In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes

One of the main causes of failure of fluorescence in situ hybridization with rRNA-targeted oligonucleotides, besides low cellular ribosome content and impermeability of cell walls, is the inaccessibility of probe target sites due to higher-order structure of the ribosome. Analogous to a study on the 16S rRNA (B. M. Fuchs, G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann, Appl. Environ. Microbiol. 64:4973–4982, 1998), the accessibility of the 23S rRNA of Escherichia coli DSM 30083T was studied in detail with a set of 184 CY3-labeled oligonucleotide probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI, 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness of 23S-2018, respectively) was as follows: class I, 3%; class II, 21%; class III, 35%; class IV, 18%; class V, 16%; and class VI, 7%. A fine-resolution analysis of selected areas confirmed steep changes in accessibility on the 23S RNA to oligonucleotide probes. This is similar to the situation for the 16S rRNA. Indeed, no significant differences were found between the hybridization of oligonucleotide probes to 16S and 23S rRNA. Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA ofE. coli, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future

Population dynamics of anaerobic microbial consortia in thermophilic granular sludge in response to feed composition change

A thermophilic UASB reactor was operated at 55°C for greater than 470 days in order to investigate the effects of feed composition on the changes in microbial community structure where thermophilic granular sludge was used as the inoculum source. The feed compositions were changed with cultivation days; phase 1 (1–70 days), alcohol distillery wastewater; phase 2 (71–281 days), artificial acetate wastewater; phase 3 (282–474 days), artificial sucrose wastewater. During the first one month of each phase, the methanogenic activity and cell density of methanogens quantified by fluorescence in situ hybridization (FISH) drastically changed as a result of shift in feed composition. When artificial acetate wastewater was used as feed, retained granular sludge was partially disintegrated due to a decrease in the number of symbiotic bacterial community members: acetogens (acidogens) and hydrogenotrophic methanogens. In contrast, when the feed was shifted to sucrose (phase 3), granulation of biomass was promoted by a remarkable proliferation of the symbiotic community. The presence of hydrogen-utilizing methanogens and acetogens (acidogens) are shown to be effective for the enhancement of thermophilic granulation. The cell density of methanogens determined by FISH was strongly correlated with the methane-producing potential of the retained thermophilic granular sludge.</jats:p

Treatment of Sugar Containing-Low Strength Wastewater at 20°C by Anaerobic Granular Sludge Bed Reactor

To investigate the feasibility of anaerobic wastewater treatment technology for low strength sugar refinery wastewater (0.4 - 0.5 g COD/L), an 8.8 L volume of anaerobic granular sludge bed reactor was operated at 20°C for 400 days. The operation mode was combination of one pass flow (UASB, 50 min) and effluent-recirculation (EGSB, 10 min) mode. The aerobic down-flow hanging sponge (DHS) reactor was installed as a post-treatment. During the started-up period, reactors were fed with synthetic wastewater at overall HRT of 3 hours (anaerobic 2 hours, aerobic 1 hour). After day 85, feed was changed to real wastewater together with supplement of nutrients, trace elements and NaHCO3. The sufficient COD removal efficiency (85% SD±6.2) and stable process performance were elicited from the granular sludge bed reactor.Also, post-treatment (DHS reactor) offered good quality of effluent (45 mg COD/L, 7 mg BOD/L) and it achieved the discharge standard. Increasing of sulfate concentration of wastewater caused higher contribution of sulfate reducing bacteria for COD removal. The sludge concentration and settleability were well maintained thoroughly. However, floatation of large size granule was observed in the later part of experiment. This phenomenon may attribute to the high growth yield of retained sludge at 20°C.</jats:p

Behavior of cellulose-degrading bacteria in thermophilic anaerobic digestion process

Previously, we found that the newly isolated Clostridium sp. strain JC3 became the dominant cellulose-degrading bacterium in thermophilic methanogenic sludge. In the present study, the behavior of strain JC3 in the thermophilic anaerobic digestion process was investigated quantitatively by molecular biological techniques. A cellulose-degrading experiment was conducted at 55 °C with a 9.5 L of anaerobic baffled reactor having three compartments (Nos. 1, 2, 3). Over 80% of the COD input was converted into methane when 2.5 kgCOD m−3 d−1 was loaded for an HRT of 27 days. A FISH probe specific for strain JC3 was applied to sludge samples harvested from the baffled reactor. Consequently, the ratio of JC3 cells to DAPI-stained cells increased from below 0.5% (undetectable) to 9.4% (compartment 1), 13.1% (compartment 2) and 21.6% (compartment 3) at day 84 (2.5 kgCOD m−3 d−1). The strain JC3 cell numbers determined by FISH correlated closely with the cellulose-degrading methanogenic activities of retained sludge. A specific primer set targeting the cellulase gene (cellobiohydrolaseA: cbhA) of strain JC3 was designed and applied to digested sludge for treating solid waste such as coffee grounds, wastepaper, garbage, cellulose and so on. The strain JC3 cell numbers determined by quantitative PCR correlated closely with the cellulose-sludge loading of the thermophilic digester. Strain JC3 is thus important in the anaerobic hydrolysis of cellulose in thermophilic anaerobic digestion processes.</jats:p

Characteristics of Granular Sludge in an EGSB Reactor for Treating low Strength Wastewater

A lab-scale expanded granular sludge bed (EGSB) reactor was operated at 20°C with low strength wastewater (0.6-0.8 g COD/L) for over 200 days. Reactor was inoculated with mesophilic granular sludge. The up-flow velocity was set to 5 m/h by effluent recirculation. The COD loading was increased up to 12 kg COD/m3/day until the day 76, resulting in hydraulic retention time of 1.5 hours. Physical properties (settleability anddiameter) of retainedsludge tended todeteriorate during the first 2-3 months, however sludge settleability kept sufficiently in the later part of experiment due to the reconstruction of granular sludge. The growth yield (Yg) of retained sludge (0.13 g VSS/g COD) was about two times higher than mesophilic and thermophilic granular sludge processes while the endogenous decay constant (Kd) is very low (0.0001/day) as compared with those processes. The sludge retention time of retained sludge reduced from 100 days to 40 days by the reduction of hydraulic retention time from 4 hours to 1.5 hours. Maintenance of 40 days of sludge retention time caused the stable retainment of biomass and the significant increase of methanogenic activity of the retained sludge

Quantification of methanogen cell density in anaerobic granular sludge consortia by fluorescence in-situ hybridization

Whole cell fluorescence in-situ hybridization (FISH) with 16S rRNA targeted oligonucleotides was applied to reveal the microbial ecological structure of UASB-grown granular sludge. The FISH analysis indicated that the members of the domain Archaea accounted for 28 to 53% of the total cells in various granular sludge sources, while Methanosaeta and Methanobacteriaceae cells accounted for 13 to 38%, and 4 to 27%, respectively. Methanosaeta cell density and Methanobacteriaceae cell density were strongly correlated, respectively, with acetate-utilizing methane production activity and with hydrogen-utilizing methane production activity.</jats:p

Intermittent Effluent Recirculation for the Efficient Treatment ofLow Strength Wastewater by an EGSB Reactor

In order to establish the appropriate methane fermentation technology for low strength wastewater, a 2.0 L EGSB reactor was operated at 20°C with 0.3 - 0.4 g COD/L of sucrose-based synthetic wastewater for 500 days. At the start up period, the reactor was operated in EGSB mode with a 5 m/h up flow velocity by continuous effluent recirculation. However, the EGSB reactor exhibited insufficient COD removal (50-60%) at COD loading of 7.2 - 9.6 kg COD/m3·day due to the low COD concentration in the sludge bed. Therefore, we proposed the new operation mode by switching to UASB mode (without recirculation, 0.7 m/h up flow velocity) for 30 minutes and EGSB mode for 10 minutes in 40 minutes cycle. Moreover we added sodium sulfide to make the low ORP condition. In this operation, COD removal increased dramatically, from 65% to 91%. Additionally, physical properties of the retained sludge were well maintained in this operation mode. Furthermore, the retained sludge possessed appropriate levels of methanogenic activity (0.2-0.4 g COD/g VSS/ day) at 20°C

Characteristics of Granular Sludge in an EGSB Reactor for Treating low Strength Wastewater

A lab-scale expanded granular sludge bed (EGSB) reactor was operated at 20°C with low strength wastewater (0.6-0.8 g COD/L) for over 200 days. Reactor was inoculated with mesophilic granular sludge. The up-flow velocity was set to 5 m/h by effluent recirculation. The COD loading was increased up to 12 kg COD/m3/day until the day 76, resulting in hydraulic retention time of 1.5 hours. Physical properties (settleability anddiameter) of retainedsludge tended todeteriorate during the first 2-3 months, however sludge settleability kept sufficiently in the later part of experiment due to the reconstruction of granular sludge. The growth yield (Yg) of retained sludge (0.13 g VSS/g COD) was about two times higher than mesophilic and thermophilic granular sludge processes while the endogenous decay constant (Kd) is very low (0.0001/day) as compared with those processes. The sludge retention time of retained sludge reduced from 100 days to 40 days by the reduction of hydraulic retention time from 4 hours to 1.5 hours. Maintenance of 40 days of sludge retention time caused the stable retainment of biomass and the significant increase of methanogenic activity of the retained sludge

Intermittent Effluent Recirculation for the Efficient Treatment ofLow Strength Wastewater by an EGSB Reactor

In order to establish the appropriate methane fermentation technology for low strength wastewater, a 2.0 L EGSB reactor was operated at 20°C with 0.3 - 0.4 g COD/L of sucrose-based synthetic wastewater for 500 days. At the start up period, the reactor was operated in EGSB mode with a 5 m/h up flow velocity by continuous effluent recirculation. However, the EGSB reactor exhibited insufficient COD removal (50-60%) at COD loading of 7.2 - 9.6 kg COD/m3·day due to the low COD concentration in the sludge bed. Therefore, we proposed the new operation mode by switching to UASB mode (without recirculation, 0.7 m/h up flow velocity) for 30 minutes and EGSB mode for 10 minutes in 40 minutes cycle. Moreover we added sodium sulfide to make the low ORP condition. In this operation, COD removal increased dramatically, from 65% to 91%. Additionally, physical properties of the retained sludge were well maintained in this operation mode. Furthermore, the retained sludge possessed appropriate levels of methanogenic activity (0.2-0.4 g COD/g VSS/ day) at 20°C