NOVEL ANAMMOX BACTERIA FOR WASTEWATER TREATMENT MMatias ti Vanotti V tti1, TTakao k FujiiF jii2, AiAriel lS Szögi ö i1, MichaelMi h l Rothrock R th k1, MariaMiC Cruz GGarcia i3, Airton Kunz4, Albert Magri5, Kenji Furukawa6, Jose Martinez7 1USDA1. USDA -ARSARS, Florence,Fl SouthS th Carolina,C li USAUSA; 2.2 SjSojo UiUniversity, itJ Japan; 3.3 ITACITACyL L, Valladolid,V ll d lid SSpain; i 4EMBRAPAC4. EMBRAPA,,,;,,p; Concordia, diB Brazil; il5GIROB 5. GIRO, Barcelona, l SSpain; i 6K6. Kumamoto t UiUniversity, itJy,p;,, Japan; 7CEMAGREFR7. CEMAGREF, Rennes, FFrance

A novell anammox btibacteria,, CdidtCandidatus BdiBrocadia caroliniensisli i i , hihavingg AiAccession DitDepositp NbNumber NRRL B-50286 andd ththe characteristicshtitioff oxidizingidi i g ammoniai andd releasinglig di-nitrogenit g underd anaerobicbi conditionsconditions, has been discovered. It can be used for effective treatment of wastewater having undesirable levels of ammoniaammonia, including agriculturalagricultural, industrialindustrial, or municipal wastewaters. The obtained anammox sludge has been used for quick start-up of bio-reactors using the deammonification process.

Deammonification process: Partial nitritation + anaerobic oxidation (anammox) Anammox bacteria characterization

CONCEPT • DNA analyses lyp iindicated di t d novel l species i of f anammox ithUSAin the USA  TtllTotallyy autotrophicttp hi bibioprocessp ffor ththe removall off nitrogenit g • Candidatus “Brocadia caroliniensis” Sequence Similarity to Candidatus “B. caroliniensis” ()(JF487828) TARGET • Isolated from livestock manure sludge • Specific molecular probe FLO1 and Fish protocol developed Candidatus “Brocadia 96%  LLow costt treatmentttoff wastewatersttcontainingtiig hihighg h ammoniai nitrogenit g flidfulgida” 81 Candidatus Brocadia sp.AM285341) ( 100 Uncultured planctomycetey( Pla 5GA-8GQ356125) ( ) Confocal FISH imageg of Anammox Candidatus “Brocadia 94% ADVANTAGES (relative to conventional -) 97 Uncultured bacterium Asahi BRW(AB456583) ( ) bacteria clusters using FLO1 anammoxidans” 96 CandidatusCa d datus Brocadiaocad acaroliniensis ca o e s s  Reduces 58% of the oxygen requirements and 83% of biosolids production Candidatus “Jettenia 92% 87 CandidatusBrocadia Brocadia fulgida fulgidaEU478693) ( ( Uncultured planctomycete HAuD-MB/2-35HAuD MB/2AB176696) 35 ( asiatica”

 Reduces 100% off the carbon needs forf denitrificationf 99 89 CandidatusBrocadia Brocadia anammoxidans anammoxidansAF375994) ( ( CandidatusCdidt “Kuenia“K i 90% 100 Uncultured anoxic sludge bacterium KU1AB054006) ( stuttgartiensis”  Does not produce nitrous oxide,oxide a powerful greenhouse gas CandidatusAnammoxoglobus propionicusDQ317601) ( Candidatus 90% 100 CdidtCandidatusJttJettenia i asiatica itiDQ301513) (  NitrogenNit g removall ratest are fivefi tot tent timesti higherhig h 100 UltdltUncultured planctomycete tKSU1( KSU-1AB057453) ( “Anammoxoglobus Candidatus Kuenenia stuttgartiensisAF375995) ( propionicuspp ” Anammox Candidatus Scalinduasorokinii AY257181) ( + － CandidatusCdid “ScalinduaSlid 86% NH4 + NO2 N2 + 2H2O 0.005 sorokinii”

Isolation of anammox Stoichiometry and treatment performance • AAnammox btibacteria were isolatedl t d from f lilivestock t k manure sludge l dggy using i synthetic th ti Stoichiometry of Brocadia caroliniensis + - - mediamedia. PVA porous beads were used for biomass attachment (Exp 1 & 2) • Average removal rate obtained during 1 year 100NH1.00 NH4 + NO2  N + NO3 • A pilot reactor that used non-woven carrier was inoculated with Up-flow pilot reactor using non-woven carrier at HRT of 33.9 9 hours was 1477 mg N/L -reactor/day 2000 tes tes Total N RemoRemoval al y = 2.12x the isolated anammox; it showed quick start-up and high activity (Exp 3) NO2 Removal 2 • StoichiometrySt i hi t y off BrocadiaBdicaroliniensisli i i was dtdetermined i d Rat NO3 PProduction d ti R = 00.99 99 1500 on R on PVA carrier beads (4 mm) under wide range of N loading rates during 33.8 8-yr period y]

ctio y = 1.30x day R2 = 0.98 L/d 1000

N Removal Rates in Pilot Reactor Seeded with oduc Pro ASld(E3)Anammox Sludgeg( (Exp. p 3) ) N/ & P 500 non-woven carrier mg NrN removal rates in continuous up -flow pilot reactor [m y = 0.18x Experiment 1 val R2 = 0.71

600 Experiment 2 mov 0

Rem 0 200 400 600 800 1000 e Eperiment3Experiment 3 Mass Nitrogen + NH4 Removal [mg N/L/day] ate 500 b y] Ra

ay Influent Effluent Removal

/da 40000 al R al Removal 10-L Continuous Up-flow Anammox Reactor va /L/ StStoichiometric i hiCtiContinuously t i coefficients l ffioperated i tdf38 t of for f anammox 3.8 yrs N/ 300 Efficiencyffi i y mov

g -1 -1 c

em ------mg N L d ------% Consumed mol Produced mol

m 200 Re [ d NH + NO - NO - N Total N 1,735 ± 137 260 ± 122 1,477 ± 168 85 ± 7 4 2 3 2 N RN STROUS et al. (1998) 100 1 1.32 0.26 1.02 Ammonia 804 ± 80 92 ± 71 709 ± 88 89 ± 9 The Netherlands VAN DE GRAAF et alal. 11.310.221.05 0 NiNitrite i 930 ± 80 22 ± 47 915 ± 90 98 ± 5 (1996)() The Netherlands FURUKAWA et al. (2009)() 1 1311.31 0170.17 1031.03 0 300 600 900 Ammonia + 1,735 ± 138 114 ±1± 101 1,621 ± 155 94 ± 6 Japan KUNZ et al. (2008) - Brazil 1 1.21 0.25 0.98 Time [days] NiNitrite i VANOTTI et al. (2011)() - USA 1 1.30 0.18 1.06 IImmobilized bili d anammox pelletsp ll t Anammox coupled with partial nitritation (two-stage process)

• Mix of anammox and PVA was polymerized with the freezing techniquetechnique. • Used two sequencing batch reactors PPellets ll t were used d in i continuous ti stirred ti d reactors t (CSTR) ()att 20% v/v / (SBR)(SBR), one for partial nitritation (PN) and • The immobilized anammox recovered 81% of the potential activity the other for anammox reaction to treat swinei wastewater t t with ith 1400 mgg NH 4-N L-1 • The PN used suspended High PPerformance f NitNitrifying ifyg i SlSludge d g( (HPNS) ) (NRRL B -50298) and produced an

effluent with aNOa NO2-N:NH4-N ratio of 1.4 1 4 • ThThe anammox reactortd used suspended,p dd granular Brocadia caroliniensis (NRRL B-50286). Influent NO -N+NHN NH -NwasN was 2 4 NtN transformations f ti in i batch b t h reactor t removed at rates up to 1,340 1 340 mg/L - using suspended anammox reactor/dayreactor/day.

SiSingle l tank k N removal l using i anammox anddil partial nitritationii i ((one-stage process)) LLongg-tterm anammox preservationp ti Deammonification stoichiometry theory: • Used one continuous flow aerated reactor containing (1) Partial nitritation: • Long-term preservation was needed to + + - + suspended nitrifying sludge (NRRL B-50298) and 2 NH4 + 1.5 O2 → NH4 + NO2 + H2O + 2 H ditdepositp BdiBrocadia caroliniensisli i i iin suspendedp dd anammox BdiBrocadia caroliniensisli i i (NRRL( (2) Anammox: international depositary authority under NH + + 132NO1.32 NO - → 102N1.02 N + 026NO0.26 NO - + 2H2 H O B-50286)50286). 4 2 2 3 2 Budapest Treaty of United Nations Single-stage theory (1+2): BiBiofilm fil plasticlti carriers i • The reactor contained plastic carriers (1200 + - + (WIPO)(WIPO).() NH4 +085O+ 0.85 O2 → 0440.44 N2 +011+ 0.11 NO3 +143H+ 1.43 H2O+114HO + 1.14 H m2//m3 ))pg at t 30% v/v / packing ki ratio ti that th t were fluidized fl idi d by by Singleg -stagegy stoichiometry (data( shown below):) + - + • A preservation protocol was successfully the aerationaeration. NH4 +088O+ 0.88 O2 → 0.44044 N2 +011+ 0.11 NO3 +143H+ 1.43 H2O+114HO + 1.14 H o -1 100 1000 developed using lyophilization after • The water temperature was 22 C and DO <0.3 0.3 mg L NiNitrate

-1 ) 90 Ammonia 900 o • Influent contained 200 mg NH -NLN L and 1500 mg Mixing nitrifying sludge freezing with skim milk at -200 CC. 4 80 Alkalinityy 800 ) -1 g/L) with anammox mg carbonate alkalinity L 70 700 g/L) mg n (m 60 600 • This protocol was used to deposit Brocadia

• TTotal t l N removal l rates t of f 750 to t 1000 mg/Lg /L were tion 50 500 ty ( nit obtained during the first 3 months trat 40 400 caroliniensis under Budapest Treaty ent 30 300 kali provisionspgii in i the thA Agriculture i lt Research R h AlkA • The stoichiometry of the reaction obtained (batch once 20 200 Co Culture Collection in Peoria, Peoria ILIL, under ttest) t)) was consistent i t t with ith ddeammonification ifi ti process.p N 10 100 0 0 One-stage experimental setup Accession Deposit NRRL B-5028650286. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Time (hours)