Thermophilic biohydrogen production from reservoir residual hydrocarbons using palm oil mill effluent-derived microbial consortia.

Residual crude oil remaining in depleted reservoirs represents a largely untapped carbon source for biological hydrogen generation. Previous studies have relied on indigenous bacteria present in oil reservoirs but reported low hydrogen yields, as not all reservoir microorganisms are hydrogen-producing. Therefore, in this study, external mixed culture bacterial consortia obtained from palm oil mill effluent (POME) were used to degrade crude oil for hydrogen production. Morphological changes in microbial communities were assessed using field emission scanning electron microscopy. Metagenomic profiling was conducted to identify the dominant microbial taxa capable of producing biohydrogen. Thereafter, a high-temperature and high-pressure (800 °C/30 MPa) stainless-steel bioreactor containing crude oil was inoculated with mixed culture consortia to simulate an oilfield reservoir for hydrogen production. Box-Behnken design was applied to systematically examine the effects of exposure time (6-90 h), crude oil volume (10-40 mL), and temperature (35-70 °C) on continuous hydrogen production. Statistical analysis of variance was used to evaluate model parameters. Heat pretreatment selectively enriched hydrogenogenic spore-formers (Clostridium and Bacillus), resulting in a ~ 4-fold increase (97.40 ± 0.02 mL/L) in hydrogen yield compared to 25.68 ± 0.04 mL/L POME for untreated sludge. In the presence of crude oil, the optimum hydrogen production was 152.50 ± 0.01 mL/L at 50 °C, compared to 125.45 ± 0.03 mL/L and 29.95 ± 0.01 mL/L crude oil at 35 °C and 70 °C, respectively. Predicted hydrogen production, with R2 value of 97.4% close to unity, indicates that the model was highly consistent with the experimental results, with high precision and reliability. Thermodynamic analysis shows negative Gibbs free energy changes of -122 to -236 kJ/mol, demonstrating that hydrocarbon-to‑hydrogen conversion was energetically favorable and feasible across all tested temperatures. Overall, the experimental, statistical, and thermodynamic analyses establish the technical and energetic feasibility of microbial enhanced hydrogen recovery in depleted oil reservoirs.