Carbon persistence of soils with long-term biosolids amendments in California agroecosystems

doi:10.21203/rs.3.rs-2706293/v1

Biosolids can build soil organic matter, but their ability to increase carbon and nitrogen in persistent fractions in deep soil is not well understood. We aimed to assess the mechanisms that influence soil carbon and nitrogen dynamics at three sites: Sacramento (irrigated, grazed grassland), Solano (rainfed, grazed grassland), and Merced (feed cropping system with alfalfa-corn rotation), where soils were amended with biosolids for 20 years using density fractionations, organo-mineral extractions, and correlations between calcium and soil organic carbon at three depths (0–10 cm, 30–50 cm, 75–100 cm). We found that amended soils had higher carbon and nitrogen content in the free-and occluded light fractions at all depths relative to the control in the Sacramento and Solano sites; however, the Merced site had a greater relative increase of carbon and nitrogen associated with the heavy fraction. Effect sizes show that biosolids increase carbon and nitrogen content in free- and occluded light fractions in the surface soil (0–10 cm), and in both light and heavy fractions in the deep soil layer (75–100 cm). Ratios of carbon to iron and aluminum show that chelation is an important mechanism of carbon stabilization in Sacramento and Solano sites throughout the soil profile. No (0–10 cm) to negative (75–100 cm) correlations were observed between calcium and soil carbon in the amended soils in the Merced site. Our results indicate that, while biosolids are typically incorporated at shallow depths, long-term application of biosolids can increase the amount of free- and occluded-light carbon fractions in deep soil.

Deep soil contains about 68% of global soil organic carbon (SOC) from 30 to 200 cm (Jackson et al., 2017). Research has shown that deep soil carbon (C) may also be mobile under certain conditions, indicating that studying topsoil is no longer sufficient to understand how soils would respond to climate change (Berhe et al., 2008). The magnitude and direction of deep soil C changes with management and global change remains (Hicks Pries et al., 2017; Soong et al., 2021). Neglecting to sample deep soil C can lead to misleading conclusions on the climate change mitigation potential of soil (Harrison et al., 2011). The history of sampling topsoil is likely a reflection of agricultural research, which focuses on the top 30 cm where plant roots and nutrients are found. There has been interest in managing agricultural soils in a way that would promote C sequestration; however, little is known about deep soil C in these soils.

Managed-induced soil C sequestration has been studied in agricultural contexts, including conservation tillage, improved grazing, fertilizer use efficiency, cover cropping, and organic matter amendment applications (Brar et al., 2013; Buckley Biggs and Huntsinger, 2021; Deen and Kataki, 2003; Mazzoncini et al., 2011; Powlson et al., 2012; Ryals et al., 2014). Organic matter amendment application has been proposed as a way to increase soil organic carbon (SOC) stocks through C inputs and indirectly through the promotion of plant production (Ryals et al., 2014). However, not all organic amendments have the same effects on soil C dynamics. Differences in organic material, treatment processes, and management strategies can influence soil biogeochemical cycling; thus, influencing the affects the magnitude of changes to soil organic matter (SOM) and the potential benefit of this practice.

The production of biosolids has increased, and their accumulation and disposal pose environmental challenges (Kominko et al., 2017; Torri et al., 2014). Hence, a lot of attention has been paid to recycling biosolids and using them as a soil organic amendment due to its valuable source of plant nutrients (Brown et al., 2011; Kominko et al., 2017). Long-term application of biosolids to agroecosystems has been shown to increase soil organic C, which in turn increases and improves soil fertility (Brown et al., 2011; Charlton et al., 2016; Cogger et al., 2013; F. Nicholson et al., 2018; Villa and Ryals, 2021). Although long-term biosolids application has been shown to provide benefits for soil fertility and increasing soil organic C content, little is known about the persistence of C in agroecosystems soils, particularly in deep soils (Tian et al., 2009; Torri et al., 2014). Some studies have emphasized the importance of sampling deeper depths due to the nature of subsoils having larger concentrations of clays and organo-mineral complexes that can contribute to the persistence of soil C over long periods of time (Kögel-Knabner et al., 2008; Poeplau and Don, 2015; Tautges et al., 2019; Villa and Ryals, 2021; Yost and Hartemink, 2020). There have been some studies that have focused on biosolids application and assessing the persistence of these carbon pools through fractionation techniques; however, these studies sampled soils in the top 30 cm of soil (Chiu and Tian, 2011; Pan et al., 2017; Silva et al., 2015; Wijesekara et al., 2017). Thus, there is little information on assessing how biosolids application may influence C sequestration of soils, specifically the soil dynamics that may play a role in the persistence of that C. Understanding these mechanisms would provide insight into the vulnerability of C and how to properly manage these soils to get the maximum benefits in terms of soil fertility and climate benefits (Tautges et al., 2019; Wang et al., 2021).

The rate at which SOC is mineralized to the atmosphere is determined by soil physiochemical stabilization mechanisms of C; such as occlusion of organic C within aggregates, sorption to mineral surfaces, and co-precipitation of organics with dissolved metals (Heckman et al., 2018). Soil fractionation techniques can improve our ability to determine how management-induced changes influence pools of C and nitrogen (N) and provide an indication of the physical location of C and N in the soil matrix and how easily mineralizable C and N are to microbes (Powlson et al., 2012; Ryals et al., 2014; Swanston et al., 2005). For this study, bulk soil C and N were partitioned into three fractions using a physical density fractionation procedure (Ryals et al., 2014; Swanston et al., 2005). These fractions are the free-light fraction (FLF), the physically protected occluded-light fraction (OLF), and the mineral-associated heavy fraction (HF).

In addition, current understanding suggests other physico-chemical parameters may be better predictors than clay content in order to determine the stability of SOC (Rasmussen et al., 2018). Studies have indicated that SOC content may be related to extractable metals and exchangeable cations, emphasizing that clay content is not always correlated with the persistence of SOC (Kaiser and Guggenberger, 2000; Rasmussen et al., 2006). Polyvalent cations, such as calcium (Ca²⁺), can play a significant role in the stabilizing SOC by creating bridges between organic compounds and mineral surfaces (Rasmussen et al., 2018; Rowley et al., 2021; Solly et al., 2020). In addition, Ca²⁺ is also thought to indirectly contribute to SOC associated with OLF through the promotion of soil aggregation through the bridging of organic matter to calcium (Oades, 1988; Rowley et al., 2021, 2018). Biosolids contain high levels of iron (Fe) and aluminum (Al), and studies have shown that long-term applications of biosolids could increase soil amorphous Fe and Al, leading to increased levels of SOC stability (Chen et al., 2019; Chiu and Tian, 2011; Silva et al., 2015; Tian et al., 2013; Wang et al., 2021). The properties of these organic materials such as, Fe and Al concentrations, may raise some additional questions and challenges on how to properly manage them. Hence, it is important to understand how biosolids influence C permanence in agricultural soils with different soil physicochemical properties, such as soils with a high concentration of Ca²⁺.

We determined the changes in the amount and distribution of C in soil fractions from three agroecosystems that have received twenty-years of biosolids application. We assessed these changes in the surface soil (0–10 cm), as well as in subsoil layers (30–50 cm and 75–100 cm). We also evaluated potential mechanisms that influence soil C dynamics across these fractions, including organo-mineral associations at all sites (referred to as Merced, Sacramento, and Solano) and Ca²⁺ concentrations in the HF at the site with high pH (Merced). We hypothesized that: 1) The application of biosolids C and N would increase C and N present in the FLF in surface soil across the three sites; 2) Treatment effects on C and N will be dependent on fractions and depth; where less persistent fractions would have positive effects at the surface soils and more persistent fractions would have positive effects in deeper soil depths. 3) Both amended and control soils from sites that have a lower pH will have stronger organo-mineral C content than the site with a high pH; 4) At the Merced site, Ca²⁺ concentration in the HF will be the primary stabilization mechanism for C regardless of treatment due to the high concentrations of calcium carbonate (CaCO₃).

Study sites and experimental design

The study sites were situated in Northern California, including locations in Sacramento County, CA (38°20’06.3”N, 121°10’06.5”W), Solano County, CA (38°11’52.3”N, 121°45’38.0W), and Merced County, CA (37°04’20.3”N, 120°31’43.0”W). Briefly, transects were selected using information from records of biosolids application to the site, USDA-NRCS soil maps, and discussions with the land manager at each site. Neither the biosolids treatment nor the control received inorganic fertilizer. Biosolids were applied at each site according to local regulations for biosolids land application. The Sacramento site soils that are classified as Alfisols from the Hicksville and Corning series. This site consisted of flood-irrigated annual grasslands managed for grazing beef cattle. Livestock was rotated every 60–70 days depending on feed availability. Soils at the Solano site are classified as Alfisols from the Antioch and Pescadero series and Vertisols from the Altamont soil series. The Solano site consists of rainfed annual grasslands used for grazing lambs and beef cattle. Livestock are rotated seasonally and based on feed availability, the grazing intensity is 0.3 animal units ha^− 1. The Merced site soils are classified as Mollisols from the Pozo series and Alfisols from the Fresno series. The site is flood irrigated and managed for livestock feed crops consisting of 1 y corn and 4 y alfalfa rotations for livestock silage and were tilled after each rotation. Sampling at these sites consisted of establishing an unamended control and an amendment treatment where biosolids were applied for twenty years as described in detail in Villa and Ryals (2021). Sampling was replicated at three fields within each of the three locations (n = 9). Soil samples were collected using a 57 mm-diameter auger at five depth increments, 0–10 cm, 10–30 cm, 30–50 cm, 50–75 cm, and 75–100 cm, at every ten meters along a 100-m transect (n = 10). Transect starting points and bearings were randomly selected from within each field, excluding a 5 m buffer from edges. A total of 900 soil samples were collected and transported to UC Merced for laboratory analysis. Total organic C and N concentrations were measured for all 900 samples as described in Villa & Ryals (2021). One paired transect (amended treatment and an unamended control) with the highest relative change was selected per site for density fractionations (n = 3).

Density fractionations

Soils from each paired transect and from depths 0–10 cm, 30–50 cm, and 75–100 cm were density separated at 1.85 g cm^− 3 as described in Swanston et al. (2005). Each of these fractions provides information on the potential turnover times of C and N in soils. The organic matter in the FLF closely resembles plant material which is “free floating” in the soil matrix and not protected by mineral surfaces. This material is more easily mineralizable to microbes and often has the fastest turnover time (Marín-Spiotta et al., 2008; Swanston et al., 2005). The OLF contains C and N that is physically protected in soil aggregates. This makes it more difficult for decomposers to access it; hence, this fraction has a slower turnover time than that of FLF. The HF contains C and N that are bound to mineral surfaces and typically has the longest residence time compared to FLF and OLF.

Briefly, 20 g of soil was gently mixed with 75 ml of sodium polytungstate (SPT). After centrifuging for one hour at 3,500 rpm, the FLF was aspirated off, filtered and rinsed with DI water five times, then dried at 50ºC for 48 hrs and weighed. The remaining sample was then mixed, sonicated at 70% pulse for 3 minutes, then centrifuged for one hr at 4,000 rpm. The OLF was aspirated off, filtered, and rinsed five times with DI water, dried at 50ºC for 48 hrs and weighed. The remaining soil pellet, known as the HF, was mixed with DI water, centrifuged 5 times, and dried at 150ºC for 24 hrs and weighed.

Total C and N of fractions

Fractions were analyzed for total organic C and N on the Costech ECS 4010 CHNS-O Elemental Analyzer (Valencia, CA, USA) coupled to a Thermo Scientific Delta-V Plus continuous flow isotope ratio mass spectrometer (Waltham, MA, USA) at the UC Merced Stable Isotope Laboratory. The HF from the Merced site was treated further using the acid fumigation method due to effervescence upon the addition of 4M HCl only in the HF, indicating the presence of inorganic C, the FLF and OLF did not effervesce following the addition of HCl (Harris et al., 2001). Acid-treated HFs were analyzed on the elemental analyzer to determine the concentration of SOC. Soil inorganic C (SIC) was calculated by subtracting the acid-treated C concentrations from total soil C concentrations.

Organo-metal complexes in heavy fractions

The HF was used for organo-mineral extractions following Heckman et al. (2018). Briefly, 40 ml of 0.1 M sodium pyrophosphate was added to one gram of air-dried ground sample and shaken for 16 hours. Then samples were centrifuged at 20,000 rpm for 20 minutes. Then the extraction was decanted and filtered through a 0.2 µm Whatman GD/X gradient syringe nylon membrane filter into 50 ml falcon tubes. Filtered extracts were then stored at -20 °C until they were ready for analyses. Extractants were diluted with DI at a 1:50 ratio and analyzed on an Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) for iron (Fe) and aluminum (Al) at the UC Davis Analytical Laboratory.

Calcium

The HF from the Merced site was analyzed for Ca²⁺ concentrations using the barium method (buffered to pH 7.0) to measure extractable at the UC Davis Analytical Laboratory (https://anlab.ucdavis.edu/analysis/Soils/430; Rible and Quick, 1960). This site was only selected due to its high levels of inorganic C and effervescence.

Statistical analyses

The results of this study are presented as treatment means (± standard errors). Shapiro-Wilk tests were used to determine the normality of data for all analyses. A three-way repeated measures analysis of variance (ANOVA) was performed on density fraction samples and organo-metal extractions between depths to determine the effects of treatment, fraction, and site between depths. A Tukey’s post hoc test was used to determine interactions between these factors. The determination of contributions of total organic C and total N from each fraction was assessed by dividing the C and N by the total for each depth within a site. Treatment effects were analyzed using a two-way ANOVA with treatment and fractions as factors, followed by Tukey’s post hoc tests to determine the interactions between these factors. We used a Pearson’s correlation (r) test to determine if %SOC and Ca²⁺, Fe, and Al were correlated in the HF. A three-way repeated measures ANOVA was used to examine the effects of treatment, depth, and fraction within a site for density fractions and organo-metal complexes. A Tukey’s post hoc test was used to determine interactions. The Pearson coefficient (R²) quantifies a correlation; a positive value closer to 1 is a strong correlation, 0 is no correlation, and negative correlations (closer to -1.0) is a strong inverse relationship. P-values from the correlation test demonstrates whether the correlation is statistically significant. Effect sizes of C and N content in fractions were calculated by taking the average of the percent change across sites within a treatment, depth, and fraction. The average of the biosolids’ percent change was divided by the control’s percent change, and the natural log was taken of that value. Statistical tests were performed using RStudio 3.3.1.

Mass distribution of C and N among fractions and depths

In the surface soil (0–10 cm), no significant differences in total organic C content were observed in the fractions between treatment (p = 0.36) or sites (p = 0.97); indicating that the surface soil at these three sites, regardless of treatment, have similar C contents. However, all fractions within a treatment and site were significantly different from each other; thus, demonstrating that the mass of C differs among fractions (p < 0.001; Fig. 1a). There was a significant interaction between treatment and site (p = 0.01). Similar to C, total N content in fractions was not significantly different between sites (p = 0.43) or treatments (p = 0.52), but significant differences were observed between fractions (p < 0.001; Fig. 3a) from 0–10 cm. Interaction between these factors were observed between treatment and site (p = 0.02), treatment and fraction (p = 0.01), and all three factors of treatment, site, and fraction (p < 0.001).

C content in the fractions in the 30–50 cm layer were not significantly different between treatment (p = 0.69) and site (p = 0.75), but the C content in fractions were significantly different from each other (p < 0.001; Fig. 1b). Similarly, N content in fractions from the 30–50 cm showed no significant differences between sites (p = 0.96) and treatment (0.91; Fig. 3b). Significant differences in mass of C and N content were observed in fractions (p < 0.01). No interactions were observed between factors.

The C content in the deep soil layer (75–100 cm) showed significant differences between treatment and fractions (p = 0.01; p < 0.001) with a marginal significance between sites (p = 0.08; Fig. 1c). The total N content at the soil depth of 75–100 cm had marginally significant treatment (p = 0.09) effects, with significant site (p < 0.001) and fraction (p < 0.001) differences, no interactions were observed between factors (Fig. 3c).

Proportions of fractions contributing to total C and N content varied depending on treatment and site. Biosolids application increased the amount of C and N content in the FLF relative to the control in the Sacramento, Solano sites (Tables 2&3). In contrast, biosolids amended soils at the Merced site had more C and N content associated with the HF across all depths. Control soils had more C and N content associated with the OLF compared to the biosolids amended soils, where most of the total C and N is associated with the FLF, indicating that biosolids application adds more C and N content that is attributed to FLF.

Treatment effects of biosolids application on C and N

Negative treatment effects (biosolids-control) are present between fractions and depths in many cases, indicating shifts on where SOC is present in the soil depending on site and depth (Fig. 4). When assessing the effect size from compiling C content associated with fractions across all sites, we found that biosolids application increases C in the FLF across all depths (Fig. 2a). The magnitude of the positive treatment effect on C in fractions increased with depth. In the deepest soil layer sampled (75–100 cm), all soil fractions had positive effect sizes.

Soil from 0–10 cm at the Sacramento site showed significant treatment effects in all three fractions where the FLF increased from 7.84 ± 0.22 Mg C/ha to 22.00 ± 5.86 Mg C/ha (p = 0.03); however, both the OLF (Control: 8.86 ± 1.44 Mg C/ha, Biosolids: 2.29 ± 0.29 Mg C/ha) and HF (Control: 3.84 ± 0.21 Mg C/ha, Biosolids: 1.38 ± 0.38 Mg C/ha) decreased significantly (both p < 0.001; Fig. 4a). No significant differences across fractions, treatment or depths were observed at the Solano site from 0–10 cm (Table 4a). The only significant treatment effects at the Merced site from 0–10 cm were observed in the FLF where the application of biosolids decreased FLF C to 1.55 ± 0.76 Mg C/ha from 10.18 ± 8.71 Mg C/ha in the control (p = 0.01; Table 4a). At the Solano site, only the FLF from 30–50 cm showed a considerable significant increase of 1609% (Control: 3.40 ± 1.40 Mg C/ha, Biosolids: 58.10 ± 39.64 Mg C/ha; p = 0.09; Fig. 4b). From 30–50 cm at the Merced site showed there were no significant treatment effects, the FLF and OLF decreased, however the HF increased by 85% (Control: 10.49 ± 2.01 Mg C/ha, Biosolids: 19.39 ± 4.57 Mg C/ha). Treatment effects both Sacramento and Solano demonstrated remarkable increases of C content in the deeper soil depth of 75–100 cm in the FLF (Sacramento: 1531%, p < 0.001; Solano: 712%, p = 0.90) and OLF (Sacramento: 172%, p = 0.04; Solano: 1054%, p = 1.00). However, the HF increased significantly at the Merced site from 5.74 ± 0.71 Mg C/ha in the control to 15.79 ± 11.06 Mg C/ha in the amended soils.

Treatment effects on soil total N have shown in some cases negative treatment difference in fractions, indicating loss of total soil N (Fig. 5). Effect sizes on N follows a similar trend as C, where the FLF has increased across all depths, while the effect size of the FLF and OLF remain relatively small and in some cases negative from 0–10 cm and 30–50 cm (Fig. 2b). Deep soil shows all fractions have positive or zero effect size, similar to C. From 0–10 cm at the Sacramento site, the FLF experienced an increase in treatment effect for total N (p = 0.01; Table 4b), but a significant loss of 48% in the OLF (p = 0.07) and 69% in the HF (p < 0.001; Fig. 4a). Marginal significance was shown in the FLF at the Solano site from 0–10 cm (p = 0.05; Table 4b). From 30–50 cm, the Solano site was the only site with a marginally significant increase of 1526% of total soil N content in the FLF (p = 0.06), but the Merced site experienced a loss of 85%, though not significant. All the HF in all three sites showed a decrease in total N from 30–50 cm (all p = 0.99). From 75–100 cm, total N has shown a significant increase of 2820% in the FLF at the Sacramento site (Control: 0.01 ± 0.00 Mg N/ha; Biosoilds: 0.36 ± 0.06 Mg N/ha, p < 0.001). The Merced site decreased in the FLF and OLF from 75–100 cm except in the HF where it increased from 0.80 ± 0.12 Mg N/ha to 0.96 ± 0.22 Mg N/ha. (Fig. 4c).

Correlations of organo-mineral complexes

At the Sacramento site, TOC was positively associated with each other along all depths to Fe and Al, though not significant (all p > 0.05; Table 5). Only from 75–100 cm was there a negative association between Fe and TOC though not significant (r=-0.397, p = 0.74). Biosolids amended soils at the Sacramento site had stronger correlations between TOC to Fe and Al from 0–10 cm and 30–50 cm though not significant except for Al from 30–50 cm (p = 0.04). From the 75–100 cm depth, negative correlations were observed for both Fe and Al associated with TOC in the biosolids amended soils (both p > 0.05, Fe: r= -0.77; Al: r= -0.40). TOC and Fe from 0–10 cm in the control at the Solano site were moderately correlated (r = 0.41, p = 0.73) and from 75–100 cm (r = 0.228, p = 0.924), with weaker correlations observed from the 30–50 cm depth (r = 0.02, p = 0.98). Correlations between TOC and Al in the Solano control soils show a moderately negative correlation from 0–10 cm (r= -0.70, p = 0.52) and an increasing positive correlation along depth (30–50 cm: r = 0.05; 75–100 cm: r = 0.397; both p > 0.05). TOC and Fe are weakly correlated from 0–10 cm (r = 0.1), negatively correlated in the biosolids amended soils at the Solano site from 30–50 cm (r= -0.996; p = 0.052), and weakly correlated again from 75–100 cm (r = 0.228, p = 0.92). The TOC and Al correlations in the biosolids amended soils at Solano also had negative correlations from the 0–10 cm (r=-0.05, p = 0.96) and from 30–50 cm with a significant negative Pearson’s correlation (r=-1, p < 0.001). Deep soils that were amended with biosolids had a weak correlation between TOC and Al (r = 0.30, p = 0.81). The Merced site had weak correlations between TOC to Fe and Al for both treatments along depths, except for the correlation between TOC and Al in the biosolids amended soil from 0–10 cm (r = 0.99, p = 0.03). Some concentrations of Fe and Al were undetectable and Pearson correlations could not be performed (Controls: TOC:Al from 0–10 cm; TOC:Al from 30–50 cm; TOC:Fe/Al from 75–100 cm; Biosolids: TOC:Al from 30–50 cm; TOC:Fe from 75–100 cm).

Correlations between soil organic C and Ca²⁺ in Merced

Stronger correlations between Ca²⁺ and SOC% were observed from the control HF compared to the long-term amended soils across all depths (Fig. 6). Correlations were strongest at the 0–10 cm for each treatment compared to other depths where the control had a correlation coefficient of 0.94 (p = 0.22) and the biosolids amended had an r of 0.03 (p = 0.98). The 30–50 cm showed a slight negative correlation in the amended soils (R²=-0.09, p = 0.93), but positive correlations in the control soils (r = 0.47, p = 0.70). Finally, the 75–100 cm depth showed strong negative correlations in the biosolids soil with an r of -0.88 (p = 0.31), and the control showed a minimal correlation between Ca²⁺ and SOC% though not negative (r = 0.31, p = 0.80).

Effects of long-term biosolids applications on soil density fractionations

Fractionation techniques are a valuable tool that can provide indications on the fate of the C and N from organic matter amendments and the long-term C storage potential of a soil (Grunwald et al., 2017; Ryals et al., 2014). Our study found that relative proportions of C and N found in each fraction differed along depth, sites, and treatment. Effect sizes across our study shows the impact of how biosolids application and measuring deep soil C can provide a better assessment on the C sequestration potential of a soil. The effect size of both C and N, the FLF increased in surface soils, and strengthens in deeper soil layers even though biosolids are only incorporated to 30 cm. Across all fractions, the effect sizes increased in the 75–100 cm depth, indicating a stronger relationship between depth, C, and N (Fig. 2a-b). As SOC and total N moves to subsoils, the turnover rate tends to decrease; thus, remaining for longer periods of time (Hicks Pries et al., 2017; Jackson et al., 2017; Shi et al., 2020; Tautges et al., 2019). Subsoils also typically contain higher concentrations of clays and organo-mineral complexes that can contribute to C stabilization mechanisms (Kögel-Knabner et al., 2008).

As expected, total organic C and N content associated with the FLF from 0–10 cm and 30–50 cm increased in the biosolids amended soil, but only in the Sacramento and Solano site. The higher proportions of FLF in the amended soils from the Sacramento and Solano site are likely from two sources. The first likely source is the C and N from the biosolids that persisted over time. One 547-day incubation study looked at the chemical composition of organic amendments, including biosolids, and how the decomposition rates were influenced by this factor (Baldock et al., 2021). They found that biosolids tended to emit less CO₂ and that most of the total C from biosolids was allocated in slow mineralizing pools. The second likely source of additional FLF C and N is from above- and -belowground plant litter through the promotion of plant production (Ryals and Silver, 2013). Several studies have found that biosolids increase SOC and plant production after a one-time application (Bolan et al., 2013; Brown et al., 2011; Wijesekara et al., 2017). Grasses are known to have deep roots that can reach down to deep soil depths; here, C and N may also be contributed through root turnover (Silver et al., 2010). One study found similar results on the distribution of C following the application of biosolids, particularly in the biologically active layer of the soil (0–10 cm; Silva et al., 2015). In addition, they found active vertical transport into deeper soil layers showing increased input from plant roots (Silva et al., 2015).

Deeper soil depths had higher proportions of C and N in FLF and OLF only at the Sacramento and Solano sites. The C and N present in the control soils at the Sacramento and Solano sites had organic C and total N associated with HF at relatively high proportions. Similarly, many cases have shown that the N associated with the HF in the control soils were higher in proportion than the amended soil. The higher proportions of C and N associated with HF in the control soils may be attributed to less accumulation of FLF C directly from biosolids application and the promotion of plant production (Brown et al., 2011; Silva et al., 2015).

The Merced site did not experience an increase in C and N associated with FLF from 0–10 cm or from 30–50 cm. Similarly N did not increase from 0–10 cm, but the 30–50 cm remained unchanged. This may be attributed to a coarser soil texture at the Merced site which can facilitate the movement of SOC to deeper soil. When comparing the unamended control and the biosolids amended soils at Merced, a shift in C and N associated with fractions differ. Unamended controls had higher proportions of C and N associated with the FLF than the amended soil where higher proportions of C and N was associated with the HF (Tables 2 & 3). This shift from FLF to HF indicates that C and N from 0–10 cm and 30–50 cm in amended soils is changing chemically and binding to mineral surfaces instead of free-floating, as observed in the control (Duddigan et al., 2019). Carbon inputs from biosolids and increasing plant production have been shown to can enhance soil microbial activity (Fernandes et al., 2005; Hamdi et al., 2019). This produces microbially-derived compounds that are the primary constituents of stable SOM, which can further promote the formation of long-term SOM storage (Marín-Spiotta et al., 2008; Poeplau et al., 2018). Soil management may also influence this more C and N associated with HF through practices such as flood irrigation and tillage. The Merced site has a coarse texture, has had the fewest frequency of biosolids application, soils are used for an alfalfa-corn rotation resulting in tillage, and is flood irrigated. Tillage breaks up soil structure and reduces aggregates, resulting in C loss in surface soils from erosion or through mineralization from microbes that have access to C that were previously protected in aggregates (Stewart et al., 2012).

Organo-mineral complexes is dependent on pH

Complexation of SOC to Al and Fe was more strongly correlated at the Sacramento and Solano sites than the Merced site (Fig. 7). We calculated the C:M ratio (Fe + Al mols) to determine whether there is enough stock of metal ions in the soil for chelation to have significant influence on C storage in soils across the three sites, treatments and by depth. Following Oades (1989), Masiello et al. (2004), and Berhe et al. (2012), we assumed each organic C functional group represents SOC C atoms associated with one negative charge. If every OC functional group was bound to a metal ion and metal ions existed in the + 1 charge state, the molar C:M ratio would be six (Masiello et al., 2004). Any ratio above 6 would indicate that there is not enough stock for Al and Fe to bind to SOC. However, it can be assumed that not all SOC and metal ions will fall under these assumptions. Some OC can have many functional groups that can interact with one metal ion, and some metal ions exist as polycations (raising the C:M ratio; (Berhe et al., 2012; Masiello et al., 2004). Hence, bonding of all OC to M falls within a ratio range between 2 to 10 (Oades, 1989).

At Sacramento and Solano, the C:M ratio generally falls within the 2 to 10 range (Fig. 6), except in the biosolids amended soil at the Solano site from 30–50 cm and 75–100 cm, where ratios were higher than 10. This may be due to more C to deeper soil layers at the Solano site. Thus, indicating there are not enough Fe and Al ions to bind to excess C present in the soil. The Merced site has a very large C:M ratio, ranging from 100–350. One possible explanation for this finding is that the soil at the Merced site has a pH of 8.0, the solution used for this procedure, sodium pyrophosphate, acts as a chelating agent and is used to estimate the amount of metal ions involved in soil via alkali-induced organic matter dissolution (Coward et al., 2018, 2017). Both the Sacramento and Solano site had a pH of 5.9 in the top 30 cm of soil, making these sites more ideal candidates for estimating organo-mineral complexes. The alkalinity of the soil at the Merced site could interfere with the extraction procedure since acidic pH drives the sorption of organic matter onto metal nanoparticles, hence, potentially causing inconsistent results (Coward et al., 2017).

Biosolids have been shown to increase Fe and Al in soils due to the direct contribution of these metal ions to soil. One long-term study found that thirty years of biosolids application significantly increased amorphous Fe and Al in mined soils (Tian et al., 2013). They also found increases in mineral-associated organic matter in biosolids amended soils, indicating that the C and N in these plots may remain for longer periods of time. Mikutta et al. (2006) found positive correlations between mineral-associated organic matter and soil amorphous Fe and Al. Biosolids contain high Fe and Al in amorphous form, thus introducing more metal ions into the soil (Mikutta et al., 2007; Tian et al., 2013). In certain conditions, the introduction of metal oxides found in biosolids and SOC can become closely related due to organic matter carrying negative charges and metals having positive charges (Chiu and Tian, 2011). At the Sacramento site, the biosolids amended soils had a lower C:M ratio than the control, indicating that biosolids application increased the total amount of Fe and Al bound to SOC. In the Solano site, the biosolids amended soils had a higher C:M ratio, indicating that there is more C than Fe or Al in the soil. Since the composition of the biosolids applied was similar across sites, it could be deduced that due to the yearly, high frequency of biosolids application, there was more Fe and Al present in the Sacramento soils compared to the Solano and Merced site.

Biosolids application influences C associations to Ca²⁺ in the Merced site

Mechanisms that influence how Ca²⁺ interacts with SOC are influenced by high pH in soils where positively charged ions are increasingly adsorbed to surfaces with variable charges such as clays, iron and aluminum oxides, and SOM (Solly et al., 2020; Weil and Brady, 2017). At the Merced site, soils had a pH of 8 from 0-100 cm and a high percentage of soil inorganic carbon (SIC; ~22% of total C) in the form of calcium carbonate (CaCO₃) (Villa and Ryals, 2021). Studies in unmanaged soils have found strong correlations between Ca²⁺ and SOC content and stabilization (Martí-Roura et al., 2019; Rowley et al., 2021, 2018; Solly et al., 2020). Hence, we considered the role Ca²⁺ may play in C sequestration at the Merced site.

We hypothesized that the increasing SOC content from biosolids application would be correlated with Ca²⁺; hence, indicating that the Ca²⁺ may be associated with SOC chemically or physically. However, we found the opposite to be true (Fig. 7). In fact, there is an ascending negative correlation moving down the soil profile in the amended soil compared to the control, where there is a weaker positive correlation moving down in depth. Soil organic matter has a high abundance of negatively charged sites which can improve cation exchange capacity, thus increasing soil C content in soils by reacting as a surface area in soil where SOC may be adsorbed (Solly et al., 2020). Some studies have found that the application of organic matter amendments increases SOC and CEC (Diacono and Montemurro, 2010). However, CEC is strongly associated with the available surface area of the soil (Farrar and Coleman, 1967; Solly et al., 2020), effectively influencing the ability of SOC to adsorb to reactive soil surfaces (Solly et al., 2020). One study found no significant increase of CEC in a coarse textured almond orchard after application of green waste compost and composted wood-chip manure (Villa et al., 2021). The Merced site has a uniform soil texture of sandy loam from 0-100 cm, and the percentage of sand ranged from 53% (0–10 cm)-73% (Villa and Ryals, 2021).

The chemical composition of the biosolids could also contribute to the negative correlation with Ca²⁺. Biosolids are rich in Fe, and the biosolids applied in this study had a concentration of 35 ± 19 g/kg (Villa and Ryals, 2021). It is possible that the Fe in the biosolids interacts with the C that is already present in the biosolids, indicating preferential binding. The strength of metal cations in binding organic compounds largely depends on the size of the hydration shell and the valence of electrons, Fe has a 3 + charge; therefore, a larger hydration shell than Ca²⁺ (Solly et al., 2020).

Our study assesses the mechanisms that influence deep soil C sequestration in three managed soils that have had biosolids application for twenty-years. Density fractionations demonstrated that amended soils had more C and N associated with the microbially accessible, free-floating fraction in surface and deep soils at the Sacramento and Solano site. This was likely due to the promotion of plant production and vertical movement of C and N to deeper soil depths. The Merced site had more C and N associated in HF in amended soils relative to the control suggesting the chemical composition of the C and N from biosolids application is changing. Organo-complexes was a stabilization mechanism in the Sacramento and Solano site, indicating that the C associated with these metal ions will remain stabilized for long periods of time. Negative correlations between SOC and Ca²⁺ along depth relative to the control imply that the biosolids themselves may be interacting with the C from the biosolids and soil. Our study indicates that biosolids increase SOC not only in shallow soils, but also in deep soils where it can stay for long periods of time across different agroecological contexts. We demonstrated the importance of accounting for deep soil C when assessing the climate change mitigation potential of managed soils.

The authors declare no conflict of interest.

Funding

Funding for this research was provided by a grant to R. Ryals from the Bay Area Clean Water Agencies and the Jena and Michael King Foundation. Funding was also provided by the Department of Life and Environmental Sciences at the University of California, Merced.

Data availability

Data generated for this study can be found at (DOI: https://doi.org/10.6071/M33X22). Any other information required is available from the corresponding author on reasonable request.

Acknowledgements:

Funding for this research was provided by a grant to R. Ryals from the Bay Area Clean Water Agencies and the Jena and Michael King Foundation. Funding was also provided by the Department of Life and Environmental Sciences at the University of California, Merced. We would like to thank Dr. Asmeret A. Berhe, Dr. Stephen C. Hart, and Dr. Samantha Ying for their feedback on the paper. We thank F. Gamiño, E., B. Alrawi, C. De Leon, K. Porterfield, S. Cousineau, N. Goncalves, D. Davenport, and Ryan Batjiaka; our farming partners; and members of the San Francisco Public Utilities Commission.

Author Contributions

YBV and RR contributed to the study conception, design, and field collection. Material preparation, data collection, and analysis were performed by YBV with assistance from EP. The first draft of the manuscript was written by YBV, and RR commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Table 1: The chemical composition of C, N, C:N, and Fe of biosolids from ten wastewater treatment plants. Values were averaged, standard deviations are shown. Values in brackets are the minimum and maximum value. One-way ANOVA was used to determined significant differences as described in Villa and Ryals, 2021. The biosolids composition of the elements below were not statistically different from each other across the wastewater treatment plants (p<0.05).

Nutrients

Biosolids

Composition

C

(mg/kg)

381.2±32.6

(338-443)

N

(mg/kg)

58.3±9.5

(49.2- 74.7)

C/N

6:1

(5:1-7:1)

Fe

(mg/kg)

34.8±19.2

(7.3- 64)

Table 2: Proportions of fractions associated with the total SOC between treatments along depths.

		Carbon
Site	Treatment	Depth	FLF%	OLF%	HF%
	Biosolids		85%	12%	3%
Sacramento		0-10
	Control		19%	28%	53%
	Biosolids		88%	2%	10%
Solano		0-10
	Control		71%	16%	13%
	Biosolids		12%	28%	60%
Merced		0-10
	Control		58%	21%	21%
	Biosolids		47%	36%	17%
Sacramento		30-50
	Control		19%	28%	53%
	Biosolids		88%	2%	10%
Solano		30-50
	Control		27%	14%	59%
	Biosolids		11%	6%	83%
Merced		30-50
	Control		34%	33%	32%
	Biosolids		29%	34%	37%
Sacramento		75-100
	Control		4%	28%	68%
	Biosolids		27%	55%	17%
Solano		75-100
	Control		15%	21%	64%
	Biosolids		5%	3%	92%
Merced		75-100
	Control		13%	8%	80%

Table 3: Proportions of fractions associated with the total N between treatments along depths.

		Nitrogen
Site	Treatment	Depth	FLF%	OLF%	HF%
	Biosolids		82%	12%	5%
Sacramento		0-10
	Control		39%	35%	26%
	Biosolids		71%	16%	13%
Solano		0-10
	Control		53%	12%	35%
	Biosolids		12%	26%	62%
Merced		0-10
	Control		54%	26%	20%
	Biosolids		36%	30%	34%
Sacramento		30-50
	Control		10%	14%	76%
	Biosolids		88%	2%	14%
Solano		30-50
	Control		17%	7%	75%
	Biosolids		18%	12%	69%
Merced		30-50
	Control		18%	12%	69%
	Biosolids		30%	19%	51%
Sacramento		75-100
	Control		2%	25%	73%
	Biosolids	75-100	28%	41%	31%
Solano
	Control	75-100	9%	10%	81%
	Biosolids		10%	8%	81%
Merced		75-100
	Control		13%	11%	75%

Table 4: Treatment differences between depth, fraction and site in a) C and b) N.

a)

		Carbon
Site	Treatment	Depth	FLF (MgC/ha)	OLF (MgC/ha)	HF (MgC/ha)
	Biosolids		22.00±5.86	2.29±0.29	1.38±0.38
Sacramento		0-10
	Control		7.84±0.22	8.86±1.44	3.84±0.21
	Biosolids		21.61±1.18	8.95±7.35	4.11±0.61
Solano		0-10
	Control		8.45±3.77	1.85±0.31	5.53±0.30
	Biosolids		1.55±0.76	3.56±1.13	7.74±2.89
Merced		0-10
	Control		10.18±8.71	4.87±0.53	4.83±0.39
	Biosolids		16.76±14.46	12.82±5.24	6.12±2.38
Sacramento		30-50
	Control		6.34±3.74	9.29±5.28	17.49±13.33
	Biosolids		58.10±39.64	1.96±0.63	8.78±1.59
Solano		30-50
	Control		3.40±1.40	1.73±0.37	7.37±1.54
	Biosolids		2.64±1.16	1.41±0.39	19.39±4.57
Merced		30-50
	Control		8.73±8.58	10.94±7.81	10.49±2.01
	Biosolids		2.97±1.49	3.46±1.15	3.84±1.18
Sacramento		75-100
	Control		0.18±0.05	1.27±0.10	3.13±0.44
	Biosolids		10.65±9.57	21.71±21.34	6.82±0.92
Solano		75-100
	Control		1.31±0.36	1.88±0.92	5.64±0.90
	Biosolids		0.83±0.24	0.58±0.20	15.79±11.06
Merced		75-100
	Control		0.92±0.25	0.56±0.32	5.74±0.71

b)

		Nitrogen
Site	Treatment	Depth	FLF (MgN/ha)	OLF (MgN/ha)	HF (MgN/ha)
	Biosolids		2.47±0.47	0.37±0.09	0.16±0.03
Sacramento		0-10
	Control		0.80±0.19	0.70±0.10	0.53±0.04
	Biosolids		3.29±0.40	0.72±0.59	0.62±0.08
Solano		0-10
	Control		0.73±0.33	0.18±0.04	0.77±0.05
	Biosolids		0.19±0.13	0.40±0.23	0.95±0.15
Merced		0-10
	Control		1.80±1.66	0.88±0.21	0.66±0.03
	Biosolids		1.08±0.73	0.92±0.55	1.03±0.39
Sacramento		30-50
	Control		0.47±0.29	0.64±0.33	3.44±3.30
	Biosolids		6.10±1.94	0.16±0.05	1.05±0.08
Solano		30-50
	Control		0.38±0.12	0.16±0.05	1.65±0.36
	Biosolids		0.31±0.12	0.21±0.09	1.20±0.13
Merced		30-50
	Control		2.09±1.62	2.44±1.87	1.50±0.27
	Biosolids		0.36±0.06	0.23±0.06	0.61±0.15
Sacramento		75-100
	Control		0.01±0.00	0.21±0.03	0.60±0.12
	Biosolids	75-100	1.09±0.94	1.62±1.59	1.22±0.25
Solano
	Control	75-100	0.17±0.06	0.17±0.06	1.22±0.25
	Biosolids		0.12±0.05	0.10±0.04	0.96±0.22
Merced		75-100
	Control		0.14±0.05	0.12±008	0.80±0.12

Table 5: Concentrations of TOC, Fe and Al from pyrophosphate extractions. Ratios of TOC:Fe and TOC:Al and Pearson correlations are shown. NA values are due to undetectable concentrations of Fe or Al. Asterisks represent significance (p<0.05*; p<0.01**; p<0.001***).

Site	Treatment	Depth (cm)	Al (mg/g)	Fe (mg/g)	TOC (mg/g)	TOC:Fe	Pearson Correlations	TOC:Al	Pearson Correlations
		0-10	0.48±0.00	1.25±0.00	1.47±0.19	1.17	r=0.95	3.08	r=0.88
	Biosolids	30-50	0.84±0.00	0.57±0.00	0.65±0.17	1.14	r=0.98	0.77	r=0.99*
Sacramento		75-100	0.41±0.02	0.27±0.00	0.62±0.17	2.31	r=-0.84	1.50	r=-0.8
		0-10	0.91±0.00	0.82±0.00	2.03±0.02	2.47	r=0.88	2.23	r=0.99
	Control	30-50	0.64±0.00	0.37±0.00	0.96±0.13	2.59	r=0.629*	1.49	r=0.99*
		75-100	0.64±0.00	0.46±0.00	0.25±0.04	0.55	r=0.39	0.40	r=0.39
		0-10	0.44±0.19	0.54±0.20	2.05±0.13	3.78	r=-0.11	4.69	r=-0.05
	Biosolids	30-50	0.22±0.20	0.21±0.15	1.73±0.10	8.38	r=-0.99*	7.83	r=-1.00***
Solano		75-100	0.1±0.14	0.07±0.10	0.730.10	9.88	r=0.761	7.42	r=0.23
		0-10	0.31±0.32	0.36±0.29	2.15±1.73	5.97	r=0.41	7.00	r=-0.69
	Control	30-50	0.26±0.44	0.19±0.28	0.85±0.27	4.56	r=0.02	3.29	r=0.05
		75-100	0.22±0.05	0.03±0.06	0.61±0.04	18.28	r=0.23	2.77	r=0.40
		0-10	0.01±0.14	0.03±0.12	1.8±0.22	53.79	r=0.45	217.41	r=0.359
	Biosolids	30-50	0.00±0.04	0.01±0.02	0.86±0.08	63.84	r=0.62	NA	NA
Merced		75-100	0.02±0.03	0.01±0.02	0.55±0.17	50.52	NA	30.74	r=0.94
		0-10	0.01±0.01	0.03±0.02	1.93±0.15	56.89	NA	177.60	NA
	Control	30-50	0.00±0.09	0.01±0.06	0.82±0.18	56.50	r=-0.78	593.95	NA
		75-100	0.00±0.04	0.01±0.00	0.41±0.16	42.46	NA	149.55	NA

Carbon persistence of soils with long-term biosolids amendments in California agroecosystems

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Conclusions

Declarations

References

Tables

Status:

Version 1