The study was conducted in the Kadiogo region of Burkina Faso. This area is characterized by a Sudano-Sahelian climate, savanna vegetation, a dry season from November to May, and a rainy season from June to October with an average annual rainfall of 935 mm [16]. Jatropha curcas cake was collected from the Belwet industrial unit in Ouagadougou (12˚22'33.8"N - 1˚32'31.0"W), forest litter from the classified forest of Gonsé (12˚26'20"N, 1˚19'40"W), and millet bran. Water was collected from the Loumbila dam (12˚29'14"N, 1˚24'28"W), and the source of lactic acid bacteria was from the “La Vache Enchantée” dairy (12˚23'5"N - 1˚31'30"W).

The collection sites were chosen based on the absence of risk of chemical contamination that could inhibit microbial growth during fermentation. The Gonsé forest is classified and is characterized by an absence of human activities likely to generate chemical contamination. The Belwet unit extracts oil from J. curcas seeds using a pressing technique that does not involve the use of chemicals. The Faso cereal processing unit specializes in the processing of agricultural products without the use of chemicals. The “La Vache Enchantée” dairy specializes in the collection and sale of cow’s milk in the city of Ouagadougou. The Loumbila dam is a source of surface water used for drinking water production in the town of Ouagadougou, outside the influence of human and agricultural activities.

Forest litter is the basic material used in the production of FFL [13]. Jatropha curcas cake was used as a carbon source due to its high protein content. Fresh milk was used as a source of lactic acid bacteria to ensure the natural quality of the lactic acid bacteria source. Millet bran was used as a source of starch.

The process of fermented forest litter preparation was carried out in two successive fermentation stages [13]. The first stage is based on an anaerobic batch fermentation, and the second stage consists of activating the solid fermented mixture obtained from the first step by fermentation in a liquid medium. To this end, the various components were mixed in appropriate proportions (Table 1).

Table 1. FFL production substrates and their proportions.

Following a specific order and in a container disinfected with 70% alcohol, 200 g/L of forest litter was mixed with 270 g/L of Jatropha seed cake and 67 g/L of millet bran before adding a mixture of 67 ml/L of fresh milk and 370 ml/L of dam water (Figure 1).

For the activation step, the substrates used are solid LFF and sugarcane molasses, each at a concentration of 25 g/L, and dam water. Thus, to prepare 250 mL of activated LFF, 6.25 g of sugarcane molasses was mixed with 100 mL of dam water before adding 6.25 g of solid LFF. The reaction medium volume was composed of dam water. After homogenization, the bottle was tightly sealed and placed at room temperature for a one-week fermentation period. In accordance with the activation period, solid FFL was activated in seven (07) 250 mL bottles. Each day, one bottle was used to measure pH and electrical conductivity.

Figure 1. Order of mixing substrates for FFL production.

2.3.1. Sampling

Samples of NFS, the solid FFL, and the activated FFL were collected for analysis. For the microbiological analysis, 100 g of each sample was collected in a sterile bag and stored at 4˚C. The samples for chemical analysis (100 g) were taken and stored at −20˚C to avoid any biochemical activity that could modify the chemical parameters. For the activated FFL, 50 mL was collected in a sterile glass bottle and stored at 4˚C for microbiological analysis, and in a polyethylene bottle stored at −20˚C to determine the chemical parameters.

2.3.2. Chemical Analysis

The pH and the electrical conductivity of the samples were determined using the method described by Marois et al. [13]. Changes in pH and electrical conductivity were assessed during the seven-day FFL activation period. A sample/demineralized water suspension in a ratio of 1/5 (m/v) was analyzed using a portable pH/EC/TDS/temperature meter (Hanna instrument, Romania).

The organic carbon was determined using the organic matter incineration method [17].

The total nitrogen, total phosphorus, and total potassium of the samples were also determined using the Kjeldahl method [18]. Total nitrogen and phosphorus contents were determined by colorimetry using the SKALAR flow analyzer (segment flow analyzer, model SANplus 4000-02, Skalar Holland) [19]. Total potassium content was determined using a flame spectrophotometer (Jenway PFP7 flame photometer) [20].

The water-soluble nutrients were determined from a distilled water extract of NFS and FFL samples in a 1:20 (m/v) ratio [21]. From the extracts, nitrates were measured using a spectrophotometer at 410 nm [22]. Soluble phosphorus and ammonium were measured using the colorimetric method with the SKALAR automatic analyzer, and the total potassium was measured using a flame spectrophotometer.

2.3.3. Microbiological Analysis

The microbiological quality of NFS and FFL was assessed through the determination of total coliforms, thermotolerant coliforms, and Salmonella spp. Total and thermotolerant coliforms were determined using Chromocult Coliform agar [23]. Salmonella spp. were evaluated in accordance with AFNOR standard NF 08.052.

In addition, daily monitoring of lactic acid bacteria, yeasts, and molds was carried out during the activation of FFL.

To assess the phytotoxicity of activated FFL, crops commonly grown in Burkina Faso, such as maize (Zea mays), tomatoes (Solanum lycopersicum L.), and okra (Abelmoschus esculentus) were used [24].

The activated FFL is filtered using a 0.5 mm sieve, and the filtrate was diluted to concentrations of 2% and 5%, the concentrations commonly used in agricultural practices [9], for the germination test. A randomized complete block design was adopted and carried out under laboratory, day, and night temperature and light conditions based on three treatments with two (02) replicates each: activated FFL at 2%, activated FFL at 5%, and a sterile distilled water solution (control) [25]. Ten (10) seeds from each crop were placed in a 90 mm diameter Petri dish containing a Whatman paper disc. The dishes were watered every two (02) days with 5 ml of each irrigation solution for 07 days.

Germination rate (GR), radicle elongation (RE), and germination index (GI) were determined according to the following formulas.

A paired t-test, with a significance threshold of p < 0.05, was performed on Jamovi software to compare the means of the chemical parameters before and after fermentation. The relationship between the different parameters was established according to Pearson’s correlation. An ANOVA was performed using XLSTAT software version 2016.02.27444 at a significance level of p < 0.05 to compare the mean values of the germination indices and root elongation for the different dilutions of activated FFL for each species.

The chemical characteristics of the non-fermented substrate (NFS) and fermented forest litter (FFL) are shown in Table 2. The average electrical conductivity of NFS and FFL is 2.53 to 4.64 mS/cm and 6.38 to 4.36 for the pH, respectively. The paired t-test indicated that the variation in pH was not statistically significant (p = 0.072 > 0.05). At the same time, the variation in EC is statistically significant (p = 0.018 < 0.05). The paired t-test showed that these variations are not statistically significant.

The average organic matter and carbon content, as well as the average C/N ratio, changed from 766.229 to 736.644 (g/kg), from 445.482 to 439.277 (g/kg), and from 16.03 to 14.94. According to the paired t-test, only the variation in organic matter content was statistically significant (p = 0.042).

The average contents of ammonium, nitrate, soluble phosphorus, and soluble potassium ranged from 241.17 to 1948.48 (mg/kg), from 672.60 to 2003.25 (mg/kg), and from 4662.13 to 4864.83 (mg/kg). The t-test showed that the increase in ammonium and soluble potassium content was statistically significant (p = 0.004 for NH 4 + and p < 0.001 for K). The average nitrate content varied from 582.0 to 66.2 (mg/kg). The t-test showed that this decrease was statistically significant (p = 0.023).

The relationships between the different parameters are shown in Table 3. Positive and statistically significant correlations were found between ammonium content and EC (Pearson’s r = 0.994 and p = 0.004), nitrate content and pH (Pearson’s r = 0.996 and p = 0.004), and soluble phosphorus and ammonium contents (Pearson’s r = 0.965 and p = 0.035). The relationship test also showed a negative correlation between pH and EC (Pearson’s r = −0.996 and p = 0.004), pH and ammonium content (Pearson’s r = −0.990 and p = 0.010), between nitrate content and EC, and nitrate content and ammonium content (with Pearson’s r = −0.998 and p = 0.002), between soluble phosphorus content and pH, and soluble phosphorus content and nitrate content (Pearson’s r = −0.956 and −0.960 and p = 0.044 and 0.040, respectively).

Table 2. Chemical characteristics of the unfermented mixture and FFL.

Legend: NFS (Non-Fermented Substrate), FFL (fermented forest litter), E.C. (electrical conductivity), O.C. (organic carbon), C/N (carbon/nitrogen ratio), and O.M. (organic matter). NH 4 + (ammonium), P (phosphorus), NO 3 − (nitrate), K (potassium).

Table 3. Correlation matrix between the chemical parameters of solid FFL.

During the activation of FFL, the pH gradually decreased from 5.09 to 3.98 (Figure 2). The electrical conductivity increased slightly, from 3.52 to 3.77 mS/cm.

Figure 2. Evolution of pH and electrical conductivity during the process of FFL activation.

Figure 3. Evolution of pH and electrical conductivity during the process of FFL activation.

The results of microbiological analyses of NFS showed a total coliform concentration of 3.75 × 10⁵ CFU/g of substrate and no Salmonella. After fermentation, the results of microbial analyses showed a total reduction in the total coliform load and no Salmonella in FFL.

The results of the evolution in lactic acid bacteria, yeast, and mold loads are shown in Figure 3. These results show that on the first day of activation, no growth in lactic acid bacteria, yeast, or mold was observed. From 24 hours after activation until the last day, the concentration of lactic acid bacteria increased from 1.44 × 10⁸ to 4.5 × 10⁹ CFU/ml, and the concentration of yeast and mold varied from 3.7 × 10⁵ to 5.1 × 10⁴ CFU/ml.

Relationships between the parameters of lactic acid bacteria load and the chemical parameters of active FFL are shown in Table 4. A negative correlation is found between the evolution of pH during activation and changes in electrical conductivity (Spearman’s rho = −0.703, p = 0.039), as well as the evolution of lactic bacteria concentration (Spearman’s rho = −0.750, p = 0.033). These correlations suggest that the gradual decrease in pH during activation is associated with the fermentation activity of lactic acid bacteria. Indeed, the growth of lactic acid bacteria generates lactate, which decreases the pH.

Table 4. Correlation matrix between the parameters of lactic acid bacteria load and the chemical parameters of active FFL.

The results of the germination tests are reported in Table 5.Figure 4(A) shows the radicle elongation of the germinated seeds, and Figure 4(B) shows the germination index of the seeds. At a concentration of 2%, the activated FFL showed a germination rate of 95% for okra and tomato and 100% for maize. The average root elongation was 5.25 ± 0.04 mm, 3.15 ± 0.3 mm, and 12.67 ± 0.7 mm for okra, tomato, and maize, respectively. The germination index was 429% ± 29.7% for okra, 92.5% ± 1.6% for tomato, and 127.6% ± 8.93% for maize. At a concentration of 5%, the activated FFL showed a germination rate of 90% for okra, 95% for tomato, and 100% for maize. The root elongation was 8.2 ± 0.24 mm for maize, while no elongation was noticed for okra and tomato. The germination index was 82.24% ± 3.54% for maize.

After adjusting the acidity to the neutral pH (pH = 7), FFL diluted to 2% showed a germination rate of 85% for okra, 95% for tomato, and 100% for maize. The average root elongation was 6.4 ± 0.19 mm for okra, 5.2 ± 0.07 mm for tomato, and 16.3 ± 0.47 mm for maize. The germination index was 465% ± 0.35% for okra, 151.8% ± 13.14% for tomato, and 164.5% ± 7.09% for maize. FFL diluted to 5% showed a germination rate of 100% for okra, 95% for tomato, and 100% for maize. The average root elongation was 7.1 ± 0.47 mm for okra, 2.9 ± 0.11 mm for tomato, and 13.6 ± 0.35 mm for maize. The germination index was 612.63% ± 22.83%, 85.5% ± 8.31%, and 136.73% ± 1.61% for okra, tomato, and maize, respectively.

Figure 4. Root elongation of seedlings (A) and seed germination index (B).

With the control, the germination rate was 95% for okra and 100% for tomato and maize. The root elongation was 1.23 mm, 3.23 mm, and 9.93 mm for okra, tomato, and maize, respectively. The germination index was 100% for all crops. The comparison of the germination indices showed a significant difference between the different FFL solutions and the control for okra seeds (0.01 < p < 0.001). For tomato, only the germination index of the 2% FFL solution at neutralized pH showed a significant difference compared to the control (p = 0.004). For maize, the 2% FFL solution at neutral pH and the 2% solution at acidic pH showed a significant difference compared to the control (p = 0.019 and p < 0.001). No significant difference was observed between the seeds’ germination rate in the controls and the values obtained when the seeds were watered with the various activated FFL solutions (Table 6).

Table 5. Root elongation and germination index (A: acidic; NA: non-acidic).

Table 6. Correlation between the parameters of lactic acid bacteria load and the chemical parameters of active FFL.

The results showed a non-significant decrease in the pH and a significant increase in the electrical conductivity following the fermentation of the substrates. Marois et al. [13] reported a decrease in pH and an increase in electrical conductivity after fermentation of FFL based on forest litter from two different climates. In addition, the results revealed a negative correlation between pH and conductivity. This negative correlation indicates that an acidic pH promotes the mineralization of the organic matter and the release of ions (phosphate ions, potassium ions, ammonium ions, bicarbonate ions, etc.) by the endogenous microorganisms during the fermentation process. This release of ions increases the electrical conductivity. Indeed, Ajaweed et al. [26] reported that a high ion concentration leads to an increase in electrical conductivity. The decomposition of organic matter by endogenous microorganisms, which can lead to increased electrical conductivity, contributes to making nutrients available to plants [27]. However, increased electrical conductivity can lead to increased salinity, which would negatively impact nutrient absorption by plants. Indeed, Nikiema et al. [28] reported that when it exceeds 4 dS/m, electrical conductivity can reduce plants’ ability to absorb water. The EC value obtained with the FFL (4.64 mS/cm) was roughly equal to this threshold value. Adverse effects on soil physicochemical properties and soil microbial community metabolism associated with high soil salinity have been reported [29]. Diluting activated FFL before application can help mitigate the adverse effects of high salinity. The production of organic acids by lactic acid bacteria can lead to a decrease in pH in the fermentation medium. Lowering the pH has many advantages for FFL. An acidic pH during fermentation promotes the elimination of pathogenic microorganisms by weakening the cell wall and inhibiting several metabolic activities. In addition, a decrease in pH due to the production of organic acids promotes the solubilization of nutrients like inorganic phosphates by chelating metal cations [30]. The chemical composition shows that the average total nitrogen, total phosphorus, and total potassium contents decreased from 30.30 to 30.21 (g/kg), from 5.05 to 4.73 (g/kg), and from 14.60 to 15.54 (g/kg), respectively; but these variations are not statistically significant. These results show that the fermentation did not have any significant effect on the total nitrogen, total phosphorus, and total potassium. Organic matter, organic carbon, and C/N ratio content decreased by 29.585 (g/kg), 6.205 (g/kg), and 1.09, respectively, but not statistically significant. However, the NPK content of FFL may contribute to enhancing its fertilizing capacity. The organic matter content (75.5%) and C/N ratio of the solid matter of the produced FFL show that it is of good quality for composting [26]. For the water-soluble nutrients, the fermentation significantly increased the ammonium and the soluble potassium contents, representing 1707.31 (mg/kg) and 202.70 (mg/kg), respectively. The nitrate content decreased, corresponding to a decrease of 516.0 (mg/kg). The increase in ammonium content could result from the mineralization of organic nitrogen during the fermentation process. Indeed, Chen et al. [31] reported that during the degradation of organic matter, certain microorganisms convert organic nitrogen (osamines and amino acids) into ammonium. The high ammonium concentration at the end of fermentation could be explained by the high protein content of the cake, coupled with the high enzymatic activity of microorganisms capable of degrading organic nitrogen. In fact, Jatropha seed cake has a protein content of 57.3 to 64.4% [32]. On the other hand, the loss of nitrate can be explained by denitrification during the fermentation process, a phenomenon in which nitrates are converted into molecular nitrogen under anaerobic conditions by denitrifying bacteria. Mahmoud et al. [33] reported a very significant loss of nitrate during anaerobic fermentation in connection with the addition of organic matter to the fermentation medium. Ammonium and nitrate are the forms of nitrogen that can be assimilated by plants. A mixed supply of ammoniacal nitrogen and nitrates is very favorable for nitrogen absorption by plants, which improves photosynthesis and carbon metabolism [34]. Ammonium, which is present in high concentrations in the developed FFL, could be converted into nitrate in the presence of oxygen, which would help regulate the NH 4 + / NO 3 − ratio. The increase in soluble potassium content could result from the endogenous microbial activity of the biomass mixture. Microorganisms mineralize organic matter and convert the potassium present in the medium into a water-soluble form [35]. The increase in soluble phosphorus content after fermentation is not statistically significant but could be explained by the mineralization of organic phosphorus during fermentation by certain microbial groups [36]. Organic phosphorus is converted into water-soluble forms such as hydrogen phosphate (HPO₄) and phosphoric acid (H₂PO₄) by biological means so that it can be assimilated by plants. Positive correlations were observed between ammonium content and electrical conductivity, and between soluble phosphorus content and ammonium content. The positive correlation between ammonium content and electrical conductivity may indicate that as the ammonium content increases, the soluble ion content also increases. The positive correlation between ammonium content and soluble phosphorus content may indicate simultaneous release of these elements following mineralization. Ammonium and soluble phosphorus contents were negatively correlated with pH. These correlations show that acidic conditions promote the availability of ammonium and soluble phosphorus. This justifies biological activities such as fermentation and the degradation of organic matter by the endogenous microbial community. The developed FFL showed better levels of soluble elements (ammonium, soluble phosphorus, and soluble potassium) after the fermentation. These elements are the main nutrients for plants. Additionally, the microorganisms responsible for the various changes during fermentation may have several properties that help plants grow. Furthermore, FFL supplemented with Jatrophaseed cake exhibited higher electrical conductivity compared to FFL supplemented with wheat bran produced by Marois et al. [13]. The FFL supplemented with Jatropha seed cake may be richer than the FFL supplemented with wheat bran.

The prepared FFL is characterized by the total absence of coliforms and Salmonella. Valdes et al. [37] reported the absence of coliforms and Salmonella in fermented forest litter in Cuba. The total reduction in coliform concentration may be related to the acidic pH of the medium, as well as the chemical composition of the medium. These results suggest that natural selection occurred during the fermentation. This demonstrates the safety of fermented forest litter and guarantees its sanitary quality.

The activated FFL showed a high concentration of lactic acid bacteria compared to yeasts and molds. The presence of lactic acid bacteria in high concentration, yeasts, and molds in activated FFL is a key factor in FFL’s effectiveness as a biofertilizer. Thanks to their metabolic capacity, these microbial groups have many properties that promote plant growth. The mechanisms of the plant growth-promoting properties of lactic acid bacteria are based on the degradation of organic matter, the production of growth hormones, bioactive molecules, and other secondary metabolites [38].

Furthermore, the presence of these microorganisms may explain the changes observed in the physicochemical characteristics of the FFL.

A comparison of the different FFL solutions and the control showed a significant difference between the germination indexes for okra. For tomato, only the germination index of the 2% FFL solution at neutral pH showed a significant difference compared to the control. For maize, the 2% FFL solutions at acidic and neutral pH showed a significant difference compared to the control. For all crops, there was no significant difference between the seed germination rate of the controls and the germination rate of seeds watered with the various activated FFL solutions. According to Finch-Savage & Footitt [39], temperature, humidity, light, and oxygen are factors that can influence seed germination by modulating enzyme activity during the germination process. At acidic pH, only 2% FFL improved the average root elongation and the germination index compared to the control for okra and maize seedlings. At a concentration of 5%, activated FFL inhibits the development or causes root rot in seedlings, resulting in a germination index of zero. Our results show that at a concentration of 2%, active FFL improves the germination index of okra, compared to the control. For tomato and maize, at the same concentration, activated FFL only improved the germination index after adjusting the acidity of the solution to neutral pH. At a concentration of 5%, activated FFL only improved the germination index of okra after adjusting the acidity to neutral pH. Given that the germination index was determined based on the germination rate and root elongation, the active FFL solutions used had effects on root development. These effects may be related to the salinity, the pH, ammonium content, etc., of the activated FFL solutions used. However, no statistically significant correlation was observed between pH and germination index or root elongation. Milon et al. [40] reported that acidity can hurt root growth. This acidity alters the roots’ ability to absorb nutrients and thus reduces root elongation. The effect of active FFL solutions may be mainly based on chemical characteristics other than pH. According to the work of Kong et al. [41], electrical conductivity, organic carbon, dissolved nitrogen, ammonium content, potassium, zinc, and copper content are factors that can negatively influence the germination index. In our study, the chemical characteristics of the FFL solutions used were not determined. In fact, laboratory germination tests have shown promising results. However, the approach used in Petri dish experiments does not necessarily reflect performance in complex soil conditions.