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Abstract Members of the familyTrichomeriaceae,belonging to theChaetothyrialesorder and theAscomycotaphylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genusKnufiaencompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain ofKnufia obscura(CBS 148926^T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926^T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed asK. obscura.The morphological analysis and description of the genomic characteristics ofK. obscuraFJI-L2-BK-P2 may contribute to refining the taxonomy ofKnufiaspecies. Key morphological features are reported in thisK. obscurastrain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments. 

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