Nutritional content and growth ability of aquatic plant Azolla pinnata on wastewater of catfish

Azolla pinnata is a floating aquatic plant with several benefits as a source of feed in aquaculture and animal as well as a phytoremediator. Research with two replications was carried out at RC for Limnology and Water Resources laboratory on May 2022 to explore information on its nutritional content, growth ability and function as a phytoremediator. The growth ability research was carried out in-door using media of catfish cultivation wastewater varied in concentration (100%, 60%, and 30%), and the observation period of 3 days for 15 days. Azolla pinnata contains nutrition (% dry weight): ash: 31.20; fat: 12.37; protein: 27.16; fibre: 11.15; and Nitrogen Free Extract: 18.12, respectively. There were 15 types of amino acids, as much as 1.59 % w/w. On the 15th day, the highest growth was seen in the media with 100% concentration, where the culture density was 310.47 g/m2, specific growth rate (SGR) was 15.38%, and productivity was 35.93 g/m2/day. At the same media, Total Phosphate (TP) elimination rates were 46.06%, and Total Nitrogen (TN) was 79.474%. Azolla pinnata can be cultivated in catfish wastewater to provide nutritious feed as well as to reduce the content of TP and TN.


Introduction
Inland ecosystems have high biological diversity, including various types of aquatic plants floating on the surface, submerged under the water, or embedded roots to the bottom.Azolla pinnata is one among various popular floating plants, such as water hyacinth (Eichornia crassipes), group of Salviniaceae, and duckweed (Lemnaceae).The name Azolla comes from the Yunani language, which means azo (to dry) and allyo (to kill), meaning the plant dies when it dries up.
The genus Azolla was discovered by JB Lamark in 1783 of the family Salviniaceae (order Salviniales).Still, following the development of biosystematic science, it recently grouped into a new family named Azollaceae.Azolla is also commonly known as Mosquito Fern [1][2][3][4].Azolla pinnata is a small fern with a triangular stem up to 2.5 centimetres long that floats on the water.The stems have rounded or overlapping angular leaves, each 1 or 2 millimetres long (Figure 1).They are green, and blue-green, covered with tiny hairs, giving a velvety appearance, and could even turn red on certain varieties (var.imbricata)[2,3].
Azolla lives in association with Anabaena algae as a floating plant in stagnant waters such as lakes, ponds, rice fields, canals, or slow-flowing waters.Azolla reproduces using buds and has rapid growth.In certain areas, it is declared as an invasive species that has to be controlled, but in other areas, such as India, Azolla is used for bio fertilizers and animal feed [1, 2, 4 ,8 ,9].Azolla has various benefits, such as agricultural fertilizer, source of bioethanol, mosquito population control, phytoremediator in areas experiencing eutrophication and can absorb metal pollutants.In addition, Azolla is also used as a source of feed for livestock [10−12] and even has the potential to be a food source [2].
In Asia, Azolla is commonly grown in rice fields not just to increase rice yield but also simultaneously to feed aquatic animal species such as fish (notably Tilapia, Loach, and Grass Carp), shrimp, ducks, and geese [2,4,13,14].Azolla has been nicknamed a tiny super plant, and according to Raja et al., Lumpkin and Plucknett [4,6], Azolla is a great potential aquatic plant.Azolla also exhibits high bioremediation potential for nutrient and metal pollutants such as Pb, Cd, Cr, Cu, and Zn [15][16][17].
According to the above information, it is suggested to utilize this plant benefit for aquaculture, particularly in Indonesia.As has been known, aquaculture activities face two main problems, namely high feed prices and poor water quality related to contamination, both from industrial, domestic, agricultural and even from the aquaculture waste itself.Applying Azolla in aquaculture systems can give double advantages: providing the plant biomass for a low-cost feed source and improving the water quality, which is very useful for healthy fish growth.As has been reported by Said et al. [18,19], an application of minute duckweed (Lemna perpusilla) in an Integrated Multitrophic Aquaculture (IMTA) has given such double advantages that the Azolla would.
This research aims to study the growth and nutritional value of Azolla pinnata grown on catfish aquaculture wastewater, as well as the ability of the plant to improve the wastewater quality, particularly in terms of the Nitrogen (N) and Phosphate (P) content.

Materials and methods
The Azolla pinnata used in this research is a collection of Planktonology Laboratory, Research Center for Limnology and Water Resources, BRIN.It is a small frond of 1.0−2.5 cm long, consists of a main rhizomes, branching into several secondary rhizomes which each has alternately arranged smal leaves.Some unbranched roots hang down from nodes on the ventral side of the rhizomes [20].The stock culture was maintained on a channel of a tilapia recirculation aquaculture system that was placed outside but under the roof.It receives enough sunlight while preventing the system from rainwater.

Growth potential of Azolla pinnata
The research on the growth ability of A. pinnata was carried out indoors on May 2022 at the Laboratory of RC for Limnology and Water Resources-BRIN in two replications.The research used a plastic basin (container) sized 40x30x15 cm in a simple greenhouse constructed of a translucent polycarbonate roof and insect net walls.
Each basin was cleaned and then filled with culture media as required for the experimental treatment, which consisted of 3 various concentrations of catfish cultivation wastewater treatment, namely 100%, 60%, and 30%.The culture media was given once at the beginning of the experiment.The wastewater was obtained from the greenhouse's closed system catfish culture tank.Measurements using the Water Quality Checker [WQC-Toshiba] show the waste characteristics are as follows: temperature according to room temperature, pH: 7.13±0.18,conductivity: 0.59 ± 0.41 mS/cm, Total Organic Matter (TOM): 527.77 ± 29.50 mg/L, and Total Phosphate (TP): 6.19±0.11mg/L.The catfish culture wastewater is diluted using tap water with pH 6.15 and conductivity of 0.12 mS/cm content.
Each container is stocked with 20 g of the Azolla, equivalent to 166.67 g/m 2 , and let grow for 15 days.The observation was carried out every three days by harvesting using a scoop net and then swinging away to remove the water before weighing (Wt) on a digital balance at two digits reading.After observation, 20 g of the harvested Azolla was stocked back into each container (Figure 2).Analysis of growth ability was carried out descriptively.Observation parameters include the growth density (g/m 2 ), specific growth rate (SGR-%) and daily productivity (P) per unit area per day [21][22][23][24].
The density (D) is the total biomass (g) of Azolla at time t, per unit area (m 2 ), obtained according to an equation as follows: The specific growth rate (SGR) is calculated using the following equation Daily productivity (P) (g/m 2 /day) is the total of yields (g) of Azolla per unit area (m 2 ) per unit time (day), calculated according to the following equation P (g/m 2 /day) = Wt -W0 (4) t.A where: Wt is the Azolla biomass at time t; W0 is the Azolla initial biomass; t is the cultivation times (days); and A is the culture area (m 2 )

Proximate and Amino Acid Analysis
Proximate and amino acids were analyzed at the Laboratory of Research Center for Biological Resources and Biotechnology, Bogor Agriculture University (RCBIO, IPB).The analysis method for proximate refers to AOAC and SNI [26,27].There are consists of content of protein, lipid, ash, crude fibre, moisture, and Nitrogen Free Extract (NFE).While to analyze 15 types of amino acids, the HPLC (high-performance liquid chromatography) Post-Column derivatization method was used [28].The sample was taken to the laboratory in a chilled condition.

2.3.Water Quality of Culture Media
Water quality observations was focused on the ability of Azolla to absorb nutrients from the media, including Total Nitrogen (TN) and Total Phosphate (TP).Analysis of TN and TP using the colorimetric method [29].N uptake was calculated according to a formulation as follow: U= (Cnt-Cn0) A.t (5) where: U is N uptake rate by Azolla culture (g/m 2 /day), Cnt and Cn0 are N concentration in the medium at t day t and day 0, A is surface wide of the experimental container, and it is time (day).
N conversion rate into Azolla biomass (N-Biomass CR) determined following a formula: CR = (Cnt-Cn0) (Bt-B0) (6) where: CR is N-Biomass convertion rate biomass ( g/g B/N), Cnt and Cn0 are N concentration in the medium at day t and day 0, Bt and B0 are total Azolla biomass at day t and day 0 At the the same time, determination of N elimination rate by Azolla culture was according to as follow x 100 (7) where ER is N ellimination rate (%), Cnt and Cn0 are N concentration in the medium at day t and day 0.
The same formulae were employed to determine the P uptake rate, P-Biomass conversion rate, and P elimination rate, with P replacement of the N entity.

Growth and productivity
Azolla exhibited good growth in catfish cultivation wastewater, although unfavourable growth was observed initially with the higher waste concentration (Figure 3).More adaptation processes were needed when a higher waste concentration was applied to the growth media.As the acclimatization was successful, the plant enjoyed the more beneficial nutrients and performed better growth in the late culture time.As shown in Figure 3, while the plant grew with 30% waste concentration had a subsiding culture density during the 15 days of experimental time, the opposite growth pattern was found in the plant with 100 % waste concentration in which the culture density tended to increase with the time up to the end of the experiment.
Based on this phenomenon, it is suggested that there is a correlation between the optimal growth of Azolla to the concentration of the media (catfish waste).[30] reported that the giant duckweed Spirodela polyrhiza grew well on supernatant filtered media of catfish waste, resulting in higher production than using media made of a mixture of fertilizers (1.0 g Urea + 1.0 g NPK and 0.1 g Gandasil).Consistent with the above phenomenon, as high as 20.19 %/day SGR was achieved in Azolla culture with 30 % waste concentration at the early culture period and decreased gradually to 11.17 %/day after 15 days (Figure 4).At the same time, the culture with 100 % waste concentration exhibited only 4.19 %/day in the beginning.Eventually, it increased to 17.93 %/day after 12 days, and even though it decreased to 15.38 %/day in the last phase of this experiment, the value was still remarkably higher than that with 30 % waste concentration.A relatively constant growth rate was performed with 60 % waste concentration, indicating the optimal waste concentration is around this level.The growth value was in the range reported [31] for A. pinnata grown in Hoagland medium with the variation of N concentration, which was around 10−20 %/day.
The decrease in growth rate with culture time, such that occurred in the culture with 30 % waste concentration, can generally be explained in terms of nutrient depletion, as shown in Figure 6 and 8; during the experimental time, there was a decrease both in TN and TP concentration in the media.Azolla is generally known to live symbiotically with the N-fixative blue-green algae Anabaena azollae.However, the degreasing growth rate is likely attributed to TP exhaustion.
Figure 4, however, shows that the highest culture productivity was achieved in the early stage of culture with 30% waste concentration, in which the value was 46.25 g/m 2 /day.This value is equivalent to the fresh weight biomass of 168.91 ton/ha/ year, and with the moisture content of 90.87 % (Table 1), it accounted to be 15,41 ton/ha/year dry biomass.In this lower waste concentration media, however, the culture productivity rapidly dropped, which means a continual lower waste concentration input has to apply in the Azolla culture to achieve maximum biomass productivity.
At the same time, the culture productivity of the 60% waste concentration media was relatively constant in the range of 37.64−39.17g/m 2 /day up to day 12.It decreased to 30.83 g/m 2 /day in the last experimental stage.In the meantime, the culture with 100 % waste concentration media had a constant productivity value after day 4 in the 34.27−39.58g/m 2 /day range.

Nutritional content
Proximate analysis was carried out to find out the composition of the main components of the nutritional content, namely the moisture, ash, lipid, protein, lipid, crude fiber, and Nitrogen Free Extract (NFE) expressed in percent of the dry weight.According to Cherryl et al. [32] that the analysis of the component composition is commonly used for the analysis of feed content or food ingredients.The result of the investigation of nutritional content of A. pinnata showed at Table 1.The total ash content of A. pinnata in this study was 31.26±0.877(30.64−31.88)%,remarkably higher than the results of A. pinnata in India with a value of around 23-24.% [33,34].It is also higher than ash in duckweed Wolffia globosa by 20.43% [35].The high ash content is thought to be related to the mineral content in this plant.Azolla pinnata is an aquatic plant rich in minerals, including calcium, magnesium, potassium, iron, manganese, and others [1,33,34].
In this study, the protein content of A. pinnata was 27.16±0.019(27.15−27.17)%.This value is higher than that reported by [32] and [33] by about 23%.It is also higher than the protein content of duckweed Spirodella polyrhiza, which was around 22.4−25% [30], and similar to duckweed W. arrizha which is about 28.6% [34].The lipid content of A. pinnata is also quite high, namely 12.37±1.079(11.61−13.12)%.It is remarkably higher than the lipid content of duckweed W. globosa, about 5% [34] and also higher than the lipid content of Lemna perpussilla [36].The fat content of A. pinnata has exceeded the fat requirement of fish feed by 5−10% [38].The crude fiber was   [32,33] but higher than the crude fiber content of Lemna perpussila and W. globosa [21,35].While the NFE content was 18.06±2.353(16.40−19.73)%.This value is similar to W. globosa [35], but lower than reported by [32] around 33%.While the moisture of A. pinnata in this study was 90.87% (table 1), it is similar to the [32] which is 89.73%.Amino Acid analysis showed in table 2. The number of amino acids (15 types) of A.pinnata, which are 1.60% w/w consisting of 50.625 % are nine types of Essential Amino Acid (EAA), and 49.375% of them are six types of Non-Essential Amino Acid (NEAA).The total amount of Amino Acids and composition of EAA to NEAA in A. pinnata is similar to the amount and composition of amino acids of W. globossa (1.51 %), and the EAA percentage (52.318%) is higher than NEAA (47.682%) [35].The NEAA can be synthesized by the body itself.However, the EAA cannot be synthesized in the human body, so it must be met through food intake.Therefore, to get EAA we have to consume food or supplements.[31,38,39] stated that EAA is needed for the balance of the human diet, especially, cysteine + methionine, tyrosine, lysine, threonine, phenylalanine, and leucine, are necessary for human nutrition.Based on the results, it was indicated that A.pinnata in Indonesia has the potential to be a source of protein and Amino Acid.These potentials could be very helpful for animal livestock or aquaculture, such as for IMTA systems like the application of duckweed Lemna [21,32].Azolla could be developed as a source of protein for animal feed, such as in the farming of laying duck and buffalo [10,11].According to Small and Darbyshire [2] that may even be developed for human food.In general, it can be concluded that some aquatic weed contains high protein and Amino Acids.According to Cherryl et al. [32], plants when reared at optimum conditions, will be a source of various minerals, such as Calcium (Ca), Phosphor (P), and various vitamins such as A precursor, B12, and beta-carotene.

Water quality
Table 3 shows the medium water properties of the Azolla culture during the experimental time.It was characterized by a pH value range of 7.08−8.65 with a trend of increasing along with the culture time but no remarkable difference between those three waste concentration media.In the meantime, the conductivity value tended to be higher with the waste concentration, and it lowered during the experimental time in all the treatments.
In this study, the water quality conditions can support the A. pinnata to growth.Azolla is thought to be similar to other aquatic plants such as duckweed.Duckweed is able to grow well in a fast time if the conditions of light, temperature, nutrient content in the growing media and several other factors are met properly [40].In addition, the type of plant or strain, growth environment and development stage were play a very important role to produce biomass or other components [41].The TN in the media was subsequently decreased along with the Azolla growth (Figure 5), which can be meant that the substance was taken up and converted into the plant biomass.The decreasing pattern can be explained conveniently in terms of logistic models against time, with a higher rate magnitude toward the higher N initial content or waste concentration.This investigation is consistent with [17] that reported a logistic decrease pattern of TN in domestic wastewater treated by Azolla pinnata.In addition, [42] has also reported the ability of Azolla filiculoides to remove N-NO3 and N-NH3 from sewage water.It is also observed that the initial TN concentration had a remarkable impact on the phytoremediation performance of the plant.As shown in Figure 6, the TN uptake rate decreased from 1.918 g/m 2 /day at culture with 100 % wastewater to 0.850 g/m 2 /day when the wastewater diluted down to 30%.The TN initial concentration, however, had no remarkable impact when the phytoremediation This elimination rate value is comparable with that reported for Azolla pinnata, which had TN elimination rate value of 74.521 % during 20 day phytoremediation trial [17].At the same time, Figure 5 also shows a more luxurious amount of TN used by A. pinnata to build up the biomass under excessive TN availability in the growth media.This phenomenon is consistent with the fact that TN uptake rate of the plant is higher with the TN concentration in the growth media and might have a consequence on the biomass properties, which determine the potential uses.
Such as reported by [30], under N limitation A. pinnata biomass was characterized by a low C/N ratio and high sugar content while less chlorophyll was synthesized.While [43] reported the higher protein content of Azolla microphylla directly proportional with nitrogen concentration in the growth media.The nitrogenous content is important in biomass use for biofertilizers, while protein content is the major concern when biomass is proposed for feed materials.
The TP content in the growth media was also observed to decrease along with the plant's growth (Figure 5).It can be explained in terms of logistic models.The relative content of TN to TP (N/P ratio) tended to be higher toward the lower waste concentration and culturing time, indicating the role of TP as the growth limiting factor rather than TN.These observations were also in accord with [17] and give an indication of the potential of Azolla plant genera to be used as phytoremediation agents to control water eutrophication by absorbing phosphorous substance in the water and converting it into biomass.The TP uptake rate and the biomass conversion rate appeared to have a similar pattern to the TN (Figure 7) but with remarkably higher magnitudes which can be attributed to the TP role as the growth limiting factor mentioned above.So, the plant's TP elimination performance tended to be remarkably higher under lower TP initial concentration.However, the highest TP elimination rate of 83.447% in this experiment was slightly lower than that reported by [17] for A. pinnata, which accounts for as high as 94.366 %, possibly related to the species variation, different wastewater properties, or also the experimental conditions.

Conclusion
In Indonesia, Azolla pinnata has the potential to be a source of protein as well as Amino Acid and high ash content as a mineral source.For this reason, the Azolla could be a good source of protein and minerals as an alternative natural feed in aquaculture, animal husbandry, or even for humans.Azolla pinnata can be cultivated in catfish wastewater to provide nutritious feed and reduce the content of TN and TP in the wastewater.

Figure 3 .
Figure 3. Culture density of A. pinnata culture grown under various concentration of catfish cultivation wastewater media.

Figure 4 .
Figure 4. Specific growth rate and biomass productivity of A. pinnata culture grown under various concentration of catfish cultivation wastewater media.

Figure 5 .
Figure 5. Medium TN and TP dynamics in A. pinnata culture grown under various concentration of catfish cultivation wastewater media.
plant was evaluated in terms of the TN elimination rate calculated based on the final and initial TN concentrations, in which the values ranged from 79.078−83.132%.

Figure 6 .
Figure 6.N-uptake, N-biomass convertion rate, and N-elimination rate in A. pinnata culture grown under various concentration of catfish cultivation wastewater media.

Figure 7 .
Figure 7. P-uptake, P-biomass convertion rate, and P-elimination rate in A pinnata culture grown under various concentration of catfish cultivation wastewater media.

Table 1 .
The nutritional content of Azolla pinnata.

Tabel 3 .
Water quality of A pinnata culture during the experimental time.