Pyrolysis is a widely used method to produce biochar and/or bio-oil. During this process,synthesis gas (syngas) is also formed with a main composition of a mixture of carbon monoxide(CO), hydrogen (H2), and carbon dioxide (CO2). Syngas can be converted by a biologicalwater-gas shift reaction, where CO is converted to H2 without the necessity of hightemperatures and expensive catalysts, as opposed to the traditional water-gas shift reaction.This process not only meets the high demand for biohydrogen in various industrial applications(e.g., chemical, food, and pharmaceutical processes) but also utilizes off-gases from industrialand thermochemical processes as energy and carbon sources for microorganisms. Whilebatch processes have been successfully demonstrated, scaling up to continuous operationsremains challenging. This study aims to identify the key challenges in scaling up from batch tocontinuous processes by applying parameters that were efficient in batch experiments to atrickle bed reactor (TBR). In batch experiments, suitable environmental factors were proposedusing pretreated wastewater treatment granules after evaluating pH values, temperatures, andinoculum concentrations. Using a mixed culture of microorganisms from a wastewatertreatment plant as inoculum and a chemical-free methanogenesis inhibition approach, abiohydrogen yield of up to 0.07 g H2/g CO was achieved. The same parameters, inoculumsource, and growth medium were then applied to a TBR to enhance gas-liquid mass transferand process control. The biohydrogen yield achieved was 0.05 g H2/g CO/h during seven daysin continuous mode. However, process stability and product selectivity were key challenges.Despite using pretreated inoculum and in-situ heat treatments, methane production eventuallyoccurred, causing fluctuations in biohydrogen production. This shift in metabolic activitysuggests that microbial community dynamics in continuous operation differ significantly frombatch conditions, despite using identical environmental parameters. Methanogenic activity inbatch mode may not have been detected due to the shorter duration of the experiments. Thesefindings highlight critical considerations for scaling up biohydrogen production with mixedculture, contributing to our understanding of microbial metabolic activities and the processchallenges in syngas fermentation.