Inhibition Of Methane Oxidation Research Articles

A perspective on how glyphosate and 2,4-D in wetlands may impact climate change

An increase in herbicide use is occurring due to a growing population and herbicide-resistant crops in agriculture, which has resulted in more herbicide tolerant target species. Glyphosate and 2,4-Dichlorophenoxyacetic acid (2,4-D) are two of the most commonly used herbicides worldwide and are more recently being used in combination in pre-mixed commercial formulas. Subsequently, herbicide contamination of wetlands will increase exposure of microorganisms to multiple chemical stressors. Methane is a potent greenhouse gas naturally emitted from wetlands, but herbicides may disrupt biogeochemical processes leading to an unbalanced methane cycle. We review the impacts of these herbicides on aquatic microbial communities from glyphosate-derived nutrient enrichment and 2,4-D inhibition of methane oxidation, and examine how these altered metabolic processes may lead to increased methane production in wetlands. The response of wetland ecosystems to herbicide contamination will vary across regions, in part due to the complexity of microbial communities, however, this perspective gives a glimpse into the potential global implications of continuing herbicide use on wetlands and demonstrates the importance for research on ecosystem-level co-stressors.

Response of a methane-driven interaction network to stressor intensification.

Microorganisms may reciprocally select for specific interacting partners, forming a network with interdependent relationships. The methanotrophic interaction network, comprising methanotrophs and non-methanotrophs, is thought to modulate methane oxidation and give rise to emergent properties beneficial for the methanotrophs. Therefore, microbial interaction may become relevant for community functioning under stress. However, empirical validation of the role and stressor-induced response of the interaction network remains scarce. Here, we determined the response of a complex methane-driven interaction network to a stepwise increase in NH4Cl-induced stress (0.5-4.75 g L-1, in 0.25-0.5 g L-1 increments) using enrichment of a naturally occurring complex community derived from a paddy soil in laboratory-scale incubations. Although ammonium and intermediates of ammonium oxidation are known to inhibit methane oxidation, methanotrophic activity was unexpectedly detected even in incubations with high ammonium levels, albeit rates were significantly reduced. Sequencing analysis of the 16S rRNA and pmoA genes consistently revealed divergent communities in the reference and stressed incubations. The 16S rRNA-based co-occurrence network analysis revealed that NH4Cl-induced stress intensification resulted in a less complex and modular network, likely driven by less stable interaction. Interestingly, the non-methanotrophs formed the key nodes, and appear to be relevant members of the community. Overall, stressor intensification unravels the interaction network, with adverse consequences for community functioning.

Importance of Methane Oxidation for Microbial Degradation of the Herbicide Bentazone in Drinking Water Production

Bentazone is a herbicide, which is frequently detected in groundwater due to its mobility and persistence in aquifers. Groundwater is used as a drinking water source all over the world, and sustainable methods to remove pesticides at low concentrations are urgently needed since pesticide contaminations can adversely affect human health. The aim of this study was to investigate whether microbial bentazone degradation was associated with methane oxidation in full-scale drinking water treatment plants. To this end, we investigated bentazone biodegradation in microcosms with water and filter material from rapid sand filters, or biomass from aeration systems, and we investigated the statistical relation between the presence of methane and bentazone in groundwater abstraction wells. An array of evidence supported an association between bentazone degradation and methane oxidation in the biological treatment process. The biodegradation potential of bentazone was associated with the presence of methane in the raw water at 14 different water works. In contrast, no association was observed with any of the other investigated inorganic energy sources, e.g. ammonium. Addition of acetylene inhibited methane oxidation and the bentazone degradation in filter material from two investigated waterworks. Biomass from the aeration tanks degraded bentazone, but only while oxidizing methane. Bentazone removal rates and methane removal rates correlated significantly across all the experiments with biomass or filter material, with an overall transformation yield of 15×10-5 moleBTZ/moleCH4. This demonstrated that the bentazone degradation was conducted by the same type of process in all the investigated communities, governed by methane oxidation. Furthermore, based on more than 10.000 water analyses from waterworks abstraction wells in Denmark, bentazone was detected significantly less frequent in wells with high methane concentrations (> 1 mg/L) than in wells without methane. This suggests that biological treatment of bentazone contamination in drinking water may be achieved using methanotrophs.

Evidence of co-metabolic bentazone transformation by methanotrophic enrichment from a groundwater-fed rapid sand filter

The herbicide bentazone is recalcitrant in aquifers and is therefore frequently detected in wells used for drinking water production. However, bentazone degradation has been observed in filter sand from a rapid sand filter at a waterworks with methane-rich groundwater. Here, the association between methane oxidation and removal of bentazone was investigated with a methanotrophic enrichment culture derived from methane-fed column reactors inoculated with that filter sand. Several independent lines of evidence obtained from microcosm experiments with the methanotrophic enrichment culture, tap water and bentazone at concentrations below 2 mg/L showed methanotrophic co-metabolic bentazone transformation: The culture removed 53% of the bentazone in 21 days in presence of 5 mg/L of methane, while only 31% was removed in absence of methane. Addition of acetylene inhibited methane oxidation and stopped bentazone removal. The presence of bentazone partly inhibited methane oxidation since the methane consumption rate was significantly lower at high (1 mg/L) than at low (1 μg/L) bentazone concentrations. The transformation yield of methane relative to bentazone normalized by their concentration ratio ranged from 58 to 158, well within the range for methanotrophic co-metabolic degradation of trace contaminants calculated from the literature, with normalized substrate preferences varying from 3 to 400. High-resolution mass spectrometry revealed formation of the transformation products (TPs) 6-OH, 8-OH, isopropyl-OH and di-OH-bentazone, with higher abundances of all TPs in the presence of methane. Overall, we found a suite of evidence all showing that bentazone was co-metabolically transformed to hydroxy-bentazone by a methanotrophic culture enriched from a rapid sand filter at a waterworks.

The differential effects of ammonium and nitrate on methanotrophs in rice field soil

It has been known that nitrogenous fertilizers can either stimulate or inhibit methane oxidation in soils. The mechanism, however, remains unclear. Here we conducted laboratory incubation experiments to evaluate the effects of ammonium versus nitrate amendment on CH4 oxidation in a rice field soil. The results showed that both N forms stimulated CH4 oxidation. But nitrate stimulated CH4 oxidation to a greater extent than ammonium per unit N base. The 16S rRNA genes and the pmoA genes were analyzed to determine the dynamics of total bacterial and methanotrophic populations, respectively. The methanotrophic community consisted of type I and type II methanotrophs and was dominated by type I group after two weeks of incubation. Nitrate promoted both types of methanotrophs, but ammonium promoted only type I. DNA-based stable isotope probing confirmed that ammonium stimulated the incorporation of 13CH4 into type I methanotrophs but not type II, while nitrate caused almost homogenous distribution of 13CH4 in type I and type II methanotrophs. Our study suggests that nitrate can promote CH4 oxidation more significantly than ammonium and is probably a better N source for both types of methanotrophs in rice field soil. More investigations, e.g. using 15N labeling, are necessary to elucidate this possibility.

Inhibition of Methane Oxidation in a Slurry Surface Crust by Inorganic Nitrogen: An Incubation Study

Livestock slurry is an important source of methane (CH). However, depending on the dry matter content of the slurry, a floating crust may form where methane-oxidizing bacteria (MOB) and CH oxidation activity have been found, suggesting that surface crusts may reduce CH emissions from slurry. However, it is not known how MOB in this environment interact with inorganic nitrogen (N). We studied inhibitory effects of ammonium (NH), nitrate (NO), and nitrite (NO) on potential CH oxidation in a cattle slurry surface crust. At headspace concentrations of 100 and 10,000 ppmv, CH oxidation was assayed at salt concentrations up to 500 mM. First-order rate constants were used to evaluate the strength of inhibition. Nitrite was the most potent inhibitor, reducing methanotrophic activity by up to 70% at only 1 mM NO. Methane-oxidizing bacteria were least sensitive to NO, tolerating up to 30 mM NO at 100 ppmv CH and 50 mM NO at 10,000 ppmv CH without any decline in activity. The inhibition by NH increased progressively, and no range of tolerance was observed. Methane concentrations of 10,000 ppmv resulted in 50- to 100-fold higher specific CH uptake rates than 100 ppmv CH but did not change the inhibition patterns of N salts. In slurry surface crusts, MOB maintained activity at higher concentrations of NH and NO than reported for MOB in soils and sediments, possibly showing adaptation to high N concentrations in the slurry environment. Yet it appears that the effectiveness of surface crusts as CH sinks will depend on inorganic N concentrations.

Inhibition of methane oxidation by nitrogenous fertilizers in a paddy soil.

Nitrogenous fertilizers are generally thought to have an important role in regulating methane oxidation. In this study, the effect of ammonium on methane oxidation activity was investigated in a paddy soil using urea at concentrations of 0, 50, 100, 200, and 400 μg N per gram dry weight soil (N/g.d.w.s) and ammonium sulfate at concentrations of 0, 50, and 200 μg N/g.d.w.s. The results of this study demonstrate that urea concentrations of 200 μg N/g.d.w.s. and above significantly inhibit methane oxidation activity, whereas no statistically significant difference was observed in methane oxidation activity among soil microcosms with urea concentrations of less than 200 μg N/g.d.w.s after incubation for 27 days. Similar results were obtained in a sense that methane oxidation activity was inhibited only when the ammonium sulfate concentration was 200 μg N/g.d.w.s in soil microcosms in this study. Phylogenetic analysis of pmoA genes showed that nitrogen fertilization resulted in apparent changes in the community composition of methane-oxidizing bacteria (MOB). Type I MOB displayed an increased abundance in soil microcosms amended with nitrogenous fertilizers, whereas type II MOB dominated the native soil. Furthermore, although no statistically significant relationship was observed between pmoA gene and amoA gene abundances, methane oxidation activity was significantly negatively correlated with nitrification activity in the presence of urea or ammonium sulfate. Our results indicate that the methane oxidation activity in paddy soils might be inhibited when the concentration of ammonium fertilizers is high and that the interactions between ammonia and methane oxidizers need to be further investigated.

A Chemical Approach to Controlling High Temperature Oxidation(Effect of NO Doping on High Temperature Oxidation Reactions)

The effect of NO doping on high temperature oxidation reaction was investigated to have insights into the control of HCCI combustion. Numerical analysis based on GRI-Mech methane oxidation scheme showed that the doped NO inhibited methane oxidation. The engine experiments were conducted to verify the calculation results using a single cylinder HCCI test engine. The experimental results showed that the doped NO enhanced the oxidation reaction, which were completely contrary to the calculation results. The largest cause for the error was in CH3 oxidation path in GRI-Mech.

The Response of Nitrogen Deposition to Methane Oxidation Availability and Microbial Enzyme Activities in Forest Soils

Forest soils are often nitrogen-limited, and nitrogen input to forest soils can cause substantial changes in the structure and functions of a soil ecosystem. To determine the effects of nitrogen input on methane oxidation and the microbial enzyme activities, manipulation experiments were conducted using nitrogen addition to soil samples from Mt. Jumbong. Our findings suggested that the addition of nitrogen to the soil system of Mt. Jumbong did not affect the microbial enzyme activities. Conversely, the addition of nitrogen affected the rate of methane oxidation. Inorganic nitrogen in soils can inhibit methane oxidation via several mechanisms, such as substrate competition, toxic effects, and competition with other microbes, but the inhibitory effects are not always the same. In this research, seasonal changes were found to produce different inhibitory factors, and these different responses may be caused from differences in the methantrophic bacteria community structure.

Stability of trifluoromethane in forest soils and methanotrophic cultures

Trifluoromethane (TFM) has been reported as an endproduct of trifluoroacetate degradation under oxic conditions. Although other halomethanes, such as chloroform, methyl bromide, and methyl fluoride, inhibit methane oxidation or are degraded by methanotrophs, the fate of TFM is unknown. TFM had no affect on atmospheric methane consumption when added to forest soils at either 10 ppm or 10, 000 ppm. No degradation of TFM was observed at either concentration for incubations of 6 days. Cultures of Methylobacter albus BG8 and Methylosinus trichosporium OB3b grown with and without added copper were also used to assay TFM degradation at 10–10 000 ppm levels. TFM did not inhibit methane oxidation under any growth conditions, including those inducing expression of soluble methane monooxygenase, nor was it degraded at measurable rates. In contrast, parallel assays showed that both methyl fluoride and chloroform inhibited methane oxidation in M. trichosporium OB3b. Our results suggest that TFM may be relatively inert with respect to methanotrophic degradation. Although TFM has a negligible ozone depletion potential, it absorbs infrared radiation and has a relatively long atmospheric residence time. Thus, accumulation of TFM in the atmosphere as a consequence of the decomposition of hydrochlorofluorocarbons may have significant unpredicted climate impacts.

Investigation of the effect of ammonium sulfate on populations of ambient methane oxidising bacteria by 13C-labelling and GC/C/IRMS analysis of phospholipid fatty acids

The oxidation of atmospheric methane by methanotrophic bacteria residing in soils constitutes an important terrestrial methane sink with previous studies having revealed the inhibition of microbially mediated methane oxidation in the presence of salt ions. The bacteria responsible for ambient methane oxidation are not amenable to currently available methods of culturing, resulting in the need for a method of in situ analysis. A combination of phospholipid fatty acid (PLFA) analysis and stable isotopic labelling has been employed in this investigation as a means of cultivation-independent bacterial analysis. Soil samples were treated with an ammonium sulfate solution at a concentration that was known to inhibit methane oxidation or with distilled water, serving as a control, and incubated with 13C-labelled methane. PLFAs were analysed by GC/C/IRMS in order to determine their 13C content and, hence, the PLFA distribution of the methane oxidising bacteria. Ammonium sulfate treatment reduced the amount of 13C incorporated into the majority of PLFAs except the i17:0 PLFA in the presence of high concentrations of methane. These results implied a shift in the composition of the methane oxidising bacterial community in the soils treated with ammonium ions, with the treatment appearing to suppress one group of organisms more than another.

Facultative Methanotrophs Revisited

Facultative Methanotrophs Revisited

Stereospecific effect of hexachlorocyclohexane on activity and structure of soil methanotrophic communities

In the past decades, large amounts of non-insecticidal hexachlorocyclohexane (HCH) isomers (alpha-, beta-, delta- and epsilon-HCH) have been dumped as side-products of the insecticide gamma-HCH (lindane). This study investigates the effect of HCH isomers on methane oxidation, an important soil function performed by methanotrophic bacteria. Both activity and structure of the methanotrophic community were assessed, using methane oxidation assays and PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) respectively. Methane oxidation assays with historically polluted soils revealed that on the long-term methane oxidation was inhibited by HCH pollution. PCR-DGGE and diversity analysis based on Lorenz curves showed that the type I methanotrophic community was less evenly distributed in historically HCH-polluted soils compared with less polluted reference soils. Short-term experiments with methane-enriched consortia further demonstrated that only gamma- and delta-isomers inhibited methane oxidation. Type I methanotrophs of methane-enriched microbial consortia that received gamma- or delta-HCH evolved towards higher species richness. Apparently, for historically HCH-polluted soils, a narrow community remained after long-term exposure while in case of short-term exposures, methane-enriched consortia were converted into less active, but richer communities when they were stressed by the presence of gamma- or delta-HCH. This work demonstrates the importance of incorporating all isomers and possible other side-products in risk assessment studies of persistent organic pollutants and the use of structural analysis of type I methanotrophic communities as evaluating tool.

Microbiological activities contributing to nitrogen removal with methane: effects of methyl fluoride and tungstate

When methane (CH 4) and O 2 are present, nitrogen can be removed from wastewater that does not contain other organic carbon sources. In this study, microbial activities during methane-dependent denitrification (MDD) were investigated by adding inhibitors of methane-oxidation and denitrification. Sludge susceptible to MDD showed methane oxidation activity in the presence of CH 4 and O 2, and denitrification activity with methanol and acetate under anoxic conditions. Methyl fluoride (CH 3F) is known to inhibit methane oxidation. When CH 3F was present, MDD did not occur, perhaps because methane oxidation was inhibited. Tungstate (WO 4 2−), a known inhibitor of nitrate reduction, also lowered denitrification activity in the sludge, and partly inhibited methane oxidation. When WO 4 2− was added to the medium, MDD almost ceased, perhaps because of a synergic inhibitory effect on denitrification and methane oxidation. These results show that both methane oxidation and denitrification contribute to MDD.

Pollution induced community tolerance (PICT) and analysis of 16S rRNA genes to evaluate the long‐term effects of herbicides on methanotrophic communities in soil

SummaryThere is an increasing interest in agricultural systems in which the use of herbicides is forbidden. Therefore, soils treated with herbicides atrazine and metolachlor for the last 20 years were compared with soil samples from the same field that had never been treated (control soil). We determined the pollution induced community tolerance (PICT) by evaluating the methane oxidation capacity of soil samples after adding increasing amounts of a methane oxidation inhibitor, 2,4‐dichlorophenoxyacetic acid (2,4‐D). Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes assessed whether the soil methanotrophic community differed between the two treatments. Addition of 60 µg 2,4‐D per g soil clearly inhibited methane oxidation in both soils but increased the time needed to oxidize 5% methane in the headspace by 250% for the control soil compared with 175% for the herbicide‐treated soil. This indicates that the soil with a long‐term herbicide history had a greater tolerance to the methane oxidation inhibitor than did the control soil. The DGGE of 16S rRNA genes amplified directly from soil community DNA could also distinguish the two treatments. The banding patterns of the Type I methanotrophs contained fewer bands in the herbicide‐treated soil. It seems that both the PICT approach and DGGE analysis are effective assays to distinguish a long‐term herbicide‐treated soil from an untreated soil.

Inhibition of methane oxidation by volatile sulfur compounds (CH3SH and CS2) in landfill cover soils.

Methanethiol and carbon disulphide were investigated for their ability to inhibit methane oxidation in two landfill cover soils. Methanethiol was found to be a competitive inhibitor, and at concentrations occurring in landfills, both these VSCs (volatile sulfur compounds) had inhibitory effects on the methane oxidation rates. Analysis of the phospholipid fatty acid contents in the soils indicated that type I-methanotrophs were more affected than type II. These effects of VSCs on methane oxidation are likely to have implications both for the establishment and the selectivity of a methane oxidizing microflora in landfills.

Short-term kinetic response of enhanced methane oxidation in landfill cover soils to environmental factors

This paper aims at a better understanding of methane oxidation under conditions that are representative of landfill cover soils. The kinetics of methane oxidation were studied in landfill cover soils that had been exposed to high methane mixing ratios. This was done in batch experiments, under various environmental conditions. Vmax increased exponentially with temperature in the range 5–35 °C, with a Q10 value of 2.8. Km increased approximately linearly in this range from 1.2 μM to 7 μM. Consequently, the influence of temperature on methane consumption was more pronounced at high concentrations than at low concentrations. The inhibition by ammonium of methane consumption was much stronger after 6–7 months of exposure to high methane mixing ratios than after 5–7 weeks of exposure, indicating that there was a shift of dominating methanotrophic species in soils after long exposure times. Additions of nitrifying sludge or compost to soils initially inhibited methane oxidation, followed by a stimulation after a few days.

Methane production in aerated marshland and model soils: effects of microflora and soil texture

Under oxic conditions the importance of the indigenous microflora and the soil texture on methane production was investigated using marshland soils (clayey silt) and different textured model soils (clay, sand, gravel). Under an oxic partial pressure >2.5% O 2, soil slurries had a low methane production rate over a period of 50–70 h with acetate (0.35±0.1 nmol g −1 h −1) as well as with hydrogen (2.43±0.51 nmol g −1 h −1) as substrate. The rates amounted to 1 and 5%, respectively, compared to methane production rates under anoxic conditions. As soon as the oxygen concentration decreased to 2.5% (microaerophilic conditions) the methane production increased significantly (9.5±3.3 nmol g −1 h −1 with acetate; 16.3±3.3 nmol g −1 h −1 with hydrogen). In the absence of the indigenous microflora, the inoculated culture of Methanosarcina mazei (DSM 2053) did not produce any methane in soil slurries under oxic conditions. To inhibit methane oxidation, all samples (oxic and anoxic) were supplied with 60 nl acetylene ml −1 headspace. Furthermore, methane production in different textured model soils demonstrated that a high amount of negative surface charges increased methane production under oxic as well as under anoxic conditions. Consequently the methane production rates increased in the following sequence: sand<gravel<clayey silt (marshland soil)≤clay. Our results show that the indigenous microflora in combination with the sorptive quality of soil particles (clay, silt, organic matter) enables methanogenic activity in the presence of oxygen, promoting microscale anoxia within the slurries.

Dichloromethane and trichloroethylene inhibition of methane oxidation by the membrane-associated methane monooxygenase of Methylosinus trichosporium OB3b

Whole-cell assays were used to measure the ef- fect of dichloromethane and trichloroethylene on methane oxidation by Methylosinus trichosporiumOB3b synthesiz- ing the membrane-associated or particulate methane mono- oxygenase (pMMO). For M. trichosporium OB3b grown with 20 μM copper, no inhibition of methane oxidation was observed in the presence of either dichloromethane or trichloroethylene. If 20 mM formate was added to the re- action vials, however, methane oxidation rates increased and inhibition of methane oxidation was observed in the presence of dichloromethane and trichloroethylene. In the presence of formate, dichloromethane acted as a competi- tive inhibitor, while trichloroethylene acted as a noncom- petitive inhibitor. The finding of noncompetitive inhibi- tion by trichloroethylene was further examined by mea- suring the inhibition constants KiE and KiES. These constants suggest that trichloroethylene competes with methane at some sites, although it can bind to others if methane is al- ready bound. Whole-cell oxygen uptake experiments for ac- tive and acetylene-treated cells also showed that provision of formate could stimulate both methane and trichloroeth- ylene oxidation and that trichloroethylene did not affect formate dehydrogenase activity. The finding that different chlorinated hydrocarbons caused different inhibition pat- terns can be explained by either multiple substrate binding sites existing in pMMO or multiple forms of pMMO with different activities. The whole-cell analysis performed here cannot distinguish between these models, and further work should be done on obtaining active preparations of the pu- rified pMMO.

Effect of selected monoterpenes on methane oxidation, denitrification, and aerobic metabolism by bacteria in pure culture.

Selected monoterpenes inhibited methane oxidation by methanotrophs (Methylosinus trichosporium OB3b, Methylobacter luteus), denitrification by environmental isolates, and aerobic metabolism by several heterotrophic pure cultures. Inhibition occurred to various extents and was transient. Complete inhibition of methane oxidation by Methylosinus trichosporium OB3b with 1.1 mM (-)-alpha-pinene lasted for more than 2 days with a culture of optical density of 0.05 before activity resumed. Inhibition was greater under conditions under which particulate methane monooxygenase was expressed. No apparent consumption or conversion of monoterpenes by methanotrophs was detected by gas chromatography, and the reason that transient inhibition occurs is not clear. Aerobic metabolism by several heterotrophs was much less sensitive than methanotrophy was; Escherichia coli (optical density, 0.01), for example, was not affected by up to 7.3 mM (-)-alpha-pinene. The degree of inhibition was monoterpene and species dependent. Denitrification by isolates from a polluted sediment was not inhibited by 3.7 mM (-)-alpha-pinene, gamma-terpinene, or beta-myrcene, whereas 50 to 100% inhibition was observed for isolates from a temperate swamp soil. The inhibitory effect of monoterpenes on methane oxidation was greatest with unsaturated, cyclic hydrocarbon forms [e.g., (-)-alpha-pinene, (S)-(-)-limonene, (R)-(+)-limonene, and gamma-terpinene]. Lower levels of inhibition occurred with oxide and alcohol derivatives [(R)-(+)-limonene oxide, alpha-pinene oxide, linalool, alpha-terpineol] and a noncyclic hydrocarbon (beta-myrcene). Isomers of pinene inhibited activity to different extents. Given their natural sources, monoterpenes may be significant factors affecting bacterial activities in nature.