16S Metagenomics investigation of Bacterial diversity and ﻿prediction of its functionalities in Moroccan phosphate mine ecosystem.
Authors: Azaroual Salah Eddine , Kasmi Yassine , Aasfar Abderrahim , Arroussi Hicham El , Zeroual Youssef , Kadiri Youssef El , Zrhridri Abdelali , Elfahime Elmostafa , Sefiani Abdelaziz , KADMIRI Issam MEFTAH 
Published: June 2021

Abstract
Background

Native plants in extreme environments may harbor some unique microbial communities with particular functions to sustain their growth and tolerance to harsh conditions. The aim of this study was to investigate the bacterial communities profiles in the Moroccan phosphate mine using 16S metagenomics sequencing. Taxonomic resolution of six hypervariable regions of the 16S rRNA was assessed in the collected samples and mock standard community. The bacterial functions enriched in the samples were also predicted to assess the contribution of the proposed low cost approach in the description of bacterial diversity and functionality in soil and plant.
Methods

The Ion 16S™ metagenomics kit was used to compare the discriminatory potential of the six targeted hypervariable regions of the 16S rRNA gene in the description of the bacterial communities. Alpha and beta diversity analysis were also performed accordingly in the studied samples. The later were collected from the bulk phosphate mine and the soils attached to roots of three different wild plants, that natively grow in this environment. Samples from the rhizosphere of wheat plants in cropping system were also studied. Actinobacteria related sequences were compared between the samples to investigate their possible origin between the bulk mine and the wild plants rhizosphere. The prediction of 82 bacterial functions related to nitrogen and phosphate metabolism along with stress was performed using Tax4fun bioinformatics tool.
Results

The rhizosphere of the three wild plants in the Moroccan phosphate mine is characterized by interesting bacterial diversity including Proteobacteria (62,24%, 71,15% and 65,61%), Actinobacteria (22,53%, 15,24%, 22,30%), Bacteroidetes (7,57% ; 4,23% ; 7,63%), and Firmicutes (5,82%; 1,17% ; 2,83%). Broad taxonomic diversity of minor phyla was also found in the native plants samples and included Acidobacteria, Armatimonadetes, Chloroflexi, Cyanobacteria, Gemmatimonadetes and Verrucomicrobia. The bulk phosphate mine samples were dominated by Actinobacteria with average relative abundance of 97,73% and Proteobacteria was Less abundant phylum in these samples. V3, V4 and V67 regions performed better in the taxonomic resolution at different levels e.g., phylum, family and genus. However, V8 and V9 regions gave the lesser taxonomic resolution and V9 region identified only Proteobacteria phylum in the mock standard sample. Alpha and beta diversity analysis indicated low abundance of observed OTUs and diversity in the bulk samples compared with the rhizosphere samples of the native plants in the phosphate mine. Moreover, significant (p = 0,021) differences in bacterial diversity were observed among at least three kind of samples : The bulk phosphate mine, the rhizosphere of the native wild plants of the phosphate mine and the rhizosphere of the wheat crop. The results indicated that both plant genotype and mainly soil conditions may be involved in the shaping of bacterial diversity. Such indication was also confirmed by the prediction of functional profiles that showed enrichment of many functions related to biological nitrogen fixation in the rhizosphere of native plants and the stress related functions in the bulk phosphate mine in comparison with the wheat rhizosphere samples
Conclusion

The proposed amplicon sequencing approach combined to bioinformatic prediction of bacterial functions could be considered as low-cost method to study the bacterial communities in soils and plants. The application of such approach to samples from the phosphate mines and wild plants revealed the need to consider at least V3, V4 and V6-7 hypervariable regions of the 16S rRNA gene in the taxonomic resolution of bacterial diversity in soil samples. This approach revealed also interesting bacterial diversity governed by the plant genotypes and the local conditions with interesting predicted functionalities involved in plant growth and development and stress tolerance in extreme environments. Further studies could target some interesting identified phyla and predicted bacterial functionalities, such studies could also deeply investigate the origin of the bacterial diversity in these native plants to develop new generation of bacterial inoculants for sustainable agriculture.