WO2021040088A1 - Method for pretreating sewage sludge using mixed enzyme, and method for treating sewage sludge using anaerobic digestion - Google Patents

Method for pretreating sewage sludge using mixed enzyme, and method for treating sewage sludge using anaerobic digestion Download PDF

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WO2021040088A1
WO2021040088A1 PCT/KR2019/011039 KR2019011039W WO2021040088A1 WO 2021040088 A1 WO2021040088 A1 WO 2021040088A1 KR 2019011039 W KR2019011039 W KR 2019011039W WO 2021040088 A1 WO2021040088 A1 WO 2021040088A1
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sewage sludge
amyloliquefaciens
mixture
culture
burkholderia vietnamiensis
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PCT/KR2019/011039
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French (fr)
Korean (ko)
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김상민
황석환
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포항공과대학교 산학협력단
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Priority to PCT/KR2019/011039 priority Critical patent/WO2021040088A1/en
Publication of WO2021040088A1 publication Critical patent/WO2021040088A1/en

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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00—Treatment of sludge; Devices therefor
    • C02F11/02—Biological treatment
    • C02F11/04—Anaerobic treatment; Production of methane by such processes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/28—Anaerobic digestion processes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used

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  • the present invention relates to a method of pretreatment of sewage sludge and a method of treating sewage sludge using the same, and more specifically, a sewage sludge suitable for anaerobic digestion using a mixture of specific microorganisms present in sewage sludge or enzymes secreted therefrom. It relates to a method of pretreatment and a method of treating pretreated sewage sludge by anaerobic digestion.
  • Anaerobic digestion refers to a series of biochemical processes in which organic matter is converted into biogas containing methane and carbon dioxide as main components through hydrolysis and acid fermentation by anaerobic microorganisms in an anaerobic state without oxygen.
  • the anaerobic digestion process is a hydrolysis step in which organic substances composed of carbohydrates, proteins, fats, etc. are hydrolyzed by the extracellular enzyme of anaerobic microorganisms and split into monomers such as disaccharides, monosaccharides, amino acids, fatty acids, etc.
  • Acid fermentation step in which monomers are used as food for acidogens and are converted into various organic acids (Volatile fatty acids (VFA)) by the metabolism of the microorganisms;
  • Acetogenesis in which organic acids are converted into acetic acid, hydrogen and carbon dioxide by microorganisms using organic acids;
  • It is composed of a methane generation step (Methanogenesis) that is finally converted into methane and carbon dioxide by the methanogenic bacteria, and the methane and carbon dioxide finally generated are summed and generally referred to as biogas. Therefore, anaerobic digestion can produce energy called biogas at the same time while treating sewage sludge rich in organic matter, so studies on this are actively being conducted.
  • Organic matter can be largely divided into carbohydrates, proteins, and fats, and it has been reported that most proteins and fats have a low treatment rate in the anaerobic digestion process for sewage sludge (Castillo-Hernandez et al., 2015; Elbeshbishy & Nakhla, 2011; Jia et al., 2013; Lee et al., 2016).
  • Korean Patent Publication No. 10-1279135 discloses fermentation strain after adding 10 to 12 parts by weight of pearlite and 5 to 6 parts by weight of loess ceramic balls to a mixing tank based on 100 parts by weight of sludge.
  • a mixing step of adding and mixing A fermentation step of fermenting the sludge for 60 to 100 hours while stirring under aerobic conditions after putting the mixture obtained in the mixing step into a fermentation tank; A first transfer step of returning the fermentation mixture discharged from the fermentation tank to the mixing tank after completion of the fermentation step; A discharge adjustment step of adjusting discharge by introducing the remaining fermentation mixture excluding the fermentation mixture returned to the mixing tank into a discharge control tank; A sorting step of separating the loess ceramic balls contained in the fermentation mixture by introducing the fermentation mixture discharged from the discharge control tank into a separator; A second conveying step of conveying the loess ceramic balls separated in the sorting step to the mixing tank; Disclosed is a fermentation treatment method of sewage sludge comprising a; carrying out step of carrying out the fermented sludge from which the loess ceramic balls are removed in the sorting step.
  • Korean Laid-Open Patent Publication No. 10-2016-0081254 discloses that a complex microorganism stock solution is diluted with water, oxygen and molasses are added to the diluted complex microorganism, and the temperature is controlled, while stirring for prevention of precipitation and uniform mixing.
  • 10-1870797 includes the steps of culturing aerobic and thermophilic microorganisms; Mixing the sludge, the dry sludge conveyed product, and the ventilation improver in a mixing tank; And an aerobic and thermophilic microorganism using a stirring facility to decompose and dry organic matter, and a reaction tank that is divided into first, second, and third sectors, an odor collection line in the reaction tank, a biofiltering system, a recirculating air supply line, and air.
  • a sewage sludge treatment method comprising: a supply device, an air supply line, and a recirculating air discharge line further comprising the steps of drying sludge and removing odors.
  • the present invention was derived from the conventional technical background, and an object of the present invention is to provide a method for pretreating sewage sludge to be suitable for anaerobic digestion using a mixture of specific microorganisms present in sewage sludge or enzymes secreted by them. have. In addition, an object of the present invention is to provide a method of treating the pretreated sewage sludge through an anaerobic digestion process.
  • the inventors of the present invention believe that when the sewage sludge is pretreated using a combination of enzymes produced from certain strains screened from the sewage sludge, the content of volatile suspended solids in the sewage sludge is greatly reduced, and the pretreated sewage sludge is It was confirmed that the treatment with an anaerobic digestion process can promote biogas production, and the present invention was completed.
  • culture supernatant used in the present invention refers to a liquid phase in which a solid/liquid separation method such as centrifugation or filtration is used to remove a solid phase such as cells.
  • culture solution used in the present invention is a product obtained by inoculating and culturing a microorganism (or strain) in a liquid medium, and is a concept including a microorganism (or strain).
  • sewage sludge used in the present invention is a sludge generated and collected in each process of sewage or wastewater treatment, such as raw sludge, excess sludge, industrial wastewater sludge, domestic sewage sludge, human manure, etc. It is a comprehensive concept that includes.
  • biogas used in the present invention is a mixture of gases generated by the decomposition of organic matter in an anaerobic state in the absence of oxygen, and is mainly composed of methane and carbon dioxide, and may contain a small amount of hydrogen or hydrogen sulfide in some cases. I can.
  • an example of the present invention provides a sewage sludge pretreatment method comprising the step of adding a crude enzyme solution mixture to sewage sludge and reacting under aerobic conditions.
  • another example of the present invention provides a sewage sludge pretreatment method comprising the step of adding a microbial culture solution mixture to sewage sludge and reacting under aerobic conditions.
  • the sewage sludge contains proteins and fats.
  • the sewage sludge preferably contains 5 to 20 g/L of volatile suspended solids, and more preferably 8 to 15 g/L of volatile suspended solids. Includes.
  • the crude enzyme solution mixture is a mixture of amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant and Burkholderia vietnamiensis (Burkholderia vietnamiensis) culture supernatant.
  • the microbial culture solution mixture is a mixture of amyloliquefaciens (Bacillus amyloliquefaciens ) culture solution and a Burkholderia vietnamiensis culture solution.
  • the amyloliquefaciens (Bacillus amyloliquefaciens ) is preferably a strain screened from sewage sludge, and a protease having excellent proteolytic activity can be produced in the form of an extracellular enzyme (exoenzyme).
  • the Burkholderia vietnamiensis is preferably a strain screened from sewage sludge, and a lipase having excellent lipolytic activity can be produced in the form of an extracellular enzyme (exoenzyme).
  • the Bacillus amyloliquefaciens strain is a clear ring formed around colonies after smearing and culturing sewage sludge on a solid medium containing a protein substrate such as skim milk. zone) by comparing the size of the first selection of strains having high proteolytic activity; Inoculating and culturing the first selected strain in a liquid medium containing a protein substrate such as Tryptone, and then obtaining a supernatant of the culture medium; And measuring the proteolytic enzyme activity of the supernatant and secondly selecting a strain having high proteolytic activity.
  • amyloliquefaciens (Bacillus amyloliquefaciens ) strain screened by the above method is preferably amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA.
  • the Burkholderia vietnamiensis strain is a clear zone formed around the colonies after smearing and culturing sewage sludge on a solid medium containing a fatty substrate such as Tributyrin.
  • the Burkholderia vietnamiensis strain screened by the above method is preferably Burkholderia vietnamiensis LA-96 comprising the nucleotide sequence of SEQ ID NO: 2 as 16S rDNA.
  • the proteolytic enzyme activity of the amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant constituting the crude enzyme solution mixture is preferably 400 to 450 unit/mL, and more preferably Is 410 ⁇ 440 unit/mL.
  • the lipolytic enzyme activity of the Burkholderia vietnamiensis culture supernatant constituting the crude enzyme solution mixture is preferably 30 to 40 unit/mL, More preferably, it is 32-38 unit/mL.
  • the crude enzyme solution mixture is preferably amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant and Burkholderia vietnamiensis (Burkholderia vietnamiensis) when considering the reduction rate of volatile suspended solids contained in sewage sludge.
  • amyloliquefaciens Bacillus amyloliquefaciens
  • Burkholderia vietnamiensis Burkholderia vietnamiensis
  • the culture supernatant is mixed in a volume ratio of 5:1 to 1:10, more preferably amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant and Burkholderia vietnamiensis (Burkholderia vietnamiensis) culture supernatant is 3: It is mixed in a volume ratio of 1 to 1:6, most preferably amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant and Burkholderia vietnamiensis (Burkholderia vietnamiensis) culture supernatant of 1:2 to 1:5 It is mixed by volume ratio.
  • the proteolytic enzyme activity of the amyloliquefaciens (Bacillus amyloliquefaciens ) culture medium constituting the microbial culture medium mixture is preferably 400 to 450 unit/mL, and more preferably It is between 410 and 440 units/mL.
  • the lipolytic enzyme activity of the culture medium Burkholderia vietnamiensis constituting the microbial culture medium mixture is preferably 30 to 40 unit/mL, and further It is preferably 32 to 38 unit/mL.
  • microorganism culture solution mixture is preferably amyloliquefaciens (Bacillus amyloliquefaciens ) culture solution and Burkholderia vietnamiensis (Burkholderia vietnamiensis) when considering the reduction rate of volatile suspended solids contained in sewage sludge.
  • amyloliquefaciens Bacillus amyloliquefaciens
  • Burkholderia vietnamiensis Burkholderia vietnamiensis
  • the culture solution is mixed in a volume ratio of 5:1 to 1:10, more preferably amyloliquefaciens (Bacillus amyloliquefaciens ) culture solution and Burkholderia vietnamiensis (Burkholderia vietnamiensis) culture solution is 3:1 to 1: It is mixed in a volume ratio of 6, and most preferably, amyloliquefaciens (Bacillus amyloliquefaciens ) culture solution and Burkholderia vietnamiensis culture solution are mixed in a volume ratio of 1:2 to 1:5.
  • Bacillus amyloliquefaciens culture solution and Burkholderia vietnamiensis culture solution are components of a microbial culture solution mixture used in the sewage sludge pretreatment method according to another example of the present invention.
  • the amyloliquefaciens (Bacillus amyloliquefaciens ) culture medium can be obtained by inoculating amyloliquefaciens (Bacillus amyloliquefaciens ) in various known liquid media containing a protein substrate and culturing under predetermined conditions.
  • the protein substrate includes, but is not limited to, skim milk, tryptone, yeast extract, and the like.
  • the Burkholderia vietnamiensis culture solution can be obtained by inoculating Burkholderia vietnamiensis in various known liquid media containing a fatty substrate and culturing under predetermined conditions.
  • the fat substrate includes various vegetable oils such as Tributyrin and olive oil, and various fatty acids such as oleic acid, but is not limited thereto.
  • the culture temperature may be selected in a variety of ranges depending on the strain, for example, at 25-35°C. Can be chosen.
  • the culture time is preferably 8 to 48 hr in terms of ensuring sufficient enzyme production, and It is more preferable that it is 12 to 24 hr.
  • the amount of the coenzyme liquid mixture added is preferably 0.1 to 5 g per 1 liter of sewage sludge in consideration of the reduction rate and economical efficiency of volatile suspended solids, and 1 liter of sewage sludge It is more preferable that it is 0.5 to 2.5 g of sugar.
  • the reaction temperature is preferably 30 to 45 °C and more preferably 35 to 43 °C in consideration of the reduction rate and economy of volatile suspended solids. Do.
  • the reaction time is preferably 48 to 96 hr, and more preferably 60 to 84 hr, considering the reduction rate and economic efficiency of volatile suspended solids. desirable.
  • the amount of the microbial culture medium mixture added is preferably 0.1 to 5 g per 1 liter of sewage sludge in consideration of the reduction rate and economic efficiency of volatile suspended solids, and 1 liter of sewage sludge. It is more preferable that it is 0.5 to 2.5 g of sugar.
  • the reaction temperature is preferably 30 to 45 °C and more preferably 35 to 43 °C in consideration of the reduction rate and economic efficiency of volatile suspended solids. Do.
  • the reaction time is preferably 48 to 96 hr, and more preferably 60 to 84 hr, considering the reduction rate and economic efficiency of volatile suspended solids. desirable.
  • proteins and fats contained in the sewage sludge are hydrolyzed by proteolytic enzymes and lipolytic enzymes to be converted into amino acids and fatty acids.
  • the decomposition of proteins and fats was indirectly measured as a decrease in volatile suspended solids in sewage sludge. Accordingly, the sewage sludge pretreated by the method according to an example or another example of the present invention has a state very suitable for production of biogas by anaerobic digestion afterwards.
  • an example of the present invention is a step of culturing under anaerobic conditions in which oxygen is completely removed by putting a planting source composed of anaerobic microorganisms and sewage sludge pretreated with the above-described coenzyme solution mixture or microbial culture solution mixture in an anaerobic digester. It provides a method for treating sewage sludge by anaerobic digestion comprising a.
  • the culture temperature is preferably 30 to 45° C., and preferably 35 to 40° C., considering the amount of biogas generation and economical efficiency.
  • the cultivation time is preferably 3 to 30 days, more preferably 5 to 15 days, and more preferably 6 to 12 days when considering the amount of biogas generation and economical efficiency. It is most preferable to be work.
  • the crude enzyme solution mixture used in the sewage sludge pretreatment method according to an embodiment of the present invention is a mixture of the culture supernatant of strains screened from sewage sludge and has high proteolytic enzyme activity and lipolytic enzyme activity. Sewage sludge pretreated according to the method according to the method is very suitable for biogas production by anaerobic digestion because the content of volatile suspended solids is greatly reduced.
  • the crude enzyme solution mixture used in the sewage sludge pretreatment method according to an embodiment of the present invention is composed of enzymes produced by microorganisms screened from sewage sludge, it is free from GMO problems and can be mass-produced. Superior economics can be secured. Therefore, the sewage sludge pretreatment method according to an example of the present invention has an advantage applicable to a full-scale facility for economically treating sewage sludge.
  • 1 is a phylogenetic tree of the PA-21 strain finally selected as a strain having protease activity from sewage sludge in an embodiment of the present invention.
  • FIG. 2 is a phylogenetic tree of the LA-96 strain finally selected as a strain having lipolytic enzyme activity from sewage sludge in an embodiment of the present invention.
  • Figure 3 is a coenzyme solution produced from amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 strain of the sewage sludge sample (a) in the embodiment of the present invention, a mixture of three types of coenzyme, Burgholderia vietnamiensis ( Burkholderia vietnamiensis ) is a graph showing the reduction rate of VSS according to the reaction time when pretreated with a crude enzyme solution produced from the LA-96 strain.
  • FIG. 5 is a graph showing the reduction rate
  • FIG. 5 is a coenzyme solution produced from amyloliquefaciens PA-21 strain, a mixture of three coenzymes, and Burg Holderia vietnamiensis (Burkholderia vietnamiensis) is a graph showing the reduction rate of VSS according to the reaction time when pre-treatment with a coenzyme solution produced from LA-96 strain.
  • FIG. 6 is a graph showing the cumulative amount of biogas generation by cultivation time when an unpretreated sewage sludge sample (a) or a pretreated sewage sludge sample (a) is treated by anaerobic digestion in an embodiment of the present invention
  • FIG. 7 is In the embodiment of the present invention, a graph showing the cumulative amount of biogas generation by cultivation time when the unpretreated sewage sludge sample (b) or the pretreated sewage sludge sample (b) is treated by anaerobic digestion
  • FIG. 8 is an embodiment of the present invention. This is a graph showing the cumulative amount of biogas generation by cultivation time when the unpretreated sewage sludge sample (c) or the pretreated sewage sludge sample (c) is treated by anaerobic digestion.
  • Liquid restricted medium containing 1% olive oil main ingredients and content: K 2 HP0 4 0.3g/L, KH 2 P0 4 0.3g/L, (NH 4 ) 2 S0 4 0.5g/L, MgS0 4 7H 2 0 0.4g/L, MgCl 2 6H 2 O 0.7g/L, CaCl 2 2H 2 O 0.5g/L, yeast extract 1g/L) in an amount of 0.1% (w/v)
  • the supernatant was recovered by centrifugation.
  • the recovered strain culture supernatant was regarded as a coenzyme solution, and lipolytic enzyme activity was measured by a microplate assay method.
  • p-nitrophenyl ester pNP-ester
  • the amount of p-nitrophenol released by reaction with the coenzyme solution was determined as an increase in absorbance measured at a wavelength of 405 nm.
  • two methods were mixed according to the type of substrate.
  • each of the first selected strains was inoculated in a liquid LB medium (main ingredients and content: Tryptone 10g/L, Yeast extract 5g/L, NaCl 10g/L) in an amount of 1% (w/v) and After incubation for 24 hr under a stirring condition of temperature and 180 rpm, the supernatant was recovered by centrifugation. Thereafter, the recovered strain culture supernatant was regarded as a crude enzyme solution, and protease activity was measured.
  • Table 1 shows the names of a total of 10 strains having the proteolytic enzyme activity first selected from sewage sludge, the 16S rDNA gene homology analysis results, and the proteolytic enzyme activity measurement results of the strain culture supernatant.
  • FIG. 1 is a phylogenetic tree of the PA-21 strain finally selected as a strain having protease activity from sewage sludge in the embodiment of the present invention.
  • SEQ ID NO: 1 16S rDNA nucleotide sequence (SEQ ID NO: 1) and phylogenetic tree, the species of the PA-21 strain was confirmed as Bacillus amyloliquefaciens, The PA-21 strain was named as Bacillus amyloliquefaciens PA-21.
  • liquid restriction medium containing 1% olive oil main ingredients and content: K 2 HP0 4 0.3g/L, KH 2 P0 4 0.3g/L, (NH 4 ) 2 S0 4 0.5g/L, MgS0 4 7H 2 0 0.4g/L, MgCl 2 6H 2 O 0.7g/L, CaCl 2 2H 2 O 0.5g/L, yeast extract 1g/L) 1% (w/ v) and incubated for 24 hr at a temperature of 25° C. and stirring conditions at 180 rpm, and then centrifuged to recover the supernatant. Thereafter, the recovered strain culture supernatant was regarded as a crude enzyme solution, and the lipolytic enzyme activity was measured.
  • Table 2 shows the names of a total of 10 strains having the lipolytic enzyme activity first selected from sewage sludge, the 16S rDNA gene homology analysis results, and the lipolytic enzyme activity measurement results of the strain culture supernatant.
  • the LA-96 and LA-18 strains showed excellent lipolytic enzyme activity, and among them, the LA-96 strain having the most excellent lipolytic enzyme activity was finally selected. It is a phylogenetic tree of LA-96 strain finally selected as a strain having lipolytic enzyme activity from sewage sludge in Based on the mycological properties of the LA-96 strain, 16S rDNA nucleotide sequence (SEQ ID NO: 2) and phylogenetic tree, the species of the LA-96 strain was confirmed as Burkholderia vietnamiensis, and LA The -96 strain was named Burkholderia vietnamiensis LA-96.
  • Liquid basic medium (main ingredients and content: Fructose 20 g/L, K 2 HPO 4 0.1 g/L, KH 2 PO 4 0.1 g/L, yeast extract 5 g/L, CaCl 2 2H 2 O 1 g/L )
  • 3L amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 strain was inoculated in an amount of 300 g (inoculation amount: 10% w/v) and a temperature of 30° C., pH 6.5, stirring conditions of 200 rpm, and 1.0 vvm The supernatant was recovered by centrifugation after shaking culture for 12 hr under the condition of the amount of air injected.
  • the recovered strain culture supernatant was regarded as a crude enzyme solution, and protease activity was measured.
  • the protease activity of the coenzyme solution produced from the Bacillus amyloliquefaciens PA-21 strain was about 417.7 unit/mL.
  • Liquid basic medium containing 1% oleic acid main ingredients and content: K 2 HP0 4 0.3g/L, KH 2 P0 4 0.3g/L, (NH 4 ) 2 S0 4 0.5g/L, MgS0 4 7H 2 0 0.4g/L, MgCl 2 6H 2 O 0.7g/L, CaCl 2 2H 2 O 0.5g/L, yeast extract 1g/L) Burkholderia vietnamiensis in 3L LA-96 strain was inoculated in an amount of 300g (inoculation amount: 10% w/v) and cultured with shaking for 18 hr at a temperature of 30°C, agitation condition of pH 7.0, 600 rpm, and an air injection amount of 1.66 vvm, followed by centrifugation.
  • main ingredients and content K 2 HP0 4 0.3g/L, KH 2 P0 4 0.3g/L, (NH 4 ) 2 S0 4 0.5g/L, MgS0 4 7H 2
  • the lipolytic enzyme activity of the crude enzyme solution produced from the Burkholderia vietnamiensis LA-96 strain was about 33.6 unit/mL.
  • the coenzyme solution produced from the Bacillus amyloliquefaciens PA-21 strain and the coenzyme solution produced from the Burkholderia vietnamiensis LA-96 strain were 3:1, 1:1 and 1: Each mixture was mixed at a volume ratio of 3 to prepare a mixture of three types of coenzyme solutions.
  • Each raw sludge collected from Busan, Ulsan and Pohang which is a metropolitan area in the Yeongnam area, is filtered through a sieve of 60 mm mesh to remove substances not related to enzyme reactions such as gravel, and volatile suspended solids (Volatile Suspended Solids, hereinafter referred to as'VSS')
  • VSS Volatile Suspended Solids
  • Coenzyme solution produced from Bacillus amyloliquefaciens PA-21 strain in sewage sludge samples, a mixture of three coenzyme solutions, and a coenzyme solution produced from Burkholderia vietnamiensis LA-96 strain Each was added in an amount of 1 g per 1 L of sewage sludge sample, the pH was adjusted to 7.0, and reacted for about 72 hr under aerobic conditions at a temperature of 40° C. and a stirring speed of 120 rpm to pretreat the sewage sludge sample.
  • Figure 3 is a coenzyme solution produced from amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 strain of the sewage sludge sample (a) in the embodiment of the present invention, a mixture of three types of coenzyme, Burgholderia vietnamiensis ( Burkholderia vietnamiensis ) is a graph showing the reduction rate of VSS according to the reaction time when pretreated with a crude enzyme solution produced from the LA-96 strain.
  • FIG. 5 is a graph showing the reduction rate
  • FIG. 5 is a coenzyme solution produced from amyloliquefaciens PA-21 strain, a mixture of three coenzymes, and Burg Holderia vietnamiensis (Burkholderia vietnamiensis) is a graph showing the reduction rate of VSS according to the reaction time when pre-treatment with a coenzyme solution produced from LA-96 strain.
  • a sewage sludge sample was produced from a coenzyme solution produced from amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 strain and a Burkholderia vietnamiensis (Burkholderia vietnamiensis) LA-96 strain.
  • the crude enzyme solution was pretreated with a crude enzyme solution mixture prepared by mixing in a volume ratio of 1:3, the highest VSS reduction rate was shown.
  • Sewage sludge samples were treated by putting a planting source composed of anaerobic microorganisms and a predetermined sewage sludge sample in an anaerobic digester, completely removing oxygen, and incubating under anaerobic conditions at pH 7 and 37.5°C for about 30 days. The amount of gas generated was measured.
  • 6 is a graph showing the cumulative amount of biogas generation by cultivation time when an unpretreated sewage sludge sample (a) or a pretreated sewage sludge sample (a) is treated by anaerobic digestion in an embodiment of the present invention, and FIG.
  • FIG. 7 is In the embodiment of the present invention, a graph showing the cumulative amount of biogas generation by cultivation time when the unpretreated sewage sludge sample (b) or the pretreated sewage sludge sample (b) is treated by anaerobic digestion
  • FIG. 8 is an embodiment of the present invention. This is a graph showing the cumulative amount of biogas generation by cultivation time when the unpretreated sewage sludge sample (c) or the pretreated sewage sludge sample (c) is treated by anaerobic digestion.
  • “Inoculum (Ctrl)” denotes a case where only a planting source composed of anaerobic microorganisms is put in an anaerobic digestion tank and cultivated
  • “1S” denotes a planting source composed of anaerobic microorganisms in an anaerobic digester and unpretreated sewage sludge sample
  • “1S+P1L3(1)” and “1S+P1L3(2)” refer to the sequence of the results of repeated experiments under the same process conditions.
  • sewage sludge sample (a) when the pretreated sewage sludge was anaerobic digested, biogas production increased by about 13.2% compared to the case of anaerobic digestion of unpretreated sewage sludge, and in the case of sewage sludge sample (b), pretreatment When the treated sewage sludge was anaerobic digested, biogas production increased by about 54.7% compared to the anaerobic digestion of untreated sewage sludge, and the sewage sludge sample (c) was not pretreated when the pretreated sewage sludge was anaerobic digested. Compared to the case of anaerobic digestion of sludge, biogas production increased by about 35.1%.

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Abstract

An embodiment of the present invention provides a method for pretreating sewage sludge, the method comprising a step for adding a coenzyme mixture to sewage sludge and reacting the resultant mixture under aerobic conditions. Since the coenzyme mixture contains a culture supernatant of strains screened from sewage sludge and has high protease activity and lipase activity, sewage sludge pretreated by the method according to an embodiment of the present invention contains significantly reduced amounts of volatile suspended solids, and is thus highly suitable for biogas production using anaerobic digestion. Therefore, the method for pretreating sewage sludge according to an embodiment of the present invention has the advantage of being applicable to a full-scale facility for economically treating sewage sludge.

Description

혼합 효소를 이용한 하수 슬러지 전처리 방법 및 혐기소화에 의한 하수 슬러지 처리방법Sewage sludge pretreatment method using mixed enzyme and sewage sludge treatment method by anaerobic digestion
본 발명은 하수 슬러지의 전처리 방법 및 이를 이용한 하수 슬러지의 처리방법에 관한 것으로서, 더 상세하게는 하수 슬러지에 존재하는 특정 미생물들 또는 이들이 분비하는 효소들의 혼합물을 이용하여 혐기소화에 적합하도록 하수 슬러지를 전처리하는 방법과, 전처리된 하수 슬러지를 혐기소화에 의해 처리하는 방법에 관한 것이다.The present invention relates to a method of pretreatment of sewage sludge and a method of treating sewage sludge using the same, and more specifically, a sewage sludge suitable for anaerobic digestion using a mixture of specific microorganisms present in sewage sludge or enzymes secreted therefrom. It relates to a method of pretreatment and a method of treating pretreated sewage sludge by anaerobic digestion.
혐기소화는 유기물이 산소가 없는 혐기 상태에서 혐기소화 미생물에 의해 가수분해, 산 발효 과정을 거쳐 최종적으로 메탄과 이산화탄소를 주성분으로 하는 바이오가스로 전환되는 일련의 생화학적 과정을 말한다. 구체적으로, 혐기소화 공정은 크게 탄수화물, 단백질, 지방 등으로 구성된 유기물이 혐기 미생물의 체외효소(extracellular enzyme)에 의해 가수분해되어 이당류, 단당류, 아미노산, 지방산 등과 같은 단량체로 쪼개지는 가수분해 단계(Hydrolysis); 단량체가 산생성 미생물(acidogen)의 먹이로 이용되고 미생물의 신진대사 작용에 의해 다양한 유기산(Volatile fatty acids, VFA)로 전환되는 산발효 단계(Acidogenesis); 유기산을 이용하는 미생물들에 의해 유기산이 아세트산, 수소 및 이산화탄소로 전환되는 아세트산 생성 단계(Acetogenesis); 및 최종적으로 메탄 생성균에 의해 메탄과 이산화탄소로 전환되는 메탄 생성 단계(Methanogenesis)로 구성되고, 최종적으로 생성된 메탄과 이산화탄소를 합하여 일반적으로 바이오가스(Biogas)라 한다. 따라서, 혐기 소화는 유기물이 풍부한 하수 슬러지를 처리함과 동시에 바이오가스라는 에너지를 생산할 수 있어 이에 대한 연구가 활발히 진행되고 있다. 유기물은 크게 탄수화물, 단백질, 지방으로 나뉠 수 있으며, 하수 슬러지에 대한 혐기소화 공정에서 대부분 단백질과 지방의 처리율이 낮은 것으로 보고되고 있다(Castillo-Hernandez et al., 2015; Elbeshbishy & Nakhla, 2011; Jia et al., 2013; Lee et al., 2016).Anaerobic digestion refers to a series of biochemical processes in which organic matter is converted into biogas containing methane and carbon dioxide as main components through hydrolysis and acid fermentation by anaerobic microorganisms in an anaerobic state without oxygen. Specifically, the anaerobic digestion process is a hydrolysis step in which organic substances composed of carbohydrates, proteins, fats, etc. are hydrolyzed by the extracellular enzyme of anaerobic microorganisms and split into monomers such as disaccharides, monosaccharides, amino acids, fatty acids, etc. ); Acid fermentation step (Acidogenesis) in which monomers are used as food for acidogens and are converted into various organic acids (Volatile fatty acids (VFA)) by the metabolism of the microorganisms; Acetogenesis, in which organic acids are converted into acetic acid, hydrogen and carbon dioxide by microorganisms using organic acids; And it is composed of a methane generation step (Methanogenesis) that is finally converted into methane and carbon dioxide by the methanogenic bacteria, and the methane and carbon dioxide finally generated are summed and generally referred to as biogas. Therefore, anaerobic digestion can produce energy called biogas at the same time while treating sewage sludge rich in organic matter, so studies on this are actively being conducted. Organic matter can be largely divided into carbohydrates, proteins, and fats, and it has been reported that most proteins and fats have a low treatment rate in the anaerobic digestion process for sewage sludge (Castillo-Hernandez et al., 2015; Elbeshbishy & Nakhla, 2011; Jia et al., 2013; Lee et al., 2016).
하수 슬러지의 생물학적 처리에 관한 최근 기술을 살펴보면, 대한민국 등록특허공보 제10-1279135호에는 슬러지 100중량부에 대하여 펄라이트 10 내지 12중량부, 황토 세라믹볼 5 내지 6중량부를 혼합조에 투입한 후 발효 균주를 첨가하여 혼합하는 혼합단계와; 상기 혼합단계에서 수득한 혼합물을 발효조에 투입한 후 호기조건에서 교반하면서 상기 슬러지를 60 내지 100시간 동안 발효시키는 발효단계와; 상기 발효단계 완료 후 상기 발효조에서 배출되는 발효 혼합물을 상기 혼합조로 반송시키는 제 1반송단계와; 상기 혼합조로 반송되는 발효 혼합물을 제외한 나머지 발효 혼합물을 배출조정조에 투입하여 배출을 조정하는 배출조정단계와; 상기 배출조정조에서 배출되는 발효 혼합물을 선별기에 투입하여 상기 발효 혼합물 중에 포함된 상기 황토 세라믹볼을 분리하는 선별단계와; 상기 선별단계에서 분리된 상기 황토 세라믹볼을 상기 혼합조로 반송시키는 제 2반송단계와; 상기 선별단계에서 상기 황토 세라믹볼이 제거된 발효 슬러지를 반출시키는 반출단계;를 포함하는 것을 특징으로 하는 하수 슬러지의 발효 처리방법이 개시되어 있다. 또한, 대한민국 공개특허공보 제10-2016-0081254호에는 물을 이용하여 복합미생물 원액을 희석하고, 희석된 복합미생물에 산소 및 당밀을 투입하고 온도를 조절하며 침전방지와 균일한 혼합을 위해 교반하면서 복합미생물을 배양하는 단계; 복합미생물과 슬러지를 혼합조에 투입한 후 균일한 농도로 혼합하기 위해 교반하는 단계; 균일한 농도로 혼합된 복합미생물과 슬러지를 저류조 또는 별도의 저장조 또는 소화조에 투입하는 단계; 저류조 또는 별도의 저장조 또는 소화조에 복합미생물의 대사 촉진을 위해 일정 온도로 유지하고 산소를 공급하여 교반하면서 슬러지를 감량하는 단계를 포함하는 복합미생물을 이용한 하수처리장의 슬러지 감량방법이 개시되어 있다. 또한, 대한미국 등록특허공보 제10-1870797호에는 호기 및 호열성 미생물을 배양하는 단계; 혼합조에서 슬러지, 건조 슬러지 반송물 및 통기개량제를 혼합하는 단계; 및 교반시설을 이용하여 호기 및 호열성 미생물로 유기물을 분해하여 건조시키고, 제1, 2, 3 섹터로 구역이 나누어지는 반응조, 상기 반응조에 악취포집라인, 바이오 필터링 시스템, 재순환 공기 공급라인, 공기 공급장치, 공기 공급라인 및 재순환 공기 배출라인이 더 포함되어 슬러지 건조 및 악취를 제거하는 단계;로 이루어지는 하수슬러지 처리방법이 개시되어 있다.Looking at the latest technology for biological treatment of sewage sludge, Korean Patent Publication No. 10-1279135 discloses fermentation strain after adding 10 to 12 parts by weight of pearlite and 5 to 6 parts by weight of loess ceramic balls to a mixing tank based on 100 parts by weight of sludge. A mixing step of adding and mixing; A fermentation step of fermenting the sludge for 60 to 100 hours while stirring under aerobic conditions after putting the mixture obtained in the mixing step into a fermentation tank; A first transfer step of returning the fermentation mixture discharged from the fermentation tank to the mixing tank after completion of the fermentation step; A discharge adjustment step of adjusting discharge by introducing the remaining fermentation mixture excluding the fermentation mixture returned to the mixing tank into a discharge control tank; A sorting step of separating the loess ceramic balls contained in the fermentation mixture by introducing the fermentation mixture discharged from the discharge control tank into a separator; A second conveying step of conveying the loess ceramic balls separated in the sorting step to the mixing tank; Disclosed is a fermentation treatment method of sewage sludge comprising a; carrying out step of carrying out the fermented sludge from which the loess ceramic balls are removed in the sorting step. In addition, Korean Laid-Open Patent Publication No. 10-2016-0081254 discloses that a complex microorganism stock solution is diluted with water, oxygen and molasses are added to the diluted complex microorganism, and the temperature is controlled, while stirring for prevention of precipitation and uniform mixing. Culturing complex microorganisms; Agitating the complex microorganisms and sludge in a mixing tank and then mixing them to a uniform concentration; Injecting the complex microorganisms and sludge mixed at a uniform concentration into a storage tank or a separate storage tank or digester; Disclosed is a method for reducing sludge in a sewage treatment plant using a complex microorganism comprising the step of reducing sludge while maintaining a storage tank or a separate storage tank or a digester at a constant temperature for promoting the metabolism of complex microorganisms, supplying oxygen, and stirring. In addition, Korean US Patent Publication No. 10-1870797 includes the steps of culturing aerobic and thermophilic microorganisms; Mixing the sludge, the dry sludge conveyed product, and the ventilation improver in a mixing tank; And an aerobic and thermophilic microorganism using a stirring facility to decompose and dry organic matter, and a reaction tank that is divided into first, second, and third sectors, an odor collection line in the reaction tank, a biofiltering system, a recirculating air supply line, and air. Disclosed is a sewage sludge treatment method comprising: a supply device, an air supply line, and a recirculating air discharge line further comprising the steps of drying sludge and removing odors.
본 발명은 종래의 기술적 배경하에서 도출된 것으로서, 본 발명의 목적은 하수 슬러지에 존재하는 특정 미생물들 또는 이들이 분비하는 효소들의 혼합물을 이용하여 혐기소화에 적합하도록 하수 슬러지를 전처리하는 방법을 제공하는데에 있다. 또한, 본 발명의 목적은 전처리된 하수 슬러지를 혐기소화 공정으로 처리하는 방법을 제공하는데에 있다.The present invention was derived from the conventional technical background, and an object of the present invention is to provide a method for pretreating sewage sludge to be suitable for anaerobic digestion using a mixture of specific microorganisms present in sewage sludge or enzymes secreted by them. have. In addition, an object of the present invention is to provide a method of treating the pretreated sewage sludge through an anaerobic digestion process.
본 발명의 발명자들은 하수 슬러지에서 스크리닝된 소정의 균주들로부터 생산된 효소들의 조합을 이용하여 하수 슬러지를 전처리하면 하수 슬러지 내의 휘발성 부유물질(Volatile Suspended Solids) 함량이 크게 감소되고, 전처리된 하수 슬러지를 혐기소화 공정으로 처리하면 바이오가스 생산을 촉진시킬 수 있다는 점을 확인하고 본 발명을 완성하였다.The inventors of the present invention believe that when the sewage sludge is pretreated using a combination of enzymes produced from certain strains screened from the sewage sludge, the content of volatile suspended solids in the sewage sludge is greatly reduced, and the pretreated sewage sludge is It was confirmed that the treatment with an anaerobic digestion process can promote biogas production, and the present invention was completed.
본 발명에서 사용하는 용어 "배양 상등액"은 미생물(또는 균주)의 배양액을 원심분리, 여과 등과 같은 고체/액체 분리 방법을 사용하여 균체 등과 같은 고상을 제거한 액상을 의미한다.The term "culture supernatant" used in the present invention refers to a liquid phase in which a solid/liquid separation method such as centrifugation or filtration is used to remove a solid phase such as cells.
본 발명에서 사용하는 용어 "배양액"은 액상 배지에 미생물(또는 균주)을 접종하고 배양하여 얻은 산물로서, 미생물(또는 균주)를 포함하는 개념이다.The term "culture solution" used in the present invention is a product obtained by inoculating and culturing a microorganism (or strain) in a liquid medium, and is a concept including a microorganism (or strain).
본 발명에서 사용하는 용어 "하수 슬러지"는 하수 또는 폐수 처리의 각 공정에서 발생하여 집수된 슬러지로서, 생슬러지(raw sludge), 잉여슬러지(waste sludge), 산업폐수오니, 생활하수오니, 인분뇨 등을 포함하는 포괄적인 개념이다.The term "sewage sludge" used in the present invention is a sludge generated and collected in each process of sewage or wastewater treatment, such as raw sludge, excess sludge, industrial wastewater sludge, domestic sewage sludge, human manure, etc. It is a comprehensive concept that includes.
본 발명에서 사용하는 용어 "바이오가스"는 산소가 존재하지 않는 혐기 상태에서 유기물의 분해에 의해 발생하는 가스들의 혼합물로서, 주로 메탄과 이산화탄소로 구성되고 경우에 따라 소량의 수소, 황화수소 등을 포함할 수 있다.The term "biogas" used in the present invention is a mixture of gases generated by the decomposition of organic matter in an anaerobic state in the absence of oxygen, and is mainly composed of methane and carbon dioxide, and may contain a small amount of hydrogen or hydrogen sulfide in some cases. I can.
상기 목적을 달성하기 위하여, 본 발명의 일 예는 하수 슬러지에 조효소액 혼합물을 첨가하고 호기 조건에서 반응시키는 단계를 포함하는 하수 슬러지 전처리 방법을 제공한다. 또한, 본 발명의 다른 예는 하수 슬러지에 미생물 배양액 혼합물을 첨가하고 호기 조건에서 반응시키는 단계를 포함하는 하수 슬러지 전처리 방법을 제공한다.In order to achieve the above object, an example of the present invention provides a sewage sludge pretreatment method comprising the step of adding a crude enzyme solution mixture to sewage sludge and reacting under aerobic conditions. In addition, another example of the present invention provides a sewage sludge pretreatment method comprising the step of adding a microbial culture solution mixture to sewage sludge and reacting under aerobic conditions.
본 발명의 일 예 또는 다른 예에 따른 하수 슬러지 전처리 방법에서 상기 하수 슬러지는 단백질 및 지방을 함유한다. 또한, 상기 하수 슬러지는 바람직하게는 5~20 g/L 농도의 휘발성 부유물질(Volatile Suspended Solids)을 포함하고, 더 바람직하게는 8~15 g/L 농도의 휘발성 부유물질(Volatile Suspended Solids)을 포함한다.In the sewage sludge pretreatment method according to one or another example of the present invention, the sewage sludge contains proteins and fats. In addition, the sewage sludge preferably contains 5 to 20 g/L of volatile suspended solids, and more preferably 8 to 15 g/L of volatile suspended solids. Includes.
본 발명의 일 예에 따른 하수 슬러지 전처리 방법에서 상기 조효소액 혼합물은 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양 상등액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양 상등액의 혼합물이다. 또한, 본 발명의 다른 예에 따른 하수 슬러지 전처리 방법에서 상기 미생물 배양액 혼합물은 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액의 혼합물이다. 상기 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens)는 바람직하게는 하수 슬러지에서 스크리닝된 균주로서 단백질 분해 활성이 우수한 프로테아제(protease)를 세포외효소(exoenzyme) 형태로 생산할 수 있다. 또한, 상기 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis)는 바람직하게는 하수 슬러지에서 스크리닝된 균주로서 지방 분해 활성이 우수한 리파제(lipase)를 세포외효소(exoenzyme) 형태로 생산할 수 있다. 예를 들어, 상기 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 균주는 하수 슬러지를 스킴밀크(Skim milk) 등과 같은 단백질 기질이 함유된 고체 배지에 도말하고 배양한 후 콜로니 주위에 형성되는 투명환(clear zone)의 크기를 비교하여 단백질 분해 활성이 높은 균주를 1차 선별하는 단계; 상기 1차 선별된 균주를 트립톤(Tryptone) 등과 같은 단백질 기질이 함유된 액상 배지에 접종하고 배양한 후 배양액의 상등액을 수득하는 단계; 및 상기 상등액의 단백질 분해 효소 활성을 측정하여 단백질 분해 활성이 높은 균주를 2차 선별하는 단계로 구성되는 방법을 통해 스크리닝될 수 있다. 상기 방법에 의해 스크리닝된 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 균주는 바람직하게는 16S rDNA로 서열번호 1의 염기서열을 포함하는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21이다. 또한, 상기 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 균주는 하수 슬러지를 트리부티린(Tributyrin) 등과 같은 지방 기질이 함유된 고체 배지에 도말하고 배양한 후 콜로니 주위에 형성되는 투명환(clear zone)의 크기를 비교하여 지방 분해 활성이 높은 균주를 1차 선별하는 단계; 상기 1차 선별된 균주를 올리브 오일 등과 같은 지방 기질이 함유된 액상 배지에 접종하고 배양한 후 배양액의 상등액을 수득하는 단계; 및 상기 상등액의 지방 분해 효소 활성을 측정하여 지방 분해 활성이 높은 균주를 2차 선별하는 단계로 구성되는 방법을 통해 스크리닝될 수 있다. 상기 방법에 의해 스크리닝된 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 균주는 바람직하게는 16S rDNA로 서열번호 2의 염기서열을 포함하는 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96이다.In the sewage sludge pretreatment method according to an embodiment of the present invention, the crude enzyme solution mixture is a mixture of amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant and Burkholderia vietnamiensis (Burkholderia vietnamiensis) culture supernatant. In addition, in the sewage sludge pretreatment method according to another example of the present invention, the microbial culture solution mixture is a mixture of amyloliquefaciens (Bacillus amyloliquefaciens ) culture solution and a Burkholderia vietnamiensis culture solution. The amyloliquefaciens (Bacillus amyloliquefaciens ) is preferably a strain screened from sewage sludge, and a protease having excellent proteolytic activity can be produced in the form of an extracellular enzyme (exoenzyme). In addition, the Burkholderia vietnamiensis is preferably a strain screened from sewage sludge, and a lipase having excellent lipolytic activity can be produced in the form of an extracellular enzyme (exoenzyme). For example, the Bacillus amyloliquefaciens strain is a clear ring formed around colonies after smearing and culturing sewage sludge on a solid medium containing a protein substrate such as skim milk. zone) by comparing the size of the first selection of strains having high proteolytic activity; Inoculating and culturing the first selected strain in a liquid medium containing a protein substrate such as Tryptone, and then obtaining a supernatant of the culture medium; And measuring the proteolytic enzyme activity of the supernatant and secondly selecting a strain having high proteolytic activity. The amyloliquefaciens (Bacillus amyloliquefaciens ) strain screened by the above method is preferably amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 comprising the nucleotide sequence of SEQ ID NO: 1 as 16S rDNA. In addition, the Burkholderia vietnamiensis strain is a clear zone formed around the colonies after smearing and culturing sewage sludge on a solid medium containing a fatty substrate such as Tributyrin. Comparing the size of the first selection of a strain having high lipolytic activity; Inoculating and culturing the first selected strain in a liquid medium containing a fatty substrate such as olive oil, and then obtaining a supernatant of the culture medium; And by measuring the lipolytic enzyme activity of the supernatant may be screened through a method comprising the step of secondary selection of a strain having a high lipolytic activity. The Burkholderia vietnamiensis strain screened by the above method is preferably Burkholderia vietnamiensis LA-96 comprising the nucleotide sequence of SEQ ID NO: 2 as 16S rDNA.
본 발명의 일 예에 따른 하수 슬러지 전처리 방법에서 상기 조효소액 혼합물을 구성하는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양 상등액의 단백질 분해 효소 활성은 바람직하게는 400~450 unit/mL이고 더 바람직하게는 410~440 unit/mL이다. 또한, 본 발명의 일 예에 따른 하수 슬러지 전처리 방법에서 상기 조효소액 혼합물을 구성하는 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양 상등액의 지방 분해 효소 활성은 바람직하게는 30~40 unit/mL이고, 더 바람직하게는 32~38 unit/mL이다. 또한, 상기 조효소액 혼합물은 하수 슬러지에 함유된 휘발성 부유물질(Volatile Suspended Solids)의 감소율을 고려할 때 바람직하게는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양 상등액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양 상등액을 5:1 내지 1:10의 부피비로 혼합한 것이고, 더 바람직하게는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양 상등액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양 상등액을 3:1 내지 1:6의 부피비로 혼합한 것이며, 가장 바람직하게는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양 상등액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양 상등액을 1:2 내지 1:5의 부피비로 혼합한 것이다.In the sewage sludge pretreatment method according to an embodiment of the present invention, the proteolytic enzyme activity of the amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant constituting the crude enzyme solution mixture is preferably 400 to 450 unit/mL, and more preferably Is 410~440 unit/mL. In addition, in the sewage sludge pretreatment method according to an embodiment of the present invention, the lipolytic enzyme activity of the Burkholderia vietnamiensis culture supernatant constituting the crude enzyme solution mixture is preferably 30 to 40 unit/mL, More preferably, it is 32-38 unit/mL. In addition, the crude enzyme solution mixture is preferably amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant and Burkholderia vietnamiensis (Burkholderia vietnamiensis) when considering the reduction rate of volatile suspended solids contained in sewage sludge. ) The culture supernatant is mixed in a volume ratio of 5:1 to 1:10, more preferably amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant and Burkholderia vietnamiensis (Burkholderia vietnamiensis) culture supernatant is 3: It is mixed in a volume ratio of 1 to 1:6, most preferably amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant and Burkholderia vietnamiensis (Burkholderia vietnamiensis) culture supernatant of 1:2 to 1:5 It is mixed by volume ratio.
본 발명의 다른 예에 따른 하수 슬러지 전처리 방법에서 상기 미생물 배양액 혼합물을 구성하는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액의 단백질 분해 효소 활성은 바람직하게는 400~450 unit/mL이고 더 바람직하게는 410~440 unit/mL이다. 또한, 본 발명의 다른 예에 따른 하수 슬러지 전처리 방법에서 상기 미생물 배양액 혼합물을 구성하는 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액의 지방 분해 효소 활성은 바람직하게는 30~40 unit/mL이고, 더 바람직하게는 32~38 unit/mL이다. 또한, 상기 미생물 배양액 혼합물은 하수 슬러지에 함유된 휘발성 부유물질(Volatile Suspended Solids)의 감소율을 고려할 때 바람직하게는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액을 5:1 내지 1:10의 부피비로 혼합한 것이고, 더 바람직하게는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액을 3:1 내지 1:6의 부피비로 혼합한 것이며, 가장 바람직하게는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액을 1:2 내지 1:5의 부피비로 혼합한 것이다.In the sewage sludge pretreatment method according to another embodiment of the present invention, the proteolytic enzyme activity of the amyloliquefaciens (Bacillus amyloliquefaciens ) culture medium constituting the microbial culture medium mixture is preferably 400 to 450 unit/mL, and more preferably It is between 410 and 440 units/mL. In addition, in the sewage sludge pretreatment method according to another example of the present invention, the lipolytic enzyme activity of the culture medium Burkholderia vietnamiensis constituting the microbial culture medium mixture is preferably 30 to 40 unit/mL, and further It is preferably 32 to 38 unit/mL. In addition, the microorganism culture solution mixture is preferably amyloliquefaciens (Bacillus amyloliquefaciens ) culture solution and Burkholderia vietnamiensis (Burkholderia vietnamiensis) when considering the reduction rate of volatile suspended solids contained in sewage sludge. The culture solution is mixed in a volume ratio of 5:1 to 1:10, more preferably amyloliquefaciens (Bacillus amyloliquefaciens ) culture solution and Burkholderia vietnamiensis (Burkholderia vietnamiensis) culture solution is 3:1 to 1: It is mixed in a volume ratio of 6, and most preferably, amyloliquefaciens (Bacillus amyloliquefaciens ) culture solution and Burkholderia vietnamiensis culture solution are mixed in a volume ratio of 1:2 to 1:5.
본 발명의 일 예에 따른 하수 슬러지 전처리 방법에서 상기 조효소액 혼합물을 구성하는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양 상등액은 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액에서 원심분리, 여과 등과 같은 고체/액체 분리 방법을 사용하여 균체 등과 같은 고상을 제거한 것이다. 또한, 본 발명의 일 예에 따른 하수 슬러지 전처리 방법에서 상기 조효소액 혼합물을 구성하는 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양 상등액도 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액에서 원심분리, 여과 등과 같은 고체/액체 분리 방법을 사용하여 균체 등과 같은 고상을 제거한 것이다. 또한, 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액은 본 발명의 다른 예에 따른 하수 슬러지 전처리 방법에서 사용되는 미생물 배양액 혼합물의 구성요소이다. 상기 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액은 단백질 기질이 함유된 공지의 다양한 액상 배지에 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens)를 접종하고 소정의 조건에서 배양하여 얻어질 수 있다. 상기 단백질 기질에는 스킴밀크(Skim milk), 트립톤(Tryptone), 효소추출물(Yeast extract) 등이 있으나, 반드시 이에 제한되지 않는다. 또한, 상기 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액은 지방 기질이 함유된 공지의 다양한 액상 배지에 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis)를 접종하고 소정의 조건에서 배양하여 얻어질 수 있다. 상기 지방 기질에는 트리부트린(Tributyrin), 올리브 오일 등과 같은 다양한 식물성 오일, 올레산(Oleic acid) 등과 같은 다양한 지방산 등이 있으나, 반드시 여기에 제한되지 않는다. 상기 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액 또는 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액을 제조할 때 배양 온도는 균주에 따라 다양한 범위에서 선택될 수 있고, 예를 들어 25~35℃에서 선택될 수 있다. 또한, 상기 상기 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액 또는 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액을 제조할 때 배양 시간은 충분한 효소 생산을 담보하는 측면에서 8~48 hr인 것이 바람직하고 12~24 hr인 것이 더 바람직하다.In Riku as amyl constituting the crude enzyme mixture from the sewage sludge pre-treatment method according to an embodiment of the present invention Pacific Enschede (Bacillus amyloliquefaciens) culture supernatant on Riku as amyl Pacific Enschede (Bacillus amyloliquefaciens) centrifugation in culture, such as filtration The solid/liquid separation method is used to remove solid phases such as cells. In addition, in the sewage sludge pretreatment method according to an embodiment of the present invention, the Burkholderia vietnamiensis culture supernatant constituting the crude enzyme solution mixture is also centrifuged and filtered in the Burkholderia vietnamiensis culture solution. Solid phases such as cells are removed using a solid/liquid separation method such as. In addition, Bacillus amyloliquefaciens culture solution and Burkholderia vietnamiensis culture solution are components of a microbial culture solution mixture used in the sewage sludge pretreatment method according to another example of the present invention. The amyloliquefaciens (Bacillus amyloliquefaciens ) culture medium can be obtained by inoculating amyloliquefaciens (Bacillus amyloliquefaciens ) in various known liquid media containing a protein substrate and culturing under predetermined conditions. The protein substrate includes, but is not limited to, skim milk, tryptone, yeast extract, and the like. In addition, the Burkholderia vietnamiensis culture solution can be obtained by inoculating Burkholderia vietnamiensis in various known liquid media containing a fatty substrate and culturing under predetermined conditions. The fat substrate includes various vegetable oils such as Tributyrin and olive oil, and various fatty acids such as oleic acid, but is not limited thereto. When preparing the amyloliquefaciens ( Bacillus amyloliquefaciens ) culture solution or Burkholderia vietnamiensis culture solution, the culture temperature may be selected in a variety of ranges depending on the strain, for example, at 25-35°C. Can be chosen. In addition, when preparing the amyloliquefaciens (Bacillus amyloliquefaciens ) culture solution or Burkholderia vietnamiensis culture solution, the culture time is preferably 8 to 48 hr in terms of ensuring sufficient enzyme production, and It is more preferable that it is 12 to 24 hr.
본 발명의 일 예에 따른 하수 슬러지 전처리 방법에서 상기 조효소액 혼합물의 첨가량은 휘발성 부유물질(Volatile Suspended Solids)의 감소율 및 경제성 등을 고려할 때 하수 슬러지 1L 당 0.1~5g인 것이 바람직하고, 하수 슬러지 1L 당 0.5~2.5g인 것이 더 바람직하다. 또한, 본 발명의 일 예에 따른 하수 슬러지 전처리 방법에서 상기 반응 온도는 휘발성 부유물질(Volatile Suspended Solids)의 감소율 및 경제성 등을 고려할 때 30~45℃인 것이 바람직하고 35~43℃인 것이 더 바람직하다. 또한, 본 발명의 일 예에 따른 하수 슬러지 전처리 방법에서 상기 반응 시간은 휘발성 부유물질(Volatile Suspended Solids)의 감소율 및 경제성 등을 고려할 때 48~96 hr인 것이 바람직하고, 60~84 hr인 것이 더 바람직하다.In the sewage sludge pretreatment method according to an embodiment of the present invention, the amount of the coenzyme liquid mixture added is preferably 0.1 to 5 g per 1 liter of sewage sludge in consideration of the reduction rate and economical efficiency of volatile suspended solids, and 1 liter of sewage sludge It is more preferable that it is 0.5 to 2.5 g of sugar. In addition, in the sewage sludge pretreatment method according to an embodiment of the present invention, the reaction temperature is preferably 30 to 45 °C and more preferably 35 to 43 °C in consideration of the reduction rate and economy of volatile suspended solids. Do. In addition, in the sewage sludge pretreatment method according to an embodiment of the present invention, the reaction time is preferably 48 to 96 hr, and more preferably 60 to 84 hr, considering the reduction rate and economic efficiency of volatile suspended solids. desirable.
본 발명의 다른 예에 따른 하수 슬러지 전처리 방법에서 상기 미생물 배양액 혼합물의 첨가량은 휘발성 부유물질(Volatile Suspended Solids)의 감소율 및 경제성 등을 고려할 때 하수 슬러지 1L 당 0.1~5g인 것이 바람직하고, 하수 슬러지 1L 당 0.5~2.5g인 것이 더 바람직하다. 또한, 본 발명의 다른 예에 따른 하수 슬러지 전처리 방법에서 상기 반응 온도는 휘발성 부유물질(Volatile Suspended Solids)의 감소율 및 경제성 등을 고려할 때 30~45℃인 것이 바람직하고 35~43℃인 것이 더 바람직하다. 또한, 본 발명의 다른 예에 따른 하수 슬러지 전처리 방법에서 상기 반응 시간은 휘발성 부유물질(Volatile Suspended Solids)의 감소율 및 경제성 등을 고려할 때 48~96 hr인 것이 바람직하고, 60~84 hr인 것이 더 바람직하다.In the sewage sludge pretreatment method according to another example of the present invention, the amount of the microbial culture medium mixture added is preferably 0.1 to 5 g per 1 liter of sewage sludge in consideration of the reduction rate and economic efficiency of volatile suspended solids, and 1 liter of sewage sludge. It is more preferable that it is 0.5 to 2.5 g of sugar. In addition, in the sewage sludge pretreatment method according to another example of the present invention, the reaction temperature is preferably 30 to 45 °C and more preferably 35 to 43 °C in consideration of the reduction rate and economic efficiency of volatile suspended solids. Do. In addition, in the sewage sludge pretreatment method according to another example of the present invention, the reaction time is preferably 48 to 96 hr, and more preferably 60 to 84 hr, considering the reduction rate and economic efficiency of volatile suspended solids. desirable.
본 발명의 일 예 또는 다른 예에 따른 방법으로 하수 슬러지를 전처리하는 경우 하수 슬러지에 함유된 단백질 및 지방은 단백질 분해 효소와 지방 분해 효소에 의해 가수분해되어 아미노산 및 지방산으로 전환된다. 본 발명에서는 이러한 단백질 및 지방의 분해를 하수 슬러지 내의 휘발성 부유물질(Volatile Suspended Solids)이 감소하는 것으로 간접적으로 측정하였다. 따라서, 본 발명의 일 예 또는 다른 예에 따른 방법으로 전처리된 하수 슬러지는 이후 혐기소화에 의한 바이오가스 생산에 매우 적합한 상태를 가지게 된다.When the sewage sludge is pretreated by a method according to an example or another example of the present invention, proteins and fats contained in the sewage sludge are hydrolyzed by proteolytic enzymes and lipolytic enzymes to be converted into amino acids and fatty acids. In the present invention, the decomposition of proteins and fats was indirectly measured as a decrease in volatile suspended solids in sewage sludge. Accordingly, the sewage sludge pretreated by the method according to an example or another example of the present invention has a state very suitable for production of biogas by anaerobic digestion afterwards.
상기 목적을 달성하기 위하여, 본 발명의 일 예는 혐기 소화조에 혐기 미생물로 구성된 식종원과 전술한 조효소액 혼합물 또는 미생물 배양액 혼합물에 의해 전처리된 하수 슬러지를 넣고 산소를 완전히 제거한 혐기 조건에서 배양하는 단계를 포함하는 혐기소화에 의한 하수 슬러지 처리방법을 제공한다. 본 발명의 일 예에 따른 혐기소화에 의한 하수 슬러지 처리방법에서 상기 배양 온도는 바이오가스 생성량 및 경제성 등을 고려할 때 30~45℃인 것이 바람직하고 35~40℃인 것이 바람직하다. 또한, 본 발명의 일 예에 따른 혐기소화에 의한 하수 슬러지 처리방법에서 상기 배양 시간은 바이오가스 생성량 및 경제성 등을 고려할 때 3~30일 인 것이 바람직하고 5~15일인 것이 더 바람직하고 6~12일인 것이 가장 바람직하다.In order to achieve the above object, an example of the present invention is a step of culturing under anaerobic conditions in which oxygen is completely removed by putting a planting source composed of anaerobic microorganisms and sewage sludge pretreated with the above-described coenzyme solution mixture or microbial culture solution mixture in an anaerobic digester. It provides a method for treating sewage sludge by anaerobic digestion comprising a. In the method for treating sewage sludge by anaerobic digestion according to an embodiment of the present invention, the culture temperature is preferably 30 to 45° C., and preferably 35 to 40° C., considering the amount of biogas generation and economical efficiency. In addition, in the method for treating sewage sludge by anaerobic digestion according to an embodiment of the present invention, the cultivation time is preferably 3 to 30 days, more preferably 5 to 15 days, and more preferably 6 to 12 days when considering the amount of biogas generation and economical efficiency. It is most preferable to be work.
본 발명의 일 예에 따른 하수 슬러지 전처리 방법에서 사용하는 조효소액 혼합물은 하수 슬러지에서 스크리닝된 균주들의 배양 상등액을 혼합한 것으로서 높은 단백질 분해 효소 활성 및 지방 분해 효소 활성을 가지기 때문에 본 발명의 일 예에 따른 방법으로 전처리된 하수 슬러지는 휘발성 부유물질(Volatile Suspended Solids)의 함량이 크게 감소되어 혐기소화에 의한 바이오가스 생산에 매우 적합하다. 또한, 본 발명의 일 예에 따른 하수 슬러지 전처리 방법에서 사용하는 조효소액 혼합물은 하수 슬러지에서 스크리닝된 미생물이 생산하는 효소로 구성되기 때문에 GMO 문제에서 자유롭고 대량생산이 가능하여 하수 슬러지 처리시 상업적인 관점에서 우위의 경제성을 확보할 수 있다. 따라서, 본 발명의 일 예에 따른 하수 슬러지 전처리 방법은 하수 슬러지를 경제적으로 처리하기 위한 실규모 설비에 적용가능한 장점이 있다.The crude enzyme solution mixture used in the sewage sludge pretreatment method according to an embodiment of the present invention is a mixture of the culture supernatant of strains screened from sewage sludge and has high proteolytic enzyme activity and lipolytic enzyme activity. Sewage sludge pretreated according to the method according to the method is very suitable for biogas production by anaerobic digestion because the content of volatile suspended solids is greatly reduced. In addition, since the crude enzyme solution mixture used in the sewage sludge pretreatment method according to an embodiment of the present invention is composed of enzymes produced by microorganisms screened from sewage sludge, it is free from GMO problems and can be mass-produced. Superior economics can be secured. Therefore, the sewage sludge pretreatment method according to an example of the present invention has an advantage applicable to a full-scale facility for economically treating sewage sludge.
도 1은 본 발명의 실시예에서 하수 슬러지로부터 단백질 분해 효소 활성을 가지는 균주로 최종 선별된 PA-21 균주의 계통수(phylogenetic tree)이다.1 is a phylogenetic tree of the PA-21 strain finally selected as a strain having protease activity from sewage sludge in an embodiment of the present invention.
도 2는 본 발명의 실시예에서 하수 슬러지로부터 지방 분해 효소 활성을 가지는 균주로 최종 선별된 LA-96 균주의 계통수(phylogenetic tree)이다.2 is a phylogenetic tree of the LA-96 strain finally selected as a strain having lipolytic enzyme activity from sewage sludge in an embodiment of the present invention.
도 3은 본 발명의 실시예에서 하수 슬러지 시료 (a)를 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액, 3종의 조효소액 혼합물, 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액으로 전처리하였을 때 반응 시간에 따른 VSS의 감소율을 나타낸 그래프이고, 도 4는 본 발명의 실시예에서 하수 슬러지 시료 (b)를 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액, 3종의 조효소액 혼합물, 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액으로 전처리하였을 때 반응 시간에 따른 VSS의 감소율을 나타낸 그래프이고, 도 5는 본 발명의 실시예에서 하수 슬러지 시료 (c)를 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액, 3종의 조효소액 혼합물, 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액으로 전처리하였을 때 반응 시간에 따른 VSS의 감소율을 나타낸 그래프이다. Figure 3 is a coenzyme solution produced from amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 strain of the sewage sludge sample (a) in the embodiment of the present invention, a mixture of three types of coenzyme, Burgholderia vietnamiensis ( Burkholderia vietnamiensis ) is a graph showing the reduction rate of VSS according to the reaction time when pretreated with a crude enzyme solution produced from the LA-96 strain. ( Bacillus amyloliquefaciens ) Coenzyme solution produced from PA-21 strain, 3 kinds of coenzyme solution mixture, Burkholderia vietnamiensis ) VSS according to reaction time when pretreated with coenzyme solution produced from LA-96 strain. Fig. 5 is a graph showing the reduction rate, and FIG. 5 is a coenzyme solution produced from amyloliquefaciens PA-21 strain, a mixture of three coenzymes, and Burg Holderia vietnamiensis (Burkholderia vietnamiensis) is a graph showing the reduction rate of VSS according to the reaction time when pre-treatment with a coenzyme solution produced from LA-96 strain.
도 6은 본 발명의 실시예에서 전처리되지 않은 하수 슬러지 시료 (a) 또는 전처리된 하수 슬러지 시료(a)를 혐기소화에 의해 처리하였을 때 배양 시간별 바이오가스 발생 누적량을 나타낸 그래프이고, 도 7은 본 발명의 실시예에서 전처리되지 않은 하수 슬러지 시료 (b) 또는 전처리된 하수 슬러지 시료(b)를 혐기소화에 의해 처리하였을 때 배양 시간별 바이오가스 발생 누적량을 나타낸 그래프이고, 도 8은 본 발명의 실시예에서 전처리되지 않은 하수 슬러지 시료 (c) 또는 전처리된 하수 슬러지 시료(c)를 혐기소화에 의해 처리하였을 때 배양 시간별 바이오가스 발생 누적량을 나타낸 그래프이다.6 is a graph showing the cumulative amount of biogas generation by cultivation time when an unpretreated sewage sludge sample (a) or a pretreated sewage sludge sample (a) is treated by anaerobic digestion in an embodiment of the present invention, and FIG. 7 is In the embodiment of the present invention, a graph showing the cumulative amount of biogas generation by cultivation time when the unpretreated sewage sludge sample (b) or the pretreated sewage sludge sample (b) is treated by anaerobic digestion, and FIG. 8 is an embodiment of the present invention. This is a graph showing the cumulative amount of biogas generation by cultivation time when the unpretreated sewage sludge sample (c) or the pretreated sewage sludge sample (c) is treated by anaerobic digestion.
이하, 본 발명을 실시예를 통하여 구체적으로 설명하고자 한다. 그러나 하기 실시예는 본 발명의 기술적 특징을 명확하게 예시하기 위한 것일 뿐, 본 발명의 보호범위를 한정하는 것은 아니다.Hereinafter, the present invention will be described in detail through examples. However, the following examples are only intended to clearly illustrate the technical features of the present invention, and do not limit the protection scope of the present invention.
1. 분석 방법1. Analysis method
(1) 균주의 16S rDNA 분석 및 계통수(phylogenetic tree)(1) 16S rDNA analysis and phylogenetic tree of strain
균주로부터 분리한 유전체 DNA(Genomic DNA)을 주형으로 하고, 27F primer(염기서열 : 5'-AGA GTT TGA TCM TGG CTC AG-3') 및 1492R primer(염기서열 : 5'-TAC GGY TAC CTT GTT ACG ACTT-3')를 사용하여 PCR(polymerase chain reaction)을 실시하고, 16S rRNA를 코딩하는 DNA 단편을 증폭시켰다. 상기 PCR은 94℃에서 30초, 55℃에서 30초, 72℃에서 1분간 반응으로 구성되는 사이클ㄹ을 30회 반복하여 실시하였다. 이후, PCR 산물의 염기서열을 분석하여 균주의 16S rDNA 염기서열을 결정하였다. 또한, GeneBank database 검색을 통하여 16S rDNA 유전자의 상동성을 조사하고, CLUSTAL W 프로그램과 neighbor-joining method를 이용하여 균주의 계통수를 작성하였다.Using genomic DNA isolated from the strain as a template, 27F primer (base sequence: 5'-AGA GTT TGA TCM TGG CTC AG-3') and 1492R primer (base sequence: 5'-TAC GGY TAC CTT GTT) ACG ACTT-3') was used to perform a polymerase chain reaction (PCR), and a DNA fragment encoding 16S rRNA was amplified. The PCR was performed by repeating a cycle D consisting of a reaction at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, 30 times. Thereafter, the nucleotide sequence of the PCR product was analyzed to determine the 16S rDNA nucleotide sequence of the strain. In addition, the homology of the 16S rDNA gene was investigated through the GeneBank database search, and the phylogenetic tree of the strain was created using the CLUSTAL W program and the neighbor-joining method.
(2) 단백질 분해 효소(protease) 활성의 측정(2) Measurement of protease activity
액상 LB 배지(주요성분 및 함량 : Tryptone 10g/L, Yeast extract 5g/L, NaCl 10g/L)에 균주를 0.1%(w/v)의 양으로 접종하고 25℃의 온도 및 180 rpm의 교반 조건에서 24 hr 동안 배양한 후 원심분리하여 상등액을 회수하였다. 이후, 회수한 균주 배양 상등액을 조효소액으로 간주하고 시그마알드리치에서 제공하는 단백질 분해 효소의 활성도 측정 방법(Sigma’s non-specific protease activity assay using casein as a substrate)을 이용하여 단백질 분해 효소(protease) 활성을 측정하였다. 구체적으로, 반응 튜브에 Hammarsten casein(0.6%) 1mL와 조효소액 0.1mL을 첨가하고 혼합한 후 50℃에서 30분간 효소반응을 실시하였다. 이후, 반응액에 1mL의 TCA 용액(주요성분 및 함량 : 18g/L TCA, 18.1g/L anhydrous sodium acetate, 18.8g/L acetic acid)을 첨가하고 혼합함으로써 반응을 중지시키고 37℃에서 30분간 방치한 후 실온에서 10분간 원심분리 하였다. 이후, 원심분리된 상등액 0.25mL를 취하여 새 튜브에 옮기고 여기에 0.55M 농도의 Na 2CO 3 용액 0.625mL와, 증류수로 5배 희석된 Folin-Ciocalteus phenol 시약 0.125 mL를 첨가하여 37℃에서 30분간 발색시킨 후 660nm의 파장에서 흡광도를 측정하였다. Protease 활성 1 unit는 반응시간 1분동안 1μg tyrosine을 생성하는 효소의 양으로 정의하였다. Inoculate the strain in an amount of 0.1% (w/v) in liquid LB medium (main ingredients and content: Tryptone 10g/L, Yeast extract 5g/L, NaCl 10g/L), and stirring conditions at a temperature of 25°C and 180 rpm. After incubation for 24 hr at, the supernatant was recovered by centrifugation. Thereafter, the recovered strain culture supernatant was regarded as a coenzyme solution, and the protease activity was measured using the protease activity measurement method provided by Sigma-Aldrich (Sigma's non-specific protease activity assay using casein as a substrate). It was measured. Specifically, 1 mL of Hammarsten casein (0.6%) and 0.1 mL of coenzyme solution were added to the reaction tube and mixed, followed by enzymatic reaction at 50° C. for 30 minutes. Thereafter, 1 mL of TCA solution (main components and content: 18g/L TCA, 18.1g/L anhydrous sodium acetate, 18.8g/L acetic acid) was added to the reaction solution, and the reaction was stopped by mixing and allowed to stand at 37℃ for 30 minutes. And then centrifuged at room temperature for 10 minutes. Thereafter, 0.25 mL of the centrifuged supernatant was taken and transferred to a new tube, and 0.625 mL of a 0.55 M Na 2 CO 3 solution and 0.125 mL of Folin-Ciocalteus phenol reagent diluted 5 times with distilled water were added thereto at 37°C for 30 minutes. After color development, absorbance was measured at a wavelength of 660 nm. 1 unit of protease activity was defined as the amount of enzyme that produced 1 μg tyrosine during 1 minute of reaction time.
(3) 지방 분해 효소(lipase) 활성의 측정(3) Measurement of lipolytic enzyme activity
1% 올리브 오일이 함유된 액상 제한배지(주요성분 및 함량 : K 2HP0 4 0.3g/L, KH 2P0 4 0.3g/L, (NH 4) 2S0 4 0.5g/L, MgS0 4·7H 20 0.4g/L, MgCl 2·6H 2O 0.7g/L, CaCl 2·2H 2O 0.5g/L, yeast extract 1g/L)에 균주를 0.1%(w/v)의 양으로 접종하고 25℃의 온도 및 180 rpm의 교반 조건에서 24 hr 동안 배양한 후 원심분리하여 상등액을 회수하였다. 이후, 회수한 균주 배양 상등액을 조효소액으로 간주하고 마이크로플레이트 어세이(microplate assay) 방법으로 지방 분해 효소(lipase) 활성을 측정하였다. 구체적으로, p-nitrophenyl ester(pNP-ester)를 기질로 사용하고, 조효소액과의 반응에 의해 유리되는 p-nitrophenol의 양을 405nm의 파장에서 측정한 흡광도 증가량으로 결정하였다. 또한, 기질의 종류에 따라 두가지 방법을 혼용하였다. Ester carbon의 길이가 12개 이하의 탄소수를 가진 기질(pNP-acetate)을 사용하는 경우, acetonitrile로 녹인 10mM pNP-acetate 5μL, ethanol 5μL, 50mM phosphate buffer(pH 7.4) 180μL와 조효소액 10μL를 섞은 다음 37℃에서 10분간 반응 후 405nm에서 흡광도를 측정하였다. 또한, long chain인 pNP-palmitate를 기질로 사용하는 경우 10μL의 조효소액과 170μL의 buffer 혼합액(50mM phosphate buffer containing 0.1% gum arabic and 0.2% deoxycholate) 그리고 isoprophanol로 녹인 8mM의 기질 20μL를 혼합하고 37℃에서 10분간 반응 후 405nm에서 흡광도를 측정하였다. Lipase 활성 1 unit는 반응시간 1분동안 1 μmol의 p-nitrophenyl를 생성하는 효소의 양으로 정의하였다.Liquid restricted medium containing 1% olive oil (main ingredients and content: K 2 HP0 4 0.3g/L, KH 2 P0 4 0.3g/L, (NH 4 ) 2 S0 4 0.5g/L, MgS0 4 7H 2 0 0.4g/L, MgCl 2 6H 2 O 0.7g/L, CaCl 2 2H 2 O 0.5g/L, yeast extract 1g/L) in an amount of 0.1% (w/v) After incubation for 24 hr at a temperature of 25° C. and a stirring condition of 180 rpm, the supernatant was recovered by centrifugation. Thereafter, the recovered strain culture supernatant was regarded as a coenzyme solution, and lipolytic enzyme activity was measured by a microplate assay method. Specifically, p-nitrophenyl ester (pNP-ester) was used as a substrate, and the amount of p-nitrophenol released by reaction with the coenzyme solution was determined as an increase in absorbance measured at a wavelength of 405 nm. In addition, two methods were mixed according to the type of substrate. When using a substrate (pNP-acetate) having a carbon number of less than 12 Ester carbon, 5 μL of 10 mM pNP-acetate dissolved in acetonitrile, 5 μL of ethanol, 180 μL of 50 mM phosphate buffer (pH 7.4) and 10 μL of coenzyme solution are mixed. After reaction at 37° C. for 10 minutes, absorbance was measured at 405 nm. In addition, when long chain pNP-palmitate is used as a substrate, 10 μL of coenzyme solution, 170 μL of buffer mixture (50 mM phosphate buffer containing 0.1% gum arabic and 0.2% deoxycholate), and 20 μL of 8 mM substrate dissolved in isoprophanol are mixed and then mixed at 37°C. After reacting for 10 minutes, absorbance was measured at 405 nm. Lipase activity 1 unit was defined as the amount of enzyme that produced 1 μmol of p-nitrophenyl during 1 minute of reaction time.
2. 하수 슬러지로부터 단백질 분해 효소 활성을 가지는 균주 및 지방 분해 효소 활성을 가지는 균주의 선별2. Selection of strains having proteolytic enzyme activity and strains having lipolytic enzyme activity from sewage sludge
(1) 단백질 분해 활성을 가지는 균주의 선별(1) Selection of strains having proteolytic activity
국내의 다수 하수 처리장으로부터 채수된 슬러지 샘플을 각각 스킴밀크(skim milk)가 첨가된 고체배지(주요 성분 및 함량 : skim milk 1%(w/w), nutrient broth 0.8%(w/w) 및 agar 2%(w/w))에 도말하고 25℃에서 2일 동안 배양한 후 콜로니 주위에 형성되는 투명환(clear zone)의 크기를 비교하여 단백질 분해 활성이 높은 균주 총 10종을 1차 선별하였다. 1차 선별된 총 10종의 균주에 대해 16S rDNA 염기서열을 분석하고, GenBank database 검색을 통하여 공지 균주와의 16S rDNA 유전자 상동성을 조사하였다. 이후, 액상 LB 배지(주요성분 및 함량 : Tryptone 10g/L, Yeast extract 5g/L, NaCl 10g/L)에 1차 선별한 균주 각각을 1%(w/v)의 양으로 접종하고 25℃의 온도 및 180 rpm의 교반 조건에서 24 hr 동안 배양한 후 원심분리하여 상등액을 회수하였다. 이후, 회수한 균주 배양 상등액을 조효소액으로 간주하고 단백질 분해 효소 활성을 측정하였다.Sludge samples collected from a number of domestic sewage treatment plants were used in solid medium with added skim milk (main ingredients and content: skim milk 1% (w/w), nutrient broth 0.8% (w/w) and agar). 2% (w/w)) and incubated at 25°C for 2 days, the size of the clear zone formed around the colony was compared, and a total of 10 strains with high proteolytic activity were first selected. . The 16S rDNA nucleotide sequence was analyzed for a total of 10 strains selected first, and homology of 16S rDNA genes with known strains was investigated through a GenBank database search. Thereafter, each of the first selected strains was inoculated in a liquid LB medium (main ingredients and content: Tryptone 10g/L, Yeast extract 5g/L, NaCl 10g/L) in an amount of 1% (w/v) and After incubation for 24 hr under a stirring condition of temperature and 180 rpm, the supernatant was recovered by centrifugation. Thereafter, the recovered strain culture supernatant was regarded as a crude enzyme solution, and protease activity was measured.
하기 표 1에 하수 슬러지로부터 1차 선별한 단백질 분해 효소 활성을 가지는 총 10종의 균주명, 16S rDNA 유전자 상동성 분석 결과 및 균주 배양 상등액의 단백질 분해 효소 활성 측정 결과를 나타내었다.Table 1 below shows the names of a total of 10 strains having the proteolytic enzyme activity first selected from sewage sludge, the 16S rDNA gene homology analysis results, and the proteolytic enzyme activity measurement results of the strain culture supernatant.
Strain nameStrain name Highly matched type strainHighly matched type strain GenBank Accession No.GenBank Accession No. 16S rDNA Similarity(%)16S rDNA Similarity(%) Protease activity(uint/mL)Protease activity(uint/mL)
PA-1PA-1 Bacillus aerophilus 28K (T)Bacillus aerophilus 28K (T) AJ831844 AJ831844 100100 0.750.75
PA-6PA-6 Bacillus cereus ATCC 14579 (T)Bacillus cereus ATCC 14579 (T) AE016877 AE016877 100100 0.320.32
PA-12PA-12 Bacillus licheniformis ATCC 14580 (T)Bacillus licheniformis ATCC 14580 (T) AE017333AE017333 99.2299.22 0.850.85
PA-13PA-13 Bacillus subtilis subsp. subtilis NCIB 3610 (T)Bacillus subtilis subsp. subtilis NCIB 3610 (T) ABQL01000001ABQL01000001 99.9399.93 0.230.23
PA-15PA-15 Bacillus sonorensis NBRC 101234 (T)Bacillus sonorensis NBRC 101234 (T) AYTN01000016AYTN01000016 99.8699.86 0.280.28
PA-17PA-17 Bacillus amyloliquefaciens subsp. plantarum FZB42 (T)Bacillus amyloliquefaciens subsp. plantarum FZB42 (T) CP000560CP000560 99.9399.93 1.531.53
PA-18PA-18 Bacillus atrophaeus JCM 9070 (T)Bacillus atrophaeus JCM 9070 (T) AB021181AB021181 99.9399.93 0.880.88
PA-21PA-21 Bacillus amyloliquefaciens subsp. amyloliquefaciens DSM7 (T)Bacillus amyloliquefaciens subsp. amyloliquefaciens DSM7 (T) FN597644FN597644 99.7999.79 1.821.82
PA-31PA-31 Aeromonas hydrophila subsp. hydrophila ATCC 7966 (T)Aeromonas hydrophila subsp. hydrophila ATCC 7966 (T) CP000462CP000462 99.9399.93 1.001.00
PA-35PA-35 Bacillus subtilis subsp. inaquosorum KCTC 13429 (T)Bacillus subtilis subsp. inaquosorum KCTC 13429 (T) AMXN01000021AMXN01000021 99.9399.93 0.720.72
상기 표 1에서 보이는 바와 같이 PA-17, PA-21 및 PA-31 균주가 우수한 단백질 분해 효소 활성을 보였고, 그 중 단백질 분해 효소 활성이 가장 우수하고 공시된 균주와 16S rDNA 유전자 상동성이 가장 낮은 PA-21 균주를 최종 선별하였다.도 1은 본 발명의 실시예에서 하수 슬러지로부터 단백질 분해 효소 활성을 가지는 균주로 최종 선별된 PA-21 균주의 계통수(phylogenetic tree)이다. 상기 PA-21 균주의 균학적 성질, 16S rDNA 염기서열(서열번호 1) 및 계통수(phylogenetic tree)에 기초하여 PA-21 균주의 종을 바실러스 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens)로 확정하고, PA-21 균주를 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21로 명명하였다.As shown in Table 1 above, PA-17, PA-21, and PA-31 strains showed excellent protease activity, among which the proteolytic enzyme activity was the best, and 16S rDNA gene homology was the lowest. The PA-21 strain was finally selected. FIG. 1 is a phylogenetic tree of the PA-21 strain finally selected as a strain having protease activity from sewage sludge in the embodiment of the present invention. Based on the mycological properties of the PA-21 strain, 16S rDNA nucleotide sequence (SEQ ID NO: 1) and phylogenetic tree, the species of the PA-21 strain was confirmed as Bacillus amyloliquefaciens, The PA-21 strain was named as Bacillus amyloliquefaciens PA-21.
(2) 지방 분해 활성을 가지는 균주의 선별(2) Selection of strains having lipolytic activity
국내의 다수 하수 처리장으로부터 채수된 슬러지 샘플을 각각 트리부티린(tributyrin)이 포함된 고체배지(주요 성분 및 함량 : tributyrin 1%(w/w), nutrient broth 0.8%(w/w) 및 agar 2%(w/w))에 도말하고 25℃에서 3일 동안 배양한 후 콜로니 주위에 형성되는 투명환(clear zone)의 크기를 비교하여 지방 분해 활성이 높은 균주 총 10종을 1차 선별하였다. 1차 선별된 총 10종의 균주에 대해 16S rDNA 염기서열을 분석하고, GenBank database 검색을 통하여 공지 균주와의 16S rDNA 유전자 상동성을 조사하였다. 이후, 1% 올리브 오일이 함유된 액상 제한배지(주요성분 및 함량 : K 2HP0 4 0.3g/L, KH 2P0 4 0.3g/L, (NH 4) 2S0 4 0.5g/L, MgS0 4·7H 20 0.4g/L, MgCl 2·6H 2O 0.7g/L, CaCl 2·2H 2O 0.5g/L, yeast extract 1g/L)에 1차 선별한 균주 각각을 1%(w/v)의 양으로 접종하고 25℃의 온도 및 180 rpm의 교반 조건에서 24 hr 동안 배양한 후 원심분리하여 상등액을 회수하였다. 이후, 회수한 균주 배양 상등액을 조효소액으로 간주하고 지방 분해 효소 활성을 측정하였다.Sludge samples collected from many domestic sewage treatment plants were collected in solid medium containing tributyrin (main components and content: tributyrin 1% (w/w), nutrient broth 0.8% (w/w) and agar 2). % (w/w)) and cultured at 25° C. for 3 days, a total of 10 strains having high lipolytic activity were first selected by comparing the size of a clear zone formed around the colony. The 16S rDNA nucleotide sequence was analyzed for a total of 10 strains selected first, and homology of 16S rDNA genes with known strains was investigated through a GenBank database search. Afterwards, liquid restriction medium containing 1% olive oil (main ingredients and content: K 2 HP0 4 0.3g/L, KH 2 P0 4 0.3g/L, (NH 4 ) 2 S0 4 0.5g/L, MgS0 4 7H 2 0 0.4g/L, MgCl 2 6H 2 O 0.7g/L, CaCl 2 2H 2 O 0.5g/L, yeast extract 1g/L) 1% (w/ v) and incubated for 24 hr at a temperature of 25° C. and stirring conditions at 180 rpm, and then centrifuged to recover the supernatant. Thereafter, the recovered strain culture supernatant was regarded as a crude enzyme solution, and the lipolytic enzyme activity was measured.
하기 표 2에 하수 슬러지로부터 1차 선별한 지방 분해 효소 활성을 가지는 총 10종의 균주명, 16S rDNA 유전자 상동성 분석 결과 및 균주 배양 상등액의 지방 분해 효소 활성 측정 결과를 나타내었다.Table 2 below shows the names of a total of 10 strains having the lipolytic enzyme activity first selected from sewage sludge, the 16S rDNA gene homology analysis results, and the lipolytic enzyme activity measurement results of the strain culture supernatant.
Strain nameStrain name Highly matched type strainHighly matched type strain GenBank Accession No.GenBank Accession No. 16S rDNA Similarity(%)16S rDNA Similarity(%) Lipase activity(unit/mL)Lipase activity(unit/mL)
LA-96LA-96 Burkholderia vietnamiensis LMG 10929 (T)Burkholderia vietnamiensis LMG 10929 (T) AF097534AF097534 99.8699.86 3.803.80
LA-11LA-11 Bacillus methylotrophicus KACC 13105Bacillus methylotrophicus KACC 13105 JTKJ01000077JTKJ01000077 99.9399.93 0.330.33
LA-17LA-17 Bacillus amyloliquefaciens subsp. plantarum FZB42 (T)Bacillus amyloliquefaciens subsp. plantarum FZB42 (T) CP000560CP000560 99.9399.93 1.711.71
LA-18LA-18 Aeromonas hydrophila subsp. hydrophila ATCC 7966 (T)Aeromonas hydrophila subsp. hydrophila ATCC 7966 (T) CP000462CP000462 99.8699.86 2.312.31
LA-19LA-19 Acinetobacter parvus DSM 16617 (T)Acinetobacter parvus DSM 16617 (T) AIEB01000124AIEB01000124 98.7998.79 0.500.50
LA-22LA-22 Aeromonas veronii PRJEB7044 (T)Aeromonas veronii PRJEB7044 (T) CDDK01000015 CDDK01000015 100100 0.560.56
LA-23LA-23 Acidovorax temperans, partial sequenceAcidovorax temperans, partial sequence AF078766AF078766 99.8599.85 1.411.41
LA-26LA-26 Bacillus subtilis subsp. subtilis, partial sequenceBacillus subtilis subsp. subtilis, partial sequence ABQL01000001ABQL01000001 99.9399.93 1.531.53
LA-27LA-27 Bacillus subtilis subsp. inaquosorum KCTC 13429 (T)Bacillus subtilis subsp. inaquosorum KCTC 13429 (T) AMXN01000021AMXN01000021 99.8699.86 1.661.66
LA-24LA-24 Aeromonas media PRJEB7032 (T)Aeromonas media PRJEB7032 (T) CDBZ01000012CDBZ01000012 99.0199.01 0.760.76
상기 표 2에서 보이는 바와 같이 LA-96 및 LA-18 균주가 우수한 지방 분해 효소 활성을 보였고, 그 중 지방 분해 효소 활성이 가장 우수한 LA-96 균주를 최종 선별하였다.도 2는 본 발명의 실시예에서 하수 슬러지로부터 지방 분해 효소 활성을 가지는 균주로 최종 선별된 LA-96 균주의 계통수(phylogenetic tree)이다. 상기 LA-96 균주의 균학적 성질, 16S rDNA 염기서열(서열번호 2) 및 계통수(phylogenetic tree)에 기초하여 LA-96 균주의 종을 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis)로 확정하고, LA-96 균주를 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96으로 명명하였다.As shown in Table 2, the LA-96 and LA-18 strains showed excellent lipolytic enzyme activity, and among them, the LA-96 strain having the most excellent lipolytic enzyme activity was finally selected. It is a phylogenetic tree of LA-96 strain finally selected as a strain having lipolytic enzyme activity from sewage sludge in Based on the mycological properties of the LA-96 strain, 16S rDNA nucleotide sequence (SEQ ID NO: 2) and phylogenetic tree, the species of the LA-96 strain was confirmed as Burkholderia vietnamiensis, and LA The -96 strain was named Burkholderia vietnamiensis LA-96.
3. 최종 선별된 균주들로부터 조효소액의 생산 및 효소 활성 측정3. Production of crude enzyme solution and measurement of enzyme activity from the final selected strains
(1) 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 조효소액의 생산 및 단백질 분해 효소 활성 측정(1) Production of coenzyme solution and measurement of protease activity from Bacillus amyloliquefaciens PA-21 strain
액상 기본배지(주요성분 및 함량 : Fructose 20 g/L, K 2HPO 4 0.1 g/L, KH 2PO 4 0.1 g/L, yeast extract 5 g/L, CaCl 2·2H 2O 1 g/L) 3L에 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주를 300g의 양으로 접종하고(접종량 : 10% w/v) 30℃의 온도, pH 6.5, 200 rpm의 교반 조건 및 1.0 vvm의 공기 주입량 조건에서 12 hr 동안 진탕배양한 후 원심분리하여 상등액을 회수하였다. 이후, 회수한 균주 배양 상등액을 조효소액으로 간주하고 단백질 분해 효소 활성을 측정하였다. 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액의 단백질 분해 효소 활성은 약 417.7 unit/mL 이었다.Liquid basic medium (main ingredients and content: Fructose 20 g/L, K 2 HPO 4 0.1 g/L, KH 2 PO 4 0.1 g/L, yeast extract 5 g/L, CaCl 2 2H 2 O 1 g/L ) In 3L amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 strain was inoculated in an amount of 300 g (inoculation amount: 10% w/v) and a temperature of 30° C., pH 6.5, stirring conditions of 200 rpm, and 1.0 vvm The supernatant was recovered by centrifugation after shaking culture for 12 hr under the condition of the amount of air injected. Thereafter, the recovered strain culture supernatant was regarded as a crude enzyme solution, and protease activity was measured. The protease activity of the coenzyme solution produced from the Bacillus amyloliquefaciens PA-21 strain was about 417.7 unit/mL.
(2) 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 조효소액의 생산 및 지방 분해 효소 활성 측정(2) Burkholderia vietnamiensis (Burkholderia vietnamiensis) Production of coenzyme solution from LA-96 strain and measurement of lipolytic enzyme activity
1% 올레산(oleic acid)이 함유된 액상 기본배지(주요성분 및 함량 : K 2HP0 4 0.3g/L, KH 2P0 4 0.3g/L, (NH 4) 2S0 4 0.5g/L, MgS0 4·7H 20 0.4g/L, MgCl 2·6H 2O 0.7g/L, CaCl 2·2H 2O 0.5g/L, yeast extract 1g/L) 3L에 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주를 300g의 양으로 접종하고(접종량 : 10% w/v) 30℃의 온도, pH 7.0, 600 rpm의 교반 조건 및 1.66 vvm의 공기 주입량 조건에서 18 hr 동안 진탕배양한 후 원심분리하여 상등액을 회수하였다. 이후, 회수한 균주 배양 상등액을 조효소액으로 간주하고 지방 분해 효소 활성을 측정하였다. 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액의 지방 분해 효소 활성은 약 33.6 unit/mL 이었다.Liquid basic medium containing 1% oleic acid (main ingredients and content: K 2 HP0 4 0.3g/L, KH 2 P0 4 0.3g/L, (NH 4 ) 2 S0 4 0.5g/L, MgS0 4 7H 2 0 0.4g/L, MgCl 2 6H 2 O 0.7g/L, CaCl 2 2H 2 O 0.5g/L, yeast extract 1g/L) Burkholderia vietnamiensis in 3L LA-96 strain was inoculated in an amount of 300g (inoculation amount: 10% w/v) and cultured with shaking for 18 hr at a temperature of 30°C, agitation condition of pH 7.0, 600 rpm, and an air injection amount of 1.66 vvm, followed by centrifugation. And the supernatant was recovered. Thereafter, the recovered strain culture supernatant was regarded as a crude enzyme solution, and the lipolytic enzyme activity was measured. The lipolytic enzyme activity of the crude enzyme solution produced from the Burkholderia vietnamiensis LA-96 strain was about 33.6 unit/mL.
4. 최종 선별된 균주들로부터 생산한 조효소액의 혼합물 제조 및 이를 이용한 하수 슬러지의 전처리4. Preparation of a mixture of crude enzyme solution produced from the finally selected strains and pretreatment of sewage sludge using the same
(1) 최종 선별된 균주들로부터 생산한 조효소액의 혼합물 제조(1) Preparation of a mixture of coenzyme solution produced from the finally selected strains
아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액을 3:1, 1:1 및 1:3의 부피비로 각각 혼합하여 3종의 조효소액 혼합물을 제조하였다.The coenzyme solution produced from the Bacillus amyloliquefaciens PA-21 strain and the coenzyme solution produced from the Burkholderia vietnamiensis LA-96 strain were 3:1, 1:1 and 1: Each mixture was mixed at a volume ratio of 3 to prepare a mixture of three types of coenzyme solutions.
(2) 조효소액 혼합물을 이용한 하수 슬러지의 전처리(2) Pretreatment of sewage sludge using crude enzyme solution mixture
영남권 대도시인 부산, 울산, 포항에서 채수된 각각의 생슬러지를 60 mm 메쉬 크기의 체로 걸러 자갈 등과 같이 효소 반응과 관계없는 물질을 제거하고 휘발성 부유물질(Volatile Suspended Solids, 이하 'VSS'라 함)의 농도가 서로 다른 총 3종의 하수 슬러지 시료를 제조하였다. 하수 슬러지 시료 (a)에는 8.6 g/L 농도의 VSS가 포함되어 있고, 하수 슬러지 시료 (b)에는 11.4 g/L 농도의 VSS가 포함되어 있고, 하수 슬러지 시료 (c)에는 13.5 g/L 농도의 VSS가 포함되어 있다. 하수 슬러지 시료에 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액, 3종의 조효소액 혼합물, 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액 각각을 하수 슬러지 시료 1L 당 1g의 양으로 첨가하고 pH를 7.0으로 조정한 후 40℃의 온도, 120 rpm의 교반 속도로 호기 조건에서 약 72 hr 동안 반응시켜 하수 슬러지 시료를 전처리하였다.Each raw sludge collected from Busan, Ulsan and Pohang, which is a metropolitan area in the Yeongnam area, is filtered through a sieve of 60 mm mesh to remove substances not related to enzyme reactions such as gravel, and volatile suspended solids (Volatile Suspended Solids, hereinafter referred to as'VSS') A total of three sewage sludge samples with different concentrations were prepared. Sewage sludge sample (a) contains 8.6 g/L concentration of VSS, sewage sludge sample (b) contains 11.4 g/L concentration of VSS, and sewage sludge sample (c) contains 13.5 g/L concentration. VSS is included. Coenzyme solution produced from Bacillus amyloliquefaciens PA-21 strain in sewage sludge samples, a mixture of three coenzyme solutions, and a coenzyme solution produced from Burkholderia vietnamiensis LA-96 strain Each was added in an amount of 1 g per 1 L of sewage sludge sample, the pH was adjusted to 7.0, and reacted for about 72 hr under aerobic conditions at a temperature of 40° C. and a stirring speed of 120 rpm to pretreat the sewage sludge sample.
도 3은 본 발명의 실시예에서 하수 슬러지 시료 (a)를 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액, 3종의 조효소액 혼합물, 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액으로 전처리하였을 때 반응 시간에 따른 VSS의 감소율을 나타낸 그래프이고, 도 4는 본 발명의 실시예에서 하수 슬러지 시료 (b)를 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액, 3종의 조효소액 혼합물, 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액으로 전처리하였을 때 반응 시간에 따른 VSS의 감소율을 나타낸 그래프이고, 도 5는 본 발명의 실시예에서 하수 슬러지 시료 (c)를 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액, 3종의 조효소액 혼합물, 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액으로 전처리하였을 때 반응 시간에 따른 VSS의 감소율을 나타낸 그래프이다. 도 3 내지 도 5에서 "Protease only"는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액으로 전처리한 경우를 나타내고, "Lipase only"는 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액으로 전처리한 경우를 나타낸다. 또한, "Pro:Lip=3:1", "Pro:Lip=1:1" 및 "Pro:Lip=1:3"는 각각 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액을 3:1, 1:1 및 1:3의 부피비로 혼합하여 제조한 조효소액 혼합물로 전처리한 경우를 나타낸다. Figure 3 is a coenzyme solution produced from amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 strain of the sewage sludge sample (a) in the embodiment of the present invention, a mixture of three types of coenzyme, Burgholderia vietnamiensis ( Burkholderia vietnamiensis ) is a graph showing the reduction rate of VSS according to the reaction time when pretreated with a crude enzyme solution produced from the LA-96 strain. ( Bacillus amyloliquefaciens ) Coenzyme solution produced from PA-21 strain, 3 kinds of coenzyme solution mixture, Burkholderia vietnamiensis ) VSS according to reaction time when pretreated with coenzyme solution produced from LA-96 strain. Fig. 5 is a graph showing the reduction rate, and FIG. 5 is a coenzyme solution produced from amyloliquefaciens PA-21 strain, a mixture of three coenzymes, and Burg Holderia vietnamiensis (Burkholderia vietnamiensis) is a graph showing the reduction rate of VSS according to the reaction time when pre-treatment with a coenzyme solution produced from LA-96 strain. In FIGS. 3 to 5, "Protease only" represents a case of pre-treatment with a coenzyme solution produced from the Bacillus amyloliquefaciens PA-21 strain, and "Lipase only" indicates a case of pre-treatment with a coenzyme solution produced from the Bacillus amyloliquefaciens PA-21 strain, and "Lipase only" is a Burkholderia vietnamiensis vietnamiensis ) shows a case of pretreatment with a crude enzyme solution produced from the LA-96 strain. In addition, "Pro:Lip=3:1", "Pro:Lip=1:1" and "Pro:Lip=1:3" are respectively amyloliquefaciens (Bacillus amyloliquefaciens ) produced from PA-21 strains It shows a case of pretreatment with a crude enzyme solution mixture prepared by mixing the crude enzyme solution and the crude enzyme solution produced from the Burkholderia vietnamiensis LA-96 strain at a volume ratio of 3:1, 1:1, and 1:3. .
도 3 내지 도 5에서 보이는 바와 같이 하수 슬러지 시료를 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21 균주로부터 생산한 조효소액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96 균주로부터 생산한 조효소액을 1:3의 부피비로 혼합하여 제조한 조효소액 혼합물로 전처리하였을 때 가장 높은 VSS 감소율을 보였다. 특히, 하수 슬러지 시료 (c)를 "Pro:Lip=1:3"의 조효소액 혼합물로 72 hr 동안 전처리하였을 때 약 42.4%의 VSS 감소율을 나타내었다.As shown in Figures 3 to 5, a sewage sludge sample was produced from a coenzyme solution produced from amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 strain and a Burkholderia vietnamiensis (Burkholderia vietnamiensis) LA-96 strain. When the crude enzyme solution was pretreated with a crude enzyme solution mixture prepared by mixing in a volume ratio of 1:3, the highest VSS reduction rate was shown. In particular, when the sewage sludge sample (c) was pretreated with a crude enzyme solution mixture of "Pro:Lip=1:3" for 72 hr, it showed a VSS reduction rate of about 42.4%.
5. 혐기소화에 의한 전처리된 하수 슬러지의 처리 및 바이오가스의 생산5. Treatment of pretreated sewage sludge and production of biogas by anaerobic digestion
혐기소화조에 혐기 미생물로 구성된 식종원과 소정의 하수 슬러지 시료를 넣고 산소를 완전히 제거한 후 pH 7 및 37.5℃의 온도에서 약 30일 동안 혐기 조건으로 배양하여 하수 슬러지 시료를 처리하였고, 배양 시간별로 바이오가스 발생량을 측정하였다. 도 6은 본 발명의 실시예에서 전처리되지 않은 하수 슬러지 시료 (a) 또는 전처리된 하수 슬러지 시료(a)를 혐기소화에 의해 처리하였을 때 배양 시간별 바이오가스 발생 누적량을 나타낸 그래프이고, 도 7은 본 발명의 실시예에서 전처리되지 않은 하수 슬러지 시료 (b) 또는 전처리된 하수 슬러지 시료(b)를 혐기소화에 의해 처리하였을 때 배양 시간별 바이오가스 발생 누적량을 나타낸 그래프이고, 도 8은 본 발명의 실시예에서 전처리되지 않은 하수 슬러지 시료 (c) 또는 전처리된 하수 슬러지 시료(c)를 혐기소화에 의해 처리하였을 때 배양 시간별 바이오가스 발생 누적량을 나타낸 그래프이다. 도 6 내지 도 8에서 "Inoculum (Ctrl)"은 혐기소화조에 혐기 미생물로 구성된 식종원만을 넣고 배양한 경우를 나타내고, "1S"는 혐기소화조에 혐기 미생물로 구성된 식종원과 전처리되지 않은 하수 슬러지 시료를 넣고 배양한 경우를 나타내고, "1S+P1L3"는 혐기소화조에 혐기 미생물로 구성된 식종원과 "Pro:Lip=1:3"의 조효소액 혼합물로 72 hr 동안 전처리된 하수 슬러지 시료를 넣고 배양한 경우를 나타낸다. 또한, "1S+P1L3(1)" 및 "1S+P1L3(2)"는 동일한 공정 조건에서 반복 실험한 결과의 순서를 의미한다.Sewage sludge samples were treated by putting a planting source composed of anaerobic microorganisms and a predetermined sewage sludge sample in an anaerobic digester, completely removing oxygen, and incubating under anaerobic conditions at pH 7 and 37.5°C for about 30 days. The amount of gas generated was measured. 6 is a graph showing the cumulative amount of biogas generation by cultivation time when an unpretreated sewage sludge sample (a) or a pretreated sewage sludge sample (a) is treated by anaerobic digestion in an embodiment of the present invention, and FIG. 7 is In the embodiment of the present invention, a graph showing the cumulative amount of biogas generation by cultivation time when the unpretreated sewage sludge sample (b) or the pretreated sewage sludge sample (b) is treated by anaerobic digestion, and FIG. 8 is an embodiment of the present invention. This is a graph showing the cumulative amount of biogas generation by cultivation time when the unpretreated sewage sludge sample (c) or the pretreated sewage sludge sample (c) is treated by anaerobic digestion. In FIGS. 6 to 8, "Inoculum (Ctrl)" denotes a case where only a planting source composed of anaerobic microorganisms is put in an anaerobic digestion tank and cultivated, and "1S" denotes a planting source composed of anaerobic microorganisms in an anaerobic digester and unpretreated sewage sludge sample In the anaerobic digestion tank, "1S+P1L3" is a mixture of a seed source composed of anaerobic microorganisms and a coenzyme solution of "Pro:Lip=1:3". Indicate the case. In addition, "1S+P1L3(1)" and "1S+P1L3(2)" refer to the sequence of the results of repeated experiments under the same process conditions.
도 6 내지 도 8에서 보이는 바와 같이 전처리한 하수 슬러지 시료를 혐기소화 공정으로 분해하였을 때 전처리되지 않은 하수 슬러리 시료를 혐기소화 공정으로 분해하였을 때보다 바이오가스 발생량이 훨씬 높게 나타났다. 구체적으로, 하수 슬러지 시료 (a)의 경우 전처리된 하수 슬러지를 혐기소화하였을 때 전처리되지 않은 하수 슬러지를 혐기소화한 경우보다 바이오가스 생산량이 약 13.2% 증가하였고, 하수 슬러지 시료 (b)의 경우 전처리된 하수 슬러지를 혐기소화하였을 때 전처리되지 않은 하수 슬러지를 혐기소화한 경우보다 바이오가스 생산량이 약 54.7% 증가하였고, 하수 슬러지 시료 (c)의 경우 전처리된 하수 슬러지를 혐기소화하였을 때 전처리되지 않은 하수 슬러지를 혐기소화한 경우보다 바이오가스 생산량이 약 35.1% 증가하였다.As shown in FIGS. 6 to 8, when the pretreated sewage sludge sample was decomposed by the anaerobic digestion process, the amount of biogas generated was much higher than when the unpretreated sewage slurry sample was decomposed by the anaerobic digestion process. Specifically, in the case of sewage sludge sample (a), when the pretreated sewage sludge was anaerobic digested, biogas production increased by about 13.2% compared to the case of anaerobic digestion of unpretreated sewage sludge, and in the case of sewage sludge sample (b), pretreatment When the treated sewage sludge was anaerobic digested, biogas production increased by about 54.7% compared to the anaerobic digestion of untreated sewage sludge, and the sewage sludge sample (c) was not pretreated when the pretreated sewage sludge was anaerobic digested. Compared to the case of anaerobic digestion of sludge, biogas production increased by about 35.1%.
이상에서와 같이 본 발명을 상기의 실시예를 통해 설명하였지만 본 발명의 보호범위가 반드시 여기에만 한정되는 것은 아니며 본 발명의 범주와 사상을 벗어나지 않는 범위 내에서 다양한 변형실시가 가능함은 물론이다. 따라서, 본 발명의 보호범위는 최상의 양식으로서 개시된 특정 실시 형태로 국한되는 것이 아니며, 본 발명에 첨부된 특허청구의 범위에 속하는 모든 실시 형태를 포함하는 것으로 해석되어야 한다.As described above, the present invention has been described through the above embodiments, but the scope of protection of the present invention is not necessarily limited thereto, and various modifications may be made without departing from the scope and spirit of the present invention. Accordingly, the scope of protection of the present invention is not limited to the specific embodiments disclosed as the best mode, and should be construed as including all embodiments falling within the scope of the claims appended to the present invention.

Claims (16)

  1. 하수 슬러지에 조효소액 혼합물을 첨가하고 호기 조건에서 반응시키는 단계를 포함하는 방법으로서,A method comprising the step of adding a crude enzyme solution mixture to sewage sludge and reacting under aerobic conditions,
    상기 조효소액 혼합물은 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양 상등액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양 상등액의 혼합물인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The crude enzyme mixture is amyloliquefaciens (Bacillus amyloliquefaciens ) culture supernatant and Burkholderia vietnamiensis (Burkholderia vietnamiensis) culture supernatant, characterized in that it is a mixture of, sewage sludge pretreatment method.
  2. 제1항에 있어서, 상기 하수 슬러지는 5~20 g/L 농도의 휘발성 부유물질(Volatile Suspended Solids)을 포함하는 것을 특징으로 하는, 하수 슬러지 전처리 방법.The method of claim 1, wherein the sewage sludge contains volatile suspended solids having a concentration of 5 to 20 g/L.
  3. 제1항에 있어서, 상기 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 및 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis)는 하수 슬러지에서 스크리닝된 균주인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The method of claim 1, wherein the amyloliquefaciens (Bacillus amyloliquefaciens ) and Burkholderia vietnamiensis are strains screened from sewage sludge.
  4. 제3항에 있어서, 상기 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 균주는 16S rDNA로 서열번호 1의 염기서열을 포함하는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21이고, 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis)는 16S rDNA로 서열번호 2의 염기서열을 포함하는 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The method of claim 3, wherein the amyloliquefaciens (Bacillus amyloliquefaciens ) strain is amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 containing the nucleotide sequence of SEQ ID NO: 1 in 16S rDNA, and Burgholderia Viet N-Sys Nami (Burkholderia vietnamiensis), the method pre-treatment of sewage sludge, characterized in that Burkholderia Viet Nami N-Sys (Burkholderia vietnamiensis) LA-96 comprising the nucleotide sequence of SEQ ID NO: 2 with 16S rDNA.
  5. 제1항에 있어서, 상기 조효소액 혼합물을 구성하는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양 상등액의 단백질 분해 효소 활성은 400~450 unit/mL이고 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양 상등액의 지방 분해 효소 활성은 30~40 unit/mL인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The method of claim 1, wherein the protease activity of the culture supernatant of Bacillus amyloliquefaciens constituting the crude enzyme solution mixture is 400 to 450 unit/mL, and Burkholderia vietnamiensis culture supernatant Lipolytic enzyme activity of, characterized in that 30 to 40 unit / mL, sewage sludge pretreatment method.
  6. 제6항에 있어서, 상기 조효소액 혼합물은 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양 상등액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양 상등액을 5:1 내지 1:10의 부피비로 혼합한 것인, 하수 슬러지 전처리 방법.The method of claim 6, wherein the crude enzyme solution mixture is a mixture of amyloliquefaciens culture supernatant and Burkholderia vietnamiensis culture supernatant in a volume ratio of 5:1 to 1:10. Phosphorus, sewage sludge pretreatment method.
  7. 제1항에 있어서, 상기 조효소액 혼합물의 첨가량은 하수 슬러지 1L 당 0.1~5g인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The method of claim 1, wherein the amount of the crude enzyme solution mixture is 0.1 to 5 g per 1 L of sewage sludge.
  8. 제1항에 있어서, 상기 반응 온도는 30~45℃이고, 반응 시간은 48~96 hr인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The method of claim 1, wherein the reaction temperature is 30 to 45°C, and the reaction time is 48 to 96 hr.
  9. 하수 슬러지에 미생물 배양액 혼합물을 첨가하고 호기 조건에서 반응시키는 단계를 포함하는 방법으로서,A method comprising the step of adding a microbial culture solution mixture to sewage sludge and reacting under aerobic conditions,
    상기 미생물 배양액 혼합물은 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액의 혼합물인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The microbial culture medium mixture is a mixture of a culture medium of Bacillus amyloliquefaciens and a culture medium of Burkholderia vietnamiensis.
  10. 제9항에 있어서, 상기 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 및 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis)는 하수 슬러지에서 스크리닝된 균주인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The method of claim 9, wherein the amyloliquefaciens (Bacillus amyloliquefaciens ) and Burkholderia vietnamiensis are strains screened from sewage sludge.
  11. 제10항에 있어서, 상기 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 균주는 16S rDNA로 서열번호 1의 염기서열을 포함하는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) PA-21이고, 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis)는 16S rDNA로 서열번호 2의 염기서열을 포함하는 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) LA-96인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The method of claim 10, wherein the amyloliquefaciens (Bacillus amyloliquefaciens ) strain is amyloliquefaciens (Bacillus amyloliquefaciens ) PA-21 containing the nucleotide sequence of SEQ ID NO: 1 in 16S rDNA, and Burgholderia Viet N-Sys Nami (Burkholderia vietnamiensis), the method pre-treatment of sewage sludge, characterized in that Burkholderia Viet Nami N-Sys (Burkholderia vietnamiensis) LA-96 comprising the nucleotide sequence of SEQ ID NO: 2 with 16S rDNA.
  12. 제9항에 있어서, 상기 미생물 배양액 혼합물을 구성하는 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액의 단백질 분해 효소 활성은 400~450 unit/mL이고 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액의 지방 분해 효소 활성은 30~40 unit/mL인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The fat of claim 9, wherein the proteolytic enzyme activity of the amyloliquefaciens (Bacillus amyloliquefaciens ) culture medium constituting the microbial culture medium mixture is 400-450 unit/mL, and Burkholderia vietnamiensis culture medium Degrading enzyme activity is characterized in that 30 to 40 unit / mL, sewage sludge pretreatment method.
  13. 제12항에 있어서, 상기 미생물 배양액 혼합물은 아밀로리쿠에파시엔스( Bacillus amyloliquefaciens) 배양액과 부르크홀데리아 비엣나미엔시스( Burkholderia vietnamiensis) 배양액을 5:1 내지 1:10의 부피비로 혼합한 것인, 하수 슬러지 전처리 방법.The method of claim 12, wherein the microbial culture mixture is a mixture of amyloliquefaciens culture solution and a Burkholderia vietnamiensis culture solution in a volume ratio of 5:1 to 1:10. Sewage sludge pretreatment method.
  14. 제9항에 있어서, 상기 미생물 배양액 혼합물의 첨가량은 하수 슬러지 1L 당 0.1~5g이고, 반응 온도는 30~45℃이고, 반응 시간은 48~96 hr인 것을 특징으로 하는, 하수 슬러지 전처리 방법.The method of claim 9, wherein the addition amount of the microbial culture medium mixture is 0.1 to 5 g per 1 L of sewage sludge, the reaction temperature is 30 to 45°C, and the reaction time is 48 to 96 hr.
  15. 혐기 소화조에 혐기 미생물로 구성된 식종원과 제1항 내지 제14항 중 어느 한 항의 방법으로 전처리된 하수 슬러지를 넣고 산소를 완전히 제거한 혐기 조건에서 배양하는 단계를 포함하는, 혐기소화에 의한 하수 슬러지 처리방법.Sewage sludge treatment by anaerobic digestion, comprising putting a planting source composed of anaerobic microorganisms in an anaerobic digester and sewage sludge pretreated by the method of any one of claims 1 to 14 and culturing under anaerobic conditions to completely remove oxygen Way.
  16. 제15항에 있어서, 상기 배양 온도는 30~45℃이고, 배양 시간은 3~30일 인 것을 특징으로 하는, 혐기소화에 의한 하수 슬러지 처리방법.The method of claim 15, wherein the culture temperature is 30 to 45°C, and the culture time is 3 to 30 days.
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US20150336831A1 (en) * 2013-03-01 2015-11-26 Paradigm Environmental Technologies Inc. Wastewater treatment process and system

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