Biodetoxification of organic contaminants using microorganisms isolated from gold (Au) mine tailings

Abstract

Benzonitrile (C6H5C≡N) is a synthetic nitrile with vast applications as a solvent and intermediate in the manufacturing of drugs, perfumes, dyes, rubber, textiles, agrochemicals, resins, and specialty lacquers. Benzonitrile is the most used form of synthetic nitriles. The discharge of these synthetic nitriles directly into the environment via wastewater is an alarm for possible health hazard, pointing to the toxicity of these compounds some of which possess carcinogenic and mutagenic traits. Therefore benzonitrile (synthetic nitrile) waste requires treatment before being discharged into the environment. The physical and chemical treatment methods to treat nitrile contaminated waste is usually expensive and could lead to the production of toxic by-products that require further treatment. Hence bioremediation process which is an environmentally friendly, cost effective and does not produce toxic by-products is an effective alternative and preferred method of decontamination. The hypothesis of the study was that microorganisms isolated from Au mine tailings are able to degrade environmental contaminants (nitriles). The study approach included isolating and identifying microorganisms from gold (Au) mine tailings. Followed by the extraction and identification of organic compounds foun in Au mine tailings. A solid-liquid extraction method was used with dichloromethane (DCM), ethyl acetate and hexane as extraction solvents. Compounds were identified via Liquid Chromatography quadrupole –Time-of-Flight Mass Spectrometry (LC-QTOF-MS), for non-volatile compounds, and Gas Chromatography Quadrupole Time of Flight Mass Spectrometry (GC-QTOF-MS), for volatile compounds. This study focused on the degradation of benzonitrile as the contaminant of interest. Preliminary biodegradation trials were conducts whereby the microorganisms were incubated in a minimal media (M.M) which contained aminobenzonitrile, as the sole source of carbon and nitrogen, for 72 hrs., shaking at 180 rpms, pH 7 and 30 °C. Ammonia was monitored as a product of nitrile degradation using Merck ammonium (NH4+) (00683) test kit and quantified using a Merck Spectroquant Nova 60. A bacterial consortium was formed which consisted of bacterial species capable of degrading aminobenzonitrile thereby yielding ammonium. The bacterial consortium was subjected to optimization studies using response surface methodology (RSM) to achieve optimal physiochemical parameters. Biodegradation products were identified using GC-MS and allowed for pathway determination based on products formed. The isolated microorganisms from Au mine tailings belonged to the Bacillus and Paenibacillus genus as identified by the 16S rRNA gene sequencing. The microorganisms isolated from Au mine tailings were identified as P. shunpengii, B. pumilus, B. safensis, B. thuringiesis and B. cereus. A total of 27 organic compounds of interest were extracted and identified, compound classes with environmental significance such as; ethers, pyridines, pyrimidines, phenols and nitriles were amongst these compounds of interest. Preliminary biodegradation trials conducted indicated that all bacterial species isolated from Au mine tailings possess the ability to biodegrade aminobenzonitrile by detecting ammonium on the Merck spectroquant. The physical and chemical parameters of biodegradation of aminobenzonitrile were optimised using RSM with optimised conditions for temperature, pH and substrate (aminobenzonitrile) concentration being 35 °C, 9.46 and 50 mg/L respectively, with the maximum biological removal efficiency (BRE %) of 73% over a 72 hrs. period. 2-piperidinone was identified as a biodegradation product of aminobenzonitrile by the bacterial consortium, furthermore, 2-piperidinone is a cyclic amide which indicates that the bacterial consortium utilises the NHase enzymatic pathway of nitrile degradation.