Freshwater Macrophytes: A Potential Source of Minerals and Fatty Acids for Fish, Poultry, and Livestock.

The freshwater macrophytes are abundant in tropical and subtropical climates. These macrophytes may be used as feed ingredients for fish and other animals. The nutritional value of twelve freshwater-cultured macrophytes was evaluated in the present study. Significantly higher crude protein (36.94-36.65%) and lipid (8.13-7.62%) were found in Lemna minor and Spirodela polyrhiza; ash content was significantly higher in Hydrilla verticillata, Wolffia globosa, and Pistia stratiotes (20.69-21.00%) compared with others. The highest levels of sodium, magnesium, chromium, and iron levels were recorded in P. stratiotes. H. verticillata was a rich source of copper, manganese, cobalt, and zinc; the contents of calcium, magnesium, strontium, and nickel were highest in S. polyrhiza. Selenium and potassium contents were higher in Salvinia natans and W. globosa, respectively. The n-6 and n-3 polyunsaturated fatty acids (PUFAs) contents were significantly higher in W. globosa and Ipomoea aquatica, respectively compared with others. Linoleic and α-linolenic acids were dominant n-6 and n-3 PUFAs. The highest value (4.04) of n-3/n-6 was found in I. aquatica. The ratio ranged from 0.61 to 2.46 in other macrophytes. This study reveals that macrophytes are rich sources of minerals, n-6 and n-3 PUFAs.

Introduction

Freshwater macrophytes, the fastest growing aquatic plants, are abundant in tropical and subtropical countries. They grow profusely in nutrient-rich water. These macrophytes are broadly classified into four groups based on their occurrence in the water body: the surface floating (e. g. Azolla spp.), submerged (e. g. Hydrilla spp.), emergent (e. g. Potamogeton spp.), and marginal (e. g. Ipomoea spp.). The nutritional value of freshwater macrophytes has been recognized globally. The unchecked propagation of freshwater macrophytes creates problems in many water bodies. The judicial exploitation of these nutrient-rich plants may open a new avenue from a nutritional view point for humans and animals. The leaf protein extracted from freshwater macrophytes may be used for human or non-ruminant animals (1). Macrophytes are a rich source of protein, lipid, amino acids, fatty acids, and minerals (2). The amino acid and fatty acid profiles of duckweeds Lemna minor and Spirodela polyrhiza have been documented recently (3, 4). The mineral composition of macrophytes is different from the usual terrestrial vegetation. Calcium (Ca), iron (Fe), and manganese (Mn) contents are higher in aquatic plants compared with the terrestrial ones (1). Minerals are important catalysts for various biochemical reactions. These are essential components for metabolism, growth, and development and help the animals to cope with the variable environmental conditions (5). There is an optimum dose for each mineral. Low/high concentrations may affect the physiology of the organisms. Toxic minerals like arsenic (As), mercury (Hg), antimony (Sb), cadmium (Cd) etc., are required by the body in little amounts, whereas excess levels of useful minerals like, sodium (Na), potassium (K), magnesium (Mg), Ca, Fe etc., may be harmful (5). Dietary inclusions of polyunsaturated fatty acids (PUFAs) have several health benefits for humans and other animals. The study of the profiles of fatty acids of feed ingredients ensures the quality of diets. Fish are unable to synthesize two essential fatty acids like n-6 (derived from linoleic acid, LA) and n-3 (derived from alpha-linolenic acid, ALA). So these fatty acids should be supplied to the diets of fishes (6).

The evaluation of minerals and fatty acids' compositions of aquatic macrophytes is essential for their selection as potential feed ingredients for fish and other animals. Some of the commonly occurring freshwater macrophytes are: Azolla microphylla, A. pinnata, Enhydra fluctuans, Hydrilla verticillata, Ipomoea aquatica, Lemna minor, Marsilea quadrifolia, Pistia stratiotes, Salvinia molesta, S. natans, Spirodela polyrhiza, and Wolffia globosa. These macrophytes are distributed throughout the temperate, sub-tropical, and tropical regions of the world. Some of these macrophytes like, E. fluctuans, I. aquatica, and M. quadrifolia, are consumed as vegetables by humans in India and Bangladesh (7), and W. arrhiza has been consumed in Thailand (8). Most of these macrophytes, except H. verticillata (submerged plant), M. quadrifolia, and E. fluctuans (marginal plants) are surface floating macrophytes. All these macrophytes propagate through vegetative reproduction. Mosquito fern Azolla spp. (Azollaceae) are heterosporous free-floating ferns. It lives symbiotically with nitrogen-fixing blue-green algae Anabaena azollae. Watercress Enhydra fluctuans (Asteraceae) is a hydrophytic plant and it grows in canals and marshy places. Waterthyme Hydrilla verticillata (Hydrocharitaceae) is a submerged, rooted aquatic plant. It can grow in water up to a depth of 6 m, and in transparent water it can survive up to a depth of 12 m. The water spinach Ipomoea aquatica (Convolvulaceae) with hollow roots floats in water easily. Three members of the family Lemnaceae, namely Lemna spp., Spirodela spp., and Wolffia spp. are known as duckweeds. The plant consists of a single leaf or frond with one or more roots. Water clover Marsilea quadrifolia (Marsileaceae) is a deciduous, aquatic fern. Each green and thin stalk rises from the rhizome to the water surface; it contains a single shamrock-like leaf with four leaflets. Water cabbage Pistia stratiotes (Araceae) is a perennial monocotyledon with thick, soft, and light green leaves that form a rosette. It floats on the surface of the water and roots are hanging beneath the leaves. The short stolon connects both the mother and daughter plants. Water fern Salvinia spp. (Salviniaceae) is a perennial free-floating macrophyte. During the period of high growth, leaf size decreases and both leaves and stems fold, doubling and layering to cover more of the water surface. The nutritional value of macrophytes in terms of proteins, lipids, ash etc. varies greatly (2). The culture medium influences the mineral contents of the macrophytes (8). The extracts of seven freshwater macrophytes show no cytotoxic and anti-proliferative effects on human cell lines (9). Therefore, macrophytes should be considered as useful feed ingredients. Production of macrophytes using a standard technique may help to maintain the nutritional value of the plant and also maximize the health benefits.

The aim of the present study is to evaluate the nutritional value, viz. proximate composition, minerals and fatty acids profiles of twelve cultured freshwater macrophytes. This study will help to evaluate the suitability of these macrophytes as feed ingredients for fish, poultry, and livestock.

Materials and Methods

Culture of Macrophytes

Freshwater macrophytes were collected from water bodies of Delhi, Uttar Pradesh, and West Bengal and then identified. Macrophytes were cultured in outdoor cemented tanks (1.2 × 0.35 m) with clean dechlorinated tap water (3). A 10-cm layer of soil was used for the culture of H. verticillata, M. quadrifolia, and E. fluctuans. All other macrophytes were cultured without soil base. The depth of water was 30 cm in all culture tanks. A combination of organic manures viz. cattle manure, poultry droppings, and mustard oil cake was used (1:1:1) at the rate of 1.052 kg/m3. All manures were decomposed for 5 days and then macrophytes were introduced individually in the outdoor cemented tanks. Three replicates were used for each macrophyte. For the steady supply of nutrients for the growth of macrophytes, the same combination of manures (at one-fourth dose of the initial one) was applied in the culture tanks. Manures were decomposed for 5 days in separate containers and then applied on day 6. This schedule was followed throughout the culture period. Culture tanks were monitored regularly and macrophytes were harvested when the whole surface of the tank was covered with plants. The freshly harvested macrophytes were washed twice with tap water and then with distilled water. After air drying, macrophytes were kept at 40°C for 3 h. Then the ground, sieved, and fine powders were kept in air-tight containers and stored in a refrigerator at 4°C for further assay.

Proximate Composition Analysis

The proximate composition of the macrophytes was analyzed (10). Three replicates were used for each assay. Moisture content was estimated after drying the sample at 105°C for 24 h. The dried samples were kept in a muffle furnace at 550°C for 8 h for the determination of ash contents. The crude protein contents were analyzed by measuring the nitrogen content (N x 6.25) with an automated micro-Kjeldhal apparatus (Pelican Instruments, Chennai, India). Crude lipid contents of the macrophytes were assayed gravimetrically (11). Carbohydrate contents were estimated by the subtraction method.

Mineral Assay

The mineral compositions of macrophytes were assayed using Inductively-Coupled Plasma Mass Spectrometer (ICP-MS, Agilent 7900, USA) following standard protocol at the Instrumentation Facility of Indian Institute of Technology, New Delhi. The powdered macrophyte sample (150 mg) was taken in a closed digestion vessel and 8 ml of suprapure 69% nitric acid (HNO3, Merck, USA) was added to this. The sample was digested in Microwave digestion system (Multiwave PRO; Anton Paar, Austria). The digested sample was cooled at room temperature and transferred into a measuring cylinder; Milli-Q ultrapure water was added to make the volume 40 ml. Then the sample was filtered through a 0.2 μM syringe filter (Thermo Scientific, USA) and was collected in a glass vial. A 20 μL sample was injected through autosampler in the ICP-MS. The standard solution for each mineral was supplied with the equipment (Agilent Technologies, USA). It was diluted with Milli-Q ultrapure water containing 1% HNO3 to make concentrations of 20, 40, 60, 80, 100, 250, 500, 1000 μg/l. The calibration (standard) curve was prepared. The blank was prepared with Milli-Q ultrapure water containing HNO3 (1%). Minerals are divided into three major groups based on their concentrations in the mammal/human body viz. macro, trace, and ultra-trace minerals (5).

Fatty Acid Analysis

The fatty acid profiles of the macrophytes were analyzed using Gas Chromatograph (GC)–Flame Ionization Detector, Clarus 580 (Perkin Elmer, USA). The total lipid extracted from plants (11) was used to prepare fatty acid methyl esters (FAME) by transesterification using sulfuric acid in methanol at 50°C for 16 h (12). After extraction and purification of FAME (13), 1 ml sample was kept in a glass vial of autosampler of GC. The sample was separated and quantified in a GC column (60 m × 0.32 mm i.d. × 0.25 μm ZB-wax, Phenomenex, UK). The data were collected from pre-installed program software (TotalChrom Workstation Ver6.3, Perkin Elmer). The FAME was identified with the help of standards (Supelco FAME 37 mix, Sigma-Aldrich, USA).

Statistical Analysis

The compositions of twelve macrophytes are given as means ± standard error (SE). The differences in nutritional values of various macrophytes were tested using one-way analysis of variance (ANOVA) and Duncan's multiple range test (14). Statistical analyses were performed using the SPSS program (version 25.0). Statistical significance was accepted at p < 0.05.

Results

Proximate Composition

The moisture content was highest (11.86%) and lowest (6.26%) in E. fluctuans and W. globosa, respectively (Figure 1). Significantly higher crude protein contents were found in two duckweeds, namely L. minor and S. polyrhiza, compared with others. The highest lipid content was also recorded in L. minor, followed by S. polyrhiza. The lipid content was minimum in E. fluctuans. Ash content was significantly higher in H. verticillata, W. globose, and P. stratiotes compared with other macrophytes. The ash content was minimum in M. quadrifolia. Carbohydrates levels were minimum and maximum in L. minor and M. quadrifolia, respectively.

Figure 1

Proximate composition of twelve freshwater macrophytes cultured with organic manures. Bars with different superscripts are significantly different (n = 3).

Mineral Composition

Macrominerals

Among these twelve freshwater macrophytes, Na content was significantly higher in P. stratiotes compared with others (Figure 2A). This group was followed by S. natans and E. fluctuans. A significantly higher K level was found in W. globosa compared with others. This was followed by L. minor, E. fluctuans, H. verticillate, and P. stratiotes. Ca content was highest in S. polyrhiza, followed by P. stratiotes. A significantly higher Mg level was found in P. stratiotes and S. polyrhiza compared with others. The Na, Ca, and Mg contents were minimum in M. quadrifolia compared with other macrophytes. It indicates the nutritional value of the macrophytes.

Figure 2

(A) Macrominerals, (B) trace minerals, and (C) ultra-trace minerals contents of twelve freshwater macrophytes cultured with organic manures. Bars with different superscripts are significantly different (n = 3).

Trace Minerals

A total of nine trace minerals were found in these macrophytes (Figure 2B). Molybdenum (Mo) content was significantly higher in A. microphylla, A. pinnata, and P. stratiotes compared with others. Mn, zinc (Zn), copper (Cu), and Cd contents were significantly higher in H. verticillata compared with others. In P. stratiotes, significantly higher levels of Fe and aluminum (Al) were found compared with others. Among these macrophytes, A. pinnata ranked second for both Fe and Al. A. microphylla ranked third for Fe and fourth for Al contents. Maximum strontium (Sr) level was recorded in S. polyrhiza followed by P. stratiotes. In all these macrophytes lead (Pb) was found.

Ultra-Trace Minerals

Five ultra-trace minerals were found in these macrophytes (Figure 2C). A significantly higher level of selenium (Se) was found in S. natans compared with others. This plant was followed by H. verticillata and P. stratiotes. Se was absent in S. molesta, E. fluctuans, I. aquatica, and S. polyrhiza. Chromium (Cr) content was significantly higher in P. stratiotes, A. microphylla, and A. pinnata compared with others. Cobalt (Co) content was significantly higher in H. verticillata compared with others. This was followed by P. stratiotes and S. natans. Nickel (Ni) and tin (Sn) levels were significantly higher in S. polyrhiza and A. microphylla, respectively compared with others. Among these macrophytes, P. stratiotes ranked third for Ni content.

Fatty Acid Profile

The fatty acid profiles of twelve freshwater macrophytes were documented in the present study (Tables 1–3). The saturated fatty acids (SFAs) content was significantly higher in W. globosa compared with others. This was followed by A. pinnata, L. minor, and I. aquatica. SFA content was minimum in P. stratiotes. Among, SFA, palmitic acid (C16:0) was the dominant one in all these plants. Monounsaturated fatty acids (MUFA) content was significantly higher in M. quadrifolia compared with others. Among various MUFAs, oleic acid (C18:1n-9) was present in most of the plants and the amount was also higher compared with the others (Supplementary Tables 1A–C). MUFA content was also minimum in P. stratiotes. Though in small amounts two other monounsaturated fatty acids like, palmitoleic acid (C16:1n-9) and nervonic acid (C24:1), were present in all macrophytes, except E. fluctuans and A. pinnata. Another isomer of palmitoleic acid (C16:1n-7) was absent in two species of Azolla and S. natans.

Table 1

Saturated fatty acids (SFA) profiles of freshwater macrophytes cultured with organic manures (mg/100 g, dry weight).

Macrophytes /Fatty acids	C14:0	C15:0	C16:0	C18:0	C20:0	C22:0	C24:0	∑SFA
Azolla microphylla	6.55 ± 0.06fg	-	541.84 ± 18.38de	1.76 ± 0.89h	1.39 ± 0.23h	-	5.65 ± 0.23ef	557 ±19.79fg
Azolla pinnata	16.99 ± 0.69e	17.76 ± 8.72a	815.78 ± 3.39b	27.06 ± 0.36cd	4.08 ± 0.42ef	17.58 ± 1.45a	16.23 ± 1.09a	915.51 ±8.08b
Enhydra fluctuans	9.46 ± 0.29f	6.90 ± 2.29bc	377.47 ± 0.07f	29.34 ± 0.06c	2.01 ± 0.17gh	0.07 ± 0.03d	5.63 ± 1.02ef	431.01 ±0.83h
Hydrilla verticillata	20.03 ± 1.90de	4.40 ± 1.49cde	377.33 ± 16.67f	21.53 ± 1.68ef	17.91 ± 0.17a	0.89 ± 0.12cd	8.03 ± 0.57de	450.14 ±5.075h
Ipomoea aquatica	14.97 ± 0.73ef	3.64 ± 0.89def	622.79 ± 0.87c	68.69 ± 0.80a	4.54 ± 0.33de	0.07 ± 0.01d	14.47 ± 0.41ab	729.22 ±1.90d
Lemna minor	40.11 ± 0.05a	8.25 ± 0.13b	753.13 ± 0.05b	17.69 ± 0.06fg	5.65 ± 0.11d	5.04 ± 0.10b	12.81 ± 0.15bc	842.69 ±0.55c
Marsilea quadrifolia	14.50 ± 0.46e	1.92 ± 0.36efg	542.58 ± 6.39de	16.81 ± 0.47fg	1.38 ± 0.10h	0.84 ± 0.02cd	10.37 ± 0.81cd	588.42 ±5.72ef
Pistia stratiotes	5.29 ± 0.40g	3.85 ± 0.19cde	268.75 ± 0.06g	14.07 ± 1.16g	8.11 ± 0.10c	0.57 ± 0.04cd	10.10 ± 0.45cd	310.77 ±0.70i
Salvinia molesta	34.04 ± 5.04b	6.14 ± 0.85bcd	574.15 ± 19.84cd	23.35 ± 0.72de	2.99 ± 1.18fg	5.13 ± 0.02b	10.28 ± 0.48cd	656.11 ±16.58e
Salvinia natans	4.04 ± 0.37g	-	486.68 ± 2.72e	3.26 ± 0.07h	2.39 ± 0.18gh	-	5.08 ± 0.41f	501.45 ±4.02gh
Spirodela polyrhiza	24.26 ± 2.07cd	0.81 ± 0.04fg	509.94 ± 16.48e	24.84 ± 1.58cde	5.51 ± 0.62d	1.42 ± 0.03cd	7.85 ± 0.89de	574.66 ±22.02fg
Wolffia globosa	28.24 ± 0.60c	3.08 ± 0.29def	1084.31 ± 63.38a	54.25 ± 4.57b	11.31 ± 0.90b	2.07 ± 0.01c	10.87 ± 0.46c	1194.14 ±71.13a

Values having the means (n = 3) in each row with different superscript are significantly (p < 0.05) different.

Table 3

Polyunsaturated fatty acids (PUFA) profiles of freshwater macrophytes cultured with organic manures (mg/100 g, dry weight).

Macrophytes /Fatty acids	C18:2 n-6	C18:3 n-6	C20:2 n-6	C20:3 n-6	C20:4 n-6	∑n-6 PUFA	C18:3 n-3	∑n-3 PUFA	n-3/n-6
Azolla microphylla	96.72 ± 4.71j	-	-	-	16.71 ± 0.14d	113.43 ±4.85i	149.05 ± 4.98i	149.05 ±4.98i	1.31 ± 0.01f
Azolla pinnata	443.29 ± 1.13c	10.57 ± 0.31b	-	3.17 ± 0.62de	81.32 ± 0.42a	538.36 ±2.49c	591.33 ± 0.66e	591.33 ±0.66e	1.09 ± 0.01h
Enhydra fluctuans	329.01 ± 3.35e	-	1.11 ± 0.10bc	-	5.24 ± 0.24e	335.38 ±3.01e	608.37 ± 5.12e	608.37 ±5.12e	1.81 ± 0.01d
Hydrilla verticillata	168.35 ± 2.01g	-	0.97 ± 0.08bc	-	1.20 ± 0.17g	170.53 ±1.89g	405.91 ± 5.50g	405.91 ±5.50g	2.38 ± 0.06b
Ipomoea aquatica	384.86 ± 1.85d	-	0.95 ± 0.63bc	-	2.87 ± 0.09f	388.69 ±1.31d	1572.23 ± 2.87a	1572.23 ±2.87a	4.04 ± 0.01a
Lemna minor	601.47 ± 0.10b	-	3.35 ± 0.10a	5.33 ± 0.07c	-	610.16 ±0.07b	1505.63 ± 10.10b	1505.63 ±10.10b	2.46 ± 0.02b
Marsilea quadrifolia	255.25 ± 1.33f	-	0.91 ± 0.49bc	55.44 ± 0.55a	0.06 ± 0.01i	311.68 ±2.31f	473.56 ± 1.79f	473.56 ±1.79f	1.51 ± 0.01e
Pistia stratiotes	132.20 ± 0.67h	-	0.71 ± 0.04c	1.03 ± 0.03f	0.27 ± 0.01h	134.22 ±2.11h	322.97 ± 0.32h	322.97 ±0.32h	2.40 ± 0.03b
Salvinia molesta	131.23 ± 1.29h	-	2.39 ± 0.54ab	23.88 ± 1.30b	0.21 ± 0.01h	157.72 ±0.56g	97.09 ± 1.18j	97.09 ±1.18j	0.61 ± 0.01j
Salvinia natans	118.57 ±0.78i	-	-	-	19.21 ± 0.22c	137.78 ±1.00h	116.51 ± 0.89j	116.51 ±0.89j	0.85 ± 0.01i
Spirodela polyrhiza	368.28 ± 7.45d	-	1.00 ± 0.01bc	3.80 ± 0.34cd	0.93 ± 0.02g	374.03 ±6.06d	724.41 ± 12.66d	724.41 ±12.66d	1.93 ± 0.02c
Wolffia globosa	728.27 ± 17.19a	16.54 ± 1.18a	2.70 ± 0.05a	1.65 ± 0.01ef	22.44 ± 1.57b	771.63 ±17.37a	909.28 ± 16.17c	909.28 ±16.17c	1.17 ± 0.01g

Values having the means (n = 3) in each row with different superscript are significantly (p < 0.05) different.

The n-6 PUFA content was significantly higher in W. globosa compared with others. This was followed by L. minor and A. pinnata. The minimum level was found in A. microphylla. Among n-6 PUFA, LA (C18:2n-6) was the dominant one and was present in all macrophytes. Arachidonic acid (C20:4n-6) was the second dominant n-6 PUFA found in all macrophytes, except in L. minor. ALA (C18:3n-3) was the only member of n-3 PUFA present in all these macrophytes. ALA content was significantly higher in I. aquatica compared with others. This was followed by L. minor and W. globosa. The highest (4.04) n-3/n-6 was found in I. aquatica (Supplementary Table 2). The ratio ranged from 0.61 (S. molesta)−2.46 (L. minor) in other macrophytes.

Discussion

A wide variation in the composition of freshwater macrophytes was recorded in the present study. The advantage of this study is that plants were cultured in the outdoor systems following a standard protocol (3). Therefore, almost the same quality of products is expected in a further study. There is scope for improvement in the nutritional value as the quality of the culture medium influences the composition of the plants.

In the present study, crude protein levels in three members of Lemnaceae family and E. fluctuans were above 30%, and protein contents of other macrophytes (except P. stratiotes, S. natans, and M. quadrifolia) were above 20%. The present study confirms the previous finding that macrophytes are rich sources of protein. The protein contents of L. minor and S. polyrhiza were 36.07 and 35.82%, respectively (3, 4). A previous study in Bangladesh reported that the protein contents of E. fluctuans and I. aquatica were 16.69 and 21.45%, respectively; macrophytes were collected from natural water bodies (15). In the present study, protein contents of E. fluctuans and I. aquatica were 16.35 and 7.51% higher compared with the same macrophytes studied in Bangladesh. Lipid contents of I. aquatica, S. polyrhiza, and L. minor ranged from 7.16 to 8.13% in the present study. The lipid contents of E. fluctuans and I. aquatica were 1.90 and 3.82% higher in the present study compared with the previous study (15). Ash contents of these two macrophytes were also higher in the present study compared with the previous one. Higher levels of ash contents of H. verticillata, W. globosa, and P. stratiotes compared with other macrophytes enhanced the nutritional value of these plants as feed ingredients for fish, poultry, and livestock. In the present study, lower levels of carbohydrates were observed in macrophytes compared with the plants harvested from the wild (15). Culture of macrophytes with organic manures enhanced the nutritional value of plants.

Among these macrophytes, highest levels of macrominerals, Na and Mg, were found in P. stratiotes. K and Ca were highest in W. globosa and S. polyrhiza, respectively. In the present study, among various macrophytes, P. stratiotes ranked second and fifth for Ca and K, respectively. A previous study reported the highest Ca level in Hydrilla sp., followed by P. stratiotes and E. crassipes. There was no variation in Mg level among these three macrophytes (16). Macromineral profile of leaves and roots of P. stratiotes collected from a natural water body of Nigeria was documented (17). This study showed that Na, K, Ca, and Mg contents were 3.73, 32.83, 2.30, and 3.70 g/kg of leaves, respectively. In the present study, Na, Ca, and Mg contents were 47, 20, and 30%, respectively, higher in P. stratiotes compared with the plants studied in Nigeria. K content was almost the same in the plants grown in two different conditions. The Na, Ca, Zn, and Cu contents were higher in I. aquatica grown in Bangladesh compared with the macrophytes assayed in the present study (15). Although, Mg, K, and Fe contents were higher in the I. aquatica assayed in the present study compared with the plants studied in Bangladesh, Na, Mg, and K contents were higher in E. fluctuans evaluated in the present study compared with the previous study in Bangladesh. The Na, K, Mg, and Ca contents were higher in A. filiculoides and S. molesta grown in swine lagoons compared with the present study (18). In A. filiculoides, Na, K, Mg, and Ca contents were 2.77, 22.5, 5.04, and 9.3 g/kg (dry matter), respectively. In S. molesta Na, K, Mg, and Ca contents were 4.44, 34.7, 5.18 and 10.6 g/kg (dry matter), respectively.

In the present study, Na to K ratio ranged from 0.038 (M. quadrifolia) −0.276 (I. aquatica). The ratio was 0.063, 0.109, 0.121, 0.160, 0.177, 0.182, 0.185, 0.205, 0.218, and 0.238 in L. minor, W. globosa, S. polyrhiza, A. pinnata, E. fluctuans, H. verticillata, A. microphylla, S. molesta, P. stratiotes, and S. natans, respectively. In all these macrophytes, the ratio of Na to K is less than the WHO/FAO-recommended ratio for an adult human, i. e., <0.49 (19). Various studies showed the effect of culture medium on the mineral profile of macrophytes (8, 20, 21). In different species of duckweeds Na: K varied from 0.027–1.49 (K: Na = 0.67–37). In Wolffia, the ratio was 0.025 (K: Na = 40) and in another species, W. microscopica it was 0.003 (K: Na = 276).

In the present study, the Mg: Ca varied from 1.20 (L. minor)−4.65 in (E. fluctuans). Ca has been serving as the main structural mineral and helps in metabolism. It serves as a signal for vital physiological processes. Mg, the fourth most abundant cation in the body, is a co-factor for 350 cellular enzymes, most of which are involved in energy metabolism (22), hence, the ratio of Mg: Ca should be maintained. The Mg: Ca ratio was 0.4 in duckweed (21) and 0.5 in other species, W. microscopica (8). In the present study, the ratio was 1.28 for W. globosa.

The trace minerals analysis showed that among these macrophytes, P. stratiotes was a rich source for Mo, Fe, and Al. This macrophyte also has considerable amounts of Mn, Zn, and Sr. A. microphylla and A. pinnata were also rich sources of Fe and Mo. In a different strain of W. arrhiza, Fe contents ranged from 0.16–0.29 μg/g freeze-dried sample (23). The Fe content of W. globosa was 254.12 μg/g in the present study. Higher levels of Zn and Cu were found in I. aquatica grown in Bangladesh compared with the present study; Fe content was higher in the present study compared with the previous one (15). Fe content of E. fluctuans grown in two different environments was the same. Zn and Cu contents were lower in the plants assayed in the present study compared with the plants studied in Bangladesh. The Cu content of S. molesta grown in swine lagoons was 13 g/kg, dry weight (18). In the present study, Cu content of S. molesta was less compared with the previous study.

In the present study, the highest level of ultra-trace mineral Se was found in S. natans. This important mineral was also present in H. verticillata and P. stratiotes. It was interesting to record that Se was absent in S. molesta, S. polyrhiza, E. fluctuans, and I. aquatica. The Se content of freeze-dried W. arrhiza was <0.03 μg/g (23). In the present study, Se content of W. globosa was higher compared with the previous study. Significantly higher Cr levels were found in P. stratiotes, A. microphylla, and A. pinnata compared with other macrophytes. A significantly higher Co level was found in H. verticillata compared with the others. This macrophyte was followed by P. stratiotes and S. natans. Co content in all these macrophytes was >0.50 μg/g (dry weight). Among these macrophytes, the highest Ni content was found in S. polyrhiza, and this macrophyte was followed by H. verticillata and P. stratiotes. In the present study, the contents of heavy metals viz. Cd, Cu, Pb, and Sn of macrophytes were within the permissible limits (Cd: 0.2, Cu: 73.3, Pb: 0.3, Sn: 250 Zn: 99.40; mg/kg of wet weight) of WHO/FAO (24). In the present study, the mineral composition was evaluated in the dry sample. Therefore, the moisture (minimum 90%) contents of the samples should be considered at the time of comparison with the permissible limit of WHO/FAO for humans (where fresh plants were considered). In seaweeds, there is no regulation on the maximum heavy metals contents (25).

Various studies showed the dietary requirements of different macro, trace, and ultra-trace minerals for different animals (Supplementary Tables 3A,B). Na requirements of grass carp (Ctenopharyngodon idella), poultry, cattle, and humans are 2, 0.012–0.200, 0.96 g/kg diet, and 2.4 g/day, respectively. Among various fishes and prawns (Pinneaus indicus), Mg requirements vary from 0.4 to 0.946 g/kg of diets. K requirements recorded for common carp (Cyprinus carpio), grass carp, and Nile tilapia (Orechromis niloticus) are as follows: 0.9–12.4, 4.6, and 2.1–3.3 g/kg diets. K requirements for poultry, cattle, and humans are 0.3 and 2.4 g/kg diet and 3.5 g/day, respectively. Among different groups of fishes, rohu (Labeo rohita), common carp, grass carp, catla (Catla catla), and Nile tilapia require 1.9, 0.1, 2, 1.9 and 7 g Ca/kg diet, respectively. Ca requirements for poultry, cattle, and humans are 8 and 5.12 g/kg diet and 1.0 g/day, respectively. Among various fishes, Mn, Fe, Zn, and Co requirements vary from 12–25, 30–200, 15–79, and 0.01–0.5 mg/kg diet, respectively. Nile tilapia requires Se and Cr at the rate of 0.4 and 139.6 mg/kg diet, respectively. Fe, Zn, Cu, Se, Cr, and Co requirements are also evaluated for poultry, cattle, and humans. In channel catfish Ictaluraus punctatus, Fe, Cu, Mn, Zn, Se, and Co requirements were 30, 5, 25, 200, 0.1, and 0.05 mg/kg feed, respectively (26). In fish, Fe deficiency causes hypochromic microcytic anemia, Co and Mn deficiencies result in poor growth; Zn deficiency causes growth depression, cataract, and caudal fin and skin erosion; Se deficiency results in muscular dystrophy. In fish nutrition, Co plays a significant role. In common carp, the addition of cobalt chloride/cobalt nitrate enhanced the growth and hemoglobin formation (27). Therefore, supplementation of freshwater macrophytes may help to overcome the mineral deficiency in fish and other animals without showing any negative impact (9).

The fatty acid compositions of the two duckweeds L. minor and S. polyrhiza showed similarity with the previous study (3, 4). In the present study, palmitic acid and oleic acid were the dominant SFA and MUFA, respectively. Similar results were also found in four duckweeds, Landoltia, Lemna, Wolffiella, and Wolffia (8). The fatty acid compositions of four aquatic plants S. cuculata, Trapa natans, L. minor, and I. reptans showed that cis-15 tetracosenoic acid and 9-hexadecenoic acid were the dominant fatty acids, and highly unsaturated fatty acids contents were higher compared with the saturated fatty acids (28). In the present study, LA was the major contributor for n-6 PUFA in all plants, and except in L. minor, arachidonic acid was also found in all macrophytes. ALA was the only member of n-3 PUFA present in these macrophytes. The presence of LA and ALA were recorded in duckweeds (8). The freshwater teleosts are capable of converting ALA to long-chain polyunsaturated fatty acids (LC-PUFA) like eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) (29–31). Therefore, the feeding of fish with freshwater macrophytes-based diets helps to fulfill the LC-PUFA requirements of cultured fish (32, 33). The n-6/n-3 PUFA was always <1; it ranged from 0.48–0.94 in different Wolffia species (23). A similar result was also found in the present study, except in two species of Salvania, where the ratio was >1.0.

Conclusion

Among these macrophytes, Na, Mg, Cr, and Fe contents were maximum in P. stratiotes; this macrophyte ranked second for Co, Sr, and Ca. H. verticillata was the richest source for Cu, Mn, Co, and Zn, and it ranked second for Se. Ca, Mg, Sr, and Ni contents were higher in S. polyrhiza compared with the others. S. natans and W. globosa were rich sources for Se and K, respectively. All these macrophytes were rich sources of n-6 and n-3 fatty acids. This study shows that macrophytes have an immense potential to be used as rich sources of minerals, as well as n-6 and n-3 PUFA for fish, poultry, and livestock.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

Author Contributions

JS, RC, and DRT designed the study. GK, RG, AS, NK, JS, and RC grown the macrophytes and analyzed samples. RC, DRT, GK, and JS wrote the manuscript. GK, AS, RG, NK, RC, DRT, and JS prepared tables. All authors contributed to the article and approved the submitted version.

Funding

This study has been supported by the Department of Biotechnology, DBT, New Delhi, India (Dy. No.102/IFD/SAN/4678/2015-2016) and Biotechnology and Biological Science Research Council, BBSRC (BB/N005031/1). GK is thankful to the Indian Council of Medical Research (3/1/3/JRF-2017/HRD-LS/55340/01).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Table 2

Monounsaturated fatty acids (MUFA) profiles of freshwater macrophytes cultured with organic manures (mg/100 g, dry weight).

Macrophytes /Fatty acids	C16:1n-9	C16:1n-7	C17:1	C18:1n-9	C20:1n-9	C22:1n-9	C24:1	∑MUFA
Azolla microphylla	9.85 ± 0.70cde	-	12.49 ± 0.75c	77.22 ± 1.20e	-	-	2.83 ± 0.74b	102.39 ±1.91e
Azolla pinnata	9.39 ± 0.52cde	-	91.52 ± 0.17b	106.75 ± 0.34c	-	-	-	207.67 ±0.35b
Enhydra fluctuans	-	25.29 ± 0.52c	-	44.99 ± 0.21g	0.28 ± 0.13cd	-	0.18 ± 0.13c	70.75 ±0.73f
Hydrilla verticillata	0.13 ± 0.02g	8.81 ± 0.42d	-	48.97 ± 1.60g	14.15 ± 0.14a	-	0.21 ± 0.03c	72.28 ±3.67f
Ipomoea aquatica	0.33 ± 0.02f	27.25 ± 0.29c	-	24.16 ± 1.20h	-	-	3.45 ± 0.62b	55.20 ±1.73g
Lemna minor	51.60 ± 0.08a	86.47 ± 0.15a	-	9.67 ± 0.10i	2.39 ± 0.04b	-	7.01 ± 0.11a	157.17 ±0.12c
Marsilea quadrifolia	7.86 ± 0.26de	24.61 ± 0.42c	168.24 ± 2.69a	127.93 ± 2.59b	0.54 ± 0.40cd	7.58 ± 0.76b	1.14 ± 0.74c	337.94 ±7.36a
Pistia stratiotes	0.65 ± 0.08f	10.59 ± 0.63d	-	30.66 ± 0.03h	0.25 ± 0.08cd	5.39 ± 0.25c	0.82 ± 0.03c	48.38 ±0.14g
Salvinia molesta	6.51 ± 0.89e	10.41 ± 2.38d	9.53 ± 0.49d	94.04 ± 0.02d	0.39 ± 0.05cd	3.63 ± 0.25d	0.51 ± 0.01c	125.06 ±4.06d
Salvinia natans	16.32 ± 0.74b	-	4.71 ± 0.34e	68.48 ± 1.18f	-	-	0.65 ± 0.01c	90.16 ±0.28e
Spirodela polyrhiza	13.81 ± 1.55bc	38.93 ± 2.70b	-	49.23 ± 5.09g	0.13 ± 0.02cd	3.30 ± 0.59d	0.06 ± 0.01d	105.49 ±5.59e
Wolffia globosa	12.00 ± 0.69bcd	40.67 ± 0.54b	-	137.22 ± 0.61a	0.64 ± 0.03c	17.65 ± 0.92a	0.20 ± 0.02c	208.40 ±2.60b

Values having the means (n = 3) in each row with different superscript are significantly (p < 0.05) different.