Effects of the toxin 3-chloroaniline at low concentrations on microbial community dynamics and membrane bioreactor performance

The effects of toxins at ambient concentrations on microbial activity and community dynamics are poorly understood. We operated 4 membrane bioreactors (MBRs) in parallel; two reactors were continuously exposed to the toxin 3-chloroaniline (3-CA) at environmentally relevant levels, representing 25% of the total chemical oxygen demand (COD; Total COD = 400 mg l⁻¹ d⁻¹), and two reactors received no 3-CA. During the 70 d exposure to 3-CA the microbial communities never adapted as evidenced by a 48% and 14% reduction in COD and ammonia removal, respectively, compared to over 92% reduction for both measurements in the controls. The bacterial 16S rRNA gene was monitored using terminal restriction fragment length polymorphism (T-RFLP) analysis (n = 15 temporal grab samples per reactor) over the 70 d period. T-RFLP spectra analysis compared the rapid species turnover rate (STR) approach with the more computationally intensive non-metric multi-dimensional scaling (NMS) complemented with multi-response permutation procedure (MRPP). The methods revealed comparable findings and the presence of 3-CA selected for a more convergent community with less bacterial turnover. In contrast, the control MBRs were more divergent as evidenced by greater bacterial turnover variability. The importance of studying replicate reactors is highlighted by the fact that one of the two controls was significantly different from the treatment MBRs (p-value = 0.01, α = 0.05) whereas the other one was not (p-value = 0.24, α = 0.05). The study suggests that analysis of community dynamics with the rapid STR approach and with NMS/MRPP can lead to comparable results when targeting the 16S rRNA gene. The use of replicate bioreactors is essential for meaningful interpretation of microbial community patterns.     

Quantitative analysis of methanogenic community dynamics in three anaerobic batch digesters treating different wastewaters

Quantitative changes in methanogenic community structures, associated with performance data, were investigated in three anaerobic batch digesters treating synthetic glucose medium, whey permeate, and liquefied sewage sludge. All digesters were initially seeded with anaerobic sludge obtained from a local municipal wastewater treatment plant. Dynamics of methanogenic populations were monitored, at order and family levels, using real-time PCR based on the 16S rRNA gene. The molecular monitoring revealed that, in each digester, the quantitative structure of methanogenic community varied continuously over treatment time and the variation corresponded well to the changes in chemical profiles. Biphasic production of methane, associated with successive increases in aceticlastic (mainly Methanosarcinaceae) and hydrogenotrophic (mainly Methanomicrobiales) methanogenic groups, was observed in each digester. This corresponded to the diauxic utilization of acetate and longer-chain volatile fatty acids (C₃-C₆), mainly propionate. Additionally, the non-metric multidimensional scaling (NMDS) analysis of the quantification results demonstrated that the community shift patterns in three digesters were totally different from each other. Considering that the operating conditions in all trials were identical except substrates, the differences in quantitative shift profiles were suggested to be due to the different substrate compositions. This implied that the composition of wastewater could affect the evolution of quantitative methanogenic community structure in an anaerobic process. Overall, our results suggested that more attention to quantitative as well as qualitative approaches on microbial communities is needed for fundamental understanding of anaerobic processes, particularly under dynamic or transitional conditions.     

Development of microbial community structure and actvity in a high-rate anaerobic bioreactor at 18 degrees C

Anaerobic digestion in the psychrophilic (< 20 degrees C) or sub-mesophilic temperature range has recently been proven as an effective treatment option for the mineralization of a wide variety of problematic wastewaters. In this study, an expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactor was seeded with a full-scale, mesophilic sludge and employed to evaluate the long-term operational potential, and underlying microbial ecology, of this approach for the treatment of a medium-strength (5 g chemical oxygen demand [COD] l(-1)), synthetic, volatile fatty acid-based wastewater. Throughout the trial period of 625 days, extended intervals of consistently stable and efficient wastewater treatment were sustained. These results were highlighted by a short start-up period (21 d), low hydraulic retention times (4.88h), high organic (up to 24.64kg CODm(-3)d(-1)), and volumetric loading rates (up to 4.92 m3 m(-3) d(-1)). A stable, well-settling granular sludge bed was maintained in the bioreactor for the majority of the trial; however, reduced treatment efficiency and biomass washout were observed at an imposed OLR of 36.96 kg COD m(-3) d(-1). The microbial biomass in the bioreactor was investigated using maximum specific methanogenic activity assays and polymerase chain reaction-denaturing gradient gel electrophoresis. A temporal succession of both the bacterial and archaeal populations was noted during the trial, compared to the seed sludge, in response to bioreactor operation at lower temperatures, loading rate increases and to VFA accumulation in the bioreactor. During the trial, an increased contribution of hydrogenotrophic methanogenesis as a pathway of methane production was observed, along with the overall emergence of a highly active psychrotolerent-though still mesophilic biomass.     

Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester

Functional Bacteria and Archaea community structures of a full-scale anaerobic sludge digester were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescent in situ hybridization (FISH) technique and micromanipulation. FISH analysis with a comprehensive set of 16S and 23S rRNA-targeted oligonucleotide probes based on 16S rRNA clone libraries revealed that the Gram-positive bacteria represented by probe HGC69A-hybridized Actinobacteria (8.5+/-1.4% of total 4', 6-diamidino-2-phenylindole (DAPI)-stained cells) and probe LGC354-hybridized Firmicutes (3.8+/-0.8%) were the major phylogenetic bacterial phyla, followed by Bacteroidetes (4.0+/-1.2%) and Chloroflexi (3.7+/-0.8%). The probe MX825-hybridized Methanosaeta (7.6+/-0.8%) was the most abundant archaeal group, followed by Methanomicrobiales (2.8+/-0.6%) and Methanobacteriaceae (2.7+/-0.4%). The functional community structures (diversity and relative abundance) of major trophic groups were quantitatively analyzed by MAR-FISH. The results revealed that glucose-degrading microbial community had higher abundance (ca. 10.6+/-4.9% of total DAPI-stained cells) and diversity (at least seven phylogenetic groups) as compared with fatty acid-utilizing microbial communities, which were more specialized to a few phylogenetic groups. Despite the dominance of Betaproteobacteria, members of Chloroflexi, Smithella, Syntrophomonas and Methanosaeta groups dominated the [(14)C]glucose-, [(14)C]propionate-, [(14)C]butyrate- and [(14)C]acetate-utilizing microorganism community, and accounted for 27.7+/-4.3%, 29.6+/-7.0%, 34.5+/-7.6% and 18.2+/-9.5%, respectively. In spite of low abundance (ca. 1%), the hitherto unknown metabolic functions of Spirochaeta and candidate phylum of TM7 as well as Synergistes were found to be glucose and acetate utilization, respectively.     

Natural organic matter (NOM) removal and structural changes in the bacterial community during artificial groundwater recharge with humic lake water

This study evaluated the removal of natural organic matter (NOM) and structural changes in the microbial community during infiltration of humic lake water at three artificial groundwater recharge (AGR) sites in Finland. The three sites were at waterworks in H?meenlinna, Jyv?skyl? and Tuusula, sites A, B and C, respectively. Site A used groundwater recharge by both basin and sprinkling infiltration, site B used only sprinkling infiltration, and site C used only basin infiltration. Reductions of total organic carbon at sites A, B and C were 91%, 84% and 74%, respectively, in the winter, and 88%, 77% and 73%, respectively, in the summer. The Finnish national recommended value of 2 mg/l for TOC was achieved at all sites and the TOC of natural groundwater at site C was much lower, at 0.6 mg/l. Large molecular fractions of NOM were removed more efficiently than the smaller ones. Total amount of DAPI-stained cells decreased during infiltration at sites A, B and C in winter by 94%, 94% and 75% and in summer by 96%, 97% and 94%, respectively. Bacterial communities in raw waters and extracted groundwaters were diverse with changes occurring during infiltration, which was shown by DNA extraction followed by PCR of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) fingerprinting. While the natural groundwater microbial community was diverse, it was different from that of the extracted groundwater in the AGR area. Simultaneous organic carbon removal and the decrease of bacterial counts during infiltration indicated biodegradation. In addition, the changing DGGE profiles during the process of infiltration, demonstrated that changing environmental conditions were reflected by changes in bacterial community composition.     

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.     

Seasonal dynamics of bacterial community structure and composition in cold and hot drinking water derived from surface water reservoirs

In temperate regions, seasonal variability of environmental factors affects the bacterial community in source water and finished drinking water. Therefore, the bacterial core community and its seasonal variability in cold and the respective hot drinking water was investigated. The bacterial core community was studied by 16S rRNA-based SSCP fingerprint analyses and band sequencing of DNA and RNA extracts of cold and hot water (60 °C). The bacterial communities of cold and hot drinking water showed a highly different structure and phylogenetic composition both for RNA and DNA extracts. For cold drinking water substantial seasonal dynamics of the bacterial community was observed related to environmental factors such as temperature and precipitation affecting source and drinking water. Phylogenetic analyses of the cold water community indicated that the majority of phylotypes were very closely affiliated with those detected in former studies of the same drinking water supply system (DWSS) in the preceding 6 years, indicating a high stability over time. The hot water community was very stable over time and seasons and highly distinct from the cold water with respect to structure and composition. The hot water community displayed a lower diversity and its phylotypes were mostly affiliated with bacteria of high temperature habitats with high growth rates indicated by their high RNA content. The conversion of the cold to the hot water bacterial community is considered as occurring within a few hours by the following two processes, i) by decay of most of the cold water bacteria due to heating, and ii) rapid growth of the high temperature adapted bacteria present in the hot water (co-heated with the cold water in the same device) using the nutrients released from the decaying cold water bacteria. The high temperature adapted bacteria originated partially from low abundant but beforehand detected members of the cold water; additionally, the rare members (“seed bank “) of the cold water are considered as a source.