Screening, isolation and molecular identification of biodegrading mycobacteria from Iranian ecosystems and analysis of their biodegradation activity

Abstract

Anthropogenic origin pollutants including pesticides, heavy metals, pharmaceuticals and industry chemicals impose many risks to human health and environment and bioremediation has been considered the strategy of choice to reduce the risk of hazardous chemicals. In the current study, we aimed to screen and characterize mycobacteria from the diverse range of Iranian aquatic and terrestrial ecosystems with harsh and unfavorable environmental conditions that can be utilized for biodegradation of target pollutants. Mycobacteria were isolated from a collection of 90 environmental samples and identified to the species level using conventional microbiological and molecular methods including the PCR amplification of hsp65 and sequence analysis of, 16S rRNA genetic markers. The growth rate of the isolates in presence of pollutants, chromatography, Gibbs and turbidometric methods were used to assess their biodegradation activity. A total of 39 mycobacterial isolates (43.3%) were recovered from 90 samples that belonged to 21 various species consisting of M. fortuitum; 6 isolates, M. flavescens and M. paragordonae; 4 isolates each, M. monacense, M. fredriksbergense and M. aurum; 2 isolates each, 7 single isolates of M. conceptionense, M. porcinum, M. simiae, M. celeriflavum, M. novocastrense, M. neoaurum, M. obuense and 12 isolates that belonged to 8 unknown potentially novel mycobacterial species. The isolates were categorized in three groups based on their bioremediation activity, i.e., 5 (12.8%) organisms without biodegradation activity, 20 (51.2%) organisms with previously reported biodegradation activity, and 14 (35.9%) organisms that showed biodegradation activity but not previously reported. Our results showed that the Iranian ecosystems harbor a good reservoir of diverse mycobacterial species with biodegrading potentiality for neutralizing environmental chemical pollutants.

Keywords: 16S rRNA sequencing; Bioremediation; Mycobacterium; Phylogeny.

Fig. 1

Geographic distribution of sampling site from Iran’s ecosystems

Fig. 2

16S rRNA sequence based phylogenetic tree for Iranian biodegrading NTM isolates and the nearest validated species of mycobacteria by using the neighbor-joining method. The figures at each node represent bootstrapping values. The tree was rooted with N. asteroides

Fig. 3

Growth curves of Iranian isolates of mycobacteria over a 24 h. Incubation period at 30 °C in the presence of PAHs

Fig. 4

HPLC chromatograms of PAHs mix solution by selected mycobacterial isolates, a control samples, b after 144 incubation at 30 °C. (1) Naphthalene, (2) acenaphthylene, (3) acenaphthene, (4) fluorene, (5) phenanthrene, (6) anthracene, (7) fluoranthene, (8) pyrene, (9) benzo[a] anthracene, (10) chrysene, (11) benzo[b]fluoranthene, (12) benzo[k]fluoranthene, (13) benzo[a]pyrene, (14) indeno[1,2,3-cd]pyrene, (15) dibenzo[a,h]anthracene

Fig. 5

Growth curves of Iranian mycobacterium isolates over a 24 h. Incubation period at 30 °C in the presence of phenol

Fig. 6

Growth curves of Iranian isolates of mycobacteria over a 24 h. Incubation period at 30 °C in the presence of sodium sulfate