JP2015039348A - Microorganism variant, and method and equipment for treating waste water using thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new microorganism variant that has excellent capacity of synthesizing ethanol from glycerol and has resistance to formic acid.SOLUTION: Microorganism variant is provided, the variant belonging to Klebsiella variicola, having the capacity of generating ethanol from glycerol, growing in a formic acid concentration of 4 to 5.5 wt.% in culture medium.

Description

  The present invention relates to a microbial mutant obtained from a microorganism capable of producing ethanol from glycerol contained in wastewater, for example, and further relates to a method for producing ethanol and a wastewater treatment apparatus using the microbial mutant.

  Biodiesel fuel is a fuel for diesel engines mainly made from plant-containing fats and oils, and can be used as a substitute for light oil currently used. In particular, biodiesel fuel has attracted attention as a carbon neutral energy source. In order to purify biodiesel fuel, first, methanol and a catalyst (acid catalyst or alkali catalyst) are added to fats and oils such as palm oil to cause a transesterification reaction, which is then neutralized with acid or alkali. Then, glycerol is separated from the fatty acid methyl ester as a residual component. A catalyst component is removed from the separated fatty acid methyl ester, and methanol is removed by further distillation, whereby a biodiesel fuel mainly composed of the fatty acid methyl ester can be purified.

  As described above, glycerol is always obtained as a residual component in the process of purifying biodiesel fuel, but this residual component is usually discarded without particularly useful use. As an example of utilizing glycerol obtained as a residue component, hydrogen and ethanol are produced from glycerol using Enterobacter bacteria, as disclosed in Patent Document 1 (Japanese Patent Laid-Open No. 2006-180782). To do.

More specifically, Patent Document 1 discloses a technique in which Enterobacter aerogenes is fixed to a porous carrier, and hydrogen and ethanol are produced from glycerol by fermentation using this. However, Patent Document 1 states that when using waste liquid containing glycerol produced in the process of refining biodiesel fuel as a raw material, fermentation efficiency is extremely low if Enterobacter aerogenes is used as it is.

Then, the present inventors have isolated and identified a novel microorganism excellent in the ability to synthesize ethanol from glycerol (Patent Document 2). Patent Document 2 discloses Klebsiella variicola having the ability to produce ethanol from glycerol. Novel microorganism that has been disclosed in Patent Document 2, in Klebsiella Barikora (Klebsiella variicola) independent as TB-83 share National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (currently, the successor to the National Institute of Technology and Evaluation) It has been deposited. Patent Document 2 discloses that a mutagen treatment was performed on this TB-83 strain to obtain a mutant strain with improved ethanol resistance. This mutant strain is named TB-83D and is also deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

JP 2006-180782 A JP 2012-139216 A

  However, the TB-83 strain and the TB-83D strain disclosed in Patent Document 2 have a problem that resistance to formic acid produced along with glycerol metabolism is poor. That is, in the system that synthesizes ethanol from glycerol using the TB-83 and TB-83D strains, the growth of the fungus is inferior due to the generated formic acid, and it is difficult to achieve sufficient glycerol metabolism and excellent ethanol synthesis. there were. Therefore, in view of such circumstances, the present invention aims to provide a microbial mutant excellent in formic acid resistance, and further provides an ethanol production method and a wastewater treatment apparatus using the microbial mutant. With the goal.

  In order to achieve the above-mentioned object, the present inventors searched for a mutant strain excellent in formic acid resistance using the known Klebsiella baricola TB-83D strain. The present inventors have succeeded in producing an excellent mutant and have completed the present invention.

  The microbial mutant according to the present invention belongs to Klebsiella variicola, has the ability to produce ethanol from glycerol, and grows at a formic acid concentration in the medium of 4 to 5.5% by weight. is there.

  In particular, the microbial mutant according to the present invention is a Klebsiella baricola mutant identified by the deposit number FERM P-22039 (TB-83 strain) or the deposit number FERM P-22203 (TB-83D strain). preferable.

  The microbial mutant produced by the present inventors was named TB-83H, and was incorporated by the National Institute of Technology and Evaluation Technology Patent Microorganism Depositary Center (Room 2-5-8 Kazusa Kamashitsu, Kisarazu City, 292-0818, Japan) ) As deposit number NITE AP-01688 on August 12, 2013.

  On the other hand, the method for producing ethanol according to the present invention is a method for producing ethanol from glycerol using the above-described microbial mutant according to the present invention. That is, the ethanol production method according to the present invention can be applied to a system using a waste liquid containing glycerol. Examples of the waste liquid containing glycerol include glycerol waste liquid produced as a residue component in the process of refining biodiesel fuel. In the ethanol production method according to the present invention, hydrogen gas can be recovered together with ethanol. In other words, a method for producing hydrogen from glycerol using the above-described microbial mutant according to the present invention can also be provided.

  Moreover, the wastewater treatment apparatus according to the present invention includes a fermenter for bringing the above-described microbial mutant according to the present invention into contact with wastewater containing glycerol. In the wastewater treatment apparatus according to the present invention, wastewater obtained in the process of refining biodiesel fuel can be used as wastewater containing glycerol. Moreover, the wastewater treatment apparatus according to the present invention can include a hydrogen recovery apparatus that recovers hydrogen generated in the fermenter.

  According to the present invention, a useful substance such as ethanol can be obtained using glycerol contained in wastewater and the like, and a novel microbial mutant excellent in formic acid resistance can be provided. In particular, the microbial mutant according to the present invention is excellent in ethanol-producing ability and excellent in formic acid resistance, so that it can be applied to, for example, a system for producing ethanol from glycerol contained in waste liquid generated in the purification process of biodiesel fuel. it can.

  Moreover, according to this invention, the waste-liquid processing apparatus which has a fermenter of manufacturing ethanol using the glycerol contained in wastewater etc. by a microorganism mutant can be provided. According to the wastewater treatment apparatus of the present invention, it is possible to efficiently produce substances such as ethanol and hydrogen that are useful using, as a raw material, glycerol-containing waste liquid that is produced, for example, during the purification process of biodiesel fuel.

It is a photograph which shows the result of having culture | cultivated the formic acid tolerance mutant strain and TB-83D strain which were produced in Example 1 in the formic acid containing medium of various density | concentrations.

Hereinafter, the present invention will be described in detail.
The microbial mutant according to the present invention belongs to Klebsiella variicola, has the ability to produce ethanol from glycerol, and grows at a formic acid concentration in the medium of 4 to 5.5% by weight. is there. That is, the microbial mutant according to the present invention is characterized by excellent formic acid resistance. Since the microbial mutant according to the present invention has the ability to produce ethanol from glycerol even in the presence of formic acid at a concentration of 4 to 5.5% by weight,
Even if formic acid is produced in a system for synthesizing ethanol from glycerol, sufficient glycerol metabolism and excellent ethanol synthesis can be maintained.

  In particular, the microbial mutant according to the present invention should be prepared from the Klebsiella baricola mutant identified by the deposit number FERM P-22039 (TB-83 strain) or the deposit number FERM P-22203 (TB-83D strain). Can do. The Klebsiella baricola mutant strain identified by the accession number FERM P-22039 (TB-83 strain above) or the accession number FERM P-22203 (TB-83D strain) is the National Institute for Product Evaluation Technology Patent Microorganism Deposits. You can get sales from the center (NITE-IPOD).

  Specifically, the mutagen treatment is performed on the TB-83 strain or TB-83D strain, and the microbial mutant strain according to the present invention can be produced by introducing the mutation. Here, the mutagen treatment is not particularly limited as long as it is a treatment that induces a mutation in the target microorganism, for example, a treatment that irradiates electromagnetic waves such as X-rays, radiation, and ultraviolet rays, nitrosoguanidine, nitrosamine, Examples include treatment of contacting compounds such as bromodeoxyuridine, N-ethyl-N-nitrosourea, methyl ethanesulfonate, benzopyrene, ethidium bromide, treatment of introducing nucleic acids using viruses, transposons, etc. .

  Further, among the deposited microorganisms, the TB-83D strain can be subjected to mutagen treatment to obtain a microbial mutant strain having improved formic acid resistance and improved ethanol synthesis ability. TB-83D strain is the parent strain, and the microbial mutant with improved formic acid resistance and ethanol synthesizing ability is named TB-83H. Independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (〒292-0818 Chiba Prefecture) The deposit was made on August 12, 2013 as receipt number NITE AP-01688. The microorganisms according to the present invention also include the TB-83H strain with improved formic acid resistance.

  Moreover, the microbial variant which concerns on this invention is equipped with the characteristics of a parent strain except the characteristics that formic acid tolerance improves compared with a parent strain, and is excellent in ethanol synthesis ability. In particular, when the TB-83D strain is used as the parent strain, the microbial mutant according to the present invention has the characteristics of the TB-83D strain. As disclosed in JP 2012-139216 A, the TB-83D strain has the ability to produce ethanol from glycerol, and is subjected to mutagen treatment on the TB-83 strain having ethanol resistance, It is a mutant that has streptomycin resistance and improved ethanol resistance. Therefore, when the TB-83D strain is used as the parent strain, the microbial mutant according to the present invention has resistance to streptomycin and can grow in the presence of ethanol at a high concentration of 7% by weight.

  However, the microbial mutant according to the present invention is not limited to the deposited microbial mutant TB-83H. In addition to the deposited microorganism, the microbial mutant according to the present invention can be produced, for example, by subjecting the TB-83 strain or TB-83D strain to mutagen treatment. As a specific method, first, the strain TB-83 or TB-83D was subjected to ultraviolet irradiation treatment, and then a strain capable of growing in a glycerol-containing medium containing 4 to 5.5% by weight of formic acid was used. It can be identified as a microbial mutant according to the invention.

  Furthermore, the microbial mutant according to the present invention includes a mutant in which the mutagen treatment is further performed on the deposited microbial mutant TB-83H and the mutation is introduced. Usually, when producing a mutant strain by mutagen treatment, the purpose is often to give a phenotype different from that of the parent strain. Therefore, it is preferable that the mutant strain produced from the deposited microorganism (parent strain) has a phenotype different from that of the parent strain for this purpose, but it is a mutant strain that does not have a significantly different phenotype. There may be. In addition, the mutant which gave the drug resistance to the parent strain, and the mutant which has the alcohol tolerance superior to the parent strain can be produced by the mutagen treatment.

  By using the microbial mutant according to the present invention, a method for producing ethanol using glycerol can be constructed. That is, by using the microorganism, an ethanol production system using a solution containing glycerol can be constructed. The solution containing glycerol is not particularly limited, and examples thereof include waste liquid generated in the process of producing biodiesel fuel. When producing biodiesel fuel, first, oil and fat (also known as triglyceride, triacylglycerol) and methanol produce methyl ester and glycerol by chemical reaction (methyl esterification). And the biodiesel fuel which has methyl ester as a main component can be manufactured by isolate | separating the produced | generated glycerol.

  In addition, it does not specifically limit as a method of methyl-esterifying fats and oils when manufacturing biodiesel fuel, What is necessary is just to use a well-known method. For example, it can be performed using a method of reacting oil and fat in the presence of a catalyst such as lipase, or a method of reacting supercritical methanol and oil.

  The fats and oils used when producing this biodiesel fuel are not particularly limited, and vegetable fats and oils, animal fats and oils or waste oils thereof can be used as fats and oils. Examples of the vegetable oil include palm oil, rapeseed oil, sesame oil, and olive oil. Examples of the animal oil include lard, beef tallow, and fish oil. Moreover, even if the said fats and oils contain impurities etc., reagent grade fats and oils may be used.

  Moreover, what is necessary is just to use well-known methods, such as a decantation, as a method of removing methyl ester (biodiesel fuel) from the product obtained by methyl-esterifying fats and oils. More specifically, after the methyl esterification (after methanolysis), the produced biodiesel fuel is separated into an upper layer and the biodiesel waste liquid portion containing glycerol is divided into a lower layer by allowing the reaction solution to stand. Therefore, biodiesel fuel and biodiesel waste liquid can be easily separated by a method such as decantation.

  The waste liquid after separating such biodiesel fuel contains a large amount of glycerol as a main component. The waste liquid may contain lactic acid, acetic acid, ethanol or the like, although it varies depending on the components contained in the fat used as a raw material. Further, the waste liquid after separating the biodiesel fuel may be used as it is in the method for producing ethanol, or may be used after adding a solvent such as water as appropriate to obtain a desired glycerol concentration. Furthermore, other substances can be added to the waste liquid after separating the biodiesel fuel.

  In addition, when producing ethanol from glycerol using the microbial mutant according to the present invention, the concentration of glycerol contained in the solution is not particularly limited, and can be 10 to 200 (g / l), In particular, it is preferably 10 to 100 (g / l), more preferably 20 to 50 (g / l). When cultivating the microbial mutant according to the present invention, in addition to the glycerol described above, a medium containing a material serving as a carbon source and a substance serving as a nitrogen source can be appropriately selected and used. Moreover, you may use the culture medium composition generally used for the culture medium for microorganisms, such as enzyme extract of proteins, such as a yeast extract and casein.

  Furthermore, when producing ethanol from glycerol using the microbial mutant according to the present invention, the culture time of the microbial mutant is not particularly limited, and can be 3 to 7 (days). 4 to 5 (days) is preferable. Although it does not specifically limit as culture | cultivation temperature, It can be set to 20-35 (degreeC), and it is preferable to set it as 25-30 (degreeC) especially.

  On the other hand, according to the above-described microbial mutant, hydrogen is produced when ethanol is produced from glycerol. Therefore, a hydrogen production method and a hydrogen production system can be constructed by using the microbial mutant described above.

  By the way, the culture apparatus used for the production method of the present invention is not particularly limited, and can be used after appropriately selecting a known culture apparatus. The culture method may be any of batch culture, continuous culture, and feed culture. Although not shown in the drawings, the culture apparatus is not particularly limited as long as it includes a fermenter for producing ethanol from glycerol using the above-described microbial mutant. For example, the culture device may include a recovery device for recovering ethanol or a hydrogen recovery device for recovering hydrogen.

  In particular, since the microbial mutant according to the present invention has formic acid resistance, it can avoid inconveniences such as growth inhibition due to formic acid produced in the process of producing ethanol from glycerol, or reduction in glycerol metabolic capacity. it can. Specifically, the microbial mutant according to the present invention grows until glycerol contained in the culture broth consumes glycerol and begins to biosynthesize ethanol and then the formic acid concentration contained in the broth exceeds 4 to 5.5% by weight. Can continue.

  In addition, when the microbial mutant according to the present invention is produced using TB-83 or TB-83D as a parent strain, the alcohol resistance of the parent strain is maintained. Therefore, the microbial mutant according to the present invention can avoid such disadvantages that the growth inhibition is caused by ethanol produced from glycerol and the ability of glycerol metabolism is reduced. Specifically, the novel microorganism according to the present invention grows until glycerol contained in the culture solution is consumed to start biosynthesis of ethanol and then the ethanol concentration contained in the culture solution exceeds 5% by weight, preferably over 2%. Can continue.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, Klebsiella baricola identified by the deposit number FERM P-22203 (TB-83D strain) was obtained, and an attempt was made to obtain a mutant strain excellent in formic acid resistance using the TB-83D strain as a parent strain. First, in order to investigate the formic acid resistance of the parent strain TB-83D, growth was examined on LB agar medium at each formic acid concentration (3%, 4% and 5%). It became clear that it grew to%.

  Next, as a result of repeated mutagenesis by UV irradiation, 768 mutant strains were obtained in the first screening. Next, as in the formic acid tolerance test of the TB-83D strain, on the agar medium of each formic acid concentration (3%, 4% and 5%), the obtained 768 strains were replicated and their growth was examined. We obtained 152 strains that grew at a formic acid concentration of 5%. Among them, 27 strains with relatively good growth could be selected.

  These 27 strains were statically cultured at 25 ° C. for 4 days using a 24-well multiplate dispensed with 1 ml of 2% glycerol basic medium. After centrifuging the culture and performing simple ethanol measurement using 4-aminoantipyrine on the supernatant, 5 strains (3B12, 3F12, 4A6, 4A9 and 5C11) showed higher absorbance values than the TB-83D strain. It was. From these results, 5 strains 3B12, 3F12, 4A6, 4A9 and 5C11 were designated as microbial mutants having formic acid resistance. When the growth of these 5 strains was examined on the agar medium of each formic acid concentration, including the examination of the reverse mutation in the subculture, it showed good growth on the agar medium containing 5% formic acid (FIG. 1). On the other hand, the parent strain TB-83D did not grow (FIG. 1).

  In FIG. 1, A has a formic acid concentration of 3%, B has a formic acid concentration of 4%, C has a formic acid concentration of 5%, and D has a formic acid concentration of 5.5%. In FIG. 1, “1” is a TB-83D strain, “2” is 3B12, “3” is 3F12, “4” is 4A6, “5” is 4A9, 6 ”is 5C11.

[Example 2]
The ethanol production test was performed on the formic acid resistant strain obtained in Example 1. A mini jar fermenter was used for the experiment.

  First, the 5 strains (3B12, 3F12, 4A6, 4A9, and 5C11) obtained in Example 1 were inoculated with 2 mL of 2% glycerol-containing basic medium (pH 9) at 30 ° C. The cells were cultured at 120 rpm for 3 days in advance. Next, 1 mL of the culture solution was inoculated into 25 mL of a basic medium containing 1% glycerol, and precultured by standing at 25 ° C. for 3 days. Then, add the whole amount of the preculture to 500 mL of 5% glycerol-containing basic medium containing 5 times the yeast extract and casamino acid in the basic medium (see Table 1 below), and cultured at 25 ° C with gentle stirring with a stirrer. . Moreover, 6N NaOH solution was dripped using the pH controller and the peristaltic pump, and pH was maintained at 8. About 1 mL of the culture solution was collected over time, and the growth amount OD580 was measured and the supernatant was analyzed by HPLC. Further, 50 mL and 20 mL of 50% glycerol solution were replenished on the 3rd and 10th days of culture, respectively.

  Table 2 shows the results of quantification of ethanol contained in the medium on the third and seventh days from the start of the culture.

  As shown in Table 2, among the five formic acid resistant strains produced in the Examples, 4A6 strain showed higher ethanol production than the parent strain, TB-83D strain. Mutant 4A6, to which formic acid tolerance was imparted to the parent strain and ethanol production ability was improved, was named TB-83H strain. That is, according to this example, it was shown that a microbial mutant strain having formic acid resistance and excellent ability to produce ethanol from glycerol can be produced.

Claims (8)

  A microbial mutant belonging to Klebsiella variicola, capable of producing ethanol from glycerol, and growing at a formic acid concentration of 4 to 5.5% by weight in the medium.   The microbial mutant according to claim 1, which is a mutant strain of Klebsiella baricola identified by accession number FERM P-22039 or accession number FERM P-22203.   The microorganism mutant according to claim 1, which has a receipt number NITE AP-01688. Contacting the microbial mutant according to any one of claims 1 to 3 with wastewater containing glycerol;
And a step of recovering the produced ethanol after contacting the microbial mutant.   The method for producing ethanol according to claim 4, further comprising a step of recovering the produced hydrogen after contacting the microbial mutant.   The method for producing ethanol according to claim 4, wherein the waste water is waste water containing a residue after producing biodiesel fuel.   A wastewater treatment apparatus comprising a fermenter for contacting the microbial mutant according to any one of claims 1 to 3 with wastewater containing glycerol.   The wastewater treatment apparatus according to claim 7, further comprising a hydrogen recovery apparatus that recovers hydrogen generated in the fermenter.

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