LibroMicrobiological Methods for Assessing Soil Qualit

7/30/2019 LibroMicrobiological Methods for Assessing Soil Qualit

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CAB Intemational2006. All rights reserved. No part of this publication may bereproduced in any form or by any means, electronically, mechanically, byphotocopying, recording or otherwise, without the prior permission of thecopyright owners.A catalogue record for this book is available from the British Library,London, UK.

Library of Congress Cataloging-in-Publication DataMicrobiological methods for assessing soil quality / edited by [aap Bloem, DavidW. 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.M392005579'.1757--dc22 2005001632

ISBN-13: 978 O0851 99 098 9ISBN-lO: O85199 098 3Typeset by Columns Design Ud, ReadingPrinted and bound in the UK by Biddles Ud, King's Lynn


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Contents

Editors ix

Abbreviations xiPart1 : Approaches toDefining, Monitoring, Evaluating andManaging Soil Quality

1

1 IntroductionAnna Benedetti and O live r D illy

3

2 Defining Soil Quality 15Richard G. Burns, Pao lo Nannipieri, Anna Bened etti and David W Hopkins

3 Monitoring and Evaluating Soil Quality 23Ja ap Bloem , Anton J. Schouien, Seren J. Serensen, M ichiel Rut ge rs,Adri van der W erf and Anton M. Breur e

4 Managing Soil Quality 50Micha el Schloter, Je an C ha rles Mun ch and Fabio T itt arell i

5 Concluding Remarks 63Anna Ben edetti , Philip C. Brookes and James M. Lyn ch

Part11 : Selected Methods 716 Microbial Biomass and Numbers

6.1 Estimating Soil Microbial BiomassAndreas Flief3bach a nd Franco W idmer

7373

v


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vi Contents Contents

6.2 Microbial Biomass Measurements by Fumigation-Extraction 77Philip C. B ro oke s a nd R ain er G eo rg Joergensen

9 .2

6.3 Substrate-induced RespirationHein ric h H o pe r

6.4 Enumeration and Biovolume Determination ofMicrobial CellsManfr ed Bo lie r, Jaap B loem , K laus M einers and R olf M oller

84

93 9 .3

7 Soil Microbial Activity7.1 Estimating Soil Microbial Activity

O liv er D il ly7.2 Soil Respiration

Mikael Pel l, [o hn S tenstrom and Ulf G ra nh all7.3 Soil Nitrogen Mineralization

S tefa no C an ali a nd A nn a B en ed etti7.4 Nitrification in Soil

Ann ette B ollm a nn7.5 Thymidine and Leucine Incorporation to Assess 142

Bacterial Growth Rate[aap B lo em and P opko R. Bolhuis

7.6 N20 Emissions and Denitrification from Soil 150U lr ik e S eh y, M ic ha el Sc hlo te r, H e rmann Bothe and Je an C ha rles M unch7.7 Enzyme Activity Profiles and Soil Quality 158

Liz J . S ha w a nd R ich ard G. Burns

114114 9 .4

1179. 5127

136 10 CenO li

Index

8 Soil Microbial Diversity and Community Composition 1838.1 Estimating Soil Microbial Diversity and Community 183

CompositionJan D irk van E lsa s an d M ichiel R utg ers

8.2 Soil Microbial Community Fingerprinting Based on Total 187Community DNA orRNAJan D irk van EIs as, E va M. Top a nd K orn elia Smalla

8.3 Phospholipid Fatty Acid (PLFA)Analyses 204Ansa Pa lo jii rvi

8.4 Substrate Utilization in Bolog'" Plates for Analysis of CLPP 212M ic hiel R utge rs , Anton M. Br eu re and He rib er t Insam

9 Plant-Microbe Interactions and Soil Quality 2289.1 Microbial Ecologyof the Rhizosphere 228

Phi lipp e L em a nc ea u, P ierre O ffre, Chri stophe Mou gel, E lisa Gam al ero,Yves De ssa ux , Y va n Moenne-Loccoz an d G raziella Ber ta


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Contents v i i

9.2 Nodulating Symbiotic Bacteria and Soil Quality 231A lain H artm ann, Sylvie M azurier, D ulce N. Rodrguez-Navarro,F ra nc is co T emp ra no V er a, J ea n-Cl au de Cleyet-Marel, Yves P rin, A ntoineG a lia na , Ma nu el F er n nd ez-Lpez, N icols Toro and Yvan M oenne-Loccoz

9.3 Contribution of Arbuscular Mycorrhiza to Soil Quality and 248Terrestrial EcotoxicologyS ilv io G ia nin az zi, Emman ue lle P lum ey -J ac qu ot, V ivi enne Gianina z zi -Pea rs on a nd C or in ne Leyval

9.4 Concepts and Methods to Assess the Phytosanitary Quality 257of SoilsClaude Alabouvette, [o s R aa ijm ak er s, W iets e d e B oe r, R g in a N ot z,Gene uioe Dfa go , C hr is ti an S te in be rg a nd P hil ip pe L ema nc ea u

9.5 Free-living Plant-beneficial Microorganisms and Soil Quality 270Y va n Mo en ne -L oc co z, S he ri da n L. Woo, Ya acov O kon, R en B ally,Ma tt eo L or ito, Phi lippe L em anceau and A nton H artm ann

10 Census of Microbiological Methods for Soil QualityOliver D ill y

296

Index 301


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6 .2M i c ro b i a l B i o m as s M eas u rem en ts byFumiga t i on -Ex t r ac t i onPHILlPC . BROOKES 1 ANO RAINER GEORG JOERGENSEN2

1So il S c ien c e O ep ar tm en t, IACR-Ro thams ted, HarpendenAL52JQ , UK ; 20epar tm en t o f So i l B io lo g y and P lan t Nu t r i t io n,Un ive rs i t y o f Kasse l , No rdbahnho f s t r aBe , 0-37213Wi~enhausen,Germany

I n t r o d u c t i o nThesoilmicrobial biomass responds much more quickly than most other soilfractionsto changing environmental conditions, such as changes in substrateinputs(e.g.Powlson et al., 1987)or increases in heavy metal content (Brookesand McGrath, 1984). This, and much other similar, research supports theoriginalidea of Powlson and Jenkinson (1976)that biomass is a much moresensitiveindicator of changing soil conditions than, for example, the totalsoilorganicmatter content. Thus, the biomass can serve as an 'early warn-ing'ofsuch 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). Thismay permit interpretation of measurements in the natural environment,where, unlike in controlled experirnents, there may not be suitable non-contaminated soil (for example) to provide good 'control' or 'background'measurements.Herewe provide experimental details of two measurements of biomass

which have proved useful in environmental studies, particularly at lowlevels (i.e. around European Union limits) of pollution by heavy metals,namelysoil microbial biomass C and biomass ninhydrin N.

P r i n c i p i e o f the MethodFollowingchloroform fumigation of soil, there is an increase in the amountofvarious components coming from the cells of soil rnicroorganisms whichare lysed by the fumigant and made partially extractable (Jenkinson andPowlson,1976b).Organic C (Vanceet al., 1987),total N and NH4-N (Brookeset al., 1985),and ninhydrin-reactive N (Amato and Ladd, 1988;Joergensenand Brookes, 1990) can be measured in the same 0.5M ~S04 extract.Further information on furnigation--extraction and other rnicrobiologicalmethods is given byAlef and Nannipieri (1995).

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78 P Brookes and R. G. Joergensen

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)

Chemicals and Reagents Ethanol-free chloroform (CHCl3)Soda lime0.5MPotassium sulphate (~S04) (87.1 gil)

ProcedureFumigation--extraction

A moist soil sample of 50 g is divided into two subsamples of 25 g. The non-fumigated control samples are placed in 250 ml conical flasks and thenimmediately 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 at40 rpm) and then filtered through a folded filter paper. For the fumigatedtreatment, 50 ml glass vials containing the moist soils are placed in a desicca-tor containing wet tissue paper and a vial of soda lime. A beaker containing25ml ethanol-free CHCl3 and a few boiling chips is added and the desiccatorevacuated until the CHCl3 has boiled vigorously for 2 mino The desiccator isthen incubated in the dark at 25C for 24 h. After fumigation, CHCl3 isremoved by repeated (sixfold) evacuation and the soils are transferred to250 ml bottles for extraction with 0.5 M ~S04' All treatments are replicatedthree times. All ~SO 4extracts are stored at -15C prior to analysis.

Biomass e estimated by dichromate oxi dat ionPrincipIe of the methodIn the presence of strong acid and dichromate, organic matter is oxidizedand Cr(+VI) reduced to Cr(+III). The amount of dichromate left is back-titrated with iron 11 ammonium sulphate (Kalembasa and Jenkinson, 1973)and the amount of carbon oxidized is calculated.

Biomass Measurements b

Additional met Liebig conc 250ml roui Burette

Additional chef 66.7mMp Concentra1 Concentra1 40.0mMir 25mM 1.1All chemicals is used througl Digestion nH3P04 (vi Titration s edistlled v1000ml w

ProcedureTo 8 ml soil e)~Crp7and 1gently refluxewater, addedmeasured byusing 25mMindicator.Calculation 01CALCULATIOf\DIGESTIONC (..g/ml)= [where: S =cosumption ofsumption ofnormality of 1tion (ml): VSCr(+VI) to CrC ( ..g l g soil)


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Biomass Measurements by Fumigation-Extraction 79

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

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 solutionAll chemicals are analytical reagent grade and distilled or de-ionized wateris used throughout. D ig es tio n m ix tu re : two parts conc. H2S04 are mixed with one part conc.H3P04 (vIv). T i tra ti on so lu t ion : iron II ammonium sulphate (15.69 gil) is dissolved in

distilled water, acidified with 20 ml conc. H2S04 and made up to1000 ml with distilled water.

PracedureTo 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 isgently refluxed for 30min, allowed to cool and diluted with 20-25 rnlwater, added through the condenser as a rinse. The excess dichromate ismeasured by back-titration with 40.0 mM iron II ammonium sulphate,using 25 mM 1.10-phenanthroline-iron II sulphate complex solution as anindicator.

Calculatian ot resultsCALCULATION OF EXTRACTABLE ORGANIC C FOLLOWING DICHROMATEDIGESTIONC (Ilg/rnl) =[(HB - S) I CB] x N x [VD/VS] x E x 1000where: S = consumption of titration solution by the sample (ml): HB = con-sumption of titration solution by the hot (refluxed) blank (ml): CB =con-sumption of titration solution by the cold (unrefluxed) blank (ml): N =normality of the KzCr207 solution; VD =added volume of the KzCr207 solu-tion (ml): VS = added volume of the sample (ml): and E = 3, conversion ofCr(+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


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80 P. Brookes and R.G. Joergensen

where: VK = volume of ~S04 extractant (ml): SW = volume of soil water(ml): and DW =dry weight of sample (g).CALCULATION OF BIOMASS CBiomass C (Bc) =Ec!kECwhere: Ec = (organic C extracted from fumigated soils) - (organic Cextracted from non-fumigated soils) and kEC =0.38 (Vance et al., 1987).

Biomass e by UV-persulphate oxidat ionPrincipIe ot the methadIn the presence of potassium persulphate (~S208)' extractable soil organiccarbon is oxidized by ultraviolet (UV) light to CO2, which is measuredusing infrared (IR) or photo-spectrametric detection.Additianal materials and apparatusAutomatic carbon analyser with IR-detection (e.g. Dohrman DC 80) or con-tinuous-flow systems with colourimetric detection (Skalar, Perstorp).Additianal chemicals and reagents ~S208Concentrated H3P04Sodium 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 mlAcidification buffer: 50 g sodium hexametaphosphate are dissolved in

900 ml distilled water, acidified to pH 2 with conc. H3P04 and made upto 1000 ml

ProcedureFor the automated UV-persulphate oxidation method, 5 ml ~S04 soilextract are mixed with 5 ml acidification buffer. Any precipita te of CaS04 inthe soil extracts is dissolved by this pracedure. The ~S208 is automaticallyfed into the UV oxidation chamber, where the oxidation to CO2 is activatedby UV light. The resulting CO2 is measured by IR absorption.Calculatian ot resultsCALCULATION OF EXTRACTABLE ORGANIC CC (Ilg!g soil) =[(S x DS) - (B x DB)] x (VK +SW)!DW

Biomass Measurement

where: S = e= dilution owith the acivolurne ofsoCALCULATIOBiornasse ( Bwhere: E c =extracted frJoergensen, I

Biomass e by ovenExtractable s eplatinurn catautornatic ar uoven systernextracts con taautornateddiluted withhexarnetapho:and biornassoxidation rne

Determinat ion of n inPrincipIe of thNinhydrin fonitragen andgraups, such The presencequantitative eand Ladd (19from the micn2 M KCl, is clrcontent.

Additianal app Boiling W Photo-spe


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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 blankwith the acidification buffer; VK = volume of KzS04extractant (ml): SW =volume of soil water (ml): and OW = dry weight of sample (g).CALCULATION OF BIOMASS CBiomassC (Bc) =Ec/k ECwhere: Ec = (organic C extracted from fumigated soils) - (organic Cextracted from non-fumigated soils) and kEC = 0.45 (Wu et al., 1990;Joergensen, 1996a).

Biomass e by oven oxidat ionExtractable soil organic C is oxidized to CO2 at 850C in the presence of aplatinum catalyser. The CO2 is measured by infrared absorption using anautomatic analyser (Shimadzu 5050, Dimatoc 100,Analytic [ena), The newoyen systems use small sample volumes - so they are able to measure C inextracts containing large amounts of salts. The procedure is similar to theautomated UV-persulphate oxidation method, except that the samples arediluted with water and acidified using a few drops of HCl instead of thehexametaphosphate acidification buffer. The calculations of extractable Cand biomass C are identical to those used in the automated UV-persulphateoxidation method.

Determinat ion 01 ninhydr in-reactive nit rogenPrincipIe of the methodNinhydrin forms a purple complex with molecules containing o-amnonitrogen and with ammonium and other compounds with free u-aminogroups, such as amino acids, peptides and proteins (Moore and Stein, 1948).The presence of reduced ninhydrin (hydrindantin) is essential to obtainquantitative colour development with ammonium. According to Amatoand Ladd (1988), the amount of ninhydrin-reactive compounds, releasedfrom the microbial biomass during the CHC13fumigation and extraction by2M KCl, is closely correlated with the initial soil microbial biomass carboncontent.

Additional apparatus Boiling water bath Photo-spectrophotometer


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82 P Brookes and R.G. Joergensen

Additianal chemicals and solutions 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 buf fer : lithium acetate (408 g) is dissolved in water

(400 ml), adjusted to pH 5.2 with acetic acid and finally made up to 1 1with water N inh ydrin reagen t: ninhydrin (2 g) and hydrindantin (0.3 g) are dis-solved in dimethyl sulphoxide (75 ml), 25 ml of 4 M lithium acetatebuffer at pH 5.2 are then added (Moore, 1968) C itric acid buffer : citric acid (42 g) and NaOH (16 g) are dissolved inwater (900 ml), adjusted to pH 5 with 10 M NaOH if required, then

finally made up to 11 with water

ProcedureThe procedure is described according to Joergensen and Brookes (1990) formeasuring biomass C and microbial ninhydrin-reactive N in ~S04 soilextracts. 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 anddiluted within the range 0-1000 ..MN. The standard solutions, ~S04 soilextracts or blank (0.6 ml) and the citric acid buffer (1.4 ml) are added to20 ml test tubes. The ninhydrin reagent (1 ml) is then added slowly, mixedthoroughly and closed with loose aluminium lids. The test tubes are thenheated for 25 rnin in a vigorously boiling water bath. Any precipita teformed during the addition of the reagents then dissolves. After heating, anethanol:water mixture (4 ml 1:1) is added, the solutions are thoroughlymixed again and the absorbance read at 570 nm (1 cm path length).

Calculatian ot resultsCALCULATION OF EXTRACTED NINHYDRIN-REACTIVE N (NNIN)Nnin(..g/gsoil) =(S - B)/L x N x (VK+SW)/DWwhere: S = absorbance of the sample; B = absorbance of the blank; L = mil-limolar absorbance coefficient of leucine; N = 14 (atomic weight of nitro-gen); VK =volume of ~S04 extractant (ml): SW =volume of soil water(ml): and DW = dry weight of the sample (g).

Biomass Measuremen

CALCULATICBnin=(Nninon-fumigaCALCULATICBiomass CBiomass eThe conversC and Bninithe fumigati

DiscussionBiomass rruThey have tas being ra]that these rrsoil ecosystover bioma:This makesSecondly, inthe biomassWith bioma:hydrin N iterror in its ciever, so bio idescribed aquently cauwhite preC!However, tlsafely ig non

AcknowledgemIACR recenSciences Re!


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Bomass Measurements by Fumgation-Extraction 83

CALCULATION OF MICROBIAL NINHYDRIN-REACTIVE NBnin=(Nninextracted from the fumigated soil) - (Nninextracted from thenon-fumigated soil)CALCULATION OF MICROBIAL BIOMASS CARBONBiomassC=Bninx 22 (soils pH-H20 > 5.0)BiomassC = Bninx 35(soils pH-H20 ~ 5.0)The conversion factors were obtained by correlating the microbial biomassCand Bninin the same extracts of 110arable, grassland and forest soils bythe fumigation-extraction method (Joergensen, 1996b).

D i s cu s s i o nBiomass measurements are certainly useful in studies of soil protection.They have the advantage that they are relatively cheap and simple, as wellas being rapid. There is now a considerable amount of literature to showthat these measurements are useful in determining effects of stresses on thesoil ecosystem. Biomass ninhydrin measurements have two advantagesover biomass C. First, a reflux digestion is not required for ninhydrin N.Thismakes it very suitable for situations with minimallaboratory facilities.Secondly,in both biomass C and N measurements the fraction coming fromthe biomass is determined following subtraction of an appropriate 'control'.Withbiomass C this value is often half of the total, while with biomass nin-hydrin N it is commonly about lOor less. This causes considerably lesserror in its determination. Both parameters are very closely correlated, how-ever, so biomass C may be readily estimated from biomass ninhydrin N, asdescribed above. One feature of the fumigation-extraction method fre-quently caused concern. Upon thawing of frozen ~S04 soil extracts, awhite precipita te of CaS04 occurs in near-neutral or alkaline soils.However, this causes no analytical problems in either method and may besafely ignored.

A cknow l e dgemen t sIACRreceives grant-aided support from the Biotechnology and BiologicalSciencesResearch Council of the United Kingdom.

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LibroMicrobiological Methods for Assessing Soil Qualit