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.

How long can tardigrades survive in the anhydrobiotic state? A search for tardigrade anhydrobiosis patterns.

Anhydrobiosis is a desiccation tolerance that denotes the ability to survive almost complete dehydration without sustaining damage. The knowledge on the survival capacity of various tardigrade species in anhydrobiosis is still very limited. Our research compares anhydrobiotic capacities of four tardigrade species from different genera, i.e. Echiniscus testudo, Paramacrobiotus experimentalis, Pseudohexapodibius degenerans and Macrobiotus pseudohufelandi, whose feeding behavior and occupied habitats are different. Additionally, in the case of Ech. testudo, we analyzed two populations: one urban and one from a natural habitat. The observed tardigrade species displayed clear differences in their anhydrobiotic capacity, which appear to be determined by the habitat rather than nutritional behavior of species sharing the same habitat type. The results also indicate that the longer the state of anhydrobiosis lasts, the more time the animals need to return to activity.

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.

Extraordinary Multi-Organismal Interactions Involving Bacteriophages, Bacteria, Fungi, and Rotifers: Quadruple Microbial Trophic Network in Water Droplets.

Our observations of predatory fungi trapping rotifers in activated sludge and laboratory culture allowed us to discover a complicated trophic network that includes predatory fungi armed with bacteria and bacteriophages and the rotifers they prey on. Such a network seems to be common in various habitats, although it remains mostly unknown due to its microscopic size. In this study, we isolated and identified fungi and bacteria from activated sludge. We also noticed abundant, virus-like particles in the environment. The fungus developed absorptive hyphae within the prey. The bacteria showed the ability to enter and exit from the hyphae (e.g., from the traps into the caught prey). Our observations indicate that the bacteria and the fungus share nutrients obtained from the rotifer. To narrow the range of bacterial strains isolated from the mycelium, the effects of bacteria supernatants and lysed bacteria were studied. Bacteria isolated from the fungus were capable of immobilizing the rotifer. The strongest negative effect on rotifer mobility was shown by a mixture of Bacillus sp. and Stenotrophomonas maltophilia. The involvement of bacteriophages in rotifer hunting was demonstrated based on molecular analyses and was discussed. The described case seems to be an extraordinary quadruple microbiological puzzle that has not been described and is still far from being understood.

Tolerance to Anhydrobiotic Conditions Among Two Coexisting Tardigrade Species Differing in Life Strategies.

Water availability is one of the most important factors for terrestrial life. Terrestrial habitats may periodically become dry, which can be overcome by an organism's capability to undergo anhydrobiosis. In animals, this phenomenon has been reported for invertebrates, with tardigrades being the best-known. However, different tardigrade species appear to significantly differ in their anhydrobiotic abilities. While several studies have addressed this issue, established experimental protocols for tardigrade dehydration differ both within and among species, leading to ambiguous results. Therefore, we apply unified conditions to estimate intra-and interspecies differences in anhydrobiosis ability reflected by the return to active life. We analysed Milnesium inceptum and Ramazzottius subanomalus representing predatory and herbivorous species, respectively, and often co-occur in the same habitat. The results indicated that the carnivorous Mil. inceptum displays better anhydrobiosis survivability than the herbivorous Ram. subanomalus. This tendency to some degree coincides with the time of "waking up" since Mil. inceptum showed first movements and full activity of any first individual later than Ram. subanomalus. The movements of all individuals were however observed to be faster for Mil. inceptum. Differences between the experimental groups varying in anhydrobiosis length were also observed: the longer tun state duration, the more time was necessary to return to activity.

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.

Integrative description of bisexual Paramacrobiotus experimentalis sp. nov. (Macrobiotidae) from republic of Madagascar (Africa) with microbiome analysis.

In a moss samples collected on Madagascar two populations of Paramacrobiotus experimentalis sp. nov. were found. Paramacrobiotus experimentalis sp. nov. with the presence of a microplacoid and areolatus type of eggs is similar to Pam. danielae, Pam. garynahi, Pam. hapukuensis, Pam. peteri, Pam. rioplatensis and Pam. savai, but it differs from them by some morphological and morphometric characters of the eggs. The p-distance between two COI haplotypes of Pam. experimentalis sp. nov. was 0.17%. In turn, the ranges of uncorrected genetic p-distances of all Paramacrobiotus species available in GenBank was from 18.27% (for Pam. lachowskae) to 25.26% (for Pam. arduus) with an average distance of 20.67%. We also found that Pam. experimentalis sp. nov. is bisexual. This observation was congruent on three levels: (i) morphological - specimen size dimorphism; (ii) structural (primary sexual characteristics) - females have an unpaired ovary while males have an unpaired testis and (iii) molecular - heterozygous and homozygous strains of the ITS-2 marker. Although symbiotic associations of hosts with bacteria (including endosymbiotic bacteria) are common in nature and these interactions exert various effects on the evolution, biology and reproductive ecology of hosts, there is still very little information on the bacterial community associated with tardigrades. To fill this gap and characterise the bacterial community of Pam. experimentalis sp. nov. populations and microbiome of its microhabitat, high throughput sequencing of the V3-V4 hypervariable regions in the bacterial 16S rRNA gene fragment was performed. The obtained 16S rRNA gene sequences ranged from 92,665 to 131,163. In total, 135 operational taxonomic units (OTUs) were identified across the rarefied dataset. Overall, both Pam. experimentalis sp. nov. populations were dominated by OTUs ascribed to the phylum Proteobacteria (89-92%) and Firmicutes (6-7%). In the case of samples from tardigrades' laboratory habitat, the most abundant bacterial phylum was Proteobacteria (51-90%) and Bacteroides (9-48%). In all compared microbiome profiles, only 16 of 137 OTUs were shared. We found also significant differences in beta diversity between the partly species-specific microbiome of Pam. experimentalis sp. nov. and its culturing environment. Two OTUs belonging to a putative bacterial endosymbiont were identified - Rickettsiales and Polynucleobacter. We also demonstrated that each bacterial community was rich in genes involved in membrane transport, amino acid metabolism, and carbohydrate metabolism.

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.