Model-aided targeted volatile fatty acid production from food waste using a defined co-culture microbial community.

The production of volatile fatty acids (VFA) is gaining momentum due to their central role in the emerging carboxylate platform. Particularly, the production of the longest VFA (from butyrate to caproate) is desired due to their increased economic value and easier downstream processing. While the use of undefined microbial cultures is usually preferred with organic waste streams, the use of defined microbial co-culture processes could tackle some of their drawbacks such as poor control over the process outcome, which often leads to low selectivity for the desired products. However, the extensive experimentation needed to design a co-culture system hinders the use of this technology. In this work, a workflow based on the combined use of mathematical models and wet experimentation is proposed to accelerate the design of novel bioprocesses. In particular, a co-culture consisting of Pediococcus pentosaceus and Megaphaera cerevisiae is used to target the production of high-value odd- and even­carbon VFA. An unstructured kinetic model was developed, calibrated and used to design experiments with the goal of increasing the selectivity for the desired VFA, which were experimentally validated. In the case of even­carbon VFA, the experimental validation showed an increase of 38 % in caproate yield and, in the case of enhanced odd­carbon VFA experiments, the yield of butyrate and caproate diminished by 62 % and 94 %, respectively, while propionate became one of the main end products and valerate yield value increased from 0.007 to 0.085 gvalearte per gconsumed sugar. The workflow followed in this work proved to be a sound tool for bioprocess design due to its capacity to explore and design new experiments in silico in a fast way and ability to quickly adapt to new scenarios.

Microbial invasions in sludge anaerobic digesters.

Among processes that control microbial community assembly, microbial invasion has received little attention until recently, especially in the field of anaerobic digestion. However, knowledge of the principles regulating the taxonomic and functional stability of microbial communities is key to truly develop better predictive models and effective management strategies for the anaerobic digestion process. To date, available studies focus on microbial invasions in digesters feed with activated sludge from municipal wastewater treatment plants. Herein, this review summarizes the importance of invasions for anaerobic digestion management, the ecological theories about microbial invasions, the traits of activated sludge microorganisms entering the digesters, and the resident communities of anaerobic reactors that are relevant for invasions and the current knowledge about the success and impacts of invasions, and discusses the research needs on this topic. The initial data indicate that the impact of invasions is low and only a small percentage of the mostly aerobic microorganisms present in the activated sludge feed are able to become stablished in the anaerobic digesters. However, there are still numerous unknowns about microbial invasions in anaerobic digestion including the influence of anaerobic feedstocks or process perturbances that new approaches on microbial ecology could unveil. KEY POINTS: • Microbial invasions are key processes to develop better strategies for digesters management. • Knowledge on pathogen invasions can improve anaerobic digestion microbial safety. • To date, the number of successful invasions on anaerobic digesters from activated sludge organisms is low. • Feed organisms detected in digesters are mostly inactive residual populations. • Need to expand the range of invaders and operational scenarios studied.

Steering the conversion of protein residues to volatile fatty acids by adjusting pH.

This study investigates the influence of pH on protein conversion into volatile fatty acids by anaerobic mixed-culture fermentation, a topic that, in contrast to glucose fermentation, only had scarce and contradictory information available. Several experiments were performed with two model proteins (casein and gelatin) at three different pH values (5, 7 and 9) using chemostats and batch tests. Highest conversion was reached at neutral pH although complete acidification was never achieved. Longer chain carboxylates production was favoured at low pH, while acetic acid was the main product at pH 7 and 9. Amino acids preferential consumption also varied with pH and protein composition. In fact, protein conversion stoichiometry is mainly driven by energetic yields and amino acid molecular configuration. Overall, this study identifies pH adjustment as a way to steer volatile fatty acid production during mixed-culture fermentation of proteins.

Minimization of dissolved methane, nitrogen and organic micropollutants emissions of effluents from a methanogenic reactor by using a preanoxic MBR post-treatment system.

The use of a hybrid membrane bioreactor (MBR) post-treatment system is proposed as a cost-efficient technology in order to minimize the environmental impact of anaerobic effluents, treating low-strength sewage at room temperature, such as their high nitrogen content and the presence of dissolved methane. In this research, nitrite was externally added at different concentrations into the anoxic compartment, providing an extra electron acceptor besides the existing nitrate, to evaluate its effect on denitrification, methane oxidation and OMPs removal processes. The nitrite addition significantly improved the denitrification potential of the system, achieving nitrogen removals up to 35 mg TN L-1. Moreover, higher nitrite concentrations clearly promoted an increase in the removal of some organic micropollutants (OMPs) such as diclofenac (DCF), ethinylestradiol (EE2), triclosan (TCS) and ibuprofen (IBP). Nevertheless, methane removal efficiencies or rates were not affected by this fact. Finally, COD and ammonium removals higher than 99 and 91% were observed during the entire operation, respectively. Based on the results, a future strategy in which ammonium is partially oxidized to nitrite could result in better nitrogen and OMPs removals for the proposed technology.

The time response of anaerobic digestion microbiome during an organic loading rate shock.

Knowledge of connections between operational conditions, process stability, and microbial community dynamics is essential to enhance anaerobic digestion (AD) process efficiency and management. In this study, the detailed temporal effects of a sudden glycerol-based organic overloading on the AD microbial community and process imbalance were investigated in two replicate anaerobic digesters by a time-intensive sampling scheme. The microbial community time response to the overloading event was shorter than the shifts of reactor performance parameters. An increase in bacterial community dynamics and in the abundances of several microbial taxa, mainly within the Firmicutes, Tenericutes, and Chloroflexi phyla and Methanoculleus genera, could be detected prior to any shift on the reactor operational parameters. Reactor acidification already started within the first 24 h of the shock and headed the AD process to total inhibition in 72 h alongside with the largest shifts on microbiome, mostly the increase of Anaerosinus sp. and hydrogenotrophic methanogenic Archaea. In sum, this work proved that AD microbial community reacts very quickly to an organic overloading and some shifts occur prior to alterations on the performance parameters. The latter is very interesting as it can be used to improve AD process management protocols.

A systematic methodology for the robust quantification of energy efficiency at wastewater treatment plants featuring Data Envelopment Analysis.

This article examines the potential benefits of using Data Envelopment Analysis (DEA) for conducting energy-efficiency assessment of wastewater treatment plants (WWTPs). WWTPs are characteristically heterogeneous (in size, technology, climate, function …) which limits the correct application of DEA. This paper proposes and describes the Robust Energy Efficiency DEA (REED) in its various stages, a systematic state-of-the-art methodology aimed at including exogenous variables in nonparametric frontier models and especially designed for WWTP operation. In particular, the methodology systematizes the modelling process by presenting an integrated framework for selecting the correct variables and appropriate models, possibly tackling the effect of exogenous factors. As a result, the application of REED improves the quality of the efficiency estimates and hence the significance of benchmarking. For the reader's convenience, this article is presented as a step-by-step guideline to guide the user in the determination of WWTPs energy efficiency from beginning to end. The application and benefits of the developed methodology are demonstrated by a case study related to the comparison of the energy efficiency of a set of 399 WWTPs operating in different countries and under heterogeneous environmental conditions.

Electron bifurcation mechanism and homoacetogenesis explain products yields in mixed culture anaerobic fermentations.

Anaerobic fermentation of organic wastes using microbial mixed cultures is a promising avenue to treat residues and obtain added-value products. However, the process has some important limitations that prevented so far any industrial application. One of the main issues is that we are not able to predict reliably the product spectrum (i.e. the stoichiometry of the process) because the complex microbial community behaviour is not completely understood. To address this issue, in this work we propose a new metabolic network of glucose fermentation by microbial mixed cultures that incorporates electron bifurcation and homoacetogenesis. Our methodology uses NADH balances to analyse published experimental data and evaluate the new stoichiometry proposed. Our results prove for the first time the inclusion of electron bifurcation in the metabolic network as a better description of the experimental results. Homoacetogenesis has been used to explain the discrepancies between observed and theoretically predicted yields of gaseous H2 and CO2 and it appears as the best solution among other options studied. Overall, this work supports the consideration of electron bifurcation as an important biochemical mechanism in microbial mixed cultures fermentations and underlines the importance of considering homoacetogenesis when analysing anaerobic fermentations.

Environmental assessment of alternative treatment schemes for energy and nutrient recovery from livestock manure.

The application of livestock manure on agricultural land is being restricted due to its significant content of phosphorus (P) and nitrogen (N), leading to eutrophication. At the same time, the growing demand for N and P mineral fertilizers is increasing their production costs and causing the depletion of natural phosphate rock deposits. In the present work, seven technologically feasible treatment schemes for energy (biogas) and nutrient recovery (e.g., struvite precipitation) and/or removal (e.g., partial nitritation/anammox) were evaluated from an environmental perspective. In general, while approaches based solely on energy recovery and use of digestate as fertilizer are commonly limited by community regulations, strategies pursuing the generation of high-quality struvite are not environmentally sound alternatives. In contrast, schemes that include further solid/liquid separation of the digestate improved the environmental profile, and their combination with an additional N-removal stage would lead to the most environmental-friendly framework. However, the preferred scenario was identified to be highly dependent on the particular conditions of each site, integrating environmental, social and economic criteria.

What happens with organic micropollutants during UV disinfection in WWTPs? A global perspective from laboratory to full-scale.

The phototransformation of 18 organic micropollutants (OMPs) commonly detected in wastewater treatment plant (WWTP) effluents was examined attempting to explain their fate during UV disinfection in WWTPs. For this purpose, a lab-scale UV reactor (lamp emitting at 254nm) was used to study the influence of the operational conditions (UV dose, temperature and water matrix) on OMPs abatement and disinfection efficiency. Chemical properties of OMPs and the quality of treated effluent were identified as key factors affecting the phototransformation rate of these compounds. Sampling campaigns were carried out at the inlet and outlet of UV systems of three WWTPs, and the results evidenced that only the most photosensitive compounds, such as sulfamethoxazole and diclofenac, are eliminated. Therefore, despite UV treatment is an effective technology to phototransform OMPs, the UV doses typically applied for disinfection (10-50mJ/cm2) are not sufficient to remove them. Consequently, small modifications (increase of UV dose, use of catalysts) should be applied in WWTPs to enhance the abatement of OMPs in UV systems.

Assessing the feasibility of two hybrid MBR systems using PAC for removing macro and micropollutants.

The removal of 10 organic micropollutants (OMPs) was studied in two MBRs using different types of membrane (flat sheet microfiltration, MF, and hollow fiber ultrafiltration, UF) operated under aerobic conditions with direct dosing of powdered activated carbon (PAC) in the mixed liquor. In both reactors high COD degradation and nitrification were achieved (>95%), while nitrate removal was only observed after PAC addition. The adsorbent improved the operation of both systems (sludge properties and microbial diversity) which resulted in an enhancement of the quality of the final effluent. The operation with both types of membrane was feasible being the UF system slightly better in terms of the quality of the final effluent. The strategy of 250 mg/L of PAC additions every 35 days was validated according to the results obtained for the removal of the most recalcitrant OMPs, such as diclofenac and carbamazepine. Concerning the type of membrane, only significant differences were observed for diclofenac and roxithromycin, which were better removed in the UF configuration. These differences were attributed to sorption and/or further biotransformation processes occurring in the cake layer.

Is anaerobic digestion effective for the removal of organic micropollutants and biological activities from sewage sludge?

The occurrence of emerging organic micropollutants (OMPs) in sewage sludge has been widely reported; nevertheless, their fate during sludge treatment remains unclear. The objective of this work was to study the fate of OMPs during mesophilic and thermophilic anaerobic digestion (AD), the most common processes used for sludge stabilization, by using raw sewage sludge without spiking OMPs. Moreover, the results of analytical chemistry were complemented with biological assays in order to verify the possible adverse effects (estrogenic and genotoxic) on the environment and human health in view of an agricultural (re)use of digested sludge. Musk fragrances (AHTN, HHCB), ibuprofen (IBP) and triclosan (TCS) were the most abundant compounds detected in sewage sludge. In general, the efficiency of the AD process was not dependent on operational parameters but compound-specific: some OMPs were highly biotransformed (e.g. sulfamethoxazole and naproxen), while others were only slightly affected (e.g. IBP and TCS) or even unaltered (e.g. AHTN and HHCB). The MCF-7 assay evidenced that estrogenicity removal was driven by temperature. The Ames test did not show point mutation in Salmonella typhimurium while the Comet test exhibited a genotoxic effect on human leukocytes attenuated by AD. This study highlights the importance of combining chemical analysis and biological activities in order to establish appropriate operational strategies for a safer disposal of sewage sludge. Actually, it was demonstrated that temperature has an insignificant effect on the disappearance of the parent compounds while it is crucial to decrease estrogenicity.

Effect of oxygen on the microbial activities of thermophilic anaerobic biomass.

Low oxygen levels (µgO2L(-1)) in anaerobic reactors are quite common and no relevant consequences are expected. On the contrary, higher concentrations could affect the process. This work aimed to study the influence of oxygen (4.3 and 8.8mgO2L(-1), respectively) on the different microbial activities (hydrolytic, acidogenic and methanogenic) of thermophilic anaerobic biomass and on the methanogenic community structure. Batch tests in presence of oxygen were conducted using specific substrates for each biological activity and a blank (with minimum oxygen) was included. No effect of oxygen was observed on the hydrolytic and acidogenic activities. In contrast, the methane production rate decreased by 40% in all oxygenated batches and the development of active archaeal community was slower in presence of 8.8mgO2L(-1). However, despite this sensitivity of methanogens to oxygen at saturation levels, the inhibition was reversible.

Understanding the fate of organic micropollutants in sand and granular activated carbon biofiltration systems.

In this study, sand and granular activated carbon (GAC) biofilters were comparatively assessed as post-treatment technologies of secondary effluents, including the fate of 18 organic micropollutants (OMPs). To determine the contribution of adsorption and biotransformation in OMP removal, four reactors were operated (two biofilters (with biological activity) and two filters (without biological activity)). In addition, the influence of empty bed contact time (EBCT), ranging from 0.012 to 3.2d, and type of secondary effluent (anaerobic and aerobic) were evaluated. Organic matter, ammonium and nitrate were removed in both biofilters, being their adsorption higher on GAC than on sand. According to the behaviour exhibited, OMPs were classified in three different categories: I) biotransformation and high adsorption on GAC and sand (galaxolide, tonalide, celestolide and triclosan), II) biotransformation, high adsorption on GAC but low or null adsorption on sand (ibuprofen, naproxen, fluoxetine, erythromycin, roxythromycim, sulfamethoxazole, trimethoprim, bisphenol A, estrone, 17ß-estradiol and 17α-ethinylestradiol), and, III) only adsorption on GAC (carbamazepine, diazepam and diclofenac). No influence of EBCT (in the range tested) and type of secondary effluent was observed in GAC reactors, whereas saturation and kinetic limitation of biotransformation were observed in sand reactors. Taking into account that most of the organic micropollutants studied (around 60%) fell into category II, biotransformation is crucial for the elimination of OMPs in sand biofilters.

The potential of the innovative SeMPAC process for enhancing the removal of recalcitrant organic micropollutants.

SeMPAC is an innovative process based on a membrane sequential batch reactor to which powdered activated carbon (PAC) is directly added. It was developed with the aim of obtaining a high quality effluent in terms of conventional pollutants and organic micropollutants (OMPs). High COD removal and nitrification efficiencies (>95%) were obtained already during the operation without PAC, although denitrification was enhanced by PAC addition. OMPs were followed in the solid and liquid matrixes so that biotransformation, sorption onto the sludge and adsorption onto the PAC could be assessed. Recalcitrant compounds, such as carbamazepine and diazepam, were readily removed only after PAC addition (>99%). Progressive saturation of PAC was observed, with increasing concentrations of OMPs in the solid phase. Removal efficiencies for recalcitrant compounds were used as indicators for new additions of PAC. An improvement in the moderately biodegradable OMPs removal was observed after PAC addition (e.g. fluoxetine, trimethoprim) which was attributed to the biofilm that grew onto the sorbent, as well as to adsorption onto PAC.

A UASB reactor coupled to a hybrid aerobic MBR as innovative plant configuration to enhance the removal of organic micropollutants.

An innovative plant configuration based in an UASB reactor coupled to a hybrid aerobic membrane bioreactor designed for sustainable treatment of municipal wastewater at ambient temperatures and low hydraulic retention time was studied in terms of organic micropollutants (OMPs) removal. OMPs removal mechanisms, as well as the potential influence of biomass activity and physical conformation were assessed. Throughout all periods of operation (150 days) high organic matter removals were maintained (>95%) and, regarding OMPs removal, this innovative system has shown to be more efficient than conventional technologies for those OMPs which are prone to be biotransformed under anaerobic conditions. For instance, sulfamethoxazole and trimethoprim have both shown to be biodegradable under anaerobic conditions with similar efficiencies (removal efficiencies above 84%). OMPs main removal mechanism was found to be biotransformation, except in the case of musk fragrances which showed medium sorption onto sludge. OMPs removal was strongly dependent on the efficiency of the primary metabolism (organic matter degradation and nitrification) and the type of biomass.

Biotransformation of pharmaceuticals under nitrification, nitratation and heterotrophic conditions.

The effect of nitrification, nitratation and heterotrophic conditions on the biotransformation of several pharmaceuticals in a highly enriched nitrifying activated sludge was evaluated in this study by selective activation of ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and heterotrophic bacteria. Nitrifiers displayed a noticeable capacity to process ibuprofen due to hydroxylation by ammonia monooxygenase (AMO) to produce 2-hydroxy-ibuprofen. Naproxen was also biotransformed under nitrifying conditions. On the other hand, heterotrophic bacteria present in the nitrifying activated sludge (NAS) biotransformed sulfamethoxazole. In contrast, both nitrifying and heterotrophic activities were ineffective against diclofenac, diazepam, carbamazepine and trimethoprim. Similar biotransformation rates of erythromycin, roxithromycin and fluoxetine were observed under all conditions tested. Overall, results from this study give more evidence on the role of the different microbial communities present in activated sludge reactors on the biological removal of pharmaceuticals.

Fostering the action of versatile peroxidase as a highly efficient biocatalyst for the removal of endocrine disrupting compounds.

Response surface methodology (RSM) was used to optimize the removal of five endocrine disrupting compounds (EDCs) by the enzyme versatile peroxidase (VP): bisphenol A (BPA), triclosan (TCS), estrone (E1), 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2). The optimal variables of enzyme activity (90-100 U L(-1)), sodium malonate (29-43 mM) and MnSO4 (0.8-1 mM) led to very high removal rates of the five pollutants (2.5-5.0 mg L(-1) min(-1)). The structural elucidation of transformation products arising from the enzymatic catalysis of the EDCs was investigated by Gas Chromatography coupled to Mass Spectrometry (GC-MS) and Liquid Chromatography Electrospray Time-of-Flight Mass Spectrometry (LC-ESI-TOF-MS). The presence of dimers and trimers, indicative of oxidative coupling, was demonstrated.

Assessing the use of nanoimmobilized laccases to remove micropollutants from wastewater.

Enzymes immobilization is a useful way to allow enzyme reuse and increase their stability. A high redox potential laccase from Trametes versicolor (TvL) and a low redox potential, but commercially available low-cost laccase from Myceliophthora thermophila (MtL), were successfully immobilized and co-immobilized onto fumed silica nanoparticles (fsNP). Enzyme loads of 1.78 ± 0.07, 0.69 ± 0.03, and 1.10 ± 0.01 U/mg fsNP were attained for the optimal doses of TvL, MtL, and co-immobilized laccases, respectively. In general, the laccase-fsNP conjugates showed a higher resistance against an acidic pH value (i.e., pH 3), and a higher storage stability than free enzymes. In addition, immobilized enzymes exhibited a superior long-term stability than free laccases when incubated in a secondary effluent from a municipal wastewater treatment plant (WWTP). For instance, the residual activity after 2 weeks for the co-immobilized laccases and the mixture of free laccases were 40.2 ± 2.5% and 16.8 ± 1.0%, respectively. The ability of the laccase-fsNP to remove a mixture of (14)C-bisphenol A (BPA) and (14)C-sodium diclofenac (DCF) from spiked secondary effluents was assessed in batch experiments. The catalytic efficiency was highly dependent on both the microbial source and state of the biocatalyst. The high redox potential TvL in free form attained a four-fold higher percentage of BPA transformation than the free MtL. Compared to free laccases, immobilized enzymes led to much slower rates of BPA transformation. For instance, after 24 h, the percentages of BPA transformation by 1000 U/L of a mixture of free laccases or co-immobilized enzymes were 67.8 ± 5.2 and 27.0 ± 3.9%, respectively. Nevertheless, the use of 8000 U/L of co-immobilized laccase led to a nearly complete removal of BPA, despite the unfavorable conditions for laccase catalysis (pH ~ 8.4). DCF transformation was not observed for any of the enzymatic systems, showing that this compound is highly recalcitrant toward laccase oxidation under realistic conditions.

Enzymatic technologies for remediation of hydrophobic organic pollutants in soil.

Worldwide there are numerous contaminated sites as a result of the widespread production and use of chemicals in industrial and military activities as well as poor schemes of waste disposal and accidental spillages. The implementation of strategies for decontamination and restoration of polluted sites has become a priority, being bioremediation with biological agents a promising alternative. Enzyme-based technologies offer several advantages over the use of microbial cells, provided that the biocatalyst meets specific requirements: efficiency to remove the target pollutant/s, non-dependency on expensive coenzymes or cofactors, enzyme stability, and an affordable production system. In this mini-review, the direct application of enzymes for in situ soil bioremediation is explored, and also novel ex situ enzymatic technologies are presented. This new perspective provides a valuable insight into the different enzymatic alternatives for decontamination of soils. Examples of recent applications are reported, including pilot-scale treatments and patented technologies, and the principles of operation and the main requirements associated are described. Furthermore, the main challenges regarding the applicability of enzymatic technologies for remediation of hydrophobic organic pollutants from soil are discussed.