细菌脱色酶TpmD对三苯基甲烷类染料脱色的酶学特性研究

从嗜水气单胞菌DN322中分离纯化出能够对三苯基甲烷类染料结晶紫、碱性品红、灿烂绿及孔雀绿进行有效脱色的脱色酶,命名为TpmD。该酶的亚基分子量为29.4kDa,等电点为5.6。该酶催化上述4种三苯基甲烷类染料脱色反应的适合温度为40~60℃,适合pH范围为5.5~9.0。动力学参数测定结果显示TpmD对结晶紫、碱性品红、灿烂绿及孔雀绿的Km值分别为24.3、40.65、4.2、68.5μmol-1.L-1,Vmax值分别为19.6、74.1、82.8、115.6μmol.L-1.s-1。结晶紫为该酶的最适反应底物。TpmD催化的脱色反应依懒于NADH/NADPH及分子氧的存在,显示该酶属于NADH/NADPH依赖型的氧化酶类。这是国内外首次关于细菌中三苯基甲烷类染料脱色酶酶学性质的描述。     

细菌脱色酶TpmD的酶学特性研究

从嗜水气单胞菌DN322中分离纯化出能够对三苯基甲烷类染料结晶紫、碱性品红、灿烂绿及孔雀绿进行高效脱色的脱色酶,命名为TpmD。在测定TpmD分子量、等电点及对不同三苯基甲烷染料脱色的动力学参数、脱色过程对分子氧及NADH/NADPH具有依赖性的基础上,又进一步从黄素FAD/FMN对酶活力的影响、酶抑制剂、酶蛋白N-末端测序及酶溶液的特征吸收光谱等方面对TpmD的酶学本质进行了分析。结果表明,TpmD不含核黄素,其脱色活性也不因加入FAD或FMN而提高。TpmD的N-末端氨基酸序列与多种氧化还原酶具有同源性。甲吡酮及维生素C(Vc)对TpmD的脱色活性具有明显的抑制作用。TpmD酶蛋白的溶液在408nm处有一特征吸收峰,但在连二亚硫酸钠的还原条件下通入CO气体后,该酶却不具有P450酶在450nm处的特征吸收峰。上述结果显示脱色酶TpmD是一种新的氧化酶。     

Accelerated decolorization of structurally different azo dyes by newly isolated bacterial strains

Wastewater effluents from the textile and other dye-stuff industries contain significant amounts of synthetic dyes that require treatment to prevent groundwater contamination. In research aimed at biotechnology for treatment of azo dyes, this study examined 288 strains of azo-dye degrading bacteria to identify efficient strains and determine incubation times required for decolorization. Initial enrichment cultures were carried out using a mixture of four structurally different dyes (Acid Red 88, Reactive Black 5, Direct Red 81, and Disperse Orange 3) as the sole source of C and N to isolate the bacteria from soil, activated sludge, and natural asphalt. Six strains were selected for further study based on their prolific growth and ability to rapidly decolorize the dyes individually or in mixtures. Treatment times required by the most efficient strain, AS96 (Shewanella putrefaciens) were as short as 4 h for complete decolorization of 100 mg l⁻¹ of AR-88 and DR-81 dyes under static conditions, and 6 and 8 h, respectively, for complete decolorization of RB-5 and DO-3. To our knowledge, these bacterial strains are the most efficient azo-dye degrading bacteria that have been described and may have practical application for biological treatment of dye-polluted wastewater streams.     

Genetic diversity and composition of a plasmid metagenome from a wastewater treatment plant

Plasmid metagenome nucleotide sequence data were recently obtained from wastewater treatment plant (WWTP) bacteria with reduced susceptibility to selected antimicrobial drugs by applying the ultrafast 454-sequencing technology. The sequence dataset comprising 36,071,493 bases (346,427 reads with an average read length of 104 bases) was analysed for genetic diversity and composition by using a newly developed bioinformatic pipeline based on assignment of environmental gene tags (EGTs) to protein families stored in the Pfam database. Short amino acid sequences deduced from the plasmid metagenome sequence reads were compared to profile hidden Markov models underlying Pfam. Obtained matches evidenced that many reads represent genes having predicted functions in plasmid replication, stability and plasmid mobility which indicates that WWTP bacteria harbour genetically stabilised and mobile plasmids. Moreover, the data confirm a high diversity of plasmids residing in WWTP bacteria. The mobile organic peroxide resistance plasmid pMAC from Acinetobacter baumannii was identified as reference plasmid for the most abundant replication module type in the sequenced sample. Accessory plasmid modules encode different transposons, insertion sequences, integrons, resistance and virulence determinants. Most of the matches to Transposase protein families were identified for transposases similar to the one of the chromate resistance transposon Tn5719. Noticeable are hits to beta-lactamase protein families which suggests that plasmids from WWTP bacteria encode different enzymes possessing beta-lactam-hydrolysing activity. Some of the sequence reads correspond to antibiotic resistance genes that were only recently identified in clinical isolates of human pathogens. EGT analysis thus proofed to be a very valuable method to explore genetic diversity and composition of the present plasmid metagenome dataset.     

Impact of operational parameters on bacterial community in a full-scale municipal wastewater treatment plant

A bacterial community in activated sludge from a full-scale municipal wastewater treatment plant was monitored throughout the year with the use of FISH, RISA and DGGE techniques. In the investigated range of temperatures (11.9-21.6 degrees C), a rise in temperature resulted in a lower total bacteria richness, while organic load rate changes from 0.09 to 0.21 g COD x g TSS(-1) x d(-1) were positively correlated with the number of bands in RISA patterns. The most diverse pattern (29 different bands) was characteristic for the activated sludge sample collected at the end of January at wastewater temperature of 11.9 degrees C. The ammonia-oxidising bacteria community did not change during the study, and comprised of 4 different bacterial populations with one dominant species closely related to Nitrosospira sp. REGAU (GenBank accession number AY635572.1). The percentage of ammonia-oxidising bacteria in the activated sludge varied from 6.2 to 19.5% and depended on temperature (R = 0.61, p = 0:02) and organic load rate (R = -0.55, p = 0.04).     

Carbapenem-hydrolyzing GES-5-encoding gene on different plasmid types recovered from a bacterial community in a sewage treatment plant

Plasmids pRSB113 and pRSB115 were recovered from an activated sludge bacterial community of a municipal wastewater treatment plant in Germany. Both plasmids carry the same bla(GES-5) carbapenemase gene, located within two distinct class 1 integrons. These plasmids have different backbones, belong to different incompatibility groups, and could replicate in both Pseudomonas aeruginosa and Escherichia coli.     

All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant

The presence and diversity of IncP-1 plasmids in the influent of a Danish wastewater treatment plant was studied by PCR amplification of the trfA gene in community DNA followed by sequencing. Three sets of PCR primers were designed to amplify a 281 bp fragment of trfA from all currently sequenced IncP-1 plasmids. A neighbor-joining tree, based on a multiple alignment of 72 obtained sequences together with homologous sequences of previously published IncP-1 plasmids, revealed that all established subgroups of IncP-1 plasmids, α, β, γ and δ, were present in the wastewater treatment plant influent. Also sequences representing the recently described fifth subgroup, the ε subgroup, were detected in the wastewater. Thus, these results confirm the presence of at least five phylogenetically distinct subgroups of IncP-1 plasmids and represent the first time that sequences associated with plasmids of all of these five subgroups have been detected in a single setting. Additionally, the results confirm that wastewater constitutes a reservoir for the conjugative IncP-1 plasmids, which often harbor multiple antibiotic resistance genes.     

Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool

The dramatic spread of antibiotic resistance is a crisis in the treatment of infectious diseases that affect humans. Several studies suggest that wastewater treatment plants (WWTP) are reservoirs for diverse mobile antibiotic resistance elements. This review summarizes findings derived from genomic analysis of IncP-1 resistance plasmids isolated from WWTP bacteria. Plasmids that belong to the IncP-1 group are self-transmissible, and transfer to and replicate in a wide range of hosts. Their backbone functions are described with respect to their impact on vegetative replication, stable maintenance and inheritance, mobility and plasmid control. Accessory genetic modules, mainly representing mobile genetic elements, are integrated in-between functional plasmid backbone modules. These elements carry determinants conferring resistance to nearly all clinically relevant antimicrobial drug classes, to heavy metals, and quaternary ammonium compounds used as disinfectants. All plasmids analysed here contain integrons that potentially facilitate integration, exchange and dissemination of resistance gene cassettes. Comparative genomics of accessory modules located on plasmids from WWTP and corresponding modules previously identified in other bacterial genomes revealed that animal, human and plant pathogens and other bacteria isolated from different habitats share a common pool of resistance determinants.     

IncP-1β Plasmid pGNB1 Isolated from a Bacterial Community from a Wastewater Treatment Plant Mediates Decolorization of Triphenylmethane Dyes

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1β plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the α-proteobacterium Sinorhizobium meliloti and the γ-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1β plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.     

Anaerobic biodegradation of triphenylmethane dyes in a hybrid UASFB reactor for wastewater remediation

Anaerobic digestions have been proved more successful than aerobic systems for the degradation and destruction of dye-containing wastewaters. The performance of a hybrid up flow anaerobic sludge-filter bed (UASFB) reactor was tested with a synthetic wastewater containing Crystal violet (CV) as a carbon source and sodium acetate as a co-substrate. Continuous feeding of the reactor started with an initial OLR of 0.9 g COD/l-d and then it was increased step wise to 4 g COD l⁻¹ d⁻¹, while maintaining constant HRT (24 h). The optimum pH value and temperature for decolorization of crystal violet by this mixed culture species under anaerobic conditions were found to be 8–9 and 30–35°C respectively. N,N-dimethylaminophenol and N,N-bis (dimethylamino) benzophenone (Michler’s Ketone) were detected as the degradative metabolites of Crystal Violet. Subsequently, N,N-dimethylaminophenol was further degraded to aniline in the reactor whereas Michler’s ketone was not degraded under anaerobic conditions. The UASFB bioreactor was able to remove the CV completely up to a loading rate of 100 mg CV l⁻¹d⁻¹.     

Impact of conjugal transfer on the stability of IncP-1 plasmid pKJK5 in bacterial populations

The intrinsic stability of IncP-1 plasmid pKJK5 was assessed in both an Escherichia coli and a Kluyvera sp. population maintained in bacterial mats and in liquid nutrient broth without selective pressure. A fluorescence tagging/flow cytometry approach was used to detect and quantify plasmid loss from populations harboring either conjugation-proficient or -deficient pKJK5 derivatives. The results show that the plasmid's ability to conjugate plays an important role in its stable maintenance in populations of both species. This effect was most pronounced in dense bacterial populations and to a far lesser extent during growth in liquid broth. Furthermore, conjugation-proficient plasmids were able to spread infectiously in the bacterial mats initiated with various ratios of plasmid-harboring cells, resulting in a nearly exclusively plasmid-harboring population.     

Peroxidase mediated decolorization and remediation of wastewater containing industrial dyes: a review

In this article an effort has been made to review literature based on the role of peroxidases in the treatment and decolorization of a wide spectrum aromatic dyes from polluted water. Peroxidases can catalyze degradation/transformation of aromatic dyes either by precipitation or by opening the aromatic ring structure. Peroxidases from plant sources; horseradish, turnip, tomato, soybean, bitter gourd, white radish and Saccharum uvarum and microbial sources; lignin peroxidases, manganese peroxidases, vanadium haloperoxidases, versatile peroxidases, dye decolorizing peroxidases have been employed for the remediation of commercial dyes. Soluble and immobilized peroxidases have been successfully exploited in batch as well as in continuous processes for the treatment of synthetic dyes with complex aromatic molecular structures present in industrial effluents at large scale. However, recalcitrant dyes were also decolorized by the action of peroxidases in the presence of redox mediators.     

Modification of norfloxacin by a Microbacterium sp. strain isolated from a wastewater treatment plant

Antimicrobial residues found in municipal wastewater may increase selective pressure on microorganisms for development of resistance, but studies with mixed microbial cultures derived from wastewater have suggested that some bacteria are able to inactivate fluoroquinolones. Medium containing N-phenylpiperazine and inoculated with wastewater was used to enrich fluoroquinolone-modifying bacteria. One bacterial strain isolated from an enrichment culture was identified by 16S rRNA gene sequence analysis as a Microbacterium sp. similar to a plant growth-promoting bacterium, Microbacterium azadirachtae (99.70%), and a nematode pathogen, "M. nematophilum" (99.02%). During growth in medium with norfloxacin, this strain produced four metabolites, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) analyses as 8-hydroxynorfloxacin, 6-defluoro-6-hydroxynorfloxacin, desethylene norfloxacin, and N-acetylnorfloxacin. The production of the first three metabolites was enhanced by ascorbic acid and nitrate, but it was inhibited by phosphate, amino acids, mannitol, formate, and thiourea. In contrast, N-acetylnorfloxacin was most abundant in cultures supplemented with amino acids. This is the first report of defluorination and hydroxylation of a fluoroquinolone by an isolated bacterial strain. The results suggest that some bacteria may degrade fluoroquinolones in wastewater to metabolites with less antibacterial activity that could be subject to further degradation by other microorganisms.     

Ammonia oxidizing bacteria community dynamics in a pilot-scale wastewater treatment plant

Background

Chemoautotrophic ammonia oxidizing bacteria (AOB) have the metabolic ability to oxidize ammonia to nitrite aerobically. This metabolic feature has been widely used, in combination with denitrification, to remove nitrogen from wastewater in wastewater treatment plants (WWTPs). However, the relative influence of specific deterministic environmental factors to AOB community dynamics in WWTP is uncertain. The ecological principles underlying AOB community dynamics and nitrification stability and how they are related are also poorly understood.

Methodology/Principal Findings

The community dynamics of ammonia oxidizing bacteria (AOB) in a pilot-scale WWTP were monitored over a one-year period by Terminal Restriction Fragment Length Polymorphism (T-RFLP). During the study period, the effluent ammonia concentrations were almost below 2 mg/L, except for the first 60 days, indicting stable nitrification. T-RFLP results showed that, during the test period with stable nitrification, the AOB community structures were not stable, and the average change rate (every 15 days) of AOB community structures was 10%±8%. The correlations between T-RFLP profiles and 10 operational and environmental parameters were tested by Canonical Correlation Analysis (CCA) and Mantel test. The results indicated that the dynamics of AOB community correlated most strongly with Dissolved Oxygen (DO), effluent ammonia, effluent Biochemical Oxygen Demand (BOD) and temperature.

Conclusions/Significance

This study suggests that nitrification stability is not necessarily accompanied by a stable AOB community, and provides insight into parameters controlling the AOB community dynamics within bioreactors with stable nitrification.     

Bacterial community dynamics in a functionally stable pilot-scale wastewater treatment plant

To determine whether functional stability was correlated with a stable microbial community structure in a functionally stable pilot-scale wastewater treatment plant, bacterial communities in the system were monitored over a one-year period. Bacterial community dynamics was characterized by the terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes. During the study period, the effluent BOD concentrations were very stable, with the average BOD concentration below 10 mg/L. The effluent TN concentrations were always below 20 mg/L, except for the first 40 days. T-RFLP results showed that, during the test period, the bacterial community structures were not stable, with an average change rate (every 15 days) of 20.4% ± 11.2%. Based on Lorenz distribution curves, it was observed that 20% of the species corresponded with 40-77% of cumulative relative abundances. Results clearly revealed that, in the pilot-scale wastewater treatment plant, functional stability did not correlate with stable bacterial communities.     

Microbial community origin and fate through a rural wastewater treatment plant

Conventional wastewater treatment relies on a complex microbiota; however, much of this community is still to be characterized. To better understand the origin, dynamics and fate of bacteria within a wastewater treatment plant: untreated primary wastewater, activated sludge and post-treatment effluent were characterized. From 3163 exact sequence variants (ESVs), 860 were annotated to species-level. In primary wastewater, 28% of ESVs were putative bacterial species previously associated with humans, 14% with animals and 5% as common to the environment. Differential abundance analysis revealed significant relative reductions in ESVs from potentially human-associated species from primary wastewater to activated sludge, and significant increases in ESVs from species associated with nutrient cycling. Between primary wastewater and effluent, 51% of ESVs from human-associated species did not significantly differ, and species such as Bacteroides massiliensis and Bacteroides dorei increased. These findings illustrate that activated sludge increased extracellular protease and urease-producing species, ammonia and nitrite oxidizers, denitrifiers and specific phosphorus accumulators. Although many human-associated species declined, some persisted in effluent, including strains of potential health or environmental concern. Species-level microbial assessment may be useful for understanding variation in wastewater treatment efficiency as well as for monitoring the release of microbes into surface water and the wider ecosystem.     

Exploring characteristics of bioelectricity generation and dye decolorization of mixed and pure bacterial cultures from wine-bearing wastewater treatment

This study uncovered microbial characteristics of bioelectricity generation and dye decolorization in single-chamber microbial fuel cells (MFCs) using activated sludge for wine-containing wastewater treatment. Phylogenetic tree analysis on 16S rRNA gene fragments indicated that the predominant strains on anodic biofilm in acclimatized MFCs were Gamma-Proteobacteria Aeromonas punctata NIU-P9, Pseudomonas plecoglossicida NIU-Y3, Pseudomonas koreensis NIU-X8, Acinetobacter junii NIU-Y8, Stenotrophomonas maltophila NIU-X2. Our findings showed that the current production capabilities of these pure strains were only ca. 10% of those of their mother activated sludge, indicating that synergistic interactions among microbes might be the most influential factor to maximize power generation in MFCs. Plus, these electrochemically active strains also performed reductive decolorization of C.I. reactive blue 160, suggesting that bioelectricity generation might be directly associated to azo dye decolorization to deal with electron transfer on anodic biofilm in MFCs.     

Antimicrobial resistance patterns in Enterobacteriaceae isolated from an urban wastewater treatment plant

Over 18 months, enterobacteria were isolated from the raw (189 isolates) and treated (156 isolates) wastewater of a municipal treatment plant. The isolates were identified as members of the genera Escherichia (76%), Shigella (7%), Klebsiella (12%) and Acinetobacter (4%). Antimicrobial susceptibility phenotypes were determined using the agar diffusion method for the antibiotics amoxicillin, gentamicin, ciprofloxacin, sulfamethoxazole/trimethoprim, tetracycline and cephalothin, the disinfectants hydrogen peroxide, sodium hypochlorite, quaternary ammonium/formaldehyde and iodine, and the heavy metals nickel, cadmium, chromium, mercury and zinc. Class 1 integrons were detected by PCR amplification using the primers CS5 and CS3. Compared with the raw influent, the treated wastewater presented higher relative proportions of Escherichia spp. isolates resistant to ciprofloxacin and cephalothin (P<0.0001 and P<0.05, respectively). Except for mercury, which showed a positive correlation with tetracycline and sulfamethoxazole/trimethoprim, no significant positive correlations were observed between antibiotic, disinfectant and heavy metal resistance. The variable regions of class 1 integrons, detected in c. 10% of the Escherichia spp. isolates, contained predominantly the gene cassettes aadA1/dhfrI.     

Sequence of the 68,869 bp IncP-1alpha plasmid pTB11 from a waste-water treatment plant reveals a highly conserved backbone, a Tn402-like integron and other transposable elements

To analyse the significance of conjugative broad-host-range IncP-1alpha plasmids for the spread of antibiotic resistance determinants in waste-water treatment plants we isolated and characterised five different IncP-1alpha plasmids from bacteria of activated sludge and the final effluents of a municipal waste-water treatment plant. These plasmids mediate resistance to ampicillin, cefaclor, cefuroxime, gentamicin, kanamycin, spectinomycin, streptomycin, tetracycline, tobramycin, and trimethoprim. The complete 68,869 bp DNA-sequence of the IncP-1alpha plasmid pTB11 was determined. The pTB11 backbone modules for replication (Rep), mating pair formation (Trb), multimer resolution (Mrs), post-segregational killing (Psk), conjugative DNA-transfer (Tra), plasmid control (Ctl), and stable maintenance and inheritance (KilA, KilE, and KilC) are highly conserved as compared to the 'Birmingham' IncP-1alpha plasmids. In contrast to the 'Birmingham' plasmids pTB11 carries an insert of a Tn402-derivative integrating a class 1 integron in the intergenic region between the multimer resolution operon parCBA and the post-segregational killing operon parDE. The integron comprises the resistance gene cassettes oxa2 (beta-lactamase), aacA4 (aminoglycoside-6'N-acetyltransferase), and aadA1 (aminoglycoside-3'-adenylyltransferase) and a complete tniABQR transposition module. Integron-specific sequences were also identified on other IncP-1alpha plasmids analysed in this work. In contrast to the 'Birmingham' plasmids the pTB11 tetracycline resistance module carries a pecM- and a pncA-like gene downstream of the tetracycline resistance gene tetA and contains an insertion of the new insertion sequence element ISTB11. The transposable elements IS21 and Tn1 which disrupted, respectively, orf7 and klcB on the 'Birmingham' plasmids are not present on pTB11. Identification of IncP-1alpha plasmids in bacteria of the waste-water treatment plant's final effluents indicates that bacteria carrying these kind of plasmids are released into the environment.     

Comamonas granuli sp. nov., isolated from granules used in a wastewater treatment plant

A Gram-negative, motile, rod-shaped, non-spore-forming bacterial strain, designated as Ko03(T), was isolated from microbial granules, and was characterized, using a polyphasic approach, in order to determine its taxonomic position. The isolate was positive for catalase and oxidase, but negative for gelatinase and beta-galactosidase. Phylogenetic analyses using the 16S rRNA gene sequence showed that the strain formed a monophyletic branch towards the periphery of the evolutionary radiation occupied by the genus Comamonas, its closest neighbors being Comamonas koreensis KCTC 12005(T) (95.9% sequence similarity), Comamonas nitrativorans DSM 13191(T) (95.7%), and Comamonas odontotermitis LMG 23579(T) (95.7%). Strain Ko03(T) had a genomic DNA G+C content of 68.4 mol% and the predominant respiratory quinone was Q-8. The major fatty acids were C(16:1) omega7c (44.7%), C(16:0) (28.1%), C(18:1) (16.1%), and C(10:0) 3-OH (3.5%). These chemo-taxonomic results supported the affiliation of strain Ko03(T) to the genus Comamonas. However, low 16S rRNA gene sequence similarity values and distinguishing phenotypic characteristics allowed genotypic and phenotypic differentiation of strain Ko03(T) from recognized Comamonas species. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Ko03(T) represents a novel species of the genus Comamonas, for which the name Comamonas granuli sp. nov. is proposed. The type strain is Ko03(T) (= KCTC 12199(T) = NBRC 101663(T)).