Long-term effect of organic amendments on soil enzymatic activities
Cheviron Nathalie1,P, Marrauld Christelle1, Vecten Helen1, Houot Sabine2, Bispo Antonio3,
Grand Cécile3 , Galsomiès Laurence3, Mougin Christian1
(1) INRA, Unité PESSAC 78026 Versailles, FR
(2) INRA/AGROPARISTECH, UMR Environnement et Grandes Cultures, 78850 Thiverval-Grignon,
FR
(3) ADEME, 49004 ANGERS cedex 01, FR
[email protected]
Abstract
Microbial ecology provides tools to understand the effects of organic matter application on soil
microbial functions, following soil amendment with organic waste products. Here we studied the longterm effect of soil amendment with composts and manure on different soil enzyme activities involved
in the C (β-glucosidase), N (urease), S (arylsulphatase) and P (phosphatase) biogeochemical cycles.
The amended plots showed higher enzymatic activity than the unamended ones. Our results
demonstrate that organic amendments had a noticeable long-term effect on enzyme activities,
depending on the cycle considered. Clearly, the dynamics of microorganism response is closely related
to organic matter nature, and more precisely to its degradability.
Introduction
For many decades, soil has been considered as a reactor for recycling human and agricultural
wastes. Since 2006, it is considered as a non-renewable resource to protect (CEE, 2006). Threats
affecting soils, such as organic matter loss or contaminations by chemical pollutants, have been
pointed out by EEC. The use of organic waste products (OWP) seems a good alternative to reduce soil
organic matter (OM) losses, to improve soil fertility, and thus to limit ecological deleterious impacts
of intensive agriculture. These actions stimulate the activity of soil microorganisms and reactive the
biogeochemical cycles [1]. Amendments allow the storage of Carbon in soil as a remediation of
agricultural practices.
In France, the SOERE-PRO is a long-term experiment network to observe and understand the
effects of OWP amendments on cropped soils. The use of long-term field experiments is very useful to
investigate the environmental impacts of recycling organic residues on soil biochemical properties, as
well as the possible resilience of the agrosystems, to assess cost/benefit balance, and to improve
agricultural practices. Since 1998, one site of the SOERE-PRO, Qualiagro (Feucherolles - Yvelines;
France), allows to study the effects of several types of organic amendments (composts and manure) in
relation with two levels of nitrogen.
Soil microorganisms are involved in the transformation and recycling of OM [2] and contaminants
[3, 4, 5]. They are the main contributors to biogeochemical cycles and are key actors responsible for
optimal soil functioning [6]. Soil functions are maintained by the production of extra or intracellular
enzymes [7]. Several authors have shown that OM amendments [8] and cultural practices [6] affect
soil enzymatic activities. The changes observed are linked to modifications of gene and protein
expression under the pressure of environmental factors [9, 10], thus governing the final enzymatic
activities. In our laboratory, previous studies have shown that enzymatic activities were relevant to
assess the impacts of human perturbations, namely urease, β-glucosidase, arylsulfatase, and
phosphatase. In addition, numerous organisms including filamentous fungi are known to secrete
oxidases [11]. The production of laccase, a phenoloxidase, is known as regulated by lignin availability
and induced by pesticides at environmental concentration [12]. Enzymatic activities are relevant
indicators of soil functioning [14] and soil functional biodiversity [15]. Here we investigated the
enzymatic activities in the Qualiagro site, in relation with OM amendments.
Materials and methods
Experimental site
For each treatment, the Qualiagro site (INRA and Veolia Environment collaboration) comprised
four replicates of plots (45 m x 10 m, 450 m2). The treatments were: no organic amendment (CON),
farmyard manure (FYM), biowaste compost (BIO), co-compost of green wastes and sludge (GWS)
and municipal solid waste compost (MSW)(Figure 1). The present study was managed on plots with
optimal mineral nitrogen fertilization. In the site, the luvisol was a silt loam comprising 15% clay,
78% silt, and 7% sand. Its initial pH value was 6.9 and initial organic matter was 1.8%. During the
past twenty years, the average annual precipitations amounted to 582 mm, and the average annual
temperature was 11°C. The experimental design
CON FYM
BIO MSW GWS MSW CON
GWS CON MSW BIO
MSW BIO
FYM
BIO
FYM GWS
bloc 4
FYM GWS FYM CON
BIO MSW
bloc 3
FYM MSW GWS
bloc 2
BIO GWS MSW CON FYM
Bloc 1
CON FYM GWS CON
BIO GWS MSW CON
low mineral N low
BIO
optimal mineral N
Figure 1: Map of the Qualiagro site
The physic-chemical characteristics of the amendments were listed in Table 1.
Tab 1: Average physico-chemical characteristics of organic amendments applied in 1998, 2000, 2002, 2004,
2006, 2007, 2009 and 2011,
GWS
MSW
BIO
FYM
Dry matter, g kg-1 fw
633 (±9)
678 (±129)
700 (±91)
396 (±97)
OC, g kg-1 dw
308 (±48)
208 (±50)
320 (±72)
265 (±48)
C/Ntot
10.7 (±2.2)
15.6 (±2.8)
11.7 (±1.9)
14.0 (±3.0)
pH (in H2O)
7.7 (±0.8)
7.4 (±0.4)
8.5 (±0.2)
9.0 (±0.2)
Potentially Mineralized Carbonb, %OC
44 (±11)
16 (±6)
20 (±10)
12 (±5)
a
fw, fresh weight; dw, dry weight; OC, organic carbon content; ON, organic nitrogen content ; C/Ntot, organic C to total N ratio
b
The excess mineralized carbon after 91 days in the soil-organic amendment mixtures was expressed as a percentage of the amendment
organic C applied during incubation (after subtracting mineralized C of the control treatment)
Enzymatic activity measurements
The soils were collected at different times (at T0, then 6 and 18 months after amendments
from) 2011 to 2013 at a depth of 0-10 cm. Samples were sieved through a 5 mm mesh, and stored at
12 °C before use. Soil water content was determined by drying 5 g of soil at 105 °C for 48 H.
Tab 2: Characteristics of hydrolase activity measurements
Substrates
Incubation
Incubation T°C
λ (nm)
Phosphatase
4-nitrophenylphosphate (0,05M)
30 min
37
405
β -Glucosidase
4-nitrophenyl β-D-glucopyranoside (0,05M)
1h
37
405
Arylsulfatase
Potassium 4-nitrophenyl sulfate (25 mM)
4h
37
405
Urease
Urea (0,4 M)
3h
25
650
Phosphatase, β-glucosidase, arylsulfatase, and urease activities were performed in microplate
according to modified protocols [16, 17, 18, 19] less than 48 hours after sample collection. Briefly,
hydrolases were measured in triplicates in 4 g soil samples mixed during 10 min at 250 rpm with 25
ml water. For phosphatase, β-glucosidase and arylsulfatase activities, soil solutions were incubated
with specific substrates (Sigma chemicals) (Tab.2). Reaction was stopped with 0,5 M CaCl2 and 0,1 M
Tris at pH 12. For urease activity, soil solutions were mixed with 0,4 M urea. NH4 formed was
revealed with ammonium salicylate and ammonium cyan rate (Hach reagents). After the reactions,
each plate was centrifuged 5 min at 2000 g, and absorbance was measured on a microplate reader
Genius (SAFAS). The amounts of p-nitrophenol (PNP) and NH4Cl formed were obtained by the
absorbance at 405 nm and 610 nm.
Results and discussion
Our results show that OWPs modify the soil organic matter status and microbial biomass
during the months following OWP inputs (data not shown). Soil enzymatic activities are modified for
P, C and N cycles with brought of organic amendment, according to OM status, as well with microbial
biomass, water retention capacity and soil aggregate stability. The level of increase depends on the
nature of the amendment, more specially on the status of organic matter added, its availability and
degradability. Moreover, these organic amendments promote immobilization of enzymes linked to
humic colloids, which could strongly affect soil quality.
Fig 2: Time-course of hydrolase activities (expressed in % of control plot): A: phosphatase activity, B: arylsulfatase activity,
C: urease activity, D: β-glucosidase activity. Amendments were: farmyard manure: FYM (purple lines), biowaste compost:
BIO (red lines), municipal solid waste compost: MSW (blue line), co-compost of green wastes and sludge: GWS (green line).
Significant differences between control and amended plots are with (*).
Dick [7] and many researchers reported that phosphatase activity was increased by the
addition of organic materials. Our results are in agreement with these previous studies, but the
responses vary according to amendment and duration after application (figure 1A). Just after
amendment, FYM increases the activity by 40%, probably because of its high biodegradability and
micro-organisms addition. Incorporation and stimulation of phosphatase activity occurs later for MSW
and GWS, and no effect is observed for BIO after 18 months.
Whatever the amendment considered (Figure 1B), arylsulfatase activity does not show
contrasted results. That lack of noticeable effects is in agreement with previous studies [20], which
reported that arylsulfatase activity was inversely affected by elevated urban and industrial wastes
amendments.
Just after amendments, urease activity increases slightly (BIO) or highly (FYM and MSW).
No effect of GWS is noticed. For 18 months, the activity is increased by BIO, FYM and GWS. The
low effect of MSW may be due to accessibility of OM, N content and low NH4+ concentration. High
NH4+ concentration induces low urease activity in GWS. Nevertheless, progressive incorporation of
recalcitrant OM with GWS and BIO induces a high increase of activity with time.
Several months after amendments, BIO and GWS do not modify β-glucosidase activity with
respect to the control. These two amendments present high amounts of recalcitrant OM and the
impacts of amendment appear later, after incorporation to the soil. The highest response occurs with
MSW, the most biodegradable compost exhibiting a high OC content. Then, FYM increases highly
enzymatic activity.
Conclusion
Our results demonstrate that organic amendments have noticeable long-term-effects on
enzymatic activities. They suggest that soil microbial processes are sensitive indicators for the
monitoring of soil biological properties changes due to amendments by organic wastes.
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Acknowledgments. The SOERE-PRO and Qualiagro programmes are acknowledged for their
interdisciplinary research framework.