*2.1. Coccomyxa acidophila enhanced growth on urea*

It was mentioned above that acidophile microalgae have so far never been used for massive production. Massive production requires fast growing microalgal strains. Most acidophile microalgae are slow growth strains, as reported in the literature [5,17]. However, the growth of acidophile microorganisms in acidic culture media becomes advantageous for biomass production, as under such conditions the growth of other microorganisms becomes difficult. Therefore, we attempted to find culture conditions under which *Coccomyxa acidophila* cultivation is enhanced, such that growth rates and productivity values approached those of common microalgae. In such a situation, the acidophile microalga should show fastgrowth and could hopefully be grown in outdoor systems with limited risks for microbial contamination, in comparison to common microalgae. 

One of the main growth conditions assayed was nitrogen source. In previous experiments, we first tested the effect of adding ammonium, nitrite or nitrate to photoautotrophically growing *Coccomyxa acidophila* cells. Growth on ammonium and nitrate resulted in the highest productivities. Unlike common microalgae, nitrite became toxic for *Coccomyxa acidophila*. In the experiments here, we also used urea as a combined source for C and N, with high CO2 concentration (5% v.vƺ1) as the main carbon source where indicated. Urea has been widely used instead of high CO2ƺ for sustaining microalgae growth and is also a cheap N source. As shown in Figure 1, urea promoted enhanced growth of *Coccomyxa acidophila*, both in terms of chlorophyll content (Figure 1A) and cell density (Figure 1B). This resulted in an increased growth rate with respect to control cultures (photoautotrophically grown on nitrate), as shown in Table 1. In addition, culture productivity was higher when the microalga was grown on urea (the highest productivity) and ammonium. Specifically, the highest productivity was reached in cultures grown on 0.67 g·L 1 urea ("control; air" in Figure 1). This urea concentration provided cultures with the same molar concentration of nitrogen than the nitrate added to control cultures. More interestingly, the best productivity values obtained from the microalgal growth on urea did not differ from those usually obtained for most of common microalgae (0.2–0.4 g·L 1·d 1). Unexpectedly, the simultaneous presence of urea and nitrate limited *Coccomyxa acidophila* growth. This will be discussed further. 

**Figure 1.** Time-course of chlorophyll ( **A**) and cell density ( **B**) in *Coccomyxa acidophila* cultures grown on nitrate, urea or nitrate plus urea. Air alone or CO2 in air (5% v/v) were used as carbon source, as indicated for each culture within the Figure legend. 

**Table 1.** Growth rates and productivity of *Coccomyxa acidophila* grown on different N-sources. 


The previous results were obtained by means of using mixotrophic or photoautotrophic cultures, *i.e.*, either urea or nitrate were added to culture media as N sources while high CO2 concentration (5% v/v in air) was supplied as a carbon source (first set of experiments). It has also been discussed that the microalga cells could make use of urea as an additional carbon source [18,19], perhaps being one of the reasons behind the improved microalgal productivity of urea grown cultures. Low CO2 solubility at acidic pHs makes carbon uptake more difficult than at pH 7 (standard pH for most common microalgal cultures). Therefore, the supply of additional carbon in a soluble form at low pH (e.g., urea, glucose) could help to increase microalgal productivity. This raises the question of whether addition of glucose as a carbon source to cultures of an acidophila microalga should also increased microalgal productivity. Such a question was investigated by our group in previous research [1] and the answer was "no". Urea should by far allow maximum productivity in *Coccomyxa acidophila* cultures when used as a carbon source.

From the results above, *Coccomyxa acidophila* apparently prefers urea to nitrate as nitrogen source. Therefore, another question we addressed was whether such consumption preference indeed occurred. For this purpose, nitrate and urea consumption were followed in time in photoautotrophic cultures to which nitrate (control culture) or urea and nitrate (with the same molar nitrogen concentration to that used in control cultures, 22.7 mM), were added. Results are shown in Figure 2. If urea and nitrate are added simultaneously, nitrate only started to be consumed at late exponential growth phase while urea was first consumed as the only nitrogen source. A decreasing time-course trend in urea concentration is observed from the beginning of the experiment, whereas the nitrate concentration time-course trend remains stable. Inhibition of nitrate consumption by the presence of urea has been reported to occur in microalgae, though not many references dealing with the subject have been published. Cochland and Harrison [20] reported about 30% inhibition of nitrate consumption by urea in the eukaryotic picoflagellate *Microsomas pusilla*. Following consumption, assimilatory reduction of nitrate also could be inhibited. One of the first classic references was published by Smith and Thompson [21] who observed 70% nitrate reductase inhibition by urea in *Chlorella*, evidencing nitrate assimilatory reduction down regulation to be behind nitrate consumption inhibition by urea. 

As already mentioned, simultaneous addition of urea and nitrate as nitrogen sources slightly limits cell growth. Merigout *et al.* [22] evidenced in *Arabidopsis* plants that urea uptake was stimulated by urea but was reduced by the presence of nitrate in the growth medium. Such conclusions from their recent study on physiological and transcriptomic aspects of urea uptake and assimilation are in good concordance with the following observations from our results: (a) urea increased *Coccomyxa acidophila* growth and (b) simultaneous presence of urea and nitrate resulted in a decreased uptake of urea and culture productivity. These observations related to nitrogen uptake regulation in *Coccomyxa acidophila* are for the first time reported in acidophile microalgae and suggest that urea uptake and assimilation patterns in extreme acidophile microalgae (living in fully urea-free environments) and plants are similar. Further experiments in nitrogen assimilatory enzymes and gene expression are currently being developed in our group. 

**Figure 2.** Time course of nitrogen consumption in *Coccomyxa acidophila* cultures grown on nitrate, urea or nitrate plus urea. Air alone or CO2 in air (5% v/v) were used as carbon source, as indicated for each culture within the Figure legend. Dotted line with triangles corresponds to timecourse of nitrate consumption of cultures incubated with nitrate plus urea. 

To determine whether simultaneous addition of urea and nitrate to the algal cultures has any impact on photosynthesis, relative electron transport rates were determined in each of the cultures (namely, control –nitrate ; urea; urea and nitrate, as nitrogen sources, respectively). Results are shown in Figure 3. Surprisingly, there was no nitrogen source-dependent impact on PS2 and on photosynthetic energy production chain, if the light intensity remained approximately below 150 μE·mƺ2·sƺ1. However, in urea grown cultures incubated under higher light intensities, photosynthetic energy production is shown to be clearly limited, up to the point that the electron transport chain becomes inhibited. This dramatically influences carbon assimilation and culture productivity. 

According to our results, urea appears to be a suitable nitrogen source for *Coccomyxa acidophila* growth at relatively low light intensity; however, it has a dramatic impact on the photosynthetic energy production chain when exposed to high light intensity, which has never been reported for any other microalga. This is currently under study in our laboratories. 

**Figure 3.** Light-dependent electron transport rates in *Coccomyxa acidophila* cultures grown on nitrate, urea or nitrate plus urea. Air alone or CO2 in air (5% v/v) were used as carbon source, as indicated for each culture within the Figure legend.

In addition, physiological responses of acidophile microalgae to urea and nitrate uptake processes anyhow differ, according to the observed pH changes in the culture media which tend to increase if nitrate (2.3 g·L 1) is the only added nitrogen source and to decrease if urea (0.67 g·L 1) is used (data not shown). So far, we have no evidence for antyport/symport mechanism details that help to elucidate the different physiological behavior. 
