Juliana Calábria de Araújo

Effect of phenol on the nitrogen removal performance and microbial community structure and composition of an anammox reactor.

The effects of phenol on the nitrogen removal performance of a sequencing batch reactor (SBR) with anammox activity and on the microbial community within the reactor were evaluated. A phenol concentration of 300 mg L(-1) reduced the ammonium-nitrogen removal efficiency of the SBR from 96.5% to 47%. The addition of phenol changed the microbial community structure and composition considerably, as shown by denaturing gradient gel electrophoresis and 454 pyrosequencing of 16S rRNA genes. Some phyla, such as Proteobacteria, Verrucomicrobia, and Firmicutes, increased in abundance, whereas others, such as Acidobacteria, Chloroflexi, Planctomycetes, GN04, WS3, and NKB19, decreased. The diversity of the anammox bacteria was also affected by phenol: sequences related to Candidatus Brocadia fulgida were no longer detected, whereas sequences related to Ca. Brocadia sp. 40 and Ca. Jettenia asiatica persisted. These results indicate that phenol adversely affects anammox metabolism and changes the bacterial community within the anammox reactor.

Impact of inocula and operating conditions on the microbial community structure of two anammox reactors

The microbial community structure of the biomass selected in two distinctly inoculated anaerobic oxidation of ammonium (anammox) reactors was investigated and compared with the help of data obtained from 454-pyrosequencing analyses. The anammox reactors were operated for 550 days and seeded with different sludges: sediment from a constructed wetland (reactor I) and biomass from an aerated lagoon part of the oil-refinery wastewater treatment plant (reactor II). The anammox diversity in the inocula was evaluated by 16S rRNA gene-cloning analysis. The diversity of anammox bacteria was greater in the sludge from the oil-refinery (three of the five known genera of anammox were detected) than in the wetland sludge, in which only Candidatus Brocadia was observed. Pyrosequencing analysis demonstrated that the community enriched in both reactors had differing compositions despite the nearly similar operational conditions applied. The dominant phyla detected in both reactors were Proteobacteria, Chloroflexi, Planctomycetes, and Acidobacteria. The phylum Bacteroidetes, which is frequently observed in anammox reactors, was not detected. However, Acidobacteria and GN04 phyla were observed for the first time, suggesting their importance for this process. Our results suggest that, under similar operational conditions, anammox populations (Ca. Brocadia sinica and Ca. Brocadia sp. 40) were selected in both reactors despite the differences between the two initial inocula. Taken together, these results indicated that the type of inoculum and the culture conditions are key determinants of the general microbial composition of the biomass produced in the reactors. Operational conditions alone might play an important role in anammox selection.

How to use molecular biology tools for the study of the anaerobic digestion process

Anaerobic digestion is used with success for the treatment of solid waste, urban and industrial effluents with a concomitant energy production. The process is robust and stable, but the complexity of the microbial community involved in the process is not yet fully comprehensive. Nowadays, the study of this complex ecosystem is facilitated by the availability of different molecular tools, but it is very important to choose the adequate tool to answer specific questions. The aim of this review is to describe different molecular techniques, indicate the questions that can be addressed by each technique, enumerate their limitations and give practical advices for their use. Examples of how the molecular tools have been used to address various questions in anaerobic digestion are presented. The key point now is to apply all this information to improve anaerobic digestion. The integration of concepts of microbial-ecology, environmental-engineering, modeling and bioinformatics is currently necessary.

Anammox for nitrogen removal from anaerobically pre-treated municipal wastewater: Effect of COD/N ratios on process performance and bacterial community structure.

Long-term effects of COD/N ratios on the nitrogen removal performance and bacterial community of an anammox reactor were evaluated by adding a synthetic medium (with glucose) and real anaerobic effluent to a SBR. At a COD/N ratio of 2.8 (COD, 390mg·L(-1)) ammonium removal efficiency was 66%, while nitrite removal remained high (99%). However, at a COD/N ratio of 5.0 (COD, 300mg·L(-1)), ammonium and nitrite removal efficiencies were high (84% and 99%, respectively). High COD, nitrite, and ammonium removal efficiencies (80%, 90% and 95%, respectively) were obtained on adding anaerobically pre-treated municipal wastewater (with nitrite) to the reactor. DGGE revealed that the addition of anaerobic effluent changed the bacterial community structure and selected for DNA sequences related to Brocadia sinica and Chloroflexi. Adding glucose and anaerobic effluent increased denitrifiers concentration threefold. Thus, the possibility of using the anammox process to remove nitrogen from anaerobically pre-treated municipal wastewater was demonstrated.

Organic loading rate and food-to-microorganism ratio shape prokaryotic diversity in a demo-scale up-flow anaerobic sludge blanket reactor treating domestic wastewater

We investigated the microbial community in an up-flow anaerobic sludge blanket (UASB) reactor treating domestic wastewater (DW) during two different periods of organic loading rate (OLR) and food-to-microorganism (F/M) ratio. 16S rDNA clone libraries were generated, and quantitative real-time PCR (qPCR) analyses were performed. Fluctuations in the OLR and F/M ratio affected the abundance and the composition of the UASB prokaryotic community, mainly at the species level, as well as the performance of the UASB reactor. The qPCR analysis suggested that there was a decrease in the bacterial cell number during the rainy season, when the OLR and F/M ratio were lower. However, the bacterial diversity was higher during this time, suggesting that the community degraded more diversified substrates. The diversity and the abundance of the archaeal community were higher when the F/M ratio was lower. Shifts in the methanogenic community composition might have influenced the route of methane production, with methane produced by acetotrophic methanogens (dry season), and by hydrogenotrophic, methylotrophic and acetotrophic methanogens (rainy season). This study revealed higher levels of bacterial diversity, metabolic specialization and chemical oxygen demand removal efficiency of the DW UASB reactor during the rainy season.

Bacterial community involved in the nitrogen cycle in a down-flow sponge-based trickling filter treating UASB effluent

The bacterial community composition of a down-flow sponge-based trickling filter treating upflow anaerobic sludge blanket (UASB) effluent was investigated by pyrosequencing. Bacterial community composition considerably changed along the reactor and over the operational period. The dominant phyla detected were Proteobacteria, Verrucomicrobia, and Planctomycetes. The abundance of denitrifiers decreased from the top to the bottom and it was consistent with the organic matter concentration gradients. At lower loadings (organic and nitrogen loading rates), the abundance of anammox bacteria was higher than that of the ammonium-oxidizing bacteria in the upper portion of the reactor, suggesting that aerobic and anaerobic ammonium oxidation occurred. Nitrification occurred in all the compartments, while anammox bacteria prominently appeared even in the presence of high organic carbon to ammonia ratios (around 1.0-2.0 gCOD gN(-1)). The results suggest that denitrifiers, nitrifiers, and anammox bacteria coexisted in the reactor; thus, different metabolic pathways were involved in ammonium removal in the post-UASB reactor sponge-based.

Microbial communities in anammox reactors: a review

ABSTRACT The anammox (anaerobic ammonium oxidation) process has been used to remove nitrogen from wastewaters and is considered a promising approach due to its advantages over conventional processes (nitrification and denitrification). The development of molecular biology tools has allowed for great advances in describing the microbial communities in anammox reactors, which is important to understand the complex reactions and interactions that occur inside these systems. This study presents a review of the microbial diversity studies in anammox reactors. Several researchers have investigated the microbial community composition in anammox reactors, in order to elucidate the roles that the different microbial groups (besides the anammox bacteria) play in these systems. The microbial communities of anammox reactors appear to be affected by several factors, such as the configuration of the reactors, the biomass growth mode inside of the reactors, operational conditions and type of inoculum used for start-up. However, independent of reactors characteristics, some microbial groups such as Proteobacteria and Chloroflexi have always been found with Planctomycetes in anammox reactors. Moreover, other microbial groups such as Chlorobi, Acidobacteria and Bacteroidetes are often detected in these systems. Identifying the roles and interactions of such microorganisms inside anammox reactors is a great challenge for future studies.

Enrichment and activity of methanotrophic microorganisms from municipal wastewater sludge

In this study, methanotrophic microorganisms were enriched from a municipal wastewater sludge taken from an Upflow Anaerobic Sludge Blanket reactor. The enrichment was performed in a sequencing batch reactor (SBR) with an autotrophic medium containing nitrite and nitrate. The microbial community composition of the inoculum and of the enrichment culture after 100 days of SBR operation was investigated and compared with the help of data obtained from 454 pyrosequencing analyses. The nitrite and nitrate removal efficiencies were 68% and 53%, respectively, probably due to heterotrophic denitrification. Archaeal cells of the anaerobic methanotrophic Archaic (ANME)-I and ANME-II groups were detected by polymerase chain reaction throughout the whole cultivation period. Pyrosequencing analysis showed that community composition was different among the two samples analysed. The dominant phyla found in the inoculum were Synergistestes, Firmicutes and Euryarchaeota, while Planctomycetes, Verrucomicrobia, Chloroflexi and Proteobacteria prevailed in the enriched biomass. The cultivation conditions decreased Methanobacterium abundance from 8% to 1%, and enriched for methanotrophic bacteria such as Methylocaldum, Methylocistis and Methylosinus. Sequences of Methylocaldum sp. accounted for 2.5% of the total reads. The presence and high predominance of Verrucomicrobia in the enriched biomass suggested that other unknown methanotrophic species related to that phylum might also have occurred in the reactor. Anaerobic methane oxidation activity was measured for both samples, and showed that the activity of the enrichment culture was nearly three times higher than the activity of the inoculum. Taken together, these results showed that the inoculum type and cultivation conditions were properly suited for methanotrophic enrichment.

Effect of temperature on microbial diversity and nitrogen removal performance of an anammox reactor treating anaerobically pretreated municipal wastewater

The effects of temperature reduction (from 35 °C to 20 °C) on nitrogen removal performance and microbial diversity of an anammox sequencing batch reactor were evaluated. The reactor was fed for 148 days with anaerobically pretreated municipal wastewater amended with nitrite. On average, removal efficiencies of ammonium and nitrite were high (96%) during the enrichment period and phases 1 (at 35 °C) and 2 (at 25 °C), and slightly decreased (to 90%) when the reactor was operated at 20 °C. Deep sequencing analysis revealed that microbial community structure changed with temperature decrease. Anammox bacteria (Ca. Brocadia and Ca. Anammoximicrobium) and denitrifiers (Burkholderiales, Myxococcales, Rhodocyclales, Xanthomonadales, and Pseudomonadales) were favoured when the temperature was lowered from 35 °C to 25 °C, while Anaerolineales and Clostridiales were negatively affected. The results support the feasibility of using the anammox process for mainstream nitrogen removal from anaerobically pretreated municipal wastewater at typical tropical temperatures.

Biological sulphide removal from anaerobically treated domestic sewage: reactor performance and microbial community dynamics

We developed a biological sulphide oxidation system and evaluated two reactors (shaped similar to the settler compartment of an up-flow anaerobic sludge blanket [UASB] reactor) with different support materials for biomass retention: polypropylene rings and polyurethane foam. The start-up reaction was achieved using microorganisms naturally occurring on the open surface of UASB reactors treating domestic wastewater. Sulphide removal efficiencies of 65% and 90% were achieved with hydraulic retention times (HRTs) of 24 and 12 h, respectively, in both reactors. However, a higher amount of elemental sulphur was formed and accumulated in the biomass from reactor 1 (20 mg S0 g−1 VTS) than in that from reactor 2 (2.9 mg S0 g−1 VTS) with an HRT of 24 h. Denaturing gradient gel electrophoresis (DGGE) results revealed that the the pink and green biomass that developed in both reactors comprised a diverse bacterial community and had sequences related to phototrophic green and purple-sulphur bacteria such as Chlorobium sp., Chloronema giganteum, and Chromatiaceae. DGGE band patterns also demonstrated that bacterial community was dynamic over time within the same reactor and that different support materials selected for distinct bacterial communities. Taken together, these results indicated that sulphide concentrations of 1–6 mg L−1 could be efficiently removed from the effluent of a pilot-scale UASB reactor in two sulphide biological oxidation reactors at HRTs of 12 and 24 h, showing the potential for sulphur recovery from anaerobically treated domestic wastewater.