Do Antarctic bivalves present microdebris? The case of Livingston Island.

Marine microdebris (MD) seem to be widespread in benthic invertebrates, even in the most remote areas of the planet such as Antarctica, although the information available is still very scarce. Here we provide a detailed quantification and characterization of the MD found on three common bivalve species (Aequiyoldia eightsii, Thracia cf. meridionalis, and Cyclocardia astartoides) inhabiting shallow areas in Johnsons' Bay, Livingston Island (South Shetland Islands, Antarctica) as a snapshot of the MD present. On average, these bivalves contained 0.71 ± 0.89 items per individual and 1.49 ± 2.35 items per gram, being comparable to the few previous existing studies in other Antarctic areas. Nearly half of the organisms analysed here (45.6 %), contained at least one item. No significant differences were found in the three bivalve species. As far as we know, this is the first study to analyse and compare MD in three bivalve species in the Antarctic Peninsula. Although our results indicate bivalves are as not as polluted as in other areas of the planet, this is remarkable since this is considered one of the last pristine areas of the world. Our results point to local activities as the main source of MD pollution in Livingston Island, although global pollution cannot be discarded. We believe this research provides a useful baseline for future studies and will contribute to develop policies and strategies to preserve Antarctic marine ecosystems from MD pollution.

Niche differentiation drives microbial community assembly and succession in full-scale activated sludge bioreactors.

Network models and community phylogenetic analyses are applied to assess the composition, structure, and ecological assembly mechanisms of microbial communities. Here we combine both approaches to investigate the temporal dynamics of network properties in individual samples of two activated sludge systems at different adaptation stages. At initial assembly stages, we observed microbial communities adapting to activated sludge, with an increase in network modularity and co-exclusion proportion, and a decrease in network clustering, here interpreted as a consequence of niche specialization. The selective pressure of deterministic factors at wastewater treatment plants produces this trend and maintains the structure of highly functional and specialized communities responding to seasonal environmental changes.

Effects of two submerged macrophyte species on microbes and metazoans in rooftop water-storage ponds with different labile carbon loadings.

Nature-based solutions including rooftop-water storage ponds are increasingly adopted in cities as new eco-designs to address climate change issues, such as water scarcity and storm-water runoff. Macrophytes may be valuable additions for treating stored rooftop waters and provisioning other services, including aquaponics, esthetic and wildlife-conservation values. However, the efficacy of macrophyte treatments has not been tested with influxes of different labile carbon loadings such as those occurring in storms. Moreover, little is known about how macrophytes affect communities of metazoans and microbes, including protozoans, which are key players in the water-treatment process. Here, we experimentally investigated the effectiveness of two widely distributed macrophytes, Ceratophyllum demersum and Egeria densa, for treating drained rooftop water fed with two types of leaf litter, namely Quercus robur (high C lability) and Quercus rubra (low C lability). C. demersum was better than E. densa at reducing water conductivity (by 10 ̶ 40 µS/cm), TDS (by 10-18 mg/L), DOC (by 4-5 mg/L) and at increasing water transparency (by 4-9%), water O2 levels (by 19-27%) and daylight pH (by 0.9-1.3) compared to leaf-litter only microcosms after 30 days. Each treatment developed a different community of algae, protozoa and metazoa. Greater plant mass and epiphytic chlorophyll-a suggested that C. demersum was better at providing supporting habitat than E. densa. The two macrophytes did not differ in detritus accumulation, but E. densa was more prone to develop filamentous bacteria, which cause sludge bulking in water-treatment systems. Our study highlights the superior capacity of C. demersum and the usefulness of whole-ecosystem experiments in choosing the most adequate macrophyte species for nature-based engineered solutions.

Strategies to Optimize Microalgae Conversion to Biogas: Co-Digestion, Pretreatment and Hydraulic Retention Time.

This study aims at optimizing the anaerobic digestion (AD) of biomass in microalgal-based wastewater treatment systems. It comprises the co-digestion of microalgae with primary sludge, the thermal pretreatment (75 °C for 10 h) of microalgae and the role of the hydraulic retention time (HRT) in anaerobic digesters. Initially, a batch test comparing different microalgae (untreated and pretreated) and primary sludge proportions showed how the co-digestion improved the AD kinetics. The highest methane yield was observed by adding 75% of primary sludge to pretreated microalgae (339 mL CH4/g VS). This condition was then investigated in mesophilic lab-scale reactors. The average methane yield was 0.46 L CH4/g VS, which represented a 2.9-fold increase compared to pretreated microalgae mono-digestion. Conversely, microalgae showed a low methane yield despite the thermal pretreatment (0.16 L CH4/g VS). Indeed, microscopic analysis confirmed the presence of microalgae species with resistant cell walls (i.e., Stigioclonium sp. and diatoms). In order to improve their anaerobic biodegradability, the HRT was increased from 20 to 30 days, which led to a 50% methane yield increase. Overall, microalgae AD was substantially improved by the co-digestion with primary sludge, even without pretreatment, and increasing the HRT enhanced the AD of microalgae with resistant cell walls.

Characterization of nutrient-removing microbial communities in two full-scale WWTP systems using a new qPCR approach.

Biological wastewater treatment processes involve very complex microbial communities. Culture-independent molecular methods are feasible tools used to analyze and control the structure of different microbial communities, such as bacterial communities that remove nutrients. Here, we used the gBlocks gene fragments method, a new real-time PCR approach for the development of DNA standards, to quantify total bacterial cells, AOB, NOB, and Archaeal genes at two different WWTPs. PAOs were also quantified using the FISH technique. Our findings highlight a significant improvement in real-time PCR detection for the microorganisms studied. The qPCR and FISH technique applied allowed characterization of the microbial composition of two WWTPs operated as a conventional WWTP and a biological nutrient-removal WWTP. The results revealed a significant difference in the microbial profiles of the WWTPs, with a higher abundance of nitrifying bacterial communities and PAOs in the nutrient removal plant, which were in accordance with operational performance.

Microeukaryote community in a partial nitritation reactor prior to anammox and an insight into the potential of ciliates as performance bioindicators.

An in-depth, long-term, multidisciplinary study was conducted in order to study the microeukaryote community in a partial nitritation (PN) reactor prior to anammox. The PN reactor operated with moving bed biofilm reactor (MBBR) technology, using plastic supports (carriers) for biofilm development. The microeukaryote community from the biofilm (BF) and the surrounding media (mixed liquor or ML) were analysed separately. Despite the physicochemical conditions under which the PN-MBBR operated (an average of 305.9±117mg TAN l-1 and 328.4±131.9mg N-NO2- l-1), up to 24 microeukaryotic taxa were observed by microscope. Microeukaryote species showed an uneven distribution in the PN-MBBR, thus suggesting the existence of two habitats: the BF, preferred by species with specific structures for adhering to a substrate, such as the stalked Peritrichia, and the ML, preferred by free-swimming or non-substrate dependent species. The results indicated that most ciliate population dynamics mainly responded to the nitrous acid and free ammonia concentrations and, to a lesser extent, to sCOD values. In the BF, variations in the population of Epistylis camprubii and Opercularia coarctata suggest the existence of competition between these species due to niche overlap. A V4 18S rDNA molecular survey (Illumina) was carried out for some samples with the aim of obtaining maximum coverage of the main eukaryote species that were microscopically detected throughout the study. The diversity and abundance data provided by both detection methods were compared. The study helped identify broader tolerance ranges of the microeukaryote taxa to the physicochemical parameters analysed.

Inactivation of microbiota from urban wastewater by single and sequential electrocoagulation and electro-Fenton treatments.

This work aims at comparing the ability of two kinds of electrochemical technologies, namely electrocoagulation (EC) and electro-Fenton (EF), to disinfect primary and secondary effluents from municipal wastewater treatment plants. Heterotrophic bacteria, Escherichia coli, enterococci, Clostridium perfringens spores, somatic coliphages and eukaryotes (amoebae, flagellates, ciliates and metazoa) were tested as indicator microorganisms. EC with an Fe/Fe cell at 200 A m-2 and natural pH allowed >5 log unit removal of E. coli and final concentration below 1 bacteria mL-1 of coliphages and eukaryotes from both effluents in ca. 60 min, whereas heterotrophic bacteria, enterococci and spores were more resistant. A larger removal was obtained for the primary effluent, probably because the flocs remove higher amount of total organic carbon (TOC), entrapping more easily the microbiota. EF with a boron-doped diamond (BDD) anode and an air-diffusion cathode that produces H2O2 on site was first performed at pH 3.0, with large or even total inactivation of microorganisms within 30 min. A more effective microorganism removal was attained as compared to EC thanks to •OH formed from Fenton's reaction. A quicker disinfection was observed for the secondary effluent owing to its lower TOC content, allowing the attack of greater quantities of electrogenerated oxidants on microorganisms. Wastewater disinfection by EF was also feasible at natural pH (∼7), showing similar abatement of active microorganisms as a result of the synergistic action of generated oxidants like active chlorine and coagulation with iron hydroxides. A sequential EC/EF treatment (30 min each) was more effective for a combined decontamination and disinfection of urban wastewater.

Microalgae recycling improves biomass recovery from wastewater treatment high rate algal ponds.

Microalgal biomass harvesting by inducing spontaneous flocculation (bioflocculation) sets an attractive approach, since neither chemicals nor energy are needed. Indeed, bioflocculation may be promoted by recycling part of the harvested microalgal biomass to the photobioreactor in order to increase the predominance of rapidly settling microalgae species. The aim of the present study was to improve the recovery of microalgal biomass produced in wastewater treatment high rate algal ponds (HRAPs) by recycling part of the harvested microalgal biomass. The recirculation of 2% and 10% (dry weight) of the HRAPs microalgal biomass was tested over one year in an experimental HRAP treating real urban wastewater. Results indicated that biomass recycling had a positive effect on the harvesting efficiency, obtaining higher biomass recovery in the HRAP with recycling (R-HRAP) (92-94%) than in the control HRAP without recycling (C-HRAP) (75-89%). Microalgal biomass production was similar in both systems, ranging between 3.3 and 25.8 g TSS/m2d, depending on the weather conditions. Concerning the microalgae species, Chlorella sp. was dominant overall the experimental period in both HRAPs (abundance >60%). However, when the recycling rate was increased to 10%, Chlorella sp. dominance decreased from 97.6 to 88.1%; while increasing the abundance of rapidly settling species such as Stigeoclonium sp. (16.8%, only present in the HRAP with biomass recycling) and diatoms (from 0.7 to 7.3%). Concerning the secondary treatment of the HRAPs, high removals of COD (80%) and N-NH4+ (97%) were found in both HRAPs. Moreover, by increasing the biomass recovery in the R-HRAP the effluent total suspended solids (TSS) concentration was decreased to less than 35 mg/L, meeting effluent quality requirements for discharge. This study shows that microalgal biomass recycling (10% dry weight) increases biomass recovery up to 94% by selecting the most rapidly settling microalgae species without compromising the biomass production and improving the wastewater treatment in terms of TSS removal.

Improvement of the intersection method for the quantification of filamentous organisms: basis and practice for bulking and foaming bioindication purposes.

Bulking and foaming phenomena in activated sludge wastewater treatment plants are in most cases related to the abundance of filamentous microorganisms. Quantifying these microorganisms should be a preliminary stage in their control. In this paper, the simplicity of quantifying them based on the intersection method is demonstrated, by redescribing the theory and applying a new improved protocol; new data of interest are also provided. The improved method allows us to use it with stained smears, including epifluorescence techniques. The error that could be made, when considering the distribution of filamentous bacteria in fresh microscope preparations in two dimensions rather than three is negligible. The effect of the different types of filamentous microorganisms on the settleability was also studied. The effect of the total extended filament length on the sludge settleability was shown to depend on the type of filamentous organism and how it aggregates. When these groups of filamentous organisms are found in small aggregations and there is an increase in the number of filamentous organisms, the sludge volume index (SVI) increases proportionally to the filament length. However, when aggregation increases, the impact on the SVI is significantly lower.

Tracking and quantification of nitrifying bacteria in biofilm and mixed liquor of a partial nitrification MBBR pilot plant using fluorescence in situ hybridization.

A moving bead biofilm reactor (MBBR) pilot plant was implemented as a partial nitrification process for pre-treatment of ammonium-rich liquors (676 ± 195 mg L(-1)), and studied for 479 days under variations in hydraulic retention time. The main purpose of this work, was the study of dynamics abundance of total bacteria and single-cells nitrifying bacteria belonging to ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in biofilms and mixed liquor of the plant. The microbial monitoring was successfully achieved using fluorescence in situ hybridization combined with flocs disaggregation protocol as a useful microbial monitoring tool. A partial nitrification process with a N-NH4(+) removal rate of about 38.6 ± 14.8% was successfully achieved at 211 days after start-up, with a clear dominance of AOB, which accounted for 11.3 ± 17.0% of total bacterial cells compared with only 2.1 ± 4.0% of NOB. The effluent obtained was subsequently supplied to an Anammox reactor for complete ammonium treatment.

Chronic effects of temperature and nitrate pollution on Daphnia magna: Is this cladoceran suitable for widespread use as a tertiary treatment?

Effluent clarification and disinfection are major challenges in wastewater management. The cladoceran Daphnia magna has been proposed as a cost-effective and ecosystem-friendly option to clarify and disinfect secondary effluents, but its efficacy has not been fully tested under different sewage conditions. The present study explores the effects of temperature and nitrate on the efficacy of D. magna as a tertiary treatment at two different scales (individual assays and microcosms). Individual assays were employed to determine direct effects of temperature and/or nitrate on D. magna cultured in a suspension of organic matter. Using microcosms under the same environmental conditions, we explored the clearing efficacy of D. magna interacting with a natural microbial community. Individual assays revealed that D. magna mortality increased by 17% at 26 °C, 21% at >250 mg NO3(-)/l and by 60% at 26 °C and at >250 mg NO3(-)/l, and individuals displayed reduced body size, filtering rates and fecundity when compared to those at 21 °C and <40 mg NO3(-)/l. Improved performance under these conditions was also mirrored in the microcosms, with a higher density of D. magna (>100 ind/l) at 21 °C and <40 mg NO3(-)/l compared to the number (0-21 ind/l) at 26 °C and/or >250 mg NO3(-)/l. In the microcosms at 21 °C and <40 mg NO3(-)/l, turbidity and the density of bacteria, protists and micro-metazoa decreased in relation to those at 26 °C and/or >250 mg NO3(-)/l. Each treatment developed a unique and characteristic microbial assemblage, and D. magna was identified as the major driver of the community structure of protists and micro-metazoa. This enabled us to determine taxa vulnerability to D. magna grazing, and to re-define their tolerance thresholds for nitrate. In conclusion, this study increases our knowledge of how microbes respond to temperature and nitrate pollution, and highlights that D. magna efficacy as a tertiary treatment can be seriously compromised by variable environmental conditions.

Assessment of total bacterial cells in extended aeration activated sludge plants using flow cytometry as a microbial monitoring tool.

The extended aeration activated sludge (EAAS) process is one of the most applied biological processes in small towns. Here, we study the abundance and viability of total bacterial cells in two wastewater treatment plants (WWTPs) operating with an EAAS process. We use flow cytometry (FCM) combined with SYTO13 and propidium iodide (PI) dyes as a rapid, easy, reliable and accurate microbial monitoring tool. A disaggregation procedure with an ultrasonic bath was designed to detach total bacterial cells from activated sludge flocs for subsequent FCM analysis. This procedure permitted the recovery of total bacterial cells from sludge flocs without affecting bacterial viability, as indicated by bacterial strain controls. Since FCM is a multi-parameter technique, it was possible to determine total bacterial abundance and their viability in the activated sludge. As a comparative method, epifluorescence microscopy was also used to quantify total bacterial cells; both methods produced similar results. The FCM analysis revealed relative microbial stability in both the WWTPs. The total bacterial abundance quantified by FCM in the two plants studied was 1.02-6.23 × 10(11) cells L(-1) with 70-72% viability, one logarithm less than that reported in the literature for WWTPs using the conventional activated sludge process. This can be explained by the difference in the operational parameters between the conventional plant and EAAS, mainly the organic loading rate.

Effect of chlorine and temperature on free-living protozoa in operational man-made water systems (cooling towers and hot sanitary water systems) in Catalonia.

In recent decades, free-living protozoa (FLP) have gained prominence as the focus of research studies due to their pathogenicity to humans and their close relationship with the survival and growth of pathogenic amoeba-resisting bacteria. In the present work, we studied the presence of FLP in operational man-made water systems, i.e. cooling towers (CT) and hot sanitary water systems (HSWS), related to a high risk of Legionella spp. outbreaks, as well as the effect of the biocides used, i.e. chlorine in CT and high temperature in HSWS, on FLP. In CT samples, high-chlorine concentrations (7.5 ± 1.5 mg chlorine L(-1)) reduced the presence of FLP by 63.8 % compared to samples with low-chlorine concentrations (0.04 ± 0.08 mg chlorine L(-1)). Flagellates and amoebae were observed in samples collected with a level of 8 mg chlorine L(-1), which would indicate that some FLP, including the free-living amoeba (FLA) Acanthamoeba spp., are resistant to the discontinuous chlorine disinfection method used in the CT studied. Regarding HSWS samples, the amount of FLP detected in high-temperatures samples (53.1 ± 5.7 °C) was 38 % lower than in low-temperature samples (27.8 ± 5.8 °C). The effect of high temperature on FLP was chiefly observed in the results obtained by the culture method, in which there was a clear reduction in the presence of FLP at temperatures higher than 50 °C, but not in those obtained by PCR. The findings presented here show that the presence of FLP in operational man-made water systems should be taken into account in future regulations.

The use of rotifers for limiting filamentous bacteria Type 021N, a bacteria causing activated sludge bulking.

The excessive growth of filamentous bacteria and the resultant bulking of activated sludge constitute a serious problem in numerous wastewater treatment plants. Lecane inermis rotifers were previously shown to be capable of reducing the abundance of Microthrix parvicella and Nostocoida limicola in activated sludge. In the present study, the effectiveness of four Lecane clones in reducing the abundance of Type 021N filamentous bacteria was investigated. Three independent experiments were carried out on activated sludge from three different treatment plants. We found that Lecane rotifers are efficient consumers of Type 021N filaments.

Microbial and physicochemical parameters associated with Legionella contamination in hot water recirculation systems.

Hot water recirculation systems (HWRS) in hotels and nursing homes, which are common in countries such as Spain, have been related to outbreaks of legionellosis. To establish the relationships of microbial and physicochemical parameters, especially protozoa, with the occurrence of Legionella in HWRS, 231 samples from hotels and nursing homes were analysed for Legionella, protozoa, heterotrophic plate counts (HPC) at 22 and 37 °C, Pseudomonas, metals, temperature and others. Legionella pneumophila was the dominant species isolated, and 22 % were sg. 1. The sampling method became particularly important in order to define which factors were involved on the occurrence of Legionella. Results showed that the bacteria and the accompanying microbiota were more abundant in the first flush water whose temperature was lower. The bacteria occurred in those samples with high HPC and were inversely correlated with high temperatures. Multivariate regression showed that a concentration above 1 × 10(5) CFU/100 mL of HPC at 37 °C, Fe above 0.095 ppm and the presence of protozoa increased significantly the risk of Legionella colonization, while univariant regression showed that the presence of Cu above 0.76 ppm and temperature above 55 °C diminished it. Therefore, to reduce the risk associated with Legionella occurrence in HWRS these parameters should be taken into consideration.

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.

Effects of particulate and soluble substrates on microfauna populations and treatment efficiency in activated sludge systems.

To determine the effects of particulate and soluble compounds on microfauna populations and treatment efficiency in activated sludge systems, two experimental wastewater treatment plants were set up and evaluated for a period of five months. The plants were fed with pre-flocculated domestic sewage enriched with starch or glucose as model substrates of particulate and soluble organic matter, respectively. It was observed that the starch-enriched system presented lower abundance of filamentous bacteria that turned into a better sludge sedimentation. Mean sludge volume index (SVI) values for the starch and glucose-enriched systems were 54+/-24 and 885+/-845 mL g(-1), respectively. Although no differences in organic matter removal were detected between the systems, nitrification and denitrification were higher in the starch-enriched system, which is likely to have been the result of its more compact flocs. The mean ammonia-N effluent concentrations for the starch and glucose-enriched systems were 4.7+/-5.7 and 16.2+/-9.7 mg L(-1), respectively, whereas the nitrate-N concentrations were 20.1+/-10.8 and 30.8+/-12.2 mg L(-1), respectively. Concerning microfauna analysis, ciliated protozoa specifically, attached ciliates were the dominant microfauna group in both treatment systems, whereas metazoa, particularly Lecanidae rotifera, were more abundant in the starch-enriched system. Lecanidae rotifera abundances above 400 ind mL(-1) reduced the mean floc area from 60 to 20mm(2) without affecting sludge settleability in the starch-enriched system. Finally, the reduction in floc area caused by metazoa feeding led to effluents of lower nitrogen quality, although no differences in sludge production were detected.

Comparison of microfauna communities in full scale subsurface flow constructed wetlands used as secondary and tertiary treatment.

In order to evaluate the microfauna composition and distribution in two horizontal subsurface flow constructed wetlands used as secondary and tertiary treatment a full-scale wastewater treatment plant was monitored during five months. Results indicate that total microfauna abundance in the wetland treating primary influents is around five times higher than that found in the wetland treating secondary influents. Ciliated protozoa and microflagellates are the most important microfauna groups in both wetlands; microflagellates in terms of abundance and ciliates in terms of biomass. The most abundant ciliate species in the wetland treating primary influents are polysaprobic organisms as Dexiostoma campylum, Trimyema compressum, and to a lesser extend Metopus spp. On the other hand, the most important ciliate species found in the wetland treating secondary influents are mainly aerobic ciliates as Vorticella comvallaria-complex, Aspidisca cicada, Litonotus lamella and some ciliates belonging to the group of the scuticociliates and Hypotrichidae. The sort of the organic matter treated (particulated or dissolved) is at least as important as the amount of it in order to explain microfauna dynamics in constructed wetlands.

Short-term harmful effects of ammonia nitrogen on activated sludge microfauna.

The response of activated sludge microfauna in terms of abundance and diversity has been analysed to evaluate both the toxic effect of ammonia nitrogen and the acclimatisation capacity of these microorganisms to its toxicity. The harmful effect of ammonia nitrogen was studied by means of two toxicological tests. The ammonia concentrations tested were: 9, 20, 30 and 50mg NH4+-N/l in the first toxicological test and 30, 40, 50 and 80 mg NH4+-N/l in the second. The results suggest that ammonia nitrogen causes a clear but reversible toxic effect on microfauna abundance when its concentrations are around three times higher than that which the microfauna is used to. Chilodonella uncinata and Acineria uncinata were the ciliates least affected by the ammonia nitrogen toxicity. Furthermore, the majority of microfauna groups analysed (gymnamoebae and ciliates) showed capability for acclimatisation to ammonia nitrogen in terms of abundance.

Dynamics of nematodes in a high organic loading rotating biological contactors.

Nematode diversity and dynamics of a full-scale rotating biological contactor plant (RBC) has been studied. Analysis of biofilm composition showed a well-established zoning of microfauna among the three RBC sections analysed. Nematodes appeared to be the dominant group within the larger microfauna populations with average abundances between 200 and 300ind/mg or 8000 and 17000ind/cm(2). The most abundant nematode species were Diplogasteritus nudicapitatus and Paroigolaimella coprophages and, to a lesser extent, Paroigolaimella bernensis and Steinernema intermedia. The relationship between nematodes and filamentous bacteria (specifically the genus Beggiatoa) was the most significant biotic relationship found, and to a lesser extent, nematodes with ciliates. The relationship between the abundance of nematode species and the physical-chemical variables suggests that nematodes may be good indicators of low pollutant load levels in the entry of the RBC system. Finally, the results indicate that nematodes may have a relevant role for a good biofilm development.