Appl Microbiol Biotechnol

DOI 10.1007/s00253-010-2858-y

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Correlations between molecular and operational parameters

in continuous lab-scale anaerobic reactors
Marta Carballa & Marianne Smits &
Claudia Etchebehere & Nico Boon & Willy Verstraete

Received: 19 May 2010 / Revised: 2 August 2010 / Accepted: 19 August 2010

# Springer-Verlag 2010

Abstract In this study, the microbial community character-
istics in continuous lab-scale anaerobic reactors were
correlated to reactor functionality using the microbial
resource management (MRM) approach. Two molecular
techniques, denaturing gradient gel electrophoresis (DGGE)
and terminal-restriction fragment length polymorphism (T-
RFLP), were applied to analyze the bacterial and archaeal
communities, and the results obtained have been compared.
Clustering analyses showed a similar discrimination of
samples with DGGE and T-RFLP data, with a clear
separation between the meso- and thermophilic communi-
ties. Both techniques indicate that bacterial and mesophilic
communities were richer and more even than archaeal and
thermophilic communities, respectively. Remarkably, the
community composition was highly dynamic for both
Bacteria and Archaea, with a rate of change between 30%
Marta Carballa and Marianne Smits have equally contributed to this
paper.
M. Carballa (*)
Department of Chemical Engineering, School of Engineering,
University of Santiago de Compostela,
Rúa Lope Gómez de Marzoa s/n, E-15782,
Santiago de Compostela, Spain
e-mail: marta.carballa@usc.es
M. Carballa : M. Smits : N. Boon : W. Verstraete
Laboratory of Microbial Ecology and Technology (LabMET),
Faculty of Bioscience Engineering, University Ghent,
Coupure Links 653,
9000 Ghent, Belgium
C. Etchebehere
Microbiology Department,
School of Science and School of Chemistry,
University of the Republic,
General Flores 2124,
Montevideo, Uruguay
and 75% per 18 days, also in stable performing periods. A
hypothesis to explain the latter in the context of the
converging metabolism in anaerobic processes is proposed.
Finally, a more even and diverse bacterial community was
found to be statistically representative for a well-
functioning reactor as evidenced by a low Ripley index
and high biogas production.
Keywords Anaerobic digestion . Community
organization . Diversity . Dynamics . Microbial community
structure . Richness
Introduction
The waste treatment concept will change drastically in the
coming years from a pollution control point of view to a
recovery of valuable resources perspective. In this context,
anaerobic digestion (AD) can make an important contribu-
tion, since it offers the opportunity to stabilize organic
wastes and to recover energy simultaneously. However,
despite the well-known advantages of this process, AD is
restrained by some obstacles like the presence of poorly
biodegradable substrates (Liu et al. 2007; Hecht and Griehl
2009) and the occurrence of process instabilities (Verstraete
et al. 2005). Therefore, a considerable monitoring and
optimization is still required.
The common process parameters used to characterize the
performance of anaerobic reactors are the volatile fatty acid
(VFA) concentrations, the biogas production, and the
Ripley index, defined as the ratio between the free fatty
acids and the buffer capacity of the reactor (Ripley et al.
1986; Boe et al. 2008). Propionic acid (HPr) tends to
accumulate when a reactor is overloaded, and the maximum
acceptable HPr concentrations as total acid vary from 0.8 to

2.2 g/L, depending on the type of waste and reactor
(Barredo and Evison 1991; Mosche and Jordening 1998).
Biogas production and biogas composition allow a rapid
evaluation of anaerobic reactors performance (Dearman et
al. 2006; Boe et al. 2008; Rincón et al. 2008), and a Ripley
index lower than 0.3 is an indication of a well-functioning
reactor (Ripley et al. 1986). These parameters suffice to
evaluate the ongoing performance of anaerobic reactors, but
their power to predict the stability and future performance
of the reactors is limited.
Anaerobic digesters are characterized by complex
microbial consortia (Riviere et al. 2009), and culture-
independent molecular techniques, such as denaturing
gradient gel electrophoresis (DGGE) and terminal restric-
tion fragment length polymorphism (T-RFLP), have dem-
onstrated that the microbial community characteristics
(“who is there doing what with whom”) can play an
important role for a good reactor performance (McHugh et
al. 2004). Many studies have postulated that biomonitoring
of the microbial community characteristics could lead to an
early detection of operational problems, making preventive
action possible (McHugh et al. 2004; Lee et al. 2008; Malin
and Illmer 2008; Rincón et al. 2008; Talbot et al. 2008). In
general, a higher diversity of the bacterial community,
mainly at mesophilic temperature (Leven et al. 2007), is
observed compared to the archaeal community (Rincón et
al. 2008). Microbial diversity was shown to be not
important in the development of a functionally successful
anaerobic microbial community (Dearman et al. 2006). In
contrast herewith, a high rate of change of the microbial
community composition (Miura et al. 2007) and an initial
community evenness (Wittebolle et al. 2009) are important
factors for stable operation of mixed microbial cultures.
Thus far, no direct relationships between such microbial
community characteristics and process parameters have
been established (Bouallagui et al. 2005).
The main objective of this work was the application of
the microbial resource management (MRM) approach to
correlate microbial community characteristics with the
process performance in continuous lab-scale anaerobic
digesters. The MRM concept describes the microbial
community by three parameters (Verstraete et al. 2007;
Marzorati et al. 2008): (a) the richness (Rr), which reflects
the number of dominant species; (b) the dynamics of
change (Dy), which reflects the specific rate of species
coming to significance; and (c) the community organization
(Co), which relates the task distribution of the microbial
community. To estimate these parameters, two different
molecular techniques, DGGE and T-RFLP, have been used,
and the results obtained have been compared. To our
knowledge, this is the first study applying not only MRM
to AD processes but also the MRM concept with T-RFLP
data.
Appl Microbiol Biotechnol
Materials and methods
Lab-scale anaerobic reactors
In the first experiment, four continuous tank reactors with a
volume of 18 L were used: two reactors (M) were operated
in mesophilic range (34±2°C) and the other two (T) in
thermophilic conditions (53±2°C). At each temperature,
both reactors were operated similarly till the organic
loading rate (OLR) reached 2 g chemical oxygen demand
(COD) per liter per day. Afterwards, this OLR was kept
constant in one reactor (M1 and T1), while in the other
reactor (M2 and T2), the OLR was gradually increased to
determine the maximum applicable load.
At the onstart, 16 L of anaerobic sludge was used as
inoculum with an initial in-reactor biomass concentration of
35 g volatile suspended solids (VSS) per liter. The
thermophilic inoculum was taken from a thermophilic
digester treating pig manure and organic co-substrates
(Bio Electric, Beernem, Belgium) and the mesophilic one
from a mesophilic digester treating sewage sludge
(Ossemeersen, Ghent, Belgium). Mixed kitchen waste
(Trans Vanheede, Wervik, Belgium) diluted with sewage
to obtain the desired OLR was used as substrate. The main
characteristics of the mixed kitchen waste were: 215 g
CODtotal/kilogram, 75 g CODsoluble/kilogram, 166 g total
solids (TS)/kilogram, 155 g volatile solids (VS)/kilogram
and pH 4. The reactors were stirred before effluent
discharge and after feeding addition and operated at a
hydraulic retention time (HRT) of 18 days. During the last
days of each experiment, when the reactors suffered from
acidification, the pH was maintained at around 7.5 by the
addition of NaOH (10 N). The theoretical gas productions
were based on the COD measurements performed on the
concentrated feed on the one hand and the volumetric gas
production on the other hand.
In the second experiment, only the mesophilic reactors
(M1 and M2) were operated, and their performance was
optimized by installing mechanical mixers (IKA Labor-
technik, Staufen, Germany), which worked 5 min/h, and by
enhancing the biomass retention in the reactors (the mixed
liquor was allowed to settle for at least 30 min before
effluent discharge).
The pH and the biogas production were monitored daily,
and influent and effluent samples were taken thrice a week
for solids, COD, and VFA analyses. Further on, the percent
biogas production refers to the amount of biogas produced
relative to the theoretical amount, which would be
produced based on the COD supplied, assuming a constant
methane content of 70% (0.5 L biogas/gram COD
removed). From days 54 and 80 for the thermophilic and
mesophilic reactors, respectively, a sample of the anaerobic
biomass was taken every HRT to examine the microbial
Appl Microbiol Biotechnol
community. These samples were stored at −20°C until
DNA extraction was performed.
Analytical techniques
TS, VS, and COD were measured according to standard
methods (Greenberg et al. 1992). VFAs (acetic, propionic,
i-butyric, and i-valeric) were extracted with diethyl ether
as described by Holdeman et al. (1977) and measured in a
capillary gas chromatograph (GC 8000 Carlo Erba
Fractovap 4160, CE Instruments, Wigan, UK) equipped
with a flame-ionization detector and a Delsi-Nermag
integrator (Thermo Separation Products, Wilrijk, Bel-
gium). The biogas production was followed by liquid
displacement. The pH was measured with a C532 pH
meter (Consort, Turnhout, Belgium), and the partial and
total alkalinities were determined according to Ripley et
al. (1986). The Ripley index was calculated as the ratio
between the intermediate alkalinity (total alkalinity −
partial alkalinity) and the total alkalinity.
Molecular techniques
Conditions and references for deoxyribonucleic acid (DNA)
extraction, polymerase chain reaction (PCR), and DGGE
were previously described in detail by Boon et al. (2003)
and Rooney-Varga et al. (2007) and were based on the
primers 338F-GC and 518R for Bacteria and the primers
915F and 1352aR-GC for Archaea. DGGE analyses were
performed in a Bio-Rad DGenesystem (BioRad, Hercules,
CA, USA) according to Muyzer et al. (1993).
For T-RFLP, the 16S rRNA genes were amplified by
PCR using universal primers (27F and 907R for Bacteria
and 21F and 915R for Archaea). The forward primers
were fluorescently labeled with the dye 5-(6-carboxy-
fluorescein). The amplification reactions were carried out
as described by Lueders et al. (2004). The PCR products
were purified using PCR purification kit (Qiagen, Venlo,
The Netherlands) and quantified using the ND-1000
spectrophotometer (Nanodrop, Isogen Life Sciences,
Sint-Pieters-Leeuw, Belgium). One hundred nanograms
of the purified PCR product was digested with the MspI
and the TaqI restriction enzymes (Fermentas, St. Leon-
Rot, Germany) for Bacteria and Archaea analyses,
respectively (Lueders et al. 2004). DNA fragments were
precipitated with 95% ethanol and washed with 70%
ethanol. After drying in a Savant SpeedVac system
(ThermoFischer Scientific, Tournai, Belgium), the DNA
fragments were re-suspended with 8 μL formamide and
0.3 μL of internal standard (GeneScan-500 Liz Size
Standard, Applied Biosystems, Halle, Belgium) and
separated on an 3130 Genetic Analyzer (Applied Bio-
systems, Halle, Belgium).
Statistical analysis
Normalization and analysis of the obtained DGGE patterns
were done with BioNumerics software v.2.0 (Applied
Maths, Sint-Martens-Latem, Belgium). The chromatograms
obtained with T-RFLP were analyzed using Peak Scanner
Software v.1.0 (Applied Biosystems, Halle, Belgium). In
both cases, bands or peaks with more than 1% intensity or
abundance, respectively, were considered.
For the interpretation of the results, the three levels of
analysis analogous to those described by Marzorati et al.
(2008) were calculated. Rr was determined as the number
of bands or peaks in each DGGE pattern and electrophe-
rogram, respectively. To calculate the Dy, profile similari-
ties were obtained by determination of the Jaccard
coefficient, and cluster analyses were constructed using
UPGMA algorithm (Hammer et al. 2001) with PAST
software. The Co was calculated as the percentage of the
Gini coefficient, a value that describes a specific degree of
evenness of a microbial community by measuring the
normalized area between a given Pareto–Lorenz curve and
the perfect evenness line (Wittebolle et al. 2009).
The open-source statistical environment R (http://www.r-
project.org/) was used to determine the correlation coef-
ficients among the molecular parameters and between the
molecular and the operational coefficients as well as to
conduct significance tests in order to check if the
relationship/correlation was significant. Statistical signifi-
cance was established at the p<0.05 level.
Results
Anaerobic reactors operation
The results of the operation of the thermophilic and
mesophilic reactors are shown in Figs. 1 and 2, respective-
ly. Significant differences in performance (biogas produc-
tion and COD removal) were observed neither between the
two mesophilic reactors (M1 and M2) nor between the
thermophilic ones (T1 and T2). A good performance of the
thermophilic reactors was obtained until days 55–60
(Fig. 1) when the OLR was kept below or around 2 g
COD per liter per day. However, it can be noted that HPr
concentrations already showed an increasing trend from
day 50 on. During this period, the biogas production was on
average 75% of the maximum theoretical production based
on the OLR applied, and the CODtotal and HPr concen-
trations remained below 30 g COD/liter and 0.5 g HPr-
COD/liter, respectively. However, when the applied OLR
surpassed 2 g COD per liter per day, the CODtotal and HPr
levels in the reactors increased up to 45–70 g COD/liter and
3.0–3.5 g HPr-COD/liter, respectively, with the concomi-
 Appl Microbiol Biotechnol

a b
3,5 3,5
Organic loading rate (g COD/L·d) ( )
and biogas production (L/L·d) ( )

3,0 3,0

2,5 2,5

2,0 2,0

1,5 1,5

1,0 1,0

0,5 0,5

0,0 0,0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

c d
80 4,0
80 4,0

Propionic acid concentration

70 3,5
70 3,5
Total COD concentration

60 3,0

(g HPr-COD/L) ( )
60 3,0
(g COD/L) ( )

50 2,5
50 2,5

40 2,0 40 2,0

30 1,5 30 1,5

20 1,0 20 1,0

10 0,5 10 0,5

0 0,0 0 0,0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
Time (d) Time (d)

Fig. 1 Performance of the thermophilic reactors T1 (a, c) and T2 (b, d). Arrows indicate the sludge sampling points

tant decrease of the biogas production to negligible values.
The reactors failed, and the experiment was terminated on
day 84.
The mesophilic reactors showed a similar behavior as the
thermophilic ones (Fig. 2). When the OLR was kept below
2 g COD per liter per day, the biogas production was 85%
of the maximum theoretical value, approximately, and the
CODtotal and HPr concentrations remained below 25 g
COD/liter and 2.0 g HPr-COD/liter, respectively. The
increase in the OLR to 2.2 g COD per liter per day
(day 37) caused the failure of the reactors, since the
CODtotal and HPr accumulated in the reactors (up to 50 g
COD/liter and 6–7 g HPr-COD/liter, respectively) and the
biogas production decreased to 0.2 L/Lday. The reactors
failed and were stopped on day 72.
The transition of the mesophilic reactors between experi-
ments 1 and 2 (day 80) was done by diluting 9 L of mixed
liquor from experiment 1 with 5 L of mesophilic inoculum
and 4 L of tap water. Since the HPr concentrations were
still high after dilution (around 3.5 g HPr-COD/liter, data
not shown), the digesters were operated without feeding
until the residual HPr levels decreased, and biogas was
produced again. On day 108 (Fig. 2), the mixing and the
feeding started.
The results of experiment 2 confirmed those of exper-
iment 1. Until day 151, in which the OLR was stepwise
increased to 2 g COD per liter per day in M1 and to 2.4 g
COD per liter per day in M2, the performance of the
reactors was quite good, with constant biogas production
rates (0.8 L/Lday) and CODtotal (<30 g COD/liter) and HPr
concentrations (around 3 g HPr-COD/liter). However, the
increase in the OLR to 2.5–2.7 resulted in the irreversible
decrease of the biogas production to negligible values.
Concomitantly, the CODtotal concentrations rose up to 40–
50 g COD/L. The reactors were stopped on day 162.
Microbial community results
Clustering analyses
Figure 3 shows the cluster analysis for Bacteria in the lab-
scale anaerobic digesters based on the DGGE and the T-
RFLP profiles. A similar discrimination of the samples was
obtained with DGGE and T-RFLP data, with a clear
Appl Microbiol Biotechnol

a b
3,0
Organic loading rate (g COD/L·d) ( )

3,0
and biogasproduction (L/L·d) ( )

2,5
2,5

2,0
2,0

1,5 1,5

1,0 1,0

0,5 0,5

0,0 0,0
0 15 30 45 60 75 90 105 120 135 150 165 0 15 30 45 60 75 90 105 120 135 150 165

c d
60 8,0 60 8,0

Propionic acid concentration

7,0 7,0
Total COD concentration

50 50
6,0

(g HPr-COD/L) ( )
6,0
(g COD/L) ( )

40 40
5,0 5,0

30 4,0 30 4,0

3,0 3,0
20 20
2,0 2,0
10 10
1,0 1,0

0 0,0 0 0,0
0 15 30 45 60 75 90 105 120 135 150 165 0 15 30 45 60 75 90 105 120 135 150 165
Time (d) Time (d)

Fig. 2 Performance of the mesophilic reactors M1 (a, c) and M2 (b, d). Arrows indicate the sludge sampling points

segregation between the mesophilic and the thermophilic Microbial community organization
communities. Among the mesophilic samples, those from
the first experiment stirred manually twice a day (days 55 The microbial community organization was assessed by
and 72) could be clearly differentiated from those from the calculating the community organization coefficient (Co,
second experiment (days 128, 144, and 158) with mechan- Fig. 4c, d). The higher is the Co, the more uneven is the
ical stirring and enhanced biomass retention. The samples community. In general, higher Co values were obtained
from day 108 were situated in between since they with DGGE (between 42 and 76 for Bacteria and between
corresponded with the end and beginning of experiments 25 and 89 for Archaea) than with T-RFLP data (between 28
1 and 2, respectively. Similar results were obtained with the and 51 for Bacteria and between 42 and 62 for Archaea).
archaeal profiles (data not shown). However, both techniques indicated that the archaeal
organization was more uneven (Co values of 69±22 with
Richness DGGE and 54±7 with T-RFLP) than the bacterial organi-
zation (Co values of 60±9 with DGGE and 41±6 with T-
For Bacteria (Fig. 4a), a similar or even higher number of RFLP) and that both bacterial and archaeal communities
bands were achieved with DGGE analyses (22±8) than were more even in mesophilic reactors (average Co values
with T-RFLP (17±5), while the opposite occurred for of 60 and 45, respectively) than in the thermophilic ones
Archaea (Fig. 4b), 4±3 and 11±4, respectively. However, (average Co values of 70 and 50, respectively).
both techniques indicate that the bacterial community was
richer than the archaeal one independently of temperature, Dynamics of change
and that mesophilic communities were richer than the
thermophilic ones (except for Archaea with T-RFLP). No The values of the rate of change of the bacterial and
clear conclusions can be made about the evolution of the archaeal community were calculated for each reactor per
richness of bacterial and archaeal communities over time. HRT (18 days). The average rates of change were in the
 Appl Microbiol Biotechnol

a Similarity for the archaeal community), but the evolution of the

0,12

0,24

0,36

0,48

0,72

0,84

0,96
dynamics over time was more variable (higher standard

0,6
0

deviations) in mesophilic reactors and for the archaeal

0

M1-55 community.
M2-55
2,5

M2-72 Correlation among process parameters and microbial

M1-128 community
M1-144
5

M1-158 Figure 5 shows the correlations among the three molecular

M2-128
parameters (Co, Rr, and Dy) for Bacteria and Archaea. No
7,5

M2-144
clear correlations among the parameters could be obtained
M2-158
from T-RFLP profiles (data not shown). However, the
10

M1-72
DGGE profiles show some general trends for both Bacteria
M1-108
M2-108
and Archaea. The more even the community was (lower
12,5

T2-84
Co), the higher the richness (Fig. 5a, b) was. The higher the
T1-84 dynamics, the lower the richness (Fig. 5c, d) and the more
uneven the community (Fig. 5e, f). These correlations were
15

T1-72
T2-72 statistically significant at 95% confidence interval for
T2-54 Bacteria (p<0.05), while for Archaea, only the correlation
17,5

T1-54 between Co and R was significant.

Figure 6 shows the correlations between the molecular
(Co, Rr, and Dy) and the operational (Ripley index and
b Similarity biogas production) parameters for Bacteria, while no
correlations could be obtained for Archaea (data not
0,12

0,24

0,36

0,48

0,72

0,84

0,96
0,6
0

shown). Some statistically significant trends could be

0

M1-55 observed for Bacteria (p ranging from 0.0013 and

M2-55 0.0052). The more uneven the microbial community was,
2,5

M1-72 the poorer the reactor performance was, i.e., the higher the
M2-72 Ripley index (Fig. 6a), and the lower the biogas production
M1-108
5

(Fig. 6b). On the contrary, the higher the richness, the better
M2-108
the performance, i.e., the lower the Ripley index (Fig. 6c)
M1-128
7,5

and the higher the biogas production (Fig. 6d). Although

M1-144
M1-158
some trends were observed between Dy and reactor
performance (Fig. 6e, f), the correlations were weak (R2 <
10

M2-128
M2-144 0.30) and not statistically significant (p>0.05).
M2-158
12,5

T2-54
T1-54 Discussion
15

T1-84
T2-72
An ecological interpretation of the behavior of microbial
T2-84
17,5

communities under particular conditions in function of time

T1-72
Fig. 3 Cluster analysis of Bacteria in thermophilic (T) and meso-
philic (M) reactors based on DGGE (a) and T-RFLP (b) profiles
same range when comparing the DGGE (47–63% for
Bacteria and 43–53% for Archaea) and T-RFLP (48–61%
for Bacteria and 35–74% for Archaea) data (Table 1).
Overall, there were no significant differences between the
two reactors operated at the same temperature, but the
mesophilic communities were somewhat less dynamic.
At each temperature, the rates of change of Bacteria and
Archaea were in the same range (with a trend to be lower
can provide useful information about process performance
and efficiency and can facilitate the comparison between
different reactors situations. This ecological interpretation
should be independent of the fingerprinting method used,
and in this study, the later statement was evaluated using
two molecular techniques (DGGE and T-RFLP) to charac-
terize the bacterial and archaeal communities in continuous
lab-scale anaerobic reactors.
Cluster analyses were independent of the molecular
technique used. Recently, Smalla et al. (2007) showed that,
although the amplified PCR fragments comprised different
variable regions and lengths, DGGE and T-RFLP analyses
Appl Microbiol Biotechnol

Fig. 4 Comparison between mi-

crobial parameters from DGGE
a

Number of bands/peaks
(black) and T-RFLP (white). 40
Richness Bacteria (a), richness
Archaea (b), community orga- 30
nization Bacteria (c), and com-
munity organization Archaea 20
(d). Five samples of Archaea are
10
missing due to problems in the
analyses
0

T1-54

T1-72

T1-84

T2-54

T2-72

T2-84

M1-55

M1-72

M2-55

M2-72

M1-108

M1-128

M1-144

M1-158

M2-108

M2-128

M2-144

M2-158
b

Number of bands/peaks
25

20

15

10

0
T1-84

T2-54

T2-84

M1-55

M2-55

M1-108

M1-128

M1-144

M1-158

M2-108

M2-128

M2-144

M2-158
c
100

80

60
Co

40

20

0
T1-54

T1-72

T1-84

T2-54

T2-72

T2-84

M1-55

M1-72

M2-55

M2-72

M1-108

M1-128

M1-144

M1-158

M2-108

M2-128

M2-144

M2-158
d
100

80

60
Co

40

20

0
T1-84

T2-54

T2-84

M1-55

M2-55

M1-108

M1-128

M1-144

M1-158

M2-108

M2-128

M2-144

M2-158

led to similar findings in terms of clustering of the
fingerprints and replicate variability. A clear segregation
was obtained between mesophilic and thermophilic commu-
nities, but the microbial communities were not distinct enough
to distinguish between the two reactors operated at the same
temperature, probably due to the similar operational con-
ditions and the same type of substrate. This finding is also in
accordance with previous studies, which stressed the predom-
inant influence of temperature (Karakashev et al. 2005; Leven
et al. 2007) and type of substrate or operational conditions
(Leclerc et al. 2004; Lee et al. 2009) on the composition of
the microbial community.
R values are an estimation of “who is there” and
inform on the carrying capacity of an environment, i.e.,
whether an environment is very habitable or adverse/
exclusive (Verstraete et al. 2007). Some differences were

Table 1 Average dynamics between bacterial and archaeal profiles
obtained with DGGE and T-RFLP using an 18-day window
Reactor Bacteria Archaea
DGGE T-RFLP DGGE T-RFLP
T1 61.6±1.6 54.1±7.4 nd 74.2±5.8
T2 62.5±2.5 61.2±5.6 nd 46.7±13.4
M1 47.4±18.1 48.4±10.1 42.7±16.8 34.9±13.1
M2 47.9±13.4 48.2±16.6 53.4±13.5 40.4±13.5
nd no data due to problems with the analyses
observed between DGGE and T-RFLP when evaluating
the richness of the bacterial and archaeal communities. It
is well known that each technique has its own sensitivity
and accuracy. In the case of Bacteria, the region of the gen
targeted by DGGE (positions 338–518, Escherichia coli
16S rRNA gene numbering) is included in the region
targeted by T-RFLP (positions 27–907) and known as the
most variable region of the gen for Bacteria (Li et al.
2009). In contrast, the targeted regions for Archaea are
quite different. Since a higher diversity of Archaea was
observed with T-RFLP, it can be concluded that the region
targeted by T-RFLP (positions 21–915) is more diverse/
variable than the one targeted by DGGE (positions 915–
1352), as previously reported (Yu et al. 2008). However,
both molecular techniques showed that bacterial commu-
nities are richer than archaeal communities, as previously
judged (Fernández et al. 1999; Tang et al. 2004; Dearman
et al. 2006; Hori et al. 2006; Malin and Illmer 2008). This
appears logical since the first group relates to a multitude
of substrates, while the second group is confined to the
narrow niches of methane production from acetate and
hydrogen. It was also observed from both techniques that
mesophilic communities are richer than thermophilic
communities, a finding in agreement with previous
reported data (Karakashev et al. 2005; Leven et al. 2007).
Co values are an estimation of “who is doing what with
whom” and inform on the functional organization of the
microbial community (Marzorati et al. 2008). Low (20–25)
and high (>80) Co values indicate a highly even or
specialized community, respectively, and consequently, a
long lag phase resp. longer recovery times could be needed
to counteract a sudden stress. Average Co values (45–60)
represent balanced communities, which can potentially deal
with changing environmental conditions. Both techniques
indicate that the archaeal community is more uneven than the
bacterial community, regardless the temperature of operation.
In addition, mesophilic communities are more even than
thermophilic communities. The more even bacterial commu-
nities (lower Co values) in mesophilic reactors are in
accordance with previous studies (LaPara et al. 2002).
Appl Microbiol Biotechnol
However, the higher evenness of archaeal communities in
mesophilic temperature has not been well-documented yet.
Dy values are an estimation of the rate of change in a
microbial community and inform on the number of species
that on average come to significant dominance at a given
habitat, during a defined time interval (Marzorati et al.
2008). Stable communities (low Dy values) represent small
reservoirs that limit the influx of new propagules, and they
might be useful in terms of technical performance but
dangerous in terms of overall adaptability (Verstraete et al.
2007). In this study, the community composition of both
Bacteria and Archaea was highly dynamic (30–75% per
18 days) during the whole experiment. The high dynamics
during well-functioning periods can be explained by the
functional redundancy among diverse phylogenetic groups
allowing oscillations of their populations with no effects on
the reactor function (Zumstein et al. 2000; Briones and
Raskin 2003). Transitions between deteriorative and stable
reactor conditions and considerable process events (e.g.,
increase in OLR) are commonly related to significant shifts
in microbial populations (Hori et al. 2006; Lee et al. 2008).
Overall, it was observed that archaeal and mesophilic
communities were less dynamic than bacterial and thermo-
philic communities, respectively. This finding is in agree-
ment with previous studies showing that bacterial
populations display a highly variable pattern of temporal
variation, even with stable reactor performance, while
archaeal populations displayed less temporal variability
(Fernández et al. 1999, Zumstein et al. 2000).
Regardless of the nature of community dynamics in a
constant environment, a stable system must possess the
ability to maintain process stability in response to dis-
turbances. This does not imply that the microbial commu-
nity be stably maintained. In this study, higher dynamics
(>30%) were observed during well- and poor-functioning
periods, a finding in agreement with previous reported data
(Fernández et al. 1999, 2000). However, in reactors with
relatively long sludge residence times (>50 days) and very
slow growing anaerobic microbial communities (cell
doubling times of a week or more), it is surprising to find
different subpopulations emerging to dominance and
becoming subsequently less prominent again in a matter
of weeks. However, it is well known that AD does not
entail a single set of food chains but represents a multitude
of parallel converging bioconversions. Indeed, a wide array
of metabolites (higher and lower fatty acids, alcohols,
amines, etc.) has, each by a specific pathway, to be
converted to the two ultimate principal end products, i.e.,
CH4 and CO2. In such a situation of a “funneling” of
bioconversions, it is conceivable that constantly all routes
are experiencing hinder, e.g., because critical metabolites
(such as hydrogen or HPr) of the one conversion influence
the kinetics of the other conversion. In this context, it stems
Appl Microbiol Biotechnol

a b
80 100

70 y = -0,8342x + 77,867
R² = 0,5143 80
60 y = -8,3595x + 100,16
R² = 0,9832
50 60

Co
Co

40

30 40
p = 0.0008 p = 6.3·10-13
20
20
10

0 0
0 5 10 15 20 25 30 35 40 0 2 4 6 8 10
Richness Richness

c d
70 80

60 70

60 y = -2,8544x + 59,941
50
Dynamics (%)

Dynamics (%)
y = -1,1187x + 77,981 R² = 0,0832
R² = 0,4059 50
40
40
30
30
20
20
p = 0.0350 p = 0.5305
10 10

0 0
0 5 10 15 20 25 30 35 40 0 2 4 6 8 10
Richness Richness

e f
70 80

60 70
y = 1,0491x -9,3789
R² = 0,3938 60
50
Dynamics (%)
Dynamics (%)

50
40
40
30
30 y = 0,3367x + 25,154
20 R² = 0,0833
20
p = 0.0393
p = 0.5294
10 10

0 0
30 40 50 60 70 80 30 40 50 60 70 80 90
Co Co

Fig. 5 Correlations between DGGE-based molecular parameters for Bacteria (a, c, e) and Archaea (b, d, f)

to reason that the multi-routing toward the common funnel
results in a non-occurrence of a dominant and stable team
of organisms that governs the overall process. Hence,
according to the latter hypothesis, the high Dy values may
be a most important characteristic of a well-functioning AD
system in which all of the processes and their responsible
cross-feeding microbial associations properly come to
action. Obviously, further work is required to corroborate
to what extent high dynamics in microbial patterns are
congruent with “equal opportunity” microbial population
dynamics in converging metabolic systems.
One of the novel aspects of this study was the attempt to
correlate the overall behavior of the anaerobic microbial
community to process efficiency by means of the generic
tools/parameters proposed by MRM. Two sets of interesting
and statistically significant trends could be observed with
DGGE-based data of Bacteria and the Ripley index
(Fig. 6a, c). Indeed, for this important index of process
stability, reactors with low Ripley values (and hence good
performance) typically had also low Co resp. high Rr
values (R2 values above 0.75, p<0.006) indicating that an
even and rich association of Bacteria corresponded with
 Appl Microbiol Biotechnol

a b 80
y = -0,2229x + 70,741
R² = 0,5619
70 70
y = 78,479x + 23,273
R² = 0,8273 60
60

Co
50
Co

50
40
p = 0.0017
p = 0.0013
40
30

30 20
0,30 0,35 0,40 0,45 0,50 0,55 0 20 40 60 80 100
Ripley index Biogas production

c d 40
40 35 y = 0,1825x + 14,001
y = -58,373x + 50,848 R² = 0,4773
30
R² = 0,753
30

Richness
25
Richness

20
20
15
p = 0.0052 p = 0.0030
10 10
5
0 0
0,30 0,35 0,40 0,45 0,50 0,55 0 20 40 60 80 100
Ripley index Biogas production

e 70
f
70

60 60
Dynamics (%)

Dynamics (%)

50 50
y = 202,61x -29,992 y = -0,2473x + 63,774
R² = 0,2869 R² = 0,297
40 40

30 30
p = 0.2734 p = 0.0669
20 20
0,25 0,30 0,35 0,40 0,45 0 20 40 60 80 100
Ripley index Biogas production
Fig. 6 Correlations between DGGE-based molecular and operational parameters for Bacteria. Biogas production is shown as the percentage of
the theoretical value based on the COD supplied

good conversion of fatty acids to methane. Wittebolle et al.
(2009) also found that the initial community evenness is a
key factor in preserving the functional stability of an
ecosystem against environmental stress. Although weaker
linear trends (R2 between 0.3 and 0.6) were observable
between Co and Rr and the biogas production (Fig. 6b, d),
these correlations were statistically significant (p<0.003),
and therefore, these positive trends detected between
richness and community evenness and biogas yield certain-
ly warrants further exploration.
To conclude, despite slight differences in the “absolute”
values, the same ecological interpretation was derived from
DGGE and T-RFLP: (a) temperature has a strong effect on the
microbial composition of both Archaea and Bacteria; (b)
bacterial and mesophilic communities are richer than the
archaeal and thermophilic ones, respectively; (c) archaeal and
thermophilic populations are more uneven than bacterial and
mesophilic ones, respectively; (d) The community composi-
tion of both Bacteria and Archaea was highly dynamic (rate
of change between 30% and 75% per 18 days) in well- and
Appl Microbiol Biotechnol
also poor-functioning periods; and (5) a more even (low Co
values) and diverse (high richness) bacterial community was
indicative of a well-functioning reactor.
Acknowledgments This research was funded by the project Sewage
Plus 180B12A7 (MIP project, Milieu- & Energietechnologie–Innova-
tieplatform, Berchem, Belgium), the Geconcerteerde Onderzoeksactie
(GOA) of Ghent University contract grant (BOF09/GOA/005), the
fellowship of CSIC-UdelaR (Uruguay) for Dr. Claudia Etchebehere,
and by a postdoctoral contract for Dr. Marta Carballa from the Xunta
de Galicia (Isidro Parga Pondal program, IPP-08-37). The authors
acknowledge Dr. Jingxing Ma and Varvara Tsilia for their support and
cooperation with the lab work.
References
Barredo MS, Evison LM (1991) Effect of propionate toxicity on
methanogen-enriched sludge, Methanobrevibacter smithii and
Methanospirillum hungatii at different pH values. App Environ
Microbiol 57:1764–1769
Boe K, Steyer JP, Angelidaki I (2008) Monitoring and control of the
biogas process based on propionate concentration using online
VFA measurement. Water Sci Technol 57:661–666
Boon N, Top EM, Verstraete W, Siciliano SD (2003) Bioaugmentation
as a tool to protect the structure and function of an activated-
sludge microbial community against a 3-chloroaniline shock
load. App Environ Microbiol 69:1511–1520
Bouallagui H, Touhami Y, Ben Cheikh R, Hamdi M (2005) Bioreactor
performance in anaerobic digestion of fruit and vegetable wastes.
Process Biochem 40:989–995
Briones A, Raskin L (2003) Diversity and dynamics of microbial
communities in engineered environments and their implications
for process stability. Curr Opin Biotechnol 14:270–276
Dearman B, Marschner P, Bentham RH (2006) Methane production
and microbial community structure in single-stage batch and
sequential batch systems anaerobically co-digesting food waste
and biosolids. App Microbiol Biotechnol 69:589–596
Fernández A, Huang S, Seston S, Xing J, Hickey R, Criddle C, Tiedje
J (1999) How stable is stable? Function versus community
composition. App Environ Microbiol 65:3697–3704
Fernández AS, Hashsham SA, Dollhopf SL, Raskin L, Glagoleva O,
Dazzo FB, Hickey RF, Criddle CS, Tiedje JM (2000) Flexible
community structure correlates with stable community function
in methanogenic bioreactor communities perturbed by glucose.
App Environ Microbiol 66:4058–4067
Greenberg A, Clesceri L, Eaton A (1992) Standard methods for the
examination of water and wastewater, 18th edn. American Public
Health Association Publications, Washington
Hammer Ø, Harper DAT, Ryan PD (2001) PAST: paleontological
statistics software package for education and data analysis.
Palaeontol Electron 4:1–9
Hecht C, Griehl C (2009) Investigation of the accumulation of
aromatic compounds during biogas production from kitchen
waste. Biores Technol 100:654–658
Holdeman L, Cato E, Moore W (1977) Anaerobic laboratory
manual. Virginia Polytechnic Institute and State University,
Blacksberg
Hori T, Haruta S, Ueno Y, Ishii M, Igarashi Y (2006) Dynamic
transition of a methanogenic population in response to the
concentration of volatile fatty acids in a thermophilic anaerobic
digester. App Environ Microbiol 72:1623–1630
Karakashev D, Batstone DJ, Angelidaki I (2005) Influence of
environmental conditions on methanogenic compositions in
anaerobic biogas reactors. App Environ Microbiol 71:331–338
LaPara TM, Nakatsu CH, Pantea LM, Alleman JE (2002) Stability of
the bacterial communities supported by a seven-stage biological
process treating pharmaceutical wastewater as revealed by PCR-
DGGE. Water Res 36:638–646
Leclerc M, Delgenes JP, Godon JJ (2004) Diversity of archaeal
community in 44 anaerobic digesters as determined by single
strand conformation polymorphism analysis and 16S rDNA
sequencing. Environ Microbiol 6:809–819
Lee C, Kim J, Shin SG, Hwang S (2008) Monitoring bacterial and
archaeal community shifts in a mesophilic anaerobic batch
reactor treating a high-strength organic wastewater. FEMS
Microbiol Ecol 65:544–554
Lee C, Kim J, Hwang K, O’Flaherty V, Hwang S (2009) Quantitative
analysis of methanogenic community dynamics in three anaero-
bic batch digesters treating different wastewaters. Water Res
43:157–165
Leven L, Eriksson ARB, Schnürer A (2007) Effect of process
temperature on bacterial and archaeal communities in two
methanogenic bioreactors treating organic household waste.
FEMS Microbiol Ecol 59:683–693
Li H, Zhang Y, Li DS, Xu H, Chen GX, Zhang CG (2009)
Comparisons of different hypervariable regions of rrs genes for
fingerprinting of microbial communities in paddy soils. Soil Biol
Biochem 41(5):954–968
Liu G, Zhang R, Sun Z, Li X, Dong R (2007) Research progress in
anaerobic digestion of high moisture organic solid waste. Agric
Eng Int 9 (e-journal)
Lueders T, Manefield M, Friedrich MW (2004) Enhanced sensitivity
of DNA- and rRNA-based stable isotope probing by fractionation
and quantitative analysis of isopycnic centrifugation gradients.
Environ Microbiol 6:73–78
Malin C, Illmer P (2008) Ability of DNA content and DGGE analysis
to reflect the performance condition of an anaerobic biowaste
fermenter. Microbiol Res 163:503–511
Marzorati M, Wittebolle L, Boon N, Daffonchio D, Verstraete W
(2008) How to get more out of molecular fingerprints: practical
tools for microbial ecology. Environ Microbiol 10:1571–
1581
McHugh S, Collins G, Mahony T, O’Flaherty V (2004) Biofilm
reactor technology for low temperature anaerobic waste treat-
ment: microbiology and process characteristics. IWA Internation-
al Conference on Biofilm Structure and Activity. IWA, Las
Vegas, pp 107–113
Miura Y, Hiraiwa MN, Ito T, Itonaga T, Watanabe Y, Okabe S (2007)
Bacterial community structures in MBRs treating municipal
wastewater: relationship between community stability and reactor
performance. Water Res 41:627–637
Mosche M, Jordening HJ (1998) Detection of very low saturation
constants in anaerobic digestion: influences of calcium
carbonate precipitation and pH. App Microbiol Biotechnol
49:793–799
Muyzer G, de Waal EC, Uitterlinden A (1993) Profiling of complex
microbial populations using denaturing gradient gel electropho-
resis analysis of polymerase chain reaction-amplified genes
coding for 16S rRNA. App Environ Microbiol 59:695–700
Rincón B, Borja R, González JM, Portillo MC, Sáiz-Jiménez C (2008)
Influence of organic loading rate and hydraulic retention time on
the performance, stability and microbial communities of one-
stage anaerobic digestion of two-phase olive mill solid residue.
Biochem Eng J 40:253–261
Ripley LE, Boyle WC, Converse JC (1986) Improved alkalimetric
monitoring for anaerobic digestion of high-strength wastes. J
WPCF 58:406–411

Riviere D, Desvignes V, Pelletier E, Chaussonnerie S, Guermazi S,
Weissenbach J, Li T, Camacho P, Sghir A (2009) Towards the
definition of a core of microorganisms involved in anaerobic
digestion of sludge. ISME J 3:700–714
Rooney-Varga JN, Giewat MW, Duddleston KN, Chanton JP, Hines
ME (2007) Links between archaeal community structure,
vegetation type and methanogenic pathway in Alaskan peatlands.
FEMS Microbiol Ecol 60:240–251
Smalla K, Oros-Sichler M, Milling A, Heuer H, Baumgarte S, Becker
R, Neuber G, Kropf S, Ulrich A, Tebbe CC (2007) Bacterial
diversity of soils assessed by DGGE, T-RFLP and SSCP
fingerprints of PCR-amplified 16S rRNA gene fragments: do
the different methods provide similar results? J Microbiol
Methods 69:470–479
Talbot G, Topp E, Palin MF, Massé DI (2008) Evaluation of molecular
methods used for establishing the interactions and functions of
microorganisms in anaerobic bioreactors. Water Res 42:513–537
Tang Y, Shigematsu T, Ikbal MS, Kida K (2004) The effects of micro-
aeration on the phylogenetic diversity of microorganisms in a
thermophilic anaerobic municipal solid-waste digester. Water Res
38:2537–2550
Appl Microbiol Biotechnol
Verstraete W, Morgan-Sagastume F, Aiyuk S, Waweru M, Rabaey K,
Lissens G (2005) Anaerobic digestion as a core technology in
sustainable management of organic matter. Water Sci Technol 52
(1–2):59–66
Verstraete W, Wittelbolle L, Heylen K, Vanparys B, de Vos P, van de
Wiele T, Boon N (2007) Microbial resource management: the
road to go for environmental biotechnology. Eng Life Sci 7:117–
126
Wittebolle L, Marzorati M, Clement L, Balloi A, Daffonchio D,
Heylen K, De Vos P, Verstraete W, Boon N (2009) Initial
community evenness favours functionality under selective stress.
Nature 458:623–626
Yu Z, García-González R, Schanbacher FL, Morrison M (2008)
Evaluations of different hypervariable regions of archaeal 16S r
RNA genes in profiling of methanogens by archaea-specific PCR
and denaturing gradient gel electrophoresis. App Environ Micro-
biol 74(3):889–893
Zumstein E, Moletta R, Godon JJ (2000) Examination of two years of
community dynamics in an anaerobic bioreactor using fluores-
cence polymerase chain reaction (PCR) single-strand conforma-
tion polymorphism analysis. Environ Microbiol 2:69–78