CHAPTER THREE
3.1 Introduction
Lignocellulosic biomass, primarily composed of cellulose, hemicellulose, and lignin, constitutes
a significant portion of agricultural waste. The efficient extraction of these natural components
ensures their utilization in biofuels, biochemicals, nano plastics, and other valuable products. The
extraction of lignin and cellulose from agricultural biomass, such as peanut shells, has gained
significant attention due to the increasing demand for sustainable and renewable resources.
However, it is important that this extraction should be environmentally friendly and relatively
cheap. Traditional extraction methods, including acid, alkali, and organic solvent treatments, had
been widely employed; however, they often presented several drawbacks, such as high energy
consumption, environmental toxicity, and the generation of hazardous by-products (Silveira et
al., 2015; Yoshida et al., 2019).
In this research work, three major extraction and treatment methods were synergistically
combined to obtain an optimal extraction of lignin and cellulose from peanut shells: steam
explosion treatment, DES (Deep Eutectic Solvent) method and enzymatic method.
The justifications for employing these extraction methods are highlighted below:
3.2 Overview of the Deep Eutectic Solvent Method
The DES method, first recognized in 2003, involved the use of a mixture of hydrogen bond
donors (HBDs) and hydrogen bond acceptors (HBAs) that formed a eutectic mixture with a
significantly lower melting point than the individual components. This unique property allowed
DESs to operate at lower temperatures, thus minimizing thermal degradation of biomass
components (Malaeke et al., 2020). DESs could be easily synthesized from natural and
inexpensive compounds, making them an attractive option for large-scale applications.
Environmental Friendliness of DES
One of the primary benefits of the DES method is its environmental friendliness. Unlike
conventional organic solvents, which are often volatile and toxic, DES exhibits low volatility,
non-flammability, and low toxicity (Hu et al., 2023). This characteristic has no negative
environmental impact associated with biomass extraction processes.
Extraction Efficiency of DES
In terms of extraction efficiency, the DES method demonstrates superior performance compared
to traditional methods. Research showed that DESs could effectively solubilize lignin and
cellulose by disrupting the hydrogen bonds and covalent linkages that bound these components
together. Hu et al. proved in their study that DESs composed of choline chloride and various
HBDs, such as urea or glycerol, achieved lignin extraction rates of up to 90% from peanut shells
(Chen and Sui, 2016; Hu et al., 2023). In contrast, traditional methods often yielded lower
extraction efficiencies due to the recalcitrance of lignocellulosic structures.
Versatility of DES
Another significant advantage of the DES method is its versatility. The composition of DESs
could be tailored to optimize extraction conditions for specific biomass types. By adjusting the
molar ratio of HBA to HBD and selecting appropriate components, researchers could enhance
the solvation power of the solvent, thereby improving lignin and cellulose recovery rates
(Muzamal et al., 2017). This flexibility was particularly beneficial when dealing with diverse
agricultural biomass sources, as it allowed for the development of customized extraction
protocols.
Improved Selectivity in Extraction
According to Li et al. 2023, the DES method also offered improved selectivity in the extraction
of lignin and cellulose. Traditional extraction methods often resulted in the co-extraction of
unwanted components, leading to complex mixtures that required additional purification steps. In
contrast, DES can selectively target lignin based on their solvation characteristics, facilitating the
separation of desired products from undesired impurities (Li et al., 2023). This selectivity not
only streamlines the extraction process, but also enhances the purity of the final products.
Cost-Effectiveness of DES
In addition to these benefits, the DES method is cost-effective. The use of natural and
inexpensive materials for DES synthesis, the lower energy requirements for DES extraction,
coupled with the potential for solvent recovery and reuse, all contributes to the economic
feasibility of this method (Zhang et al., 2023). Studies demonstrated that DESs could be recycled
multiple times without significant loss of extraction efficiency, further enhancing their cost-
effectiveness.
3.3 Comparison with Acid and Alkaline Extraction Methods
Acid and alkali treatments, while effective in lignin and cellulose extraction, often lead to the
generation of inhibitory by-products that could hinder downstream processing. For instance, acid
hydrolysis could produce furfural and other degradation products that inhibits fermentation
processes in biofuel production (Auxenfans et al., 2017). The DES method, on the other hand,
minimized the formation of such by-products, promoting a cleaner extraction process.
Additionally, many organic solvents, such as methanol and acetone, pose risks to both human
health and the environment due to their toxicity and volatility. The DES method, with its low
toxicity and biodegradability, provides a safer alternative that aligns with the growing emphasis
on sustainable practices in biomass processing (Muzamal et al., 2016).
Deep Eutectic Solvents Acid Hydrolysis Alkaline Hydrolysis Organic Solvents
(DES)
ADVANTAGES ADVANTAGES ADVANTAGES ADVANTAGES
1. Environmentally 1. Effective in 1. Effective lignin 1. Effective in
friendly and breaking removal and extracting
biodegradable. down cellulose specific
2. Low toxicity and lignocellulos- solubilization compounds.
non-flammability. sic structures. 2. Less formation of 2. High solubility
3. High extraction 2. High yields inhibitory by- for certain
efficiency (up to of products biomolecules.
90% lignin fermentable compared to the DISADVANTAGES
recovery). sugars. acid method. 1. Toxicity and
4. Versatile solvent DISADVANTAGES DISADVANTAGES volatility raise
composition can be 1. Generates 1. High energy and environmental
tailored for specific inhibitory chemical costs. and health
biomass. by-products 2. Environmental concerns.
5. Improved (e.g., concerns related 2. Requires
selectivity reduces furfural). to chemical careful
purification needs. 2. High energy disposal. handling and
6. Cost-effective due consumption 3. Potential disposal.
to natural and corrosion degradation of 3. Often leads to
components and of cellulose complex
solvent equipment. structure. mixtures
recyclability. 3. Requires needing
DISADVANTAGES neutralization further
1. Limited research on and purification.
large-scale additional
applications. purification
2. Potential variability steps.
in extraction
efficiency
depending on
biomass type and
DES composition.
Table 3.1 Comparison of the Different Extraction Methods
3.4 EXPERIMENTATION
Method for Extracting Lignin and Cellulose from Peanut Shells Using Deep Eutectic
Solvent
Step 1: Preparation of Peanut Shells
Peanut shells were collected, washed and dried at room temperature to reduce moisture content
as excess moisture can interfere with the solvation capacity of the DES (Li et al., 2022).
Step 2: Steam Explosion Treatment
The peanut shells were dried and ground to a uniform particle size to increase surface area and
enhance the efficiency of steam explosion. The ground biomass was subjected to high-pressure
steam treatment at a temperature of 160°C for 5 minutes. Following the steam treatment, the
reactor was rapidly depressurized to atmospheric pressure, leading to explosive decompression.
This step created shear forces that fragmented the biomass, enhancing the accessibility of
cellulose and lignin (Auxenfans et al., 2017).
Step 3: Synthesis of Deep Eutectic Solvent
DES was prepared by mixing choline chloride (HBA) with urea (HBD) in a 1:2 molar ratio, and
was heated until a homogenous clear liquid was formed. The pre-treated peanut shell from the
steam explosion was then soaked in the prepared DES solution at a solid-to-liquid ratio of 1:10
(w/v), at a controlled temperature of 70°C for 3 hours. The mixture was stirred continuously to
enhance mass transfer and solubilization of lignin.
Step 4: Separation of Lignin and Cellulose
The mixture was then filtered to separate the solid residue from the liquid phase containing
dissolved lignin. The solid residue was washed with distilled water to recover any residual DES,
and the solid was dried to obtain cellulose which was to be subjected to enzymatic treatment to
obtain high purity cellulose. On the other hand, the filtrate was collected and subjected to
centrifugation at 5000 rpm for 10 minutes to separate the dissolved components (impurities)
based on their densities (Malaeke et al., 2018).
Step 5: Recovery of the Deep Eutectic Solvent
The liquid containing lignin was then pushed to the rotary evaporator at 60°C to remove the
DES. This step was crucial to recover the solvent for potential reuse in subsequent extraction
processes, and to efficiently separate the dissolved lignin from the deep eutectic solvent mixture.
Step 6: Enzymatic Hydrolysis
After the primary treatments have removed the bulk of lignin from the biomass, an unknown
portion of hemicellulose still remained within the cellulose matrix. The presence of
hemicellulose—an amorphous, branched polymer of various sugars—could hinder the purity and
quality of the extracted cellulose. Hence a specialized enzyme treatment using hemicellulases, a
group of enzymes specifically tailored to break down the complex structures of hemicellulose
without affecting the cellulose fibers, was applied.
A hemicellulase solution was prepared in a buffer, typically at a pH of around 4.5-6.0, which is
optimal for the enzyme’s activity. The hemicellulase solution was then added to the washed
cellulose material obtained from step 4. The mixture was incubated overnight at 40°C, during
which the hemicellulase enzymes selectively targeted hemicellulose, breaking it down into small,
water-soluble sugar molecules.
When the enzymatic hydrolysis was complete, the mixture was filtered to separate the cellulose-
rich solid from the dissolved sugars. The solid cellulose is then thoroughly washed with distilled
water to ensure all soluble sugar fragments are removed.
Step 7: Purity Test
The Fourier Transform Infrared Spectroscopy (FTIR) was used to characterize the quality and
purity of the experimentally obtained cellulose and lignin, confirming that the DES treatment
yielded high purity lignin and cellulose, suitable for industrial applications.
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