Understanding the differential impacts of two antidepressants on locomotion of freshwater snails (Lymnaea stagnalis).

There is growing evidence of negative impacts of antidepressants on behavior of aquatic non-target organisms. Accurate environmental risk assessment requires an understanding of whether antidepressants with similar modes of action have consistent negative impacts. Here, we tested the effect of acute exposure to two antidepressants, fluoxetine and venlafaxine (0-50 µg/L), on the behavior of non-target organism, i.e., freshwater pond snail, Lymnaea stagnalis. As compounds interact with chemical cues in the aquatic ecosystems, we also tested whether the effects altered in the presence of bile extract containing 5α-cyprinol sulfate (5α-CPS), a characterized kairomone of a natural predator, common carp (Cyprinus carpio). Behavior was studied using automated tracking and analysis of various locomotion parameters of L. stagnalis. Our results suggest that there are differences in the effects on locomotion upon exposure to venlafaxine and fluoxetine. We found strong evidence for a non-monotonic dose response on venlafaxine exposure, whereas fluoxetine only showed weak evidence of altered locomotion for a specific concentration. Combined exposure to compounds and 5α-CPS reduced the intensity of effects observed in the absence of 5α-CPS, possibly due to reduced bioavailability of the compounds. The results highlight the need for acknowledging different mechanisms of action among antidepressants while investigating their environmental risks. In addition, our results underline the importance of reporting non-significant effects and acknowledging individual variation in behavior for environmental risk assessment.

Impact of mixed microalgal and bacterial species on organic micropollutants removal in photobioreactors under natural light.

Single microalgae species are effective at the removal of various organic micropollutants (OMPs), however increased species diversity might enhance this removal. Sixteen OMPs were added to 2 continuous photobioreactors, one inoculated with Chlorella sorokiniana and the other with a microalgal-bacterial community, for 112 d under natural light. Three media were sequentially used in 3 Periods: I) synthetic sewage (d 0-28), II) 10x diluted anaerobically digested black water (AnBW) (d 28-94) and III) 5x diluted AnBW (d 94-112). Twelve OMPs were removed > 30 %, while 4 were < 10 % removed. Removal efficiencies were similar for 9 OMPs, yet the mixed community showed a 2-3 times higher removal capacity (µg OMP/g dry weight) than C. sorokiniana during Period II pseudo steady state. The removal decreased drastically in Period III due to overgrowth of filamentous green algae. This study shows for the first time how microbial community composition and abundance are key for OMPs removal.

Impact of wastewater characteristics on the removal of organic micropollutants by Chlorella sorokiniana.

Microalgae-based technologies can be used for the removal of organic micropollutants (OMPs) from different types of wastewater. However, the effect of wastewater characteristics on the removal is still poorly understood. In this study, the removal of sixteen OMPs by Chlorella sorokiniana, cultivated in three types of wastewater (anaerobically digested black water (AnBW), municipal wastewater (MW), and secondary clarified effluent (SCE)), were assessed. During batch operational mode, eleven OMPs were removed from AnBW and MW. When switching from batch to continuous mode (0.8 d HRT), the removal of most OMPs from AnBW and MW decreased, suggesting that a longer retention time enhances the removal of some OMPs. Most OMPs were not removed from SCE since poor nutrient availability limited C. sorokiniana growth. Further correlation analyses between wastewater characteristics, biomass and OMPs removal indicated that the wastewater soluble COD and biomass concentration predominantly affected the removal of OMPs. Lastly, carbon uptake rate had a higher effect on the removal of OMPs than nitrogen and phosphate uptake rate. These data will give an insight on the implementation of microalgae-based technologies for the removal of OMPs in wastewater with varying strengths and nutrient availability.

High resolution functional analysis and community structure of photogranules.

Photogranules are spherical aggregates formed of complex phototrophic ecosystems with potential for "aeration-free" wastewater treatment. Photogranules from a sequencing batch reactor were investigated by fluorescence microscopy, 16S/18S rRNA gene amplicon sequencing, microsensors, and stable- and radioisotope incubations to determine the granules' composition, nutrient distribution, and light, carbon, and nitrogen budgets. The photogranules were biologically and chemically stratified, with filamentous cyanobacteria arranged in discrete layers and forming a scaffold to which other organisms were attached. Oxygen, nitrate, and light gradients were also detectable. Photosynthetic activity and nitrification were both predominantly restricted to the outer 500 µm, but while photosynthesis was relatively insensitive to the oxygen and nutrient (ammonium, phosphate, acetate) concentrations tested, nitrification was highly sensitive. Oxygen was cycled internally, with oxygen produced through photosynthesis rapidly consumed by aerobic respiration and nitrification. Oxygen production and consumption were well balanced. Similarly, nitrogen was cycled through paired nitrification and denitrification, and carbon was exchanged through photosynthesis and respiration. Our findings highlight that photogranules are complete, complex ecosystems with multiple linked nutrient cycles and will aid engineering decisions in photogranular wastewater treatment.

Enhancing phosphorus removal of photogranules by incorporating polyphosphate accumulating organisms.

Photogranules are a novel wastewater treatment technology that can utilize the sun's energy to treat water with lower energy input and have great potential for nutrient recovery applications. They have been proven to efficiently remove nitrogen and carbon but show lower conversion rates for phosphorus compared to established treatment systems, such as aerobic granular sludge. In this study, we successfully introduced polyphosphate accumulating organisms (PAOs) to an established photogranular culture. We operated photobioreactors in sequencing batch mode with six cycles per day and alternating anaerobic (dark) and aerobic (light) phases. We were able to increase phosphorus removal/recovery by 6 times from 5.4 to 30 mg/L/d while maintaining similar nitrogen and carbon removal compared to photogranules without PAOs. To maintain PAOs activity, alternating anaerobic feast and aerobic famine conditions were required. In future applications, where aerobic conditions are dependent on in-situ oxygenation via photosynthesis, the process will rely on sunlight availability. Therefore, we investigated the feasibility of the process under diurnal cycles with a 12-h anaerobic phase during nighttime and six short cycles during the 12 h daytime. The 12-h anaerobic phase had no adverse effect on the PAOs and phototrophs. Due to the extension of one anaerobic phase to 12 h the six aerobic phases were shortened by 47% and consequently decreased the light hours per day. This resulted in a decrease of phototrophs, which reduced nitrogen removal and biomass productivity up to 30%. Finally, we discuss and suggest strategies to apply PAO-enriched photogranules at large-scale.

N2 -fixation can sustain wastewater treatment performance of photogranules under nitrogen-limiting conditions.

Wastewater characteristics can vary significantly, and in some municipal wastewaters the N:P ratio is as low as 5 resulting in nitrogen-limiting conditions. In this study, the microbial community, function, and morphology of photogranules under nitrogen-replete (N+) and limiting (N-) conditions was assessed in sequencing batch reactors. Photogranules under N- condition were nitrogen deprived 2/3 of a batch cycle duration. Surprisingly, this nitrogen limitation had no adverse effect on biomass productivity. Moreover, phosphorus and chemical oxygen demand removal were similar to their removal under N+ conditions. Although performance was similar, the difference in granule morphology was obvious. While N+ photogranules were dense and structurally confined, N- photogranules showed loose structures with occasional voids. Microbial community analysis revealed high abundance of cyanobacteria capable of N2 -fixation. These were higher at N- (38%) than N+ (29%) treatments, showing that photogranules could adjust and maintain treatment performance and high biomass productivity by means of N2 -fixation.

Removal processes of individual and a mixture of organic micropollutants in the presence of Scenedesmus obliquus.

Organic micropollutants (OMPs) need to be removed from wastewater as they can negatively affect aquatic organisms. It has been demonstrated that microalgae-based technologies are efficient in removing OMPs from wastewater. In this study, the removal processes and kinetics of six persistent OMPs (diclofenac, clarithromycin, benzotriazole, metoprolol, carbamazepine and mecoprop) were studied during cultivation of Scenedesmus obliquus in batch mode. These OMPs were added as individual compounds and in a mixture. Short experiments (8 days) were performed to avoid masking of OMP removal processes by light and nutrient limitation. The results show that diclofenac, clarithromycin, and benzotriazole were mainly removed by photodegradation (diclofenac), biodegradation (benzotriazole), or a combination of these two processes (clarithromycin). Peroxidase was involved in intracellular and extracellular biodegradation when benzotriazole was present as individual compound. Carbamazepine, metoprolol and mecoprop showed no biodegradation or photodegradation, and neglectable removal (<5%) by bioadsorption and bioaccumulation. Using an OMP mixture had an adverse effect on the photodegradation of clarithromycin and diclofenac, with reduced first-order kinetic constants compared to the individual compounds. Benzotriazole biodegradation was inhibited by the presence of the OMP mixture. This indicates that the presence of OMPs inhibits the photodegradation and biodegradation of some individual OMPs. These results will improve our understanding of removal processes of individual and mixtures of OMPs by microalgae-based technologies for wastewater treatment.

On-Site Blackwater Treatment Fosters Microbial Groups and Functions to Efficiently and Robustly Recover Carbon and Nutrients.

Wastewater is considered a renewable resource water and energy. An advantage of decentralized sanitation systems is the separation of the blackwater (BW) stream, contaminated with human pathogens, from the remaining household water. However, the composition and functions of the microbial community in BW are not known. In this study, we used shotgun metagenomics to assess the dynamics of microbial community structure and function throughout a new BW anaerobic digestion system installed at The Netherlands Institute of Ecology. Samples from the influent (BW), primary effluent (anaerobic digested BW), sludge and final effluent of the pilot upflow anaerobic sludge blanket (UASB) reactor and microalgae pilot tubular photobioreactor (PBR) were analyzed. Our results showed a decrease in microbial richness and diversity followed by a decrease in functional complexity and co-occurrence along the different modules of the bioreactor. The microbial diversity and function decrease were reflected both changes in substrate composition and wash conditions. Our wastewater treatment system also decreased microbial functions related to pathogenesis. In summary, the new sanitation system studied here fosters microbial groups and functions that allow the system to efficiently and robustly recover carbon and nutrients while reducing pathogenic groups, ultimately generating a final effluent safe for discharge and reuse.

Enhancement of co-production of nutritional protein and carotenoids in Dunaliella salina using a two-phase cultivation assisted by nitrogen level and light intensity.

Microalga Dunaliella salina is known for its carotenogenesis. At the same time, it can also produce high-quality protein. The optimal conditions for D. salina to co-produce intracellular pools of both compounds, however, are yet unknown. This study investigated a two-phase cultivation strategy to optimize combined high-quality protein and carotenoid production of D. salina. In phase-one, a gradient of nitrogen concentrations was tested. In phase-two, effects of nitrogen pulse and high illumination were tested. Results reveal optimized protein quantity, quality (expressed as essential amino acid index EAAI) and carotenoids content in a two-phase cultivation, where short nitrogen starvation in phase-one was followed by high illumination during phase-two. Adopting this strategy, productivities of protein, EAA and carotenoids reached 22, 7 and 3 mg/L/d, respectively, with an EAAI of 1.1. The quality of this biomass surpasses FAO/WHO standard for human nutrition, and the observed level of ß-carotene presents high antioxidant pro-vitamin A activity.

Toward an Ecologically Optimized N:P Recovery from Wastewater by Microalgae.

Global stores of important resources such as phosphorus (P) are being rapidly depleted, while the excessive use of nutrients has led to the enrichment of surface waters worldwide. Ideally, nutrients would be recovered from wastewater, which will not only prevent eutrophication but also provide access to alternative nutrient stores. Current state-of-the-art wastewater treatment technologies are effective in removing these nutrients from wastewater, yet they can only recover P and often in an insufficient way. Microalgae, however, can effectively assimilate P and nitrogen (N), as well as other macro- and micronutrients, allowing these nutrients to be recovered into valuable products that can be used to close nutrient cycles (e.g., fertilizer, bioplastics, color dyes, and bulk chemicals). Here, we show that the green alga Chlorella sorokiniana is able to remove all inorganic N and P present in concentrated toilet wastewater (i.e., black water) with N:P ratios ranging between 15 and 26. However, the N and P uptake by the algae is imbalanced relative to the wastewater N:P stoichiometry, resulting in a rapid removal of P but relatively slower removal of N. Here, we discuss how ecological principles such as ecological stoichiometry and resource-ratio theory may help optimize N:P removal and allow for more effective recovery of N and P from black water.

Micropollutant removal in an algal treatment system fed with source separated wastewater streams.

Micropollutant removal in an algal treatment system fed with source separated wastewater streams was studied. Batch experiments with the microalgae Chlorella sorokiniana grown on urine, anaerobically treated black water and synthetic urine were performed to assess the removal of six spiked pharmaceuticals (diclofenac, ibuprofen, paracetamol, metoprolol, carbamazepine and trimethoprim). Additionally, incorporation of these pharmaceuticals and three estrogens (estrone, 17ß-estradiol and ethinylestradiol) into algal biomass was studied. Biodegradation and photolysis led to 60-100% removal of diclofenac, ibuprofen, paracetamol and metoprolol. Removal of carbamazepine and trimethoprim was incomplete and did not exceed 30% and 60%, respectively. Sorption to algal biomass accounted for less than 20% of the micropollutant removal. Furthermore, the presence of micropollutants did not inhibit C. sorokiniana growth at applied concentrations. Algal treatment systems allow simultaneous removal of micropollutants and recovery of nutrients from source separated wastewater. Nutrient rich algal biomass can be harvested and applied as fertilizer in agriculture, as lower input of micropollutants to soil is achieved when algal biomass is applied as fertilizer instead of urine.