Simultaneous Production of Antimicrobial Agent (2- hydroxyacetohydrazide) and Phenol Degradation Via Microbial Technology

Abstract

Toxic pollutants of varying anthropogenic sources have been showing adverse effects on growth and survival of biological systems. Microorganisms play a vital role in the biotransformation of these toxic pollutants to less complex chemical compounds which can be used in various industrial applications. These organisms have been evolved to degrade pollutants in different metabolic pathways, and some of them have adapted to consume these chemicals as either growth supplements or elemental sources. The identification and investigation of such organisms is a great challenge and will be beneficial for recycling of pollutants to useful products, thereby reducing the need for expensive substrates. Phenol degrading organisms were effectively isolated from petroleum-contaminated soil. Based on their resistivity to phenol up to 1500 mg l-1, the bacterial strains were isolated as R1, R2, R3, R4, and R5. Morphological studies showed that most of these organisms as Grampositive, rod-shaped, and aerobic. These five isolated organisms showed maximum similarity with Pseudomonas stutzeri NCG1, Bacillus flexus strain MS14-1, Bacillus thuringiensis strain 2PR56-10, Bacillus anthracis strain IHB B 18197, and Bacillus thuringiensis strain Bt 2 based on 16S rRNA sequencing. Preliminary experimental conditions such as pH, temperature, aeration, inoculum age, and inoculum volume were studied for maximum phenol degrading organisms along with the mixed culture. Under optimized conditions, Gram-negative P. stutzeri (Genbank Ac. No. MG230258) showed the maximum capability to degrade phenol up to 81.8% at 500 mg l-1 initial concentration within 84 h.
Moreover, phenol degradation by Pseudomonas stutzeri was optimized using response surface methodology (RSM) for various parameters viz. pH, temperature, phenol concentration, and yeast extract concentration. Optimized conditions of pH, temperature, and yeast extract concentration were used to determine the kinetic parameters of phenol degradation using various models. The analysis of kinetic data suggests that the Haldane substrate inhibitory model can be used to fit experimental data. Additionally, phenol tolerant Pseudomonas stutzeri produced cis, cis-muconic acid, which is a metabolic intermediate signifying the organism followed ortho pathway. Pseudomonas stutzeri also exhibited antimicrobial activity against Gram-positive bacteria like Bacillus cereus (MTCC 430), Bacillus subtilis (MTCC 1133), etc. The culture medium of Pseudomonas stutzeri containing various metabolites was analyzed using Gas chromatography-mass spectrometry (GCMS), and 2-hydroxyacetohydrazide (HAH) was found to be active antimicrobial substance in the culture medium after 72 h of fermentation. The organism P. stutzeri is capable of producing HAH in the culture medium supplemented with 500 mg l-1 of phenol.
The HAH was purified using silica gel column chromatography by subsequent collection of fractions, and the purified HAH was analyzed using Fourier transform infrared spectroscopy (FTIR). Additionally, the concentration of HAH present in the culture medium was assayed using HPLC and found to be 357 μg ml-1. The action of HAH against B. subtilis (MTCC 1133) was determined using tunneling electron microscopy (TEM) analysis. The minimum inhibitory concentration (MIC) of the HAH was found to be 40 μg ml-1 against B. subtilis (MTCC 1133). The optimum parameters for the production of HAH and degradation of phenol were determined using strain improvement techniques. The resultant strain obtained from strain improvement was employed in a 2-liter batch reactor for phenol degradation and HAH production simultaneously.