CN103820360B

CN103820360B - One strain can effectively be degraded the degradation bacteria of propyzamide and application thereof

Info

Publication number: CN103820360B
Application number: CN201410017230.9A
Authority: CN
Inventors: 王鸣华; 赵百萍; 华修德; 施海燕
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2014-01-16
Filing date: 2014-01-16
Publication date: 2016-04-20
Anticipated expiration: 2034-01-16
Also published as: CN103820360A

Abstract

The invention belongs to microorganism field, relate to a strain propyzamide degradation bacteria W1, do you belong to Comamonas (Comamonas? sp.), be preserved in China typical culture collection center, preservation date is on October 11st, 2013, does is preserving number CCTCC? NO:M2013464,16S? rRNA gene order is 1680505 in Genebank accession number.This bacterium can degrade in 8h 100mg/L this acyl of alkynes grass amine reach 90%.Purposes is: this bacterial strain is used for degrading herbicide propyzamide, can be used for the biopurification of water body, soil and the agricultural-food polluted by propyzamide.

Description

One strain can effectively be degraded the degradation bacteria of propyzamide and application thereof

Technical field

The invention belongs to microorganism field, relate to strain propyzamide degradation bacteria and an application thereof.

Background technology

Propyzamide is Rhom and Hass of the U.S. (Rohm & Haas) a kind of inner sucting conduction type selectivity acetamide-group herbicides of developing in 1969, by the mitotic division of Root Absorption conducted interference cell, applicable crops is small-sized seed, leguminous crop, peanut, soybean, lettuce, some orchard, turf and some ornamental plants.Have annual grassy weeds and some perennial weeds and significantly suppress and root out effect.

Biological degradation is the agricultural chemicals important conversion process of a class in the environment.Microorganism, owing to being present in nature in a large number and having of a great variety, breeding rapidly with to features such as environmental compatibility are strong, serves vital role in the biodegradation process of agricultural chemicals.Not yet find the report of biological degradation propyzamide at present, therefore screen the bacterial strain of propyzamide of effectively degrading, to the exploitation of degrading genes resource, there is good realistic meaning.

Summary of the invention

The object of the present invention is to provide a strain can effectively to degrade the degradation bacteria of propyzamide, this bacterium is isolated from the soil near Jiangsu Province's Yancheng City propyzamide production plant.

Object of the present invention realizes by following technical scheme:

One strain propyzamide degradation bacteria Comamonassp.W1, is preserved in China typical culture collection center, and preservation date is on October 11st, 2013, and preserving number is CCTCCNO:M2013464.

The morphological feature of Comamonassp.W1 (CCTCCNO:M2013464): Comamonassp.W1 (CCTCCNO:M2013464) belongs to Gram-negative bacteria, on solid LB culture machine, bacterium colony is creamy white, circular, smooth, neat in edge, projection (Figure 1A).Thalline becomes shaft-like (0.58*1.07 μm), has pod membrane, amphitrichous (Figure 1B).

The physio-biochemical characteristics of bacterial strain W1 are in table 1:

The physio-biochemical characteristics of table 1 bacterial strain W1

Note: "+" is positive, "-" is negative

The cultivation characteristic of bacterial strain W1:

W1 (CCTCCNO:M2013464) can use LB culture medium culturing.W1 (CCTCCNO:M2013464) well-grown within the scope of pH5.0-9.0, when pH is lower than 4.0 or be obviously suppressed (Fig. 2) higher than the growth of 10.0 bacterial strains; Bacterial strain temperature 25-37 DEG C of growth better (Fig. 3); When NaCl concentration is 0-2%, better, when NaCl concentration is greater than 3%, thalli growth is suppressed (Fig. 4) in growth;

Beneficial effect:

The invention provides a strain propyzamide degradation bacteria Comamonassp.W1, can degrade in the 8h propyzamide of 100mg/L of this bacterium reaches more than 90%, is the alternative bacterium that biological degradation propyzamide provides.

Accompanying drawing explanation

Figure 1A: bacterial strain W1 bacterium colony photo B: bacterial strain W1W1 transmission electron microscope photo on LB flat board, scale is 1.0 μm

The impact that Fig. 2 pH grows bacterial strain W1

The impact that Fig. 3 temperature grows bacterial strain W1

The impact that Fig. 4 NaCl concentration grows bacterial strain W1

Fig. 5 bacterial strain W1 is to the degraded UV scanning figure of propyzamide

HPLC collection of illustrative plates (RT:5.148 is parent propyzamide, and 6.316 is product) in Fig. 6 bacterial strain W1 degraded propyzamide process

The genomic dna of Fig. 7 bacterial strain W1 and 16SrRNA gene PCR product

A schemes: the genomic dna of W1, wherein, and 1: λ-HindIIIMarker, 2:W1 genomic dna;

B schemes: the 16SrRNAPCR product of bacterial strain W1, wherein 1:marker, the 16SrRNA gene amplification product of 2: negative control, 3: bacterial strain W1

The phylogenetic tree of Fig. 8 bacterial strain W1

Biomaterial preservation information

Comamonassp.W1, is preserved in China typical culture collection center, preservation address Wuhan, China Wuhan University, and preservation date is on October 11st, 2013, and preserving number is CCTCCNO:M2013464.

Embodiment

The screening of embodiment W1

Get 5g soil sample (picking up from Yancheng City) and be placed in the enrichment medium (g/L:NH of 100ml containing 50mg/L propyzamide ₄nO ₃1.0g, KH ₂pO ₄0.5g, K ₂hPO ₄1.5g, NaCl1.0, MgSO ₄.7H ₂o0.1g, pH7.0) in, in 30 DEG C, 180r/min cultivates 5d.The degraded situation of propyzamide in pregnant solution is measured with ultraviolet scanner, after finding that propyzamide is degraded, inoculum size with 10% is linked in 100mg/L propyzamide substratum, continue enrichment and measure degraded situation, according to said method until propyzamide concentration is increased to 400mg/L, and go down to posterity 3 times.Propyzamide pregnant solution is coated after gradient dilution on the LB flat board containing 200mg/L propyzamide, in 30 DEG C of incubators, cultivate 2d.Single bacterium different for the colonial morphology that flat board grows is lined respectively the dull and stereotyped purifying of LB, and be inoculated in the liquid inorganic salt culture medium (g/L:NH that 50mg/L propyzamide is sole carbon source ₄nO ₃1.0g, KH ₂pO ₄0.5g, K ₂hPO ₄1.5g, NaCl1.0, MgSO ₄.7H ₂o0.1g, pH7.0) in, in 30 DEG C, 180r/min shaking table cultivates after 2d, isopyknic methylene dichloride is added, thermal agitation 1min, sucking-off aqueous phase after stratification in sample to be determined, add enough anhydrous sodium sulphate and remove moisture remaining in methylene dichloride, within the scope of wavelength 200-350nm, carry out UV scanning, the maximum absorption band of propyzamide is 230nm, determines that pregnant solution and bacterial strain are to the degradation capability of propyzamide according to the changing conditions of charateristic avsorption band.Found by UV scanning, the bacterial strain being numbered W1 can make the ultraviolet absorption peak of propyzamide change, as shown in Figure 5.Utilize HPLC to carry out analysis to the nutrient solution before and after bacterial strain W1 degraded propyzamide to find, the retention time not connecing propyzamide in the contrast nutrient solution of bacterium is 5.144 (Fig. 6).And inoculate W1 bacterial strain and after cultivating 2h, the content in nutrient solution declines, and is that 6.316 places have new absorption peak to occur (Fig. 6) in retention time.

Adopt DNA extracting method extraction thalline STb gene (Fig. 7 A) in a small amount, utilize 16SrRNA to identify (Fig. 7 B) it.Be tested and appraised, this bacterial strain is Comamonassp. called after Comamonassp.W1, and gene accession number is 1680505, and the systematic evolution tree of this bacterium is shown in Fig. 8.

Bacterial strain W1 is delivered China typical culture collection center (CCTCC) preservation, preservation date is 2013.5.21, and preserving number is CCTCCNO:M2013464.

Embodiment 2

By Comamonassp.W1 (CCTCCNO:M2013464) single colony inoculation in 20ml LB liquid medium (g/L: yeast powder 5.0, Tryptones 10.0, NaCl10.0, pH7.0), 30 DEG C, 180r/min cultivate 16h make OD _600nmthe seed liquor of=1.0.At the minimal medium (g/L:NH that propyzamide final concentration is 100mg/L ₄nO ₃1.0g, KH ₂pO ₄0.5g, K ₂hPO ₄1.5g, NaCl1.0, MgSO ₄.7H ₂o0.1g, pH7.0) in, by 1% inoculum size access seed liquor, with 30 DEG C, 180r/min shaking table cultivates, every 2h sampling once, the concentration of mensuration propyzamide, can degrade in the 8h propyzamide of 100mg/L of bacterial strain W1 reaches 90%.

Claims

1. a strain propyzamide degradation bacteria Comamonassp.W1, be preserved in China typical culture collection center, preservation date is on October 11st, 2013, preserving number is CCTCCNO:M2013464, Main Biological is: Gram-negative, thalline is shaft-like, size is about 0.58 × 1.07 μm, polar flagella, oxydase, catalase, Citrate trianion utilizes and is determined as the positive, urase, arginine dihydrolase, Starch Hydrolysis, gelatine liquefication, hydrogen sulfide, glucose, lactose, sucrose, fructose fermentation is determined as feminine gender, the 16SrRNA gene order of this bacterial strain is 1680505 in Genebank accession number.

2. the purposes of the effective degradation bacteria of weedicide propyzamide as claimed in claim 1, the purification of water body, soil and agricultural-food of this bacterial strain for being subject to propyzamide and polluting.