By Ole Pedersen, Claus Christensen and Troels Andersen

Poor growth in plant aquaria has usually been attributed to insufficient light over the tank and when asking the experts, the advice has always been to increase the light availability before any other action is taken. New research shows that this may be poor advice, in particular, for an aquarium without CO2 fertilisation
 
Even in modern textbooks, you may still meet the allegation that only one resource may limit plant growth at a time. This has also been known as the principle of Liebig. Justus Liebig was a famous German chemist that among many things worked with the nutrition of agricultural plants. He postulated that one and only one factor could limit plant growth at the time. It is unclear whether Liebig himself developed the ancestor to Figure 1, but the simplicity of the barrel, which is partly filled with water, has greatly contributed to maintaining this perception of resource limitation. For terrestrial plants this has been known to be wrong for several decades and also within the aquatic plant sciences co-limitation of resources has been an accepted principle for at least twenty years. Few aquatic studies have shown that the interacting effect of light and CO2 may translate from photosynthesis into effects on growth (Maberly 1985, Madsen and Sand-Jensen 1994). In this paper we show data from experiments where we have created co-limitation of CO2 and light, which in nature are the two main limiting factors for aquatic plant growth. 
 
To understand how plants respond to incident light, the general light utilisation of an aquatic plant is schematically shown in Figure 2A. At very low light intensities, the incident light is insufficient to sustain a positive photosynthesis and the net oxygen budget of the plant is negative. In other words, the respiration processes exceed the photosynthesis. At a certain light level, however, the two processes equal each other and we have then defined the light compensation point of the plant. By illuminating the plant with still higher light intensities the photosynthesis is also positively stimulated in linear way. At high light, the resulting outcome from the photosynthesis becomes less until it finally levels out at a point where we have the maximum photosynthesis. From this point on, more light will not translate into a greater photosynthesis.
In nature, growth of aquatic plants is often limited by the availability of light. Light is efficiently absorbed by water itself where light energy is transformed into heat and if the water also contains some dissolved organic substances - for example humic acids in black waters - the light absorption from the water itself can be very efficient. The light absorption of water and of the substances dissolved herein sets the depth limit of aquatic plant growth in nature and the transparency of water can sometimes turn so bad that it excludes all submerged vegetation so that only floating and emergent plants can thrive. Since light is such an important competitive parameter, evolution has developed a very efficient system of light utilisation in submerged plants. If the plant has sufficient nutrients available, it often invests more energy into light capturing pigments carotenoids, Xanthophylls and more important, Chlorophyll. Chlorophyll is the green pigment that absorbs light and transforms it into chemical energy, which can be used for growth in the cells. By doing that the plant ensures that the light, which is actually reaching the plant surface, is absorbed and used for energy purposes instead of being merely transmitted through the plant tissue. It is also necessary to contain a lot of Chlorophyll to obtain a large maximum photosynthesis but a large pool of Chlorophyll is not of much use if the energy produced cannot be used to fix inorganic carbon into sugars and carbohydrates.

Aquatic plants usually have access two sources of inorganic carbon: carbon dioxide (CO2) and bicarbonate (HCO3-). Most aquatic plants prefer CO2 rather than bicarbonate because it can be taken up from the surroundings without any energetic expenses and many aquatic plants are not able to directly utilise bicarbonate in the photosynthesis. The general CO2 utilisation of an aquatic plant is schematically shown in Figure 2B. The curvature of the curve is a little different from the light curve in that it is not linear even a very low CO2 concentrations. Apart from that, we can also define a CO2 compensation point as a point where CO2 concentrations below this point result in a negative net photosynthesis whereas concentrations above this point result in a positive net photosynthesis. One of the curves has a CO2 point of zero. This illustrates the bicarbonate user, which can continue to carry out positive photosynthesis even at zero CO2 because it can use bicarbonate as a source of inorganic carbon.

In nature, the concentration of CO2 is often larger in water than in air but in spite of that the actual availability to the aquatic plant is lower. This is because of the slow movement of gasses in water where the diffusion is 10,000 times lower in water than in air. Hence, although the concentration of CO2 in many streams and rivers may be larger than in air, the slow movement of gasses in water eventually leads to CO2 limitation of aquatic plant growth. The thin leaves typical of submerged plants greatly alleviate the CO2 limitation. This is partly because thin leaves hold thinner boundary layers through which CO2 must diffuse and partly because once the CO2 enters the leaf it does not have to travel long before it is fixed to carbohydrates in the photosynthesis. Of much more importance is the ability of the plant to up- or down-regulate the different pools of enzymes (fx Rubisco and Pepcarboxylase) that take part in the carbon fixation. At low CO2 availability, the plant may invest more energy in enzymes, which help in process of CO2 uptake or CO2 fixation and thereby alleviate the effect of carbon limitation. Some plants are also able to produce isoenzymes, which are enzymes with different chemical optima and so the affinity for CO2 may be changed to favour CO2 uptake. It is unclear how important isoenzymes are in the process of CO2 uptake and in the scientific literature isoenzymes are often associated with temperature acclimatisation in plants. 
 
The ecological implications of interactions between light and CO2 are obvious. If for example enhanced CO2 availability increases the light use efficiency it may allow aquatic plants to penetrate to greater depth where the light is scarce but concentrations of CO2 are higher due to remineralization in the sediment. High light availability may also allow aquatic plants to lower the CO2 compensation point (Maberly 1983, Maberly 1985). This may be particular advantageous for mat-forming photoautotrophs in shallow water. In such systems, the light is often abundant whereas concentrations of CO2 inside the mat are low due to low intra-mat water exchange. Here, the interactions between light and CO2 may allow the photosynthesising organisms to extract CO2 more efficiently as a result of a lowered CO2 compensation point. 

Adaptation to resource limitation is, however, a costly affair. Whether the plant invests in more chlorophyll or more enzymes it results in higher nutrient requirements and higher energy use. The higher energy use comes from the fact that proteins require constant maintenance in the cell to work properly and these maintenance processes absorb valuable energy and carbohydrates, which might otherwise be use for growth purposes. Therefore, not all the inorganic carbon that is fixed to carbohydrates in photosynthesis can be used for growth purposes.
We were interested in determining which factor - CO2 or light - was the more important limiting growth factor for aquatic plants. In addition, we set out to answer the question of whether CO2 or light could act as a substitute for one another and thereby increase growth by fx. Adding more CO2 even under strong light limitation or vice versa. To answer these questions experimentally, we grew Riccia fluitans in a cross-factorial design under saturating nutrient conditions (by nutrients we mean nitrogen and phosphorus and all the micro nutrients). We designed the experiment so that we could regulate light and CO2 availability independently and Table 1 shows the experimental matrix where we had a total of 9 treatments differing solely in light and CO2 availability. Low light corresponds to the conditions found near the depth limit of aquatic plants and in many standard equipment aquaria. High light corresponds to light levels found in nature near the water surface or in an extremely well illuminated aquaria with high-pressure mercury lamps or halogen lamps. In fact, the medium light intensity in this study corresponds to a standard well illuminated plant aquarium. Low CO2 corresponds to the CO2 concentration found in many lakes or in an aquarium without CO2 fertilisation - but with an aerating pump running. High CO2 is 40 mg/l, which can be found in many small groundwater feed streams and it is also the maximum level recommended by most experienced plant aquarium keepers. We used Riccia fluitans as a model plant in our experiments mainly because it is easy to grow. Hence, we could produce a large number of replicates, which subsequently allows us to draw more rigid conclusions from our experiments.

Table 1 shows the results of these experiments expressing the growth rate of Riccia fluitans in percent per day, while assuming exponential growth over the period (1). We see that at low light and low CO2 Riccia fluitans is barely able to maintain a positive growth rate whereas at low CO2 and high light the growth rate is almost 6 fold higher. More importantly, at low light levels the addition of CO2 is able to stimulate the growth by a factor of almost 4!
Maybe a stimulation of the growth rate by a factor of 4 to 6 does not seem much but because of the exponential nature of the growth rate it really makes a difference over a period of, for example, two weeks. Figure 3 shows how 1gram of Riccia fluitans develops over two weeks with four different growth rates. Low light and low CO2 barely result in positive growth over the two weeks, whereas the treatment with high CO2 and low light translate into almost a doubling of the tissue weight. For comparison, the high light and low CO2 result in 2.5 gram of tissue after two weeks. It is needless to say, that the benefit from increasing both light and CO2 surpass the effect of raising only one parameter. At the highest light and CO2 availability, 1 gram of Riccia grows into astonishing 6.9 gram after two weeks. Surprisingly, the stimulation observed on the growth rate when both light and CO2 are increased is larger than the additive contribution from each individual parameter. Example: The growth rate at low light and low CO2 is 1.1% per day. By increasing light, the plant grows 3.3% per day or an additional 2.2% compared to the starting condition. Similarly, by increasing CO2 the growth rate is now 3.8% per day or an additional 2.7%. An additive relationship would then translate into 6.0% per day (1.1 + 2.2 + 2.7) but the resulting growth rate from combining light and CO2 is 9.2% per day, which is significantly larger. This tendency also holds when moving even higher up in light and CO2 (see Table 1).

(1) By assuming exponential growth, we can use this formula to calculate the growth rate: µ = (ln W1 - ln W0) / t, where W0 is the weight of the plant tissue when the experiments begins, W1 is the weight after the incubation and t is the incubation time in days. The background for assuming exponential growth is the fact that all new tissue formed during the incubation in principle may form new tissue and so on.

Figure 4 shows a conceptual explanation of our findings. At low light and low CO2 there is not much energy to play around with for up or down-regulation of the pools of Chlorophyll or enzymes. If we then add a little more CO2 to the system the plant can afford to invest less energy and resources in CO2 uptake and that leaves more energy for optimising the light utilisation - more Chlorophyll can be produced without fatal consequences for the energy budget. Hence, although we have not raised the light, the plant can now utilise the available light more efficiently. Exactly the same explanation can be used to explain why increased light can stimulate growth even at very low CO2 concentrations. With more light available, less investment in the light utilisation system is necessary and the free energy can be invested into a more efficient CO2 uptake system so that the CO2, which is present in the water, can be more efficiently extracted.

We believe that our findings for Riccia can be extrapolated to most aquatic plants and the last decade has also brought more scientific evidence supporting this idea. There have been experiments with Elodea canadensis and Callitriche sp. showing the same tendency (see the literature list) suggesting that resource limitation is not as simple as Liebig suggested. Many resources are able to substitute for each other or at least alleviate the symptoms of resource limitation. Seeing these data in a more global aspect, we may expect that increasing atmospheric CO2 may actually lead to increased plant production on the earth. However, we can also foresee serious side effects. Plants growing at elevated CO2 could dilute their pools of carbon fixing enzymes and that would lower the nutrition value of crops since enzymes are proteins. For aquatic plants, a doubling in atmospheric CO2 probably won't have any effect on plant production because most aquatic plants already grow under supersaturated CO2 conditions. For those few that do not, namely the submerged lake plants, it is difficult to predict the influence from increased CO2 availability because here we have competition from lake phytoplankton.
One may ask how we can use all the above information in the plant aquarium hobby! In many ways, the modern plant aquarium resembles our experimental set-up with Riccia. Although all the individual resources are difficult to control perfectly, we are able to determine how much light, how much CO2 and how much nutrients in the form of nitrogen, phosphorus, iron and micronutrients we would like to offer our plants. Starting with the nutrients, an average plant aquarium with a decent fish population usually has sufficient nitrogen and phosphorus. When it comes to iron, potassium, manganese and other micronutrients it is often a trickier thing. Some aquaria are well planned from the beginning with, for example, laterite and other fertilizers in the substrate whereas others are not. In most cases, however, an aquarium plant fertilizer without nitrogen and phosphorus may safely be added to maintain healthy growth. It is often a much more difficult and expensive task to provide adequate light over the plant aquarium. Both fluorescent light and highpressure-quicksilver lamps may produce sufficient light if supplied with effective reflectors but in deep aquaria (more than 50 cm) is very difficult to offer enough light to small light demanding foreground plants. Based on our experiments, we suggest commencing CO2 addition before any other action is taken! We believe that even at very modest light intensities you will experience a conspicuous change in plant performance in your aquarium. The exact amount CO2 may always be discussed but if you do not have very sensitive fishes in your fish stock, concentrations from 25 and up to 50 mg/l will only improve plant growth. You will probably see that plants, which were barely able to survive before now, thrive in the presence of CO2.
 
 
Literature
Andersen (1999) Interactions between light and inorganic carbon stimulate the growth of Riccia fluitans L. Report from The Freshwater Biological Laboratory, University of Copenhagen (e-mail tandersen@zi.ku.dk), in Danish.
Maberly (1983) The interdependence of photon irradiance and free carbon dioxide or bicarbonate concentrations on the photosynthetic compensation points of freshwater plants. New Phytologist 93: 1-12.

Maberly (1985) Photosynthesis by Fontinalis antipyretica. I. Interaction between photon irradiance, concentration of carbon dioxide and temperature. New Phytologist 100, 127-140.

Madsen (1993) Growth and photosynthetic acclimation by Ranunculus aquatilis L. in response to inorganic carbon availability. New Phytologist 125: 707-715.

Madsen and Sand-Jensen (1994) The interactive effect of light and inorganic carbon on aquatic plants growth. Plant, Cell and Environment 17: 955-962.
 

Figure 1 The figure shows the classical illustration of Liebig's principle. In this particular case, the element Bor is limiting plant production and water is running out of the barrel when the growth limitation is reached.

Figure 2A and B The figure shows the theoretical relationship between light and photosynthesis (A) and CO2 and photosynthesis (B). In both situations, a saturation functions describes the relationship although the actual shape of the function differs.

For both resources a compensation point is defined as the level where the net photosynthesis or growth is zero. Below this point, the plant cannot maintain its biomass. In a saturation curve, there is also a point at which an increase in resource availability does not result in increased photosynthesis. See the text for further explanation.

Table 1 The experimental matrix showing the experimental design and the resulting growth rates as percent per day. When moving horizontally from left the right the light availability increases and when moving vertically from top to bottom the CO2 availability increases. Low light corresponds to the situation in many standard aquaria and low CO2 corresponds to an aquarium with an aerating pump (air saturation). For comparison, full sunlight in Northern Europe is about 70000 lux and a natural small stream may contain up to 50 mg CO2 per litre. Both lux and mg/l is rather old units for light and CO2 and these units are not found in scientific literature. For an exact conversion from scientific units to these general units the following factors may be used: for light (400 to 700 nm) 1 µmol photons m-2 s-1 = 60.6 lux and for CO2 1 mmol l-1 = 44 mg / l.

Figure 3 The figure shows how 1 gram of Riccia develops over two weeks under the given light and CO2 levels. At low light and low CO2, Riccia is barely able to maintain the biomass and 1 gram turns into 1.16 gram after two weeks (the white line). At low light and high CO2, 1 gram turns into 1.75 (the green line) and at high light but low CO2, 1 gram turns into 2.41 gram. The combination, however, paramounts the effect from the individual resources and at high light and high CO2, 1 gram turns into 6.90 gram.

Figure 4 This conceptual diagram shows how the external resource availability affects the internal expenses bound to light capture and CO2 assimilation. Put in this simple way, the balance between income and expenses determines the outcome.