CN103834588B

CN103834588B - One strain has bacillus cereus and the application thereof of degrading polycyclic aromatic hydrocarbons ability

Info

Publication number: CN103834588B
Application number: CN201410005967.9A
Authority: CN
Inventors: 徐莉; 张振; 李辉信; 张言诚; 陈雄; 李伟明; 焦加国; 刘满强; 陈小云; 胡锋
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2014-01-07
Filing date: 2014-01-07
Publication date: 2016-04-13
Anticipated expiration: 2034-01-07
Also published as: CN103834588A

Abstract

The invention discloses bacillus cereus and application thereof that a strain has degrading polycyclic aromatic hydrocarbons ability.Bacillus cereus (Bacillus? Cereus) Z21, on July 3rd, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number CGMCC? No.7875.This bacterial strain can be sole carbon source and energy growth with fluoranthene, and under laboratory shake flask condition, the fluoranthene degradation rate of this bacterial strain to 50mg/L can reach 57.63%, and also has certain degradation effect to some other polycyclic aromatic hydrocarbons (as phenanthrene, anthracene and benzo [a] pyrene).

Description

One strain has bacillus cereus and the application thereof of degrading polycyclic aromatic hydrocarbons ability

Technical field

The present invention relates to a kind of microorganism, be specifically related to bacillus cereus and application thereof that a strain has degrading polycyclic aromatic hydrocarbons ability.

Background technology

Polycyclic aromatic hydrocarbons (PolycyclicAromaticHydrocarbon is called for short PAHs) is a hydrocarbon with two or more phenyl ring, is mainly derived from the incomplete combustion of material, in the environment ubiquity.In physical environment, PAHs has hundreds of kind, wherein has 28 kinds of PAHs, due to the certain teratogenesis of tool, mutagenesis and carinogenicity, is classified as a class persistence organic pollutant of priority acccess control by U.S. EPA (EnvironmentalProtectionAgency).

The current conventional process mode for PAHs mostly is peripheral doses and chemical remediation technology, its maximum drawback is that pollutant removal is not thorough, easily cause the generation of secondary pollution, biological restoration especially microorganism remediation then has plurality of advantages, and as processing costs is low, treatment effect is good, little to environmental influence, can not secondary pollution be caused, and simple to operate, can treatment in situ.Therefore, from physical environment, screen the efficient bacterium obtaining removing polycyclic aromatic hydrocarbons to be significant.

Summary of the invention

The object of this invention is to provide a kind of bacillus cereus, efficiently can remove the various polycyclic aromatic hydrocarbonss in environment.

Object of the present invention realizes by following technical scheme:

A kind of bacillus cereus (BacillusCereus) Z21, on July 3rd, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number CGMCCNo.7875.

Described bacillus cereus bacterium colony is less, becomes faint yellow, swells, translucent, neat in edge, and smooth surface, in irregular shaft-like arrangement, produces gemma.

The physio-biochemical characteristics of described bacillus cereus are: Gram-negative, facultative aerobic, chemoheterotrophy, and catalase is positive, M.R and VP negative, and Starch Hydrolysis is positive, gelatin liquefaction positive, and nitrate reduction is positive, and Citrate trianion utilizes negative.

Bacillus cereus of the present invention has the ability of stronger degrading polycyclic aromatic hydrocarbons, and by the Z21 inoculation of degrading polycyclic aromatic hydrocarbons in LB culture media shaking vase, shaking culture is to logarithmic phase; By above-mentioned cultured bacterial classification by a certain amount of be inoculated in polycyclic aromatic hydrocarbons be sole carbon source and the energy minimal medium in grow.Take fluoranthene as target contaminant, fluoranthene starting point concentration be 50mg/L 30 DEG C, the degradation rate of BacillusCereusZ21CGMCCNo.7875 to this polycyclic aromatic hydrocarbons reaches 57.63% under the condition of 180r/min.

As further optimization of the present invention, described bacillus cereus initial fluoranthene concentration be 25mg/L, the initial pH of substratum is 8, culture temperature is 35 DEG C, liquid amount is 60mL/150mL time degradation effect best, add glucose and to degrade fluoranthene for this bacterium can be promoted.

Bacillus cereus of the present invention also has certain degradation effect to other polycyclic aromatic hydrocarbonss such as phenanthrene, anthracene and benzo [a] pyrenes.

Beneficial effect: a kind of bacillus cereus of the present invention, efficiently can remove fluoranthene, and have certain degradation effect to other polycyclic aromatic hydrocarbonss under the condition of laboratory shake flask.

Accompanying drawing explanation

Fig. 1 is the bacterium colony figure of bacterial strain Z21 of the present invention;

Fig. 2 represents the degradation capability of Z21 bacterium to fluoranthene, anthracene, phenanthrene and benzo [a] pyrene;

Fig. 3 represents the impact of different culture condition on Z21 bacterium fluoranthene degradation capacity;

Fig. 4 represents that different Co metabolism substrate is on the impact of Z21 bacterium fluoranthene degradation capacity.

Biomaterial preservation information

Z21, Classification And Nomenclature is bacillus cereus BacillusCereus, be preserved in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation date is on July 3rd, 2013, preserving number CGMCCNo.7875.

Bacillus cereus ZZ13, Classification And Nomenclature is bacillus cereus BacillusCereus, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on March 15th, 2013, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number CGMCCNo.7322.

Embodiment

Embodiment 1: the separation of fluoranthene degradation bacteria, Purification and Characterization

1, substratum: 1. LB substratum; 2. minimal medium (MS): often liter containing K ₂hPO ₄6.0g, KH ₂pO ₄5.5g, NH ₄nO ₃2.0g, Na ₂sO ₄2.0g, KCl2.0g, MgSO ₄7H ₂o0.2g, trace metal salts solution 1.0mL, distilled water, pH7.0; 3. containing the minimal medium of fluoranthene: prepare fluoranthene solution with DMF, add the minimal medium of sterilizing, fluoranthene final concentration is 50mg/L.

2, the isolation and purification of PAHs degradation bacteria

Take 10g fresh soil, add and be equipped with in the triangular flask of 100mL containing the minimal medium of fluoranthene, 180r/min shaking culture 1 week at 30 DEG C.Be transferred to by 10% inoculum size fresh in the minimal medium of fluoranthene, continue cultivation 1 week, 4 times repeatedly.Finally draw 0.1mL nutrient solution to dilute different gradient and coat on LB culture medium flat plate, picking list bacterium colony, single bacterium colony after plate streaking purifying is inoculated in the inorganic salt liquid substratum containing fluoranthene, choose the fastest bacterial strain of the speed of growth as research bacterial strain, called after Z21.

3, morphologic observation and Determination of Physiological And Biochemical Indices

By the inoculation of fully activation in LB substratum, cultivate 24h for 30 DEG C, observation of cell form, thalline size under an optical microscope after gramstaining.By activated spawn streak inoculation on LB plate, be inverted and cultivate 24h, observe colonial morphology (Fig. 1) in 30 DEG C of incubators, bacterium colony is less is faint yellow, protuberance, and neat in edge, smooth surface is moistening, opaque, irregular shaft-like arrangement, produces gemma.After the mensuration of bacterial strain physiological and biochemical index, show that this bacterium is Gram-negative again, facultative aerobic, chemoheterotrophy, catalase is positive, M.R and VP negative, and Starch Hydrolysis is positive, gelatin liquefaction positive, and nitrate reduction is positive, and Citrate trianion utilizes negative.

4, the structure of the pcr amplification of 16SrDNA, sequential analysis and phylogenetic tree

The bacterial strain of screening and separating, through Shanghai, the order-checking of handsome biotechnology company limited is according to the sequencing result of 16SrDNA, analyze in http://www.ncbi.nlm.nih.gov online query, utilize Blast software to carry out tetraploid rice with other 16SrDNA sequence in GenBank, select the 16SrDNA systematic evolution tree of the sequence MEGAversion3 software building Z21 of close sequence and Z21.According to the physiological and biochemical property of this bacterial strain, be accredited as bacillus cereus (BacillusCereus), homology is 99%.By this bacterial strain on July 3rd, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number CGMCCNo.7875.

Embodiment 2:Z21 bacterium is to the degradation effect of polycyclic aromatic hydrocarbons

Z21 bacterial classification (CGMCCNo.7875) is inoculated in 30 DEG C of activation 24h on LB substratum solid plate, enlarged culturing in rear access 30ml/150mL liquid seeds LB substratum, then to be again transferred in 30ml LB liquid medium 30 DEG C with the inoculum size of 1%, 180r/min cultivates 24h.Again by the bacterial strain collected after centrifugation thalline of fermentation culture, three times are washed afterwards with the 0.05M phosphoric acid buffer that pH is 7.0, in the PAHs minimal medium of equal volume of transferring, add polycyclic aromatic hydrocarbons, the ultimate density of fluoranthene and anthracene is 50mg/L, and luxuriant and rich with fragrance ultimate density is 100mg/L, and the ultimate density of benzo [a] pyrene is 25mg/L, 30 DEG C, under 180r/min condition, cultivate 7d.After cultivation terminates, with isopyknic extraction into ethyl acetate 3 times, after be 40 DEG C in water temperature rotary evaporation thinks highly of spin concentration to dry, then with methanol constant volume, measure the concentration of polycyclic aromatic hydrocarbons in solution with high performance liquid chromatography (HPLC).Because invention one strain before me has the bacillus cereus ZZ13(CGMCCNo.7322 of degrading polycyclic aromatic hydrocarbons ability) also there is certain degradation effect to fluoranthene, Gu Te is by ZZ13 to the degradation effect of fluoranthene also in contrast.

Obtain Z21 bacterium by above-mentioned experiment and all have good degradation effect to multiple polycyclic aromatic hydrocarbons, after cultivating 7d, the degradation rate of Z21 to fluoranthene, anthracene, phenanthrene, benzo [a] pyrene is respectively 57.63%, 31.43%, 22.27% and 24.92%, 30.29% is exceeded especially than the degradation rate of ZZ13 to fluoranthene, there were significant differences, as shown in Figure 2.And ZZ13 does not almost have degradation capability, Z21(CGMCCNo.7875 to except other polycyclic aromatic hydrocarbonss of fluoranthene) then can degrade except removing other polycyclic aromatic hydrocarbonss of fluoranthene.

Embodiment 3:Z21 bacterium is to the degradation condition optimization of fluoranthene

In order to optimize bacillus cereus Z21 that example 1 obtains to the degradation capability of fluoranthene, explore its optimum condition to fluoranthene degraded from different fluoranthene starting point concentrations, pH, temperature, liquid amount and Co metabolism substrate.

In bacterial strain conversion process, different fluoranthene concentration (12.5mg/L is set respectively, 25mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L), different conversion fluid pH(4, 5, 6, 7, 8, 9, 10), different culture temperature (20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C), different liquid amounts (is equipped with 15mL in the triangular flask of 150mL respectively, 30mL, 45mL, 60mL, 75mL, 90mL substratum) with add different Co metabolism substrate (50mg/L biphenyl, 50mg/L sodium succinate, 50mg/L pyrocatechol, 100mg/L glucose, 50mg/L Whitfield's ointment, do not add as CK) carry out degradation capability analysis.

Obtain as shown in Figure 3, bacterial strain Z21 is to the degradation rate of fluoranthene along with the raising of fluoranthene starting point concentration presents the rear downward trend that first rises, and when fluoranthene concentration is 25mg/L, its degradation rate is up to 63.82%; Degradation rate is lower in acid condition for this bacterial strain simultaneously, all has good degradation effect, wherein when pH is 8, preferably reach 59.39% to the degradation effect of fluoranthene under neutrality, weakly alkaline and alkaline condition; And under different culture temperature condition, 20 ~ 25 DEG C time, the degradation rate of this bacterial strain to fluoranthene is lower, time between 25 ~ 35 DEG C, degradation rate increases along with the rising of temperature, and when temperature is 35 DEG C, degradation rate is maximum reaches 67.43%, but when temperature continues to be increased to 40 DEG C, degradation rate then starts to decline; When bacterial strain is at different liquid amount, then the change along with liquid amount in triangular flask is large, and the degradation rate of bacterial strain to fluoranthene presents the trend of first increases and then decreases, maximum to fluoranthene degradation rate when 60mL substratum is wherein housed in bottle, reaches 71.09%.

Add pyrocatechol, glucose and Whitfield's ointment and all improve the degradation rate of bacterial strain to fluoranthene as Co metabolism substrate, wherein the promoter action of glucose is the most obvious, degradation rate reaches 74.79%, improves 18.04% compared with CK, and the facilitation effect of biphenyl and sodium succinate not obvious (Fig. 4).

Claims

1. a strain have degraded fluoranthene ability bacillus cereus ( bacillusCereus) Z21, on July 3rd, 2013 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number CGMCCNo.7875.

2. the application of bacillus cereus Z21 according to claim 1 in degraded fluoranthene.