The metagenome of a biogas-producing microbial community of a production-scale biogas plant fermenter analysed by the 454-pyrosequencing technology

Composition and gene content of a biogas-producing microbial community from a production-scale biogas plant fed with renewable primary products was analysed by means of a metagenomic approach applying the ultrafast 454-pyrosequencing technology. Sequencing of isolated total community DNA on a Genome Sequencer FLX System resulted in 616,072 reads with an average read length of 230 bases accounting for 141,664,289 bases sequence information. Assignment of obtained single reads to COG (Clusters of Orthologous Groups of proteins) categories revealed a genetic profile characteristic for an anaerobic microbial consortium conducting fermentative metabolic pathways. Assembly of single reads resulted in the formation of 8752 contigs larger than 500 bases in size. Contigs longer than 10kb mainly encode house-keeping proteins, e.g. DNA polymerase, recombinase, DNA ligase, sigma factor RpoD and genes involved in sugar and amino acid metabolism. A significant portion of contigs was allocated to the genome sequence of the archaeal methanogen Methanoculleus marisnigri JR1. Mapping of single reads to the M. marisnigri JR1 genome revealed that approximately 64% of the reference genome including methanogenesis gene regions are deeply covered. These results suggest that species related to those of the genus Methanoculleus play a dominant role in methanogenesis in the analysed fermentation sample. Moreover, assignment of numerous contig sequences to clostridial genomes including gene regions for cellulolytic functions indicates that clostridia are important for hydrolysis of cellulosic plant biomass in the biogas fermenter under study. Metagenome sequence data from a biogas-producing microbial community residing in a fermenter of a biogas plant provide the basis for a rational approach to improve the biotechnological process of biogas production.     

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.     

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.     

Bacterial communities in different sections of a municipal wastewater treatment plant revealed by 16S rDNA 454 pyrosequencing

In this study, we successfully demonstrated that 454 pyrosequencing was a powerful approach for investigating the bacterial communities in the activated sludge, digestion sludge, influent, and effluent samples of a full scale wastewater treatment plant treating saline sewage. For each sample, 18,808 effective sequences were selected and utilized to do the bacterial diversity and abundance analysis. In total, 2,455, 794, 1,667, and 1,932 operational taxonomic units were obtained at 3 % distance cutoff in the activated sludge, digestion sludge, influent, and effluent samples, respectively. The corresponding most dominant classes in the four samples are Alphaproteobacteria, Thermotogae, Deltaproteobacteria, and Gammaproteobacteria. About 67 % sequences in the digestion sludge sample were found to be affiliated with the Thermotogales order. Also, these sequences were assigned into a recently proposed genus Kosmotoga by the Ribosomal Database Project classifier. In the effluent sample, we found high abundance of Mycobacterium and Vibrio, which are genera containing pathogenic bacteria. Moreover, in this study, we proposed a method to differentiate the “gene percentage” and “cell percentage” by using Ribosomal RNA Operon Copy Number Database.     

Phosphate removal from the returned liquor of municipal wastewater treatment plant using iron-reducing bacteria

AIM: The application of iron-reducing bacteria (IRB) to phosphate removal from returned liquor (liquid fraction after activated sludge digestion and anaerobic sludge dewatering) of municipal wastewater treatment plant (WWTP) was studied. METHODS AND RESULTS: An enrichment culture and two pure cultures of IRB, Stenotrophomonas maltophilia BK and Brachymonas denitrificans MK identified by 16S rRNA gene sequencing, were produced using returned liquor from a municipal WWTP as carbon and energy source, and iron hydroxide as oxidant. The final concentration of phosphate increased from 70 to 90 mg l(-1) in the control and decreased from 70 to 1 mg l(-1) in the experiment. The mass ratio of removed P to produced Fe(II) was 0.17 g P g(-1) Fe(II). The strain S. maltophilia BK showed the ability to reduce Fe(III) using such xenobiotics as diphenylamine, m-cresol, 2,4-dichlorphenol and p-phenylphenol as sole sources of carbon under anaerobic conditions. CONCLUSIONS: Bacterial reduction of ferric hydroxide enhanced the phosphate removal from the returned liquor. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of the facultative anaerobes S. maltophilia BK and B. denitrificans MK to reduce Fe(III) was shown. These micro-organisms can be used for anaerobic removal of phosphate and xenobiotics by bacterial reduction of ferric ions.     

Acetylation of fluoroquinolone antimicrobial agents by an Escherichia coli strain isolated from a municipal wastewater treatment plant

Aims:  To isolate environmental bacteria capable of transforming fluoroquinolones to inactive molecules.
Methods and Results:  Bacteria were isolated from the aerobic liquor of a wastewater treatment plant on a medium containing norfloxacin (100 mg l⁻¹). Twenty-two isolates were highly resistant (minimal inhibitory concentration: 6·25−200 μg ml⁻¹) to five fluoroquinolones and six of them were positive by PCR amplification for the aminoglycoside resistance gene aac(6')-Ib. Of these, only Escherichia coli strain LR09 had the ciprofloxacin-acetylating variant gene aac(6')-Ib-cr ; HPLC and mass spectrometry showed that this strain transformed both ciprofloxacin and norfloxacin by N -acetylation. This bacterium also had mutations in the quinolone-resistance determining regions of the gyrA and parC genes.
Conclusions:  An E. coli isolate from wastewater, which possessed at least two distinct fluoroquinolone resistance mechanisms, inactivated ciprofloxacin and norfloxacin by N -acetylation.
Significance and Impact of the Study:  This is the first report of N -acetylation of fluoroquinolones by an aac(6')-Ib-cr -containing bacterium from an environmental source.     

The complete genome sequences of four new IncN plasmids from wastewater treatment plant effluent provide new insights into IncN plasmid diversity and evolution

The dissemination of antibiotic resistance genes among bacteria often occurs by means of plasmids. Wastewater treatment plants (WWTP) were previously recognized as hot spots for the horizontal transfer of genetic material. One of the plasmid groups that is often associated with drug resistance is the incompatibility group IncN. The aim of this study was to gain insights into the diversity and evolutionary history of IncN plasmids by determining and comparing the complete genome sequences of the four novel multi-drug resistance plasmids pRSB201, pRSB203, pRSB205 and pRSB206 that were exogenously isolated from the final effluent of a municipal WWTP. Their sizes range between 42,875 bp and 56,488 bp and they share a common set of backbone modules that encode plasmid replication initiation, conjugative transfer, and plasmid maintenance and control. All plasmids are transferable at high rates between Escherichia coli strains, but did not show a broad host range. Different genes conferring resistances to ampicillin, streptomycin, spectinomycin, sulfonamides, tetracycline and trimethoprim were identified in accessory modules inserted in these plasmids. Comparative analysis of the four WWTP IncN plasmids and IncN plasmids deposited in the NCBI database enabled the definition of a core set of backbone genes for this group. Moreover, this approach revealed a close phylogenetic relationship between the IncN plasmids isolated from environmental and clinical samples. Phylogenetic analysis also suggests the existence of host-specific IncN plasmid subgroups. In conclusion, IncN plasmids likely contribute to the dissemination of resistance determinants between environmental bacteria and clinical strains. This is of particular importance since multi-drug resistance IncN plasmids have been previously identified in members of the Enterobacteriaceae that cause severe infections in humans.     

Using phenotype microarrays in the assessment of the antibiotic susceptibility profile of bacteria isolated from wastewater in on-site treatment facilities

The scope of the study was to apply Phenotype Biolog MicroArray (PM) technology to test the antibiotic sensitivity of the bacterial strains isolated from on-site wastewater treatment facilities. In the first step of the study, the percentage values of resistant bacteria from total heterotrophic bacteria growing on solid media supplemented with various antibiotics were determined. In the untreated wastewater, the average shares of kanamycin-, streptomycin-, and tetracycline-resistant bacteria were 53, 56, and 42%, respectively. Meanwhile, the shares of kanamycin-, streptomycin-, and tetracycline-resistant bacteria in the treated wastewater were 39, 33, and 29%, respectively. To evaluate the antibiotic susceptibility of the bacteria present in the wastewater, using the phenotype microarrays (PMs), the most common isolates from the treated wastewater were chosen: Serratia marcescens ss marcescens, Pseudomonas fluorescens, Stenotrophomonas maltophilia, Stenotrophomonas rhizophila, Microbacterium flavescens, Alcaligenes faecalis ss faecalis, Flavobacterium hydatis, Variovorax paradoxus, Acinetobacter johnsonii, and Aeromonas bestiarum. The strains were classified as multi-antibiotic-resistant bacteria. Most of them were resistant to more than 30 antibiotics from various chemical classes. Phenotype microarrays could be successfully used as an additional tool for evaluation of the multi-antibiotic resistance of environmental bacteria and in preliminary determination of the range of inhibition concentration.     

Identification and antimicrobial susceptibility testing of clinical isolates of nonfermenting gram-negative bacteria by the Phoenix Automated Microbiology System

The Phoenix Automated Microbiology System (Becton Dickinson, Sparks, MD) was evaluated for its ability to identify nonfermenting gram-negative pathogens and measure their drug susceptibility. Isolates producing rare extended-spectrum beta-lactamases (PER-1, IMP-2, VIM-1, and VIM-2) were included in the study. Species identification was compared to that given by the ATB System (bio-Mérieux, Marcy l'Etoile, France), whereas susceptibility results were compared to those produced by a reference broth microdilution test (panels manufactured by Pasco Laboratories, Becton Dickinson). The Phoenix system consistently identified all isolates of Pseudomonas aeruginosa (n = 55) and Stenotrophomonas maltophilia (n = 28), while in other cases species agreement was obtained for 47/53 isolates (Acinetobacter baumannii, 29/31; Pseudomonas putida, 10/11; Burkholderia cepacia, 6/7; and Pseudomonas fluorescens, 2/4). Overall, the Phoenix and ATB systems gave equal results in 130/136 cases (95.6%). For two isolates, consistent identification was obtained at the genus level, thus bringing the cumulative agreement to 97.1%. MIC values (interpreted according to NCCLS guidelines) gave essential and categorical agreement in 94.2% and 93.1% of cases, respectively. Minor and major errors were 5.1% and 5.2%, respectively. No very major errors were produced. The mean time to results (TTR) for the Phoenix system was 14.8 +/- 1.6 h (mean +/- SD), with the shortest TTR being observedfor A. baumannii (13.0 +/- 1.8 h) and the longest one for P. aeruginosa (15.6 +/- 1.2 h). In conclusion, the Phoenix system performed rapidly and correctly in the identification of clinical isolates of important opportunistic pathogens and in measuring their susceptibility to antipseudomonal drugs.     

Molecular diversity and anammox activity of novel planctomycete-like bacteria in the wastewater treatment system of a full-scale alcohol manufacturing plant

In this study, the diversity of planctomycete-like bacteria and the presence of anaerobic ammonia-oxidizing bacteria in the aerobic and anaerobic basins of a full-scale alcohol manufacturing wastewater treatment plant were examined by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Two clone libraries were constructed by using a 16S rRNA-targeted universal reverse primer and a forward PCR primer specific for planctomycetes. Phylogenetic analysis of 63 16S rRNA gene sequences defined 52 operational taxonomic units (OTUs). The majority of these sequences (51.6%) clustered in two separated monophyletic groups distantly within the planctomycete tree. 15.9% sequences were related to some uncultured planctomycetes and branched as a monophyletic cluster from the described four genera (planctomyces, Pirellula, Isosphaera and Gemmata) of Planctomycetales with similarity of 81–89% to the cultured Planctomyces. More interestingly, 17.5% sequences (most retrieved from the anaerobic basin) formed a separated group close to the anammox cluster with similarity of 82–90% to the described anaerobic ammonia-oxidizing species despite of the unsuitable environments for these bacteria in the wastewater treatment plant. However, the following batch experiment results indicated low level of anammox activity (anaerobic ammonia-oxidizing rate of 0.8 mg NH₄⁺-N g⁻¹ dry biomass day⁻¹) existing in the anaerobic biomass of alcohol manufacturing wastewater treatment plant.     

Role of lime treatment in the removal of bacteria, enteric viruses, and coliphages in a wastewater reclamation plant.

Lime flocculation/sedimentation in the first process unit of a 4,500-m3/day wastewater reclamation plant reduced numbers of microorganisms extensively when operated at pH 11.2. The efficiency was much less at lower pH values, and some bacteria even multiplied at pH 9.6. Data on reduction in the number of microorganisms in the lime treatment and subsequent units indicate that inactivation by hydroxide alkalinity plays an important role in the efficiency of lime treatment. Reductions in the numbers of enteric viruses were higher than those of coliphages, enterococci, and total plate and coliform bacteria, which indicate that lime treatment can be monitored by means of coliphage and conventional bacteriological tests. This paper illustrates the valuable role of high-pH lime treatment in reducing the load of pathogenic microorganisms on subsequent units, including ultimate disinfection processes, which is important in the multiple safety barrier concept of wastewater reclamation processes.     

Role of lime treatment in the removal of bacteria, enteric viruses, and coliphages in a wastewater reclamation plant.

Lime flocculation/sedimentation in the first process unit of a 4,500-m3/day wastewater reclamation plant reduced numbers of microorganisms extensively when operated at pH 11.2. The efficiency was much less at lower pH values, and some bacteria even multiplied at pH 9.6. Data on reduction in the number of microorganisms in the lime treatment and subsequent units indicate that inactivation by hydroxide alkalinity plays an important role in the efficiency of lime treatment. Reductions in the numbers of enteric viruses were higher than those of coliphages, enterococci, and total plate and coliform bacteria, which indicate that lime treatment can be monitored by means of coliphage and conventional bacteriological tests. This paper illustrates the valuable role of high-pH lime treatment in reducing the load of pathogenic microorganisms on subsequent units, including ultimate disinfection processes, which is important in the multiple safety barrier concept of wastewater reclamation processes.     

Optimization of sulfide production by an indigenous consortium of sulfate-reducing bacteria for the treatment of lead-contaminated wastewater

Bioprocess and Biosystems Engineering - Biological treatment with sulfate-reducing bacteria (SRB) is considered to be an excellent option to remove heavy metals from wastewater. In this study, the...     

Pseudomonas laurylsulfatovorans sp. nov., sodium dodecyl sulfate degrading bacteria,isolated from the peaty soil of a wastewater treatment plant

Pseudomonas are known from their flexible degradation capabilities and their engagement in xenobiotic biotransformation and bioremediation in habitats like soil, active sludge, plant surfaces, and freshwater or marine environments. Here we present taxonomic characterization of three efficient sodium dodecyl sulfate degrading strains: AP3_10, AP3_20 and AP3_22^T belonging to the genus Pseudomonas, recently isolated from peaty soil used in a biological wastewater treatment plant. Sequence analyses of 16S rRNA and housekeeping genes: gyrB, rpoD and rpoB showed that the three closely related isolates classify within the Pseudomonas jessenii subgroup. ANIb or dDDH genomic comparisons of AP3_22^T (type strain DSM 105098^T = PCM 2904^T) supported by biochemical tests showed that the isolates differ significantly from their closest relatives. The combined genotypic, phenotypic and chemotaxonomic data strongly support the classification of the three strains: AP3_10, AP3_20 and AP3_22^T as a novel species of Pseudomonas, for which we propose the name Pseudomonas laurylsulfatovorans sp. nov. with AP3_22^T as the type strain.     

A method to increase the bioactivity of plasmid DNA by heat treatment

A method to increase the bioactivity of plasmid DNA by heat treatment has been developed. The structure of the heat treated plasmid DNA was investigated by electrophoresis assay and atomic force microscope (AFM) observation. Electrophoresis assay showed that the heat treated DNA consisted of three components: the supercoiled DNA (component I), the open circular DNA (component II) and the heat denatured DNA component. The bioactivity of the heat treated plasmid DNA was investigated by both DNA condensation experiments and gene transfection experiment with mammal cells. DNA condensation experiments showed that the heat denatured DNA component owned higher sensitivity to spermidine and polyethylenimine (PEI) than component I and component II DNA. Gene transfection experiment with PEI indicated that the heat treated DNA had higher gene transfection efficiency than untreated DNA. Our experiment not only shows an effective approach to increase the bioactivity of plasmid DNA but also leads a way to improve the bioactivity of DNA by physically modifying their structure.     

Measurement and prediction of the susceptibility of lager beer to spoilage by lactic acid bacteria

J.L. FERNANDEZ AND W.J SIMPSON. 1995. The ability of 14 strains of hop-resistant lactic acid bacteria ( Lactobacillus spp., Pediococcus spp.) to grow in 17 different lager beers was assessed using a biological challenge test. All beers were spoiled by at least one strain: all strains could grow in at least one beer. The sensitivity of different beers to spoilage by lactic acid bacteria varied. Spoilage potential could be described in the form of a 'Resistance to Spoilage Value' (RSV) based on the ability of the organisms to grow in each beer. Parameters which correlated to RSV included pH, beer colour and the content of free amino nitrogen, total soluble nitrogen, a range of individual amino acids, maltotriose and the undissociated forms of SO₂ and hop bitter acids. Predictive models based on the technique of partial least squares regression and constructed using values for undissociated SO₂ content, undissociated hop bitter acids content, polyphenol content, maltotriose content, free amino nitrogen content and a colour intensity component allowed RSV to be predicted.     

厌氧处理低浓度污水颗粒污泥的形成和生物特性研究

用UASB反应器在室温条件下处理模拟低浓度污水,研究了颗粒污泥的形成过程和特性。UASB反应器进水浓度为100～200mgCOD／L,水温为25℃～9℃,60天内形成了成熟的颗粒污泥。颗粒污泥中以丝状菌为主,存在少量球状菌。     

Relationship between the susceptibility of various bacteria to active oxygen species and to intracellular killing by macrophages

The susceptibilities of six micro-organisms to active oxygen species generated in the xanthine oxidase-mediated bactericidal system were as follows: Escherichia coli 81 greater than or equal to Listeria monocytogenes EGD greater than or equal to Salmonella typhimurium HKB-1 greater than or equal to Staphylococcus aureus Smith much greater than Mycobacterium tuberculosis H37Rv approximately equal to Candida albicans NIH A207 (the last two organisms were essentially resistant to this system). The H2O2-Fe-mediated halogenation system exhibited a higher microbicidal activity. When the micro-organisms were compared for their sensitivity to bactericidal activity of resident mouse peritoneal macrophages (M phi s), C. albicans, Staph. aureus and E. coli were killed rapidly, whereas M. tuberculosis, L. monocytogenes and S. typhimurium were more resistant. In tests for the ability to trigger an oxidative burst in mouse peritoneal M phi s (as measured by chemiluminescence), Staph. aureus showed the highest activity followed by the other organisms in the following order: C. albicans greater than E. coli greater than L. monocytogenes congruent to M. tuberculosis. S. typhimurium exhibited no triggering activity. The high susceptibility of Staph. aureus and E. coli to M phi bactericidal activity, and the partial resistance of L. monocytogenes and M. tuberculosis, correlated with their susceptibility to active oxygen and the H2O2-Fe-mediated halogenation reaction.