Organic Fertilizer Enriched with Humic Acids and Plant Growth-Promoting Bacteria Applied in a Mandarin Orchard

Fertilizante orgânico enriquecido com ácidos húmicos e bactérias promotoras do crescimento vegetal aplicado num pomar de tangerinas

Abstract:

The expansion of large-scale organic fruit production in Brazil is constrained by the scarcity of biological inputs. One potential solution is to enrich organic fertilizers with humic acids and plant growth-promoting bacteria, which could increase plant nutrition efficiency. A compost produced on an industrial scale from pruning residues of urban trees and cattle slaughterhouse waste was enriched with humic acids extracted from the organic compost itself and inoculated with a microbial consortium formed by the bacteria Herbaspirillum seropedicae strain HRC54, Bacillus safensis strain 77, Bacillus pumilus strain J1.1,and Gluconacetobacter diazotrophicus strain PR2. Chemical and spectroscopic characterization of the organic fertilizer was performed, and the resulting product was applied to three plots with a total of 200 g of nitrogen per plant per year. Solid-state ¹³C nuclear magnetic resonance (NMR) analysis revealed the presence of lignocellulosic materials and smaller but significant amounts of hydrophobic alkyl molecules. Gas chromatography coupled to mass spectrometry of the pyrolysis products of the compound indicated the presence of nitrogen, lignin, carbohydrates, and lipids. There were no significant differences in biometric parameters, such as plant height and stem diameter, among the different mandarin trees. However, plants treated with the enriched fertilizer showed significantly higher productivity in the first year of production, with fruitweights ranging from 3.8 to 6.2 kg per plant, compared to plants receiving the unenriched fertilizer, which produced between 2.2 and 3.8 kg of fruit per plant. Biological enrichment is a viable alternative to increase the efficiency of organicfertilizers.

Index terms
Organic fertilization; biofertilizers; organic citrus farming; humic substances

Resumo:

A fruticultura orgânica, em larga escala no Brasil, é limitada pela carência de insumos biológicos. O enriquecimento de fertilizantes orgânicos com ácidos húmicos e com bactérias promotoras do crescimento vegetal é uma alternativa para aumentar a eficiência na nutrição das plantas. Um composto produzido em escala industrial, a partir de restos de poda da arborização urbana e de resíduos de abatedouro bovino, foi enriquecido com ácidos húmicos extraídos do próprio composto e inoculado com um consórcio microbiano formado pela bactérias Herbaspirillum seropedicae estirpe HRC54, Bacillus safensis estirpe 77, Bacillus pumilus estirpe J1.1 e Gluconacetobacter diazotrophicusestirpe PR2. Foi realizada a caracterização química e espectroscópica do fertilizante orgânico que foi aplicado em 3 parcelas, totalizando 200 g de nitrogênio por planta, por ano, em 10 diferentes copas de tangerinas. A análise espectroscópica de Ressonância Magnética Nuclear ¹³C revelou a presença de materiais lignocelulósicos equantidades menores, mas significativas, de moléculas alquílicas hidrofóbicas. Acromatografia gasosa, acoplada à espectrometria de massas dos produtos da pirólise do composto,indicou a presença de unidades derivadas principalmente de ligninas, carboidratos e delipídios. Não houve diferença significativa nos parâmetros biométricos, como altura da planta e diâmetro do caule das diferentes copas, porém as plantas tratadas com o fertilizante enriquecido apresentaram maior produtividade no primeiro ano de produção, compeso de frutos variando entre 3,8 a 6,2 Kg por planta, enquanto as plantas que receberam o fertilzante sem enriquecimento produziram entre 2,2 a 3,8 Kg de frutos porplanta. O enriquecimento biológico é uma alternativa viável para aumentar a eficiênciade fertilizantes orgânicos.

Termos para indexação
Fertilização orgânica; biofertilizantes; citricultura orgânica; substâncias húmicas

Introduction

The global market for organic products is growing and, according to the International Federation of Organic Agriculture Movements (IFOAM/FIBL), it is valued at more than 106 billion euros annually (IFOAM/FIBL 2022).

In addition to its export potential, a significant increase in the demand for organic fruits was observed during the pandemic (VAN DER PLOEG, 2021). However, less than 1% of Brazil’s cultivated area is dedicated to organic food production (LOURENÇO et al., 2023).

Plant nutrition in organic systems is achieved through integrated biological processes such as nutrient cycling, mineralization of organic matter, biological nitrogen fixation, and others (COSTABEBER et al., 2013). Organic fertilizers, therefore, play a fundamental role in maintaining nutrient supply in commercial organic orchards.

Improving efficiency is necessary, especially considering the high production and transportation costs. In this context, the biofortification of organic fertilizers, i.e., enriching composts and vermicomposts with biological products, can be a viable alternative to improve nutrient use efficiency.

For example,the addition of rock phosphate along with phosphorus (P)-solubilizing bacteria during the maturation of a vermicompost has been shown to increase water-soluble P, allowing for a reduction in the amount applied (BUSATO et al., 2012).

In addition to the use of plant growth-promoting bacteria (PGPB), the incorporation of humic substances (HS) in fertilizers can also lead to yield increases in various crops (GAO et al., 2022; JING et al., 2022), as these substances release highly bioactive compounds from their supramolecular arrangement. These compounds are mobilized by short-chain organic acids exuded by plant roots into the rhizosphere, leading to chemical changes (CANELLAS et al., 2002; 2012; 2019).

This promotes the formation of stable soil aggregates, which enhances water retention, increases nutrient availability, and stimulates microbial activity.

The well-recognized individual effects of PGPB and HS highlight the potential of their combined use, and the synergistic mechanisms involved in their simultaneous application have been documented for a vast number of agriculturally important crops (OLIVARES et al., 2017).

For example,in pineapple cultivation, this combined application contributed to an increase in the number of shoots and roots and in the levels of N, P, K, Ca, and Mg . In tomato plants, it improved plant growth at all stages, including fruit production (OLIVARES et al., 2015), with increased Fe and K content, greater shoot and root development (GALAMBOS et al., 2020).

In sugarcane, foliar application promoted a 37% increase in stem yield compared to untreated plants (SILVA et al., 2017). It also increased total tuber production (EKIN, 2019) and improved fruiting and fruit quality in guava (ASHWINIA et al., 2022).

In maize, it increased photosynthetic pigment levels and overall plant growth. Bio-based agricultural inputs have also been studied in polycultures of tropical fruit species, showing increased productivity, especially in environments with low natural fertility (CANELLAS et al., 2022).

Therefore, the objectives of this study were to determine the ideal concentration of PGPB and HS for the enrichment of organic compost, to characterize its chemical properties, and to evaluate the effect of fertilization with the enriched compost on the agronomic performance of mandarin trees.

Materials and Methods

1. Organic Fertilizer Production

1.1 Composting

To produce the compost, branches from urban pruning in the region of Campos dos Goytacazes - RJ were shredded with an industrial knife shredder to an average particle size of 3 cm. The material was then piled in a brick shed measuring 60 m long, 6 m wide, and 1.2 m high. Rumen content from cattle slaughterhouses was added to the pile at a 3:1 (volume: volume) ratio.

The pile was turned mechanically using a self-propelled auger system driven by an electric motor that moved on rails. Blood was applied to the pile by spraying during the turning process at a rate of 1500 L h^-1 for 4 hours. This phase lasted 30 days, during which the pile was turned for 8 hours a day.

Moisture content was monitored using the method developed by Silva (2008), which determines the dryness or runoff index of the sample. Temperature was controlled daily with a digital thermometer (Minipa - MT401A).

After this period, the material was moved for maturation and arranged in windrows measuring 50 m long, 2.5 m wide, and 1.2 m high in a shed covered with agricultural plastic.

During this 60-day process, the windrows were turned daily for the first week using a tractor-drawn compost turner to regulate temperature and moisture. After maturing, the compost was mechanically collected, homogenized with 6 mm sieves, and packed in 25 kg bags.

1.2 Chemical Characterization of the Final Compost

Following the methodology established by BRASIL (2017), samples were collected from 10 points spaced 5 meters apart along the windrow, at a depth of 0.6 meters, for the chemical characterization of the compost.

These samples were then combined and homogenized to form a composite sample.

The pH was determined in a 0.01 mol L^-1 CaCl₂ solution, using a 1:2.5 (w/v) ratio, and electrical conductivity was measured in an aqueous suspension.

Organic carbon was determined by oxidation with potassium dichromate and concentrated sulfuric acid under external heating.

The remaining potassium dichromate was quantified by titration with ammoniacal ferrous sulfate solution. Nitrogen (N) content was determined using the Kjeldahl method, and phosphorus (P₂O₅) was quantified by the molybdovanadophosphoric acid spectrophotometric method.

Potassium (K) was extracted with hot water and quantified by flame photometry, while sulfur (S) was extracted based on the oxidation of various sulfur forms to sulfate using hydrogen peroxide in an alkaline medium, followed by precipitation as barium sulfate.

Calcium (Ca) and magnesium (Mg) were extracted using a nitroperchloric acid mixture and determined by atomic absorption spectrometry, as were the micronutrients iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), and boron (B).

The content of humic substances (HS), fulvic acids (FA), and humic acids (HA) was determined by the volumetric potassium dichromate method, after extraction with NaOH at concentrations between 0.1 and 0.5 mol L^-1. The extract was acidified to pH 1 to precipitate the humic acids.

2. Compost Enrichment

2.1 Extraction of Humic Substances

The soluble humic substances were extracted from the compost using 0.1 M KOH at a solid-liquid ratio of 1:20, with mechanical stirring for 6 hours. Afterward, the suspension was left to settle overnight (~16 hours).

The supernatant was collected and acidified to pH 2.0 with phosphoric acid (H₃PO₄) and left to rest for another 16 hours to precipitate the humic acids (HA). The supernatant containing fulvic acids (FA) was collected separately, and the precipitated material was centrifuged at 5000 g to enhance separation between FA and HA.

The precipitate was washed three times until no reaction occurred with silver nitrate, and the pH was adjusted to 6.5 with 0.1 mol L^-1 H₃PO₄. Finally, the samples were dialyzed using a 1000 Da molecular weight cutoff membrane (Spectrapore) against deionized water. Subsequently, carbon content was quantified in the different fractions: humic substances (HS), fulvic acids (FA), and humic acids (HA).

2.2 Bacterial Inoculum Production

Four plant growth-promoting bacterial (PGPB) strains were used: Herbaspirillum seropedicae strain HRC54, Bacillus safensis strain 77, Bacillus pumilus strain J1.1, and Gluconacetobacter diazotrophicus strain PR2, kindly provided by The Microbial Culture Collection of the Laboratório de Biologia Celular e Tecidual (LBCT-UENF).

Bacterial suspensions were prepared in liquid nutrient broth (NB) medium supplemented with 10 g/L glycerol and incubated in a rotary shaker at 180 rpm and 30°C for 24 hours. Cells were pelleted by centrifugation (4000 x g for 15 minutes) and resuspended in sterilized water at a final density of 10⁹ cells mL^-1.

The organic fertilizer was prepared with different concentrations of HA (0, 0.1, 1, 10, 100, and 1000 mg C L^-1), and bacteria were inoculated at a final concentration of 5 x 10⁸ cells mL^-1.

2.3 Addition of Humic Acids and Bacteria to the Fertilizer

The HA and PGPB suspensions were diluted in deionized water and added to the organic compost until reaching a maximum moisture content of 40%. Preliminary tests of drying and moistening were conducted to calculate the necessary volume of solution to be added.

2.4. Preliminary Test for Determination of Humic Acid (HA) Concentration

The preliminary test used hybrid corn seeds UENF 506-11 (Zea mays L.) germinated on germination paper moistened with distilled water and kept in a growth chamber at 25 °C in the dark, with a relative humidity of 85%. After 4 days, the seedlings were transferred to Petri dishes (9 cm in diameter) lined with germination paper and moistened with 10 mL of either distilled water (control) or extracts of enriched fertilizers at different humic acid (HA) concentrations (0, 0.1, 1, 10, 100, and 1000 mg C L^-1) and bacteria.

The extracts were prepared at a 1:10 ratio in distilled water, stirred for 40 minutes at 200 RPM, centrifuged for 20 minutes at 600 RPM, and filtered through 0.45 μm paper. All treatments were performed in triplicate, with 10 seedlings per dish. After 4 days of incubation, the roots were scanned and analyzed using the WinRHIZO software (MONDA et al., 2017).

Since the treatment with 100 mg C L^-1 of HA showed effects on root length and lateral root emission, this concentration was adopted for use in the field experiment with tangerine plants.

3. Chemical Characterization of the Enriched Organic Fertilizer

3.1. Solid-State ¹³C NMR Spectroscopy (CP/MAS)

The ¹³C CP/MAS NMR spectrum of the fertilizer enriched with HA at a concentration of 100 mg C L^-1 was recorded on a Bruker AV- 300 spectrometer equipped with a 4 mm MAS probe, using the following acquisition parameters: 13,000 Hz rotor spinning rate; 2 s recycle delay; ¹H power for CP: 92.16 W; ¹H 90° pulse: 2.85 μs; ¹³C power for CP: 150.4 W; contact time: 1 ms; acquisition time: 30 ms; 4000 scans. Samples were packed in 4 mm zirconium rotors with Kel-F caps.

The cross-polarization pulse sequence was applied using a ramped pulse shape on the ¹H channel to account for the inhomogeneity of the Hartmann-Hahn condition at high spinning frequencies.

Fourier transform was performed on 4k data points with an exponential apodization corresponding to a 100 Hz line broadening. For interpretation of the ¹³C NMR spectra, the general chemical shift range was divided into the following main regions: aliphatic-C (0–45 ppm); methoxyl-C and N-alkyl-C (45–60 ppm); O-alkyl-C (60–110 ppm); aromatic-C (110– 145 ppm); O-aryl-C (145–160 ppm); carboxyl- C (160–200 ppm).

The area of each functional group region (Riabs) was divided by the sum of all spectral regions to obtain a relative proportion (using MestreNova 6.2.0 software, Mestrelab Research, 2010).

3.2. Pirólise off-line – cromatografia gasosa acoplada à espectrometria de massas

Approximately 200 mg of dried organic fertilizer was placed in a quartz container and moistened with 2 mL of tetramethylammonium hydroxide (TMAH) solution (25%in methanol).

After drying, the sample was introduced into a Pyrex tubular reactor (50 cm x 3.5 cm i.d.) and heated (ramp of 20 °C min^-1) to 400 °C (10 min isothermal) for a total run time of approximately 30 minutes in a Barnstead Thermolyne 21100 oven.

The products released by thermochemolysis were continuously transferred by a helium flow (20 mL/min) into two successive flasks containing chloroform (50 mL), kept in an ice bath. The chloroform solutions were combined in a round-bottom flask and concentrated by rotary evaporation under reduced pressure. The sample was dissolved in 200 μL of hexane and analyzed using a PerkinElmer Autosystem XL (GC) equipped with a PE Turbomass-Gold quadrupole mass spectrometer.

Chromatographic separation was performed using a 60 m Supelco capillary column (SLB-5 ms).

Helium was used as the carrier gas (1 mL/min), and the sample (2.5 μL) was injected in splitless mode with the injector maintained at 250 °C. The oven temperature was initially set to 100 °C and held for 5 minutes, then increased to 210 °C at a rate of 2 °C/min, followed by an increase from 210 to 250 °C at a rate of 5 °C/min, and held at 250 °C for 12 minutes.

Mass spectra were obtained in EI mode (70 eV), scanning in the m/z range of 50–600, with a cycle time of 1 second. Compound identification was based on comparison of the mass spectra with the NIST library database and with previously obtained spectra (SPACCINI; PICCOLO, 2007, 2012, and 2013; MARTINEZBALMORI et al., 2013 and 2014).

3.3. Fourier Transform Infrared Spectroscopy with Diffuse Reflectance (DRIFT)

The DRIFT-IR spectra were recorded using a Perkin Elmer 1720-X FT-IR spectrometer (Waltham, MA, USA) equipped with a Perkin Elmer Diffuse Reflectance accessory, accumulating up to 8 scans at a resolution of 4 cm^-1.

4. Orchard Establishment, Fertilization, and Inoculation with Microorganisms

The experimental orchard was established in Campos dos Goytacazes, in the northern region of the State of Rio de Janeiro, Brazil (21° 45’16” S, 41° 19’28” W), on the mid-slope of a half-orange-shaped hill, in a typical Dystrophic Yellow Argisol, according to the Brazilian Soil Classification System (Santos et al., 2018).

Routine soil analysis was carried out by a certified laboratory following EMBRAPA’s methodology, using a composite sample of 20 subsamples collected from the 0–0.20 m soil layer (Table 1).

The climate is classified as type Aw according to the Köppen classification, meaning a humid tropical climate with a rainy summer and a hot, dry winter (Figure 1).

Figure 1
Climatic data: monthly accumulated rainfall (mm) and average maximum and minimum temperatures (°C) during the period from the establishment of the experiment to the evaluations.

Table 1
Chemical properties and texture of the surface layer of the Yellow Argisol soil.

pH	P		Al3+		H+Al	K+	Ca2+	Mg2+	Na+	CEC	N	C	sand		silt	clay		m	V
H2O	mg Kg-1		cmolcKg-1									(%)
5,0	2	0,9		9,7		0,15	2,3	0,5	0,04	13,0	0,2	2,2	56,0	7,0		37,0	6,4		30

In November 2019, the orchard was established using five rootstocks (Citrandarin ‘Riverside’, Citrandarin Indio, Citrandarin ‘San Diego’, BRS Sunki Tropical, and Swingle Citrumelo) and ten mandarin grafting varieties (Ponkan, Fremont, Piemonte, Swatow, Kincy, Cleopatra Mandarin, South Africa, Oneco, Murcott BRS Salibe, and Span Americana), all selected by the Citrus Breeding Program (PMG Citros) of the Brazilian Agricultural Research Corporation (EMBRAPA).

The trees were arranged in a rectangular layout with a 3 x 4 m spacing. Planting holes measured 50 x 50 x 50 cm and received 200 g of dolomitic limestone, 5 L of well-composted cattle manure, and 100 g of natural rock phosphate.

The area was divided into two strips containing three replications of the scion varieties. One strip received an application of composted organic fertilizer, while the other was treated with the fertilizer enriched with humic acids (HA) and plant-growth-promoting bacteria (PGPB), applied to the canopy projection area.

Fertilization was performed at planting, with a second application two months later. Subsequent topdressings were performed using the same organic fertilizer according to the treatments, based on the recommendation of 200 g of nitrogen per plant per year, divided into applications at four-month intervals.

In the area treated with the enriched and inoculated fertilizer, plants were also sprayed with 200 mL of a solution containing the same microorganisms used in the enriched fertilizer.

5. Evaluations

After two and a half years from orchard planting, the following parameters were evaluated: plant height, measured with a 3-meter graduated ruler; stem diameter, measured with a caliper at 10 cm above the soil surface; and the number and weight of fruits, determined with a digital scale.

The data were subjected to analysis of variance (ANOVA) in a strip-plot design using R Studio, version 4.8 (ExpDes package), and means were compared using the t-test (LSD) at a significance level of p ≤ 0.05.

Results and Discussion

Chemical Analysis of the Organic Fertilizer

The main chemical characteristics of the organic compost fertilizer used, without adding humic acids and without bacterial inoculation, are presented in Table 2.

Table 2
Main chemical characteristics of the organic compost fertilizer.

N	P	K	Ca	Mg	S	Fe	Cu	Zn	Mn	B	O.C.	FA	HA	pH
%
1,76	0,73	0,61	2,73	0,71	0,16	0,639	0,034	0,026	0,11	0,012	16,9	3,6	6,7	8,2

• ^*
  O.C. – Organic Carbon, FA – Fulvic Acids, HA – Humic Acids

Based on the raw materials used in the composting process and the results obtained in the analytical report, the fertilizer was classified as a Class A organic compost fertilizer, since all parameters complied with the requirements of the Normative Instruction No.61, dated July 8, 2020, from the Ministry of Agriculture, Livestock and Supply (BRASIL (2020)).

The minimum required nitrogen (N) content is 0.5%, and the fertilizer contained 1.76% N. Organic carbon must not be less than 15%, and was found to be 17%, with a pH of 8.2, while the minimum required is 6.0. The carbon/nitrogen (C/N) ratio was 9.6, whereas the normative instruction states that it should be less than 20. These parameters indicate that the composting process was effective.

Souza et al. (2019) studied the composting of goat and sheep slaughter waste, evaluated the chemical, physical, and microbiological characteristics of the compost obtaining results that met MAPA standards, including pH 7.7; organic carbon 15.1%; N 1.8%; P 0.53%; K 1.91%; Ca 1.83%, and a C/N ratio of 8.4%. The authors concluded that, besides meeting regulatory standards, the composting process efficiently reduced environmental liabilities.

Characteristics of the organic matter in the enriched compost (infrared spectroscopy – IR region)

The IR spectrum of the fortified compost fertilizer is shown in Figure 2. The well-defined absorption band at 3691 cm^-1 can be attributed to the presence of free OH or NH groups, while those at 2921 cm^-1 are due to the stretching of C-H bonds in aliphatic CH₂ and CH₃ groups, as well as the absorption band at 2850 cm^-1 (CH₂n).

Figure 2
Infrared spectrum of the organic fertilizer enriched with 100 mg C L^-1 of humic acid (HA) and plant growth-promoting bacteria (PGPB).

The presence of the absorption band around 1413 cm^-1 is due to both the C–O stretching of phenolic OH and the C–H bending of CH₂ and CH₃ groups.

The absorption around 1260 cm^-1 is related to guaiacyl ring breathing and out-of-plane C–H vibrations in the guaiacyl units (C2, C5, and C6), respectively, while the signal at 1123 cm^-1 can be attributed to syringyl ring breathing. The absorption around 1028 cm^-1 is typical for C–O bonds in carbohydrates. Finally, the band at 908 cm^-1 is attributed to angular C–H deformation in aromatic groups or from contaminating silicates.

Spaccini and Piccolo (2007) carried out a molecular characterization of composts at different stages of maturity and reported a high content of aliphatic compounds in the IR spectra of composts after 60 days of composting.

This was evident from the strong absorptions in the 2926-2853 cm^-1 region and the intense peaks in the 1452-1368 cm^-1 region, which represent the symmetric stretching and bending vibrations of CH₂ groups in long-chain aliphatic molecules, respectively.

The authors also observed that the IR spectra suggested a progressive decrease in biolabile compounds with compost maturity, such as alkyl groups, carbohydrates, and proteinaceous materials.

Cross-Polarization Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance Spectroscopy (CPMAS ¹³C-NMR)

The CPMAS ¹³C-NMR spectrum of the organic fertilizer enriched with 100 mg C L^-1 of humic acid (HA) and plant growth-promoting bacteria (PGPB) is shown in Figure 3, while the relative distribution of carbon types is listed in Table 3.

Figure 3
Solid-state ¹³C Cross-Polarization Magic Angle Spinning (CPMAS) NMR spectrum of the organic fertilizer enriched with 100 mg of C L^-1 of Humic Acids and Plant Growth-Promoting Bacteria.

The distribution of carbon species in the NMR spectrum shows that the fertilizer contained mainly lignocellulosic materials and smaller but significant amounts of hydrophobic alkyl molecules.

The main carbon source in the composting process consisted of urban pruning residues and rumen contents, including partially undegraded grass, as evidenced by resonance signals within the typical range of O-alkyl-C groups (carbohydrates), which accounted for 43% of the total spectral area (Table 3).

Table 3
Relative distribution (percentage) of signal area over chemical shift (ppm) in ¹³C-CPMAS-NMR spectra of the organic fertilizer enriched with 100 mg C L^-1 of Humic Acids and Plant Growth-Promoting Bacteria.

Carboxylic (190-160)		Phenolic (160-140)		Aromatic (140-110)		Carbohydrates (110-60)		Methoxyls/C-N (60-45)		Alkyl (45-0)		HB	AR	LR
6.06	8.61		17.03		43.33		12.30		12.67		0.80		0.29	1.43

• ^*
  HB hydrophobicity index: [(0–60) + (110–160)/(45–60) + (60–110) + (160–200)]; LR lignin ratio: (45–60)/(145–160); AR alkyl ratio (Alkyl-C/O-alkyl-C): (0–45)/(60–110)

In this chemical shift region (60-110 ppm), monomeric units of polysaccharide chains, such as cellulose and hemicellulose from plant tissues, are found.

The signal at 65 ppm relates to the C nucleus at position 6, followed by a strong signal band at 72 ppm formed by overlapping carbons 2, 3, and 5 resonances in pyranoside structures. The signal band at 104 ppm is associated with anomeric di-O-alkyl C^-1 structures of glucose units (Spaccini et al., 2016).

The signals at 62-65 ppm and the shoulders at 83-87 ppm represent the C6 and C4 carbons of the monomeric units, respectively.

The higher and lower chemical shift resonances within these ranges are attributed to the crystalline and amorphous forms of cellulose, respectively (Atalla;VanderHart, 1999).

The signal around 56 ppm may correspond to methoxyl substituents on the aromatic rings of guaiacyl and syringyl units in lignin components and C-N bonds in amino acid fractions.

In addition, various O-alkyl regions may also contain signals related to ether and epoxy groups in plant biopolyesters (Deshmukh et al., 2005), whose resonances at 56, 62-67, and 74 ppm in complex matrices may indeed be masked by lignin and polysaccharides.

The results for methoxyl groups at 56 ppm should be consistent with signals in the phenolic C region (145-160 ppm), which are associated with O-substituted carbons in aromatic rings (Spaccini et al., 2012).

Indeed, resonances around 148 and 154 ppm are attributed to C3 and C5 ring carbons coupled to methoxyl substituents and O-substituted C4 carbons in lignin monomers (Preston et al., 2009). According to Spaccini et al. (2012), the signal between 172-174 ppm indicates a high content of carboxyl groups in aliphatic acids of plant and microbial origin and the presence of amide groups in peptide moieties.

The spectrum shown in Figure 3 is similar to that of several recycled biomasses used as organic fertilizers, such as biodynamic preparations (Spaccini et al., 2012), vermicompost (Aguiar et al., 2013; Aquino et al., 2019), and organic compost (Verrillo et al., 2021).

According to Spaccini et al.(2012), less recalcitrant organic matter resulted in a higher content of labile, bioavailable molecules and aromatic compounds derived from lignin, which could potentially allow plant growth due to the promoting effect.

The results obtained for the HB index are identical to those obtained by Aquino et al. (2019) in vermicompost with 75% and 100% cotton.

The relative amount of carbohydrates was also very similar; however, the aromatic C percentage was higher than that of vermicompost.

Thermochemolysis reaction assisted by tetramethylammonium (TMAH) (Offline pyrolysis followed by gas chromatography coupled with mass spectrometry)

The total ion chromatogram derived from the compounds identified after thermochemolysis of the organic fertilizer enriched with 100 mg C L^-1 of humic acids (HA) and plant growth promoting bacteria (PGPB) is shown in Figure 4.

Figure 4
Representative total ion chromatogram of the THM product released from the organic fertilizer enriched with 100 mg C L^-1 of Humic Acids (HA) and Plant Growth-Promoting Bacteria (PGPB), LIG = Lignin, nitrogen (circle), lipids (triangle), microbial lipids (square).

In contrast, the identification of the compounds present is listed in Table 4. The main compounds identified were derived from lignin, nitrogenous compounds, lipids, and microbial lipids (Table 4).

Table 4
Identified products released by THM from the organic fertilizer enriched with 100 mg C L^-1 of HA and BPCV.

Tempo (min)	Compostos	Origem
12.92	Benzene, 4-Ethenyl-3,4-Dimethoxy	Lig G3
13.21	Benzoic Acid, 4-Methoxy-, Methyl Ester	Lig P6
14.51	1H-Isoindole-1,3(2H)-dione, 2-Methyl	N-comp.
16.23	Benzaldehyde, 3,4-Dimethoxy	Lig G4
18.58	1-(3,4-Dimethoxyphenyl)ethanone	Lig G5
19.18	Benzoic Acid, 3,4-Dimethoxy-, Methyl Ester	Lig G6
19.49	Benzaldehyde, 3,4,5-Trimethoxy-	Lig S4
22.58	Benzoic Acid, 3,4,5-Trimethoxy-, Methyl Ester	Lig S6
24.46	Pentadecanoic Acid, 14-Methyl-, Methyl Ester	Mic
24.66	Pentadecanoic Acid, 13-Methyl-, Methyl Ester	Mic
27.99	Hexadecanoic Acid, Methyl Ester	Lip
29.58	Heptadecanoic Acid, 16-Methyl-, Methyl Ester	Mic
29.88	Methyl 15-methylheptadecanoate	Mic
32.28	Octadecenoic Acid, Methyl Ester	Lip
33.01	Octadecanoic Acid, Methyl Ester	Lip

Aquino et al. (2019) and Verrillo et al. (2021) noted that significantly lower amounts of carbohydrates were found among the pyrolysis products compared to the results obtained from NMR analyses.

According to these authors, this phenomenon is related to the lower efficiency of the offline pyrolysis technique in detecting carbohydrate moieties of polysaccharides in complex matrices. This fact has been observed in pyrograms of woody plant tissues, organic soils, organic matter, and biomass (Monda et al., 2017; Aquino et al., 2019; Verrillo et al., 2021).

In addition to the thermal and pyrolytic behavior that causes the rearrangement of polyhydroxy compounds, the TMAH reagent solution can negatively interfere with diagnosing polysaccharide components (Spaccini et al., 2013).

The lignin products (Table 4) are identified by the symbols currently used for the basic lignin structures: P for p-hydroxyphenyl, G for guaiacyl (3-methoxy, 4-hydroxyphenyl), and S for syringyl (3,5-dimethoxy, 4-hydroxyphenyl) (Spaccini et al., 2013; Aquino et al., 2019; Verrillo et al., 2021).

Similar to the results of Verrillo et al. (2021), the predominant lignin monomers were the oxidized forms of di- and tri-methoxyphenylpropanoids, such as benzaldehydes (G4, S4) and benzoic acids (P6, G6, and S6).

When characterizing the humic acids extracted from vermicompost made from cattle manure and cotton, Aquino et al. (2019) also found that the main products of thermochemolysis were derived from lignin, confirming the selective incorporation of O-aryl-C compounds shown in the NMR spectra. A lesser but noticeable release of nitrogen derivatives was also observed in this material.

Effects of Composted Organic Fertilizer Application in a Mandarin Orange Orchard

There was no effect of treatments on vegetative growth parameters of the plants, such as height and stem diameter. No interaction between graft types and inoculation factors was observed for average fruit weight. Thus, when analyzing the individual effects, plants fertilized with inoculated fertilizer showed a higher average fruit weight regardless of the graft type (Figure 5).

Figure 5
Effect of organic fertilization with enrichment of HS and PGPB inoculation, and pure organic fertilizer, on the average fruit weight of mandarins.

Among the grafting types used, the highest average fruit weight per plant was obtained with the Murcott BRS Salibe, Oneco, and Ponkan scions (Table 5).

Table 5
Fruit weight (kg), number of fruits (NF), height (H), and stem diameter (SD) at 10 cm above the soil.

Mandarin cultivars	FW (Kg)	NF	H (m)	DC (cm)
Murcott BRS Salibe	8.457 a	93±36,00	2.05±0,26	5.55±1,07
Oneco	7.64 ab	88±34,27	1.86±0,63	4.78±0,98
Ponkan	4.828 abc	54±37,92	2.37±0,49	4.88±1,08
Swatow	4.203 bc	38±48,26	1.80±0,65	4.38±1,13
Fremont	3.492 c	45±48,47	1.85±0,61	5.18±1,68
Piemonte	3.369 c	39±37,75	1.99±0,32	10.06±12,31
Africa do Sul	3.33 c	36±48,15	1.89±0,70	4.93±2,18
Kincy	2.293 c	43±35,84	1.60±0,71	4.30±1,50
Span Americana	1.895 c	60±44,40	1.92±0,21	5.08±1,51
TCOP	1.376 c	24±30,94	1.90±0,61	4.28±1,14

• ^*
  Averages followed by the same letter do not differ statistically according to Fisher's LSD test at a 5% probability level.

This fact may be related to the number of fruits per plant, since no thinning was done during this harvest. Therefore, although Ponkan usually has larger fruits with thicker skins, the average weight was lower than that of Murcott and Oneco because the number of fruits per plant was higher in Murcott (93 fruits), followed by Oneco (88 fruits) and Ponkan (54 fruits on average) (Table 5).

Gowda et al. (2024), who evaluated a five-year-old Murcott mandarin orchard during two growing cycles (2021 and 2022), found that a fertilizer combination of 50% NPK + 25% compost + 25% yeast vinasse resulted in the most significant vegetative growth, yield and fruit quality, with an average fruit weight of 181.66 g and productivity of 72.86 kg/ tree.

According to the authors, the exclusive use of mineral fertilizers showed inferior results for the same parameters. These variables showed higher values than those obtained in the present study, possibly due to the age difference between the orchards.

According to Stuchi et al. (2008), a common problem in Mandarin cultivation is low yield associated with production alternation.

Fruit retention must be carefully adjusted in the “on” years (higher production years) through fruit thinning, pruning, branch ringing, and exogenous applications of plant growth regulators (SPÓSITO et al., 1998).

The least productive canopy was TCOP, but no statistical difference was observed between the following canopies: Span Americana, Kincy, South Africa, Piemonte, Fremont, Swatow, and Ponkan (Table 5).

When comparing the enrichment of organic fertilizer with HA and PGPB inoculation, the average fruit weight of the plants fertilized with the enriched fertilizer was higher, highlighting the positive effect of the addition of HA and the bacterial consortium on tangerine productivity in general, regardless of the grafting type.

Fikry et al. (2022) observed that the nitrogen fertilizer significantly influenced the weight of Murcott mandarins. These authors also reported that the best results were obtained with 75% ammonium nitrate + 25% poultry litter, followed by 50% ammonium nitrate + 50% poultry litter + biofertilizer with effective microorganisms (EM).

However, when the percentage of chicken manure + biofertilizer increased to 75%, the weight and size of the fruits decreased compared to the other treatments. Olivares et al. (2015) reported that maximum fruit production was obtained when tomato plants were grown with vermicompost enriched with H. seropedicae strain HRC 54, followed by two foliar applications of bacteria combined with SH extracted from vermicompost.

In the same farm where the mandarin experiment was installed, i.e. in conditions with similar soil and climate, Canellas et al. (2022), in a case study on the effects of HA applied together with plant growth promoting bacteria as an efficient tool in the transition to organic farming systems, observed that there was an increase in banana production (50%), papaya production (90%) and passion fruit production (25%), with significantly higher yields over four production cycles.

Conclusion

The composting process of slaughterhouse residues, such as blood and intestinal contents, combined with plant material from urban pruning, proved effective in transforming this material, thus reducing the environmental liability generated by this activity.

After chemical analysis and laboratory and field tests, it was confirmed that the organic compost produced can be used as fertilizer, including with official registration by the Brazilian Ministry of Agriculture (MAPA).

Spectroscopic characterization by CPMAS 13C-NMR and TMAH-assisted thermochemolysis revealed the hydrophobic properties of the organic matter and showed that the compost product inherited molecular characteristics from the original biomasses, such as stable and less decomposed lignin fragments.

Enriching the fertilizer with humic acids (HA) and inoculating with plant growth-promoting bacteria (PGPB) resulted in a higher productivity of mandarins, regardless of the graft cultivar evaluated. When comparing the cultivars’ productivity in general, i.e., without considering the fertilization effect, Murcott, Oneco and Ponkan had the best performance.

These results show that biological enrichment of organic fertilizers is a viable alternative to increase the efficiency of biological inputs in agriculture.

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Edited by

• Scientific Editor
  Alexandre Pio Viana

• Associate Editor
  Marta Simone M. Freitas

Publication Dates

• Publication in this collection
  13 Oct 2025
• Date of issue
  2025

History

• Published
  28 Aug 2025
• Received
  14 May 2024
• Accepted
  14 Apr 2025