1. Introduction
The compound 2,3-Butanediol (2,3-BD) is an industrial platform chemical with vast industrial applications, particularly for its potential use in the synthesis of 1,3-butadiene (1,3-BD), a monomer of synthetic rubber. Other applications of 2,3-BD include the synthesis of methyl ethyl ketone (MEK), a fuel additive with a higher heat of combustion than ethanol, and as solvents for lacquers and resins [
1]. Furthermore, 2,3-BD finds applications as an antifreeze due to its low freezing temperature of −60 °C [
2], an ink additive, as a chemical feedstock for the production of acetoin and diacetyl, vital flavor enhancers in the food industry [
3], and as an additive in aviation fuel. Due to the finite nature of petroleum and the need to reduce society’s dependence on petroleum-derived feedstocks for industrial processes, it has become imperative to develop sustainable feedstocks such as 2,3-BD from renewable resources. At present, 2,3-BD is produced from hydrocarbons by the cracking of butane and 2-butene in which the resulting product is further hydrolyzed to 2,3-BD [
4,
5]. Recently, the often encountered instabilities in crude oil price have re-ignited interest in fermentative 2,3-BD production from cheap renewable feedstocks. To reach this goal, multifarious research efforts are currently underway to increase the yield, titer, and productivity of microbe-derived 2,3-BD. These include metabolic engineering of producer organisms to produce and tolerate higher 2,3-BD concentrations, and the optimization of fermentation media components and conditions for maximal 2,3-BD accumulation in the broth. In this study, we sought to optimize 2,3-BD production by
Paenibacillus polymyxa DSM 365 (hereafter referred to as
P. polymyxa) by assessing the impacts of both medium components and fermentation conditions on 2,3-BD accumulation.
P. polymyxa was chosen for this study due to its non-pathogenicity and capacity to produce 98% levo 2,3-BD; the industrially preferred 2,3-BD isomer due to its properties, which make it amenable to important chemical reactions that generate key industrially applicable products, such as dehydration to 1,3-BD (for synthetic rubber production), dehydrogenation to acetoin or diacetyl (flavor enhancers and essential components in fragrances), ketalization to Methyl tert-butyl ether (fuel additive), and esterification to 2,3-BD diester (used as a precursor in the synthesis and compounding of cosmetics, drugs, and thermoplastic polymers [
1,
6].
To assess the 2,3-BD production capacity of
P.
polymyxa, we first conducted batch fermentations in 100 mL Pyrex bottles, which resulted in a maximum 2,3-BD concentration of 24 g/L [
7]. Batch fermentation in the bioreactor (6-L) produced 27 g/L 2,3-BD, whereas fed-batch fermentation (in the bioreactor) resulted in 47 g/L, despite excess glucose supply [
7]. Therefore, we rationalized that in addition to other factors, 2,3-BD-mediated feedback inhibition might pose a significant roadblock to the accumulation of 2,3-BD during fermentation, and this assumption was confirmed by assaying 2,3-BD toxicity against
P. polymyxa in fermentation cultures [
7]. We observed that 2,3-BD exerts a concentration-dependent toxicity on
P. polymyxa with ~50 g/L 2,3-BD as the toxic threshold above which cell growth stalls considerably and the accumulated 2,3-BD is converted backwards to acetoin, the precursor of 2,3-BD; most plausibly to alleviate 2,3-BD-mediated toxicity [
7]. In addition, a significant portion of sugar substrates are diverted to exopolysaccharides (EPS) production during 2,3-BD fermentation, thereby lowering 2,3-BD yield and complicating its recovery from the fermentation broth [
7,
8]. Therefore, if
P. polymyxa 2,3-BD fermentation is to reach an industrial-scale, it is critical to determine the optimal conditions and medium components necessary for marked 2,3-BD accumulation and tolerance during fermentation. Further, cheaply available substrates such as glycerol, which is currently accumulated in excess as a by-product of biodiesel production [
9,
10], holds significant promise towards improving the economics of 2,3-BD fermentation, either as a sole carbon source or as a supplement to glucose or other sugars. In fact, glycerol has been shown to support 2,3-BD production by
Klebsiella pneumoniae as a sole carbon source [
11,
12]. Thus, we investigated the optimal conditions and medium components for high 2,3-BD production by
P. polymyxa using a glycerol-supplemented medium. In addition to lowering the overall substrate cost, glycerol catabolism furnishes the cell with additional NADH [
13,
14], which supplies extra reducing power for 2,3-BD dehydrogenase, the final enzyme of the 2,3-BD pathway, which consumes NADH during the conversion of acetoin to 2,3-BD [
15].
Previous optimization studies focused largely on enhancing 2,3-BD production. These studies either targeted medium components only, or fermentation conditions without a holistic evaluation of both parameters (medium components and fermentation conditions; [
8,
16]). Medium components and fermentation conditions such as temperature, inoculum size, pH, and aeration rate most reasonably interact during fermentation to engender 2,3-BD production. Therefore, in this study, select medium components and fermentation conditions were assessed collectively for their capacity to enhance 2,3-BD production by employing various optimization strategies. Plackett-Burman experimental design, path of steepest ascent method, Box-Behnken experimental design, and response surface methodology strategies were employed to optimize 2,3-BD production by
P. polymyxa. The medium components tested in this study include yeast extract, tryptone, ammonium acetate, ammonium sulfate, and crude glycerol; whereas the fermentation conditions that were extensively investigated include temperature and inoculum size. These factors were shown to influence 2,3-BD production by
P. polymyxa from our one-factor-at-a-time experiments.
4. Conclusions
Based on the results from the Box-Behnken design and response surface methodology, an optimized medium (7 g/L crude glycerol included) and culture conditions for enhanced 2,3-BD production by
P. polymyxa were developed. The optimized conditions were validated in batch and fed-batch fermentations, leading to the production of 51.10 and 68.54 g/L, respectively, of 2,3-BD. These account for 47% and 31% increases in 2,3-BD production in batch and fed-batch cultures, respectively, with attendant diminished generation of competing co-products, especially EPS, relative to the non-optimized fermentations. The results presented here underline the interplay between medium components, culture conditions, and product-mediated toxicity (feedback inhibition), as the earlier determined toxic threshold of 2,3-BD (50 g/L) on
P. polymyxa in a non-optimized medium [
7] was significantly exceeded in this work (68.54 g/L). However, it is worth mentioning that glycerol was incorporated in the fermentation medium used in this study, which may contribute to 2,3-BD biosynthesis via improved NADH regeneration, especially in the optimized medium, relative to the un-optimized control medium. Collectively, we demonstrate that lower amounts of the expensive organic nitrogen sources, tryptone and yeast extract, can be used for optimal 2,3-BD production. This represents a significant reduction in operating costs in the efforts to commercialize biological production of 2,3-BD.