3.1. Mineralization
The addition of calcium carbonate and biochar to the composted mass changed the dynamics of the transformation processes of plant residues. Regardless of the enrichment of plant material with nitrogen under the influence of the additives, the activation of mineralization of organic substances was observed (
Table 2).
At the same time, this effect appeared not only in the initial stage of composting, when the plant material is still rich in compounds more accessible for biodegradation, but it also remained until the end of the experiment. Evidently, both calcium carbonate and biochar intensified the decomposition of less immobilized organic compounds. The greatest effect during the all the experiment was observed with the joint composting of biochar and plant residues, regardless of their chemical composition. The mineralization of rye and clover residues was by 18%, and oats by 16%, more than in the control variant, while the addition of calcium carbonate in rye and oat compost increased mineralization by 10%, and in clover compost, by 6%.
Considering the possible mechanism of the observed effect, it can be assumed that the intensification of the plant material mineralization processes during composting, together with biochar, occurs due to the introduction of an additional source of mineral nutrition for microorganisms into the system. However, the green mass of clover is rich in easily degradable compounds of both carbon and nitrogen. Nevertheless, even during the decomposition of this plant material, the stimulation of the mineralization process was observed. Therefore, without excluding the possible influence of biochar as a material source of nutrition and energy for microorganisms, it should also be assumed that there is another as yet unexplored mechanism for activating this process.
3.2. Humification
Before assessing the peculiarities of the humification processes, it should be noted that no HAs were detected in the initial plant materials (before composting) (
Table 3).
This is evidenced by the insignificant amount of organic compounds soluble in 0.1 M NaOH, and the fraction precipitated by acid, as well as their very low optical density index, which is uncharacteristic for HAs [
26,
43].
The additives introduced significantly changed the nature and volumes of HAs newly formed, as well as their further transformation (
Figure 1).
First, in the presence of calcium carbonate and biochar, the HAs formation was significantly more active than in the control variants. Against the background of additives, more organic substances were involved in humification (by 20–30%). The composts with additives practically did not differ from each other by the total amount of formed HAs (extracted by sodium pyrophosphate solution from the decomposing plant residues). Secondly, during the decomposition of plant residues together with both CaCO
3 and biochar, the formation of calcium humates (
Figure 2), forms of HAs that are highly resistant to the decomposing action of microorganisms and completely uncharacteristic for young weakly humified compounds, were observed [
24,
27,
28].
Significant differences in the effect of the studied reagents were evident in the assessment of the structure and properties of the newly formed HAs, as evidenced by the results of their optical density and elemental composition (
Figure 3,
Table 4). It should be noted that all compounds formed during the decomposition of plant residues, despite the low value of the optical density index, can be reasonably classified as brown HAs [
28,
41].
It should be emphasized that in all composts with added calcium carbonate, the optical density index of HAs extracted with sodium pyrophosphate (HAs1 + HAs2) practically did not change during the whole composting period, and was significantly lower than in composts with added biochar (
Figure 3). In addition, there were no differences in the values of optical density HAs1 + HAs2 when compared with labile forms of HAs1. Thus, the appearance of stable forms in variants with the introduction of CaCO
3 did not lead to a deepening of the humification processes, that is, it did not fundamentally change their nature. This allowed the probable mechanism of calcium humates formation under the influence of CaCO
3 to consider the regrouping of some fractions similar to the processes occurring during soil liming [
30]. With the process of HAs regrouping under soil liming, the part of HAs1 transit to HAs2, however, such conversion does not practically affect the properties of the resulting fraction. Such a state will be maintained only with a significant excess of Ca in the system, and with the decrease of its content the reverse regrouping will be observed.
On the contrary, in the variants with the addition of biochar, the humification process actively continued throughout the entire composting time. In all compost with a biochar, the sum of HAs (HAs1 + HAs2) optical density was significantly higher than of only labile forms (HAs1) and increased during the experiment. The maximum value of the HAs optical density index was reached in composts with clover (
Figure 3); all this indicates a higher degree of HAs aromaticity, an increase in their chemical “maturity”, and a deepening of the humification process during the composting period. This generally increases the resistance of newly formed HAs to microbiological and biochemical actions. Probably, the mechanism of formation of stabilized HAs is associated both with the rearrangement of fractions (since Ca is also present in large quantities in the composition of biochar) and with the positive biochar influence on microbiological activity in the compostable material.
Discussing the mechanism of stable HAs fractions formation at the early stages of humification, it is impossible to exclude sorption of newly formed compounds directly by biochar, which can increase their resistance to extraction. In addition, the role of biochar as a material source of HAs formation should not be ruled out when biochar is included in the humic complex. However further careful research is required to confirm these assumptions.
3.3. Elemental Composition and 13C-NMR Data of HAs
In addition to optical density characteristics, the elemental composition analysis was used to assess the quality of newly formed compounds. For this, at the final stage of the experiment (90 days), HAs preparations were isolated from all the obtained composts. To obtain HAs (HAs1 + HAs2) the extraction was performed with 0.1 M NaOH after pretreatment with 0.1 M HCl. The results of the elemental composition of the studied HAs are presented in
Table 4.
The elemental composition of all the studied preparations fully corresponded to the parameters of the HAs group, regardless of the plant material type and the added reagent [
23,
24]. Their composition is specific for newly formed HAs with a reduced carbon and oxygen content, as well as a high H/C ratio [
5,
7,
47]. Such compounds are characterized by a benzoidicity low degree, an insignificant content of acidic functional groups, and as a consequence a lack of affinity for calcium. Therefore, the presence of calcium humates in their composition is not obvious [
26,
48].
The analysis of the results obtained in this experiment showed that both plant residues and the tested reagents influenced the formation of the HAs elemental composition.
The influence of the plant residues composition on the HAs elemental composition in all experiment variants was manifested very clearly. In the HAs elemental composition the maximum differences were noted in the nitrogen content, which increased in clover compost and decreased in cereal compost. The clover plant residue compost was characterized by the highest HAs humification of all those studied. This is evidenced by a relatively high content of C, N, and O, and a lower content of H, as well as the increased atomic ratio of O/H and the reduced ratios of H/C and C/N. With that, according to the considered results, the HAs formed in all three variants of clover compost (control, with calcium carbonate, and with biochar) not only exceed the HAs of rye and oat compost but are quite comparable with soil HAs [
24]. Thus, the high nitrogen enrichment of the initial plant residues was of great importance for all stages of humification (mineralization and humus formation), and contributed to a more active and efficient flow of this process.
Evaluating the results of the studied reagents’ influence on the HAs elemental composition, it should be noted that the addition of calcium carbonate to compost did not provide a noticeably positive effect. More significant changes were observed under the influence of biochar. In the newly formed HAs obtained in the presence of biochar, an increase in C, but a decrease in the H proportion in the molecules, was observed. Additionally, the active influence of the biochar on the HAs formation has been clearly manifested when determining the oxidation degree, which is one of the most informative indicators characterizing the HAs elemental composition [
24]. In general, the process of humification is characterized by an increase in the oxidation degree of the formed compounds. According to this indicator, it is possible to assess the intensity and direction of the process. The increase in this indicator was observed in this experiment in all three compost variants under the influence of both calcium carbonate and biochar. Consequently, with the joint composting of all three plant residues types and reagents, the humification processes are taking place more actively that in the control variants. Apparently, the noted changes in the preparations’ elemental composition are caused by the replenishment of HAs with more humified and relatively chemically mature compounds.
The data obtained may indicate a partial restructuring of the HAs molecules’ carbon skeleton, a decrease in the proportion of aliphatic chains as an effect of their partial destruction, an increase in the aromatization degree, and in the oxygen-containing functional groups content. This is consistent with the study of the HAs optical density results (
Figure 3). In general, the noted changes indicate an increase in the stabilization and stability of the HAs. The maximum positive changes in the HAs elemental composition were revealed during the joint humification of clover and biochar.
It should also be noted that, for all the studied parameters of the elemental composition, biochar significantly differs from the HAs (
Table 4).
To obtain additional information about changes in the molecular structure of the formed under the influence of reagents, HAs were selected for analysis preparations isolated from the compost of clover mass. Additionally, with the aim of evaluating if it had a possible direct participation in the formation of Has, the initial biochar was stated. There samples were investigated using the
13C-NMR spectroscopy method (
Figure 4,
Table 5).
The obtained data are characteristic of the HAs molecular composition [
46,
49,
50]. The content of aromatic fragments in the newly formed HAs is significantly lower than in the initial biochar, but it still remains quite high (
Table 5). The organic matter decomposition degree varies in a wide range. The content of quinone, aldehyde, and ketone groups, is approximately comparable in all variants, which means that this part of the HAs is not affected by the experiment. At the same time, the carboxyl groups content is minimal in the variant, with the addition of biochar.
Furthermore, the content of aliphatic groups, double-substituted heteroatoms (including carbohydrates), and esters and ethers, is increased in the variant with biochar. However, in general, there were no significant differences between HAs in the results of compost 13C-NMR analysis, which apparently is associated with the active replenishment of HAs by forming labile compounds. For a deeper characterization of newly formed Has, further separation of them, according to the resistance to microbiological effects degree, is required (separation of fractions 1 and 2).
As for the comparative analysis of the composition and molecular structure of the HAs obtained in the process of composting, and the original biochar used for application to plant residues, the data obtained indicate their completely different nature (
Table 4 and
Table 5). The elemental composition of the original biochar differed significantly from the composition of the newly formed HAs. It was characterized by very high carbon content, extremely low oxygen content, and practical absence of nitrogen. The high content of polycyclic aromatic rings in the original biochar, the increased degree of aromaticity of the material, and the extremely low degree of the organic matter decomposition, also indicate a fundamentally different molecular structure of HAs and biochar. This does not give argument to consider the biochar as a material source of the HAs formation.