Responsible Science, Engineering and Education for Water Resource Recovery and Circularity

Water resource recovery is central to the circular economy framework. It underlies the transition of environmental engineering from pollution prevention to responsible innovation for sustainable systems engineering. In order to speed this transition, resource recovery and circularity need integration into new higher education curricula to train the next generation of young professionals. However, training of new concepts requires the development of new course materials and books, while integrating substantial illustrations and problems on circularity and resource recovery in new editions of existing textbooks in environmental science and engineering. Moreover, university–utility–industry partnerships are important mechanisms to bridge theoretical fundamentals to concepts for engineering practice, and to promote knowledge exchange and technology adoption between practitioners and academics. Interactive platforms should be designed to facilitate the integration and development of resource recovery and circularity concepts from science and practice into education. Consensus was built on this perspective article from interaction with the members of the Association of Environmental Engineering and Science Professors in a workshop that we organized at the AEESP Research and Education Conference 2017. Overall, this paper gives actionable roadmaps to (i) apprehend how new science and technological findings need to get integrated to sustain resource recovery and circularity in practice, along with the fact that (ii) skills sets can be engineered with relatively minor changes to existing lecture material that will have maximal impact on the scope of the thought material. It lays out (iii) how partnership with engineering practitioners can make a lecture more vivid by giving students reasoning for why the learned material is important, and (iv) how a platform for an integrated science, education, and practice can deliver them with concrete tools for practical implementation for benefits at community level.BT/Environmental Biotechnolog

Got Whey? Sustainability Endpoints for the Dairy Industry through Resource Biorecovery

Whey has applications in food, beverages, personal care products, pharmaceuticals, and the medical sector. However, it remains a massive dairy residue worldwide (160.7 million m3 year−1), with high organic and nutrient loads. About 42% is used for low-value products such as animal feed and fertilizers or is even directly discharged into water streams, leading to ecosystem damage via eutrophication. We reviewed the uses and applications of cheese whey, along with associated environmental impacts and innovative ways to mitigate them using affordable and scalable technologies. Recycling and repurposing whey remain challenges for remote locations and poor communities with limited access to expensive technology. We propose a closed-loop biorefinery strategy to simultaneously mitigate environmental impacts and valorize whey resources. Anaerobic digestion utilizes whey to produce biogas and/or carboxylates. Alternative processes combining anaerobic digestion and low-cost open photobioprocesses can valorize whey and capture organic, nitrogenous, and phosphorous nutrients into microalgal biomass that can be used as food and crop supply or processed into biofuels, pigments, and antioxidants, among other value-added products. The complete valorization of cheese whey also depends on facilitating access to relevant information on whey production, identifying stakeholders, reducing technology gaps among countries, enforcing legislation and compliance, and creating subsidies and fostering partnerships with industries and between countries.BT/Environmental Biotechnolog

Got Whey? The significance of cheese whey at the confluence of dairying, environmental impacts, energy and resource biorecovery

Milk discovery and processing enabled human settling and thriving in various settings. The discovery of cheese led to the production of whey as dairy by-product. Although it can find application in food, beverages, personal care products, pharmaceuticals and medical treatment, cheese whey is a massive dairying residue world-wide (154 Mm3·y-1) with high organic and nutrient loads. About 42% is used as low-value products as animal feed and fertilisers or even directly discharged in water streams, leading to ecosystem damage by eutrophication. Recycling and repurposing whey remains a challenge for remote locations and poor communities with limited access to expensive technology. Anaerobic digestion is proven and accessible for utilizing whey as substrate to produce biogas and/or carboxylates. Alternative processes combining anaerobic digestion and low-cost open photobioprocesses can foster the valorisation of cheese whey and capture of organics and nitrogen and phosphorus nutrients into a microalgal biomass that can be used as food and crop supply or processed into biofuels, pigments, antioxi-dants, among other value-added products. Awareness should be raised about the economic potential of cheese whey surplus by developing an action plan that (i) identifies stakeholders, (ii) sets goals and achieves solutions, (iii) decreases technology gaps among countries, (iv) enforces legislation and compliance, and (v) creates subsidies and foments partnerships with industries and other countries for the full valorisation of whey. We propose a closed-loop biorefinery implementation strategy to simultaneously mitigate environmental impacts and valorise whey resources.BT/Environmental Biotechnolog

Free-floating extracellular DNA: systematic profiling of mobile genetic elements and antibiotic resistance from wastewater

The free-floating extracellular DNA (exDNA) fraction of microbial ecosystems harbors antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Natural transformation of these xenogenetic elements can generate microbial cells resistant to one or more antibiotics. Isolating and obtaining high yield exDNA is challenging due to its low concentration in wastewater environments. Profiling exDNA is crucial to unravel the ecology of free-floating ARGs and MGEs and their contribution to horizontal transfer. We developed a method using chromatography to isolate and enrich exDNA without causing cell lysis from complex wastewater matrices like influent (9 µg exDNA out of 1 L), activated sludge (5.6 µg), and treated effluent (4.3 µg). ARGs and MGEs were metagenomically profiled for both the exDNA and intracellular DNA (iDNA) of activated sludge, and quantified by qPCR in effluent water. qPCR revealed that ARGs and MGEs are more abundant in the iDNA fraction while still significant on exDNA (100-1000 gene copies mL−1) in effluent water. The metagenome highlighted that exDNA is mainly composed of MGEs (65%). According to their relatively low abundance in the resistome of exDNA, ARGs uptake by natural transformation is likely not the main transfer mechanism. Although ARGs are not highly abundant in exDNA, the prevalence of MGEs in the exDNA fraction can indirectly promote antibiotic resistance development. The combination of this method with functional metagenomics can help to elucidate the transfer and development of resistances in microbial communities. A systematic profiling of the different DNA fractions will foster microbial risk assessments across water systems, supporting water authorities to delineate measures to safeguard environmental and public health.BT/Environmental BiotechnologyPattern Recognition and Bioinformatic

Monitoring SARS-CoV-2 in sewage: Toward sentinels with analytical accuracy

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effects of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested: electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.BT/Environmental Biotechnolog

Operational Strategies to Selectively Produce Purple Bacteria for Microbial Protein in Raceway Reactors

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 m2 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. Accepted Author ManuscriptBT/Environmental Biotechnolog

Light intensity defines growth and photopigment content of a mixed culture of purple phototrophic bacteria

Purple bacteria (PPB), anoxygenic photoorganoheterotrophic organisms with a hyper-versatile metabolism and high biomass yields over substrate, are promising candidates for the recovery of nutrient resources from wastewater. Infrared light is a pivotal parameter to control and design PPB-based resource recovery. However, the effects of light intensities on the physiology and selection of PPB in mixed cultures have not been studied to date. Here, we examined the effect of infrared irradiance on PPB physiology, enrichment, and growth over a large range of irradiance (0 to 350 W m−2) in an anaerobic mixed-culture sequencing batch photobioreactor. We developed an empirical mathematical model that suggests higher PPB growth rates as response to higher irradiance. Moreover, PPB adapted to light intensity by modulating the abundances of their phototrophic complexes. The obtained results provide an in-depth phylogenetic and metabolic insight the impact of irradiance on PPB. Our findings deliver the fundamental information for guiding the design of light-driven, anaerobic mixed-culture PPB processes for wastewater treatment and bioproduct valorization.BT/Environmental Biotechnolog

The impact of mixtures of xylose and glucose on the microbial diversity and fermentative metabolism of sequencing-batch or continuous enrichment cultures

Efficient industrial fermentation of lignocellulosic waste containing a large part of glucose and xylose is desirable to implement a circular economy. Mixed culture biotechnologies can aid in realizing this goal. The effect of feeding equivalent substrates to a microbial community, such a xylose and glucose, is not well understood in terms of the number of dominant species and how these species compete for the substrates. We compared the metabolism and microbial community structure in a continuous-flow stirred tank reactor (CSTR) and a sequencing batch reactor (SBR) fed with a mixture of xylose and glucose, inoculated with bovine rumen at pH 8, 30°C and a hydraulic retention time of 8 h. We hypothesised that a CSTR will select for generalist species, taking up both substrates. We used 16S rRNA gene sequencing and fluorescent in situ hybridisation to accurately determine the microbial community structures. Both enrichments were stoichiometrically and kinetically characterised. The CSTR enrichment culture was dominated by Clostridium intestinale (91% ± 2%). The SBR showed an abundance of Enterobacteriaceae (75% ± 8%), dominated by Citrobacter freundii and a minor fraction of Raoultella ornithinolytica. C. freundii ferments xylose and glucose in a non-diauxic fashion. Clearly, a non-diauxic generalist outcompetes specialists and diauxic generalists in SBR environments.Accepted Author ManuscriptBT/Environmental Biotechnolog

Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System

Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP) effluent and at one fixed location in the drinking water distribution network (NET). The samples were analysed for heterotrophic plate counts (HPC), Aeromonas plate counts, adenosine-tri-phosphate (ATP) concentrations, and flow cytometric (FCM) total and intact cell counts (TCC, ICC), water temperature, pH, conductivity, total organic carbon (TOC) and assimilable organic carbon (AOC). Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 103 cells mL-1 in winter time up to 455 × 103 cells mL-1 in summer time) and in bacterial ATP concentrations (<1–3.6 ng L-1), which were congruent with water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 103 cells mL-1 on average) compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson’s correlation factor r = -0.35), and positively correlated with water temperature (r = 0.49). Collecting a large dataset at high frequency over a two year period enabled the characterization of previously undocumented seasonal dynamics in the distribution network. Moreover, high-resolution FCM data enabled prediction of bacterial cell concentrations at specific water temperatures and time of year. The study highlights the need to systematically assess temporal fluctuations in parallel to spatial dynamics for individual drinking water distribution systems.BT/Environmental Biotechnolog

Selecting for a high lipid accumulating microalgae culture by dual growth limitation in a continuous bioreactor

A dual-growth-limited continuous operated bioreactor (chemostat) was used to enhance lipid accumulation in an enrichment culture of microalgae. The light intensity and nitrogen concentration where both limiting factors resulting in high lipid accumulation in the mixed culture. Both conditions of light and nitrogen excess and deficiency were tested. Strategies to selectively enrich for a phototrophic lipid-storing community, based on the use of different nitrogen sources (ammonium vs. nitrate) and vitamin B supplementation in the growth medium, were evaluated. The dual limitation of both nitrogen and light enhanced the accumulation of storage compounds. Ammoniacal nitrogen was the preferred nitrogen source. Vitamin B supplementation led to a doubling of the lipid productivity. The availability of vitamins played a key role in selecting an efficient lipid-storing community, primarily consisting of Trebouxiophyceae (with an 82 % relative abundance among eukaryotic microorganisms). The obtained lipid volumetric productivity (387 mg L−1 d−1) was among the highest reported in literature for microalgae bioreactors. Lipid production by the microalgae enrichment surpassed the efficiencies reported for continuous microalgae pure cultures, highlighting the benefits of mixed-culture photo-biotechnologies for fuels and food ingredients in the circular economy.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BT/Environmental Biotechnolog