Biological control of predatory fungi inhabiting activated sludge in wastewater treatment.

The unfavorable phenomenon of activated sludge bulking that occurs in sewage treatment plants (WWTPs) is caused by the over-proliferation of filamentous bacteria that should be limited by the Lecane rotifers that feed on them; however, predatory, rotiferovorous fungi that often inhabit WWTPs pose a real threat to these organisms. To solve this problem, we investigated the interaction of the fungus Clonostachys rosea, which is a known Biological Control Agent (BCA) and the predacious Zoophagus sp. in simplified laboratory culture conditions. The presence of C. rosea in the cultures reduced the number of active traps, thus translating into a much smaller number of rotifers being caught. The mycelium of C. rosea was labeled with a red fluorescent protein (RFP). The life cycle of C. rosea that were attacking Zoophagus sp. (hunting for rotifers) is described. C. rosea spores germinate into single-celled forms and penetrate the interior of the Zoophagus mycelium where they feed on the cytoplasm. Then is the mycelium produced abundantly and forms conidiophores. This type of life strategy has not been known before. The obtained results demonstrated the potential of C. rosea as a BCA that can be used to protect rotifers in the event of an infection of activated sludge by the predatory fungi that threaten the rotifer population.

The potential of Lecane rotifers in microplastics removal.

Dealing with hard-to-degrade plastics pollution of terrestrial and aquatic environments is one of the most urgent problems of the modern world. The smallest fraction (<5 mm) called micro-plastics (MP) has been found everywhere from ice in Greenland, streams, rivers, soil and even in the human placenta. The goal of our research was to assess the ability of rotifers Lecane inermis to remove micro-plastics suspended in the water column. In the experiments we investigated specific interactions between MP, biofilm and rotifers specialized in feeding on biofilm. We hypothesized that MP adhere to the biofilm and after ingestion by rotifers could be extracted from the water in the form of compact conglomerates excreted with fecal pellets. In these experiments, we demonstrated that: (i) the rotifers preferentially ingest microplastics embedded in biofilm, (ii) the presence of microplastics does not affect growth and fecundity of rotifers, and (iii) that MP aggregation is significantly improved by the presence of biofilm, additionally enhanced in the presence of rotifers. Our findings will help to understand the role of micro-grazers, such as L. inermis feeding on biofilm, in the fate of MP in nature. In the longer term, our results could help to develop biotechnological tools for MP removal from the aquatic environment.

Effects of polyaluminum chloride (PAX-18) on the relationship between predatory fungi and Lecane rotifers.

PAX-18 (polyaluminum chloride) is frequently used in WWTPs (wastewater treatment plants) to overcome sludge bulking. An alternative biological method is the usage of Lecane rotifers, which can be endangered by predacious fungi. We investigated the influence of different PAX-18 concentrations on the relationship between Lecane inermis and predacious fungi (Zoophagus and Lecophagus) differing in feeding mode. High PAX concentration (6 mg Al3+ L-1) strongly limited the number of the rotifers, which in low concentration (1.2 mg Al3+ L-1), after an initial decline, increased, but significantly slower than in control. Under the simultaneous influence of Lecophagus and PAX, rotifers were driven almost extinct at the high concentration, but survived at the lower concentration and increased in the control. When treated with Zoophagus, only one or two rotifers survived in treatments and control. High concentrations of PAX significantly restricted the growth of fungi, whereas in low concentrations and control conditions, their length increased, with Zoophagus growing much quicker than Lecophagus. Zoophagus was significantly more efficient in trapping rotifers regardless of PAX concentration. The trapping ability of mycelium following extended exposure to PAX was strongly limited at high concentrations, in comparison to control. Conidia of Zoophagus turned out to be considerably more resistant to PAX-18 and starvation than Lecophagus conidia.

Multivariate analysis of activated sludge community in full-scale wastewater treatment plants.

We investigated changes in protozoa and metazoa community in relation to process parameters in activated sludge from four wastewater treatment plants (WWTPs) throughout the period of 1 year. Principal component analysis (PCA) showed that activated sludge from investigated treatment plants had different dominating species representatives and community composition mainly depends on individual features of the treatment plants. Redundancy analysis (RDA) showed that the temperature in bioreactors was the most relevant factor explaining changes in the microorganism community, whereas reduction rate of chemical oxygen demand (COD), biological oxygen demand (BOD5), suspended solids (SS), and total nitrogen (TN) did not sufficiently explain the variation in protozoa and metazoan community composition. The results indicate that in stable working WWTP it is difficult to find a pronounced link between activated sludge species composition, process parameters, and plant configuration. Applied multivariate analysis can be a valuable tool for the exploration of the relations between community composition and WWTP process parameters.

Rotifers weaken the efficiency of the cyanobacterium defence against ciliate grazers.

Cyanobacteria can protect themselves through limited dispersion and by increasing the compactness of the mucilage-covered cyanobacterial mat as well as by producing sheaths covering their trichomes. These features have been used in research to measure their degree of inducible defence. The influence of the presence of the rotifers Lecane inermis on the effectiveness of Phormidium sp. (Ph2) cyanobacterium defence was investigated. Experiments were conducted on the ciliates Pseudomicrothorax dubius and Furgasonia blochmanni, specialised in the ingestion of filamentous cyanobacteria. The most compact were cyanobacterial mats that were subjected exclusively to ciliates and the most dispersed were mats in the presence of rotifers alone. The presence of rotifers feeding on cyanobacterial mucilage led to the decreased effectiveness of the defence in two ways, by increasing the dispersion of cyanobacterial trichomes, thus loosening the cyanobacterial mat, and through the ingestion of the exopolysaccharide material covering the trichomes. As a result, in the presence of rotifers and the high density of ciliates, almost all the trichomes were removed. Moreover, in comparison with other treatments, a higher number of ciliates and rotifers remained active until the end of the experiments. This is the first report to show how rotifers can weaken the defence of cyanobacteria.

The Relations Between Predatory Fungus and Its Rotifer Preys as a Noteworthy Example of Intraguild Predation (IGP).

Intraguild predation (IGP) is a widespread interaction combining predation and competition. We investigated a unique IGP example among predacious fungus Zoophagus sp. and two rotifers, the predacious Cephalodella gibba and the common prey Lecane inermis. We checked the influence of the fungus on its competitor C. gibba and their joint influence on shared prey L. inermis, and the impact of the competitive predator on the growth of predacious fungus. The experiment on grown mycelium showed that Zoophagus strongly, negatively influences the growth of C. gibba (intermediate consumer) whose number did not increase throughout the experiment. The intermediate consumer was also trapped by Zoophagus and become extinct when it was its only prey, whereas in the absence of the fungus and with unlimited access to prey, its number grew quickly. As only few C. gibba were trapped by fungi when common preys were present, competition for food seems to have stronger effect on intermediate consumer population than predation. The experiment with conidia of the fungus showed that intermediate consumer significantly limits the growth of Zoophagus by reducing the number of available prey. It was observed that although the fungus can trap C. gibba, the latter does not support its growth. Trapping the intermediate consumer might serve to eliminate a competitor rather than to find a source of food. The chances of survival for L. inermis under the pressure of the two competing predators are scarce. It is the first example of IGP involving representatives of two kingdoms: Fungi and Animalia.

Diversity and function of the microbial community under strong selective pressure of rotifers.

We unearthed some interesting microecological discoveries while selecting for the most beneficial bacterial strains to be used as probiotics in Lecane inermis rotifer mass culture. For 3 years, we maintained the cultures of L. inermis, with selection for the highest growth rate and resistance to potential contamination. Then, we conducted further selection and isolation in two groups: rotifers inoculated with the bacterial consortium isolated from the rotifer cultures, and rotifers fed with a commercial bioproduct. Selection was conducted in demanding conditions, with particulate matter suspended in spring water as a substrate, without aeration and under strong consumer pressure, and led to selection of two cultivable strains isolated from the optimal rotifers culture. According to molecular analysis, these strains were Aeromonas veronii and Pseudomonas mosselii. Biolog® ECO plate tests showed that both investigated bacterial communities metabolized wide but similar range of substrates. Therefore, intensely selective conditions led to considerable reduction in bacterial community regarding taxonomy, but not in metabolic activity, showing a functional composition decoupling. Aside from this result, our novel selection method dedicated to the sustainable culture of two trophic levels, a directed selection procedure (DSC), could potentially lead to the development of biotechnologically valuable strains with high metabolic activity and the ability to metabolize different sorts of substrate without harmful impact on higher trophic levels.

Temperature-Dependence of Predator-Prey Dynamics in Interactions Between the Predatory Fungus Lecophagus sp. and Its Prey L. inermis Rotifers.

Temperature is considered an important factor that influences the bottom-up and top-down control in water habitats. We examined the influence of temperature on specific predatory-prey dynamics in the following two-level trophic system: the predatory fungus Lecophagus sp. and its prey Lecane inermis rotifers, both of which originated from activated sludge obtained from a wastewater treatment plant (WWTP). The experiments investigating the ability of conidia to trap rotifers and the growth of fungal mycelium were performed in a temperature range that is similar to that in WWTPs in temperate climate. At 20 °C, 80% of the conidia trapped the prey during the first 24 h, whereas at 8 °C, no conidium was successful. The mycelium growth rate was the highest at 20 °C (r = 1.44) during the first 48 h but decreased during the following 24 h (r = 0.98), suggesting the quickest use of resources. At a medium temperature of 15 °C, the tendency was opposite, and the r value was lower during the first 48 h. At 8 °C, the growth rate was very low and remained at the same level even though numerous active rotifers were potentially available for the fungus. The temperature also influences the production of new conidia; on the 7th day, new conidia were observed in 96% of the wells at 20 °C, but no new conidia were observed at 8°C. These results show that the prey (rotifers)-predator (Lecophagus) dynamics in WWTPs is temperature-dependent, and a temperature of 8 °C is a strongly limiting factor for the fungus. Moderate temperatures ensure the most stable coexistence of the fungus and its prey, whereas the highest temperature can promote the prevalence of the predator.

Evaluation of the accuracy of two simple methods for microscopic activated sludge analysis.

Biological microscopic analysis is a popular method employed in wastewater treatment plants worldwide for evaluating activated sludge condition. However, many operators still have reservations regarding its reliability. In this study, we evaluated and compared two methods of microscopic sludge investigation: the sludge index (SI) and the Eikelboom-van Buijsen method (EB). We investigated 79 activated sludge samples from nine treatment plants located in southern Poland over a 1-year period. For each sample, sludge volume index values were calculated and compared with the results of evaluation made on the basis of microscopic analysis. Additionally, the effluent quality was analysed in 45 of 79 cases, including investigation of suspended solids, biochemical oxygen demand, chemical oxygen demand, total nitrogen and total phosphorous. The sign test and Wilcoxon matched pairs test showed that a significant difference existed between the two investigated methods. General conclusions from both methods do not provide reliable information concerning nitrogen and phosphorus removal. The EB method had a tendency to be more conservative in its general conclusions than the SI method. Both are highly reliable for estimating activated sludge quality and solid separation properties.

Interaction Between a Bacterivorous Ciliate Aspidisca cicada and a Rotifer Lecane inermis: Doozers and Fraggles in Aquatic Flocs.

Activated sludge is a semi-natural habitat composed of macroaggregates made by flocculating bacteria and inhabited by numerous protozoans and metazoans, creating a complicated interactome. The activated sludge resembles the biological formation of naturally occurring floc habitats, such as "marine snow." So far, these two types of habitat have been analyzed separately, despite their similarities. We examined the effect of a bacterivorous ciliate, Aspidisca cicada, on the quality of the macroaggregate ecosystem by estimating (i) the floc characteristics, (ii) the proliferation of other bacterivores (rotifers), and (iii) the chemical processes. We found that A. cicada (i) positively affected floc quality by creating flocs of larger size; (ii) promoted the population growth of the rotifer Lecane inermis, an important biological agent in activated sludge systems; and (iii) increased the efficiency of ammonia removal while at the same time improving the oxygen conditions. The effect of A. cicada was detectable long after its disappearance from the system. We therefore claim that A. cicada is a very specialized scavenger of flocs with a key role in floc ecosystem functioning. These results may be relevant to the ecology of any natural and engineered aggregates.

Foam-forming bacteria in activated sludge effectively reduced by rotifers in laboratory- and real-scale wastewater treatment plant experiments.

Lecane inermis rotifers were shown to diminish sludge bulking due to their ability to ingest the filamentous bacteria in activated sludge. To determine if rotifers are also able to control branched actinomycetes, we investigated three other Lecane species (Monogononta). In a week-long experiment, only Lecane tenuiseta significantly reduced the density of Microthrix parvicella and Type 0092 filaments, but in a 2-week experiment, actinomycetes were significantly reduced by most of the tested monogonont rotifers: L. inermis, Lecane decipiens and Lecane pyriformis. Rotifers L. inermis originating from the mass culture were artificially introduced into real-scale wastewater treatment plant (WWTP) in two series. The WWTP was monitored for 1 year. Rotifer inoculation resulted in diminishing of M. parvicella and actinomycete abundance. The experiments showed that different species of rotifers vary in their effectiveness at limiting various types of filamentous organisms. This is the first report demonstrating that one of the most troublesome bacteria, branched actinomycetes, which cause heavy foaming in bioreactors, can be controlled by rotifers. Knowledge of the consumers of filamentous bacteria that inhabit activated sludge could help WWTP operators overcome bulking and foaming through environmentally friendly methods.

The effect of three different predatory ciliate species on activated sludge microfauna.

Bacterivorous ciliates play important roles in the functioning of activated sludge by reducing dispersed bacteria and enhancing flocculation. There are, however, no data on the resistance of this functional group of microorganisms to predation. Our experiment was conducted with activated sludge subsamples subjected to artificially introduced three predatory ciliates species. The two predator species originating from activated sludge were Oxytricha sp. and Spathidium spathula. Dileptus margaritifer was a "foreign" predator species. The latter was introduced to compare its effect with the influence of predators naturally occurring in activated sludge on the ciliates community potentially adapted to certain predation strategies. Results showed that introduction of predatory ciliates into the activated sludge did not significantly change the total abundance of protozoa but rebuilt bacterivorous ciliate communities. Introduced predators significantly affected the most numerous ciliate species from the genera Epistylis and Cyclidium. In the presence of D. margaritifer, the abundance of sessile, colonial ciliates (Epistylis sp.) was significantly lower compared to the control treatment and to the treatments with the other predators. The activated sludge ciliate community was the most affected by the introduction of the "foreign" predator - D. margaritifer, a large ciliate armed with toxicysts.

Effect of high levels of the rotifer Lecane inermis on the ciliate community in laboratory-scale sequencing batch bioreactors (SBRs).

Due to its ability to feed on filamentous bacteria, the rotifer Lecane inermis has already been recognized as a potential control agent of activated sludge bulking, which is usually caused by the excessive growth of filamentous microorganisms. However, their effectiveness depends, in part, on their abundance. We studied the influence of high densities of L. inermis on the protozoan community in activated sludge from a wastewater treatment plant (WWTP) in 4 laboratory-scale sequencing batch bioreactors (SBRs). Two treatments and two controls were subjected to nutrient removal system in process similar to that used in a WWTP. The experiment lasted 9 days and was repeated in 24-h cycles, including phases of agitation with feeding, aeration and agitation and sedimentation with decantation at the end of the cycle. In total, 32 taxa were identified, among which 25 were ciliated protozoa, 4 were amoebae, 2 were flagellates, and one was a nematode. Rotifers were then introduced to 2 bioreactors at a final concentration of 500ind.mL(-1), and the taxonomic composition and abundance of the activated sludge microfauna were assessed 2, 5 and 8 days thereafter. The mean density of ciliates on the first day of experiment was 12,610ind.mL(-1) and diminished to 4868±432ind.mL-±432ind.mL(-1) in the control and 5496±638ind.mL(-1) in the rotifer-treated group on the last day. Thus, even extremely high densities of artificially introduced rotifers did not negatively affect the protozoan community. On the contrary, the protozoan community was more diverse in the treatment group than in the control.

Experimental attempt at using Lecane inermis rotifers to control filamentous bacteria Eikelboom Type 0092 in activated sludge.

In laboratory experiments, the authors tested the efficacy of Lecane inermis rotifers in reducing the abundance of filamentous bacteria in activated sludge samples dominated by one filamentous bacterium, Eikelboom Type 0092. The experiment was conducted in three replicates and repeated three times at 1-month intervals. In all repetitions, rotifers were able to significantly reduce the density of bacterial filaments through predation. It was also shown that the influence of rotifers on filament abundance depends on the duration of grazing pressure. The data show that L. inermis, already reported to be effective in reducing the density of Microthrix parvicella, Nostocoida limicola, and Type 021N in activated sludge, can be equally helpful in limiting Type 0092, another troublesome filamentous bacterium found in low-loaded water resource recovery facilities.

Chemical and mechanical signals in inducing Phormidium (Cyanobacteria) defence against their grazers.

The nature of stimuli evoking cyanobacterium defence was investigated in experiments on Phormidium sp. strain able to defend itself against ciliate grazers. Limited dispersion of trichomes in reaction to Pseudomicrothorax dubius separated from cyanobacterium with a mesh insert indicates the existence of a chemical cue originating from the ciliates. Grazers released into the wells where trichomes' dispersion was already limited by the cue initially had no difficulty finding food, but started to starve 24 h later. Similar situation was observed in control wells. Direct observations of trichomes attacked by the ciliates showed a distinct difference between the trichomes previously subjected to mesh-separated ciliate and the control ones. The former withdrew more frequently into a rigid sheath, whereas the latter usually withdrew into elastic tubes. This suggests that both chemical and mechanical stimuli are necessary to express cyanobacterium defence to the fullest extent. Further investigations showed that ciliates specialised in ingesting filamentous Cyanobacteria limit trichomes' dispersion, whereas filter-feeding Euplotes and Cyanobacteria-feeding rotifer do not. The cyanobacterium can detect grazer presence even without direct contact and modify its morphology in a way enabling full expression of defence reaction. This is the first report on ciliate-cyanobacterium chemical mediation.

Seasonal changes in the body size of two rotifer species living in activated sludge follow the Temperature-Size Rule.

Temperature-Size Rule (TSR) is a phenotypic body size response of ectotherms to changing temperature. It is known from the laboratory studies, but seasonal patterns in the field were not studied so far. We examined the body size changes in time of rotifers inhabiting activated sludge. We hypothesize that temperature is the most influencing parameter in sludge environment, leading sludge rotifers to seasonally change their body size according to TSR, and that oxygen content also induces the size response. The presence of TSR in Lecane inermis rotifer was tested in a laboratory study with two temperature and two food-type treatments. The effect of interaction between temperature and food was significant; L. inermis followed TSR in one food type only. The seasonal variability in the body sizes of the rotifers L. inermis and Cephalodella gracilis was estimated by monthly sampling and analyzed by multiple regression, in relation to the sludge parameters selected as the most influential by multivariate analysis, and predicted to alter rotifer body size (temperature and oxygen). L. inermis varied significantly in size throughout the year, and this variability is explained by temperature as predicted by the TSR, but not by oxygen availability. C. gracilis also varied in size, though this variability was explained by both temperature and oxygen. We suggest that sludge age acts as a mortality factor in activated sludge. It may have a seasonal effect on the body size of L. inermis and modify a possible effect of oxygen. Activated sludge habitat is driven by both biological processes and human regulation, yet its resident organisms follow general evolutionary rule as they do in other biological systems. The interspecific response patterns differ, revealing the importance of taking species-specific properties into account. Our findings are applicable to sludge properties enhancement through optimizing the conditions for its biological component.

Effect of the rotifer Lecane inermis, a potential sludge bulking control agent, on process parameters in a laboratory-scale SBR system.

The influence of a high density of rotifers, which is known to be able to control filamentous bacteria, on the parameters of an activated sludge process was examined in four professional laboratory batch reactors. These reactors allow the imitation of the work of a wastewater treatment plant with enhanced nutrient removal. The parameters, including oxygen concentration, pH and temperature, were constantly controlled. The experiment showed that Lecane rotifers are able to proliferate in cyclically anaerobic/anoxic and aerobic conditions and at dissolved oxygen concentrations as low as 1 mg/L. In 1 week, rotifer density increased fivefold, exceeding the value of 2,200 ind./mL. The grazing activity led to an improvement in settling properties. Extremely high numbers of rotifers did not affect the main parameters, chemical oxygen demand (COD), N-NH(4), N-NO(3), P-PO(4) and pH, during sewage treatment. Therefore, the use of rotifers as a tool to limit the growth of filamentous bacteria appears to be safe for the entire wastewater treatment process.

The toxicity of selected trace metals to Lecane inermis rotifers isolated from activated sludge.

The aim of the study was to assess the toxicity of a range of trace metals to the rotifer Lecane inermis, a species tested as a potential biological tool to control activated sludge bulking caused by overgrowth of filamentous bacteria in wastewater treatment plants. LC50 values (concentration lethal to 50 % of individuals, mg dm(-3)) were ranked in the following order: Cu < Al < Fe < Zn < Sn < Mn. L. inermis apparently is more sensitive to metals than other aquatic species widely used as model organisms in ecotoxicological testing, making it potentially useful for quick ecotoxicological tests.

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.

The influence of temperature on the effectiveness of filamentous bacteria removal from activated sludge by rotifers.

We investigated the feeding of the rotifer Lecane inermis on filamentous bacteria to determine if the ability of rotifers to remove filaments depends on temperature. The bacteria originated from two treatment plants, one of which was dominated by Microthrix parvicella and the other by Nostocoida limicola-like organisms. The experiments showed that the number of rotifers increased with temperature, and thus the ability of rotifers to reduce the number of filaments also increased with temperature. At 8 degrees C, their removal effectiveness was low, but the rotifers were able to survive at this temperature. When presented with sludge containing N. limicola-like microorganisms at 20 degrees C, the rotifers reduced the number of bacteria by 95%. In the case of M. parvicella, the reduction reached 50%. The results confirmed that Lecane are capable of controlling the growth of bacteria responsible for sludge bulking. This is the first report indicating that the seasonality of bulking may be the result of the activity of filamentous bacteria grazers, which is temperature dependent.