Role of Microbial Community of Sludge from a Pilot-Scale Reactor Treating Real Wastewater in Simultaneous Biological Removal of Nitrogen and Sulfur (sulphur)

Fonseca, D. F.1 and Foresti E,

1Laboratory of Biological Processes, Department of Hydraulics and Sanitation, São Carlos School of Engineering, University of São Paulo


Nutrients levels reduction from wastewater can be accomplished by biological processes as autotrophic denitrification. Microbial communities in denitrifying reactor treating real sewage with nitrate as electron acceptor and sulfide as electron donor were analyzed by DGGE of PCR-amplified 16S rRNA gene fragments and sequencing. Bacterial populations were changed in two 30-day period conditions according to substrate composition: 100% non-nitrified anaerobic effluent (C1) and 20 – 30% anaerobic plus 70 – 80% nitrified effluent (C2). Sequences were clustered within Acidobacteria, Chlroroflexi, Ralstonia and Cupriavidus genera. Morphologies included: rods, cocci, filaments, and sulfate-reducing bacteria-like. Denitrifying bacteria were estimated by MPN (Most Probable Number) at the end of conditions: 9.2 x 1021 and 1.6 x 1022 cells/100 mL of culture, respectively. These results provide insights into dynamics of bacterial communities in autotrophic denitrification and showed that 16S rRNA-based molecular approach provides identification of denitrifiers in environments samples.


Nutrients levels reduction from waste water can be accomplished by a variety of physical, chemical and biological processes. Nitrate is a common pollutant in water bodies and may causes eutrophication. Hydrogen sulfide is regarded as an environmental problem due to its toxicity, odor, corrosive properties, and chemical oxygen demand (Wang and Chapman, 1999).

Dissimilatory reduction of nitrate or nitrite to N2 with energy conservation is termed denitrification and constitutes the global nitrogen cycle in combination with nitrogen fixation, nitrification, and ammonification. Autotrophic denitrification consists on oxidation of inorganic sources coupled to N oxide reduction and shows its significant impacts on ecosystems by linking the three important elemental cycles: sulfur, nitrogen, and carbon (Shao et al., 2010).

The process presents ecological significance and important applications in environmental technology especially if it driven by reduced sulfur compounds. Sulfide could be oxidized to insoluble elemental sulfur or sulfate by sulfide oxidizing bacteria (SOB) communities using nitrate or nitrite as electron acceptor. Elemental sulfur can be physically removed from effluents for reuse by sedimentation or slow rate sand filtration, while nitrate and nitrite introduced were converted to nitrogen gas that would not cause secondary pollution. Nitrate and nitrite generated after nitrification can be circulated to these waste waters containing nitrogenous contaminants to promote sulfide oxidation. Sulfide-utilizing denitrification can remove sulfide and prevents emission of denitrification byproducts such as nitrous oxide (Li et al., 2009).

Denitrifying bacteria strains have been isolated to anaerobically oxidize inorganic sulfur compounds such as sulfide, sulfur, thiosulfate and sulfite by nitrate reduction to nitrogen gas (Kim et al., 2004; Moon et al., 2004).

Denitrification using reduced sulfur compounds offers a great biotechnology potential. The process provides simultaneous removal of nitrogen and sulfur in a single phase system and transforms such contaminants in environmentally acceptable forms (nitrogen gas and sulfate or elemental sulfur). It has been studied for drinking water treatment (Sierra-Alvarez et al., 2007), simultaneous removal of nitrogen and sulfur from petrochemical industries effluents (Cardoso et al., 2006) and nitrogen removal from waste water (Foresti et al., 2006) specially for treatment of low concentration organic matter sewage and H2S removal from biogas.

The aim of this work was to study the application of autotrophic denitrification process to post-treatment of anaerobic reactors applied in waste water treatment by operating a pilot-scale reactors system and characterization of its microbial diversity. This system design was based on employing generated sulfide in the anaerobic compartment as electron donor for denitrification and others electron donors alternative sources.

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