Analysis of the phylogenetic diversity of estrone-degrading bacteria in activated sewage sludge using microautoradiography-fluorescence in situ hybridization

In situ uptake of [2,4,6,7-3H(N)]estrone ([3H]E1) by the major phylogenetic groups present in activated sludge samples from two different municipal wastewater treatment plants was investigated using microautoradiography-fluorescence in situ hybridization (MAR-FISH). Approximately 1-2% of the total cells confined in the samples by an EUB probe mix contributed to E1 assimilation. Almost all the detected E1-assimilating cells involved in the early phase of E1 degradation were affiliated with the Beta- and Gammaproteobacteria. In the early phase of E1 degradation, no E1-assimilating cells affiliated with the Alphaproteobacteria, Actinobacteria, the Cytophaga-Flavobacterium cluster of phylum Bacteroidetes, or the phyla Chloroflexi, Nitrospira and Planctomycetes were detected. Bacteria affiliated with the Betaproteobacteria in the shape of long rods or chains of rods were found to contribute most to in situ E1 degradation. They contributed 61% and 82% of total E1-assimilating cells in cultures from two sources of activated sludge spiked with [3H]E1. The E1-degrading bacteria related to the Betaproteobacteria differed phylogenetically from the aerobic E1-degrading bacterial isolates reported in previous studies. In addition, MAR-FISH revealed the significant contribution of E1-degrading bacteria affiliated with the Gammaproteobacteria in the degradation of E1 in activated sludge.     

Eco-physiological characterization of fluorescence in situ hybridization probe-targeted denitrifiers in activated sludge using culture-independent methods

AIMS: This study proposes the application of a culture-independent method [fluorescence in situ hybridization (FISH)] and a bioreactor operation control strategy to characterize environmental micro-organisms according to their survival strategies in a mixed suspension culture. Eco-physiological characteristics of two 16S rRNA probe-targeted denitrifiers (DEN581 and DEN124) were investigated against the availability of two resources. METHODS AND RESULTS: Four sequencing batch reactors were operated with manipulation of the sludge retention times to enforce limited and excess availability of two nutrients, namely acetate and nitrite, to the biomass. DEN581 FISH probe-targeted denitrifiers demonstrated dominance when the ratio of either acetate or nitrite to biomass was in excess, while DEN124-targeted organisms dominated when the above were limited. CONCLUSIONS: The study demonstrated that microbial populations in mixed cultures can be selected by changing the substrate availability (Rs) to biomass (X) ratio. The manipulation of the specific resource availability (Rs/X) determined which one of the studied probe-targeted denitrifiers (DEN124 or DEN581) became dominant. Rs/X provides a basis to study the physiology of micro-organisms that cannot be isolated in pure culture from activated sludge. SIGNIFICANCE AND IMPACT OF THE STUDY: The eco-physiological characterization of micro-organisms responsible for biological nutrient removal is anticipated to assist process designers and operators to optimize a specific biological process, such as denitrification.     

Phosphate uptake by immobilizedAcinetobacter calcoaceticus cells in a full scale activated sludge plant

Anin situ study of the P-uptake ability ofAcinetobacter calcoaceticus was carried out using the alginate immobilization technique. ImmobilizedA. calcoaceticus cells displayed a high P-uptake ability (>97% P-accumulating cells) when immersed in the aerobic zone of an activated sludge system for 30–240 min. The overall P-accumulation pattern of the anaerobic zone depicted a typical P-release mechanism. However, limited P-accumulation was also observed at this stage. Growth and anaerobiosis were not prerequisites for P-uptake. The immobilized cell retention time in the anaerobic zone did not affect remarkably the inherent P-uptake ability of immobilizedA. calcoaceticus when exposed to the aerobic stage. P-uptake and release were reversible and depended on the environmental conditions to which immobilized cells were exposed. Immobilization ofA. calcoaceticus using alginate can be regarded as a reliable method of studying pure cultures in the activated sludge process.     

Effects of Nais elinguis on the performance of an activated sludge plant

The oligochaete worm Nais elinguis was counted during a year and a half in a full-scale, completely mixed, municipal activated sludge plant consisting of four aeration tanks connected in parallel. Simultaneously the operating variables, i.e. effluent quality, energy costs in kWh for oxygen supply in the aeration tanks, and sludge-disposal were measured. The number of worms varied both seasonally and among the aeration tanks. A major worm bloom resulted in a low sludge volume-index, lower energy consumption for oxygen supply expressed in kWh and, depending on the temperature, less sludge-disposal. The worms had no influence on the effluent quality.     

Electricity generation using chocolate industry wastewater and its treatment in activated sludge based microbial fuel cell and analysis of developed microbial community in the anode chamber

Feasibility of using chocolate industry wastewater as a substrate for electricity generation using activated sludge as a source of microorganisms was investigated in two-chambered microbial fuel cell. The maximum current generated with membrane and salt bridge MFCs was 3.02 and 2.3 A/m², respectively, at 100 Ω external resistance, whereas the maximum current generated in glucose powered MFC was 3.1 A/m². The use of chocolate industry wastewater in cathode chamber was promising with 4.1 mA current output. Significant reduction in COD, BOD, total solids and total dissolved solids of wastewater by 75%, 65%, 68%, 50%, respectively, indicated effective wastewater treatment in batch experiments. The 16S rDNA analysis of anode biofilm and suspended cells revealed predominance of β-Proteobacteria clones with 50.6% followed by unclassified bacteria (9.9%), α-Proteobacteria (9.1%), other Proteobacteria (9%), Planctomycetes (5.8%), Firmicutes (4.9%), Nitrospora (3.3%), Spirochaetes (3.3%), Bacteroides (2.4%) and γ-Proteobacteria (0.8%). Diverse bacterial groups represented as members of the anode chamber community.     

Characterization of Thiobacillus thioparus isolated from an activated sludge bioreactor used for hydrogen sulfide treatment

AIMS: To compare Thiobacillus thioparus population dynamics in a control and a test activated sludge (AS) bioreactor, used for hydrogen sulfide (H(2)S) degradation. METHODS AND RESULTS: Denaturing gradient gel electrophoresis (DGGE) was used to confirm the presence of T. thioparus, and real-time PCR was used to quantify the level of this bacterium in the AS samples. The DGGE analysis showed a band for T. thioparus in all samples, with the band being more prominent in the test sample with H(2)S diffusion. It also showed that although a change occurred in the diversity of the microbial population in the test sludge after 6 weeks of H(2)S diffusion, the microbial community structure of the test and control was still similar. Thiobacillus thioparus-specific PCR primers confirmed that 50% of the isolates from both the test and control bioreactors were T. thioparus. The thiobacilli population became more efficient at degrading the diffused H(2)S. This increase in efficiency was confirmed by a significant increase in the number of isolates from the test sludge compared with those from the control sludge, when they were grown in a thiosulfate-rich liquid medium. CONCLUSIONS: It was concluded that the use of AS process for H(2)S removal encourages the population of T. thioparus to increase even at times when the total biomass concentration shows a decrease. SIGNIFICANCE AND IMPACT OF THE STUDY: The research results give an insight into the dynamics of the microbial population in an AS pilot plant used in a dual role, to treat the wastewater and H(2)S.     

Detection of nitrifying bacteria in activated sludge by fluorescent in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for eubacteria (EUB338), ammonium-oxidizing bacteria (Nsm156) and nitrite-oxidizing bacteria (Nb1000) were used for the rapid detection of nitrifying bacteria in the activated sludge of a pilot nitrifying reactor by whole-cell, fluorescent in situ hybridization (FISH). Emission scanning and synchronous scanning fluorescence spectrometry were used to measure the hybridization. The binding of the probes at a temperature significantly lower than the melting temperature of the hybrids was conventionally considered as non-specific. Total binding of the probes at a temperature significantly higher than the melting temperature of the hybrids was conventionally considered as the sum of non-specific and specific binding (hybridization). Non-specific binding of the oligonucleotide probes with a biomass of activated sludge was 37% of the total binding of the EUB338 probe, 54% of the total binding of the Nsm156 probe, and 69% of the total binding of the Nb1000 probe. The ratio of the specific binding of the Nsm156 and Nb1000 probes was 2.3:1. The ratio of the numbers of ammonium-oxidizing bacteria to nitrite-oxidizing bacteria, determined by microbiological methods, was 2.4:1. Measuring fluorescent in situ hybridization by fluorescence spectrometry appears to be a practical tool for monitoring the microbial communities that contain nitrifying bacteria. However, a method that accounts for the non-specific binding of the probes more easily and reliably should be developed for practical application.     

Variation of 16S-23S rRNA intergenic spacer regions (ISRs) in Acinetobacter baylyi (strain B2) isolated from activated sludge

To determine the variability of the 16S-23S rRNA intergenic spacer region (ISR) of the newly described Acinetobacter baylyi, 88 clones containing ISR amplicons were screened and 14 chosen for further analysis. Two different sized 16S-23S rRNA ISRs were distinguished comprising five variable and four conserved nucleotide blocks. The major regions of heterogeneity between the different sized ISRs were due to blocks of substitutions with unique secondary structures interspersed with nucleotide substitutions, rather than differences caused by presence or absence of tRNA genes, which is often the case. Recombination events causing shuffling of nucleotide blocks are considered the most likely explanation for the mosaic structure observed between the different copies of the ISR. Single base differences present in the long ISR (LISR) were then exploited in attempts to detect possible heterogeneity between rrn copies in Acinetobacter baylyi but variability was not detected by RFLP analysis of LISR-specific PCR products. These primers were shown to be highly specific for 3 Acinetobacter baylyi strains based on LISR sequence homogeneity.     

Biodegradation of formaldehyde and its derivatives in industrial wastewater with methylotrophic yeast Hansenula polymorpha and with the yeast-bioaugmented activated sludge

Methylotrophic yeast Hansenula polymorphawere shown to cooperate with activated sludgefrom biological wastewater treatment stations,enhancing substantially its potential tobiodegrade formaldehyde in industrial wastewater. After integration with yeast cells the modified sludge retained its original structure and activity whereas its resistance to elevated formaldehyde concentrations was significantly improved. The applicability of the yeast in the utilization of formaldehyde derivatives, as exemplified by urotropine and trioxane, was also investigated. The treatment of urotropine-containing wastewater with methylotrophic yeast was found to be effective at acidic conditions (pH below 5.5). Trioxane was not degraded due to the stability of an ether bond which made the molecule recalcitrant to oxidation via methylotrophic pathway reactions. It is concluded that the yeast species may be applied to treat wastewater containing formaldehyde and some of its derivatives as either monocultures or as an integrated, specialized element of the activated sludge biocenosis.     

Implementation of a multiple biomonitoring approach to evaluate the potential for impact from an industrial discharge

Over a 2-year period, an industrial discharger implemented a program to determine if there was a potential for in-stream impact from its discharge, and, if necessary, to eliminate that potential. Six basic study designs were used. These included: (1) ambient toxicity tests using indicator organisms; (2) in-stream waste concentration (IWC) chronic testing using indicator organisms; (3) on-site flow-through toxicity testing using indicator and resident species with receiving stream water as the diluent; (4) in situ acute toxicity studies using indicator and resident species; (5) biological surveys of the receiving stream; and (6) artificial stream studies. The outcome of the studies resulted in conclusive data on which to base the design of a diffuser to dilute the effluent 1:20. This concentration was well below the lowest acute no-observed-effect concentration (10% effluent) determined using sensitive resident test species. In this manner, impact from the effluent on the James River had been reduced so that even the most sensitive resident species were protected. As a result of the study, the facility's permit was modified so that toxicity tests were made only on effluent diluted with receiving stream water to represent dilution at 1Q10 rather than 100 percent effluent. Follow-up studies have concentrated on a series of toxicity tests which were designed to identify the toxicants in the final effluent.     

Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.     

Isolation and characterization of Dechlorospirillum anomalous strain JB116 from a sewage treatment plant

The dissimilatory perchlorate reducers mainly belong to two monophyletic groups, viz. Dechloromonas and Azospira in the beta subclass of Proteobacteria. The present study describes isolation and genetic characterization of Dechlorospirillum anomalous strain JB116 that belongs to alpha subclass of Proteobacteria. The strain JB116 was isolated under facultative anaerobic conditions on a growth medium containing sodium perchlorate and sodium acetate as electron (e(-)) acceptor and e(-) donor, respectively. The strain is a spirillum shaped, dissimilatory perchlorate and nitrate reducer that prefers nitrate to perchlorate. It grows heterotrophically with acetate at temperatures between 25-35 degrees C, NaCl concentrations between 0-0.5% and pH of 7-7.8. The strain JB116 is the second only representative strain within D. anomalous that shares 99% 16S rDNA sequence similarity with the type strain D. anomalous strain WD.     

Effect of arbuscular mycorrhizae on soil microbial populations and associated plant performance of the salt marsh grass Spartina patens

The effect of arbuscular mycorrhizae (AM) on soil microbial populations and on growth performance of the high salt marsh plant Spartina patens was investigated in a AM suppression study on field-collected soil cores with S. patens. The application of benomyl resulted in a significant reduction of AM colonization on roots of S. patens, but did not completely suppress AM. Non-treated cores had significantly greater colonization (26 ± 6%) than either benomyl- (12 ± 7%) or benomyl-phosphorus-treated (7 ± 3%) cores at a depth of 2.5 cm. Colonization differences between cores declined with depth (5.0 and 7.5 cm), however, so that at 7.5 cm there was no difference between treatments. This decline was attributed to a reduction in oxygen availability with depth as evidenced by decreasing redox potential. Basic environmental conditions generally resembled those found at the field site. There were no environmental differences between treatments at the depths examined. Cell numbers and specific biomass of DAPI-stained organisms as well as members of the Domain Bacteria were significantly higher when AM colonization was suppressed, while those of the Domains Eucarya and Archaea were not significantly influenced. The increase in both microbial and bacterial population size and biomass in the presence of lower levels of AM colonization is most likely due to increases in carbon exudation to soil and rhizosphere populations that accompany AM suppression. PCR-RFLP analysis of nifH amplicons in bulk soil and rhizosphere at varying depths through the soil cores showed differences in banding patterns between rhizosphere and soil material in the presence of AM. The lack of such strong differences in the benomyl-treated cores suggests that AM colonization more strongly affects the nitrogen-fixing population than do physicochemical conditions (e.g. redox potential) alone. Plant growth performance assessed by analyzing root and leaf biomass, as well as excitation transfer efficiency of open photosynthesis system II (PS II) reaction centers (Fv/Fm) was not significantly influenced by AM. Significant differences were found between treatments for C/N ratios and nitrogen content in leaf tissue, indicating that suppression of AM increased plant nitrogen acquisition.     

Bacterial community structure of a full-scale biofilter treating pig house exhaust air

Biological air filters represent a promising tool for treating emissions of ammonia and odor from pig facilities. Quantitative fluorescence in situ hybridization (FISH) and 16S rRNA gene sequencing were used to investigate the bacterial community structure and diversity in a full-scale biofilter consisting of two consecutive compartments (front and back filter). The analysis revealed a highly specialized bacterial community of limited diversity, dominated by a few groups of Betaproteobacteria (especially Comamonas) and diverse Bacteroidetes. Actinobacteria, Gammaproteobacteria, and betaproteobacterial ammonia oxidizers (Nitrosomonas eutropha/Nitrosococcus mobilis-lineage) were also quantitatively important. Only a few quantitative differences existed between the two filter compartments at the group level, with a lower relative abundance of Actinobacteria and a higher relative abundance of the Cytophaga-Flavobacteria group in the back filter compared to the front filter. These results confirmed the N. eutropha/Nc. mobilis-lineage as the main ammonia oxidizers in pig house air filters and allowed first hypotheses for the key organisms involved in odor removal.     

Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide spring in the north Caucasus

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide spring (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Sphaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism’s population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or “threads” of natural sulfide springs.     

The nitrite-oxidizing community in activated sludge from a municipal wastewater treatment plant determined by fatty acid methyl ester-stable isotope probing

Metabolically-active autotrophic nitrite oxidizers from activated sludge were labeled with ¹³C-bicarbonate under exposure to different temperatures and nitrite concentrations. The labeled samples were characterized by FAME-SIP (fatty acid methyl ester-stable isotope probing). The compound cis-11-palmitoleic acid, which is the major lipid of the most abundant nitrite oxidizer in activated sludge, Candidatus Nitrospira defluvii, showed ¹³C-incorporation in all samples exposed to 3 mM nitrite. Subsequently, the lipid cis-7-palmitoleic acid was labeled, and it indicated the activity of a nitrite oxidizer that was different from the known Nitrospira taxa in activated sludge. The highest incorporation of cis-7-palmitoleic acid label was found after incubation with a nitrite concentration of 0.3 mM at 17 and 22 °C. While activity of Nitrobacter populations could not be detected by the FAME-SIP approach, an unknown nitrite oxidizer with the major lipid cis-9 isomer of palmitoleic acid exhibited ¹³C-incorporation at 28 °C with 30 mM nitrite. These results indicated flexibility of nitrite-oxidizing guilds in a complex community responding to different conditions. Labeled lipids so far not described for activated sludge-associated nitrifiers indicated the presence of unknown nitrite oxidizers in this habitat. The FAME-SIP-based information can be used to define appropriate conditions for the enrichment of nitrite-oxidizing guilds from complex samples.     

The community composition of root-associated bacteria of the tomato plant

The objective of this study was to characterize the bacterial community composition in the bulk soil, rhizosphere soil and root tissue of the tomato plant (Lycopersicum esculentum Mill). 16S ribosomal DNA (rDNA) from the bacterial community was amplified using PCR, and sequence analysis of 16S rDNA clones was subsequently used for bacterial identification and phylogenetic classification. Phylogenetic analysis of clones (total of 68) from the bulk soil, rhizosphere and root tissues showed that about 50% of the bacteria belonged to the α-, β-, γ-, and δ-Proteobacteria or Cytophaga–Flavobacterium–Bacteroides (CFB) phyla, with only one high G+C clone identified. A number of diverse bacteria were identified within Proteobacteria, while 87% of the bacteria belonged to the genus Flavobacterium within the CFB phylum, which is a unique finding for tomato plants. Our results will be of interest to those wanting to identify bacteria that can promote plant growth or resistance to diseases.     

植物内生细菌测定方法的研究进展

伴随全功能体(holobiont)和全基因组(hologenome)概念的出现,植物微生物群落被看作植物全功能体的重要组成部分,其结构和功能逐渐得到研究和解析.内生细菌是植物微生物群落的成员之一,由于其定殖在组织内部而与宿主的接触更为紧密,因而其与植物的相互作用也更加直接、高效且不容易受到环境条件变化的影响.本文介绍了...