Engineering topics

Hunt, Kristopher A.

Publications and source records attributed to Hunt, Kristopher A..

8 records

DOE OSTI2024

Origin of biogeographically distinct ecotypes during laboratory evolution

Resource partitioning is central to the incredible productivity of microbial communities, including gigatons in annual methane emissions through syntrophic interactions. Previous work revealed how a sulfate reducer (Desulfovibrio vulgaris, Dv) and a methanogen (Methanococcus maripaludis, Mm) underwent evolutionary diversification in a planktonic context, improving stability, cooperativity, and productivity within 300-1000 generations. Here, we show that mutations in just 15 Dv and 7 Mm genes within a minimal assemblage of this evolved community gave rise to co-existing ecotypes that were spatially enriched within a few days of culturing in a fluidized bed reactor. The spatially segregated communities partitioned resources in the simulated subsurface environment, with greater lactate utilization by attached Dv but partial utilization of resulting H 2 by low affinity hydrogenases of Mm in the same phase. The unutilized H 2 was scavenged by high affinity hydrogenases of planktonic Mm, producing copious amounts of methane. Our findings show how a few mutations can drive resource partitioning amongst niche-differentiated ecotypes, whose interplay synergistically improves productivity of the entire mutualistic community.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Contribution of Microorganisms with the Clade II Nitrous Oxide Reductase to Suppression of Surface Emissions of Nitrous Oxide

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. Further, to better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Ammonia-oxidizing bacteria and archaea exhibit differential nitrogen source preferences

Ammonia-oxidizing microorganisms (AOM) contribute to one of the largest nitrogen fluxes in the global nitrogen budget. Four distinct lineages of AOM: ammonia-oxidizing archaea (AOA), beta- and gamma-proteobacterial ammonia-oxidizing bacteria (β-AOB and γ-AOB) and complete ammonia oxidizers (comammox), are thought to compete for ammonia as their primary nitrogen substrate. In addition, many AOM species can utilize urea as an alternative energy and nitrogen source through hydrolysis to ammonia. How the coordination of ammonia and urea metabolism in AOM influences their ecology remains poorly understood. Here we use stable isotope tracing, kinetics and transcriptomics experiments to show that representatives of the AOM lineages employ distinct regulatory strategies for ammonia or urea utilization, thereby minimizing direct substrate competition. The tested AOA and comammox species preferentially used ammonia over urea, while β-AOB favoured urea utilization, repressed ammonia transport in the presence of urea and showed higher affinity for urea than for ammonia. Characterized γ-AOB co-utilized both substrates. Furthermore, these results reveal contrasting niche adaptation and coexistence patterns among the major AOM lineages.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Cross-Feedings, Competition, and Positive and Negative Synergies in a Four-Species Synthetic Community for Anaerobic Degradation of Cellulose to Methane

A synthetic community was designed using four microbial species that together performed distinct key metabolic processes in the anaerobic degradation of cellulose to methane and CO 2 . The microorganisms exhibited expected interactions, such as cross-feeding of acetate from a cellulolytic bacterium to an acetoclastic methanogen and competition of H 2 between a sulfate reducing bacterium and a hydrogenotrophic methanogen.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Ecophysiological and genomic analyses of a representative isolate of highly abundant Bacillus cereus strains in contaminated subsurface sediments

Abstract Bacillus cereus strain CPT56D‐587‐MTF (CPTF) was isolated from the highly contaminated Oak Ridge Reservation (ORR) subsurface. This site is contaminated with high levels of nitric acid and multiple heavy metals. Amplicon sequencing of the 16S rRNA genes (V4 region) in sediment from this area revealed an amplicon sequence variant (ASV) with 100% identity to the CPTF 16S rRNA sequence. Notably, this CPTF‐matching ASV had the highest relative abundance in this community survey, with a median relative abundance of 3.77% and comprised 20%–40% of reads in some samples. Pangenomic analysis revealed that strain CPTF has expanded genomic content compared to other B. cereus species—largely due to plasmid acquisition and expansion of transposable elements. This suggests that these features are important for rapid adaptation to native environmental stressors. We connected genotype to phenotype in the context of the unique geochemistry of the site. These analyses revealed that certain genes (e.g. nitrate reductase, heavy metal efflux pumps) that allow this strain to successfully occupy the geochemically heterogenous microniches of its native site are characteristic of the B. cereus species while others such as acid tolerance are mobile genetic element associated and are generally unique to strain CPTF.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Microbial maintenance energy quantified and modeled with microcalorimetry

Refining the energetic costs of cellular maintenance is essential for predicting microbial growth and survival in the environment. In this work, we evaluate a simple batch culture method to quantify energy partitioning between growth and maintenance using microcalorimetry and thermodynamic modeling. The constants derived from the batch culture system were comparable to those that have been reported from meta-analyses of data derived from chemostat studies. The model accurately predicted temperature-dependent biomass yield and the upper temperature limit of growth for Desulfovibrio alaskensis G20, suggesting the method may have broad application. An Arrhenius temperature dependence for the specific energy consumption rate, inferred from substrate consumption and heat evolution, was observed over the entire viable temperature range. By combining this relationship for specific energy consumption rates and observed specific growth rates, the model describes an increase in nongrowth associated maintenance at higher temperatures and the corresponding decrease in energy available for growth. This analytical and thermodynamic formulation suggests that simply monitoring heat evolution in batch culture could be a useful complement to the recognized limitations of estimating maintenance using extrapolation to zero growth in chemostats.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

An automated multiplexed turbidometric and data collection system for measuring growth kinetics of anaerobes dependent on gaseous substrates

Standard methods of monitoring the growth kinetics of anaerobic microorganisms are generally impractical when there is a protracted or indeterminate period of active growth, and when high numbers of samples or replications are required. As part of our studies of the adaptive evolution of a simple anaerobic syntrophic mutualism, requiring the characterization of many isolates and alternative syntrophic pairings, here we developed a multiplexed growth monitoring system using a combination of commercially available electronics and custom designed circuitry and materials. This system automatically monitors up to 64 sealed, and as needed pressurized, culture tubes and reports the growth data in real-time through integration with a customized relational database. The utility of this system was demonstrated by resolving minor differences in growth kinetics associated with the adaptive evolution of a simple microbial community comprised of a sulfate reducing bacterium, Desulfovibrio vulgaris, grown in syntrophic association with Methanococcus maripaludis, a hydrogenotrophic methanogen.

59 BASIC BIOLOGICAL SCIENCES↗