KR20000056878A - Production of Pichia guilliermondii Biomass Using Saline Medium and Process for Treating Waste by Employing Same - Google Patents

Abstract

PURPOSE: A preparation method of cell mass from new strain of Pichia guilliermondii A9 (KCTC8932P) growing at high salts condition is provided which produces single cell protein. The cell mass can be used to treat waste water from Kimchi plant. CONSTITUTION: Waste water from Kimchi plant, soil or fermented foods is mixed with sterile water and plated on YM agar plate containing 8% of NaCl at 30°C for 2-5 days. 70 colonies are selected and the growth rate in waste water from Kimchi plant is compared to that of salts tolerant yeast by measuring optical density at 540nm. The finally selected yeast is identified as Pichia guilliermondii. The optimal temperature for growth is 20-40°C and optimal pH is 2-11. The yeast can grow well at 18% of NaCl. Sigle cell protein is obtained by culturing in the medium containing 0.5-18% of NaCl at 20-4-°C. The crude protein content of cell mass reaches about 40% of total mass. Waste water treatment experiment shows that the BOD of waste water decreases by 90% in 24 hours and added organic materials from waste water is removed in 48 hours.

Description

Production of Pichia guilliermondii Biomass Using Saline Medium and Process for Treating Waste by Employing Same}

The present invention is a single-cell protein by culturing yeast Pichia cullimondi, especially the novel yeast strain Pichia guilliermondii A9, which can be grown in a salt-containing medium, especially in the cabbage pickling wastewater generated during kimchi production. The present invention relates to a method for producing microbial cells that can be used as a method for treating wastes such as Chinese cabbage pickled wastewater using the strain, and Pichia ceriamondi A9.

Kimchi is Korea's representative traditional fermented food, and industrialization started in earnest from the 1980s. Kimchi produced at the factory was surveyed at 140,000 tons per year, but it is increasing by 15 ~ 20% every year due to the increase in exports and domestic demand. Choi, TD, An Analysis on the Market Organization of Kimchi Industry, Doctoral Thesis, Seoul National University, Seoul, 1994). As a result of the industrialization of kimchi, researches for scientific production, processing, and preservation of kimchi have been actively conducted. However, there is no research on pickling wastewater generated as a by-product during pickling during kimchi production.

The amount and composition of pickled wastewater at the Kimchi plant has not been investigated and reported, but it is estimated to generate about 80,000 to 100,000 tons per year. Pickled wastewater contains sugar, which is extracted from cabbage during pickling, and has a high BOD and COD, and a very high salt content of about 7-10%. It is a serious problem and can also be seen as a wastewater that is worth recycling the organic components of pickled wastewater as a substrate for cell production and useful material production.

Until now, as a study on the production of cells using wastewater in food factories, studies have been reported in Korea to produce yeast using distillation wastewater from alcohol fermentation plants and at the same time obtain the wastewater treatment effect. Lee et al. (Lee, HY et al., Kor. J. Appl. Microbiol. Bioeng., 10, 95-100 (1982)), were used to culture yeast Torulopsis candida in alcoholic liquor wastewater from intercalated sweet potatoes. BOD reported a 39% reduction, and Kim et al. (Kim, et al., Kor. J. Appl. Microbiol. Biotechnol., 21, 281-287 (1993)) reported Candida lugosa in rice barley alcohol fermentation distillation wastewater. rugosa) showed higher cell production but lower COD reduction. Lee et al. (Lee, CY et al., Korean J. Biotechnol. Boeng., 8, 172 to 177 (1993)) also disclose alcohol fermentation distillation of Rhodotolula glutinis as a yeast suitable for single cell lipid production. It was reported that culturing in the waste solution resulted in 4 g / L single cell lipids and 88.7% BOD reduction. On the other hand, Cho et al. (Cho, SH et al., J. of Korean Agric. Chem. Soc., 32, 424-434 (1989)) investigated the characteristics of food plant wastewater and cultivated the microorganisms in alcohol factory wastewater. As a result, more than 90% reported BOD and COD reduction effect.

In foreign countries, BOD (Hang, YD et al., J. of Milk and Food Technol., 35, 432-435 (1972)) produces wastewater from the production of sauerkraut, a vegetable fermented food similar to kimchi. Is very high, pH is low, NaCl concentration is about 3-4% (w / v), and wastewater treatment by conventional methods is inadequate. Candida utilis and Aspergillus na A method of reducing BOD and producing cells was reported by culturing Aspergillus niger (Hang, YD, et al., Progress in Water Technol., 8, 381-384 (1976)). It has also been reported that cultivating Candida utilities in Sourkraut wastewater can reduce the BOD by more than 90% and produce cells that can be used for food or feed or as an enzyme source of invertase (Hang, YD). , Process Biochem., 12, 27-28 (1977). On the other hand, Welsh and Fal (Welsh, F. W. and R. R. Zall, Process Biochem., 19, 122-123 (1984)) reported a method for producing cells by culturing Candida utility in pickled aquatic wastewater. Japanese Patent Laid-Open No. 4-173089 discloses a bacterium belonging to the Rhizobiaceae family capable of decomposing phenol in the salt concentration range of 0.2-7% (w / w).

However, no pickling wastewater from the Kimchi plant with higher salt concentration has been investigated until now, and there is no research on cell production using the wastewater.

Accordingly, it is an object of the present invention to provide novel strains that can grow in an environment containing high concentrations of salt and can be used as single cell proteins.

Another object of the present invention is to provide a method for producing cells that can be used as a single-cell protein by culturing Pichia cerimondi in a salt-containing medium.

It is another object of the present invention to provide a method for treating waste containing high concentrations of salts using Pichia ceralimondi.

Figures 1a to 1c show the effect of temperature, pH and NaCl concentration on the growth of Pichia guilliermondii A9 when incubated in pickled cabbage wastewater, respectively.

Figure 2 shows the effect of aeration on the growth of Pichia ceriamondi A9 when incubated at 30 ℃ in pickled waste water (pH 4.0, 8% NaCl).

3A and 3B show the effects of ammonium sulfate and potassium phosphate on the growth of Pichia cerimondi A9 when incubated at 30 ° C. in Chinese cabbage pickled wastewater (pH 4.0, 8% NaCl).

Figure 4 shows the effect of micronutrients on the growth of Pichia ceriamondi A9 when incubated at 30 ℃ in pickled waste water (pH 4.0, 8% NaCl).

FIG. 5 shows the effect of zinc sulfate concentration on the growth of Pichia ceriamondi A9 when incubated at 30 ° C. in Chinese cabbage pickled wastewater (pH 4.0, 8% NaCl).

Figure 6 shows the BOD reduction of the Chinese cabbage pickling wastewater during the growth of Pichia ceriamondi A9 at 30 ℃.

Figure 7 shows the growth in pickled wastewater of Chinese cabbage pickles of Pichia ceriamondi A9 and other saline tolerant yeast.

In accordance with the above object, the present invention provides a novel yeast strain Pichia guilliermondii A9 capable of growing in an environment containing a high concentration of salt such as Chinese cabbage pickled waste water.

blood. Cullimondi A9 shows excellent growth at a temperature of about 20 ° C. to 42 ° C., and a pH of about 2 to 11, particularly at about 18% NaCl. In particular, the strain can be grown in a variety of medium containing high concentrations (7-12%) of NaCl, such as Chinese cabbage pickled waste water, and the strain is excellent in the use of organic matter contained in the Chinese cabbage pickled waste water, such that the strain is cultured in pickled cabbage waste water, etc. This allows for efficient production of single cell proteins and at the same time removing organics. Since the strain has a wide range of growth pH, growth temperature and growth salt concentration in the Chinese cabbage pickling wastewater, etc., it can grow without any additional treatment in any pickling wastewater having different ingredients according to different pickling processes. Moreover, the strains of the present invention are known blood when cultured in Chinese cabbage pickled wastewater. It shows higher growth than vilimondi strain, especially known blood at high temperature of 40 ° C. It is much industrially advantageous in terms of reducing the cooling cost against temperature rise due to fermentation heat generated during industrial microbial growth because it shows much better growth than vilimondi strains.

In the present invention also p. Blood containing Cerealmondy A9. It has been found that the C. mori species can be cultured in a medium containing a high concentration of salt to produce cells that can be used as single cell proteins. Specifically, blood. After inoculating Villimondi into a medium having an initial pH of 2 to 11 and a NaCl concentration of 0.5 to 18%, shaking culture at a temperature of 20 ° C to 42 ° C, cells were recovered from the culture by a method such as centrifugation. Single cell proteins can be obtained by washing and drying to obtain dry cells. blood. It is most preferable to cultivate the cultivar shaker at 30 ° C. in a medium having a pH of 4 and NaCl of 12% or less, and culturing by adjusting the initial pH of the culture to a low level of about 3 to 4 is advantageous because it can avoid contamination by bacteria. Aeration does not significantly affect growth, and 0.5 vvm is sufficient.

The cells cultured by the above method were recovered, dried and pulverized as in the general unicellular protein production process (Solomons, GL, CRC Cirtical Reviews in Biotechnology, 1 (1), 21-58 (1982)). It can be commercialized.

The cells produced according to the method of the present invention exhibit a high crude protein content of about 40% and can be usefully used as single cell proteins. Pichia ceriamondi includes Yoshida, A. and Hashimoto, K., Agric. Biol. Chem., 50, 2117-2120 (1986a) and Mauersberger et al. (1996). As reported by Candida maltosa, pp. 411-480, In Nonconventional Yeasts in Biotechnology, Wolf, K. ed.Springer-Verlag, Berlin, Germany), there is little or no pathogenicity. In addition, the risk of toxicity can be further reduced if the cells that have been sterilized through heat treatment by the drying process of the cells are used as single cell proteins.

Blood in the present invention. Any medium containing a high concentration of salt can be used as a medium for culturing vili mondi, and in particular, pickled wastewater and wastes from a kimchi manufacturing plant, pickled wastewater from pickled vegetable processing plants (pickles, pickled radish production plants, etc.) and Wastes containing high concentrations of salts can be used, such as waste, enteric processed wastewater and waste, aquatic salted processed wastewater and waste, salted meat processing (ham, etc.) wastewater and waste.

If necessary, any nutrient can be added to the medium to enhance the growth of the cells, and the addition of about 0.5 mM of zinc as a trace element enhances the growth of the cells. However, the effect of cell growth by the addition of 0.5 mM zinc is not so large, and may not be added.

In addition, in the present invention, a method for treating waste by using microorganisms, blood, which is salt-tolerant as a strain in the waste containing salt. There is provided a waste treatment method comprising inoculating Valimonty and culturing the strain to decompose organic matter. The culture method of the strain is the same as described above with respect to the cell production method, wastes that can be treated by the method of the present invention. Wastewater and wastes containing high concentrations of salts exemplified above in connection with cell production of Zurimondi.

In particular, when the kimchi pickled wastewater discharged from the kimchi manufacturing plant is introduced, the process of adding the cabbage trash juice to the kimchi pickled wastewater simultaneously treats the two wastes that are discharged from the kimchi manufacturing plant: Chinese cabbage pickled waste and Chinese cabbage waste. It is effective.

When the cabbage pickled wastewater is treated using the strain of the present invention, since the BOD is reduced by about 90% or more within 24 hours, the wastewater treatment method of the present invention is very simple and excellent in removing organic matters. Even when the cabbage waste juice was added to the cabbage pickling waste water, the reduced organic sugar content was completely consumed within 24 hours when added within 20% and within 48 hours when added within 20%.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following Examples are only for illustrating the present invention, and the scope of the present invention is not limited to these. In this embodiment, the pickled wastewater and waste in the kimchi manufacturing plant, pickled wastewater and waste from pickled vegetable processing plants (pickles, pickled radish production plants, etc.), enteric processed wastewater and waste, aquatic salted processed wastewater and waste, salted meat processing (ham, etc.) ) Chinese cabbage pickled wastewater was used.

Example 1 Characterization of Chinese Cabbage Pickling Wastewater

(1) Pickled wastewater

Chinese cabbage pickled wastewater samples were collected from four kimchi factories with different production scales in Gyeonggi-do.

In addition, in order to obtain a pickled wastewater with a uniform composition, the pickled wastewater was prepared in the laboratory as follows, similar to the process of pickling cabbage at the Kimchi factory. In other words, the cabbage purchased in the market was cut into 4 pieces and then marinated for 15 hours while maintaining 15 ° C in 10% brine. At this time, the cabbage weight and brine ratio were about 2: 3 (w: v), and the pickled water was filtered using filter paper (Whatman No. 2). In addition, in consideration of the reuse of the brine used for pickling once or twice in the Kimchi plant, some of the pickled cabbages were pickled twice under the same conditions.In this case, the ingredients of pickled wastewater and reused pickled wastewater The differences were also compared.

In order to investigate the growth characteristics of yeast in pickled wastewater, laboratory-made pickled wastewater was used. The pickled wastewater was made with NaCl so that the NaCl concentration was 8%, and the reducing sugar concentration was about 1.0 g / l. Standardized.

(2) Characterization of Pickled Wastewater

Using the cabbage pickled wastewater prepared in (1), the main characteristics such as biological oxygen demand (BOD), chemical oxygen demand (COD), NaCl concentration, pH, total sugar and reducing sugar content, and nitrogen content were analyzed. BOD and COD of pickled wastewater were measured according to standard methods (APHA, AWWA and WPCF (1992), Standard Methods for the Examination of Water and Waste Water, 18th ed., American Public Health Association, Washington, DC). Was measured by Mohr's method (AOAC (1995), Official Methods of Analysis, 16th ed., Association of Official Analytic Chemists, Washington, DC). Reducing sugar content was measured by the dinitrosalicyric acid (DNS) method; Miller, GL, Anal. Chem., 31, 426-428 (1959), and the Kjeldahl nitrogen content was measured by standard methods (APHA, AWWA). and WPCF (1992), Standard Methods for the Examination of Water and Waste Water, 18th ed., American Public Health Association, Washington, DC) and AOAC Act (AOAC (1995), Official Methods of Analysis, 16th ed., Association of Official Analytic Chemists, Washington, DC).

The results are shown in Table 1.

sample pH NaCl (%) Reducing Sugar (g / ℓ) BOD (mg / ℓ) COD (mg / l) Kjeldahl Nitrogen (mg / ℓ) A1 5.36 11.6 0.64 1,100 1,300 25 A2 4.91 9.5 1.00 1,200 1,800 28 A3 5.48 8.4 0.60 1,100 1,600 20 A4 5.80 7.0 0.50 1,100 1,300 25 B1 6.65 7.5 0.50 900 1,000 10 B2 6.55 7.4 0.51 900 1,100 10 B3 6.25 6.2 1.10 1,300 1,800 17 B4 6.70 6.2 0.75 1,100 1,300 16 B5 6.50 5.8 0.80 1,100 1,300 15 A1 to A4: Kimchi factory cabbage pickled waste water B1, B2: Lab-made cabbage pickled waste water pickled once B3 to B5: Lab-made cabbage pickled waste water pickled twice

Pickling wastewater (A1 ~ A4) of the Kimchi plant ranged from pH 5-6, and NaCl concentration was very high (7-12%). In addition, the reducing sugar concentration was about 0.5 to 1.0 g / l, BOD was about 1,100 to 1,200 mg / l, and the COD was 1,300 to 1,800 mg / l, and the total nitrogen content was 20 to 28 mg / l with Kjeldahl nitrogen. It was found to contain significant amounts of organic matter.

As such, the pickled wastewater of the Kimchi plant was found to have a difference in ingredients depending on the plant, which may be due to the fact that the ingredients of Chinese cabbage are different depending on the season, and the ratio of the brine and pickling conditions were not consistent.

In addition, the pH of the cabbage pickled wastewater prepared in the laboratory was about 6.2-6.7, NaCl concentration was around 6-7%, and it contained 0.5 g / l to 1.1 g / l reducing sugar. Pickled wastewater and its characteristics were similar. However, as a result of comparing the pickled wastewater to the two times, the pickled wastewater was found to increase the reducing sugar content by about 50 to 100%, and the NaCl concentration was lowered by about 1%.

The pickled wastewater at the Kimchi plant has a low organic matter content compared with the very high BOD of alcoholic liquor in the alcoholic distillation plant, as high as tens of thousands of mg / l. SH, et al., J. of Korean Agric. Chem. Soc., 32, 424-434 (1989)), were similar to wastewater from beer plants, fruit canning plants, and seafood processing plants. In particular, sauerkraut wastewater (Hang, YD, et al., J. of Milk and Food Technol., 35, 432-435 (1972)) has a high NaCl concentration of 3-4%, which can be treated by conventional wastewater treatment methods. In view of the fact that it is not suitable for the pickling wastewater of the Kimchi Plant, which has much higher NaCl concentration than this, it would be difficult to treat by conventional wastewater treatment methods.

Example 2: Isolation, Selection and Identification of Yeast Strains

As samples for strain isolation, foods such as pickled wastewater, soil and soy sauce, miso, and red pepper paste were collected. YM medium to which 8% NaCl was added as a strain separation medium was used, and the sample for bacterial separation was appropriately diluted and plated and incubated at 30 ° C. for 2 to 5 days. About 70 strains of yeast isolated from colonies formed on the plate medium were cultured in the standardized laboratory-made cabbage pickled wastewater described in Example 1, and the absorbance at 540 nm was measured to select strains superior to the known salt tolerant yeasts. . The selected saline tolerant yeast strains were incubated in YM broth for 2 days, and then inoculated with 1% in a standard laboratory pickled wastewater medium and incubated at 30 ° C. The strain named A9 showed the best growth. A9 was analyzed by Biolog Inc., Hayward, Calif., And identified as Pichia guilliermondii.

This yeast strain was named Pichia ceriamondi A9 and was deposited on February 18, 1999 to KCTC (KCTC) affiliated with the Biotechnology Research Institute of Korea Institute of Science and Technology as Accession No. KCTC 8932P.

Example 3: Growth Characteristics of Salt-Resistant Yeast Pichia Verriamondi A9 in Pickled Wastewater

(1) Effect of Optimal Culture Temperature, pH and NaCl Concentration

To investigate the optimum incubation temperature suitable for cell production, blood was added to standardized laboratory-made cabbage pickled wastewater adjusted to pH 6, NaCl concentration of 8%. After inoculating Zurimondi A9 and incubated for 24 hours while shaking at 180 rpm at each temperature of 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ and then determined the optimal culture temperature by measuring the degree of growth. As a result, as can be seen in Figure 1a, the degree of growth was high in the range of the culture temperature 25 ℃ to 35 ℃, 30 ℃ showing the best growth was determined as the optimum culture temperature.

In addition, in order to examine the optimum pH for cell production, blood was collected from standardized laboratory-made cabbage pickled wastewater with a NaCl concentration of 8% and a pH range of 2-8. The optimum pH was determined by incubating the Zerimondy A9 at 30 ° C. with shaking at 180 rpm for 24 hours and then measuring the growth. As a result, as can be seen in FIG. Zurimondi A9 showed a very wide growth range in the range of pH 3 to pH 8 with very little difference, and showed the highest growth at pH 4. As confirmed in Example 1, the pH of the kimchi factory pickled wastewater was 5 to 6, so the pickled wastewater can be used for cell production without pH adjustment, but the strain of the present invention has excellent growth even at low pH, so the initial pH of the pickled wastewater is When the culture is adjusted to a low level of 3 to 4 it can be seen that it is advantageous to avoid contamination by bacteria.

Meanwhile, blood was added to a standardized laboratory prepared cabbage pickled wastewater at pH 6 with a change in NaCl concentration of 6-15%. After inoculating Zerimondi A9 and incubating at 180 rpm at 30 ° C for 24 hours, the growth degree was measured to investigate the effect of salt concentration on the growth of yeast. As a result, as can be seen in FIG. Zerimondi A9 showed excellent growth without any influence up to 9% NaCl concentration, slightly lower growth from 10% and significantly inhibited at more than 12%. Therefore, blood. Zurimondi A9 is a saline tolerant yeast that can be grown with little inhibition in pickled wastewater with a NaCl concentration of 7-12%.

(2) optimum ventilation

To investigate the optimal aeration in fermenter culture for cell production, the standardized laboratory cabbage pickled wastewater with 8% NaCl and an initial pH of 4 was added to YM broth containing 5% NaCl. Blood incubated at 180 rpm for an hour. After inoculating 2% of Zurimondi A9, the fermentation capacity of the fermenter was set to 1 L, the stirring speed was fixed at 300 rpm, and the growth rate was adjusted to 0.5 vvm, 1.0 vvm, 2.0 vvm, and the growth was compared.

As a result, the growth of yeast is almost no difference depending on the amount of aeration in the growth rate and degree, it was found that even with a ventilation amount of 0.5 vvm (Fig. 2).

(3) Effect of Addition of Ammonium Salt, Phosphate and Trace Elements

Pickled waste water has a lower nutrient content than the other food factories' waste water, which is required for growth of cells including carbon sources. A small amount of ammonium salt and phosphate was added to the culture of Zurimondi A9 to investigate the effect of increasing the cell production. Specifically, after adding (NH ₄ ) ₂ SO ₄ and K ₂ HPO ₄ to 0%, 0.2% and 0.5%, respectively, in a standard laboratory cabbage pickled wastewater with pH 4.0 and 8% NaCl concentration, p. Inoculation with Zurimondi A9 and shaking growth at 180 ℃ at 30 ℃ for 24 hours to measure the growth of the cell production was investigated by the addition of ammonium salt and phosphate. As a result, as can be seen in Figures 3a and 3b, (NH ₄ ) ₂ SO ₄ and K ₂ HPO ₄ did not show a significant difference compared to the control even when added up to 0.5%.

Meanwhile, copper (CuSO) as a trace element₄), Manganese (MnSO₄), Magnesium (MgSO₄), Calcium (CaSO₄), Iron (FeSO₄), Zinc (ZnSO₄) And then added to the pickled wastewater at a concentration of 0.5 mM. In Vilermondi A9 was incubated for 24 hours at 30 ℃ to investigate the effect of increasing the cell production. The results are shown in Figure 4, where 1 is a control group without addition of trace elements and 2 to 7 CuSO as a trace element.₄, MnSO₄, MgSO₄, CaSO₄, FeSO₄And ZnSO₄It is the test group which added each. As can be seen in Figure 4, ZnSO₄Showed a slight increase, but there was no effect. ZnSO₄In order to determine the optimal concentration of addition, ZnSO₄Blood in pickled wastewater added at 0-5 mM concentration. As a result of culturing Zerimondi A9, the growth was the highest at 0.5 mM as shown in FIG. However, 0.5 mM ZnSO₄The effect of cell growth by the addition of is not so great that it is not necessary to add.

Example 4: BOD Removal Effect

Blood to the standardized laboratory prepared cabbage pickled wastewater described in Example 1. The change of BOD in pickled wastewater was measured when culturing Zurimondi A9 to investigate the effect of organic matter removal by yeast culture treatment.

Blood in pickled wastewater with an adjusted NaCl concentration of 8% and an initial pH of 4. The effect of BOD reduction was investigated by inoculating 1% of ZeriMondi A9 and shaking culture at 180 rpm at 30 ° C. As a result, the pickled wastewater, which had an initial BOD of 1,200 mg / l, decreased to 120 mg / l of BOD within 24 hours.

As a result of removing more than 90% of the BOD in pickled wastewater, Lee, HY, et al., Kor, who reduced 38.9% of BOD in Tolulopsis candida culture in the interlaced sweet potato raw liquor liquor J. Appl.Microbiol.Bioeng., 10, 95-100 (1982)), much higher than that of 94% BOD by culturing Aspergillus fumigatus in brewery wastewater. Results of Cho et al. (Cho, SH, et al., J. of Korean Agric. Chem. Soc., 32, 424-434 (1989)) and cultured Candida utility in Sourkraut waste saline to obtain more than 90% BOD. This is similar to the result of the removed row (Hang, YD, et al., Progress in Water Technol., 8, 381-384 (1976)).

Example 5: Component Analysis of Cells

Blood in pickled wastewater adjusted to 8% NaCl, initial pH 4. Inoculated with ZeriMondi A9 and incubated for 24 hours while shaking at 180 rpm at 30 ℃. The culture solution was centrifuged to recover the cells, washed twice with distilled water and lyophilized to obtain dried cells, and the protein, lipid, carbohydrate and ash content were investigated. Crude protein content of dry cells was determined by the micro-Kjeldahl method (APHA, AWWA and WPCF (1992), Standard methods for the examination of water and waste water, 18th ed., American Public Health Association, Washington, DC). The carbohydrate content was measured by the phenol-sulfuric acid method (Dubois, M., et al., Anal. Chem., 28, 350-356 (1956)), and the crude fat content was the method of Bligh, EG and WJ. Dyer, Can. J. Biochem. Physiol., 37, 911-917 (1959)). The ash content was placed in a crucible of known quantity, weighed, placed in an electric furnace, burned for several hours at 500 ° C., and then cooled to room temperature after being transferred to a desiccator. The ash content was calculated by repeating until the dosage was reached.

The results of the analysis of the components of the yeast cells are shown in Table 2, and the protein content of the yeast cells cultured in pickled wastewater was about 6% less than that of the culture medium under the same conditions in YM broth, a nutrient medium, and lipids The content was about 6% and the ash content was the same.

Furtherance(%) Waste saline YM Broth Crude protein 40.0 46.1 Landscape geology 11.7 4.4 carbohydrate 45.4 47.4 Ash 5.9 5.9

This result showed that 48% of Tolulopsis Candida (Lee, HY, et al., Kor. J. Appl. Microbiol. Bioeng., 10, 95-100 (1982)) was cultured in interlaced sweet potato alcoholic liquor. Compared to that of Saccharomyces fragilis, 30-40.25% (El-Samragy, TA, et al., Process Biocem. 23, 28-30) (1988), which is much higher than Candida guilliermondii, which is 26.7% when cultured in hydrocarbon (Sabry, SA, et al., Biomed. Lett. 46, 615-618 (1991)). appear.

Example 6: Chinese cabbage trash juice addition effect

During the process of trimming Chinese cabbage, which is a kimchi raw material, more than 10% of Chinese cabbage waste is generated, and since the cabbage contains 1.6-2.6% of sugars that can be easily used by microorganisms such as yeast (Shim, ST, et al., Korean J. Food Sci. Technol., 22, 278-284 (1990); and Yu, HG, et al., Korean J. Food Sci. Technol., 24, 107-110 (1992)) Is an excellent organic waste resource with very high value as a microbial medium. Therefore, in the present embodiment, a method of increasing the growth of yeast by adding cabbage waste, which is difficult to process due to a large amount of kimchi production during kimchi production, was added to the pickled wastewater.

The cabbage waste generated during the process of trimming cabbage during kimchi production was collected and crushed with a blender (Waring blender), and filtered to obtain cabbage waste juice. Juices of Chinese cabbage waste were added to standardized laboratory cabbage pickled wastewater adjusted to 8% NaCl and initial pH 4 at 5%, 10%, 20% and 30%, respectively. Cell inoculum was measured by inoculating Varianmondi A9 and shaking culture at 180 ° C at 30 ° C. Cell production was measured by taking a certain amount of the culture medium, centrifuged (4,000 rpm, 15 minutes) to obtain the cells, washed with distilled water and centrifuged again and dried until the cell reaches a constant in 105 ℃ dryer.

The results are shown in Table 3. As can be seen in Table 3, when the Chinese cabbage waste was not added, the maximum cell production was 0.69 g / L, but the dry cell weight was 1.87 when the cabbage waste juice was added. g / l, 20% added 2.75 g / l, 30% added 3.4 g / l, 2.7 times, 4.0 times and 4.9 times increased, respectively, compared to the case without addition, the cell production is greater than the use of pickled waste water only Increased. At this time, reducing sugar was completely consumed within 24 hours when 5%, 10% and 20% were added, and within 48 hours when 30% was added.

Chinese cabbage juice Reducing Sugar (g / ℓ) Dry cell mass (g / ℓ) Yield (g / g reducing sugar) 0 % 1.08 0.69 0.64 5% 2.15 1.35 0.63 10% 3.06 1.87 0.61 20% 4.87 2.75 0.57 30% 6.80 3.40 0.50

Therefore, by adding a large amount of cabbage waste, which is a problem in the Kimchi factory, added to the pickled waste water of the Kimchi factory, the two types of waste can be effectively treated simultaneously.

Example 7: Growth of Pichia verriamondi A9 and some salt tolerant yeasts in Chinese cabbage pickled wastewater

Strain P of the Invention. The effect of BOD removal in pickled wastewater of Pichia verriamondi ATCC 6260, which is known to be the same species as VeriMondi A9, was investigated by the method described in Example 4, and strain P of the present invention. Comparison was made with Zerimondi A9. blood. The removal rate of BOD of pickled wastewater was 86%, which was weaker than A9, which was 90%.

Meanwhile, the blood of the present invention. Zerimondi A9, blood of this kind. Examples using Zulmondi ATCC 6260 and flame resistant yeast Candida halonitratophila IFO 1595, Zygosaccharomyces rouxii KFRI 204, and Candida versatilis IFO 1228 The growth of the cabbage pickled wastewater was investigated by the method of 4. As a result, as can be seen in FIG. The highest growth level of Zerimondy ATCC 6260 was found to be 1.3 when absorbed at 540 nm when incubated at 30 ° C. It was 90% of the maximum growth level of ZeriMondi A9. In particular, as the incubation temperature increases, the difference in growth degree is increased. After 24 hours of incubation with Zerimondi ATCC 6260, the degree of growth was 21% of that of A9. That is, in the case of 40 ° C. culture, strain A9 of the present invention was about 4.6 times better than ATCC 6260. Strain P of the Invention. The excellent growth characteristics of Zurimondi A9 are very industrially advantageous in that the cooling cost of the temperature rise due to the fermentation heat generated during the culture of industrial microorganisms can be reduced. Also, strain P. of the present invention. Varianmondi A9 was much better in the Chinese cabbage pickled waste compared to other salt resistant strains.

However, blood. Zerimondi ATCC 6260 also shows a very good growth and BOD removal effect in pickled cabbage wastewater, strain of the present invention. Strains of the same species as Zurimondi A9 were found to be suitable for the production of cells in Chinese cabbage pickled wastewater.

The strain of the present invention is a salt-tolerant yeast capable of growing even in an environment containing 18% NaCl, which can be grown in pickled wastewater containing NaCl of high concentration (7-12%), and has the ability to use organic matter contained in the pickled wastewater. Excellent by culturing the strain in the Chinese cabbage pickled waste water can effectively produce a single-cell protein while at the same time remove the organic matter. Since the strain A9 is a strain having a wide range of growth pH, growth temperature and growth salt concentration in the Chinese cabbage pickling wastewater, it is unnecessary to perform additional treatment for the difference in pickling wastewater components according to different pickling processes. Also, p. By culturing Curiarmond in wastes containing salt, it is possible to produce cells and process wastes. As a specific example, the method of the present invention can be used for pickling wastewater and waste in kimchi manufacturing plant, pickled vegetable processing plant (pickles, radish production plant, etc.) (Ham, etc.) can be used for the treatment of waste water and waste. In particular, in the case of cabbage pickled wastewater, the cabbage waste juice is added to the pickled wastewater to avoid blood. Cultivation of vitreous mondy greatly increases cell production, and can effectively treat two kinds of wastes from the Kimchi plant at the same time.

Claims (13)

In the method of treating wastes using microorganisms, waste-containing wastes are inoculated with a salt-tolerant Pichia guilliermondii as a strain, and the strain is cultured to decompose organic matter. Way. The method of claim 1, The waste is selected from the group consisting of pickled wastewater and waste of kimchi manufacturing plant, pickled wastewater and waste of pickled vegetable processing plant, enteric processing wastewater and waste, aquatic salt processing wastewater and waste, and salted meat processing wastewater and waste. Waste disposal method. The method of claim 2, Said waste is Chinese cabbage pickled wastewater, and the Chinese cabbage waste juice is added thereto. The method according to any one of claims 1 to 3, Waste treatment method characterized in that the strain is Pichia ceriamondi A9 (Accession Number: KCTC 8932P). The method of claim 4, wherein PH of the strain is 2 to 11, NaCl concentration of 0.5 to 18%, the temperature is 20 to 42 ℃ waste treatment method characterized in that the culture. The method of claim 5, Waste treatment method characterized in that the pH is 3 to 6. (a) inoculating a salt-tolerant medium with salt-resistant Pichia ceriamondi as a strain, (b) culturing the strain, (c) recovering the cells from the culture, and (d) washing and drying the recovered cells Cell production method comprising a. The method of claim 7, wherein In the group consisting of the pickling wastewater and waste of the kimchi manufacturing plant, pickled wastewater and waste of the pickling vegetable processing plant, enteric processing wastewater and waste, aquatic salt processing wastewater and waste, and salted meat processing wastewater and waste of the step (a) Cell production method, characterized in that selected. The method of claim 8, The medium is a Chinese cabbage pickling waste water, characterized in that the cabbage waste juice is added to the cell production method. The method according to any one of claims 7 to 9, Cell strain production method characterized in that the strain is Pichia ceriamondi A9 (Accession Number: KCTC 8932P). The method of claim 10, PH of the strain is 2 to 11, NaCl concentration of 0.5 to 18%, the temperature of the cell production method, characterized in that the culture at 20 ℃ to 42 ℃. The method of claim 11, Cell production method characterized in that the pH is 3 to 6. Pichia ceriamondi A9 (Accession No .: KCTC 8932P) capable of growing in a high concentration of salt environment.