PhD dissertation by Alexandre Sànchez Melsió “From inocula to biological reactors: molecular characterization of N-cycle bacterial assemblages in a PANAMMOX® process”

The thesis has been directed by UdG professors Dr Jesús Colprim from LEQUIA research group (The Laboratory of Chemical and Environmental Engineering) and Dr Xavier Vila from gEMM (Group of Molecular Microbial Ecology). The defence will take place next Friday 5th May at 11:30h, at University of Girona LEAR building (entrance at Aulari comú), Sala d’Actes – Campus Montilivi.


The PANAMMOX® process

Wastewater treatments plants (WWTPs) commonly use two sequential processes (nitrification and denitrification) for the nitrogen compounds removal, which are converted to dinitrogen gas and transferred to the atmosphere. It is an expensive process, because it requires a high contribution of oxygen in the former step, and organic matter in the latter. In the mid 90s a shortcut in the nitrogen cycle was discovered, in which ammonium and nitrite are converted to dinitrogen gas without oxygen and organic matter requirements. This novel pathway, called anaerobic ammonium oxidation, is carried out by a single group of bacteria (anammox). However, a previous step conducted by ammonium-oxidizing bacteria (AOB) is required to convert part of the ammonium into nitrite, to be able to use anammox bacteria for nitrogen removal in an efficient way. Thus, ammonium and nitrite levels are regulated and a suitable proportion is provided to anammox bacteria. The PANAMMOX® process, developed in the Laboratory of Chemical and Environmental Engineering of the University of Girona, allows to successfully treat wastewater through a sequential combination of both processes, in separate compartments.


Optimization of anammox inocula isolation and identification of new bacteria

The main goals of the doctoral thesis “From inocula to biological reactors: molecular characterization of N-cycle bacterial assemblages in a PANAMMOX® process” by Alexandre Sànchez Melsió have been the research of a method to easily find suitable inocula for an anammox reactor, since these bacteria are not easily found in nature and they are slow-growers (they have a duplication time of 15-30 days), and the characterization by molecular techniques of the different types of bacteria involved in both compartments of the PANAMMOX® process.


Several enrichments from different origins were performed to find the required inocula for the anammox reactor and to optimize the procedure to achieve them. Anammox bacteria were detected by molecular and chemical techniques after a long enrichment period in 5 of these enrichments, belonging to natural systems (brackish coastal lagoon), modified environments (artificial wetland system) and artificial environments (anoxic reactors and WWTPs). The application of molecular techniques to the enrichments allowed the development of protocols for an early detection of anammox bacteria in low-concentrated samples. Thus, some bacterial species were identified to be useful as potential indicators of the capacity of a sample to further develop dense anammox bacterial populations.


Molecular techniques also allowed the detection of some bacterial groups that often go along with anammox bacteria, using data obtained from enrichments of different origins. A bacterial group pattern has been identified, which appears repeatedly in several studies, thus suggesting that they could have some kind of relationship with anammox bacteria in a structural or metabolic level. Finally, a molecular characterization of the N-oxidizing bacteria was performed in the reactor where ammonium is partially oxidized to nitrite, as a previous step for the anammox reaction. The changes in these bacterial populations were analyzed throughout the adaptation process of the reactor for treating wastewater with high nitrogen concentrations, going from Nitrosomonas species common8 in environments with low-nitrogen concentrations to other species already known from highly-nitrogenated environments. Besides, it was determined that some species that theoretically should be washed out by the reactor performance (such as Nitrospira species) not only remained but even were able to grow. However, their presence was not detrimental for the reactor performance.


Thus, a better comprehension of bacterial groups that are involved in the so-called PANAMMOX® process developed by LEQUIA has been achieved through molecultar techniques: another step to optimize the sustainable treatment of wastewater with high nitrogen contents.


Photo: Confocal microphotography of a sample belonging to an anammox enrichment, treated with specific fluorescent markers. Anammox bacteria are stained in pink, whereas the rest of bacteria are depicted in blue.

Additional Info

  • Date: 2015-6-5

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Laboratory of Chemical and Enviromental Engineering

Institut de Medi Ambient
Universitat de Girona
Campus Montilivi
17003 Girona

Parc Científic i Tecnològic de la UdG
Edifici Jaume Casademont, Porta B
Pic de Peguera, 15
17003 Girona
Tel. +34 972 41 98 59




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