CN107151635B - Rhodococcus ruber capable of degrading phthalate and application thereof - Google Patents
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Abstract
The invention provides a Rhodococcus ruber capable of degrading phthalate and application thereof. The strain is YC-YT1 with the preservation number of CGMCC No.13959, which can degrade the di (2-ethylhexyl) phthalate of 100mg/L contained in inorganic salt by 85 percent within 12 hours and degrade the di (2-ethylhexyl) phthalate, dipropyl phthalate and butyl benzyl phthalate of 100 percent contained in an inorganic salt culture medium within 5 days; has high degradation effect on other phthalates. The Rhodococcus ruber YC-YT1 has wider tolerance range for salt ion concentration, pH and temperature, can be applied to bioremediation of phthalate ester substance environmental pollution and industrial wastewater treatment, and has better economic value and application prospect.
Description
Technical Field
The invention relates to the field of microorganisms and biodegradation, in particular to Rhodococcus ruber (Rhodococcus ruber) YC-YT1 and application thereof.
Background
Phthalate Esters (PAEs), also known as phthalates, are a generic name for Esters formed from phthalic Acid. The phthalate is an environmental endocrine disrupter which is artificially synthesized as a main source, is mainly used for preparing polyvinyl chloride materials and plays a role of a plasticizer. As a plasticizer, PAEs are present in plastic products in amounts of about 20% to 30%, even up to 50%. Since the advent of polyvinyl chloride (PVC) in the 50's last century, PAEs have been produced on a large scale. Currently, it accounts for more than 80% of the global plasticizer market. In addition to their use as plasticizers, PAEs are widely used as raw materials for the production of paints, adhesives, insect repellents, cosmetics, fragrances and lubricants. In germany, the annual consumption of PAEs reaches forty thousand tons, and the worldwide annual use of PAEs is over 820 million tons, of which over 1% enters the environment through leakage. Currently, PAEs have been detected universally in the ecological environment of major industrial countries worldwide. PAEs have become one of the most common pollutants worldwide. Therefore, how to realize the efficient degradation of the phthalate ester pollutants in the environment becomes a problem to be solved urgently.
The phthalate esters are more than 30 kinds, are mostly colorless transparent oily liquids, are generally insoluble in water, are easily soluble in organic solvents, belong to medium polar substances, and can enter human bodies and animal bodies through breathing, diet and skin contact. The effects of phthalates on human health are a chronic process that takes a long time to occur and can have a cross-generation effect through placenta and lactation, so that toxicity is mainly studied through animal experiments at present. Animal experiments show that phthalate has low acute toxicity and negative reaction to Ames test (pollutant mutagenicity detection), but has teratogenic, carcinogenic and mutagenic effects on animals under the condition of large dose. The subacute toxicity is mainly manifested by damaging liver, kidney and testis, inhibiting spermatogenesis, affecting reproductive function, etc. Phthalates contain weaker female hormone active ingredients. Recent studies have indicated that PAEs are primarily detrimental to environmental hormonal effects and can interfere with the endocrine system of humans and animals at very low concentrations. Its disruption of the endocrine system is through an Estrogen receptor (Estrogen receptor) -mediated response, which, by binding to the Estrogen receptor, acts on an Estrogen responsive element (Estrogen responsive element) in the DNA to activate transcription of the gene, producing an estrogenic effect. Animal experiments show that the biochemical effects of PAEs as endocrine disruptors are represented by peroxisome proliferation, podocyte toxicity, liver promoting effect, antiandrogen, in-vitro estrogen activity and the like. The disturbance of animal reproduction is mainly seminal vesicle atrophy, sperm quantity reduction to stop sperm formation, reproductive capacity reduction, offspring quantity reduction, weight reduction, uterine mucosa tissue hyperplasia and the like. There are dozens of Phthalate-based substances having an environmental hormonal effect, of which dimethyl Phthalate (DMP), diethyl Phthalate (DEP), Dipropyl Phthalate (DPP), di-n-butyl Phthalate (DBP), Dioctyl Phthalate (DOP) and di (2-ethylhexyl) Phthalate (DEHP) are among the priority pollutants recognized by the U.S. environmental protection agency.
The rate of hydrolysis and photolysis of PAEs in the environment is very slow, and microbial degradation is the main route of mineralization. In recent years, bacterial degradation of PAEs has been extensively studied, and a large number of strains that efficiently degrade PAEs have been isolated from various environments. Meanwhile, the bacterial metabolic pathway of PAEs and the genetic mechanism of phthalic acid degradation are also deeply researched. However, the research on the genetic mechanism of degradation mainly focuses on cloning and identifying the enzyme gene participating in the degradation from phthalic acid to protocatechenol, and the research on the protein aspect is not deeply carried out; and the enzyme gene involved in the two-step phthalate-to-phthalate ester removal process is less studied. Moreover, reports of application in practical environments are lacking at present, such as harsh environmental conditions of industrial wastewater, high salt, extreme pH, abundant pollutant types and the like, which all put higher requirements on the tolerance of microorganisms. Therefore, the search for the bacterial strain which still maintains the degradation capability to various phthalate ester pollutants under the high salt concentration has important economic value and practical significance for treating the environmental pollution.
Disclosure of Invention
The invention aims to provide a rhodococcus ruber YC-YT1 capable of degrading phthalate and application thereof in degradation of phthalate substances.
The strain YC-YT1 is a bacterium which can degrade a plurality of phthalic acid esters such as di (2-ethylhexyl) phthalate (DEHP), dicyclohexyl phthalate (DCHP), dimethyl phthalate (DMP), di-n-butyl phthalate (DBP), diethyl phthalate (DEP) and the like and is separated from a ditch near the Wangwao industrial area of Xinbucun village in western Li town in Nanshan region of Shenzhen, Guangdong province.
Observed under an electron microscope (figure 1), the strain has various morphological cells, sprouts from a round ball shape into a short rod shape, and has no flagella and spores. The colony is round and smooth, and the mucilaginous pigment is orange yellow pigment (figure 2). Gram staining of the strain, catalase activity and urease reaction are all positive; oxidase activity and indole reaction were negative. Based on morphological characteristics, physiological and biochemical characteristics, the strain was identified as Rhodococcus ruber, named YC-YT 1. The strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 of West Lu No.1 of Beijing university Hokko-sunny district, microbiological research institute of Chinese academy of sciences, zip code 100101) in 31.3.2017.3.D., the preservation number is CGMCC No.13959, and the classification name is Rhodococcus ruber.
The invention provides a microbial inoculum containing erythrococcus ruber YC-YT 1.
The invention provides a biological cleanser containing erythrococcus ruber YC-YT 1.
The invention provides application of Rhodococcus ruber YC-YT1 or microbial inoculum containing the same or biological cleaning agent containing the same in cleaning environment.
Further, the invention provides an application of the Rhodococcus ruber YC-YT1 or a microbial inoculum containing the same or a biological cleaning agent containing the same in degrading pollutants.
Wherein the organic pollutant is phthalate ester. The phthalate-based substances include di (2-ethylhexyl) phthalate (DEHP), dicyclohexyl phthalate (DCHP), dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dipropyl phthalate (DPrP), butylbenzyl phthalate (BBP), dipentyl phthalate (DAP), dihexyl phthalate (DHP), diheptyl phthalate (DHPP), dioctyl phthalate (DOP), dinonyl phthalate (DNP), didecyl phthalate (DDP).
The invention further provides application of the Rhodococcus ruber YC-YT1 in preparation of a biodegradation agent for phthalate esters.
The invention provides application of Rhodococcus ruber YC-YT1 in preparation of a biological cleanser.
The Rhodococcus ruber YC-YT1 can efficiently degrade DEHP, DCHP, DMP, DBP and DEP simultaneously contained in an inorganic salt ion culture medium (containing 70g/LNaCl) in simulated wastewater treatment, and the degradation rate is more than 90% within 7 days.
The Rhodococcus ruber YC-YT1 can completely degrade DEHP, DPrP and BBP which are respectively 100mg/L in an inorganic salt ion culture medium, and the degradation capability is determined by continuously transferring strains, which shows that the degradation capability of the Rhodococcus ruber is stable. The strain has wider tolerance range on salt ion concentration, pH and temperature, and the degradation rate of 100mg/L DEHP contained in an inorganic salt ion culture medium with NaCl concentration of 0-70g/L and 80-120g/L in 3 days is more than 98% and 85% respectively; the DEHP can be efficiently degraded within the range of pH4-10, when the pH is 5.0-10.0, the degradation rate of 100mg/L DEHP in the inorganic salt ion culture medium within 3 days is 100%, the DEHP can be efficiently degraded within the temperature range of 10-50 ℃, and 100mg/L DEHP in the inorganic salt ion culture medium can be completely degraded within 60 hours at the temperature of 16 ℃ and 30 ℃. DEHP degradation was significantly inhibited when the NaCl concentration was greater than 120g/L, or the temperature was greater than 50 ℃, or the pH was greater than 11 or less than 4.
The Rhodococcus ruber YC-YT1 and the microbial inoculum thereof provided by the invention have no pollution and public nuisance in the use process, can be applied to bioremediation of various phthalate ester environmental pollution and treatment of phthalate ester production wastewater with higher concentration, have wide degradation spectrum, can carry out bioremediation at lower and higher temperature, can be widely applied to the field of environmental soil cleaning and the clean treatment of industrial wastewater, and have better economic value and application prospect.
Drawings
FIG. 1 is a structural diagram of the Rhodococcus ruber YC-YT1 under electron microscope.
FIG. 2 shows the colony morphology of Rhodococcus ruber YC-YT1 on LB solid medium.
FIG. 3 is a phylogenetic tree diagram of 16S rRNA gene of Rhodococcus ruber YC-YT1 according to the present invention.
FIG. 4 shows the degradation rate of Rhodococcus ruber YC-YT1 in the presence of different phthalate substrates in the presence of inorganic salt ion medium at 100 mg/LDEHP.
FIG. 5 is a graph showing the ability of Rhodococcus ruber YC-YT1 to degrade DDP, DNP, DOP, DCHP, DEHP, BBP, DHPP, DHP, DAP, DBP, DPrP, DEP, DMP at a concentration of 100mg/L, respectively, by GC assay in example 2 of the present invention.
FIG. 6A is a standard curve graph of the relationship between the concentrations of DDP, DOP, DBP, DPeP and DHP and the area of the peak at 302nm in example 2 of the present invention; FIG. 6B is a standard curve graph of the relationship between the concentrations of DMP, DEP, DPrP and DNP and the area of the absorption peak at 302nm in example 2 of the present invention; FIG. 6C is a standard graph of BBP, DEHP, DCHP and DHPP concentrations versus the area of the absorption peak at 302nm in example 2 of the present invention.
FIG. 7 is a graph showing the degradation rate of 100mg/L DEHP by Rhodococcus ruber YC-YT1 in mineral salt ion media of various pH values.
FIGS. 8A and 8B are graphs showing the degradation rate of Rhodococcus ruber YC-YT1 in the presence of different concentrations of NaCl in the presence of inorganic salt ion medium to 100mg/L DEHP in example 2 of the present invention.
FIG. 9 shows the degradation rate of Rhodococcus ruber YC-YT1 in the presence of different temperature concentrations of inorganic salt ion medium to 100mg/L DEHP in example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.
The inorganic salt medium used in this application had the following composition: 1.0g/L NH4NO3,0.5g/L NaCl,0.5g/L(NH4)2SO4,0.5g/L KH2PO4,1.5g/L K2HPO4And 0.005g/L yeast extract, pH 7.0 ± 0.2.
Slant culture medium: 10.0g/L peptone, 5.0g/L NaCl, 10.0g/L yeast extract, pH 7.0 ± 0.2.
The plate culture medium is corresponding culture medium added with 1.5% agar.
Example 1 isolation and characterization of Rhodococcus ruber YC-YT1
1. Isolation of the Strain
The activated sludge sample is collected in a canal near the West Lizhou Wangchun birchleaf industrial area in the south China of Shenzhen, Guangdong province. A10 g sample of activated sludge was inoculated into 100mL of a medium containing 100mg/L of DEHP in inorganic salt ion under aseptic conditions, and cultured at 30 ℃ and 180 rpm. After each 7 days of culture, 5% of the culture volume was transferred to fresh mineral salts medium and the concentration of DEHP was increased by 100mg/L each time, and transfer was continued 3 times until the concentration of DEHP in the mineral salts medium was increased to 300 mg/L.
The acclimatized bacterial liquid is streaked on an inorganic salt culture medium plate containing 100mg/L DEHP, and cultured in an incubator at 30 ℃ for 3 days. Single colonies on the plates were picked for streaking and cultured three times until purified strains were isolated. And (3) selecting the purified single colony, transferring the single colony into an inorganic salt liquid culture medium with the concentration of 100mg/LDEHP, culturing for 7 days, and performing slant preservation on the strain which grows well, is stable in passage and has better degradation capacity. The strain was named YC-YT 1.
2. Morphological characteristics of the Strain
The bacillus is gram-positive, round and germinates into brevibacterium, has various forms, and has no flagellum and no spore (see figure 1); the colony on LB culture medium is orange yellow, wet and soft, round and convex, regular in edge, opaque and smooth in surface (see figure 2).
3. 16S rDNA identification
Inoculating strain YC-YT1 into LB culture medium, culturing overnight at 30 deg.C and 180rpm, collecting 3mL bacterial liquid, centrifuging, collecting thallus, extracting genome DNA with bacterial genome extraction kit, detecting the obtained gene DNA with 0.8% agarose gel electrophoresis, and storing at-20 deg.C for use.
A pair of universal primers was designed for amplification of 16s rDNA sequences: 27F5 '-AGAGAGTTTGATCCTGGCTCAG-3' and 1492R5'-GGTTACCTTGTTACG ACTT-3', using genomic DNA asTemplate, Premix Taq additionTMPCR amplification is carried out, PCR products are detected by 0.8% agarose gel electrophoresis, purified by a DNA purification recovery kit, connected to a pGM-T vector, transformed into escherichia coli DH5 α competent cells, coated on an LB solid medium plate containing ampicillin, cultured for 12h at 37 ℃, white colonies are picked up to be put into a liquid LB medium, subjected to shaking culture at 37 ℃ and 180rpm overnight, plasmids are extracted by a plasmid extraction kit and sent to a Haishengsheng Bionshinbo company for sequencing, the sequence is subjected to Blast comparison analysis on NCBI websites (http:// www.ncbi.nlm.nih.gov /), and a phylogenetic tree (figure 3) is constructed by MEGA6.0 software, so that the strain YC-YT1 is rhodococcus and has higher similarity with the currently published rhodococcus ruber sequence.
4. Biolog identification
Inoculating strain YC-YT1 into LB culture medium plate, culturing for 5 days at 30 deg.C by inversion, picking single colony to Biolog A liquid and B liquid, respectively reducing the light transmittance from 100% to 98% and 95%, adding A liquid and B liquid into Biolog culture plate according to 100 μ L per well, culturing for 33h at 30 deg.C, detecting and analyzing with Biolog microorganism identifier, and showing that YC-YT1 is Rhodococcus ruber (Rhodococcus ruber).
From the combination of the morphology of the cells, the 16S rDNA gene sequence and the Biolog result, strain YC-YT1 was identified as Rhodococcus ruber.
The strain YC-YT1 was identified as Rhodococcus ruber (Rhodococcus ruber) by combining the morphological, physiological and biochemical characteristics and 16S rDNA gene sequence. The strain YC-YT1 is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of Siro 1 of Beijing city facing Yang district, Microbiol research institute of Chinese academy of sciences, postal code 100101) in 31.03.2017.31.A preservation number is CGMCC No.13959, and a classification name is Rhodococcus ruber.
Example 2 degradation Performance test of Rhodococcus ruber YC-YT1
1. The ability of Rhodococcus ruber YC-YT1 to degrade di (2-ethylhexyl) phthalate (DEHP), dipropyl phthalate (DPrP), butylbenzyl phthalate (BBP), dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dipentyl phthalate (DAP), dihexyl phthalate (DHP), diheptyl phthalate (DHPP), dicyclohexyl phthalate (DCHP), dioctyl phthalate (DOP), dinonyl phthalate (DNP), didecyl phthalate (DDP) at a concentration of 100mg/L is shown in FIG. 4.
High performance gas chromatography detection shows that Rhodococcus ruber YC-YT1 has simultaneous degradation effect on di (2-ethylhexyl) phthalate (DEHP), dipropyl phthalate (DPrP), butylbenzyl phthalate (BBP), dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), diamyl phthalate (DAP), dihexyl phthalate (DHP), diheptyl phthalate (DHPP), dicyclohexyl phthalate (DCHP), dioctyl phthalate (DOP), dinonyl phthalate (DNP) and didecyl phthalate (DDP) in an inorganic salt culture medium.
Inoculating strain YC-YT1 into liquid LB culture medium for activation, and culturing to logarithmic growth phase OD6000.8 percent by volume, the inoculated amount was inoculated into an inorganic salt medium containing a mixture of 100mg/L of each of di (2-ethylhexyl) phthalate (DEHP), dipropyl phthalate (DPrP), butylbenzyl phthalate (BBP), dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dipentyl phthalate (DAP), dihexyl phthalate (DHP), diheptyl phthalate (DHPP), dicyclohexyl phthalate (DCHP), dioctyl phthalate (DOP), dinonyl phthalate (DNP), and didecyl phthalate (DDP), as a treatment group, di (2-ethylhexyl) phthalate (DEHP), dipropyl phthalate (DPrP), butylbenzyl phthalate (BBP) of a non-inoculated strain, Dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), diamyl phthalate (DAP), dihexyl phthalate (DHP), diheptyl phthalate (DHPP), dicyclohexyl phthalate (DCHP), dioctyl phthalate (DOP), dinonyl phthalate (DNP), dioctyl phthalate (DOP)Didecyldimethylester (DDP) was added to 100mg/L of each of the mixtures in mineral salt medium to prepare a control group, and the control group and the treated group were each prepared in triplicate. The control group and the treated group were incubated at 30 ℃ with shaking and light shielding at 180rpm, the incubation was stopped on the 7 th day of the incubation, and the concentration of each substance was measured.
Adding equal volume of n-hexane into the obtained sample, performing ultrasonic oscillation extraction for 10 min, standing for 1 hr, collecting upper layer organic solvent, volatilizing the organic solvent, re-dissolving with equal volume of methanol, filtering with 0.22 μm organic filter membrane, and performing GC analysis.
The GC analysis conditions were: a SHIMADZU GC-2010 high performance gas chromatograph, the apparatus configured to: RTX-1301 capillary column (30.0 m.times.0.25 mm.times.0.25 μm) and ion trap detector (ECD), detection conditions: the results were analyzed using the software GC solution (v 2.32.00, SHIMADZU) at a sample inlet temperature of 300 ℃, a detector temperature of 300 ℃, a column box temperature of 280 ℃, a carrier gas of nitrogen (purity ≥ 99.999%, flow rate of 30ml/min), and an injection volume of 1.0 μ L, and retention times of DDP, DNP, DOP, DCHP, DEHP, BBP, DHPP, DHP, DAP, DBP, DPrP, DEP, DMP were determined as follows: 16.358min, 14.173min, 12.296min, 9.995min, 9.608min, 8.161min, 7.732min, 6.393min, 5.377min, 4.575min, 3.970min, 3.624min and 3.515 min; (FIG. 5); phthalate standards were plotted as a standard curve between concentration and absorption peak area at 302nm (FIGS. 6A-6C).
And (3) calculating the degradation rate: the daily residual concentration of each substrate in the inorganic salt medium is calculated according to the standard curve of different substrates, and the degradation rate of the strain YC-YT1 to the substrate is obtained according to the degradation rate calculation formula (Table 1).
Percent degradation = (final concentration of substrate in control-final concentration of substrate in treated group)/final concentration of substrate in control × 100%
Percent natural degradation = (initial substrate concentration-substrate concentration in control group)/initial substrate concentration × 100%
TABLE 1 degradation rate of various strains YC-YT1 on the seed substrate and the natural degradation rate of the substrate
2. Tolerance of Rhodococcus ruber YC-YT1 to pH
Preparing inorganic salt ion culture media with different pH values (4, 5, 6, 7, 8, 9 and 10) respectively, and sterilizing for later use. DEHP was added to the prepared inorganic salt ion media to a substrate concentration of 100 mg/L. Inoculating strain YC-YT1 into liquid LB culture medium for activation, and culturing to logarithmic growth phase OD600The medium was inoculated with an inoculum size of 5% by volume (0.8), and the resulting mixture was subjected to shaking at 30 ℃ and 180rpm in the dark to obtain a treatment group.
The same culture medium with DEHP added to the concentration of 100mg/L at the same time of different pH of the non-inoculated strain was used as a control group, and the same was subjected to shaking and light-shielding culture at 30 ℃ and 180rpm for 3 days, and the DEHP concentration in the solution was measured and the degradation rate was calculated.
FIG. 7 shows the DEHP degradation rate of Rhodococcus ruber YC-YT1 under different pH conditions for 3 days. When the pH value is increased from 4.0 to 5.0, the degradation rate of DEHP is gradually increased from 40% to more than 99%. When the pH value is increased from 7.0 to 8.0, the degradation efficiency of YC-YT1 to DEHP is gradually reduced from 100% to 90%. When the pH value is more than 8 and the pH value is 9 or 10.0, the DEHP degradation rate of YC-YT1 is increased to 100 percent again.
3. Tolerance of Rhodococcus ruber YC-YT1 to salt concentration
Respectively preparing inorganic salt ion culture media with different NaCl concentrations (1, 3, 5, 7, 10, 15, 20, 25, 40, 50, 60, 70, 80, 90, 100, 110 and 120g/L), and sterilizing for later use. DEHP was added to the prepared inorganic salt ion medium to a substrate concentration of 100mg/L, respectively. Inoculating strain YC-YT1 into liquid LB culture medium for activation, and culturing to logarithmic growth phase OD600The medium was inoculated with an inoculum size of 5% by volume (0.8), and the resulting mixture was subjected to shaking at 30 ℃ and 180rpm in the dark to obtain a treatment group.
The same inorganic salt ion medium containing different NaCl concentrations without inoculated strain and simultaneously adding DEHP to the concentration of 100mg/L was used as a control group, and the culture was carried out at 30 ℃ and with shaking at 180rpm in a shaking table in a dark place. After 60 hours of incubation, the DEHP concentration in the solution was measured and the degradation rate was calculated.
FIGS. 8A and 8B show the DEHP degradation rate of Rhodococcus ruber YC-YT1 at different salt concentrations over 60 hours. The result shows that when the NaCl concentration is between 1 and 100g/L, the DEHP degradation rate of the strain is between 90 and 98 percent in 60 hours, and when the NaCl concentration is 110g/L, the substrate degradation rate is reduced to 60 percent; when the NaCl concentration is increased to 120g/L, the substrate degradation rate is increased back to more than 85%. Therefore, the strain YC-YT1 has better salt tolerance and can efficiently degrade DEHP under the condition of higher salt concentration (less than or equal to 100 g/L). When the NaCl concentration reaches a certain concentration, the metabolism regulation and control system of the strain makes certain adjustment so as to adapt to more extreme environmental conditions.
4. Temperature tolerance of Rhodococcus ruber YC-YT1
Inoculating strain YC-YT1 into liquid LB culture medium for activation, and culturing to logarithmic growth phase OD600The cells were inoculated in an inorganic salt ion medium (DEHP concentration: 100mg/L) at an inoculum size of 5% by volume, and were subjected to shaking and light-shielding culture at 10 ℃ C., 16 ℃ C., 30 ℃ C., 40 ℃ C., 50 ℃ C., respectively, and 180 rpm.
The same inorganic salt ion culture medium containing 100mg/L DEHP without inoculated strain was used as a control group, and the DEHP concentration in the solution was measured and the degradation rate was calculated after shaking and light-shielding cultivation at 180rpm at 10 ℃, 16 ℃, 30 ℃, 40 ℃ and 50 ℃ for 60 hours.
FIG. 9 shows the DEHP degradation rate of Rhodococcus ruber YC-YT1 under different temperature conditions over 60 hours. At 10 ℃, the degradation rate can reach more than 90 percent, which indicates that the strain can tolerate a lower temperature condition, and the degrading enzyme secreted by the strain is probably a low-temperature starting temperature; the DEHP degradation rate can reach a maximum value of 100% along with the temperature rise to 30 ℃; when the temperature is increased to 40-50 ℃, the degradation rate is reduced to some extent, but the degradation rate can still reach more than 90 percent, thereby showing that the strain YC-YT1 has wide tolerance range to the temperature and higher degradation rate under both low temperature and high temperature conditions.
Example 3 application of Rhodococcus ruber YC-YT1 to treatment of wastewater containing various phthalates
In this example, a medium containing 70g/L NaCl in inorganic salt ion (unsterilized)) As a model wastewater (hereinafter, referred to as "wastewater"), a model wastewater treatment was carried out using a 5.0L fermenter (BioFlo 115, New Brunswick Scientific Co., N.J., USA). Culturing Rhodococcus ruber YC-YT1 in LB liquid medium to logarithmic phase (OD)600The cell concentration was about 1.6X 10 at 0.88CFU/mL), adding the prepared bacterial liquid to the wastewater to final concentrations of 8X 107CFU/L、1.6×108CFU/L、3.2×108CFU/L、4.8×108CFU/L、6.4×108CFU/L and 8.0X 108CFU/L wastewater (each 2L wastewater), and DEHP, DPrP, DBP, DOP and BBP were added to the wastewater simultaneously to respective concentrations of 100mg/L as treatment groups; meanwhile, wastewater which is added with pollutant substrates with the same concentration and is not inoculated with bacteria under the same condition is used as a control group. The stirring speed was 150rpm, the aeration ratio was 0.8 (air), and the culture temperature was 30 ℃. The pH and Dissolved Oxygen (DO) levels of the reaction solution were monitored by the reactor operating system. Samples were taken on day 7 of treatment to determine various substrate concentrations. Control and treatment groups were performed in 3 replicates.
The sampling method comprises the following steps: after each treatment, 2L of n-hexane was added to the fermentor, stirred by the motor of the fermentor, left to stand for 30 minutes after 1 hour (all lines of the fermentor were closed throughout the process), and 20mL of the upper organic phase was taken for each substrate concentration measurement. The degradation rate of each substrate in the wastewater by the strain YC-YT1 was calculated based on the substrate concentration in the final measurement treatment group and the control group.
The degradation rate of Rhodococcus ruber YC-YT1 on each substrate in wastewater is shown in Table 2, and the degradation rate of each substrate is gradually increased with the increase of the added bacterial quantity, when the bacterial quantity reaches 4.8X 108The degradation rate of each substrate reaches the maximum value basically when CFU/L waste water is used.
TABLE 2 degradation rate of Rhodococcus ruber YC-YT1 on various substrates in wastewater
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
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<120> Rhodococcus ruber capable of degrading phthalate and application thereof
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Claims (9)
1. Rhodococcus ruber (C)Rhodococcus ruber) YC-YT1 with preservation number of CGMCC No. 13959.
2. A microbial agent comprising the Rhodococcus ruber YC-YT1 as claimed in claim 1.
3. A biological detergent prepared from the Rhodococcus ruber YC-YT1 of claim 1 or the microbial agent of claim 2.
4. Use of the Rhodococcus ruber YC-YT1 as claimed in claim 1, the microbial inoculum as claimed in claim 2 or the biological cleaning agent as claimed in claim 3 for biodegradation of phthalate esters.
5. The use according to claim 4, wherein the phthalate-based material comprises di (2-ethylhexyl) phthalate, dicyclohexyl phthalate, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipropyl phthalate, butylbenzyl phthalate, dipentyl phthalate, dihexyl phthalate, diheptyl phthalate, dioctyl phthalate, dinonyl phthalate, didecyl phthalate.
6. Use of the Rhodococcus ruber YC-YT1 according to claim 1, the microbial inoculum according to claim 2 or the biological cleaning agent according to claim 3 for bioremediation of phthalate-based environmental pollution.
7. Use of the Rhodococcus ruber YC-YT1 as claimed in claim 1, the microbial inoculum as claimed in claim 2 or the biological cleaning agent as claimed in claim 3 in the treatment of industrial wastewater from phthalate production.
8. The use of Rhodococcus ruber YC-YT1 in claim 1 for the preparation of a biodegradable phthalate.
9. The use according to any one of claims 6 to 8, wherein the phthalate-based material comprises di (2-ethylhexyl) phthalate, dicyclohexyl phthalate, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipropyl phthalate, butylbenzyl phthalate, dipentyl phthalate, dihexyl phthalate, diheptyl phthalate, dioctyl phthalate, dinonyl phthalate, didecyl phthalate.
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| CN108130288B (en) * | 2017-12-15 | 2020-10-09 | 浙江工业大学 | Rhodococcus ruber and application thereof in degrading organic pollutants |
| CN108410771A (en) * | 2018-04-08 | 2018-08-17 | 宁夏大学 | The application of Rhodococcus sp YC915 and its absorption microbial inoculum in soil phthalic acid ester of degrading |
| CN109207400B (en) * | 2018-09-26 | 2021-09-28 | 东北农业大学 | Composite microbial inoculum for efficiently degrading phthalic acid ester in black soil and degradation method |
| US12102654B2 (en) | 2019-01-15 | 2024-10-01 | Liaoning Greatest Bio-Pharmaceutical Co., Ltd. | Product derived from Rhodococcus ruber, and pharmaceutical use thereof |
| KR20220004091A (en) | 2019-04-24 | 2022-01-11 | 랴오닝 그레이티스트 바이오-파마슈티컬 컴퍼니 리미티드 | Use of Rhodococcus louver products in the treatment of thermal injury |
| WO2021147899A1 (en) | 2020-01-21 | 2021-07-29 | 辽宁格瑞仕特生物制药有限公司 | Use of rhodococcus ruber cell wall skeleton in regenerative medicine |
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