CN104630104A - Novel strain with azo decoloration capacity and application of novel strain - Google Patents
Novel strain with azo decoloration capacity and application of novel strain Download PDFInfo
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- CN104630104A CN104630104A CN201510053388.6A CN201510053388A CN104630104A CN 104630104 A CN104630104 A CN 104630104A CN 201510053388 A CN201510053388 A CN 201510053388A CN 104630104 A CN104630104 A CN 104630104A
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Abstract
The invention discloses a strain Novibacillus thermophilus CGMCC No.1.12771 and an application thereof in azo dye decoloration. The Novibacillus thermophilus strain SG-1 T is separated from soil and purified, the polyphasic taxonomy result proves that SG-1 T is a novel variety (Novibacillus thermophilus gen.nov.sp.nov.) in the taxonomy, and the novel variety (Novibacillaceae fam.nov.) is established by taking Novibacillus as a type genus. The strain SG-1 T has the effects of decolorizing azo dye golden oranges I under high-temperature anaerobic conditions by utilizing various electron donors, the decoloration rate can be 99 percent, and the strain is an effective biosystem for treating azo dye wastewater and has wide application prospects in the aspect of water environment remediation.
Description
Technical field
The present invention relates to a strain novel bacterial and the application in environmental engineering thereof, be specifically related to a facultative anaerobic bacterium SG-1
tisolation identification, and the application in Azo dye decol, belongs to microflora classification and field of environmental biotechnology.
Background technology
Azoic dyestuff refers to that azo bond (-N=N-) is as chromophoric group, and two ends connect the compound of aromatic group.Ice color is widely used in weaving, leather, plastics, makeup and foodstuffs industry dyeing.Due to these azoic dyestuffs be the organism of poisonous difficult degradation, strong, the persistence of chemical stability be distributed widely in environment, azoic dyestuff is considered to main environmental pollutant.Even if concentration is very low, azoic dyestuff enters water body also can affect light to the perviousness of water and hydrobiological growth, destroys water ecology balance.
The technique of early time treatment waste water from dyestuff mainly utilizes physical/chemical method, and the processing power of these methods to waste water from dyestuff is limited, and energy consumption is large, cost is high, and easily causes secondary pollution.The microbial method treatment of dyeing and printing that development in recent years is got up effectively can not only carry out Azo dye decol process and also cost low, paid close attention to widely.Although the existing certain research of the decolouring of bacterium azo dyes, but in biological treatment process, the broad spectrum of most of bacterial strain is poor, and environmental compatibility ability is more weak, and decolorization rate can not meet the requirement of actual waste water process.Therefore, the emphasis that more efficient decolorizing bacterial strains become Azo dye decol research is sought.
In recent years, some thermophile bacteria due to industrially there is important use, and pay close attention to by more and more investigator.In sewage disposal, the thermophile bacteria in anaerobic environment can improve biodegradation rate, and high temperature kills pathogenic micro-organism, improves organic matter degradation load.But, very few to the azo reduction-decolor report of thermoduric bacteria at present, rare especially to the development and application of the thermophile bacteria with Azo dye decol ability.The discharge water temperature of dyeing waste water is generally between 40 ~ 70 DEG C, can utilize the heat of waste water, reduce the running cost that waste water cools, more effectively carry out the process of pollutent the high temperature anaerobic biological treatment of dyeing waste water.Therefore, azo decolouring discussion is carried out to thermophilic microorganism novel species not only there is important researching value, and all there is directive significance in the practice for the treatment of of dyeing wastewater.
Summary of the invention
The object of the present invention is to provide a strain novel bacterial:
novibacillus thermophilus gen. nov. sp. nov.sG-1
t.
Another object of the present invention is the application providing above-mentioned bacterial strains in Azo dye decol.
The technical solution used in the present invention is:
Bacterial strain of the present invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center by applicant, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preservation date is on January 21st, 2015, and has identified that the bacterial strain of preservation is survival.The preserving number that preservation center gives this culture is CGMCC No.1.12771, and the Classification And Nomenclature of suggestion is Novibacillus thermophilus.
The invention has the beneficial effects as follows:
Bacterial strain of the present invention
novibacillus thermophilus gen. nov. sp. nov.sG-1
t, under high temperature anaerobic condition, (50 DEG C) utilize methyl alcohol, formic acid, acetic acid, ethanol, lactic acid, glucose and pyruvic acid as electron donor, and azo dyes gold orange I has reduction-decolor effect, and percent of decolourization can up to 99%.The processing power of bacterial strain of the present invention to high temperature waste water from dyestuff is large, energy consumption is little, cost is low, be not easy to cause secondary pollution, can not water column structure be destroyed, be a kind of living things system of effective process azo dye wastewater, be with a wide range of applications in aquatic ecological restoration.
Accompanying drawing explanation
Fig. 1 is bacterial strain SG-1
tthe scanning electron microscope (SEM) photograph of cell;
Fig. 2 is bacterial strain SG-1
tthe transmission electron microscope picture of cell ultrathin section(ing);
The bacterial strain SG-1 that Fig. 3 adopts Neighbor-joining method to build
tphylogenetic tree;
Fig. 4 bacterial strain is at SG-1
tthe situation of anaerobic reduction gold orange I at different temperatures;
Fig. 5 bacterial strain SG-1
tto the decoloring ability of gold orange I under different electron donor.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited thereto.
embodiment 1
one, bacterial strain SG-1
t
separation and enrichment
1) pedotheque 10g being taken from Guangdong Province Xuwen County tropical rain forest adds in 100 mL 2216E substratum, cultivates 2 weeks for 50 DEG C; Nutrient solution is carried out gradient dilution, coats on 2216E substratum solid plate, cultivate under conditions.
2) picking list bacterium colony, and coat on new 2216E substratum, further abstraction and purification thalline.
3) step poly-2 is repeated), until obtain mono-clonal bacterium colony SG-1
t.
two, bacterial strain SG-1
t
morphological specificity
1) thalli morphology characteristic
Bacterial strain SG-1
tthalline two ends are blunt round, and thalline is slightly curved, arrange (as shown in Figure 1) in Bamboo-shaped long-chain.Bacterial strain SG-1
tfor Gram-negative bacteria, the general 0.4-0.5 of somatic cells μm wide, and 0.7-2.3 μm long, atrichia, do not produce gemma (as shown in Figure 2), and the CW in Fig. 2 refers to cell walls (Cell Wall).
2) colonial morphology characteristic
Bacterial strain SG-1
tafter activated, under 50 DEG C of aerobic conditions, forming diameter after 24 hours at LB slat chain conveyor is that 1-1.5 mm is dark yellow, circle raises up, opaque bacterium colony.
three, bacterial strain SG-1
t
classification position
Extract invention bacterial strain SG-1
tsTb gene.With the DNA extracted for template, with the 16S rRNA gene of universal primer 27F and 1492R amplification bacterium, check order after pcr amplification product being reclaimed purifying, gained sequence (1489bp) carries out sequence similarity calculating by the online comparison instrument of EzTaxon server 2.1 website.By bacterial strain SG-1
tand after the 16S rRNA sequence of close bacterial strain carries out Multiple Sequence Alignment analysis by CLUSTALX.1.8, Mega (Version5.0) software is utilized to make Phylogenetic Analysis, adopt Neighbor-joining method constructing system evolutionary tree, the evolutionary tree bootstrap method validation of 1200 repeated samplings.
Result shows, with SG-1
tthe highest bacterial strain of 16s rRNA sequence similarity be
mechercharimyces asporophorigenens(90.8%), be secondly
lihuaxuella thermophila(90.7%),
m. mesophilus(90.6%) and
polycladomyces abyssicola(90.5%).Be greater than in the pattern bacterium of 90% in 16s rRNA similarity,
microaerobacter geothermalisnad S1
t(similarity 90.4%) is that a unique strain does not exist
thermoactinomycetaceaepattern bacterium in section.Evolutionary analysis shows, the pattern bacterium the most close with SG-1T sees
thermoactinomycetaceaein section (
m. asporophorigenensyM11-542T and
m. mesophilusyM3-251T), other two strain phase plesiotype bacterium
microaerobacter geothermalisnad S1
twith
caldalkalibacillus uzonensisjW/WZ-YB58
tdo not exist
thermoactinomycetaceaein section.But, in evolutionary tree (Fig. 3), SG-1
t?
bacillalesnow an obvious branch is formed.Generally speaking, exist
bacillalesnow, SG-1
twith
thermoactinomycetaceaesection the most close to but form branch independent and far away.
four, bacterial strain SG-1
t
physiological and biochemical analysis
In order to understand bacterial strain SG-1 further
t, test this bacterial strain relevant physiological biochemical character according to standard program.
In LB liquid nutrient medium, SG-1
tcan grow under the temperature condition of 30-65 DEG C, optimum growth temperature is 50 DEG C, and this bacterium can grow under the pH condition of 6.5-11.0, and the most suitable growth pH is 7.5-8.0.Moderate is addicted to salt, and growth scope is 0-10% NaCl, and under the salt concn of 5.0-7.0%, growing way is best.A series of Physiological Appraisal shows, SG-1
tthere is oxydase, catalase and betagalactosidase activity, can caseinhydrolysate, gelatin and polychrom, but TYR, tween 80, polysorbas20 can not be hydrolyzed.Gluconate can be utilized.Negative in the growth of Mai Kangkai culture plate, the growth of 2216E substratum is positive, and globulolysis is positive, and nitrate reduction is positive.Bacterial strain SG-1
tand the difference characteristic between the type strain of relative genus is in table 1.
Table 1 bacterial strain SG-1
tand the difference characteristic between the type strain of relative genus
+: positive;-: negative
SG-1
tcarbon under anaerobism/nitrogenous source utilization power is in table 2, and utilization of carbon source is with (NH4)
2sO
4as unified nitrogenous source, nitrogenous source utilizes using glucose as unified carbon source.
Table 2 bacterial strain SG-1
tsugar is utilized to produce comparing of sour situation with the type strain of relative genus
+: positive;-: negative
five, bacterial strain SG-1
t
cYTOCHEMICAL ANALYSIS
For peptidoglycan analytical results shows, SG-1
tcell walls hydrolysate mainly comprise: amino acid (Ala), L-glutamic acid (Glu) and identify meso diaminopimelic acid (
meso-DAP); Partial water hydrolysis products comprises: polypeptide class L-Ala-D-Glu and
meso-DAP-D-Ala.These results all show the peptidoglycan type of SG-1T be A1 γ (
meso-DAP direct).
SG-1
tmain breathing quinone be MK-7 (92.1%), be secondly MK-8 (7.9%), this and its at the most close genus of 16s rRNA sequence
mechercharimycescomplete difference (
mechercharimycesthe main breathing quinone belonged to is MK-9).SG-1
tg+C content be 50.5%; Main fatty acid contained when SG-1T cultivates under 50 ° of C conditions is iso-C15:0 (52.9%), iso-C17:0 (15.0 %), anteiso-C15:0 (10.0 %).Further, to bacterial strain SG-1
tpolar lipid analyze, by the analysis of two-dimensional TLC (TLC) method display SG-1
tpolar lipid component mainly comprise diphosphatidylglycerol (DPG), phosphatidyl glycerol (PG), phosphatidylethanolamine (PE) and a small amount of APL.
Based on SG-1
tthe Phylogenetic Analysis of 16s rRNA similarity, and the serial identification experiment such as physiological and biochemical property shows, SG-1
tmany important difference are had: 1. there is gram-negative bacteria cell wall feature with the most close pattern bacterium; 2. can at aerobic and grown under anaerobic conditions; 3. iso-C15:0 is as main lipid acid, and MK-7 is main breathing quinone; 4. with
thermoactinomycetaceaesection compares, and does not produce mycelia, does not produce gemma.SG-1
talso serious difference is had in growth scope, cell aggregation form, mobility etc. with close type strain.In conjunction with SG-1
tin 16s rRNA gene order and the low similarity (90.8%) of nearest bacterial strain, polyphase sort result qualification SG-1
tbe new genera and species on taxonomy (
novibacillus thermophilus gen. nov. sp. nov.), and set up new (
novibacillaceaefam. nov.), by bacterial classification SG-1 of the present invention
tcalled after
novibacillus thermophilus gen. nov. sp. nov.sG-1
t.
six, bacterial strain SG-1
t
the summation of feature
Comprehensive above-mentioned bacterial strains SG-1
tand the various distinguishing characteristicss between existing bacterial strain, and bacterial strain SG-1
tdetected result in other respects, to bacterial strain SG-1
tfeature be summarized as follows:
1. morphological specificity: gram negative bacillus, without mobility, slight catenation between thalline.Thalline is straight or slightly curved, and 0.4-0.5 μm wide, and 0.7-2.3 μm long.2216E agar plate forms white, little and round bacterium colony; LB agar plate forms dark yellow bacterium colony.
2. growth characteristics: amphimicrobian, can anaerobic growth in the substratum containing different electron donor and electron acceptor(EA).Can not grow in Mai Kangkai substratum.Growth scope is: 30-65 DEG C (the suitableeest 50 DEG C), pH 6.5-10.5 (optimal pH 7.5-8.0), 0-10% (w/v) NaCl salinity (the suitableeest 5.0-7.0%).
3. there is hemolytic action: under 50 DEG C of conditions, blood agar is cultivated after 2 days and can observe hemolytic action;
4. enzymic activity situation: oxydase, catalase, betagalactosidase activity, nitrate reduction; Alkaline phosphatase, acid phosphatase, N-acetyl-2-amino glucosaccharase activity are positive; Urase, arginine hydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophane deaminase active are negative.Hydrolyzable gelatin, polychrom and casein, be not hydrolyzed tween 80, polysorbas20 hydrolysis and TYR.
5. chemical feature: whole cell peptidoglycan type is A1 γ.Main breathing quinone is MK-7, and dominant polarity fat is DPG, PG and PE.DNA G+C component is 50.5 mol%, and main fatty acid is iso-C15:0, iso-C17:0 and anteiso-C15:0.
seven, bacterial strain SG-1
t
the reduction-decolor of azo dyes
1) bacterial strain SG-1
t
azo decolouring at different temperatures
1. materials and methods
Azoic dyestuff: gold orange I (Orange I, 1-naphthols azo P-TOLUENE SULFO ACID 99 sodium) is purchased from Shanghai Reagent Company (chemical pure).
Basic medium (BAS): often liter containing NaHCO
32.5g, (NH4)
2sO
40.25g, NaH
2pO
40.6g, KCl 0.1g, yeast extractive substance 0.2 g, a small amount of vitamin stock and trace element.
Temperature experiment is placed in 25,30,35,40,45,50,55,60,65,70 DEG C of incubators and leaves standstill lucifuge cultivation.
Method: add in BAS substratum by electron donor acetic acid, bacteria suspension, gold orange I, final concentration is respectively 5.0 mmol/L, 3 × 10
6cFU/mL, 0.2 mmol/L.Then thalline is placed in respectively 25,30,35,40,45,50,55,60,65,70 DEG C of incubators and leaves standstill lucifuge cultivation, and detect the concentration of gold orange I at different incubation time point.
Adopt ultraviolet-visible spectrophotometry measure each group of gold orange I concentration: first by sample in centrifugal 10 min of 6800 r/min, be the absorption spectrum curve surveying supernatant liquor (or dilution after) within the scope of 200 ~ 600 nm at wavelength, determine the characteristic wavelength λ of the azo bond of gold orange I
max=480 nm.Characterize the decolouring intensity to gold orange I with percent of decolourization, percent of decolourization (D) calculation formula is: D=(C
0– C
t)/C
0× 100%, C
0for gold orange I starting point concentration, C
tfor gold orange I is in the concentration of reaction t time.
2. results and discussions
Under condition of different temperatures, bacterial strain SG-1
tfig. 4 is shown in the anaerobic decolorization effect of gold orange I.After 32h, 25,30,35,40,45,50,55,60,65,70 DEG C of percent of decolourizations are respectively 7%, 13.5%, 20%, 40%, 89%, 99%, 90%, 37.5%, 15%, 5.5%.The absorption of general thalline accounts for 8%, therefore thinks to think at 25,70 DEG C biochemical reaction does not occur.Percent of decolourization under differing temps and bacterial strain SG-1
tgrowth scope contact directly, at optimum growth temperature 50 DEG C, its percent of decolourization is also the highest.Illustrate that suitable growth temperature makes bacterial strain be bred fast, and then improve decolorizing efficiency.
2) bacterial strain SG-1
t
azo decolouring under different electron donor
1. materials and methods
Azoic dyestuff and basic medium are with reference to above-mentioned 1).
Electron donor: methyl alcohol (Methanol), formic acid (Formate), ethanol (Alcohol), acetic acid (Acetate), lactic acid (Lactate), glucose (Glucose), pyruvic acid (Pyuvate) are purchased from Shanghai Reagent Company (chemical pure).
The method that under different electron donor, bacterial strain decolours to gold orange I: join in BAS substratum by different electron donor, gold orange I, bacteria suspension successively, final concentration is with above-mentioned 1).The electron donor selected has methyl alcohol, formic acid, ethanol, acetic acid, lactic acid, glucose, pyruvic acid.Not add donor (Without e-donor) and not add bacterium (Without strain SG-1) in contrast, measure gold orange I change in concentration in each process.More than test and all adopt strict aseptic and Anaerobic culturel operation, each process arranges 3 repetitions.Gold orange I concentration determination is with reference to above-mentioned 1) experimental technique.
2. results and discussions
Bacterial strain SG-1
tutilize the situation of different electron donor vat golden orange I as Fig. 5.Result shows, and through 32h reaction, in the process of methyl alcohol, formic acid, acetic acid, ethanol, lactic acid, glucose and pyruvic acid, reduction ratio is respectively 96.9%, 99.1%, 99.2%, 77.3%, 88.2%, 99.0% and 79.9%.Do not add the contrast of electron donor or SG-1T, be showed no decolouring phenomenon.Thus, bacterial strain SG-1T can utilize all for examination electron donor vat golden orange I, and when formic acid, acetic acid, glucose are donor, reduction effect is better, has certain broad spectrum.
For those skilled in the art's easy understand; the foregoing is only the preferred embodiment of patent of the present invention; not in order to limit the present invention, any amendment done within all the spirit and principles in the present invention, equivalent to replace and improvement etc., within the protection domain all dropping on application claims.
Claims (3)
1. a novel bacterial Novibacillus thermophilus gen. nov. sp. nov. SG-1
t, be deposited in General Microbiological Culture preservation center, its preserving number is
cGMCC 1.12771.
2. novel bacterial Novibacillus thermophilus gen. nov. sp. nov. SG-1 according to claim 1
tapplication in Azo dye decol.
3. an Azo dye decol biotechnological formulation, is characterized in that: containing new bacterium Novibacillus thermophilus gen. nov. sp. nov. SG-1 according to claim 1 in described preparation
t.
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Cited By (2)
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| CN106399175A (en) * | 2016-09-28 | 2017-02-15 | 甘肃农业大学 | Novel application of dipeptide meso-DAP-D-Ala and/or tetrapeptide L-Ala-D-Glu-L-Lys-D-Ala |
| CN108395002A (en) * | 2017-02-08 | 2018-08-14 | 广州中国科学院先进技术研究所 | Degradation of Azo Dyes Decolourization Bacteria and its application |
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| CN101880639A (en) * | 2010-01-29 | 2010-11-10 | 清华大学 | Staphylococcus pasteuri and application thereof in decolorization |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399175A (en) * | 2016-09-28 | 2017-02-15 | 甘肃农业大学 | Novel application of dipeptide meso-DAP-D-Ala and/or tetrapeptide L-Ala-D-Glu-L-Lys-D-Ala |
| CN108395002A (en) * | 2017-02-08 | 2018-08-14 | 广州中国科学院先进技术研究所 | Degradation of Azo Dyes Decolourization Bacteria and its application |
| CN108395002B (en) * | 2017-02-08 | 2021-04-20 | 广州中国科学院先进技术研究所 | Azo dye degradation decolorizing bacterium and application thereof |
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| CN104630104B (en) | 2018-08-21 |
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