JPH0426836B2 - - Google Patents

Description

[Detailed description of the invention]

"Industrial Application Field" This invention is applicable to Rhodococcus erythrophores (formerly known as Nocardia erythrophores) KR-
As represented by S-1 stock (FERM No. 3530),
The present invention relates to a microbial flocculant production enhancement medium and a production enhancement method for culturing Rhodococcus microorganisms having flocculant production ability. This flocculant is used in various wastewater treatment fields,
It is applied to the treatment of various fermentation liquids and also to the flocculation (sedimentation) process in the food field. "Conventional technology" Conventionally, as a turbid water treatment agent and civil engineering dredging water treatment agent,
Inorganic flocculants (mainly composed of metal salts such as iron and aluminum) and polymeric flocculants (mainly composed of synthetic polymer flocculants such as polyacrylamide) have been used. These flocculants are widely used due to their high flocculating ability, but because they are made of metals such as aluminum or synthetic polymer compounds such as acrylamide, problems have been pointed out in terms of biological toxicity and environmental pollution. ing. In addition, as a flocculant that has improved these problems,
Biologically derived polymer compounds (for example, polyalginic acid extracted from seaweed, agar, etc.) are sometimes used, but their use is limited due to their high costs. Furthermore, chitosan extracted from crab and shrimp shells has good biodegradability and is attracting attention as a flocculant without the risk of environmental pollution. The problem is that the cost is high because it requires alkali treatment, separation and purification, and pulverization during the manufacturing process. On the other hand, it has long been known that substances secreted by certain microorganisms exhibit flocculating activity against various suspended solids, and one such microbial flocculant is Rhodococcus erythropores ( Former name: Nocardia erythropores) KR-S-
An agglutinating active component secreted by one strain (FERM No. 3530) has been discovered. The former name, Nocardia erythropores, was reorganized and reclassified as Rhodococcus erythropores by the International Committee on Nomenclature of Microorganisms in 1980. Rhodococcus erythropores KR-S-1
The flocculating active ingredient secreted by the strain was discovered at the Institute of Microbial Technology, Agency of Industrial Science and Technology, and was published in Japanese Patent Publication No. 56-39633 (Manufacturing method of microbial flocculant NOC-1: Patent
1096062) as NOC-1, and has excellent productivity, flocculation ability, and biodegradability.
Moreover, it has been revealed that it is a non-pathogenic and non-toxic aggregating active ingredient. For example, the flocculation activity of NOC-1 is effective against various substances such as inorganic substances (kaolin, activated carbon, coal ash, etc.), organic polluted wastewater (sludge, pig urine, licorice wastewater, etc.), and microorganisms (E. coli, yeast, activated sludge, etc.). It is also known to be effective for drainage, and is excellent as a flocculant. Patents related to the above-mentioned microbial flocculant NOC-1 include the above-mentioned Japanese Patent Publication No. 56-39633 (method for producing microbial flocculant NOC-1); JP-A-62-195288 (Method for enhancing production of microbial flocculant),
Patent Application No. 61-272583 (decolorization method for soluble pigments using microorganisms), etc. By the way, the medium used for culturing microorganisms and for fermentation production by microorganisms usually contains trace amounts of organic compounds such as carbon sources, nitrogen sources, inorganic salts (minerals), vitamins and hormones as nutritional sources for the microorganisms. However, some of these ingredients are
May be supplied simultaneously by the same substance. For example, by adding yeast extract, casein, casamino acids, peptone, beef extract, etc., multiple of the above components can be supplied simultaneously, and as shown in Table 1, for NOC-1 production. Yeast extract is also added to each culture medium (medium 1 to medium 4) that has been commonly used in the past as an (organic) nitrogen source and vitamin/hormone source.

【table】

[Table] In addition, in normal culture, it is necessary to sterilize the medium before culturing in order to eliminate the negative effects of germs. sterilization (for example, through a membrane filter with a pore size of 0.22 μm) is often applied. Of these, the latter, over-sterilization, can clog the pores on the filter surface if the medium contains solids, so in many cases, the former, high-temperature pressurization, is used. Sterilization is applied. In other words, conventional
The NOC-1 production method was no exception to this, and high temperature autoclaving was applied. For this reason, when producing NOC-1 using the conventional method, two steps are required: adding at least one of yeast extract, peptone, and casamino acid as a nutrient source to the medium, and sterilizing the medium prior to culturing. I needed the requirements. ``Problem to be solved by the invention'' However, substances such as the above-mentioned yeast extract are very expensive and account for a large proportion of the total cost of the culture medium.In particular, yeast extract is not effective in producing NOC-1. However, when considering scale-up, the cost of this yeast extract accounts for 70 to 80% of the total cost of the culture medium.
Cost has been a major hindrance in the practical application of the microbial flocculant NOC-1. In addition, the culture medium is usually sterilized by high-temperature autoclaving (for example, 121°C for 15 to 20 minutes), which requires initial costs for equipment and running costs such as electricity and gas required for heating. Therefore, the cost of NOC-1 has increased, which has become a major obstacle to its practical application. This invention was made in view of the above circumstances, and by using a medium to which wastewater or residue containing blood components as a nutrient source is used as a medium for a microbial flocculant production machine, it is possible to achieve low cost and Moreover, the aim is to obtain a microbial flocculant with high flocculating activity.Furthermore, by using the above-mentioned medium and culturing without high-temperature autoclaving, the initial cost of equipment and running energy for heating can be reduced. The aim is not only to eliminate costs, but also to increase aggregation activity and shorten culture time. "Means for Solving the Problems" The microbial flocculant production enhancement medium of the present invention is a medium for culturing microbial flocculant-producing bacteria belonging to the genus Rhodococcus, and uses blood components from household stock or fish as a nutrient source. or wastes such as household stock or processing residues containing blood components from fish, etc., are added. Here, waste water containing blood components of household stocks or fish, etc., or waste such as residues containing blood components of household stocks or fish, etc., refers to household stocks (including domestic animals and domestic birds) and fish. Wastes include blood or wastewater containing blood, food processing residue containing blood, organic substances of animal origin (excluding excrement and filth), animal processed products, residues, and by-products. Furthermore, the method for enhancing the production of a microbial flocculant of the present invention involves culturing microbial flocculant-producing bacteria belonging to the genus Rhodococcus using the above-mentioned microbial flocculant production enhancement medium without performing sterilization. It is. "Examples" Hereinafter, the microbial flocculant production enhancement medium and production enhancement method of the present invention will be described in detail with reference to Examples. (Example 1) In this experiment, Rhodococcus erythropores strain KR-S-1 was used as a flocculant-producing bacterium, and a fishery product collected from a fish canning factory was used as wastewater containing blood components that served as a nutrient source for the flocculant-producing bacterium. Using medium A (the composition of which is shown in Table 2) to which processing wastewater was added, the microbial flocculant was cultured and produced using this medium A. However, the concentration of the fishery processing wastewater in this medium A is 80%. This seafood processing wastewater contains blood produced during the fish slaughtering process. The results of water quality analysis of the above seafood processing wastewater are shown in Table 3.
Shown below.

【table】

【table】

[Table] Then, culture was carried out for a predetermined period using such medium A, and the flocculation activity value was measured as an index representing the amount of microbial flocculant produced by this medium A, and changes over time in the flocculation activity value were investigated. . In addition, culture was performed using a conventional medium containing yeast extract, and the change in aggregation activity value over time was measured in the same manner as above and compared with the above measurement results. In this case, the conventional medium is heated at 121°C and 15°C before incubation.
Autoclaving was performed for 1 minute. In addition, medium A is
Culture medium autoclaved at 121℃ for 15 minutes (in the table,
(indicated as (sterilized)) and a medium that was not autoclaved (indicated as (non-sterilized) in the table) were simultaneously cultured to examine the effect of non-sterilization. The initial pH of each medium was set to 8.0. In addition, in the measurement of the aggregation activity value, kaolin was selected as the substance to be agglomerated, and the activity was expressed as 1/turbidity of the supernatant. That is, put 80 ml of 5000 ppm kaolin suspension and 0.5 ml of culture solution in a 100 ml graduated cylinder, fill up to 90 ml with distilled water, add 10 ml of 10% calcium chloride, stir, and measure the absorbance of the supernatant liquid after 5 minutes at 550 nm. Measure at The aggregation activity value was calculated as the value obtained by subtracting the reciprocal of the absorbance of the control from the reciprocal of the absorbance of the supernatant when the test solution was used, and was used as the titer. That is, the larger the aggregation activity value, the greater the transparency, indicating the higher aggregation ability. The results of this experiment are shown in Table 4. However, the maximum aggregation activity value in the table indicates the maximum agglutination activity value during the experimental period (6 days), and the number of culture days indicates the number of culture days when the maximum agglutination activity value was obtained.

[Table] From this table, it is clear that the culture method using medium A containing seafood processing wastewater and the same non-sterilized medium has the following advantages. Fishery processing wastewater can be used as a nutrient source in place of yeast extract, which is the main cause of high cost of culture medium components. By culturing using medium A containing seafood processing wastewater, the maximum flocculation activity value is
It reached 3.4 times the conventional method. Comparing the maximum flocculation activity value when cultured after sterilizing medium A containing fisheries processing wastewater and the maximum flocculation activity value when cultured using the same medium without sterilization, the non-sterilized one is better. A high agglutination activity value was obtained (approximately twice that of the sterilized case). When culture medium A containing seafood processing wastewater was cultured in a non-sterile system, the culture time could be significantly shortened compared to the conventional method (the culture time to obtain the maximum flocculation activity was 1/2 that of the conventional method). 6). (Example 2) An experiment similar to the above Example 1 was conducted using medium B to which fishery processing wastewater having the water quality shown in Table 5 was added.

【table】

[Table] The results of this experiment are shown in Table 6. However, the maximum aggregation activity value and number of culture days in the table are the same as in Example 1.
These are the maximum aggregation activity value during the experimental period (8 days) and the number of culture days at which the maximum agglutination activity value was obtained.

[Table] As a result of this experiment, it was again confirmed that culture medium B using fishery processing wastewater with different water quality analysis results from Example 1 had the same advantages as Example 1. (Example 3) In this example, in order to investigate whether substances to be disposed of, such as wastewater or processing residue from slaughterhouses and meat processing plants, can be used as a nutrient source for a microbial flocculant production enhancement medium, household blood was used. An experiment was carried out using this as a nutrient source. In slaughterhouses and meat processing plants, blood and gastrointestinal contents from slaughtered animals are mixed into wastewater. in this case,
At the time of slaughter, 40-60% of the blood is usually collected and used as fertilizer, feed, or raw material for medicine.
Or it will be disposed of. Most of the remaining blood then gets mixed into the wastewater and is drained away. and,
The BOD of blood stored in these households is extremely high at approximately 165,000 mg/l (Water and Wastewater Handbook (revised 2nd edition), Maruzen Co., Ltd., p. 701). As the temperature increases, the color of the wastewater becomes blood red (or reddish brown). For this reason,
Blood is the main source of pollution in these wastewaters;
Therefore, problems often arise in the treatment of these wastewaters. As shown in Table 7, this experiment consisted of four types of media (medium, medium,
The test was carried out using a conventional culture medium (medium) and a conventional culture medium (medium) for comparison.

【table】

[Table] Table 9 shows the experimental results. However, the maximum aggregation activity value and the number of culture days in the table are the maximum agglutination activity value during the experimental period (12 days) and the number of culture days in which the maximum agglutination activity value was obtained.

[Table] As a result, the agglutination activity value when cow blood is used as a culture medium depends on the concentration, when sterilization is better, and when it is better to sterilize.
In some cases, it is better to use non-sterile products. this is,
This is thought to be because if the blood concentration is high and it is used non-sterile, the enzymes and immune system cells contained therein will inhibit the growth of the producing bacteria. For this reason, it is better to set the blood concentration to 10% or less when using non-sterile products. When culturing in medium (non-sterilized) and medium (sterilized), the maximum aggregation activity value was 1.3 times and 1.9 times higher, respectively, compared to the conventional method, and the culture time at which the maximum agglutination activity value was obtained was were reduced to 3/5 and 3/10, respectively, compared to the conventional method.
In addition, for culture medium (sterile) and culture medium (non-sterile),
The maximum aggregation activity value was similar to that of the conventional culture medium of Example 1 and Example 2, and the culture time at which the maximum agglutination activity value was obtained was about the same as that of the conventional method or longer. It was short. These results showed that cow blood can be used as a nutritional source in place of yeast extract in conventional microbial flocculant production. Furthermore, it has been found that by setting the blood concentration to an appropriate concentration, this culture solution can be used for culture without sterilization, and high agglutination activity can be obtained in a short culture time. "Effects of the Invention" Table 10 summarizes the experimental results conducted in the three examples described above.

[Table] As explained above, the following effects can be achieved by the present invention. (1) As a nutrient source for the culture medium for culturing microbial flocculant-producing bacteria belonging to the genus Rhodococcus, as an alternative to expensive nutrient sources such as yeast extract, the culture medium can be grown by adding waste such as wastewater or residue containing blood components. The cost can be significantly reduced. (2) By setting an appropriate addition amount according to the type of waste water or waste to be added, the work of sterilizing the culture medium can be omitted. (3) Higher flocculation activity than before can be obtained without adding expensive nutrients such as yeast extract and without sterilization. (4) The time required to obtain aggregation activity equivalent to or higher than conventional methods can be significantly shortened.

Claims (1)

[Scope of Claims] 1. A medium for culturing flocculant-producing microorganisms belonging to the genus Rhodococcus, which contains wastewater containing blood components from household stock or fish as a nutrient source, or blood components from household stock or fish, etc. A culture medium for enhancing the production of a microbial flocculant, characterized in that it contains waste such as processing residue. 2. Enhancement of production of a microbial flocculant, characterized in that when culturing a microbial flocculant-producing bacterium belonging to the genus Rhodococcus, the microbial flocculant production enhancement medium described in item 1 is used to culture without sterilization. Method.