Bioprospecting and Bioresources for Next-Generation Biofuel Production

Introduction

The sustainable utilization of waste organic biomass is an attractive option for the production of carbon-neutral biofuels to mitigate greenhouse gas emissions and address the rising global energy demands (Nanda et al. 2016c). Biofuels from renewable and biogenic materials (e.g. agricultural crop residues, forestry biomass, algae, energy crops, municipal solid waste, food waste and cattle manure) provide a wide range of advantages such as mitigation of greenhouse gas emissions, supplementing energy security, waste valorization and reinvigorating rural economy (Nanda et al. 2015b; Okolie et al. 2020a). Lignocellulosic biomass comprises agricultural crop residues, forestry biomass and energy crops, which contain cellulose, hemicellulose and lignin as their key biopolymeric components (Azargohar et al. 2019; Okolie et al. 2020b).

Waste biomass can be converted to biofuels and biochemicals through biological conversion technologies (e.g. fermentation and anaerobic digestion) and thermochemical conversion technologies (e.g. pyrolysis, gasification, liquefaction, transesterification, torrefaction and carbonization) to process specific liquid, gaseous and solid biofuels (Azargohar et al. 2013; Nanda et al. 2016b; Parakh et al. 2020). A wide variety of biofuels such as bioethanol, biobutanol, biohydrogen, biomethane, syngas, bio-oil, biodiesel and biochar are gaining attention for research and development worldwide for their potential use as sustainable fuels for automobiles, industries as well as combined heat and power (Nanda et al. 2014b). This chapter aims to discuss different generations of biofuels along with their composition and properties. The production of biofuels utilizing lignocellu- losic biomass and conversion processes are also described in this chapter.

Different Generations of Biofuels

Bioethanol production from different starch-based food crops and grains (e.g. potato, cassava, corn, wheat, etc.) via fermentation technologies is considered as the first- generation biofuel (Nanda et al. 2018). Bioethanol and biodiesel are two liquid biofuels, which can be blended with petrol and diesel, respectively, to reduce greenhouse gas emissions (Nayak et al. 2019; Nayak et al. 2020). The supply chain of food crops and grains as well as the global economy suffer dramatically because of the reduced food supply and rising food prices owing to their diversion to first-generation biofuel refineries (Nanda et al. 2015b). Moreover, the arable or cultivable land area is also found to be competitive in such a scenario of food versus fuel debate associated with first- generation biofuel production.

The second-generation biofuels, on the contrary, can be generated from lignocel- lulosic feedstocks, which have no direct competition with the human food supply chain or animal feed (Okolie et al. 2019). These materials are easy to procure, abundantly available and relatively cheap to sustain second-generation biofuel refineries. Moreover, second-generation biofuels produced from lignocellulosic biomass are more enviable as they are non-edible, renewable and pose no threat to food crops and arable lands. A few examples of lignocellulosic biomass are bagasse, stalk, peel, straw, shell, husk, stem, wood shavings, sawdust, etc., which originate as residues from agricultural harvesting and forests. Depending on the crop variety, geography, weather and climatic conditions, agricultural and harvesting practices operate round the year across the globe. Hence, enormous amounts of waste plant residues tend to be generating globally and annually. It has been reported that the global production of agricultural wastes reaches 1.4 billion tons annually (Saini et al. 2015).

The third-generation biofuels are produced from algal biomass (Yadav et al. 2019). Cultivable land is not required for the production of third-generation biofuel feedstocks (i.e. algae), which is a major advantage of third-generation biorefineries. Moreover, algae can grow on wastewater while leading to bioremediation, heavy metal removal, bioenergy production (i.e. in situ lipid accumulation), carbon sequestration and greenhouse gas mitigation (Ankit et al. 2020). Last but not least, fourth-generation biorefining is devoted to the applications of genetic engineering to alter biofuel feedstocks and microorganisms for techno-economically efficient biofuel production (Sarangi and Nanda 2019b). Although fourth-generation biorefineries aim to achieve higher biofuel production rates in short durations with less energy and cost input, secondary markets aim for byproducts while posing relatively low carbon footprints. Figure 1.1 illustrates all the four generations of biofuels.

FIGURE 1.1 Different generations of biofuels

Lignocellulosic Biomass and Pretreatment

Lignocellulosic biomass is the most predominant organic matter on earth and has a wide range of industrial applications such as in the production of biofuels, biomaterials, biomaterials, nutraceuticals, pharmaceuticals, cosmeceuticals and carbon-based specialty materials (Okolie et al. 2020b). As mentioned earlier, lignocellulosic biomass constitutes agricultural crop residues, forestry residues, energy crops and invasive crops (Nanda et al. 2016a; Singh et al. 2020). Such biomasses are a ubiquitous source of renewable biobased energy, which can favorably affect the sustainability matrix in terms of economics, employment, environmental concerns and energy security (Isikgor and Becer 2015). Considering their massive applications and utility, lignocellulosic biomasses are considered least deployed reservoirs of renewable natural polymers such as cellulose, hemicel- lulose and lignin. Lignocellulosic biomass typically contains 35-55 wt% cellulose, 20-40 wt% hemicellulose and 10-25 wt% lignin along with certain amounts of extractives and mineral matter (ash) (Nanda et al. 2013). Cellulose is composed of glucose (hexose sugar) monomers linked with P-1,4 glycosidic bonds, hydrogen bonds and Van der Waals force. On the other hand, hemicellulose comprises pentose sugars (e.g. xylose and arabinose) and hexose sugars (e.g. glucose, rhamnose, galactose and mannose) as well as sugar acids (e.g. glucuronic acid and galacturonic acid) (Nanda et al. 2015a). In contrast, lignin is a phenylpropane polymer having p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol as its basic building blocks (Fougere et al. 2016; Rana et al. 2018).

Due to the complex chemistry and interconnected network of cellulose, hemi- cellulose and lignin in lignocellulosic biomass, a pretreatment process is necessary before biological conversion. The pretreatment technologies involve physical agents (e.g. grinding, ozonolysis, ultrasound, microwave, etc.), chemical agents (e.g. acids, alkalis, organosolv, ionic liquids, liquid ammonia, steam, etc.) and biological agents (e.g. cellulolytic, lignin-degrading enzymes, lignin-modifying enzymes and microorganisms) (Nanda et al. 2014a; Nanda et al. 2014b).

Microorganisms consume organic substrates and utilize them in their metabolic processes, thereby generating useful products (metabolites), which can be further recovered as fuels, chemicals, nutraceuticals, pharmaceuticals, cosmeceuticals, flavoring agents, pigments, aromatic compounds and other value-added products with vast commercial applications (Sarangi and Nanda 2019a: Bhatia et al. 2020; Sarangi and Nanda 2020). Among the several microbial-assisted biofuels and biochemicals produced, bioethanol, biobutanol, biohydrogen, biomethane (biogas) and biomethanol are the most widely explored ones (Nanda et al. 2014a; Nanda et al. 2017a; Nanda et al. 2017b; Nanda et al. 2017c; Nanda et al. 2017d; Sarangi et al. 2018; Sarangi and Nanda 2018; Sarangi et al. 2020; Nanda et al. 2020a).

The growth of soil microbial heterotrophs is supported by their efficiencies to undergo natural enzymatic hydrolysis of lignocellulosic materials as plant debris, which is also an important process for terrestrial carbon cycling. Moreover, this process plays an important role in plant-microbial interactions to convert lignocellulosic materials to carbonaceous materials as well as C0₂ and CH₄. A variety of glycoside hydrolases are involved in enzymatic hydrolysis of complex lignocelluloses. The glycoside hydrolases family includes cel- lulases, hemicellulases, pectin-degrading enzymes and lignin-degrading enzymes.

Following biomass pretreatment, the recovered sugars are fermented using specific microorganisms to produce the desired alcohol-based biofuels and biochemicals. While both fungal and bacterial species are involved in ethanol fermentation, butanol production is achieved by Clostridium-aided acetone-butanol-ethanol (ABE) fermentation (Nanda et al. 2017b). Similarly, certain methanogenic bacteria (e.g. Methanobacterium sp„ Methanobrevibacter sp., Methanococcus sp., Methanoculleus sp., Methcmofollis sp„ Methanogenium sp., Methanomicrobium sp., Methanosarcina sp., etc.) are involved in biomethane production through anaerobic digestion (Rana and Nanda 2019), whereas anaerobic bacteria such as Clostridium sp., Enterobacter sp. and Bacillus sp. are involved in dark fermentation to produce biohydrogen (Sarangi and Nanda 2020).

Microbial Biomass in Bioenergy Production

Basler et al. (2018) have reported on the efflux pump i.e. on native resistance - nodulation-cell division (RND) acting on short-chain alcohols. Pseudomonas putida has gained attention as a potential microorganism in biorefinery owing to diversified catabolism and elaborated stability to various lethal materials (Udaondo et al. 2012). Furthermore, due to the diversity in features such as compliant metabolism, suppleness to noxious substances and flexibility for metabolic engineering, P. putida is considered as the benign microorganism for the fourth-generation biofuels production. P. putida has also the ability for the production of «-butanol after expressing the biosynthetic pathway from Clostridium acetobutylicum (Nielsen et al. 2009). Moreover, the engineered strain of P. putida was employed in a biphasic liquid extraction system to aid in the formation and down streaming of toxic compounds in fermenters (Schmitz et al. 2015; Basler et al. 2018).

P. putida is considered as a potential microorganism for industrial production of bioethanol because of the following factors (Dos Santos et al. 2004):

i. Native elaborated defense to different stressors including several solvents.

ii. Genetic stability.

iii. Competence to develop vigorously on complex substrates.

iv. Generally regarded as safe (GRAS).

Certain species of Clostridium and Pseudomonas are also employed to produce platform biochemicals. Pseudomonas, Enterobacter and Bacillus also have the potential to express transesterification activities leading to biodiesel production (Singh et al. 2008; Escobar-Nino et al. 2014). Implementing the genetic engineering tools, P. putida is confirmed as a powerful biocatalyst for the production of a wide range of value-added compounds such as non-ribosomal peptides, rhamnolipids, polyketides as well as aromatic and non-aromatic compounds (Loeschcke and Thies 2015).

Considering the versatile activities of microorganisms, a consolidated bioprocessing system for the production of biofuels, biochemicals and other essential bioproducts can lead to a circular economy with maximum utility and marketability of desirable products and coproducts while ensuring sustainability (Sarangi and Nanda 2019b). For example, activated sludge can provide effective support and flourish the growth of microalgae for third-generation biofuel production due to the consortia of a few plant growth-promoting bacteria (PGPB), e.g.. Azospirillum sp„ Pseudomonas sp.. Bacillus sp., Rhodococcus sp. and Acinetobacter sp. (Cea et al. 2015). This can be accomplished by two methods such as the cultivation of microalgae and bacteria in a single process and pretreatment of waste- water with bacteria for better growth of microalgae. Bacterial pretreatment of wastewater provides a suitable environment for the growth of algal biomass.