Characterization of melanin from Exophiala mesophila with the prospect of potential biotechnological applications.

Introducion: Fungal melanin is an underexplored natural biomaterial of great biotechnological interest in different areas. This study investigated the physical, chemical, electrochemical, and metal-binding properties of melanin extracted from the metallotolerant black fungus Exophiala mesophila strain IRTA-M2-F10. Materials and methods: Specific inhibitory studies with tricyclazole and biochemical profiling of whole cells by synchrotron radiation-based Fourier-transform infrared spectral microscopy (SR-FTIRM) were performed. An optimized extraction protocol was implemented, and purified fungal melanin was characterized using an array of spectrophotometric techniques (UV-Vis, FTIR, and EPR) and by cyclic voltammetry (CV) experiments. The metal-binding capacity of melanin extracts was also assessed by using Cr(VI) as a model heavy metal. Results: Inhibitory studies indicated that 1,8-dihydroxynaphthalene may be the main precursor molecule of E. mesophila melanin (DHN-melanin). The biochemical characterization of fungal melanin extracts were benchmarked against those from two melanins comprising the precursor molecule L-3,4-dihydroxiphenylalanine (DOPA-melanin): extracts from the ink of the cephalopod Sepia officinalis and DOPA-melanin synthesized in the laboratory. The CV results of melanin extracts incubated with and without cell suspensions of the electroconductive bacterium Geobacter sulfurreducens were indicative of novel semiquinone/hydroquinone redox transformations specific for each melanin type. These interactions may play an important role in cation exchange for the adsorption of metals and in microbial interspecies electron transfer processes. Discussion: The obtained results provided further evidence for the DHN-nature of E. mesophila melanin. The FTIR profiling of melanin extracts exposed to Cr(VI), compared to unexposed melanin, resulted in useful information on the distinct surface-binding properties of fungal melanin. The parameters of the Langmuir and Freundlicht isotherms for the adsorption of Cr(VI) were determined and compared to bibliographic data. Altogether, the inherent properties of fungal melanin suggest its promising potential as a biomaterial for environmental applications.

The Metallotolerance and Biosorption of As(V) and Cr(VI) by Black Fungi.

A collection of 34 melanized fungi isolated previously from anthropogenic contaminated sites were assessed for their tolerance to toxic concentrations of As(V) and Cr(VI) anions. Three strains of the species Cyphellophora olivacea, Rhinocladiella similis, and Exophiala mesophila (Chaetothyriales) were identified as hyper-metallotolerant, with estimated IC50 values that ranged from 11.2 to 16.9 g L-1 for As(V) and from 2.0 to 3.4 g L-1 for Cr(VI). E. mesophila and R. similis were selected for subsequent assays on their biosorption capacity and kinetics under different pH values (4.0 and 6.5) and types of biomass (active and dead cells and melanin extracts). The fungal biosorption of As(V) was relatively ineffective, but significant removal of Cr(VI) was observed from liquid cultures. The Langmuir model with second-order kinetics showed maximum sorption capacities of 39.81 mg Cr6+ g-1 for R. similis and 95.26 mg Cr6+ g-1 for E. mesophila on a dry matter basis, respectively, while the kinetic constant for these two fungi was 1.32 × 10-6 and 1.39 × 10-7 g (mg Cr6+ min)-1. Similar experiments with melanin extracts of E. mesophila showed maximum sorption capacities of 544.84 mg Cr6+ g-1 and a kinetic constant of 1.67 × 10-6 g (mg Cr6+ min)-1. These results were compared to bibliographic data, suggesting that metallotolerance in black fungi might be the result of an outer cell-wall barrier to reduce the diffusion of toxic metals into the cytoplasm, as well as the inner cell wall biosorption of leaked metals by melanin.

The potential of a combination of pungent spices as a novel supplement in gilthead seabream (Sparus aurata) diets to aid in the strategic use of fish oil in aquafeeds: a holistic perspective.

This work studied the potential of a combination of pungent spices (capsicum, black pepper, ginger, and cinnamaldehyde) to be used as a supplement in diets of gilthead seabream (Sparus aurata; 44.1 ± 4.2 g). During 90 days, fish were fed three experimental diets with low inclusion of fish oil and containing poultry fat as the main source of lipids, supplemented with graded levels of the tested supplement: 0 (control), 0.1 (SPICY0.1%), and 0.15% (SPICY0.15%). As a result, the pungent spices enhanced the growth performance, the activity of the bile-salt-activated lipase in the intestine, and decreased fat deposit levels within enterocytes. The SPICY0.1% diet reduced the feed conversion ratio and the perivisceral fat index and lipid deposits in the liver. Moreover, the ratio of docosahexaenoic acid/eicosapentaenoic acid in fillet increased in fish fed the SPICY0.1% diet, while the hepatic levels of docosahexaenoic acid and total n-3 polyunsaturated fatty acids increased in fish fed the SPICY0.15% diet. Furthermore, there was an effect on the expression of some biomarkers related to lipid metabolism in 2-h postprandial fish (fasn, elovl6, scd1b, cyp7a1, lpl, and pparß), and in 48 h fasted-fish fed with the SPICY0.1% diet, a regulation of the intestinal immune response was indicated. However, no significant differences were found in lipid apparent digestibility and proximate macronutrient composition. The spices did not affect biomarkers of hepatic or oxidative stress. No differences in microbial diversity were found, except for an increase in Simpson's Index in the posterior intestine of fish fed the SPICY0.1% diet, reflected in the increased relative abundance of the phylum Chloroflexi and lower relative abundances of the genera Campylobacter, Corynebacterium, and Peptoniphilus. In conclusion, the supplementation of gilthead seabream diets with pungent spices at an inclusion of 0.1% was beneficial to enhance growth performance and feed utilization; reduce fat accumulation in the visceral cavity, liver, and intestine; and improve the fish health status and condition. Results suggest that the tested supplement can be used as part of a nutritional strategy to promote a more judicious use of fish oil in fish diets due to its decreasing availability and rising costs.

Modulation of gut microbiota and intestinal immune response in gilthead seabream (Sparus aurata) by dietary bile salt supplementation.

Given their role in lipid digestion, feed supplementation with bile salts could be an economic and sustainable solution to alterations in adiposity and intestinal inflammation generated by some strategies currently used in aquaculture. An important part of the metabolism of bile salts takes place in the intestine, where the microbiota transforms them into more toxic forms. Consequently, we aimed to evaluate the gut immune response and microbial populations in gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of bile salts with proven background as a regulator of lipid metabolism and fat content. After the 90-day feeding trial, a differential modulation of the microbiota between the anterior and posterior intestine was observed. While in the anterior intestine the relative abundance of Desulfobacterota doubled, in the posterior intestine, the levels of Firmicutes increased and Proteobacteria, Actinobacteriota, and Campylobacterota were reduced when supplementing the diet with bile salts. Even so, only in the anterior intestine, there was a decrease in estimated richness (Chao1 and ACE indices) in presence of dietary bile salts. No significant differences were displayed in alpha (Shannon and Simpson indices) nor beta-diversity, showing that bile sales did not have a great impact on the intestinal microbiota. Regarding the gene expression profile in 2 h postprandial-fish, several changes were observed in the analyzed biomarkers of epithelial integrity, nutrient transport, mucus production, interleukins, cell markers, immunoglobulin production and pathogen recognition receptors. These results may indicate the development of an intestinal immune-protective status to tackle future threats. This work also suggests that this immune response is not only regulated by the presence of the dietary bile salts in the intestine, but also by the microbial populations that are in turn modulated by bile salts. After a fasting period of 2 days, the overall gene expression profile was stabilized with respect to fish fed the unsupplemented diet, indicating that the effect of bile salts was transient after short periods of fasting. On the balance, bile salts can be used as a dietary supplement to enhance S. aurata farming and production without compromising their intestinal health.

Use of wood and cork in biofilters for the simultaneous removal of nitrates and pesticides from groundwater.

About 13% and 7% of monitored groundwater stations in Europe exceed the permitted levels of nitrates (50 mg NO3- L-1) or pesticides (0.1 µg L-1), respectively. Although slow sand filtration can remove nitrates via denitrification when oxygen is limited, it requires an organic carbon source. The present study evaluates the performance of the use of wood pellets and granulated cork as carbon sources in bench-scale biofilters operated under water-saturated and water-unsaturated conditions for more than 400 days. The biofilters were monitored for nitrate (200 mg L-1) and pesticide (mecoprop, diuron, atrazine, and bromacil, each at a concentration of 5 µg L-1) attenuation, as well as for the formation of nitrite and pesticide transformation products. Microbiological characterization of each biofilter was also performed. The water-saturated wood biofilter achieved the best nitrate removal (>99%), while the cork biofilters lost all denitrification power over time (from 38% to no removal). The unsaturated biofilter columns were not effective for removing nitrates (20-30% removal). As for pesticides, all the biofilters achieved high removal rates of mecoprop and diuron (>99% and >75%, respectively). Atrazine removal was better in the wood-pellet biofilters than the cork ones (68-96% vs. 31-38%). Bromacil was only removed in the water-unsaturated cork biofilter (67%). However, a bromacil transformation product was formed there. The water-saturated wood biofilter contained the highest number of denitrifying microorganisms, with Methyloversatilis as the characteristic genus. Microbial composition could explain the high removal of pesticides and nitrates achieved in the wood-pellet biofilter. Overall, the results indicate that wood-pellet biofilters operated under water-saturated conditions are a good solution for treating groundwater contaminated with nitrates and pesticides.

The use of recovered struvite and ammonium nitrate in fertigation in a horticultural rotation: agronomic and microbiological assessment.

Phosphorus and nitrogen recovery from wastewater as struvite and ammonium nitrate (AN) may be viable alternative fertilizers to boost circularity in horticulture. A 2-year fertigated crop rotation in soil under greenhouse conditions was evaluated to determine the efficiency of both recovered products as raw materials for a nutrient solution (NS) manufacture. The effects of these treatments versus synthetic fertilizers were compared in terms of crop performance, plant nutrient uptake, soil chemistry and microbiota. This is the first study to implement struvite through fertigation as the sole source of P in soil crops. Results showed that both recovered products can be used as fertilizers in NS, due to the similar response to the control for different parameters and crops (tomato, lettuce, and cauliflower). However, the AN treatment showed lower yield in the first tomato crop, which results may depend on the cultivar ammonium tolerance. Besides, the concentration of heavy metals in fruits/leaves was below the permissible limits. Total and Olsen phosphorus soil analysis revealed no differences among treatments, resulting in a similar performance of P-struvite to commercial phosphate. Bulk soil bacteria structure, richness and relative dominance were increased over time, while archaea only showed lower evenness, both despite the fertilization strategy. Shannon diversity was not significantly affected. A predominance of ammonia-oxidizing bacteria (AOB) versus archaea (AOA) was observed, while nitrite-oxidizing bacteria (NOB), dominated by Nitrospira, increased with fertigation. Our results demonstrate that fertilizer blends for NS containing recovered nutrients are a feasible alternative to synthetic fertilizers.

Assessment of a novel microalgae-cork based technology for removing antibiotics, pesticides and nitrates from groundwater.

Groundwater pollution has increased in recent years due to the intensification of agricultural and livestock activities. This results in a significant reduction in available freshwater resources. Here, we have studied the long term assessment of a green technology (1-4 L/day) based on a photobioreactor (PBR) containing immobilised microalgae-bacteria in polyurethane foam (PF) followed by a cork filter (CF) for removing nitrates, pesticides (atrazine and bromacil), and antibiotics (sulfamethoxazole and sulfacetamide) from groundwater. The prototype was moderately effective for removing nitrates (58%) at an HRT of 8 days, while its efficiency decreased at a HRT of 4 and 2 days (<20% removal). The combined use of PBR-CF enabled antibiotics and pesticides to be attenuated by up to 95% at an HRT of 8 days, but their attenuation decreased with shorter HRT, with pesticides being the compounds most affected (reducing from 97 to 98% at an HRT of 8 days to 23-45% at an HRT of 2 days). Pesticide transformation products were identified after the CF, supporting biodegradation as the main attenuation process. A gene-based metataxonomic assessment linked the attenuation of micropollutants to the presence of specific pesticide biodegradation species (e.g. genus Phenylobacterium, Sphingomonadaceae, and Caulobacteraceae). Therefore, the results highlighted the potential use of microalgae and cork to treat polluted groundwater.

Bioaugmentation of Native Fungi, an Efficient Strategy for the Bioremediation of an Aged Industrially Polluted Soil With Heavy Hydrocarbons.

The concurrence of structurally complex petroleum-associated contaminants at relatively high concentrations, with diverse climatic conditions and textural soil characteristics, hinders conventional bioremediation processes. Recalcitrant compounds such as high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) and heavy alkanes commonly remain after standard soil bioremediation at concentrations above regulatory limits. The present study assessed the potential of native fungal bioaugmentation as a strategy to promote the bioremediation of an aged industrially polluted soil enriched with heavy hydrocarbon fractions. Microcosms assays were performed by means of biostimulation and bioaugmentation, by inoculating a defined consortium of six potentially hydrocarbonoclastic fungi belonging to the genera Penicillium, Ulocladium, Aspergillus, and Fusarium, which were isolated previously from the polluted soil. The biodegradation performance of fungal bioaugmentation was compared with soil biostimulation (water and nutrient addition) and with untreated soil as a control. Fungal bioaugmentation resulted in a higher biodegradation of total petroleum hydrocarbons (TPH) and of HMW-PAHs than with biostimulation. TPH (C14-C35) decreased by a 39.90 ± 1.99% in bioaugmented microcosms vs. a 24.17 ± 1.31% in biostimulated microcosms. As for the effect of fungal bioaugmentation on HMW-PAHs, the 5-ringed benzo(a)fluoranthene and benzo(a)pyrene were reduced by a 36% and 46%, respectively, while the 6-ringed benzoperylene decreased by a 28%, after 120 days of treatment. Biostimulated microcosm exhibited a significantly lower reduction of 5- and 6-ringed PAHs (8% and 5% respectively). Higher TPH and HMW-PAHs biodegradation levels in bioaugmented microcosms were also associated to a significant decrease in acute ecotoxicity (EC50) by Vibrio fischeri bioluminiscence inhibition assays. Molecular profiling and counting of viable hydrocarbon-degrading bacteria from soil microcosms revealed that fungal bioaugmentation promoted the growth of autochthonous active hydrocarbon-degrading bacteria. The implementation of such an approach to enhance hydrocarbon biodegradation should be considered as a novel bioremediation strategy for the treatment of the most recalcitrant and highly genotoxic hydrocarbons in aged industrially polluted soils.

Breeding farm, level of feeding and presence of antibiotics in the feed influence rabbit cecal microbiota.

BACKGROUND: The effect of the production environment and different management practices in rabbit cecal microbiota remains poorly understood. While previous studies have proved the impact of the age or the feed composition, research in the breeding farm and other animal management aspects, such as the presence of antibiotics in the feed or the level of feeding, is still needed. Characterization of microbial diversity and composition of growing rabbits raised under different conditions could help better understand the role these practices play in cecal microbial communities and how it may result in different animal performance. RESULTS: Four hundred twenty-five meat rabbits raised in two different facilities, fed under two feeding regimes (ad libitum or restricted) with feed supplemented or free of antibiotics, were selected for this study. A 16S rRNA gene-based assessment through the MiSeq Illumina sequencing platform was performed on cecal samples collected from these individuals at slaughter. Different univariate and multivariate approaches were conducted to unravel the influence of the different factors on microbial alpha diversity and composition at phylum, genus and OTU taxonomic levels. The animals raised in the facility harboring the most stable environmental conditions had greater, and less variable, microbial richness and diversity. Bootstrap univariate analyses of variance and sparse partial least squares-discriminant analyses endorsed that farm conditions exerted an important influence on rabbit microbiota since the relative abundances of many taxa were found differentially represented between both facilities at all taxonomic levels characterized. Furthermore, only five OTUs were needed to achieve a perfect classification of samples according to the facility where animals were raised. The level of feeding and the presence of antibiotics did not modify the global alpha diversity but had an impact on some bacteria relative abundances, albeit in a small number of taxa compared with farm, which is consistent with the lower sample classification power according to these factors achieved using microbial information. CONCLUSIONS: This study reveals that factors associated with the farm effect and other management factors, such as the presence of antibiotics in the diet or the feeding level, modify cecal microbial communities. It highlights the importance of offering a controlled breeding environment that reduces differences in microbial cecal composition that could be responsible for different animal performance.

Rabbit Microbiota Changes Throughout the Intestinal Tract.

To gain insight into the importance of carefully selecting the sampling area for intestinal microbiota studies, cecal and fecal microbial communities of Caldes meat rabbit were characterized. The animals involved in the study were divided in two groups according to the feed intake level they received during the fattening period; ad libitum (n = 10) or restricted to 75% of ad libitum intake (n = 11). Cecum and internal hard feces were sampled from sacrificed animals. Assessment of bacterial and archaeal populations was performed by means of Illumina sequencing of 16S rRNA gene amplicons in a MiSeq platform. A total of 596 operational taxonomic units (OTUs) were detected using QIIME software. Taxonomic assignment revealed that microbial diversity was dominated by phyla Firmicutes (76.42%), Tenericutes (7.83%), and Bacteroidetes (7.42%); kingdom Archaea was presented at low percentage (0.61%). No significant differences were detected between sampling origins in microbial diversity or richness assessed using two alpha-diversity indexes: Shannon and the observed number of OTUs. However, the analysis of variance at genus level revealed a higher presence of genera Clostridium, Anaerofustis, Blautia, Akkermansia, rc4-4, and Bacteroides in cecal samples. By contrast, genera Oscillospira and Coprococcus were found to be overrepresented in feces, suggesting that bacterial species of these genera would act as fermenters at the end of feed digestion process. At the lowest taxonomic level, 83 and 97 OTUs in feces and cecum, respectively, were differentially represented. Multivariate statistical assessment revealed that sparse partial least squares discriminant analysis (sPLS-DA) was the best approach for this purpose. Interestingly, the majority of the most discriminative OTUs selected by sPLS-DA were found to be differentially represented between sampling origins in univariate analysis. Our study provides evidence that the choice of intestinal sampling area is relevant due to important differences in some taxa's relative abundance that have been revealed between rabbits' cecal and fecal microbiota. An appropriate sampling intestinal area should be chosen in each microbiota assessment.

Correction: Neglecting the fallow season can significantly underestimate annual methane emissions in Mediterranean rice fields.

[This corrects the article DOI: 10.1371/journal.pone.0198081.].

Neglecting the fallow season can significantly underestimate annual methane emissions in Mediterranean rice fields.

Paddy rice fields are one of the most important sources of anthropogenic methane. Improving the accuracy in the CH4 budget is fundamental to identify strategies to mitigate climate change. Such improvement requires a mechanistic understanding of the complex interactions between environmental and agronomic factors determining CH4 emissions, and also the characterization of the annual temporal CH4 emissions pattern in the whole crop cycle. Hence, both the growing and fallow seasons must be included. However, most of the previous research has been based on single-factor analyses that are focused on the growing season. In order to fill this gap, a study was conducted in a Mediterranean rice agrosystem (Ebre Delta, Catalonia) following a farm-to-farm approach with the purpose of 1) evaluating the cumulative and temporal pattern of CH4 emission, and 2) conducting a multi-variate analyses to assess the associative pattern, relative contribution and temporal variation of the main explanatory variables concerning the observed CH4 emissions. Measurements of CH4 emissions and agronomic and environmental parameters in 15 commercial rice fields were monitored monthly, during a whole crop field cycle. The temporal pattern of CH4 emission followed a bi-modal distribution peaking in August and October. The cumulative annual CH4 emissions from rice fields amounted 314 kg CH4 kg ha-1, of which ca. 70% were emitted during the fallow season. The main controlling factors of the CH4 emission rate in the growing season were positive related to water level and plant cover, while soil redox was negatively related. The main controlling factors in the fallow season were water level (negatively related, conversely to the growing season), as well as straw incorporation and soil temperature (positively related). The results of this study highlight the importance of the often neglected fallow season in the accurate estimation of CH4 emissions and, thus, the necessity of measurement programs that cover the whole crop field cycle. This information is the first step for setting effective mitigation strategies based on straw and water management.

Effects of partially saturated conditions on the metabolically active microbiome and on nitrogen removal in vertical subsurface flow constructed wetlands.

Nitrogen dynamics and its association to metabolically active microbial populations were assessed in two vertical subsurface vertical flow (VF) wetlands treating urban wastewater. These VF wetlands were operated in parallel with unsaturated (UVF) and partially saturated (SVF) configurations. The SVF wetland exhibited almost 2-fold higher total nitrogen removal rate (5 g TN m-2 d-1) in relation to the UVF wetland (3 g TN m-2 d-1), as well as a low NOx-N accumulation (1 mg L-1 vs. 26 mg L-1 in SVF and UVF wetland effluents, respectively). After 6 months of operation, ammonia oxidizing prokaryotes (AOP) and nitrite oxidizing bacteria (NOB) displayed an important role in both wetlands. Oxygen availability and ammonia limiting conditions promoted shifts on the metabolically active nitrifying community within 'nitrification aggregates' of wetland biofilms. Ammonia oxidizing archaea (AOA) and Nitrospira spp. overcame ammonia oxidizing bacteria (AOB) in the oxic layers of both wetlands. Microbial quantitative and diversity assessments revealed a positive correlation between Nitrobacter and AOA, whereas Nitrospira resulted negatively correlated with Nitrobacter and AOB populations. The denitrifying gene expression was enhanced mainly in the bottom layer of the SVF wetland, in concomitance with the depletion of NOx-N from wastewater. Functional gene expression of nitrifying and denitrifying populations combined with the active microbiome diversity brought new insights on the microbial nitrogen-cycling occurring within VF wetland biofilms under different operational conditions.

Vitamin B12 effects on chlorinated methanes-degrading microcosms: Dual isotope and metabolically active microbial populations assessment.

Field-derived anoxic microcosms were used to characterize chloroform (CF) and carbon tetrachloride (CT) natural attenuation to compare it with biostimulation scenarios in which vitamin B12 was added (B12/pollutant ratio of 0.01 and 0.1) by means of by-products, carbon and chlorine compound-specific stable-isotope analysis, and the active microbial community through 16S rRNA MiSeq high-throughput sequencing. Autoclaved slurry controls discarded abiotic degradation processes. B12 catalyzed CF and CT biodegradation without the accumulation of dichloromethane, carbon disulphide, or CF. The carbon isotopic fractionation value of CF (ƐCCF) with B12 was -14±4‰, and the value for chlorine (ƐClCF) was -2.4±0.4‰. The carbon isotopic fractionation values of CT (ƐCCT) were -16±6 with B12, and -13±2‰ without B12; and the chlorine isotopic fractionation values of CT (ƐClCT) were -6±3 and -4±2‰, respectively. Acidovorax, Ancylobacter, and Pseudomonas were the most metabolically active genera, whereas Dehalobacter and Desulfitobacterium were below 0.1% of relative abundance. The dual C-Cl element isotope slope (Λ=Δδ13C/Δδ37Cl) for CF biodegradation (only detected with B12, 7±1) was similar to that reported for CF reduction by Fe(0) (8±2). Several reductive pathways might be competing in the tested CT scenarios, as evidenced by the lack of CF accumulation when B12 was added, which might be linked to a major activity of Pseudomonas stutzeri; by different chlorine apparent kinetic isotope effect values and Λ which was statistically different with and without B12 (5±1 vs 6.1±0.5), respectively. Thus, positive B12 effects such as CT and CF degradation catalyst were quantified for the first time in isotopic terms, and confirmed with the major activity of species potentially capable of their degradation. Moreover, the indirect benefits of B12 on the degradation of chlorinated ethenes were proved, creating a basis for remediation strategies in multi-contaminant polluted sites.

Effectiveness of a full-scale horizontal slow sand filter for controlling phytopathogens in recirculating hydroponics: From microbial isolation to full microbiome assessment.

The microbial disinfestation efficiency of an innovative horizontal-flow slow sand filter (HSSF) for treating nutrient solution spent from an experimental closed-loop nursery was evaluated by means of a combination of culture-dependent and independent molecular techniques. A dense inoculum of the fungal plant pathogen Fusarium oxysporum f.sp. lycopersici was applied in the fertigation system (106 cells per mL). Indigenous and introduced populations of eubacteria and fungi were assessed in the nutrient solution, the HSSF influent/effluent, and a sand bed transect by isolation on selective media, as well as by quantitative qPCR and next-generation sequencing (NGS) on target ribosomal genes. The HSSF effectively reduced viable Fusarium propagules and fungal gene content with an efficiency consistently above 99.9% (5 orders of magnitude down). On the other hand, Fusarium cells accumulated in the sand bed, indicating that physical entrapment was the main removal mechanism. The viability of retained Fusarium cells tended to decrease in time, so that treatment efficiency might be enhanced by antagonistic species from the genera Bacillus, Pseudomonas, and Trichoderma, also identified in the sand bed. Indigenous bacterial populations from the HSSF effluent were reduced by 87.2% and 99.9% in terms of colony forming units and gene counts, respectively, when compared to the influent. Furthermore, microbial populations from the HSSF effluent were different from those observed in the sand bed and the influent. In summary, the HSSF microbial disinfestation efficiency is comparable to that reported for other more intensive and costly methodologies, while allowing a significant recovery of water and nutrients.

Unraveling the active microbial populations involved in nitrogen utilization in a vertical subsurface flow constructed wetland treating urban wastewater.

The dynamics of the active microbial populations involved in nitrogen transformation in a vertical subsurface flow constructed wetland (VF) treating urban wastewater was assessed. The wetland (1.5m2) operated under average loads of 130gCODm-2d-1 and 17gTNm-2d-1 in Period I, and 80gCODm-2d-1 and 19gTNm-2d-1 in Period II. The hydraulic loading rate (HLR) was 375mmd-1 and C/N ratio was 2 in both periods. Samples for microbial characterization were collected from the filter medium (top and bottom layers) of the wetland, water influent and effluent at the end of Periods I (Jun-Oct) and II (Nov-Jan). The combination of qPCR and high-throughput sequencing (NGS, MiSeq) assessment at DNA and RNA level of 16S rRNA genes and nitrogen-based functional genes (amoA and nosZ-clade I) revealed that nitrification was associated both with ammonia-oxidizing bacteria (AOB) (Nitrosospira) and ammonia-oxidizing archaea (AOA) (Nitrososphaeraceae), and nitrite-oxidizing bacteria (NOB) such as Nitrobacter. Considering the active abundance (based in amoA transcripts), the AOA population revealed to be more stable than AOB in both periods and depths of the wetland, being less affected by the organic loading rate (OLR). Although denitrifying bacteria (nosZ copies and transcripts) were actively detected in all depths, the denitrification process was low (removal of 2gTNm-2d-1 for both periods) concomitant with NOx-N accumulation in the effluent. Overall, AOA, AOB and denitrifying bacteria (nosZ) were observed to be more active in bottom than in top layer at lower OLR (Period II). A proper design of OLR and HLR seems to be crucial to control the activity of microbial biofilms in VF wetlands on the basis of oxygen, organic-carbon and NOx-N forms, to improve their capacity for total nitrogen removal.

Unravelling the active microbial community in a thermophilic anaerobic digester-microbial electrolysis cell coupled system under different conditions.

Thermophilic anaerobic digestion (AD) of pig slurry coupled to a microbial electrolysis cell (MEC) with a recirculation loop was studied at lab-scale as a strategy to increase AD stability when submitted to organic and nitrogen overloads. The system performance was studied, with the recirculation loop both connected and disconnected, in terms of AD methane production, chemical oxygen demand removal (COD) and volatile fatty acid (VFA) concentrations. Furthermore, the microbial population was quantitatively and qualitatively assessed through DNA and RNA-based qPCR and high throughput sequencing (MiSeq), respectively to identify the RNA-based active microbial populations from the total DNA-based microbial community composition both in the AD and MEC reactors under different operational conditions. Suppression of the recirculation loop reduced the AD COD removal efficiency (from 40% to 22%) and the methane production (from 0.32 to 0.03 m3 m-3 d-1). Restoring the recirculation loop led to a methane production of 0.55 m3 m-3 d-1 concomitant with maximum MEC COD and ammonium removal efficiencies of 29% and 34%, respectively. Regarding microbial analysis, the composition of the AD and MEC anode populations differed from really active microorganisms. Desulfuromonadaceae was revealed as the most active family in the MEC (18%-19% of the RNA relative abundance), while hydrogenotrophic methanogens (Methanobacteriaceae) dominated the AD biomass.

Assessment of active methanogenic archaea in a methanol-fed upflow anaerobic sludge blanket reactor.

Methanogenic archaea enrichment of a granular sludge was undertaken in an upflow anaerobic sludge blanket (UASB) reactor fed with methanol in order to enrich methylotrophic and hydrogenotrophic methanogenic populations. A microbial community assessment, in terms of microbial composition and activity-throughout the different stages of the feeding process with methanol and acetate-was performed using specific methanogenic activity (SMA) assays, quantitative real-time polymerase chain reaction (qPCR), and high-throughput sequencing of 16S ribosomal RNA (rRNA) genes from DNA and complementary DNA (cDNA). Distinct methanogenic enrichment was revealed by qPCR of mcrA gene in the methanol-fed community, being two orders of magnitude higher with respect to the initial inoculum, achieving a final mcrA/16S rRNA ratio of 0.25. High-throughput sequencing analysis revealed that the resulting methanogenic population was mainly composed by methylotrophic archaea (Methanomethylovorans and Methanolobus genus), being also highly active according to the RNA-based assessment. SMA confirmed that the methylotrophic pathway, with a direct conversion of methanol to CH4, was the main step of methanol degradation in the UASB. The biomass from the UASB, enriched in methanogenic archaea, may bear great potential as additional inoculum for bioreactors to carry out biogas production and other related processes.

Removal of volatile fatty acids and ammonia recovery from unstable anaerobic digesters with a microbial electrolysis cell.

Continuous assays with a microbial electrolysis cell (MEC) fed with digested pig slurry were performed to evaluate its stability and robustness to malfunction periods of an anaerobic digestion (AD) reactor and its feasibility as a strategy to recover ammonia. When performing punctual pulses of volatile fatty acids (VFA) in the anode compartment of the MEC, simulating a malfunction of the AD process, an increase in the current density was produced (up to 14 times, reaching values of 3500mAm(-2)) as a result of the added chemical oxygen demand (COD), especially when acetate was used. Furthermore, ammonium diffusion from the anode to the cathode compartment was enhanced and the removal efficiency achieved up to 60% during daily basis VFA pulses. An AD-MEC combined system has proven to be a robust and stable configuration to obtain a high quality effluent, with a lower organic and ammonium content.

Overcoming organic and nitrogen overload in thermophilic anaerobic digestion of pig slurry by coupling a microbial electrolysis cell.

The combination of the anaerobic digestion (AD) process with a microbial electrolysis cell (MEC) coupled to an ammonia stripping unit as a post-treatment was assessed both in series operation, to improve the quality of the effluent, and in loop configuration recirculating the effluent, to increase the AD robustness. The MEC allowed maintaining the chemical oxygen demand removal of the whole system of 46±5% despite the AD destabilization after doubling the organic and nitrogen loads, while recovering 40±3% of ammonia. The AD-MEC system, in loop configuration, helped to recover the AD (55% increase in methane productivity) and attained a more stable and robust operation. The microbial population assessment revealed an enhancement of AD methanogenic archaea numbers and a shift in eubacterial population. The AD-MEC combined system is a promising strategy for stabilizing AD against organic and nitrogen overloads, while improving the quality of the effluent and recovering nutrients for their reutilization.