Long-term effects of operating temperature and sulphate addition on the methanogenic community structure of anaerobic hybrid reactors

The diversity, population dynamics, and activity profiles of methanogens in anaerobic granular sludges from two anaerobic hybrid reactors treating a molasses wastewater both mesophilically (37 degrees C) and thermophilically (55 degrees C) during a 1081 day trial were determined. The influent to one of the reactors was supplemented with sulphate, after an acclimation period of 112 days, to determine the effect of competition with sulphate-reducing bacteria on the methanogenic community structure. Sludge samples were removed from the reactors at intervals throughout the operational period and examined by amplified ribosomal DNA (rDNA) restriction analysis (ARDRA) and partial sequencing of 16S rRNA genes. In total, 18 operational taxonomic units (OTUs) were identified, 12 of which were sequenced. The methanogenic communities in both reactors changed during the operational period. The seed sludge and the reactor biomass sampled during mesophilic operation, both in the presence and absence of sulphate, was characterised by a predominance of Methanosaeta spp. Following temperature elevation, the dominant methanogenic sequences detected in the non-sulphate supplemented reactor were closely related to Methanocorpusculum parvum. By contrast, the dominant OTUs detected in the sulphate-supplemented reactor upon temperature increase were related to the hydrogen-utilising methanogen, Methanobacterium thermoautotrophicum. The observed methanogenic community structure in the reactors correlated with the operational performance of the reactors during the trial and with physiological measurements of the reactor biomass. Both reactors achieved chemical oxygen demand (COD) removal efficiencies of over 90% during mesophilic operation, with or without sulphate supplementation. During thermophilic operation, the presence of sulphate resulted in decreased reactor performance (effluent acetate concentrations of >3000 mg/l and biogas methane content of <25%). It was demonstrated that methanogenic conversion of acetate at 55 degrees C was extremely sensitive to inhibition by sulphide (50% inhibition at 8-17 mg/l unionised sulphide at pH 7.6-8.0), while the conversion of H(2)/CO(2) methanogenically was favoured. The combination of experiments carried out demonstrated the presence of specific methanogenic populations during periods of successful operational performance.     

Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors

To promptly establish anaerobic ammonium oxidation (anammox) reactors, appropriate seeding sludge with high abundance and activity of anammox bacteria was selected by quantifying 16S rRNA gene copy numbers of anammox bacteria by real-time quantitative PCR (RTQ-PCR) and batch culture experiments. The selected sludge was then inoculated into up-flow fixed-bed biofilm column reactors with nonwoven fabric sheets as biomass carrier and the reactor performances were monitored over 1 year. The anammox reaction was observed within 50 days and a total nitrogen removal rate of 26.0 kg-Nm(-3)day(-1) was obtained after 247 days. To our knowledge, such a high rate has never been reported before. Hydraulic retention time (HRT) and influent NH(4)(+) to NO(2)(-) molar ratio could be important determinant factors for efficient nitrogen removal in this study. The higher nitrogen removal rate was obtained at the shorter HRT and higher influent NH(4)(+)/NO(2)(-) molar ratio. After anammox reactors were fully developed, the community structure, spatial organization and in situ activity of the anammox biofilms were analyzed by the combined use of a full-cycle of 16S rRNA approach and microelectrodes. In situ hybridization results revealed that the probe Amx820-hybridized anaerobic anammox bacteria were distributed throughout the biofilm (accounting for more than 70% of total bacteria). They were associated with Nitrosomonas-like aerobic ammonia-oxidizing bacteria (AAOB) in the surface biofilm. The anammox bacteria present in this study were distantly related to the Candidatus Brocadia anammoxidans with the sequence similarity of 95%. Microelectrode measurements showed that a high in situ anammox activity (i.e., simultaneous consumption of NH(4)(+) and NO(2)(-)) of 4.45 g-N of (NH(4)(+)+NO(2)(-))m(-2)day(-1) was detected in the upper 800 microm of the biofilm, which was consistent with the spatial distribution of anammox bacteria.     

A new fluorescent oligonucleotide probe for in situ detection of s-triazine-degrading Rhodococcus wratislaviensis in contaminated groundwater and soil samples

A bacterial strain (FPA1) capable of using terbuthylazine, simazine, atrazine, 2-hydroxysimazine, deethylatrazine, isopropylamine or ethylamine as its sole carbon source was isolated from a shallow aquifer chronically contaminated with s-triazine herbicides. Based on its 16S rDNA sequence analysis, the strain FPA1 was identified as Rhodococcus wratislaviensis. The disappearance time of 50% of the initial terbuthylazine concentration in the presence of this strain (DT₅₀) was 62 days. This strain was also able to mineralise the [U-ring ¹⁴C] triazine-ring, albeit at a slow rate. A 16S rRNA target oligonucleotide probe (RhLu) was designed, and the FISH protocol was optimised, in order to detect R. wratislaviensis in s-triazine-contaminated sites. The RhLu probe gave a positive signal (expressed as % of total DAPI-positive cells) in both the groundwater (2.19 ± 0.41%) and soil (2.10 ± 0.96%) samples analysed. Using the RhLu probe, R. wratislaviensis can be readily detected, and its population dynamics can be easily monitored, in soil and in water ecosystems contaminated with s-triazine. To the best of our knowledge, this is the first report showing the isolation, from groundwater, of a bacterial strain able to degrade s-triazines.     

Methanogenic population dynamics during startup of a full-scale anaerobic sequencing batch reactor treating swine waste

Changes in methanogenic population levels were followed during startup of a full-scale, farm-based anaerobic sequencing batch reactor (ASBR) and these changes were linked to operational and performance data. The ASBR was inoculated with anaerobic digester sludge from a municipal wastewater treatment facility. During an acclimation period of approximately 3 months, the ASBR content was diluted to maintain a total ammonia-N level of approximately 2000mg l(-1). After this acclimation period, the volatile solids loading rate was increased to its design value of 1.7g l(-1) day(-1) with a 15-day hydraulic retention time, which increased the total ammonia-N level in the ASBR to approximately 3,600 mg l(-1). The 16S ribosomal RNA (rRNA) levels of the acetate-utilizing methanogens of the genus Methanosarcina decreased from 3.8% to 1.2% (expressed as a percentage of the total 16S rRNA levels) during this period, while the 16S rRNA levels of Methanosaeta concilii remained low (below 2.2%). Methane production and reactor performance were not affected as the 16S rRNA levels of the hydrogen-utilizing methanogens of the order Methanomicrobiales increased from 2.3% to 7.0%. Hence, it is likely that during operation with high ammonia levels, the major route of methane production is through a syntrophic relationship between acetate-oxidizing bacteria and hydrogen-utilizing methanogens. Anaerobic digestion at total ammonia-N levels exceeding 3500mg l(-1) was sustainable apparently due to the acclimation of hydrogen-utilizing methanogens to high ammonia levels.     

Identification of microorganisms involved in reductive dehalogenation of chlorinated ethenes in an anaerobic microbial community

In this study, we report on phylogenetic and physiological characterization of an anaerobic culture capable of reductive dehalogenation of tetrachloroethene (PCE) obtained from a PCE-contaminated site. The culture was enriched using different combinations of electron donors (hydrogen and acetate) and electron acceptors (PCE, cis-1,2-dichloroethene (cDCE) and controls without chlorinated ethenes). The resulting subcultures were analyzed using three different approaches: chemical analysis to document conversion of chlorinated ethenes; polymerase chain reaction (PCR) of 16S rRNA gene fragments and denaturing gradient gel electrophoresis (DGGE) to compare community compositions; fluorescence in situ hybridization (FISH) to quantify specific groups of microorganisms using oligonucleotide probes previously designed or newly designed based on the sequences retrieved from sequence analysis of specific DGGE bands. Members of two genera which contain bacteria capable of reductive dehalogenation were detected in the culture: Dehalococcoides and Desulfitobacterium. The combined analyses suggested that Dehalococcoides-like bacteria are associated with complete dehalogenation of chlorinated ethenes to ethene with hydrogen as electron donor; and Desulfitobacterium-like bacteria, in contrast, are associated with incomplete PCE dehalogenation to cDCE and appear to be able to use acetate as electron donor. In addition, Sporomusa-like bacteria were identified, which most likely act as homoacetogens. The results demonstrated that combination of culture enrichment with different substrates, DGGE, and FISH allowed a detailed qualitative and quantitative characterization of the dominant microorganisms associated with reductive dehalogenation.     

Microbial community analysis of thermophilic contact oxidation process by using ribosomal RNA approaches and the quinone profile method

Microbial community structure of a lab scale thermophilic aerobic wastewater treatment reactor was analyzed by a combination of culture-independent methods. Quinone profile method provides for chemical analysis of respiratory quinone molecular species, which corresponds to bacterial groups. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA partial sequences (PCR-DGGE) clarifies community changes at species level, as DGGE can separate DNA fragments of different sequences. Certain phvlogenetic groups of bacterial cells can be labeled by fluorescence in situ hybridization (FISH). Quinone profile showed a predominant presence of MK-7. PCR-DGGE revealed that constituents of the community were unchanged during the stable phase. FISH demonstrated the existence of the relatives of Bacillus lentus and B. thermocloacae in considerable proportions. The community was mainly composed of Bacillaceae, and obligate thermophilic and mesophilic Bacillus appeared in spite of the temperature fluctuation from 35 degrees C to 60 degrees C. The combination of these culture-independent methods revealed the community precisely enough to evaluate the reactor performance.     

Sulfidogenic fluidized-bed treatment of metal-containing wastewater at 8 and 65 degrees C temperatures is limited by acetate oxidation

Acetate utilization in sulfidogenic fluidized-bed reactors (FBRs) was investigated for the treatment of iron containing wastewater at low (8 degrees C) and high (65 degrees C) temperatures. The FBRs operated at low and high temperatures were inoculated with cultures of sulfate-reducing bacteria (SRB) originally enriched from arctic and hot mining environments, respectively. Acetate was not utilized as a carbon and electron source for SRB at 8 degrees C. With ethanol, hydrogen sulfide was produced from ethanol to acetate oxidation, which precipitated the iron. Then, several attempts were made to obtain acetate oxidation at 8 degrees C. Inoculation of two different low temperature enrichments and operating the FBR for a long period of time (321 days) did not result in enrichment of acetate oxidizing SRB. Due to the absence of acetate oxidation at 8 degrees C, external alkalinity addition was required to keep the pH neutral. At 65 degrees C, average acetate and sulfate removals were 52+/-12% and 24+/-8% at 670 mg/Ld acetate and 1500 mg/Ld sulfate loadings, respectively. The produced alkalinity from acetate oxidation increased the pH from 6.4 to around 7.5 and electron flow to sulfate reduction averaged 65%. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed quite stable SRB community at 8 degrees C, whereas, at 65 degrees C SRB community was dynamic. In the FBRs, Desulfomicrobium apsheronum and Desulfosporosinus sp. at 8 degrees C and Desulfotomaculum sp. at 65 degrees C were detected.     

Nutritional status and the serum levels of Mg, Ca, Zn and Cu

Serum Mg, Ca, Zn and Cu levels of 65 Indian children aged 5 months to 3 y 7 months, living in Bombay city, were estimated by atomic absorption spectrometry. Twenty-five well nourished controls exhibited the following serum levels: Mg 2.5 +/- 0.3 mg%, Ca 9.7 +/- 0.6 mg%, Zn 103.2 +/- 9.7 micrograms % and Cu 148.7 +/- 26 micrograms %. The remaining 40 patients were divided into four categories depending on the status of their nutrition; Group I was minimally undernourished, whereas Group IV was the worst effected. Group I (n = 6) had serum values of 2.5 +/- 0.2 mg% Mg, 8.9 +/- 0.6 mg% Ca, 85 +/- 18 micrograms % Zn and 108 +/- 25.8 micrograms % Cu. Group II (n = 20) levels were 2.2 +/- 0.3 mg% Mg, 8.3 +/- 0.9 mg% Ca, 82.6 +/- 16.5 micrograms %, Zn and 89.2 +/- 27.6 micrograms % Cu. Group III (n = 9) were 2.1 +/- 0.3 mg% Mg, 8.3 +/- 0.8 mg% Ca, 88.3 +/- 22.6 micrograms % Zn and 93 +/- 26.9 micrograms % Cu, whereas the most severely undernourished (Group IV) children had serum levels of 2.2 +/- 0.4 mg% Mg, 7.6 +/- 0.8 mg% Ca, 78.4 +/- 15.7 micrograms % Zn and 62.8 +/- 17.5 micrograms % Cu.     

Bacteria from drinking water supply and their fate in gastrointestinal tracts of germ-free mice: A phylogenetic comparison study

Microorganisms in drinking water sources may colonize in gastrointestinal (GI) tracts and this phenomenon may pose a potential health risk especially to immunocompromised population. The microbial community diversity of the drinking water was compared with the GI tracts of the mice using phylogenetic and statistical analyses of 16S rRNA gene sequences. A group of germ-free mice were fed with drinking water from public water supply that passed through an automated watering system with documented biofilm accumulation. From drinking water and GI tracts of the germ-free mice, 179 bacteria were isolated and 75 unique 16S rRNA gene phylotypes were sequenced as operational taxonomic unit (OTU, >97% similarity). Three major groups of the genus Acidovorax (21%), Variovorax (42%) and Sphingopyxis (15%) were found in drinking water. Three major groups of the genus Ralstonia (24%), Staphylococcus (20%) and Bosea (22%) were found in GI tracts. Ralstonia (6%, 24%), Sphingopyxis (15%, 2%), Bacillus (3%, 5%), Escherichia coli (3%, 2%) and Mesorhizobium (3%, 5%) were found in both sources - drinking water and GI tract. A lineage-per-time plot shows that the both bacterial communities have convex shape lines, suggesting an excess of closely related ecotypes. A significant F_ST test (0.00000-0.00901) coupled with an insignificant P test (0.07-0.46) implies that the tree contained several clades of closely related bacteria. Both phylogenetic and statistical results suggest a correlation between the bacterial communities originating in the drinking water and those associated with the GI tracts. The GI tract showed a higher genetic diversity than the drinking water, but a similar lineage-per-time plot was obtained overall. It means a sudden evolutionary transformation and colonization occurred with high selective forces.