In situ nitrogen mineralization and nutrient release by soil amended with black soldier fly frass fertilizer

Although black soldier fly frass fertilizer (BSFFF) is effective on crop performance, information on nitrogen (N) mineralization and nutrient release capacity of soils amended with BSFFF is lacking. This study utilized field incubation experiments to investigate the ammonification, nitrification, microbial populations, and quantities of nutrients released by soils amended with BSFFF and commercial organic fertilizer (SAFI) for a period equivalent to two maize cropping seasons. For the control treatment, no BSFFF or SAFI was added. Results indicated that most of the N in BSFFF amended soils was available in the ammonium form, while soils treated with SAFI had higher nitrate concentration. The BSFFF amended soils experienced shorter net immobilization periods of N (30–60 days) compared to SAFI treated soils (60–95 days). Increased rates of mineralization (3–10 times) and nitrification (2–4 times) were observed in soils treated with BSFFF during the second season of application. The BSFFF treated soils showed significantly higher N, phosphorus, and magnesium release than the control. Repeated application of BSFFF led to increased N release by three-folds in the soil. Furthermore, soil amendment with BSFFF increased the populations of bacteria and fungi, reduced soil acidity, and increased phosphorus (two-folds) and magnesium (two–four-folds) release than SAFI treated soils. Our findings highlight the crucial role of BSFFF in improving soil health by addressing the challenges of soil acidity, phosphorus fixation and nutrient mining, which is characteristic of most tropical soils.

. Concentrations of ammonium and nitrates, and ammonium-nitrate ratio of soil amended with BSF frass and commercial organic fertilizers. *** p < 0.001, **p < 0.01, *p < 0.05, ns = not significant at p ≥ 0.05, BSFFF = Black soldier fly frass fertilizer, SAFI = commercial organic fertilizer, control = unamended soil. Within the same row and per parameter, means (± standard error) with the same letters are not significantly different at p ≥ 0.05, n = 3. www.nature.com/scientificreports/ incubation during the long rainy season (p < 0.001). Soil amended with BSFFF had significantly higher ammonium concentration than the control treatment throughout experiments, except at 60 days after incubation during both seasons. Likewise, soil treated with BSFFF achieved significantly higher ammonium concentration than SAFI treatments, except at 15 and 60 days after incubation during the short and long rainy season experiments, respectively. At the end of experiments, soil amended with BSFFF had the highest ammonium concentration, which was 34 and 40 times higher than those of the control and SAFI treatments, respectively (Table 1). Significant differences in soil nitrate concentration due to fertilizer treatments (short rainy season: χ 2 = 242.0 df = 2, p < 0.001, long rainy season: χ 2 = 299.0, df = 2, p < 0.001) and the interaction of treatment and incubation time (short rainy season: χ 2 = 23.2, df = 10, p = 0.01, long rainy season: χ 2 = 31.3, df = 10, p < 0.001) were observed. The effect of incubation time was significant during the long rainy season only (short rainy season: χ 2 = 11.0, df = 5, p = 0.05, long rainy season: χ 2 = 13.0, df = 5, p = 0.02). Initial nitrate concentrations ranged between 0.2 and 400 mg kg −1 , while the soil in the control treatment and that treated with SAFI had the lowest and highest values, respectively ( Table 1). The nitrate concentration of the control treatment increased to peak values (14-18 mg kg −1 ) at the 90th day after incubation during both seasons and decreased afterwards.
The nitrate concentrations of soil amended with BSFFF and SAFI did not follow a uniform trend. Soil treated with BSFFF reached peak nitrate concentration 125 days after incubation, while SAFI amended soil achieved the highest nitrate values after 90 and 15 days of incubation during the short and long rainy seasons, respectively. The nitrate concentration of soil amended with SAFI fertilizer significantly (p = 0.02) decreased from the 30th to 60th day after incubation during the long rainy season. The peak nitrate concentration of SAFI treated soil was 17-27 and 26-28 times higher than those achieved using the control and BSFFF treatments, respectively. Soil amended with SAFI fertilizer maintained significantly higher nitrate concentrations than the control and BSFFF treatments, except at 60 days after incubation during both seasons. At 125 days after incubation, the nitrate concentration soil amended with SAFI was 20 and 19 times higher than those of the control and BSFFF treatments, respectively ( Table 1).
Amendment with BSFFF triggered net mineralization rates at 60 and 125 days after incubation during the short rainy season and most of the long rainy season, except at 90 days after incubation. Soil amended with SAFI achieved net mineralization in 90 days after incubation during both seasons and at 15 days after incubation during the long rainy season. For the unamended soil, net mineralization rates were recorded at 15 and 60 days after incubation during the short rainy season, and most of the long rainy season, except at 90 days after incubation ( Table 2).
Soil amended with SAFI experienced longer periods (60-95 days) of N immobilization during both seasons than BSFFF treatments (30-60 days) ( Table 2). During the short rainy season, the highest immobilization rate (− 11 mg N kg −1 day −1 ) was recorded at 30 days after incubation in soil amended with SAFI, and this was significantly (p < 0.038) higher than that of BSFFF and control treatments. Soil amended with BSFFF experienced the highest immobilization rate (− 12 mg N kg −1 day −1 ) during the long rainy season, which was not significantly different (p < 0.01) from that of the untreated soil.
All treatments achieved peak N mineralization rates (0.5-5.2 mg N kg −1 day −1 ) between 60 and 90 days after incubation ( Table 2). Soil amended using SAFI achieved the highest net mineralization rate during the short rainy season at 90 days after incubation. During the long rainy season, the unamended soil had the highest mineralization rate (7.4 mg N kg −1 day −1 ) 60 days after incubation, which was not significantly different from that of BSFFF treatment. Generally, all treatments experienced rises in the rates of N mineralization during the long rainy seasons, with higher increases (three-ten folds) observed in BSFFF treated soils.
The soil nitrification rate was significantly influenced by incubation time (short rainy season: χ 2 = 21.6, df = 4, p < 0.001, long rainy season: χ 2 = 10.9, df = 4, p = 0.03) and the interaction of incubation time and  Table 2). The net nitrification rates of soils amended with BSFFF were significantly higher than those achieved using SAFI at 30 and 60 days after incubation during the short (p < 0.001) and long rainy seasons (p = 0.019), respectively. The net nitrification rates of BSFFF amended soils increased by two-four folds during the long rainy season.
Soil pH, and populations of bacteria and fungi. The soil pH was significantly influenced by fertilizer treatments (short rainy season: χ 2 = 733.7, df = 2, p < 0.001, long rainy season: χ 2 = 192.8, df = 2, p < 0.001), incubation time (short rains season: χ 2 = 119.2, df = 5, p < 0.001, long rainy season: χ 2 = 387.2, df = 5, p < 0.001) and their interaction (short rainy season: χ 2 = 127.1, df = 10, p < 0.001, long rainy season: χ 2 = 315.4, df = 10, p < 0.001). The initial pH of soils amended with different fertilizer treatments ranged between 5.2 and 8.7. Thereafter, the soil pH decreased until the end of the experiments (Fig. 1a,d). Soil amended with BSFFF maintained significantly (p < 0.001) higher pH values than control soil up to 90 days of incubation and throughout experiments for SAFI treated soil. The unamended soil had significantly higher pH than that of soil treated with SAFI, except at 90 days after incubation during the short rainy season (Fig. 1a). At the end of experiments, soil pH ranged between 4.8 and 6.2, whereby the control soil had the highest pH while soil amended with SAFI had the lowest pH.
The soil bacteria population was significantly influenced by incubation time during both seasons (short rainy season: χ 2 = 24.7, df = 5, p < 0.001, long rainy season: χ 2 = 73.9, df = 5, p < 0.001) and fertilizer amendment during the short rainy season only (χ 2 = 19.9, df = 2, p < 0.001). However, the interaction of incubation time and fertilizer amendments was not significant (short rainy season: χ 2 = 8.9, df = 10, p = 0.541, long rainy season: χ 2 = 8.1, df = 10, p = 0.618). The bacterial population significantly increased from initial values to peak levels (9.5-10.3 log 10 CFU g −1 ) between 30 and 60 days after incubation and decreased afterwards (Fig. 1b,e). Soil treated with BSFFF achieved the highest bacterial populations during both seasons. Its peak bacterial population during the long rainy season was equivalent to the highest value obtained using SAFI (Fig. 1e). Amendment with BSFFF caused significantly higher bacterial populations than other treatments at 30 and 90 days during the short rainy season and at 125 days after incubation during both seasons. At the end of experiments, the bacterial populations ranged between 7.3 and 9.3 log 10 CFU g −1 , whereby BSFFF and control treatments had the highest and lowest values, respectively.
The different fertilizer amendments caused significant differences in soil fungi populations during the short rainy season only (χ 2 = 15.5, df = 2, p < 0.001). The effect of incubation time was significant during both seasons (short rainy season: χ 2 = 11.9, df = 5, p = 0.04, long rainy season: χ 2 = 53.7, df = 5, p < 0.001) while the interaction of fertilizer treatments and incubation time was significant during the long rainy season only (χ 2 = 18.7, df = 10, p = 0.044). Minimal changes in fungi populations were observed in the first 30 days of incubation (Fig. 1c,f). The soil fungi populations significantly increased to peak values (7.9-9.0 log 10 CFU g −1 ) between 60 and 90 days after incubation and decreased afterwards. However, SAFI treated soil achieved peak fungi populations after 125 days during the long rainy season. Soil amended with BSFFF maintained significantly (p < 0.001) higher fungi populations than other treatments during the entire short rainy season and 125 days after incubation during the long rainy season. At the end of the experiments, the control treatment had the lowest fungi population (7.1 log 10 Table 2. Nitrogen mineralization and nitrification rates of soil amended with BSF frass and commercial organic fertilizers. ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant at p ≥ 0.05, BSFFF = Black soldier fly frass fertilizer, SAFI = commercial organic fertilizer, control = unamended soil. Within the same row and per parameter, means (± standard error) followed by the same letters are not significantly different at p ≥ 0.05, n = 3. www.nature.com/scientificreports/ CFU g −1 ) while BSFFF treated soil had the highest (9.0 log 10 CFU g −1 ). Also, soils treated with BSFFF and SAFI had a significantly (p < 0.001) higher fungi population than the unamended soil at the end of the experiments.
The total N concentration of soil treated with BSFFF significantly increased to peak values (0.74-0.83%) at 30 and 60 days after incubation during the short and long rainy season, respectively, and decreased afterwards (Fig. 2a,d). On the other hand, there was minimal changes in the total N concentration in soil amended with SAFI during experiments, while total N concentration in unamended soil was observed to increase from day 60 after incubation and peaked on the 125th day. Soils amended with BSFFF maintained significantly (p < 0.001) higher total N concentration than other treatments throughout experiments. The total N concentration of soil treated with BSFFF was 2.5-5.4 and 2.5-4.4 times higher than soil amended with SAFI during the short and long rainy seasons, respectively. Likewise, soil amended with SAFI achieved significantly (p < 0.001) higher total N concentration than the unamended soil, except at 30 days during the long rainy season and on the 125th day during both seasons. At the end of the experiments, soils treated with BSFFF had the highest N concentration (0.14-0.34%), which was significantly (p < 0.001) higher than that of the SAFI and control treatments by 149-152% and 32-44%, respectively.
The Mg concentration increased to peak values of 0.68 to 3.10 cmol kg −1 between 30 and 60 days of incubation, after which the concentrations kept decreasing up to the end of the experiments. The Mg concentration of soil amended with BSFFF was 2.6-3.7 and 3.4-4.3 times higher than those of SAFI treated and unamended soil, respectively. Also, soils treated with BSFFF maintained significantly (p < 0.001) higher Mg concentration than other treatments, except the control at 125 days of incubation during the long rainy season. At the end of the experiments, soil treated with BSFFF had the highest Mg concentration (2.4-2.9 cmol kg −1 ), which was 3.7 and 4.3 times higher than those of SAFI and control treatments.
Nutrients released by unamended soil and soils amended with organic fertilizers. The amount of N (short rainy season: F = 5.5, df = 2, 6, p = 0.044, long rainy season: F = 6.2, df = 2, 6, p = 0.035), that of available P (short rainy season: F = 740.9, df = 2, 6, p < 0.001, long rainy season: F = 740.2, df = 2, 6, p < 0.0001) and exchangeable Mg (short rainy season: F = 135.7, df = 2, 6, p < 0.001, long rainy season: F = 28.6, df = 2, 6, p < 0.001) released by the unamended soil and soils amended with different organic fertilizers varied significantly during the experiments (Table 4). However, the quantities of K (short rainy season: F = 3.4, df = 2, 6, p = 0.102, long rainy Table 3. Concentrations of exchangeable calcium and magnesium in soil alone and soil amended by BSF frass and commercial organic fertilizers during the short and long rainy seasons. ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant at p ≥ 0.05 BSFFF = Black soldier fly frass fertilizer, SAFI = commercial organic fertilizer, control = unamended soil. Within the same row and per parameter, means (± standard error) followed by the same letters are not significantly different at p ≥ 0.05, n = 3.  Table 4. Nutrients released from unamended soil and soils amended with organic fertilizers during the short and long rains cropping seasons. ***p < 0.001, **p < 0.01, *p < 0.05, ns = nonsignificant at p ≥ 0.05, BSFFF = Black soldier fly frass fertilizer, SAFI = commercial organic fertilizer, control = unamended soil. Within the same column and per parameter, means (± standard error) followed by the same letters are not significantly different at p ≥ 0.05, n = 3. www.nature.com/scientificreports/ season: F = 4.0, df = 2, 6, p = 0.078) and Ca (short rainy season: F = 4.2, df = 2, 6, p = 0.073, long rainy season: F = 4.1, df = 2, 6, p = 0.074) released were not significantly influenced by fertilizer amendments. Soil amended with SAFI and BSFFF released significantly (p < 0.001) higher N than unamended soil during the short and long rainy seasons, respectively ( Table 4). The N released by soil treated with SAFI and BSFFF was 7-16 times and 8-11 times higher than N released by the control soil, respectively. Soils treated with SAFI released 107% higher N than BSFFF treated soil during the short rainy season. On the other hand, soil treated with BSFFF released the highest N during the long rainy season, 58 and 998% higher than SAFI amended soil and the control soil, respectively.
The quantity of P released by BSFFF amended soils was significantly (p < 0.001) higher than those of SAFI and control treatments by about two-and forty-folds, respectively. In contrast, the P released by soil treated with SAFI was 22 times higher than that released by the control soil (Table 4). Soil treated with SAFI had the highest K, which was 1.9 times and 1.4-1.6 times higher than those of BSFFF and control treatments, respectively. The Ca released by soil amended with SAFI was 44 and 18% higher than that released by BSFFF and the control treatments, respectively.
The highest quantity of Mg was released by soil amended with BSFFF, and this was significantly (p < 0.001) higher than those of SAFI and control treatments by 82-268% and 319-326%, respectively (Table 4). However, soil amendment with SAFI released significantly higher Mg than the control treatment.

Discussion
The patterns of soil mineralization and nitrification rates, and seasonal differences observed during this study (Tables 1 and 2) have been previously reported 35,40 . The high ammonium and low nitrate concentrations observed in BSFFF treated soil could be attributed to the quality of the frass fertilizer used in the study (Table 5). Ammonium nitrogen is one of the major N fractions that influence N released from organic fertilizers 32 . The BSF frass contains high levels of ammonium nitrogen 15,18,41 , which requires time to be converted into plant-available form (NO 3 − ) through mineralization and nitrification processes. At the same time, ammonium is preferred by soil microorganisms, making it prone to immobilization 25,42 . The high pH values (8.0-8.8) (Fig. 1a,d) observed in soil amended with BSFFF could have triggered gaseous losses through ammonia volatilization and reduced the nitrification process as indicated by the higher ratios of ammonium to nitrate 43 . The pH values above 7.5 favour nitrogen loss through ammonia volatilization 44 because an increase in pH has been shown to increase the dissociation of ammonium to ammonia gas, thus shifting the equilibrium to ammonia, which eventually evaporates 45 .
The higher soil nitrate concentration (Table 1) and nitrification rates ( Table 2) realized using SAFI could be partly attributed to initially low ammonium/nitrate ratio (Table 1) and lower soil pH values (< 7.0) during experiments (Fig. 1a,d). This agrees with previous studies that reported higher autotrophic nitrification under conditions of low ammonium and low pH 46 . Therefore, the low nitrification rates observed in BSFFF amended soil could also be attributed to the toxic effects of high ammonium and alkaline conditions on the enzymatic activities of nitrifying bacteria 47 . Although, the current studies found slightly higher populations of bacteria (Fig. 1b,e) and fungi (Fig. 1c,f) in BSFFF treated soil than SAFI treatments, the strains of nitrifiers present, their functional roles, and influence on enzymatic activities were not determined. Future studies would be necessary to investigate the effect of the BSFFF amendment on the abundance and diversity of nitrifying bacteria, and enzymatic activities, to generate accurate conclusions on the nitrification process.
Conversely, the net N immobilization associated with SAFI could be attributed to the lignin and polyphenols present in the biochar used to make SAFI 31,48,49 . The recalcitrant carbon in biochar is resistant to microbial decomposition and consequently causes delays in N mineralization 50,51 . The low total N concentration, such as that observed in soil amended with SAFI (Fig. 2a,d), has also been found to stimulate immobilization and slow down N release 40 . Our sister paper 21 suggested supplementation with mineral N fertilizers to compensate for N immobilization and improve synchrony of N supply for optimal crop production. This agrees with Kaleem Abbasi and Khaliq 34 , who reported accelerated mineralization and nitrification rates in soils amended with a combination of mineral N and organic fertilizers. However, the higher and net positive values of nitrification and nitrification rates observed during the long rains (Table 2) suggest that continued amendment of soil using BSFFF could gradually increase the nitrification rate and mineral N release for plant uptake. Table 5. Selected physical and chemical characteristics of the experimental soil, and organic fertilizers. TOC total organic carbon, SOM soil organic matter, BSFFF black soldier fly frass fertilizer, SAFI commercial organic fertilizer, n = 3. www.nature.com/scientificreports/ The current study revealed higher nutrients concentrations ( Fig. 3 and Table 3). Nutrient release (Table 4) in fertilizer amended soils compared to unfertilized soil is consistent with previous studies that reported the resultant effect of low soil fertility in Kenya 3,52,53 and most countries in SSA 1,2 . It recommended regular fertilizer inputs for soil fertility improvement 23,[54][55][56] . Therefore, the application of high-quality fertilizers (such as the BSFFF) can improve soil health and crop productivity 20,21 by increasing the availability of nutrients (such as nitrogen and phosphorus), which are the most limiting nutrients to crop production in SSA 2 .
The amount of N released using BSFFF was higher than that achieved using farmyard manure and tithonia in the soils of central Kenya 57 . The higher concentrations and nutrients release capacity associated with BSFFF could be attributed to the good quality of the fertilizer and its high mineralization rate, as previously reported 27 . The increased phosphorus availability achieved in soil treated with BSFFF could be partly attributed to the high soil pH values (8.0-8.9) (Fig. 1a,d) that do not favour P fixation, which is common in the acidic soils of Kenya 52,53 and SSA 2 . Acidic soils (< pH 6) have high iron and aluminium oxides that react with phosphate ions to form insoluble compounds, thus reducing the quantity of P available for plant growth 58,59 . Findings from the current study concur with those of von Arb et al. 60 , who reported an increase of available soil P in organic farming systems of Central Kenya. Therefore, this study highlights the role of the BSFFF amendment in ameliorating P availability by causing a liming effect in soils with low pH.
The high concentration and release of potassium observed in SAFI amended soil could be attributed to the initially high K concentration in SAFI (Table 5). This is in agreement with previous studies that reported high potassium concentration in biochar-fortified organic fertilizers 48,61 . Nevertheless, the quantities of potassium released by soil amended with BSFFF were sufficient to produce most cereals, legumes, and vegetable crops. www.nature.com/scientificreports/ The increased release of N, P and K observed during the long rainy season (Table 4) indicates the role of soil moisture in nutrients release from organic fertilizer. With continued application, BSFFF could be solely relied on for providing nutrients for crop production. This would save farmers from the expensive mineral fertilizer that is less effective in soils with multiple degradation challenges 4,23 , and cater for organic farmers who exclusively rely on organic fertilizers for crop production. Our sister paper supported this assertation, which reported higher growth, yield and nutrient uptake associated with maize grown using BSFFF compared to commercial organic (SAFI) and mineral (urea) fertilizers 20 .
The higher populations of soil bacteria (Fig. 1b,e) and fungi (Fig. 1c,f) achieved using BSFFF indicate that in addition to increasing nutrient supply, this frass fertilizer has the potential to increase soil microbial abundance and diversity, which is key for improving biological soil fertility. The current findings agree with previous studies that have reported the additional benefits of using insect frass fertilizer (such as increased microbial abundance and activity, deeper plant root growth and suppression of plant pathogens) 9,11,12,22 . Furthermore, the BSFFF takes a shorter time to generate (5 weeks) than conventional composts (8-24 weeks) 18 . Therefore, findings from this study are crucial in changing attitudes towards organic fertilizer use, with the advantages of higher nutrient release capacity, less bulkiness and shorter production time associated with BSFFF.

Conclusion
The current study has demonstrated that BSFFF has a high potential to supply adequate nutrients for optimal crop production. The higher ammonification and low nitrification rates indicate that nitrogen immobilization could occur at the initial stages of crop growth. Therefore, mineral N supplementation would be necessary to compensate for net immobilization and improve synchrony of N release for plant growth. Nevertheless, the increase in nutrient release and nitrification rates during the long rainy season implies that the continued application of BSFFF would increase N mineralization and nutrient release. The significantly higher amounts of P released by soil amended with BSFFF indicate its key role in enhancing P availability through its liming effect.
Furthermore, the higher population of soil bacteria and fungi associated with BSFFF amended soil underline its potential for improving biological soil fertility. Therefore, BSFFF is recommended for a sustainable enhancement of soil health and crop productivity, thus reducing overdependence on low-quality organic fertilizers and expensive mineral fertilizers. Future studies should determine mid-and long-term patterns in nutrient release, abundance, diversity and functional roles of bacteria and fungi in soils amended with BSFFF.

Description of the study site. Field experiments were set up for two seasons (April-September 2019 and
October 2019-March 2020) at the Kenyatta University Teaching and Demonstration farm (1°10′59″ S, 36°55′34″ E, 1580 m above sea level), Nairobi County, Kenya. Nairobi County receives bimodal rainfall with annual averages of 925 mm and mean monthly temperatures of 21-28 °C (www. meteo.go.ke). The first rainfall season (short rains) starts from March to June, while the second season (long rains) runs from October to January. Cumulative rainfall totals of 252 and 281 mm were received during the short and long rainy seasons experiments, respectively (Fig. 3a). Mean monthly rainfall of 40 and 87 mm, and 31-102 mm were received during the short and long rainy seasons experiments, respectively (Fig. 3c). Mean daily temperatures of 22-29 °C, and 22-28 °C were recorded during the short and long rainy seasons, respectively (Fig. 3b). The short rainy season had higher mean monthly temperatures than the long rainy season (Fig. 3c).
Soils in the study site are Acric Ferralsols 62 characterized by low organic matter, shallow depths, and low pH levels. Before the onset of experiments, soils were sampled (0-20 cm) for determination of total organic N, total organic carbon, available phosphorus (P), exchangeable cations [potassium (K), calcium (Ca), and magnesium (Mg)], pH, and soil texture following procedures described by Okalebo et al. 63 . Table 5 presents the characteristics of the experimental soil.
Source of organic fertilizers. The experiment involved two organic fertilizers: the black soldier fly frass fertilizer (BSFFF) and commercial organic fertilizer (SAFI). The BSFFF was a product obtained from the feeding of BSF larvae on brewery spent grain (sourced from Kenya Breweries Limited, Nairobi) at the Animal Rearing and Quarantine Unit of the International Centre of Insect Physiology and Ecology (icipe), Nairobi. The BSF larvae were reared in metallic trays using a rearing substrate hydrated to approximately 70 ± 1% moisture content, following procedures described by Beesigamukama et al. 18 . The frass obtained was composted inside a greenhouse using the heap method. During composting, frass heaps of 1 m height and 4 m long were built on surfaces lined with polythene sheets and hydrated to 55-65% moisture content. After 5 weeks, a mature and stable frass product was obtained and used in the experiments as BSFFF. Details of the entire composting process up to the compost maturity stage are described in our sister paper 21 .
The SAFI was sourced from Safi Organics Limited (http:// safio rgani cs. co. ke/) located in Mwea town, Kirinyaga County, Kenya. It was a mixture of composted chicken manure, biochar, and rock phosphate. Table 5 presents selected physical-chemical characteristics of the organic fertilizers used in the experiments.
Experimental set up. This study was carried out alongside agronomic experiments aimed at determining the nitrogen fertilizer equivalence value of BSFFF and synchrony of N release for maize production 21 . The BSFFF and SAFI were applied at rates of 0 and 5 t ha −1 , according to an organic fertilizer rate that had been previously used in central Kenya 31,57,64 . Thus, parallel experiments were carried out for a period equivalent to two maize cropping seasons (April-September 2019 and October 2019-March 2020) to investigate the ammonification and nitrification processes, rates of N mineralization and nitrification, and the quantities of nutrients released by soil amended with BSFFF and SAFI. www.nature.com/scientificreports/ Soil sampling and incubation. From each plot, the soil was collected from 0 to 20 cm depth before applying organic fertilizers. The soil was manually sorted to remove objects, stones, and clods bigger than 2 mm. The soil was then homogenized by hand mixing in a basin. The two organic fertilizers were separately mixed with the soil at the rate of 5 t ha −1 . The moisture content of the mixture (soil-organic fertilizer) was adjusted to 60% soil water holding capacity using distilled water. Two hundred grams of the mixture were placed in an air-permeable ziplock bag sealed to prevent water entry and nutrient loss 36,40 . The ziplock bags were buried at 10-20 cm depth in respective plots in the field. At the same time, 200 g of unamended soil from each plot (as control) were placed in ziplock bags and buried at the same depths (10-20 cm) in each of the respective plots. Five bags were randomly buried per replicate, giving a total of 15 bags per treatment at the beginning of each cropping season. The bags were retrieved at 15, 30, 70, 91, and 125 days of incubation corresponding to seedling, vegetative, tasseling, silking, and harvesting stages of the maize crop growth. The positions of the bags were marked using pegs to avoid disturbance during weeding and for easy retrieval. The retrieved bags were labelled on each sampling date, placed in airtight polythene bags, and carried in a cool box containing ice blocks to reduce microbial activities during transportation. The samples were used to determine soil mineral N (ammonium and nitrate) concentration, pH, fungi and bacterial populations, and other nutrient concentrations.
Nitrogen mineralization. The concentrations of nitrate (NO 3 -1 ) and ammonium (NH 4 + ) in unamended soil and soil amended with organic fertilizers were used to calculate N mineralization and nitrification rates at each sampling time using Eqs. (1) and (2) 40 . The nitrification ratio was calculated by dividing the concertation of ammonium (mg kg −1 ) at each sampling period by concentrating nitrate (mg kg −1 ) during the same period.
where, t i represents sampling times i = 0, 1, 2, 3, … t i + k is t i plus time k intervals where k = 1, 2, 3, 4,… Nutrient released by unamended soil and soils amended with organic fertilizers. The concentrations of nutrients (N, P, K, Ca, and Mg) released by unamended soil and soil amended with organic fertilizers at the maturity stage (125th day of incubation) of each cropping season were used to calculate the cumulative quantities of nutrients released throughout the maize growing season. The amounts of nutrients (N, P, K, Ca, and Mg) released were expressed on a kg per hectare basis using Eq. (3). The amount of N released was computed using the mineral N (ammonium and nitrate) data.
where, mass of soil layer kg = soil bulk density kgm −3 × volume of soil layer m 3 Laboratory analysis methods. The pH and electrical conductivity (EC) were determined using extracts of 1:10 and 1:2.5 (w/v) for organic fertilizer to distilled water and soil to distilled water, respectively. The contents were then shaken for 1 h, 180 revolutions min −1 , on an orbital and linear shaker (MI0103002, Foure's Scientific, Guangdong, China). Then, the pH and EC were read directly using a pH (AD1000, Adwa, Bucharest, Romania) and EC meter (AVI, Labtech, Mumbai, India), respectively Okalebo et al. 63 .
The mineral N (nitrate and ammonium) was extracted from organic fertilizers and soil using 0.5 M potassium sulphate at a ratio of 1:10 (w/v). The nitrate and ammonium concentrations in solutions after filtration were determined by colourimetric methods at 419 and 655 nm, respectively, as described by Okalebo et al. 63 . The populations of soil bacteria and fungi were determined by culturing using nutrient agar for bacteria and potato dextrose agar for fungi. The number of colony-forming units (CFU) per treatment were counted after 24-48 h, and data were expressed as CFU g −1 . Total organic carbon of organic fertilizers and soil was determined using the wet oxidation method 65 .
The total N, P, K, Ca, and Mg of organic fertilizers were extracted using the acid digestion method 63 . From this extract, total N, P, and K were determined using the Kjeldahl digestion and distillation method 66 , UV-Vis spectrometry 63 , and flame photometry 63 , respectively. The total Ca and Mg concentrations were determined using atomic absorption spectrometry (AAS) 63 at 422.7 and 285.2 nm, respectively (iCE 3300 AA system, Thermo Scientific, Shanghai, China). Available P and exchangeable Ca and Mg in soil were determined using Bray 2 63 and AAS, respectively, while exchangeable K was determined using flame photometry. Total N in soil was determined using Kjeldahl digestion and distillation method 66 , while soil texture and bulk density determined using the Bouyoucos and core sampling methods, respectively 63 . Data analysis. Before statistical analysis, data were tested for normality using the Shapiro-Wilk test. Analysis of variance tests was performed on data on soil pH and concentrations of ammonium, nitrate, nutrients (N, P,