Biodegradation of Alprazolam in Pharmaceutical Wastewater Using Mesoporous Nanoparticles-Adhered Pseudomonas stutzeri.

The release of pharmaceutical wastewaters in the environment is of great concern due to the presence of persistent organic pollutants with toxic effects on environment and human health. Treatment of these wastewaters with microorganisms has gained increasing attention, as they can efficiently biodegrade and remove contaminants from the aqueous environments. In this respect, bacterial immobilization with inorganic nanoparticles provides a number of advantages, in terms of ease of processing, increased concentration of the pollutant in proximity of the cell surface, and long-term reusability. In the present study, MCM-41 mesoporous silica nanoparticles (MSN) were immobilized on a selected bacterial strain to remove alprazolam, a persistent pharmaceutical compound, from contaminated water. First, biodegrading microorganisms were collected from pharmaceutical wastewater, and Pseudomonas stutzeri was isolated as a bacterial strain showing high ability to tolerate and consume alprazolam as the only source for carbon and energy. Then, the ability of MSN-adhered Pseudomonas stutzeri bacteria was assessed to biodegrade alprazolam using quantitative HPLC analysis. The results indicated that after 20 days in optimum conditions, MSN-adhered bacterial cells achieved 96% biodegradation efficiency in comparison to the 87% biodegradation ability of Pseudomonas stutzeri freely suspended cells. Kinetic study showed that the degradation process obeys a first order reaction. In addition, the kinetic constants for the MSN-adhered bacteria were higher than those of the bacteria alone.

Aerobic Degradation of Clothianidin to 2-Chloro-methyl Thiazole and Methyl 3-(Thiazole-yl) Methyl Guanidine Produced by Pseudomonas stutzeri smk.

Overuse of pesticides in agriculture may harm environmental and agricultural yields. Sustainable maintenance of soil fertility and management of the environment have become a concern due to the persistence of pesticides in the soil. Microbes have various mechanisms for the bioremediation of persistent organic pollutants from the environment. A bacterium that degrades clothianidin was isolated from the pesticide and applied to agricultural soil by the enrichment technique. The identity of the bacterium was determined by studying morphological, cultural, and biochemical characteristics and 16S rRNA gene sequences. The ability to metabolize clothianidin was confirmed using UV-visible spectrophotometric, chromatographic, and spectroscopic analyses. A Gram-negative bacterium, designated smk, isolated from clothianidin-contaminated soil was confirmed to be a member of Pseudomonas stutzeri. The biodegradation of clothianidin was studied using P. stutzeri smk. Approximately 62% degradation of clothianidin was achieved within two weeks when grown at 30°C and pH 7. The effects of various physicochemical parameters, including pH, temperature, and clothianidin concentrations, on catabolic rates were studied. The biodegradation studies using UV-Vis spectrophotometry, HPLC, FTIR, and LC-MS indicated the production of the following metabolites: 2-chloro-5-methyl thiazole (CMT), methyl nitroguanidine (MNG), methyl 3-[thiazole-yl], and methyl guanidine (TMG). Identification of specific degradation metabolites indicates that bioremediation of toxic neonicotinoid insecticides may be achieved by application of P. stutzeri smk.

Simultaneous biodegradation of phenol and cyanide present in coke-oven effluent using immobilized Pseudomonas putida and Pseudomonas stutzeri

ABSTRACT Discharge of coke-oven wastewater to the environment may cause severe contamination to it and also threaten the flora and fauna, including human beings. Hence before dumping it is necessary to treat this dangerous effluent in order to minimize the damage to the environment. Conventional technologies have inherent drawbacks however, biological treatment is an advantageous alternative method. In the present study, bacteria were isolated from the soil collected from the sites contaminated by coke-oven effluent rich in phenol and cyanide. Nucleotides sequence alignment and phylogenetic analysis showed the identity of the selected phenol and cyanide degrading isolates NAUN-16 and NAUN-1B as Pseudomonas putida and Pseudomonas stutzeri, respectively. These two isolates tolerated phenol up to 1800 mg L-1 and cyanide up to 340 mg L-1 concentrations. The isolates were immobilized on activated charcoal, saw dust and fly ash. The effluent was passed through the column packed with immobilized cells with a flow rate of 5 mL min-1. The isolates showed degradation of phenol up to 80.5% and cyanide up to 80.6% and also had the ability to reduce biological oxygen demand, chemical oxygen demand and lower the pH of effluent from alkaline to near neutral. The study suggests the utilization of such potential bacterial strains in treating industrial effluent containing phenol and cyanide, before being thrown in any ecosystem.

Simultaneous biodegradation of phenol and cyanide present in coke-oven effluent using immobilized Pseudomonas putida and Pseudomonas stutzeri.

Discharge of coke-oven wastewater to the environment may cause severe contamination to it and also threaten the flora and fauna, including human beings. Hence before dumping it is necessary to treat this dangerous effluent in order to minimize the damage to the environment. Conventional technologies have inherent drawbacks however, biological treatment is an advantageous alternative method. In the present study, bacteria were isolated from the soil collected from the sites contaminated by coke-oven effluent rich in phenol and cyanide. Nucleotides sequence alignment and phylogenetic analysis showed the identity of the selected phenol and cyanide degrading isolates NAUN-16 and NAUN-1B as Pseudomonas putida and Pseudomonas stutzeri, respectively. These two isolates tolerated phenol up to 1800mgL-1 and cyanide up to 340mgL-1 concentrations. The isolates were immobilized on activated charcoal, saw dust and fly ash. The effluent was passed through the column packed with immobilized cells with a flow rate of 5mLmin-1. The isolates showed degradation of phenol up to 80.5% and cyanide up to 80.6% and also had the ability to reduce biological oxygen demand, chemical oxygen demand and lower the pH of effluent from alkaline to near neutral. The study suggests the utilization of such potential bacterial strains in treating industrial effluent containing phenol and cyanide, before being thrown in any ecosystem.

Characterization of a novel bioemulsifier from Pseudomonas stutzeri.

This study describes a novel and efficient alasan-like bioemulsifier produced by Pseudomonas stutzeri NJtech 11-1, which was isolated from the Shengli Oilfield. The strain was found to produce a new and interesting emulsion stabilizer. The crude bioemulsifier showed super stability with 50% salinity and broad pH 3-10. The emulsion index (EI24) was increased to 100% after heating from 45 to 95 °C and the emulsion could be stable for at least 30 days. The yield of Ps-bioemulsifier (pure bioemulsifier) was 0.68 ± 0.05 mg mL-1. The Ps-bioemulsifier was composed of carbohydrates (80 ± 2.6%) and proteins (9.5 ± 0.5%). A low concentration (0.2 mg mL-1) of the Ps-bioemulsifier was obtained maximum emulsifying activity at pH 7.1 and its emulsifying activity strengthened by suitable salinity. Furthermore, Ps-bioemulsifier could also emulsify cyclohexane, hexadecane, kerosene, xylene hydrocarbons efficiently. Therefore, the Ps-bioemulsifier showed emulsifying characteristics which make it a good candidate for potential applications in bioremediation and microbial enhanced oil recovery.

Partial structure and immunological properties of lipopolysaccharide from marine-derived Pseudomonas stutzeri KMM 226.

The partial structure and immunology of the lipopolysaccharide (LPS) of Pseudomonas stutzeri KMM 226, a bacterium isolated from a seawater sample collected at a depth of 2000 m, was characterised. The O-polysaccharide was built up of disaccharide repeating units constituted by L-Rhap and D-GlcpNAc: →2)-α-L-Rhap-(1→3)-α-D-GlcpNAc-(1→. The structural analysis of the lipid A showed a mixture of different species. The major species were hexa-acylated and penta-acylated lipids A, bearing the 12:0(3-OH) in amide linkage and 10:0(3-OH) in ester linkage, while the secondary fatty acids were present only as 12:0. The presence of 12:0(2-OH) was not detected. The immunology experiments demonstrated that P. stutzeri KMM 226 LPS displayed a low ability to induce TNF-α, IL-1ß, IL-6, IL-8 and IL-10 cytokine production and acted as an antagonist of hexa-acylated Escherichia coli LPS in human blood in vitro.

Microbial community shift in a suspended stuffing biological reactor with pre-attached aerobic denitrifier.

Bioaugmentation is substantially determined by pre-attached communities in biological stuffing systems. However, the inevitable changes of microbial community shift occurred between pre-attached microorganisms on stuffing material and other existing communities in wastewater. Targeting at nitrogen removal in aerobic denitrification reactors, biological augmentation was built by polyurethane supporting material and aerobic denitrification bacteria of Pseudomonas stutzeri strains were primarily colonized. The total nitrogen removal reached a high efficiency of 77 ± 6%, resulting from a relative high nitrate removal (90%) and a low nitrite production of 24 mg l-1. The nitrate removal was kept 10% higher using preattached strains than that using wastewater communities. During the bioaugmentation process, abundant bacteria related to nitrogen removal were evolutively enriched to compete with preattached Pseudomonas stutzeri. The most abundant bacteria growing up in the biofilm belonged to various Classes of Proteobacteria Phylum. A noticeable nitrite production with a relative low TN removal efficiency occurred when Brucella sp. and Brevundimonas sp. were simultaneously enriched in place of Pseudomonas, because Brevundimonas also accumulated nitrite during denitrification under an aerobic condition. The results indicated that pre-attached denitrifiers in comprehensive communities on stuffing material can be established for the efficient nitrogen and COD removal in aerobic denitrification reactors.

Functional redundancy in phenol and toluene degradation in Pseudomonas stutzeri strains isolated from the Baltic Sea.

In the present study we describe functional redundancy of bacterial multicomponent monooxygenases (toluene monooxygenase (TMO) and toluene/xylene monooxygenase (XylAM) of TOL pathway) and cooperative genetic regulation at the expression of the respective catabolic operons by touR and xylR encoded regulatory circuits in five phenol- and toluene-degrading Pseudomonas stutzeri strains. In these strains both toluene degradation pathways (TMO and Xyl) are active and induced by toluene and phenol. The whole genome sequence of the representative strain 2A20 revealed the presence of complete TMO- and Xyl-upper pathway operons together with two sets of lower catechol meta pathway operons, as well as phenol-degrading operon in a single 292,430bp contig. The much lower GC content and analysis of the predicted ORFs refer to the plasmid origin of the approximately 130kb region of this contig, containing the xyl, phe and tou genes. The deduced amino acid sequences of the TMO, XylA and the large subunit of phenol monooxygenase (LmPH) show 98-100% identity with the respective gene products of the strain Pseudomonas sp. OX1. In both strains 2A20 and OX1 the meta-cleavage pathways for catechol degradation are coded by two redundant operons (phe and xyl). We show that in the strain 2A20 TouR and XylR are activated by different effector molecules, phenol and toluene, respectively, and they both control transcription of the xyl upper, tou (TMO) and phe catabolic operons. Although the growth parameters of redundant strains did not show advantage at toluene biodegradation, the functional redundancy could provide better flexibility to the bacteria in environmental conditions.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium.

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Aerobic denitrification by Pseudomonas stutzeri C3 incapable of heterotrophic nitrification.

An aerobic denitrifier was isolated from activated sludge and the isolate possessed an average nitrate removal efficiency of 95.8% in 24 h, with the maximum nitrite accumulation of 25.6 mg/l. The bacterium could not conduct heterotrophic nitrification. The results of PCR indicated that the strain lacked the gene of amoA. The phylogenetic tree indicated a clear evolutionary divergence that Pseudomonas stutzeri C3 belonged to the group of strains that could not exhibit ammonia oxidation.

Biotransformation of Tributyltin chloride by Pseudomonas stutzeri strain DN2

A bacterial isolate capable of utilizing tributyltin chloride (TBTCl) as sole carbon source was isolated from estuarine sediments of west coast of India and identified as Pseudomonas stutzeri based on biochemical tests and Fatty acid methyl ester (FAME) analysis. This isolate was designated as strain DN2. Although this bacterial isolate could resist up to 3 mM TBTCl level, it showed maximum growth at 2 mM TBTCl in mineral salt medium (MSM). Pseudomonas stutzeri DN2 exposed to 2 mM TBTCl revealed significant alteration in cell morphology as elongation and shrinkage in cell size along with roughness of cell surface. FTIR and NMR analysis of TBTCl degradation product extracted using chloroform and purified using column chromatography clearly revealed biotransformation of TBTCl into Dibutyltin dichloride (DBTCl2) through debutylation process. Therefore, Pseudomonas stutzeri strain DN2 may be used as a potential bacterial strain for bioremediation of TBTCl contaminated aquatic environmental sites.

Genomovar assignment of Pseudomonas stutzeri populations inhabiting produced oil reservoirs.

Oil reservoirs are specific habitats for the survival and growth of microorganisms in general. Pseudomonas stutzeri which is believed to be an exogenous organism inoculated into oil reservoirs during the process of oil production was detected frequently in samples from oil reservoirs. Very little is known, however, about the distribution and genetic structure of P. stutzeri in the special environment of oil reservoirs. In this study, we collected 59 P. stutzeri 16S rRNA gene sequences that were identified in 42 samples from 25 different oil reservoirs and we isolated 11 cultured strains from two representative oil reservoirs aiming to analyze the diversity and genomovar assignment of the species in oil reservoirs. High diversity of P. stutzeri was observed, which was exemplified in the detection of sequences assigned to four known genomovars 1, 2, 3, 20 and eight unknown genomic groups of P. stutzeri. The frequent detection and predominance of strains belonging to genomovar 1 in most of the oil reservoirs under study indicated an association of genomovars of P. stutzeri with the oil field environments.

Biotransformation of Tributyltin chloride by Pseudomonas stutzeri strain DN2.

A bacterial isolate capable of utilizing tributyltin chloride (TBTCl) as sole carbon source was isolated from estuarine sediments of west coast of India and identified as Pseudomonas stutzeri based on biochemical tests and Fatty acid methyl ester (FAME) analysis. This isolate was designated as strain DN2. Although this bacterial isolate could resist up to 3 mM TBTCl level, it showed maximum growth at 2 mM TBTCl in mineral salt medium (MSM). Pseudomonas stutzeri DN2 exposed to 2 mM TBTCl revealed significant alteration in cell morphology as elongation and shrinkage in cell size along with roughness of cell surface. FTIR and NMR analysis of TBTCl degradation product extracted using chloroform and purified using column chromatography clearly revealed biotransformation of TBTCl into Dibutyltin dichloride (DBTCl2) through debutylation process. Therefore, Pseudomonas stutzeri strain DN2 may be used as a potential bacterial strain for bioremediation of TBTCl contaminated aquatic environmental sites.

Whole-cell MALDI-TOF mass spectrometry and multilocus sequence analysis in the discrimination of Pseudomonas stutzeri populations: three novel genomovars.

Pseudomonas stutzeri is a widely distributed species with very high genetic diversity and metabolic capacities, occupying many diverse ecological niches. A collection of 229 P. stutzeri strains isolated from different habitats and geographical locations has been previously characterised phylogenetically by rpoD gene sequencing analysis and in the present study 172 of them phenotypically by whole-cell MALDI-TOF mass spectrometry. Fifty-five strains were further analysed by multilocus sequencing analysis to determine the phylogenetic population structure. Both methods showed coherence in strain grouping; 226 strains were allocated in the 18 genomovars known presently. The remaining three strains are proposed as references for three novel genomovars in the species. The correlation and usefulness of sequence-based phylogenetic analysis and whole-cell MALDI-TOF mass spectrometry, which are essential for autoecological studies in microbial ecology, is discussed for the differentiation of P. stutzeri populations.

Application potential of a newly isolated indigenous aerobic denitrifier for nitrate and ammonium removal of eutrophic lake water.

The aim of this work was to evaluate the utilization potential of a newly isolated indigenous aerobic denitrifier, Pseudomonas stutzeri strain T1, for nitrogen removal from the eutrophic Lake Taihu in China. The strain was capable of conducting heterotrophic nitrification-aerobic denitrification and had both excellent nitrate and ammonium removal without nitrite build-up. The characteristics of P. stutzeri strain T1 were studied under different cultural conditions. Furthermore, under the optimized cultivation conditions, strain T1 was added into the water samples from Lake Taihu, the ammonium and nitrate removal rates of the strain reached to 60% and 75%, respectively. Via adding this strain, the water qualities of the sample ameliorated from Grade V to Grade II. Thus, the strain T1 should be an useful biological tool to remediate eutrophic lakes and do not meet acclimation problems.

Removal of 2,4,5-trichlorophenol by bacterial isolates from the secondary sludge of pulp and paper mill.

2,4,5-trichlorophenol (2,4,5-TCP) mineralizing bacteria were isolated from the secondary sludge of pulp and paper industry. These isolates used 2,4,5-TCP as a source of carbon and energy and were capable of degrading this compound, as indicated by stoichimetric release of chloride and biomass formation. Based on 16S rRNA sequence analysis, these bacteria were identified as Kocuria sp. (CL2), Bacillus pumillus (CL5), Pseudomonas stutzeri (CL7). HPLC analysis revealed that these isolates were able to degrade 2,4,5-TCP at higher concentrations (600 mg/l or 3.0 mM). A consortia of these isolates completely removed 2,4,5-TCP from the sludge obtained from pulp and paper mill within 2 weeks when supplemented at a rate of 100 mg l(-1) . Bacterial consortium also significantly reduced absorbable organic halogen (AOX) and extractable organic halogen (EOX) by 61% and 63%, respectively from the sludge. These isolates have high potential to remove 2,4,5-TCP and may be used for remediation of pulp paper mill waste containing 2,4,5-TCP.

Coal induced production of a rhamnolipid biosurfactant by Pseudomonas stutzeri, isolated from the formation water of Jharia coalbed.

A strain of Pseudomonas stutzeri was isolated form an enrichment of perchlorate reducing bacteria from the formation water collected from an Indian coalbed which solubilized coal and produced copious amount of biosurfactant when coal was added to the medium. It produced maximum biosurfactant with lignite coal followed by olive oil and soybean oil which was able to emulsify several aromatic hydrocarbons including kerosene oil, diesel oil, hexane, toluene etc. Haemolytic test, growth inhibition of Bacillus subtilis and FTIR analysis showed rhamnolipid nature of the biosurfactant. The stability of the coal induced biosurfactant in pH range of 4-8 and up to 25% NaCl concentration and 100 °C temperature suggests that due to its ability to produce biosurfactant and solubilize coal P. stutzeri may be useful in the coalbed for in situ biotransformation of coal into methane and in the bioremediation of PAHs from oil contaminated sites including marine environments.

Nicotine degradation enhancement by Pseudomonas stutzeri ZCJ during aging process of tobacco leaves.

Nicotine is a key harmful component of tobacco and cigarettes, and the development of low-nicotine cigarettes is of increasing importance in the market. The objectives of this study are to isolate native nicotine-degrading strains and evaluate their feasibility for nicotine reduction during the aging (or fermentation) of tobacco leaves. A novel nicotine-degrading strain was isolated and identified as Pseudomonas stutzeri ZCJ based on its 16S rDNA sequence and morphological-biochemical characteristics. In submerged cultures, P. stutzeri ZCJ could tolerate 4.5 g/L nicotine and completely degrade 1.5 g/L nicotine within 24 h at 37°C and pH 7.4. The addition of glucose (1 g/L) could improve nicotine degradation by P. stutzeri ZCJ in submerged cultures. After submerged culturing, the cell suspension of P. stutzeri ZCJ could be utilized to improve nicotine reduction in tobacco leaves during solid-state fermentation. The nicotine content of tobacco leaves decreased by as much as 32.24% after 7 days of solid-state fermentation by P. stutzeri ZCJ, suggesting the industrial application potential of the native strain to enhance nicotine degradation during the aging of tobacco leaves.

Identification and genomovar assignation of clinical strains of Pseudomonas stutzeri.

The identification of Pseudomonas stutzeri clinical isolates through conventional phenotypic methods was compared with identification through partial rpoD gene sequencing. We observed that commercial phenotypic systems easily confuse P. stutzeri with other Pseudomonas species. We also demonstrated that most of the clinical strains of P. stutzeri herein studied (79%) belonged to genomovar 1 of the species. We propose the use of partial rpoD gene sequence analysis as a complementary molecular tool for the precise routine identification and genomovar assignation of P. stutzeri clinical isolates, as well as for typing and epidemiological studies.