JP2016214232A - Microorganisms, method for producing lipid and wastewater treatment method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of efficiently producing a material that can consume lower organic acid to use it.SOLUTION: Nitratireductor microorganisms are used to produce lipid from lower organic acid. The microorganisms are, for example, Nitratireductor sp.OM-1 strain. The lower organic acid may be, for example, at least one of propionic acid and acetic acid.SELECTED DRAWING: Figure 1

Description

  The technology disclosed in the present specification relates to a method for producing lipids using microorganisms.

  Organic industrial wastewater is treated with activated sludge containing microorganisms, but surplus sludge generated at that time is legally required to be treated in the facility. When the activated sludge is anaerobically fermented, organic acids such as propionic acid are produced, and other bacteria are killed. Such sludge containing organic acids must be dewatered and incinerated, and the treatment is costly. For this reason, reduction of the amount of sludge to be incinerated and reuse of sludge are desired.

  Patent Document 1 discloses a microorganism that can use propionic acid or the like as a carbon source. Patent Document 2 discloses a microorganism capable of producing hydrocarbons using a lower organic acid such as propionic acid as a carbon source.

JP 2003-245066 A JP 2003-229 A

  Degradation of organic acids contained in sludge is desired, but if the substances produced by decomposing organic acids are not useful, the products will eventually be discarded, reducing sludge. I can't plan.

  The microorganism described in Patent Document 1 generates gluconic acid from glucose. However, Patent Document 1 does not disclose any substance that is useful when propionic acid is used as a carbon source.

  Although the microorganisms described in Patent Document 2 can produce hydrocarbons, the microorganisms have different substrates and suitable growth conditions depending on the type, so that it is still desirable to discover other microorganisms that can produce substances that can be used as fuels. ing. In particular, the search for microorganisms capable of producing a useful substance with a lower organic acid as a substrate with higher efficiency has been continued.

  In view of the above problems, an object of the present invention is to provide a method for efficiently producing a usable substance by consuming a carbon source such as a lower organic acid.

  This specification discloses microorganisms of the genus Nitratireductor that produce lipids from lower organic acids. This microorganism may be, for example, Nitratireductor sp. OM-1 strain.

  The method disclosed in the present specification is a method for producing lipid from a lower organic acid using Nitratireductor sp. OM-1 strain belonging to the genus Nitratireductor having lower organic acid assimilation property.

  According to a method using a microorganism of the genus Nitratireductor having a lower organic acid assimilation property (for example, Nitratireductor sp. OM-1 strain), lipids such as biodiesel can be efficiently produced from the lower organic acid.

FIG. 1 is a flowchart showing a screening procedure for bacteria having organic acid assimilation ability. FIG. 2 is a diagram showing the results of measuring the fluorescence intensity per cell density of each strain. FIG. 3A is a diagram showing the time at which peaks of palmitic acid, stearic acid, and squalene were detected in GC. FIG. It is a figure which shows the analysis result by GC of the lipid contained in a microbial cell about 1 microbe. FIG. It is a figure which shows the analysis result by GC of the lipid contained in a microbial cell about 2 microbes. FIG. It is a figure which shows the analysis result by GC of the lipid contained in a microbial cell about 3 microbes. FIG. It is a figure which shows the analysis result by GC of the lipid contained in a microbial cell about 4 microbes. FIG. It is a figure which shows the analysis result by GC of the lipid contained in a microbial cell about 5 microbes. FIG. 4 is a diagram showing bacterial growth curves when the medium is changed from a medium with a limited carbon source and passaged without changing the medium. FIG. 5 is a graph showing changes in the total lipid amount (mg / g) per dry cell weight when the nitrogen source concentration in the medium is changed. FIG. 6 is a graph showing a growth curve of Nitratireductor sp. OM-1 when the carbon source is only glycerin.

(Embodiment)
-Screening for bacteria with organic acid utilization ability-
The inventors of the present application searched for a bacterium having resistance to a lower organic acid and capable of degrading the lower organic acid. In the present specification, the “lower organic acid” refers to an organic acid having 6 or less carbon atoms. The inventors of the present application searched for bacteria capable of producing lipid among bacteria having resistance to lower organic acids. In this specification, unless otherwise specified, lipids include not only alcohol and fatty acid esters (simple lipids) but also complex lipids such as phospholipids and derived lipids such as fatty acids.

  FIG. 1 is a flowchart showing a screening procedure for bacteria having organic acid assimilation ability.

Seawater and gravel were collected as samples in the sea area near Osaki Kamijima in Hiroshima Prefecture, and the bacteria contained in this sample were accumulated and cultured using ATCC # 1409 medium to which propionic acid was added (step S1). Here, the ATCC # 1409 medium is 30 g / L NaCl, 1 mL / L SLA trace elements, 1 mL / L VA vitamins, 1.0 g / L KH ₂ PO ₄ , 0.5 g / L MgCl _2. 6H ₂ O, 0.1 g / L CaCl ₂ · H ₂ O, 1.0 g / L NH ₄ Cl, 3.0 g / L NaHCO ₃ , 0.7 g / L Na ₂ SO ₄ , 1.0 g / L sodium acetate (amount equivalent to 10 mM as acetic acid), 0.5 g / L sodium ascorbate, 0.1 g / L Yeast extract, 0.156 g / L sodium sulfate, pH 6.9 -7.0.

SLA trace elements are 250 mg CoCl ₂ · 6H ₂ O, 10 mg NiCl ₂ · 6H ₂ O, 10 mg CuCl ₂ · 2H ₂ O, 70 mg MnCl ₂ · 4H ₂ O, 100 mg ZnCl ₂ , 500 mg H ₃ It was prepared by dissolving BO ₃ , 30 mg Na ₂ MoO ₄ .2H ₂ O, 10 mg Na ₂ SeO ₃ .5H ₂ O, 1.8 g FeCl ₂ .4H ₂ O in 1 L distilled water.

  VA vitamins are 100 mg biotin, 350 mg nicotinamide, 300 mg thiamine-HCL, 200 mg paraaminobenzoic acid, 100 mg pyridoxine-HCL, 100 mg calcium D-pantothenate, 50 mg cyanocobalamin (B12) in 1 L distilled water. Created by dissolving.

  In this step, the above-mentioned ATCC # 1409 medium was added with propionic acid so as to have a concentration of 10 mM and cultured at 28 ° C. in a dark place under aerobic conditions for 168 hours.

  Next, the bacterial solution obtained in the previous culture was applied to an agar medium (agarose concentration 1.5%) of ATCC # 1409 medium containing 10 mM acetic acid and propionic acid, respectively. The cells were cultured for 216 hours under aerobic conditions (step S2).

  Next, colonies generated on the agar medium were picked, the bacteria were inoculated into a 96-well plate, and cultured in ATCC # 1409 medium containing 10 mM propionic acid (step S3). The amount of ATCC # 1409 medium was 600 μL per well. Incubation was performed at 25 ° C., 250 rpm, in the dark, under aerobic conditions.

  Next, the absorbance in each well was measured using a plate reader (Spectra MAX M5, manufactured by Molecular Devices). Here, a part of the cultured bacterial solution was transferred to a 96-well plate, and the absorbance at 660 nm was measured. In this step, a large absorbance means that the bacteria are growing.

  Next, each culture solution is centrifuged to collect the cells, and after washing the cells with Phosphate Buffered Saline (PBS), 50 μL of PBS and 2 μL of Nile Red (manufactured by Wako Pure Chemical Industries, Ltd., concentration 1 mg / mL) are added. The cells were stained. The sample was transferred to a 96-well plate, and the fluorescence intensity at an excitation wavelength of 540 nm and a fluorescence wavelength of 590 nm was measured using a fluorescence / luminescence microplate reader (Fluoroskan Ascent FL, manufactured by Thermo). The fluorescence intensity per cell density of each strain was calculated, and a strain having a relatively high value was selected as a candidate strain for organic acid resistant bacteria (step S4).

  FIG. 2 is a diagram showing the results of measuring the fluorescence intensity per cell density of each strain. In the figure, each of the bar graphs shows the measurement result of the sample. By this measurement, No. A total of 5 strains of 1 to 5 were selected as candidate strains of organic acid resistant bacteria.

Next, the total lipid amount contained in the bacterium selected in the previous step was measured (step S5). This measurement was performed by gas chromatography (GC) using GC-17A and CBM-102 (manufactured by Shimadzu Corporation). GCSolution version 2.4 (manufactured by Shimadzu Corporation) was used as analysis software. As the column, a DB-5HT capillary GC column (Agilent Technologies) having an inner diameter of 0.25 mm, a length of 30 m, and a film thickness of 0.1 μm was used. The analysis conditions were that the column oven was heated from 100 ° C. to 380 ° C. at 10 ° C./min, N ₂ was used as the carrier gas, the sample vaporization chamber was 300 ° C., the detector was 380 ° C., the pressure was 107 kPa, The total flow rate was 16 mL / min, the column flow rate was 1.19 mL / min, and the split ratio was 1:10.

  In this step, wet cells collected by centrifugation of each sample were stored frozen at -80 ° C. Next, the cells were freeze-dried using a freeze dryer. To the dried cells, 500 μL of a chloroform / methanol mixture (2: 1 by volume) was added and suspended. This operation was repeated to obtain 2 mL of cell extract. Further, 500 μL of milli-Q water was added to the extract and suspended, followed by centrifugation. The chloroform layer was transferred to a test tube and dried. 250 μL of chloroform was added to the extract after drying, transferred to a glass vial, and dried under a nitrogen stream. Next, 25 μL of chloroform was added to the extract after drying to obtain an extraction sample. GC measurement was performed using 1 μL of this extracted sample.

  Moreover, the product was identified by the gas chromatography mass spectrometer (GC / MS). ZB-1HT (Zebron) having an inner diameter of 0.25 mm, a length of 30 m and a film thickness of 0.25 μm was used as the column, and JMS-T100GCV “AccuTOF GCv 4G” (manufactured by JEOL) was used as the GC / MS. Analysis conditions were as follows: the column oven was heated from 100 ° C. to 380 ° C. at 10 ° C./min, He was used as the carrier gas, the sample vaporization chamber was 300 ° C., the detector was 270 ° C., and the injection volume was 1.0 μL. The column flow rate was 1.2 mL / min, and the split ratio was 1:10.

  3A is a diagram showing the time at which peaks of palmitic acid, stearic acid, and squalene were detected in GC, and FIGS. It is a figure which shows the analysis result by GC of the lipid contained in a microbial cell about five of 1-5. Table 1 summarizes the results of this analysis.

  From the results shown in FIG. 3B to FIG. 3F and Table 1, the yield of lipid per dry cell was sample No. It was found that there were many strains of 1, 4 and 5. Further, it was confirmed that lipids such as palmitic acid and stearic acid and hydrocarbons such as squalene were produced by each bacterium. The “total lipid” shown in Table 1 includes hydrocarbons such as squalene for convenience.

Next, bacteria having lower organic acid assimilation properties were separated by culturing using a medium containing only NaHCO ₃ as a carbon source (step S6). Specifically, each selected bacterium was transferred to a medium containing only NaHCO ₃ as a carbon source and cultured at room temperature for 7 days. It is considered that bacteria that can use lower organic acids consumed the lower organic acids, so that it was possible for bacteria that cannot use lower organic acids to survive. It was expected to be. The “medium containing only NaHCO ₃ as a carbon source” used in this step is obtained by removing a carbon source other than NaHCO ₃ from the above-described ATCC # 1409 medium, and includes a yeast extract.

In a medium containing only NaHCO ₃ as a carbon source, the number of bacteria reached a saturation state in about 2 days, and the number of bacteria was smaller than that in a culture using a normal ATCC # 1409 medium supplemented with 10 mM propionic acid. As a result of this culture, the sample No. shown in FIG. 3 successfully obtained a microbial strain (Nitratireductor sp. OM-1 strain described later) having lower organic acid assimilation ability. Next, the bacterium monocultured from a medium containing only NaHCO ₃ as a carbon source to ATCC # 1409 medium was inoculated and cultured at 28 ° C. for 7 days. Subsequently, the bacterial cells were treated by the above-described method, and the production amounts of lipids and hydrocarbons were measured by GC analysis. On the other hand, culture in a medium containing only NaHCO ₃ as a carbon source (that is, culture under carbonic acid fixation conditions) was continued for 7 days.

  Table 2 shows the total lipid amount per dry cell and palmiticin obtained when the medium was changed to the normal ATCC # 1409 medium supplemented with propionic acid from the carbonic acid fixation condition and the carbonic acid fixation condition. It is a figure which shows the result of having measured the yield of an acid, a stearic acid, and squalene. Table 3 shows the total lipid and palmitic acid per volume of the medium obtained under the conditions of carbonic acid fixation and when the medium was changed from the carbonic acid fixation condition to a normal ATCC # 1409 medium supplemented with propionic acid. It is a figure which shows the result of having measured the yield of stearic acid and squalene. FIG. 4 is a diagram showing bacterial growth curves when the medium is changed from a medium with a limited carbon source and passaged without changing the medium. The increase in absorbance shown in the figure represents an increase in cell density. The “total lipid” shown in Tables 2 and 3 also includes hydrocarbons such as squalene for convenience.

  From the results shown in FIG. 4, when planted in an ATCC # 1409 medium with a limited carbon source, the bacterial cell concentration was saturated in about 2 days, whereas it was planted in an ATCC # 1409 medium containing propionic acid. In some cases, it took 5-7 days for the bacterial cell concentration to saturate, and significant bacterial growth was observed. In addition, as shown in Tables 2 and 3, the cells per plant weight in the ATCC # 1409 medium containing propionic acid was larger than those in the ATCC # 1409 medium with a limited carbon source. Both the total lipid amount and the total lipid amount per medium volume were significantly increased.

  As shown in Tables 2 and 3, lipids such as palmitic acid and stearic acid and hydrocarbons such as squalene were produced in the cells. In addition, as can be seen from the comparison between Table 2 and Table 1, the yield of total lipid and each lipid per dry cell weight is greatly increased by planting from the carbonic acid fixation condition to ATCC # 1409 medium containing propionic acid. It was confirmed that it increased. It is presumed that this was because the bacteria were switched to give priority to the metabolism for storing lipids in the cells by placing them in oligotrophic conditions.

  If the ATCC # 1409 medium containing propionic acid was subcultured to the same medium, the total lipid amount per cell weight gradually decreased from 177.964 mg / g and became almost constant, The yield was similar. However, the total lipid per dry cell weight was increased compared to that before singularization shown in Table 1.

  Here, since the carbon source is limited in the ATCC # 1409 medium, and when the bacterium before isolation as a single strain was cultured, the concentration of propionic acid in the medium was reduced, these lipids were observed. Was considered to have been converted from propionic acid.

On the other hand, from the results shown in FIG. 4 and Tables 2 and 3, this bacterium can grow to some extent even in a medium containing only NaHCO ₃ , and hydrocarbons and lipids such as squalene are stored in the microbial cells after culturing. I understood. NaHCO ₃ although divided into a Na ion and HCO ₃ ions when dissolved in water, because the CO ₂ is produced in the same manner is HCO ₃ ions be soluble in water, immobilized the CO ₂ dissolved in the liquid in the fungus It is thought that there is an ability to do. Therefore, if this bacterium is used, it is considered that lipids and hydrocarbons can be produced from CO ₂ even in a liquid not containing a carbon source containing an organic acid.

-Identification of bacterial species-
Next, in order to identify the species of the obtained strain, the partial sequence (about 0.5 kbp) of 16S rDNA was analyzed by a known PCR method. As a result, it showed 99% homology with 16S rDNA of Nitratireductor aquimarinus strain CL-SC21, and was clarified to be a bacterium belonging to the genus Nitratireductor. This bacterium is an aerobic short gonococcus having heterotrophic properties and also having propionic acid utilization. This bacterium also has a carbonic acid fixing ability and produces triacylglycerol from a lower organic acid. On the other hand, it has not been reported that known bacteria of the genus Nitratireductor produce triacylglycerol from lower organic acids. In addition, this bacterium can synthesize lipids and hydrocarbons from various substances compared to known bacteria such as sugars and alcohols as well as lower organic acids.

  Next, the present inventors also examined the assimilation properties of lower organic acids other than propionic acid for the obtained strain. Specifically, various lower organic acids having 5 or less carbon atoms as a carbon source were added to ATCC # 1409 medium and cultured at 28 ° C. for 7 days under dark and aerobic conditions. The concentration of each lower organic acid in the medium was 10 mM. The acetic acid assimilation property was examined using a medium in which acetic acid was added at a concentration of 10 mM to ATCC # 1409 medium from which sodium acetate was removed.

  The test results are shown in Table 4. In Table 4, on the basis of the growth state when cultured for 7 days using an ATCC # 1409 medium supplemented with 10 mM propionic acid, the growth state exceeding the standard is judged as A, and growth comparable to the standard If it was in the state, it was determined as determination B, and the case where it was able to grow but was below the standard was determined as determination C.

  As shown in Table 4, it was confirmed that the isolated bacterium can assimilate a lower organic acid having at least 5 carbon atoms. It was also confirmed that the bacteria can grow more than the medium containing propionic acid in the medium containing acetic acid, citric acid, butyric acid and valeric acid among the lower organic acids. The Nitratireductor aquimarinus strain CL-SC21 strain, which is considered to be a related species, has almost no malic acid assimilation, whereas the bacterium isolated by the present inventors has malic acid assimilation. However, the strain was significantly different from the Nitratireductor aquimarinus strain CL-SC21 strain (see International Journal of Systematic and Evolutionary Microbiology (2011), 61, 2676-2681).

  Nitratireductor aquibiodomus NL21 (see International Journal of Systematic and Evolutionary Microbiology (2004), 54, 269-273), which is a representative species of the genus Nitratireductor, has 97% homology of 16S rDNA with the isolated bacterium. The utilization of lower organic acids was different.

  It was also confirmed that the bacteria isolated here can use not only nitrates but also ammonium salts and glutamic acids as nitrogen sources.

  From the above properties, the bacteria obtained here were found to be a new species of the genus Nitratireductor, and were named Nitratireductor sp. OM-1 as described above.

  Nitratireductor sp. OM-1 strain has been deposited with the Patent Microbiology Depositary Center of the National Institute of Technology and Evaluation, 2-5-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture, under the accession number NITE P-02043. Yes.

-Analysis of products by Nitratireductor sp. OM-1-
Nitratireductor sp. OM-1 was cultured for 7 days at 28 ° C. using the above ATCC # 1409 medium containing 10 mM propionic acid. Next, after methyl esterifying not only fatty acids but also all lipids including diacylglycerol and triacylglycerol, GC analysis and GC / MS analysis are performed to determine the total amount of fatty acids per dry cell and the amount of each fatty acid. Was measured. Further, in order to examine the influence of the presence of the nitrogen source on the amount of lipid production, the culture was performed while changing the concentration of the nitrogen source between 0 mM and 20 mM. NH ₄ Cl was used as an example of the nitrogen source.

  Here, the ATCC # 1409 medium containing 10 mM sodium acetate and 10 mM propionic acid is transferred from the above-mentioned ATCC # 1409 medium limited in carbon source, and subcultured several times using the ATCC # 1409 medium containing propionic acid. After that, GC analysis and the like were performed. The analysis results are shown in Table 5. FIG. 5 shows the total lipid amount per dry cell weight obtained by GC analysis. The “total fatty acid amount” shown in Table 5 does not include the yield of hydrocarbons such as squalene. The yield of these hydrocarbons is included in the total lipid amount per dry cell weight shown in FIG.

According to the results shown in Table 5, in the culture of Nitratireductor sp. OM-1, the amount of fatty acid produced per dry cell weight, particularly triacylglycerol, increased as the NH ₄ Cl concentration in the medium decreased. When the cultured cells were stained with Nile Red, it was confirmed that the oil droplets (oil drop) in the cells were large. This was thought to be because metabolism was switched to store lipids in the cells by depleting the nitrogen source. Further, as shown in FIG. 5, the total lipid amount per dry cell weight also increased as the NH ₄ Cl concentration decreased.

However, it was also confirmed that Nitratireductor sp. OM-1 can synthesize lipids from lower organic acids when the NH ₄ Cl concentration in the medium is at least 20 mM or less. Here, since the assimitable carbon source in the culture medium is focused on the lower organic acid, the lipid is considered to be synthesized from the lower organic acid.

In addition, as a result of GC analysis, as the concentration of nitrogen source (NH ₄ Cl) in the medium decreases, 2-butenoic acid 1-metylethyl ester is produced in large quantities. I understand. The production amount of 2-butenoic acid 1-methylethyl ester per dry cell weight was about 50% of the total lipid amount when the concentration of the nitrogen source was 0 mM. 2-Butenoic acid 1-methylethyl ester is flammable and can be used as a fuel as it is. Furthermore, 2-butenoic acid as a derivative can be obtained by cleaving the ester bond of 2-butenoic acid 1-methylethyl ester. If this 2-butenoic acid is used as light oil, higher combustion energy than ethanol can be obtained.

In addition, it is thought that the tendency of the change of metabolism does not change with the kind of nitrogen source. For this reason, Nitratireductor sp. OM-1 removes lipids from lower organic acids when the concentration as a nitrogen source is at least 20 mM or less even when ammonium salt, nitrate, or glutamic acid other than NH ₄ Cl is used as the nitrogen source. It is thought that it can be synthesized.

  From the results shown in Table 5, it was found that triacylglycerol produced by Nitratireductor sp. OM-1 contains linoleic acid as a main component and contains pentadecenoic acid, palmitoleic acid and the like in the molecule.

From the above results, the culture of Nitratireductor sp. OM-1 is preferably performed in an environment where the concentration of a nitrogen source such as NH ₄ Cl is low, and at least 20 mM or less, the total lipid per cell weight It can be said that the amount of 2-butenoic acid 1-methylethyl ester, which is more useful as a fuel, can be increased while increasing the amount, which is preferable.

  Nitratireductor sp. OM-1 can generate lipids from lower organic acids such as acetic acid and propionic acid. For example, Nitratireductor sp. OM-1 not only removes lower organic acids from activated sludge, but also uses the generated lipids as biodiesel. It becomes possible. In addition, if Nitratireductor sp. OM-1 bacterial solution is used instead of activated sludge, after processing the object containing the lower organic acid, the pellets obtained by collection and drying should be used as fuel. You can also.

Further, during the processing of the object by using a bacterial solution cultured in nutrient-poor medium restrict advance carbon source only NaHCO ₃ to the process, it is possible to greatly improve the yield of lipids.

  Thus, when Nitratireductor sp. OM-1 is used, the amount of sludge to be discarded can be effectively reduced and fuel can be obtained.

  Most of the lipids produced by Nitratireductor sp. OM-1 are stored as oil droplets in the cells and are not released very much into the medium. For this reason, after producing lipids in a liquid containing a lower organic acid as described above, the produced lipids can be easily and efficiently obtained by recovering the cells by a known method such as centrifugation. it can.

  According to the method using Nitratireductor sp. OM-1, there is no need to use toxic chemicals or increase the temperature compared to the case of removing propionic acid etc. by chemical methods. Treatment of sludge etc. can be performed. Moreover, the treatment target containing a lower organic acid is not limited to activated sludge.

  In the present embodiment, Nitratireductor sp. OM-1 is cultured at about 20 ° C. to 26 ° C., but lipids can be grown from lower organic acids by culturing at temperatures that allow growth outside this range. It is possible to synthesize. The rotation speed at the time of shaking culture is not particularly limited, and the stirring method and stirring conditions may be appropriately changed depending on the volume of the object to be treated. Although the pH of a culture medium is not specifically limited, For example, 4 or more and 8 or less may be sufficient.

  In the above example, an example in which acetic acid and propionic acid are contained in the medium as substrates as the lower organic acid is shown. However, only one of acetic acid and propionic acid may be contained in the medium, or acetic acid and propion may be contained in the medium. Lipids can be synthesized even if lower organic acids other than acids are contained in the medium.

  FIG. 6 is a graph showing a growth curve of Nitratireductor sp. OM-1 when the carbon source is only glycerin. Table 6 shows the total lipid amount and the amount of various fatty acids per dry cell or volume of the culture solution when the carbon source is glycerol only. FIG. 6 shows the results of culturing Nitratireductor sp. OM-1 for 7 days under the above medium at 28 ° C. in the dark and under aerobic conditions. Moreover, the measurement of the amount of total lipid and the amount of various fatty acids was performed by GC and GC / MS.

  As shown in FIG. 6 and Table 6, it can be seen that Nitratireductor sp. OM-1 can grow using glycerol as a carbon source and can produce lipids from glycerol.

  Triacylglycerol produced by Nitratireductor sp. OM-1 is converted to biodiesel by methyl esterification, and glycerin is produced in this process. As described above, since Nitratireductor sp. OM-1 can also use glycerin as a substrate, it is also possible to treat glycerin discharged in the production process of biodiesel.

  Thus, Nitratireductor sp. OM-1 can assimilate not only lower organic acids but also a wide range of substances such as sugars and alcohols, so that composite wastewater containing multiple types of carbon sources can be treated alone. For this reason, management of conditions etc. becomes easy compared with the case where waste water treatment is performed using a plurality of kinds of microorganisms. The type of wastewater to be treated is not particularly limited as long as it is a wastewater capable of growing microorganisms of the present embodiment. The wastewater to be treated may be food wastewater such as wastewater containing waste molasses.

Further, Nitratireductor sp. OM-1 is not only available for wastewater treatment, as described above by limiting the carbon source in the liquid, the CO ₂ contained in the air is fixed, Ya lipids useful as a fuel or the like Hydrocarbons can be produced.

  Furthermore, lipids can be synthesized from acetic acid and propionic acid even when the concentration of lower organic acids such as acetic acid and propionic acid is other than 10 mM. For example, the concentration of the lower organic acid may be greater than 0 mM and about 50 mM or less. Since the strain obtained this time is highly resistant to lower organic acids, it is considered that growth is possible even if the concentration of the lower organic acid in the medium is considerably high. On the other hand, even if the concentration of the lower organic acid is low, the growth of the bacteria is possible if there is a carbon source.

  Nitratireductor sp. OM-1 has been cited as a hydrocarbon that can be produced by Nitratireductor sp. OM-1, but Nitratireductor sp. OM-1 can also produce hydrocarbons other than squalene.

  Further, if a method including steps S1 to S6 shown in FIG. 1 is used, it is possible to isolate a new strain of the genus Nitratireductor or another genus capable of producing lipids from lower organic acids in the same manner as Nitratireductor sp. OM-1. It is. As in Nitratireductor sp. OM-1, such strains of the genus Nitratireductor and other genera are treated with substances containing lower organic acids, or complex wastewater containing lower organic acids and carbon sources other than lower organic acids, etc. Can be used. In addition, microorganisms obtained by appropriately modifying Nitratireductor sp. OM-1 strain or strains having the same microbiological properties by known genetic engineering techniques, etc., can be used for substances containing lower organic acids. It may be used for processing.

  As described above, the microorganism according to an example of the present disclosure and the lipid production method using the microorganism are applied to treatment of activated sludge, waste liquid, and the like.

Claims (13)

A microorganism belonging to the genus Nitratireductor,
A microorganism that produces lipids from lower organic acids. In the microorganism according to claim 1,
A microorganism characterized by being Nitratireductor sp. OM-1 strain (Accession No. NITE P-02043).   A method for producing lipids from a lower organic acid using a microorganism belonging to the genus Nitratireductor having lower organic acid assimilation properties. The method of claim 3, wherein
A method for producing lipids from lower organic acids using Nitratireductor sp. OM-1 strain (Accession Number NITE P-02043) as the microorganism. The method of claim 4, wherein
The method wherein the lower organic acid is at least one of propionic acid and acetic acid. The method according to claim 4 or 5, wherein
Nitratireductor sp. OM-1 strain is cultured in a liquid containing the lower organic acid and having a nitrogen source concentration of less than 20 mM. The method of claim 6, wherein
Nitratireductor sp. OM-1 strain further produces 2-butenoic acid 1-methylethyl ester from the lower organic acid. The method according to any one of claims 4-7,
A method comprising culturing Nitratireductor sp. OM-1 strain in a liquid containing only NaHCO ₃ as a carbon source and then subculturing in a liquid containing the lower organic acid to produce lipid. The method according to any one of claims 4 to 8, wherein
Nitratireductor sp. OM-1 strain further produces hydrocarbons. 10. The method according to any one of claims 4-9,
A method comprising culturing Nitratireductor sp. OM-1 strain in waste water containing at least a lower organic acid and a carbon source other than the lower organic acid. The method according to any one of claims 4 to 10, wherein
The lipid produced by Nitratireductor sp. OM-1 strain contains at least one of linoleic acid, palmitic acid and stearic acid. The Nitratireductor microorganisms of the genus have a lower organic acid assimilation, it is limited carbon source, and by culturing in a liquid medium containing CO _2, the method of producing lipid while fixing the CO _2.   A method of treating wastewater containing a lower organic acid and a carbon source other than the lower organic acid, using a microorganism belonging to the genus Nitratireductor having lower organic acid utilization.