Biodegradation of phenanthrene using adapted microbial consortium isolated from petrochemical contaminated environment

Abstract

In developing countries like India, there are many industrial areas discharging effluent containing large amount of polyaromatic hydrocarbon (PAH) which causes hazardous effect on the soil-water environment. The objective of this study was to isolate and characterize high-efficiency PAH-degrading microbial consortium from 3 decade old petrochemical refinery field located in Nagpur, Maharashtra with history of PAH disposal. Based on biochemical tests and 16S rDNA gene sequence analysis the consortium was identified as Sphingobacterium sp., Bacillus cereus and a novel bacterium Achromobacter insolitus MHF ENV IV with effective phenanthrene-degrading ability. The biodegradation data of phenanthrene indicates about 100%, 56.9% and 25.8% degradation at the concentration of 100 mg/l, 250 mg/l and 500 mg/l respectively within 14 days. The consortium and its monoculture isolates also utilized variety of other hydrocarbons for growth. To best of our knowledge this is the first time that Achromobacter insolitus has been reported to mineralize phenanthrene effectively. GC–MS analysis of phenanthrene degradation confirmed biodegradation by detection of intermediates like salicylaldehyde, salicylic acid and catechol. All the results indicated that the microbial consortium have a promising application in bioremediation of petrochemical contaminated environments and could be potentially useful for the study of PAH degradation and for bioremediation purposes.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) constitute a group of priority environmental pollutants in soils and sediments which are of concern because of their toxic, mutagenic and carcinogenic effects [1]. Some of these PAHs are considered to be possible or probable human carcinogens, and their distributions in the environment causes hazard to human beings [2]. Exposure to PAHs constitutes a significant health risk for people living in industrialized areas of the world [3]. For pollution abatement, these organic compounds are generally removed through physico-chemical methods. These Conventional chemical or physical technologies have inherent drawbacks due to high operating cost, difficulty in operation and production of secondary pollutants [4]. Biodegradation involving the use of naturally occurring microorganisms presents an alternative, yet better approach for the degradation of PAH [5].

Bacteria of the genera Sphingomonas, Burkholderia, Pseudomonas, Acinetobacter, Rhodococcus and Mycobacterium are well-known PAH-degraders. They successfully mineralized both low molecular weight PAHs and high molecular weight PAHs [6], [7]. Although pure cultures of several PAH-degrading bacteria can readily utilize PAHs as a carbon source [8], [9], degradation results may improve if a mixed bacterial culture or bacterial–fungal consortium was used [10], [11], [12]. Generally, biodegradation using a pure strain does not represent the actual behavior of environmental microorganisms during bioremediation in natural PAH-contaminated soils, because in nature, bioremediation depends on cooperative metabolic activities of mixed microbial populations. One important advantage of using microbial consortia is they possess multiple metabolic capacities that increase the efficiency of the bioremediation process [13].

Phenanthrene is among the 16 PAH listed as primary pollutants by US Environmental Protection Agency (EPA) [14] and is known to be a human skin photosensitizer and mild allergen [15]. In the present study we have evaluated the capacity of microbial consortium (Sphingobacterium sp., Bacillus cereus and a novel bacterium Achromobacter insolitus MHF ENV IV) enriched from a petrochemical refinery field to degrade and mineralize different concentrations of solid phenanthrene as a model PAH in liquid medium. The potential phenanthrene degraders were isolated and identified by different morphological, physiological, biochemical assays and 16S rDNA technology. Further the phenanthrene biodegrading capability of the consortium was assessed and the potential pathway inducers, which are produced as intermediates during PAH degradation such as salicylaldehyde, salicylic acid and catechol was detected by GC–MS to determine biodegradation. The increase in the microbial count and the changes in Chemical Oxygen Demand (COD) and Biological Oxygen Demand (BOD) were monitored and quantified as an indicator for growth and proliferation of microorganisms along the degradation of the phenanthrene in the mineral medium. The growth of the isolates on other hydrocarbon sources was also evaluated to determine the cooperative metabolic activity of mixed microbial population and the adaptability of microorganisms to survive in hydrocarbon contaminated area.