While often obvious for macroscopic organisms, determining whether a microbe is dead or alive is fraught with complications. Fields such as microbial ecology, environmental health, and medical microbiology each determine how best to assess which members of the microbial community are alive, according to their respective scientific and/or regulatory needs. Many of these fields have gone from studying communities on a bulk level to the fine-scale resolution of microbial populations within consortia. For example, advances in nucleic acid sequencing technologies and downstream bioinformatic analyses have allowed for high-resolution insight into microbial community composition and metabolic potential, yet we know very little about whether such community DNA sequences represent viable microorganisms. In this review, we describe a number of techniques, from microscopy- to molecular-based, that have been used to test for viability (live/dead determination) and/or activity in various contexts, including newer techniques that are compatible with or complementary to downstream nucleic acid sequencing. We describe the compatibility of these viability assessments with high-throughput quantification techniques, including flow cytometry and quantitative PCR (qPCR). Although bacterial viability-linked community characterizations are now feasible in many environments and thus are the focus of this critical review, further methods development is needed for complex environmental samples and to more fully capture the diversity of microbes (e.g., eukaryotic microbes and viruses) and metabolic states (e.g., spores) of microbes in natural environments.

Given that epiphytic microbes are often found in large population sizes on plants, we tested the hypothesis that plants are quantitatively important local sources of airborne microorganisms. The abundance of microbial communities, determined by quantifying bacterial 16S RNA genes and the fungal internal transcribed spacer (ITS) region, in air collected directly above vegetation was 2- to 10-fold higher than that in air collected simultaneously in an adjacent nonvegetated area 50 m upwind. Nonmetric multidimensional scaling revealed that the composition of airborne bacteria in upwind air samples grouped separately from that of downwind air samples, while communities on plants and downwind air could not be distinguished. In contrast, fungal taxa in air samples were more similar to each other than to the fungal epiphytes. A source-tracking algorithm revealed that up to 50% of airborne bacteria in downwind air samples were presumably of local plant origin. The difference in the proportional abundances of a given operational taxonomic unit (OTU) between downwind and upwind air when regressed against the proportional representation of this OTU on the plant yielded a positive slope for both bacteria and fungi, indicating that those taxa that were most abundant on plants proportionally contributed more to downwind air. Epiphytic fungi were less of a determinant of the microbiological distinctiveness of downwind air and upwind air than epiphytic bacteria. Emigration of epiphytic bacteria and, to a lesser extent, fungi, from plants can thus influence the microbial composition of nearby air, a finding that has important implications for surrounding ecosystems, including the built environment into which outdoor air can penetrate.IMPORTANCE This paper addresses the poorly understood role of bacterial and fungal epiphytes, the inhabitants of the aboveground plant parts, in the composition of airborne microbes in outdoor air. It is widely held that epiphytes contribute to atmospheric microbial assemblages, but much of what we know is limited to qualitative assessments. Elucidating the sources of microbes in outdoor air can inform basic biological processes seen in airborne communities (e.g., dispersal and biogeographical patterns). Furthermore, given the considerable contribution of outdoor air to microbial communities found within indoor environments, the understanding of plants as sources of airborne microbes in outdoor air might contribute to our understanding of indoor air quality. With an experimental design developed to minimize the likelihood of other-than-local plant sources contributing to the composition of airborne microbes, we provide direct evidence that plants are quantitatively important local sources of airborne microorganisms, with implications for the surrounding ecosystems.

Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients.

Knowledge of the factors controlling the diverse chemical emissions of common environmental bacteria and fungi is crucial because they are important signal molecules for these microbes that also could influence humans. We show here not only a high diversity of mVOCs but that their abundance can differ greatly in different environmental contexts. Microbial volatiles exhibit dynamic changes across microbial growth phases, resulting in variance of composition and emission rate of species-specific and generic mVOCs. In vitro experiments documented emissions of a wide range of mVOCs (>400 different chemicals) at high time resolution from diverse microbial species grown under different controlled conditions on nutrient media, or residential structural materials ( N = 54, N = 23). Emissions of mVOCs varied not only between microbial taxa at a given condition but also as a function of life stage and substrate type. We quantify emission factors for total and specific mVOCs normalized for respiration rates to account for the microbial activity during their stationary phase. Our VOC measurements of different microbial taxa indicate that a variety of factors beyond temperature and water activity, such as substrate type, microbial symbiosis, growth phase, and lifecycle affect the magnitude and composition of mVOC emission.