1. Introduction
Meeting nutritional demands of a continuously growing human population while maintaining a human-friendly environment poses a global problem today [
1]. The growing demand for protein derived from animal products—meat, milk, eggs, has contributed to the intensification of animal production across the world [
2,
3]. The livestock population has increased, and 90% of these animals are reared in the industrial farming system and fed with standardized feedstuffs [
4]. It is therefore necessary to ensure greater availability of plant-derived feed materials capable of covering energy and protein demands for the production of feed mixtures used in animal feeding. Among the many components commonly used in animal feed mixtures, soybean meal serves as the most valuable source of protein [
5]. Other meals, like those made of rapeseed, sunflower, or legume seeds, are used less frequently and rather as additives to soybean meal [
6]. The storage and disposal of animal waste, i.e., manure, dung, and slurry, is a serious issue for environmental protection strategies [
7]. The European Union (EU) promotes an initiative to minimize waste and greenhouse gas emissions by converting waste into energy and other re-usable sources [
8]. Therefore, the possibility of combining individual components of animal production into one closed circuit, namely using farm animal excreta to produce feed mixtures that can further be used in animal feeding/fattening [
9], is currently extensively sought.
As part of the recovery of fertilizing substrates from various growth media, aquatic plants can be used for the treatment of various types of wastewater [
10,
11,
12]. Duckweed has been found to be important in sustainable production and has spurred social interest due to its unique morphological characteristics and phytoremediating potential. It is a small plant composed of several leaves that is free-floating—it floats on the surface, quickly multiplies, and easily adapts to different environments [
13,
14]. Duckweed is commonly used for wastewater treatment at Lemna-type treatment plants [
15]. Its application for water purification has been driven by its morphological features: a well-developed root system and rapid vegetative growth as well as ease of harvesting and further management [
16]. The growth rate of duckweed in various growth media is determined by the media’s abundance in nutrients (nutrient concentrations) and environmental conditions (temperature and pH of the medium, insolation, day length, and wind speed) [
17,
18]. Under optimal growth conditions, duckweed produces large amounts of biomass rich in protein and nutrients and can therefore be used in commercial livestock and aquaculture feeding [
19]. Large-scale production of duckweed takes place in tropical countries, where natural conditions are appropriate for its rapid growth and high biomass production within a relatively short time span [
20]. In animal feeding, it can be used as a meal (dried) or in a natural form (fresh green biomass). Duckweed production can provide 4–5 times more protein per hectare than soybean cultivation [
21]. Other advantages of this aquatic plant include: (1) it is not genetically modified; (2) it does not contain gluten; and (3) it does not require arable land or the use of mineral (artificial) fertilizers. In contrast, potential threats include heavy metals, dioxins, phenols, pesticides, and pathogens it can accumulate [
22].
Natural fertilizers are very valuable nutrient-rich by-products of animal production and are useful for crop fertilization. The content of nutrients in fertilizers varies and depends on many factors, including animal species and breed, feed mixture type, and bedding in terms of both its type and use. In order to preserve the highest fertilizing value, certain methods are recommended regarding the collection, storage, and use of natural fertilizers [
23]. Slurry is a mixture of feces and urine with an admixture of water and is generated during pig and cattle rearing. The problem faced by producers of these animals is to provide appropriate tanks for its storage and sufficiently large areas of arable land for its agricultural management. Excess volumes of slurry applied to arable land lead to excessive accumulation of nitrogen and phosphorus in the soil, which leach into surface and underground waters [
24]. Incorrect use of slurry can also lead to its direct runoff into water bodies and their local contamination [
25]. Algae bloom (proliferation of blue-green algae—cyanobacteria) and the toxic effects of ammonia and nitrites cause the death of aquatic plant and animal species [
26,
27]. Additionally, slurry is a source of greenhouse gases, such as nitrous oxide and methane [
28].
The aim of this research was to determine the effect of different concentrations of pig slurry added to the growth media used to produce duckweed as well as to determine the chemical composition and nutritional value of duckweed.
4. Discussion
Duckweed, the growth rate of its green mass, and its ability to absorb and accumulate nutrients from various growth media, have been discussed in the literature before [
14,
34,
35,
36,
37,
38]. Duckweed was grown on culture media prepared under laboratory conditions [
14,
35], wastewater [
34,
37], and animal excreta [
36,
38], like the present study.
In the present study, the course of duckweed growth observed in Groups 2–5 was deemed appropriate. Our previous study [
11] with effluent from a biorefinery demonstrated that its small addition (0.39%, 0.60%, and 0.78% concentration in the growth media) had a positive effect on the growth of duckweed (
Lemna minuta). Stadtlander et al. [
39] drew a similar conclusion from their study with bovine slurry, wherein a decreasing concentration of the natural fertilizer promoted higher yields of duckweed (
Spirodela polyrhiza and
Lemna punctata) fresh mass. As noticed by the aforementioned authors, duckweed growth decline could have been mainly due to the use of high slurry concentrations. The slurry has a high concentration of NH
3, exerting a toxic effect on live organisms, duckweed included. In addition, high slurry concentration in the growth media may contribute to the unfavorable increase in their pH—towards alkaline values—and these high pH values also inhibit duckweed growth. In the present study, between days 20 and 30 of observations, a moderate pH increase was noted in Group 5, which ultimately led to duckweed yield decrease compared to groups 2–4, which is consistent with the findings reported by other authors [
39,
40,
41].
The appropriate growth of duckweed and its capability to accumulate nutrients are affected by multiple factors. One of the key factors is the already mentioned pH. As demonstrated by Ullah et al. [
40], the pH optimal for growth and maximal green mass yield of duckweed (
Lemna minor) is 7 ± 1, whereas pH values exceeding 8 and lower than 4 were observed to inhibit duckweed growth. In the present study, the pH values either fell within the recommended range or were exceeded periodically (in Group 5). Similar conclusions regarding acidification of growth and culture media used to produce duckweed (
L. minor) were reported by Jones et al. [
41]. They demonstrated that pH > 8.2 inhibited its growth and that better yields could be achieved at pH < 8.
The total content of compounds dissolved in water (TDS) and the electrical conductance of liquid (EC) should be considered together. The TDS index is based on the electrical conductance measurement; therefore, when EC values decrease/increase, the TDS values respectively do the opposite, i.e., increase/decrease. The EC values are additionally affected by temperature. Conductance can only be recorded when inorganic metal ions, such as N, P, K, Ca, and Mg, are present in the solution. In a study by Wendeou et al. [
42], the best growth of duckweed (
S. polyrhiza) was observed at EC values of 800, 1200, and 1400 μS/cm. In the present research, the values of the EC index were similar, indicating good conditions for duckweed growth, which indeed grew well as seen by its new, large, green leaves. Similar observations related to the content of soluble compounds and the electrical conductance of the medium were made by Iqbal et al. [
43]. They recorded the best growth of duckweed (
L. minor) and its nutrient accumulation capability at an EC value of the growth medium approximating 1000 μS/cm.
Salinity, which is a measure of the salt concentration in a solution, provides information about the mass of all dissolved substances, excluding gases, colloids, suspended solids, and organic matter. The results of investigations conducted by Tkalec et al. [
44], Wendeou et al. [
42], and Ullah et al. [
40] indicate that excessively high salinity of the culture/growth medium negatively affects the growth and proliferation of duckweed green mass. In the present research, the level of media salinity was low; no problems were observed in the subsequent stages of the experiment (days 10, 20, 30)—namely, no inhibition of duckweed growth. The worse duckweed growth results recorded in Group 5 could be due to the aforementioned increased pH and a too low nutrient concentration (concentration 0.50%) in the growth medium used.
Physicochemical factors affect the growth and development of organisms, while rapid and strong changes in abiotic factors can inhibit these processes. One of the important factors is temperature, which has a significant impact on the growth, development and metabolism of organisms, as it determines the rate and amount of absorbed and accumulated nutritionally important nutrients. Different duckweed species have adapted to a broad range of ambient temperatures from 5 to 35 ℃ [
45]. According to Vymazal [
46], the optimum temperature of the growth medium for duckweed production should be between 20 and 30 °C. Air temperature, which was stable in the present study, and—more importantly—water temperature or culture medium/growth medium temperature under experimental conditions are important for the organisms living in water and partly on its surface. The study conducted by Chakrabarti et al. [
20] demonstrated that the temperature of the growth medium enriched with manure or chemical fertilizers was lower than 18.5 °C, which inhibited duckweed (
L. minor) growth. Intensive growth of duckweed was observed again when it increased to 19.4 °C. In the present study, the temperature of the growth media at the beginning of observations was relatively low (17.6–17.8 °C) for the growth needs of duckweed; hence, the increase in its green mass in the first stage of the experiment was slow. On day 10 of observations, the growth media temperature was 18.7 °C and in the final phase, i.e., on day 30, it was higher by 0.2–0.5 °C and amounted to 18.9–19.2 °C. This ensured a good growth of green mass, without compromising the values of the remaining important parameters tested, i.e., media pH, TDS, EC, and salinity.
Quantitative and qualitative parameters were assessed in duckweed from the
Lemnaceae family, which is used as human food [
32] as well as feedstuffs for animals [
19,
20]. The chemical composition of duckweed depends on many factors, including the type of growth medium, species of duckweed, place of cultivation, availability of nutrients, and environmental conditions [
47]. These factors allow modifying duckweed composition through the use of various types, concentrations, and solution forms of the growth media. In a study by Devlamynck et al. [
48] with pig slurry used as a growth medium, the protein content of duckweed (
L. minor) was approximately 35% that of dry matter. In another study by Mohedano et al. [
49] investigating duckweed (
Lemna punctata) growth media with animal excreta, the manure was first subjected to biofermentation and then, the leachate was discharged to the retention tank, from where it was pumped to the ponds where duckweed was grown. The media used were fed with 1 m
3/day of leachate, and the crude protein content in the produced duckweed ranged from 28 to 35%. In the present study, the protein content of the duckweed produced was higher or comparable to the results reported by the aforementioned authors [
49]. Our previous study [
11] showed that biogas plant effluent could also serve as a good medium for the growth of duckweed (
L. minuta) and allowed us to conclude that the installation used would enable the recovery of valuable fertilizing materials (struvite and ammonia) and the production of high-quality animal feed on the leachate. Compared to our previous study [
11], the present research results indicate a comparable or higher content of protein, fat, fiber, and ash in the produced plant material—duckweed. The study conducted by Stadltander et al. [
39] with duckweed (
Spirodela polyrhiza and
Lemna punctata) produced using bovine slurry also confirmed a high content of crude protein per mass unit, i.e., from 30 to 38 g 100 g
−1 DM. Duckweed produced in the present growth study had a high total protein content, which indicates its potential suitability for commercial production and use for feedstuff-production purposes; however, the variability of results reported in the available literature [
50,
51] makes this area ripe for further research.
In the present study, the contents of macroelements, microelements, and heavy metals determined in duckweed produced on growth media with different concentrations of pig slurry turned out to be lower than those in the research by Devlamynck et al. [
48]. The regulation of the European Commission [
52] specifies the maximum levels of certain contaminants, including heavy metals, in various foodstuffs. In the group of food products including leafy vegetables and seaweed, the highest permissible levels are 0.10 for lead and 0.20 mg/kg fresh weight for cadmium. Like in the present study, Devlamynck et al. [
48] also performed their experiment with pig slurry, but it had been first subjected to centrifugation and bio-treatment. In our previous experiment [
11], the contents of mineral elements and heavy metals in duckweed (
L. minuta) were similar to those reported in the present study. In another experiment carried out by Appenroth et al. [
32], wherein duckweed (genus
Wolffia) was grown on a medium prepared under laboratory conditions (KNO
3, KH
2PO
4, K
2HPO
4, MgSO
4, Ca(NO
3)
2, H
3BO
3, ZnSO
4, Na
2MoO
4, MnCl
2, Fe(III)NaEDTA, EDTA-Na
2), the contents of Ca, P, K, Cd, and Pb in the plant material were higher than in the present study. It should be noted, however, that different
Wolffia species used for the study had various contents of minerals even though they were grown under the same conditions. This finding allows us to conclude that there is a need and even a necessity to control the heavy metal content of commercially produced duckweed for feed or nutritional purposes considering the variety of duckweed species and different types and concentrations of growth media used to this end.
Carotenoids are an important group of compounds responsible for the pigmentation of plants and animal products (egg yolk, broiler carcass) that also exhibit antioxidant properties. The analyzed duckweed was found to contain six representatives of this group of compounds, i.e., α-tocopherol, β-carotene, α-carotene, violaxanthin, zeaxanthin, and lutein. These carotenoids occur naturally in feedstuffs for animals. For instance, carrot contains β-carotene and α-carotene, alfalfa contains lutein and zeaxanthin, whereas marigold flower and maize both contain lutein and zeaxanthin [
53]. These compounds are nutritionally important for livestock; hence, their presence in duckweed produced in the present research additionally confirms its usefulness for animal feeding purposes. Investigations conducted by Appenroth et al. [
32,
54] and our previous study [
11] demonstrated similar levels of the analyzed carotenoids. According to Polutchko et al. [
55] and Stewart et al. [
56], duckweed production can provide a significant amount of green mass rich in nutrients, containing an attractive mixture of carotenoids and polyphenols, which supports its viability as a feed supplement for animals.
Duckweed is also a source of amino acids. Their average and diversified contents were determined in the duckweed samples analyzed in the present study. In turn, Stadtlander et al. [
39] showed higher contents in duckweed (
L. punctata and
S. polyrhiza) produced on media with a cattle slurry addition compared to our own research. The cited authors showed no tendencies of changes in the contents of amino acids in the plant material samples. No similar relationships were found in the present study either, as the contents of amino acids were similar in all analyzed groups. Alike results were reported by Chakrabarti et al. [
20]. The present study results confirm the value of duckweed as a source of valuable amino acids.
The fatty acid profile of the analyzed duckweed samples seems interesting because the contents of SFAs and PUFAs turned out to be comparable. According to previous studies, the content of SFAs was usually lower than that of PUFAs [
20,
32]. The results of a study conducted by Appenroth et al. [
32] show the following percentages of individual groups of fatty acids in the total fatty acid profile: SFA—33.9, MUFA—3.5, and PUFA—62.6 (% fatty acid methyl esters). Chakrabarti et al. [
20] also determined a lower content of SFAs (22.72% of total fatty acids) and a higher content of PUFAs (63.38% of total fatty acids).
High levels of nitrates in plants can indirectly lead to increased intake of nitrites and N-nitroso compounds, increasing the risk of development of human and animal diseases [
57]. According to standards in force, the permissible content of nitrates in feed materials ingested with via feed by ruminants is 9.3 g NO
3 kg
−1 DW, whereas in for human diets, it is 46 g NO
3 kg
−1 DW [
52,
58]. In a study by Devlamynck et al. [
57], the nitrate content of duckweed was higher than in the present study. These authors [
57] demonstrated that the level of nitrates increased with the increasing content of macronutrients in the growth medium. Similar correlations were noted in our own research, which indicates the need to precisely control the quality of the media used for duckweed production.