edf4d8235067d2d27962850dfe082c44 appmic_a2022v2n1p272.pdf d96d5fe35269d4b11678d2f40b5823290197619c appmic_a2022v2n1p272.pdf 04945fe115b3fc42daddacfe83e314c4299cca262727410d73edef3cf66fd0ef appmic_a2022v2n1p272.pdf Title: Metaproteomics, Heterotrophic Growth, and Distribution of Nitrosomonas europaea and Nitrobacter winogradskyi after Long-Term Operation of an Autotrophic Nitrifying Biofilm Reactor Subject: Bioregenerative life support systems (BLSS) are currently in development to tackle low recovery efficiencies, high energy demands, as well as food, water, and oxygen production challenges through the regeneration of nutrients from waste streams. The MELiSSA pilot plant has been developed as a testbed for regenerative life support system bioreactor operation and characterization. As nitrogen is a vital resource in such systems, we studied the functional composition of a new packed-bed nitrifying bioreactor inoculated with a co-culture of Nitrosomonas europaea (ATCC 25978) and Nitrobacter winogradskyi (ATCC 25391). After 840 days of autotrophic continuous cultivation, the packed-bed was sampled at five vertical positions, each with three horizontal positions, and the biomass at each position was characterized via qPCR, 16S amplicon sequencing, and liquid chromatography tandem mass spectrometry. The total number of cells within the different sections fluctuated around 8.95 5.10 107 cells/mL of beads. Based on 16S amplicons and protein content, N. europaea and N. winogradskyi constituted overall 44.07 11.75% and 57.53 12.04% of the nitrifying bioreactor, respectively, indicating the presence of a heterotrophic population that, even after such a long operation time, did not affect the nitrification function of the bioreactor. In addition, DNA-based abundance estimates showed that N. europaea was slightly more abundant than N. winogradskyi, whereas protein-based abundance estimates indicated a much higher abundance of N. europaea. This highlights that single-method approaches need to be carefully interpreted in terms of overall cell abundance and metabolic activity. Keywords: Nitrosomonas europaea; Nitrobacter winogradskyi; nitrification; bioreactor; life support system; BLSS; RLSS Author: Felice Mastroleo, Carolina Arnau, Tom Verbeelen, Mohamed Mysara, Francesc Gòdia, Natalie Leys and Rob Van Houdt Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Mar 9 09:11:32 2022 CET ModDate: Wed Mar 9 11:05:17 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2261269 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- KPNHET+VnURWPalladioL Type 1 Custom yes yes no 10 0 BYKBQH+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 UMFPNQ+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 VESTII+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 LVEJMA+URWPalladioL-BoldItal Type 1 Custom yes yes no 28 0 OJYQXC+CMSY10 Type 1 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CPMNPD+PalatinoLinotype CID TrueType Identity-H yes yes yes 166 0 CPMNPE+PalatinoLinotype TrueType WinAnsi yes yes no 172 0 CPMNPG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 175 0 CPMNPC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 185 0 CPMNPD+PalatinoLinotype CID TrueType Identity-H yes yes yes 188 0 CPMNPE+PalatinoLinotype TrueType WinAnsi yes yes no 194 0 CPMNPG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 197 0 CPMNPC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 211 0 CPMNPE+PalatinoLinotype TrueType WinAnsi yes yes no 214 0 CPMNPG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 217 0 CPMOGG+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 220 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-04-01 02:30:58 CEST RepresentationInformation: appmic_a2022v2n1p272.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-03-22 11:09:08 CET Size: 2261269 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 427 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Bacterial Strains and Culture Medium Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Nitrifying Packed-Bed Reactor Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Bioreactor Disassembly Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Processing of Bioreactor Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: ATP Measurements Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: qPCR and 16S rRNA Gene Amplicon Sequencing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Metaproteomic Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: Nitrifying Packed-Bed Operation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Cell Abundance in the Biofilm Biomass Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Relative Abundance of N. europaea and N. winogradskyi Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Composition of the Microbial Community Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Metaproteomics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Children: Item: Title: Database Search Restricted to Nitrosomonas and Nitrobacter Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Database Search Broadened to Bacteria (Eubacteria) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Info: Title: Metaproteomics, Heterotrophic Growth, and Distribution of Nitrosomonas europaea and Nitrobacter winogradskyi after Long-Term Operation of an Autotrophic Nitrifying Biofilm Reactor Author: Felice Mastroleo, Carolina Arnau, Tom Verbeelen, Mohamed Mysara, Francesc Gòdia, Natalie Leys and Rob Van Houdt Subject: Bioregenerative life support systems (BLSS) are currently in development to tackle low recovery efficiencies, high energy demands, as well as food, water, and oxygen production challenges through the regeneration of nutrients from waste streams. The MELiSSA pilot plant has been developed as a testbed for regenerative life support system bioreactor operation and characterization. As nitrogen is a vital resource in such systems, we studied the functional composition of a new packed-bed nitrifying bioreactor inoculated with a co-culture of Nitrosomonas europaea (ATCC 25978) and Nitrobacter winogradskyi (ATCC 25391). After 840 days of autotrophic continuous cultivation, the packed-bed was sampled at five vertical positions, each with three horizontal positions, and the biomass at each position was characterized via qPCR, 16S amplicon sequencing, and liquid chromatography tandem mass spectrometry. The total number of cells within the different sections fluctuated around 8.95 5.10 107 cells/mL of beads. Based on 16S amplicons and protein content, N. europaea and N. winogradskyi constituted overall 44.07 11.75% and 57.53 12.04% of the nitrifying bioreactor, respectively, indicating the presence of a heterotrophic population that, even after such a long operation time, did not affect the nitrification function of the bioreactor. In addition, DNA-based abundance estimates showed that N. europaea was slightly more abundant than N. winogradskyi, whereas protein-based abundance estimates indicated a much higher abundance of N. europaea. This highlights that single-method approaches need to be carefully interpreted in terms of overall cell abundance and metabolic activity. 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