Preparation and Characterization of Liquid Fertilizers Produced by Anaerobic Fermentation

Abstract

Biol is a liquid product, obtained by anaerobic fermentation of local inputs, which improves the health of agroecosystems, which is an emerging area in agronomy. The aim of this study consists of the preparation of two biols from inoculums of cow dung (BCD) and native forest duff (BNF) by using specific biodigesters and commercial inputs. The biol characterization was made in terms of mineral (ionic and complex forms), amino acids, hormones and volatile compounds, along with Pfeiffer circular chromatography during fermentation monitoring. The results showed a pH acidic in both biols (4.5–5.5), which is higher for BCD. Also, this biol had higher content in several macro- and micronutrients in ionic (nitrates, phosphates, calcium, iron and sodium) and complex forms (calcium, iron and potassium). Both have interesting content in amino acids and hormones. The absence of microorganisms in the final products could be due to the presence of volatile compounds such as pyrazines and sulfoxides. Along with this, other volatile compounds such as esters were identified, which can be responsible for their pleasant odor. The novelty of this work is to provide a protocol for obtaining biols and to demonstrate their potential to be used as biofertilizers.

1. Introduction

Intensive food production for animal and human consumption, for which conventional agricultural systems have been adopted, has led to the haphazard and promiscuous use of agrochemical products, generating several negative and dangerous effects for the agroecosystem, including the conservation of the biodiversity connected to these agricultural systems [1]. Currently, chemical fertilizers are applied excessively to provide the plant nutrient requirement for increasing agriculture productivity worldwide, as part of the high-input farming systems. However, their application is often characterized by low nutrient use efficiency, resulting in substantial nutrient loss and environmental degradation, thereby compromising their economic viability [2]. These pollution problems leading to public health hazards necessitated the development of technologies that are sustainable and eco-friendly, which could reduce the application of synthetic fertilizers [3]. To overcome the side effects of inorganic fertilizers on the soil, the application of organic manures is highly recommended and, in addition, the application of fermented liquid bio-formulations [4], as many of these products are made with cultures of microorganisms [5].

The use of liquid ferments in small-scale agriculture occurs worldwide, with different regional and historical influences [6]. These preparations have roots in South Asia, with dung-, milk- and fish-based preparations described in Vedic texts dating back to before 1000 b.c. [7]. It was during the 1990s that the use of liquid fertilizers exclusively as agronomic inputs to support small producers gained adoption by the implementation of development projects carried out by non-governmental organizations (NGOs) worldwide. Currently, liquid fertilizer based on manure, also known as Supermagro (SUP), is one of a range of biologically made fertilizers, the use of which is being promoted in developing countries around the world. The Food and Agriculture Organization (FAO) of the United Nations (UN) describes SUP as a “biofertilizer enriched with mineral salts” developed by Delvino Magro in Argentina, used to address micronutrient deficiencies and crop pests and diseases [8]. In Brazil, the Ministry of Agriculture, Fisheries and Food produced a leaflet describing the manufacture and use of SUP [9], where a mixture of mineral and organic fertilizers is defined by law as an organo-mineral fertilizer [10]. Later, Pinheiro and Restrepo provided tuition in the preparation and use of biofertilizers around the world, including the publication in 2007 of a manual [11], which includes other techniques such as liquid fertilizers based on forest litter microbiomes. Currently, these techniques are getting recognition and adoption by farmers worldwide. However, scarcely published research has been developed on the preparation and characterization of these liquid fertilizers.

These liquid fertilizers are made using anaerobic fermentation, mainly through an inoculum, milk, a carbon source such as molasses or water and supplemented with inorganic minerals. The acidic conditions created during fermentation assist in the extraction of minerals, which can be in solution and suspended solids [12]. Moreover, anaerobic fermentation can produce phytohormones [13] and inhibitors of plant pathogen infection [14]. To understand the properties of liquid ferments, it is necessary to know the role of microorganisms and the transformations that occur during anaerobic digestion, which is a complex process from a microbiological point of view, where the decomposition of organic matter results in the production of inorganic compounds, such as CO₂, CH₄, N₂ and NH₃, some of which leave the ferment in gaseous form, giving rise the biogas production. Therefore, when this transformation takes place, part of the gaseous nutrients is released, but others can be concentrated such as phosphorus and potassium, among others. The steps of anaerobic digestion are well studied and are a useful guide to understanding liquid ferments, including those originating from small batches made on farms [6]. Anaerobic digestion is carried out in three main stages: (1) hydrolysis, in which the organic matter is broken down by the action of a group of anaerobic bacteria that hydrolyze water-soluble molecules, such as fats, proteins and carbohydrates, and transform them into monomers and simple soluble compounds; (2) acetogenesis and dehydrogenation, where alcohols, fatty acids and aromatic compounds are degraded to produce acetic acid, CO₂ and hydrogen, which are the substrates for the bacteria methanogenic; and (3) methanogenic, where methane is produced from CO₂ and hydrogen, from the activity of methanogenic bacteria [15].

Since there is limited research about biols, it is interesting to introduce these practices into academic contexts to study their advantages on agroecosystem health in a “good agricultural practices” scenario as an alternative to conventional agricultural practices, which are used broadly at present. For this reason, the present work has been undertaken with the aim of providing new knowledge on the composition of two standard biols, produced according to the criteria recommended to small-holding farmers in rural areas, using homemade biodigesters. For this purpose, cow dung and leaf litter inoculums will be used and, to standardize the results, the local inputs (commonly used by farmers) have been replaced by commercial products of known composition. To achieve this, characterization of the fermented liquids has been performed in terms of their quantitative (physical, chemical and biological analysis) and qualitative (Pfeiffer’s circular chromatography) composition. Pfeiffer’s circular chromatography is a physical method of separating the different components that characterize complex substances and, at the same time, provides an integrated analysis of the quality rather than numeric values [16]. Although the procedure for obtaining a chromatogram is well described in soils [11], a robust and standardized procedure for the interpretation of biols is scarce [17].

For all these reasons, and in view of the lack of enlightening information on the preparation and composition of the two main types of biols used in the context of traditional and environmentally friendly agriculture, this work is proposed.

2. Materials and Methods

2.1. Study Localization

The preparation and fermentation process of the liquid fertilizers was carried out at “Norivia’s farm” in Valdepeñas, Spain, between December 2023 and January 2024. The place is located at coordinates 38°47′38.7″ N, 3°17′17.7″ W and 796 m above sea level. The climate of the area according to the Köppen–Geiger classification [18] is Csa type, i.e., a Mediterranean climate with cold winters (reaching −4 °C) and hot, dry summers (temperatures over 40 °C), with frequent rainfall in autumn and spring (470 mm³).

2.2. Biodigester Design

Fermentations were prepared under controlled conditions in two specific biodigesters according to Hernández-Chontal [19] and Restrepo [20], with some modifications. Briefly, tanks of 220 L of high-density polyethylene (PET, diameter of 590 mm, height of 975 mm, lid diameter of 460 mm and inner mouth of 410 mm) with lids were used. The lids were connected to a hose to condition the exit of gas and sealed at the exit to avoid leaks. The hose was finally led to the escape valve using a 1 L PET bottle, filled three quarters with water. Perfect sealing of the gas outlet is necessary, as well as the monitoring of the exhaust valve, always taking care to contain the water. In the exhaust valve with water, the gas is released safely when there is high pressure inside the digester. To obtain the liquid fertilizer samples, a tap was connected to the side of the tanks, avoiding the entrance of oxygen and maintaining anaerobic conditions inside the biodigesters.

To maintain the fermentation temperature (35 ± 1 °C), heating blankets (HPD, Noxman, Granada, Spain) covering the entire surface of the biodigesters were used, keeping the environment protected from the outside.

2.3. Components of Biols

2.3.1. Common Components

The common components used for the preparation of biols were water, milk, basaltic rock dust and sugar cane molasses. Water, which comes from the well of the plot previously indicated, and the initial chemical composition, are summarized in Table S1. Milk from cow was used and its composition is summarized in Table S2. Basaltic rock dust was supplied by Ecoforte S.L. (Alicante, Spain) and its composition is included in Table S3. Beet molasses was supplied by Poballe (Barcelona, Spain) and its composition can be observed in Table S4.

The following chemical components were used: zinc sulfate heptahydrate (99.5% pure), magnesium sulfate heptahydrate (99% pure), manganese (II) sulfate monohydrate (98% pure), sodium molybdate dihydrate (99.5% pure), iron (II) sulfate heptahydrate (99% pure), copper (II) sulfate pentahydrate (98% pure) and potassium sulfate (99% pure), supplied by Scharlau (Sentmenat, Barcelona, Spain); cobalt (II) chloride hexahydrate (98% pure), supplied by Sigma-Aldrich (Darmstadt, Germany); calcium chloride (95% pure), supplied by ChemLab (Zedelgem, Belgium); boric acid (99.8% pure), supplied by Honeywell (Charlotte, NC, USA); and calcium anhydrous phosphate (96% pure), supplied by Thermo Fisher Scientific (Waltham, MA, USA).

As a starter of the anaerobic fermentation, a commercial yeast (Mondelez International, Chicago, IL, USA) was used.

2.3.2. Specific Components: Microbe Inoculums

Two inoculums were used on each biol and obtained from two different sources: (1) from the leaf litter of the forestry Quercus environment (hereinafter referred to as native forest duff inoculum, NFI); and (2) from the fresh cattle manure that is antibiotic-free (hereinafter referred to as cow dung inoculum, CDI).

The NFI was obtained by mixing homogeneously 40 kg of cereal bran and 20 kg of local forest duff with 12 L of molasses and 5 L of water, to reach a final moisture content of approximately 40%, and then leaving it in anaerobic conditions above 30 days according to Dantinne [21] and Restrepo [20]. CDI was obtained by filtering fresh cow dung (free antibiotics) from a cattle farm nearby, according to Restrepo [20].

2.4. Preparation and Fermentation Process of Biols

Two different procedures were followed in the preparation of the biols according to Restrepo [20], and depending on the two inoculums used.

2.4.1. Biol from Native Forest Duff (BNF)

BNF was prepared with NFI added at once to the mixture of components shown in Table S5, and then the mixture was stirred and left in anaerobic conditions until the fermentation process was finished.

2.4.2. Biol from Cow Dung (BCD)

BCD was prepared with the CDI added every three days to the mixture of components shown in Table S6, and then the mixture was stirred and left in anaerobic conditions until the fermentation process was finished.

2.4.3. Fermentation Monitoring Parameters

The monitored parameters during the biols fermentation were pH, conductivity (mS/cm), total dissolved solids (ppt) and °Brix, measured with the multiparameter instrument HI98195, software version 1.08 of Hanna Instruments© (Gipuzkoa, Spain). Monitoring was performed every two days during fermentation and every three days during biofertilizer stabilization.

Also, Pfeiffer circular chromatography was measured according to the methodology proposed by Restrepo and Pinheiro [11]. Whatman round filter papers N° 41 with a diameter of 150 mm (Cytiva, Shanghai, China) were used to obtain the chromatograms. Briefly, pure liquid fertilizers were pushed with a solution of sodium hydroxide (1%) through the round filter paper that has been treated with silver nitrate (0.5%). The liquid fertilizer and the sodium hydroxide were poured into two different Petri dishes and drawn up: first, the liquid fertilizers and then, the sodium hydroxide through a wick inserted through the middle of the filter paper. The different elements in the liquid fertilizer were soaked up by the paper at different rates through capillary action, resulting in distinctive patterns.

2.5. Analytical Methods

The analyses carried out in this research, except for volatile compounds determination, were conducted by the AGRAMA laboratory (Seville, Spain), according to the ISO standards [22].

2.5.1. Organic Matter

Organic matter was analyzed by calcination, attending to the Official Methods of Analysis of the Ministry of Agriculture, Fisheries and Food of Spain (MAPA, 2024). Carbon was analyzed by dry combustion [23].

2.5.2. Mineral Composition

Chlorides, fluorides, nitrates, nitrites, phosphates and sulfates were analyzed using ion chromatography (HPLC-IC) according to the method proposed by Amin et al. [24]. Borates, calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, potassium, sodium and zinc were determined by inductively coupled plasma–optical emission spectrometry (ICP-OES) according to the method established by UNE standards [25].

2.5.3. Amino Acids

Amino acids were analyzed using HPLC MSMS QqQ Agilent equipment (Agilent Technologies, Palo Alto, CA, USA) according to the method established by Han et al. [26].

2.5.4. Plant Hormones

Auxins (abscisic acid, indole-3-acetic acid and 1-naphthylacetamide), cytokinins (kinetin), gibberellins (gibberellic acid) and salicylic acid were analyzed by using HPLC MSMS QqQ Agilent equipment (Agilent Technologies, Palo Alto, CA, USA) according to the method established by Hou et al. [27].

2.5.5. Microbiome: Metagenomic Analysis

The methodology used in the metagenomic analysis of microbial populations was using a MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, United Kingdom) according to Morrison et al. [28] and Benítez-Páez et al. [29].

2.5.6. Microbiological Count

At the end of the production of the biols, plate counts of the colony-forming units were made at 37 °C, both for aerobic and anaerobic organisms.

2.5.7. Determination of Volatile Compounds by HS-SBSE-GC-MS

Wine volatiles were determined according to the methodology of Sánchez-Gómez et al. [30]. The extraction of these compounds was carried out using Stir Bar Sorptive Extraction of PDMS coating (10 mm length; 0.5 mm film thickness). Wines were stirred at 500 rpm for 60 min at room temperature (21 ± 3 °C). After the extraction period, Stir Bars were washed with distilled water and dried with tissue. Analysis was performed using an automated thermal desorption unit (TDU, Gerstel, Mülheim and der Ruhr, Germany) mounted on an Agilent 7890A gas chromatograph (GC) system coupled to a quadrupole Agilent 5975C electron ionization mass spectrometric detector (MS, Agilent Technologies, Palo Alto, CA, USA). The GC system was equipped with a fused silica capillary column (BP21 stationary phase; 30 m length; 0.25 mm I.D.; and 0.25 μm film thickness) sourced from SGE (Ringwood, Australia). Helium was employed as the carrier gas, maintaining a constant column pressure of 20.75 psi.

The Stir Bars were thermally desorbed within a helium carrier gas, flowing at a rate of 75 mL/min with the TDU programmed to increase from 40 to 295 °C (maintained for 5 min) at a ramp rate of 60 °C/min at splitless desorption mode. The analytes were concentrated within a programmed temperature vaporizing injector (PTV) (CIS-4, Gerstel, Mülheim, Germany), incorporating a packed liner (20 mg tenax TA), held at −10 °C with cryo cooling prior to injection. Following desorption and concentration, the CIS-4 was programmed from −10 °C to 260 °C (held for 5 min) at a ramp rate of 12 °C/min to transfer the captured volatiles onto the analytical column. The GC oven temperature was programmed to 40 °C (held for 2 min), gradually increased to 80 °C (5 °C/min, held for 2 min), further elevated to 130 °C (10 °C/min, held for 5 min), then to 150 °C (5 °C/min, held for 5 min) and, finally, to 230 °C (10 °C/min, held for 5 min). MS analysis was performed using scan acquisition (m/z 27–300) with an ionization energy of 70 eV. The temperature of the MS transfer line was maintained at 230 °C. MS data acquisition was operated in positive scan mode to avoid matrix interferences, and the MS quantification was executed in single-ion-monitoring mode using the characteristic m/z values of each compound. Detailed information regarding the analyzed compounds and their corresponding m/z vales can be found in the work of Sánchez-Gómez et al. [30]. Compound identification was achieved using the NIST library. The internal standard used was 3-methyl-1-pentanol. All analyses were performed in triplicate.

2.6. Statistical Analysis

Statistical analyses were performed with the Statgraphics Centurion statistical program (version 19.4.02; StatPoint, Inc., The Plains, VA, USA). The data analysis was examined using one-way analysis of variance (ANOVA) at a 95% probability level, according to Tukey’s HSD test, to determine the differences between biols.

3. Results and Discussion

3.1. Biols Preparation and Evolution

Table 1. Metagenomic analysis of microbial populations (fungi and bacteria) in the two inoculums.

Biological Function	Number of Species (×105)
	NFI	CDI
Bacteria producing siderophores	76.62	99.51
Biocontrol	72.44	29.52
Exopolysaccharides	71.13	43.18
Nitrogen fixation	52.37	72.82
Pathogenic bacteria to crops	74.07	12.76
Phosphate solubilizing bacteria	0.49	120.34
Phytase production	1.16	11.33
Phytohormones	74.10	29.73
Potassium-solubilizing bacteria	71.96	115.10

NFI: inoculums from native forest duff; CDI: inoculums from cow dung.

summarizes the metagenomic results of the inoculums used in this study: from native forest duff (NFI) and from cow dung (CDI), where the species have been classified by their biological function, considering that some of them have been included in different groups. The total microbial community in CDI was composed of 1890 genera and 8120 species and a total of 1275 genera and 4103 species were found on NFI. Two indices were calculated from the data: Shannon’s and Simpson’s. The first one indicates the heterogeneity of a community based on two factors (the number of species present and their relative abundance) and the second one is the dominance. For the first, in which a value less than 2 is considered low heterogeneity, both inoculums, with values of 0.67 for NFI and 1 for CDI, showed low heterogeneity (few species and low relative abundance). According to Simpson’s index, which varies between 0 and 1, being 0 the maximum dominance of species over others and 1 minimum, NFI and CDI show a high dominance in genera/species with values of 0.65 and 0.68, respectively.

Within the total number of species of each inoculum, the group of siderophore-producing bacteria was more numerous in the CDI than in NFI (Table 1). With respect to the species associated with biocontrol, on NFI, the number was higher than twice in CDI, representing 8.01% and 0.67% of the total, respectively. The exopolysaccharides production bacteria were higher on NFI, which belong to 8.12% within the total of bacteria, and 0.86% for CDI. The species of microorganism directly related to fertilization (nitrogen fixation, phosphate and potassium-solubilizing bacteria) were higher on CDI, with a total amount of 308.26 × 10⁵, rather than the 124.82 × 10⁵ observed in NFI. These results suggest a greater fertilizing capacity of CDI compared to NFI, especially in producing phosphates that could be assimilated by plants. The number of species that produce phytases on CDI, was above that in NFI, whereas for bacteria pathogenic to crops and phytohormones, production was higher on NFI.

The dominant species on CDI were Bacillus spp. and Pseudomones spp., as previously reported by some authors [31]. However, for NFI, Pseudomonas spp. was the majority species. The diversity and total amount of microorganisms on NFI could suggest the microbial richness of the forest duff, where the inoculum comes from, in this case, Quercus forest [32]. Bacillus subtilis strains isolated from CDI had several beneficial attributes, which included biocontrol, plant growth promotion, sulfur oxidation, phosphorus solubilization and production of industrially important enzymes (amylase and cellulase) [33]. Pseudomonas, for its part, possesses many traits to act as a biocontrol agent and to promote the plant growth ability [34]. About the species associated with stress adaptation, phytohormone production and biocontrol functions were higher in NFI than in CDI, according to previous studies [35]. Previous research has shown the antifungal and antiseptic properties of fresh cow dung and urine in nature, which might be due to the secretion of antimicrobial metabolites by cow dung microflora [36].

The evolution of the fermentation process to produce bios is represented in Figure 1, where the pH and °Brix are shown. The duration of this process was 84 days for cow dung biol (BCD) and 24 days for native forest duff biol (BNF). The longer period on BCD than BNF is an indicator of an optimum anaerobic digestion step. Although the fertilizer quality depends on the inputs with different physical and chemical characteristics [37], another indicator of quality is a proper fermentation process. In this line, an optimal period could be considered for 40 days according to Oliveira et al. [38], since this time allows hydrolysis, acidogenesis and methanogenesis processes for a complete fermentation process. de Oliveira Neto et al. [39] point out that fermentation period is an indicator of the quality of the biol produced, and, also, the greatest availability of nutrients occurs along this process. In the present study, fermentation was carried out for 24 and 84 days on BNF and BCD, respectively, so it could be established better quality for BCD than BNF. The nature of the different inoculums and the procedure when making biols may be the reason for the longer fermentation period in BCD. Fermentations are highly dependent on the chemical composition of the organic material [40], so a greater number of species on CDI (Table 1) and the different procedures of the BCD (Table S6), suggest the longer period in the fermentation in this biol (Figure 1).

Regarding pH, it is observed that the average value was higher in biol from native forest duff (BNF) than in biol from cow dung (BCD) at the end of fermentation, with a slight decrease at the beginning of the fermentation, probably due to the accumulation of volatile fatty acids, which have deleterious effect in the anaerobic digestion process [41]. Regarding the °Brix, the mean values at the end of the fermentation were lower in BCD compared to BNF. It is worth highlighting a different behavior during the fermentation process, since in BCD the addition of components was progressive, which did not take place in BNF, causing a sharp decrease in the °Brix. At the end of the addition, values were reached that remained constant until the end of the fermentation. Visually, this process can be monitored in numerous ways, odor indicates microbial processes (ranging from sour to rancid) and color change has also been used to assess fermentation [6]. On BNF and BCD, once they were stabilized, bad odors were eliminated, suggesting successful fermentation.

Also, for the biols fermentation evolution, Pfeiffer’s circular chromatography was realized (Figure 2). For a correct understanding of the information on the circular chromatograms, Figure S1 shows the identification of the different zones of a characteristic chromatogram of a biol, where the shapes and colors of the different rings (a total of five) are related to the presence of different organic components in the biols. This chromatography technique allows constant monitoring of the progress of fermentation and provides diagnosis and useful information on the physical, chemical and biological characteristics along the production process of liquid fertilizers. The different characteristics and patterns to consider in the chromatograms to interpret the results are as follows: (1) the colors and their development; (2) the characteristics of the different zones or rings formed; and (3) the presence and characteristics of the strips and channels (Figure S1). For biol from native forest duff (BNF), one chromatogram is shown at the beginning of fermentation, another at the end and the last once the biol has been stabilized (Figure 2). In this Figure, for biol from cow dung (BCD), one chromatogram is shown at the beginning of the addition of the components, another at the beginning of fermentation and another at the end of it. The chromatograms for both biols at the end of fermentation show that they were “integrated”, since the diversification of colors from the center (amber, orange) to the outer ring (darker) means the diversity of compounds and, depending on their nature, from inorganic compounds in the center (such as minerals, mainly the different forms of nitrogen) to organic compounds towards the outer ring. These different rings have more channels and sinuous bands than at the beginning. The presence of dark areas suggests a diversity of compounds such as enzymes, hormones and organic acids, among other compounds produced during fermentation [11].

3.2. Biols Characterization

Table 2. General parameters, organic richness and count of microorganisms of biols from native forest duff (BNF) and from cow dung (BCD) at the end of their production.

	BNF	BCD	Fp-value
General Parameters
°Brix	9.4 ± 0.21 a	9.4 ± 0.50 a	0.00
Conductivity (mS/cm)	31.54 ± 0.52 a	32.76 ± 0.98 a	3.63
pH	5.26 ± 0.09 b	4.47 ± 0.08 a	129.12 ***
TDS (ppt)	22.10 ± 0.36 a	22.94 ± 0.69 a	3.49
Organic Matter and Microorganisms
Carbon (%)	2.82 ± 0.32	2.56 ± 0.29	1.09
Organic matter (%)	4.87 ± 0.52	4.41 ± 0.47	1.29
Colony-forming units/g #	<1.0 × 101	<1.0 × 101	0.00

TDS = total dissolved solids. The mean values (n = 3) are shown with their standard deviation. For each parameter, different letters indicate significant differences between both biofertilizers according to Tukey’s HSD test (*** p value < 0.001) and typed in bold. ^# Total count of aerobic and anaerobic microorganisms at 37 °C.

shows the general parameters, organic richness and colony-forming units in biol from native forest duff (BNF) and in biol from cow dung (BCD) at the end of their production. The general parameters measured were °Brix, conductivity, pH and total dissolved solids (TDS). Only for pH, significant differences were observed among the two biols, which is higher in BNF, as expected. These pH values were within the acid ranges in these types of anaerobic liquid fertilizers (3.7–5.9) [19]. The values of °Brix were also within the range previously reported by Orellana et al. [42] for this type of preparations and input used (around 8.0 °Brix). The conductivity in BNF and BCD was higher than reported previously by other authors [13,19,42], which could be due to the different inputs added, mainly the chemical components. Measuring conductivity could also be used to assess how much to dilute liquid ferments prior to addition to crops [43]. For TDS, since the inputs were the same (Tables S5 and S6), a similar value was expected in both biols. The % carbon was similar on BNF and BCD and for % organic matter. These results agree with other previous studies where a liquid fertilizer was prepared using cow dung inoculums [19], but lower than those reported by Cano-Hernández et al. [13], and higher than those reported by Criollo et al. [44], which suggests that this is due to the different nature of the inoculums used. Finally, in BNF and BCD, the number of colonies forming units was lower than the quantification limit of the method. Mei et al. [45] suggested that microbes introduced into digests are generally consumed during fermentation, and microbial diversity tends to decrease. The composition of microbes varies greatly among different anaerobic digests, especially based on inputs [46], but mainly on the nature of inoculums; liquid manure-based ferments can contain thousands of different microbial species [31,47]. The previous finding in the biols can assume that the presence of E. colli and Samonella were within the safety ranges on BCD and BNF. The previous favors the elimination of coliforms [48], as fermentation may also reduce their abundance [49].

The mineral content of the two biols at the end of their production is summarized in

Table 3. Mineral content (mg/L) of the two biols: from native forest duff (BNF) and from cow dung (BCD) at the end of their production.

	BNF	BCD	Fp-value
Anions
Borates	120 ± 8.0 a	110 ± 7.4 a	2.50
Chlorides	7114 ± 948 a	6775 ± 903 a	0.20
Fluorides	0.50 ± 0.07 a	0.50 ± 0.07 a	0.00
Nitrates	1800 ± 192 a	2590 ± 276 b	16.54 *
Nitrites	0.10 ± 0.01 a	0.10 ± 0.01 a	0.00
Phosphates	75.7 ± 10.1 a	312.0 ± 41.6 b	91.42 ***
Sulfates	7785 ± 1038 a	8200 ± 10,913 a	0.23
Cations
Ammonium	58.6 ± 5.23 a	67.5 ± 6.03 a	3.73
Calcium #	1200 ± 151 a	1900 ± 240 b	18.41 *
Cobalt	58.0 ± 7.7 a	58.0 ± 7.7 a	0.00
Copper	210 ± 28 a	210 ± 28 a	0.00
Iron	100.0 ± 12.7 a	240.0 ± 30.4 b	54.21 **
Magnesium #	2500 ± 316 a	2300 ± 291 a	0.65
Manganese	510 ± 68 a	490 ± 65 a	0.13
Molybdenum	170 ± 22 a	170 ± 22 a	0.00
Potassium #	12,000 ± 1520 a	13,000 ± 1646 a	0.60
Sodium #	1800 ± 228 a	2700 ± 342 b	14.38 *
Zinc	1800 ± 216 a	1700 ± 204 a	0.34
Complex forms
Calcium #	1000 ± 126 a	1600 ± 202 b	18.91 *
Cobalt	381 ± 51 a	359 ± 48 a	0.30
Copper	110.0 ± 14.7 a	130.0 ± 17.3 a	2.33
Iron	49.0 ± 5.9 a	110.0 ± 13.2 b	53.46 **
Magnesium #	1500 ± 190 a	1400 ± 177 a	0.44
Manganese	260.0 ± 34.7 a	240.0 ± 32.0	0.54
Molybdenum	130 ± 15 a	130 ± 17 a	0.00
Potassium #	6900 ± 874 a	18,200 ± 2305 b	63.02 **
Sodium #	9900 ± 1254 a	11,700 ± 1482 a	2.58
Zinc	880 ± 82 a	920 ± 86 a	0.34

^# Cations expressed as their respective oxides. The mean values (n = 3) are shown with their standard deviation. For each parameter, different letters indicate significant differences between both biofertilizers according to Tukey’s HSD test (* p value < 0.05; ** p value < 0.01; *** p value < 0.001) and typed in bold.

, where the minerals were classified by anions (borates, chlorides, fluorides, nitrates, nitrites, phosphates and sulfates), cations (ammonium, calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, potassium, sodium and zinc) and complexed forms (calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, potassium, sodium and zinc). Related to anions, only significant differences were observed for nitrates and phosphates, both in a higher concentration in biol from cow dung (BCD). In terms of cations, significant differences were observed for ammonium, calcium, iron and sodium, which were higher also in BCD. Finally, regarding the complex forms significant differences were observed only for calcium, iron and potassium, which is higher in BCD, rather than biol from native forest duff (BNF).

The composition of the liquid ferment depends on the nutrients in the starting material including the chemical composition of the organic material, mineral inputs, temperature, pH and the presence of inhibitors [44,50]. In the biols preparation, the same amounts of inputs were added (Tables S1–S6), except for the inoculums (Tables S5 and S6). Furthermore, de Oliveira Neto et al. [39] mention that the greatest availability of nutrients in biofertilizers occurs with a longer fermentation period, so it would be expected to have some variability in the contents and forms of the minerals, as the fermentation process was longer in BCD than BNF (Figure 1).

In a short period, there is not enough decomposition in the biofertilizer to release nutrients, such as nitrogen, phosphorous, potassium, sulfur, calcium, magnesium and iron, which can be concentrated in the remaining material [51]. Furthermore, since BCD was more acidic than BNF during fermentation (Figure 1) and at the end (Table 2), a different ion content in both biofertilizers can be expected. The most abundant anions were sulfates, chlorides and nitrates, and, among the cations, potassium, magnesium and calcium stand out (Table 3). It should be noted that among all these ionic forms, potassium was the most abundant, with contents around 12 g/L. The complex forms of cations are of great agronomic interest since such forms are not subject to the precipitation conditions that can be undergone when they are found as cations in the solution. These forms are easily absorbed by plants at both the foliar and root levels [52]. For all these reasons, taking into account only the mineral composition, these biols could be considered as a good source of mineral nutrients for plants, and, in the case of potassium, as a commercial chemical preparation [53].

At the end of biols production, the content of borates, chlorides, fluorides, nitrites and sulfates did not show significant differences between both biols (Table 3). However, the content of nitrates and phosphates was higher in BCD than in BNF, especially of the latter anions. These results could be attributed to several factors: on the one hand, to the higher number of microbial populations in CDI than in NFI (Table 1), and, on the other, to the higher amount of inoculum used in BCD than in BNF, but also it could be due to the longer fermentation period and the lower pH in BCD.

The fermentation behavior can be assessed with that of nitrogen compounds [6], since the degradation of organic compounds, especially proteins, produces ammonia during the anaerobic process [48]. However, although part of the ammonia produced in fermentation can be lost as gas, most of it dissolves in the liquid, thus becoming easily available for plants [54]. At low pH, part of the ammonium can precipitate as carbonate and, furthermore, in the presence of oxygen, it is converted into nitrates [43]. It should also be taken into account that an important source of nitrogen is immobilized by microorganisms and released into the environment when microorganisms die [55].

The content of phosphates in BCD was four times higher than in BNF, which could be attributed to the higher number of phosphorus-solubilizing bacteria species in CDI than in NFI (Table 1). In addition, both the lower pH and the shorter fermentation period in NFI could have influenced the lower phosphate concentration in this biol. Phosphorus is not released from organic matter during anaerobic fermentation as gas, but it can be precipitated in acidic media in the form of struvite [43], which is a hydrated salt of ammonium, magnesium and phosphorus [6].

Regarding macronutrient cations, significant differences were found between both biols only for calcium and sodium, for which it could be argued that both have a lower content in biol from native forest duff (BNF), rather than biol from cow dung (BCD), due to the higher pH value, which has favored greater precipitation. So, Criollo et al. [44] point out that calcium solubility directly depends on the pH, then creating a dynamic equilibrium between dissolved forms in the liquid and solids, such as carbonates and phosphates [43]. Also, the longer fermentation process on BCD allowed calcium and sodium to be released into the liquid [51]. Furthermore, the organic acid content in the fermentation medium would be closely related to the amount of free calcium released by acidolysis of CaCO₃ contained in the water. The higher iron content in BCD than in BNF may be because the number of siderophore bacteria was higher in CDI than in NFI (Table 1), but is also due to the fact that a more acidic pH favors its solubility [44].

Viewing the complex forms, significant differences were only found between the two biols for potassium, iron and calcium, with higher contents in BCD than in BNF (Table 3). The temperature and fermentation time are closely related to the phenomenon of complex formation [56], and ammonia and derivatives, such as amino acids, can form stable chemical complexes with various metal ions [57]. On the other hand, Gurd et al. [58] argued that the nature of metal–protein complexes varies widely depending on the metal, and the side chains of the amino acids, among others. Thus, in the case of iron, the results were as expected due to the greater tendency of its corresponding inoculum to release iron, but also due to the higher amino acid content (

Table 4. Amino acids and hormones content (mg/L) of the two biols: from native forest duff (BNF) and from cow dung (BCD) at the end of their production.

	BNF	BCD	Fp-value
Amino acids
Alanine	143.0 ± 19.1 a	203.0 ± 27.1 b	9.85 *
β-Alanine	3.08 ± 0.41 a	3.87 ± 0.51 a	4.27
γ-Aminobutyric acid	4.53 ± 0.60 a	6.21 ± 0.82 b	8.07 *
Glycine	19.5 ± 2.6 a	14.7 ± 1.9 a	6.52
Glutamic acid	73.0 ± 9.7 a	85.7 ± 11.4 a	2.15
Glutamine	2.57 ± 0.34 b	0.82 ± 0.11 a	71.95 **
Histamine	0.22 ± 0.03 b	0.03 ± 0.01 a	111.64 ***
Histidine	0.51 ± 0.07 b	0.15 ± 0.02 a	73.36 **
Phenylalanine	11.50 ± 1.53 b	5.17 ± 0.69 a	42.67 **
Hormones
Abscisic acid	0.20 ± 0.026	0.19 ± 0.025	0.22
Indole-3-acetic acid	0.009 ± 0.0012	0.050 ± 0.0066	109.91 ***
Salicylic acid	0.036 ± 0.005 a	0.23 ± 0.03 b	126.95 ***

The mean values (n = 3) are shown with their standard deviation. For each parameter, different letters indicate significant differences between both biofertilizers according to Tukey’s HSD test (* p value < 0.05; ** p value < 0.01; *** p value < 0.001) and typed in bold.

). It is surprising that potassium in its cationic form was found in similar concentrations in both products, but that, in its complex form, it has a higher content in BCD than in BNF. This fact would be attributed to a greater number of potassium-solubilizing species in CDI than in NFI and, as in the case of iron and calcium, to its higher content of amino acids (Table 4). In addition, the higher contents of the complexes in BCD may also be due to its longer fermentation period (Figure 1), since the temperature was similar throughout the fermentation in both biols [56].

As stated, the amino acid content is summarized in Table 4. The production of amino acids in biols depends on the quality of the inoculum [59] but pH, feed rate, aeration intensity and process temperature are also crucial in the amino acids production [60]. D’Este et al. [61] mention that the process temperature must be carefully chosen, taking into consideration the target compound to be produced, converting the sugars present in a substrate into a broad spectrum of amino acids by several microorganisms [62]. Moreover, it has been reported that Saccharomyces cerevisiae can easily produce alanine [63] as the content of biol from cow dung (BCD) when preparation was higher than BNF (Tables S5 and S6), suggesting the different values of alanine between biols. Furthermore, the concentration of ammonium at the end of the fermentation as a reduced product has been shown to affect alanine formation [63]. The synthesis of γ-aminobutyric acid is catalyzed by the enzyme glutamic acid, which has been identified in Saccharomyces cerevisiae. The addition of this starter on BCD but none in BNF suggests the production of γ-aminobutyric acid in BCD over BNF during the fermentation process. Sahab et al. [64] argue that pH, temperature and a longer period of fermentation influence the synthesis of γ-aminobutyric, although some authors [60] suggest that effective pH value for the maximum of γ-aminobutyric acid production is species-dependent. Even though it has been reported that a high number of amino acids in manures [65], the contents of the amino acids related to defense response and stress adaption functions were higher on BNF than BCD at the end of the fermentation process. The reason could be the higher number of species related to biocontrol and exopolysaccharides function on NFI rather than CDI (Table 1), allowing a higher synthesis of these compounds contained in the inoculums, such as glutamine [66]. For instance, Pseudomonas aeruginosa is an abundant species on NFI, which acts as a precursor in the production of a wide range of free amino acids, especially on biocontrol capacity [67]. The values of histidine and histamine were higher in BNF than in BCD, which is normal considering that histidine is the precursor for histamine [68]. Some amino acids such as phenylalanine, higher in BNF than BCD, have a unique role in plants as a source of a wide range of specialized metabolites that contribute to the adjustment of plants to changing developmental and environmental conditions [69].

In the biols, several hormones have been analyzed, although it was only possible to quantify indole-3-acetic acid and salicylic acid (Table 4). Both have shown values much higher in biol from cow dung (BCD) than biol from native forest duff (BNF) (Table 4). Cai et al. [70] mentioned the ability of various microorganisms to produce indole-3-acetic acid, such as bacteria, actinomycetes, fungi or yeast. Therefore, the different values of indole-3-acetic acid between biols could be due to the higher number of species of microorganisms in CDI rather than NFI. It is known that salinity, pH and temperature during the fermentation process also affect microbial indole-3-acetic acid production [71]. Many bacterial species (Pseudomonas, Bacillus, Azospirillum, Salmonella, Achromobacter, Vibrio, Yersinia and Mycobacteria) have been reported to synthesize salicylates [72], and to a greater or lesser extent, almost all of them were represented in NFI and CDI. This bacterial salicylate production is often linked to the biosynthesis of small ferric-ion-chelating molecules, known as catecholate (also termed salicylic-derived siderophores). Pseudomonas spp. has been documented as the main synthesizer for the catecholate siderophore, whose value on CDI was higher than NFI (Table 1), which was derived from the major content of salicylic acid on BCD.

The volatile composition of biols at the end of the preparation process is summarized in

Table 5. Volatile relative response of the two biols (×10³): from native forest duff (BNF) and from cow dung (BCD) at the end of their production.

	BNF	BCD	Fp-value
Acids
Acetic acid	49.6 ± 15.7 a	055.5 ± 7.0 a	0.23
Decanoic acid	1.9 ± 0.0 a	5.2 ± 0.1 b	4225.00 ***
Hexanoic acid	4.7 ± 0.1 a	61.0 ± 3.2 b	625.80 **
Octanoic acid	3.4 ± 0.3 a	27.3 ± 1.8 b	354.96 **
Alcohols
Acetol	129.1 ± 41.9 a	127.8 ± 48.8 a	0.00
Benzyl alcohol	1.4 ± 0.1 a	2.4 ± 0.0 b	441.00 **
Cyclopentanol	71.0 ± 19.8 a	79.2 ± 25.6 a	0.13
Isobutyl alcohol	9.9 ± 0.2 a	29.7 ± 0.4 b	4612.24 ***
3-Methyl-1-butanol	9.6 ± 0.1 a	353.5 ± 8.7 b	3177.52 ***
1-Octen-3-ol	0.8 ± 0.1 a	1.0 ± 0.0 b	25.00 *
Nonanol	0.8 ± 0.0 a	1.4 ± 0.4 a	4.00
Phenylethyl alcohol	6.6 ± 1.1 a	81.9 ± 4.4 b	553.18 **
2,4-di-tert-Butylphenol	45.0 ± 2.1 a	58.0 ± 1.0 b	65.69 *
Aldehydes
Benzaldehyde	15.8 ± 2.5 a	12.9 ± 2.5 a	1.36
Decanal	4.5 ± 1.6 a	4.6 ± 1.1 a	0.01
Nonanal	35.8 ± 6.0 a	36.3 ± 0.7 a	0.02
Octanal	3.6 ± 0.6 a	3.2 ± 0.0 a	0.60
Esters
Ethyl acetate	92.1 ± 2.3 a	1021.6 ± 16.9 b	5942.92 ***
Ethyl butyrate	3.8 ± 0.0 a	299.2 ± 3.3 b	15,795.68 ***
Ethyl decanoate	0.6 ± 0.1 a	5.5 ± 0.0 b	2401.00 ***
Ethyl dihydrocinnamate	0.1 ± 0.0 a	27.7 ± 1.0 b	1552.33 ***
Ethyl hexanoate	3.5 ± 2.1 a	306.8 ± 4.8 b	6661.18 ***
Ethyl lactate	5.0 ± 0.3 a	173.2 ± 5.3 b	2004.92 ***
Ethyl octanoate	2.8 ± 0.6 a	65.7 ± 0.3 b	19,782.05 ***
Diethyl succinate	1.6 ± 0.7 a	4.2 ± 0.2 b	23.86 *
Isoamyl acetate	2.3 ± 1.8 a	36.9 ± 1.7 b	382.48 **
Isovaleric acid, ethyl ester	21.0 ± 1.6 b	10.6 ± 0.1 a	81.63 *
Phenylacetaldehyde	9.9 ± 0.1 a	10.8 ± 0.1 b	162.00 **
Norisoprenoids
β-Damascenone	2.3 ± 0.2 a	4.0 ± 0.0 b	115.60 **
Terpenes
Elemol	61.7 ± 42.1 a	48.6 ± 9.7 a	0.18
D-Limonene	3.4 ± 0.4 a	3.9 ± 0.4 a	1.39
Linalool	1.1 ± 0.1 a	2.4 ± 0.0 b	729.00 **
Nerol	1.4 ± 0.4 a	2.3 ± 0.8 a	1.80
Pinene	18.7 ± 1.4 a	17.0 ± 4.8 a	0.23
α-Terpineol	1.7 ± 0.2 a	3.3 ± 0.1 b	102.40 **
Pyrazines
Pyrazine, 2-ethyl-6-methyl-a	14.4 ± 0.0 a	26.6 ± 1.3 b	183.75 **
Pyrazine, 2-ethyl-6-methyl-b	10.3 ± 0.1 a	17.0 ± 0.6 b	264.06 **
Pyrazine, 2-ethyl-3,5-dimethyl	9.3 ± 0.3 a	17.6 ± 0.5 b	418.85 **
Pyrazine, trimethyl	87.6 ± 1.2 a	143.1 ± 4.9 b	243.98 **
Sulfides
Dimethyl disulfide	27.1 ± 0.4 b	15.0 ± 0.5 a	791.41 **
Dimethyl tetrasulfide	14.8 ± 1.2 b	4.6 ± 0.1 a	101.50 **
Dimethyl trisulfide	129.4 ± 9.1 b	53.9 ± 9.4 a	66.01 *
Volatile phenols
Guaiacol	1.7 ± 0.2 a	2.5 ± 0.4 a	6.42
Syringol	0.3 ± 0.0 a	0.5 ± 0.1 a	9.00
4-Vinylguaiacol	3.0 ± 1.2 a	2.8 ± 0.6 a	0.03
Others
2(5H)-Furanone	16.0 ± 4.4 a	17.8 ± 4.6 a	0.15

The mean values (n = 3) are shown with their standard deviation. For each parameter, different letters indicate significant differences between both biols according to Tukey’s HSD test (* p value < 0.05; ** p value < 0.01; *** p value < 0.001) and typed in bold.

. Most of the identified compounds are characteristic of the different stages of an anaerobic fermentation, which includes hydrolysis, acidogenesis, acetogenesis and methanogenesis of organic matter formed by carbohydrates, proteins and lipids, which are transformed into monomers such as sugars, amino acids and long chain fatty acids [15]. Significant differences were observed for some compounds and families, except for aldehydes, volatiles phenols and others, and some individual volatiles, such as acetic acid, acetol, cyclopentanol, nonanol, elemol, D-limonene, nerol and pinene. For the volatile compounds that showed statistical differences, their response was higher in the BCD, except for isovaleric acid, ethyl ester and the three sulfides identified, where the response was higher in BNF. It is worth highlighting the high values obtained for the esters, where the higher significant differences were observed for ethyl butyrate. Although the bibliography related to the volatile composition of biols is scarce, some references and coincidences have been detected in existing research. For example, ethyl acetate has previously been identified in forest litter-based biofertilizers and has been related to as responsible for the pleasant odor, indicating a successful fermentation process [73]. It is worth noting that the two biols studied have agreeable odors at the end of fermentation, possibly due to the abundance of esters. The presence of some sulfides and pyrazines, whose formation via fermentation could be related to the lower colony-forming unit detected in both biols at the end of the anaerobic fermentation, since sulfides have fungicidal activity [74] and pyrazines have demonstrated their effective antimicrobial action [75].

4. Conclusions

The two biols prepared in the present study have an acidic pH (4.5–5.5), which is higher than those obtained for cow dung. Both have an interesting mineral content, amino acids and hormones, which confirm their agronomic potential. Regarding cow dung biol, a higher content of ionic forms such as nitrates, phosphates, calcium, iron and sodium, and, as for soluble complexes of calcium, iron and potassium were observed, which supports its greater fertilizing value than native forest duff biol. It should be noted that, only because of their potassium content, both could be considered potassium fertilizers like current commercial ones. The volatile compounds suggest that the pleasant odors of both biols may be due to the presence of esters, and the absence of microorganisms in the final products could be explained by pyrazines and sulfides.

These results contribute to the scarce existing knowledge on the preparation, fermentation monitoring and composition of liquid ferments and will be useful to compare with the variants introduced when local inputs are used in biols preparation.