3.1. Choice of the Optimal Bioreactor Loading
In the first stage, the optimal loading had to be chosen for this type of bioreactor. The results obtained in the measurements from series one were used for this purpose. The total volume of the biogas obtained for each loading value during the three week incubation is presented in Figure 1
. The content of methane in the biogas for all the bioreactor-loading values on the successive incubation days is presented in Figure 2
As might be expected, the highest biogas yield was found in the highest bioreactor loading, i.e., 13% (Figure 1
). However, it is worth noting that the difference in biogas yields between 7% and 13% was significantly lower than that between 2% and 7% (244 mL and 937 mL, respectively). This may indicate that the biogas production process at 13% loading was inhibited by pressure formed in the bottle and that the daily reduction thereof was insufficient. Another cause may lie in the fact that microorganisms are not capable of a quicker and efficient reduction of biomass and the production of biogas under excessive loading. This hypothesis is supported by the results presented in the paper by Betts et al. [45
]. If the bioreactor is loaded with excessive amounts of biomass, slow growth of methanogenic bacteria can result in a rapid decline in pH throughout the process. This situation may be caused by intermediates produced in previous phases that have not been completely decomposed. Regardless of the actual cause of the decrease in dynamics of gas yields, 13% loading had to be excluded as it was too high. The primary reason for discarding 7% loading was the fact that, similar to 13% loading, the septa sealing the bottles were always bulging and, in some cases, the pressure was so high that the septum lost tightness and gas leaked out.
The total amount of biogas produced at 2% loading was lower than in the case of higher loads (Figure 1
). This seems to be obvious: the lower the substrate input; the lower the biogas yield. However, it is worth analysing the methane content in the produced biogas. Analysis of trends on the graph presented in Figure 2
indicates that, at 2% loading, methane concentration in the first biofermentation stage (up to day 11) was lower than at other loading values. However, the analysis did not show statistically significant differences. Moreover, in the second stage (from day 14) of the experiments, concentrations of CH4
for the 2% loading were highest (there was still no statistical significance between the individual values of loaded biomass). The content of methane at the level of ~ 60% volume was high and comparable with other investigations in both laboratory [46
] and industrial [16
] bioreactors. Therefore, since this provides indirect information about the quality of the biofermentation process, the content of methane in the biogas implied the similar efficiency of the process, irrespective of the loading selected for the experiment. Thus, 2% (per dry weight) was assumed to be the best loading in the case of septum-sealed bottle bioreactors and, therefore, only the results obtained at this loading will be presented and discussed below.
Biogas composition (i.e., primarily methane content) derived in the biofermentation process in bottles with 2% loading did not raise any objections; in contrast, there was a problem of high divergence of results obtained in the parallel replications (Figure 2
). This may have been caused by the relatively high variability of the pH value of the suspension.
As indicated in Table 2
, in some cases the pH of the suspension dropped below 6.7, i.e., a value regarded as a threshold below which the biofermentation process is inhibited [48
]. Carbon dioxide, which accumulated in the bottle and dissolved in the solution, forming HCO3
-ions, was found to be a direct cause of suspension acidification [50
]. In such cases, methane content in the biogas decreased. Since the same substrate and inoculum mixture was used in all of the replications, the suspension acidification in some bottles can be explained by the heterogeneity of the input mixture, even though it had been vigorously stirred to achieve homogenization [51
3.2. Inhibition of the Biofermentation Process by Excessive Pressure of Biogas
Gas yields obtained during biofermentation in the bottles in which the gas was removed daily to equalize the pressure to the atmospheric value, and in the bottles without gas removal and increasing pressure, are presented in Figure 3
. In both cases, the biomass load was 2%. The content of methane in the biogas on successive days of the series two experiments is shown in Figure 4
. The points in the graph correspond to the points in Figure 3
. Table 2
presents the pH of the suspension measured in the bottles without gas removal. The measurements were carried out immediately after determination of the biogas yield and composition for the 2% loading.
The structure of industrial bioreactors and large-scale laboratory bioreactors ensure the continuous collection of produced biogas. On one hand, this provides safety (elimination of the possibility of an explosion), on the other hand, it shifts the reaction equilibrium towards biogas production. According to Strömberg et al. [52
], gas pressure can be one of the most important factors influencing biogas production. The use of small-volume bottles, tightly sealed with septa, gave rise to the problem of excessive pressure. The standard solution adopted in the experiments involved the aforementioned and discussed procedure of the daily removal of gas from the headspace by piercing the septum with a syringe needle (with simultaneous measurements of the volume of the produced biogas). However, we decided to determine the dynamics of the process under excessive pressure. In fact, the probability of eliminating the need for time-consuming pressure equalization was low. Nevertheless, an exploration of the rate of biofermentation inhibition appeared attractive, particularly given the fact that experiments that can provide an answer to this question are not feasible in industrial bioreactors and large-scale laboratory bioreactors for two reasons: the first reason is related to safety (explosion of such a bioreactor would pose a real threat to the environment); the other is economics (the cost of the bioreactor).
Since the pressure in the bottle at the higher bioreactor loadings (7% and 13%) caused bulging of the septum rubber (or unsealed it), even after daily biogas removal, this stage of the investigation was carried out only at 2% loading. Analysis of Figure 3
reveals that biogas production under excessive pressure at this loading was already inhibited from day four. It can be claimed that biogas yield remains virtually unchanged throughout the incubation period and reaches a level of several tens of per cent per gram of dry weight. When pressure is reduced daily, the amount of produced biogas increases throughout the incubation period. A similar relationship can be noted in Figure 4
. The amount of methane produced under excessive pressure hardly changed and remained at a level of several per cent. In turn, when pressure was reduced daily, methane production yield had already exceeded 50% by incubation day 11 and had reached almost 60% by the end of the experiment (day 21).
3.3. Stabilisation of the Biogas Production Process with Bicarbonate
The content of methane in variants with and without the addition of a sodium bicarbonate solution to the input mixture is presented in Figure 5
Analysis of the graph presented in Figure 5
allows the unambiguous conclusion that the addition of bicarbonate not only stabilized the results (with a substantially lower standard deviation for the variant with NaHCO3
supplementation) but also ensured higher methane yields. By day 12 of incubation, methane concentration had already exceeded 60%, i.e., a yield obtained within circa 14 days in the experiment variant presented in Figure 1
. Stabilization of pH was confirmed by the results obtained during measurements of the suspension after opening the bottles; pH ranged from 7.04 to 7.48 and did not drop below the threshold of 6.7. The idea of adding a sodium bicarbonate solution to the input suspension was borrowed from the paper by Esposito et al. [14
]. The substance is designed to prevent critical pH lowering during the fermentation process, which in extreme cases may lead to the inhibition of methanogenesis [53
The paper by Mittweg et al. [54
] should be mentioned when comparing the results presented above with those reported by other researchers who have used bioreactors with similar volumes. The authors used 100 mL syringes as fermentation chambers. Their results indicate that the process carried out in such bioreactors can be used in laboratory investigations. The only drawback of this solution is the technical difficulty in reproducing the structure described in their paper. The solution proposed in this study seems to be considerably simpler.