JPH0767619A - Screening method of highly affinitive microorganism and highly phenol-affinitive microorganism - Google Patents

Abstract

PURPOSE:To conduct efficient screening of highly affinitive microorganisms having a carbon compound decomposition activity satisfactory even in the low concentration areas by gradually increasing the concentration of the carbon compound in the culture medium to be fed till reaching a certain level. CONSTITUTION:As single carbon source in the culture medium to be fed, a carbon compound such as phenol, benzene or the like is added to increase the concentration gradually till a certain level to effect screening of the microorganisms having high potency of biodegradation of the carbon compound as a substrate. Accordingly to this process, a microorganism having a high affinity to phenol originating from Alcaligenes-sp. R2 strain (FERM-P-13805) is obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention provides a method for screening bacteria (microorganisms) having a high affinity for a substrate compound.

[0002]

2. Description of the Related Art Techniques for applying microorganisms have progressed and developed along with their culture methods. Among them is the technology of utilizing (biodegrading) compounds in industrial waste with microorganisms. Especially, a biological method that uses activated sludge as a method of treating wastewater discharged in large quantities. Are considered to be advantageous in terms of operability and cost.

In the assimilation technique using such microorganisms,
Substrate affinity or specificity of microorganisms becomes a problem, and it is necessary to select a microorganism having a high affinity for each substrate. In order to select these microorganisms, a screening method by a batch culture method has been conventionally used.

However, when the substrate to be biodegraded has bactericidal activity, the microorganism is required to have resistance to survive, proliferate and maintain its function even in the presence of the substrate having bactericidal activity. However, it has been difficult to achieve both substrate affinity and resistance by the conventional batch culture method.

A representative of these substrates having bactericidal activity is a phenol compound. Phenolic compounds are used in coal gas and coke production plants and phenol resin production plants,
COD components contained in wastewater from organic chemical factories and the like. Conventionally, methods such as chemical oxidation, solvent extraction, and activated carbon adsorption have been adopted for treating wastewater containing these.

[0006] Phenol compounds are themselves regarded as one of the hardly biodegradable pollutants because they have a growth inhibitory effect or a bactericidal action against many microorganisms.

Therefore, establishing a biological treatment tank using a phenol-assimilating bacterium is significant in industrial and environmental pollution prevention.

[0008] Conventionally, microorganisms having the ability to decompose phenol include, for example, bacteria such as Pseudomonas, Nocardia, Bacillus and Acinetobacter, fungi such as Aureobasidium and Fusarium, and yeasts such as Trichosporon and Candida. It has been known.

However, these phenol-degrading bacteria can be treated by the conventional batch culture method (initial phenol concentration, hundreds of pp
Since it was screened in step m), it was highly resistant to high-concentration phenol, but it was not a high-affinity bacterium capable of efficiently decomposing low-concentration phenol [ P. putida WAS2 (JP-A-3-67581), P. putida WAS202
(Japanese Patent Application No. 4-341430), and Acinetoba
cter (Hashimoto et al., Journal of Fermentation Engineering 70 70 267-271
1992)]. That is, it can be seen that these batch culture methods merely screened bacteria having high resistance to phenol, and were not necessarily compatible with the phenol degrading ability of the selected bacteria.

Attempts have also been made to microbially formulate these bacteria into an activated sludge or the like, but no example has succeeded in allowing these bacteria to remain at a high concentration. The cause is considered to be that these bacteria have low phenol affinity.

[0011]

As described above, the bacteria screened by the batch culture method have low phenol affinity, and the maximum load of continuous phenol treatment with these bacteria is 5.5 gr obtained by WAS202. p
The best is about henol / liter and day.

In addition, the concentration of phenol that can be decomposed in an inorganic salt medium is low, and it has the drawbacks that it takes a long time to complete the decomposition and that it requires a long acclimation period to phenol. Application to the pharmaceutical field was difficult.

[0013]

The present invention has been made for the purpose of solving such problems. That is,
The present inventors have developed a screening method for obtaining a bacterium with a high affinity for a substrate and a high biodegradability,
The present invention has been completed.

The present invention provides a screening method applicable to various compounds, and a microorganism having a high affinity for the compound, particularly a bacterium (microorganism) having a high affinity for a phenol compound. Useful to earn.

That is, the present invention is a method for screening a bacterium having a high ability to biodegrade a carbon compound as a substrate by continuously culturing a mixture of bacteria, the carbon being used as the sole carbon source in a feed medium. The concentration of the compound is gradually increased to bring the concentration of the carbon compound in the feed medium to a certain value, and preferably, a portion of the cultivated bacterial mixture is further removed and cultivated again in a non-selective medium. Among the formed colonies, there are the above-mentioned screening method for selecting the one having the largest number of colonies, and the bacterium obtained by the method. The present invention is characterized by using only a carbon compound as a substrate as a carbon source in a continuous culture medium. An ordinary medium contains sugars such as glucose, but the base of the present invention is an inorganic salt medium.
Examples of the carbon compound as a substrate include phenol,
Examples thereof include, but are not limited to, benzene, toluene, cresol, chlorobenzene, catechol, aniline, pyridine, chlorophenol, dimethylphenol and chlorocresol.

Any non-selective medium may be used as long as it is used for ordinary culture, but it is also possible to contain a carbon compound or a similar compound as a substrate, and various antibiotics may be added if necessary. You may include additive components, such as an agent.

Further, in the present invention, unlike a usual batch culture method such as shaking culture, a continuous culture method in which a fresh medium is continuously supplied is carried out. That is, in the batch culture method, the concentration of the substrate in the medium changes (decreases) due to biodegradation of bacteria, but in the continuous culture method, since the fresh medium is continuously supplied, the substrate concentration can be appropriately controlled. The present invention is characterized in that the culture is carried out while gradually increasing the substrate concentration in the feed medium, and more preferably while gradually increasing the substrate concentration with a constant increasing rate.

Bacteria obtained by the screening method using activated sludge as the mixture of bacteria and phenol as the carbon compound are industrially particularly useful and preferred.

In this case, the activated sludge is put into a continuous culture tank and continuously cultured in a medium containing phenol as the sole carbon source, and then the phenol concentration in the feed medium is gradually increased while keeping the dilution rate constant. The substrate concentration was allowed to reach a constant high load state (at this time, the phenol concentration in the continuous culture tank was always low at 1 ppm or less).

Furthermore, the screened bacteria can be grown by taking out a part of the solution or the bacterial mixture therein and plating it on a non-selective natural medium plate.

From the colonies formed on the plate as described above, a high affinity bacterium can be selected by selecting the largest colony in consideration of the colony area, the number of colonies and the colony morphology.

The bacteria obtained by the screening method of the present invention preferably have the largest number of colonies formed on a non-selective natural medium plate, but the number of colonies is 2
It is possible that these bacteria can be used because it is possible that they have a certain level of affinity for the bacteria after the th.

The following method may be mentioned as a preferred embodiment of the method of the present invention.

1. Furthermore, the screening method including the step of taking out a part of the cultured bacterial mixture and culturing again in a non-selective medium.

2. A screening method comprising the step of selecting a colony having the largest number of colonies among the colonies formed by the culture in the non-selective medium.

3. A screening method, characterized in that the non-selective medium contains a carbon compound as a substrate or a compound similar thereto.

4. A screening method, characterized in that the rate of increase is kept constant when gradually increasing the concentration of carbon compounds in the supply medium.

5. A screening method wherein the bacterial mixture is activated sludge.

The present invention is described in detail below with reference to examples, but the present invention is not limited to this range.

[0030]

【Example】

1. Screening for high-affinity bacteria Using an activated sludge tank for experiments, take the activated sludge conditioned with synthetic phenol wastewater into a continuous culture tank, and feed it with a medium of 1500 p
pm MP medium (2.75 g of K ₂ HPO ₄ , KH ₂ PO
_{4 2.25g, (NH 4) 2} SO 4 1.0g, MgCl
_{2 · 6H 2 O0.2g, NaCl0.1g,} FeCl 3
6H ₂ O 0.02 g, CaCl ₂ 0.01 g, and phenol 1.500 g were included in 1 l) to start continuous culture. During culturing, the temperature is 25 ° C and the dissolved oxygen concentration is 5 to 7
It was defined as ppm. The phenol loading was varied by the dilution rate and the experiment was started with a loading of 0.6 g / l, day.
Thereafter, the load was increased by 0.6 g / l and day every 4 to 5 days, and a part of the solution therein was collected at 3.0 g / l and day. This solution was diluted 1 × 10 ⁸ times and
Orient Broth Medium Plate (Bactop
epton 5g, Beefextract 3g, N
5 g of aCl and 15 g of Bactoagar were contained in 1 l), and cultured at 30 ° C. for 4 days. The colonies formed here were classified into 5 types in terms of their morphology. Isolate each by agar plate method, R1, R2, R
3, R4 and R5 strains. These identifications to Berg
ey's Manual of Systematic
The results obtained by the method described in Bacteriology are shown in Table 1.

[0031]

[Table 1]

Among these, Nutrient B
The most abundant on the plate of roth medium was R2.

2. Identification of high-affinity bacterium R2 Thus, the phenol-decomposing bacterium R2 strain obtained by screening by the method of the present invention using phenol as a substrate compound was examined by the taxonomic properties of the bacterium, and as a result, Alcalige
nes sp. (See Table 1).

The present bacterium is Alcaligenes sp.
R2 (deposit number FERM P-1 deposited on August 18, 1993 at the Institute of Biotechnology, Institute of Biotechnology, AIST)
3805).

3. Phenol-degrading activity of R2 Inorganic salt medium (M
(P medium) was inoculated into 5 ml and cultured at 30 ° C. with batch shaking. As a result, as shown in FIG. 1, this bacterium was able to grow up to the initial phenol concentration of 500 ppm.

Similarly, the present bacterium was cultivated in an MP medium containing 200 ppm of phenol, and the microbial cells were collected at the latter stage of the logarithmic growth phase. The phenol-degrading activity of this bacterium was measured as follows using an Oxygraph oxygen electrode manufactured by Gilson Company.

First, 2 ml of MP medium was placed in the reaction cell to stabilize the dissolved oxygen concentration, and then 5 mg of cells was added to the cell. Next, 20 μl of 1 M potassium cyanide was added to suppress oxygen consumption due to the respiration of bacteria, and then phenol was added to measure specific oxygen consumption associated with the decomposition of phenol. The amount of phenol added was adjusted to determine oxygen consumption at various substrate concentrations.

Assuming that one molecule of oxygen is required to decompose one molecule of phenol, the phenol decomposing activity can be obtained by dividing the specific oxygen consumption rate associated with the phenol decomposition by the amount of added cells.

When the substrate concentration is low, this decomposition reaction is Mic.
Since it is considered to follow the Haelis-Menten equation, the Km (substrate affinity) value and the maximum degradation rate Vmax value are calculated using the Michaelis-Menten equation below.

[0040]

[Equation 1]

It was determined by plotting 1 / S on the X axis and 1 / V on the Y axis by a Lineweaver-burk plot.

FIG. 2 shows the results of investigating the relationship between the substrate concentration and the degrading activity, which are shown in Pseudomonas putida WA.
It is shown together with the result of S2.

As described above, this bacterium has a P. As compared with putida WAS2, the degrading activity was high at a low concentration, and the maximum activity value was about four times as large.

The results are analyzed based on the Michaelis-Menten equation, and shown in Table 2.

[0045]

[Table 2]

From this, the Km value (substrate affinity) of this bacterium R2 is 0.34 ppm, which is about 1/7 of that of WAS2.
It is understood that this bacterium has a very high affinity for phenol.

The Vmax value of this bacterium R2 (maximum decomposition rate, measured by oxygen electrode method) was 28.1 μmol O ₂ / mi.
n. , Gram cells, and about 2.
It was five times as large.

Substrates capable of decomposing R2 were examined using the same cells by the above-mentioned method using an oxygen electrode. The results are shown in Table 3.

[0049]

[Table 3]

As described above, the present bacterium comprises benzene, toluene,
It was also found that cresol, chlorophenol, catechol, aniline and pyridine can also be assimilated and that they have relatively low substrate specificity.

A conventional phenol-utilizing bacterium P. pu
tida WAS2, the Km value is 2.5 ppm, and Vmax values 7.9μmolO ₂ / min. , Gra
It is mcells. Thus, it can be said that the bacterium of the present invention is characterized by a low Km value (high affinity) and a high decomposition rate.

4. Phenol continuous treatment experiment A phenol continuous treatment experiment was performed using entrapped and immobilized cells. For comprehensive fixation of bacterial cells, PVA developed by Hashimoto et al.
Using the freezing method, cell concentration 20 mg / ml, gel concentration 15
It went in%. This is molded into a cube with a side of about 5 mm and 3
It was charged into the treatment tank at a concentration of 0% (V / V). M with 1500 ppm phenol as the only carbon source as treated water
P medium was used, and the load was changed by the dilution rate. The result is shown in FIG.

From this, P. putida WAS2 and Alcaligenes sp. It can be seen that the limit maximum load of R2 is 3 g / l, day and 7 g / l, day, respectively. Like this, Alcaligene
s sp. It was revealed that a phenol high load type reactor can be produced by using R2.

[0054]

INDUSTRIAL APPLICABILITY A screening method for obtaining a microorganism having a high affinity for various substrates by changing the substrate which is a carbon source in the medium has been established. For example, the phenol-assimilating bacterium obtained by using phenol as the substrate is
It can be used as a microbial preparation for improving the capacity of biological wastewater treatment (for example, activated sludge) when a large amount of a phenol compound is contained in industrial wastewater such as in the steel industry.

[Brief description of drawings]

FIG. 1 shows the growth behavior of the bacterium of the present invention when shake-cultured in an inorganic salt medium containing phenol as the sole carbon source. It can be seen that growth is possible up to an initial concentration of 500 ppm.

FIG. 2 is a graph showing the phenol-degrading activity of the bacterium of the present invention at various phenol concentrations. In particular, it can be seen that it has about 4 times the activity of conventional bacteria (WAS2) at low concentrations.

FIG. 3 shows the behavior when the bacterial cells of the present invention are entrapped and fixed and loaded with phenol. By following the phenol concentration in the treated water, it can be seen that the maximum limit load value has increased to 7 g / l, day.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12N 1/20 F 7236-4B // (C12N 1/20 C12R 1:05) (72) Inventor Yasushi Onodera Saitama 1-3-1, Nishitsurugaoka, Oi-cho, Iruma-gun, Tochigi Research Institute

Claims (3)

[Claims] 1. A method for screening a bacterium having a high ability to biodegrade a carbon compound as a substrate by continuously culturing a bacterial mixture, wherein the concentration of the carbon compound as a sole carbon source in a feed medium is determined. The screening method, which comprises the step of gradually increasing the concentration of the carbon compound in the feed medium to a certain value. 2. A high-affinity bacterium capable of assimilating or degrading a substrate carbon compound, which can be selected from the screening method according to claim 1. 3. Alcaligenes sp. A high-affinity phenol bacterium characterized by being derived from the R2 strain.