Procesos microbianos en biorreactores de membrana con lechos fluidificados en tratamientos de aguas residuales

Abstract

ABSTRACT Nowadays, there is certainly no doubt that improving of biological wastewater treatments is a key goal to get better quality of the effluents, which not only contribute to protect the environment, but also to enable their reuse. In recent years, the idea of combining suspended and attached growth to improve biological process on well established wastewater treatment technologies represents an interesting solution. In this sense, different combinations of biofilm-based and membrane filtration technologies (hybrid membrane bioreactors) have been proposed. From a biological point of view, the incorporation of biofilm process confers several advantages to the membrane bioreactor system (MBR) such as higher biomass activity, higher biomass resistance to toxic or organics loads socks and improving of nitrification and denitrification processes, since the biofilm favours the development of slow growing bacteria, such as nitrifying bacteria. One of the most innovative hybrid MBRs systems is the moving bed membrane bioreactor (MBMBR) described as a combination of the moving bed biofilm rector (MBBR) and the membrane technology. In essence, MBMBRs systems are based on the addition of freely moving support material (carriers) to the biological reactor of MBRs. Biofilm development takes place in the carriers. The biological wastewater treatments feed on the metabolic activity of microorganisms for the transformation of toxic substances, the degradation of organic pollutants and the removal of nutrients from urban and industrial effluents. In particular, the biodegradation process begins with the hydrolysis of macromolecules performed by extracellular microbial enzymes. This initial process is considered as the main rate-limiting step in organic matter degradation, since an important fraction of organic matter, present in the influent, must be hydrolysed by enzymes before it can be utilised by bacterial metabolism. Consequently, it is generally accepted that the investigation of extracellular hydrolases, as well as the study of structure and dynamics of microbial communities are essential to understand the environmental or operational factors affecting efficiency and stability of the biological process, as well as to develop strategies to improve the performance of removing organic contaminants and nutrients. In this context, this research has focussed on the microbiology characterization of the suspended biomass (SB) and attached biofilm (AB) developed in a moving bed membrane bioreactor (MBMBR) as well as on the establishment of an adequate link between operational parameters and biological data. Specifically, the analysis of microbial hydrolytic enzymatic activities (acid phosphatase, alkaline phosphatase and ¿-glucosidase), the study of structure and dynamic of total Bacteria and AOB (ammonia-oxidizing bacteria) communities, and the quantitative study of N-cycle bacteria were the goals of the research. In order to establish a rough link between operational conditions and biological data, four experimental phases with 20% and 35% (v/v) Carrier Filling Ratios (CFRs) were conducted, which combined two Mixed Liquor Total Suspended Solid (MLTSS) concentrations (c.a. 2,500 and 4,500 mg/L) and two Hydraulic Retention Times (HRTs) (10 and 24 h). Regardless of the operational conditions, hydrolytic activities (acid phosphatase, alkaline phosphatase and ¿-glucosidase) in the mixed liquor (suspended biomass) were higher than in the biofilm under all the experimental conditions tested, it was possibly due to better substrate diffusion in the mixed liquor. A redundancy analysis (RDA) was performed to evaluate the relationship between enzymatic activities and operational parameters: Chemical Oxygen Demand (COD), Biological Oxygen Demand at 5 days (BOD5), Mixed Liquor Volatile Suspended Solid concentration (MLVSS), Biofilm Total Solids (BTS), HRT, pH, CFR and temperature. According to the results obtained with the Monte Carlo permutation test, CFR, MLVSS, BTS, COD, temperature and HRT significantly contributed to the variation of enzymatic activities in the MBMBR. Fingerprinting and next generation sequencing methodologies (TGGE and 454-pyrosequencing, respectively), were used to approach the study of the structure and dynamic of total Bacteria and ammonia-oxidizing bacteria (AOB) communities. These molecular techniques revealed that the structure of both total Bacteria and AOB communities was greatly similar in both SB and AB samples under all the experimental conditions tested. This fact could be connected with the free and continuous movement of carriers inside bioreactor, which could favour interactions between both SB and AB fractions. Another hypothesis was based on the residence time of carriers inside bioreactor. For better understanding about population richness and evenness in the samples, several theoretical indices (richness, functional organization, dynamics and diversity) were calculated for total Bactria and AOB communities. These indices described a total Bacteria community characterized by a high degree of diversity and optimal functional organization. By contrast, AOB community was characterized as more specialized and probably more fragile to environmental/operational conditions changes. TGGE bands sequencing and 454-pyrosequencing, showed that Proteobacteria and Actinobacteria were the predominant phyla in both sample types (SB and AB), followed by Bacterioidetes, Chloroflexi and Firmicutes. Within the phylum Proteobacteria, ¿- and ß- proteobacteria were the most predominant classes. Acidimicrobiia and Actinobacteria were the predominant classes related to phylum Actinobacteria. As for taxonomic composition of AOB community, all the TGGE-bands sequenced and belong to ammonium oxidizing ß-proteobacteria, were related to genus Nitrosomonas (N. oligotropha/ureae, N. Cryotolerans and N. europaea). The order Nitrosomonadales and the family Nitrosomonadaceae were detected by 454-pyrosequencing, but the members related to this family were unclassified at genus level. Multivariate analysis indicated that the structure and dynamic of total Bacteria and AOB communities in both SB and AB samples were mainly affected by changes in MLVSS, BTS, temperature, CFR and BOD5. The abundance of target genes of total Bacteria (16S rRNA), ammonia-oxidizing bacteria (16S rRNA and amoA of AOB), nitrite-oxidizing bacteria (16S rRNA of Nitrospira, NOB) and denitrifying bacteria (nosZ) was evaluated by quantitative real-time PCR (qPCR). The results obtained showed that the abundance and dynamics of total Bacteria, AOB, NOB and denitrifiers were fairly similar in both SB and AB fractions, suggesting that SB and AB could play equally important roles in the nitrification-denitrification process in the MBMBR. RDA analysis and Monte Carlo permutation test confirmed a significant effect of MLVSS, BTS, COD, temperature and CFR on the abundance values of total Bacteria, AOB, NOB and denitrifiers in both samples (SB and AB).