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.

Startup strategy for nitrogen removal via nitrite in a BAF system.

A biological aerated filter (BAF) pilot plant consisting of two reactors (aerobic and anoxic one) was used to determine a strategy to remove nitrogen via nitrite. RNA/DNA analysis was performed to assess microbial activity and support chemical results. In less than 13 days the pilot plant was able to remove COD and suspended solids. Nitrogen removal via nitrite pathway could not be observed until day 130 when the empty bed contact time (EBCT) was set at 0.71 h. Nitrite was detected in the aerated BAF effluent but never nitrate. qPCR of amoA gene from RNA and DNA extracts of the aerobic biofilm confirmed that ammonia oxidizing bacteria (AOB) were present from the beginning of the operation but not active. AOB activity increased with time, reaching stability from operational day 124. The combination of both, low EBCT together with high OLR, has been demonstrated to be a feasible strategy to startup a BAF to achieve nitrogen removal via nitrite.

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.

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.

Fast acquisition of a polysaccharide fermenting gut microbiome by juvenile green turtles Chelonia mydas after settlement in coastal habitats.

BACKGROUND: Tetrapods do not express hydrolases for cellulose and hemicellulose assimilation, and hence, the independent acquisition of herbivory required the establishment of new endosymbiotic relationships between tetrapods and microbes. Green turtles (Chelonia mydas) are one of the three groups of marine tetrapods with an herbivorous diet and which acquire it after several years consuming pelagic animals. We characterized the microbiota present in the feces and rectum of 24 young wild and captive green turtles from the coastal waters of Brazil, with curved carapace length ranging from 31.1 to 64.7 cm, to test the hypotheses that (1) the ontogenetic dietary shift after settlement is followed by a gradual change in the composition and diversity of the gut microbiome, (2) differences exist between the composition and diversity of the gut microbiome of green turtles from tropical and subtropical regions, and (3) the consumption of omnivorous diets modifies the gut microbiota of green turtles. RESULTS: A genomic library of 2,186,596 valid bacterial 16S rRNA reads was obtained and these sequences were grouped into 6321 different operational taxonomic units (at 97% sequence homology cutoff). The results indicated that most of the juvenile green turtles less than 45 cm of curved carapace length exhibited a fecal microbiota co-dominated by representatives of the phyla Bacteroidetes and Firmicutes and high levels of Clostridiaceae, Prophyromonas, Ruminococaceae, and Lachnospiraceae within the latter phylum. Furthermore, this was the only microbiota profile found in wild green turtles > 45 cm CCL and in most of the captive green turtles of any size feeding on a macroalgae/fish mixed diet. Nevertheless, microbial diversity increased with turtle size and was higher in turtles from tropical than from subtropical regions. CONCLUSIONS: These results indicate that juvenile green turtles from the coastal waters of Brazil had the same general microbiota, regardless of body size and origin, and suggest a fast acquisition of a polysaccharide fermenting gut microbiota by juvenile green turtles after settlement into coastal habitats.

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.

Operational, design and microbial aspects related to power production with microbial fuel cells implemented in constructed wetlands.

This work aimed at determining the amount of energy that can be harvested by implementing microbial fuel cells (MFC) in horizontal subsurface constructed wetlands (HSSF CWs) during the treatment of real domestic wastewater. To this aim, MFC were implemented in a pilot plant based on two HSSF CW, one fed with primary settled wastewater (Settler line) and the other fed with the effluent of a hydrolytic up-flow sludge blanket reactor (HUSB line). The eubacterial and archaeal community was profiled on wetland gravel, MFC electrodes and primary treated wastewater by means of 16S rRNA gene-based 454-pyrosequencing and qPCR of 16S rRNA and mcrA genes. Maximum current (219 mA/m(2)) and power (36 mW/m(2)) densities were obtained for the HUSB line. Power production pattern correlated well with water level fluctuations within the wetlands, whereas the type of primary treatment implemented had a significant impact on the diversity and relative abundance of eubacteria communities colonizing MFC. It is worth noticing the high predominance (13-16% of relative abundance) of one OTU belonging to Geobacter on active MFC of the HUSB line that was absent for the settler line MFC. Hence, MFC show promise for power production in constructed wetlands receiving the effluent of a HUSB reactor.

Role of Thiobacillus thioparus in the biodegradation of carbon disulfide in a biofilter packed with a recycled organic pelletized material.

This study reports the biodegradation of carbon disulfide (CS2) in air biofilters packed with a pelletized mixture of composted manure and sawdust. Experiments were carried out in two lab-scale (1.2 L) biofiltration units. Biofilter B was seeded with activated sludge enriched previously on CS2-degrading biomass under batch conditions, while biofilter A was left as a negative inoculation control. This inoculum was characterized by an acidic pH and sulfate accumulation, and contained Achromobacter xylosoxidans as the main putative CS2 biodegrading bacterium. Biofilter operation start-up was unsuccessfully attempted under xerophilic conditions and significant CS2 elimination was only achieved in biofilter A upon the implementation of an intermittent irrigation regime. Sustained removal efficiencies of 90-100 % at an inlet load of up to 12 g CS2 m(-3) h(-1) were reached. The CS2 removal in this biofilter was linked to the presence of the chemolithoautotrophic bacterium Thiobacillus thioparus, known among the relatively small number of species with a reported capacity of growing on CS2 as the sole energy source. DGGE molecular profiles confirmed that this microbe had become dominant in biofilter A while it was not detected in samples from biofilter B. Conventional biofilters packed with inexpensive organic materials are suited for the treatment of low-strength CS2 polluted gases (IL <12 g CS2 m(-3) h(-1)), provided that the development of the adequate microorganisms is favored, either upon enrichment or by inoculation. The importance of applying culture-independent techniques for microbial community analysis as a diagnostic tool in the biofiltration of recalcitrant compounds has been highlighted.

Fungal/bacterial interactions during the biodegradation of TEX hydrocarbons (toluene, ethylbenzene and p-xylene) in gas biofilters operated under xerophilic conditions.

The treatment of air contaminated with toluene, ethylbenzene, and p-xylene was assayed in three laboratory-scale biofilters, each consisting of two modules connected in series, packed with a pelletized organic fertilizer and inoculated with a toluene-degrading liquid enrichment culture. Biofilters were operated in parallel for 185 days in which the volumetric organic loading rate was progressively increased. The operation regime was subjected to drying out, so that packing humidity generally remained below 40%. Significant process failure occurred with ethylbenzene and p-xylene, but the toluene biofilter comparatively sustained a significant elimination capacity. Microbial community characterization by quantitative PCR and denaturing gradient gel electrophoresis showed substantial fungal enrichment in the toluene biofilter. Ribotypes identical to the well-known toluene-degrading black yeast Exophiala oligosperma (Chaetotyriales) were found among the dominant species. The microbial community structure was similar in the biofilters loaded with toluene and ethylbenzene but with p-xylene was quite specific and encompassed other chaetothyrialean fungi. Several species of Actinomycetales were found in the packing while the inoculum was dominated by representatives of the Burkholderiales and Xanthomonadales. One single fungal ribotype homologous to Acremonium kiliense was detected in the inoculum. The implications of xerophilic biofilter operation on process biosafety and efficiency are discussed.