Ecological observations on Heterotrophic,methane oxidizing and sulfate reducing bacteria in Pond

Numbers of heterotrophic, methane oxidizing and sulfate reducing bacteria were counted in Lake Vechten. A dynamic distribution pattern was found in the stratified lake. A maximum of heterotrophs (numbers of 10⁹ bact./l) occured in the deepest part of the lake in spring and in the metalimnion during summer-stratification. These bacteria use nearly all available oxygen in the hypolimnion. It was found that the concentration of available organic material and the oxygen tension caused the numbers of heterotrophs in the metalimnion to be high.The maximal numbers of methane oxidizers (numbers of 5.10⁵ bact./l) were found at a depth of maximal methane concentration: the de-oxygenated hypolimnion. Preliminary evidence indicated that these organisms were facultative methane oxidizers and must be regarded as micro-aerophyllics. By oxidizing methane they removed the residual oxygen under the metalimnion.The sulfate reducing bacteria could be observed in the hypolimnion only. Decreased SO _inf4 ^sup–2 concentration and increased numbers, of bacterai were found in the bottom water. An association between the methane oxidizers and the sulfate reducers could be deduced. It was assumed that favourable redox requirements for obligate anaerobic sulfate reducers were the results of the activities of the methane oxidizing bacteria.The dynamic distribution equilibrium of the investigated groups of bacteria was disturbed by the autumn turn-over. The heterotrophic and methane oxidizing bacteria decreased in number at that period and were equally distributed, no sulfate reducers could be detected in the free water of Lake Vechten.     

Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. I. Field observations

Observations on the seasonal periodicity in bottom deposits of Lake Vechten indicated an ecological relationship between sulfate-reducing and methane-producing bacteria. Sulfate reducers are most abundant at depths of 0 to 2 cm in the mud at pS^2- values of about 11 and redox potential values of-100 to-150 mV. Maximum number of methane producers are situated at depths of 3 to 6 cm in the mud at pS^2- values of about 14, redox potential values of-250 to-300 mV and maximum values of the methane concentration. During summer stratification the numbers of bacteria increased considerably. However the number of methane producers rose much more than that of the sulfate reducers. Sulfate in the interstitial water of the sediments is reduced by the sulfate reducers and the sulfate concentration limited the latter's abundance. Methane producers are found deeper in the mud at lower concentrations of hydrogen sulphide. Therefore the different localities of the two bacterial groups may be due to sensitivity of methane producers to hydrogen sulphide. Differential counting of the mixed population of methane-producing bacteria showed that acetate-and methyl-alcohol-fermenting types are most abundant at a depth of 5, and formate-and CO₂/H₂-fermenting types at a depth of 3 cm in the mud.     

Sedimentation and breakdown kinetics of organic matter in the anaerobic zone of Lake Vechten

The turnover and exchange rates, as well as the sedimentation rates concerning the input and output of carbon in the anaerobic hypolimnion and the sediment in Lake Vechten were studied. The carbon input data were derived from those on sedimentation. By comparison of C-fixation data, sedimentation rates measurements and microbial reduction rates of observed fluxes of electron acceptors a mineralization of 20–30% of the limnetic C-fixation was found in the hypolimnion. The rate-limiting step of methanogenesis in anaerobic mineralization is the breakdown of algal cell wall components to their main product, namely acetate. This intermediate has a relatively fast turnover rate to the end-products, namely the gases CH₄ and CO₂. Finally, from the data on the diffusion of low-molecular weight metabolic intermediates and on the rates of formation of CH₄ and CO₂ the output of carbon and its cycling in Lake Vechten are discussed.     

Methane emission from natural wetlands: interplay between emergent macrophytes and soil microbial processes. A mini-review

Background

According to the Intergovernmental Panel on Climate Change (IPCC) 2007, natural wetlands contribute 20–39 % to the global emission of methane. The range in the estimated percentage of the contribution of these systems to the total release of this greenhouse gas is large due to differences in the nature of the emitting vegetation including the soil microbiota that interfere with the production and consumption of methane.

Scope

Methane is a dominant end-product of anaerobic mineralization processes. When all electron acceptors except carbon dioxide are used by the microbial community, methanogenesis is the ultimate pathway to mineralize organic carbon compounds. Emergent wetland plants play an important role in the emission of methane to the atmosphere. They produce the carbon necessary for the production of methane, but also facilitate the release of methane by the possession of a system of interconnected internal gas lacunas. Aquatic macrophytes are commonly adapted to oxygen-limited conditions as they prevail in flooded or waterlogged soils. By this system, oxygen is transported to the underground parts of the plants. Part of the oxygen transported downwards is released in the root zone, where it sustains a number of beneficial oxidation processes. Through the pores from which oxygen escapes from the plant into the root zone, methane can enter the plant aerenchyma system and subsequently be emitted into the atmosphere. Part of the oxygen released into the root zone can be used to oxidize methane before it enters the atmosphere. However, the oxygen can also be used to regenerate alternative electron acceptors. The continuous supply of alternative electron acceptors will diminish the role of methanogenesis in the anaerobic mineralization processes in the root zone and therefore repress the production and emission of methane. The role of alternative element cycles in the inhibition of methanogenesis is discussed.

Conclusions

The role of the nitrogen cycle in repression of methane production is probably low. In contrast to wetlands particularly created for the purification of nitrogen-rich waste waters, concentrations of inorganic nitrogen compounds are low in the root zones in the growing season due to the nitrogen-consuming behaviour of the plant. Therefore, nitrate hardly competes with other electron acceptors for reduced organic compounds, and repression of methane oxidation by the presence of higher levels of ammonium will not be the case. The role of the iron cycle is likely to be important with respect to the repression of methane production and oxidation. Iron-reducing and iron-oxidizing bacteria are ubiquitous in the rhizosphere of wetland plants. The cycling of iron will be largely dependent on the size of the oxygen release in the root zone, which is likely to be different between different wetland plant species. The role of the sulfur cycle in repression of methane production is important in marine, sulfate-rich ecosystems, but might also play a role in freshwater systems where sufficient sulfate is available. Sulfate-reducing bacteria are omnipresent in freshwater ecosystems, but do not always react immediately to the supply of fresh sulfate. Hence, their role in the repression of methanogenesis is still to be proven in freshwater marshes.     

Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. II. Inhibition experiments

A possible substrate interrelationship between methane-producing and sulfate-reducing bacteria has been studied in bottom deposits of Lake Vechten. Inhibition of methanogenesis in mud samples by chlorine-containing analogues of methane resulted in accumulation of acetate. Fluoroacetate reduced the concentration of methane by about 75%. With carbon tetrachloride, accumulation of hydrogen gas was observed. These results indicate that acetate is the main precursor of methanogenesis in mud. After addition of β-fluorolactate, lactate accumulated and H₂S was no longer produced, which indicates that lactate is the main source of energy for sulfate reduction in mud. At the same time the concentration of methane increased possibly due to the lower concentration of H₂S, which has a toxic effect on methanogenesis. Experiments with intact mud cores provide evidence that the described phenomena occur also in situ.     

The relationship between planktonic algae and bacteria in a small lake

The seasonal changes and vertical distribution of the aerobic and anaerobic bacteria in a small edaphically eutrophic lake which exhibited thermal and chemical stratification are described. There was some correspondence between the phytoplankton and particularly the aerobic bacteria but this was not consistent. Increases in the numbers of anaerobic bacteria coincided with the low dissolved oxygen concentrations in the hypolimnion when algal populations were first senescent and then increasing actively in size.     

Seasonal changes in the chemistry and biology of a meromictic lake (Big Soda Lake,Nevada, U.S.A.)

Big Soda Lake is an alkaline, saline lake with a permanent chemocline at 34.5 m and a mixolimnion that undergoes seasonal changes in temperature structure. During the period of thermal stratification, from summer through fall, the epilimnion has low concentrations of dissolved inorganic nutrients (N, Si) and CH₄, and low biomass of phytoplankton (chlorophyll a ca. 1 mgm ^-3). Dissolved oxygen disappears near the compensation depth for algal photosynthesis (ca. 20 m). Surface water is transparent so that light is present in the anoxic hypolimnion, and a dense plate of purple sulfur photosynthetic bacteria (Ectothiorhodospira vacuolata) is present just below 20 m (Bchl a ca. 200 mgm^-3). Concentrations of N H₄ ⁺, Si, and CH₄ are higher in the hypolimnion than in the epilimnion. As the mixolimnion becomes isothermal in winter, oxygen is mixed down to 28 m. Nutrients (NH₄ ⁺, Si) and CH₄ are released from the hypolimnion and mix to the surface, and a diatom bloom develops in the upper 20 m (chlorophyll a > 40 mgm^-3). The deeper mixing of oxygen and enhanced light attenuation by phytoplankton uncouple the anoxic zone and photic zone, and the plate of photosynthetic bacteria disappears (Bchl a ca.10mgm^-3). Hence, seasonal changes in temperature distribution and mixing create conditions such that the primary producer community is alternately dominated by phytoplankton and photosynthetic bacteria: the phytoplankton may be nutrient-limited during periods of stratification and the photosynthetic bacteria are light-limited during periods of mixing.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^?1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^?1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^-1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^-1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

Microbial activities in soil near natural gas leaks

Summary From the present experiments it may be concluded that in the surroundings of natural gas leaks, methane, ethane and possibly some other components of the natural gas are oxidized by microbial activities as long as oxygen is available. This is demonstrated by an increased oxygen consumption and carbon dioxide production, as well as by increased numbers of different types of bacteria. The resulting deficiency of oxygen, the excess of carbon dioxide, and perhaps the formation of inhibitory amounts of ethylene, are considered to be mainly responsible for the death of trees near natural gas leaks. Also the long period of time needed by the soil to recover, may be due to prolonged microbial activities, as well as to the presence of e. g. ethylene.The present experiments suggest that especially methane-oxidizing bacteria of the Methylosinus trichosporium type were present in predominating numbers and consequently have mainly been responsible for the increased oxygen consumption. However, some fungi oxidizing components of natural gas, including methane and ethane may also have contributed to the increased microbial activities in the soil. The same will be true of a possible secondary microflora on products derived from microorganisms oxidizing natural gas components.     

Bacterioplankton Abundance and Activity in a Small Hypertrophic Stratified Lake

Bacterioplankton abundance and production were followed during one decade (1991–2001) in the hypertrophic and steeply stratified small Lake Verevi (Estonia). The lake is generally dimictic. However, a partly meromictic status could be formed in specific meteorological conditions as occurred in springs of 2000 and 2001. The abundance of bacteria in Lake Verevi is highly variable (0.70 to 22 × 10⁶ cells ml⁻¹) and generally the highest in anoxic hypolimnetic water. In 2000–2001, the bacterial abundance in the hypolimnion increased probably due to meromixis. During a productive season, heterotrophic bacteria were able to consume about 10–40% of primary production in the epilimnion. Our study showed that bacterioplankton in the epilimnion was top-down controlled by predators, while in metalimnion bacteria were dependent on energy and carbon sources (bottom-up regulated). Below the thermocline hypolimnetic bacteria mineralized organic matter what led to the depletion of oxygen and created anoxic hypolimnion where rich mineral nutrient and sulphide concentrations coexisted with high bacterial numbers.     

Methane oxidation in anoxic lake water stimulated by nitrate and sulfate addition

Methanotrophic bacteria play a key role in limiting methane emissions from lakes. It is generally assumed that methanotrophic bacteria are mostly active at the oxic-anoxic transition zone in stratified lakes, where they use oxygen to oxidize methane. Here, we describe a methanotroph of the genera Methylobacter that is performing high-rate (up to 72 μM day⁻¹) methane oxidation in the anoxic hypolimnion of the temperate Lacamas Lake (Washington, USA), stimulated by both nitrate and sulfate addition. Oxic and anoxic incubations both showed active methane oxidation by a Methylobacter species, with anoxic rates being threefold higher. In anoxic incubations, Methylobacter cell numbers increased almost two orders of magnitude within 3 days, suggesting that this specific Methylobacter species is a facultative anaerobe with a rapid response capability. Genomic analysis revealed adaptations to oxygen-limitation as well as pathways for mixed-acid fermentation and H₂ production. The denitrification pathway was incomplete, lacking the genes narG/napA and nosZ, allowing only for methane oxidation coupled to nitrite-reduction. Our data suggest that Methylobacter can be an important driver of the conversion of methane in oxygen-limited lake systems and potentially use alternative electron acceptors or fermentation to remain active under oxygen-depleted conditions.     

Methane production by microbial mats under low sulphate concentrations

Cyanobacterial mats collected in hypersaline salterns were incubated in a greenhouse under low sulphate concentrations ([]) and examined for their primary productivity and emissions of methane and other major carbon species. Atmospheric greenhouse warming by gases such as carbon dioxide and methane must have been greater during the Archean than today in order to account for a record of moderate to warm palaeoclimates, despite a less luminous early sun. It has been suggested that decreased levels of oxygen and sulphate in Archean oceans could have significantly stimulated microbial methanogenesis relative to present marine rates, with a resultant increase in the relative importance of methane in maintaining the early greenhouse. We maintained modern microbial mats, models of ancient coastal marine communities, in artificial brine mixtures containing both modern [] (c. 70 mm ) and ‘Archean’[] (<0.2 mm ). At low [], primary production in the mats was essentially unaffected, while rates of sulphate reduction decreased by a factor of three, and methane fluxes increased by up to 10‐fold. However, remineralization by methanogenesis still amounted to less than 0.4% of the total carbon released by the mats. The relatively low efficiency of conversion of photosynthate to methane is suggested to reflect the particular geometry and chemical microenvironment of hypersaline cyanobacterial mats. Therefore, such mats were probably relatively weak net sources of methane throughout their 3.5 Ga history, even during periods of low environmental levels oxygen and sulphate.     

Biomarker evidence for widespread anaerobic methane oxidation in Mediterranean sediments by a consortium of methanogenic archaea and bacteria. The Medinaut Shipboard Scientific Party

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in (13)C (delta(13)C values are as low as -95 per thousand). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of (13)C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.     

Development of respirometry methods to assess the microbial activity of thermophilic bioleaching archaea

Respirometry methods have been used for many years to assess the microbial activity of mainly heterotrophic bacteria. Using this technique, the consumption of oxygen and evolution of carbon dioxide for heterotrophic carbon catabolism can be used to assess microbial activity. In the case of autotrophic bioleaching bacteria, carbon dioxide is used as a carbon source resulting in the consumption of both oxygen and carbon dioxide. The use of such respirometry techniques at high temperatures (up to 80 degrees C) for the investigation of bioleaching Archaea, however, poses particular difficulties. At these elevated temperatures, the solubility of oxygen into the liquid phase is particularly poor. This work details specific methods by which high temperature constraints are overcome while monitoring the activity of thermophilic Archaea using a Micro-Oxymax respirometer (Columbus Instruments). The use of elevated headspace oxygen concentrations, in order to overcome low oxygen solubility, is demonstrated as well as the effect of such elevated oxygen concentrations on microbial oxygen consumption rates. The relative rates of oxygen and carbon dioxide consumption are also illustrated during the oxidation of a chalcopyrite concentrate. In addition, this paper details generic methods by which respirometry data can be used to quantify inhibitory effects of a compound such as Na(2)SO(4). The further use of such data in predicting minimum hydraulic reactor retention times for continuous culture bioleaching reactors, as a function of concentration of potentially inhibitory compounds, is also demonstrated.     

Inhibition Experiments on Anaerobic Methane Oxidation

Anaerobic methane oxidation is a general process important in controlling fluxes of methane from anoxic marine sediments. The responsible organism has not been isolated, and little is known about the electron acceptors and substrates involved in the process. Laboratory evidence indicates that sulfate reducers and methanogens are able to oxidize small quantities of methane. Field evidence suggests anaerobic methane oxidation may be linked to sulfate reduction. Experiments with specific inhibitors for sulfate reduction (molybdate), methanogenesis (2-bromoethanesulfonic acid), and acetate utilization (fluoroacetate) were performed on marine sediments from the zone of methane oxidation to determine whether sulfate-reducing bacteria or methanogenic bacteria are responsible for methane oxidation. The inhibition experiment results suggest that methane oxidation in anoxic marine sediments is not directly mediated by sulfate-reducing bacteria or methanogenic bacteria. Our results are consistent with two possibilities: anaerobic methane oxidation may be mediated by an unknown organism or a consortium involving an unknown methane oxidizer and sulfate-reducing bacteria.     

The carbon cycle and associated redox processes through time

Earth's biogeochemical cycle of carbon delivers both limestones and organic materials to the crust. In numerous, biologically catalysed redox reactions, hydrogen, sulphur, iron, and oxygen serve prominently as electron donors and acceptors. The progress of these reactions can be reconstructed from records of variations in the abundance of 13C in sedimentary carbonate minerals and organic materials. Because the crust is always receiving new CO2 from the mantle and a portion of it is being reduced by photoautotrophs, the carbon cycle has continuously released oxidizing power. Most of it is represented by Fe3+ that has accumulated in the crust or been returned to the mantle via subduction. Less than 3% of the estimated, integrated production of oxidizing power since 3.8 Gyr ago is represented by O2 in the atmosphere and dissolved in seawater. The balance is represented by sulphate. The accumulation of oxidizing power can be estimated from budgets summarizing inputs of mantle carbon and rates of organic-carbon burial, but levels of O2 are only weakly and indirectly coupled to those phenomena and thus to carbon-isotopic records. Elevated abundances of 13C in carbonate minerals ca 2.3 Gyr old, in particular, are here interpreted as indicating the importance of methanogenic bacteria in sediments rather than increased burial of organic carbon.     

硝酸盐和硫酸盐厌氧氧化甲烷途径及氧化菌群

甲烷属于温室气体,厌氧氧化甲烷有效地减少了大气环境中甲烷的含量。依据吉布斯自由能变,以SO42、Mn4+、Fe3+、NO3等作为电子受体,厌氧条件下甲烷可以转化为CO2。重点阐述以SO42和NO3为电子受体时甲烷厌氧氧化的机理、反应发生的环境条件以及甲烷厌氧氧化菌的特点。针对目前研究存在的主要问题,提出了今后的发展方向。SO42为电子受体时,甲烷厌氧氧化的可能途径包括:逆甲烷生成途径、乙酰生成途径以及甲基生成途径。甲烷的好氧或厌氧氧化协同反硝化是以NO3为电子受体的甲烷氧化的可能途径。环境中的甲烷、硫酸盐或硝酸盐的浓度,有机质的数量,以及环境条件对甲烷的厌氧氧化有显著影响。     

Methane oxidation and the competition for oxygen in the rice rhizosphere

A mechanistic approach is presented to describe oxidation of the greenhouse gas methane in the rice rhizosphere of flooded paddies by obligate methanotrophic bacteria. In flooded rice paddies these methanotrophs compete for available O(2) with other types of bacteria. Soil incubation studies and most-probable-number (MPN) counts of oxygen consumers show that microbial oxygen consumption rates were dominated by heterotrophic and methanotrophic respiration. MPN counts of methanotrophs showed large spatial and temporal variability. The most abundant methanotrophs (a Methylocystis sp.) and heterotrophs (a Pseudomonas sp. and a Rhodococcus sp.) were isolated and characterized. Growth dynamics of these bacteria under carbon and oxygen limitations are presented. Theoretical calculations based on measured growth dynamics show that methanotrophs were only able to outcompete heterotrophs at low oxygen concentrations (frequently < 5 microM). The oxygen concentration at which methanotrophs won the competition from heterotrophs did not depend on methane concentration, but it was highly affected by organic carbon concentrations in the paddy soil. Methane oxidation was severely inhibited at high acetate concentrations. This is in accordance with competition experiments between Pseudomonas spp. and Methylocystis spp. carried out at different oxygen and carbon concentrations. Likely, methane oxidation mainly occurs at microaerophilic and low-acetate conditions and thus not directly at the root surface. Acetate and oxygen concentrations in the rice rhizosphere are in the critical range for methane oxidation, and a high variability in methane oxidation rates is thus expected.     

Hydrogen as a substrate for methanogenesis and sulphate reduction in anaerobic saltmarsh sediment

Hydrogen gas stimulated sulphate reduction in a saltmarsh sediment and the importance of H₂ transferred from organotrophic bacteria to the sulphate-reducers is discussed. -fluorolactate inhibited sulphate reduction whether lactate, ethanol or hydrogen was being used as growth substrate. When added to sediment -fluorolactate inhibited sulphate reduction with a consequent increase in methane production.Addition of H₂ stimulated methanogenesis in sediment and this stimulation was greater if CO₂ was also present. Hydrogen availability was the primary limitation of methanogenesis but the low concentration of dissolved CO₂ in seawater may limit methane production even if H₂ is available.The removal of inhibition of methanogenesis by the use of fluorolactate to suppress sulphate reduction or by the provision of hydrogen indicates competitive inhibition of methanogens by sulphate reducers utilizing transferred hydrogen.Abbreviations HSRB hydrogen utilizing sulphate reducing bacteria - HDO hydrogen donating organism