 2.44 A2 +
2.35 B2 + 2.5C2………………………………………………………………………………………………… eqn.4.3

The statistical significance of eqn (4.1), eqn (4.2) and eqn (4.3) were tested by analysis of
variance (ANOVA) result obtained from the quadratic CCD model as shown in the above table
4.2. As suggested by the model F value and low probability value (p-value) which is less than
0.05, it is evidenced that the model is highly significant for this study.

44
4.5 Statistical Analysis of the Experimental Results
4.5.1 ANOVA( analysis of variance) for pretreatment process
Table 4. 3 Sequential Model Sum of Squares for lignin removal

Source Sum of Squares df Mean Square F-value p-value

Mean vs Total 2099.38 1 2099.38

Linear vs Mean 1.98 3 0.6593 1.40 0.2624

2FI vs Linear 1.01 3 0.3373 0.6940 0.5640

Quadratic vs 2FI 12.60 3 4.20 3251.19 < 0.0001 Suggested

Cubic vs Quadratic 0.0084 4 0.0021 1.88 0.1548 Aliased

Residual 0.0213 19 0.0011

Total 2115.00 33 64.09

Table 4. 4 Sequential model sum of squares for holo-cellulose recovery

Source Sum of Squares df Mean Square F-value p-value

Mean vs Total 1.856E+05 1 1.856E+05

Linear vs Mean 293.89 3 97.96 3.99 0.0171

2FI vs Linear 4.44 3 1.48 0.0544 0.9829

Quadratic vs 2FI 704.63 3 234.88 1631.37 < 0.0001 Suggested

Cubic vs Quadratic 0.6224 4 0.1556 1.10 0.3854 Aliased

Residual 2.69 19 0.1415

Total 1.866E+05 33 5654.72

45
 Table 4. 5 Sequential model sum of squares for reduced sugar

Source Sum of Squares df Mean Square F-value p-value

Mean vs Total 22370.44 1 22370.44

Linear vs Mean 99.79 3 33.26 7.49 0.0007

2FI vs Linear 100.94 3 33.65 31.31 < 0.0001

Quadratic vs 2FI 22.51 3 7.50 31.78 < 0.0001 Suggested

Cubic vs Quadratic 2.01 4 0.5027 2.79 0.0558 Aliased

Residual 3.42 19 0.1800

Total 22599.12 33 684.82

Sequential Model Sum of Squares": Select the highest order polynomial where the additional
terms are significant and the model is not aliased. In this case, the highest order polynomial is
not needed since additional terms are not significant and the model is aliased. The model
summary from the design expert v11 were shown as table 4.3, 4.4, and 4.5 above for the three
responses respectively.

Lack of Fit Test

Table 4. 6 Results of Lack of Fit test for lignin removal

Source Sum of Squares df Mean Square F-value p-value

Linear 13.63 11 1.24 1203.43 < 0.0001

2FI 12.62 8 1.58 1531.86 < 0.0001

Quadratic 0.0112 5 0.0022 2.17 0.1027 Suggested

Cubic 0.0028 1 0.0028 2.67 0.1194 Aliased

Pure Error 0.0185 18 0.0010

46
 Table 4. 7 Results of Lack of Fit test For holocellulose recovery

Source Sum of Squares df Mean Square F-value p-value

Linear 710.30 11 64.57 559.05 < 0.0001

2FI 705.86 8 88.23 763.89 < 0.0001
Quadratic 1.23 5 0.2465 2.13 0.1079 Suggested
Cubic 0.6100 1 0.6100 5.28 0.0338 Aliased
Pure Error 2.08 18 0.1155

Table 4. 8 Results of Lack of Fit test For reduced sugar

Source Sum of Squares df Mean Square F-value p-value

Linear 125.46 11 11.41 60.06 < 0.0001

2FI 24.52 8 3.06 16.14 < 0.0001

Quadratic 2.01 5 0.4024 2.12 0.1099 Suggested

Cubic 0.0014 1 0.0014 0.0073 0.9329 Aliased

Pure Error 3.42 18 0.1899

Model Summary Statistics Test

This model summary statistics test provides an information for the value of squred R from the
experiment as adjusted as well as predicted value inorder to determine either the factors are
closer to the suggested model or not.

Table 4. 9 Results Model Summary Statistics Test for lignin removal

Std. Dev. R² Adjusted R² Predicted R² PRESS

Linear 0.6860 0.1266 0.0362 -0.1165 17.44

2FI 0.6971 0.1914 0.0047 -0.3239 20.68

Quadratic 0.0359 0.9981 0.9974 0.9962 0.0590 Suggested

Cubic 0.0335 0.9986 0.9977 0.9960 0.0623 Aliased

47
 Table 4. 10 Results of model summary statistics test for holocellulose recovery

Source Std. Dev. R² Adjusted R² Predicted R² PRESS

Linear 4.96 0.2921 0.2188 0.0993 906.34

2FI 5.22 0.2965 0.1341 -0.1953 1202.79

Quadratic 0.3794 0.9967 0.9954 0.9939 6.13 Suggested

Cubic 0.3762 0.9973 0.9955 0.9925 7.54 Aliased

Table 4. 11 Results of model summary statistics test for reduced sugar

Source Std. Dev. R² Adjusted R² Predicted R² PRESS

Linear 2.11 0.4364 0.3781 0.1882 185.64

2FI 1.04 0.8778 0.8496 0.7978 46.24

Quadratic 0.4859 0.9763 0.9670 0.9505 11.31 Suggested

Cubic 0.4242 0.9850 0.9748 0.9621 8.66 Aliased

The predicted R-squared indicates the closeness of the factors for the model. As it approaches to
unity means a good fit for the model selected which is quadratic model in this work. The
standard deviation indicates the difference between each factors and grouped factor difference.

In general, the significant of the coefficient term is determined by the value of F and P. The
larger the value of F and the smaller the value of P, the more significant is the model. The Model
F-value of 1340.79 for lignin removal, 774.03 for holo-cellulose recovery and 105.06 for
reducing sugar yield implies the model is significant. There is only a 0.010% chance that a
"Model F-Value" this large could occur due to noise. Values of "Prob > F" less than 0.0500
indicate model terms are significant. In this case A, C, AB, AC, BC, A2 , B2 , and C2 for lignin
content, A, B, C, BC, A2 , B2 , C2 for holocellulose recovery and A, B, C, AB, AC, BC, and A2
for reducing sugar yield are significant model terms for the three responses consecutively.
Values greater than 0.1000 indicate the model terms are not significant. The "Lack of Fit F-
value" of 2.17, 2.13, and 2.12 implies there is a 10.27%, 10.79%, and 10.99% chance that a

48
"Lack of Fit F-value" this large could occur due to noise respectively for those target. Lack of fit
is good due to insignificant based on its P-value greater than 0.05.
Table 4. 12 The ANOVA result for the significance of main and interaction effects on lignin
removal

Source Sum of Squares df Mean Square F-value p-value

Model 15.59 9 1.73 1340.79 < 0.0001 Significant

A-T 1.67 1 1.67 1292.60 < 0.0001

B-t 0.0000 1 0.0000 0.0348 0.8536

C-LSR 0.3075 1 0.3075 237.96 < 0.0001

AB 0.7353 1 0.7353 568.99 < 0.0001

AC 0.2576 1 0.2576 199.30 < 0.0001

BC 0.0189 1 0.0189 14.63 0.0009

A² 2.20 1 2.20 1705.32 < 0.0001

B² 4.56 1 4.56 3530.80 < 0.0001

C² 0.1948 1 0.1948 150.73 < 0.0001

Residual 0.0297 23 0.0013

Lack of Fit 0.0112 5 0.0022 2.17 0.1027 not significant

Pure Error 0.0185 18 0.0010

Cor Total 15.62 32

The ANOVA analysis was done the partial sum of squares of coded factor, degree of freedom, F
–value, mean square and P- value for each main factors and their interaction inorder to observe
their significance on the lignin removal or content in the water hyacinth biomass. The Model has
F-value of 1340.79 which implies the model is significant. There is only a 0.01% chance that an
F-value greater than this could occur due to noise effect. P-values less than 0.0500 indicate
model terms are significant. In this case A, C, AB, AC, BC, A², B² and C² are significant model
terms. Values greater than 0.1000 indicate the model terms are not significant. In this case the

49
liquid solid ratio and its second order are insignificant terms on the delignification rate. The
Lack of Fit F-value of 2.17 implies the Lack of Fit is not significant. There is only have a 10.27
% chance that a Lack of Fit F-value large to this could happened due to noise.
Table 4.13 The ANOVA result for the significance of main and interaction effects on
holocellulose recovery

Source Sum of Squares df Mean Square F-value p-value

Model 1002.96 9 111.44 774.03 < 0.0001 Significant

A-T 29.74 1 29.74 206.59 < 0.0001

B-t 65.63 1 65.63 455.85 < 0.0001

C-LSR 198.51 1 198.51 1378.81 < 0.0001

AB 0.3969 1 0.3969 2.76 0.1104

AC 0.3025 1 0.3025 2.10 0.1607

BC 3.74 1 3.74 26.01 < 0.0001

A² 209.58 1 209.58 1455.66 < 0.0001

B² 94.81 1 94.81 658.53 < 0.0001

C² 1.68 1 1.68 11.66 0.0024

Residual 3.31 23 0.1440

Lack of Fit 1.23 5 0.2465 2.13 0.1079 not significant

Pure Error 2.08 18 0.1155

Cor Total 1006.27 32

From ANOVA analysis, the coded factor sum of squares, mean squares, degree of freedom, F-
value and P- values for all factors were observed and recorded to know their significancy on
lignocellulosic waste recovery. In this study, the Model has an F-value of 774.03 that implies
the model is significant. There is only a 0.01% chance that an F-value this large could occur due
to noise. P-values less than 0.0500 indicate model terms are significant. In this case A, B, C, BC,
A², B², C² are significant model terms. Values greater than 0.1000 indicate the model terms are
not significant. The Lack of Fit has F-value of 2.13 implies the Lack of Fit is not significant.

50
There is only a 10.79 % chance that a Lack of Fit F-value higher than this could happened due to
noise. We want the model to fit.
Table 4.14 The ANOVA result for the significance of main and interaction effects on reduced
sugar

Source Sum of Squares df Mean Square F-value p-value

Model 223.24 9 24.80 105.06 < 0.0001 significant

A-T 78.69 1 78.69 333.27 < 0.0001

B-t 1.37 1 1.37 5.79 0.0245

C-LSR 19.74 1 19.74 83.61 < 0.0001

AB 83.45 1 83.45 353.44 < 0.0001

AC 6.60 1 6.60 27.97 < 0.0001

BC 10.89 1 10.89 46.12 < 0.0001

A² 12.27 1 12.27 51.96 < 0.0001

B² 0.7225 1 0.7225 3.06 0.0936

C² 0.0311 1 0.0311 0.1317 0.7200

Residual 5.43 23 0.2361

Lack of Fit 2.01 5 0.4024 2.12 0.1099 not significant

Pure Error 3.42 18 0.1899

Cor Total 228.67 32

From ANOVA analysis, the coded factor sum of squares, mean squares, degree of freedom, F-
value and P- values for all factors were observed and recorded to know their significancy on
reducing sugar yield. In this study, the Model F-value of 105.06 implies the model is significant.
There is only a 0.01% chance that an F-value this large could occur due to noise. P-values less
than 0.0500 indicate model terms are significant. In this case A, B, C, AB, AC, BC, and A² are
significant model terms. Values greater than 0.1000 indicate the model terms are not significant.
If there are many insignificant model terms (not counting those required to support hierarchy),
model reduction may improve your model. The Lack of Fit F-value of 2.12 implies the Lack of

51
Fit is not significant relative to the pure error. There is a 10.99% chance that a Lack of Fit F-
value this large could occur due to noise. Non-significant lack of fit is good -- we want the model
to fit.
Squared R-Test
This test can be done with the parameters like PRESS, R2 , standard deviation, mean value and
coefficient variance to identify the suggested model is significant or not.
Table 4. 15 Results of R- squared test for lignin removal

Std. Dev. 0.0359 R² 0.9981

Mean 7.98 Adjusted R² 0.9974

C.V. % 0.4507 Predicted R² 0.9962

PRESS 0.0590 Adeq Precision 125.5198

The Predicted R² of 0.9962 is in reasonable agreement with the Adjusted R² of 0.9974; i.e. the
difference is less than 0.2. Adeq Precision measures the signal to noise ratio that is greater than
4 to be desirable. In this study, the ratio is 125.5198 which indicates an adequate signal. This
model can be used to navigate the design space.

Table 4. 16 Results of R- squared test For holocellulose recovery

Std. Dev. 0.3794 R² 0.9967

Mean 74.99 Adjusted R² 0.9954

C.V. % 0.5060 Predicted R² 0.9939

PRESS 6.13 Adeq Precision 95.8432

The Predicted R² of 0.9939 is in reasonable agreement with the Adjusted R² of 0.9954; i.e. the
difference is less than 0.2. So, the the predicted residual value is acceptable and recommended
for this model. And also the signal to noise ratio was measured by the Adequate Precision with
desirable ratio greater than 4. In this case the ratio is 95.8432 that indicates an adequat signal and
therefore the model can be used in the design space navigation.
Table 4. 17 Results of R- squared test for reduced sugar

52
 Std. Dev. 0.4859 R² 0.9763

Mean 26.04 Adjusted R² 0.9670

C.V. % 1.87 Predicted R² 0.9505

PRESS 11.31 Adeq Precision 45.5039

The Predicted R² of 0.9505 is in reasonable agreement with the Adjusted R² of 0.9670; i.e. the
difference is less than 0.2. Adeq Precision measures the signal to noise ratio. A ratio greater
than 4 is desirable. In this study, the ratio of 45.5039 indicates an adequate signal. This model
can be used to navigate the design space. The adequacy of the model was also evaluated by the
residuals which is difference between the observed and the predicted response values. Residuals
are thought of as elements of variation unexplained by the fitted model and then it is expected
that they occur according to a normal distribution.

Generally, Multiple regression coefficients R2 is calculated from the second-degree polynomial

equation given in eqn (4.1, 4.2, and 4.3) is 0.9981, 0.9967, and 0.9763 respectively. This
indicates that the predicted values are closer to experimental data and the quadratic polynomial
was capable of representing the system for the given experimental domain. The closer the R 2
value to unity, the stronger the model and the better it predicts the response. A lower value of
coefficient of variation, CV(%) = 0.4507, 0.5060, and 1.87 respectively indicates the precision
with which the experiments were conducted. Similarly, the smaller predicted residual sum of
squares (PRESS) statistic shows the better data points fit the model.

Diagnostic plots for pretreatment process

Normal probability plots

Normal probability plots were also a suitable graphical method for judging the normality of the
residuals. A normal plot of residuals between the normal probability (%) and the externally
studentized residuals was obtained to determine how better the model satisfies the assumptions
of ANOVA. The externally studentized residuals can also be used to measure the standard
deviations separating the experimental and predicted values. Figure 4.3a, 4.4a, and 4.5a below
shows the relationship between the normal probability (%) and the externally studentized

53
residuals for the three responses respectively. The straight line means that no response
transformation was required and that there was no apparent problem with normality.
Predicted versus actual plots
The plot which shows how much the model was precised by detecting the value or groups of
values that are not easily predicted. The points at the plot determines the actual and predicted
values of each run approach to the straight line. This line indicates the closeness of the actual and
predicted values each other. The points which are above the straight line demonstrates that the
predicted value is greater than actual value and vice versa. And also the scatter of plots are on the
diagonal line, the experimental data is closely related to the data predicted from the designed
model i.e the model is well designed. The figure 4.3b,4.4b,and 4.5b below shows the relationship
between the experimental and predicted values.

a)

54
b)
Figure 4. 6 a) normal plot of residuals, b) predicted vs actual lignin response of the experiment.

a) Normal plot of residuals

55
b) Predicted vs actual plot
Figure 4. 7 a) normal plot of residuals, b) predicted vs actual plot for holocellulose response of
the experiment.
a) normal plots of residuals

56
b) predicted vs actual plot

Figure 4. 8a) normal plot of residuals, b) predicted vs actual plot for reduced sugar yield

The Residual vs Predicted Plots and Residual vs Run Plot

The observed residuals were also plotted against the expected values, given by a normal
distribution or a constant range of residuals across the graph which is justifiable no requirement
of transformation to minimize the personal error was shown in Figure 4.6(a,b,c and d) below.
The approximate straight lines obtained on the plot indicates that, the residuals are normally
distributed and appeared to be randomly scattered.
a) Residual vs predicted plot for lignin content

57
b) Residual vs run plot

c) residual vs predicted plot for reduced sugar

58
 d) Residual vs run plot

Figure 4. 9 Residual vs predicted and residual vs run plots for pretreatment process

4.6 Effects of Parameters on Lignin Removal, and Holocellulose Recovery

Pretreatment temperature, time and liquid-solid ratio are considered the main operational
parameters influencing the removal of lignin and recovery of holocellulose. The effect of
pretreatment temperature, time and liquid - solid ratio on the lignin removal as well as reduced
sugar yield from water hyacinth are shown in the figure 4.10 and 4.11 respectively. Higher
temperatures, longer contact times and higher liquid-solid ratios all contribute to higher lignin
removal, while generally reducing holocellulose yields. This is due to increased cleavage of
glycosidic bonds in cellulose during hot pretreatment causing cellulose hydrolysis and lower
yield. Additionally, increasing operating temperatures can cause excessive organic compound
degradation resulting in the heighest carbohydrate losses. Increasing pretreatment times can
provide further decrystallization of the substrate allowing for better accessibility and improved
chemical hydrolysis yield; however overlong treatment times may result in excessive
solubilization leading to reduced carbohydrate yield. Higher liquid-solid ratios enhance heat
transfer capabilities by minimizing any "cold spots" resulting from unequal solid distribution on
the untreated feedstock surface for more consistent reactor thermal conditions which can

59
improve both lignin removal and carbohydrate yield. The longr time and high temperature also
cuases the decrease in deligninfication due to condensation of lignin tha resulted in a decrease in
its solubility as reported by wangietal et.al (Umai, R.D.; Jacob, S.; Kumar, 2022)

The interaction between pretreatment parameter affects the delignification rate and holo-
cellulose recovery in several ways, such as by altering chemical challenges posed to lignin,
influencing the accessibility of lignin to microbial attack or other biodegradation processes. For
example, changes in temperature and reactant concentration/ solvent cocentration could
influence the solubility of lignin and its bond strength with cellulose fibers, affecting degradation
rates and the efficiency of the reaction. Additionally, a combination of factors may affect the
mechanisms through which biomass is decomposed such as chemical catalytic impacting holo-
cellulose recovery levels as well as delignification rate. The interaction effect of pretreatment
parameters on lignin removal and holocellulose recovery depends on the specific parameters
used during pretreatment. Generally, higher temperatures and longer reaction times can lead to
increased lignin removal; however, this may come at the expense of holo-cellulose recovery.
Lower temperatures, short reaction times and low doses of acid are typically better for holo-
cellulose recovery. And also the interaction effect of pretreatment parameters on lignin removal
and holo-cellulose recovery depends on the type of biomass and the chemical composition of
lignin. In general, higher temperatures tend to result in higher levels of lignin removal, but an
increase in temperature could also lead to higher rates of degradation of glucan that are also
found in biomass. Time affects the efficiency of delignification but has a lesser impact on overall
carbohydrate yields. Higher liquid-solid ratios can help increase both lignin removal and
carbohydrate recovery, although there may be some dilution effects due to additional water
present in the reaction condition. Based on the effect of factor observed from the experimental
analysis, the optimum pretreatment condition were suggested and acceptable to maximize the
response of the study at medium temperature level and medium level of time as well as liquid-
solid ratio.

4.6.1 Interaction effect of pretreatment factors

The figure 4.10 below presents the effect of temperature, time and liquid-solid ratio on lignin
removal and reduced sugar yield respectively. As it can be seen in figure 4.10a, lignin removal
increases with temperature upto 120℃ and then becomes level off at higher temperature at time

60
of 90 minutes. This could be due evaporization of the solvent which decreases the interaction
with the solid biomass. Similarly the holocellulisse recovery also increases with temperature
upto 120℃ and then decrease at maximum temperature. This could be due to the degradation of
the most common carbohydrate components into sugars and furfurals. In this case thr reduced
sugar yield reachs it maximum value.

From the result of design expert v11 analysis as well as the figure prepared from an excel, the
interaction effect and main effect of pretreatment parameters were observed and investigated
with respect to the target or responses of this study. In this study, the lignin removal and holo-
cellulose recovery from water hyacinth pretreatment were decreased at higher level of liquid-
solid ratio (15 l/g) from the pretreatment temperature of 120⁰c to 140℃ and from the
pretreatment time of 90 minute to 120 minute. At this temperature and liquid-solid ratio, the rate
of lignin removal from the biomass is decreased because the chemical reactions that break down
lignin are slower. This is due to the fact that not enough heat is being transferred into the
biomass for these reactions to occur rapidly. The over increase in solvent results decrease in
delignification due to the reason that, the solvent concentration causes a high viscous nature and
limit the interaction of solvent on biomass as repoted in the literatures(Umai, R.D.; Jacob, S.;
Kumar, 2022). Additionally, this ratio does not allow for adequate contact time between the
pretreatment solution and biomass. As a result, there would be inadequate diffusion time for
holocellulose recovery.

61
 10 10

9.5 9.5

Lignin removal (%)

9
Lignin removal (%)

8.5 8.5

8 8 LSR=10

7.5 7.5
LSR=12.5
7 t=60 7
LSR=15
t=90
6.5 6.5
t=120
6 6
100 120 140 100 120 140
Temperature (℃) Temperature (℃
a) b)

8.8 LSR=10
8.6 LSR=12.5
8.4 LSR=15
8.2
8
Lignin removal (%)

7.8
7.6
7.4
7.2
7
60 90 120
Time (min)

c)

Figure 4. 10 The Interaction Effect of pretreatment parameters on lignin removal

From the graph above, in all cases, medium temperature level (T= 120⁰c) on the response reach
their maximum value compared to low and high temperature level. Generally, the response reach
their maximum value at the medium level of the pretreatment parameters which were observed
and analyzed from the design expert v 11 as well as the graph constructed using an excel from

62
the experimental data study. As reported in the previous study, 57.59% of holo-cellulose and
1.43% of lignin content were recorded through water bath pretreatment method and the
holocellulose content increased with an increase in ratio and decrease in lignin content that
leading to the expelling of cellulose and hemicellulose from their matrices. Another previous
researcher reports that 52.8% of lignin were removed by acid pretreatment method, 68% and
55% of holocellulose were recovered through alkali and oxidative pretreatment method (Singh &
Bishnoi, 2013). And also through micro-wave pretreatment method, 60.42% of holo-cellulose
recovery and 0.63% of lignin content were recorded according to Xia (Xia et al., 2013). In this
study 85.35% of holocellulose recovery and 60.07% of lignin removal were achieved at higher
pretreatment temperature, in 90 miuntes and 12.5 l/g of liquid-solid ratio.

4.6.2 Effects of pretreatment parameters on reduced sugar yield

Effect of temperature
As observed from the experiment, the temperature has a significant effect on the reduced sugar
yielding from WHB. By keeping both time and solvent concentration lower (time=60 minute,
and LSR=10), as temperature increase, the RS yield was increased from 20.54 to 25.97 g/L.
Also, at lower reaction time with higher solvent concentration, the RS yield was increased as
temperature increased i.e, temperature has higher significance. Additionally, when the time and
solvent concentration keeping high (t=120min, and LSR=15 ), the RS yield was decreased as
temperature increases to its maximum value due to over degradation of the biomass to organic
matter resulting in the loss of necessary carbohydrate.

Effect of time
The effect of pretreatment time on the RS yield was investigated from the design expert v11
software. Time have negative effect on RS yield when keeping the temperature and solvent
concentration higher. As keeping temperature high and low solvent concentration, the RS yield
has a slight change due to poor biomass distribution causes in the formation of cold spot. Also,
time has a positive significance as it increases to its max resulting maximum RS yield when
keeping the temperature and solvent concentration low due to enough space and time for the
reactants.

Effect of solvent concentration

63
As keeping time and temperature lower, the RS yield was decreased as solvent concentration
reaches maximum due to lack of thermal heat to degrade or time to solubilize. Also, when
keeping temperature and time higher, the RS yield has increased slightly as solvent concentration
increased compared to RS yield when keeping time and temperature lower. Additionally, as
temperature keeping higher and time lower, the RS yield was higher as solvent concentration
increased due to biomass get enough heat to decompose.

32 32

Reduced sugar conc. (g/L)

30 30
Reduced sugar conc. (g/L)

28 28
26 t1 =60 26
LSR=10
24 t2=90 24 LSR=12.5
t3=120
22 22 LSR=15
20 20

18 18
100 120 140 100 120 140
a Temperature (℃) Temperature (℃) b

32
Reduced sugar conc. (g/L)

30

28

26
LSR=10
24
LSR=12.5
22

20 LSR=15

18
60 90 120
Time (min)
c

Figure 4.11 Interaction effect of pretreatment parameters on reduced sugar yield

64
4.7 Effects of hydrolysis Operating Conditions on total amount of lactic acid
4.7.1 Individual effect of Operating Conditions on lactic acid yield
Effect of Catalyst Load
The catalyst load was one of the major factors that affect the yield of lactic acid dierectly. In this
study, as the catalyst load increase the lactic acid was increased from 0.3172g/g to 0.846g/g and
it was decreased as catalyst load decreased. In general, lactic acid yield is directly proportional to
the catalyst load.

Effect of Hydrolysis Temperature

From this study, as observed from the experimantl result, temperature has dierect effect on the
yield. As temperature increased from 90℃ to 120℃, the yield also increased from 0.283g/g to
0.746g/g. When it was observed from the study, catalyst load and temperature has dierect effect
on the yield and it was highly significant factor depends on the ANOVA result analysis from
design expert 11 version software.

Effect of Hydrolysis Time

Based on the present study, the hydrolysis time has a negative effect on the lactic acid yield as
observed from the result organized from the experiment. As hydrolysis time increased from 30
minte to 60 minute, the lactic acid yield decreased from 0.7835g/g to 0.521g/g. Also lactic acid
yield increased from 0.205g/g to 0.8395g/g as time decreased from 60 minute to 30 minute. So
lactic acid is inversly proportional to the hydrolysis time unlike to catalyst load and hydrolysis
temperature.

a)

65
 b)

c)

Figure 4. 12 Graphical represention fot the individual effect of hydrolysis condition on LA yield
The above figure 4.12 (a,b, and c) shows the the main effects of catalyst load, hydrolysis
temperature and hydrolysis reaction time respectively on the total amount of lactic acid yield
produced from the water hyacinth biomass. As observed from the figure, catalyst load and
temperature are more significant positively and time has negative effect on the yield of lactic
acid.

66
4.7.2 Interaction Effect of Hyadrolysis Operating Conditions on Lactic Acid Yield
Temperature-time effect on total lactic acid yield
Figure 4.13 represents the effect of temperature on total amount of lactic acid produced from
water hyacinth biomass by solid acid graphene oxide catalysis. The maximum amount of lactic
acid produced during water hyacinth hydrolysis, at the center point of hydrolysis time
experimental level (t = 45 min), were 0.378g/L, 0.605g/L, and 0.8395g/L at 90℃, 105℃, and
120 ℃ respectively. On the other hand, the minimum amount of lactic acid produced were
0.205g/L, 0.498g/L, and 0.521g/L with the respective hydrolysis temperatures at (t= 60 min)
from lower level to higher level. In both case, the results clearly show that the total amount of
lactic acid increase with temperature from 90℃ to 120 ℃. However, it may starts to decrease
with temperature too be high due to the formation of unwanted byproducts or decomposition of
reactants and products. Also, temperature is dependable on the hydrolysis time as shown in the
experimental result. Individually, the lactic acid yield were increased as the hydrolysis
temperature increase due to the increase in kinetic energy of the reacting molecules and having
lower activation energy of the reaction because of the reaction process is endothermic. Lactic
acid production in the hydrolysis reactor depended on the prevailing conditions of temperature.
For this reason the control of hydrolysis temperature were necessary during lactic acid
production. And from the design expert result, the optimum hydrolysis temperature was decided
based on the acceptable catalyst load needed and time consumption as maximum result was
happened.

The temperature-time interaction has a strong effect on the lactic acid yield as anlyzed from the
design expert result based on the experimental work. In this case, as the temperature and time
increased, the reduction of an interesting product yield was observed. This due to the over
degradation of biomass to furfural and depolymerization reaction of lactic acid to formic acids
and formaldhydes. In addition to this, the major sugar units involved in the biomass like glucose
and fructose were converted to other by-products such as organic acids (acetic acid, glycolic and
e.t.c).

67
 1

concentration(g/L)
0.8

Lactic acid
t=30
0.6 t=45
t=60
0.4

0.2

0
90 105 120
Temperature (℃)

Figure 4. 13 Temperature -Time interaction effect on total lactic acid yield

Effect of catalyst load-time interaction on total lactic acid yield

From the figure 4.14 below, Increasing catalyst load from 0.25g to 0.75g at constant time and
temperature can increase the yield of lactic acid from 0.2907g/L to 0.93 g/L and from 0.152g/L
to 1.144g/L respectively. This is because the catalyst increases the rate of reaction by lowering
the activation energy needed for the reaction to take place. This means that at the same
temperature, more reactants are able to undergo the reaction in a given amount of time leading to
a greater yield of desired product. However it is important to note that increasing the catalyst
load beyond a certain point may not necessarily lead to further increases in yield and may have
negative effects like formation of side reaction and decreasing of selectivity. As keeping
hydrolysis temperature actual factor, the interaction between CL and hydrolysis time have
significant effect i.e as they were in their lower value, the yield obtained was lower and as they
increased yield also increased but the highest yield of lactic acid was obtained at hihest CL with
in 45 minute both at lower and higher temperature range. But as compared to the CL-HT
interaction effect, the CL–time interaction has slight significance effect on the lactic acid yield.
In this case, the highest yield was obtained at lower catalyst load with longer time and at lower
time with highest catalyst load. Generally, their interaction should be opposite to achieve the
maximum yield of lactic acid. According to previous researcher report, the maximum yield from
water hyacinth was obtained at low concentration with higher hydrolysis time and at high
cocentration with short period of time. This was due to the reason that the complete hydrolysis of
hemicellulose(xylose) to furfural and charing of the remaining cellulose or formation of humins.
68
Figure 4.14 indicates the effect of time on total amount of lactic acid produced from
water hyacinth biomass by solid acid graphene oxide catalysis. The minimum amount of lactic
acid produced during water hyacinth hydrolysis, at constant temperature and catalyst load (T=90
- 120℃ and CL= 0.25g – 0.75g) were 0.169g/L, 0.378g/L, and 0.205g/L at 30min, 45min and
60min at lower temperature and the maximum lactic acid yield of 0.7835g/L, 0.8395g/L and
0.521g/L from lower to higher time level at higher temperature respectively. On the other hand,
the lactic acid yield at constant catalyst load (CL= 0.5g) where 0.4763g/L, 0.605g/L, and
0.363g/L from lower to higher time respectively was recorded from the experimental result. In
both case, the results clearly show that the total amount of lactic acid increase with time from 30
min to 45 min. However, it starts to decrease with time increase from 45 min to 60 min. These
result clearly indicates the optimum acid hydrolysis time might be around 45 minute. The
decrease lactic acid yield with increasing time above its optimum value resulted due to product
degradation or further reaction may not lead to more yield and may forms unwanted byproduct
like furfural and 5-HMF. For this reason, the control of hydrolysis time were necessary during
lactic acid production.

1.4
t1=30
1.2
LA concentration(g/L)

t2=45
1
t3=60
0.8

0.6

0.4

0.2

0
0.25 0.5 0.75
Catalyst load (g)

Figure 4.14 Catalyst load -Time interaction effect on total lactic acid yield

69
Catalyst load -Temperature effect on total lactic acid yield
The interaction between catalyst load and hydrolyssis temperature have strong significant effect
on the yield making the hydrolysis time as an actual factor shown below figure 4.15. As analyzed
from the result, the effect of CL and HT interaction have direct relation to the hydrolysis yield
but inverse to time i.e at low residence time with higher CL and HT, a highest lactic acid yield
(1.144g/g) was achieved and vice versa. This were due to the reason that the biomass get enough
heat to decompose and solublization as well as fast chemical rection with the best catalyatic
performance of the catalyst due to presence of wide catalytic surface for biomass degradation.

1.2
T1=90
1
Lactic acid concenntration(g/L)

T2=105

0.8 T3=120

0.6

0.4

0.2

0
0.25 0.5 0.75

Catalyst load (g)

Figure 4. 15 Catalyst load -Temperature interaction effect on total lactic acid yield

4.8 Countor and 3-D Response Surface for Yield with Parameter Interaction
The individual and cumulative effects as well as the mutual interactions between the parameters
on the dependent variables were described using response surface. In this study, the response
surfaces were described by the regression model for CCD and BBD which was developed using
Design-expert version 11 software. Figure below from the Appendix 6 and 7 shows the
interaction effect of all pretreatment parameters on the yield of lignin removal, holo-cellulose
recovery, and reduced sugar yield by fixing value of one parameter and two of the left

70
interchangeably. Their effect were observed for each yield in appendences listed before. At the
higher and lower temperature and time level, the yield of lignin removal is lower as compared to
the yield at the medium level of the temperature and time and the yield of holocellulose recovery
was lower due to the over degradation of cellulose to undesired products at higher time and
temperature. As time increase, biomass residue decrease due to degradation of cellulose into
another products according to previous researcher (Cheng et al., 2015). The response surface for
LC and RS were described by the regression model inorder to describe or investigate the
interaction effect of parameters on the target using color identification for minimum or
maximum value of the response graphically.

4.9 FTIR Analysis for Produced Lactic Acid

The FT-IR spectroscopy analysis for the determination of lactic acid quantity, structure and
functional groups were done by the FT-IR JASCO 6600 instrument in research laboratory. The
figure 4.16 below was the FT-IR spectra of lactic acid to observe the peak intensity by the data
displayed from the instrument.

Lactic acid
100 O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n
Lactic acid (Trancmitance(%))

O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n
80

O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n

60
O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n

40
O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n

20 O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n

0 O r i g i n P r o 8 E v a l u a t i o n O r i g i n P r o 8 E v a l u a t i o n

0 500 1000 1500 2000 2500 3000 3500 4000 4500

wavelength (1/CM)

Figure 4. 16 FTIR analysis for lactic acid

From the figure above, the peak intensity at 1069 cm-1, 1180 cm-1, 1251cm-1, 1532cm-1, 1631cm-
1
, 1846cm, 2078cm-1, 2606.8 cm-1, and 3201.5cm-1 - 3632.4 cm-1 were observed. The peak at
1069 cm-1 is due to CH- stretching of C-CH3 group as well as C-O stretching of alcohol that
contribute the substantial noise in the spectra. The peak intensity at 1118 cm -1 is due to the
stretching modes of C-C, C-O functional groups, as the previous reporter said that the peak
intensity occurred below 1200 cm-1 represents different kinds of C-C, C-O, and CH3 vibration

71
(rocking, deformation, or stretching) (Zhao et al., 2015). The peak intensity at 1251 cm which
occurred in the region between (1210-1321) cm-1 is due to C-O stretching in the sample and O-
H bending. The peak region (1500-2000) cm-1, i.e at 1532 cm-1, 1631cm-1, and at 1846cm-1 was
observed due to double bond manifestation assigned to the carbonyl of carboxylic acid
group(C=O) and based on the region (1543 - 1716) cm-1 the peak at 1631 cm-1 is caused by water
according to previous researcher (Tohamy et al., 2022). The peak at 2606.8 cm-1, 3201.5 cm-1
and between (3201.5 - 3632.4 ) cm-1 is due to the O-H stretching of the acid component and
caused by water.

4.10 Design expert for acid hydrolysis process

Table 4. 18 Build Information of experimental analysis of hydrolysis process

File Version 11.1.2.0

Study Type Response Surface Subtype Randomized

Design Type Box-Behnken Runs 15

Design Model Quadratic Blocks No Blocks

Table 4. 19 Fit Summary test for lactic acid yield

Source Sequential p-value Lack of Fit p-value Adjusted R² Predicted R²

Linear < 0.0001 0.0018 0.8538 0.7642

2FI 0.3657 0.0017 0.8619 0.6223

Quadratic < 0.0001 0.8999 0.9998 0.9997 Suggested

Cubic 0.8999 0.9997 Aliased

Table 4. 20 Sequential Model Sum of Squares

Source Sum of Squares df Mean Square F-value p-value

Mean vs Total 4.42 1 4.42

Linear vs Mean 1.01 3 0.3379 28.26 < 0.0001

72
 2FI vs Linear 0.0411 3 0.0137 1.21 0.3657

Quadratic vs 2FI 0.0903 3 0.0301 2242.20 < 0.0001 Suggested

Cubic vs Quadratic 0.0000 3 4.823E-06 0.1832 0.8999 Aliased

Residual 0.0001 2 0.0000

Total 5.57 15 0.3713

Select the highest order polynomial where the additional terms are significant and the model is
not aliased.

Model Summary Statistics test for hydrolysis process

Table 4. 21 Model Summary Statistics

Source Std. Dev. R² Adjusted R² Predicted R² PRESS

Linear 0.1093 0.8852 0.8538 0.7642 0.2701

2FI 0.1063 0.9211 0.8619 0.6223 0.4326

Quadratic 0.0037 0.9999 0.9998 0.9997 0.0004 Suggested

Cubic 0.0051 1.0000 0.9997 * Aliased

This model summary statistics test provides an information for the value of squred R from the
experiment as adjusted as well as predicted value inorder to determine either the factors are
closer to the suggested model or not. In this study, the astrix (*) indicates that the Predicted sum
of square is not determined or defined and the model should focuses on the maximizing the
adjusted and predicted R squared values. So the factors are closer to the designed model for this
study.
Table 4. 22 Lack of Fit Tests

Source Sum of Squares df Mean Square F-value p-value

Linear 0.1315 9 0.0146 554.73 0.0018

2FI 0.0903 6 0.0151 571.74 0.0017

Quadratic 0.0000 3 4.823E-06 0.1832 0.8999 Suggested

73
 Cubic 0.0000 0 Aliased

Pure Error 0.0001 2 0.0000

The selected model should have insignificant lack-of-fit. In this case the selected model is
quadratic model and it have insignificant lack of fit p- value of 0.8999 which is greater than that
of 0.05.
Analysis of variance for hydrolysis process

Table 4. 23 ANOVA for Quadratic model of Lactic Acid

Source Sum of df Mean F-value p-value

Squares Square

Model 1.15 9 0.1272 9475.81 < Significant

0.0001

A-catalyst 0.5587 1 0.5587 41611.40 <

concentration 0.0001

B-hydrolysis 0.4295 1 0.4295 31985.44 <

temperature 0.0001

C-hydrolysis time 0.0254 1 0.0254 1895.21 <

0.0001

AB 0.0062 1 0.0062 459.52 <

0.0001

AC 0.0127 1 0.0127 945.09 <

0.0001

BC 0.0223 1 0.0223 1658.97 <

0.0001

A² 0.0176 1 0.0176 1308.09 <

0.0001

B² 0.0159 1 0.0159 1182.61 <

0.0001

C² 0.0533 1 0.0533 3968.30 <0.0001

Residual 0.0001 5 0.0000

Lack of Fit 0.0000 3 4.823E-06 0.1832 0.8999 Not

significant

74
 Pure Error 0.0001 2 0.0000

Cor Total 1.15 14

Factor coding is coded. Sum of squares is Type III – Partial. The Model F-value of 9475.81
implies the model is significant. There is only a 0.01% chance that an F-value this large could
occur due to noise. P-values less than 0.0500 indicate model terms are significant. In this case A,
B, C, AB, AC, BC, A², B², C² are significant model terms. Values greater than 0.1000 indicate
the model terms are not significant. If there are many insignificant model terms (not counting
those required to support hierarchy), model reduction may improve your model. The Lack of Fit
F-value of 0.18 implies the Lack of Fit is not significant relative to the pure error. There is a
89.99% chance that a Lack of Fit F-value this large could occur due to noise. Non-significant
lack of fit is good -- we want the model to fit.

Fit Statistics
Coefficient of Variation (C.V), the standard deviation expressed as a percentage of the mean;
Predicted Residual Error Sum of Squares, which is a measure of how the model fits each point in
the design; the R-Squared, measure of the amount of variation around the mean explained by the
model; Adjusted R² that is a measure of the amount of variation around the mean explained by
the model, Predicted R², a measure of the amount of variation in new data explained by the
model, and Adequate Precision, this is a signal to disturbance ratio due to random error,
presented in the table below, are used to decide whether the model can be used or not.

Table 4. 24 Fit statstics test table

Std. Dev. 0.0037 R² 0.9999

Mean 0.5431 Adjusted R² 0.9998

C.V. % 0.6747 Predicted R² 0.9997

PRESS 0.0004 Adeq Precision 331.5435

The Predicted R² of 0.9997 is in reasonable agreement with the Adjusted R² of 0.9998; i.e. the
difference is less than 0.2. Adeq Precision measures the signal to noise ratio. A ratio greater

75
 than 4 is desirable. In this study the ratio 331.544 indicates an adequate signal. This model can
be used to navigate the design space.
Regression model equation

Table 4. 25 Coefficients in Terms of Coded Factors

Factor Coefficient Estimate df Standard Error 95% CI Low 95% CI High VIF

Intercept 0.6053 1 0.0021 0.5999 0.6108

A-catalyst 0.2643 1 0.0013 0.2609 0.2676 1.0000

concentration

B-hydrolysis 0.2317 1 0.0013 0.2284 0.2350 1.0000

temperature

C-hydrolysis -0.0564 1 0.0013 -0.0597 -0.0531 1.0000

time

AB 0.0393 1 0.0018 0.0346 0.0440 1.0000

AC -0.0563 1 0.0018 -0.0610 -0.0516 1.0000

BC -0.0746 1 0.0018 -0.0793 -0.0699 1.0000

A² 0.0690 1 0.0019 0.0641 0.0739 1.01

B² -0.0656 1 0.0019 -0.0705 -0.0607 1.01

C² -0.1201 1 0.0019 -0.1250 -0.1152 1.01

The coefficient estimatation represents the expected change in response per unit change in factor
value when all remaining factors are held constant. The intercept in an orthogonal design is the
overall average response of all the runs. The coefficients are adjustments around that average
based on the factor settings. When the factors are orthogonal the VIFs are 1; VIFs greater than 1
indicate multi-co-linearity, the higher the VIF the more severe the correlation of factors. As a
rough rule, VIFs less than 10 are tolerable.

76
Equation for lactic acid yield in terms of coded and actual factor
Final equation in terms of coded factors:
The equation in terms of coded factors can be used to make predictions about the response for
given levels of each factor. By default, the high levels of the factors are coded as +1 and the low
levels are coded as -1. The coded equation is useful for identifying the relative impact of the
factors by comparing the factor coefficients.
Lactic acid yield 6 53 + 2643 A + 23 7 B − 564 C + 393 AB − 563 AC −
0.0746 BC +0.069 A2 − 656 B2−0.1201 C2 …………………………………………………………… (4.4)

Final equation in terms of actual factors:

The equation in terms of actual factors can be used to make predictions about the response for
given levels of each factor. Here, the levels should be specified in the original units for each
factor. This equation should not be used to determine the relative impact of each factor because
the coefficients are scaled to accommodate the units of each factor and the intercept is not at the
center of the design space.
Lactic acid Yield =– 6.7498 – 0.470233CL + 0.086342HT + 0.086627Ht + 0.010473CL*HT
– 0.01502CL*Ht – 0.000332HT*Ht + 1.10353CL2 – 0.000291HT2 – 0.000534Ht2…………(4.5)

4.10.1 Graphical analysis (diagnostic plot) for lactic acid yield

Normal probability plot
A normal probability plot of the raw data is used to check the postulation of normality. In the
Analysis of variance, it is usually more effective and straight forward to do this with the
residuals. If the underlying error distribution is normal, this plot will resemble a straight line. In
envisioning the straight line, place more prominence or emphasis on the central values of the plot
than on the extremes. In addition, the normal probability plot indicates the residuals succeeding a
normal distribution. In the case of this experiment, the points in the figure 4.17(a) below shows
fit to a straight line, this demonstrations that the quadratic polynomial model fulfills the
assumptions of analysis of variance (ANOVA) i.e. the error distribution is approximately
normal. The points are coded by color to the level of response and they represent going from
cool blue for lowest values to hot red for the highest.

77
 a) Normal plot of residuals

Actual versus predicted plot

The figure 4.17 (b) below predicted versus actual value of lactic acid yield was plotted. The plot
indicates how precisely the model demonstrated or fitted. The purpose is to detect a value, or
group of values, that are challenging to estimate by the model. The point shows how the
predicted value and actual values of each run approach the straight line. The straight line shows
how the predicted and actual values are nearer or closer to each other. When the point is above
the straight-line, predicted value is greater than the actual value and the reverse is also true. If the
scatter of the plot lies almost on the diagonal line, this shows that the model is intended or
designed very well i.e. the trial data is closely related to the data predicted from the model.

78
 b) Predicted versus actual
Figure 4. 17 (a) Normal plots of residuals and (b) predicted versus actual plots for lactic acid
yield

Residual versus predicted plot

If the model is correct and the predictions are satisfied, the residuals should be structure less; in
particular, they should be unrelated to any other variable including the predicted response. A
simple check is to plot the residuals versus the fitted (predicted) values. This is a plot of the
residuals versus the ascending predicted response values. It tests the assumption of constant
variance. The plot should be a random scatter (constant range of residuals across the graph). The
plot shows random scatter which justifying no need transformation for an alteration to minimize
personal error. The figure 4.18 (a) bleow shows the plot between residual versus predicted value
of the experimental data.

79
 a) Residual versus predicted plot for lactic acid yield
Residual versus run number plot
A scheme of the residuals versus the experimental run order checks for skulking variables that
may have influenced the response during the experiment. The plot should show a random
distribute. Trends indicate a time-related variable lurking/ skulking in the background. Blocking
and randomization provide insurance against trends debasement/ ruining the analysis. The
random scatter/distribution makes zigzag along the origin line. The figure 4.18 (b) below shows
the residual versus run.

b) Residual versus run number plot for lactic acid yield

Figure 4. 18 (a) Residual versus predicted and (b) Residual versus run number plots for lactic
acid yield.

Countor and 3-D Response Surface Plot for Interaction Effect of Hydrolysis Parameters

The interaction effect of hydrolysis parameters on the lactic acid was determined briefly on the
response countor and 3-d response surface plot from the figure 4.19 and 4.20 below. From the
result, figure 4.19a shows the interaction effect hydrolysis temperature and hydrolysis time on
the lactic acid yield by countor plot surface. In this case the effect of temperature-time
interaction on the yield has a positive effect in some cases at their medium level as observed in

80
the figure shown and have a negative effect at their lower and higher level simultaneously at a
constant catalyst load of 0.75g. But, when the effect is investigated individually, the yield
reaches its maximum at lower time and maximum temperature level i.e as time decrease the yield
increased as temperature increased. The same is true for the effect of catalyst load-time
interaction on the yield shown in figure 4.19b. In the case of figure 4.19c, the effect of catalyst
load-temperature interaction on the yield has direct effect i.e as temperature and catalyst load
increase, the yield also increased and vice versa at time of 60 minute but not that much as
indicated from figure. But at the time of 30 minute, their effect is more significant. The
interaction effect on 3-D surface were as shown in figure 4.20 and transfer the same
interpretation or information as countor polt surface by using color indication blue for minimum
and hot red for maximum result of the yield.

a) b)

81
c)

Figure 4.19 Countor plot response surface plot for (a) HT-Ht,(b) CL-Ht, and (c) CL-HT
interaction effect on lactic acid yield

a) The Temperature-Time interaction effect on lactic acid yield

82
 b) the catalyst load- time interaction effect on lactic acid yield

c) the catalyst load- temperature effect on lactic acid yield

Figure 4. 20 The interaction effect of hydrolysis parameters on lactic acid yield by 3-D surface

83
Diagnostics report for the hydrolysis process
This sector contains explanations of each case statistic. The values in the report table are
used to produce the diagnostics graphs. The actual values are the measured response data for the
particular run. Predicted values are the value predicted from the model, generated using the
prediction equation. Residuals are difference between actual and predicted values for each point.
The residual values are close to each other. This shows there is small difference between actual
and predicted values and normal error distribution.
Table 4. 26 Diagnostics report of Lactic Acid yield

Run Actu Predict Residu Levera Internally Externall Cook's Influen Standa
Ord al ed al ge Studentiz y Distan ce on rd
er Valu Value ed Studentiz ce Fitted Order
e Residuals ed Value
Residuals DFFIT
S

1 0.604 0.6053 - 0.333 -0.446 -0.407 0.010 -0.288 15

0 0.0013

2 0.603 0.6020 0.0019 0.750 1.023 1.029 0.314 1.783 2

9

3 0.290 0.2900 0.0007 0.750 0.396 0.360 0.047 0.623 5

7

4 0.783 0.7824 0.0011 0.750 0.628 0.585 0.118 1.013 10

5

5 0.601 0.6053 - 0.333 -1.448 -1.700 0.105 -1.202 13

0 0.0043

6 1.14 1.14 0.0000 0.750 0.014 0.012 0.000 0.021 4

7 0.705 0.7057 - 0.750 -0.396 -0.360 0.047 -0.623 8

0 0.0007

8 0.169 0.1697 - 0.750 -0.382 -0.347 0.044 -0.601 9

0 0.0007

9 0.205 0.2061 - 0.750 -0.628 -0.585 0.118 -1.013 11

0 0.0011

10 0.152 0.1520 - 0.750 -0.014 -0.012 0.000 -0.021 1

0 0.0000

84
 11 0.930 0.9312 - 0.750 -0.641 -0.599 0.123 -1.037 6
0.0012

12 0.291 0.2898 0.0012 0.750 0.641 0.599 0.123 1.037 7

13 0.611 0.6053 0.0057 0.333 1.894 3.187 0.179 2.253 14

14 0.521 0.5203 0.0007 0.750 0.382 0.347 0.044 0.601 12

15 0.535 0.5369 - 0.750 -1.023 -1.029 0.314 -1.783 3

0.0019

4.11 Optimization of Acid Hydrolysis Production of LA from Water Hyacinth Plant

Table 4.27 presents the optimization of maximum lactic acid production in a given constriants
and the maximum lactic acid produced was 1.134 g/L at 115.169℃ of temperature, 39.841
minute of time and 0.75g of catalyst load with 0.994 desirability which is selected optimum
solution. However, there are a number of feasible solutions that satisfies the given constraints
which are listed in the table 4.27 below.

Table 4. 27 Optimization for The Maximum Lactic Acid Production in a Given Constraints and
its solutions

Name Goal Lower Limit Upper Lower Upper Importa

Limit Weight Weight nce

A:catalyst is in range 0.25 0.75 1 1 3

load

B: is in range 90 120 1 1 3
temperature

C:time is in range 30 60 1 1 3

lactic acid maximize 0.152 1.144 1 1 5

Std Err is target = 0.0021156 0.00317 1 1 3

0.0026445 341
(lactic acid)

Solutions
Number catalyst hydrolysis hydrolysis lactic Std Err Desirability
load temperature time acid
(lactic acid)

85
 1 0.750 115.169 39.841 1.134 0.003 0.994 Selected

2 0.750 115.123 39.724 1.134 0.003 0.994

3 0.750 114.755 38.916 1.133 0.003 0.993

4 0.740 116.304 40.129 1.130 0.003 0.991

5 0.750 106.361 55.996 0.810 0.003 0.774

6 0.750 94.039 43.251 0.711 0.003 0.699

7 0.750 97.780 54.062 0.703 0.003 0.692

8 0.750 95.642 51.781 0.690 0.003 0.682

9 0.750 95.239 51.072 0.689 0.003 0.681

From the result, the predicted and the actual values depend on the design expert soft ware and
doing experiment by the constraints to validate the model were determined as 1.134g/L and
1.385 g/L respectively at optimized catalyst load of 0.75g, hydrolysis temperature of 115.2℃
and hydrolysis time of 39.8 minutes. This indicates that the model for this study is in a better
agreement with the experiment and it is valid & suggested to use.

The experimental result of 1.144 g/L or 34.2% yield of lactic acid was recorded and it is an
acceptable yield as compared to other previous researchers report. This makes the solid GO
catalyst is the better catalyst due to its greater acid site surface concentration for the hydrolysis
reaction of lactic acid compared to other solid acid catalysts like ZrO2 and Al2O3 with the yield
of lactic acid 8.02% and 6.63% at a temperature of 200℃ for 6hr reaction process respectively
(Doungsri et al., 2019). This result indicates that GO may effect on LA production significantly.

86
 CHAPTER FIVE

CONCLUSION AND RECOMMENDATION

5.1 Conclusion

This study were carried out inorder to utilize water hyacinth plant for the production of lactic
acid by using GO catalyst under different conditions of calayst load, temperatue, and reaction
time of chemical hydrolysis process this process were performed in a 2L galss pressure vessel.or
reactor( GLSS/SS316). It can be concluded that ethanol pretreated water hyacinth biomass can
be an effective method for lignin removal and recovery reduced sugar yield. The ethanol
pretreatment process removes lignin effectively while retaining a high holocellulose yield, which
in turn leads to a higher yield of reduced sugars when hydrolyzing the biomass. In this study the
sugar concentration of hydrolysate was determined using DNS method and UV
spectrophotometry absorbance with the maximum yield of 32.13g/L or 64.26% recording. The
lignin- depleted biomass also allows for better catalytic hydrolysis among other benefits.
Therefore, the ethanol pretreatment provides a promising approach for maximizing bio-refinery
usage of water hyacinth biomass and could potentially be used in larger scale applications.

Due to its simplicity Box-Behnken design of response surface was used in this study to decide
the experimental runs of hydrolysis process. The optimal values of tested variables were found to
be 0.75g, 115.2℃, 39.8 minute for CL, HT, and contact time respectively. The optimum values
of lactic acid with the given constraints was predicted to be 1.134g/L and compared to the actual
or experimental value of 1.1385g/L with 0.994 desirability. The maximum lactic acid yield was
found to be 1.144g/L which obtained at temperature of 120℃, catalyst load of 0.75g, and
reaction time of 45 minute. These makes the selected model was adequate to fit the data. In FTIR
and LA standard method, it was confirmed that the presence of lactic acid in the product.
Generally, conversion of water hyacinth biomass in to lactic acid with optimized solid catalyst
hydrolysis condition can be an excellent alternative of carbon source for the production of lactic
acid.

87
5.2 Recommendation
Ethanol pretreatment of water hyacinth biomass offers promising results for lignin removal and
holocellulose recovery. Compared to other pretreatment, ethanol pretreatment shows a notably
high degree of delignification and provides good conditions for further hydrolysis processes.
Also, due to the mild temperature requirements, the process can be used in both commercial-
scales as well as home-scale markets at affordable costs. Moreover, it does not produce
hazardous byproducts such as furfural and acetic acid that are oftentimes associated with other
pretreatment techniques. Thus, ethanol pretreatment is highly recommended for lignin removal
and recovery of holocellulose for high yield reduced sugar production from water hyacinth
biomass.

In this study, 2L pressure vessel that the temperature not greater than 120 ℃ were used during
each run of hydrolysis processes. However, it may not have the same result during scale up
work. Since, the experimental outcomes of this investigation work shows a great possibility of
using ethanol pretreated water hyacinth biomass for the effective production of lactic acid.
Additional study shoud be directed in order to tell the effect of scale up on the yield of lactic
acid before it becomes commercialized. In addtion to that, the effect of major hydrolysis
parameters such as catalyst load, temperature, and time alone were analysed on total amount of
lactic acid. However, there are other minor parameters such as biomass load, agitation speed,
oxygen source, vapor pressure, and others, which will affect the total amount of lactic acid.

In this study, the total amount of lactic acid was determined by using spectrophotometer with its
specific wave length and there is no way to know the other compostions of hydrolysate. So, it is
better to know the whole compostion of hydrolysate with HPLC and also the kinetics of lactic
acid production, which was not incorporated with this work should be studied in future
researches.

88
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Appendeces
Appendex 1 Design Combination of Pretreatment Process
Factor 1 Factor 2 Factor 3
Run A:T(℃) B:t (min) C:LSR(L/g)
1 100 60 10
2 120 120 12.5
3 120 90 10
4 140 60 10
5 120 90 12.5
6 140 60 10
7 140 120 10
8 140 90 12.5
9 140 120 10
10 120 60 12.5
11 120 120 12.5
12 100 120 10
13 100 120 15
14 100 60 10
15 120 90 15
16 100 120 15
17 140 60 15
18 140 60 15
19 120 90 15
20 120 90 12.5
21 120 90 10
22 140 120 15
23 100 60 15
24 100 90 12.5
25 120 90 12.5
26 100 60 15
27 140 90 12.5
28 100 120 10
29 120 90 12.5
30 100 90 12.5
31 120 60 12.5
32 120 90 12.5
33 140 120 15

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Appendex 2 Organosoly pretreatment process of raw water hyacinth biomass
sample

99
Appendex 3 improved hammers method synthesis of graphene oxide from fine
graphite powder

Appendex 4 Standard curve of lactic acid

2.5

2
absorbance

1.5
y = 0.7122x + 1.6567
R² = 0.988
1

0.5

0
0 0.2 0.4 0.6 0.8 1 1.2

concentration of lactic acid standard solution

100
Appendex 5 The BOX bohnken design for the lactic acid hydrolysis
Factor 1 Factor 2 Factor 3 Response 1
Run A:catalyst load(g) B: temperature (℃) C: time (min) LA (g/L)
1 0.5 105 45 0.604
2 0.75 90 45 0.6039
3 0.25 105 30 0.2907
4 0.5 120 30 0.7835
5 0.5 105 45 0.601
6 0.75 120 45 1.144
7 0.75 105 60 0.705
8 0.5 90 30 0.169
9 0.5 90 60 0.205
10 0.25 90 45 0.152
11 0.75 105 30 0.93
12 0.25 105 60 0.291
13 0.5 105 45 0.611
14 0.5 120 60 0.521
15 0.25 120 45 0.535
Appendex 6 The Interaction effect of factors on lignin content and reduced sugar
yield using countor surface plot

101
a) Countor surface plot for lignin content

102
103
b) countor surface plot for reduced sugar yield

104
Appendix 7 The Interaction effect of factors on lignin content and reduced sugar
yield using 3-D Surface

105
a) 3-D response surface plot for lignin content

106
b) 3-D response surface plot ofa reduced sugar yield

107
Appendix 8 the main components of reactors used both in pretreatment and
hydrolysis process

The main components of Pressure vessel or reactor SS316

1 Body holder 8 Gas inlet valve

2 Gas outlet valve 9 Sample inlet holder

3 Reactor body 10 Motor

4 Liquid outlet 11 Temperature sensor

5 Magnetic drive 12 Binder

6 Condenser 13 Pressure gauge

7 Control panel 14 Gas outlet wire

The main components of reactor (GLASS/ SS316)

1 Nitrogen and oxygen gas 9 Magnetic drive with motor

2 Reactor 10 Pressure gauge

3 Stirrer 11 Gas inlet

4 Cooling coil 12 Liquid outlet

5 Pump 13 Safety valve

6 Cooling water tank 14 Pressure sensor

7 Control panel 15 Temperature sensor

8 Gas outlet

108