CA2083253A1 - Production of metabolites by methanogens - Google Patents

Abstract

ABSTRACT
The present invention relates to the preparation of isotope labelled metabolites such as amino acids and carbohydrates by growing methanogenic bacteria on labelled acetate. Methano-genic bacteria useful for this purpose are acid tolerant, require acetate in addition to CO2 and H2 for growth, use essentially non-cyclic biosynthetic pathways, demonstrate a low level of carbon exchange between the carboxyl group of acetate and CO2, and have a low level of acetate synthesis from CO2 in the presence of exogeneous acetate.

Description

20832~3 Metabolites labelled with isotopes are in demand for many research, diagnostic and medical applications, especially with the development of powerful mass spectrometry and nuclear magnetic resonance (NMR) instrumentation. For example, labelled amino acids are useful in the study of protein structure by NMR.
Labelled amino acids can be prepared by either chemical or biolo-gical means. Chemical means usually involve long, multistep organic synthesis that are generally inefficient. Most organic methods generally involve the use of expensive precursors, the isolation of intermediates, intricate purification schemes as well as optical resolution of the products since a mixture of optical isotopes is often produced. Furthermore, organic chemical methods are generally limited to introducing a carbon label to a terminal position such as the terminal carboxyl group. These labels are often unsuitable for biological applications as they are suscep-tible to decarboxylation reactions or similar modes of metabo-lism.
The preparation of labelled amino acids by biological methods seems a more practical alternative. These biological methods usually involve the growth of an organism on a labelled precursor. An example of this is growing algae on 13Co2. This technique i9 not always practical for producing labelled amino acids for ~MR work as uniformly labelled samples yield highly complex spectral data due to the adjacency of two or more 13C
nuclei which causes splitting of the resonances. Uniform label-ling also results in an enormous conversion of 13C into unwanted biomass. Therefore, it is highly desirable to produce site speci-2~32~3 fic labelled species. One of the methods to date involves thedevelopment of mutants in order to decrease the randomization of the label and to increase the efficiency of labelled precursor utilization. LeMaster and Cronan (in the Journal of Biological Chemistry, Volume 257, Page 1224 to 1230, 1982) developed two Escherichia coli (E. coli) strains for the production of specifi-cally labelled amino acids. The strains that they prepared had metabolic lesions so that no carbon interchange could occur between the intermediates of glycolysis and the tricarboxylic acid cycle. The amino acids synthesized by these E. coli mutants showed relatively few instances of adjacently labelled carbons thus resulting in a more simplified NMR spectra. However, there are problems with this method. Firstly, it is highly cumbersome to produce such mutants. Secondly, the degree of the label randomization is usually higher than that which can be obtained by chemical methods. In the case of the E. coli mutants, label randomization occurs because of the presence of the pentose phos-phate shunt common to many microbes. Label randomization, as used herein, denotes the distribution of label to carbon atoms that are relatively unlabelled compared to the predominantly labelled carbon atom. This can occur by the transfer of label from the carboxyl group of acetate to CO2 or by the presence of cyclic pathways.
The applicants have shown that certain methanogenic bacteria can be used to produce specifically-labelled amino acids and carbohydrates. Methanogenic bactexia are obligate anaerobes which derive energy by the metabolism of simple compounds, includ-20832~3 ing C02 and H2 gases, to methane. This mode of energy ~enerationis unique. Also unique is their mode of assimilating simple Cl or C2 compounds into cell constituents. Methanogens capable of grow-ing with Co2 as sole carbon source synthesize acetate (acetyl Coenzyme A) from 2 CO2 molecules via a CO-dehydrogenase reaction.
However, certain methanogens are incapable of this synthesis, and consequently require for growth H2/C02 plus acetate supplied to the medium. Such auxotrophy for acetate was acquired in Methano-spirillum hungatei (M. hungatei) strains GPl and JFl during multi-ple passages over several years in a medium containing acetate(Sprott and Jarrell in Can. J. Microbiol, Volume 27, Page 444-451, 1981). In view of this property M. hungatei was grown on isotopi-cally labelled acetate to produce labelled metabolites such as amino acids and carbohydrates. The extent of label randomization was very low in M. hungatei. Other methanogens examined were inferior to M. hungatei with respect to label randomization, especially with ~ 3C]acetate precursor.
Labelled amino acids have previously been prepared in methanogens. However, some of the problems with the known methanogen systems for preparing labelled amino acids include low percentage utilization of the acetate precursor and randomization of the label. These are discussed below.
The applicants have shown that M. hungatei and other methanogens require for optimal growth high concentrations of acetate and utilize a low percentage of the acetate. This was confirmed recently by Jetten et al. (in FEMS Microbiology Ecology, Volume 73, Page 339-344, 1990) for strains GPl and JFl. These 20832~3 problems are generally due to the fact that _. hungatei strains GPl and JFl, and certain other methanogens, cannot grow below pH
6.6, making them difficult organisms to grow in the C02/HC03 buffered medium which is required for this technique. Also, since there appears to be no active transport system for acetate in these methanogens, acetate utilization at neutral pH is less effi-cient due to a difficulty in diffusion of the labelled-compound into the cell at neutral pH. The protonated form of weak acids is known to penetrate cell membranes, hence penetration occurs best at pH values too acidic for growth of these methanogens.
Most methanogens have a considerable degree of exchange, or randomization between C02 and the C-l or carboxyl group of acetate. The result is that growth in media containing (1-13C)-acetate results in production of amino acids labelled not only in carbon atom positions originating from the C-l of acetate, but also in positions where C02 is incorporated. An example of this (described in the Journal of Bacteriology Vol. 162 p. 905-908, Ekiel et al. 1985) is the methanogen Methanosaeta concilii GP6 ("Methanothrix conc~ ") which demonstrates a considerable degree of carbon exchange between C02 and the C-l of acetate.
In view of all of the above, it was desirable to try and isolate acetate-requiring methanogens capable of metabolizing H2/C02 which could grow at acidic pH conditions (acid-tolerant methanogens). Growth of such methanogens at acidic pH should ensure sufficient acetate in protonated form for its more complete removal from the medium, and hence give higher efficiencies of acetate precursor conversion. It was also desirable to isolate a 20~3253 methanogen that has a very low level of randomization or carbon exchange between CO2 and the C-l of acetate.
SUMMARY OF THE INVENTION
It is a feature of the present invention to provide a method for producing isotope labelled metabolites comprising in-cubating an acid tolerant methanogen in the presence of isotope labelled acetate. Acid tolerant methanogens as used herein, refers to methanogens capable of growth at pH values less than 6Ø
It is a feature of the present invention whereby the metabolites are amino acids or carbohydrates.
It is another feature of the present invention whereby the methanogen has a very low level of acetate synthesis from CO2 in the presence of exogenous acetate.
It is another feature of the present invention whereby said methanogen exhibits a low level of carbon exchange between C2 and the carboxyl group of acetate. It is also a feature whereby the metabolic pathways of the methanogen are non-cyclic.
It is also a feature of this invention whereby the isotope is 13Carbon.
It is yet another feature of the present invention whereby said methanogen is Methanobacterium espanolae.
DESCRIPTION OF THE DRAWINGS

_ _ Figure 1 is a schematic diagram illustrating the key metabolic reactions of strain GP9 (M. espanolae).
Figure 2 is a flow chart showing the fractionation of strain GP9 cells labelled with 13C acetate.

Figure 3 is a 13C NMR spectrum of the protein hydro-lysate obtained from cultures grown on 13C acetate.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
The applicants have isolated a new strain of methanogen named Methanobacterium espanolae and designated as strain GP9, that has a broad pH range of 5.6 to 6.4 for optimum growth and is capable of growth at pH levels at least as low as 4.7.
Figure 1 illustrates the key metabolic reactions of strain GP9 indicating its inability to synthesize acetate from CO2 and showing the presence of a reductive, partial citric acid path-way. Ingredients supplied in the medium are shown in boxes. All these elements of biosynthesis lead to very unique labelling patterns of amino acids, carbohydrates and nucleotides. By way of example, as discussed below, we will describe the isotope label-ling of both amino acids and carbohydrates in strain GP9.
Isolation, stock cultures, and inoculum.
This new strain was isolated from the primary sludge obtained from the waste treatment facility of a pulp mill in Canada, which is described in the International Journal of Systemic Bacteriology, January 1990, page 12-18 (Patel et al.).
This methanogen is deposited with the German Collection of Micro-organisms and Cell Cultures as DSM 5982, with the National Research Council of Canada culture collection as NRC 5912, and with the oregon Collection of Microorganisms as OCM 178.
A 10-ml sludge sample, that was collected anaerobically from the anoxic zone of the primary settling basin of the E. B.
Eddy Forest Products Ltd. (Espanola, Ontario, Canada) bleach 20~32~3 kraft mill, was transferred to a 60-ml serum vial under 100~ N2.
The cellulolytic and methanogenic activity of this mixed culture was maintained (incubation temperature, 35C) by transferring it (10% vol/vol) to fresh primary sludge (under N2, supplemented with cysteine-Na2S and 5 mM NH4Cl) every 6 weeks. This primary sludge enrichment culture was inoculated into SA medium (80% H2-20% CO2) at pH 5Ø After we detected large quantities (10% vol/vol) of CH4 gas in a headspace gas analysis, the methanogenic cultures were maintained in similar media by transferring them (10%
vol/vol) at l-week intervals. Such a culture was serially diluted into SA broth (pH 5.0) and plated onto SA agar (pH 5.5) (SA medium supplemented with 2.2% [wt/vol] Noble agar [Difco Laboratories, Detroit, Mich.]). The agar medium was prepared just as the broth medium was, but the prereduced and autoclaved medium (20-ml portions in 60-ml vials) was poured into Petri plates inside an anaerobic chamber tCoy Manufacturing Co., Ann Arbor, Mich.) containing a 5% CO2-10% H2-85% N2 atmosphere. After overnight drying in the chamber, the plates were streaked, introduced into Brewer anaerobic iars which were then flushed out with 80% H2-20 CO2, and incubated at 35C. Representative colonies from agar plates were transferred into vials containing SA broth (pH 5.5) inside the anaerobic chamber. Methanogenic broth cultures were serially diluted and plated for colony picking. This procedure was repeated several times until culture purity was established.
Stock cultures of methanogenic isolates were maintained in SA broth at pH 5.5 and 5.0 by transferring them (10%, vol/vol) every 7 to 10 days into fresh media at the appropriate initial pH.

The stock culture vials were repressurized every 4 days by inject-ing 80~ H2-20% C02.
Unless stated otherwise, the inoculum for tests consis-ted of 1% (vol/vol) of a l-week-old culture in SA medium (pH 5.5) which was anaerobically and aseptically concentrated 10 times into the appropriate medium to avoid carry-over of the nutrients or compounds under investigation. All incubations were static, except when indicated otherwise.
Preparation of the Cells For analytical purposes herein GP9 cells were grown an-aerobically in 100 ml aliquots of citrate/phosphate, pH 5.5, S-medium (SA medium lacking acetate) supplemented with either [1-13C]acetate or [2-13C]acetate as sodium salts to give an initial concentration of 465 mg acetic acid per liter. The inoculum, when grown in unlabelled-acetic acid medium, was washed once with acetate-free citrate/phosphate S-medium and resuspended (10-fold concentrated) prior to inoculating with 1 ml/lOOml of medium.
Incubation was at 35C with a 100 rpm shake rate. Growth flasks were pressurized to 10 psi twice daily with 80% H2/20% C02 after the first 24h of incubation. At 48 and 72h the headspace gases were removed by flushing and replaced with fresh H2/C02. Two-ml of fresh cysteine/sodium sulfide reducing agent was added per 100 ml at 72 and 79h incubation. Figure 2 is a flow chart that illustrates the preparation of the GP9 cells for NMR analysis of the amino acids or carbohydrates. Cells were harvested by centri-fugation (10,500xg, 15 min) at 96h yielding ca 0.36 mg cell dry weight/ml culture. The cells were washed, and resuspended into 10 20~32~3 ml deionized water.
Lysis was achieved mechanically by passing twice through a French pressure cell (ca. 16,Q00 psi) with ca. 99% breakage.
[Treatment of the cells with dithiothreitol and sodium dodecylsul-fate at pH 8 also causes lysis]. The lysate was treated for 30 min at 30C with deoxyribonuclease (0.05 mg/ml) and centrifuged (15,000xg, 30min).
Protein was precipitated from the supernatant by adding ethanol to 70% (vol/vol) and storing for lh at -5C. The protein was collected by centrifugation and hydrolyzed in vacuo in 6N HCl (24 or 48 h at 110C). The labelled amino acids were recovered from the crude bacterial protein hydrolysate and sometimes subjec-ted to an ion exchange system similar to that described by Le Masters and Richards in Analytical Biochemistry, Volume 122, pages 238-247, 1982. Hydrolysates were dried in vacuo for quantitation with an amino acid analyzer. Dried samples hydrolyzed for 48h were resuspended in O.lM HCl solution in D20 for NMR analysis.
Carbohydrates were recovered from the pellet (obtained after centrifuging the cell lysate) by hydrolyzing with 1 ml of 2 M H2S04 (110C for 4 h). Samples were neutralized with BaC03, centrifuged and the supernatant passed through columns of Dowex 50 and AGl-X2 (BioRad). Monosaccharides were eluted with water, lyophilized and resuspended in D20 solution.
NMR spectra were recorded with a Bruker AM-500 spectro-meter operating at 125 MHZ in the Fourier transform mode, at 26C
in 5-mm tubes.

AMINO ACIDS

Labelling patterns of amino acids . _ 13C NMR spectra of protein hydrolysates obtained from cultures grown on [1-13C] and [ -13C]acetate are shown in Figure 3; chemical shift values are included in Table I. Similar to the results for _. hunyatei (discussed in Journal of Bacteriology, p. 316-326, 1983, Ekiel et al), a very high specificity of label-ling is observed, with labelling patterns virtually identical to those in M. hun~atei (discussed in Journal of Bacteriology, p. 316-326, 1983, Ekiel et al). Therefore, the same biosynthetic pathways are operating in both GP9 and M. hun~atei including the incomplete tricarboxylic acid pathway operating in the reductive direction and a pathway to isoleucine via citramalate. In GP9, as in most other methanogenic bacteria, acetate is incorporated and converted to acetyl-CoA, which is then reductively carboxylated to pyruvate (Figure 1). However, with M. espanolae we avoided the difficulties experienced with known methanogen systems such as Methanosaeta concilii, Methanosarcina barkeri or M. hungatei, ___ _ wherein either label randomization occurs through interchange of the carboxyl group of acetate with CO2 (and visa versa), or much of the acetate precursor remains in the medium following growth.

Label randomization -Label randomization is essentially the distribution of label to the carbon atoms that are relatively unlabelled as compared to the predominantly labelled carbon atom. Randomization can be caused, for example, by the transfer of label from the carboxyl group of acetate to C02 (scrambling) or by the presence of cyclic pathways. Random labelling can be easily detected in 20832~3 13C NMR spectra, since the signal intensities of the randomly labelled carbon atoms will be sma:Ll in relation to the predomi-nantly labelled positions, but greater than the intensity result-ing from a 1.1~ natural abundance of 13C. In protein hydrolysates from strain GP9, we could estimate the intensities (measured as heights) of these peaks as below 2% (of the total intensity) in the case of the culture grown on [1-13C]acetate, and below 1.5%
for the culture grown on [2-13C]acetate. Therefore, there is very little, if any, randomization of label between positions primarily labelled from carboxyl and methyl groups of acetate, and very little transfer of label to positions primarily labelled from CO2 .
Enrichment level _ _ _ _ . _ _ The level of carbon atom enrichment with 13C was estima-ted in two ways: by monitoring protons covalently bound to label-led positions by lH NMR, and from 13C NMR spectra of those amino acids which have two adjacent carbons simultaneously labelled.
In lH NMR spectra, hydrogen directly bound to 13C are observed as doublets, because of carbon-proton coupling.
Additionally, in the center of each doublet, a signal will be present for protons in these molecules which have no isotope en-richment. The level of labelling can be calculated from the in-tensiLy ratio of the central peak to the doublet. Because of the signal overlap in lH NMR spectra of amino acids, only a few signals can be measured accurately in this way. Five signals (mostly of methyl groups of branched-chain amino acids) were measured in a spectrum of amino acids labelled from [2-3C]acetate, ~nd two signals for amino acids labelled from ~l-l3C]acetate. Obtained levels of l3C labelling were 92 and 9l~, respectively. Similar measurements, using l3C NMR, were performed for Ile, Leu and Val signals, and labelling from [2-l3C]acetate, and gave 94~ l3C enrichment.
Figure 3 illustrates a l3C-NMR spectrum of the protein hydrolysate obtained from the cultures grown on, A) [l-l3C]acetate, B) [2-l3C]acetate. Signals were assigned according to Table I.
The experiment shown in Figure 3 was repeated with citrate excluded from the medium to test whether or not citrate could affect the quality of the labelled-amino acid products. As found previously (Figure 3), the degree of labelling was similar (89.5% based on l3C NMR of Ile, Leu, Val), the level of label randomization was low (< 2~1, and the labelling patterns of the amino acids were identical to those with citrate in the medium.
Reproducibillty All NMR analysis were repeated for three growth experi-ments, with the ~ame results.
Amino acid yields In addition to l3C NMR spectroscopy, the amino acid hydrolysates obtained by the growth of strain GP9 on ~l-l3C]acetate and [2-l3C]acetate were subjected to amino acid analy-sis. This is illustrated in Table II. The amino acid yield on a dry wt. basis was about 40~ of the cell dry weight, with indivi-dual amino acid yields as shown. Hydrolysis for 24 or 48 ~ gave similar results.

CARBOHYDRATES
Labelling of Carbohydrates In addition to the carbon atoms of amino acids, other cell constituents of GP9 are labelled selectively (Fig.l). The 13C chemical shifts for the carbon atoms of ~ and ~ anomers of glucose, galactose, and mannose, the three most abundant sugars, are presented in Table III. Growth of GP9 in media containing 3C)acetate resulted in label incorporation in hexose sugars to carbons 2 and 5, whereas (2-13C)acetate resulted in incorporation in carbons 1 and 6.
Summary of results The overall dilution of the C-l or C-2 acetate label is exceptionally low, compared to other bacteria, and no mutation was necessary to block pathways leading to label dilution. Acetate precursor enriched to the level of 99% was used, and some dilution was caused by the use of unlabelled inoculum, which combined can account for 4% of carbon positions unlabelled. The additional 2-5% of carbons having 12C most probably originate from low levels of acetate synthesis from CO2. Amino acids labelled from both C-l and C-2 of acetate gave very similar enrichment levels. This result means that, unlike many methanogenic bacteria, carbon ex-change between the C-l of acetate and C2 was not active in strain GP9.
The proposed technology has several advantages over the known techniques for producing labelled amino acids and carbo-hydrates. These are as follows:
1. Strain GP9 grows over a broad pH range in the acidic region.

In a bicarbonate buffered medium an appropriate acidic pH is readily achieved without the need for pH control during growth.
Often methanogens such as M. hungatei can be difficult to grow, because growth will not occur at pH values more acidic than approximately 6.6.
2. High level of acetate utilisation by strain GP9 and low unit cost of acetate. Certain members of the methanogenic bacteria (ex, strain GP9) selectively incorporate acetate and C02 into biomass, and convert CO2 into methane. This unique coupling of energetic and biosynthetic pathways allows very economic use of acetate. Simultaneously, acetate is one of the cheapest sources of label, much cheaper than other sources used for other bacteria, such as E. coli, described in The Journal of Biological Chemistry, Vol 257, p. 1224-1230 LeMaster and Cronan, 1982. Unlike M. hungatei, strain GP9 utilizes acetate completel~; i.e. after 2 days of growth 45% of the acetate precursor had been taken up by the strain GP9 cells and after the 3rd day all of the acetate had been taken up.
3. Very high level of specificity. Strain GP9, like other methanogenic bacteria, lacks cyclic biosynthetic pathways, and in strain GP9 the exchange between CO2 and the carboxyl group of the acetate is negligible. As a consequence, much higher levels of enrichment can be obtained than in other systems, for example the E. coli mutants of LeMaster and Cronan, 1982 (> 90 versus 70-85%).

4. Because of the presence of unique biosynthetic pathways, methanogens produce patterns of labelling which cannot be mimicked 20832~3 by any other bacteria, in particular they are very different from those obtained for E. coli (LeMaster and Cronan, 1982).

5. Labeling patterns for each amino acid can be varied according to the carbon atom, or combination of carbon atoms, in the precur-sors fed to the cells (i.e. [1-13C]acetate, [2-13C]acetate, [1,2-13C]acetate or 13Co2). Other more costly precursors such as pyru-vate could be used as an alternative, ex. ~1-13C]pyruvate could be used to label other positions.

6. Production of a broad spectrum of site-specific labelled amino acid species. The procedure described allows recovery of all amino acids but cys, trp and his, which can be obtained as well, using modified hydrolysis conditions as described in Analytical Biochemistry, 1982, p. 238-247 LeMaster and Richards.

7. The specificity of labeling applies also to cell products other than amino acids and carbohydrates, such as purine/pyrimidine bases and lipids.
The foregoing embodiment of the present invention is an example of this invention meant to illustrate and not limit the present invention. It will be evident to those skilled in the art that this invention can be practiced with other acid tolerant methanogens and other isotopes (such as 14Carbon).

20~3253 Table I. 13C NMR chemical shifts of amino acids from a protein hydrolysate of GP9a.

_ Amino acids Chemical shifts (ppm) forb aOOH C-2 C-3 C-4 C-5 C-6 Other Alanine 49.73* 15.31 Serine 55.20* 59.43 Glycine 40.33*
Aspartate 49.87* 33.89 Threonine 59.01* 65.31 24.93 29.46 Glutamate 24.98* 29.50*
. . _ . _ .
27.02 23.77 Arginine 27.08* 23.75*
_ _ .. . . _ . _ _ 28.45 23.44 Proline 28.45* 23.40*
Leucine 173.20* 51.70 39.01* 23.96* 20.88 C-5' 21.64 Valine 58.85* 28.95* 16.76 C-4' 17.43 Isoleucine 171.99* 57.60 35.70* 24.57* 10.91 C-6' 14.2 Phenylalanine 54.68* 35.69 C-5' 129.38 C-6' 129.11*
Tyrosine 54.68* 34.84 C-5' 130.82 C-6' 115.87*
Lysine 53.33* 26.27 29.35 39.00*
MRthionine 28.98 _ _ a Reference TMS capillary, pH=1Ø
b The sources of carbon atoms are designated by *, C-l acetate; C-2 acetate.

Table II. Yield of amino acids from 24 and 48 h hydrolysates of protein recovered from strain GP9 grown on [1-13C] or [2-13C]acetate.

[1-13C]acetate [2-13C]acetate Yield Amino acid mol % mol ~ ~g/mg cell dry wt.
24H 48H 24H 48h 48h .
Aspartic11.52 11.11 11.3111.21 46.65 Threonine6.07 6.19 6.11 6.20 23.09 Serine 4.99 4.79 4.95 4.65 15.28 Glutamic12.50 11.96 12.6412.33 56.73 Proline 3.88 4.00 4.17 4.12 14.84 Glycine 9.47 10.03 8.67 8.98 21.08 Alanine 9.03 9.15 9.26 9.32 25.97 Valine 6.90 7.59 6.81 7.51 27.51 Methionine1.81 1.52 2.13 1.58 7.36 Isoleucine6.67 7.41 6.60 7.35 30.14 Leucine 7.61 7.42 7.58 7.79 31.94 Tyrosine2.60 2.49 2.64 2.46 13.91 Phenylalanine 3.69 3.66 3.71 3.70 19.09 Histidine1.59 1.55 1.70 1.58 7.73 Lysine 8.07 7.79 8.15 7.89 36.04 Arginine3.58 3.35 3.56 3.36 18.27 395.57 20832~
TABLE III. 13C NMR chemical shifts for carbon atoms of most abundant carbohydrates labelled by growth of GP9 in media contain-ing (1-13C) or (2-13C)acetate.

Anomer Carbon atom 13cprecursorglc gal man 1 (2-13C)acetate 92.19 92.38 94.14 2 (1-13C)acetate 71.58 68.45 70.79 (1-13C)acetate 71.54 70.58 72.50 6 (2-13C)acetate 60.68 61.28 61.3-61.4 (2-13C)acetate 96.01 96.56 93.77 2 (1_13C)acetate 74.24 71.98 71.32 (1-13C)acetate 76.05 75.26 76.27 6 (2-13C~acetate 60.85 61.09 61.3-61.4 _ _ . _ _ ,: .

.
;

. ,

Claims (30)

1. A method for producing isotope labelled metabolites comprising incubating an acid tolerant methanogen in the presence of isotope labelled carbon source.

2. The method of claim 1 wherein said metabolite is an amino acid.

3. The method of claim 1 wherein said metabolite is a carbohydrate.

4. The method of claim 1 whereby said carbon source is acetate.

5. The method of claim 1 wherein said methanogen is capable of growth at a pH less than about 6Ø

6. The method of claim 1 wherein said methanogen is capable of growth at a pH less than about 5.5.

7. The method of claim 1 wherein said methanogen exhibits a low level of carbon exchange between CO2 and the carboxyl group of acetate.

8. The method of claim 1 wherein said methanogen has a low level of acetate synthesis from CO2 in the presence of exogeneous acetate.

9. The method of claim 1 wherein said methanogen's anabolic pathways are essentially non-cyclic.

10. The method of claim 1 wherein said isotope is 13Carbon.

11. The method of claim 1 wherein the said isotope is 14carbon.

12. The method of claim 4 wherein said amino acid is select-ed from the group comprising alanine, serine, glycine, aspartate, threonine, glutamate, arginine, proline, leucine, valine, iso-leucine, phenylalanine, tyrosine, lysine and methionine.

13. The method of claim 3 wherein said carbohydrate is selected from the group consisting of glucose, galactose and mannose.

14. A method of claim 12 wherein said methanogen is Methano-bacterium espanolae.

15. A method of claim 13 wherein said methanogen is Methano-bacterium espanolae.

16. A use of an acid tolerant methanogen to prepare isotope labelled metabolites.

17. The use according to claim 16 wherein said metabolite is an amino acid.

18. The use according to claim 16 wherein said metabolite is a carbohydrate.

19. The use according to claim 16 wherein said methanogen is capable of growth at a pH less than about pH 6Ø

20. The use according to claim 16 whereby said methanogen is capable of growth at a pH less than about pH 5.5.

21. The use according to claim 16 whereby said methanogen exhibits a low level of exchange between CO2 and a carboxyl group of acetate.

22. The use according to claim 16 whereby said methanogen has a low level of acetate synthesis from CO2 in the presence of exogeneous acetate.

23. The use according to claim 16 wherein said methanogen's anobolic pathways are essentially non-cyclic.

24. The use according to claim 16 wherein said isotope is 13-carbon .

25. The use according to claim 16 wherein the said isotope is 14-Carbon.

26. The use according to claim 17 wherein said amino acid is selected from the group comprising alanine, serine, glycine, aspartate, threonine, glutamate, arginine, proline, leucine, valine, isoleucine, phenylalanine, tyrosine, lysine and methio-nine.

27. The use according to claim 18 wherein said carbohydrate is selected from the group consisting of glucose, galactose and mannose.

28. The use according to claim 16 whereby said methanogen is Methanobacterium espanolae.

29. An isotope labelled amino acid prepared by the method described in claim 2.

30. An isotope labelled carbohydrate prepared by the method described in claim 3.