One-Week-Ahead Prediction of Cyanobacterial Harmful Algal Blooms in Iowa Lakes.

Cyanobacterial harmful algal blooms (CyanoHABs) pose serious risks to inland water resources. Despite advancements in our understanding of associated environmental factors and modeling efforts, predicting CyanoHABs remains challenging. Leveraging an integrated water quality data collection effort in Iowa lakes, this study aimed to identify factors associated with hazardous microcystin levels and develop one-week-ahead predictive classification models. Using water samples from 38 Iowa lakes collected between 2018 and 2021, feature selection was conducted considering both linear and nonlinear properties. Subsequently, we developed three model types (Neural Network, XGBoost, and Logistic Regression) with different sampling strategies using the nine selected variables (mcyA_M, TKN, % hay/pasture, pH, mcyA_M:16S, % developed, DOC, dewpoint temperature, and ortho-P). Evaluation metrics demonstrated the strong performance of the Neural Network with oversampling (ROC-AUC 0.940, accuracy 0.861, sensitivity 0.857, specificity 0.857, LR+ 5.993, and 1/LR- 5.993), as well as the XGBoost with downsampling (ROC-AUC 0.944, accuracy 0.831, sensitivity 0.928, specificity 0.833, LR+ 5.557, and 1/LR- 11.569). This study exhibited the intricacies of modeling with limited data and class imbalances, underscoring the importance of continuous monitoring and data collection to improve predictive accuracy. Also, the methodologies employed can serve as meaningful references for researchers tackling similar challenges in diverse environments.

Insights on biotic and abiotic 2,4-dichlorophenoxyacetic acid degradation by anaerobic iron-cycling bacteria.

The use of the phenoxy herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has been steadily increasing in recent years due to its selectivity against broad-leafed weeds and use on genetically modified crops resistant to 2,4-D. This increases the likelihood of 2,4-D persisting in agriculturally impacted soils, sediments, and aquatic systems. Aerobic microorganisms are capable of degrading 2,4-D enzymatically. Anaerobic degradation also occurs, though the enzymatic pathway is unclear. Iron-reducing bacteria (FeRB) have been hypothesized to augment anaerobic degradation through the production of a chemically reactive Fe(II) adsorbed to Fe(III) oxyhydroxides. To test whether this iron species can catalyze abiotic degradation of 2,4-D, an enrichment culture (BLA1) containing a photosynthetic Fe(II)-oxidizing bacterium (FeOB) "Candidatus Chlorobium masyuteum" and the FeRB "Candidatus Pseudopelobacter ferreus", both of which lacked known 2,4-D degradation genes was investigated. BLA1 produces Fe(II)-adsorbed to Fe(III) oxyhydroxides during alternating photoautotrophic iron oxidation and dark iron reduction (amended with acetate) cycles. No 2,4-D degradation occurred during iron oxidation by FeOB Ca. C. masyuteum or during iron reduction by FeRB Ca. P. ferreus under any incubation conditions tested (i.e., +/-Fe(II), +/-cells, and +/-light), or due to the presence of Fe(II) adsorbed to Fe(III) oxyhydroxides. Our results cast doubt on the hypothesis that the mineral-bound Fe(II) species augments the anaerobic degradation of 2,4-D in anoxic soils and waters by iron-cycling bacteria, and further justify the need to identify the genetic underpinnings of anaerobic 2,4-D degradation.

Farming on Mars: Treatment of basaltic regolith soil and briny water simulants sustains plant growth.

A fundamental challenge in human missions to Mars is producing consumable foods efficiently with the in situ resources such as soil, water, nutrients and solar radiation available on Mars. The low nutrient content of martian soil and high salinity of water render them unfit for direct use for propagating food crops on Mars. It is therefore essential to develop strategies to enhance nutrient content in Mars soil and to desalinate briny water for long-term missions on Mars. We report simple and efficient strategies for treating basaltic regolith simulant soil and briny water simulant for suitable resources for growing plants. We show that alfalfa plants grow well in a nutrient-limited basaltic regolith simulant soil and that the alfalfa biomass can be used as a biofertilizer to sustain growth and production of turnip, radish and lettuce in the basaltic regolith simulant soil. Moreover, we show that marine cyanobacterium Synechococcus sp. PCC 7002 effectively desalinates the briny water simulant, and that desalination can be further enhanced by filtration through basalt-type volcanic rocks. Our findings indicate that it is possible to grow food crops with alfalfa treated basaltic regolith martian soil as a substratum watered with biodesalinated water.

Seasonal phytoplankton and geochemical shifts in the subsurface chlorophyll maximum layer of a dimictic ferruginous lake.

Subsurface chlorophyll maxima layers (SCML) are ubiquitous features of stratified aquatic systems. Availability of the micronutrient iron is known to influence marine SCML, but iron has not been explored in detail as a factor in the development of freshwater SCML. This study investigates the relationship between dissolved iron and the SCML within the dimictic, ferruginous lake Grosses Heiliges Meer in northern Germany. The occurrence of the SCML under nonferruginous conditions in the spring and ferruginous conditions in the fall are context to explore temporal changes in the phytoplankton community and indicators of primary productivity. Results indicate that despite more abundant chlorophyll in the spring, the SCML sits below a likely primary productivity maximum within the epilimnion, inferred based on colocated dissolved oxygen, δ13 CDIC , and pH maxima. The peak amount of chlorophyll in the SCML is lower in the fall than in the spring, but in the fall the SCML is colocated with elevated dissolved iron concentrations and a local δ13 CDIC maximum. Cyanobacteria and Chlorophyta have elevated abundances within the SCML in the fall. Further investigation of the relationship of iron to primary productivity within ferruginous SCML may help to understand the environmental controls on primary productivity in past ferruginous oceans.

Corrigendum: "Candidatus Chlorobium masyuteum," a Novel Photoferrotrophic Green Sulfur Bacterium Enriched From a Ferruginous Meromictic Lake.

[This corrects the article DOI: 10.3389/fmicb.2021.695260.].

"Candidatus Chlorobium masyuteum," a Novel Photoferrotrophic Green Sulfur Bacterium Enriched From a Ferruginous Meromictic Lake.

Anoxygenic phototrophic bacteria can be important primary producers in some meromictic lakes. Green sulfur bacteria (GSB) have been detected in ferruginous lakes, with some evidence that they are photosynthesizing using Fe(II) as an electron donor (i.e., photoferrotrophy). However, some photoferrotrophic GSB can also utilize reduced sulfur compounds, complicating the interpretation of Fe-dependent photosynthetic primary productivity. An enrichment (BLA1) from meromictic ferruginous Brownie Lake, Minnesota, United States, contains an Fe(II)-oxidizing GSB and a metabolically flexible putative Fe(III)-reducing anaerobe. "Candidatus Chlorobium masyuteum" grows photoautotrophically with Fe(II) and possesses the putative Fe(II) oxidase-encoding cyc2 gene also known from oxygen-dependent Fe(II)-oxidizing bacteria. It lacks genes for oxidation of reduced sulfur compounds. Its genome encodes for hydrogenases and a reverse TCA cycle that may allow it to utilize H2 and acetate as electron donors, an inference supported by the abundance of this organism when the enrichment was supplied by these substrates and light. The anaerobe "Candidatus Pseudopelobacter ferreus" is in low abundance (∼1%) in BLA1 and is a putative Fe(III)-reducing bacterium from the Geobacterales ord. nov. While "Ca. C. masyuteum" is closely related to the photoferrotrophs C. ferroooxidans strain KoFox and C. phaeoferrooxidans strain KB01, it is unique at the genomic level. The main light-harvesting molecule was identified as bacteriochlorophyll c with accessory carotenoids of the chlorobactene series. BLA1 optimally oxidizes Fe(II) at a pH of 6.8, and the rate of Fe(II) oxidation was 0.63 ± 0.069 mmol day-1, comparable to other photoferrotrophic GSB cultures or enrichments. Investigation of BLA1 expands the genetic basis for phototrophic Fe(II) oxidation by GSB and highlights the role these organisms may play in Fe(II) oxidation and carbon cycling in ferruginous lakes.

Months-long spike in aqueous arsenic following domestic well installation and disinfection: Short- and long-term drinking water quality implications.

Exposure to high concentration geogenic arsenic via groundwater is a worldwide health concern. Well installation introduces oxic drilling fluids and hypochlorite (a strong oxidant) for disinfection, thus inducing geochemical disequilibrium. Well installation causes changes in geochemistry lasting 12 + months, as illustrated in a recent study of 250 new domestic wells in Minnesota, north-central United States. One study well had extremely high initial arsenic (1550 µg/L) that substantially decreased after 15 months (5.2 µg/L). The drilling and development of the study well were typical and ordinary; nothing observable indicated the very high initial arsenic concentration. We hypothesized that oxidation of arsenic-containing sulfides (which lowers pH) combined with low pH dissolution of arsenic-bearing Fe (oxyhydr)oxides caused the very high arsenic concentration. Geochemical equilibrium considerations and modeling supported our hypothesis. Groundwater equilibrium redox conditions are poised at the Fe(III)(s)/Fe(II)(aq) stability boundary, indicating arsenic-bearing Fe (oxyhydr)oxide mineral sensitivity to pH and redox changes. Changing groundwater geochemistry can have negative implications for home water treatment (e.g., reduced arsenic removal efficiency, iron fouling), which can lead to ongoing but unrecognized hazard of arsenic exposure from domestic well water. Our results may inform arsenic mobilization processes and geochemical sensitivity in similarly complex aquifers in Southeast Asia and elsewhere.

An evolving view on biogeochemical cycling of iron.

Biogeochemical cycling of iron is crucial to many environmental processes, such as ocean productivity, carbon storage, greenhouse gas emissions and the fate of nutrients, toxic metals and metalloids. Knowledge of the underlying processes involved in iron cycling has accelerated in recent years along with appreciation of the complex network of biotic and abiotic reactions dictating the speciation, mobility and reactivity of iron in the environment. Recent studies have provided insights into novel processes in the biogeochemical iron cycle such as microbial ammonium oxidation and methane oxidation coupled to Fe(III) reduction. They have also revealed that processes in the biogeochemical iron cycle spatially overlap and may compete with each other, and that oxidation and reduction of iron occur cyclically or simultaneously in many environments. This Review discusses these advances with particular focus on their environmental consequences, including the formation of greenhouse gases and the fate of nutrients and contaminants.

Microaerophilic Oxidation of Fe(II) Coupled with Simultaneous Carbon Fixation and As(III) Oxidation and Sequestration in Karstic Paddy Soil.

Microaerophilic Fe(II)-oxidizing bacteria are often chemolithoautotrophs, and the Fe(III) (oxyhydr)oxides they form could immobilize arsenic (As). If such microbes are active in karstic paddy soils, their activity would help increase soil organic carbon and mitigate As contamination. We therefore used gel-stabilized gradient systems to cultivate microaerophilic Fe(II)-oxidizing bacteria from karstic paddy soil to investigate their capacity for Fe(II) oxidation, carbon fixation, and As sequestration. Stable isotope probing demonstrated the assimilation of inorganic carbon at a maximum rate of 8.02 mmol C m-2 d-1. Sequencing revealed that Bradyrhizobium, Cupriavidus, Hyphomicrobium, Kaistobacter, Mesorhizobium, Rhizobium, unclassified Phycisphaerales, and unclassified Opitutaceas were fixing carbon. Fe(II) oxidation produced Fe(III) (oxyhydr)oxides, which can absorb and/or coprecipitate As. Adding As(III) decreased the diversity of functional bacteria involved in carbon fixation, the relative abundance of predicted carbon fixation genes, and the amount of carbon fixed. Although the rate of Fe(II) oxidation was also lower in the presence of As(III), over 90% of the As(III) was sequestered after oxidation. The potential for microbially mediated As(III) oxidation was revealed by the presence of arsenite oxidase gene (aioA), denoting the potential of the Fe(II)-oxidizing and autotrophic microbial community to also oxidize As(III). Thisstudy demonstrates that carbon fixation coupled to Fe(II) oxidation can increase the carbon content in soils by microaerophilic Fe(II)-oxidizing bacteria, as well as accelerate As(III) oxidation and sequester it in association with Fe(III) (oxyhydr)oxides.

Improved detection of mcyA genes and their phylogenetic origins in harmful algal blooms.

Microcystins, a group of cyanotoxins produced by cyanobacterial strains, have become a significant microbial hazard to human and animal health due to increases in the frequency and intensity of cyanobacterial harmful algal blooms (CyanoHABs). Many studies have explored the correlation between microcystin concentrations and abundances of toxin-producing genes (e.g., mcyA genes) measured using quantitative PCR, and discrepancies between toxin concentrations and gene abundances are often observed. In this study, the results show that these discrepancies are at least partially due to primer sets that do not capture the phylogenetic diversity of naturally present toxin-producers. We designed three novel primer gene probes based on known mcyA genes to improve the detection and quantification of these genes in environmental samples. These primers were shown to improve the identification of mcyA genes compared to previously published primers in freshwater metagenomes, cyanobacterial isolates, and lake water samples. Unlike previously published primers, our primer sets could selectively amplify and resolve Microcystis, Anabaena, and Planktothrix mcyA genes. In lake water samples, abundance estimations of mcyA genes were found to correlate strongly with microcystin concentrations. Based on our results, these primers offer significant improvements over previously published probes to accurately identify and quantify mcyA genes in the environment. There is an increasing need to develop models based on microbial information and environmental factors to predict CyanoHABs, and improved primers will play an important role in aiding monitoring efforts to collect reliable and consistent data on toxicity risks.

Biogeochemical and physical controls on methane fluxes from two ferruginous meromictic lakes.

Meromictic lakes with anoxic bottom waters often have active methane cycles whereby methane is generally produced biogenically under anoxic conditions and oxidized in oxic surface waters prior to reaching the atmosphere. Lakes that contain dissolved ferrous iron in their deep waters (i.e., ferruginous) are rare, but valuable, as geochemical analogues of the conditions that dominated the Earth's oceans during the Precambrian when interactions between the iron and methane cycles could have shaped the greenhouse regulation of the planet's climate. Here, we explored controls on the methane fluxes from Brownie Lake and Canyon Lake, two ferruginous meromictic lakes that contain similar concentrations (max. >1 mM) of dissolved methane in their bottom waters. The order Methanobacteriales was the dominant methanogen detected in both lakes. At Brownie Lake, methanogen abundance, an increase in methane concentration with respect to depths closer to the sediment, and isotopic data suggest methanogenesis is an active process in the anoxic water column. At Canyon Lake, methanogenesis occurred primarily in the sediment. The most abundant aerobic methane-oxidizing bacteria present in both water columns were associated with the Gammaproteobacteria, with little evidence of anaerobic methane oxidizing organisms being present or active. Direct measurements at the surface revealed a methane flux from Brownie Lake that was two orders of magnitude greater than the flux from Canyon Lake. Comparison of measured versus calculated turbulent diffusive fluxes indicates that most of the methane flux at Brownie Lake was non-diffusive. Although the turbulent diffusive methane flux at Canyon Lake was attenuated by methane oxidizing bacteria, dissolved methane was detected in the epilimnion, suggestive of lateral transport of methane from littoral sediments. These results highlight the importance of direct measurements in estimating the total methane flux from water columns, and that non-diffusive transport of methane may be important to consider from other ferruginous systems.

Oxic Fe(III) reduction could have generated Fe(II) in the photic zone of Precambrian seawater.

Many marine Precambrian iron formations (IF) record deep anoxic seawater enriched in Fe(II) (i.e. ferruginous) overlain by mildly oxygenated surface water. This is reflected by iron-rich sediments forming in deep basins, and relatively iron-poor sediments forming in shallow, sunlit waters. Such an iron gradient is often interpreted as a redox interface where dissolved Fe(II) was oxidized and precipitated as Fe(III)-bearing minerals. As such, sedimentary iron enrichments are proxy to the progressive oxidation of the oceans through geological time. However, this interpretation is founded on the assumption that Fe(II) could not persist within an oxygenated water column. Here, we cultivated cyanobacteria in an illuminated column supplied with Fe(II)-rich seawater medium in a laboratory-scale analog of a continental margin supporting IF deposition. We first observed Fe(II) oxidation with oxygen, then biologically-mediated reduction of Fe(III) (oxyhydr)oxides, which maintained a pool of Fe(II) in the presence of oxygen. Such steady-state iron redox cycling may have maintained dissolved, and hence mobile Fe(II) in oxygenated seawater above ferruginous deep basins such as those inferred for many Precambrian IF.

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria.

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments - including shallow marine or lacustrine sediments.

The interplay of microbially mediated and abiotic reactions in the biogeochemical Fe cycle.

Many iron (Fe) redox processes that were previously assumed to be purely abiotic, such as photochemical Fe reactions, are now known to also be microbially mediated. Owing to this overlap, discerning whether biotic or abiotic processes control Fe redox chemistry is a major challenge for geomicrobiologists and biogeochemists alike. Therefore, to understand the network of reactions within the biogeochemical Fe cycle, it is necessary to determine which abiotic or microbially mediated reactions are dominant under various environmental conditions. In this Review, we discuss the major microbially mediated and abiotic reactions in the biogeochemical Fe cycle and provide an integrated overview of biotic and chemically mediated redox transformations.

Characterization of the physiology and cell-mineral interactions of the marine anoxygenic phototrophic Fe(II) oxidizer Rhodovulum iodosum--implications for Precambrian Fe(II) oxidation.

Anoxygenic phototrophic Fe(II)-oxidizing bacteria (photoferrotrophs) are suggested to have contributed to the deposition of banded iron formations (BIFs) from oxygen-poor seawater. However, most studies evaluating the contribution of photoferrotrophs to Precambrian Fe(II) oxidation have used freshwater and not marine strains. Therefore, we investigated the physiology and mineral products of Fe(II) oxidation by the marine photoferrotroph Rhodovulum iodosum. Poorly crystalline Fe(III) minerals formed initially and transformed to more crystalline goethite over time. During Fe(II) oxidation, cell surfaces were largely free of minerals. Instead, the minerals were co-localized with EPS suggesting that EPS plays a critical role in preventing cell encrustation, likely by binding Fe(III) and directing precipitation away from cell surfaces. Fe(II) oxidation rates increased with increasing initial Fe(II) concentration (0.43-4.07 mM) under a light intensity of 12 µmol quanta m(-2) s(-1). Rates also increased as light intensity increased (from 3 to 20 µmol quanta m(-2) s(-1)), while the addition of Si did not significantly change Fe(II) oxidation rates. These results elaborate on how the physical and chemical conditions present in the Precambrian ocean controlled the activity of marine photoferrotrophs and confirm the possibility that such microorganisms could have oxidized Fe(II), generating the primary Fe(III) minerals that were then deposited to some Precambrian BIFs.

Potential for Nitrogen Fixation and Nitrification in the Granite-Hosted Subsurface at Henderson Mine, CO.

The existence of life in the deep terrestrial subsurface is established, yet few studies have investigated the origin of nitrogen that supports deep life. Previously, 16S rRNA gene surveys cataloged a diverse microbial community in subsurface fluids draining from boreholes 3000 feet deep at Henderson Mine, CO, USA (Sahl et al., 2008). The prior characterization of the fluid chemistry and microbial community forms the basis for the further investigation here of the source of NH(4) (+). The reported fluid chemistry included N(2), NH(4) (+) (5-112 µM), NO(2) (-) (27-48 µM), and NO(3) (-) (17-72 µM). In this study, the correlation between low NH(4) (+) concentrations in dominantly meteoric fluids and higher NH(4) (+) in rock-reacted fluids is used to hypothesize that NH(4) (+) is sourced from NH(4) (+)-bearing biotite. However, biotite samples from the host rocks and ore-body minerals were analyzed by Fourier transform infrared (FTIR) microscopy and none-contained NH(4) (+). However, the nitrogenase-encoding gene nifH was successfully amplified from DNA of the fluid sample with high NH(4) (+), suggesting that subsurface microbes have the capability to fix N(2). If so, unregulated nitrogen fixation may account for the relatively high NH(4) (+) concentrations in the fluids. Additionally, the amoA and nxrB genes for archaeal ammonium monooxygenase and nitrite oxidoreductase, respectively, were amplified from the high NH(4) (+) fluid DNA, while bacterial amoA genes were not. Putative nitrifying organisms are closely related to ammonium-oxidizing Crenarchaeota and nitrite-oxidizing Nitrospira detected in other subsurface sites based upon 16S rRNA sequence analysis. Thermodynamic calculations underscore the importance of NH(4) (+) as an energy source in a subsurface nitrification pathway. These results suggest that the subsurface microbial community at Henderson is adapted to the low nutrient and energy environment by their capability of fixing nitrogen, and that fixed nitrogen may support subsurface biomass via nitrification.

Paenibacillus phoenicis sp. nov., isolated from the Phoenix Lander assembly facility and a subsurface molybdenum mine.

A novel Gram-positive, motile, endospore-forming, aerobic bacterium was isolated from the NASA Phoenix Lander assembly clean room that exhibits 100 % 16S rRNA gene sequence similarity to two strains isolated from a deep subsurface environment. All strains are rod-shaped, endospore-forming bacteria, whose endospores are resistant to UV radiation up to 500 J m(-2). A polyphasic taxonomic study including traditional phenotypic tests, fatty acid analysis, 16S rRNA gene sequencing and DNA-DNA hybridization analysis was performed to characterize these novel strains. The 16S rRNA gene sequencing convincingly grouped these novel strains within the genus Paenibacillus as a separate cluster from previously described species. The similarity of 16S rRNA gene sequences among the novel strains was identical but only 98.1 to 98.5 % with their nearest neighbours Paenibacillus barengoltzii ATCC BAA-1209(T) and Paenibacillus timonensis CIP 108005(T). The menaquinone MK-7 was dominant in these novel strains as shown in other species of the genus Paenibacillus. The DNA-DNA hybridization dissociation value was <45 % with the closest related species. The novel strains had DNA G+C contents of 51.9 to 52.8 mol%. Phenotypically, the novel strains can be readily differentiated from closely related species by the absence of urease and gelatinase and the production of acids from a variety of sugars including l-arabinose. The major fatty acid was anteiso-C(15 : 0) as seen in P. barengoltzii and P. timonensis whereas the proportion of C(16 : 0) was significantly different from the closely related species. Based on phylogenetic and phenotypic results, it was concluded that these strains represent a novel species of the genus Paenibacillus, for which the name Paenibacillus phoenicis sp. nov. is proposed. The type strain is 3PO2SA(T) ( = NRRL B-59348(T)  = NBRC 106274(T)).

Phylogenetic Relationships and Functional Genes: Distribution of a Gene (mnxG) encoding a putative manganese-oxidizing enzyme in Bacillus species.

Several Bacillus and Paenibacillus species were isolated from Fe and Mn oxide minerals precipitating at a deep subsurface oxic-anoxic interface at Henderson Molybdenum Mine, Empire, CO. The isolates were investigated for their Mn(II)-oxidizing potential and interrogated for possession of the mnxG gene, a gene that codes for a putative Mn(II)-oxidizing enzyme in Bacillus species. Seven of eight Bacillus species were capable of Mn(II) oxidation; however, the mnxG gene was detected in only one isolate. Using sequences of known Bacillus species both with and without amplifiable mnxG genes and Henderson Mine isolates, the 16S rRNA and mnxG gene phylogenies were compared to determine if 16S rRNA sequences could be used to predict the presence or absence of an amplifiable mnxG gene within the genomes of the isolates. We discovered a strong correspondence between 16S rRNA sequence similarity and the presence/absence of an amplifiable mnxG gene in the isolates. The data revealed a complex phylogenetic distribution of the mnxG gene in which vertical inheritance and gene loss influence the distribution of the gene among the Bacillus species included in this study. Comparisons of 16S rRNA and functional gene phylogenies can be used as a tool to aid in unraveling the history and dispersal of the mnxG gene within the Bacillus clade.

Subsurface microbial diversity in deep-granitic-fracture water in Colorado.

A microbial community analysis using 16S rRNA gene sequencing was performed on borehole water and a granite rock core from Henderson Mine, a >1,000-meter-deep molybdenum mine near Empire, CO. Chemical analysis of borehole water at two separate depths (1,044 m and 1,004 m below the mine entrance) suggests that a sharp chemical gradient exists, likely from the mixing of two distinct subsurface fluids, one metal rich and one relatively dilute; this has created unique niches for microorganisms. The microbial community analyzed from filtered, oxic borehole water indicated an abundance of sequences from iron-oxidizing bacteria (Gallionella spp.) and was compared to the community from the same borehole after 2 weeks of being plugged with an expandable packer. Statistical analyses with UniFrac revealed a significant shift in community structure following the addition of the packer. Phospholipid fatty acid (PLFA) analysis suggested that Nitrosomonadales dominated the oxic borehole, while PLFAs indicative of anaerobic bacteria were most abundant in the samples from the plugged borehole. Microbial sequences were represented primarily by Firmicutes, Proteobacteria, and a lineage of sequences which did not group with any identified bacterial division; phylogenetic analyses confirmed the presence of a novel candidate division. This "Henderson candidate division" dominated the clone libraries from the dilute anoxic fluids. Sequences obtained from the granitic rock core (1,740 m below the surface) were represented by the divisions Proteobacteria (primarily the family Ralstoniaceae) and Firmicutes. Sequences grouping within Ralstoniaceae were also found in the clone libraries from metal-rich fluids yet were absent in more dilute fluids. Lineage-specific comparisons, combined with phylogenetic statistical analyses, show that geochemical variance has an important effect on microbial community structure in deep, subsurface systems.