CN112063550A - For reducing Fe in complex stateⅢAnaerobic strain, culture method and application thereof - Google Patents

For reducing Fe in complex stateⅢAnaerobic strain, culture method and application thereof Download PDF

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CN112063550A
CN112063550A CN202010855065.XA CN202010855065A CN112063550A CN 112063550 A CN112063550 A CN 112063550A CN 202010855065 A CN202010855065 A CN 202010855065A CN 112063550 A CN112063550 A CN 112063550A
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刘楠
陈润
吴超
郭瑞
李营营
陈亚辉
赵继红
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Zhengzhou University of Light Industry
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Abstract

The invention discloses a method for reducing complex state FeIIIThe strain is named as Klebsiella oxytoca LF-1 with the deposition number GDMCC NO. 60977. The invention is used for reducing complex state FeIIIThe strain is anaerobic gram-negative bacteria, through researching the biological and degradation characteristics of the strain, the strain can grow by using glucose as a carbon source and an energy source, and can lead Fe in a complex stateIIIReduction to L-FeII. Under pure culture conditions, the strain is cultured at 35-40 deg.C and pH of 6.7-6.9For L-FeIIIThe reduction efficiency is highest. The strain has good substrate adaptability and electrochemical activity, and is resistant to L-FeIIIThe maximum reduction efficiency of the method reaches 95 percent, and the method can provide a technical support in the biological aspect for strengthening treatment of nitrogen oxides in industrial flue gas by a chemical absorption-biological process.

Description

For reducing Fe in complex stateⅢAnaerobic strain, culture method and application thereof
Technical Field
The invention relates to the field of microbial strains, in particular to a method for reducing complex-state FeIIIThe anaerobic strain and the culture method and the application thereof.
Background
With the rapid development of economy and the recent acceleration of industrialization, Nitrogen Oxide (NO)x) Has resulted in serious environmental problems such as acid rain, ozone depletion, and also PM2.5And photochemical smog, etc. Flue gas of industrial boiler (kiln) is considered as NO in atmospherexIs a significant source of. According to the prediction of related research, NO in China currentlyxThe emission amount increases year by year, and is expected to reach 2660-4t, control of NO in the atmospherexEmissions have received high attention from both academia and government.
At present, the main flue gas denitration technologies comprise Selective Catalytic Reduction (SCR) and selective non-catalytic reduction (SNCR), an absorption method, an adsorption method, a biological method and the like. SCR and SNCR have been widely used in industrial industries such as coal-fired power plants, but these two technologies have high cost and risk of secondary pollution. The biological process of industrial waste gas research began in the last 80 th century, in which biological reduction could utilize denitrifying bacteria to reduce NO to non-polluting N2. But due to NO in the flue gasxMore than 90% is present in the form of NO, and the solubility of NO in the liquid phase is minimal. And Fe in a complex stateII(e.g., EDTA-Fe)II、Citrate-FeIIEtc., EDTA: ethylene diamine tetraacetic acid; citrate: citric acid; hereinafter referred to as L-FeII) Can quickly complex NO, solves the problems of low gas-liquid mass transfer efficiency of NO in flue gas, short reaction time on the surface of microorganism and the like, and therefore, adopts a complex absorption methodCombined with biological reduction method and reduction and regeneration of L-Fe by heterotrophic bacteriaIIThereby effectively improving the biological denitration efficiency. Meanwhile, the oxygen (about 9 percent) in the industrial flue gas can convert the L-FeIIOxidation to complex state Fe without the ability to complex NOIII(e.g., EDTA-Fe)III、Citrate-FeIIIEtc., hereinafter abbreviated as L-FeIII) And flue gas temperature is in the pair of L-FeIIIThere is an adverse effect of the bioreduction of (a). Therefore, for L-FeIIIThe reduced efficient microorganism strain breeding pair efficiently reduces NO by the technologyxOf crucial importance, exclusively for the reduction of L-FeIIIThe screening and separation of anaerobic strains are the research hotspots of the current flue gas denitration.
At present, strains capable of reducing complex state high valence iron have been found in many cases. Klebsiella oxytoca IMFRCUG-1 (CCTCC NO: M2015332) separated from inner Mongolia high-arsenic groundwater by Liping et al has strong dissimilatory reduction of FeIIIThe ferric citrate reduction rate can reach 97.3 percent under proper conditions. Rhodococcus ruber DIF2 (CCTCC NO: M2018275) separated from Metronite and underground iron ore soil by Chimonanthus mollis can efficiently reduce Fe in ferric citrate and ferric oxideIIIReducing Fe in 96h under proper conditionsIIIThe capacity of (A) is about 0.46 mmol. multidot.L-1. The bacterium can also reduce hexavalent chromium and has better electricity generation capability. Klebsiella oxytoca Z6 is separated from anode biomembrane of microbial fuel cell with ferric citrate as substrate by cinnanmart (CCTCC NO: M2012446), has strong electrochemical activity and maximum power density of 287.4 mW.m when citrate is used as matrix-2. However, the analysis of the current research situation of the high-valence iron reducing bacteria in the current complex state shows that the currently screened L-FeIIIThe reduction-like bacteria have similarity at the first level of the genus, but have larger difference in gene sequence structure at the first level of the species; and the reported degradation effect of the strain under various complexing ligands and various concentration gradients thereof has no deep comparative study, and has no specific requirement on the culture time of the strain, so that the culture time is different, and the optimal degradation efficiency is different.
Therefore, screening and separating and using the directional domestication method to obtain reducible L-FeIIIThe high-efficiency strain can provide technical support and theoretical basis for the development of a chemical absorption-biological reduction flue gas denitration process by researching the biological characteristics and the reduction characteristics of the high-efficiency strain.
Disclosure of Invention
The invention provides a method for reducing L-FeIIIThe anaerobic strain of (1), which is capable of efficiently reducing L-FeIII。
For reducing complex state FeIIIIs named as Klebsiella oxytoca (Klebsiella oxytoca LF-1) with the deposition number: GDMCC No. 60977.
The specific preservation information of the strain is as follows:
name: klebsiella oxytoca LF-1
The preservation unit: GDMCC for short
The address of the depository: guangzhou city first furious Zhonglu No. 100 large yard No. 59 building No. 5 building
Preservation time: 2020.3.11
The preservation number is: GDMCC No.60977
Wherein the Klebsiella oxytoca (Klebsiella oxytoca LF-1) belongs to the genus Klebsiella oxytoca; the bacterial colony is anaerobic gram-negative bacteria, is round, milky white, convex, smooth in surface, and has a faint yellow soluble pigment, and the diameter of the bacterial colony is about 2 mu m.
The invention also provides a method for reducing complex state FeIIIThe method for culturing the anaerobic strain comprises the steps of extracting return sludge from Zhengzhou Wulongkou water affair branch company, adding liquid culture fluid for a long time to drive oxygen for directional acclimation, repeatedly culturing in the liquid culture medium, performing flat plate scribing on a solid culture medium on an aseptic operation platform, culturing in a constant temperature box at 40 ℃, and purifying to obtain the dominant strain with efficient reduction characteristics through multiple separation.
For reducing complex state FeIIIThe method for culturing the anaerobic strain comprises the following steps:
s1), selecting Zhengzhou Wulongkou water divisionReflux sludge of a company, adding a liquid culture medium for anaerobic culture, and then adding a centrifugal substrate, the liquid culture medium and L-Fe of an enriched bacterial liquidIIIAdding the complex into a serum bottle, and culturing on a shaking table to obtain a microbial colony.
S2), purifying and separating the target strain by utilizing a solid medium combined with a plate marking method to obtain the L-Fe for reductionIIIThe anaerobic strain of (4).
In step S1, the liquid culture medium is composed of the following components by weight, based on 1L of the liquid culture medium:
0.2g MgCl2·6H2O，0.008g ZnCl2，0.0176g Na2MoO4·2H2O，0.08g CaCl2·2H2O，0.02g CuSO4·5H2O，0.0192g CoCl2·6H2O，0.14g Na2SO3，0.25g NaNO22g of glucose, 0.0152g of NiCl2·6H2O，1.2g K2HPO4·2H2O，10.8g NaHCO3，0.0792g MnCl·4H2O，0.00112g H3BO3Then, 1L of distilled water was added to prepare a solution.
L-FeIIIPreparation of the complex: from FeCl3And EDTANA2(or Citrate, glutamic acid, lysine and the like) in a molar ratio of 1: 1.
In step S2, the solid medium is composed of the following components by weight, based on 1L of the solid medium:
glucose 2.5g, K2HPO4·3H2O 1g，KH2PO4 0.625g，NaSO3 0.07g，MgSO4 0.1g，CaCl20.002g，MnSO4 0.0005g，Na2MoO4·2H2O 0.0001g，CuSO4·5H2O 0.0001g，1g NaNO2Agar 1.5-2.0%.
The purification and separation of the target strain are carried out by combining a solid culture medium with a plate streaking method, namely 100 mu L of microbial liquid in a liquid culture medium after repeated purification is taken, and then the plate streaked on the solid culture medium by using a solid culture medium experimental method step. Culturing in a 40 deg.C incubator. After 5 days, picking out single colony for culture by using a solid medium test method, carrying out amplification culture in a liquid medium to obtain a reduced strain, adding the strain liquid into a sterilized 50mL centrifuge tube, centrifuging in a centrifuge with the operating conditions of 5000r/min, 15min and 15 ℃, and removing the supernatant.
The invention is used for reducing complex state FeIIIThe anaerobic strain can be applied to chemical absorption-biological process for strengthening treatment of nitrogen oxides in industrial flue gas. For reducing L-FeIIIInoculating anaerobic strain into a biofilm reactor to carry out NO-containingxTreating the smoke (waste gas), and acclimating to a stable stage to obtain good L-FeIIIAnd (4) reducing effect.
The invention is used for reducing L-FeIIIReduction of L-Fe by anaerobic strainsIIIThe strain is carried out at the temperature of 35-40 ℃ and under the condition that the pH value is 6.7-6.9, and experiments show that the strain can be used for leading different initial concentrations (5-20mmol/L calculated by the volume of a serum bottle) of L-Fe in a liquid culture medium to be stored in a liquid culture medium within 96hIIICompletely reducing; has stronger substrate adaptability.
A comparison document publication (publication) No. CN1793332A describes a bacterial strain for reducing ferric iron and its use (publication) date: 2006-06-28), the method of which includes the breeding of the strain and the testing of the reduction effect of the strain (Klebsiella Trevisan sp.) on Fe (III) -EDTA on a solid selective medium. The reduction efficiency of the bacterium on Fe (III) -EDTA can reach 70% under proper conditions, and the bacterium has the characteristic of taking ferric iron as a final electron acceptor. However, biological information such as a single colony map and a specific sequence related to the strain is not shown, and the research on the reducing capability of the strain on Fe (III) -EDTA under different influencing factors (such as carbon source concentration) and specific actions of the strain and the reduction effect of the strain on different iron compounds is lacked.
Compared with the prior art, the invention has the beneficial effects that:
the invention is used for reducing L-FeIIIThe anaerobic strain of (4) is named as Klebsiella oxytoca (Klebsiella oxytoca LF-1) belonging to the genus Klebsiella oxytoca; is anaerobic gramThe bacterial colony of the negative bacterium is round, milky white, convex, smooth in surface, generated by a light yellow soluble pigment and about 2 microns in diameter. Having L-FeIIIThe efficient reduction capacity can be realized, and the glucose can be used as a carbon source and an energy source for growth and efficiently reduce substrates with different initial concentrations; the strain can efficiently degrade L-FeIIIA complex compound; the invention can provide a technical support in the biological aspect for the enhanced treatment of nitrogen oxides in industrial flue gas by a chemical absorption-biological process.
Drawings
FIG. 1 is a tree of the phylogenetic tree of Klebsiella oxytoca of the present invention;
FIG. 2 is a photograph of gram stain of the strain of the present invention;
FIG. 3 is a photomicrograph of Klebsiella oxytoca (Klebsiella oxytoca LF-1) produced in the present invention;
FIG. 4 shows the reduction of L-Fe by Klebsiella oxytoca (Klebsiella oxytoca LF-1) in the present invention at different pH valuesIIIThe efficiency impact;
FIG. 5 shows different concentrations of L-Fe in the present inventionIIIReducing L-Fe by Klebsiella oxytoca (Klebsiella oxytoca LF-1) under complex conditionIIIThe efficiency impact;
FIG. 6 shows the reducing power of Klebsiella oxytoca (Klebsiella oxytoca LF-1) on various substrates in the present invention.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1: separation and identification of strains:
1. domestication and breeding of strains
Performing aeration domestication (2d) on activated sludge (added with liquid culture solution) taken from a sewage discharge port of a water utilities company (five-dragon mouth water utilities of China environmental protection limited company, 5 months in 2019), and then performing aeration domestication on a centrifugal substrate (5 mL) of enriched bacteria liquid, a liquid culture medium and L-Fe with different concentrationsIIIAdding the complex into a serum bottle, and culturing for 5 days in a shaking table to obtain reducible target substrate L-FeIIIThe microbial community of (a); method for carrying out target bacteria by solid medium plate markingThe strains were purified and isolated by streaking 100. mu.L of the two microbial cultures from previously repeatedly purified liquid media onto solid media according to the previous procedure for the experimental procedures on solid media. Placing in a constant temperature incubator, culturing at 40 deg.C for 5 days, selecting single colony in liquid culture medium, performing amplification culture in the liquid culture medium to obtain reduced strain, and centrifuging (5000r/min, 15min, 15 deg.C) the strain liquid to obtain substrate for strain identification. The strain is named as Klebsiella oxytoca (Klebsiella oxytoca LF-1).
The strain is preserved for a short time, inoculated into a slant solid culture medium and preserved in a refrigerator at 4 ℃. Long-term preservation in Guangdong province microbial strain preservation center, address: guangzhou city, Jielizhou 100 college, building 59, floor 5, preservation date: 2020.3.11, accession number: GDMCC No. 60977.
2. Culture conditions
The liquid culture medium is calculated by 1L of liquid culture medium, and consists of the following components in parts by weight:
0.2g MgCl2·6H2O，0.008g ZnCl2，0.0176g Na2MoO4·2H2O，0.08g CaCl2·2H2O，0.02g CuSO4·5H2O，0.0192g CoCl2·6H2O，0.14g Na2SO3，0.25g NaNO22g of glucose, 0.0152g of NiCl2·6H2O，1.2g K2HPO4·2H2O，10.8g NaHCO3，0.0792g MnCl·4H2O，0.00112g H3BO3Then, 1L of distilled water was added to prepare a solution.
L-FeIIIPreparation of the complex: from FeCl3And EDTANA2(or Citrate, glutamic acid, lysine and the like) in a molar ratio of 1: 1.
The solid culture medium is calculated by 1L of the solid culture medium and comprises the following components in parts by weight:
glucose 2.5g, K2HPO4·3H2O 1g，KH2PO4 0.625g，NaSO3 0.07g，MgSO4 0.1g，CaCl20.002 g，MnSO4 0.0005g，Na2MoO4·2H2O 0.0001g，CuSO4·5H2O 0.0001g，1g NaNO2Agar 1.5-2.0%.
The culture conditions are as follows: the optimum growth pH is 6.7-6.9; the optimum growth temperature is 35-40 ℃.
3. Strain morphology and molecular biology identification
Obtained for reduction of L-FeIIIThe 16S rRNA gene sequence of the anaerobic strain is determined to be compared with the existing nucleic acid sequence in Genbank by homology comparison by using a BLAST program, and the similarity of the sequence of the anaerobic strain and the sequence of a plurality of strains of Klebsiella oxytoca is found to be more than 99.9 percent. Selecting several strains, performing homology comparison between the corresponding sequences and the strain sequence by using DNASAR software, establishing a phylogenetic tree to obtain the phylogenetic tree of the strain (as shown in figure 1), uploading a gene sequence to Genbank, and obtaining a gene sequence number (MT 032493); by constructing a phylogenetic relationship, the genetic relationship between the bacillus and Klebsiella oxytoca is determined to be nearest, and the similarity is more than 99.9 percent. The strain was thus assigned to the genus Klebsiella oxytoca and named Klebsiella oxytoca LF-1.
The strain belongs to Klebsiella oxytoca, is anaerobic gram-negative bacteria, and has round, milky white, convex, smooth surface, yellowish soluble pigment, and diameter of about 2 μm (as shown in FIG. 2 and FIG. 3).
EXAMPLE 2 Klebsiella oxytoca (Klebsiella oxytoca LF-1) reducing Properties
1. Klebsiella oxytoca reduction characteristics under different pH conditions (5.6-8.4)
Taking 25mL of each of the basic culture medium and the trace element culture medium in the liquid culture medium, and taking 100mL of L-Fe in 4 serum bottlesIII(100mmol·L-1) 5mL of the bacterial suspension was added to the serum bottle. With NaHCO3The pH of 1#, 2#, 3#, and 4# flasks were adjusted to 6.6, 6.8, 7.0, and 7.2, respectively, and the flasks were placed at 30 ℃ and 180 r.min-1The culture was continued in a constant temperature shaker with sampling analysis at regular intervals (results are shown in FIG. 4). The results show that the optimum reduction pH of the strain is 6.8 at L-FeIIIReduction process ofIn addition, the reduction characteristic is obviously superior to other pH value conditions; the strain can reduce L-Fe to different degrees in different pH environmentsIIIProvides guarantee for the application of the strain in different pH environments, but the reduction process of the strain (incomplete substrate reduction) can be influenced by the over-high or over-low pH value (less than 6.6 or more than 7.2).
2. Different concentrations of L-FeIIIReduction characteristics of Klebsiella oxytoca LF-1 under complex
Glucose is used as the only carbon source of the strain, and L-Fe with gradient concentration is added into bottles No. 1, No. 2, No. 3 and No. 4IIIAnd 5mL of bacterial suspension. Respectively placing the mixture at a current density of 216.4 mW.m-2The electrode biofilm reaction period is continuously cultured, and samples are taken for analysis at certain time intervals (as shown in figure 5). The results show that the strain is suitable for L-FeIIIThe concentration of the complex reaches 5-20 mmol.L-1And for L-FeIIIThe reduction rate of the strain is kept above 80 percent, which indicates that the strain has stronger electrochemical activity and can also be used for treating L-FeIIIHas efficient and stable reduction capability.
Analysis of Klebsiella oxytoca LF-1 reduction on various substrates
Glucose is used as carbon source for Klebsiella oxytoca LF-1, and about 15 mmol. L are added into the carbon sources 1#, 2#, 3#, and 4#, respectively-1EDTA-FeIII，Citrate-FeIII，NTA-FeIII(triacetic acid), DTPA-FeIII(Ethanetriaminepentaacetic acid) and 5mL Klebsiella oxytoca LF-1. Respectively standing at 38 deg.C for 180 r.min-1The culture was continued in a constant temperature shaker with sampling analysis at regular intervals (results are shown in FIG. 6). The test result shows that Klebsiella oxytoca LF-1 is opposite to Citrate-FeIIIThe method has the advantages that the best reduction efficiency is achieved, and the maximum reduction efficiency reaches 94%; second EDTA-FeIIIThe maximum reduction efficiency was 91%.

Claims (7)

1. For reducing complex state FeIIIThe anaerobic strain of (4), which is named Klebsiella oxytoca LF-1 with the deposition number: GDMCC No.60977, deposit time: year 2020, 3, 11.
2. Use according to claim 1 for reducing Fe in complexed stateIIIThe method for culturing an anaerobic strain of (1), comprising the steps of:
s1, extracting return sludge, adding liquid culture solution for aeration culture, and then adding the centrifugal substrate, liquid culture medium and L-Fe of the enriched bacterial liquidIIIAdding the complex into a serum bottle, and culturing in a shaking table to obtain a microbial community;
s2, purifying and separating the target strain by utilizing a solid medium combined with a plate marking method to obtain the L-Fe for reductionIIIThe anaerobic strain of (4).
3. Use according to claim 2 for the reduction of L-FeIIIThe method for culturing an anaerobic strain according to (1), wherein the liquid medium in step S1 comprises the following components in parts by weight:
0.2g MgCl2·6H2O，0.008g ZnCl2，0.0176g Na2MoO4·2H2O，0.08g CaCl2·2H2O，0.02g CuSO4·5H2O，0.0192g CoCl2·6H2O，0.14g Na2SO3，0.25g NaNO22g of glucose, 0.0152g of NiCl2·6H2O，1.2g K2HPO4·2H2O，10.8g NaHCO3，0.0792g MnCl·4H2O，0.00112g H3BO3Adding distilled water to 1L to prepare solution;
L-FeIIIpreparation of the complex: is prepared from EDTANA2Citrate, glutamic acid, lysine and FeCl respectively3Prepared in a molar ratio of 1:1, reduced L-FeIIIThe complex is carried out at 35-40 deg.C and pH 6.7-6.9.
4. Use according to claim 2 for the reduction of L-FeIIIThe method for culturing an anaerobic strain according to (1), wherein the solid medium comprises the following components in parts by weight, based on 1L of the solid medium in step S2:
glucose 2.5g, K2HPO4·3H2O 1g，KH2PO4 0.625g，NaSO3 0.07g，MgSO4 0.1g，CaCl20.002g，MnSO4 0.0005g，Na2MoO4·2H2O 0.0001g，CuSO4·5H20.0001g of O, 1.5-2.0% of agar, and 1g of L-1NaNO2。
5. Use according to claim 2 or 4 for the reduction of L-FeIIIThe method for culturing an anaerobic strain according to (1), wherein the step S2 is as follows:
purifying and separating the target strain by using a solid culture medium and a plate streaking method, namely taking 100 mu L of microbial liquid in a liquid culture medium which is repeatedly purified, and streaking the microbial liquid on the solid culture medium by using a plate through a solid culture medium experimental method; culturing in a constant temperature incubator at 40 deg.C; after 5 days, picking out single colony for culturing by using a solid medium test method, carrying out amplification culture in a liquid medium to obtain a reducing strain, adding the strain liquid into a sterilized 50mL centrifuge tube, centrifuging in a centrifuge with the operating conditions of 5000r/min, 15min and 15 ℃, and removing the supernatant to obtain the L-Fe for reductionIIIThe anaerobic strain of (4).
6. Use according to claim 1 for reducing Fe in complexed stateIIIThe anaerobic strain is applied to chemical absorption-biological process for strengthening treatment of nitrogen oxides in industrial flue gas.
7. Use according to claim 6 for reducing Fe in complex state according to claim 1IIIInoculating anaerobic strain into a biofilm reactor to carry out NO-containingxTreating flue gas/waste gas, and acclimating to a stabilization stage.
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