Biochimie (1992) 74, 897-901 © Soci6t6 fran~;aise de biochimie et biologie mol~culaire / Elsevier, Paris

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N M R studies of bacterial fermentations J P G r i v e t 1, M D u r a n d 2, J L T h o l o z a n 3 1Centre de Biophysique Moldculaire, CNRS, and Universitd d'Orl~ans, 1A avenue de ia Recherche, 45071 Orleans Cedex; 2Laboratoire de Nutrition et Sdcuritd Alimentaire, INRA, 78352 Jouy-en-Josas Cedex; 3Station de Technologie Alimentaire, INRA, 369 rue Jules Guesde, BP 39, 59651 Villeneuve d'Ascq Cedex, France (Received 1 June 1992; accepted 7 July 1992)

Summary n We describe the experimental methods used and the constraints that apply in studies of anaerobic cell metabolism by t3C NMR. We review some of the results of our recent work in this area. Clostridium neopropionicum was shown to ferment ethanol into propionate by the acrylate, non-randomizing pathway. The same metabolic route accounts for 50% of the propionate formed in the complex ecosystem that inhabits the pig's large intestine. The rest is formed via the randomizing succinate pathway. Reductive, hydrogenotrophic acetogenesis was studied in several ecosystems. Although it is usually overshadowed by methanogenesis in the competition for hydrogen, it may become an efficient electron sink when methane biosynthesis is blocked by a specific inhibitor. tsC NMR / anaerobic metabolism / bacteria / reductive acetogenesis

Introduction Anaerobic organisms

Twenty years ago, Eakin el al [ 1] published the first example of an in vivo NMR spectrum and showed how the catabolism of a substrate (glucose) and the appearance of a metabolite could be followed, in real time, on living cells. Since then, the field of metabolic NMR has grown enormously and encompasses studies of cells and organs, both in vivo and ex wvo, from procaryotes to human beings. Many types of cells have been studied, from many points of view: primary metabolism, energetic metabolism, intracellular pH and regulation, influx and efflux of ions, etc. Most studies have been concerned with aerobic organisms, for instance E coli and S cerevisiae. In this report, we will describe our recent work on the metabolism of anaerobic bacteria. Since these microorganisms are much less known than their respiring counterparts, a few words of presentation may be useful [2]. Many diverse microbes are anaerobic: fungi, archaebacteria (such as the methanogens), or bacteria. They can be either Gram-negative or Gram-positive. Some, such as the Clostridiae, can form endospores. Strict anaerobes do not tolerate oxygen, presumably because they lack superoxide dismutase and catalase and are killed by oxygen free radicals. These organisms derive energy by fermentation. The ATP yield is about 2-4 mol of ATP per mol of substrate consumed, that is 10 or 20 times less than what could be

derived from respiration. The growth rate is correspondingly small, at most 0.15 h-i, and the phrase fastiduous organisms aptly describes this type of growth. Anaerobic organisms are usually quite specialized, fermenting a narrow range of substrates into a few products. Their natural habitats are oxygen-free ecosystems or consortia, as can be found in lake sediments, marshes, in the digestive tracts of most animals, and in sewage plants. These ecosystems effect the complete degradation of organic matter into methane and carbon dioxide. Since each bacterium requires a particular substrate, interspecies exchange of metabolites plays an important role. This last point is important to the NMR spectroscopist, because much information can be obtained from an examination of the culture medium. Knowledge about anaerobic microorganisms is quite recent. Two metabolic pathways characteristic of these organisms have been described in general terms in the last few years: methanogenesis [3] and acetogenesis [4]. Reports on the discovery of original metabolisms are not uncommon [5]. Other aspects of the physiology of anaerobic microbes have received much less attention. NMR as a tool in the context of metabolic studies

Given the current state of knowledge, NMR should be a very convenient technique in the investigation of the

898 primary metabolism of anaerobic organisms. Since many simple organic compounds are involved, 13C NMR will be the preferred method. Provided the metabolites are present at sufficient concentrations, all species are detected at once, whatever their chemical nature. Furthermore, labelling patterns are easily established, in contrast with the tedious sequential degradations necessary with radioactive tracers. Since NMR is not destructive or invasive, experiments can be done on live cell suspensions, yielding kinetic data which are difficult to obtain otherwise. We now examine briefly some general aspects of metabolic NMR studies.

Cell density NMR sensitivity is low, so that spectroscopists will constantly strive to use as much material as possible. Since the useful sample volume is limited (about 2 ml for the usual 10-mm diameter tubes), this means using the highest cell density as possible. In this respect, anaerobic bacteria have an advantage because they naturally grow to very high densities: up to 10 t2 cells per ml in the bovine rumen or in the human large intestine. Many different units are in use to indicate the number of cells present in the NMR sample, and the foLowing approximate conversion factors may be useful in order to compare literature values. Anaerobic bacteria are about 1 lam in diameter. When such cells are packed, eg in a centrifugation pellet, the cell density is close to 10 )2 ml-~, and the total cell volume is 0.5 ml. Taking the specific mass as 1 g ml-~, we find that these cells weigh 0.5 g: this is the wet weight. The corresponding dry weight is a third less, or 150 mg. Nearly half of this mass is protein, so that 10~2 cells contain 75 mg protein or 12 mg nitrogen. The internal cell volume has been estimated as 3.2 lal per mg of protein [61. In the work reported here, we used, for each sample, 2 ml of cell suspension, at densities of 1--4 1011 cells/ml.

NMR sensitivity, accuracy, and precision The sensitivity of an NMR experiment is a function of many parameters, some easily determined, such as field strength, others quite difficult to quantify, like probe design. We will give some typical results for our spectrometer, operating at 7.05 T, with 10-mm sample tubes. In the case of an homogeneous solution (culture supernatant), 5 mM of ~C are recorded with a signal-to-noise ratio of 10 in a single scan (0.5 s measuring time, filter equivalent to 2 Hz broadening). Deuterium at natural abundance in tap water (16.5 mM) yields a signal-to-noise ratio of 20 (3 s acquisition time), while 1 mM phosphate is just detected (signal/noise ratio about 2, 0.3-s acquisition). Since sensitivity is proportional to peak height, it will be inversely related to the linewidth. ~3C spectra of cell suspensions display reasonable linewidths (5-

15 Hz), and sensitivity is reduced by a factor of about 10, with respect to supematant spectra. Obviously, if an intracellular solute is being examined, the effective intracellular volume replaces the sample volume. As mentioned in the previous paragraph, the highest practical cell density corresponds to some 75 mg protein per ml, so that at most a fourth of the sample volume is useful. In general, a single scan will not suffice to gather a useful spectrum, so we must resort to accumulation. Assuming that the sample does not evolve in time, we sum N~ signals, for a gain of x/-N~in sensitivity. Calling Tp the time between consecutive pulses, the duration of the experiment becomes NsTp. A new problem arises in this connexion, because the NMR sample (in contrast to what happens for other spectroscopies) displays a kind of memory, for a duration of the order of T~, the spin-lattice relaxation time (a few seconds for 13C). It is recommended [7] that, for optimal sensitivity, the pulse angle 0 be set at the Ernst value 0E = arccos[exp(-T~/Tp)], the pulse repetition time Tp being the smallest value compatible with the desired resolution and spectral width. The operating parameters which maximize sensitivity will also lead to severe intensity distorsions for nuclei having relaxation times very different from the average. For ~ccurate, quantitative results, they must be abandoned in favor of the prescriptions of Gillet and Delpuech [8]: Tp > 10 T~, 0 = 90 °, gated proton decoupling in order to suppress the nuclear Overhauser enhancements. A very long pulse repetition time is obviously incompatible with high sensitivity or useful time resolution. A way out of this wellknown dilemna is the following. The time evolution of the cell suspension is studied at optimum sensitivity. At a convenient time, the cells are killed, for instance by addition of mercuric chloride. Two spectra are then recorded, one using the previous parameters, another under quantitative conditions. Assuming that cellular death does not significantly alter the relaxation rates, the ratio of true to apparent intensity will give a correction factor (also called a saturation factor) for each line, which must then be used to correct every intensity obtained during the kinetic experiment. The precision of NMR measurements is a function of the signal-to-noise ratio. In the case of integrals, which are the most common means of determining concentrations, it has been shown [9] that precision and sensitivity are inversely related, almost independently of any filter or window function that can be used. A precision of 1% requires a signal-to-noise ratio of 100. For ~3C work, we estimate that 10 min of accumulation will allow the assay of 1 mM at a precision of l0 %. The sample temperature is regulated, for instance at a value corresponding to the organisms' optimal growth;

899 a lower setting may be chosen in order to slow the metabolism. It is important to note that proton decoupling may heat the cell suspension. Although this was a critical problem with older spectrometers, recent machines incorporate very efficient decoupling schemes (such as coherent pulse decoupling), needing 1-2 W of radiofrequency power. If sample heating becomes a problem, a two-level decoupling schedule can be used: low power pre-irradiation to allow the build-up of the nuclear Overhauser enhancement followed by a spell of high power decoupling during the actual recording of the signal.

Labelled precursors The natural abundance of 13C is 1.1%, making mandatory the use of enriched starting material, except in special cases [10]. This situation has the obvious disadvantage that the experimenter is critically dependent on the availability and cost of labelled compounds. On the other hand, useful signals are seldom obscured by the background.

metabolic pathway to propionate has been investigated with the help of ~3C NMR [15]. When X4 bacteria are fed with [1-~3C]-ethanol, the substrate is rapidly exhausted. The main fermentation products are [2-13C]-propionate, [1-13C]-acetate, and [ 2 - 1 3 C ] propanol, along with traces of [3-13C]-butyrate.The first steps of this metabolism involve the conversion of ethanol to acetyl CoA and pyruvate. At this stage, two routes may lead to propionate: a well-known pathway through methylmalonyl CoA and succinate, and a rare pathway through lactate and acrylate. The former sequence, implying the symmetrical intermediates fumarate and succinate, will randomize the label between the 2 and 3-positions of propionate. The latter is non-randomizing. The NMR spectra conclusively show that only the acrylate pathway is active in this organism. A confirmation can be obtained by loading the culture with acrylate [16]. A transient spectrum assigned to [2-~3C]-lactate (the precursor of acrylate) was observed.

Ecosystems: rumen and large intestine

Materials and methods Sample preparation for NMR In order that NMR in vivo metabolic experiments be both possible and meaningful, one must be able to maintain a healthy and dense cell population for a rather long time in a NMR tube. Many techniques have been devised to this end [ 11]. In the case of anaerobic organisms, only the simplest method has been used. Cells are harvested by centrifugation, resuspended in the NMR medium and sealed in an NMR tube, with rigourous exclusion of oxygen. The final suspension medium should contain as few paramagnetic ions as possible. A low concentration of EDTA may be added as a further precaution against line broadening.

NMR spectroscopy Typically, spectra were recorded at 75.4 MHz, using 16 K data points and a 200 ppm spectral width. In the case of supernatants, the sample contained 10% 2H20; dioxane (114 mM, equivalent to 5 mM 13C) was used both as an intensity and frequency reference (8 = 67.4 ppm). Quantitative data were obtained with long relaxation delays (30 s) and inverse gated decoupling. Fully coupled and DEPT 1121 spectra, addition of known compounds, and spectra of perchloric acid extracts were used to identify resonances [131. Dioxane was omitted when working on cell suspensions, and chemical shifts were determined with respect to a resonance of the starting material (for instance 13-[1-13C]-glucose at 96.8 ppm). True intensities were obtained as described above.

Results and discussion A pure culture: Clostridium neopropionicum, strain X4 Some years ago, a new bacterium was described, which fermented ethanol into propionic acid [14]. The

Because knowledge of the physiology of the complex ecosystems found in the digestive tracts of animals and man is important to improve nutrition, many studies have been directed to these types of systems, even though their analysis can never be as detailed or accurate as those performed on pure cultures. The microorganisms present are traditionally divided in three different trophic groups: i) hydrolytic bacteria; ii) saccharolytic bacteria; and iii) methanogenic bacteria. Hydrolytic bacteria ferment waste material to monomers, organic acids and alcohols, and these p.roducts are converted to acetate and reducing eqmvalents (XH2) by saccharolytic bacteria. Methanogens cleave acetate to CH4 and CO: and use XH2 to reduce CO: to CH4. In the digestive tract, short chain organic acids are absorbed by the host, to be metabolized in the Krebs cycle. Thus, reprocessing of metabolites is minimal in these systems.

Formation of propionate Among saccharolytic bacteria, many species ferment glucose to propionate. The initial steps of glyco!ysis are identical to those found in aerobic microorgamsms and lead to pyruvate. Depending on the ecosystem being examined, the relative activities of bacteria utilizing the succinate or the acrylate pathways will be different. The relative fluxes through each path can be estimated from the relative intensities of the C2 and C.~ signals of propionate. It was thus found [17] that the acrylate pathway accounted for about 20 % of the propionate formed by anaerobic organisms isolated from the sheep rumen. This proportion rose to 50% in the case of bacteria isolated from the pig hindgut.

900

N B

A ,,,,O ! 186

,

1 184

,

I 182

~

I 180

~ I 128 PPM

~

I 126

,

I 124

,

I 122

I 40

I 35

I 30

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I 25

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Fig 1. Sections from spectra of supernatants after labelled bicarbonate metabolism. Cells were obtained anaerobically from a pig large intestin, washed and resuspended in NMR buffer. Spectra labelled 'A': incubation under a hydrogen atmosphere; spectra labelled 'B': incubation under hydrogen, in the presence of 20 mM bromoethanesulfonate. Assignments: 185.8: propionate-C~; 182.2: acetate-Cj; 172.2: formiate; 40.5: butyrate-C:; 31.6: propionate-C.,; 29.0: valerate-C3; 24.9: bromoethanesulfohate; 24.2: acetate-C:; 20.1: butyrate-C.~; 14.1: butyrate-C4; 11.0: propionate-C3. Replacing the glucose substrate by lactate did not significantly alter these results.

Acetogenesis As mentioned in a previous paragraph, acetate, a major end product of fermentation, is usually produced from more complex molecules by an oxidative process, the reducing equivalents released simul!aneously being used for methanogenesis. However, it is also possible to form acetate through a reductive process [4, 18-20]. There are, in fact, two versions of this process: heterotrophic (homoacetate fermentation of multicarbon compounds) and autotrophic (acetogenesis on one-carbon compounds). In the latter pathway, exogenous carbon dioxide and molecular hydrogen are coupled in an energy yielding reaction: 2 CO2 + 4 H: ~ CH3COOH + 2 H20 Most acetogenic bacteria can utilize both pathways [20]. In an ecosystem, acetogenic bacteria have to

compete for hydrogen with methanogens and sulfatereducing bacteria. In pig large-intestinal flora (DeGraeve KG, Grivet JP, Durand M, Beaumatin P, Cordelet C, Demayer D, to be published), acetogenesis and methanogenesis may coexist. Figure 1 shows sections of spectra of a supernatant recorded after the cells have consumed labelled carbon dioxide. Spectrum A is characteristic for the normal activity of the ecosystem: it shows some acetate, both singly labelled (originating from the heterotrophic pathway) and doubly labelled (autotrophic pathway). Spectrum B was acquired after methanogenesis was blocked by adding bromoethanesulfonate to the culture: this compound is analogous to coenzyme M and is a specific inhibitor of methane biosynthesis. The incorporation of label is much higher in this incubation, and doubly labelled acetate is also more abundant. Thus, when production of methane is inhibited, acetogenesis becomes the principal electron sink. Carboxylases are active in this system and catalyze the exchange of

901

carbon dioxide with the carbonyl group of propionate (signal at 185.8 ppm). This compound is apparently used in the biosynthesis of valerate: the line at 29.0 ppm is assigned to the valerate C-3 position. The production of both compounds is decreased in the presence of bromoethanesulfonate.

7 8 9 10

Conclusion and outlook

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We hope to have convinced the reader that 13C NMR applied to bacterial fermentations can be interesting and useful. Much remains to be done in order to bring the level of knowledge and expertise in this field on a par with current practice in studies of aerobic cells and perfused organs. In vivo enzyme mechanisms and kinetics have hardly been examined (for an exception, see [21]). The low concentrations of phosphorylated metabolites found in anaerobic organisms make it difficult to study the energetic metabolism, except in favourable cases [22]. Ion exchanges with the medium have not been studied. Some of the above studies are now being extended to human flora in our laboratories. The question whether significant work can be done in vivo on the intact intestine remains unformulated. An affirmative answer would probably be well received since knowledge of biochemical mechanisms in this organ is mainly indirect [23].

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