Process performance and biogas upgrading of in-situ BM
The characteristics of the inoculum and the applied substrates are given in Table 1. Operating parameters and performance data for the 10 L control and upgrading reactors (CR, UR) under steady-state conditions are summarized in Table 2 and 3, respectively. The experiment was conducted for 172 days and divided into six phases. Figures 2 and 3 illustrate the changes in methane yield, pH, and VFAs over the experimental period for upgrading and control reactors.
Phase I: Initial phase – without H2 addition
In this phase, the two reactors were operated identically and showed very similar performance in terms of biogas production (241-245 mL g-1VS) and CH4 yield (144-145 mL g-1VS) (Table 3). The average CH4 content of the reactors (58 to 59%) and the pH (7.9) were also similar. The total VFA content was around 18 mM, with acetic acid (AA) accounting for more than 60% of the total VFAs. The ratio of propionic acid (PA) to AA of both reactors was below 1.4, indicating a stable AD process according to [18]. The TAN concentration was around 2.5 g L-1. The values align well with those obtained by [19], who observed that a TAN value of 2.5g L-1 (pH 7.9) resulted in stable biogas production during thermophilic (55oC) anaerobic digestion of cow manure.
Phase II: Initial H2 phase
H2 was added in UR from day 64 at a flow rate of 3 mL min-1, corresponding to a H2:CO2 ratio of 2:1. As shown in Figure 2, CH4 yield increased immediately after H2 addition and stabilized from day 70. The average CH4 yield of UR was 185 mL g-1VS, which was approximately 27% higher than the average CH4 yield of CR (Table 3). A similar observation was reported by Treu et al., [20] where H2 addition into a CSTR at a 2:1 ratio resulted 13% increase in CH4 yield. The pH of UR increased from 7.94 to 8.10, while the pH of CR remained the same as in phase 1. BM resulted in a rise in pH due to the removal of CO2 from the liquid phase. Bicarbonate ions (HCO3-) are produced during the AD process when CO2 reacts with OH in the liquid phase, contributing to the buffering capacity of the reactor. Addition of H2 to the system resulted in CO2 consumption and thus loss of buffering capacity [14]. Similar findings have been reported in previous studies [11,20,21]. Total VFA levels in UR rose to more than double the amount in phase I. In contrast to our study, Treu et al., [20] reported relatively low and stable VFA levels after H2 addition.
In CR, the average AA concentration was 21 mM, while in UR, it was 36 mM. PA levels were slightly higher in both reactors than in phase 1. TAN concentrations were also elevated, with 2.57 g L-1 for CR and 2.77 g L-1 for UR. The H2 consumption rate of UR was calculated to be 25%, corresponding to a CH4 production rate of 0.04 mL L-1 d-1.
Phase III: Increased stirring speed
In phase III, the stirring speed of both reactors was increased from 80 to 140 rpm (day 79) in an attempt to improve the transfer of H2 to the liquid phase in UR. As shown in Figure 2, the CH4 yield from UR decreased significantly as the stirring speed increased. The CH4 yield of UR was reduced from 185 (day 78) to 126 mL g-1VS (day 85) for UR. The decrease in CH4 yield of UR was corroborated by the accumulation of acetate (67 mM on average), which was nearly double of what was measured in phase II (Figure 3b). Besides, the propionate concentration was slightly increased from 9 to 13 mM. These observations could indicate that parts of the microbial community were negatively affected by the higher share forces at 140 rpm.
Regardless of the fact that the total CH4 yield decreased as the stirring speed increased, the H2 consumption rate in UR increased from 25% to 46%. This observation was in agreement with our previous study [22]. The rate of CH4 production from H2 and CO2 conversion was increased from 0.04 to 0.08 mL L-1 d-1. For the CR, the CH4 yield was reduced from 143 to 131 mL g-1VS. Ghanimeh et al., [23] observed a decrease in CH4 yield when stirring speed was increased from 80 to 120 rpm. No AA accumulation was observed in the CR, whereas the PA level was slightly higher than in phase II (12 mM) (Figure 3a and Table 3). The pH in both reactors was higher than in phase II, with pH of 8.15 and 8.28 for CR and UR, respectively. The elevated pH in UR can be attributed to greater CO2 consumption in the liquid as a result of the increased H2 gas-liquid mass transfer rate at higher stirring speeds and thus higher BM activity [1].
Phase IV: Change of feedstock blend ratio
On day 86, the stirring speed was again reduced to 80 rpm (return to Phase II conditions), and the CH4 yield rose significantly until it reached a plateau from day 90 (Figure 2). From day 92 the CW fraction was increased from 10% to 20% on day 93 (Phase IV), resulting in an OLR of 0.78 gVS L-1 d-1. The CH4 yield increased in both reactors, with maximum values being 195 mL g-1VS (CR) and 276 mL g-1VS (UR) (Figure 2). After day 102, however, the CH4 yield gradually decreased until it reached a stable period around day 111. During the stable period, the average CH4 yields of CR and UR were 142 mL g-1VS and 204 mL g-1VS, respectively (Table 3). The average CH4 yield of CR measured in this study was lower than that measured by Comino et al., [24] (similar feedstock blend, 80% CM:20% whey), despite the fact that both studies had comparable CH4 content (53%). Longer HRT (41 days) and higher OLR (3.33 gVS L-1d-1) were used by Comino et al., which may explain the difference in performance. The average CH4 content of UR was 39%. The H2 consumption rate was around 17%, which was 31% lower than the consumption rate when CW fraction was set at 10%. The total VFA content of CR was slightly higher towards the end of phase IV (Figure 3a), while the total VFA content of UR was relatively stable (Figure 3b). The pH of both reactors was lower than in phase III, with an average pH of 7.91 for CR and 8.11 for UR. Increased CW ratio to 20% resulted in higher TAN values (both reactors) compared to phase II, suggesting more thorough CW degradation as TAN is a product of protein degradation.
Phase V: Feeding frequency
In phase V, the CW fraction was reduced to 10% and the feeding frequency was changed to once every 48 hours (instead of once per 24 hours). In term of CH4 yield for CR, no changes were observed, while CH4 yield for UR was gradually reduced until a stable period was achieved (day 134). The average CH4 yield for CR was 139 mL g-1VS and 194 mL g-1VS for UR. The CH4 yield of UR in phase IV was slightly higher than in phase II (feeding every 24 hours). The H2 consumption rate was higher than phase II (24 h feeding) when the reactor was fed every 48 hours (25% vs 32%). The increased CH4 yield and H2 consumption rate in UR could be attributed to enrichment of hydrogenotrophic methanogens in less frequent feeding. According to Piao et al., [25], reducing feeding frequency tended to increase the abundance of H2-utilizing methanogens. In the Piao study, the abundance of hydrogenotrophic methanogens increased from 45% to 53% when feeding frequency was reduced from every 24 hours to every 48 hours. The average total VFA content for CR and UR were 26 and 50mM, respectively. The pH of both reactors was slightly lower than in phase II.
Phase VI: Increased H2:CO2 ratio
Substrate feeding was changed to once daily starting on day 141, and the H2 flow rate was increased to 6 mL min-1, equivalent to a 4:1 H2:CO2 ratio (Phase VI). The increased H2:CO2 ratio initially boosted CH4 yield in UR with a maximum at day 151. However, the yield fell after day 163. The average CH4 yield in this period was 165 mL g-1VS, about 11% lower than the value in phase II (H2:CO2 ratio = 2:1). Despite the lower CH4 yield, the H2 consumption rate was doubled (54%) compared to phase II (25%) due to the increased H2:CO2 ratio, which probably stimulated H2-consuming anaerobic microbes.
AA accumulated toward the end of the phase, reaching a maximum concentration of 84.5 mM. The increase in AA levels may be explained by the inhibition of acetoclastic methanogens (e.g. Methanosarcina) caused by high H2 partial pressure [26] or by the enrichment of particular microbial pathways such as homoacetogenesis (Wood-Ljungdahl pathway) [6]. PA content was also increased from 15 to 18 mM when the H2:CO2 ratio was increased. The rise in total VFA content coincided with a drop in pH from 8.01 to 7.91. For CR, the CH4 yield remained consistent throughout phase VI, with an average of 134 mL g-1VS. The average total VFA concentration was 21 mM, with a pH of 7.82. AA concentration accounted for 58% of the total VFA content. The TAN concentration was 2.65 g L-1, which was similar to the value observed in phase II (2.57 g L-1).
In-situ vs. hybrid configurations
A hybrid configuration was tested at the end of the experiment (after day 172). An additional 2 L reactor filled with packing materials was used as an ex-situ biogas upgrading reactor (HR) for the biogas from UR (Figure 1b). Initially, the operating parameters of UR were adjusted to the same as in phase II with a H2:CO2 ratio of 2:1.
When the hybrid setup was used instead of an in-situ (phase II), 39% extra CH4 was obtained (Figure 4). The average CH4 yield rose from 185 to 257 mL g-1VS. Furthermore, the H2 consumption rate increased by twofold compared to in-situ (phase II), and the average CH4 content increased from 40% to 63% (Tables 3 & 4). The CH4 content without considering H2 from hybrid system was around 80%. When compared to the control reactor (Figure 4), the hybrid configuration resulted in a 76% higher CH4 yield, while in-situ configuration resulted in 27% more CH4 (Figure 4). HR had an average pH of 8.07 and an AA concentration of approximately 4.12 mM. The TAN concentration of HR was around 1.09 g L-1.
The H2:CO2 ratio was increased to 4:1 after a stable condition was observed. The average CH4 yield fell from 257 mL g-1VS to 234 mL g-1VS (approximately 9% less CH4). The average CH4 content was reduced from 63 to 51%. Nonetheless, the H2 consumption rate (62%) was slightly higher than at the 2:1 H2:CO2 ratio (60%), indicating that acetate-oxidizing bacteria had the capacity to consume more H2 to produce acetate, as observed in phase VI. Compared to in-situ configuration (phase VI), about 42% extra CH4 was measured and approximately 75% more CH4 was produced when compared to control (Figure 4). The concentrations of AA and TAN were equivalent to those found at a 2:1 H2:CO2 ratio.
Compared to Corbellini et al., [15] our study resulted in lower upgraded CH4 content of in-situ BM. This may be attributed to differences in reactor working volume, as a larger working volume (6L) was used in the present study compared to 3L in [15]. Our findings were more comparable to those of [17], who used a 9L working volume for in-situ testing. Furthermore, when a 4:1 H2:CO2 ratio was added to UR in our study, AA accumulation (> 4 g L-1) was observed, leading to a decrease in pH, while VFA level observed in [15] was maintained at 2 g L-1.
To prevent process instability in in-situ BM reactor, we propose that the amount of H2 added to the in-situ reactor should be kept at a relatively low H2:CO2 ratio (e.g. 2:1). This will minimize the increase in pH caused by bicarbonate removal as well as the possible inhibition of some anaerobic bacteria that are sensitive to high H2 partial pressure. Our study discovered residual H2 in the in-situ and hybrid BM reactors, indicating that further optimization is required. A pressurized reactor may be a solution. Increased operating pressure enhances the solubility of gases and decreases bubble size. Smaller bubble size is beneficial since it maximizes the contact area between bacteria and gaseous substrates while slowing gas upflow through the reactor [1,27]. Previous research found that increasing reactor pressure during in-situ and ex-situ BM resulted in improved conversion efficiency [28,29]. A very high CH4 concentration (> 98%) in the biogas was reported when reactor pressure was set between 5 and 15 bars for a 5 m3ex-situ CSTR [30]. Additionally, the design of the ex-situ reactor used in our study can be improved, for example, by using a long column design like trickle-bed reactor.
Microbial community composition
Microbial analysis of the reactor feed (80% CM:20% CW) showed that Firmicutes and Proteobacteria were the two dominant bacterial phyla, accounting for approximately 50 and 18 % of the abundance, respectively (Figure 5a). Other phyla present in the feed included Actinobacteria (9%) and Bacteriodetes (8 %). Analysis of the inoculum microbiology showed that Firmicutes was the dominating phylum (71%), followed by Synergistetes (7%), Actinobacteria, and Euryarchaeota (both phyla accounted 3% abundance) (Figure 5b). Atribacteria and Thermotogae were also detected in the inoculum, but they were not found in the feed sample.
The taxonomic classification of the microbial community revealed that Firmicutes were the most abundant phyla n the reactors, accounting for 57 to 72% of relative abundance depending on the time points (Figure 5c). This is in agreement with the findings of [31] where Firmicutes dominated a thermophilic biogas reactor digesting cow manure. Firmicutes engages in a variety of metabolic processes for carbohydrate and fatty acid degradation, including the Wood–Ljungdahl pathway (homoacetogenesis) and syntrophic acetate oxidation, which explains their abundance in the reactors [11]. Clostridia, which belong to the Firmicutes, was the most abundant class (representing more than 33% of all bacterial sequences). Other bacterial phyla, such as Synergistetes and Bacteriodetes, were present in both reactors at first, but their numbers declined over time. In terms of methanogenic population, the abundance of Euryarchaeota varied over time, between 13 – 33% for CR, and 18 – 38% for UR (Figure 5c).
Some bacteria, such as HAW-R60, an Atribacteria phyla, was clearly negatively affected by H2 addition (Figure 6a). Their abundance declined over time and was nearly non-existent in phase VI.Atribacteria have been found previously in thermophilic biogas reactors and are involved in hydrolysis of polysaccharides [32]. Another hydrolytic bacterium, Halocella, behaved differently, reaching highest abundance when the H2:CO2 ratio was increased to 4:1 (phase VI) (Figure 6b). Their abundance in UR increased from 6.7 (without H2 addition) to 14.6%. The increase in stirring speed in phase II (day 79-85) seemed to negatively affect Halocella, with decreased abundance in both CR and UR. The cellulolytic bacteria Halocella belong to the class Clostridia and is responsible for cellulose degradation and produces ethanol and H2 from lignocellulosic substrates [33]. Halocella have mainly been found in manure-based samples and their presence in thermophilic biogas reactor has been reported previously [34].
Within the domain archaea, Methanosarcina was the only detected methanogen capable of acetoclastic methanogenesis, although it can also carry out hydrogenotrophic methanogenesis [35]. Methanosarcina was clearly negatively affected by H2 addition and disappeared from UR after 108 days (Figure 6c). High H2 partial pressure has previously been shown to be detrimental to Methanosarcina [36]. Furthermore, the observed accumulation of AA in UR (Figure 3b) is in agreement with inhibition of Methanosarcina.
In contrast to Methanosarcina, the hydrogenotrophic methanogen Methanothermobacter increased in abundance over time and responded positively to H2 addition. Methanothermobacter are typical hydrogenotrophic methanogens that are commonly found in thermophilic biogas reactors [37]. As shown in Figure 6d, their abundance in UR got higher than the abundance in CR over time, suggesting that they were enriched as a result of H2 addition. The high abundance of Methanothermobacter found in this study is consistent with previous research that found this genus to be dominant in thermophilic biogas upgrading systems [6,14,38]. According to [39], Methanothermobacter expand rapidly when H2 is abundant and are adaptable to different concentrations of dissolved H2.
Syntrophaceticus abundance increased rapidly in UR when H2-supplementation was initiated but was greatly reduced after day 140 when the 48h feeding regime was introduced (Figure 6e). Syntrophaceticus is a well-known syntrophic acetate-oxidizing (SAO) bacterium that was discovered in a biogas reactor that relied on the energy from acetate oxidation to produce H2 and CO2 [15,34]. SAO bacteria, which are syntrophic with hydrogenotrophic methanogens (Methanothermobacter in our case), can be inhibited by short or long-term H2 addition to their living atmosphere [20,35]. Increased H2 partial pressure can inhibit SAO from a thermodynamic perspective because syntrophic sustainability is dependent on the H2/formate concentration, which is usually kept low by the methanogenic partners [40]. Interestingly, our study revealed that H2 addition at an H2:CO2 ratio of 2:1 promotes the growth of Syntrophaceticus while increasing the H2:CO2 ratio to 4:1 significantly reduces their abundance. In addition, the abundance of Syntrophaceticus of was maximum when the CW ratio was increased from 10 to 20%.
Similar to Halocella, f_Hydrogenisporaceae_OTU_28, was also affected by the increased stirring speed, seen as reduced abundance after 64 h in both reactors (Figure 6f). f_Hydrogenisporaceae_OTU_28, a member of the OPB54 class, have previously been reported to be involved in the fermentation of carbohydrates to produce acetate and H2 [41].
Our findings revealed that the H2:CO2 ratio, stirring speed, CM:CW ratio, and feeding frequency all had an effect on in-situ BM, either on overall CH4 production or on CH4 production from H2 and CO2 conversion. However, it was only the H2:CO2 ratio and stirring speed that strongly affected the microbial community profile of the reactors.