INTRODUCTION
Lactic acid is currently one of the essential chemical commodities due to widespread commercial applications in the pharmaceutical, cosmetics, chemical, and food industries (Martinez et al., 2013). With a worldwide production of 400,000 tons per year, lactic acid is considered a top-value platform chemical (Becker et al., 2015; Choi et al., 2015). Lactic acid is the precursor for poly-lactic acid–a popular biodegradable plastic with physicochemical, thermal, and mechanical properties that are competitive with traditional petroleum-based polymers, such as polypropylene (PP) and low-density polypropylene (LDPE) (Ajala et al., 2020; Nduko & Taguchi, 2019). In recent years, about 90% of industrial lactic acid has been manufactured via fermentation rather than chemical synthesis (Dusselier et al., 2013). The former strategy is more environmentally friendly, less energy-intensive, and yields an optically pure product.
Production of lactic acid is even more lucrative when low-cost feedstock is employed in the process. Due to its abundant availability, lignocellulosic biomass has been a prominent material for producing of various bio-based chemicals (Chen et al., 2020; Kim et al., 2019; Tian et al., 2020). Moreover, sugarcane bagasse (SCB) generated by the sugar and alcohol industry is ideal for this objective. Data show that more than 1.8 billion tons of sugarcane were produced worldwide in 2017 (de Matos et al., 2020). About 31.8% composition of sugarcane is bagasse–a residual fraction obtained after the milling process (Cortez et al., 2020). Considering its availability, the utility of SCB has become the subject of numerous studies related to establishing a circular bioeconomy and sustainable industries.
Despite the compelling benefits, the utilization of lignocellulose as a feedstock for bioprocessing has several bottlenecks. The generation of various by-products during the pre-treatment process is one of the challenging issues. These by-products, which include furan derivatives (furfural, 5-HMF, etc.), weak organic acids (formic acid, acetic acid, etc.), and phenolic compounds (vanillin, syringaldehyde, ferulic acid, etc.) (Ling et al., 2014), inhibit microbial metabolism, which renders fermentation and diminishes productivity. Biological, physical, and chemical methods have been explored in a quest to detoxify this process (Palmqvist & Hahn-Hägerdal, 2000). However, these methods necessitate additional equipment, which drives up cost. These processes also decrease the quantity of fermentable sugar (Hahn-Hägerdal et al., 2007). Therefore, employing a stress-tolerant microorganism in the fermentation step would undoubtedly be more desirable than modifying the operational process.
During lactic acid bioproduction, the product itself may cause additional stress for a microbial host. Many microorganisms, particularly bacteria, suffer growth-rate inhibition under highly acidic conditions. Commonly, neutralizing agent, such as calcium carbonate (Yang et al., 2015), is added to maintain the pH of the medium. In addition to increasing cost, neutralizing agents are often toxic for microorganisms. In addition, calcium salts generally have low solubility in water. Therefore, they can easily mix with biomass and complicate the subsequent downstream process. Calcium salts also react with lactic acid to form calcium lactate. An acidification step using sulfuric acid followed by subsequent purification is needed to obtain the free form of lactic acid. This acidification process generates a harmful by-product known as gypsum, which must be disposed of in landfills (Komesu et al., 2017) and could cause additional environmental issues. Approximately one ton of gypsum is formed per ton of lactic acid production (Dusselier et al., 2013). Hence, from both industrial and environmental perspectives, eliminating the use of neutralizing agents would be advantageous. It can be achieved by employing a robust microbial host during the fermentation process.
Various approaches to obtain a robust microorganism have been proposed. Tolerance engineering by genetic modification is an example of common tools to enhance strain robustness. For instance, the co-expression of both TAL1 and ADH1 in Saccharomyces cerevisiaeenhances ethanol production in a medium containing furfural (Hasunuma et al., 2014). Co-overexpression of both HAA1 and PRS3 and disruption of FPS1 also improve acetic acid tolerance (Cunha et al., 2018; Zhang et al., 2011). Nevertheless, due to the complexity of biomass chemical composition, the successive introduction of a large number of tolerance-related genes could be cumbersome.
On the contrary, rather than performing extensive genetic engineering, the strategy proposed here focuses on increasing the lactic acid production of an originally robust microorganism via a handful of genetic modifications. In the present study, we selected newly isolated yeast, identified as S. cerevisiae BTCC3, obtained from the screening of Ascomycota yeasts deposited in Indonesian Culture Collection (InaCC). This strain has the ability to survive at relatively low pH (up to 2.5), as well as the presence of lignocellulose-derived inhibitors such as furfural, formic acid, acetic acid, etc. However, akin to other yeasts, this strain lacks the metabolic pathway for lactic acid production. Therefore, we introduced an exogenous L-LDH gene to enable lactic acid fermentation from glucose as an example. This experiment intended to construct a microbial strain with phenotypes suitable for utilizing lignocellulosic biomass as a carbon feedstock, such as SCB. Also, we considered the potential of this recombinant strain to ferment glucose to lactic acid without the use of a neutralizing agent.