Environmental proteomics as a useful methodology for early-stage detection of stress in anammox engineered systems.

Anammox bacteria are widely applied worldwide for denitrification of urban wastewater. Differently, their application in the case of industrial effluents has been more limited. Those frequently present high loads of contaminants, demanding an individual evaluation of their treatability by anammox technologies. Bioreactors setting up and recovery after contaminants-derived perturbations are slow. Also, toxicity is frequently not acute but cumulative, which causes negative macroscopic effects to appear only after medium or long-term operations. All these particularities lead to relevant economic and time losses. We hypothesized that contaminants cause changes at anammox proteome level before perturbations in the engineered systems are detectable by macroscopic analyses. In this study, we explored the usefulness of short-batch tests combined with environmental proteomics for the early detection of those changes. Copper was used as a model of stressor contaminant, and anammox granules were exposed to increasing copper concentrations including previously reported IC50 values. The proteomic results revealed that specific anammox proteins involved in stress response (bacterioferritin, universal stress protein, or superoxide dismutase) were overexpressed in as short a time as 28 h at the higher copper concentrations. Consequently, EPS production was also increased, as indicated by the alginate export family protein, polysaccharide biosynthesis protein, and sulfotransferase increased expression. The described workflow can be applied to detect early-stage stress biomarkers of the negative effect of other metals, organics, or even changes in physical-chemical parameters such as pH or temperature on anammox-engineered systems. On an industrial level, it can be of great value for decision-making, especially before dealing with new effluents on facilities, deriving important economic and time savings.

Assessing labelled carbon assimilation from poly butylene adipate-co-terephthalate (PBAT) monomers during thermophilic anaerobic digestion.

PBAT (poly butylene adipate-co-terephthalate) is a widely used biodegradable plastic, but the knowledge about its metabolization in anaerobic environments is very limited. In this study, the anaerobic digester sludge from a municipal wastewater treatment plant was used as inoculum to investigate the biodegradability of PBAT monomers in thermophilic conditions. The research employs a combination of 13C-labelled monomers and proteogenomics to track the labelled carbon and identify the microorganisms involved. A total of 122 labelled peptides of interest were identified for adipic acid (AA) and 1,4-butanedio (BD). Through the time-dependent isotopic enrichment and isotopic profile distributions, Bacteroides, Ichthyobacterium, and Methanosarcina were proven to be directly involved in the metabolization of at least one monomer. This study provides a first insight into the identity and genomic potential of microorganisms responsible for biodegradability of PBAT monomers during anaerobic digestion under thermophilic conditions.

Thermal hydrolysis pre-treatment has no positive influence on volatile fatty acids production from sewage sludge.

The study compares the potential to produce volatile fatty acids (VFA) from sewage sludge, both raw and thermally pre-treated in two modes of operation. In batch mode, raw sludge at pH 8 obtained the highest maximum VFA yield (0.41 g COD-VFA/g CODfed) whereas pre-treated sludge achieved a lower value (0.27 g COD-VFA/g CODfed). The operation of 5-L continuous reactors showed that thermal hydrolysis pre-treatment (THP) did not have any significant influence on VFA yields, averaging 15.1 % g COD-VFA/g COD with raw sludge and 16.6 % g COD-VFA/g COD with pre-treated one. Microbial community analysis showed that phylum Firmicutes was predominant in both reactors and that the enzymatic profiles involved in VFA production were very similar regardless of the substrate fed.

Sulfamethoxazole Enhances Specific Enzymatic Activities under Aerobic Heterotrophic Conditions: A Metaproteomic Approach.

The growing concern about antibiotic-resistant microorganisms has focused on the sludge from wastewater treatment plants (WWTPs) as a potential hotspot for their development and spread. To this end, it seems relevant to analyze the changes on the microbiota as a consequence of the antibiotics that wastewater may contain. This study aims at determining whether the presence of sulfamethoxazole (SMX), even in relatively low concentrations, modifies the microbial activities and the enzymatic expression of an activated sludge under aerobic heterotrophic conditions. For that purpose, we applied a metaproteomic approach in combination with genomic and transformation product analyses. SMX was biotransformed, and the metabolite 2,4(1H,3H)-pteridinedione-SMX (PtO-SMX) from the pterin-conjugation pathway was detected at all concentrations tested. Metaproteomics showed that SMX at 50-2000 µg/L slightly affected the microbial community structure, which was confirmed by DNA metabarcoding. Interestingly, an enhanced activity of the genus Corynebacterium and specifically of five enzymes involved in its central carbon metabolism was found at increased SMX concentrations. Our results suggest a role of Corynebacterium genus on SMX risks mitigation in our bioreactors.

Enzymatic cometabolic biotransformation of organic micropollutants in wastewater treatment plants: A review.

Biotransformation of trace-level organic micropollutants (OMPs) by complex microbial communities in wastewater treatment facilities is a key process for their detoxification and environmental impact reduction. Therefore, understanding the metabolic activities and mechanisms that contribute to their biotransformation is essential when developing approaches aiming to minimize their discharge. This review addresses the relevance of cometabolic processes and discusses the main enzymatic activities currently known to take part in OMPs removal under different redox environments in the compartments of wastewater treatment plants. Furthermore, the most common methodologies to decipher such enzymes are discussed, including the use of in vitro enzyme assays, enzymatic inhibitors, the analysis of transformation products and the application of several -omic techniques. Finally, perspectives on major challenges and future research requirements to improve OMPs biotransformation are proposed.

Elimination of SARS-CoV-2 along wastewater and sludge treatment processes.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is shed in the feces of infected people. As a consequence, genomic RNA of the virus can be detected in wastewater. Although the presence of viral RNA does not inform on the infectivity of the virus, this presence of genetic material raised the question of the effectiveness of treatment processes in reducing the virus in wastewater and sludge. In this work, treatment lines of 16 wastewater treatment plants were monitored to evaluate the removal of SARS-CoV-2 RNA in raw, processed waters and sludge, from March to May 2020. Viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in 5 different laboratories. These laboratories participated in proficiency testing scheme and their results demonstrated the reliability and comparability of the results obtained for each one. SARS-CoV-2 RNA was found in 50.5% of the 101 influent wastewater samples characterized. Positive results were detected more frequently in those regions with a COVID-19 incidence higher than 100 cases per 100,000 inhabitants. Wastewater treatment plants (WWTPs) significantly reduced the occurrence of virus RNA along the water treatment lines. Secondary treatment effluents showed an occurrence of SARS-CoV-2 RNA in 23.3% of the samples and no positive results were found after MBR and chlorination. Non-treated sludge (from primary and secondary treatments) presented a higher occurrence of SARS-CoV-2 RNA than the corresponding water samples, demonstrating the affinity of virus particles for solids. Furthermore, SARS-CoV-2 RNA was detected in treated sludge after thickening and anaerobic digestion, whereas viral RNA was completely eliminated from sludge only when thermal hydrolysis was applied. Finally, co-analysis of SARS-CoV-2 and F-specific RNA bacteriophages was done in the same water and sludge samples in order to investigate the potential use of these bacteriophages as indicators of SARS-CoV-2 fate and reduction along the wastewater treatment.

Heterotrophic enzymatic biotransformations of organic micropollutants in activated sludge.

While heterotrophic microorganisms constitute the major fraction of activated sludge biomass, the role of heterotrophs in the biotransformation of organic micropollutants (OMPs) has not been fully elucidated. Yet, such knowledge is essential, particularly when conceiving novel wastewater treatment plants based on a two-stage process including an A-stage under heterotrophic conditions and a B-stage based on anammox activity. Biotransformation of OMPs in activated sludge is thought to mostly occur cometabolically thanks to the action of low specificity enzymes involved in the metabolism of the primary substrates. For a better understanding of the process, it is important to determine such enzymatic activities and the underlying mechanisms involved in OMPs biotransformation. This task has proven to be difficult due to the lack of information about the enzymatic processes and the complexity of the biological systems present in activated sludge. In this paper, a continuous aerobic heterotrophic reactor following 20 OMPs at environmental concentrations was operated to (i) assess the potential of heterotrophs during the cometabolic biotransformation of OMPs, (ii) identify biotransformation reactions catalyzed by aerobic heterotrophs and (iii) predict possible heterotrophic enzymatic activities responsible for such biotransformations. Contradicting previous reports on the dominant role of nitrifiers in OMPs removal during activated sludge treatment, the heterotrophic population proved its capacity to biotransform the OMPs to extents equivalent to reported values in nitrifying activated sludge plants. Besides, 12 transformation products potentially formed through the activity of several enzymes present in heterotrophs, including monooxygenases, dioxygenases, hydrolases and transferases, were identified.

Microbial inefficient substrate use through the perspective of resource allocation models.

Microorganisms extract energy from substrates following strategies that may seem suboptimal at first glance. Beyond the so-called yield-rate trade-off, resource allocation models, which focus on assigning different functional roles to the limited number of enzymes that a cell can support, offer a framework to interpret the inefficient substrate use by microorganisms. We review here relevant examples of substrate conversions where a significant part of the available energy is not utilised and how resource allocation models offer a mechanistic interpretation thereof, notably for open mixed cultures. Future developments are identified, in particular, the challenge of considering metabolic flexibility towards uncertain environmental changes instead of strict fixed optimality objectives, with the final goal of increasing the prediction capabilities of resource allocation models. Finally, we highlight the relevance of resource allocation to understand and enable a promising biorefinery platform revolving around lactate, which would increase the flexibility of waste-to-chemical biorefinery schemes.

Chemical and thermal stabilization of CotA laccase via a novel one-step expression and immobilization in muNS-Mi nanospheres.

A methodology that programs eukaryotic or bacterial cells to encapsulate proteins of any kind inside micro/nanospheres formed by muNS-Mi viral protein was developed in our laboratory. In the present study such "in cellulo" encapsulation technology is utilized for immobilizing a protein with an enzymatic activity of industrial interest, CotA laccase. The encapsulation facilitates its purification, resulting in a cost-effective, one-step way of producing immobilized enzymes for industrial use. In addition to the ability to be recycled without activity loss, the encapsulated protein showed an increased pH working range and high resistance to chemical inactivation. Also, its activity was almost unaffected after 30 min incubation at 90 °C and 15 min at the almost-boiling temperature of 95 °C. Furthermore, the encapsulated laccase was able to efficiently decolorate the recalcitrant dye RB19 at room temperature.

The fate of SARS-COV-2 in WWTPS points out the sludge line as a suitable spot for detection of COVID-19.

SARS-CoV-2 genetic material is detectable in the faeces of a considerable part of COVID-19 cases and hence, in municipal wastewater. This fact was confirmed early during the spread of the COVID-19 pandemic and prompted several studies that proposed monitoring its incidence by wastewater. This paper studies the fate of SARS-CoV-2 genetic material in wastewater treatment plants using RT-qPCR with a two-fold goal: i) to check its presence in the water effluent and in the produced sludge and ii) based on the understanding of the virus particles fate, to identify the most suitable spots for detecting the incidence of COVID-19 and monitor its evolution. On the grounds of the affinity of enveloped virus towards biosolids, we hypothesized that the sludge line acts as a concentrator of SARS-CoV-2 genetic material. Sampling several spots in primary, secondary and sludge treatment at the Ourense (Spain) WWTP in 5 different days showed that, in effect, most of SARS-CoV-2 particles cannot be detected in the water effluent as they are retained by the sludge line. We identified the sludge thickener as a suitable spot for detecting SARS-CoV-2 particles thanks to its higher solids concentration (more virus particles) and longer residence time (less sensitive to dilution caused by precipitation). These findings could be useful to develop a suitable strategy for early warning of COVID-19 incidence based on WWTP monitoring.

Resource allocation explains lactic acid production in mixed-culture anaerobic fermentations.

Lactate production in anaerobic carbohydrate fermentations with mixed cultures of microorganisms is generally observed only in very specific conditions: the reactor should be run discontinuously and peptides and B vitamins must be present in the culture medium as lactic acid bacteria (LAB) are typically auxotrophic for amino acids. State-of-the-art anaerobic fermentation models assume that microorganisms optimise the adenosine triphosphate (ATP) yield on substrate and therefore they do not predict the less ATP efficient lactate production, which limits their application for designing lactate production in mixed-culture fermentations. In this study, a metabolic model taking into account cellular resource allocation and limitation is proposed to predict and analyse under which conditions lactate production from glucose can be beneficial for microorganisms. The model uses a flux balances analysis approach incorporating additional constraints from the resource allocation theory and simulates glucose fermentation in a continuous reactor. This approach predicts lactate production is predicted at high dilution rates, provided that amino acids are in the culture medium. In minimal medium and lower dilution rates, mostly butyrate and no lactate is predicted. Auxotrophy for amino acids of LAB is identified to provide a competitive advantage in rich media because less resources need to be allocated for anabolic machinery and higher specific growth rates can be achieved. The Matlab™ codes required for performing the simulations presented in this study are available at https://doi.org/10.5281/zenodo.4031144.

The organic loading rate affects organic micropollutants' cometabolic biotransformation kinetics under heterotrophic conditions in activated sludge.

Several studies have shown that organic micropollutants (OMPs) are biotransformed cometabolically in activated sludge systems. However, the individual role of heterotrophs in the microbial consortium is still not clear, i.e., there is still a gap regarding the influence of the heterotrophic activity on the cometabolic biotransformation kinetics and yield of the OMPs. Aiming to answer these questions, experiments with increasing primary substrate concentrations were performed under aerobic heterotrophic conditions in a continuous stirred tank reactor operated at several organic loading rates (OLR) with fixed hydraulic retention time. Moreover, the individual kinetic parameters were determined in batch assays with different initial substrate concentrations using the sludges from the continuous reactor. A set of 15 OMPs displaying a variety of physicochemical properties were spiked to the feeding in the ng L-1 - µg L-1 range. Results reveal that the biodegradation of the primary carbon source and the biotransformation of the OMPs occur simultaneously, in clear evidence of cometabolic behavior. Moreover, we conclude that the OMPs biotransformation kinetic constant (kbiol) shows a linear dependence with the OLR of the primary substrate for most of the compounds studied, suggesting that the heterotrophic activity seriously affects the OMPs biotransformation kinetics. However, under typical activated sludge systems operating conditions (hydraulic retention times above 8 h), their biotransformation yield would not be significantly affected.

"Who Cares?": The Acceptance of Decentralized Wastewater Systems in Regions without Water Problems.

There is a growing interest in decentralized wastewater treatment systems, especially in regions with water scarcity problems or water management issues. This study aims to determine whether the perceived advantages and disadvantages (leading to acceptance) of decentralized wastewater plants in such regions are the same in regions where the population is not aware of these water issues. Firstly, this study systematically reviews previous findings on public perceptions of the acceptance of decentralized wastewater treatment systems. Then, the study details the results of a focus group study to determine whether the elements identified in the literature are also relevant in a region where people are unaware of water problems. The results show that a lack of awareness of water issues seems to be a critical factor influencing acceptance. Reframing the usefulness of these systems by focusing on other aspects, such as environmental sustainability, is key.

Energy requirements and economics of acetone-butanol-ethanol (ABE) extractive fermentation: a solvent-based comparative assessment.

The reindustrialization of acetone-butanol-ethanol (ABE) fermentation is hampered by its significant production cost, linked to high product inhibition and low product yield. ABE fermentation can be significantly enhanced by integrating in situ liquid-liquid extraction. In this study, hybrid simulations using Excel® and ASPEN Plus® were performed based on solvent-dependent experimental data (product titer, yield and productivity) to consider the physiological response of the microorganism in specific extractive ABE fermentations, and to quantify the energy requirements and the economic improvement of the overall process. Four scenarios, based on two different solvents (2-butyl-1-octanol, 2B1O, and a vegetable oil, VO) applied in batch or fed-batch operation, were compared with the batch conventional process. Total energy demand decreased in all extractive configurations and the greatest energy savings (61%) were reached with the VO-based fed-batch operation. However, the highest profit increase was achieved with 2B1O in fed-batch mode, reducing the minimum butanol selling price by 29% over the base case, along with 34% savings in raw materials and 80% wastewater reduction. The techno-economical solvent-based comparative evaluation is a useful tool to identify key challenges to be tackled when revisiting ABE extractive fermentation.

Protein composition determines the preferential consumption of amino acids during anaerobic mixed-culture fermentation.

The valorisation of protein-rich residual streams by anaerobic mixed-culture fermentation (MCF) has been barely studied in contrast to carbohydrate-rich wastes. The aim of this work was, therefore, to investigate how protein composition, i.e. the amino acid (AA) profile, affects the individual consumption of amino acids and, consequently, the outcome of the process. Mixed-culture fermentations were performed with two model proteins (casein and gelatin) using continuous and batch reactors at neutral pH values and 25 °C. The acidification was incomplete for both proteins, with casein achieving a higher value than gelatin. Albeit dominated by acetic acid, product spectra were different as well, with n-butyric acid as the second major product for casein and propionic acid for gelatin. The preferential consumption of amino acids was demonstrated, which interestingly depends on protein composition. The previously accepted stoichiometry accurately describes iso and n-butyric acid production, but it fails for propionic, iso and n-valeric acid generation. Overall, this study offers a better understanding of protein fermentation mechanisms, which will help to improve degradation models and to design fermentation processes, based on optimal substrate selection.

Purple phototrophic bacteria for resource recovery: Challenges and opportunities.

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.

Assessment of the fate of organic micropollutants in novel wastewater treatment plant configurations through an empirical mechanistic model.

Novel wastewater treatment plants (WWTPs) are expected to be less energetically demanding than conventional ones. However, scarce information is available about the fate of organic micropollutants (OMPs) in these novel configurations. Therefore, the objective of this work is to assess the fate of OMPs in three novel WWTP configurations by using a plant-wide simulation that integrates multiple units. The difference among the three configurations is the organic carbon preconcentration technology: chemically enhanced primary treatment (CEPT), high-rate activated sludge (HRAS) combined or not with a rotating belt filter (RBF); followed by a partial-nitritation (PN-AMX) unit. The simulation results show that the three selected novel configurations lead mainly to comparable OMPs removal efficiencies from wastewater, which were similar or lower, depending on the OMP, than those obtained in conventional WWTPs. However, the presence of hydrophobic OMPs in the digested sludge noticeably differs among the three configurations. Whereas the configuration based on sole HRAS to recover organic carbon leads to a lower presence of OMPs in digested sludge than the conventional WWTP, in the other two novel configurations this presence is noticeable higher. In conclusion, novel WWTP configurations do not improve the OMPs elimination from wastewater achieved in conventional ones, but the HRAS-based WWTP configuration leads to the lowest presence in digested sludge so it becomes the most efficient alternative.

Metabolic modeling for predicting VFA production from protein-rich substrates by mixed-culture fermentation.

Proteinaceous organic wastes are suitable substrates to produce high added-value products in anaerobic mixed-culture fermentations. In these processes, the stoichiometry of the biotransformation depends highly on operational conditions such as pH or feeding characteristics and there are still no tools that allow the process to be directed toward those products of interest. Indeed, the lack of product selectivity strongly limits the potential industrial development of these bioprocesses. In this work, we developed a mathematical metabolic model for the production of volatile fatty acids from protein-rich wastes. In particular, the effect of pH on the product yields is analyzed and, for the first time, the observed changes are mechanistically explained. The model reproduces experimental results at both neutral and acidic pH and it is also capable of predicting the tendencies in product yields observed with a pH drop. It also offers mechanistic insights into the interaction among the different amino acids (AAs) of a particular protein and how an AA might yield different products depending on the relative abundance of other AAs. Particular emphasis is placed on the utility of this mathematical model as a process design tool and different examples are given on how to use the model for this purpose.

A new decentralized biological treatment process based on activated carbon targeting organic micropollutant removal from hospital wastewaters.

Although hospital wastewaters (HWWs) are usually discharged in urban sewage systems, their separate treatment has several benefits, such as the specific treatment of potential toxics as well as avoidance of further dilutions. In this work, an integrated industrial pilot plant (2200 L) corresponding to the technology SeMPAC® is proposed and validated for such purpose. The process consists of a sequential batch reactor (SBR) connected to an external submerged microfiltration membrane, in which powdered activated carbon (PAC) is directly added into the biological reactor to enhance the removal of the organic micropollutants (OMPs). The combination of different redox conditions in the SBR, as well as the operation at long sludge retention times (SRTs) and high biomass concentrations favored OMP biotransformation in the SBR, being their final removal efficiencies enhanced clearly after PAC addition, especially for the recalcitrant compounds. A periodical renewal of the adsorbent is necessary to overcome its gradual saturation. The main operational conditions were influenced by (i) the recalcitrant OMP carbamazepine, which defines the PAC dosage; (ii) the easily degradable OMP ibuprofen, which can be used to optimize the duration of the aerobic cycle; and (iii) the denitrification efficiency, which defines the correct time length of the anoxic period.