Bioconversion of Waste Biomass to Biobutanol
There has been a growing interest in the production and utilization of synthetic renewable transportation fuels due to rising crude oil prices, mounting demand for fossil fuels, the adverse impact of greenhouse gas emissions and the resulting global warming (Nanda et al. 2015b; Nanda et al. 2016a; Nanda et al. 20l6g; Nanda et al. 2017d). Hence, the worldwide interest in deploying biofuels and biochemicals in addition to their production from renewable biomass and wastes is gaining momentum (Sarangi and Nanda 2018; Sarangi and Nanda 2019a; Sarangi and Nanda 2019b; Sarangi and Nanda 2020; Sarangi et al. 2020). First-generation biofuels are criticized severely as far as their sustainability and competition to food supply chain and cultivable lands are concerned (Nanda et al. 2018a). On the other hand, next-generation biofuels provide a more sustainable platform ensuring both energy security and food security as their production relies on mostly inedible plant residues including agricultural crop refuse (Nanda et al. 2018b; Sun et al. 2020; Okolie et al. 2020c), forestry biomass (Nanda et al. 2016f; Nanda et al. 2017c), dedicated energy crops (Nanda et al. 2016c; Singh et al. 2020), cattle manure (Nanda et al. 2016b), municipal solid wastes (Okolie et al. 2020a), food waste (Nanda et al. 2015c; Nanda et al. 2016d; Nanda et al. 2019a), industrial effluents (Nanda et al. 2015d), sewage sludge (Gong et al. 2017a; Gong et al. 2017b), polymeric wastes (Nanda et al. 2019b) and petroleum residues (Rana et al. 2018a; Rana et al. 2019; Rana et al. 2020).
Lignocellulosic biomass, consisting of agricultural crop residues, dedicated energy crops, invasive plants and forestry biomass, is a storehouse of renewable natural polymers (i.e. lignin) and polysaccharides (i.e. cellulose and hemicellulose) that can be converted through thermochemical and biological technologies to solid (e.g. biochar, torrefied biomass and fuel pellets), liquid (e.g. bio-oil, bioethanol, biobutanol, biodiesel, etc.) and gaseous biofuels (e.g. biohydrogen, biomethane, syngas, etc.) (Nanda et al., 2016e; Azargohar et al. 2019; Kang et al. 2020; Parakh et al. 2020; Okolie et al. 2020b).
Hence, there are different alternatives to seek future biofuel solutions for the transportation sectors through the utilization of next-generation bioenergy feedstocks.
In contrast to bioethanol (C2H5OH), biobutanol (C4H9OH) appears to be a superior fuel with advanced properties, a few of which are less corrosiveness, higher calorific value (29.2 MJ/L), lower volatility and less hygroscopic (7.3% soluble in water), gasoline-equivalent research octane number (96), motor octane number (78) and air-to-fuel ratio (11.2), lower oxygen content (22%), less flammability and reduced hazardousness for handling (Nanda et al. 2014a; Nanda et al. 2017a; Nanda et al. 2017b; Sarangi and Nanda 2018; Nanda et al. 2020). Due to its lower vapor pressure and less hygroscopic nature, biobutanol can be transported in the gasoline supply chain pipelines to the fueling stations even in the cold weather (Qureshi and Ezeji 2008). Owing to its fuel properties similar to gasoline, biobutanol can either be blended with gasoline in flexible ratios or be used as a drop-in fuel without blending in the current gasoline-fueled automobile engines. Biobutanol can be produced biologically through the traditional acetone- butanol-ethanol (ABE) fermentation, although there are a few technical bottlenecks associated with its fermentative production such as lower product yields, bacteriophage contamination, product separation, expensive process, etc. This chapter discusses a few of such attributes in the biobutanol production through ABE fermentation from ligno- cellulosic biomass.
Pretreatment of Lignocellulosic Biomass
Although lignocellulosic biomasses are found to be promising next-generation bioenergy feedstocks, there are many challenges in their direct utilization because of their recalcitrant chemistry. Lignocellulosic biomass primarily contains cellulose, hemicellulose and lignin in the ranges of 35-55 wt%, 20-40 wt% and 10-25 wt%, respectively (Nanda et al. 2013). A pretreatment step coupled with enzymatic hydrolysis and saccharification is necessary to denature the complex cellulose- hemicellulose-lignin framework, the matrix associated between lignin and various sugars so that microbial enzymes can access cellulose and hemicelluloses (Nanda et al. 2014c). Biomass pretreatment is achieved by a wide variety of mechanical (e.g. particle size reduction), physical (e.g. ultrasound, ozonolysis, microwave, irradiation, etc.), chemical (e.g. steam explosion, subcritical and supercritical fluids, acids, alkalis, liquid ammonia, ionic liquids, organosolv, etc.) and biological agents (e.g. cellulases, hemicellulases, lignin-modifying enzymes and lignin-degrading enzymes) (Nanda et al. 2014b).
During biomass pretreatment, the configuration of cellulosic fibers and highly branched arrangement of lignin is altered, thus facilitating the admittance of hydrolytic enzymes for saccharification and release of fermentable pentose and hex- ose sugars (Fougere et al. 2016; Rana et al. 2018b). There are many other benefits associated with pretreating lignocellulosic biomass for fermentative production of bioethanol and biobutanol, such as (i) faster hydrolysis, (ii) high product yields, (iii) reduced cellulose crystallinity, (iv) easier hemicellulose separation and (v) alteration and increase of biomass pore size for easy accessibility of cellulolytic enzymes (Nanda et al. 2015a).
Acetone-Butanol-Ethanol (ABE) fermentation, mostly performed by Clostridium bacterium, has been explored for the conversion of several complex carbohydrates into biobutanol. Saccharomyces cerevisiae, which is a model fungus responsible for ethanol fermentation, lacks the natural ability to metabolize pentose sugars (i.e. hemi- celluloses). Instead, it is efficient in metabolizing hexose sugars, mainly glucose (i.e. cellulose) through the glycolytic pathway (Walfridsson et al. 1995). On the contrary, Clostridium spp. can metabolize both pentose and hexose sugars, suggesting the utilization of hydrolyzed cellulose and hemicellulose from lignocellulosic biomass. This is another significant advantage of ABE fermentation over ethanol fermentation.
Clostridium is a rod-shaped gram-positive obligatory anaerobic bacterium that grows on a wide range of sugar substrates including starch, hemicellulose and cellulose. An array of enzymatic systems in Clostridium spp. enhances the production of biobutanol utilizing glucose, cellobiose, galactose, arabinose, mannose and xylose to butanol (Ezeji et al. 2007a). A few Clostridium spp. such as C. acetobutylicum, C. beijerinckii, C. aurantibutyricum, C. butylicum, C. saccharobutylicum, C. saccharoperbutylace- tonicum, etc. have been explored for production of biobutanol through ABE fermentation (Durre 2007; Nanda et al. 2017b). Some starch-based (first-generation) feedstocks like molasses, potatoes, corn, millet, rice, wheat and whey have the potential for butanol production, but their industrial usage is obsolete due to food versus fuel debate.
ABE fermentation is a biphasic bioconversion process consisting of acidogenic phase and solventogenic phase (Figure 4.1). In the acidogenic phase, the bacterium grows exponentially producing acetic acid and butyric acid from the sugars, whereas in the solventogenesis phase, the formation of acetone, butanol and ethanol takes place in a typical ratio of 3:6:1 (Durre 2007). Some inhibition of the metabolic pathway occurs in the acetogenesis called as acidic stress, which favors the acid production rather than sugar consumption (Xue et al. 2013). The acidogenic phase is characterized by an increase in the acidity of the fermentation medium because of the formation of organic acids, which causes the bacterium to undergo the stationary growth phase and the subsequent solventogenic phase. The shift from the acetogenesis phase to solventogenic phase by Clostridium is characterized by a decelerated growth rate, formation of endospores and an increase in the levels of solvents (i.e. acetone, butanol and ethanol). For an obligate anaerobic bacterium, the acidogenic phase has a great role to play in its energy metabolism. As the pH level is reduced, the bacterium abridges acid formation
FIGURE 4.1 Simplified version of ABE fermentation by Clostridium
converting butyric acid and acetic acid to butanol and acetone, respectively. In the acidogenic phase, the redox equilibrium of butyric acid creates a balanced environment so that more butyric acid is formed than acetic acid (Zheng 2009).
According to the study conducted by Garcia et al. (2011), shifting of phase during Clostridium metabolism occurs by the sporulation of 70-80% of the viable cells. A pH level of 5.5 facilitates the phase shifting from acidogenic phase to solventogenic phase. It should be noted that the decrease in the pH occurs in the late acidogenic phase by the accumulation of acetic acid and butyric acid (Lee et al. 2008). The role of pH in fermentation conditions is important for determining the production of acids and solvents. Nevertheless, by increasing the buffering capacity of the fermentation medium, the bacterial growth increases to support the utilization and conversion of remaining sugars, thereby producing more butanol (Nanda et al. 2017b).
The non-dissociation of butyric acid favors the phase-shifting (Garcia et al. 2011) while the induction of butyrate kinase and acetate kinase occurs by butyric acid and acetic acid, respectively (Ballongue et al. 1986). Furthermore. butyryl-CoA along with butyryl phosphate facilitates the shifting of the acidogenic phase to the solventogenic phase. The ABE fermentation is accomplished by producing three types of products: (i) solvents such as acetone, butanol and ethanol, (ii) organic acids such as acetic acid, butyric acid and lactic acid and (iii) gases such as H2 and CO, (Xue et al. 2013). The ABE fermentation period typically persists for 36-72 h producing approximately 20-25 g/L of total ABE (Qureshi and Ezeji 2008).
Challenges and Opportunities of Acetone-Butanol-Ethanol Fermentation
At different stages of ABE fermentation, some technical challenges are encountered. Although waste biomass is found to be an economical source of sugar substrates for fermentation, the main limitations are its total utilization by Clostridium, which is impacted due to butanol toxicity (inhibitory to the bacteria) and endospore formation (because of higher acid accumulation in the acidogenic phase). These limitations result in lower butanol yields by the bacteria (Ezeji et al. 2007b). The butanol yields and expensive solvent recovery methods (e.g. perstraction, pervaporation, gas stripping, liquid-liquid extraction, adsorption, distillation and supercritical fluid separation) make the ABE fermentation process expensive over bioethanol fermentation. The removal of inhibitors resulting from biomass pretreatment by detoxification is an important issue to address.
Butanol toxicity is a vital limitation of the industrial ABE fermentation process. Clostridium spp. seldom has a tolerance level of more than 2% butanol, which hampers the final yields of butanol through ABE fermentation. Butanol level up to 12-13 g/L is considered as the maximum limit for the wild-type Clostridium strains (Garcia et al.
2011). For genetically modified C. beijerinckii ВАЮ1, the maximum yield of butanol reported is 19.6 g/L (Qureshi and Blaschek 1999). Apart from butanol concentration, the butanol recovery level is dependent on the bacteriophage infection. Bacteriophage such as Siphoviridae and Podoviridae have been reported to infect C. madisonii and C. beijerinckii P260, respectively (Jones et al. 2000).
The implementation of genetic and metabolic engineering is supported for overcoming some bottlenecks such as increased butanol tolerance by Clostridium. Another advancement like antisense RNA technology can be used for enhancing the microbial effectiveness for butanol production (Tummala et al. 2003). Therefore, synthetic biology approach can be applied for the development of novel microbial strains posing resistance to higher levels of butanol and contamination to bacteriophages, thereby sustaining the near-complete utilization of sugars and resulting in greater butanol yields.