Hydrogen gas (H₂) is an energy carrier that does not generate carbon dioxide emissions during combustion, but several processes in use for its production demand high energy inputs associated with fossil fuels and greenhouse emissions. Biological processes, such as dark fermentation (DF), have the potential to remove the dependency on fossil fuels in H₂ production. DF is a process that encourages fermentative bacteria to ferment organic substrates to produce H₂ as a truly clean energy carrier, but its success depends on removing the presence of competing H₂₋consuming microorganisms in the inoculum consortia. This paper addresses a strategy to enhance H₂ production from different types of substrates by testing inoculum pre-treatment processes to inactivate H₂₋consuming bacteria, including acid-shock (pH 3), basic-shock (pH 10) and heat-shock (115 °C) methods. Digestate from anaerobic digesters processing sewage sludge was used to produce pre-treated inocula, which were subsequently tested in a batch bio-H₂ potential (BHP) test using glucose as a substrate. The results show that heat-shock pre-treatment was the best method, reporting a H₂ yield of 191.8 mL-H₂/gVS added (the untreated inoculum reported 170.91 mL-H₂/gVS added). Glucose conversion data show a high concentration of butyric acid in both treated and untreated inocula during BHP tests, which indicate that the butyrate pathway for H₂ production was dominant; shifting this to the formate route could further enhance net H₂ production. A standardised inoculum-conditioning method can help to consistently assess the biohydrogen potential of suitable feedstock for DF and maximise H₂ yields.

The increasing rate of food waste (FW) generation around the world is a growing environmental concern, notwithstanding, its valorisation through anaerobic digestion (AD) makes it a potential resource. Moreover, there is a growing demand to optimise the biomethane from AD for gas‐to‐grid (GtG) and vehicular applications. This has spurred researches on hydrogen gas (H₂) injection into AD systems to enhance the biological conversion of H₂ and carbon dioxide (CO₂) to methane (CH₄), a process known as biomethanation. A simplistic approach for biomethanation is to add H₂ directly into working AD reactors (in situ biomethanation). However, a competition for the injected H₂ towards other biological reactions besides H₂/CO₂ conversion to CH₄ could follow, thus, reducing the efficiency of the system. Hence, this study was conducted to understand how different H₂ injection points would affect H₂/CO₂ conversion to CH₄ during FW in situ biomethanation, to identify an optimal injection point. Experiments were designed using H₂ equivalent to 5% of the head‐space of the AD reactor at three injection points representing different stages of AD: before volatile fatty acids (VFA) accumulation, during VFA accumulation and at depleted VFA intermediates. Lower potential for competitive H₂ consumption before the accumulation of VFA enabled a high H₂/CO₂ conversion to CH₄. However, enhanced competition for soluble substrates during VFA accumulation reduced the efficiency of H₂/CO₂ conversion to CH₄ when H₂ was added at this stage. In general, 12%, 4% and 10% CH₄ increases as well as 39%, 25% and 34% CO₂ removal were obtained for H₂ added before VFA accumulation, during VFA accumulation and at depleted VFA intermediates, respectively. For immediate integration of biomethanation with existing AD facilities, it is suggested that the required H₂ be obtained biologically by dark fermentation.

Dark fermentation of food waste for biohydrogen production is a progressive technology that can contribute to low-cost solutions to the global clean energy need. As dark fermentation research progresses, it is important to evaluate opportunities for real-life application such as integration into existing anaerobic digestion systems. The present study proposes a novel approach of combined inoculum and food waste heat shock pretreatment for biohydrogen production through dark fermentation. It evaluates the effect of the heating duration (at 115°C) and starting pH on the biohydrogen yield and system profiles in two stages, namely, Exp1 and Exp2. Exp1 investigated the optimal heating duration and starting pH for a combined inoculum/food waste heat shock pretreatment. A 24-h biohydrogen production test at four heating durations (15, 30, 45 and 60 min) was assayed at acidic and alkaline pH ranges (4, 5, 6 and 8, 9, 10). The optimal starting pH from these experiments across all four heating durations was pH 5. Biohydrogen yield increased linearly from 37.4 to 63.8 mL/gVS, with an increase in heating duration from 15 to 60 min at pH 5. However, an optimal heating duration was not reached, therefore, additional tests were conducted at pH 5 for extended heating durations of 75–120 min. Biohydrogen yield was similar at 75, 90 and 105-min heating durations in a range of 69.7–73.5 mL/gVS. Above 105 min of heating duration, the overall gas production starts to decline, making it the maximum allowable heating duration. In Exp2, a comparative analysis of the system profiles between the combined inoculum/food waste pretreatment (Test) and inoculum-only pretreatment (Control) was investigated using the optimal heating duration range (75 and 105 min) and starting pH of 5. The peak biohydrogen yield from the Control was achieved following a 75-min heating duration (84.5 mL/gVS, 58.6%), while this was achieved following a 90-min heating duration for the Test (81.3 mL/gVS, 53.3%). Higher volatile fatty acids fermentation and pH recovery were achieved in the Test in addition to potential economic savings compared to the Control. Therefore, the innovative approach of combined food waste/inoculum heat shock pretreatment (Test) presents opportunities to integrate dark fermentation into existing anaerobic digestion systems as a step to scale up the dark fermentation technology from lab to real-life application.