NOVEL ANAMMOX BACTERIA FOR WASTEWATER TREATMENTNOVEL ANAMMOX BACTERIA FOR WASTEWATER TREATMENTNOVEL ANAMMOX BACTERIA FOR WASTEWATER TREATMENTM ti V tti1 T k F jii2 A i l S ö i1 Mi h l R th k1 M i C G i 3Matias Vanotti1 Takao Fujii2 Ariel Szögi1 Michael Rothrock1 Maria Cruz Garcia3Matias Vanotti , Takao Fujii , Ariel Szögi , Michael Rothrock , Maria Cruz Garcia ,

Airton Kunz4 Albert Magri5 Kenji Furukawa6 Jose Martinez7Airton Kunz4, Albert Magri5, Kenji Furukawa6, Jose Martinez7Airton Kunz , Albert Magri , Kenji Furukawa , Jose Martinez1 USDA ARS Fl S th C li USA 2 S j U i it J 3 ITAC L V ll d lid S i1. USDA-ARS, Florence, South Carolina, USA; 2. Sojo University, Japan; 3. ITACyL, Valladolid, Spain;1. USDA ARS, Florence, South Carolina, USA; 2. Sojo University, Japan; 3. ITACyL, Valladolid, Spain;

4 EMBRAPA C di B il 5 GIRO B l S i 6 K t U i it J 7 CEMAGREF R F4. EMBRAPA, Concordia, Brazil; 5. GIRO, Barcelona, Spain; 6. Kumamoto University, Japan; 7. CEMAGREF, Rennes, France, , ; , , p ; y, p ; , ,

A l b t i C did t B di li i i h i A i D it N b NRRL B 50286 d th h t i ti f idi i i d l i di it d biA novel anammox bacteria, Candidatus Brocadia caroliniensis, having Accession Deposit Number NRRL B-50286 and the characteristics of oxidizing ammonia and releasing di-nitrogen under anaerobic, , g p g g gconditions has been discovered It can be used for effective treatment of wastewater having undesirable levels of ammonia including agricultural industrial or municipal wastewaters The obtained anammoxconditions, has been discovered. It can be used for effective treatment of wastewater having undesirable levels of ammonia, including agricultural, industrial, or municipal wastewaters. The obtained anammoxsludge has been used for quick start-up of bio-reactors using the deammonification process.sludge has been used for quick start up of bio reactors using the deammonification process.

Deammonification process: Partial nitritation + anaerobic ammonium oxidation (anammox) Anammox bacteria characterizationDeammonification process: Partial nitritation + anaerobic ammonium oxidation (anammox) Anammox bacteria characterizationDNA l i di t d l i f i th USACONCEPT • DNA analyses indicated novel species of anammox in the USACONCEPT

T t ll t t hi bi f th l f ity p

• Candidatus “Brocadia caroliniensis” Totally autotrophic bioprocess for the removal of nitrogen • Candidatus Brocadia caroliniensis Sequence Similarity to Candidatus “B. y p p g• Isolated from livestock manure sludge caroliniensis” (JF487828)

TARGET • Isolated from livestock manure sludge ( )

TARGET L t t t t f t t t i i hi h i it • Specific molecular probe FLO1 and Fish protocol developed Candidatus “Brocadia 96%

Low cost treatment of wastewaters containing high ammonia nitrogen Specific molecular probe FLO1 and Fish protocol developedf l id ”

g g g Brocadia sp. (Candidatus AM285341)81

fulgida”

ADVANTAGES (relative to conventional nitrification-denitrification) Confocal FISH image of Anammox Uncultured planctomycete Pla 5GA-8 (GQ356125)100 Candidatus “Brocadia 94%ADVANTAGES (relative to conventional nitrification denitrification) gbacteria clusters using FLO1

y ( )

Uncultured bacterium Asahi BRW (AB456583)97anammoxidans”

Reduces 58% of the oxygen requirements and 83% of biosolids production( )

Brocadia caroliniensisCandidatus 96anammoxidans

Reduces 58% of the oxygen requirements and 83% of biosolids production ocad a ca o e s sCa d datusBrocadia fulgida (Candidatus EU478693)87

Candidatus “Jettenia 92%

100% f f f Brocadia fulgida (Candidatus EU478693)

Uncultured planctomycete HAuD-MB/2-35 (AB176696)

87

asiatica”

Reduces 100% of the carbon needs for denitrification Uncultured planctomycete HAuD MB/2 35 (AB176696)

Brocadia anammoxidans (Candidatus AF375994)8999 C did t “K i 90%% Brocadia anammoxidans (Candidatus AF375994)

Uncultured anoxic sludge bacterium KU1 (AB054006)1008999 Candidatus “Kuenia 90%

Does not produce nitrous oxide a powerful greenhouse gas Uncultured anoxic sludge bacterium KU1 (AB054006)

Anammoxoglobus propionicus (Candidatus DQ317601)

100stuttgartiensis” Does not produce nitrous oxide, a powerful greenhouse gas Anammoxoglobus propionicus (Candidatus DQ317601)

J tt i i ti (C did t DQ301513) Candidatus 90%

Nit l t fi t t ti hi h Jettenia asiatica (Candidatus DQ301513)

U lt d l t t KSU 1 (AB057453)100100

Candidatus 90%

Nitrogen removal rates are five to ten times higher Uncultured planctomycete KSU-1 (AB057453)100 “Anammoxoglobusg g Kuenenia stuttgartiensis (Candidatus AF375995)

propionicus” Scalindua sorokinii (Candidatus AY257181)

p p

C did “S li d 86%Anammox Candidatus “Scalindua 86%NH4+ + NO2

－ N2 + 2H2O0.005 sorokinii”

4 2 2 2

Isolation of anammoxIsolation of anammox Stoichiometry and treatment performanceA b t i i l t d f li t k l d i th ti

Stoichiometry and treatment performance• Anammox bacteria were isolated from livestock manure sludge using synthetic Stoichiometry of Brocadia caroliniensisg g y

media PVA porous beads were used for biomass attachment (Exp 1 & 2) • Average nitrogen removal rate obtained during 1 yearStoichiometry of Brocadia caroliniensis

1 00 NH + + NO N + NOmedia. PVA porous beads were used for biomass attachment (Exp 1 & 2) • Average nitrogen removal rate obtained during 1 year 1.00 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 3 9 hours was 1477 mg N/L-reactor/dayA pilot reactor that used non woven carrier was inoculated withthe isolated anammox; it showed quick start up and high activity (Exp 3)

Up flow pilot reactor using non woven carrier at HRT of 3.9 hours was 1477 mg N/L reactor/day St i hi t f B di li i i d t i d

2000tes

Total N Remo althe isolated anammox; it showed quick start-up and high activity (Exp 3) • Stoichiometry of Brocadia caroliniensis was determined y = 2.12xR2 = 0 99R

at Total N Removal

NO2 RemovalNO3 P d ti

PVA carrier beads (4 mm)

yunder wide range of N loading rates during 3 8 yr period

R = 0.991500o

n R

y]

NO3 Production

PVA carrier beads (4 mm) under wide range of N loading rates during 3.8-yr periody = 1.30xc

tio

day

N Removal Rates in Pilot Reactor Seeded withyR2 = 0.981000o

du

cL/

d

N Removal Rates in Pilot Reactor Seeded with A Sl d (E 3)

1000

Pro

N/

Anammox Sludge (Exp. 3) N removal rates in continuous up flow pilot reactornon-woven carrier 500 & P

mg

g ( p )Experiment 1 N removal rates in continuous up-flow pilot reactor non-woven carrier

y = 0.18x2

500

val [m

600Experiment 1Experiment 2

R2 = 0.710m

ov

600

e

Experiment 2E periment 3 Mass Nitrogen

00 200 400 600 800 1000R

em

500ate

y]

Experiment 3 Mass Nitrogen b NH4

+ Removal [mg N/L/day]500

Ra

ay Influentb Effluent Removal NH4 Removal [mg N/L/day]

400al R /da

Removal 10 L Continuous Up flow Anammox Reactor00

va /L/ Removal

ffi i10-L Continuous Up-flow Anammox Reactor

C ti l t d f 3 8St i hi t i ffi i t f300

mov N/ Efficiency Continuously operated for 3.8 yrsStoichiometric coefficients of anammox

200em g

ymg N L-1 d-1 c %200R

e m -----------------mg N L d ------------- % d

Consumed mol Produced mol

100N R [ Total Nd 1,735 ± 137 260 ± 122 1,477 ± 168 85 ± 7 NH4

+ NO2- NO3

- N2

100N , ,Ammonia 804 ± 80 92 ± 71 709 ± 88 89 ± 9 STROUS et al. (1998)

The Netherlands 1 1.32 0.26 1.02

0Ammonia 804 ± 80 92 ± 71 709 ± 88 89 ± 9 Ni i 930 80 22 47 915 90 98 5

The NetherlandsVAN DE GRAAF et al0 Nitrite 930 ± 80 22 ± 47 915 ± 90 98 ± 5 VAN DE GRAAF et al. (1996) The Netherlands 1 1.31 0.22 1.05

0 300 600 900 Ammonia + 1 735 ± 138 114 ± 101 1 621 ± 155 94 ± 6( )FURUKAWA et al. (2009) 1 1 31 0 17 1 030 300 600 900 Ammonia +

Ni i1,735 ± 138 114 ± 101 1,621 ± 155 94 ± 6 ( )

Japan 1 1.31 0.17 1.03

Time [days] Nitrite KUNZ et al. (2008) - Brazil 1 1.21 0.25 0.98Time [days]VANOTTI et al. (2011) - USA 1 1.30 0.18 1.06 ( )

I bili d ll tImmobilized anammox pellets Anammox coupled with partial nitritation (two-stage process)p Anammox coupled with partial nitritation (two stage process)

• Mix of anammox and PVA was polymerized with the freezing technique • Used two sequencing batch reactorsMix of anammox and PVA was polymerized with the freezing technique. P ll t d i ti ti d t (CSTR) t 20% /

Used two sequencing batch reactors (SBR) one for partial nitritation (PN) andPellets were used in continuous stirred reactors (CSTR) at 20% v/v (SBR), one for partial nitritation (PN) and ( )

• The immobilized anammox recovered 81% of the potential activity the other for anammox reaction to treat• The immobilized anammox recovered 81% of the potential activity the other for anammox reaction to treat i t t ith 1400 NH Nswine wastewater with 1400 mg NH4-N g 4

L-1L 1

• The PN used suspended HighThe PN used suspended High P f Nit if i Sl d (HPNS)Performance Nitrifying Sludge (HPNS) y g g ( )(NRRL B 50298) and produced an(NRRL B-50298) and produced an effluent with a NO2-N:NH4-N ratio of 1 4effluent with a NO2 N:NH4 N ratio of 1.4 Th t d d d• The anammox reactor used suspended, pgranular Brocadia caroliniensis (NRRLgranular Brocadia caroliniensis (NRRL

N t f ti i b t h tB-50286). Influent NO2-N +NH4-N was N transformations in batch reactorB 50286). Influent NO2 N NH4 N was removed at rates up to 1 340 mg/L using suspended anammoxremoved at rates up to 1,340 mg/L- using suspended anammoxreactor/dayreactor/day.

L t tiSi l k N l i d i l i i i ( ) Long-term anammox preservation Single tank N removal using anammox and partial nitritation (one-stage process) g pSingle tank N removal using anammox and partial nitritation (one stage process)Deammonification stoichiometry theory:

• Long-term preservation was needed to• Used one continuous flow aerated reactor containingDeammonification stoichiometry theory:(1) Partial nitritation: Long term preservation was needed to

d it B di li i i i• Used one continuous flow aerated reactor containing (1) Partial nitritation:

deposit Brocadia caroliniensis in suspended nitrifying sludge (NRRL B-50298) and 2 NH4+ + 1.5 O2 → NH4

+ + NO2- + H2O + 2 H+ p

international depositary authority undersuspended nitrifying sludge (NRRL B 50298) and

d d B di li i i (NRRL4 2 4 2 2

(2) Anammox: international depositary authority under suspended anammox Brocadia caroliniensis (NRRL (2) Anammox:NH + 1 32 NO 1 02 N 0 26 NO 2 H O Budapest Treaty of United Nations

p (B 50286) NH4

+ + 1.32 NO2- → 1.02 N2 + 0.26 NO3

- + 2 H2O Budapest Treaty of United Nations (WIPO)

B-50286). Bi fil l ti i

4 2 2 3 2Single-stage theory (1+2): (WIPO).• The reactor contained biofilm plastic carriers (1200 Biofilm plastic carriersSingle stage theory (1+2):NH + + 0 85 O 0 44 N + 0 11 NO - + 1 43 H O + 1 14 H+ ( )The reactor contained biofilm plastic carriers (1200

2/ 3 ) t 30% / ki ti th t fl idi d bNH4

+ + 0.85 O2 → 0.44 N2 + 0.11 NO3- + 1.43 H2O + 1.14 H+

m2/m3 ) at 30% v/v packing ratio that were fluidized by Single-stage stoichiometry (data shown below):• A preservation protocol was successfully

) p g ythe aeration

g g y ( )NH + + 0 88 O → 0 44 N + 0 11 NO - + 1 43 H O + 1 14 H+ A preservation protocol was successfully

developed using lyophilization afterthe aeration.

1NH4 + 0.88 O2 → 0.44 N2 + 0.11 NO3 + 1.43 H2O + 1.14 H

developed using lyophilization after • The water temperature was 22oC and DO <0.3 mg L-1 1000100 NitriteNi freezing with skim milk at -200oC

The water temperature was 22 C and DO 0.3 mg LInfluent contained 200 mg NH N L-1 and 1500 mg 90090)

NitrateAmmonia freezing with skim milk at -200 C.• Influent contained 200 mg NH4-N L-1 and 1500 mg 80080 )g/

L) Ammonia

Alkalinity Mixing nitrifying sludge 4carbonate alkalinity L-1 700

8007080

g/L

)

mg y

with anammox

• This protocol was used to deposit Brocadiacarbonate alkalinity LT t l N l t f 750 t 1000 /L 600

7006070

mg

n (m

• This protocol was used to deposit Brocadia• Total N removal rates of 750 to 1000 mg/L were 500600

5060

ty (tio

n

caroliniensis under Budapest Treatyg

obtained during the first 3 months50050

nit

trat caroliniensis under Budapest Treaty

i i i th A i lt R hobtained during the first 3 months 40040

kali

ent

provisions in the Agriculture Research • The stoichiometry of the reaction obtained (batch 30030

Alk

once p g

Culture Collection in Peoria IL underThe stoichiometry of the reaction obtained (batch t t) i t t ith d ifi ti

20020 A

Co

Culture Collection in Peoria, IL, under test) was consistent with deammonification process. 10010N

Accession Deposit NRRL B-50286) p

00 One-stage experimental setup Accession Deposit NRRL B 50286. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

One stage experimental setup

Time (hours)