Chapter IV.

BACTERIA

The bacteria of natural waters include multitudinous unnamed species. All bacteria are aquatic in that they feed and reproduce in water [1]. (Even the bacteria in dry terrestrial soils live within the soil’s pore water.) However, few bacteria live freely suspended in water; most live attached to surfaces– rocks, sediment, plants, etc– within the water. Thus, there may be 100,000 more bacteria in the sediment than in the overlying water [2]. Often these bacteria don’t live as individual cells or in pure colonies, but rather they live in biofilms– complex associations with other bacteria, algae, and protozoa.

Bacteria that are important in aquariums can be compared with other organisms by the chemicals they use for their metabolic processes (Table IV-1). Animals and heterotrophic bacteria use organic compounds for energy, while chemoautotrophic bacteria use inorganic chemicals. Most organisms use oxygen to accept electrons for respiration.¹

Table IV-1. Organisms Classified by Chemicals Required to Sustain Life.

Organisms	Energy Source	Carbon Source	Electron Acceptor (for respiration)
Man, Animals, and Fish	Organic cpds	Organic cpds	oxygen
Plants	Light	CO2 and HCO3-	oxygen
Chemoautotrophic Bacteria	Inorganic cpds	CO2 and HCO3-	oxygen
Heterotrophic Bacteria: Aerobes Anaerobes	Organic cpds Organic cpds	Organic cpds Organic cpds	oxygen NO3-, NO2-, Mn4+, Fe3+, SO42-, organic cpds

ABBREVIATIONS: CO₂ = carbon dioxide; cpds = compounds; HCO₃^- = bicarbonate; Fe = iron; Mn = manganese; NO_2^- = nitrite; NO_3^- = nitrate; and SO₄² = sulfate

The metabolic processes of bacteria also result in the conversion of one chemical to another. Some of the chemical conversions important to aquariums are shown in Table IV-2. For example, in the bacterial process of nitrification, ammonium is converted to nitrate.

Table IV-2. Input and Output of Chemicals during Bacterial Metabolism.

Process	Input	Output
Nitrification	NH4+	NO3-
H2S oxidation	H2S	SO42-
Methane oxidation	CH4	CO2
Aerobic decomposition	organic cpds	CO2, NH3, PO43-, H2S, etc
Anaerobic decomposition	organic cpds	organic acids, ethanol, NH3, H2S, etc
*Denitrification	NO3-	N2O, N2
*Nitrate respiration	NO3-	NO2-
*Manganese reduction	Mn4+, Mn3+	Mn2+ (soluble manganese)
*Iron reduction	Fe3+	Fe2+ (soluble iron)
*Sulfate reduction	SO42-	H2S
*Fermentation	organic cpds	organic acids, alcohols, CO2
*Methanogenisis	acetic acid, CO2, H2	CO2, CH4

ABBREVIATIONS: CH₄ = methane; H₂S = hydrogen sulfide; N₃= nitrogen gas; NH₄⁺ = ammonium; NH₃ = ammonia; N₂O = nitrous oxide; PO₄^3- = phosphate. See also Table IV-I.

*Forms of anaerobic decomposition by heterotrophic bacteria.

All metabolism, including decomposition of organic matter, generates electrons. For example, the sugar glucose provides four electrons when bacteria break it down to pyruvic acid:

C₆H₁₂O₆ ⇒ 2 CH₃COCOOH + 4 H⁺ + 4 electrons

Every electron generated by metabolism requires an electron acceptor. Otherwise, metabolism (and life) stops.

Anaerobic metabolism differs from aerobic metabolism in that oxygen is not the electron acceptor. Under anaerobic conditions, bacteria must find other, less desirable compounds. Instead of oxygen, bacteria use nitrates, manganese, iron, sulfates, etc. Thus, when bacteria use sulfates to accept electrons, sulfates are converted to hydrogen sulfide.

A.Bacteria Processes

1.Decomposition by Heterotrophic Bacteria

The decomposition of organic matter by ordinary (i.e., heterotrophic) bacteria is important to planted aquariums. Organic matter contains all the elements that plants require, but the elements are ‘locked up’ in large organic compounds. Heterotrophic bacteria convert organic matter, whether in the form of fishfood, plant debris, dead bacteria, etc, into the nutrients that plants can use. Some of the conversions that occur are:

Organic Matter	⇒	Inorganic Compounds (Plant Nutrients)
organic N	⇒	ammonia + CO2
organic P	⇒	phosphates + CO2
organic S	⇒	sulfides + CO2

Because organic matter invariably contains carbon, CO₂ is always released during decomposition. Moreover, other elements, not just N, P, S, and C, are converted from their organic forms to plant nutrients by heterotrophic bacteria.

Organic matter that heterotrophic bacteria feed on comes in two physical forms– particulate organic carbon (POC) and dissolved organic carbon (DOC). POC, which includes fish feces and fibrous plant matter, is harder for bacteria to digest than the much smaller DOC. (Here is where fungi and snails are useful, because they reduce particle size, thereby speeding up the decomposition process [3,4].

Ironically, DOC, which we can’t see, is usually a much larger reservoir of carbon in natural systems [5], plus it is the form of organic matter from which plant nutrients will be most rapidly released. The average DOC concentration for the world’s rivers is 5.8 mg/l, while the average for 500 Wisconsin lakes is 15.2 mg/l. (For all natural waters the range is 1-30 mg/l [5].)

Almost all DOC and debris in aquariums is in various stages of decay, but the rate of nutrient release may vary considerably. (Heterotrophic bacteria have their own preferences in terms of what constitutes desirable food and a suitable environment.) DOC includes proteins, organic phosphates, and simple sugars, which are metabolized rapidly, probably within hours at the warm temperatures and neutral pH of most aquariums. The less-digestible portion of DOC, such as humic substances, may take months or longer for bacteria to digest.2 Finally, complete digestion of POC in the anaerobic substrate environment may be impossible, resulting in the gradual accumulation of sediment humus (‘fish mulm’).

Bacteria understandably divert part (20-60%) of the nutrients released by decomposition to synthesize their own cellular material [8]. However, these bacteria also die and decompose themselves. Indeed, in lake water over a 20 day period, four separate and sequential bacteria populations were associated with reed decomposition [9]. There may be several of these recyclings before a nutrient is finally taken up by plants.

Aerobic decomposition, which requires oxygen, is much faster than anaerobic decomposition. Thus, air/water mixing and plant photosynthesis stimulate decomposition by adding oxygen to the water.

Most bacteria require a neutral pH, such that pH can have a major impact on decomposition. For example, swamps containing Sphagnum (‘peat’) mosses are often very acidic (pH 3 to 4.5), because the plants themselves are acidic [6]. Bacterial activity and decomposition slow considerably in this acidic environment. Organic matter accumulates, because bacteria are not converting it to gases such as methane, CO₂, and hydrogen. The end result is that a Sphagnum swamp gradually fills in with the undigested organic matter.

In the final analysis, decomposition in an ecosystem is a summation of many separate, on-going metabolic processes. Thus, in lakes as well as in the established aquarium, decomposition and the release of plant nutrients is typically a steady, stable, and continuous process.

a)      Decomposition in the Sediment as a CO₂ Source

The decomposition of sediment organic matter by heterotrophic bacteria releases CO₂ and methane into the water. Almost all lakes have more CO₂ than what would result solely from their equilibration with atmospheric CO₂ [10]. Much of this CO₂ surplus comes from decomposition in the sediments.

CO₂ release by sediments depends on the amount and type of organic matter it contains. For example, investigators [11] compared the decomposition rates of different types of organic matter mixed with lake sediment. Sediment containing a 5% addition of fresh aquatic plant matter generated large amounts of CO₂ (1,000 µg/g dry sediment/day). In contrast, sediment containing a 5% addition of dead oak tree leaves gave off CO₂ much less rapidly (150 µg/g/day).³ Chemical analysis confirmed that the fresh aquatic plant matter was richer in nutrients than the dead tree leaves. Bacteria activity was greater on the richer organic matter, so that CO₂ was more rapidly released.

Sphagnum cuspidatum. S. cuspidatum has long (5”-16”) feather-like stems and often grows fully submerged. Like other sphagnum mosses, it forms dense, spongy mats in swamps and bogs.

Sphagnum mosses are inherently acidic and the main ingredient of ‘peat’. Some hobbyists use peat filtration to naturally soften and acidify the water. (Ca and Mg exchange for acidic protons on the peat’s numerous binding sites.)

Drawing from Watson [7] and reprinted with the permission of Cambridge University Press.

b)      Production of Humic Substances (HS)

The recycling of organic matter into CO₂ and nutrients that plants can use is not decomposition’s only benefit. Incomplete decomposition of plant matter results in humic substances, which accumulate both in the water and substrate [12].

Humic substances (HS) are non-specific molecules or particles originating from the random decomposition of plant material, especially lignin, by non-specific bacteria. Often HS are phenolic in their chemical nature, because they retain some of the phenolic groups of the original lignin. Exactly, how bacteria form HS from a ‘chemical soup’ of proteins, polyphenols, and other plant material is still a mystery. However, it may involve the polymerization of phenols (after their oxidation to quinones) with proteins [13]. Because HS formation inevitably involves bacterial oxidation of the plant molecules to obtain energy, HS carry multiple carboxylic acid groups. Even at neutral pH the carboxylic acid groups are negatively charged (R-COO^-). Multiple negative charges increase the water solubility of HS. They also bind positively charged ions, such as iron (Fe³⁺) and manganese (Mn⁴⁺). After the metals are bound, they can be released into the water in a light-induced process that simultaneously reduces (chemically) the metal and oxidizes the organic matter (see pages 167-169).

Humic substances, which sometimes add a yellowish or brownish color to natural waters, make up about 50% of the DOC in natural freshwaters (Fig. IV-1).

Figure IV-1. DOC Composition in an ‘Average’ River’.

Fulvic, humic, and hydrophilic acids are all humic substances and have a similar molecular weight (~1,000 to 2,000). They differ mainly in their solubility, with humic acids the least soluble and the hydrophilic acids the most soluble. ‘Simple compounds’ include amino acids, phospholipids, peptides, etc whose chemical structure and origins are well-known.

Fig. 4.1 from Thurman [12] used with kind permission from Kluwer Academic Publishers.

The humic substances found in the aquatic environment are different than those found in the terrestrial environment. Aquatic HS tend to have less phenolic groups, less color, and are more water-soluble than soil HS [12,15]. Sometimes they can only be detected by their strong absorption of UV light [16].

Humic substances benefit aquariums in two major ways. First, they help keep micronutrients in solution and available to plants. (Without HS, many metals, especially iron and manganese, would precipitate out of solution and be unavailable for plant uptake.) Second, the binding and chelating of metals by HS helps counteract metal toxicity in fish and plants (see pages 14-16). Both of these effects would occur both in the substrate and in the water.

2.Nitrification

Nitrification is the two-step process whereby bacteria convert ammonia, which is toxic (see page 20), to nitrate, which is not toxic.⁴ In new aquariums nitrifying bacteria gradually colonize aquarium filters where they are provided with lots of attachment sites and plentiful oxygen from the moving water. [In tanks containing soil, which already contains nitrifying bacteria, the process starts much sooner (see page 138).]

The bacteria responsible for nitrification consist of many different species, but they can be grouped into those that oxidize ammonia and those that oxidize nitrite.⁵ Although nitrifying bacteria play a secondary, non-critical role in natural ecosystems, they are found in all soils, sediments, and natural waters.⁶

Nitrifying bacteria are chemoautotrophic and differ from heterotrophic bacteria in that they oxidize inorganic chemicals (ammonium and nitrite) to obtain their energy. (Other chemoautotrophic bacteria are H₂S-oxidizing bacteria.) Chemoautotrophic bacteria differ from the vast majority of bacteria, which are heterotrophic (heterotrophic bacteria obtain their energy from the decomposition of organic compounds, such as proteins and sugars).

Because the requirements of nitrifying bacteria are so different than ordinary (i.e., heterotrophic) bacteria, early scientists had trouble cultivating them in the laboratory. Nitrifying bacteria simply would not grow on the organic nutrient media that had worked for other bacteria; in fact, organic compounds inhibited them. It was not until 1890 that the Russian scientist Winogradsky discovered that if he used a simple inorganic media containing mainly ammonium and calcium carbonate, nitrifying bacteria would grow. Winogradsky had hypothesized correctly that these bacteria required an inorganic carbon source such as bicarbonate [22].

Actually, nitrifying bacteria are similar to plants in that they synthesize the large organic compounds they are made of (proteins, sugars, etc.) from small inorganic compounds like CO₂, iron, phosphates, etc. Plants use light energy to fuel the process (photosynthesis); nitrifying bacteria use chemical energy to fuel the process (chemosynthesis).

In the first step of nitrification one bacterial group converts ammonium to nitrite:

NH₄⁺ + 1½ O₂ ⇒ 2 H⁺ + NO_2^- + H₂O

In the second step another bacterial group converts nitrite to nitrate:

NO_2^- + ½ O₂ ⇒ NO_3^-

The overall nitrification reaction (NH₄⁺ + 2 O₂ = NO_3^- + H₂O + 2 H⁺) generates acid and consumes oxygen. Indeed, nitrifying bacteria require more oxygen than ordinary bacteria, up to 100 oxygen atoms per carbon atom fixed [22]. Thus, nitrifying bacteria may capriciously interfere with municipal water purification; during sewage treatment, if ammonium levels reach 2 mg/l, nitrification may consume all oxygen [23].

Nitrifying bacteria are helpful, if not essential, in tanks without plants. However, in planted tanks they compete with plants for ammonia. The energy nitrifying bacteria gain from oxidizing ammonium to nitrates is an equivalent energy loss to plants (see page 111).

3.Denitrification

Denitrification is a common process in soils and sediments that converts nitrate to N₂ gas:

Many ordinary bacteria (Pseudomonas, Achromobacter, Escherichia, Bacillus, Micrococcus, etc.) can denitrify [26,27]. The most common organisms are various species of Pseudomonas, Flavobacterium, and Alcaligenes [28].

Although denitrification occurs wherever there are nitrates, organic matter, and anaerobic conditions, it is often linked to nitrification [24,29]. Nitrification provides the nitrates, and by consuming oxygen, provides the anaerobic environment.

Nitrification-denitrification can result in substantial losses of N to aquatic ecosystems. In aquaculture ponds, one investigator found that only 43% of the added fishfood nitrogen could be recovered in water, soil, and fish; the remaining 57% of added N was believed to be lost through denitrification [30]. Lake Tanganyika is believed to be N-limited due to linked nitrification-denitrification [31]. Other investigators [32] studying nitrogen cycling in a Rhode Island bay concluded that denitrification reduced about 50% of the N loading from rivers, land, and sewage.

One investigator [33] closely followed N losses in nutrient-rich wastewater. Nitrate and ammonium were added to 100 gal treatment tanks containing sediment, wastewater, and various aquatic plants. Nitrogen distribution between water, substrate, and plants was measured at the end of 27 days (Table IV-3).

Table IV-3. Recovery of N Fertilizers in Tank Systems after 27 Days [33] (or “Where did the N that was added to the tanks go?”)

Both nitrates and ammonium (0.010 ppm N of each) were added to all the tanks. In the first set of 4 tanks, ammonium was labeled with ¹⁵N (radioactive nitrogen), while in the second set of 4 tanks, nitrates were labeled with ¹⁵N. By measuring the radioactivity in the water, soil, and plants, the investigators were able to monitor the fate of the additions.

Each treatment was done in duplicate. (I reported values for different tanks that were not significantly different as ranges of reported values.)

N Source Monitored	Tank System	N in Water	N in Sediment	N in Plants (or algae)	Lost N
NH4+	Pennywort	0-3 %	8-9 %	67 %	24%
	Water hyacinth	0-3	8-9	41-44	47-54
	Cattail-Elodea	0-3	8-9	41-44	47-54
	Control (algae)	21	21	5	47-54
NO3-	Pennywort	0-0.1	6	13	81
	Water hyacinth	12	6	39	43-48
	Cattail-Elodea	0-0.1	29-31	24	43-48
	Control (algae)	36	29-31	4	29

Even though plants took up some of the added N, much of it could not be accounted for. For example, 24-54% of added ammonium (NH₄⁺) was lost in the first set of 4 tanks (where ammonium-N was monitored). Some of the N loss was attributed to the escape of ammonia gas (“ammonia volatilization”). (During heavy photosynthesis when the pH climbed above 8.0 in these tanks, considerable NH₄⁺ would convert to NH₃ gas.) In the second set of tanks where nitrate (NO_3^-) was monitored, N losses were even greater– 29 to 81%. The investigators attributed most nitrate losses to denitrification, which probably occurred in the sediment.

Denitrification can also reduce nitrogen levels in aquariums like mine that have a soil layer. I did some experiments [34] to see if soil alone (no plants) could remove nitrates from the water. I used duplicate plastic bottles with tapwater and a soil/sand substrate. I added nitrates to the water and then measured both nitrate and nitrite levels twice a week for 32 days. I found that even in bottles containing a hefty 250 mg/l nitrates, nitrates started declining substantially within one week and were completely gone within 1 month. During this experiment, nitrites appeared in the water at 3 days indicating that some nitrate respiration was also occuring. In similar bottles without the soil/gravel substrates, which would have very little bacterial activity, nitrate, levels remained high.

Nitrates diffuse into the anaerobic soil layer where the abundant bacteria quickly use the nitrates. Thus, for aquarium hobbyists, denitrification is a harmless bacterial process that helps prevent nitrate accumulation.

4.Nitrite Accumulation

Nitrites, which are quite toxic to fish (see page 22), may accumulate from several different bacterial processes, not just one. The most probable candidates for causing nitrite accumulation are nitrate respiration and incomplete nitrification. However, two other bacterial processes (DAP and denitrification) might also release nitrites. All of these separate bacterial processes could contribute to nitrite accumulation in aquariums.

Q. Can you suggest some rapid growing, nitrate-consuming aquatic plants. My tanks need nitrate reduction, which I cannot seem to accomplish by changing 25 % of the water weekly.

A. I wouldn’t count on plants alone to prevent nitrate accumulation. Even hobbyists with phenomenal plant growth report nitrate accumulation (see Q&A on page 111).

Rather I would focus on denitrification, a bacterial process. Denitrification occurs in soil, clogged filters, and other anaerobic environments that contain debris and organic matter.

Nitrates are undetectable in most of my aquariums even after months of heavy fish feeding and virtually no water changes. Even if nitrates accumulate, they are not toxic (see page 62). I have had nitrates levels as high as 90 mg/l in my Rainbow tanks without problems.

The only problem with high nitrates in an aquarium is that under anaerobic conditions, bacteria readily convert nitrates to nitrites, which are toxic. Nitrates diffusing into an anaerobic substrate (e.g., freshly submerged potting soil) would be readily converted to nitrites via nitrate respiration by soil bacteria. Tanks with high nitrate levels have a greater potential for nitrite toxicity.

I would advise hobbyists to focus their concerns on nitrite levels in their aquariums and worry less about nitrates.

a)      Nitrate Respiration

Nitrate respiration is a common bacterial process carried out by a variety of ordinary bacteria under anaerobic conditions. The reaction whereby bacteria use nitrate (NO_3^-) for respiration is:

NO_3^- + 2 H⁺ + 2 e^- ⇒ NO_2^- + H₂O

Unlike denitrification where nitrite is further converted to the gases (N₂O and N₂), nitrites are the endproduct of this reaction. Nitrate respiration is a major anaerobic process carried out by a wide variety of ordinary bacteria. Thus, in an extensive survey [28] of sediment and soil bacteria, about 80% of the bacteria capable of growing under anaerobic conditions were nitrate-respiring bacteria (produced nitrites when isolated and cultured). The remaining 20% of the anaerobic bacteria were denitrifying bacteria (i.e., produced N₂ but no nitrites when isolated and cultured with nitrate).

b)      Incomplete Nitrification

Sometimes nitrification is incomplete resulting in nitrite accumulation. This can happen when environmental stresses (acidity, low temperature, etc) inhibit nitrite-oxidizing bacteria more than the sturdier ammonia-oxidizing bacteria. Nitrite accumulation occurs when the second step of nitrification (NO_{2^- ⇒ NO3^-) no longer processes the nitrites produced by the first step (NH4⁺ ⇒ NO2^-).}

When aquariums are first set up, there may be several weeks during which nitrites accumulate. This is because ammonia-oxidizing bacteria must first establish themselves and generate enough nitrites to encourage the nitrite-oxidizing bacteria. Thus, nitrification in the typical beginner’s new and ultra-clean tank is usually incomplete until after about 6-8 weeks [19, 24, 25].) The hobbyist can speed up the process greatly by adding a soil underlayer to the tank or seeding the tank with used gravel or filter material taken from an established tank.

c)      Incomplete DAP and Incomplete Denitrification

Bacteria use nitrates in yet another pathway besides denitrification and nitrate respiration. Apparently, numerous bacteria convert nitrates to ammonium by a pathway called ‘dissimilatory ammonium production’ or DAP. This pathway is linked to fermentation and energy production; therefore, it occurs even when there is adequate ammonium.⁷ The reaction for DAP is:

DAP produces substantial ammonium in some sediments. Investigators tracing the fate of added nitrates have found that DAP often rivals denitrification in nitrate processing [35, 36, 37]. Although much of the ammonium produced by DAP is recycled back to nitrates (via nitrification), DAP appears to be a major bacterial process in the nitrogen cycle.

Sometimes DAP does not go to completion; when this happens, nitrites may accumulate. Thus, one soil bacterium (Citrobacter sp) converted 97% of added nitrates to nitrite under certain conditions [38]. (Under other conditions, it produced N₂O and NH₄⁺.)

Similarly, denitrification (see page 63) does not always go to completion either. Incomplete denitrification may result in transient nitrite accumulation [26]. Under the right conditions, both DAP and denitrification, could contribute to nitrite accumulation.

5.Reduction of Iron and Manganese

When oxygen and nitrates are gone, many soil and sediment bacteria can use iron (Fe) or manganese (Mn) to accept the electrons generated by their metabolism. This ‘biological reduction’ of Fe and Mn solubilizes the two metals allowing them to be taken up by plant roots. Thus, anaerobic bacteria are critical in providing plants with Fe and Mn.

Although there is less Mn than Fe in soils, oxidized Mn is a better electron acceptor than oxidized Fe (see page 128). Therefore, if Mn is available, it will be used before Fe. The following reaction describes Mn reduction by the electrons generated by bacterial metabolism:

MnO₂ + 4 H⁺ + 2 e^- ⇒ Mn²⁺ + 2 H₂O

In the above reaction, Mn goes from an oxide precipitate (MnO₂) to a soluble cation (Mn²⁺) that can now enter plant roots. Apparently, a wide range of bacteria and microfungi can use MnO₂ as an electron acceptor [4]. When MnO₂ is exhausted, bacteria use ferric iron to accept electrons:

Fe(OH)₃ + 3 H⁺ + e^- ⇒ Fe²⁺ + 3 H₂O

As with Mn, an insoluble oxide precipitates, in this case Fe(OH)₃, is converted to a soluble ion (Fe²⁺). Plant roots readily take up the Fe²⁺ form of iron.

6.Hydrogen Sulfide Production

Hydrogen sulfide (H₂S), which is readily formed in aquarium substrates, is a foul-smelling gas that is extremely toxic (see page 133).

There are two sources of H₂S. One is from the ordinary decomposition of proteins by heterotrophic bacteria during which the protein’s SH group is released as H₂S:

Protein-SH + H⁺ + e^- ⇒ H₂S

The second source of H₂S is the specialized reduction of sulfates by Desulfovibrio, Desulfotomaculm and other bacteria genera. Sulfate is used as an electron acceptor by these bacteria during the anaerobic decomposition of organic matter:

SO₄^2- + 10 H⁺ + 8 e^- ⇒ H₂S + 4 H₂O

Sulfate-reducing bacteria are associated with high sulfate levels and severely anaerobic conditions (sediment redox below -120 mvolts) [39]. (See page 128 for an explanation of Redox.) Marine sediments are particularly conducive to H₂S production. This is because seawater contains an extremely high level of sulfates (2,700 mg/l), which is over 200 times that of most freshwaters [40]. The diffusion of plentiful sulfates from seawater into marine sediments, especially if the sediment contains plentiful organic matter, promotes H₂S production.

7.Hydrogen Sulfide Destruction

In the presence of oxygen, various bacteria rapidly oxidize hydrogen sulfide (H₂S) to sulfates. (This reaction is analagous to the nitrification reaction where a very toxic molecule is converted to a harmless salt.) The overall reaction for H₂S oxidation is:

H₂S + 2 O₂ ⇒ HSO₄^- + H⁺

H₂S oxidation can be carried out anaerobically in the presence of light by photosynthetic bacteria. Some contain green pigments (Chlorobacteriaceae), and others contain purple pigments (Thiorhodaceae) [4,40]. In aquariums with a soil substrate, sometimes photosynthetic bacteria will appear as a strange horizontal and purple-colored line next to the glass.

Aerobic H₂S oxidation by chemoautotrophic bacteria, such as Thiobacillus and Beggiatoa, is probably more common in aquariums.

Bacteria that oxidize H₂S protect plant roots by destroying toxic H₂S in the root zone (see page 152). These bacteria also protect fish. H₂S-oxidizing bacteria would remove H₂S gas generated within the substrate or pockets of anaerobic debris. These bacteria would be expected to colonize the surface layer of sediments and aquarium substrates (see page 129).

8.Fermentation and Methanogenisis

Under severely anaerobic conditions, organic matter is only partially metabolized by bacteria resulting in the accumulation of ethanol and various organic acids. (In contrast, when oxygen is present, bacteria metabolize organic matter to CO₂ and water.) In lake sediments large quantities of organic matter are degraded by the linked processes of fermentation and methanogenisis [4, 42]. This happens when inorganic electron acceptors (NO_3^-, Fe³⁺, Mn⁴⁺, SO₄^2-) are no longer available. After oxygen and inorganic electron acceptors are depleted, the organic matter itself releases and receives electrons. Bacteria oxidize one portion of the organic molecule, while reducing another portion of the same molecule (or another organic molecule).

Fermentation involves the breakdown of organic matter into various fatty acids, alcohols, acetic acid, hydrogen gas, and CO₂ by fermentative bacteria. Some of the organic acids and alcohols moderately inhibit plant roots (see page 133).

Methanogenisis is carried out by four major genera: Methanobacterium, Methanobacillus, Methanococcus, and Methanosarcina. These bacteria, which are strictly anaerobic, use the acetic acid, hydrogen gas, and CO₂ produced during fermentation to produce methane, CO₂, and water. The two reactions they use are:

CO₂ + 4 H₂ ⇒ CH₄ (methane) + 2 H₂O

CH₃COOH (acetic acid) ⇒ CH₄ (methane) + CO₂

In the aquarium, methanogenisis and fermentation occur in the substrate. Methane is released from the substrate by diffusion into the water as well as by gas bubbling [43]. Some of the methane produced will be oxidized into CO₂ that plants can use.

9.Methane Oxidation

Methane-oxidizing bacteria, such as Methanomonas methanica, Pseudomonas methanica, and Thioploca species, are common and widely distributed [44,45]. They are located in the surface layer of sediments and quickly convert methane released from anaerobic sediments into CO₂. For example, approximately 91% of the methane produced in a peat sediment of the Florida Everglades was oxidized to CO₂ and water [4]. The overall reaction for methane oxidation is:

5 CH₄ + 8 O₂ ⇒ 2 (CH₂O) + 3 CO₂ + 8 H₂O

Aquatic plants undoubtedly enhance methane oxidation by providing a home for these bacteria. Thus, one investigator [46] showed that in the emergent plant Pontederia cordata, methane-oxidizing bacteria were not only attached to the root surface but also lived within the roots.

Q. I added potting soil to my tank as an underlayer, and I noticed that a lot of gas bubbled from the substrate. Are these gases harmful to the fish?

A. Possibly. If the fish have lost their appetites, or rooted plants are not doing well, I would be concerned. The H₂ S (and other decomposition products) released from fresh potting soil substrates can sometimes cause problems in newly setup tanks.

You can keep a new and highly organic substrate from becoming too anaerobic by gently poking the substrate with a knife, etc. Make sure to keep the water sufficiently oxgenated.

If plants and fish are okay, I would not worry about substrate bubbling. Gas bubbling stirs the soil layer allowing in oxygenated water.

In aquariums, methane oxidation insures that methane generated in the substrate is made available to plants. Methane, which plants cannot use, is converted to CO₂, which plants can use. Since carbon is often the limiting plant nutrient in aquariums, methane-oxidizing bacteria are helpful.

B.Biofilms

Many ideas about bacteria are based on laboratory studies where bacteria exist as individuals suspended in nutrient-rich media. However, the same bacteria in the natural world behave much differently than those in the laboratory. This is because nature, where predation is common and nutrients are not so plentiful, is a much harsher environment than the laboratory. To survive, bacteria have learned to attach themselves to surfaces, to associate cooperatively with other species, and to protect themselves from their enemies. This microcosm, which is held together by polysaccharide ‘gums’ produced by the bacteria, is called a biofilm (or periphyton).

Biofilms are the norm in the natural world. Aquarium hobbyists are familiar with filter debris or scum on the water surface. These are examples of biofilms. The most well-studied ones are, of course, those that create problems: (1) dental plaque; (2) chronic lung infections of cystic fibrosis patients; (3) the corrosion of water pipes and ship hulls, and (4) the contamination of contact lenses, artificial hearts and other medical implant devices [47,51].

The reason bacteria attach and form biofilms on surfaces is that surfaces are where nutrients congregate. This is because all surfaces have a negative charge that attracts cations and dissolved organic carbon (DOC). The congregation of positively charged compounds, in turn, attracts negatively charged compounds. Thus, even in nutrient-depleted water, often enough organic compounds will adhere to surfaces to support some bacterial growth [48]. When organic compounds collect at the water surface, they attract various feeding bacteria, algae and protozoa, which may over time develop into a biofilm, sometimes called ‘neuston’ [49].

Bacteria stick to surfaces by various strategies. Some bacteria are sticky to begin with; they are essentially ‘glueballs’ covered with sticky lipopolysaccharide capsules or proteinaceous appendages. Other bacteria only synthesize the attachment components when a surface is present. For example, within 15 min of Pseudomonas aeruginosa’s encountering a glass surface, a gene (AlgC) critical for polysaccharide synthesis was stimulated [50].

Once bacteria attach to a surface, they divide and continuously produce large quantities of polysaccharides to form a ‘mature’ biofilm. A mature biofilm may be 600 to 900 µm thick [24], which is several hundred times thicker than an individual bacterium. (A bacterium is about 1 µm long [51].) The biofilm is not an amorphous, gelatinous mass of polysaccharides and bacteria as was once supposed; it has organization and structure. Even the densest area of a biofilm is permeated by water channels. Water flows through mushroom-like structures of clumped bacteria thereby bringing the inhabitants food and carrying away their wastes [47].

Apparently, the internal structure of biofilms does not happen by chance. Investigators [53] showed that active communication between bacteria insures that the biofilm develops properly. (Mutant bacteria that were unable to communicate formed abnormal biofilms.)

Nor do biofilms consist of uniform layers of aerobic bacteria on top of uniform layers of anaerobic bacteria. Because of the water channels, anaerobic and aerobic bacteria coexist in microniches throughout biofilms. Thus, investigators [24] were surprised to find dentrification occuring in a supposedly aerobic filter used for wastewater treatment. (This filter would be similar to a ‘trickle filter’.) They found similar proportions of aerobic heterotrophs, nitrifying bacteria, denitrifiers, and anaerobic heterotrophs at both the bottom and the top (Fig. IV-2). And in additional experiments, they found the metabolic activities of nitrifying (aerobic) and denitrifying bacteria (anaerobic) were the same in the bottom layer as in the top layer.

Fig. IV-2. Bacterial Populations in a Wastewater Biofilm.

Investigators sliced a mature biofilm of about 730 µm thickness into three horizontal layers. (Thickness of each layer was: top layer = 400 µm; middle layer = 200 µm; and bottom layer = 130 µm.). The layers were homogenized and the numbers of aerobic hetertrophs, facultative heterotrophs, nitrifying bacteria (Nitrosomonas sp., Nitrobacter sp.), and denitrifying bacteria were counted.

(Facultative heterotrophs are bacteria that can metabolize under both aerobic and anaerobic conditions.)

Figure from Masuda [24] redrawn and used with permission from Elsevier Science.

Probably nitrifying bacteria and other bacteria have worked out tight and mutually beneficial relationships in the biofilms of biological filters. As ordinary heterotrophs release ammonia during the decomposition of organic compounds, nitrifying bacteria can use the ammonia as its energy source. In turn, denitrifying bacteria, which consume acid, probably protect nitrifying bacteria, which are particularly sensitive to acidity.

Bacteria in biofilms have many advantages over those suspended freely in water. First, they share genetic information and metabolites. For example, in dental plaque biofilms, Veillonella bacteria use the lactate generated by Streptococcus bacteria [52]. Second, biofilm bacteria are protected from predators and destructive chemicals. In aquatic systems, biofilms protect bacteria from protozoa, various predatory algae (dinoflagellates) and predatory bacteria (Myxobacteria).

Q. New problem: Surface scum. The tank is now completely covered by a scummy film thick enough that oxygen bubbles from the plants are getting trapped under it. Water current is evident just below the surface, but the surface itself is held motionless. What is this film? What can I do about it?

A. The scum is an ecosystem of bacteria, algae, and protozoa. It is basically harmless, but it sounds like it’s gotten out of control in your tank.

You could temporarily increase surface agitation. Or you can quickly “mop up” the layer by laying pieces of newspaper on the water surface. I try to “scoop up” surface scum when I change water.

In human disease, biofilms protect bacteria from antibiotics, chemicals, antibodies, immune cells, etc. Thus, suspended cells of Pseudomonas aeruginosa were killed by 0.050 mg/ml of the antibiotic tobramycin, whereas 20 times more (1.0 mg/ml) could not kill this same bacterium when it was part of a biofilm [51]. When the nitrifier Nitrosomonas europaea was exposed to 5 µg/ml of the inhibitory chemical nitrapyrin, bacterial growth in biofilm cultures was unaffected, whereas bacteria growth in suspended cultures was reduced 82% [54]. The investigators used their results to explain why nitrapyrin has not been as effective in blocking nitrification for farmers as predicted by laboratory studies. Thus, while nitrapyrin might be a potent inhibitor of N. europaea growing as suspended cells in nutrient media in the laboratory, it would not work as well under field conditions where the bacteria would attach to soil particles and reside within a protective biofilm.

Q. Why did you write about biofilms? It doesn’t seem very relevant to aquarium hobbyists.

A. The subject of biofilms gives us an interesting glimpse into the natural world of bacteria. However, biofilms are relevant to hobbyists for two reasons.

First, biofilms explains why denitrification readily takes place alongside nitrification in ordinary aquarium filters and fish mulm (debris on the gravel). There is no need for hobbyists to buy ‘denitrators’ for denitrification.

The second reason is that biofilms prevent turbidity when ordinary garden soil is used in the aquarium. As bacteria within the soil spin their polysaccharide webs, they bind soil particles together. This binding of soil particles keeps even the smallest soil particles from entering and clouding the water (see page 135).

C.Bacteria Processes in the Aquarium

Bacteria affect nutrient cycling and the production (and destruction) of inhibitory compounds, such as ammonia, nitrites, acetic acid, and hydrogen sulfide. The fact that we cannot easily see bacteria should not discount their importance in aquariums.

Probably the most important bacterial process in the planted aquarium is simply the decomposition of organic matter. The gradual decomposition of organic matter by heterotrophic bacteria into plant nutrients is a natural and continuous process. It seems to work well in my aquariums. While CO₂ and other nutrients may be added artificially to obtain good plant growth, controlled decomposition by heterotrophic bacteria converts excess fishfood and debris into nutrients that plants can use. Without recycling by heterotrophic bacteria, organic matter would simply accumulate and be unavailable for plants.

In aquariums containing soil, the decomposition of the soil’s organic matter by bacteria provides plants with a generous supply of CO₂. Indeed, I calculated that an ‘average’ soil substrate provides plants with enough CO₂ for about 11 months (see page 83).

Table IV-4 lists the main effects that bacterial processes have in the planted aquarium.

Table IV-4. Effects of Bacterial Processes on Aquarium Ecosystems.

Bacterial Process	Where Found	Asset(s)	Drawback(s)
Nitrification	surface of filter, substrate, plants, etc.	detoxifies ammonia and nitrite	competes with plants for ammonium; may cause water to acidify and to accumulate nitrates
H2S oxidation	surface of substrate and plant roots	detoxifies H2S
Methane oxidation	surface of substrate	converts methane to CO2 that plants can use
Aerobic decomposition	surface of filter, substrate, plants, etc.	converts organic matter to plant nutrients
Anaerobic decomposition	substrate and filters	converts organic matter to plant nutrients and humus
Nitrate respiration*	substrate and filters		generates nitrites
Denitrification*	filter & substrate	removes nitrates from the tank
Manganese reduction*	anaerobic soil substrate	provides manganese for plants
Iron reduction*	anaerobic soil substrate	provides iron for plants, detoxifies H2S
Sulfate Reduction*	severely anaerobic substrate		produces toxic H2S
Fermentation*	severely anaerobic substrate	provides CO2 for plants	produces acetic acid and other inhibitory organic compounds
Methanogenisis*	severely anaerobic substrate	removes inhibitory acetic acid

*Processes that occur along with anaerobic decomposition by heterotrophic bacteria.

Bacteria and fish both use oxygen. During the aerobic decomposition of organic matter, bacteria consume one oxygen molecule (O₂) for every CO₂ molecule they release. Oxygen consumption can cause problems in deep ponds without water circulation and which contain large quantities of fresh organic matter (fallen leaves, freshly submerged soils, etc). Sudden and large influxes of highly labile (readily digestible) organic matter can cause acute problems. For example, a power outage may cause a canister filter’s internal media to become anaerobic. The bacteria and protozoa inside suffocate and die. When the power resumes, dead and decomposing microorganisms spew into the water and may kill the fish.

Q. How clean do you keep your planted tanks?

A. Tanks with good plant growth do not need much cleaning. I change 25 to 50% of the water about once every 3-6 months. If the water surface contains a thick scum, I remove it along with the water change. I don’t vacuum the gravel, but I do siphon off fish mulm that gets more than ¼” deep.

I clean the filters when the flow rate decreases. Usually, that’s every 1-2 months for the submerged cartridge filters I use. I wring and rinse out the filter material with tapwater and then re-insert the material.

Once every month or so, I remove excess plant growth. I snip off old leaves and remove excess moss and plants. I consider pruning to be vital to insure continuous plant growth. Plant growth that has stagnated from overcrowding will not purify the water for fish. Indeed, decaying plants may pollute the water.

I hand-remove any pockets of algae.

I use signs of labored fish breathing in the early morning, when oxygen levels are lowest, to gauge whether oxygen is sufficient for the tank. For providing fish with oxygen, I would use only the amount of aeration that is necessary. For excessive aeration can remove all CO₂ from the water and deprive the plants of a much needed nutrient. In the beginning, I had to reduce the number of fish in each aquarium so that oxygen would be adequate for the way I feed and maintain my tanks.8 Adjustment is rarely necessary now. There appears to be an innate stability to the aquariums whereby the oxygen needs of fish and heterotrophic bacteria are matched by oxygen inputs from plant photosynthesis and air/water mixing.

Q. I try to control nutrient levels in the tank by feeding my fish sparingly. I would like to feed them more, but I don’t want to pollute the water.

A. In tanks with good plant growth, you don’t have to choose between feeding your fish well and keeping the water pure for them.

All my fish get fed well twice a day. I consider excess fishfood and meat juices not taken up by the fish to be a rich source of nutrients for plants, thanks to decomposition by heterotrophic bacteria. Thus, when I feed my fish, I generally toss in a little extra for the plants. Invariably, these extra scraps of food are gone the next day. (While I cannot see the bacteria, I know that they are there.)

REFERENCES

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3.Mann KH. 1972. Macrophyte production and detritus food chains in coastal waters. Mem. Ist. Ital. Idrobiol., 29 (Sup.): 353-383.

4.Westermann P. 1993. Wetland and swamp microbiology. In: Ford TE (ed.). Aquatic Microbiology. An Ecological Approach, pp 205-238.

5.Wetzel 1983, p. 667-668.

6.Wetzel 1983, p. 743.

7.Watson EV. 1981. British Mosses and Liverworts (3^rd Ed). Cambridge University Press (Cambridge, England), p. 132.

8.Rheinheimer G. 1985. Aquatic Microbiology (3rd ed.). John Wiley (NY), p. 147.

9.Wetzel 1983, p. 610.

10.Cole JJ, Caraco NF, Kling GW, and Kratz TK. 1994. Carbon dioxide supersaturation in the surface waters of lakes. Science 265: 1568-1570.

11.Barko JW and Smart RM. 1983. Effects of organic matter additions to sediment on the growth of aquatic plants. J. Ecol. 71: 161-175.

12.Thurman EM. 1985. Organic Geochemistry of Natural Waters. Martinus Nijhoff (Boston).

13.Haslam E. 1989. Plant Polyphenols. Vegetable Tannins Revisited. Cambridge Univ Press (NY), p 191.

14.Graneli W, Lindell M, and Tranvik L. 1996. Photo-oxidative production of dissolved inorganic carbon in lakes of different humic content. Limnol. Oceanogr. 41: 698-706.

15.Gundersen DT, Bustaman S, Seim WK, and Curtis LR. 1994. pH, hardness, and humic acid influence aluminum toxicity to rainbow trout (Oncorhynchus mykiss) in weakly alkaline waters. Can. J. Fish. Aquat. Sci. 51: 1345-1355.

16.Graneli W, Lindell M, and Tranvik L. 1996. Photo-oxidative production of dissolved inorganic carbon in lakes of different humic content. Limnol. Oceanogr. 41: 698-706.

17.Spotte S. 1979. Fish and Invertebrate Culture. Wiley-Interscience Publications (NY), p. 117.

18.Hamlin HJ. 2006. Nitrate toxicity in Siberian sturgeon (Acipenser baeri). Aquaculture 253: 688-693.

19.Hovanec TA, Taylor LT, Blakis A, and DeLong EF. 1998. Nitrospira-like bacteria associated with nitrite oxidation in freshwater aquaria. Appl. Environ. Microbiol. 64: 258-264.

20.Burrell PC, Phalen CM and Hovanec TA. 2001. Identification of bacteria responsible for ammonia oxidation in freshwater aquariua. Appl. Environ. Microbiol. 67: 5791-5800.

21.Stephen JR et al. 1998. Analysis of β-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing. Appl. Environ. Microbiol. 64: 2958-2965.

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24.Masuda S, Watanabe Y, and Ishiguro M. 1991. Biofilm properties and simultaneous nitrification and denitrification in aerobic rotating biological contactors. Water Sci. Technol. 23: 1355-1363.

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26.Payne WJ. 1973. Reduction of nitrogenous oxides by microorganisms. Bacteriol. Rev. 37: 409-452.

27.Wetzel 1983, p. 237.

28.Gamble TN, Betlach MR, and Tiedje JM. 1977. Numerically dominant denitrifying bacteria from world soils. Appl. Environ. Microbiol. 33: 929-939.

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30.Boyd, CE. 1995. Bottom Soils, Sediment, and Pond Aquaculture. Chapman & Hall (NY), p. 141.

31.Edmond JM, Stallard RF, Craig H, Craig V, Weiss RF, and Coulter GW. 1993. Nutrient chemistry of the water column of Lake Tanganyika. Limnol. Oceanogr. 38: 725-738.

32.Seitzinger SP, Nixon SW, and Pilson MEQ. 1984. Denitrification and nitrous oxide production in a coastal marine ecosystem. Limnol. Oceanogr. 29: 73-83.

33.Reddy KR. 1983. Fate of nitrogen and phosphorus in a waste-water retention reservoir containing aquatic macrophytes. J. Environ. Qual. 12: 137-141.

34.Walstad D. 2001. Nitrates in the planted aquarium. The Aquatic Gardener 14(1).

35.Sorensen J. 1978. Capacity for denitrification and reduction of nitrate to ammonia in a coastal marine sediment. Appl. Environ. Microbiol. 35: 301-305.

36.Gilbert F, Souchu P, Bianchi M, and Bonin P. 1997. Influence of shellfish farming activities on nitrification, nitrate reduction to ammonium and denitrification at the water-sediment interface of the Thau lagoon, France. Mar. Ecol. Prog. Ser. 151: 143-153.

37.Jones JG and Simon BM. 1981. Differences in microbial decomposition processes in profundal and littoral lake sediments, with particular reference to the nitrogen cycle. J Gen Microbiol 123: 297-312.

38.Smith MS. 1982. Dissimilatory reduction of NO_2^- to NH₄⁺ and N₂O by a soil Citrobacter sp. Appl. Environ. Microbiol. 43: 854-860.

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1Oxygen provides much more energy than other electron acceptors. For example, aerobic bacteria gain 26.5 kcal/mole of energy using oxygen as compared to the 18 and 3.4 kcal/mole that anaerobic bacteria gain when using nitrates and sulfates (respectively) [26].

²While some DOC is not easily digested by bacteria, it is quite susceptible to decomposition by light (i.e., photo-oxidation). Thus, DOC photo-oxidation in several unpolluted Swedish lakes released 0.086 to 0.41 mg of C/l/day as compared to 0.1 to 0.27 mg of C/l/day for bacterial metabolism [16]. Metals like iron, manganese and copper act as catalysts for DOC photo-oxidation (see page 167).

³Methane was also released from the two experimental sediments, 310 µg/g/day for the sediment spiked with aquatic plant matter, and 15 µg/g/day for the sediment spiked with dead oak tree leaves.

⁴Spotte [17] reports that NO_3^-N levels even as high as 400 mg/l did not affect the growth or mortality of largemouth bass (Micropterus salmoides) and channel catfish (Ictalurus punctatos). Nitrate was only lethal to the Siberian sturgeon (Acipenser baeri) at 400 to 1,000 mg/l (3 day LC50s depending on the size of tested fish) [18].

⁵Investigators recently studied the nitrifying bacteria of several freshwater aquarium filters using highly sensitive genetic probes (e.g., PCR amplification of ribosomal DNA) to detect bacterial genetic material. (Previous studies that required culturing the bacteria beforehand probably missed the species that could not grow under laboratory conditions.) Nitrite oxidation, which most investigators had previously attributed to Nitrobacter species, appeared to be mediated instead by two unknown species related to Nitrospira moscoviensis and Nitrospira marina [19]. Ammonia oxidation, appeared to be carried out by an unknown species related to Nitrosomonas marina [20]. Fluorescent staining of genetic material clearly shows these Nitrospira and Nitrosomonas species growing side by side each other in an aquarium filter [20]. The investigators [19] explain that “starter cultures” of nitrifying bacteria sold to aquarium hobbyists are usually ineffectual, because they contain the wrong species.

⁶For example, when a cultivated soil was analyzed with sophisticated genetic probes (i.e., PCR amplification of ribosomal DNA) for ammonia-oxidizing bacteria, several strains of Nitrosomonas and Nitrosospira were detected [21].

⁷DAP differs from ‘assimilatory nitrate reduction’ whereby bacteria convert nitrates to ammonium, which can then be assimilated (incorporated) into amino acids and proteins [35]. Bacteria use this pathway when ammonium is not available.

⁸I generally keep my aquariums lightly or moderately stocked with fish. For several years, I kept six Tropheus duboisi (about 4” in length) in my 45 gal tank. However, I had no problems raising hundreds of juvenile Tanganyikans (See Color Plate 3) in a 20 gal tank with fluorishing plant growth.