Production of added value bacterial lipids through valorisation of hydrocarbon-contaminated cork waste

This work demonstrates that cork used as oil-spill sorbents, contaminated with liquid hydrocarbons, herein demonstrated with hexadecane, can be biologically treated by Rhodococcus opacus B4 with concomitant lipids production. R. opacus B4 consumed up to 96% of hexadecane (C16) impregnated in natural and regranulated cork sorbents after 48 h incubation, producing 0.59 ± 0.06 g of triacylglycerol (TAG) g 1 of C16 consumed with a TAG content of 0.60 ± 0.06 g g 1 of cellular dry weight (CDW) and 0.54 ± 0.05 g TAG g 1 of C16 consumed with a TAG content of 0.77 ± 0.04 g g 1 (CDW), respectively. TAG was mainly composed by fatty acids of 16 and 18 carbon chains demonstrating the feasibility of using it as raw material for biodiesel production. In addition, the obtained lipid-rich biomass (whole cells) can be used for biomethane production, at a yield of 0.4 L CH4 g 1 (CDW). The obtained results support a novel approach for management of oil-spill contaminated cork sorbents through its valorisation by producing bacterial lipids, which can be used as feedstocks for biofuels production.This work was financially supported by the Portuguese Science Foundation (FCT) and European Social Fund (ESF, POPH-QREN) through the grant given to A.R. Castro (SFRH/BD/64500/2009), the FCT Strategic Project of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) 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. The authors gratefully acknowledge Cortiçeira Amorim, S.A., Portugal, for providing the CORKSORB materials used in this work.info:eu-repo/semantics/publishedVersio

Garden and food waste co-fermentation for biohydrogen and biomethane production in a two-step hyperthermophilic-mesophilic process

Co-fermentation of garden waste (GW) and food waste (FW) was assessed in a two-stage process coupling hyperthermophilic dark-fermentation and mesophilic anaerobic digestion (AD). In the first stage, biohydrogen production from individual substrates was tested at different volatile solids (VS) concentrations, using a pure culture of Caldicellulosiruptor saccharolyticus as inoculum. FW concentrations (in VS) above 2.9 g L-1 caused a lag phase of 5 days on biohydrogen production. No lag phase was observed for GW concentrations up to 25.6 g L-1. In the co-fermentation experiments, the highest hydrogen yield (46±1 L kg-1) was achieved for GW:FW 90:10% (w/w). In the second stage, a biomethane yield of 682±14 L kg-1 was obtained using the end-products of GW:FW 90:10% co-fermentation. The energy generation predictable from co-fermentation and AD of GW:FW 90:10% is 0.5 MJ kg-1 and 24.4 MJ kg-1, respectively, which represents an interesting alternative for valorisation of wastes produced locally in 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-FEDER006684), Project SAICTPAC/0040/2015 (POCI-01-0145-FEDER016403) 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. The authors also acknowledge the financial support of FCT and European Social Fund through the grant attributed to A.A. Abreu (SFRH/BPD/82000/2011). Research of A.J. Cavaleiro was supported by the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement No 323009.info:eu-repo/semantics/publishedVersio

The protective effect of Pseudomonas in syntrophic fatty acids degradation under microaerophilic conditions

Micro-aeration has been used in anaerobic digestion (AD) systems to improve some aspects of this multifunctional and flexible technology. The addition of vestigial amounts of oxygen is usually associated with an increase of the relative abundance of facultative anaerobic bacteria (FAB) in the microbial community. Besides being involved in fermentation/acidogenesis, FAB have been referred to act as a protective shield against the damaging effects of oxidative environments to the strict anaerobic organisms of these communities. The microbial relationships between FAB, syntrophic bacteria and methanogens were investigated, during the degradation of short (C4), medium (C8) and long (C16) chain fatty acids by two syntrophic cultures (Syntrophomonas species and a methanogenic partner) in the presence of facultative bacteria (two Pseudomonas sp.). Pseudomonas were added to the pre-grown syntrophic pair, along with the substrate and a range of O2 concentrations (0-10% v/v). The grown cultures then were transferred a second time, with the same substrate and to all the O2 conditions previously tested. The cultures were followed through CH4, VFA, LCFA and pH measurements. In the presence of O2 (up to 2%) an effective syntrophic relationship, as well as the maintenance of methanogenic activity, was only possible in the presence of FAB. Cultures exposed to O2 in the first incubation performed well when transferred back to anaerobic conditions. Moreover, at the second phase, Pseudomonas maintained its protective shield effect and most of the cultures previously developed under micro-aerobic conditions could also maintain its activity. This work demonstrates that the presence of Pseudomonas contributes for a more resilient and functional syntrophic consortium degrading fatty acids under micro-aerobic conditions.info:eu-repo/semantics/publishedVersio

Inhibition studies with 2-bromoethanesulfonate reveal a novel syntrophic relationship in anaerobic oleate degradation

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/publishedVersio

Molecular assessment of complex microbial communities degrading long chain fatty acids in methanogenic bioreactors

Microbial diversity of anaerobic sludge after extended contact with long chain fatty acids (LCFA) was studied using molecular approaches. Samples containing high amounts of accumulated LCFA were obtained after continuous loading of two bioreactors with oleate or with palmitate. These sludge samples were then incubated in batch assays to allow degradation of the biomass-associated LCFA. In addition, sludge used as inoculum for the reactors was also characterized. Predominant phylotypes of the different samples were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Fingerprinting analysis showed changes in bacterial and archaeal communities during LCFA accumulation and degradation. Full-length 16S rRNA gene sequences of 22 clones, representing the predominant bacteria and archaea, were determined. Most bacterial clones (80%) clustered within the Clostridiaceae. Two major groups of methanogens were identified: hydrogen- and formate-utilizing organisms, closely related to Methanobacterium, and acetoclastic organisms closely related to Methanosaeta and Methanosarcina. Quantification by FISH and real-time PCR showed that the relative abundance of archaea increased during degradation of biomass-accumulated LCFA. These results provide insight into the importance and dynamics of balanced communities of bacteria and methanogens in LCFAaccumulation/ degradation cycles.Fundação para a Ciência e a Tecnologia (FCT); Fundo Social Europeu (FSE)

Endurance of methanogenic archaea in anaerobic bioreactors treating oleate-based wastewater

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

Study of 16 Portuguese activated sludge systems based on filamentous bacteria populations and their relationships with environmental parameters

A survey in 16 activated sludge waste water treatment plants (WWTP) was conducted to contribute to the knowledge of the environmental parameters that determine the composition of the filamentous community. A total of 128 samples of mixed liquor from municipal WWTP were collected during 2 years, and 22 filamentous morphotypes were identified. The most frequent and abundant filamentous bacteria were, in both cases and by this order, type 0041/0675, type 0092, Microthrix parvicella and 1851, nocardioforms and Haliscomenobacter hydrossis. Concerning dominance, type 1851 was the most frequently dominant morphotype, followed by M. parvicella and types 0092 and 0041/0675. These were also, and by this order, the dominant morphotypes during bulking occurrences. Significant correlations were obtained between the abundance of filamentous bacteria and environmental parameters, but multivariate statistical analysis only confirmed the correlation between type 0092 and Sludge Volume Index (SVI), emphasizing the association of this filament with bulking. The discussion of the results in light of published works was complicated by the random use of terms such as frequency, abundance, and dominance with different and often unclear meanings. This reinforces the need of clarifying these terms when discussing the causes of filamentous overgrowth in WWTP.Portuguese Foundation for Science and Technology (FCT) and the European Community fund FEDER, through Program COMPETE, in the ambit of the Projects FCOMP-01-0124-FEDER-007025 (PTDC/AMB/68393/2006), PEst-OE/EQB/LA0023/2013, RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and the Project BBioEnv - Biotechnology and Bioengineering for a sustainable world,REF. NORTE-07-0124- FEDER-000048, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. PhD grant SFRH/BD/64848/200

Continuous fungal treatment of non-sterile veterinary hospital effluent: pharmaceuticals removal and microbial community assessment

Source point treatment of effluents with a high load of pharmaceutical active compounds (PhACs), such as hospital wastewater, is a matter of discussion among the scientific community. Fungal treatments have been reported to be successful in degrading this type of pollutants and, therefore, the white-rot fungus Trametes versicolor was applied for the removal of PhACs from veterinary hospital wastewater. Sixty-six percent removal was achieved in a non-sterile batch bioreactor inoculated with T. versicolor pellets. On the other hand, the study of microbial communities by means of DGGE and phylogenetic analyses led us to identify some microbial interactions and helped us moving to a continuous process. PhAC removal efficiency achieved in the fungal treatment operated in non-sterile continuous mode was 44 % after adjusting the C/N ratio with respect to the previously calculated one for sterile treatments. Fungal and bacterial communities in the continuous bioreactors were monitored as well.Authors want to acknowledge the UAB veterinary hospital staff for their kind permission and help for the samplings. This work has been funded by the Spanish Ministry of Economy and Competitiveness and FEDER (projects CTM2013-48545-C2 and AIB2010PT-00169) and supported by the Generalitat de Catalunya (Consolidated Research Groups 2014-SGR-476 and 2014-SGR-291). The Department of Chemical Engineering of the Universitat Autonoma de Barcelona (UAB) is a member of the Xarxa de Referencia en Biotecnologia de la Generalitat de Catalunya. M. Badia-Fabregat and D. Lucas acknowledge the predoctoral grants from UAB and from the Spanish Ministry of Education, Culture and Sports (AP-2010-4926), respectively. The authors also thank the Portuguese Foundation for Science and Technology (FCT) Strategic Project PEst-OE/EQB/LA0023/2013, Project FCOMP-01-0124-FEDER-027462 co-funded by Operational Competitiveness Programme, FEDER, and Project "BioEnv-Biotechnology and Bioengineering for a sustainable world," REF. NORTE-07-0124-FEDER-000048, co-funded by Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER