Optimizing the dosing and trickling of nutrient media for thermophilic biomethanation in a biotrickling filter

Ex-situ biomethanation in a biotrickling filter is being developed as a robust technology for the conversion of CO2 to CH4 using H2 generated from renewable power. The technology uses hydrogenotrophic methanogens in a biofilm as the catalyst and the optimumsupply of nutrients in the biofilm is imperative for stable and long-term operation. In this study, essential nutrients for the methanogens in the biotrickling filter were highlighted along with a systemic investigation of the nutrient supply methods. The results showed that ammonium and iron were the critical nutrients with a minimum required concentration of 0.3 g/L and 1.5 mg/L, respectively. Furthermore, thoroughly wetting the biofilm with nutrient media, either using flooding or trickling at a high-rate, was needed to maintain an active biofilm; although, these methods temporarily reduce the conversion for a duration of 18 to 24 h due to the formation of the thicker water film.


Introduction
Power-to-methane (PtM) is a potential solution for the storage of excess renewable power [1] and it is a source of low carbon gaseous fuel [2]. The interest in PtM is supported by the fact that the product, methane (CH 4 ), can be utilized in the existing natural gas infrastructure. For example, the EU has a network of approx. 2 million km of transmission and distribution with 800 TWh of underground storage for natural gas [2]. PtM can be applied for the conversion of industrial and/or biogenic CO 2 to CH 4 . Overall, application of PtM can integrate the renewable power grid with biomethane/nautral gas grid, which will offer flexibility to the electricity grid while aiding decarbonization of other sectors [3]. It is estimated that by 2050 about 150 TWh (14 bcm natural gas equivalent) of PtM could be produced in the EU using biogenic CO 2 captured from biogas plants combined with locally produced H 2 using electricity from solar and wind power [4]. The application of PtM at a biogas plant is also a substitute for the conventional biogas upgrading process, where CO 2 is removed from the biogas to increase its heating value. Whereas, using PtM the CO 2 is utilized to produce CH 4 that increases the heating value and the overall CH 4 output from biogas plant.
Several studies have shown the ex-situ biomethanation using biotrickling filter (BTF) as a feasible technology for PtM and for the biogas upgrading [5][6][7][8]. The BTF is a 3 phase reactor where the HM are grown over a packing material as biofilm, the gaseous substrates (H 2 and CO 2 ) pass over the biofilm, and the liquid nutrient media is trickled over the packed bed to supply the nutrients. The solubility of H 2 is approx. 40 times lower than CO 2 and the overall process is mass transfer limited [9]. The arrangement of HM over a packing material offers a relatively large surface area for the mass transfer of gaseous substrates that results in higher mass transfer rates. The main advantage of BTF is the relatively higher volumetric rate of CH 4 production as compared to the other biomethanation technologies. For example, CH 4 production rate of 15.4 Nm 3 /m 3 d was achieved using a lab scale BTF [10] as compared to 3.7 Nm 3 /m 3 d using CSTR [11] and 0.25 Nm 3 /m 3 d using upflow reactor [12].
In addition, the BTF does not require stirring or gas recirculation and hences offers a lower operating cost. The BTF technology can also be applied with other sources of CO 2 , e.g. for biomethanation of syngas [13].
In BTF the conversion of CO 2 and H 2 to CH 4 is carried out by the hydrogenotrophic methanogens (HM), a class of anaerobic archaea found in the anaerobic digester for the biogas production. The HM are enriched and immobilized on a packing material in the form of biofilm and the substrate gases pass over it. The optimal supply of the nutrients is essential for the continuous operation of BTF because Ashraf et al. the methanogens are dependent on their availability. Major elements found in the methanogenic microorganism are C, H, N, Na, K, S, P, Ca, and trace elements are Fe, Ni, Co, Mn, Zn, Cu, and Mo [14]. Central to the hydrogenotrophic methanogenesis pathway are enzyme complexes that catalyze redox reactions, and these enzymes contain transition metals as cofactors for electron transport or as catalytic centers at active sites. Important transition metals for methanogenic archaea are Fe, Ni, Co, Mn, and Zn and low bioavailability of transition metals limits the methanogenesis in pure and mixed cultures [15,16]. The depletion of trace metals results in process imbalances because they are essential in enzyme complexes [17]. Specifically, for the hydrogenotrophic methanogenesis, the hydrogenases are Ni-Fe enzymes that oxidize H 2 and reduce ferredoxin (coenzyme F 420 ) [18]. These energy converting hydrogenases are abundant in Fe and contain a cluster of Fe 4 S 4 with a Ni-Fe active site. For example, the membrane-associated hydrogenases contain three Fe 4 S 4 clusters and a Ni-Fe active site [14], and can contain additional polyferredoxin subunits with 6, 10, or 14 Fe 4 S 4 clusters and a Ni-Fe active site [19]. Another type of hydrogenase contains four Fe 4 S 4 clusters and a Ni-Fe active site [20]. Physiological studies of pure cultures have shown that optimal metal concentrations for microbial metabolism are orders of magnitude higher than in situ concentrations [16]. In the case of biofilm reactors, such as BTF, the nutrient concentration required could be higher due to the diffusion resistance in the water and biofilm. In addition, for the biofilm reactors the supply method of the nutrients over the packing materials plays a vital role.
The availability of nutrients is needed for the stability of anaerobic digester for biogas production which is operated with energy crops, animal excreta, crop residues, organic fraction of municipal solid wastes or any other type of organic waste [21], and have been studied in detail [22,23]. As compared to the anaerobic digestion, the substrate for the ex-situ biomethanation in BTF is simple, i.e. H 2 and CO 2 , hence nutrients supplementation is more essential. However, to the best of our knowledge, analytical investigation of nutrient consumption and the analysis of nutrient dosing and supply methods have not been carried out for BTF, specifically for the long-term continuous operation of the reactor. In this study, an optimization of the nutrients dosing and supply method was studied for stable operation of ex-situ biomethanation in a BTF. Essential nutrients were identified along with their minimum concentration levels, followed by a systematic approach to find the effective method of supplying these nutrients. The dosing method relates to the frequency of supplementation of the nutrients in the nutrient media sump and the supply method relates to the method of trickling the nutrient media over the biofilm in the BTF. After the optimization of the nutrient dosing and supply methods, the maximization of methane production was investigated.

Experimental setup
The schematic layout of the BTF reactor and the connections to ancillary equipment are shown in Figure 1. The reactor body was built using a clear polyvinyl chloride pipe. The inner diameter was 5.4 cm, packed length was 22 cm, working volume (V r ) was 503.8 mL, and media sump volume (V s ) was 400 mL. The reactor had a heating jacket that was made by wrapping a silicon tube (6.4 mm inner diameter) around the whole length of the reactor. On top of the heating jacket, a layer of thermal foam insulation was wrapped. Water at 57°C was circulated from a water bath in the silicon tube to maintain thermophilic conditions in the reactor. The reactor was packed with finned Raschig rings made of polyethylene and had the size of 7 mm x 10 mm (PE08, Tongxiang Small Boss Special Plastic Products Ltd. (http://www.smallboss.com/). One piece of the packing materials had a vendor quoted surface area of 3500 m 2 /m 3 . The bed void fraction of the packing material was measured to be 76%.
The feed gas used was a mix of N 2 , CO 2 , and H 2 . The inlet flowrate of these gas components was controlled using thermal conductivity-based mass flow controllers (EL-FLOW Select series, Bronkhorst ® ).  Nutrient media was trickled from the sump to the top of the reactor using a peristaltic pump (Watson Marlow Qdos30 Universal Pump). A 125-micron mesh filter was installed in the media line connecting the peristaltic pump and the reactor. The filter stops the larger particle size solids from entering the reactor, which was the modification made after the clogging of the BTF reactor in an earlier study [24]. The filter was cleaned regularly. A relief valve with a set point of 100 mbar was installed before the filter. The relief valve provided safety in case the peristaltic pump was running, and the filter got clogged. The pH in the nutrient media sump was measured using a digital pH meter with the probe (INTELLICAL PHC735, HACH Lange) mounted in the sump. The outlet flowrate of the gas was measured using a positive displacement type gas flowmeter (BPC ® µFlow, BPC Instruments AB, Sweden).

Nutrient media
Pasteurized cow manure (PCM) was used as the nutrient media. The use of PCM has been validated as an effective nutrient media in the earlier study [8], however, the presence of solids in PCM led to eventual clogging of the BTF, as reported in [24]. In this study, an improved method of pretreating the manure was adopted that consisted of the following steps: (i) coarse sieving the cow manure to remove large solids and fibers, (ii) filtering through 500 µm sieve, (iii) centrifugation at 8000 rpm for 10 min, (iv) filtering the supernatant through 125 µm sieve, (v) and lastly heating the filtrate from 125 µm sieve till 70°C for 1 hr. Two different batches of PCM were prepared and their analysis is given in Table 1. PCM Batch 1 was used from the start of reactor operation till day 280 and PCM Batch 2 was used afterward till day 450.

Inoculation
The reactor was started using the direct inoculation methods as was reported in the earlier study [25].
Mixed anaerobic culture was used as inoculum that was obtained from the primary digester of Nature Energy Midtfyn biogas plant, Denmark. The inoculum was coarsely sieved to remove large solids and straw, afterwards, it was filtered through 125 µm sieve. The filtered inoculum was degassed for one week at 55°C in an incubator. The analysis of the filtered and degassed inoculum is given in Table 1.

Analytical methods
Gas samples were collected in triplicates at the gas outlet of the reactor in 5.9 ml vacuum vials (Exetainer ® Evacuated Flat Bottom Vial). The composition of H 2 , CO 2 , N 2 , and CH 4 was analyzed using gas chromatography (GC, 7890A, Agilent Technologies, Santa Clara, CA, USA) equipped with a thermal conductivity detector and a fused silica capillary column (Carboxen ® 1010 PLOT, 30 m × 0.53 mm). The Ashraf et al.

Performance parameters
Gas retention time (GRT) was calculated based on the total gas flowrate at the inlet of the reactor and empty bed, as shown in Eq. 1. Here, V r is the empty packed bed volume and F H 2 , F CO 2 , and F N 2 are the inlet flowrate of the gas components H 2 , CO 2 , and N2, respectively, in NmL/min.
To analyze the reactor performance in terms of methane production, specific methane production capacity (PC, Nm 3 /m 3 d) was calculated as shown in Eq. 2. Here, F out (NmL/min) is the total gas flowrate at the reactor outlet and C CH 4 is the CH 4 composition at the reactor outlet.
To analyze the conversion of H 2 removal efficiency (RE, %) was calculated, as shown in Eq. 3. Here,

Startup and operating procedures
Details about the input parameters and experimental trials conducted are given in Table 2. The reactor was started using the direct inoculation methods as outlined in [25]. A mix of H 2 , CO 2 , and N 2 was used as the inlet gas to the reactors. N 2 was added in the inlet gas to simulate the feed gas as biogas, where N 2 asserts an equivalent partial pressure in the feed gas as that of CH 4 in the case of a biogas feed. However, during the startup and enrichment period, only H 2 and CO 2 were fed as inlet gas to the reactors. This enables a higher partial pressure of substrate H 2 and CO 2 that assists the enrichment and development of biofilm in the BTF reactors. After the startup period, GRT reduction trials were conducted, where the objective was the incremental increase in the gas feed flowrate and in turn to maximize the PC. After that, the nutrient consumption and supply trials were conducted.
Here a distinction needs to be made, the term nutrient dosing is used for the addition and/or supple-

Enrichment and GRT reduction trail
The reactor started producing CH 4 on the second day and on day 5 its concentration reached a maximum value of 28.5 mole% (C CH 4 at the reactor outlet. This showed that the enrichment of the hydrogenotrophic methanogens has started. After that N 2 feed to the reactor was started along with the start of GRT reduction trial. The results of the performance parameters are shown in Figure 2 relatively improved the H 2 conversion, and C H 2 decreased from 42% to 35%, however, this effect was short-lived and the C H 2 increased again to < 40% on day 114.
The objective of the GRT reduction trial was to find the limits for stable conversion and maximum PC.
During this trial the GRT was reduced stepwise and it showed that the supply of the nutrient could be the bottleneck in increasing the PC and maintaining the quality of the upgraded biogas.

Estimation of nutrient limitation
From day 116 to 281 nutrient limitation trials were conducted, the objective was to find whether the trickling or supply method is the limiting factor or the concentration level of the nutrients in the media sump are the limiting factor. To stabilize the reactor the gas feed flowrate to the reactor was reduced and GRT was increased from 25.7 min to 43.2 min. This temporality improved the H 2 conversion but overall, the C H 2 kept increasing at the outlet of the reactor, as shown in Figures 2a and 2b.

Nutrient supply method
The nutrient media was trickled continuously from the top of the reactor at a flowrate of 10 mL/min procedure: 100 mL of was removed and 200 mL of the fresh media was added in the sump. As can be seen in Figure 2b, the H 2 conversion improved significantly after media addition on days 150 and 175, the C H 2 reduced to almost 0. However, the C H 2 remained stable for approx. 10 days and afterward it started increasing again. The routine of flushing the reactor every second day was carried out during this period ensured supply over a wider fraction of the packing. Considering this, the decrease in conversion showed that the concentration of nutrients could have decreased over time that resulted in lowering the activity of methanogens. Hence, the concentration levels of the nutrients were measured over time.

Critical nutrients
From day 191 to 281 nutrient concentrations were routinely measured to identify the essential nutri- and Fe could be the significant parameter for the methanogens in BTF. However, this does not mean that other trace metals are not essential; their significance for the methanogens and for the overall anaerobic digestion process have been reviewed in detail [16,21,26].
In this study, the critical level of NH 4 + and Fe was estimated as a minimum concentration below which the activity of the hydrogenotrophic methanogenesis starts getting affected. For NH 4 + and Fe, the

Optimizing the nutrient supply method
As was shown in section 3.

Effect of flowrate of continuous trickling
Flowrate of the continuous media trickling was varied from 10 mL/min to 200 mL/min. Each setting of the flowrate was maintained for 7 days and the average values of performance parameters measured are given in Table 3.  Comparing the flushing and flooding, no relative difference was found between these two methods of nutrient supply in terms of their efficacy of nutrient supply. However, there are aspects of operating and capital cost that needs to be determined to select among these methods of nutrient supply. The flooding method will require a separate vessel of at least equal volume of the BTF reactor to store the nutrient media for flooding. This will increase the capital cost of the biomethanation system. The flushing method does not require additional vessels however, a high flowrate pump will be required.

Flushing vs flooding
Moreover, the flooding method ensures a complete wetting of the packing material and ensures the supply of nutrients over all the packing material. Hence, inoculation of the BTF with flooding is recommended as it will ensure the formation of the biofilm over all the packing material.
As was discussed in section 3.2.1, the method of supplying the nutrients with only continuous trickling at a relatively low flowrate could lead to channeling and reduced activity of the biofilm. Hence, ensuring the nutrient media supply using the relatively vigorous method of flushing or flooding is necessary. However, they cause the conversion to reduce for the duration of the transient period.
This means that a biomethanation system consisting of a single BTF reactor cannot maintain a steady operation or CH 4 productivity. To overcome this shortcoming, a system of two or more BTF can be designed that undergo the vigorous nutrient supply method of flooding or flushing at alternative times. This system can be operated using a feed gas flowrate control scheme that shifts the feed gas flowrate to the reactor operating at a stable period and reduces the feed gas flowrate from the reactor that is under the transient period. Overall, the feed gas control scheme can ensure a steady conversion and outlet flowrate from the biomethanation system.
The effect of the thickness of water film on BTF performance was investigated by [9] where they compared the PC with continuous nutrient trickling at a flux of 1.36 m 3 /m 2 h and no trickling. They reported a 20% increase in PC without trickling as compared to the trickling. This improvement effect was attributed to direct gas to biofilm contact where the mass transfer resistance due to water film is not present. The results of this study also show the same effect where PC decrease with increasing the trickling rate. It was also shown in this study that the case of no trickling is not sustainable for long period. However, intermittent supply of the nutrient can be adopted, where the reactor is either flushed or flooded at controlled intervals.

Biomethanation trial
During the biomethanation trial, the feed gas consisted only of H 2 and CO 2 . The objective was to investigate the effect of relatively higher partial pressure of reacting gases, H 2 and CO 2 , in the absence of N2 which is an inert gas component. The relatively higher partial pressure of the reacting gases could lead to higher absorption of reacting components in the biofilm and higher rates of reaction.
The biomethanation trial was conducted at a high gas feed rate to GRT of 24.9 and 16.7 min and the results are shown in Figure 5. On average PC was 8.51 ± 0.56 Nm 3 /m 3 d at GRT of 24.9 min, which was 21% higher than the PC of 7.0 ± 0.17 Nm 3 /m 3 d achieved at a similar GRT of 25.7 min but had (b) C H 2 (mole%) -H 2 gas composition at the reactor outlet, C CO 2 (mole%) -CO 2 gas composition at the reactor outlet, and C CH 4 (mole%) -CH 4 gas composition at the reactor outlet.
During the biomethanation trial, there was unreacted H 2 and CO 2 at the reactor outlet, RE was 96.1% and 67.6% at GRT of 24.9 and 16.7 min, respectively. This indicated that the feed gases were available over the whole length of the packing in the reactor, hence the CH 4 formation was not limited by the unavailability of the substrate. This also means that the PC value achieved at GRT of 16.7 was the maximum possible for this size and type of packing material and reactor, considering that the whole packing contained an active biofilm of hydrogenotrophic methanogens. The reason for unconverted H 2 and CO 2 at the same GRT compared to when the feed was H 2 , CO 2 , and N2, is the increased H 2 and CO 2 flow due to the same GRT.

Overall reactor performance
In an earlier study BTF reactor using the PCM and polyurethane foam as packing material was tested.
The PCM used was only coarse filtered through 500 µm mesh, which resulted in clogging the reactor after 215 days of operation. During this study pretreatment of PCM was carried to filter it through 125 µm. Also, an external filter was installed in the nutrient media line, as shown in Figure 1, which was cleaned routinely. With these modifications, the reactor was successfully operated for 450 days.
This showed that the continuous operation of the BTF could be extended by adopting a pretreatment method for PCM and with the use of an inline filter. The same approach applies to other solid containing nutrient media, for example, anaerobic digestate.
The maximum average PC achieved in this study was 7.0 ± 0.17 Nm 3 /m 3 d with the inert gas present in the feed gas and an average outlet gas quality of C N 2 +CH 4 of 91% ± 1.7% was achieved. During the biomethanation trail, the maximum average PC was 9.44 Nm 3 /m 3 d, however, this was achieved at GRT of 16 min and without complete conversion of H 2 and CO 2 . These values of PC are lower as compared to the maximum reported in the literature for BTF, for example, PC of 15.4 Nm 3 /m 3 d was reported by [10]. The relatively lower PC achieved in this study could be due to the packing material, as in the earlier study a PC of 8.54 ± 0.47 Nm 3 /m 3 d was achieved [24], that used a similar BTF design and PCM as nutrient media but had polyurethane foam as the packing material.

Conclusion
The installation of an external filter and pretreatment of the pasteurized cow manure led to 450 days of continuous BTF operation without clogging. NH 4 + and Fe were essential nutrients and their concentration of nutrients can be controlled using either a routine or continuous replacement of the nutrient media. Continuously trickling at a low flowrate together with periodical flushing or flooding was found to be an effective strategy for the nutrient supply; although both flushing and flooding affect the CH 4 productivity in the hours after. This effect needs to be considered at a system level design of BTF.