Lucía Arregui

Assessment of plausible bioindicators for plant performance in advanced wastewater treatment systems

Three full-scale advanced biological systems for nitrogen removal showing different efficiencies were assessed during one year, to investigate the protist communities supported in these wastewater treatment plants (WWTP). The main goal of this research was to explore the differences of these communities from those observed in conventional activated sludge systems. The final objective was to provide background support for the proposal of bioindicators in this type of biological systems, where scarce information was available until now, since only conventional systems had been previously studied from this point of view. Results obtained indicate that, in fact, protist population density and diversity in advanced systems for N-elimination are quite different from other wastewater systems studied before. A statistical approach through multivariate analysis was developed to search for association between protist species and physical-chemical system performance, and specifically N-removal efficiencies. The original hypothesis proposing that previous indicators from conventional systems are not adequate in advanced N-removal mechanisms was proved to be correct. Efficient processes on N-removal, despite what it had been usually found in conventional systems, show important flagellate and amoeba populations and these populations tend to reduce their abundances as nitrogen removal performance decreases (moderate to low). Ciliates are however less abundant in these N-removal efficient systems. Certain groups and genera of protist such as flagellates and small amoebae are thus proposed as indicative of high performance N-removal, while in this case the appearance of certain ciliates were indicative of low performance on N- or high organic matter removal (as COD) efficiencies.

Direct Visualization of the Microtubular Cytoskeleton of Ciliated Protozoa with a Fluorescent Taxoid

Abstract Visualization of the infraciliature, which is an essential tool for the identification of ciliate species, has traditionally been obtained with silver proteinate methods. Since infraciliature is mainly composed of microtubules, we used the synthetic fluorescent taxoid FLUTAX as a method for ciliate identification. The main advantages of this method are the facility and rapidity of its application and the fact that no previous fixation and permeabilization processes are required. FLUTAX may also be used as a probe to follow morphogenetical changes in the microtubular cytoskeleton during the ciliate life cycle.

Involvement of Crawling and Attached Ciliates in the Aggregation of Particles in Wastewater Treatment Plants

The biological community in activated sludge wastewater plants is organized within this ecosystem as bioaggregates or flocs, in which the biotic component is embedded in a complex matrix comprised of extracellular polymeric substances mainly of microbial origin. The aim of this work is to study the role of different floc-associated ciliates commonly reported in wastewater treatment plants-crawling Euplotes and sessile Vorticella- in the formation of aggregates. Flocs, in experiments with ciliates and latex beads, showed more compactation and cohesion among particles than those in the absence of ciliates. Ciliates have been shown to contribute to floc formation through different mechanisms such as the active secretion of polymeric substances (extrusomes), their biological activities (movement and feeding strategies), or the cysts formation capacity of some species. Staining with lectins coupled to fluorescein showed that carbohydrate of the matrix contained glucose, manose, N-acetyl-glucosamine and galactose. Protein fraction revealed over the latex beads surfaces could probably be of bacterial origin, but nucleic acids represented an important fraction of the extracellular polymeric substances of ciliate origin.

Unravelling the interactions among microbial populations found in activated sludge during biofilm formation.

Microorganisms colonize surfaces and develop biofilms through interactions that are not yet thoroughly understood, with important implications for water and wastewater systems. This study investigated the interactions between N-acyl homoserine lactone (AHL)-producing bacteria, yeasts and protists, and their contribution to biofilm development. Sixty-one bacterial strains were isolated from activated sludge and screened for AHL production, with Aeromonas sp. found to be the dominant AHL producer. Shewanella xiamenensis, Aeromonas allosaccharophila, Acinetobacter junii and Pseudomonas aeruginosa recorded the highest adherence capabilities, with S. xiamenensis being the most effective in surface colonization. Additionally, highly significant interactions (i.e. synergic or antagonistic) were described for dual and multistrain mixtures of bacterial strains (P. aeruginosa, S. xiamenensis, A. junii and Pseudomonas stutzeri), as well as for strongly adherent bacteria co-cultured with yeasts. In this last case, the adhered biomass in co-cultures was lower than the monospecific biofilms of bacteria and yeast, with biofilm observations by microscopy suggesting that bacteria had an antagonist effect on the whole or part of the yeast population. Finally, protist predation by Euplotes sp. and Paramecium sp. on Aeromonas hydrophila biofilms not only failed to reduce biofilm formation, but also recorded unexpected results leading to the development of aggregates of high density and complexity.

Microtubular Elements of the Marine Antarctic Ciliate Euplotes focardii (Ciliophora, Hypotrichia)

Summary: Microtubules are the major cytoskeletal component in Euplotes focardii . The spatial organization of the microtubular system of this marine antarctic ciliate is described. Three different methods were employed: immunofluorescence using antitubulin antibodies, scanning electron microscopy, and transmission electron microscopy. The general arrangement of the cytoskeletal microtubular system is similar to that described for temperate species of Euplotes . Basal bodies, microtubular derivatives associated with the kinetosomes and superficial microtubular networks in both dorsal and ventral sides are stable at the low temperatures this species must bear. Slight differences are reported in the disposition of the microtubules on the somatic cortex, As for the oral cytoskeleton the left side infraciliature is reinforced by a complex microtubular system: on the one hand, a developed microtubular network spreading under and among the paramembranelles, and on the other hand microtubules reinforcing the oral crests.

Membrane bioreactor wastewater treatment plants reveal diverse yeast and protist communities of potential significance in biofouling

The yeast community was studied in a municipal full-scale membrane bioreactor wastewater treatment plant (MBR-WWTP). The unexpectedly high diversity of yeasts indicated that the activated sludge formed a suitable environment for them to proliferate, with cellular concentrations of 2.2 ± 0.8 × 103 CFU ml−1. Sixteen species of seven genera were present in the biological reactor, with Ascomycetes being the most prevalent group (93%). Most isolates were able to grow in a synthetic wastewater medium, adhere to polyethylene surfaces, and develop biofilms of variable complexity. The relationship between yeast populations and the protists in the MBR-WWTP was also studied, revealing that some protist species preyed on and ingested yeasts. These results suggest that yeast populations may play a role in the food web of a WWTP and, to some extent, contribute to membrane biofouling in MBR systems.

FLUTAX facilitates visualization of the ciliature of oxytrichid hypotrichs

The fluorescent taxoid FLUTAX that specifically binds to microtubules was employed to stain cells of the oxytrichid ciliate Sterkiella cavicola. Due to their cortical peculiarities, ciliates of this group have traditionally been difficult to stain, and in consequence their identification presents uncertainty. Here, a variant of the employment of FLUTAX is proposed with the aim of simplifying the visualization of oxytrichid ciliature and other microtubular structures. As a result, key characters for the identification of these organisms are revealed.

A New Species of the Genus Metacystis (Ciliophora, Prostomatida, Metacystidae) from a Wastewater Treatment Plant

ABSTRACT. Unusual prostomatid specimens were found in the biological reactor of a wastewater treatment plant in a health resort in Valencia, Spain. These ciliates were attached to flocs unlike other free‐swimming prostomatid ciliates described to date in the mixed liquor of activated sludge plants. The morphological study of this species led to a typically different combination of characteristics: elongated cell shape, 20–30 somatic kineties, 2 perioral kineties, and 1 circumoral kinety, 1 large vacuole protruding at the terminal end, a lorica tapered toward the aperture with a smooth neck, and 11–16 annular ridges. These characteristics place this representative as a new species of the genus Metacystis—Metacystis galiani n. sp. This species became the dominant population within the biological reactor when high values of conductivity (4,244 mS/cm) and temperature (26.8 °C) were recorded.

Quorum Sensing versus Quenching Bacterial Isolates Obtained from MBR Plants Treating Leachates from Municipal Solid Waste

Quorum sensing (QS) is a mechanism dependent on bacterial density. This coordinated process is mediated by the synthesis and the secretion of signal molecules, called autoinducers (AIs). N-acyl-homoserine lactones (AHLs) are the most common AIs that are used by Gram-negative bacteria and are involved in biofilm formation. Quorum Quenching (QQ) is the interference of QS by producing hydrolyzing enzymes, among other strategies. The main objective of the present study was to identify QS and QQ strains from MBR wastewater treatment plants. A total of 99 strains were isolated from two Spanish plants that were intended to treat leachate from municipal solid waste. Five AHL producers were detected using AHL biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1). Fifteen strains of seventy-one Gram-positive were capable of eliminating or reducing at least one AHL activity. The analysis of 16S rRNA gene sequence showed the importance of the Pseudomonas genus in the production of biofilms and the relevance of the genus Bacillus in the disruption of the QS mechanism, in which the potential activity of lactonase or acylase enzymes was investigated with the aim to contribute to solve biofouling problems and to increase the useful lifespan of membranes.