Enzymatic Activities and Soil Carbon Sequestration Strategies

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The expression "enzymatic activities" is usually preferred to "enzymes," the reason being that applied methods do not isolate and measure the enzyme itself in soils, but quantify (mainly by absorbance or fluorescence) the rate of transformation of an enzyme substrate added to soil. Already in the 1940s a paper was published reporting the effect of copper nitrogen complex on soil potential nitrification, as assessed by this substrate-induced approach (Lees 1946), and it was followed by a series of pioneering works on the location and activities of enzymes in soils

Table 7.1 Number of published literature on enzymes and PLFAs in soil as related to compost and tillage treatments

Search word

<2008

2008

2009

2010

2011

Total

Soil* AND enzym*

10,388

1,188

1,290

1,337

135

14,338

Soil* AND enzym* AND compost

220

48

55

56

3

382

Soil* AND enzym* AND tillage

124

16

27

33

3

203

Soil* AND PLFA*

502

113

95

109

18

819

Soil* AND PLFA* AND compost

19

4

1

5

0

29

Soil* AND PLFA* AND tillage

20

4

3

6

3

36

The enquiry was carried out on Scopus database in February 2011 among titles, abstracts and keywords

The enquiry was carried out on Scopus database in February 2011 among titles, abstracts and keywords

(McLaren 1954; McLaren et al. 1957). From then on, the assessment of enzymatic activities (or more generally biological activities, as in the case of nitrification, where a series of enzymatic activities is involved) has become widely popular. A search on the Scopus scientific database using "soil*" and "enzym*" as search words among titles, abstracts and keywords gives a total 14,332 published papers, with more than a thousand papers per year (Table 7.1). If the search is restricted adding either "compost" or "tillage," the number of published papers is, respectively, 382 and 203, with a slight increase in the last years.

Numerous reviews about enzymatic activities have been published in the last years (e.g., Dick 1992; Tabatabai et al. 2002; Nannipieri et al. 2003; Caldwell 2005), as well as a number of books (Burns 1978; Dick and Burns 2002). Thus, the aim of this chapter is not to provide a further review on the topic but to analyse the evidence available in literature on the effects of organic fertilization and tillage on soil enzymatic activities and to assess whether some general conclusions can be drawn from this critical bibliographic investigation.

Soil enzymatic activities are related to the majority of ecological processes in soils such as soil organic matter decomposition, cycling of nutrients, and detoxification of undesired compounds, such as pesticides and other organic contaminants. Enzymes play a main role in relation to presence and activities of soil microorganisms, since their catalytic activity toward transformation of organic substrates allows liberation of the necessary energy for their activities (Kiss et al. 1978) and promotes soil fertility by releasing nutrients for plants growth. Soil enzyme activities have been suggested as suitable indicators of soil quality for a number of reasons: (1) they are an index of soil microbial activity and, thus, they are strictly related to nutrient cycles and transformations; (2) they may rapidly respond to changes in soil caused by both natural and anthropogenic factors; (3) they are easy to measure (Calderon et al. 2000; Drijber et al. 2000; Nannipieri et al. 2002). The information given by a single enzymatic activity is of course important but limited. This is why most works usually consider a range of enzymatic activities, eventually condensing all information in numerical indices obtained by different approaches. In a recent review paper, 13 indexes based on soil enzymatic activities were discussed (Bastida et al. 2008).

Here we present the outcomes of an extended bibliographic review carried out in order to assess, identify, and interpret possible trends in the response of the main experimental factors studied in MESCOSAGR project: organic fertilization (Table 7.2) and reduced or no tillage (Table 7.3). Among the papers cited in Table 7.1, only the ones which allowed extrapolation of quantitative data were selected.

Fourteen studies dealing with the effects of organic fertilizers on soil enzymatic activities have been reviewed. Twelve enzymatic activities have been considered, namely arylsulphatase, p-glucosidase, phosphatase, fluorescein diacetate hydrolysis activity (FDA), urease, dehydrogenase, invertase, phenoloxidase, catalase, protease, nitrate reductase and amylase. For each study the type and amount of organic fertilizer applied is reported, together with information (when available) on soil texture and taxonomy. Soil textures ranged from sandy to clay, whereas 11 different soil types were considered (Table 7.2).

Arylsulphatase activity is usually assessed by adding a substrate such as p-nitrophenylsulphate in soil, and quantifying the amount of p-nitrophenol produced in time. Up to our knowledge, it is the only enzyme of the S cycle whose activity is assessed in soil. However, it is considered quite representative of the mineralization of organic S in soils, since sulfate esters represent a large fraction (25-93%) of the soil total S (Elsgaard et al. 2002). Six papers were considered about the effects of compost amendment on arylsulphatase (Table 7.2). Five papers indicated an increase in arylsulphatase activity as a result of organic additions, whereas only one (Abdelbasset et al. 2011) indicated no relevant effects after application of up to 80 ton ha-1 of municipal solid waste (MSW) compost to a clayey-loamy soil cropped with Triticum durum (although the use of sewage sludge had instead a positive effect). In two of the works, arylsulphate activity was more than doubled as a result of application of 2-4 ton ha-1 of MSW compost (Albiach et al. 2000) or of composted red clover corresponding to 416 kg of total N ha-1 (Elfstrand et al. 2007a). Two other reports dealt with a maize field amended for several years with MSW compost and sewage sludge (Puglisi et al. 2006) and with the application of compost rates up to 45 ton ha-1 in a greenhouse and in an open field under Mediterranean conditions (Iovieno et al. 2009). Finally, Darby et al. (2006) assessed the effects of compost from dairy manure solids (56 ton ha-1) on sweet corn plots in Oregon.

p-Glucosidase is one of the enzymatic activities involved in C cycling in soils. It is usually assessed using p-nitrophenyl-b-D-galactoside as a substrate, and it thus gives an indication of the activity of enzymes involved in cellulose degradation, specifically in the hydrolysis of p-1,4 bonds in p-glucopiranosides. Eight papers were considered here (Table 7.2). Five of them dealt with MSW compost, one with municipal food waste (MFW) compost, one with compost from manure mixed with leguminous residues, and another one with an unspecified compost. Concentrations considered ranged from 5 to 80 ton ha-1. According to five studies, p-glucosidase activity was increased after amendment with compost from MSW (Garcia-Gil et al. 2000; Crecchio et al. 2004; Hojati and Nourbakhsh 2009; Abdelbasset et al. 2011), MFW (Iovieno et al. 2009) and manure mixed with leguminous residues (Laudicina

Table 7.2 Analysis of literature info on the effects of organic fertilization on soil enzymatic activities

Enzyme activity

Organic fertilizer applied

Effect

Soil texture

Soil taxonomy

S cycle

Arylsulphatase

MSW compost

** Albiach et al.

Sandy-silty loam

Xerorthent

(24 ton ha"1 year"1)

(2000)

Composted dairy manure

* Darby et al.

Silty loam

Chealis

(56 ton ha"1 year"1)

(2006)

MSW compost

* Puglisi et al.

Sandy loam

Alluvial

(25 ton ha"1 year"1)

(2006)

hydromorphic

MFW compost (45 ton ha"

_1 * Iovieno et al.

-

Calcaric Cambisol

in 3 rates)

(2009)

Composted red clover

** Elfstrand et al.

Silty clay loam

-

(15 ton ha"1)

(2007a)

MSW compost

- Abdelbasset et al.

Clay loam

Mollisol

(80 ton ha"1)

(2011)

C cycle

b-Glucosidase

MSW compost

* Garcia-Gil et al.

Sandy

Typic Haploxeralf

(80 ton ha"1)

(2000)

MSW compost

* Crecchio et al.

Clay

Typic

(24 ton ha"1 year"1)

(2004)

Chromoxerert

MSW compost

+ Puglisi et al.

Sandy loam

Alluvial

(25 ton ha"1 year"1)

(2006)

hydromorphic

Compost

+ Nayak et al.

Sandy clay loam

Aeric Endoaquept

(5 ton ha"1 year"1)

(2007)

MSW compost

* Hojati and

Silty clay loam

Typic Haplargid

(100 ton ha"1)

Nourbakhsh

(2009)

MFW compost (45 ton ha

1 - Iovieno et al.

-

Calcaric Cambisol

in 3 rates)

(2009)

Compost from manure

* Laudicina et al.

-

Hortic Cambisol

(30 ton ha"1 year"1)

(2010)

MSW compost

* Abdelbasset et al.

Clay loam

Mollisol

(80 ton ha"1)

(2011)

Invertase

MSW compost

* Puglisi et al.

Sandy loam

Alluvial

(25 ton ha"1 year"1)

(2006)

hydromorphic

Compost

+ Nayak et al.

Sandy clay loam

Aeric Endoaquept

(5 ton ha"1 year"1)

(2007)

Phenoloxidase

MSW compost

* Puglisi et al.

Sandy loam

Alluvial

(25 ton ha"1 year"1)

(2006)

hydromorphic

Amylase

MSW compost

* Pramanik et al.

Clay

Aqualfs

(15 ton ha"1)

(2010)

P cycle

Phosphatase

MSW compost

** Albiach et al.

Sandy-silty loam

Xerorthent

(24 ton ha"1 year"1)

(2000)

MSW compost

+ Garcia-Gil et al.

Sandy

Typic Haploxeralf

(80 ton ha"1)

(2000)

MSW compost

* Crecchio et al.

Clay

Typic

(24 ton ha"1 year"1)

(2004)

Chromoxerert

MFW compost

** Lee et al.

-

-

(27 ton ha"1)

(2004)

MSW compost

** Puglisi et al.

Sandy loam

Alluvial

(25 ton ha"1 year"1)

(2006)

hydromorphic

Composted red clover

** Elfstrand et al.

Silty clay loam

(15 ton ha"1)

(2007a)

Table 7.2 (continued)

Enzyme activity

Organic fertilizer applied

Effect

Soil texture

Soil taxonomy

MFW compost (45 ton ha-1

- Iovieno et al.

-

Calcaric Cambisol

in 3 rates)

(2009)

Compost

* Laudicina et al.

-

Hortic Cambisol

(30 ton ha-1 year-1)

(2010)

MSW compost

* Pramanik et al.

Clay

Aqualfs

(15 ton ha-1)

(2010)

MSW compost

* Abdelbasset et al.

Clay loam

Mollisol

(80 ton ha-1)

(2011)

N cycle

Urease

MSW compost

* Albiach et al.

Sandy-silty loam

Xerorthent

(24 ton ha-1 year-1)

(2000)

MSW compost

- Garcia-Gil et al.

Sandy

Typic Haploxeralf

(80 ton ha-1)

(2000)

MSW compost

* Crecchio et al.

Clay

Typic

(24 ton ha-1 year-1)

(2004)

Chromoxerert

MSW compost (6 ton ha-1)

* Bhattacharyya

-

Typic fluvaquent

et al. (2005)

MSW compost

- Puglisi et al.

Sandy loam

Alluvial

(25 ton ha-1 year-1)

(2006)

hydromorphic

Compost

* Nayak et al.

Sandy clay loam

Aeric Endoaquept

(5 ton ha-1 year-1)

(2007)

Compost

** Laudicina et al.

-

Hortic Cambisol

(30 ton ha-1 year-1)

(2010)

MSW compost

* Pramanik et al.

Clay

Aqualfs

(15 ton ha-1)

(2010)

MSW compost

* Abdelbasset et al.

Clay loamy

Mollisol

(80 ton ha-1)

(2011)

Protease

MSW compost

- Garcia-Gil et al.

Sandy

Typic Haploxeralf

(80 ton ha-1)

(2000)

MSW compost

- Crecchio et al.

Clay

Typic

(24 ton ha-1 year-1)

(2004)

Chromoxerert

Composted red clover

** Elfstrand et al.

Silty-clay-loam

-

(15 ton ha-1)

(2007a)

MSW compost

* Pramanik et al.

Clay

Aqualfs

(15 ton ha-1)

(2010)

Nitrate

MSW compost

* Crecchio et al.

Clay

Typic

reductase

(24 ton ha-1 year-1)

(2004)

Chromoxerert

Total microbial activity

FDA

Composted dairy manure

* Darby et al.

Silt loam

Chealis

(56 ton ha-1 year-1)

(2006)

Compost

- Nayak et al.

Sandy clay loam

Aeric Endoaquept

(5 ton ha-1 year-1)

(2007)

MFW compost (45 ton ha-1

* Iovieno et al.

-

Calcaric Cambisol

in 3 rates)

(2009)

Dehydrogenase

MSW compost

* Albiach et al.

Sandy-silty loam

Xerorthent

(24 ton ha-1 year-1)

(2000)

MSW compost

** Garcia-Gil et al.

Sandy

Typic Haploxeralf

(80 ton ha-1)

(2000)

MSW compost

* Crecchio et al.

Clay

Typic

(24 ton ha-1 year-1)

(2004)

Chromoxerert

MFW compost

** Lee et al.

-

-

(27 ton ha-1)

(2004)

Table 7.2 (continued)

Enzyme

Organic fertilizer applied

Effect

Soil texture

Soil taxonomy

activity

MSW compost

* Puglisi et al.

Sandy loam

Alluvial

(25 ton ha-1 year-1)

(2006)

hydromorphic

Compost

* Nayak et al.

Sandy clay loam

Aeric Endoaquept

(5 ton ha-1 year-1)

(2007)

Compost

* Laudicina et al.

-

Hortic Cambisol

(30 ton ha-1 year-1)

(2010)

MSW compost

* Abdelbasset et al.

Clay loamy

Mollisol

(80 ton ha-1)

(2011)

Catalase

MSW compost

* Garcia-Gil et al.

Sandy

Typic Haploxeralf

(80 ton ha-1)

(2000)

MSW compost

* Abdelbasset et al.

Clay loamy

Mollisol

(80 ton ha-1)

(2011)

For each enzymatic activity the referred biogeochemical cycle is reported. (*, + Increase or decrease of less than 100%; ++ increase or decrease of more than 100%; - no effect)

For each enzymatic activity the referred biogeochemical cycle is reported. (*, + Increase or decrease of less than 100%; ++ increase or decrease of more than 100%; - no effect)

et al. 2010). Differently from what reported above for arylsulphatase, no differences were found in p-glucosidase activity according to Iovieno et al. (2009). Conversely, Puglisi et al. (2006) and Nayak et al. (2007) even found a slight but significant decrease, possibly due to presence of toxic trace elements in MSW compost and to the low amount applied (5 ton ha-1), respectively.

Another important enzymatic activity involved in soil C cycling is invertase. As for urease, for which urea is the substrate, invertase is the only other hydrolase assessed using its natural substrate, namely sucrose (Speir et al. 2002). Only two papers dealing with effects of compost on invertase activity were found in literature (Table 7.2). Puglisi et al. (2006) found that in a sandy loam soil amended with 25 ton ha-1 of MSW compost, invertase was significantly enhanced, while according to Nayak et al. (2007) invertase activity was instead reduced in a soil of similar texture (sandy clay loam). In the latter, however, only 5 ton ha-1 of compost were tested, and the presence of clays might as well played a role in adsorbing the enzyme and thus reducing its activity (Gianfreda et al. 1991).

Another C cycling enzymatic activity considered here was phenoloxidase, involved in organic matter degradation. Only one study (Puglisi et al. 2006) was found that showed a significant increase in phenoloxidase activity after compost amendment. It was also found that another C cycling enzyme (amylase, responsible for starch degradation) was increased by compost addition at 15 ton ha-1 (Pramanik et al. 2010).

Phosphatases are key enzymes controlling phosphorus turnover and availability for plants. Assayed after addition of the synthetic substrate p-nitrophenylphosphate to soil samples, phosphatases control the transformation of organic P to inorganic P through dephosphorylation processes. These enzymes can be assessed under either alkaline or acidic conditions. Alkaline phosphatases are mostly of microbial origin, while acid phosphatases are more of plant or fungal origin, though this is not a strict difference and conditions may differ from soil to soil. Ten scientific papers were considered here (Table 7.2): seven reports analysed acid phosphatase, two of them discussed alkaline phosphatase (Albiach et al. 2000; Abdelbasset et al. 2011), while only one (Lee et al. 2004) evaluated both forms. Most papers (eight) analysed the effects of MSW compost (rates ranging from 15 to 80 ton ha"1), one paper described the effect of composted red clover (Elfstrand et al. 2007a) and another one reported effects of an unspecified compost (Laudicina et al. 2010). In eight out of ten papers, phosphatase activities were significantly induced by compost addition. A significant reduction was instead found by Garcia-Gil et al. (2000) for a low organic matter sandy soil amended with an MSW compost, though contaminated with significant levels of Zn (1325 mg kg"1), Cu (548 mg kg"1), Ni (81 mg kg"1) and Pb (681 mg kg"1). The authors attributed this phosphatase inhibition to trace elements level and to the large content of soluble P in the amended soil, in agreement with evidence showing an inhibitory effect of inorganic P on phosphatases (Spiers and McGill 1979).

Urease activity is at the basis of nitrogen turnover and soil fertility. Being assessed through determination of the ammonium liberated after soil addition with urea, this enzyme plays a central role in organic nitrogen mineralization, as it is the gateway for nitrification. Urease is sensitive to a number of environmental parameters such as oxygen and trace elements, and it is well correlated with soil quality (Badiane et al. 2001; Coppolecchia et al. 2010). Nine studies were considered here to assess general trends on the effects of organic amendments on urease activity (Table 7.2). Most of these studies have been already cited for the enzymatic activities discussed above. Seven out of nine studies used MSW compost as fertilizer, two of them used an unspecified compost. Amendment rates ranged from 5 to 80 ton ha"1, and, as for other enzymes, a wide range of soil textural types were considered. In most cases, urease activity was significantly increased by compost, and in one case (Laudicina et al. 2010) more than doubled. In one case (Garcia-Gil et al. 2000; Puglisi et al. 2006), no significant effect was found, while in another work (Garcia-Gil et al. 2000) a significant inhibition of urease from MSW compost was even found. These effects did not seem to be related to compost rates, while Nayak et al. (2007) showed significant increase with a rate of only 5 ton ha"1. The inhibition of urease activity can be due to the presence of trace elements as for other enzymes, or, as suggested by Garcia-Gil et al. (2000), to the large content of NH4+ (a urease inhibitor) produced by the activity of proteases present in the MSW compost-amended soil.

Proteases belong to a large family of soil enzymes, and, depending on the substrate used for determination, different protease activities can be assayed. We considered four papers about the effects of compost on soil protease activities (Table 7.2). The used substrates were Na-benzoyl-argininamide (Garcia-Gil et al. 2000; Crecchio et al. 2004), Na-caseinate (Pramanik et al. 2010), and caseine (Elfstrand et al. 2007a). It was found that protease activity was induced by 15 ton ha"1 of composted red clover (Elfstrand et al. 2007a) and 15 ton ha"1 of MSW compost (Pramanik et al. 2010), while according to Crecchio et al. (2004) and Garcia-Gil et al. (2000), the rates of 80 and 24 ton ha"1 of MSW compost did not cause any significant change in protease activities, respectively.

The assessment of nitrate reductase activity in soil can give an important indication of denitrification. Also this activity is enhanced by compost amendment, thus confirming that organic matter addition stimulates microorganism involved in very different cycles (Crecchio et al. 2004).

Fluorescein diacetate (FDA) is a substrate that can be hydrolysed by a variety of nonspecific enzymes and the assessment of FDA hydrolysis activity is thus used as an indicator of total microbial activity in soil (Perucci 1992). Three papers on the effects of composts on FDA in soil were considered here (Table 7.2). It was found that 56 ton ha-1 of composted dairy manure (Darby et al. 2006) and 45 ton ha-1 of MFW compost (Iovieno et al. 2009) significantly increased soil FDA, while a lower dose of 5 ton ha-1 of compost induced no significant changes (Nayak et al. 2007).

Dehydrogenases are ubiquitous in all intact, viable microbial cells: the estimation of dehydrogenase activity as determined by the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride (INT) to iodo-nitrophenyl formazan (INTF) is thus widely used as an estimation of soil total microbial activity (Lagomarsino et al. 2009a). In accordance with the results reported above for the other enzymatic activity used to estimate total microbial activity such as FDA, it was found also through the analysis of papers dealing with the dehydrogenase that compost has a general effect of increase of soil microbial activity. Eight papers were considered (Table 7.2), and all of them showed a significant increase (in some cases more than a doubling) of dehydrogenase as the result of the application of doses of compost ranging from 5 to 80 ton ha-1, on different textures and soil types.

Catalase is the third enzymatic indicator of total microbial activity considered here, and it consists of an oxido-reductase associated with aerobic microbial activity (Rodriguez-Kabana and Truelove 1982). Two papers dealing with the effects of compost on soil catalase were found (Garcia-Gil et al. 2000; Abdelbasset et al. 2011), and in line with FDA and dehydrogenase results, they confirmed the stimulation of microbial activity by compost.

Similarly to the effects of organic amendments, also the effects of tillage on soil enzymatic activities were searched in the scientific literature (Table 7.3). Most of the enzymatic activities discussed above in detail have been also considered for tillage effects. Eleven papers in total have been reviewed, covering different soil textural types. Though both no tillage and reduced tillage were included in the bibliographic query, the vast majority of studies considered solely no tillage. Only one (Ramos et al. 2011) assessed the effect of reduced tillage by chisel ploughing on arylsulphatase, p-glucosidase, phosphatase and dehydrogenase activities.

The effect of no tillage in inducing enzymatic activities was even greater and clearer than that observed for amendment of organic materials. Among ten enzymatic activities, nine were generally increased by no tillage: arylsulphatase (5/7), p-glucosidase (6/8), phenoloxidase (1/1), catalase (1/1), phosphatase (8/8), urease (3/4), invertase (1/1), dehydrogenase (6/8) and protease (2/2). In all other cases, no significant difference was reported. In the case of FDA, only one study was found (Nsabimana et al. 2004) that showed no significant differences between control and no till plots for a clay Rhodic Ferrisol.

Table 7.3 Analysis of literature info on the effects of tillage on soil enzymatic activities

Enzyme activity

Tillage

Effect

Soil texture

Soil taxonomy

S cycle

Arylsulphatase

No tillage

* Nsabimana et al.

Clay

Rhodic Ferisol

(2004)

No tillage

- Mijangos et al.

Clay loam

-

(2006)

No tillage (direct

* Melero et al.

Clay

Chromic

drilling)

(2008)

Haploxeret

No tillage

* Lagomarsino et al.

Silt-clay

Calcaric Gleyic

(2009b)

Cambisol

No tillage

* Mikanova et al.

Clay loam

Orthic Luvisol

(2009)

No tillage

* Zhang et al. (2010)

-

-

Reduced tillage (chisel

- Ramos et al. (2011)

Clay loam

Hypercalcic

ploughing)

Calcisol

C cycle

b-Glucosidase

No tillage

* Mijangos et al.

Clay loam

-

(2006)

No tillage (direct

** Melero et al.

Clay

Chromic

drilling)

(2008)

Haploxeret

No tillage

- Mina et al. (2008)

Sandy clay

-

loam

No tillage

** Lagomarsino

Silt-clay

Calcaric Gleyic

et al. (2009b)

Cambisol

No tillage (mouldboard

* Lopez-Garrido

-

Eutric Leptosol

ploughing)

et al. (2010)

No tillage

** Ulrich et al.

Sandy

Stagnic Luvisol

(2010)

loam

No tillage

- Zhang et al. (2010)

-

-

Reduced tillage (chisel

* Ramos etal. (2011)

Clay loam

Hypercalcic

ploughing)

Calcisol

Phenoloxidase

No tillage (mouldboard

* Lopez-Garrido

-

Eutric Leptosol

ploughing)

et al. (2010)

Invertase

No tillage

* Mikanova et al.

Clay loam

Orthic Luvisol

(2009)

P cycle

Phosphatase

No tillage

* Nsabimana et al.

Clay

Rhodic Ferisol

(2004)

No tillage

* Mijangos et al.

Clay loam

-

(2006)

No tillage

* Roldan et al.

Clay

Vertisol

(2007)

No tillage (direct

* Melero et al.

Clay

Chromic

drilling)

(2008)

Haploxeret

No tillage

* Mina et al. (2008)

Sandy clay

-

loam

No tillage

** Lagomarsino

Silt-clay

Calcaric Gleyic

et al. (2009b)

Cambisol

Table 7.3 (continued)

Enzyme

Tillage

Effect

Soil texture

Soil taxonomy

activity

No tillage

** Zhang et al.

-

-

(2010)

Reduced tillage (chisel

* Ramos etal. (2011)

Clay loam

Hypercalcic

ploughing)

Calcisol

N cycle

Urease

No tillage

- Mina et al. (2008)

Sandy clay

-

loam

No tillage

* Mikanova et al.

Clay loam

Orthic Luvisol

(2009)

No tillage

* Zhang et al. (2010)

-

-

No tillage

* Qin et al. (2010)

Silt loam

Haplic Cambisol

Protease

No tillage

* Mina et al. (2008)

Sandy clay

-

loam

No tillage

** Zhang et al.

-

-

(2010)

Total microbial activity

FDA

No tillage

- Nsabimana et al.

Clay

Rhodic Ferisol

(2004)

Dehydrogenase

No tillage

- Nsabimana et al.

Clay

Rhodic Ferisol

(2004)

No tillage

* Mijangos et al.

Clay loam

-

(2006)

No tillage

* Roldan et al.

Clay

Vertisol

(2007)

No tillage (direct

** Melero et al.

Clay

Chromic

drilling)

(2008)

Haploxeret

No tillage

* Mina et al. (2008)

Sandy clay

-

loam

No tillage

- Mikanova et al.

Sandy clay

-

(2009)

loam

No tillage (mouldboard

* Lopez-Garrido

-

Eutric Leptosol

ploughing)

et al. (2010)

Reduced tillage (chisel

* Ramos etal. (2011)

Clay loam

Hypercalcic

ploughing)

Calcisol

Catalase

No tillage

** Ulrich et al.

Sandy

Stagnic Luvisol

(2010)

loam

For each enzymatic activity the referred biogeochemical cycle is reported. (*, + Increase or decrease of less than 100%; ++ increase or decrease of more than 100%; - no effect)

For each enzymatic activity the referred biogeochemical cycle is reported. (*, + Increase or decrease of less than 100%; ++ increase or decrease of more than 100%; - no effect)

7.3 PLFAs and Soil Carbon Sequestration Strategies

The determination of soil phospholipid fatty acids (PLFAs) is a powerful and still widely used method to assess the structure of viable microbial communities in soils. Phospholipids are at the basis of life itself, since they represent the structural skeleton of most living cells. The double layer of phospholipids found in most cells (exception represented by Archaea, where tetraether lipids substitute phospholipids) allows the separation of living cells from the surrounding environment, and regulates, together with proteins, sterols and glycolipids, the exchanges between cells and their outside. Phospholipids are made up by a hydrophilic head constituted by a negatively charged phosphate group, usually substituted with a choline, and a hydrophobic tail, usually constituted by two fatty acids. These fatty acids belong to different classes, the most common ones being saturated, monounsaturated, polyunsaturated, branched, and cyclopropanic (Fig. 7.1). As explained below, fatty acids composition of cell membranes differs from species to species (and thus can also be used for community structure assessments), and, within a single species, it is sensitive to a number of environmental parameters (e.g., temperature, nutrients, pollutants).

Bligh and Dyer (1959) published the first method for the identification and estimation of individual phospholipids in biological samples. The rationale of the method is based on isolation of the phospholipidic fraction, and removal from

Fig. 7.1 Schematic representation of a microbial cell, its double layer phospholipidic membrane and examples of the most important classes of phospholipids fatty acids

phospholipids of single fatty acids by an alkaline hydrolysis. Then, a method was devised for the conversion of fatty acids in methyl esters and their easy determination by silica chromatographic analyses (Luddy et al. 1960). Marr and Ingraham (1962) assessed the composition of individual PLFAs in Escherichia coli cells grown at 10, 15, 20, 25, 30, 35, 40 and 43°C. They found that the increase of temperature resulted in an increase in hexadecanoic acid (16:0) and a decrease in unsaturated acids such as hexadecenoate (16:1). Furthermore, they found that the composition in fatty acids reflects the composition of the growth medium, but does not correlate with the culture growth stage. This was a milestone paper since it casted the basis of PLFAs use as ecological biomarkers of environmental conditions. Moreover, derived concepts such as the ratio between specific saturated and unsaturated fatty acids as index of specific conditions are still fundamental in the environmental extension of PLFA studies. PLFAs have been also used for the ecological assessment of microbial communities in marine and estuarine sediments (White et al. 1979), and, later, for the more complex soil microbial communities (Tunlid et al. 1985; Nichols et al. 1986; Vestal and White 1989).

Despite the introduction in later years of more advanced methods (especially those based on nucleotides), PLFAs analysis remains a widely used and useful method for the analyses of soil microbial communities. An enquire in SCOPUS using "soil*" and "PLFA*" as a search key among titles, keywords and abstracts gives a total of 819 papers between 1987 and today, with the number of papers per year almost constant in the last years (Table 7.1). Similarly to the case of enzymatic activities, Table 7.1 shows how tillage and organic fertilization represent only a part (around 10%) of the effects studied in the scientific literature.

A number of reasons make PLFAs still a very useful and informative method. First, fatty acids are easily degraded after cells death and, thus, their analyses give a snapshot on the total viable microbial communities. This is a full advantage, since in DNA-based analyses it is often difficult if not impossible to distinguish between living and dead cells. The second feature is related to the fact that specific PLFAs analysis (Table 7.4) can provide reliable information about microbial groups such as Gram-positive and Gram-negative bacteria, actinomycetes, fungi, protozoa, arbuscural mycorrhiza and total fungi (Zelles 1999; Bougnom et al. 2010).

Table 7.4 PLFA biomarkers and their use for the analysis of specific groups and conditions

Bacterial group/environmental

Biomarker PLFAs

condition

Gram-positive bacteria

Sum of i14:0, i15:0, a15:0, i16:0, i17:0 and a17:0

Gram-negative bacteria

Sum of cy17:0, cy19:0, 18:1rn9c, 16:1rn9c, 18:1rn9c, 15:1rn4c

and 18:107c

Arbuscural mycorrhiza

16:105c

Protozoa

Sum of 20:2m6,9,c, 20:3m6,9,12c and 20:4m6,9,12,15c

Actinomycetes

10Me16:0 and 10Me18:0

Fungal biomass

18:2rn6c and 18:1rn9

Total microbial biomass

16:0

Stress conditions

cy19:0/18:1rn7c ratio

Furthermore, some ratios between specific PLFAs have been proposed as indicators of stress conditions, and a numerical index of soil alteration based on PLFA has also been proposed (Puglisi et al. 2005).

A bibliographic search on PLFA values, as the one described above for enzymatic activities, is reported for the effects of organic amendments to soil (Table 7.5) and for those of soil tillage practices (Table 7.6). As it is indicated in each retrieved paper, aggregated PLFA data have been reported in order to refer to specific groups (i.e., actinobacteria, Gram-positive, Gram-negative, fungi and arbuscural mycorrhiza).

Five papers dealt with the effects of organic fertilizers on PLFAs (Table 7.5). The organic fertilizer additions were: composted red clover at 15 ton ha-1 (Elfstrand et al. 2007a), farmyard manure at 4 ton of carbon ha-1 (Elfstrand et al. 2007b), wood ash compost mixed with soil at 33% v:v ratio (Bougnom et al. 2010), compost at 10 ton ha-1 (Treonis et al. 2010), and composted manure at 373 kg of nitrogen ha-1 (Kong et al. 2011). Actinobacteria was the only microbial group that was not affected by organic fertilization, whereas significant changes in the community structure were found for all other groups. Specifically, fungi were always increased (five out of five studies), arbuscural mycorrhiza were increased in four studies and unaffected in one report, while total microorganisms (as estimated by the sum of all PLFAs) were significantly increased in four out of four studies.

Contrasting results about Gram-positive and Gram-negative bacteria were shown in these works. No significant differences for both groups were found in the study of Elfstrand et al. (2007b), while both groups were increased after the organic fertilization conducted by Treonis et al. (2010) and Bougnom et al. (2010). Finally, Kong et al. (2011) reported a decrease in both bacterial groups after addition of composted manure.

Five papers dealing with the effects of soil tillage on PLFAs have also been reviewed (Table 7.6). All these papers compared no tillage versus conventional tillage. No significant reductions on specific microbial groups were experimentally found, although results indicated an increasing trend in some cases. These findings were common for each microbial group, thus suggesting that effects of no tillage on microbial communities structure was quite site-specific, and most probably affected by general variables such as climate, soil type and cultivation. An exception was represented by Gram-positive bacteria, whose numbers were never affected by soil tillage treatments.

7.4 The MESCOSAGR Case Study

The MESCOSAGR project aimed at assessing the sustainability of methods for soil carbon sequestration (see Foreword) and their effects on soil physical, chemical, biological parameters as well as crop productivity. Two innovative methods have been compared with traditional and reduced tillage methods (see Chap. 1). The innovative methods were: the soil amendment with mature humified compost to promote a hydrophobic protection against microbial degradation of the more easily

Table 7.5 Analysis of literature info on the effects of organic fertilizers on soil PLFAs

Microbial group

Organic fertilizer applied

Effect

Soil texture

Soil taxonomy

Total microorganisms

Composted red clover

* Elfstrand et al.

Silty clay loam

-

(15 ton ha"1)

(2007a)

Farmyard manure (4 ton

* Elfstrand et al.

Clay loam

Eutric Cambisol

Cha"1)

(2007b)

Wood ash compost

** Bougnom

Clay

-

(33%)

et al. (2010)

Compost (10 ton ha"1)

* Treonis et al.

Loamy sand

-

(2010)

Actinobacteria

Composted manure

- Kong et al.

Silt loam

Typic Xerorthent

(373 kg N ha"1)

(2011)

Compost (10 ton ha"1)

- Treonis et al.

Loamy sand

-

(2010)

Gram-positive

Farmyard manure (4 ton

- Elfstrand et al.

Clay loam

Eutric Cambisol

Cha"1)

(2007b)

Compost (10 ton ha"1)

* Treonis et al.

Loamy sand

-

(2010)

Wood ash compost

** Bougnom

Clay

-

(33%)

et al. (2010)

Composted manure

+ Kong et al.

Silt loam

Typic Xerorthent

(373 kg N ha"1)

(2011)

Gram-negative

Farmyard manure (4 ton

- Elfstrand et al.

Clay loam

Eutric Cambisol

Cha"1)

(2007b)

Compost (10 ton ha"1)

* Treonis et al.

Loamy sand

-

(2010)

Wood ash compost

** Bougnom

Clay

-

(33%)

et al. (2010)

Composted manure

+ Kong et al.

Silt loam

Typic Xerorthent

(373 kg N ha"1)

(2011)

Fungi

Composted manure

* Kong et al.

Silt loam

Typic Xerorthent

(373 kg N ha"1)

(2011)

Composted red clover

* Elfstrand et al.

Silty clay loam

-

(15 ton ha"1)

(2007a)

Farmyard manure (4 ton

* Elfstrand et al.

Clay loam

Eutric Cambisol

Cha"1)

(2007b)

Wood ash compost

** Bougnom

Clay

-

(33%)

et al. (2010)

Compost (10 ton ha"1)

** Treonis et al.

Loamy sand

-

(2010)

Arbuscural mycorrhiza

Composted red clover

- Elfstrand et al.

Silty clay loam

-

(15 ton ha"1)

(2007a)

Farmyard manure (4 ton

* Elfstrand et al.

Clay loam

Eutric Cambisol

Cha"1)

(2007b)

Wood ash compost

** Bougnom

Clay

-

(33%)

et al. (2010)

Compost (10 ton ha"1)

* Treonis et al.

Loamy sand

-

(2010)

+ Increase or decrease of less than 100%; ++ increase or decrease of more than 100%; - no effect

Table 7.6 Analysis of literature info on the effects of tillage on soil PLFAs

Microbial group

Tillage

Reduced/no tillage effects

Soil texture

Soil taxonomy

Total microorganisms

No tillage No tillage

* Helgason et al.

(2010b)

Loamy

Typic Fragiudults

No tillage

- Treonis et al. (2010)

Loamy sand

-

Actinobacterial

No tillage

- Treonis et al. (2010)

Loamy sand

-

Gram-positive

No tillage No tillage

- Muruganandam et al.

- Helgason et al. (2010a)

Sandy clay loam

Typic Kanhapludult

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