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Applied and Environmental Microbiology, October 1999, p. 4497-4505, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Localization and In Situ Activities of Homoacetogenic Bacteria in
the Highly Compartmentalized Hindgut of Soil-Feeding Higher
Termites (Cubitermes spp.)
Anne
Tholen and
Andreas
Brune*
Fakultät für Biologie,
Mikrobielle Ökologie, Universität Konstanz, 78457 Konstanz,
Germany
Received 4 June 1999/Accepted 4 August 1999
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ABSTRACT |
Methanogenesis and homoacetogenesis occur simultaneously in the
hindguts of almost all termites, but the reasons for the apparent predominance of methanogenesis over homoacetogenesis in the hindgut of
the humivorous species is not known. We found that in gut homogenates of soil-feeding Cubitermes spp., methanogens outcompete
homoacetogens for endogenous reductant. The rates of methanogenesis
were always significantly higher than those of reductive acetogenesis,
whereas the stimulation of acetogenesis by the addition of exogenous
H2 or formate was more pronounced than that of
methanogenesis. In a companion paper, we reported that the anterior gut
regions of Cubitermes spp. accumulated hydrogen to high
partial pressures, whereas H2 was always below the
detection limit (<100 Pa) in the posterior hindgut, and that all
hindgut compartments turned into efficient H2 sinks when
external H2 was provided (D. Schmitt-Wagner and A. Brune,
Appl. Environ. Microbiol. 65:4490-4496, 1999). Using a microinjection
technique, we found that only the posterior gut sections P3/4a and P4b,
which harbored methanogenic activities, formed labeled acetate from
H14CO3
. Enumeration of
methanogenic and homoacetogenic populations in the different gut
sections confirmed the coexistence of both metabolic groups in the same
compartments. However, the in situ rates of acetogenesis were strongly
hydrogen limited; in the P4b section, no activity was detected unless
external H2 was added. Endogenous rates of reductive
acetogenesis in isolated guts were about 10-fold lower than the in vivo
rates of methanogenesis, but were almost equal when exogenous
H2 was supplied. We conclude that the homoacetogenic populations in the posterior hindgut are supported by either substrates other than H2 or by a cross-epithelial H2
transfer from the anterior gut regions, which may create microniches
favorable for H2-dependent acetogenesis.
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INTRODUCTION |
In the absence of other electron
acceptors, CO2 is the terminal electron sink in
anoxic environments. Formate and molecular hydrogen are typical
products formed in the fermentative degradation of organic
compounds. Their reducing equivalents are used either by methanogenic
archaea for the direct reduction of CO2 to CH4 or by homoacetogenic bacteria for the reduction of
CO2 to acetate, which is subsequently converted to
CO2 and CH4 by aceticlastic methanogens
(32, 34).
In most environmental situations, the free-energy change (per carbon)
of CO2 reduction to CH4 is larger than that of
CO2 reduction to acetate (32). It has been shown
that the minimum H2 partial pressures in pure cultures of
methanogens are roughly 1 order of magnitude lower (3 to 10 Pa) than
those achieved by homoacetogens (50 to 100 Pa) under similar conditions
(12, 34). This probably explains why methanogenesis usually
predominates over homoacetogenesis as the terminal electron sink
reaction (32). Only at lower temperatures does the
thermodynamic advantage change in favor of homoacetogenesis, and also
in slightly acidic environments, methanogens may not be as competitive
as homoacetogens as a hydrogen sink (for reviews, see references
31, 32, and 34).
The intestinal tracts of animals are generally characterized by the
coexistence of homoacetogenic and methanogenic microorganisms (5,
39). In principle, homoacetogenesis is considered advantageous for the host organism, which is able to use acetate as a carbon and/or
energy source (7). However, the numbers of
H2-oxidizing methanogenic archaea cultivated from rumen or
cecum contents of different mammals are often orders of magnitude
higher than those of homoacetogenic bacteria (23).
Especially in ruminants, the energy loss via methane production is
considerable, and many efforts have been made to shift the ruminal
fermentation towards acetogenesis in order to reduce methanogenesis and
thereby increase feed exploitation by sheep and cattle (22,
25).
Although in situ rates of H2-dependent homoacetogenesis are
generally low in ruminal samples and pig hindgut, they increase when
methanogenesis is inhibited by bromoethanesulfonate (BES) or when the
H2 partial pressure is increased (13, 25).
Inhibition of methanogens by BES also allowed the demonstration of
significant numbers of H2-oxidizing homoacetogens in
rumen and hindgut contents of animals or in human feces
(14). The competition for H2 seems to be a
decisive factor: in rumen samples incubated under an H2 headspace, reductive acetogenesis was significantly stimulated by the
addition of the homoacetogenic Acetitomaculum ruminis or Peptostreptococcus productus only when the methanogens were
simultaneously inhibited with BES (22, 25).
Unfortunately, not much is known about the hydrogen partial pressures
in the intestinal tracts of animals in which homoacetogenesis has been
identified as the dominating process (7). Considering the
metabolic versatility of homoacetogens, which are capable of utilizing
other substrates in the absence of H2 or mixotrophic with
H2 (5), and the apparently genetic determination
in different animal lineages of the ability to host methanogens in
their intestinal tracts (17), the actual reasons governing
the coexistence of both metabolic groups are complex and may differ in
each case.
Termite guts are an excellent example of this situation (6).
Breznak and coworkers showed that CO2 reduction to acetate in gut homogenates of most wood-feeding termites exceeds methane emission rates by an order of magnitude (3, 8). In
Reticulitermes flavipes, the explanation for the
predominance of homoacetogens was found in the high hydrogen partial
pressures in the hindgut, which may exceed the typical threshold values
of homoacetogenic bacteria by more than 2 orders of magnitude
(16). Since the methanogenic population in this termite is
restricted almost exclusively to the microoxic gut periphery (16,
20), it has been postulated that in this case, the predominance
of homoacetogenesis is founded on a spatial separation of the
methanogenic and homoacetogenic populations (11, 16).
In the much more abundant and globally very important group of
soil-feeding termites (40), which exhibit a strong hindgut compartmentalization (1, 24) and an extreme alkalinity (>pH 12) in the anterior hindgut (2, 10), the digestive
physiology is still largely obscure (11). In an earlier
study, Brauman et al. (3) had shown that in contrast to
wood-feeding termites, the soil-feeding species emit relatively large
amounts of CH4, while the rates of homoacetogenesis in
their gut homogenates are an order of magnitude lower. At that time,
the authors had already cautioned that homogenization and dilution of
the gut contents inevitably disrupt the physical interaction of
hydrogen-producing and hydrogen-consuming microorganisms within the
hindgut, which might lead to a serious underestimation of
homoacetogenesis in situ (3).
Our current study of soil-feeding Cubitermes spp. tries to
resolve this issue by addressing the spatial distribution of
homoacetogenic and methanogenic microorganisms in the different gut
compartments and their respective activities under in situ conditions.
In a companion paper, we reported that (i) H2 accumulates
only in the anterior gut regions, while H2 partial
pressures in the posterior hindgut are always below the detection limit
(<100 Pa); (ii) only the anterior gut regions represent hydrogen
sources, whereas all hindgut regions turn into H2 sinks
when external H2 is provided; and (iii) methanogenic
activities are localized only in the posterior gut regions
(33).
In the present paper, we used radiotracer techniques to estimate the
distribution and numerical abundance of H2-oxidizing methanogenic and homoacetogenic populations in the hindgut. By microinjection of minute amounts of radiotracers, we determined the
localization and the in situ activities of reductive acetogenesis in the individual hindgut compartments. Finally, we discuss the results
together with those of the microsensor studies described in the
companion paper (33).
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MATERIALS AND METHODS |
Termites.
Cubitermes orthognathus Emerson and
Cubitermes umbratus Williams (Termitidae: Termitinae) were
collected near Busia (Kenya) and in the Shimba Hills Natural Reserve
(Kenya), respectively. Nest fragments with termites were brought to the
laboratory in polypropylene containers together with soil from the
collection site; measurements were generally performed within 1 to 2 months of collection. Worker caste termites were used for all experiments.
14CO2 reduction by gut homogenates.
Termites were dissected and guts were homogenized (10 guts
ml1) in anoxic buffered salt solution (BSS
[37]), reduced with 1 mM dithiothreitol (DTT), by
using a glass tissue homogenizer. Aliquots of the suspension (0.75 ml)
were dispensed into 5-ml glass serum vials and sealed with butyl rubber
stoppers. The whole procedure was performed in an anoxic glove box
under N2 (2 to 5% H2); all equipment was
preincubated in the glove box for 48 h.
In the experiments with C. umbratus, the vials were gassed
with H2 or N2 for 2 min, and 25 µl of an
anoxic aqueous solution of NaH14CO3 (0.71 µmol; 0.15 MBq) was immediately injected. Considering the internal
CO2 pool of C. umbratus hindguts (78 nmol
termite1 [36]), the final amount of
CO2 in the assay was 1.3 µmol. In the case of C. orthognathus, bicarbonate buffer (30 mM) was added to the BSS, and
the vials were gassed with H2-CO2 or
N2-CO2 gas mixtures (both 80/20 [vol/vol]),
increasing the total amount of CO2 to 57.1 µmol. Fifty
microliters of an anoxic aqueous solution of
Na214CO3 (1.44 µmol; 2.8 MBq) or
[14C]Na-formate (4.1 µmol; 7.8 MBq) was injected. The
final pH of the assays was between 7.2 and 7.4.
Vials were incubated at 30°C on a rotary shaker (200 rpm), and
samples were taken every hour by using syringes equipped with
gas-tight
valves and flushed with N
2. Headspace samples (50 µl)
were analyzed for
14CH
4 and
14CO
2 by gas chromatography as described below.
Liquid-phase samples
(50 µl) were combined with 50 µl of a solution
containing nonradioactive
standards (acetate, formate, lactate,
propionate, butyrate, isobutyrate,
and ethanol at 5 mM each) in NaOH
(0.4 M). After centrifugation
(5 min at 14,000 ×
g),
the supernatant was analyzed for radioactivity
and label distribution
by liquid scintillation counting (LSC)
and by high-performance liquid
chromatography (HPLC) (see below).
The pellet was washed and analyzed
by LSC after heat treatment
(15 min at 80°C), and a complete
radioactivity balance was
performed.
Microinjection of 14C-labeled compounds.
For
microinjection of radiolabeled compounds, we used a hydraulic system
consisting of a 25-µl Hamilton syringe actuated by means of a
micrometer drive (10-µm minimum step increment) and connected to a
50-µl glass micropipette via PEEK high-pressure capillaries and
fittings. The system was filled with silicone oil, and great care was
taken to purge out all air bubbles. The micropipette was drawn to a
fine tip with a pipette puller, broken back to a tip diameter of 7 to
12 µm, and heat polished. The inner surface was coated with paraffin
by filling the tip with a mixture of toluene and paraffin oil (95%/5%
[vol/vol]) and drying the micropipette for 24 h at 100°C.
Micropipettes were positioned by means of a manual micromanipulator,
and the position of the tip was controlled visually with a
stereomicroscope; the whole setup was essentially the same as that used
for the microsensor studies (33).
The micropipette was filled with 1 to 2 µl of an aqueous solution of
the respective radioactive metabolite kept under silicone
oil to avoid
evaporation. Immediately before injection, termites
were dissected and
hindguts were embedded flat and fully extended
(see Fig.
1 in the
companion paper [
33]) in a glass microchamber
by using
agarose made up with insect Ringer's solution (
33)
and
preincubated under a controlled headspace of air or H
2; the
setup was as described by Ebert and Brune (
16). The
micropipette
was inserted into individual gut sections, with the tip
positioned
at the center of the respective compartment, and minute
volumes
of the label (40 to 80 nl) were injected via hydraulic
pressure.
Micropipettes were calibrated before and after each
experiment
by delivering aliquots of the respective tracer solution
directly
into LSC vials filled with scintillation cocktail. Over all
pipettes
tested, the deviation from the average of the injected label
was
3.8% ± 1.7% (
n = 111).
At different time intervals after injection, the guts were removed from
the agarose and immediately disrupted by sonication
in 0.1 ml of NaOH
(0.2 M) containing nonlabeled standards (see
above), by using an
ultrasonic probe with a microtip (60 W; Ultraschallprozessor
50 H; Dr.
Hielscher GmbH, Teltow, Germany). After centrifugation
(14,000 ×
g for 5 min), aliquots of the supernatant
were analyzed
for radioactivity and label distribution by LSC and HPLC.
The
pellet was analyzed as described above. The agarose block (~0.5
cm
3) which had surrounded the guts was transferred into 0.5 ml of
0.2 M NaOH containing nonlabeled standards (see above) and was
cut into pieces. After 3 days of equilibration at 4°C, aliquots
of
the liquid were analyzed by LSC and HPLC. For a complete radioactivity
balance of the injected label, the remaining mixture was added
quantitatively to scintillation vials, and the recovery was determined
by comparing the sum of the radioactivities in the pellet, supernatant,
and
agarose.
Enumeration of bacteria.
Three-tube most-probable-number
(MPN) determinations were performed essentially as described before
(37), with anoxic, bicarbonate-buffered mineral medium AM 4 (37) containing bovine rumen fluid (2% [vol/vol]), acetate (1 mM), and resazurin (10 mg liter1) as a redox
indicator and a headspace of H2-CO2 (80/20
[vol/vol]). All tubes contained a palladium catalyst (5% palladium
on activated carbon; Aldrich, Steinheim, Germany; final catalyst
concentration, 50 mg liter1) to ensure reducing
conditions; hydrogen-free controls were reduced with DTT (1 mM). When
indicated, a mixture of additional unlabeled substrates
(D-glucose, 5 mM; methanol, 10 mM; formate, 10 mM) or BES
(3 mM) was added. All tubes received
Na214CO3 (10.3 kBq
ml1, 0.22 GBq mmol1), and were incubated
for 10 weeks at 30°C on a rotary shaker. Tubes were scored as
positive for H2-dependent acetogenesis, methanogenesis, or
formate production when the amount of labeled product was above that of
H2-free controls. Background growth was followed
photometrically by measuring the turbidity of the cultures at 600 nm.
MPNs were computed with the universal equation for MPN calculation
(19).
Analytical methods.
To analyze the total amount of
radioactivity in liquid samples, 10-µl aliquots were added to 3.5 ml
of Pico Aqua liquid scintillation cocktail (Canberra Packard,
Frankfurt, Germany) and were analyzed with a liquid scintillation
counter (LS 1801; Beckman Instruments, München, Germany).
Duplicate assays were performed; all values were quench corrected by
using n-[14C]hexadecane as internal standard.
Liquid samples (50 µl) were analyzed by HPLC with a system equipped
with an ion-exclusion column and a refractive-index detector
(
37); radioactivity was measured with an on-line flow
scintillation
analyzer (Ramona 2000; Raytest, Straubenhardt, Germany)
with a
cell volume of 1.2 ml. The scintillation cocktail (Quicksafe
Flow
2; Zinsser Analytic, Eschborn, Germany) was used at a
buffer/cocktail
ratio of 1:3. Radioactive gases
(
14CH
4 and
14CO
2) were
detected with a gas chromatographic system equipped
with a molecular
sieve column (
29), a methanizer (for the catalytic
reduction
of CO
2 to CH
4), a flame-ionization detector,
and a radioactivity
monitor (RAGA 2026/2028; Raytest). The detection
limit was between
2 and 5 Bq per peak for soluble organic compounds and
was 25 Bq
for radioactive gases. Radiolabeled products were identified
by
cochromatography with labeled and unlabeled
standards.
Chemicals.
Radiochemicals were purchased from Sigma,
Deisenhofen, Germany, with the following radiochemical purities and
specific activities, respectively: [U-14C]Na-acetate,
98.7% and 2.3 GBq mmol1; [14C]Na-formate,
97.4% and 2.1 GBq mmol1; [14C]polyethylene
glycol 4000, 99.7% and 1.9 GBq mmol1; and
NaH14CO3, 210 MBq mmol1.
Na214CO3 (1.9 GBq
mmol1) was from Moravek Biochemicals (Brea, Calif.). All
other chemicals were of the highest available purity. Gases were
supplied by SWF, Friedrichshafen, Germany, and were 99.999% pure.
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RESULTS |
14CO2 reduction by gut homogenates.
Gut homogenates of both Cubitermes spp. reduced
14CO2 to methane, acetate, and formate. Methane
and acetate accumulated linearly with time, whereas formate accumulated
only transiently under an N2 atmosphere, but reached a
steady-state concentration when hydrogen was present. The time courses
of product formation were basically identical for both termite species;
therefore, the time course is shown in detail only for C. orthognathus (Fig. 1).
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FIG. 1.
Formation of acetate ( ), formate ( ), and
CH4 ( ) from H14CO3
in gut homogenates of C. orthognathus under
N2-CO2 atmosphere (A), under
H2-CO2 atmosphere (B), or under
N2-CO2 atmosphere in the presence of 5 mM
unlabeled formate (C). Values are means of two replicate assays. In the
case of acetate, it was assumed that both C atoms stemmed from
CO2. Similar results were obtained for C. umbratus (Table 1).
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Methane formation rates were always much higher than those of acetate
formation, but homoacetogenesis was stimulated much
more than
methanogenesis when external H
2 was provided (Table
1). Considering the differences in fresh
weight (10.6 mg per
termite for
C. umbratus and 6.8 mg per
termite for
C. orthognathus),
there were only small
differences in the potential rates of H
2-dependent
methanogenesis (0.303 and 0.391 µmol g
1
h
1) and acetogenesis (0.025 and 0.034 µmol
g
1 h
1) between the two species.
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TABLE 1.
Formation of labeled acetate and CH4 from
14CO2 in gut homogenates of
Cubitermes spp. in the presence and absence of external
electron donors.
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In contrast to CH
4 and acetate, formate did not accumulate
with a linear rate. The initial rates of formate formation under
an
N
2-CO
2 or H
2-CO
2
atmosphere were quite high (4.2 to 5.4 and
3.2 to 5.1 nmol
gut
1 h
1 for
C. umbratus and
C. orthognathus, respectively), but declined
already within
the first hour of incubation (Fig.
1A and B).
14CO
2 reduction to acetate and methane was
stimulated as well by
the addition of unlabeled formate as an electron
donor (Fig.
1C
[tested only with gut homogenates of
C. orthognathus] and Table
1). When labeled formate was injected,
however, labeled methane
was formed only at very low rates (<0.01 nmol
gut
1 h
1), in both the presence and absence
of external H
2, and labeled
acetate was never
detected.
No labeled products other than CH
4, acetate, and formate
were detected. The recovery of added radioactive label was close
to
unity in all assays (95.5 to 99.7%) and did not differ significantly
between the two incubation atmospheres. Less than 0.5% of the
radioactivity was recovered in the particulate fraction. All rates
were
proportional to the amount of gut homogenate added, and product
formation could be abolished by boiling the homogenates for 10
min (not
shown).
Microinjection of radiotracers.
In order to evaluate the in
situ rates and the localization of homoacetogenic activities, we
injected minute amounts of radiolabeled metabolites into individual
sections of agarose-embedded guts of C. umbratus and
C. orthognathus. Figure 2A
gives an overview of the different experimental series and the specific
incubation conditions and illustrates the distribution of injected
radioactivity between gut and agarose at the end of the incubation
period.
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FIG. 2.
Microinjection of various 14C-labeled
metabolites into individual sections of agarose-embedded guts of
Cubitermes spp. (C. umbratus and C. orthognathus) incubated under air or H2 atmosphere.
(A) Distribution of radioactivity between guts and agarose at the end
of the incubation period. Incubation times were 45 min, except for
series 6 (20 min) and 7 and 8 (15 min). Each bar represents the mean of
three to four injections into separate guts, except for PEG (six to
eight injections per section). Error bars indicate standard deviations
of the total recovery. (B) Product formation rates from labeled
substrates. (C) Efflux rates of labeled metabolites into the
surrounding agarose. In the case of the unidentified product (series
6), the rates were calculated for a C1 compound. Bar
definition in panel C is the same as in panel B. For definitions of the
sections, see Fig. 1 in the companion paper (33).
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When polyethylene glycol (PEG) was injected, the radiolabel was always
retained within the gut. The amount of radiolabel recovered
from the
surrounding agarose directly after microinjection (2.9%
± 1.0%;
n = 20) was not significantly different from that
after
45 min of incubation (3.4% ± 1.0%;
n = 20).
There was also no
significant difference between the individual gut
sections (Fig.
2A; series 1). Since PEG does not diffuse through
epithelia, this
series represented an important control which ensured
that (i)
the injected label was indeed located within the gut, (ii) the
initially injected label could be completely recovered, and (iii)
radioactivity could escape into the surrounding agarose only if
there
was a selective permeability of the gut epithelium for the
respective
compound. Since there was no reason to assume a different
situation in
C. orthognathus, these controls were performed only
with
C. umbratus.
Also after microinjection of radiolabeled
HCO
3, acetate, or formate, recovery of
radioactivity at the end of the incubation
period was complete for most
sections. Only in the case of the
P4b segment were considerable amounts
of radiolabel missing when
HCO
3 was injected
and the guts were incubated under H
2 (Fig.
2A, series
3 and
5), or when formate was injected and the guts were incubated
under air
(Fig.
2A, series 6). Since the P4b segment of
C. orthognathus has been shown to form substantial amounts of
CH
4 when incubated
in the presence of H
2 or
formate (
33), this gap in the recovery
is most likely
attributable to the formation of methane from
14CO
2. Unfortunately, the microinjection assay
procedure did not
allow the detection of
14CH
4.
In all series, substantial amounts of the injected label
were recovered
from the agarose at the end of the incubation period,
indicating that
the respective gut sections were permeable for
the injected compounds
or their metabolites (see
below).
Product formation rates.
In order to calculate the product
formation rates (Fig. 2B) and, when applicable, the efflux rates of the
injected substances or their metabolites into the agarose (Fig. 2C),
the radioactivity values were transformed into absolute amounts of
metabolites by using the specific radioactivity of the injected
substrate and correcting for the pool of unlabeled substrate already
present in the respective gut section (Table
2). Due to the large internal pools of
CO2 and acetate in all gut sections, pool sizes were not
significantly affected by the injection. The amount of labeled products
increased linearly with incubation time, as exemplified by the results
obtained for series 5 (Fig. 3), and
product formation rates (Fig. 2B) were calculated by linear regression.
Only in the case of formate were the internal pools so small that the pool concentrations were significantly increased by the injected label
(Table 2). Nevertheless, injected formate was turned over already
within the first incubation interval of 20 min, and only the minimal
rates could be reported.
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TABLE 2.
Pool sizes of selected metabolites in the different gut
sections of Cubitermes spp. and the injected amounts of
the corresponding radiolabeled tracersa
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FIG. 3.
Formation of labeled acetate after microinjection of
H14CO3 into the P1 (), P3/4a
(), and P4b (C) sections of C. orthognathus hindguts
incubated under H2 (Fig. 2B, series 5). Each data point
represents an injection into a separate gut. For definitions of the
sections, see Fig. 1 in the companion paper (33).
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When HCO
3 was injected into
C. umbratus and
C. orthognathus hindguts incubated under
air, no labeled products could be detected
(results with
C. umbratus are shown in Fig.
2B, series 2). After
we had realized
that intestinal H
2 production in
Cubitermes spp.
decreased progressively during the first weeks after collection,
but
could be restored by feeding fresh soil (
33), we repeated
the experiment with a batch of
C. orthognathus fed with
topsoil
from the collection site 24 h before the experiment. The
hindguts
now formed labeled formate in the P1 section and acetate in
the
P3/4a section, albeit at low rates (Fig.
2B, series
4).
The apparent H
2 limitation of reductive acetogenesis was
confirmed when the guts were incubated under an H
2
atmosphere, which
increased the rate of acetate production from
14CO
2 considerably. Acetate was the major
product in the P3/4a and
P4b sections of both termites (Fig.
2B, series
3 and 5); the combined
in situ rates of all gut sections (Table
3) were roughly 3 to
4 times higher than
the rates observed in gut homogenates under
external H
2
(Table
1). The specific rates of homoacetogenesis
under an
H
2 atmosphere were slightly higher in
C. orthognathus than in
C. umbratus (0.141 and 0.091 µmol g
1 h
1, respectively).
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TABLE 3.
In situ rates of product formation after microinjection
of H14CO3 into the major gut
sections of agarose-embedded hindguts of Cubitermes spp.
incubated under air or an
H2 atmospherea
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Also formate formation from
14CO
2 in
C. orthognathus increased when the guts were incubated under
H
2, and formate was formed
in both the P1 and the P3/4a
compartments (Fig.
2B, series 5).
These gut sections (incubated under
air) rapidly oxidized injected
formate to CO
2 (Fig.
2B,
series 6). A consistent discrepancy between
the total radioactivity
recovered in the supernatant and that
detected by HPLC analysis
indicated the production of further,
so far unidentified product(s) in
all gut sections when formate
was injected. It should be considered
that microinjection of formate
increased the intestinal pool
concentrations considerably (Table
2), which might cause overestimation
of in situ rates, whereas
the complete turnover of injected formate
already within the first
incubation interval (see above) led to
underestimated values.
Therefore, both the rates of formate oxidation
and the formation
of the unidentified product(s) should be regarded
with
caution.
Hindguts of both termites (incubated under air) oxidized injected
acetate to CO
2 (Fig.
2B, series 7 and 8), although the
rates
were lower than those for acetate formation from CO
2
in the respective
gut sections. The only exception was the P3/4a
section of
C. umbratus,
which showed an exceptionally high
rate of acetate oxidation,
while acetate oxidation in the P1 section of
this termite was
at the detection limit. In the P4b compartment of both
termites,
injected acetate was also converted to formate at a low
rate.
Efflux of metabolites from the gut.
After microinjection of
CO2 or acetate into any of the hindgut compartments, the
labeled substrates were always recovered as well from the agarose, and
efflux rates (Fig. 2C) were determined by the calculation procedure
described above. However, in the cases in which acetate or
CO2 was formed as a labeled product within the gut (Fig.
2B), they were never detected in the agarose, probably due to the
trapping of the respective product label in the large internal pools of
acetate and CO2 (Table 2). Apparently, this does not apply
to the unidentified product(s) formed from formate, since slight but
significant discrepancies between total and HPLC-detectable
radioactivity were also found in the agarose samples (Fig. 2C, series 6).
In general, CO
2 efflux rates were significantly higher than
those for acetate. When compared between the gut sections, the
efflux
rates were similar, except for the low rate of acetate
efflux from the
P4b section of
C. orthognathus (series 7). Injected
formate
was not recovered from the agarose, presumably due to
its rapid
turnover. The apparent discrepancy between the relative
amounts of
label recovered from the agarose (Fig.
2A) and the
rates of acetate
efflux from the different sections (Fig.
2C)
is explained by the
different pool sizes of the respective metabolites
in each compartment
(Table
2).
Enumeration of CO2-reducing bacteria.
The MPNs of
microorganisms capable of H2-dependent CO2
reduction in the different gut sections of C. umbratus were
determined by serial dilution. We used 14CO2 to
increase sensitivity and to reliably distinguish reductive acetogenesis
from acetate formation by fermentative processes, especially when
additional substrates were included. The highest MPNs of homoacetogenic
and methanogenic bacteria were obtained in the posterior hindgut, i.e.,
in the P3/4a and the P4b/5 sections (Fig.
4A). When the MPNs are converted to
population densities, using the average volume of the respective
sections (Table 2), the concentrations of homoacetogenic and
methanogenic bacteria in the two sections are similar (4.9 × 106 and 2.0 × 106 cells ml1
in the P3/4a section and 1.1 × 106 and 4.4 × 106 cells ml1 in the P4b/5 section).
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 4.
Comparison of the MPNs of CO2-reducing
methanogenic, homoacetogenic, and formate-producing microorganisms in
the different hindgut sections of C. umbratus (A) with the
potential rates of H2-dependent methanogenesis,
acetogenesis, and formicogenesis from CO2 in the respective
hindgut sections of C. orthognathus (B). The rates for
methanogenesis are from the companion paper (33). n.d., not
determined. For definitions of the sections, see Fig. 1 in the
companion paper (33).
|
|
In the anterior gut sections, the MPNs of homoacetogens and methanogens
were negligible (Fig.
4A). Instead, both the M and
the P1 sections
harbored large numbers of bacteria that reduced
CO
2 to
formate, which corresponded to cell densities of 1.3 ×
10
7 and 3.1 × 10
6 cells
ml
1, respectively. Such bacteria may be responsible
for the high
rates of H
2-dependent
14CO
2 reduction to formate observed in
the P1 section of
C. orthognathus (Fig.
4B).
The MPNs did not increase when glucose, methanol, and formate were
included as additional substrates. H
2-dependent
acetogenesis
and formicogenesis from CO
2 were not detected
when BES, an inhibitor
of methanogenesis, was omitted or when the tubes
were incubated
under an N
2-CO
2 atmosphere. In
the latter case, no methanogenesis
was
detected.
| |
DISCUSSION |
This paper represents the first report on the localization and in
situ activities of homoacetogenic bacteria in the highly compartmentalized hindgut of soil-feeding termites and, together with
the studies of the wood-feeding termites Nasutitermes
walkeri (38) and Reticulitermes flavipes
(35), is one of the few studies addressing the metabolic
rates of the gut microbiota within the intact intestinal tract of insects.
It has been suggested that methanogenesis outprocesses homoacetogenesis
in soil-feeding termites (3). To date, the activities of
homoacetogens had been measured only in gut homogenates and were found
to vary strongly, depending on the hydrogen supply, and information on
methanogenesis in homogenates was scarce (3). Our results
demonstrate that methanogens outcomplete homoacetogens in gut
homogenates of Cubitermes spp., but addition of
H2 or formate stimulated homoacetogenesis much more than
methanogenesis (Table 1). In view of the considerable axial and radial
differences in H2 partial pressure in the hindgut of these
termites (33), homogenization is likely to cause
considerable artifacts, and the possibility of a strong homogenization
bias underlines the necessity for measuring the rates of reductive
acetogenesis under more realistic conditions.
In situ activities and their localization.
Using a
microinjection assay originally developed to study metabolic fluxes in
wood-feeding termites (35), we found that the potential
rates of H2-dependent acetogenesis in intact hindguts of
Cubitermes umbratus and C. orthognathus were
roughly three times higher than those obtained in homogenates. A
similar discrepancy exists between the potential rates of
methanogenesis in homogenates (Table 1) and in living termites
(33), indicating that homogenization has a negative effect
on both processes.
Also the endogenous rates of reductive acetogenesis were as severely
H
2 limited in intact hindguts as in homogenates, which
is
apparent from the stimulation with exogenously supplied H
2,
but is also indicated by the differences observed between starved
and
freshly fed termites. Most interestingly, the homoacetogenic
activities
in
Cubitermes spp. are not located in gut regions with
high
H
2 partial pressures, as previously shown for the
wood-feeding
R. flavipes (
16,
35). Instead, they
are restricted to the
posterior hindgut, a region which does not
accumulate H
2 from
endogenous sources (except for the P3
compartment) and which also
harbors all methanogenic activities
(
33).
The coexistence of methanogens and homoacetogens in the posterior
hindgut compartments is unexpected, but is supported by
the results of
the differential enumeration of homoacetogenic
and methanogenic
microorganisms, which demonstrated high numbers
for both metabolic
groups in these gut sections, although the
absolute numbers have to be
regarded with caution and are by no
means realistic estimates of the
total populations. In the P4b/5
section, the MPN of methanogens is
fourfold higher than that of
the homoacetogens, which is in good
agreement with the potential
rates of methanogenesis and
homoacetogenesis in the P4b compartment
(Fig.
4). In the P3/4a section,
the MPN of homoacetogens exceeds
that of methanogens, but the potential
rate of methanogenesis
is more than threefold higher than that of
homoacetogenesis in
this compartment. This may merely reflect a
cultivation bias,
i.e., an underestimation of the methanogenic
population, but might
also indicate that due to differences in the
spatial distribution
of the two populations within this gut section,
homoacetogens
are still limited for H
2 even when
H
2 is supplied in the
headspace.
Notably, there are distinct differences in the in situ rates of
methanogenesis and homoacetogenesis between the P3/4a and
P4b
compartments in the absence of external electron donors. While
the
P3/4a compartment forms methane already from endogenous sources,
methanogenesis is virtually absent in the P4b compartment unless
exogenous H
2 is provided (
33). The same is true
for H
2-dependent
acetogenesis, although the endogenous
rates in the P3/4a compartment
were above the detection limit only when
the termites were supplied
with fresh soil 24 h before the
experiment (Table
3). It has
already been suggested that the close
contact of anterior and
posterior hindgut within the abdomen would
allow a cross-epithelial
H
2 transfer which would drive
methanogenesis and homoacetogenesis
in the posterior compartments,
especially in the P4b compartment,
which seems to have small or no
endogenous sources of reductants
(
33).
Another exogenous electron donor for methanogenesis and
homoacetogenesis could be formate. Formate is present in the hemolymph
of
C. orthognathus in appreciable concentrations (2.6 mM
[
36])
and is most probably a product of microbial
fermentations in the
anterior gut compartments (see below). The
stimulation of methane
emission of isolated P3/4a and P4b sections by
formate is even
stronger than that by exogenous H
2
(
33), and H
2-oxidizing and
formate-oxidizing
methanogens have been found to occur in similar
numbers in
Cubitermes speciosus and other soil-feeding members
of the
subfamily Termitinae (
4,
30). It is not clear whether
formate also functions as an electron donor of homoacetogenesis
in
situ. Labeled formate was not converted to acetate when injected
into
C. orthognathus hindguts, and in gut homogenates of this
termite, it was only oxidized to CO
2 and never reduced to
methane
or acetate. However, when formate was added as an electron
donor,
it stimulated both homoacetogenesis and methanogenesis from
14CO
2 almost as strongly as the addition of
H
2 (Table
1). It is
possible that formate serves only as
electron donor for CO
2 reduction,
but is itself not reduced
in the C1
pathway.
Hindgut homogenates of both
C. orthognathus and
C. umbratus also formed formate from
14CO
2 at high rates when H
2 was
present (Fig.
1B). The same reaction
had been previously observed in
gut homogenates of the closely
related
C. speciosus
(
4) and of the wood-feeding termites
R. flavipes,
Zootermopsis angusticollis,
Prorhinotermes
simplex,
Nasutitermes costalis, and
N. nigriceps (
8). However, formate
did not accumulate in
intact guts of
R. flavipes (
35),
Nasutitermes walkeri (
38), and
C. umbratus (this study). Only in
C. orthognathus hindguts
did we find low rates of formate formation after microinjection
of
14CO
2 into the alkaline P1 compartment and, in
the presence of exogenous
H
2, also into the P3/4a
compartment, whereas injected formate
was rapidly oxidized to
CO
2 in both compartments (Fig.
2A). It
is possible that
formate accumulation is an artifact caused by
homogenization or by high
H
2 partial pressures. Also the large
microbial
population(s) catalyzing the H
2-dependent reduction
of
CO
2 to formate in the anterior gut region (Fig.
4B) may
catalyze
the observed reactions only under high H
2 partial
pressures and
may actually represent fermenting bacteria involved in
the formation
of H
2 and formate in situ. They are most
likely also responsible
for the high consumption rates of externally
added H
2 in the anterior
gut regions observed with hydrogen
microsensors (
33).
Importance of reductive acetogenesis in vivo.
The metabolic
rates of soil-feeding termites are much lower than those of
wood-feeding species (26, 28, 30). If one bases the in vivo
rates of methanogenesis and the in situ rates of homoacetogenesis on
the overall electron flow during oxidation of organic matter, it
becomes apparent that methanogenesis is of similar importance as a
terminal electron sink in hindgut metabolism in the soil-feeding
C. orthognathus as homoacetogenesis is in the wood-feeding
R. flavipes; both contribute almost 10% to the overall
electron flow (Table 4). Conversely, the
contributions of methanogenesis in R. flavipes and of
homoacetogenesis (from endogenous substrates) in C. orthognathus are hardly significant.
View this table:
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|
TABLE 4.
Comparison of the relative contributions of
methanogenesis and homoacetogenesis to the overall electron flow in
a soil-feeding termite and a wood-feeding termite
|
|
However, in contrast to
R. flavipes, in which reductive
acetogenesis is not strongly stimulated by exogenous H
2 due
to the
high intestinal H
2 concentration (
35),
the activities in
C. orthognathus will strongly depend on
the actual H
2 partial pressures
in the microniches occupied
by the homoacetogens. Considering
the strong possibility of a transfer
of H
2 or other reductants
between the compartments (see
above), the low endogenous rates
of homoacetogenesis obtained with
isolated guts probably underestimate
the true in vivo rates. The
endogenous rates of methanogenesis
in isolated gut sections of
C. orthognathus (0.047 µmol g
1 h
1) are
almost four times lower than the respective in vivo rates,
but surpass
them when exogenous electron donors are added (
33).
Therefore, the in vivo rate of homoacetogenesis can also be expected
to
lie within the range spanned by the in situ rates in the absence
and in
the presence of exogenous reductants, implying that in
living termites,
the electron flow towards homoacetogenesis might
be as high as that
towards methanogenesis (Table
4). In such
a scenario, homoacetogenesis
would contribute as much to host
nutrition in the soil-feeding species
as in the wood-feeding
species.
Metabolic versatility as a basis for coexistence.
Under
starvation conditions, when the P3 compartment does not accumulate
H2 to significant concentrations (33),
homoacetogenesis in the P3/4a compartment of both Cubitermes
spp. was below the detection limit, unless exogenous H2 was
added. In freshly fed C. orthognathus, however, the
H2 partial pressure in the P3 compartment increased
considerably (33), and homoacetogenesis in the P3/4a compartment occurred at significant albeit moderate rates (Table 3).
The metabolic versatility of homoacetogens, which are generally capable
of utilizing a wide variety of substrates (5, 15), including
many of the fermentation products found in the hindgut fluid of
soil-feeding species of Termitinae (36) and methoxylated aromatic compounds derived from lignins or humic substances,
might help them to maintain an active metabolism during phases of
low H2 partial pressure. Also their being capable of
mixotrophic growth, i.e., the simultaneous utilization of
H2 and organic substrates, as demonstrated for
Sporomusa termitida isolated from the wood-feeding Nasutitermes nigriceps (9), would add to their
competitiveness in an environment where H2 is only
temporarily available.
Unfortunately, there is only little information on the physiology of
homoacetogens in soil-feeder hindguts. To date,
Clostridium mayombei from the hindgut of
C. speciosus
(
18) remains the only
homoacetogen isolated from
soil-feeding termites. It has been
recently discovered that
spirochetes isolated from the wood-feeding
Zootermopsis
angusticollis are homoacetogenic (
21), but the
metabolic properties of the spirochetal morphotypes present in
the P4b
section of
C. umbratus (
36) remain to be
established.
More isolates and physiological studies are urgently
needed, but
it is of equal importance to gain insight into the radial
distribution
of the homoacetogenic population(s) within the posterior
hindgut
compartments. In contrast to methanogenic archaea, where
information
on the spatial distribution can be derived already from the
localization
of microbial cells with F
420-like
autofluorescence within the
respective compartments (
36),
the in situ identification of
homoacetogens calls for specific
molecular
probes.
Conclusions and outlook.
Together with the results presented
in the companion paper (33), it is now evident that
methanogens and homoacetogens coexist within the posterior hindgut of
soil-feeding Cubitermes spp. and that the importance of
reductive acetogenesis to the overall electron flow might be larger
than previously expected. An intercompartment transfer of
H2 or formate and fluctuations of H2 partial
pressures in the anterior hindgut emerge as important factors
sustaining the substantial homoacetogenic populations. Our findings
underline the fact that it is necessary to determine microbial
activities within their environmental context in order to correctly
assess the functional ecology of a microbial population. In situ rates will be affected not only by the metabolic interaction of organisms located within different microniches, but also by temporal fluctuations of metabolite concentrations. Therefore, only an exact knowledge of the
spatial distribution of microbial populations and their orientation in
the metabolic gradients will allow us to understand diversity and
coexistence on the microscale.
| |
ACKNOWLEDGMENTS |
This study was supported by a grant of the Deutsche
Forschungsgemeinschaft (DFG) within the program "Structural and
Functional Analysis of Natural Microbial Communities."
We thank Hamadi Boga, Lucie Rogo, Nixon Onyimbo, and Patrick Muthama
for helping to collect the termites used in this study and Bernhard
Schink for continuing support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Fakultät
für Biologie, Mikrobielle Ökologie, Universität
Konstanz, Fach M 654, 78457 Konstanz, Germany. Phone: 49-7531-883282. Fax: 49-7531-882966. E-mail:
Andreas.Brune{at}uni-konstanz.de.
| |
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Abstract
Introduction
