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Item Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04(Elsevier BV, 2023-01) Goemann, Calvin L.C.; Wilkinson, Royce; Henriques, William; Bui, Huyen; Goemann, Hannah M.; Carlson, Ross P.; Viamajala, Sridhar; Gerlach, Robin; Wiedenheft, BlakeAlgae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments – high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Collectively, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequence-based methods.Item Development of Martian saline seep models and their implications for planetary protection(Elsevier BV, 2023-12) Mettler, Madelyn K.; Goemann, Hannah M.; Mueller, Rebecca C.; Vanegas, Oscar A.; Lopez, Gabriela; Singh, Nitin; Venkateswaran, Kasthuri; Peyton, Brent M.While life on Mars has not been found, Earth-based microorganisms may contaminate the Red Planet during rover expeditions and human exploration. Due to the survival advantages conferred by the biofilm morphology to microorganisms, such as resistance to UV and osmotic stress, biofilms are particularly concerning from a planetary protection perspective. Modeling and data from the NASA Phoenix mission indicate that temporary liquid water might exist on Mars in the form of high salinity brines. These brines could provide colonization opportunities for terrestrial microorganisms brought by spacecraft or humans. To begin testing for potential establishment of microbes, results are presented from a simplified laboratory model of a Martian saline seep inoculated with sediment from Hailstone Basin, a terrestrial saline seep in Montana (USA). The seep was modeled as a sand-packed drip flow reactor at room temperature fed media with either 1 M MgSO4 or 1 M NaCl. Biofilms were established within the first sampling point of each experiment. Endpoint 16S rRNA gene community analysis showed significant selection of halophilic microorganisms by the media. Additionally, we detected 16S rRNA gene sequences highly similar to microorganisms previously detected in two spacecraft assembly cleanrooms. These experimental models provide an important foundation for identifying microbes that could hitch-hike on spacecraft and may be able to colonize Martian saline seeps. Future model optimization will be vital to informing cleanroom sterilization procedures.Item Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04(Elsevier BV, 2023-01) Goemann, Calvin L.C.; Wilkinson, Royce; Henriques, William; Bui, Huyen; Goemann, Hannah M.; Carlson, Ross P.; Viamajala, Sridhar; Gerlach, Robin; Wiedenheft, BlakeAlgae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments – high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Collectively, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequence-based methods.Item Climate mitigation potential and soil microbial response of cyanobacteria‐fertilized bioenergy crops in a cool semi‐arid cropland(Wiley, 2022-10) Gay, Justin D.; Goemann, Hannah M.; Currey, Bryce; Stoy, Paul C.; Christiansen, Jesper Riis; Miller, Perry R.; Poulter, Benjamin; Peyton, Brent M.; Brookshire, E. N. JackBioenergy carbon capture and storage (BECCS) systems can serve as decarbonization pathways for climate mitigation. Perennial grasses are a promising second-generation lignocellulosic bioenergy feedstock for BECCS expansion, but optimizing their sustainability, productivity, and climate mitigation potential requires an evaluation of how nitrogen (N) fertilizer strategies interact with greenhouse gas (GHG) and soil organic carbon (SOC) dynamics. Furthermore, crop and fertilizer choice can affect the soil microbiome which is critical to soil organic matter turnover, nutrient cycling, and sustaining crop productivity but these feedbacks are poorly understood due to the paucity of data from certain agroecosystems. Here, we examine the climate mitigation potential and soil microbiome response to establishing two functionally different perennial grasses, switchgrass (Panicum virgatum, C4) and tall wheatgrass (Thinopyrum ponticum, C3), in a cool semi-arid agroecosystem under two fertilizer applications, a novel cyanobacterial biofertilizer (CBF) and urea. We find that in contrast to the C4 grass, the C3 grass achieved 98% greater productivity and had a higher N use efficiency when fertilized. For both crops, the CBF produced the same biomass enhancement as urea. Non-CO2 GHG fluxes across all treatments were low and we observed a 3-year net loss of SOC under the C4 crop and a net gain under the C3 crop at a 0–30 cm soil depth regardless of fertilization. Finally, we detected crop-specific changes in the soil microbiome, including an increased relative abundance of arbuscular mycorrhizal fungi under the C3, and potentially pathogenic fungi in the C4 grass. Taken together, these findings highlight the potential of CBF-fertilized C3 crops as a second-generation bioenergy feedstock in semi-arid regions as a part of a climate mitigation strategy.Item Root exudate composition reflects drought severity gradient in blue grama (Bouteloua gracilis)(Springer Nature, 2022-07) Ulrich, Danielle E. M.; Clendinen, Chaevien S.; Alongi, Franklin; Mueller, Rebecca C.; Chu, Rosalie K.; Toyoda, Jason; Gallegos‑Graves, La Verne; Goemann, Hannah M.; Peyton, Brent; Sevanto, Sanna; Dunbar, JohnPlant survival during environmental stress greatly affects ecosystem carbon (C) cycling, and plant–microbe interactions are central to plant stress survival. The release of C-rich root exudates is a key mechanism plants use to manage their microbiome, attracting beneficial microbes and/or suppressing harmful microbes to help plants withstand environmental stress. However, a critical knowledge gap is how plants alter root exudate concentration and composition under varying stress levels. In a greenhouse study, we imposed three drought treatments (control, mild, severe) on blue grama (Bouteloua gracilis Kunth Lag. Ex Griffiths), and measured plant physiology and root exudate concentration and composition using GC–MS, NMR, and FTICR. With increasing drought severity, root exudate total C and organic C increased concurrently with declining predawn leaf water potential and photosynthesis. Root exudate composition mirrored the physiological gradient of drought severity treatments. Specific compounds that are known to alter plant drought responses and the rhizosphere microbiome mirrored the drought severity-induced root exudate compositional gradient. Despite reducing C uptake, these plants actively invested C to root exudates with increasing drought severity. Patterns of plant physiology and root exudate concentration and composition co-varied along a gradient of drought severity.Item Aboveground and belowground responses to cyanobacterial biofertilizer supplement in a semi-arid, perennial bioenergy cropping system(Wiley, 2021-08) Goemann, Hannah M.; Gay, Justin D.; Mueller, Rebecca C.; Brookshire, E. N. Jack; Miller, Perry; Poulter, Benjamin; Peyton, Brent M.The need for sustainable agricultural practices to meet the food, feed, and fuel demands of a growing global population while reducing detrimental environmental impacts has driven research in multi‐faceted approaches to agricultural sustainability. Perennial cropping systems and microbial biofertilizer supplements are two emerging strategies to increase agricultural sustainability that are studied in tandem for the first time in this study. During the establishment phase of a perennial switchgrass stand in SW Montana, USA, we supplemented synthetic fertilization with a nitrogen‐fixing cyanobacterial biofertilizer (CBF) and were able to maintain aboveground crop productivity in comparison to a synthetic only (urea) fertilizer treatment. Soil chemical analysis conducted at the end of the growing season revealed that late‐season nitrogen availability in CBF‐supplemented field plots increased relative to urea‐only plots. High‐throughput sequencing of bacterial/archaeal and fungal communities suggested fine‐scale responses of the microbial community and sensitivity to fertilization among arbuscular mycorrhizal fungi, Planctomycetes, Proteobacteria, and Actinobacteria. Given their critical role in plant productivity and soil nutrient cycling, soil microbiome monitoring is vital to understand the impacts of implementation of alternative agricultural practices on soil health.