Nitrilase gene detection and nitrile metabolism in two bacterial strains associated with waste streams in Lagos, Nigeria
Authors: Ogunyemi Adewale Kayode , Buraimoh Olanike Maria , Ogunyemi Bukola Caroline , Samuel Titilola Aderonke , Ilori Matthew Olusoji , Amund Olukayode Oladipo 
Published: May 2022

Abstract
Background
The use of nitrile compounds is usually high, particularly in chemical industries, which calls for serious concern because of their relevance to the environment. The essential role of nitrilases in the bioremediation of harmful nitriles from environmental wastes cannot be overemphasized. The study aimed to unveil the biodegradative potentials of bacterial strains associated with the degradation of nitrile pollutants.

Methods
Bacterial strains capable of utilizing glutaronitrile as the sole source of carbon and nitrogen were isolated from solid waste leachates by a selective enrichment culture technique. The test organisms were grown in mineral salts medium (MSM), and the metabolic products were determined using gas chromatography-flame ionization detection (GC-FID). The nitrilase gene was amplified by polymerase chain reaction (PCR) and by using appropriate primers.

Results
The growth studies showed that the test organisms grew on the two nitriles. The doubling times of 12.16 d and 9.46 d (specific growth rate, µ=0.082 d−1, 0.106 d−1) were obtained for each pure culture of Bacillus sp. srain WOD8 and Corynebacterium sp. srain WOIS2 on glutaronitrile (as single substrate), respectively. While the same strains had doubling times of 11.11 d and 10.00 d (µ=0.090 d−1, 0.100 d−1) on benzonitrile (as single substrate). However, the mixed culture (comprising the two strains) had doubling times of 7.40 d and 7.75 d (µ=0.135 d−1, 0.129 d−1) on glutaronitrile (as single and mixed substrates), respectively. While doubling times of 8.09 d and 8.71 d (µ=0.124 d−1, 0.115 d−1) were obtained for the same mixed culture on benzonitrile (as single and mixed substrates). Based on gas chromatographic analysis, the residual glutaronitrile concentrations at day 16 for strains WOD8 and WOIS2 were 35.77 g L−1 (72.2%) and 9.30 g L−1 (92.5%), respectively, whereas the residual benzonitrile concentrations for the same strains were 27.39 g L−1 (78.8%) and 13.79 g L−1 (89.2%), respectively. For the mixed culture, residual glutaronitrile and benzonitrile concentrations at day 16 were 13.40 g L−1 (88.5%) and 10.42 g L−1 (91.5%), respectively, whereas for the mixed substrates (glutaronitrile and benzonitrile), 7.21 g L−1 (91.7%) and 4.80 g L−1 (94.2%) of residual glutaronitrile and benzonitrile concentrations were obtained by the same consortium. The gene for nitrilase involved in nitrile degradation was detected in the genome of the bacterial strains. The amplified nitrilase gene gave PCR products of sizes 1400 bp and 1000 bp, as expected for strains WOD8 and WOIS2, respectively. 4-Cyanobutyric acid (4CBA), glutaric acid (GA), and benzoic acid (BA) were obtained as metabolites following nitrile degradation in vitro.

Conclusion
These results revealed that strains WOD8, WOIS2 and the mixed culture (consisting of the two strains) have proven to have the capacity to metabolize nitriles (glutaronitrile and benzonitrile) as the carbon and nitrogen sources. However, the mixed culture had higher nitrile degradation rate as compared to each pure culture of the two test organisms. These results also provide insight into the evolutionary genetic origin of a nitrilase gene that encodes an enzyme that catalyzes nitrile degradation in these strains. Hence, the bacterial strains that harbor this kind of gene may be used as promising biological agents for the remediation of sites polluted with nitriles, thereby opening new perspectives for encouraging data for a bioremediation bioprocess.