Selected Properties of Bioconversion Products of Lignocellulosic Biomass and Biodegradable Municipal Waste as a Method for Sustainable Management of Exogenous Organic Matter

Abstract

In recent decades, there has been a significant decrease in humus resources in the terrestrial environment, including in agriculturally used soils, due to increased mineralisation of soil organic matter (SOM). Using composting as a method for recycling lignin-cellulosic biomass, the application of innovative microbial stimulation seems reasonable for obtaining the most useful product. The aim of this study was the qualitative and quantitative analysis of humic acids during the composting of exogenous organic matter (EOM) of energy willow biomass (WBC) and biodegradable municipal waste (MSWC). Samples were collected at different maturity stages, and the following determinations were performed: total organic carbon (TOC) and total nitrogen (TN), carbon of fulvic (CFA) and humic (CHA) acids, carbon of residual fraction (CR), the elemental composition of humic acids, humification index (HR₁), and ω (oxidation) ratios. Results indicated the direction and intensity of biotransformation processes were determined by the availability of nitrogen compounds. The innovative use of microbial preparation has resulted in a more useful final product. Inoculation of lignocellulosic substrates stimulated the synthesis of humic acids, and the decomposition of the CR fraction, as well as HR₁ and ω ratios, may be used to assess the recycling efficiency.

1. Introduction

The presence of soil organic matter (SOM) is considered to be a key element that influences nutrient cycling, determines soil productivity, and directly or indirectly affects other environmental components [1,2,3,4,5,6,7]. The observed increase in the intensity of soil humus (HS) decline is a phenomenon of concern to the scientific community. Furthermore, many authors indicate that qualitative and quantitative changes in soil organic matter (SOM) are among the most recognisable symptoms of degradation and ask for sustainable strategies to prevent actual soil organic matter decline rates [8,9,10,11]. It is estimated that 70% of the SOM resource is in the form of humic substances, which are in constant transformation [11,12,13,14,15]. Current trends and guidelines related to the proper use of soils indicate that efforts should be made to increase the soil organic matter content [4,10,15,16,17,18]. The most reasonable solutions seem to be those suggesting the sustainable management of humus compound resources, especially in intensively used and/or modified areas; optimisation of the humus content by using components of sufficient quality and/or products made from exogenous organic matter (EOM); and developing optimal and reasonable methods for the use of SOM resources based on comprehensive, integrated long-term management [6,7,8,10,14,15,19,20,21,22].

Due to the heterogeneous and polymorphic nature and the mechanism of formation, HS form a complex system in the environment, consisting of materials in varying degrees of decomposition and transformation [5,13,16,23,24,25,26]. A large number of microorganisms are involved in their formation processes, as well as a number of chemical compounds that are either direct substrates or indirect products of humification [13,23,26,27,28]. Among the most important of these are the following organic components, defined qualitatively and quantitatively: lignin, carbohydrates (cellulose, other sugars), protein component (proteins, amino acids), and hydrophobic component (fats, fatty acids, waxes, resins). For the identification of humification products, the method [26] most commonly used is that recommended by the International Humic Substances Society (IHSS), which allows quantitative and qualitative characterisation of individual groups and fractions based on susceptibility to extraction and isolation using different extractants [22,23,26,29]. On this basis, three main fractions are separated: (1) humic acids (HA), (2) fulvic acids (FA), and (3) humins (HU). HA and FA are extracted from soil and other solid phase sources using alkali (usually NaOH or KOH). Fulvic acids are soluble in the full pH range, while humic acids coagulate at low pH (separation by acidification of an alkaline solution), and humins are stable and insoluble in both acids and alkalis.

The agricultural or environmental use of bioconversion products of different biomass types requires caution and adaptation to specific conditions. The crucial parameters are the quantity and quality of HS, determining directly or indirectly the expected economic (agriculture, horticulture) or environmental effects [4,11,16,17,19,20]. Primarily, the different abilities of HS to form bonds and complexes with mineral components of the environment should be taken into consideration [8,22,24,26,27,30,31,32,33,34]. Fulvic acids, whose molecules have a high share of aliphatic components and a poorly developed and condensed aromatic core, show a very high affinity for bond formation. Due to their reactivity, they determine the availability and mobility of nutrients—mainly cations—for plants and very intensively modify the populations of microorganisms inhabiting the ecosystem [1,2,3,8,13,22,23]. Due to their susceptibility to degradation, leaching, and mineralisation processes, the influence of FA is very intense, short-term, and dependent on the pH of the environment. The more stable HS fractions are represented by humic acids. They present a high degree of polymerisation and condensation of the aromatic core, a low share of aliphatic structures, and a lower susceptibility to oxidation and degradation [13,23,24,35,36,37]. Furthermore, the bonds present in HA are not only chemical in nature but are also determined by specific molecular interactions, less dependent on soil properties, such as pH and salinity.

The significant progress in the production and use of biologically processed exogenous biomass for agricultural and non-agricultural purposes has demonstrated the need to revise previous views on humification processes and the interpretation of humus formation phenomena based on quantitative and qualitative changes in individual HS fractions [10,19,20,27]. An official system is currently in use based on legislation that regulates the quality of the EOM substrate, controls the processing, and allows certification of the final product [27,38,39,40]. Additionally, chemical maturity indices for composts are used to assess the degree of product maturity, the direction and intensity of aerobic EOM biotransformation processes, the quantitative characterisation of the humic compounds formed and the effectiveness of the technical and technological solutions applied [26,37,38,39,40]. As the regulatory framework neglects the aspect of quantity and quality of humic compounds, a ‘hybrid’ system, including formal guidelines supplemented by additional physico-chemical parameters, seems to be the most rational solution when assessing and qualifying EOM biotransformation products.

Despite decades of research, the metabolic processes of humification have not been described. On the other hand, the conditions for these phenomena are very well understood, and methods have been developed to stimulate them. It is generally known which chemical, biological and technical methods can provide the best results. In addition, there are specially developed procedures for specific types of biodegradable substrates [12,13,14,22,25,26,40]. Using composting as a method to recycle lignin-cellulosic biomass by obtaining the most useful product possible, the application of microbial stimulation seems to be justified. It should be noted that the lack of knowledge of metabolic processes should not be a barrier to this type of research, but on the contrary, it should be an invitation to use biological solutions in biomass composting.

Inappropriate substrate selection and defective aerobic EOM processing technology result in products of poor quality. Their application to the environment or use as a horticultural substrate is highly controversial and undermines the ecological and economic sense of these efforts. Therefore, stimulating the process through a well-balanced chemical composition of the composted mass and the optional use of supporting microbiological preparations is very important. Thus, the aim of this study was to determine the qualitative and quantitative changes in organic matter resulting from the aerobic bioconversion of different types of biomass using microbial support through the innovative application of a stimulating microbial skeleton to lignin-cellulosic substrates. It was assumed that (a) regardless of the biomass used and the microbial inoculation, the most organic carbon is contained in the residual fractions represented by humins (CHU); (b) aliphatic structures will predominate in the molecules of the HA studied; (c) the inoculation of lignin-cellulosic biomass influences the quantitative changes and the chemical composition of HA. The results obtained may improve the existing knowledge on the quality of EOM humification products and the possibility of their rational and sustainable use.

2. Materials and Methods

The objects of the research were three composts from biodegradable substances produced according to the semi-dynamic open pile system [40,41] with the following composition and share (% by mass) of individual components (Figure 1):

• MSWC (Municipal Solid Waste Compost): 50% of green waste and 50% domestic kitchen waste, m:m = 1:1;
• WHBC (Willow and Hay Biomass Compost): 50% of willow chips (Salix viminalis L., chips diameter 8–30 mm) and 50% of hay residues from feed production (share of main plant groups: 50% grasses, 25% legumes, and 25% herbaceous plants), m:m = 1:1;
• WHIBC (Willow and Hay and Inoculated Biomass Compost): 50% of willow chips (Salix viminalis L., chips diameter 8–30 mm) and 50% of hay residues from feed production (share of main plant groups: 50% grasses, 25% legumes, and 25% herbaceous plants), m:m = 1:1, inoculated with a single application (beginning of experiment) of Peniophora gigantea using the commercial preparation Pg-POSZWALD Eko (Wiązowna, Poland) according to the manufacturer’s requirements; the ratio of vaccine/willow biomass is covered by patent application No. P.448767—a legal procedure is currently underway.

2.1. Experiment Design and Sampling

The total duration of the experiment was 14 weeks. The components collected for each variant were mixed in suitable mass proportions to increase homogenisation (Figure 1a,c). Additionally, the willow biomass was fragmented into chips with the following percentages (in mass) of the individual fractions: above 1.9 cm (46.3%), 1.9–0.8 cm (42.1%), and below 0.8 cm (11.7%). The materials were watered (Figure 1d) to an optimum level of 40–60% of H₂O formed into piles with dimensions L × W × H = 5.0 m × 2.0 m × 1.0 m. Each pile was mechanically turned every 5 days for the first 8 weeks and every 10 days between weeks 9 and 13 of the experiment.

During the experiment, the temperature (daily) and moisture content (every 5 days) of the composted mass were monitored using a Vaisala HUMICAP HM42 mobile device (Vaisala GmbH, Hamburg, Germany). Measurements were performed at three randomly selected locations at a depth of approximately 30 cm. Due to the different intensities of the biotransformation processes of the tested variants and the time required to obtain stable final products, samples were taken based on the thermal composting phase of the tested variants (

Table 1. Sampling scheme during the experiment.

Variant	Composting Phase	Composting Day	Temperature [°C]
MSWC	initial	1	21.7
	thermophilic	7	56.2
	stabilisation	80	22.5
WHBC	initial	1	20.4
	thermophilic	42	55.2
	stabilisation	102	22.1
WHIBC	initial	1	20.2
	thermophilic	42	57.3
	stabilisation	105	21.7

).

Based on moisture measurements, water deficiencies were supplemented, while temperature changes determined the time of sampling. Samples for laboratory analyses were taken directly from the piles from three randomly selected locations at the defined composting phases, and the total number of samples was 27 (three samples per phase for the three variants). The collected materials were dried, ground, and sieved through a 2.0 mm sieve.

2.2. Basic Laboratory Analyses

In the prepared samples, the following chemical parameters were determined: pH (1 mol dm⁻³ KCl) using a potentiometric method (S20 SevenEasy™ pH meter, Mettler-Toledo Inc., Greifensee, Switzerland), total nitrogen (TN), and total organic carbon (TOC) using a Vario Macro Cube CN analyser (Elementar Analysensysteme GmbH, Langenselbold, Germany), and the TOC/TN ratio was calculated. The effect of the carbon content of inorganic compounds (mainly carbonates) was eliminated by applying the procedure recommended by the device manufacturer [File S1]. The determined TOC and TN contents and changes in the TOC TN⁻¹ ratio were used to assess the quality of the tested materials based on chemical parameter standards for product qualification in EU countries [40,42].

2.3. Humic Substance Analyses

From the collected compost samples, humic substances (HS) were extracted (C_ALK fraction) and separated into fulvic (FA) and humic acid (HA) fractions according to the method [23,28,43] recommended by the International Humic Substances Society (IHSS). The organic carbon of humic and fulvic acids (CHA and CFA, respectively) was determined with a Vario Macro Cube CN analyser, and the CHA/CFA ratio (HR₁), the share of CHA and CFA (P_HA and P_FA) in TOC, and the non-hydrolysing/residual organic carbon (CR) content—represented (P_CR) mainly by humins—were calculated according to the following formulas:

• HR₁ = CHA CFA⁻¹;
• P_HA = (CHA 100) TOC⁻¹;
• P_FA = (CFA 100) TOC⁻¹;
• P_CR = (CR 100) TOC⁻¹.

The obtained hydrated humic acids were purified from minerals (0.1 mol dm⁻³ HCl + 0.3 mol dm⁻³ HF), then dialysed (7 Spectra/Por Dialysis MWCO: 10,000 membranes) and freeze-dryed to obtain HA preparations in solid phase. Subsequently, the following determinations were made in the solid HA samples: ash content (combustion 550 °C 5 h⁻¹), elemental composition with 2400 CHN Perkin Elmer (Waltham, MA, USA) analyser, and the atomic ratios H/C, N/C, O/C, and O/H and oxidation ratio “ꞷ” were calculated according to the formula

ω = (2 × O + 3 × N − H) × C⁻¹,

where O, N, H, and C are the shares (atomic %) of O, N, H, and C in HA molecules.

According to a number of papers [4,30,33,34,41], the interpretation of the results obtained using chemical maturity indices refers to a specific moment in the experiment and is commonly used, especially for open-pile composting.

2.4. Statistical Analysis

The obtained results of the studied chemical parameters were statistically processed using Statistica 13 software. Differences between subjects were checked with the Tukey test at a significance level of p < 0.05 using the ANOVA programme, correlation matrices were performed for selected parameters (StatSoft Inc., Tulsa, OH, USA, ver. 12 pl), and the standard error for selected parameters was determined (EXCEL, MS Office, Redmond, WA, USA, ver. 16 pl).

3. Results and Discussion

3.1. Changes in the Temperature and Moisture of the Investigated Composting Materials

Temperature and moisture of the composted mass are the key physical parameters conditioning the intensity and direction of the bioconversion of the substrates used. They affect directly and indirectly the microbiological and physico-chemical activity and, consequently, determine the proper course of the process and the quality of the product obtained, especially the quantity and quality of the HS and the hygienic safety [26,44]. Therefore, it is very important to determine the start time of the thermophilic/hygienisation phase (over 55 °C), its duration, and the maximum temperature reached by the composted mass. The results of the measurements showed differences between the variants used in the experiment (Figure 2).

The average temperature of the initial mixture was about 21 °C in all investigated variants. The thermophilic phase in the WSWC variant was reached on the 7th day of the process and continued until the 18th day (duration: 12 days), and the highest temperature of 69.7 °C was noted on the 11th day of composting. In the WHBC variant, the thermophilic phase was initiated on the 41st day and lasted for 4 days, with a maximum temperature of 56.1 °C on the 43rd day of composting (Figure 1).

In the inoculated material (WHIBC), the thermophilic phase started after 36 days of composting and lasted until the 54th day with a peak temperature of 58.3 °C on the 49th day of composting. Based on the temperature changes during the experiment, it should be concluded that the MSWC variant reached thermal stability after approximately 78 days, the WHBC after 100 days, and the inoculated WHIBC variant after 105 days.

The moisture content of the composted mass is commonly recognised as a key physical parameter of composting, determining the efficiency of the whole process. It is acknowledged [26,44] that maintaining a water content of 40–60% H₂O in the composted mass, particularly in the thermophilic phase, is the most optimal for the proper intensity of biotransformation. Due to the regular control of the values of this parameter during the experiment, the moisture content of the composts studied was in this range, regardless of the thermal phase of the process (Figure 2).

It should be noted, however, that during the period of highest temperatures in the thermophilic phase, the moisture content of the composted mass decreased below 40% H₂O, with the water deficiency being immediately replenished to a level of about 50% in each compost (Figure 3). Additionally, the final products of all variants investigated showed an optimum moisture content of approximately 40%.

Considering both the changes in temperature and water content of the tested composts, it can be concluded that the composting process was correctly conducted. All variants tested have undergone a hygienisation phase and achieved thermal stability while maintaining optimum moisture conditions. Moreover, the applied open-pile technology, supported by regular monitoring and optimisation procedures (watering, mixing), meets the requirements for biological, aerobic recycling [26,40,45] for the tested biomass variants.

3.2. Changes in the pH_KCl, TOC, TN Contents and the TOC/TN Ratio During Composting of Investigated Biomass

Changes in pH during composting are directly related to the quality of the substrate and the process conditions. Values of pH between 5.5 and 8.0 in the initial material are considered to be the most optimal, while the final product should be slightly acidic or neutral: a pH between 6.0 and 7.0 is recommended [26,40,41]. This study showed changes in the values of this parameter, and their dynamics varied according to the types of organic substrates in the particular composting variants (Figure 4).

It should be noted that the observed pH changes during the composting of MSWC biomass (Figure 4a) were characteristic of this type of substrate [26,33,41,45], clearly indicating both an optimal substrate composition and a well-conducted composting process. Changes in this parameter occurred differently in both variants with willow biomass. The analysis of the results obtained (Figure 4a) indicates acidification in the initial phase of both WHBC and WHIBC. Differences became evident in the thermophilic phase. In the inoculated variant, pH = 6.21, while in WHBC, pH = 5.68. This difference remained until the end of the experiment. In the WHBC variant, the pH reached 5.26, while in the WHIBC, pH = 5.88, which was close to the optimum for the final products of the biomass composting process. Statistical analysis of the correlation between the parameters (

Table 2. Correlation coefficients between the investigated chemical properties and composting phase in the investigated composts.

Parameter	Composting Phase	TOC	TN	pH	CHA	CFA	CR	C	H	O	N	ꞷ
MSWC
Composting phase	1.000	−0.813	0.938	−0.179	0.931	0.811	−0.836	−0.800	−0.996	0.961	0.981	0.979
TOC		1.000	−0.955	−0.419	−0.965	−0.974	0.999	0.947	0.842	−0.912	−0.877	−0.886
TN			1.000	0.152	0.991	0.956	−0.966	−0.910	−0.949	0.966	0.977	0.963
pH				1.000	0.185	0.377	−0.383	−0.374	0.127	0.052	−0.035	−0.020
CHA					1.000	0.952	−0.975	−0.939	−0.948	0.981	0.963	0.971
CFA						1.000	−0.977	−0.914	−0.828	0.886	0.879	0.866
CR							1.000	0.950	0.863	−0.927	−0.896	−0.904
C								1.000	0.834	−0.930	−0.863	−0.901
H									1.000	−0.978	−0.982	−0.990
O										1.000	0.970	0.997
N											1.000	0.981
ꞷ												1.000
WHBC
Composting phase	1.000	−0.863	0.967	−0.052	0.040	0.938	−0.896	−0.927	−0.803	0.836	0.973	0.859
TOC		1.000	−0.825	−0.086	−0.165	−0.754	0.996	0.805	0.900	−0.885	−0.909	−0.908
TN			1.000	0.069	0.100	0.905	−0.860	−0.906	−0.820	0.850	0.908	0.865
pH				1.000	0.671	−0.227	−0.066	−0.204	−0.463	0.446	−0.093	0.399
CHA					1.000	−0.085	−0.167	−0.318	−0.419	0.437	−0.017	0.389
CFA						1.000	−0.804	−0.819	−0.656	0.690	0.921	0.720
CR							1.000	0.837	0.903	−0.894	−0.933	−0.917
C								1.000	0.864	−0.914	−0.903	−0.918
H									1.000	−0.993	−0.808	−0.993
O										1.000	0.831	0.998
N											1.000	0.860
ꞷ												1.000
WHIBC
Composting phase	1.000	−0.954	0.889	0.000	0.640	−0.845	−0.888	−0.642	−0.939	0.754	0.917	0.829
TOC		1.000	−0.973	−0.256	−0.799	0.662	0.982	0.813	0.893	−0.876	−0.969	−0.921
TN			1.000	0.437	0.873	−0.528	−0.987	−0.884	−0.860	0.923	0.982	0.948
pH				1.000	0.741	0.479	−0.426	−0.679	−0.115	0.567	0.382	0.478
CHA					1.000	−0.183	−0.891	−0.931	−0.713	0.927	0.865	0.907
CFA						1.000	0.518	0.161	0.757	−0.323	−0.593	−0.438
CR							1.000	0.896	0.854	−0.932	−0.973	−0.955
C								1.000	0.683	−0.981	−0.834	−0.950
H									1.000	−0.810	−0.924	−0.876
O										1.000	0.901	0.991
N											1.000	0.940
ꞷ												1.000

●—significant at p < 0.05.

) also showed that, in all the samples analysed, changes in pH were not significantly influenced by the composting phase. Therefore, it can be assumed that the differences observed in the willow biomass samples were due to the fact that biochemical transformations in the WHIBC were stimulated by inoculation with microbial preparations.

Quantitative changes in TOC and TN and the resulting changes in the TOC/TN ratio are the most commonly used chemical parameters when describing biotransformation processes during composting [46,47,48,49]. It is assumed that, regardless of the total carbon and nitrogen content, the most optimal TOC TN⁻¹ values in the initial phase are 30 to 40, and in the stabilisation phase, they are less than 12. Based on the results, a decrease in TOC content, an increase in TN, and a decrease in TOC TN⁻¹ values were observed in all variants tested (Figure 4b–d). Willow is a very specific substrate, rich in lignin, and therefore highly resistant to biotransformation processes. This explains the differences in the rate of decomposition between the variants. These trends are significantly negatively correlated with composting time for TOC, positively for TN and not correlated with pH (Table 2).

During the composting process, a transformation of N can be observed, leading to both a decrease and an increase in content. One possible reason for this phenomenon can be an incorporation of N-containing compounds (of fungal and microbial origin) into structures of humic acids. Those humus structures protect bounded nitrogen against microbial degradation [49,50]. In our experiment, a significant correlation coefficient between TN and N content in the HA molecules (Table 2 and

Table 3. Changes in the selected chemical properties in the investigated composts and humic acids analysed using the Tukey post hoc test (Statistica 13).

Variant	Composting Phase	Compost					Humic Acids
		TOC	TN	CHA	CFA	CR	C	H	O	N	ꞷ
		g kg−1					Atomic %
MSWC	initial	343.80 a	11.23 a	62.80 a	66.70 a	216.23 a	33.74 a	44.50 a	18.90 a	2.87 a	0.05 a
	thermophilic	276.92 b	15.32 b	72.90 b	69.05 b	134.27 b	30.88 b	42.79 b	22.88 b	3.46 b	0.41 b
	stabilisation	244.61 c	20.56 c	83.05 c	70.10 c	92.99 c	30.22 b	36.22 c	28.58 c	4.99 c	1.24 c
WHBC	initial	416.40 a	4.91 a	33.42 a	60.17 a	337.82 a	35.00 a	44.97 a	19.16 a	0.87 a	−0.13 a
	thermophilic	403.30 ab	5.30 a	35.18 a	59.44 a	308.69 a	34.70 b	42.83 b	21.57 b	0.91 a	0.09 b
	stabilisation	374.90 c	6.17 b	34.17 a	67.74 b	273.00 b	33.93 c	42.04 c	22.83 c	1.21 b	0.20 c
WHIBC	initial	409.14 a	4.90 a	33.42 a	70.17 a	305.56 a	34.15 a	43.57 a	21.38 a	0.90 a	0.05 a
	thermophilic	382.40 b	7.90 b	43.72 b	74.81 b	263.88 b	31.21 b	43.43 a	24.17 b	1.20 b	0.29 b
	stabilisation	326.89 c	10.31 c	47.50 b	63.34 c	216.05 c	30.25 b	42.08 b	26.08 b	1.59 c	0.48 c

For each variant tested, means followed by the same letter are not significantly different at p  <  0.05.

) confirmed this theory. Some authors suggest that composting can concentrate N-rich structures in stable structures with time of the process [51,52], eventually affecting a decrease of the C/N parameter. However, it should be noted that the intensity of TOC and TN changes varied among the variants studied. The results were nearly optimal in the MSWC, while they differed significantly in the willow and hay biomass variants [26,31,52,53]. The differences resulted from the type of substrates used and, in particular, from relatively low TN content and, consequently, high TOC TN⁻¹ in the WHBC and WHIBC variants at each stage of the experiment (Table 3, Figure 4c,d).

3.3. Quantitative Changes in Humus Compounds in the Investigated Composts

It is widely recognised that any process for converting biomass into products for environmental purposes should increase the content of the so-called useful SOM fractions [1,19,37,44,47]. Parameters based on qualitative and quantitative analysis of humic substances produced during the composting process seem to be a useful tool for assessing the recycling efficiency of biodegradable compounds in the composts studied [6,7,8,10,22,23,24,25,26].

Regardless of substrate composition and composting time, the non-hydrolysing fraction (CR), represented by the most stable organic and organic-mineral compounds referred to as humins, was the predominant fraction in all samples investigated (Figure 5c and Figure 6). In all samples tested, the share of CR in TOC exceeded 50% in the initial and thermophilic phases, while in the stabilised material, it was 45% in the MSWC variant and around 70% in the willow variants, respectively (Figure 5 and Figure 6). However, as the composting process progressed, there was a decrease in the content and share of this fraction (Figure 5 and Figure 6), and the intensity of these changes depends on the chemical composition of the composted mass. The results obtained, confirmed using statistical analyses (Table 2 and Table 3), indicate that the biotransformation of the CR fraction is stimulated by both nutrient content (MSWC variant) and the inoculation of willow-containing material (WHIBC variant). Although many authors confirm the dominant role of CR in TOC content [14,22,23,30,34], the conclusive interpretation of changes in the content and share of this fraction is still a major scientific problem.

The results obtained indicated significant changes in the content (Figure 5a,b and Figure 6) and proportion of humic acids (CHA and CFA), and the dynamics of these changes depended on the chemical composition of the composted mass and microbial stimulation in the WHIBC variant. An increase in the content and share of CKH was observed in all samples, which corresponds to the results obtained by other authors [1,2,8,20,26,32]. The highest contents and contributions were found in the stabilised materials in the MSWC and WHIBC variants: 83.05 and 47.50 g kg⁻¹, respectively, corresponding to approximately 30% and 20% of TOC. The biggest differences were related to the transformations of CFA. While in the MSWC and WHBC variants, the content of this fraction increased with composting time, in the WHIBC, a significant increase was observed in the thermophilic phase, followed by a decrease in stabilised material (Figure 5b). While in the MSWC and WHBC variants, the content of this fraction increased significantly with composting time (Table 2 and Table 3), in WHIBC, a clear increase was observed in the thermophilic phase, followed by a decrease (Figure 5b). Furthermore, despite the different intensities of transformations, the share of CFA in TOC in the willow variants (WHBC and WHIBC) was very comparable and reached a value of approximately 19% of TOC in the stable material (Figure 5).

A consequence of the observed quantitative changes in CHA and CFA is changes in the HR₁ index (Figure 5d). Many studies [2,23,24,26,30,31,33,34] highlight its versatility and usefulness in determining the direction of organic matter transformation processes in soils and composts, the effectiveness of methods used to stimulate composting processes, and in assessing the stability of humic substances. It is generally accepted that a value of HR1 ≥ 1 indicates optimum properties of humic substances.

Based on the results obtained (Figure 5b), it should be noted that the MSWC variant achieved optimal parameters due to the interrelationship of CHA and CFA (HR₁ = 1.2), while the WHBC and WHIBC variants were characterised by an excessively high content and share of the CFA fraction: HR₁ = 0.5 and 0.7, respectively. Furthermore, the quantitative changes in the ‘useful’ humic fractions CHA and CFA in the willow variants, and in particular the stimulation of these changes by inoculation in the WHIBC variant, indicate a reasonable use of aerobic technologies for the processing and recycling of lignocellulosic biomass [31,46,51].

3.4. Elemental Composition of Humic Acids Extracted from Investigated Composts

Previous research indicates that the nutrient richness of the composted mass and the technology used have a direct impact on the chemical composition of the humic acids formed [26,27,30,33,34,40]. It is assumed that as the maturation and stabilisation processes progress, the share (atomic %) of oxygen and nitrogen should increase, while that of hydrogen should decrease.

The obtained results indicate that the observed changes in elemental composition were in accordance with the existing state of knowledge, while the dynamics of these changes differed between the variants (Table 3, Figure 7). The lowest dynamics were observed in the carbon changes, which decreased from about 35 (initial phase) to about 30 (stable product) atomic % in all samples tested. The most significant differences were observed in changes in the share of N and O. Although an increasing trend was observed in all samples, the values obtained indicated a significant dependence (Table 2 and Table 3) on the abundance of composted matter (MSWC) and microbial stimulation (WHIBC). In the stable compost, the nitrogen share was 4.99, 1.21, and 1.59 atomic %, respectively, for MSWC, WHBC, and WHIBC. The results indicate that inoculation of willow composts stimulates nitrogen and oxygen incorporation processes in humic acid molecules.

The atomic ratios calculated from the elemental composition (Figure 7 and Figure 8) showed similar values and trends in changes in O/C and O/H ratios. Differences in trends were most apparent in the H/C ratio in the MSWC composite, which initially increased from 1.3 to 1.4 and decreased to 1.2 in the stable composite (Figure 7a). In contrast, differences in values were associated with the C/N ratio (Figure 8). They were caused mainly by a significantly higher share of N atoms (Table 2 and Table 3) in the HA molecules in the MSWC variant: C/N in the range 12.1–5.99 and C atoms in the WHBC (C/N 40.7 to 28.5) and WHIBC (C/N 39.2–19.0) variants. The effect of inoculation of the WHIBC variant on the values and intensity of the decrease in the C/N parameter with the progress of the composting process was also observed (Figure 8).

Humic acids extracted from the composts studied were characterised by an increase in the value of the ‘ꞷ’ parameter with progressing composting processes (Table 3). In stable composts, it reached 1.24, 0.20, and 0.48 for MSWC, WHBC and WHIBC, respectively. The observed difference in the willow composts was due to the stimulation of N and O incorporation processes in the HA structure, resulting from the inoculation of the WHIBC variant (Table 3). While an increasing trend indicates progressive humification processes, the intensity of these changes in time indicates qualitative differences in the humic acids formed. The humification processes occurring in the MSWC variant led to HA molecules with a higher oxidation index, suggesting that in composts containing lignocellulosic substrates (WHBC and WHIBC), humification promotes the formation of fractions less stable and more susceptible to further microbial transformations [23,24,25,26,30,31,54].

Studies have confirmed that the use of composting to recycle organic matter is one of the most sensible and widely used methods [4,13,14,15,26,30]. Although the use of biodegradable waste fractions as a substrate is well established and has been practised for many years, the use of lignocellulosic substances also appears to be reasonable. The results obtained from qualitative and quantitative analyses indicate the potential application of both municipal waste composts and those produced from willow. However, it should be noted that the content and properties of the useful fractions, mainly humic substances, can considerably determine the use of composts. Moreover, the interactions among CHA, CFA, CR, elemental composition, and the HR₁ and ‘ꞷ’ parameters can be useful not only to assess the direction and intensity of humification but also the effectiveness of the recycling of organic compounds from the EOM applied. It must, therefore, be considered that the product with the widest range of application potential and containing the most optimal content of active humic compounds was the MSWC compost [32,41,42]. Additionally, the use of lignocellulosic materials also seems to be a reasonable method of biological recycling [26,47,53,54], while the efficiency of this process was clearly higher in the inoculated WHIBC variant.

4. Conclusions

Based on the results of the presented studies, the following conclusions can be drawn: (1) The direction and intensity of biotransformation processes were determined by the availability of nitrogen compounds. (2) Inoculation of lignocellulosic substrates stimulated the synthesis of humic acids and the decomposition of the CR fraction. (3) All investigated composts meet the legal and ecological criteria for products of biological recycling. (4) The physico-chemical parameters of the MSWC variant allow its direct use as an organic amendment to improve the soil organic matter balance and quality parameters of the soil environment. (5) Considering the properties investigated, WHBC and WHIBC materials are more suitable as fertiliser components or inert growing media. (6) Studies demonstrated that HR1 and ꞷ ratios may be used to assess the recycling efficiency of organic compounds during the composting of different biomasses.

5. Patents

Some of the results obtained for WHIBC were used in patent application No. P.448767 registered on 6 June 2024 in the Polish Patent Office. A legal procedure is currently underway.