## Thursday, 18 August 2016

### Article - CO2 Q&A - The science behind

 Fig 1.: CO2 diffuser.
During my time on internet, I have noticed many questions about the use of CO2 and its interactions inside the ecosystem of the aquarium. They are not only related to its role into the photosynthesis and how to dose it, but also about some of its chemical and physical properties, or effect in livestock. As usual, many of the answers I have read are quite misleading and lacking of scientific basis.

One of the goals of The Living Tank is to provide solid and scientific-based information, and make it accessible to all the hobbyist of the Aquascaping and Planted Aquarium. Because of that, and considering the confusion about this nutrient into the aquarium, I have decided to prepare a "not so short" article about it.

The article is long, and it has a few sections. If you are not interested in all the aspects, then I recommend to move towards the points of your interest. This is intended to be a reference, and not a text requiring full reading, even if I think is rather interesting. I have split the text in a few parts:

1. Physical and Chemical Properties of CO2: A summary of the "inorganic" role of CO2 into aquariums. it includes important concepts to understand other sections., like the carbonates equilibrium.
2. CO2 and plants: Usage of CO2 by the plants and environmental factors affecting to the way in which plants make use of it.
3. CO2 and fishes/shrimps: Effects of environmental CO2 into the fishes and shrimps, justified from a physiological point of view.
4. Questions & Answers -The CO2 Quiz: Common questions and their answers, as interpreted from the previous text.
I hope you will enjoy the articles as much as I did writing it. Feel free to share it or make me any questions about it.

## Physical and Chemical Properties of CO2

The inorganic properties of the CO2 are important to understand. They explain how its concentration evolves into the tank and what kind of interaction has with other chemical components dissolved in water. This section is very helpful to answer many questions like which the role of carbonates hardness into the CO2 dissolution is, or about what happens when increasing/reducing the temperature of the tank.

### Basic properties

 Fig.2: Representation of CO2 molecule.
The CO2, or carbon dioxide is a gas under normal conditions (25°C, 1 atm of pressure). It has a melting temperature of -78°C and a boiling temperature of -57°C. Over such temperature is always a gas, so for the common range of daily conditions, all the Physics associated to this molecule are related to such state of the matter.

It has a molecular mass of 44,01 gr/mol, which means is a quite light and small molecule. This is not surprising as it is composed by only three atoms, 1 atom of carbon (centre of Fig. 2) and 2 of oxygen (red sides of Fig. 2), all of them light and small. For those not knowing it, the molecular mass is expressed as the total mass (or weight for simplicity) accumulated by a mol of such atoms or molecules. A mol is just a way to count things, and it is represented by the Avogadro's number, equal to 6,022 x 10^23 entities. This represents that such number of CO2 molecules weight up to 44.01 grams. The fact that is small is important because large molecules are much difficult of being used by plants, for example.

CO2 is soluble in water, with a characteristic solubility of 1.45 gr/l (1450 ppm). I revisit this value below, as it is dependent of different parameters, like temperature,  one of the topics of discussion I have seen in forums and social networks.

### Solubility in water

As previously indicated, the solubility of CO2 in water is of about 1.45 gr/l under normal conditions (25°C, 1 atm of pressure). However, solubility of gases is strongly dependent of the temperature of the medium in which they are dissolved, as well as dependent of other factors associated to the ions already presented into the solution.

Moreover, many people also confuse solubility with saturation, as well as the role of the water dynamics with the fugacity or degasification process. I  try to clarify all these points in this article.

#### Saturation

An important point for those people using CO2 injection is the point of saturation. Each substance being dissolved in water has one, and refers to the maximum concentration of it that can be mixed with the water molecules. Beyond such a point, no further dissolution is produced. With solids the idea is very easy to understand: If you have a glass of water and you add salt, stirring it with a spoon, the saturation point is reached when does not matter how long you stir the water with the spoon that no more salt is dissolved. I will go back a few times around this example, as it has keys for many of the concepts.

In gases, the idea is the same: there is a physical limit for a given gas being dissolved into the water. That limit is dependent of temperature (mentioned above and explained below) but also from other factors also indicated in this article.

Meanwhile the concept is easy, there are many details that are not so obvious. In our example, there is a reason why we need the spoon to stir the water. If you think about it, when you add the salt to the water, this goes to the bottom and most of it remains there, still visible. It is only when we stir that most or all of the salt dissapears. Reality is that part of the salt has really dissolved without stirring. And, in fact, even if you do not stir, leaving it alone, all the salt will eventually dissolve (if no saturation point has been reached).

What is then the difference between stirring and not? The speed of the process, which is dependent of the dynamics of the water. When we do not stir, salt dissolves very quickly only in the surrounding water. This surrounding water or boundary layer will be significantly thin. Probably just a few milliliters away from the salt grains. This thin layer becomes saturated very fast, so now salt is just in contact with water already saturated, i.e. no more dissolution happens in such moment. However, there is a diffusion process (depending on the gradient or differences of concentration) that promotes the mix of the saturated water with the non-saturated surrounding water (see Fig. 3 for an example of this). This phenomena is explained by the Fick's law, which has two components: a molecular one or Brownian movement, which explains the diffusion originated just due to the molecular collisions between particles, and other term related to the turbulence of the medium (i.e. agitation), in its extended version for fluid dynamics. With no agitation (so no stirring), the molecular process is dominant, which is rather small and slow, as well as strongly dependent of the local concentrations. In other words, with no agitation, a structure of concentric layers with progressive less salt concentration forms around the salt grains, and then diffusion in water is very slow. But still happens. On the other hand, when we stir, we are introducing lot of turbulence. This breaks this structure in layers and allows to the grain salts to be exposed to water with lower concentrations of salt, then speeding up the dissolution process. Going back to the Fick's law, turbulent term is considerably much higher than molecular one, so when stirring, we significantly accelerate the dissolution process in a few orders of magnitude.

 Fig. 3 : Example of molecular diffusion.
There is also a second important concept involved in the speed of the dissolution. As indicated above, this speed is related to how close or far away we are of the saturation point. In plain words, the closest to the saturation point, the slowest the dissolution process will be. For most cases, there is no noticeable effect of this unless you get quite close to such a point, or if you do not have enough agitation. Hence, speed can be improved by increasing agitation, as explained above. This is easy to check in the glass of water and salt example I have just done, and trying different stirring speeds and salt additions.

This is important to understand when talking about CO2 injection into tanks. The learning of the saturation is that we can reach right values locally but not globally, if the agitation in the tank is not good enough. The lack of mixing in the water of the tank promotes stratification (formation of layers) which interferes into establishing the right CO2 concentrations in the tank.

#### Solubility and Temperature

As with any other gas, temperature is a key factor to determine the amount of CO2 can be dissolved into water. This is important because it means that the temperature of our aquariums has, a priori, a say in the way in which we should handle CO2, if we purely base our answer in the physical properties.

The solubility of ideal gases into water is physically explained by the Henry's law. This law establishes that a gas will dissolve into water is proportional to a solubility constant (or Henry's constant) and the partial pressure of that gas. In other words, the highest the concentration of the gas into the air, the higher will be the total amount of gas dissolved into the water. In the same way, the higher the value of the Henry's constant associated to such a gas, the higher the resulting concentration into water will be. Typically, each gas has its own Henry's constant. There are a few different approaches to this law, but the interpretation does not change.

The dependency of the temperature appears not in the Henry's equation but rather in the formulation of the constant. Even if it sounds a bit contradictory, many physical and chemical constants are just fix for a series of conditions. If such conditions change, the value of the constant will change. This is the case of Henry's constants, which are temperature-dependent. These dependency is expressed by the Van't Hoff equation, which indicates that the Henry's constant for a given gas will be exponentially reduced as the temperature increases respect to a temperature of reference. In simpler words, the more we increment the temperature, the larger will be the reduction of solubility. This inverse link is not fix, which means that the reduction of solubility associated to 1 degree of temperature increment varies depending on the range of temperatures in which the change happens. I have mentioned a temperature of reference, which in our case refers to 25°C.

This behavior means that when the temperature is reduced from such value of reference, the solubility of CO2 will increase respect to the value provided above. On the other hand, when over passing it, the solubility will be minor. But now the question is... how much less will the CO2 dissolve into water when increasing the temperature? Well, this is expressed in the following Fig. 4:

 Fig. 4: Change in solubility of CO2 into water depending on temperature.

The horizontal axis includes the temperature (in Celsius degrees), meanwhile the vertical axis shows the solubility, expressed in grams of CO2 in 100 grams of water (i.e. the solubility in gr/l is equal to the values multiplied by 10, and in ppms multiplying by 10000). As you can see, at 25°C, the solubility is of 0.145 which corresponds to 1450 ppm as said above. However, when we reduce the temperature, the solubility increases. For example, at 20°C reaches a value of 1700 ppm. Oppositely, when we increment the temperature, the solubility reduces. For example, at 30°C, the maximum concentration of CO2 in water we can reach goes up to 1250 ppm, approximately.

It is clear that the change in solubility is significant in function of the temperature. Note that, however, that the provided values and what is shown in the Fig. 1 are related to saturation values, so they are maximum values of concentrations. Whenever the dissolved CO2 is below such values, no negative effect will appear from the temperature, apart from a change in the speed that the dissolution process happens, which will be faster as larger is the gradient (difference of concentrations) between air and water, as also reflected in the Henry's law under the partial pressure term, as explained also above.

#### Solubility and (bi)carbonates

The properties explained above are extracted by considering that CO2 is an ideal gas (i.e. no interactions further the physical ones). However, CO2 is far beyond behaving like an ideal gas. In this case, CO2 reacts with water, producing H2CO3 (carbonic acid) that frees a proton reducing the pH and becoming into bicarbonate (HCO3-), which is part of a chemical equilibrium related to carbonates. As a result, part of the dissolved CO2 becomes into a different chemical form, a non-gaseous one, which allows further dissolution of CO2 is gas form.

 $CO_{2}+H_{2}O\rightleftharpoons HCO_{3}^{-}+H^{+}$ Eq. 1: Dissolution of CO2 into water and resulting generation of protons

The process described in Eq. 1 means that dissolved CO2 is gas form and in bicarbonate form are related, and the proportion between them depends on pH.

This kind of reactions change the value of the Henry's constant, which also becomes dependent of the concentration of related chemical components. In the case of the CO2, the higher the (bi)carbonates concentration (for a given pH), the larger the amount of additional CO2 in gas form must be present to preserve the equilibrium. Note that this does not alter the physical limit imposed by a specific gas before reaching saturation; it just means that, at high pHs, more of such gas will transform in something else, so additional injection is required before observing the dissolved concentrations of the gas being incremented. On the other hand, at low  (bi)carbonates concentration, small injections of CO2 can translate into a significant reduction of pH.

The effective constant is then computed by considering in addition to the dissolved gas concentration the other chemical species associated to it. This basically implies that the prior presence of this secondary species in water will effectively affect to the concentration of the gas in dissolved form. In the case of the CO2 the problem is even harder, as the concentration of the resulting species are involved in a three component equilibrium which also strongly depends on pH. In order to simplify the question, I will just interpret the following diagram (Fig. 5), which includes the effect of pH into the carbonate equilibrium:

 Fig. 5: Carbonates equilibrium as a function of pH

The figure shows precisely this pH dependency between CO2 in solution and the pH. Note that the plot refers to proportions between the chemicals, and not actual absolute concentrations. A good point to consider in the discussion is that most of the CO2 remains as gas dissolved into water at low pH, and just a small amount becomes into carbonic acid. This is important because, as we will see later, plants prefer to use CO2 as carbon source. In pure water, the addition of CO2 then leads to a fraction of it being carbonic acid, which decomposes into bicarbonates and reduces the pH. As pure water has a pH of around 7.1, when CO2 injection starts, part of it becomes into bicarbonate (see Fig. 5). However, when injecting CO2, the decomposition into bicarbonate reduces the pH (see Eq. 1), so eventually (with pH below 4.5), all the CO2 being dissolved remains as dissolved gas instead of migrating to other chemical form. In aquariums with CO2 being injected, the pH reduction also happens but can be buffered due to pre-existing (bi)carbonates concentration.

In this direction, the injection of CO2 in pure water is closely following Henry's law, so the effective Henry's constant is quite close to the physical one when reaching a steady situation at pH below 4.5. However, this is not true when we start from water with a some carbonate hardness. The carbonate hardness is a measure of the amount of carbonates and bicarbonates dissolved into water. When we inject CO2 into water with a certain carbonate hardness, then things are different due to the buffer effect of them. This buffer effect means that the solution is able to absorb protons originated by the CO2 dissolution in water, and hence, preserving the pH from varying. Essentially, the buffer effect favors the formation of bicarbonate (HCO3) due to the absorption of protons by the carbonates. By doing so, the equilibrium is displaced to the left, and the concentration of bicarbonate ions increases.

The process consumes part of the carbonates, increasing bicarbonates and also preserving the pH. The dissolution of CO2 does increase also the bicarbonates by disassociation of the carbonic acid (H2CO3) into HCO3- and protons. Those are the protons that added to the carbonates (CO3(2-)) generate further bicarbonate. Bicarbonate is highly soluble  (in the range of 100000 ppm for saturation levels) in comparison to carbonates or CO2, so there is lot of room to this process happening. This phenomena implies that pH does not change as far as there are enough carbonates ions able to take the protons originated during the dissolution of CO2. However, the availability of carbonates is rather small at normal range of pH in aquariums, especially if there are other factors altering the pH (like ammonium, nitrites or nitrates). In Fig. 5, it is possible to see that, at most common pHs of aquariums, level of carbonates in solution is rather small in favor of bicarbonates. When the concentration of dissolved carbonates drops to zero, the excess of protons starts to be absorbed by bicarbonate ions, generating H2CO3. As we said above H2CO3 is not much stable in water, and the increasing concentration will propitiate the decomposition into CO2, due to the fact that dissociation into H2CO3 is inhibited by the pH. In the plot you can see this effect happening when pH is below 8.2. From that point bicarbonates are being consumed to generate more CO2, and hence, the CO2 concentration does not increase only due to the injection but also due to this chemical reaction.

One important conclusion at this point is that, CO2 injection in planted tanks, with a common target of pH below 8, it diminishes the hardness of the water within the time. This effect is desired, as prevent the pH dropping too much during the CO2 injection, but it involves the hobbyist should have always an eye into the water hardness, especially if working with soft waters, in order to prevent a crash of pH which could kill all the fishes and inverts in the tank. Slightly carbonated rocks are then desirable for planted tanks in order to preserve a good buffering effect.

Back to the topic, from the last two paragraphs, the message is that CO2 dissolution into water is dependent of the pH of the dissolution and carbonate hardness. Above pH = 8.2, more CO2 needs to be injected in order to see the dissolved CO2 in gas form increasing. Below that pH, less CO2 injection is required to increase the dissolved CO2 in gas form. This idea is one to take into account, as it means that more efficient use of the gas can be achieved in soft waters respect to hard waters. However, as mentioned, too low levels of (bi)carbonates means a lack of buffer effect, so CO2 injection can cause significant and dangerous drops of pH in short periods of time.

During this part of the article, I have been talking about dissolved CO2 in gaseous form. This is not casual, as it makes a difference for the plants. The next part of the article is devoted to explaining that part.

## CO2 and Plants

As many readers will already know, CO2 is one of the main nutrients plants need to grow. In high-tech aquariums, dense-planted tanks, or in most aquascapes, CO2 is in deficit so injection of the gas is required. The plants are really interested into the Carbon atoms rather than the oxygen. In fact, the photosynthesis process allows the plants to fix the carbon in organic molecules and get rid of the oxygen, which is then expelled to the medium. The oxygen we breath comes from that residual generated by the plants and algae all around the world.

I am not going to thoroughly explain here the photosynthesis process: It would be too long and there is no need for that. However, I will focus the discussion in what form of inorganic carbon the aquatic plants are using, and also explaining the effect of the temperature in this process.

### Use of CO2 by plants

The plants are called autotrophic organisms, which means they are able to build up their organic matter from inorganic elements. On the other side, heterotrophic organisms employ existing organic matter to obtain the materials they need to grow. In the case of the plants, there a variety of inorganic molecules that they require to synthesize organic matter from scratch: CO2, water, inorganic nitrogen, phosphates, and other elements like potassium, iron, sodium, etc. From all of these, CO2 is the one needed in more quantities. This makes sense, as carbon is the atom which compounds the structure of most of the organic molecules. The carbon is extracted by the plants from the CO2 that they adsorb from the medium in which they are growing.

The mechanism that allows the plant to transform CO2 into organic molecules is known ad photosynthesis, and as mentioned above, it is really complex. In a very simplistic way, it is possible to say that plants bind together 6 molecules of CO2, with another 6 molecules of water, to generate one molecule of glucose and 6 molecules of oxygen. This type of reactions cannot happen spontaneously, so there is a need of adding energy into it. The plants solve the issue by using the energy of the sun. I clarified this because there are other autotrophic organisms that they employ energy coming from chemical reactions, instead of the sun. But in plants, the sun is the source of energy to do so. The reaction can be summarized as follows:

 $6CO_2+6H_{2}O+Light\rightarrow C_{6}H_{12}O_{6}+6O_2$ Eq. 2: Stoichiometry of the photosynthesis process

The reaction seems simple, but the actual way in which this happens is very complicated, until such a point that even after being investigated for long years, the process is not still fully clear for the science. But right now, the key point is that the reaction consumes significant amounts of CO2 in order to generate a single molecule of glucose. Oxygen is also produced in large amounts and released as a residual to the atmosphere or water. Part of these so-generated glucose is used as fuel by the plants to generate energy, which is then employed in other catabolic and anabolic processes, like building proteins, pigments, cellulose, etc.

At this point, it is worth to mention that aquatic plants and terrestrial plants differ in several aspects when goes in how they intake CO2. In the case of the terrestrial plants, the leaves are full of small holes called stoma that allow the inflow of air in the leave, putting in contact the leave´s cells with the CO2 and other gases found in the air. The mechanism in which this happens is called diffusion, and naturally happens from areas with higher concentration of a given gas, to areas with lower concentration. As plants are consuming CO2, there is a tendency to this gas pervade inside the leaves. Terrestrial plants then work using the CO2 concentration into the air, which nowadays is about 400 ppm, but before industry era was about 280 ppm. Try to keep these figures in mind, because they are important.

On the other hand, aquatic plants take the CO2 from the water. The process is also driven by diffusion, and plants still have stomas playing this role. However, the availability of CO2 is rather different, as is dependent on the alkalinity of the water and the pH, as previously shown, as well as solubility is driven by Henry's law, as also explained above. This means that soft fresh waters have much less CO2 dissolved into it than hard waters, for the same pH. Consequently, CO2 concentration for freshwater environments are commonly found between 1 to 5 ppm (in pure water and equilibrium conditions, with no alkalinty, value would ve around 0.6 ppm). These values are significantly smaller than in air, which means aquatic plants have much less access to CO2 than terrestrial ones.

However, the problem is even more complex than that, as diffusion of CO2 within the water is about 10.000 times slower than in air. This is due to the highest viscosity and density of water than air. Meanwhile in the atmosphere the degree of turbulence is very high and air circulates a good rates, water has much less movement in comparison, and water molecules also are a stronger barrier to diffusion. This situation means that a limited system, as an aquarium, can be depleted of CO2 due to the consumption of it at faster rates than the gas is able to diffuse into water. Why is this a problem? Next paragraphs try to explain this.

### Aquatic plants and the boundary layer

The depicted problem happens due to the increase of the size of the Prandtl boundary layer. A boundary layer originates whenever a fluid is in contact with a different medium, surface, or even other fluid. This layer is formed by the reduction of kinetic energy associated to the fluid when interacting with the other medium, mainly caused by friction, which rates to viscosity between other things. This explains, for example, why we can feel a stronger wind when we climb to the top of a tree, in comparison with the situation in the base. The air in movement is blocked by the grass, bushes, rocks, every element from molecular level to big elements, which reduces the speed. As we go higher, however, there are less elements causing this interruption and wind speed increases (see Fig. 6 for graphical details). The Prandtl boundary layer follows the same idea but is caused at molecular level, and viscosity as such scales has also a major role.

 Fig. 6: Prandtl or viscous boundary layer respect to velocity in fluid.

In a dramatic example, both water and honey are fluids, each one with a different viscosity. If you put honey into a spoon and invert it, the honey will eventually fall down, as gravity demands. However, it is going to do it a much less pace than the same action with water. This difference of speed in the falling is due to the difference in viscosity, which helps to keep a physical integrity into the fluid. In order to gravity forcing the honey to fall down, it has firstly to overcome the viscosity that keeps the honey attached to the spoon. Going to a diffusion example, a person can dive and swim in water, but will probably drown if tries the same in a swimming pool filled with honey: Both viscosity and density slow the swimmer down and become any movement in it much harder than in water.

The indicated boundary layers are important because they have some specific properties. For instance, they have a laminar behavior rather than turbulent. This implies that diffusion into them is mainly driven by molecular processes, as the Brownian movement. In the Fick's law, this means that turbulence of the equation is negligible, and the speed of diffusion significantly reduces. Now think in a leaf. As driven by Physics, this leaf will have a boundary layer associated to it. This means that the fluid around the leaf will behave as described above. The leaf consumes CO2 from its surroundings, by diffusion, i.e. from inside the boundary layer. Now, as the diffusion is much slower in the layer, the leaf has a risk of using the CO2 at higher rate than it diffuses inside the layer. In that situation, eventually, the maximum rate of CO2 the plant could make use of will be identical to the diffusion rate, which is much less than the needs of the plant, and then, the plant can starve, literally.

For terrestrial plants this is not an issue because the thickness of the Prandtl boundary layer is very small, CO2 concentrations are larger, and then diffusion of CO2 happens at higher rates than the plant's needs. But in water, diffusion is itself much smaller and the boundary layer is also thicker, about 10 times the size in air, and with a representative thickness of 0.5 mm. This situation creates a problem to the aquatic plants, as they have then much less access to CO2 than terrestrial ones. How do the aquatic plants solve this? Well, here is where the magic of nature happens.

### Adaptation of plants to improve CO2 uptake

As it should be expected, the aquatic plants have some specific mechanisms to tackle this issue. I will explain some of them, but there are more. It is quite common to find aquatic plants with more than one of those adaptations, which gives more plasticity to the species to survive at various environmental conditions.

#### Using strategies to reduce the thickness of the boundary layer

Obviously, if the problem is the boundary layer...why not trying to reduce it? And there are ways to do so. For instance, many plants have developed spiky and/or dissected leaves. The reason for that is double. On one hand, the spike leaves reduce the size of the boundary layer, as they reduce resistance to water flow and, hence, minimizing the viscosity effect. On the other hand, this strategy also increase the ratio between surface and volume of the plant, and this is important. The larger the surface in contact with water, respect to the volume of the plant, the more proportion of CO2 can be absorbed for the same biomass, as CO2 intake happens through the surface of the plant.

There are many plants with this approach, like Cabomba aquatica, Limnophila sessiliflora or Myriophyllum mattogrossense. The adaptation can also be observed in some plants that are able to grow in emerged forms. In fact, some of these species actually change the shape of the leaves when they are under water, moving from more round-shaped leaves to more spiky ones. Some examples of that is Ludwigia arcuata, which submersed form develops leaves in that shape (Fig. 7).

 Fig. 7: Change of shape in the leaves in Ludwigia arcuata: Emersed form (left) and submersed form (right).

Other ways for plants reducing the boundary layer consist on growing just in areas with higher water flow. Turbulence in water reduces the thickness of the layer, improving the capability of aquatic plants to get more CO2. Some plants are adapted to grow in rapids and/or quicker rivers to have advantage of this.

#### Internal morphological changes

In certain number of species, the plants have developed internal hollow channels filled with air, called aerenchyma (Fig. 8). The function of these channels is multiple: They allow the gases to move freely through the plant, what includes produced O2 and CO2. As the CO2 inside the channels is not any longer into water, the effect associated to the boundary layer in water is eliminated, as plant can now capture CO2 from the inner airy part. Additionally, aerenchyma are also a storage point for CO2 generated by the plant during their metabolic activity (especially during nights), and in some cases, they are connected to the roots so the plant can also absorb and take advantage of CO2 existing in the sediment, usually generated by heterotrophic bacteria.

 Fig. 8: Aerenchyma in plant.
Some examples of aquatic plants with this adaptation are Eleocharis sp., Cabomba aquatica Ludwigia repens or Myriophyllum mattogrossense. In general, most of stem plants have this characteristic, as it has shown special efficiency in solving the issue.

Plants with this morphological characteristic are easy to identify in the aquarium, as the cutting points start to bubble after trimming. Some hobbyist confuse this phenomena as a good response of the plants to trimming, but reality is that is only the gases stored in the aerenchyma scaping to water through that point. We can say then the plant is "bleeding" gases through it, mainly oxygen.

There is, however, other approach to the problem. So, if the difficulty is the availability of CO2 dissolved into water...why not then changing the source of carbon? This is, precisely, what many aquatic plants are really doing. As previously explained, the amount of CO2 present into water is strongly linked to pH. At the normal pH values in planted tanks, most of the CO2 is, in fact, in bicarbonate form (see Fig. 5). Hence, it seems much better idea to exploit these bicarbonates as an alternative source, which usually complements the normal CO2 intake.

The way in which plants do so can vary, but there are three main ways to do so:

1. By active uptake of bicarbonate: The plant absorbs bicarbonates from water into the cells, and there is processed to remove an ion hydroxide (OH-) by using the ubiquitous carbon anhidrase enzyme, becoming the bicarbonate into CO2 that can be normally used.

2. By proton extrusion:  The plant pumps protons into the boundary layer, which reduces the pH in it. As shown in Fig. 5, this reduction of pH favors the formation of CO2 from bicarbonates, which now then can be used normally by the plant. This is a very smart solution, which takes advantage of the boundary layer itself.

3. By enzymatic reactions at surface level: The plant has carbonic anhydrase enzyme at surface level, in contact with water, removing OH- from bicarbonates and uptaking CO2.

The counter side of those methods is that requires the plant investing energy into it. These processes are not spontaneous, and it involves active ionic exchanges between the plant cells and the water. Because of that, efficiency of photosynthesis when working in this way is minor, which means the plants grow slower when using such mechanisms. However, availability of bicarbonates in water tends to be much higher than CO2, so these strategies become in a difference, especially in waters with high density of plants and low water flow, when most of the CO2 in gas form is consumed very quickly.

The problem with the first solution is that generates OH- ions that must be removed, somehow. The third method fix that problem by performing the reaction in the water side but then increases locally the pH, interfering into the natural absorption of CO2. In some cases, plants have these mechanism in different parts of the plants to avoid interference between them. For instance, they use the active uptake of carbonates in the upper part of the leave, meanwhile using the proton extrusion in the lower part.

Nonetheless, most aquatic plants prefer CO2 uptake rather than using bicarbonates, so the detailed mechanisms are only a main contribution to their metabolisms when CO2 concentration is below certain thresholds and then investing the energy in these processes makes sense.

### Effects of temperature in the photosynthesis and CO2 uptake

As derived from the text, photosynthesis is highly dependent on the CO2 availability. By increasing the CO2 concentration in water, plants will also increase the photosynthesis rate if no other factors are limiting the process. The same applies to light: The higher availability of light, the higher the photosynthesis will be.

In high-tech planted tanks, this is the idea behind the set up and fertilizing mechanism: We add light and CO2 at high enough doses, making sure that other nutrients are not in shortage, and then we get the maximum speed of growth which derives in healthy plants.

However, these are not the only parameters affecting the process. In order to photosynthesis giving a positive balance of energy, the needed chemical reactions must happen as much spontaneously as possible. This is not just a matter of putting the ingredients all together and wait for this to happen. In order to optimize the process, all life forms employ catalysts that promote the reactions. These catalysts are substances of many types (e.g. metals, vitamins, proteins) but with the same characteristic: They improve the possibilities of a specific reaction happening spontaneously.

The way in which these catalysts work is related to the concept of enthalpy of reaction (H). Each chemical reaction involves a change in the energy levels associated to the system. In general terms, a reaction is spontaneous when the final levels of energy are lower than the ones found in the original situation. The difference of energy is emitted as heat or light. When a reaction leads to a higher level of energy than the original one, then the reaction is not spontaneous and requires injection of energy to happen. Enthalpy is just a measure of such energies: The amount released in the first case, the amount needed in the second case. It is straight forward to see that the lower the enthalpy is, the more likely is the reaction to naturally happen without requiring energy. That is why photosynthesis, eventually, cannot be profitable in terms of energy if the addition of the enthalpies of all the chemical reactions happening in the process is positive. This is important, because it means that a process composed by a series of reactions can naturally take place, even if some of the intermediate steps require to invest energy, as far as the global frame of reactions ends in a system with lower energy than the starting situation. This is what the plants do during the photosynthesis.

The catalysts play a role here by reducing the enthalpy of the reaction. The catalyzed reaction has then more probabilities to happen than the normal one. The way in which catalysts work can be very complex and different at each case, but they usually play a role into an intermediate reaction that smooths the process. As mentioned above, each reaction has an enthalpy associated to it. Let's say then that we want the reaction P1+P2=P3 to happen. However, this reaction requires a certain level of energy, H1, that gives a low chance to the reaction happening naturally. Now, we know of a substance Q that is a catalyst of the reaction. Q has capability to react with P1, in a reaction like P1+Q = P1Q in an spontaneous way, with an enthalpy significantly lower than H1. On the other side, the product of the reaction, P1Q, is also able to react with P2 in a spontaneous way, so P1Q+P2 = P3+Q. Both reactions driven by the catalyst Q are spontaneous, which means that the addition of the enthalpy of the two intermediate reactions is smaller than the enthalpy associated to the direct reaction P1+P2=P3. Moreover, the component Q is recycled in the process, and then is available again to catalyze other reaction of the same type. Because of this, small amounts of a catalyst can propitiate a much faster and efficient reaction chain, by speeding them up and reducing the energy needs.

 Fig. 9: Example of effect of a catalyst into a chemical reaction.

This is not normally like this, as shown in Fig. 9. In most occasions, the role of the catalyst is not exactly consisting in becoming the reactions as spontaneous; in many cases, catalysts reduce the amount of energy that must be added to the reaction happening, factor extensively employed by any life form to optimize their organic processes.

Explained this, photosynthesis is a process which is catalyzed at many levels. In no few cases, the intermediate reactions are controlled or catalyzed by enzymes generated by the plant. The reason why this is so important is because the catalyzed process can make use of the energy from the sun to trigger the reaction, in a way that otherwise would not be possible because the energy obtained from it would not be enough. Without this particularity, photosynthesis would not be possible.

Besides the role of the catalysts, temperature also has an impact. Depending on the kind of reaction, a higher temperature reduces the need of input of additional energy, making easier the process and speeding it up. This is also true for the intermediate reactions associated to catalysts, which by definition require already less energy. Combining then temperature and catalysts, the speed and efficiency of a catalyzed reaction will significantly improve. As most living things employ enzymes to catalyze their organic processes, temperature is critical in explaining the velocity of reaction. Most enzymes have an optimal temperature to work with. Theoretically, with an ideal catalyst, the speed of the reaction will increase as temperature raises. Reality is that organic catalysts have a range of temperatures in which they can work. Below or above such range, they do not work and/or lose their properties. This is mainly due to the fact that enzymes are proteins and its structure is both pH and temperature dependent. By increasing too much the temperature, the protein loses its fundamental structure and properties as catalyst. This is known as thermal stress and explains why too high or too low temperatures kill to a given species: There is a point in which metabolism just stop working. Some animals have temperature regulation mechanisms, like all the animals of warm blood, which helps them to keep similar levels of activity in a wider range of temperatures.

For aquatic plants, the effects of the temperature also apply. Increasing temperature means higher rate of photosynthesis, which involves larger CO2 uptake. The range of temperature in which most plants can work is between 10° and 40°. Outside such range, photosynthesis is not possible for most plants. The range is not absolute and there are species with specific adaptations to very high or very low temperatures, which allows them to thrive in extreme environments. However, for tropical plants, the mentioned range works well.

 Fig 10: Evolution of photosynthesis rate as function of time and temperature.
Within such limits, the optimal range of temperature is not easy to determine. To start with, not only the temperature plays a role, but also the time that such temperature is applying. Enzymes will not degrade immediately after the change of temperature: they will work for a while and plant will try to replace the harmed ones, but eventually, the accumulated damage happened to them due to the heat will affect to the photosynthesis rate. This is what Fig. 10 shows. The higher the temperature is, the higher the initial photosynthetic rate is, but then also the faster the rate decays as result of the thermal stress. For example, at 40°C, the rate is maximum but just during 15 minutes. After that, the destruction of the enzymes makes the rate to quickly fall down. When reducing the temperature, however, maximum rates are lower than before, but it takes longer to show a thermal stress and even so the decay is slower than before. At 30°C, there is practically no thermal stress and none at 25°C.

Hence, the optimal temperature is the one that involves no thermal stress and maximizes the production. In the plot this values is situated at 25°C, but, as said, this is just representative of the global set of plants, and not the rule.

Some tropical aquatic plants have a higher tolerance to heat as they live in areas in which waters can have higher values of temperature with frequency. More specifically, some studies identify values between 28-32°C, depending on the species, as top limit for photosynthesis efficiency in tropical aquatic plants. Most hobbyist try to keep plants in around 25°C, because it is good for most of tropical/subtropical aquatic plants and livestock, but there is not always an easy way to keep such temperature when the aquarium is in a hot country. In such cases, CO2 injection must be adjusted to keep the nutritional balance in the tank.

But...how much can vary the uptake of CO2 under higher temperatures? This question has not an easy answer. The exact effect will rely on the specific species included in the tank and their amounts. However, it is possible to say that hobbyist should expect a higher requirement of CO2 when temperature in the aquarium raises. As a rule of the thumb, firstly, it is a good idea to try to preserve the water temperature below 30°C to avoid thermal stress. In average for tropical aquariums, 30°C is the temperature of maximum rate of photosynthesis, so the requirements of CO2 will be higher than the ones associated to 25°C.

In general terms, enzymatic-catalyzed reactions double the rate for every 10°C of temperature increment, roughly. For instance, this is what we observe when comparing, in Fig. 10, the line associated to 25°C with the one of 35°C, or the one at 30°C with the one at 40°C. The relation between temperature and photosynthesis rate is not a straight line, but can be reasonably assumed like one. Hence, by increasing temperature from 25°C to 30°C, the CO2 needs of the plants in the aquarium will grow about a 50%. This essentially means that in hot countries, planted tanks require  in the range of 50% more CO2 injection to preserve the balance and to keep the CO2 concentrations into the desired or target levels. The same will apply to any hobbiyst in no so hot countries but keeping higher temperatures in the aquarium.

## CO2 and fishes/shrimps

Meanwhile for plants the CO2 is a vital component they need for their most basic functions, for animals the molecule is a residual and toxic substance that they need to get rid of. Animals generate CO2 during the breath process, which involves the consume of organic matter to produce energy, by using oxygen. This chemical reaction is called combustion and, when using the right catalyst, it can happen in a spontaneous way into the mitochondria of the cells. Plants also use this mechanism to generate energy required by their metabolism. Difference is that plants are using a part of what they synthesize, and animals rely in their food ingestion to get the fuel.

### Breathing

For most readers, breathing consist in the act of inhaling/exhaling air with their lungs. Reality is that, this basic function of our bodies, is just a part of the actual breathing. Filling the lungs with air serves just to renovate the air and get better O2 concentrations in contact with our lung cells, which are in charge of uptaking such O2 into our bodies. At the same time, the excess of CO2 is also removed in such a way. The so-acquired O2 is then used by our cells in combustion reactions, which generate CO2 that must be eliminated. Of course, there are some differences between how we do it and how other animals do it, but the idea behind it is pretty similar.

#### Role of the gills

Most aquatic animals we keep into aquariums benefit of using gills. Meanwhile, in terrestrial vertebrates, the air is actively forced into the lungs for the exchange of gases (note that in insects this is different), in aquatic animals the gills play such a role. The idea is the same as depicted above: Absorb oxygen and expel CO2. The efficiency of this process relies in several factors:
1. The relative concentrations of O2 and CO2 in body respect to the environment.
2. The surface/volume ratio associated to the gills.
3. The flow of water through the gills.
Gases have a natural tendency to move from higher concentrations to lower concentrations. Animals take advantage of this and the gills are the perfect place for that. Molecular diffusion can be easily blocked by physical barriers, so certain layer of cells can block it. Amphibians have a very thin skin due to this, so they favor the diffusion of gases between their bodies and water, but on the other hand, they are much more sensible to toxics in water, as well as bacterial and fungi diseases. Many other aquatic animals are adapted to reduce these risks, having thicker skins and focusing the gas exchange into the gills. There, the animals have a very thin layer of cells between the internal circulation system and the water. Thanks to that, gases can travel from the animal to outside and vice-versa, following the gradient of concentration. The gills are conformed by a set of sheets, each one subdivided into arches, formed by filaments, which are composed by sets of lamellae. The full structure is thought to maximize the surface of contact between gills and water (Fig. 11).

 Fig. 11: Gills structure and physical exchange of gases.

The lamellae have a shape and construction aiming to maximize diffusion, by having high surface/volume ratio and really thin walls. Each one has blood vessels, one of input (afferent), other as output (efferent), linked between them by even smaller vessels very close to the surface of the lamellae. The idea is that blood charged with CO2 travels through the lamellae, getting rid of the CO2 and uptaking O2. This exchange is not purely physical and involves also biochemical reactions that are driven by the hemoglobin in the blood cells. The oxygenated blood then travels back to the body through the efferent vessel.

The blood flows in opposite direction than the water flow. The reason for this is to optimize the time in which the exchange happens at maximum O2/CO2 gradients. The strongest the differences between water and blood are, the more efficient the exchange is. Water flow is the result of both water speed and volume, so many aquatic animals with gills have adaptations to ensure such flow. Both fishes and inverts literally pump water into the gills to maximize the exchange. In the case of fishes, the method is even smarter: They are not only pumping it but also ensuring the speed of the water flowing in the gills is as high as possible. To do so, they open the mouths to allow water going inside, then compress it into their equivalent to our throats, and then opening the operculum of the gills to allow water go out through them. As the water is compressed and the gills allow a small space between them, water is then affected by a Ventury effect and increase its speed of flow in its way out. Amazing.

Th three elements above explained are very important to understand the exchange of gases between these animals and the water of the aquarium.

#### The exchange of gases

Thus, the gills are then in charge of this gas exchange. However, this is not as simple as purely Physics. Most animals are not making use of just gases dissolved into the blood, as that becomes them difficult to control, it is limited by ohysical saturation levels and do not grant delivery into the right spots. Instead, hemoglobin is used as vehicle of oxygen so it can be delivered exactly in the points the tissues need it and covering the demand of oxygen of the different biological processes. Such molecule is in large quantities in the blood cells (erythrocytes) and has a special characteristic: It is a complex protein but with an atom of iron on it. Iron has a really high affinity by oxygen to become into FeO/Fe2O3 (ferrous/ferric oxide). In fact, blood is red due, precisely, to the colour produced by the iron in the hemoglobin when oxidized. Animals take advantage of this to transport oxygen. Inside aquatic fishes and inverts, there is a full universe of variations of hemoglobin, each one with some specific properties and mechanisms. I am not going to enter there (too complex and long) but I am going to provide some generalities.

The summary of the breathing of the fishes is explained by the Bohr-Haldane effects, which are connected to each other and explain the way in which oxygen is transported by hemoglobin and released, with CO2 conversely removed from the system.

The Bohr effect determines that an increment of CO2 in blood involves a reduction of pH. Under such acidic conditions, hemoglobin reacts with the protons in solution, which drives the release of the oxygen being transported by the hemoglobin. This is quite convenient, because it means that an increase of CO2 in blood forces a release of oxygen, and then erythrocytes provide the oxygen in the places in which is actively being used.

On the other hand, the Haldane effect is just the opposite: Under presence of oxygen in blood at higher concentrations, with the right pH, the hemoglobin releases protons to blood and kidnaps oxygen. This phenomena is also pH-driven and happens when pH rises after removal of CO2 from blood in the gills. Both effects are just opposite sides of the same coin, reason while is named Bohr-Haldane effect. It is easy to see that the key point is where each of these processes are happening. Haldane effect is only possible at gill level because is the point in which CO2 can be released to the water, increasing the pH of the blood. On the other hand, Bohr effect can take place only in areas in which the CO2 accumulates into the blood, i.e. in the vessels in contact with the tissues. The largest the generation of CO2, the largest the amount of transported oxygen is released. This connection allow the covering of oxygen demand coming from the activity of the organism where is needed.

 Fig. 12: Gas exchange mechanism or Bohr-Haldane effects.

The process is better detailed in Fig. 12. When pH increases, erythrocytes uptake bicarbonate from blood, which also increases the pH inside these cells (6). When oxygen is present, the higher pH allows the release of protons by the hemoglobin with the corresponding kidnapping of oxygen (5). These released protons down the pH, which displace the carbonates equilibrium towards the formation of CO2. The increased CO2 concentration then produces a flow by diffusion towards the blood, and from the blood towards the water at gill level (4, bottom side of the figure). On the other hand, at tissue level (top side of the figure), CO2 generated by the cells flows towards the blood, due to the favoring differences of concentration. There, it is absorbed by the erythrocytes, and thanks to the carbon anhydrase (an enzyme which catalyze the process), is practically fully conversed into bicarbonates. The bicarbonates are pumped to the blood, increasing the pH outside the cell(3). These two mechanisms generate protons (2) and reduce the pH, which favors the release of oxygen from hemoglobin as exchange for protons. The oxygen then diffuses, favored by difference of concentration, from the erythrocytes to blood and from there to tissues, completing the cycle.

In summary, the removal of CO2 from blood allows to the erythrocytes the absorption of oxygen in one side, and the accumulation of CO2 causes the release of oxygen into blood, in the other side.

#### The problem of excess of CO2 in water

There are, however, some important aspects from this situation related to the environmental conditions. For example, removal of CO2 from blood is only possible by a diffusive mechanism at gill level. This means that if the right combination of low pH and high dissolved CO2 happens in the water, this diffusion will happen at less rate or even inverse the direction of flow, at certain levels. Aquatic animals show relatively low levels of CO2 concentrations in blood, in the order of 10 times less than terrestrial ones. The reason behind it is that CO2 is far more soluble in water than oxygen and concentrations are also far smaller, so meanwhile fishes have more difficulties to oxygenate, they have much less problem in eliminating CO2. Because of that, fishes keep CO2 concentrations at closer levels to those found in their environments, which become them much more sensitive to changes in CO2 than terrestrial organisms.

More specifically, a typical value of CO2 concentration in blood for fishes is about 4-5 ppm, which naturally obeys to about 8 times the amounts found in water in equilibrium with the atmosphere. This difference ensures the right flow of CO2 from blood to water into some range of CO2 concentrations and pH values. However, if the environmental concentration of CO2 raises, the gradient diminishes, slowing down the removal of CO2 from the fish body. As per Fig. 12, this involves the increase of the CO2 dissolved in blood, which becomes a problem as favors a reduction of pH of the blood, which at certain point can block the uptake of oxygen, as hemoglobin is then not able to oxidize.

 Fig. 8: Re-absorption of  bicarbonate and release of protons at kidney level.
The problem can be partially compensated at kidney level, at least in fishes. It has been shown that fishes are able to reabsorb bicarbonate at kidney level and release protons with the urine, as part of a cation exchange process. Both actions allow an internal regulation of the pH, which keeps working the absorption of oxygen. Additionally, cardiac and breathing rhythm are increased, in an attempt to improve the gas exchange (which, as pointed out before, is also linked to flow rates, both in blood and water). However, all these strategies involve the investment of energy into it, becoming then into a stress factor at certain levels. Moreover, the capability of the animals to regulate pH into blood has some certain limits, and beyond some point, the perturbation caused by the excess of CO2 into water cannot be compensated, causing acidosis to the animal, and eventually, the death.

In this sense, most fishes manage well in waters with CO2 concentrations up to 20 ppm. Beyond that point, internal regulation begins to be a stressing factor and is fully lethal in a couple of hours, in most of the cases, when concentrations in water reach values close to 60 ppm or higher. Of course, all these values are just a reference. Individual species can have a different behavior, with some of them being affected by CO2 levels at much higher/lower levels than others.

#### The effect of oxygen injection

This is also an interesting topic. There is a belief into the community that negative effect of CO2 increment can be partly compensated by increasing the oxygen concentration in water. Following the schema of breathing I have depicted above, this is true at certain level, but does not compensate any excess of CO2 in water.

The increment of oxygen into water improves the natural flow of the gas into the blood. Under normal circumstances, this increase means that aquatic animals need a slower flood of water/blood to get an efficient gas exchange rate, and oxygen enough to cover their needs. As a result, breathing and cardiac rhythms reduce, saving energy invested in them, and reducing the stress levels of the fish. In well oxygenated waters, fishes will be happier and with a better health. This is correct, so far we assume that there is no special or harmful situation coming from CO2 concentrations in water.

The effect of oxygen is then positive, as far as the oxygenation levels are also kept into certain thresholds. Aquatic animals have a limit to reduce the breathing and cardiac rate before being fatally affected by it. If consumption of oxygen becomes significantly lower than the uptake of it at gill level, then hemoglobin is not reaching a situation in which can get rid of the oxygen and take protons. The CO2 being generated is then converted into bicarbonates by the carbon anhydrase enzyme, increasing the alkalinity of the blood. As mentioned before, fishes have mechanisms to regulate this changes into the pH of the blood. However, at certain levels of oxygen, these mechanisms cannot cope, which derives into alkalosis,  and eventually, into death.

This is quite uncommon into aquariums, as usually the mechanisms to add oxygen are using just air. Because of that, maximum concentrations of O2 into water are determined by Henry's law, and they aim to be in equilibrium with the atmosphere. Such situation is not a problem for the aquatic animals at all. Situation could be different if we were actively injecting oxygen at partial pressures higher than the values found in the air, as we do with CO2, and reason why CO2 injection can harm the livestock.

But, in general terms, favoring a good oxygenation of water is good for the aquarium, when using the traditional methods. However, when CO2 levels are increasing, oxygenating water does not solve the issue. As explained, Fig. 12 shows that oxygen uptake happens when oxygen is available and pH is at the right values. If the water has a high concentration of CO2, the regulation of the pH in blood becomes a problem, which diminishes the capability of the aquatic animals to oxidize their hemoglobin with oxygen. In other words, oxygen availability is good, but its use by the hemoglobin is controlled by the capability of the animals to remove CO2 from their blood. If this cannot happen, breathing process is then blocked and animals suffocate, does not matter at all the amount of oxygen in water.

But then, why do fishes improve when aeration is in place, when CO2 levels in water are high? This
 Fig. 13: Example of aeration.
has nothing to do with the increase of oxygen. Instead, aeration increments the total surface of water in contact with air. The exchange of gases between water and air is related to precisely the amount of surface in contact: The larger is, the more efficient the diffusion of gases from higher to lower concentrations (i.e. usually oxygen from air to water, CO2 from water to air). Air stones generate thousands of small bubbles. The smaller the bubbles the higher is the ratio between surface and volume, so exchange of gases benefits of this. This fact speeds up the degasification of the aquarium in terms of CO2, and eventually, it helps to reduce the concentration of the gas in water.

Similar results are obtained when increasing agitation of the surface in the tank. In this case, it is not much related to the increment of surface. Rather than that, diffusion also depends on the turbulence term of the Fick's law, as pointed out in this article, so when we increment it, we also propitiate the transfer of oxygen to the tank, but more important in this case, the removal of excess of CO2 to the atmosphere.

This degasification from air stones and agitation can be measured by monitoring pH. The loss of CO2 means that water in the aquarium increase the pH, an easy way to check if aeration of the tank is correct.

Aeration is positive as helps in many aspects, like removing excess of CO2, improving the oxygen levels for aquatic animals (and plants during the night phase), and also is key to reduce the load of organic matter into the tank. Do not forget that oxygen is extensively used by both heterotrophic and autotrophic bacteria (i.e. in charge of organic matter breakdown and re-mineralization processes, like formation of NO2 and NO3). In terms of planted tanks, however, aeration will reduce the efficiency of the injected CO2 during the photo-period, which means more difficulties to achieve the desired CO2 concentrations.

## Questions & Answers - The CO2 Quiz

So far, the article has been focused in presenting mainly the basis of the role of CO2 into aquariums, as well as its dependencies of physical and chemical parameters. The gas intervenes at many levels, and a good understanding on how behaves and interacts can be the difference between success and failure.

Now, I have collected a list of common questions associated to the use of CO2 in planted aquariums and aquascapes. If you do not find your question here, please, feel free to contact us and we will add your question here.

### 1. Do I really need CO2 injection to have healthy plants in the aquarium?

The answer is "No", but with some peculiarities. CO2 injection improves the conditions of the plants, in terms of nutrients, ensuring that CO2 is not a limiting factor of growth. Essentially, by injecting CO2, it is possible to get faster and denser growth than without it. However, this will not make any good if the additional CO2 provided to the tank is not followed by right and higher lighting conditions and fertilizers. The reason why is because plants will grow limited by the factor not able to cover their needs. Light encourages photosynthesis, but the additional rate will not really happen if there is not enough CO2 and fertilizers. In the same line, CO2 will not be used in the amount of light is not enough, or if some nutrients are in deficit. Injecting CO2 under a unbalanced situation will just encourage the appearance of algae, so any time CO2 is injected, a balance of parameters must be achieved.

Hence, the answer is then linked to these other elements. For instance, people not using CO2 does not need to add stronger lights or so much fertilizers, if any. In this case, the balance must be achieved by playing with the natural concentration of CO2, which, as explained in this article, will be much smaller, so low-tech aquariums need less lights and, in many cases, no fertilizers at all.

 Fig. 14: Example of low-tech aquarium
However, CO2 demand is not only related to the conditions but also to the plants biomass in the tank. By heavily planting a low-tech tank, plants can be able of depleting CO2 from water, which interrupts their growth and, sometimes, this can derive in no healthy plants. There are ways to improve their conditions, though, as for example providing a good water circulation, agitation, and including aeration. In this way, CO2 from atmosphere will diffuse better in the tank and reduce the problem of availability. In these cases, by keeping not too strong lights, the aquariums can thrive without major problems. Despite of all of this, main difference is related to the speed of growth, though.

Other key aspect is that plant selection of species rally matters. Some of the species will not grow at all, or even die, without CO2 injection or under some poor conditions, like low light. As indicated in this article, plants prefer the use of CO2 is gas form rather than bicarbonates. They have adaptations to compensate the lack of CO2, but some species are not so well adapted and they really need higher levels of CO2 to survive in aquariums. Avoiding such plants will make easier to have a planted aquarium in low-tech.

### 2. How much CO2 should I aim to have into the aquarium?

There is an agreement that CO2 concentrations between 25 and 45 ppm are the best one for planted tanks. This, of course, is only true for high-tech tanks. It is possible to have reduced levels but then the same discussion as for the previous question applies.

Note that, however, CO2 concentrations are dependent on the carbonate hardness of the water and pH. The article thoroughly explains these dependencies, but it is not focused in how measure it and control it. For that matter, I strongly recommend you to read this other article I wrote about the use of drop checkers, a useful tool to control CO2.

### 3. Is there any standard flow of CO2 I should follow?

No, there is none. Consumption of CO2 varies in function of many factors: Amount of light, temperature of the water, density of plants into the tank. When people recommends 1 bubble per second, or 2 bubbles per second, it means nothing, really. The only term that is valid for dosage purposes is the CO2 concentration, and the best way to track it without having to spend money in a CO2 probe, is the drop checker, as pointed out in the previous question.

Nonetheless, plants will not make use of CO2 in the same way when they are just recent in the tank than when they have already settled into it. New setups are recommended to have lower level of CO2 and light, progressively increasing both parameters into a coupled way until reaching the nominal dosage of both.

### 4. Is the CO2 concentration affected by the temperature of the water?

In an strict sense, yes, but in practical sense, no. Figure 4 of this article shows the physical dependency of the CO2 concentration in water as a function of the temperature. As you can see, increasing temperature means lower maximum concentrations of CO2, so there is an effect. However, these maximum values are still far beyond the amounts we aim in aquariums.

For instance, between 25 and 30, saturation level of CO2 is reached at 1450 and 1250 ppm, respectively. We usually aim between 25 and 40 ppm, so the reduction of the solubility associated to temperature has no practical effect at all.

Nonetheless, there is a real need of increasing the dosage of CO2 when increasing the temperature, but that factor is not related to the saturation levels. In this case, we talk about the photosynthesis rate. As explained in the article (see Fig. 10), by increasing the temperature 5 degrees, we roughly increase the need of CO2 up to 50%, because that is the percentage the photosynthesis rate increases. This is mainly due to the fact the photosynthesis is an enzymatic process, and hence, dependent of temperature. Hobbyist with aquariums in hot countries need to inject more CO2 to compensate this increase of the metabolism of the plants. But this has nothing to do with the fugacity of CO2 associated to the change of temperature, and only to the acceleration of the metabolism.

### 5. What is better: a lower or a higher carbonate hardness when injecting CO2?

The answer to the question is not fully evident. The amount of dissolved CO2 depends on the CO2 injection rate, the pH, and the carbonate hardness. Carbonate hardness and pH are related because (bi)carbonates have impact into alkalinity. Alkalinity is the capability of the water to absorb protons, which in many occasions  is associated to OH- ions. However, HCO3- is also able to accept protons, so (bi)carbonates in solution tend to increase the pH of the water. As shown in Fig. 5, pH determines the relative abundance of the inorganic forms of carbon in water (CO2, bicarbonate, carbonate).

This implies that, at high pHs, there is practically no CO2 dissolved in gas form. This can create a difficult situation form some species of aquatic plants with less adaptations to use bicarbonates instead, and growth rate will be affected. As a result, it is possible to propitiate an unbalance of conditions, which unavoidably will lead to algae. This, of course, it depends on the species of plants and the other environmental conditions like light and nutrients.

On the other hand, carbonate hardness allow to more CO2 being accumulated into the water for a given pH, i.e. they stabilize the pH and minimize the acidification caused by the CO2 injection, difficulting to have a pH crash and killing the fishes for that, or just killing the fishes due to an excess of CO2 gas in water.

The contrary situation, so low carbonate hardness, allows the increase of CO2 in form of dissolved gas, which benefits the plants. At lower dkH values, CO2 levels will increase much faster, but the pH will also be reduced much faster, with the subsequent risk for livestock.

A good rule of the thumb, then, is to try to have a water carbonate hardness within certain levels. A common range of carbonates hardness is between 3 and 5 dkH. Higher dkH values are also possible, but then the CO2 injection will require higher flow and time to reach the desired levels of CO2. Lower values are not advised if keeping fishes in the tank.

By targeting such range of dkH values, CO2 will be at right concentrations as dissolved gas when pH reaches about 6.5 value.

### 6. How can I improve the use of CO2 in the aquarium?

There are a few ways to optimize the use of CO2 and ensure that there is no shortage. To start with, aiming the right concentrations, as commented din Question 2 of this article. Secondly, by controlling the carbonates hardness of the water, as also commented in Question 5. But, even with those things well done, other subtle elements can have a play on this.

 Fig. : Example of lily pipe.
For instance, it is good idea to have limited agitation during the photoperiod, if you are injecting CO2. Agitation helps into the gas exchange and diffusion processes, which means that propitiates the removal of CO2 from water. A high agitation can produce difficulties to achieve the desired CO2 levels and it will require than higher rates of injection into water to compensate it. Limiting it will make easier the task and also much faster. In this sense, the use of lily pipes is highly recommended in planted aquariums, as they give us some control over the agitation.

On the other hand, it is also crucial having a good circulation rate and distribution. Even if it seems otherwise, a water with a given properties does not mix very fast with a water with other properties (e.g. if the two water masses have different temperature). This means that water circulation in the tank really matters. If not attention to this is paid, some areas of the tank can receive much less CO2 than supposed to, especially if using CO2 in-line injection, which puts all the injected gas in the output of the filter. An uneven mix of water with CO2 can have a few negative effects:
1. Appearance of algae in areas with low CO2 concentrations.
2. Undesired low growth in some planted sectors.
3. Wrong measurement of CO2 levels when using a drop checker (which can be fatal).
4. Excess of acidity in the output point, which can actually harm the plants situated close to the outlet or CO2 injection point.
A good rule of the thumb consist in having a circulation rate of 10 times the volume of the aquarium per hour. The flow per hour depends on the filter, so read the technical specifications of your filtering solution to know this data. It is not rare, in both planted aquariums and aquascapes, having more than one filter to achieve this goal. Note that, agitation and circulation are not the same thing. Agitation relates to the speed of water at surface level: The fastest is the water there, the more favorable the removal of CO2 is. However, water flow situated at different depths or not directed to surface has not to increase such speed at surface level.

In addition to these concepts, other ways to improve the CO2 usage are the user  of a timer linked to a solenoid (as CO2 is not required during the nights), and also good diffusers/reactors. CO2 dissolution is slow, as pointed out into the article, which means that the longest the time in contact with water, the better. This is what a CO2 reactor really does. Also, the gas exchange is dependent of the surfaces in contact, which increases when we reduce the size of the bubbles. A good diffuser or atomizer has this role, what also improves the solution of CO2.

### 7. Has the CO2 injection to be deactivated during nights?

In general terms, I strongly recommend to do so. There are many reasons for that:

(a) Plants do not need it during the nights because, with no light, no photosynthesis, so having CO2 being injected in such period of time is a waste and forces you to recharge the CO2 bottle more often.

(b) Plants do generate CO2 during the night (they also breath), so if you inject CO2 during that period of time, and we add the CO2 generated by the plants, CO2 concentration can reach levels high enough to kill your fishes by suffocation or a drop of pH equally fatal.

(c) Its is very difficult to keep a stable CO2 concentration all the time. The plants start using it as soon as the intensity of light is high enough for photosynthesis, but usually takes some time before CO2 injection carries the CO2 concentration at the targeted levels. We can tune the dosage in order to achieve these levels during most of the photic phase, but during nights the plants do not use it, so CO2 levels will increase and the next day the starting concentration will be higher than the previous day. Because of that, CO2 can increase more than needed, with the subsequent problems.

The best idea then is to have CO2 systems including a solenoid that can be controlled with a timer. Most recommendations are to set the timer to start injection between 1 to 2 hours before lights go on, and stopping doing it between one or half hour before the lights go off.

### 8. Do I need to introduce aeration if I dose CO2?

Not necessarily, but is highly recommended, for several reasons. Nonetheless, aeration is not required during the day or photo-period, as plants generate lots of oxygen, and aeration propitiates the removal of CO2 from water, what will difficult you to achieve the right levels of CO2. However, aeration during nights can be very useful:

(a) It will provide oxygen when the plants are not producing it (in fact, all the opposite, as the plants will be consuming it), what ensures the fishes will not suffocate by lack of it.

(b) It will provide also oxygen for the re-mineralization processes and degradation of organic matter, which avoids the appearance of oily films in the surface of the water plus reduce the presence of precursors for algae formation.

(c) It will increase pH and stabilize it during the nights.

The best ways to do this are using air stones or moving up the lily pipes during the nights to promote agitation at surface level. If you do not want to be taking care of this, best option is to add an air stone with an air compressor linked to a timer and activate it when the lights go off.

### 9. My drop checker had the right colour but my plants are still showing symptoms of CO2 deficiency. What is going on?

 Fig. : Example of drop checker.
It can be a potential case of lack of water mixing. Try to change the place in which you have the drop checker, wait a couple of hours and check the colour. If it is not right, then it would be a good idea to improve the mixing. As pointed out in Question 6, it is possible to get this by using lily pipes and increasing the circulation rate. If you already meet these criteria, then try to put the inlets/outlets in a different configuration.

Other possible cause if you are using water of your aquarium for the drop checker. The values of the drop checker are closely linked to the carbonate hardness into it, so if you are mixing indicator with water of your tank, the readings can be wrong. The proper way to use the drop checker is as explained in this article.

### 10. I have the right levels of CO2 but some of my fishes are dying since I started with it.

As explained in this article, concentrations of CO2 about 20 ppm are stressing for most species and amounts close to or over 60 pm are lethal after a couple of hours. Sensitivity to CO2 varies with the species, so even normal amounts of CO2 for planted tanks could be harmful for some of them.

This also relates to pH, as CO2 injection will reduce the pH of water. This can kill the fishes if some precautions are not kept. Monitoring the pH in regular basis to avoid this, and ensuring certain level of carbonate hardness in water to avoid sudden drops of pH is a good way to prevent this happening.

There is also a chance that has nothing to do with the CO2. If pH is correct and levels are OK, and your species are not sensitive to such levels, then try to check other parameters of the tank (e.g. nitrites, nitrates, ammonium) to see if the problem is linked to them.

### 11. I have difficulties fixing my carbonate hardness to a  certain value. What is the cause of this?

In this article, I have explained the carbonate equilibrium and the role of CO2 injection in it. When we inject CO2, we generate certain amount of carbonic acid in the water. This carbonic acid reacts first with carbonates dissolved in water (if any), producing bicarbonate, and after that, it consumes bicarbonate ions, producing CO2. The relative ratio to which this process happens depends on the pH of the water (Fig. 5).

If the pH of water reaches a value equal or below 8.2, then the consumption of bi-carbonates starts. The so-generated CO2 can leave the aquarium by pure diffusion, or being used by the plants. As a result, under pH 8.2 there is a progressive reduction of carbonate hardness. If no source of carbonates is provided, the process keeps going until the carbonates hardness is equal to zero.

Note that kH consumption will be slow, in any case. Theoretically speaking, if with the injection you are able to equal the CO2 consumption done by the plants, then there is no loss of kH unless other molecules in water are having an acid/base reaction with bicarbonates. However, it is very difficult to reach such state and kH can vary due to the CO2 injection.

Hence, corrective measurements must be taken. There a few strategies to solve the issue:

(a) Doing regular kH testing and then adjusting value by adding bicarbonates to water. There are commercial products for that but dry substances like potassium bicarbonate (KHCO3) can be used. Important: Do not add them if you do not know how to calculate the amount you need for your aquarium to reach the desired dkH value.

(b) Including slightly carbonated rocks in the aquarium. Most of rocks used in aquascaping are varieties of limestome, which contains certain amounts of carbonates. Carbonates have a very low solubility in water, but this one increases when pH is low. Carbonic acid generated by CO2 injection will attack these rocks generating bicarbonates and then, increasing the carbonate hardness. However, the effect of this can be uneven within the time. It is a good idea to check the kH value at regular basis and remove/add pieces of rock as needed. The use of coral sand is strongly discouraged, as it can cause significant and relatively quick increments of pH.

(c) If your tap water has the right carbonate hardness, then as simple as making regular water changes to correct it.

For those people with high carbonate hardness in tap water, the problem can be the opposite, as the regular water changes will make difficult to have a lower carbonate hardness. In these cases, it is possible to use commercial substances to correct it, or preferably, use reverse osmosis water re-mineralized at the right levels. This is more work but more efficient than using other methods in this case.

### 12. Will oxygenation reduce the toxic effect of CO2 when reaches high concentrations?

The answer is no. As explained in the article, the uptake of oxygen by the aquatic animals is linked to their capability to eliminate CO2 from their bodies. This capability diminishes when CO2 concentrations raise into the water. Increasing oxygen levels does not neutralize or compensate this problem in any way.

However, aeration or increasing agitation will have a beneficial effect. Reason is because these methods increase the ventilation rate of water, allowing the excess of CO2 being removed from water more efficiently. The benefit in terms of fish/inverts breathing has nothing to do with the additional oxygen, but to the establishment of a CO2-reduced environment.

Oxygenation will be good, in any case, for many reasons, already pointed out in Question 8.

### 13. Is is true that you need to reach a drop of 1 pH unit to get an increment of 30 ppms of CO2 into water?

This is also a common mistake. By principia, a drop of 1 pH unit corresponds to 30 ppm of CO2 increment, so far your water is in equilibrium, which means that your starting level of CO2 in water is just the one associated to the chemical equlibrium built by adding the equivalent bicarbonates to water than the value of kH you have, and of course, assuming that pH only depends on such chemical product.

In simpler words: It is only true if CO2 concentration is only related to kH and no other effects are taking place, like CO2 injection or plants using/producing CO2. However, this is not true in most planted aquariums. To start with, CO2 injection increases the actual values until you stop injecting CO2 in a given day. During nights, large part of such CO2 is removed by degasification processes: The concentration in water is higher than should if atmosphere were the only input of CO2, and then there is a flow of CO2 from water to air. The rate of ventilation is variable and is fully possible that CO2 levels when starting the new day are not the ones in full equilibrium.

Additionally to that, plants also breath during night, which also involves CO2 generation when no light is provided. This CO2 input will avoid CO2 levels reaching the chemical equilibrium during the night, so just before lights are on, pH will be lower than should, if only bicarbonates were producing CO2.

Because of that, a drop of 1 unit of pH can imply a much larger level of CO2 than expected, or much less, depending on pH and value of kH. Some people tend to state this idea just because using pH meters becomes in an easier way to track CO2 concentrations than trying to test also kH. However, this assumption can be dangerous because only works well if full degasification is reached at nights, which is not possible to achieve.

Only simple and trusting method is to check both parameters (pH and kH) is using a drop checker or testing water. However, note that errors in the estimation of pH can lead to large errors in the estimation of CO2. Best option is a CO2 probe, but these are not commonly available in the market of aquatics, and they can be rather expensive.