Novel major bacterial candidate division within a municipal anaerobic sludge digester

In a previous study, we analyzed the molecular diversity of Planctomycetales by PCR amplification and sequencing of 16S rRNA clone libraries generated from a municipal wastewater plant, using planctomycete-specific and universal primer sets (R. Chouari, D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir, Appl. Environ. Microbiol. 69:7354-7363, 2003). Only a small fraction (4%) of the 16S rRNA gene sequences of the digester clone library corresponded to the Planctomycetales division. Importantly, 85.9% of the digester clone sequences are grouped into two different clusters named WWE1 (81.4% of the sequences) and WWE2 (4.5%) and are distantly affiliated with unidentified bacterial sequences retrieved from a methanogenic reactor community and from a termite gut, respectively. In phylogenetic analysis using 16S rRNA gene sequence representatives of the main phylogenetic bacterial divisions, the two clusters are monophyletic, branch apart from each other, and are distantly related to Planctomycetales and other bacterial divisions. A novel candidate division is proposed for WWE1, while the WWE2 cluster strongly affiliates with the recently proposed Lentisphearae phylum. We designed and validated a 16S rRNA probe targeting WWE1 16S rRNA sequences by both fluorescent in situ hybridization (FISH) and dot blot hybridization (DBH). Results of FISH analysis show that WWE1 representative microorganisms are rods or filamentous shaped, while DBH shows that WWE1 accounts for 12% of the total bacterial rRNA within the anaerobic digester. The remaining 16S rRNA gene sequences are affiliated with Verrucomicrobia or recently described candidate divisions with no known pure culture representatives, such as OD1, BRC1, or NBL-UPA2, making up less than 3.5% of the clone library, respectively. This inventory expands the known diversity of the latter bacterial division-level lineages.     

Practical method for detecting poliovirus in anaerobic digester sludge.

A method has been developed which permits reliable detection of low numbers of poliovirus infective units in anaerobic digester sludge and dewatered composted sludge.     

Survival of Salmonella spp. in a simulated acid-phase anaerobic digester treating sewage sludge

The presence of pathogenic microorganisms in municipal waste sludge may create a serious outbreak of water borne diseases if the sludge is used for agricultural purpose. An attempt to decrease the number of pathogenic microorganisms, Salmonella spp. using a simulated acid-phase anaerobic digester was tested in a laboratory-scale batch experiment. Reduction of Salmonella spp. was demonstrated in a mixture of sludge and organic acid, simulating an acid digester of a two-phase anaerobic digestion process. A high concentration of organic acid at a pH value of 5.5-6.0 prevents a decrease in Salmonella spp. concentration. Almost complete destruction of Salmonella spp. is observed within two days if the pH value is maintained below 5.5.     

Survival of Salmonella spp. in a simulated acid-phase anaerobic digester treating sewage sludge

The presence of pathogenic microorganisms in municipal waste sludge may create a serious outbreak of water borne diseases if the sludge is used for agricultural purpose. An attempt to decrease the number of pathogenic microorganisms, Salmonella spp. using a simulated acid-phase anaerobic digester was tested in a laboratory-scale batch experiment. Reduction of Salmonella spp. was demonstrated in a mixture of sludge and organic acid, simulating an acid digester of a two-phase anaerobic digestion process. A high concentration of organic acid at a pH value of 5.5-6.0 prevents a decrease in Salmonella spp. concentration. Almost complete destruction of Salmonella spp. is observed within two days if the pH value is maintained below 5.5.     

Impacts of hydrodynamic conditions on sludge digestion in internal circulation anaerobic digester

An investigation of hydrodynamic conditions and their impacts on sludge digestion in internal circulation anaerobic digester (ICAD) was conducted. This novel sludge digester employing upflow reactor concept was composed of reaction zone, riser and downcomer, etc. Part of the generated biogas was injected to the reaction zone to intensify the circulation in ICAD in order to enhance the mass transfer. The advanced particle image velocimetry was used to explore the hydrodynamic conditions at micro scale. The results revealed that the average shear rate in the reaction zone and downcomer was linearly correlated with the Reynolds number; the shear rates in ICAD revealed an order as riser > downcomer > reaction zone. Thermophilic digestion of waste activated sludge in a pilot ICAD for 5.25 d combined with thermal pretreatment at 60 °C for 1 d was conducted under various hydrodynamic conditions. The survey at global level demonstrated that both the longer sludge retention time than the hydraulic retention time and the satisfactory mass transfer could be realized and as consequences, the maximal biogas production rate and VSS removal were obtained when the Reynolds number of the reaction zone was approximately 0.53. This digester under the optimal hydrodynamic conditions demonstrated good degradation capacity and buffer capability to resist various shock loadings.     

Phylogenetic and functional diversity of propionate-oxidizing bacteria in an anaerobic digester sludge

The phylogenetic and functional diversity of syntrophic propionate-oxidizing bacteria (POB) present in an anaerobic digester was investigated by microautoradiography combined with fluorescent in situ hybridization (MAR–FISH) that can directly link 16S rRNA phylogeny with in situ metabolic function. The syntrophic POB community in the anaerobic digester sludge consisted of at least four phylogenetic groups: Syntrophobacter, uncultured short rod Smithella (Smithella sp. SR), uncultured long rod Smithella (Smithella sp. LR), and an unidentified group. The activities of these POB groups were dependent on the propionate concentrations. The uncultured Smithella sp. SR accounted for 52–62% of the total active POB under all the propionate concentrations tested (0.5–15 mM). In contrast, uncultured Smithella sp. LR was active only at lower propionate concentrations and became a dominant active POB at 0.5 mM of propionate. Syntrophobacter accounted for 16–31% of the total active POB above 2.5 mM propionate, whereas the active Syntrophobacter population became low (ca. 6%) at 0.5 mM of propionate. The anaerobic digester was operated in a fill and draw mode, resulting in periodical changes in propionate concentration ranging from 0 to 10 mM. These phylogenetically and functionally diverse, to some extent functionally redundant, active POB communities were dynamically responding to the periodical changes in propionate concentration.     

Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge

Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope- and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with ¹⁴C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with ¹³C₆-glucose and ¹³C₃-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with ¹³C-glucose and ¹³C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with ¹⁴C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high K_m for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5–10 m). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta.     

Inhibitory effect of chlorinated guaiacols on methanogenic activity of anaerobic digester sludge

Of four chlorinated guaiacols, tetrachloroguaiacol at 62 M inhibited acetate methanogenesis, the strongest decreasing activity by 50%. 4,5,6-Trichloroguaiacol, 4,5-dichloroguaiacol, and 4-chloroguaiacol showed 50% inhibition at 0.13, 0.32, and 1.50 mM, respectively. Degradation test results of volatile fatty acids (acetic, propionic, and butyric acid) by anaerobic digester sludge (stored 5 weeks) indicated that syntrophic butyrate degraders of this sludge were more sensitive to tetrachloroguaiacol than acetoclastic methanogens and syntrophic propionate degraders.     

Paenibacillus xylaniclasticus sp. nov., a xylanolytic-cellulolytic bacterium isolated from sludge in an anaerobic digester

A mesophilic, facultative, anaerobic, xylanolytic-cellulolytic bacterium, TW1(T), was isolated from sludge in an anaerobic digester fed with pineapple waste. Cells stained Gram-positive, were spore-forming, and had the morphology of straight to slightly curved rods. Growth was observed in the temperature range of 30 to 50°C (optimum 37°C) and the pH range of 6.0 to 7.5 (optimum pH 7.0) under aerobic and anaerobic conditions. The strain contained meso-diaminopimelic acid in the cell-wall peptidoglycan. The predominant isoprenoid quinone was menaquinone with seven isoprene units (MK-7). Anteiso-C(15:0), iso-C(16:0), anteiso-C(17:0), and C(16:0) were the predominant cellular fatty acids. The G+C content of the DNA was 49.5 mol%. A phylogenetic analysis based on 16S rRNA showed that strain TW1(T) belonged within the genus Paenibacillus and was closely related to Paenibacillus cellulosilyticus LMG 22232(T), P. curdlanolyticus KCTC 3759(T), and P. kobensis KCTC 3761(T) with 97.7, 97.5, and 97.3% sequence similarity, respectively. The DNA-DNA hybridization values between the isolate and type strains of P. cellulosilyticus LMG 22232(T), P. curdlanolyticus KCTC 3759(T), and P. kobensis KCTC 3761(T) were found to be 18.6, 18.3, and 18.0%, respectively. The protein and xylanase patterns of strain TW1(T) were quite different from those of the type strains of closely related Paenibacillus species. On the basis of DNA-DNA relatedness and phenotypic analyses, phylogenetic data and the enzymatic pattern presented in this study, strain TW1(T) should be classified as a novel species of the genus Paenibacillus, for which the name Paenibacillus xylaniclasticus sp. nov. is proposed. The type strain is TW1(T) (=NBRC 106381(T) =KCTC 13719(T) =TISTR 1914(T)).     

The development of granulation in an upflow floc digester and an upflow anaerobic sludge blanket digester treating cane juice stillage

Summary The use of synthetic polyelectrolytes in the Upflow Floc digester during the treatment of high strength cane juice stillage, resulted in a more rapid accumulation of biomass and promoted granule formation at an earlier stage compared to a control upflow anaerobic sludge blanket digester (UASB). In the Upflow Floc reactor the granules were composed of rod shaped organisms, whereas in the UASB the granules were primarily filamentous. Both types of granules had good settling properties and high activities.     

Preparation techniques for the electron microscopy of granular sludge from an anaerobic digester

Several fixation and dehydration techniques for scanning and transmission electron microscopy of glycocalyx and microbial populations within granules from an upflow anaerobic sludge blanket digester purifying a brewery wastewater were compared. Sputter-cryo and freeze-drying techniques prior to scanning electron microscopy (SEM) allowed viewing of the glycocalyx. In contrast standard fixation and dehydration techniques were suitable for examination of underlying microbial populations by both SEM and transmission electron microscopy.     

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA–DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.     

Preservation characteristics of anaerobic granular sludge

Summary The preservation characteristics of granular sludges were investigated in terms of storing age and environmental condition. As a preservative index, physicochemical, microbiological, and morphological parameters were measured in order to quantify the change in granular sludge. Results from this research indicated that, as the anaerobic granular sludge had an outstanding preservation characteristic, UASB system could be effectively applied to the industries operating seasonally or intermittently.     

Operating a full-scale poultry manure anaerobic digester

A full-scale, completely-mixed digester, with a liquid capacity of 587 m³, was constructed to process the manure from 70 000 caged layers. Biogas from the digester was used as fuel for an engine/generator set. The operating temperature was maintained at 35°C using waste heat from the engine. The digester was operated on a 22–24 day HRT. Digester influent averaged 5·90% TS, 5250 ppm TKN, and 3790 ppm NH₃N. Digester effluent averaged 3·11% TS, 5090 ppm TKN, and 4060 ppm NH₃N.Sustained operation of the digester was achieved during the period of study (8/83−4/85). During this period biogas production averaged 0·38 m³ kg⁻¹ VS added (0·58 m³ kg⁻¹ VS destroyed). The CH₄ content averaged 58·0%. The major operational problem encountered was grit accumulation in the digester. This problem was reduced by settling most of the grit from the manure prior to the digester. Biogas production was reduced when concentrated lagoon-liquid was used as make-up water. Approximately 22% of the electricity produced was required for operating the system.     

Rapid startup and high rate nitrogen removal from anaerobic sludge digester liquor using a SNAP process

In this study, a single-stage autotrophic nitrogen removal reactor, packed with a novel acrylic fiber biomass carrier material (Biofix), was applied for nitrogen removal from sludge digester liquor. For rapid start-up, conventional activated sludge was added to the reactor soon after the attachment of anammox biomass on the Biofix carriers, which allowed conventional activated sludge to form a protective layer of biofilm around the anammox biomass. The Nitrogen removal efficiency reached 75% within 1 week at a nitrogen loading rate of 0.46 kg-N/m³/day for synthetic wastewater treatment. By the end of the synthetic wastewater treatment period, the maximum nitrogen removal rate had increased to 0.92 kg-N/m³/day at a nitrogen loading rate of 1.0 kg-N/m³/day. High nitrogen removal rate was also achieved during the actual raw digester liquor treatment with the highest nitrogen removal rate being 0.83 kg-N/m³/day at a nitrogen loading rate of 0.93 kg-N/m³/day. The thick biofilm on Biofix carriers allowed anammox bacteria to survive under high DO concentration of 5–6 mg/l resulting in stable and high nitrogen removal performance. FISH and CLSM analysis demonstrated that anammox bacteria coexisted and surrounded by ammonium oxidizing bacteria.     

Mixing characteristics of fixed film anaerobic reactors

Summary Since the mixing characteristics of anaerobic reactors would appear to have an effect on their operational performance, lithium tracer studies were made on two different digester types; the upflow sludge blanket and the expanded bed reactors. The mixing characteristics of both types of reactor, defined by this technique, were found to be of the intermediate type with a bias towards good mixing.     

Novel predominant archaeal and bacterial groups revealed by molecular analysis of an anaerobic sludge digester

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anaerobic sludge digester. Two 16S rRNA gene libraries were constructed using total genomic DNA, and amplified by polymerase chain reaction (PCR) using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 246 and 579 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was performed using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota and Crenarchaeota. Interestingly, we detected a novel monophyletic group of 164 clones representing 66.6% of the archaeal library. Culture enrichment and probe hybridization show that this group grows better under formate or H2-CO2. Within the bacterial library 95.6% of the operational taxonomic units (OTUs) represent novel putative phylotypes never described before, and affiliated with eight divisions. The Bacteroidetes phylum is the most abundant and diversified phylogenetic group representing 38.8% of the OTUs, followed by the gram-positives (27.7%) and the Proteobacteria (21.3%). Sequences affiliated with phylogenetic divisions represented by few cultivated representatives such as the Chloroflexi, Synergistes, Thermotogales or candidate divisions such as OP9 and OP8 are represented by <5% of the total OTUs. A comprehensive set of 15 16S and 23S rRNA-targeted oligonucleotide hybridization probes was used to quantify these major groups by dot blot hybridization within 12 digester samples. In contrast to the clone library, Firmicutes and Actinobacteria together accounted for 21.8 +/- 14.9% representing the most abundant phyla. They were surprisingly followed by the Chloroflexi representing 20.2 +/- 4.6% of the total 16S rRNA. The Proteobacteria and the Bacteroidetes group accounted for 14.4 +/- 4.9% and 14.5 +/- 4.3%, respectively, WWE1, a novel lineage, accounted for 11.9 +/- 3.1% while Planctomycetes and Synergistes represented <2% each. Using the novel set of probes we extended the coverage of bacterial populations from 52% to 85.3% of the total rRNA within the digester samples.     

Flow pattern visualization in a mimic anaerobic digester using CFD

Three-dimensional steady-state computational fluid dynamics (CFD) simulations were performed in mimic anaerobic digesters to visualize their flow pattern and obtain hydrodynamic parameters. The mixing in the digester was provided by sparging gas at three different flow rates. The gas phase was simulated with air and the liquid phase with water. The CFD results were first evaluated using experimental data obtained by computer automated radioactive particle tracking (CARPT). The simulation results in terms of overall flow pattern, location of circulation cells and stagnant regions, trends of liquid velocity profiles, and volume of dead zones agree reasonably well with the experimental data. CFD simulations were also performed on different digester configurations. The effects of changing draft tube size, clearance, and shape of the tank bottoms were calculated to evaluate the effect of digester design on its flow pattern. Changing the draft tube clearance and height had no influence on the flow pattern or dead regions volume. However, increasing the draft tube diameter or incorporating a conical bottom design helped in reducing the volume of the dead zones as compared to a flat-bottom digester. The simulations showed that the gas flow rate sparged by a single point (0.5 cm diameter) sparger does not have an appreciable effect on the flow pattern of the digesters at the range of gas flow rates used.     

Rheological characteristics of highly concentrated anaerobic digested sludge

Highly concentrated anaerobic sludge digestion is a favorable way to treat a considerable quantity of sewage sludge and recover biogas from both environmental and energetic point of view. For enhancing mass transfer and ensuring efficient anaerobic digestion, pumping and mixing are crucial operations which are directly conditioned by the rheology of sludge. The present work aims at studying the rheological characteristics of highly concentrated anaerobic digested sludge with TS (total solid) content more than 8%. The dependence on both TS content and temperature is investigated, respectively, in dynamic and flow measurements. The results show a shear-thinning behavior with a yield stress under flow measurements and a viscoelastic property in dynamic measurements, comparable to a pasty material. The Herschel–Bulkley model can describe these experimental results and a master curve is then proposed. In addition, the yield stress as well as cohesion energy increase with TS content following a power-law. The effect of temperature is relatively smaller, as a consequence of pronounced internal interactions strengthening the interior structure after the digestion, in the form of networks and steric linking.