Microbial Ecology and Activity of Anaerobic Ammonium Oxidation (ANAMMOX) Bioreactors
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1 Microbial Ecology and Activity of Anaerobic Ammonium Oxidation (ANAMMOX) Bioreactors Hongkeun Park Ph.D. Student Earth and Environmental Engineering Columbia University
2 Background: Nitrogen Removal Human Activity ethomas.web.wesleyan.edu/lisweb/wqaug02.jpg Hypoxia s/florida_eutrophication_7536.jpg Eutrophication kdaq.empas.com/qna/view.html?n= Red tide 2 Public Health
3 Background: Conventional Nitrogen Removal NH 4 + Nitrogen fixation Ammonium Oxidation NH 2 OH N 2 Nitrogen Cycle N 2 O Nitrite Oxidation NO 2 - NO Aerobic Anaerobic NO 3 - Denitrification 3
4 Background: Anaerobic Ammonium-oxidation NH 4 + N 2 NH 2 OH HZO (hydrazine oxidase) N 2 H 4 Anaerobic Ammoniumoxidation (ANAMMOX) NO 2 - Sustainable!!! No external carbon required 60% reduction of oxygen supply 40% reduction of biomass production No N 2 O-GHG emission NO Aerobic Anaerobic 4
5 Background: ANAMMOX Bacteria Nature Reviews Microbiology 6, (2008) Appl. Environ. Microbiol. 71, (4), (2005) Anammoxosome: unique morphological feature Deep branch of Planctomycetales : C. Scalindua, C. Brocadia, C. Kuenenia Challenge: Low growth rate, sensitive to oxygen and nitrite 5
6 Objectives To identify and monitor the quantification, and population dynamics of ANAMMOX bacteria using molecular genetics for enrichment To develop the molecular biomarker for ANAMMOX activity and apply to modeling as a design parameter 6
7 Fluorescence Approach ANAMMOX reactor running (CCNY) and molecular study (Columbia) Molecular Characterization of ANAMMOX bacteria using 16s rrna clone libraries Molecular Quantification of using real-time polymerase chain reaction (real-time PCR) Community analysis using Denaturing Gradient Gel Electrophoresis (DGGE) PCR Cycle 7
8 Phylogenetic tree of 16s rrna for ANAMMOX Planctomycetes seed (10/10) 5GA (10/10) C. Brocadia sp S rrna AM C. Brocadia fulgida DQ PO-55 (9/10) PO-55 (1/10) C. Brocadia anammoxidans AF Austria (10/10) KOLL2a AJ Pla1-47 AF Pla2-48 AF C. Kuenenia stuttgartiensis AF C. Scalindua wagneri AY C. Scalindua brodae AY C. Scalindua sorokinii AY C. Anammoxoglobus propionicus DQ p. KSU-1 16S rrna AB C. Jettenia asiatica DQ SBR (10/10) 100 Isosphaera pallida X64372 P. maris X62910 Planctomyces maris X62910 Pirellula limnophilus X62911 Planctomyces brasiliensis X G. obscuriglobus X85248 Gemmata obscuriglobus X85248 Pirellula Schlesner 678 X81947 Pirellula Schlesner 139 X81945 B. marina X62912 Blastopirellula marina X N. nitrosa 16S rrna N. communis 16S rrna N. ureae 16S rrna N. marina 16S rrna N. oligotropha 16S rrna N. halophila 16S rrna N. europaea M103 16S rrna N. eutropha C91 C91 Neut R0005 N. eutropha C91 Nm57 16S rrna ANAMMOX Planctomycetes Planctomycetales AOB ANAMMOX bacteria from Austria PO55 pilot plant bacteria Seed for 5GA 5GA reactor SBR reactor
9 12345 Microbial community analysis: DGGE Band No. Closest class Name Similarity 1 Planctomycetacia Candidatus Brocadia sp. 40 partial 16S rrna gene, clone 40 98% 2 Planctomycetacia Candidatus Brocadia sp. 40 partial 16S rrna gene, clone 40 97% SBR 3 Planctomycetacia Candidatus Brocadia sp. 40 partial 16S rrna gene, clone 40 99% 4 Acidobacteria Uncultured bacterium clone KIST-JJY008 16S rrna gene 97% 5 gammaproteobacteria Uncultured bacterium clone SB1\30 16S rrna gene 94% 6 gammaproteobacteria Uncultured bacterium clone ANTLV2_F07 16S rrna gene 95% 7 alphaproteobacteria Uncultured bacterium clone B3NR56D7 16S rrna gene 92% Seed, 5GA reactor Austria sludge 8 alphaproteobacteria Environmental 16s rdna sequence from Evry wastewater treatment plant anoxic basin 98% 9 Planctomycetacia Candidatus Brocadia sp. 40 partial 16S rrna gene, clone 40 97% 10 Betaproteobacteria Nitrosomonas europaea ATCC % 11 Chloroflexi Uncultured bacterium gene for 16S rrna, partial sequence, clone: 3 95% 12 Chloroflexi Uncultured bacterium clone KIST-JJY023 16S rrna gene 98% 13 Planctomycetacia Uncultured bacterium gene for 16S rrna, partial sequence, clone: AnDHS-6 82% 14 Chloroflexi Uncultured bacterium gene for 16S rrna, partial sequence, clone: YS16Us88 96% 15 Chloroflexi Uncultured bacterium clone Pol_B_17 16S rrna gene 96% 16 Chloroflexi Uncultured bacterium gene for 16S rrna, partial sequence, clone: AnDHS-2 99% PO55 sludge 17 Chloroflexi Uncultured Chloroflexi bacterium isolate PG-39 16S rrna gene 95% 18 Planctomycetacia Kuenenia stuttgartiensis genome fragment KUST_E 99% 19 Chlorobi Uncultured bacterium clone BH_B43 16S rrna gene 99% 20 Betaproteobacteria Uncultured bacterium clone KIST-JJY037 16S rrna gene 91% 21 Planctomycetacia Candidatus Brocadia fulgida 16S rrna gene 97% Chloroflexi Uncultured bacterium gene for 16S rrna, partial sequence, clone: N2B16 97% Lane 1: PO55 pilot plant bacteria Lane 2: ANAMMOX bacteria from Austria Lane 3: Seed for 5GA Lane 4: 5GA reactor Lane 5: SBR reactor
10 Quantification analysis: real-time PCR Fluorescence detection as indicator of PCR production at each cycle.(real-time detection) Molecular quantification of AOB and ANAMMOX microbes using amoa/anammox 16s rrna targeted real-time PCR AOB population ANAMMOX population 10
11 N removal (mg-n/d) marker day 1 day 19 day 23 day 42 day 48 day 52 day 55 day 60 day 69 day 74 day 98 day 117 day 126 day 135 day 140 marker Population dynamics: DGGE fingerprints C. Brocadia Lane on DGGE profile Some correlation between fingerprints and reactor performance
12 Background: Anaerobic Ammonium-oxidation NH 4 + N 2 NH 2 OH HZO (hydrazine oxidase) N 2 H 4 Anaerobic Ammoniumoxidation (ANAMMOX) NO 2 - Sustainable!!! No external carbon required 60% reduction of oxygen supply 40% reduction of biomass production No N 2 O-GHG emission NO Aerobic Anaerobic 12
13 Novel ANAMMOX biomarkers: RT-qPCR hzo (hydrazine oxygenase) and ISR (Intergenic Spacer Region between 16s and 23s rrna) target normalized with ANAMMOX 16s rrna 13
14 Findings Dominant ANAMMOX bacteria in 5GA was C. Brocadia sp. 40, with 10 7 ~10 8 copies/ml Dynamic change in ANAMMOX species (C. Kuenenia to C. Brocadia) over three years Novel sensitive biomarker of functional gene (hzo) successfully developed and to be tested on 5GA ANAMMOX reactor 14
15 Dr. Kartik Chandran Acknowledgement Joon Ahn, Huijie Lu, Ran Yu, and Sungpyo Kim (Chandran Group) Alex Rosenthal, Krish Ramalingam, John Fillos (CCNY) Allen Deur, Keith Beckmann (NYC DEP) 15
16 Ongoing projects in our group Optimization for nitritification in sidestream treatment Ecology of denitrification - methanol, ethanol, acetate Translation of -omics into usable parameters for WWTP monitoring and optimization NH NH 2 OH---NO 2 - NO NO NO 3 - NH NO 3 - Generation of energetic materials from biosolids Modeling of complex bioreactor systems Evolution and dynamics of resistance in WWTPs The global N genome
17 Thank you & Questions? 17
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