FEMS Microbiology Ecology 27 (1998) 9^20
Microbial community dynamics during composting of straw
material studied using phospholipid fatty acid analysis
Morten Klamer a; *, Erland Baîaîth
a
b
Department of Mycology, University of Copenhagen. Oester Farimagsgade 2D, DK-1353 Copenhagen K, Denmark
b
Department of Microbial Ecology, Ecology Building, Lund University, SE-223 62 Lund, Sweden
Received 18 February 1998 ; revised 14 May 1998; accepted 8 June 1998
Abstract
Microbial biomass and community structure were investigated in two composts using the phospholipid fatty acid technique.
The composts consisted of shredded straw of Miscanthus with the addition of pig slurry to give an initial C:N ratio of about 25.
Samples were taken following changes in the compost temperature (at 5³C intervals) during the first month of composting and
additionally after 2 and 3 months. The total microbial biomass, measured as total amount of phospholipid fatty acid, peaked
after 1 day with about six times the initial values. The temperature also peaked after 1 day, being above 60³C, and then slowly
declined to around 25³C over 3 months. Microbial biomass was approximately halved during this time. When the total amount
of phospholipid fatty acid was separated into indicator phospholipid fatty acids for different groups of microorganisms, these
groups showed different patterns during the composting process. Gram-positive bacteria increased rapidly with increasing
temperature and decreased with decreasing temperature. Gram-negative bacteria and fungi increased initially up to a
temperature around 50³C, but decreased during the extreme heating phase. When the temperature declined to about 50³C, the
amounts of phospholipid fatty acids indicative of these two groups increased again. The phospholipid fatty acid indicative of
actinomycetes, 10Me18:0, was at a low level during the whole experiment, but increased slightly during the last month of
composting. The development of the microbial community in the two composting systems was similar during the initial
thermophilic phase of the composting process, but the communities after 3 months differed. z 1998 Federation of European
Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Phospholipid fatty acid analysis; Microbial biomass ; Compost ; Microbial community structure; Miscanthus
1. Introduction
Composting is not only a way to reduce human
waste and recycle nutrients, but can also produce
useful components, e.g. compost for mushroom production [1,2], soil conditioners [3,4], or growth media
* Corresponding author. Tel.: +35 322322;
Fax: +35 322321; E-mail: [email protected]
for horticultural plants [5^8]. Since bacteria and fungi are the main organisms responsible for the decomposition, much e¡ort has been put into understanding the changes in the microbial biomass, community
structure and activity during the composting process.
A variety of methods have so far been used to
investigate the microorganisms during composting.
These include traditional plating and identi¢cation
of the culturable microorganisms [9^14], and more
0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 5 1 - 8
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recent techniques measuring ATP content [15], microbial biomass [16,17] and potential metabolic abilities determined by the BIOLOG sole carbon utilization test [18].
One technique, that gives an indication of the microbial community composition without the culturing of organisms on agar media, is the direct analysis
of the phospholipid fatty acid (PLFA) pattern of a
sample. The PLFA analysis is based on the fact that
di¡erent subsets of a microbial community di¡er in
their fatty acid composition. Although few PLFAs
can be considered to be absolute signature substances for a single species or even a speci¢c group of
organisms, it is possible to get indications of changes
in major groups, like fungi, actinomycetes, and
Gram-positive and Gram-negative bacteria [19,20].
The PLFA pattern of a compost sample can therefore be regarded as an integrated measurement of all
organisms in that sample. The PLFA analysis has
been used to study changes in the microbial community in a variety of natural terrestrial environments [21^25], but to our knowledge, only two studies of changes in the microbial community in
composts have been carried out using PLFA analysis
[26,27]. Both these studies concentrated on the period after peak temperatures had been reached, and
thus did not test if the PLFA technique is sensitive
enough to detect rapid changes during the initial
temperature increase. Furthermore, neither of these
two studies investigated composts consisting of straw
material and a nitrogen source.
We have investigated the changes in the microbial
biomass and community structure by analysing the
composition of the PLFAs during 3 months of composting with special emphasis on the early, thermophilic phase. This was achieved by taking samples
following the changes in temperature (at about 5³C
intervals) until the temperature reached the level of
the surroundings. In particular, we wanted to follow
the development of di¡erent taxonomic groups of
microorganisms (actinomycetes, fungi, Gram-positive and Gram-negative bacteria). Furthermore, since
it is well known that there would be rapid changes,
¢rst to a thermophilic and then to a mesophilic community, with presumably di¡erent PLFA patterns,
this would enable us to estimate turnover rates of
PLFAs from dead/inactive groups of organisms
under natural conditions.
2. Materials and methods
2.1. Compost set-up and sampling
The material consisted of shredded straw (2^5 cm
pieces) of MiscanthusUogiformis Honda `Giganteus'
(Andropogoninae: Poaceae) to which pig slurry was
added to give an initial C:N ratio of about 25. The
moisture content was adjusted to about 70%. The
compost was packed loosely in an open 800 l wooden box (48 kg dry weight of straw) and in a closed
500 l reactor (32 kg dry weight) with forced areation
(5 l h31 ). Both types of containers were insulated.
Water was added twice a week to keep the moisture
content stable and the compost in the box was
turned every 14 days to insure aerobic conditions.
The compost containers were placed in a greenhouse
where the temperature was kept between 15 and
20³C during the experiment.
The temperature in the center of the containers
was monitored continuously and sampling was
done with temperature intervals of about 5³C during
the heating phase, beginning just after mixing of the
compost. After the heating phase samples were taken
after 48, 51, 53, and 93 days of composting in the
reactor, and after 51, 55, 78, and 93 days in the box.
In the reactor, the samples were taken through a
hole in the middle, close to the center. In the box, the
samples were taken in the center at a depth of about
30 cm. At each sampling four subsamples were taken
at randomized positions, where the measured temperature corresponded with that of the center. The
subsamples were pooled, mixed, and stored at minus
20³C until the extraction took place.
Dry weight was measured after drying for 24 h at
70³C. Organic matter content (OM), based on the
dried samples, was calculated as loss on ignition at
550³C for 5 h.
2.2. PLFA analysis
Prior to the extraction procedure 0.5^1 g wet
weight of the samples were ball-milled.
The lipid extraction, fractionation, mild alkaline
methanolysis and GC analysis used here were described in detail by Frostegaîrd et al. [23,28]. To summarize shortly: lipids were extracted from the compost samples in a one-phase mixture of chloroform/
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M. Klamer, E. Baîaîth / FEMS Microbiology Ecology 27 (1998) 9^20
methanol/citrate bu¡er and the polar lipids were separated using silicic acid columns, followed by a mild
alkaline methanolysis to form fatty acid methyl
esters before GC analysis. Fatty acids were designated in terms of total number of carbon atoms:
number of double bonds, followed by the position
of the double bond from the methyl end of the molecule. The pre¢xes a and i indicate anteiso and iso
branching, cy indicates a cyclopropane fatty acid and
methyl branching (Me) is indicated as the position of
the methyl group from the carboxyl end of the chain.
The sum of the following fatty acids was considered to represent Gram-positive bacteria: i15:0,
a15:0, i16:0, i17:0, a17:0 and 10Me17:0 [29]. The
Gram-negative bacteria were represented by the
sum of: 16:1g7t, 16:1g5, cy17:0, 18:1g7 and
cy19:0. 16:1g5 and 18:1g7 are also found in arbuscular mycorrhizal fungi [30,31], which, however, are
very unlikely to appear in compost. As markers for
actinomycetes and fungi 10Me18:0 and 18:2g6,9
were used, respectively [19,20]. The fungal to bacterial PLFA index ratio was calculated by dividing the
amount of 18:2g6,9 with the sum of bacterial
PLFAs. The PLFA pattern of dried straw was analyzed initially. 16:1g7 and 16:0 constituted over half
of the PLFAs found and all other PLFAs were below 10 mol%.
The mole percents of the PLFA values from both
composting systems were subjected to a principal
component analyses after scaling each variable by
dividing with its standard deviation.
3. Results
The temperature increased quickly after mixing of
the compost material, reaching peak values within 20
and 26 h for the box and the reactor, respectively.
Then the temperature gradually declined to ambient
level over about 3 months (Fig. 1A,B). The organic
matter content decreased from 92 to 72% of dry
weight, and pH decreased from 8.5 to 7.4 in both
systems during this time. Peak temperature in the
reactor system was 69³C, and in the box system
64³C. The total amount of phospholipid fatty acids
(totPLFA) covaried with the temperature, increasing
very rapidly from a starting value around 500 nmol
g31 OM to approximately 6 times higher amounts at
11
or shortly after peak temperature. Then totPLFA
decreased gradually, resulting in around 1500 nmol
g31 OM after 3 months of composting. However,
after about 30 days the totPLFA increased, without
any simultaneous increase in temperature. This was
most notable in the reactor system (Fig. 1A). At this
time, a large number of fruit bodies of Coprinus cinereus (Schae¡er: Fries) s.f. Gray were present indicating that a major fungal biomass must have been
present in the compost.
In total, 32 di¡erent PLFAs were detected during
the composting process, and 28 of these were identi¢ed. The development of three representative PLFAs
during composting in the reactor is shown in Fig. 2.
The amounts of PLFAs 18:1g9 and cy19:0 increased
until the temperature reached about 50³C, but then
they decreased during the period of highest temperature. However, when the temperature had decreased
to about 40^50³C (after about 10^20 days of composting), both these PLFAs started to increase again,
indicating growth of Gram-negative bacteria and
fungi. The PLFA i17:0 only appeared in greater
amount when the temperature was above 50³C.
The peak value of this PLFA was almost 20 times
the initial value. The temperature almost decreased it
to initial values at the end of the experiment.
Most PLFAs could be grouped into the same patterns as these illustrated in Fig. 2, both in the reactor
and the box system. Thus, PLFAs with a pattern
similar to i17:0 (that is with peak abundance during
the peak temperature) were i15:0, i16:0, and a17:0,
while a range of PLFAs had a pattern with increasing levels when the temperature was below 50³C,
including PLFAs indicating eukaryotic organisms
(18:2g6,9, 18:1g9, 20:4), Gram-negative bacteria
(cy17:0, cy19:0, 16:1g5) and actinomycetes
(10Me18:0). Three unidenti¢ed PLFAs were also
found in this group.
The major shifts in the microbial community during the composting process could be ascertained using marker PLFAs (expressed as mol%) for main
taxonomic groups of organism (see Section 2.2 for
speci¢c marker PLFAs) (Fig. 3A,B). In total, these
marker PLFAs amounted to between 72 and 89% of
the totPLFA. The PLFAs representing Gram-positive bacteria increased very rapidly with increasing
temperature, especially i16:0. As the temperature
started to decrease the amount of Gram-positive
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Fig. 1. Total concentration of PLFA and temperature (- - -) in (A) the reactor (F) and (B) the box (E) system during 3 months of composting.
PLFA also decreased. During the whole period the
amount of actinomycetes (as judged from the
amount of 10Me18:0) was at a low level, with only
a small increase towards the end of the experiment.
The fungi (represented by 18:2g6,9) increased until
temperature reached 50³C, then decreased during the
extreme heat phase, and ¢nally started to increase
again when the temperature decreased. This was es-
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Fig. 2. The concentration of PLFAs i17:0 (R), 18:1g9 (F) and cy19:0 (S) in the reactor system during 3 months of composting. The
temperature curve is superimposed. The time scale is logarithmically transformed.
pecially evident in the box system (Fig. 3B). The
PLFAs representing Gram-negative bacteria showed
the same pattern as 18:2g6,9.
In total, the PLFAs considered to be mainly of
bacterial origin accounted for between 34 and 73%
of the total amount of PLFAs during the composting
period. The changes in the relative amount of fungal
and bacterial biomass during composting could be
shown by estimating a fungal to bacterial PLFA index ratio over time. In the reactor system, this ratio
was 0.37 from start. During the heating phase, the
ratio decreased very quickly, reaching a minimum
value of 0.007 after 6 days. Then the ratio started
to increase again, but never exceeded 0.1. The box
system showed the same pattern as the reactor system until day 30, but then a much more pronounced
invasion of fungi was evident (Fig. 3B), resulting in a
maximum ratio of 0.34 after about 50 days of composting.
To compare the development of the microbial
communities of the two composting systems, a principal component analysis of the whole data set (using
mol% PLFA) was made (Fig. 4). The ¢rst and last
sampling (the latter after 3 months), as well as the
samplings at peak temperatures are shown in the
graph. The lines are graphical representations of
the timing of the changes of the whole microbial
community. The two ¢rst principal components
(PC) appeared to represent di¡erent communities.
The second PC (characterized by high amounts of
i15:0, i16:0, i17:0, a17:0, and 17:0) re£ected the
thermophilic community. PC1 represented the mesophilic community that developed after temperatures
had fallen below 50³C. Samples with high values for
this PC were characterized by relatively high
amounts of especially 10:Me16:0, 10:Me17:0,
10:Me18:0, cy17:0, cy19:0, 16:1g5, 18:1g9 and
20:4.
The degree of fatty acid unsaturation in both composting systems decreased during the heating phase
and increased during the following phase with lower
temperature, although the degree of unsaturation
was not completely restored to initial values in the
reactor system even after 3 months of composting
(Fig. 5A). In addition, the ratio between PLFAs
with 16 and 18 carbon atoms showed the expected
changes, with almost equal amounts of C16 and C18
in the mesophilic phases, but relatively high amounts
of C16 during and just after the thermophilic phase
(Fig. 5B). In Fig. 5C, the changes in the ratio be-
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Fig. 3. The development of Gram-positive (R) and Gram-negative bacteria (S), fungi (F) and actinomycetes (b) during 3 months of
composting. Fungi is represented by the PLFA 18:2g6,9, actinomycetes by 10Me18:0, and Gram-positive and Gram-negative bacteria by
the sum of several characteristic PLFAs (see Section 2.2). The temperature curves are superimposed. The time scale is logarithmically
transformed. (A) The reactor system. (B) The box system.
tween iso and anteiso forms of 15:0 and 17:0 are
shown. The ratio for 15:0 increased until the temperature reached maximum levels, in both the reactor
and the box system. When the temperature started to
decrease, the iso/anteiso ratio also decreased slightly,
but during the last 2 months, the ratio increased
again, ending with a clear dominance of the iso
form. The ratio for 17:0 in the box system showed
the same pattern as 15:0, but this was not the case in
the reactor system. Until the temperature reached
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Fig. 4. Principal component analysis showing the variation in phospholipid fatty acid patterns in the reactor (F) and in the box (E) system during 3 months of composting. First (F), and last (L) samplings, as well as the sampling at peak temperatures (P), are shown. The
lines from F to L thus represent the timing of the changes in the microbial community structure.
around 50³C the iso/anteiso ratio was 0.8:1.0, but
then the anteiso form dominated during the rest of
the experiment.
4. Discussion
TotPLFA has been suggested as a measure of the
total microbial biomass [25], and has been shown to
correlate well with the biomass measurements using
the fumigation extraction technique during the period after peak temperature in a municipal solid waste
compost [26]. In the present experiment, the gradual
decrease in biomass, estimated as totPLFA, after
peak temperatures (Fig. 1A,B) is consistent with
results obtained by others [18,26], although we did
not ¢nd the decrease to be as drastic as in these
studies.
TotPLFA increased rapidly during the ¢rst day of
composting, indicating a doubling time of the microbial biomass in both systems of 6^7 h. However, this
is certainly an underestimation of the growth rate for
the thermophilic part of the community during this
period, since totPLFA in the initial samplings included PLFAs that might partly be of plant origin
(e.g. 16:0) or PLFAs from microorganisms not
growing at higher temperatures (e.g. 18:1g7, Fig.
2). Using only PLFAs indicative of Gram-positive
bacteria (assuming these to be the main part of the
thermophilic community, see below), a doubling time
of approximately 4 h was found during the ¢rst day
of composting.
The pattern of a fast increase in microbial biomass
immediately after the composting process started,
followed by a gradual decrease might be explained
by the availability of nutrients. Initially fresh substrate was colonized rapidly, but as easily degradable
nutrients became exhausted a gradual decrease in
biomass took place. Although the availability of nutrients thus determined the development of the total
microbial biomass over time, di¡erent subsets of microorganisms were clearly selected for by the temperature regime during the composting process (Fig.
3A,B). The thermophilic community developing
above 50³C appeared to consist of Gram-positive
bacteria, as judged from the increase in iso- and anteiso-branched PLFAs (Figs. 2 and 3A,B). These
PLFAs are common in the genus Bacillus, a genus
well known to be dominant in composts at high temperatures [13,14,32]. Strom [14], for example, identi-
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Fig. 5. The development of di¡erent ratios of PLFAs indicating adaption to di¡erent temperatures during three months of composting in
the reactor (S) and in the box (P) system. The temperature curves are superimposed (reactor, 9 - 9; box: - - -). The time scale is logarithmically transformed. (A) Degree of unsaturation of PLFAs calculated as: (1.0 (% monoene)+2.0 (% diene)+3.0 (% triene))/100).
(B) C16/C18 ratio. (C) Iso/anteiso ratio of the PLFAs 15:0 (reactor, S ; box, P) and 17:0 (reactor, R ; box, O).
¢ed 87% of isolated bacteria at 40^69³C as Bacillus
spp. Iso- and anteiso-branched PLFAs were also
prevalent during the thermophilic phase of municipal
waste composts [27], and branched-chain fatty acids
increased much during the initial phase with high
temperatures in an open windrow compost [26].
Gram-negative bacteria and fungi appeared to
grow only below 50³C. An increase in PLFAs, indi-
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17
Fig. 5. Continued.
cative of these groups of microorganisms, was observed before the temperature had reached peak values, and especially during the cooling phase, where
they partly outcompeted the thermophilic, Grampositive bacteria (Figs. 2 and 3A,B). The increase
in totPLFA after about 30 days was assumed to be
caused by the invasion of Coprinus cinereus. The increase was partly caused by an increase in 18:2g6,9
and 18:1g9, PLFAs common in fungi. Species of
Coprinus are often observed on composts [33].
Actinomycetes (as judged from the proportion of
the PLFA 10Me18:0) only increased in the later
phase, when the compost temperature was relatively
low. This di¡ered from the municipal waste compost
studied by Herrmann and Shann [27], where high
relative amounts of 10Me18:0 were found already
during the phase with the highest temperature regime.
One must bear in mind that the development of
di¡erent groups of microorganisms during composting inferred from the changes in the PLFA pattern
does not give absolute amounts of biomass of di¡erent groups, since conversion factors from the
amounts of PLFAs characteristic for the di¡erent
microorganism groups to actual biomass are lacking.
Another problem could be the contribution of PLFA
from the straw material, but since the main fraction
of PLFA in Miscanthus straw are 16:1g7c and 16:0,
which are not used as marker PLFAs, and the other
PLFAs present are divided on all the groups of microorganisms except actinomycetes, the error made
should be minor. Furthermore, this error will decrease with time as the straw material is decomposted. Thus, the patterns shown in Fig. 3 must be
interpreted with caution, although the main dynamics of the di¡erent groups are well-supported by data
obtained with traditional isolation techniques [9,34^
37].
Some conclusions on dominant groups can, however, be drawn from the changes in the fungal to
bacterial PLFA index ratio. This ratio varied from
0.02 to 0.04 in di¡erent agricultural soils low in organic matter to between 0.3 and 0.5 in forest soils
[20]. The latter are believed to be dominated by fungal biomass. Since these high ratios are similar to
that found in the box system at the end of the
composting process, it appears that fungi were the
dominant microorganism group at the end of the
composting process. On the other hand, at peak
temperatures values for the fungal to bacterial
PLFA index ratio was below 0.01. This was lower
than found in any soil, indicating that, at this point,
it was a bacterial-dominated system. Overall, the
variation in the fungal to bacterial PLFA index
ratio during the composting was as large or even
larger than that found in a range of very di¡erent
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soils [20], emphasizing the drastic changes in microbial community structure that occurred during composting.
An altered PLFA pattern during composting may
be due to changes in species composition or due to
adaptation to the di¡erent temperature regimes by
one and the same community. Although changes in
e.g. chain lengths or degree of unsaturation of the
PLFAs were consistent with changes expected from
the di¡erent temperature regimes ([38^41], Fig.
5A,B), we believe that the altered PLFA pattern
was mostly due to changes in speci¢c groups of organisms. The changes found during the ¢rst day of
composting, for example, were changes from a mesophilic to a completely di¡erent thermophilic community (Figs. 3A,B and 4), and the major changes in
the PLFA pattern during the decrease in temperature
was also found between PLFAs from di¡erent
groups of organisms. A similar conclusion regarding
interpretation of the PLFA data was drawn by Hellman et al. [26].
An increase in a speci¢c PLFA must be related to
an increased growth of a species or a group of organisms having that speci¢c PLFA. However, a decrease in a certain PLFA can be caused by at least
two mechanisms. First, the organisms are outcompeted and die, and second, the phospholipids have
to be broken down in order not to be included in the
analyses. Thus, a decrease in a PLFA characteristic
of a certain group of microorganisms will always
underestimate the real death rate of that organism
group. We lack information about the rate at which
phospholipids are broken down both in soils and in
composts, although studies in marine sediments indicated a fast turnover rate [42]. However, our data
suggests a fast turnover rate in the compost. The
PLFA 18:1g9 (common in fungi) that initially increased to around 200 nmol g31 OM, decreased to
less than 50 nmol g31 OM in 5 days time when
temperatures were to high for fungal growth (Fig.
2). This suggests a half-life of phospholipids with
this fatty acid of about 1 day during this period.
Likewise, i17:0 decreased from a maximum of 600
nmol g31 OM to around 100 nmol g31 OM in about
2 weeks, indicating a half life of less than a week.
Thus, the decrease in PLFAs during the composting
process probably would indicate the death rate of the
di¡erent groups of organisms rather well.
One objective of the present work was to study if
the PLFA technique was sensitive and reproducible
enough to be used to monitor the rapid development
of the microbial community during the initial phase
of the composting process. The very similar development in the two composting systems before, during
and shortly after the thermophilic phase (e.g. Fig. 4)
indicated that this was the case. Furthermore, the
PLFA analysis can also be used to indicate di¡erences during the later stages, since di¡erences in the
microbial community in the two composting systems
could be detected after three months of composting
using PCA (Fig. 4). These di¡erences were caused by
di¡erences in the two systems. First, the reactor, as a
closed system with forced aeration, remained moist
and well aerated, and the material was therefore not
disturbed. The open box system without aeration, on
the other hand, could become anaerobic and it also
tended to dry out due to evaporation, and therefore
the material had to be turned and watered regularly.
This procedure is known to speed up the composting
process [18]. The microbial community in the box
system was more altered than in the reactor system
(more to the right at the end of composting in Fig.
4), showing that the PLFA pattern can be used to
indicate the maturity of the compost. This will be
investigated further in the future.
Acknowledgments
We thank Thomas L×essoe for identi¢cation of
the Coprinus species, Erling Hyldig for sampling,
and Gosha Afshar and Else M. Andersen for technical assistance. We also thank Ulrik Soechting for
valuable comments, and Andrea Gargas for revising
the English in this paper. This study was supported
by a grant from The Danish Research Councils
(M.K.).
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