JP5219069B2 - Water treatment method and water treatment material - Google Patents

Description

The present invention relates to a water treatment method and a water treatment material for decomposing oil from waste oil containing oil discharged from the food service industry, food industry, and the like using photosynthetic bacteria.

  Many kitchen oils such as vegetable oils and animal oils are used in restaurant kitchens and food processing factories. A lot of oil-containing wastewater is generated from such facilities, but if this wastewater is poured into sewage as it is, environmental degradation such as bad odor and pollution will be caused. For this reason, wastewater is treated to decompose oil and then drained into sewage. In recent years, high-temperature wastewater at 45 ° C. or more tends to increase due to the spread of dishwashers and kitchen wastewater drained from restaurants and the like.

  As wastewater treatment, a wastewater treatment method for treating oil in wastewater using microorganisms is conventionally known, and various microorganisms are used.

  Among them, it is known that photosynthetic bacteria can decompose many kinds of organic substances relatively quickly and have high cell safety. In Non-Patent Document 1, Rhodobacter sphaeroides S, Rhodobacter sphaeroides NR-3, which is a Rhodobacter genus having a broad substrate utilization and a relatively high degree of proliferation, is fixed to sodium alginate or agar to remove oil in wastewater. Is going. Since Rhodobacter genus has low heat resistance and 20-35 ° C is inhabiting conditions, photosynthetic bacteria are used at a wastewater temperature of 30 ° C.

Moreover, in patent document 1, the microorganisms of the Burkholderia genus with high heat resistance are used, and the oil content in wastewater is decomposed | disassembled by making it contact under the waste water temperature about 50 degreeC high temperature.
"Treatment of oil-containing sewage wastewater using immobilized photosynthetic bacteria: World J Microbiol, Biotechnol. Vol. 21, No. 8/9, Page 1385-1391, issued December 2005" JP-A-9-85283

  As typified by Non-Patent Document 1, the Rhodobacter genus conventionally used has low heat resistance and is all killed at 45 ° C. or higher. For this reason, photosynthetic bacteria represented by the genus Rhobacter cannot treat discharged high-temperature wastewater at 45 ° C. or higher.

  In addition, since Rhodobacter genus has low heat resistance, it is necessary to cool high-temperature wastewater to about 30 ° C in advance, which requires equipment such as a cooling device, which increases costs and requires cooling time. There is a problem that the time becomes longer.

  In Patent Document 1, treatment can be performed even at a wastewater temperature of 50 ° C. or higher, but Burkholderia microorganisms are not photosynthetic bacteria, so there is a problem with the safety of the microorganisms themselves. Therefore, microorganisms may flow with the treated wastewater, which may cause environmental problems. In addition, microorganisms cannot be recycled for fertilizer or feed.

  The present invention has been made in view of the above-mentioned matters, and uses oil-synthetic bacteria having heat resistance, and decomposes oil components at high temperatures even in high-temperature oil-containing wastewater of 45 ° C. or higher discharged from kitchens and the like. And it aims at providing the processing method of the oil containing wastewater which does not impair safety even if it flows into sewage.

  The present invention is characterized by decomposing oil in oil-containing wastewater using Rhodobacter sphaeroides NAT (reception number: FERM AP-21486).

  Moreover, this invention uses the said oil-containing waste water of 45 to 55 degreeC, It is characterized by the above-mentioned.

  Furthermore, the present invention is characterized in that lipase is released from the Rhodobacter sphaeroides NAT into the oil-containing wastewater to decompose the oil.

  Furthermore, the present invention is characterized in that the Rhodobacter sphaeroides NAT is immobilized on an alginate and used.

  According to the present invention, Rhodobacter sphaeroides NAT has heat resistance and can inhabit even at 45 ° C. or higher, so that oil can be decomposed even in high temperature wastewater at 45 ° C. to 55 ° C.

  Moreover, according to this invention, since waste water can be processed with high temperature as mentioned above, it is not necessary to cool waste water in advance. For this reason, facilities, such as a cooling device, become unnecessary and an installation cost can be made cheap.

  Furthermore, according to the present invention, since the cooling time is not required as described above, the processing time can be shortened.

  Furthermore, according to the present invention, Rhodobacter sphaeroides NAT releases lipase into the wastewater, so that not only the oil contacting the surface but also the oil content of the whole wastewater is efficiently decomposed, so that the processing time is shortened. Have.

  Furthermore, according to the present invention, Rhodobacter sphaeroides NAT can be used by immobilizing it on alginate, so that each bacterium can be dispersed and arranged, and the surface area increases, so that the oil can be efficiently decomposed. Since the treatment time can be further shortened and the bacteria can be prevented from flowing, it can be used for a long time.

  Furthermore, according to the present invention, Rhodobacter sphaeroides NAT is a photosynthetic bacterium and a highly safe bacterium, so that even if it flows into sewage, there is no problem in safety and there is an advantage that there is no risk of causing environmental problems. .

  Furthermore, according to the present invention, Rhodobacter sphaeroides NAT can also remove COD and phosphorus at the same time as the decomposition of oil.

  Furthermore, according to the present invention, the oil content can be decomposed only by interposing the NAT strain in the waste water, so that there is an advantage that the waste water treatment can be easily performed only by interposing the net or the like containing the NAT strain in the existing septic tank. There is no need for new capital investment.

  The present invention uses Rhodobacter sphaeroides NAT (reception number: FERM AP-21486) (hereinafter referred to as NAT strain), which is a newly isolated photosynthetic bacterium, and a treatment capable of decomposing oil by simply adding this NAT strain to oil-containing wastewater. Is the method. NAT strains inhabit at 45 ° C or higher and decompose oil, so even high-temperature oil-containing wastewater that comes out of a restaurant kitchen can be treated as it is without being cooled.

  First, the NAT strain used in the present invention will be described. The NAT strain is a novel photosynthetic bacterium isolated from soil in Kasetsart University (Bangkok, Thailand). It was verified that this photosynthetic bacterium was a novel bacterium.

  The bacteria isolated from the soil were cultured for about one week at about 5 klux illuminance (tungsten lamp) in a static culture with a liquid paraffin overlay. Plate culture was performed under the same conditions from the culture medium grown in red, and this was repeated. Among them, the strain having the highest growth rate was screened to obtain a NAT strain. This was subjected to preculture.

  As a screening and preculture medium, glutamate maleate (GM) medium was used. The pre-culture was performed by static culture as described above, and was carried out for 2 days at 40 ° C. under an illuminance of about 5 klux using a 1.5-liter culture bottle.

  Since the cells after the pre-culture contained many remaining medium components, the cells were washed. The washing method is to collect cells grown in GM medium by centrifugation (10,000 × g (5,000 rpm), 15 min), suspend in deionized water once sterilized, and then centrifuge again under the same conditions. It was. The washed bacterial cells obtained here were adjusted to OD666≈10 with pure water and used.

  The washed cells were identified by 16S rDNA analysis. DNA extraction was performed by Insta Matrix (BIO RAD, CA, USA), and MicroSeq 500 16S rDNA Bacterial Identification PCR Kit (Applied Biosystems, California, USA) and MicroSeq 500 16S rDNA Bc. Used to perform PCR and sequencing. The results are shown in Table 1.

表１に示すように、国際塩基配列データベース検索（Ｇｅｎｂａｎｋ／ＤＤＢＪ／ＥＭＢＬ）により、Ｒｈｏｄｏｂａｃｔｅｒ ｓｐｈａｅｒｏｉｄｅｓ ＡＴＣＣ１７０２９株や２．４．１株、野生株であるＩＦＯ１２２０３株などの菌株と本ＮＡＴ株のＤＮＡは１００％の相同率を示している。この結果から、本ＮＡＴ株はＲｈｏｄｏｂａｃｔｅｒ ｓｐｈａｅｒｏｉｄｅｓ属に属する光合成細菌であることを同定した。

As shown in Table 1, DNA of the present NAT strain and strains such as Rhobacter sphaeroides ATCC17029 strain, 2.4.1 strain, and wild-type IFO12203 strain were determined by international base sequence database search (Genbank / DDBJ / EMBL). % Homology rate. From this result, this NAT strain was identified as a photosynthetic bacterium belonging to the genus Rhodobacter sphaeroides.

  Next, the heat resistance of the NAT strain was examined. A 500 ml shake flask was charged with 200 ml of GM medium, and the above washed cells were inoculated with 1% (w / v) for examination. Cultivation was performed at 30 ° C. to 55 ° C. under aerobic dark conditions using a shake incubator TAITEC bio-shaker BR-40LF (130 rpm, 7 cm amplitide) (Shimadzu Co. Ltd. Japan). As a reference example, Rhodobacter sphaeroides S (hereinafter referred to as S strain) was cultured at 45 ° C.

  The result is shown in FIG. It can be seen that the NAT strain inhabited and grew in all temperature ranges over 30 ° C to 55 ° C. At 30 ° C., the growth of red cells with a dry cell mass of about 3 g / l was observed after 48 hours of culture. Also, under high temperature conditions of 45 to 55 ° C., the growth was less than that at 30 ° C., but it was inhabited in red to pink, and heat resistant growth of the NAT strain was confirmed. From this, it can be seen that it can be used for wastewater treatment in a wide temperature range of 30 ° C to 55 ° C. In particular, it can be found in high temperature wastewater of 45 ° C to 55 ° C and can decompose oil.

  On the other hand, the S strain has been killed immediately. Thus, it is known that the photosynthetic bacteria such as S strains known so far are generally low in heat resistance, cannot live in high temperature wastewater at 45 ° C. or higher, and cannot decompose oil. .

  Then, using this NAT strain, the oil in the liquid was decomposed in a pure culture system. 200 ml of oil-containing artificial sewage was dispensed into a 500 ml shake flask (Sakaguchi flask), and 10% (w / v) of the aforementioned washed cells were inoculated. The temperature was 10 days under the condition of 45 ° C., and the oil and lipase activity were analyzed. The oil content is n-Hex. Analysis was performed based on JIS K 0102 as extract. The treated sewage was centrifuged (5000 × g, 10 mim), and the resulting supernatant was analyzed as the extracellular lipase activity by an emulsifier-free method (JIS K 0601). As a reference example, the same procedure was carried out for a sample to which no NAT strain was added (control experiment).

The oil-containing artificial sewage was used by adding oil to synthetic artificial sewage mainly composed of glucose and peptone. The oil used was a salad oil for cooking (manufactured by Nissin Foods Co., Ltd.), which is a mixed oil of soybean oil and rapeseed oil. The fatty acid composition of the oil is classified as olein / linoleic acid, and is composed of unsaturated fatty acids having 22 to 28 carbon atoms. The composition of the oil-containing artificial sewage (each g / l) was: D-glucose: 4.0, K ₂ HPO ₄ : 0.015, KH ₂ PO ₄ : 0.006, Na ₂ HPO ₄ · 12H ₂ O: 0 0.030, NH ₄ Cl: 0.117, MgSO ₄ .7H ₂ O: 0.056, CaCl ₂ : 0.010, pettone: 0.150, KNO ₃ : 0.069, Oil (salad oil): 3. .

  FIG. 2 is a graph showing changes over time in lipase activity and oil content. 2A shows the oil content, and FIG. 2B shows the lipase activity.

  Referring to FIG. 2 (A), in a control experiment (Control) in which no NAT strain was added, about 10% of the oil content decreased in 8 days. This is considered due to thermal denaturation or decomposition at 45 ° C.

  On the other hand, in the plant inoculated with the NAT strain, the oil content of about 66% contained in the oil-containing artificial sewage decreased on the 8th. It was confirmed that the oil can be decomposed and removed by the NAT strain even at a high temperature of 45 ° C.

  In addition, as shown in FIG. 2B, in the control experiment (Control), there is no lipase activity. However, when the NAT strain was added, the lipase activity increased to about 1.8 units / ml one day later. It can be confirmed that the strain releases lipase, an oil-degrading enzyme, in sewage. Thus, it can be seen that the oil content is not decomposed only on the surface of the NAT strain, but the lipase is diffused into the sewage to efficiently promote the decomposition of the oil in the entire sewage. It can be confirmed that the lipase activity is not deactivated even at a high temperature of 45 ° C., and oil removal of the high temperature wastewater can be efficiently performed. No examples of lipase release have been reported so far in photosynthetic bacteria, indicating the specific properties of NAT strains. In addition, although the lipase activity is low after the 2nd day, it is considered that the lipase was subjected to the decomposition of the oil and the activity was lowered.

  So far, no photosynthetic bacteria such as Rhodobacter sphaeroides have been reported on heat resistance, lipase activity, and oil resolution. As described above, this NAT strain is different from the conventional Rhodobacter sphaeroides strain because it has heat resistance that can live even under high temperature conditions of 45 ° C. or higher, exhibits lipase activity, and has oil resolution. It was confirmed to be a novel photosynthetic bacterium.

  And it is known that photosynthetic bacteria are highly safe bacteria. Therefore, the NAT strain, which is a photosynthetic bacterium, is harmless. For example, even if it flows into sewage during wastewater treatment, environmental problems do not occur, and the safety is very high. It can also be used as fish and animal data after wastewater treatment and can be reused.

  Next, the NAT strain was immobilized on alginate, and the above-described treatment of oil-containing artificial sewage was performed.

100 ml of the aforementioned washed cells and 4% sodium alginate solution were mixed and added dropwise to an aqueous CaCl ₂ solution (6.62 g / l). The beaded NAT strain was immobilized on calcium alginate by Na / Ca exchange (hereinafter alginate beads). The alginate beads were adjusted to have a diameter of about 1 cm. Alginate beads are immobilized at about 1.36 g per cell, and about 4.25 mg / cell in dry cell mass. Moreover, in the control experiment of the open experimental system, sterile alginate beads prepared without mixing bacterial cells were prepared and used for the experiment.

  In the batch treatment experiment of oil-containing artificial sewage, aerobic treatment in a wastewater treatment facility is considered, and the above-mentioned alginate beads are placed in a 5-liter culture apparatus MDL (BE Marubishi. Co. Ltd) with 0, 5, Each of 15, 25, 50, and 75 g / l was introduced into the treatment tank, and the optimum input amount was examined. Of these, 0 g was obtained when only aeration was performed in the treatment tank as a control experiment (Control). The experiment was performed under the conditions of 45 ° C. under ventilation of 1 vvm, the rotation speed was set to 100 rpm, and the oil was dispersed as much as possible. In this experiment, an air filter was not attached, and the treatment was performed for 4 days (96 hours) in an open system. The pH was controlled at 7.0 ± 0.1. If pH control is not performed, it will shift to acidity immediately by the decomposition of artificial sewage, and the growth of bacteria will stop, making it difficult to remove oil.

表２はアルギン酸ビーズを用いて廃水処理した結果である。アルギン酸ビーズを投入した全ての処理槽内において、ビーズによる油除去効果が確認され、本アルギン酸ビーズが油含有高温廃水に有効であることがわかる。特に５０ｇのアルギン酸ビーズを投入した場合では、４５℃の高温条件下においても、油除去およびＣＯＤ除去が同時に行われ、４日で油が約９２％、ＣＯＤが約９６％除去できた。なお、ＣＯＤ除去率は、対照実験（Ｃｏｎｔｒｏｌ）の処理開始時の３６００ｍｇ／ｌをベースに算出している。

Table 2 shows the results of wastewater treatment using alginate beads. The oil removal effect by the beads was confirmed in all treatment tanks charged with the alginate beads, and it can be seen that the present alginate beads are effective for oil-containing high-temperature wastewater. In particular, when 50 g of alginate beads were added, oil removal and COD removal were performed simultaneously even under high temperature conditions of 45 ° C., and about 92% of oil and about 96% of COD could be removed in 4 days. The COD removal rate is calculated based on 3600 mg / l at the start of the control experiment (Control).

  This result is rapidly removed even when compared with the removal results (removal rate: oil 65%, COD 60%) in 4 days under the condition of 30 ° C. reported in Non-Patent Document 1. In addition, the present alginic acid beads have a phosphorus removal effect and are useful for the treatment of oil-containing high-temperature wastewater.

  In the control experiment (0 g), the oil removal rate is about 46%, and COD is also about 39% removed. This is considered to have been removed by contamination with various bacteria because the pH was controlled in an open experimental system. However, even when compared with the control experiment, the treatment effect of the NAT strain in which rapid water purification was observed is clear. In addition, the oil removal rate of 50 g of the immobilized cells was calculated to be 688.5 mg oil / l / day, which was 285.8 mg oil / l / reported in Comparative Patent Document 1 treated at 30 ° C. Compared with, the removal rate is twice or more, and it can be seen that the oil can be decomposed quickly.

  In addition, since the calcium alginate of this alginate bead may adsorb | suck oil, the oil adsorption amount of the alginate bead was measured. Sterile alginate beads were used in the experiment, calculated to be equivalent to the waste water contact surface area of 50 g / l of alginate beads, and put into oil-containing artificial sewage. The experimental conditions were aseptically performed at 45 ° C. for 4 days as in the previous pure culture experiment. After shaking, the beads were collected with a simple net, and n-Hex immersed in hexane for about 30 minutes was detected. The experiment was performed three times and the average was calculated.

  As a result, about 93% of the oil content was detected in the shake flask and about 3% of the total amount from the surface of the sterile alginate beads, and a small amount of oil adsorption by calcium alginate was confirmed. The remaining 4% is thought to be due to thermal denaturation or decomposition. This oil adsorption amount is about 3%, which is almost negligible, so that the high oil resolution of the NAT strain could be demonstrated.

  Subsequently, 50 g / l of alginate beads, which showed the best results in Example 2, were filled in the treatment tank, and the practical treatment temperature range was examined under the temperature conditions of 45, 50, 55 and 60 ° C. In this experiment, as a target experiment, aseptic alginate beads were placed in a treatment tank and treated under the same conditions. The results are shown in Table 3.

４５、５０、５５℃の温度条件下において、それぞれ約９１％、８０％、７６％の油が９６時間で除去され、ビーズ投入による油除去効果が顕著に示されている。４５℃の条件では、先の実験と同等の結果が得られている。また、ＣＯＤにおいても、それぞれ９３％、８７％、８１％の高い除去率を示した。

Under the temperature conditions of 45, 50, and 55 ° C., about 91%, 80%, and 76% of oil were removed in 96 hours, respectively, and the oil removing effect by introducing beads was remarkably shown. Under the condition of 45 ° C., a result equivalent to the previous experiment is obtained. Also, COD showed high removal rates of 93%, 87%, and 81%, respectively.

  Accordingly, it was confirmed that oil removal at 45 ° C. to 50 ° C. was sufficiently possible and oil decomposition by NAT strain was possible even at 55 ° C. In addition, under the condition of 60 ° C., the effects of oil decomposition, COD removal and phosphorus removal are almost equal as compared with the control experiment, and the NAT strain does not act much. It is considered that at 60 ° C., the NAT strain was killed and the activity was reduced accordingly. For this reason, it is preferable to use with respect to waste water of 55 degrees C or less.

  FIG. 3 shows an example of an apparatus for treating oil-containing wastewater using a NAT strain. The processing device 11 is connected between a drain pipe and a sewer pipe from a restaurant kitchen. A basket-like net 21 is provided in the processing apparatus 11, and alginate beads 22 on which NAT strains are immobilized are dispersedly placed in the net 21.

  Oil-containing wastewater 23 flows into the treatment device 11 from the wastewater inlet 11 through a water distribution pipe from the kitchen. First, the oil-containing wastewater 23 flows into the inflow tank 12, and then proceeds to the anaerobic tank 13 and the aeration tank 14 while the flow is suppressed by the dam plate 16. A net 21 is provided in the anaerobic tank 13 and the aeration tank 14. Since the net 21 contains the alginate beads 22 on which the NAT strain is immobilized, the oil content of the oil-containing wastewater 23 is decomposed and removed by the NAT strain. The decomposed oil accumulates in the sedimentation tank as sludge. Then, the treated waste water flows into the sewage through the discharge port 17.

  Since the NAT strain has heat resistance as described above, even high-temperature waste water at 45 ° C. or higher can be treated as it is without cooling. Since the NAT strain releases lipase into the oil-containing wastewater 23, the oil content can be removed not only on the surface of the NAT strain but also in the entire processing apparatus 11.

  In addition, the NAT strain is immobilized and used as alginate beads 22, and each NAT strain is in a dispersed form. Therefore, the surface area is increased, and the oil can be decomposed and removed efficiently and rapidly. Further, as described above, the NAT strain also removes COD and phosphorus simultaneously with the decomposition of the oil.

  Further, the present processing apparatus 11 is provided with an aeration tank 14, and further COD removal is promoted by aeration.

  Note that the NAT strain is a photosynthetic bacterium, but has activity and can decompose oil even in places where it is not exposed to light. Therefore, the septic tank is usually installed in a place where light is not exposed, but the oil-containing wastewater can be treated even in such a place.

  In the present invention, since Rhodobacter sphaeroides NAT having heat resistance is used, oil can be decomposed as it is even in high temperature waste water at 45 ° C. or higher. For this reason, it can be widely used for the treatment of high temperature wastewater discharged from facilities that use a lot of oil, such as the restaurant industry and the food industry.

It is a graph which shows the relationship between culture | cultivation time and culture | cultivation temperature of photosynthetic bacteria used for this invention. It is a graph which shows the relationship between reaction time by the processing method of this invention, lipase activity, and hexane concentration. It is the schematic which shows an example of the apparatus used for the oil containing wastewater processing method by this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Treatment apparatus 11 Wastewater inflow port 12 Inflow tank 13 Anaerobic tank 14 Aeration tank 15 Final sedimentation tank 16 Scallop 17 Waste water discharge port 21 Net 22 Alginic acid beads

Claims (6)

The water treatment method characterized by decomposing | disassembling the oil component in oil-containing wastewater using Rhodobacter sphaeroides NAT (Accession number: FERM P- 21486).   The water treatment method according to claim 1, wherein the oil-containing waste water at 45 ° C to 55 ° C is used.   The water treatment method according to claim 1, wherein the oil is decomposed by releasing lipase from the Rhodobacter sphaeroides NAT into the oil-containing wastewater.   The water treatment method according to claim 1, wherein the Rhodobacter sphaeroides NAT is immobilized on an alginate and used. A water treatment material comprising a photosynthetic bacterium Rhodobacter sphaeroides NAT (accession number: FERM P- 21486) that decomposes oil contained in water, a COD component, and phosphorus. A water treatment material comprising a photosynthetic bacterium Rhodobacter sphaeroides NAT (accession number: FERM P- 21486) that decomposes oil contained in water, a COD component, and phosphorus, fixed to an alginate.

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