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From the Aquarium Lab

Substrate Denitrification

Substrate Size Experiment

12 gallon lab tanks

Published - 20230921, Revised:

The substrates' role in reducing nitrate in aquatic systems has been known to science for decades. The substrate provides an anoxic environment for anaerobic heterotrophic and autotrophic bacteria and archaea. Some notable beneficial anaerobic processes in the substrate are hydrogen sulfide production, fermentation, methane production, decomposition of detritus, manganese reduction, iron reduction, and denitrification.

Nitrogen Cycle
Freshwater Nitrogen Cycle. The teal arrow points to the denitrification process that happens in the anoxic environment in the substrate. Original Credit: University of Florida/IFAS. Modified to include information from recent aquatics research.

In microbiology, anoxic is used to describe environments without molecular oxygen. Anaerobic refers to microorganisms that can live without molecular oxygen. The metabolism bacteria use is also called anaerobic. Easy distinction, anoxic refers to environments, and anaerobic refers to microorganisms and processes.

To achieve an anoxic environment in the substrate, water must flow through at a very slow rate. Aerobic bacteria and organisms need to use up the oxygen in the water before it reaches the anoxic region. If water flows through the substrate too quickly, an anoxic environment will not develop.

This experiment looks at substrate size and depth and its ability to aid in nitrate reduction within a closed system. The research shows that the substrate size matters to achieve denitrification in aquariums.

Three 12 gallow lab tanks.
Three 12 gallon lab tanks. Tank #1 (left) with 60 mesh sand, Tank #2 with 10 mesh gravel, and Tank #3 with pea gravel.

Three 12-gallon cubes (14" x 14" x 14" [53 L, 35.5 x 35.5 x 35.5 cm]) aquariums were initially set up (2022.11.06) and coupled to provide the same water chemistry and similar microbe biodiversity. Each aquarium was set up with 2 inches (5 cm) of substrate. The first system has 60 mesh (grit) sand, the second 10 mesh sand, and the third set up with pea gravel.

Each aquarium held about 8.25 gallons (31.23 L) of water above the substrate. Evaporation was replaced with tap water which contains no detectable nitrate (Hanna), no detectable phosphate (Hanna), 22 ppm KH (Hanna), 2 dGH (API), 37 ppm TDS (Hanna).

Tank #1.
Tank #1 with 60 mesh sand (Cemex Monterey sand). Aquired from a local landscape rock yard.
Tank #2.
Tank #2 with 10 mesh gravel. Aquired from a local landscape rock yard.
Tank #3.
Tank #3 pea gravel. Aquired from a local hardware store garden center.
Substrate comparison.
Substrate comparison above a centimeter ruler. Left to right, 60 mesh (6 grains per mm), 10 mesh (about 1 mm wide), and pea gravel (3 to 10 mm).

The systems were stocked with fish, shrimp, snails, and aquatic plants to help develop a diverse biome cycle within the aquariums. All fish, shrimp, and plants were removed by 2023.07.07, and the lights were turned off. Three species of snails were allowed to remain in the system:

Substrate comparison.
At the bottom is a ruler in centimeters for size comparison. Physella acuta (acute bladder snail) left, Melanoides tuberculata (Malaysian trumpet snail [MTS] or Malaysian livebearing snail), Planorbella duryi (Seminole ramshorn snail) right.

Temperature in the systems averaged around 80°F (26.7°C) during the experiment.

Hygger Aquarium Internal Power Filter HG-009-S
The three filters supplied by Hygger (sponsor of this research) are the 85 gph Aquarium Internal Power Filter HG-009-S (SKU: HGB009).

Filtration and current in each aquarium were supplied by Hygger (sponsor of this research) with the Aquarium Internal Power Filter HG-009-S (SKU: HGB009). The Hygger HG-009-S filter mounts in the corner of the aquarium. In the test systems, it was mounted in the upper left corner.

The filters were configured with the optional spray bar, and the carbon-infused polyester mechanical filtration pads and the sponge were installed. The biological media was left out of the filters to force biological activity within the substrate, consistent with the Planted Aquarium Keep it Simple (PAKIS) method. The spray bars were configured to create a current from left to right and angled towards the surface to aid gas exchange.

The systems were run coupled, and the nitrate, phosphate, KH, GH, TDS, and pH values were collected on 2023.07.30.

Hygger filters were installed in all systems on 2023.07.31.

Systems were decoupled on 2023.08.01.

Demonstration of the Hygger HG-009-S Aquarium Internal Power Filter (SKU: HGB009). Corner-mounted internal power filter for aquariums in the 5 (20 L) to 15 gallon (60 L) size.

Testing Equipment

The following is a list of the equipment and test kits used to collect data:

Starting Values 2023.07.31

The three tanks were couped with 3/4 in PVC J-tubes to allow the water chemistry values to be consistent across all tanks. Starting values were collected before the three tanks were decoupled.

Test Value
Nitrate Hanna 40.6 ppm
Phosphate Hanna 1.32 ppm
KH Hanna 43 ppm
TDS Hanna 202 ppm
Temperature AMES 79° F (26.1 C)
pH at 10:00 AM. 7.5
End Values of the Three Aquariums 2023.09.04

Below are the tested end values of the three tanks.

Tank #1 60 Mesh Sand (Very Fine Sand)

Test Value
Nitrate Hanna 22.2 ppm
Phosphate Hanna 2.23 ppm
KH Hanna 70 ppm
TDS Hanna 217 ppm
Temperature AMES 79° F (26.1 C)
pH at 11:30 AM. 7.8

Tank #2 10 Mesh Gravel (Fine Gravel)

Test Value
Nitrate Hanna 55.3 ppm
Phosphate Hanna 1.41 ppm
KH Hanna 59 ppm
TDS Hanna 232 ppm
Temperature AMES 77° F (25 C)
pH at 11:30 AM. 7.8

Tank #3 Pea Gravel

Test Value
Nitrate Hanna 48.8 ppm
Phosphate Hanna 1.08 ppm
KH Hanna 56 ppm
TDS Hanna 223 ppm
Temperature AMES 78° F (25.5 C)
pH at 11:30 AM. 7.8
Value Change of the Three Aquariums 2023.09.04

Nitrate dropped in Tank #1 18.4 ppm 34 days, while it increased 14.7 ppm in Tank #2, and 8.2 ppm. Phosphate increased 0.91 ppm in Tank #1, 0.11 ppm in Tank #2, and decreased 0.24 in Tank #3. KH, GH, TDS, and pH increased in all tanks.

Test Tank #1 Difference Tank #2 Difference Tank #3 Difference
Nitrate Hanna -18.4 ppm 14.7 ppm 8.2 ppm
Phosphate Hanna 0.91 ppm 0.11 ppm -0.24 ppm
KH Hanna 27 ppm 16 ppm 13 ppm
TDS Hanna 15 ppm 30 ppm 21 ppm
GH API 2 ppm 2 ppm 2 ppm
pH at 11:30 AM. Milwaukee .3 .3 .3
Summary of the Results

The results demonstrate that the very fine 60 mesh sand at 2 inches (5 cm) in depth in Tank #1 provides an anoxic environment for anaerobic bacteria to reduce nitrate. The 2 inches (5 cm) of substrate depth of Tanks #2 and #3 may need to be deeper to reduce nitrate, or it is less efficient than 60 mesh sand.

The increase in KH, GH, TDS, and pH can be attributed to the top-off water not being distilled.

New Questions

This experiment created more questions than it resolved. Here is a list of unresolved questions:

Contact Tony about this article.