Fe(II)Cl2 amendment suppresses pond methane emissions by stimulating iron-dependent anaerobic oxidation of methane.

Aquatic ecosystems are large contributors to global methane (CH4) emissions. Eutrophication significantly enhances CH4-production as it stimulates methanogenesis. Mitigation measures aimed at reducing eutrophication, such as the addition of metal salts to immobilize phosphate (PO43-), are now common practice. However, the effects of such remedies on methanogenic and methanotrophic communities-and therefore on CH4-cycling-remain largely unexplored. Here, we demonstrate that Fe(II)Cl2 addition, used as PO43- binder, differentially affected microbial CH4 cycling-processes in field experiments and batch incubations. In the field experiments, carried out in enclosures in a eutrophic pond, Fe(II)Cl2 application lowered in-situ CH4 emissions by lowering net CH4-production, while sediment aerobic CH4-oxidation rates-as found in batch incubations of sediment from the enclosures-did not differ from control. In Fe(II)Cl2-treated sediments, a decrease in net CH4-production rates could be attributed to the stimulation of iron-dependent anaerobic CH4-oxidation (Fe-AOM). In batch incubations, anaerobic CH4-oxidation and Fe(II)-production started immediately after CH4 addition, indicating Fe-AOM, likely enabled by favorable indigenous iron cycling conditions and the present methanotroph community in the pond sediment. 16S rRNA sequencing data confirmed the presence of anaerobic CH4-oxidizing archaea and both iron-reducing and iron-oxidizing bacteria in the tested sediments. Thus, besides combatting eutrophication, Fe(II)Cl2 application can mitigate CH4 emissions by reducing microbial net CH4-production and stimulating Fe-AOM.

Seasonal dynamics of the microbial methane filter in the water column of a eutrophic coastal basin.

In coastal waters, methane-oxidizing bacteria (MOB) can form a methane biofilter and mitigate methane emissions. The metabolism of these MOBs is versatile, and the resilience to changing oxygen concentrations is potentially high. It is still unclear how seasonal changes in oxygen availability and water column chemistry affect the functioning of the methane biofilter and MOB community composition. Here, we determined water column methane and oxygen depth profiles, the methanotrophic community structure, methane oxidation potential, and water-air methane fluxes of a eutrophic marine basin during summer stratification and in the mixed water in spring and autumn. In spring, the MOB diversity and relative abundance were low. Yet, MOB formed a methane biofilter with up to 9% relative abundance and vertical niche partitioning during summer stratification. The vertical distribution and potential methane oxidation of MOB did not follow the upward shift of the oxycline during summer, and water-air fluxes remained below 0.6 mmol m-2 d-1. Together, this suggests active methane removal by MOB in the anoxic water. Surprisingly, with a weaker stratification, and therefore potentially increased oxygen supply, methane oxidation rates decreased, and water-air methane fluxes increased. Thus, despite the potential resilience of the MOB community, seasonal water column dynamics significantly influence methane removal.

CO2, CH4, and N2O emissions from dredged material exposed to drying and zeolite addition under field and laboratory conditions.

Dredging, the removal of sediment from water courses, is generally conducted to maintain their navigability and to improve water quality. Recent studies indicate that dredging can significantly reduce aquatic greenhouse gas (GHG) emissions. These studies, however, do not consider the potential emission from the dredged material (sludge) in the depot. In addition, it is unknown if and how GHG emissions from sludge depots can be reduced. Here we present spatiotemporal variations of carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) fluxes, as well as environmental variables from a sludge depot located in the Netherlands. Measurements were conducted monthly from the time the depot was filled until the sludge was dry and the depot was abolished. We also experimentally assessed the GHG mitigation potential of 1) keeping the sludge permanently inundated, and 2) the addition of different amounts of zeolite to increase sludge nitrogen binding capacity to reduce N2O emissions. In the depot and in the laboratory, a decrease in moisture content coincided with increased CO2 and N2O emissions while CH4 emissions decreased. We observed that permanent inundation reduced emissions (∼4 times less CO2-eq than in drying sludge). Adding zeolite lowered N2O fluxes from permanently inundated sludge but did not reduce total GHG emissions. During the depot's operational period, average CO2, CH4, and N2O fluxes were 5078, 27, and 5 mg m-2 d-1, respectively. GHG emissions from drying sludge occurred mainly in the form of CO2 (73% of the total CO2-eq emissions), with average GHG emission rates comparable to those reported for ditches and ponds. We estimate that approximately 14 tons of CO2-eq were emitted from the 0.011 km2 depot, which contained ∼20,000 m3 of sludge, during its entire operational period, and we argue that more studies are needed, considering different sludge origins, to expand our understanding of sludge depots.

Polishing wastewater effluent using plants: floating plants perform better than submerged plants in both nutrient removal and reduction of greenhouse gas emission.

While research on aquatic plants used in treatment wetlands is abundant, little is known about the use of plants in hydroponic ecological wastewater treatment, and its simultaneous effect on greenhouse gas (GHG) emissions. Here, we assess the effectiveness of floating and submerged plants in removing nutrients and preventing GHG emissions from wastewater effluent. We grew two species of floating plants, Azolla filiculoides and Lemna minor, and two species of submerged plants, Ceratophyllum demersum and Callitriche platycarpa, on a batch of domestic wastewater effluent without any solid substrate. In these systems, we monitored nitrogen and phosphorus removal and fluxes of CO2, CH4 and N2O, for 2 weeks. In general, floating plants produced the most biomass, whereas submerged plants were rapidly overgrown by filamentous algae. Floating plants removed nutrients most efficiently; both floating species removed 100% of the phosphate while Lemna also removed 97-100% of the inorganic nitrogen, as opposed to a removal of 81-88% in submerged plants with algae treatments. Moreover, aquaria covered by floating plants had roughly three times higher GHG uptake than the treatments with submerged plants or controls without plants. Thus, effluent polishing by floating plants can be a promising avenue for climate-smart wastewater polishing.

Versatile methanotrophs form an active methane biofilter in the oxycline of a seasonally stratified coastal basin.

The potential and drivers of microbial methane removal in the water column of seasonally stratified coastal ecosystems and the importance of the methanotrophic community composition for ecosystem functioning are not well explored. Here, we combined depth profiles of oxygen and methane with 16S rRNA gene amplicon sequencing, metagenomics and methane oxidation rates at discrete depths in a stratified coastal marine system (Lake Grevelingen, The Netherlands). Three amplicon sequence variants (ASVs) belonging to different genera of aerobic Methylomonadaceae and the corresponding three methanotrophic metagenome-assembled genomes (MOB-MAGs) were retrieved by 16S rRNA sequencing and metagenomic analysis, respectively. The abundances of the different methanotrophic ASVs and MOB-MAGs peaked at different depths along the methane oxygen counter-gradient and the MOB-MAGs show a quite diverse genomic potential regarding oxygen metabolism, partial denitrification and sulphur metabolism. Moreover, potential aerobic methane oxidation rates indicated high methanotrophic activity throughout the methane oxygen counter-gradient, even at depths with low in situ methane or oxygen concentration. This suggests that niche-partitioning with high genomic versatility of the present Methylomonadaceae might contribute to the functional resilience of the methanotrophic community and ultimately the efficiency of methane removal in the stratified water column of a marine basin.

Nitrate-dependent anaerobic methane oxidation (N-DAMO) as a bioremediation strategy for waters affected by agricultural runoff.

Agricultural drainage ditches are subjected to high anthropogenic nitrogen input, leading to eutrophication and greenhouse gas emissions. Nitrate-dependent anaerobic methane oxidation (N-DAMO) could be a promising remediation strategy to remove methane (CH4) and nitrate (NO3-) simultaneously. Therefore, we aimed to evaluate the potential of N-DAMO to remove excess NO3- and decrease CH4 release from agricultural drainage ditches. Microcosm experiments were conducted using sediment and surface water collected from three different sites: a sandy-clay ditch (SCD), a freshwater-fed peatland ditch (FPD), and a brackish peatland ditch (BPD). The microcosms were inoculated with an N-DAMO enrichment culture dominated by Candidatus Methanoperedens and Candidatus Methylomirabilis and supplemented with 13CH4 and 15NO3-. A significant decrease in CH4 and NO3- concentration was only observed in the BPD sediment. In freshwater sediments (FPD and SCD), the effect of N-DAMO inoculation on CH4 and NO3- removal was negligible, likely because N-DAMO microorganisms were outcompeted by heterotrophic denitrifiers consuming NO3- much faster. Overall, our results suggest that bioaugmentation with N-DAMO might be a potential strategy for decreasing NO3- concentrations and CH4 emission in brackish ecosystems with increasing agricultural activities where the native microbial community is incapable of efficient denitrification.

Phosphorus control and dredging decrease methane emissions from shallow lakes.

Freshwater ecosystems are an important source of the greenhouse gas methane (CH4), and their emissions are expected to increase due to eutrophication. Two commonly applied management techniques to reduce eutrophication are the addition of phosphate-binding lanthanum modified bentonite (LMB, trademark Phoslock©) and dredging, but their effect on CH4 emissions is still poorly understood. Here, this study researched how LMB and dredging affected CH4 emissions using a full-factorial mesocosm design monitored for 18 months. The effect was tested by measuring diffusive and ebullitive CH4 fluxes, plant community composition, methanogen and methanotroph activity and community composition, and a range of physicochemical water and sediment variables. LMB addition decreased total CH4 emissions, while dredging showed a trend towards decreasing CH4 emissions. Total CH4 emissions in all mesocosms were much higher in the summer of the second year, likely because of higher algal decomposition and organic matter availability. First, LMB addition lowered CH4 emissions by decreasing P-availability, which reduced coverage of the floating fern Azolla filiculoides, and thereby prevented anoxia and decreased surface water NH4+ concentrations, lowering CH4 production rates. Second, dredging decreased CH4 emissions in the first summer, possibly it removed the methanogenic community, and in the second year by preventing autumn and winter die-off of the rooted macrophyte Potamogeton cripsus. Finally, methanogen community composition was related to surface water NH4+ and O2, and porewater total phosphorus, while methanotroph community composition was related to organic matter content. To conclude, LMB addition and dredging not only improve water quality, but also decrease CH4 emissions, mitigating climate change.

Warming and eutrophication interactively drive changes in the methane-oxidizing community of shallow lakes.

Freshwater ecosystems are the largest natural source of the greenhouse gas methane (CH4), with shallow lakes a particular hot spot. Eutrophication and warming generally increase lake CH4 emissions but their impacts on the sole biological methane sink-methane oxidation-and methane-oxidizer community dynamics are poorly understood. We used the world's longest-running freshwater climate-change mesocosm experiment to determine how methane-oxidizing bacterial (MOB) abundance and composition, and methane oxidation potential in the sediment respond to eutrophication, short-term nitrogen addition and warming. After nitrogen addition, MOB abundance and methane oxidation potential increased, while warming increased MOB abundance without altering methane oxidation potential. MOB community composition was driven by both temperature and nutrient availability. Eutrophication increased relative abundance of type I MOB Methyloparacoccus. Warming favoured type II MOB Methylocystis over type I MOB Methylomonadaceae, shifting the MOB community from type I dominance to type I and II co-dominance, thereby altering MOB community traits involved in growth and stress-responses. This shift to slower-growing MOB may explain why higher MOB abundance in warmed mesocosms did not coincide with higher methane oxidation potential. Overall, we show that eutrophication and warming differentially change the MOB community, resulting in an altered ability to mitigate CH4 emissions from shallow lakes.

The Water Hyacinth Microbiome: Link Between Carbon Turnover and Nutrient Cycling.

Water hyacinth (WH), a large floating plant, plays an important role in the biogeochemistry and ecology of many freshwaters globally. Its biogeochemical impact on wetland functioning is strongly mediated by the microbiome associated with its roots. However, little is known about the structure and function of this WH rhizobiome and its relation to wetland ecosystem functioning. Here, we unveil the core and transient rhizobiomes of WH and their key biogeochemical functions in two of the world's largest wetlands: the Amazon and the Pantanal. WH hosts a highly diverse microbial community shaped by spatiotemporal changes. Proteobacteria lineages were most common, followed by Actinobacteria and Planctomycetes. Deltaproteobacteria and Sphingobacteriia predominated in the core microbiome, potentially associated with polysaccharide degradation and fermentation of plant-derived carbon. Conversely, a plethora of lineages were transient, including highly abundant Acinetobacter, Acidobacteria subgroup 6, and methanotrophs, thus assuring diverse taxonomic signatures in the two different wetlands. Our findings point out that methanogenesis is a key driver of, and proxy for, community structure, especially during seasonal plant decline. We provide ecologically relevant insights into the WH microbiome, which is a key element linking plant-associated carbon turnover with other biogeochemical fluxes in tropical wetlands.

Local Functioning, Landscape Structuring: Drivers of Soil Microbial Community Structure and Function in Peatlands.

Agricultural peatlands are essential for a myriad of ecosystem functions and play an important role in the global carbon (C) cycle through C sequestration. Management of these agricultural peatlands takes place at different spatial scales, ranging from local to landscape management, and drivers of soil microbial community structure and function may be scale-dependent. Effective management for an optimal biogeochemical functioning thus requires knowledge of the drivers on soil microbial community structure and functioning, as well as the spatial scales upon which they are influenced. During two field campaigns, we examined the importance of different drivers (i.e., soil characteristics, nutrient management, vegetation composition) at two spatial scales (local vs. landscape) for, respectively, the soil microbial community structure (determined by PLFA) and soil microbial community functional capacity (as assessed by CLPP) in agricultural peatlands. First, we show by an analysis of PLFA profiles that the total microbial biomass changes with soil moisture and relative C:P nutrient availability. Secondly, we showed that soil communities are controlled by a distinct set of drivers at the local, as opposed to landscape, scale. Community structure was found to be markedly different between areas, in contrast to community function which showed high variability within areas. We further found that microbial structure appears to be controlled more at a landscape scale by nutrient-related variables, whereas microbial functional capacity is driven locally through plant community feedbacks. Optimal management strategies within such peatlands should therefore consider the scale-dependent action of soil microbial community drivers, for example by first optimizing microbial structure at the landscape scale by targeted areal management, and then optimizing soil microbial function by local vegetation management.

Resistance and Recovery of Methane-Oxidizing Communities Depends on Stress Regime and History; A Microcosm Study.

Although soil microbes are responsible for important ecosystem functions, and soils are under increasing environmental pressure, little is known about their resistance and resilience to multiple stressors. Here, we test resistance and recovery of soil methane-oxidizing communities to two different, repeated, perturbations: soil drying, ammonium addition and their combination. In replicated soil microcosms we measured methane oxidation before and after perturbations, while monitoring microbial abundance and community composition using quantitative PCR assays for the bacterial 16S rRNA and pmoA gene, and sequencing of the bacterial 16S rRNA gene. Although microbial community composition changed after soil drying, methane oxidation rates recovered, even after four desiccation events. Moreover, microcosms subjected to soil drying recovered significantly better from ammonium addition compared to microcosms not subjected to soil drying. Our results show the flexibility of microbial communities, even if abundances of dominant populations drop, ecosystem functions can recover. In addition, a history of stress may induce changes in community composition and functioning, which may in turn affect its future tolerance to different stressors.

Environmental legacy contributes to the resilience of methane consumption in a laboratory microcosm system.

The increase of extreme drought and precipitation events due to climate change will alter microbial processes. Perturbation experiments demonstrated that microbes are sensitive to environmental alterations. However, only little is known on the legacy effects in microbial systems. Here, we designed a laboratory microcosm experiment using aerobic methane-consuming communities as a model system to test basic principles of microbial resilience and the role of changes in biomass and the presence of non-methanotrophic microbes in this process. We focused on enrichments from soil, sediment, and water reflecting communities with different legacy with respect to exposure to drought. Recovery rates, a recently proposed early warning indicator of a critical transition, were utilized as a measure to detect resilience loss of methane consumption during a series of dry/wet cycle perturbations. We observed a slowed recovery of enrichments originating from water samples, which suggests that the community's legacy with a perturbation is a contributing factor for the resilience of microbial functioning.

Successive DNA extractions improve characterization of soil microbial communities.

Currently, characterization of soil microbial communities relies heavily on the use of molecular approaches. Independently of the approach used, soil DNA extraction is a crucial step, and success of downstream procedures will depend on how well DNA extraction was performed. Often, studies describing and comparing soil microbial communities are based on a single DNA extraction, which may not lead to a representative recovery of DNA from all organisms present in the soil. The use of successive DNA extractions might improve soil microbial characterization, but the benefit of this approach has only been limitedly studied. To determine whether successive DNA extractions of the same soil sample would lead to different observations in terms of microbial abundance and community composition, we performed three successive extractions, with two widely used commercial kits, on a range of clay and sandy soils. Successive extractions increased DNA yield considerably (1-374%), as well as total bacterial and fungal abundances in most of the soil samples. Analysis of the 16S and 18S ribosomal RNA genes using 454-pyrosequencing, revealed that microbial community composition (taxonomic groups) observed in the successive DNA extractions were similar. However, successive DNA extractions did reveal several additional microbial groups. For some soil samples, shifts in microbial community composition were observed, mainly due to shifts in relative abundance of a number of microbial groups. Our results highlight that performing successive DNA extractions optimize DNA yield, and can lead to a better picture of overall community composition.

Biotic Interactions in Microbial Communities as Modulators of Biogeochemical Processes: Methanotrophy as a Model System.

Microbial interaction is an integral component of microbial ecology studies, yet the role, extent, and relevance of microbial interaction in community functioning remains unclear, particularly in the context of global biogeochemical cycles. While many studies have shed light on the physico-chemical cues affecting specific processes, (micro)biotic controls and interactions potentially steering microbial communities leading to altered functioning are less known. Yet, recent accumulating evidence suggests that the concerted actions of a community can be significantly different from the combined effects of individual microorganisms, giving rise to emergent properties. Here, we exemplify the importance of microbial interaction for ecosystem processes by analysis of a reasonably well-understood microbial guild, namely, aerobic methane-oxidizing bacteria (MOB). We reviewed the literature which provided compelling evidence for the relevance of microbial interaction in modulating methane oxidation. Support for microbial associations within methane-fed communities is sought by a re-analysis of literature data derived from stable isotope probing studies of various complex environmental settings. Putative positive interactions between active MOB and other microbes were assessed by a correlation network-based analysis with datasets covering diverse environments where closely interacting members of a consortium can potentially alter the methane oxidation activity. Although, methanotrophy is used as a model system, the fundamentals of our postulations may be applicable to other microbial guilds mediating other biogeochemical processes.

Recovery rates reflect distance to a tipping point in a living system.

Tipping points, at which complex systems can shift abruptly from one state to another, are notoriously difficult to predict. Theory proposes that early warning signals may be based on the phenomenon that recovery rates from small perturbations should tend to zero when approaching a tipping point; however, evidence that this happens in living systems is lacking. Here we test such 'critical slowing down' using a microcosm in which photo-inhibition drives a cyanobacterial population to a classical tipping point when a critical light level is exceeded. We show that over a large range of conditions, recovery from small perturbations becomes slower as the system comes closer to the critical point. In addition, autocorrelation in the subtle fluctuations of the system's state rose towards the tipping point, supporting the idea that this metric can be used as an indirect indicator of slowing down. Although stochasticity prohibits prediction of the timing of critical transitions, our results suggest that indicators of slowing down may be used to rank complex systems on a broad scale from resilient to fragile.

Warming can boost denitrification disproportionately due to altered oxygen dynamics.

BACKGROUND: Global warming and the alteration of the global nitrogen cycle are major anthropogenic threats to the environment. Denitrification, the biological conversion of nitrate to gaseous nitrogen, removes a substantial fraction of the nitrogen from aquatic ecosystems, and can therefore help to reduce eutrophication effects. However, potential responses of denitrification to warming are poorly understood. Although several studies have reported increased denitrification rates with rising temperature, the impact of temperature on denitrification seems to vary widely between systems. METHODOLOGY/PRINCIPAL FINDINGS: We explored the effects of warming on denitrification rates using microcosm experiments, field measurements and a simple model approach. Our results suggest that a three degree temperature rise will double denitrification rates. By performing experiments at fixed oxygen concentrations as well as with oxygen concentrations varying freely with temperature, we demonstrate that this strong temperature dependence of denitrification can be explained by a systematic decrease of oxygen concentrations with rising temperature. Warming decreases oxygen concentrations due to reduced solubility, and more importantly, because respiration rates rise more steeply with temperature than photosynthesis. CONCLUSIONS/SIGNIFICANCE: Our results show that denitrification rates in aquatic ecosystems are strongly temperature dependent, and that this is amplified by the temperature dependencies of photosynthesis and respiration. Our results illustrate the broader phenomenon that coupling of temperature dependent reactions may in some situations strongly alter overall effects of temperature on ecological processes.

Predicting microbial nitrogen pathways from basic principles.

Nitrogen compounds are transformed by a complicated network of competing geochemical processes or microbial pathways, each performed by a different ecological guild of microorganisms. Complete experimental unravelling of this network requires a prohibitive experimental effort. Here we present a simple model that predicts relative rates of hypothetical nitrogen pathways, based only on the stoichiometry and energy yield of the performed redox reaction, assuming competition for resources between alternative pathways. Simulating competing pathways in hypothetical freshwater and marine sediment situations, we surprisingly found that much of the variation observed in nature can simply be predicted from these basic principles. Investigating discrepancies between observations and predictions led to two important biochemical factors that may create barriers for the viability of pathways: enzymatic costs for long pathways and high ammonium activation energy. We hypothesize that some discrepancies can be explained by non-equilibrium dynamics. The model predicted a pathway that has not been discovered in nature yet: the dismutation of nitrite to the level of nitrate and dinitrogen gas.

ALGAL RESPONSE TO NUTRIENT ENRICHMENT IN FORESTED OLIGOTROPHIC STREAM(1).

Nutrient input in streams alters the density and species composition of attached algal communities in open systems. However, in forested streams, the light reaching the streambed (rather than the local nutrient levels) may limit the growth of these communities. A nutrient-enrichment experiment in a forested oligotrophic stream was performed to test the hypothesis that nutrient addition has only minor effects on the community composition of attached algae and cyanobacteria under light limitation. Moderate nutrient addition consisted of increasing basal phosphorus (P) concentrations 3-fold and basal nitrogen (N) concentrations 2-fold. Two upstream control reaches were compared to a downstream reach before and after nutrient addition. Nutrients were added continuously to the downstream reach for 1 year. Algal biofilms growing on ceramic tiles were sampled and identified for more than a year before nutrient addition to 12 months after. Diatoms were the most abundant taxonomic group in the three stream reaches. Nutrient enrichment caused significant variations in the composition of the diatom community. While some taxa showed significant decreases (e.g., Achnanthes minutissima, Gomphonema angustum), increases for other taxa (such as Rhoicosphenia abbreviata and Amphora ovalis) were detected in the enriched reach (for taxonomic authors, see Table 2). Epiphytic and adnate taxa of large size were enhanced, particularly during periods of favorable growth conditions (spring). Nutrients also caused a change in the algal chl a, which increased from 0.5-5.8 to 2.1-10.7 µg chl ·â€Šcm(-2) . Our results indicate that in oligotrophic forested streams, long-term nutrient addition has significant effects on the algal biomass and community composition, which are detectable despite the low light availability caused by the tree canopy. Low light availability moderates but does not detain the long-term tendency toward a nutrient-tolerant community. Furthermore, the effects of nutrient addition on the algal community occur in spite of seasonal variations in light, water flow, and water chemical characteristics, which may confound the observations. [Table: see text].

Adaptive divergence in pigment composition promotes phytoplankton biodiversity.

The dazzling diversity of the phytoplankton has puzzled biologists for decades. The puzzle has been enlarged rather than solved by the progressive discovery of new phototrophic microorganisms in the oceans, including picocyanobacteria, pico-eukaryotes, and bacteriochlorophyll-based and rhodopsin-based phototrophic bacteria. Physiological and genomic studies suggest that natural selection promotes niche differentiation among these phototrophic microorganisms, particularly with respect to their photosynthetic characteristics. We have analysed competition for light between two closely related picocyanobacteria of the Synechococcus group that we isolated from the Baltic Sea. One of these two has a red colour because it contains the pigment phycoerythrin, whereas the other is blue-green because it contains high contents of the pigment phycocyanin. Here we report theory and competition experiments that reveal stable coexistence of the two picocyanobacteria, owing to partitioning of the light spectrum. Further competition experiments with a third marine cyanobacterium, capable of adapting its pigment composition, show that this species persists by investing in the pigment that absorbs the colour not used by its competitors. These results demonstrate the adaptive significance of divergence in pigment composition of phototrophic microorganisms, which allows an efficient utilization of light energy and favours species coexistence.