Biology subjects

Siles, J. A.

Publications and source records attributed to Siles, J. A..

3 recordsLinked to original sources

bioRxiv2025

Accessing rare bacterial biosphere of soil through culturing: a comparative study of culture media effectiveness integrated with metataxonomics

Soil harbors a vast bacterial diversity, including a "rare biosphere" of low-abundance taxa increasingly recognized as crucial for ecosystem functioning. Traditional plate-based culturing can facilitate access to this fraction. Here, we investigated how culture medium type influences the recovery of rare soil bacteria. To do this, culture-independent bacterial diversity of agricultural, forest, and contaminated soils was compared with culture-dependent diversity on four media (TSA (tryptic soy agar), SEM (soil extract medium), R2A (Reasoners 2A), and 20-fold diluted R2A) using a high-throughput approach in which all colonies were collected and analyzed via 16S rRNA gene metabarcoding (metataxonomics). Across all media and soils, ~30% of the culture-independent taxa were recovered through culturing, of which ~40% were members of the rare community. Culturability of bacterial communities declined across soil types in the order: contaminated > agricultural > forest, but the community isolated from forest soil contained a higher proportion of rare taxa. SEM, characterized by its low nutrient content and chemical composition resembling that of soil, proved to be the most effective, yielding higher colony-forming unit counts, greater overall and rare richness, and recovering a more taxonomically diverse rare community. The rare community retrieved by SEM was dominated by non-spore-forming taxa, in contrast to the other media, including diluted R2A. Culturing revealed a unique fraction of culturable taxa, representing ~17% of the culture-independent community on average, likely members of the rare biosphere that escape detection in metabarcoding analyses. SEM was also the medium that best captured this fraction. Our study demonstrates that low-nutrient, soil-like media such as SEM are powerful tools for accessing and characterizing the rare soil biosphere, complementing metabarcoding and providing isolates to explore the ecological and functional significance of rare taxa.

microbiology↗

bioRxiv2024

A comparison among EL-FAME, PLFA, and quantitative PCR methods to detect changes in the abundance of soil bacteria and fungi

EL-FAME (ester-linked fatty acid methyl ester), PLFA (phospholipid fatty acid) and qPCR (quantitative PCR) of ribosomal genes are three of the most common methods used to quantify soil microbial communities. The reliability of these three methods has not been simultaneously compared in situations of rapid changes in soil microbial abundances. For this purpose, we (i) incubated badland, cropland, and forest soils with nutrients or antibiotics for 2, 7, 14, and 28 days, (ii) quantified total, bacterial, and fungal abundances through EL-FAME, PLFA, and qPCR methods, and (iii) measured soil basal respiration. The general dynamic patterns of the three soil microbial fractions in response to soil addition of nutrients and antibiotics were captured by the three methods, which led to strong and positive associations between the abundances of total microorganisms, bacteria, and fungi measured by the three techniques. However, these relationships were found to be stronger between the EL-FAME and PLFA results, indicating that reliability of the fatty-acid based methods is higher than that of the qPCR. Further, soil basal respiration was associated to a higher extent with total, bacterial, and fungal abundances captured by EL-FAME and PLFA analyses than with those measured by qPCR, which suggests that the first two methods are most closely related to the soil living microbial community. In general, dynamics in the abundance of total and bacterial communities were better captured than those of fungi. The PLFA analysis seems to perform better than the EL-FAME method in forest soil and in detecting the small antibiotic-induced decreases in microbial abundances. Since the EL-FAME method is cheaper and allows a much faster processing of samples than the PLFA method, and the reliability of both methods is similar in detecting rapid changes of soil microbial abundances, choosing EL-FAME over PLFA may be advantageous in most cases.

microbiology↗

bioRxiv2022

Coupling of metataxonomics and culturing improves description of bacterial communities from a multi-contaminated waste sediment and identifies a novel Rhizorhapis sp. with metal resistance potential

Long-term contaminated environments have been recognized as potential hotspots for bacterial discovery in taxonomic and functional terms for bioremediation purposes. Here, bacterial diversity in waste sediment collected from a former industrial dumpsite and contaminated with petroleum hydrocarbon and heavy metals was investigated through the parallel application of culture-independent (16S rRNA gene amplicon sequencing) and -dependent (plate culturing followed by colony picking and identification of isolates by 16S rRNA gene Sanger sequencing) approaches. The bacterial diversities retrieved by both approaches greatly differed. Bacteroidetes and Proteobacteria were dominant in the culture-independent community, while Firmicutes and Actinobacteria were the main culturable groups. Only 2.7% of OTUs (operational taxonomic units) in the culture-independent dataset were cultured. Most of the culturable OTUs were absent or in very low abundances in the culture-independent dataset, revealing that culturing is a useful tool to study the rare bacterial biosphere. One culturable OTUs (comprising only the isolate SPR117) was identified as a potential new species in the genus Rhizorhapis (class Alphaproteobacteria) and was selected for further characterization. Phytopathogenicity tests showed that Rhizorhapis sp. strain SPR117 (ATCC TSD-228) is not pathogenic for lettuce, despite the only described species within this genus, Rhizorhapis suberifaciens, is causal agent of the lettuce corky root disease. The genome of the strain SPR117 was sequenced, assembled in 256 contigs, with a length of 4,419,522 bp and a GC content of 59.9%), and its further annotation revealed the presence of genes related to the resistance to arsenic, copper, iron, and mercury, among other metals. Therefore, the coupling of metataxonomics and culturing is a useful tool to obtain not only an improved description of bacterial communities in contaminated environments, but also to isolate microorganisms with bioremediation potential.

microbiology↗