Ann Microbiol (2017) 67:727–737 DOI 10.1007/s13213-017-1300-4 ORIGINAL ARTICLE Statistically optimized production and characterization of vanillin from creosol using newly isolated Klebsiella pneumoniae P27 Geetanjali T. Mali1 & Pramod J. Kasabe1 & Padma Babulal Dandge1 Received: 8 March 2017 /Accepted: 19 September 2017 /Published online: 4 October 2017 # Springer-Verlag GmbH Germany and the University of Milan 2017 Abstract The current research study deals with the screening Keywords Response surface methodology . Vanillyl alcohol of a potent vanillin-producing microorganism among 96 iso- oxidase . 16S rDNA sequencing . FTIR . HRMS lated strains. Biochemical characterization and molecular identification confirmed that the isolated strain belongs to the Klebsiella pneumoniae bacteria, so it was denoted as Introduction Klebsiella pneumoniae P27. The optimization of medium components for the enhanced production of vanillin was car- Vanillin is one of the important flavoring agents and is a major ried out using two-stage statistical experimental designs, in constituent of the natural vanilla (Walton et al. 2003). The which the significant medium components for vanillin pro- most traditional source of natural vanilla is bean or pod of duction were screened using a Plackett-Burman experimental the tropical vanilla orchid (Vanilla planifolia). Vanillin has design. And the optimal levels of those noteworthy factors tremendous applications in the food, pharmaceutical, bever- were determined by using central composite design. The sta- age and perfume industries. More than 12,000 tons of vanillin tistical optimization of medium components resulted in in- are produced every year. Of that, <1% of it is extracted from creases in vanillin production and vanillyl alcohol oxidase vanilla pods; the remainder is synthesized via chemical pro- activity of 2.05-fold and 3.055-fold, respectively. The highest cesses (Priefert et al. 2003). Extraction of vanillin from vanilla vanillin production (30.88 mg/L) and vanillyl alcohol oxidase pods is a very expensive and laborious process. Hence, the activity (0.044 U/mL) was observed after 16 h of incubation in average cost of natural vanillin ranges from US $1200 to the presence of 0.26 mL/L creosol, 8.06 g/L yeast extract and $4000/kg and that of the synthetic vanillin is <US $15/kg 2.77 g/L NH4NO3 in the production medium. The optimally (Walton et al. 2003). Consumers are very conscious about produced vanillin was extracted and confirmed using FTIR their health and therefore prefer natural products over synthet- and LCMS spectral analysis. The results of the current study ic despite the higher market price (Schrader et al. 2004). To support a statistical process optimization approach as a poten- fulfill the enhanced industrial demand for natural vanillin, tial technique for the enhanced production of vanillin from biotechnological researchers are busy exploring ways to creosol by using newly isolated Klebsiella pneumoniae P27 biosynthesize vanillin. Biotechnology based approaches to bacterial strain. vanillin synthesis use readily available and economically fea- sible natural feedstocks (Walten et al. 2000). Several potential feedstocks, such as curcumin, phenolic stilbenes, eugenol, Electronic supplementary material The online version of this article isoeugenol, ferulic acid, have been reported. The controlled (https://doi.org/10.1007/s13213-017-1300-4) contains supplementary oxidation of lignin is another possible path to vanillin synthe- material, which is available to authorized users. sis by utilizing lignin waste (Yoon et al. 2005; Zhao et al. * Padma Babulal Dandge 2005;Overhageetal.2006; Shakeri et al. 2013). [email protected] Furthermore, eugenol and isoeugenol are essential oil compo- nents that are used widely for vanillin synthesis. Isoeugenol 1 Department of Biochemistry, Shivaji University, has been reported to be biotransformed into vanillin by Kolhapur, Maharashtra 416004, India Bacillus subtillis B2 (Shimoni et al. 2000). 728 Ann Microbiol (2017) 67:727–737 Extraction from natural vanilla pods and biotransformation further optimized by using central composite design (CCD) using natural feedstocks are quite expensive methods, so re- (Omran 2014;Facchinietal.2015; Jhample et al. 2015). After searchers turned to vanillin synthesis by a biocatalytic method successful production optimization, the produced vanillin was employing microbial enzyme vanillyl alcohol oxidase (VAO) extracted from fermentation broth for identification and (EC 1.1.3.38). VAO is an oxidoreductase enzyme, and was characterization. first reported in the fungus Penicillium simplicissimum CBS 170.90 (Jong et al. 1992), then purified and characterized from Byssochlamys fulva V107 (Furukawa et al. 1999). The recom- Material and methods binant strain Amycolatopsis sp. HR 167, containing the VAO gene (vaoA), has been reported as a very efficient Chemicals and reagents biotransformer to convert eugenol to coniferyl alcohol with the production of trace amount of vanillin (Overhage et al. Standard vanillin, vanillyl alcohol, creosol and horse radish 2006). peroxidase were purchased from Sigma (Bangalore, India). 4- VAO is a versatile biocatalyst for vanillin biosynthesis, Aminoantipyrine, phenol, hydrogen peroxide (H2O2) and oth- with a broad range of substrate specificity. It catalyzes vanillin er media components used in this study were purchased from synthesis using natural feedstocks, like creosol (2-methoxy-p- HiMedia and SD Fine Chemicals, Chennai, India. All the cresol), as a substrate. Creosol is a major component of creo- organic solvents (ethyl acetate, dichloromethane, chloroform, sote, which is obtained from heating wood or coal tar (van den formic acid and methanol) were procured from Qualigens Heuvel et al. 2001, 2004). Another potential feedstock for (Mumbai, India) and all other reagents were of analytical vanillin synthesis is vanillylamine derived from capsaicin (8- grade and obtained from local suppliers. methyl-N-vanillyl-6-nonenamide)—a pungent principle of hot red pepper (van den Heuvel et al. 2001). Isolation and culture conditions As the demand for vanillin is increasing day by day, most researchers have been in search of natural sources for vanillin The sampling was done at various locations involved in the production. Some earlier reports stated that certain fungal and wood processing and preservation industries as well as chem- bacterial species possess the ability to produce VAO—an en- ical and steel industrial sites of Kolhapur district, (M.S.), zyme responsible for vanillin biosynthesis. So it was hypoth- India, in order to isolate the vanillin-producing microorgan- esized that these active microbial cultures could be screened isms. The samples were subjected to a five-stage enrichment from the environment for natural vanillin production, and such technique, as described in our earlier reports (Kasabe et al. cultures could be directed to produce enhanced vanillin con- 2015). Briefly, the enrichment was achieved by maintaining tent during their growth by optimizing their metabolic condi- various vanillyl alcohol concentrations ranging from 0.02% to tions. Accordingly, the aim of the present study was to hunt for 0.1% in three repeated cycles for each concentration stage. 1% some environmental microorganisms with a potent ability to of each sample was inoculated in the 100 mL mineral salt produce vanillin. The study was also intended to optimize the medium containing g/L: KH2PO4,1.8;Na2HPO4,1.0; medium components for the increased production of vanillin NH4Cl, 2.0; (NH4)2SO4,0.1;MgCl2⋅6H2O, 0.07; 0.2 mL trace using these environmental isolates. With the designed objec- elements solution (Jong et al. 1990) and vanillyl alcohol as a tives and hypothesis, the screening, isolation and identifica- sole carbon source with a particular concentration. After the tion of the efficient bacterium were carried out here. The au- enrichment process, 100 μL serially diluted broth was spread thors have successfully screened and identified an proficient on the same medium containing 2.2% agar in sterile Petri VAO-producing bacterial strain from Klebsiella pneumoniae. plates. The inoculated plates were incubated at 30 °C for This newly isolated bacterial isolate has been designated as 24–48 h. After completion of incubation period, each isolated Klebsiella pneumoniae P27. This is the first report of vanillin colony was further streaked out separately on the sterile Petri production by using an environmental isolate, K. pneumoniae plates containing same solid medium and incubated at 30 °C P27. Microbial screening for the potency of VAO production for 24 h. Then, the grown colonies of individual isolated bac- was achieved by a rapid colony staining method (Kasabe et al. teria were further subjected to screening for VAO production 2015). This method was modified by us and applied for the as this enzyme plays a key role in vanillin biosynthesis. first time to the screening of VAO producers. The pre- optimization medium components and conditions were taken Screening of VAO-producing bacteria from earlier reports (Van Rooyen 2012). Statistical optimiza- tion studies were carried out using response surface method- VAO-producing potent bacteria were screened using a ology (RSM). The significant medium components affecting H2O2-based rapid colony staining method with certain vanillin synthesis by this strain were selected by employing modifications (Kasabe et al. 2015). In order to select a Plackett-Burman