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Novel physiological and metabolic insights into the beneficial gut microbe
Faecalibacterium prausnitzii
Khan, Muhammad Tanweer
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Chapter 6
Metabolic switch upon transition from an electrogenic
phase to fermentation in the human gut bacterium
Faecalibacterium prausnitzii
M. Tanweer Khan1, Wesley R. Browne2, Jan Maarten van Dijl1,
Hermie J.M. Harmsen1
1
Department of Medical Microbiology, University of Groningen, University
Medical Center Groningen, Groningen, the Netherlands
2
Stratingh Institute for Chemistry, Faculty of Mathematics and Natural
Sciences, University of Groningen, Nijenborgh 4, 9747AG Groningen, The
Netherland
Submitted to Energy Environ. Sci
Chapter 6
Abstract
Faecalibacterium prausnitzii is one of the most abundant commensal microbes in
the gut of healthy humans, and this bacterium is well-known for its beneficial
effects on gut inflammation. Previous studies revealed that F. prausnitzii can
efficiently exploit flavins as redox mediator to shuttle electrons to external electron
acceptors. The present studies were aimed at elucidating the mechanism and
impact of riboflavin-mediated extracellular electron transfer (EET) on the growth
and metabolism of F. prausnitzii in microbial fuel cells (MFCs). Using
chronoamperometry, bacterial cells were cultured at different anodic potentials and
their metabolic profiles were assessed. Furthermore, resting cells were used to
investigate possible connections between metabolic flux and current-producing
pathways. The results show that at highly oxidizing potentials, F. prausnitzii
exploits riboflavin as electron shuttle to dissipate the reducing equivalents that is
generated by glycolysis and a presumed pyruvate:ferredoxin oxidoreductase
system. The growth profile in the MFCs further reveals an initial current-producing
electrogenic phase that is followed by a fermentation phase. Notably, carbon flux
is regulated during the electrogenic phase with a part of the glycolytic flux being
utilized for the biosynthesis of an extracellular polymeric substance (EPS).
Altogether, the present study shows that the metabolic flux in F. prausnitzii is
modulated towards EPS production in response to oxidizing environments, and
that the internal redox balance is maintained via EET. Based on these observations,
we hypothesize that F. prausnitzii uses EPS and EET for colonization of the gut
mucosa and protection against the oxidative conditions that can be encountered in
this niche.
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Electrogenic metabolism in Faecalibacterium prausnitzii
Introduction
The ‘gut microbiota’ is of fundamental importance to human health and well-being
due to its critical roles in the recovery of energy from undigested food, immune
regulation and colonization resistance against pathogens. Faecalibacterium
prausnitzii is one of the most abundant gut microbes, representing about 20% of
the total faecal microbiota in healthy individuals 1. Recently, metabolic and
physiological studies have revealed major contributions of F. prausnitzii to gut
health, relating to the production of butyrate and potentially strong antiinflammatory compounds 2-4.
The major product of glucose fermentation by F. prausnitzii is butyrate, but
formate and lactate are also produced at lower levels 5. In this case, the glycolytic
pathway catabolizes glucose to pyruvate, generating ATP and the reduced electron
carrier NADH (equation 1). The subsequent conversion of pyruvate to acetyl-CoA
is either catalysed by pyruvate:ferredoxin oxidoreductases (PFOR; equation 2) that
generate reduced ferredoxin (Fdred) and CO2 as additional products, or by pyruvate
formate lyase that generates formate as an additional product (equation 3). AcetylCoA is then converted into butyrate through a series of enzymatic reactions
involving the uptake of extracellular acetate (equation 4)
6,7
. Thus, glucose
consumption and butyrate production proceed in a 1:1 stoichiometric ratio if no
lactate or acetate are produced through the respective activities of lactate
dehydrogenase (LDH) and acetate kinase (ACK) 8. A tentative model for the
metabolic fluxes in F. prausnitzii is presented in Figure 1. This model takes into
account the established biochemistry of the central carbon metabolism in bacteria
belonging to the Clostridium group to which F. prausnitzii belongs 6,9.
133 | P a g e
Chapter 6
134 | P a g e
Electrogenic metabolism in Faecalibacterium prausnitzii
Figure 1. Proposed Metabolic flux network of F. prausnitzii based on Patni &
Alexander 19719 and Seedorf et al., 20086. The EPS biosynthetic pathway was
included in this network based on the observations presented in this study and blast
searches, which revealed the presence of genes for EPS synthesis (data not shown).
Etf, electron transport flavoproteins; EPS, extracellular polymeric substance; F1F0,
ATP synthase; F-6-P, fructose 6-phosphate; G-1-P, Glucose 1-phosphate; G-6-P,
Glucose 6-phosphate; IDP-G, Isoprenoid diphosphate glucose; PEP, Phosphoenol
pyruvate; RnF complex, electron transferring ferredoxin:NAD oxidoreductase;
a
UDP-G, uridine diphosphate glucose; , indicates hypothetical redox enzymes that
mediate EET and proton translocation across the membrane.
135 | P a g e
Chapter 6
Theoretically, butyrate production disposes of around 83% of the total electrons
derived from glucose catabolism, while generating NAD+ and Fdred (equation 5).
The proton-translocating NAD+:ferredoxin oxidoreductase complex (NAD+: FOR
or Rnf complex) in the membrane is then exploited to regenerate oxidized
ferrodoxin (Fdox) from Fdred at the expense of NAD+ (Fig. 1)
7,10
. This NAD+
dissipation might influence the glycolytic flux, since NAD+ is the crucial oxidant in
this pathway 11. Alternatively, Fdox could be regenerated from Fdred by the activities
of a hydrogenase, which would result in hydrogen production 10,12,13. However, the
generation of hydrogen gas was thus far not detected in in vitro fermentations of F.
prausnitzii and, therefore, Fdox recycling via this route remains ambiguous 5.
Importantly, electron disproportionate conditions where excess of reduced
products, such as Fdred or NADH, might accrue must be firmly regulated. To cope
with such conditions, microbes have evolved subtle mechanisms to couple their
endogenous oxidative metabolism (i.e. the regeneration of NAD+) to external
reductive reactions, such as the reduction of metal complexes, via a process called
‘extracellular electron transfer’ (EET) 14. Since electrons cannot traverse the lipid
bilayer spontaneously, EET relies on distinct components, such as electron
shuttles/mediators, membrane bound cytochromes, or nanowires
. Biological
15-17
electrochemical systems, such as microbial fuel cells (MFCs), in which electrodes
are used as the extracellular electron acceptor, represent highly effective tools to
study EET. Interestingly, previous investigations with MFC systems have revealed
changes in microbial metabolic profiles when different anodic potentials were
applied 18,19.
Electron mediators can be employed in MFCs to facilitate the electron transfer
between microbial cells and the electrode 20. Accordingly, we have recently shown
that riboflavin can be used to study EET by F. prausnitzii in MFC systems 21. This
was important, because our earlier studies had revealed that F. prausnitzii employs
riboflavin-mediated EET to consume oxygen in moderately oxygenated
environments, such as the gut mucosa. By doing so, this highly oxygen-sensitive
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Electrogenic metabolism in Faecalibacterium prausnitzii
bacterium creates a niche in which it can both survive and thrive 22. In the present
studies, we performed combined EET and metabolic investigations on F.
prausnitzii cells growing in MFCs to obtain novel insights in the mechanisms that
determine fermentative end-product changes. Essentially, F. prausnitzii was used
as a biocatalyst with glucose as electron donor and riboflavin as electron mediator
to couple the bacterium's oxidative metabolism to the MFC electrode. Electronic
parameters and fermentation profiles were then compared under electrogenic and
non-electrogenic conditions. Interestingly, production of an extracellular polymeric
substance (EPS), typically composed of polysaccharides, was observed under
oxidizing conditions. Since it has been proposed previously that EPS production
by commensal gut microbes may have a beneficial role in the maintenance of guthealth
23
, and since EPS production and central carbon metabolism are directly
connected
24
, we focused particular attention on the modulation of the metabolic
flux towards EPS production by F. prausnitzii. Altogether, our findings show that
the internal redox balance of F. prausnitzii is maintained via EET and that the
metabolic flux is directed towards EPS production in response to oxidizing
environments.
137 | P a g e
Chapter 6
Materials and Methods
Bacterial strains and culturing conditions
F. prausnitzii strain A2-165 (DSM 17677) was maintained at 37o C on yeast
extract, casitone, fatty acid and glucose (YCFAG) agar in an anaerobic tent. For
the microbial fuel cell experiments, the bacterial cells were grown anaerobically in
5 ml of YCFAG broth to an optical density at 600 nm (OD600) of ~0.8 5. Cells
were harvested by centrifugation, washed with fresh YCFAG medium and injected
(2.5% v/v inoculum) into the anode chamber using a gas tight syringe. Modified
YCFAG medium (mYCAG) was used as anolyte. The mYCAG medium was
similar to the YCFAG medium, but all short-chain fatty acids (SCFAs) except
acetate were omitted, cysteine was reduced to 3 mM and 200 µM riboflavin was
included as a redox mediator. For experiments with resting cells, cell cultures were
prepared as described previously 21.
Microbial fuel cell and electrical parameters
A custom-made two-chambered, MFC was fabricated from borosilicate glass
bottles with 250 ml and 100 ml working volumes for the anode and cathode
chambers, respectively (Supplementary Fig. 1). The two compartments were
separated by a CMI-7000S cation exchange membrane (Membranes International,
Inc.), using a 20 cm diameter septum. A graphite slab (dimensions of 8.5 x 2.5 x
0.5 cm3) was used as anode, a 0.5 mm diameter platinum wire as auxiliary
electrode and an Ag/AgCl wire was used as reference electrode (in the anode
chamber). The anode was connected to the external circuit via copper wires and the
bare ends were sealed with non-conducting epoxy resin. The self-resistance
between anode and copper wire was less than 2 Ω. The mYCAG medium, which
contains 200 µM riboflavin as a redox mediator, was used as anolyte while 100
mM potassium phosphate buffer was used as catholyte. A potentiostat (model #
600C, CH Instruments, Austin, TX, USA) was used to apply external anodic
potentials. The selected potentials were +0.62 V, +0.35 V, +0.15 V and -0.35 V
138 | P a g e
Electrogenic metabolism in Faecalibacterium prausnitzii
versus Ag/AgCl, corresponding to +0.82 V,+0.55 V, +0.35 V and -0.15 V versus
the standard hydrogen electrode (SHE), respectively. The potentiostat was
operated in bulk electrolysis mode and the catalytic current was measured at
intervals of 30 s for 24 h. After 24 h of growth in the MFC, cyclic voltammetry
was conducted using the afore-mentioned potentiostat without exchange of the
spent broth. The anode was used as working electrode, the Ag/AgCl wire as
reference electrode, and the platinum wire cathode as auxiliary electrode. The
selected scanning potential range was -0.5 V to -0.8 V versus Ag/AgCl and a scan
rate of 50 mV s-1 was employed. MFC experiments with resting cells were
performed as previously described 21. However, these experiments were conducted
at an applied anodic potential of +0.15 V versus Ag/AgCl. Furthermore, nonelectrogenic controls (i.e. without electrode) were also executed. Live/dead
staining for detection of biofilm formation on the working electrode was
performed as previously described25. For energizing the resting cells, glucose,
glucose plus acetate, and pyruvate were used. The 200 µM riboflavin was added as
a redox mediator. The resting cells were incubated for 1.5 h.
Analysis of short-chain fatty acids and carbohydrates
A high-performance liquid chromatography (HPLC) Ion-Chromatography system
(MetrohmAG, Herisau, Switzerland) equipped with a conductivity detector was
used for measuring SCFA concentrations including lactate, formate, butyrate and
acetate. A 6.1005.200 Metrosep Organic Acids column (Metrohm AG) with 9 µ m
particle size and dimensions of 7.8 mm x 250 mm was used for SCFA separation.
The eluent was 0.5 mM sulfuric acid and acetone (98:2 v/v), and the flow rate was
0.7 ml/min. Glucose consumption was estimated using the 3,5-dinitrosalicylic acid
(DNSA) method
26
. Secreted EPS was extracted from the growth medium as
described previously
27
and total carbohydrate content was determined by the
phenol-sulphuric acid method 28.
139 | P a g e
Chapter 6
Results and Discussion
Metabolite profiles of F. prausnitzii cells grown at different redox potentials
indicate a metabolic switch upon the transition from an electrogenic phase to
fermentation
To investigate the influence of environmental redox states on the metabolism of F.
prausnitizii, cells were grown in a MFC at four different anodic potentials. In
addition, a control experiment was performed in a MFC without electrode. When
grown on mYCAG medium in the absence of an electrode, F. prausnitzii
fermented glucose into lactate, formate and butyrate as previously described for the
batch fermentation of this bacterium in YCFAG medium 8. Figure 2a summarizes
the observed bacterial growth (right panel) as well as the profiles for glucose
consumption and the production of butyrate, formate and acetate (left panel).
These data show that the production of butyrate and formate was proportional to
glucose consumption. It was furthermore observed that the formate production rate
(1.45 mM. h-1) during the exponential phase was around 1.6-fold higher than the
butyrate production rate (0.95 mM. h-1). Importantly, it was previously established
that bacterial cells harvested from exponentially growing or stationary phase
cultures exhibit no significant differences in the activities of enzymes required for
the final steps in butyrate formation 8. Therefore, the higher rate of formate
production is indicative of a high glycolytic flux at the pyruvate node (Fig. 1) that
will cause an accumulation of glycolytic intermediates and reduced electron
carriers, such as NADH 29. The reoxidation of NADH through butyrate production
requires Fdox 7. In general, bacteria of the Clostridium group produce high Fd
levels, and the reduced Fd forms are commonly recycled via hydrogenases or
hydrogenase-bifurcating enzymes, generating Fdox and NAD+ simultaneously 30.
140 | P a g e
Electrogenic metabolism in Faecalibacterium prausnitzii
Figure 2. Metabolic profiles under electrogenic and non-electrogenic
growth conditions in a Microbial Fuel Cell system. a, growth in the absence
of an electrode; b, growth at -0.35 V; c, growth at +0.15 V; d, growth at
+0.35 V; e, growth at +0.62 V. All applied voltages were measured against
an Ag/AgCl reference electrode. Left panels show extracellular
concentrations of glucose and short-chain fatty acids (in mM) and catalytic
current production (in mA). Right panels show growth measured by OD600
readings and the production of EPS (in mg/ml).
141 | P a g e
Chapter 6
However, faecalibacteria do not seem to exploit such hydrogenases for the
regeneration of Fdox, which implies that the NAD+:FOR route is used for this
purpose (Fig. 1, depicted as Rnf complex). This recycling of Fdox via the
NAD+:FOR route can result in high NADH/NAD+ ratios during the anaerobic
growth of F. prausnitzii
10,30
. During cultivation of F. prausnitzii in the MFC
without an electrode, acetate consumption in the early exponential growth phase
was followed by acetate production in the late exponential and early stationary
growth phases. The initial acetate consumption has been linked to the activity of a
butyryl-CoA:acetate-CoA transferase (Fig. 1)31,32. In contrast, the subsequent
acetate production is catalyzed by ACK, which generates additional ATP via
substrate level phosphorylation
6,8
. Previous studies have shown that ACK was
activated or produced during the stationary phase, which may relate to an increased
flux at the acetyl-CoA node in central carbon metabolism 8. Furthermore, during,
the early stationary phase, low levels of lactate were detected in the growth
medium, indicating a build-up of intracellular glycolytic intermediates and reduced
electron carriers (Figs. 1 and 2a) 30. Intriguingly, the bacteria secreted substantial
amounts of EPS into their growth medium during the lag- and exponential growth
phases (Fig 2a, right panel). This EPS was subsequently degraded, but it
reappeared during the stationary phase, which may relate to hindrance in the
carbon and electron flow as discussed in the following sections. The overall carbon
and electron recoveries under these conditions were ~90% after 16 h of growth
(Supplementary Table 1a).
Next, the effects of different applied electrochemical potentials on the growth and
metabolic profiles of F. prausnitzii were investigated. To this end, the bacteria
were grown in the MFC at applied potentials of -0.35 V, 0.15 V, 0.35 V or 0.62 V
versus Ag/AgCl. When an anodic potential of -0.35 V was applied, no catalytic
current was observed. Instead, a negative current was observed (Fig. 2b, left panel).
This was due to the reduction riboflavin as determined by cyclic voltammetry (-0.2
V versus Ag/AgCl; Fig. 3c). Under these conditions, the growth rate of F.
142 | P a g e
Electrogenic metabolism in Faecalibacterium prausnitzii
prausnitzii was slower than in the MFC without an electrode (Fig. 2a and b,
compare right side panels), but the profiles for glucose, formate, butyrate, acetate
and lactate remained typical for fermentative growth (Fig. 2b, left panel;
Supplementary Table 1b). Also, EPS was produced during the exponential growth
phase, consumed during the early stationary phase and produced again in the later
stationary phase (Fig. 2b, right panel).
Figure 3. Electrode pacification by biofilm formation. a, Photograph of a graphite
working electrode pacified via biofilm formation. b, Live/dead staining of a F.
prausnitzii biofilm on a working electrode; the green and orange fluorescence
indicates live and dead cells, respectively. c, Cyclic voltammogram of a working
electrode before (i) and after (ii) chronoamperometry.
143 | P a g e
Chapter 6
When a potential of +0.15 V (versus Ag/AgCl) was applied, a catalytic current was
observed (Fig. 2c, left panel) that represents the riboflavin-mediated flow of
electrons from the bacterial cells to the anode as previously described 21. After ~15
h of growth, a maximum current of 17.6 mA was observed and, subsequently, the
current dropped to lower levels. During the 'electrogenic' phase, before the
maximum current was reached, the current was correlated to glucose consumption
(Fig. 2c). It is presently not clear why the catalytic current declined after 15 h of
growth, but this could be due acidification of the growth medium, or pacification
of the electrode via the formation of a biofilm that interrupts electron transfer
between bacterial cells and the electrode 33, or to a combination of these effects.
The possible pacification of the working electrode was probed using live/dead
staining, which revealed the presence of a biofilm (~15 µm thick) that entraps the
projected surface area of electrode (Fig. 3, a and b). Analysis of the anode by
cyclic voltammetry revealed a shift of ~60 mV in the potential difference between
the oxidation and reduction peaks of riboflavin, indicating that the phenomenon of
flavin adsorption on the anode may have occurred 34. Additionally, the pH of the
medium was not controlled during MFC operation. Thus, the acidification of the
growth medium may have contributed to the shift in the redox peaks of riboflavin
possibly due to the floating potential. Interestingly, in previous studies it was
shown that other electrogenic bacteria, such as Geobacter spp. or Shewenella spp.,
form anodic biofilms that positively correlate to power generation through direct
electron transfer to the anode or through self-secreted redox mediators 35,15. On the
other hand, Geobacter spp. and Shewenella spp. lack the ability to use glucose as
an electron donor. Thus, the behaviour of F. prausnitzii in MFCs is very different
from that of Geobacter spp. and Shewenella spp. as no direct electron transfer to
the anode was observed in our present experiments. Moreover our previous studies
have shown that F. prausnitzii requires glucose and externally added riboflavin for
the generation of a catalytic current 21.
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Electrogenic metabolism in Faecalibacterium prausnitzii
Notably, at a potential of +0.15 V, glucose consumption, EPS production and
current generation by F. prausnitzii started long before SFA production was
detectable (Fig. 2c, left panel). Moreover, the overall carbon/electron recoveries
remained low (~50%) as compared to the control experiment in a MFC without
electrode, where they amounted ~90% (Supplementary Table 1a, b and c).
Together, these observations reveal an important switch in the metabolism of F.
prausnitzii between the predominantly electrogenic initial growth phases and the
subsequent fermentative growth phases. The increasing current generated by EET
before the start of the fermentative phase indicates a dissipation of the reducing
power generated by glycolysis due to NADH oxidation
10,19
. This would explain
the retardation of SCFA production since both butyrate and lactate production
require NADH as reducing equivalent (Fig. 1) 6. Furthermore, since no formate
was detectable before the onset of the fermentative growth phase, it seems that
glycolytic intermediates were shuttled away from the pyruvate node of central
carbon metabolism. This is in line with the previous observation that the pyruvate
formate lyase enzyme complex catalyses formate production independent of
reducing energy consumption
6,22
. Together, these findings suggest that, at a
potential of +0.15 V, glycolytic intermediates are directed into the EPS
biosynthetic pathway during the early electrogenic growth phase, as depicted in
Figure 1. This view is consistent with studies on EPS formation by Staphylococcus
epidermidis, where it was shown that inhibition of the TCA cycle resulted in a
modulation of the redox state of the cells, possibly due to higher NADH/NAD+
ratios, and a concomitant accumulation of glycolytic intermediates that facilitated
the biosynthesis of EPS 36. Furthermore, the data in Figure 2c reveal that during the
fermentative phase, starting after ~15 h of growth, formate and butyrate production
occurred at similar rates without the simultaneous production of lactate. These
SCFA productions rates are thus very different from those that were observed for
the control conditions (no electrode present or -0.35 V), where the formate
production rate was higher than the butyrate production rate, and where
145 | P a g e
Chapter 6
lactate was also produced (Fig. 2, compare panels a, b and c). This underscores the
view that, during electrogenic growth, the accumulation of glycolytic intermediates
is lower than under fermentative growth, and that reduced electron carriers are
reoxidized with the help of EET while glycolytic intermediates are used for the
biosynthesis of EPS 18,19,37.
When potentials of +0.35 V or +0.62 V were applied, concomitant with the growth
of F. prausnitzii a rapid increase in catalytic current was observed (Fig. 2, d and e).
As noted for growth at +0.15 V, rapid glucose consumption was observed before
significant levels of fatty acid production were detectable. The maximum catalytic
currents observed at potentials of +0.35 V and +0.62 V were 12 mA (at ~9 h) and 9
mA (at ~6 h), respectively. Thus, the peak values of the currents obtained were
lower than the peak value of 17.6 mA observed at a potential of +0.15 V (Fig. 2).
These findings indicate that at relatively high potentials, rapid EET mediates an
earlier peak in current generation and allows the faecalibacteria to grow at higher
rates. Furthermore, at high potentials the cells acquired glucose relatively rapidly,
allowing them to regulate their internal redox state by producing more reducing
equivalents. In turn, this would allow a concomitantly increased generation of
current, biomass and EPS, resulting in an earlier pacification of the electrode (Fig.
2, d and e) and preventing the MFC system from attaining
higher catalytic
currents. Most likely, the pacification of the electrode causes an increased
intracellular level of glycolytic intermediates and reducing equivalents, which
triggers a more rapid onset of fermentation (Fig 2, d and e; Supplementary Table 1,
d and e). Indeed, the observed formate to butyrate production ratios at potentials of
+0.35 V and +0.62 V were 1.3 and 1.8, respectively, which is close to the ratio of
1.6 observed in the control experiment where no electrode was present and the
cells could grow exclusively fermentatively. Taken together, these observations
indicate that a major metabolic switch occurs in F. prausnitzii upon the transition
from the electrogenic phase to fermentation, which impacts directly on the
formation of EPS.
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Electrogenic metabolism in Faecalibacterium prausnitzii
Resting cells of F. prausnitzii show differential metabolic fluxes under
electrogenic and non-electrogenic conditions
To obtain a better understanding of the current-generating pathway, resting cells of
F. prausnitzii were used for MFC experiments. In these experiments, cells grown
in YCFAG broth were harvested in the stationary growth phase and injected into a
MFC system. Subsequently, 25 mM glucose, 25 mM glucose plus 50 mM acetate,
or 50 mM pyruvate were added to energize the cells. As shown in Figure 4A, the
steady state current generated in the presence of glucose was 1.6-fold higher than
when the cells were incubated with glucose plus acetate (Fig. 4a). Under both of
these current-producing conditions, so irrespective of the presence of acetate, the
production of formate, acetate and butyrate was observed, whereas lactate
production remained undetectable (Fig. 4, a, b and c). This indicates that NADH,
which is needed as a reductant for lactate generation (Fig. 1), was dissipated via
EET. Moreover, the cells avoided a possible blockage in carbon flux at the
pyruvate node through the production of formate, acetate and butyrate.
Interestingly, the lactate homofermentor Lactococcus lactis shows a similar
metabolic shift towards mixed acid fermentation when grown under electrogenic
conditions, which is possibly due to a high carbon flux at the pyruvate node
18
.
When glucose plus acetate were added to the MFC, fatty acid production by F.
prausnitzii was several folds higher than when only glucose was added (Fig 4, b
and c). This indicates that a higher flux of carbon and reducing energy was
directed towards SCFA production when acetate was present
8,38
, which would
explain the lower steady-state current production by cells incubated with glucose
and acetate (Fig. 4, a, b and c). Interestingly, when cells were incubated with
glucose only under non-electrogenic conditions (i.e. no electrode was present in
the MFC), the production of formate, acetate and butyrate was significantly
reduced while lactate production was increased (Fig. 4, b and c). This indicates that
the accumulation of reducing equivalents in the absence of EET hinders the
glycolytic flux and that the cells started to produce lactate for NAD+ recycling 18,39.
When pyruvate was added to the resting F. prausnitzii cells, an almost 3-fold lower
147 | P a g e
Chapter 6
steady state current was observed than when glucose was added (Fig. 4a). This is
in line with the fact that pyruvate generates four-fold less reducing equivalents
than glucose (Fig. 1) 6. Furthermore, under electrogenic conditions, pyruvate was
found to generate less lactate and more formate and acetate as compared to the
non-electrogenic control conditions where no electrode was present (Fig. 4d).
Importantly, under both electrogenic and non-electrogenic conditions, butyrate
production was halted, even when 50 mM of acetate was added (data not shown).
These observations indicate that the NADH, which is critical for butyrate
production, was dissipated either via EET and lactate production under
electrogenic conditions, or via lactate production under non-electrogenic
conditions 10,12,13. Furthermore, the efficient lactate and acetate production prevents
the blockage of central carbon metabolism at the pyruvate and acetyl-CoA nodes,
thereby diverting the carbon flux away from butyrogenesis. Under electrogenic
conditions, the lower level of lactate production due to the utilization of NADH for
EET seems to enhance the carbon flux at the pyruvate node towards acetyl-CoA
production, which would explain the enhanced production of formate and acetate
(Fig. 4d). Together, these findings show that fluxes in the central carbon
metabolism of F. prausnitzii are very different under electrogenic and nonelectrogenic conditions.
148 | P a g e
Electrogenic metabolism in Faecalibacterium prausnitzii
Figure 4. Current production and metabolic profiles of resting F. praunitzii
cells in the microbial fuel cell. Panel a. Cells were fed with either: i, glucose
(25 mM); ii, glucose (25 mM) plus acetate (50 mM); or iii, pyruvate (50
mM), chronoamperometry was carried out at a potential of +0.15 V versus
Ag/AgCl. Panels b, c & d. Metabolic profiles of resting cells using glucose,
glucose/acetate or pyruvate as electron donors under electrogenic (dark
bars) or fermentative conditions (light bars). Note that in the experiment
where glucose plus acetate were added, the net acetate production was
calculated by subtracting the base line value at t=0.
149 | P a g e
Chapter 6
Energetics of riboflavin-coupled Extracellular Electron Transport
To assess the possible advantage of EET for F. prausnitzii, the energetics of EET
and ATP gain were calculated from the Gibb’s free energy of the electron transfer
between NADH and riboflavin based upon the following considerations. During
glucose fermentation, F. prausnitzii produced mainly formate and butyrate, as well
as some lactate (Fig. 2). In contrast, SCFA production was retarded during the
electrogenic phase, despite glucose consumption. Thus a major proportion of
energy must have been derived from glycolysis, while EET mediated the recycling
of NAD+ (Fig. 1). Consequently, during the metabolism of glucose to acetyl-CoA,
eight electrons were respired to the anode of the MFC, thereby potentially
generating ATP via the generation of a proton-motive force and an F-type ATP
synthase
7,13
. Since the electrons generated during the conversion of glucose to
acetyl-CoA are bound to electron carriers, such as NADH (Eo’ -0.32 V), a Gibbs
free energy of ~85 kJ/mol can be estimated for the reduction of riboflavin (Eo’ 0.21 V). This free energy change can generate 1.5 mol of ATP per mole of glucose
assuming an ATP synthesis cost of ~60 kJ/mol ATP. Thus, at a current of 17 mA,
F. prausnitzii can synthesize ATP at a rate of ~33 nmol/s 10,13,15,34. A similar rate of
ATP synthesis was estimated for Shewanella spp., which transfers electrons to the
anode with the help of secreted riboflavin during the metabolic conversion of
lactate to acetate 34. It thus seems that faecalibacteria can potentially gain 3.5 mol
of ATP/mol of glucose through glycolysis combined with EET, without producing
SCFAs.
Implications of flavin-mediated electron shuttling
The present studies provide novel insights into riboflavin-mediated extracellular
respiration and the modulation of metabolic fluxes in F. prausnitzii. As shown in
previous studies, this bacterium exploits an NAD+:FOR system to regenerate Fdox
rather than hydrogenases. Consequently, the recycling of produced NADH to
NAD+ via EET is an attractive option (Fig. 1) 7,13. However, faecalibacteria do not
produce extracellular riboflavin and, therefore, they need riboflavin from external
150 | P a g e
Electrogenic metabolism in Faecalibacterium prausnitzii
sources as electron mediator
21,22
. This external riboflavin can then be used to
reduce locally available electron acceptors, such as Fe3+ complexes, oxidized thiols
or oxygen
34,21,22
. In MFCs, this phenomenon allows for catalytic current
production, since the anode acts as a terminal electron acceptor. Interestingly, at
relatively high positive anodic potentials, flavin-mediated respiration allows for
rapid consumption of glucose, growth and pacification of the electrode, which
causes a switch back towards the fermentative pathway. Additionally, during
electrogenic growth the faecalibacteria produce EPS as a metabolic by-product,
which possibly facilitates regulation of the glycolytic flux. Our present
experiments with resting cells clearly show that the reducing energy for EET
during the electrogenic phase was mainly derived from glycolysis and the
subsequent conversion of pyruvate to acetyl-CoA (Figs. 1 and 4). Based on these
findings, we propose the model depicted in Figure 5 to explain how faecalibacteria
can undergo a metabolic switch from an electrogenic to a fermentative phase. A
central element in this model is the view that riboflavin-mediated EET provides an
efficient means for electron disposal and Fdox recycling. This is in line with the fact
that F. praunitzii is a common inhabitant of the human gut, where considerable
amounts of flavins derived from either diet or the local microbiota are readily
available. Similarly, this explains why F. prausnitzii requires yeast extract as an
obligatory component in its growth media
, since the flavins contained in yeast
8,38
extract can be exploited as redox mediator for EET
40
. During the electrogenic
growth, the cells produce no SCFAs indicating that the fermentative metabolism is
shut down due to the dissipation of NADH for EET. This is energetically feasible,
because our calculations imply that the Gibbs free energy available from
riboflavin-coupled respiration can provide an extra gain of 1.5 mol of ATP per mol
of glucose. Importantly, this will give faecalibacteria a competitive advantage over
other gut bacteria (or other faecalibacteria) that grow purely fermentatively. In fact,
this is exactly what we observed in our previous experiments where, compared to
anaerobically growing faecalibacteria, the faecalibacteria in microaerobic
151 | P a g e
Chapter 6
environments showed significantly enhanced growth, provided that EET to oxygen
was possible due to the presence of riboflavin and oxidized thiols
21,22
. When EET
in the MFC system is blocked due to pacification of the electrode by bacterial cells
and EPS, F. prausnitzii switches to fermentative growth that results in the
formation of SCFAs (Figure 5, right panel). Thus, our findings explain how F.
prausnitzii deals with the external oxidative stress via EET and how it manipulates
the central carbon flow by EPS production. It is tempting to translate these
findings to the in vivo situation, where EET can be facilitated by riboflavin and
thiols that are present in the gut lumen and oxygen diffuses from epithelial cells
into the gut mucosa. This situation would facilitate electrogenic growth of F.
prausnitzii, giving this bacterium a competitive advantage over other gut bacteria
that are incapable of EET and creating a protective niche for host microbial
interaction. Once EET would become impaired, for example due to EPS formation,
the faecalibacteria would switch to fermentative growth thereby producing
butyrate, which is the preferred energy source
for colonocytes and has a
potentially protective role against colitis and colorectal cancers.
152 | P a g e
Electrogenic metabolism in Faecalibacterium prausnitzii
Figure 5. Proposed metabolic switch during the transition from electrogenic
to fermentative growth as based on the present studies. See text for details.
Acknowledgements: M.T.K. was supported by a grant from the Graduate
School GUIDE of the University of Groningen.
153 | P a g e
Chapter 6
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158 | P a g e
Electrogenic metabolism in Faecalibacterium prausnitzii
Supplementary information
Potentiosat
Nitrogen
Ag/AgCl reference
electrode
Graphite anode
Platinum wire
auxiliary electrode
s
Cathode Chamber
Proton exchange
membrane
Sampling port
Anode Chamber
Supplementary Figure 1. Microbial fuel cell design implemented in the
present study
159 | P a g e
Chapter 6
Supplementary Table 1a: Anaerobic control of MFC growth experiment using F. prausnitzii
as biocatalyst and riboflavin as redox mediater. The values shown are the consumption of
glucose and acetate, the production of lactate, formate, butyrate and EPS, and the carbon
and electron recoveries.
Time (h)
Glucose (mM)
mM C
mM eAcetate (mM)
mM C
mM eLactate (mM)
3
0.26
1.56
6.24
0.05
0.09
0.74
0.00
6
1.80
10.81
43.23
-1.49
-2.98
-23.82
0.00
9
2.42
14.52
58.08
0.25
0.50
4.00
0.00
12
2.49
14.94
59.77
0.07
0.15
1.19
0.00
16.5
2.96
17.76
71.05
-0.69
-1.39
-11.12
0.02
19
3.11
18.66
74.62
-0.92
-1.84
-14.69
0.07
22
3.48
20.86
83.46
-1.14
-2.28
-18.26
0.10
25
3.49
20.91
83.65
-1.34
-2.67
-21.36
0.10
mM C
mM eFormate (mM)
mM C
mM eButyrate (mM)
mM C
mM eEPS (mg/ml)
mM C
0.00
0.00
0.15
0.15
0.29
0.07
0.29
1.47
0.05
0.29
0.00
0.00
1.18
1.18
2.37
0.57
2.30
11.49
0.06
0.38
0.00
0.00
2.39
2.39
4.78
1.18
4.71
23.55
0.08
0.49
0.00
0.00
3.20
3.20
6.40
1.89
7.56
37.81
0.04
0.21
0.05
0.20
3.89
3.89
7.77
3.30
13.21
66.04
0.03
0.16
0.22
0.89
4.11
4.11
8.22
3.63
14.52
72.59
0.07
0.43
0.29
1.14
4.11
4.11
8.23
3.85
15.40
77.01
0.09
0.54
0.29
1.17
4.04
4.04
8.08
4.15
16.61
83.04
0.03
0.18
mM eRecoveries (%)
Carbon
Electron
1.16
1.50
1.98
0.85
0.65
1.71
2.15
0.70
53
59
28
25
56
59
74
77
90
91
93
93
87
84
88
86
160 | P a g e
Electrogenic metabolism in Faecalibacterium prausnitzii
Supplementary Table 1b: MFC-growth experiment using F. prausnitzii as
biocatalyst and riboflavin as redox mediater with an applied potential of
-0.35Volts vs Ag/AgCl . The values shown are the consumption of glucose and
acetate, the production of lactate, formate, butyrate and EPS, and the carbon
and electron recoveries.
Time (h)
Glucose (mM)
mM C
mM eAcetate (mM)
mM C
mM eLactate(mM)
mM C
mM eFormate (mM)
mM C
mM eButyrate (mM)
mM C
mM eEPS (mg/ml)
mM C
3
0.91
5.49
21.95
0.26
0.52
4.15
0.00
0.00
0.00
0.10
0.10
0.21
0.07
0.29
1.44
-0.002
-0.01
6
1.20
7.20
28.82
0.11
0.23
1.83
0.00
0.00
0.00
0.38
0.38
0.76
0.28
1.14
5.69
0.003
0.02
9
1.72
10.31
41.22
0.01
0.03
0.22
0.00
0.00
0.00
1.34
1.34
2.68
1.03
4.14
20.69
0.02
0.13
12
2.07
12.40
49.62
0.26
0.52
4.15
0.00
0.01
0.04
2.69
2.69
5.38
1.75
6.99
34.96
0.01
0.06
16.5
2.36
14.17
56.68
0.04
0.07
0.57
0.00
0.00
0.01
3.07
3.07
6.13
2.22
8.88
44.41
0.00
0.01
19
2.42
14.50
58.02
-0.22
-0.45
-3.58
0.01
0.04
0.15
3.27
3.27
6.54
2.71
10.84
54.21
0.01
0.07
22
2.57
15.41
61.64
-0.03
-0.05
-0.43
0.04
0.11
0.43
3.41
3.41
6.83
2.65
10.58
52.91
0.01
0.09
mM eRecoveries (%)
Carbon
Electron
-0.05
0.07
0.51
0.23
0.03
0.27
0.35
17
26
25
29
55
58
83
90
85
90
94
98
92
97
161 | P a g e
Chapter 6
Supplementary Table 1c: MFC-growth experiment using F. prausnitzii as biocatalyst and
riboflavin as redox mediater with an applied potential of +0.15 Volts vs Ag/AgCl . The
values shown are the consumption of glucose and acetate, the production of lactate,
formate, butyrate, EPS and current, and the carbon and electron recoveries.
Time (h)
Glucose (mM)
mM C
mM eAcetate (mM)
mM C
mM eFormate (mM)
mM C
mM eButyrate (mM)
mM C
mM eEPS (mg/ml)
mM C
mM eCurrent
mM eRecoveries (%)
Carbon
Electron
162 | P a g e
3
0.68
4.09
16.36
-0.17
-0.35
-2.80
0.05
0.05
0.10
0.00
0.00
0.00
0.03
0.17
0.66
6
0.67
4.00
16.01
0.05
0.10
0.80
0.09
0.09
0.17
0.15
0.59
2.96
0.02
0.14
0.55
9
1.32
7.92
31.67
0.06
0.12
0.96
0.13
0.13
0.26
0.20
0.80
4.00
0.00
0.00
0.02
12
1.69
10.14
40.55
-0.11
-0.21
-1.68
0.31
0.31
0.62
0.54
2.17
10.85
-0.01
-0.06
-0.24
16.5
2.02
12.14
48.55
-0.09
-0.18
-1.43
0.93
0.93
1.86
1.00
4.00
20.00
-0.01
-0.04
-0.15
19
2.41
14.49
57.95
-0.34
-0.67
-5.39
1.54
1.54
3.08
1.80
7.20
36.00
0.00
0.02
0.08
22
2.49
14.97
59.86
-1.20
-2.39
-19.15
1.88
1.88
3.76
2.86
11.42
57.12
-0.01
-0.03
-0.14
25
2.89
17.31
69.26
-0.65
-1.30
-10.39
2.16
2.16
4.32
2.80
11.20
55.99
-0.01
-0.05
-0.18
0.09
0.38
0.92
2.08
4.91
5.98
6.86
7.53
5
4
23
30
13
19
24
26
40
40
58
56
76
71
72
69
Electrogenic metabolism in Faecalibacterium prausnitzii
Supplementary Table 1d: MFC-growth experiment using F. prausnitzii as
biocatalyst and riboflavin as redox mediater with an applied potential of +0.35
Volts vs Ag/AgCl . The values shown are the consumption of glucose and
acetate, the production of lactate, formate, butyrate, EPS and current, and the
carbon and electron recoveries..
Time (h)
Glucose (mM)
mM C
mM eAcetate (mM)
mM C
mM eFormate (mM)
mM C
mM eButyrate (mM)
mM C
mM eEPS (mg/ml)
mM C
mM eCurrent
mM eRecoveries (%)
Carbon
Electron
3
1.00
5.99
23.96
-0.08
-0.17
-1.34
0.06
0.06
0.13
0.00
0.00
0.00
6
1.16
6.95
27.80
0.09
0.17
1.40
0.32
0.32
0.65
0.22
0.89
4.43
9
1.24
7.45
29.82
0.10
0.19
1.53
1.08
1.08
2.15
0.65
2.59
12.97
12
1.63
9.79
39.15
0.17
0.33
2.66
2.11
2.11
4.23
1.46
5.85
29.24
15
2.13
12.76
51.04
0.29
0.59
4.72
2.69
2.69
5.39
1.69
6.75
33.74
18
2.29
13.72
54.88
0.15
0.31
2.48
2.86
2.86
5.71
1.89
7.57
37.86
24
2.36
14.18
56.71
0.14
0.29
2.29
3.01
3.01
6.01
2.15
8.61
43.07
-0.002
-0.01
-0.05
0.003
0.02
0.07
0.02
0.13
0.51
0.01
0.06
0.23
0.001
0.01
0.03
0.01
0.07
0.27
0.01
0.09
0.35
0.22
0.81
1.96
3.35
4.44
5.16
5.65
1
1
20
26
54
64
85
101
79
95
79
94
85
101
163 | P a g e
Chapter 6
Supplementary Table 1e: MFC-growth experiment using F. prausnitzii as biocatalyst
and riboflavin as redox mediater with an applied potential of +0.62 Volts vs Ag/AgCl .
The values shown are the consumption of glucose and acetate, the production of
lactate, formate, butyrate, EPS and current, and the carbon and electron recoveries.
Time (h)
Glucose (mM)
mM C
mM eAcetate (mM)
mM C
mM eFormate (mM)
3
0.71
4.23
16.94
0.23
0.47
3.73
0.09
6
1.40
8.42
33.69
0.30
0.60
4.79
1.44
9
1.84
11.01
44.04
0.67
1.35
10.77
3.14
12
2.82
16.89
67.57
0.42
0.84
6.71
4.15
16.5
2.99
17.93
71.71
0.11
0.21
1.70
4.24
19
3.16
18.96
75.85
0.18
0.35
2.81
4.30
22
3.25
19.50
78.00
0.22
0.45
3.60
4.29
25
3.30
19.81
79.23
0.11
0.21
1.70
4.20
mM C
mM eButyrate (mM)
mM C
mM eEPS (mg/ml)
mM C
mM eCurrent
mM eRecoveries (%)
Carbon
0.09
0.18
0.00
0.00
0.00
0.03
0.16
0.66
1.44
2.88
0.80
3.22
16.09
0.04
0.22
0.89
3.14
6.27
1.48
5.91
29.57
0.03
0.19
0.78
4.15
8.31
2.42
9.68
48.39
0.03
0.18
0.71
4.24
8.49
2.84
11.38
56.89
0.01
0.04
0.15
4.30
8.60
2.94
11.76
58.80
0.01
0.06
0.24
4.29
8.58
2.93
11.71
58.54
0.00
0.01
0.03
4.20
8.41
2.91
11.65
58.23
0.01
0.06
0.25
0.90
1.74
2.72
3.74
5.01
5.66
6.38
7.02
17
32
65
78
96
114
88
100
89
101
87
100
84
99
81
95
Electron
164 | P a g e