25316 Recirculating Aquaculture Systems

8. Denitrification

Mathis von Ahnen

Technical University of Denmark
DTU Aqua
Section for Aquaculture

Spring 2022

DTU Title 2
 Basics of water treatment in RAS

Feed

N OM, P

Fish Tank
Biofilter
(Nitrification;
NH4→NO2→NO3)
(N-conversion but no N removal)

Effluent with NO3 Mechanical

Filter
Removal of nitrate from RAS only through:
Removal of
-Water exchange
Effluent with particulate
organic matter organic -Denitrification
matter
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 Removal efficiencies in recirculating Model Trout Farms

Nitrogen is the least retained

nutrient in Model Trout Farms and
in RAS in general! → N discharge
often limits the production.

(Svendsen et al., 2008)

Farms are regulated by their discharges of total BOD5, Total-N and Total-P.

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”…given the new legislation there is no longer any upper limit to the amount of feed that
a farmer may apply, and hence to the amount of fish that may be produced, as long as
permitted nutrient emissions are not exceeded.” (Dalsgaard et al., 2013)

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 What is Denitrification?

1. Aerobic respiration of organic matter (e.g. C10H19O3N) by heterotrophic bacteria:

Heterotrophic bacteria
Organic Matter + O2 CO2 + H2O + Energy + new cells

2. Anoxic respiration of organic matter (when no free dissolved O2 is present but NO3)

Organic Matter + NO3 Heterotrophic bacteria CO2 + H2O + Energy + new cells + N2 (nitrogen gas!)

→ NO3 is denitrified!

(Respiration of organic matter using NO3 yields only ~70% of the energy compared to respiration using O2!
(Payne, 1970) Thus, oxygen rather than nitrate is the preferred terminal oxidant! → Most denitrifying bacteria are
heterotrophic aerobes/facultative denitrifiers.)

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 Aerobic respiration

Energy
Denitrification
gained
by
bacteria

Sulfate reduction

Methanogenisis

Korom, 1992

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Nitrogen Cycle

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 Denitrification Process

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 Denitrification Process

- 3.57g of alkalinity as CaCO3 produced for each gram of NO3-N reduced

- about one half of the alkalinity consumed by nitrification (7.14 mg CaCO3/l consumed
per g NH3-N nitrified) is regained through denitrification

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Denitrification Process

Heterotrophic denitrifiers are fast-growing microorganisms with maximum specific

growth rates of 3-6 d-1 and doubling times of up to 15-30 minutes under controlled
conditions.
(→ generally denitrification reactors require less time to reach stable
performance as compared to biofilters relying on the relatively slow-
growing autotrophic nitrifiers!)

Heterotrophic denitrification produces a consideral amount of bacterial biomass due to

the relatively high maximum yield constants (Ymax = 0.5-0.65 kg COD/kg COD or 1.6-
1.8 kg COD/kg NO3-N; Henze et al., 2002).
(→ build up of excessive sludge within a heterotrophic denitrification
system can become a problem and must therefore be controlled)

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 Why is denitrification becoming more important?

• Implementation of denitrification-units in RAS most often due to:

- Economical advantages (warm water species, limited water supply)

- Environmental constrains (discharge regulations)

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 Factors affecting Denitrification

• Oxygen: under aerobic conditions denitrification will not occur

(denitrification may start below 0.8-1.2 mg O2/L)

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 Factors affecting Denitrification

• Temperature: (Arrhenius)

μAmT = μ Am20 (Ѳn)(T-20)

Q10= ~2
(increase in temperature by 10 degrees yields about a doubling in the reaction rate)

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 Factors affecting Denitrification
• Carbon source: Type of carbon source

Carbon for denitrification can come from two sources:

• External
e.g.
methanol CH3OH,
acetate C2H3O2-

• Internal (single-sludge)
Carbon sources come from the fish waste/sludge
- Not commonly applied in commercial scale
- Not much information about it
- sludge needs a pre-hydrolysis step to form
more simple organic compounds that can
easily be consumed by denitrifiers

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 CARBON REQUIREMENTS (CALCULATIONS)

Excercise 1: How much methanol is needed for achieving anoxic

conditions for a flow of 1000 m3/d with an O2 concentration of 5 mg/L?

-1 mg/L of O2 to achieve anoxic condition

Assumption: 1.5 KgMeth/KgO2

How much methanol is needed for denitrifying a flow of 1000 m3/d with an
inlet concentration of 6 mg nitrate/L and an expected nitrate effluent
concentration of 1 mg/L?

Assumption: C:N = 2.3 KgMeth/KgNO3-N

- water is already anoxic

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 Factors affecting Denitrification

• Carbon source: C/N ratio

Different types of carbon sources have different optimal C/N ratios for denitrification:

Organic matter C/N Unit

Organic matter in activated 4 -5 Kg COD/Kg N
sludge
Methanol 2.3-2.7 Kg COD/Kg N

Acetate 2.9-3.5 Kg COD/Kg N

RAS sludge 4-7 Kg COD/Kg NO3--N

C/N ratio
Too high → dissimilatory nitrate reduction to ammonium (DNRA) NO3 → NH4, sulfate
reduction
Too low → incomplete denitrification, NO2 production, etc..

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 INCOMPLETE DENITRIFICATION

If insufficient electron donors (C-sources) are available and/or little amounts of

dissolved oxygen are present, incomplete denitrification may occur!

• Intermediates:
- NO2  toxic to fish

- NO  poison gas

- N2O  greenhouse gas (298 x CO2)

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Exercise 2: You have been asked to estimate the Nitrate and Carbon masses required
to pursue denitrification for a RAS effluent using endogenous carbon sources and
external carbon sources

Conditions: Assumptions

- Effluent flow: 2419 m3/d - C:N using methanol: 2.3

- Nitrate before the treatment: 6 mg/L - C:N using TCOD: 6

- Nitrate after the treatment: 1 mg/L - Methanol for reducing O2: 1.5
KgMeth/KgO2
- Oxygen before the treatment: 5 mg/L
- Maximum O2 concentration for
- Oxygen after the treatment: 0 mg/L denitrification: 1 mg/L

- TCOD (total chemical oxygen demand)

produced by the system: 15 KgTCOD/d
Flow: 2419 m3/d
Calculate: Nitrate (6 mg/L) Nitrate (1 mg/L)
O2 (5 mg/L) O2 (0 mg/L)
- Mass of O2 to remove for pursuing denitrification

- Mass NO3 to remove

- Methanol required for (NO3 and O2 removal)

- NO3 that can be reduced using endogenous carbon sources (consider water is in anoxic state)

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 Sludge management

An average to large land-based fish farm (1000 tons feed/year) can produce up
to 15 tons of sludge (dry matter) each month equivalent to 150 m3 wet sludge
(10% TS in wet sludge) – with approximately 200g of suspended solids per
kilogram of fish feed!

Sludge Tank
HRT: 3-4 days

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Sludge hydrolysis/fermentation

”complex” Hydrolysis Short molecules

organic (2 - 6 days) (VFAs) that can
matter in readily fuel the
particles denitrification
process

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Hydrolysis of sludge to gain easily degradable organic matter as
an energy source for denitrification

Sludge retention time

inside hydrolysis reactor
(~2-6 days)

Volatile fatty acids (VFAs)

are the desired, simple
organic compounds for
denitrification

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 Soluble organics/VFA formation during hydrolysis

VFA
4500
4000
3500
VFA mg/L 3000
2500
2000
1500
1000
500
0
0 1 2 3 4 5 6 7
Days

(Suhr et al., 2013)

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 Other things that happen during hydrolysis….
NH4-N
pH
50
8.0 45

NH4-N (mg/L)
7.5 40
7.0 35
6.5 30
25
pH

6.0
20
5.5
15
5.0
10
4.5 5
4.0 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Days
Days

PO4-P • pH drop due to production of VFA, H+

100
and CO2.
90
80
PO4-P (mg/L)

70 • Ammonia released from proteins

60
50
40
30 • Bound orthophospate is released
20
10 (pH dependent)
0
0 1 2 3 4 5 6 7
Days

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 Sludge Treatment
- Geotextile Bags → Sludge dewatering

- Flocculant + Belt filter → Sludge dewatering

Application of sludge as fertilizer on land? Use for biogas?

29

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 Denitrification Reactor Designs

Suspended Activated sludge

growth systems

UASB
Upflow anaerobic sludge-blanket
Biofilters Biodegradeble carriers
plastic polymers, PHA,MicroC TM ,
etc.

Moving bed
Fixed-film
Fluidized bed
Fixed bed

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 Activated Sludge
 Not applied in aquaculture but fully applied in wastewater treatment plants

 Efficiency depends on mantained bacterial biomass

 Control over bacterial mass needed (mantain SRT)

(Henze et al., 2013)

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Upflow Sludge Blanket (USB) Reactor

 Sludge bed in the bottom → up flow velocity lower

than the settling velocity of the granule

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 Packed media
Bioblocks (200 m2/m3)

RK bioelements (750 m2/m3) Bio beads (solid organic carbon polymer)

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 Moving Bed
• Frequently used in aquaculture

• Channeling can occur

• Good for high flows since

bacteria are attached.

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 Fluidized Reactor

• Not frequently used in

aquaculture.

• Carriers can provide carbon

source

• Good for high flows since

bacteria are attached.

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 Denitrification Removal Rates

• Can be expressed as:

- g NO3-N removed /reactor volume/ time

- g NO3-N removed / media area/ time
- g NO3-N removed/ bacterial mass/ time

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Exercise 3

A denitrifying reactor with a flow of 2700 m3/d and a volume of 400 m3 has an inlet
and outlet Nitrate concentration of 50 and 2 gNO3-N/m3 respectively . The reactor is
filled with biofilter media with a specific surface area of 750 m2/m3.

Find the volumetric and surface specific nitrate removal rates.

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 Autotrophic Denitrification

As opposed to heterotrophic denitrification, some autotrophic bacteria use

inorganic C sources (CO2 or HCO3-) together with electron donors other than
organic C for denitrification, such as:
reduced manganese (Mn2+),
reduced iron (Fe2+),
hydrogen (H2),
elemental sulfur (S), or reduced sulfur compounds (e.g. HS-, H2S, S2O32-, SO32-)

Among the different autotrophic denitrification pathways, sulfur-based

denitrification is most commonly used for wastewater treatment including
aquaculture wastewater.

Denitrification with elemental sulfur:

Consumes 4.57g CaCO3 and produces 2.5 g SO4 per g NO3-N reduced

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Autotrophic Denitrification

(Batchelor and Lawrence, 1978)

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Denitrification in Constructed Wetlands

N removal rates in constructed wetlands

connected to outdoor RAS in Denmark are
about 1.5-1.9 g N/m2/d.

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Nitrogen removal from Agricultural Drainage

Nitrogen source

Nitrate

stream soil surface

groundwater level

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 Nitrogen removal from Agricultural Drainage

Nitrogen source

Nitrate Nitrogen
gases

stream soil surface

groundwater level

http://www.bwsr.state.mn.us/news/webnews/June%202013/woodchip_bioreactor_illinois.png https://waiber.files.wordpress.com/2012/01/img_1546-e1326692785301.jpg?w=1200

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Woodchip characteristics

Woodchips as a source of organic matter for denitrification to

compensate for the lack of carbon in RAS effluents.

Wood has a C/N ratio of ~ 500:1

And is a long-lasting source of carbon, which is mainly composed of
cellulose (~40%), hemicellulose (~20-30%) and lignin (~20-30%)

Woodchips remain in shape for many years if kept under anoxic

conditions (water can pass through voids)

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 Microbial interactions in denitrifying woodchip bioreactors

Source:

Sulfur-based
Heterotrophic
Autotrophic
Denitrification
Denitrification

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 Average removal rates in woodchip bioreactors:
Nitrogen 5.3 – 8.5 gN/m3/d

BOD5 0.2 – 2.0 gBOD5/m3/d

Phosphorus 0.1 gP/m3/d

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 6000m3 of woodchips (4 x 1500m3)
100 l/s (4 x 25l/s)
(17h hydraulic retention time)
Inlet: 9.0 mgNO3-N/l Outlet: 0.8 mgNO3-N/l
90% removal of NO3-N (10.9 gNO3-N/m3/d)

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