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.