Ozone, a strong oxidant and disinfectant, seems ideal to cope with future challenges of water treatment, such as micropollutants, multiresistant bacteria (MRB) and even intracellular antibiotic resistance genes (ARG), but information on the latter is scarce. In ozonation experiments we simultaneously determined kinetics and dose-dependent inactivation of Escherichia coli and its plasmid-encoded sulfonamide resistance gene sul1 in different water matrixes. Effects in E. coli were compared to an autochthonous wastewater community. Furthermore, resistance elimination by ozonation and post-treatment were studied in full-scale at a wastewater treatment plant (WWTP). Bacterial inactivation (cultivability, membrane damage) and degradation of sul1 were investigated using plate counts, flow cytometry and quantitative real-time PCR. In experiments with E. coli and the more ozone tolerant wastewater community disruption of intracellular genes was observed at specific ozone doses feasible for full-scale application, but flocs seemed to interfere with this effect. At the WWTP, regrowth during postozonation treatment partly compensated inactivation of MRB, and intracellular sul1 seemed unaffected by ozonation. Our findings indicate that ozone doses relevant for micropollutant abatement from wastewater do not eliminate intracellular ARG.

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Desert surface soils devoid of plant cover are populated by a variety of microorganisms, many with yet unresolved physiologies and lifestyles. Nevertheless, a common feature vital for these microorganisms inhabiting arid soils is their ability to survive long drought periods and reactivate rapidly in rare incidents of rain. Chemolithotrophic processes such as oxidation of atmospheric hydrogen and carbon monoxide are suggested to be a widespread energy source to support dormancy and resuscitation in desert soil microorganisms. Here, we assessed the distribution of chemolithotrophic, phototrophic, and desiccation-related metabolic potential among microbial populations in arid biological soil crusts (BSCs) from the Negev Desert, Israel, via population-resolved metagenomic analysis. While the potential to utilize light and atmospheric hydrogen as additional energy sources was widespread, carbon monoxide oxidation was less common than expected. The ability to utilize continuously available energy sources might decrease the dependency of mixotrophic populations on organic storage compounds and carbon provided by the BSC-founding cyanobacteria. Several populations from five different phyla besides the cyanobacteria encoded CO2 fixation potential, indicating further potential independence from photoautotrophs. However, we also found population genomes with a strictly heterotrophic genetic repertoire. The highly abundant Rubrobacteraceae (Actinobacteriota) genomes showed particular specialization for this extreme habitat, different from their closest cultured relatives. Besides the ability to use light and hydrogen as energy sources, they encoded extensive O2 stress protection and unique DNA repair potential. The uncovered differences in metabolic potential between individual, co-occurring microbial populations enable predictions of their ecological niches and generation of hypotheses on the dynamics and interactions among them. IMPORTANCE This study represents a comprehensive community-wide genome-centered metagenome analysis of biological soil crust (BSC) communities in arid environments, providing insights into the distribution of genes encoding different energy generation mechanisms, as well as survival strategies, among populations in an arid soil ecosystem. It reveals the metabolic potential of several uncultured and previously unsequenced microbial genera, families, and orders, as well as differences in the metabolic potential between the most abundant BSC populations and their cultured relatives, highlighting once more the danger of inferring function on the basis of taxonomy. Assigning functional potential to individual populations allows for the generation of hypotheses on trophic interactions and activity patterns in arid soil microbial communities and represents the basis for future resuscitation and activity studies of the system, e.g., involving metatranscriptomics.

<p>As the global hydrological cycle intensifies with future warming, more severe droughts will alter the terrestrial biogeochemical carbon (C) cycle. As soil microbial physiology controls the large fluxes of C from soil to the atmosphere, improving our ability to accurately quantify microbial physiological parameters in soil is essential. However, currently available methods to determine microbial C metabolism in soil require the addition of water, which makes it practically impossible to measure microbial physiology in dry soil samples without stimulating microbial growth and respiration (namely, the “Birch effect”).</p> <p>We developed a new method based on in vivo <sup>18</sup>O water vapor equilibration to minimize soil re-wetting effects. This method allows the isotopic labelling of soil water without any liquid water or dissolved substrate addition to the sample. This was compared to the main current method (<sup>18</sup>O-water application method) in soil samples either at near-optimal water holding capacity or in air dry soils. We generated time curves of the isotopic equilibration between liquid soil water and water vapor and calculated the average atom percent <sup>18</sup>O excess over incubation time, which is necessary to calculate microbial growth rates. We tested isotopic equilibration patterns in nine different soils (natural and artificially constructed ones) covering a wide range of soil texture and organic matter content. We then measured microbial growth, respiration and carbon use efficiency in three natural soils (either dry or at near-optimal water holding capacity). The proposed <sup>18</sup>O vapor equilibration method provides similar results as the currently widely used method of liquid <sup>18</sup>O water addition to determine microbial growth when used a near-optimal water holding capacity. However, when applied to dry soils the liquid <sup>18</sup>O water addition method overestimated growth by up to 250%, respiration by up to 500%, and underestimated carbon use efficiency by up to 40%.</p> <p>Finally, we applied the new method to undisturbed biocrust samples, at field water content (1-3%), and show for the first time real microbial growth rates and CUE values in such arid ecosystems. We describe new insights into biogeochemical cycling of C that the new method can help uncover and consider the wide range of questions regarding microbial physiology and its response to global change that can now be proposed and addressed.</p>