The role of microbial ecology during biogas production from renewable energy sources. Characterizing microbial community structures in bioenergy production systems for future water resource recovery facilities

La Microbiología tiene un rol fundamental en la integración de procesos para la recuperación de nutrientes, energía y agua durante el tratamiento biológico del agua residual. La identificación de los grupos microbiológicos clave, así como de sus dinámicas, ecología y estructuras microbianas, mejorará el entendimiento de los procesos que integran las futuras plantas de recuperación de recursos, conocidas por sus siglas en inglés como Water Resource Recovery Facilities (WRRF). Este conocimiento podría ser de gran utilidad durante la optimización de sistemas anaerobios que recuperan energía a partir de fuentes de biomasa renovables. Hasta la fecha, diferentes estudios han demostrado que la combinación de los biorreactores anaerobios de membranas (AnMBR) para el tratamiento de agua residual y los fotobiorreactores de membranas para el cultivo de microalgas producen un efluente de alta calidad y son una opción sostenible, enmarcada en un concepto de economía circular. Además, el aumento del potencial hidrolítico de las comunidades microbianas ha demostrado ser una prometedora estrategia para incrementar el potencial de recuperación de metano a partir de fuentes de biomasa renovables, que se generan en las áreas municipales (como la fracción orgánica de los residuos sólidos urbanos) o durante los tratamientos del agua residual (biomasa de microalgas o fangos de depuradora). Este trabajo evalúa la ecología microbiana de un total de siete reactores anaerobios empleados en la recuperación de bioenergía a partir de fuentes de biomasa renovables. La secuenciación masiva del biomarcador de microorganismos procariotas (gen 16S rDNA) ha sido aplicada en todos estos sistemas para detectar la influencia de los parámetros operacionales sobre la ecología microbiana de los biorreactores durante la optimización del proceso. Los estudios han sido llevados a cabo a escala de laboratorio y de planta piloto. La complejidad de estos estudios de secuenciación ha motivado el desarrollo de una metodología en este trabajo para el análisis de datos de bioinformática y su posterior tratamiento con técnicas de bioestadística. En este contexto, la aplicación de técnicas de análisis multivariante ha permitido comprender el efecto de parámetros operacionales clave tales como la temperatura, la fuente de inóculo, los tiempos de retención hidráulico y celular, la velocidad de carga orgánica y la composición del afluente. Además, se han comparado diversas configuraciones de reactores, incluyendo el reactor AnMBR por su alto potencial de integración en las futuras WRRF. En este estudio se demuestra que la temperatura es el parámetro con la mayor influencia sobre las comunidades microbianas. Los phyla más abundantes en condiciones mesofílicas fueron 15-30% Chloroflexi, 14-27% Proteobacteria, 2-19% Bacteroidetes, 2-15% Firmicutes, y 1-7% Synergistes. En los sistemas termofílicos destacaron 17-32% Firmicutes y 6-44% Thermotoga. Cabe destacar que los sistemas mesofílicos de degradación de microalgas compartían un 57% de su diversidad microbiana y que las diferencias observadas se atribuían a los tiempos de retención hidráulico y celular. El análisis de rDNA y rRNA se recomienda para sistemas termofílicos con el fin de eliminar los grupos de microorganismos de fondo que se asocian a la diversidad microbiana intrínseca de la biomasa. Finalmente, en este trabajo se concluye que el uso de comunidades aclimatadas a altos tiempos de retención celular en reactores AnMBR es una mejor alternativa que el uso de cultivos externos hidrolíticos, ya que tienen una mayor resistencia ante cambios en las condiciones operacionales. Además, las microalgas Scenedesmus y Chlorella, que crecen en efluentes anaerobios, pueden ser degradadas por comunidades microbianas sin aplicar pretratamientos.Microbiology has a fundamental role in the integration of nutrients, energy and water recovery in biological wastewater treatment processes. The knowledge about key microbial groups composition, structure, dynamics, and ecology, will improve the comprehension of processes integrated into future Water Resource Recovery Facilities (WRRF) and could be helpful to optimize the anaerobic systems for bioenergy recovery from renewable sources. The combination of anaerobic membrane bioreactors (AnMBR) for sewage treatment and membrane photobioreactors (MPBR) for microalgae cultivation produces high-quality reclaimed water and is a sustainable solution on a circular economy frame. Also, the enhance of hydrolytic groups in microbial communities is a feasible strategy to boost biomethanization when using renewable sources that are produced in municipalities (e.g. food waste) or during the treatment of wastewater (microalgae, sewage sludge). This work evaluates the microbial ecology of seven anaerobic reactors for bioenergy recovery from renewable sources during performance optimization. Massive sequencing of 16S rDNA biomarker has been applied in these systems to detect the influence of the operational parameters on the bioreactor microbiology. The studies have been carried out at both the laboratory and the pilot plant scales. The complexity of the information retrieved through high-throughput sequencing has required the development of bioinformatics and biostatistics knowledge. The application of multivariate analysis techniques has allowed the full comprehension of the effect of operational parameter selection such as temperature, inoculum source, hydraulic and solids retention time, organic loading rate and influent composition. Besides, different bioreactor configurations have been explored, including the AnMBR because of its potential integration in future WRRF. This study demonstrates that the temperature is the most influencing parameter over microbial communities. The most remarkable mesophilic phyla of anaerobic systems were 15-30% Chloroflexi, 14-27% Proteobacteria, 2-19% Bacteroidetes, 2-15% Firmicutes, and 1-7% Synergistes; and 6-44% Thermotoga and 17-32% Firmicutes for thermophilic systems. Mesophilic systems for microalgae degradation through digestion or co-digestion share 57% of their microbial diversity. The differences were mainly attributed to solids (SRT) and hydraulic (HRT) retention times. The rDNA and rRNA sequencing strategy is especially recommended for thermophilic systems to remove the background groups associated with the feedstock biomass. Finally, it is concluded in this work that the use of acclimated communities at high SRT using AnMBR systems is a better alternative than the use of exogenous hydrolytic consortia since they are more resistant to changes in the operational conditions. Moreover, both Scenedesmus and Chlorella microalgae can be degraded by similar communities without pre-treatments

Understanding the performance of an AnMBR treating urban wastewater and food waste via model simulation and characterization of the microbial population dynamics

[EN] An anaerobic membrane bioreactor (AnMBR) pilot plant treating kitchen food waste (FW) jointly with urban wastewater was run for 536 days. Different operational conditions were tested varying the sludge retention time (SRT), the hydraulic retention time (HRT) and the penetration factor (PF) of food waste disposers. COD removal efficiency exceeded 90% in all tested conditions. The joint treatment resulted in an almost 3-fold increase in methane production (at 70 days of SRT, 24 h HRT and 80% PF) in comparison with the treatment of urban wastewater only. Mathematical model simulations and Illumina technology were used to obtain in-depth information of this outstanding process performance. Both the PF and SRT factors increased influent biodegradability. The experimental results were accurately reproduced via model simulations modifying only the influent biodegradability. The high SRT and the presence of ground FW in the influent resulted in higher hydrolytic activity. Not only did the Archaea population increase 3-fold but Levilinea genera was also significantly raised. Three new genera characterised by anaerobic fermentation of amino acids (Leptolinea, Aminomonas and Aminobacterium) were among the ten most abundant of the total sequences identified during the joint treatment, indicating an improvement in the hydrolysis step of anaerobic degradation. Influent biodegradability remained at high values when FW addition stopped.This research work has been financially supported by the Generalitat Valenciana (PROMETEO/2012/029 PROJECT), which is gratefully acknowledged.Durán Pinzón, F.; Zamorano -López, N.; Barat, R.; Ferrer, J.; Aguado García, D. (2018). Understanding the performance of an AnMBR treating urban wastewater and food waste via model simulation and characterization of the microbial population dynamics. Process Biochemistry. 67:139-146. https://doi.org/10.1016/j.procbio.2018.02.010S1391466

Thermophilic anaerobic conversion of raw microalgae: Microbial community diversity in high solids retention systems

[EN] The potential of microbial communities for efficient anaerobic conversion of raw microalgae was evaluated in this work. A long-term operated thermophilic digester was fed with three different Organic Loading Rates (OLR) (0.2, 0.3 and 0.4¿g·L¿1·d¿1) reaching 32¿41% biodegradability values. The microbial community analysis revealed a remarkable presence of microorganisms that exhibit high hydrolytic capabilities such as Thermotogae (~44.5%), Firmicutes (~17.6%) and Dictyoglomi, Aminicenantes, Atribacteria and Planctomycetes (below ~5.5%) phyla. The suggested metabolic role of these phyla highlights the importance of protein hydrolysis and fermentation when only degrading microalgae. The ecological analysis of the reactor suggests the implication of the novel group EM3 in fermentation and beta-oxidation pathways during microalgae conversion into methane. Scenedesmus spp. substrate and free ammonia concentration strongly shaped thermophilic reactor microbial structure. Partial Least Square Discriminant Analysis (PLS-DA) remarked the resilient role of minor groups related to Thermogutta, Armatimonadetes and Ruminococcaceae against a potential inhibitor like free ammonia. Towards low-cost biogas production from microalgae, this study reveals valuable information about thermophilic microorganisms that can strongly disrupt microalgae and remain in high solids retention anaerobic digesters.This research work has been supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Project CTM2011-28595-C02-02) jointly with the European Regional Development Fund (ERDF), which are gratefully acknowledged. The authors are thankful to Fernando Fdz-Polanco research team (University of Valladolid, Spain) for providing the thermophilic sludge from their pilot plant to inoculate the bioreactor and Llúcia Martínez and Giusseppe D'Aria from FISABIO sequencing service (Valencia, Spain) for their technical support during the Illumina sequencing design.Zamorano-López, N.; Greses-Huerta, S.; Aguado García, D.; Seco Torrecillas, A.; Borrás Falomir, L. (2019). Thermophilic anaerobic conversion of raw microalgae: Microbial community diversity in high solids retention systems. Algal Research. 41:1-9. https://doi.org/10.1016/j.algal.2019.101533S194

Acclimatised rumen culture for raw microalgae conversion into biogas: Linking microbial community structure and operational parameters in anaerobic membrane bioreactors (AnMBR)

[EN] Ruminal fluid was inoculated in an Anaerobic Membrane Reactor (AnMBR) to produce biogas from raw Scenedesmus. This work explores the microbial ecology of the system during stable operation at different solids retention times (SRT). The 16S rRNA amplicon analysis revealed that the acclimatised community was mainly composed of Anaerolineaceae, Spirochaetaceae, Lentimicrobiaceae and Cloacimonetes fermentative and hydrolytic members. During the highest biodegradability achieved in the AnMBR (62%) the dominant microorganisms were Fervidobacterium and Methanosaeta. Different microbial community clusters were observed at different SRT conditions. Interestingly, syntrophic bacteria Gelria and Smithella were enhanced after increasing 2-fold the organic loading rate, suggesting their importance in continuous systems producing biogas from raw microalgae.This research work has been supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Projects CTM2011-28595-C02-01 and CTM2011-28595-C02-02), which is gratefully acknowledged. The Education, Investigation, Culture and sports Council from the Valencian Generality for the Post-Doctoral fellowship of the third co-author is also acknowledged (APOSTD/2016/104). The authors are thankful to Ion Pérez Baena from the Universitat Politècnica de Valencia, Institut de Ciència I Tecnología Animal for gently providing the ruminal fluid use in this work.Zamorano-López, N.; Borrás Falomir, L.; Giménez, JB.; Seco Torrecillas, A.; Aguado García, D. (2019). Acclimatised rumen culture for raw microalgae conversion into biogas: Linking microbial community structure and operational parameters in anaerobic membrane bioreactors (AnMBR). Bioresource Technology. 290:1-9. https://doi.org/10.1016/j.biortech.2019.121787S1929

Effect of long residence time and high temperature over anaerobic biodegradation of Scenedesmus microalgae grown in wastewater

[EN] Anaerobic digestion of indigenous Scenedesmus spp. microalgae was studied in continuous lab-scale anaerobic reactors at different temperatures (35 degrees C and 55 degrees C), and sludge retention time - SRT (50 and 70 days). Mesophilic digestion was performed in a continuous stirred-tank reactor (CSTR) and in an anaerobic membrane bioreactor (AnMBR). Mesophilic CSTR operated at 50 days SRT only achieved 11.9% of anaerobic biodegradability whereas in the AnMBR at 70 days SRT and 50 days HRT reached 39.5%, which is even higher than the biodegradability achieved in the thermophilic CSTR at 50 days SRT (30.4%). Microbial analysis revealed a high abundance of cellulose-degraders in both reactors, AnMBR (mainly composed of 9.4% Bacteroidetes, 10.1% Chloroflexi, 8.0% Firmicutes and 13.2% Thermotogae) and thermophilic CSTR (dominated by 23.8% Chloroflexi and 12.9% Firmicutes). However, higher microbial diversity was found in the AnMBR compared to the thermophilic CSTR which is related to the SRT. since high SRT promoted low growth-rate microorganisms, increasing the hydrolytic potential of the system. These results present the membrane technology as a promising approach to revalue microalgal biomass, suggesting that microalgae biodegradability and consequently the methane production could be improved operating at higher SRT. (C) 2018 Elsevier Ltd. All rights reserved.This research work has been supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Project CTM2011-28595-C02-01/02) jointly with the European Regional Development Fund (ERDF), which are gratefully acknowledged. The authors are thankful to Fernando Fernandez-Polanco for providing the thermophilic sludge to inoculate the reactor.This research work has been financially supported by the Generalitat Valenciana (PROMETEO/2012/029 PROJECT), which is gratefully acknowledged.Greses-Huerta, S.; Zamorano -López, N.; Borrás Falomir, L.; Ferrer, J.; Seco Torrecillas, A.; Aguado García, D. (2018). Effect of long residence time and high temperature over anaerobic biodegradation of Scenedesmus microalgae grown in wastewater. Journal of Environmental Management. 218:425-434. https://doi.org/10.1016/j.jenvman.2018.04.086S42543421

Influence of Food Waste addition over microbial communities in an Anaerobic Membrane Bioreactor plant treating urban wastewater

[EN] Notorious changes in microbial communities were observed during and after the joint treatment of wastewater with Food Waste (FW) in an Anaerobic Membrane Bioreactor (AnMBR) plant. The microbial population was analysed by high-throughput sequencing of the 16S rRNA gene and dominance of Chloroflexi, Firmicutes, Synergistetes and Proteobacteria phyla was found. The relative abundance of these potential hydrolytic phyla increased as a higher fraction of FW was jointly treated. Moreover, whereas Specific Methanogenic Activity (SMA) rose from 10 to 51 mL CH4 g(-1) VS, Methanosarcinales order increased from 34.0% over 80.0% of total Archaea, being Methanosaeta the dominant genus. The effect of FW over AnMBR biomass was observed during the whole experience, as methane production rose from 49.2 to 144.5 L CH4 . kg(-1) influent COD. Furthermore, biomethanization potential was increased over 82% after the experience. AnMBR technology allows the established microbial community to remain in the bioreactor even after the addition of FW, improving the anaerobic digestion of urban wastewater. (C) 2018 Elsevier Ltd. All rights reserved.This research work has been financially supported by the Generalitat Valenciana (PROMETEO/2012/029 PROJECT), which is gratefully acknowledged.Zamorano -López, N.; Moñino Amoros, P.; Borrás Falomir, L.; Aguado García, D.; Barat, R.; Ferrer, J.; Seco Torrecillas, A. (2018). Influence of Food Waste addition over microbial communities in an Anaerobic Membrane Bioreactor plant treating urban wastewater. Journal of Environmental Management. 217:788-796. https://doi.org/10.1016/j.jenvman.2018.04.018S78879621

Exploring the limits of anaerobic biodegradability of urban wastewater by AnMBR technology

[EN] Anaerobic membrane bioreactors (AnMBRs) can achieve maximum energy recovery from urban wastewater (UWW) by converting influent COD into methane. The aim of this study was to assess the anaerobic biodegradability limits of urban wastewater with AnMBR technology by studying the possible degradation of the organic matter considered as non-biodegradable as observed in aerobic membrane bioreactors operated at very high sludge retention times. For this, the results obtained in an AnMBR pilot plant operated at very high SRT (140 days) treating sulfate-rich urban wastewater were compared with those previously obtained with the system operating at lower SRT (29 to 70 days). At 140 days SRT the organic matter biodegraded by the AnMBR system accounted for 64.4% of the influent COD (45.9% was removed by sulfate reducing bacteria (SRB), and only 18.5% was converted into methane, highlighting the strong competition between SRB and methanogenic archaea (MA) when treating sulfate-rich wastewater). Almost half of the methane produced (46%) was dissolved in the permeate and most of it was recovered by a degassing membrane. The organic matter biodegraded by the AnMBR system was similar to the influent anaerobic biodegradability determined by wastewater characterization assays (68.5% of the influent COD), indicating that nearly all the influent's biodegradable organic matter had been removed. This percentage of degraded COD was similar to that obtained in previous studies working at 70 days SRT, showing that the limit of anaerobic biodegradability was already reached in this SRT. The organic matter considered as non-biodegradable according to wastewater characterization assays therefore was not seen to degrade in the AnMBR pilot plant, even at very high SRT. Once the biodegraded COD is close to the influent's anaerobic biodegradability, increasing the SRT is not justified as it only leads to higher operational costs for the same biogas production. These findings support the use of mathematical models for AnMBR design since they accurately represent the behaviour of these systems in a wide range of operating conditions.This research project was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Project CTM2014-54980-C2-2-R). The authors are also grateful for the support received from the Generalitat Valenciana via CPI-16-155 fellowships.Seco Torrecillas, A.; Mateo-Llosa, O.; Zamorano-López, N.; Sanchis-Perucho, P.; Serralta Sevilla, J.; Martí Ortega, N.; Borrás Falomir, L.... (2018). Exploring the limits of anaerobic biodegradability of urban wastewater by AnMBR technology. Environmental Science: Water Research & Technology. 4(11):1877-1887. https://doi.org/10.1039/c8ew00313kS18771887411Li, W.-W., & Yu, H.-Q. (2011). From wastewater to bioenergy and biochemicals via two-stage bioconversion processes: A future paradigm. Biotechnology Advances, 29(6), 972-982. doi:10.1016/j.biotechadv.2011.08.012Shin, C., & Bae, J. (2018). Current status of the pilot-scale anaerobic membrane bioreactor treatments of domestic wastewaters: A critical review. Bioresource Technology, 247, 1038-1046. doi:10.1016/j.biortech.2017.09.002EEA , Performance of water utilities beyond compliance (Technical report No. 5/2014) , Luxemburg , 2014Martin, I., Pidou, M., Soares, A., Judd, S., & Jefferson, B. (2011). Modelling the energy demands of aerobic and anaerobic membrane bioreactors for wastewater treatment. 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Maximizing resource recovery from urban wastewater through an innovative facility layout

[EN] This research work proposes an innovative layout for urban wastewater treatment based on anaerobic technology, microalgal cultivation and membrane technology. The proposed Water Resource Recovery Facility (WRRF) system can treat urban wastewater efficiently, complying with legal discharge limits and allowing for resource recovery, i.e. energy, nutrients and reclaimed water. In addition, the proposed layout produces less solid wastes than a conventional wastewater treatment plant (WWTP) and it is possible to recover energy as biogas, not only from the original wastewater sources but also from the biomass generated in the WRRF system

Resource recovery from sulphate-rich sewage through an innovative anaerobic-based water resource recovery facility (WRRF)

[EN] This research work proposes an innovative water resource recovery facility (WRRF) for the recovery of energy, nutrients and reclaimed water from sewage, which represents a promising approach towards enhanced circular economy scenarios. To this aim, anaerobic technology, microalgae cultivation, and membrane technology were combined in a dedicated platform. The proposed platform produces a high-quality solid- and coliform-free effluent that can be directly discharged to receiving water bodies identified as sensitive areas. Specifically, the content of organic matter, nitrogen and phosphorus in the effluent was 45 mg COD.L-1 , 14.9 mg N.L-1 and 0.5 mg P.L-1 , respectively. Harvested solar energy and carbon dioxide biofixation in the form of microalgae biomass allowed remarkable methane yields (399 STP L CH 4.kg(-1) CODinf ) to be achieved, equivalent to theoretical electricity productions of around 0.52 kWh per m 3 of wastewater entering the WRRF. Furthermore, 26.6% of total nitrogen influent load was recovered as ammonium sulphate, while nitrogen and phosphorus were recovered in the biosolids produced (650 +/- 77 mg N.L-1 and 121.0 +/- 7.2 mg P.L-1).This research was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Projects CTM2014-54980-C2-1-R and CTM2014-54980-C2-2-R) jointly with the European Regional Development Fund (ERDF), which are gratefully acknowledged. This research was also supported by the Spanish Ministry of Education, Culture and Sport via two pre-doctoral FPU fellowships (FPU14/05082 and FPU15/02595) and by the Spanish Ministry of Economy and Competitiveness via two pre-doctoral FPI fellowships (BES-2015-071884, BES-2015-073403) and one Juan de la Cierva contract (FJCI-2014-21616). The authors would also like to acknowledge the support received from Generalitat Valenciana via two VALithornd post-doctoral grants (APOSTD/2014/049 and APOSTD/2016/104) and via the fellowships APOTI/2016/059 and CPI-16-155, as well as the financial aid received from the European Climate KIC association for the 'MAB 2.0' Project (APIN0057_ 2015-3.6-230_ P066-05) and Universitat Politecnica de Valencia via a pre-doctoral FPI fellowship to the seventh author.Seco Torrecillas, A.; Aparicio Antón, SE.; Gonzalez-Camejo, J.; Jiménez Benítez, AL.; Mateo-Llosa, O.; Mora-Sánchez, JF.; Noriega-Hevia, G.... (2018). Resource recovery from sulphate-rich sewage through an innovative anaerobic-based water resource recovery facility (WRRF). Water Science & Technology. 78(9):1925-1936. https://doi.org/10.2166/wst.2018.492S19251936789Bair, R. A., Ozcan, O. O., Calabria, J. L., Dick, G. H., & Yeh, D. H. (2015). 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Genome-wide pathway analysis identifies VEGF pathway association with oral ulceration in systemic lupus erythematosus

Background: Systemic lupus erythematosus (SLE) is a genetically complex rheumatic disease characterized by heterogeneous clinical manifestations of unknown etiology. Recent studies have suggested the existence of a genetic basis for SLE heterogeneity. The objective of the present study was to identify new genetic variation associated with the clinically relevant phenotypes in SLE. Methods: A two-stage pathway-based approach was used to identify the genetic variation associated with the main clinical phenotypes in SLE. In the discovery stage, 482 SLE patients were genotyped using Illumina Human Quad610 microarrays. Association between 798 reference genetic pathways from the Molecular Signatures Database and 11 SLE phenotypes was tested using the set-based method implemented in PLINK software. Pathways significantly associated after multiple test correction were subsequently tested for replication in an independent cohort of 425 SLE patients. Using an in silico approach, we analyzed the functional effects of common SLE therapies on the replicated genetic pathways. The association of known SLE risk variants with the development of the clinical phenotypes was also analyzed. Results: In the discovery stage, we found a significant association between the vascular endothelial growth factor (VEGF) pathway and oral ulceration (P value for false discovery rate (P FDR) < 0.05), and between the negative regulation signaling pathway of retinoic acid inducible gene-I/melanoma differentiation associated gene 5 and the production of antinuclear antibodies (P FDR < 0.05). In the replication stage, we validated the association between the VEGF pathway and oral ulceration. Therapies commonly used to treat mucocutaneous phenotypes in SLE were found to strongly influence VEGF pathway gene expression (P = 4.60e-4 to 5.38e-14). Analysis of known SLE risk loci identified a strong association between PTPN22 and the risk of hematologic disorder and with the development of antinuclear antibodies. Conclusions: The present study has identified VEGF genetic pathway association with the risk of oral ulceration in SLE. New therapies targeting the VEGF pathway could be more effective in reducing the severity of this phenotype. These findings represent a first step towards the understanding of the genetic basis of phenotype heterogeneity in SLE