Manganese reductive dissolution coupled to Sb mobilization in contaminated shooting range soil.

A "redox-stat" RMnR bioreactor was employed to simulate moderately reducing conditions (+ 420 mV) in Sb-contaminated shooting range soils for approximately 3 months, thermodynamically favoring Mn(IV) reduction. The impact of moderately reducing conditions on elemental mobilization (Mn, Sb, Fe) and speciation [Sb(III) versus Sb(V); Fe2+/Fe3+] was compared to a control bioreactor RCTRL without a fixed redox potential. In both bioreactors, reducing conditions were accompanied by an increase in effluent Sb(V) and Mn(II) concentrations, suggesting that Sb(V) was released through microbial reduction of Mn oxyhydroxide minerals. This was underlined by multiple linear regression analysis showing a significant (p < 0.05) relationship between Mn and Sb effluent concentrations. Mn concentration was the sole variable exhibiting a statistically significant effect on Sb in RMnR, while under the more reducing conditions in RCTRL, pH and redox potential were also significant. Analysis of the bacterial community composition revealed an increase in the genera Azoarcus, Flavisolibacter, Luteimonas, and Mesorhizobium concerning the initial soil, some of which are possible key players in the process of Sb mobilization. The overall amount of Sb released in the RMnR (10.40%) was virtually the same as in the RCTRL (10.37%), which underlines a subordinate role of anoxic processes, such as Fe-reductive dissolution, in Sb mobilization. This research underscores the central role of relatively low concentrations of Mn oxyhydroxides in influencing the fate of trace elements. Our study also demonstrates that bioreactors operated as redox-stats represent versatile tools that allow quantifying the contribution of specific mechanisms determining the fate of trace elements in contaminated soils. KEY POINTS: • "Redox-stat" reactors elucidate Sb mobilization mechanisms • Mn oxyhydroxides microbial reductive dissolution has a major role in Sb mobilization in soils under moderately reducing conditions • Despite aging the soil exhibited significant Sb mobilization potential, emphasizing persistent environmental effects.

Pollutant profile complexity governs wastewater removal of recalcitrant pharmaceuticals.

Organic pollutants are an increasing threat for wildlife and humans. Managing their removal is however complicated by the difficulties in predicting degradation rates. In this work, we demonstrate that the complexity of the pollutant profile, the set of co-existing contaminants, is a major driver of biodegradation in wastewater. We built representative assemblages out of one to five common pharmaceuticals (caffeine, atenolol, paracetamol, ibuprofen, and enalapril) selected along a gradient of biodegradability. We followed their individual removal by wastewater microbial communities. The presence of multichemical background pollution was essential for the removal of recalcitrant molecules such as ibuprofen. High-order interactions between multiple pollutants drove removal efficiency. We explain these interactions by shifts in the microbiome, with degradable molecules such as paracetamol enriching species and pathways involved in the removal of several organic pollutants. We conclude that pollutants should be treated as part of a complex system, with emerging pollutants potentially showing cascading effects and offering leverage to promote bioremediation.

Analyzing microbial communities and their biodegradation of multiple pharmaceuticals in membrane bioreactors.

Pharmaceuticals are of concern to our planet and health as they can accumulate in the environment. The impact of these biologically active compounds on ecosystems is hard to predict, and information on their biodegradation is necessary to establish sound risk assessment. Microbial communities are promising candidates for the biodegradation of pharmaceuticals such as ibuprofen, but little is known yet about their degradation capacity of multiple micropollutants at higher concentrations (100 mg/L). In this work, microbial communities were cultivated in lab-scale membrane bioreactors (MBRs) exposed to increasing concentrations of a mixture of six micropollutants (ibuprofen, diclofenac, enalapril, caffeine, atenolol, paracetamol). Key players of biodegradation were identified using a combinatorial approach of 16S rRNA sequencing and analytics. Microbial community structure changed with increasing pharmaceutical intake (from 1 to 100 mg/L) and reached a steady-state during incubation for 7 weeks on 100 mg/L. HPLC analysis revealed a fluctuating but significant degradation (30-100%) of five pollutants (caffeine, paracetamol, ibuprofen, atenolol, enalapril) by an established and stable microbial community mainly composed of Achromobacter, Cupriavidus, Pseudomonas and Leucobacter. By using the microbial community from MBR1 as inoculum for further batch culture experiments on single micropollutants (400 mg/L substrate, respectively), different active microbial consortia were obtained for each single micropollutant. Microbial genera potentially responsible for degradation of the respective micropollutant were identified, i.e. Pseudomonas sp. and Sphingobacterium sp. for ibuprofen, caffeine and paracetamol, Sphingomonas sp. for atenolol and Klebsiella sp. for enalapril. Our study demonstrates the feasibility of cultivating stable microbial communities capable of degrading simultaneously a mixture of highly concentrated pharmaceuticals in lab-scale MBRs and the identification of microbial genera potentially responsible for the degradation of specific pollutants. KEY POINTS: • Multiple pharmaceuticals were removed by stable microbial communities. • Microbial key players of five main pharmaceuticals were identified.

Functional diversity can facilitate the collapse of an undesirable ecosystem state.

Biodiversity may increase ecosystem resilience. However, we have limited understanding if this holds true for ecosystems that respond to gradual environmental change with abrupt shifts to an alternative state. We used a mathematical model of anoxic-oxic regime shifts and explored how trait diversity in three groups of bacteria influences resilience. We found that trait diversity did not always increase resilience: greater diversity in two of the groups increased but in one group decreased resilience of their preferred ecosystem state. We also found that simultaneous trait diversity in multiple groups often led to reduced or erased diversity effects. Overall, our results suggest that higher diversity can increase resilience but can also promote collapse when diversity occurs in a functional group that negatively influences the state it occurs in. We propose this mechanism as a potential management approach to facilitate the recovery of a desired ecosystem state.

Predicting the effects of multiple global change drivers on microbial communities remains challenging.

Microbial communities in many ecosystems are facing a broad range of global change drivers, such as nutrient enrichment, chemical pollution, and temperature change. These drivers can cause changes in the abundance of taxa, the composition of communities, and the properties of ecosystems. While the influence of single drivers is already described in numerous studies, the effect and predictability of multiple drivers changing simultaneously is still poorly understood. In this study, we used 240 highly replicable oxic/anoxic aquatic lab microcosms and four drivers (fertilizer, glyphosate, metal pollution, antibiotics) in all possible combinations at three different temperatures (20, 24, and 28°C) to shed light into consequences of multiple drivers on different levels of organization, ranging from species abundance to community and ecosystem parameters. We found (i) that at all levels of ecological organization, combinations of drivers can change the biological consequence and direction of effect compared to single drivers, (ii) that effects of combinations are further modified by temperature, (iii) that a larger number of drivers occurring simultaneously is often quite closely related to their effect size, and (iv) that there is little evidence that any of these effects are associated with the level of ecological organization of the state variable. These findings suggest that, at least in this experimental ecosystem approximating a stratified aquatic ecosystem, there may be relatively little scope for predicting the effects of combinations of drivers from the effects of individual drivers, or by accounting for the level of ecological organization in question, though there may be some scope for prediction based on the number of drivers that are occurring simultaneous. A priority, though also a considerable challenge, is to extend such research to consider continuous variation in the magnitude of multiple drivers acting together.

Contrasting resistance and resilience to light variation of the coupled oxic and anoxic components of an experimental microbial ecosystem.

Understanding how microbial communities of aquatic ecosystems respond to environmental change remains a critical challenge in microbial ecology. In this study, we used light-dependent oxic-anoxic micro-ecosystems to understand how the functioning and diversity of aerobic and anaerobic lake analog communities are affected by a pulse light deprivation. Continuous measurements of oxygen concentration were made and a time series of full-length 16S rRNA sequencing was used to quantify changes in alpha- and beta diversity. In the upper oxic layer, oxygen concentration decreased significantly under light reduction, but showed resilience in daily mean, minimum, and maximum after light conditions were restored to control level. Only the amplitude of diurnal fluctuations in oxygen concentrations did not recover fully, and instead tended to remain lower in treated ecosystems. Alpha diversity of the upper oxic layer communities showed a delayed increase after light conditions were restored, and was not resilient in the longer term. In contrast, alpha diversity of the anoxic lower layer communities increased during the light reduction, but was resilient in the longer term. Community composition changed significantly during light reduction, and showed resilience in the oxic layer and lack of resilience in the anoxic layer. Alpha diversity and the amplitude of daily oxygen fluctuations within and among treatments were strongly correlated, suggesting that higher diversity could lead to less variable oxygen concentrations, or vice versa. Our experiment showed that light deprivation induces multifaceted responses of community function (oxygen respiration) and structure, hence focusing on a single stability component could potentially be misleading.

Large and interacting effects of temperature and nutrient addition on stratified microbial ecosystems in a small, replicated, and liquid-dominated Winogradsky column approach.

Aquatic ecosystems are often stratified, with cyanobacteria in oxic layers and phototrophic sulfur bacteria in anoxic zones. Changes in stratification caused by the global environmental change are an ongoing concern. Increasing understanding of how such aerobic and anaerobic microbial communities, and associated abiotic conditions, respond to multifarious environmental changes is an important endeavor in microbial ecology. Insights can come from observational and experimental studies of naturally occurring stratified aquatic ecosystems, theoretical models of ecological processes, and experimental studies of replicated microbial communities in the laboratory. Here, we demonstrate a laboratory-based approach with small, replicated, and liquid-dominated Winogradsky columns, with distinct oxic/anoxic strata in a highly replicable manner. Our objective was to apply simultaneous global change scenarios (temperature, nutrient addition) on this micro-ecosystem to report how the microbial communities (full-length 16S rRNA gene seq.) and the abiotic conditions (O2 , H2 S, TOC) of the oxic/anoxic layer responded to these environmental changes. The composition of the strongly stratified microbial communities was greatly affected by temperature and by the interaction of temperature and nutrient addition, demonstrating the need of investigating global change treatments simultaneously. Especially phototrophic sulfur bacteria dominated the water column at higher temperatures and may indicate the presence of alternative stable states. We show that the establishment of such a micro-ecosystem has the potential to test global change scenarios in stratified eutrophic limnic systems.

A multi-omic screening approach for the discovery of thermoactive glycoside hydrolases.

Next-generation sequencing and computational biology have facilitated the implementation of new combinatorial screening approaches to discover novel enzymes of biotechnological interest. In this study, we describe the successful establishment of a multi-omic approach for the identification of thermostable hydrolase-encoding genes by determination of gene expression levels. We applied this combinatorial approach using an anaerobic enrichment culture from an Azorean hot spring sample grown on green coffee beans as recalcitrant substrate. An in-depth analysis of the microbial community resulted in microorganisms capable of metabolizing the selected substrate, such as the genera Caloramator, Dictyoglomus and Thermoanaerobacter as active and abundant microorganisms. To discover glycoside hydrolases, 90,342 annotated genes were screened for specific reaction types. A total number of 106 genes encoding cellulases (EC 3.2.1.4), beta-glucosidases (EC 3.2.1.21) and endo-1,4-beta-mannosidases (EC 3.2.1.78) were selected. Mapping of RNA-Seq reads to the related metagenome led to expression levels for each gene. Amongst those, 14 genes, encoding glycoside hydrolases, showed highest expression values, and were used for further cloning. Four proteins were biochemically characterized and were identified as thermoactive glycoside hydrolases with a broad substrate range. This work demonstrated that a combinatory omic approach is a suitable strategy identifying unique thermoactive enzymes from environmental samples.

Biomass-degrading glycoside hydrolases of archaeal origin.

During the last decades, the impact of hyperthermophiles and their enzymes has been intensively investigated for implementation in various high-temperature biotechnological processes. Biocatalysts of hyperthermophiles have proven to show extremely high thermo-activities and thermo-stabilities and are identified as suitable candidates for numerous industrial processes with harsh conditions, including the process of an efficient plant biomass pretreatment and conversion. Already-characterized archaea-originated glycoside hydrolases (GHs) have shown highly impressive features and numerous enzyme characterizations indicated that these biocatalysts show maximum activities at a higher temperature range compared to bacterial ones. However, compared to bacterial biomass-degrading enzymes, the number of characterized archaeal ones remains low. To discover new promising archaeal GH candidates, it is necessary to study in detail the microbiology and enzymology of extremely high-temperature habitats, ranging from terrestrial to marine hydrothermal systems. State-of-the art technologies such as sequencing of genomes and metagenomes and automated binning of genomes out of metagenomes, combined with classical microbiological culture-dependent approaches, have been successfully performed to detect novel promising biomass-degrading hyperthermozymes. In this review, we will focus on the detection, characterization and similarities of archaeal GHs and their unique characteristics. The potential of hyperthermozymes and their impact on high-temperature industrial applications have not yet been exhausted.

Microbial biofilm formation and degradation of octocrylene, a UV absorber found in sunscreen.

Octocrylene is a widely used synthetic UV absorber of sunscreens and found in several environments. Ecological consequences of the accumulation of UV filters are widely discussed. This is the first report revealing the microbial potential to transform octocrylene. A microbial community comprising four bacterial species was enriched from a landfill site using octocrylene as carbon source. From these microorganisms Mycobacterium agri and Gordonia cholesterolivorans were identified as most potent applying a new "reverse discovery" approach. This relies on the possibility that efficient strains that are already isolated and deposited can be identified through enrichment cultures. These strains formed massive biofilms on the octocrylene droplets. GC-MS analysis after cultivation for 10 days with M. agri revealed a decrease in octocrylene concentration of 19.1%. LC-MS/MS analysis was utilized in the detection and quantification of transformation products of octocrylene. M. agri thus represents an ideal candidate for bioremediation studies with octocrylene and related compounds.

Characterization of an extremely thermo-active archaeal ß-glucosidase and its activity towards glucan and mannan in concert with an endoglucanase.

A metagenome from an enrichment culture of a hydrothermal vent sample taken at Vulcano Island (Italy) was sequenced and an endoglucanase-encoding gene (vul_cel5A) was identified in a previous work. Vul_Cel5A with maximal activity at 115 °C was characterized as the most heat-active endoglucanase to date. Based on metagenome sequences, genomes were binned and bin4 included vul_cel5A as well as a putative GH1 ß-glycosidase-encoding gene (vul_bgl1A) with highest identities to sequences from the archaeal genus Thermococcus. The recombinant ß-glucosidase Vul_Bgl1A produced in E. coli BL21 pQE-80L exhibited highest activity at 105 °C and pH 7.0 (76.12 ± 5.4 U/mg, 100%) using 4NP ß-D-glucopyranoside as substrate and 61% relative activity at 120 °C. Accordingly, Vul_Bgl1A represents one of the most heat-active ß-glucosidases to date. The enzyme has a broad substrate specificity with 155% activity towards 4NP ß-D-mannopyranoside in comparison with 4NP ß-D-glucopyranoside. Moreover, nearly complete hydrolysis of cellobiose was demonstrated. The enzyme exhibited a high glucose tolerance with 26% residual activity in presence of 2 M glucose and was furthermore activated at glucose concentrations of up to 0.5 M. When the endoglucanase Vul_Cel5A and the ß-glucosidase Vul_Bgl1A were applied simultaneously at 99 °C, 158% activity towards barley ß-glucan and 215% towards mannan were achieved compared with the activity of Vul_Cel5A alone (100%). Consequently, a significant increase in glucose formation was observed when both enzymes were incubated with ß-glucan and mannan suggesting a synergistic effect. Hence, the two archaeal extremozymes are ideal candidates for complete glucan and mannan saccharification at temperatures above the boiling point of water.

Enrichment of anaerobic heterotrophic thermophiles from four Azorean hot springs revealed different community composition and genera abundances using recalcitrant substrates.

DGGE analysis combined with a metagenomic approach was used to get insights into heterotrophic anoxic enrichment cultures of four hot springs of Vale das Furnas, Portugal, using the recalcitrant substrate spent coffee ground (SCG). Parallel enrichment cultures were performed using the major components of spent coffee ground, namely arabinogalactan, galactomannan, cellulose, and proteins. DGGE revealed that heterotrophic thermophilic bacteria are highly abundant in the hydrothermal springs and significant differences in community composition depending on the substrate were observed. DNA, isolated from enrichment cultures of different locations that were grown on the same substrate were pooled, and the respective metagenomes were analyzed. Results indicated that cultures grown on recalcitrant substrate SCG consists of a totally different thermophilic community, dominated by Dictyoglomus. Enrichments with galactomannan and arabinogalactan were dominated by Thermodesulfovibrio, while cultures with casein and cellulose were dominated by Thermus. This study indicates the high potential of thermophilic bacteria degrading recalcitrant substrate such as SCG and furthermore how the accessibility to complex polymers shapes the bacterial community.

Extremely thermoactive archaeal endoglucanase from a shallow marine hydrothermal vent from Vulcano Island.

Already-characterized microbial cellulases have proven to be highly useful for industrial processes, since they can withstand harsh industrial conditions with characteristics such as high thermo- and acid stability. These properties provide promising features for the process of plant biomass degradation and biofuel generation. Nevertheless, the number of known extremely thermoactive archaeal cellulases is low. Hence, the discovery of archaeal cellulases with different characteristics is crucial for the development of efficient and sustainable biorefinery. In this work, the metagenome of a high-temperature enrichment culture from marine environment of Vulcano Island was screened for the presence of novel endoglucanase-encoding genes of archaeal origin. The ORF vul_cel5A was detected, and the deduced protein was characterized as the most thermoactive endoglucanase described to date. Vul_Cel5A was identified as a thermoactive glycoside hydrolase family 5 endoglucanase, with the highest sequence identity (72-75%) to putative endoglucanases from archaeal genera. Vul_Cel5A showed the highest activity at notable 115 °C towards barley ß-glucan (210.7 U/mg), and lichenan (209.9 U/mg), and further towards carboxymethyl cellulose (38.6 U/mg) and locust bean gum (83.0 U/mg). The endoglucanase exhibited a half-life time of 46 min at 100 °C and did not show any loss of activity after incubation for 48 h at 75 °C. Furthermore, Vul_Cel5A showed high affinity to barley ß-glucan with a Km of 0.52 mg/mL and showed tolerance against various chemical reagents. Due to the outstanding high thermoactivity and thermostability and tolerance to acidic conditions, Vul_Cel5A represents a promising novel archaeal endo-ß-glucanase for application in biorefineries for an efficient biomass pre-treatment.

Diversity of bacteria and archaea from two shallow marine hydrothermal vents from Vulcano Island.

To obtain new insights into community compositions of hyperthermophilic microorganisms, defined as having optimal growth temperatures of 80 °C and above, sediment and water samples were taken from two shallow marine hydrothermal vents (I and II) with temperatures of 100 °C at Vulcano Island, Italy. A combinatorial approach of denaturant gradient gel electrophoresis (DGGE) and metagenomic sequencing was used for microbial community analyses of the samples. In addition, enrichment cultures, growing anaerobically on selected polysaccharides such as starch and cellulose, were also analyzed by the combinatorial approach. Our results showed a high abundance of hyperthermophilic archaea, especially in sample II, and a comparable diverse archaeal community composition in both samples. In particular, the strains of the hyperthermophilic anaerobic genera Staphylothermus and Thermococcus, and strains of the aerobic hyperthermophilic genus Aeropyrum, were abundant. Regarding the bacterial community, ε-Proteobacteria, especially the genera Sulfurimonas and Sulfurovum, were highly abundant. The microbial diversity of the enrichment cultures changed significantly by showing a high dominance of archaea, particularly the genera Thermococcus and Palaeococcus, depending on the carbon source and the selected temperature.

Draft Genome Sequence of Donghicola sp. Strain KarMa, a Model Organism for Monomethylamine-Degrading Nonmethylotrophic Bacteria.

The C1-compound monomethylamine can serve as a nitrogen, carbon, and energy source for heterotrophic bacteria. The marine alphaproteobacterium Donghicola sp. strain KarMa can use monomethylamine as a source only for nitrogen and not for carbon. Its draft genome sequence is presented here and reveals putative gene clusters for the methylamine dehydrogenase and the N-methylglutamate pathways for monomethylamine metabolism.

Potential N2O Emissions from the Tanks of Bromeliads Suggest an Additional Source of N2O in the Neotropics.

We studied the propensity of the tank bromeliad Werauhia gladioliflora to emit the greenhouse gas nitrous oxide (N2O) at current and at increased N deposition levels in the range of predicted future scenarios. Potential production rates and net accumulation of N2O from tank substrate corresponded to N availability. N2O was produced in excess at all N levels due to a low level of N2O reductase activity which agreed well with a low abundance of N2O reducers compared to nitrite reducers. Transcriptional activation, however, indicated that expression of denitrification genes may be enhanced with increasing N supply eventually leading to more efficient N2O turnover with potential for adaptation of denitrifier communities to higher N levels. Our findings indicate that tank bromeliads may constitute a novel source of N2O in Neotropical forest canopies but further studies are required to understand the size and significance of in situ N2O fluxes from tank bromeliads to the environment.

Interkingdom Cross-Feeding of Ammonium from Marine Methylamine-Degrading Bacteria to the Diatom Phaeodactylum tricornutum.

Methylamines occur ubiquitously in the oceans and can serve as carbon, nitrogen, and energy sources for heterotrophic bacteria from different phylogenetic groups within the marine bacterioplankton. Diatoms, which constitute a large part of the marine phytoplankton, are believed to be incapable of using methylamines as a nitrogen source. As diatoms are typically associated with heterotrophic bacteria, the hypothesis came up that methylotrophic bacteria may provide ammonium to diatoms by degradation of methylamines. This hypothesis was investigated with the diatom Phaeodactylum tricornutum and monomethylamine (MMA) as the substrate. Bacteria supporting photoautotrophic growth of P. tricornutum with MMA as the sole nitrogen source could readily be isolated from seawater. Two strains, Donghicola sp. strain KarMa, which harbored genes for both monomethylamine dehydrogenase and the N methylglutamate pathway, and Methylophaga sp. strain M1, which catalyzed MMA oxidation by MMA dehydrogenase, were selected for further characterization. While strain M1 grew with MMA as the sole substrate, strain KarMa could utilize MMA as a nitrogen source only when, e.g., glucose was provided as a carbon source. With both strains, release of ammonium was detected during MMA utilization. In coculture with P. tricornutum, strain KarMa supported photoautotrophic growth with 2 mM MMA to the same extent as with the equimolar amount of NH4Cl. In coculture with strain M1, photoautotrophic growth of P. tricornutum was also supported, but to a much lower degree than by strain KarMa. This proof-of-principle study with a synthetic microbial community suggests that interkingdom cross-feeding of ammonium from methylamine-degrading bacteria is a contribution to phytoplankton growth which has been overlooked so far. IMPORTANCE: Interactions between diatoms and heterotrophic bacteria are important for marine carbon cycling. In this study, a novel interaction is described. Bacteria able to degrade monomethylamine, which is a ubiquitous organic nitrogen compound in marine environments, can provide ammonium to diatoms. This interkingdom metabolite transfer enables growth under photoautotrophic conditions in coculture, which would not be possible in the respective monocultures. This proof-of-principle study calls attention to a so far overlooked contribution to phytoplankton growth.