JPH0357745B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the use of Pseudomonas bacteria, which rapidly assimilates, grows and emulsifies in an oligotrophic environment consisting only of seawater and crude oil or heavy oil, and rapidly dies upon completion of assimilation. The Pseudomonas bacterium according to the present invention has the characteristic that it rapidly dies in approximately the same period of time required for proliferation, which is contrary to the conventional wisdom in bacteriology.
The present invention relates to a preparation containing this bacterium that utilizes the properties of this bacterium, and a method for using the same. Furthermore, when the endogenous nature is analyzed according to the present invention, bacteria naturally exhibit good growth in a field of good nutrition and environment, especially in the logarithmic phase. By the way, if crude oil is added in this logarithmic phase,
Crude oil is quite harsh and toxic for bacteria, so the growth rate quickly deteriorates soon. The present invention, however, relates to the development and utilization of a bacterium that grows even more vigorously and exhibits accelerated growth when crude oil, which should originally be toxic, is added during the logarithmic growth phase. In general, regarding marine pollution caused by oil, we will discuss oil spill accidents into the ocean, the amount thereof, the fate of the spilled oil,
Detailed reports have been made regarding the dispersion, decomposition, and dissipation of spilled oil into the sea, and the contamination of land resources.
Also, fairly detailed knowledge has been reported regarding the decomposition of contaminated oils by microorganisms. For example, above sea level
Among the oil that has spread as a thin film of 0.1 to 0.4μ, various bacteria will attack it within 1 to 2 weeks, and a considerable portion of the dispersed oil will decompose or disappear within 2 to 3 months. There is a theory that It is also said that the amount lost by other organisms and natural oxidation is about 1/10 of the amount lost by microorganisms. The genus of bacteria that contributes to the oxidation of contaminated oil has also been identified. However, some experts believe that the proportion of bacteria capable of oxidizing liquid hydrocarbon components among marine microorganisms is larger than expected. In any case, knowledge about the evolution of crude oil in the natural world and the organisms related to it is still being accumulated, and an extremely detailed and specific report is the "Environmental Impact Comprehensive Report on the Mizushima Heavy Oil Spill" released by Japan's Environment Agency. According to this investigation report, it appears that most of the oil spilled into the sea will be decomposed and dissipated within a year.
On the contrary, the United Nations Educational, Scientific and Cultural Organization (UNESCO)
A survey on oil pollution jointly conducted between 1975 and 1979 by the Intergovernmental Oceanographic Commission (IOC) and the World Meteorological Organization (WMO) found that the actual state of oil pollution in the ocean was serious, and that bacterial The conventional theory that almost all the oil is digested within a few years is considered to be unconvincing. Just then, on March 3rd of this year, it was reported that sludge was being dumped into the ocean regarding the cleaning of the inner tanks of supertankers, and that a number of domestic and international oil tankers were experiencing serious problems related to this, resulting in marine pollution. This shocked the public and led to the issue being brought up in the Diet. Furthermore, even on the coast, the treatment of oil with a low concentration is becoming increasingly viewed as a problem by specialists as it is extremely difficult. By the way, there are two conventional methods for separating petroleum-degrading bacteria or petroleum component-degrading bacteria. One method uses marine inorganic salts and _NH3 salts (or urea).
and petroleum or petroleum components; another method is to use a medium to which the above-mentioned medium is supplemented with highly nutritious substances such as vitamins or casamino acids or yeast extract. It is known that when competent complex oxidizing bacteria proliferate in a highly nutritious environment as in the latter method, the medium gradually becomes cloudy after 10 days of shaking culture at 20°C. Taking advantage of these properties, small amounts of organic nutrients are also being introduced into polluted oceans as growth agents. The present inventors have completed the present invention by repeating a large number of experimental studies regarding the above-mentioned removal of oil-contaminated seawater by microorganisms. That is, the present invention provides that when a petroleum-based hydrocarbon is added during the logarithmic growth phase of a Pseudomonas petroleum-utilizing bacterial strain in a Stephenson-Wessam medium, the hydrocarbon transforms the original Pseudomonas petroleum An oil purification preparation containing a Pseudomonas strain that has an increased ability to assimilate oil than an assimilating strain, and the preparation can be grown in a sea area or place contaminated with petroleum-based hydrocarbons to prevent contamination of the sea area or place. It provides a method of purification. The details of the invention will be made clear by the following description. Purification of petroleum-contaminated seawater using a single bacterium that decomposes and assimilates petroleum-based hydrocarbons is known, for example, from JP-A-49-61376 and JP-A-51-142865. In the former case, a unicellular microorganism containing a first energy-generating plasmid defining a decomposition pathway and a unicellular microorganism containing at least one energy-generating plasmid that cannot coexist with the first plasmid are selected as a donor bacterium and a recipient bacterium, respectively. , conjugating the donor bacteria and recipient bacteria,
The aim is to stabilize the unstable super microorganisms that have been created in this way and obtain new microorganisms with desired properties, and in particular, to obtain microorganisms that have the ability to decompose and assimilate hydrocarbon complexes in a single cell. Experiments were conducted with the aim of developing and biologically controlling marine oil pollution caused by a single bacterial species of Pseudomonas bacteria by utilizing the latest genetics. The idea was to obtain a single new bacterium that can decompose oil, which could be used to clean up oil-polluted oceans. On the other hand, the latter is achieved by growing a single bacterium in a medium consisting entirely of inorganic substances except for the heavy oil component.
This is based on the knowledge that it was possible to reduce 1000 ppm of heavy oil components to the point where only traces remained on thin layer chromatography in 5 days. However, in all of these prior art techniques, bacterial cells are cultured in the presence of _an artificially added nitrogen nutrient source;
NH ₂ CONH ₂ is present, and crude oil or heavy oil is decomposed and utilized in a rich nutrient medium rather than just seawater. In other words, these prior art techniques use bacteria grown in an environment significantly different from the oil-polluted ocean, and the former uses NH ₄ as a nitrogen source without containing salts such as NaCl, which are present in large amounts in seawater.
A medium containing 2.1 g/and further 10 mg/vitamin C was used, the latter containing 0.25 NH ₂ CONH ₂
Both media are rich in nutrients, which is different from the oil-polluted marine environment, which consists only of petroleum and seawater. The Pseudomonas bacterium according to the present invention has characteristics completely different from those of the prior art fungi as described above, and can survive under extreme oligotrophic conditions with only seawater and crude oil, during severe temperature fluctuations, and even under fluctuations in the amount of oil. It can assimilate crude oil in a large medium and show extremely good throughput; after inoculation, intense growth and emulsification occur rapidly in a place with only seawater and oil, and it rapidly dies when the assimilation of crude oil is finished. There is. In particular, the process from growth to death far exceeds conventional knowledge of microorganisms, and the number of days required for growth is approximately equal to the number of days from the end of growth to death. Specifically, when the amount of crude oil is 30 ppm, the number of days required for proliferation is 1.5 days, the steady period is 0.5 days, and the number of days from which it will die is 2.5 days, and when the amount of crude oil is 500 ppm, the number of days required for proliferation is 2.5 days. , the steady-state period is 1 day, and the period leading to death is 4 days. The Pseudomonas bacterium according to the present invention can be obtained as follows. However, the method described here is just one example, and the method described here is based on the culture medium composition,
It goes without saying that various modified methods can be adopted depending on the culture conditions. 1st step: As a bacterial cell, FERM-P No. (FERM-P number)
2927 and 2928, the bacteria were inoculated into a medium with the following composition and cultured. Medium composition Seawater 1000ml Heavy oil A 1g Nutrient source ^* small amount Culture temperature 18℃ * (NH ₄ ) ₂ SO ₄ 0.5g/, starch 0.2g/,
Vitamin 0.01g/. When they were able to grow sufficiently in the above medium, they were repeatedly subcultured in a medium in which the addition of nutrients was gradually reduced, and they became able to grow and proliferate only with seawater and heavy oil A without adding any nutrients. Second Step Using the bacteria obtained in the first step, the bacteria were inoculated into a medium with the following composition and cultured. Medium composition Seawater 1000ml Crude oil 30ppm Vitamin 0.01g/Culture temperature 21℃ When cultured in the above medium at a temperature of 21℃, a product that becomes cloudy in 10 to 15 days is obtained, but the vitamin content of this product is gradually reduced. Through subculture, we obtained bacteria that can sufficiently grow in seawater using only crude oil (residual crude oil amount: 4 ppm). Third step In subculturing the bacteria obtained in the second step, passages were repeatedly carried out when the bacteria had consumed all of the crude oil or when the bacteria were at their most vigorous growth period, a little before that. In this way, bacteria with a residual crude oil amount of 0.15 ppm were obtained 4 days after the addition of the bacteria. Fourth step: The bacteria obtained in the third step were continued to be grown through acclimatization to obtain a strain with high growth and emulsifying ability. This bacterium rapidly assimilates and emulsifies crude oil and has an abnormally high crude oil consumption rate, with a consumption rate of 30 ppm in the third step.
A bacterial strain that could once assimilate 100 liters of crude oil in four days has now become a bacterium capable of almost complete assimilation in just one and a half days.
Bacteria that can emulsify crude oil and rapidly completely assimilate crude oil in oligotrophic conditions are highly dependent on crude oil. In other words, this bacterium is healthy if crude oil is present, but when the crude oil is gone, the bacterium undergoes self-fusion and rapidly dies out. It was found that this bacterium dies in an oligotrophic environment consisting only of seawater and crude oil in approximately the same period as its own growth period, but this phenomenon has traditionally been caused by lysis by bacteriophages and an excess amount of antibiotics produced by self-production. This is something that was not known other than death, and was discovered for the first time by the present inventor. 5th step Amount of crude oil dissolved in the sea by the bacteria obtained in the 4th step 420
Acclimatization was repeated to show the greatest assimilation ability for each crude oil concentration, from ~30 ppm) to the amount floating on the sea surface (1500 ppm). Initially, bacteria are inoculated at a low crude oil concentration, and emulsification begins in 4 to 5 hours, but after confirming complete assimilation, the crude oil concentration is gradually increased to achieve the desired concentration from low to high. We obtained bacteria that grew well at certain concentrations.
For example, at a crude oil concentration of 30 ppm, the bacteria obtained in this way are 1
Completely assimilated in a day and a half (using 30ppm adapted bacteria; the same applies hereinafter),
100ppm for 2 days, 300ppm for 2 and a half days, 500ppm
At 1000ppm, complete assimilation could be achieved in 2 and a half days, and in about 3 days at 1000ppm. 6th step Next, so that the bacteria obtained in this way can fully demonstrate its ability at both high temperatures (e.g. 37 degrees Celsius) and low temperatures (about 8 degrees Celsius), the bacteria are moved from the optimal temperature for growth (18 to 30 degrees Celsius) to higher or lower temperatures. The cells were passaged and acclimatized gradually so as not to weaken their proliferation, emulsification, and assimilation effects. Thus, if the titer at the optimum growth temperature is 100, then at high temperature (37℃)
115, and was able to acclimate to a low temperature (8℃) until it showed a score of 50. For example, we obtained bacteria that can almost completely assimilate bacteria adapted to a crude oil concentration of 30 ppm in one and a half days at moderate and high temperatures, and in three days at low temperatures. The bacteria obtained in this way were initially only strong enough to assimilate some components in crude oil, but were placed in seawater in the presence of petroleum without adding anything else. It utilizes and consumes this and shows an oil removal rate of over 99.5%, but it has the novel feature of quickly dying out once the oil disappears. In addition, the bacteria obtained in this way will quickly lose its ability to assimilate oil and return to its original form in a favorable environment where no petroleum is present, and will quickly die if exposed to seawater alone, so it is not safe from a safety point of view. It has extremely great practical value for the purpose of removing contaminated petroleum. Furthermore, the live bacteria and culture supernatant of this bacterium were examined using a prescribed method for acute, subacute, and chronic conditions, and it was confirmed that they were highly safe. In particular, no abnormalities were observed in experiments in which live bacteria were mixed with feed and administered orally to mice, and a large amount of bacterial mass was applied to the eyes, demonstrating a high level of safety. Figure 1 shows one of the characteristics of the bacterium according to the present invention, in which the growth/death curve A of the bacterium in a medium containing seawater and 30 ppm of crude oil shows rapid rises and falls. The growth and death curves of the bacteria disclosed in JP-A-49-61376 are as shown in a to d, and it can be seen that the characteristics of the bacteria according to the present invention are significantly different from those of the conventional ones. Even the product described in the above publication in the final stage of genetic engineering with 50,000 ppm of petroleum added is still 10 ⁹ /ml.
The bacteria has clearly passed the logarithmic phase and is entering the stationary phase. In comparison, the bacteria of the present invention still showed a yield of 3.5 billion cells even with the addition of only 500 ppm of the above-mentioned amount of petroleum added, and the time to reach this cell yield was very quick, and the growth rate was fast in a highly nutritious medium. This is similar to the growth and proliferation rate when culturing E. coli. Moreover, although the growth environment described in the above publication is eutrophic, the one of the present invention is extremely oligotrophic, consisting only of seawater and crude oil. In other words, the rapid rise in the growth and death curve of the bacteria according to the present invention should be interpreted as one manifestation of the growth-promoting effect of petroleum on the bacteria, and such an effect has not been observed at all with conventional bacteria. It is distinctive. Therefore, the most important feature of the bacterium according to the present invention will be further explained. When the bacteria grown through the above steps 1 to 6 are subcultured in a Stephenson-Wesham medium (S-W) having the following composition, the degree of growth promotion due to crude oil addition and emulsifying power increase with the passage number. It has become clear that assimilation capacity declines, but based on this phenomenon, rapid growth in oligotrophic conditions is a particularly desired characteristic for cleaning petroleum contaminants, which has been often mentioned. We will explain below what characteristics of bacteria are closely related to the abilities such as rapid killing, near-complete devouring, etc. Here, Stephenson-Wesham medium (hereinafter referred to as S
-W medium) has the following composition: KH ₂ PO ₄ 1 g; MgSO _{4.7H 2} O 0.7 g; NaCl 1
g; (NH ₄ ) ₂ HPO ₄ 4 g; glucose 5 g;
A medium containing 0.03 g of FeSO ₄ .7H ₂ O in 1 part of water (the composition of the medium below indicates the amount in 1 part of water). Regression of the growth promotion phenomenon depending on the number of passages in the (S-W) medium (a) In the case of strains acclimated to 500 ppm crude oil; Strains with different passage numbers were cultured in the [S-W] medium, and their growth was in the logarithmic phase. 10ppm of crude oil,
When dropping amounts of 30 ppm, 100 ppm, 300 ppm, and 500 ppm, the strength and presence of promotion of the growth of the bacteria were observed. The measurement results are shown in Table 1 group. Measurement method: Hexane extraction; Continuously measure with UV (200-400nm) and calculate the area. Turbidity is UV
(460nm). Those showing accelerated growth are underlined.

【table】

【table】

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【table】

【table】

【table】

[Table] As is clear from Group 1 in Table 1 above, 1) The strain, which has just been passaged for the first generation in the (S-W) medium and is not weakened yet, is 120% after adding crude oil.
It can be seen that after 10 minutes, the growth promotion is quite strong, and that the higher the amount added (addition amounts of 100 ppm or more show the same results as 100 ppm addition), the stronger the growth promotion. (See the underlined numbers in the table) 2) A somewhat weakened strain that has been passaged for 4 generations in the (S-W) medium receives growth promotion from 180 minutes after addition when the amount added is 10 ppm. 30ppm, 100ppm,
When adding 300ppm and 500ppm, growth was promoted after 120 minutes, but as in case 1), the degree of growth promotion became stronger as the amount added increased. 3) A weakened strain that has been passaged for 6 generations in (S-W) medium does not undergo growth promotion when the added amount is 10 ppm, but only after 180 minutes when the added amount is 30 ppm, 100 ppm, 300 ppm, and 500 ppm. It can be seen that growth is promoted. 4) The strain of 8th passage in (S-W) medium is no longer
When small amounts of 10 ppm and 30 ppm were added, growth was not promoted, but when 110 ppm, 300 ppm, and 500 ppm were added, growth was slightly promoted after 180 minutes. 5) The bacterial strain that had been passaged for 10 generations on the (S-W) medium experienced growth promotion only when the amount added was 300 ppm, and this only slightly appeared after 180 minutes. 6) The strain that had been passaged for 12 generations on the (S-W) medium not only no longer experienced growth promotion in any case, but also showed some tendency for growth inhibition at the addition amount of 500 ppm. 7) Furthermore, we also measured the bacterial strains from the 14th generation to the 18th generation, and all of them showed similar results to the 12th generation strain, and not only did no growth promotion occur at any of the added amounts, but For the 13th and 14th generation strains, the growth of the bacteria was suppressed when 500ppm of crude oil was added, and for the 15th, 16th, 17th, and 18th generation strains, when 300ppm and 500ppm were added. Although strain calculations revealed that this bacterium promotes growth, only the case of the 1st generation strain will be described here.

[Table] As is clear from the table, the same results as the nephelometer measurements were observed, that is, growth promotion was observed from 120 minutes after adding crude oil, and after 240 minutes, compared to the comparative sample, there was a 12% increase in the case of 10 ppm addition, and a 12% increase in the case of 30 ppm addition. 20% increase if
When adding 100ppm, 300ppm, and 500ppm, the amount increased by 24%. (b) In the case of bacteria adapted to 30 ppm crude oil The same experiment as in the case of bacteria adapted to 500 ppm was conducted (However, the amount of crude oil added during the logarithmic growth phase was
The amount was set to 10ppm, 30ppm, 100ppm, and 300ppm. ) The experimental results are shown in Table 3.

【table】

【table】

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【table】

[Table] As is clear from the above Table 3 group, even if the amount of crude oil added was small, the performance of the 500 ppm-acclimated strain and the presence or absence of growth promotion, strength, etc. showed similar trends. In addition, we also measured the strains from the 14th generation to the 18th generation, and not only did they not experience any growth promotion like the 12th generation strain, but the addition of 300 ppm also inhibited the growth to some extent compared to the comparison sample. It was done. The above experiment was performed using (S-W) medium, but even when using more nutritious peptone or meat extract in the medium, the results are essentially different from those in (S-W) medium. However, from about 120 minutes after addition, a gradual growth phenomenon was observed in those with a small number of passages in the medium. In addition, regarding the killing of the present invention bacteria with a high degree of growth promotion, when 500 ppm and 30 ppm of the adapted bacteria were introduced into seawater or fresh water, the highly oil-dependent bacteria up to 3rd generation were rapidly killed. There is. However, as the degree of growth promotion weakens, its lifespan gradually increases. (S-W) Changes in emulsification phenomenon depending on the number of passages in culture medium (a) Using 500ppm adapted bacteria (medium: seawater + crude oil
500ppm)

【table】

[Table] As is clear from Table 4 above, the emulsifying power of the bacteria gradually decreases as the number of passages increases. In addition to this, there is a stage where the emulsification phenomenon occurs from very early bacteria to a stage where it is delayed. Also, more importantly,
Even in the 12th to 14th generation bacteria, whose growth was no longer stimulated by the addition of crude oil, it was recognized that a slight emulsification phenomenon occurred 48 hours after inoculation. This is an important key to elucidating the relationship between growth promotion and emulsification. (b) Using 30ppm adapted bacteria (medium; seawater + crude oil
30ppm)

[Table] As is clear from Table 5 above, the results were similar to those of the 500 ppm-acclimated bacteria, and it can be seen that the emulsification phenomenon occurs even with the strain that is no longer stimulated to grow by the addition of crude oil. (S-W) Degree of crude oil removal power depending on the number of passages in the medium (a) Using 500 ppm adapted bacteria (medium: seawater + crude oil
500ppm)

【table】

[Table] As is clear from Table 6 above, when 500 ppm of crude oil is added, the remaining amount of crude oil at 168 hours after addition is only 1 ppm for the first generation strain (1/500 of the original amount)
In addition, the fourth generation strain is only 2 ppm (1/250 of the original amount)
5 ppm (1/100 of the original amount) for the 6th generation strain, and 25 ppm (1/20 of the original amount) for the 8th generation strain.
In strains showing minimal growth promotion at passage 10,
Although the removal rate gradually deteriorates to 50 ppm (1/10 of the original amount), the remaining amount of crude oil will still be one order of magnitude smaller. On the other hand, in the 12th generation strain that no longer promotes growth, the removal ability suddenly decreases to 1/2 of its original level.
2.5, making it impossible for it to be a practical bacterium as a crude oil cleaning agent. 250ppm for the 14th generation strain (1/2 the original amount)
Next, the removal rate of the 16th generation strain, which had lost its emulsifying ability, deteriorated to 300 ppm (2/3 of the original amount), and the 18th generation strain was placed in a medium of (seawater + crude oil 500 ppm). could not grow at all, and the crude oil removal rate was 0 (experimental errors have been corrected). It should be noted that if culture is continued for 240 hours, the concentration will be less than 2 ppm even for strains in the 6th generation, which have a weak degree of growth promotion, and that strains that no longer receive growth promotion and only show emulsification, such as strains in the 12th generation, It was found that the crude oil removal rate of the strain decreased only to around 198 ppm, which indicates that the growth-promoting bacteria according to the present invention has a more remarkable crude oil removal rate than the bacteria that only exhibits emulsification. In other words, it can be seen that growth-promoting bacteria have a much better performance in terms of cutting through than simple emulsifying bacteria, and exhibit an order of magnitude better effect, and the difference from simple assimilating bacteria is even more remarkable. (b) Using 30ppm adapted bacteria (medium: seawater + crude oil
30ppm)

[Table] As is clear from Table 7 above, when adding 30 ppm of crude oil as well as when adding 500 ppm of crude oil, the removal of crude oil gradually worsens as the number of passages passes, but growth is not promoted by adding crude oil. It became clear that the situation with the mature strains suddenly changed and the situation in which they were eaten became worse. Furthermore, if the culture is continued for 240 hours,
Even in the 6th generation strain, which has a weak degree of growth promotion, the crude oil removal rate is 0.8 ppm or less, and strains that no longer receive growth promotion, e.g.
In the 12th generation strain, it is the same as 10ppm, which means that there is a significant difference in terms of throughput between growth-promoting bacteria and emulsifying bacteria that do not promote growth, and an even more significant difference from mere assimilating bacteria. Ta. Correlation between growth promotion, presence or absence of emulsification, and ability to remove crude oil The results of ~ above can be summarized as follows: (a) In the case of 500 ppm-acclimated bacteria (medium: seawater + crude oil
500ppm)

[Table] ×: Not recognized
(b) In the case of 30ppm-acclimated bacteria (medium: seawater + crude oil
30ppm)

[Table] The results of Tables 8 and 9 are shown in graphs as shown in FIGS. 2 and 3. The above results are for FERM-P No. 5490, but in FERM-P No. 5491 deposited at the same time as well as in other bacteria, the correlation between growth promotion, emulsification, and assimilation by crude oil is essentially the same. It's the same. This was confirmed not only when the titer of powerful growth-promoting bacteria was decreased, but also when the titer was increased by induction. The results shown in the tables and figures are the results of looking at the various characteristics that microorganisms should possess in microbial purification of petroleum contamination, on the one hand from the perspective of crude oil, and on the other from the characteristics of microorganisms, and relating them. This is a valuable achievement. The experimental results of conventional bacteria are that they can only assimilate crude oil,
Even with the remarkable advances in research, emulsification was finally within reach, and even the existence of growth-promoting bacteria was unexpected. Example 1 A culture medium 50 consisting of 0.5 g of crude oil and 1000 ml of seawater was injected into an aquarium (90 cm x 50 cm x 40 cm), and the bacteria according to the present invention were cultured in seawater + crude oil medium in advance to obtain a strain.
20ml was inoculated and statically cultured at 18°C for 42 hours. The thus obtained culture solution was cooled and centrifuged to obtain 60 g of a mixture of bacterial cells and emulsion. (Formulation A) Add 0.44 g of starch and 0.001 g of crude oil to 10 g of the above mixture and mix well. The mixture thus obtained is dried in vacuo to a moisture content of 8%, and 2.9 g of the resulting dry product is poured into a glass bottle. For example, fill with CO ₂ gas, N ₂ gas, etc.
The shelf life of the thus obtained formulation when stored at room temperature was 20 days or more, and when stored refrigerated at 5-8°C, it was 3 months or more. (Formulation B) 0.44 g of starch in 10 g of the above mixture,
Add 0.001g of crude oil and 0.11g of cystine and mix well to make a formulation with a water content of 8% as in Formulation A.
Make 3.01g and seal in a glass bottle using _N2 gas. The shelf life of the thus obtained preparation at room temperature is
The storage period was 20 days or more, and the storage period was 3 months or more when refrigerated at 5-8°C. (Formulation C) 0.4 g of starch was added to 10 g of the above mixture and mixed well, and the mixture thus obtained was vacuum dried to obtain 2.5 g of a preparation. The thus obtained formulation was effective for a long period of time when stored at room temperature. (Formulation D) 0.1 g of cystine and 0.001 g of crude oil were added to 10 g of the above mixture and mixed well, and the mixture thus obtained was vacuum dried to obtain 2.1 g of the preparation. The thus obtained formulation was effective for a long period of time when stored at room temperature. Example 2 12.5 ml of crude oil (mixed) was dropped into an acrylic resin water tank with a length of 26 cm, a width of 45 cm, a depth of 30 cm, and a capacity of approximately 33 cm.
After thoroughly applying the crude oil to the bottom and walls of the tank, 25 ml of seawater was added. The amount of crude oil in the seawater at this time is 500 ppm. Adjust the seawater temperature to 18℃,
An aeration device was installed. Next, a culture solution of the crude oil assimilating bacterium (FERM-P No. 5490) according to the present invention (cultivated for 48 hours in a medium of seawater + crude oil) 9
ml (1/3000 of seawater was added. This 9 ml consisted of 3 ml of bacteria adapted to 500 ppm of crude oil, 3 ml of bacteria adapted to 100 ppm, and 3 ml of bacteria adapted to 30 ppm. Aeration was carried out at an aeration rate of 2/min from 24 hours after the start of the experiment. After starting the experiment, we conducted water tests over time to visually observe the emulsification state of water in the water tank and the state of crude oil adhesion on the tank wall, and to calculate the dissolved crude oil content (UV measurement) and the number of bacteria per c.c. The results are shown in Table 10.

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[Table] As is clear from Table 10, the emulsification phenomenon started 4 hours after the addition of the bacteria, rapidly became stronger after 7 hours, and was most noticeable after 48 to 72 hours. After that, it gradually declined, and after 144 hours, the transparency rapidly increased and became almost transparent after 192 hours. On the other hand, the crude oil that had initially adhered to the wall surface began to gradually become thinner after about 24 hours, and after 96 hours, almost no crude oil could be seen adhering to it. Also,
The limit oil content in seawater also rapidly decreased to 180ppm after 24 hours, 30ppm after 48 hours, 0.5ppm after 72 hours, 0.3ppm after 96 hours, and 0.3ppm after 120 hours.
Although it gradually decreased to 0.2 ppm, the results were the same as after 120 hours (99.96% removal rate). Next, the evolution of crude oil-utilizing bacteria in seawater is initially 1×10 ⁶
8 pieces/cc in 24 hours, ^3.5 in 48 hours
The bacteria increased to × ¹⁰⁹ . After that, it started to gradually decrease to 2.5×10 ⁹ at 72 hours, 3×10 ⁸ at 96 hours, and 6× at 120 hours.
10 ⁷ , and 1×10 ⁷ in 144 hours. After that, it rapidly died out and only 2 × 10 ⁵ , 192 in 168 hours
No live specimens were detected at this time. The progress of the experiment has been briefly described above, but after 192 hours (8 days) from the start of the experiment, the aquarium was as beautiful as the aquarium before the oil pollution, and the seawater was also very slightly cloudy. Other than that, the water was as clean as normal seawater. Example 3 The same procedure as in Example 2 was performed except that the crude oil content was 30 ppm and bacteria adapted to 30 ppm crude oil were used.
The results of the experiment are shown in Table 11.

[Table] As is clear from Table 11, the emulsification phenomenon started from 4 hours after the start of the experiment, gradually strengthened over time, became most noticeable after 36 to 48 hours, then declined, and rapidly increased from around 96 hours. A sense of transparency arises,
After 144 hours, it became almost transparent. On the other hand, the crude oil content of seawater also increased to 3ppm after 24 hours.
48 hours later, it rapidly decreased to 0.3 ppm, 1/100 of the original value. After 72 hours
0.2ppm, which gradually decreased to 0.15ppm after 96 hours, but thereafter showed the same results as after 96 hours (99.5%
removal rate). Next, regarding the evolution of assimilating bacteria, the initial population of 3 x 10 ⁵ increased to 4 x 10 ⁸ in 24 hours, and to 1.2 x 10 ⁹ in 48 hours. After that, the number continued to decrease, reaching 5 x 10 ⁸ in 72 hours and 2 x 10 ⁷ in 96 hours. After that, it begins to die rapidly, and in 120 hours, only 3 × 10 ⁵ , 144
When the time came, all of them died. The progress of the experiment has been briefly described above, but the aquarium and aquarium water were extremely beautiful 144 hours (6 days) after the start of the experiment. Furthermore, when we examined how the bacteria whose growth was promoted by crude oil reacted to various components in crude oil, we found that they grew vigorously with very little acclimatization to light oil. In contrast, slightly more difficult acclimatization difficulties were encountered with B fuel oil and even more severe acclimatization difficulties were encountered with C fuel oil.
However, by continuing induction similar to the above-mentioned steps 1 to 6, growth assimilation, emulsification, and promotion phenomena will appear in the same opening order as in crude oil, and once the promotion phenomenon appears, from then on, it will become more concentrated. Acclimatization was easier with B heavy oil and C heavy oil than with crude oil. In addition, the fact that it is well eaten and there is an abnormally small amount of remaining components means that most of the components have been taken up by the bacterial cells. Conventional “petroleum protein”
However, no matter what kind of bacteria is used in this case, bacteria whose growth is promoted by the petroleum components used have never been used. It goes without saying that growth promotion will result in better yields. Another example of the application of the bacteria according to the present invention is a method of treating oil by adding bacteria whose growth is promoted by oil to activated sludge, that is, creating reinforced activated sludge. It has the advantage of efficiently discharging beautiful water during cleaning, etc. Next, an example of application of bacteria to activated sludge according to the present invention will be shown. Example 4 (1) A culture solution of bacteria whose growth is promoted by crude oil that has been acclimated to 500 ppm, 100 ppm, and 30 ppm of crude oil is added to activated sludge in an amount of 1/10 of the amount of activated sludge, and petroleum-containing wastewater is introduced into the activated sludge. Increased speed. The treatment capacity of activated sludge strengthened in this way was 70 times that of activated sludge without added bacteria. In addition, the processing capacity was 15 times that of activated sludge containing known petroleum-utilizing bacteria. (2) The adaptive ability of protozoa in activated sludge is significantly inferior to that of bacteria, so it may be better to treat petroleum-containing wastewater with high load fluctuations first using bacteria alone. So crude oil 500ppm, 100ppm
Then, 30 ppm growth-promoting bacteria were cultured in a water tank with a capacity of 30 to obtain a bacterial count of 10 ⁹ /ml, and crude oil-containing wastewater was flowed into this at a rate of 10 ml/min. It was poured into a water tank and subjected to normal purification treatment. The amount of crude oil detected in the water obtained by separating the treated liquid was less than 1/250 of the initial amount. Example 5 A petroleum mixture was applied to the walls and bottom of a small drum with a capacity of 50, and after being left for 3 days, as much of the adhering oil was removed as much as possible with seawater under hydraulic pressure. After removing by suction the substances and components that have peeled off from walls and floated on the seawater surface, the bacteria according to the present invention are removed.
FERM-P No. 5490) culture solution (cultivated for 48 hours in a medium of seawater + crude oil) was added in an amount of 1/3000 of the amount of seawater. 120 hours after adding the bacteria, both the seawater and the walls and bottom of the drum became extremely clean. In addition, the present invention bacteria FERM-P No. 5490 and
5491 is the registration number of the bacteria produced from FERM-P No. 2927 and 2928, respectively, by the growth method described in this specification. And FRI No. 2927 is given to the biological findings of the culture of these present invention bacteria in normal culture medium.
And 2928 is the same as when registered, but only crude oil 0.5
g, After 48 hours of culture in seawater 1 and agar-only media, colonies are white, opaque, and 1.5 mm in diameter.
reach.

[Brief explanation of drawings]

FIG. 1 is a chart showing the growth/death curve of the strain according to the present invention, and FIGS. 2 and 3 are charts showing the growth promotion, emulsification, and assimilation characteristics of the strain according to the present invention.

Claims (1)

[Scope of Claims] 1. When a petroleum hydrocarbon of the genus Pseudomonas is cultured in a Stephenson-Wessam medium and a petroleum-based hydrocarbon is added during its logarithmic growth phase,
An oil purification preparation containing a Pseudomonas strain whose oil assimilation ability is increased by the hydrocarbons compared to the original Pseudomonas strain. 2. The petroleum-utilizing bacterial strain of the genus Pseudomonas is a microorganism strain of the Microtechnical Research Institute No. 2927, No. 2928, No. 5490, or No. 5491.
The petroleum purification formulation according to claim 1, which is a strain of No. 3. The petroleum purification formulation according to claim 1, wherein the petroleum-based hydrocarbon is crude oil, heavy oil, or other petroleum-containing product. 4. The petroleum purification preparation according to claim 1 or 2, wherein the Pseudomonas strain is sealed in a container as is or together with crude oil or petroleum-based hydrocarbons or/and a nutrient source under an inert gas atmosphere. . 5. When petroleum-based hydrocarbons were added during the logarithmic growth phase of Pseudomonas oil-utilizing bacterial strains in the Stephenson-Wetham medium,
An oil purification preparation containing a Pseudomonas strain whose oil assimilation ability is increased by the hydrocarbons compared to the original oil assimilating strain of the Pseudomonas genus is grown in a sea area or polluted place contaminated by petroleum-based hydrocarbons. A method of decontaminating the area or location. 6. The method according to claim 5, wherein the contaminated sea area is the ocean or the coast. 7. The method according to claim 5, wherein the contaminated site is an oil tanker, an oil storage tank, or an activated sludge treatment plant.