Organic matter mineralisation in contrasting agricultural soils amended with sewage sludge

The mineralisation of organic matter (OM) when sewage sludge was used as amendment in 70 contrasting agricultural soils from Spain was analysed. Soils received a single dose of sewage sludge (equivalent to 50t dry weight ha-1) and the O2 consumption was continuously monitored for 30 days using a multiple sensor respirometer in a laboratory experiment. The cumulative O2 consumption and rates after 8 and 30 days of incubation (O2 cum 8d, 30d and O2 rate 8d, 30d), the respiratory quotient (RQ), the maximum O2 rates over the incubation period (O2 max) and time from the beginning of the incubation when O2 max occurred (Tmax), were determined in both amended and non-amended soils. Sewage sludge application resulted in increased values for O2 max, O2 rate 8d, and O2 cum 30d. Differences were minor for Tmax, RQ 8d and O2 rate 30d. A considerable amount of the initial OM applied was mineralised during the first 8 days. Organic matter decomposition (as expressed by O2 cum 30d) was favoured in soils with high values of pH, carbonates, soil organic carbon and low values of amorphous Mn. Soils with these characteristics may potentially lose soil C after sewage sludge application.


Introduction
The production of sewage sludge in Europe has increased dramatically in the last decades.With the increment of economic restraints and environmental concerns about landfilling and incineration (Liang et al. 2011), there has been increased interest in the land application of these wastes (O'Connor et al. 2005).Land application enhances the recycling of nutrients and organic matter to soil and can contribute to the reduction of the atmospheric concentration of CO 2 , increasing the levels of soil organic carbon (SOC) and associated fertility (Ajwa and Tabatabai 1994;Bernal et al. 1998;Tian et al. 2009).The conversion of natural to agricultural ecosystems has resulted in the depletion of SOC, as much as 60% in soils of temperate regions and 75% or more in the tropics.The land application of sewage sludge represents an opportunity for carbon (C) sequestration (Lal 2004;Lal 2008).This practice is of particular relevance in soils from arid and semi-arid regions, where soil erosion and decline of organic matter are major threats.
However, recycling sewage sludge onto agricultural land poses risks for humans and the environment due to the presence of high levels of nutrients, labile organic matter and pathogens as well as organic pollutants and toxic metals (McBride 2003;Basta et al. 2005;Cesar et al. 2012).The intense microbial activity is enhanced by the land application of waste materials with high levels of labile organic matter (Saviozzi et al. 2002;Belyaeva and Haynes 2012), which may promote the mineralisation of the native SOC (Bernal et al. 1998) known as the priming effect (Dalenberg and Jager 1989).
The decomposition of exogenous organic matter (EOM) in soil following the application of sewage sludge is thought to play a crucial role in the balance between the potential positive or negative effects derived from these applications.In particular, this process drives C, N and other important elements cycles in agroecosystems (Curtin et al. 1998;Spargo et al. 2011) and conditions potential adverse effects and the contribution to soil C storage of sewage sludge applications (Navarro-Pedreño et al. 1996;Kaur et al. 2008).The mineralisation of EOM following the land application of organic wastes is dependent on a myriad of biotic (Blagodatskaya and Kuzyakov 2008;Blagodatsky et al. 2010) and abiotic factors including EOM composition (Levi-Menzi et al. 1990;Bernal et al. 1998), environmental conditions (Hsieh et al. 1981) and soil properties (Pedra et al. 2007;Huang and Chen 2009), which affect the microbial activity that drives this process (Blagodatskaya and Kuzyakov 2008).Studies evaluating the mineralisation of EOM following sewage sludge application in a range of soils are sparse and the soil mechanisms responsible for this process still remain unclear (Bradford et al. 2008).In particular, there is a considerable controversy about the effects of sewage sludge application on the SOC pool (Jones et al. 2006;Soriano-Disla et al. 2010;Tian et al. 2013).

KEY WORDS
Studies evaluating the mineralisation of EOM in soil should involve the combined information about short-(hours to days) and long-term (weeks to months) respiratory parameters since they reveal different mechanisms of the decomposition of EOM (Smith et al. 1985;Adani et al. 2004;Blagodatskaya and Kuzyakov 2008;Ponsá et al. 2010).
The objective of this study was to assess the mineralisation of OM when sewage sludge was applied in 70 contrasting agricultural soils and the influence of the soil factors responsible for short-term mineralisation.

Material and methods
Seventy agricultural soils from different parts of Spain were chosen.The soils were sampled from the ploughed layer (0-30 cm) and then airdried, homogenised and sieved (< 2 mm).A synthesis of the selected properties of the soils is shown in Table 1 (more detailed information in Supplementary Material 1).
Selected soil properties were analysed as follows: pH (1:2.5 wv -1 , distilled water), electrical conductivity (1:5 wv -1 , distilled water), equivalent calcium carbonate (CO 3 2-) determined using the Bernard calcimeter (Hulseman 1966), soil organic carbon (SOC) determined by potassium Simultaneous analysis of CO 2 release using an infrared sensor was done.In order to know the percentage of organic C release from the mineralisation of the added EOM, the cumulative CO 2 release after 30 d for both amended and non-amended soils was calculated: CO 2 cum 30d and CO 2 cum_na 30d.In addition, the cumulative CO 2 release after 8 d was also determined for both amended and non-amended soils for the calculation of the respiratory quotient (RQ), expressed as the ratio of mol CO 2 evolution over mol O 2 consumption (Dilly 2003).
For the determination of the percentage of organic C from EOM mineralised after 30d (% EOM 30d), the CO 2 cum_na 30d was subtracted from the CO 2 cum 30d and the result expressed as C released after 30d.This value was compared (ms cm -1 ) (g kg -1 ) (g kg -1 ) (g kg -1 ) (mg kg -1 ) (mg kg -1 ) (mg kg -1 ) (mg kg -1 ) (mg kg -1 ) (mg kg to the amount of organic carbon added with the sewage sludge.The cumulative values of CO 2 released after 30 days for the non-amended soils were transformed to C and compared to the SOC, determining the % of SOC mineralised after 30 days (% SOC 30d).
The influence of soil properties on the respiratory parameters from the soils amended with sewage sludge was determined by Pearson correlation coefficients (r).Amorphous Fe and Mn were log 10 transformed in order to normalise the variable distributions and reduce asymmetry (Maindonald and Braun 2010).In the case of electrical conductivity, soil samples 21, 40, 50, 61 corresponding with concentration outliers (samples that appear to be isolated from the main body of data) were detected and not considered in the analysis in order to fulfil model conditions (Maindonald and Braun 2010).Statistical analyses were performed using the software SPSS v.21.0 and Microsoft Excel 2007.

Results
The mean values (± standard deviation) and ranges of the respiratory parameters for the amended and non-amended soils can be found in Table 3. Figure 1 shows the evolution for selected soils (45, 55 and 57).Oxygen consumption rates were considerably high at the beginning of the incubation experiment for the soils amended with sewage sludge (O 2 max and O 2 rate 8d), but they declined rapidly thereafter (Figure 1 and Table 3).Maximum rates were reached, on average, after 23 hours of incubation with samples ranging from 10 to 43 hours (Table 3).Consumed O 2 was mainly accumulated during the first days of incubation and a gradual and slight increase was observed over the incubation time (Figure 1).On average, more than 60% of the total cumulative O 2 at the end of the incubation period (64±4%, 50-74%) was consumed in the first 8 days (Figure 2).The average values for RQ was 0.52 and weakly varied across the soils (Table 3). [

ORGANIC MATTER MINERALISATION IN CONTRASTING AGRICULTURAL SOILS AMENDED WITH SEWAGE SLUDGE ]
Table 3. Mean±standard deviation (SD), range and percentiles of the respiratory parameters a for the sewage sludge amended and non-amended soils  Mineralisation patterns for the non-amended soils were similar to those described for the amended soils but observing expectable lower mineralisation rates and cumulative O 2 consumption values (Figure 1 and Table 3).Poor differences were found for the T max , RQ and O 2 rate 30d values as a consequence of sewage sludge application (Table 3).The variation across soils for the respiratory parameters in the non-amended soils was higher than for the amended soils (an example of contrasting soils in Figure 1).
Respiratory parameters were related to soil properties through Pearson correlations.Poor correlation coefficients were found between the O 2 max , T max , RQ and soil properties (data not shown).On the other hand, significant relationships were observed between the O 2 cum 30d and soil properties (Table 4).
The highest levels of correlation were observed between O 2 cum 30d and pH, carbonates and SOC.After these results, soils were ranked according to the O 2 cum 30d values and divided into low (lower than the 25th percentile) and high (higher than the 75th percentile) values.Soils with low O 2 cum 30d values were characterised by relatively low average values of pH (6.9), carbonates (4.1%), SOC (4.2 g kg -1 ) and high values of amorphous Mn (119.1 mg kg -1 ).
Contrasting average values for these variables were found in the soils with high O 2 cum 30d: pH (8.5), carbonates (45%), SOC (12.4 g kg -1 ) and amorphous Mn (42.7 mg kg -1 ).According to these results, the cumulative O 2 cum 30d following In an attempt to evaluate if the relationships between soil properties and O 2 cum 30d were affected by the SOC mineralised from the nonamended soils, the differences between O 2 cum and O 2 cum_na 30d across soils were also related to soil properties through Pearson correlations (Table 4).The contribution of SOC was weaker while the contribution of the rest of soil properties was similar.

Discussion
Since addition of sewage sludge means addition of a source of available C, an initial short-term O 2 flush during the first 2-3 days of incubation is characteristic of the mineralisation dynamics of the labile fraction of EOM applied in soils (Bernal et al. 1998;Haynes and Naidu 1998;Pedra et al. 2007;Huang and Chen 2009).Our results showed that a relatively high amount of O 2 was consumed during the mineralisation of available substrates.High microbial activity following the land application of sewage sludge can potentially result in negative effects for the soilplant system (e.g.deprivation of O 2 and release of toxic substances), which can be reduced by adding organic wastes to soil some weeks before sowing (Bernal et al. 1998).The mineralisation patterns of sewage sludge in soils during the incubation time (Figure 1) were typical for organic wastes applied in soils (e.g.Hsieh et al. 1981;Bernal et al. 1998).Contrasting respiration rates at different stages of the mineralisation (Figure 1 and Table 3) were accounted for the degradation of different types of organic matter (easily and slowly biodegradable).
Oxygen uptake increases exponentially as the microbial population grows, but diminishes when the availability of the most highly biodegradable substrates becomes limited or the oxygen reduced.Thus, O 2 max provides information regarding the availability of easily biodegradable C.
T max is dependent on the lag-time, i.e. time between the addition of substrate and the start of the exponential increase of the respiration rate (Nordgren et al. 1988) which is assumed to be indicative of changes in the physiological status of soil microorganisms (Mochizuki and Hattori 1987).Once this labile pool is decomposed, the mineralisation rates decline, revealing why weak differences were found in the O 2 rate 30d for amended and non-amended soils.
The RQ can reveal useful information on C metabolic pathway -organic matter mineralisation patterns (Dilly 2003).The RQ may vary depending on the composition of available substrates and the current physiology of the soil microbial communities (Dilly 2001).
As expressed by RQ, both SOC and the EOM were mineralised with the same efficiency, and so RQ may be controlled by the soil microbial community of the soil.
The lower variability within the mineralisation parameters found for the amended soils, in contrast with non-amended soils, is indicative of the importance of the great mineralisation of the organic matter following the application of sewage sludge.Despite the lower variability, considerable differences were still observed across the amended soils.These differences are dependent on the mineralisation of the SOC and the different mineralisation of the EOM as conditioned by soil properties.
The lower variability found within the mineralisation parameters for the amended soils is also reflected by the % EOM 30d, which was high irrespective of the soil, contrasting with the average % of SOC mineralised after 30 days.The % EOM 30d represents an approximate value, since the increment of the CO 2 release is a consequence of the application of EOM and the mineralisation of this organic waste, but it could be potentially affected by an enhanced degradation of SOC following the application of sewage sludge (previously defined as priming effect).
The O 2 max is indicative of the amount of easily biodegradable C.This probably conditioned the absence of relationships between O 2 max and soil properties since the amount and type of EOM applied was the same in all the soils.
As ) and microbial activity was conditioned by the also positive relationship between carbonates and soil pH (r = 0.76, p < 0.001).Soils with high pH and calcium carbonate content have been found to favour the contribution of inorganic C to CO 2 production (Stevenson and Verburg 2006) due to the chemical response of carbonates to the addition of organic matter forming CO 2 .
The O 2 cum 30d values were related (negatively) to the amorphous Mn, possibly due to a mechanism of protection of the organic matter by minerals, thus preventing mineralisation (Baldock and Skjemstad 2000;Kleber et al. 2005).The positive relationship between SOC and O 2 cum 30d can be explained by the increased availability of labile substrates in native SOC (Steenwerth et al. 2005).This is supported by the reduction of the contribution of SOC to the O 2 cum 30d once the SOC mineralised in the controls was subtracted (Table 4).

Conclusions
The mineralisation of sewage sludge (EOM) in soils can be described by respiratory parameters obtained after a short period from the application and offer complementary information concerning environmental problems.Considering that high microbial activity was observed within the first days of incubation (with maximum O 2 flushes within the first 2 days), care must be taken in sewage sludge applications, especially during the first days following the addition to soils.
It was observed that O 2 max , T max and RQ were not related to the physical and chemical properties of this pool of contrasting agricultural soils, and that the last two parameters, together with organic matter mineralisation rates after 30 days, were weakly affected by the sludge application.In terms of the potential contribution of the sewage sludge application to the SOC pool, and irrespective of the soil, a large proportion of the EOM added to the soil was mineralised in a short period of time.Despite this fact, considerable differences were still observed across soils.Low levels of soil pH, SOC, carbonates and high levels of amorphous Mn contributed to a lower mineralisation of EOM (as expressed by O 2 cum 30d).Thus, soils with these characteristics may be beneficial in terms of C conservation following sewage sludge application.
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Figure 1 .
Figure 1.Cumulative O 2 consumption (A, C) and O 2 consumption rates (B, D) over 30 days of incubation for soils amended (top plots, A and B) and non-amended (bottom plots C and D) with sewage sludge.

Table 1 .
Selected properties of the soils (n=70)

Table 2 .
Selected properties of the sewage sludge

Table 4 .
Pearson correlations (r value) between cumulative O 2 consumption after 30 days of incubation and soil properties for soils amended with sewage sludge and after subtracting the cumulative O 2 consumption by the non-amended soils **, **, *: the probability level of p<0.001, 0.01 and 0.05, respectively. *

Table .
ORGANIC MATTER MINERALISATION IN CONTRASTING AGRICULTURAL SOILS AMENDED WITH SEWAGE SLUDGE ]Respiratory parameters: O 2 max , maximum rates; T max , time to reach the maximum rate; O 2 cum 30d, cumulative O 2 consumption after 30 days of incubation; RQ 8d, respiratory quotient after 8 days of incubation.