Using RNA-Stable isotope probing to investigate methane oxidation metabolites and active microbial communities in methane oxidation coupled to denitrification.

The active denitrifying communities performing methane oxidation coupled to denitrification (MOD) were investigated using samples from an aerobic reactor (∼20% O2 and 2% CH4) and a microaerobic reactor (2% O2, 2% CH4) undertaking denitrification. The methane oxidation metabolites excreted in the reactors were acetate, methanol, formate and acetaldehyde. Using anaerobic batch experiments supplemented with exogenously supplied 13C-labelled metabolites, the active denitrifying bacteria were identified using 16S rRNA amplicon sequencing and RNA-stable isotope probing (RNA-SIP). With the aerobic reactor (AR) samples, the maximum NO3- removal rates were 0.43 mmol g-1 d-1, 0.40 mmol g-1 d-1, 0.33 mmol g-1 d-1 and 0.10 mmol g-1 d-1 for exogenously supplied acetate, formate, acetaldehyde and methanol batch treatments respectively, while with the microaerobic reactor (MR) samples, the maximum NO3- removal rates were 0.41 mmol g-1 d-1, 0.33 mmol g-1 d-1, 0.38 mmol g-1 d-1 and 0.14 mmol g-1 d-1 for exogenously supplied acetate, formate, acetaldehyde and methanol batch treatments respectively. The RNA-SIP experiments with 13C-labelled acetate, formate, and methanol identified Methyloversatilis, and Hyphomicrobium as the active methane-driven denitrifying bacteria in the AR samples, while Pseudoxanthomonas, Hydrogenophaga and Hyphomicrobium were the active MOD bacteria in the MR samples. Collectively, all the data indicate that formate is a key cross-feeding metabolite excreted by methanotrophs and consumed by denitrifiers performing MOD.

Microaerobic methane-driven denitrification in a biotrickle bed - Investigating the active microbial biofilm community composition using RNA-stable isotope probing.

A microaerobic (2% O2 v/v) biotrickle bed reactor supplied continuously with 2% methane to drive nitrate removal (MAME-D) was investigated using 16S rDNA and rRNA amplicon sequencing in combination with RNA-stable isotope probing (RNA-SIP) to identify the active microorganisms. Methane removal rates varied from 500 to 1000 mmol m-3h-1 and nitrate removal rates from 25 to 58 mmol m-3h-1 over 55 days of operation. Biofilm samples from the column were incubated in serum bottles supplemented with 13CH4. 16S rDNA analysis indicated a simple community structure in which four taxa accounted for 45% of the total relative abundance (RA). Dominant genera included the methanotroph Methylosinus and known denitrifiers Nubsella and Pseudoxanthomonas; along with a probable denitrifier assigned to the order Obscuribacterales. The 16S rRNA results revealed the methanotrophs Methylocystis (15% RA) and Methylosinus (10% RA) and the denitrifiers Arenimonas (10% RA) and Pseudoxanthomonas (7% RA) were the most active genera. Obscuribacterales was the most active taxa in the community at 22% RA. Activity was confirmed by the Δ buoyant density changes with time for the taxa, indicating most of the community activity was associated with methane oxidation and subsequent consumption of methanotrophic metabolic intermediates by the denitrifiers. This is the first report of RNA stable isotope probing within a microaerobic methane driven denitrification system and the active community was markedly different from the full community identified via 16S-rDNA analysis.

RNA stable isotope probing and high-throughput sequencing to identify active microbial community members in a methane-driven denitrifying biofilm.

AIMS: Aerobic methane oxidation coupled to denitrification (AME-D) is a promising process for removing nitrate from groundwater and yet its microbial mechanism and ecological implications are not fully understood. This study used RNA stable isotope probing (RNA-SIP) and high-throughput sequencing to identify the micro-organisms that are actively involved in aerobic methane oxidation within a denitrifying biofilm. METHODS AND RESULTS: Two RNA-SIP experiments were conducted to investigate labelling of RNA and methane monooxygenase (pmoA) transcripts when exposed to 13 C-labelled methane over a 96-hour time period and to determine active bacteria involved in methane oxidation in a denitrifying biofilm. A third experiment was performed to ascertain the extent of 13 C labelling of RNA using isotope ratio mass spectrometry (IRMS). All experiments used biofilm from an established packed bed reactor. IRMS confirmed 13 C enrichment of the RNA. The RNA-SIP experiments confirmed selective enrichment by the shift of pmoA transcripts into heavier fractions over time. Finally, high-throughput sequencing identified the active micro-organisms enriched with 13 C. CONCLUSIONS: Methanotrophs (Methylovulum spp. and Methylocystis spp.), methylotrophs (Methylotenera spp.) and denitrifiers (Hyphomicrobium spp.) were actively involved in AME-D. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to use RNA-SIP and high-throughput sequencing to determine the bacteria active within an AME-D community.

Development of a feedback control system for a differential biofilter degrading toluene contaminated air.

In this study, a proportional - integral feedback control system was implemented on a lab-scale differential biofilter to control the gas phase toluene concentration in the soil bed through online manipulation of the inlet toluene concentration. The feedback control system was based on a cascade controller that manipulated the setpoint of an air bath diffusion system to manipulate the inlet toluene concentration. The controller performed well for toluene concentrations in the reactor of 10 - 300 ppm for both setpoint changes and disturbance rejections; however, the system was nonlinear requiring different tuning parameters at different outlet concentrations. Feedback control of the toluene concentration in the differential reactor was used to explore the impact of concentration on start-up and long-term biofilter operation in a rigorous fashion. Starting at an reactor concentration of 20 ppm and then increasing to 65 ppm increased the toluene removal rate (33 ± 1.6 g m-3h-1) compared to starting the reactor at an outlet concentration of 81 ppm before settling at 65 ppm (42 ± 0.9 g m- 3h-1). The toluene removal rate increased with increasing outlet toluene concentration and then eventually decreased when reaching the inhibitory toluene concentration (ranged from 80 to 250 ppm).

The impact of environmental parameters on the conversion of toluene to CO2 and extracellular polymeric substances in a differential soil biofilter.

The fraction of pollutant converted to CO2 versus biomass in biofiltration influences the process efficacy and the lifetime of the bed due to pressure drop increases. This work determined the relative quantitative importance and potential interactions between three critical environmental parameters: toluene concentration (Tol), matric potential (ψ) and temperature (T) on % CO2, elimination capacity (EC) and the production rate of non-CO2 products. These parameters are the most variable in typical biofilter operation. The data was fit to a non-linear model of the form y=a(Tol)bTcψd. A rigorous carbon balance (100.5 ±â€¯7.0%) tracked the fate of degraded toluene as CO2 and non-CO2 carbon endpoints. The % CO2 mineralization varied from (34-91%) with environmental parameters: temperature (20-40 °C), matric potential, (-10 to -100 cmH2O) and residual toluene, (20-180 ppm). The highest conversion to CO2 was at the wettest conditions (-10 cmH2O) and lowest residual toluene concentration (18 ppm). Matric potential had twice the impact of toluene concentration on % CO2, while temperature had less impact. The elimination capacity varied from 11 to 50 gC⋅m-3h-1 and was highest at 40 °C, the wettest conditions with limited impact by toluene concentrations. Temperature increased the EC and non-CO2 production rates strongly while matric potential and toluene concentration had less influence (4x - 10x less). This study illustrated the quantitative significance and simultaneous interaction between critical environmental parameters on carbon endpoints and biofilter performance. This kind of multivariable parameter study provides valuable insights which can address performance and clogging issues in biofilters.

Fate of degraded pollutants in waste gas biofiltration: An overview of carbon end-points.

The fate of the carbon from degraded pollutants in biofiltration is not well understood. The issue of missing carbon needs to be addressed quantitatively to better understand and model biofilter performance. Elucidating the various carbon end-points in various phases should contribute to the fundamental understanding of the degradation kinetics and metabolic pathways as a function of various environmental parameters. This article reviews the implications of key environmental parameters on the carbon end-points. Various studies are evaluated reporting carbon recovery over a multitude of parameters and operational conditions with respect to the analytical measurements and reported distribution of the carbon end-points.

Effects of hydraulic and solids retention times on productivity and settleability of microbial (microalgal-bacterial) biomass grown on primary treated wastewater as a biofuel feedstock.

High biomass productivity and efficient harvesting are currently recognized challenges in microbial biofuel applications. To produce naturally settleable biomass, combined growth of native microalgae and bacteria was facilitated in laboratory sequencing batch reactors (SBRs) using primary treated wastewater from the Christchurch Wastewater Treatment Plant (CWTP) in New Zealand. SBRs were operated under a simulated, local, summer climate (i.e., 925 µmol/m(2)/s of photosynthetically active radiation for 14.7 h per day at 21 °C mean water temperature) using 1.4- to 8-day hydraulic retention times (HRTs) to optimize growth. Solids retention times (SRTs) were varied from 4 to 40 days by discharging different ratios of supernatant and completely mixed culture. Biomass productivity up to 31 g/m(2)/day of solids was obtained, and it generally increased as retention times decreased. Biomass settleability was typically 70-95%, and the microbes aggregated into compact flocs as cultures aged up to four months. Due to a low lipid content of 10.5%, anaerobic digestion appeared to be the most appropriate biofuel conversion process with potential to generate 19,200 m(3)/ha/yr of methane based on settleable mixture productivity.

Fungal inoculum properties and its effect on growth and enzyme activity of Trametes versicolor in soil.

The effect of fungal inoculum properties on colonization of nonsterile soil by three isolates of the white-rot fungus Trametes versicolor was investigated. Fungal inoculum properties were examined in separate experiments and were fungal inoculum composition, age of fungal inoculum, concentration of the inoculum and inoculation method. The fungal inoculum composition study compared pine versus poplar sawdust as the basic carrier with varying amounts of corn grit, corn meal and starch. The age of the fungal inoculum studied ranged from 3 to 21 days. The inoculum concentration gradually increased from 0 to 50% (v/v). The study assessing inoculation method compared mixing with layering techniques. The effect of moisture conditions of soil, sawdust and sand in combination with two inoculation methods (mixing versus point source inoculation) on colonization by T. versicolor was also determined. Colonization of soil was always assessed visually and enzymatically monitoring mycelial growth, biological potential (fluorescein diacetate assay) and laccase levels. Generally, the three different assessment methods correlated (P < 0.05) with each other. A fungal inoculum based on pine sawdust supported white-rot fungal growth in soil better than a poplar sawdust basis. Colonization of soil by T. versicolor was improved by increasing the corn content of the fungal inoculum. Younger (<7 days old) fungal inoculum resulted in better soil colonization than older (>10 days). A strong correlation (P < 0.001) was observed between the amount of fungal inoculum used in the soil augmentation and white-rot fungal colonization of soil. Inoculation of the fungal inoculum into soil by mixing was preferable over application in layers or point source inoculation. Moisture level did not influence biological potential measurements, but affected mycelial growth and laccase expression.