Extremely halophilic methanogenic archaebacteria and process for the production of methane

Abstract: Methanohalococcus alcaliphilum bacteria having an optimum growth sodium chloride concentration ranging from about 2.5 to about 3 mole are described. The bacteria are halophilic or halophilic and alkalophilic and can be used as microorganisms for methane fermentation to enhance the efficiency of such a fermentation since their use makes it possible to carry out the fermentation at an alkaline pH in the presence of salts in a high concentration and at a low temperature.

Description: The present invention relates to extremely halophilic methanogenic (methane producing) bacteria and more particularly to new species of the new genus Methanohalococcus having an optimum growth concentration of sodium chloride ranging from 2.5 M to 3.0 M. Examples of the halophilic methanogenic bacteria conventionally known include those having an optimum growth at a sodium chloride concentration which ranges from 1 to 2 M (Applied and Environmental Microbiology, 1985, Vol. 50, No. 4, pp. 877 - 881); those having an optimum growth sodium chloride concentration which ranges from 1.2 to 1.5 M (Microbiology (U.S.S.R.), 1983, Vol. 52, pp. 290 - 297); and those having an optimum sodium chloride concentration of 2.1 M (Applied and Environmental Microbiology, 1985, Vol. 50, No. 1, pp. 140 - 143). Although either of these bacteria is halophilic, the sodium chloride concentration among the optimum growth conditions thereof is not more than 2.1 M and thus they are moderately halophilic methanogenic bacteria. The principal object of the present invention is to provide new species of the new genus Methanohalococcus having an optimum growth condition at high sodium chloride concentrations. Another object of the present invention is to eliminate disadvantages encountered in carrying out methods of methane gas production (methane fermentation) in order to improve the efficiency thereof. Consequently, the present invention relates to Methanohalococcus alcaliphilum having an optimum growth condition at a sodium chloride concentration ranging from 2.5 M to 3.0 M. The microorganisms of the present invention will hereunder be explained in more detail with reference to the accompanying drawings, wherein: Figs. 1 to 4 are diagrams in which the amount of methane gas produced by the methanogenic bacteria NY-218 of the present invention is plotted against NaCl concentration, pH, temperature and caltivation days respectively; and Figs. 5 to 8 are diagrams similar to Figs. 1 to 4 provided that the methanogenic bacteria NY-844 of the present invention are used instead of NY-218. Methanohalococcus alcaliphilum of the present invention can be classified into two groups: one of which is represented by NY-218 (FERM BP-2079) whose optimum growth pH ranges from 8.0 to 8.5 and the other by NY-844 (FERM BP-2080) whose optimum growth pH ranges from 7.5 to 8.0. The means for obtaining Methanohalococcus alcaliphilum of the present invention (the screening method or the like) will now be explained in more detail below. (1) Screening Method In the present invention, the screening of methanogenic bacteria was carried out in accordance with the methods for separating anaerobic bacteria disclosed in Japanese Patent Unexamined Publication No. Sho 63-49074 and Japanese Utility Model Unexamined publication No. Sho 63-31799. More specifically, the screening method employed herein comprises the steps of first exploring soil in extreme environments such as soil under a strong alkali condition or a high salt concentration to collect bacteria under the desired special environment; then immediately establishing conditions suitable for the collected bacteria such as anaerobic conditions and low redox potential conditions utilizing a diluent for pretreatment which is prepared on the basis of the solution disclosed in J.P.A. No. 61-191541; preparing a solution for dilution while taking into consideration the characteristics of the soil, from which the bacteria have been collected, such as pH and salt concentration thereof; and finally immediately suspending the soil into the solution for dilution. The suspension thus obtained should be stored in a container as disclosed in J.U.M.A. No. 61-125086 so that the methanogenic bacteria, i.e., strictly anaerobic bacteria do not die out and simultaneously it should be transported while maintaining it so that the objective methane producing bacteria in a special environment do not die out. The transported sample is used as the sample for isolation culture. With regard to the soil as a source for screening, soil in special environment is widely explored in Japan and abroad. (2) Isolation Culture As the medium for isolation culture, an isolation medium which was originally developed by the inventors of the present invention is employed. The composition of the isolation medium used is summarized in Table I given below. In this connection, the composition of the solution of trace elements or that of the solution of vitamin included in Table I are listed in Table II or III respectively. Regarding the trace element solution shown in Table II, the requirement of the bacteria for trace elements has not yet been clear and, therefore, a trace element solution having an original composition in which the kinds of trace elements used and the amount thereof are increased. As the substrates to be added to the culture medium, there can be used, as shown in Table I, such substances as sodium formate, sodium acetate, methanol and methylamines (e.g., trimethylamine) and the amount thereof is independently adjusted to 0.5 to 1.0%. Sodium bicarbonate is used to control the pH value of the culture medium when culture is commenced. In addition, methanogenic bacteria are archaebacteria and they do not contain peptidoglycan in their cell wall. Therefore, they exhibit no sensitivity to a peptidoglycan synthesis inhibitor. Moreover, they do not have sensitivity to protein synthesis inhibitors effective to bacteria which have procaryotic cell type 70S ribosome system, though their ribosome are 70S type one. Thus, effective isolation of the desired methanogenic bacteria was carried out by adding, to the culture medium, an antibiotic which could suppress the growth of only non-methane producing bacteria during isolation culture. More specifically, vancomycin which is an inhibitor for peptidoglycan synthesis in the cell wall and kanamycin which is a protein synthesis inhibitor effective to bacteria having procaryotic cell type 70S ribosome system were practically employed as shown in Table I. As the gas phase during culture, there were used three kinds of gases, i.e., H₂/CO₂ mixed gas (80/20); N₂/CO₂ mixed gas (80/20) and N₂ gas which were deoxygenated prior to use. Such a gas was charged into a container for culture so that the pressure therein was equal to positive 2 atms. during culture irrespective of the kinds of the culture medium which may be either liquid medium or agar medium, whereby the cultivation is carried out under press. Moreover, the culture was principally carried out by stationary culture or culture in the dark. As described above, the methanogenic bacteria growing under special conditions have widely been explored. Conditions other than those disclosed above during the isolation culture were as follows: pH = 6.5 to 9.0; temperature = 10 to 60°C; and the sodium chloride concentration = 1 to 4 M. When the isolation culture is conducted at a sodium chloride concentration ranging from 1 to 4 M as a condition for isolation culture, the isolation medium is prepared by changing the concentration of magnesium chloride or potassium chloride within the range shown in Table I since the requirements of the bacteria concerned for substances such as magnesium and potassium is unclear. (3) Identification of the Objective Bacteria The identification of the methanogenic bacteria of the present invention growing under extreme environments was effected as follows. The culture is effected according to the aforementioned isolation culture technique at a desired cultivation temperature utilizing a culture medium in which conditions such as pH, the concentration of sodium chloride and kinds of substrates used and to which there are added the aforesaid inhibitor for peptidoglycan synthesis in the cell wall and an antibiotic as a protein synthesis inhibitor effective to bacteria having procaryotic cell type 70S ribosome system in order to isolate only the methane producing bacteria belonging to archaebacteria. The bacteria which would grow during such isolation culture are considered to be one of the following bacteria, so far as can be judged from the composition of the culture medium and the like: (a) objective methanogenic bacteria; (b) strictly anaerobic bacteria, which are antibiotic resistant, other than methane producing bacteria; or (c) archaebateria other than methanogenic bacteria, which are strictly anaerobic or resistant to anaerobic conditions. The following two methods are adopted to identify only the objective methane producing bacteria from a product of the isolation culture: one of which comprises analyzing gases present in the head space of the container for culture by a means such as gas chromatography to confirm the production of methane gas; and the other of which comprises observing the grown bacteria by a fluorescence microscope to examine the product. The methanogenic bacteria have a factor F₄₂₀ inherent thereto and emit fluorescent rays of bluish green originated from the factor when they are irradiated with ultraviolet rays of long wave length. Therefore, it can be confirmed whether the grown bacteria are methane producing ones or non-methanogenic ones on the basis of the presence or absence of the emission of fluorescent rays upon irradiating them with ultraviolet rays and examining them by a fluorescence microscope. In this regard, some of the non-methanogenic bacteria emit fluorescent rays, but the fluorescence rays emitted by them are not bluish green but yellowish white or bluish white. Thus, in such cases, the non-methanogenic bacteria may certainly be distinguished from the methanogenic ones. The methanogenic bacteria of the present invention can be isolated in the pure condition by identifying them according to the foregoing methods and repeating procedures such as dilution and agar plating methods to purify the same. (4) Collection of the Objective Microorganisms The inventors of the present invention tried to isolate desired methanogenic bacteria from soil in special environmental conditions widely collected from Japan and abroad in accordance with the aforementioned methodology. As a result, the inventors could isolate a variety of halophilic or halophilic and alkalophilic methanogenic bacteria from the soil of high salt content collected at the West of United State of America. These methanogenic bacteria were cultured and examined. As a result, there were isolated two kinds of extremely halophilic methanogenic bacteria having different optimum growth pH range. These microorganisms grow at wide sodium chloride concentration which ranges at least from 1 to 4 M and have the optimum growth concentration thereof ranging from 2.5 to 3 M and good methane gas production activity. Thus, these bacteria were confirmed to be extremely halophilic. It was confirmed that the first methanogenic bacteria NY-218 of the present invention are also alkalophilic since it grow at least at a pH ranging from 7.0 to 9.0 and has the optimum growth pH ranging from 8.0 to 8.5 and good methane gas production activity. It was likewise confirmed that these bacteria show the methane gas production activity even under the culture condition of 15 to 45°C and that their optimum growth temperature is in the range of 35 to 37°C. It was confirmed that the bacteria NY-218 are gram-negative and strictly anaerobic bacteria which are a motilecoccus with flagella having a diameter of 0.5 to 1.5 micron and which never cause sporulation and that unlike the general bacteria, they are archaebacteria exhibiting no sensitivity to antibiotics or the like which inhibit the peptidoglycan synthesis of cell wall. The bacteria NY-218 are capable of assimilating methanol and methylamine at least at a cultivation temperature of 37°C, sodium chloride concentration of 2.5 to 3 M and pH of 8.0 to 8.5. The inventors of this invention deposited the bacteria NY-218 to Fermentation Research Institute (FRI) in accordance with the Budapest Treaty under the accession number of FERM BP-2079 on October 7, Showa 62 (1987). It is confirmed that the second bacteria NY-844 of the present invention grow at least at a pH ranging from 6.5 to 8.5 and that they have the optimum growth pH ranging from 7.5 to 8.0 and good methane gas production activity. Moreover, It was likewise confirmed that the bacteria NY-844 show the methane gas production activity even at a cultivation temperature of 15 to 45°C and that its optimum growth temperature is in the range of 35 to 37°C. It was confirmed that the bacteria NY-844 are gram-negative and strictly anaerobic bacteria which are a motilecoccus with flagella having a diameter of 0.5 to 1.5 micron and which never cause sporulation and that unlike the general bacteria, they are archaebacteria exhibiting no sensitivity to antibiotics or the like which inhibit the peptidoglycan synthesis in the cell wall. The bacteria NY-844 are capable of assimilating methanol and methylamine at least at a cultivation temperature of 37°C, sodium chloride concentration ranging from 2.5 to 3 M and pH ranging from 7.5 to 8.0. The inventors of this invention deposited the bacteria NY-844 to Fermentation Research Institute (FRI) in accordance with the Budapest Treaty under the accession number of FERM BP-2080 on October 7, Showa 62 (1987). The present invention will hereunder be explained in more detail with reference to the following non-limitative working Examples. Example 1-1 This Example was carried out to examine the influence of the sodium chloride concentration on the amount of methane gas produced by the bacterium NY-218 of the present invention collected in the manner as discussed above when they were cultured in a culture medium whose composition was shown in Table I and in which the substrate was 0.8% trimethylamine and the sodium chloride concentration was changed within the range of 1 to 4 M. The cultivation was effected at 37°C for 15 days. The results obtained are plotted in Fig. 1, with the sodium chloride concentration (M) in the culture medium as abscissa and the amount of methane gas (m mole) generated as ordinate. As seen from the results shown in Fig. 1, it is appreciated that the bacteria NY-218 grow at a sodium chloride concentration ranging from 1 to 4 M and that their optimum growth concentration of sodium chloride ranges from 2.5 to 3 M. Example 1-2 This Example was carried out to examine the influence of pH on the amount of methane gas produced by the bacteria NY-218 of the present invention collected by the manner as discussed above when they were cultured in a culture medium whose composition was shown in Table I and in which the substrate was 0.8% trimethylamine and the pH value was changed within the range of 6.5 to 9.0. The cultivation was effected at 37°C for 15 days. The results obtained are plotted in Fig. 2, with the pH value of the culture medium as abscissa and the amount of methane gas produced (m mole) as ordinate. As seen from the results shown in Fig. 2, it is appreciated that the bacteria NY-218 grow at a pH value ranging from 7.0 to 9.0 and that the optimum growth pH condition thereof ranges from 8.0 to 8.5. Example 1-3 This Example was carried out to examine the influence of temperature on the amount of methane gas produced by the bacteria NY-218 of the present invention collected in the manner as discussed above when they were cultured in a culture medium whose composition was shown in Table I, in which the substrate was 0.8% trimethylamine, and whose temperature was changed within the range of 15 to 60°C. The cultivation was continued for 15 days. The results obtained are plotted in Fig. 3, with the temperature of the culture medium as abscissa and the amount of methane gas (m mole) generated as ordinate. As seen from the results shown in Fig. 3, it is appreciated that the bacteria NY-218 grow at a temperature of the culture medium ranging from 15 to 45°C and that the optimum growth temperature condition thereof approximately ranges from 35 to 37°C. Example 1-4 This Example was carried out to examine the influence of the kinds of substrates used on the amount of methane gas generated by the bacteria NY-218 of the present invention collected in the manner as discussed above when they were cultured in a culture medium whose composition was shown in Table I and in which the substrate used was one member selected from the group consisting of sodium formate, sodium acetate, methanol, trimethylamine and H₂/CO₂. In these cases, the gas phase used for the culture was N₂ (2 atm.; positive) except for the case where H₂/CO₂ was used as the substrate. When the substrate was H₂/CO₂, it was not added to the culture medium but was used as the gas phase (2 atm.; positive). In this Example, pH was 8.2, the sodium chloride concentration was 3 M and the cultivation temperature was 37°C. The results obtained are plotted in Fig. 4, with the cultivation days as abscissa and the amount of methane gas (m mole) generated as ordinate and are also summarized in Table IV given below. Example 1-5 The bacteria NY-218 of the present invention were cultured in a culture medium whose composition was shown in Table I and in which the substrate was 0.8% of trimethylamine. Then, the generation time thereof was estimated from the amount of methane gas generated and it was found to be 5 to 10 hours. Example 2-1 This Example was carried out to examine the influence of the sodium chloride concentration on the amount of methane gas generated by the bacteria NY-844 of the present invention collected in the manner as discussed above when they were cultured in a culture medium whose composition was shown in Table I and in which the substrate was 0.8% trimethylamine and the sodium chloride concentration was changed within the range of 1 to 4 M. The cultivation was effected at 37°C for 15 days. The results obtained are plotted in Fig. 5, with the sodium chloride concentration (M) in the culture medium as abscissa and the amount of methane gas produced (m mole) as ordinate. As seen from the results shown in Fig. 5, it is appreciated that the bacteria NY-844 grow at a sodium chloride concentration ranging from 1 to 4 M and that their optimum growth concentration of sodium chloride ranges from 2.5 to 3 M. Example 2-2 This Example was carried out to examine the influence of pH on the amount of methane gas generated by the bacteria NY-844 of the invention collected in the manner as discussed above when they were cultured in a culture medium whose composition was shown in Table I and in which the substrate was 0.8% trimethylamine and the pH value was changed within the range of 6.5 to 8.5. The cultivation was effected at 37°C for 15 days. The results obtained are plotted in Fig. 6, with the pH value of the culture medium as abscissa and the amount of methane gas produced (m mole) as ordinate. As seen from the results shown in Fig. 6, it is appreciated that the bacteria NY-844 grow at a pH value ranging from 6.5 to 8.5 and that their optimum growth pH condition ranges from 7.5 to 8.0. Example 2-3 This Example was carried out to examine the influence of temperature on the amount of methane gas generated by the bacteria NY-844 of the present invention collected in the manner as discussed above when they were cultured in a culture medium whose composition was shown in Table I, in which the substrate was 0.8% trimethylamine and whose temperature was changed within the range of 15 to 60°C. The cultivation was continued for 20 days. The results obtained are plotted in Fig. 7, with the temperature of the culture medium as abscissa and the amount of methane gas (m mole) generated as ordinate. As seen from the results shown in Fig. 7, it is appreciated that the bacteria NY-844 grow at a temperature of the culture medium ranging from 15 to 45°C and that their optimum growth temperature condition approximately ranges from 35 to 37°C. Example 2-4 This Example was carried out to examine the influence of the kinds of substrates used on the amount of methane gas generated by the bacteria NY-844 of the present invention collected in the manner as discussed above when they were cultured in a culture medium whose composition was shown in Table I and in which the substrate used was one member selected from the group consisting of sodium formate, sodium acetate, methanol, trimethylamine and H₂/CO₂. In these cases, the gas phase for culture was N₂ (2 atms.; positive) except for the case where H₂/CO₂ was ysed as the substrate. When the substrate was H₂/CO₂, it was not added to the culture medium but was used as the gas phase (2 atms.; positive). In this Example, pH was 7.8, the sodium chloride concentration was 3 M and the cultivation temperature was 37°C. The results obtained are plotted in Fig. 8, with the cultivation days as abscissa and the amount of methane gas (m mole) generated as ordinate and are also summarized in Table IV given below. Example 2-5 The bacteria NY-844 of the present invention were cultured in a culture medium whose composition was shown in Table I and in which the substrate was 0.8% of trimethylamine. Then, the generation time thereof was estimated from the amount of methane gas generated and it was found to be 5 to 10 hours. In order to make it clear that the highly halophilic methane producing bacteria of the present invention are novel methane producing bacteria which can clearly be distinguished from known ones, properties of the bacteria NY-218 and NY-844 such as assimilation of substrates, morphological features, gram-staining properties, generation time, optimum growth temperature, optimum growth pH and optimum growth sodium chloride concentration are summarized in Table IV in comparison with those of the known methane producing bacteria. As seen from the results listed in Table IV, it is found, in the light of the following facts, that the bacteria NY-218 of the present invention are novel methanogenic bacteria belonging to the new genus Methanohalococcus: (i) The methanogenic bacteria NY-218 of the present invention are extremely halophilic methanogenic bacteria having an optimum growth sodium chloride concentration higher than that of most of the known methanogenic bacteria. (ii) There are not any conventional methanogenic bacteria being gram-negative, strictly anaerobic motile cocci with flagella known which simultaneously exhibit alkalophilic and halophilic properties. On the contrary, the methanogenic bacteria NY-218 of the invention are highly halophilic and alkalophilic. (iii) There is not conventionally known any halophilic or alkalophilic methanogenic bacteria which show methane gas production activity even at a low temperature of the order of 15 to 20°C. However, the methanogenic bacteria NY-218 of the present invention can withstand such a low temperature and exhibit the methane production activity even at a temperature ranging from 15 to 20°C. (iv) All the known alkalophilic methanogenic bacteria are not coccus but bacillus. Nevertheless, the methanogenic bacteria NY-218 of the present invention are coccus shaped. (v) There are not known any conventional alkalophilic methanogenic bacteria which have an ability of assimilating substrates other than hydrogen. Nevertheless, the methanogenic bacteria NY-218 of the invention can assimilate at least methanol and methylamine as a substrate. (vi) The methanogenic bacteria NY-218 differ from previously reported moderately or extremely halophilic methanogenic bacteria in their ability to grow at higher pH values and by the presence of flagella. Likewise, it is found, from the following facts, that the bacteria NY-844 of the present invention are novel methanogenic bacteria belonging to genus Methanohalococcus: (i) No extremely halophilic methanogenic gram-negative, strictly anaerobic motile bacteria with flagella are known so far. However, the methanogenic bacteria NY-844 of the invention are extremely halophilic and have an optimum sodium chloride concentration which is higher than that of the known methanogenic bacteria; (ii) All the known halophilic methanogenic bacteria have the optimum growth pH ranging from 7.0 to 7.5. On the contrary, the extremely halophilic methanogenic bacteria NY-844 of the present invention have the optimum growth pH ranging from 7.5 to 8.0, in other word they have a pH-dependency different from that of the known ones and unlike the known methane producing bacteria they maintain high activity even at higher pH condition. (iii) There are not known any conventional extremely halophilic methanogenic bacteria which show methane gas production activity even at a low temperature of the order of 15 to 20°C. However, the methanogenic bacteria NY-844 of the present invention can withstand such a low temperature and exhibit the methanogenic activity even at a temperature of 15 to 20°C. (iv) The methanogenic bacteria NY-844 differ from previously reported moderately or extremely halophilic methanogenic bacteria in their ability to grow at higher pH values and by the presence of flagella. Methane producing bacteria are used as the microorganisms playing central roll in the methane fermentation (bio-methanation) widely employed for the purposes of efficient use of biomass and biological gas production. If the novel methane producing bacteria of the present invention are applied in the industrial methane fermentation, such a fermentation makes rapid progresses since the bacteria of the present invention exhibit properties such as extremely halophilic and alkalophilic properties as well as resistance to low temperatures. More specifically, the methods of methane fermentation are commonly carried out at around neutral pH values, a medium temperature of the order of 37°C and a low salt concentration. The methanogenic bacteria of the present invention permit to carry out said methods at an alkaline pH, a high salt concentration and a lower temperature. This, in turn, makes it possible to use a wide variety of biomass waste in such a method of methane fermentation. Moreover, the conventional methods of methane fermentation must be effected at neutral pH values and for this reason, a large amount of pH adjusting agents are in general added to the bioreactor. As a result, it is sometimes observed that the methane fermentation is inhibited by the presence of large amounts of salts in the bioreactor. Such a disadvantage may effectively be eliminated by the use of the methane producing bacteria of the invention. In addition, if the methane producing bacteria of this invention are used in breeding by a genetic engineering technique as the source of genes encoding properties favorable for special environments or resistances to special environmental conditions such as halophilic and alkalophilic properties and resistance to low temperature, it would be possible to develop an effective methane fermentation or to efficiently produce other useful substances.

Claims: Methanohalococcus alcaliphilum characterized in having an optimum growth concentration of sodium chloride ranging from 2.5 to 3.0 mole per Liter; and an optimum growth pH ranging from 7.5 to 8.0 or 8.0 to 8.5; and being a gram-negative, strictly anaerobic motile coccus with flagella. Methanohalococcus alcaliphilum of claim 1 wherein it has methane gas production activity at a temperature ranging from 15 to 45°C. Methanohalococcus alcaliphilum of claim 1 or 2 wherein it has an optimum growth temperature ranging from 35 to 37°C. Methanohalococcus alcaliphilum of claim 3 wherein it is Methanohalococcus alcaliphilum NY-218 deposited with the FRI as FERM BP-2079. Methanohalococcus alcaliphilum of claim 1 wherein it has methane gas production activity at a pH ranging from 6.5 to 8.5. Methanohalococcus alcaliphilum of claim 1 wherein it is Methanohalococcus alcaliphilum NY-844 deposited with the FRI as FERM BP-2080. Process for the production of methane wherein a Methanohalococcus alcaliphilum according to any one of claims 1 to 6 is cultivated under anaerobic conditions in presence of a suitable substrate being fermentable by said bacterium, preferably in presence of sodium formate, sodium acetate, methanol or of a methylamine, and wherein the methane produced during the fermentation is recovered.

Inventors: HORIKOSHI KOKI, NAKATSUGAWA NAOKI, HORIKOSHI, KOKI, NAKATSUGAWA, NAOKI
Applicants: JAPAN RES DEV CORP, MITSUBISHI ELECTRIC CORP, RIKAGAKU KENKYUSHO, MITSUBISHI ELECTRIC CORPORATION, RESEARCH DEVELOPMENT CORPORATION OF JAPAN
Application year: 1988
Main classification: C12N1
IPC codes: C12N5, C12R1, C12P5, C12N1, C02F11