Phycobiliprotein recovery coupled to the tertiary treatment of wastewater in semi-continuous photobioreactors. Tracking contaminants of emerging concern.

This study evaluated a tertiary wastewater treatment technology using cyanobacteria to recover value-added phycobiliproteins. The presence of contaminants of emerging concern (CECs) in wastewater, cyanobacterial biomass and pigments recovered were also analyzed. For this, a wastewater-borne cyanobacterium (Synechocystis sp. R2020) was used to treat secondary effluent from a municipal wastewater treatment plant, with and without nutrients supplementation. Then, the stability of phycobiliprotein production was assessed by operating the photobioreactor in semi-continuous mode. Results showed similar biomass productivity with and without nutrients supplementation (153.5 and 146.7 mg L-1 d-1, respectively). Upon semi-continuous operation, the phycobiliprotein content was stable and reached up to 74.7 mg gDW-1. The phycocyanin purity ratio ranged from 0.5 to 0.8, corresponding to food grade (>0.7). Out of 22 CECs detected in secondary effluent, only 3 were present in the phycobiliprotein extracts. In order to identify applications, prospective research should focus on CECs removal during pigment purification.

The potential of wastewater grown microalgae for agricultural purposes: Contaminants of emerging concern, heavy metals and pathogens assessment.

In the coming years, the use of microalgal biomass as agricultural biofertilizers has shown promising results. The use of wastewater as culture medium has resulted in the reduction of production costs, making microalgae-based fertilizers highly attractive for farmers. However, the occurrence of specific pollutants in wastewater, like pathogens, heavy metals and contaminants of emerging concern (CECs), such as pharmaceuticals and personal care products may pose a risk on human health. This study presents an holistic assessment of the production and use of microalgal biomass grown in municipal wastewater as biofertilizer in agriculture. Results showed that pathogens and heavy metals concentrations in the microalgal biomass were below the threshold established by the European regulation for fertilizing products, except for cadmium. Regarding CECs, 25 out of 29 compounds were found in wastewater. However, only three of them (hydrocinnamic acid, caffeine, and bisphenol A) were found in the microalgae biomass used as biofertilizer. Agronomic tests were performed for lettuce growth in greenhouse. Four treatments were studied, comparing the use of microalgae biofertilizer with a conventional mineral fertilizer, and also a combination of both of them. Results suggested that microalgae can help reducing the mineral nitrogen dose, since similar fresh shoot weights were obtained in the plants grown with the different assessed fertilizers. Lettuce samples revealed the presence of cadmium and CECs in all the treatments including both negative and positive controls, which suggests that their presence was not linked to the microalgae biomass. On the whole, this study revealed that wastewater grown microalgae can be used for agricultural purposes reducing mineral N need and guaranteeing health safety of the crops.

Production of phycobiliproteins, bioplastics and lipids by the cyanobacteria Synechocystis sp. treating secondary effluent in a biorefinery approach.

Cyanobacteria have been identified as promising organisms to reuse nutrients from waste effluents and produce valuable compounds such as lipids, polyhydroxyalkanoates (PHAs), and pigments. However, almost all studies on cyanobacterial biorefineries have been performed under lab scale and short cultivation periods. The present study evaluates the cultivation of the cyanobacterium Synechocystis sp. in a pilot scale 30 L semi-continuous photobioreactor fed with secondary effluent for a period of 120 days to produce phycobiliproteins, polyhydroxybutyrate (PHB) and lipids. To this end, the harvested biomass from the semi-continuous photobioreactor was transferred into 5 L vertical column batch photobioreactors to perform PHB and lipid accumulation under nutrient starvation. Three hydraulic retention times (HRT) (6, 8 and 10 days) were tested in the semi-continuous photobioreactor to evaluate its influence on biomass growth and microbial community. A maximum biomass concentration of 1.413 g L-1 and maximum productivity of 173 mg L-1 d-1 was reached under HRT of 8 days. Microscopy analysis revealed a shift from Synechocystis sp. to Leptolyngbya sp. and green algae when HRT of 6 days was used. Continuous, stable production of phycobiliproteins in the semi-continuous photobioreactor was obtained, reaching a maximum content of 7.4%dcw in the biomass. In the batch photobioreactors a PHB content of 4.8%dcw was reached under 7 days of nitrogen and phosphorus starvation, while a lipids content of 44.7%dcw was achieved under 30 days of nitrogen starvation. PHB and lipids production was strongly dependent on the amount of nutrients withdrawn from the grow phase. In the case of lipids, their production was stimulated when there was only phosphorus depletion. While Nitrogen and phosphorus limitation was needed to enhance the PHB production. In conclusion, this study demonstrates the feasibility of cultivating cyanobacteria in treated wastewater to produce bio-based valuable compounds within a circular bioeconomy approach.

Can microalgae grown in wastewater reduce the use of inorganic fertilizers?

Alternatives to conventional inorganic fertilizers are needed to cope with the growing global population and contamination due to the production and use of those inorganic compounds. The recovery of nutrients from wastewater and organic wastes is a promising option to provide fertilization in a circular economy approach. In this context, microalgae-based systems are an alternative to conventional wastewater treatment systems, reducing the treatment costs and improving the sustainability of the process, while producing nutrient-rich microalgal biomass. The aim of the present study is to evaluate the use of microalgal biomass produced during domestic wastewater treatment in high rate algal ponds as a biofertilizer in basil crops (Ocimum basilicum L.). Wastewater was successfully treated, with removal efficiencies in the secondary treatment of 69, 91 and 81% in terms of chemical oxygen demand (COD), total inorganic nitrogen (TIN) and phosphates (PO43-P), respectively. The microalgal biomass, composed mainly by Scenedesmus, presented the following composition: 12% of dry weight and nutrients concentration of 7.6% nitrogen (N), 1.6% phosphorus (P) and 0.9% potassium (K). The study compared the performance of 3 different fertilizers: 1) microalgae fertilizer (MF), 2) inorganic fertilizer (IF) as positive control and 3) the combination of both microalgae and inorganic fertilizer (MF + IF). Comparable plant growth (i.e., number of leaves, shoot fresh and dry weight and leaf fresh weight) was observed among treatments, except for leaf dry weight, which was significantly higher in the IF + MF and MF treatments (28 and 27%, respectively) in comparison with the control. However, the microalgae treatment provided the lowest chlorophyll, N and K leaf content. In conclusion, this study suggests that combining microalgae grown in wastewater with an inorganic fertilizer is a promising nutrients source for basil crops, enhancing the circular bioeconomy.

Monitoring PHB production in Synechocystis sp. with hyperspectral images.

Microalgae wastewater treatment systems have the potential for producing added-value products. More specifically, cyanobacteria are able to accumulate polyhydroxybutyrates (PHBs), which can be extracted and used for bioplastics production. Nonetheless, PHB production requires proper culture conditions and continue monitoring, challenging the state-of-the-art technologies. The aim of this study was to investigate the application of hyperspectral technologies to monitor cyanobacteria population growth and PHB production. We have established a ground-breaking measurement method able to discern spectral reflectance changes from light emitted to cyanobacteria in different phases. All in all, enabling to distinguish between cyanobacteria growth phase and PHB accumulation phase. Furthermore, first tests of classification algorithms used for machine learning and image recognition technologies had been applied to automatically recognize the different cyanobacteria species from a complex microbial community containing cyanobacteria and microalgae cultivated in pilot-scale photobioreactors (PBRs). We have defined three main indicators for monitoring PHB production: (i) cyanobacteria specific-strain density, (ii) differentiate between growth and PHB-accumulation and (iii) chlorosis progression. The results presented in this study represent an interesting alternative for traditional measurements in cyanobacteria PHB production and its application in pilot-scale PBRs. Although not directly determining the amount of PHB production, they would give insights on the undergoing processes.

Inorganic carbon stimulates the metabolic routes related to the polyhdroxybutyrate production in a Synechocystis sp. strain (cyanobacteria) isolated from wastewater.

Cyanobacteria are capable of transforming CO2 into polyhydroxybutyrate (PHB). In this study, different inorganic carbon concentrations (0-2 gC L-1) were evaluated for a Synechocystis sp. strain isolated from wastewater. Quantitative RT-qPCR was also performed to decipher the links between inorganic carbon and PHB and glycogen metabolism. 2 gC L-1 of bicarbonate stimulated cell growth, nutrients consumption and production of PHB. Using this concentration, a 14%dcw of PHB and an average productivity of 2.45 mgPHB L-1 d-1 were obtained. Gene expression analysis revelated that these conditions caused the overexpression of genes related to glycogen and PHB synthesis. Moreover, a positive correlation between the genes codifying for the glycogen phosphorylase, the acetyl-CoA reductase and the poly(3-hydroxyalkanoate) polymerase was found, meaning that PHB synthesis and glycogen catabolism are strongly related. These results provide an exhaustive evaluation of the effect of carbon on the PHB production and cyanobacterial metabolism.

Advanced biokinetic and hydrodynamic modelling to support and optimize the design of full-scale high rate algal ponds.

High rate algal ponds (HRAP) are known for their suitability to treat wastewater and to produce microalgal biomass, which can be converted into bioproducts. However, full-scale application of HRAP is still limited to few cases, and design procedures are not consolidated or standardized. In this study, a demonstrative-scale HRAP system for secondary wastewater treatment to be implemented in India (treatment capacity of 50 m3·d-1) has been designed combining conventional dimensioning techniques and advanced modelling tools. The objective of the study was to assist, verify and optimize the conventional dimensioning of the secondary HRAP by means of simulations predicting the behaviour of the system in the specific local conditions under different configurations and operational strategies. Biokinetic modelling and hydrodynamic analysis using Computational Fluid Dynamics (CFD) were carried out. The simulations performed with the biokinetic model showed that the optimal hydraulic retention time to enhance nutrient removal and biomass production is 4 days. For the hydrodynamic modelling, a 3D model of the HRAP was built to simulate the hydrodynamic behaviour of 36 different designs. Simulations allowed quantifying the presence of low velocity zones as well as the land use efficiency of the different designs in terms of the useful area vs. the total occupied area. Two baffles and tear-shapes with a diameter equal to » of the channel width was the most efficient configuration. Moreover, a technical-economic assessment of the system was carried out, resulting in an investment cost of 483 € per population equivalent and an operational cost of 0.19 € per m3 of treated wastewater.

Removal and environmental risk assessment of contaminants of emerging concern from irrigation waters in a semi-closed microalgae photobioreactor.

The present study evaluated the efficiency of a semi-closed, tubular, horizontal photobioreactor (PBR) to treat a mixture of irrigation and rural drainage water, focusing in the removal of different contaminants of emerging concern (CECs), and evaluating the environmental impact of the resulting effluent. Target CECs included pharmaceuticals, personal care products and flame retardants. Of the 13 compounds evaluated, 11 were detected in the feed water entering the PBR, and diclofenac (DCF) (1107 ng L-1) and N,N-diethyl-toluamide (DEET) (699 ng L-1) were those present at the greatest concentrations. The best removal efficiencies were achieved for the pharmaceuticals diazepam (94%), lorazepam (LZP) (83%) and oxazepam (OXA) (71%), and also for ibuprofen (IBU) (70%). For the rest of the CECs evaluated, attenuation was similar to that obtained after conventional wastewater treatment, ranging from basically no elimination (carbamazepine (CBZ) and tris-(2-chloroethyl) phosphate (TCEP)) to medium efficiencies (DCF and tributyl phosphate (TBP) (50%)). Environmental risk assessment based on hazard quotients (HQs) resulted in HQ values < 0.1 (no risk associated) for most of the compounds and most of the trophic levels considered. Values between 1 and 10 (moderate risk) were obtained for tonalide (AHTN) (fish) and CBZ (invertebrates). The most sensitive trophic level was green algae, whereas fish and aquatic plants were the most resilient. Our results suggest that microalgae-based treatments could become a green, cost-effective alternative to conventional wastewater treatment regarding the efficient elimination of these contaminants.

Microalgae-based bioremediation of water contaminated by pesticides in peri-urban agricultural areas.

The present study evaluated the capacity of a semi-closed, tubular horizontal photobioreactor (PBR) to remove pesticides from agricultural run-off. The study was carried out in summer (July) to study its efficiency under the best conditions (highest solar irradiation). A total of 51 pesticides, including 10 transformation products, were selected and investigated based on their consumption rate and environmental relevance. Sixteen of them were detected in the agricultural run-off, and the estimated removal efficiencies ranged from negative values, obtained for 3 compounds, namely terbutryn, diuron and imidacloprid, to 100%, achieved for 10 compounds. The acidic herbicide MCPA was removed by 88% in average, and the insecticides 2,4-D and diazinon showed variable removals, between 100% and negative values. The environmental risk associated to the compounds still present in the effluent of the PBR was evaluated using hazard quotients (HQs), calculated using the average and highest measured concentrations of the compounds. HQ values > 10 (meaning high risk) were obtained for imidacloprid (21), between 1 and 10 (meaning moderate risk) for 2,4-D (2.8), diazinon (4.6) and terbutryn (1.5), and <1 (meaning low risk) for the remaining compounds diuron, linuron and MCPA. The PBR treatment yielded variable removals depending on the compound, similarly to conventional wastewater treatment plants. This study provides new data on the capacity of microalgae-based treatment systems to eliminate a wide range of priority pesticides under real/environmental conditions.

Bioremediation of emerging micropollutants in irrigation water. The alternative of microalgae-based treatments.

The present study evaluated the efficiency of a semi-closed horizontal tubular photobioreactor (PBR) at demonstrative scale to remove a total of 35 target compounds, including benzotriazoles, benzophenones, antibiotics and different pharmaceuticals present in irrigation water in a peri-urban rural area. This water run through an open channel and was a mixture of reclaimed wastewater from a nearby wastewater treatment plant (WWTP) and run-off from the different agricultural fields in the area. Most of the compounds studied are usually not fully eliminated during conventional wastewater treatment, which justifies the need to investigate alternative treatment strategies. A total of 21 of these compounds were detected in the irrigation water. Benzotriazoles were only partially removed after the microalgae treatment, with elimination rates similar to those of conventional WWTPs. The UV filter benzophenone-3 (BP3) showed variable removals, ranging from no elimination to 51%, whereas 4-methylbenzilidenecamphor (4MBC) was completely eliminated. Regarding pharmaceuticals, average removals were higher, in the range of 60-100%, with the exception of the antibiotics sulfamethoxazole (46%) and sulfapyridine, which was not removed. Despite the low biomass productivity of the PBR, parameters such as the size of the reactors, the specific mixed cultures developed and the high temperatures and pH in the closed system may account for the overall good results, The efficiency and sustainability of these systems make them a solid, feasible treatment choice.

Free ammonia inhibition in microalgae and cyanobacteria grown in wastewaters: Photo-respirometric evaluation and modelling.

The inhibitory effects of free ammonia (FA) on microalgae/cyanobacteria in wastewater-treating photobioreactors (PBR) can strongly reduce their treatment efficiency, increasing the operational costs and undermining the stability of the system. Although FA-promoting conditions (high pH, temperature and ammoniacal nitrogen concentration) are commonly met in outdoor PBRs, photosynthesis inhibition from FA has been scarcely explored and is rarely considered in microalgae-bacteria growth models. Two pilot systems and a series of lab-scale monocultures were tested using a photo-respirometry approach, to evaluate the effects of FA (8.5-136 mg NH3 L-1) on photosynthesis. Two mathematical inhibition models were compared, with the aim of selecting best-fitting equations to describe photo-respirometric experiments. A set of calibrated inhibition parameters was obtained for microalgae and cyanobacteria, growing in monocultures or in mixed algae-bacteria consortia. Cyanobacteria were more sensitive to FA than green microalgae and mixed phototrophs-bacteria consortia showed a higher resistance compared to monocultures. Estimated inhibition parameters were used to describe common operational/environmental conditions in algae-bacteria systems, demonstrating the potential drop in photosynthetic activity under those relevant operational conditions.

Upflow anaerobic sludge blanket in microalgae-based sewage treatment: Co-digestion for improving biogas production.

Upflow anaerobic sludge blanket (UASB) reactors are widely used to treat domestic sewage and frequently require post-treatment. Little is known about the use of high rate algal ponds (HRAP) for post-treating UASB reactors' effluent. This study aimed to evaluate a UASB reactor followed by a HRAP in terms of sewage treatment efficiency and biogas production, during one year at demonstration-scale. The UASB reactor co-treated raw sewage and the harvested microalgal biomass from the HRAP, which was recirculated to the reactor. An identical UASB reactor, treating only raw sewage, was used as control. The results showed an overall removal of 65% COD and 61% N-NH4 in the system. Furthermore, methane yield was increased by 25% after anaerobic co-digestion with microalgae, from 156 to 211 NL CH4 kg-1 VS. An energy assessment was performed and showed a positive energy balance, with a net ratio of 2.11 to the annual average.

Polyhydroxybutyrate and glycogen production in photobioreactors inoculated with wastewater borne cyanobacteria monocultures.

The aim of this study was to investigate the PHB and glycogen accumulation dynamics in two photobioreactors inoculated with different monocultures of wastewater-borne cyanobacteria, using a three-stage feeding strategy (growth phase, feast-famine phase and feast phase). Two cyanobacterial monocultures containing members of Synechocystis sp. or Synechococcus sp. were collected from treated wastewater and inoculated in lab-scale photobioreactors to evaluate the PHB and glycogen accumulation. A third photobioreactor with a complex microbial community grown in real wastewater was also set up. During each experimental phase different concentrations of inorganic carbon were applied to the cultures, these shifts allowed to discern the accumulation mechanism of carbon storage polymers (PHB and glycogen) in cyanobacteria. Conversion of one into the other was directly related to the carbon content. The highest PHB and glycogen contents (5.04%dcw and 69%dcw, respectively) were achieved for Synechocystis sp.

Influence of liquid-to-biogas ratio and alkalinity on the biogas upgrading performance in a demo scale algal-bacterial photobioreactor.

The influence of the liquid-to-biogas ratio (L/G) and alkalinity on methane quality was evaluated in a 11.7 m3 outdoors horizontal semi-closed tubular photobioreactor interconnected to a 45-L absorption column (AC). CO2 concentrations in the upgraded methane ranged from <0.1 to 9.6% at L/G of 2.0 and 0.5, respectively, with maximum CH4 concentrations of 89.7% at a L/G of 1.0. Moreover, an enhanced CO2 removal (mediating a decrease in CO2 concentration from 9.6 to 1.2%) and therefore higher CH4 contents (increasing from 88.0 to 93.2%) were observed when increasing the alkalinity of the AC cultivation broth from 42 ±â€¯1 mg L-1 to 996 ±â€¯42 mg L-1. H2S was completely removed regardless of the L/G or the alkalinity in AC. The continuous operation of the photobioreactor with optimized operating parameters resulted in contents of CO2 (<0.1%-1.4%), H2S (<0.7 mg m-3) and CH4 (94.1%-98.8%) complying with international regulations for methane injection into natural gas grids.

Model-based evaluation of a trickling filter facility upgrade to biological nutrient removal.

This article presents the feasibility evaluation and preliminary design of a wastewater treatment plant upgrade supported by simulation. The existing facility was based on trickling filters, and the objective of the upgrade was to achieve nutrients removal. The proposed solution modifies the existing primary clarifier to host an anaerobic-anoxic suspended growth reactor, which is an alternative that, to our knowledge, has not been proposed or explored so far. The trickling filters would remain as aerobic reactors. In this study, the novel treatment scheme has been assessed for the first time, through model simulations. The modified treatment train was simulated, showing that the anoxic zone is able to denitrify satisfactorily achieving the required effluent nitrogen concentration. However, to promote biological phosphorus removal, an additional aerobic zone combined with a bypass of activated sludge from the anoxic zone to the first trickling filter is needed, in order to provide aerobic conditions to the phosphate accumulating organisms. Several combinations of additional aerobic volume and sludge bypass flowrate were found to successfully achieve both nitrogen and phosphorus removal, using the existing facilities without the need for new reactors neither implementing modifications that could put the trickling filters' physical integrity at risk. The novel treatment scheme could be applied in other cases with similar flowsheet in the same context.

Performance evaluation of a novel anaerobic-anoxic sludge blanket reactor for biological nutrient removal treating municipal wastewater.

A novel anaerobic-anoxic sludge blanket reactor, AnoxAn, unifies the non-aerated zones of the biological nutrient removal treatment train in a single upflow reactor, aimed at achieving high compactness and efficiency. The environmental conditions are vertically divided up inside the reactor with the anaerobic zone at the bottom and the anoxic zone above. This contribution presents the performance evaluation of the novel reactor in the removal of organic matter and nutrients from municipal wastewater, coupled with an aerobic hybrid MBR. The overall system achieved total nitrogen and phosphorus removal with average efficiencies of 75% and 89%, respectively. Separate anoxic and anaerobic conditions were maintained in AnoxAn, allowing anaerobic phosphate release and nearly complete anoxic denitrification in the single reactor operating with an HRT of 4.2h. Biomass was retained in the reactor achieving TSS concentration up to 10gL(-1) and partial hydrolysis of influent particulate organic matter.

Feasibility of hydraulic separation in a novel anaerobic-anoxic upflow reactor for biological nutrient removal.

This contribution deals with a novel anaerobic-anoxic reactor for biological nutrient removal (BNR) from wastewater, termed AnoxAn. In the AnoxAn reactor, the anaerobic and anoxic zones for phosphate removal and denitrification are integrated in a single continuous upflow sludge blanket reactor, aiming at high compactness and efficiency. Its application is envisaged in those cases where retrofitting of existing wastewater treatment plants for BNR, or the construction of new ones, is limited by the available surface area. The environmental conditions are vertically divided up inside the reactor with the anaerobic zone at the bottom and the anoxic zone above. The capability of the AnoxAn configuration to establish two hydraulically separated zones inside the single reactor was assessed by means of hydraulic characterization experiments and model simulations. Residence time distribution (RTD) experiments in clean water were performed in a bench-scale (48.4 L) AnoxAn prototype. The required hydraulic separation between the anaerobic and anoxic zones, as well as adequate mixing in the individual zones, were obtained through selected mixing devices. The observed behaviour was described by a hydraulic model consisting of continuous stirred tank reactors and plug-flow reactors. The impact of the denitrification process in the anoxic zone on the hydraulic separation was subsequently evaluated through model simulations. The desired hydraulic behaviour proved feasible, involving little mixing between the anaerobic and anoxic zones (mixing flowrate 40.2 % of influent flowrate) and negligible nitrate concentration in the anaerobic zone (less than 0.1 mgN L(-1)) when denitrification was considered.