Coupled effects of methane monooxygenase and nitrogen source on growth and poly-β-hydroxybutyrate (PHB) production of Methylosinus trichosporium OB3b

The coupled effects of nitrogen source and methane monooxygenase(MMO) on the growth and poly-β-hydroxybutyrate(PHB) accumulation capacity of methanotrophs were explored.The ammonia-supplied methanotrophs expressing soluble MMO(s MMO) grew at the highest rate, while N_2-fixing bacteria expressing particulate MMO(p MMO) grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing p MMO, which might cause their slightly lower growth rate. The highest PHB content(51.0%) was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing p MMO. Ammoniasupplied bacteria also accumulated a higher content of PHB(45.2%) with the expression of p MMO, while N_2-fixing bacteria containing p MMO only showed low PHB production capacity(32.1%). The maximal PHB contents of bacteria expressing s MMO were low, with no significant change under different nitrogen source conditions. The low MMO activity,low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N_2 with the expression of p MMO were greatly improved in the cyclic NO_3-N_2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing p MMO.     

Isolation and characterization of a thermophilic Bacillus shackletonii K5 from a biotrickling filter for the production of polyhydroxybutyrate

Polyhydroxyalkanoates（PHAs） are aliphatic polyesters accumulated intracellularly by both Gram-negative and Gram-positive bacteria. However, compared to the PHAs of Gramnegative bacteria, few endotoxins（lipopolysaccharides, LPS）, which would be co-purified with PHAs and cause immunogenic reactions, are found in the PHAs produced by Gram-positive bacteria. A thermophilic Gram-positive bacterium K5, which exhibited good growth and polyhydroxybutyrate（PHB）-accumulating ability, has been isolated and characterized from a biotrickling filter designed for the removal of NOx from flue gas in a coal-fired power plant in China. Based on the biochemical characterization and 16 S rRNA gene sequence（Genbank accession no. JX437933）, the strain K5 has been identified as Bacillus shackletonii, which has rarely been reported in the literature, and this report is the first time that B. shackletonii has been found to accumulate PHB. The strain K5 was able to utilize glucose as carbon source to synthesize PHB at a broad range of temperatures（from 35 to 50 °C）, and the ideal temperature was 45 °C. The strain K5 could effectively yield PHB of up to 69.9% of its cell dry weight（CDW）（2.28 g/L） in flask experiments employing glucose as carbon source at 45 °C, followed by 56.8% and 52.3% of its CDW when using sodium succinate and glycerol as carbon source, respectively. For batch cultivation, the strain K5 was able to produce PHB of up to 72.6% of its cell dry weight（9.76 g/L） employing glucose as carbon source at 45 °C and pH 7.0.     

Diversity of methanotrophs in a simulated modified biocover reactor

A simulated landfill biocover microcosm consisting of a modifying ceramsite material and compost were investigated. Results show that the mixture can improve the material porosity and achieve a stable and highly efficient (100%) methane oxidation over an extended operating period. The diversity of the methanotrophic community in the microcosm was assessed. Type I methanotrophs were enhanced in the microcosm due to the increased air diffusion and distribution, whereas the microbial diversity and population density of type II methanotrophs were not significantly affected. Moreover, the type I methanotrophic community structure significantly varied with the reactor height, whereas that of type II methanotrophic communities did not exhibit a spatial variation. Phylogenetic analysis showed that type I methanotroph-based nested polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) resulted in the detection of eight different populations, most of which are related to Methylobacter sp., whereas that of type II resulted in the detection of nine different populations, most of which are related to Methylocystaceae. Methanotrophic community analysis also indicated that a number of new methanotrophic genera not closely related to any known methanotrophic populations were present.     

Bulking sludge for PHA production:Energy saving and comparative storage capacity with well-settled sludge

Two acetate-fed sequencing batch reactors (SBR) were operated under an aerobic dynamic feeding (ADF) model (SBR #2) and with anaerobic phase before aerobic phase (SBR #1) to select mixed cultures with a high polyhydroxyalkanoates (PHA) storage response. Although kinetic selection based on storage response should bring about a predominance of floc-formers, a bulking sludge with storage response comparable to well-settled sludge was steadily established. An anaerobic phase was introduced before the aerobic phase in the ADF model to improve the sludge settleability (SBR #1), however, due to the consequent increased feast/famine ratio, the performance of SBR #1, in terms of both the maximum PHB (polyhydroxybutyrate) cell content and ΔPHB, was lower than that of SBR #2. SBR #2 gradually reached a steady state while SBR #1 failed suddenly after 50 days of operation. The maximum specific substrate uptake rate and storage rate for the selected bulking sludge were 0.4 Cmol Ac/(Cmol X.hr) and 0.18 Cmol Ac/(Cmol PHB.hr), respectively, resulting a yield of 0.45 Cmol PHB/(Cmol Ac) in SBR #2 in the culture enrichment phase. A maximum PHB content of 53% of total suspended solids and PHB storage rate of 1.36 Cmol Ac/(Cmol PHB.hr) was achieved at 10.2 hr in batch accumulation tests under nitrogen starvation. The results indicated that it was feasible to utilize filamentous bacteria to accumulate PHA with a rate comparable to well-settled sludge. Furthermore, the lower dissolved oxygen demand of filamentous bacteria would save energy required for aeration in the culture enrichment stage.     

Comparative study of polyhydroxyalkanoates production from acidified and anaerobically treated brewery wastewater using enriched mixed microbial culture

The production of polyhydroxyalkanoates(PHA) from wastewaters using microbial mixed cultures(MMC) has been attracting increased interest because of PHA's biodegradability characteristics. Production of PHA by an MMC enriched with PHA-accumulating bacteria was compared using anaerobically treated and acidified brewery wastewaters under various feeding strategies, namely pulse and batch feed addition. To obtain an enriched MMC, a sequencing batch reactor was inoculated with activated sludge fed with acetate and subjected to aerobic dynamic feeding. The enriched MMC was able to accumulate PHA up to 72.6% of cell dry weight(CDW) with pulse addition of acetate controlled by the dissolved oxygen(DO) concentration in the reactor. In a batch accumulation experiment with acetate,the PHA content achieved(28.5% CDW) was less than that of the pulse feeding strategy with the same amount of acetate(～2000 mg C/L). Using anaerobically treated and acidified brewery wastewater fed in pulses, the maximum PHA accumulated by the enriched MMC was similar for both wastewaters(45% CDW), in spite of the higher volatile fatty acid concentration in acidified brewery wastewater. The pulse feed addition controlled by the DO concentration was difficult to implement for wastewater as compared to acetate because the difference in DO concentration between substrate availability and depletion was low. For the batch addition of acidified wastewater, a slightly lower PHA content(39%CDW) was obtained. These results show that both brewery wastewaters can be utilized for PHA production with a similar maximum PHA storage capacity.     

Distribution of Cu and Pb in particle size fractions of urban soils from different city zones of Nanjing, China

Soil samples from 4 defined city zones of Nanjing were randomly collected at 0-5 cm and 5-20 cm intervals and size fractions of soil particles were separated from undisturbed bulk soils by low energy dispersion procedure. The total contents of Cu and Pb in the different particle size fractions of the urban soils were analyzed by HNO3-HF-HClO4 digestion and flame atomic absorption spectrophotometer determination. The total content of Cu and Pb in soil particle size fractions varied with their size and with city zones as well. Both the content and variation with the size fractions of Pb was bigger than of Cu supporting our previous finding that there was Pb pollution to different degrees in the urban soils although the two elements were generally enriched in clay-sized fraction. Contaminated Pb tended to be preferentially enriched in the size fraction of 2000-250 μm and clay-sized fraction. While the size fractions of the soils from newly developed and preserved area contained smaller amount of Cu and Pb, the partitioning of them in coarse and fine particle size fractions were insignificant compared to that from inner residence and commercial area. The very high Pb level over 150 mg/kg of the fine particle fractions from the soils of the inner city could be a cause of high blood Pb level reported of children from the city as acute exposure to Pb of fine particles of the urban soil might occur by soil ingestion and inhalation by young children. Thus, much attention should be paid to the partitioning of toxic metals in fine soil particles of the urban soils and countermeasures against high health risk of Pb exposure by soil ingestion and dust inhalation should be practiced against the health problem of blood Pb for young children from the cities.     

Instrumental and bio-monitoring of heavy metal and nanoparticle emissions from diesel engine exhaust in controlled environment

In the present article we characterized the emissions at the exhaust of a Common Rail (CR) diesel engine, representative of lightduty class, equipped with a catalyzed diesel particulate filter (CDPF) in controlled environment. The downstream exhausts were directly analyzed (for PM, CO, CO2, O2, HCs, NOx) by infrared and electrochemical sensors, and SEM-EDS microscope; heavy metals were chemically analyzed using mosses and lichens in bags, and glass-fibre filters all exposed at the engine exhausts. The highest particle emission value was in the 7–54 nm size range; the peak concentration rose until one order of magnitude for the highest load and speed. Particle composition was mainly carbonaceous, associated to noticeable amounts of Fe and silica fibres. Moreover, the content of Cu, Fe, Na, Ni and Zn in both moss and lichen, and of Al and Cr in moss, was significantly increased. Glass-fibre filters were significantly enriched in Al, B, Ba, Cu, Fe, Na, and Zn. The role of diesel engines as source of carbonaceous nanoparticles has been confirmed, while further investigations in controlled environment are needed to test the catalytic muffler as a possible source of silica fibres considered very hazardous for human health.     

Effect of bio-column composed of aged refuse on methane abatement – A novel configuration of biological oxidation in refuse landfill

An experimental bio-column composed of aged refuse was installed around the exhaust pipe as a new way to mitigate methane in refuse landfill.One of the objectives of this work was to assess the effect of aged refuse thickness in bio-column on reducing CH 4 emissions.Over the study period,methane oxidation was observed at various thicknesses,5 cm (small size),10 cm (middle size) and 15 cm (large size),representing one to three times of pipeline diameters.The middle and large size both showed over 90% methane conversion,and the highest methane conversion rate of above 95% occurred in the middle-size column cell.Michaelis-Menten equation addressed the methanotrophs diffusion in different layers of the bio-columns.Maximum methanotrophic activity (V max) measured at the three thicknesses ranged from 6.4 × 10 3 to 15.6 × 10 3 units,and the half-saturation value (K M) ranged from 0.85% to 1.67%.Both the highest V max and K M were observed at the middle-size of the bio-column,as well as the largest methanotrophs population,suggesting a significant efficiency of methane mitigation happened in the optimum zone with greatest affinity and methanotrophic bacteria activities.Therefore,bio-column is a potential style for methane abatement in landfill,and the aged refuse both naturally formed and artificially placed in the column plays a critical role in CH 4 emission.     

Universally improving effect of mixed electron donors on the CO2 fixing efficiency of nonphotosynthetic microbial communities from marine environments

The universality of improved CO2 fixing upon the addition of mixed electron donors（MEDs）composed of Na2 S,NO2-,and S2O32-to non-photosynthetic microbial communities（NPMCs）obtained from 12 locations in four oceans of the world was validated. The CO2 fixing efficiencies of NPMCs were universally enhanced by MED compared with those obtained using H2 alone as electron donor,with average increase of about 276%. An increase in microbial inoculation concentration could increase the net amount of CO2 fixing to853.34 mg/L in the presence of MED. NO2-and S2O32-may play the roles of both electron acceptor and electron donor under aerobic conditions,which may improve the energy utilization efficiency of NPMC and enhance the CO2 fixation efficiency. The sequence determination of 16 S ribosomal deoxyribonucleic acid（rDNA） from 150 bacteria of NPMC showed that more than 50% of the bacteria were symbiotic and there were many heterotrophic bacteria such as Vibrio natriegens. These results indicate that NPMC acts as a symbiotic CO2 fixing system. The interaction between autotrophic and heterotrophic bacteria may be a crucial factor supporting ladder utilization and recycling of energy/carbon source.     

Effect of media heights on the performance of biological aerated filter

The optimum media height of carbon oxidation and nitrification in a down-flow biological aerated filter was determined, and the distribution of the heterotrophic and nitrifying populations through studying the changes of organic carbon contents and ammonia concentration at different media height was got. The results showed that as a down flow BAF with granular media, the active layer of nitrifiere was deeper than heterotrophs in BAF. And the optimum media height for the removal of SS, CODcr and NH4^＋ -N was 40 cm,60cm and 80 cm respectively. The removal efficiency of SS, CODcr and NH4^＋ -N was 79.1%, 63.9% and 96.4% respectively under theinfluent CODcr and NH4^＋ -N of 122.1 mgCODcr/L and 14.84 mgNH4^＋ -N/L, the influent flux of 15.8 L/h, air to liauid ratio of 3 : 1.     

Contributions of root cell wall polysaccharides to Cu sequestration in castor (Ricinus communis L.) exposed to different Cu stresses

Cell wall polysaccharides play a vital role in binding with toxic metals such as copper(Cu)ions.However, it is still unclear whether the major binding site of Cu in the cell wall varies with different degrees of Cu stresses.Moreover, the contribution of each cell wall polysaccharide fraction to Cu sequestration with different degrees of Cu stresses also remains to be verified.The distribution of Cu in cell wall polysaccharide fractions of castor(Ricinus communis L.) root was investigated with various Cu concentrations in the hydroponic experiment.The results showed that the hemicellulose1(HC1) fraction fixed 44.9%–67.8% of the total cell wall Cu under Cu stress.In addition, the pectin fraction and hemicelluloses2(HC2) fraction also contributed to the Cu binding in root cell wall,accounting for 11.0%–25.9% and 14.1%–26.6% of the total cell wall Cu under Cu treatments, respectively.When the Cu levels were ≤ 25 μmol/L, pectin and HC2 contributed equally to Cu storage in root cell wall.However, when the Cu level was higher than 25 μmol/L, the ability of the pectin to bind Cu was easy to reach saturation.Much more Cu ions were bound on HC1 and HC2 fractions, and the HC2 played a much more important role in Cu binding than pectin.Combining fourier transform infrared(FT-IR) and twodimensional correlation analysis(2 D-COS) techniques, the hemicellulose components were showed not only to accumulate most of Cu in cell wall, but also respond fastest to Cu stress.     

Methane oxidation and attenuation of sulphur compounds in landfill top cover systems: Lab-scale tests

In this study,a top cover system is investigated as a control for emissions during the aftercare of new landfills and for old landfills where biogas energy production might not be profitable.Different materials were studied as landfill cover system in lab-scale columns:mechanical–biological pretreated municipal solid waste(MBP);mechanical–biological pretreated biowaste(PB);fine(PBS_f)and coarse(PBS_c)mechanical–biological pretreated mixtures of biowaste and sewage sludge,and natural soil(NS).The effectiveness of these materials in removing methane and sulphur compounds from a gas stream was tested,even coupled with activated carbon membranes.Concentrations of CO_2,CH_4,O_2,N_2,H_2S and mercaptans were analysed at different depths along the columns.Methane degradation was assessed using mass balance and the results were expressed in terms of methane oxidation rate(MOR).The highest maximum and mean MOR were observed for MBP(17.2 g CH_4/m~2/hr and 10.3 g CH_4/m~2/hr,respectively).Similar values were obtained with PB and PBS_c.The lowest values of MOR were obtained for NS(6.7 g CH_4/m~2/hr)and PBS_f(3.6 g CH_4/m~2/hr),which may be due to their low organic content and void index,respectively.Activated membranes with high load capacity did not seem to have an influence on the methane oxidation process:MBP coupled with 220 g/m~2and 360 g/m~2membranes gave maximum MOR of 16.5 g CH_4/m~2/hr and 17.4 g CH_4/m~2/hr,respectively.Activated carbon membranes proved to be very effective on H_2S adsorption.Furthermore,carbonyl sulphide,ethyl mercaptan and isopropyl mercaptan seemed to be easily absorbed by the filling materials.     

Digestion performance and microbial community in full-scale methane fermentation of stillage from sweet potato-shochu production

Sweet potato shochu is a traditional Japanese spirit produced mainly in the South Kyushu area in Japan. The amount of stillage reaches approximately 8 × 105tons per year. Wastewater mainly containing stillage from the production of sweet potato-shochu was treated thermophilically in a fullscale treatment plant using fixed-bed reactors(8 reactors × 283 m3). Following the addition of Ni2+ and Co2+, the reactors have been stably operated for six years at a high chemical oxygen demand(COD) loading rate of 14 kg/(m3·day). Analysis of coenzyme content and microbial communities indicated that similar microbial communities were present in the liquid phase and on the fiber carriers installed in reactors. Bacteria in the phyla Firmicutes as well as Bacteroidetes were dominant bacteria, and Methanosarcina thermophila as well as Methanothermobacter crinale were dominant methanogens in the reactors. This study reveals that stillage from sweet potato-shochu production can be treated effectively in a full-scale fixed-bed reactor under thermophilic conditions with the help of Ni2+and Co2+. The high diversity of bacterial community and the coexistence of both aceticlastic and hydrogenotrophic methanogens contributed to the excellent fermentation performance.     

Polycyclic aromatic hydrocarbons in soils of Beijing and Tianjin region： Vertical distribution, correlation with TOC and transport mechanism

The contents and distribution of 20 polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic hydrocarbons (HAHs) were investigated in 16 soil profiles of Beijing and Tianjin region. Transport of high molecular weight PAHs (HMWPAHs) and the correlation between total organic carbon (TOC) and their concentrations were also discussed. The results indicated that highly contaminated sites were located at urban or wastewater irrigation areas and pollutants mainly accumulated in topsoil (< 40 cm), with a sharp content decrease at the vertical boundary of 30–40 cm. Total PAHs/HAHs concentrations in soils from Tianjin were markedly greater than those from Beijing. Even the contents at bottoms of soil profiles in Tianjin were higher than those in topsoils of Beijing soil profile. HMWPAHs dominated the PAH profiles, exhibiting a uniform distribution of pyrogenic origin between topsoils and deep layers. Furthermore, the percentages of HMWPAHs remained relative constant with the depth of soil profiles, which were consistent with the distribution of particulate matter-associated PAHs in the local atmospheric environments. Therefore, HMWPAHs transport with particulates might be the predominant source found in soil profiles.     

Adsorption of benzene, cyclohexane and hexane on ordered mesoporous carbon

Ordered mesoporous carbon(OMC) with high specific surface area and large pore volume was synthesized and tested for use as an adsorbent for volatile organic compound(VOC)disposal. Benzene, cyclohexane and hexane were selected as typical adsorbates due to their different molecular sizes and extensive utilization in industrial processes. In spite of their structural differences, high adsorption amounts were achieved for all three adsorbates, as the pore size of OMC is large enough for the access of these VOCs. In addition, the unusual bimodal-like pore size distribution gives the adsorbates a higher diffusion rate compared with conventional adsorbents such as activated carbon and carbon molecular sieve. Kinetic analysis suggests that the adsorption barriers mainly originated from the difficulty of VOC vapor molecules entering the pore channels of adsorbents. Therefore, its superior adsorption ability toward VOCs, together with a high diffusion rate, makes the ordered mesoporous carbon a promising potential adsorbent for VOC disposal.     

New microbial electrosynthesis system for methane production from carbon dioxide coupled with oxidation of sulfide to sulfate

Microbial electrosynthesis system (MES) is a promising method that can use carbon dioxide, which is a greenhouse gas, to produce methane which acts as an energy source, without using organic substances. However, this bioelectrical reduction reaction can proceed at a certain high applied voltage when coupled with water oxidation in the anode coated with metallic catalyst. When coupled with the oxidation of HS^– to SO₄²⁻, methane production is thermodynamically more feasible, thus implying its production at a considerably lower applied voltage. In this study, we demonstrated the possibility of electrotrophic methane production coupled with HS^– oxidation in a cost-effective bioanode chamber in the MES without organic substrates at a low applied voltage of 0.2 V. In addition, microbial community analyses of biomass enriched in the bioanode and biocathode were used to reveal the most probable pathway for methane production from HS^– oxidation. In the bioanode, electroautotrophic SO₄²⁻ production accompanied with electron donation to the electrode is performed mainly by the following two steps: first, incomplete sulfide oxidation to sulfur cycle intermediates (SCI) is performed; then the produced SCI are disproportionated to HS^– and SO₄²⁻. In the biocathode, methane is produced mainly via H₂ and acetate by electron-accepting syntrophic bacteria, homoacetogens, and acetoclastic archaea. Here, a new eco-friendly MES with biological H₂S removal is established.     

Organic carbon source and burial during the past one hundred years in Jiaozhou Bay, North China

Organic carbon (OC), total nitrogen (TN), and 210Pb in core sediment were measured to quantify the burial of organic carbon and the relative importance of allochthonous and autochthonous contributions during the past one hundred years in Jiaozhou Bay, North China. The core sediment was dated using 210Pb chronology, which is the most promising method for estimation of sedimentation rate on a time scale of 100–150 years. The variation of the burial flux of organic carbon in the past one hundred years can be divided into the following three stages: (1) relatively steady before 1980s; (2) increasing rapidly from the 1980s to a peak in the 1990s, and (3) decreasing from the 1990s to the present. The change is consistent with the amount of solid waste and sewage emptied into the bay. The OC:TN ratio was used to evaluate the source of organic carbon in the Jiaozhou Bay sediment. In the inner bay and bay mouth, the organic carbon was the main contributor from terrestrial sources, whereas only about half of organic carbon was contributed from terrestrial source in the outer bay. In the inner bay, the terrestrial source of organic carbon showed a steady change with an increase in the range of 69%–77% before 1990 to 93% in 2000, and then decreased from 2000 because of the decrease in the terrestrial input. In the bay mouth, the percentage of organic carbon from land reached the highest value with 94% in 1994. In the outer bay, the sediment source maintained steady for the past one hundred years.     

Insights into metals in individual fine particles from municipal solid waste using synchrotron radiation-based micro-analytical techniques

Excessive inter-contamination with heavy metals hampers the application of biological treatment products derived from mixed or mechanically-sorted municipal solid waste(MSW). In this study, we investigated fine particles of 2 mm, which are small fractions in MSW but constitute a significant component of the total heavy metal content, using bulk detection techniques. A total of 17 individual fine particles were evaluated using synchrotron radiation-based micro-X-ray fluorescence and micro-X-ray diffraction. We also discussed the association, speciation and source apportionment of heavy metals. Metals were found to exist in a diffuse distribution with heterogeneous intensities and intense hot-spots of 10 μm within the fine particles. Zn–Cu, Pb–Fe and Fe–Mn–Cr had significant correlations in terms of spatial distribution. The overlapped enrichment, spatial association, and the mineral phases of metals revealed the potential sources of fine particles from size-reduced waste fractions(such as scraps of organic wastes or ceramics) or from the importation of other particles. The diverse sources of heavy metal pollutants within the fine particles suggested that separate collection and treatment of the biodegradable waste fraction(such as food waste) is a preferable means of facilitating the beneficial utilization of the stabilized products.     

Effect of air masses motion on the rapid change of aerosols in marine atmosphere

The impact of air masses motion on marine aerosol properties was investigated using an on-board single particle mass spectrometer(SPAMS) deployed for the determination of single particle size resolved chemical composition over Southeast China Sea. Two aerosol blooms(E1 and E2) were observed during the cruise. High average particle number count occurred in E1(7320), followed by E2(5850), which was more than 100–150 times of the average particle number count during normal periods. Particles were classified as four major sources, including continental source, shipping source, marine source, and transport source based on the mass spectral similarity. Transport source was identified as those particles with high particle number count occurred only during aerosol bloom period. Three sub-types of EC-Ca, OC-Ca, and Al-rich were classified as transport source.EC-Ca was the dominant particles of the transport source, accounting for more than 70%of the total particles in aerosol bloom events. A uni-modal size distribution in the size range of 0.1–2.0 μm was observed during normal period, while a bimodal distribution with a tiny mode(0.3 μm) and a coarse mode between 0.4 and 0.6 μm was present during aerosol bloom. The variation of aerosol source is consistent with air masses back trajectories, for the reason that most of the long-range air trajectories are from the ocean,while short air trajectories originate in the continental regions, which means that air masses have a significant impact on the aerosol physical–chemical properties along their tracks.     

Methanotrophic community structure of aged refuse and its capability for methane bio-oxidation

Aged refuse from waste landfills closed for eight years was examined and found to contain rich methanotrophs capable of biooxidation for methane. Specially, community structure and methane oxidation capability of methanotrophs in the aged refuse were studied. The amount of methanotrophs ranged 61.97 103–632.91 103 cells/g (in dry basis) in aged refuse from Shanghai Laogang Landfill. Type I and II methanotrophs were found in the aged refuse in the presence of sterilized sewage sludge and only Type I methanotrophs were detected in the presence of nitrate minimal salt medium (NMS). The clone sequences of the pmoA gene obtained from the aged refuse were similar to the pmoA gene of Methylobacter, Methylocaldum, and Methylocystis, and two clones were distinct with known genera of Type I methanotrophs according to phylogenetic analysis. Aged refuse enriched with NMS was used for methane biological oxidation and over 93% conversions were obtained.