Cultivating efficiency: high-throughput growth analysis of anaerobic bacteria in compact microplate readers.

Anaerobic microbes play crucial roles in environmental processes, industry, and human health. Traditional methods for monitoring the growth of anaerobes, including plate counts or subsampling broth cultures for optical density measurements, are time and resource-intensive. The advent of microplate readers revolutionized bacterial growth studies by enabling high-throughput and real-time monitoring of microbial growth kinetics. Yet, their use in anaerobic microbiology has remained limited. Here, we present a workflow for using small-footprint microplate readers and the Growthcurver R package to analyze the kinetic growth metrics of anaerobic bacteria. We benchmarked the small-footprint Cerillo Stratus microplate reader against a BioTek Synergy HTX microplate reader in aerobic conditions using Escherichia coli DSM 28618 cultures. The growth rates and carrying capacities obtained from the two readers were statistically indistinguishable. However, the area under the logistic curve was significantly higher in cultures monitored by the Stratus reader. We used the Stratus to quantify the growth responses of anaerobically grown E. coli and Clostridium bolteae DSM 29485 to different doses of the toxin sodium arsenite. The growth of E. coli and C. bolteae was sensitive to arsenite doses of 1.3 µM and 0.4 µM, respectively. Complete inhibition of growth was achieved at 38 µM arsenite for C. bolteae and 338 µM in E. coli. These results show that the Stratus performs similarly to a leading brand of microplate reader and can be reliably used in anaerobic conditions. We discuss the advantages of the small format microplate readers and our experiences with the Stratus. IMPORTANCE: We present a workflow that facilitates the production and analysis of growth curves for anaerobic microbes using small-footprint microplate readers and an R script. This workflow is a cost and space-effective solution to most high-throughput solutions for collecting growth data from anaerobic microbes. This technology can be used for applications where high throughput would advance discovery, including microbial isolation, bioprospecting, co-culturing, host-microbe interactions, and drug/toxin-microbial interactions.

Cultivating efficiency: High-throughput growth analysis of anaerobic bacteria in compact microplate readers.

Anaerobic microbes play crucial roles in environmental processes, industry, and human health. Traditional methods for monitoring the growth of anaerobes, including plate counts or subsampling broth cultures for optical density measurements, are time and resource intensive. The advent of microplate readers revolutionized bacterial growth studies by enabling high-throughput and real-time monitoring of microbial growth kinetics but their use in anaerobic microbiology has remained limited. Here, we present a workflow for using small-footprint microplate readers and the Growthcurver R package to analyze the kinetic growth metrics of anaerobic bacteria. We benchmarked the small-footprint Cerillo Stratus microplate reader against a BioTek Synergy HTX microplate reader in aerobic conditions using Escherichia coli DSM 28618 cultures. The growth rates and carrying capacities obtained from the two readers were statistically indistinguishable. However, the area under the logistic curve was significantly higher in cultures monitored by the Stratus reader. We used the Stratus to quantify the growth responses of anaerobically grown E. coli and Clostridium bolteae DSM 29485 to different doses of the toxin sodium arsenite. The growth of E. coli and C. bolteae was sensitive to arsenite doses of 1.3 µM and 0.4 µM, respectively. Complete inhibition of growth was achieved at 38 µM arsenite for C. bolteae, and 338 µM in E. coli. These results show that the Stratus performs similarly to a leading brand of microplate reader and can be reliably used in anaerobic conditions. We discuss the advantages of the small format microplate readers and our experiences with the Stratus.

Conductive Particles Enable Syntrophic Acetate Oxidation between Geobacter and Methanosarcina from Coastal Sediments.

Coastal sediments are rich in conductive particles, possibly affecting microbial processes for which acetate is a central intermediate. In the methanogenic zone, acetate is consumed by methanogens and/or syntrophic acetate-oxidizing (SAO) consortia. SAO consortia live under extreme thermodynamic pressure, and their survival depends on successful partnership. Here, we demonstrate that conductive particles enable the partnership between SAO bacteria (i.e., Geobacter spp.) and methanogens (Methanosarcina spp.) from the coastal sediments of the Bothnian Bay of the Baltic Sea. Baltic methanogenic sediments were rich in conductive minerals, had an apparent isotopic fractionation characteristic of CO2-reductive methanogenesis, and were inhabited by Geobacter and Methanosarcina As long as conductive particles were delivered, Geobacter and Methanosarcina persisted, whereas exclusion of conductive particles led to the extinction of Geobacter Baltic Geobacter did not establish a direct electric contact with Methanosarcina, necessitating conductive particles as electrical conduits. Within SAO consortia, Geobacter was an efficient [13C]acetate utilizer, accounting for 82% of the assimilation and 27% of the breakdown of acetate. Geobacter benefits from the association with the methanogen, because in the absence of an electron acceptor it can use Methanosarcina as a terminal electron sink. Consequently, inhibition of methanogenesis constrained the SAO activity of Geobacter as well. A potential benefit for Methanosarcina partnering with Geobacter is that together they competitively exclude acetoclastic methanogens like Methanothrix from an environment rich in conductive particles. Conductive particle-mediated SAO could explain the abundance of acetate oxidizers like Geobacter in the methanogenic zone of sediments where no electron acceptors other than CO2 are available.IMPORTANCE Acetate-oxidizing bacteria are known to thrive in mutualistic consortia in which H2 or formate is shuttled to a methane-producing Archaea partner. Here, we discovered that such bacteria could instead transfer electrons via conductive minerals. Mineral SAO (syntrophic acetate oxidation) could be a vital pathway for CO2-reductive methanogenesis in the environment, especially in sediments rich in conductive minerals. Mineral-facilitated SAO is therefore of potential importance for both iron and methane cycles in sediments and soils. Additionally, our observations imply that agricultural runoff or amendments with conductive chars could trigger a significant increase in methane emissions.

Molecular phylogeny of phyllopharyngean ciliates and their group I introns.

We analyzed small subunit ribosomal DNA (ssu-rDNA) sequences to evaluate both the monophyly of the ciliate class Phyllopharyngea de Puytorac et al. (1974), and relationships among subclasses. Classifications based on morphology and ultrastructure divide the Phyllopharyngea into four subclasses, the Phyllopharyngia, Chonotrichia, Rhynchodia, and Suctoria. Our analyses of ssu-rDNA genealogies derived from sequence data collected from diverse members representing three of the four subclasses of Phyllopharyngea (Suctoria: Ephelota spp., Prodiscophyra collini, Acineta sp.; Phyllopharyngia: Chlamydodon exocellatus, Chlamydodon triquetrus, Dysteria sp.; and Chonotrichia: Isochona sp.) provide strong support for the monophyly of the Phyllopharyngea, and show that the Chonotrichia emerge from within the Phyllopharyngia. Based on this initial sampling, suctorian budding types are monophyletic, and exogenous budding appears to be basal to evaginative and endogenous budding. Further, we report the discovery of a group I intron at position 891 in the Suctoria Acineta sp. and Tokophrya lemnarum, and a second group I intron at position 1506 in T. lemnarum. These introns represent only the second examples of group I introns in a ciliate ribosomal gene, since the discovery of ribozymes in the LSU rRNA gene of Tetrahymena thermophila. Phylogenetic analyses of Group I introns suggest a complex evolutionary history involving either multiple loses or gains of introns within endogenously budding Suctoria.

Structure of the micronuclear alpha-tubulin gene in the phyllopharyngean ciliate Chilodonella uncinata: implications for the evolution of chromosomal processing.

Ciliates are a group of microbial eukaryotes defined by the presence of dimorphic nuclei-each cell contains both a transcriptionally active macronucleus and a germline micronucleus. During the development of the macronucleus, germline chromosomes are rearranged through extensive fragmentation, removal of internally excised sequences (IESs) and DNA amplification. We have characterized three IESs in the gene that encodes alpha-tubulin in the phyllopharyngean ciliate Chilodonella uncinata. The IESs are located within the coding domain, range in size from 81 to 107 bp, and are flanked by direct repeats that vary in length from 6 to 8 bp. All three IESs are moderately AT-rich and each contains two copies of a conserved sequence motif. These data provide evidence for the existence of IESs in phyllopharyngean ciliates and suggest that IES processing in C. uncinata may rely on a novel cis-acting sequence. Comparisons of the IESs in C. uncinata with those of 'model' ciliates-Paramecium, Tetrahymena, Euplotes, Oxytricha and Stylonychia-reveal considerable variation in chromosomal processing among ciliates.

Insights into the diversity of choreotrich and oligotrich ciliates (Class: Spirotrichea) based on genealogical analyses of multiple loci.

To examine relationships among spirotrich ciliates using multi-locus sequence analyses and to provide preliminary insights into molecular diversity within species, we sequenced the small subunit rDNA (SSU rDNA), 5.8S rDNA, alpha-tubulin and the internally transcribed spacer regions (ITS1 and ITS2) of the rDNA genes from seven choreotrich (Class: Spirotrichea) and three oligotrich (Class: Spirotrichea) taxa. Genealogies constructed from SSU rDNA and ITS sequences are concordant and broadly support current classifications based on morphology. The one exception is the freshwater oligotrich Halteria grandinella, which, as has been previously noted, falls outside of the clade containing the other oligotrichs. In contrast, analyses of alpha-tubulin sequences are discordant with traditional taxonomy and rDNA genealogies. These analyses also indicate that considerably more genetic variation exists among choreotrich and oligotrich genera than among stichotrich genera. To explore the level of genetic variation among individuals in temporally isolated populations, we collected additional samples of a subset of planktonic choreotrichs and oligotrichs and characterized polymorphisms in ITS1, ITS2 and 5.8S rDNA. Analyses of these data indicate that, at least for some ciliate lineages, DNA polymorphisms vary temporally, and that genetic heterogeneity underlies some very similar morphological types.