Better understanding of the activated sludge process combining fluorescence-based methods and flow cytometry: A case study.

This study aims to demonstrate the validity of fluorescence-based methods, together with flow cytometry, as a complementary tool to conventional physicochemical analyses carried out in wastewater treatment plants (WWTPs), for the control of the currently largely unknown activated sludge process. Staining with SYTO 9, propidium iodide and 5-(and 6)-carboxy-2',7'-difluorodihydrofluorescein diacetate (carboxy-H2DFFDA) was used for cell viability and oxidative stress monitoring of the bacterial population forming the activated sludge of a WWTP. Throughout the period of research, several unstable periods were detected, where the non-viable bacteria exceeded the 75% of the total bacterial population in the activated sludge, but only in one case the cells with oxidative stress grew to 9%, exceeding the typical values of 2%-5% of this plant. These periods coincided in two cases with high values of total suspended solids (SST) and chemical oxygen demand (COD) in the effluent, and with an excess of ammonia in other case. A correlation between flow cytometric and physicochemical data was found, which enabled to clarify the possible origin of each case of instability in the biological system. This experience supports the application of bacterial fluorescence staining, together with flow cytometric analysis, as a simple, rapid and reliable tool for the control and better understanding of the bacteria dynamics in a biological wastewater treatment process.

Functional diversity and dynamics of bacterial communities in a membrane bioreactor for the treatment of metal-working fluid wastewater.

An extensive microbiological study has been carried out in a membrane bioreactor fed with activated sludge and metal-working fluids. Functional diversity and dynamics of bacterial communities were studied with different approaches. Functional diversity of culturable bacterial communities was studied with different Biolog™ plates. Structure and dynamics of bacterial communities were studied in culturable and in non-culturable fractions using a 16S rRNA analysis. Among the culturable bacteria, Alphaproteobacteria and Gammaproteobacteria were the predominant classes. However, changes in microbial community structure were detected over time. Culture-independent analysis showed that Betaproteobacteria was the most frequently detected class in the membrane bioreactor (MBR) community with Zoogloea and Acidovorax as dominant genera. Also, among non-culturable bacteria, a process of succession was observed. Longitudinal structural shifts observed were more marked for non-culturable than for culturable bacteria, pointing towards an important role in the MBR performance. Microbial community metabolic abilities assessed with Biolog™ Gram negative, Gram positive and anaerobic plates also showed differences over time for Shannon's diversity index, kinetics of average well colour development, and the intensely used substrates by bacterial community in each plate.

Establishment of toxicity thresholds in subpopulations of coelomocytes (amoebocytes vs. eleocytes) of Eisenia fetida exposed in vitro to a variety of metals: implications for biomarker measurements.

Coelomocytes comprise the immune system of earthworms and due to their sensitivity responding to a wide range of pollutants have been widely used as target cells in soil ecotoxicology. Recently, in vitro assays with primary cultures of coelomocytes based in the neutral red uptake (NRU) assay have been developed as promising tools for toxicity assessment chemical in a reproducible and cost-effective manner. However, NRU showed a bimodal dose-response curve previously described after in vivo and in vitro exposure of earthworm coelomocytes to pollutants. This response could be related with alterations in the relative proportion of coelomocyte subpopulations, amoebocytes and eleocytes. Thus, the aims of the present work were, first, to establish the toxicity thresholds that could be governed by different cell-specific sensitivities of coelomocytes subpopulations against a series of metals (Cu, Cd, Pb, Ni), and second to understand the implication that coelomocyte population dynamics (eleocytes vs. amoebocytes) after exposure to pollutants can have on the viability of coelomocytes (measured by NRU assay) as biomarker of general stress in soil health assessment. Complementarily flow cytometric analyses were applied to obtain correlative information about single cells (amoebocytes and eleocytes) in terms of size and complexity, changes in their relative proportion and mortality rates. The results indicated a clear difference in sensitivity of eleocytes and amoebocytes against metal exposure, being eleocytes more sensitive. The bimodal dose-response curve of NRU after in vitro exposure of primary cultures of coelomocytes to metals revealed an initial mortality of eleocytes (decreased NRU), followed by an increased complexity of amoebocytes (enhanced phagocytosis) and massive mortality of eleocytes (increased NRU), to give raise to a massive mortality of amoebocytes (decrease NRU). A synergistic effect on NRU was exerted by the exposure to high Cu concentrations and acidic pH (elicited by the metal itself), whereas the effects on NRU produced after exposure to Cd, Ni and Pb were due solely to the presence of metals, being the acidification of culture medium meaningless.

Application of ecological risk assessment based on a novel TRIAD-tiered approach to contaminated soil surrounding a closed non-sealed landfill.

The Ecological Risk Assessment (ERA) is a reliable tool for communicating risk to decision makers in a comprehensive and scientific evidence-based way. In this work, a site-specific ERA methodology based on the TRIAD approach was applied to contaminated soil surrounding a closed non-sealed landfill, as a case study to implement and validate such ERA methodology in the Basque Country (northern Spain). Initially, the procedure consisted of the application of a Parameter Selection Module aimed at selecting the most suitable parameters for the specific characteristics of the landfill contaminated soil, taking into consideration the envisioned land use, intended ecosystem services and nature of contaminants. Afterwards, the selected parameters were determined in soil samples collected from two sampling points located downstream of the abovementioned landfill. The results from these tests were normalized to make them comparable and integrable in a risk index. Then, risk assessment criteria were developed and applied to the two landfill contaminated soil samples. Although the lack of a proper control soil was evidenced, a natural land use was approved by the ERA (at Tier 2) for the two landfill contaminated soils. However, the existence of a potential future risk resulting from a hypothetical soil acidification must be considered.

Congruence in the performance of model nitrifying activated sludge plants located in Germany, Scotland and Spain.

A laboratory model nitrifying activated sludge plant treating OECD synthetic sewage was designed and constructed by each of three laboratories in Germany, Scotland and Spain in order to produce a sludge inoculum for 5 rapid toxicity bioassays. The plants were run for 3 years and produced sludge for the microbially based bioassays Vibrio fischeri bioluminescence, ATP luminescence and respiration, and, nitrification and enzyme inhibition. Although the initial sludge inoculum for the plants differed, as did some of the running conditions such as temperature regime, the sludge produced within the different countries had similar characteristics with respect to sludge age, total suspended solids and volatile suspended solids. Nitrification was generally maintained over the 3-year period although there were occasions when the process was inconsistent. Nitrification recovery was afforded by reseeding with a nitrifying sludge from a local wastewater treatment works (WWTW) or imposition of starvation conditions for a period of time. The sludge produced was used to carry out toxicity testing and results compared well with those using sludge from a WWTW. Overall, the use of sludge generated in the laboratory could be used for toxicity testing negating the need to resort to the use of natural WWTW sludge, which may contain a range of toxic substances due to uncontrolled industrial and domestic inputs and an unbalanced microbial consortium.