Li Shunpeng

Evaluation of Biological Characteristics of Bacteria Contributing to Biofilm Formation

Abstract Biofilm-associated microorganisms play crucial roles in terrestrial and aquatic nutrient cycling and in the biodegradation of environmental pollutants. Biofilm formation was determined for a total of 18 bacterial isolates obtained from the biofilms of wastewater treatment systems and of little carpolite in soil. Among these isolates, seven showed strong biofilm-forming capacity. The phylogenetic affiliation of the isolates showing high biofilm formation capacity was determined through 16S rDNA sequencing and the isolates were grouped into 7 bacterial species including Pseudomonas sp., Pseudomonas putida, Aeromonas caviae, Bacillus cereus, Pseudomonas plecoglossicida, Aeromonas hydrophila , and Comamonas testosteroni . The biofilm-forming capacity was closely related with flagella, exopolysaccharide, and extracellular protein. According to the coefficient of determination, the relative importance of the five biological characteristics to biofilm formation was, in order from greatest to least, exopolysaccharide > flagella >N -acyl-homoserine lactones (AHLs) signaling molecules > extracellular protein > swarming motility.

Isolation and characterization of a carbendazim-degrading Rhodococcus sp. djl-6

Bacterium djl-6, capable of degrading carbendazim, was isolated by continuous enrichment culture originating from carbendazim-treated soil. The isolate was identified as Rhodococcus sp. according to its phenotypic features, physiologic and biochemical characteristics, and phylogenetic analysis. The strain could use carbendazim as sole carbon or nitrogen source. It showed a high average degradation rate of 55.56 mg · L−1 · d−1 in M9 medium amended with carbendazim. High-pressure liquid chromatography–mass spectrometry (HPLC-MS) analysis showed the presence of 2-aminobenzimidazole, benzimidazole, and an unknown metabolite with molecular ions (M+) of m/z 104.8 and 118.5. The degradation in the isolate djl-6 seems to be initiated with the cleavage of the methyl carbemate side chain, resulting in the formation of 2-aminobenzimidazole and benzimidazole. This is the first report of the intermediates benzimidazole and 2-aminobenzimidazole found together in the culture filtrate of pure bacterium.

Biodegradation of Ethametsulfuron-Methyl by Pseudomonas sp. SW4 Isolated from Contaminated Soil

A soil bacterium SW4, capable of degrading the sulfonylurea herbicide ethametsulfuron-methyl (ESM), was isolated from the bottom soil of a herbicide factory. Based on physiological characteristics, biochemical tests and phylogenetic analysis of the 16S rRNA gene sequence, the strain was identified as a Pseudomonas sp. The total degradation of ESM in the medium containing glucose was up to 84.6% after 6 days of inoculation with SW4 strain. The inoculation of strain SW4 to soil treated with ESM resulted in a higher degradation rate than in noninoculated soil regardless of the soil sterilized or nonsterilized. Five metabolites of ESM degradation were analyzed by liquid chromatography/mass spectrometry. Based on the identified products, strain SW4 seemed to degrade ESM after two separate and different pathways: one leads to the cleavage of the sulfonylurea bridge, whereas the other to the dealkylation and opening of the triazine ring of ESM.

Characterization of a fenpropathrin-degrading strain and construction of a genetically engineered microorganism for simultaneous degradation of methyl parathion and fenpropathrin.

A gram-negative fenpropathrin-degrading bacterial strain Sphingobium sp. JQL4-5 was isolated from the wastewater treatment sludge of an insecticide factory. Strain JQL4-5 showed the ability to degrade other pyrethroid insecticides, but it was not able to degrade methyl parathion. To enhance its degrading range of substrate, a methyl parathion hydrolase gene (mpd) was successfully introduced into the chromosome of strain JQL4-5 with a mini-Tn-transposon system. A genetically engineered microorganism (GEM) named JQL4-5-mpd resulted, which was capable of simultaneously degrading methyl parathion and fenpropathrin. Soil treatment results indicated that JQL4-5-mpd is a promising multifunctional bacterium in the bioremediation of multiple pesticide-contaminated environments.

Biodegradation of Pentachloronitrobenzene by Labrys portucalensis pcnb-21 Isolated from Polluted Soil

Abstract A bacterial strain, pcnb-21, capable of degrading pentachloronitrobenzene (PCNB) under aerobic and anoxic conditions, was isolated from a long-term PCNB-polluted soil by an enrichment culture technique and identified as Labrys portucalensis based upon its morphological, physiological and biochemical properties, as well as 16S rRNA gene sequence analysis. Effects of different factors, such as temperature and pH, on PCNB biodegradation were studied. Strain pcnb-21 efficiently degraded PCNB at temperatures from 20 to 30 °C and initial pH values from 4 to 7, which might be the first time that a Labrys strain was found capable of efficiently degrading PCNB. The degradation of PCNB was affected by oxygen, and the degradation decreased with increasing aeration. Exogenous electron donors such as glucose, lactic acid and succinic acid promoted the biodegradation of PCNB, while electron acceptors such as sodium nitrite, sodium sulfate, sodium nitrate and sodium sulfate inhibited PCNB biodegradation. The degradation of PCNB in sterile and non-sterile soils by a green fluorescent protein (GFP)-labeled strain, pcnb-21- gfp , was also studied. Cells of pcnb-21- gfp efficiently degraded 100 mg kg −1 PCNB in sterile and non-sterile soils and could not be detected after 42 days. Strain pcnb-21 might be useful in bioremediating PCNB-polluted soils and environment.

Isolation and Characterization of Pseudomonas sp. Strain ONBA-17 Degrading o-Nitriobenzaldehyde

Aerobic bacteria degrading o-nitrobenzaldehyde (ONBA) were isolated from activated sludges. One of the isolates, ONBA-17, was identified as Pseudomonas sp. The isolate could grow on ONBA as its sole source of carbon and nitrogen. Further studies demonstrated that the strain was a moderately halophilic bacterium and capable of degrading benzoic acid, 2-nitrophenol, 2-aminophenol, 4-hydroxybenzoic acid, and 4-dimetylaminobenzaldehyde. It could completely degrade 100 mg L−1 ONBA at a range of pH 6–8 in 48 h at 30°C, and up to 400 mg L−1 after 288 h. The strain showed potential to be a good candidate for biotreatment of industrial wastewaters containing ONBA due to its salt-tolerance ability, multiresistance to some heavy metals and antibiotics, and the abilities of degradation of aromatic compounds. These findings may help in developing a process for ONBA-containing industrial wastewater treatment.

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh)

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.

Isolation and Characterization of a Methomyl-Degrading Paracoccus sp mdw-1

Methomyl, an extremely toxic pesticide, is widely used in agriculture. A strain named mdw-1 capable of degrading methomyl rapidly was successfully isolated from activated sludge in this study. It could utilize methomyl as the sole carbon or nitrogen source. The optimal temperature and medium pH for its growth and methomyl biodegradation were 30 degrees C and 7.0, respectively. It was identified as a Paracoccus sp. according to its morphological features, physiological and biochemical characteristics, and phylogenetic analysis based on the sequence of 16S rDNA. Gas chromatography-mass spectrometry (GC-MS) analysis showed that methomyl could be completely transformed to S-methyl-N-hydroxythioacetamidate in 10 h of incubation with the isolate mdw-1.

Biodegradation of malachite green by strain Pseudomonas sp. K9 and cloning of the tmr2 gene associated with an ISPpu12

A bacterial strain K9 capable of degrading malachite green was isolated from the sludge of the wastewater treatment system of a chemical plant. It was identified preliminarily as Pseudomonas sp. Strain K9 was also able to degrade other triphenylmethane dyes, such as Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase, was cloned from strain K9, and functionally expressed in E. coli. A 5946-bp DNA fragment including the tmr2 gene was cloned from the genomic DNA of strain K9 by chromosome walking. Its sequence analysis showed that tmr2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase) and orf1 (encoding a putative MerR family regulator), orf2 (encoding a CDF family heavy metal/H+ antiporter). This association was also found in another malachite green-degrading strain Pseudomonas sp. MDB-1, which indicated that the tmr2 gene might be a horizontally transferable gene.

Isolation,Identification and Degradation of Carbofuran-degrading Strain CFDS-1

Carbofuran(2,3-dihydro-2,2-dimethyl-7-benzofuranoyl N-methylcarbamate) was first introduced into China in 1979 as a pesticide and is extensively used as a soil-incorporated N-methylcarbamate insecticide to control a variety of insect pests that infest many kinds of crops,such as corn and potato.Carbofuran is of a concern due to its relatively high mammalian toxicity and relatively high mobility in soil.Some N-methylcarbamate compounds and their metabolites can be sufficiently mobile in certain soil,thus considered as potential pollutants of ground and surface waters.One carbofuran-degrading strain named CFDS-1 was isolated from soil polluted by carbofuran for a long time.According to its physiological biochemical characters,and the homology analysis of its 16S rDNA sequence(GenBank accession No.AY702969),CFDS-1 was primarily identif ied as Sphingomonas sp.CFDS-1 was able to completely degrade carbofuran in medium within 48 hours at the initial concentration of 100 mg L-1.CFDS-1 could well degrade carbofuran at concentrations from 20 mg L-1 to 300 mg L-1.The degradation speed was related positively to initial inoculation amount.The optimal pH and aeration for the degradation were 8.0～9.0 and 100 ～250 mL,respectively.The temperature(between 25 ℃ and 42 ℃) had no notable effect on the degradation of carbofuran by CFDS-1.CFDS-1 could well degrade carbofuran not only in medium,but also in soil.