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.

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.

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.

Potentiality of a ceramic membrane reactor for the laccase-catalyzed removal of bisphenol A from secondary effluents.

In this study, the removal of bisphenol A (BPA) by laccase in a continuous enzymatic membrane reactor (EMR) was investigated. The effects of key parameters, namely, type of laccase, pH, and enzyme activity, were initially evaluated. Once optimal conditions were determined, the continuous removal of the pollutant in an EMR was assessed in synthetic and real biologically treated wastewaters. The reactor configuration consisted of a stirred tank reactor coupled to a ceramic membrane, which prevented the sorption of the pollutant and allowed the recovery and recycling of laccase. Nearly complete removal of BPA was attained under both operation regimes with removal yields above 94.5 %. In experiments with real wastewater, the removal of BPA remained high while the presence of colloids and certain ions and the formation of precipitates on the membrane potentially affected enzyme stability and made necessary the periodic addition of laccase. Polymerization and degradation were observed as probable mechanisms of BPA transformation by laccase.

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.

Solvent screening methodology for in situ ABE extractive fermentation.

Solvent screening for in situ liquid extraction of products from acetone-butanol-ethanol (ABE) fermentation was carried out, taking into account biological parameters (biocompatibility, bioavailability, and product yield) and extraction performance (partition coefficient and selectivity) determined in real fermentation broth. On the basis of different solvent characteristics obtained from literature, 16 compounds from different chemical families were selected and experimentally evaluated for their extraction capabilities in a real ABE fermentation broth system. From these compounds, nine potential solvents were also tested for their biocompatibility towards Clostridium acetobutylicum. Moreover, bioavailability and differences in substrate consumption and total n-butanol production with respect to solvent-free fermentations were quantified for each biocompatible solvent. Product yield was enhanced in the presence of organic solvents having higher affinity for butanol and butyric acid. Applying this methodology, it was found that the Guerbet alcohol 2-butyl-1-octanol presented the best extracting characteristics (the highest partition coefficient (6.76) and the third highest selectivity (644)), the highest butanol yield (27.4 %), and maintained biocompatibility with C. acetobutylicum.

Operational strategies for producing bioethanol in a continuous single-stage reactor.

Novel strategies to facilitate the transition from batch to continuous simultaneous saccharification and fermentation were studied in this work. Implementing these strategies in bioethanol production plants to change production to a continuous mode will avoid large modifications in the process configuration. Therefore, experiments were carried out in a single-stage reactor applying strategies that favour a priori viability of yeast and stability of the process. The effects of (a) hydraulic residence time (HRT), (b) anaerobic and microaerobic operation, (c) inoculation strategy and (d) growth inhibition due to high ethanol concentrations were evaluated. The highest ethanol concentration (6.3 % w/w) was achieved during anaerobic operation, with reinoculations every 3-4 days and an HRT of 60 h; however, the processes suffered severe instability under these conditions. The greatest productivity and stability of the process was achieved using periodic microaeration and an HRT of 36 h (0.169 % ethanol weight/h), overcoming the result obtained during batch operation (0.128 % ethanol weight/h).

Optimisation of the biological pretreatment of wheat straw with white-rot fungi for ethanol production.

The biological pretreatment of lignocellulosic biomass for the production of bioethanol is an environmentally friendly alternative to the most frequently used process, steam explosion (SE). However, this pretreatment can still not be industrially implemented due to long incubation times. The main objective of this work was to test the viability of and optimise the biological pretreatment of lignocellulosic biomass, which uses ligninolytic fungi (Pleurotus eryngii and Irpex lacteus) in a solid-state fermentation of sterilised wheat straw complemented with a mild alkali treatment. In this study, the most important parameters of the mechanical and thermal substrate conditioning processes and the most important parameters of the fungal fermentation process were optimised to improve sugar recovery. The largest digestibilities were achieved with fermentation with I. lacteus under optimised conditions, under which cellulose and hemicellulose digestibility increased after 21 days of pretreatment from 16 to 100 % and 12 to 87 %, respectively. The maximum glucose yield (84 %) of cellulose available in raw material was obtained after only 14 days of pretreatment with an overall ethanol yield of 74 % of the theoretical value, which is similar to that reached with SE.

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.

Biotransformation of three pharmaceutical active compounds by the fungus Phanerochaete chrysosporium in a fed batch stirred reactor under air and oxygen supply.

White-rot fungi are a group of microorganisms capable of degrading xenobiotic compounds, such as polycyclic aromatic hydrocarbons or synthetic dyes, by means of the action of extracellular oxidative enzymes secreted during secondary metabolism. In this study, the transformation of three anti-inflammatory drugs: diclofenac, ibuprofen and naproxen were carried out by pellets of Phanerochaete chrysosporium in fed-batch bioreactors operating under continuous air supply or periodic pulsation of oxygen. The performance of the fungal reactors was steady over a 30-day treatment and the effect of oxygen pulses on the pellet morphology was evidenced. Complete elimination of diclofenac was achieved in the aerated and the oxygenated reactors, even with a fast oxidation rate in the presence of oxygen (77% after 2 h), reaching a total removal after 23 h. In the case of ibuprofen, this compound was completely oxidized under air and oxygen supply. Finally, naproxen was oxidized in the range of 77 up to 99% under both aeration conditions. These findings demonstrate that the oxidative capability of this microorganism for the anti-inflammatory drugs is not restricted to an oxygen environment, as generally accepted, since the fungal reactor was able to remove these compounds under aerated and oxygenated conditions. This result is very interesting in terms of developing viable reactors for the oxidation of target compounds as the cost of aeration can be significantly reduced.

Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed reactor.

Laccase from Myceliophthora thermophila was covalently immobilised on Eupergit C and Eupergit C 250L yielding specific activities of up to 17 and 80 U/g, respectively. Due to its superior activity, Eupergit C 250L was chosen for further research. The somewhat lower catalytic efficiency (based on the ratio between the turnover number and the Michaelis constant, k(cat)/K(M)) of the immobilised enzyme in comparison with that of the free enzyme was balanced by its increased stability and broader operational window related to temperature and pH. The feasibility of the immobilised laccase was tested by using a packed bed reactor (PBR) operating in consecutive cycles for the removal of Acid Green 27 dye as model substrate. High degrees of elimination were achieved (88, 79, 69 and 57% in 4 consecutive cycles), while the levels of adsorption on the support varied from 18 to 6%, proving that dye removal took place mainly due to the action of the enzyme. Finally, a continuous PBR with the solid biocatalyst was applied for the treatment of a solution containing the following endocrine disrupting chemicals: estrone (E1), 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2). At steady-state operation, E1 was degraded by 65% and E2 and EE2 were removed up to 80% and only limited adsorption of these compounds on the support, between 12 and 22%, was detected. In addition, a 79% decrease in estrogenic activity was detected in the effluent of the enzymatic reactor while only 14% was attained by inactivated laccase.

Evaluation of natural zeolite as microorganism support medium in nitrifying batch reactors: influence of zeolite particle size.

An evaluation of natural zeolite as a microorganism carrier in nitrifying reactors operated in batch mode was carried out. Specifically, the influence of zeolite particle sizes of 0.5, 1.0 and 2.0 mm in diameter on microorganism adherence to zeolite, ammonium adsorption capacity and the identification of microbial populations were assessed. The greatest amount of total biomass adhered was observed for a zeolite particle size of 1 mm (0.289 g) which was achieved on the 12th day of operation. The highest ammonium adsorption capacity was observed for a zeolite particle size of 0.5 mm, which was 64% and 31% higher than that observed for particle sizes of 1.0 and 2.0 mm, respectively. The maximum de-sorption values were also found for a zeolite particle size of 0.5 mm, although when equilibrium was reached the ammonium concentrations were similar to those observed for a zeolite particle size of 1.0 mm. It was also found that the experimental data on ammonium adsorption fitted very well to the Freundlich isotherm for the three particle sizes studied. Finally, the nitrifying reactors showed similar microbial populations independently of the particle size used as microorganism carrier. The dominant bacterial community was Gammaproteobacteria making up 80% of the total population found. Betaproteobacteria were also identified and made up 12% approx. of the total population. Ammonium Oxidant Betaproteobacteria and Nitrobacter were also detected.

Comparison of PPCPs removal on a parallel-operated MBR and AS system and evaluation of effluent post-treatment on vertical flow reed beds.

The presence in the aquatic environment of xenobiotics such as Pharmaceutical and Personal Care Products (PPCPs) has emerged as an issue of concern. Upgrading sewage treatment quality with modern technologies such as Membrane Bioreactors (MBRs) and/or implementing a further posttreatment might mitigate the release of xenobiotics into surface waters. The performance of two processes treating municipal sewage, a MBR and an Activated Sludge (AS) unit, have been compared in terms of PPCPs removal. Moreover, their effluents were treated using vertical flow reed beds. Both systems were operated under similar conditions, more specifically Hydraulic Retention Time (HRT), maintained at 8 hours, and Sludge Retention Time (SRT) set at 6 and 20 days. Pharmaceuticals belong to therapeutic groups such as antiepileptics (carbamazepine) and analgesics (ibuprofen, naproxen, diclofenac), whereas the personal care products are musk fragrances (galaxolide and tonalide). Xenobiotics removals achieved in the MBR showed better results, particularly for the acidic drugs ibuprofen (87% vs. 50%) and naproxen (56% vs. 6%) operating at low SRT. After filtration through vertical flow reed-beds, PPCPs content in effluents was decreased, below 1 ppb in most cases, improving the effluent quality and confirming reed-beds as an interesting low cost alternative in order to attenuate xenobiotics contamination.

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.

Influence of the employment of adsorption and coprecipitation agents for the removal of PPCPs in conventional activated sludge (CAS) systems.

Three activated sludge reactors were operated to improve the removal of organic micropollutants such as Pharmaceutical and Personal Care Products (PPCPs). Reactor 1 (R1) was operated as a Conventional Activated Sludge reactor (CAS), Reactor 2 (R2) consisted of a CAS unit that was continuously fed with FeCl3 whereas granular activated carbon (GAC) was fed directly into the mixed liquor of Reactor 3 (R3) in order to attain concentrations in the range 100-1,000 mg/L. PPCPs removal rates varied depending on the compound present in each reactor during the entire 220 days of operation. Some substances showed the same behaviour in all reactors, such as the acidic pharmaceuticals naproxen and ibuprofen, which were almost completely removed (> 90%). More hydrophobic organic substances, like musk fragrances, were about 90% removed after 40 days of operation in all of the reactors. The main difference between the three reactors was obtained in R3 when the GAC concentrations in the aeration tank were around 500-1,000 mg/L. Under these conditions, the more recalcitrant compounds like diazepam and carbamazepine could be removed by up to 40%, and diclofenac up to 85%. Adsorption isotherms for PPCPs were obtained with activated carbon, and the results were successfully fitted to the Freundlinch equation. The more recalcitrant compounds (carbamazepine, diazepam and diclofenac) had the highest adsorption capacities onto GAC, which is consistent with the behaviour observed in R3 and helps to identify the removal mechanism (adsorption for these compounds, whereas absorption for fragrances).

Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus hosts.

Production of recombinant versatile peroxidase in Aspergillus hosts was optimized through the modification of temperature during bioreactor cultivations. To further this purpose, the cDNA encoding a versatile peroxidase of Pleurotus eryngii was expressed under control of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans. A dependence of recombinant peroxidase production on cultivation temperature was found. Lowering the culture temperature from 28 to 19 degrees C enhanced the level of active peroxidase 5.8-fold and reduced the effective proteolytic activity twofold. Thus, a maximum peroxidase activity of 466 U L(-1) was reached. The same optimization scheme was applied to a recombinant Aspergillus niger that bore the alcohol dehydrogenase regulator (alcR), enabling transformation with the peroxidase cDNA under the same alcA promoter. However, with this strain, the peroxidase activity was not improved, while the effective proteolytic activity was increased between 3- and 11-fold compared to that obtained with A. nidulans.

Selection of variables for on-line monitoring, diagnosis, and control of anaerobic digestion processes.

This work aims to systematize the study of indicators for two types of wastewaters: carbohydrate-based and protein-based synthetic wastewaters. Characterization of steady states and dynamic response analysis against disturbances were carried out using both a factorial discriminant analysis (FDA) and a phenomenological analysis, respectively. This research seeks reconciling both sets of indicators in order to optimize resources and provide a minimal cost in instrumentation for its implementation at industrial scale. According to the results of this research, the best indicators for the two types of wastewaters, considering both process steady states and organic load perturbations are: Biogas flow rate or Methane flow rate, and Hydrogen concentration in the biogas; Volatile fatty acids and Partial alkalinity in the liquid phase.

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.

Enzymatic degradation of low soluble compounds in monophasic water: solvent reactors. Kinetics and modeling of anthracene degradation by MnP.

Polycyclic aromatic hydrocarbons (PAHs) are toxic compounds presenting low water solubility and high hydrophobicity, which greatly hampers their natural biodegradation. The enzymatic degradation of a model compound, anthracene, was evaluated in presence of a miscible solvent for an increased solubility. Manganese peroxidase, a ligninolytic enzyme from white-rot fungi, was used as biocatalyst in a medium containing acetone. The kinetic parameters of the enzymatic degradation of anthracene, obtained from fed-batch experiments, were applied to model the operation of a continuous reactor. Kinetics comprised a Michaelis-Menten equation, modified with an autocatalytic term, assumed to the effect of quinones acting as electron carriers, and a logistic function related to enzyme activity. The continuous reactor has been operated for 108 h, attaining a 90% of anthracene degradation, which demonstrated the feasibility of the system for its application in the removal of poorly soluble compounds. The model of this reactor permitted to predict accurately anthracene degradation in different conditions, such as external addition of anthraquinone and different enzymatic activities.

Characterization of anaerobic granular sludge developed in UASB reactors that treat ethanol, carbohydrates and hydrolyzed protein based wastewaters.

In this work, granules developed from UASB reactors that treat different types of wastewaters (ethanol, carbohydrates and protein-based synthetic wastewaters) were studied. Granule parameters (size distribution; density; settlement characteristics; elemental composition; acidogenic and methanogenic activities) were analyzed along with micro-organisms identified by FISH to better understand granule behavior and its formation process. Micro-organisms distributions in anaerobic granules are highly dependent on the type of treated wastewater. Granules developed in a UASB reactor that treats wastewater with a high content of carbohydrates presented high acidogenic bacteria colonization. Members of Methanosaetaceae were the dominant methanogens in the studied granules, and Methanobacteriales appear to be co-dominant in the granules developed with carbohydrates and protein-based wastewaters.