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Developing Guidelines for Azolla microphylla Production as Compost for Sustainable Agriculture

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27 September 2024

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29 September 2024

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Abstract
Azolla is a substitute compost that has the potential to enhance nutrient cycling in agricultural systems for sustainable development. In this study, four experiments were conducted to compare the Department of Agriculture (DOA, Thailand)'s methodology for determining the suitable type and rate of animal manure and the optimal light intensity for the growth and yield of Azolla (Azolla microphylla). The results revealed that applying 100% pig manure gave the highest yield of Azolla compared to the other manure. However, there was no discernible difference in yield across the various doses (20.16, 30.16, and 40.16 gN m-2) of pig manure treatments, for which the minimal pig manure dosage of 20.16 gN m-2 was chosen. For further experimentation in the optimal light in-tensity, the 40% shading gave the highest yield of Azolla compared to no shading or 20 and 60% shading. When compared with the DOA Thailand methodology (1.27 kg m-2 of cow manure and covered with a size 32 mesh net), the findings indicated that the modified method (20.16 gN m-2 of pig manure + 40% shading) gave a 16% greater Azolla yield than that under the DOA Thailand methodology.
Keywords: 
Subject: Environmental and Earth Sciences  -   Soil Science

1. Introduction

Azolla (Azolla sp.) is an aquatic floating fern that can be grown in swampy areas of both temperate and tropical regions. It can be grown quickly in varied environments globally, and its proper soils are clay, sandy, and nutrient-rich, as well as stagnant water with little flow [1,2]. The genus Azolla sp. is an excellent biofertilizer due to its ability to harvest atmospheric nitrogen through a symbiotic association with cyanobacteria or blue-green algae (specifically Anabaena azollae), which resides in its dorsal leaves [3,4]. Azolla sp. is known for being a natural source of green manure and is composted for agricultural use in many areas [5,6]. Specifically, Azolla sp. has been widely utilized for nitrogen fertilization; this plant's nitrogen content has been observed to range from 3.3% to 6.0% dry weight [7,8]. Azolla also contains various macronutrients and micronutrients, such as Ca, Mg, Fe, Cu, and Zn, considered essential nutrients for plant growth. In addition, it is well established that Azolla released the nutrients quickly, probably due to the comparatively low or narrow C:N ratio of Azolla. A study by Watanabe et al. [9] showed that around 50% of the N content in Azolla tissues is released within the first 6-8 weeks after soil application and entirely within 13 weeks. Widiastuti et al. [10] suggested that the optimal time to harvest these plants is 1-2 weeks after cultivation, which accounts for the most nutrients stored in its tissues.
The abiotic characteristics, both structural habitat and physicochemical factors, influenced the growth of Azolla [11]. Numerous studies have been conducted on the optimal water depth and conditions (e.g., temperature and pH) for Azolla growth. There were indicated that Azolla thrives in conditions of stagnant or gently flowing water, and the ideal water depth is between 10 and 30 cm [12] since shallow water depths might slow down the Azolla growth and hence reduce its biomass production [13]. Furthermore, Serag et al. [14] reported that Azolla is capable of surviving in environments with pH values between 3.5 and 10, in which the growth rate is regulated by the interacting effect of temperature, light intensity, and the amount of nutrients [15,16]. According to Kathirvelan et al. [17] and Cary and Weerts [18], the optimum temperature for growing Azolla is 18–28 °C with a pH of 4.5–7.5. However, it has been discovered that the ideal pH range for Azolla microphylla is between 4.0 and 4.5 [19]. In addition, Azolla sp. generally requires 25-50% full sunlight for regular growth and multiplication, although the reduction in the nitrogen fixation would occur when the light intensity was lower than 10,000–13,000 lux [20] and a considerable decrease in Azolla yield occurs when light intensity falls below 1500 lux [21]. Nevertheless, the nutrients directly impact Azolla's development and increase sporulation-based multiplication. According to Costa et al. [22], if there is sufficient phosphorus in the aquatic environment, Azolla can grow without the need for a combined form of nitrogen, such as NH4NO3, as numerous studies have shown that phosphorus is a major limiting nutrient for Azolla growth and sporulation [23,24,25].
Several studies have revealed that organic fertilizer as a nutrition source was more efficient for Azolla sp. growth than inorganic fertilizer [2,26]. For instance, Azab and Soror [2] found that the growth and protein content of Azolla sp. was higher in the treatment of organic fertilizer used as poultry manure rather than that of inorganic fertilizer treatment (urea and pure phosphorus), which attributed to the high contents of N and P in the tissues of Azolla sp. exposed to organic treatment. However, the supply of organic fertilizer made from animal manure generally affected the productivity of Azolla biomass, despite the stability and quality of the manure varying depending on animal species, diet composition, manure storage, type of bedding, and moisture content [27]. Some literature has reported the utilization of animal manure, such as cattle [28], poultry, goat, rabbit [29], sheep, vermicompost [30], as well as the wastewater from catfish pond [31] and piggery farm [32], for cultivating Azolla sp. However, cow, swine, and chicken manures are often the most readily available animal manures for Azolla production in the local region of Thailand, which would reduce the production cost of Azolla farming. It is evident that most nutrition supplies from animal manures are local and have varying nutrient concentrations.
Additionally, no research has been conducted on using open-farm animal manure, such as cow or pig manure, following organic farming regulations. The objectives of this study were to establish the type and rates of open-farm animal manures, including cow and pig manure, as well as the optimal shading for the growth and production of A. microphylla. This knowledge can be applied to Azolla cultivation, which has been extensively used for compost or soil amendment for organic plant production, a sustainable alternative protein source for animal feed, and medicinal supplements.

2. Materials and Methods

2.1. Experimental Layout and Treatments

A. microphylla, which is a high-potential fern in biomass productivity and tolerance to various environmental stresses, was selected in this study. It was obtained from the Agricultural Production Sciences Research and Development Division, Department of Agriculture, Bangkok, Thailand. Firstly, A. microphylla was vegetatively multiplied in plastic containers before being injected into the experiments at a small farm in Chai Nat province (Latitude: 14.913466, Longitude: 99.963741), Thailand, following the Department of Agriculture. In Brief, a 20 cm depth of soil was mixed with 1.27 kg m-2 of cow manure uniformly spread over, and the water depth from the soil layer was raised to 10 cm using tap water. A 100 g m-2 of A. microphylla was placed into plastic containers and shaded with a blue nylon net having 70% light transmittance, which was determined by comparing the light intensity under full sunlight with that under the blue nylon net. A. microphylla was harvested after ten days for use in setting up the studies.
Four experiments were conducted in concrete ponds, each with a volume of 0.105 m3 (55 cm length, 55 cm width, 35 cm height). The purpose was to investigate the most suitable type and rate of animal manure and the optimal shading conditions to evaluate the maximum biomass growth and nutrient compositions of A. microphylla. This modified cultivation method was then compared with the conventional method the Department of Agriculture referenced. All the experimental designs and layouts were described as follows.

2.1.1. Effect of different manure types on growth development and chemical compositions of A. microphylla

The experiment used a completely randomized design (CRD) with three replicates. The treatments were T1: control (no manure), T2: cow manure, T3: pig manure, T4: 25:75 v/v of cow manure and pig manure, T5: 50:50 v/v of cow manure and pig manure, and T6: 75:25 v/v of cow manure and pig manure. The identical amounts of manure per each concrete pond were 380 g (1.27 kg m-2).
The chemical properties of cow manure used in this experiment were as follows: pH 9.30, EC 6.63 dS m−1, 41.63% organic matter, 1.16% total N, 0.34% total P, 3.31% total K, and a C:N ratio of 20.82. In addition, the chemical properties of pig manure were as follows: pH 7.50, EC 1.12 dS m−1, 15.90% organic matter, 0.52% total N, 0.64% total P, 0.72% total K, and a C:N ratio of 17.73.

2.1.2. Effect of different manure application rates on growth development and chemical compositions of A. microphylla

The selected manure from the previous experiment (2.1.1) was used to evaluate the appropriate rate for A. microphylla cultivation. A completely randomized design (CRD) with three replications (0.105 m3 of concrete ponds) was conducted in this trial. Four treatments of applying rates were consisted of T1: no manure (control), T2: 20.16 g N m-2, T3: 30.16 g N m-2, and T4: 40.16 g N m-2.

2.1.3. Effect of different shading levels on growth development and chemical compositions of A. microphylla

The selected manure type indicated in experiment 2.1.1 and the optimal manure rate discovered in experiment 2.1.2 were used to evaluate the appropriate shading levels for A. microphylla cultivation continuously. The experiment was laid out in a completely randomized design (CRD) with three replications (0.105 m3 of concrete ponds). Four treatments consisted of T1: no shading (control), T2: 20% shading net, T3: 40% shading net, and T4: 60% shading net. The light transmittances of the three shading nets (20%, 40%, and 60% black shading nets, respectively) were 80%, 71%, and 50%, respectively, which was determined by comparing the light intensity under full sunlight with that under the shade nets.

2.1.4. Comparison between the developed method and the conventional method

The developed method from the previous experiments (2.1.1-2.1.3) was used to compare the conventional method referenced by the Department of Agriculture, Thailand. The management of manure type, rate, and shading level of the traditional method consisted of 1.27 kg m-2 of cow manure shaded with the blue nylon net, with the light transmittance being 70%. The developed method was called 3.86 kg m-2 (20.16 g N m-2) of pig manure and shaded with 40% black shading nets.
The entire set of experiments was designed to cultivate A. microphylla with a density of 100 g m-2. Fertile soil was evenly distributed into the pits at a depth of 20 cm from the soil layer, and freshwater was added during the experimentation to a 10 cm depth from the soil layer, which the water's pH ranged from 7.13 to 7.57. Daily readings of the water's temperature (measured 10 cm depth from the water surface), air temperature (measured 20 cm above the canopy), and relative humidity were made.

2.2. Data Collection and Analysis

2.2.1. Growth rate analysis

Three samples of A. microphylla were harvested every ten days for each treatment (one sample from each concrete pond). They were then thoroughly washed with tap water to remove dirt particles and carefully blotted dry on a paper towel before weighing them to determine the fresh weight. They were dried in the oven at 50 °C for 72 h until a stable dry weight was achieved, and subsequently, their dry weight was determined. The total fresh and dry weight of harvested A. microphylla each month (three harvesting per month) under different experimental treatments was also calculated. The relative growth rate and doubling time expressed as g g-1 per day and day, respectively, were calculated using the formula as follows [2,33,34]
RGR = (lnW2 − lnW1) / (t2 − t1)
where
W1 and W2 represent the plant fresh weight at times t1 and t2 of the sampling period.
Doubling time = t/r
where
t = the duration of Azolla growth; r = [log (Wt/Wo)] / 0.301; Wt = weight of Azolla at time t; Wo = weight of initial inoculums.
For the determination of the dry matter from the fresh and dry weight of harvested A. microphylla, the equation below with the modification [35] was used:
Dry matter (%) = 100 − {[(fresh weight − dry weight) / fresh weight] x 100}

2.2.2. Chemical analysis of A. microphylla

The total N concentration was determined by using the Kjeldahl method. The levels of phosphorus (P) and potassium (K) in the samples were measured using a modified version of the standard protocol of the Association of Official Analytical Chemists [36]. In Brief, each sample (1.0 g) was mixed with a solution of nitric-perchloric acid (HNO3:HClO4 in a ratio of 2:1 v/v) and digested. The digestion process was carried out in 15 mL of the solution. After digestion, the samples were diluted with distilled water to a final volume of 50 mL and stored in plastic tubes at room temperature. The determination of P in distilled samples was analyzed by using a spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan) at 420 nm. The K concentration was analyzed by an atomic absorption spectrometer (PinAAcle900F, Perkin-Elmer, Waltham, MA, USA). The Walkley and Black [37] method quantified the organic carbon and organic matter. The ratio of C and N of each sample was then calculated.
The protein content was determined using the Kjeldahl method [36] for comparison between the developed method and the conventional method in Experiment 2.1.4, which was calculated by the following formula:
Protein (%) = % N x 6.25

2.3. Statistical Analysis

The experimental treatment effects were analyzed using a completely randomized design (CRD) with three replications. Data were analyzed through one-way analysis of variance (ANOVA). The means of different manure types and rates, as well as the different shading levels (2.1.1-2.1.3), were evaluated by Duncan's multiple range test (p ≤ 0.05). For the comparison between the modified cultivated method and the conventional method (2.1.4), the mean values were compared by using the Independent-Samples T-test using SPSS statistic, Version 26.0 software (IBM Corp., Armonk, NY, USA).

3. Results

The average temperature in the Azolla culture area (an open environment) ranged from 22.5–34.3 ᴼC. The water temperature was slightly lower than the surrounding temperature and varied between 21.6–33.9 ᴼC. The relative humidity in the air ranged from 60.19 to 76.09%. In addition, the water pH stayed between 7.03–7.85 during the experiment. The cultivation of A. microphylla in each experiment was uniformly set up using a 20 cm depth of soil, and the water depth from the soil layer was raised daily to 10 cm using tap water. A 30 g of A. microphylla was placed into the concrete ponds and harvested every ten days for the experiment (Figure 1).

3.1. Effect of Different Manure Types on Growth Development and Chemical Compositions of A. microphylla

Growth responses of A. microphylla under the different manure treatments are presented in Table 1. The average relative growth rate of A. microphylla was much higher in the presence of manure than in the absence, ranging from 0.11 to 0.13 g g-1 day-1. In terms of the doubling time, it was seen that all manure treatments quadrupled the amount of A. microphylla, with an average doubling time of 5.86 to 8.50 days, whereas the lack of manure necessitated 12.79 days for the amount to double. Meanwhile, treatment with pig manure revealed the highest relative growth rate and the shortest doubling time compared to the other fertilizer treatments. Furthermore, compared to no manure or other manure treatments, the application of pig manure produced the highest fresh weight of A. microphylla, 360.16 g month-1. The highest dry weight of A. microphylla was found under pig manure treatment (16.98 g month-1), which did not significantly differ from the treatment of cow manure and 25:75 w/w of cow and pig manure. Conversely, A. microphylla produced the least amount of dry matter (4.89%) when the pig manure was added, without any significant difference with the ratio of 25:75, 50:50, and 75:25 w/w of cow and pig manure (5.37%, 5.19% and 5.48%, respectively) (Table 1).
The nutritional contents of A. microphylla with several kinds of manure use can be seen in Table 2. The different fertilizer treatments unaffected the total nitrogen and phosphorus content, ranging from 2.30 - 2.55% and 0.09 - 0.14%, respectively. Additionally, the organic matter content of A. microphylla, which was found to fluctuate between 59.32 and 61.94%, was unaffected by the presence or absence of manure. As a result, there was no significant difference in the carbon-to-nitrogen ratio (C:N ratio), with values ranging from 13.86 to 15.10 (Table 2). However, the significantly highest potassium content of A. microphylla occurred in the pig manure treatment (1.14%), which did not differ considerably from the 1.02% potassium content seen in the cow manure treatment. In this case, potassium content was increased by 44% and 29%, respectively, compared with the control treatment (no fertilizer).
As a result of this experiment, the pig manure was chosen to assess the appropriate rate in the following studies because, in comparison to the other treatments, it produced the highest fresh weight yield and relative growth rate, the shortest doubling time, and the highest potassium content in A. microphylla tissues.

3.2. Effect of Different Manure Application Rates on Growth Development and Chemical Compositions of A. microphylla

Based on the observation, the average growth rate of A. microphylla grown under the pig manure application at all rates was considerably greater than without, which showed an increment of 57% (Table 3). Whereas, all rates of pig manure treatment resulted in almost two days shorter than that with no manure applied, varying from 3.10 to 3.22 days. Additionally, all rates of pig manure application led to noticeably greater fresh and dry weight increments for A. microphylla when compared to no manure treatment; these increments were enhanced by 2.1-2.3 and 1.6-1.7 times, respectively. Concerning fresh and dry weight, A. microphylla had the highest dry matter at 5.46 % when no manure was applied. In contrast, all rates of pig manure application had no statistical effect on A. microphylla 's dry matter, which was between the range of 4.05 to 4.18 % (Table 3).
Then, the nutrient content of A. microphylla cultivated with various amounts of pig manure was discovered to be significantly higher when compared to cultivation without pig manure, which contained the total phosphorus and potassium between 0.84-1.01% and 4.21-4.33%, respectively. In addition, A. microphylla tissue also had a 24-46% increase in total nitrogen content after the pig manure was exposed. However, there was no discernible difference between the pig manure treatment at a rate of 40.16 gN m-2 and the control group. In terms of organic matter, there was no significant difference between the treatments, with results falling between 55.05 and 57.85%. The control plants without N supply showed the highest C:N ratio, which was not significantly different from the pig manure treatment at a rate of 40.16 gN m-2. The C:N ratio was only somewhat decreased by adding N from the pig manure at a rate of 20.16 to 30.16 gN m-2, and this effect was not statistically significant (Table 4).
Therefore, considering the experiment results, pig manure applied at a rate of 20.16 gN m-2 was found to be the lowest rate of application for promoting A. microphylla growth, yield, fresh and dry weight, and reducing doubling time. Even with the higher rates of pig manure application, the increase in A. microphylla tissue nutrients was not statistically different. For the next experiment, pig manure was selected at a rate of 20.16 gN m-2.

3.3. Effect of Different Shading Levels on Growth Development and Chemical Compositions of A. microphylla

Although there was no difference in the relative growth rate of A. microphylla among the shading levels in a range of 0.22-0.23 g g-1 day-1, different shading levels significantly influenced its doubling time. Among the A. microphylla grown in the control (no shading) and those under varying shading levels, the shortest doubling time was observed in the 40% shading treatment, which did not differ noticeably from the non-shading (control) and 20% shading treatments.
The 40% shading and non-shading treatments observed the greatest fresh and dry weight. However, A. microphylla grown under the 60% shading showed the significantly lowest fresh and dry weight, which decreased by 11% and 15%, respectively, compared to the control. In terms of dry matter, it was discovered that the different shading did not affect the dry matter of A. microphylla, that were in a range of 4.10-4.44% (Table 5).
The nutrient contents in A. microphylla tissues varied across different shading treatments. There were no significant differences among the shading treatments in the total nitrogen content and organic matter, which ranged from 3.27% to 3.81% and 59.92% to 63.66%, respectively. However, the total phosphorus and potassium contents in A. microphylla tissues followed a similar trend, with the highest values observed under the 20% shading treatment. There were no discernible differences between the 20% and 40% shading treatments for total phosphorus content and between the 40% and 60% shading treatments for total potassium content. Specifically, the lowest total phosphorus and potassium contents were observed in A. microphylla tissues cultured with non-shading (control) treatment. In addition, the C:N ratio decreased with increasing levels of shading to 40% and 60%. Control plants without shading applied showed an increase of the C:N ratio to 11.09, without any significant difference with 20% shading plants (Table 6).
Based on experimental outcomes, the growth development of A. microphylla was not statistically different, whether it was slightly shaded (20% and 40% shading) or not. However, this experiment revealed that 40% shading was considered as an optimal shading for promoting both A. microphylla’s development and the highest macronutrient content (3.81% N + 0.67% P + 1.88% K = 6.36% N+P+K). In addition to the observation under non-shaded situations, rainwater pouring directly on A. microphylla can harm the plant by smashing and dispersing it. The 40% shaded allows A. microphylla to be grown in all seasons, including rainy ones, under various environmental conditions. The results of the three experiments led to the conclusion that applying 20.16 gN m-2 of pig manure along with 40% shading was a suitable management method for A. microphylla culture. This resulted in A. microphylla having the shortest doubling time and the highest fresh weight yield, dry weight yield, and nutrient accumulation contents. Consequently, this developed method was selected to compare with the approach advised by the Department of Agriculture, using 1.27 kg m-2 of cow manure (23.37 gN m-2) and shaded with a blue nylon net (30% shading) to avoid insect infestation.

3.4. Comparison Between the Developed Method and the Conventional Method

The comparison between the conventional method advised by the Department of Agriculture, Thailand (using 23.37 gN m-2 of cow manure and shaded with a blue nylon net at 30% shading) and the developed method (using 20.16 gN m-2 of pig manure and shaded with the black shading net at 40% shading) for A. microphylla production is displayed in Figure 2. According to the study results, the developed method increased the relative growth rate of A. microphylla from 0.24 to 0.25 g g-1 day-1. It reduced the doubling time from 2.95 to 2.77 days compared to the conventional method. In addition, the modified method enhanced the fresh weight of A. microphylla by almost 16% compared to the conventional method. More specifically, the developed method elicited an increment in the total nitrogen, total potassium, protein, and organic matter (0.61, 28.30, 0.46, and 6.85%) of A. microphylla tissues. Its total phosphorus was 28.57% lower in the developed method compared to the conventional method. Regarding the C:N ratio, it was discovered that the developed method provided a higher C:N ratio than the conventional method, which showed an increment of 6.13% (Table 7).

4. Discussion

Azolla is one of the fastest-growing aquatic macrophytes globally [11]. Because of its broad distribution and quick biomass production, Azolla has excellent potential as a green manure and biofertilizer that could be used to replace part or all of the inorganic nitrogenous fertilizer required for plant production [26] and could encourage the sustainable plant production, especially in the local farmers [38,39]. However, Azolla's growth directly affects the optimal ecological systems, including temperature (air and water), relative humidity, water quality and availability, nutrition, and light intensity. To investigate the appropriate type and rate of animal manure as well as the ideal light intensity in this study, the temperature of air (22.5–34.3 ᴼC) and water (21.6–33.9 ᴼC), as well as the relative humidity estimated at 60.19–76.09%, were observed to confirm that these ecological factors were optimized. Some literature reported that the suitable temperature should not exceed 35 ᴼC [40], and a mean relative humidity for allowing Azolla growth was estimated at 55-83% [11]. Additionally, the water pH (7.03–7.85) during these experiments was within the optimal growth pH of Azolla species that various literatures identified as a range of pH 5–8 [18,41].
From the research finding of the growth responses of A. microphylla plants exposed to different treatments of cow manure and pig manure, only the 100% pig manure treatment revealed the shortest doubling time (5.86 days), which was nearly the previous study, which was found in a range of 2–5 days [42]. In addition to the appearance of A. microphylla among the different manure treatments, the reddish-brown coloration was more observed in A. microphylla grown under the 100% cow manure treatment, compared to that grown under the 100% pig manure treatment (Figure 3). The presence of anthocyanin pigments shows that the Azolla plants were under stress, usually because of high light intensity or nutrient deficiency, especially the phosphorus [11,41]. Furthermore, Temmink et al. [43] stated that the most crucial and frequently limiting component for Azolla growth is phosphorus, which is red, which could indicate its phosphorus deficiency. The current results are consistent with this general statement, reflecting the nutritional content of different manures, in which the phosphorus content in cow manure was two times less than in pig manure.
Furthermore, previous research has shown that Azolla plants can rapidly grow during the early stages of development due to their ability to absorb phosphorus [44]. Phosphorus is important for increasing plants' dry weight, which helps them form the necessary pyrophosphate compounds, which serve as the primary energy source for plant growth and development [45]. Phosphorus is also a component of phospholipids, essential for the structure of cell membranes [29].
Although nitrogen is also an essential nutrient that Azolla needs in large amounts, it was found that the cyanobacteria (Anabaena sp.), which reside in the Azolla's leaf sheaths, play a significant role in fixing nitrogen from the air and converting it into a form that plants can use [20]. Additionally, several researchers found that external N supply did not increase Azolla growth, and it can double its biomass within one week under N-free and P-rich conditions, entirely relying on the symbiosis with diazotrophs for its N supply [43,46]. Our results are consistent with this finding that supplying pig manure at different N rates had no positive or negative effects on the fresh weight of A. microphylla. Notably, A. microphylla tended to accumulate lower nitrogen when pig manure was treated at the highest rate. It seems likely that the negative impact of the high pig manure rate on nitrogen accumulation of A. microphylla was due to excessive salinity in the manure released into water, which agreed with Arora and Singh [47], who reported that salinity drastically decreased biomass production in all 6 Azolla species (A. filiculoides, A. mexicana, A. microphylla, A. pinnata, A. rubra, and A. caroliniana). Based on this observation, however, A. microphylla showed higher tolerance to salinity than other species, and our results concerning saline tolerance of this Azolla species may be similar to that previous report.
The light intensity has a direct effect on leaf growth and fresh biomass yield of Azolla species which previous studies with A. pinnata reported the optimal average natural light varied from 47,500 - 75,000 lux [48,49,50]. Nevertheless, Effendi et al. [51] stated that the optimal growth rate of A. microphylla was found under the 30% shaded level, compared to the full sunlight (0% shade level) or the other shade levels (50%, 70%, and 100% shaded levels). In this study, the measured light intensity in daily (12 p.m.) throughout the different shading studies was measured that the average illuminations under different shading conditions from no shading, 20% shading, 40% shading, and 60% shading were 88,300-114,167 lux (Av=114,167 lux), 82,567–112,200 lux (Av= 97,384 lux), 69,767–98,733 lux (Av= 84,250 lux), and 45,633–70,133 lux (Av=57,883 lux), respectively. From the results, it seems that the full sunlight caused A. microphylla to produce higher final biomass, which was not significantly different from that grown under 20% and 40% shading. However, the 60% shading with the lowest intensity of sunlight than 57,883 lux affected the fresh and dry weight of A. microphylla. This finding agreed with the previous reports, which stated that the light intensity had a strong correlation to the growth rate of Azolla species and usually required 25-50% full sunlight for regular growth. In addition, the increase would decrease quickly under heavy shade (more than 50% of full sunlight or the light intensity range of 1202 to 44,945 lux) by reducing photosynthesis [41,42,52].
In general, Azolla plants have long been used in agriculture as a green manure or soil amendment product because they contain a lot of plant nutrients, such as nitrogen (N), phosphorus (P), and magnesium (Mg), as well as a high organic matter, thereby improving the soil chemical properties and increasing the crop yield [42,53]. An increase in crop yield has been found for a variety of crops, including rice [54], maize [55], and beans [56]. Using Azolla compost in agriculture is also considered a sustainable environmental practice because it reduces methane emissions and slows global warming [57]. To confirm the potential of A. microphylla plants that could be used as compost for crop production, some selected chemical properties of its dry sample were measured to compare with the compost specifications of Thai Agricultural Standard, Thailand, as shown in Table 8. It was evident that the level of organic matter, as well as the macronutrients (N, P, K) in A. microphylla tissues were in accordance with the compost quality standards according to TAS 9503-2005, which the organic matter, total N, total P and total K were 67.42% (≥ 30.0%), 4.92% (≥ 1.0%), 0.75% (≥ 0.5%) and 4.08% (≥ 0.5%), respectively. Additionally, the C:N ratio of A. microphylla tissues (7.96) was less than 20:1, indicating the decomposition process ran ideally [58]. Furthermore, several reports have demonstrated that Azolla species with a low C:N could mineralize within 2-5 days, and about 40–60% of available N and P were released by 20-40 days after application [5,59,60]. Notably, the addition of Azolla compost led to the enhancement of the soil organic matter and soil microbial activity and thus can improve nutrient recycling in treated soil, as well as the formation of both macro and micro aggregates [61,62]. Based on the previously mentioned advantages of Azolla compost and the current results of the chemical composition of A. microphylla tissues compared to Thai Agricultural Standard, A. microphylla can be used as a compost with a beneficial source of nutrients for crop production.

5. Conclusions

Managing the right fertilizer (types and rates) and the optimal light intensity significantly influenced the yield production of A. microphylla. The results reported here demonstrated that the developed method, indicating 20.16 gN m-2 of pig manure application with 40% shading by the black shading net, can increase the relative growth rate (4.17%) and fresh biomass (15.89%), as well as decrease the doubling time (6.10%), compared to the conventional method advised by the Department of Agriculture, Thailand (using 23.37 gN m-2 of cow manure and shaded with the blue nylon net at 30% shading). The current finding method can produce a monthly fresh biomass of 3.7 kg m-2 projected as 40.7 t ha-1 year-1, with two rest days after fertilizer application and harvesting every ten days (three harvesting per month). Nevertheless, the currently developed method for A. microphylla production can be a viable alternative to the management of compost production on their farm for smallholder farmers to decrease crop production costs.

Author Contributions

Conceptualization, O.T.; methodology, O.T.; formal analysis, O.T.; investigation, O.T., and N.S.; resources, O.T.; data curation, O.T.; writing—original draft preparation, O.T.; writing—review and editing, O.T., P.C., and R.M.; supervision, H.E.; funding acquisition, O.T. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by Thammasat University Research Fund, Contract No TUFT 46/2566, and partially supported by the Thailand Science Research and Innovation Fundamental Fund fiscal year 2023.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors thank the Department of Agricultural Technology, Faculty of Science and Technology, Thammasat University, for providing experimental and laboratory facilities.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the study’s design, data collection, analysis, interpretation, manuscript writing, or decision to publish the results.

References

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Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and the editor(s). MDPI and the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.
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Figure 1. The cultivation of A. microphylla in each experiment was uniformly set up using a 20 cm depth of soil (a), and the water depth from the soil layer was raised to 10 cm with tap water. A 30 g of A. microphylla was placed into the concrete ponds (b) and harvested every ten days (c).
Figure 1. The cultivation of A. microphylla in each experiment was uniformly set up using a 20 cm depth of soil (a), and the water depth from the soil layer was raised to 10 cm with tap water. A 30 g of A. microphylla was placed into the concrete ponds (b) and harvested every ten days (c).
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Figure 2. Characteristics of A. microphylla cultivated by conventional (a) and developed (b) methods.
Figure 2. Characteristics of A. microphylla cultivated by conventional (a) and developed (b) methods.
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Figure 3. Morphology of A. microphylla in response to different cow and pig manure treatments.
Figure 3. Morphology of A. microphylla in response to different cow and pig manure treatments.
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Table 1. Growth responses of A. microphylla plants exposed to different cow and pig manure treatments.
Table 1. Growth responses of A. microphylla plants exposed to different cow and pig manure treatments.
Treatment Relative Growth Rate (g g-1 day-1) Doubling time
(day)		 Fresh Weight
(g month-1)		 Dry
Weight
(g month-1)		 Dry Matter
(%)
T1: Control (no fertilizer) 0.09 b1 12.79 a 229.83 d 13.56 c 6.13 a
T2: Cow manure, 100% 0.11 a 7.61 b 301.65 c 16.06 ab 5.62 ab
T3: Pig manure, 100% 0.13 a 5.86 b 360.16 a 16.98 a 4.89 c
T4: Cow manure: Pig manure, 25:75% w/w 0.12 a 6.56 b 305.09 bc 16.02 ab 5.37 bc
T5: Cow manure: Pig manure, 50:50% w/w 0.12 a 7.83 b 312.71 b 15.48 b 5.19 bc
T6: Cow manure: Pig manure, 75:25% w/w 0.11 a 8.50 b 297.25 c 15.29 b 5.48 bc
Significance ** ** ** ** **
C.V. (%) 9.58 18.91 1.58 3.97 5.88
1 Mean with different letters in the same column indicates a significant difference according to Duncan’s multiple range test. ** refer to p ≤ 0.01. Fresh and dry weight was calculated from the volume of each pond at 0.105 m3.
Table 2. The chemical properties of A. microphylla plants exposed to different cow and pig manure treatments.
Table 2. The chemical properties of A. microphylla plants exposed to different cow and pig manure treatments.
Treatment Total N
(%)		 Total P
(%)		 Total K
(%)		 Organic
Matter (%)		 C:N
Ratio
T1: Control (no fertilizer) 2.30 0.09 0.79 b1 59.32 14.95
T2: Cow manure, 100% 2.55 0.10 1.02 a 60.37 13.86
T3: Pig manure, 100% 2.43 0.14 1.14 a 60.12 14.38
T4: Cow manure: Pig manure, 25:75% w/w 2.38 0.10 0.54 c 61.94 15.10
T5: Cow manure: Pig manure, 50:50% w/w 2.52 0.12 0.50 c 61.72 14.36
T6: Cow manure: Pig manure, 75:25% w/w 2.49 0.10 0.33 d 61.71 14.55
Significance ns ns ** ns ns
C.V. (%) 7.51 15.07 9.58 3.24 13.86
1 Mean with different letters in the same column indicates a significant difference according to Duncan’s multiple range test. **, ns refer to p ≤ 0.01 and non-significant, respectively.
Table 3. Growth responses of A. microphylla plants exposed to different rates of pig manure.
Table 3. Growth responses of A. microphylla plants exposed to different rates of pig manure.
Treatment Relative Growth Rate (g g-1 day-1) Doubling time
(day)		 Fresh Weight
(g month-1)		 Dry
Weight
(g month-1)		 Dry Matter
(%)
T1: Control (no fertilizer) 0.14 b1 5.06 a 375.73 b 20.67 b 5.46 a
T2: Pig manure 20.16 gN m-2 0.22 a 3.22 b 792.06 a 32.49 a 4.18 b
T3: Pig manure 30.16 gN m-2 0.22 a 3.15 b 834.08 a 33.60 a 4.05 b
T4: Pig manure 40.16 gN m-2 0.22 a 3.10 b 865.32 a 35.07 a 4.07 b
Significance ** ** ** ** **
C.V. (%) 3.33 5.42 6.69 7.61 3.33
1 Mean with different letters in the same column indicates a significant difference according to Duncan’s multiple range test. ** refer to p ≤ 0.01. Fresh and dry weight was calculated from the volume of each pond at 0.105 m3.
Table 4. Chemical properties of A. microphylla plants exposed to different rates of pig manure.
Table 4. Chemical properties of A. microphylla plants exposed to different rates of pig manure.
Treatment Total N
(%)		 Total P
(%)		 Total K
(%)		 Organic
Matter (%)		 C:N
Ratio
T1: Control (no fertilizer) 2.67 b1 0.09 b 2.11 b 57.75 12.60 a
T2: Pig manure 20.16 gN m-2 3.69 a 0.84 a 4.21 a 55.05 8.75 b
T3: Pig manure 30.16 gN m-2 3.90 a 0.96 a 4.34 a 55.94 8.36 b
T4: Pig manure 40.16 gN m-2 3.32 ab 1.01 a 4.33 a 57.85 10.31 ab
Significance * ** ** ns *
C.V. (%) 11.26 19.74 5.62 17.20 13.07
1 Mean with different letters in the same column indicates a significant difference according to Duncan’s multiple range test. **, *, ns refer to p ≤ 0.01, 0.05 and non-significant, respectively.
Table 5. Growth responses of A. microphylla plants exposed to different shading conditions.
Table 5. Growth responses of A. microphylla plants exposed to different shading conditions.
Treatment Relative Growth Rate (g g-1 day-1) Doubling time
(day)		 Fresh Weight
(g month-1)		 Dry
Weight
(g month-1)		 Dry Matter
(%)
T1: Control (no shading) 0.23 3.12 ab1 903.80 ab 39.15 a 4.44 a
T2: Shading 20% 0.22 3.07 ab 890.47 b 36.63 b 4.14 b
T3: Shading 40% 0.23 3.00 b 922.13 a 37.51 ab 4.10 b
T4: Shading 60% 0.22 3.18 a 806.68 c 32.91 c 4.10 b
Significance ns * ** ** **
C.V. (%) 2.86 1.98 1.58 2.66 2.10
1 Mean with different letters in the same column indicates a significant difference according to Duncan’s multiple range test. **, *, ns refer to p ≤ 0.01, 0.05 and non-significant, respectively. Fresh and dry weight was calculated from the volume of each pond at 0.105 m3
Table 6. Chemical properties of A. microphylla plants exposed to different shading conditions.
Table 6. Chemical properties of A. microphylla plants exposed to different shading conditions.
Treatment Total N
(%)		 Total P
(%)		 Total K
(%)		 Organic
Matter (%)		 C:N
Ratio
T1: Control (no shading) 3.27 0.30 c1 1.56 b 62.29 11.09 a
T2: Shading 20% 3.40 0.75 a 2.11 a 63.66 10.92 a
T3: Shading 40% 3.81 0.67 a 1.88 a 59.92 9.20 b
T4: Shading 60% 3.80 0.44 b 2.06 a 60.81 9.29 b
Significance ns ** ** ns *
C.V. (%) 8.43 11.68 7.07 3.01 8.41
1 Mean with different letters in the same column indicates a significant difference according to Duncan’s multiple range test. **, *, ns refer to p ≤ 0.01, 0.05 and non-significant, respectively.
Table 7. Comparison of A. microphylla productivity between the conventional and developed methods.
Table 7. Comparison of A. microphylla productivity between the conventional and developed methods.
Parameters Conventional Method * Developed Method Compared (%)
Relative growth rate (g g-1 day-1)
Doubling time (day)
Fresh weight (g month-1)		 0.24 0.25 +4.17
2.95 2.77 -6.10
1,589.94 1842.60 +15.89
Dry weight (g month-1) 74.64 71.27 -4.52
Dry matter (%) 4.65 3.87 -16.77
Total N (%) 4.89 4.92 +0.61
Total P (%) 1.05 0.75 -28.57
Total K (%) 3.18 4.08 +28.30
Protein (%) 30.58 30.72 +0.46
Organic matter (%) 63.10 67.42 +6.85
C:N ratio 7.50 7.96 +6.13
* Conventional method referenced from the Department of Agriculture, Thailand.
Table 8. The chemical properties of A. microphylla are compared with Thailand's organic fertilizer standard.
Table 8. The chemical properties of A. microphylla are compared with Thailand's organic fertilizer standard.
Parameters A. microphylla Compost Specifications of Thai Agricultural Standard: Compost (TAS 9503-2005),
Thailand
Organic matter (%) 67.42 ≥ 30.0
C:N ratio 7.96 ≤ 20:1
Total Nitrogen (%) 4.92 ≥ 1.0
Total Phosphorus (%) 0.75 ≥ 0.5
Total Potassium (%) 4.08 ≥ 0.5
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Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.
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