Ecophysiology of microbial populations from novel, Horizontal Flow Biofilm Reactors (HFBRs) treating methane-contaminated air S. Gerrity1, C. Kennelly2, R. Walsh3

, E. O. Reilly2, G. Collins1, E. Clifford2 1. School of Natural Sciences, National University of Ireland, Galway 2. School of Engineering, National University of Ireland, Galway 3. Irish Seaweed Centre, Ryan Institute, National University of Ireland, Galway Email: s.gerrity1@nuigalway.ie

Abstract

Three Horizontal Flow Biofilm Reactors (HFBRs) were used to treat contaminated airstreams containing low concentrations of methane (CH4) at 10°C. The HFBRs were seeded with a highly enriched, mixed biomass culture capable of CH4 oxidation. During the trial, which lasted 177 days, average CH4 removal rates of up 47.9% were observed in the HFBRs. A series of perturbations was applied to the HFBRs during the trial, including a low temperature period when the temperature was reduced to between 1°C and 5°C, and a shutdown period lasting 14 days when the CH4 supply was turned off. Reduced CH4 removal rates were observed in response to perturbations, but the HFBRs recovered after normal conditions were restored. Temporal biofilm samples were taken for fingerptinting of 16S rRNA genes, which indicated slightly reduced diversity over the trial. Fluorescent in-situ Hybridizations detected both Type I and Type II methanotrophs, although in dispersed, small clusters along the biofilm. Maximum potential methane oxidation potential assays were used to characterise biofilm from vertically-resolved zones of sheets in the HFBRs and indicated excellent potential of the HFBRs to biologically treat low concentrations of CH4 at 10°C.

Keywords: HFBR, methane oxidation, methanotrophs, TRFLP.

Introduction

Methane (CH4) is an important greenhouse gas (IPCC, 2007), which is produced when organic matter is degraded by microorganisms under anaerobic conditions. Anthropogenic sources of methane include landfills, wastewater treatment facilities and agriculture, which contribute significantly to global CH4 emissions. In the EU, legislation dealing with the generation and treatment of odours (EC, 2005), is becoming increasing stringent. Traditional technologies used to treat foul airstreams have been physical and chemical; however, the growing popularity of biological treatment of foul airstreams can be attributed to the ability of the microorganisms involved to destroy pollutants rather that just transferring them from the gas to liquid phase (Burgess et al., 2001).

Horizontal Flow Biofilm Reactors (HFBRs) have previously been used to treat domestic-strength wastewater (Rodgers and Clifford, 2009) and dairy wastewater (Rodgers et al., 2007). The unique flow regime in the HFBR ensures good contact between the wastewater and the biofilm in the reactor and alleviates problems that can be associated with conventional biofilm reactors, such as clogging, channelling, compaction and pressure drop. In this study, three HFBRs were designed to treat low concentrations of CH4 at 10°C. Air flow and methane loading rates were recorded; the methane concentration through zones of the HFBRs was determined; and in situ removal rates and the removal efficiency (RE) of each HFBR were calculated. Biofilm samples from various HFBR zones were used to determine microbial ecology using terminal restriction fragment length polymorphism (TRFLP) analyses and fluorescence in situ hybridizations (FISH). Maximum potential methane oxidation activity of biofilm samples from the different zones were determined using batch activity assays. Materials and methods

HFBR design and set up

The three pilot-scale HFBR units each comprised a stack of 57 horizontal plastic sheets positioned one above the other (Fig. 1). Each sheet measured 0.2 m x 0.2 m in plan and had integrated frustums 8 mm high and 8 mm diameter at 15 mm centres. For each HFBR, the stack of sheets was enclosed in an airtight, sealed reactor fitted with a removable panel to allow for easy access to the biofilm growing on the sheets. The working volume of each reactor was 20 l and the top plan surface area (TPSA) of the plastic media was 0.04 m2 (0.2 m x 0.2 m), giving a total medium top plan area of 2.28 m2 (57 sheets x 0.04 m2/sheet). The air mixture was introduced at the top of each reactor, just above Sheet 1, and flowed horizontally across each sheet before moving to the sheet below and flowing along that sheet in the opposite direction. This pattern continued throughout the reactor. Synthetic synthetic wastewater (SWW; (Odegaard and Rusten, 1980) was added to the reactors in a step-feed manner; 75% were added onto Sheet 1 and 25% onto Sheet 30. Six sampling ports located vertically along the side of each reactor permitted intermediate gas and SWW sampling. Thus, the reactors were each divided into 7 distinct sampling regions for gas analyses (Fig. 1), such that removal ‘profiles’ could be established.

Figure 1. Schematics illustrating (A) the HFBR setup; and (B) biofilm sampling zones and gas sample ports.

Operating conditions and perturbations

The HFBRs were operated for a total of 177 d. The average CH4 influent loading rate for each HFBR was 12.84 g CH4/m

3/h (0.8 mol CH4/m3/h) with an average influent air CH4

concentration of 1.55%. The gas loading rate for each HFBR was 1.2 m3/m3/h. An intermittent supply of SWW was pumped into the reactors for 10 min each hour. The total volume of SWW added to each reactor was 8 l/d, comprising 6 l through the main influent line (Sheet 1) and 2 l at the step-feed (Sheet 30). The purpose of the SWW was to provide necessary nutrients to facilitate biofilm growth, to hydrate the biofilm and to allow for the diffusion of CH4 across the biofilm. The HFBRs were operated at 10°C for the duration of the trial, except for during a ‘Cold Period’, which comprised 17 d between day 90 and day 107. During this period, air temperature in the laboratory fluctuated between 1°C and 5°C. The HFBRs were also subjected to a 14-d ‘Shutdown period’ between day 120 and day 133, during which the CH4 supply was turned off. The biofilm was hydrated during this period with an SWW supply.

Seeding and enrichment cultures

The HFBRs were initially seeded with activated sludge sourced from full-scale sewage treatment plants. Even after one month of operation, no methane oxidation could be detected. Therefore, an enrichment strategy was employed to cultivate a methanotroph-rich biomass capable of methane oxidation, which could be used to re-seed the HFBRs. A biomass mix comprising landfill cover soil, landfill leachate, composted OFMSW and compost leachate in a 1:1:1:1 ratio was used for the enrichment culture. Briefly, 2 ml of the biomass mix were

A.  B. 

placed in each of several 40-ml crimp-top, glass vials with 8 ml Adapted Whittenbury Medium (AWM; Whittenbury et al., (1970). The vials were sealed and the headspace was adjusted to a methane concentration of 10% at atmospheric pressure. Vials were incubated in the dark at 10°C shaking at 80 rpm. The headspace methane concentration was monitored by gas chromatography (GC; Varian CP-3800 Gas Chromatograph) analysis of twice-weekly headspace samples. Once the headspace methane concentration was <0.5%, the headspace was flushed with air and a 10% methane headspace was re-instated. Over the course of 4 months, the cultures were subcultured (c. 10% inoculum) to fresh medium and eventually were scaled to 2-l enrichment cultures to cultivate sufficient biomass for HFBR inoculation. The enriched culture was added to the HFBRs (‘day 0’), along with the SWW, and then re-circulated around the systems for several days to encourage biofilm formation and to bio-augment the biofilm already present, before the feeding regime was resumed.

Gas analyses

Influent, effluent and profile samples of the gas mixtures were taken from each reactor. The profile samples were taken from the sampling ports located along the vertical profile of each reactor (Fig. 1) using a gas-tight syringe and analysed using a Varian CP-3800 Gas Chromatograph (GC).

Biofilm sampling

The HFBR biofilms were sampled 5 times during the trial: day 0 (immediately prior to HFBR re-seeding with the enriched methanotrophic biomass); and days 44, 84, 108 and 177. For sampling, each reactor was notionally divided into six distinct zones (Fig. 1). Sheets from each zone were briefly detached from the system and composite samples of biofilm (c. 4 g) were sampled from across each respective zone. Biofilm samples were immediately placed in liquid nitrogen and then transferred to -80°C for storage until further analysis. A total of 89 biofilm samples were taken from the HFBRs over the course of the trial.

DNA extraction

Samples were centrifuged at 3,500 X g for 2 min and the supernatant was discarded. For each sample, a total mass of 20 mg was then suspended in 80 µl DEPC H2O. The mixtures were passed through a pipette tip several times and vortexed for 2 min to disassociate the biofilm structure. DNA was extracted using an automatic extractor and a Magtration Genomic DNA Whole Blood Kit 100 (VhBio). Separately, DNA from the landfill/compost/leachate mixture (used for enrichment culture) was extracted using a PowerSoil DNA isolation kit (MoBio). This sample was pre-treated by freezing with liquid nitrogen and crushing with a pestle and mortar to free cells from other organic material. All DNA extractions were visualised on 1%

(w/v) agarose gels containing SYBR Gold under UV light, and quantified spectrophotometrically using a nano-drop (Thermo Scientific, UK).

PCR-TRFLP analyses

Polymerase chain reaction (PCR) assays were performed using all DNA extracts with the universal bacterial 16S rRNA gene primers; 27F (labeled with FAM 6-carboxyfluorescein) and HEX-labeled 1392R (Lane et al., 1985). An aliquot of 2 µl of the DNA template was added to a PCR mixture of 5 µl PCR buffer, 3 µl MgCl2 (25 mM), 1 µl dNTPs (10 µM), 1 µl 27F, 1 µl 1392R and 0.5 µl BioTaq polymerase. The PCR amplification protocol was as follows: 2 min at 94°C, and 30 cycles consisting of 1 min at 94°C, 1 min at 57°C and 1 min at 72°C, followed by 10 min at 72°C.

Clone library construction

The bacterial community in the landfill seed mix (used for the enrichment culture) was characterised by clone library construction using the unlabeled universal bacterial primers, 27F and 1392R. Following PCR amplification of the extracted DNA, the PCR template was cloned (TOPO TA; Invitrogen) into competent E. coli cells. One-hundred-and-seventy clones were randomly selected for screening, comprising PCR-amplification of the insert and flanking plasmid regions, using an M13-specific primer set, as described previously by Collins et al., (2003). Amplified ribosomal DNA restriction analysis (ARDRA) was used to compare genetic polymorphisms of the clones and to calculate the relative abundance of 119 unique operational taxonomic units (OTUs), which were identified based on restriction patterns. Inserts from representative clones from each OTU were sequenced and used for phylogenetic analyses, as outlined by Collins et al., (2003).

TRFLP

The virtual TRFLP restriction profiles of clonal sequences were determined, and compared, in-silico using six different enzymes; Alu I, Hae III, HhaI, Mbo I, Mse I, Msp I using NEBcutter software. Based on their restriction patterns; Alu I, Hae III and Hha I were selected for in vitro assays with the PCR-TRFLP amplicons. All restriction enzymes were sourced from Fermantas and the PCR products were digested according to the manufacturer’s instructions. The restriction digests were analysed using ABI sequencers and software by MWG, Berlin, Germany and data were processed using the ABI GeneScan Application.

Fluorescence in-situ Hybridization (FISH)

Several HFBR biofilm samples from day 177 were analysed using FISH. Biofilm samples were carefully removed from the HFBRs ensuring that the integrity of the biofilm structure was not destroyed. The samples were incubated in 4% paraformaldehyde (w/v in 1X PBS) at 4°C overnight. The PFA was discarded and the biofilm was washed in 1X PBS three times. The sample were stored in ethanol:1X PBS (1:1) at -20°C until required. The samples were washed in ddH2O to remove the ethanol/PBS solution and incubated in tissue freezing medium at -20°C overnight. Vertical cross-sections of the biofilm were cut using a cryomicrotome (Bright 5030 microtome model P143) at -20°C. Ten-micrometre-thick sections were transferred to gelatine-coated, Amann-type slides. Hybridization of the sections was done using CY5-labelled Type I methanotroph probes; MG84 (5’-CCACTCGTCAGCGCCCGA) and MG705 (5’-CTGGTGTTCCTTCAGATC), and Cy3-labelled Type II methanotroph probe; MA450 (5’-ATCCAGGTACCGTCATTATC) as described by (Eller et al., 2001). Hybridizations were visualised using an epi-fluorescent microscope (Nikon Eclipse E600, Japan).

Maximum methane oxidation potential activity tests

The maximum methane oxidation potential of biofilm samples from the HFBRs was calculated on day 177. The six biofilm zones from each HFBR were compared. The concentration of volatile suspended solids of each sample was calculated, based on loss of mass on ignition of volatile compounds, and 500 mg VSS of biofilm were placed in 40-ml hypo-vials with 8 ml AWM. The headspace was adjusted to 10% methane concentration at atmospheric pressure and the vials were incubated at 10°C in the dark on a shaker at 80 rpm. The rate of reduction in headspace methane concentrations was measured using GC analyses until <0.5%.

Results and Discussion

Removal rates and perturbations

After the HFBRs were seeded with the enriched methane-oxidising biomass, an initial ‘start up’ period of 55 d was observed. During the start up period, removal rates steadily increased until an average removal rate of 23.6% in R1, 27.8% in R2 and 26.3% in R3 was achieved. Removal rates in the ‘start up’ period showed significant fluctuations from day to day, indicating that the HFBRs were unstable during this period. This start up phase was then followed by a ‘steady-state’ phase lasting 35 d until day 90. Average removal rates of the three HFBRs were similar throughout this period; R1 (30.7%), R2 (33.9%) and R3 (34.5%) (Fig. 2).

On day 90, when the HFBRs were subjected to a ‘cold shock’ perturbation lasting 17 d until day 107, whereby the air temperate in the laboratory varied between 1°C and 5°C, a sharp

reduction in reactor performance was observed in all three HFBRs. The removal rates decreased by 46%, to an average of 17.7% removal efficiency across R1 (14.9%), R2 (19.2%) and R3 (19.2%).

When the air temperature in the laboratory was restored to 10°C on day 107, the methane removal efficiency of the HFBRs improved almost immediately. However, in the 13-d period following the temperature change, the removal efficiencies fluctuated and did not recover to the rates observed in the steady state phase from days 55-90 (Fig. 2).

On day 120, the HFBRs were stopped for 14 d. During this ‘shut-down’ period, the CH4 supply was turned off and a reduced volume of SWW was fed to the HFBRs. On day 134, the CH4 supply was turned on and CH4 oxidation was observed almost instantaneously. This second recovery period (Recovery Period 2; Fig. 2) comprised 14 d, and the HFBRs reached a steady-state by day 148. During Recovery Period 2, a rapid R1 recovery was observed and an average removal rate of 39.2% was achieved. The recovery of R2 and R3 was slower, with removal rates of 16.0 % and 19.1%, respectively.

Figure 2. Methane removal efficiency (%) of the three HFBRs.

The HFBRs were operated at steady state for a further 30 d (‘steady state 2’), before the trial was terminated on day 177. By the end of the trial, highest removal rates were recorded from R1 (Fig. 2). The performance of R1 and R2 was higher toward the conclusion of the trial than at any other stage (Fig. 2). In contrast, removal rates of only c. 28% were observed at this stage in R3.

DATA FROM 177 ONWARDS

In-situ HRBR removal rates during ‘Steady state 1’ and ‘Steady state 2’

Cumulative CH4 removal by the HFBRs, as well as discrete CH4 removal in individual zones, from ‘steady state 1’ (SS1) and ‘steady state 2’ (SS2) are presented (Fig. 3). Cumulative removal followed a similar trend during SS1 and SS2. Low removal was observed in the first gas sample zone (sheets 1-4), but removal rates were linear throughout the rest of the sheets. Cumulative and discrete removal data indicated that the efficiency of removal improved with time (Fig. 3). The volume (moles) of methane oxidised during SS1 was higher than during SS2, but this was a result of higher influent methane concentrations during SS1.

Figure 3. CH4 removal efficiencies of HFBRs during (A) ‘steady state 1’ and (B) ‘steady State 2’. (i) Cumulative CH4 removal of HRBRs, (ii) total moles of CH4 removed in HFBRs, (iii) discrete removal within gas sample zone (mmol CH4/m

2sheet/day) and (iv) removal efficiency of gas sample zone.

Maximum methane oxidation potential activity tests

Maximum methane oxidation activity varied between zones and HFBRs (Fig. 4). Assays of R1 and R3 samples indicated a similar trend, comprising high potential for oxidation in zones 1-6 and 7-12, but lower potential in zones 13-18 and 19-24. At zone SF 1-6 (i.e. the step-

feed), the potential of R1 and R2 samples increased to close to activity in the upper zones, but significantly lower potential was observed again in zone SF 7-12.

Figure 4. Mamaimum CH4 oxidation potential (mol CH4/gVSS/d) of biofilm samples from HFBRs.

Phylogenetic analyses of clonal sequences

A very diverse bacterial community was apparent (Fig. 4). Of the 170 clones selected for analyses, 119 individual OTUs were detected. The library predominantly comprised Proteobacteria (38.2%), Firmicutes (31.2%) and Bacteroidetes (25.3%) (Fig. 4). No methanotroph sequences were detected in the library, probably due to low abundance.

DO WE NEED TO SHOW TREES? NEED TO SUBMIT SEQUENCES?

Figure 4. (A) Maximum potential methane oxidation rates (mol CH4/gVSS/d) of biofilm samples from HFBRs; (B) landfill-compost clone library composition; (C) typical FISH image of biofilm (R1, day 177) showing small clusters of Type I methanotrophs (circled) and larger clusters of Type II methanotrophs (arrows).

A B C

Temporal community succession indicated by TRFLP analyses

Community profiles indicated that the diversity of bacterial species decreased during the trial from day 0 (immediately prior to re-seeding with the enriched medium) to day 177 (Fig. 5). Similar profiles were observed vertically down through each HFBR, although in some cases, moderately reduced diversity was observed in lower layers. Nonetheless, it appears that the main peaks (c. 500 bp) evident on day 0 persisted as the predominant groups by the end of the trial (Fig. 5). It may be the case that uncultured methanotrophs were abundant already on day 0, or, indeed, that the increased activity by day 177 (SS2) was supported by a small population of very active bacteria.

Distribution of methanotrophs observed by FISH experiments

Type I methanotrophs were present in low concentrations and clumped together in small, well-defined clusters. Type II methanotrophs were more widely and evenly distributed in larger cell clusters. The majority of the biofilm consisted of other unidentified prokaryotes. From the FISH data, it was clear that Type II methanotrophs were present in much higher numbers than Type I methanotrophs, which may indicate that they were more important in terms of overall methane oxidation in the HFBRs. The spatial arrangement of the biofilm showed that both Type I and Type II methanotrophs clumped in individual clusters, indicating that methane oxidation only have occurred in localised regions of the biofilm (Fig. 4).

Figure 5. Electropherograms illustrating the bacterial community present in the HFBRs (i) R1, (ii) R2 and (iii) R3 on (A) day 0 and (B) day 177. For illustrative purposes, the sampling zones are labelled in electropherogram A(i). The zones are in the same order for the rest of the electropherograms (however, for HFBR2 SF7-12 Day 0, no data are available, so only five samples shown).

Conclusions

1. The efficacy of HFBR technology was demonstrated for abatement of low concentrations of methane at 10°C.

2. An average steady state removal rate of 38.6% was achieved at loading of >12 g CH4/m

3/h. A cold temperature perturbation resulted in significantly reduced RE. 3. Maximum CH4  oxidation potential rates varied significantly between HFBR zones

and across the three HFBRs. 4. Genetic fingerprinting of temporal biofilm samples indicated stability over time but

some reduction in diversity. FISH analyses of biofilm samples from HFBRs confirmed the presence of Type I and Type II methanotrophs.

5. HFBR technology can be effectively applied for the treatment of CH4 emissions in a simple, cost-effective manner.

Further optimisation of the HFBRs is now being investigated and new trials to treat H2S and NH4 have also commenced.

Acknowledgements

This publication has emanated from research conducted with the financial support of Science Foundation Ireland and a European Research Council Starting Grant (‘3C-BIOTECH’, Number 261330).

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