Degradation of furazolidone by bacteria Acinetobacter calcoaceticus T32, Pseudomonas putida SP1 and Proteus mirabilis V7

Furazolidone (FZD) has been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle and farmed fish. Since FZD has been shown to have mutagenic, genotoxic and potentially carcinogenic properties when tested in a variety of systems, there is an increasing need to find a way to remove FZD from contaminated environments. In this report, three bacterial strains Acinetobacter calcoaceticus T32, Pseudomonas putida SP1 and Proteus mirabilis V7 capable of degrading FZD effectively were isolated, identified and characterized. The reduced FZD concentration after degradation was determined by HPLC. After bacterial cells were grown in the media containing 5 mg l⁻¹ FZD for 5 days, almost all FZD was degraded by A. calcoaceticus T32, and more than 50% of FZD was degraded by P. putida SP1 and P. mirabilis V7, respectively. Bacterial GST activity of A. calcoaceticus T32, P. putida SP1 and P. mirabilis V7 was determined to be influenced by different FZD concentrations. Cytotoxicity analysis showed that FZD was degraded to the metabolites with far less cytotoxicity compared to FZD. The inoculation of bacterial strains A. calcoaceticus T32, P. putida SP1 and P. mirabilis V7 into FZD-contained media resulted in a higher degradation efficiency than natural degradation, which indicated the potential application of these strains in treatment of FZD-polluted freshwater or seawater environments.     

Simultaneous degradation of the pesticides methyl parathion and chlorpyrifos by an isolated bacterial consortium from a contaminated site

The simultaneous degradation of the pesticide methyl parathion and chlorpyrifos was tested using a bacterial consortium obtained by selective enrichment from highly contaminated soils in Moravia (Medellin, Colombia). Microorganisms identified in the consortium were Acinetobacter sp, Pseudomonas putida, Bacillus sp, Pseudomonas aeruginosa, Citrobacter freundii, Stenotrophomonas sp, Flavobacterium sp, Proteus vulgaris, Pseudomonas sp, Acinetobacter sp, Klebsiella sp and Proteus sp. In culture medium enriched with each of the pesticides, the consortium was able to degrade 150 mg l⁻¹ of methyl parathion and chlorpyrifos in 120 h. When a mixture of 150 mg l⁻¹ of both pesticides was used the percentage decreased to 72% for methyl parathion and 39% for chlorpyrifos. With the addition of glucose to the culture medium, the consortium simultaneously degraded 150 mg l⁻¹ of the pesticides in the mixture. 4 treatments were carried out in soil that included the addition of glucose with microorganisms, the addition of sugar cane with microorganisms, microorganisms without nutrient addition and without the addition of any item. In the treatment in which glucose was used, degradation percentages of methyl parathion and chlorpyrifos of 98% and 97% respectively were obtained in 120 h. This treatment also achieved the highest percentage of reduction in toxicity, monitored with Vibrio fischeri.     

Biodegradation of chlorpyrifos by bacterial consortium isolated from agriculture soil

Organophosphorous pesticides are widely used in agriculture to control major insect pests. Chlorpyrifos is one of the major organophosphorous pesticides which is used to control insects including termites, beetles. The widespread use of these pesticides is hazardous to the environment and also toxic to mammals, thus it is essential to remove the same from the environment. From the chlorpyrifos contaminated soil nine morphologically different bacterial strains, one actinomycete and two fungal strains were isolated. Among those isolates four bacterial strains which were more efficient were developed as consortium. The four bacterial isolates namely Pseudomonas putida (NII 1117), Klebsiella sp., (NII 1118), Pseudomonas stutzeri (NII 1119), Pseudomonas aeruginosa (NII 1120) present in the consortia were identified on the basis of 16S rDNA analysis. The intracellular fractions of the consortium exhibited more organophosphorus hydrolase activity (0.171 ± 0.003 U/mL/min). The degradation studies were carried out at neutral pH and temperature 37°C with chlorpyrifos concentration 500 mg L⁻¹. LC-mass spectral analysis showed the presence of metabolites chlopyrifos-oxon and Diethylphosphorothioate. These results highlight an important potential use of this consortium for the cleanup of chlorpyrifos contaminated pesticide waste in the environment.     

Targeting Acyl Homoserine Lactones (AHLs) by the quorum quenching bacterial strains to control biofilm formation in Pseudomonas aeruginosa

Navigating novel biological strategies to mitigate bacterial biofilms have great worth to combat bacterial infections. Bacterial infections caused by the biofilm forming bacteria are 1000 times more resistant to antibiotics than the planktonic bacteria. Among the known bacterial infections, more than 70% involve biofilms which severely complicates treatment options. Biofilm formation is mainly regulated by the Quorum sensing (QS) mechanism. Interference with the QS system by the quorum quenching (QQ) enzyme is a potent strategy to mitigate biofilm. In this study, bacterial strains with QQ activity were identified and their anti-biofilm potential was investigated against the Multidrug Resistant (MDR) Pseudomonas aeruginosa. A Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136-based bioassays were used to confirm the degradation of different Acyl Homoserine Lactones (AHLs) by QQ isolates. The 16S rRNA gene sequencing of the isolated strains identified them as Bacillus cereus strain QSP03, B. subtilis strain QSP10, Pseudomonas putida strain QQ3 and P. aeruginosa strain QSP01. Biofilm mitigation potential of QQ isolates was tested against MDR P. aeruginosa and the results suggested that 50% biofilm reduction was observed by QQ3 and QSP01 strains, and around 60% reduction by QSP10 and QSP03 bacterial isolates. The presence of AHL degrading enzymes, lactonases and acylases, was confirmed by PCR based screening and sequencing of the already annotated genes aiiA, pvdQ and quiP. Altogether, these results exhibit that QQ bacterial strains or their products could be useful to control biofilm formation in P.aeruginosa.     

Response of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> F1 cultures to fluctuating toluene loads and operational failures in suspended growth bioreactors

The response of Pseudomonas putida F1 to process fluctuations and operational failures during toluene biodegradation was evaluated in a chemostat suspended growth bioreactor. The ability of P. putida F1 to rapidly increase its specific toluene degradation capacity resulted in no significant variation in process removal efficiency when toluene load was increased from 188 to 341 g m⁻³ h⁻¹. Likewise, bacterial activity rapidly reached steady state performance (in less than 1.5 h after the restoration of steady state operational conditions) following an 8 h process shutdown, or after episodes of toluene or mineral nutrients deprivation. Process performance was however highly sensitive to pH, as pH levels below 4.5 dramatically inhibited bacterial activity, decreasing severely process robustness and inducing a cycle of periodic process collapses and recoveries. This pH mediated deterioration of bacterial activity was confirmed by further respirometric tests, which revealed a 50–60% reduction in the O₂ consumption rate during the degradation of both toluene and 3-methyl catechol when pH decreased from 5.05 to 4.55. Finally, process robustness was quantified according to methods previously described in literature.     

Comparison of in-situ biodegrading abilities of Pseudomonas putida mutants: leuB− auxotroph,fliC− non-motility,and cheA− non-chemotaxis

Bioremediation of pollutants in natural environments is affected by many factors, such as bacterial survival, motility, and chemotaxis. However, these roles in in-situ biodegradation of organophosphorus pesticides have not been examined extensively. In this paper, a highly effective methyl-parathion (MP) degrading strain, Pseudomonas putida DLL-1, which also demonstrates motile ability and chemotactic response toward MP, was selected as the research material. A leuB^? auxotroph mutant A3-27 and fliC^? non-motility mutant a4-8 were first constructed by random insertion of the kanamycin gene into the chromosome of P. putida DLL-1 with the mini-transposon system. Biodegradation of MP in liquid medium and soil microcosms by A3-27, a4-8 and a previously constructed cheA^? non-chemotaxis mutant P. putida DAK were compared. The kinetic parameters for MP degradation were all similar in the well-mixed liquid systems. However, in soil microcosms, all the three mutants had lower degrading rates compared with wild-type P. putida DLL-1. The auxotroph mutant A3-27 had the lowest degrading rate and could only degrade 25.7–34.2% MP in 5 days, and the non-motility mutant a4-8 and non-chemotaxis mutant DAK could only degrade 53.5–68.1% and 64.3–85.7% MP, respectively. This paper emphasizes, for the first time, the use of non-auxotroph bacteria for efficient removal of organophosphorus pesticides in contaminated sites, and also points out the importance of select microorganisms with specific motile or chemotactic affinities in optimizing pesticide bioremediation.     

Biodegradation of photo-degraded mulching films based on polyethylenes and stearates of calcium and iron as pro-oxidant additives

Polyethylene film materials persist in the environment for a long time. Several bacterial species have been isolated from films buried in soil located in Murcia, Spain. Bacterial strains were characterized with a combination of culture-dependent methods and sequencing of part of the 16S ribosomal RNA gene (rDNA) after amplification by polymerase chain reaction (PCR). Three bacterial species common in soil were found attached to the polymer and identified as Bacillus. cereus, B. megaterium, and B. subtilis. These microorganisms, as well as Brevibacillus borstelensis, were tested for biodegradation susceptibility at 30 and 45 °C on highly photo-degraded polyethylene films (500 h under irradiation of Xe-Lamp-solar filter) that contained calcium and iron stearates as pro-oxidant additives. Biofilm formation developed on the photo-degraded materials after one week of bacterial treatment. Biodegradation of the polyethylene films was studied by chemiluminescence, ATR-FTIR, and GC-product analysis and the data confirm a more efficient biodegradation on the bioassays carried out at higher temperature. The CL emissions due to decomposition of oxidation species take place at lower temperatures; the decrease of carbonyl index and the disappearance of photogenerated low-molecular products with biodegradation were more efficient on the biodegraded films at 45 °C. Also, mineralization was evaluated by carbon dioxide measurements using an indirect impedance technique. Biodegradation by B. borstelensis and MIX at 30 °C was slow and in the range of 0.7-1.2% of mineralization after 90 days of bacterial bioassay. At 45 °C biodegradation was more efficient and in particular in the more photo-degraded films containing Ca and Fe stearates where mineralization extents reached values of 11.5% with B. borstelensis and 7-10% with the mixture of Bacillus (MIX).     

Influence of inoculation with plant growth promoting rhizobacteria (PGPR) on tomato plant growth and nematode reproduction under greenhouse conditions

Numerous species of soil bacteria which flourish in the rhizosphere of plants or around plant tissues stimulate plant growth and reduce nematode population by antagonistic behavior. These bacteria are collectively known as PGPR (plant growth promoting rhizobacteria). The effects of six isolates of PGPR Pseudomonas putida, Pseudomonas fluorescens, Serratia marcescens, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus cereus, were studied on tomato plant growth and root knot nematode reproduction after 45 days from nematode infection. The highest number of shoot dry weight/g (43.00 g) was detected in the plant treated with S. marcescens; then P. putida (34.33 g), B. amyloliquefaciens (31.66 g), P. fluorescens (30.0 g), B. subtilis (29.0 g), B. cereus (27.0 g) and nematode alone (untreated) 20 g/plant. While the highest number of plant height was observed when plant was treated with S. marcescens, P. fluorescens, P. putida, B. amyloliquefaciens and P. putida 52.66, 50.66, 48 and 48 cm respectively. No significant differences were seen between previous treatments but only had significant differences compared with untreated plant. The highest number of fruit/plant was observed when plants were treated with S. marcescens (10.66), then B. amyloliquefaciens (8.66), P. putida (8), P. fluorescens (8) and B. cereus (7.66). No significant differences between the last 4 treatments, but all had significant differences compared with untreated plants. The highest weight of plant yield (g) was observed with S. marcescens (319.6 g/plant) and the lowest weight of plant yield was observed in plants treated with nematode alone (untreated). On the other hand, the lowest numbers of J₂/10 g of soil (78), galls/root, (24.33) galls/root, egg masses/root (12.66) and egg/egg masses were observed in the plants treated with S. marcescens.     

Biodegradation of Chlorpyrifos in Soil by Enriched Cultures

Three aerobic bacterial consortia, AC, BC, and DC, developed from pesticide-contaminated soils of Punjab were able to degrade chlorpyrifos after 21 days of incubation in basal medium by 54, 46, and 61% and chlorpyrifos (50 mg/L) in soil after 30 days by 50, 56, and 64%. Pseudomonas aeruginosa, Bacillus cereus, Klebsiella sp., and Serratia marscecens obtained from these consortia showed 84, 84, 81, and 80% degradation of chlorpyrifos (50 mg/L) in liquid medium after 20 days and 92, 60, 56, and 37% degradation of chlorpyrifos (50 mg/L) in soil after 30 days. Populations of Bacillus cereus, Klebsiella sp., and Serratia marscecens remained steady in soil experiments except for P. aeruginosa, where the population showed a substantial increase. Formation of 3,5,6-trichloro-2-pyridinol, the major metabolite of chlorpyrifos degradation, was observed during the degradation of chlorpyrifos by P. aeruginosa, which disappeared to negligible amounts.     

Determination of 17 Organophosphate Pesticide Residues in Mango by Modified QuEChERS Extraction Method Using GC-NPD/GC-MS and Hazard Index Estimation in Lucknow,India

A total of 162 samples of different varieties of mango: Deshehari, Langra, Safeda in three growing stages (Pre-mature, Unripe and Ripe) were collected from Lucknow, India, and analyzed for the presence of seventeen organophosphate pesticide residues. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method of extraction coupled with gas chromatography was validated for pesticides and qualitatively confirmed by gas chromatography- mass spectrometry. The method was validated with different concentrations of mixture of seventeen organophosphate pesticides (0.05, 0.10, 0.50 mg kg⁻¹) in mango. The average recovery varied from 70.20% to 95.25% with less than 10% relative standard deviation. The limit of quantification of different pesticides ranged from 0.007 to 0.033 mg kg⁻¹. Out of seventeen organophosphate pesticides only malathion and chlorpyriphos were detected. Approximately 20% of the mango samples have shown the presence of these two pesticides. The malathion residues ranged from ND-1.407 mg kg⁻¹ and chlorpyriphos ND-0.313 mg kg⁻¹ which is well below the maximum residues limit (PFA-1954). In three varieties of mango at different stages from unpeeled to peeled sample reduction of malathion and chlorpyriphos ranged from 35.48%–100% and 46.66%–100% respectively. The estimated daily intake of malathion ranged from 0.032 to 0.121 µg kg⁻¹ and chlorpyriphos ranged from zero to 0.022 µg kg⁻¹ body weight from three different stages of mango. The hazard indices ranged from 0.0015 to 0.0060 for malathion and zero to 0.0022 for chlorpyriphos. It is therefore indicated that seasonal consumption of these three varieties of mango may not pose any health hazards for the population of Lucknow, city, India because the hazard indices for malathion and chlorpyriphos residues were below to one.     

Gene expression profiling of Pseudomonas putida F1 after exposure to aromatic hydrocarbon in soil by using proteome analysis

Pseudomonas putida F1 can metabolize toluene, ethylbenzene, and benzene for growth. Previously, we identified proteins involved in the utilization of these compounds by P. putida F1 through culture in liquid media. However, it was unclear whether laboratory analysis of bacterial activity and catabolism accurately reflected the soil environment. We identified proteins involved in the degradation of toluene, ethylbenzene, and benzene growth in soil using two-dimensional gel electrophoresis (2-DE) or standard SDS-PAGE combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS). According to 2-DE/LC–MS/MS analysis, 12 of 22 key enzymes involved in the degradation of toluene, ethylbenzene, and benzene were detected. In standard SDS-PAGE/LC–MS/MS analysis of soil with ethylbenzene, approximately 1,260 cellular proteins were identified in P. putida F1. All key enzymes and transporter and sensor proteins involved in ethylbenzene degradation were up-regulated similar to that noted in liquid cultures. In P. putida F1, aromatic hydrocarbon response in soil is the same as that observed in liquid media.     

Carbon and Hydrogen Stable Isotope Fractionation during Aerobic Bacterial Degradation of Aromatic Hydrocarbons

¹³C/¹²C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.     

Bacterial Endosymbionts of Pyrodinium bahamense var. compressum

The study presents evidence in support of the bacterial theory associated with the toxicity of Pyrodinium bahamense var. compressum. Bacterial endosymbionts from Philippine P. bahamense var. compressum strain Pbc MZRVA 042595 were isolated and identified via 16S rDNA sequence analysis. Taxonomic diversity of the identified culturable intracellular microbiota associated with Philippine P. bahamense var. compressum was established to be limited to the Phyla Proteobacteria, Actinobacteria, and Firmicutes. Major endosymbionts identified included Moraxella spp., Erythrobacter spp., and Bacillus spp., whereas Pseudomonas putida, Micrococcus spp., and Dietzia maris were identified as minor isolates. All identified strains except D. maris, P. putida, and Micrococcus spp. were shown to contain either saxitoxin or neo saxitoxin or both at levels ≤73 ng/10⁷ bacterial cells based on high-performance liquid chromatography analysis. Paralytic shellfish poisoning-like physiologic reactions in test animals used in the mouse assay were recorded for the endosymbionts except for P. putida. The study is the first to elucidate the possible contribution of bacterial endosymbionts in the toxicity of P. bahamense var. compressum isolated in the Philippines.     

Isolation and characterization of potential aerobic bacteria capable for pyridine degradation in presence of picoline,phenol and formaldehyde as co-pollutants

Pyridine, heterocyclic aromatic compound is known to be toxic, carcinogenic and teratogenic to several living organisms. In this study, two aerobic bacteria ITRCEM1 and ITRCEM2 capable for pyridine degradation were isolated and characterized as Bacillus cereus (DQ435020) and Alcaligenes faecalis (DQ435021), respectively. For pyridine degradation, mixed bacterial culture was found more effective compared to axenic culture ITRCEM1 and ITRCEM2 degrading 94.23, 67.84, and 83.35% pyridine, respectively, at 144 h incubation period at pH 7.0 ± 0.1, temp 37 ± 2°C and shaking rate 125 rpm in MSM containing 1% glucose and 0.2% peptone as carbon and nitrogen source, respectively. The presence of phenol and formaldehyde in MSM has shown inhibitory effect on pyridine degradation whereas picoline has favored the bacterial growths and pyridine degradation. Further, the HPLC analysis has shown the reduction in peaks compared to controls, indicating that reduction in peak area might be largely attributed to the bacterial degradation of pyridine by bacterial catabolic enzymes.     

Efficiency of natural and engineered bacterial strains in the degradation of 4-chlorobenzoic acid in soil slurry

Two bacterial strains, the natural isolate Arthrobacter sp. FG1 and the engineered strain Pseudomonas putida PaW340/pDH5, were compared for their efficiency in the degradation of 4-chlorobenzoic acid in a slurry phase system. The recombinant strain was obtained by cloning the Arthrobacter sp. FG1 dehalogenase encoding genes in P. putida PaW340. In the slurry inoculated with pre-adapted cultures of Arthrobacter sp. FG1, the 4-chlorobenzoic acid degradation was found to be slower than that observed in the slurry inoculated with the recombinant strain P. putida PaW340/pDH5, regardless of the presence or absence of soil indigenous bacteria. Slurry inoculated with mixed cultures of Arthrobacter sp. FG1 and the 4-hyroxybenzoic acid degrader P. putida PaW340 did not show any improvement in 4-chlorobenzoic acid degradation.     

Effect of Trichloroethylene on the Competitive Behavior of Toluene-Degrading Bacteria

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida GJ31, and Burkholderia cepacia G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight]⁻¹ h⁻¹). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While P. putida mt-2 took over the culture, mutants of this strain which could no longer grow on p-xylene arose. Most of them had less or no meta-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.     

Biodegradation of alkyl branched aromatic alkanoic naphthenic acids by Pseudomonas putida KT2440

The majority of the world’s crude oil reserves consist of highly biodegraded heavy and super heavy crude oils and oil sands that have not yet been fully exploited. These vast resources contain complex mixtures of carboxylic acids known as naphthenic acids (NAs). NAs cause major environmental and economic problems, as they are recalcitrant, corrosive and toxic. Although aromatic acids make up a small proportion of most NA mixtures, they have demonstrable toxicities to some organisms (e.g. some bacteria and algae) and ideally need to be removed or reduced by remediation. The present study analysed the ability of Pseudomonas putida KT2440 to degrade highly recalcitrant aromatic acids, as exemplified by the alkyl phenylalkanoic acid (4′-t-butylphenyl)-4-butanoic acid (t-BPBA) and the more degradable (4′-n-butylphenyl)-4-butanoic acid (n-BPBA). n-BPBA was completely metabolized after 14 days, with the production of a persistent metabolite identified as (4′-n-butylphenyl)ethanoic acid (BPEA) which resulted from removal of two carbon atoms from the carboxyl side chain (beta-oxidation) as observed previously with a mixed consortium. However, when n-BPBA concentration was increased two-fold, degradation decreased by 56% with a concomitant six-fold decrease in cell numbers, suggesting that at greater concentrations, n-BPBA may be toxic to P. putida KT2440. In contrast, P. putida KT2440 was unable to degrade the highly recalcitrant t-BPBA even after 49 days. These findings have implications for NA bioremediation in the environment.     

Biodegradation potential of oily sludge by pure and mixed bacterial cultures

The biodegradation capacity of aliphatic and aromatic hydrocarbons of petrochemical oily sludge in liquid medium by a bacterial consortium and five pure bacterial cultures was analyzed. Three bacteria isolated from petrochemical oily sludge, identified as Stenotrophomonas acidaminiphila, Bacillus megaterium and Bacillus cibi, and two bacteria isolated from a soil contaminated by petrochemical waste, identified as Pseudomonas aeruginosa and Bacillus cereus demonstrated efficiency in oily sludge degradation when cultivated during 40 days. The bacterial consortium demonstrated an excellent oily sludge degradation capacity, reducing 90.7% of the aliphatic fraction and 51.8% of the aromatic fraction, as well as biosurfactant production capacity, achieving 39.4% reduction of surface tension of the culture medium and an emulsifying activity of 55.1%. The results indicated that the bacterial consortium has potential to be applied in bioremediation of petrochemical oily sludge contaminated environments, favoring the reduction of environmental passives and increasing industrial productivity.     

Benzene, toluene, and o-xylene degradation by free and immobilized P. putida F1 of postconsumer agave-fiber/polymer foamed composites

Benzene, toluene, and o-xylene (BTX) degradation by immobilized Pseudomonas putida F1 of postconsumer agave-fiber/polymer foamed-composites (AFPFC) and suspended cultures was studied under controlled conditions. Analyses using FTIR-ATR and SEM showed that P. putida F1 adhered onto the composite surface and developed a biofilm. In this sense, the AFPFC were successfully used as a support for bacterial immobilization. Both systems, immobilized and suspended cells of P. putida F1, were able to completely degrade benzene and toluene from initial concentrations of 15, 30, 60, and 90 mg l⁻¹. An inhibitory effect of the intermediary catechol from benzene degradation was observed in suspended cultures but it was not presented in the immobilized system. The degradation of o-xylene was partially accomplished in both systems. The Monod equation was used to model the experimental data obtained from the biodegradation kinetics, and they were adequately described with this model.