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Soil Biology & Biochemistry 41 (2009) 1301–1310

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Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lb io

Effect of biochar amendment on soil carbon balance and soil microbial activity

S. Steinbeiss a,*, G. Gleixner a, M. Antonietti b

a Max Planck Institute for Biogeochemistry, Hans-Knoell-Str. 10, 07745 Jena, Germanyb Max Planck Institute of Colloids and Interfaces, Am Muehlenberg 1, 14476 Potsdam-Golm, Germany

a r t i c l e i n f o

Article history:Received 28 July 2008Received in revised form16 March 2009Accepted 21 March 2009Available online 17 April 2009

Keywords:Biochar13C labelingPLFAResidence timesGreenhouse experiment

* Corresponding author at: Institute of GroundwatMunich, Ingolstaedter Landstr. 1, 85764 Neuherberg, G2916; fax: þ49 (0) 89 3187 3361.

E-mail address: sibylle.steinbeiss@helmholtz-mue

0038-0717/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.soilbio.2009.03.016

a b s t r a c t

We investigated the behavior of biochars in arable and forest soil in a greenhouse experiment in orderto prove that these amendments can increase carbon storage in soils. Two qualities of biochar wereproduced by hydrothermal pyrolysis from 13C labeled glucose (0% N) and yeast (5% N), respectively. Wequantified respiratory losses of soil and biochar carbon and calculated mean residence times of thebiochars using the isotopic label. Extraction of phospholipid fatty acids from soil at the beginning andafter 4 months of incubation was used to quantify changes in microbial biomass and to identifymicrobial groups utilizing the biochars. Mean residence times varied between 4 and 29 years,depending on soil type and quality of biochar. Yeast-derived biochar promoted fungi in the soil, whileglucose-derived biochar was utilized by Gram-negative bacteria. Our results suggest that residencetimes of biochar in soils can be manipulated with the aim to ‘‘design’’ the best possible biochar fora given soil type.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

There is a large imbalance between carbon release to theatmosphere and carbon uptake by other compartments that leadsto a continued increase in atmospheric CO2 equivalent to a rate of4.1�109 tons of carbon per year (IPCC, 2007). Thus, it should be ofutmost importance to develop new methods to retain carbon ina stable form that can be stored outside of the atmosphere forlonger time periods. In this context, biochars have attracted a lotof research within the last years basically with focus on theapplication of biochars to soils, where they not only contribute tocarbon storage but at the same time act as fertilizers (Glaser et al.,2001; Marris, 2006). Although a positive effect of biocharamendments on crop yields was already known to ancientcultures (Glaser, 2007), to date little is known about the effects ofbiochar addition on soil microorganisms and consequently on thesoil carbon balance.

There is a huge variability in physical biochar structuresdepending on the parent material and the conditions present attheir formation, which leads to quite different turnover times insoils (Czimczik and Masiello, 2007). Large charcoal particles origi-nated from forest wildfires have been shown to remain in soils for

er Ecology, Helmholtz Centreermany. Tel.: þ49 (0) 89 3187

nchen.de (S. Steinbeiss).

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thousands of years (Pessenda et al., 2001; Gouveia et al., 2002;Gavin et al., 2003), however, smaller particles as derived fromgrassland burning can hardly be detected in steppe ecosystems(Forbes et al., 2006). The physical and chemical structure, e.g.surface area and condensation grade, and the particle size ofsynthetic biochars can be modified in technical processes (Titiriciet al., 2007a,b) opening the question about the stability of syntheticbiochars in soils.

There have been developed numerous chemical and technicalmethods to produce charcoals from a variety of biomass materials(Antal and Gronli, 2003; Marris, 2006; Titirici et al., 2007a). Eachproduction method needs a certain energy supply to activate thereactions and results in completely different biochar structures.However, hydrothermal carbonization looks especially promisingenergy- and process-wise. Once activated in a continuous process,20–30% of the energy bound to the original biomass are liberatedin the process, while keeping practically all carbon bound to thefinal structure (Titirici et al., 2007b). No extensive biomassmaterial preparation or costly product isolation procedures arerequired. Also soft, wet and low grade biomass can be carbonized,making industrial biowaste, sludges or green household waste aptto carbonization. A crude estimate of such directly accessible andmostly already collected biowaste sums up to about 25�106 tonsper year in Germany, or to 10�109 tons per year worldwide.Thus, we deal with a potential measure to cure at least significantparts of the CO2 problem, appropriate biological stability in soilsand an added biological benefit provided. The optimal biochar

S. Steinbeiss et al. / Soil Biology & Biochemistry 41 (2009) 1301–13101302

combining fertilizer and carbon storage function in soils wouldactivate the microbial community leading to nutrient release andfertilization and would add to the decadal soil carbon pool. Thestructural and chemical properties of biochars that are drivingtheir decomposition or stabilization in soils still have to beidentified.

In our current study, we added two types of hydrothermallysynthesized biochar, a highly condensed, nitrogen free biocharexpected to be stable in soil, and a nitrogen containing biocharwith low condensation grade expected to be easily degradable,to different soils with the aim to answer the followingquestions:

1) How stable are biochars produced by this method in differentsoils?

2) How do inherited soil microorganisms react on the addition ofsuch biochars?

3) Is the stability of these biochars tuneable by varying thecondensation grade and chemical composition of the biochar?

2. Materials and methods

2.1. Soil sampling and characterization

Soils used for the greenhouse experiment were sampled at thecontinued arable plot of the Jena Experiment (Roscher et al., 2004)and at the old growth forest field site of the Hainich National Park(Knohl et al., 2003), respectively. The soil of the Jena Experimentwas classified as Eutric Fluvisol (FAO, 1998) and had a texture of 23%clay, 64% silt and 13% sand (Kreutziger, personal communication).The soil of the Hainich field site was a fertile Cambisol containing40% clay, 56% silt and 4% sand (Knohl et al., 2003). In September2007 the top 5 cm of soil were sampled at both field sites, passedthrough a sieve with a mesh size of 2 mm and partitioned for PLFAextraction (fresh soil), for soil column filling and for chemicalanalyses (dried at 40 �C), respectively.

Soil carbon and nitrogen concentrations were measured fromball-milled sub-samples by elemental analysis (Elementar-analysator vario Max CN, Elementar Analysensysteme GmbH,Hanau, Germany) before and after incubation. Organic carbonconcentration was determined by calculating the differencebetween elemental analyses of the total carbon concentration andsoil inorganic carbon concentration (Steinbeiss et al., 2008b). Ford13C analysis of the soil organic carbon, 3 mg ground sample wasweighed in small tin capsules. The arable soil contained about 1.6%inorganic carbon, which was removed by treatment with 120 ml ofsulfurous acid (5–6% SO2, Merck, Darmstadt, Germany) prior toisotope analysis (Steinbeiss et al., 2008a). Isotope ratios weremeasured by a coupling of an elemental analyzer (EA 1110) with anisotope ratio mass spectrometer (DeltaPlusXL, Thermo Finnigan,Bremen, Germany). All values represent repeated measurementswith a standard deviation of less than 0.3& and were calibratedversus V-PDB using CO2 as reference gas (Werner and Brand, 2001).Soil analyses were summarized in Table 1.

Table 1Basic characterization of biochars and soils; sd refers to standard deviation of replicatedcalculated y-intercept was given instead (see Methods section for details).

Glucose-derived biochar sd Yea

C content (%) 64.6 0.5 67N content (%) 0.0 0.0 5d13C value (&) 3.6 0.15 �2d13C value of CO2 gas in the controls (&)

2.2. Biochar production and characterization

Stable carbon isotopes have proven to reliably trace the flow ofcarbon in various soil organic matter pools (Gleixner et al., 2001)and into soil microorganisms using the compound-specific 13Ccontent of phospholipid fatty acids (Rubino et al., 2007; Kramer andGleixner, 2008). Moreover, changes in the13C content of variouspools enable the determination of mean residence times (Balesdentand Mariotti, 1996; Gleixner et al., 2002). Consequently, theproduction of isotopically labeled biochars from simple isotopicprecursors is most promising investigating synthetic biochars inthe soil system. Biochars were produced by hydrothermal pyrolysis(Titirici et al., 2007a,b) using glucose (signature G) and yeast(signature Y) as parent material, respectively. A 13C label wasintroduced to both biochars adding uniformly 13C labeled glucose(99 atom%, Sigma Aldrich, Seelze, Germany) to the parent materialsprior to biochar synthesis.

Glucose should be seen as model compound for cellulose, the majorstructural component of plant biomass. Several investigations haveshown that charcoal produced from very different types of biomassalways show similar chemical structures (Schmidt and Noack, 2000;Gleixner et al., 2001; Titirici et al., 2007b, in press). Heterocyclic (O-containing) pyran and furan ring systems of carbohydrates or phenoltype structures that are the backbone of lignin form for examplebenzene and other polyaromatic hydrocarbons (PAH) due to thearomatization reactions in the charring process. Solid state 13C NMRexaminations proof this remarkable structural and compositionalsimilarity of all charcoals made from different sources of biomass(Titirici et al., in press) and therefore we do not expect serious differ-ences between charcoals produced from model compounds and frombiomass (Baccile et al., submitted for publication).

Yeast acted as a protein, i.e. nitrogen, rich model waste materialresulting from bioethanol, beer and wine production (pomace,draff, brewer grains, distiller’s grain or distiller’s wash (Belyea et al.,1998; Pfeffer et al., 2007; Maas et al., 2008; Quintero et al., 2008))and thus represented a probable commercial source material forthe synthesis of nitrogen rich biochars. The yeast we used wasprovided by a local beer brewery and represents the brewer grainsthat were separated from the beer product as described in theliterature above. The grains were basically made up of the yeastactive in the fermentation and additionally contain some rest ofbarley glume and wheat bran.

Element composition and d13C values of the biochars weredetermined by elemental analysis and EA-IRMS as was described forthe soil samples, respectively (Table 1). Thermogravimetry(TGA851e, Mettler-Toledo, Gießen, Germany) was applied to char-acterize the thermal stability and thus the carbonization grade of thebiochars (Meszaros et al., 2007; Pastor-Villegas et al., 2007; Strezovet al., 2007). Samples, biochars and their respective parent materials,were introduced into the oven at 60 �C and heated with a rate of1 �C min�1 to 850 �C in an Argon atmosphere. Scanning electronmicroscopy (SEM) was performed on a DSM 940 A (Zeiss, Oberko-chen, Germany). Infrared-spectra were measured with an IFS 66 FTIRspectrometer (Bruker Optik GmbH, Ettlingen, Germany). Spectrawere obtained averaging 128 scans, with a resolution of 4 cm�1.

measurements. For d13C values of CO2 gas in the controls the standard error of the

st-derived biochar sd Arable soil sd Forest soil sd

.4 0.5 2.5 0.01 5.5 0.06.0 0.03 0.3 0.0003 0.5 0.005.8 0.30 �27.7 0.14 �27.1 0.06

�24.0 0.8 �27.6 0.6

S. Steinbeiss et al. / Soil Biology & Biochemistry 41 (2009) 1301–1310 1303

2.3. Experimental design and regular measurements

Soil columns were filled with 150 g soil (dry weight); 15columns were filled with arable soil (signature A) and 15 columnswere filled with forest soil (signature F). The soil of six columns ofeach soil type was mixed with glucose-derived biochar (signaturesAG and FG) and further six columns of each soil type were mixedwith yeast-derived biochar (signatures AY and FY). Three columnsof each soil type were left as control without biochar (signatures Aand F). The amount of biochar added to the soil was calculated tocorrespond to a carbon addition of 30% of the initial soil organiccarbon content. Initial soil properties including PLFA analyses weredetermined from soil samples without incubation (signatures AIand FI) with triple replicates.

Soil columns were incubated at 25 �C during the day and 20 �Cduring the night. No artificial lighting was applied. Soil moisturewas adjusted every three to four days in all columns. No waterleached out from the columns.

Soil respiration was measured using a carbon dioxide probe(GMP 343, Vaisala, Helsinki, Finland) from week 1 to week 25 witha temporal resolution of 1 week in the beginning (up to week 7) and2–3 weeks afterwards. The respired gas was collected in weeks 7,12, 15, 17, 20, 23 and 26 using 2.3 l gas flasks connected viaa capillary to the soil columns (filling time 4 h per sample). Sampleair was dried chemically using magnesium perchlorate (FisherScientific, Loughborough, UK). Each time two flasks were filled thesame way with greenhouse air to correct ambient CO2 concentra-tion and isotope ratios of the treatments (d13Ctreatment,korr)(Amundson et al., 1998). Gas CO2 concentration was measured byGC-FID (Agilent technologies, Santa Clara, USA) and stable carbonisotope ratios were determined by isotope ratio mass spectrometry(Finnigan MAT 252, Bremen, Germany).

Based on the isotopic difference between the respired CO2 frombiochars and from soil organic carbon it was possible to calculatethe proportion of biochar-derived carbon in the respired gas forevery sampling date (Equation (1)) (Balesdent et al., 1998; Waldropand Firestone, 2004). To overcome possible treatment effects wedetermined the control values used in Equation (1) from the gasmeasurements of the control treatments (A and F), assuming thesame processes in the treatments as in the controls. We madeKeeling plots combining the measured d13C values for the completetime series of the gas collected from the respective control columnsand the reciprocal CO2 concentration (1/CO2). The y-intercept of thelinear fit (R2¼ 0.96 for A and R2¼ 0.97 for F) can be interpreted asthe d13C value of biologically produced CO2 during respiration(Amundson et al., 1998) (Table 1).

Fð%Þ ¼ d13Ctreatment;corr � d13Ccontrol

d13Cbiochar � d13Ccontrol� 100 (1)

Mean residence times (T) for the biochars were calculated usingmeasured carbon contents before (ct0) and after incubation (ct)combined with the calculated proportion of biochar carbon in therespiration gas assuming a first order reaction mechanism (Equa-tion (2)) (Gregorich et al., 1996; Gleixner et al., 2002).

T ¼ ðt � t0Þlnðct=ct0Þ

(2)

2.4. Phospholipid fatty acid (PLFA) extraction

Phospholipid fatty acids were extracted from fresh sieved soilbefore incubation and from all treatments after 4 months in thegreenhouse. We extracted three replicates of each treatment exceptof the controls (signatures A, F). There, only one sample per soil

type was extracted to leave the other two replicates for furthercontinuous measurements.

PLFA extraction was performed after standard methodsdescribed in the literature (Bligh and Dyer, 1959; Zelles and Bai,1993). Briefly, soils were shaken for 2 h in a mixture of chloroform,methanol and phosphate buffer. The lipid extracts (chloroformphase) were transferred to silica-filled solid phase extractioncolumns (SPE). Phospholipids were separated from neutral lipidsand glycolipids by eluting with chloroform, acetone and methanol,respectively. The phospholipids in the methanol fraction werehydrolyzed and methylated using a methanolic KOH solutionleading to phospholipid fatty acid methyl esters.

Quantification of the PLFAs per soil dry weight was performedon a GC-FID system (Agilent Technologies, Santa Clara, USA) usinga fused silica column (HP ultra 2, 50 m length� 0.32 mm ID,0.52 mm film thickness). The temperature program started at 140 �C(1 min isotherm) followed by a heating rate of 2 �C min�1 to 270 �C,which was held for 9 min, and followed by a final heating rate of30 �C min�1 to 320 �C. For peak identification retention times ofstandard measurements were used.

Compound-specific isotope ratios of the identified PLFAs weremeasured by GC–IRMS (DeltaPlusXL, Thermo Finnigan, Bremen,Germany). Gas chromatographic separation was performed withthe same parameters as the quantification (see above). To obtaind13C values of the PLFAs, measured isotope values were correctedfor the methyl carbon added during methylation (Kramer andGleixner, 2006).

Identified PLFAs were assigned to certain microbial groups, i.e.fungi (C18:2u6,9, C18:1u9), Gram-positive bacteria (branchedsaturated fatty acids), Gram-negative bacteria (monounsaturatedfatty acids) and bacteria in general (straight chain saturated fattyacids) (Zelles, 1997; Baath and Anderson, 2003; Waldrop andFirestone, 2004; Kramer and Gleixner, 2006; Allison et al., 2007).

2.5. Statistical evaluation

Statistical evaluation of the data sets was performed with SPSSversion 16.0 (SPSS Inc., Chicago, USA). For direct comparison oftreatments simple t-tests were used. To test for systematic effects ofthe soil type, the charcoal type or any interaction of both variablesin complete data sets of all treatments analyses of variance(ANOVA) were calculated. Principal component analyses using theproportion of microbial groups in the soil were performed tocompare the structure of the microbial community in the differenttreatments and the respective initial soil samples. Statisticalsignificance was assigned at the p� 0.05 level.

3. Results

3.1. Biochar characterization

Glucose-derived biochar was highly carbonized and thusthermally stable. Thermogravimetry to a temperature maximumof 850 �C in an inert atmosphere led to a total mass loss of about50% with the highest rate of volatilization at temperaturesbetween 380 and 390 �C. In contrast, the parent material, glucose,lost 86% of its initial mass under the same experimental condi-tions and thermal degradation occurred in several processes withmaximum reaction rates already at temperatures of 200 �C and280 �C, respectively.

The degree of condensation in the yeast-derived biochar wasmuch lower than that of the glucose-derived biochar, indicated bya total mass loss of 72%, which is only 10% less than that of theparent material, yeast, under the same conditions. Thermaldegradation of the yeast-derived biochar already started at 200 �C,

4500 4000 3500 3000 2500 2000 1500 1000 500 0

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

CH2, CH3 aliphaticC-O-C

C-H aromatic

C=C aromaticC=O

OH

Ab

so

rb

an

ce

Wavenumber in cm-1

CHaliphatic

Fig. 2. Infrared-spectra of glucose-derived biochar (straight line) and yeast-derivedbiochar (dashed line).

S. Steinbeiss et al. / Soil Biology & Biochemistry 41 (2009) 1301–13101304

reached the next maximum reaction rate at 325 �C and showeda last process similar to the glucose-derived biochar degradationbetween 380 and 390 �C.

Scanning electron microscopy (SEM) showed similar structuresof our model biochars (carbonaceous spheres and continuousnanopore systems) as were observed for charcoals produced fromdifferent biomass types (e.g. rice grains, oak leafs, pine needles) byhydrothermal pyrolysis (Cui et al., 2006; Titirici et al., 2007b)(Fig. 1).

Infrared-spectra reveal a polar surface structure of the modelbiochars containing phenolic, carbonyl and hydroxyl functionalgroups. The aromatic structure was more pronounced for the highlycondensed glucose-derived biochar, whereas the low condensationgrade of the yeast-derived biochar resulted in larger proportions ofsaturated and unsaturated aliphatic structures (Fig. 2). Infrared-spectra of charcoals produced from biomass verify hydroxyl groups,phenolic residues, carbonyl functions, aliphatic double bonds anda certain degree of aromaticity as the typical biochar structurecharacteristics (Titirici et al., 2007b).

3.2. Soil respiration and gas measurements

Initial respiration rates differed strongly between the treat-ments (Fig. 3) but did not correlate to the initial carbon content.In arable soil, both biochar treatments (AG, AY) showed similarrespiration rates to the control despite the carbon addition(p¼ 0.64 and 0.50, respectively). In forest soil, the highest initialrespiration was measured in the treatment with labile yeast-derived biochar (FY), which was significantly higher (p< 0.001)than both the control (F) and the treatment with glucose-derivedbiochar (FG). No difference was observed between the treatmentwith stable glucose-derived biochar in forest soil and therespective control (p¼ 1.00). Respiration rates strongly decreasedwithin 4 weeks of incubation and had leveled off after 12 weeksto a constant median value of 2 mg C d�1 for all treatments. Nosystematic differences in respiration rates were observed betweenthe treatments, although the FY treatment showed significantlyhigher respiration rates at some occasions within the first 10weeks of the experiment (i.e. weeks 4 and 10: p< 0.001 andp¼ 0.062).

Labeling with 13C led to isotopic differences of 24–31&

between biochar carbon and soil organic carbon (Table 1) whichwas used to quantify the proportion of biochar-derived carbon in

Fig. 1. SEM picture of (a) glucose-derived biochar a

the respired CO2. Two major differences were observed betweenthe soil types. First, data variability as well between replicates asbetween repeated samplings was much higher in arable soiltreatments than in forest soil treatments (p< 0.001). Second, theproportion of biochar carbon in the respiration gas was generallylower in forest soil treatments compared to arable soil treatments(p¼ 0.025). In detail, variability between replicates for arable soiltreatments were 5.4% (AG) and 3.3% (AY) on average, whilestandard deviations of 0.9% were observed for replicates of bothforest soil treatments (FG and FY). The proportion of biochar-derived carbon in the respiration gas was 28% (sd¼ 7.9%) in theAG treatment and 22% (sd¼ 24.3%) in the AY treatment. In forestsoil 8% (sd¼ 1.2%) of the respired carbon derived from biochar inthe FG treatment and 12% (sd¼ 3.6%) derived from biochar in theFY treatment. In the AG treatment, the proportion of biochar-derived CO2 showed an increasing trend with time, reaching themaximum of 43% in week 20. The AY treatment showed the mostinconsistent pattern in the composition of the respiration gasduring incubation, varying between 0 and 53% biochar carbon inthe gas without any regularity. In contrast, in the forest soiltreatments the proportion of biochar carbon in the respiration gas

nd (b) yeast-derived biochar. Scale bar 10 mm.

0 5 10 15 20 25 0 5 10 15 20 250

10

20

30

40

50

60

0

10

20

30

40

50

60

resp

iratio

n rate in

m

g C

d

-1

week after experiment start

A AG AY

week after experiment start

F FG FY

Fig. 3. Respiration rates for arable soil treatments (left) and forest soil treatments (right) including the respective controls during incubation. Error bars represent standarddeviations between three replicates per treatment.

S. Steinbeiss et al. / Soil Biology & Biochemistry 41 (2009) 1301–1310 1305

was constant at 8% for FG, while FY showed a decreasing trendfrom 19% in week 7 down to 9% in week 17 and later on.

3.3. Development of soil carbon stocks and mean residence times

Measured respiration rates reflect temporary carbon losses andvaried over time due to temperature and soil moisture variability.The total carbon budget was thus determined by elemental analysisfrom soil sub-samples before and after four months of incubation(Table 2). Total carbon content decreased in controls and all treat-ments over the incubation time. Despite different absolutenumbers in the carbon budget due to the different initial carboncontent of the two soil types, the relative amount of carbonremaining in the soils after biochar incubation only depended onthe type of biochar added but did not depend on the soil type itselfor any interaction between soil type and biochar type (Table 3).Thus, of the initially added 3% of charcoal carbon, both treatmentswith more stable glucose-derived biochar still contained 27% morecarbon than the respective control, while both treatments withlabile yeast-derived biochar still contained 23% more carbon thanthe respective control (Table 2).

To quantify losses of biochar carbon and soil organic carbonwe used the isotopic signature of the respiration gas. Wenormalized all losses to the respective initial carbon amounts of

Table 2Carbon budget in g C and relative to the respective controls for all treatments beforeand after incubation; sd refers to standard deviation of replicated measurements.

Treatment Cinitial

(g)sd Amount C relative

to control (%)Cfinal

(g)sd Amount C relative

to control (%)

A 3.79 0.02 100 3.63 0.02 100AG 4.93 0.02 130 4.61 0.03 127AY 4.93 0.02 130 4.45 0.04 123F 8.23 0.04 100 7.75 0.04 100FG 10.70 0.05 130 9.87 0.01 127FY 10.70 0.05 130 9.53 0.06 123

the treatments for better comparability (Fig. 4). Losses of soilorganic carbon were generally smaller in the arable soil treat-ments compared to the respective forest soil treatments andbiochar addition always increased the loss of carbon from the soilorganic carbon pool (Table 4). In both soil types, soil organiccarbon losses were largest when labile yeast-derived biochar wasadded (Fig. 4, Table 4). In these treatments, twice the amount ofsoil organic carbon was respired compared to the controls.Moreover, yeast-derived biochar seemed to be better degradablethan glucose-derived biochar as indicated by larger biocharcarbon losses in both soil types (Fig. 4), although the differencewas significant only in forest soil (p¼ 0.003, p¼ 0.147 for arablesoil). Most interestingly, normalized losses of soil and biocharcarbon in the arable soil were almost identical (p¼ 0.544), whilein forest soil less biochar carbon than soil organic carbon was lost(p< 0.001).

The mean residence times (Equation (2)) for biochar carbonwere calculated from measured total carbon losses and theproportions of biochar carbon in the respiration gas (Equation(1)). In total, the controls lost 4% (A) and 6% (F) of their initialcarbon content, treatments with glucose-derived biochar lost 7%(AG) and 8% (FG) carbon, and addition of yeast-derived biocharcaused total carbon losses of 10% (AY) and 11% (FY), respectively.Consequently, mean residence times ranged between 4 (AG) and29 (AY) years (Fig. 5). The 29 years were calculated for yeast-derived biochar in arable soil and give just a rough estimate dueto the huge uncertainty caused by the high variability in thedetected proportion of biochar carbon in the respiration gas.

Table 3Summary of analysis of variance (ANOVA) of the carbon budget relative to therespective controls for all biochar treatments after incubation.

Parameter Sum of squares F-value Significance

Soil 0.80 1.07 0.332Charcoal 56.77 75.52 <0.001Soil� charcoal 0.01 0.01 0.923

-22

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0A AG AY F FG FY

treatment

% carb

on

lo

st

soil organic carbonbiochar carbon

Fig. 4. Losses of biochar carbon and soil organic carbon after 4 months of incubationgiven relative to the respective initial amounts in the treatments. Error bars reflect theuncertainties in the proportion of biochar carbon in the respiration gas used forcalculation and were determined according to error propagation laws.

AG AY FG FY0

10

20

30

40

50

60

70

80

90

mean

resid

en

ce tim

e in

years

treatment

Fig. 5. Calculated mean residence times for the biochars in the different treatments.Error bars reflect the uncertainty caused by the variability of the proportion of biocharcarbon in the respiration gas. They were determined according to error propagationlaws.

S. Steinbeiss et al. / Soil Biology & Biochemistry 41 (2009) 1301–13101306

Glucose-derived biochar remained longer in forest soil (12years) than in arable soil (p< 0.001), where it would bemineralized after 4 years, assuming a continuously ongoingdecomposition as observed in the 4 months of incubation. Foryeast-derived biochar it would take about 6 years to be miner-alized in the forest soil.

Table 5

3.4. Microbial community in the soils

To get an estimate of microbial adaptation to the new carbonsource the total amount of phospholipid fatty acids in the soils wasdetermined before and after incubation in all treatments. Asalready found for the total carbon budget, biochar type was thedriving parameter for any effects on the microbial communitybetween the treatments. Arable soil generally contained smalleramounts of microorganisms than forest soil as well for the initialvalues as in every treatment after incubation (p< 0.001). Additionof glucose-derived biochar to both soil types caused a significantreduction of microbial biomass (p< 0.001) during incubation. Incontrast, yeast-derived biochar addition did not change the PLFAcontent in the soils (p¼ 0.39), which still was as high as beforeincubation in both soil types. We found no interaction between soiltype and biochar type (p¼ 0.15).

The identified PLFAs were assigned to four main groups ofmicroorganisms in soil, i.e. fungi, Gram-positive bacteria, Gram-negative bacteria and bacteria in general. The proportions ofthese groups in the microbial community were calculated for theinitial soils and for all treatments after incubation (Table 5). Theinitial microbial community composition in both soil types was

Table 4p-Values resulting from multiple comparison t-tests of soil organic carbon losses(normalized to the initial carbon content) for all treatments after incubation.Numbers smaller than 0.05 reflect significant differences between the treatments.

A AG AY F FG FY

A 0.120 0.031AG 0.120 0.004 0.001AY 0.031 0.004 0.018F 0.005 0.013FG 0.001 0.005 0.001FY 0.018 0.013 0.001

quite similar (Fig. 6). Again, major effects of biochar additionwere the same in both soil types and depended only on thebiochar type (Fig. 6, Table 6). While the addition of glucose-derived biochar rarely changed the composition of the soilmicrobial community, the yeast-derived biochar stronglypromoted fungi in both soils (p< 0.001). The majority of presentmicroorganisms belonged to the group of usually root associatedGram-negative bacteria and made up 42% (AI) and 44% (FI) of themicrobial biomass. In both soils, 27% of the microbes belonged tothe Gram-positive bacteria, 11% were assigned to bacteria ingeneral and only 11% (FI) to 12% (AI) of the microbial communitywas made up of fungal biomass. The addition of glucose-derivedbiochar led to shifts along PC 2 in the principal componentanalyses (PCA) (Fig. 6), which only explained 4–11% of thevariance and reflected non-systematic changes in the proportionof all microbial groups. In treatments with yeast-derived biochar,the proportion of fungal biomass increased by 16% in both soiltypes, while Gram-positive and Gram-negative bacteriadecreased by 7–14%. The increase in fungal biomass wasexpressed in the large shifts along PC 1 of the PCA (p< 0.001)(Fig. 6), which explained 89% of the variance in forest soil and94% in arable soil. The microbial community composition of thecontrols without biochar addition showed the same pattern afterincubation as the initial soil samples (Table 5).

Proportion of the amount of PLFAs assigned to different microbial groups, i.e. fungi,Gram-negative bacteria, Gram-positive bacteria and bacteria in general, before andafter incubation; sd refers to standard deviation between three replicates. There wasno replicate extracted for the controls (A, F).

treatment Fungi sd Gram(�)bacteria

sd Gram(þ)bacteria

sd Bacteria sd

AI 11.9 0.1 41.9 0.1 26.9 0.1 11.5 0.1AG 10.8 0.3 41.8 2.1 25.0 2.4 13.0 0.3AY 28.0 0.9 30.7 0.4 18.8 0.5 15.7 0.2FI 11.0 0.2 43.6 0.5 26.8 0.4 10.8 0.0FG 10.7 0.2 37.7 0.3 29.9 0.4 13.8 0.1FY 27.6 0.8 28.9 0.3 20.3 0.7 16.8 0.1A 12.0 41.9 26.9 11.6F 10.9 39.0 27.3 14.5

-1,5 -1,0 -0,5 0,5 1,0 1,5 2,0 2,5

-0,5

-1,0

-1,5

0,5

1,0

1,5

2,0

2,5

AI

AG

AY

FI

FG

FY

PC 2

PC 1

Fig. 6. Principal component analyses of the microbial community composition for bothsoil types. PC 1 explained 89% (forest soil) and 94% (arable soil) of the variance and wasdriven by the proportion of fungi and bacteria in both soil types. PC 2 explained 11%(forest soil) and 4% (arable soil) of the variance.

S. Steinbeiss et al. / Soil Biology & Biochemistry 41 (2009) 1301–1310 1307

Finally, we determined compound-specific isotope ratios ofthe PLFAs to identify the major carbon sources of the differentgroups of soil microorganisms (Appendix 1). PLFA enriched in13C compared to the initial value indicated the uptake of biocharcarbon by the respective microbial group, while unchanged d13Cvalues prove the continuous uptake of soil organic carbon.Control treatments generally showed small shifts in all microbialgroups as decomposition of soil organic carbon led to anenrichment in 13C in the remaining soil organic carbon. Althoughthe microbial group specific enrichments somehow depended onthe type of biochar added to the soils, e.g. significant enrich-ments of Gram-negative and Gram-positive bacteria for glucose-derived biochar treatments (2.1–4.7&, 0.001< p< 0.027) andstrong yeast-derived biochar uptake by fungi in both soils(isotopic shift 9.5 and 14.8&, 0.003< p< 0.062), several differ-ences were observed between the soil types (Fig. 7). In contrastto arable soil, where fungi were less involved in the decompo-sition of glucose-derived biochar (isotopic shift¼ 1.6&,p¼ 0.350), in forest soil fungal biomarkers showed an averageenrichment of 4.8& (p¼ 0.417). Especially the d13C value ofC18:2u6,9 increased by 8.4& (p¼ 0.007), while other fungibiomarkers (C18:1u9) remained unchanged (isotopicshift¼ 1.1&). Beside the obvious utilization of yeast-derivedbiochar by fungi in arable soil, also all bacterial groups were ableto decompose this biochar to a certain extent in this soil type.Significant isotopic enrichments were measured in all bacterialbiomarkers (p� 0.002). In forest soil, Gram-negative bacteriawere the only microbial group beside fungi that took up yeast-derived biochar carbon in a significant amount (p¼ 0.001).

Table 6Summary of analysis of variance (ANOVA) of the principal component analyses ofmicrobial community composition.

Parameter Sum of squares F-value Significance

PC 1 Soil 0.00 0.00 1.000Charcoal 15.94 7.97 <0.001Soil� Charcoal 0.00 0.00 0.741

PC 2 Soil 0.00 0.00 1.000Charcoal 10.87 13.92 0.001Soil� Charcoal 0.44 0.57 0.581

Assuming biochar and soil organic carbon as only carbonsources for the soil microorganisms, the proportion of biocharcarbon incorporated in the microbial biomass can be calculated.For instance, the observed enrichment in fungal biomarkers of14.8& in the AY treatment would equal a utilization of 60%biochar carbon for fungal biomass production. In forest soil,fungi used 40% yeast-derived biochar carbon to build up theirbiomass. Bacteria in arable soil used between 10% and 15%glucose-derived biochar carbon as carbon source and theproportion of glucose-derived biochar carbon in Gram-negativebacteria in forest soil amounted to 13%.

4. Discussion

It has been observed in several studies that biochar addition tosoils improved soil fertility and thus increased crop yields onagricultural lands (Marris, 2006; Chan et al., 2007). This fertilizereffect could be explained by a stimulation of soil microorganismsthat consequently led to an increased recycling of nutrientstrapped in biomass residues. The fertilizer function is additionallysupported by an increased water retention and cation exchangecapacity of the soils caused by the huge surface area of the bio-chars. An aspect of biochar amendments that got more attentionrecently is that additional photosynthetically fixed carbon isbrought into the soil, where it could contribute to longer termcarbon storage and thus mitigates increasing atmospheric CO2

concentrations (Schmidt and Noack, 2000; Lehmann, 2007).However, little is known about turnover times of biochars in soilsand a long-term storage function contradicts the fertilizer func-tion of biochars that requires a certain biodegradability of thebiochar material. The major question to solve will be to designbiochars that fulfil both functions with the best possiblecompromise. Biochar structure, e.g. the condensation grade, couldeasily be managed in production processes, but studies arenecessary to check, whether the condensation grade is a tool tocontrol the turnover of biochars in soils.

In our greenhouse experiment we investigated the conse-quences of the addition of biochar with different condensationgrades (high¼ glucose-derived, low¼ yeast-derived) to two soiltypes (arable and forest soil) for inherited carbon stocks and for soilmicrobial communities.

Soil respiration measurements indicated a strong stimulationof soil microorganisms by yeast-derived biochar in the beginningof the incubation, whereas treatments that received glucose-derived biochar showed respiration rates similar to the respec-tive controls. After 12 weeks no differences between anytreatments could be observed anymore and respiration rates hadgenerally decreased to very low levels. As a result of theincreased respiration total carbon losses were always higher intreatments that received biochar than in the controls. The typeof biochar clearly showed a systematic influence on soil organiccarbon losses. Yeast-derived biochar, the model compound foreasily degradable biochar stimulated soil microorganisms mostin both soils leading to soil organic carbon losses twice as highas in the controls, whereas glucose-derived biochar led tointermediate soil organic carbon losses in both soil types.Increasing soil organic carbon losses caused by charcoal inputhave been observed also in other studies (Wardle et al., 2008).The mechanism behind still has to be resolved. However, even ifcarbon turnover was increased by the biochar treatments in ourexperiment we want to point out that the total carbon content inthe soil still was 27% higher in glucose-biochar treatments and23% in yeast-biochar treatments compared to the controls at theend of our experiment regardless of the soil type. Calculatedmean residence times of 4–29 years lead to the conclusion that

0

2

4

6

8

10

12

14

16

18

AGAYA

fungi gram(+) gram(-) bacteria fungi gram(+) gram(-) bacteria0

2

4

6

8

10

12

14

16

18

FGFYF

iso

to

pic sh

ift in

‰

(treatm

en

t - in

ital)

Fig. 7. Isotopic shift of PLFA biomarkers (treatment after incubation – initial values) for certain microbial groups, i.e. fungi, Gram-negative bacteria, Gram-positive bacteria andbacteria in general. Error bars reflect the standard deviation in isotopic shift within a group of microorganisms.

S. Steinbeiss et al. / Soil Biology & Biochemistry 41 (2009) 1301–13101308

biochars produced by hydrothermal pyrolysis would probablyadd to the decadal soil carbon pool. The fast decrease in soilrespiration rates during incubation indicated that microbialstimulation and thus decomposition processes remarkablyslowed down after 4 months. Concluding from this observation,we assume that any fertilizer effect of these biochars will bebiggest directly after biochar addition to the soils and that thecarbon storage function will gain importance on the longer term.Mean residence times given here represent results from theinitial phase of biochar degradation and might increase withtime.

As has been shown for the total carbon stocks, also thereaction of soil microorganisms on biochar addition was drivenby the biochar type and largely independent of the soil type.Yeast-derived biochar strongly increased the proportion of fungiin both soils, which consequently turned out as the microbialgroup that most utilized this type of biochar. Glucose-derivedbiochar was used as carbon source for the build up of bacterialbiomass. Bacteria in arable soil, probably better adapted tocarbon limitation events and more complex remaining soilcarbon, respired more biochar carbon than bacteria in forest soil.Consequently, calculated mean residence times of glucose-derived biochar in arable soil were slightly shorter than in forestsoil. Microorganisms in the forest soil seemed to be lessspecialized on certain carbon sources. The adaptation of certainmicrobial groups to biochar degradation was less pronouncedthan in arable soil.

To finally answer the questions that should be solved with ourexperiment:

1) How stable are biochars produced by this method in differentsoil?

Biochars produced by hydrothermal pyrolysis would add to thedecadal soil carbon pool.

2) How do inherited soil microorganisms react on the addition ofsuch biochars?

Inherited soil microorganisms adapted to the new carbon sourceand utilized both types of biochar. The biochar type determined,which group of microorganisms were involved in the decomposi-tion process. Yeast-derived biochar strongly promoted fungi, whileglucose-derived biochar primarily was utilized by Gram-negativebacteria.

3) Is the stability of these biochars tuneable by varying thecondensation grade and chemical composition of thebiochar?

Our results clearly show that the type of biochar, i.e. conden-sation grade and chemical structure, is the main driver for alldifferences observed between our treatments. All patternsobserved for the biochar types were the same in both soils. We thusconclude that the condensation grade and the chemical structure ofbiochars produced by this method could serve as ‘‘tuning param-eter’’ to design biochars that act as fertilizers but simultaneouslyadd to the soil carbon pool on a decadal time scale.

In our current experiment we manipulated the condensationgrade and the nitrogen content of the biochars with promisingresults. Several other elements (phosphorus, sulphur, cations)could be introduced to the biochar structure helping to fill specialnutrient demands of arable lands. Further studies are necessary todesign the best possible soil amendments and to investigate thelong-term behavior of these biochars in natural systems.

Acknowledgements

This investigation was financially supported by the Max PlanckSociety within the scope of the EnerChem project house. Wethank Maria M. Titirici for her help with SEM measurements atthe Max Planck Institute of Colloids and Interfaces in Potsdam-Golm. Valerian Ciobota and Petra Roesch kindly recorded theInfrared-spectra at the Institute of Physical Chemistry of theFriedrich Schiller University in Jena.

Appendix 1

Amounts of identified PLFAs in mg g�1 soil dw and d13C values in & in initial soils, all treatments and the controls after incubation.Standard deviations are given in parentheses. Source specific summaries can be found at the end of each section.

PLFA Source AI (mg g�1 dw) AG (mg g�1 dw) AY (mg g�1 dw) A (mg g�1 dw) FI (mg g�1 dw) FG (mg g�1 dw) FY (mg g�1 dw) F (mg g�1 dw)

C14:0 br Gram(þ) 1.8 (0.1) 0.4 (0.2) 0.7 (0.2) 2.5 5.1 (0.7) 2.5 (0.4) 2.3 (0.1) 2.1C14:0 Bacteria 1.1 (0.1) 0.4 (0.1) 0.7 (0.1) 1.5 1.9 (0.2) 1.3 (0.2) 1.5 (0.1) 1.2C15:1 Gram(�) 1.4 (0.1) 0.4 (0.2) 0.6 (0.1) 1.9 2.4 (0.2) 0.9 (0.1) 0.9 (0.0) 0.8C15:0 br Gram(þ) 6.6 (0.6) 2.8 (1.2) 4.3 (0.7) 9.1 10.9 (0.9) 7.7 (1.2) 8.2 (0.4) 6.2C15:0 br Gram(þ) 5.5 (0.5) 2.3 (0.9) 3.1 (0.5) 7.6 14.8 (1.0) 10.6 (1.7) 10.5 (0.6) 8.5C15:0 Bacteria 0.6 (0.0) 0.4 (0.1) 0.6 (0.1) 0.8 0.8 (0.1) 0.7 (0.1) 0.7 (0.0) 0.5C16:0 br Gram(þ) 0.6 (0.1) 0.4 (0.1) 0.4 (0.1) 0.8 0.9 (0.2) 0.9 (0.1) 0.7 (0.0) 0.7C16:1 Gram(�) 1.2 (0.1) 0.5 (0.2) 0.6 (0.1) 1.7 1.8 (0.1) 1.0 (0.2) 0.9 (0.1) 0.8C16:0 br Gram(þ) 3.6 (0.4) 1.8 (0.7) 2.1 (0.5) 5.1 5.4 (0.3) 4.2 (0.8) 4.2 (0.2) 3.1C16:1 Gram(�) 2.4 (0.2) 1.1 (0.3) 1.7 (0.3) 3.4 4.2 (0.2) 1.7 (0.3) 2.0 (0.2) 1.5C16:1 Gram(�) 10.6 (0.9) 4.6 (1.2) 5.6 (1.1) 14.6 11.8 (0.5) 4.9 (0.7) 5.6 (0.4) 4.8C16:1 Gram(�) 6.0 (0.5) 3.1 (0.6) 3.4 (0.7) 8.3 8.5 (0.4) 3.1 (0.4) 3.2 (0.2) 2.8C16:0 Bacteria 11.4 (1.2) 7.7 (1.5) 15.9 (3.3) 16.1 20.5 (0.9) 15.6 (2.4) 31.1 (2.9) 14.3C17:0 br Gram(þ) 1.3 (0.1) 0.8 (0.2) 0.8 (0.1) 1.7 2.1 (0.1) 1.1 (0.1) 1.2 (0.1) 0.9C17:1 Gram(�) 4.0 (0.4) 2.3 (0.6) 2.8 (0.6) 5.5 5.5 (0.3) 2.7 (0.4) 2.8 (0.2) 2.3C17:0 br Gram(þ) 5.9 (0.5) 3.9 (0.7) 3.6 (0.9) 8.2 11.2 (0.5) 5.2 (0.9) 4.7 (0.3) 4.2C17:0 br Gram(þ) 1.3 (0.1) 0.9 (0.1) 0.9 (0.1) 1.7 1.9 (0.1) 0.9 (0.1) 0.9 (0.1) 0.8C17:0 br Gram(þ) 1.2 (0.1) 0.8 (0.1) 0.7 (0.2) 1.6 1.8 (0.1) 0.9 (0.1) 0.9 (0.0) 0.8C17:0 br Gram(þ) 2.3 (0.2) 1.7 (0.4) 1.9 (0.3) 3.1 4.2 (0.2) 3.1 (0.5) 3.2 (0.2) 2.6C17:0 br Gram(þ) 2.5 (0.2) 1.6 (0.3) 1.7 (0.4) 3.6 4.1 (0.2) 2.8 (0.4) 2.9 (0.2) 2.3C17:1 Gram(�) 2.0 (0.2) 1.1 (0.3) 0.9 (0.2) 2.8 1.5 (0.1) 0.9 (0.1) 0.8 (0.0) 0.7C17:1 Gram(�) 2.8 (0.2) 2.0 (0.3) 2.0 (0.4) 3.8 5.9 (0.2) 3.4 (0.5) 4.1 (0.3) 3.2C17:0 Bacteria 0.5 (0.1) 0.4 (0.1) 0.5 (0.1) 0.7 0.7 (0.1) 0.7 (0.1) 0.7 (0.1) 0.5C18:0 br Gram(þ) 2.3 (0.2) 1.8 (0.9) 1.7 (0.5) 3.2 3.0 (0.2) 2.7 (0.5) 2.4 (0.1) 1.9C18:0 br Gram(þ) 0.5 (0.0) 0.3 (0.1) 0.3 (0.1) 0.6 0.7 (0.0) 0.4 (0.1) 0.4 (0.0) 0.3C18:0 br Gram(þ) 1.0 (0.1) 0.8 (0.3) 0.7 (0.2) 1.5 1.4 (0.1) 1.5 (0.3) 1.2 (0.1) 1.0C18:2u6,9 Fungi 3.4 (0.3) 1.6 (0.3) 15.9 (3.3) 4.7 3.5 (0.2) 1.8 (0.3) 27.3 (3.4) 1.5C18:1u9 Fungi 13.5 (1.4) 7.4 (1.3) 22.0 (4.6) 19.0 25.7 (1.0) 15.5 (2.6) 39.7 (4.1) 13.6C18:1u11 Gram(�) 21.2 (2.1) 14.1 (2.2) 16.6 (3.9) 29.3 46.7 (1.6) 26.2 (3.9) 31.1 (2.7) 23.2C18:1 Gram(�) 1.8 (0.1) 1.2 (0.1) 1.4 (0.3) 2.8 2.9 (0.3) 1.3 (0.1) 1.7 (0.1) 1.2C18:0 Bacteria 2.7 (0.3) 2.1 (0.4) 3.3 (0.8) 3.8 4.8 (0.3) 3.7 (0.6) 6.0 (0.5) 3.5C18:0 cyc Gram(�) 1.7 (0.2) 1.3 (0.2) 1.6 (0.3) 2.3 3.2 (0.1) 3.0 (0.4) 3.7 (0.3) 2.8C19:0 br Gram(þ) 2.6 (0.3) 1.8 (0.6) 2.3 (0.7) 3.6 4.8 (0.3) 4.0 (0.8) 4.6 (0.3) 2.9C19:0 br Gram(þ) 0.7 (0.0) 0.6 (0.1) 0.5 (0.1) 1.1 1.3 (0.1) 1.0 (0.1) 1.0 (0.1) 0.7C18:0 cyc Gram(�) 5.5 (0.6) 4.5 (0.5) 4.2 (1.0) 7.7 22.9 (0.9) 12.2 (2.0) 12.3 (0.9) 10.7C20:1u9 Gram(�) 1.2 (0.1) 0.7 (0.2) 1.1 (0.2) 1.6 2.2 (0.2) 1.1 (0.2) 1.7 (0.2) 1.0C20:0 Bacteria 0.7 (0.0) 0.4 (0.0) 0.6 (0.1) 0.9 1.0 (0.1) 0.8 (0.1) 1.1 (0.1) 0.8

Sum Gram(�) 61.8 36.9 42.5 85.7 119.5 62.4 70.8 55.8Gram(þ) 39.7 22.7 25.7 55.0 73.5 49.5 48.3 39.0

Fungi 16.9 9.0 37.9 23.7 29.2 17.3 67.0 15.1Bacteria 16.5 11.4 21.6 23.8 29.7 22.8 41.1 20.8

d13C (&) d13C (&) d13C (&) d13C (&) d13C (&) d13C (&) d13C (&) d13C (&)

C14:0 br Gram(þ) �29.4 (1.4) �26.6 (1.3) �29.4 (0.4) �28.5 (1.5) �30.6 (3.4) �28.1 (0.3)C14:0 Bacteria �28.8 (0.3) �22.6 (0.5) �24.6 (1.3) �23.2 (0.9) �23.1 (0.9) �25.6 (1.2) �24.3 (0.0)C15:1 Gram(�) �26.6 (2.1) �23.0 (0.6) �24.3 (0.5) �24.2 (0.3) �24.2 (0.2)C15:0 br Gram(þ) �25.0 (0.6) �25.0 (1.1) �21.7 (0.2) �23.6 (1.4) �24.7 (0.3) �23.1 (1.0) �23.1 (1.2) �23.3 (0.5)C15:0 br Gram(þ) �21.2 (0.2) �19.1 (1.2) �19.0 (0.3) �21.5 (0.7) �20.4 (0.2) �18.8 (0.2) �16.8 (0.6) �19.3 (0.3)C15:0 Bacteria �27.8 (1.6) �23.0 (1.2) �25.9 (1.3) �25.7 (0.5) �26.0 (0.5)C16:0 br Gram(þ) �22.5 (3.3) �22.8 (0.4) �25.3 (1.0) �28.0 (1.9) �24.3 (1.8) �26.4 (0.9)C16:1 Gram(�) �27.1 (1.5) �21.4 (1.8) �24.5 (1.4) �26.3 (0.5)C16:0 br Gram(þ) �25.2 (0.3) �23.7 (1.4) �22.5 (0.4) �25.9 (1.3) �23.8 (0.9) �25.5 (0.1)C16:1 Gram(�) �21.3 (0.3) �19.0 (0.9) �17.1 (0.3) �20.8 (0.2) �23.4 (0.9) �20.3 (0.4) �19.5 (0.8) �20.2 (0.1)C16:1 Gram(�) �26.1 (0.5) �20.5 (1.0) �21.6 (1.7) �24.9 (0.5) �25.9 (0.5) �22.4 (0.7) �21.3 (0.6) �25.1 (0.3)C16:1 Gram(�) �20.7 (0.1) �18.8 (0.3) �18.7 (0.3) �21.3 (0.4) �21.5 (0.3) �18.6 (1.9) �17.4 (0.9) �20.2 (0.5)C16:0 Bacteria �24.8 (1.4) �20.8 (0.8) �17.8 (1.0) �23.9 (0.5) �23.8 (0.1) �21.0 (1.1) �18.4 (0.2) �23.2 (0.2)C17:0 br Gram(þ) �19.0 (1.1) �9.7 (0.8)C17:1 Gram(�) �27.1 (1.0) �21.2 (0.6) �24.8 (1.5) �24.6 (0.4) �22.9 (1.6) �24.2 (1.7) �22.1 (0.5)C17:0 br Gram(þ) �23.9 (0.3) �22.0 (0.2) �21.8 (0.9) �23.0 (0.5) �22.6 (0.4) �21.4 (1.1) �21.1 (1.1) �22.4 (0.6)C17:0 br Gram(þ) �18.9 (0.7) �15.4 (0.7) �20.2 (0.0) �17.6 (1.7) �19.5 (1.3) �18.1 (0.1) �20.0 (1.3) �17.9 (1.3)C17:0 br Gram(þ) �22.3 (0.3) �20.3 (0.6) �21.1 (1.1) �22.6 (0.2) �20.6 (1.4) �21.8 (1.3) �21.8 (0.4)C17:0 br Gram(þ) �22.2 (0.3) �22.3 (0.6) �18.7 (0.4) �20.6 (0.4) �20.4 (0.2) �19.1 (1.1) �19.5 (0.7) �19.4 (0.5)C17:1 Gram(�) �23.5 (0.6) �15.9 (1.3) �20.7 (0.6) �22.4 (0.7) �23.7 (0.3) �17.5 (1.7) �19.0 (0.4) �22.2 (0.5)C17:0 Bacteria �24.4 (0.9) �20.6 (0.6) �22.0 (1.6) �16.6 (0.8) �19.4 (0.6) �21.0 (0.5)C18:0 br Gram(þ) �25.5 (0.1) �24.2 (0.3) �24.0 (0.7) �26.1 (1.4) �24.3 (0.2) �22.3 (1.6) �22.7 (1.2) �23.9 (0.6)C18:0 br Gram(þ) �21.6 (1.0) �24.0 (0.3) �20.5 (0.6)C18:0 br Gram(þ) �27.3 (1.1) �23.9 (1.1) �26.6 (1.6) �22.9 (1.7) �17.0 (2.0) �23.6 (2.5) �20.7 (1.7)C18:2u6,9 Fungi �31.6 (1.1) �31.0 (0.6) �15.4 (1.2) �29.7 (0.7) �28.0 (0.5) �19.6 (1.3) �18.6 (0.2) �23.2 (1.0)C18:1u9 Fungi �27.1 (0.7) �24.6 (1.0) �13.8 (0.5) �27.2 (1.6) �23.3 (0.3) �22.2 (0.3) �13.8 (0.3) �22.7 (0.5)C18:1u11 Gram(�) �24.8 (0.5) �16.7 (1.2) �17.6 (0.4) �24.7 (1.0) �24.6 (0.2) �19.5 (0.5) �18.2 (0.4) �23.3 (0.2)

(continued on next page)

S. Steinbeiss et al. / Soil Biology & Biochemistry 41 (2009) 1301–1310 1309

Appendix 1 (continued )

PLFA Source AI (mg g�1 dw) AG (mg g�1 dw) AY (mg g�1 dw) A (mg g�1 dw) FI (mg g�1 dw) FG (mg g�1 dw) FY (mg g�1 dw) F (mg g�1 dw)

C18:1 Gram(�) �14.9 (0.5) �10.1 (0.8) �13.0 (2.0) �15.2 (1.3) �14.5 (1.1) �8.2 (2.5) �5.5 (1.3) �10.8 (1.0)C18:0 Bacteria �20.9 (0.7) �18.5 (0.3) �17.5 (0.3) �21.4 (1.2) �21.3 (0.5) �18.9 (0.5) �14.9 (0.3) �20.9 (0.4)C18:0 cyc Gram(�) �21.1 (0.9) �16.6 (0.4) �19.4 (0.4) �20.5 (0.6) �23.7 (0.5) �16.9 (0.3) �15.3 (0.4) �21.9 (0.6)C19:0 br Gram(þ) �29.1 (0.8) �23.3 (1.0) �25.8 (0.8) �22.1 (0.9) �21.7 (1.4) �18.7 (0.2) �21.7 (0.9)C18:0 cyc Gram(�) �28.3 (0.5) �25.4 (1.0) �25.6 (1.2) �28.5 (0.8) �27.9 (0.4) �26.4 (0.4) �25.3 (0.5) �27.5 (0.3)C20:0 Bacteria �21.6 (1.9) �23.6 (1.0) �25.4 (0.1) �20.9 (0.6) �21.5 (1.0)

Average Gram(�) �23.8 (4.0) �17.9 (4.4) �19.9 (3.3) �22.9 (3.4) �23.4 (3.5) �19.1 (5.1) �19.3 (6.2) �22.2 (4.4)Gram(þ) �24.2 (3.1) �21.4 (3.3) �22.0 (2.4) �23.4 (2.9) �22.8 (3.2) �20.6 (4.3) �21.8 (3.8) �22.0 (3.5)Fungi �29.4 (3.2) �27.8 (4.5) �14.6 (1.1) �28.5 (1.8) �25.7 (3.3) �20.9 (1.8) �16.2 (3.4) �23.0 (0.4)Bacteria �25.3 (3.1) �19.7 (1.6) �20.5 (2.4) �24.0 (1.9) �23.3 (1.5) �21.0 (3.4) �19.8 (3.9) �22.8 (2.1)

S. Steinbeiss et al. / Soil Biology & Biochemistry 41 (2009) 1301–13101310

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