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Article

Azolla Biofertilizer Is an Effective Replacement for Urea Fertilizer in Vegetable Crops

by
Aisha Jama
1,2,
Dwi P. Widiastuti
1,3,
Sutarman Gafur
4 and
Jessica G. Davis
1,*
1
Department of Soil & Crop Sciences, Colorado State University, Fort Collins, CO 80523, USA
2
TUMI Genomics, LLC, Fort Collins, CO 80524, USA
3
National Research and Innovation Agency, Jakarta Pusat 10340, Indonesia
4
Faculty of Agriculture, Tanjungpura University, Pontianak 78124, Indonesia
*
Author to whom correspondence should be addressed.
Sustainability 2023, 15(7), 6045; https://doi.org/10.3390/su15076045
Submission received: 2 March 2023 / Revised: 23 March 2023 / Accepted: 27 March 2023 / Published: 31 March 2023
(This article belongs to the Special Issue Organic Fertilizers: Applications and Research)
Browse Figures
Review Reports Versions Notes

Abstract

:
Azolla spp. is a water fern that hosts Anabaena azolla, an N-fixing cyanobacterium, in its dorsal leaf cavities. Azolla occurs naturally in freshwater bodies in warm-temperate and tropical regions, and they have commonly been grown in rice paddies as a living fertilizer, providing N to the rice crop. We evaluated the potential use of Azolla harvested from freshwater bodies and applied as a biofertilizer to dryland vegetable crops. Two-thirds of the greenhouse gas emissions from crop production is attributed to N fertilizer, including fossil fuels used in fertilizer production and transportation. Azolla grown in on-farm ponds could remove CO2 from the atmosphere and minimize the use of fossil fuels in fertilizer production and transport. A 140-d laboratory incubation was used to compare the N mineralization of Azolla biofertilizer with compost and cyanobacterial biofertilizer treatments. Azolla treatments had the greatest N availability at the end of the incubation (73.0%), with compost demonstrating the least N availability (15.5%), and the cyanobacterial biofertilizers moderate in N release (31.6%). A greenhouse study evaluated the N uptake and yield of kale (Brassica oleracea) receiving Azolla biofertilizer compared to urea and organic fertilizers. The nitrogen uptake by kale followed the same pattern as in the incubation study, with the Azolla treatments highest among the organic fertilizers, and urea the greatest overall. Compost yielded better than the control but was the lowest yielding among the fertilizer treatments. Finally, we compared the agronomic effect of Azolla biofertilizer with urea and manure applied at the same N rates to spinach (Amaranthus cruentus) and radish (Raphanus sativus) crops grown in the field on alluvial and peat soils. Fertilizer treatments affected the spinach yield at both locations but did not affect the radish yield. The manure treatment resulted in the highest spinach yields (18–27 t ha−1), and the Azolla treatment applied at the same N rate as the manure yielded the same as the manure treatment on the peat soil and had the highest leaf and branch numbers. Azolla shows promise as a biofertilizer for dryland vegetable crops; however, an economic feasibility analysis is needed prior to encouraging the widespread adoption of on-farm Azolla production and use.

1. Introduction

The production of NH3-based fertilizer is dependent upon fossil fuels as an ingredient and as an energy source to achieve the needed high temperatures and pressures to create NH3 gas. In addition, it is then transported to fields, and after application a portion of the N is lost as nitrous oxide, a potent greenhouse gas. Overall, it is estimated that two-thirds of the greenhouse gas emissions of crop production can be attributed to N fertilizer including its production, transport, and field emissions [1]. A fertilizer that could be produced on-farm without fossil fuels would dramatically reduce the C footprint of agriculture, and if that fertilizer also provided C to the soil, potentially increasing C sequestration and improving soil health, that would address many of the key challenges of our times.
The aquatic pteridophyte (water fern) Azolla has excellent potential as a biofertilizer and green manure because of its widespread distribution and rapid biomass production [2,3]. Azolla can be found in both tropical and temperate climates and grows prolifically in freshwater ponds, paddy fields, and ditches [3]. The ability of this fern to multiply rapidly primarily by vegetative reproduction ensures that year-round biomass can be sustained.
Azolla’s ability to fix N is due to the presence of a heterocystous (containing specialized cells where N fixation takes place) cyanobacterium (Anabaena azolla) that grows inside the dorsal leaf cavity of the fern [4]; without this symbiosis with cyanobacteria, no atmospheric N would be fixed or incorporated into the Azolla plant material. The Azolla–Anabaena system’s ability to fix atmospheric N makes it an outstanding agronomic choice for the cultivation of paddy rice under tropical conditions [3]. The N fixation potential of the Azolla–Anabaena system has been estimated to be 1.1 kg N ha−1 day−1, and one crop of Azolla was documented as providing 20–40 kg N ha−1 to a rice crop in about 20–25 days [5,6]. In another study, Azolla pinnata was reported to fix 75 mg N g−1 dry weight day−1 and generate 353 metric ton fresh weight ha−1 of biomass per year. The biomass contained 868 kg N, equivalent to 1900 kg urea [7]. Depending on the frequency and time of application, Azolla can provide 30–60 kg of N ha−1 [8] or 40–60 kg N ha−1 to a rice crop where Azolla is used as a dual crop grown along with rice [9]. The ability of the endosymbiont Anabaena azollae to fix the atmospheric N2 inside the leaf cavities of Azolla has made the association useful in rice ecosystems in many countries in Asia [5,10,11,12,13,14,15], the Americas [16,17,18], and West Africa [19].
Although research on optimizing the use of Azolla in paddy rice is ongoing [20], there are many other potential uses for Azolla; for example, in aquaculture [21] and phytoremediation [22]. The focus of this study is the potential use of Azolla as a fertilizer for vegetable crops grown in soil (not in paddies such as rice). Studies have elucidated the potential use of Azolla for enhancing the yield of crops in addition to rice, such as corn [18,23], wheat [24], and mung beans [25], in addition to taro (Colocasia esculenta) in China [26] and the Cook Islands [27]. In Senegal, Azolla is harvested from ponds and incorporated into the soil in vegetable crop fields, whereas, on bananas, Azolla is used as a mulch surrounding the base of the plants [28].
Azolla commonly has a low C/N ratio that allows it to mineralize faster than other organic fertilizers [29]. Approximately 60–80% of N in Azolla mineralizes within two weeks when Azolla is incorporated into water-logged soils [30]. Azolla may require 30–60 days in tropical climates or 60 days or more in temperate climates to mineralize the organic N to inorganic forms [8,31,32,33,34]. However, little is known about the mineralization of N in Azolla applied as a biofertilizer in dryland vegetable cropping systems.
In order to evaluate the potential of Azolla for use as a fertilizer for vegetable crops, we designed laboratory, greenhouse, and field experiments to address the following hypotheses:
(1) Laboratory Incubation—Based on their C:N ratios, we hypothesize that compost and Azolla treatments will have similar N availability and that inorganic N release will be more rapid in cyanobacterial biofertilizer using Anabaena spp. without Azolla.
(2) Greenhouse Evaluation—We hypothesize that plant N uptake and yields of the fertilizer treatments will follow the same pattern as the inorganic N release in the laboratory incubation and that urea, being quickly soluble, will result in the maximum N uptake and yield compared to the organic sources.
(3) Field Testing—Assuming that N is the primary yield-limiting nutrient, we hypothesize that Azolla applied at the same N rate as urea (Azolla-U) will produce similar agronomic results to urea, and Azolla applied at the same N rate as manure (Azolla-M) will produce similar agronomic results to manure.

2. Materials and Methods

2.1. Laboratory Incubation

The soil used in the mineralization study was collected from the Agricultural Research Development and Education Center (ARDEC), Colorado State University (40°39′21.2″ N, 104°59′45.2″ W) and classified as a fine-loamy, mixed, mesic Aridic Haplustalf of the Fort Collins series [35]. The soil was sieved through 8.0 mm and then 2.0 mm sieves to obtain a uniform soil particle size. The soil pH and electrical conductivity (EC) were 7.2 and 1.6 dS m−1, measured in a supernatant suspension of 1:1 soil to water using a Mettler Toledo pH/EC meter (Thermo Fischer Scientific, Waltham, MA, USA). Soil organic matter was 2.5%, as determined by the Loss on Ignition method [36].
A liquid medium for the Azolla culture was prepared in order to maintain Azolla in the vegetative state in preparation for the study [5]. The liquid medium (to be referred to as Watanabe solution) was prepared in the laboratory before being added to a shallow, circular, open container that had a diameter of 111 cm, depth of 12.7 cm, and volume of 122 L. The medium consisted of CaCl2·2H2O (40 mg/L), NaH2PO4·H2O (20 mg/L), MgSO4·7H2O (40 mg/L), and K2SO4 (40 mg/L) as macronutrients and MnCl2·4H2O (0.5 mg/L), Na2MoO4·2H2O (0.15 mg/L), H3BO3 (0.2 mg/L), ZnSO4·7H2O (0.01 mg/L), CuSO4·5H2O (0.01 mg/L), CoCl2·6H2O (0.01 mg/L), and FeSO4·7H2O (0.50 mg/L) as micronutrients [5]. These nutrients were then diluted with deionized H2O to make a final volume of 55 L.
The Azolla plants used in this study were Azolla mexicana obtained from Alpine Koi and Reef, Fort Collins, CO, USA. Prior to their dispersal by man, A. mexicana were endemic to northern South America through western North America [6,37,38]. Since A. mexicana is native to the Great Plains, it has the potential to be grown in this region and used as a fertilizer. The Azolla was grown in 68 L shallow, circular pools in either Watanabe solution (Azolla + Watanabe) or dechlorinated tap water (Azolla) [39].
A plant-based compost was obtained from Hageman Earth Cycle located in Fort Collins, CO, USA. The cyanobacteria species used in this study was free-living Anabaena cylindrica, and the cyanobacteria (Cyano) were cultured in a hoop house at ARDEC following the method of Barminski et al. (2016), settled by gravity, and then air-dried and ground to a powder [40]. The use of Moringa oleifera seeds as a coagulate or flocculate has given satisfactory results in reducing the number of cyanobacterial cells in water-treatment processes [41]. In this study, some cyanobacteria were flocculated in raceways by sprinkling ground M. oleifera seeds (Cyano + Moringa) on the surface of the nutrient media to flocculate the cyanobacteria and subsequently hasten the settling, drying, and grinding processes.
The soil incubation study was carried out at the Soil, Water and Plant Testing Laboratory, Colorado State University, USA, in an incubation room kept at a constant 25 °C. The treatments in this study were no fertilizer (Control), compost (Compost), Azolla grown in dechlorinated tap water (Azolla), Azolla grown in Watanabe solution (Azolla + Watanabe), cyanobacteria (Cyano), and cyanobacteria flocculated with moringa (Cyano + Moringa) (Table 1). All the fertilizers were applied at a rate of 50 kg N ha−1. Fifty-gram sub-samples of air-dried soil were mixed with the fertilizers and placed in 1 L Mason jars according to the treatment assigned. All the Mason jars were loosely lidded to prevent anaerobic conditions. Each treatment had four replications. The water content was adjusted once a week to 60% Water-Filled Pore Space (WFPS) [42]. The treatments were randomized within blocks to represent each sampling date.
The soils were incubated for 140 days, and Inorganic N (NH4+-N and NO3− -N) was analyzed on the initial sampling date (t = 0) after the fertilizer application and on 7, 14, 28, 56, 84, 112, and 140 days after treatment. Twenty-four experimental units were destructively sampled on each sampling date throughout the incubation period. Inorganic N for each sample was determined by extracting a 5 g sub-sample in 25 mL 2M KCl, shaking for 60 min on a reciprocating shaker at 180 cycles per minute, and filtering to obtain a clear extract [43]. Leachates were filtered using Whatman No. 42 filter paper. The extracts were analyzed immediately for NH4+-N and NO3−-N using an Alpkem Flow Solution IV Auto Analyzer (OI Analytical, College Station, TX, USA). If immediate analysis was not possible, the extracts were frozen at −20 °C to prevent further microbial processes.
The data were analyzed using R version 3.2.2 (The R Foundation for Statistical Computing). The experimental units were arranged in a factorial design with six treatments, eight dates, and four replications. Boxplot procedures were used to evaluate the normality of data distribution. Interaction plots were generated to observe the interaction effects when the effect of one variable depends on the value of another. Analysis of variance (ANOVA) was performed on the data using the linear models function since there were replicates for each day and treatment combination. Pairwise comparisons with Tukey adjustment were obtained to determine whether the main effects or interactions were significant (p < 0.05).

2.2. Greenhouse Evaluation

A pot experiment was carried out from November 2016 to January 2017 in a greenhouse at the Colorado State University Horticulture Center (40 °56′54” N, 105 °08′43” W). Certified organic kale seeds (Brassica oleracea ‘Toscano’) from Johnny’s Selected Seeds (Waterville, ME, USA) were grown in a greenhouse equipped with an evaporative cooling system in natural light during the day and LED top lighting for 8 hours during the night.
A soilless potting mixture known as PRO-MIX® BX (Premier Horticulture Inc., Quakertown, PA, USA) was used. It is a general purpose, peat-based growing medium typically used in greenhouse and transplanting applications. Ingredients include sphagnum peat moss (75–85%), horticultural grade perlite, horticultural grade vermiculite, calcitic limestone, dolomitic limestone, macronutrients, micronutrients, and a wetting agent. The pH (6.1) and EC (1.5 dS m−1) of the potting mixture were measured as described above, and the water holding capacity was determined to be 67 mg L−1 [44].
The fertilizers used for the greenhouse experiment were the same as those used in the mineralization experiment with the addition of urea (Table 1). The experiment consisted of seven treatments (Azolla, Azolla + Watanabe, Cyano, Cyano + Moringa, Compost, Urea, and Control) with four replications. All treatments were applied at a rate of 56 kg N ha−1. The pots measured 15.24 cm by 15.24 cm and were watered to maintain an equal weight twice weekly throughout the entire experiment. The treatments were arranged in a Randomized Complete Block Design (RCBD).
Seeds of ‘Toscano’ kale were planted in plastic trays containing 7.62 cm of well-mixed PRO-MIX® BX in the second week of November 2016. After four weeks, the seedlings were transplanted into pots. Fertilizers were applied when the seedlings were being transplanted into each pot (1 seedling/pot), and the harvest took place 56 d later.
At harvest, the total fresh yield consisting of the leaf blade, stem, and petioles were placed in labeled paper bags after measuring the fresh weight. The roots were carefully separated from the potting mix and washed with deionized water. All plant material was then dried at 70 °C for 72 h and then weighed to determine dry matter. The aboveground plant samples were then ground and sieved to pass an 80-mesh sieve prior to analysis, and N concentrations were measured using a LECO Tru-SPEC elemental analyzer (Leco Corp., St. Joseph, MI, USA). Plant N uptake was calculated by multiplying the N concentration by the dry weight of the aboveground plant material.
Data were analyzed using R version 3.2.2 (The R Foundation for Statistical Computing). Boxplot procedures were used to evaluate the normality of data distribution. ANOVA was performed on the data using the linear models function. Pairwise comparisons with Tukey adjustment were obtained from mean square errors to determine whether main effects or interactions were significant (p < 0.05).

2.3. Field Testing

Field studies were located on two soils (alluvial and peat) in West Kalimantan, Indonesia. Indonesia is reported to have the second largest greenhouse gas emissions from agriculture globally, following Brazil [1]. The alluvial site was located at the Agricultural Research Station of the Assessment Institute for Agricultural Technology of West Kalimantan in Pal Sembilan Village, Sei Kakap (0°03′32.5″ S, latitude and 109°15′27.9″ E longitude); and the peat site was located in a farmer’s field in Siantan, Pontianak (0°00′57.2″ N, latitude and 109°20′13.6″ E longitude).
Based on the USDA soil taxonomy, the soil type for the alluvial site was Sulfic Endoaquepts, and for the peat soil, it was Terric Sulfihemists [45]. The climate is a tropical moist climate with III C and IV C classification [46]. The average temperature is greater than 18 °C, and annual precipitation ranges from 2000–4000 mm with an average relative humidity of 80.8%.
Two vegetables were selected for this study, a leafy vegetable (red spinach) and a bulb vegetable (daikon radish). The spinach variety (Amaranthus tricolor) used was Red “Giti” Spinach (Indonesian Vegetable Research Institute), and the daikon radish (Raphanus sativus var. longipinnatus) variety was No. 22 short leaves (GL seeds, China). Red “Giti” Spinach was selected due to its higher yield, antioxidant activity, and flavonoid content compared to other spinach varieties [47,48]. Daikon radish was selected for its high market value and health benefits [49].
The spinach was transplanted 21 days after seeding, and the radish was direct seeded; the planting distance used for both spinach and radish was 20 × 15 cm.
Field studies were conducted in 2015. At both sites, there were separate spinach and radish experiments, each arranged in an RCBD with three replicates and fertilizer treatments as follows: no fertilizer (Control), urea applied at 23 kg N ha−1 (Urea), dried Azolla applied at the urea N rate of 23 kg N ha−1 (Azolla-U), chicken manure applied at 5 t ha−1 (108 kg N ha−1) (Manure), and dried Azolla applied at 4.69 t ha−1 (at the chicken manure N rate of 108 kg N ha−1)(Azolla-M). The average N concentration was 2.88% in Azolla and 3.19% in manure.
Soil samples from 0–20 cm depth were air dried, sieved (2 mm), and analyzed for chemical and physical properties prior to the study and again by plot after harvest (Table 2). Organic amendments were analyzed using standard methods (Table 3).
Azolla pinnata was used for this field study since it is native to Indonesia. Azolla was grown in a natural pond at the alluvial site; whereas, in the peat site, it was grown in an artificial pond lined with polyethylene. Azolla is sensitive to heat and light. Therefore, when we grew Azolla, we aimed to maintain the temperature of the growing media below 30 °C and protected the Azolla from high light intensity. The inoculation rate of Azolla for the production ponds was 100–200 g m–2 based on a previous greenhouse study result [50]. Azolla was harvested 3–4 weeks after inoculation.
Water was analyzed prior to the field study to identify the pre-existing conditions. The water resources for the Azolla ponds and the Azolla field study at the alluvial site were from surface water (pH 6.4) and rainwater (pH 4.8), respectively. At the peat site, surface water was used for the artificial Azolla pond (pH 2.8) and a mix of surface and rain water were used for the Azolla field study. In the peat site, plant ash was applied into the Azolla pond at the rate of 2.68 t ha−1 to increase the water pH.
Organic fertilizer was applied as a basal application, i.e., manure and Azolla were applied 3 days after transplanting (DAT) for spinach and 12 days after planting (DAP) for radish. The urea application was split into two applications with 50% applied at each time. Urea was applied at 3 and 14 DAT for spinach; whereas, for radish, it was applied at 12 and 27 DAP. Additional P and K fertilizers were applied to radish [250 kg superphosphate (36% P2O5) ha−1 and 180 kg muriate of potash (62% K2O) ha−1] on 12 DAP. Plant ash was applied to the peat soil at 3 t ha−1 following farmers’ common practice.
The crops were harvested at 45 days for spinach and 49 days for radish. The agronomic parameters measured were as follows: yield, plant height, leaf number, branch number (spinach), leaf N content (spinach), and bulb N content (radish). Plant nutrients were analyzed from the harvested vegetable crops (red spinach leaf and radish bulb) including Total N using the Kjeldahl method [51,52]; P, K, Fe, and Zn utilizing high-temperature oxidation (dry ashing) to digest organic matter followed by dissolution with 1 N HCl [53]; P was measured using a spectrophotometer, whereas K was measured using a flame photometer; and Fe and Zn were measured using atomic absorption.
All agronomic parameters were analyzed using SAS version 9.4 (SAS Institute, 2016). ANOVA was performed on the data by using the mixed procedure (Proc Mixed). Treatment means were compared using Tukey’s honestly significant difference (Tukey’s HSD) post hoc test (n = 3, p < 0.10).

3. Results

3.1. Laboratory Incubation

In the incubation trial, soil NH4+-N concentrations generally increased up to 56 d after initiation and then declined (Figure 1). On the other hand, NO3−-N concentrations increased gradually over the entire 140 d incubation. This trend is expected since N mineralization requires two steps: ammonification (conversion of organic N to NH4+-N) followed by nitrification (conversion of NH4+-N to NO3−-N). The control which received no fertilizer addition was consistently lowest in both forms of inorganic N throughout the incubation period. The compost treatment started out highest in NH4+-N and declined after day 56.
The Azolla and cyano-fertilizer treatments were indistinguishable throughout the study without much difference among them. However, soil NH4+-N concentrations were significantly higher in the Azolla + Watanabe treatment than in the Azolla treatment on days 28, 56, 112, and 140. In addition, the Azolla + Watanabe treatment was significantly higher in soil NO3−-N concentrations than the Azolla treatment from day 7 through to day 140. The Cyano and Cyano + Moringa treatments were never significantly different in soil NH4+-N concentrations, although soil NO3−-N concentrations were significantly higher in the Cyano + Moringa treatment on days 7, 28, 56, 84, 112, and 140.
At the end of the experiment, the N availability, defined as the sum of the inorganic N concentrations in a treatment minus the sum of the inorganic N concentrations in the control, expressed as the % of the amount of N applied, varied by treatment as follows: Azolla + Watanabe (82.3%) > Azolla (63.6%) > Cyano + Moringa (36.5%) = Cyano (26.6%) > Compost (15.5%).

3.2. Greenhouse Evaluation

All fertilizer treatments significantly increased fresh aboveground kale yield and root dry weight (Figure 2) and plant height as compared to the control. The Azolla + Watanabe (13.6 g pot−1) and Urea (12.9 g pot−1) treatments had the highest yields, followed by the Azolla, Cyano, and Cyano + Moringa treatments. Compost was the lowest yielding of the fertilizer treatments (10.2 g pot−1). On the other hand, root mass was highest for the Azolla + Watanabe and Cyano treatments, followed by the Cyano + Moringa and Azolla treatments. The Compost and Urea treatments had the lowest root masses compared to the other fertilizer treatments, but they were greater than the Control.
Nitrogen uptake was greatest in the Urea treatment, followed by the Azolla + Watanabe and Azolla treatments (Figure 3). N uptake was lowest in the Cyano, Cyano + Moringa, and Compost treatments, although all of these treatments had a N uptake greater than the Control.

3.3. Field Testing

Fertilizer treatments significantly affected the spinach yield at both locations (Figure 4) but had no effect on the radish yield. In both locations, the Manure treatment resulted in the highest spinach yields. Azolla applied at the same N rate as the Manure yielded the same as the Manure treatment on the peat soil but yielded significantly lower on the alluvial soil. In both locations, the Urea and Azolla-U treatments yielded the same as the Control.
On the peat soil, the Azolla-M treatment had the greatest spinach height and highest spinach leaf and branch numbers, and the Manure treatment was not significantly different from Azolla-M for any of these parameters (Figure 5). The Urea and Azolla-U treatments had the same spinach leaf and branch numbers as the control, and the Urea treatment resulted in the same spinach height as the Control. In addition, the plant heights of radish were only higher than the Control in the Manure treatment.
In the radish plots grown on peat soil, all of the treatments significantly increased the soil pH from below 5.0 up to around 5.5. However, on the alluvial soil, fertilizer treatments had no effect on soil pH. Fertilizer treatments impacted other soil fertility parameters, such as the plant-available soil P concentration, where the Manure treatment resulted in the highest Bray-1 P concentration (Figure 6). Although the plant P concentration was not generally impacted by treatment (spinach on the peat soil was the exception), the plant K concentration was influenced, in particular on the peat soil, where plant K concentration was higher in the Manure and Azolla-M treatments than in the Control (Figure 6).

4. Discussion

4.1. Laboratory Incubation

Higher C:N ratios of fertilizers or soil amendments (especially those over 18 C: 1N) tend to result in greater immobilization of N and a slower release of inorganic N for plant uptake. Therefore, we predicted that the Compost (C:N=13.1), Azolla (C:N=13.9), and Azolla + Watanabe (C:N=11.1) treatments would have slower N mineralization and less N availability than the Cyano (C:N=4.9) and Cyano + Moringa (C:N=5.8) treatments (Table 1). However, the results showed that Azolla treatments had the greatest N availability at the end of the 140 d incubation period, Compost had the lowest N availability, and the Cyano treatments were moderate in N release (Figure 1). The greatest net N mineralization of organic amendments applied to soils is often attributed to amendments with a low C:N ratio and high N content [54]. However, it is clear that C:N ratios alone cannot predict relative N mineralization rates since Azolla had similar C:N ratios as Compost but recorded much higher N availability over time.
The quantity of N released to crops depends on the chemical composition of the organic matter [55]. N content, C:N ratio, lignin, and the contents of cellulose, hemicelluloses, and polyphenols are some of the factors that affect the amount of N released to soils [56]. Organic fertilizers with high N contents and low C:N ratios typically mineralize sufficient N to meet the demands of plant growth [57,58].
In a mineralization study using composted manure and two different forms of cyanobacteria as fertilizers, the C:N ratio of composted manure was higher than that of cyanobacteria [59]. The composted manure and control treatments both resulted in lower soil inorganic N (NH4+-N and NO3−-N) concentrations and lower soil N availability than the other treatments, in agreement with our results. The Cyano treatments had higher N availability than Compost in both studies.
A comparable incubation study on loam soil demonstrated immobilization between days 1 and 63, reaching the highest immobilization on day 35 in the composted manure [60]. Net mineralization was observed from days 84 to 140. In our study, the soil NH4+-N reached its peak on day 56 and then declined due to conversion to soil NO-3-N. Biosolid incubation experiments have shown similar trends, whereby NH4+-N was the dominant form of soil inorganic N during the first half of the incubation and then NO3−-N constituted more than 50% of the inorganic N during the latter half of the study [61]. These findings confirm that Azolla decomposes rapidly in soil and thus, can provide available N for rice plants [62]. In addition, the results of this study, like the others described, show that when the soil NH4+-N concentration declined, there was a corresponding increase in soil NO3--N concentration, demonstrating that NH4+-N formed during ammonification was then nitrified into the NO3--N form [59].

4.2. Greenhouse Evaluation

Nitrogen uptake was the highest in the Urea treatment as predicted, followed by the Azolla treatments, the Cyano treatments, and finally the Compost (Figure 3). This trend follows the pattern in N mineralization documented in the laboratory incubation above (Figure 1). The yield results followed a similar pattern with a few exceptions; the Azolla + Watanabe treatment yielded equally to the Urea, and the Cyano treatments yielded equally to the Azolla (Figure 2).
An interconnection exists between the N availability of the organic fertilizers measured in the incubation study and leaf N concentrations found in the greenhouse study. This is seen on day 56 whereby the Azolla + Watanabe treatment resulted in both the highest N availability (Figure 1) and leaf blade N concentration of the organic fertilizer treatments. In a study on rice, inoculation with either Azolla or cyanobacteria, even with urea fertilizer at 144 kg N/ha, led to an increase in N accumulation in grain and straw, but the effect of Azolla was superior to that of cyanobacteria alone [63]. Furthermore, the higher N availability and leaf blade N concentrations in Azolla +Watanabe could be attributed to the addition of essential nutrients in the Watanabe solution. Essential nutrient availability influences the effectiveness of N fixation [64]. Essential nutrients such as Fe and Mo are required to produce the nitrogenase enzyme and fix atmospheric N [65].
Comparatively, the N uptake in the Azolla treatments was significantly lower than that of Urea (Figure 3). Inorganic fertilizers such as urea are soluble [66], and therefore, N is immediately available for uptake when the material is incorporated into the soil [67]. Although the Urea treatment resulted in taller kale compared to all other treatments after day 35, we noticed that kale in all the Urea treatment pots began to shrivel and turn yellow after day 48. Previous studies indicate that NO3-N leaching losses are higher from quick-release-N sources such as urea and are enhanced by well-drained sandy soils [68,69,70,71,72,73]. Since this greenhouse study used a potting mixture which contained vermiculite and drained well, we could expect N losses through leaching as well. As a result, there were no significant differences in kale height at the end of the study between the Urea treatment and the Azolla +Watanabe treatment.
Compost has been shown to release limited available nutrients even though it can produce good results for vegetable transplant production [74,75,76,77]. The Compost treatment in this study showed both lower mineralization rates and smaller yields compared to the other organic fertilizers. Not all composts are the same, and not all show potential as good potting media, mainly because of their low N mineralization rate and high N immobilization and salinity levels [78].

4.3. Field Testing

Urea had no effect on the yield or plant height of radish or spinach in either the alluvial or peat soils. In addition, since urea provides only N and no other nutrients, it also had no effect on soil or plant nutrient concentrations. Apparently, both soil types had adequate N levels prior to fertilization to support the production of these short season vegetable crops. Impacts due to other treatments are probably due to broader impacts beyond N alone, such as macro- and micronutrients.
The spinach yield was responsive to treatment, with the Manure treatment resulting in the highest yield on both soil types (Figure 4). The Azolla-M treatment yielded the same as the Manure on the peat soil but less than the Manure on the alluvial soil. In addition, plant growth components such as the leaf and branch numbers of spinach were substantially influenced by the Azolla-M treatment on the peat soil, and the N concentration in spinach leaves was significantly higher in the Azolla-M and Manure treatments in the peat soil, as well. Many other studies have reported on the comparative impact of fertilizer and manure applications on Amaranthus species [79,80]; however, comparisons of manure or fertilizer with Azolla applications in Amaranthus are rare, although the scientific literature contains numerous reports on the impact of Azolla on paddy rice [81,82].
Treatment responses varied by soil type, which is not unexpected due to the very different natures of these two soils. Overall, total N in the peat soil was about 10 times greater than in the alluvial soil. Peat soils store considerable amounts of N compared to alluvial soils. However, due to the lower bulk density of peat soil (0.1 g cm–3), the average 2.2% N only contains 2200 kg N ha–1 in its upper 10 cm layer. In contrast, although the alluvial soil contained a lower N concentration (0.2%), with the higher bulk density for mineral soil of 1–1.2 g cm–3, the alluvial soil contained 2000–2400 kg N ha–1, roughly the same N content as the peat soil [83].
Fertilizer treatment had a significant effect on available soil P concentration in both crops and soils, except in the radish–peat site (Figure 6). Manure had the greatest soil P concentration in all four sites. The fertilizer treatments were applied based on N rate; therefore, the P/N ratio could be used to predict the impact of a treatment on soil P. The P/N ratio was highest in the chicken manure (0.23), while the Azolla biofertilizer had a 0.08–0.10 P/N ratio (Table 3). Phosphorus had apparently not been fully released through the decomposition of the Azolla treatments (Azolla-U and Azolla-M) [84]. The Azolla-U was not different from the Control and contained the same soil P concentration as Azolla-M, despite the application rate being about ¼ of the Azolla-M rate. Other studies have reported that at higher A. pinnata application rates, there was a higher soil available P concentration [34,81,85].
Overall, macronutrient concentrations (N, P, and K) in either spinach leaves or radish bulbs in both soil types were the highest in the manure treatment and in the Azolla treatment applied at the manure N rate. Our results correspond with Zeid et al. (2015) who also found that chicken manure was more effective at enhancing nutrient concentrations in radish leaves and tubers compared to other organic fertilizers and a control [86]. Although the plant P concentration was affected by the fertilizer treatment in the spinach–peat site only, the plant K concentration was significantly affected by the treatment in radish in both soil types, and in the spinach–peat site. Interestingly, in the spinach–peat site, the pattern of plant P was comparable to soil P, in which the manure treatment held the highest P concentration both in the soil and in the plant tissue across soil and crop types. Plant K concentrations were also consistently highest in the Manure treatment. In addition, Azolla-M increased the plant K concentration in both crops grown on the peat soil. Azolla has been reported to be a good biofertilizer source for low K soil, since when it decomposes, Azolla provides K for plants, and thus it performed as a K fertilizer substitution [28,87]. On the other hand, Azolla-U and Urea had plant K concentrations comparable to the control in all cases. The pattern of fertilizer treatment effect on plant K generally followed the soil K pattern. Manure and Azolla-M had the highest K concentrations in soil and plant tissue, regardless of soil and crop types.

5. Conclusions and Future Recommendations

These studies demonstrate that Azolla is an effective fertilizer for vegetable crops. Despite Azolla having similar C:N ratios as compost, Azolla mineralized N much more rapidly than compost. Nitrogen uptake by kale in the greenhouse study followed the same trend, with N uptake being higher in the Azolla + Watanabe treatment than in the Compost treatment, although Urea resulted in the highest N uptake. In addition, the Azolla + Watanabe treatment resulted in a kale yield equal to the Urea treatment. In field studies, the Manure treatment resulted in the highest spinach yields. Azolla applied at the same N rate as the Manure yielded the same as the Manure treatment on the peat soil but yielded significantly lower on the alluvial soil. In both locations, the Urea and Azolla-U treatments had the same spinach yield as the Control. Overall, Azolla was equally effective as Urea as a fertilizer for vegetable production.
The long-term sustainability of urea fertilizer is in question. With increasing oil prices resulting in increasing fertilizer prices since the 1970s, many concerns over the stability of world chemical fertilizer prices have been raised [88]. In addition, climate change and the long-term adverse effects of the heavy use of chemical fertilizers on crop productivity, soil structure, and off-farm pollution call for new solutions. Organic fertilizers and green manures are seen as having many agronomic and environmental advantages [89], and Azolla grown on-farm and utilized as a soil amendment in dryland crops shares many of those advantages.
However, labor costs and the high opportunity costs of land use could be major constraints to the economic feasibility of organic fertilizers produced on-farm, such as Azolla. Capital requirements for Azolla cultivation are quite small [90], since Azolla can be grown in small catchments, tanks, or canals of irrigation systems and can be floated onto fields and incorporated into the soil [91]. These methods of growing Azolla could provide substantial savings in land and labor costs [91]. Additional expenses to optimize Azolla production might include ash, P fertilizer, and pesticides to protect the Azolla from pests [90], such as snails and frogs.
An economic feasibility analysis is critical prior to encouraging the widespread adoption of on-farm Azolla production and use. In a study conducted in 1987 in the Philippines, where low-resource farmers are rarely able to purchase chemical fertilizers without government credit, the use of Azolla was relatively attractive, especially in farms with poor irrigation systems [89]. It is critical to update this analysis to determine under what environmental and economic conditions Azolla could practically replace urea.
Table
Table 2. Baseline soil properties of alluvial and peat soils.
Table 2. Baseline soil properties of alluvial and peat soils.
AlluvialPeat
pH-H2O 14.705.27
pH-KCl 13.894.65
Organic C 2 (%)2.1550.3
NH4+-N 3 (mg kg−1)6.60.8
NO3–-N 3 (mg kg−1)54.1198.1
Total N 4 (%)0.252.29
C/N ratio8.6022.0
P 5 (mg kg−1)359236
Exchangeable cations 6:
- K (cmol(+) kg−1)0.760.62
- Na (cmol(+) kg−1)0.600.29
- Ca (cmol(+) kg−1)1.8058.36
- Mg (cmol(+) kg−1)0.619.19
Cation exchange capacity 6 (cmol(+) kg−1)10.4108.8
Base saturation (%)36.463.0
Exchangeable H 7 (cmol(+) kg−1)0.150.13
Exchangeable Al 7 (cmol(+) kg−1)0.160.27
Fe 8 (mg kg−1)727528
Cu 8 (mg kg−1)68.830.9
Zn 8 (mg kg−1)43.379.0
B (mg kg−1)193.632.3
Texture 9:ClayNA
Sand (%)5.3NA
Silt (%)41.5NA
Clay (%)53.2NA
1 pH was measured in a soil:DI water extraction and soil:1 N KCl extraction with a ratio of 1:5 [92,93,94]. 2 Soil organic C was determined using the Walkley–Black method [95]. 3 NH4+-N and NO3–-N were analyzed using a 2 M KCl extraction and measured with an automated analyzer (Flow Solution IV, O-I-Analytical) [96]. 4 Total N using Kjeldahl method [97]. 5 Available P was extracted with Bray-1 solution and measured with a spectrophotometer [98]. 6 Exchangeable cations (K, Na, Ca, and Mg) and cation exchange capacity (CEC) were measured using ammonium acetate 1 N pH 7.0 extraction and measured with a flame photometer for K and atomic absorption spectroscopy for Na, Ca, and Mg [99]. 7 Exchangeable H and Al were determined using 1 N KCl extraction and titrated with standardized 0.1 M NaOH [100]. 8 Total Fe, Cu, and Zn were measured using nitric acid (HNO3) and perchloric acid (HClO4) extraction and then analyzed by atomic absorption spectroscopy [101,102]. 9 Soil texture was analyzed using the pipette method [103].
Table
Table 3. Soil amendment and biofertilizer analysis used in field studies.
Table 3. Soil amendment and biofertilizer analysis used in field studies.
Plant Ash 1Chicken Manure 2Azolla 3
AlluvialPeat
pH-H2O9.136.886.757.15
Organic C (%)-32.245.148.1
NH4+-N (%)-0.330.250.25
NO3–-N (%)-0.200.200.20
Total N (%)-3.192.762.99
C/N ratio-10.116.416.1
P (%)1.280.740.230.31
K (%)5.495.083.914.10
Ca (%)5.513.27--
Mg (%)0.590.22--
Fe (mg kg−1)13,00018.9992897
Zn (mg kg−1)31.17.45108132
Moisture (%)6.132.120.219.7
CaCO3 equivalent (%)38.5---
1 Analysis of plant ash included pH [92] and moisture content [104,105]. Plant ash was oven dried at 105 °C for 3 h, extracted with 1 N HCl, and colorimetry was used to measure P (spectrophotometer); a flame photometer was used for measuring K, and atomic absorption spectrometry for Ca, Mg, Fe, and Zn [106,107]. CaCO3 equivalency was also determined after plant ash was extracted with 0.5 N HCl and titrated with 0.1 N NaOH [100]. 2 Manure was analyzed for its moisture content (gravimetric) [104,105], pH [108], organic C [95], total Kjeldahl N, NH4+-N, and NO3–-N [109,110]. The manure sample was dry ashed and then extracted with 1 N HCl [106]. Spectrophotometer and flame photometer were used to determine P and K, while atomic absorption spectrometry was used for Ca, Mg, Fe, and Zn [107]. 3 Azolla tissue was analyzed for its dry matter [104,105] and nutrient concentrations. pH-H2O was measured using a pH meter with a dual electrode system [108]; organic C was determined by the Walkley–Black method [95]; and total N, NH4+-N, and NO3–-N were analyzed using the Kjeldahl and distillation methods [109,110]. The other nutrients (P, K, Fe, and Zn) were determined following dry ashing and digestion with sulfuric acid, then reacted with hydrogen peroxide. P was measured with a spectrophotometer, K using a flame photometer, and Fe and Zn were measured by an atomic absorption spectrophotometer (AAS) [107,111].

Author Contributions

Conceptualization, J.G.D.; Methodology, J.G.D.; Investigation, A.J. and D.P.W.; Resources, S.G.; Writing–original draft, A.J. and D.P.W.; Writing–review & editing, J.G.D.; Supervision, S.G. and J.G.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the USDA Western Sustainable Agriculture Research and Education Program and the Indonesian Agency for Agricultural Research and Development.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data available on request.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Soil NH4+-N (A) and NO3−-N (B) concentrations as a function of time during the 140-day incubation period. Bars represent standard errors of mean.
Figure 1. Soil NH4+-N (A) and NO3−-N (B) concentrations as a function of time during the 140-day incubation period. Bars represent standard errors of mean.
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Figure 2. Total fresh yield (leaf blade, stem, and petiole weight) (A) and root dry weight (B) of kale tissue 56 days after a one-time fertilizer application of 56 N kg/ha. Bars represent standard errors of mean. Bars with a common letter indicate no significant difference based on Tukey’s honest significant difference (HSD) test (p < 0.05).
Figure 2. Total fresh yield (leaf blade, stem, and petiole weight) (A) and root dry weight (B) of kale tissue 56 days after a one-time fertilizer application of 56 N kg/ha. Bars represent standard errors of mean. Bars with a common letter indicate no significant difference based on Tukey’s honest significant difference (HSD) test (p < 0.05).
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Figure 3. Total N uptake in kale tissue 56 days after a one-time fertilizer application at 56 N kg/ha. Bars represent standard errors of mean. Bars with a common letter indicate no significant difference based on Tukey’s honest significant difference (HSD) test (p < 0.05).
Figure 3. Total N uptake in kale tissue 56 days after a one-time fertilizer application at 56 N kg/ha. Bars represent standard errors of mean. Bars with a common letter indicate no significant difference based on Tukey’s honest significant difference (HSD) test (p < 0.05).
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Figure 4. Yield of spinach grown on alluvial and peat soils. † Values followed by a common letter indicate no significant difference within the same crop and soil based on Tukey’s honest significant difference (HSD) test (p < 0.10). Control: no N fertilizer, Urea: 23 kg N ha−1, Azolla-U: Azolla applied at the urea N rate (23 kg N ha−1), Manure: 108 kg N ha−1, Azolla-M: Azolla applied at the manure N rate (108 kg N ha−1).
Figure 4. Yield of spinach grown on alluvial and peat soils. † Values followed by a common letter indicate no significant difference within the same crop and soil based on Tukey’s honest significant difference (HSD) test (p < 0.10). Control: no N fertilizer, Urea: 23 kg N ha−1, Azolla-U: Azolla applied at the urea N rate (23 kg N ha−1), Manure: 108 kg N ha−1, Azolla-M: Azolla applied at the manure N rate (108 kg N ha−1).
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Figure 5. Plant height (A), leaf (B), and branch numbers (C) of spinach grown on peat soil. Values followed by a common letter indicate no significant difference within the same crop and soil based on Tukey’s honest significant difference (HSD) test (p < 0.10). Control: no N fertilizer, Urea: 23 kg N ha−1, Azolla-U: Azolla applied at the urea N rate (23 kg N ha−1), Manure: 108 kg N ha−1, Azolla-M: Azolla applied at the manure N rate (108 kg N ha−1).
Figure 5. Plant height (A), leaf (B), and branch numbers (C) of spinach grown on peat soil. Values followed by a common letter indicate no significant difference within the same crop and soil based on Tukey’s honest significant difference (HSD) test (p < 0.10). Control: no N fertilizer, Urea: 23 kg N ha−1, Azolla-U: Azolla applied at the urea N rate (23 kg N ha−1), Manure: 108 kg N ha−1, Azolla-M: Azolla applied at the manure N rate (108 kg N ha−1).
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Figure 6. Soil P concentration (A) and plant K concentration (B) affected by N fertilizer treatments on radish and spinach crops grown in alluvial and peat soils. Values followed by a common letter indicate no significant difference within the same crop and soil based on Tukey’s honest significant difference (HSD) test (p < 0.10). Control: no N fertilizer, Urea: 23 kg N ha−1, Azolla-U: Azolla applied at the urea N rate (23 kg N ha−1), Manure: 108 kg N ha−1, Azolla-M: Azolla applied at the manure N rate (108 kg N ha−1).
Figure 6. Soil P concentration (A) and plant K concentration (B) affected by N fertilizer treatments on radish and spinach crops grown in alluvial and peat soils. Values followed by a common letter indicate no significant difference within the same crop and soil based on Tukey’s honest significant difference (HSD) test (p < 0.10). Control: no N fertilizer, Urea: 23 kg N ha−1, Azolla-U: Azolla applied at the urea N rate (23 kg N ha−1), Manure: 108 kg N ha−1, Azolla-M: Azolla applied at the manure N rate (108 kg N ha−1).
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Table
Table 1. Nutrient values and C/N ratio of fertilizers evaluated in the incubation and greenhouse studies.
Table 1. Nutrient values and C/N ratio of fertilizers evaluated in the incubation and greenhouse studies.
Treatment †NPKCC/N Ratio ‡
--%----%----%----%--
Compost1.530.630.5220.0613.1
Cyano8.811.401.0443.184.9
Cyano + Moringa7.382.040.7242.785.8
Azolla3.270.350.9645.4413.9
Azolla + Watanabe4.041.112.8744.8011.1
Urea4600--
† Treatments consisted of compost (Compost), cyanobacteria (Cyano), cyanobacteria and moringa (Cyano + Moringa), Azolla (Azolla), and Azolla grown in Watanabe solution (Azolla + Watanabe). ‡ In order to determine the C/N ratio of each fertilizer, the total C and N concentrations of the five different organic fertilizers (excluding urea) were analyzed using a LECO Tru-SPEC elemental analyzer (Leco Corp., St. Joseph, MI, USA) [42].
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Jama, A.; Widiastuti, D.P.; Gafur, S.; Davis, J.G. Azolla Biofertilizer Is an Effective Replacement for Urea Fertilizer in Vegetable Crops. Sustainability 2023, 15, 6045. https://doi.org/10.3390/su15076045

AMA Style

Jama A, Widiastuti DP, Gafur S, Davis JG. Azolla Biofertilizer Is an Effective Replacement for Urea Fertilizer in Vegetable Crops. Sustainability. 2023; 15(7):6045. https://doi.org/10.3390/su15076045

Chicago/Turabian Style

Jama, Aisha, Dwi P. Widiastuti, Sutarman Gafur, and Jessica G. Davis. 2023. "Azolla Biofertilizer Is an Effective Replacement for Urea Fertilizer in Vegetable Crops" Sustainability 15, no. 7: 6045. https://doi.org/10.3390/su15076045

APA Style

Jama, A., Widiastuti, D. P., Gafur, S., & Davis, J. G. (2023). Azolla Biofertilizer Is an Effective Replacement for Urea Fertilizer in Vegetable Crops. Sustainability, 15(7), 6045. https://doi.org/10.3390/su15076045

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