Soil Biology & Biochemistry 40 (2008) 2977–2991

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Soil Biology & Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Quantitative assessment of the fungal contribution to microbial tissue in soil

Rainer Georg Joergensen*, Florian Wichern 1
Department of Soil Biology and Plant Nutrition, University of Kassel, Nordbahnhofstrasse 1a, 37213 Witzenhausen, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The fungi-to-bacteria ratio in soil ecological concepts and its application to explain the effects of land use
Received 11 April 2008 changes have gained increasing attention over the past decade. Four different main approaches for
Received in revised form 13 August 2008 quantifying the fungal and bacterial contribution to microbial tissue can be distinguished: (1) micro-
Accepted 16 August 2008
scopic methods, (2) selective inhibition, (3) specific cell membrane components and (4) specific cell wall
Available online 24 September 2008
components. In this review, the different methods were compared and we hypothesized that all these
approaches result in similar values for the fungal and bacterial contribution to total microbial biomass,
Keywords:
activity, and residues (dead microbial tissue) if these methods are evenly reliable for the estimation of
Direct microscopy
Selective inhibition fungal biomass. The fungal contribution to the microbial biomass or respiration varied widely between 2
Ergosterol and 95% in different data sets published over the past three decades. However, the majority of the
PLFA literature data indicated that fungi dominated microbial biomass, respiration or non-biomass microbial
16:1u5 residues, with mean percentages obtained by the different methodological approaches varying between
18:1u9 35 and 76% in different soil groups, i.e. arable, grassland, and forest soils and litter layers. Microscopic
18:2u6,9 methods generally gave the lowest average values, especially in arable and grasslands soils. Very low
Saprotrophic fungi ratios in fungal biomass C-to-ergosterol obtained by microscopic methods suggest a severe underesti-
Biotrophic fungi
mation of fungal biomass by certain stains. Relatively consistent ratios of ergosterol to linoleic acid
Glucosamine
(18:2u6,9) indicate that both cell membrane components are useful indicators for saprotrophic and
Muramic acid
ectomycorrhizal fungi. More quantitative information on the PLFA content of soil bacteria and the 16:1u5
content of arbuscular mycorrhizal fungi is urgently required to fully exploit the great potential of PLFA
measurements. The most consistent results have been obtained from the analysis of fungal glucosamine
and bacterial muramic acid in microbial residues. Component-specific d13C analyses of PLFA and amino
sugars are a promising prospect for the near future.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction
Soil microorganisms encompass archaea, bacteria, fungi, and
protozoa. They maintain the majority of enzymatic processes in soil
and preserve energy and nutrients in their biomass (Jenkinson and
Ladd, 1981). The diversity of soil microorganisms is enormous
(Torsvik et al., 1990) and the majority of species is still unknown,
although microbial diversity has received much research interest
and activity over the past decade (Torsvik and Øvreås, 2007). The
main approach is to measure the large diversity on a functional,
phenotype or genotype level (Emmerling et al., 2002). This has
been done by measuring soil enzymes (Kandeler et al., 1997),
phospholipid fatty acids (Tunlid and White, 1992; Zelles and Bai,
1993) or DNA sequences (Torsvik et al., 1990; Torsvik and Øvreås,
2002), respectively. For managing the large microbial diversity in
* Corresponding author. Tel.: þ49 5542 98 1591; fax: þ49 5542 98 1596.
E-mail address: joerge@uni-kassel.de (R.G. Joergensen).
1
Present address: KþS KALI GmbH, 34131 Kassel, Germany.
soil on the genotype level, the operational taxonomic units such as
the sequences for 16S rRNA (rDNA) seem to be replacing the species
concept (Torsvik and Øvreås, 2007), which has not been clearly
defined for microorganisms (Roselló-Mora and Amann, 2001).
Soil ecological concepts, for example to describe the interactions
of soil animals and soil microorganisms in food webs, prefer to
separate the microbial community into fungi and bacteria (Hedlund
et al., 2004; van der Putten et al., 2004; Coleman, 2008; Holtkamp
et al., 2008), which are the two largest functional microbial
subgroups in soil. Archaea and protozoa contribute only small
percentages to the soil microbial biomass, i.e. approximately 1 and
2%, respectively (Gattinger et al., 2002; Bardgett and Griffiths,
1997). The reason for separating the microbial community into
fungi and bacteria as important functional units is their different
behaviour in soil.
Fungal energy channels are considered as slow cycles, because
they are favoured by acidic soils low in available nutrients, recal-
citrant organic materials and high C/N ratio in soil, leading to
relatively long generation times (Blagodatskaya and Anderson,
1998; Högberg et al., 2007). Fungi have been shown to use organic

0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2008.08.017
2978 R.G. Joergensen, F. Wichern / Soil Biology & Biochemistry 40 (2008) 2977–2991
substrates more efficiently (Holland and Coleman, 1987; Sakamoto
and Oba, 1994), i.e. they form more biomass from the same amount
of substrate than bacteria. Also, increasing heavy metal contami-
nation, especially with Zn, Pb, and Ni, has been repeatedly observed
to promote fungi (Frostegård et al., 1996; Chander et al., 2001). The
same is true for a reduction in tillage and the resulting mulch layer
on the surface of arable soils (Beare et al., 1997; Frey et al., 1999;
Thiet et al., 2006). Most work has shown that fungal hyphae are
more resistant against further microbial decomposition than
bacterial polymers (Webley and Jones, 1971; Guggenberger et al.,
1999). Consequently, hyphae contribute significantly to soil
aggregation (Hu et al., 1995). For this reason, the promotion of fungi
might be an important tool to foster C sequestration in soil (Bailey
et al., 2002; Jastrow et al., 2007).
Belowground energy channels with a strong bacterial compo-
nent mainly occur in nutrient-rich soils that contain readily
decomposable substrate, promoting a rapid growth response,
turnover of carbon and a fast cycling of nutrients (Holtkamp et al.,
2008; Ingwersen et al., 2008). This might be a reason for the lower
substrate use efficiency of bacteria in comparison with fungi
(Holland and Coleman, 1987; Sakamoto and Oba, 1994). Relatively
more bacteria have been found in alkaline and saline soils, espe-
cially under semi-arid or arid conditions (Pankhurst et al., 2001).
On the other hand, waterlogging also promotes bacteria (Nakamura
et al., 2003). Increasing nutrient availability to the plants is often
combined with a higher organic matter input by plants with
a lower C/N ratio, which can promote bacterial biomass (Högberg
et al., 2003). Bacterial biomass is promoted by increasing land use
intensity, for example by fertilization (Högberg et al., 2003), tillage
(Beare et al., 1997; Frey et al., 1999) and livestock grazing of the
aboveground biomass (Bardgett et al., 1993).
The fungi-to-bacteria ratio in soil ecological concepts and its
application to explain the effects of land use changes have gained
increasing attention over the past decade (Hedlund et al., 2004; van
der Putten et al., 2004; Bardgett, 2005). For this reason, the
methods used to quantify the fungal and bacterial contribution to
the microbial biomass and to the microbial turnover demand
thorough evaluation. An uncritical use of the different methodo-
logical approaches may lead to misinterpretation of human influ-
ence on soil biological processes and wrong practices for
maintaining soil fertility in agricultural and forest ecosystems. Four
different approaches for quantifying the fungal and bacterial
contribution to the soil microbial community are distinguished in
this review: (1) microscopic methods, (2) selective inhibition, (3)
specific cell membrane components, and (4) specific cell wall
components. In this review, the different methods were compared
and we hypothesized that all these approaches result in similar
values for the fungal and bacterial contribution to total microbial
biomass, activity, and non-biomass microbial residues if these
methods are evenly reliable for the estimation of fungal biomass.
Possible deviations between the approaches might indicate varia-
tion in fungal and bacterial species, methodological flaws or
systematic errors, which should be considered or eliminated in the
future.
2. Approaches for estimating fungal and bacterial
biomass in soil
2.1. Direct microscopic methods
For decades, direct microscopic methods represented the only
possibility for the determination of bacterial and fungal biomass in
soil (Jenkinson and Ladd, 1981). These methods are all based on the
principle that a known amount of a homogenized soil suspension is
placed on a known area of a microscopic slide, the bacteria or fungi
are stained and numbers are counted using a microscope.
Biovolumes and biomass must be estimated from lengths and
widths of the microorganisms. Extensive reviews of microscopic
methods in microbial ecology have been given recently by Hart-
mann et al. (2004) and Bölter et al. (2006).
Soil bacteria have been counted after staining using soil smears
on glass slides (Bloem et al., 1994) or membrane filters (Sakamoto
and Oba, 1994). The stains used solely for bacteria were acridine
orange, which stains nucleic acids (West, 1986), fluorescein iso-
thiocyanate (FITC) (Shields et al., 1973) and 5-(4,6-dichlorotriazin-
2-yl)-aminofluorescein (DTAF), both of which stain proteins (Bloem
et al., 1995). Soil fungi have been counted after staining in agar films
(Jones and Mollison, 1948; Lin and Brookes, 1999a) or on membrane
filters (Bloem et al., 1995). The fluorescent brightener calcofluor
white (West, 1986) has been used as a most specific stain for fungi.
Several stains have been used for bacteria and fungi such as phenol
aniline blue (Arao et al., 2001) and phenolic tryptophan blue (Lin
and Brookes, 1999a), which stain the polysaccharides of cell walls,
fluorescein diacetate (West, 1986), and europium chelate/fluores-
cent brightener (calcofluor white) differential stain (DFS) (Bloem
et al., 1994). Europium chelate stains DNA and RNA, and calcofluor
white stains the polysaccharides of cell walls (Bloem et al., 1995).
Most of the methods can be applied to conventional visual
microscopy, although in most cases an epifluorescence microscope
was used, in some cases combined with differential interference
phase-contrast microscopy (Klein et al., 1995). The use of a confocal
laser-scanning microscope (CLSM) offers an extended depth of
focus, better contrast due to the suppression of stray light and less
bleaching of fluorescent dye (Bloem et al., 1995).
Weighted means for the fungal part of the total microbial
biomass are 37% for arable soils, 35% for grassland soils, 47% for
forest soils and 64% for litter layers (Table 1; Fig. 1a). The coefficient
of variation for litter layers was exceptionally low at 2%, but only
very few scientists contributed data to this group. A larger variety of
methods apparently led to a much larger range for the fungal part
of the total microbial biomass. In the group of arable soils the range
was 2 (Bloem et al., 1994) to 86% (Shields et al., 1973) and in the
group of grassland soils 2 (Hassink et al., 1993) to 85% (Lin and
Brookes, 1999a). Lowest values in these two soil groups were
always obtained from scientists working on soils in the Nether-
lands. This has been explained by the general low level of fungi in
the clayey soils of the country, due to their recent formation from
marine sediments (Velvis, 1997). This might be true, but indirect
methods such as selective inhibition, although rarely applied, led to
more than 15-fold higher percentages (Table 2: Velvis, 1997) than
the direct microscopic approach (Table 1: Velvis, 1997). Also the
ratio of fungal biomass C-to-ergosterol using DSF staining for
estimating fungal biomass suggests a strong 10-fold underestima-
tion of this eukaryotic functional group based on microscopic
counts (Table 3: van der Wal et al., 2006; de Vries et al., 2007).
These deviations from other methods often remained unrecognised
and the underlying methodological constraints have often been
neglected, although the basic problems of direct microscopic
methods have been carefully presented by Domsch et al. (1979) and
Jenkinson and Ladd (1981).
However, soil microbiologists still face the same problems as in
the late seventies that microbial biomass estimates by direct
microscopy suffer from the separation of live and dead cells, with
a strong tendency nowadays towards exclusion of an unknown
proportion of living cells. Dormant microorganisms may react
differently to certain stains than actively growing microorganisms.
Other sources of error are the differentiation between living
microorganisms and soil particles, the conversion of area to volume
and especially to dry matter (Domsch et al., 1979; Jenkinson and
Ladd, 1981) as investigator effects on the data (Stahl et al., 1995). For
these reasons, the total microbial biomass levels obtained by direct
microscopy can be very low in comparison with those obtained by
 R.G. Joergensen, F. Wichern / Soil Biology & Biochemistry 40 (2008) 2977–2991 2979

Table 1
Contribution of fungi to the soil microbial biomass in percent based on microscopic methods; in brackets: number of observations as taken from figures and tables of the listed
references multiplied by the respective replicate samples

Author Arable Grassland Forest Litter Staining (fungi/bacteria)

Shields et al. (1973) 86 (15) – – – PAB/FITC
West (1986) 75 (5) – 58 (10) – CW/AO
Ingham and Horton (1987) 9 (10) – – – FDA/FITC
Neely et al. (1991) – – – 64 (72) FDA/FITC
Hassink et al. (1991) 5 (36) – – – CW/DFS
Hassink et al. (1993) – 2 (54) – – CW/DFS
Bloem et al. (1994) 2 (6) – – – CW/DFS
Sakamoto and Oba (1994) 61 (60) – – – PAB/AO
Klein et al. (1995) – 59 (96) – – FDA/FITC
Velvis (1997) 12 (6) – – 83 (1) CW/DTAF
Klein et al. (1998) – 5 (64) – – FDA/FITC
Frey et al. (1999) 30 (72) – – – CW/DTAF
Lin and Brookes (1999a) – 85 (6) – – PTB/PTB
Morris and Boerner (1999) – – 33 (209) – DFS/DFS
Tiunov and Scheu (1999) – – 73 (48) – CW/AO
Tiunov and Scheu (2000) – – 77 (10) 64 (10) CW/AO
Tiunov et al. (2001) – – 81 (20) – CW/AO
Lipson et al. (2002) – 89 (9) – – FDA/FDA
Tiunov and Dobrovolskaya (2002) – – 90 (12) – CW/AO
van der Wal et al. (2006) – 38 (29) – – DFS/DTAF
de Vries et al. (2007) – 23 (48) – – DFS/DTAF
Holtkamp et al. (2008) – 51 (64) – – DFS/DTAF
Weighted mean (number) 37 (210) 35 (370) 47 (309) 64 (83)
Standard deviation 26 25 20 2

– ¼ No information; CW ¼ calcofluor white; AO ¼ acridine orange; FDA ¼ fluorescein diacetate; FITC ¼ fluorescein isothiocyanate; DFS ¼ europium chelate/fluorescent
brightener (¼CW) differential stain; PAB ¼ phenol aniline blue; PTB ¼ phenolic tryptophan blue; DTAF ¼ 5-(4,6-dichlorotriazin-2-yl)-aminofluorescein.

fumigation extraction (Klein et al., 1995, 1998). However, Lin and
Brookes (1999a), like others, achieved concordance between these
two approaches. It should be considered that small modifications in
sample handling or experimental conditions can have unexpected
effects on the results of a certain method (Powlson, 1994).
The combination of microscopic methods with automated
image analysis has considerably increased the use of this approach
over the past years, especially in the Netherlands (van der Wal et al.,
2006; de Vries et al., 2007; Holtkamp et al., 2008). Microscopic
techniques are able to give important information about the in situ
localization of soil microorganisms, creating a strong and contin-
uous motivation for many soil biologists in the future (Hartmann
et al., 2004).
2.2. Selective respiratory inhibition
The selective inhibition method (Anderson and Domsch, 1973)
has been widely used to estimate the relative contribution of fungi
and bacteria to glucose induced respiration in soil. This assay is
based on the stimulation of the overall metabolism of soil micro-
organisms by adding glucose. Simultaneously, subsamples of soil
are amended with inhibitors specific for fungi and bacteria. In most
experiments (Table 2), cycloheximide was used to inhibit fungi and
streptomycin to inhibit bacteria. The basic idea of the selective
inhibition method is persuasive and elegant. Although the method
was initially described for a system continuously monitoring soil
respiration (Anderson and Domsch, 1973), modifications have been
developed using simple titration equipment (Lin and Brookes,
1999a).
The initial maximal respiration induced by glucose is propor-
tional to the size of the original soil microbial biomass (Anderson
and Domsch, 1978) but the quantity of glucose added to achieve
maximal respiration rate varies greatly between 500 and
8000 mg g1 soil, depending on soil chemical and physical proper-
ties, and should be determined for each soil (Anderson and
Domsch, 1973, 1975; Lin and Brookes, 1999a). Also the concentra-
tion of inhibitors to achieve optimum inhibition varies over a large
range between 500 and 4000 mg g1 soil. Furthermore, an inhibitor
has to be used which is selective to a targeted community and not
unspecifically inhibiting with increasing inhibitor concentration.
Then, the percentage inhibition of the respiration can be calculated
as follows: (A  D)/A  100, where A is the respiratory response of
the soil after addition of glucose in the absence of inhibitors and D
is the respiration of the samples with the combined fungal and
bacterial inhibitors. The inhibitor additivity ratio (IAR) is calculated
as ((A  B) þ (A  C))/(A  D), where B is the respiratory response
with the fungal inhibitor and C that with the bacterial inhibitor
(Beare et al., 1990). The IAR should be 1.0  5% to be able to exclude
synergistic (IAR < 1.0) as well as overlapping and non-target effects
of the antibiotics (IAR > 1.0) (Anderson and Domsch, 1975; Scheu
and Parkinson, 1994).
However, the IAR was often not tested or information on IAR
was not presented. Streptomycin and cycloheximide have repeat-
edly been associated with antagonistic and overlapping effects, but
also with problems regarding selectivity (Velvis, 1997; Bailey et al.,
2002, 2003). Incomplete inhibition might be due to adsorption of
the inhibitors on the soil colloids (Badalucco et al., 1994), especially
soil organic matter (Alphei et al., 1995) or due to resistance against
the inhibitors (Heilmann et al., 1995). If fungal respiration and
bacterial respiration are considered as indices for biomass, the
assumptions should be valid that fungi and bacteria respond
similarly to glucose, and the inhibitor insensitive part of the
microbial population has a similar fungi-to-bacteria biomass ratio
to the inhibitor sensitive part. Roughly 54% of the soil microbial
biomass has been shown to be responsive to glucose addition (Wu
et al., 1993). Less is known about differences in the respiratory
response of bacteria and fungi to glucose additions (Anderson and
Domsch, 1978).
Weighted means of the fungal respiration to the total substrate
induced respiration are 51% for arable and grassland soils, 65% for
forest soils and 68% for litter layers (Table 2, Fig. 1b). It is surprising
that similar percentages were found in arable and grassland soils,
because tillage and fertilization are usually considered as reasons
for a decline in fungal biomass. Due to the methodological
constraints explained above, Bailey et al. (2003) suggested pre-
testing combinations of different antibiotics in various
2980 R.G. Joergensen, F. Wichern / Soil Biology & Biochemistry 40 (2008) 2977–2991
Fig. 1. Box-plots (median, 25% quartiles, and outliers) for the contribution of fungi in
percent (a) to the soil microbial biomass by microscopic methods, (b) to the soil
microbial respiration by selective inhibition, and (c) to the soil microbial residue C,
recalculated according to Appuhn and Joergensen (2006) and Engelking et al. (2007),
based on the measurements of glucosamine and muramic acid.
concentrations as an improvement. However, the results of Bailey
et al. (2002) for arable soils in North America with captan as an
inhibitor of fungal respiration and oxytetracycline hydrochloride as
an inhibitor of bacterial respiration are not conclusively better in
comparison to others (Table 2). Cycloheximide not only reduced the
respiratory response of soil microorganisms, but also nitrification
(Landi et al., 1993), deamination of arginine (Lin and Brookes,
1999b) and the ATP concentration as well as the adenylate energy
charge (Raubuch et al., 2006). However, none of the cycloheximide
effects on these processes was clearly related to the percentage of
fungal biomass in soil.
The selective inhibition technique is the only approach of the
four presented that is not biased by the difficulties in separating live
and dead microorganisms. For this reason, the selective inhibition
technique is still useful for giving a first approximate estimate of
the fungal and bacterial part of the microbial biomass on the basis
of a close relationship between biomass and glucose induced
respiration (Anderson and Domsch, 1978; Miura et al., 2008).
Anderson and Domsch (1975) presented their data in 5% steps,
indicating that the precision of the data was not very high. This
means that the selective inhibition method is unable to detect small
shifts in the microbial community structure. This is an important
drawback, as environmental variables such as tillage (Frey et al.,
1999), climate (Guggenberger et al., 1999) or changes in land use
(Amelung et al., 2002) have only small effects on the percentage of
fungal and bacterial components, but a strong influence on
ecosystem functions.
2.3. Ergosterol
Ergosterol is the predominant sterol in fungal cell membranes,
highly specific and endogenous only in higher fungal phyla, i.e.
Basidiomycota, Ascomycota, and the majority of Zygomycota
(Weete and Ghandi, 1996; Weete and Gandhi, 1999; Klamer and
Bååth, 2004). As a constituent of cell membranes, ergosterol
controls their permeability, micro-viscosity and the activity of
membrane-bound enzymes (Peacock and Goosey, 1989). Ergosterol
has been used to detect fungal invasion and spoilage of food and
cereal grains (Seitz et al., 1977), fungal infection of wood and other
plants (Newell, 1992), and the development of ectomycorrhiza
(Martin et al., 1990). Ergosterol has been increasingly used as
a biomarker for fungi in soil over the past two decades (West et al.,
1987; Montgomery et al., 2000; Engelking et al., 2008). Ergosterol
does not occur in vascular plants and in most algae (Newell, 1992;
Newell et al., 1996). Beside fungal cell membranes, ergosterol has
been found only in rare microalgae such as Prototheca wickerhamii
(Sud and Feingold, 1979). Consequently, ergosterol has also been
successfully used as an indicator for fungal biomass in marine
ecosystems in the presence of higher algae populations (Newell
et al., 1996; Collier et al., 2004).
A large variety of different methods are available for the
extraction of ergosterol using alkaline mixtures of methanol and
ethanol (Zelles et al., 1987), pure methanol (Gong et al., 2001), pure
ethanol (Grant and West, 1986; Djajakirana et al., 1996) followed,
for example, by liquid extraction with hexane (Davis and Lamar,
1992) or solid phase extraction (Gessner and Schmitt, 1996).
Attempts have been made to improve extraction efficiency by ultra-
sonification (Grant and West, 1986), physical disruption with glass
beads (Gong et al., 2001), or microwave irradiation (Young, 1995).
However, the differences in the results between the extraction
procedures are small if the methods have been carried out properly.
After extraction, filtration and concentration, the majority of the
data was obtained by isocratic reversed-phase HPLC followed by UV
detection at 282 nm, where no other substances are co-chroma-
tographing. Ergosterol can also be determined according to Bligh
and Dyer (1959) together with PLFA and other fatty acids from soil
lipids, purified with silicic acid chromatography, recovered from
the neutral lipid fraction, and analysed by capillary gas chroma-
tography (Tunlid and White, 1992; McKinley et al., 2005). This
procedure is especially useful for component-specific d13C analysis
(Malosso et al., 2004). Not only the simple measurement tech-
niques, but also the possibility of storing samples frozen for certain
 R.G. Joergensen, F. Wichern / Soil Biology & Biochemistry 40 (2008) 2977–2991 2981

Table 2
Contribution of fungi to the soil microbial respiration in percent based on selective inhibition; in brackets: number of observations as taken from figures and tables of the listed
references multiplied by the respective replicate samples

Author Arable Grassland Forest Litter Inhibitor

Anderson and Domsch (1973) 78 (6) – – – C/S
Anderson and Domsch (1975) 76 (20) – 70 (10) 70 (5) C/S
Vančura and Kunc (1977) 73 (8) – – – C/S
Parkinson et al. (1978) – – – 75 (20) C/S
Domsch et al. (1979) 75 (18) 70 (6) 72 (12) – C/S
Nakas and Klein (1980) – 41 (16) – – C/S
Bewley and Parkinson (1984) – – 73 (14) 89 (16) C/S
West (1986) – – 52 (8) – C/S
Wardle and Parkinson (1990) 75 (6) – – – C/S
Beare et al. (1990) – – – 68 (18) C/S
Yang and Insam (1991) – – 27 (9) – C/S
Neely et al. (1991) – – – 61 (72) C/S
Tate (1991) – – 69 (3) 82 (3) C/S
Scheu and Parkinson (1994) – – 53 (3) 62 (12) C/S
Alphei et al. (1995) – – 56 (6) – C/S
Bardgett et al. (1996) – 36 (12) – – C/S
Johnson et al. (1996) 95 (6) – – – C/S
Velvis (1997) 35 (3) – 22 (1) – C/S
Vishnevetsky and Steinberger (1997) – 67 (72) – – C/S
Blagodatskaya and Anderson (1998) – – 86 (40) – C/S
Lin and Brookes (1999a) – 80 (6) – – C/S
Mamilov et al. (2001) – – 81 (3) – C/S
Imberger and Chiu (2001) – 58 (12) 61 (8) 56 (32) C/S
Imberger and Chiu (2002) – – 52 (32) – C/S
Bailey et al. (2002) 64 (6) 55 (12) 67 (12) – CA/OT
Lipson et al. (2002) – 79 (6) – – C þ N/E þ S
Bailey et al. (2003) – 60 (9) 44 (3) – CA/BR
Nakamoto and Wakahara (2004) 55 (18) – – – C/CL
Miura et al. (2008) 53 (120) – – – C/CL
Weighted mean (number) 61 (211) 61 (151) 65 (164) 68 (146)
Standard deviation 12 12 16 9

– ¼ No information; fungal inhibitors: C ¼ cycloheximide, CA ¼ captan; N ¼ nystatin; bacterial inhibitors: BR ¼ bronopol, CL ¼ chloramphenicol, E ¼ erythromycin,
OT ¼ oxytetracycline–HCl, S ¼ streptomycin..

periods before measurement is an important advantage of ergos- probably large, especially in grassland soils (Oehl et al., 2003). The
terol analysis (Davis and Lamar, 1992; Gessner and Schmitt, 1996). mycelium of the Glomeromycota also does not contain ergosterol
The family of Mortierellaceae within the fungal phylum Zygo- (Olsson et al., 2003). This is a serious drawback for the estimation of
mycota, pioneer colonisers of litter, does not produce ergosterol total fungal biomass in soil, but might be useful for separating
(Weete and Gandhi, 1999), but their biomass in the C-limited soil is biotrophic and saprotrophic fungi in arable and grassland soils
probably small. In contrast, the biomass of the fungal phylum (Scheller and Joergensen, 2008). Other drawbacks repeatedly
Glomeromycota, i.e. the arbuscular mycorrhizal fungi (AMF), is mentioned are the species-specific variation in ergosterol content
(Djajakirana et al., 1996) due to differences in hyphal morphology
and hyphal diameter (Klamer and Bååth, 2004) as well as due to
Table 3 growth conditions and mycelium age (Newell et al., 1987). In pre-
Ratio of fungal biomass C-to-ergosterol in cultivated fungi and soils from different incubated and C-limited soils, where only a small part of the
land use forms; number of observations as taken from figures and tables of the listed complex fungal community is actively growing, the effects of
references without replicates species-specific hyphal morphology and growth conditions on the
Authors Fungal biomass Method Number of fungal ergosterol content are probably smaller (Montgomery et al.,
C/ergosterol observations, 2000). This is most likely also true for the risk of incomplete
(g g1) land use form extraction, which was observed by Montgomery et al. (2000) after
Stahl et al. (1995) 90 DM, CW 3, Grassland the addition of cultured fungi to the soil.
Stahl and Parkin (1996) 50 DM, FDA 7, Arable, grassland, forest
Although ergosterol is not accumulated in organic matter in the
Djajakirana et al. (1996) 90 Cultivation 42
Nemec et al. (1997) 135 Cultivation 6, Aspergillus sp. long-term (Davis and Lamar, 1992; Nylund and Wallander, 1992;
Montgomery et al. (2000) 120 Cultivation 6, Soil and plant fungi Engelking et al., 2008), there is a possibility that a significant amount
Imberger and Chiu (2001) 100a SI 17, Forest, grassland of ergosterol is accumulated in dead fungal cells for certain periods
Imberger and Chiu (2002) 95a SI 18, Forest, litter in incubation experiments with large additions of organic substrates
Klamer and Bååth (2004) 190 Cultivation 24, Compost fungi
Appuhn et al. (2006) 410 FG 6, Arable, grassland
and a resulting strong turnover of the fungal biomass (Mille-Lind-
Zhao et al. (2005) 250 DM 28, Arable blom et al., 2004; Zhao et al., 2005; Engelking et al., 2008). Ergosterol
Zhao et al. (2005) 290 SI 28, Arable concentrations exceeding 3% of microbial biomass C in litter layers of
van der Wal et al. (2006) 16 DM, DSF 3, Grassland forest soils (Smolander et al., 1994; Joergensen and Scheu, 1999)
de Vries et al. (2007) 13 DM, DSF 2, Grassland
might be attributed to the very fine fungal mycelium in litter layers,
DM ¼ direct microscopy, CW ¼ calcofluor white, FDA ¼ fluorescein diacetate, but may also indicate a certain accumulation of dead fungal cells.
DFS ¼ europium chelate/fluorescent brightener (¼CW) differential stain; Also the spatial variation of ergosterol in soil can be a problem, as
SI ¼ selective inhibition; FG ¼ fungal glucosamine; fungal biomass was recalculated
into fungal biomass C assuming a C content in fungi of 47% (Jenkinson and Ladd,
patches occur with high fungal colonization and high ergosterol
1981). contents close to those with low fungal colonization and low
a
Geometric mean. ergosterol contents (Möttönen et al., 1999).
 2982 R.G. Joergensen, F. Wichern / Soil Biology & Biochemistry 40 (2008) 2977–2991

The average fungal biomass C-to-ergosterol ratio ranged from 13
to 410 (Table 3). This huge range is mainly caused by the different
approaches to estimate fungal biomass C. As explained earlier, the
direct microscopic approach of van der Wal et al. (2006) and de
Vries et al. (2007) led to severe underestimation of fungal biomass
C, and the amino sugar approach of Appuhn et al. (2006) includes
arbuscular mycorrhizal fungi, which do not contain ergosterol
(Olsson et al., 2003). The average ratio of ergosterol-to-18:2u6,9
ranged from 0.5 to 2.6 (Table 4). As explained in Section 2.4, diffi-
culties sometimes occur in gas chromatographic separation and
detection of 18:2u6,9, which results in unsound low 18:2u6,9
values (Joergensen and Potthoff, 2005). This might be the reason for
the three values above 1.9 (Table 4). The exclusion of these values
resulted in a weighted mean of 0.6, varying in a rather narrow range
from 0.5 to 0.8.
Due to the unique specificity, ergosterol will remain an impor-
tant indicator for changes in fungal biomass and shifts in the fungal
community structure, especially if the interactions of plants, sap-
rotrophic fungi and soil animals are to be analysed (Bonkowski,
2004). However, for estimating the exact contribution of fungi to
the total microbial biomass, ergosterol data are less useful on their
own, especially due to the unknown contribution of AMF to the soil
fungal biomass. For this reason, ergosterol as a fungal biomass
indicator in soil needs to be strengthened by other biomarkers such
as 16:1u5c, 18:1u9, 18:2u6,9 or fungal glucosamine.
2.4. Phospholipid fatty acids
In all organisms except archaea, phospholipid fatty acids (PLFA)
are the main components of cell membranes and do not occur in
storage components (Tunlid and White, 1992; Zelles and Bai, 1993).
In archaea, PLFA are replaced by phospholipid ether lipids (PLEL)
(Gattinger et al., 2002). PLFA are rapidly synthesized during
microbial growth and quickly decomposed after microbial cell
death (Tunlid and White, 1992; Zelles and Bai, 1993), indicating
a fast turnover of microbial PLFA. For this reason, they are usually
not accumulated in organic matter (Tunlid and White, 1992). In soil,
the total concentration of PLFA gives information on the microbial
biomass (Frostegård et al., 1991; Joergensen and Emmerling, 2006),
and composition of signature components gives information on the
microbial community structure (Tunlid and White, 1992; Fros-
tegård et al., 1993).
PLFA are in most cases extracted with different modifications of
Bligh and Dyer (1959) procedure followed by purification with
silicic acid chromatography (Frostegård et al., 1991) and detection
with capillary gas chromatography (Tunlid and White, 1992).
Depending on the concentration of the PLFA and the extent of
extract fractionation, between 30 and 80 PLFA have been identified
and summarized as total PLFA. In contrast to microbial biomass
measurements, limited advice is available concerning the sample
pre-treatment, such as moisture adjustment, sieving and pre-
incubation (Federle and White, 1982; Petersen and Klug, 1994).
Sieving or even grinding has been found to be advisable to
homogenise soil, to remove interfering plant residues and to ach-
ieve maximum PLFA yields (Allison and Miller, 2005; Grigera et al.,
2007). Also the soil moisture content is most likely a critical point
for extraction efficiency, but experimental evidence is missing. In
the majority of the references listed in Table 6, information on the
soil conditions during PLFA extraction was vague or lacking. The
most likely explanation is that no further pre-treatment was
carried out after sampling in these cases. Similar to ergosterol
analysis, the soil samples might be stored frozen before extraction
(Federle and White, 1982; McKinley et al., 2005; Wagner et al.,
2007), but also the extracts can be stored for extended periods in
the freezer (Allison and Miller, 2005).
One major advantage of PLFA measurements is the possibility of
separating large functional groups within the soil microbial
community. Gram-negative bacteria contain more hydroxylated
and cyclopropyl fatty acids (Zelles, 1997). Gram-positive bacteria
contain more fatty acids that are branched on positions other than
iso and anteiso (Zelles, 1997). Actinomycetes can be characterized
by ester-linked 10-methyl branched saturated fatty acids (McKinley
et al., 2005; Williams and Rice, 2007). As a signature fatty acid for
saprotrophic and ectomycorrhizal fungi belonging to phyla Zygo-
mycota, Ascomycota and Basidiomycota, mainly linoleic acid
(18:2u6) has been used (Table 5). Also, oleic acid (18:1u9) and
sometimes g-linolenic acid (18:3u6) have been used as fungal
biomarkers (Bååth and Anderson, 2003; Potthoff et al., 2006).
However, oleic acid (Ruess et al., 2007) and g-linolenic acid (Arao
et al., 2001) have been used to estimate the error introduced by
plant material in other cases. As an indicator for arbuscular
mycorrhizal fungi (AMF, phylum Glomeromycota), 16:1u5 has been
successfully applied (Olsson et al., 1995).
The data in Table 5 give a weighted mean of 41 to convert nmol
total fungal PLFA to mg fungal biomass C, a weighted mean of 107 to
Table 5
Ratios of fungal biomass C to 16:1u5, indicator for arbuscular mycorrhizal fungi, to
linoleic acid (18:2u6,9), indicator for fungal biomass, or to total PLFA extracted from
soil fungi and different cultivated fungi; number of observations as taken from
figures and tables of the listed references without replicates
Fungal Number of
biomass observations (n),
C/PLFA (mg nmol1) fungal group

Table 4 16:1u5
Ratio of ergosterol to the phospholipid fatty acid linoleic acid (18:2u6,9), indicators Johansen et al. (1996) 313 1, AMF hyphae
for fungal biomass, in different soils and in cultivated fungi; number of observations Olsson and Johansen (2000) 323 2, AMF hyphae
as taken from figures and tables of the listed references without replicates Olsson and Wilhelmsson (2000) 390 4, AMF hyphae
Weighted mean 366 7
Authors Ergosterol Number of
/18:2u6,9 observations 18:2u6,9
(mg nmol1) (n), land use form Arao et al. (2001) 110 38, soil fungi
a Klamer and Bååth (2004) 85 24, cultured fungi
Frostegård and Bååth (1996) 0.8 15, Forest, grassland
Korkama et al. (2007) 160 8, ECMF hyphae
Bååth and Anderson (2003) 0.6 35, Forest
Weighted mean 107 70
Klamer and Bååth (2004) 0.5 24, Cultivated fungi
Hackl et al. (2005) 0.6 54, Forest Total PLFA
Joergensen and Potthoff (2005) 2.6 3, Arable Johansen et al. (1996) 77 1, AMF hyphae
Högberg (2006) 2.0a 9, Forest Olsson and Johansen (2000) 56 2, AMF hyphae
van der Wal et al. (2006) 0.5 3, Grassland Olsson and Wilhelmsson (2000) 57 1, AMF hyphae
Butenschoen et al. (2007) 1.9 6, Arable Klamer and Bååth (2004) 38 24, Cultured fungi
Demoling et al. (2008) 0.5 6, Forest Korkama et al. (2007) 46 8, ECMF hyphae
Weighted meanb 0.6 137 Weighted mean 42 36
a
Geometric mean. AMF ¼ arbuscular mycorrhizal fungi; ECMF ¼ ectomycorrhizal fungi; fungal biomass
b
Without the data of Joergensen and Potthoff (2005), Högberg (2006), and was recalculated into fungal biomass C assuming a C content in fungi of 47% (Jen-
Butenschoen et al. (2007). kinson and Ladd, 1981).
 R.G. Joergensen, F. Wichern / Soil Biology & Biochemistry 40 (2008) 2977–2991 2983

convert nmol linoleic acid (18:2u6,9) to the biomass C of sapro-
trophic (þ ectomycorrhizal) fungi and a weighted mean of 345 to
convert nmol 16:1u5c to biomass C of arbuscular mycorrhizal fungi.
The weighted mean concentration of linoleic acid (18:2u6,9) was
2.5 mol% in arable soils (Table 6, Fig. 2a), 3.1 mol% in grassland soils
and markedly higher at 6.2 mol% in the A horizon or at 10.7 mol% in
litter layers of forest soils. The standard deviation of each mean is
large, leading to an average coefficient of variation of 55%. The
weighted mean concentration of oleic acid (18:1u9) was 7.7 mol%
in arable soils (Table 6; Fig. 2b), 4.6 mol% in grassland soils,
8.4 mol% in the A horizon and 10.9 mol% in litter layers of forest
soils. It has to be mentioned that the weighted means of these four
soil groups might be affected by the fact that scientists are
specialized in investigation of specific ecosystems. No comparisons
are available on the PLFA structure of different land use forms in
contrast to the selective inhibition technique or direct microscopy
(Domsch et al., 1979).
Joergensen and Emmerling (2006) presented a weighted mean
of 5.8 for converting nmol PLFA into mg microbial biomass C. Then,
the plausibility of the mean values presented above can be tested
by the following calculation: if a soil contains 580 mg microbial
biomass C g1 soil, independently of the form of land use or soil
horizon, the total PLFA content would be 100 nmol g1 soil. Then,
the mean mol% of 18:2u6,9 in the four different soil groups (Table 6)
could be converted by multiplying with the mean fungal biomass
C/18:2u6,9 ratio of 107 (Table 5), which would result in 270 (arable
soils), 330 (grassland soils), 660 (forest soils), and 1140 (litter layers)
mg fungal biomass C g1 soil or 47, 57, 114 and 197% of total microbial
biomass C, respectively. If the sum of 18:2u6,9 and 18:1u9 (Table 6)
is converted with the mean fungal biomass C/total PLFA ratio of 42

Table 6
Concentrations of 16:1u5, indicator for arbuscular mycorrhizal fungi, oleic acid (18:1u9) and linoleic acid (18:2u6,9), indicators for fungal biomass, in mol% of total phos-
pholipid fatty acids (PLFAs) extracted from soil and cultivated fungi; in soil, peat and litter samples: number of observations as taken from figures and tables of the listed
references multiplied by the respective replicate samples; in cultivated fungi: number of observations as taken from figures and tables of the listed references without
replicates

Authors 16:1u5 (mol% total PLFA) 18:1u9 (mol% total PLFA) 18:2u6,9 Number of observations (n), land use form
Frostegård et al. (1993) 3.8 12.3 13.2 28, Forest
Borga et al. (1994) – 10.9 2.4 22, Peat
Bardgett et al. (1996) – – 3.3 12, Grassland
Frostegård and Bååth (1996) – – 4.4 15, Arable, grassland, forest
Frostegård et al. (1996) 3.5 – 8.6 6, Arable, forest
Bardgett et al. (1997) – – 5.6 128, Grassland
Yeates et al. (1997) – – 0.9 30, Grassland
Bardgett et al. (1999) – 3.6 1.0 64, Grassland
Pennanen et al. (1999) – – 16.2 44, Litter
Calderón et al. (2000) – – 2.7 8, Arable, grassland
Kandeler et al. (2000) – – 6.7 6, Arable
Schmidt et al. (2000) – – 6.9 60, Litter
Yao et al. (2000) – – 3.8 24, Arable, grassland, forest
Bargdett et al. (2001) – – 2.9 36, Grassland
Grayston et al. (2001) – – 4.8 27, Grassland
Zeller et al. (2001) – – 2.0 18, Grassland
Gattinger et al. (2002) – – 4.4 9, Arable
Merilä et al. (2002) 1.8 7.0 4.8 20, Forest
Ponder and Tadros (2002) 1.4 0.4 5.1 15, Forest
Perkiömäki et al. (2003) 1.9 13.2 18.3 20, Forest
Certini et al. (2004) – – 3.9 24, Forest
Grayston et al. (2004) – – 2.8 90, Grassland
Leckie et al. (2004) – – 6.7 24, Litter
Spedding et al. (2004) – – 2.3 144, Arable
Hackl et al. (2005) 3.6 7.4 3.1 120, Forest
Hernesmaa et al. (2005) – – 8.5 18, Forest
Janus et al. (2005) – 6.4 2.5 12, Forest
Joergensen and Potthoff (2005) 4.1 8.3 0.5 12, Arable
McKinley et al. (2005) 4.6 7.3 2.9 24, Grassland
Smithwick et al. (2005) 0.7 2.3 0.7 20, Forest
Högberg et al. (2007) – 12.5 9.1 39, Forest
Toyota and Kuninaga (2006) 1.8 6.7 2.6 8, Arable
Elfstrand et al. (2007) – – 2.0 40, Arable
Marhan et al. (2007) – – 2.5 12, Forest
Ruess et al. (2007) – 11.7 14.9 18, Litter, forest
Williams and Rice (2007) – – 1.4 40, Grassland
Demoling et al. (2008) – – 7.4 48, Forest
Gordon et al. (2008) – – 0.2 16, Grassland
Arable, mean (SD) 3.5 (1.3) 7.7 (0.8) 2.5 (1.1) 23, 20, 193
Grassland, mean (SD) 4.6 (0.0) 4.6 (1.7) 3.1 (1.8) 24, 88, 508
Forest, mean (SD) 2.9 (1.1) 8.4 (3.6) 6.2 (4.4) 224, 283, 397
Litter, mean (SD) – 10.9 (0.0) 10.7 (5.1) 0, 9, 137
Dembitsky et al. (1992) – 11.3 51.3 9, BAM
Stahl and Klug (1996) a – 41.7 13.2 6, ZYM
Stahl and Klug (1996) a – 22.0 50.8 17, ASM, BAM
Nemec et al. (1997) a – 15.6 50.6 6, Aspergillus sp
Ruess et al. (2002) – 27.2 50.4 15, ASM, BAM
Klamer and Bååth (2004) – 39.0 15.2 2, ZYM
Klamer and Bååth (2004) – 15.4 47.9 22, ASM, BAM
Korkama et al. (2007) – – 28.8 8, ECMF hyphae

– ¼ No information.
a
Total fatty acids, SD ¼ standard deviation; BAM ¼ Basidomycota; ZYM ¼ Zygomycota; ASM ¼ Ascomycota; ECMF ¼ ectomycorrhizal fungi.
2984 R.G. Joergensen, F. Wichern / Soil Biology & Biochemistry 40 (2008) 2977–2991
Fig. 2. Box-plots (median, 25% quartiles, and outliers) for the concentrations in mol%
of total phospholipid fatty acids (PLFAs) extracted from soil (a) for linoleic acid
(18:2u6,9), indicator for soil fungi, (b) for oleic acid (18:1u9), indicator for soil fungi,
and (c) for 16:1u5, indicator for arbuscular mycorrhizal fungi.
(Table 5), fungal biomass C would be 430 (arable soils), 320
(grassland soils), 610 (forest soils), and 910 (organic layers) mg g1
soil, equivalent to 74, 55, 105 and 157% of total microbial biomass C,
respectively. Similarly to ergosterol, the PLFA contents in forest
A horizons, but especially in litter layers, suggest the occurrence of
very fine fungal mycelium, but may also indicate a certain accu-
mulation of dead fungal cells as observed by Arao et al. (2001) in
an incubation experiment. In this calculation approach, the
contribution of arbuscular mycorrhizal fungi to arable and grass-
land soils has been neglected. The weighted mean concentration of
16:1u5 was 3.1 mol% in all soils (Table 6, Fig. 2c). If this PLFA
were converted to AMF biomass C using the conversion factor of
366 (Table 5), AMF biomass C would be 1130 mg g1 soil, suggesting
a 5- to 10-fold overestimation. This indicates the urgent need for the
determination of more accurate concentrations of 16:1u5 in AMF
hyphae.
If the microbial biomass of an arable soil consists of 74%
(¼430 mg fungal biomass C g1 soil; see previous paragraph) fungi
with a total mol% of 10.2 (18:2u6,9 þ 18:1u9) and 26%
(¼150 mg bacterial biomass C) bacteria with a mol% of 89.8, the ratio
of mg bacterial biomass C to bacterial nmol PLFA would be 1.8 and
that of mg fungal biomass C to fungal nmol PLFA would be 42, i.e.
bacteria would contain 23 times higher concentrations of PLFA than
fungi. The above calculation is in line with the fact that bacteria
have a roughly 25-fold smaller ratio of cell volume to cell surface.
However, fungi have different cell-internal organelles, which are
surrounded by membranes containing PLFA. This means that the
difference between fungal PLFA and bacterial PLFA should be
smaller. Ratios of bacterial PLFA-to-fungal PLFA of between 14 and
40 have been repeatedly reported (Yeates et al., 1997; Pennanen
et al., 1999; Kandeler et al., 2000; Grayston et al., 2004). However,
no conversion values are currently available for total PLFA to soil
bacterial C or any specific PLFA to Gram-negative bacteria, or Gram-
positive bacteria, such as Firmicutes and Actinobacteria.
White et al. (1979) presented an average conversion factor of 43
from mmol total PLFA to mg bacterial biomass C on the basis of 13
bacterial species cultured in the laboratory. This factor is almost
identical to the average conversion factor of mmol total PLFA to mg
fungal biomass C. However, cultured bacteria probably have
a considerably larger ratio of cell volume to cell surface than
bacteria living in soil. A further problem for analysing bacteria-
specific PLFA contents is the fact that many soil bacteria are not
culturable (Torsvik et al., 1990; Torsvik and Øvreås, 2002). Although
this type of analysis is usually focussed on bacterial PLFA, it is more
difficult to get quantitative information on this group of prokary-
otes than on fungal PLFA. Gram-negative bacteria have a roughly
50% higher PLFA content than Gram-positive bacteria, as the inner
part of the outer membrane consists of PLFA. Quantitative infor-
mation on the concentration of total PLFA or that of indicator PLFA
is inevitably necessary for exact biomass estimates of these two
important bacterial groups in soil. Somewhat different assignations
for bacterial PLFA were used, for example, by Gattinger et al. (2002)
and Marschner et al. (2003), indicating the urgent need for more
basic data on bacteria.
The main problem in converting membrane components such as
ergosterol and PLFA specific for fungi or bacteria to the respective
biomass is the variation in the ratio biomarker to biomass within
a species and between different species. For this reason only
approximate values exist for the ratios of PLFA to particular
microbial groups. In laboratory cultures of bacteria, the total PLFA
content varied 10-fold between 12 and 120 mmol g1 biomass
(White et al., 1979). In laboratory cultures of fungi, the total PLFA
content varied 17-fold between 2.6 and 43.5 mmol g1 biomass, not
much less than the 24-fold range of ergosterol (Klamer and Bååth,
2004). The interspecific variation of the membrane components
was mainly explained by differences in the diameter of the hyphae,
i.e. the ratio cell surface to cell volume (Klamer and Bååth, 2004).
The effect of the growth stage on total PLFA concentration was
relatively small (Klamer and Bååth, 2004) and also the concentra-
tion of fungal indicator PLFA in mol% varied in a reasonably small
range in the cultivated fungi (Table 5). In Ascomycota and Basi-
diomycota, the average concentration of 18.2u6,9 was 48 mol% and
that of 18:1u9c was 19 mol%. In contrast, Zygomycota contained
less 18.2u6,9 at 14 mol% and more 18:1u9c at 41 mol%.
 R.G. Joergensen, F. Wichern / Soil Biology & Biochemistry 40 (2008) 2977–2991
A major disadvantage of PLFA analysis is the fact that none of the
indicator PLFA is fully specific for a certain microbial group. As
indicated by the trivial names, plants may contain high concen-
trations of oleic (18:1u9c) and linoleic acid (18:2u6,9) (Zelles,
1997). The presence of plant debris might lead to strong interfer-
ences and it is not fully known whether sieving, tweezers picking
and pre-incubation is able to eliminate these interferences. This
problem is exacerbated by the fact that many soil ecologists do not
like this pre-treatment and tend to do without it. Also soil animals
such as nematodes and collembola contain high concentrations of
18:1u9c and 18:2u6,9 (Ruess et al., 2002, 2007). The problem of
specificity exists also for 16:1u5, which has been found in some
Gram-negative bacteria (Zelles, 1997). For this reason, it has been
used repeatedly as an indicator PLFA for Gram-negative bacteria
(Vestal and White, 1989; Butler et al., 2003). Another problem may
be analytical constraints in detecting 18:2u6,9. Unreasonably high
ergosterol/18:2u6,9 ratios suggest insufficient detection of linoleic
acid for unknown reasons (Joergensen and Potthoff, 2005; Hög-
berg, 2006; Butenschoen et al., 2007). Insufficient gas chromato-
graphic separation of 18:2u6,9 from other PLFA, for example 18:0
anteiso (Potthoff, personal communication), and co-chromato-
graphing with other PLFA are possible explanations. This may
explain the low mol% of 18:2u6,9 (Smithwick et al., 2005; Potthoff
et al., 2006; Gordon et al., 2008). For this reason, the weighted
mean in Table 6 might underestimate the mol% of 18:2u6,9 for
arable and grassland soils.
In conclusion, the PLFA method has clear advantages, but also
serious drawbacks, such as the accumulation in dead microbial cells,
which need more attention by soil microbiologists. The reliability
and accuracy of gas chromatographic separation need to be checked
regularly with control samples against unintentional variations.
Even more important is the investigation of the quantitative effects
of soil properties on the extractability of PLFA. Moreover, more
information on the PLFA content of soil bacteria and arbuscular
mycorrhizal fungi is urgently required. If these data are forthcoming
in the near future, PLFA analysis will be the most important tool for
measuring the biomass of large functional groups, such as sapro-
trophic fungi, arbuscular mycorrhizal fungi, Gram-positive and
Gram-negative bacteria. The power of PLFA analysis will be further
strengthened by the increasing use of component-specific d13C fatty
acid analysis (Olsson and Johnson, 2005).
3. Amino sugars for estimating fungal and bacterial residues
in soil
Amino sugar containing polymers are mainly of microbial
origin contribute a significant amount of between 5 and 12% to soil
organic N (Stevenson, 1982). The most important amino sugars in
soil are glucosamine, galactosamine, muramic acid and man-
nosamine, although a variety of other amino sugars also exist
(Amelung, 2001). Fungal cell walls, except that of the Per-
onosporomycetes, contain large amounts of glucosamine, mainly
as chitin, and are the major source of this component in soil
(Parsons, 1981). The contribution of chitin from the exoskeleton of
microarthropods to the glucosamine content of soils is probably
minimal, as their biomass is typically below 0.5% of the fungal
biomass (Beare, 1997; Simpson et al., 2004). The contribution of
bacterial cell wall murein to the glucosamine content can be
simply calculated, assuming that muramic acid and glucosamine
occur at a molar 1-to-2 ratio in bacteria (Engelking et al., 2007).
Then, fungal glucosamine can be converted to fungal C by multi-
plying by 9. The narrow range of 8–11 of the confidence limits
indicates that most fungi cultured in the laboratory had rather
constant glucosamine concentrations. This is probably even truer
for soil fungi, as their ratio of cell wall to cytoplasm is usually more
constant. Muramic acid occurs exclusively in bacterial cell walls,
2985
especially in the murein skeleton of Gram-positive species (Millar
and Casida, 1970). Gram-positive bacteria contain on average
13.9 mg muramic acid g1 dry weight and Gram-negative only
3.7 mg g1 dry weight (Appuhn and Joergensen, 2006). Within
these two groups, especially in the Gram-negative bacteria, the
muramic acid concentration is relatively constant. Bacterial C can
be calculated by multiplying muramic acid by 45, assuming a ratio
of 66% for Gram-positive bacteria to 33% for Gram-negative
bacteria (Joergensen and Potthoff, 2005).
Little is known about the origin of galactosamine in soil, which
has been sometimes used as indicator for bacterial tissue (Liang
et al., 2007a,b), although it accounts for 30–50% of the amino sugar
content. Bacteria, but also fungi, produce galactosamine during
growth (Glaser et al., 2004; Engelking et al., 2007), but the function
of galactosamine in bacterial or fungal cells or exudates is still
unclear (Amelung, 2001). It is probably a component of microbial
mucous substances (Distler and Roseman, 1960).
The basis of most amino sugar analysis used today is the
hydrolysis of samples with 6 M HCl for 3–6 h (Zelles, 1988; Ame-
lung, 2001; Appuhn et al., 2004) although sometimes alkaline
hydrolysis with 5 M NaOH has been used (Greenfield, 2001). A
variety of different methods are available for the determination of
the amino sugars. Gas chromatographic methods are very sensitive
and have a high specificity, but the derivatization procedure of the
hydrolysis products into volatile components, e.g. to alditol acetates
or aldononitrile acetates, is time consuming and needs experience
in laboratory work (Zhang and Amelung, 1996). With the exception
of amperometric detection (Benner and Kaiser, 2003), high
performance liquid chromatography also needs derivatization steps
to determine amino sugars (Zelles, 1988; Chantigny et al., 1997).
However, this can be carried out automatically by the HPLC system,
for example, as pre-column derivatisation with ortho-phthaldial-
dehyde (Appuhn et al., 2004).
In freshly colonized organic material, such as excised roots, the
highly specific cell wall components fungal glucosamine and
bacterial muramic acid are useful indicators of fungal biomass C
and bacterial biomass C, because they are not significantly affected
by the presence of roots (Kortemaa et al., 1997; Appuhn and Joer-
gensen, 2006). In soil, glucosamine and muramic acid accumulate
in soil organic matter (Amelung, 2001) and form the main
component of the microbial residues fraction, which comprises
non-biomass microbial metabolites such as exoenzymes, mucous
substances and dead cell remains. However, a minor percentage of
amino sugars are still part of the soil microbial biomass. The
weighted means of fungal C in percent of total microbial residues
were 75% for arable soils, 68% for grassland soils, 70% for forest soils,
and 76% for litter layers (Table 7, Fig. 1c). The average coefficient of
variation was 12% and much lower than those obtained for the
other methods. The amino sugar data are apparently not affected by
methodological differences, giving confidence in the reliability of
the methods used.
Differences in turnover of fungal glucosamine and bacterial
muramic acid should be reflected by a disproportionate decrease or
increase in one of these two amino sugars, comparing their
concentration in freshly colonized soil. However, differences were
not observed by Appuhn et al. (2006). This is in line with the
observation that fungal and bacterial residues added to soil were
decomposed at similar rates (Jenkinson, 1976). However, this
contrasts the view that the turnover of bacterial cell wall remains is
faster than that of fungal cell walls, especially those containing
melanins (Amelung, 2001; Greenfield, 2001). However, Luther and
Lipke (1980) found melanins easy to degrade. Appuhn et al. (2006)
observed a significant relationship between the ergosterol
concentration and the glucosamine content of grass roots and
rhizosphere soil. This suggests a relationship between fungal
biomass and fungal residues. Legacy effects of soil organic matter
2986 R.G. Joergensen, F. Wichern / Soil Biology & Biochemistry 40 (2008) 2977–2991

Table 7
Contribution of fungal C to the total microbial residue C in percent, recalculated according to Appuhn and Joergensen (2006) and Engelking et al. (2007), based on the
measurements of glucosamine and muramic acid; number of observations as taken from figures and tables of the listed references multiplied by the respective replicate
samples; litter includes data on microbial root and straw colonization

Author Arable Grassland Forest Litter Method

Zelles (1988) 76 (4) 87 (2) – 90 (2) Z
Zelles et al. (1990) – – 73 (60) 87 (90) Z
Zelles et al. (1991) 77 (6) – 81 (3) 88 (6) Z
Zhang et al. (1997) 76 (17) – – – ZA
Chantigny et al. (1997) 62 (9) 58 (9) – – CþZ
Amelung et al. (1999) – 67 (36) – – ZA
Guggenberger et al. (1999) 81 (36) – – – ZA
Zhang et al. (1999) 84 (2) 71 (2) 79 (2) – ZA
Kandeler et al. (2000) 78 (6) – – – ZA
Amelung et al. (2001) – – – 88 (30) ZA
Solomon et al. (2001) 84 (12) – 79 (8) – ZA
Amelung et al. (2002) 81 (40) 77 (5) – – ZA
Dai et al. (2002) – – 71 (20) – ZA
Turrión et al. (2002) 72 (5) – 31 (5) 34 (15) ZA
Glaser et al. (2004) 60 (5) – – – ZA
Simpson et al. (2004) 73 (16) – – – ZA
Appuhn and Joergensen (2006) 71 (6) – – 69 (90) AþZ
Appuhn et al. (2006) 66 (24) 68 (18) – 73 (42) AþZ
Jolivet et al. (2006) 83 (9) 86 (3) 88 (6) – CþZ
Wichern et al. (2006) – 74 (8) – 95 (4) AþZ
Engelking et al. (2007) 70 (12) – – – AþZ
Formowitz et al. (2007) 74 (32) – – – AþZ
van Groenigen et al. (2007) 77 (8) 65 (12) 64 (6) – ZA
Liang et al. (2007a) 74 (18) – – 45 (6) ZA
Liang et al. (2007b) – – 59 (15) – ZA
Raubuch et al. (2007) – – – 87 (16) AþZ
Weighted mean (number) 75 (267) 68 (95) 70 (125) 76 (301)
Standard deviation 6 6 10 14

– ¼ No information; Z ¼ Zelles (1988); ZA ¼ Zhang and Amelung (1996); A þ Z ¼ Appuhn et al. (2004) and Zelles (1988); C þ Z ¼ Chantigny et al. (1997) and Zelles (1988).

accumulation periods in the past may control or mask microbial
responses to recent management changes (Stromberger et al.,
2007; Holtkamp et al., 2008). The use of amino sugar specific
analysis of 15N and 13C will make it possible to elucidate the turn-
over of fungal and bacterial residues in the near future (Glaser and
Gross, 2005; He et al., 2006).
In conclusion, fungal and bacterial residue C are important
independent measures for testing the reliability of other methods.
More quantitative information on the proportion of Gram-negative
to Gram-positive bacteria, such as Firmicutes and Actinomycetes,
will reduce existing uncertainties with the conversion of muramic
acid to bacterial residue C.
4. Methodological remarks on the ratio of fungi-to-bacteria
The soil microbial biomass of all terrestrial ecosystems is
generally dominated by fungi, as suggested by the presented data
obtained with different methods. Fungal dominance was, in
general, higher in forest A horizons and litter layers. All of the
evaluated methods are able to provide similar and valid ratios of
fungal to bacterial tissue if properly carried out. The most variable
and difficult approaches belong apparently to the group of direct
microscopic methods (Table 1). It is rather disappointing that the
development of new fluorescent dyes and automated image anal-
ysis has not resulted in an improvement of data over the past two
decades. At the moment, PLFA analysis seems to have the greatest
potential for providing a quantitative insight into the microbial
community, indicated by the rapidly growing number of publica-
tions on the basis of this method over the past decade. However,
the most consistent information on the ratio of fungal to bacterial
tissue can be obtained from amino sugar data.
In arable soils, a reduction in tillage, i.e. no tillage versus disc
ploughing or chisel ploughing versus mouldboard ploughing, has
usually been found to promote fungi independently of the meth-
odological approach used (Hu et al., 1995; Frey et al., 1999;
Guggenberger et al., 1999; Scheller and Joergensen, 2008). The
maximum increase in soil fungal C was from 81% of microbial
residue C under disc ploughing to 89% under no tillage (Guggen-
berger et al., 1999) based on glucosamine and muramic acid data,
recalculated as suggested by Appuhn and Joergensen (2006) and
Engelking et al. (2008). However, the difference was much smaller
in most comparisons (Guggenberger et al., 1999; Scheller and
Joergensen, 2008) or even absent (Simpson et al., 2004; Miura
et al., 2008). A shift of the soil fungal community towards sapro-
trophic fungi, which are able to decompose organic material
rapidly, was accompanied by a considerably higher metabolic
quotient and a lower soil organic C content in comparison with
a similar soil receiving less C in the form of farmyard manure
(Scheller and Joergensen, 2008). Consequently, the promotion of
saprotrophic fungi in arable systems may lead to significant soil
organic matter losses, which contradicts the general meaning
stated in Section 1.
In contrast to arable soils with annual crops, soils under
perennial grassland vegetation revealed consistently the lowest
contribution of fungi to the microbial biomass and to the microbial
residues soils, independently of the methodological approach used.
This is surprising, as grassland soils do not receive tillage. However,
the colonization of roots with AMF under perennial grassland
vegetation is most likely much stronger than that under annual
arable crops due to the absence of mechanical disturbance and the
permanent presence of host plants. For this reason also, the
contribution of AMF to the soil microbial biomass is probably much
higher in grassland than in arable soils. Olsson and Wilhelmsson
(2000) suggested that AMF may comprise 30% of the soil microbial
biomass. As their conversion value from 16:1u5 to AMF biomass C
is apparently not appropriate (Table 5), the real contribution of AMF
to the microbial biomass should be a focus of future research.
Additionally, information on the turnover of biotrophic AMF
hyphae in the absence of their host plants is scarce and conflicting
(Olsson and Johnson, 2005).
 R.G. Joergensen, F. Wichern / Soil Biology & Biochemistry 40 (2008) 2977–2991
It is sad that in the majority of PLFA data presented on arable and
grassland soils (Table 6), 16:1u5c was not used as an indicator for
the biomass of AMF fungi as suggested by Olsson et al. (1995). The
main reason for not using 16:1u5 as an indicator for AMF is prob-
ably the possibility, stated by Zelles (1997, 1999), that 16:1u5 occurs
in Gram-negative bacteria. Högberg et al. (2007) used the ratio
between NLFA and PLFA 16:1u5 to distinguish between AMF and
Gram-negative bacteria as the ratio is high in AMF (1–200) and low
in bacteria (<1). The PLFA cy17:0 and cy19:0 (Zelles, 1999; Bååth,
2003) could be used to estimate the interference of Gram-negative
bacteria in the AMF estimation by the PLFA 16:1u5.
In arable and grassland systems, the long-term shifts from
arbuscular mycorrhizal fungi to saprotrophic fungi can be moni-
tored by increases in the ratio of ergosterol-to-fungal glucosamine
(Scheller and Joergensen, 2008). The same shift could probably also
be observed by an increased ratio of 18:2u6,9 or 18:1u9c to fungal
glucosamine. However, the relative shift of the fungal contribution
to the microbial C might be small, as demonstrated by Amelung
et al. (2002), if their glucosamine and muramic acid data are
recalculated into fungal C and bacterial C (Appuhn and Joergensen,
2006; Engelking et al., 2008). After this recalculation, Amelung
et al. (2002) observed an increase in fungal C from 79% in native
grassland to 84% after 98 years of arable use, neglecting the shift
from biotrophic AMF to saprotrophic fungi (Preger et al., 2007).
The large contribution of biotrophic AMF to the microbial
biomass may partly explain decreasing metabolic quotients of
microbial communities with increasing fungal biomass (Sakamoto
and Oba, 1994). AMF starve under the incubation conditions for
basal respiration although they may have a limited degradative
capability of organic matter (Hodge et al., 2001). Consequently,
a shift from biotrophic AMF to saprotrophic fungi might also
explain the increased microbial turnover of nutrients (Saito et al.,
2004), repeatedly observed by aboveground animal grazing
(Bardgett et al., 1993, 1996; Grayston et al., 2004) or artificial
defoliation (Mawdsley and Bardgett, 1997). This explanation would
not be in line with the repeatedly stated explanation that grazing
and defoliation are accompanied by a shift from a bacteria domi-
nated to a fungi dominated microbial community (Bardgett et al.,
1993, 1996; Grayston et al., 2004). Grazing and defoliation keeps
plants in the vegetative growth phase. The rhizodeposits are then
characterized by a higher percentage of labile and easily decom-
posable components than those of mature plants (Wichern et al.,
2007).
The observation of strong seasonal variations is a specific detail
of many results obtained by PLFA measurements evaluated by
principal or canonical component analysis (Waldrop and Firestone,
2006; Toyota and Kuninaga, 2006), which override tillage and
organic fertilizer treatments. These strong seasonal effects clearly
contrast the observation by others that climatic conditions have
only small and variable effects on the ratio of fungi-to-bacteria
(Vishnevetsky and Steinberger, 1997; Frey et al., 1999; Imberger and
Chiu, 2001, 2002). The ratio of fungi-to-bacteria sums up the
functional groups ‘‘bacteria’’ and ‘‘fungi’’, whereas the whole PLFA
pattern represents the structural diversity within these groups.
Diversity may change at different time points of the year, whereas
different microbes will fulfil similar ecological functions
throughout the year. This occurrence of functional groups is more
resource driven, whereas individual species are additionally
affected by environmental changes such as temperature or
moisture.
However, the reasons seasonal effects may be unintentional
variations of the method, caused, for example, by differences in the
interferences of plant material and rhizodeposits, by differences in
the water content on the extractability of PLFA or by other differ-
ences in sample handling. Several small and insignificant shifts in
the content of different PLFA may be bundled to significant shifts by
2987
multivariate statistics with doubtful ecological significance. It
should be considered that shifts in the microbial community
structure are a result of death and growth processes, which are
combined with severe energy losses and energy demands,
respectively. However, it cannot be excluded that only small frac-
tions of the microbial community are relevant for carrying out the
actual process within a year, leading to the observed seasonal
variations in the PLFA structure.
5. Conclusions
All methodological approaches give reasonable approximate
answers, but no one is perfect. The ratio of fungal to bacterial
biomass, respiration or residues is relatively stable and dominated
by fungi, especially in forest A horizons and litter layers. Shifts
within the soil fungal community, especially from biotrophic
mycorrhizal fungi to saprotrophic fungi apparently have
a stronger influence on soil biological processes than a general
shift from fungi-to-bacteria contrasting generally accepted
concepts. It would be interesting if shifts within the soil bacterial
community have similar effects on soil biological processes, for
example shifts from Gram-positive bacteria (Actinobacteria and
Firmicutes) to Gram-negative bacteria. The most consistent
answers have been obtained from amino sugar analysis, but PLFA
analysis seems to have the greatest potential. However, more
basic research on the concentration of specific indicator PLFA in
soil bacteria is necessary to obtain the full quantitative informa-
tion from this method.
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