A Real-Time Multiplexed Microbial Growth Intervalometer for Capturing High-Resolution Growth Curves.

Batch cultures are a low maintenance and routine culturing method in microbiology. Automated tools that measure growth curves from microorganisms grown in traditional laboratory glassware, such as Balch-type tubes, are not commercially available. Here, we present a new MicrobiAl Growth Intervalometer (MAGI) that measures optical density as it correlates to microbial growth by utilizing photo-conduction as opposed to photo-attenuation used by traditional OD measurement equipment. Photo-attenuation occurs when biomass in suspension within a medium blocks and/or diffuses light directed at the detector, such that an increase in biomass results in a decrease in the measured signal. Photo-conduction differs in which the biomass contained in a medium conducts light from the emitter to the detector, where an increase in the biomass results in a corresponding increase in the measured signal. MAGI features software-driven automation that provides investigators with a highly sensitive, low-background noise growth measurement instrument with added capabilities for remote visualization and data acquisition. It is a low maintenance, cost effective, versatile, and robust platform for aerobic/anaerobic cultivation. We demonstrate the versatility of this device by obtaining growth curves from two common laboratory organisms Escherichia coli K-12 and Bacillus subtilis. We show that growth rates and generation times in E. coli K-12 are reproducible to previously published results and that morphological changes of B. subtilis during growth can be detected as a change in the slope of the growth curve, which is a function of the effects of cell size on photo-conduction through the medium. We also test MAGI to capture growth curves from an environmental organism, Intrasporangium calvum C5, under various media compositions. Our results demonstrate that the MAGI platform accurately measures growth curves in media under various redox conditions (aerobic, microaerobic, and anaerobic), complex and minimal medias, and resolving diauxic growth curves when I. calvum is grown on a disaccharide. Lastly, we demonstrate that the device can resolve growth curves for µM concentrations of resources that yield low biomass. This research advances the tools available to microbiologists aiming to monitor growth curves in a variety of disciplines, such as environmental microbiology, clinical microbiology, and food sciences.

Effects of Organic Compounds on Dissolution of the Phosphate Minerals Chlorapatite, Whitlockite, Merrillite, and Fluorapatite: Implications for Interpreting Past Signatures of Organic Compounds in Rocks, Soils and Sediments.

Phosphate is an essential nutrient for life on Earth, present in adenosine triphosphate (ATP), deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and phospholipid membranes. Phosphorus does not have a significant volatile phase, and its release from minerals is therefore critical to its bioavailability. Organic ligands can enhance phosphate release from minerals relative to release in inorganic solutions, and phosphorus depletion in paleosols has consequently been used as a signature of the presence of ligands secreted by terrestrial organisms on early Earth. We performed batch dissolution experiments of the Mars-relevant phosphate minerals merrillite, whitlockite, chlorapatite, and fluorapatite in solutions containing organic compounds relevant to Mars. We also analyzed these phosphate minerals using the ChemCam laboratory instrument at Los Alamos, providing spectra of end-member phosphate phases that are likely present on the surface of Mars. Phosphate release rates from chlorapatite, whitlockite, and merrillite were enhanced by mellitic, oxalic, succinic, and acetic acids relative to inorganic controls by as much as >35 × . The effects of the organic compounds could be explained by the denticity of the ligand, the strength of the complex formed with calcium, and the solution saturation state. Merrillite, whitlockite, and chlorapatite dissolution rates were more strongly enhanced by acetic and succinic acids relative to inorganic controls (as much as >10 ×) than were fluorapatite dissolution rates (≲2 ×). These results suggest that depletion of phosphate in soils, rocks or sediments on Mars could be a sensitive indicator of the presence of organic compounds.

Aerobic and Anaerobic Thiosulfate Oxidation by a Cold-Adapted, Subglacial Chemoautotroph.

Geochemical data indicate that protons released during pyrite (FeS2) oxidation are important drivers of mineral weathering in oxic and anoxic zones of many aquatic environments, including those beneath glaciers. Oxidation of FeS2 under oxic, circumneutral conditions proceeds through the metastable intermediate thiosulfate (S2O3 (2-)), which represents an electron donor capable of supporting microbial metabolism. Subglacial meltwaters sampled from Robertson Glacier (RG), Canada, over a seasonal melt cycle revealed concentrations of S2O3 (2-) that were typically below the limit of detection, despite the presence of available pyrite and concentrations of the FeS2 oxidation product sulfate (SO4 (2-)) several orders of magnitude higher than those of S2O3 (2-). Here we report on the physiological and genomic characterization of the chemolithoautotrophic facultative anaerobe Thiobacillus sp. strain RG5 isolated from the subglacial environment at RG. The RG5 genome encodes genes involved with pathways for the complete oxidation of S2O3 (2-), CO2 fixation, and aerobic and anaerobic respiration with nitrite or nitrate. Growth experiments indicated that the energy required to synthesize a cell under oxygen- or nitrate-reducing conditions with S2O3 (2-) as the electron donor was lower at 5.1°C than 14.4°C, indicating that this organism is cold adapted. RG sediment-associated transcripts of soxB, which encodes a component of the S2O3 (2-)-oxidizing complex, were closely affiliated with soxB from RG5. Collectively, these results suggest an active sulfur cycle in the subglacial environment at RG mediated in part by populations closely affiliated with RG5. The consumption of S2O3 (2-) by RG5-like populations may accelerate abiotic FeS2 oxidation, thereby enhancing mineral weathering in the subglacial environment.

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity.

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88±11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.