JPH10272490A - Treatment of pectin-containing waste water - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible to extremely efficiently treat pectin-contg. waste water by using the S-2 strain belonging to Cryptococcus sp. which is a specific microorganism or specific yeast bacterium confluent strain ANY-180 strain. SOLUTION: The Cryptococcus sp. S-2 strain to be used produces a large amt. of pectinase outside the microbial cells, has high pectin decompsability and low nutritional property and exhibits vigorous growth even at a low temp. Hansenula anomala J224 and Hansenula fabrianii J640 and the confluent strain ANY-180 are subjected to the formation of the confluent strain by executing cell fusion by using a J-224 strain as a parent strain having aggregation property/adhesiveness and a J-640 as a parent strain having high pectin decomposability, respectively. The colonies grown in a selected medium are selected as the confluent strain. Waste water is supplied into a treating vessel where the contact materials adhered with the confluent strain ANY-180 or S-2 is adhered are installed, by which the continuous treatment or batch treatment is executed in order to treat the waste water.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for treating pectin-containing wastewater, and more particularly, the present invention is characterized in that a specific yeast is used. The yeast used in the present invention is not only excellent in pectin decomposability, but also excellent in cohesiveness and adhesiveness, so that it adheres to the treatment tank and the contact material disposed therein, and may run away. Therefore, high-concentration wastewater treatment and efficient wastewater treatment can be achieved.

[0002]

2. Description of the Related Art Waste water for refining fibers contains a large amount of pectin, and direct discharge of the pectin into rivers and the like is prohibited. In addition, various wastewaters containing a large amount of pectin are known as food production wastewaters, and development of an efficient treatment method thereof is strongly desired.

[0003] For example, in recent years, the unique texture of pectin has been favored, and pectin-containing confections have been manufactured.
In the production process, pectin-containing wastewater is discharged in large quantities. In addition, fruit processing wastewater discharged from fruit processing plants such as apples and citrus fruits contains a large amount of organic acids, sugars, pulp, cellulose, etc. in addition to pectin. For example, in the orange canning process, after peeling the epicarp with salt water, the endocarp is treated with an acid and then an alkali to peel the endocarp to dissolve and separate pectin and cellulose, etc. Waste water (alkaline waste water) is not only alkaline, but also has high viscosity.
It contains a large amount of hardly decomposable pectin and sugar, and its COD load is very high. Of course, it cannot be discharged into rivers as it is, and an enormous amount of water is required for dilution. Is not a practical method.

[0004] Further, since the pectin-containing wastewater has a high viscosity as described above, stirring and transfer cannot be carried out smoothly even if mechanical treatment is performed. Has been extremely difficult.

At present, activated sludge treatment and calcium treatment are performed as pectin decomposition treatment.
In the former treatment, the COD removal rate is low, and the sludge condition is not good. In the latter treatment, a method is also used in which a large amount of calcium is put into wastewater to convert pectin into a calcium salt and settle and remove it. Therefore, it is hard to say that it is a realistic method on a factory scale.

As described above, a method for industrially treating a pectin-containing waste liquid has not been established. Usually, a pectin-containing waste liquid contains a large amount of organic acids and sugars in addition to pectin, such as a citrus factory waste liquid. It is more practical to be able to handle them as well, but the state of the art is that no such method has yet been established.

[0007]

DISCLOSURE OF THE INVENTION In view of the above-mentioned state of the art, the present invention provides a method having a higher treatment efficiency than the above-mentioned conventional method, and in particular, a novel method capable of treating pectin-containing wastewater extremely efficiently. The purpose was to develop a new method.

[0008]

DISCLOSURE OF THE INVENTION The present invention has been made to achieve the above object, and as a result of various studies, it has been found that such a type of wastewater can be produced at low cost, safely, and on the smallest possible scale. It has been concluded that a target microorganism is screened and a method utilizing the obtained microorganism is optimal for efficient mass treatment.

Therefore, the present inventors have developed a microorganism which can not only directly assimilate pectin but also withstand high viscosity and can withstand severe physical and chemical changes in the wastewater treatment process. Screening was performed from a huge number of natural or artificial microorganisms such as bacteria, filamentous fungi, yeast, basidiomycetes, and incomplete bacteria.

As a result, the S-2 strain belonging to the genus Cryptococcus, which is a specific microorganism (FERM P-1515)
5) (hereinafter also referred to as S-2 strain) and a specific yeast cell fusion strain ANY-180 strain (FERM P-152).
71) (hereinafter sometimes referred to as ANY180 strain)
All discovered that they had excellent pectin decomposability and could efficiently treat pectin-containing wastewater, and based on this new finding, completed the present invention.

The strains created and isolated here can treat pectin-containing wastewater very efficiently under extremely severe conditions, and can efficiently treat citrus processing plant wastewater, confectionery plant wastewater and other various pectin-containing plant wastewater. These are strains having a conventionally unknown novel utility of chemically purifying, each of which is named as follows, and all of them are deposited with the National Institute of Advanced Industrial Science and Technology as follows.

Crytococcus sp. S-2 (FERM P-1)
5155); Hansenula anomala J224 and Hansenula f
abianii J640, fusion strain ANY-180 (FERM P
-15271).

[0013] In practicing the present invention, it may be Seshimere contacting the strain and pectin-containing waste water described above, about 10 ^7-1
It is preferable to maintain a cell density of 0 ⁸ cells / ml or more. By treating wastewater at such a cell density, COD, TOC, BOD, total sugar, and viscosity are significantly reduced. And efficient purification of wastewater becomes possible.

The S-2 strain used in the present invention produces a large amount of pectinase extracellularly and has a strong pectin decomposing ability. In addition, since the present bacterium is hypotrophic and shows vigorous growth even at low temperatures, it can be said that the bacterium is suitable for use in wastewater treatment. Further, it is also possible to obtain an aggregated mutant from the S-2 strain and attach it to a contact material before use. By using the contact material to which the microorganisms are attached in this way, a high microorganism density can be maintained, and wastewater can be treated more efficiently.

[0015] The fusion yeast used in the present invention can be prepared as follows. That is, in order to create a fusion strain having both excellent cohesiveness / adhesiveness and excellent pectin degradability, first, Hansenula anomala J-224 (hereinafter referred to as J-224) is used. As a result of various studies on the strain or J-224), the J-224 strain was newly focused on having a property of strongly adhering to the inner wall of the culture vessel in addition to the aggregating property. Fixed floor (contact material,
(Fixed on a carrier), a new finding that a better drainage treatment effect can be obtained.

As a result of research from various fields, the results show that the J-224 strain is a flocculant yeast and the Hansenula fabianii J-640 is a pectin-resolving yeast.
A strain (hereinafter, sometimes referred to as J-640 strain to J-640) was used as a parent strain, and this was subjected to cell fusion by a polyethylene glycol method (PEG method). Cell fusion may be performed by a conventional method. As a preferred cell fusion accelerator, for example, polyethylene glycol having an average molecular weight of about 1,000 to 6000 is used at a concentration of 20 to 50%, preferably 25 to 50%, and room temperature. ~ 37 ° C, pH
Under the conditions of 5.5 to 8.0, the protoplast-converted parent strain is added, and the mixture is stirred and fused for about 0.5 to 1 hour. Then, a fusion strain having both properties may be selected and bred using a selective medium. . In addition, the cell fusion treatment can be performed by an electric pulse in addition to the polyethylene glycol (PEG) method.

As a parent strain having cohesive / adherent properties, J-2
24 strains, J-64 as parent strain having high pectin resolution
Using 0 strains, cell fusion was performed as described above to prepare a fusion strain, and colonies grown on a selective medium were selected as fusion strains. These fusion strains are previously unknown excellent strains having both agglutinating / adhering properties and high pectin-degrading property. As described above, these strains are newly added to Hansenul.
a anomala J224 and Hansenula fabianii J640, a fusion strain ANY-180, and deposited with the National Institute of Advanced Industrial Science and Technology as a FERM P-15271.

The new fused strain successfully bred in this manner exhibits excellent cohesiveness, high pectin-degrading property, and adherence to a contact material (fixed bed member), as is apparent from the examples described later. On the other hand, the contact material to which the fusion strain is immobilized enables extremely excellent and efficient wastewater treatment.

Accordingly, another aspect of the present invention is to provide a fixed or partially fixed contact material or a non-fixed contact material in a treatment tank, to which a cohesive and / or adherent yeast is attached. At the very least, various wastewater treatments such as high pectin-containing wastewater are performed.

Examples of the contact material include a granular contact material, a rod-like contact material, a cylindrical contact material, a plate-like contact material, a grid-like contact material, a mat-like or mesh-like contact material, a flexible contact material, and a small cylindrical body. ,
Molded resinous fillers and / or Raschig rings or leasing rings or Kura type fillers can be used widely and freely, and single or two or more types can be used in combination.

Specific examples of the contact material are as follows. In the present invention, any type of contact material can be used as long as it is within the above-mentioned range.

Granular contact material: Various sizes of carriers such as glass, plastic beads, and granular activated carbon, as well as gravel are also included.

Bar-shaped contact material: Square bars, round bars having various lengths, and rods or rods having cross sections of various shapes are widely included.

Tubular contact material: A cylindrical contact material formed by hollowing out a rod-like contact material.

Plate contact material: flat, corrugated, curled or bent flat, curled or bent corrugated,
A wide variety of other deformed plates are included.

Grid-like contact material: Various deformed contact materials based on a lattice, such as a long and thin carrier formed into a lattice shape and a well-formed shape.

Matte or reticulated contact material: A contact material composed of matte, coarse, cloth-to-reticulated fibers, synthetic resin, or other materials, and a plate-like body of these materials or spaced apart from each other And a plate-shaped body formed into a cylinder or a square tube, a three-dimensional net-like body, and the like.

Flexible contact material: fibrous, cloth, sheet,
A contact material made of a flexible material having a string shape, a band shape, a ribbon shape, a flake shape, or the like is included.

Small cylindrical body: All relatively small cylindrical bodies are included, but the cylindrical body is not only an accurate cylindrical body but also deformed like a small container for a lactic acid bacteria beverage with a bottom removed. The above-mentioned cylindrical body is also included.

Various packings used in the chemical industry, such as packings for distillation towers, can also be used. A Raschig ring, which is one of the above-mentioned small cylindrical bodies, and a dressing with a partition attached thereto Rings, clads, reticulated McMahon packings, Stedman packings and the like can be advantageously used.

Molded resinous filler: Plastic fillers in chemical industry equipment such as drying towers, washing towers, cooling towers, etc. are widely used. For example, a wheel-shaped circular body in which a large number of rings are radially arranged, in which the cross section of the ring is circular, round, square or elliptical, or any other shape,
Similarly, a space or perforation is provided in the center part, an outer ring circle is also provided in the outer periphery, or a circular body in which a number of spokes are radially arranged, and an outer ring circle is provided in the outer periphery to form a wheel. What is done can be used.

For this purpose, commercially available products can also be used as appropriate, and for example, a flower-shaped filler TELLERETTE (registered trademark of Nippon Steel Chemical Co., Ltd.) is exemplified. In the present packing, in addition to the structure in which the rings are radially arranged on a horizontal plane as described above, a structure in which the rings are radially arranged in a three-dimensional manner can also be used.

In the present invention, these contact materials may be formed into a predetermined shape, or may be variously deformed or irregularly shaped. Further, the surface may be roughened, provided with irregularities or undulations, made porous, formed with relatively large holes, or formed into a net-like structure.

In the present invention, the contact material can be used alone or in combination of the same type or different types. Furthermore, it is also possible to stack, collect, or condense individual contact materials and use them collectively. As an example thereof, for example, a plurality of plate-like contact members are laminated at intervals, and in this case, a flat plate or a corrugated contact member may be laminated of the same kind, or both may be mixed and laminated. The corrugated plate-like contact members may be alternately focused in the opposite direction to form a kind of honeycomb structure, or the corrugated plate and the flat plate-like contact material may be directly and alternately directly bundled to form a deformed honeycomb structure.
Furthermore, a contact material having a honeycomb structure can be obtained even if various kinds of rectangular cylinders are bundled, and a porous cylindrical structure can be obtained if the cylinders are bundled. The porous body structured as described above is also very effective as the contact material in the present invention.

When installing these contact materials in the treatment tank, the individual contact materials or several contact materials may be arranged in the tank in a non-fixed state, that is, in a free state, Several or many contact materials may be collectively accommodated in the support, and the support may be arranged in the tank. At this time, a mesh or perforated container is used as the support, and one or more shelves are provided in the container to improve the efficiency of the treatment, and the contact material is disposed while leaving a space there. as well as/
Alternatively, a shelf in which the contact material is not provided may be provided so that the wastewater and the yeast sufficiently contact each other. In addition, in order to prevent individual contact materials from flowing out, it is preferable to provide a net or baffle plate for preventing flow-off on the upper surface of the treatment tank,
Alternatively, a relatively large support accommodating the contact material may be disposed in a processing tank provided with a support pedestal at a lower portion and a contact material levitation prevention table at an upper portion. At this time, it is preferable that a space for drainage transfer, a rectifier, an air diffuser, and the like are arranged in the tank so that the yeast and the wastewater are sufficiently in contact with each other.

The present invention also includes fixing the contact material to the processing tank. To fix the contact material, the contact material may be directly fixed to the bottom and / or side surface of the processing tank, or a support, a partition, or the like may be arranged in the tank, and the contact material may be fixed thereto. In the latter case, for example, a large number of molded resin fillers, for example, Terralet (registered trademark) may be fixed at regular intervals to one or more pillars arranged in a tank. Also, for example, converging or gathering, laminated contact material,
Normally, this is fixed upright in the tank, but a space is provided at the bottom and a draft tube or space is provided at the center or at an appropriate place, and an air diffuser is further provided to improve efficiency. Wastewater treatment can be made possible.

Further, in the present invention, a partially fixed contact material can also be used. As the partially fixed contact material, in addition to the above-mentioned flexible contact material, a fibrous, rod-like, tubular, string-like, band-like, ribbon-like and / or flaky contact material can be widely used. it can.

In actual use, for example, one end of the string-like contact material is fixed to the bottom surface, wall surface and / or top surface of the processing tank, and the string-like body does not flow out of the tank, and is kept in the tank. It swings or floats in wastewater without being tightly fixed inside. Therefore, both ends of the long string-shaped contact material may be fixed to both wall surfaces of the processing tank while holding the slack, or if necessary, an appropriate position of the intermediate portion may be fixed to the bottom surface or the wall surface of the processing tank. Is also good.

As the partially fixed contact material according to the present invention,
Any material made of a material having flexibility can be used, and a rod-shaped body, a tubular body, and other contact materials can be widely used.

Still further, even if the material itself does not have flexibility, it can be used as a partially fixed contact material in the present invention by imparting flexibility. For example, if a rigid contact material is fixed to the treatment tank via a flexible material, flexibility can be imparted, and various immobilized carriers can be freely used in the present invention.

As a specific example thereof, a glass fiber, a string or a hollow pipe made of synthetic resin may be fixed on a carrier having a high ability to immobilize microorganisms, and this may be fixed in a tank, or may be made of vinyl chloride or other synthetic resin. Strings, rods, plates, cylinders, etc.
Fibers or the like may be attached and fixed to the tank. Also,
Carriers such as beads may be passed through a string, and a part or both ends of the string may be fixed to the tank, or instead of fixing the beads one by one to the string, several beads may be packed in a mesh bag. , And this may be hung on one or more bags.

As described above, according to any one of the above-mentioned methods according to the present invention, not only the flexible material but also all the other materials can be freely used as a partly fixed contact material.

To treat wastewater according to the present invention,
Wastewater may be supplied into a treatment tank provided with a contact material to which yeast such as a fused yeast or the S-2 strain is adhered, and a conventional method may be used. That is, wastewater is stored in a raw water tank, and the wastewater is directly or diluted, and then sent to a treatment tank (yeast tank). The pH and temperature in the treatment tank are adjusted and maintained at a level suitable for yeast growth. If so, continuous stirring or batch treatment may be performed by performing aeration stirring with an air pump or the like or mechanical stirring with a stirrer. Since the contact material is used, the yeast adheres and agglutinates to the contact material in the meantime to increase the bacterial cell concentration, and effective and rapid treatment is performed. At this time, since the contact material does not flow out of the tank, there is no run-off of the yeast cells, and a high cell concentration is maintained, while, for example, the non-fixed or partially fixed contact material swings in the wastewater. This not only does not hinder the stirring, but also improves the air permeability, thereby further increasing the bacterial cell concentration and further promoting the wastewater treatment. The yeast used in the present invention is not only excellent in assimilating starch but also excellent in cohesiveness and / or adhesion. Therefore, even when no contact material is used, the yeast itself can be used. The density of yeasts increases due to mutual cohesion or adhesion to the vessel wall of the treatment tank, and wastewater treatment can be performed very efficiently even in a small treatment tank.

The treated water thus obtained can be discharged to a public sewer as it is. If necessary, the treated water is subjected to physical or chemical treatment such as sedimentation or coagulation, or activated, according to a conventional method. It may be subjected to a biological treatment such as a sludge treatment and then discharged. Hereinafter, examples of the present invention will be described.

[0045]

Example 1: Preparation of fusion strain Hansenula anomala J-224
(FERM P-7671) and Hansenula fabianii J-640 (FERMP-1
5154) was prepared as follows. That is, first, both the deposited parent strains were mutated according to a conventional method, and the auxotrophic mutant Hansenula anomala J-
224, AM82 (arg-) and Hansenula fabianii J-6
40-4 (thr-met-) was separated, and these were used for cell fusion according to the PEG method.

Cell fusion by the PEG method was performed according to a conventional method. After protoplastization of both strains, protoplast fusion was performed using PEG. That is, the parent strain was inoculated into 5 ml of YPD medium (peptone 2%, yeast extract 1%, glucose 2%), cultured with shaking at 30 ° C. for 24 hours, and 0.1 ml of the culture solution was further added to 5 ml of YPD medium. The cells were inoculated and cultured with shaking at 30 ° C. for 17 hours. After collecting and washing the cultured yeast, the yeast count was 2 × 10 5 ml in 60 mM EDTA containing 0.2% β-mercaptoethanol.
^The cells were suspended at ⁷ cells / ml and shaken at 30 ° C for 30 minutes. After shaking, the cells were collected, 0.2% β-mercaptoethanol, 100 μg of Zymolyase 20T
The reaction was performed at 30 ° C. for 60 minutes in a 0.1 M phosphate buffer (pH 7.5) containing 1 / ml and 1.2 M sorbitol, and each yeast was protoplasted. After the reaction, the yeast was washed with 1.2 M sorbitol, resuspended in 1.2 M sorbitol, mixed with protoplasts, and centrifuged. Next, the cells were suspended in a 30% polyethylene glycol 4000 solution containing 50 mM calcium chloride, and subjected to a cell fusion treatment at 30 ° C. for 30 minutes. The treated cells were transformed into a selective medium (Difc) containing 1.2 M sorbitol.
o: yeast nitrogen-based W / O amino acid, glucose 1%, agar 2%) and overlaid the same medium.
After culturing at 30 ° C. for 7 to 10 days, the grown colonies were used as fusions.

[0047] As a result of the protoplast fusion, strain seems to fusion can be obtained 11 strains, fusion frequency became 2.3 × 10- ^7. Among the 11 strains, 5 strains having cohesiveness were selected.
Furthermore, from the five strains, a yeast strain having a strong cohesive adhesion and a high starch degradability, which meets the intended purpose, is finally selected,
This was designated as a fusion strain ANY-180, and deposited with Life Research Institute (FERM P-15271).

[0048]

Example 2: Measurement of agglutination rate For each yeast (parent strain, fusion strain), each absorbance of OD (T) and OD (S) was measured.
The aggregation ratio (Flocculation ratio; FR) was calculated from the following equation, and the obtained results are shown in Table 1 below. FR = {OD (T) -OD (S)} / OD (T) FR: flocculation ratio OD (T): 0.02 M Tris of yeast suspension (about 5 × 10 ⁷ cells / ml) Absorbance measured at 660 nm, 10 mm cell after treatment with pronase E (2.5 mg / 10 ml) in hydrochloric acid buffer (pH 7.6) at 35 ° C. for 1 hour. OD (S): After centrifuging the yeast suspension of the same concentration in a 0.01 M acetate buffer (pH 5.0) at 300 rpm for 5 minutes,
Absorbance measured at 00 nm, 10 mm cell.

[0049]

[Table 1]

[0050]

Example 3 Adhesion to Fixed Bed Member Adhesion to the fixed bed member of each yeast (parent strain and fusion strain) was measured as follows. That is, in a 500 ml Erlenmeyer flask, a 5 cm piece of fixed bed member (ring lace: made of vinylidene chloride, string shape) and a YPD medium (1% yeast extract,
100 ml of a cell suspension cultured with 2% peptone and 2% glucose) was added thereto, shaken at 30 ° C., and the absorbance at 660 nm of the suspension was measured over time, and the adhesion rate was calculated from the following equation. A. R = (O.DI-O.DT) /O.R. DI × 100 where A.I. R: Adhesion ratio DI: absorbance at 660 nm before shaking DT: absorbance at 660 nm after shaking

FIG. 1 shows the obtained results. In the figures, open circles indicate H. anomala J-224, closed triangles indicate H. fabianii J-640, and closed circles indicate fusion strain ANY-180.

[0052]

[Example 4: Wastewater treatment (1)] Pectin (made of apple) 1
%, Yeast extract 0.5%, 100 ml of artificial wastewater having a composition of pH 4.8 was placed in a 500 ml Erlenmeyer flask (with a baffle), and the preculture of the next strain was used.
Each treatment was carried out at 48 ° C. for 48 hours in a rotary shaking incubator. Strain used: Cryptococcus sp. S-2 strain FERM P-15155 Hansenula anomala J224 and Hansenula fabianii J640, fusion strain ANY-180 strain FERM P-15271

The analytical values of the artificial wastewater are as shown below. In addition, TOC is TOC-500 (Shimadzu Corporation),
BOD is BOD tester (Taiyo Scientific), CO
D was measured using JIS K0102, the total sugar was measured using a phenol-sulfuric acid method, and the viscosity was measured using a rotary B-type viscometer. The measurement results are shown in Table 2 below.

[0054]

[Table 2]

A 1 L jar fermenter was fitted with a ring race of 30 cm as a fixed floor, and 200 ml of artificial wastewater having the above composition was treated. As a strain, the fusion strain ANY
Although -180 was used, this strain was added to a fermenter, and was easily attached and fixed to a carrier (ring race) simply by stirring it. As a result of treatment for 20 days, the treated water (TOC removal rate 79%, BOD removal rate 8
2%). This treated water can be reduced to a BOD of 30 mg / L or less by performing an activated sludge treatment, and can be discharged to a river.

[0056]

Example 5: Wastewater treatment (2) 0.5% ammonium sulfate and 0.1% monopotassium phosphate were added to pectin-containing food factory wastewater having the composition shown in Table 3 below, and the S-2 strain and ANY were added. −
After inoculating each of the 180 strains, they were shaken at 27 ° C. for 48 hours.

[0057]

[Table 3]

The supernatant of the obtained yeast-treated water was analyzed, and the results are shown in Table 4 below. As is evident from the results, both strains exhibited excellent treatment effects.

[0059]

[Table 4]

[0060]

According to the present invention, a large amount of pectin-containing wastewater can be treated very efficiently despite the use of a small treatment tank. Therefore, the present invention provides an efficient pollution control technology on a factory scale. It plays an important role in various food processing factories.

[Brief description of the drawings]

FIG. 1 shows the adhesion rate of yeast to a fixed bed (ring race).

Claims (2)

[Claims] [Claim 1] Crytococcus sp. S-2 strain or Hansenu
A method for treating pectin-containing wastewater, characterized by using la anomala J224 and Hansenula fabianii J640, a fusion strain ANY-180. 2. A method for treating pectin-containing wastewater, comprising using the contact material to which the yeast according to claim 1 is adhered.