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Opencast mining removes topsoil and associated bacterial communities that play crucial roles in soil ecosystem functioning. Understanding the community composition and functioning of these organisms may lead to improve mine-rehabilitation practices. We used a culture-dependent method, combined with Illumina sequencing, to compare the taxonomic richness and composition of living bacterial communities in opencast mine substrates and young mine-rehabilitation plots, with those of soil in adjacent remnant forest at a limestone mine in northern Thailand. We further investigated the effects of soil physico-chemical factors and ground-flora cover on the same. Although, loosened subsoil, brought in to initiate rehabilitation, improved water retention and facilitated plant re-establishment, it did not increase the population density of living microbes substantially within 9 months. Planted trees and sparse ground flora in young rehabilitation plots had not ameliorated the micro-habitat enough to change the taxonomic composition of the soil bacteria compared with non-rehabilitated mine sites. Viable microbes were significantly more abundant in forest soil than in mine substrates. The living bacterial community composition differed significantly, between the forest plots and both the mine and rehabilitation plots. Proteobacteria dominated in forest soil, whereas Firmicutes dominated in samples from both mine and rehabilitation plots. Although, several bacterial taxa could survive in the mine substrate, soil ecosystem functions were greatly reduced. Bacteria, capable of chitinolysis, aromatic compound degradation, ammonification and nitrate reduction were all absent or rare in the mine substrate. Functional redundancy of the bacterial communities in both mine substrate and young mine-rehabilitation soil was substantially reduced, compared with that of forest soil. Promoting the recovery of microbial biomass and functional diversity, early during mine rehabilitation, is recommended, to accelerate soil ecosystem restoration and support vegetation recovery. Moreover, if inoculation is included in mine rehabilitation programs, the genera: Bacillus, Streptomyces and Arthrobacter are likely to be of particular interest, since these genera can be cultivated easily and this study showed that they can survive under the extreme conditions that prevail on opencast mines., Competing Interests: The authors declare no competing interests. The Siam Cement (Lampang) Co., Ltd. allowed access to the experimental sites and funded field work and lab analyses. However, the company played no role in experimental design, data collection, analysis, and interpretation, drafting the manuscript and decision to submit the work for publication. None of the authors were paid by the company. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected.

Stingless bees play a crucial role in the environment and agriculture as they are effective pollinators. Furthermore, they can produce various products that can be exploited economically, such as propolis and honey. Despite their economic value, the knowledge of microbial community of stingless bees, and their roles on the bees' health, especially in Thailand, are in its infancy. This study aimed to investigate the composition and the functions of bacterial community associated with Tetragonula pagdeni stingless bees using culture-independent and culture-dependent approaches with emphasis on lactic acid bacteria. The culture-independent results showed that the dominant bacterial phyla were Firmicutes, Proteobacteria and Actinobacteria. The most abundant families were Lactobacillaceae and Halomonadaceae. Functional prediction indicated that the prevalent functions of bacterial communities were chemoheterotrophy and fermentation. In addition, the bacterial community might be able to biosynthesize amino acid and antimicrobial compounds. Further isolation and characterization resulted in isolates that belonged to the dominant taxa of the community and possessed potentially beneficial metabolic activity. This suggested that they are parts of the nutrient acquisition and host defense bacterial functional groups in Thai commercial stingless bees. [ABSTRACT FROM AUTHOR]