Subsurface environments contain a large proportion of planetary microbial biomass and harbor diverse communities responsible for mediating biogeochemical cycles important to groundwater used by human society for consumption, irrigation, agriculture and industry. Within the saturated zone, capillary fringe and vadose zones, microorganisms can reside in two distinct phases (planktonic or biofilm), and significant differences in community composition, structure and activity between free-living and attached communities are commonly accepted. However, largely due to sampling constraints and the challenges of working with solid substrata, the contribution of each phase to subsurface processes is largely unresolved. Here, we synthesize current information on the diversity and activity of shallow freshwater subsurface habitats, discuss the challenges associated with sampling planktonic and biofilm communities across spatial, temporal and geological gradients, and discuss how biofilms may be constrained within shallow terrestrial subsurface aquifers. We suggest that merging traditional activity measurements and sequencing/-omics technologies with hydrological parameters important to sediment biofilm assembly and stability will help delineate key system parameters. Ultimately, integration will enhance our understanding of shallow subsurface ecophysiology in terms of bulk-flow through porous media and distinguish the respective activities of sessile microbial communities from more transient planktonic communities to ecosystem service and maintenance.

Bacterial biofilms are more resilient to standard killing methods than free-living bacteria. Pseudomonas aeruginosa PAO1 biofilms grown on borosilicate coupons were treated with gas-discharge plasma for various exposure times. Almost 100% of the cells were inactivated after a 5-min plasma exposure. Atomic force microscopy was used to image the biofilms and study their micromechanical properties. Results show that the adhesiveness to borosilicate and the thickness of the Pseudomonas biofilms are reduced upon plasma treatment.

Course-based undergraduate research experiences (CUREs) are an effective means of transforming the learning and teaching of science by involving students in the scientific process. The potential importance of the microbiome in shaping both environmental health and disease makes investigations of microbiomes an excellent teaching tool for undergraduate microbiology. Here, we present a CURE based on the microbiome of the bean beetle ( Callosobruchus maculatus ), a model system for undergraduate laboratory education. Despite the extensive research literature on bean beetles, little is known about their microbiome, making them an ideal system for a discovery-based CURE. In the CURE, students acquire microbiological technical skills by characterizing both culturable and unculturable members of the beetle gut-microbial community. Students plate beetle gut homogenates on different media, describe the colonies that are formed to estimate taxonomic diversity, extract DNA from colonies of interest, PCR amplify the16S rRNA gene for Sanger sequencing, and use the NCBI-nBLAST database to taxonomically classify sequences. Additionally, students extract total DNA from beetle gut homogenates for high-throughput paired-end sequencing and perform bioinformatic and statistical analyses of bacterial communities using a combination of open-access data processing software. Each activity allows students to engage with studies of microbiomes in a real-world context, to apply concepts and laboratory techniques to investigate either student or faculty-driven research questions, and to gain valuable experiences working with large high-throughput datasets. The CURE is designed such that it can be implemented over either 6-weeks (half semester) or 12-weeks (full semester), allowing for flexibility within the curriculum. Furthermore, student-generated data from the CURE (including bacterial colony phenotypic data, full-length 16S rRNA gene sequences from cultured isolates, and bacterial community sequences from gut homogenates) has been compiled in a continuously curated open-access database on the Bean Beetle Microbiome Project website, facilitating the generation of broader research questions across laboratory classrooms.