Molecular study of the dynamics and structure of the biomass in different wastewater and off-gases treatment systems

  1. Rodriguez Rodriguez, Elisa Maria
Supervised by:
  1. Pedro A. García Encina Director

Defence university: Universidad de Valladolid

Fecha de defensa: 19 December 2011

  1. Alfons J. M. Stams Chair
  2. María Fernández-Polanco Secretary
  3. Manel Poch Espallargas Committee member
  4. María del Mar Peña Miranda Committee member
  5. Ana Segura Carnicero Committee member

Type: Thesis

Teseo: 318753 DIALNET lock_openTESEO editor


The adequate management of wastewater produced by humans, and off-gases coming from industries is crucial in order to prevent environmental and human health problems. During years engineers have designed systems in order to achieve this need. Systems which utilize the advantages of microbial community's amazing metabolic potential are among the most important biotechnological applications. Anaerobic and aerobic activated sludge wastewater treatments are key technologies in environmental biotechnology. Biofiltration is indisputably the most commonly employed biotechnology for odor treatment in wastewater treatment plants, but other techniques such as the activated sludge air diffusion bioreactors are emerging as reliable biotechnologies. However, knowledge of the eco-physiology of the microorganisms governing these systems, which is crucial for a good reactor design, operation and control, is limited. Cultivation-independent techniques (molecular techniques) can be used to improve this knowledge; solving some of the limitations imposed by the classical microbiological methods, e.g. only a minor part of the prokaryota present in an environmental sample is revealed. This work presents results from molecular microbiological investigations of several lab-scale wastewater and off-gases treatment systems. Anaerobic fluidized bed reactor (FBR) treating vinasse: the molecular analysis of the biomass (Archaea and Bacteria) of a fluidized bed reactor treating synthetic vinasse (acetate, propionate, butyrate, glucose and betaine) operated under different COD to sulfate ratios (8, 4 and 2) and anaerobic or micro-aerobic conditions was assessed by means of DGGE (Denaturing Gradient Gel Electrophoresis) technique, followed by sequencing and phylogenetic analysis. Results indicated that acetogens seemed to be mainly involved in glucose and betaine fermentation in the reactor, and they were substituted by other potential sugar and betaine degraders after oxygen application. Typical butyrate and propionate degraders were not detected, suggesting that sugar and betaine degraders outnumbered them in the DGGE analysis. The introduction of oxygen into the reactor led to the formation of elemental sulfur, and probably other sulfur compounds by sulfide-oxidizing bacteria (Gammaproteobacteria). Likely, the sulfur intermediates coming from sulfide oxidation were used by SRB, which belonged to the hydrogen-utilizing Desulfovibrio in the reactor, and other microorganism, as suggested by the detection of the sulfur-respiring Wolinella succiongenes. The archaeal communities detected along the whole reactor operation belonged to Methanomethylovorans and Methanosaeta, while hydrogenotrophic methanogens in the Methanobacterium genus were only detected at the highest substrate to sulfate ratio. Activated sludge (AS) air diffusion bioreactor treating gaseous streams: the shifts and composition of the microbial communities (Bacteria and Fungi) established in a lab-scale activated sludge air diffusion bioreactor treating a synthetic odorous emission composed by low concentrations of VOCs (toluene, butanone and alpha-pinene) and hydrogen sulfide, was studied by the DGGE molecular technique. The reactor was operated at different empty bed residence times (EBRTs), and under different operation fluctuations and failures. Despite the different EBRTs and the low inlet concentrations applied to the reactor, diversity and dynamics bacterial and fungal communities were detected, which linked with high removal efficiencies (REs) observed for all the compounds in the fed, except for the alpha-pinene. The low REs observed for alpha-pinene was linked to the absence of an alpha-pinene degrading community into the AS bioreactor. Seven different bacterial phyla were retrieved from the sequenced DGGE bands, while Proteobacteria followed by Actinobacteria were the dominant groups. Bacteria closely related to Comamonas, Nitrospira, Pseudonocardia, Rhodanobacter and Ensifer (Sinorhizobium) genera, which are characterized to be slow-growing organisms, could have an important role in the elimination of butanone or toluene in the system. Fungal communities were affiliated to the Ascomycota phylum, and species in the Fusarium and Fonsecaea genera might played a role in the degradation of VOCs. Although hydrogen sulfide REs remained high along the entire reaction run, typical sulfide oxidizers were not detected. Finally, the reactor showed a high robustness against the distinct operation fluctuations and failures. Under these conditions, bacterial and fungal diversity remained high, but the pH failure induced in the system caused the major decrease in fungal and bacterial biodiversity, as well as the worst removal efficiencies for the compounds in the feed. Anaerobic/anoxic/oxic activated sludge (AS) nutrient removal bioreactor: an activated sludge nutrient (phosphorus (P) and nitrogen (N)) removal bioreactor with an A2O configuration was constructed trying to mimic the real conditions found in the WWTP of Valladolid, Spain. The reactor was fed with real domestic wastewater and operated at different conditions, in order to observe its effect on the performance of the bioreactor and on the microbial communities. Different sludge retention times (SRTs) (19, 15 and 11 days), the addition of different external carbon sources (acetate, propionate and glucose) and the addition of high concentrations of heavy metals (Cd, Cr, Pb, Zn, Cr, Ni) were tested. The bacterial communities were studied by DGGE and FISH (Fluorescent in Situ Hybridization). Although statistically significant differences were observed in N and P removal efficiencies (REs) under different SRTs, shifts in bacterial community profiles were not very pronounced. The addition of an external carbon source and alkalinity was essential to obtain a good N and P performance, and a clear shift in bacterial profiles was observed after its addition. Phosphorus REs clearly increased with acetate as external carbon source, which linked with the detection of members on the Rhodocyclaceae, a group that includes the well-known PAO, Accumulibacter. The presence of Accumulibacter was further confirmed by FISH, and the higher biovolume fraction of Accumulibacter-PAOs, as well as the best P removal, was obtained with propionate as external carbon source. Known glycogen-accumulating organisms (GAOs) were not detected in this work by FISH or DGGE techniques. DGGE showed other PAOs or putative PAOs in the Actinobacteria (Tetrasphaera, Tessaracoccus) and Gemmatimonadetes (Gemmatimonas) phyla. Glucose addition led to a profound shift in bacterial profiles and the worse P removal into the reactor. The addition of acetate enhanced denitrification and this compound seemed to be the more favorable for this process. Denitrifying organisms (Zoogloea) were detected in this work, while other Actinobacteria and Proteobacteria detected could have participated in this process. Nitrification seemed to be the limiting factor for N removal along the whole reactor run. Moreover, nitrifiers were not detected. Likely its poor growth rates led to other bacteria outnumbered them in the DGGE technique. Finally, the addition of heavy metals caused a clear inhibition of nitrification and lower P REs. Putative PAOs in the Gematimonas genus and denitrifiers in the Zoogloea genus prevailed under these conditions.