WO1996004366A1

WO1996004366A1 - Non-sulfato-reducing strict anaerobic thermophilic and hyperthermophilic bacteria culture method

Info

Publication number: WO1996004366A1
Application number: PCT/FR1995/001022
Authority: WO
Inventors: Bernard Ollivier; Marie-Laure Fardeau; Hervé ROBERT; Gilles Ravot; Jean-Luc Cayol; Michel Magot; Jean-Louis Garcia
Original assignee: Institut Francais De Recherche Scientifique Pour Le Developpement En Cooperation (O.R.S.T.O.M.)
Priority date: 1994-07-29
Filing date: 1995-07-28
Publication date: 1996-02-15
Also published as: FR2723103A1; FR2723103B1

Abstract

Non-sulfato-reducing strict anaerobic thermophilic and hyperthermophilic bacteria culture method for the production of metabolites, in particular enzymes. A culture medium is used in strict anaerobiosis conditions, in the absence of an external hydrogen supply, said culture medium containing at least 1 g/l of a peptidic and/or amino acid source, at least one sugar and thiosulfate as the growth stimulating agent.

Description

BACTERIA CULTURE PROCESS

THERMOPHILES AND HYPERTHERMOPHILIC BACTERIA ANAEROBIC STRICT NON-SULFATO-REDUCING.

The subject of the invention is a process for the cultivation of thermophilic bacteria and strict anaerobic hyperthermophilic bacteria, which are not sulfato-reducing. It also relates to the use of this process for producing metabolites, more especially enzymes, and for selecting thermophilic or even hyperthermophilic bacterial strains, in particular strains producing thermostable enzymes.

Today there is an important scientific dynamic around thermophilic and more particularly hyperthermophilic bacteria, given the thermostability nature of their enzymes, many of which are of industrial interest, such as xylanases, proteases, cellulases or amylases. .

It is recalled that thermophilic bacteria are capable of growing at optimal temperatures between 45 ° C and 80 ° C.

Bacteria that grow optimally at higher temperatures, especially between 80 and 110 ° C are called hyperthermophiles.

Most often, thermophilic bacteria of the genus Thermoanaerobacter and hyperthermophilic bacteria of the order of Thermotogales belonging to the Bacteria domain show weak or even slow growth even in the presence of elemental sulfur (in particular in Thermotogales). This slow growth in the presence of elemental sulfur constitutes a limiting factor, in particular for biochemical studies and the industrial production of enzymes. Of this fact, the research concerned the isolation of the gene (s) coding for thermostable enzymes in order to clone and express them in Escherichia coli or Bacill us sp.

The inventors' work on thermophilic bacteria and on hyperthermophilic bacteria of the above type enabled them to demonstrate that they were capable of using, during their metabolism, under given culture conditions, a particular sulfur compound. , and that the latter played a role of great interest as a growth-stimulating agent.

The object of the invention is therefore to provide a new method for cultivating these bacteria which makes it possible to increase cell yields, and thus the quantity of metabolites produced, more especially enzymes.

The object of the invention is also to apply this culture method to the selection of thermophilic, even hyperthermophilic, anaerobic, thiosulfato-reducing, non-sulfato-reducing, bacterial strains and to the production of thermostable enzymes.

It also targets the new bacterial strains and the enzymes obtained by using these culture, selection and isolation techniques.

The process for the cultivation of thermophilic and hyperthermophilic, strict anaerobic, thiosulphate-reducing, non-sulphate-reducing bacteria of the invention is characterized in that it is carried out, under strict anaerobic conditions, in the absence of external supply of hydrogen, a culture medium containing a peptide source and / or amino acids, at a rate of at least 1 g / l of culture medium, at least one sugar and thiosulfate as a growth stimulant, which is recovered and purified, if desired, the metabolites produced, in particular the enzymes.

The peptide source is advantageously used in an amount of at least 2 g / l of culture medium, or even more than 4 to 5 g / l.

By operating under these conditions, thiosulfate is reduced to sulfides during the metabolism of these bacteria.

This effect of the thiosulfate on which the invention is based translates into a significant improvement in bacterial kinetics as well as in cell yields. We thus observe a reduction in generation times of the order of 50 to 100% and improved cell yields of about 100%.

The results are always much higher than those observed in the presence of elemental sulfur.

This beneficial effect is manifested on all of the sugars used by these bacteria, and in particular those in C6, such as sugars of the glucose type (cellulose or starch monomer), or those in C5, such as ribose. or of the xylose type (xylan monomer).

This stimulation, which can also be observed when the bacteria are cultivated on a biological polymer such as xylan, is the result of a metabolic deviation which leads to a greater production of acetate in the presence of thiosulfate, in particular at the expense lactate and / or ethanol, in particular for the species of the genus Thermoanaerobacter, or to phosphorylations not linked to the substrate, in particular in hyperthermophilic bacteria of the order Thermotogales (T. mari tima and T. neapoli tana, respectively deposited with the DSM under N ° 3109 and 4539).

In general, this deviation from metabolism is accompanied by greater use of the substrate.

In addition, a notable improvement in growth is observed on glucose in the presence of thiosulfate and results here from a greater production of acetate to the detriment of L-alanine in Thermosipho africanus (DSM N ° 5309), Fervido bacteri um islandicum (DSM N ° 9442), and Thermotoga elfii (DSM N ° 5733). The production of L-alanine from glucose in Thermotogales and in the field of Bacteria in general has been demonstrated for the first time by the inventors.

In addition, sugars, peptides and / or amino acids are also used as culture substrate, in accordance with the invention, in an amount of at least 1 g / l of culture medium. Their use is improved in the presence of thiosulfate, under the conditions of the invention. An increase in the cell density of the culture medium and / or a higher production of volatile fatty acids is thus observed.

Bacteria capable of using thiosulfate under the conditions defined above, and thus of being used in the process of the invention, are chosen from Bacteria:

Thermotogales and the genus Thermoanaerobacter.

Bacteria of the order Thermotogales advantageously used include in particular the genera Fervidobacterium, Thermosipho and Thermotoga. By extension, the Geotoga bacteria and

Petrotoga are likely to have the same properties.

Thermotogales have as their habitat volcanic thermal springs of marine or terrestrial origin, deep hydrothermal systems, large artesian basins and water from petroleum deposits.

These bacteria of the order Thermotogales, as demonstrated according to the invention, are capable of reducing thiosulfate to sulfides, which had not been reported in the scientific literature to date. This allows the use of a product more soluble than elemental sulfur and therefore able to be easily separated from the bacterial culture.

Among the Bacteria, we will mention the genus

Thermoanaerobacter.

Bacteria of the genus Thermoanaerobacter are thermophilic, heterotrophic, saccharolytic anaerobes and are capable of reducing thiosulfate to sulfide.

Their habitat is soil, sugar beet extraction juice, sugar cane juice, volcanic hot springs and oil wells.

Among these bacteria, mention will be made of T. finnii, T. brockii, T. ethanolicus T. thermohydrosulfuricus and Thermoanaerobacter SEBR 5268.

The addition of thiosulfate to cultures of these bacteria significantly increases cell yields, by at least 50% and even up to 300% depending on the strains. Likewise, cell productivity, in mg of cells / liter / hour, is considerably increased.

Advantageous conditions for modifying, under the effect of thiosulfate, the metabolism of these bacteria towards more energetic pathways, include carrying out the culture at a temperature of the order of 45 to 110 ° C., in particular from 60 to 90 °. C at a pH of 3 to 10, in particular from 6.5 to 8.0 for neutrophilic species, 3 to 6.5 for acidophilic species, and 8 to 10 for alkalophilic species.

Preferably thiosulfate is used in an amount of 1 to 100 mM, in particular 15 to 25 mM.

The culture medium is inoculated with approximately at least 10% w / v of culture medium.

The culture method of the invention can be carried out continuously or batchwise.

The invention also relates to the application of the culture method as defined above to the selection or screening of thermophilic or hyperthermophilic, anaerobic, non-sulfato-reducing bacterial strains from a medium containing them, and more especially the selection of bacteria of the above-mentioned type producing thermostable enzymes.

This process for selecting thermophilic, even hyperthermophilic bacterial strains, is characterized by the culture of strains taken from a given medium, under strict anaerobic conditions, in the absence of external hydrogen supply, in the presence of thiosulfate. , on a support for peptides and / or amino acids, and sugars, under conditions suitable for their growth.

The strains will be selected from enrichments carried out in a liquid medium under given conditions (pH and temperature corresponding to the properties sought for the strain) on substrates bringing about given enzymatic activities.

These enrichments make it possible to make a selection within a bacterial population to bring out a given microorganism.

The colonies recovered in agar medium will be purified by successive dilution techniques, in anaerobiosis, developed by Hungate RE, 1969, in "Methods in Microbiology", JR, Norris and DW Ribbons, p.117-132, vol-3B, Académie. Press Inc. New York.

Thanks to this process, it is possible to select, from a given medium, thermophilic or hyperthermophilic, alkalotolerant or alkalophilic, or acidotolerant or acidophilic strains, according to the pH conditions chosen.

The invention makes it possible in particular to have alkalophilic thermotogals, while only neutrophil thermotogals have been described to date.

The invention thus makes it possible to isolate strictly thiosulfato-reducing, sulfuro-reducing and non-sulfato-reducing bacterial strains, of the order of Thermotogales in particular.

According to an aspect of great interest, the screening method of the invention makes it possible to select strictly thiosulfato-reducing, non-sulfuro-reducing, and non-sulfato-reducing bacteria, of the order of Thermotogales in particular.

Such strains are new and fall, as such, within the scope of the invention.

According to another aspect of major interest, the selection process of the invention makes it possible to search for bacterial strains having a given enzymatic activity, in particular bacterial xylanolytic, proteolytic, lipase, amylolytic or cellulolytic strains.

It will be noted that the obtaining of such bacterial populations is all the more different from the state of the art that to date, the Thermotogales have been described as being sulfuro-reducing, except for the species Thermotoga thermarum incapable of '' use the sulfur, and whose thiosulfato-reducing properties had not been demonstrated.

The selection of bacterial strains operated in accordance with the invention is carried out from various biotopes, such as the large artesian, Australian and Indian basins, deep hydrothermal systems, water from petroleum deposits or ecosystems of volcanic, terrestrial or Marine.

In general, the stimulating effect on bacterial growth, in the presence of thiosulfate, reported above, is also accompanied by an improvement in the total enzymatic activity.

The invention therefore also provides means for obtaining enzymes which are thermostable in high quantities, which makes it possible to envisage industrial exploitation, in the numerous sectors which use enzymatic technologies at high temperature and to meet non-specific needs. satisfied to date.

The invention therefore also relates to a process for the production of thermostable enzymes, characterized in that it comprises the culture of thermophilic or hyperthermophilic bacteria endowed with a given enzymatic activity, under strict anaerobic conditions, in the absence of external supply of hydrogen, in the presence of thiosulfate, on peptide substrate and / or amino acids and sugars, so as to obtain the production of the desired enzymes, and the recovery of these enzymes from the culture medium, followed by their purification if desired.

With the amplifying systems constituted by the culture media containing thiosulfate, it is thus possible to produce, in large quantities, various thermostable enzymes of interest in various sectors. Alternatively, these enzymes can be expressed, according to conventional molecular biology techniques in microorganisms, for example mesophilic microorganisms such as Escherichia coli and Bacillus sp.

The enzymes obtained are advantageously subjected to at least one purification step, with a view to their use.

For example, the purification of xylanases from cell pellets or supernatants obtained after centrifugation will include the steps of precipitation with ammonium sulphate, passage through DEAE-Sepharose, elution of the xylanase activity by gradient NaCl, passing through an ion exchange column and, where appropriate, passing through a hydroxyapatite column.

These steps will naturally be modified according to the proper properties of the enzyme and other methods are readily accessible to those skilled in the art for purifying the various enzymes obtained.

The invention thus provides enzymes, such as amylases, glucoamylases, glucoisomerases and pullulanases, usable for the transformation of starch (starch manufacture and glucosery), xylanases for the bleaching of paper pulp, proteases, pectinases, amylases , cellulases, lipases for detergency, amylases and proteases for desizing in the textile industry and proteases for scouring silk.

Other examples of enzymes will also be mentioned: glucose isomerase, xylose isomerase, cellobio hydrolase, pullulanase, amylosaccharidase, hydrogenases and alcohol dehydrogenases, the latter making it possible to modify the chirality of isomers and therefore to lead to active products for example in pharmacy. It will be appreciated that some of these enzymes are new products, in particular due to their characteristics of stability at high temperature and, where appropriate, their alkaline or acidic properties. Such enzymes are therefore also part of the invention.

By operating under acidic conditions, acidic thermostable enzymes are isolated, for example acid amylases. On the contrary, the use of alkaline pH conditions makes it possible, from cultures of alkalotolerant or alkalophilic strains, to have thermostable alkaline enzymes of great interest in certain application sectors.

Thus, the culture, under the conditions of the invention, of alkalophilic thermotogals makes it possible to produce large quantities of alkalophilic xylanases which can be used for bleaching paper pulp or also for the release of simple sugars at C5 in animal feed.

The invention allows in particular the production of thermostable enzymes which, if necessary, cannot be expressed in their active form via genetic cloning in other organisms.

It should be noted in this regard that even if cloning is possible, the proteins synthesized by recombinant microorganisms may no longer possess the enzymatic properties of the original organism.

In addition, it has been demonstrated, in the context of the invention, as regards for example the xylanases of the thermophilic organisms studied, that they were largely linked to the bacterial wall, which facilitates their recovery.

To illustrate the invention defined above, examples of culture of thermophilic and hyperthermophilic bacteria in the presence of thiosulfate.

In these examples, reference is made to FIGS. 1 to 7, which respectively represent,

FIGS. 1 and 2, the growth of T. finnii respectively on glucose and xylose, with or without thiosulfate,

- Figure 3, the effect of thiosulfate on the growth of several bacteria of the order Thermotogales,

FIG. 4, the xylanolytic activities of bacteria of the order Thermotogales and of the genus Thermoanaerobacter, and

- Figure 5, xylanolytic activities on xylan and xylose, with or without thiosulfate in

Thermotoga SEBR 2665, and

- Figures 6 and 7, the effect of thiosulfate on the oxidation of bio-trypcase ^R by T. brockii and T. ethanolicus, respectively.

Example 1: Culture of bacteria of the genus Thermoanaerobacter on glucose or xylose.

The results obtained with the strains T. finnii (DSM nº 3389) and Thermoanaerobacter SEBR 5268 are reported.

T. finnii was obtained from the German Collection of Microorganisms DSM (Deutsche Sammlung von Mikroorganismen in Braunschweig (Germany).

The Thermoanaeroiacter SEBR 5268 strain was isolated from an oil field water in France using the following culture medium (g / l): NH4CI, 1; K ₂ HPO ₄ , 0.3; KH ₂ PO ₄ , 0.3; MgCl ₂ , 6H ₂ O, 1.3; CaCl ₂ . 2H ₂ O, 0.1; KCl, 0.2; CH ₃ COONa 3H ₂ O, 0.5; NaCl, 2; biotrypease R (bioMérieux, France), 5; yeast extract (Difco Lab., Detroit, Mien.), 5; glucose 10

Resazurin, 1 mg; Cysteine-HCl, 0.5; Balch mineral solution, 10 ml. The pH was adjusted to 7.0 with a 10 M KOH solution. The medium was prepared anaerobically with N2: CO ₂ (80/20) in the gas phase. After sterilization (110ºC, 40 min), 0.2 ml of Na ₂ S. 9H ₂ O (2%), 1 ml of NaHCO ₃ (10%), 0.1 ml of a solution of Widdel vitamins and 0 , 1 ml of a sodium dithionite solution (0.2%) are added sterile for 20 ml of medium.

The purification of this strain was carried out using the method of dilution in agar medium developed by Hungate.

All the experiments were carried out at 60 ° C. in a base medium supplemented with yeast extract 1 g / l, as described by Fardeau et al in FEMS Microbiol. Lett., 1993, 113, 327-332.

Hungate's anaerobic techniques were used to carry out all of these experiments. These techniques are described in Methods in Microbiology (Norris JR, Ribbons DW, Eds.) Vol 3B, pp 117-132. Academy Press, London.

After preparing the anaerobic medium, 5 ml or 20 ml of the liquid medium are distributed in Hungate tubes or in 50 ml serum bottles respectively under a stream of nitrogen devoid of oxygen.

Analysis techniques.

The optical density, hydrogen sulfide, hydrogen and volatile fatty acid measurements are analyzed as described in the article by Fardeau et al above.

The times of division and the maximum optical density obtained with these are reported in Table 1.

Examination of this table shows that the intake of thiosulfate makes it possible to reduce the division time in T. finnii from 6 h to 3 h 30. Cell yields of the genus Thermoanaerobacter increase by 30 to 200%, with an average of l 'order of 100%.

This beneficial effect is observed with the two types of substrates in the genus Thermoanaerobacter, as illustrated by FIGS. 1 and 2 which give the optical density at 580 nm as a function of time in hours. The curves

- - correspond to the use of glucose or xylose with thiosulfate and the curves

- - to that of glucose or xylose alone.

The study of the metabolites produced in a culture of T. finnii on glucose with or without thiosulfate shows the changes in the metabolism of the bacteria.

The results obtained are given in Table 2 where "M" in the first column means

"culture medium" and "Thio", thiosulfate at 20 mM.

The metabolic deviation induced by thiosulfate leads to a higher production of acetate to the detriment of lactate and / or ethanol. Example 2. Culture of bacteria belonging to Thermotogals.

The culture is carried out in a medium consisting of 5 g / l of yeast extract, 5 g / l of biotrypcase R, 20 mM / l of glucose and 5g / l of NaCl when using Thermosipho africanus, Thermotoga maritima, Thermotoga neapolitana and Thermotoga SEBR 2665.

With Fervidoiacterium islandicum, a medium containing 1 g / l of NaCl is used instead of 5 g / l as before, the other constituents being used in the same proportions.

All these strains come from the German collection of microorganisms (DSM), except Thermotoga

SEBR 2665 isolated from petroleum reservoir water, anaerobically, using the successive dilution techniques in solid medium developed by Hungate.

Is indicated in Table 3 according to the amount in mM products sulfides, the maximum optical densities and cell productivities mg cell weight sec.1 ^-1 .h ^-1.

For the calculation of cellular productivity, we assume that a unit D.0. corresponds to 0.5 g of dry weight cells. per liter.

u a u 3 2 2 3 8

Examination of this table shows that in this case also the addition of thiosulfate leads to a significant increase in cell yields of bacteria, from 50 to 300% depending on the strains and cell productivity.

The effect of thiosulfate on the growth of T. marltima is illustrated in FIG. 3 which gives the variation of the optical density at 580 nm as a function of time in hours.

The curve

- ■ - corresponds to the use of a base medium containing 5 g / l of yeast extract, 5 g / l of biotrypcase and 20 mmol / l of glucose, the other curves corresponding to the use of this medium added with 2% elemental sulfur for the curve -

- of thiosulfate at a rate of 20 mmol / l for the curve

- ♦ - or sulfate at a rate of 20 mmol / l for the curve -

- ♢ -

The incubation temperature is in each case 70ºC.

This figure highlights the increase in cell yields. These results when using thiosulfate are always superior to those observed in the presence of elemental sulfur.

Example 3: Study of the effect of the addition of thiosulfate on the enzymatic activity.

In this example illustrated by FIGS. 4 and 5, the results obtained with bacteria of the order of Thermotogals and of the genus rJienπoanaeroiacter are reported.

These results relate to xylanolytic activity in u / ml of resuspended pellets.

Figure 4 gives the values obtained after 92 hours of culture on xylan at 10 ºC, without thiosulfate

and with 20 mM thiosulfate

with Thermotoga maritima, Thermotoga neapolltana, Thermotoga sp. SEBR 2665, and Thermoanaerobacter brockll.

In FIG. 5, the xylanolytic activities, measured at pH 6.5 and at 75 ° C. are given as a function of time for cultures of Thermotoga SEBR 2665 on xylose

☐), on xylose with thiosulfate at a rate of

20 mM

on xylane ◇

on xylan with thiosulfate at a rate of 20 mM ♦)

Examination of these curves shows an improvement in xylanolytic activity, expressed in u / ml of resuspended cells of the order of 100% and even more for certain strains, in particular with regard to Thermotoga neapolltana.

These results clearly show that the improvement of enzymatic activities, within the framework of studies carried out on xylose, is directly linked to that of bacterial growth.

Example. 4: Culture of thermophilic bacteria on peptide and / or amino acid substrates.

Strains

Thermoanaerobacter thermohydrosulf urlcus (DSM Nº 567), T. brockll (DSM nº 1457), T. flnnli (DSM Nº 3389) and T. ethanollcus (DSM Nº 2246) were obtained from DSM.

The SEBR 5268 strain corresponds to that indicated in Example 1.

Culture media

The operation is carried out under the conditions reported in Example 1, but the following products have been added: thiosulfate, casamino acids (Difco, EUA), gelatin (Sigma, EUA) and the bio-Trypcase R peptones (bioMérieux, France), peptone pancreatic, (Prolabo, France), bactopeptone R (Difco, EUA), bio-gelytone ^R (bio Mérieux, France), papain soy peptone (Institut Pasteur, Production, France).

The results obtained relating to the effect of thiosulfate on the oxidation of peptides and amino acids by the strains SEBR 5268, T. flnnll and Ther moanaero-iacter sp brockll, T. Thermohydrosulfuricus are reported in Tables 4 to 6. and T. ethanolicus.

These results correspond to a 3-day incubation at 60ºC with the addition of peptides, casamino acids or gelatin at a rate of 5 g per liter. The basic medium used contains one gram per liter of yeast extract as indicated above and thiosulfate at a rate of 20 mM.

In these tables, Thio means thiosulfate

20 mM; Casa, casamino acids, Biot, bio trypcase R, Pap, a papain soy peptone, Pan, a pancreatic peptone, Bact, bacteropeptone, Biogel, biogelyton; and Gel, gelatin.

As the results reported in Tables 4 and 5 show, amino acids and peptides from different biological sources are used more efficiently when thiosulfate is added to the medium.

In the presence of thiosulfate, an increase in optical density and / or in the production of volatile fatty acids such as acetate, propionate, iso-butyrate, and iso-valerate is observed. On the contrary, these activities are limited in the absence of thiosulfate.

Tables 4, 5 and 6 show that all of the isolates are capable of making significant use of peptides and amino acids.

The examination of these three tables shows for these strains of Thermoanaerobacter, at the same time an improved growth and an increase in the production of fatty acids, also a production of sulfide and a decrease in the levels of hydrogen.

The effect of thiosulfate on the oxidation of bio-trypcase R by T. brockll and T. ethanolicus is illustrated by Figures 6 and 7. These figures give respectively, as a function of time in hours, the variation in the production of fatty acids (acetate☐; isobutyrate ■; isovalerate ▲; propionate (O) and that of the production of H ₂ (O) and H2S

(☐).

These results therefore demonstrate for the first time that members of the genus The rmoanaerobacter make better use of peptides and amino acids in the presence of thiosulfate.

Their ability to grow on substrates such as amino acids and / or peptides in the presence of thiosulfate indicates that they can grow by mixotrophy, since using both hydrogen and an organic substrate. All members of the genus Thermoanaerobacter studied are indeed, stimulated by thiosulfate and the subsequent reduction of thiosulfate during the fermentation of amino acids and / or peptides, which results in an increase in hydrogen sulfide. It has further been reported that members of the genus Thermoanaerobacter are capable of oxidizing hydrogen in the presence of thiosulfate, which means that hydrogenases could play a role in the reduction process of thiosulfate.

Claims

1) Process for the culture of thermophilic bacteria and hyperthermophilic bacteria, strict anaerobes, non-sulfato-reducing, characterized in that one implements, under strict anaerobic conditions, in the absence of external hydrogen supply , a culture medium containing a peptide and / or amino acid source, in an amount of at least lg / 1 of culture medium, at least one sugar, and thiosulfate as growth stimulating agent.

2) Culture method according to claim 1, characterized in that the peptide and / or amino acid source is used in an amount of at least 2 g / l.

3) Method according to claim 1 or 2, characterized in that it further comprises a step of recovering the metabolites produced during the culture, in particular enzymes, followed where appropriate by a purification step.

4) Method according to one of claims 1 to 3, characterized in that bacteria selected from Bacteria are used with members of the genus Thermoanaerobacter or of the order Thermotogales.

5) Method according to claim 4, characterized in that one implements Thermotogales, in particular like those of the genera Fervidobacterium, Thermosipho and Thermotoga, Geotoga and Petrotoga.

6) Method according to one of claims 1 to 5, characterized in that the culture is carried out at a temperature of about 45 to 110 ° C, in particular from 60 to 90 ° C, at a pH of 3 to 10 , in particular from 6.5 to 8 for neutrophilic species, from 3 to 6.5 for acidophilic species and from 8 to 10 for alkalophilic species, the thiosulfate being used at a rate of 1 to 100 mM, in particular from 15 to 25 mM.

7) Method according to one of claims 1 to 6, characterized in that it is carried out continuously or discontinuously.

8) Method for selecting thermophilic or hyperthermophilic, anaerobic, non-sulfato-reducing bacterial strains, characterized by the culture of an isolate of strains from a given medium, under strict anaerobic conditions, in the absence of external supply of hydrogen, in a culture medium containing a peptide source and / or amino acids / at least one sugar, under conditions suitable for their growth, in particular as defined in claim 6, isolation strains selected from enrichments in liquid medium on substrates bringing given enzymatic activities, the recovery of the colonies formed, and their purification if desired.

9) Method according to claim 8, characterized by the isolation of alkalotolerant or alkalophilic, or acidotolerant or acidophilic bacterial strains, depending on the pH of the culture medium.

10) Method according to claim 9, characterized by the isolation of strains from the field of

Bacteria, in particular of the genus Thermoanaerobacter or of the order Thermotogales.

11) A method according to claim 9, characterized in that it is applied to the isolation of strictly thiosulfato-reducing, non-sulfuro-reducing and non-sulfato-reducing bacteria, of the order Thermotogales.

12) Method according to claim 9, characterized in that it is applied to the isolation of strictly thiosulfato-reducing, sulfur-reducing and non-sulfato-reducing bacteria, of the order of Thermotogales.

13) Method according to one of claims 8 to 12, characterized in that it is applied to the isolation of bacterial strains having an enzymatic activity such as xylanolytic, proteolytic, amylolytic, lipase or cellulolytic.

14) Method for producing thermostable enzymes, characterized in that it comprises the culture of thermophilic or hyperthermophilic bacteria in the presence of thiosulfate, on peptide substrate and / or amino acids and sugars, in the absence of intake hydrogen exterior, in particular, under the conditions defined in claim 6, where appropriate from bacterial strains as isolated according to the method of one of claims 8 to 12, and the recovery of these enzymes from the medium culture, followed by their purification if desired.

15) The enzymes as obtained by the process according to claim 14, where appropriate purified.