This is an environment friendly and sustainable method for the conversion of agricultural biomass into fuel especially bioethanol. This technique is more suitable for wet wastes containing lignocellulosic biomass. Three principal methods of biochemical conversion route are pretreatment, hydrolysis, and fermentation (Figure 13.3).

Schematic representation of biochemical conversion of biomass into ethanol

FIGURE 13.3 Schematic representation of biochemical conversion of biomass into ethanol.


Pretreatment is the process which aims to decrease the crystalline nature of cellulose to make it more accessible to enzymes. During pretreatment lignin cellulose interaction is broken, hemicellulose is removed, and cellulose structure is opened up to increase its exposed surface area. This enables efficient and rapid bioconversion of cellulose into free sugar molecules (Panday et al., 2015). Several biowaste pretreatments are done which includes:

  • • Physical treatment;
  • • Dilute acid pretreatment;
  • • Dilute alkali treatment;
  • • Hot water treatment; and
  • • Steam explosion treatment.

Physical pretreatment aims to reduce the particle as small as possible. It includes techniques like milling and grinding. All these preliminary treatments are meant to increase hydrolysis efficiency. The pretreatment varies with the availability and type of biomass (Murthy et al., 2011). Dilute acid pretreatment is one of the extensively used treatments. In it, the biomass is subjected to acid treatment at high temperature whose concentration varies with the type and composition of waste. This method is more suitable for large-scale treatments owing to its characteristics of short residence which makes biomass ready for next treatment quickly. In the presence of dilute acid, hemicellulose present in the biomass gets solubilized by means of hydrolyzation, while cellulose and lignin components are left behind in solid fraction. This allows one to separate the hemicellulose from cellulose. At high temperatures or high acid concentrations the hemicellulose gets converted into furans which inhibit the subsequent treatments (Nguyen et al., 1999). Dilute alkali pretreatment is rare though it also denatures the biomass structure. In presence of alkali, at high temperature, lignin is degr aded. The lignin-cellulose interaction is broken down due to the reaction of alkali with the esters of lignin. This makes lignin soluble and removable from the mixture and sugar-containing compounds remain in solution. The degradation of biomass varies with concentration of alkali and a good balance is required to achieve the good result (Murthy et al., 2011). Hot water pretreatment is an auto catalyzed process in which biomass is dissolved in high temperature water. This allows the degradation of branched strucnire of hemicellulose. In this process acetic acid is generated which lowers the pH thus facilitates structural degradation of biomass. This method has not been used on commercial scale since it requires high pressure for the water to be kept liquid (Kotiranta et al., 1999). The steam explosion treatment is more suitable for large-sized biomass and is energy saving since no energy is needed to reduce the size of biomass, hi it, biomass is heated under high pressure with water vapor. The pressure is released at once which facilitates the degradation of biomass (Sim et al., 2002). All the aforementioned pretreatment methods have its merits and drawbacks. Once the agricultural biomass undergoes pretreatment, it is subsequently subjected to hydrolysis by enzymes.


The enzymatic hydrolysis or saccharification is the second step involved in the process of ethanol production from agricultural waste. This step is carried out with the aid of certain enzymes like cellulases and hemicel- lulases that are produced from a variety of fungal strains. These cellulases are enzyme complexes of three main enzymes as: endocellulase, exocellu- lase, beta-glucosidase. All three enzymes cooperate to hydrolyze cellulose into monomeric sugars (Singhania et al., 2009). Besides this, enzymes like xylanase and ligninase are also found to work in association. It has been found that all of these three enzymes are produced by several fungal microorganisms among Trichoderma reesei is one mostly studied cellu- lase producer strain of fungi. There are several other filamentous fungal strains that had been adopted for the commercial production of cellulases and all other components required for hydrolysis of waste (Gusakov et ah, 2013). Depending upon the type of waste and fermentation process, different fungal species are used for cellulase production, as shown in Table 13.3.

TABLE 13.3 Different Fimgal Species Used in Fermentation Process


Waste Category




Aspergillus terreus

Rice straw


Nana et al. (2012)

Aspergillus fumigates .TBK9

Wheat bran, rice straw


Das et al. (2013)

Aspergillus protuberus

Rice husk


Yadav et al. (2016)

T. asperellum

Wheat brau


Ragliuwanshi et al. (2014)


Wheat straw


Pensupa et al. (2013)

Aspergillus fumigates NITDGPKA3

Rice straw


Sarkar and Aikat (2012)

Aspergillus niger KK2

Rice straw


Kang et al. (2004)

Aspergillus niger NCIM548

Wheat bran. Com bran, Kiimow peel


Kumar et al. (2011)

Penicillium echinulatum

Sugar cane bagasse


Camassola and Dillon (2014)

Trichoderma viride YKF3

Sugarcane bagasse


Nathan et al. (2014)

Rhizopus oryzae CCT7560

Rice husk and rice bran


Kupski et al. (2014)

In the whole process of ethanol production from biomass this step accounts maximum part of the cost of technology because of cost of enzyme (Mosier et ah, 2005). In order to reduce the cost and make this technology economically feasible recycling of enzyme is an important strategy that needs to be adopted (Tu et al., 2007). Zhao et al. (2012) reviewed certain chemical and physical factors that had been found to affect enzyme hydrolysis. Some of the key factors are:

  • • Amount of lignin content in the pretreated waste;
  • • Activity of the cellulases and hemicellulases;
  • • Optimum temperature and pH for action of enzyme;
  • • Optimal concentration and amount of enzyme and substrate loading;
  • • Time duration of the enzymatic hydrolysis;
  • • Inhibitory compounds (Furfurals) generated from lignin degradation.

All the above-mentioned factors should be overcome to maximize the efficiency and yield of the process. In order to tackle the problem of enzyme loading, it has been seen that addition of surfactants during the hydrolysis had reduced the surface tension and changed the surface properties of soil (Taherzadeh and Karimi, 2007).

Currently separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) are highly adopted technologies for bioethanol production from biomass. However, Onsite production of enzyme using low substrate could be the forward step to develop economically feasible technique (Singhania et al., 2014).


The hydrolyzed or saccharified AWs are subsequently subjected to incubation with yeast and bacteria and this process is known as fermentation. This is the thu d and final step of ethanol production from biomass. In this process, several microorganisms, especially yeasts and some bacteria, as shown in Table 13.4, are used to reduce hexoses and pentoses to ethanol, hi most of the cases, this process is carried out at moderate temperature (25-37°C), but it has been found that high temperatures (45-55°C) yield good results (Antoni et al., 2007). Since the microorganisms lack the ability to ferment both pentoses and hexoses, this makes the process more costly and is one of the hurdles in lowering ethanol prices.

TABLE 13.4 Different Microorganisms Which Produce Ethanol by Fermentation





1. 24860-5. cerevisiae

Valet et al. (1996)

2. 27774-Khtyveromycesfragilis

3. 30016-Kluyveromyces marxianus

4. 30091 -Candida utilis


1. Mucor sp. M105

Ingram et al. (1998)

2. Fusarium sp. F5


1. Clostridium sporogenes

Miyamoto et al. (1997)

2. Clostridium indoli

3. Clostridium sphenoides

4. Clostridium sordelli

5. Spirochaeta stenostrepia

6. Spirochaeta litorali

7. Erwinia amylovora

8. Streptococcus lactis

9. Leuconosroc meseuteroides

10. Escherichia coli KOI

Fermentation can be carried out through two different ways as:

  • 1. Separate Hydrolysis and Fermentation (SHF): In SHF, the two processes of hydrolysis and fermentation are earned out separately in two different units, each at its own optimal conditions of temperature, pH, and enzyme substrate concentration. This accounts for efficiency of both of the steps. However, main demerit of this method is that glucose formed as a result of hydrolysis inhibits further activity of enzymes. Moreover, this process is time consuming which leads to the risk of contamination (Taherzadeh and Karimi, 2007).
  • 2. Simultaneous Saccharification and Fermentation (SSF): In this method both the time and labor are reduced. In SHF the two steps of hydrolysis and feimentation are merged and earned out in one-step. As a result pentoses or hexoses formed from hydrolysis gets quickly converted into ethanol which does not inhibit any enzyme activity. Over SHF this process is more advantageous owing to its low cost, low labor, less time, minimum equipment requirement and less risk of contamination (Brethauer et al., 2010).


Anaerobic digestion (AD) is a sequence of biological processes that occur in oxygen-free conditions in which microorganisms break down biodegradable material. The anaerobic process is economical that can also produce energy at low cost treatments. The process is accomplished in four steps as hydrolysis, acidogenesis, acetogenesis, and methanogenesis (Singhania etal., 2014).

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