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At least 847 records · Page 47

Coassembly and binning of a twenty-year metagenomic time-series from Lake Mendota

Abstract The North Temperate Lakes Long-Term Ecological Research (NTL-LTER) program has been extensively used to improve understanding of how aquatic ecosystems respond to environmental stressors, climate fluctuations, and human activities. Here, we report on the metagenomes of samples collected between 2000 and 2019 from Lake Mendota, a freshwater eutrophic lake within the NTL-LTER site. We utilized the distributed metagenome assembler MetaHipMer to coassemble over 10 terabases (Tbp) of data from 471 individual Illumina-sequenced metagenomes. A total of 95,523,664 contigs were assembled and binned to generate 1,894 non-redundant metagenome-assembled genomes (MAGs) with ≥50% completeness and ≤10% contamination. Phylogenomic analysis revealed that the MAGs were nearly exclusively bacterial, dominated by Pseudomonadota (Proteobacteria, N = 623) and Bacteroidota (N = 321). Nine eukaryotic MAGs were identified by eukCC with six assigned to the phylum Chlorophyta. Additionally, 6,350 high-quality viral sequences were identified by geNomad with the majority classified in the phylum Uroviricota. This expansive coassembled metagenomic dataset provides an unprecedented foundation to advance understanding of microbial communities in freshwater ecosystems and explore temporal ecosystem dynamics.

59 BASIC BIOLOGICAL SCIENCES↗

Rainforest-to-pasture conversion stimulates soil methanogenesis across the Brazilian Amazon

The Amazon rainforest is a biodiversity hotspot and large terrestrial carbon sink threatened by agricultural conversion. Rainforest-to-pasture conversion stimulates the release of methane, a potent greenhouse gas. The biotic methane cycle is driven by microorganisms; therefore, this study focused on active methane-cycling microorganisms and their functions across land-use types. We collected intact soil cores from three land use types (primary rainforest, pasture, and secondary rainforest) of two geographically distinct areas of the Brazilian Amazon (Santarém, Pará and Ariquemes, Rondônia) and performed DNA stable-isotope probing coupled with metagenomics to identify the active methanotrophs and methanogens. At both locations, we observed a significant change in the composition of the isotope-labeled methane-cycling microbial community across land use types, specifically an increase in the abundance and diversity of active methanogens in pastures. We conclude that a significant increase in the abundance and activity of methanogens in pasture soils could drive increased soil methane emissions. Furthermore, we found that secondary rainforests had decreased methanogenic activity similar to primary rainforests, and thus a potential to recover as methane sinks, making it conceivable for forest restoration to offset greenhouse gas emissions in the tropics. These findings are critical for informing land management practices and global tropical rainforest conservation.

59 BASIC BIOLOGICAL SCIENCES↗

FeGenie: a comprehensive tool for the identification of iron genes and iron gene neighborhoods in genomes and metagenome assemblies

Iron is a micronutrient for nearly all life on Earth. It can be used as an electron donor and electron acceptor by iron-oxidizing and iron-reducing microorganisms and is used in a variety of biological processes, including photosynthesis and respiration. While it is the fourth most abundant metal in the Earth’s crust, iron is often limiting for growth in oxic environments because it is readily oxidized and precipitated. Much of our understanding of how microorganisms compete for and utilize iron is based on laboratory experiments. However, the advent of next-generation sequencing and surge in publicly available sequence data has made it possible to probe the structure and function of microbial communities in the environment. To bridge the gap between our understanding of iron acquisition, iron redox cycling, iron storage, and magnetosome formation in model microorganisms and the plethora of sequence data available from environmental studies, we have created a comprehensive database of hidden Markov models (HMMs) based on genes related to iron acquisition, storage, and reduction/oxidation in Bacteria and Archaea. Along with this database, we present FeGenie, a bioinformatics tool that accepts genome and metagenome assemblies as input and uses our comprehensive HMM database to annotate provided datasets with respect to iron-related genes and gene neighborhood. An important contribution of this tool is the efficient identification of genes involved in iron oxidation and dissimilatory iron reduction, which have been largely overlooked by standard annotation pipelines. We validated FeGenie against a selected set of 28 isolate genomes and showcase its utility in exploring iron genes present in 27 metagenomes, 4 isolate genomes from human oral biofilms, and 17 genomes from candidate organisms, including members of the candidate phyla radiation. We show that FeGenie accurately identifies iron genes in isolates. Furthermore, analysis of metagenomes using FeGenie demonstrates that the iron gene repertoire and abundance of each environment is correlated with iron richness. While this tool will not replace the reliability of culture-dependent analyses of microbial physiology, it provides reliable predictions derived from the most up-to-date genetic markers. FeGenie’s database will be maintained and continually updated as new genes are discovered.

59 BASIC BIOLOGICAL SCIENCES↗

Defensive symbiosis against giant viruses in amoebae

Protists are important regulators of microbial communities and key components in food webs with impact on nutrient cycling and ecosystem functioning. In turn, their activity is shaped by diverse intracellular parasites, including bacterial symbionts and viruses. Yet, bacteria–virus interactions within protists are poorly understood. Here, we studied the role of bacterial symbionts of free-living amoebae in the establishment of infections with nucleocytoplasmic large DNA viruses (Nucleocytoviricota). To investigate these interactions in a system that would also be relevant in nature, we first isolated and characterized a giant virus (Viennavirus, family Marseilleviridae) and a sympatric potential Acanthamoeba host infected with bacterial symbionts. Subsequently, coinfection experiments were carried out, using the fresh environmental isolates as well as additional amoeba laboratory strains. Employing fluorescence in situ hybridization and qPCR, we show that the bacterial symbiont, identified as Parachlamydia acanthamoebae, represses the replication of the sympatric Viennavirus in both recent environmental isolates as well as Acanthamoeba laboratory strains. In the presence of the symbiont, virions are still taken up, but viral factory maturation is inhibited, leading to survival of the amoeba host. The symbiont also suppressed the replication of the more complex Acanthamoeba polyphaga mimivirus and Tupanvirus deep ocean (Mimiviridae). Our work provides an example of an intracellular bacterial symbiont protecting a protist host against virus infections. The impact of virus–symbiont interactions on microbial population dynamics and eventually ecosystem processes requires further attention.

59 BASIC BIOLOGICAL SCIENCES↗

Weaning transition, but not the administration of probiotic candidate Kazachstania slooffiae , shaped the gastrointestinal bacterial and fungal communities in nursery piglets

As in-feed antibiotics are phased out of swine production, producers are seeking alternatives to facilitate improvements in growth typically seen from this previously common feed additive. Kazachstania slooffiae is a prominent commensal fungus in the swine gut that peaks in relative abundance shortly after weaning and has beneficial interactions with other bacteriome members important for piglet health. In this study, piglets were supplemented with K. slooffiae to characterize responses in piglet health as well as fungal and bacterial components of the microbiome both spatially (along the entire gastrointestinal tract and feces) and temporally (before, during, and after weaning). Litters were assigned to one of four treatments: no K. slooffiae (CONT); one dose of K. slooffiae 7 days before weaning (day 14; PRE); one dose of K. slooffiae at weaning (day 21; POST); or one dose of K. slooffiae 7 days before weaning and one dose at weaning (PREPOST). The bacteriome and mycobiome were analyzed from fecal samples collected from all piglets at day 14, day 21, and day 49, and from organ samples along the gastrointestinal (GI) tract at day 21 and day 49. Blood samples were taken at day 14 and day 49 for cytokine analysis, and fecal samples were assayed for antimicrobial resistance. While some regional shifts were seen in response to K. slooffiae administration in the mycobiome of the GI tract, no remarkable changes in weight gain or health of the animals were observed, and changes were more likely due to sow and the environment. Ultimately, the combined microbiome changed most considerably following the transition from suckling to nursery diets. This work describes the mycobiome along the piglet GI tract through the weaning transition for the first time. Based on these findings, K. slooffiae administered at this concentration may not be an effective tool to hasten colonization of K. slooffiae in the piglet GI tract around the weaning transition nor support piglet growth, microbial gut health, or immunity. However, diet and environment greatly influence microbial community development.

60 APPLIED LIFE SCIENCES↗

Depth-specific distribution of bacterial MAGs in permafrost active layer in Ny Ålesund, Svalbard (79°N)

Arctic soil microbial communities may shift with increasing temperatures and water availability from climate change. We examined temperature and volumetric liquid water content (VWC) in the upper 80 cm of permafrost-affected soil over 2 years (2018–2019) at the Bayelva monitoring station, Ny Ålesund, Svalbard. We show VWC increases with depth, whereas in situ temperature is more stable vertically, ranging from -5°C to 5°C seasonally. Prokaryotic metagenome-assembled genomes (MAGs) were obtained at 2–4 cm vertical resolution collected while frozen in April 2018 and at 10 cm vertical resolution collected while thawed in September 2019. The most abundant MAGs were Acidobacteriota, Actinomycetota, and Chloroflexota. Actinomycetota and Chloroflexota increase with depth, while Acidobacteriota classes Thermoanaerobaculia Gp7-AA8, Blastocatellia UBA7656, and Vicinamibacteria Vicinamibacterales are found above 6 cm, below 6 cm, and below 20cm, respectively. All MAGs have diverse carbon-degrading genes, and Actinomycetota and Chloroflexota have autotrophic genes. Further, genes encoding β -glucosidase, N-acetyl-β-D-glucosaminidase, and xylosidase increase with depth, indicating a greater potential for organic matter degradation with higher VWC. Acidobacteriota dominate the top 6 cm with their classes segregating by depth, whereas Actinomycetota and Chloroflexota dominate below ~6 cm. This suggests that Acidobacteriota classes adapt to lower VWC at the surface, while Actinomycetota and Chloroflexota persist below 6cm with higher VWC. This indicates that VWC may be as important as temperature in microbial climate change responses in Arctic mineral soils. Here we describe MAG-based Seqcode type species in the Acidobacteriota, Onstottus arcticum, Onstottus frigus, and Gilichinskyi gelida and in the Actinobacteriota, Mayfieldus profundus.

54 ENVIRONMENTAL SCIENCES↗

Enabling Experimental Evolution: Multi-Parameter Sensor System Integration into a Culture/Stressor Biofluidics System

Experimental evolution (EE) exposes microbial communities to ecological stressors, simulating dynamics up to near-extinction events. Combined with comparative sequencing and other molecular tools, such data can inform the genetic and other biological mechanisms underlying extremophile adaptation, and other observed effects. Automating this type of experiment using biofluidics can mitigate many traditional obstacles, including delays in assay results and environment adjustment and the need for many replicates. A first-generation device for automating EE procedures, the Automated Adaptive Directed Evolution Chamber (AADEC), was developed at NASA Ames. UV-C radiation was the stressor, an LED-photodiode array measured optical density, magnetic agitation and peristaltic pump systems ensured nutrient availability, and Arduino microcontrollers provided control. Escherichia coli in LB kanamycin media was used for testing and performance verification. A manual laboratory procedure with timed exposure to UV-C was performed to typify tolerance acquisition. Approximately a 106 factor increase in survival ratio was recorded over multiple iterations. Currently, a second-generation device is being developed integrating more real-time sensors: redox potential (ORP), indicating available/consumed metabolic energy; dissolved oxygen (DO), indicating aerobic/anaerobic growth; pH, indicating metabolic products; and electrical conductivity (EC), another indicator of metabolic products. The EC sensor system was constructed and calibrated in-house and matched commercial sensors in the required range. A Raspberry Pi computer automated the electrical system, allowing real-time data acquisition. The fluidics card was made of CNC-milled polycarbonate for biocompatibility. Each sensor parameter can also be used as a selection pressure alone or in combination with others to create extreme microbial environments. As a proof of concept, this work demonstrated sensor operation in one pair of growth-sensor chambers. It can be expanded to a multi-chamber system to enable inter-culture comparisons and multi-population studies. The prior Arduino system will be ported to the RPi system. Future stressors to be added include thermal, reactive oxygen species, and varying nutrient availability.

Govinda Raj, Chinmayee↗

Polysaccharide niche partitioning of distinct Polaribacter clades during North Sea spring algal blooms

Massive releases of organic substrates during marine algal blooms trigger growth of many clades of heterotrophic bacteria. Algal polysaccharides represent the most diverse and structurally complex class of these substrates, yet their role in shaping the microbial community composition is poorly understood. We investigated, whether polysaccharide utilization capabilities contribute to niche differentiation of Polaribacter spp. (class Flavobacteriia; known to include relevant polysaccharidedegraders) that were abundant during 2009–2012 spring algal blooms in the southern North Sea. We identified six distinct Polaribacter clades using phylogenetic and phylogenomic analyses, quantified their abundances via fluorescence in situ hybridization, compared metagenome-assembled genomes, and assessed in situ gene expression using metaproteomics. Four clades with distinct polysaccharide niches were dominating. Polaribacter 2-a comprised typical first responders featuring small genomes with limited polysaccharide utilization capacities. Polaribacter 3-a were abundant only in 2010 and possessed a distinct sulfated α-glucoronomannan degradation potential. Polaribacter 3-b responded late in blooms and had the capacity to utilize sulfated xylan. Polaribacter 1-a featured high numbers of glycan degradation genes and were particularly abundant following Chattonella algae blooms. These results support the hypothesis that sympatric Polaribacter clades occupy distinct glycan niches during North Sea spring algal blooms.

59 BASIC BIOLOGICAL SCIENCES↗

Functional insights of novel Bathyarchaeia reveal metabolic versatility in their role in peatlands of the Peruvian Amazon

ABSTRACT The decomposition of soil organic carbon within tropical peatlands is influenced by the functional composition of the microbial community. In this study, building upon our previous work, we recovered a total of 28 metagenome-assembled genomes (MAGs) classified as Bathyarchaeia from the tropical peatlands of the Pastaza-Marañón Foreland Basin (PMFB) in the Amazon. Using phylogenomic analyses, we identified nine genus-level clades to have representatives from the PMFB, with four forming a putative novel family (“CandidatusPaludivitaceae”) endemic to peatlands. We focus on theCa. Paludivitaceae MAGs due to the novelty of this group and the limited understanding of their role within tropical peatlands. Functional analysis of these MAGs reveals that this putative family comprises facultative anaerobes, possessing the genetic potential for oxygen, sulfide, or nitrogen oxidation. This metabolic versatility can be coupled to the fermentation of acetoin, propanol, or proline. The other clades outsideCa. Paludivitaceae are putatively capable of acetogenesis andde novoamino acid biosynthesis and encode a high amount of Fe 3+ transporters. Crucially, theCa. Paludivitaceae are predicted to be carboxydotrophic, capable of utilizing CO for energy generation or biomass production. Through this metabolism, they could detoxify the environment from CO, a byproduct of methanogenesis, or produce methanogenic substrates like CO 2 and H 2 . Overall, our results show the complex metabolism and various lineages of Bathyarchaeia within tropical peatlands pointing to the need to further evaluate their role in these ecosystems. IMPORTANCE With the expansion of theCandidatusPaludivitaceae family by the assembly of 28 new metagenome assembled genomes, this study provides novel insights into their metabolic diversity and ecological significance in peatland ecosystems. From a comprehensive phylogenic and functional analysis, we have elucidated their putative unique facultative anaerobic capabilities and CO detoxification potential. This research highlights their crucial role in carbon cycling and greenhouse gas regulation. These findings are essential for resolving the microbial processes affecting peat soil stability, offering new perspectives on the ecological roles of previously underexplored and underrepresented archaeal populations.

Microbiology↗

Harnessing Fermentation May Enhance the Performance of Biological Sulfate-Reducing Bioreactors

Biological sulfate reduction (BSR) represents a promising strategy for bioremediation of sulfate-rich waste streams, yet the impact of metabolic interactions on performance is largely unexplored. Here, genome-resolved metagenomics was used to characterize 17 microbial communities in reactors treating synthetic sulfate-contaminated solutions. Reactors were supplemented with lactate or acetate and a small amount of fermentable substrate. Of the 163 genomes representing all the abundant bacteria, 130 encode 321 NiFe and FeFe hydrogenases and all genomes of the 22 sulfate-reducing microorganisms (SRM) encode genes for H 2 uptake. We observed lactate oxidation solely in the first packed bed reactor zone, with propionate and acetate oxidation in the middle and predominantly acetate oxidation in the effluent zone. The energetics of these reactions are very different, yet sulfate reduction kinetics were unaffected by the type of electron donor available. We hypothesize that the comparable rates, despite the typically slow growth of SRM on acetate, are a result of the consumption of H 2 generated by fermentation. This is supported by the sustained performance of a predominantly acetate-supplemented stirred tank reactor dominated by diverse fermentative bacteria encoding FeFe hydrogenase genes and SRM capable of acetate and hydrogen consumption and CO 2 assimilation. Thus, addition of fermentable substrates to stimulate syntrophic relationships may improve the performance of BSR reactors supplemented with inexpensive acetate.

59 BASIC BIOLOGICAL SCIENCES↗

Chemolithoautotroph distributions across the subsurface of a convergent margin

Subducting oceanic crusts release fluids rich in biologically relevant compounds into the overriding plate, fueling subsurface chemolithoautotrophic ecosystems. To understand the impact of subsurface geochemistry on microbial communities, we collected fluid and sediments from 14 natural springs across a ~200 km transect across the Costa Rican convergent margin and performed shotgun metagenomics. The resulting 404 metagenome-assembled genomes (MAGs) cluster into geologically distinct regions based on MAG abundance patterns: outer forearc-only (25% of total relative abundance), forearc/arc-only (38% of total relative abundance), and delocalized (37% of total relative abundance) clusters. In the outer forearc, Thermodesulfovibrionia, Candidatus Bipolaricaulia, and Firmicutes have hydrogenotrophic sulfate reduction and Wood-Ljungdahl (WL) carbon fixation pathways. In the forearc/arc, Anaerolineae, Ca. Bipolaricaulia, and Thermodesulfovibrionia have sulfur oxidation, nitrogen cycling, microaerophilic respiration, and WL, while Aquificae have aerobic sulfur oxidation and reverse tricarboxylic acid carbon fixation pathway. Transformation-based canonical correspondence analysis shows that MAG distribution corresponds to concentrations of aluminum, iron, nickel, dissolved inorganic carbon, and phosphate. While delocalized MAGs appear surface-derived, the subsurface chemolithoautotrophic, metabolic, and taxonomic landscape varies by the availability of minerals/metals and volcanically derived inorganic carbon. Furthermore, the WL pathway persists across all samples, suggesting that this versatile, energy-efficient carbon fixation pathway helps shape convergent margin subsurface ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolite release by nitrifiers facilitates metabolic interactions in the ocean

Microbial chemoautotroph-heterotroph interactions may play a pivotal role in the cycling of carbon in the deep ocean, reminiscent of phytoplankton-heterotroph associations in surface waters. Nitrifiers are the most abundant chemoautotrophs in the global ocean, yet very little is known about nitrifier metabolite production, release, and transfer to heterotrophic microbial communities. To elucidate which organic compounds are released by nitrifiers and potentially available to heterotrophs, we characterized the exo- and endometabolomes of the ammonia-oxidizing archaeon Nitrosopumilus adriaticus CCS1 and the nitrite-oxidizing bacterium Nitrospina gracilis Nb-211. Nitrifier endometabolome composition was not a good predictor of exometabolite availability, indicating that metabolites were predominately released by mechanisms other than cell death/lysis. Although both nitrifiers released labile organic compounds, N. adriaticus preferentially released amino acids, particularly glycine, suggesting that its cell membranes might be more permeable to small, hydrophobic amino acids. We further initiated co-culture systems between each nitrifier and a heterotrophic alphaproteobacterium, and compared exometabolite and transcript patterns of nitrifiers grown axenically to those in co-culture. In particular, B vitamins exhibited dynamic production and consumption patterns in nitrifier-heterotroph co-cultures. We observed an increased production of vitamin B 2 and the vitamin B 12 lower ligand dimethylbenzimidazole by N. adriaticus and N. gracilis, respectively. In contrast, the heterotroph likely produced vitamin B 5 in co-culture with both nitrifiers and consumed the vitamin B 7 precursor dethiobiotin when grown with N. gracilis. Our results indicate that B vitamins and their precursors could play a particularly important role in governing specific metabolic interactions between nitrifiers and heterotrophic microbes in the ocean.

54 ENVIRONMENTAL SCIENCES↗

Soil minerals affect taxon-specific bacterial growth

Abstract Secondary minerals (clays and metal oxides) are important components of the soil matrix. Clay minerals affect soil carbon persistence and cycling, and they also select for distinct microbial communities. Here we show that soil mineral assemblages—particularly short-range order minerals—affect both bacterial community composition and taxon-specific growth. Three soils with different parent material and presence of short-range order minerals were collected from ecosystems with similar vegetation and climate. These three soils were provided with 18O-labeled water and incubated with or without artificial root exudates or pine needle litter. Quantitative stable isotope probing was used to determine taxon-specific growth. We found that the growth of bacteria varied among soils of different mineral assemblages but found the trend of growth suppression in the presence of short-range order minerals. Relative growth of bacteria declined with increasing concentration of short-range order minerals between 25–36% of taxa present in all soils. Carbon addition in the form of plant litter or root exudates weakly affected relative growth of taxa (p = 0.09) compared to the soil type (p < 0.01). However, both exudate and litter carbon stimulated growth for at least 34% of families in the soils with the most and least short-range order minerals. In the intermediate short-range order soil, fresh carbon reduced growth for more bacterial families than were stimulated. These results highlight how bacterial-mineral-substrate interactions are critical to soil organic carbon processing, and how growth variation in bacterial taxa in these interactions may contribute to soil carbon persistence and loss.

59 BASIC BIOLOGICAL SCIENCES↗

Deep Basalt Aquifers in Orcus Patera, Elysium Basin Mars: Perspectives for Exobiology Exploration

Direct indicators of shorelines, spillways, and terraces allowed to determine the extent of the Elysium Paleolake between the contour-lines 1000 and 500 m below the Martian datum. The Elysium Paleolake is bordered north by Orcus Patera (14N/181W), which lies west of the Tartarus Montes and Tartarus Colles. The Orcus Patera displays an ellipse-shaped collapsed caldera of 360-km long and 100-km wide. Viking topographic data show that the bottom of the caldera is located at 2500 below the Martian datum, and surrounded by a steep-walled ram art which crest is located at about 0 m elevation. Considering the localization of Orcus Patera in the Elysium paleolake, its altimetry, and the magmatic origin of this caldera, we propose the existence of a paleolake in Orcus Patera generated (a) by juvenile water from magma during the Noachian period, and (b) by intermittent influx of the Elysium Basin from Hesperian to Amazonian. Results are encouraging to consider this site as a potential high-energy source environment for microbial communities. are circumscribed by a 50-km wide lava field mapped as Noachian material. The structure of Orcus Patera represents the record of material erupted from a magmatic reservoir. The caldera is enclosed by steep inner walls (25% measured from topographic data), values which could be in agreement with the presence of a deep magmatic reservoir, as suggested by the typology of Crumpler et.al. The depth of the caldera might be due to the collapse of the magma reservoir, and the release of gases accompanying the magma thermal evolution. Origins of water for the paleolake(s): The water that generated a paleolake in Orcus Patera may have come from two origins: (1) Juvenile water: Plescia and Crips estimated a magma H20 content by weight between 0.5% and 1.5% using for the first value a comparison with terrestrial basalt, and for the second values from a Martian meteorite. The amount of H20 can be estimated by the volume of erupted lava, and the lava content of the caldera. In this study, we adopt a water content of 1%. The total volume of magma that has been contained in the caldera, and the volume of lava contained in the observed lava field is about 110 x 10(exp 6) cubic km, that gives a total volume of 1.10 x 10(exp 6) cubic km of water. The juvenile water expelled by the overpressure within the magma chamber charged with desolved water-vapor may have moved into the crust. The decrease in overburden pressure led to bubble formation. The ascent of these bubbles generated a pressurization of the magma, which was sufficient to fracture the overlaying magma layer, (2) Water from Elysium paleolake. During the Amazonian, the rise of the Elysium paleolake level generated an overspilling that supplied the caldera with water. The southern portion of the crest shows a deep gap 12-km wide at -1500 m elevation, locating the gap between 500 to 1000 in below the assumed water of Elysium paleolake, thus facilitating the influx of Elysium paleolake water into Orcus Patera. Bathymetric calculations give a floor area of 25,500 sq km at -2000 m elevation, and a water volume of 42,000 cubic km, with a lake-level at -1500 m. A substantial amount of water may have percolated through the fractured lava, and part of the volume may have overspilled the northern crest of Orcus Patera to debouch in the Tartarus Montes region. We envision the formation of a subsurface aqueous environment in basaltic rocks at the contact of the two water-source origins, possibly the percolating surface lake water, and more likely the juvenile water. Similarly to terrestrial calderas, Orcus Patera might be surrounded by ring-fractures caused by the collapse of the magma chamber that followed the release of gases. These ring-fractures may have been covered later by sedimentation in the caldera (lacustrine, aeolian, and volcanic), and by mass wasting. The detumescence of the magma in the caldera, and the vesiculation of the juvenile water may have operated simultaneously. Comparatively to terrestrial melts, Martian iron-rich melts are denser. This greater density implies greater effusion rates (eight-times terrestrial values), and larger fissuration widths (two-times terrestrial ones). With increasing vesiculation of magma, the bubbles interact with one-another because there are of similar pressure. They make a magma froth at the contact with the caldera surface, and on the walls of the fractures. In the saturated magma, froth, where the volume ratio of gases-to-liquid is about 4:1, the bubbles form a huge surface area of interconnected spaces. Bubbles near the caldera surface disrupt the magma, and fragmentation takes place, which moves downward through the magma column. On Earth, the bubbles are likely to grow between 1 and 50 mm in diameter due to the difference between the magma surface tension, and the bubble supersaturation pressure. The Martian low-pressure at surface level is likely to accelerate the expansion of the bubbles, and increase their final diameter and number, creating more voids in the magma. The strong magma froth with enclosed juvenile water bubbles interconnected with exsolved gas bubbles constitute a potential geothermal environment for geochemical energy production from basalt and water that does not require excessive temperatures. This process can start at +20C. Similar types of environments have been shown on Earth as potential energy sources for microbial metabolism, and could have provided deep aqueous basaltic niches for possible Martian microorganisms, even geologically recently. During the Amazonian, combination of volcanism and water activity still existed on Mars. Moreover, this type of potential niches open ways for investigation of possible oases of extinct or extant life, not only on paleolakes, and surface hydrothermalism spring areas, but also all large systems of fossae, which combine hydrologic and volcanic activities, and which provide an energy source, and an underground shelter to prevent surface UV bombardment. Additional information contained in the original.

Grin, E. A.↗

Does Collection Time Bias the Ecology of Cleanroom Air Samples?

Microbial monitoring of astromaterials collections has taken on increased importance with the return of biologically sensitive samples from the asteroids Ryugu and Bennu and the initiation of the Mars Sample Return Program. Terrestrial bacteria and fungi can alter the mineralogy and organic composition of our collections causing irreversible contamination of pristine samples and increasing the risk of false positives for life detection measurements. NASA has conducted routine microbial monitoring of its existing collections since 20181. Initial monitoring focused on surface samples collected with foam swabs. Although, airborne microbiology is often decoupled from surface microbiology in the built environment2 culture-based air sampling techniques like impactors were not compliant with existing contamination control requirements. Bringing organic rich media, gelatin or liquids into curation cleanrooms presents an unacceptable risk to pristine samples. In 2022 NASA purchased a materials complaint air sampler and began collecting air samples from the cleanrooms in addition to surface samples3. The new instrument uses an electret filter to collect samples that are suitable for cultivating organisms or for direct DNA sequencing. Preliminary DNA sequencing results appeared to indicate that longer sampling times biased the microbial community in favor of hearty, spore-forming bacteria3. We present the results of a study comparing overnight sampling (17 hours) to short (1 hour) sampling of unoccupied curation cleanrooms. The results will help us optimize our monitoring protocols and develop a more detailed inventory of the ecology of astromaterials curation cleanrooms. Methods: We analyzed 72 paired air samples from six different cleanrooms including the meteorite processing lab (ISO 7 equivalent, 16 samples), the lunar lab (ISO 6 equivalent, 10 samples), the stardust lab (ISO 5 equivalent 14 samples), the OSIRIS-REx lab (ISO 5 equivalent, 12 samples), the Hayabusa2 lab (ISO 5 equivalent, 14 samples), and the Genesis lab (ISO 4 equivalent, 6 samples). All the samples were collected with an InnovaPrep Bobcat air sampler operating at a sampling rate of 200 L/min. The sampler operates for 5 minutes out of every 20 minute period. Half of the samples were collected by filtering 3,000L (15 min. of active sampling) of air across an electret filter for one hour. The rest of the samples were collected by filtering approximately 51,000 L air across the filter overnight (~17 hours, 255 min. of active sampling). Cells were eluted from the filter using 6-7 ml of pressurized 0.15% tween 20 in PBS (phosphate buffered saline). This liquid was used to cultivate bacteria according to previously published methods1,4,5 and for DNA extraction and next generation sequencing. DNA was extracted with a Qiagen MagAttract PowerMicrobiome kit6. To identify bacteria and archaea, the 16S rRNA gene was amplified using Earth Microbiome primers for the V4 region 7. The amplified DNA was sequenced on an Illumina MiSeq using a V3 reagent kit. The resulting sequences were processed using DADA2 and QIIME2 as implemented on the EDGE bioinformatics platform8–10. Results: Only two of the 72 samples had no amplifiable DNA. Amplified DNA concentrations ranged from 2.67 – 0.272 ng/µl. The median concentration of amplified DNA for the 1 hour samples was 0.770 ± 0.368 ng/µl. The median concentration of amplified DNA for the overnight samples was 0.877 ± 0.434 ng/µl. On average the overnight samples had slightly more sequences (58,960 vs. 59,456) and ASV’s (amplicon sequence variants) (60 vs 64.5) than the one hour samples, but these differences are not statistically significant. The most abundant ASV in every sample mapped to the genus Cupravidus. ASV’s mapping to the genuses Bacillus, Schlegelella, Thermus, and Staphylococcus were also common. Discussion and Future Work: Alpha diversity statistics like Shannon Entropy and Faith Phylogenetic Diversity are used to describe the diversity of organisms in a single sample. If a longer sampling time was biasing the data, we would expect to see a change in these diversity statistics vs. sample time. However, we did not observe this in our data. The median Shannon entropy was slightly higher for the overnight samples (3.773 vs 3.611) as was the Faith Phylogenetic Diversity (4.042 vs 3.596), but both values were within a standard deviation of each other for the two sampling times (Fig. 1). It is unlikely, that the longer sampling time is introducing bias into our data. We do observe a significant decrease in diversity when comparing the air samples by lab. The Genesis lab (ISO 4 equivalent) has a lower median number of ASV’s (45.5) than the other labs (62). Median values for Shannon Entropy (3.717 vs. 3.430) and Faith Phylogenetic Diversity (3.796 vs. 3.548) are also lower for Genesis, but those values are with one standard deviation of each other for the different sampling times. This is consistent with previous culture-based results suggesting that the environment in cleanrooms tends to select for a core group of organisms capable of surviving under dry, low nutrient, conditions. The presence of the ASV’s mapping to Cupravidus and Thermus in our sequencing blanks and controls suggests that several of the most common organisms in our samples represent contaminants from the reagents used to perform the DNA extractions and sequencing. Further work is needed to identify these contaminants, remove them from our data and recalculate the diversity statistics. This is a systematic error. Therefore, we do not expect removing the sequencing contaminants to change our conclusions. Longer air sample collection times appear to result in slightly higher diversity and do not bias the results towards “hardy” bacteria like spore-formers. Based on these preliminary results we conclude that sampling at least 3,000 liters of air is sufficient to capture the microbial diversity of cleanrooms, and that air samples can also be collected overnight without negatively impacting diversity. These results allow us to be flexible when designing microbial monitoring plans so that they do not interfere with routine lab activity. References: 1. Regberg, A. B. et al. 49th Lunar and Planetary Science Conference (2018). 2. The United States Pharmacopeial Convention. USP General Chapter <1116> (2013). 3. Regberg, A. B., et al. 54th Lunar and Planetary Science Conference (2023). 4. Regberg, A. B. et al. 53rd Lunar and Planetary Science Conference ( 2022). 5. Davis, R. E.,et al. 50th Lunar and Planetary Science Conference (2019). 6. Qiagen. MagAttract® PowerMicrobiome® DNA/RNA EP Kit Handbook. (2018). 7. Walters, W. et al. mSystems 1, (2015). 8. Callahan, B. J. et al. Nat. Methods 13, 581–583 (2016). 9. Hall, M. & Beiko, R. G. Microbiome Analysis: Methods and Protocols113–129 (Springer, 2018). 10. Philipson, C. et al. Bio-Protoc. 7, e2622 (2017).

A. B. Regberg↗

Characterizing suburban soil and microbial properties along a soil age chronosequence

Abstract Rapid urbanization is drastically altering ecosystem processes in landscapes around the world. In particular, suburban residential neighborhoods comprise novel ecosystems with water and nutrient inputs that differ greatly from the surrounding land area. These impacts generate concern over the sustainability of urban ecosystems, especially whether they will be characterized by net carbon gain or loss over time. To address this knowledge gap, we established a chronosequence of residential yards in Southern California to test how urban soils change after development. We predicted that urbanized soils would experience shifts in physical characteristics and microbial function over time consistent with ecological succession theory, but residential soils would maintain novel moisture and nutrient regimes compared to undeveloped soils, never “recovering” to a pre‐developed state. We compared different vegetation types to quantify impacts of homeowner landscaping choices and characterized yard soils and their microbial communities. We found that yard soils were nutrient‐ and moisture‐enriched compared to an adjacent undeveloped ecosystem, and turfgrass was associated with higher levels of water and nitrogen. Despite high respiration rates, yard soils accumulated carbon and nitrogen over time. We conclude that suburban residential soils comprise dynamic and heterogeneous ecosystems that are highly influenced by landscaping choices and management practices, and warrant closer study at small management‐relevant scales.

Suratt, Andie [Department of Ecology and Evolution↗

Combined effects of lipase and Lactiplantibacillus plantarum 1‐24‐LJ on physicochemical property, microbial succession and volatile compounds formation in fermented fish product

Abstract BACKGROUND Studies have shown that either the addition of starter culture or enzyme can improve fermentation in fish or other products. However, little research has been carried out on the effects of coupling starter cultures with lipase on the microbial community and product quality. Suanzhayu is a Chinese fermented fish product that mainly relies on spontaneous fermentation, resulting in an unstable flavor and quality. The present study investigated the impact of lipase and Lactiplantibacillus plantarum 1‐24‐LJ on the quality of Suanzhayu . RESULTS Inoculation decreased pH and 2‐thiobarbituric acid reactive substances (TBARS) values, and also helped the dominance of the strain in the ecosystem, whereas lipase addition raised TBARS values and had little effect on pH, water activity ( a w ) and microbiota. The addition of lipase and/or Lpb. plantarum increased the content of alcohols, aldehydes, ketones, esters and umami amino acids. The co‐additions with the most significant effect and the total contents of volatile compounds (VCs) and free amino acids (FAAs) were 1801.92 g per 100 g and 21 357.05 mg per 100 g, respectively. Former‐ Lactobacillus was negatively correlated with pH, a w and Prevotella , but positively with VCs (ethyl ester of heptanoic acid, ethyl ester of octanoic acid) and FAAs (Tyr, Phe). Furthermore, adding Lpb. plantarum 1‐24‐LJ alone or in combination with lipase shortened the fermentation process. CONCLUSION The present study provides a recommended Suanzhayu process approach for improving product quality and flavor, as well as shortening fermentation time, by adding Lpb. plantarum 1‐24‐LJ with or without lipase. © 2023 Society of Chemical Industry.

Zhang, Zuoli↗

Unveiling the hidden world of microorganisms and their impact on the Earth's ecosystems

Microorganisms are the unseen drivers of the Earth, impacting everything from the air we breathe to the soil beneath our feet. The roles they play in maintaining ecosystem functioning, particularly in the biogeochemical cycling of carbon, nitrogen, phosphorus, sulfur, and metals, are profound. Microbial communities are critical in mediating organic carbon mineralization, methane cycling, denitrification, and sulfate reduction, all of which regulate carbon storage and greenhouse gas emissions.

59 BASIC BIOLOGICAL SCIENCES↗