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Biosorbent materials are effective in the removal of spilled oil from water, but their effect on hydrocarbonoclastic bacteria is not known. Here, we show that corksorb, a cork-based biosorbent, enhances growth and alkane degradation by Rhodococcus opacus B4 (Ro) and Alcanivorax borkumensis SK2 (Ab). Ro and Ab degraded 96 ± 1% and 72 ± 2%, respectively, of a mixture of n-alkanes (2 g L1) in the presence of corksorb. These values represent an increase of 6 and 24%, respectively, relative to the assays without corksorb. The biosorbent also increased the growth of Ab by 51%. However, no significant changes were detected in the expression of genes involved in alkane uptake and degradation in the presence of corksorb relative to the control without the biosorbent. Nevertheless, transcriptomics analysis revealed an increased expression of rRNA and tRNA coding genes, which confirms the higher metabolic activity of Ab in the presence of corksorb. The effect of corksorb is not related to the release of soluble stimulating compounds, but rather to the presence of the biosorbent, which was shown to be essential. Indeed, scanning electron microscopy images and downregulation of pili formation coding genes, which are involved in cell mobility, suggest that cell attachment on corksorb is a determinant for the improved activity. Furthermore, the existence of native alkane-degrading bacteria in corksorb was revealed, which may assist in situ bioremediation. Hence, the use of corksorb in marine oil spills may induce a combined effect of sorption and stimulated biodegradation, with high potential for enhancing in situ bioremediation processes. © Copyright © 2021 Martins, Freitas, Castro, Silva, Gudiña, Sequeira, Salvador, Pereira and Cavaleiro., This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of project MORE (POCI-01-0145-FEDER-016575) and Salt Oil+ (POCI-01-0145-FEDER-030180) and of the strategic funding of UIDB/04469/2020 unit. Research of RS and JS was supported by Ph.D. grants SFRH/BD/116154/2016 and SFRH/BD/147271/2019, respectively, funded by FCT., info:eu-repo/semantics/publishedVersion

In anaerobic bioreactors, the electrons produced during the oxidation of organic matter can potentially be used for the biological reduction of pharmaceuticals in wastewaters. Common electron transfer limitations benefit from the acceleration of reactions through utilization of redox mediators (RM). This work explores the potential of carbon nanomaterials (CNM) as RM on the anaerobic removal of ciprofloxacin (CIP). Pristine and tailored carbon nanotubes (CNT) were first tested for chemical reduction of CIP, and pristine CNT was found as the best material, so it was further utilized in biological anaerobic assays with anaerobic granular sludge (GS). In addition, magnetic CNT were prepared and also tested in biological assays, as they are easier to be recovered and reused. In biological tests with CNM, approximately 99% CIP removal was achieved, and the reaction rates increased ?1.5-fold relatively to the control without CNM. In these experiments, CIP adsorption onto GS and CNM was above 90%. Despite, after applying three successive cycles of CIP addition, the catalytic properties of magnetic CNT were maintained while adsorption decreased to 29 ± 3.2%, as the result of CNM overload by CIP. The results suggest the combined occurrence of different mechanisms for CIP removal: adsorption on GS and/or CNM, and biological reduction or oxidation, which can be accelerated by the presence of CNM. After biological treatment with CNM, toxicity towards Vibrio fischeri was evaluated, resulting in ? 46% detoxification of CIP solution, showing the advantages of combining biological treatment with CNM for CIP removal., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte. Ana Rita Silva holds an FCT grant SFRH/BD/131905/2017. Cátia S.N. Braga holds an FCT grant SFRH/BD/132003/2017. This work was also financially supported by: Base Funding-UIDB/50020/2020 of the Associate Laboratory LSRE LCM-funded by national funds through FCT/MCTES (PIDDAC). OSGPS acknowledges FCT fund ing under the Scientific Employment Stimulus-Institutional Call CEECINST/00049/2, info:eu-repo/semantics/publishedVersion

Polyhydroxyalkanoates (PHAs) are polymers with widespread applications, from medical devices to packaging. PHAs can be biodegradable in natural environments, such as soil, but the blend of PHA with other materials can change the polymer properties and consequently affect the biodegradation process. The composition of the microbial communities in soil also significantly affects the biodegradation, but other factors such as temperature, pH, and soil moisture, can also be determinant. These ecological and physic/chemical factors change in different seasons and in different soil layers. It is essential to know how these factors influence the PHAs biodegradation to understand the impact of PHAs in nature. This review compiles the results on PHA polymers and PHA blends biodegradation, with focus on laboratory tests. The main factors affecting PHA's biodegradation in soil, both in laboratory tests and in the environment are also discussed., Miguel Fernandes acknowledges the grant PD/BD/146195/2019 provided by the Portuguese Foundation for Science and Technology (FCT). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Long-chain fatty acids (LCFA) are common contaminants in municipal and industrial wastewater that can be converted anaerobically to methane. A low hydrogen partial pressure is required for LCFA degradation by anaerobic bacteria, requiring the establishment of syntrophic relationships with hydrogenotrophic methanogens. However, high LCFA loads can inhibit methanogens, hindering biodegradation. Because it has been suggested that anaerobic degradation of these compounds may be enhanced by the presence of alternative electron acceptors, such as iron, we investigated the effect of sub-stoichiometric amounts of Fe(III) on oleate (C18:1 LCFA) degradation by suspended and granular methanogenic sludge. Fe(III) accelerated oleate biodegradation and hydrogenotrophic methanogenesis in the assays with suspended sludge, with H2-consuming methanogens coexisting with iron-reducing bacteria. On the other hand, acetoclastic methanogenesis was delayed by Fe(III). These effects were less evident with granular sludge, possibly due to its higher initial methanogenic activity relative to suspended sludge. Enrichments with close-to-stoichiometric amounts of Fe(III) resulted in a microbial community mainly composed of Geobacter, Syntrophomonas, and Methanobacterium genera, with relative abundances of 83&ndash, 89%, 3&ndash, 6%, and 0.2&ndash, 10%, respectively. In these enrichments, oleate was biodegraded to acetate and coupled to iron-reduction and methane production, revealing novel microbial interactions between syntrophic LCFA-degrading bacteria, iron-reducing bacteria, and methanogens.

Pharmaceutical compounds end up in wastewater treatment plants but little is known on their effect towards the different microbial groups in anaerobic communities. In this work, the effect of the antibiotic Ciprooxacin (CIP), the non-steroidal anti-inammatory drugs Diclofenac (DCF) and Ibuprofen (IBP), and the hormone 17-ethinylestradiol (EE2), on the activity of acetogens and methanogens in anaerobic communities, was investigated. Microbial communities were more affected by CIP, followed by EE2, DCF and IBP, but the response of the different microbial groups was dissimilar. For concentrations of 0.01 to 0.1 mg/L, the specic methanogenic activity was not affected. Acetogenic bacteria were sensitive to CIP concentrations above 1 mg/L, while DCF and EE2 toxicity was only detected for concentrations higher than 10 mg/L, and IBP had no effect in all concentrations tested. Acetoclastic methanogens showed higher sensitivity to the presence of these micropollutants, being affect by all the tested pharmaceutical compounds although at different degrees. Hydrogenotrophic methanogens were not affected by any concentration, indicating their lower sensitivity to these compounds when compared to acetoclasts and acetogens., e Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Ana Rita Silva holds a Grant from FCT, reference SFRH/BD/131905/2017, info:eu-repo/semantics/publishedVersion

Conductive nanomaterials affect anaerobic digestion (AD) processes usually by improving methane production. Nevertheless, their effect on anaerobic communities, and particularly on specific trophic groups such as syntrophic bacteria or methanogens, is not extensively reported. In this work, we evaluate the effect of multi-walled carbon nanotubes (MWCNT) on the activity of two different anaerobic microbial communities: an anaerobic sludge and a river sediment. Methane production by anaerobic sludge was assessed in the presence of different MWCNT concentrations, with direct methanogenic substrates (acetate, hydrogen) and with typical syntrophic substrates (ethanol, butyrate). MWCNT accelerated the initial specific methane production rate (SMPR) from all compounds, with a more pronounced effect on the assays with acetate and butyrate, i.e., 2.1 and 2.6 times, respectively. In the incubations with hydrogen and ethanol, SMPR increased 1.1 and 1.2 times. Experiments with the river sediment were performed in the presence of MWCNT and MWCNT impregnated with 2% iron (MWCNT-Fe). Cumulative methane production was 10.2 and 4.5 times higher in the assays with MWCNT-Fe and MWCNT, respectively, than in the assays without MWCNT. This shows the high potential of MWCNT toward bioenergy production, in waste/wastewater treatment or ex situ bioremediation in anaerobic digesters., This research was funded by the Portuguese Foundation for Science and Technology(FCT) under the scope of project MORE (POCI-01-0145-FEDER-016575), of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte, as well as FCT/MCTES trough national funds (PIDDAC) and Base Funding-UIDB/50020/20 of the Associate Laboratory LSRE-LCM-funded by national funds rough FCT/MCTES (PIDDAC). Research of O.S.G.P.S. was funded by FCT under the Scientific Employment Stimulus-Institutional Call EECINST/00049/2018., info:eu-repo/semantics/publishedVersion

Anaerobic degradation of longchain fatty acids (LCFA) involves syntrophic bacteria and methanogens, but facultative anaerobic bacteria (FAB) might have a relevant role as well. Here we investigated oleate degradation by a syntrophic synthetic coculture of Syntrophomonas zehnderi (Sz) and Methanobacterium formicicum (Mf) and FAB (two oleatedegrading Pseudomonas spp. I1 + I2). Sz + Mf were first cultivated in a continuous bioreactor under strict anaerobic conditions. Thereafter, I1 + I2 were inoculated and microaerophilic conditions were provided. Methane and acetate were the main degradation products by Sz + Mf in anaerobiosis, and by Sz + Mf + I1 + I2 in microaerophilic conditions. However, acetate production from oleate was higher in microaerophilic conditions (5% O2) with the four microorganisms together (0.41 ±0.07mmol d1) than in anaerobiosis with Sz + Mf (0.23 ±0.05mmol d1). Oleate degradation in batch assays was faster by Sz + Mf + I1 + I2 (under microaerophilic conditions) than by Sz + Mf alone (under strict anaerobic conditions). I1 + I2 were able to grow with oleate and with intermediates of oleate degradation (hydrogen, acetate and formate). This work highlights the importance of FAB, particularly Pseudomonas sp., in anaerobic reactors treating oleatebased wastewater, because they accelerate oleate conversion to methane, by protecting strict anaerobes from oxygen toxicity and also by acting as alternative hydrogen/formate and acetate scavengers for LCFAdegrading anaerobes., This study was supported by the Portuguese Foundation forScience and Technology (FCT) under the scope under thescope of project MORE (PTDC/AAG-TEC/3500/2014; POCI-01-0145-FEDER-016575), of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Carbon materials have been reported to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens improving methane production in anaerobic processes. In this work, the effect of increasing concentrations of carbon nanotubes (CNT) on the activity of pure cultures of methanogens and on typical fatty acid-degrading syntrophic methanogenic coculture was evaluated. CNT affected methane production by methanogenic cultures, although acceleration was higher for hydrogenotrophic methanogens than for acetoclastic methanogens or syntrophic coculture. Interestingly, the initial methane production rate (IMPR) by Methanobacterium formicicum cultures increased 17 times with 5 g·L-1 CNT. Butyrate conversion to methane by Syntrophomonas wolfei and Methanospirillum hungatei was enhanced (∼1.5 times) in the presence of CNT (5 g·L-1 ), but indications of DIET were not obtained. Increasing CNT concentrations resulted in more negative redox potentials in the anaerobic microcosms. Remarkably, without a reducing agent but in the presence of CNT, the IMPR was higher than in incubations with reducing agent. No growth was observed without reducing agent and without CNT. This finding is important to re-frame discussions and re-interpret data on the role of conductive materials as mediators of DIET in anaerobic communities. It also opens new challenges to improve methane production in engineered methanogenic processes.

Degradation of long-chain fatty acids (LCFAs) in methanogenic environments is a syntrophic process involving the activity of LCFA-degrading bacteria and hydrogen-utilizing methanogens. If methanogens are inhibited, other hydrogen scavengers are needed to achieve complete LCFA degradation. In this work, we developed two different oleate (C18:1 LCFA)-degrading anaerobic enrichment cultures, one methanogenic (ME) and another in which methanogenesis was inhibited (IE). Inhibition of methanogens was attained by adding a solution of 2-bromoethanesulfonate (BrES), which turned out to consist of a mixture of BrES and isethionate. Approximately 5 times faster oleate degradation was accomplished by the IE culture compared with the ME culture. A bacterium closely related to Syntrophomonas zehnderi (99\% 16S rRNA gene identity) was the main oleate degrader in both enrichments, in syntrophic relationship with hydrogenotrophic methanogens from the genera Methanobacterium and Methanoculleus (in ME culture) or with a bacterium closely related to Desulfovibrio aminophilus (in IE culture). A Desulfovibrio species was isolated, and its ability to utilize hydrogen was confirmed. This bacterium converted isethionate to acetate and sulfide, with or without hydrogen as electron donor. This bacterium also utilized BrES but only after 3 months of incubation. Our study shows that syntrophic oleate degradation can be coupled to desulfonation.IMPORTANCE In anaerobic treatment of complex wastewater containing fat, oils, and grease, high long-chain fatty acid (LCFA) concentrations may inhibit microbial communities, particularly those of methanogens. Here, we investigated if anaerobic degradation of LCFAs can proceed when methanogens are inhibited and in the absence of typical external electron acceptors, such as nitrate, iron, or sulfate. Inhibition studies were performed with the methanogenic inhibitor 2-bromoethanesulfonate (BrES). We noticed that, after autoclaving, BrES underwent partial hydrolysis and turned out to be a mixture of two sulfonates (BrES and isethionate). We found out that LCFA conversion proceeded faster in the assays where methanogenesis was inhibited, and that it was dependent on the utilization of isethionate. In this study, we report LCFA degradation coupled to desulfonation. Our results also showed that BrES can be utilized by anaerobic bacteria., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004), funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. We also acknowledge Project MultiBiorefinery (SAICTPAC/0040/2015 [POCI-01-0145-FEDER-016403]), funded by Sistema de Apoio à Investigação Científica e Tecnológica (SAICT), Programas de Atividades Conjuntas (PAC), and the financial support of the European Research Council under the European Union Seventh Framework Programme (FP/2007-2013)/ERC (grant agreement 323009), info:eu-repo/semantics/publishedVersion

Conductive materials (CM) have been extensively reported to enhance methane production in anaerobic digestion processes. The occurrence of direct interspecies electron transfer (DIET) in microbial communities, as an alternative or complementary to indirect electron transfer (via hydrogen or formate), is the main explanation given to justify the improvement of methane production. Not disregarding that DIET can be promoted in the presence of certain CM, it surely does not explain all the reported observations. In fact, in methanogenic environments DIET was only unequivocally demonstrated in cocultures of Geobacter metallireducens with Methanosaeta harundinacea or Methanosarcina barkeri and frequently Geobacter sp. are not detected in improved methane production driven systems. Furthermore, conductive carbon nanotubes were shown to accelerate the activity of methanogens growing in pure cultures, where DIET is not expected to occur, and hydrogenotrophic activity is ubiquitous in full-scale anaerobic digesters treating for example brewery wastewaters, indicating that interspecies hydrogen transfer is an important electron transfer mechanism in those systems. This paper presents an overview of the effect of several iron-based and carbon-based CM in bioengineered systems, focusing on the improvement in methane production and in microbial communities changes. Control assays, as fundamental elements to support major conclusions in reported experiments, are critically revised and discussed., This study was supported by the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement no. 323009 and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145FEDER-006684) and project PAC MultiBiorefinery SAICTPAC/0040/2015 (POCI-01-0145-FEDER-016403), and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. We acknowledge the fellowships awarded to Gilberto Martins (SFRH/BPD/80528/2011) and Luciana Pereira (SFRH/BPD/110235/2015) under the scope of the program POPH/ESF., info:eu-repo/semantics/publishedVersion

Metagenomics (MG) and Metatranscriptomics (MT) approaches open new perspectives on the interpretation of biological systems composed by complex microbial communities. Dealing with large sequencing datasets, to extract the desired information and interpret the results are big challenges associated with meta-omics studies. There are several bioinformatics pipelines for MG data analysis and less to MT. Up to date, none performs a complete analysis integrating both MG and MT data, including the assembly of reads into contigs, functional and taxonomic annotation of identified genes, differential gene expression analysis and the comparison of multiple samples. Here, we present Meta-Omics Software for Community Analysis (MOSCA) that was designed with this purpose. It integrates RNA-Seq analysis with Whole Genome Sequencing as reference. Raw sequencing reads are submitted to preprocessing for quality trimming and rRNA removal, and assembled into contigs, which afterwards are annotated by using a reference database. MOSCA performs differential gene expression and provides graphical visualization of the results and comparison of multiple samples. Validation and reproducibility of the pipeline was obtained by using simulated MG and MT datasets.

1-Hexadecene-contaminated wastewater is produced in oil refineries and can be treated in methanogenic bioreactors, although generally at low conversion rates. In this study, a microbial culture able to degrade 1-hexadecene was enriched, and different stimulation strategies were tested for enhancing 1-hexadecene conversion to methane. Seven and three times faster methane production was obtained in cultures stimulated with yeast extract or lactate, respectively, while cultures amended with crotonate lost the ability to degrade 1-hexadecene. Methane production from 1-hexadecene was not enhanced by the addition of extra hydrogenotrophic methanogens. Bacteria closely related to Syntrophus and Smithella were detected in 1-hexadecene-degrading cultures, but not in the ones amended with crotonate, which suggests the involvement of these bacteria in 1-hexadecene degradation. Genes coding for alkylsuccinate synthase alpha-subunit were detected in cultures degrading 1-hexadecene, indicating that hydrocarbon activation may occur by fumarate addition. These findings are novel and show that methane production from 1-hexadecene is improved by the addition of yeast extract or lactate. These extra electron donors may be considered as a potential bioremediation strategy of oil-contaminated sites with bioenergy generation through methane production., European Research Council (323009); Fundação para a Ciência e a Tecnologia (FCOMP-01-0124-FEDER-027462, POCI-01-0145-FEDER006684, POCI-01-0145-FEDER-016575, SFRH/BPD/104837/2014, UID/BIO/04469/2013); European Regional Development Fund (NORTE-01-0145-FEDER-000004)., info:eu-repo/semantics/publishedVersion

Polymerase chain reaction (PCR) is central to methods in molecular ecology. Here, we describe PCR-dependent approaches useful for investigating microbial diversity and its function in various natural, human-associated, and built environment ecosystems. Protocols routinely used for DNA extraction, purification, cloning, and sequencing are included along with various resources for the statistical analysis following gel electrophoresis-based methods (DGGE) and sequencing. We also provide insights into eukaryotic microbiome analysis, sample preservation techniques, PCR troubleshooting, DNA quantification methods, and commonly used ordination techniques.

The effects of dietary short chain fructooligosaccharides (scFOS) incorporation on hematology, fish immune status, gut microbiota composition, digestive enzymes activities, and gut morphology, was evaluated in gilthead sea bream (Sparus aurata) juveniles reared at 18 °C and 25 °C. For that purpose, fish with 32 g were fed diets including 0, 0.1, 0.25 and 0.5% scFOS during 8 weeks. Overall, scFOS had only minor effects on gilthead sea bream immune status. Lymphocytes decreased in fish fed the 0.1% scFOS diet. Fish fed the 0.5% scFOS diet presented increased nitric oxide (NO) production, while total immunoglobulins (Ig) dropped in those fish, but only in the ones reared at 25 °C. Red blood cells, hemoglobin, bactericidal activity and NO were higher at 25 °C, whereas total white blood cells, circulating thrombocytes, monocytes and neutrophils were higher at 18 °C. In fish fed scFOS, lymphocytes were higher at 18 °C. Total Ig were also higher at 18 °C but only in fish fed 0.1% and 0.5% scFOS diets. No differences in gut bacterial profiles were detected by PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) between dietary treatments. However, group's similarity was higher at 25 °C. Digestive enzymes activities were higher at 25 °C but were unaffected by prebiotics incorporation. Gut morphology was also unaffected by dietary prebiotic incorporation. Overall, gut microbiota composition, digestive enzymes activities and immunity parameters were affected by rearing temperature whereas dietary scFOS incorporation had only minor effects on these parameters. In conclusion, at the tested levels scFOS does not seem worthy of including it in gilthead sea bream juveniles diets., This research was partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT Foundation for Science and Technology, under the projects "PEst-C/MAR/LA0015/2013 and UID/Multi/04423/2013". I. Guerreiro, P. Enes, A. Couto and B. Costas were supported by grants (SFRH/BD/76139/2011; BPD/39688/2007, BD/47495/2008 and BPD/77210/2011, respectively) from FCT, Portugal. C. Serra and A. Salvador were recipient of grants within the Project AQUAIMPROV (reference NORTE-07-0124-FEDER-000038) and Project BioEnv (REF. NORTE-07-0124-FEDER-000048), co-financed by the North Portugal Regional Operational Programme (ON.2 - O Novo Norte). We would like to express our thanks to Pedro Correia for the assistance during the growth trial. Authors would also thank to Jefo Species-specific additives France, for providing the prebiotic.

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.watres.2016.06.004., The catalytic effect of commercial microporous activated carbon (AC) and macroporous carbon nanotubes (CNT) is investigated in reductive bioreactions in continuous high rate anaerobic reactors, using the azo dye Acid Orange 10 (AO10) as model compound as electron acceptor and a mixture of VFA as electron donor. Size and concentration of carbon materials (CM) and hydraulic retention time (HRT) are assessed. CM increased the biological reduction rate of AO10, resulting in significantly higher colour removal, as compared to the control reactors. The highest efficiency, 98%, was achieved with a CNT diameter (d) lower than 0.25 mm, at a CNT concentration of 0.12 g per g of volatile solids (VS), a HRT of 10 h and resulted in a chemical oxygen demand (COD) removal of 85%. Reducing the HRT to 5 h, colour and COD removal in CM-mediated bioreactors were above 90% and 80%, respectively. In the control reactor, thought similar COD removal was achieved, AO10 decolourisation was just approximately 20%, demonstrating the ability of CM to significantly accelerate the reduction reactions in continuous bioreactors. AO10 reduction to the correspondent aromatic amines was proved by high performance liquid chromatography (HPLC). Colour decrease in the reactor treating a real effluent with CNT was the double comparatively to the reactor operated without CNT. The presence of AC in the reactor did not affect the microbial diversity, as compared to the control reactor, evidencing that the efficient reduction of AO10 was mainly due to AC rather than attributed to changes in the composition of the microbial communities., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01- 0145-FEDER-006684). Raquel Pereira had a fellowship (SFRH/BD/ 72388/2010) and Luciana Pereira has the fellowship (SFRH/BPD/ 110235/2015) from FCT. The authors thank the FCT exploratory EXPL/AAG-TEC/0898/2013 project.

Long-chain fatty acids (LCFA) can inhibit methane production by methanogenic archaea. The effect of oleate and palmitate on pure cultures of Methanosaeta concilii and Methanosarcina mazei was assessed by comparing methane production rates from acetate before and after LCFA addition. For both methanogens, a sharp decrease in methane production (> 50%) was observed at 0.5 mmol L1 oleate, and no methane was formed at concentrations higher than 2 mmol L1 oleate. Palmitate was less inhibitory than oleate, and M. concilii was more tolerant to palmitate than M. mazei, with 2 mmol L1 palmitate causing 11% and 64% methanogenic inhibition respectively. This study indicates that M. concilii and M. mazei tolerate LCFA concentrations similar to those previously described for hydrogenotrophic methanogens. In particular, the robustness of M. concilii might contribute to the observed prevalence of Methanosaeta species in anaerobic bioreactors used to treat LCFA-rich wastewater., The authors thank the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement no. 323009 and the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and Project RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462).

Methanogenic archaea are reported as very sensitive to lipids and long chain fatty acids (LCFA). Therefore, in conventional anaerobic processes, methane recovery during LCFA-rich wastewater treatment is usually low. By applying a start-up strategy, based on a sequence of step feeding and reaction cycles, an oleate-rich wastewater was efficiently treated at an organic loading rate of 21 kg COD m(-3) day(-1) (50 % as oleate), showing a methane recovery of 72 %. In the present work, the archaeal community developed in that reactor is investigated using a 16S rRNA gene approach. This is the first time that methanogens present in a bioreactor converting efficiently high loads of LCFA to methane are monitored. Denaturing gradient gel electrophoresis profiling showed that major changes on the archaeal community took place during the bioreactor start-up, where phases of continuous feeding were alternated with batch phases. After the start-up, a stable archaeal community (similarity higher than 84 %) was observed and maintained throughout the continuous operation. This community exhibited high LCFA tolerance and high acetoclastic and hydrogenotrophic activity. Cloning and sequencing results showed that Methanobacterium- and Methanosaeta-like microorganisms prevailed in the system and were able to tolerate and endure during prolonged exposure to high LCFA loads, despite the previously reported LCFA sensitivity of methanogens., This study has been financially supported by FEDER funds through the Operational Competitiveness Programme (COMPETE) and by national funds through the Portuguese Foundation for Science and Technology (FCT) in the frame of the projects FCOMP-01-0124-FEDER-007087 and FCOMP-01-0124-FEDER-014784. Financial support from FCT and the European Social Fund (ESF) through PhD grants SFRH/BD/48960/2008 and SFRH/BD/24256/2005 attributed to Andreia Salvador and Ana Julia Cavaleiro is also acknowledged.

Lipids can be anaerobically digested to methane, but methanogens are often considered to be highly sensitive to the long-chain fatty acids (LCFA) deriving from lipids hydrolysis. In this study, the effect of unsaturated (oleate [C18:1]) and saturated (stearate [C18:0] and palmitate [C16:0]) LCFA toward methanogenic archaea was studied in batch enrichments and in pure cultures. Overall, oleate had a more stringent effect on methanogens than saturated LCFA, and the degree of tolerance to LCFA was different among distinct species of methanogens. Methanobacterium formicicum was able to grow in both oleate- and palmitate-degrading enrichments (OM and PM cultures, respectively), whereas Methanospirillum hungatei only survived in a PMculture. The two acetoclastic methanogens tested, Methanosarcina mazei and Methanosaeta concilii, could be detected in both enrichment cultures, with better survival in PM cultures than inOMcultures. Viability tests using live/dead staining further confirmed that exponential growth-phase cultures of M. hungatei are more sensitive to oleate than are M. formicicum cultures; exposure to 0.5 mMoleate damaged 99% 1% of the cell membranes of M. hungatei and 53% 10% of the cell membranes of M. formicicum. In terms of methanogenic activity, M. hungatei was inhibited for 50% by 0.3, 0.4, and 1mMoleate, stearate, and palmitate, respectively. M. formicicum was more resilient, since 1mMoleate and>4mMstearate or palmitate was needed to cause 50% inhibition on methanogenic activity., This study was financially supported by FEDER funds through the Operational Competitiveness Programme (COMPETE) and by national funds through the Portuguese Foundation for Science and Technology (FCT) in the frame of the projects FCOMP-01-0124-FEDER-007087 and FCOMP-01-0124-FEDER-014784. Financial support from the FCT and the European Social Fund (POPH-QREN) through Ph.D. grant SFRH/BD/48960/2008 to A.F.S. is also acknowledged. A.J.M.S. was supported by grants from ALW-TOP (project 700.55.343) and ERC (project 323009).

Mineralization of a synthetic effluent containing 50% COD as oleic acid was achieved in a continuous anaerobic reactor at organic loading rates up to 21 kg COD m−3 day−1, HRT of 9 h, attaining 99% of COD removal efficiency and a methane yield higher than 70%. A maximum specific methane production rate of 1170 ± 170 mg COD-CH4 g VS−1 day−1 was measured during the reactor’s operation. A start-up strategy combining feeding phases and batch degradation phases was applied to promote the development of an anaerobic community efficient for long chain fatty acids (LCFA) mineralization. Through the start-up period, the methane yield increased gradually from 67% to 91%, and LCFA accumulated onto the sludge only during the first 60 days of operation. For the first time, it is demonstrated that a step feeding start-up is required to produce a specialized and efficient anaerobic community for continuous high rate anaerobic treatment of LCFA-rich wastewater., Fundação para a Ciência e a Tecnologia (FCT) - POCTI/CTA/46328/2002, PTDC/BIO/69745/ 2006, SFRH/24256/2005, European Commission (EC) - LIFE03 ENV/P/000501