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Library of Congress Cataloging-in-Publication

Data

Microbiological methods for assessing soil quality / edited by [aap Bloem, David W. Hopkins, and Anna Benedetti. p.cm. Includes index. ISBN 0-85199-098-3 (alk. paper) 1. Soil microbiology. 2. Soils--Quality. 3. Soils--Analysis. I. Bloem, [aap, 1958- II. Hopkins, David W., Dr. III. Benedetti, Arma, Dr. IV. Title. QR111.M392005 579'.1757--dc22 2005001632 ISBN-13: 978 O0851 99 098 9 ISBN-lO: O85199 098 3 Typeset by Columns Design Ud, Reading Printed and bound in the UK by Biddles Ud, King's Lynn

Contents

Editors Abbreviations Part 1: Approaches to Defining, Monitoring, Evaluating and Managing Soil Quality 1 Introduction Anna Benedetti and Oliver Dilly

ix xi

Defining Soil Quality 15 Richard G. Burns, Paolo Nannipieri, Anna Benedetti and David W Hopkins Monitoring and Evaluating Soil Quality Jaap Bloem, Anton J. Schouien, Seren J. Serensen, Michiel Rutgers, Adri van der Werf and Anton M. Breure Managing Soil Quality Michael Schloter, Jean Charles Munch and Fabio Tittarelli Concluding Remarks Anna Benedetti, Philip C. Brookes and James M. Lynch
23

50

63

Part 11: Selected Methods

6

71 73 73

Microbial Biomass and Numbers 6.1 Estimating Soil Microbial Biomass Andreas Flief3bach and Franco Widmer

vi

Contents

Contents

6.2 Microbial Biomass Measurements by Fumigation-Extraction Philip C. Brookes and Rainer Georg Joergensen 6.3 Substrate-induced Respiration Heinrich Hoper 6.4 Enumeration and Biovolume Determination of Microbial Cells Manfred Bolier, Jaap Bloem, Klaus Meiners and Rolf Moller 7 Soil Microbial Activity
7.1 Estimating Soil Microbial Activity

77 84 93

9.2

9.3

114 114 9.4

Oliver Dilly 7.2 Soil Respiration Mikael Pell, [ohn Stenstrom and Ulf Granhall 7.3 Soil Nitrogen Mineralization Stefano Canali and Anna Benedetti 7.4 Nitrification in Soil Annette Bollmann 7.5 Thymidine and Leucine Incorporation to Assess Bacterial Growth Rate [aap Bloem and Popko R. Bolhuis 117
9.5

127 136
142

10 Cen Oli Index

7.6 N20 Emissions and Denitrification from Soil 150 Ulrike Sehy, Michael Schloter, Hermann Bothe and Jean Charles Munch 7.7 Enzyme Activity Profiles and Soil Quality Liz J. Shaw and Richard G. Burns 8 Soil Microbial Diversity and Community Composition
8.1 Estimating Soil Microbial Diversity and Community 158

183 183

Composition Jan Dirk van Elsas and Michiel Rutgers 8.2 Soil Microbial Community Fingerprinting Based on Total Community DNA or RNA Jan Dirk van EIsas, Eva M. Top and Kornelia Smalla 8.3 Phospholipid Fatty Acid (PLFA)Analyses Ansa Palojiirvi 187

204

8.4 Substrate Utilization in Bolog'" Plates for Analysis of CLPP 212 Michiel Rutgers, Anton M. Breure and Heribert Insam 9 Plant-Microbe Interactions and Soil Quality 228

9.1 Microbial Ecology of the Rhizosphere 228 Philippe Lemanceau, Pierre Offre, Christophe Mougel, Elisa Gamalero, Yves Dessaux, Yvan Moenne-Loccoz and Graziella Berta

Contents

vii

9.2 Nodulating Symbiotic Bacteria and Soil Quality 231 Alain Hartmann, Sylvie Mazurier, Dulce N. Rodrguez-Navarro, Francisco Temprano Vera, Jean-Claude Cleyet-Marel, Yves Prin, Antoine Galiana, Manuel Fernndez-Lpez, Nicols Toro and Yvan MoenneLoccoz 9.3 Contribution of Arbuscular Mycorrhiza to Soil Quality and 248 Terrestrial Ecotoxicology Silvio Gianinazzi, Emmanuelle Plumey-Jacquot, Vivienne GianinazziPearson and Corinne Leyval 9.4 Concepts and Methods to Assess the Phytosanitary Quality of Soils Claude Alabouvette, [os Raaijmakers, Wietse de Boer, Rgina Notz, Geneuioe Dfago, Christian Steinberg and Philippe Lemanceau 9.5 Free-living Plant-beneficial Microorganisms and Soil Quality Yvan Moenne-Loccoz, Sheridan L. Woo, Yaacov Okon, Ren Bally, Matteo Lorito, Philippe Lemanceau and Anton Hartmann 10 Census of Microbiological Methods for Soil Quality Oliver Dilly Index
257

270

296

301

6.2Microbial Biomass Measurements by Fumigation-Extraction

PHILlP C. BROOKES 1 ANO RAINER GEORG JOERGENSEN2

1 Soil Science Oepartment, IACR-Rothamsted, Harpenden AL52JQ, UK; 20epartment of Soil Biology and Plant Nutrition, University of Kassel, NordbahnhofstraBe, 0-37213 Wi~enhausen,Germany

Introduction
Thesoil microbial biomass responds much more quickly than most other soil fractionsto changing environmental conditions, such as changes in substrate inputs (e.g.Powlson et al., 1987)or increases in heavy metal content (Brookes and McGrath, 1984). This, and much other similar, research supports the originalidea of Powlson and Jenkinson (1976)that biomass is a much more sensitive indicator of changing soil conditions than, for example, the total soilorganic matter content. Thus, the biomass can serve as an 'early warning' of such changes, long before they are detectable in other ways. Linked parameters (e.g. biomass-specific respiration or biomass as a percentageof soil organic C) are also useful as they have their own intrinsic 'internal controls' (see Barajas et al., 1999 for a discussion of this). This may permit interpretation of measurements in the natural environment, where, unlike in controlled experirnents, there may not be suitable noncontaminated soil (for example) to provide good 'control' or 'background' measurements. Here we provide experimental details of two measurements of biomass which have proved useful in environmental studies, particularly at low levels (i.e. around European Union limits) of pollution by heavy metals, namelysoil microbial biomass C and biomass ninhydrin N.

Principie of the Method

Followingchloroform fumigation of soil, there is an increase in the amount of various components coming from the cells of soil rnicroorganisms which are lysed by the fumigant and made partially extractable (Jenkinson and Powlson,1976b).Organic C (Vance et al., 1987),total N and NH4-N (Brookes et al., 1985),and ninhydrin-reactive N (Amato and Ladd, 1988; Joergensen and Brookes, 1990) can be measured in the same 0.5 M ~S04 extract. Further information on furnigation--extraction and other rnicrobiological methods is given by Alef and Nannipieri (1995).
77

78

P Brookes and R. G. Joergensen

Biomass Measurements b

Materials and Apparatus

A room or incubator adjustable to 25C An implosion-protected desiccator A vacuum line (water pump or electric pump) A horizontal or overhead shaker A deep-freezer at -15C Folded filter papers (e.g. Whatman 42 or Schleicher & Schue1l5951/2) Glass conical flasks (250 ml)

Additional met

Liebig conc 250 ml roui Burette

Additional chef

Chemicals and Reagents

66.7mMp Concentra1 Concentra1 40.0mMir 25 mM 1.11

Ethanol-free chloroform (CHCl3) Soda lime 0.5 M Potassium sulphate (~S04) (87.1 gil)

All chemicals is used througl Digestion

n

H3P04 (vi

Procedure
Fumigation--extraction

Titration se distlled v 1000 ml w

A moist soil sample of 50 g is divided into two subsamples of 25 g. The nonfumigated control samples are placed in 250 ml conical flasks and then immediately extracted with 100 ml 0.5 M ~S04 (ratio extractant:soil is 4:1) for 30 min by oscillating shaking at 200 rpm (or 45 min overhead shaking at 40 rpm) and then filtered through a folded filter paper. For the fumigated treatment, 50 ml glass vials containing the moist soils are placed in a desiccator containing wet tissue paper and a vial of soda lime. A beaker containing 25 ml ethanol-free CHCl3 and a few boiling chips is added and the desiccator evacuated until the CHCl3 has boiled vigorously for 2 mino The desiccator is then incubated in the dark at 25C for 24 h. After fumigation, CHCl3 is removed by repeated (sixfold) evacuation and the soils are transferred to 250 ml bottles for extraction with 0.5 M ~S04' All treatments are replicated three times. All ~SO 4 extracts are stored at -15C prior to analysis.

Procedure

To 8 ml soil e) ~Crp7and 1 gently refluxe water, added measured by using 25mM indicator.
Calculation 01
CALCULATIOf\ DIGESTION

C (..g/ml)=
Biomass e estimated by dichromate oxidation PrincipIe of the method

In the presence of strong acid and dichromate, organic matter is oxidized and Cr(+VI) reduced to Cr(+III). The amount of dichromate left is backtitrated with iron 11 ammonium sulphate (Kalembasa and Jenkinson, 1973) and the amount of carbon oxidized is calculated.

where: S = co sumption of sumption of normality of 1 tion (ml): VS Cr( +VI) to Cr C (..gl g soil)

en

Biomass Measurements

by Fumigation-Extraction

79

Additianal materials and apparatus Liebig condenser 250 ml round-bottom Burette

flask

Additianal chemicals and reagents 66.7 mM potassium chromate (KzCr207) (19.6125 gil) Concentrated phosphoric acid (H3P04) Concentrated sulphuric acid (H2S04) 40.0 mM iron II ammonium sulphate [(NH4)2[Fe(S04)2] x 6H20] 25 mM 1.10-phenanthroline-ferrous sulphate complex solution water

All chemicals are analytical reagent grade and distilled or de-ionized is used throughout.

Digestion mixture: two parts conc. H2S04 are mixed with one part conc.
H3P04 (v Iv).

Titration solution: iron II ammonium sulphate (15.69 gil) is dissolved in distilled water, acidified with 20 ml conc. H2S04 and made up to 1000 ml with distilled water.

Pracedure To 8 ml soil extract in a 250 ml round-bottom flask, 2 ml of 66.7 mM (0.4 N) KzCr207 and 15 ml of the H2S04/H3P04 mixture are added. The mixture is gently refluxed for 30 min, allowed to cool and diluted with 20-25 rnl water, added through the condenser as a rinse. The excess dichromate is measured by back-titration with 40.0 mM iron II ammonium sulphate, using 25 mM 1.10-phenanthroline-iron II sulphate complex solution as an indicator. is o d Calculatian ot results
CALCULATION DIGESTION OF EXTRACTABLE ORGANIC C FOLLOWING DICHROMATE

C (Ilg/rnl)

= [(HB - S)

I CB] x N x [VD/VS] x E x 1000

where: S = consumption of titration solution by the sample (ml): HB = consumption of titration solution by the hot (refluxed) blank (ml): CB = consumption of titration solution by the cold (unrefluxed) blank (ml): N = normality of the KzCr207 solution; VD = added volume of the KzCr207 solution (ml): VS = added volume of the sample (ml): and E = 3, conversion of Cr(+VI) to Cr(+III), assuming that, on average, all organic C is as [C(O)]. C (Ilg/gsoil)
= C (Ilg/ml)

x (VK + SW)/DW

80

P. Brookes and R.G. Joergensen

Biomass Measurement~

where: VK = volume of ~S04 extractant (ml): SW (ml): and DW = dry weight of sample (g).
CALCULATION OF BIOMASS C

volume of soil water

where: S = e dilution of with the acid volurne of soi

Biomass C (Bc) = Ec!kEC where: Ec = (organic C extracted from fumigated soils) - (organic C extracted from non-fumigated soils) and kEC = 0.38 (Vance et al., 1987).

CALCULATIOr

Biornass e (B,

Biomass e by UV-persulphate PrincipIe ot the methad

oxidation

where: Ec = extracted fro Joergensen, I'

In the presence of potassium persulphate (~S208)' extractable soil organic carbon is oxidized by ultraviolet (UV) light to CO2, which is measured using infrared (IR) or photo-spectrametric detection.
Additianal materials and apparatus

Biomass e by oven ,

Automatic carbon analyser with IR-detection (e.g. Dohrman DC 80) or continuous-flow systems with colourimetric detection (Skalar, Perstorp).
Additianal chemicals and reagents

~S208 Concentrated H3P04 Sodium hexametaphosphate [(Na(P04)6)n] Oxidation reagent: 20 g ~S208 are dissolved in 900 ml distilled water, acidified to pH 2 with conc. H3P04 and made up to 1000 ml Acidification buffer: 50 g sodium hexametaphosphate are dissolved in 900 ml distilled water, acidified to pH 2 with conc. H3P04 and made up to 1000 ml

Extractable se platinurn cat autornatic aru oven systerns extracts conta autornated U diluted with . hexarnetapho: and biornass ( oxidation rnet

Determination

of nin

PrincipIe of th.

Procedure

For the automated UV-persulphate oxidation method, 5 ml ~S04 soil extract are mixed with 5 ml acidification buffer. Any precipita te of CaS04 in the soil extracts is dissolved by this pracedure. The ~S208 is automatically fed into the UV oxidation chamber, where the oxidation to CO2 is activated by UV light. The resulting CO2 is measured by IR absorption.
Calculatian ot results
CALCULATION OF EXTRACTABLE = ORGANIC C

Ninhydrin fo nitragen and graups, such The presence quantitative e and Ladd (19 from the micn 2 M KCl, is clr content.

Additianal app

C (Ilg!g soil)

[(S x DS) - (B x DB)] x (VK + SW)!DW

Boiling W Photo-spe

ensen water

Biomass Measurements

by Fumigation-Extraction

81

where: S = C in sample extract (llg/mI); B = C in blank extract (llg/mI); OS = dilution of sample with the acidification buffer; OB = dilution of blank with the acidification buffer; VK = volume of KzS04 extractant (ml): SW = volume of soil water (ml): and OW = dry weight of sample (g).
CALCULATION OF BIOMASS C =

nic

Biomass C (Bc)

Ec/kEC

where: Ec = (organic C extracted from fumigated soils) - (organic C extracted from non-fumigated soils) and kEC = 0.45 (Wu et al., 1990; J oergensen, 1996a).

ganic sured

Biomass

by oven oxidation

r con-

Extractable soil organic C is oxidized to CO2 at 850C in the presence of a platinum catalyser. The CO2 is measured by infrared absorption using an automatic analyser (Shimadzu 5050, Dimatoc 100, Analytic [ena), The new oyen systems use small sample volumes - so they are able to measure C in extracts containing large amounts of salts. The procedure is similar to the automated UV-persulphate oxidation method, except that the samples are diluted with water and acidified using a few drops of HCl instead of the hexametaphosphate acidification buffer. The calculations of extractable C and biomass C are identical to those used in the automated UV-persulphate oxidation method.

ater, ed in deup

Determination 01 ninhydrin-reactive PrincipIe of the method

nitrogen

4 soil 04 in ically vated

Ninhydrin forms a purple complex with molecules containing o-amno nitrogen and with ammonium and other compounds with free u-amino groups, such as amino acids, peptides and proteins (Moore and Stein, 1948). The presence of reduced ninhydrin (hydrindantin) is essential to obtain quantitative colour development with ammonium. According to Amato and Ladd (1988), the amount of ninhydrin-reactive compounds, released from the microbial biomass during the CHC13 fumigation and extraction by 2 M KCl, is closely correlated with the initial soil microbial biomass carbon content.
Additional apparatus

Boiling water bath Photo-spectrophotometer

82

P Brookes and R.G. Joergensen

Biomass Measuremen

Additianal chemicals and solutions

CALCULATIC

Ninhydrin Hydrindantin Dimethyl sulphoxide (DMSO) Lithium acetate dihydrate Acetic acid (96%) Citric acid Sodium hydroxide (NaOH) Ethanol (95%) L-Leucine Ammonium sulphate NH4)2S04) Lithium acetate buffer: lithium acetate (408 g) is dissolved in water (400 ml), adjusted to pH 5.2 with acetic acid and finally made up to 1 1 with water Ninhydrin reagent: ninhydrin (2 g) and hydrindantin (0.3 g) are dissolved in dimethyl sulphoxide (75 ml), 25 ml of 4 M lithium acetate buffer at pH 5.2 are then added (Moore, 1968) Citric acid buffer: citric acid (42 g) and NaOH (16 g) are dissolved in water (900 ml), adjusted to pH 5 with 10 M NaOH if required, then finally made up to 11 with water

Bnin= (Nnin non-fumiga

CALCULATIC

Biomass C = Biomass e = The convers the fumigati

C and Bnin i

Discussion

Procedure

The procedure is described according to Joergensen and Brookes (1990) for measuring biomass C and microbial ninhydrin-reactive N in ~S04 soil extracts. A 10 mM L-leucine (1.312 gil) and a 10 mM ammonium-N [(NH4)2S04 0.661 gil] solution are prepared separately in 0.5 M ~S04 and diluted within the range 0-1000 ..MN. The standard solutions, ~S04 soil extracts or blank (0.6 ml) and the citric acid buffer (1.4 ml) are added to 20 ml test tubes. The ninhydrin reagent (1 ml) is then added slowly, mixed thoroughly and closed with loose aluminium lids. The test tubes are then heated for 25 rnin in a vigorously boiling water bath. Any precipita te formed during the addition of the reagents then dissolves. After heating, an ethanol:water mixture (4 ml 1:1) is added, the solutions are thoroughly mixed again and the absorbance read at 570 nm (1 cm path length).
Calculatian ot results
CALCULATION OF EXTRACTED = NINHYDRIN-REACTIVE N (NNIN)

Biomass rru They have t as being ra] that these rr soil ecosyst over bioma: This makes Secondly, in the biomass With bioma: hydrin N it error in its ci ever, so bioi described a quently cau white preC! However, tl safely ignon

Acknowledgem
IACR recen Sciences Re!

Nnin(..g/gsoil)

(S - B)/L x N x (VK + SW)/DW

where: S = absorbance of the sample; B = absorbance of the blank; L = millimolar absorbance coefficient of leucine; N = 14 (atomic weight of nitrogen); VK = volume of ~S04 extractant (ml): SW = volume of soil water (ml): and DW = dry weight of the sample (g).

Bomass Measurements

by Fumgation-Extraction

83
N

CALCULATION

OF MICROBIAL

NINHYDRIN-REACTIVE

Bnin= (Nninextracted from the fumigated soil) - (Nnin extracted from the non-fumigated soil)
CALCULATION OF MICROBIAL BIOMASS CARBON

Biomass C = Bnin x 22 (soils pH-H20 > 5.0) Biomass C = Bnin x 35 (soils pH-H20 ~ 5.0) The conversion factors were obtained by correlating the microbial biomass C and Bninin the same extracts of 110 arable, grassland and forest soils by the fumigation-extraction method (Joergensen, 1996b).

Discussion
Biomass measurements are certainly useful in studies of soil protection. They have the advantage that they are relatively cheap and simple, as well as being rapid. There is now a considerable amount of literature to show that these measurements are useful in determining effects of stresses on the soil ecosystem. Biomass ninhydrin measurements have two advantages over biomass C. First, a reflux digestion is not required for ninhydrin N. This makes it very suitable for situations with minimallaboratory facilities. Secondly, in both biomass C and N measurements the fraction coming from the biomass is determined following subtraction of an appropriate 'control'. With biomass C this value is often half of the total, while with biomass ninhydrin N it is commonly about lOor less. This causes considerably less error in its determination. Both parameters are very closely correlated, however, so biomass C may be readily estimated from biomass ninhydrin N, as described above. One feature of the fumigation-extraction method frequently caused concern. Upon thawing of frozen ~S04 soil extracts, a white precipita te of CaS04 occurs in near-neutral or alkaline soils. However, this causes no analytical problems in either method and may be safely ignored.

Acknowledgements
IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.