Anammox for nutrient nitrogen removal from sludge liquors

Dept. Chemical & Environmental EngineeringUniversity of Arizona

Jim Field, Guangbin Li and Reyes Sierra

Industrial Partners: Pima County Regional Wastewater Reclamation Department (PCRWRD)

Los Angeles County Sanitation District (LACSD)

1

The N Cycle

2

NH4+N2

NO2-

NO3-

Org-Nmineralizationas

sim

ilatio

n

O2

O2e-

e-

N-fixation

nitrification-denitrification

Costs N Removal

Conventional Nutrient N Removal Costs

Nitrification:

Denitrification: NO3- + 5 e- eq → 0.5 N2

64 g O2 / mol N

40 g COD/ mol N

3

Conventional Nutrient N Removal

DenitrificationAOBNitrification

NOB

N2

NH4+ NO3-

AOB

NOB

O2 COD

ammonia nitrate

dinitrogen gas

The Missing Link in the N Cycle

4

NH4+N2

NO2-

NO3-

Org-N

anammox

mineralizationas

sim

ilatio

n

O2

O2e-

e-

N-fixation

Anammox

Discovered in Late 1980’s in The Netherlands

Simplified Stoichiometry

5

N2NH4+ NO2-ammonia nitrate dinitrogen gas

+

Anaerobic Ammonium Oxidation

Nitrate was mixed with anaerobic effluent at Gist Brocadus to oxidize hydrogen sulfide and anammox was discovered due to NH4

+ in wastewater

Costs and Savings of AnammoxAnammox Reaction (Detailed Stoichiometry)

NH4+

(100% N)

57% flow

43% flow

57% less O2

no O2 needed for anammox

NitritationAnammox

N2 89% NNO3

- 11% NAOB

NO2-

no COD needed for anammox

6

Anammox is combined with AOB as a technology

O2 No COD

Issues with Anammox

pH optima are critical

Sensitive to Substrate (NO2-) Toxicity

H2S is very toxic

Sensitivity depends heavily on physological status cells

Low pH exacerbates NO2- stress

High pH slowly deactivates anammox

Slow growthe rate

Arizona has high sulfate levels in municipal wastewater 7

Doubling times range from 5 to 15 d

Toxicity of NO2-

starved/NO2- pre-exposure

NO2- pre-exposure

active

starved/active

Physiological status

8

Is it Possible to Cultivate Anammox from Return Activated Sludge (RAS)?

9

RAS anammox enrichment

Enrichment Procedure

10

minerals + Medium:

NH4+ + NO2

-

NH4+ + NO2

- NH4+ + NO2

- 10% v/v--------- spikes --------- --------------- transfers ------------

90% vol new medium

10% v/v

time

Establish Enrich

Controls: Just NH4+ (no NO2

-) “no nitrite control”Just NO2

- (no NH4+) “endogenous control”

NO2- + NH4

+ (no inoculum) “abiotic control”

Enrichment ExperimentExample: Anammox Enrichment (RAS, Ina Rd)

NH4+ Graph

abiotic

no nitrite control

11

Enrichment ExperimentExample: Anammox Enrichment (RAS, Ina Rd)

NO2- Graph

abiotic

12

Activity Experiment RAS Anammox Enrich.

0.0

1.0

2.0

3.0

4.0

5.0

0 5 10 15 20 25 30 35Time (hr)

NO

2- con

cent

ratio

n (m

M)

0.0

1.0

2.0

3.0

4.0

0 5 10 15 20 25 30 35Time (hr)

NH

4+ con

cent

ratio

n (m

M)

0.0

1.0

2.0

3.0

4.0

0 5 10 15 20 25 30 35Time (hr)

N2 c

once

ntra

tion

(mm

ole

L liq

uid-1

)

0.0

0.2

0.4

0.6

0 5 10 15 20 25 30 35Time (hr)

NO

3- con

cent

ratio

n (m

M)

NO2-

NH4+

N2

NO3-

abiotic

endogenous

abiotic

no nitrite control

no nitrite control

no nitrite control

13

N-Balance RAS Anammox Enrichment

Final Balance

NO2- = 3.89 mM

NH4+ = 2.75 mM

Sum N = 6.64 mM

Consumed

NO3- = 0.57 mM

N2 = 3.02 mmol gas/Lliq

Sum N = 6.61 mM

Produced

Ratios

Measured: NO2-/NH4

+ = 1.40

Theoretical: NO2-/NH4

+ = 1.3214

DNA Evidence of RAS Anammox EnrichmentPCR forward primer Pla46F, reverse primer AMX-820R

Lane 1: MW markers

Lane 2: demiwater

Lane 3: anammox enrichment, Univ. Santiago de Compostela, Spain

Lane 4: anammox sludge Bioreactor Paques, The Netherlands

Lane 7: Activated Sludge High O2

Lane 8: RAS Ina Rd, original sample

Lane 5: RAS oxidation ditch

Lane 6: RAS Ina Rd, anammox enrichment

In theory PCR product 774 bp

15

Conclusions Enrichment RAS

First consistent evidence of Anammox activity = 45 d

Starting Material = Ina Road, RAS

Anammox the dominant reaction = 65 d

Genus of enriched anammox = Brocadia

16

Doubling time = 9.2 d

Details of enrichment: Sun et al. Chemosphere 2011, 84:1262-1269

Expanded Granular Sludge Bed (EGSB)

17influent

effluent

N2 gas

sludge bed

gas cap

settler

baffles

sludge granulegas bubble

weir3 phaseseparator

recirculationeffluent

18

Expanded Granular Sludge Bed (EGSB)

19

Expanded Granular Sludge Bed (EGSB)

HRT = 5.11 hInfl NO2- = 29 mM (1340 mg NO2-/L)Infl NH4+ = 25 mM (450 mg NH4+ /L)pH range effluent = 7.2-7.7

Statistics Field-Sierra Research Group with the Anammox Process

7 oral presentations (inter)national conferences

3 research projects

3 PhD dissertations, 3 MS theses, 4 post docs

15 journal articles on Anammox (incl. 1 pending)

NSF-CBET $337,340 (2013-2017)USDA (US-Egypt STDF) $180,000 (2010-2012)WSP $83,450 (2008-2009)

WEF/IWA Nutrient Removal and Recovery, Jul. 12th, 2016 Denver

20

Questions

21

Biochemistry

22

Cell Morphology

23

Bioaugmentation Strategy

24

Strategy to Recover Bioreactor Experienceing NO2

- Stress

25

Bioaugmentation Strategy

26

Experience Research Group Field-Sierra with the Anammox Process

Molecular ecology

Reactor technology experience

Operating anammox under “optimal conditions”

Experience with upward-flow anaerobic sludge bed for annamoxMembrane bioreactor

Preventing inhibitionReversing reactor failure

Clone librariesPrimer sets for functional genes

Granular biomass

pH optimumStress markers

27

What Field-Sierra Group Can do for Pima Co.

Operate bench-scale reactors with Tres Rios reject water

Develop, test and monitor strategies for rapid cultivation of anammox biomass with reject water

Advise and provide monitoring program pilot scale reactors

Compare reactor technology strategies

Monitoring program

Advise full-scale plant

28

pH Optima versus Biomass Types

29

Toxicity of H2S

Granular biomass

Suspended biomass

IC50 suspended = 0.03 mM (1 ppm)

30

pH Optima versus Biomass Types

31

Toxicity of H2S

Granular biomass

Suspended biomass

IC50 suspended = 0.03 mM (1 ppm)

32