CO₂ in planted aquaria

CO2 – the most important plant nutrient
CO2 is beyond comparison the most important of all plant nutrients because of its role in photosynthesis eventually leading to formation of new leaves and roots. Photosynthesis is a process that is only mastered by photoautotrophs, i.e. organism that can live with light as the sole source of energy. In photosynthesis, carbondioxide (CO2) and water (H2O) are converted into energy rich sugar (C6H12O6) and oxygen (O2) by means of light energy.

It is evident from the photosynthesis equation, that only CO2, water and light energy are needed to fuel the photosynthesis process. Hence it follows that if one of the three main ingredients are missing, photosynthesis will not take place. This seems odd, because we all know of people who are perfectly able to maintain a beautiful planted aquarium without artificial supply of CO2. Consequently, CO2 must be naturally present in the water or else, this would be impossible. In biological systems, CO2 comes from respiration. You could say that respiration is the opposite process of photosynthesis. In respiration, energy is released when sugars are converted into CO2 and water. Aquatic plants also respire and they do so 24 hours a day. However, while illuminated, most aquatic plants are producing much more organic carbon in photosynthesis than they are burning in respiration. At nighttime, however, there is no photosynthesis because there is no light and thus, respiration dominates and CO2 is produced by both plants, invertebrates, fish and microorganisms.

Box 1 When CO2 dissolves in water, it forms an equilibrium between carbonic acid (H2CO3), bicarbonate (HCO3-) and carbonate (CO32-) according to eq. 1: Equation 1               H2O + CO2 ↔* H2CO3 ↔ H+ + HCO3- ↔ H+ + CO32- It follows from general chemical principles that if CO2 is utilized in photosynthesis by the plants, pH increases because protons are removed from the solution. Protons are removed because the equilibrium tends to flow towards the left when CO2 is replenished from bicarbonate (HCO3-) and carbonate (CO32-). The * denotes that this particular process is often catalyzed by carbonic anhydrase, an enzyme which has evolved in plants as well as in animals several times during the evolution of life on earth. On the other hand, during nighttime when respiration dominates, pH will decrease because more protons are formed, when CO2 is constantly added to the left side of the equilibrium.   CO2 is a function of pH. At low pH, most of the inorganic carbon is present as CO2. At neutral pH, most is present as bicarbonate and at high pH, the equilibrium is shifting towards carbonate. The sum of bicarbonate and carbonate is called the carbonate hardness and is measured in degrees (dKH). A better and more correct term is the carbonate alkalinity and this is measured in milli equivalents per liter (meq/L). Milli equivalents refer to how many milli equivalents of acid that is needed to titrate bicarbonate and carbonate that both act as weak bases. Once CO2 has diffused into the cells and further into the chloroplasts, where photosynthesis takes place, CO2 is converted from inorganic carbon (carbondioxide, CO2) to organic carbon (sugar, C6H12O6) in the photosynthesis according to eq. 2: Equation 2               6CO2 + 12H2O → 6C6H12O6 + 6 O2 Eq. 2 is very simplified and in reality, it contains several chemical cycles. To focus on each one of them, is beyond the scope of this article. It is important to note, however, that the process must be fuel by light energy and thus, it follows that photosynthesis only takes place during the day. The first step in photosynthesis is the trapping CO2, where the carboxylating enzyme, RuBisCO, catalyzes the first step in a long array of biochemical processes. Like all other enzymes, RuBisCO is made from protein and thus, it has a high content of organic nitrogen. This is the reason why we observe the strong interactions between CO2, nutrients and light as described elsewhere in the text.

The water chemistry of CO2
CO2 is easily dissolved in water and the solubility is high. The solubility is almost 1:1 meaning that 1 L of water can contain almost the same amount of CO2 as 1 L of air when in equilibrium. When CO2 dissolves in water, it forms an equilibrium between carbonic acid (H2CO3), bicarbonate (HCO3-) and carbonate (CO32-), see Box 1. The balance between carbondioxide, bicarbonate and carbonate is strongly dependant on pH, i.e. at low pH, carbondioxide dominates and virtually no bicarbonate and carbonate are present, whereas at neutral pH bicarbonate dominates over the two other carbon species. Only at high pH, there is a dominance of carbonate. We can take advantage of this fact and manipulate pH to a level that suits us and thereby obtain a desirable concentration of CO2 in our planted aquaria.

Uptake of carbondioxide in aquatic plants
All aquatic plants can take up CO2 directly from the water. When terrestrial plants take up CO2 from the surrounding air, they do so via their stomata. True aquatic plants do not form stomata and their cuticles are also reduced compared to their terrestrial relatives. Consequently, when aquatic plants take up CO2 from the surrounding water, they do so by passive diffusion of CO2 from the water over the reduced cuticle and into the photosynthetic cells. In aquatic plants, even the epidermis cells contain chloroplast in order to reduce the distance from the CO2 source to the sink.

In water, the uptake of CO2 is limited by slow diffusion. Diffusion of gasses in water is almost 10,000 fold slower than in air. We can partly compensate for that by raising the CO2 concentration in the planted aquarium. In most cases, however, we only raise the concentration 100 fold compared to air equilibrium meaning that aquatic plants can still be limited by slow gaseous diffusion in our aquaria. There are alternative photosynthetic pathways and alternative sources of CO2 including the use of bicarbonate and these are all described in Box 2.

Interactions with other plant nutrients and light
High supply of CO2 can help the plant to conserve other essential nutrients, and if CO2 is plentiful, aquatic plants can grow even with less light. This phenomenon has been discussed by us in TAG 2001, where we used submerged Riccia fluitans as a study plants. In brief, our study showed that elevated CO2 in planted aquaria could maintain the same plant growth but at lower light and nitrogen supply. We concluded that it is often easier to raise the CO2 level in the tank than to increase the light and thus, we recommend aiming for the higher end of the CO2 intervals in Box 3 in particular if the aquarium is not already well illuminated.

Another aspect of the interactions between CO2 and other nutrients is that the nutrient level can be lowered without loosing the benefits of CO2 fertilization. High CO2 levels in the planted aquarium allow the plants to use less nitrogen for Rubisco, which is the most common enzyme in plants. Rubisco is an enzyme that catalyzes the first step in the Calvin cycle where CO2 is added to ribulose 1,5 bisphosphate. All enzymes are made from proteins and proteins are very rich in nitrogen. Thus, if less enzyme is needed because the CO2 concentration is high, the proteins can be used in other processes in the plants leading to formation of new biomass.

CO2 fertilization in the planted aquarium
If you have an air pump, switch it off! If you have two air pumps, switch both of them off! It cannot be stated too often that an air pump should never be part of a planted aquarium. The function of an air pump is to supply oxygen (O2) to fish and invertebrates in aquaria that have no sustainable oxygen production from aquatic plants. In all planted aquaria, there should be more than sufficient oxygen for both fish and invertebrates also at nighttime when there is no photosynthesis. When plants, fish and invertebrates respire during the night, CO2 is produced and dissolves readily in the water. This CO2 can then be used in photosynthesis by the plants once the light is switched on the following day. If an air pump is running, the CO2 is degassed to the air in the same way carbonic acid is lost from a soda or a beer when shaken. Thus, offer your air pump a well deserved retirement!

In order to routinely follow the CO2 concentration in the aquarium we recommend using a continuous CO2 test. It is an ingenious little device that is placed inside the tank visible from the outside. It works with a chemical color indicator (brom-thymol-blue), which should always be green if the CO2 level is within the recommended range. In fact the device does not measure CO2 but pH and thus, it can only be taken as an indication that the CO2 level is just about right. The dKH vs. pH table elsewhere in this article shows how much the CO2 level is changing at a given pH as a function of carbonate hardness. The continuous CO2 test is just an easy way to monitor the CO2 level (pH) and not a CO2 fertilizer in itself.

A yeast reactor is perhaps the cheapest alternative if CO2 fertilization is required to boost the CO2 level in the planted aquarium. The basic principle of a CO2 reactor is based on yeast cells that in the absence of oxygen ferment sugars or starch into CO2. The CO2 gas is then supplied to the water by means of a bubble stone, a mister or a CO2 reactor. The waste product from the fermentation process is alcohol of some sort (there could be methanol in it too, so don’t drink it). There are numerous designs of well functioning yeast reactors to be found on the web but yeast reactors are also manufactured and sold in aquarium shops. A yeast reactor is certainly better than nothing but there is one major disadvantaged with the yeast reactor and that is that it cannot be easily controlled. Sometimes the yeast cells are happy and ferment a lot of sugar, which leads to huge amounts of CO2 being dissolved in the water. Other times, the yeast cells are less active and too little CO2 is supplied to the tank. Some people feel that these CO2 fluctuations that lead to similarly high pH fluctuations have adverse effects on invertebrates, fish and even plants. It may be so in some cases but both lakes and streams in nature may experience huge diel fluctuations in CO2 and pH. For example, lowland streams in Denmark may have as much as 20 mg CO2 per L in the morning and only 5 mg/L in the late afternoon. The natural density of aquatic plants is so high that they can extract all this CO2 during the daytime although it is constantly supplied from the CO2 rich groundwater. Plants, invertebrate and fish all live happily with these dramatic changes in CO2 over the day. However, some invertebrates and fish may be more sensitive to such pH changes so one should always check their sensitivity to pH fluctuations in the literature before installing a yeast reactor.

Box 2 Some aquatic plants can use bicarbonate (HCO3-) if CO2 is scarce. In water with a reasonable carbonate alkalinity, bicarbonate (HCO3-) is present at large amount at pH 7 to 10 (see Box 1). On the other hand, gaseous CO2 is scarce when pH is above 8 regardless of the carbonate alkalinity and thus, aquatic plants that are able to utilize bicarbonate as a source of inorganic carbon have a great competitive advantage over strict CO2 user. The uptake of bicarbonate by aquatic plants is a science on its own but basically only two different models may explain how it works in most bicarbonate users. One model, first proposed by Prins and Elzenga (1989), deals with plants that have polarized leaves when bicarbonate is utilized. In these plants, protons are pumped out on the lower side of the leaves (abaxial side) resulting in very low pH down to 4. Here, bicarbonate is converted into CO2 that subsequently diffuses into the leaves where it is fixed in photosynthesis. Negative loadings in the form of hydroxyl ions are pumped out on the upper leaf surface (adaxial side) where pH rises to above 10. Sometimes the high pH leads to precipitation of calcium carbonate on the leaf surfaces, giving these plants a whitish look. Good examples of these bicarbonate users are Elodea canadensis, Egeria densa and most species of pondweeds. At intensive photosynthesis, pH is rising on the leaf surfaces and in some case it may lead to the precipitation of calcium carbonate. Here, biogenic calcium carbonate has formed on the leaves of Anubias. Other bicarbonate using plants do not form polarized leaves, for example Vallisneria species, and bicarbonate is taken up by the leaves by ion pumps and converted into CO2 inside the leaves. Regardless of the model in use, bicarbonate uptake is an energy consuming process and thus, even good bicarbonate users do not produce the necessary enzymes unless needed. Thus, in environments with high CO2, these bicarbonate users cannot use bicarbonate without going though a period of time with low CO2 during which the necessary enzymes are produced. One of the most important enzymes in bicarbonate using plants is the carbonic anhydrase that catalyzed the slow formation of carbonic acid from water and CO2, or vice versa, which is a critical step when going from bicarbonate to CO2 (see Box 1). There are a few other tricks that aquatic plants can play in order to compensate for the slow diffusion of CO2 in water. One is the use of C4 photosynthesis, which is a very common type of photosynthesis in terrestrial plants with corn as the most well known example. In C4 plants, the oxygen evolving processes are spatially separated from the CO2 fixing processes. Such plants can photosynthesize at lower CO2 concentrations because oxygen is kept away from Rubisco. If too much oxygen is present around Rubisco it becomes very inefficient because Rubisco turns into an oxygenase resulting in photorespiration and loss of organic carbon. C4 photosynthesis has only been described for one aquatic plant (Hydrilla verticillata) and here it seems to work without the Kranz anatomy which is always characteristic of terrestrial C4 plants. Another strategy that may alleviate the slow diffusion of CO2 is the dark fixation of respiratory CO2 in CAM plants. Here, CO2 is trapped in malate during darkness and then subsequently released as CO2 during light when photosynthesis needs the substrate. Finally, some aquatic plants have specialized in using sediment derived CO2 in their photosynthesis. Here, CO2 diffuses from the sediment, where CO2 always is present at high concentrations, into the roots and via the aerenchyma further up to the leaves where it is fixed in photosynthesis. Previously, it was thought that this would only be of significant importance in aquatic isoetids (Lobelia dortmanna, Littorella uniflora and species of Isoetes) but recent research by Anders Winkel from the Freshwater Biological Laboratory has shown that sediment derived CO2 is also important for photosynthesis in Vallisneria americana. This model shows how bicarbonate uptake works in polarized leaves. Protons are pumped out of the leaves and acidify the abaxial leaf surfaces where bicarbonate is converted into CO2. The adxial sides are strongly basic often leading to precipitation of calcium carbonate.

Various lime tablets that dissolve and release CO2 when added to the aquarium may also be used as CO2 fertilizer. We have no personal experiences using these products and you can find all kinds of observations on the web reporting either positive or no effects using calcium carbonate tablets in planted aquaria. A few years back, Carbo Plus was marketed. Carbo Plus produces CO2 electrolytically from solid carbon and it works reasonably well in well buffered aquaria (8-12 dKH carbonate hardness).

Compressed CO2 is the best alternative to a yeast reactor. When compressed and stored in a tank, CO2 is liquid and the pressure is approximately 58 bars. CO2 can be bought on various gas cylinders that are not necessarily custom built for CO2 fertilization because CO2 is also used for soda (fx. SodaStream), welding or as a “propelling system” for tap beer. In principle, all these CO2 types can be used but in practice, we are limited by the threading of our pressure reducer. In its most simple form, a CO2 fertilization system using compressed CO2 consists of a CO2 cylinder, a pressure reducer with manometer and low pressure regulator connected to a bubble stone, a mister or a CO2 reactor. It is a recurrent discussion whether or not to switch off the CO2 supply at nighttime. As explained above, many plants, invertebrates and fish are used to diel fluctuations in CO2. So, switching CO2 off during the night is mainly a matter of avoiding wasting CO2 when the plants cannot use it in photosynthesis.

A somewhat more advanced system may include a solenoid valve that can switch off the supply during nighttime by using a timer. Very advanced systems also include a pH electrode and a pH meter that can control the solenoid valve. In this way, CO2 can be automatically controlled and switched on and off according to a set point. When photosynthesis dominates over respiration, CO2 is consumed and pH rises. When pH rises above the set point, the pH meter opens the solenoid valve and CO2 is supplied to the water. When sufficient CO2 is supplied, pH drops and the pH meter switches off the CO2 supply via the solenoid valve. Using such a system, conserves CO2 and keeps pH very stable. However, the pH electrode must be regularly calibrated in order to avoid electrode drift that results in CO2 concentrations far from the desired levels.

In planted nano aquaria, sodawater (obviously without lemon and sugar) has been used to boost CO2 in the water. It is not easy to administer and it takes a little experience to get the quantum right. We have seen a few examples where fish were hanging gasping in the surface following too much sodawater. Also, some plants (fx. Cryptocorynes) may be sensitive to dramatic pH changes, so sodawater as a CO2 fertilizer should be used with caution.

Recently, various organic carbon fertilizer compounds have been marketed. We have tested two of the common products on Hygrophila corymbosa “Siamensis” (a widespread and popular aquarium plant only using CO2) and Egeria densa (another common aquarium plant able to use bicarbonate) and we found neither positive nor negative effects on the photosynthesis measured as oxygen evolution. Nevertheless, some people have reported positive observations on plant growth when using such organic carbon fertilizers and a more detailed study is probably required in order to sort out pros and cons in these products.

Literature
Bowes G (1987) Aquatic plant photosynthesis: strategies that enhance carbon gain. In RMM Crawford (ed) Plant life in amphibious habitats, pp 99-112
Madsen TV & Sand-Jensen K (1991) Photosynthetic carbon assimilation in aquatic macrophytes. Aquatic Botany 41: 5-40
Prins HBA & Elzenga JTM (1989) Bicarbonate utilization: function and mechanism. Aquatic Botany 34: 59-83