Effects of Azolla (Azolla pinnata) biofertilizer on soil nitrogen mineralization in soils collected from Hawassa Zuria and Wondo Genet Districts Incubated under laboratory conditions

doi:10.21203/rs.3.rs-4859448/v1

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Effects of Azolla (Azolla pinnata) biofertilizer on soil nitrogen mineralization in soils collected from Hawassa Zuria and Wondo Genet Districts Incubated under laboratory conditions

https://doi.org/10.21203/rs.3.rs-4859448/v1

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Knowledge of soil nitrogen (N) mineralization in response to different amendments is crucial for improving the efficiency of N use and improving crop productivity. The present study aimed to determine the effects of Azolla pinnata biofertilizer on soil N mineralization under laboratory conditions. The experiment was carried out in the soil laboratory of the College of Agriculture at Hawassa University. Soil samples were collected from the Hawassa Zuria and Wondo Genet districts and then incubated for 56 days at a room temperature of 25°C. Four levels of Azolla pinnata biofertilizer (0, 15, 30, and 45 g kg^− 1) and eight incubation periods (0, 7, 14, 21, 28, 42, 49, and 56 days) were arranged in a factorial order and laid out in a completely randomized design (CRD) with three replications. The total available NH₄⁺-N and NO₃⁻-N were determined every seventh day following standard laboratory methods. The results of this study revealed highly significant interactions (P < 0.01) between the effects of the Azolla biofertilizer concentration and the incubation period under laboratory conditions on the total mineralized N, NH₄⁺ and NO₃⁻. The maximum mineralization of N occurred after 42 days of incubation with 45 g kg^− 1 Azolla pinnata biofertilizer in soils from both locations (Hawassa Zuria and Wondo Genet). The highest total mineralized N was recorded after 42 days of incubation (39.88 and 48.57 mg kg^− 1 soil, respectively). However, the lowest total mineral N content was obtained from the control group. Nitrogen mineralization varied significantly between treatments (P < 0.01 or P < 0.001). Using Azolla pinnata as a biofertilizer significantly increased the mineralization rate of N in the soil, potentially reducing the need for inorganic N fertilizer. By studying Azolla pinnata biofertilizers, researchers can estimate mineralization during incubation, crop nutrient availability, and optimal timing. Field research is necessary to validate these laboratory findings and provide sound recommendations.

Agronomy

Ammonium

Incubation

Mineralization Potential

Net Mineralization and Nitrification

The environment has lost nearly half of the nitrogen (N) applied to agricultural fields worldwide, which has reduced the efficiency of N usage and harmed the environment. Determining the ideal fertilizer N rate is challenging due to the variability in soil N availability. Soil nitrogen mineralization is a crucial process that influences soil nitrogen availability and balance (Wang et al., 2019). Nitrogen mineralization is a key step in soil organic N transformation and soil nitrogen cycling. Organic nitrogen in soils is mineralized into inorganic nitrogen, mainly as ammonium N (NH₄⁺-N) and nitrate N (NO₃⁻-N), or as a transitional fraction, such as soluble organic N (SON), which is then mineralized into inorganic nitrogen (Zhang et al., 2017). Low soil fertility in sub-Saharan Africa has been linked to reduced agricultural productivity and insufficient knowledge on maintaining soil fertility through balanced nutrient application, soil conservation, and fertility management within fields (Amede and Taboge, 2007; Edwin et al., 2012). The use of organic fertilizers for growing crops, fruits and vegetables has increased due to consumer demand, awareness of organically grown products, and the desire of farmers for health.

Nitrogen (N) is the most difficult nutrient to manage for organic crop production, as N uptake depends on the plant-available N supplied by the soil. OM must breakdown into NH₄⁺ or NO₃⁻ ions before plants can absorb nitrogen through their roots. Nutrient management in organic systems should consider the roles of plants, soil organic matter (SOM), and soil organisms in the regulation of N availability (Sukor, 2013). Managing nitrogen is complex due to potential losses in both oxidized and reduced soil layers, floodwater, outflow, leaching, and biological forms in the soil (Anggria et al., 2012). The application of manure is crucial for nutrient control, as excessive application can lead to surface or groundwater contamination, while inadequate application can reduce crop yield (Van Kessel and Reeves, 2002).

Nitrogen mineralization, a biological process, depends on the chemical composition of organic matter and the properties of soil microbes. A high nitrogen content and low carbon-to-nitrogen (C: N) ratio in organic amendments can mineralize enough nitrogen for plant growth, while a low nitrogen content and high C:N ratio can immobilize nitrogen (Manojlovic et al., 2010). Nitrogen mineralization is a slow process that is influenced by aeration and moisture. Nitrate nitrogen is mobile and can contaminate groundwater, leading to degradation (Strebel et al., 1989; Spalding and Exner, 1993). The decomposition rates of organic amendments in soils provide information on nutrient release and changes in soil nitrogen stocks, which are usually determined by soil sample incubation (Rochette et al., 2006).

Nitrogen mineralization converts organic N to plant-available inorganic forms, involving the microbial conversion of complex organic N to simpler mineral N forms (NH₄⁺-N and NO₃⁻ -N). Soil microorganisms play an important role in ecosystem functions by regulating litter decomposition and nutrient cycling (Abera et al., 2012). Soil N mineralization and nitrification rates not only affect mineral N availability for plants but also indicate the soil's ability to release N (Haynes, 1986). The beneficial effects of Azolla include increasing soil organic matter, improving soil quality, and providing fixed nitrogen. Azolla improves nutrient uptake and promotes soil aeration and drainage (Sinha et al., 2002; Bhuvaneshwari and Kumar, 2013). Studying the effects of Azolla pinnata biofertilizers and inorganic N fertilizers on soil N mineralization and N release can aid in understanding decomposition rates relative to crop N requirements, improving N use efficiency, and reducing inorganic N fertilizer costs and environmental losses.

Due to the scarcity of inorganic nitrogen fertilizers, high prices, and concerns about pollution, researchers have explored alternative technologies to minimize the use of synthetic nitrogen fertilizers (Bhuvaneshwari and Kumar, 2013). The rate of decomposition of Azolla in soil depends on the C/N ratio, temperature, and soil properties. Adding Azolla to the soil improves properties such as aggregation, structure, permeability, and water retention capacity and reduces evapotranspiration. Azolla also increases soil nitrogen and phosphorus contents, enhancing crop yields in a manner similar to the application of mineral or organic nitrogen. Incorporating Azolla lowers soil pH and CE, promoting microbial growth and increasing soil organic matter (Abd-el Rasoul et al., 2004).

Furthermore, Azolla mineralization leads to the release of nitrogen into the soil, resulting in an increase in soil nitrogen content (EL-Zeky et al., 2005; Girgis et al., 2006). Compared with fresh Azolla, dry Azolla increases the water holding capacity more (Herzalla et al., 2002). Azolla has a low C:N ratio, allowing it to mineralize faster than other species (Wang et al., 1987) and provide nitrogen to crop plants. It also contributes to an adequate supply of phosphorus, potassium, sulfur, zinc, iron, and molybdenum. The application of Azolla has improved soil biological health, leading to increased mineralization and microbial status (Yadav et al., 2014). Humic substances produced during the decomposition of Azolla also impact soil fertility. Applying it as a green manure to agricultural lands can increase nutrient availability and improve soil physical properties, particularly soil porosity.

Azolla typically has a low C/N ratio, allowing it to mineralize faster than other organic fertilizers (Jama et al., 2023). Approximately 60 to 80% of the nitrogen in Azolla is mineralized in two weeks when it is incorporated into waterlogged soils (Jama et al., 2023). Azolla may take 30 to 60 days in tropical climates or 60 days or more in temperate climates to mineralize organic nitrogen into inorganic forms (Bhuvaneshwari and Kumar, 2013; Jama et al., 2023). However, little is known about the mineralization of nitrogen in Azolla when applied as a biofertilizer in dry-land vegetable cropping systems. Soil incubation experiments have previously been used to estimate potentially mineralizable nitrogen in soils (Stanford and Smith, 1972). The mineralization process for several organic fertilizers has been investigated under controlled laboratory conditions in a number of studies (Griffin and Honeycutt, 2000). Many studies have investigated the mineralization process for various organic fertilizers under controlled laboratory conditions (Griffin and Honeycutt, 2000). Although many studies have been conducted on the soil nitrogen mineralization of organic fertilizers, none have examined soil nitrogen mineralization in Azolla pinnata biofertilizers. Research on how the Azolla pinnata biomass used as a biofertilizer affects soil nitrogen mineralization rates at different incubation times is insufficient. Thus, the objectives of this study were to determine the effects of Azolla pinnata biofertilizer on N mineralization under laboratory conditions, to evaluate the maximum amount of mineralized nitrogen released during a period of incubation and to determine the amount of nitrogen mineralized during different incubation periods and to investigate the effect of Azolla pinnata biofertilizers on organic carbon and total N in soil.

2.1 Description of the study areas

The laboratory experiment was conducted at Hawassa University, College of Agriculture, School of Plant and Horticultural Sciences, Soil Science Laboratory. The soil samples were collected at a depth of 0 to 20 cm from the Hawassa Zuria and Wondo Genet districts of the Sidama Region, southern Ethiopia. In the Hawassa Zuria district, a soil sample was collected from a specific village, Jara Gelalicha, which is located 270 km south of Addis Ababa and 19 km from Hawassa town. Geographically, the area is located at 7°0'13.7" to 7°48'11.5" N and 38°21'21.1" to 38°63'30.5" E, with a mean altitude of 1712 meters above sea level (ma sl). It receives a mean annual rainfall of 952 mm, with mean minimum and maximum temperatures of 12.1°C and 26.7°C, respectively (Hawassa Meteorological Center, 2018). The main crops grown in the study area were maize (Zea mays L.), common bean (Phaseolus vulgaris L.), cabbage (Brassica oleracea L.), tomato (Lycopersicon esculentum L.), wheat (Triticum aestivum L.) and enset (Ensete ventircosum Welw. ), beet root (Beta vulgaris L.), lettuce (Lactuca sativa L.), sweet potato (Ipomea batata L.), khat (Khat edulis L.), and chili (Capsium annum L.).

The second location was at Aruma Kebele on a private farm in the Wondo Genet district, which is located 264 km south of Addis Ababa and 27 km south west of Hawassa town at 6°16' to 7°45' N and 38°31' to 38°63' E. The district's altitude varies from 1001 to 1780 meters above sea level. The mean annual rainfall in the district ranges from 801 to 1221 mm, and the mean annual temperature ranges from 17.6 to 22.5°C (SNNPRS, BoFED, 2018). The soil samples were collected at a depth of 0–20 cm from Wondo Genet (Aruma) and Hawassa Zuria (Jara Gelalicha) kebeles. The laboratory incubation experiment was conducted at Hawassa University, School of Plant and Horticultural Sciences, Soil Science Laboratory.

2.2. Soil Sampling

The soil samples were collected from the surface at a depth of 0–20 cm in the Wondo Genet (Aruma) and Hawassa Zuria (Jara Gelalicha) kebeles. At each location, soil samples were randomly taken from different locations within the 0–20 cm layer and mixed to create a composite soil sample. At each location, we composited the collected soil samples separately and sieved them through 2.0 mm sieves to achieve a uniform particle size. The soil was collected from the cultivated fields at depths of 0 to 20 cm from the selected locations and incubated for 56 consecutive days after the application of Azolla pinnata. After incubation, the amount of nitrogen mineralized from the soil (ammonium and nitrate) was measured after 7 days. All the samples were subsequently transported to the Hawassa University Soil Science Laboratory, School of Plant and Horticultural Sciences, where sieving was carried out using a 2-mm sieve for all the nutrients except for organic carbon and total nitrogen, which were sieved through a 0.5 mm mesh.

2.3. Experimental design, treatments, and procedures

The laboratory experiment was carried out at Hawassa University, School of Plant and Horticultural Sciences, Soil Science Laboratory. The experiment consisted of 32 treatments for the nitrogen mineralization study. These treatments were combined with four concentrations of dry Azolla pinnata (0, 15, 30, and 45 g/kg; A0 = 0, A1 = 15, A2 = 30, and A3 = 45) and eight incubation times (0, 7, 14, 21, 28, 42, 49, and 56 days). The 32 combinations of treatments were arranged in a factorial manner with three replications. Altogether, a total of 96 jars (at 4 levels of Azolla, 8 incubation times, and 3 replications) were arranged in accordance with a completely randomized design. The samples were incubated at room temperature in the laboratory at 25°C, after which the samples were analyzed in accordance with a completely randomized design (CRD) with 3 replicates. After the treatments were added, all the jars were weighed, and their weights were recorded. The soil moisture was checked and adjusted every 2 days by weighing the glass jars and adding the required amount of distilled water when the soil was dry and felt with the finger. A soil sample was collected from the field at a depth of 0–20 cm after the application of Azolla pinnata and inorganic nitrogen fertilizers from the fields (Hawassa Zuira and Wondo Genet districts). The samples were air-dried and sieved through a 2 mm sieve prior to incubation. The mineralization of the soils was tested in a laboratory at room temperature for 56 days. The mineralization of N was evaluated by periodic quantification of the mineral N in the mixtures. The observation of nitrogen mineralization was carried out at 0, 7, 14, 21, 28, 42, 49, and 56 days after the Azolla biomass was used for the application and incubation of biofertilizers. Mineral nitrogen was measured in fresh soil samples immediately after sampling. Ammonium (NH₄⁺) was extracted from 20 g of soil using 50 ml of 2 M KCl. The samples were shaken for 1 hour on a reciprocal shaker and filtered, and the extracts were analyzed using a visible spectrophotometer at 636 nm (Anggria et al., 2012). Nitrate ions (NO3) were determined with 0.01 M CaCl₂ using an ultraviolet spectrophotometer at 210 and 275 nm (Anggria et al., 2012).

2.4. Soil analysis from the preincubation experiment

The bulk density of the above soil series was determined using the cylindrical core method (Arshad et al., 1996). The particle size distribution (soil texture) was determined following the Bouyoucous hydrometer method (Bouyoucous, 1962). The soil textural class names were then assigned based on the relative percentages of sand, silt, and clay separated using the soil textural triangle of the USDA. The soil pH was measured potentiometrically using a combined pH meter with a glass electrode in water, and a 0.01 M KCl solution was measured at a 1:2.5 soil-to-water ratio (Van Reeuwijk, 1992) for each experimental unit after the incubation. Exchangeable bases (Ca, Mg, Na, and K) were extracted with 1 M NH₄OAc at pH 7, and then Ca and Mg were determined by EDTA titration, while K and Na were determined by flame photometry. To determine the cation exchange capacity (CEC) of the soil, the soil sample was first leached with a 1 M NH₄OAc solution buffered at pH 7. The ammonium-saturated soil sample was washed with ethanol, after which the adsorbed ammonium was replaced by sodium ions (Chapman et al., 1961). The quantity of ammonium ions replaced by sodium ions was estimated by distilling ammonia into dilute sulfuric acid. The distillate was retitrated with dilute acid, and the amount of ammonium ions was expressed as the CEC of the soil. Organic carbon (OC) was determined by the wet digestion method through the chromic acid digestion method described by Walkley and Black (1934), while the Kjeldahl procedure was followed for the determination of total nitrogen as described by Jackson (1958). The available phosphorus (AP) concentration was determined using standard Olsen et al. (1954) extraction methods.

2.5. Soil incubation and sampling procedure

The incubation methods used in this study were performed according to our previous methods (Kaleeem et al., 2015). The experiment consisted of 32 treatments for the nitrogen mineralization study. These treatments were combined with four concentrations of dry Azolla pinnata (0, 15, 30, and 45 g/kg; A0 = 0, A1 = 15, A2 = 30, and A3 = 45) and eight incubation times. The four levels of Azolla and eight incubation timing combinations were arranged factorially with three replications. Taken together, a total of 96 jars (32 treatments with 3 replications) were arranged in a completely randomized design. The samples were incubated at room temperature in the laboratory at 25°C, after which the samples were analyzed in accordance with a completely randomized design (CRD) with 3 replicates. After the treatments were added, all the jars were weighed, and their weights were recorded. The soil moisture content was checked and adjusted every 2 days by weighing the glass jars and adding the required amount of distilled water when the soil was dry and felt with the finger.

Soil samples were taken at seven-day intervals from the field for the subsequent eight weeks (56 days) after the biofertilizers of Azolla pinnata were incorporated into the soil. Soil samples were collected at soil depths of 0–20 cm using an auger from four randomly selected locations in each plot of the two selected districts. The time intervals involved 8 incubation periods, that is, 0, 7, 14, 21, 28, 42, 49, and 56 days (after the application of the amendments). Soil sampling was carried out after 0, 7, 14, 21, 28, 42, 49, and 56 days of incubation (for each sampling time, replicate treatments were taken for analysis). A 10 g soil sample was taken from each treatment after thoroughly mixing the soil in the polyethylene bag, and 50 ml of 1 M KCl was added to the soil in a plastic tube. The tube was capped and shaken for 1 hour on a mechanical shaker, and the mixture was filtered using Whatman No. 42 filter paper. The method of Ogoshi et al. (1999) was used to determine both ammonium and nitrate N using 10 ml of the above filtrate.

2.6. Measurements of mineral nitrogen

To measure the mineral N content, 10 g of soil was extracted in 50 ml of 2 M KCl with shaking for 1 h. The mixture was filtered through mineral N filter paper (MN 616), and the extract was subsequently stored at -18 ̊̄C until analysis. The mineral NNO₃⁻ -NN and NH₄-N concentrations were determined with a continuous UV spectrophotometer (Abbasi and Khizar, 2012). The net mineralization of N in response to each amendment was calculated as the difference in the amount of mineral N released between the amended and control soils (Mohanty et al., 2011). The percentage of total N mineralized from each Azolla application at each sampling time was calculated as described by Azeez and Van Averbeke (2010) and Abbasi et al. (2007) as follows:

% mineralization =\(\:=\frac{\text{N}\text{M}\left(\text{a}\text{m}\text{e}\text{n}\text{d}\text{e}\text{d}\right)-\text{N}\text{M}\left(\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}{\text{N}\:\text{t}\text{o}\text{t}\text{a}\text{l}\:\left(\text{a}\text{p}\text{p}\text{l}\text{i}\text{e}\text{d}\right)}\)x100

where MN is the total mineral N and N is the total N in the applied Azolla biofertilizer and N-fertilizer.

2.7. Nitrogen mineralization and N availability calculation

The net mineralization rates of N in the soil were determined by subtracting the final inorganic N (day 56) from the initial inorganic N (day 0) of the soil and dividing the resulting number by the number of days of incubation (Jama, 2018).

The nitrogen mineralization rate was calculated as follows:

Net N mineralization = (NH₄⁺-N treatment + NO₃⁻-N treatment) – (NH₄⁺-N control + NO3–N control) ................................................................................................................................ (Eq. 1)

Nitrogen availability % = (net N mineralization/amount N applied as fertilizer) × 100 ………………………………………………………………. ……………. (Eq. 2)

2.8. Soil Extraction and Analysis

Samples from all the treatments were incubated at different intervals for total mineral nitrogen and ammonium-N (NH₄⁺-N) analysis. The initial concentration of total mineral nitrogen and NH₄ C-N on day zero was determined by extracting soil samples with 50 ml of 1 M KCl added directly to the flask immediately after the incorporation of each amendment. Subsequently, soil samples from each treatment were removed randomly from the incubator after different incubation periods. The soil was extracted by shaking for 1 hour with 50 ml of 1 M KCl, followed by filtration using Whatman's No. 40 filter paper. The mineral N content of the extract was determined using steam distillation and titration methods (Keeney and Nelson, 1982). Nitrate N was calculated by subtracting NH₄⁺-N from total mineral N via the procedure or method used in our previous studies (Abbasi and Khizar, 2012). The C and N contents (total C and total N) of the soil were also monitored during incubation. For this purpose, a 10 g subsample of incubated soil was removed from each jar during each sampling period and placed in dictated Petri dishes to determine changes in total N and C with time. The soil organic carbon content was determined according to Nelson and Sommers (1982), while the total soil N content was determined using the Kjeldahl method (Bremmer, 1982). The net cumulative mineralization of N expressed as the mineralization of N (or total mineral N) and the net cumulative N nitrified as NO3-N accumulation at each sampling time (t) were calculated as previously described (Griffin et al., 2005).

2.9. Data collection

The laboratory incubation data collected from all the treatments included ammonium N and nitrate N concentrations during different incubation periods, total nitrogen (TN), and soil organic carbon (SOC).

2.10. Data analysis

The data obtained from the soil nitrogen mineralization analysis were subjected to analysis of variance (ANOVA) using the GLM (General Linear Model) of SAS version 9.4 statistical analysis software (SAS Institute, 2016). A simple correlation analysis was performed to measure the release of nutrients (NH₄⁺ and NO₃⁻ and organic carbon and total nitrogen) on the days of soil incubation and N mineralization. Significant differences between and/or among treatment means were delineated using the least significant difference (LSD) test when the F test was significant (5%).

3.1. Effects of Azolla Pinnata Biofertilizer on Soil N Mineralization during Different Incubation Periods

In the Hawassa Zuria and Wondo Genet districts, the application of Azolla pinnata biofertilizers alone gradually increased NH₄⁺-N concentrations after up to 42 days of incubation, after which the concentrations decreased (Tables 1a and 1b). At both locations, the NO₃⁻-N concentration gradually increased over the entire 42-day incubation. This trend is expected since N mineralization involves two steps: ammonification (conversion of organic N to NH₄⁺-N) and nitrification (conversion of NH₄⁺-N to NO₃⁻ -N). The control, which did not receive fertilizer addition, consistently had the lowest levels of inorganic nitrogen throughout the incubation period. The biomass of Azolla plants that were used as biofertilizers initially had the highest concentrations of NH₄⁺-N and NO₃--N after 42 days of incubation, after which the concentrations decreased after 42 days (Fig. 1a and 1b, Fig. 2a and 2b, respectively). Various authors have reported similar findings for vegetable crops, demonstrating the effects of Azolla biofertilizers and inorganic N fertilizers on soil nitrogen mineralization during different incubation periods (Jama et al., 2023).

The Azolla pinnata biofertilizer treatments had distinct effects on the plants throughout the study, revealing differences among them. Ammonium and nitrate levels increased after 0 to 42 days of incubation, but after 42 days, the ammonium and nitrogen concentrations decreased. However, after 42 days of incubation, the soil NH₄⁺-N concentrations were significantly greater in the 45 g/kg treatment than in the other treatments on days 28, 42, 49, and 56. Furthermore, after 42 days of incubation, the 45 g/kg treatment had significantly greater soil NO₃⁻ ₋N concentrations than did the other treatments from day 7 to day 56. At the Hawassa Zuria and Wondo Genet locations, although the soil NO₃⁻-N concentrations were significantly greater in the 30 and 45 g/kg treatment groups, the highest levels of nitrate were recorded after 42 days of incubation (Tables 3 and 4). At the end of the experiment, the availability of N, 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 a percentage of the amount of N applied, varied by treatment as follows: Azolla (45 g/kg) (82.3%) > Azolla (30 g/kg) (63.6%) > Azolla (15 g/kg) > control (36.5%) (best of best minera88 pdf).

3.2 Nitrogen mineralization

Analysis of variance revealed that N mineralization was significantly (p < 0.001) affected by the Azolla pinnata biofertilizer treatment and incubation time, while the interaction effect between the Azolla treatment and incubation time was also highly significant. The results indicated that at the Hawassa Zuria and Wondo Genet locations, the control soil without any amendments after 42 days of incubation and 45 g/kg of Azolla biofertilizer released the maximum amount of nitrogen mineralization (NH₄⁺-N + NO3-N) (10.03 and 36.12 mg Nkg^− 1 and 14.07 and 43.45 mg Nkg^− 1, respectively) (Tables 2 and 4, Fig. 4(a) and (b)).

At the Hawassa Zuria and Wondo Genet locations, on day 0 of incubation, the NH₄⁺-N contents in mg kg^− 1 soils were 0.61, 0.57, 0.60, and 0.63 mg kg^− 1 and 0.86, 0.86, 0.85, and 0.86 mg kg^− 1 in soils treated with Azolla biofertilizers; control, 15, 30, and 45 gkg^− 1 Azolla; and 0.58, 2.26, 8.86, and 11.44 mg kg^− 1 and 0.89, 3.34, 12.87, and 17.52 mg kg^− 1 NH₄⁺-N, respectively (Tables 1 and 2). At the Hwassa Zuria and Wondo Genet locations, after 42 days of incubation, the highest NH₄⁺-N concentration (20.27 mg kg^− 1) was obtained for 45 g kg-1 Azolla, followed by 30 g kg-1 Azolla (16.85 mg kg^− 1), 45 g kg-1 Azolla (24.31 mg kg^− 1) and 30 g kg^− 1 Azolla (21.35 mg kg^− 1) (Tables 1 and 2 and Figs. 1a and b). With respect to the N application rate, the content of NH4 + -N progressively increased after up to 42 days of incubation, after which it decreased in the same pattern. After 56 days of incubation, the temperature gradually decreased after 42 days of incubation (Fig. 2b).

NO₃⁻ -N and NH4 + -N gradually increased in soils amended with different Azolla biofertilizers from 0 to 42 days of incubation and then declined in a similar way until 56 days of incubation (Fig. 5a and b). The soil was collected from the Hawassa Zuria location, and after 42 days of incubation, the maximum concentrations of NO₃⁻ N in the soil were recorded at 45 g kg-1 (19.61 mg kg^− 1), followed by 30 g kg^− 1 (17.87 mg kg-1) (Table 3). The soil was collected at the Wondo Genet site, and after 42 days of incubation, the maximum concentrations of NO₃^−–N in the soil were recorded at 45 g kg-1 (24.26 mg kg^− 1), followed by g kg^− 1 (20.89 mg kg^− 1) (Table 4). Conversely, at 56 days of incubation, NO₃⁻N was detected to be 13.44, 9.77, 3.14, and 0.75 mg kg^{− 1 in the Hawassa Zuria} and 17.73, 12.91, 3.79, and 1.07 mg kg⁻1 in the soils amended with 45, 30, 15, and the control (g kg1), respectively (Tables 3 and 4).

The interaction effects of Azolla pinnata biofertilizers and incubation duration on the availability of NH₄⁺-N and NO₃⁻ -N exhibited significant variations at both locations in the soils (Tables 1 and 2). Azolla biofertilizers exhibited superior release of available N to soils after different durations of incubation. However, at both locations, a higher Azolla biofertilizer rate of 45 g kg^− 1 always had the greatest effect on the release of both NH₄⁺-N and NO₃⁻ -N into the soils. However, the interaction of 45 g kg^− 1 Azolla biofertilizer soil had the highest concentration of NH₄⁺-N (20.27 and 24.31 mg kg^− 1) and NO₃⁻-N (19.61 and 24.26 mg kg^− 1) after 42 days of incubation (Tables 1–2 and 3–4). Additionally, soil NH₄⁺-N concentrations were significantly affected by the rate of treatment, the duration of incubation, and all possible interactions between these factors (p < 0.001; Tables 1–2). At the Hawassa Zuria and Wondo Genet locations, after 56 days of incubation, 45 g kg^− 1 Azolla pinnata biofertilizer soil provided the maximum amount of NH₄⁺ -N (11.44 and 17.52 mg kg^− 1) and the maximum amount of NO₃⁻ -N (13.4 and 17.73 mg kg^− 1) on this day, respectively (Tables 1–2 and 3–4).

3.2. Ammonium nitrogen (NH₄⁺-N) release affected by Azolla pinnata biofertilizers during different incubation periods

The data in Tables 1 and 2 show the conversion of nitrogen in the soil due to the use of Azolla biomass as a biofertilizer in addition to ammonium (NH₄⁺ mg kg^− 1) on different incubation days. In the soils collected from the two locations, all the treatments gradually increased the ammonium nitrogen concentration from 0 to 42 days, but after 42 days, the concentration decreased (Fig. 3a and b). The patterns of ammonium nitrogen mineralization in response to the different treatments on the Azolla biomass are shown in Figs. 3a and b. Treatments with 30 and 45 g kg^− 1 ammonium nitrogen at the Hawassa Zuria and Wondo Genet locations exhibited high ammonium nitrogen concentrations after 42 days of incubation. Generally, the concentrations of released ammonium nitrogen were lower than the concentrations of nitrate nitrogen (Fig. 1ab and 2ab). The ammonium nitrogen release pattern was low from day 0 to day 28 of incubation. From days 42 to 49, a high release of ammonium nitrogen was observed, resulting in a high total nitrogen release (Fig. 1a–b). From day 42 to day 49, high levels of mineralized nitrogen were observed for 45 and 30 g kg^− 1 Azolla, as well as in the other treatments (Tables 1–2, Figs. 3a and b). Other authors have reported similar findings, demonstrating that Azolla can maximally mineralize organic nitrogen to its inorganic form after 30–60 days of incubation (Singh et al., 2000; Widiastuti, 2017). At both locations, the lowest ammonium concentration was zero for all the Azolla biomasses used as biofertilizers for the treated soil. At the Hawassa Zuria and Wondo Genet locations, at 42 days of incubation, the effects of the six Azolla biomass biofertilizer treatments on the ammonium content were as follows: control (0.66 mg kg-1, 0.88 mg kg^− 1), < 15 g kg^− 1 (3.44 mg kg^− 1, 4.61 mg kg^− 1), < 30 g kg-1 (16.85 mg kg^− 1, 21.35 mg kg^− 1), and < 45 g kg^− 1 (20.27 mg kg^− 1, 24.58 mg kg^− 1) (Tables 1 and 2). The authors reported similar findings for other soil types, showing that organic fertilizers affected the ammonification of organic nitrogen during different incubation periods (El-Ghamry et al., 2015).

Organic forms (plant or animal residues) are the source of nitrogen and, under biological processes, can be transformed into inorganic forms as follows:

NH₄ + NH₂OH NOH NO₂- NO₃⁻ (Ang gria et al., 2012)

. Mineralization of organic nitrogen in the soil begins with ammonification. The ammonium concentrations in Hawassa Zuria and Wondo Genet are shown in Tables 1 and 2 and Figs. 1a and 1b, respectively. In general, the soils exhibited the lowest mineralization rates at the beginning of the incubation, followed by those at the maximum concentration on day 42, in the order of 45 g/kg > 30 g/kg > 15 g/kg > control. Higher rates of Azolla biofertilizer application resulted in increased mineralization of inorganic nitrogen. Different authors have reported similar findings, showing that higher rates of nitrogen fertilizer application and equivalent organic manure use resulted in increased nitrogen mineralization (Woods, Cole, Porter and Coleman (1987), Patil and Sarkar (1993), and Murugan and Swarnam (2013)). At 42 days of incubation, at the Hawassa Zuria and Wondo Genet locations, the maximum concentration ranged from 16.85 to 20.27 and 21.35 to 24.31 mg N-NH₄⁺ kg^{− 1, respectively}. At the end of the incubation, the amount of ammonium decreased for all the treatments (Tables 1 and 2). The results indicated that at both locations, the maximum amount of nitrogen was mineralized during the period between 42 and 49 days of incubation of Azolla pinnata biomass for biofertilizers. In Hawassa and Wondo Genet, the maximum concentration of ammonium nitrogen was mineralized after 42 days of incubation (20.27 and 24.31 mg kg^− 1 of soil) when the biofertilizer biomass of the Azolla pinnata biomass biofertilizers was 45 g kg^− 1. The maximum concentration of Azolla pinnata was detected on day 42. At the end of the incubation, the ammonium concentration decreased as the biomass of Azolla pinnata was used as a biofertilizer. Different authors have reported these findings. Approximately 60 to 80% of the nitrogen in Azolla mineralizes within two weeks when Azolla is incorporated into waterlogged soils (Ito et al., 1985). Azolla may take 30 to 60 days in tropical climates or 60 days or more in temperate climates to mineralize organic nitrogen into inorganic forms (Asuming-Brempon et al., 2008; Bhubaneswar and Kumar, 2013; and Jama et al., 2023). However, little is known about the mineralization of nitrogen in Azolla when applied as a biofertilizer in dryland vegetable cropping systems.

Table 1

NH₄⁺ -N contents (mg kg^− 1) of soils after different incubation periods in the laboratory from the Hawassa Zuria district
			Incubation Days
Azolla rate(g kg^− 1)	0	7	14	21	28	42	49	56	Mean
0	0.61r	0.62r	0.61r	0.59r	0.60r	0.66r	0.62r	0.58r	0.61
15	0.57r	1.12q	2.26p	2.13p	3.12o	3.44n	3.08o	2.26p	2.25
30	0.60r	4.97 m	4.92 m	6.34k	6.71j	16.85b	13.23d	8.68f	7.87
45	0.63r	5.87 l	7.27i	7.76 h	8.15^g	20.27a	14.41c	11.44e	9.50
Means	0.60	3.15	3.76	4.21	4.61	10.31	7.84	5.74
LSD(0.05)	0.80
CV (%)	9.14

The mean values followed by the same letter (s) in the same column are not significantly different at the 5% level of significance.

Table 2

NH4+-N contents (mg kg^− 1) of soils after different incubation periods in the laboratory. The soil was collected from the Wondo Genet district.
			Incubation days
Azolla rate(g kg^− 1)	0	7	14	21	28	42	49	56	Mean
0	0.86r	0.85r	0.86r	0.88r	0.86r	0.88r	0.86r	0.89r	0.87
15	0.86r	2.64q	2.64q	2.73o	3.73o	4.61n	3.72o	3.34p	3.04
30	0.85r	7.00 m	7.34 m	8.22k	9.13j	21.35b	17.96d	12.87f	10.59
45	0.86r	7.77 l	8.85i	11.03 g	10.78 h	24.31a	20.29c	17.52e	12.67
Means	0.86	4.57	4.92	5.72	6.13	12.67	10.71	8.66
LSD(0.05)	0.90
CV (%)	7.62

The mean values followed by the same letter (s) in the same column are not significantly different at the 5% level of significance.

Figure 3a. Effects of Azolla pinnata biofertilizers on the NH₄⁺-N content (mg kg^− 1) in soils collected from the Hawassa Zuria district after different incubation periods in the laboratory.

Figure 3b. Effects of Azolla pinnata biofertilizers on the NH4+-N content (mg kg^− 1) in soils collected from the Wondo Genet district after different incubation periods in the laboratory.

Tables 1 and 2 at the Hawassa and Wondo Genet locations show the mean concentrations of NH₄⁺-N (mg kg^− 1 soil) in the soils treated with the Azolla biofertilizers for the 56 incubation periods, respectively. As shown in Table 1, there was a lower concentration of NH₄⁺-N in the soil on day 0, which was the day of application in all the treatments, than on subsequent sampling days. The NH₄⁺-N concentration in the soil was increased by the addition of Azolla biofertilizers, with or without a control. At both locations, the difference in the concentration of NH₄⁺-N after treatment with the azolla biofertilizers for several days was highly significant (P < 0.001). In all the treatments, with the exception of the control, the lowest concentrations of NH4+-N were detected on day 0, although the ammonium concentrations were greater on days 7, 14, 21, 28, 42, 49, and 56. The ammonium concentrations on these days were significantly (P < 0.05) greater than those on day 0 (Tables 1 and 2). A comparison of the concentrations of NH₄⁺-N on days 0 and 42 revealed (Tables 1 and 2) that the soil alone, which had almost the lowest concentration of NH₄⁺-N (the last except one for 0 among the combined treatments), had the highest concentration of NH₄⁺-N on day 42. The concentration of NH₄⁺-N increased from day 0 to day 42 in all the treatments, but after 42 days of incubation, the concentration of NH4+-N decreased, and the maximum concentration of ammonium was recorded after 42 days of incubation compared to that of the other treatments (Tables 1 and 2).

Figure 4 (a) Effect of incubation time on the mineralization of nitrogen forms (NH₄⁺ mg kg^− 1) in the laboratory in the soil collected from the Hawassa Zuria district. The bars represent the standard errors of the mean.

Figure 4 (b) Effect of incubation time on the mineralization of nitrogen forms (NH₄⁺ mg kg^− 1) in the laboratory in the Wondo Genet district. The bars represent the standard errors of the mean.

3.3. Nitrate-N Release Affected by Azolla pinnata Biofertilizers and Inorganic N Fertilizers at Different Incubation Periods

The concentrations of NO₃⁻-N in the Hawassa Zuria and Wondo Genet locations decreased at the end of the incubation period (Tables 3 and 4). Mineralization rates peaked on day 42, at which time the highest amount of NO₃⁻ -N was frequently recorded 42 days after the application of biofertilizers containing Azolla pinnata. After 42 days of incubation, the NO₃⁻ -N concentrations in the soil at both locations decreased in all the treatments and in the control (Fig. 7a and b). Glinski et al. (2007) reported that NO₃⁻ -N concentrations increased near the end of the incubation period.

In the Hawassa Zuria and Wondo Genet districts, the results showed that the conversion of N in the soil was due to the addition of azolla biofertilizers to the nitrate (NO₃⁻ -N mg kg-1) in the soil after different incubation durations. The different rates of Azolla biofertilizer application to the soil strongly differed (P < 0.001) in terms of the concentration of nitrate nitrogen at both locations between the treatments with Azolla pinnata. At both locations, the nitrate-N content (mg kg-1) was strongly significantly different due to the difference in the Azolla rate. The difference in nitrate-N content increased from 0 to 42 days after incubation. After 42 days of incubation, the amount of nitrate N decreased. At the Hawassa Zuria and Wondo Genet locations, after 42 days of incubation, the maximum concentrations of nitrate N (19.61 mg kg-1 and 24.26 mg kg-1, respectively) were recorded, and the lowest concentrations were recorded after 0 days of incubation (0.72 mg kg^− 1 and 1.08 mg kg^− 1, respectively) (Tables 3 and 4). When the percentage of Azolla pinnata increased, the concentration of nitrate N increased (Tables 3–4). Similar findings were reported for vegetable crops by different authors, who showed the effects of Azolla biofertilizers on nitrate N concentrations (Jama et al., 2023). The maximum difference in nitrate-N concentrations among all the nitrate-N concentrations was also observed at 45 mg kg^− 1 in Azolla. Similarly, the interaction effect between treatment level and incubation period (days) was highly significant (P < 0.001) (Table 1).

Figure 5a and b show that the NO₃⁻ -N concentrations at the two soil incubation locations generally increased significantly with increasing incubation period. The incubation periods investigated can be organized according to their effects on the NO₃⁻-N content in the following order: zero < 7 < 14 < 21 < 28 < 42 > 49 > 56 days of incubation. The highest values of the (NO₃⁻ -N) concentration at the Hawassa Zuria and Wondo Genet locations in the soil were 8.77 and 14.57 mg kg-1, respectively, after 42 days of incubation (Tables 3 and 4, Figs. 5a and b).

The production of nitrate by nitrification is highly dependent on other N-transformation processes in the soil, especially the accumulation of nitrification substrates (i.e., NH₄⁺). At both locations, the pattern of nitrate accumulation in response to the biofertilizers applied to Azolla pinnata was similar to that of mineralization. In Hawassa Zuria, the accumulation of NO₃⁻-N was 5.10, 17.89, and 19.61 mg kg^− 1 soil relative to 0.69 mg NO₃- -N kg^− 1 in the control, showing that Azolla 15, 30, and 45 g kg^− 1 accumulated NO₃- -N almost seven times and more than seven times more than in the control soil without amendments, respectively (Table 3). At the Wondo Genet location, the accumulations of NO₃^−–-N were 6.46, 20.89, and 24.26 mg kg^− 1 soil relative to 1.05 mg NO₃^−–-N in the control, showing that the 15 g kg^− 1 treatment resulted in a nearly sixfold increase in NO3–N and that the 30 and 45 g kg-1 treatments resulted in more than six times more NO3-N than did the control soil without amendments (Table 4). Similarly, the accumulation of NO₃⁻-N in the Hawassa Zuria and Wondo Genet locations in soils treated with 45 g kg^− 1 Azolla was 4 and 4.48 times greater than that in the control, respectively (Tables 4–5). In the soil collected from the Hawassa Zuria and Wondo Genet sites, the maximum nitrate N content was recorded at the 45 g/kg treatment (19.61 and 24.26 mg kg^− 1, respectively) after 42 days of incubation. At Hawassa Zuria, after 42 days of incubation, the proportions of total mineral N relative to NO₃⁻-N were 52, 60, 51, and 49% of the total mineral N in the control group and 15, 30, and 45 g kg^− 1, respectively. At the Wondo Genet location, the proportions of total mineral N relative to NO₃⁻-N were 54, 58, 49, and 50%, respectively, of the total mineral N in the control group and 15, 30, and 45 g kg^− 1, respectively, indicating that nitrifiers could not completely oxidize the available NH₄⁺ substrate even under favorable conditions of moisture, temperature, pH and absence of plants for competition with microbes.

Table 3

NO₃⁻ -N contents (mg kg^− 1) of soils after different incubation periods in the laboratory from the Hawassa Zuria district
				Incubation Days
Dry Azolla rate(g/kg)	0	7	14	21	28	42	49	56	Mean
0	0.72^o	0.71o	0.72o	0.74 ^o	0.73^o	0.69^o	0.71 ^o	0.75^o	0.72
15	0.76^o	1.37 ⁿ	2.65^m	3.15^l	4.36^k	5.10 ^j	3.85^kl	3.14^l	3.02
30	0.73^o	6.58 i	7.27^hi	8.17^g	8.96^fh	17.87^b	14.13^d	9.77^e	9.18
45	0.70^o	7.41^h	9.15^f	9.11^f	10.08^e	19.61^a	15.50^c	13.44^de	10.63
Mean	0.73	4.02	4.95	5.30	6.04	10.82	8.47	6.77
LSD(0.05)	1.44
CV (%)	13.85

The mean values followed by the same letter (s) in the same column are not significantly different at the 5% level of significance.

Table 4

NO₃⁻ -N contents (mg kg^− 1) of soils collected from the Wondo Genet district during different incubation periods in laboratory soil
				Incubation Days
Dry Azolla rate(g/kg)	0	7	14	21	28	42	49	56	Mean
0	1.08^r	1.09^r	1.07^r	1.08r	1.08^r	1.05^r	1.05^r	1.07^r	1.07
15	1.08^r	3.35^p	3.54ⁿ	3.12	5.51^kl	6.46^k	4.25 m	3.79ⁿ	3.88
30	1.09^r	9.09^j	10.06	10.78^h	12.39f	20.89^b	17.71c	12.91^de	11.86
45	1.07^r	10.25ⁱ	11.88^g	13.31^d	12.53de	24.26a	20.82b	17.73c	13.89
Means	1.08	5.95	6.64	7.07	7.88	13.17	10.96	8.87
LSD	1.15
CV (%)	8.16

The mean values followed by the same letter (s) in the same column are not significantly different at the 5% level of significance.

3.4. Total Mineral N (NH₄⁺ -N + NO₃^- -N) Release

At the Hawassa Zuria and Wondo Genet locations, the availability of N (NH4 + -N + NO3 - N) was significantly influenced by the combination of Azolla pinnata biofertilizers and the number of days of incubation (p < 0.001; Tables 5 and 6). The mean total mineral N content was greater in the Wondo Genet soil than in the Hawassa Zuria soil, with values of 48.57 and 39.88 mg N kg^− 1, respectively (Tables 5 and 6). Furthermore, different incubation periods also had an impact on the mean mineral N (p < 0.001; Tables 5 and 6).

At both the Hawassa Zuria and Wondo Genet locations, the total mineral N content was significantly affected by the combination of treatments (p < 0.001; Tables 5 and 6). After 42 days of incubation at both locations, the highest mean mineral N content was 45 g/kg, followed by 30 g/kg, with the lowest total mineral N found in the control treatments (Tables 5 and 6, Figs. 6a and b). The effects of the interaction between the Azolla pinnata biofertilizer and the number of days of incubation on the total mineral N concentration were significant at both locations (Tables 5 and 6). When considering the incubation period and the interactions of the combination treatment, the mean total mineral N was significantly greater in both the Hawassa Zuria and Wondo Genet soils treated with 45 g/kg N than in the other treatments. The lowest value was observed in the control treatments. Similarly, treatment combination and incubation day had significant effects on the mean total mineral N. The total changes in total inorganic N, NH₄⁺-N and NO₃⁻-N in soil treated with Azolla pinnata biofertilizers and inorganic N fertilizers compared to those in the control without amendments are shown in Figs. 6a and b. After adding biofertilizers of Azolla pinnata and inorganic N fertilizers, the total amount of inorganic N in the control soil increased significantly (p ≤ 0.05) to 16.7 mg kg^− 1 during incubation. After 42 days of incubation, the maximum mineralization potential of total mineralized N was shown by the 45 g/kg Azolla pinnata biofertilizer in Hawassa and Wondo Genet, which released 39.88 and 48.57 mg of mineral N kg-1 soil, respectively (Tables 5 and 6). A high amount of N was mineralized from days 42 to 49 of incubation, with significant differences between treatments. This finding was consistent with the results of Asuming-Brempong et al. (2008).

Table 5

Total mineral nitrogen (NH₄⁺ + NO₃⁻) concentration (mg/kg soil) of the treatments for the various incubation periods (days) of the soil collected from the Hawassa Zuria district
			Incubation days
Dry Azolla rate(g/kg)	0	7	14	21	28	42	49	56	Mean
Control	1.33^u	1.33^u	1.34^u	1.33^u	1.33^u	1.32^u	1.33^u	1.36^u	1.33
15	1.33^u	2.50^t	4.91^rs	5.28^r	7.48^op	8.55^mn	6.63^q	5.40^qr	5.26
30	1.33^u	11.55^m	12.19^l	14.51^k	15.67j	34.71b	27.36^d	18.45^ef	17.64
45	1.33^u	13.28^k	16.42^h	16.87^g	18.23^f	39.88^a	29.91^c	24.88^e	20.68
Mean	1.33	7.17	8.72	9.50	10.68	21.11	12.31	12.52
LSD(0.05)	2.02
CV (%)	10.54

The mean values followed by the same letter (s) in the same column are not significantly different at the 5% level of significance.

Table 6

Total mineral nitrogen (NH₄⁺ + NO₃⁻) concentration (mg/kg soil) of the treatments for the various incubation periods (days) of the soil collected from the Wondo Genet Zuria district
				Incubation Days
Dry Azolla rate(g/kg)	0	7	14	21	28	42	49	56	Mean
Control	1.94 ^v	1.94v	1.93v	1.96v	1.94v	1.93v	1.94v	1.96v	1.94
15	1.93v	5.99u	6.18 s	5.85t	9.24p	11.07o	7.97q	7.13r	6.92
30	1.94v	16.09n	17.40 m	19.00k	21.52i	42.24b	35.67^d	25.78f	23.35
45	1.93v	18.02 l	20.73j	24.34 h	23.31 g	48.57a	41.11c	35.25e	27.56
Mean	1.94	10.51	11.56	12.79	14.00	25.95	17.00	17.52
LSD(0.05)	1.78
CV (%)	6.85

The mean values followed by the same letter (s) in the same column are not significantly different at the 5% level of significance.

3.5. Mineralization dynamics of soil nitrogen under different fertilization treatments

Soil collected from both locations showed a pattern of increasing soil nitrogen mineralization, followed by a gradual decrease and a final stabilization during the incubation process, with a peak occurring on the 42nd day of incubation (Fig. 2(a)). In the same treatment, the soil nitrogen mineralization rates were greater in the Azolla pinnata fertilizer treatment than in the inorganic N fertilizer treatment. The amended treatments significantly induced soil nitrogen mineralization, with Azolla pinnata fertilization having a stronger effect than inorganic N fertilization. Additionally, nitrogen mineralization increased more rapidly in the Azolla pinnata treatment group than in the control treatment group. The difference in soil nitrogen mineralization between the fertilization treatments was minimal in the early stage of incubation but became more significant with time. The highest rate of soil nitrogen mineralization was 45g/kg of Azolla (0.421 and 0.595 mg N kg^− 1 day^− 1), respectively, on day 56 in the districts of Hawassa and Wondo Genet (Table 7).

The soils collected from the Hawassa Zuria and Wondo Genet locations showed that at the end of incubation, the accumulated mineralization of inorganic nitrogen in the treatments with the application of Azolla biofertilizer was in the order of control < 15 < 30 < 45 g kg^− 1 (Tables 1–2 and 3–4). A similar order was observed for treatments with inorganic nitrogen at the end of the incubation experiment. The statistical results indicated that the Hawassa Zuria district had less accumulated mineralized nitrogen than the Wondo Genet district, which may be due to the higher pH of the soil in the Hawassa Zuria district. These findings align with the results reported by Pathak and Rao (1998), who noted that total inorganic N decreased in soils with high pH during incubation.

Table 7

Soil N mineralization rates among fertilizers as a function of time period during the 56-day incubation study.
Hawassa Zuria district
Azolla treatments(g kg^− 1)	N mineralization rates(mg N kg^− 1 day^− 1)	N mineralization rates(mg N kg^− 1 day^− 1)
0	0.041d	0.099d
15	0.073c	0.094c
30	0.306b	0.426b
45	0.421a	0.595a
Means	0.168	0.226
CV (%)	8.12	9.01
LSD(0.05)	1.65	2.02

The soil net N mineralization rates were determined by subtracting the final (day 56) soil inorganic N from the initial (day 0) soil inorganic N and dividing the resulting number by the number of incubation days. Values followed by common small letters are not significantly different (P < 0.05) according to the LSD test for mean separation.

3.6. Changes in the organic carbon content, total nitrogen content, and C:N ratio in Azolla pinnata biofertilizers

3.6. 1. Release of organic carbon and total nitrogen

The patterns of organic carbon and total nitrogen release for the different treatments involving Azolla pinnata and inorganic N fertilizers are shown in Tables 8–9 for both locations. Various patterns of organic carbon and total nitrogen release were observed for nearly all the treatments. The 45 g kg^− 1 treatment exhibited a high release of OC and TN on day 14 after incubation at both locations. Generally, in the soils collected from Hawassa Zuria, the amount of OC and TN released was lower than that in Wondo Genet (Tables 8–9). Throughout the incubation experiment, the total nitrogen and organic carbon contents in the Azolla pinnata biofertilizer treatment groups differed significantly during each incubation period (Tables 8–9). The interaction effects of the treatments and the duration of the incubation were significantly different for the release of organic carbon and the total nitrogen content in the soils of both districts. An increasing trend was observed in the soils at both locations during the mineralization process from days 7 to 14 (Tables 8–9). A decreasing trend was observed from 14 to 56 days of mineralization. The nitrogen and organic carbon contents of each treatment exhibited known variations. A strong correlation was observed between organic carbon and total nitrogen during the mineralization of Azolla pinnata biofertilizers at both soil collection locations. The correlation coefficient (r = 0.866, P < 0.001) was found to be high. According to laboratory analysis, the 30 and 45 g kg^− 1 treatments had the highest total nitrogen during mineralization. Research by Fu et al. (1987), who focused primarily on initial soil organic matter, the addition of organic residues, and other factors, supports these findings by demonstrating that the mineralization process is influenced by the N supply capacity, which is primarily dependent on initial soil organic matter, the addition of organic residues, and various soil environmental factors.

Table 8

Interaction effects of the Azolla rate and incubation duration on organic carbon
Incubation days	Hawassa district				Wondo Genet district
	Azolla rate(g kg^− 1)				Azolla rate(g kg^− 1)
	Control	15	30	45	Control	15	30	45
0	2.00^m	2.01ⁱ	2.01ⁱ	2.00ⁱ	3.89^de	4.00^d	4.02^d	4.01^bc
7	2.01^m	2.42^f	2.65^c	2.74^b	4.01^d	4.15^bc	4.05^c	4.12^bc
14	2.01^m	2.48^de	2.71^bc	3.21^a	4.00^d	4.19^b	4.13^b	4.53^a
21	2.00^m	2.21^g	2.36^d	2.51^d	3.00^fg	3.38^f	3.41^f	3.86^de
28	2.01^m	2.13i	2.20^ef	2.34^d	2.55^h	2.42ⁱ	2.54^h	2.83^g
42	2.00^m	2.10^jk	2.14^h	2.29^e	2.25^k	2.22^k	2.34^ij	2.51^h
49	2.00^m	2.06^ij	2.00^ij	2.15^ik	2.11l^m	2.13^l	2.20^kl	2.34^ij
56	2.01^m	2.02^m	2.01^m	2.10^kl	2.00^o	2.10^lm	2.14^l	2.29^jk
Means	2.00	2.18	2.26	2.42	2.98	3.08	3.11	3.31
LSD (5%)	0.79				0.83
CV (%)	2.39				2.44

Means in a column followed by the same superscript letters are not significantly different.

Table 9

Interaction effects of the Azolla rate and incubation duration on total nitrogen
Incubation days	Hawassa district(Cambisols)				Wondo Genet districts(Luvisols)
	Azolla rate(g kg^− 1)				Azolla rate(g kg^− 1)
	Control	15	30	45	Control	15	30	45
0	0.15i	0.17e	0.17e	0.165ef	0.33 fg	0.35e	0.35e	0.35e
7	0.17e	0.18cd	0.175d	0.19c	0.35e	0.38cd	0.39c	0.39c
14	0.16 g	0.19c	0.25a	0.25a	0.34ef	0.40bc	0.41b	0.44a
21	0.14ij	0.16 g	0.181cd	0.21b	0.34ef	0.35e	0.35e	0.37cd
28	0.14ij	0.15i	0.165ef	0.19c	0.27hi	0.28j	0.25 h	0.30 h
42	0.12ik	0.12jk	0.156gh	0.15ij	0.20kl	0.21k	0.17n	0.24 h
49	0.11kl	0.10lm	0.150i	0.13jk	0.17n	0.16no	0.15p	0.21k
56	0.10 m	0.10 m	0.12ik	0.11kl	0.12	0.14pq	0.12r	0.15p
Means	0.14	0.15	0.18	0.18	0.26	0.28	0.28	0.31
LSD (5%)	0.14
CV (%)	2.39

Means in a column followed by the same superscript letters are not significantly different.

3.6.2. Organic carbon to total nitrogen ratio of Azolla pinnata biofertilizers

Soils collected from locations treated with 45 or 30 g kg^− 1 released the most NH₄⁺-N and NO3-N due to the relatively high levels of Azolla. The application of inorganic N increases the activity of ureases in the soil and has a positive linear relationship with the rate of nitrogen application (Jahangir et al., 2021). A favorable environment for urease activity promotes rapid hydrolysis of urea fertilizer and the breakdown of Azolla pinnata biofertilizers, leading to increased levels of NH₄⁺ -N in all situations. Despite the higher N content in the biofertilizers of Azolla pinnata, the plants in the control treatment had the lowest levels of mineral NH4+ -N. N-rich biofertilizers such as Azolla pinnata have potential as nitrogen fertilizers with delayed release (Jama et al., 2023).

The accumulation of NO₃⁻-N was significantly greater in the 45 g kg^− 1 treatment than in the other treatments due to higher NH₄⁺-N production, which led to rapid nitrification. The amount of nitrogen released by organic additions depends on the chemical composition of the organic components (e.g., N content, C:N ratio and cellulose, hemicellulose, lignin and polyphenol contents) (Maitlo et al., 2022), the application rate of manure and the properties of the soil microorganisms (Manojlovic et al., 2010). Azolla pinnata biofertilizers with high nitrogen contents and low C:N ratios provide more mineral N through mineralization (Cordovil et al., 2005). Similar findings by other authors showed that low C:N ratios of Azolla biofertilizers and inorganic N fertilizers release more mineral N during soil nitrogen mineralization (Jama et al., 2023). Organic agriculture with lower levels of N and higher C:N ratios may lead to the immobilization of N (Manojlovic et al., 2010). The organic materials used in the study, which had low C:N ratios and high N contents, led to organic N mineralization in Azolla pinnata and all the other treatments. Several authors have reported N release rates ranging from 64 to 91% from farmyard manure, dhaincha and legume residues (Rahaman et al., 2013) and from 64 to 83% from cow dung, poultry manure, cow dung slurry, and poultry manure slurry (Haque et al., 2015), supporting the conclusion that nitrogen release depends on the soil type, moisture status, and organic amendments. The total nitrogen concentration remained higher than that in the initial unamended soil after 56 days. A lower C:N ratio in response to soil amendments improved mineralization, resulting in a decrease in the total nitrogen concentration after 56 days compared to the initial total nitrogen (TN) concentration, consistent with the findings of other authors (Uddin et al., 2021). The significant decreases in the TN and OC contents varied on each sampling day from 21 to 56 days, showing similar trends at both locations. Consequently, the C:N ratio decreased during mineralization. Perez-Harguindeguy et al. (2000) reported that the C:N ratio was a good indicator of the mineralization rate, with higher C:N values often associated with compounds exhibiting greater C enrichment.

At Wondo Genet, mineralization occurred over an incubation period of 49–56 days with a wide C:N ratio (12–18:1), followed by 42 days (10–14:1) and 0–28 days (9–12:1), indicating a narrowing C:N ratio (Fig. 8b). Mary et al. (1996) confirmed that organic residues with low C:N ratios exhibit more mineralization of N, while wide C:N ratios may cause immobilization of N. Hawassa Zuria has a higher C:N ratio during mineralization than Wondo Genet does, possibly due to differences in precipitation, soil properties, and microbial factors. A higher C:N ratio of fertilizer or soil amendment (especially above 18 C:1 N) can lead to greater immobilization of N and slower inorganic N release for plant uptake (Jama et al., 2023). Therefore, at the end of the 56-day incubation period, treatments with slower mineralization of N and less availability of N were observed in the control (C:N = 20:1, 17:1), 15 g kg-1 (C:N = 20:1, 13:1), 30 g kg-1 (C:N = 17:1, 18:1), and 45 g kg^− 1 (C:N = 19:1, 15:1) treatments at both the Hawassa Zuria and Wondo Genet sites (Fig. 8a and b). The net available N decreased exponentially with the C/N ratio recorded after 56 days of incubation under laboratory conditions, indicating that higher C/N ratios result in lower N availability in the soil and vice versa. Different authors have reported similar findings, showing that higher C/N ratios lead to lower N availability in the soil (Islam et al., 2021). The highest net mineralization of N in soils in which organic amendments are applied is often attributed to amendments with a low C:N ratio and a high N content (Flavel et al., 2006). However, C:N ratios alone cannot predict relative N mineralization rates, as Azolla has a similar C:N ratio as compost but has a much greater N availability over time. The quantity of N released into crops depends on the chemical composition of the organic matter (Calderón et al., 2005). The N content, C:N ratio, lignin content, cellulose content, hemicellulose content, and polyphenol content are factors that affect the amount of N released into soils (Mohanty et al., 2011). Organic fertilizers with high N contents and low C:N ratios typically mineralize sufficient N to meet plant growth demands (Cordovil et al., 2005; Seneviratne et al., 2000).

The mineralization of Azolla pinnata in Hawassa Zuria revealed that its initial C:N ratio was influenced by the mineralization process (Fig. 8). The mineralization of Azolla pinnata biofertilizers significantly negatively correlated with the C/N ratio (r = -0.7621; p < 0.001) and with the number of days of incorporation, indicating that as the days of incorporation progressed, the C/N ratio increased. The highest C:N ratio was observed after 56 days of incubation. Perez-Harguindeguy et al. (2000) noted that the C:N ratio was a good indicator associated with compounds showing greater C enrichment, particularly lignin. The high organic carbon content may be due to the initially high C:N ratio and the difference in total nitrogen content among the treatments. Stemmer et al. (1999) reported that stabilized organic products with an adequate C:N ratio (< 20) enhance the mineralization process, while products with a high C:N ratio promote immobilization. The low C:N ratio may have facilitated the rapid mineralization of the Azolla pinnata biofertilizers at the Wondo Genet location.

4.1. Summaries AND CONCLUSIONS

The findings of the study revealed that the rates at which Azolla pinnata biomass increased mineral nitrogen varied depending on the availability period. The use of Azolla pinnata biofertilizers in conjunction with inorganic fertilizers increased the organic carbon, total N, total P, and total K contents of the soil. Although they are more available than inorganic nitrogen fertilizers, Azolla pinnata biofertilizers can help reduce the loss of nutrients to the environment. This is because the nutrients in the fertilizer mineralize more slowly. Despite having C:N ratios similar to those of compost, Azolla mineralizes faster than other organic fertilizers. The findings of this study indicate that the best way to increase the soil mineralization rate and N availability is to apply Azolla pinnata biofertilizers in a laboratory setting. Similarly, compared with those in the other treatments, the total mineralized N in the Azolla pinnata biofertilizer treatment (45 g kg^− 1) increased during the 42-day incubation period. The high C/N ratio and low mineralization of Azolla pinnata in response to the inorganic N fertilizers may have contributed to the low specific mineralization rate in the untreated control. Although the biomass of Azolla pinnata and the rates of N addition used as biofertilizers varied, the mineralization of N in the other treatments changed over the course of several days of incubation. All the modified treatments produced significantly greater total N mineralization than did the controls at both locations. The net N mineralization and specific N mineralization rates significantly decreased over time. The findings revealed that when Azolla pinnata was incubated for 42 to 49 days, the highest amount of N was mineralized at both locations. The amount of nitrogen that can be extracted from the soil is a measure of its quality. Naturally, fertile soils have a high potential for nitrogen mineralization. According to the present study, the rate at which mineral nitrogen is increased by Azolla pinnata biofertilizer increases, as does the duration of nitrogen availability. The loss of nutrients to the environment can be minimized even though Azolla pinnata biofertilizers have a longer availability period and slower mineralization of nutrients. The application of Azolla pinnata biofertilizers is the most effective method for improving crop nutrient availability and increasing N availability in agricultural ecosystems. In addition to increasing soil N availability and total N availability, the application of Azolla pinnata biofertilizer may help decrease the dependence on inorganic N fertilizer.

4.2. RECOMMENDATIONS

Based on the current study, the following key recommendations have been developed.

The maximum amount of inorganic nitrogen (NH₄⁺-N and NO₃^- -N) was observed under laboratory conditions for 42 days with 45 g kg^-1 Azolla. However, further testing under actual field conditions is needed to confirm these findings and determine the maximum levels of mineral nitrogen.
Additional research is needed to explore the potential of Azolla pinnata biofertilizers for slow mineralization. However, further studies are needed under field conditions to validate the results obtained in the laboratory.

Funding Declaration: This research did not receive any external funding, as I am currently working on my PhD dissertation and the country is still in a political crisis and is considered a least developed country.

Data availability: All the data are presented in the manuscript

Declarations Ethics approval and consent to participate: The collection of the plant materials complied with all relevant institutional, national, and international guidelines. Because Azolla pinnata R.BR. is not an endangered wild plant, plant collection does not require specific permits.

Consent for publication: Not applicable.

Acknowledgements: We greatly appreciate Hawassa University soil and soil laboratory assistants. Hawassa, helping me at incubated periods and data recording.

Authors’ contributions Mr. Habtamu Alemayehu; wrote the original manuscript, data collected, data analysis: Dr. Wassie Haile and Dr. Alemayehu Kiflu; review and improved the manuscript and advised team. All authors read and approved the final manuscript.

Conflicting Interests: The authors declare no conflicts of interest, as all the authors have read and carefully reviewed the manuscript and agree to its submission.

Authors Detailed: ^{1, 2}Samara University, College of Dryland Agriculture, P.O. Box:132, Afar, Ethiopia, ² Hawassa University, College of Agriculture P.O. Box:05, Hawassa,Ethiopia

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Effects of Azolla (Azolla pinnata) biofertilizer on soil nitrogen mineralization in soils collected from Hawassa Zuria and Wondo Genet Districts Incubated under laboratory conditions

Status:

Version 1

Abstract

Figures

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 Description of the study areas

2.2. Soil Sampling

2.3. Experimental design, treatments, and procedures

2.4. Soil analysis from the preincubation experiment

2.5. Soil incubation and sampling procedure

2.6. Measurements of mineral nitrogen

2.7. Nitrogen mineralization and N availability calculation

2.8. Soil Extraction and Analysis

2.9. Data collection

2.10. Data analysis

3. RESULTS AND DISCUSSION

3.1. Effects of Azolla Pinnata Biofertilizer on Soil N Mineralization during Different Incubation Periods

3.2 Nitrogen mineralization

3.2. Ammonium nitrogen (NH₄⁺-N) release affected by Azolla pinnata biofertilizers during different incubation periods

3.3. Nitrate-N Release Affected by Azolla pinnata Biofertilizers and Inorganic N Fertilizers at Different Incubation Periods

3.4. Total Mineral N (NH₄⁺ -N + NO₃^- -N) Release

3.5. Mineralization dynamics of soil nitrogen under different fertilization treatments

3.6. 1. Release of organic carbon and total nitrogen

3.6.2. Organic carbon to total nitrogen ratio of Azolla pinnata biofertilizers

4. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

4.1. Summaries AND CONCLUSIONS

4.2. RECOMMENDATIONS

Declarations

References

Additional Declarations

Status:

Version 1

Effects of Azolla (Azolla pinnata) biofertilizer on soil nitrogen mineralization in soils collected from Hawassa Zuria and Wondo Genet Districts Incubated under laboratory conditions

Status:

Version 1

Abstract

Figures

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 Description of the study areas

2.2. Soil Sampling

2.3. Experimental design, treatments, and procedures

2.4. Soil analysis from the preincubation experiment

2.5. Soil incubation and sampling procedure

2.6. Measurements of mineral nitrogen

2.7. Nitrogen mineralization and N availability calculation

2.8. Soil Extraction and Analysis

2.9. Data collection

2.10. Data analysis

3. RESULTS AND DISCUSSION

3.1. Effects of Azolla Pinnata Biofertilizer on Soil N Mineralization during Different Incubation Periods

3.2 Nitrogen mineralization

3.2. Ammonium nitrogen (NH4+-N) release affected by Azolla pinnata biofertilizers during different incubation periods

3.3. Nitrate-N Release Affected by Azolla pinnata Biofertilizers and Inorganic N Fertilizers at Different Incubation Periods

3.4. Total Mineral N (NH4+ -N + NO3- -N) Release

3.5. Mineralization dynamics of soil nitrogen under different fertilization treatments

3.6. 1. Release of organic carbon and total nitrogen

3.6.2. Organic carbon to total nitrogen ratio of Azolla pinnata biofertilizers

4. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

4.1. Summaries AND CONCLUSIONS

4.2. RECOMMENDATIONS

Declarations

References

Additional Declarations

Status:

Version 1

3.2. Ammonium nitrogen (NH₄⁺-N) release affected by Azolla pinnata biofertilizers during different incubation periods

3.4. Total Mineral N (NH₄⁺ -N + NO₃^- -N) Release