Abstract: Resource recovery from source-separated urine can shorten nutrient cycles on Earth and is essential in regenerative life support systems for deep-space exploration. In this study, a robust two-stage, energy-efficient, gravity-independent urine treatment system was developed to transform fresh real human urine into a stable nutrient solution. In the first stage, up to 85% of the COD was removed in a microbial electrolysis cell (MEC), converting part of the energy in organic compounds (27-46%) into hydrogen gas and enabling full nitrogen recovery by preventing nitrogen losses through denitrification in the second stage. Besides COD removal, all urea was hydrolysed in the MEC, resulting in a stream rich in ammoniacal nitrogen and alkalinity, and low in COD. This stream was fed into a membrane-aerated biofilm reactor (MABR) in order to convert the volatile and toxic ammoniacal nitrogen to non-volatile nitrate by nitrification. Bio-electrochemical pre-treatment allowed to recover all nitrogen as nitrate in the MABR at a bulk-phase dissolved oxygen level below 0.1 mg O2 L-1. In contrast, feeding the MABR directly with raw urine (omitting the first stage), at the same nitrogen loading rate, resulted in nitrogen loss (18%) due to denitrification. The MEC and MABR were characterised by very distinct and diverse microbial communities. While (strictly) anaerobic genera, such as Geobacter (electroactive bacteria), Thiopseudomonas, a Lentimicrobiaceae member, Alcaligenes and Proteiniphilum prevailed in the MEC, the MABR was dominated by aerobic genera, including Nitrosomonas (a known ammonium oxidiser), Moheibacter and Gordonia. The two-stage approach yielded a stable nitrate-rich, COD-low nutrient solution, suitable for plant and microalgae cultivation.

Abstract: Livestock production is utilizing large amounts of protein-rich feed ingredients such as soybean meal. The proven negative environmental impacts of soybean meal production incentivize the search for alternative protein sources. One promising alternative is Microbial Protein (MP), i.e. dried microbial biomass. To date, only few life cycle assessments (LCAs) for MP have been carried out, none of which has used a consequential modelling approach nor has been investigating the production of MP on food and beverage wastewater. Therefore, the objective of this study is to evaluate the environmental impact of MP production on a food and beverage effluent as a substitute for soybean meal using a consequential modelling approach. Three different types of MP production were analysed, namely consortia containing Aerobic Heterotrophic Bacteria (AHB), Microalgae and AHB (MaB), and Purple Non-Sulfur Bacteria (PNSB). The production of MP was modelled for high-strength potato wastewater (COD = 10 kg/m3) at a flow rate of 1,000 m3/day. LCA results were compared against soybean meal production for the endpoint impact categories human health, ecosystems, and resources. Soybean meal showed up to 52% higher impact on human health and up to 87% higher impact on ecosystems than MP. However, energy-related aspects resulted in an 8–88% higher resource exploitation for MP. A comparison between the MP production systems showed that MaB performed best when considering ecosystems (between 13 and 14% better) and resource (between 71 and 80% better) impact categories, while AHB and PNSB had lower values for the impact category human health (8–12%). The sensitivity analysis suggests that the conclusions drawn are robust as in the majority of 1,000 Monte Carlo runs the initial results are confirmed. In conclusion, it is suggested that MP is an alternative protein source of comparatively low environmental impact that should play a role in the future protein transition, in particular when further process improvements can be implemented and more renewable or waste energy sources will be used.

Abstract: High-rate activated sludge (HRAS) systems suffer from high variability of effluent quality, clarifier performance, and carbon capture. This study proposed a novel control approach using bioflocculation boundaries for wasting control strategy to enhance effluent quality and stability while still meeting carbon capture goals. The bioflocculation boundaries were developed based on the oxygen uptake rate (OUR) ratio between contactor and stabilizer (feast/famine) in a high-rate contact stabilization (CS) system and this OUR ratio was used to manipulate the wasting setpoint. Increased oxidation of carbon or decreased wasting was applied when OUR ratio was <0.52 or >0.95 to overcome bioflocculation limitation and maintain effluent quality. When no bioflocculation limitations (OUR ratio within 0.52–0.95) were detected, carbon capture was maximized. The proposed control concept was shown for a fully automated OUR-based control system as well as for a simplified version based on direct waste flow control. For both cases, significant improvements in effluent suspended solids level and stability (<50-mg TSS/L), solids capture over the clarifier (>90%), and COD capture (median of 32%) were achieved. This study shows how one can overcome the process instability of current HRAS systems and provide a path to achieve more reliable outcomes.

Abstract: Purple non‑sulfur bacteria (PNSB) possess significant potential for bioresource recovery from wastewater. Effective operational tools are needed to boost productivity and direct the PNSB biomass towards abundant value-added substances (e.g., protein and co-enzyme Q10, CoQ10). This study aimed to investigate the impact of light, oxygen and volatile fatty acids (VFAs) on PNSB growth (i.e., Rhodobacter sphaeroides) and productivity of protein and CoQ10. Overall, the biomass yields and specific growth rates of PNSB were in the ranges of 0.57–1.08 g biomass g−1 CODremoved and 0.48–0.71 d−1, respectively. VFAs did not influence the biomass yield, yet acetate and VFA mixtures enhanced the specific growth rate with a factor of 1.2–1.5 compared to propionate and butyrate. The most PNSB biomass (1.08 g biomass g−1 CODremoved and 0.71 d−1) and the highest biomass quality (protein content of 609 mg g−1 dry cell weight (DCW) and CoQ10 content of 13.21 mg g−1 DCW) were obtained in the presence of VFA mixtures under natural light and microaerobic (low light alternated with darkness; dissolved oxygen (DO) between 0.5 and 1 mg L−1) conditions (vs. light anaerobic and dark aerobic cultivations). Further investigation on VFAs dynamics revealed that acetate was most rapidly consumed by PNSB in the individual VFA feeding (specific uptake rate of 0.76 g COD g−1 DCW d−1), while acetate as a co-substrate in the mixed VFAs feeding might accelerate the consumption of propionate and butyrate through providing additional cell metabolism precursor. Enzymes activities of succinate dehydrogenase and fructose-1,6-bisphosphatase as well as the concentration of photo pigments confirmed that light, oxygen and VFAs regulated the key enzymes in the energy metabolism and biomass synthesis to boost PNSB growth. These results provide a promising prospect for utilization of fermented waste stream for the harvest of PNSB biomass, protein and CoQ10.

Abstract: Production of struvite (MgNH4PO4·6H2O) from waste streams is increasingly implemented to recover phosphorus (P), which is listed as a critical raw material in the European Union (EU). To facilitate EU-wide trade of P-containing secondary raw materials such as struvite, the EU issued a revised fertilizer regulation in 2019. A comprehensive overview of the supply of struvite and its quality is presently missing. This study aimed: i) to determine the current EU struvite production volumes, ii) to evaluate all legislated physicochemical characteristics and pathogen content of European struvite against newly set regulatory limits, and iii) to compare not-regulated struvite characteristics. It is estimated that in 2020, between 990 and 1250 ton P are recovered as struvite in the EU. Struvite from 24 European production plants, accounting for 30% of the 80 struvite installations worldwide was sampled. Three samples failed the physicochemical legal limits; one had a P content of <7% and three exceeded the organic carbon content of 3% dry weight (DW). Mineralogical analysis revealed that six samples had a struvite content of 80–90% DW, and 13 samples a content of >90% DW. All samples showed a heavy metal content below the legal limits. Microbiological analyses indicated that struvite may exceed certain legal limits. Differences in morphology and particle size distribution were observed for struvite sourced from digestate (rod shaped; transparent; 82 mass% < 1 mm), dewatering liquor (spherical; opaque; 65 mass% 1–2 mm) and effluent from upflow anaerobic sludge blanket reactor processing potato wastewater (spherical; opaque; 51 mass% < 1 mm and 34 mass% > 2 mm). A uniform soil-plant P-availability pattern of 3.5–6.5 mg P/L soil/d over a 28 days sampling period was observed. No differences for plant biomass yield were observed. In conclusion, the results highlight the suitability of most struvite to enter the EU fertilizer market.

Abstract: Organic fertilizers and especially microbial biomass, also known as microbial fertilizer, can enable a paradigm shift to the conventional fertilizer-to-food chain, particularly when produced on secondary resources. Microbial fertilizers are already common practice (e.g. Bloom® and Synagro); yet microbial fertilizer blends to align the nutrient release profile to the plant’s needs are, thus far, unexplored. Moreover, most research only focuses on direct fertilization effects without considering added value properties, such as disease prevention. This study has explored three promising types of microbial fertilizers, namely dried biomass from a consortium of aerobic heterotrophic bacteria, a microalga (Arthrospira platensis) and a purple non-sulfur bacterium (Rhodobacter sphaeroides). Mineralization and nitrification experiments showed that the nitrogen mineralization profile can be tuned to the plant’s needs by blending microbial fertilizers, without having toxic ammonium peaks. In a pot trial with perennial ryegrass (Lolium perenne L.), the performance of microbial fertilizers was similar to the reference organic fertilizer, with cumulative dry matter yields of 5.6–6.7 g per pot. This was confirmed in a pot trial with tomato (Solanum lycopersicum L.), showing an average total plant length of 90–99 cm after a growing period of 62 days for the reference organic fertilizer and the microbial fertilizers. Moreover, tomato plants artificially infected with powdery mildew (Oidium neolycopersici), a devastating disease for the horticultural industry, showed reduced disease symptoms when A. platensis was present in the growing medium. These findings strengthen the application potential of this novel class of organic fertilizers in the bioeconomy, with a promising match between nutrient mineralization and plant requirements as well as added value in crop protection.

Abstract: The transition to sustainable agriculture and horticulture is a societal challenge of global importance. Fertilization with a minimum impact on the environment can facilitate this. Organic fertilizers can play an important role, given their typical release pattern and production through resource recovery. Microbial fertilizers (MFs) constitute an emerging class of organic fertilizers and consist of dried microbial biomass, for instance produced on effluents from the food and beverage industry. In this study, three groups of organisms were tested as MFs: a high-rate consortium aerobic bacteria (CAB), the microalga Arthrospira platensis (‘Spirulina’) and a purple non-sulfur bacterium (PNSB) Rhodobacter sp. During storage as dry products, the MFs showed light hygroscopic activity, but the mineral and organic fractions remained stable over a storage period of 91 days. For biological tests, a reference organic fertilizer (ROF) was used as positive control, and a commercial organic growing medium (GM) as substrate. The mineralization patterns without and with plants were similar for all MFs and ROF, with more than 70% of the organic nitrogen mineralized in 77 days. In a first fertilization trial with parsley, all MFs showed equal performance compared to ROF, and the plant fresh weight was even higher with CAB fertilization. CAB was subsequently used in a follow-up trial with petunia and resulted in elevated plant height, comparable chlorophyll content and a higher amount of flowers compared to ROF. Finally, a cost estimation for packed GM with supplemented fertilizer indicated that CAB and a blend of CAB/PNSB (85%/15%) were most cost competitive, with an increase of 6% and 7% in cost compared to ROF. In conclusion, as biobased fertilizers, MFs have the potential to contribute to sustainable plant nutrition, performing as good as a commercially available organic fertilizer, and to a circular economy.

Abstract: Cost simulations provide a strong tool to render the production of microalgae economically viable. This study evaluated the unexplored effect of harvesting time and the corresponding microalgal biomass composition on the overall production cost, under both continuous light and light/dark regime using techno-economic analysis (TEA). At the same time, the TEA gives evidence that a novel product “proteinaceous salt” from Dunaliella microalgae production is a promising high-value product for commercialization with profitability. The optimum production scenario is to employ natural light/dark regime and harvest microalgal biomass around late exponential phase, obtaining the minimum production cost of 11 €/kg and a profitable minimum selling price (MSP) of 14.4 €/kg for the “proteinaceous salt”. For further optimization of the production, increasing microalgal biomass concentration is the most effective way to reduce the total production cost and increase the profits of microalgae products.

Abstract: Nitrite oxidizing bacteria (NOB) and nitrous oxide (N2O) hinder the development of mainstream partial nitritation/anammox. To overcome these, endogenous free ammonia (FA) and free nitrous acid (FNA), which can be produced in the sidestream, were used for return-sludge treatment for two integrated-film activated sludge reactors containing biomass in flocs and on carriers. The repeated exposure of biomass from one reactor to FA shocks had a limited impact on NOB suppression but inhibited anammox bacteria (AnAOB). In the other reactor, repeated FNA shocks to the separated flocs failed to limit the system’s nitrate production since NOB activity was still high on the biofilms attached to the unexposed carriers. In contrast, the repeated FNA treatment of flocs and carriers favored aerobic ammonium-oxidizing bacteria (AerAOB) over NOB activity with AnAOB negligibly affected. It was further revealed that return-sludge treatment with higher FNA levels led to lower N2O emissions under similar effluent nitrite concentrations. On this basis, weekly 4 h FNA shocks of 2.0 mg of HNO2-N/L were identified as an optimal and realistic treatment, which not only enabled nitrogen removal efficiencies of ∼65% at nitrogen removal rates of ∼130 mg of N/L/d (20 °C) but also yielded the lowest cost and carbon footprint.

Abstract: Purple non-sulfur bacteria (PNSB) show potential for microbial protein production on wastewater as animal feed. They offer good selectivity (i.e., low microbial diversity and high abundance of one species) when grown anaerobically in the light. However, the cost of closed anaerobic photobioreactors is prohibitive for protein production. Although open raceway reactors are cheaper, their feasibility to selectively grow PNSB is thus far unexplored. This study developed operational strategies to boost PNSB abundance in the biomass of a raceway reactor fed with volatile fatty acids. For a flask reactor run at a 2 day sludge retention time (SRT), matching the chemical oxygen demand (COD) loading rate to the removal rate in the light period prevented substrate availability during the dark period and increased the PNSB abundance from 50-67 to 88-94%. A raceway reactor run at a 2 day SRT showed an increased PNSB abundance from 14 to 56% when oxygen supply was reduced (no stirring at night). The best performance was achieved at the highest surface-to-volume ratio (10 m(2) m(-3) increased light availability) showing productivities up to 0.2 g protein L-1 day(-1) and a PNSB abundance of 78%. This study pioneered in PNSB-based microbial protein production in raceway reactors, yielding high selectivity while avoiding the combined availability of oxygen, COD, and darkness.

Abstract: Thermophilic nitrification has been proven in lab-scale bioreactors at 50 °C. The challenge is now to develop a solution for thermophilic nitrogen removal, integrating nitrification with denitrification and aerobic carbon removal. This pioneering study aimed at a single-sludge nitrification/denitrification process at 50 °C, through exposing nitrification in a step by step approach to anoxia and/or organics. Firstly, recurrent anoxia was tolerated by a nitrifying community during long-term membrane bioreactor (MBR) operation (85 days), with high ammonium oxidation efficiencies (>98%). Secondly, five organic carbon sources did not affect thermophilic ammonium and nitrite oxidation rates in three-day aerobic batch flask incubations. Moving to long-term tests with sequencing batch reactors (SBR) and MBR (>250 days), good nitrification performance was obtained at increasing COD/Ninfluent ratios (0, 0.5, 1, 2 and 3). Thirdly, combining nitrification, recurrent anoxia and presence of organic carbon resulted in a nitrogen removal efficiency of 92–100%, with a COD/Nremoved of 4.8 ± 0.6 and a nitrogen removal rate of 50 ± 14 mg N g−1 VSS d−1. Overall, this is the first proof of principle thermophilic nitrifiers can cope with redox fluctuations (aerobic/anoxic) and the aerobic or anoxic presence of organic carbon, can functionally co-exist with heterotrophs and that single-sludge nitrification/denitrification can be achieved.

Abstract: Sustainable development is driving a rapid focus shift in the wastewater and organic waste treatment sectors, from a “removal and disposal” approach towards the recovery and reuse of water, energy and materials (e.g. carbon or nutrients). Purple phototrophic bacteria (PPB) are receiving increasing attention due to their capability of growing photoheterotrophically under anaerobic conditions. Using light as energy source, PPB can simultaneously assimilate carbon and nutrients at high efficiencies (with biomass yields close to unity (1 g CODbiomass·g CODremoved−1)), facilitating the maximum recovery of these resources as different value-added products. The effective use of infrared light enables selective PPB enrichment in non-sterile conditions, without competition with other phototrophs such as microalgae if ultraviolet-visible wavelengths are filtered. This review reunites results systematically gathered from over 177 scientific articles, aiming at producing generalized conclusions. The most critical aspects of PPB-based production and valorisation processes are addressed, including: (i) the identification of the main challenges and potentials of different growth strategies, (ii) a critical analysis of the production of value-added compounds, (iii) a comparison of the different value-added products, (iv) insights into the general challenges and opportunities and (v) recommendations for future research and development towards practical implementation. To date, most of the work has not been executed under real-life conditions, relevant for full-scale application. With the savings in wastewater discharge due to removal of organics, nitrogen and phosphorus as an important economic driver, priorities must go to using PPB-enriched cultures and real waste matrices. The costs associated with artificial illumination, followed by centrifugal harvesting/dewatering and drying, are estimated to be 1.9, 0.3–2.2 and 0.1–0.3 $·kgdry biomass−1. At present, these costs are likely to exceed revenues. Future research efforts must be carried out outdoors, using sunlight as energy source. The growth of bulk biomass on relatively clean wastewater streams (e.g. from food processing) and its utilization as a protein-rich feed (e.g. to replace fishmeal, 1.5–2.0 $·kg−1) appears as a promising valorisation route.

Abstract: Implementation of mainstream partial nitritation/anammox (PN/A) can lead to more sustainable and cost-effective sewage treatment. For mainstream PN/A reactor, an integrated fixed-film activated sludge (IFAS) was operated (26 °C). The effects of floccular aerobic sludge retention time (AerSRT_floc), a novel aeration strategy, and N-loading rate were tested to optimize the operational strategy. The best performance was observed with a low, but sufficient AerSRTfloc (~7d) and continuous aeration with two alternating dissolved oxygen setpoints: 10 min at 0.07–0.13 mg O2 L−1 and 5 min at 0.27–0.43 mg O2 L−1. Nitrogen removal rates were 122 ± 23 mg N L−1 d−1, and removal efficiencies 73 ± 13%. These conditions enabled flocs to act as nitrite sources while the carriers were nitrite sinks, with low abundance of nitrite oxidizing bacteria. The operational strategies in the source-sink framework can serve as a guideline for successful operation of mainstream PN/A reactors.

Abstract: The strong effect of low temperatures on anammox challenges its mainstream application over the winter in temperate climates. Winter bioaugmentation with stored summer surplus sludge is a potential solution to guarantee sufficient nitrogen removal in winter. Firstly, the systems for which nitrogen removal deteriorated by the temperature decrease (25 °C → 20 °C) could be fully restored bioaugmenting with granules resp. flocs stored for 6 months at 118 resp. 220% of the initial biomass levels. Secondly, the reactivation of these stored sludges was tested in lower temperature systems (15.3 ± 0.4/10.4 ± 0.4 °C). Compared to the activity before storage, between 56% and 41% of the activity of granules was restored within one month, and 41%–32% for flocs. Additionally, 85–87% of granules and 50–53% of flocs were retained in the systems. After reactivation (15.3 ± 0.4/10.4 ± 0.4 °C), a more specialized community was formed (diversity decreased) with Candidatus Brocadia still dominant in terms of relative abundance. Capital and operating expenditures (CAPEX, OPEX) were negligible, representing only 0.19–0.36% of sewage treatment costs.

Abstract: The urgency for a protein transition towards more sustainable solutions is one of the major societal challenges. Microbial protein is one of the alternative routes, in which land- and fossil-free production should be targeted. The photohydrogenotrophic growth of purple bacteria, which builds on the H2– and CO2-economy, is unexplored for its microbial protein potential. The three tested species (Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas palustris) obtained promising growth rates (2.3–2.7 d−1 at 28°C) and protein productivities (0.09–0.12 g protein L−1 d−1), rendering them likely faster and more productive than microalgae. The achieved protein yields (2.6–2.9 g protein g−1 H2) transcended the ones of aerobic hydrogen oxidizing bacteria. Furthermore, all species provided full dietary protein matches for humans and their fatty acid content was dominated by vaccenic acid (82–86%). Given its kinetic and nutritional performance we recommend to consider Rhodobacter capsulatus as a high-potential sustainable source of microbial food.