Ensifer adhaerens strain OV14 seed application enhances Triticum aestivum L. and Brassica napus L. development.

Given the challenges imposed by climate change and societal challenges, the European Union established ambitious goals as part of its Farm to Fork (F2F) strategy. Focussed on accelerating the transition to systems of sustainable food production, processing and consumption, a key element of F2F is to reduce the use of fertilisers by at least 20% and plant protection products by up to 50% by 2030. In recent years, a substantial body of research has highlighted the potential impact of microbial-based applications to support crop production practices through both biotic/abiotic stresses via maintaining or even improving yields and reducing reliance on intensive chemical inputs. Here, we have characterised the ability of a new soil-borne free-living bacterium strain Ensifer adhaerens OV14 (EaOV14) to significantly enhance crop vigour index by up to 50% for monocot (wheat, Triticum aestivum L., p < 0.0001) and by up to 40% for dicot (oilseed rape, Brassica napus L., p < 0.0001) species under in-vitro conditions (n = 360 seedlings/treatment). The beneficial effect was further studied under controlled glasshouse growing conditions (n = 60 plants/treatment) where EaOV14 induced significantly increased seed yield of spring oilseed rape compared to the controls (p < 0.0001). Moreover, using bespoke rhizoboxes, enhanced root architecture (density, roots orientation, roots thickness etc.) was observed for spring oilseed rape and winter wheat, with the median number of roots 55% and 33% higher for oilseed rape and wheat respectively, following EaOV14 seed treatment compared to the control. In addition, EaOV14 treatment increased root tip formation and root volume, suggesting the formation of a more robust root system architecture post-seed treatment. However, like other microbial formulations, the trade-offs associated with field translation, such as loss or limited functionality due to inoculum formulation or environmental distress, need further investigation. Moreover, the delivery method requires further optimisation to identify the optimal inoculum formulation that will maximise the expected beneficial impact on yield under field growing conditions.

Enzymatic response to cadmium by Impatiens glandulifera: A preliminary investigation.

This paper aims to develop our understanding of the effect of cadmium (Cd) on Impatiens glandulifera, a recently identified potential Cd hyperaccumulator. Impatiens glandulifera plants were exposed to three concentrations of Cd (20, 60 and 90 mg/kg) and were sampled at two timepoints (one and seven days) to investigate the stress response of I. glandulifera to Cd. Cd can induce oxidative stress in plants, triggering overproduction of reactive oxygen species (ROS). The level of activity of catalase (CAT) and ascorbate peroxidase (APX), two crucial antioxidant enzymes responsible for detoxifying ROS, were found to increase in a concentration dependent manner. Though there was no change observed in the level of superoxide dismutase (SOD) activity, the activity of glutathione S-transferase (GST), involved in detoxifying and sequestering Cd, increased after exposure to Cd. Cd did not appear to impact the levels of proline and photosynthetic pigments, indicating the plants weren't stressed by the presence of Cd. These results suggest that the rapid response observed in enzyme activity aid the efficacious mitigation of the toxic effects of Cd, preventing significant physiological stress in I. glandulifera.

Low-cost physicochemical treatment for removal of ammonia, phosphate and nitrate contaminants from landfill leachate.

Four low-cost materials, oyster shells, pumice stone, sand and zeolite were employed as adsorbents in an adsorption batch assays investigating the removal of ammonia, phosphate and nitrate from an aqueous solution. These compounds were chosen as they represent typical compounds found in landfill leachate (LFL). Assay performance was evaluated by the Langmuir and Freundlich adsorption isotherms. The top two materials, oyster shells and pumice stone, were employed as adsorbents in a fixed-bed column trial examining the effect of bed height and flow rate on the treatment of a synthetic LFL. The trial concluded that the highest rates of adsorption were achieved using bed heights of 20 cm with a flow rate of 5 mL min-1. After optimization, the system was employed for the treatment of LFL from Powerstown landfill, Carlow, Ireland. Ammonia and nitrate were effectively removed by both adsorption materials resulting in a reduction of influent ammonia and nitrate concentrations to below the national discharge limits set for these compounds of ≤4 mg L-1 and ≤50 mg L-1, respectively. In contrast, although similar high removal efficiencies were observed for phosphate, these rates were not maintained during the test period with overall results indicating reduced phosphate adsorption in comparison to the other compounds tested.

Microbes a Tool for the Remediation of Organotin Pollution Determined by Static Headspace Gas Chromatography-Mass Spectrometry.

Tributyltin (TBT) is one of the most toxic anthropogenic compounds introduced into the marine environment. Despite its global ban in 2008, TBT is still a problem of great concern due to its high affinity for particulate matter, providing a direct and potentially persistent route of entry into benthic sediments. Bioremediation strategies may constitute an alternative approach to conventional physicochemical methods, benefiting from the microorganism's potential to metabolize anthropogenic compounds. In this work, a simple, precise and accurate static headspace gas chromatography method was developed to investigate the ability of TBT degrading microbes in sedimentary microcosms over a period of 120 days. The proposed method was validated for linearity, repeatability, accuracy, specificity, limit of detection and limit of quantification. The method was subsequently successfully applied for the detection and quantification of TBT and degradation compounds in sediment samples on day 0, 30, 60, 90 and 120 of the experiment employing the principles of green chemistry. On day 120 the concentration of TBT remaining in the microcosms ranged between 91.91 ng/g wet wt for the least effective microbial inoculant to 52.73 ng/g wet wt for the most effective microbial inoculant from a starting concentration of 100 ng/g wet wt.

Impact of trichloroethylene exposure on the microbial diversity and protein expression in anaerobic granular biomass at 37°C and 15°C.

Granular biomass from a laboratory-scale anaerobic bioreactor trial was analysed to identify changes in microbial community structure and function in response to temperature and trichloroethylene (TCE). Two bioreactors were operated at 37°C, while two were operated at 15°C. At the time of sampling, one of each temperature pair of bioreactors was exposed to process failure-inducing concentrations of TCE (60 mg L(-1)) while the other served as a TCE-free control. Bacterial community structure was investigated using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis. Temperature was identified as an important factor for bacterial community composition, while minor differences were associated with trichloroethylene supplementation. Proteobacteria was the dominant phylum in all bioreactors, while clone library analysis revealed a higher proportion of Bacteroidetes-, Chloroflexi-, and Firmicutes-like clones at 15°C than at 37°C. Comparative metaproteomics in the presence and absence of TCE was carried out by two-dimensional gel electrophoresis (2-DGE), and 28 protein spots were identified, with putative functions related to cellular processes, including methanogenesis, glycolysis, the glyoxylate cycle, and the methyl malonyl pathway. A good agreement between metaproteomic species assignment and phylogenetic information was observed, with 10 of the identified proteins associated with members of the phylum Proteobacteria.

Temperature dependent (37-15°C) anaerobic digestion of a trichloroethylene-contaminated wastewater.

The impact of a trichloroethylene (TCE) contaminated wastewater on the microbial community structure of an anaerobic granular biomass at 15°C compared to 37°C was investigated. Four expanded granular sludge bed (EGSB) bioreactors (R1-R4) were employed in pairs at 37 and 15°C. The influents of one of each pair were supplemented with increasing concentrations of TCE (max. 60 mgl(-1)). At 37°C, stable operation was maintained with 88% COD removal and >99% TCE removal at maximum influent TCE concentrations. R3 performance decreased at influent TCE concentration of 60 mgl(-1), although TCE removal rates of >97% were recorded. Archaeal community analysis via clone library and quantitative polymerase chain reaction (qPCR) analysis, and bacterial community analysis via denaturing gradient gel electrophoresis (DGGE), indicated that temperature resulted in a greater change in community structure than the presence of TCE, and clones related to cold adaptation of biomass were identified at 15°C.

Low-temperature (7 °C) anaerobic treatment of a trichloroethylene-contaminated wastewater: microbial community development.

The feasibility of low-temperature (7 °C) anaerobic digestion for the treatment of a trichloroethylene (TCE) contaminated wastewater was investigated. Two expanded granular sludge bed (EGSB) bioreactors (R1 and R2) were employed for the mineralisation of a synthetic volatile fatty acid based wastewater at an initial organic loading rate (OLR) of 3 kg COD m(-3) d(-1), and an operating temperature of 15 °C. Successive reductions in OLR to 0.75 kg COD m(-3) d(-1), and operational temperature to 7 °C, resulted in stable bioreactor operation by day 417, with COD removal efficiency and biogas CH(4) content ≥ 74%, for both bioreactors. Subsequently, the influent to R1 was supplemented with increasing concentrations (10, 20, 30 mg l(-1)) of TCE, while R2 acted as a control. At an influent TCE concentration of 30 mg l(-1), although phase average TCE removal rates of 79% were recorded, a sustained decrease in R1 performance was observed, with COD removal of 6%, and % biogas CH(4) of 3% recorded on days 595 and 607, respectively. Specific methanogenic activity (SMA) assays identified a general shift from acetate- to hydrogen-mediated methanogenesis in both R1 and R2 biomass, while toxicity assays confirmed an increased sensitivity of the acetoclastic community in R1 to TCE and dichloroethylene (DCE), which contributed to acetate accumulation. Quantitative Polymerase Chain Reaction (qPCR) analysis of the methanogenic community confirmed the dominance of hydrogenotrophic methanogens in both R1 and R2, representing 71-89% of the total methanogenic population, however acetoclastic Methanosaeta were the dominant organisms, based on 16S rRNA gene clone library analysis of reactor biomass. The greatest change in the bacterial community, as demonstrated by UPGMA analysis of DGGE banding profiles, was observed in R1 biomass between days 417 and 609, although 88% similarity was retained between these sampling points.

Methanogenic community development in anaerobic granular bioreactors treating trichloroethylene (TCE)-contaminated wastewater at 37 °C and 15 °C.

Four expanded granular sludge bed (EGSB) bioreactors were seeded with a mesophilically-grown granular sludge and operated in duplicate for mesophilic (37 °C; R1 & R2) and low- (15°; R3 & R4) temperature treatment of a synthetic volatile fatty acid (VFA) based wastewater (3 kg COD m(-3) d(-1)) with one of each pair (R1 & R3) supplemented with increasing concentrations of trichloroethylene (TCE; 10, 20, 40, 60 mg l(-1)) and one acting as a control. Bioreactor performance was evaluated by % COD removal efficiency and % biogas methane (CH(4)) content. Quantitative Polymerase Chain Reaction (qPCR) was used to investigate the methanogenic community composition and dynamics in the bioreactors during the trial, while specific methanogenic activity (SMA) and toxicity assays were utilized to investigate the activity and TCE/dichloroethylene (DCE) toxicity thresholds of key trophic groups, respectively. At both 37 °C and 15 °C, TCE levels of 60 mg l(-1) resulted in the decline of % COD removal efficiencies to 29% (Day 235) and 37% (Day 238), respectively, and in % biogas CH(4) to 54% (Day 235) and 5% (Day 238), respectively. Despite the inhibitory effect of TCE on the anaerobic digestion process, the main drivers influencing methanogenic community development, as determined by qPCR and Non-metric multidimensional scaling analysis, were (i) wastewater composition and (ii) operating temperature. At the apical TCE concentration both SMA and qPCR of methanogenic archaea suggested that acetoclastic methanogens were somewhat inhibited by the presence of TCE and/or its degradation derivatives, while competition by dechlorinating organisms may have limited the availability of H(2) for hydrogenotrophic methanogenesis. In addition, there appeared to be an inverse correlation between SMA levels and TCE tolerance, a finding that was supported by the analysis of the inhibitory effect of TCE on two additional biomass sources. The results indicate that low-temperature anaerobic digestion is a feasible approach for the treatment of TCE-containing wastewater.

Perturbation-independent community development in low-temperature anaerobic biological wastewater treatment bioreactors.

The reproducibility and stability of low- temperature anaerobic wastewater treatment systems undergoing transient perturbations was investigated. Three identical anaerobic expanded granular sludge bed-based bioreactors were used to degrade a volatile fatty acid and glucose-based wastewater under sub-ambient (15 degrees C) conditions. The effect of a variety of environmental perturbations on bioreactor performance was assessed by chemical oxygen demand removal. Temporal microbial community development was monitored by denaturation gradient gel electrophoresis (DGGE) of 16S rRNA genes extracted from sludge granules. Methanogenic activity was monitored using specific methanogenic activity assays. Bioreactor performance and microbial population dynamics were each well replicated between both experimental bioreactors and the control bioreactor prior to, and after the implementation of most of the applied perturbations. Gene fingerprinting data indicated that Methanosaeta sp. were the persistent, keystone members of the archaeal community, and likely were pivotal for the physical stability and maintenance of the granular biofilms. Cluster analyses of DGGE data suggested that temporal shifts in microbial community structure were predominantly independent of the applied perturbations.

Psychrophilic methanogenic community development during long-term cultivation of anaerobic granular biofilms.

Granular biomass was temporally sampled from a cold (4-15 degrees C) anaerobic bioreactor, which was inoculated with mesophilic biomass and used to treat industrial wastewater in a long-term (3.4 year) study. Data from 16S rRNA gene clone libraries, quantitative PCR and terminal restriction fragment length polymorphism analyses indicated that microbial community structure was dynamic, with shifts in the archaeal and bacterial communities' structures observed following start-up and during temperature decreases from 15 to 9.5 degrees C (phase 1). Specifically, the relative abundance of architecturally important Methanosaeta-like (acetoclastic) methanogens decreased, which was concomitant with granule disintegration and the development of a putatively psychrophilic hydrogenotrophic methanogenic community. Genetic fingerprinting suggested the development of a psychroactive methanogenic community between 4 and 10 degrees C (phase 2), which was dominated by acetogenic bacteria and Methanocorpusculum-like (hydrogenotrophic) methanogens. High levels of Methanosaeta-like acetoclastic methanogens and granular biofilm integrity were maintained during phase 2. Overall, decreasing temperature resulted in distinctly altered microbial community structure during phase 1, and the development of a less dynamic psychroactive methanogenic consortium during phase 2. Moreover, psychrophilic H(2)-oxidizing methanogens emerged as important members of the psychroactive consortia after >1200 days of low-temperature cultivation. The data suggest that prolonged psychrophilic cultivation of mesophilic biomass can establish a well-functioning psychroactive methanogenic consortium, thus highlighting the potential of low-temperature anaerobic digestion technology.

Bioaugmentation of UASB reactors with immobilized Sulfurospirillum barnesii for simultaneous selenate and nitrate removal.

Whole-cell immobilization of selenate-respiring Sulfurospirillum barnesii in polyacrylamide gels was investigated to allow the treatment of selenate contaminated (790 microg Se x L(-1)) synthetic wastewater with a high molar excess of nitrate (1,500 times) and sulfate (200 times). Gel-immobilized S. barnesii cells were used to inoculate a mesophilic (30 degrees C) bioreactor fed with lactate as electron donor at an organic loading rate of 5 g chemical oxygen demand (COD) x L(-1) day(-1). Selenate was reduced efficiently (>97%) in the nitrate and sulfate fed bioreactor, and a minimal effluent concentration of 39 microg Se x L(-1) was obtained. Scanning electron microscopy with energy dispersive X-ray (SEM-EDX) analysis revealed spherical bioprecipitates of

Effect of seed sludge and operation conditions on performance and archaeal community structure of low-temperature anaerobic solvent-degrading bioreactors.

Two laboratory-scale expanded granular sludge bed (EGSB) anaerobic bioreactors (R1 and R2) were inoculated with biomass from different mesophilic (37 degrees C) treatment plants, and used for the treatment of an organic solvent-based wastewater at 9-14 degrees C at applied organic loading rates (OLRs) of 1.2-3.6kg chemical oxygen demand (COD)m(-3)d(-1). Replicated treatment performance was observed at 10-14 degrees C, which suggested the feasibility of the process at pilot-scale. Stable and efficient COD removal, along with high methane productivity, was demonstrated at 9 degrees C at an applied OLR of 2.4kgCODm(-3)d(-1). Clonal libraries and fluorescence in situ hybridization (FISH) indicated that the seed sludges were dominated (>60%) by acetoclastic Methanosaeta-like organisms. Specific methanogenic activity (SMA) profiles indicated shifts in the physiological profiles of R1 and R2 biomass, including the development of psychrotolerant methanogenic activity. Acetoclastic methanogenesis represented the primary route of methane production in R1 and R2, which is in contrast with several previous reports from low-temperature bioreactor trials. A reduction in the abundance of Methanosaeta-like clones (R2), along with the detection of hydrogenotrophic methanogenic species, coincided with altered granule (sludge) morphology and the development of hydrogenotrophic SMA after prolonged operation at 9 degrees C.

Temporal microbial diversity changes in solvent-degrading anaerobic granular sludge from low-temperature (15 degrees C) wastewater treatment bioreactors.

Anaerobic sludge granules were obtained from laboratory-scale anaerobic bioreactors used to treat pharmaceutical-like (methanol-, acetone- and propanol-contaminated) wastewater under low-temperature conditions (15 degrees C). The microbial diversity and diversity changes of the sludge samples were ascertained by applying 16S rRNA gene cloning and terminal restriction fragment length polymorphism (TRFLP) analyses, respectively, and using sludge samples from the inoculum, throughout and at the conclusion of the bioreactor trial. Data from genetic fingerprinting correlated well with those from physiological activity assays of the reactor biomass. Specifically, for example, TRFLP profiles indicated the dominance of hydrogenotrophic methanogens within the archaeal community, thus supporting the findings of specific methanogenic activity measurements. TRFLP data supported the hypothesis that the deviation between the replicated reactors, in terms of treatment efficiency, was associated with succession within the microbial communities present, and indicated that community development was linked to both operating temperature and wastewater composition. Fluorescence in situ hybridization (FISH) was also applied, to quantitatively assess the abundance of selected microbial groups, and revealed the underestimation of the abundance Methanosarcina by gene cloning analysis and demonstrated the spatial arrangement of these organisms within the architecture of the low-temperature solvent-degrading anaerobic biofilms.

Low-temperature anaerobic biological treatment of toluene-containing wastewater.

Two expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactors, R1 and R2, were operated at 15 degrees C for the treatment of toluene-contaminated volatile fatty acid-based wastewater. The seed inoculum and the R1 reactor were unexposed to toluene, prior to and during the trial, respectively. Both reactors were operated at a hydraulic retention time of 24h at applied organic loading rates of 0.71-1.43kg chemical oxygen demand (COD)m(-3)d(-1). Toluene was supplemented to the R2 influent at concentrations of 5-104 mg toluenel(-1) (solubilised in ethanol). Bioreactor performance was evaluated by COD and toluene removal efficiency, and the methane content of biogas (%). Specific methanogenic activity and toxicity assays were employed to investigate the activity and toluene toxicity thresholds of key trophic groups, respectively, within the seed and reactor biomass samples. COD and toluene removal efficiencies of 70-90% and 55-99%, respectively, were achieved during the 630-d trial. Metabolic assays suggested that a psychrotolerant H(2)/CO(2)-utilizing methanogenic community developed in the toluene-degrading biomass. The results indicate the viability of low-temperature anaerobic digestion for the treatment of wastewater containing toluene.

New low-temperature applications of anaerobic wastewater treatment.

Low-temperature or psychrophilic (<20 degrees C) anaerobic biological treatment of simple industrial wastewaters has recently been proven feasible as an alternative to more expensive mesophilic (ca. 37 degrees C) technology. We implemented novel expanded granular sludge bed (EGSB)-based bioreactor designs for 27 psychrophilic anaerobic digestion (PAD) trials for the treatment of a broad range of simple and complex synthetic wastewaters representing dairy, food-processing and pharmaceutical sector effluents. A variety of operating parameters, such as hydraulic retention time, organic and volumetric loading rates and upflow velocity, were tested. Chemical oxygen demand (COD) removal efficiencies were recorded, which were comparable to previous mesophilic trials. Specific methanogenic activity, toxicity and biodegradability batch assays were employed to monitor the metabolic capabilities of microbial consortia in anaerobic reactors. The prevalence of psychrotolerant communities was observed and psychrophilic populations were detected in two of the reactors. The potential of PAD with respect to global sustainable development is discussed.

Accessing the black box of microbial diversity and ecophysiology: recent advances through polyphasic experiments.

The microbial ecology of a range of anaerobic biological assemblages (granular sludge) from full- and laboratory-scale wastewater treatment bioreactors, and of crop-growing and peat soils, was determined using a variety of 16S rRNA gene-based techniques, including clone library, terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis (DGGE) analyses. Fluorescent in situ hybridization (FISH) using 16S rRNA gene-targeted probes was employed to complete a "full-cycle rRNA approach" with selected biomass. Genetic fingerprinting (TRFLP and DGGE) was effectively used to elucidate community structure-crop relationships, and to detect and monitor trends in bioreactor sludge and specific enrichment cultures of peat soil. Greater diversity was resolved within bacterial than within archaeal communities, and unexpected reservoirs of uncultured Crenarchaeota were detected in sludge granules. Advanced radiotracer incubations and micro-beta imaging were employed in conjunction with FISH to elucidate the eco-functionalism of these organisms. Crenarchaeota clusters were identified in close associated with methanogenic Archaea and both were localised with acetate uptake in biofilm structure.

Low-temperature anaerobic biological treatment of solvent-containing pharmaceutical wastewater.

Low-temperature or psychrophilic (<20 degrees C) anaerobic digestion (PAD) has recently been demonstrated as a cost-effective option for the treatment of a range of wastewater categories. The aim of this work was 2-fold: (1) to screen three anaerobic sludges, obtained from full-scale reactors, with respect to suitability for PAD of pharmaceutical-like, solvent-contaminated wastewater; (2) to assess the feasibility of PAD of this wastewater category. Toxicity thresholds of key trophic groups within three candidate biomass samples were assessed against solvents prevalent in pharmaceutical wastewaters (propanol, methanol and acetone). Specific methanogenic activity (SMA) assays indicated that the metabolic optimum of each candidate biomass was within the mesophilic range. One biomass sample exhibited higher SMA assays than the other candidate samples and was also the sample least methanogenically inhibited by the addition of solvents to batch cultures. This sludge was selected as the biomass of choice for laboratory-scale trials. Two identical expanded granular sludge bed (EGSB)-based anaerobic reactors were used for the treatment of solvent-contaminated wastewater at 15 degrees C, and at applied organic loading rates (OLRs) of 5-20 kg chemical oxygen demand (COD) m(-3)d(-1). COD removal efficiencies of 60-70% were achieved during the 450 day trial. In addition, SMA assays carried out at the conclusion of the trial indicated the development of a putatively psychrophilic hydrogenotrophic methanogenic community.