CN101096645A - Gas production enterobacteria and uses thereof - Google Patents

Abstract

The invention discloses an enterobacteria aerogenes and the application. The inventor screens Enterobacter aerogenes XM02 by a great deal of experimental researches. The bacterial has ferrum redactum active, facultative and electron donor, which can survive in the aerobic or anaerobic environment, wherein the electron donor in the anaerobic environment has the wide application, the electron donor which can electron donor comprises glycerin, citrate, dextrose, sucrose or the like, the ferric iron which can deacidize comprises szomolnokite, goethite, lepidocrocite, bloodstone, yellow copiapite or the like.

Description

Enteroaerogen and application thereof

Technical field

The present invention relates to novel bacterial triage techniques field, specifically, relate to a kind of enteroaerogen and application thereof.

Background technology

The average content of ferro element in the earth's crust is 5.6%, is the metal that abundance is number two in the earth's crust.Usually with the form existence of insoluble Fe (III) oxide compound, the reduction of iron is that the bacterium with iron reducing power is a ferriferous oxide reductive power by the enzymatic reaction of specified microorganisms mediation to iron in physical environment.Studies show that, the iron reduction reaction not only influences the biomass geochemistry circulation of iron, and participate in multiple edatope process, comprise: the soil gleying process, nutritive element (as P, K, Ca, Mg etc.) discharges with the migration of micro-metals, the anaerobic degradation of organic pollutant, the reduction and detoxication of metal and inhibition discharge of methane etc. are with a wide range of applications.

Alienation iron reduction (Dissimilatory Iron Reduction) be meant microorganism by respiration with transfer transport to extracellular ferriferous oxide surface, Fe (III) is reduced into the process of Fe (II).Different with assimilation is that the reduction product Fe (II) in the alienation iron reduction process accumulates in the extracellular, does not enter in the born of the same parents as cellular constituent.The iron reducing bacteria is meant can be by oxidation electron donor coupling reduction Fe (III), and therefrom obtains the microorganism of energy.The Lovley metallic reducing ground bacillus (Geobacter metallireducens GS 15) that discovery has alienation iron reducing power in freshwater sediment first in 1987.At present, the separated purifying of the iron of strain more than 100 reducing bacteria has been arranged, mainly be distributed in ground Bacillaceae (Geobacter sp.), Shiva Bordetella (Shewanella sp.), Desulfobacter (Desulfitobacterium sp.), Desulfovibrio several genus such as (Desulfomicrobium sp.).But these bacterial strains with iron reducing activity remain in following defective: 1) most of bacterial strains can only be survived in the strictly anaerobic environment, are unfavorable for scale operation and practical application; 2) the electron donor utilization of bacterial strain spectrum is narrower, can only utilize small molecular weight organism such as acetate, lactic acid as electron donor, can not utilize glucose, organism that the sucrose equimolecular quantity is bigger, and is higher relatively to the environmental requirement of practical application.Enteroaerogen is the ubiquitous bacterial strain of occurring in nature, but finds to have the report of the enteroaerogen of iron reducing activity so far yet.

Summary of the invention

The objective of the invention is to overcome the deficiency that prior art exists, provide a strain to have iron reducing activity, amphimicrobian, enteroaerogen that electron donor utilization spectrum is wider.

Another object of the present invention provides the application of above-mentioned enteroaerogen in the ferric iron reduction.

The contriver has separated enteroaerogen (Enterobacter aerogenes XM02) through a large amount of experimental studies from the underground ancient forest soil in Zhaoqing, Guangdong.In on March 12nd, 2007 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, be numbered: CGMCC 1969.

This bacterial strain is a rod-short, size range is 1.0～1.7 * 0.6～0.8 μ m, colony colour is a white or yellow, amphimicrobian, its electron donor utilization spectrum is wider under the anaerobic environment, organism such as energy oxidation citric acid, glycerol, glucose, sucrose, ferriferous oxides such as reductive water iron ore, pyrrhosiderite, lepidocrocite, rhombohedral iron ore, ihleite simultaneously.

One, bacterial strain of the present invention from Zhaoqing, Guangdong underground ancient forest soil separate, the purifying gained, its separation purification method is as follows:

1) liquid separation culture medium:

Contain in every liter of substratum: 1.0g glucose, 16g ironic citrate, 2.5g NaHCO ₃, 0.25g NH ₄Cl, 0.678g NaH ₂PO ₄2H ₂O, 0.1g KCl, vitamin solution 10.0mL, trace element solution 10.0mL; PH 5.0～6.0.Wherein, vitamin solution is to contain 2.0mg vitamin H, 2.0mg folic acid, 10.0mg vitamin B6,5.0mg VitB1,5.0mg riboflavin, 5.0mg nicotinic acid, 5.0mg calcium pantothenate, 0.1mg vitamin B12,5.0mg para-amino benzoic acid, 5.0mg Thioctic Acid in every premium on currency; Trace element solution is to contain 1.5g nitrilotriacetic acid(NTA), 3.0g MgSO in every premium on currency ₄7H ₂O, 0.5g MnSO ₄H ₂O, 1.0g NaCl, 0.1gFeSO ₄7H ₂O, 0.1g CoCl ₂6H ₂O, 0.1g CaCl ₂, 0.1g ZnSO ₄7H ₂O, 0.01gCuSO ₄5H ₂O, 0.01g AlK (SO ₄) ₂12H ₂O, 0.01g H ₃BO ₃, 0.01gNa ₂MoO ₄2H ₂O.

2) enrichment: take by weighing the ancient forest soil sample of 5g and be inoculated into the 100mL liquid separation culture medium, bulgingly in the nutrient solution fill high-purity mixed gas (N ₂/ CO ₂=80/20) 〉=1 hour; After air-blowing finishes culture placed anaerobism workstation (N ₂/ CO ₂=80/20) 30 ℃ leave standstill cultivation, the colour-change situation that the productive rate of detection Fe (II) is observed nutrient solution simultaneously.When the output of Fe (II) constantly increased, the color of nutrient solution became black at last from original orange-yellow deepening gradually.When the output of Fe (II) surpass total iron content 50% the time, the inoculum size with 10% is forwarded in another fresh liquid separation culture medium, three times so repeatedly.

3) purifying: evenly coat on the solid medium after reacted nutrient solution suitably dilutes with the 4th generation, place anaerobism workstation (N ₂/ CO ₂=80/20) cultivated 48 hours for 30 ℃, picking list bacterium colony carries out purifying.The prescription of solid medium is identical with liquid separation culture medium, is every liter and has added 15g agar.

Two, adopt following method to verify the iron reducing activity of bacterial strain of the present invention, determine its electron donor and electron acceptor(EA) utilization spectrum.

1) be seeded to the beef extract-peptone liquid nutrient medium from the inclined-plane of preserving enteroaerogen XM02, in 30 ℃, 180 rev/mins of shaking tables activate thalline 9 hours, make bacterial number reach exponential phase of growth;

2) in the aforesaid liquid isolation medium, add different electron donors (as: formic acid, acetate, propionic acid, lactic acid, citric acid, ethanol, glycerol, glucose, sucrose etc.) and electron acceptor(EA) (as: ironic citrate, ferrihydrite, pyrrhosiderite, rhombohedral iron ore, ihleite etc.), inoculum size inoculation with 10% activates bacterium liquid in the aforesaid liquid isolation medium, anaerobism workstation (N ₂/ CO ₂=80/20) following 30 ℃ leave standstill cultivation;

3) at regular intervals (about 5～10 days) get the 4mL nutrient solution in 16mL 0.5M HCl solution (M represents to contain in every premium on currency the mole number of HCl) 180 rev/mins the vibration 1.5 hours; Precipitations such as thalline are filtered, adopt adjacent phenanthroline method to give the Fe (II) in the filtrate colour developing, adopt ultraviolet-visible spectrophotometer to measure the content of Fe (II) at the 510nm place, the iron reducing activity of checking bacterial strain.

Compared with prior art, the present invention has following beneficial effect:

The contriver has filtered out enteroaerogen (Enterobacteraerogenes XM02) through a large amount of experimental studies.This bacterial strain has iron reducing activity, amphimicrobian, electron donor utilization spectrum is wider.Can survive under aerobic or anaerobic environment, its electron donor utilization spectrum is wider under the anaerobic environment, and the electron donor of energy oxidation has: organism such as glycerol, citric acid, glucose, sucrose; Reducible ferric iron has: ferrihydrite, pyrrhosiderite, lepidocrocite, rhombohedral iron ore, ihleite etc.

Description of drawings

Fig. 1 is 30000 times of electron microscope pictures of E.aerogenes XM02.

Embodiment

Embodiment 1 strains separation

1) isolation medium: contain 1.0g glucose in every liter of isolation medium; The 16g ironic citrate; 2.5g NaHCO ₃0.25g NH ₄Cl; 0.678g NaH ₂PO ₄2H ₂O; 0.1g KCl; Vitamin solution 10.0mL; Trace element solution 10.0mL; PH 5.5.Wherein, vitamin solution is to contain 2.0mg vitamin H, 2.0mg folic acid, 10.0mg vitamin B6,5.0mg VitB1,5.0mg riboflavin, 5.0mg nicotinic acid, 5.0mg calcium pantothenate, 0.1mg vitamin B12,5.0mg para-amino benzoic acid, 5.0mg Thioctic Acid in every premium on currency; Trace element solution is to contain 1.5g nitrilotriacetic acid(NTA), 3.0g MgSO in every premium on currency ₄7H ₂O, 0.5g MnSO ₄H ₂O, 1.0gNaCl, 0.1g FeSO ₄7H ₂O, 0.1g CoCl ₂6H ₂O, 0.1g CaCl ₂, 0.1gZnSO ₄7H ₂O, 0.01g CuSO ₄5H ₂O, 0.01g AlK (SO ₄) ₂12H ₂O, 0.01gH ₃BO ₃, 0.01g Na ₂MoO ₄2H ₂O.

2) enrichment: take by weighing the ancient forest soil sample of 5g and be inoculated into 100mL aforesaid liquid isolation medium; Drum fills high-purity mixed gas (N in the nutrient solution ₂/ CO ₂=80/20) 1 hour; After air-blowing finishes culture placed anaerobism workstation (N ₂/ CO ₂=80/20) 30 ℃ leave standstill cultivation.The colour-change situation that the productive rate of detection Fe (II) is observed nutrient solution simultaneously.When the output of Fe (II) constantly increased, the color of nutrient solution became black at last from original orange-yellow deepening gradually.When the content of Fe (II) surpass total iron content 50% the time, the inoculum size with 10% is forwarded in another fresh aforesaid liquid isolation medium, three times so repeatedly.

3) purifying: last, evenly coat on the above-mentioned solid separation culture medium after reacted nutrient solution suitably dilutes with the 4th generation, place anaerobism workstation (N ₂/ CO ₂=80/20) cultivated 48 hours for 30 ℃, picking list bacterium colony carries out purifying.

Embodiment 2 CHARACTERISTICS IDENTIFICATION

(1) thalli morphology characteristic

Adopt conventional bacterium electron microscope observation, this bacterial strain is a rod-short, and size range is 1.0～1.7 * 0.6～0.8 μ m.(see figure 1)

(2) colonial morphology characteristic

Aerobic cultivation is after 24 hours on beef extract-peptone solid medium flat board, and colonial morphology is circular, neat in edge; Smooth surface, low convex surface; White, colony diameter are 2～3mm; After anaerobism on the embodiment 1 described solid separation culture medium flat board was cultivated 48 hours, colonial morphology was circular, neat in edge; Smooth surface, high convex surface; Yellow, colony diameter are 2～3mm.

(3) physiology, biochemical characteristic

According to BioLog microorganism identification instrument, analyze this bacterial strain situation of utilizing to 96 kinds of different carbon sources under aerobic condition, but this bacterial strain of preliminary evaluation is an entero-bacte.Other Physiology and biochemistries the results are shown in Table 1:

Table 1. bacterial strain physio-biochemical characteristics

Characteristic	The result	Characteristic	The result
				Gramstaining	-	Oxydase	-
Indoles	-	Methyl red (M.R)	-
				Acetyl methyl carbinol (V.P.)	+	Citrate trianion	+
Ribitol	+	The D-seminose	+
				The D-arabitol	+	The D-melizitose	+
The D-cellobiose	+	Acetate	-
				Erythritol	-	The cis equisetic acid	+
D-fructose	-	Citric acid	+
				The L-trehalose	+	Formic acid	-
The D-semi-lactosi	+	Glycerin/glycerol	+
				Gentiobiose	+	α-Tong Wuersuan	-
Alpha-D-glucose	-	α-oxopentanoic acid	-
				The m-inositol	+	D, L-lactic acid	+
α-D-lactose	+	Propionic acid	-
				Lactulose	+	Sebacic acid	-
Maltose	+	Succsinic acid	-

Annotate :+be expressed as the positive maybe can utilize;-be expressed as feminine gender maybe can not utilize

(4) molecular biological characteristic

Adopt the SDS-Proteinase K, chloroform-primary isoamyl alcohol (volume ratio 24: 1) extracting, the method for 0.6 volume isopropanol precipitating is extracted bacteria total DNA.Adopt the 16S rDNA of bacterial 16 S rDNA universal primer F8 and R1542 and bacterium gyrB gene universal primer UP-1/UP-2r pcr amplification bacterium, tangible band appears near 1500bp, to carry out sequencing after the pcr amplification product recovery, with the dna sequence dna input GenBank that obtains, with the Blastn program all sequences in the database is compared analysis, found that bacterial strain of the present invention 16S rDNA sequence and with GenBank in a plurality of bacterial strains of enteroaerogen have higher homology, wherein reach 99% (homology of the 16SrDNA sequence of same bacterial classification should greater than 97.7%) with the homology of the 16SrDNA (accession number is AF395913) of the type strain (Enterobacter aerogenes) of enteroaerogen.

In conjunction with the result of above-mentioned BioLog microorganism identification result, physio-biochemical characteristics, 16S rDNA sequence, this bacterial strain should belong to enteroaerogen (Enterobacter aerogenes), called after Enterobacter aerogenes XM02.

Embodiment 3 iron reducing activities

Electron donor is a citric acid, and electron acceptor(EA) is a ferrihydrite

Substratum (a): in the 1.0L deionized water, add 2.0g citric acid, 10.0g ferrihydrite, 2.5g NaHCO ₃, 0.25g NH ₄Cl, 0.678g NaH ₂PO ₄2H ₂O, 0.1g KCl, 10.0mL vitamin solution, 10.0mL trace element solution; Wherein, vitamin solution and trace element solution prescription are with embodiment 1; The pH that regulates substratum (a) is 5.5, and sterilization is 20 minutes under 121 ℃ of conditions, and cooling back drum fills high-purity mixed gas (N ₂/ CO ₂=80/20) 1 hour.Inoculate thalline in the beef extract-peptone liquid nutrient medium from the inclined-plane of preserving enteroaerogen XM02, in 30 ℃, 180 rev/mins of shaking tables activate thalline 9 hours, make bacterial number reach exponential phase of growth, inoculum size inoculation activation bacterium liquid with 10% is in above-mentioned substratum (a), 30 ℃ leave standstill the anaerobism cultivation, and the contrast that does not add bacterium is set simultaneously.Observe the colour-change situation of nutrient solution, got the 180 rev/mins of vibrations 1.5 hours in 16mL 0.5M HCl solution (M represents to contain in every premium on currency the mole number of HCl) of 4mL nutrient solution every 5～10 days.Adopt the content of adjacent phenanthroline colorimetric method for determining Fe (II), the iron reducing power of checking bacterial strain.Fe (II) content (unit is mg/L, promptly represents the milligram number of contained Fe (II) in every liter of sample) sees Table 2:

Table 2.XM02 bacterial strain reductive water iron ore produces the change in concentration (mg/L) of Fe (II)

	0 day	5 days	10 days	20 days
					Do not add the bacterium contrast	0.02	0.79	1.93	3.98
Add E.aerogenes XM02	0.01	17.84	39.76	84.99

Embodiment 4 iron reducing activities

Electron donor is a citric acid, and electron acceptor(EA) is a pyrrhosiderite

Substratum (b): in the 1.0L deionized water, add 2.0g citric acid, 9.0g pyrrhosiderite, 2.5g NaHCO ₃, 0.25g NH ₄Cl, 0.678g NaH ₂PO ₄2H ₂O, 0.1g KCl, 10.0mL vitamin solution, 10.0mL trace element solution; Wherein, vitamin solution and trace element solution prescription are with embodiment 1; The pH that regulates substratum (b) is 5.5, and sterilization is 20 minutes under 121 ℃ of conditions, and cooling back drum fills high-purity mixed gas (N ₂/ CO ₂=80/20) 1 hour.Be seeded to the beef extract-peptone liquid nutrient medium from the inclined-plane of preserving enteroaerogen XM02, in 30 ℃, 180 rev/mins of shaking tables activate thalline 9 hours, make bacterial number reach exponential phase of growth, inoculum size inoculation activation bacterium liquid with 10% is in above-mentioned substratum (b), and 30 ℃ leave standstill the anaerobism cultivation.Observe the colour-change situation of nutrient solution, got the 180 rev/mins of vibrations 1.5 hours in 16mL0.5M HCl solution (M represents to contain in every premium on currency the mole number of HCl) of 4mL nutrient solution every 5～10 days.Adopt adjacent phenanthroline method give to measure the content of Fe (II), and be contrast, verify the iron reducing power of bacterial strain with the nutrient solution (b) that does not add bacterium.Fe (II) content (unit is mg/L, promptly represents the milligram number of contained Fe (II) in every liter of sample) sees Table 3:

Table 3.XM02 bacterial strain reduction pyrrhosiderite produces the change in concentration (mg/L) of Fe (II)

	0 day	5 days	10 days	20 days
					Do not add the bacterium contrast	0.01	0.32	1.79	2.74
Add E.aerogenes XM02	0.01	12.84	29.55	61.29

Embodiment 5 iron reducing activities

Electron donor is a glycerol, and electron acceptor(EA) is a ferrihydrite

Substratum (c): in the 1.0L deionized water, add 0.92g glycerol, 10.0g ferrihydrite, 2.5g NaHCO ₃, 0.25g NH ₄Cl, 0.678g NaH ₂PO ₄2H ₂O, 0.1g KCl, 10.0mL vitamin solution, 10.0mL trace element solution; Wherein, vitamin solution and trace element solution prescription are with embodiment 1; The pH that regulates substratum (c) is 5.5, and sterilization is 20 minutes under 121 ℃ of conditions, and cooling back drum fills high-purity mixed gas (N ₂/ CO ₂=80/20) 1 hour.Be seeded to the beef extract-peptone liquid nutrient medium from the inclined-plane of preserving enteroaerogen XM02, in 30 ℃, 180 rev/mins of shaking tables activate thalline 9 hours, make bacterial number reach exponential phase of growth, inoculum size inoculation activation bacterium liquid with 10% is in above-mentioned substratum (c), and 30 ℃ leave standstill the anaerobism cultivation.Observe the colour-change situation of nutrient solution, got the 180 rev/mins of vibrations 1.5 hours in 16mL0.5M HCl solution (M represents to contain in every premium on currency the mole number of HCl) of 4mL nutrient solution every 5～10 days.Adopt adjacent phenanthroline method to measure the content of Fe (II), and be contrast, verify the iron reducing power of bacterial strain with the nutrient solution (c) that does not add bacterium.Fe (II) content (unit is mg/L, promptly represents the milligram number of contained Fe (II) in every liter of sample) sees Table 4:

Table 4.XM02 bacterial strain reductive water iron ore produces the change in concentration (mg/L) of Fe (II)

	0 day	5 days	10 days	20 days
					Do not add the bacterium contrast	0.03	0.39	0.87	1.23
Add E.aerogenes XM02	0.02	17.27	31.36	64.85

Embodiment 6 iron reducing activities

Electron donor is a sucrose, and electron acceptor(EA) is a ferrihydrite

Substratum (d): in the 1.0L deionized water, add 3.42g sucrose, 10.0g ferrihydrite, 2.5g NaHCO ₃, 0.25g NH ₄Cl, 0.678g NaH ₂PO ₄2H ₂O, 0.1g KCl, 10.0mL vitamin solution, 10.0mL trace element solution; Wherein, vitamin solution and trace element solution prescription are with embodiment 1; The pH that regulates substratum (d) is 5.5, and sterilization is 20 minutes under 121 ℃ of conditions, and cooling back drum fills high-purity mixed gas (N ₂/ CO ₂=80/20) 1 hour.Be seeded to the beef extract-peptone liquid nutrient medium from the inclined-plane of preserving enteroaerogen XM02, in 30 ℃, 180 rev/mins of shaking tables activate thalline 9 hours, make bacterial number reach exponential phase of growth, inoculum size inoculation activation bacterium liquid with 10% is in above-mentioned substratum (d), and 30 ℃ leave standstill the anaerobism cultivation.Observe the colour-change situation of nutrient solution, got the 180 rev/mins of vibrations 1.5 hours in 16mL0.5M HCl solution (M represents to contain in every premium on currency the mole number of HCl) of 4mL nutrient solution every 5～10 days.Adopt adjacent phenanthroline method to measure the content of Fe (II), and be contrast, verify the iron reducing power of bacterial strain with the nutrient solution (d) that does not add bacterium.Fe (II) content (unit is mg/L, promptly represents the milligram number of contained Fe (II) in every liter of sample) sees Table 5:

Table 5.XM02 bacterial strain reductive water iron ore produces the change in concentration (mg/L) of Fe (II)

	0 day	5 days	10 days	20 days
					Do not add the bacterium contrast	0.03	1.26	3.18	7.97
Add E.aerogenes XM02	0.03	61.24	108.66	189.34

Claims (2)

1. enteroaerogen is characterized in that: it is rod-short, on March 12nd, 2007 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, be numbered: CGMCC 1969.

2. the application of the described enteroaerogen of claim 1 in the ferric iron reduction.

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