Soil application of meat and bone meal. Short-term effects on mineralization dynamics and soil biochemical and microbiological properties

ARTICLE IN PRESS

Soil Biology & Biochemistry 40 (2008) 462–474 www.elsevier.com/locate/soilbio

Soil application of meat and bone meal. Short-term effects on mineralization dynamics and soil biochemical and microbiological properties Claudio Mondinia,, Maria Luz Cayuelaa,b, Tania Siniccoa, Miguel Angel Sa´nchez-Monederob, Eleonora Bertolonec, Laura Bardic a

CRA-Istituto Sperimentale per la Nutrizione delle Piante, Sezione di Gorizia, Via Trieste 23, I-34170 Gorizia, Italy CEBAS-CSIC, Campus Universitario de Espinardo, Apartado de Correos 164, E-30100 Espinardo, Murcia, Spain c CRA-Istituto Sperimentale per la Nutrizione delle Piante, Sezione di Torino, Via Pianezza 115, I-10151 Turin, Italy b

Received 23 April 2007; received in revised form 1 August 2007; accepted 18 September 2007 Available online 9 October 2007

Abstract Meat and bone meal (MBM) utilization for animal production was banned in the European Union since 2000 as a consequence of the appearance of transmissive spongiform encephalopathies. Soil application could represent a lawful and effective strategy for the sustainable recycling of MBM due to its relevant content of nutritive elements and organic matter. The effectiveness of MBM as organic fertilizer needs to be thoroughly investigated since there is a lack of knowledge about the mineralization dynamics of MBM in soil and the impact of such residues, in particular the high content of lipids, on soil biochemical and microbiological properties. For this aim, a defatted (D) and the correspondent non-defatted (ND) MBM were added at two rates (200 and 400 kg N ha1) to two different moist soils and incubated at 15 and 20 1C for 14 d. MBM mineralization dynamics was studied by measuring CO2 evolution. Water extractable + organic C, K2SO4-extractable NO 3 and NH4 , microbial biomass ninhydrin-reactive N, enzymatic activities (FDA, urease, protease, alkaline phosphatase) and microbial composition (aerobic and anaerobic bacteria, fungi) were measured 2 and 14 d after MBM addition to the soil. The rate of CO2 evolution showed a maximum 2–3 d after the addition of MBM, followed by a decrease approaching the control. MBM mineralization was fast with, on average, 54% of total CO2 evolved in the first 4 d of incubation at 20 1C. The percentage of added C which was evolved as CO2 at the end of the incubation period ranged between 8% and 16% and was affected by temperature,  soil type and MBM treatment (ND 4 D). Soil amendment with MBM caused a noteworthy increase in both extractable NH+ 4 and NO3 (about 50% of added N) which was higher for ND. The addition of MBM also enhanced microbial content and activity. Microbial biomass increased as a function of the rate of application and was higher for ND with respect to D. The increase in numbers of aerobic and anaerobic bacteria and fungi caused by MBM addition was, in general, more pronounced with ND. Enzymatic activity in amended soils showed an enhancement in nutrient availability and element cycling. At the rate of application of present work, lipids did not cause adverse effects on soil microorganisms. The potential of MBM as effective organic fertilizer was supported by the large increase in available N and the enhancement of the size and activity of soil microorganisms. r 2007 Elsevier Ltd. All rights reserved. Keywords: Available N; Enzymatic activity; Lipids; Meat and bone meal; Mineralization; Soil microbial biomass

1. Introduction Meat and bone meal (MBM), obtained from processing of the residues of slaughtering operations, has been utilized Corresponding author. Tel.: +0481 522041; fax: +0481 520208.

E-mail address: [email protected] (C. Mondini). 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.09.010

for many years as valuable source of proteins and minerals in the livestock diet. Appearance of transmissive spongiform encephalopathies (TSE) in the 1990’s was associated to feeding ruminants with MBM contaminated with the infectious TSE agent. As a consequence, the utilization of MBM for animal production was banned in 2000 in the European Union (European Commission, 2000). The new

ARTICLE IN PRESS C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

legal situation created a disposal problem for this type of waste which, to date, was mainly solved by incineration. Besides the fact that this strategy poses a harmful dioxin production problem, thermal decomposition of MBM has been scarcely studied and dedicated incineration plants are only reliable when a sufficient and constant amount of MBM can be guaranteed for a long time (Conesa et al., 2005). Environmental constraints of incineration made it necessary to seek for alternative disposal strategies for MBM. Soil application is a promising strategy for a sustainable recycling of MBM. In fact, this organic residue contains a large amount of nutritive elements such as N (E 8%), P (E 5%), Ca (E 10%) mainly in organic form or organically bounded and low amounts of K and Mg. The high content of organic matter of MBM suggests a positive effect on the physical, chemical and microbiological properties of the soil. It may therefore be a useful fertilizer for various crops. The effectiveness of MBM as fertilizer has been positively evaluated by Novelo et al. (1998), Jeng et al. (2004 and 2006), Chaves et al. (2005). Soil application of MBM is an agricultural practice permitted by the current European legislation (European Commission, 2002) provided they derived from TSE free animals and were appropriately treated (heating by steam vapour at 133 1C for 20 min and 0.3 MPa) to eliminate any transmissible disease to human and animals. Such a strong thermal treatment makes MBM a safe product regarding possible harmful hygienic impacts of their utilization as fertilizer. In addition, MBM is among the fertilizers allowed by the European legislation on organic farming (European Commission, 1991). However, the mineralization dynamics of MBM in soil needs to be thoroughly evaluated since it is the main process regulating nutrient availability. In particular, because most of the N in MBM is organically bounded, it is important to investigate N mineralization, the mineral forms in which N can be present in the soil and the processes involved in their transformations for a reliable evaluation of MBM as N fertilizer. Indeed, very few studies are reported in the literature dealing with this topic (Blatt, 1991; Jeng et al., 2004, 2006; Chaves et al., 2005). The microbial pool exerts a key role in determining the degree of soil quality (Stevenson and Cole, 1999). In addition, microbiological based parameters are simple, rapid and effective indicators of modification in soil status due to pollution and variations in soil management (Brookes, 1995). For instance, microbial biomass size was shown to be an early indicator of variations in soil organic matter content due to soil environmental and management changes (Powlson, 1994). Enzyme activities related to the cycle of main nutritive elements could give indications on the rates of substrate turnover, soil metabolic potential, as well as to the resilience of the soil when subjected to various natural and anthropogenic impacts (Shaw and Burns, 2006). Finally, determination of main microbial

463

groups such as bacteria and fungi is a valid tool to elucidate shifts in microbial population following soil amendment. Soil amendment with a substrate rich in organic matter and nutritive elements, such as MBM, is likely to significantly affect soil microbial equilibria and more information is needed about the impact of MBM addition on soil biota (Pe´rez-Piqueres et al., 2006). In particular, MBM could be characterized by a significant content of lipids (Conesa et al., 2005). Lipids are usually extracted from MBM after the thermal treatment because of its potential use in the pharmaceutical industry. However, the extraction process requires sophisticated and expensive equipment and is profitable only if a high and constant amount of MBM is assured for a long time. This makes, under some circumstances, more profitable for rendering companies to produce non-defatted MBM. Addition of MBM with high lipid content to the soil could modify specific soil characteristics such as adsorption processes and hydrological properties, affecting the availability of elements and the normal functionality of microorganisms. Furthermore, lipids represent an anomalous high load of nutritive materials for microorganisms that may cause a shift on microbial populations by favouring those microorganisms that are able to utilize them more rapidly and efficiently. In this perspective, little is known about the influence of lipids on MBM mineralization and microbial equilibria. The aim of this work was to evaluate, under laboratory conditions, the short-term effects of applying MBM on mineralization dynamics and several biochemical and microbiological properties reflecting soil quality and functioning. In particular, the effect of lipid content of MBM on the chemical and biological properties of soil was investigated. 2. Materials and methods 2.1. MBMs, soils properties and incubation conditions A defatted (D) and the correspondent non-defatted (ND) MBM were utilized for incubation with soils. MBM derived from residues of slaughtering operation that were reduced to a diameter minor than 50 mm and subjected to an autoclave treatment at 133 1C for 20 min and 0.3 MPa. Defatted MBM derived from MBM treated to reduce its lipid content. The procedure involves the immersion of the MBM in a boiler containing hexane. Then, the solvent containing the dissolved lipids is separated from MBM which is treated to eliminate the residues of hexane and heated to reduce the humidity. Main properties of non-defatted MBM were: 5.1% water content; 35.4% organic C; 8.0% total N; 4.4 CORG/NTOT; 5.7% P; 0.38% S; 7.7% lipids. Selected properties of defatted MBM were: 5.2% water content; 30.7% organic C; 8.6% total N; 3.6 CORG/NTOT; 6.8% P; 0.39% S; 2.6% lipids.

ARTICLE IN PRESS 464

C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

Two arable soils with different pH (acidic and alkaline) were chosen for incubation experiments. The acidic soil was a Typic Dystrudept (USDA) with pH 5 (H2O), 55% sand, 28% silt, 17% clay, 1.59% organic C, 0.12% total N and 118 mg kg1 microbial biomass C. The alkaline soil was a Fluventic Eutrudept (USDA) with pH 8.3 (H2O), 69% sand, 28% silt, 3% clay, 740 g kg1 CaCO3, 1.05% organic C, 0.12% total N and 114 mg kg1 microbial biomass C. The soils were sampled at a depth of 5–25 cm, sieved moist through a 2 mm aperture grid, and pre-conditioned for 5 d at 40% of water holding capacity and the temperature to be used for incubation. At the end of this period, MBM (previously ground and sieved through a 0.5 mm aperture grid) was added to the soils and incubated for 14 d at both 15 and 20 1C. The rate of MBM application corresponded to 200 and 400 kg N ha1, which were established according to good agriculture practices (MIPAF, 1999). Controls consisted of non-amended soils. Aliquots of soils were taken after 2 and 14 d of incubation for chemical, biochemical and microbiological analysis, with the exception of CO2 evolution that was measured every fourth hour. 2.2. Chemical analysis Water extractable organic C was extracted by shaking a sample with degassed deionised water (water to solid ratio of 10:1) for 2 h at room temperature and determined by means of an automatic liquid C analyser (Dohrmann, DC 80).  Extractable NH+ 4 and NO3 were extracted by shaking a sample with K2SO4 0.5 M (liquid to solid ratio of 10:1) for 30 min at room temperature. The content of NH+ 4 was determined by a colorimetric method based on Berthelot’s reaction (Sommer et al., 1992). The clear supernatant (0.5 ml) was treated with 4 ml of deionized water, 0.5 ml of 0.2 M Na–EDTA, 1 ml of 1% sodium dichloroisocianide solution and 2.5 ml of a 1:1:1 mixture of 0.3 M NaOH, 4.03 mM sodium nitroprusside/1.062 M sodium salicylate solution and H2O. After 30 min the absorbance was determined at 690 nm. The content of NO 3 in the extracts was determined by reading the absorbance at 220 nm and subtracting the absorbance at 275 nm caused by the occurrence of organic matter. 2.3. Biochemical analysis Ninhydrin reactive N content of microbial biomass was determined with the fumigation-extraction method (Vance et al., 1987). Moist soil portions, each equivalent to 10 g oven dried soil, were fumigated with ethanol-free chloroform for 24 h, then extracted with 40 ml 0.5 M K2SO4 for 30 min. A set of non-fumigated soils were similarly extracted at the time fumigation commenced. The K2SO4-extractable ninhydrin reactive N was determined as described by Joergensen and Brookes (1990).

Microbial biomass ninhydrin reactive N was calculated using the following equation: BNIN ¼ [(ninhydrin-N extracted by K2SO4 from fumigated samples)(ninhydrin-N extracted by K2SO4 from non-fumigated samples)]. CO2 evolution was measured every 4 h on aliquots of moist soils (50 g oven dried basis; 40% water holding capacity) incubated at both 15 and 20 1C for 2 weeks by means of an automated system for gas sampling and measurement (Cayuela et al., 2006). The system generally operates as an ‘‘open chamber’’ system in which the plastic jars (130 ml) containing the moist soil samples are continuously aerated at a constant flow rate (20 ml min1) by means of an air pump. At regular time, a single jar is made a ‘‘dynamic close chamber’’ for a selected accumulation period (usually in the range 10–60 min) by means of two appropriate valves (Vici Valco Instruments). During the accumulation period the air is continuously recirculated in the selected jar by means of a peristaltic pump. The gas concentration in the closed chamber is automatically measured at the beginning and the end of the accumulation period by a gas chromatograph specifically fitted for gas measurements (Varian, CP2003) and the difference between the final and initial measurements provides the rate of gas production for the selected accumulation time. The system is operated by a dedicated software and can operate with up to 16 samples. The rate of hydrolysis of fluorescein diacetate (FDA) was determined according to Schnurer and Rosswall (1982). Fresh soil (1 g oven dried basis) was mixed with 20 ml of 60 mM sodium phosphate buffer pH 7.6 and 0.1 ml of FDA solution (0.2 mg ml1 dissolved in acetone). The soil mixture was incubated in a water bath at 24 1C for 2 h. The substrate solution was added to the controls after incubation. The reaction was stopped with 20 ml of acetone and then the soil suspension was centrifuged at 4000 rev min1 for 5 min. The optical sensitivity of the clear supernatant was read at 490 nm. Urease activity was measured according to Kandeler and Gerber (1988). Fresh soil (1 g oven dried basis) was mixed with 3 ml of 75 mM borate buffer pH 10 and 0.4 ml of 0.2 M urea solution. The soil mixture was incubated in a water bath for 2 h at 37 1C. The substrate solution was added to the controls after incubation. Ammonium produced during the incubation was extracted by adding 2 ml of 2 M KCl and shaking the soil mixture for 15 min. The soil suspension was then centrifuged for 5 min at 3000 rev min1. The ammonium content in the supernatant was determined as described above for the extractable NH+ 4 . Protease activity was estimated according to Alef and Nannipieri (1995). Fresh soil (1 g oven dried basis) was mixed with 2.5 ml of 50 mM TRIS(hydroxymethyl) aminomethane buffer (pH 8.1) and 5 ml of 2% Na–casein (Sigma C 8654; suspended in this buffer). The soil mixture was incubated in a water bath at 50 1C for 2 h. The substrate suspension was added to the controls after incubation. The reaction was stopped with 2.5 ml of 15% TCA and then the

ARTICLE IN PRESS C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

suspension was centrifuged for 5 min at 4500 rev min1. The clear supernatant (2 ml) was treated with 3 ml of a 50:1:1 mixture of 0.06 M NaOH/5% Na2CO3, 0.5% CuSO4  5H2O and 1% potassium sodium tartrate. After 90 min, the absorbance was determined at 700 nm. Alkaline phosphatase activity was determined according to Alef et al. (1995). Fresh soil (1 g oven dried basis) was incubated in a water bath for 1 h at 37 1C in 4 ml of modified universal buffer (MUB) pH 11.0 and 1 ml of 0.015 M disodium p-nitrophenyldiphenyl phosphate tetrahydrate. The substrate solution was added to the controls after incubation. The reaction was stopped with 1 ml of 0.5 M CaCl2 and 4 ml of 0.5 M NaOH and then the soil suspension was centrifuged at 4000 rev min1 for 5 min. The absorbance of the clear supernatant was read at 400 nm. 2.4. Microbiological analysis Composition of heterotrophic microbial populations was evaluated by determining total viable culturable cells of aerobic and anaerobic bacteria and fungi (yeasts and moulds). Microorganisms were extracted by shaking 1 g of soil with 9 ml of 0.9% NaCl solution on an orbital shaker at 250 rev min1 for 30 min. Soil suspensions were appropriately diluted with saline solution and 1 ml of each diluted suspension was inoculated on a Petrifilm AC and Petrifilm YM for bacteria and fungi determination, respectively. Cultures inoculated on Petrifilm AC were incubated in aerobiosis and anaerobiosis for aerobic and anaerobic bacteria determination, respectively, by means of an anaerobiosis generator system (Anaerogen on Anaerojar Oxoid). Incubation of all Petrifilm was performed at 30 1C for 4 d. Microbial numbers were determined indirectly by counting the colony forming units. Values obtained were reported as the number of viable cell per gram of soil. 2.5. Statistical analysis All results are expressed on an oven-dry basis (105 1C, 24 h) and are the mean of three sample replicates. Data were subjected to univariate ANOVA and treatment means were compared using the Student–Newman–Keuls and LSD multiple range test. The relationships between variables were analyzed using the Pearson correlation coefficient. All statistical analyses were performed using SPSS version 9.0 statistical package. 3. Results 3.1. Chemical analysis Soil amendment with MBM did not cause any significant changes in water extractable organic C (data not shown), while there was a noteworthy increase in both extractable

465

 NH+ 4 and NO3 (mineral N) with respect to the control (Fig. 1). N mineralization started immediately after soil amendment since the increase in extractable NO 3 and NH+ 4 was recorded already after 2 d of incubation. As incubation time elapsed the extractable NH+ 4 was readily  + converted into NO 3 . Both NO3 and NH4 contents were markedly influenced by the rate of MBM application. The extractable NH+ 4 content of soil was significantly affected by the lipid content of MBM, with higher values in soil amended with ND, although this increase was not always significant. The extent of apparent net N mineralization was calculated as the difference in the extractable inorganic N + (NO 3 and NH4 ) between MBM treatment and the control and is reported in Table 1 in both relative amount (mg N g1 soil) and percentage of added N. Between 21.5% and 33.1% of added N was already mineralized after 2 d of incubation at 20 1C in the amended soil at the highest application (data not shown). At the end of the incubation phase, about 50% of the added N was mineralized in the amended soils incubated at 20 1C, corresponding to an increase of about 200 kg of available inorganic N ha1 (Table 1). Apparent net N mineralization was significantly affected by the kind of MBM, with higher values in soils amended with ND. On average, the increase in mineral N at the end of incubation at 20 1C was 51.5% and 47.5% of added N for ND and D treated soils, respectively (Table 1).

3.2. Biochemical analysis Addition of MBM caused a significant increase in the size of soil microbial biomass after 2 d (Fig. 1). The content of microbial biomass in amended soil decreased at the end of incubation, but it was still significantly higher than the control, with the only exception of the acidic soil incubated at 15 1C. The increase in BNIN in amended soils was directly correlated with the rate of application, but it was unaffected by the temperature of incubation (Fig. 1). Regarding the effect of the different type of MBM, results indicated that on average ND promoted a higher development of microbial biomass compared to D, even if this increase was statistically significant only at the highest dose of MBM application and at 20 1C. Dynamics of microbial activity measured as rate of CO2 evolution was characterized by a peak occurring usually 2–3 d after the addition of the organic fertilizer, followed by a gradual decrease towards values approaching the control (Fig. 2). At the end of the incubation, 14 d after MBM application, the respiration rate of amended soil was on average 50% higher in respect to the control. Dynamics of soil CO2 evolution was affected by the different factors considered in the present work, such as the type of soil, MBM properties, temperature and rate of application. Pattern of CO2 evolution in the acidic soil was characterized by a higher rate of CO2 evolution throughout the whole experiment with respect to the alkaline soil. The

ARTICLE IN PRESS C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

466

alkaline soil

50 N-NH4+ (µg g-1)

15 °C

20 °C

15 °C

30

cc

b

cc 20

b

bb

b a

a

a

ab ab a

0 14 d

2d

N-NO3- (µg g-1)

a

c b

a

d a aa a b

a

2d

14 d

2d

14 d

120 15 °C

100

20 °C c

d

80 ab

bcc

cc

bb

bb b b bb

a

a

15 °C

100

20 °C

80 c

60 a

20

0

a

bb

cc

dc

c

b b

40

20

b abb aab

a

bb a

0 2d

14 d

2d

14 d

2d

14 d

2d

14 d

25

25

15 °C

20 °C

15 °C

20 °C

20

20 BNIN (µg N g-1)

10

a

e

20

14 d

120

40

b

30

0 2d

60

20 °C c

40

40

10

acidic soil

50

b 15

c

b 10 aa

a

15

a

10

b ab a a

5

b ab a

bb

b

b

ab ab a

a

a a a

5

0

0 2d

14 d

2d

14 d

2d

14 d

2d

14 d

incubation time (days) control NH+ 4

(N-NH+ 4 )

NO 3

200 D

200 ND

400 D

400 ND

(N-NO 3 ),

Fig. 1. K2SO4-extractable and and microbial biomass ninhydrin-reactive N (BNIN) in soils incubated at 15 and 20 1C. 200: meat and bone meal (MBM) addition at a rate of 200 kg N ha1; 400: MBM addition at a rate of 400 kg N ha1; D: defatted MBM; ND: non-defatted MBM. Bars represent standard deviation (n ¼ 3). Different letters indicate significant differences (SNK test; Po0.05) for any soil, temperature and incubation period.

lower temperature of incubation caused a delay of about 24 h of the peak of maximum respiration rate. In addition, the following stage of decreasing respiration rate was more gradual respect to that of soil incubated at 20 1C. Respiration rate was higher for ND applied at both doses. The significant effect of the different factors considered in this study on soil respiration was more clearly observed when considering the cumulative amount of CO2-C evolved during the whole incubation period (i.e.: acidic soil 4 alkaline soil; 20 1C 4 15 1C; 400 kg N ha1 4 200 kg N ha1; ND 4 D) (Fig. 3). Carbon mineralization of added MBM was evaluated by calculating the total amount of extra cumulative CO2-C evolved in the different treatments (cumulative CO2-C evolved from amended soil minus cumulative CO2-C evolved from control) and is reported in Table 2 as both relative values

(mg CO2-C g1 soil) and percentage of the organic C added with MBM. The cumulative extra CO2-C evolved after 14 d of incubation at 20 1C ranged from 9.9% to 15.7% of added C. The higher mineralization recorded in the case of soil amended with ND was in agreement with results obtained for extractable NH+ 4 . The C to N mineralization ratio (Dilly et al., 2003), calculated as the ratio between cumulative extra CO2-C and apparent net N mineralization, was practically constant for all the treatments at a value of 170.2 (standard deviation, n ¼ 16). In general terms, C and N mineralization followed very similar patterns during incubation and they were likewise affected by the factors studied. The rate of hydrolysis of fluorescein diacetate showed a significant increase following MBM addition and was assumed to be an index of the total microbial activity of the soil. FDA

ARTICLE IN PRESS C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

467

Table 1 Net increase in K2SO4-extractable inorganic N in soil amended with meat and bone meal at rates corresponding to 200 and 400 kg N ha1 Meal/Soil type

Incubation at 15 1C

Incubation at 20 1C

200 kg N ha1

Non-defatted Alkaline soil Acidic soil

400 kg N ha1

(% of added N)

(mg N g1)

(% of added N)

(mg N g1)

(% of added N)

(mg N g1)

(% of added N)

21.5 26.5

40.5 49.9

44.7 39.3

42.1 36.9

27.1 22.2

50.9 41.7

62.1 47.5

58.4 44.7

45.2 17.6 16.2

39.5

32.9 30.3

Average LSD

200 kg N ha1

(mg N g1)

Average Defatted Alkaline soil Acidic soil

400 kg N ha1

37.8 35.9

35.4 33.6

31.6 2.8

46.3 21.7 23.7

40.6 44.3

34.5

5.3

6.5

51.5 52.7 48.9

42.5

6.1

2.9

49.3 45.7 47.5

5.4

2.2

2.1

The net increase was calculated as the difference in inorganic N between amended and control soil. Inorganic N represents the sum of extractable N-NH+ 4 and N-NO 3 . LSD is the least significant difference at Po0.05.

20

CO2-C (µg kg-1 min-1)

15 °C 15

alkaline s. + D alkaline s. + ND acidic s. + D

10

acidic s. + ND alkaline s. (control) acidic s. (control)

5

0 0

2

4

8 6 incubation time (days)

10

12

14

20

CO2-C (µg kg-1 min-1)

20 °C 15

alkaline s. + D alkaline s. + ND acidic s. + D

10

acidic s. + ND alkaline s. (control) acidic s. (control)

5

0 0

2

4

6

8

10

12

14

incubation time (days) Fig. 2. Dynamics of the rate of CO2 evolution in soils amended with meat and bone meal (MBM) at 400 kg N ha1 and incubated at 15 and 20 1C. D: defatted MBM; ND: non-defatted MBM.

was higher in soil incubated at 20 1C, but no significant effects of the other factors investigated (soil and MBM type, rate of application) were detected (data not shown).

Protease activity was the most sensible parameter in reflecting changes in soil fertility due to the addition of MBM (Fig. 3). Soil amendment caused a marked increase

ARTICLE IN PRESS C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

468

acidic soil

alkaline soil 120

15 °C

120

20 °C

CO2-C (µg C g-1)

100 80

20 ab

cd e

a a

2d 500 Protease (µg tyr g-1 h-1)

c

c

14 d

bc

d

0 2d

15 °C c

d c

c

300 b

200

c

e

a

cd

b

c

bb

a

2d

14 d

15 °C

d

2d

14 d

20 °C c

300

b

c

200

b

c

e

d

b

d

c

c

a

100

a

a

b a

a

a

a

c

b

bb

a

a

e d

0

0 2d

Alk.Phosphatase (µg PNP g-1 h-1)

b

c

400

b

b

cd e

500

c

c

ab

14 d

20 °C

400

b

20

a

c

d

40

b

e

d

e

60

d

d b

400

14 d

2d

15 °C

14 d

b c

bb ab a

aa

ba

c

14 d

b b a

20 °C

200 b b b

c a

cd bc b

d ab a

bc d c

100

0

14 d

300

a

100

2d

15 °C

b

de

aa

2d 400

20 °C

c

300 200

e

80

40

100

20 °C

e

e

60

0

15 °C

100

c a

bbb

0 2d

14 d

2d

14 d

2d

14 d

2d

14 d

incubation time (days) control

200 D

200 ND

400 D

400 ND

Fig. 3. Cumulative CO2 respiration (CO2-C), protease activity and alkaline phosphatase (alk. Phosphatase) activity in soil incubated at 15 and 20 1C. 200: meat and bone meal (MBM) addition at a rate of 200 kg N ha1; 400: MBM addition at a rate of 400 kg N ha1 ; D: defatted MBM; ND: non-defatted MBM. Bars represent standard deviation (n ¼ 3). Different letters indicate significant differences (SNK test; Po0.05) for any soil, temperature and incubation period.

in this enzymatic activity, particularly in soils incubated at 20 1C that showed protease activity between 2 and 5 times higher than the control, even at the end of the incubation period. The increase was directly related to the application dose and to the type of MBM (D4ND), but was not significantly affected by the temperature. MBM amendment caused a general decrease of urease activity with respect to the control, even if not always statistically significant (data not shown). MBM type did not cause differences in urease activity. Also phosphatase activity was positively affected by soil amendment (Fig. 3). The increase was significant after 2 d of incubation and was sustained without significant changes until the end of incubation. The increase was correlated with temperature of incubation and, as in the case of

protease activity, with the rate of MBM application. Conversely to protease activity, higher values of protease activity were recorded in the soil amended with ND. 3.3. Microbiological analysis Soil amendment caused a significant increase in total aerobic bacterial counts, particularly in the alkaline soil. Microbial growth was proportional to the rate of application, while no differences were found between the different types of MBM. After 14 d of incubation there was a sharp and general increase of total aerobic bacterial counts in soils incubated at 15 1C, while in soils at 20 1C numbers of aerobic bacteria decreased, especially in the alkaline soil (Fig. 4).

ARTICLE IN PRESS C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

469

Table 2 Extra cumulative CO2-C (cumulative CO2-C from amended soil minus cumulative CO2-C from control) evolved from soil amended with meat and bone meal at rates corresponding to 200 and 400 kg N ha1 after 14 d of incubation Meal/Soil type

Incubation at 15 1C 200 kg N ha

Non-defatted Alkaline soil Acidic soil

400 kg N ha

200 kg N ha1

400 kg N ha1

(% of added C)

(mg C g1)

(% of added C)

(mg C g1)

(% of added C)

(mg C g1)

(% of added C)

23.3 21.7

10.3 9.6

45.3 44.4

10.0 9.8

26.7 32.7

11.8 14.5

54.4 71.0

12.1 15.7

10.0 15.9 14.6

Average LSD

Incubation at 20 1C 1

(mg C g1)

Average Defatted Alkaline soil Acidic soil

1

8.5 7.8

9.9 34.6 29.6

8.1 0.3

0.2

9.3 7.9

13.1 18.4 23.7

8.6 0.5

0.1

9.9 12.7

13.9 38.9 41.8

11.3 1.4

0.7

10.4 11.2 10.8

2.8

0.7

LSD is the least significant difference at Po0.05.

Total counts of anaerobic bacteria were lower with respect to aerobic bacteria. In the alkaline soil, differences between treatments were minimal after 2 d of incubation, while after 14 d there was an increase which was significant only in soil incubated at 20 1C. In the acidic soil differences due to incubation temperature were minimal and anaerobic bacteria multiplication at 20 1C was more stimulated by ND and higher rate of MBM application (Fig. 4). Total fungi counts in the unamended alkaline soil were quite low, probably due to the alkalinity of the soil. Fungal biomass was significantly increased by MBM addition only after 14 d of incubation. On the contrary, in the acidic soil, characterized by initial higher numbers of fungi, there was a sharp increase following MBM addition already after 2 d of incubation at both incubation temperatures. After 14 d of incubation, total counts of fungi did not change at 15 1C, while they decreased at 20 1C (Fig. 4). On the whole results from composition of microbial population agreed with those of microbial biomass content and rate of CO2 evolution for that concerning the effects of the dose of MBM application (400 kg N ha1 4 200 kg N ha1) and MBM properties (ND 4 D). 4. Discussion 4.1. Carbon and nitrogen mineralization Dynamics of CO2 evolution showed an immediate rise in the soil respiration rate, followed by a period of exponential increase, reflecting the growth of microorganisms on the added substrate (Demetz and Insam, 1999). This behaviour is consistent with previous works dealing with the addition of readily available substrate to soil (Nordgren, 1992; Dilly, 1999) indicating that MBM presents a significant quantity of easily available substances, which can be readily utilized as carbon and energy

source by microorganisms. These substances are mainly constituted by amino acids and polypeptides derived from the partial hydrolysis of the complex proteins during the thermal treatment underwent by MBM. The pattern of CO2 evolution indicated that the microbial growth following MBM addition was not affected by nutrients deficiency. Previous researches have shown that the respiratory response of glucose added to the soil was drastically modified by limitation of N and P: the exponential phase was poorly developed and followed by a period of constant respiration, until exhaustion of the source of readily available C (Nordgren, 1992; Dilly, 1999). The absence of nutrient deficiencies in the present work is not surprising due to the significant N and P content of MBM. The total extra CO2-C evolved after 14 d of incubation of MBM amended soil in the present study (10–16% of added C at 20 1C) was comparable with values recorded for poultry manure (16%) and pig slurry (19%) in a 20-day incubation study performed at 22 1C by Levi-Minzi et al. (1990) and with decomposition of several amino acids (10–25%) in an incubation performed at 18 1C (Jones, 1999). MBM mineralization was fast with, on average, 54% of total CO2 evolved in the first 4 d of incubation at 20 1C (Fig. 2). Similar to C mineralization, organic N of MBM was readily transformed into NH+ 4 and further converted into NO , indicating the good potentiality of MBM as N 3 fertilizer. Soil N mineralization represents one of the most important factors to consider in the evaluation of the agronomical value of an organic amendment. The extent of N mineralization ranged between 30–58% of added N (Table 1), which is in agreement with results obtained by Chaves et al. (2005). Net N mineralization rate reported in the literature for other organic residues varied considerably. For instance Pansu and Thuries (2003) in a sandy soil incubated at 28 1C for 6 months found net mineralization

ARTICLE IN PRESS C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

Aerobic bacteria (cell g-1) x 108

470

18 16 14 12 10 8 6 4 2 0

alkaline soil 15 °C

Anaerobic bacteria (cell g-1) x 106

20 °C

b ab ab

c

c c bb

bb

ab a

a

b d a

a

2d

14 d

c bb

2d

12

15 °C

c

c abb a

bc aab

2d

14 d

aa a

2d

15 °C

12

c

bb

14 d

20 °C

10 c

8

8

b

6

6 b

4

0

20 °C

d

14

20 °C c

10

2

15 °C

14 d

14

ab c ab b a

a

aa a

2d

4.0 Fungi (cell g-1) x 104

acidic soil 18 16 14 12 10 8 6 4 2 0

14 d

ab

cdd

2 a

2d

15 °C

4

0

a aa

14 d

ab

2d 50

20 °C

d c a b

bc

e

14 d

a aa

bb

2d

15 °C

a aa 14 d

20 °C

40 3.0

30

2.0

20

1.0

b aa a

0.0 2d

14 d

2d

10 0

b

d

c a

14 d

cc b 2d

a

a

bb

14 d

ab aa a b 2d

14 d

incubation time (days) control

200 D

200 ND

400 D

400 ND

Fig. 4. Total viable culturable cells of aerobic bacteria, anaerobic bacteria and fungi in soils incubated at 15 and 20 1C. 200: meat and bone meal (MBM) addition at a rate of 200 kg N ha1; 400: MBM addition at a rate of 400 kg N ha1; D: defatted MBM; ND: non-defatted MBM. Bars represent standard deviation (n ¼ 3). Different letters indicate significant differences (SNK test; Po0.05) for any soil, temperature and incubation period.

values of 22% and 27% for organic fertilizers with a C/N ratio of 2.9 and 3.7, respectively. Morvan et al. (2006) reported net N mineralization ranging from 3% to 51% of added N for a range of manures in an incubation experiment of 224 d. Flavel and Murphy (2006) found gross mineralization rates in the range 29–137% of the N added with different organic amendments in a soil incubated at 15 1C for 142 d. The C and N mineralization ratio has been proposed by Dilly et al. (2003) as an indicator of the biologically active C and N pools in the soil. Application of such ratio to amended soil could give information on the quality of available organic substrate, the complex connections existing between C and N mineralization of organic

residues and the temporal dynamics of nutrients mobilization and immobilization. The C/N mineralization ratio in MBM amended soils, calculated as the ratio between cumulative extra CO2-C and cumulative apparent net N mineralization, was quite constant for every treatment at a value of around 1. Dilly et al. (2003) found values of such ratio in the range 5 –37 for a set of unamended soils. Values of C and N mineralization for soil treated with a range of organic residues showed that this ratio is generally significantly higher with respect to MBM (Flavel and Murphy, 2006; Luxhøi et al., 2006). Nevertheless, Dilly et al. (2003) found that in some biotopes N mineralization overproportionally exceeded C mineralization and addition of

ARTICLE IN PRESS C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

readily degradable substances to the soil may lead to an increase in the mineralization of soil organic N (Groffman, 1999; Kuzyakov et al., 2000). The relationship between C and N mineralization of residues is complex since on one hand residue N is a source of organic N that is subject to mineralization and immobilization. On the other hand, N residue is a constituent of the overall N availability that may affect the growth of heterotrophic microorganisms and consequently change the dynamics of C decomposition (Trinsoutrot et al., 2000). It is generally recognized that the main factors affecting this relationship are the C/N ratio and the N content of the residues, the microbial use efficiency of residue C and N and the C/N ratio of soil microbial biomass (Barraclough, 1997; Murphy et al., 2003, Luxhøi et al., 2006). The main single factor predicting soil mineralization of the N added with organic residues is the C/N ratio of the amendment (Nicolardot et al., 2001; Bruun et al., 2005; Flavel and Murphy, 2006). Generally, an inverse relationship exists between these two parameters (Khalil et al., 2005; Morvan et al., 2006) and low C/N ratio residues are regarded as highly decomposable materials (Nicolardot et al., 2001). Dilly et al. (2003) found that N mineralization was higher when a lot of soluble N compounds with a low C/N ratio were present. Also the N content of residues can affect the C to N mineralization ratio. Trinsoutrot et al. (2000) showed that the N content of the residues did not affect the amount of C mineralized, while it was positively correlated with N mineralization rate. These evidences are in agreement with the high values of apparent net N mineralization recorded in the present work, due to the high N content (8.0% and 8.6% for ND and D, respectively) and particularly low C/N ratio of MBM (4.4 and 3.6 for ND and D, respectively), representing extreme values with respect to most of the organic residues. Another important factor regulating the residue C/N mineralization ratio is the microbial use efficiency of the residue C and N. Dilly (1999) found that cumulative CO2 respiration was lower in soil amended with C and N respect to C alone, i.e. supplementary N addition caused a more efficient use of C. Similarly, it has been shown that the values of microbial C use efficiency ranged between 64-90% for amino acids added to the soil (Jones, 1999; Jones et al., 2005; van Hees et al., 2005) in comparison to values of 10–40% for organic acids and 40–80% for monosaccharide (van Hees et al., 2005). A high microbial use efficiency of C added with MBM is supported by the fact that only 10–16% of added C was evolved as CO2 and that the amount of water extractable organic C in soil was unaffected by the addition of MBM despite its fast mineralization. This could indicate that the added C is mainly utilized for microbial synthesis, as evidenced by the increase in microbial biomass and total microbial counts. For that concerning the microbial use efficiency of residue N, Dilly et al. (2003) measured high values of N mineralization rate for unit of microbial biomass C

471

(qNmin) in soils with low C/N mineralization ratio, indicating a low microbial use efficiency of added N. A possible microbial utilization of low molecular weight substrates, such as amino acids, is the so-called direct route in which low molecular weight compounds are taken up directly into the cell to meet microbial C and N requirements. If the C/N ratio of the substrate is lower with respect to that of microbes the exceeding N is excreted as NH+ 4 (Jones and Shannon, 1999; Murphy et al., 2003). Soil microbial biomass C/N is variable depending on the composition of microbial population, but there is a general consensus to a mean value at around 8 (Kuzyakov et al., 2000; Murphy et al., 2003). This value is higher than the C/N ratio of MBM and this implies that microbial utilization of MBM would lead to the liberation of mineral N in the soil and this release would be higher with increasing C/N ratio in the soil microbial biomass. 4.2. Biochemical and microbiological properties The agronomical value of MBM should be evaluated not only in terms of contribution and availability of plant nutritive elements, as previously discussed for N, but also from the point of view of the improvement of soil microbial biomass status. Indeed, MBM presented interesting properties also in this perspective. Addition of MBM caused an overall and significant increase in the size of soil microbial biomass, in agreement with the general behaviour recorded after soil application of organic residues (Rezende et al., 2004; Sa´nchez-Monedero et al., 2004; Melero et al., 2006) and in particular with findings of Novelo et al. (1998) in a soil amended with bone meal. The increase in the content of microorganisms points out to the absence of any toxic or detrimental substance that could hamper microbial maintenance and growth. Furthermore, the significant increase in BNIN after only 2 d of incubation (Fig. 1) indicates that added MBM is mainly composed by readily available compounds. This fact is supported by the dynamics of the evolution of CO2-C and by the initial increase (after 2 d of incubation) in total counts of viable culturable cells (Fig. 4). The largest increase in amended soils was measured for aerobic bacteria with respect to anaerobic bacteria and fungi. Aerobic bacteria are known to be responsible for the degradation of readily available substrate and as a consequence to grow very fast in the presence of degradable compounds (Bittman et al., 2005). MBM addition also caused a significant and fast increase in microbial activity that was sustained throughout the incubation (Fig. 3). In particular, FDA can be hydrolysed by different classes of enzymes such as lipases, esterases and proteases. Hydrolysis of FDA is not associated with specific groups of microorganisms and for this reason is assumed to be a reliable indicator of total hydrolytic activity of the soil (Schnurer and Rosswall, 1982). Total microbial activity is a good general measure of organic matter turnover since generally more than 90% of the

ARTICLE IN PRESS 472

C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

energy flow passes through microbial decomposers (Heal and McClean, 1975). Therefore, an increase in FDA activity indicates an enhanced ability of the soil to degrade and transform organic substrates and pollutants. The main effect of MBM addition on the soil biochemical properties was the noteworthy increase in protease activity (from 2 to 5 times higher than the control) in agreement with a 3- to 8- fold increase found by Rezende et al. (2004) in soils amended with distillery yeast. Protease is stimulated by the addition of organic residues (Rezende et al., 2004) and is repressed by its catabolites (Dilly and Nannipieri, 2001). In the present work, protease activity showed a fast increase after 2 d and then continued to increase (amended soils incubated at 15 1C) or remained constant (soil incubated at 20 1C) during the incubation. Protease activity was not repressed by amino acids that could be present in the added MBM as a result of thermal treatment or derived from the fast mineralization of the residues in the soil. The lack of any enzymatic activity repression points out to the presence of constitutive proteases and/or extracellular proteases stabilized by soil colloids that were not affected from the products of the enzymatic reaction (Dilly and Nannipieri, 2001). The sustained enhancement in protease activity indicates that MBM addition caused an increase of available substrate for enzymatic reaction. The availability of the added N was probably improved by the thermal process underwent by MBM, causing a partial hydrolysis of the proteins with liberation of peptides. Protein degradation is a key step in the process of mineralization of organic N (Alef and Nannipieri, 1995). The link between protease activity and N mineralization is supported by the significant correlation between extractable NH+ and protease activity after 2 d of 4 incubation (r2 ¼ 0.86; po0.01). The stimulation of protease activity caused by the MBM addition represents therefore an important improvement in the fertility status of the soil. This behaviour was not observed for the urease activity, which also plays an important role in the N cycle. Urease activity was inhibited by the addition of MBM. This could be due to the repression of enzyme synthesis caused by the  large amount of NH+ 4 and NO3 released by the fast mineralization of MBM (Fig. 1). McCarty et al. (1992), in a study on the effects of different forms of N on urease in soils amended with organic C, showed that addition of  NH+ 4 or NO3 repressed enzyme production. Similarly, a repression of urease activity by NO 3 was found by Dilly and Nannipieri (2001) in an experiment with the addition of glucose and nitrate to a beech forest soil: urease activity was increased by C alone, while combined C and N amendment repressed urease synthesis. The synthesis of repressible urease could also be affected by the amount and form of amino acids derived from the degradation of the organic N. McCarty et al. (1992) showed that several L-amino acids, but not the correspondent D-isomers, repressed urease synthesis in glucose-amended soil.

Results of enzymatic activity linked to the N cycle (protease, urease) indicate that MBM represents an important source of N readily available, as indicated by the fast mineralization of MBMs after their addition to the soil. Similarly important for the soil fertility status was the significant increase in phosphatase activity in MBM amended soils. Phosphatase activity is generally stimulated by microbial growth and from low levels of available P and repressed by the product of the enzymatic reaction. MBM may contain significant amount of mineral P (33–40% of total P; Jeng et al., 2006) and therefore soil amendment with MBM would have expected to inhibit phosphatase activity. Nevertheless, in our work phosphatase activity was significantly increased by MBM addition and such increase was sustained up to the end of incubation. The insensitivity of phosphatase activity to P addition is likely due to the stabilization of the enzymes by their interaction with soils colloids or to the presence of constitutive enzymes (Nannipieri et al., 1990). This evidence and the high P content of MBM point out to an increase of available P for plants following MBM amendment. The overall increase in the content and activity of microbial biomass represents an important enhancement of soil quality and health. A soil with an increased microbial activity is a soil with an enhanced capacity to perform fundamental ecosystem functions such as nutrient cycling, organic residues decomposition, humic substances synthesis, structure enhancement, xenobiotics degradation, N fixation, support of plant growth and regulation of water cycle (Van-Camp et al., 2004).

4.3. Effect of lipids A potential concern of soil application of MBM is represented by the high content of lipids. Lipids in the soil are known to have the potential for altering soil physical properties such as the degree of wetting (Dinel et al., 1990; Stevenson, 1994). The conditions of water-repellency or non-wettability can lower the rate of degradation of native or exogenous organic matter, since reactions involved in the transformation of organic matter occurs in aqueous solution at the solid–liquid interface. In addition some specific lipids exhibit an inhibitory action on soil activity (Dinel et al., 1990). On the other hand there are studies that refer the ability of microorganisms to degrade lipids and to utilize them as source of C (Dinel et al., 1990; Hita et al., 1996). Moucawi et al. (1981) reported high rates of decomposition of C-18 lipids in soils where the microbial populations were abundant and diversified, regardless of the chemical structure of the lipids. Evaluation of the effect of the lipids contained in the MBM on mineralization dynamics and biological properties showed that MBM with higher lipid content (ND) generally caused an increase in soil respiration, net N

ARTICLE IN PRESS C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

mineralization and size and activity of soil microbial biomass. These results therefore point out to the fact that lipids, at least in the amount present in non defatted MBM (7.7%), do not alter soil properties and/or do not have a direct inhibitory effect on microorganisms growth and activity and, on the contrary, they can be easily utilized as substrate for microbial metabolism. 5. Conclusions In conclusion the main findings of the work can be summarized as follows: (1) MBM added to the soil was characterized by a fast mineralization dynamics, indicating that MBM is a good source of readily available C and N. In particular MBM caused a higher mineralization of residue N with respect to residue C, attributable to the low C/N of the residue and to an increase in the microbial use efficiency of the residue C, that resulted in a significant increase in available mineral N. (2) Soil amendment with MBM caused an overall enhancement in the size and activity of soil microbial biomass, resulting in an increase of soil quality. In particular, respiration dynamics showed that the addition of readily available C did not cause limitation in other essential nutritive elements. The increase in enzymatic activities points out to an enhanced capacity of the soil for element cycling. (3) The presence of lipids did not show any negative effects on soil properties and soil microbial activity. On the contrary, the microbiological indexes point out to a positive role exerted by lipids, that can be utilized as source of energy and substrate by microorganisms. In view of these results, the utilization of non-defatted MBM as organic fertilizers could be recommended. The beneficial effects of MBM on soil chemical and microbiological properties makes this organic residue to have a relevant potential as organic fertilizer. Nevertheless, the rapid N mineralization would release significant amounts of N into the soil system shortly after its application. Therefore, soil amendment with MBM should be carefully considered in terms of time and rate of application in order to avoid NO 3 contamination of the environment and to optimize the efficiency of N utilization by plants. Acknowledgements The authors would like to thank Mrs. Emanuela Vida for skilful technical assistance and ILSA spa (Arzignano, Vicenza, Italy) and SAPI spa (Castelnuovo Rangone, Modena, Italy) for providing the MBMs. Thanks are due to Sandro Malmusi at SAPI spa for information on the defatting treatment of MBM. M.L. Cayuela thanks

473

Fundacio´n Se´neca (Agencia Regional de Ciencia y Tecnologı´ a. Regio´n de Murcia) for financing her postdoctoral grant. References Alef, K., Nannipieri, P., 1995. Protease activity. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London, pp. 313–315. Alef, K., Nannipieri, P., Trazar-Cepeda, C., 1995. Phosphatase activity. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London, pp. 335–344. Barraclough, D., 1997. The direct or MIT route for nitrogen immobilization: a 15N mirror image study with leucine and glycine. Soil Biology & Biochemistry 29, 101–108. Bittman, S., Forge, T.A., Kowalenko, C.G., 2005. Responses of the bacterial and fungal biomass in a grassland soil to multi-year applications of dairy manure slurry and fertilizer. Soil Biology & Biochemistry 37, 613–623. Blatt, C.R., 1991. Comparison of several organic amendments with a chemical fertilizer for vegetable production. Scientia Horticulturae 47, 177–191. Brookes, P.C., 1995. The use of microbial parameters in monitoring soil pollution by heavy metals. Biology and Fertility of Soils 19, 269–279. Bruun, S., Stenberg, B., Breland, T.A., Gudmundsson, J., Henriksen, T.M., Jensen, L.S., Korsæth, A., Luxhøi, J., Pa´lmason, F., Pedersen, A., Salo, T., 2005. Empirical predictions of plant material C and N mineralization patterns from near infrared spectroscopy, stepwise chemical digestion and C/N ratios. Soil Biology & Biochemistry 37, 2283–2296. Cayuela, M.L., Sanchez-Monedero, M.A., Roig, A., Sinicco, T., Fornasier, F., Mondini, C., 2006. A semiautomatic chromatographic system to evaluate C cycle in amended soils under laboratory conditions. In: Eckhard, K., Bidlingmaier, W., de Bertoldi, M., Diaz, L.F., Barth, J. (Eds.), Proceedings of the International Conference ORBIT 2006, Weimar ,Germany, 13–15 September 2006, pp. 983–986. Chaves, C., Canet, R., Albiach, R., Marin, J., Pomares, F., 2005. Meat and bone meal: fertilizing values and rates of nitrogen mineralization. In: Bernal, M.P., Moral, R., Clemente, R., Paredes, C. (Eds.), Proceedings of the 11th International Conference RAMIRAN, Murcia, Spain, vol. 1, 6–9 October 2004, pp. 177–180. Conesa, J.A., Fullana, A., Font, R., 2005. Dioxin production during the thermal treatment of meat and bone meal residues. Chemosphere 59, 85–90. Demetz, M., Insam, H., 1999. Phosphorus availability on a forest soil determined with a respiratory assay compared to chemical methods. Geoderma 89, 259–271. Dilly, O., 1999. Nitrogen and phosphorus requirement of the microbiota in soils of the Bornho¨ved Lake district. Plant and Soils 212, 175–183. Dilly, O., Nannipieri, P., 2001. Response of ATP content, respiration rate and enzyme activities in an arable and a forest soil to nutrient additions. Biology and Fertility of Soils 34, 64–72. Dilly, O., Blume, H.-P., Sehy, U., Jmenez, M., Munch, J.C., 2003. Variation of stabilised, microbial and biologically active carbon and nitrogen in soil under contrasting land use and agricultural management practices. Chemosphere 52, 557–569. Dinel, H., Schnitzer, M., Mehuys, G.R., 1990. Soil lipids: origin, nature, content, decomposition, and effect on soil physical properties. In: Bollag, J.M., Stotzky, G. (Eds.), Soil Biochemistry, vol. 6. Marcel Dekker, New York, pp. 397–429. European Commission, 1991. Council Regulation (EEC) N. 2092/91 of 24 June 1991on organic production of agricultural products and indications referring thereto on agricultural products and foodstuffs. Official Journal of the European Communities 22.07.1991 (L 198). European Commission, 2000. Council Decision (EC) N. 2000/766 of 4 December 2000 concerning certain protection measures with regard to

ARTICLE IN PRESS 474

C. Mondini et al. / Soil Biology & Biochemistry 40 (2008) 462–474

transmissible spongiform encephalopathies and the feeding of animal protein. Official Journal of the European Communities 07.12.2000 (L 306/32). European Commission, 2002. Regulation (EC) N. 1774/2002 of the European Parliament and of the Council of 3 October 2002 laying down health rules concerning animal by-products not intended for human consumption. Official Journal of the European Communities 10.10.2002 (L 273). Flavel, T.C., Murphy, D.V., 2006. Carbon and nitrogen mineralization rates after application of organic amendments to soil. Journal of Environmental Quality 35, 183–193. Groffman, P.M., 1999. Carbon additions increase nitrogen availability in northern hardwood forest soils. Biology and Fertility of Soils 29, 430–433. Heal, O.W., McClean Jr., S.F., 1975. Comparative productivity in ecosystems-secondary productivity. In: Dobben, W.H., Lowe-McConnell, R.H. (Eds.), Unifying Concepts in Ecology. Dr. W. Junk B.V. Publishers, The Hague, pp. 89–108. Hita, C., Parlanti, E., Jambu, P., Joffre, J., Amble`s, A., 1996. Triglyceride degradation in soil. Organic Geochemistry 25, 19–28. Jeng, A., Haraldsen, T.K., Vagstad, N., Nlund, G., Tveitnes, S., 2004. Meat and bone meal as nitrogen fertilizer to cereals in Norway. Agricultural and Food Science 13, 268–275. Jeng, A., Haraldsen, T.K., Gronlund, A., Pedersen, P.A., 2006. Meat and bone meal as nitrogen and phosphorus fertlizer to cereals and rye grass. Nutrient Cycling in Agroecosystems 76, 183–191. Joergensen, R.G., Brookes, P.C., 1990. Ninhydrin reactive nitrogen measurements of microbial biomass in 0.5M K2SO4 soil extracts. Soil Biology & Biochemistry 22, 1023–1027. Jones, D.L., 1999. Amino acid biodegradation and its potential effects on organic nitrogen capture by plants. Soil Biology & Biochemistry 31, 613–622. Jones, D.L., Shannon, D., 1999. Mineralization of amino acids applied to soils: impact of soil sieving, storage and inorganic nitrogen additions. Soil Science Society of America Journal 63, 1199–1206. Jones, D.L., Kemmitt, S.J., Wright, D., Cuttle, S.P., Bol, R., Edwards, A.C., 2005. Rapid intrinsic rates of amino acid biodegradation in soils are unaffected by agricultural management strategy. Soil Biology & Biochemistry 37, 1267–1275. Kandeler, E., Gerber, H., 1988. Short-term assay of soil urease activity using colorimetric determination of ammonium. Biology and Fertility of Soils 6, 68–72. Khalil, M.I., Hossain, M.B., Schmidhalter, U., 2005. Carbon and nitrogen mineralization in different upland soils of the subtropics treated with organic materials. Soil Biology & Biochemistry 37, 1507–1518. Kuzyakov, Y., Friedel, J.K., Stahr, K., 2000. Review of mechanisms and quantification of priming effects. Soil Biology & Biochemistry 32, 1485–1498. Levi-Minzi, R., Riffaldi, R., Saviozzi, A., 1990. Carbon mineralization in soil amended with different organic materials. Agriculture Ecosystems & Environment 31, 325–335. Luxhøi, J., Bruun, S., Stenberg, B., Breland, T.A., Jensen, L.S., 2006. Prediction of gross and net N mineralization-immobilization-turnover from respiration. Soil Science Society of American Journal 70, 1121–1128. McCarty, G.W., Shogren, D.R., Bremner, J.M., 1992. Regulation of urease production in soil by microbial assimilation of nitrogen. Biology and Fertility of Soils 12, 261–264. Melero, S., Ruiz Porras, J.C., Herencia, J.F., Madejon, E., 2006. Chemical and biochemical properties in a silty loam soil under conventional and organic management. Soil & Tillage Research 90, 162–170. MIPAF, 1999. Approvazione del codice di buona pratica agricola. Decreto Ministeriale 19 aprile 1999. Gazzetta Ufficiale della Repubblica Italiana n. 102, S.O. n. 86, 04/05/1999. Morvan, T., Nicolardot, B., Pean, L., 2006. Biochemical composition and kinetics of C and N mineralization of animal wastes: a typological approach. Biology and Fertility of Soils 42, 513–522.

Moucawi, J., Fustec, E., Jambu, P., Jacquesy, R., 1981. Decomposition of lipids in soils: free and esterified fatty acids, alcohols and ketones. Soil Biology & Biochemistry 13, 461–468. Murphy, D.V., Recous, S., Stockdale, E.A., Fillery, I.R.P., Jensen, L.S., Hatch, D.J., Goulding, K.W.T., 2003. Gross nitrogen fluxes in soil: theory, measurement and application of 15N pool dilution techniques. Advances in Agronomy 79, 69–118. Nannipieri, P., Ceccanti, B., Grego, S., 1990. Ecological significance of the biological activity in soil. In: Bollag, J.-M., Stotzky, G. (Eds.), Soil Biochemistry, vol. 6. Dekker, New York, pp. 293–355. Nicolardot, B., Recous, S., Mary, B., 2001. Simulation of C and N mineralization during drop residues decomposition: a simple dynamic model based on the C:N ratio of the residues. Plant and Soil 228, 83–103. Nordgren, A., 1992. A method for determining microbially available N and P in an organic soil. Biology and Fertility of Soils 13, 195–199. Novelo, L.P., Martı´ nez, N.S.L., Garza, V.P., 1998. Bone meal applied to soils of the coffee plantation area in los altos de Chiapas, Mexico. Terra 16, 71–77. Pansu, M., Thuries, L., 2003. Kinetics of C and N mineralization, N immobilization and N volatilization of organic inputs in soil. Soil Biology & Biochemistry 35, 37–48. Pe´rez-Piqueres, A., Edel-Hermann, V., Alabouvette, C., Steinberg, C., 2006. Response of soil microbial communities to compost amendments. Soil Biology & Biochemistry 38, 460–470. Powlson, D.S., 1994. The soil microbial biomass: before, beyond and back. In: Ritz, K., Dighton, J., Giller, K.E. (Eds.), Beyond the Biomass. Wiley, Chichester, pp. 3–19. Rezende, L.A., Assis, L.C., Nahas, E., 2004. Carbon, nitrogen and phosphorus mineralization in two soils amended with distillery yeast. Bioresource Technology 94, 159–167. Sa´nchez-Monedero, M.A., Mondini, C., De Nobili, M., Leita, L., Roig, A., 2004. Land application of biosolids. Soil response to different stabilization degree of the treated organic matter. Waste Management 24, 325–332. Schnurer, J., Rosswall, T., 1982. Fluorescine diacetate hydrolysis as a measure of total microbial activity in soil and litter. Applied and Environmental Microbiology 43, 1256–1261. Shaw, L.J., Burns, R.G., 2006. Enzyme activity profiles and soil quality. In: Bloem, D.J., Hopkins, D.W., Benedetti, A. (Eds.), Microbiological Methods for Assessing Soil Quality. CABI, Wallingford, pp. 158–172. Sommer, S.G., Kjellerup, V., Kristjansen, O., 1992. Determination of total ammonium nitrogen in pig and cattle slurry: sample preparation and analysis. Acta Agriculturae Scandinavica: Section B, Soil and Plant Science 42, 146–151. Stevenson, F.J., 1994. Humus Chemistry: genesis, Composition, Reactions. Wiley, New York, 512pp. Stevenson, F.J., Cole, M.A., 1999. Soil organic matter quality and characterization. In: Cycles of Soil. Wiley, New York, pp. 78–106. Trinsoutrot, I., Recous, S., Mary, B., Nicolardot, B., 2000. C and N fluxes of decomposing 13C and 15N Brassica napus L.: effects of residue composition and N content. Soil Biology & Biochemistry 32, 1717–1730. Van-Camp, L., Bujarrabal, B., Gentile, A.R., Jones, R.J.A., Montanarella, L., Olazabal, C., Selvaradjou, S.K., 2004. Reports of the technical working groups established under the thematic strategy for soil protection. EUR 21319 EN/3. Office for Official Publications of the European Communities, Luxembourg. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass. Soil Biology & Biochemistry 19, 703–707. van Hees, P.A.W., Jones, D.L., Finlay, R., Godbold, D.L., Lundstro¨m, U.S., 2005. The carbon we do not see—the impact of low molecular weight compounds on carbon dynamics and respiration in forest soils: a review. Soil Biology & Biochemistry 37, 1–13.