Search for: All records

ABSTRACT Stable isotope probing (SIP) experiments in conjunction with Raman microspectroscopy (Raman) or nano-scale secondary ion mass spectrometry (NanoSIMS) are frequently used to explore single cell metabolic activity in pure cultures as well as complex microbiomes. Despite the increasing popularity of these techniques, the comparability of isotope incorporation measurements using both Raman and NanoSIMS directly on the same cell remains largely unexplored. This knowledge gap creates uncertainty about the consistency of single-cell SIP data obtained independently from each method. Here, we conducted a comparative analysis of 543Escherichia colicells grown in M9 minimal medium in the absence or presence of heavy water (²H₂O) using correlative Raman and NanoSIMS measurements to quantify the results between the two approaches. We demonstrate that Raman and NanoSIMS yield highly comparable measurements of²H incorporation, with varying degrees of similarity based on the mass ratios analyzed using NanoSIMS. The¹²C²H/¹²C¹H and¹²C₂²H/¹²C₂¹H mass ratios provide targeted measurements of C-H bonds but may suffer from biases and background interference, while the²H/¹H ratio captures all hydrogen with lower detection limits, making it suitable for applications requiring comprehensive²H quantification. Importantly, despite its higher mass resolution requirements, the use of C₂²H/C₂¹H may be a viable alternative to the use of C²H/C¹H due to lower background and higher overall count rates. Furthermore, using an empirical approach in determining Raman wavenumber ranges via the second derivative improved the data equivalency of²H quantification between Raman and NanoSIMS, highlighting its potential for enhancing cross-technique comparability. These findings provide a robust framework for leveraging both techniques, enabling informed experimental design and data interpretation. By enhancing cross-technique comparability, this work advances SIP methodologies for investigating microbial metabolism and interactions in diverse systems.IMPORTANCEAccurate and reliable measurements of cellular properties are fundamental to understand the function and activity of microbes. This study addresses to what extent Raman microspectroscopy and nano-scale secondary ion mass spectrometry (NanoSIMS) measurements of single cell anabolic activity can be compared. Here, we study the relationship of the incorporation of a stable isotope (²H through incorporation of²H₂O) as determined by the two techniques and calculate a correlation coefficient to support the use of either technique when analyzing cells incubated with²H₂O. The ability to discern between the comparative strengths and limitations of these techniques is invaluable in refining experimental protocols, enhancing data comparability between studies, data interpretation, and ultimately advancing the quality and reliability of outcomes in microbiome research. 

Abstract Human chondrocytes are responsible for cartilage repair and homeostasis through metabolic production of precursors to collagen and other matrix components. This metabolism is sensitive both to the availability of media energy sources as well as the local temperature. Central carbon metabolites such as glucose and glutamine are essential not only for producing energetic compounds such as ATP and NADH, but also for assembling collagen and aggrecan from non-essential amino acid precursors. The rate at which this metabolism takes place directly relates to temperature: a moderate increase in temperature results in faster enzyme kinetics and faster metabolic processes. Furthermore, these biological processes are exothermic and will generate heat as a byproduct, further heating the local environment of the cell. Prior studies suggest that mechanical stimuli affect levels of central metabolites in three-dimensionally cultured articular chondrocytes. But these prior studies have not determined if articular chondrocytes produce measurable heat. Thus,the goal of this studyis to determine if three-dimensionally encapsulated chondrocytes are capable of heat production which will improve our knowledge of chondrocyte central metabolism and further validate in vitro methods. Here we show the results of microcalorimetric measurements of heat generated by chondrocytes suspended in agarose hydrogels over a 2-day period in PBS, glucose, and glutamine media. The results show that a significant amount of heat is generated by cells (Cells Only: 3.033 ± 0.574 µJ/cell, Glucose: 2.791 ± 0.819 µJ/cell, Glutamine: 1.900 ± 0.650 µJ/cell) versus the absence of cells (No Cells: 0.374 ± 0.251 µJ/cell). This suggests that cells which have access to carbon sources in the media or as intracellular reserves will generate a significant amount of heat as they process these metabolites, produce cellular energy, and synthesize collagen precursors. The length of the microcalorimeter experiment (48 h) also suggests that the metabolism of articular chondrocytes is slower than many other cells, such as human melanoma cells, which can produce similar quantities of heat in less than an hour. These data broadly suggest that chondrocyte metabolism is sensitive to the available nutrients and has the potential to alter cartilage temperature through metabolic activity. 

Abstract Ureolysis-induced calcium carbonate precipitation (UICP) is a biomineral solution where the urease enzyme converts urea and calcium into calcium carbonate. The resulting biomineral can bridge gaps in fractured shale, reduce undesired fluid flow, limit fracture propagation, better store carbon dioxide, and potentially enhance well efficiency. The mechanical properties of shale cores were investigated using a modified Brazilian indirect tensile strength test. An investigation of intact shale using Eagle Ford and Wolfcamp cores was conducted at varying temperatures. Results show no significant difference between shale types (average tensile strength = 6.19 MPa). Eagle Ford displayed higher strength at elevated temperature, but temperature did not influence Wolfcamp. Comparatively, cores with a single, lengthwise heterogeneous fracture were sealed with UICP and further tested for tensile strength. UICP was delivered via a flow-through method which injected 20–30 sequential patterns of ureolytic microorganisms and UICP-promoting fluids into the fracture until permeability reduced by three orders of magnitude or with an immersion method which placed cores treated with guar gum and UICP-promoting fluids into a batch reactor, demonstrating that guar gum is a suitable inclusion and may reduce the number of flow-through injections required. Tensile results for both delivery methods were variable (0.15–8 MPa), and in some cores the biomineralized fracture split apart, possibly due to insufficient sealing and/or heterogeneity in the composite UICP-shale cores. Notably in other cores the biomineralized fracture remained intact, demonstrating more cohesion than the surrounding shale, indicating that UICP may produce a strong seal for subsurface application. 

Abstract Microbially-induced calcium carbonate precipitation (MICP) is a biological process in which microbially-produced urease enzymes convert urea and calcium into solid calcium carbonate (CaCO₃) deposits. MICP has been demonstrated to reduce permeability in shale fractures under elevated pressures, raising the possibility of applying this technology to enhance shale reservoir storage safety. For this and other applications to become a reality, non-invasive tools are needed to determine how effectively MICP seals shale fractures at subsurface temperatures. In this study, two different MICP strategies were tested on 2.54 cm diameter and 5.08 cm long shale cores with a single fracture at 60 ℃. Flow-through, pulsed-flow MICP-treatment was repeatedly applied to Marcellus shale fractures with and without sand (“proppant”) until reaching approximately four orders of magnitude reduction in apparent permeability, while a single application of polymer-based “immersion” MICP-treatment was applied to an Eagle Ford shale fracture with proppant. Low-field nuclear magnetic resonance (LF-NMR) and X-Ray computed microtomography (micro-CT) techniques were used to assess the degree of biomineralization. With the flow-through approach, these tools revealed that while CaCO₃precipitation occurred throughout the fracture, there was preferential precipitation around proppant. Without proppant, the same approach led to premature sealing at the inlet side of the core. In contrast, immersion MICP-treatment sealed off the fracture edges and showed less mineral precipitation overall. This study highlights the use of LF-NMR relaxometry in characterizing fracture sealing and can help guide NMR logging tools in subsurface remediation efforts. 

Microbial diversity estimation involves extracting nucleic acids from intricate sample matrices. Preparing nucleic acid samples is time-consuming, necessitating effective cell lysis and obtaining pure, inhibitor-free nucleic acid purifications before further use. An automated system offers advantages for field deployment due to its ease of use and quick autonomous results. This is especially beneficial for rapid measurement ofin situmicrobial diversity in remote areas. Our study aimed to assess microbial diversity of Yellowstone hot springs using a field-deployable lab in a resource-limited remote setting and demonstrate on-site nucleic acid sample processing and sequencing. We collected microbial mat and sediment samples from several Yellowstone National Park hot springs, focusing on the Five Sister Springs (FSS), spring LNN010, and Octopus Spring (OS). The samples were processed for DNA extraction on-site and further sequenced in the lab for microbial diversity. In addition, DNA extracted from one sample was sequenced and analyzed on-site as proof-of-concept. Using either Illumina or Oxford Nanopore Technology sequencing, we found similar microbial diversities. Bacteria (over 90%) were predominant at the FSS and OS sites, with archaea accounting for less than 10%. Metagenomic results were taxonomically categorized based on the closest known organism with a sequenced genome. The dominant archaeal community member wasCandidatusCaldiarchaeum subterraneum, and among bacteria,Roseiflexussp. RS-1 was abundant in mat samples. Interestingly, Bacterium HR17 was also frequently found, suggesting the need for more research on this newly recognized bacterial community member. The presence of Bacterium HR17 in these hot springs suggests its potential role in nitrogen cycling, informing both ecological understanding and industrial potential. This pioneering study assessed the microbiome of Yellowstone hot springs in about 8-9 hours using an automated system for nucleic acid extraction. By its deployment, the system’s value in elucidating the microbial diversity of extreme environments without the need to bring samples to the lab for processing had been highlighted. Sample processing and sequencing had been included in the benefits of the field-deployable lab, and the Nanopore platform had been utilized. 

Arsenic is a toxic metalloid with differential biological effects, depending on speciation and concentration. Trivalent arsenic (arsenite, AsIII) is more toxic at lower concentrations than the pentavalent form (arsenate, AsV). In E. coli, the proteins encoded by the arsRBC operon are the major arsenic detoxification mechanism. Our previous transcriptional analyses indicate broad changes in metal uptake and regulation upon arsenic exposure. Currently, it is not known how arsenic exposure impacts the cellular distribution of other metals. This study examines the metalloproteome of E. coli strains with and without the arsRBC operon in response to sublethal doses of AsIII and AsV. Size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICPMS) was used to investigate the distribution of five metals (56Fe, 24Mg, 66Zn, 75As, and 63Cu) in proteins and protein complexes under native conditions. Parallel analysis by SEC-UV-Vis spectroscopy monitored the presence of protein cofactors. Together, these data reveal global changes in the metalloproteome, proteome, protein cofactors, and soluble intracellular metal pools in response to arsenic stress in E. coli. This work brings to light one outcome of metal exposure and suggests that metal toxicity on the cellular level arises from direct and indirect effects.