Links Between Two Duckweed Species (Lemna minor L. and Spirodela polyrhiza (L.) Schleid.), Light Intensity, and Organic Matter Removal from the Water—An Experimental Study

Abstract

Duckweeds—a group of floating leaf macrophytes from the family of Lemnaceae—have become a major area of interest in the fields of basic and applied aquatic sciences in recent decades, including their use in water purification. Aiming to fulfill one of the gaps in the role of light intensity in duckweed efficiency in organic matter removal, we carried out a laboratory experiment with the use of two duckweed species: Lemna minor and Spirodela polyrhiza. Our main finding was that the intensity of light has a positive effect on the process of water purification from organic compounds by Lemna minor. However, this was not applicable to Spirodela polyrhiza due to the fact that the growth of the species was inhibited by high light intensities.

1. Introduction

The biology and ecology of duckweeds have long been a topic of great interest in a wide range of fields, including fundamental and applied aquatic sciences. The past few decades has seen the rapid development of research concerning the role of Lemnaceae plants in freshwater ecosystems and their interactions with plankton; submersed macrophytes and macroalgae; fish; and macro-invertebrates [1,2,3,4,5,6,7]. For example, in the area of shallow lake ecology, the domination of lemnids within the lake ecosystem is regarded as an example of an alternative stable state [8]. Due to their capacity for rapid colonization of freshwater ecosystems by means of vegetative reproduction, there is also increasing concern about duckweeds, being native of the Americas (Lemna valdiviana Phil and Lemna minuta Kunth), which have become an invasive alien species in European freshwaters [9,10,11].

In applied sciences, duckweeds are considered a valuable raw material used for the production of biogas, biodiesel, and animal feed, as well as for the phytoremediation of polluted inland waters. Some lemnids, due to their high starch accumulation capacity and low cellulose content, can be used as a raw material for the economically advantageous production of bioethanol [12]. Moreover, by linking the usage of duckweeds in the process of treating municipal sewage and other industrial waste, biofuel production can be carried out without the risk of water pollution and with due care for waste recycling [13]. Growing duckweed in pre-treated sewage is also a low-cost way to produce valuable animal feed [14].

The use of lesser duckweed (Lemna minor L.) as a dietary supplement in broiler diets is very profitable from an economic point of view because it can be grown directly on the farm, which results in lower production costs [15]. The species also has high absorption of amino acids and other vitamins, thus being an excellent protein source for pigs [16]. The use of Lemna minor in water purification processes has also been intensively studied. Kutera and Paruch [17] and Amare et al. [18] showed that this species is distinguished by a high reduction in the concentration of nutrients and organic matter. In a biological sewage treatment plant, duckweed acts as a natural sewage purifying agent by absorbing nutrients, mainly nitrogen and phosphorus [19].

Another duckweed species, Spirodela polyrhiza (L.) Schleid., is used, among others, in the production of biofuel, as animal feed from agricultural sewage treatment plants [20], and for the removal of toxic substances from the aquatic environment. It is also used in human nutrition [21]. Due to its ability to hyperaccumulate heavy metals and high nutrient uptake from water, S. polyrhiza is used in bioremediation [22]. Spirodela has also been found to be also an effective bioaccumulator of mercuric chloride under laboratory conditions. It shows a higher accumulation coefficient compared to Lemna gibba and L. minor [23]. Due to its efficient starch production and good growth in animal wastewater, S. polyrhiza has great potential for bioethanol production [24]. In agriculture, S. polyrhiza is used on a small scale as fish or poultry feed [25]. It can also be used in the diet of cattle, pigs, and poultry; however, problems related to contamination with heavy metals in its biomass should be taken into account [26]. According to the latest research by [27], Spirodela polyrhiza can grow well in mixotrophic conditions: the addition of glucose to the medium caused a stronger increase in the biomass of this species, as well as an increase in the starch content in its tissues. It was also noticed that higher light intensity accelerates this process, which is partially contradictory to the results of this work, where it was noticed that higher light intensity had an inhibiting effect on the development of plants of this species. This shows the need for further research on the use of different lemnid species in the context of environmental factors, including light, that regulate the growth, development, and efficiency of duckweeds. Although the effect of light on the growth of various species has been the subject of ecological studies [28,29,30], new applications of duckweeds require further research in the context of their functions used in the bioeconomy.

Aiming to fulfill one of the gaps in the role of light intensity in duckweed efficiency, we carried out a laboratory experiment with the use of two duckweed species: Lemna minor and Spirodela polyrhiza. This study therefore set out to assess the effect of light intensity on the process of water purification from organic compounds by the two above-mentioned species.

2. Materials and Methods

Two lemnid species were used in the experiment: Lemna minor L. (common name: lesser duckweed) and Spirodela polyrhiza (L.) Schleid. (greater duckweed). The plants came from the culture of the Department of Hydrobiology and Ecosystem Protection at the University of Life Sciences in Lublin, Poland. Before the experiment, the plants were kept for three weeks in a culture chamber, in a Steinberg medium [31] at a temperature of 22 °C, in a light:dark cycle of 16:8, with a light intensity of average 3230 ± 200 lux (which correspond to ~60 µmol m⁻² s⁻¹). Before use in the experiment, the plants were rinsed in distilled water (to get rid of periphyton). In order to obtain organic matter for the experiment, 10 dm³ of bottom sediment was collected from a small water reservoir collecting groundwater, rainwater, and seepage water, near the Zemborzycki Reservoir in Lublin, Poland. Bottom sediments were poured in the laboratory with 10 dm³ of tap water, mixed, and then the water above the sediment was decanted for further settling. This operation was repeated. The obtained solution (5 dm³) was then diluted with 5 dm³ of tap water and boiled to sterilize the solution. After cooling, it was filtered through a standard paper filter. Next, dissolved organic carbon concentration was measured using a PASTEL UV analyzer (Secomam, Ales, France) after the sample was filtered through a Whatman GF/C glass fiber filter.

The experiment was conducted in 36 glass containers (hereinafter referred to as experimental containers). Each container was supplemented with 100 mL of modified Steinberg medium [31]. The experimental containers were then supplemented with 200 mL of organic matter solution while control containers were supplemented with tap water. Then, all of the containers were left for 24 h (for the water to settle and the chlorine to evaporate). Each experimental and control container was filled with a similar number of plant shoots, which had been weighed (by the use of analytical balance) before they were put in. The plant biomass put in each of the containers ranged from 0.0204 ± 0.0009 g to 0.0222 ± 0.0004 g (wet weight). Then, the containers with plants were placed in a growth chamber on three different shelves. The containers were divided into six groups, three for each species, which were exposed to different light intensities on each shelf. On the top shelf (marked as L), the light intensity was the lowest—3226 ± 207 lux; on the middle shelf (M), it reached a value of 8825 ± 607 lux; and on the lower shelf (H), the light intensity was as high as 13223 ± 905 lux. After recalculating from luxes to Photosynthetic Photon Flux Density [32], we attained approximate values as follows (L, M, and H, respectively): 59.7 ± 3.8 µmol m⁻² s⁻¹; 163.2 ± 11.2 µmol m⁻² s⁻¹; and 244.6 ± 16.7 µmol m⁻² s⁻¹. The light used in the experiment was thus slightly below what is optimal for Lemna minor growth, which appears to be between 250 and 300 µmol m⁻² s⁻¹ [28]. Different light intensities were obtained by using one, two, and three GLASS LED PLANT 10 W lamps (Aquastel, Prudnik, Poland) on the shelves, respectively. The lamps had an extended range of light radiation (450–700 nm) thanks to the use of additional blue and red diodes. In each group, three experimental containers were distinguished (serving as replications), and similarly three containers were distinguished as controls. The containers within a given light intensity were placed randomly.

To asses the impact of light intensity on the process of organic matter removal, we measured dissolved organic carbon (DOC, mg dm⁻³) concentration in water after 7 and 14 days and at the end of the experiment (after 21 days) using the device mentioned above. At the end of the experiment, all lemnid shoots were weighed using an analytical balance. On the last day of the experiment, chlorophyll-a concentration in water was also determined in all tanks, after removing the plant shoots (ethanol method, [33]).

Duncan’s test was used to determine the significance of differences (p < 0.05) between individual tanks. Analyses were performed using Statistica 13.3 software.

3. Results

3.1. Biomass of Duckweeds

The biomass of duckweeds at the start of the experiments ranged from 0.0209 ± 0.0008 g to 0.0222 ± 0.0004 g in the case of Lemna and from 0.0204 ± 0.0009 g to 0.0219 ± 0.0019 g regarding Spirodela (averages,

Table 1. Biomass (g) of duckweeds at the start of the experiment. Mean values ± standard deviation (L, M, and H—low, medium, and high light intensities, respectively).

	Control			Experimental
	L	M	H	L	M	H
Lemna minor	0.0222 ± 0.0004	0.0212 ± 0.0013	0.0210 ± 0.001	0.0209 ± 0.0008	0.0219 ± 0.0003	0.0218 ± 0.0006
Spirodela polyrhiza	0.02105 ± 0.00035	0.0209 ± 0.0004	0.0219 ± 0.0019	0.0212 ± 0.0009	0.0208 ± 0.0001	0.0204 ± 0.0009

).

On the last day of the experiment (after 21 days), the observed values of lemnids biomass were diverse. The fresh mass of Lemna minor ranged from 0.0548 g to 3.0020 g, and we noted huge differences between the control and experimental tanks (Figure 1). In the control tanks, a significant (p < 0.01, Duncan’s test) relationship between light intensity and the final fresh mass of plants could be observed: at low light intensity (L), the biomass value was the lowest, and in M samples—average and in H samples—it was the highest (eight times higher in H than in L). However, the relationship was not found in the experimental tanks, where there were not many variations between the samples, although a statistically significant difference was found between samples L and M (p < 0.05, Duncan’s test) (Figure 1).

Fresh mass of Spirodela polyrhiza on the last day ranged from 0.3164 g to 0.9325 g. There were small differences between the control and experimental tanks (Figure 2). In the control tanks, the biomass values at low and medium light intensity were very similar, while a much lower value (statistically significant, p < 0.05, Duncan’s test) could be observed at high light intensity. In the experimental tanks, a similar relationship was found between samples N, S, and W (Figure 2).

3.2. Chlorophyll-a Concentration in Water

The concentration of chlorophyll-a in the tanks with Lemna after 21 days of the experiment ranged from 28.8 µg dm⁻³ to 395.9 µg dm⁻³ in the control tanks, while in the experimental tanks it was much higher and ranged from 252.3 µg dm⁻³ to 652.4 µg dm⁻³ (Figure 3). However, in both sets of tanks, a similar pattern was observed: the concentration of chl-a at M and H light intensity was at an approximate level and was higher than at low light intensity. This was most visible in the control tanks, where chl-a concentrations in the L tanks were significantly and several times lower than those in the M and H tanks (p < 0.05, Duncan’s test) (Figure 3).

Much higher chl-a concentration was observed in Spirodela tanks after 21 days of the experiment, and the range was from 325.5 µg dm⁻³ to 920.0 µg dm⁻³ (Figure 4). There were significant differences between the control and experimental tanks. In the case of the control tanks, the lowest concentration of chl-a was observed in H tanks (373.6 ± 69.9 µg dm⁻³), while at L and M light intensity this concentration was statistically significantly higher (p < 0.05, Duncan’s test), and these values were at a similar level (836.6 ± 35.3–860.7 ± 83.8 µg dm⁻³, L and M, respectively). In the experimental tanks, chlorophyll-a concentrations at low, medium, and high light intensity were at a similar level as well (Figure 4).

3.3. Dissolved Organic Matter Content in Water

As a measure of the concentration of dissolved organic matter in water, we used the concentration of dissolved organic carbon (DOC, mg dm⁻³). The initial value amounted to 17.75 ± 0.64 mg dm⁻³. Then, we measured it after 7 and 14 days as well as at the end of the experiment (after 21 days). We observed a very similar pattern of DOC content dynamics in both experimental sets (Lemna and Spirodela, Figure 5 and Figure 6). In both cases, the DOC values were clearly higher for low light intensities (L), as compared to the M and H sets. We also observed clear changes over time in L sets. After seven days of the experiment, the DOC values were in the range 62.83 ± 4.48 mg dm⁻³–63.66 ± 2.56 mg dm⁻³, for Lemna and Spirodela, respectively). Then, an increase was observed after 14 days, up to 102.5 ± 6.36 mg dm⁻³–106.0 ± 11.35 mg dm⁻³ (as above). In turn, at day 21, the concentration of dissolved organic carbon dropped down and amounted to 25.86± 13.23 mg dm⁻³–31.3 ± 11.48 mg dm⁻³. In both species sets, in the case of M and H light intensity, there were no significant differences between the seventh, fourteenth, and twenty-first day (Figure 5 and Figure 6).

4. Discussion

The results of the experiment showed that Lemna minor developed better in the control tanks (only the nutrient solution) than in the experimental tanks (with the addition of organic matter). Moreover, in the control tanks the development of lesser duckweed was directly proportional to the light intensity in which the plants were grown. This is consistent with the literature data. In the publication on the use of Lemna minor in bioethanol production [34], it was found, for example, that duckweed biomass and starch production increased with increasing light intensity. Similarly, the experimental studies by Tabou et al. [28] showed that light intensity was the main factor to be taken into account for the growth of lesser duckweed. A series of other laboratory-scale experiments have shown that reductions in light intensity, temperature, and available nutrients can negatively impact duckweed growth rates, although a significant amount of biomass can still be accumulated for extended periods under these limiting environmental conditions [30]. However, no such relationships were found in the case of Spirodela polyrhiza, where plant development was similar, regardless of the addition of organic matter. Furthermore, it was observed that in the control tanks, plant development was clearly lower at the highest light intensity (244.6 ± 16.7 µmol m⁻² s⁻¹).

These findings are in agreement with literature data: in general, light saturation for the optimum growth rate in lemnids ranges from ~70 to ~200 µmol µmol m⁻² s⁻¹ [32]. It was shown in one of the experimental studies that S. polyrhiza reached the maximum growth rate at ~150 µmol m⁻² s⁻¹, while L. minor needed twice as much light intensity to do the same [35]. These findings were also validated by Strzałek and Kufel [29], who found not only that greater duckweed grew slower than Lemna minor but also that both species grew the fastest at medium light intensity. Moreover, while increasing the level of light (>236 µmol m⁻² s⁻¹), Lemna maintained a high growth rate, while the growth rate of Spirodela declined significantly. It was also hypothesized that L. minor, being a smaller species, performed better than larger S. polyrhiza in similar light conditions. Lemna minor is also regarded as a species with high ability to grow under varying light conditions [36]. Low photon flux is accompanied in this species’ leaves by a decrease in the chlorophyll a:b ratio but an increase in total chlorophyll concentration [37], which resulted in minor duckweed shade-tolerant species.

The measured concentration of chlorophyll-a in water at the end of the experiment indicates that in tanks with Lemna minor, the development of algae was faster in the experimental tanks (with organic matter) than in the control tanks (nutrient solution only). The reason for this phenomenon is certainly the additional pool of phosphorus supplied to the experimental tanks together with the organic matter, which was released into the water together with the development of microorganisms transforming particular organic matter into mineral and assimilable forms of nutrients for algae [38,39]. The biomass of duckweed in the experimental tanks was also lower compared to the control tanks, which indicates the existence of competitive relationships between both groups of primary producers, which is often observed in natural water bodies [7]. Experimental studies had shown that when the algal community was added to Lemna water culture, strong depletion of phosphorus, nitrogen, iron, and manganese was observed in the water, which in consequence slowed down the growth of plants as well as negatively influenced chlorophyll-a content in Lemna leaves [40]. On the other hand, duckweeds may outcompete algae within the area of light availability—when Lemna plants grow over almost all the surface, algal development is slowed down [41]. The competition between these two groups is thus related to nutrient content—in higher nutrient concentrations, floating plants will probably successfully suppress the algae, so the nutrient depletion does not occur. Unfortunately, we did not measure nutrient concentration during the experiment, so our conclusions on that topic are only speculative. In tanks with Spirodela, no clear pattern regarding the relationship between algal chlorophyll-a concentration and plant biomass was observed. There are some reports from the laboratory experiments in which greater duckweed showed strong capabilities in the growth suppression of cyanobacteria Microcystis—both at low and high levels of nitrogen content [42]. The difference between Lemna minor and Spirodella polirhyza in their relations with algae in the water in the experiment may be caused by the greater dimension of the last one, causing a slower growth rate, which was additionally enhanced by excessively light intensity [29].

Nevertheless, the most significant finding concerns the relationship between the concentration of DOC and the biomass of lemnids. The DOC content on the last day of the experiment was the highest in L tanks, which corresponded to the lowest values of lesser duckweed biomass recorded in the lowest light intensity. Moreover, low DOC values at medium (M) and high (H) light intensity corresponded to higher values of Lemna minor biomass. Taking these together, it should be concluded that there was a kind of relationship between the decrease in DOC content in water and the growth of lesser duckweed plants.

Also interesting, although rather unexpected, was the increase in DOC concentration at the beginning of the experiment. It occurred between day 7 and day 14, in both types of tanks (Lemna and Spirodela), but only at low light intensity. An increase in DOC in lemnid cultures during the experiments was previously reported by Ng and Chen [43]. This may be explained in different ways. It is known that higher plants may secrete organic substances to regulate microbial community or to suppress the growth of other plant species [44,45]. Although there are no reports confirming such a phenomenon observed either in Lemna nor Spirodella, it is known that smaller duckweed tissues contain a lot of potential allelopathic substances [46]. The second source of DOC in water might be connected with the death of the plants that occurred in the beginning of the experiment due to some kind of adaptation stress, especially in those plants that were exposed to low light. The release of water-soluble organic substances from decaying plant tissues is a well-known phenomenon in aquatic ecosystems [47].

As was stated before, on the basis of the results it can be proposed that the intensity of light has a positive effect on the process of water purification from organic compounds by lesser duckweed (Lemna minor) but not by greater duckweed (Spirodela polyrhiza), which was one of the main aims of the work. What could be the hypothesized mechanism leading to the decrease in DOC concentration while the increase in Lemna biomass was observed? First, species from the genus Lemna are known to have the capability to uptake organic substances. Reports within this topic covers a variety of organics, including, among others, amino acids, pesticides, or cyanobacterial toxins [48,49,50]; review in [51]. Second, the degradation of organic substances may be enhanced both by additional oxygen supply to the water and, what is probably the most important factor, creating additional surface for bacterial growth, which in consequence enhances the process of microbiological organic decomposition [52].

However, it is difficult to formulate such a hypothesis in the case of Spirodela polyrhiza due to the fact described earlier that the growth of this species was inhibited by high light intensities. Nevertheless, with a small sample size, caution must be applied to our conclusions, and further research needs to examine the links between duckweed species, light intensity, and organic matter removal from the water more closely.

5. Conclusions

• Two studied duckweed species showed different patterns of growth response to different light intensities. Lemna minor development was directly proportional to the light intensity: it grew better in better light conditions. Spirodela polyrhiza, by contrast, appeared to have had its growth suppressed in the highest light intensities;
• Lemna minor growth was weaker in tanks with added organic matter as compared to control tanks. In contrast, strong development of algae in the water was observed in these tanks, which suggests the existence of a competitive relationships between both groups of primary producers;
• Dissolved organic carbon concentration at the end of the experiment was the highest in tanks with the lowest light intensity and where the growth of Lemna minor was weaker. In reverse, low DOC values at medium and high light corresponded to higher values of this duckweed biomass there. This may suggest that light intensity played a positive role in a process of organic matter removal by Lemna minor.
• In the case of Spirodela polyrhiza, no clear pattern was observed in the context of organic matter removal due to the suppression of its growth in conditions of high light intensity.