Post-treatment of a slaughterhouse wastewater: stability of the microbial community of a sequencing batch reactor operated under oxygen limited conditions.

Slaughterhouse wastewater is a complex effluent with an important content of organic nitrogen. After an anaerobic treatment where most of the organic matter is removed, the nitrogen, remains as ammonium and post-treatment of the effluent is necessary. Sequencing batch reactor (SBR) technology has been developed to completely remove nitrogen in one single reactor combining aerobic and anoxic stages. Under oxygen limited conditions only nitrite is produced with concomitant energy saving. The stability and diversity of the microbial community from a nitrifying denitrifying SBR operated under oxygen limited conditions were studied using molecular and respirometric methods. The AOB (ammonia oxidizing bacteria) community was relatively stable Nitrosomonas being the dominant genera although Nitrosospira and Nitrosococcus were detected in low proportions. Nitrite oxidizing bacteria were out competed during the operation under oxygen-limited conditions. After an increase of the DO in the reactor Nitrobacter spp were detected suggesting that they remained in the system. Changes in the AOB and denitrifying communities were observed after the DO increase. Sedimentation problems were detected during operation, this could be related to the predominance of Thauera spp detected by FISH and T-RFLP.

Evolution of the bacterial community during granules formation in denitrifying reactors followed by molecular, culture-independent techniques.

The microbial community in two acetate-fed denitrifying reactors, inoculated with methanogenic sludge, was monitored by 16S rDNA-based methods (SSCP and FISH). Both reactors converged to similar, stable communities. The predominant organisms belonged to the genera Thauera, Paracoccus and Denitrobacter, detected both by molecular and culture-based methods.

Sludge bed development in denitrifying reactors using different inocula-performance and microbiological aspects.

Aerobic and methanogenic consortia were evaluated as inocula for laboratory scale denitrifying reactors, fed with a synthetic wastewater with acetate as the main electron donor. The denitrifying microflora of inocula and reactors was evaluated by specific denitrifying activity, enumeration and isolation of denitrifiers, which were screened by amplified ribosomal DNA restriction analysis. Reactor performance was monitored by COD and nitrate removal efficiencies and granule size. The aerobic sludge failed to form granules, probably due to the development of a filamentous, nitrate-reducing organism which was characterised by 16SrDNA sequencing as Bacillus cereus. The methanogenic sludge showed denitrifying activity and adapted very rapidly to denitrifying conditions in the two reactors seeded with granules of different sizes. Denitrifiers grew around the granules, increasing the specific denitrifying activity of the sludge over 10-fold. Exopolymer-forming organisms, belonging to the same species, were isolated from both reactors. Granule size increased during operation, but flotation of the aggregates, related to gas retention was observed.

Community analysis of a denitrifying reactor treating landfill leachate.

The bacterial community of a denitrifying reactor from a system for landfill leachate decontamination was studied applying cultivation methods, denitrifying activity measurements and characterisation of community 16S rDNA. The sludge presented a high denitrifying activity but a relatively low number of denitrifying bacteria as determined by most probable number. Over 50% of the sequences retrieved in the molecular analysis were related to genera with the capacity to denitrify in the alpha- and beta-subdivisions of the Proteobacteria. Fifteen percent of the DNAs were related to not yet cultured organisms belonging to the green non-sulphur phylum. High similarity values between sequences from isolates and clones were observed.

Comamonas nitrativorans sp. nov., a novel denitrifier isolated from a denitrifying reactor treating landfill leachate.

A group of Gram-negative denitrifying bacteria has been isolated from a denitrifying reactor treating landfill leachate. The new isolates produced both oxidase and catalase and showed growth on acetate, butyrate, n-caproate, i-butyrate, i-valerate, propionate, n-valerate, lactate, alanine, benzoate, phenylalanine and ethanol. No growth was observed on sugars. The bacteria could perform anoxic reduction of nitrate, nitrite and nitrous oxide to nitrogen, coupled to the oxidation of the same substrates as those used under aerobic conditions, except for aromatic compounds. They were very efficient denitrifiers, as estimated from the specific rate of N2 gas production. All the strains showed the same 16S rDNA restriction profile and one of them, designated 23310T, was selected for phylogenetic analysis. The organism clustered within the family Comamonadaceae, being related to Comamonas terrigena (95.8% sequence similarity). On the basis of the phylogenetic analysis, physiological characterization and the ability to efficiently reduce nitrate to N2, it is proposed that the bacterium be assigned to a new species, Comamonas nitrativorans. The type strain is 23310T (= DSM 13191T = NCCB 100007T = CCT 7062T).

Evaluation of the denitrifying microbiota of anoxic reactors.

Removal of inorganic nitrogen compounds from wastewaters can be accomplished by a combination of the biological processes of nitrification and denitrification. The information on the microbiota present in denitrifying reactors is still scarce. In the present work the evaluation of the denitrifying microbiota of different reactor sludges was performed by specific activity measurements and MPN count of denitrifiers. We also present the isolation and physiological and phylogenetic characterisation of denitrifying bacteria from the anoxic reactor of a combined system treating landfill leachate. Specific denitrifying activity measurements were faster to perform and more reliable than MPN enumerations. 16S rDNA characterisation of the isolates showed that they belonged to the genera Thauera, Acidovorax and Alcaligenes and were closely related to microorganisms retrieved from ecosystems rich in recalcitrant compounds. Two of the isolates could grow on aromatic compounds as sole carbon source.

Thiosulfate reduction and alanine production in glucose fermentation by members of the genus Coprothermobacter.

Coprothermobacter platensis is an anaerobic, proteolytic, thermophilic bacterium, which is phylogenetically related to the genera Fervidobacterium and Thermotoga. The organism was found to reduce thiosulfate to sulfide during growth on carbohydrates and proteinaceous substrates. Growth on glucose was inhibited by hydrogen, but this inhibition was overcome by thiosulfate reduction, stirring, increasing the headspace volume and coculturing with a hydrogen-consuming methanogen. Alanine was detected during glucose fermentation, its formation was influenced by the hydrogen concentration in the gas phase suggesting an electron sink mechanism, as was previously reported for the phylogenetically related Thermotogales and the archaeal hyperthermophile Pyrococcus furiosus.

Coprothermobacter platensis sp. nov., a new anaerobic proteolytic thermophilic bacterium isolated from an anaerobic mesophilic sludge.

A new anaerobic, proteolytic, moderately thermophilic bacterium, strain 3RT, was isolated from a methanogenic mesophilic reactor treating protein-rich wastewater. The cells were Gram-negative, non-spore-forming, non-motile rods. The DNA base composition was 43 mol% G + C. The optimum pH and temperature for growth were 7.0 and 55 degrees C respectively. The bacterium fermented gelatin, casein, bovine albumin, peptone and yeast extract. Glucose, fructose, sucrose, maltose and starch were poorly fermented. The major fermentation products from glucose were acetate, CO2 and H2 and, from gelatin, propionate was also detected. Growth on glucose was stimulated by thiosulfate, which was reduced to sulfide. Sulfate and nitrate were not reduced. 16S rRNA gene analysis revealed that the isolated bacterial strain was phylogenetically related to Coprothermobacter proteolyticus (96.3% sequence similarity), the only known species within the genus. DNA-DNA hybridization analysis demonstrated a very low level of homology, indicating that the isolated strain and C. proteolyticus were not related at species level. Therefore, it is proposed to classify the described strain in the genus Coprothermobacter as a new species, Coprothermobacter platensis. The type strain of C. platensis is strain 3RT (= DSM 11748T).

Caloramator proteoclasticus sp. nov., a new moderately thermophilic anaerobic proteolytic bacterium.

A new moderately thermophilic proteolytic anaerobe, strain UT, was isolated from mesophilic granular methanogenic sludge. The cells were spore-forming, motile rods that were 0.4 micron wide and 2.4 to 4 microns long and stained gram negative. Electron micrographs of thin sections revealed the presence of an atypical gram-positive cell wall. Optimum growth occurred at 55 degrees C and at pH values between 7.0 and 7.5, with a doubling time of 30 min. The DNA base ratio of guanine plus cytosine was 31 mol%. The bacterium fermented proteins mainly to acetate, hydrogen, formate, and branched-chain fatty acids. Several amino acids, including glutamate, aspartate, arginine, histidine, threonine, methionine, and branched-chain amino acids, were also utilized. Glutamate was degraded to acetate, formate, hydrogen, and alanine. In addition, the strain degraded carbohydrates, including glucose, fructose, mannose, cellobiose, and starch, to acetate, ethanol, formate, lactate, and hydrogen. The results of a 16S rRNA sequence analysis phylogenetically placed strain UT in the low-guanine-plus-cytosine-content subgroup of the gram-positive phylum. We propose to classify the described strain in the genus Caloramator as a new species, Caloramator proteoclasticus. The type strain of C. proteoclasticus, strain U, has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 10124.

Characterization of a methanogenic sludge to be used as inoculum for a high-rate reactor.

The sludge of an anaerobic lagoon treating the wastewater from a factory producing baker's yeast was evaluated as inoculum for anaerobic digestion. Specific methanogenic activity tests failed to give a good estimation of the trophic groups that were evidenced by enumerations involving Most Probable Number estimations. This failure was ascribed to the toxic effects of either the acetate concentrations used or to the ammonia content of the sludge.