7.1 Introduction

The present world economy is highly dependent on the stocked natural resources of the Earth, which are being used for the production of fuel, electricity, and other needs. The very high level of fossil fuel consumption has generated a high level of pollutants in the atmosphere, with the scenario being worse in urban areas. Because the level of greenhouse gases in the Earth’s atmosphere has drastically increased, bioethanol has received worldwide interest. Bioethanol is a major second-generation biofuel. The global market for bioethanol has entered a phase of rapid, transitional growth. Many countries around the world are shifting their focus toward renewable sources for power production because of depleted crude oil reserves. The trend is extending to transport fuel as well. Most of the environmentally aware countries across the globe consider biomass for its economic utilization, and have directed state policies regarding the same, to meet future energy demands and also to meet carbon dioxide reduction targets. The primary focus is on reducing the emissions and thereby complying with the Kyoto Protocol for specified targets and also meeting energy demands. As well as the production of bioethanol, lignocellulosic biomass is also used in the production of both power and heat through combustion. Petroleum-based fuels can be replaced by bioethanol and other biofuels if biomass materials such as sugarcane bagasse, corn stover, switchgrass, and algae are effectively utilized. As a matter of fact, lignocellulosic biomass is the most abundant biomass present on the surface of the Earth. Among biomass sources, agricultural wastes are the most plentiful and cheapest, especially wheat straw, which is the most plentiful in Europe and is second worldwide after rice straw. As well as wheat, several other crops produce plentiful waste such as corn stover, sugarcane bagasse, and rice straw.

Second-generation biofuels are those fuels that are produced from waste lignocellulosic biomass. Agricultural farms produce an abundance of lignocellulosic biomass that is considered waste. Food materials in the form of lignocellulose compose less than one third of the total lignocellulosic biomass produced on agricultural farms, which indicates the amount of waste produced as lignocellulosic biomass. Apart from waste produced in the agricultural fields, an abundance of lignocellulosic waste is produced in the forests and on uncultivated lands. Leaf litter in the urban environment, for example, is typically treated as a waste. Although the leaf litter in forest areas becomes decomposed and is processed in an environmentally sound manner, urban leaf litter tends to be burnt for disposal. The burning and disposal of lignocellulosic waste in an environmentally unsound manner tends to increase the load of atmospheric pollutants. In many urban areas, a problem of smog conditions arises with the excessive burning of this waste. The waste on agricultural fields when burnt adds to this effect by several fold.

In this chapter we discuss different forms of microbes used in the production of bioethanol along with their modification at genetic level to enhance performance. Major emphasis is given to the fungal microbes as they are the major types involved in industrial production of bioethanol. Genetic modifications carried out in fungal strains, mainly Trichoderma reesei, are discussed in detail in this chapter.

7.2 Second-Generation Biofuel Production from Fungal Strains

Second-generation biofuel production typically consists of three major steps: pre-treatment, enzymatic hydrolysis, and fermentation (Gupta and Verma 2015). Pre-treatment is the step in which the waste undergoes different types of treatment to loosen the cellulose fibers from the other components, lignin and hemicellulose. The main aim of the pre-treatment process is to cause disruption of the lignocellulosic matrix and remove lignin from the complex of cellulose and hemicellulose, thereby facilitating the hydrolytic enzymes to bring about effective degradation. One of the common pre-treatment process currently in prevalence is the steam explosion, although the severity of this process generates several by-products that hinder further steps (Alvira et al. 2010; Jurado et al. 2009). An alternative to avoid these problems is the use of biological pre-treatments, which present additional advantages as being cheaper, safer, less energy consuming, and more environmentally friendly. Bio-pre-treatment is a term used by Salvachúa et al. (2011) for the pre-treatment of lignocellulosic waste by the application of microbes. The fungal strains provide a very good solution for this purpose. Many fungal strains such as Poria subvermispora and Irpex lacteus are used for bio-pre-treatment.

7.3 Fungal Strains and Bioethanol

Agricultural waste increases in direct proportion with agricultural food crop production. The produced waste, if utilized efficiently, can provide an alternative to the dwindling oil reserves. The production of bioethanol from fungal strains is seen as a very good approach for alternative biofuel production. Fungal strains, in general, are better and more efficient than bacterial strains for the conversion of lignocellulosic biomass into bioethanol. As can be seen from the literature, many fungal strains are available that can be employed at three important steps of bioethanol production from lignocellulosic biomass. In the biological pre-treatment stage, microbes, usually rot fungus species, are used for the degradation of lignin and other hemicellulosic compounds. These fungal strains include brown rot fungi, white rot fungi, and soft rot fungi, which bring about the degradation of lignin, hemicellulose, and some amount of cellulose (Sánchez 2009).

7.3.1 Fungi for Biological Pre-treatment

Biological pre-treatment, which mainly involves fungal strains to bring about the degradation of compounds other than cellulose in the feedstock, is an eco-friendly process. Lignin degradation in nature is found to be caused by a very few microorganisms, mainly basidiomycetes. According to a study by molecular clock analyses, it is suggested that lignin degradation originated around the end of the Carboniferous period, which also coincides with decrease in the rate of organic carbon burial (Floudas et al. 2012). Biological pre-treatment has an advantage over chemical pre-treatment because it avoids the production of acid by-products, thereby preventing the inhibition caused by such by-products. The use of fungal strains for biological pre-treatment is not feasible industrially because of its slow rate of action in comparison to other methods of pre-treatment such as physical pre-treatment and chemical pre-treatment. Many known strains of white rot fungus, including Phanerochaete chrysosporium, Ceriporia lacerata, Cyathus stercoreus, Ceriporiopsis subvermispora, Pycnoporus cinnabarinus, and Pleurotus ostreatus, are widely used for the biological pre-treatment of agricultural waste (Shi et al. 2008). Among these, P. chrysosporium is considered a good candidate for the degradation of lignin. Lignin, which is chemically very strong, is connected by three dimensions, making it a very strong polymer. White rot fungi thus help in reducing the lignin content of lignocellulosic material as they produce very strong lignin-degrading enzymes, thus providing a perfect delignified substrate for other subsequent uses such as biofuel production (Rodrigues et al. 2008). The ability of white rot fungus to selectively degrade lignin from the lignocellulosic biomass, producing cattle feed as well as a fuel production source, makes it a perfect candidate for biological pre-treatment. White rot fungus are the only organisms capable of substantial lignin degradation apart from brown rot and ectomycorrhizal species (Floudas et al. 2012). One of the articles also suggests that a biorefinery coupled with a mushroom production plant can help in two ways, in sustainable biofuel production as well as fostering rural economy (Kalia and Purohit 2008). White rot fungi have other added advantages for consideration as a good pre-treatment agent as they have a nonspecific and nonstereoselective enzyme system, formed by lignin peroxidases and manganese-dependent peroxidases (Levin et al. 2008). Lignin peroxidases work by interacting directly with nonphenolic lignin structures, as they are themselves strong oxidants. Lignin peroxidases cannot penetrate the pores of strong lignocellulosic material. Manganese-dependent peroxidases, on the other hand, enter this strongly bound lignin matrix by producing small diffusible strong oxidants. Feruloyl esterase, an enzyme that brings about the conversion of ferulic acid and p-coumaric acid to hemicellulose, constitutes a key enzyme in the delignification process. This enzyme is found to show synergism with cellulases, xylanases, and pectinases, and thus it does not hinder other processes (Hermoso et al. 2004). The use of white rot fungus for the pre-treatment of wheat straw for 10 days resulted in a better process in three ways: reduction in acid loading for hydrolysis, thereby resulting in the increase of fermentable sugars, and last, reduction in amount of fermentation inhibitors (Kuhar et al. 2008).

Research on brown rot fungus is very limited and their characteristics have not been much explored, such as the ability of decolorizing wastewater and biosorption of heavy metals. Dey et al. (1994) studied the production of manganese peroxidase, which causes delignification of rice straw and has the ability to decolorize dye. Dey at al. also observed that the fungus Polyporus zosteriformis produced not only Mn peroxidase, acid protease, α-amylase, and lignin peroxidase, with maximum activities of 40, 8300, and 4200 U l−1 and 50 nkat l−1, respectively, but also brought about 99% decolorization of Congo red dye in 9 days with 18.6% lignin removal in 3 weeks from rice straw (Dey et al. 1994). During the decay of lignin caused by brown rot, a chemical alteration in lignin takes place. A nuclear magnetic resonance (NMR) study of spruce decay by brown rot showed loss of methoxy groups, and cleavage of lignin β-O-4 linkages in the decay of birch (Pandey and Pitman 2003). In the same study, it was found that in the wood decay caused by the white rot Phanerochaete chrysosporium, the lignin content decreased along with the xylan content with the progression of decay. In Coriolus versicolor, lignin degradation was preferred over carbohydrate degradation. Brown rot fungus is the most prevalent wood-decaying fungus in coniferous forests, with a significant role in the conversion of wood into coarse debris with soil organic matter (Blanchette et al. 1994), whereas white rot fungi are known to cause degradation of lignin along with cellulose, with some having the ability to selectively degrade lignin (Eriksson et al. 1990). The mechanism through which the decay of the lignin by brown rot takes place in thought to be Fenton chemistry for the production of hydroxyl anions. During the process, methoxy carbon removal from lignin takes place with the production of an aromatic hydroxyl-rich product. The process is also thought to be brought about by the oxidation of aliphatic side chains of lignin (Agosin et al. 1989). Phenolic compounds that are produced by fungi function as ferric iron chelators and sources of electrons for iron reduction (Kerem et al. 1999). The form of these low molecular weight reactants, which are small in size, makes it easier to penetrate the lignocellulosic matrix, thereby bringing about its degradation (Jellison et al. 1997).

7.3.2 Microbes for the Hydrolysis of Polysaccharides

The pre-treatment process is followed by the hydrolysis of the pre-treated substrate in the presence of hydrolytic enzymes. The main cellulose-degrading fungal species include Trichoderma, Penicillium, and Aspergillus (Galbe and Zacchi 2002). In general, cellulolytic bacteria are observed to produce lesser amounts of cellulase in comparison to the cellulolytic fungus. Among these, the best-known producer of cellulase enzyme has been found to be Trichoderma, which is known to produce a complex mixture of cellulase enzymes. The cellulase system consists of three different types of enzymes : endoglucanases, cellobiohydrolases, and β-glucosidases. A common aspect noticeable in this complex is that all these enzymes have specificity to hydrolyze β-1,4-glycosidic bonds. Also, these enzymes work in tandem and have a synergistic effect on the substrate to cause degradation. The enzyme β-glucosidase causes the cleavage of cellobiose formed by the hydrolysis carried out by endoglucanases and cellobiohydrolases. The endoglucanases bring about the degradation of the cellulose to cellodextrins, and the cellobiohydrolases subsequently convert cellodextrins into cellobiose. Cellobiose, a disaccharide, is then converted into glucose in the presence of the β-glucosidase enzyme (Gupta and Verma 2015). The process is supposed to happen in tandem, and accumulation of any of the products inhibits the activity of the respective enzymes because of product inhibition. Therefore, it becomes necessary for cellulose-degrading microbial species to produce all three specific types of enzymes in appropriate amounts to bring about the complete degradation of lignocellulosic waste or pre-treated cellulosic feedstock. The maximum activity of cellulase enzyme occurs at temperatures around 50° ± 5 °C at a pH of 4–5 (Saddler and Gregg 1998). Apart from that, the residence time of the enzymes over that substrate also are important in the production of greater amounts of glucose.

Many known fungal strains such as Sclerotium rolfsii, Phanerochaete chrysosporium, and Aspergillus spp., as well as Trichoderma and Penicillium, produce cellulase in large amounts. The use of Trichoderma reesei for the production of cellulase enzyme at the industrial level for application in different uses is credited to its ability to produce very high amounts of cellulase enzyme, up to about 100 g l–1 (Viksø-Nielsen 2008). The amount of enzyme production is also dependent on the type of inducer used in the cultures as a carbon source. It is observed that sophorose, which is a molecule containing two glucose units, linked by a β-1,2-linkage, induces maximum production of the cellulase enzyme (Mandels et al. 1962; Nisizawa et al. 1971).

In bacterial strains, Bacillus sp. are generally used for the production of cellulase enzyme on an industrial level: these are widely used in the textile industries for several purposes such as dye decolorization and preparation of fibers. Their use is preferred because of their broad range of growth conditions. Their maximum activity is obtained at a pH of 7, at which more than 85% of activity occurs (Jung et al. 1996).

In some anaerobic bacteria , a specialized mechanism is present to bring about the effective degradation of cellulosic materials, called cellulosomes (Béguin et al. 1987; Behera et al. 2014). Cellulosomes form as an alternative for the inability of anaerobic bacteria to effectively penetrate the cellulosic material: first found in Clostridium thermocellum, these were observed as large protuberances on the surface with scaffoldin protein and attached enzymatic subunits (Bayer et al. 1983, 1998; Fontes and Gilbert 2010; Lamed et al. 1983). These cellulosomes are an assembly of enzymatic subunits that aid in the successful hydrolysis of cellulosic material to monomer sugars. Apart from their ability to degrade cellulosic material, they are also found to possess the ability of gene regulation, and thereby helping in the production of new cellulosomal subunit.

7.4 Genetically Modified Microbes for Bioethanol Production

Genetically modified microbes have revolutionized the microbe-based industry. The prevalent genetic modification process is the gene cloning process, wherein a desired gene of interest from a selected microbe is inserted into a host microbe, typically Escherichia coli, with the help of a suitable vector (Fig. 7.1).

Fig. 7.1
figure 1

Typical process of gene cloning and preparation of genetically modified microbes

Full size image

The industrial production of cellulase from different microbial strains is aimed at low cost and high enzyme production levels. The use of natural isolates for the production of industrially significant enzyme levels is not practical. Therefore, to enhance the existing levels of enzyme production in the naturally occurring fungal isolates, the microbial strains are genetically modified. The process of genetic modification in the fungal strains is best done by cloning cellulase enzyme genes or specific enzyme genes. The process can be summarized in the following steps: identification and isolation of the desired enzyme gene by digesting chromosomal DNA with restriction endonucleases and DNA fragments of a suitable range ligated with the digested material. The obtained product, the ligation mixture, is used for the production of the transformed strains. A fact of significance in the cloning of the cellulase gene is that its expression is regulated at the transcriptional level, which has been confirmed by differential hybridization studies (Messner and Kubicek 1991). The end product inhibition to the enzyme by glucose can also be explained at this level. It is also seen that glucose repression of cellulase occurs at this level. The addition of sophorose to the glucose-based cultures caused transcription activity to stop (Ilmen et al. 1997).

7.4.1 Trichoderma reesei and Its Application in Bioethanol Production

The use of Trichoderma reesei in the bioethanol industry has long been prevalent . It is used for the production of beer, wine, and cheese owing basically to its hydrolyzing and fermentation capabilities. The idea of cellulase secretion from Trichoderma reesei has been explored with the possibility of a phenomenal change in the biofuel industry (Jeffries and Lindblad 2009). Research and development and the understanding of cellulase enzyme activities in detail have been possible through this microbial strain (Reese 1956; Mandels and Reese 1957, 1960). The first eukaryotic cellulase to be cloned was cellobiohydrolase CBH1, which was cloned from the same strain (Shoemaker et al. 1983; Divne et al. 1994). It has also been established that simply boosting the gene of interest cannot enhance the overall activity cellulose-degrading ability of the microbe. To overcome this, a fusion gene approach strategy can be used in which the encoded protein is also used as an expression enhancer apart from the promoter and terminator region of the gene of interest (Bischof et al. 2016). This strategy is a result of the research program at the Natick and Rutgers University in which increasing the efficiency of the extracellular protein of a Trichoderma strain QM6a by 20 fold through mutagenesis was studied. Later, the QM6a was found to be a sterile female by Seidl et al. (2009), resulting in the isolation of a novel strain, RUT-C30, which currently serves as the prototype for the hyperproduction of cellulase enzyme and is available in the public domain (Mandels et al. 1971). The titers of the enzyme reach a level of about 30 g/l with lactose used for the cellulase-inducing substrate (Durand et al. 1988).

With the advances in technology and the need to improve the industrial production of the enzyme, techniques and methods were explored for continuous improvement. Today, high-speed atomic force microscopy has enabled us to visualize cellulose degradation by cellobiohydrolase CBH1/CEL7A on the cellulose surface (Igarashi et al. 2011). With nearly all the possibilities being explored in the transformation, these techniques were considered as the best option and had become available by the early 1990s (Gruber et al. 1990). Kuhls et al. (1996) found that Hypocrea jecorina is the sexual form of Trichoderma reesei, which opened new possibilities in the field of cellulase production. Whole genome studies of Trichoderma reesei began in 2003 with the first transcriptomic study by Foreman et al. (2003) of the gene expression of T. reesei: the DNA microarray-based cDNA library corresponds to more than 5000 different transcripts of the T. reesei genome. Subsequent studies on genome analysis led to the discovery of many potential factors associated with cellulase production: nucleocytoplasmic transport, vacuolar protein trafficking, and mRNA turnover (Bischof et al. 2016; Kubicek 2013; Le Crom et al. 2009). Presently, about 80% of ethanol production through the cellulase enzyme utilizes T. reesei strains (Bischof et al. 2016). One of the major setbacks observed in the secretions of the T. reesei strain formulation was the lack of sufficient β-glucosidase activity as it is bound to the fungal cell walls (Messner et al. 1990; Ryu and Mandels 1980). Genetically modified fungal strains are found to secrete lower levels of β-glucosidase (Lynd et al. 2002; Merino and Cherry 2007). Inefficiency in producing higher levels of this enzyme means that there is inefficient saccharification at the last step, whereby cellobiose is to be converted into glucose. Subsequently, cellobiose inhibition makes a significant difference in the final production of glucose. The addition of β-glucosidase in the reaction vessel can increase the production of glucose from cellobiose, which results from the combined effects of endoglucanases and cellobiohydrolases (Sørensen et al. 2013).

Of the many factors involved in the degradation of cellulose, C2H2 type transcription factor CRE1, which mediates carbon catabolite repression, is the major one (Bischof et al. 2016). In the presence of favorable carbon sources such as glucose, it shuts down the transcription of its target genes. However, the hybridized fungal strains show considerable increase in the amount of production of the cellulase enzyme.

In cloning the β-glucosidase gene from fungal strains, Takashima et al. (1998) cloned a novel fungal β-glucosidase gene (bgl4) with its homologue (bgl2), isolated from Humicola grisea and Trichoderma reesei, respectively. The results obtained showed the recombinant H. grisea BGL4 enzyme to be thermostable. Significant levels of β-glucosidase activity were obtained in both strains, and significant β-galactosidase activity was also seen in recombinant H. grisea BGL4.

7.4.2 Genetically Modified Bacteria for Bioethanol Production

The use of bacterial strains for bioethanol production is less explored in comparison to the use of fungal strains. Some bacterial species show enzyme activity of one of the many enzymes required in the cellulase system. Bacterial strains that degrade cellulosic material can be found naturally in locations such as the gut of ruminants, the gut of insects such as wood borers, and also some bacteria that inhabit extreme environments (Kawai et al. 1987; Honda et al. 1987, 1988; Srivastava et al. 1999). In the past, many studies have involved genetic modifications in microbes to enhance cellulase enzyme production. E. coli (JM83), a transformed strain, produced results of 1.51 U/ml for endoglucanase as well as 0.32 U/ml for β-glucosidase (Srivastava et al. 1999). In another study, E. coli RI, the transformant, was produced with the help of genes isolated from a thermophilic anaerobe NA10. As a significant finding, it showed maximum activity at 80°C, which can be very beneficial in reducing the steps involved in bioethanol production and thus assist cost-effective production, because the boiling point of ethanol is 78°C (Honda et al. 1988). In this study, the transformant endoglucanase activity was found to be 307 U/mg protein and exoglucanase activity to be 0.2 U/mg protein.

7.5 Conclusions

Research on different microbes for cellulose degradation found no single natural isolate that has good activity of all three enzymes: endoglucanase, exoglucanase, and β-glucosidase. Therefore, efficient production of glucose from a lignocellulosic substrate is hindered. The use of genetically modified microbes can help in the formation of new hybrid strains capable of producing all three enzymes in good amounts and thus allow the efficient production of glucose. Apart from the pre-treatment processes, which should be standardized, the use of genetically modified microbes for the production of glucose from lignocellulosic biomass can be useful. The genetic modification in fungi has mostly been carried out with Trichoderma reesei variants. The use of fungal strains for this purpose has yielded better results in comparison with bacterial strains. However, bacterial strains offer many other advantages such as the diversity of forms present in the environment and ease of culturing. The use of extremophilic microbes for the purpose of cellulose degradation has also opened up new possibilities in this field.