3.1 Carbon content in samples of different salinity
The organic carbon content in the steppe WEOM is almost 2.5 times higher than in LTBF WEOM, which is consistent with the total carbon content in the aggregates of the studied soils: 5.7 and 2.8%, respectively (Kholodov et al. 2020a). The proportion of WEOM carbon to the total soil organic carbon is 1.1% for LTBF and 1.9% for the Steppe, indicating a higher hydrophility and diversity of the Steppe OM. These figures fall within the range reported for the share of WEOM in total soil organic carbon (Zsolnay 1996).
Analysis of variance of DOC content data showed no significant differences in its content for prepared WEOM extracts after incubation at different salinities. That is the concentration of NaCl up to 35 g/l does not lead to a noticeable coagulation of WEOM of both Chernozems. In contrast, Esteves et al. observed a decrease in DOC concentration in freshwaters after incubation with the freeze-dried sea salt (I. Esteves et al. 1999). Thus, the Chernozem WEOM appears to be quite stable in solution even at high salinity, at least, when it is due to the presence of alkali metal ions only.
3.2 Absorption spectra and spectral indexes
The absorption spectra of the LTBF and steppe WEOM samples change in different ways with increasing salinity. The UV range absorption clearly decreases for the former and shows subtle change for the latter (Fig. 1).
Two-way ANOVA showed that a254, S350 − 400 and SR depend significantly on both soil type and salinity. Since DOC does not change significantly with increasing salinity, and photobleaching is excluded by incubation in the dark, the changes in the LTBF WEOM spectra are most likely caused by the OM conformational reorganization in response to the ionic strength raise. At higher salinity, the charges of ionized functional groups are shielded, reducing the repulsion between them. Additionally, the salting out effect occurs: hydrophobic components (fragments) close to compensate for the increase in free energy of system due to water structuring by electrolyte ions. This leads to a decrease in the solubility of non-electrolytes at a high electrolyte concentrations (Endo et al. 2012). The combined action of these two mechanisms results in the contraction and increased compactness of molecules/supramolecules adopting a spherocolloidal configuration, and masking some chromophores inside it (Ghosh and Schnitzer 1980; Patel-Sorrentino et al. 2002). This is probably the reason for the drop in the absorption intensity of LTBF WEOM.
SUVA254 and SR values for the LTBF WEOM indicate its greater aromaticity, hydrophobicity and molecular weight compared to the steppe WEOM, with their values changing differently with salinity (Fig. 2). For the LTBF WEOM, a254 and SUVA254 almost monotonically decreased as salinity increased (from 25 to 19 and from 2.2 to 1.55, respectively), while no significant changes were noticed for the steppe WEOM. The different behavior seems to be due to the fact that LTBF soil OM is more mature and hydrophobic, enriched with aromatic structures (Danchenko et al. 2020, 2022), and depleted of products of microbial origin (Zhelezova et al. 2017). This evidenced by the wider C/N ratio of this sample. A study (Kholodov et al. 2020b) found that the LTBF soil organic matter differs from the steppe soil organic matter in terms of lower diversity of structural components, which is likely true for WEOM as well. As mentioned earlier, this may explain the higher sensitivity of LTBF WEOM structure to salinity variations, which is reflected in the changes in optical characteristics.
In contrast, the steppe WEOM is highly diverse due to the regular input of fresh plant residues, root exudates, and microbial products, enriching it with hydrophilic and surface-active compounds. These compounds compensate for the salting out effect and provide greater conformational and aggregation stability as salinity increases, hindering the realization of the mechanism proposed for LTBF WEOM. As a result, chromophores of Steppe WEOM are less sensitive to changes in ionic strength. In favor of the influence of the relatively young OM of probably non-humic nature on the structure/composition stability and the constancy of the optical properties of WEOM, the results of (Gao et al. 2015), where some increase in absorption was observed with an increase in the solution ionic strength of commercial preparations (i.e. purified and fractionated OM) of surface water humic substances.
The SR index, which negatively correlates with the hydrophobicity and molecular weight of the DOM (Helms et al. 2007) decreases for LTBF samples as the salt concentration rises indicating an increase in hydrophobicity and average molecular weight (hydrodynamic radius) of the DOM. The apparent increase in WEOM molecular weight may be attributed to partial aggregation.
Overall, the optical descriptors a254, SUVA254, S350 − 400, and SR and, consequently, WEOM structure of Chernozem with the most transformed OM (LTBF) respond to an increase in salinity, while the structure of the steppe WEOM is almost unaffected by the ionic strength increase.
3.3 Fluorescence spectra and structural indices calculated from them
Fluorescence emission spectra intensity decreases with increasing salinity for both samples (Fig. 3), which is coincident with the findings of (Huguet et al. 2009). However, in that study, the effect is mainly associated with the degradation of fluorophores under the UV radiation, excluded in our experiment. In our experiments, the decrease in fluorescence intensity, as well as absorption, was caused by the direct effect of salinity increase, which leads to masking of some fluorophores inside the spherocolloid DOM configuration (Patel-Sorrentino et al. 2002). That is, salinity growth itself provokes alteration of the physicochemical structure of fluorophores (Zsolnay 2003).
The shape of the fluorescence spectra depends little on salinity but clearly differs between the LTBF and Steppe samples (Fig. 3). The intensities in the regions attributed to fluorophores of different nature depend more on the origin of the organic matter than on salinity (Supporting Information), as reflected by the values and trends in the change of structural descriptors calculated from EEM data, such as HIX, BIX, and FI.
The HIX index was expectedly high for the LTBF WEOM samples ranging from 10.8 to 12.8; and much lower for the steppe ranging from 3.12 to 3.84. The higher HIX values obtained for LTBF WEOM are consistent with those reported for cultivated Chernozem WEOM (Qin et al. 2020), while the low HIX values (< 4) found for steppe WEOM are similar to those of autochthonous aquatic OM (Ohno 2002; Huguet et al. 2009). Consequently, microbial products and poorly processed substances of plant origin predominate in the WEOM of uncultivated soil (Steppe). It was noted in a number of works that the properties and/or composition of WEOM weakly depend on the land use and management (Toosi et al. 2012), while others, on the contrary, found a strong impact of these factors on WEOM (Kalbitz et al. 2000). Our findings clearly show that the shape of the fluorescence spectra and the HIX index clearly differentiate the WEOM of contrasting land use Chernozems.
The fluorescence index (FI) values are slightly higher for the LTBF samples (1.22–1.24) compared to virgin soil (1.16–1.19), and according to two-way ANOVA, the differences are significant. Similarly, the BIX index is slightly higher for LTBF samples compared to the Steppe ones. FI and BIX are considered indicators of the contribution of relatively fresh microbial OM. Unlike the expected negative correlation with the HIX index (Qin et al. 2020); FI and BIX in our case do not accurately reflect the features of the OM composition. This may be attributed to the geographical proximity of the experimental plots and the similar soil types as the sources of WEOM.
No significant dependence of HIX, BIX, and FI on salinity is observed for WEOM of both samples, but Steppe WEOM demonstrated a slight trend of HIX decreasing with increasing salinity.
Thus, the studied WEOM samples are best differentiated by the HIX index and shape of fluorescence spectra. Salinity increase leads to a decrease in the overall intensity of the fluorescence spectra of both WEOM samples, while the conventional fluorescence indices are insensitive to salinity changes.
3.4 Individual fluorescent components of WEOM
In the soil WEOM EEM spectra, 3 or 4 fluorescent components are most often identified by PARAFAC modeling (Sharma et al. 2017; Liu et al. 2019; Qin et al. 2020; Rinot et al. 2021). For both of our samples, the 5-component model best describes the array of spectral data for all salinity values. The spectra of five fluorescent components (C1, C2, C3, C4, C5) obtained by PARAFAC analysis are shown in Fig. 4. Almost all components had one main emission maximum with two distinct excitation maxima. The components identified were assigned to the fluorophores typical for DOM by comparison with spectra from the library OpenFluor (https://openfluor.lablicate.com/of/measurement) based on high values of the congruence index (Turkey index). A description of the components is given in Table 1.
The table illustrates the fact that spectra of components close to those obtained in this study are not always identified in the same way by different authors. Thus, components close to C1 are referred to as humic like M or A peak. In our opinion, this component is closer to the humic-like M-peak (Coble 1996; Coble et al. 2014).
Table 1
Description of individual fluorescent components identified by PARAFAC modeling in WEOM of chernozems at all salinity values.
Component # | Excitation wavelength, nm | Emission wavelength, nm | Description | Reference, Component ID |
C1 | 305 | 420 | marine humic-like biological activity and traditional peak M marine humic-like, peak M (Coble 1996) or microbial humic-like found in leaf leachates, potentially bioavailable similar to humic A peak (Coble 1996) humic-like M peak, autochthonous, microbial | (Chen et al. 2018) C2 (Lu et al. 2021) C5 (Gao and Gueguen 2016) C1 (Graeber et al. 2021) C4 (Søndergaard et al. 2003) C1 (Coble et al. 2014) |
C2 | 360 (270)* | 470 | terrestrial humic-like (close to peak C (Coble 1996) | (Wünsch et al. 2018) C2 (Gao and Gueguen 2016) C3 (Walker et al. 2013) C2 (Coble et al. 2014) |
terrestrial, humic-like, allochthonous |
C3 | 295 (425) | 510 (355) | biodegradable humic-like peak C+ | (Wünsch et al. 2018) C6 (Sharma et al. 2017) C4 (Coble et al. 2014) |
C4 | 275 (230) | 320 (400) | protein-like fluorescence, resembled the amino acids tryptophan and tyrosine (Coble 1996), containing fractions of autochthonous DOM (recent biological production) mixture of polycyclic aromatic hydrocarbon (PAH) and protein-like substances, similar to peak N (Coble et al. 2014) mix of humic-like component & tryptophan-like component | (Amaral et al. 2020b) C4 (Rinot et al. 2021) С4 (Amaral et al. 2020a) C5 (Amaral et al. 2021) C3 (Sharma et al. 2017) C3 |
C5 | 275 (340) | 400 | terrestrial humic-like, recalсitrant terrestrial humic-like terrestrial humic material similar to the products of oxidative degradation of lignin | (Eder et al. 2022) С3 (Chen et al. 2018) C1 (Drozdova et al. 2022) C1 |
*Figures in brackets are for secondary maxima |
The relative contributions of the PARAFAC components to the fluorescence spectra (fluorescence signature) of WEOM are little dependent on salinity, but differ markedly for Steppe and LTBF samples (Fig. 5). Noteworthy that the ratio between the contributions of fluorescent components in LTBF and Steppe samples in solution with different salinity remains almost constant. That is, the fluorescent signature of WEOM of the studied Chernozems does not change with increasing salinity.
Using PARAFAC modeling, we estimated the loadings of independent components to the fluorescence spectra of LTBF and Steppe WEOM samples with varying salinity. Component C1 was found to have the maximum loading for all experimental samples and was identified as a humic-like autochthonous OM (Table 1). Its loading for LTBF WEOM was 0.42–0.47, and of the Steppe WEOM, it was about 0.58–0.61. This component is commonly found in DOM and WEOM samples and is considered an indicator of recent microbial activity (Coble et al. 2014). Surprisingly, in the LTBF WEOM spectra, where microbial activity is attenuated, this component remains dominant, similar to the Steppe. This suggests that in Chernozems, this component may be correlated with OM of microbial origin in general. The second most abundant component, C2, was identified as humic-like allochthonous OM, which is one of the dominant components in the spectra of soil OM, as well as freshwater and deep ocean OM (Ohno and Bro 2006; Ishii and Boyer 2012; Coble et al. 2014; Sharma et al. 2017). The C2 loadings to the spectra of both soil samples were very close, ranging from 0.25 to 0.29. The closeness of these values indicates a weak dependence of C2 on the land use, which is consistent with the findings of Williams et al. (Williams et al. 2010). The third largest loading to the LTBF spectra was made by the C5 component (0.20–0.22), correlating with the most mature and stable aromatic OM (Eder et al. 2022). In the Steppe spectra, C4, representing protein-like fluorescence, was the third most significant component (0.26–0.29). The fourth most significant loading to the spectra of both soils, 0.2–0.23 - for LTBF and 0.14–0.15 for Steppe, was made by C3, a humic-like component similar to C2 (Sharma et al. 2017). This component is relatively rare in PARAFAC models and, based on the position of the maximum in the longest wavelength range, it characterizes the most decomposed OM, which is consistent with its larger fraction in the LTBF. The minor components, with fractions less than 0.1, were C4 in the LTBF WEOM spectra, which aligned with the decay of biological activity in this soil, and C5 in the Steppe WEOM. The small loading of C5 to the Steppe WEOM is likely due to its accumulation only in cultivated soils, or its more effective stabilization in the virgin soil. This stable and relatively hydrophobic OM is less sorbed on mineral particles compared to C2, and therefore, in the Steppe soil enriched with amphiphilic compounds of protein origin, C5 may be better stabilized and almost does not pass into the water extract. Furthermore, the C5 component is quite rare, as only three similar components were found in the OpenFluor database, and the similarity scores for excitation and emission were reduced to 91% and 94%, respectively. Based on the calculated loadings of the components, their dependencies on salinity were plotted (Fig. 6).
The fluorescent components of LTBF WEOM, with the exception of C4, showed a dependence on salt concentration. The contribution of C1 at low concentrations of NaCl (0–1 g/l) was significantly higher than at high concentrations (10–35 g/l), however, a further increase from 1 to 35 mg/l had no significant effect. A similar trend can be observed for the C2 component, which is sensitive to the presence of NaCl, but reacts weakly to an increase in its concentration. Components C3 and C5 exhibited a more pronounced dependence on salt concentration up to 20 g/l, but a further increase in the NaCl content to 35 g/L did not significantly reduce their loadings to the spectra. For none of the PARAFAC components of LTBF WEOM the loading decreased to negligibly small values even at a salt concentration of 35 g/l.
In contrast to fallow WEOM, the loadings of most of the fluorescent components of virgin soil WEOM did not significantly depend on salinity. Only for the C4 component, the loading in the variant with the highest concentration of 35 g/L was significantly lower compared to the variant without NaCl adding. The contribution of C5 to the Steppe WEOM fluorescence was negligible at high salinity.
It should be noted that the isolated PARAFAC components for our samples mostly correlate well with the traditionally isolated peaks: C1 - M; C2 - C; C4 - B + T and C5 – A; accordingly, the same trends are observed for them (Online Resource).