Stable-isotopic and metagenomic analyses reveal metabolic and microbial link of aerobic methane oxidation coupled to denitrification at different O2 levels.

Aerobic methane (CH4) oxidation coupled to denitrification (AME-D) can not only mitigate CH4 emission into the atmosphere, but also potentially alleviate nitrogen pollution in surface waters and engineered ecosystems, and it has attracted substantial research interest. O2 concentration plays a key role in AME-D, yet little is understood about how it impacts microbial interactions. Here, we applied isotopically labeled K15NO3 and 13CH4 and metagenomic analyses to investigate the metabolic and microbial link of AME-D at different O2 levels. Among the four experimental O2 levels of 21%,10%, 5% and 2.5% and a CH4 concentration of 8% (i.e., the O2/CH4 ratios of 2.62, 1.26, 0.63 and 0.31), the highest NO3--N removal occurred in the AME-D system incubated at the O2 concentration of 10%. Methanol and acetate may serve as the trophic linkage between aerobic methanotrophs and denitrifers in the AME-D systems. Methylotrophs including Methylophilus, Methylovorus, Methyloversatilis and Methylotenera were abundant under the O2-sufficient condition with the O2 concentration of 21%, while denitrifiers such as Azoarcus, Thauera and Thiobacillus dominated in the O2-limited environment with the O2 concentration of 10%. The competition of denitrifiers and methylotrophs in the AME-D system for CH4-derived carbon, such as methanol and acetate, might be influenced by chemotactic responses. More methane-derived carbon flowed into methylotrophs under the O2-sufficient condition, while more methane-derived carbon was used for denitrification in the O2-limited environment. These findings can aid in evaluating the distribution and contribution of AME-D and in developing strategies for mitigating CH4 emission and nitrogen pollution in natural and engineered ecosystems.

Effects of oxygen tension on the microbial community and functional gene expression of aerobic methane oxidation coupled to denitrification systems.

Aerobic CH4 oxidation coupled to denitrification (AME-D) can not only mitigate the emission of greenhouse gas (e.g., CH4) to the atmosphere, but also reduce NO3- and/or NO2- and alleviate nitrogen pollution. The effects of O2 tension on the community and functional gene expression of methanotrophs and denitrifiers were investigated in this study. Although higher CH4 oxidation occurred in the AME-D system with an initial O2 concentration of 21% (i.e., the O2-sufficient condition), more NO3--N was removed at the initial O2 concentration of 10% (i.e., the O2-limited environment). Type I methanotrophs, including Methylocaldum, Methylobacter, Methylococcus, Methylomonas, and Methylomicrobium, and type II methanotrophs, including Methylocystis and Methylosinus, dominated in the AME-D systems. Compared with type II methanotrophs, type I methanotrophs were more abundant in the AME-D systems. Proteobacteria and Actinobacteria were the main denitrifiers in the AME-D systems, and their compositions varied with the O2 tension. Quantitative PCR of the pmoA, nirS, and 16S rRNA genes showed that methanotrophs and denitrifiers were the main microorganisms in the AME-D systems, accounting for 46.4% and 24.1% in the O2-limited environment, respectively. However, the relative transcripts of the functional genes including pmoA, mmoX, nirK, nirS, and norZ were all less than 1%, especially the functional genes involved in denitrification under the O2-sufficient condition, likely due to the majority of the denitrifiers being dormant or even nonviable. These findings indicated that an optimal O2 concentration should be used to optimize the activity and functional gene expression of aerobic methanotrophs and denitrifiers in AME-D systems.

Low O2 level enhances CH4-derived carbon flow into microbial communities in landfill cover soils.

CH4 oxidation in landfill cover soils plays a significant role in mitigating CH4 release to the atmosphere. Oxygen availability and the presence of co-contaminants are potentially important factors affecting CH4 oxidation rate and the fate of CH4-derived carbon. In this study, microbial populations that oxidize CH4 and the subsequent conversion of CH4-derived carbon into CO2, soil organic C and biomass C were investigated in landfill cover soils at two O2 tensions, i.e., O2 concentrations of 21% ("sufficient") and 2.5% ("limited") with and without toluene. CH4-derived carbon was primarily converted into CO2 and soil organic C in the landfill cover soils, accounting for more than 80% of CH4 oxidized. Under the O2-sufficient condition, 52.9%-59.6% of CH4-derived carbon was converted into CO2 (CECO2-C), and 29.1%-39.3% was converted into soil organic C (CEorganic-C). A higher CEorganic-C and lower CECO2-C occurred in the O2-limited environment, relative to the O2-sufficient condition. With the addition of toluene, the carbon conversion efficiency of CH4 into biomass C and organic C increased slightly, especially in the O2-limited environment. A more complex microbial network was involved in CH4 assimilation in the O2-limited environment than under the O2-sufficient condition. DNA-based stable isotope probing of the community with 13CH4 revealed that Methylocaldum and Methylosarcina had a higher relative growth rate than other type I methanotrophs in the landfill cover soils, especially at the low O2 concentration, while Methylosinus was more abundant in the treatment with both the high O2 concentration and toluene. These results indicated that O2-limited environments could prompt more CH4-derived carbon to be deposited into soils in the form of biomass C and organic C, thereby enhancing the contribution of CH4-derived carbon to soil community biomass and functionality of landfill cover soils (i.e. reduction of CO2 emission).

Assessment of the major odor contributors and health risks of volatile compounds in three disposal technologies for municipal solid waste.

Gaseous emissions from municipal solid waste (MSW) disposal plants pose serious odor pollution and health risks. In this study, the emission of volatile organic compounds and carbon disulfide was compared in the main processing units of three disposal methods, i.e., landfilling, eco-mechanical biological treatment (EMBT) and anaerobic fermentation in a MSW disposal plant. Among the detected volatile compounds (VCs), the top ten odor compounds were methanethiol, dimethyl sulfide, dimethyl disulfide, carbon disulfide, styrene, m-xylene, 4-ethyltoluene, ethylbenzene, 2-hexyl ketone and n-hexane in the MSW disposal plant. Sulfur compounds were the main source of odor at the majority of sampling sites, and aromatic compounds were the dominant odor substrates at the tipping unit and sorting system of EMBT, while 2-hexanone was the major odor substrate at the tipping unit (AT) and sorting system (AS) of anaerobic fermentation and the landfill working surface. At AS and AT, the lifetime cancer risk values for 1,2-dichloroethane and trichloroethylene exceeded the carcinogenic risk value (>1.0E-04), and the hazard index values of naphthalene, trichloroethylene and acrolein all exceeded the acceptable level (>1). Therefore, special attention should be paid to VC emissions from MSW disposal facilities, and protection measures should be adopted for on-site workers to minimize health risks.

Characterization of toluene metabolism by methanotroph and its effect on methane oxidation.

Methanotrophs not only oxidize CH4, but also can oxidize a relatively broad range of other substrates, including trichloroethylene, alkanes, alkenes, and aromatic compounds. In this study, Methylosinus sporium was used as a model organism to characterize toluene metabolism by methanotrophs. Reverse transcription quantitative PCR analysis showed that toluene enhanced the mmoX expression of M. sporium. When the toluene concentration was below 2000 mg m-3, the kinetics of toluene metabolism by M. sporium conformed to the Michaelis-Menten equation (Vmax = 0.238 g gdry weight-1 h-1, K m = 545.2 mg m-3). The use of a solid-phase extraction technique followed by a gas chromatography-mass spectrometry analysis and molecular docking calculation showed that toluene was likely to primarily bind the di-iron center structural region of soluble methane monooxygenase (sMMO) hydroxylase and then be oxidized to o-cresol. Although M. sporium oxidized toluene, it did not incorporate toluene into its biomass. The coexistence of toluene and CH4 could influence CH4 oxidation, the growth of methanotrophs, and the distribution of CH4-derived carbon, which were related to the ratio of the toluene concentration to biomass. These results would be helpful to understand the metabolism of CH4 and non-methane volatile organic compounds in the environment.

Conversion of sulfur compounds and microbial community in anaerobic treatment of fish and pork waste.

Volatile sulfur compounds (VSCs) are not only the main source of malodor in anaerobic treatment of organic waste, but also pose a threat to human health. In this study, VSCs production and microbial community was investigated during the anaerobic degradation of fish and pork waste. The results showed that after the operation of 245 days, 94.5% and 76.2% of sulfur compounds in the fish and pork waste was converted into VSCs. Among the detected VSCs including H2S, carbon disulfide, methanethiol, ethanethiol, dimethyl sulfide, dimethyl disulfide and dimethyl trisulfide, methanethiol was the major component with the maximum concentration of 4.54% and 3.28% in the fish and pork waste, respectively. The conversion of sulfur compounds including total sulfur, SO42--S, S2-, methionine and cysteine followed the first-order kinetics. Miseq sequencing analysis showed that Acinetobacter, Clostridium, Proteus, Thiobacillus, Hyphomicrobium and Pseudomonas were the main known sulfur-metabolizing microorganisms in the fish and pork waste. The C/N value had most significant influence on the microbial community in the fish and pork waste. A main conversion of sulfur compounds with CH3SH as the key intermediate was firstly hypothesized during the anaerobic degradation of fish and pork waste. These findings are helpful to understand the conversion of sulfur compounds and to develop techniques to control ordor pollution in the anaerobic treatment of organic waste.

Methanethiol generation potential from anaerobic degradation of municipal solid waste in landfills.

Volatile sulfur compounds are the main odorants at landfills. In this study, methanethiol (CH3SH) was chosen as a typical volatile organic sulfur compound, and its generation potential was investigated during the anaerobic degradation of the organic fractions of municipal solid waste (MSW) including rice, flour food, vegetable, fish and pork, paper, cellulose textile, and yard wastes. Among the experimental wastes, gas generation was the highest in the fish and pork waste with a high CH3SH concentration of up to 2.5% (v/v). Sulfur reduction in the solid phase was mostly converted into gaseous sulfur compounds. During the whole experiment, the cumulative CH3SH generation from the fish and pork waste was 0.139 L kgdw-1, which was about 2 and 6 orders of magnitude higher than that from the other experimental wastes. The ratio of CH3SH-S to TS reduction was 31.56% in the fish and pork waste. These results would be helpful to understand the generation of volatile sulfur compounds during the anaerobic degradation of MSW and develop techniques to control odor pollution at landfills.

Response of methanotrophic activity to extracellular polymeric substance production and its influencing factors.

The accumulation of extracellular polymeric substance (EPS) is speculated to be related with the decrease of CH4 oxidation rate after a peak in long-term laboratory landfill covers and biofilters. However, few data have been reported about EPS production of methanotrophs and its feedback effects on methanotrophic activity. In this study, Methylosinus sporium was used asa model methanotroph to investigate EPS production and its influencing factors during CH4 oxidation. The results showed that methanotrophs could secret EPS into the habits during CH4 oxidation and had a negative feedback effect on CH4 oxidation. The EPS amount fitted well with the CH4 oxidation activity with the exponential model. The environmental factors such as pH, temperature, CH4, O2, NO3--N and NH4+-N could affect the EPS production of methanotrophs. When pH, temperature, CH4, O2 and N concentrations (including NO3--N and NH4+-N) were 6.5-7.5, 30-40°C, 10-15%, 10% and 20-140mgL-1, respectively, the high cell growth rate and CH4 oxidation activity of Methylosinus sporium occurred in the media with the low EPS production, which was beneficial to sustainable and efficient CH4 oxidation. In practice, O2-limited condition such as the O2 concentration of 10% might be a good way to control EPS production and enhance CH4 oxidation to mitigate CH4 emission from landfills.

[Mechanism of hypoxia-tolerance and community structure of aerobic methanotrophs in O2-limited environments: A review]. / ä½Žæ°§ç”Ÿå¢ƒä¸­å¥½æ°§ç”²çƒ·æ°§åŒ–èŒçš„ç¼ºæ°§è€å—æœºç†åŠç§ç¾¤ç»“æž„ç ”ç©¶è¿›å±•.

Methane bio-oxidation plays an important role in the global methane balance and greenhouse gases mitigation. Oxygen (O2) is one of the significant factors in methane bio-oxidation. The O2 concentration in the environments not only can affect community structure and activity of aerobic methanotrophs and the distribution of methane-derived carbon, but also aerobic methanotrophs have various ways of metabolism at different O2 concentrations. It is meaningful for carbon cycle and biodiversity of methane-driven ecosystem to understand the hypoxia-tolerance mechanisms of aerobic methanotrophs and methane bio-oxidation in O2-limited environments. In this paper, the activity and community structure of aerobic methanotrophs in O2-limited environments were summarized. The hypoxia-tolerance mechanism of aerobic methanotrophs and the relationship between methanotrophs and non-methanotrophs in O2-limited environments were reviewed. Future studies about aerobic methanotrophs were also discussed.

Ammonium conversion and its feedback effect on methane oxidation of Methylosinus sporium.

Ammonium (NH4+) is not only nitrogen source that can support methanotrophic growth, but also it can inhibit methane (CH4) oxidation by competing with CH4 for the active site of methane monooxygenase. NH4+ conversion and its feedback effect on the growth and activity of methanotrophs were evaluated with Methylosinus sporium used as a model methanotroph. Nitrogen sources could affect the CH4-derived carbon distribution, which varied with incubation time and nitrogen concentrations. More CH4-derived carbon was incorporated into biomass in the media with NH4+-N, compared to nitrate-nitrogen (NO3--N), as sole nitrogen source at the nitrogen concentrations of 10-18 mmol L-1. Although ammonia (NH3) oxidation activity of methanotrophs was considerably lower, only accounting for 0.01-0.06% of CH4 oxidation activity in the experimental cultures, NH4+ conversion could lead to the pH decrease and toxic intermediates accumulation in the their habits. Compared with NH4+, nitrite (NO2-) accumulation in the NH4+ conversion of methanotroph had stronger inhibition on its activity, especially the joint inhibition of NO2- accumulation and the pH decrease during the NH4+-N conversion. These results suggested that more attention should be paid to the feedback effects of NH4+ conversion by methanotrophs to understand effects of NH4+ on CH4 oxidation in the environments.

Conversion of methane-derived carbon and microbial community in enrichment cultures in response to O2 availability.

Methanotrophs not only play an important role in mitigating CH4 emissions from the environment, but also provide a large quantity of CH4-derived carbon to their habitats. In this study, the distribution of CH4-derived carbon and microbial community was investigated in a consortium enriched at three O2 tensions, i.e., the initial O2 concentrations of 2.5 % (LO-2), 5 % (LO-1), and 21 % (v/v) (HO). The results showed that compared with the O2-limiting environments (2.5 and 5 %), more CH4-derived carbon was converted into CO2 and biomass under the O2 sufficient condition (21 %). Besides biomass and CO2, a high conversion efficiency of CH4-derived carbon to dissolved organic carbon was detected in the cultures, especially in LO-2. Quantitative PCR and Miseq sequencing both showed that the abundance of methanotroph increased with the increasing O2 concentrations. Type II methanotroph Methylocystis dominated in the enrichment cultures, accounting for 54.8, 48.1, and 36.9 % of the total bacterial 16S rRNA gene sequencing reads in HO, LO-1, and LO-2, respectively. Methylotrophs, mainly including Methylophilus, Methylovorus, Hyphomicrobium, and Methylobacillus, were also abundant in the cultures. Compared with the O2 sufficient condition (21 %), higher microbial biodiversity (i.e., higher Simpson and lower Shannon indexes) was detected in LO-2 enriched at the initial O2 concentration of 2.5 %. These findings indicated that compared with the O2 sufficient condition, more CH4-derived carbon was exuded into the environments and promoted the growth of non-methanotrophic microbes in O2-limiting environments.