Commercial formulation amendment transiently affects the microbial composition but not the biogas production of a full scale methanogenic UASB reactor.

The treatment of dairy wastewater in methanogenic reactors cause several problems due to their high lipid content. One strategy to overcome these problems is the use of commercial formulations. Here we studied the effect of adding a commercial formulation, designed to improve fat degradation, on both the microbial community composition and reactor performance. Samples from two full-scale Up-flow Anaerobic Sludge Blanket (UASB) reactors in parallel arrangement were analysed. The commercial product was added to one of the reactors while the other was used as control. The amendment increased significantly the fat removal but an accumulation of volatile fatty acids was detected. Nevertheless, no significant differences were observed in the total Chemical Oxygen Demand (COD) removal and biogas production between reactors. A significant change in the bacterial community was not detected by 16S rRNA gene Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis probably due to the limitation of the technique. A strong change in the composition of the phylum Firmicutes was detected with 16S rRNA gene amplicon sequencing; however, it didn't persist during the whole operation period. The relative abundance of minor Operational Taxonomic Units (OTUs) with sequences related to syntrophic bacteria increased with the amendment. Although a better hydrolytic capacity was obtained when adding the commercial product, the overall process did not improve and no increase in biogas production was detected. Alternative strategies could be applied to avoid the accumulation of intermediary products and improve biogas production as intermittent addition of the commercial product or batch operation of reactors.

Preliminary analysis of Chloroflexi populations in full-scale UASB methanogenic reactors.

AIMS: The phylum Chloroflexi is frequently found in high abundance in methanogenic reactors, but their role is still unclear as most of them remain uncultured and understudied. Hence, a detailed analysis was performed in samples from five up-flow anaerobic sludge blanket (UASB) full-scale reactors fed different industrial wastewaters. METHODS AND RESULTS: Quantitative PCR show that the phylum Chloroflexi was abundant in all UASB methanogenic reactors, with higher abundance in the reactors operated for a long period of time, which presented granular biomass. Both terminal restriction fragment length polymorphism and 16S rRNA gene amplicon sequencing revealed diverse Chloroflexi populations apparently determined by the different inocula. According to the phylogenetic analysis, the sequences from the dominant Chloroflexi were positioned in branches where no sequences of the cultured representative strains were placed. Fluorescent in situ hybridization analysis performed in two of the reactors showed filamentous morphology of the hybridizing cells. CONCLUSIONS: While members of the Anaerolineae class within phylum Chloroflexi were predominant, their diversity is still poorly described in anaerobic reactors. Due to their filamentous morphology, Chloroflexi may have a key role in the granulation in methanogenic UASB reactors. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results bring new insights about the diversity, stability, dynamics and abundance of this phylum in full-scale UASB reactors which aid in understanding their function within the reactor biomass. However, new methodological approaches and analysis of bulking biomass are needed to completely unravel their role in these reactors. Combining all this knowledge with reactor operational parameters will allow to understand their participation in granulation and bulking episodes and design strategies to prevent Chloroflexi overgrowth.

Work scheme to isolate the different micro-organisms found in hydrogen-producing reactors: a study of effectiveness by pyrosequencing analysis.

AIM: The aim of this research was to create a work scheme for the isolation of the different micro-organisms commonly found in hydrogen-producing reactors and to test its effectiveness. METHODS AND RESULTS: Methods were selected to isolate anaerobic spore-forming fermenters, anaerobic fermenters that do not form spores, facultative aerobic fermenters and lactic acid bacteria. The methods were tested in two samples taken from a hydrogen-producing reactor fed with cheese whey. 16S rRNA gene sequences from isolates were compared with pyrosequencing analysis from the same samples. The isolates represented more than 88% of the abundance detected by pyrosequencing. Organisms from the genera Clostridium, Rahnella, Megasphaera, Lactobacillus, Propionibacterium, Bifidobacterium, Chryseobacterium and Acetobacter were isolated. Hydrogen-producing capacity was confirmed for the Clostridium, Rahnella and Megasphaera isolates. Coculture experiments indicate that Megasphaera prevented the total inhibition of Clostridium by Lactobacillus. CONCLUSION: The work scheme proposed was effective to isolate most of the micro-organisms detected by pyrosequencing analysis. Physiological studies suggested a key role of Megasphaera. SIGNIFICANCE AND IMPACT OF THE STUDY: We showed the high culturability of the microbial communities from hydrogen-producing bioreactors. The isolates can be used to perform physiological studies to understand the H2 -producing process.

Microbial fuel cell coupled to biohydrogen reactor: a feasible technology to increase energy yield from cheese whey.

An important pollutant produced during the cheese making process is cheese whey which is a liquid by-product with high content of organic matter, composed mainly by lactose and proteins. Hydrogen can be produced from cheese whey by dark fermentation but, organic matter is not completely removed producing an effluent rich in volatile fatty acids. Here we demonstrate that this effluent can be further used to produce energy in microbial fuel cells. Moreover, current production was not feasible when using raw cheese whey directly to feed the microbial fuel cell. A maximal power density of 439 mW/m2 was obtained from the reactor effluent which was 1000 times more than when using raw cheese whey as substrate. 16S rRNA gene amplicon sequencing showed that potential electroactive populations (Geobacter, Pseudomonas and Thauera) were enriched on anodes of MFCs fed with reactor effluent while fermentative populations (Clostridium and Lactobacillus) were predominant on the MFC anode fed directly with raw cheese whey. This result was further demonstrated using culture techniques. A total of 45 strains were isolated belonging to 10 different genera including known electrogenic populations like Geobacter (in MFC with reactor effluent) and known fermentative populations like Lactobacillus (in MFC with cheese whey). Our results show that microbial fuel cells are an attractive technology to gain extra energy from cheese whey as a second stage process during raw cheese whey treatment by dark fermentation process.

Microbial community composition and reactor performance during hydrogen production in a UASB reactor fed with raw cheese whey inoculated with compost.

This study investigated the microbial community developed in a UASB reactor for hydrogen production and correlated it to reactor performance. The reactor was inoculated with kitchen waste compost and fed with raw cheese whey at two organic loading rates, 20 gCOD/Ld and 30 gCOD/Ld. Hydrogen production was very variable, using an OLR of 30 gCOD/Ld averaged 1.0 LH(2)/Ld with no methane produced under these conditions. The hydrogen yield was also very variable and far from the theoretical. This low yield could be explained by selection of a mixed fermentative population with presence of hydrogen producing organisms (Clostridium, Ruminococcus and Enterobacter) and other non-hydrogen producing fermenters (Lactobacillus, Dialister and Prevotella). The molecular analysis of the raw cheese whey used for feeding revealed the presence of three predominant organisms that are affiliated with the genera Buttiauxella (a low-yield hydrogen producer) and Streptococcus (a lactic acid-producing fermenter). Although these organisms did not persist in the reactor, the continuous addition of these fermenters could decrease the reactor's hydrogen yield.

A time-course analysis of four full-scale anaerobic digesters in relation to the dynamics of change of their microbial communities.

This study describes the microbial community richness, -dynamics, and -organization of four full-scale anaerobic digesters during a time-course study of 45 days. The microbial community was analyzed using a Bacteria- and Archaea-targeting 16S rRNA gene-based Terminal-Restriction Fragment Length Polymorphism approach. Clustering analysis separated meso- and thermophilic reactors for both archaeal and bacterial communities. Regardless of the operating temperature, each installation possessed a distinct community profile. For both microbial domains, about 8 dominant terminal-restriction fragments could be observed, with a minimum of 4 and a maximum of 14. The bacterial community organization (a coefficient which describes the specific degree of evenness) showed a factor 2 more variation in the mesophilic reactors, compared with the thermophilic ones. The archaeal community structure of the mesophilic UASB reactor was found to be more stable. The community composition was highly dynamic for Bacteria and Archaea, with a rate of change between 20-50% per 15 days. This study illustrated that microbial communities in full-scale anaerobic digesters are unique to the installation and that community properties are dynamic. Converging complex microbial processes such as anaerobic digestion which rely on a multitude of microbial teams apparently can be highly dynamic.

Sludge deterioration in a full scale UASB reactor after a pH drop working under low loading conditions.

A full scale UASB reactor treating the effluent of a malting plant was operated during nearly two years. During 37 weeks of operation the reactor worked with a COD removal efficiency of 80% and a biogas production of nearly 300 m(3)/d with a methane content of 77%. After the start up and during these months of operation the volumetric organic load was 4 kgCOD/m(3).d and the specific organic load was between 0.2-0.4 kgCOD/kgVSS.d. The sludge SMA in this period was around 0.25 kgCOD/kg VSS.d. On week 37 as a result of a problem at the industrial process the pH in the reactor dropped to a value of 4.8. After pH recovering, the reactor worked with fluctuating COD values in the exit and showed a downward trend in the COD removal efficiency. On week 81 the presence of filaments in the granules was detected. High proportion of Chloroflexi filaments were detected by FISH in the sludge. Changes in the microbial population caused by the low pH probably destabilize the reactor performance. The presence of filamentous granules in the sludge and its further growing could be encouraged by the pH drop and the low specific organic load applied to the reactor. The low specific organic load was a consequence of the high VSS content in the UASB reactor, due to the lack of purges. The length of the filaments attached to the granules grew throughout time. In order to eliminate the sludge with poor settlement properties a recycle was applied to the reactor. As a consequence, low amount of granular sludge stayed in the reactor. At the end, COD concentration in the influent reached higher values than in normal operation; at the same time a complete sludge wash out occurred. On the other hand, using the same sludge (after the recycle implementation) in a bench scale reactor the good properties of the sludge were completely recovered.

Sequencing batch reactor as a post-treatment of anaerobically treated dairy effluent.

Wastewater from dairy industries, characterized by its high COD content and relative high COD/TKN ratio, requires post-treatment after anaerobic treatment to complete the removal of organic matter and nutrients. Due to its simplicity, robustness and low maintenance costs, sequencing batch reactors (SBR) result in an attractive system, especially in case of small dairy industries in order to comply with the emission standards. The goal of this work was to determine the operational parameters, optimize the performance, and study the stability of the microbial population of a SBR system for the post-treatment of an anaerobic pond effluent. High and stable removal of COD and TKN was achieved in the reactor, which can easily be set up in dairy industries. An active nitrifying population was selected during reactor operation and maintained relatively stable, while the heterotrophic (total and denitrifying) communities were more unstable and susceptible to changes in the operating conditions.

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.

Tc-99m MDP uptake in uterine leiomyoma.

A 47-year-old woman with adenocarcinoma of the right breast had bone scintigraphy with Tc-99m MDP. Bone imaging did not show any metastases. However, a large area of increased tracer uptake was seen extending from the abdomen to the pelvis. Abdominal ultrasound revealed a large solid and heterogeneous mass, measuring 18 x 11 x 14.3 cm, that originated in an empty uterus. A biopsy of the surgical specimen showed a leiomyoma of the uterus.

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).