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Metagenomes of Native and Electrode-Enriched Microbial Communities from the Soudan Iron Mine Jonathan P

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Metagenomes of native and electrode-enriched microbial communities from the Soudan Iron Mine Jonathan P. Badalamenti and Daniel R. Bond Department of Microbiology and BioTechnology Institute, University of Minnesota - Twin Cities, Saint Paul, Minnesota, USA Twitter: @JonBadalamenti @wanderingbond Summary Approach - compare metagenomes from native and electrode-enriched deep subsurface microbial communities 30 Despite apparent carbon limitation, anoxic deep subsurface brines at the Soudan ) enriched 2 Underground Iron Mine harbor active microbial communities . To characterize these 20 A/cm µ assemblages, we performed shotgun metagenomics of native and enriched samples. enrich ( harvest cells collect inoculate +0.24 V extract 10 Follwing enrichment on poised electrodes and long read sequencing, we recovered Soudan brine electrode 20° C from DNA biodreactors current electrodes from the metagenome the closed, circular genome of a novel Desulfuromonas sp. 0 0 10 20 30 40 filtrate PacBio RS II Illumina HiSeq with remarkable genomic features that were not fully resolved by short read assem- extract time (d) long reads short reads TFF DNA unenriched bly alone. This organism was essentially absent in unenriched Soudan communities, 0.1 µm retentate assembled metagenomes reconstruct long read return de novo complete genome(s) assembly indicating that electrodes are highly selective for putative metal reducers. Native HGAP assembly community metagenomes suggest that carbon cycling is driven by methyl-C me- IDBA_UD 1 hybrid tabolism, in particular methylotrophic methanogenesis. Our results highlight the 3 µm prefilter assembly N4 binning promising potential for long reads in metagenomic surveys of low-diversity environ- borehole N4 binning read trimming and filtering brine de novo ments. collection bottle assembly IDBA_UD Introduction Results - long reads recover a closed genome from mine enrichements on electrodes ĖŤ'#Ť.4"-Ť 1.-Ť (-#Ť(-Ť-.13'#1-Ť (--#2.3Ť/1.5("#2ŤŤ1#"(+8Ť!!#22( +#Ť/.13+Ť datasets ‘Ca. Desulfuromonas biwabikus DDH964’ into carbon cycling the deep terrestrial biosphere unenriched enriched on electrodes complete genome short read assembled contigs c-type cytochromes mapped reads > 6.5 kbp phage DNA/transposons Ť ĖŤĄĉ ŤĄ7ă㹍 /Ť ++4,(-Ť1#"2 Ť ĖŤĄć ŤĄ7ăĉĂŤ /Ť ++4,(-Ť1#"2 3,924,648 bp circular mapped long read coverage rRNA ĖŤ'#Ť"##/Ť24 241$!#Ť'1 .12Ťlow-complexity microbial communities which drive 3.9 3.8 repeat sequences >500 bp ĖŤĄćĊŤ /Ť!(.Ť+.-%Ť1#"2ŤijĄŤ Ť!#++2ďŤ 0.1 62.23% G+C 3.7 repeat sequences >2,000 bp fundamental biogeochemical cycles, including redox transformations of metals 0.2 3.6 Ť ŤŤŤ5%ĎŤ1#"Ť+#-%3'ŤċďĉăćŤ /Ĵ 3,633 CDS 0.3 genes on + strand ĖŤ1#5(.42ŤăĊŤ1Ť%#-#Ť2415#82Ť.$Ť-.7(!Ť 1(-#2Ť3Ť.4"-Ť2'.6Ť+.6Ť1#+3(5#Ť 4-- 3.5 0.4 genes on − strand imcH Gamma- 54 tRNA cbcL dance of putative metal reducers proteobacteria Delta- 3.4 0.5 proteobacteria ĖŤ+#!31."#2Ť./#13#"Ť3Ťǫ7#"Ť/.3#-3(+Ť#+(,(-3#Ť"(2"5-3%#2Ť.$Ť#-1(!'(-%Ť,#3+Ť Firmicutes 3.3 0.6 Alpha- reducers on insoluble metal oxides as electron acceptors, such as variability in DBIWA_3010 0.7 proteobacteria DBIWA_3009 crystallinity, redox potential, and adsorption of other compounds Unclassified DBIWA_3008 Bacteria Alphaproteobacteria 0.8 ĖŤ'.13Ť1#"Ť22#, +8Ĭ 2#"Ť2'.3%4-Ť,#3%#-.,(!2Ť!!#22#2Ť#5#-Ť+.6Ť 4-"-!#Ť Euryarchaeota Gammaproteobacteria DBIWA_3000 microbes in natural communities, but at the expense of assembly contiguity other other 0.9 3.2 ĖŤThere is tremendous potential for long reads in improving metagenomic assem- 1 Mb blies and downstream phylogenetic, bioinformatic, and biochemical predic- assembly comparison - electrode enrichment 2500 250 3.1 18 IDBA_UD PacBio subread filtering hgcAB 1.1 tions SPAdes hybrid 15 2000 200 SPAdes with 3 Mb 1.2 (Mbp) Illumina only 12 1500 150 Soudan Underground Iron Mine 2.9 9 1.3 1000 100 rRNA 2 rRNA 1 6 PBcR # of subreads ĖŤ4--#+2Ť!15#"Ť3Ťċ㹍,Ť"#/3'Ť(-Ť (--#2.3ġ2Ť.+"#23Ť-"Ť"##/#23Ť4-"#1%1.4-"Ť 2.8 1.4 500 50 lengthMbp > read CRISPR1 CRISPR2 mine transect massive veins of hematite embedded in an Archaean (2.7 Gya) lengthcumulative 3 2.7 1.5 banded iron formation 0 800 1600 2400 3200 4000 10 20 30 1.6 2.6 ĖŤ-.7(!Ť!+!(4,Ť!'+.1("#Ť 1(-#2Ť2##/Ť$1.,Ť#7/+.13.18Ť"1(++Ť'.+#2Ť(-Ť-Ť.3'#16(2#Ť contigs read length (kbp) 1.7 2.5 dry mine 1.8 2.4 Int’l Falls 1.9 ONT A R I O adding PacBio long reads improves metagenomic assembly CANADA M 2.3 I 2.2 2 Mb ĖŤ#2/(3#Ť!1 .-Ť+(,(33(.-ďŤ 1(-#2Ť1#Ť1(!'Ť(-Ť N long reads 2.1 N genome features E +8 short reads only short and long reads S O only reduced metals, suggesting active microbial T glyoxlyate shunt Bemidji A I O R E R U P S K E UNITED STATES L A IDBA_UD SPAdes SPAdes hybrid PBcR hybrid HGAP nitrate respiration malate synthase aceB isoctrate lyase icl metabolism 40 km Duluth contigs 2820 3816 3338 581 132 degradation of aromatics DBIWA_3278 DBIWA_3279 ĖŤSoudan is actively maintained as a Minne- total length 16,849,449 15,149,004 15,417,681 5,660,852 4,451,391 phosphonate transport 3536800 3538400 3540000 sota state park, allowing year-round N50 38,339 16,604 25,567 58,773 3,932,815 evidence for conjugative mobule element transfer (tra genes) and chromosomal integration L 107 187 80 27 1 access to the deep terrestrial biosphere 50 heavy metal resistance; mercury resistance and methylation Results - native Soudan metagenomes datasets inner rings novel lineages natural, unenriched Soudan Mine communities Firmicutes Alphaproteobacteria Class level Ť ĖŤĄć ŤĄ7ă㹍 /Ť ++4,(-Ť1#"2Ť/#1Ť .1#'.+# Bacteroidetes Gammaproteobacteria Order level Genome Comparison of Metal-Reducing Deltaproteobacteria Ť ĖŤ22#, +#"Ť6(3'Ť į Euryarchaeota Deltaproteobacteria Family level Ť ĖŤ Ť1#!.-2314!3(.-Ť.$Ť$4++Ĭ+#-%3'ŤăĊŤ1Ť%#-#2 38 unclassified Geopsychrobacter electrodophilus 24 Ť ĖŤ Ť3#31-4!+#.3("#Ť (--(-% Desulfuromusa kysingii outgroup-rooted tree based on Proteobacteria alignment of concatenated set Methanolobus unclassified of 40 conserved single-copy unclassified Clostridiales Rhodobacterales Methanolobus unclassified Dehalobacter Dehalobacter 22 6 genes generated using Phylosift Rhodobacteraceae Methanolobus Desulfuromonas thiophila Desulfosporosinus 31 (v. 1.0.1) unclassified Halocella 49 Rhodobacteraceae unclassified Roseovarius Sphaerochaeta Halocella Desulfuromonas acetoxidans Geobacter Clostridiales unclassified Pelobacter seleniigenes Rhodovulum Clostridiales 28 Rhodovulum 51 Geoalkalibacter subterraneus Red1 Pelobacter carbinolicus Desulfuromonas subbituminosa DDH DDH DDH Marinobacter 41 Geoalkalibacter ferrihydriticus Z-0531 ‘Ca. #24+$41.,.-2Ť (6 (*42ŤčĊćġ 932 944 951 unclassified ‘Ca.Ť#24+$41.,.-2Ť2.4"-#-2(2Ť ġ34 Halothiobacillus Desulfuromonas sp. TF Halothiobacillus Peptococcaceae Desulfuvibrio vulgaris Hildenborough unclassified unclassified Desulfopila 0.08 Desulfitibacter Demequina 61 Geobacter daltonii FRC-32 Alteromonadales Proteobacteria 2 multiheme* c-type Dehalobacter Pelobacter propionicus 86 Demequina unclassified Geobacter lovleyi cytochrome count Marinobacter Halanaerobium Bacteroidales 42 unclassified unclassified unclassified 86 Geobacter uraniireducens Rf4 Geobacter argillaceus Marinobacter *3 or more heme- Halanaerobiaceae Bacteroidales Peptococcaceae Geobacter sp. OR-1 Halocella 55 binding motifs GeobacterGeobacter sp. GSS01 sulfurreducens PCA unclassified Geobacter bemidjiensis 65 Geobacter metallireducens shallower/ deeper/ Peptococcaceae samples collected along redox gradient more oxidized more reduced Geobacter bremensis 58 58 pan- and core genome analysis of avail- Geobacter sp. M21 70 Geobacter sp. M18 able Geobacter/Desulfuromonas clade 76 genomes, with cytochrome plots re- 68 71 ported using default clustering param- Conclusions Acknowledgments Geobacter pickeringii G13 eters in get_homologues (3-6-2015 re- 58 59 lease) ĖŤ'.3%4-Ť,#3%#-.,(!2Ť.$Ť.4"-Ť!.,,4-(3(#2Ť1#5#+#"Ť-.5#+Ť1"#1ĬŤ-"Ť We thank the Minnesota Supercomputing Institute (MSI) for developing, implementing, and main- ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● taining the PacBio SMRT Analysis suite, and the Marine Biological Laboratory (MBL) for Illumina se- ● ● ● ● ● ● ● ● ● 4000 Family-level lineages, particularly among Firmicutes pangenome ● core genome ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● quencing under a seed grant from the Deep Carbon Observatory Census of Deep Life. Long read data all genes ● ● ● ● all genes ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 60 ● ● ● ● ● ● ● ĖŤ +(%3#+8Ť,#3'8+.31./'(!ŤMethanolobus was the only Archaeon observed in ● ● ● ● 12000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● was generously provided by Pacific Biosciences and we thank Karl Oles (Mayo Clinic Bioinformatics ● ● ● ● ● ● ● ● ● ● ● 3000 ● ● ● ● ● ● multiheme* ● ● ● ● ● unenriched metagenomes, suggesting active methyl-C metabolism in situ ● ● ● ● 1 ● ● ● ● ● ● ● ● Core) for sample preparation. We also thank Chris O’Brien (Pall Life Sciences) for guidance with TFF ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● c-type ● ● ● ● ● ● ● 30 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 100 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● cytochromes ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● -"Ť.4"-Ť1*Ť -%#1Ť (,Ť22(%Ť$.1Ť!..1"(-3(-%Ťǫ#+"Ť2,/+(-%ĎŤ'(2Ť6.1*Ť62Ť24//.13#"Ť 8Ť3'#Ť ● ● ● ĖŤ.(2#"Ť#+#!31."#2Ť#Ǫ#!3(5#+8Ť#-1(!'Ť-.5#+Ť,#3+Ĭ1#"4!(-%Ť !3#1(Ť$1.,Ť#-5(- ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 8000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 2000 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● clusters gene ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
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    Journal of Water and Environment Technology, Vol.13, No.2, 2015 Development of UASB-DHS System for Treating Industrial Wastewater Containing Ethylene Glycol Takahiro WATARI 1), Daisuke TANIKAWA2), Kyohei KURODA1), Akinobu NAKAMURA1), Nanako FUJII3), Fuminori YONEYAMA3), Osamu WAKISAKA3), Masashi HATAMOTO1), Takashi YAMAGUCHI 1) 1) Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan 2) Department of Civil and Environmental Engineering, National Institute of Technology, Kure College, 2-1-11 Agaminami, Kure, Hiroshima 737-8506, Japan 3) Fundamental Material Laboratory Office, Material Technology Research and Development Laboratory, Research and Development Headquarters, Sumitomo Riko Company Limited, 3-1 Higashi, Komaki, Aichi 485-8550, Japan ABSTRACT This study evaluated the performance of a novel treatment system consisting of an upflow anaerobic sludge blanket (UASB) and a downflow hanging sponge (DHS) for the treatment of industrial wastewater containing 8% ethylene glycol and 2% propylene glycol discharged from a rubber production unit. The system achieved high COD removal (91 ± 4.3%) and methane recovery (82 ± 20%) at an organic loading rate of 8.5 kg-COD/(m3·day). The UASB allowed an organic loading rate of 14 kg-COD/(m3·day) with a constant hydraulic retention time of 24 h. The COD of DHS effluent was 370 ± 250 mg-COD/L during the entire experimental period. Thus, the proposed system could be applicable for treating industrial wastewater containing ethylene glycol. Massively parallel 16S rRNA gene sequencing elucidated the microbial community structure of the UASB. The dominant family Pelobacteriaceae could mainly degrade the organic compounds of ethylene glycol and decomposed products of ethanol.
  • Electron Transfer Through the Outer Membrane of Geobacter Sulfurreducens a DISSERTATION SUBMITTED to the FACULTY of the UNIVE

    Electron Transfer Through the Outer Membrane of Geobacter Sulfurreducens a DISSERTATION SUBMITTED to the FACULTY of the UNIVE

    Electron transfer through the outer membrane of Geobacter sulfurreducens A DISSERTATION SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Fernanda Jiménez Otero IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR IN PHILOSOPHY Dr. Daniel R. Bond May, 2018 Fernanda Jiménez Otero, 2018, © Acknowledgements This dissertation and the degree I have gained with it, would not have been possible without the help and support from an invaluable group of people. The training I received from Chi Ho Chan and Caleb Levar continues to be essential in the way I approach scientific endeavors. The quality of genetic studies and rigor in microbiology techniques they taught me is a standard I hope to meet throughout my career. Daniel Bond has been much more than I ever expected from an advisor. I have not only gained scientific knowledge from him, but I will take with me the example of what a great mentor represents. His enthusiasm for science is only rivaled by his commitment to past and present members of his laboratory. I am extremely honored to be able to count myself in that group, and I will do my best to represent him proudly in future endeavors. Throughout these five years, Jeff Gralnick has given me numerous opportunities to explore all aspects of a scientific career. Not only is Chapter 2 a result of his vision, but I feel less intimidated by a career in science as a result of his mentoring and support. The faculty members in my committee- Carrie Wilmot, Brandy Toner, and Larry Wackett, have made sure I am well prepared for i every step through graduate school.
  • The Genus Pelobacter

    The Genus Pelobacter

    s Genu Pelobacter The Genus Pelobacter BERNHARD SCHINK The genus Pelobacter was proposed as a taxonomic entity Viable counts using the characteristic substrates consisting of strictly anaerobic, Gram-negative, nonspore- gallic acid, acetoin, polyethylene glycol, and forming, rod-shaped bacteria that use only a very limited acetylene showed that there were approximately number of substrates. The members of the genus are all 100 cells/ml of each of the Pelobacter species in unable to ferment sugars and therefore cannot be grouped sediment and up to 2,500 cells/ml in sewage with any other genus in the family Bacteroidaceae (Krieg and Holt, 1984). The genus comprises five different species, P. sludge. Since their substrate ranges are compa- acidigallici (Schink and Pfennig, 1982), P. venetianus (Schink rably small, their ecological niche in such sedi- and Sti eb, 1983), P. carbinolicus (Schink, 1984), P. propioni- ments can be understood rather well in most cus (Schink, 1984), and P. acetylenicus (Schink, 1985), which cases. P. acidigallici is restricted to the utilization all are based on 3–5 described strains. of trihydroxybenzenoids, which are probably its Comparisons of the various Pelobacter species by DNA- only energy source in its natural habitat. P. vene- DNA hybridization experiments revealed that the genus is tianus, P. carbinolicus, P. propionicus, and P. rather inhomogenous; therefore, a reorganization may per- acetylenicus were enriched and isolated with haps be necessary in the future (J. P. Touzel and B. Schink, polyethylene glycol, 2,3-butanediol, and acety- unpublished observations). Whereas the species P. vene- lene, respectively, but the ecological importance tianus, P.
  • Fermentation of Acetylene by an Obligate Anaerobe, Pelobacter Acetylenicus Sp

    Fermentation of Acetylene by an Obligate Anaerobe, Pelobacter Acetylenicus Sp

    Archives of Arch Microbiol (1985) 142: 295- 301 Microbiology Springer-Verlag 1985 Fermentation of acetylene by an obligate anaerobe, Pelobacter acetylenicus sp. nov. * Bernhard Schink Fakult/it ffir Biologie, Universit/it Konstanz, Postfach 5560, D-7750 Konstanz, Federal Republic of Germany Abstract. Four strains of strictly anaerobic Gram-negative tion reactions (Schink 1985a). No significant anaerobic rod-shaped non-sporeforming bacteria were enriched and degradation could be observed with ethylene (ethene), the isolated from marine and freshwater sediments with acety- most simple unsaturated hydrocarbon (Schink 1985 a, b). lene (ethine) as sole source of carbon and energy. Acetylene, It was reported recently that also acetylene can be metab- acetoin, ethanolamine, choline, 1,2-propanediol, and glyc- olized in the absence of molecular oxygen (Watanabe and erol were the only substrates utilized for growth, the latter de Guzman 1980). Enrichment cultures with acetylene as two only in the presence of small amounts of acetate. Sub- sole carbon source were obtained in mineral media with strates were fermented by disproportionation to acetate and sulfate as electron acceptor, and acetate could be identified ethanol or the respective higher acids and alcohols. No as an intermediary metabolite (Culbertson et al. 1981). How- cytochromes were detectable; the guanine plus cytosine ever, these enrichment cultures were difficult to maintain, content of the DNA was 57.1 _+ 0.2 tool%. Alcohol dehy- and the acetylene-degrading bacteria could not be identified drogenase, aldehyde dehydrogenase, phosphate acetyl- (C. W. Culbertson and R. S. Oremland, Abstr. 3rd Int. transferase, and acetate kinase were found in high activities Syrup.