Effects of organic amendments on soil carbon fractions, enzyme activity and humus–enzyme complexes under semi-arid conditions

European Journal of Soil Biology 53 (2012) 94e102

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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Effects of organic amendments on soil carbon fractions, enzyme activity and humuseenzyme complexes under semi-arid conditions Felipe Bastida*, Keiji Jindo, José Luis Moreno, Teresa Hernández, Carlos García Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Department of Soil Conservation and Waste Management, Campus Universitario de Espinardo, 30100 Espinardo, Murcia, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 March 2012 Received in revised form 3 September 2012 Accepted 6 September 2012 Available online 29 September 2012 Handling editor: Christoph Tebbe

The objectives of this study are to evaluate the changes in the pool of organic carbon fractions, microbial biomass, and the activity of total enzymes and humuseenzyme complexes of a semi-arid soil amended with organic wastes of distinct origin during a period 360 days. The experiment was carried out during 360 days under laboratory conditions using soil microcosms (500 g) amended with two different doses (5 and 10 g) of different materials: sewage sludge from a wastewater treatment plant (SS), compost from that sludge (CSS), the organic fraction of municipal solid wastes (MSW), and compost from MSW (CMSW). The different carbon fractions, such as the total organic carbon (TOC), water-soluble carbon (WSC), and microbial biomass carbon (MBC), increased in amended soils compared to the control soil without amendment, as well as dehydrogenase and hydrolytic enzymes (b-glucosidase and urease) activities. For instance, after 360 days the total organic carbon reached 1.41% in soil amended with the high dose of SS and water-soluble carbon content reached 56 mg kg
1 in soil amended with high dose of CMSW. The immobilized enzymes in the soil humic extracts exhibited different behaviors compared to total activity, depending on the origin of the organic material which suggests neo-formation of humiceenzyme complexes. Addition of CMSW or SS increased the activity of b-glucosidase linked to humic substances. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Semi-arid soil Organic wastes Microbial activity Humuseenzyme complexes Enzyme activity

1. Introduction Addition of organic amendments is a suitable strategy to achieve soil recuperation in semi-arid areas such as SE Spain, where the organic matter (OM) content and biological quality are low [4,15]. The status of a soil can be evaluated by assessing the state of its microbial community [3,36]. Microorganisms are largely responsible for the cycles of the elements within a soil and are involved in the decomposition of the OM at the ecosystem level via a variety of enzymes. In this sense, the addition of different organic amendments, such as solid organic wastes, sewage sludge, agricultural wastes, and animal manures, is a method of replenishing degraded soil quality through improvement of the biological status of the soil, which usually implies an increase in both microbial and enzyme activity [2,23]. Enzyme persistence in the soil environment ranges from a few days to several years depending on the location and soil conditions such as temperature, pH, soil fraction, and depth [3,13,16,20]. As

* Corresponding author. Tel.: þ34 968396259; fax: þ34 968396213. E-mail address: [email protected] (F. Bastida). 1164-5563/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejsobi.2012.09.003

soon as they are released from the cell, they can be metabolized by soil microorganisms, unless stabilization takes place through adsorption and/or incorporation into clay and clayehumic complexes [28,31]. Nannipieri et al. (1996) [28] suggested that extracellular enzymes in soil are firmly associated with humic OM. These immobilized enzymes are surrounded by a network of humic molecules with pores large enough to permit the diffusion of substrates and products but not of proteolytic enzymes [9]. Inputs of fresh or matured organic amendments contribute at different levels to soil rehabilitation by supplying different substrates [8]; fresh organic inputs are the most-readily utilized by microorganisms while matured inputs provide more-recalcitrant polymerized compounds, which are easily incorporated into soil humic substances. However, despite the huge variety of studies, including total enzyme activities, there is still scarce information on their relation to total organic carbon [37] and the formation and/or stabilization of humuseenzyme complexes [10,22] We aim to evaluate the changes in the pool of organic carbon fractions, microbial biomass, and the activity of total enzymes and humuse enzyme complexes of a semi-arid soil amended with organic wastes of distinct origin during a period 360 days. Different organic wastes will be used for these purposes: sewage sludge from

F. Bastida et al. / European Journal of Soil Biology 53 (2012) 94e102

a wastewater treatment plant (SS), compost from that sludge (CSS), the organic fraction of municipal solid wastes (MSW), and compost from MSW (CMSW). We hypothesized that: 1) carbon fractions, microbial biomass and enzyme activity would increase after the addition of organic wastes and that the trend would depend on the nature of the organic waste; 2) the effect of composted materials would last in soil more than that from fresh materials; and 3) the activity of humuseenzyme complexes should be higher in soils amended with composted materials that provide a higher amount of humic substances. 2. Materials and methods 2.1. Soil sampling and experimental design Soil is a Haplic calcisol [35], sampled in an experimental field located in Santomera (SE Spain). The climate is Mediterranean semi-arid. The mean annual rainfall is 300 mm and potential evapotranspiration reaches 1000 mm year
1. Rainfall distribution through the year is very irregular with two maxima in October and April, and the mean annual temperature is 19.2  C [1]. This area is affected by soil degradation processes, such as erosion, and deficiency of OM. Soil was sampled in the upper layer (0e15 cm), airdried, and sieved to 2 mm. The main characteristics of the soil are shown in Table 1. The soil samples were stored at 4  C for one week, prior to incubation with the organic amendments. The proposed objectives were achieved by the incubation of soil with different organic materials under laboratory conditions. Triplicate mixtures of the soil and organic materials were incubated in hermetically-closed plastic pots of 8 cm height for 360 days at 28  C in the dark. Sampling times were: 0 (20 h), 15, 30, 60, 90, 180, 270, 360 days. Independent microcosms were established for each sampling time. The mixtures were prepared with 500 g of air-dried soil and 5 g (low dose) or 10 g (high dose) of dry, ground organic material. The microcosms were maintained at 60% of water-holding capacity during the incubation. Triplicate controls, without organic amendment but with water, were run during the incubation. Four different organic residues were used in this study: a sewage sludge (SS) collected from a municipal wastewater treatment plant in El Raal-Murcia (SE Spain); the compost (CSS) produced from this sewage sludge; the organic fraction of a municipal solid waste (MSW) collected from the treatment plant of Mula (15 km from Murcia city), which receives all the household wastes produced in the metropolitan area of Murcia (300,000 inhabitants); and the compost produced from this organic material (CMSW). The MSW was obtained after manual and mechanical separation of most of

Table 1 Characteristics of the control semi-arid soil from Santomera (SE-Spain).

pH Electrical conductivity (dS m
1) Total Organic Carbon (g kg
1) Total N (g 100 g
1) NH4 þ eN (mg kg
1) Total P (mg kg
1) Available P (mg kg
1) Total K (g 100 g
1) Available K (meq 100 g
1) Cu (mg kg
1) Zn (mg kg
1) Cr (mg kg
1) Ni (mg kg
1) Pb (mg kg
1)

Soil

Standard deviation

8.07 438.50 12.0 0.15 1.95 805.60 58.30 0.81 2.60 72.55 35.10 6.15 12.45 13.55

0.19 40.31 0.11 0.00 0.49 66.04 0.14 0.04 0.08 3.46 0.99 1.20 0.35 0.35

95

the metallic, plastic, and paper materials from the waste. The composting (industrial scale) of SS and MSW was carried out in horizontal reactors, in which the material remained static but received mechanical ventilation. The maximum temperature reached (65  C) was maintained for a minimum of 48 h, after which the temperature was maintained at 53e60  C during most of the process. The moisture level of the material was kept at 60% for the maintenance of microbial processes taking place during composting. To improve oxygenation inside the sewage sludge pile during the composting process, a bulking agent (wood shavings) was added, on a volumetric basis, in the proportion of 1:2 (material:bulking agent). The composting process lasted 75 days for both SS and MSW. Three samples (each composed of 8e10 subsamples) of each organic material were collected and air-dried, then each sample was milled in order to homogenize the material. The total organic carbon (TOC) was 17.9% (SS), 20.6% (CSS), 17.0 (MSW), or 16.6% (CMSW), respectively. The total organic nitrogen (TON) was 4.6% (SS), 3.4% (CSS), 1.6% (MSW), or 2.6% (CMSW), respectively. The chemical and biochemical data of these materials are detailed by Moreno et al. (2007) [25]. 2.2. Chemical analysis The soil electrical conductivity and pH were measured in a 1/5 (w/v) aqueous extract, using a Crison mod.2001 conductometer and pH meter, respectively. The TOC was determined by oxidation with potassium dichromate in an acid medium and measurement of the excess dichromate using Mohr’s salt [40]. The microbial biomass carbon was determined by a fumigationeextraction method, with extraction of organic C by K2SO4 [38] and determination of the C content in the K2SO4 soil extracts with a liquid organic C analyzer (Shimadzu TOC-5050A). The water-extractable carbon (WSC) was obtained by shaking a mixture of soil and distilled water (1:10 soil:water ratio, w/v) for 2 h, followed by centrifugation and filtering through ashless filter paper (Albet 145 110). In this extract, the WSC was determined with a C analyzer for liquid samples (Shimadzu 5050A, Kyoto, Japan). Humic substances were extracted with a 0.1 M, pH 9.8 sodium pyrophosphate solution (w/v ratio ¼ 1:10) by mechanical shaking for 4 h. Humic substance C was determined in this extract by a C analyzer for liquid samples (Shimadzu 5050A). Humic substance suspensions were centrifuged and the filtered extracts were acidified with H2SO4 to pH 2.0 and kept for 24 h at 4  C; they were then centrifuged to separate the precipitated humic acids from the supernatant fulvic acids [18]. 2.3. Enzymatic assays The soil dehydrogenase activity was determined as reported by Von Mersi and Schinner (1991) [39], using p-iodo-nitro-tetrazolium chloride as substrate and measuring the absorbance of the iodonitrotetrazolium formazam (INTF) produced in the enzymatic reaction. The soil urease activity was determined by the method of Kandeler et al. (1999) [20], and the b-glucosidase activity was determined using the method of Eivazi and Tabatabai (1987) [12]. 2.4. Immobilized enzymatic assays and infrared spectra Immobilized enzymatic activities within humic extracts were determined using 1-ml pyrophosphate extracts obtained from the soil. Humic substances were extracted with 0.1 M sodium pyrophosphate, pH 7.1, (w/v ratio ¼ 1:10) by mechanical shaking for 24 h. The centrifuged and filtered (0.2 mm Millipore membrane, Billerica, MA, USA) extracts were dialyzed against distilled water with a membrane of 12,000e14,000 Da molecular weight cut-off

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F. Bastida et al. / European Journal of Soil Biology 53 (2012) 94e102

and a 25-Å pore diameter (ViskingÒ dialysis tube, Serva GmbH, Heidelberg, Germany), to obtain a purified humic extract. After dialysis, the humic extracts were dehydrated and concentrated through polyethylene glycol to 1/10 of the original volume of the sodium pyrophosphate solution. Using 1 ml of this extract, the immobilized enzyme activities in the humic extract were determined using the procedures mentioned above. 2.5. Statistical analysis All the results are reported as means. All ANOVA analyses were followed by Tukey’s HSD (Honestly Significant Difference) post-hoc test. Four-way ANOVA with incubation time, origin of material, stabilization process (composting), and dose as factors, was performed for the following parameters: TOC, WSC, MBC, and the dehydrogenase, urease, b-glucosidase, and phosphatase activities. Three-way ANOVA with incubation time, origin of material, and stabilization process (composting) as factors, was performed for the activity of humiceenzyme complexes and the specific enzyme activity. In order to determine pair-wise differences by post-hoc tests, the data were submitted to one-way ANOVA at each incubation time. 3. Results 3.1. Carbon fractions Soils amended with the high dose (10 g) of organic materials showed the highest values of TOC and WSC. The TOC values of soils amended with sludge were significantly (P < 0.05) higher at 360 days than for the other treatments, followed by the high doses of MSW and CMSW (Table 2). The TOC content of the control soil decreased through the incubation time, from 1.19 to 0.84 mg kg
1 (a 30% loss) (Table 2). The losses of TOC during the incubation of the soil with the different materials 0e360 days: 24% (SS5), 21% (SS10), 25% (SS10), 25% (MSW5), 29% (MSW10), 34% (CMSW5), and 26% (CMSW10). The natural TOC losses during the incubation of the control soil were in the range of those of the other treatments (30%), indicating that soil OM can be degraded even in ecosystems with low but stabilized OM contents, such as semiarid areas. The WSC showed a fast decrease during the first months, followed by stabilization during the rest of the incubation (Table 2). The soil amended with a high dose of CMSW showed the highest value of WSC (P < 0.05) after 360 days of incubation. From 0 to 360 days of incubation, the WSC losses were higher in fresh than in their respective composted organic wastes: 67% (control), 77% (SS5), 81% (SS10), 67% (CSS5), 75% (CSS10), 92% (MSW5), 94% (MSW10), 70% (CMSW5), and 73% (CMSW10). Statistically, the TOC, WSC, and MBC were affected significantly (P < 0.01) by incubation time, origin of material, stabilization process (composting), and dose. At the beginning of the incubation, all applications of the organic amendments significantly (P < 0.05) increased the carbon fractions, compared to the control soil without amendment (Table 3). Addition of the organic materials to soil significantly increased the contents of humic substances, humic acids, and fulvic acids, comparing to the control soil, at the end of the incubation. Addition of composted materials produced the highest values of fulvic acids in soil. The addition of MSW, fresh or composted, gave the highest values in the soil (P < 0.05) (Table 4). 3.2. Microbial biomass and total enzyme activity Statistically, the MBC was affected significantly (P < 0.01) by incubation time, origin of material, stabilization process

Table 2 The total organic carbon (TOC) and water-soluble carbon (WSC) contents of control and soils amended with different organic materials. Days

0

TOC (mg C kg
1) Control 11.92 0.41 SS5 15.71 0.85 SS10 17.92 1.42 CSS5 13.13 0.99 CSS10 15.61 1.40 MSW5 15.00 1.14 MSW10 17.78 1.32 CMSW5 14.87 0.74 CMSW10 16.51 2.25 WSC (mg C kg
1) Control 71.39 6.22 SS5 167.18 5.98 SS10 221.13 4.18 CSS5 134.07 5.92 CSS10 176.26 6.18 MSW5 488.60 13.01 MSW10 635.38 16.08 CMSW5 149.67 0.79 CMSW10 210.34 10.59

15

30

60

90

180

270

360

9.74 0.65 12.79 0.74 14.91 1.81 10.75 0.32 12.74 0.93 10.98 0.85 12.90 0.91 12.00 0.71 12.63 0.64

8.72 0.52 10.71 0.53 13.65 0.14 10.02 0.81 13.17 0.81 10.00 0.84 11.74 0.91 8.89 0.25 12.17 0.58

9.37 0.47 10.51 0.23 12.67 0.58 10.27 0.52 14.61 1.54 9.69 0.25 10.71 0.44 10.64 0.51 12.73 0.14

8.42 0.05 10.41 0.21 13.02 0. 71 11.32 0.43 13.04 0.74 9.25 0.32 11.03 0.14 9.72 0.44 11.92 0.15

8.32 0.21 10.11 0. 51 13.20 0.11 9.24 0.43 11.44 0.85 9.25 0.11 10.64 0.13 9.24 0.32 10.91 0.81

8.53 0.40 10.72 0.65 12.64 0.22 9.55 0.25 12.31 0.52 9.91 0.81 10.65 0.63 9.94 0.45 11.93 0.92

8.45 0.21 12.05 0.61 14.14 0.13 9.85 0.32 12.65 0.33 11.32 0.12 12.55 0.14 9.84 0.30 12.24 0.70

57.05 4.30 58.07 1.83 65.19 4.28 65.30 2.97 125.59 9.81 65.81 5.83 78.90 7.73 85.56 0.10 152.04 10.81

44.68 1.64 45.71 2.99 65.68 7.05 56.66 3.17 63.99 5.90 56.35 0.57 61.82 9.36 66.60 4.08 80.28 2.31

39.66 1.43 41.99 2.26 47.05 3.55 49.47 3.88 55.35 0.24 57.77 0.32 65.17 3.22 56.34 3.03 76.42 0.58

30.72 1.39 46.13 3.67 52.55 3.23 32.26 3.98 55.36 0.38 44.80 2.35 68.61 3.34 57.74 3.63 82.14 4.42

30.07 4.50 40.34 2.52 50.56 0.57 38.08 2.08 48.66 3.95 39.89 0.67 51.71 4.33 45.00 2.33 74.52 0.67

29.30 3.17 42.51 1.45 45.51 2.37 36.73 0.39 47.05 1.91 42.25 0.47 44.40 4.61 40.92 3.78 60.10 1.55

23.35 0.03 38.14 2.39 42.49 2.40 43.55 1.54 42.60 0.98 37.92 0.61 38.53 5.91 44.35 4.40 56.00 1.86

The bold letters are the mean values and the italic letters underneath represent the standard deviation of the mean. Sludge (SS), composted sludge (CSS), organic fraction of municipal solid wastes (MSW), composted organic fraction of municipal solid wastes (CMSW). The number following each treatment indicates doses: 5 (5 g of organic waste per 500 g soil), 10 (10 g of organic waste per 500 g soil).

(composting), and dose. At the beginning of the incubation, application of the different organic amendments significantly (P < 0.05) increased the MBC, except in the case of the low SS dose (5 g) which did not produce significant differences from the control soil without amendment (Fig. 1). At 180 days, all amended soils had significantlyhigher values than control soil (Table 3). At this time, the high doses of CSS and MSW produced the highest values and this trend was quite conserved until the end of the incubation (360 days). Statistically, the time, dose, origin, and stabilization significantly affected (P < 0.01) all the studied enzyme activities. Dehydrogenase activity was significantly higher in soil amended with fresh organic materials than in soil receiving composted materials (Fig. 2, Table 3). At 0 days, the soil amended with the high dose of sludge showed a dehydrogenase activity which was significantly (P < 0.05) the highest, followed by the soil receiving the high dose of MSW (Table 3). However, at 60 days, dehydrogenase activity was significantly higher (P < 0.05) in soil amended with the high dose of MSW or CMSW than in soil amended with sludge. At 360 days, it was significantly higher (P < 0.05) in soil amended with the high dose of sludge or CMSW than in the other soils (Fig. 2). The soils amended with fresh materials (sludge and MSW) showed higher values of b-glucosidase activity at 15 days than soils amended with composted materials (Fig. 3). This trend was

F. Bastida et al. / European Journal of Soil Biology 53 (2012) 94e102

97

Table 3 One-way ANOVA of the different parameters analyzed in the control and amended soils during incubation.

bGla

Ure

DH

bGla

Ure

DH

MBC

TOC

WSC

a cd e b d f g bc e

15 days Control SS5 SS10 CSS5 CSS10 MSW5 MSW10 CMSW5 CMSW10

a ab e ab b d f c d

a bc d ab d cd e bc d

a c g b d e h c f

a a d b c b e b d

a c d ab c ab c bc c

a a a a c a b b d

a bc f b ef b cd a de

a a c b bc b bc c d

60 days Control SS5 SS10 CSS5 CSS10 MSW5 MSW10 CMSW5 CMSW10

a c c ab ab bc d bc c

a d de c e bc de b de

a b de bc c d f bc e

b a d c f c e b d

a bc e abc f ab c bc e

a a b b c c d c e

a b e de f cd e c c

a cd g ef g b de bc f

a bc cd a d b e d f

180 days Control SS5 SS10 CSS5 CSS10 MSW5 MSW10 CMSW5 CMSW10

a ab ab c d c d b c

a f g cd e cde de b bc

a b d b e c g c g

a b f de g c g cd ef

a bc e ab d ab cd ab cd

a bc e b de b e cd f

a b e d d a f b c

a c d b d b c b d

a cd de b e cd cde bc f

360 days Control SS5 SS10 CSS5 CSS10 MSW5 MSW10 CMSW5 CMSW10

a b bc b bc bc c b bc

a b d c c b d c c

a bc e b cd b d cd e

a a de f g cd ef b bc

a d e b d c d b d

a b c c c b b c d

MBC

TOC

0 days Control SS5 SS10 CSS5 CSS10 MSW5 MSW10 CMSW5 CMSW10

a a bc a bc cd d b cd

a cd e c de b b b b

a e h a b c g d f

a a g b f e e c d

a cd e ab cd bc de bc cde

30 days Control SS5 SS10 CSS5 CSS10 MSW5 MSW10 CMSW5 CMSW10

a a cd bc bc a e ab d

a cd e b g bc fg de f

a b f c d e g cd e

a a f b g d g e f

90 days Control SS5 SS10 CSS5 CSS10 MSW5 MSW10 CMSW5 CMSW10

ab abcd bcd abc d cd d abc a

a cd e cd f b e bc de

a bc cd b d e f b g

270 days Control SS5 SS10 CSS5 CSS10 MSW5 MSW10 CMSW5 CMSW10

a cde cde de e cd cde b c

a b b b c b c a a

a d e d f b h c g

WSC

For each incubation time, data followed by the same letters are not significantly different according to the HSD test (P < 0.05). bGla (b-glucosidase activity), Ure (Urease activity), DG (dehydrogenase activity), MBC (Microbial biomass C), TOC (Total organic C), WSC (Water-soluble C). Sludge (SS), composted sludge (CSS), organic fraction of municipal solid wastes (MSW), composted organic fraction of municipal solid wastes (CMSW). The number following each treatment indicates doses: 5 (5 g of organic waste per 500 g soil), 10 (10 g of organic waste per 500 g soil).

followed by a decrease during the incubation of such fresh materials with soil. At the end of the incubation (360 days), the soil amended with the high dose of fresh MSW showed the highest bglucosidase activity, in comparison with soils amended with sludge or composts. At the beginning of the incubation (0 days), the soils amended with fresh or composted sludge showed the highest urease activities (P < 0.05) (Fig. 4). Soils amended with fresh or composted MSW

Table 4 The humic substances (HS), fulvic acid (FA), and humic acid (HA) contents of control and high-dose amended soils after 360 days of incubation. Soil treatment

HS

FA

HA

Control SS CSS MSW CMSW

1835.4 a 2080.1b 2681.4e 2169.6c 2559.0d

735.6 a 880.7b 911.0b 974.7c 1042.9d

1095.6a 1175.4b 1744.0d 1173.9b 14.66.1c

For each incubation time, data followed by the same lower-case letters are not significantly different according to the HSD test (P < 0.05). Sludge (SS), composted sludge (CSS), organic fraction of municipal solid wastes (MSW), composted organic fraction of municipal solid wastes (CMSW).

showed significantly-lower (P < 0.05) values of this activity than soils amended with sewage sludge (Table 3). However, at the end of the incubation, soils amended with the high dose of fresh sludge or MSW showed the highest (P < 0.05) urease activity (Fig. 4). 3.3. Specific enzyme activity The specific enzyme activity is defined as the enzyme activity divided by total organic C and was tested for control soil and soil amended with the high doses of organic wastes. Statistically, the activities of all the immobilized enzymes were affected significantly (P < 0.05) by incubation time, origin of material and stabilization process (Table 5). In the case of b-glucosidase, there were not significant differences between control and amended soils at the beginning of the incubation. During the incubation, a general increase in the specific b-glucosidase activity was observed compared to the initial time but still the differences between control and amended soils were not generally significant (P < 0.05) (Table 5) (except in the case of sludge amendment). In the case of specific urease activity, a marked increase was observed for soil amended with MSW and CMSW during incubation, while no noticeable differences were showed for soil amended with sludges.

98

A

F. Bastida et al. / European Journal of Soil Biology 53 (2012) 94e102

A

500

1.3 1.1

300

µmols PNP g-1 h-1

mg C/Kg soil

400

200 100

0

90 SS 5g

SS 10g

180 DAYS control

270

360 0.3

CSS 5g

0

CSS 10g

90

SS 5g

500

B

400

control

270

CSS 5g

360

CSS 10g

1.3 1.1

-1 -1

200

100 0 0

MSW 5g

90

MSW 10g

180 DAYS control

270

360

CMSW 5g

CMSW 10g

Fig. 1. The microbial biomass carbon in control and soils amended with sludge (A) and the organic fraction of municipal solid wastes (MSW) (B), both fresh and composted.

0.9 0.7 0.5 0.3 0

MSW 5g

A

SS 10g

180 DAYS

300

µmols PNP g h

mg C/Kg soil

0.7 0.5

0

B

0.9

6

90

MSW 10g

180 DAYS

control

270

CMSW 5g

360

CMSW 10g

Fig. 3. The b-glucosidase activity in control and soil amended with sludge (A) and the organic fraction of municipal solid wastes (MSW) (B), both fresh and composted.

µmols PNP g-1h-1

5

Contrarily to specific b-glucosidase activity, the initial specific urease activity was higher in soils amended with fresh and composted sludge than in control and soils amended with MSW (both fresh and composted). At the end of incubation higher urease specific activity was observed in amended soils than in control but without differences between the types of amendment (P < 0.05) (Table 5).

4 3 2

1 0

0

90 SS 5g

B

SS 10g

180 DAYS control

270 CSS 5g

360 CSS 10g

6

µmols PNP g-1h-1

5 4 3 2 1 0 0 MSW 5g

90 MSW 10g

180 DAYS control

270 CMSW 5g

360 CMSW 10g

Fig. 2. The dehydrogenase activity in control and soils amended with sludge (A) and the organic fraction of municipal solid wastes (MSW) (B), both fresh and composted.

3.4. Immobilized enzyme activity in the humic substances Statistically, the activities of all the immobilized enzymes were affected significantly (P < 0.05) by incubation time, origin of material and stabilization process. Generally, the activity of the humuseenzyme complexes extracted from soil amended with MSW was not significantly different (P < 0.05) from that of the control soil (Table 5). Initially (0 days), the CMSW (high dose) gave higher activity of immobilized b-glucosidase than the rest of the treatments, followed by amendment with the compost derived from sludge (Table 5). However, the same result was not found in the case of urease extracted in the humus complexes. The control soil showed the highest urease activity immobilized in humic substances at the beginning of the incubation. Noteworthy, at the end of the incubation, the immobilized b-glucosidase activity was significantly (P < 0.05) higher in soils amended with the high dose of sludge than for the compost treatments. However, in the case of immobilized urease activity, the result was the opposite: higher humuseenzyme activity was found in compost-treated soil than for the sludge treatment and control soil (Table 5). In general, immobilized urease activity increased during incubation of soil

F. Bastida et al. / European Journal of Soil Biology 53 (2012) 94e102

A

Table 5 Specific and immobilized enzyme activities of soil incubated with different organic materials at high dose.

3

-1 -1

1.5

µgNH4 -Ng h

+

2.5

2

Specific-enzyme activity

bG1 Days Control SS

1

CSS

0.5

MSW CMSW

0 0

90 SS 5g

B

SS 10g

180 DAYS control

270

360

Days Control SS

-1 -1

µgNH4 -Ng h

+

CSS MSW CMSW

1 0.5 0 0 MSW 5g

90 MSW 10g

180 DAYS control

UA2 180 0.55bc 0.01 0.38a 0.05 0.60bc 0.05 0.64c 0.04 0.53b 0.04

360 0.53b 0.005 0.44a 0.04 0.49ab 0.03 0.52ab 0.05 0.50ab 0.03

bG3

CSS 10g

2.5

1.5

0 0.43a 0.03 0.37a 0.05 0.43a 0.06 0.44a 0.05 0.45a 0.05

0 0.66a 0.03 1.49b 0.39 1.47b 0.045 0.82a 0.08 0.74a 0.05

180 1.50a 0.055 2.14c 0.22 1.77ab 0.21 1.85ab 0.24 1.53a 0.17

360 1.52a 0.04 1.48b 0.06 1.50b 0.02 1.62b 0.66 1.52b 0.12

180 Nd.a e 107.60d 5.04 67.85c 6.25 Nd.a e 41.98b 0

360 Nd.a e Nd.a – 75.66b 0 Nd.a e 71.53b 8.41

Humus-enzyme activity

CSS 5g

3

2

99

270 CMSW 5g

360 CMSW 10g

Fig. 4. The urease activity in control and soils amended with sludge (A) and the organic fraction of municipal solid wastes (MSW) (B), both fresh and composted.

with composted materials but decreased in soils treated with fresh materials and in control soil. The percentages of immobilized b-glucosidase and urease activity relative to the total activity in soil amended with fresh sludge were 24.5% and 0%, respectively. In the case of soil amended with CMSW, these percentages were 17.1% and 3.86%, respectively, for b-glucosidase and urease. 4. Discussion It is important to note that 30% of the TOC was lost naturally in the control soil during its incubation. This result suggests that even the stabilized OM in semi-arid areas is able to be degraded when the soil moisture (along the incubation) is adequate. Hence, organic amendments in these areas make even more sense since natural OM can be lost during the precipitation events that improve soil moisture conditions. Additions of high doses of fresh materials were responsible for the highest increments of TOC in the soil, with no influence of the stabilization process at the beginning of the experiment. These changes in TOC at the beginning of the experiment were related to an increase in the size of the microbial community (MBC) and to the highest values of WSC, which provides labile carbon sources for the microbial community [5,6]. The TOC losses during the incubation were within the range of the control soil values and indicate that mineralization processes are controlled mostly by soil conditions rather than the type of organic material added. As in the case of MBC, all the organic amendments increased the WSC content at the initial stage of incubation, and soils amended

0 16.11a 10.34 Nd.a e 27.64b 1.58 13.07a 0.43 34.54c 0.84

UA4 180 Nd.a e 41.45b 6.03 57.39c 1.03 7.11a 1.78 117.04d 7.96

360 Nd.b e 150.96d 3.56 112.12c 1.09 Nd.a e 104.37c 2.56

0 46.57c 0 21.92a 2.62 42.20bc 4.30 36.65b 8.65 21.10a 0

The bold letters are the mean values and the italic letters underneath represent the standard deviation of the mean. * PNP, p-nitro-phenyl phosphate. Nd: nondetected. Sludge (SS), composted sludge (CSS), organic fraction of municipal solid wastes (MSW), composted organic fraction of municipal solid wastes (CMSW). bG1 b-glucosidase (mmols PNP* g
1 h
1)/TOC (g 100 g
1); UA2 :Urease (mg NH4 þeN g
1 h
1)/ TOC (g 100 g
1); bG3 :b-glucosidase (nmols PNP* g
1 h
1); UA4 :Urease (mmols NH4 þeN g
1 h
1). Data followed by the same lower-case letters are not significantly different according to the HSD test (P<0.05).

with fresh material showed higher WSC than with composted materials. This could be due to the presence of higher amounts of labile OM in fresh materials than in composted ones, since it is degraded during the composting process e as shown in our previous study [25]. In contrast to the general and slow decrease of TOC during the incubation, the WSC showed a fast decline, especially in the first stages of incubation. This result suggests a change in the OM pool of amended soils, as suggested by Mondini et al. (2003) [24], which mainly affects labile fractions. At the end of the incubation, the differences in the WSC losses mainly depended on the stabilization process of the materials. Looses were more intense in soils amended with fresh materials than with composted ones. For instance, looses of WSC in soil with high dose of MSW were 94% while looses in the soil amended with CMSW were 73%. The development of microbial biomass is sustained by the biosynthesis of enzymes that provide energy and nutrients for microbial development. Dehydrogenase activity in soil has been considered as a general index for evaluating soil microbial activity [8]. Compared to compost, the application of fresh materials led to a fast increase of dehydrogenase activity followed by a concomitant reduction, which is related to the decrease of easily-degradable substrates [32,33] e as indicated by the WSC. This pattern at the beginning of the incubation was similar for the microbial biomass carbon that reached the highest values for soil amended with high doses of sludge (448 mg C kg
1) and MSW (320 mg C kg
1). Higher values of dehydrogenase activity after 360 days of incubation were found in soils amended with high dose of sludge or CMSW. However, these results do not entirely fit with the MBC values. There are conceptual and methodological differences between both parameters. Intracellular enzyme activity may increase or decrease without any change in microbial biomass.

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Moreover, certain constraints of the methodology for dehydrogenase activity, such the low electron affinity of iodo-nitrotetrazolium, makes that it does not always correlate well with microbial biomass estimations [29]. Soil is a mosaic of metabolic processes [27] and enzyme activity is governed by a high number of biotic and abiotic factors [34]. Upon their release from the cell, extracellular enzymes can be denatured or maintain their activity. The activities of b-glucosidase and urease play a vital role in the dynamics of carbon and nitrogen in soil [26], and many diverse organisms produce these enzymes: they can be derived from microorganisms such as bacteria, fungi, or protozoa and also from animals and plant residues [11]. In our study, the amended soils showed significantly-higher total activities than the control soil, in agreement with other reports on the enhancement of hydrolytic enzymes by organic amendments [19,21]. The highest peaks of all three hydrolytic activities appeared in soil amended with sludge (high dose) in the early stage of incubation. Later, a reduction of the activity in soil amended with fresh materials was found, due to the scarcity of easily-degradable compounds in the water-soluble fraction [7]. By contrast, during the whole incubation time, more-steady values of enzyme activities occurred with the composted soil amendments, caused by the existence of a more-stable OM fraction [14,17]. The values of total enzyme activity may be related to the amount of enzyme originally present in the materials or to “de novo” biosynthesis by soil microorganisms, which are stimulated by organic compounds in the added materials. In the case of bglucosidase, CMSW had higher activity than the other materials themselves [25]. However, this difference had disappeared already after 15 days of incubation of this material with soil, and at the end of the experiment no dissimilarities were found among the different treated soils. The chemical composition of the different organic wastes can explain some of the results. For instance, the higher values of urease activity in soil amended with sludge (fresh and composted) than in soil amended with MSW can be related to the higher content of nitrogen in sludge than in the organic fraction of MSW. In this study, it was clearly demonstrated that organic amendments increased organic carbon in soil. However, it is difficult to ascertain whether the observed changes in enzyme activity are due to variation in organic carbon level or to real differences in enzyme activity. One way of overcoming this difficulty is to use the values of specific activity per unit of carbon [37]. Trasar-Cepeda et al. [37] indicated that a land use disturbance is associated to a general increase of enzyme activity per unit of carbon. A proposed mechanism implies a reduction of labile carbon fractions and a progressive enrichment of induced enzymes in the most stable organic matter. In this study, an increase of total organic carbon has been shown in amended soil in comparison to control soil. However, the trend of both b-glucosidase and urease specific activities per unit of organic C was different. Particularly, at the end of the incubation amended soils showed a higher urease activity per unit of organic carbon than control, while no significant differences were generally observed in the case of specific b-glucosidase. These results point out that the specific enzyme activity is highly dependent on the type of enzyme and overall conclusions cannot be extrapolated with only one enzyme measurement. Within the total budget of organic carbon, the enzyme activity linked to humic substances may act as a reservoir of microbial activity in soil [10]. Bastida et al. (2008) [5] found that the humuslinked enzyme activity in semi-arid soils was higher after longterm organic amendment with municipal solid wastes. These results contrast with those of the current work, where MSW addition did not increase the immobilized enzyme activity measured after one year of incubation, whereas the rest of the

treatments did. This difference between the two sets of results might be explained by the time-scales involved. In the present work, the activity of the complexes was analyzed for up to one year of incubation, while Bastida et al. (2008) [5] analyzed the immobilized activity after almost 20 years of incubation under field conditions, where plants may promote enzyme production and further immobilization into the humic complex. Nevertheless, it is difficult to understand these dynamics in the MSW-amended soils, since the general activity and microbial biomass were increased significantly after incubation of the soil with MSW, relative to the control. So, we can discard an inhibitory effect of MSW on the microbial community. The behavior of this complexed activity at the end of the incubation was different to that of the activity of the total hydrolases. As indicated above, the total b-glucosidase activity was stimulated by high doses of the four materials with no significant difference among them (except that addition of MSW generated the highest bglucosidase) and total urease activity had increased more in response to fresh organic amendment than to composted amendment addition at 360 days. However, in the case of the immobilized enzymes, the highest b-glucosidase activities were in soil treated with sludge (fresh or composted) or CMSW, and the immobilized urease activity was higher in soil treated with composted materials than with fresh ones (in contrast to the trend observed for the total activity). So, it is clear that the patterns of the total and immobilized enzyme activities differed. Moreover, the patterns between the specific activity per total organic carbon and humuseenzyme activity were different. For instance, despite the specific b-glucosidase activity was not significantly different between control and amended treatments (except sludge that was lower), a higher linkage of this enzyme to humus has been found in soils amended with sludge, composted sludge and composted MSW than in control soil. Total organic carbon is structurally complex and these results can be interpreted as a preferential immobilization of bglucosidase in the most stable part of the organic matter, the humic substances. The differing behaviors of the total and immobilized enzymes at the end of the incubation could be due mostly to a preferential “de novo” linkage of enzymes within the humic compounds. Nevertheless, a chemical change in the OM of soil amended with different organic materials has been described [18] and chemical differences in organic materials of distinct origin [18] might influence enzyme linkage to the humic matrix. Sewage sludge contains easily-degradable compounds that could stimulate the synthesis of hydrolytic enzymes and their release from intracellular media into the soil. This might be the reason why, after 15 days of incubation, the addition of such fresh material had produced a high level of total b-glucosidase activity able to degrade organic compounds. Subsequently, these protein molecules could have been entrapped into the humic matrix, leading to higher values of immobilized activity than in composttreated soils. In fact, the activities of these complexes increased with the time of incubation in the soil amended with sludge. For instance, b-glucosidase reached its highest degree of immobilization, as a percentage of the total enzyme activity, in soil amended with sludge (24.5%). However, sludge did not contain the highest amount of b-glucosidase, neither total nor in immobilized complexes [25]. This fact supports our hypothesis of biosynthesis and linkage of new enzymes released by microbial cells. Addition of composted materials to soil produced higher values of humic substances and humic acids at 360 days than the addition of fresh materials. In principal, this could lead us to assume higher immobilized activities in compost-treated soil. This happened for immobilized urease, but not for b-glucosidase: soil amended with sludge showed the highest immobilized activity, suggesting that

F. Bastida et al. / European Journal of Soil Biology 53 (2012) 94e102

the immobilization process might also be partially related to the molecular weight of each enzyme [30]. In fact, the percentage immobilized urease activity was much lower than for bglucosidase. 5. Conclusions According to the suggested hypothesis we conclude that: 1) Organic amendment had positive biochemical effects on the quality of a semi-arid soil in a one-year period. The applied doses significantly influenced the microbial activity as well as total organic C, WSC and MBC in the first stages. 2) Little difference between the two doses was found at the end, reflecting a sensitive response of this semi-arid soil to low inputs of OM. The addition of composting materials produced a more stable pattern of enzyme activity than such of fresh amended soils. In general, both composted and fresh materials stimulated microbial biomass, enzyme activity and enzyme persistence within humic complexes. A higher lasting effect of composted materials than fresh ones is discarded in soil (except in the case of humuseurease activity). 3) The dynamics of enzymes immobilized into humic substances were different to those of the total enzyme activity and depended on each particular enzyme. A preferential capacity of enzymes to be entrapped into humic compounds after their synthesis has been found. This capacity increased with the addition of compost prepared from municipal solid waste or the addition of fresh sewage sludge in the case of b-glucosidase. Although some authors have claimed toxic effects of SS, we were not able to detect such effects in our semi-arid, low-OM soil. Moreover, the application of such material benefited the formation of humuseb-glucosidase complexes, to an evenhigher degree than compost amendments, during the oneyear incubation. However, the amended with composted materials (but not the addition of fresh ones) benefits the presence of humuseurease complexes. Acknowledgments This work received financial support through a grant from the SENECA foundation (K. Jindo). K. Jindo and F. Bastida have contributed equally to this study. F. Bastida thanks the JAE-Doc program of the CSIC. Authors are grateful to Marie Curie Reintegration Grant (DYNOMIWAS, PERG07-GA-2010-263897), Spanish Ministry CICYT project (AGL2010-16707) and Consolider Ingenio Program for economical supporting. K. Jindo and F. Bastida contributed equally to this work. References [1] J. Albaladejo, V. Castillo, E. Díaz, Soil loss and runoff on semiarid land as amended with urban solid refuse, Land Degrad. Dev. 11 (2000) 363e373. [2] H. Albiach, R. Canet, F. Pomares, F. Ingelmo, Microbial biomass content and enzymatic activities after the application of organic amendments to a horticultural soil, Bioresour. Technol. 75 (2000) 43e48. [3] F. Bastida, J.L. Moreno, T. Hernández, C. García, Microbiological activity in a soil 15 years after its devegetation, Soil Biol. Biochem. 38 (2006) 2503e2507. [4] F. Bastida, E. Kandeler, J.L. Moreno, M. Ros, C. García, T. Hernández, Application of fresh and composted organic wastes modifies structure, size and activity of soil microbial community under semiarid climate, Appl. Soil Ecol. 40 (2008) 318e329. [5] F. Bastida, E. Kandeler, C. García, T. Hernández, Long-term effect of municipal solid waste amendment on microbial abundance and humus-associated enzyme activities under semiarid conditions, Microb. Ecol. 55 (2008) 651e 661. [6] L. Belete, W. Eggeer, C. Neunhauserer, B. Caballero, H. Insam, Can community level physiological profiles be used for compost maturity testing? Compost Sci. Util. 9 (2001) 6e18.

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