Biological Denitrification

In Aquaria
Prepared by:
068044 Merve Ayvaz

October 27, 2009

1
 OUTLINE

Part 1 – Overview to the Biological

Denitrification
Part 2 – Principles of Denitrification
Methods
Part 3 – Comparison of ACD and HCD
Methods on an Experiment

2
PART 1
OVERVIEW
TO
BIOLOGICAL
DENITRIFICATION

3
Nitrate Accumulation
Side effects;
o Direct toxicity to organisms
o Alkanity and pH effect
o Decrease in oxygen concentration

4
Nitrate Accumulation
 Regular water exchange is impractical for large aquaria.
 Biological denitrification has been selected as a method of
choice for controlling NO3- concentration in closed systems.

5
What is Biological Denitrification?
 Reduction of nitrate to nitrogen gas in suboxic
conditions by bacteria.

6
Denitrifying Bacteria
 Denitrifying bacteria is a part of nitrogen cycel
 Facultative aerobes
 Enzymatic reduction

7
Denitrifying Bacteria
 Thiobacillus denitrificans
 Micrococcus denitrificans/Paraoccus denitrificans
 Pseudomonas

8
PART 2
PRINCIPLES OF
DENITRIFICATION
METHODS

ACD &HCD

9
Methods for Biological Denitrification
 Autotrophic columnar denitrification (ACD)
 Heterotrophic columnar denitrification (HCD)

10
 Heterotrophic and Autotrophic Columnar
Denitrification
 Heterotrophic
means obtaining
ready made organic
food from the
environment
 Autotrophic means
manufacturing food
from inorganic
compounds usually
carbon dioxide and
water.

11
Heterotrophic and Autotrophic Columnar
Denitrification

 HCD: Heterotrophic bacteria + Organic carbon source as a

food and electron donor

 ACD: Autotrophic bacteria + Inorganic carbon source to

manufacture food and sulfur as electron donor

 In both cases it is essential to provide an anaerobic

environment within the biofilter in order to allow the
reduction process to start.

12
 HCD, Heterotrophic Columnar
Denitrification

 Oxidation of different organic matters using while the

reduction of NO3- to N2 as the electron acceptor.
 Carbon source has to be added periodically to promote
growth and metabolism of heterotrophic bacteria.

- 
1) NO3  0.72C6 H12 O 6  0.62NH4  0.5N 2  0.62C5 H 7 N O 2
-
 1.62H2 O  0.38HCO3  0.86CO2

-
2) 6NO 3  5CH 3OH  3N 2  5CO 2  7H 2 O  6OH -

13
Pseudomonas Bacteria
 Gram-negative rod bacteria
 Commonly found in soil, ground water, plants and animals
 There are two type of Pseudomonas bacteria, Pseudomonas
stutzeri and Pseudomonas aeruginosa
 Pseudomonas stutzeri produced only dinitrogen;
Pseudomonas aeruginosa produced nitrous oxide as well

14
 ACD, Autotropic Columnar Denitrification
 The chemoautotrophic bacteria “Thiobacillus denitrificans”
oxidize sulphur and sulphur compounds while reducing NO3-
to free dinitrogen gas (N2).

 In this case, the metabolic reaction would progress according

to;
- -  2-
11S  10 NO3  4.1HCO3  0.5CO 2  1.71NH 4  2.5H 2 O  11SO 4
 0.92C5 H 7 NO 2 (biomass)  5.4N 2  9.62H 

15
 Thiobacillus Denitrificans
 Gram-negative, colorless, rod-shaped bacteria
 Thiobacillus are obligate autotrophic organisms, meaning they require
inorganic molecules as an electron donor and inorganic carbon (such as
carbon dioxide) as a source.
 They obtain nutrients by oxidizing iron and sulfur
with O2 or NO3-.

16
PART 3
COMPARISON
OF
ACD & HCD METHODS
ON AN EXPERIMENT

17
Aim of the Experiment
 The nitrate removal rates for ACD and HCD methods will
be compared
 pH effect and reversibility of the reaction will be discussed
Control Parameters
 Temperature is constant at 23 and 26 oC
 Salinity, pH, dissolved oxygen, temperature are followed in
each set

18
 Experimental Set-up

Glucose
injection
Outflow

Flow PUMP
regulation

Denitrification
column
19
Experimental Set-up
 The pilot plants had a total working volume of 50 L with a
circulating flow system between the glass holding tanks and
the denitrification unit.

 It is a PVC cylinder with an internal diameter of 8 cm,

packed with specific media for conditioned bacteria fixation
to a height of 40 cm.

20
Experimental Set-up
 Upward circulation of water was chosen to ease the
evacuation of gaseous nitrogen via the open top end of the
column.
o Fixation of heterotrophic bacteria was induced by periodical
addition of an organic carbon source (1 ml of 20% glucose
aqueous solution).

21
Packing Materials for Column
 The most studied packing materials are stones, clay, schist,
and plastic of various types, such as polyethylene,
polyesterene and even waste plastic materials.

22
Packing Materials in Column
 An adequate packing media should be;
inexpensive
easily available
high area/volume ratio
good mechanical resistance
suitable for microorganism attachment

 Among natural packing materials, volcanic stones

reasonably meet these criteria due to its high natural
porosity, relative resistance and inert behavior.

23
Packing Materials in HCD
Column

HCD: Porous volcanic media with 7 mm particle size.

Volcanic rocks mostly used medium in aquarium, pond
and marine denitrification systems.
o Pseudomonas bacteria attaches inherently to the volcanic
rocks porous surface.

24
Packing Materials in ACD
Column
ACD: Elemental sulphur (commercial powder) with 2–4 mm
particle size was used as bacterial fixating media.

The mechanism by which Thiobacillus

Denitrificans can use solid-phase
electron donors that cannot be taken
into the cell is of considerable interest
but that is currently unknown.

25
Start of the Experiment
 An artificial seawater solution (salinity = 36%) was
prepared with synthetic salt diluted into freshwater.

 Artificial seawater that was used in the pilot study,

purposefully contaminated with nitrate by the help of
nitrifying bacteria and NH4Cl.

 When adequate nitrate concentration was present (after the

nitrification process) bicarbonates were added to raise pH to
8.4 and create a suitable environment for denitrification to
progress.

26
Crucial Points for Denitrification
 Nitrate concentration
 Organic carbon source supply
 Oxygen concentration, pH,

27
Water Supply to the Column
 Water flow was kept low (1.5 dm3/h) in the beginning to
accelerate the creation of an anaerobic environment in
the column for both methods.

 Oxygen resistance of tolerance of autotrophic bacteria is

higher then heterotrophic bacteria. Water flow rate raised
after nitrated reduction started for ACD.

28
RESULTS
HCD ACD

29
 RESULTS
HCD ACD

- -  2-
11S  10 NO3  4.1HCO 3  0.5CO 2  1.71NH 4  2.5H 2 O  11SO 4
 0.92C5 H 7 NO 2 (biomass)  5.4N 2  9.62H 

30
Results,HCD

31
pH Effect in HCD

32
Results,ACD

33
pH Effect in ACD

• Bicarbonate addition in day 60

• Suggested pH for T. Denitrificants
is 6.2

34
Conclusion
 ACD showed double nitrate reduction rates then HCD
 Results are under effect of microbiological group that
produce N2 depending on adequate pH and oxygen resistant
 Inorganic carbon has great control on ACD
 Organic carbon has chief control on HCD

35
 THANK YOU FOR YOUR
ATTENTION

36