Continuous depletion of petroleum fuel-reserves is one of the prime concerns in this era. Despite of oil reserve availability, current estimates show that accessing them will become extremely difficult in a few decades [1,2]. Due to this, the bio-refinery systems that integrate biomass conversion processes and equipment to produce fuels, power, and chemicals from annually renewable resources are at the stage of worldwide development [3]. Many chemicals that could only be produced by chemical processes in the past can now have the potential to be generated biologically using renewable resources. Microbial production of 2,3-butanediol (2,3-BD) is one such example [4-6]. Interest in this bioprocess has increased remarkably because 2,3-BD has a large number of industrial applications, and microbial production will alleviate the dependence on oil supply.

2,3-BD can be produced efficiently via mixed acid fermentation with prokaryotes such as Klebsiella pneumonia Klebsiella oxytoca Enterobacter aerogenes Serratia, and Bacillus polymyxa. In these bacteria, pyruvate is first converted into α-acetolactate by acetolactate synthase. In anoxic state, α-acetolactate decarboxylase catalyzes the conversion of α-acetolactate into acetoin. 2,3-Butanediol is resulted from the reduction of acetoin by butanediol dehydrogenase. It exists in three stereoisomers, the dextro- (d-), levo- (l-) and meso-forms. The available reports reveal that quantitative estimation of 2,3-BD in fermentation broth is carried out only by two methods: high-performance liquid chromatography (HPLC) equipped with an Aminex HPX-87H column or by Alltech IOA-2000 organic acid column [7,8] and gas chromatography (GC) equipped with a glass column packed with Chromosorb 101 [9-11]. Although HPLC can detect 2,3-butanediol along with other by-products (1,4-propanediol, lactic acid, butyric acid, acetic acid, acetoin, 1,3-butanediol, ethanol) simultaneously; however, the process is expensive, time consuming, tedious and uneconomical and together with the limited column lifetime it fails to meet the criteria necessary to become a rapid screening procedure. Therefore it was of utmost important to have an efficient, authentic, reliable and inexpensive screening procedure to hunt for the most potent 2, 3-butanediol producer/s. Therefore, in the present investigation, the objective is to design a simple and fast protocol for 2,3-butanediol detection with wide applicability to harness the undoubted commercial potential of microorganisms to produce 2,3-butanediol as the major end product.

Materials

2, 3-butanediol, 1, 4-propanediol, 1, 3-butanediol, lactic acid, butyric acid, acetic acid, acetoin and ethanol were purchased from Sigma Chemicals (St. Louis, USA). TLC plates (Silica gel 60 F254) were purchased from E. Merck Ltd., Germany.

Fermentation conditions

Thirty two bacterial cultures, including different strains of Klebsiella pneumoniae were procured from the laboratory stock culture collection, Department of Microbiology, University of Delhi South Campus, New Delhi, India and screened for 2,3-butanediol production. The growth medium consisting of glucose 20 g/l, (NH₄)₂HPO₄ 5 g/l, MgSO₄ 0.3 g/l, KCl 1.0 g/l, pH 7.0 was used to grow and maintain the bacterial cultures.

The bacteria were grown in 250 ml Erlenmeyer flasks containing 50 ml production medium with the composition (g/L): glycerol, 25.0; Tryptone 10.0; K₂HPO₄•3H₂O 5.0; KH₂PO₄, 3.48; MgCO₃, 150 mM; CaCl2•2H2O, 0.20; CoCl2•6H2O, 0.004, MgCl2•7H2O, 0.40 and Na₂S•9H₂O, 0.02 at pH 7.0 ± 0.2. Incubation was carried out at 37°C, 150 rpm for 72 h. The samples obtained after 72 h of fermentation were centrifuged at 10,000×g for 20 min to pellet the cells, followed by filtration using syringe filters (pore size 0.22 μm, Mdi filters, India). The pellet thus obtained was used for growth estimation and supernatant was used for 2, 3-butanediol estimation.

Thin-layer chromatography

One-dimensional chromatography on pre-activated silica gel TLC plates was carried out in saturated tanks. Samples were spotted 1.5 cm from the lower edge of the plate and at least 1.0 cm from the lateral border. The chromatograms were developed by the ascending technique with the desired mobile phase. The solvent front was drawn 10.0 cm from the application line.

Analytical methods

The concentration of 2,3-butanediol and other by-products was determined by the GC method described by [9].

GC–MS analysis

GC–MS analysis was performed with the Shimadzu QP-2010 Plus with Thermal Desorption System. Compounds were separated on a capillary column, the injector temperature was 260°C, and 1 μL samples were injected in split mode. The split ratio was 100; the total flow was 125.20 mL/min. The oven temperature was maintained at 40°C for 1 min after injection, then programmed at 10°C min^-1 to 200°C, which was held for 4 min, and then at 10°C min^-1 to 200°C, which was held for 4 min. 2,3-Butanediol was identified by the use of standard database of mass spectra.

Selection of solvent system

The choice of best solvent system and the optimization of its composition are very important because the chromatographic separation is difficult to achieve. In the present investigation, 10 μl of standards of 2, 3-butanediol, 1, 4-propanediol, 1, 3-butanediol, lactic acid, butyric acid, acetic acid, acetoin and ethanol (50 mg/ml) were spotted individually and as a mixture on two TLC plates. These plates were then run in two solvent systems, separately, which were (a) chloroform:methanol 90:10 (b) hexane: ethyl acetate: glacial acetic acid 70:30:1. The solvents were allowed to run up to two-thirds of the plate height followed by its development using vanillin reagent. It was observed that with the solvent system ‘b’, except 2,3-butanediol, none of the compounds moved on to the TLC plates, however, a smear was observed with the solvent system ‘a’ and each standard spotted were not distinct and the mixture of these standards produced a smear due to overlapping. To further maximize the efficiency of the procedure and to enhance the resolution of 2,3-butanediol, different permutation combination of mobile phase were tested. Three different ratios, (A) 70:30:0.5, (B) 70:30:1.0, (C) 70:30:1.5, of the hexane: ethyl acetate: glacial acetic acid was used under the same conditions. It is clearly evident from the Figure 1 that with the ratio of (A) 70:30:0.5, no distinct resolution was achieved. Only one distinct spot corresponding to Rf 0.68 was observed. With the ratio (B) 70:30:1.0, a bit cleaner plate was observed. Finally, with the ratio (C) 70:30:1.5, much clearer plates were obtained with distinct 2,3-butanediol spots. The above edge of this solvent system is that the procedure is specific for 2,3-butanediol and other by-products were not detected.

Figure 1: Influence of solvent system on the colour development of 2,3-butanediol.

Test of sensitivity of the TLC method

A detection method is only reliable if it is able to detect even a minimal quantity of the desired product. Here, to examine the critical concentration of 2,3-butanediol required to give a visible spot on TLC plates, 10μl of different concentrations (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 mg/ml) of 2,3-butanediol was spotted on TLC plates and run in hexane: ethyl acetate: glacial acetic acid (70:30:1.5) followed by a similar process of colour development. The results obtained are presented in Figure 2 which shows that even at 10 mg ml^-1, a distinct blue spot appeared. Therefore, it can be inferred that this process is highly sensitive and can be efficiently used for the screening of 2,3-butanediol producers.

Figure 2: Sensitivity test for different concentrations of 2,3-butanediol run in the solvent.

Screening of 2,3-butanediol producers using TLC procedure

To authenticate the efficiency of the proposed TLC method, a total of 32 randomly selected bacterial cultures were used. For each culture, fermentation broth obtained at 72 h of incubation was used. Ten microliters of each of these culture supernatants (72 h) with the standard 2,3-butanediol as a reference was spotted and resolved on the TLC plates. The plates were developed under the same conditions described. The results of TLC are listed in (Table 1) which shows that out of 32 only 12 were 2,3-butanediol producers. To further authenticate the accuracy and efficiency of the proposed procedure, the samples were analysed by GC (Table 2). Among the 12 strains producing 2,3-butanediol, strain no. UDSC 6 was found to be a potent producer resulting in 2.97 g/L of 2,3-butanediol production as detected by GC. The results of prominent 2,3-butanediol producers along with a two negative ones are presented in Figure 3 which shows that in five of the culture filtrates (after 72 h), a blue coloured spot corresponding to the R_f value of standard 2,3-butanediol (0.68 ± 0.02) was observed. Two other cultures that were non 2,3-butanediol producers did not show any blue spot corresponding to 2,3-butanediol. Results obtained were coherent with the GC data. On the basis of the results, it can be concluded that the proposed method is very rapid and reliable and such a fast method has not been reported so far for screening of 2,3-butanediol producers. This TLC procedure will allow even hundreds of samples to be analysed rapidly as it requires only 15 min for each TLC run. Moreover, the method is economical as no special equipment or chemicals are required.

Table 1: Analysis of the bacterial samples for 2,3-butanediol on TLC plates.

Table 2: Evaluation of positive 2,3-butanediol producers on TLC plates and by GC method.

Figure 3: TLC analysis of the concentrated samples obtained from seven bacteria for 2,3- butanediol production after 72 h of incubation.

GC–MS analysis

2,3-butanediol scrapped from the TLC plates was confirmed by GC– MS in split mode. The mass spectrum showed it was 2,3-butanediol.

This method is simple, reliable and well suited for the routine laboratory assay of a large number of samples. Using this protocol, hundreds of samples can be analyzed in a day’s time. Moreover, this method is economical as no special equipments or chemicals are required. Realizing the importance of 2,3-butanediol and its application in different industries, devising the methods for rapid screening of 2,3-butanediol producers is of great importance.

Authors sincerely acknowledge and thank the Department of Science and Technology (DST), Govt. of India for providing the financial support to carry out this work. The infrastructural support provided by Technology Based Incubator (TBI), South Campus is also highly acknowledged