High-Performance Biogas Upgrading Using a

Author's personal copy Appl Biochem Biotechnol DOI 10.1007/s12010-017-2569-2

High-Performance Biogas Upgrading Using a Biotrickling Filter and Hydrogenotrophic Methanogens Trisha L. Dupnock 1 & Marc A. Deshusses 1

Received: 7 June 2017 / Accepted: 24 July 2017 # Springer Science+Business Media, LLC 2017

Abstract This research reports the development of a biotrickling filter (BTF) to upgrade biogas, which is achieved by adding H2 to reduce CO2. H2 and CO2 (80:20% vol.) were fed to a bench-scale BTF packed with polyurethane foam (PUF) and inoculated with hydrogenotrophic methanogens. Maximum CH4 production rates recorded were as high as 38 m3CH4 m−3reactor day−1, which is 5–30 times faster than earlier reports with other kinds of bioreactors. The high rates were attributed to the efficient mass transfer and high density of methanogens in the BTF. The removal efficiencies for H2 and CO2 were 83 and 96%, respectively. 5-Cyano-2,3-ditolyl tetrazolium chloride/DAPI staining revealed that 67% of cells were alive near the gas entrance port, while only 8.3% were alive at the exit. Furthermore, DNA sequencing showed that only 27% of the biomass was composed of Euryarchaeota, the phylum which includes methanogens. These two observations suggest that optimizing the methanogen density and activity could possibly reach even higher biogas upgrading rates. Keywords Biogas upgrade . Biomethane . Hydrogenotrophic methanogens . Biotrickling filter

Introduction Biogas shows promising potential as a renewable energy source. It can easily be produced by anaerobic digestion (AD) of organic wastes such as sewage sludge, food wastes, and animal manure [1, 2]. However, its composition (~60% CH4 and 40% CO2) limits its value and application. Therefore, upgrading biogas to natural pipeline standards (90–98% CH4 and less than 7 ppm H2S depending on location) [3] for injection into the grid is a desirable endeavor. It would allow for a clean alternative to natural gas from fossil origin. Electronic supplementary material The online version of this article (doi:10.1007/s12010-017-2569-2) contains supplementary material, which is available to authorized users.

* Marc A. Deshusses [email protected]

1

Department of Civil and Environmental Engineering, Duke University, 127C Hudson Hall; Box 90287, Durham, NC 27708-0287, USA

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Commonly practiced biogas upgrading methods include but are not limited to pressure swing adsorption, water washing, chemical scrubbing, or absorption in polyethylene glycol [4, 5]. While these technologies are effective, they are also costly and often remove small amounts of CH4 in addition to CO2 [4]. Recently, a promising biological route to biogas upgrade was proposed. It relies on the addition of H2 and metabolic action of hydrogenotrophic methanogens (HMs). These autotrophic microorganisms fix CO2 using H2 as an electron donor, thereby producing methane following the simplified stoichiometry shown in Eq. 1 or in Eq. 2, if growth of the methanogens is included [6]. In Eq. 2, C5H7O2N is the empirical composition of the methanogens (see SI for derivation of that equation). 4H2 þ CO2 →CH4 þ 2H2 O

ð1Þ

0:131CO2 þ 0:004HCO3 − þ 0:004NH4 þ þ 0:5H2 →0:115CH4 þ 0:004C5 H7 O2 N þ 0:266H2 O

ð2Þ

Early studies adding HMs and hydrogen directly to anaerobic digesters have achieved effluent CH4 purities as high as 95% [7]. However, although these studies have been conceptually validating, significant pH increases due to CO2 removal have negatively impacted the AD process. In addition, mass transfer limitations between the gas and liquid (especially for H2) have resulted in low volumetric productivities (only up to 9.2 m3 m−3 day−1) [5, 7, 8]. Thus this approach may never be economically feasible. A two-stage process with continuously stirred tank bioreactors has also been investigated in an attempt to prevent damages to the anaerobic digester microbial community caused by pH variability [8]. AD of the substrate is performed first, followed by upgrading the biogas in the second bioreactor. Again, pH variability was observed with values rising up to 8.5. Flask experiments indicated that while HMs could function in the pH range of 6.0–8.5, optimal pH was around 7. Thus, CH4 production rates in the second bioreactor were low, with a maximum rate of 0.36 m3 m−3 day−1. Possibly, interphase mass transfer of H2 was also limiting the rate of the process [5, 7, 8]. Clearly, it is crucial to separate biogas upgrading from AD and to promote fast mass transfer of H2 to a dense population of HMs. A novel approach that could resolve these drawbacks is to use biotrickling filters (BTFs), which can offer a high specific area for biofilm growth and high density of biomass and are known for their high gas to liquid and gas to biofilm mass transfer coefficients [9]. A conservative estimate of the methane production rate of a BTF based on methanotroph kinetics (see SI for details) yields rates as high as 75 m3CH4 m−3 day−1, corresponding to biogas upgrading rates of 200–250 m3biogas m−3 day−1, i.e., about 20–25 times greater than studies in CSTRs. Despite these anticipated improvements, the few studies investigating biogas upgrading with BTFs and HMs have only achieved methane production rates between 1.3 and 4 m3CH4 m−3 day−1, suggesting that important rate limitations existed. Two requirements for achieving fast rates are a high biomass density within the bioreactor packing and a high HM biomass activity. High biomass density can be achieved using packing with a high specific surface area; however, such packing can be subject to clogging and cause excessive pressure drop if biomass growth is significant [10]. Fortunately, methanogens have a low biomass yield and BTFs for biogas upgrade must be operated at relatively long gas residence times; hence, this risk is low in biogas upgrade applications. There is no consensus yet on a systematic approach for achieving a high biomass activity. Clearly, using an optimum bacterial

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seed or enrichment culture to start the BTF, providing optimum conditions for growth including sufficient supply of macro- and micronutrients and a tight pH control, and avoiding excessively thick biofilms are all factors that are thought to contribute to high primary degrader biomass activity [11–13]. Therefore, the objectives of this study were to demonstrate sustained biogas upgrade in a BTF packed with a high specific surface area packing and prove that it could upgrade biogas at a significantly higher rate compared to other biological upgrading approaches.

Materials and Methods Biotrickling Reactor and Calculations A bench-scale tubular BTF reactor was constructed from clear PVC tubing (Fig. 1), 2.5-cm ID, packed with a 46-cm-deep bed of open pore polyurethane foam (PUF) cut to fit the inner diameter. PUF was chosen because it has a high specific area (600 m2 m−3 [14]) and thus would maximize biofilm attachment and mass transfer. Three sampling ports were installed, one at the entrance, middle, and exit of the bioreactor, to monitor the gas composition throughout the length of the reactor. Heating tape and insulation were added around the bioreactor to maintain an internal temperature of 35 °C. Two peristaltic pumps served to recycle the medium over the bed and administer fresh medium daily (10.2 mL min−1 and 2.5 mL day−1 respectively). H2 and CO2 were supplied to the reactor using mass flow controllers (MFCs) (Alicat Scientific, Tucson, AZ) at initial rates of 61.9 and 17.2 mL min−1, i.e., a ratio of 3.6:1.0 which would result in 90% removal CO2 at full H2 conversion. On day 47 of reactor operation, these flows were reduced to 20 and 5 mL min−1. During days 106–177, various loading rates were tested as discussed later. For the initial inoculation, 21 mL (i.e., about 16-mg dry cell weight) of the culture enriched at 35 °C was

Fig. 1 Schematic of the biogas upgrading biotrickling filter (not to scale). MFC mass flow controller, PUF polyurethane foam packing

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added to the reactor. The influent, mid reactor, and effluent gas compositions were monitored daily as described below. Nitrogen gas (17% vol.) was also included in the influent gas as an internal standard to determine the effluent gas flow rate since it takes five gaseous molecules to create one molecule of CH4 (Eq. 1), and thus, there will be reduction of the gas flow rate through the reactor. Since N2 is an inert gas, the effluent gas flow (Fout) can be calculated using Eq. 3. F out ¼

C in;IS  F in 3 −1
m h C out;IS

ð3Þ

where Cin,IS and Cout,IS are the inlet and outlet concentrations of nitrogen and Fin is the total influent gas flow. With the effluent flow rate, the elimination capacities (ECs) of H2 and CO2 and the production capacity (PC) of CH4 in terms of gas volume per reactor volume per time (m3 m−3 day−1) are calculated using Eqs. 4 and 5, where Cin and Cout are the (mass) concentrations of the desired analytes: EC ¼

F in C in − F out C out V bed

PC ¼

F out C out V bed

m3 m−3 day−1

m3 m−3 day−1







ð4Þ

ð5Þ

Note that PC as written in Eq. 5 assumes that there is no methane in the inlet gas. The removal efficiency (RE) is defined as follows: RE ¼

F in C in −F out C out  100 F in C in

ð%Þ

ð6Þ

Mineral Medium The anaerobic mineral medium (MM) used in this study was prepared according to the American Type Culture Collection medium recipe for Methanobacterium bryantii [15]. The trace metal solution was made according to the recipe reported by Pfennig et al. [16]. An additional 200 mg L−1 KH2PO4 was added after day 25 of BTF operation to increase the buffer capacity.

Analytical Methods Gas samples were analyzed using an SRI 8610C gas chromatograph (GC) equipped with a 12′ HayeSep D column (SRI Torrance, CA), a thermal conductivity detector (TCD), and a ten-port gas injection valve. Argon served as the carrier gas [17]. The injection valve temperature was 120 °C, the detector was at 100 °C, and the initial column temperature was 60 °C and programmed to increase to 105 °C at 30 °C min−1 allowing effective separation of N2, H2, CH4, and CO2. The GC was calibrated using gas mixtures of known concentrations. The pH in the bioreactor was recorded using an Ion 510 Series meter (Oakton, Vernon Hills, IL) equipped with an Orion 9107BN pH probe (Thermo Fisher Scientific, Chelmsford, MA). Pressure in enrichment flasks was monitored using a digital pressure gauge (Dwyer Instruments, Michigan City, IN) fitted with a hypodermic needle.

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Culture Enrichment and Temperature Effects Samples for enrichment of HMs were obtained from a swine manure anaerobic digester [18]. Enrichments were conducted in 250-mL flasks that contained 1 mL of inoculum and 20 mL of MM. Flasks were sealed with gas-tight septum caps, and the headspace was flushed with an 80:20 (vol/vol) mixture of H2/CO2. This was to ensure that HMs would dominate over acetoclastic methanogens [5, 7]. To observe temperature effects, two separate enrichments were conducted at 25 and 35 °C. Both enrichments were conducted on rotary shakers set to 100 rpm. On a daily basis, 10% of the liquid volume was discarded and replenished with fresh MM and the headspace was flushed with fresh H2/CO2 gas. When the culture in the flasks became visibly turbid, 1 mL aliquots were extracted and transferred to a new flask to enrich for fast-growing cultures. Near the end of the 2-month enrichment, substrate consumption and methane production curves were determined.

5-Cyano-2,3-Ditolyl Tetrazolium Chloride/DAPI Staining 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC)/DAPI cell staining was conducted to determine the fraction of live cells in selected sections of the reactor. Swab samples (about 0.3 g wet) were collected from the entrance, middle, and exit of the reactor and placed in 2-mL microcentrifuge vials. They were prepared accordingly with CTC and DAPI stains (SigmaAldrich, St. Louis Missouri) based on a published protocol [19]. Once prepared, 5 μL of sample was trapped under a #1.5 coverslip and observed under a Leica DMI6000CS inverted confocal fluorescence microscope (Wetzlar, Germany). Samples were excited using a 405-nm diode laser, and the emission spectra were set to 415–498 nm for DAPI and 574–765 nm for CTC [20]. From each frame, three pictures were captured to evaluate total and viable cell counts. The total cell count was determined by counting both CTC- and DAPI-stained cells while the viable cell count was CTC-only-stained cells [19]. A killed control sample was also created by adding sodium azide (3.2% final concentration) 15 min before CTC addition [21].

DNA Sequencing Microbial DNA sequencing was performed for the bioreactor inoculum and the bioreactor biofilm after 177 days of operation. Inoculum samples were stored in 2-mL centrifuge tubes in 50:50 (vol/ vol) MM/glycerol and stored at − 20 °C until sequencing. DNA was isolated with the PowerSoil bacterial DNA extraction kit (MoBio). Polymerase chain reaction (PCR) was used in triplicate to amplify the V4 region of the 16S rRNA gene. Published protocols were used to amplify, clean, and quantify the resulting amplicons [22, 23]. The operational taxonomic units were chosen at 97% similarity against the Greengenes database which was constructed by QIIME developers. The PCR was checked by gel electrophoresis following the protocol reported in SI. Sequencing results were submitted to the European Nucleotide Archive (accession numbers are in the SI).

Results and Discussion Culture Enrichment During the 2-month enrichment conducted in gas-tight flasks, pH and gas composition were regularly monitored. Methane was detected after about 1 month. Cultures incubated at 35 °C

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were the first to produce significant CH4 levels; at the end of the enrichment, they were producing on average 103 mL CH4 day−1 (for a 21-mL culture) compared to 55 mL CH4 day−1 for cultures grown at room temperature (Fig. S1). These results provided the motivation to include heating to the bioreactor. Cultures incubated at 35 °C reached pH values around 9 for the entirety of the second month while producing more methane than those at a lower pH. This was unexpected since literature reports indicate that HMs are significantly less active above a pH of 8.5 [8]. The increased pH was most likely due to the microbial uptake of CO2 leading to the protonation of carbonate to restore the bicarbonate equilibrium, thus reducing the hydronium concentration [6–8]. The gas production and consumption molar ratios obtained experimentally were compared to theoretical molar ratios to confirm that the dominant biological transformation was indeed hydrogenotrophic methanogenesis (Fig. S2). The theoretical ratio for H2/CO2 and for H2/CH4 is 4.0, and the theoretical ratio for CH4/CO2 is 1.0 (Eq. 1) or 4.3 and 1.2, respectively, if considering cell growth (Eq. 2). It took 38 days for the ratios to reach steady values; H2/CH4 and CO2/CH4 were 5.3 and 1.3, respectively (at 35 °C), i.e., slightly higher than expected. This indicated that some electrons from H2 and some carbon from CO2 were used for cell growth.

Reactor Performance Figure 2 reports the elimination capacities (ECs) of H2 and CO2, the production capacity (PC) of methane, and pH values during the experiment. The figure is divided into four phases with phase I being the start-up period. PC and EC values are not reported for this phase because the internal standard method to determine the flow reduction was not yet implemented. Even so, the first detectable amounts of CH4 (~1.4% vol.) were identified 3 days after reactor inoculation and increased slowly until day 34 when effluent CH4 concentrations increased to ~6%. During this time, pH in the bioreactor was consistently decreasing due to the continuous CO2 absorption in the trickling liquid combined with the low microbial activity. On day 25, the concentration of phosphate buffer was doubled from 200 to 400 mg L−1 after two major pH declines to 5.05 and 4.16 that significantly hindered CH4 production. The increased buffer concentration resolved the pH issues; however, on day 40, the effluent methane concentration dropped and remained around 1%. In an attempt to increase conversion, the inlet H2 and CO2 flows were reduced to 20 and 5 mL min−1, respectively, on day 47 (i.e., the start of phase II) to obtain a longer gas residence time (11 min, based on inlet flow, compared to 3 min before). During phase II, CH4 effluent concentrations dramatically increased to 25% on day 52 and the PC increased to approximately 22 m3CH4 m−3 day−1 and was sustained until day 81 when it unexpectedly dropped. It is unclear what caused this sudden change in activity, but good performance was obtained again after day 94. To evaluate the effect of varying loading rates, H2 and CO2 inlet flows were then increased to 23 and 5.8 mL min−1 (phase III). This phase yielded a maximum PC of 38 m3CH4 m−3 day−1 on day 130 with corresponding removals of 87 and 82% for CO2 and H2, respectively (Fig. 3), and the methane concentration in the effluent was ~44%. During this time, there was also an overall increase in the pH of the recycled liquid, which could be related to enhanced microbial activity and increased consumption of dissolved CO2 [6, 7], as also observed during the flask experiments. This suggests that a pH controller should be installed to obtain more stable conditions. On day 140, a leak was identified in the bioreactor and the system was taken offline days 142–160 for maintenance. When operation and data collection resumed on day 161, the PC had fallen considerably to 14 m3CH4 m−3 day−1. Since these low values were consistent, load

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Fig. 2 Reactor performance during the experiment: liquid recycle pH (top), production and elimination capacities (PC and EC, middle), and CH4 concentration in the effluent gas (bottom)

testing was continued and the inlet H2 and CO2 flows were reduced to 22 and 4 mL min−1 (phase IV) and then 17 and 3.3 mL min−1 (phase V), respectively. On day 177, the BTF was stopped for biofilm analysis discussed later in the paper. As mentioned, the best performance was obtained during phase III; thus, results during this phase were examined in greater details. Figure 3 shows that excellent CO2 removal was obtained. The highest sustained REs recorded during phase III were 96% for CO2 and 83% for H2. The ratio of H2 to CO2 consumption for this phase was 3.95 m3H2 m−3CO2 ± 0.31, which is

Fig. 3 Biotrickling filter removal efficiency for hydrogen and CO2

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near the theoretical ratio of 4.0. Furthermore, some CO2 removed could simply be absorbed, resulting in an overestimation of CO2 consumed. The most probable rate limiting step during the process is the mass transfer of hydrogen from the gas to the liquid phase, which has been cited frequently in literature in other hydrogen fed bioreactor systems [6–8]. This is despite the high surface area of the packing used in this study and the low trickling rate of the liquid which should promote direct contact between the gas and the biofilm. However, the gas superficial velocity was low, because long gas residence times were required to achieve high conversion of high gas concentrations (percentage levels) as opposed to trace concentrations (ppm levels) in BTFs used for air pollution control. A yield of 0.26 m3CH4 m−3H2 ± 0.02 was observed experimentally which is not statistically different from the theoretical yield of 0.25 m3CH4 m−3H2, indicating that hydrogenotrophic methanogenesis was the dominant metabolic process in the BTF. Additionally, the yield for CO2/CH4 (0.97 m3CO2 m−3CH4 ± 0.05) was also matching its theoretical value of 1.0 m3CO2 m−3CH4. These values suggest that biomass was no longer actively growing inside the reactor; otherwise, ratios closer to those calculated with the stoichiometric coefficients of Eq. 2 would have been obtained. The effects of substrate loading are reported in Fig. 4. For both H2 and CO2, a generally increasing trend of EC vs. load is observed, suggesting that the system probably did not reach its maximum removal capacity. Even so, it is interesting to compare the ECs obtained in this study (150–400 g m−3 h−1 for H2 and 750–2400 g m−3 h−1 for CO2) with the ECs obtained in conventional BTFs used for air pollution control. Typical EC values, e.g., for the treatment of toluene vapors, rarely exceed 100 g m−3 h−1, or 200–300 g m−3 h−1 for hydrophilic volatiles such as ethanol, or 150 g m−3 h−1 for H2S [24, 25]. Clearly, the extremely high concentrations of H2 and CO2 in this study are enabling very high elimination rates despite H2 being fairly hydrophobic.

Performance Comparison Several earlier biogas upgrading studies report upgrading rates in terms of biogas volume upgraded per reactor volume per time. However, since the conditions, in particular the gas composition can vary between studies, direct comparison of rates in this manner may require some conversion. In particular, studies conducted with actual biogas or biogas mimics (i.e., ~60% vol. of gas is inert) cannot be directly compared to studies like ours, where the inert gas Fig. 4 Effects of substrate loading on EC. The dashed line represents 100% removal of H2 or CO2

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fraction was just 17%. Therefore, Table 1 was constructed to compare both the PC and biogas upgrading rates (R) in addition to the REs and methane yield (YCH4/H2) values determined for selected studies. The percentage methane upgraded was also compared. Parameters such as the specific surface area (ai) and retention time (τ) of the system were also included in Table 1 as these variables may also influence upgrading rates. The original data and detailed calculations for obtaining these values can be found in SI. Examination of Table 1 reveals that BTFs show significant promise for achieving fast methane production rates. The average methane PC obtained during phase III was 27 m3CH4 m−3 day−1, which is greater than all earlier reports listed in Table 1, even though our study included very little process optimization. Still, the removal efficiency achieved for CO2 was comparable to earlier upgrading approaches. A recent method that relies on gas sparging through a polymeric membrane [30] yielded the second highest methane PC of 8.4 m3CH4 m−3 day−1. This value was observed when feeding only an H2/CO2 gas mixture to the bioreactor; however, the authors used a simulation to predict that upgrading rates of 25 m3biogas m−3 day−1 could be achieved if actual biogas was used. The methane generation rates of the remainder of the studies listed in Table 1 were significantly lower, probably due to low mass transfer coefficients for H2 and low biomass concentrations in those bioreactors. Values for KLa and biomass concentration obtained when using a traditional gas diffuser were reported to be 0.039 h−1 [7] and 0.48 gVSS L−1 [5], respectively, while the membrane sparging approach investigated by Diaz et al. achieved significantly greater values of 430 h−1 and 3.6 gVSS L−1. Using the average EC for H2 during phase III (8.0 kg H2 m−3 day−1) and the biomass yield (Y) calculated from Eq. 2 (0.46 g VSS g H2−1) and assuming a growth rate (μ) of 0.2 to 0.5 day−1 [6], the biomass concentration in the BTF can be roughly estimated at 7.4 to 18 gVSS L−1 using Eq. 7 below:  
Y X ¼ EC gVSS L−1 ð7Þ m This is about two to five times larger than those found using gas sparging bioreactors. Furthermore, it is attached as biofilm, concentrated in a small volume fraction of the bioreactor (as opposed to a suspended growth bioreactor). The very high biomass density together with the high specific surface area of the PUF packing used in this study certainly contributed to obtaining high performances. However, using a packing with an extremely high specific surface area and small pores such as the 2–6-mm mix of vermiculite and perlite employed by Alitalo et al. [16] can be detrimental to long-term reactor performance because of the plugging. Although they observed upgrading rates of 6.4 m3CH4 m−3 day−1, clogging prevented operation after 47 days. Our BTF was in operation for close to 170 days without clogging of the open cell PUF packing. Despite the importance of interphase mass transfer in biogas upgrade studies, most studies do not present KLa values, probably because of difficulties measuring it without impacting the process culture. It is interesting to note that 4 orders of magnitude increase in KLa when using a membrane [30] versus a gas diffuser resulted only in a minor increase of the methane PC. This could be due to the fact that that bioreactor relied on suspended growth and that it was limited by the kinetics rather than by mass transfer. Limited information is available for KLa in immobilized biomass reactor configurations although it might help to better understand some apparent contradictions. The lowest methane PCs were found for other BTF configurations (Table 1), which contrasts with our study with similar operating parameters but achieved upgrading rates over 20 times faster.

d

c

b

a

n.d. 1.2d 5a 98 100 n.d. 0.25

37 4 Bioflow 40 305

n.d. 2.5 6.9 84 100 > 90 0.25

CH4 in effluent–CH4 in the feed

Reported as 4:1 with CO2 in excess

Reported as normal liters per volume per day

n.d. 1.2d 5a 94 99 n.d. 0.26

37 2.3 Bioflow 40 305

55 144 Vermiculite, perlite, wood ash 4.9 × 106 n.d. 6.4 21a 95 100 n.d. n.d. n.d. 1.3 4.3a 100 100 100 0.23

35 3.8 Polyester urethane sponge n.d.

430 8.4 25 95 95 95 0.22

55 n.d. Polymeric fibers (PVDF) 360

0.039 5.3 16 66 95 94 0.23

55 1 n.a. n.a.

80% H2 20% CO2 0% CH4

80% H2 20% CO2 0% CH4

80%c H2 20%c CO2 0% CH4

True biogas: 36–42% CO2 58–64% CH4 H2 fed 4:1 H2/CO2 37 1.3 Polypropylene packing rings 313

CSTR: in situ upgrading with H2 diffuser within the digester 60% H2 15% CO2 25% CH4

H2 sparging in a cylindrical reactor through polymeric membranes 80% H2 20% CO2 0% CH4

Up-flow fixed bed reactor with gas diffuser

Two down-flow fixed bed reactors in series

Trickling bed reactor fed in batch-wise mode

Trickling bed reactor fed in countercurrent mode

Trickling bed reactor fed in countercurrent mode

80% H2 20% CO2 0% CH4

Luo et al. [5]

Diaz et al. [26]

Lee et al. [29]

Alitalo et al. [10]

Burkhardt and Busch [28]

Rachbauer et al. [27]

Burkhardt et al. [26]

Calculation of R assumes upgrading to 90% methane (see SI for details)

n.a. not applicable, n.d. not determined

KLa (h−1) PC (m3CH4 m−3 day−1) R (m3biogas m−3 day−1) CH4 produced (%)b Max RE H2 (%) Max RE CO2 (%) Y (m3CH4/m3H2)

n.d. 27 90a 44 83 96 0.26

67% H2 16% CO2 0% CH4 17% N2 35 0.2 PUF 600

Gas feed composition

Temperature (°C) τ (h) Packing material ai (m2 m−3)

Trickling bed fed in concurrent mode

Technology

This study

Table 1 Comparison of the most pertinent biogas upgrading studies using bioreactors

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Burkhardt and Busch [28] reported methane PCs of 1.2 m3CH4 m−3 day−1 for a BTF operated in batch-wise and countercurrent modes, with a high effluent methane purity for both. Slightly higher rates (2.5 m3CH4 m−3 day−1) were reported by Rachbauer et al. [27] in a countercurrent BTF, though methane purity of the effluent gas was below 90%. In that study, the authors manage to increase the effluent gas purity to > 98% methane at the expense of a lower methane PC (1.6 m3CH4 m−3 day−1) by increasing the gas retention time to 2.3 h. It should be noted that that study included methane in the influent gas, which has been shown to decrease upgrading performance by about 50% due to the lowering of the hydrogen partial pressure [31]. With such a large potential decrease to the upgrading rates, future studies should be conducted with either real biogas or a true mimic (60% CH4, 40% CO2) to more accurately evaluate biogas upgrading feasibility for industrial implementation. Thus, while the maximum rates reported in our study are higher than earlier reports, it remains uncertain under which conditions the effluent biogas content would meet natural gas standards since our experiments were not conducted with a true biogas mimic. Generally, CO2 makes up about 40% of the biogas and 75% of this volume must be upgraded to meet standards. Rough estimates based on the PC of Table 1 suggest that about 90 m3 of biogas could be upgraded daily per cubic meter BTF to achieve a 90% methane content. This is lower than the anticipated upgrading rate of 250 m3biogas m−3 day−1, mentioned in the introduction; thus, there is room for process optimization as discussed below. Still, the markedly higher rate compared to earlier studies implies that for commercial applications, much smaller (5to 20-fold) bioreactors will be possible with obvious capital cost savings.

Biomass Analysis Upon the conclusion of bioreactor performance testing, the biofilm samples from the reactor were analyzed to determine the live cell fraction and the microbial composition in an attempt to better understand what fraction of the biomass is contributing to the upgrading process and to guide future optimization of the process.

5-Cyano-2,3-Ditolyl Tetrazolium Chloride/DAPI Staining Visual observation of the reactor packing after 177 days of operation indicated that a dense biofilm had formed. Therefore, it was hypothesized that the decrease in PC observed towards the end of the study could be due either to excessive growth or a reduction of the density of active microbes. CTC and DAPI staining which allows the determination of the fraction of live and dead cells was used to test this hypothesis. Representative images (Fig. 5) show the distribution of live and dead microorganisms from samples taken from the inlet, middle, and effluent sections of the BTF. Live cells were dominant within the first one half of the bioreactor, while the proportion of live cells markedly decreased in the middle section and near the reactor effluent. This decreasing trend was expected due to decreased substrate availability with reactor length during phase III. At the entrance, 67% of cells were active, while a mere 8.3% were active near the reactor exit. These results are consistent with the gas mole fractions observed throughout the reactor (Fig. S3). Although no sample was taken for CTC/DAPI staining while the BTF was operating at its peak performance, the low fraction of live cells in the last half of the BTF could explain the unexpected decrease in PC values observed day 161 and onwards.

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Fig. 5 CTC (live cells, shown in red) and DAPI (all cells, shown in green) staining images from inlet (a), middle (b), and effluent (c) biotrickling filter sections (Color figure online)

DNA Sequencing Figure 6 shows the major phyla identified in the inoculum (day 1) and the bioreactor (day 177). These phyla were Euryarchaeota (27.3%), Bacteroidetes (37.6%), Firmicutes (12.8%), and Proteobacteria (15.8%). This agrees with the findings of other studies that have sequenced samples from biogas reactors [5, 8, 32–34]. While the increase in Euryarchaeota from days 1– 177 was expected, since this phylum includes the methanogens, it is unclear why certain bacterial populations (Firmicutes and Bacteroidetes) also increased. Within the Euryarchaeota, 37 and 62% of the phylum were composed of an unidentified species of Methanobacterium and Methanobrevibacter arboriphilus, respectively. Both genera preferentially produce methane following Eq. 1 [35]. Interestingly, M. arboriphilus possesses a unique heme-dependant catalase that provides an effective antioxidative defense when active [36]. Studies have shown that liquid cultures of M. arboriphilus have been shown to recover from oxic desiccation periods lasting for 3 days, indicating that this particular strain is capable of surviving oxic exposures [37]. It is possible that this species became dominant in the BTF during the leak correction (after day 142) due to its unique O2 resistance capabilities, whereas other methanogenic species would have died.

Fig. 6 Major phyla identified in the inoculum (day 1) and in the biotrickling filter (day 177)

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Considerations for System Optimizing via Increased Biomass Density and Activity The combined information from the live/dead staining and DNA sequencing indicates that the fraction of active methanogens was low. At best, assuming that all Euryarchaeota were active, their live fraction would only be up to 27/67 = 40% of the total population, although the actual number is probably lower. Further detailed examination of the culture physiology, and more importantly of its specific activity and means to increase it, are warranted as it holds potential for achieving biogas upgrading rates even higher than reported in Table 1. A particular challenge will be to achieve high microbial activity throughout the packed bed, as the results of Fig. 5 indicate that the culture live fraction was markedly decreasing in the axial direction, an observation supported by many earlier biofiltration studies. Proposals to achieve greater specific methanogenic density and activity include more intensive trickling to attempt washing dead cells, working with a monoculture of methanogens, or periodically moving the feeding of the raw biogas axially in the reactor to better distribute growth.

Conclusions This study reports on a high-performance biogas upgrade system using HMs immobilized in a BTF. The high upgrading rates of 38 m3CH4 m−3 day−1 obtained were most likely the result of efficient gas to biofilm substrate mass transfer and of the successful immobilization of a dense biomass. A CO2 removal efficiency of 96% was also recorded. Kinetic experiments conducted with the BTF indicated that this system could be capable of upgrading biogas and meet pipeline standards while operating at a gas retention time of 11 min, which is markedly shorter than previous studies. Finally, a unique insight into the biofilm composition of a biogas upgrading BTF was provided by sequencing and live/dead staining. These investigations revealed that good performance was obtained despite the relatively low abundance of methanogens and a high fraction of inactive biomass. Overall, the excellent biogas upgrading performance obtained without process optimization suggests that even higher biogas upgrading rates could possibly be obtained after systematic optimization bringing this process a step closer to economic reality. Acknowledgments We would like to acknowledge the National Science Foundation’s Partnership for International Research & Education (PIRE) grant 1243433 for funding this research, the Light Microscopy Core Facility at Duke University for help with microscopy, and Drs. Heather Durand and Lawrence David for their assistance with DNA sequencing. Compliance with Ethical Standards Conflict of interest The authors declare that they have no conflicts of interest.

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