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Operation of membrane bioreactors (MBRs) for wastewater treatment is hampered by the membrane biofouling resulting from microbial activities. However, the knowledge of the microbial ecology of both biofilm and activated sludge in MBRs has not been sufficient. In this study, we scrutinized microbial communities of biofilm and activated sludge from 10 full-scale MBR plants. Overall, Flavobacterium, Dechloromonas and Nitrospira were abundant in order of abundance in biofilm, whereas Dechloromonas, Flavobacterium and Haliscomenobacter in activated sludge. Community structure was analyzed in either biofilm or activated sludge. Among MBRs, as expected, not only diversity of microbial community but also its composition was different from one another (p 0.05). Between the biofilm and activated sludge, community composition made significant difference, but its diversity measures (i.e., alpha diversity, e.g., richness, diversity and evenness) did not (p 0.05). Effects of ten environmental factors on community change were investigated using Spearman correlation. MLSS, HRT, F/M ratio and SADm explained the variation of microbial composition in the biofilm, whereas only MLSS did in the activated sludge. Microbial networks were constructed with the 10 environmental factors. The network results revealed that there were different topological characteristics between the biofilm and activated sludge networks, in which each of the 4 factors had different associations with microbial nodes. These results indicated that the different microbial associations were responsible for the variation of community composition between the biofilm and activated sludge.

DNA quantification has become a conventional method for quantifying filamentous fungi in environments. In this study, quantitative PCR was evaluated as a quantification tool by comparisons with plate count (unit, recoverable population) and microscopy with image analysis (hyphal biomass). The genetic transformant Trichoderma harzianumThzID1-M3 was used as a model organism. A soil microcosm experiment with different numbers of ThzID1-M3 alginate pellets showed that DNA was significantly correlated with biomass (P

Kinetic and enzymatic inhibition experiments were performed to investigate the effects of methanethiol (MT) and hydrogen sulfide (H2S) on methane oxidation by a methane-oxidizing consortium. In the coexistence of MT and H2S, the oxidation of methane was delayed until MT and H2S were completely degraded. MT and H2S could be degraded, both with and without methane. The kinetic analysis revealed that the methane-oxidizing consortium showed a maximum methane oxidation rate (Vmax) of 3.7 mmol g-dry cell weight (DCW)(-1) h(-1) and a saturation constant (Km) of 184.1 μM. MT and H2S show competitive inhibition on methane oxidation, with inhibition values (Ki) of 1504.8 and 359.8 μM, respectively. MT was primary removed by particulate methane monooxygenases (pMMO) of the consortium, while H2S was degraded by the other microorganisms or enzymes in the consortium. DNA and mRNA transcript levels of the pmoA gene expressions were decreased to ∼10(6) and 10(3)pmoA gene copy number g-DCW(-1) after MT and H2S degradation, respectively; however, both the amount of the DNA and mRNA transcript recovered their initial levels of ∼10(7) and 10(5)pmoA gene copy number g-DCW(-1) after methane oxidation, respectively. The gene expression results indicate that the pmoA gene could be rapidly reproducible after methane oxidation. This study provides comprehensive information of kinetic interactions between methane and sulfur compounds.

This study investigated the effects of proton exchange membranes (PEMs) on performance and microbial community of air-cathode microbial fuel cells (MFCs). Air-cathode MFCs with reactor volume of 1L were constructed in duplicate with or without PEM (designated as ACM-MFC and AC-MFC, respectively) and fed with a mixture of glucose and acetate (1:1, w:w). The maximum power density and coulombic efficiency did not differ between MFCs in the absence or presence of a PEM. However, PEM use adversely affected maximum voltage production and the rate of organic compound removal (p0.05). Quantitative droplet digital PCR indicated that AC-MFCs had a greater bacterial population than ACM-MFCs (p0.05). Likewise, ribosomal tag pyrosequencing revealed that the diversity index of bacterial communities was greater for AC-MFCs (p0.05). Network analysis revealed that the most abundant genus was Enterococcus, which comprised ≥62% of the community and was positively associated with PEM and negatively associated with the rate of chemical oxygen demand (COD) removal (Pearson correlation0.9 and p0.05). Geobacter, which is known as an exoelectrogen, was positively associated with maximum power density and negatively associated with PEM. Thus, these results suggest that the absence of PEM favored the growth of Geobacter, a key player for electricity generation in MFC systems. Taken together, these findings demonstrate that MFC systems without PEM are more efficient with respect to power production and COD removal as well as exoelectrogen growth.

The depth profiles of the CH4 oxidation potentials and the methanotrophic community were characterized in a lab-scale soil mixture biocover. The soil mixture samples were collected from the top (0–10 cm), middle (10–40 cm), and bottom (40–50 cm) layers of the biocover where most of methane was oxidized at the top layer due to consumption of O2. Batch tests using serum bottles showed that the middle and bottom samples displayed CH4 oxidation activity under aerobic conditions, and their CH4 oxidation rates were 85 and 71% of the rate of top sample (8.40 μmol g dry sample−1 h−1), respectively. The numbers of methanotrophs in the middle and bottom were not significantly different from those in the top sample. There was no statistical difference in the community stability indices (diversity and evenness) among the methanotrophic communities of the three layer samples, even though the community structures were distinguished from each other. Based on microarray analysis, type I and type II methanotrophs were equally present in the top sample, while type I was more dominant than type II in the middle and bottom samples. We suggested that the qualitative difference in the community structures was probably caused by the difference in the depth profiles of the CH4 and O2 concentrations. The results for the CH4 oxidation potential, methanotrophic biomass, and community stability indices in the middle and bottom layer samples indicated that the deeper layer in the methanotrophic biocover serves as a bioresource reservoir for sustainable CH4 mitigation.

Tobermolite was characterized as a bed material for methanotrophic biofiltration. A lab-scale biofilter packed with tobermolite was operated for different operation times under identical conditions. The three different runs showed similar acclimation patterns of methane oxidation, with methane removal efficiency increasing rapidly for the first few days and peaking within three weeks, after which the efficiency remained stable. The mean methane removal capacities ranged from 766gm(-3)d(-1) to 974gm(-3)d(-1) after acclimation. Pyrosequencing indicated that the methanotrophic proportion (methanotroph/bacteria) increased to 71-94% within three weeks. Type I methanotrophs Methylocaldum and Methylosarcina were dominant during the initial growth period, then Methylocaldum alone dominated the methanotrophic community. A community comparison showed that total bacterial and methanotrophic communities were temporally stable after the initial growth period. Quantitative PCR showed that methanotrophic density increased during the first 3-4 weeks, then remained stable over 120 days. Tobermolite can provide a special habitat for the selective growth of methanotrophs, resulting in rapid acclimation. Tobermolite also allows the microbial community and methanotrophic density to remain stable, resulting in stable methane biofiltration.

Relationship between the biocontrol fungus Trichoderma harzianum ThzID1-M3 and the plant pathogen Fusarium solani f.sp. pisi (Fsp) was determined in this study. Dual culture assay indicated the competitive interaction between ThzID1-M3 and Fsp. Alginate pellets of ThzID1-M3 and Fsp conidia were added to non-sterile soil with pea seeds. The interaction between Trichoderma and Fsp adversely affected their establishment in soil. The addition of ThzID1-M3 significantly reduced the Fsp population, as well as the colonization of roots by Fsp. In contrast, the added Fsp significantly reduced the proliferation of ThzID1-M3 and Trichoderma spp. The results suggest that the competition may be the main mechanism of the antagonistic activity of Trichoderma against Fsp.

Simultaneous removal of methane and malodor-causing volatile sulfur compounds (MVSCs), both emitted from landfills, is a desirable characteristic for methane-mitigation approaches. A methanotrophic bacterium was isolated from a microbial consortium, enriched with methane and dimethyl sulfide (DMS). It grew in the complex nutrient medium R2A without methane, and stably exhibited methanotrophic activity after facultative growth. It was identified as Sphingopyxis sp. MD2 by comparison of the 16S rRNA gene. It belongs to Sphingomonadales, whose members have not shown methanotrophic activity, phylogenetically distinct from orders of known methanotrophs. The MD2 biomass increased at a growth rate of 1.18d(-1) when methane was used as the sole growth substrate. An inhibition test with allylthiourea and PCR/sequencing confirmed the presence of particulate methane monooxygenase in MD2. DMS decreased the methane oxidation rate (2634±146 μmole g DCW(-1) h(-1)) by 12%, while H(2)S had no effect on the methane oxidation rate. Interestingly, methanethiol (MT) enhanced the methane oxidation rate by more than 50%. MD2 degraded H(2)S and MT, regardless of the presence of methane. MD2 also degraded DMS in the presence of methane, indicating co-metabolism. These combined results indicate that MD2 may be a promising biological resource for simultaneous removal of methane and MVSCs.

The biocontrol agent Trichoderma harzianum colonises sclerotia of the plant pathogenic fungus Sclerotinia sclerotiorum. Plating of sclerotia typically has been used to determine the incidence of mycoparasitism, but does not quantify the extent to which individual sclerotia are colonised. We developed a specific PCR primer/probe set for the green fluorescent protein (GFP)-transformant T. harzianum ThzID1-M3, which exhibited high precision and reproducibility. Quantitative real-time PCR was evaluated along with epifluorescence microscopy and image analysis to investigate dynamics of colonisation of sclerotia in non-sterile soil. Amounts of ThzID1-M3 DNA and S. sclerotiorum DNA from entire individual sclerotia were quantified using real-time PCR. Epifluorescence micrographs were captured from sclerotial thin-section samples, and GFP fluorescence from these was quantified using computer image analysis in order to estimate colonisation on a per-sclerotium basis. As determined by either method, ThzID1-M3 colonised sclerotia in soil, and both methods quantified colonisation dynamics over time. In a separate experiment, colonisation of sclerotia on agar plates was observed using confocal laser scanning microscopy to view the GFP-fluorescing hyphae of ThzID1-M3. This method, while highly labour-intensive, provided high spatial resolution of colonisation dynamics. Thus, each method has advantages: microscopy combined with image analysis can provide useful information on the spatial and temporal dynamics of colonisation, while real-time PCR can provide a more precise assessment of the extent of sclerotial colonisation over time and can more easily be used to sample entire sclerotia.

Some members of the fungal genus Trichoderma are able to colonize and destroy sclerotia, the thick-walled resting structures of the soilborne plant pathogenic fungus Sclerotinia sclerotiorum,thusproviding apotential means ofbiologicaldisease control. However, current methods to detect and quantify colonization of sclerotia are labor-intensive, and generally qualitative rather than quantitative in nature. Our objective was to develop quantitative real-time PCR (polymerase chain reaction) methods to detect and measure colonization of sclerotia by Trichoderma spp. Specific PCR primer/probe sets were developed for Trichoderma spp. and for S. sclerotiorum. A total of 180 ITS1 (internal transcribed spacer) and ITS2 sequences from different species in the genus Trichoderma were aligned, and consensus sequences were determined. Six candidate primer sets were based on conserved regions of the consensus sequence, and the specificity of each nucleotide sequence was examined using BLAST (Basic Local Alignment Search Tool; NCBI) software. Each candidate primer set was tested on genomic DNA of T. harzianum strain ThzID1-M3, as well as six different Trichoderma isolates from soil, and on genomic DNA of S. sclerotiorum. The optimum primer/ probe set selected, TGP4, successfully amplified genomic DNA of all Trichoderma isolates tested, and showed high precision and reproducibility over a linear range of eight orders of magnitude, from 85 ng to 8.5 fg of T. harzianum genomic DNA. TGP4 did not amplify S. sclerotiorum DNA. A specific PCR primer/probe set (TMSCL2) was developed for S. sclerotiorum, based on the calmodulin gene sequence. TMSCL2 did not amplify Trichoderma DNA. Quantitative real-time PCR with the primers then was evaluated in experiments to test differential effects of two soil moisture levels (� 50 kPa, � 1000 kPa matric potential) on biocontrol of S. sclerotiorum by indigenous Trichoderma spp. Periodically over 40 days, Trichoderma and S. sclerotiorum DNA in sclerotia were quantified by PCR with appropriate primers. Over 90% of the sclerotia were colonized by indigenous Trichoderma spp. at � 1000 kPa, over the 40-day period, compared to only 60% at � 50 kPa. In addition to determining incidence of colonization, the PCR method allowed measurement of the extent of sclerotial colonization, which also was significantly greater in the drier soil. Quantitative real-time PCR with the TGP4 primer/probe set provides a sensitive detection and measurement tool to evaluate colonization of sclerotia by Trichoderma spp.