Open access peer-reviewed chapter

Potential of Weed Biomass for Bioethanol Production

Written By

Siripong Premjet

Submitted: February 5th, 2018 Reviewed: April 23rd, 2018 Published: November 5th, 2018

DOI: 10.5772/intechopen.77507

Chapter metrics overview

1,349 Chapter Downloads

View Full Metrics


Lignocellulosic biomass from weedy plants represents a potential alternative feedstock for economic production of bioethanol. Large numbers of weedy plant species are growing all over the world. Characteristics such as high dry matter yield, low water and nutrient requirements for growth, and cellulose contents make weedy plants very attractive as feedstock for bioethanol production. However, like other lignocellulosic feedstock, the complex structure presents resistance and recalcitrance to processes of conversion to bioethanol. Several weedy plants have been studied to determine their physical characteristics and suitability for bioethanol production. Different conversion techniques have been employed to increase monomer sugars and hence bioethanol yield. This chapter discusses processes and current research activities in bioconversion of weed biomass to bioethanol.


  • bioethanol
  • fermentation
  • lignocellulosic biomass
  • pretreatment
  • weedy plants

1. Introduction

Rapid economic and population growth have resulted in drastic increase in energy consumption especially in the transportation sector. To meet growing demand for fuel energy, most countries around the world depend heavily on imported petroleum fuel [1]. However, concerns have been raised about gradual depletion of fossil fuels and environmental pollution as a result of its combustion [2]. This has necessitated the search of alternative sustainable and eco-friendly source(s) of fuel energy. As part of the search, many governments worldwide are promoting the use of biofuels such as bioethanol and biodiesel as alternative transportation fuel [3].

Bioethanol is currently the most widely used liquid biofuel [4]. It is an eco-friendly and renewable fuel produced from plant-based starches and sugars [5]. Global production of bioethanol is mainly from food-related crops such as corn, cassava, sugarcane, rice, and sweet potatoes [3]. However, these feedstock are directly consumed by humans as food or as animal feed. Continuous use of these crops for bioethanol production may put pressure on productive agricultural lands and result in higher food prices [6]. Concerns about sustainability of bioethanol production from food-related crops have raised attention to the potential of lignocellulosic biomass for bioethanol production [7].

Lignocellulosic biomass is inexpensive and abundant worldwide. It includes agricultural and forestry waste, grasses, and other nonfood plants [8]. This type of biomass is a rich source of biopolymers, chemicals, and sugars [9]. Current research into bioethanol production is mainly focused on assessing the potential of nonfood crops as feedstock and improving the efficiency of their conversion [10]. Lignocellulosic biomass from invasive weeds is a good feedstock for the economic production of bioethanol [2]. These weedy cellulosic substrates do not need extra expenses as they grow on agriculturally degraded land or water bodies [11]. Large numbers of such invasive species are found all over the world. The potential of weed biomass for the production of bioethanol has been explored and discussed in this chapter.


2. Lignocellulosic biomass from weedy plants: chemical composition and potential for bioethanol production

The major components of lignocellulosic biomass are cellulose, hemicellulose, and lignin. Cellulose and hemicellulose are the main carbohydrates in lignocellulosic biomass. The contents of these components vary significantly depending on the type of biomass and source [6]. Cellulose is a crystalline and linear structure made up of units of glucose strongly linked together by β-1-4-glycosidic bonds. These linkages give cellulose very high crystalline structure making it resistant to degradation. It is the most abundant organic polymer on earth. Hemicellulose on the other hand, consists of linear and highly branched mixture of pentoses (xylose and arabinose) and hexoses (glucose, galactose, and mannose). Lignin is a highly branched polyphenolic polymer, which gives stability to biomass structure [12]. Cellulose and hemicellulose, the major substrates for bioethanol production, form the main components of the total dry weight of lignocellulosic biomass [7]. These fractions are linked together by covalent and hydrogen bonds, which are further strongly bonded to lignin. This gives lignocellulosic biomass a very complex structure, which is very resistant to degradation. Digestibility of lignocellulosic biomass is therefore affected by the degree of complexity and composition [11]. The structure and composition of different lignocellulosic biomass differ and this greatly affects the efficiency of their conversion to bioethanol.

Lignocellulosic biomass from weedy plants is one of the most sustainable alternative feedstock for bioethanol production [12]. Annual and perennial weedy plants are found all over the world at all seasons. They invade large areas of land and water bodies causing environmental and socioeconomic problems [2]. They grow rapidly on marginal lands under extreme conditions such as drought, low nutrient and high temperatures, hence requiring no additional economic input such as fertilizer and pesticides [7]. Weed biomass contains large amounts of chemicals and materials, which can be extracted for several industrial applications [13]. These plants have been reported to produce high dry matter yield and contain high and low percentages of cellulose and lignin contents, respectively [14]. The high dry matter yield and cellulose contents of weedy plant species make them ideal feedstock for bioethanol production. They also have an added advantage as feedstock for bioethanol production since they do not compete with food crops for productive agricultural lands [15]. Moreover, due to seasonal nature of agricultural wastes, lignocellulosic biomass from weed species is very important in ensuring continuous production of bioethanol throughout the year [16]. A wide range of weedy species are grown naturally on marginal lands all over the world that can be used as feedstock for bioethanol production. Perennial grasses and short rotation forest plants are among these weedy species growing worldwide [17]. The possibility of converting biomass from invasive weeds to fuel bioethanol is currently an area of great research interest around the world. The physical characteristics and bioethanol production potential of several weedy species have been studied.

Parthenium hysterophorus, a common invasive weed species was studied in India as a potential feedstock for bioethanol production. Chemical composition analysis of this weed species revealed 53.63% holocellulose and 10.44% lignin contents, making it an attractive feedstock for production of bioethanol [18]. Cannabis sativa, a versatile weedy plant, grows naturally in large areas in Pakistan. It produces large amount of biomass due to its rapid growth rate. Cannabis sativacontains 55% cellulose and only 5% lignin. It has been reported as a potential cheap and eco-friendly feedstock for bioethanol production in Pakistan [19]. Pennisetum purpureum, commonly known as Napier grass or elephant grass, Vetiveria zizanioidesalso known as vetiver grass, Digitaria decumbens, Paspalum atratum, Cynodonsp., and Pennisetum polystachyonare all weedy species found in Asia that have been studied and proposed as feedstock for bioethanol production [12].

In an earlier research, different types of weedy plants were identified in six provinces in lower Northern Thailand (Table 1). Majority of these weed biomass were found to contain high cellulose but low lignin contents. The cellulose contents of most of these weed biomass is higher or similar compared to well-known lignocellulosic materials from agricultural residues including corn stalk bagasse (43.4%) [20], corncob (31.5 ± 1.2%) [21], wheat straw (35.2 ± 0.3%) [22], paddy straw (32.6%) [23], soybean straw (34.40%) [24], and sugarcane bagasse (27.3%) [25]. High theoretical bioethanol yields were also estimated for these weed biomass based on the contents of cellulose and hemicellulose. Bioethanol yield of between 548.4 ± 1.4 and 394.0 ± 5.3 L/ton was realized from some of the weed species [14]. Majority of these weed species are potential substrate for bioethanol production.

Scientific nameCellulose (%)Hemicellulose (%)Lignin (%)Ash (%)EtOH TY (L/Ton)
Imperata cylindrica44.4 ± 0.131.1 ± 0.06.7 ± 0.06.9 ± 0.0548.4 ± 1.4
Amaranthus viridis37.4 ± 0.134.2 ± 0.05.1 ± 0.116.9 ± 0.2521.0 ± 0.9
Sida acuta56.0 ± 0.316.0 ± 0.46.8 ± 0.15.1 ± 0.1520.3 ± 5.4
Rottboellia cochinchinensis41.6 ± 0.728.6 ± 0.47.5 ± 0.110.5 ± 0.3509.7 ± 8.1
Sorghum halepense44.4 ± 0.125.8 ± 0.26.6 ± 0.58.8 ± 0.3508.8 ± 2.6
Eragrostis amabilis39.7 ± 0.429.6 ± 0.27.2 ± 0.25.5 ± 0.1502.9 ± 4.7
Cyperus imbricatus35.6 ± 0.132.3 ± 0.34.7 ± 0.37.4 ± 0.1493.6 ± 3.2
Cenchrus echinatus35.8 ± 0.631.8 ± 0.46.3 ± 0.313.9 ± 0.2491.4 ± 7.4
Cyathula prostrata50.0 ± 0.317.0 ± 0.310.9 ± 0.110.3 ± 0.1484.8 ± 4.4
Eriochloa procera37.0 ± 0.029.5 ± 0.15.3 ± 0.313.1 ± 0.0483.2 ± 1.1
Brachiaria mutica37.7 ± 0.0128.8 ± 0.85.6 ± 0.810.9 ± 0.2482.8 ± 6.7
Sporobolus indicus35.6 ± 0.029.9 ± 0.16.6 ± 0.09.1 ± 0.3476.2 ± 1.0
Leucaena leucocephala55.2 ± 0.010.1 ± 0.116.1 ± 0.12.6 ± 0.6471.9 ± 1.2
Echinochloa crus-galli34.7 ± 0.230.1 ± 0.14.6 ± 0.08.9 ± 0.5470.8 ± 2.0
Cyperus iria33.4 ± 0.231.0 ± 0.06.3 ± 0.05.4 ± 0.1468.9 ± 1.3
Typha angustifolia47.1 ± 0.116.9 ± 0.410.0 ± 0.311.3 ± 0.1462.9 ± 3.9
Dactyloctenium aegyptium32.0 ± 0.131.6 ± 0.17.7 ± 0.09.5 ± 0.4462.4 ± 0.3
Achyranthes aspera53.7 ± 0.110.2 ± 0.18.5 ± 0.111.7 ± 0.3461.0 ± 1.5
Pennisetum polystachyon40.0 ± 0.023.3 ± 0.16.2 ± 0.27.5 ± 0.3459.2 ± 0.6
Cyperus compactus32.8 ± 0.329.0 ± 0.84.6 ± 0.511.2 ± 0.1448.9 ± 8.5
Aeschynomene Americana48.3 ± 0.213.4 ± 0.015.4 ± 0.37.4 ± 0.4446.2 ± 1.3
Celosia argentea44.3 ± 0.317.2 ± 0.29.7 ± 0.910.0 ± 0.1445.3 ± 3.2
Dicliptera roxburghiana41.9 ± 0.317.5 ± 0.38.7 ± 0.415.2 ± 0.0429.8 ± 4.3
Crotalaria pallida49.6 ± 0.29.1 ± 0.211.7 ± 0.14.3 ± 0.2423.6 ± 2.7
Scoparia dulcis36.5 ± 0.319.1 ± 0.16.6 ± 0.04.5 ± 0.6402.6 ± 2.9
Urena lobata43.5 ± 0.311.4 ± 0.79.6 ± 0.17.5 ± 0.3396.7 ± 4.4
Cyperus cyperoides29.7 ± 0.624.6 ± 0.210.9 ± 0.68.8 ± 0.1394.0 ± 5.3

Table 1.

Chemical composition and theoretical ethanol yields of weed biomass.

Source: [14] EtOH TY = Theoretical ethanol yield.


3. Biological conversion of weed biomass to bioethanol

Bioethanol is produced from three main renewable resources namely starch, sugars, and lignocellulosic biomass. The production of bioethanol from starch and sugar (first generation bioethanol production) differs significantly from that of lignocellulosic biomass. The process of bioethanol production from sugar-related crops involves direct extraction of sugars followed by fermentation to bioethanol. However, starch carbohydrates are extracted from starch-based crops and hydrolyzed into monomer sugars with subsequent fermentation of sugars to bioethanol [26]. Unlike first generation bioethanol production where carbohydrates are easily converted to bioethanol, carbohydrate portions in weed biomass are not freely available for the conversion to bioethanol. Biological conversion of weed biomass to bioethanol involves various processes (Figure 1). The major steps involved in the conversion process include pretreatment of biomass to make it easily digestible in subsequent processes. The cellulose and hemicellulose contents are then hydrolyzed to monomer sugars followed by the fermentation of sugars to bioethanol. Finally, bioethanol is purified through distillation or other processes such as dehydration to conform to world bioethanol specifications [27].

Figure 1.

Schematic diagram of major steps in weed biomass conversion to bioethanol.

3.1. Pretreatment of weed biomass

Like most lignocellulosic biomass, the recalcitrance of weed biomass is a major problem in their conversion to bioethanol. This is due to the crystalline structure of cellulose coupled with lignin and hemicellulose strongly bonded to each other and serving as a protective cover to cellulose. The pretreatment of weed biomass is thus very important in releasing fermentable sugars for bioethanol production [6]. It helps to break the bond between lignin and hemicellulose, hence destroying the protective cover of cellulose. It also helps to decrease cellulose crystallinity making it more susceptible to enzymatic hydrolysis and fermentation [12]. Different pretreatment methods can be used on various types of weed biomass for bioethanol production. However, the cost of pretreatment, production of inhibitors, type of weed biomass, energy requirements, and efficiency are major factors that need to be considered in the choice of pretreatment method [28]. Pretreatment may be physical, chemical, biological, or a combination of these [29].

3.1.1. Physical pretreatment

Physical pretreatment includes methods aimed at reducing particle size of biomass. These methods consist of mechanical operations such as chipping, milling, and grinding. These processes help to increase the porosity and surface area of biomass to enhance its conversion to bioethanol [9]. Mechanical operations are usually carried out as a preparatory step during the conversion process [12]. Other methods including different kinds of irradiation and ultrasonic pretreatment have been developed to physically enhance accessibility to cellulose during the conversion process. Physical pretreatment, however, requires high amount of energy contributing to high cost of bioethanol production [9].

3.1.2. Chemical pretreatment

Chemical pretreatment is the most common and studied pretreatment method for the conversion of lignocellulosic biomass to bioethanol. Different chemicals including alkali, ionic liquids, organic solvents, oxidizing agents, and acids can be used [30]. Acid pretreatment is one of the most promising methods and has been extensively studied. It mainly results in solubilization of hemicelluloses but less effective in lignin removal [27]. The type of acid, concentration, volume, and pretreatment temperature are some factors that affect the efficiency of this technique [9]. Acid pretreatment may be carried out with either concentrated or dilute acid. However, dilute acid is normally preferred as concentrated acid, which is toxic and corrosive, and results in the production of high levels of inhibitors including furfural derivatives, acetic acid, phenolics, and other aromatic compounds [31]. Pretreatment with acid may be conducted at high temperature for a short time or low temperature for a longer period [32]. Various types of acids including hydrochloric, phosphoric, nitric, oxalic, formic, acetic, and maleic have been studied as chemicals for pretreatment of lignocellulose biomass. Despite its effectiveness, acid pretreatment is toxic and generates inhibitory compounds that negatively affect enzymatic hydrolysis and fermentation processes [9]. It is therefore crucial to remove these compounds, a process that adds to the cost of bioethanol production.

Alkaline pretreatment on the other hand breaks the intermolecular bonds between lignin and hemicelluloses and reduces cellulose crystallinity [33]. During alkaline pretreatment, biomass is treated with alkali chemicals such as sodium, calcium, ammonium, and potassium hydroxides at varying temperatures with or without pressure [5]. Alkaline pretreatment enhances accessibility of enzymes to cellulose by mainly solubilizing lignin contents of biomass. It results in less sugar degradation and produces low inhibitors compared to acid pretreatment [20]. However, alkaline pretreatment results in the production of salts are very difficult to recover [6].

Ozone, a strong oxidizing agent is very effective for the removal of lignin in lignocellulosic biomass. This type of chemical pretreatment is normally done at room temperature and results in no inhibitor formation [30]. Organic solvents such as methanol, ethanol, ethylene glycol, glycerol, acetic acid, formic acid, phenol, and dioxane are also very effective in extracting lignin and hemicellulose [29]. Ionic liquids have been identified as promising solvents for pretreatment because of their ability to dissolve lignin and carbohydrates. A variety of ionic liquids including those containing cholinium cations and linear carboxylate anions have been identified for their ability to enhance digestibility of lignocellulosic biomass. An advantage of ionic liquid is the recovery of separate lignin and carbohydrate fractions after pretreatment. However, ionic liquids are very expensive and can inhibit enzymatic hydrolysis and fermentation processes [34].

3.1.3. Biological pretreatment

Biological pretreatment of lignocellulosic biomass involves using different types of microorganisms including fungi, bacteria, and actinomycetes [9]. These organisms have the ability to produce ligninolytic enzymes such as peroxidases (lignin peroxidase and manganese peroxidase) and laccases. These two groups of enzymes play significant role in lignin degradation during biological pretreatment. The most common microorganism for biological pretreatment is filamentous fungi. White-rot fungi have been identified as the most effective microorganism for the biological pretreatment of lignocellulosic biomass [35]. A number of white-rot fungi including Phanerochaete chrysosporium, Pleurotus ostreatus, Cyathus stercoreus, Pycnoporus cinnabarinus, Ceriporia lacerata, and Ceriporiopsis subvermisporaare able to produce lignin degrading enzymes for the effective delignification of lignocellulosic biomass. Biological pretreatment does not generate toxic substances, is mild, requires low energy, and more environmentally friendly compared to other pretreatment techniques [23]. Nonetheless, the process is very slow and requires carefully controlled conditions as well as large space making it not attractive for commercial bioethanol production. Some microorganisms also tend to degrade cellulose and hemicellulose in addition to lignin [31].

Biological pretreatment may also be carried out with ligninolytic enzyme extracts. This has been reported to prevent degradation of carbohydrates that is associated with microbial pretreatment [31]. These enzymes are extracted from lignin degrading microorganisms, purified and used for the pretreatment process. Crude enzyme extracts have, however, been reported to contain other factors such as proteins and mediators. The presence of these factors enhance the activity of these enzymes making them more effective compared to purified ones. The major problem associated with enzymatic delignification is low enzyme production and activity. Enhancing the culturing conditions may however help to increase the activity and the yield of these enzymes [36].

The effect of pretreatment on biomass varies depending on the method and type of lignocellulosic biomass. Development of effective pretreatment conditions is thus crucial for converting weed biomass to bioethanol. To release monomer sugar units from weed biomass, researchers have studied the effect of different kinds of pretreatment on different types of weed biomass (Table 2). Ratsamee [10] pretreated purple guinea grass (Panicum maximumcv. TD 53) with dilute sulfuric acid (H2SO4) and calcium hydroxide (Ca(OH)2) to improve cellulose digestibility. Pretreatment with the two chemicals resulted in a significantly higher glucose contents in the biomass after enzymatic hydrolysis. However, purple guinea grass biomass pretreated with calcium hydroxide yielded slightly higher glucose concentration after hydrolysis. Wongwatanapaiboon [17] assessed the potential of bioethanol production from different types of grasses by pretreating them with alkaline peroxide (H2O2 + NaOH). Following alkaline peroxide pretreatment and enzymatic hydrolysis with cellulase and xylanase, total reducing sugar in the range of 521–559 mg/g biomass was obtained. Chandel [37] reported maximum total reducing sugar yields of 310 ± 9.80, 541.2 ± 9.53, and 646.23 ± 8.99 mg/g biomass after enzymatic hydrolysis of wild sugarcane (Saccharum spontaneum) biomass pretreated with dilute sulfuric acid (H2SO4), dilute sodium hydroxide (NaOH), and aqueous ammonia (aq. Ammonia), respectively. In an earlier research, pretreatment of Achyranthes asperaand Sida acutawith different concentrations of phosphoric acid (H3PO4) helped to increase glucose concentration (8.0 and 8.6 g/L, respectively) of the biomass after enzymatic hydrolysis with a combination of cellulase and β-glucosidase [38]. Preliminary studies on biological pretreatment of Leucaena leucocephalawith Phanerochaete chrysosporiumalso resulted in an increase in glucose concentration (1.2 g/L) of pretreated biomass after hydrolysis with cellulase enzyme [39]. Borah [2] carried out acid hydrolysis with sulfuric acid (H2SO4) followed by delignification with sodium hydroxide (NaOH) and ultrasound irradiation of five weed species as feedstock for bioethanol production. After enzymatic hydrolysis, the average yield of total fermentable sugars (hexose and pentose) from all five weed species was reported to be 43.85 g/100 g of biomass, representing 27.36 g theoretical bioethanol yield. It can be inferred from Table 2 that the optimum conditions of pretreatment differ significantly for each weed biomass.

Weed biomassPretreatment conditionsEnzymatic hydrolysisSugars after pretreatment/hydrolysisReference
Panicum maximumcv. TD533% H2SO4, autoclave at 121°C for 30 minsAccellerase™ 1000 (9FPU/g)10.1 g/L glucose[10]
4% Ca(OH)2, autoclave at 121°C for 5 mins11.9 g/L glucose
Paspalum atratum7.5% H2O2 + NaOHCellulase (60 U/g) + xylanase (1200 U/g)506 mg/g biomass[17]
Pennisetum purpureum Schum.529 mg/g biomass
Pennisetum purpureum cv. Mott559 mg/g biomass
Pennisetum purpureum × Pennisetum americanum556 mg/g biomass
Saccharum spontaneum1.5% H2SO4 (v/v)Cellulase (15 FPU/g)310 ± 9.80 mg/g biomass[37]
1.0 M NaOHCellulase (25 FPU/g)541.2 ± 9.53 mg/g biomass
15% aq. ammoniaCellulase (25 FPU/g)646.23 ± 8.99 mg/g biomass
Achyranthes aspera80% H3PO4Cellulase (30 FPU/g) + β-glucosidase (60 U/g)8.0 g/L glucose[38]
Sida acuta75% H3PO48.6 g/L glucose
Arundo donax1% (v/v) H2SO4, autoclave at 121°C for 30 mins followed by 1.5% NaOH + ultrasound irradiationCellulase (135 FPU/g) + Cellobiase (75 FPU/g)724.0 mg/g biomass[2]
Saccharum spontaneum851.7 mg/g biomass
Mikania micrantha592.0 mg/g biomass
Lantana camara662.2 mg/g biomass
Eichhornia crassipes758.6 mg/g biomass
Leucaena leucocephalaPhanerochaete chrysosporiumCellulase (30 FPU/g)1.2 g/L glucose[39]

Table 2.

Pretreatment and enzymatic hydrolysis of weed biomass.

3.2. Enzymatic hydrolysis

Pretreatment of lignocellulosic biomass is followed by acid or enzymatic hydrolysis to break down cellulose and sometimes hemicellulose into fermentable sugars such as glucose and xylose [12]. Enzymatic hydrolysis is however eco-friendly and preferred to the noneco-friendly harsh acid hydrolysis [33]. The total amount of fermentable sugars produced is dependent on the type of lignocellulosic biomass and efficiency of pretreatment process [12]. Enzymatic hydrolysis of biomass is carried out in different forms. In some cases, pretreated biomass is initially hydrolyzed by enzymes followed by fermentation of sugars to bioethanol in a process called, separate hydrolysis, and fermentation (SHF). This process requires two separate distinct process conditions for both enzymatic hydrolysis and fermentation. A major setback back to this process is the accumulation of sugar during enzymatic hydrolysis step, which can inhibit enzymatic activities [12]. The production of monomer sugars and fermentation of these sugars may also be carried together in a process known as simultaneous saccharification and fermentation (SSF) [11]. The tendency of monomer sugar accumulation is as less as individual sugars released are converted to bioethanol at the same time. This process may however be very complex with respect to process conditions, which can lead to a decrease in bioethanol yield. Specific operating conditions must therefore be established to enhance both enzymatic hydrolysis and fermentation processes [12]. An emerging method is consolidated bioprocessing (CBP) in which a microorganism or group of microorganisms are used to convert untreated biomass to bioethanol. The microorganism(s) have special inherent abilities to secret enzymes that degrade biomass and ferment sugars released to bioethanol. This method is very promising, however, research activities is still at an infant stage [12].

Cellulase enzymes are used for enzymatic hydrolysis of cellulose after pretreatment. Enzymes for hydrolysis may be obtained from commercial enzyme producers. In some cases, the enzymes may be produced, harvested, and use for hydrolysis. These enzymes are produced by both bacteria and fungi; however, most commercial cellulases are produced from fungi [33]. Cellulases are made up of three set of enzymes including endoglucanase (1,4-β-D-glucan glucanohydrolase, EC, exoglucanase (1,4-β-D-glucan cellobiohydrolyase, EC, and cellobiase (β-glucosidase; EC Endoglucanase cuts cellulose chains into fragments of glucose, cellobiose, and cellotriose while exoglucanase cleaves it into cellobiose units [11]. Cellobiase, however, breaks cellobiose units into glucose that can be fermented to bioethanol. Majority of cellulases obtained from fungi lacks β-glucosidase and must be supplemented with β-glucosidase during enzymatic hydrolysis to enhance efficiency [33]. Cellulase activity is dependent on the concentration and source. Different dosages of cellulases are used during enzymatic hydrolysis. This may depend on the composition of pretreated biomass as well as the type of pretreatment technique used. Enzymatic hydrolysis of cellulose requires mild conditions including pH of between 4.8 and 5.0 and temperature of approximately 50°C. High hydrolysis efficiency is however achieved with an optimized temperature, time, pH, enzyme load, and biomass concentration [4].

The hemicellulose component may also be hydrolyzed with hemicellulases into monomer sugars for fermentation to bioethanol [7]. Compare to cellulose, hemicellulose hydrolysis is very complex because of its composition (mixture of pentoses and hexoses). Multiple enzyme system including endo-xylanase, exo-xylanase, and β-xylosidase together with auxiliary enzymes α-arabinofuranosidase, α-glucuronidase, acetyl xylan esterase, and ferulic acid esterase are involved in hemicellulose hydrolysis [26].

Enzymatic cocktails comprising cellulases and hemicellulases have been used to hydrolyze various pretreated weed biomass for bioethanol production (Table 2).

3.3. Fermentation

Following enzymatic hydrolysis, the supernatant containing various sugars (pentoses and hexoses) is fermented to bioethanol. Different types of microorganisms including fungi and bacteria can be used to ferment sugars from weed biomass to bioethanol. Zymomonas mobilis[40], Kluyveromycessp. [41], and Saccharomyces cerevisiae[4] are common microorganisms for fermentation of glucose to bioethanol. S. cerevisiaeis the most common microorganism for commercial bioethanol production. However, Pachysolen tannophilus, Pichia stipitis,and Candida shehataeare well-known for their ability to ferment xylose to bioethanol [33]. However, the activity of S. cerevisiaeis affected by several factors including high temperature, osmotic stress, bioethanol concentration, and contamination from bacteria [41]. These conditions inhibit microbial growth during fermentation process, thus affecting the yield of bioethanol production. Furthermore, the inability of S. cerevisiaeto ferment pentoses also affects bioethanol yield during fermentation. However, studies are continuously being conducted to isolate and identified S. cerevisiaestrains that are able to tolerate these stress conditions to improve bioethanol yield during fermentation. Microbial strains from Pichiasp., Candidasp., Schizosaccharomycessp. and Pachysolensp. have also been identified for fermentation of pentoses to bioethanol. Recombinant DNA technologies have been exploited to develop strains that are resistant to stress and also have the ability to ferment pentoses, all aimed at increasing bioethanol yield [4].

Fermentation of bioethanol is normally undertaken in a bioreactor with three major different processes namely batch, fed-batch, and continues [4]. During batch process of bioethanol production, the fermentation ingredients including substrate, culture medium, and nutrients are fed to the bioreactor only at the start of the process. No feeding is done till the process is over after which bioethanol is harvested. The substrate, medium, and nutrients may however be fed and bioethanol removed continuously during continues fermentation process. The fed-batch process is a combination of the batch and continues processes. During this process, fermentation ingredients are continuously fed to the bioreactor but bioethanol is only harvested at the end of the process [26]. Bioethanol produced after fermentation is further purified through distillation and other cutting-edge processes such as pervaporation [7]. Different types of microorganisms have been studied for their ability to ferment weed biomass to bioethanol (Table 3).

Weed biomassPretreatmentFermenting microorganismEtOH productionReference
Vetiveria zizanioidesAlkaline peroxideSaccharomyces cerevisiaeTISTR 5339 + P. stipitisCBS 57730.14 ± 0.01 g/L[17]
cv.Sri Lanka
Vetiveria zizanioides0.14 ± 0.01 g/L
cv. Ratchaburi
Parthenium hysterophorusSulfuric acidTorulaspora delbrueckiiR3DFM20.24 g/g biomass[18]
Schizosaccharomyces pombeR3DOM30.27 g/g biomass
Saccharomyces cerevisiaeR3DIM40.27 g/g biomass
Saccharum spontaneumAqueous ammoniaPichia stipitisNCIM34980.40 ± 0.01 g/g biomass[37]
Sulfuric acid0.38 ± 0.02 g/g biomass
Sodium hydroxide0.39 ± 0.02 g/g biomass
Lemna minorAlkalineSaccharomyces cerevisiae0.218 g/g biomass[13]
Lemna gibba0.197 g/g biomass
Pistia stratiotes0.215 g/g biomass
Eichhorniasp0.189 g/g biomass
Pennisetum polystachionSodium hydroxideSaccharomyces cerevisiae(TISTR 5596)16.0[42]
Panicum maximumcv. TD 53Calcium hydroxideSaccharomyces cerevisiaeTISTR 55965.9 g/L[10]

Table 3.

Ethanol production from weed biomass.

Wongwatanapaiboon [17] reported a significantly higher bioethanol yield from alkaline peroxide pretreated Vetiveria zizanioides cv.Sri Lanka and Vetiveria zizanioidescv. Ratchaburi. Using the fermenting organisms Saccharomyces cerevisiaeTISTR 5339 and P. stipitisCBS 5773, 32.72 and 30.95% of theoretical ethanol yield was reported for pretreated Vetiveria zizanioidescv. Sri Lanka and Vetiveria zizanioidescv. Ratchaburi biomass respectively. Tavva [18] reported similar bioethanol yield for Torulaspora delbrueckiiR3DFM2, Schizosaccharomyces pombeR3DOM and Saccharomyces cerevisiaeR3DIM4 fermentation of sulfuric acid pretreated Parthenium hysterophorus. The efficiency of bioethanol production by the three microbial strains was reported as 78.84, 87.82, and 87.17%, respectively. Chandel [37] used Pichia stipitisNCIM3498 to ferment hydrolyzate obtained from aqueous ammonia, sulfuric acid and sodium hydroxide pretreated Saccharum spontaneum. The results show maximum bioethanol production from hydrolyzate for all the pretreated biomass. Gusain and Suthar [13] converted alkaline pretreated aquatic weeds into bioethanol using Saccharomyces cerevisiae. Bioethanol yields of between 0.189 and 0.218 g/g biomass were reported for the four different species of aquatic weeds. Prasertwasu [42] fermented hydrolyzate from sodium hydroxide pretreated Pennisetum polystachionwith Saccharomyces cerevisiae(TISTR 5596) and reported high bioethanol yield after 24 hours. Ratsamee [10] also reported maximum bioethanol yield after fermenting hydrolyzate from calcium hydroxide pretreated Panicum maximumcv. TD 53 with Saccharomyces cerevisiaeTISTR 5596 for 48 hours.


4. Conclusion and future perspectives

Weed biomass is a promising feedstock for economic bioethanol production. The abundance of weed biomass worldwide is an assurance of its sustainability as a feedstock. Current research on the conversion of weed biomass to bioethanol is focused on pretreatment techniques. Different pretreatment techniques have been explored to convert weed biomass into bioethanol. Maximum bioethanol yields have been reported after fermentation of hydrolyzates from pretreated weed biomass. However, current technologies are still inadequate for bioethanol production from weed biomass to compete with starch and sugar based bioethanol in terms of production yield and cost. Production of cellulosic bioethanol from weedy plants is only at the laboratory scale. Further research to establish cost effective and efficient conversion processes including pretreatment technique(s) for a wide range of weed biomass is needed. Predictive models will also aid in the selection, design, optimization, and process control pretreatment technologies that match biomass feedstock with appropriate method and process configuration. On the other hand, active research is going on to ensure commercial production of bioethanol from weed biomass. This includes improvements in pretreatment technologies, specific activities of enzymes as well as isolation of new fermentation microorganism from natural environment. With strong support from various governments, bioethanol production from weed biomass will play a major role in meeting energy demand globally.



The author would like to devote this chapter to Emeritus Prof. Dr. Norio Takamura, Emeritus Prof. Dr. Kazuhiko Sameshima and Prof. Dr. Yoshito Ohtani, all of Kochi University, Nankoku, Japan, Emeritus Prof. Dr. Kazuhiko Ogino (Former Dean of United Graduate School Ehime, 1991–1993), Emeritus Prof. Dr. Sanro Tachibana, Ehime University, Matsuyama, Japan, and Emeritus Prof. Dr. Ryuichiro Kondo, Kyushu University, Fukuoka, Japan for their immense support and encouragement throughout his career. The author is also grateful to Naresuan University for the opportunity and support in getting to this level of the academic ladder.


Conflict of interest

The author has no conflict of interest to declare.


  1. 1. Chung JH, Kim DS. Miscanthus as a potential bioenergy crop in East Asia. Journal of Crop Science and Biotechnology. 2012;15(2):65-77. DOI: 10.1007/s12892-012-0023-0
  2. 2. Borah AJ, Singh S, Goyal A, Moholkar VS. An assessment of the potential of invasive weeds as multiple feedstocks for biofuel production. RSC Advances. 2016;6(52):47151-47163. DOI: 10.1039/C5RA27787F
  3. 3. Mofijur M, Masjuki HH, Kalam MA, Ashrafur Rahman SM, Mahmudul HM. Energy scenario and biofuel policies and targets in ASEAN countries. Renewable and Sustainable Energy Reviews. 2015;46(Supplement C):51-61. DOI: org/10.1016/j.rser.2015.02.020
  4. 4. Mohd Azhar SH, Abdulla R, Jambo SA, Marbawi H, Gansau JA, Mohd Faik AA, Rodri-gues KF. Yeasts in sustainable bioethanol production: A review. Biochemistry and Biophysics Reports. 2017;10:52-61. DOI: org/10.1016/j.bbrep.2017.03.003
  5. 5. Hoşgün EZ, Berikten D, Kıvanç M, Bozan B. Ethanol production from hazelnut shells through enzymatic saccharification and fermentation by low-temperature alkali pretreatment. Fuel. 2017;196:280-287. DOI: org/10.1016/j.fuel.2017.01.114
  6. 6. Xu Z, Huang F. Pretreatment methods for bioethanol production. Applied Biochemistry and Biotechnology. 2014;174(1):43-62. DOI: 10.1007/s12010-014-1015-y
  7. 7. Ullah K, Kumar Sharma V, Dhingra S, Braccio G, Ahmad M, Sofia S. Assessing the lignocellulosic biomass resources potential in developing countries: A critical review. Renewable and Sustainable Energy Reviews. 2015;51(Supplement C):682-698. DOI: org/10.1016/j.rser.2015.06.044
  8. 8. Yan Q, Wang Y, Rodiahwati W, Spiess A, Modigell M. Alkaline-assisted screw press pretreatment affecting enzymatic hydrolysis of wheat straw. Bioprocess and Biosystems Engineering. 2017;40(2):221-229
  9. 9. Behera S, Arora R, Nandhagopal N, Kumar S. Importance of chemical pretreatment for bioconversion of lignocellulosic biomass. Renewable and Sustainable Energy Reviews. 2014;36:91-106. DOI: org/10.1016/j.rser.2014.04.047
  10. 10. Ratsamee S, Akaracharanya A, Leepipatpiboon N, Srinorakutara T, Kitpreechavanich V, Tolieng V. Purple guinea grass: Pretreatment and ethanol fermentation. BioResources. 2012;7(2):1891-1906
  11. 11. Chandel AK, Singh OV. Weedy lignocellulosic feedstock and microbial metabolic engineering: Advancing the generation of ‘Biofuel’. Applied Microbiology and Biotechnology. 2011;89(5):1289-1303. DOI: 10.1007/s00253-010-3057-6
  12. 12. Banka A, Komolwanich T, Wongkasemjit S. Potential Thai grasses for bioethanol production. Cellulose. 2015;22(1):9-29. DOI: 10.1007/s10570-014-0501-2
  13. 13. Gusain R, Suthar S. Potential of aquatic weeds (Lemna gibba,Lemma minor,Pistia stratiotesandEichhorniasp.) in biofuel production. Process Safety and Environmental Protection. 2017;109:233-241. DOI: 10.1016/j.psep.2017.03.030
  14. 14. Siripong P, Duangporn P. Determining the potential of inedible weed biomass for bioenergy and ethanol production. BioResources. 2013;8(1):701-716
  15. 15. Tatijarern P, Prasertwasu S, Komalwanich T, Chaisuwan T, Luengnaruemitchai A, Wong-kasemjit S. Capability of Thai Mission grass (Pennisetum polystachyon) as a new weedy lignocellulosic feedstock for production of monomeric sugar. Bioresource Technology. 2013;143:423-430. DOI: 10.1016/j.biortech.2013.05.128
  16. 16. Borah AJ, Agarwal M, Poudyal M, Goyal A, Moholkar VS. Mechanistic investigation in ultrasound induced enhancement of enzymatic hydrolysis of invasive biomass species. Bioresource Technology. 2016;213(Supplement C):342-349. DOI: org/10.1016/j.biortech.2016.02.024
  17. 17. Wongwatanapaiboon J, Kangvansaichol K, Burapatana V, Inochanon R, Winayanu-wattikun P, Yongvanich T, Chulalaksananukul W. The potential of cellulosic ethanol production from grasses in Thailand. Journal of Biomedicine and Biotechnology. 2012;2012:1-10. DOI: 10.1155/2012/303748
  18. 18. Tavva S, Deshpande A, Durbha SR, Palakollu VAR, Goparaju AU, Yechuri VR, Bandaru VR, Muktinutalapati VSR. Bioethanol production through separate hydrolysis and fermentation ofParthenium hysterophorusbiomass. Renewable Energy. 2016;86:1317-1323. DOI: 10.1016/j.renene.2015.09.074
  19. 19. Rehman MSU, Rashid N, Saif A, Mahmood T, Han JI. Potential of bioenergy production from industrial hemp (Cannabis sativa): Pakistan perspective. Renewable and Sustainable Energy Reviews. 2013;18(Supplement C):154-164. DOI: org/10.1016/j.rser.2012.10.019
  20. 20. Cai D, Li P, Chen C, Wang Y, Hu S, Cui C, Qin P, Tan T. Effect of chemical pretreatments on corn stalk bagasse as immobilizing carrier ofClostridium acetobutylicumin the performance of a fermentation-pervaporation coupled system. Bioresource Technology. 2016;220:68-75. DOI: org/10.1016/j.biortech.2016.08.049
  21. 21. Procentese A, Johnson E, Orr V, Campanile AG, Wood JA, Marzocchella A, Rehmann L.Deep eutectic solvent pretreatment and subsequent saccharification of corncob. Biore-source Technology. 2015;192:31-36. DOI: org/10.1016/j.biortech.2015.05.053
  22. 22. Toquero C, Bolado S. Effect of four pretreatments on enzymatic hydrolysis and ethanol fermentation of wheat straw. Influence of inhibitors and washing. Bioresource Technology. 2014;157:68-76. DOI: org/10.1016/j.biortech.2014.01.090
  23. 23. Arora A, Priya S, Sharma P, Sharma S, Nain L. Evaluating biological pretreatment as a feasible methodology for ethanol production from paddy straw. Biocatalysis and Agricultural Biotechnology. 2016;8:66-72. DOI: 10.1016/j.bcab.2016.08.006
  24. 24. Wan C, Li Y. Effectiveness of microbial pretreatment byCeriporiopsis subvermisporaon different biomass feedstocks. Bioresource Technology. 2011;102(16):7507-7512. DOI: org/10.1016/j.biortech.2011.05.026
  25. 25. Eblaghi M, Niakousari M, Sarshar M, Mesbahi GR. Combining ultrasound with mild alkaline solutions as an effective pretreatment to boost the release of sugar trapped in sugarcane bagasse for bioethanol production. Journal of Food Process Engineering. 2016;39(3):273-282. DOI: 10.1111/jfpe.12220
  26. 26. Zabed H, Sahu JN, Suely A, Boyce AN, Faruq G. Bioethanol production from renewable sources: Current perspectives and technological progress. Renewable and Sustainable Energy Reviews. 2017;71:475-501. DOI: org/10.1016/j.rser.2016.12.076
  27. 27. DMd C, Sevastyanova O, Penna LS, Silva BP, Lindström ME, Colodette JL. Assessment of chemical transformations in eucalyptus, sugarcane bagasse and straw during hydrothermal, dilute acid, and alkaline pretreatments. Industrial Crops and Products. 2015;73:118-126. DOI: org/10.1016/j.indcrop.2015.04.021
  28. 28. Nieves DC, Ruiz HA, de Cárdenas LZ, Alvarez GM, Aguilar CN, Ilyina A, Martínez Hernández JL. Enzymatic hydrolysis of chemically pretreated mango stem bark residues at high solid loading. Industrial Crops and Products. 2016;83:500-508. DOI: org/10.1016/j.indcrop.2015.12.079
  29. 29. Hideno A, Kawashima A, Endo T, Honda K, Morita M. Ethanol-based organosolv treatment with trace hydrochloric acid improves the enzymatic digestibility ofJapanese cypress(Chamaecyparis obtusa) by exposing nanofibers on the surface. Bioresource Technology. 2013;132:64-70. DOI: org/10.1016/j.biortech.2013.01.048
  30. 30. Aver KR, Scortegagna AZ, Fontana RC, Camassola M. Saccharification of ionic-liquid-pretreated sugar cane bagasse usingPenicillium echinulatumenzymes. Journal of the Taiwan Institute of Chemical Engineers. 2014;45(5):2060-2067. DOI: org/10.1016/j.jtice.2014.04.017
  31. 31. Obeng AK, Premjet D, Premjet S. A review article of biological pre-treatment of agricultural biomass. Pertanika Journal of Tropical Agricultural Science. 2018;41(1):19-39
  32. 32. Kristiani A, Abimanyu H, Setiawan AH, Sudiyarmanto, Aulia F. Effect of pretreatment process by using diluted acid to characteristic of oil palm's frond. Energy Procedia. 2013;32:183-189. DOI: org/10.1016/j.egypro.2013.05.024
  33. 33. Keshwani DR, Cheng JJ. Switchgrass for bioethanol and other value-added applications: A review. Bioresource Technology. 2009;100(4):1515-1523. DOI: org/10.1016/
  34. 34. Ninomiya K, Yamauchi T, Kobayashi M, Ogino C, Shimizu N, Takahashi K. Cholinium carboxylate ionic liquids for pretreatment of lignocellulosic materials to enhance subsequent enzymatic saccharification. Biochemical Engineering Journal. 2013;71:25-29. DOI: org/10.1016/j.bej.2012.11.012
  35. 35. Hatakka A, Hammel KE. Fungal biodegradation of lignocelluloses. In: Hofrichter M, editor. Industrial Applications. The Mycota. 2nd ed. Berlin: Springer; 2010. pp. 319-340
  36. 36. Asgher M, Wahab A, Bilal M, Nasir Iqbal HM. Lignocellulose degradation and production of lignin modifying enzymes bySchizophyllum communeIBL-06 in solid-state fermentation. Biocatalysis and Agricultural Biotechnology. 2016;6:195-201. DOI: org/10.1016/j.bcab.2016.04.003
  37. 37. Chandel AK, Singh OV, Venkateswar Rao L, Chandrasekhar G, Lakshmi Narasu M. Bio-conversion of novel substrateSaccharum spontaneum, a weedy material, into ethanol byPichia stipitisNCIM3498. Bioresource Technology. 2011;102(2):1709-1714. DOI: 10.1016/j.biortech.2010.08.016
  38. 38. Siripong P, Duangporn P, Takata E, Tsutsumi Y. Phosphoric acid pretreatment ofAchyranthes asperaandSida acutaweed biomass to improve enzymatic hydrolysis. Bioresource Technology. 2016;203:303-308. DOI: org/10.1016/j.biortech.2015.12.037
  39. 39. Promkamaie A. Determination of Optimal Pretreatment Conditions for Biomass of Weeds [thesis]. Phitsanulok: Naresuan University; 2015
  40. 40. He MX, Wu B, Qin H, Ruan ZY, Tan FR, Wang JL, Shui ZX, Dai LC, Zhu QL, Pan K, Tang XY, Wang WG, Hu QC.Zymomonas mobilis: A novel platform for future biorefineries. Biotechnology for Biofuels. 2014;7(1):101. DOI: 10.1186/1754-6834-7-101
  41. 41. Costa DA, de Souza CJA, Costa PS, Rodrigues MQRB, dos Santos AF, Lopes MR, Genier HLA, Silveira WB, Fietto LG. Physiological characterization of thermotolerant yeast for cellulosic ethanol production. Applied Microbiology and Biotechnology. 2014;98(8):3829-3840. DOI: 10.1007/s00253-014-5580-3
  42. 42. Prasertwasu S, Khumsupan D, Komolwanich T, Chaisuwan T, Luengnaruemitchai A, Wongkasemjit S. Efficient process for ethanol production from Thai Mission grass (Pennisetum polystachion). Bioresource Technology. 2014;163:152-159. DOI: 10.1016/j.biortech.2014.04.043

Written By

Siripong Premjet

Submitted: February 5th, 2018 Reviewed: April 23rd, 2018 Published: November 5th, 2018