Biotechnology Letters 23: 241–248, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

241

Monitoring growth of the methanogenic archaea Methanobacterium

formicicum using an electronic nose

Jennie Brandgård1,∗∗ , Ingvar Sundh2,∗ , Åke Nordberg1 , Anna Schnürer2 , Carl-Fredrik

Mandenius3 & Berit Mathisen2
1 Swedish Institute of Agricultural Engineering, P.O. Box 7033, S-750 07 Uppsala, Sweden
2 Department of Microbiology, SLU, P.O. Box 7025, S-750 07 Uppsala, Sweden
3 Division of Biotechnology and Swedish Sensor Centre, Department of Physics and Measurement Technology,

Linköping University, S-581 83 Linköping, Sweden

∗ Author for correspondence (Fax: +46 18 673392; E-mail: ingvar.sundh@mikrob.slu.se)
∗∗ Present address: Pharmacia & Upjohn, S-112 87 Stockholm, Sweden

Received 17 November 2000; Accepted 29 November 2000

Key words: chemical gas sensors, electronic nose, Methanobacterium formicicum, methanogenic growth

Abstract
Growth of the methanogenic archaea, Methanobacterium formicicum, in pure culture was monitored by analysing
samples from the gas phase with an array of chemical gas sensors (an ‘electronic nose’). Analyses of the methane
and protein formation rates were used as independent parameters of growth, and the data obtained from the
electronic nose were evaluated using principal component analysis (PCA). We found that different growth phases
can be distinguished with the electronic nose followed by PCA evaluation. The fast response of the sensors in
combination with the high correlations with other parameters measuring growth show that the electronic nose can
be a useful tool to rapidly determine methanogenic growth.

Introduction or in the bulk of the sensor material, which leads

During the growth of methanogens in pure culture,
volatile compounds originating from the microorgan-
ism and the growth medium appear in the gas phase.
The concentrations of these compounds vary with the
medium composition and the growth phase of the cul-
ture. If the changes in the complex pattern of concen-
tration data during the growth could be monitored, it
would be possible to distinguish between the different
growth phases in real time. One way to characterise
the gas composition is to use chemical multisensor
arrays, so-called electronic noses. An electronic nose
(Gardner & Bartlett 1994) is an array of chemical gas
sensors with different selectivity, whose responses to
gas mixtures are evaluated with pattern recognition
methods. Instead of using single sensors, an array
of sensor elements with broad and partly overlapping
selectivity is advantageous. These are in most cases
based on adsorption or chemisorption on the surface,
to physical changes that are subsequently registered
(Lundström 1996). Even if the selectivity of each sen-
sor is limited, the combination of all sensors in the
electronic nose furnish a large information content.
An electronic nose is therefore particularly efficient in
measuring complex conditions, rather than a number
of single parameters.
Several applications of the electronic nose have
been reported, for example, odour classification of
grains (Börjesson et al. 1996), analysis of urine sam-
ples (Di Natale et al. 1999), monitoring sausage
fermentation (Eklöv et al. 1998), detection of pollen
(Kalman et al. 1997), characterisation of waste-water
(Stuetz et al. 1999), and monitoring of microbial and
cellular growth in bioreactors (Bachinger & Mande-
nius 2000b, Mandenius 1999). Of particular interest
to bioprocess engineering is that the electronic nose
has been shown to respond to characteristic changes
242
in microbial activity. For example, the electronic nose
has been successfully applied in strain classification
(Gibson et al. 1997, Gardner et al. 1998, Holm-
berg et al. 1998) and to identify the growth phases
of bacteria, yeasts and hamster cells typically used
in bioprocessing (Bachinger & Mandenius 2000a,
Bachinger et al. 1998, 2000, Gardner et al. 1998).
However, it has not yet been shown whether an elec-
tronic nose can detect specific metabolic activity and
growth of methanogenic archaea. If this is possible,
the electronic nose sensor arrays may be useful tools
to monitor methanogenic growth in a biogas reactor
system.
In this study, we investigated whether the re-
sponse pattern, from an array of different chemical
gas sensors, could be used to follow growth and dis-
tinguish different growth phases in a pure culture of
Methanobacterium formicicum growing on hydrogen
and carbon dioxide. The data from the electronic nose
were evaluated using principal component analysis
(PCA) and partial least square (PLS) regression. The
possibility to reduce the number and responses of the
chemical gas sensors was investigated. The responses
of the electronic nose were compared with two inde-
pendent parameters of growth (formation of methane
and protein).
Materials and methods
Organism and growth conditions
Methanobacterium formicicum has been found in
anaerobic digesters (Zellner et al. 1996, Godon et al.
1997) and in sewage sludge (Bryant & Boone 1987),
and typically forms rod-shaped cells, 0.4–0.8 µm
wide and 2–15 µm long, which are often irregularly
crooked. The organism grows with H2 and CO2 as
well as formate, 2-propanol and 2-butanol as sub-
strates for methanogenesis (Whitman et al. 1992). The
cultures of Methanobacterium formicicum (No. 1535,
supplied by DSMZ Germany), were incubated on a
shaker at 37 ◦ C in 118 ml bottles with 21 ml mineral
medium prepared as described previously (Zehnder
et al. 1980, Örlygsson et al. 1993) but with the follow-
ing modification: 1 l medium was supplemented with
1 ml of a solution containing 33.7 µg Na2 WO4 · 2H2 O
and 27.0 µg NaSeO3 · 2H2 O in 200 ml 10 mM NaOH.
An inoculum of 1 ml from cultures growing exponen-
tially were used. The bottles were sealed with butyl
rubber stoppers and aluminium caps, and a gas phase
mixture with 40% H2 , 40% N2 and 20% CO2 was
added to a pressure of 2 atm. Anaerobic methods of
Hungate (1950) as modified by Balch & Wolfe (1976)
were used to prevent air leakage when preparing the
medium and during inoculation with the methanogen.
Experimental set-up
A total of 55 sample bottles containing medium plus
the gas mixture were inoculated with Methanobac-
terium formicicum. Of these, 45 were used for protein
analysis (5 bottles were harvested each sampling), 5
were used for analysis with the electronic nose and
in the remaining 5 only methane content was mea-
sured. Furthermore, 3 uninoculated sample bottles
with medium and gas mixture were used as controls
for the analyses with the electronic nose. The growth
was followed for six days.
Measurements of protein and methane content
Samples for determination of protein content were
withdrawn at nine occasions during the six days. The
suspensions were first centrifuged (10 min, 15 800 g,
20 ◦ C) after which the cell pellets were suspended in
1 ml distilled water. Proteins were extracted by the
addition of 150 µl 5 M NaOH, followed by heating
for 3 min in boiling water (Gijzen et al. 1988). After
addition of 150 µl 5 M HCl and 130 µl 0.15% w/v
sodium desoxycholate, the samples were thoroughly
mixed and allowed to stand for 10 min at room tem-
perature. The samples were then centrifuged (15 min,
2000 g, 4 ◦ C) and the protein present in the super-
natant was precipitated with trichloracetic acid. After
centrifugation (15 min, 2000 g, 4 ◦ C) the precipi-
tates were suspended in distilled water and the protein
content was determined by the Folin Phenol method
according to Peterson (1983).
Methane concentrations were determined by gas
chromatography with flame ionisation detection and
N2 as carrier gas. The column (Porapak T, 2 m long,
2 mm inner diam) worked isothermically at 125 ◦ C
with a carrier gas flow of 30 ml min−1 . Injector and
detector temperatures were both 150 ◦ C. Gas samples
of 0.2 ml were removed from the culture headspaces
with a 1 ml pressure-lock gas syringe. The samples
were transferred to sealed (butyl rubber stoppers and
aluminium caps) 21 ml test-tubes containing 1 ml satu-
rated NaCl solution. Until analysis, the test tubes were
stored upside down to prevent gas leakage through the
stoppers.

Fig. 1. Schematic figure of the five response parameters obtained from the electronic nose.
The electronic nose
Samples of 0.3 ml for analysis with the electronic
nose were withdrawn from the headspace at regular
intervals, using a pressure-lock gas syringe. The sam-
ples were transferred to 10 ml glass bottles with teflon
covered caps. These bottles were previously flushed
and filled with N2 to atmospheric pressure. Samples
of 5 ml from these bottles were transferred to a sam-
ple chamber using a pressure-lock glass syringe. The
chamber was made of glass and teflon, and filled with
technical air. This procedure diluted the samples ap-
proximately 2500 times. Responses from the chemical
gas sensors were rapid and the time to analyse one
sample was about 10 min.
The electronic nose was assembled at the De-
partment of Physics and Measurement Technology,
Linköping University, Sweden. The instrument was
configured with two types of semiconductor sensors:
metal oxide semiconductors, MOSs (10 tin oxide sen-
sors with different specifications, purchased from Fi-
garo, Inc., Japan, and 10 tin oxide sensors from F.I.S.,
Inc., Japan); and metal oxide semiconductor field ef-
fect transistors, MOSFETs (10 sensors with different
gate metals/oxide layers, designed and manufactured
at the Department of Physics and Measurement Tech-
nology). Each sensor response curve allowed calcula-
tion of five response parameters as shown in Figure 1
(absolute response, on-derivative, off-derivative, on-
integral, off-integral), thereby producing 150 response
parameters from each analysed sample.
243
The sensors respond to a variety of volatile or-
ganic and inorganic compounds, e.g., methane, H2 ,
sulphide and acetic acid (Mandenius 1999). Although
the selectivity of the sensors is limited, qualitative
and quantitative gas analysis can be performed using
a combination of parameters from multiple chemical
gas sensors and multivariate statistical methods, e.g.,
principal component analysis, PCA (Gardner et al.
1990).
Multivariate data analysis
Multivariate pattern recognition methods were used to
study and obtain an overview of the data by reducing
the data set. This enabled us to visualise relations and
differences among the measurements and the sensor
signals. PCA has become one of the most widely used
techniques for visualising multivariate data (Jolliffe
1986). It finds the directions in space of the sensor
responses that have the largest variation, and thus
reduces the dimensionality of the data set. The am-
plitude of the responses differed substantially among
the individual chemical gas sensors. The response
parameters were therefore normalised (1/sdev ) before
performing PCA, to ensure that each scaled variable
obtained the same variance. In partial least squares
(PLS) regressions (Geladi & Kowalski 1986) the data
set contains both dependent (X-matrix, e.g., sensor
responses) and an independent (Y-matrix, e.g., gas
concentrations) variable values. PLS uses information
in the Y-space to find the Y-relevant information in
244

Fig. 2. Accumulation of methane in the headspace (a) and protein content in the liquid (b) during growth of monocultures of Methanobacterium
formicicum. Five replicates were withdrawn on nine occasions during six days.

the X-space. Plots of predicted versus measured values
gave an idea of how good the PCA model was.
Results
The methane and protein content increased until day
4 after inoculation, after which the increase quickly
levelled off (Figure 2). The rates of methane and pro-
tein increase differed among the replicate bottles, but
the overall pattern was similar. Also, both the pro-
tein and methane contents in the stationary phase were
quite similar among replicates, roughly 0.15 mg and
0.4 nmol in each bottle, respectively.
The PCA of the sensor responses shows that a
gradual change in responses occurs over time (Fig-
ure 3). As the methanogen grows, the responses move
to the left in the plot, and different growth phases can
 245

Fig. 3. (a) Score plots [principal component 1 (PC 1) versus principal component 2 (PC 2)] of the PCA (principal component analysis) where
all original sensor responses are used. (b) Different bottles are represented by different symbols. Numbers denote the sampling times in
chronological order. Samples marked H2 /CO2 only contained gas.

be distinguished (Figure 3b). The movement in the
PCA is largest during exponential growth (log phase).
At the beginning, and the end of the experiment (sta-
tionary phase) there is a small oscillation around a
state of equilibrium. The uninoculated controls and the
gas samples (H2 /CO2 ) changed little throughout the
experiment.
We could reduce the number of responses and sen-
sors from 150 to 10 without substantial loss of relevant
information. In particular, the absolute response of the
MOS-sensors gave an almost equal amount of infor-
mation as all the sensors collectively. This is illustrated
in the PCA, where the overall pattern remained and
the relationships between the different categories of
246

Fig. 4. Principal component analysis (PCA) of sensor responses with a reduced number of parameters.

Fig. 5. Partial least square regression of sensor responses versus methane content. Full cross-validation and five principal components were
used in the validation procedure. The five replicate bottles are represented by different symbols and the numbers denotes the different sampling
times in chronological order.

samples were the same as when all sensor responses Discussion

were used (Figure 4).
In the PLS regression studies the GC-analysed
methane concentration was strongly correlated to the
predicted (Figure 5), showing that the response from
the electronic nose contained components directly
related to methane concentration.
Both the methane and protein contents in the bottles
increased during incubation and they showed a strong
positive correlation. Thus, the methanogen did grow
in all bottles, albeit at different growth rates. Addi-
tionally, the PLS analysis showed that the collective
signals from the electronic nose yielded information

directly related to the methane concentration. Obvi-
ously, the electronic nose can distinguish between dif-
ferent growth phases in pure culture laboratory batches
of methanogens with accumulation of methane. This
is in accordance with earlier reports that an electronic
nose can distinguish between growth phases during
batch growth of Escherichia coli and Staphylococcus
aureus (Bachinger et al. 1998, Gardner et al. 1998,
Mandenius et al. 1998). However, these other bac-
teria were grown aerobically, and to our knowledge,
no one has yet characterised the growth dynamics of
methanogens using an electronic nose.
In the PCA, the gas samples (only H2 /CO2 ) and
the controls (H2 /CO2 and uninoculated medium) are
concentrated to a restricted area in the plot, and no ma-
jor movement with time can be discerned. The sample
bottles (H2 /CO2 and inoculated medium), on the other
hand, moved in the plot according to the same pattern,
but on slightly different time scales. The changing pat-
tern in the response from the electronic nose implies
that the gas phase composition changed with time.
The chemical gas sensors are sensitive to methane,
H2 and sulphurous compounds (Armgarth et al. 1982,
Lundström et al. 1989, Hörnsten et al. 1991) all of
which are likely to appear in fairly high concentra-
tions in the Methanobacterium cultures. We expected
that we would be able to reduce the impact of these
compounds on the response of the electronic nose,
hoping that other compounds, in lower concentrations
or with lower affinity to the sensors, could then be
detected. For example, it would be interesting if the
electronic nose could detect specific metabolites from
methanogens. Such detection of specific metabolites
would perhaps make it possible to follow the dynamics
of methanogens in complex systems, e.g., biogas reac-
tors. However, the environment in large-scale biogas
reactors differs markedly from the laboratory batch
cultures used in this experiment and the results are not
easily scaled-up. In line with this, we found no clear
connections between the results from these laboratory
batch cultures of Methanobacterium formicicum cul-
tures, and experiments using the electronic nose in our
small-scale biogas process (a mesophilic [37 ◦ C] con-
tinuously stirred tank reactor [CSTR] with 8 l active
volume and 14 l total volume [Nordberg et al. 2000]).
Clearly, more methanogens in pure culture need to
be studied. Of particular interest are the methanogens
growing on acetate, because they are very important
in biogasprocesses. For example, acetate utilisers have
been estimated to account for at least half of the
methanogenic populations in biogas reactors (Raskin
247
et al. 1995). Also, Gujer & Zehnder (1983) reported
that approximately 70% of the methane produced in
anaerobic digesters comes from the decarboxylation
of acetate.
When the data from the electronic nose were
processed and the number of chemical gas sensors and
responses were reduced, the first step was to minimise
the influence from methane. When a sensor is less sen-
sitive to methane, it is more probable that it is more
sensitive to other volatile compounds, e.g., specific
metabolites. Therefore, the sensors most sensitive to
methane (according to regression coefficients from the
PLS regression, Figure 4) were removed first. In the
second step the relative importance of the five different
responses from each sensor (Figure 1) were compared,
by their relevance for the general pattern in the PCA
plot (Figure 3a). The absolute response parameter
(Figure 1) was sufficient to build a model with little
loss of information in the plot (Figures 3a and 3b). In
the third stage the sensors were withdrawn by hand,
one at a time, and the resulting plots were inspected.
If the model remained practically the same, the sensor
had low relevance in the plot and was consequently
removed. The similarity between the PCA patterns
with all data and the reduced set (Figure 4) shows
that a less complicated electronic nose is sufficient to
characterise this system.
The gas phase composition changed during growth
of the methanogen. Particularly, the hydrogen con-
tent of the gas phase decreased since it was used as
substrate by the methanogen, and the methane concen-
tration increased. However, we could not say precisely
to what extent the responses from the electronic nose
depended on the decrease in hydrogen content, or in
the increase in methane content, or other parameters.
Our experiment shows that it is possible to fol-
low the growth of a methanogen in laboratory batch
cultures with an electronic nose. Gradually this knowl-
edge may lead to the application of this method to
production biogas reactors, where the status of the
methanogens in such a reactor may be characterised
from responses of an electronic nose. Before that,
however, it is necessary to study how the electronic
nose responds to other methanogens in pure cultures,
e.g., acetotrophic species. A major advantage of the
electronic nose to detect disturbance in microbiolog-
ical processes is that it can be connected on-line and
non-invasively to the bioreactor outlet. The fast re-
sponse of the sensors shows the electronic nose may be
used in future to rapidly determine the physiological
state of methanogenic communities.
248
Acknowledgements
We thank Anette Levin and Mikael Hansson for ex-
cellent technical assistance, and Anders Carlsson for
valuable contribution on MOS sensor evaluation. This
work was supported by the Swedish Council for
Forestry and Agricultural Research, SJFR, contract
number 30.0658/96.
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