## 2 Various Strategies for Enhancing the Production of Biofuel
### Approaches to Evade Effects of Lignocellulose-Derived Inhibitors  
In the process of the pre-treatment of lignocellulosic biomass, numerous inhibitors like hydroxybenzaldehyde, furfural, acetic acid, tannic acid, hydroxymethyl furfural, etc., are formed, which hinders the cellulolysis and fermentation processes [22]. The amount of inhibitor generated directly depends on the method of pre-treatment used and the composition of biomass [23]. Hence there is a need to design proper purification method to detoxify inhibitors from the media on a regular basis or engineer the microbes to make them tolerant to these inhibitors [24]. Genetic engineering is a reliable tool to develop tolerance towards inhibitors in microbes. One of the major inhibitors produced during the pre-treatment process is furfural. It is a more potent inhibitor when compared to the others. The presence of furfural in the microbial cells instigates the reactive oxygen species levels which in turn affects mitochondria [22; 25; 26]. Particularly, in _E. coli_, furfural presence depletes the NADPH levels because of an enzyme NADPH-dependent oxidoreductaseates (YqhD). This drop in NADPH levels eventually affects the assimilation of sulfate, leading to growth inhibition [27]. Expression of the transhydrogenase gene (pntAB) to interconvert NADH and NADPH, and supplementing cysteine, along with YqhD gene deletion, can enhance the tolerance of _E. coli_ towards furfural as well as hydroxymethyl furfural [27; 28]. Even oxidoreductaseates like FucO when overexpressed can enhance the furfural tolerance in _E. coli_[29]. [PERSON] et al. reported that overexpression of YqhD and FucO genes in _E. coli_ enhanced the production of iso-butanol and even increased the glucose utilization [30]. Most of the NADPH is generated from the pentose phosphate pathway. As the presence of furfural inhibits the pentose phosphate pathway, overexpressing glucose-6-phosphate dehydrogenase (which catalyzes the initial step of the pentose phosphate pathway) can enhance the furfural tolerance in _E. coli_[31]. In _E. coli_, with the help of new tools like genome mapping, novel genes like lpcA and thyA, associated with tolerance towards furfural, were discovered [32]. Numerous methods to remove inhibitors from the hydrolysate were already reported and some of those detoxification methods are liquid-solid extraction, chemical detoxification, vaporization, biological methods, liquid-liquid extraction, heating, etc. Chemical methods involve the addition of reducing agents like dithionite, sulphite, dithiothreitol, etc., and alkaline compounds like NaOH, NaBH\({}_{4}\), Ca(OH)\({}_{2}\), NH\({}_{4}\)OH, etc. [33; 34; 35]. In a study, a super critical liquid extraction strategy was employed to detoxify hydrolysate [36]. In a biological approach, enzymes like peroxididases can be employed to oxidize both non-phenolic and phenolic compounds, whereas lacases can be employed to oxidize phenolic compounds alone [37]. Apart from enzymes, microbes like _Pseudomonas putida Fu1_, _Ureibacillus thermosphaericus_, _Candidia Guillermodii_, and _Coniochaeta lignaria_ can also be employed for detoxification [38; 39; 40]. Depending on the type of biomass and its composition, the detoxification method has to be chosen. With the increase in the number of steps, the economic viability of the production decreases [41]. The microbial potential of inhibitor tolerance and inhibitor detoxification varies depending on their habitat. It is not necessary that the microbes that have high inhibitor tolerance also have high productivity. In a study by [PERSON] et al., a _S. cerevisiae_ strain isolated from a vineyard displayed resistance towards potent inhibitors like furan aldehyde and aliphatic carboxylic acids [42]. Emerging omics-related approaches, along with molecular adaptations, have the potential to enhance the microbial inhibitor resistance [43].