Hydrogen (H₂) is an ideal fuel that can be used to limit non-renewable energy dependence for a more sustainable society. Biological hydrogen production from industrial waste substrates can be used as a pathway to make hydrogen in a cost effective and eco-friendly nature [1]. The New Zealand dairy and brewery industries generate a considerable amount of proteinaceous waste by-products annually, consisting of dissolved air filtration (DAF) sludge, and spent yeast, hops, and grain respectively. Thermococcus waiotapuensis WT1ᵀ is a thermophilic sulphur-dependent archaeon that grows heterotrophically on complex proteinaceous substrates; and with a genomic potential suggesting near-maximal hydrogen yields, it may provide a sustainable utilisation strategy for these proteinaceous dairy and brewery by-products [2, 3, 4]. Thermophilic digestion of dairy DAF sludge reduces both the pathogen and organic load whilst producing hydrogen as a by-product in a single step of treatment. Similarly, fermentation of brewery by-products also results in biogas production. This goal of this Masters project was to determine whether waiotapuensis WT1ᵀ can be utilised to produce H₂ on substrates emulating dairy and brewery waste streams.

To achieve this goal, the ability for T. waiotapuensis WT1ᵀ to utilise a range of growth substrates was determined before testing substrates simulating common waste streams of the brewery and dairy industries. Furthermore, the range of carbon, nitrogen, and sulphur compounds that support T. waiotapuensis WT1ᵀ growth were expanded on and compared to prior findings in the literature. Confirmed T. waiotapuensis WT1ᵀ end-products included acetate, succinate, CO₂ and H₂, with H₂S production detected but not confirmed via the strain’s growth on S0. No ethanol, formate, and lactate were produced on simulated substrates of both DAF sludge and spent yeast. These results suggest T. waiotapuensis WT1ᵀ is capable of near-maximal H₂ yields on common waste substrates of brewery and dairy industries; however, further studies are required to determine the stoichiometric production of H₂. Additionally, bioreactor growth of T. waiotapuensis WT1ᵀ was successfully achieved, scaling the T. waiotapuensis WT1ᵀ cultivation from 50 mL to 2.2 L, proving that T. waiotapuensis WT1ᵀ could be employed for industrial scaled-up growth. T. waiotapuensis WT1ᵀ growth was not impacted by sulphur or H₂ concentrations. DSMZ 934 medium sulphur supplementation could be reduced by a factor of 10 without any impact on T. waiotapuensis WT1ᵀ growth or headspace moles production. This would allow for lower sulphur supplementation, resulting in reduced input costs associated with industrial fermentation, and further supporting the increased industrial potential for T. waiotapuensis WT1ᵀ to utilise these waste substrates. The media dilution series found that by increasing media dilution, gaseous by-product production was limited, thus indicating that nutrients are the limiting factor for T. waiotapuensis WT1ᵀ growth. Carbon was indicated to be the limiting nutrient as the onset of the stationary phase coincided with the exhaustion of pyruvate.

T. waiotapuensis WT1ᵀ was found to produce H₂ on the sulphur substrates of cystine, cysteine, thioglycolate, S⁰ and thiosulfate. S⁰ was determined to produce less H₂ compared to the other sulphur sources, indicating that S⁰ is not the optimal sulphur supplementation for H₂ production. The decreased H₂ production is likely due to the increased H₂S production as only media supplemented with S⁰ displayed an unconfirmed H₂S peak during GC analysis. T. waiotapuensis WT1ᵀ is hypothesised to have greater H₂S production than H₂ in the presence of S⁰, similar to P. furiosus Vc1 which produces H₂ and H₂S in a 40: 60 ratio in media with S⁰ [5]. The optimal sulphur source from those tested in this paper was found to be cysteine, however, further work should be completed to confirm this finding.