Effect of mixing
Given the experimental conditions (see “Methods” section) and the poor solubility of H2 in the aqueous phase, the optimal mixing conditions yielding the most efficient delivery of H2 from the gas phase had to be determined. The reaction vessels were incubated in an orbital shaker at various mixing speeds (rpm). Figure 1 indicates that there is an optimum value for this parameter; in our arrangement, it was 150-160 rpm. It is noteworthy that at higher mixing rates CH4 production decreased sharply in contrast to earlier observations at thermophilic temperature . In all subsequent experiments, the shaker was set at 160 rpm. It is evident that this mixing rate is valid under our conditions and henceforth was applied consistently in order to limit the number varying parameters. In other systems, the optimal mixing conditions should be determined individually. The main conclusion from these experiments was that the mixing that yields optimal H2 utilization may not be the maximum achievable mixing rate.
Optimization of H2 dosage
Next the optimal daily H2 dosage was established. Various volumes of H2 were therefore injected into the batch reactors, which were treated identically in all other known aspects. The batch fermentations were started by adding 0.3 g of α-cellulose as substrate for AD according to the VDI (Verein Deutscher Ingenieure, protocol . H2 gas was injected every day and the consumption of H2 was followed by gas chromatography. Cumulative CH4 evolution curves are plotted in Fig. 2. CH4 production proceeded steadily for 7-8 days in the control reactors, which received no daily H2 dosage, but from day 12 practically no gas evolved. In total, 6.2 ± 0.54 mmol of CH4 was generated from the residual biogas potential of the sludge and added α-cellulose substrate. 1.62 mmol of this quantity originated from the sludge and 4.58 mmol from the α-cellulose substrate. The biochemical CH4 potential of α-cellulose is 4.71  and therefore all of the added substrate was consumed by the community and was converted to CH4. Addition of a daily 0.81 ± 0.16 mmol of H2 gas into the headspace of the batch reactors dramatically increased the CH4 production (Fig. 2). The GC measurements revealed that all of the injected H2 was completely consumed by the microbes within 24 h. In separate experiments, it was established more precisely that under these conditions all the H2 had vanished from the headspace after 16 h and CH4 evolution started at hour 2 following H2 injection (data not shown). A new dosage of H2 was dispensed consistently every 24 h. Increasing the total H2 load to 43.00 ± 1.43 mmol resulted in a somewhat faster initial CH4 production, but the cumulative-specific CH4 production was lower than in the case of adding 24.42 ± 0.81 mmol of H2 in the same period of time. In line with this observation, H2 started to accumulate in the headspace on day 14 and from day 17-18 CH4 production ceased. On further increase of the overall H2 injection volume to 55.69 ± 1.85 mmol, i.e., 1.86 ± 0.38 mmol H2 day−1, even less cumulative-specific CH4 was yielded. In these reactors, H2 build-up in the headspace started sooner, i.e., on day 10 and CH4 evolution stopped completely on day 13. Overall, these results indicated that the system utilized the α-cellulose substrate within 7-8 days and the microbial community sustained its H2 conversion activity for an extended period of time if the daily H2 injection did not exceed 0.81 ± 0.16 mmol of H2 (Table 1). The concentrations of organic acids were determined every week. Acetate levels increased significantly by the end of the experimental period. 3.43 mM acetate accumulated by the end of the experiment in the reactors receiving 55.69 ± 1.85 mmol of H2, which exceeded the recommended threshold, but apparently this alone did not explain why CH4 evolution stopped in the reactors loaded with higher daily H2 injections (Fig. 3). The pH had increased considerably by the end of the 4-week experiments (Fig. 4), indicating a severe loss of the bicarbonate buffering capacity of the inoculum sludge. It is noteworthy that the pH also shifted by 1.1 units in the control reactors which were not fed with H2. In order to employ the same protocol, these vessels were also degassed and filled with N2 gas every day. It is therefore likely that the daily replacement of the headspace prompted a gradual desorption and loss of dissolved CO2 and caused a shift in the bicarbonate buffering system [27, 28]. The pH increased even further, i.e., beyond pH = 9, which is a critical upper limit for the methanogenesis . A similar exhaustion of the buffering capacity upon H2 addition was noted in previous reports [19, 20]. The system could apparently tolerate high pH fairly well when 0.81 ± 0.16 mmol of H2 was the daily dosage, but started to inhibit CH4 biosynthesis on day 13 and 10 upon addition of daily 1.43 ± 0.28 or 2.86 ± 0.38 mmol of H2, respectively. In this experimental set-up, it was not possible to determine the time points when the inhibitory pH range was attained. The results indicated that the likely reason for the obstruction of CH4 formation was the limiting buffering capacity of the system due to the low bicarbonate concentration. The optimal amount of daily H2 dosage in this system was within the range of 0.8-1.5 mmol of H2; further experiments should determine the exact value.
Effect of CO2 addition
In the next series of batch fermentations, the inoculum originated from the same mesophilic industrial biogas plant, but at different points of time, and therefore small fluctuations of organic total solid content and microbial community composition should be taken into account when the results are subjected to direct comparison. The initial addition of α-cellulose was omitted in order to avoid any disturbing effect of the CH4 generation from the substrate. The duration of these fermentations was extended to 80 days to test for sustainable CH4 production. The reactors were supplied with the optimal daily dosage of 0.81 mmol of H2 in order to check if the CO2/bicarbonate buffering capacity was indeed the major limiting factor in the previous experiments [28, 30]. The daily CH4 volumes measured in the headspace are plotted in Fig. 5. CH4 evolution progressed steadily until day 28, but dropped sharply afterwards. A warning sign of system failure was noticed already on day 27, when measurable residual H2 was detected in the headspace (Fig. 5; Table 2). As shock therapy, massive CO2 injection (25 mL) was dispensed into the reactors following the daily dosage of H2 on day 31 (Fig. 6). All of this CO2 disappeared from the gas phase within 24 h, indicating that the system was indeed severely depleted of CO2/bicarbonate. The same CO2 treatment was repeated next day, which apparently restored the functional state of the system signaled by the build-up of residual CO2 in the headspace (Fig. 6). The daily CO2 dose was then gradually decreased to the stoichiometric volume, i.e., approximately 0.25 mol of CO2/mol of H2 per day. The system responded positively, as exhibited by the restoration of CH4 production on day 32 accompanied by a gradual decrease of residual CO2 levels in the gas phase. Daily CO2 injection was stopped on day 41. H2 accumulation commenced again almost immediately and was accompanied by the loss of CH4-evolving ability from day 43, and therefore CO2 injection (25 mL) was resumed on day 47. Detectable remaining CO2 was noticed already on the next day and from this time on a daily dosage of 0.25 mol of CO2/mol of H2 of CO2 was maintained until the end of the experiment. CH4 production returned to the previous level, all of the injected daily H2 and CO2 were consumed within 24 h and this continued for an additional month. It is noteworthy that, except for pH bursts on days 31 and 45, the pH in both the control and H2-fed reactors remained within the acceptable limit of pH ≤8.5 throughout the investigated period (data not shown).
Several deductions could be drawn from this series of tests. First, the system becomes depleted of CO2 if semi-continuous H2 feeding and daily degassing are administered to the fed-batch system. This phenomenon was manifested after about 1 month in our arrangement, where daily degassing and replacement of the headspace were included to retain the same protocol in the control and experimental reactors. Clearly daily degassing is not necessary in industrial setting. Second, the residual H2 accumulation in the gas phase is a good early warning sign of upcoming system failure due to CO2 exhaustion. Third, the microbial community participating in the CH4 generation process recuperates quickly and completely even after repeated system failure if the process control is alerted in time. Fourth, the microbial community supplied only with H2 and CO2 upholds the pH within the normal operating range. Finally, stoichiometric administration of H2 and CO2 yields a practically complete conversion to pure CH4 within 24 h under mesophilic conditions.
Effect of additional substrate addition
Next, it was tested whether the addition of α-cellulose affected the CH4 productivity from H2. Two series of experiments were designed and the duration of the experimental period was shortened in order to avoid any complication due to CO2 depletion and concomitant pH elevation. In the first set of batch fermentations (Fig. 7), various amounts of α-cellulose were added only at the start of the experiments, and in the second series (Fig. 8) the addition of the same amount of α-cellulose was repeated every week. Daily replacement of the headspace with N2 and the injection of 0.81 mmol of H2 was maintained in all reactors.
There was no significant difference between the CH4 productions from H2 in the reactors receiving the substrate quantity recommended by the VDI  protocol as compared with those without substrate, i.e., the difference between the green and red curves in Fig. 7 correspond solely to the CH4 produced from α-cellulose. This suggests that the addition of substrate at the beginning of the fermentation does not assist CH4 evolution from H2. Moreover, an inhibition of CH4 productivity from H2 was noted when the substrate load was doubled, i.e., upon the addition of 0.6 g substrate, 3.47 ± 0.08 mmol of CH4 was formed from α-cellulose instead of the theoretical potential of 9.42 mmol of CH4. It should be noted that the H2 consumption rate remained unaffected by the substrate loading, i.e., the injected H2 disappeared from the headspace within 24 h. The conversion efficiency of CH4 formation from H2 was estimated from the daily CH4 levels in the headspace. The day-to-day values fluctuated considerably during the experimental period and achieved an average of 72 ± 25 %. The remainder of the H2 may have been metabolized in alternative pathways, which are the subject of future studies.
In the next set of experiments, the reactors were fed with the same amount (0.3 g) of α-cellulose every week and the daily H2 injection (0.81 mol H2) was maintained. The aim was to test whether the microbial community remained intact for an extended period of time after the expiration of its residence time in the industrial AD facility and to see whether the metabolically active community facilitated the bioconversion of H2 to CH4. The cumulative CH4 production increased almost linearly and the amount formed suggested an unchanged reaction rate for both α-cellulose and H2 when the VDI protocol  was followed (Fig. 8). It is noteworthy that increasing the weekly α-cellulose load prompted a strong inhibitory effect. The collapse of the CH4-forming activity was not associated with changes in pH. Without α-cellulose, the daily dosage of H2 caused an increase of the pH into the dangerous zone, as observed earlier (Fig. 4), due to the depletion of the buffering capacity. Weekly supply of the substrate balanced the pH; the degradation of the α-cellulose apparently yielded enough CO2 to maintain stable operation. Too much substrate, e.g., 0.6 g α-cellulose/week, shifted the pH to lower values, although it did not fall below 6.5, which is usually considered detrimental . The accumulation of acetate increased dramatically upon substrate overloading (data not shown). This might have been the likely reason for the process inhibition. It is important to note that the H2 conversion yields in this series of experiments were close to 100 %, which emphasizes the importance of the inoculum quality.