3.1 Effect of inoculum pre-treatment on biogas composition over time
The biogas composition for all treatments varied with time, as shown in the graphs (Figure 2A-E). The biogas composition of the control consisted mainly of methane (40 -62 %) and carbon dioxide gas (23-36 %). Over the course of the 15-day period, very little hydrogen was detected, as anticipated.
The biogas composition under heat inoculum pre-treatment (T1) comprised of carbon dioxide, methane and hydrogen in varying fractions (Figure 2B). At the beginning of the experiment, carbon dioxide and hydrogen were the main gas fractions at 88 % and 9 % respectively. Nevertheless, there was a shift in gas composition at the end of the experiment with the gas mainly consisting of methane (77 %) and carbon dioxide (21 %). Comparing the hydrogen and methane fractions, although the hydrogen content was low (less than 20 %) it was higher than the methane content up to Day 6, however, beyond Day 6 the hydrogen content dropped while methane increased. This indicates that heat pre-treatment had a short-term positive impact on hydrogen production and partially inhibited methane production for a short period. This is a shortcoming of inoculum pre-treatment, as reported by [26], who stated that pre-treating inoculum to inhibit hydrogen consumers is ineffective in the long run, and that repeated pre-treatment is needed to achieve sustained biohydrogen production. Scholars [13, 27] agree that inhibition of hydrogen consuming bacteria can be temporary, adding that spore forming hydrogen consuming bacteria can reactivate after a period of time, forming new stable microbial communities that are adapted to the environment. Researchers [29] ,mention that it is also possible for an inoculum pre-treatment to also suppress non-sporing hydrogen producing bacteria while some sporulating hydrogen consuming bacteria (methane producers) manage to survive the harsh pre-treatment conditions. For example, [23] states that suppression of non-spore forming HPB from the genus Enterobacter can occur while sporulating HCB like Clostridium aceticum and Clostridium thermoautotrophicum can withstand pre-treatment.
In contrast to heat pre-treatment, biogas under alkaline pre-treatment (T2) comprised mainly of carbon dioxide and hydrogen. The carbon dioxide content was high (85 %) at the start of the experiment gradually dropping to 38 %. The hydrogen content increased as the experiment progressed reaching a maximum of 48 % on Day 8, at which point gas production ceased. Throughout the experiment, small quantities of methane (7.5-10.6 %) were observed, indicating that methanogens were not fully inactivated by alkaline pre-treatment. This is similar to the results of [27] who detected small amounts of methane gas during thermophilic dark fermentation of cassava using alkaline pre-treated inoculum.
The biogas generated under acid pre-treatment (T3) consisted mainly of carbon dioxide and hydrogen. However, a lower carbon dioxide content was observed (40-50 %) compared to pre- treatment T2, and the reaction stopped after only 3 days. The hydrogen content was also higher, ranging between 46 and 54 %, the highest hydrogen content observed of all the pre-treatments investigated. Additionally, no or little methane was detected, indicating that acid pre-treatment had fully inhibited the activity of methanogens. In another study, [28] also found negligible methane after acid pre-treatment, concluding that methane-producing bacteria were effectively inhibited by acid pre-treatment. They further hypothesized that methane-producing bacteria become inactivated when culture environments become too acidic or alkaline, with acidic conditions causing more extreme inhibition.
Alkali-heat pre-treatment (T4) also produced biogas consisting mainly of carbon dioxide and hydrogen with no methane detected during the 9 day run period. However, a higher carbon dioxide content was observed compared to pre-treatment T3 (60 -84% ) and, the hydrogen content was lower (11- 34 %).
3.2 Effect of inoculum pre-treatment on hydrogen yield
The cumulative hydrogen yield obtained for each treatment is shown in Figure 3. The graph shows that inoculum pre-treatment had a positive effect on hydrogen production. This is consistent with other research findings [26, 27–30], which found that chemical and physical pre-treatment of the inoculum enhances hydrogen production. Figure 3 also shows that the different inoculum pre-treatments clearly had a varied effect on hydrogen production. This is similar to the observations made by [31–33] who found statistical differences in hydrogen yields between inoculum pre-treatment groups.
Amongst the investigated pre-treatment methods, acid pre-treatment yielded the highest hydrogen yield (142, 74 Nml/ gVS), while heat pre-treatment produced the lowest hydrogen yield (0, 90 Nml/ gVS). Other works also found that acidic pre-treatment outperformed heat pre-treatment [30, 34]. In comparison, [35–37], found inoculum heat pre-treatment to be the most effective way to boost H2 yields from organic waste such as municipal waste, ground wheat and palm oil mill effluent. While several studies have reported increased hydrogen production as a result of heat pre-treatment, literature also suggests that when temperatures above 80 oC are used, as in this research, hydrogen yields are reduced [38].
3.3 Effect of inoculum pre-treatment on VFA production
Numerous studies have shown that dark fermentation hydrogen production is accompanied by the production of VFAs and other low-molecular-weight compounds [25, 30, 33]. Figure 4 depicts the percentage composition of the VFAs produced during the dark fermentation experiments which included acetic, butyric, propionic and valeric.
The main VFA produced in all pre-treatments, including the control, was valeric acid. The highest VFA fraction (86 %) was observed under acid pre-treatment. In general, a high valeric fraction (above 60%) was observed for pre-treatments that had high hydrogen content (T2 –T4), whereas pre-treatment (T1) with negligible hydrogen production recorded low valeric acid distribution (~ 20 %).
The fact that low amounts of acetic/propionic/butyric acid was found in the pre-treatments linked to hydrogen production (T2-T4) was particularly intriguing. This is in stark contrast to other research studies that report that the key metabolites produced during dark fermentation are acetic, propionic and butyric acid [31, 30, 33,].High hydrogen yields are typically associated with butyric and acetic acid production, this is because the metabolic pathways involved in the formation of acetate and butyrate (equations 1and 2) show a positive correlation with hydrogen production and acetate [32]. However, high acetic concentration isn't always associated with high hydrogen volume, as acetic production can result from hydrogen consumption via homoacetogenesis [31].
C6H12O6 + 2 H2O → 2 CH3 COOH + 2 CO2 + 4 H2 (1)
Glucose Water Acetate carbon dioxide hydrogen
C6H12O6 → CH3 CH2 CH2 COOH + 2 CO2 + 2 H2 (2)
Glucose Propionic acid Carbon Dioxide hydrogen
3.4 Comparison of inoculum pre-treatment methods
To compare the effectiveness of the investigated inoculum pre-treatment methods for enhancing hydrogen production while inhibiting methane production, the hydrogen yield was considered as well as applying one-way ANOVA.
Hydrogen yield
Based on the mean hydrogen yields obtained from this study (Figure 5), the effectiveness of inoculum pre-treatment, ranked in descending order is: Acid >Alkaline- heat>Alkaline>Heat. Comparing the effectiveness of inoculum pre-treatment results obtained in this study other studies (Table 3), no conclusive answer can be given on the best pre-treatment method for enhancing hydrogen production from dark fermentation of FVW. This is because the effectiveness of inoculum pre-treatment is affected by substrate composition, operating conditions and inoculum type amongst other factors [31, 41]. In their works, [31] gave further insight on the variation in results of the best inoculum pre-treatment methods, explaining that the type of substrate affects the effectiveness of the inoculum pre-treatment owing to variances in substrate biodegradability. Moreover, differences in composition of the parent inoculum also influences the effectiveness of a pre-treatment method, further contributing to contradiction in results between studies. This is due to the fact that the microbial communities present in the parent inocula display different levels of tolerance towards changes in environmental conditions, thus inocula responds differently to the harsh conditions they are subjected to by pre-treatments [31].
Table 3
Comparison of literature inoculum pre-treatment ranking with study results
Inoculum | Investigated pre-treatment | Most effective pre-treatment | Ranking | Ref |
Anaerobic sludge | Acid, alkali, heat, heat-alkali | Acid | Acid>heat-alkali>alkali>control >heat | This study |
Activated sludge | Acid, alkali, BES heat, aeration, chloroform | Acid | Acid>alkali>heat> aeration>chloroform>BES | [34] |
Anaerobic granulated sludge | Chloroform, acid, alkali, heat, freeze thaw | Heat (1000C, 1h) | Heat>alkali>chloro>acid>freeze thaw>control | [36] |
Anaerobic sludge | Acid, alkali, chloroform, heat, load shocking | Untreated sludge(control) | Control>load shock>alkali>heat>acid>chloroform | [27] |
Anaerobic mixed microflora | Acid, base, heat | Heat (80 0c, 30 min) | Heat> base >acid | [38] |
However, this study notes that the majority of studies in Table 3 ranked heat pre-treatment as the most effective method. Similarly, a study paper by [42] concluded that among the pre-treatment approaches they reviewed, heat and acid pre-treatments are the most commonly investigated and most effective. Researchers [43] also stated that heat and acid-base pre-treatments are widely used because they facilitate the proliferation of HPB spores, which are better able to adapt to harsh environmental conditions (high temperature and acidic or basic pH) by removing zero spore forming hydrogen consuming bacteria. However, other novel pre-treatment approaches, such as sonication, BESA, or LA addition, have been found to be more successful in other studies [42]. In addition, new technologies including infrared and ionizing irradiation are proving to be more useful for inoculum pre-treatment [43].
While there is no consensus on the best inoculum pre-treatment method, this study and other previous research confirms that inoculum pre-treatment enhances hydrogen production.
Statistical and Kinetic Analysis
ANOVA analysis (Table 4) showed significant differences in the mean hydrogen yield between different pre-treatment groups (p <.001). The differences were also correlated with variances in metabolic products (VFAs) observed, which could be explained by the diversity of microbial populations.
Table 4
ANOVA results comparing mean hydrogen yields between pre-treatment groups
ANOVA |
| Sum of Squares | df | Mean Square | F | Sig. |
Between Groups | 45180.633 | 4 | 11295.158 | 85.750 | <.001 |
Within Groups | 1317.217 | 10 | 131.722 | | |
Total | 46497.849 | 14 | | | |