3.

4 Lignocellulosic Biomass Conversion Method

Bioethanol can be produced by using a variety of renewable carbohydrate-rich materials, which

are hydrolyse to produce fermentable sugars that are then transformed into ethanol. The major

sources of bioethanol can be divided into three which are; first-generation bioethanol which

consists of stocks such as sucrose and starch crops which include cereals, sugarcane, corn and

other similar plants. Second-generation bioethanol uses lignocellulosic biomass, which is a plant

material that is more complex and its usual source is not the food sector. Third-generation

bioethanol is about microalgae that are becoming popular because they are fast to grow and they

sample how to use the sun energy to produce biofuel in the most efficient way (Jain & Kumar,

2024).

The various feedstocks serve as examples of the multiple exploitation opportunities of bioethanol

as a renewable energy source, which could ensure the availability of several ways to produce this

clean fuel. This study will be focusing on the use of lignocellulosic biomass for the production of

bioethanol (Schubert, 2020).

Producing bioethanol from lignocellulosic biomass is an intricate and time-consuming process that

involves several key stages. This process consists of several vital stages which require a bunch of

people to perform many steps. It starts with the collection of raw materials, specifically

lignocellulosic biomass, which can come from agricultural residues such as corn cobs and rice

husks. Once got, the biomass should be transported to processing facilities. Pretreatment is the

second step which disrupts the biomass structures and makes cellulose more accessible to

enzymatic hydrolysis. Consequently, saccharification occurs when the microorganism's enzymes

perform the conversion of the polysaccharides in the biomass into fermentable sugars. The ethanol

chain starts as sugars are fermented by microorganisms. This process is then followed by

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dehydration of the ethanol produced as a means to increase the purity. Besides, the appropriate

handling of both the main and the by-products is the backbone of the cycle and thus sustainability.

This transformation path is the one that needs different resources and, in the meantime, presents

the biggest hurdles due to the natural robustness of lignocellulosic materials. The match of

elements among them including lignin and hemicellulose limits the availability of cellulose for

enzymatic breakdown, and in order to improve its output, it requires the implementation of

adequate pretreatment strategies (Broda et al., 2022).

Lignocellulosic biomass is generated from the plant types and is defined as a dry organic material.

It mainly contains 25 to 30% hemicellulose, 40 to 50% cellulose, and 15 to 20% lignin. It also

includes a very small quantity of pectin, nitrogenous compounds, and various inorganic chemicals

(Mori et al., 2015). Cellulose and hemicellulose make up two third of the dry mass of this biomass

material.

Cellulose is the main structural ingredient of the walls of plant cells, and it gives the structure's

strength and rigidity a significant boost. Cellulose generally there is about 30% to 50% of dry

weight in lignocellulosic biomass. Cellulose obtained from any source has the characteristic high

molecular weight coupled with a crystalline structure usually, which shows the result of the

formation of strong, crystalline fibers. Basically, a cellulose molecule is a type of carbohydrate

that is not only long but also straight. Its average molecular weight is about 100,000 Daltons. The

polysaccharide is synthesized via the reaction between β-D-glucopyranose units linked by β-1,4-

glycosidic bonds. The main constituent of cellulose is cellobiose, which is made of two β-glucose

molecules connected by a β(1→4) β bond (Baghaei & Skrifvars, 2020).

Hemicelluloses constitute the second largest class of plant cell wall polysaccharides and are

responsible for 15% to 30% of the dry substance weight of the lignocellulosic material. These

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complicated polymers consist of a blend of short, linear chains and highly branched hetero-

polysaccharides (Zhang et al., 2015). The polysaccharide here has a low level of polymerization

and does not have a crystalline structure, it can be easily degraded into simple sugars like fructose,

xylose, galactose, dextrose, arabinose, and mannose (Karimi & Taherzadeh, 2016).

Lignin is a very important part of lignocellulosic biomass, which accounts for around 15–30% of

its dry weight. Lignin, which is present in all vascular plants, consists of a very complicated and

irregular structure, and it is characterized as an amorphous, highly branched, and cross-linked

polymer network. This atypical structure is the result of the enzymatic free radical polymerization

of three different phenylpropane units, namely monolignols. These units include p-coumaryl,

coniferyl, and sinapyl alcohols, which bring about the production of the aromatic structural

components in lignin: p-hydroxyphenyl, guaiacyl, and syringyl respectively (Zabed et al., 2017).

3.4.1 Processes involved in the conversion of lignocellulosic biomass to ethanol

When it comes to the structural polymers found in lignocellulose, only cellulose and

hemicelluloses are suitable for bioethanol production. This is because they are long-chain

polysaccharides that can be broken down into a blend of fermentable sugars, specifically pentoses

and hexoses, which can then be transformed into ethanol. In order to generate high power and

profits from lignocellulosic feedstock to ethanol conversion, we begin with pretreatment. This is

the most important step in this process because lignocellulosic materials possess the toughest

nature ever that prevents the release of their useful parts. We can break up the complex structure

and hence we end up with a situation where cellulose and hemicelluloses are available for

enzymatic hydrolysis.

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[Link] Pretreatment process

To convert the lignocellulosic materials into ethanol, the pretreatment process is the one of the

most crucial step. This process is about the destruction of the lignocellulosic material, a structure

complicated enough to contain long polymers held together by hydrogen bonds, thus making the

material weaker throughout. The carbohydrate is then more easily separated from lignin. Besides

this, the pretreatment also breaks the crystallinity of cellulose and reduces the degree of

polymerization by tearing the cellulose chain apart along the hydrogen bonds that hold it together

(Das et al., 2021).

Pretreatment processes are the key to the whole biomass treatment and they deal with the

modification of lignocellulose's chemical, and the structure of the macro and microstructures. The

latter through this process is made more labile and, therefore, more accessible to microbial

digestion(An et al., 2015) . A proper pretreatment process consists of three key steps. First, it is

essential to break the hydrogen bonds within crystalline cellulose. Next, the cross-linked structure

of hemicelluloses and lignin needs to be dismantled. Finally, this procedure enhances the porosity

and surface area of cellulose, which is crucial for effective enzymatic hydrolysis later on (Gírio et

al., 2014). The pretreatment process needs to be straightforward, affordable, and eco-friendly. It

should generate minimal inhibitory compounds while enabling the full utilization of

lignocellulosic biomass. This approach not only enhances the efficiency of bioethanol production

but also ensures effective management of waste lignin (Das et al., 2021). The pretreatment process

method for the conversion of biomass to ethanol can be divided into four other process methods,

which are: physical pretreatment process, biological pretreatment process, chemical pretreatment

process, and physio chemical pretreatment process.

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Physical pretreatment

Physical pretreatment techniques use various mechanical and environmental forces to break down

lignocellulosic biomass. These methods involve applying mechanical energy, radiation, electric or

electromagnetic fields, heat, or pressure to reduce the size of biomass particles. As a result, the

surface area and pore volume of the material increase, making it easier to process further. Besides

size reduction, these methods often cause a decrease in the polymerization level of the biomass

components and reduced crystallinity of cellulose. The change is important as it ameliorates

accessibility in the subsequent steps of the process. The common physical pretreatment methods

include grinding, milling, chipping, extrusion, freezing, sonication, microwaving, and pulsed

electric field treatment.

It was also determined that reducing the biomass particle size to less than 0.4 mm does not affect

the yield or rate of hydrolysis. Mechanical comminution energy costs of lignocellulosic biomass

are different and depend on individual characteristics of agricultural biomass and particle target

size (Tayyab et al., 2018). Physical pre-treatment aims to result in particle breakdown of

lignocellulosic material with lower crystallinity, increasing the specific surface area and

decreasing cellulose polymerization degree so that biomass is as accessible as possible for further

treatment (Maurya et al., 2015).

Chipping, grinding, and milling are important methods that prepare the lignocellulosic biomass

into forms amenable to further treatment. The size of the biomass particles could be reduced

substantially, and sizes could range between 10 and 30 millimeters to as small as 0.2 millimeters

to 2 millimeters. Among the different mechanical methods followed, some of the most common

ones practiced in the context of bioethanol production include ball milling, colloid milling,

hammer milling, two-roll milling, and wet disk milling (Shen et al., 2020). Some of the advantages

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ascribed to milling pre-treatment are low energy requirement and prevention of inhibitor

formations of fermentation. It reduces the cost of separating the solid from the liquid because the

ratio of solid to liquid is brought down (Zhu et al., 2014).

Extrusion is a thermal/physical process whereby fast mixing, gentle heat treatment, and high shear

forces are applied to lignocellulosic biomass. The complex structure was effectively broken into

pieces and changed both physically and chemically. This kind of processing has good adaptability

and efficiency. Most importantly, no furfural or HMF is formed as a by-product during the process.

It can also be carried out in a continuous manner with high solid concentrations (Duque et al.,

2017). During this process, materials are subjected to temperature above 300 °C. Consequent to

mixing and shear, cellulose undergoes physical as well as chemical changes. It is believed that

screw speed and temperature inside the barrel contributes to the destruction of lignocellulosic

complex. This makes way for defibrillation, fibrillation, and loss in fiber length, thereby making

carbohydrate accessible enzymatically (Zheng et al., 2014). The application of enzymes during

extrusion has been considered as an innovative approach to producing Ethanol.

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Sonication, through the action of ultrasonic waves, it acts to disrupt the lignocellulosic structure

and in so doing releases cellulose and hemicelluloses for enzymatic hydrolysis. This action

increases the accessibility of cellulose to enzymatic hydrolysis, hence increasing sugar yield,

reducing the time of hydrolysis, and improving fermentation yield. This pretreatment can be

further complemented with the addition of a mild heat of about 50 °C and changing the water to

an alkaline environment (Subhedar et al., 2018).

Microwave irradiation has become quite a popular technique in the pretreatment of lignocellulosic

materials, and rightly so. It is energy-efficient, produces less by-product that may interfere with

further processing, and is easy to use. The method rather effectively degrades the complex

crystalline structure of cellulose, allowing for a fast heating rate and thereby being quite efficient

and faster in the process (Hu & Wen, 2008). The microwave irradiation technique is based on

electromagnetic fields that enhance various processes. In the case of the production of bioethanol,

this is a quite effective method of decomposing pretreated biomass. Microwaves attack polar bonds

in biomass, and such irradiation makes the polar bonds vibrate; from these vibrations, heat is

generated. The generated heat further degrades the lignocellulosic complex, increasing the

accessible surface area for the enzymes. Generally, microwave treatment is assumed to be an

enriching enhancement and supplementation of methods in biomass treatment. The duration of

microwave irradiation typically lasts between 5 to 20 minutes. This process can alter the fine

structure of cellulose by breaking down lignin and hemicelluloses, which in turn enhances the

enzymatic accessibility of lignocellulosic materials.

One of the critical advantages of the above-mentioned method is the possibility of reaching a very

short reaction time without hot spots in a mixture. Microwave-assisted pretreatment of biomass

has a great potential in saving time and energy while the inhibitor generation can be brought to a

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minimum. Among all techniques for changing the native structure of cellulose, namely for a partial

depolymerization of lignin and hemi-celluloses, this technique is one of the most promising

techniques for increasing the susceptibility of the material to enzymatic procedures (Lu et al.,

2011).

Biological pretreatment

Biological methods rely on the action of ligninolytic microorganisms-special bacteria and fungi,

or their enzymes-through which the recalcitrant nature of lignocellulosic biomass is depolymerized

into forms that are more easily hydrolyzed, hence easier to convert into ethanol.

Biological pre-treatment of lignocellulosic biomass generally uses microorganisms that degrade

cellulose and hemicellulose. Filamentous fungi represent the most commonly used organisms for

the purpose in question due to their wide distribution in nature and the possibility of their isolation

from soil, living vegetation, and waste materials of lignocellulosic origin (Vats et al., 2014).

Especially white-rot fungi are thought to be the most effective organisms in lignin degradation,

mainly due to their peculiar enzymatic machinery. The strains usually used in industrial

applications include Phanerochaete chrysosporium, Trametes versicolor, Ceriporiopsis

subvermispora, and Pleurotus ostreatus. Some bacterial strains like Clostridium, Cellulomonas,

Bacillus, Thermomonospora, and Streptomyces have also been reported to be involved in

biological pretreatment of biomass. Efficiency of these biological pretreatments depends upon the

type of microorganism employed, size of biomass particles, along with other operational

parameters like moisture, temperature, and residence time of treatment. The advantages of

biological pretreatment methods are low energy input, no addition of chemicals, lower recycling

costs, lower cost for the downstream processing, less inhibitors formation, easy operation, and

environmental-friendly. The disadvantages are that it normally requires high area, is relatively

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slow, and microbial culture and its activity have to be monitored continuously (Vasco-Correa et

al., 2016).

Biological pretreatment technique also has an added advantage since it does not produce any

unwanted byproducts. It also does not involve the use of high pressure, acids and alkalis, high

temperatures and other reactive agents (Zhang et al., 2011).

Chemical pretreatment

Chemical pretreatment process involves the use of a range of substances such as bases, acids,

gases, salts, ionic liquids, oxidizing agents, or organic solvents. This process is designed to break

down polysaccharides within the lignocellulosic structure, thereby enhancing their accessibility

for enzymatic hydrolysis (Safarian & Unnthorsson, 2018).

Among various techniques applied in the process of overcoming the recalcitrance of

lignocellulosic biomass in the production of bioethanol, acid pre-treatment is considered one of

the most commonly adopted methods. This includes the treatment of biomass with the help of

aqueous solutions of dilute strength, using hydrochloric acid (HCl) and sulfuric acid (H2SO4) as

the most commonly used mineral acids, usually under pressure conditions in the range of 1.5 bar

and temperatures between 100 °C and 290 °C, for a certain period of time that may extend to

several hours. The key parameters involved include acid concentration, solids loading,

temperature, and residence time. Dilute acids are highly effective under heat. Acid pre-treatment

mainly attacks the polysaccharide and has a slight effect on lignin; hence, hemicelluloses are

dissolved and the linkages between the polysaccharides and lignin are broken, thus increasing the

accessibility of cellulose to enzymatic attack (Rezania et al., 2020). Acid pretreatment of biomass

can be done in a somewhat economic way because H2SO4 and HCl are rather inexpensive

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compared to other acids. The process runs under high temperatures and thus high energy input is

needed, which can be quite costly. Besides that, under high temperatures, acids become corrosive,

so reaction vessels to bear such high temperatures must be used. Moreover, during the treatment

process, inhibitors are formed and have to be subsequently removed (Maurya et al., 2015).

Alkaline treatment is generally done with alkali agents such as sodium, potassium, calcium, and

ammonium hydroxides under conditions of ordinary temperature and pressure. Among the

advantages of this treatment are the high efficiency in the removal of lignin from the biomass. This

treatment also solubilizes the acetyl and uronic acid groups present in the hemicelluloses,

improving accessibility by the enzymes responsible for the degradation of hemicellulose. In

general, dilute solutions of sodium hydroxide, potassium hydroxide, calcium hydroxide, or

ammonia are commonly used in alkaline processing to disrupt and remove lignin along with some

hemicelluloses, increasing the accessibility of cellulose to enzymatic hydrolysis. Such action

causes the breaking of bonds between hemicellulose and other polymers, hence leading to swelling

of the fibrous cellulose and an increase in biomass porosity. This process can be carried out at

elevated temperatures for a short period or at lower temperatures for long times. The primary

disadvantage is that the reaction times can be significantly longer, ranging from several hours to a

full day, compared to alternative pretreatment techniques (Mood et al., 2014).

Other pre-treatment techniques that further developed included those involving organic solvents,

ionic liquids, and deep eutectic solvents. Chemicals trailed in this category trailed include but are

not limited to the following: acetone, ethanol, ethylene glycol, glycerol, methanol, n-butanol,

phenol, tetrahydrofurfuryl alcohol, and triethylene glycol (Mood et al., 2014).

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Physico-chemical pretreatment process

Physico-chemical methods use physical and chemical means; for instance, high temperature and

pressure are involved in a series of methods that effectively pretreat the lignocellulosic biomass

for further processing. Among several physico-chemical methods, steam explosion is counted

among one of the most prominent methods for bioethanol production. The process starts with the

exposure of biomass at a high temperature ranging from 160 to 260 °C with pressures ranging

from 0.7 to 4.8 MPa for a few seconds to a maximum of some minutes. The sudden drop in pressure

leads to explosive decompression of the material that raptures the cell wall structure and thus aids

in the solubilization of hemicellulose and lignin.

Among the main advantages of steam explosion, there is a large width in the biomass types this

technology operates with, independent of particle size and without previous crashing, resulting in

good yields and performances. Other positive features of steam explosion are low energy

consumption, no additional chemical costs (no recycling required), and it is environmentally

friendly. However, there are some drawbacks: this method is incapable of removing lignin

completely and may result in the formation of toxic by-products.

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The production of bioethanol from lignocellulosic biomass materials requires a lot of complex

processes after pretreatment before the final product can be finally obtained. These processes

include: detoxification, hydrolysis, fermentation, distillation, and dehydration

[Link] Detoxification

Detoxification refers to the removal of any toxic substances from previously treated biomass or

hydrolysates. This includes fermentation inhibitors such as furan aldehydes, aliphatic acids, and

phenolic compounds that may impede enzyme activities and stunt microbial growth and activity

during fermentation. It is interesting to note that, at present, several different types of in situ

techniques are directed towards trapping these inhibitors for the enhancement of bioethanol

fermentation yield and productivity. Some of these methods are membrane extraction, solvent

extraction, ion exchange, membrane bioreactors, adsorption, microbial adaptation, use of

microbial consortia or genetically modified microorganisms, and many others. There is an

increasing tendency towards specificity in the choice of technique, dictated mainly by the

pretreatment, hydrolysis, and fermentation processes adopted for ethanol production.

Detoxification can as well be a standalone process apart from its being integrated into conditions

of hydrolysis and fermentation (Robak & Balcerek, 2020).

Eliminating substances that hinder the process, such as furfural and acetic acid, significantly

enhances the effectiveness and practicality of producing bioethanol from lignocellulosic biomass.

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Extractive detoxification

Liquid-liquid extraction is indeed one of the techniques which is worthwhile for isolation of the

inhibitors because it is very safe, easy to implement, and cost-effective. Furthermore, this method

enables one to recover the inhibitors from the solvent of extraction, making it very much in tune

with the principles of bio-refinery (Kumar et al., 2020).

Membrane technology

Membrane techniques, such as nanofiltration (NF) and reverse osmosis (RO), can concentrate

fermentable sugar. Moreover, such methods eliminate unwanted by-products, such as furfural and

acetic acid, produced during the bioethanol production process (Pan et al., 2019).

[Link] Enzymatic hydrolysis

Once the pretreatment phase is finished, the raw materials undergo enzymatic hydrolysis. This step

is important for breaking down polysaccharides found in the pretreated lignocellulosic biomass

into fermentable sugars, including pentoses and hexoses. While enzymes primarily facilitate the

breakdown of cellulose and hemicellulose (specifically xylan), acids and alkalis can also be

utilized to achieve this hydrolysis (Tsai & Meyer, 2014). Cellulases and hemicellulases,

particularly xylanases, are categorized as glycoside hydrolases that facilitate the degradation of

carbohydrates, specifically in cellulose and hemicellulose, including xylan. The term "cellulase"

generally describes a group of enzymes, also known as exocellulase, endocellulase, and β-

glucosidase, which act together in cellulose decomposition to produce glucose as the end product

(Zhao et al., 2019).

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Cellulase are enzymes, which hydrolyze cellulose, and are produced from a wide range of sources

such as microorganisms–bacteria and fungi alike. They can be intrinsically produced by sources

as diverse as aerobic and anaerobic, mesophilic and thermophilic organisms. The most notable

genera of bacteria that produce cellulases are, among others, Acetovibrio, Clostridium,

Cellulomonas, Cellvibrio, Bacillus, Bacteroides, Erwinia, Ruminococcus, and many species of the

Streptomyces and Actinomycetales—like Microbispora and Thermomonospora.

In the kingdom of fungi, the most important producers of cellulase are species such as Sclerotium

rolfsii and Phanerochaete chrysosporium. There are also a number of genera involved in the

production of cellulase, including but not limited to Aspergillus, Caecomyces, Humicola,

Neocallimastix, Oprinomyces, Penicillium, Schizophyllum, and Trichoderma. These fungal

species are especially valued in industrial settings because they can excrete large amounts of

cellulases into their growth medium, thereby useful in several biotechnological processes

(Singhania et al., 2017).

Hemicelluloses constitute approximately 10-30% of the lignocellulosic biomass and lay a crucial

foundation for the production of bioethanol by being converted into fermentable sugars. Because

of the amorphous nature of hemicellulose, its hydrolysis in general proceeds more easily than that

of cellulose owing to better accessibility. However, the complex composition and structure of

hemicellulose-like many side chains of different sugar types-call for a high degree of enzyme

development.

Two types of enzymes are mainly needed for the efficient hydrolysis of hemicellulose:

depolymerizing core enzymes that cut through the main chain and debranching-auxiliary enzymes-

which remove side chains that may interfere with the core enzymes. These enzymes improve the

yields of fermentable sugars originating from lignocellulosic biomass.

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They are microorganisms that play a great role in the degradation of hemicellulose. They do the

same for cellulose. There are so many fungi and bacteria that produce enzymes for this kind of

hemicellulose degradation. The most common fungi that secrete enzymes for degradation include

Aspergillus niger, Aspergillus awamori, Trichoderma reesei, and Penicillium wortmanii, and these

groups also include genera like Cochliobacillus, Agaricus, and Sclerotium. On the bacterial side,

the most well-known important representatives are Thermotoga maritima, Clostridium

thermocellum, C. cellulovorans, and Thermobacillus xylanilyticus. Chemolithotropic soil

microorganisms comprise important components for the hydrolysis of hemicellulose, in fact, using

certain enzymes for doing so. Hemicellulose is the complex carbohydrate of plant cell walls. These

activities are responsible for the catalyzing of hemicelluloses to simple sugars that are really source

of energy for the microorganisms themselves or other organisms using these sugars. Such variety

of species does further complicate the whole process of hemicellulose degradation and reinforces

the significance of the diverse ecological and industrial scenarios (Arfi et al., 2014).

When it comes to the hydrolysis of biomass materials, there are few necessary factors that are

taken into account. These factors consist of amount of solid material introduced, resulting sugar

concentration produced, enzyme loading, shaking speed, time duration for hydrolysis, any existing

inhibitors, and how additions affect the reaction. All of these factors significantly affect the

efficiency and output of the hydrolysis process (Zhang et al., 2018).

Solid loading

Boosting the amount of biomass during enzymatic hydrolysis plays a crucial role in producing

sugar syrups with higher concentrations, typically ranging from 80 to 100 grams per liter. To make

the distillation process economically feasible, it’s essential to carry out enzymatic hydrolysis with

solid contents exceeding 15% by weight. This approach is particularly effective when the

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fermentation broth has an ethanol concentration greater than 4% by weight (Farzad et al., 2017).

Studies show that sugar production increases with substrate loading to a certain limit and then

decreases beyond that threshold. Because the levels of cellobiose and glucose are high, these

sugars may inhibit the efficacy of the enzymes. Solid concentrations above an optimum level

usually lead to a viscous broth, which becomes difficult to mix, disrupting mass and heat transfer,

and the resultant hindrance to enzyme performance and overall process efficiency (Du et al., 2014).

Shaking speed

Selecting the correct frequency of shaking or mixing becomes very important contributing factor

in most efficient heat and mass transfer, which result in higher glucose yields. If mixing speed is

too low, inadequate mixing occurs, resulting in decreased monosugar yields. Extremely high

speeds can create shearing forces that lead to damage to the involved enzymes in the process,

making it necessary to balance these speeds for maximum efficiency and output (Vasić et al.,

2021).

[Link] Fermentation

The first and foremost thing in the manufacture of bioethanol from lignocellulosic biomass is the

sugars. Both hexoses-glucose, fructose, sucrose-as well as many pentoses-xylose, mannose,

galactose, and arabinose-are fermentable into ethanol. By fermentation, a certain amount of

ethanol with co-production of carbon dioxide can be produced (Wirawan et al., 2020).

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Fermentation of glucose can be effectively done using resilient industrial host strains namely

Zymomonas mobilis and Saccharomyces cerevisiae. Such organisms boast and exhibit high rates

in production of ethanol, yields, and capability to survive on very high concentrations of ethanol

(about 120 g/l). However, these conventional ethanol fermenters were not designed specifically to

utilize pentose sugars (Mussatto et al., 2014).

With the reality that there are very few native microorganisms that can simultaneously ferment

both pentose and hexose, more attention has shifted to engineering the metabolism to develop

unique organisms with advantageous features. Microbial engineering entails the study and

modification of metabolic pathways and genetic alterations to develop new improved recombinant

microorganisms. Such an engineered organism is expected to ferment all available sugars from

biomass; resist stressful conditions; possess enhanced tolerance to inhibitors; and produce a

combination of synergistic enzymes necessary for the complete hydrolysis of substrates and

carbohydrates from lignocellulosic materials (Adegboye et al., 2021). The alternative method of

improving fermentation efficacy involves the application of immobilized recombinant microbial

cells. Essentially, the entire cells are captured by an appropriate support and thus held by a number

of techniques such as entrapment within a porous structure, adsorption onto the surface of a solid

or solid material, covalent attachment to the carrier surface, cross-linking, or even encapsulation.

This method is such that the optimum catalytic function of the cells remains undisturbed (Karagoz

et al., 2019).

There are several factors that influence in the success of monosaccharide fermentation derived

from lignocellulosic biomass for bioethanol production. Such variables include temperature, time,

pH, inoculum size, sugar concentration, solid-liquid ratio, stirring speed, oxygen content, and

rotating speed. In addition, specific operational parameters must be decided depending on whether

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saccharification occurs simultaneously or separate from the fermentation. This is difficult and

requires detailed optimization.

[Link] Purification (Distillation and dehydration)

Distillation and dehydration are the most important sections of the production process for fuel-

grade ethanol from lignocellulosic biomass. The basis of distillation is a series of selective

evaporation and condensation steps, which allows liquor mixtures such as fermentation broth to

be converted efficiently into ethanol. Separation relies on the fact that different volatilities exist

amongst the components needed in the process (Li et al., 2018).

The fermented slurry typically contains more than 80% water based on its dry weight. This fact

makes it costly and, more importantly, entailing enormous energy input to achieve a concentration

of 96% ethanol. The first step could, in general, be referred to as "the removal of alcohol." The

end product of this process, which comprises about 37% bioethanol, is purified in a rectification

column up to about 95% concentration. Finally, the dehydration process is done to obtain a high-

grade product of more than 99.5% volume ethanol (Robak & Balcerek, 2020).

A range of techniques has been established for isolating ethanol from fermentation broths in the

production of bioethanol. These methods include adsorption distillation, membrane technologies,

azeotropic distillation, diffusion distillation, extractive distillation, pervaporation, vacuum

distillation, and chemical dehydration. Each method varies in terms of the processes used,

efficiency, and associated operational expenses. With membrane distillation and pervaporation

emerging as the most economically viable method for the production of ethanol.

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3.5 Simulation of Bioethanol Production Process

The simulation of bioethanol production from sugarcane bagasse was conducted with Aspen Plus

V11. Non-Random Two-Liquid (NRTL) thermodynamic model was selected due to its ability to

handle non-ideal liquid mixture, particularly those involving polar components and electrolytes

such as water, ethanol and acetic acid. The feedstock (sugarcane bagasse) was modeled as a non-

conventional component using the proximate and ultimate analysis data obtained from Idaho

National Laboratory Bioenergy Feedstock Library ([Link] Other

components in the simulation are; cellulose, hemicellulose, lignin, enzyme (cellulase), sulphuric

acid, sodium hydroxide, sodium sulphate, glucose, xylose, cellobiose, furfural, acetic acid, water,

carbon dioxide and ethanol, which were all defined as conventional components. The last

component is Zymomonas Mobilis (Z-mobilis) which was modeled as a solid component using the

data obtained from (Wooley & Putsche, 1996).

The process began in the washer where the feed (sugarcane bagasse) at 21500 kg/hr was washed

to remove impurities. The washed bagasse was then transferred to the dryer where the moisture

was removed to improve the downstream processing efficiency. Next, the dried bagasse was sent

to the crusher for particle size reduction so that it can be easily mixed with water to form a slurry

and also to increase the surface area for subsequent reactions. In the mixer, water was added to

the crushed bagasse to form slurry. The slurry was preheated in the heater which then served as

the input for decomposition.

Decomposition occurred in the reactor 1 (RYield) where the bagasse was broken down into its

primary components; cellulose, hemicellulose and lignin. The decomposed bagasse was first

cooled in cooler1 before it was sent to reactor 2 (RStoic) where it was treated with sulphuric acid

which hydrolyzed some of the hemicellulose into fermentable sugars, primarily xylose. The acidic

90
mixture was neutralized with sodium hydroxide in reactor 3 (RStoic) to achieve a suitable pH for

enzymatic hydrolysis.

The neutralized mixture was cooled in cooler 2 to the optimal temperature required for enzymatic

hydrolysis. In the reactor 4 (RStoic), cellulase (enzyme) and fresh water were introduced to break

down cellulose and hemicellulose into glucose and xylose respectively. The resulting sugar

mixture was fermented in reactor 5 (RStoic), where Z-mobilis converted the sugars into ethanol

and carbon dioxide (CO2).

The fermentation product was purified in two consecutive distillation columns. The first

distillation was conducted in col 1 (RadFrac) followed by further distillation in col 2 (RadFrac).

Residual CO2 was removed in a flash separator (Sep), and the ethanol was dehydrated in col 3

(Sep2) to eliminate the remaining water.

At the end of the simulation, a production rate of 6445.57 kg/hr bioethanol with 99.72% purity

was achieved.

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Figure 3.1 Aspen Plus simulation flowsheet of bioethanol production

92