(536h) Towards Circular Economy: Optimisation of Co-Culture Fermentation for Biohydrogen Production from Flour Mill Waste.

Flour mill waste (FMW), a globally abundant and underutilized by-product of grain processing, presents an attractive low-cost and non-edible feedstock for biohydrogen production. On a global scale, a significant proportion of the human diet is filled with cereal grains such as wheat, rice, barley, maize, sorghum, millet, oat, and rye. According to the FAO (2016), global production of these crops is nearly 2577.85 million tons. For the production of refined flour, bran, and germ, a proportion of grain is removed and thus, nearly 8% of the production is wasted in the milling process. Its substantial carbohydrate content, primarily in the form of starch, provides a rich energy source for microbial conversion. However, the recalcitrant nature of starch poses challenges for direct fermentation by single microbial strains. This often results in inefficient substrate utilization, limited hydrogen yields, and the need for complex and costly pre-treatment processes, which not only take away from its sustainability but also pile on costs, making the biohydrogen less appealing and feasible. FMW is a waste that is generally an environmental nuisance mostly burnt leading to pollution. Thus its valorization for energy production also enables a safe and proper waste disposal methodology.

This study addresses the limitations of FMW through an innovative co-culture fermentation strategy designed to optimize both FMW hydrolysis and subsequent biohydrogen production. Initial characterization of FMW revealed a diverse sugar profile, including starch (50-70%), glucose (15-20%), maltose (15-20%), xylose (1-5%), and arabinose (1-5%). A comprehensive screening of saccharolytic Clostridia strains, including Clostridium beijerinckii G117, Clostridium butyricum NCIM 2825, Clostridium acetobutylicum NCIM 2337, and Clostridium sp. BOH3, identified Clostridium beijerinckii G117 as the most promising candidate. This strain demonstrated the ability to utilize a diverse range of sugars (including starch, glucose, maltose, xylose, and arabinose), achieving high cellular growth (1.07-1.78 OD600)and hydrogen yield (0.81-1.92 l/l) , making it well-suited for FMW-based fermentation.

Direct fermentation of FMW as a feedstock (10-100 g/l) in minimal media was carried out using Clostridium beijerinkii G117. A maximum hydrogen yield of 2.7 l/l was obtained from 50g/l FMW. It was found that increasing FMW beyond 50 g/l, hydrogen production and cellular growth declined. Also, 50-70% of the FMW was unutilized in all the cases, leading to feedstock wastage. Despite the strain showing the capacity of amylase production (1.15U/ml), the hydrogen yield remained significantly low despite the available sugar in the fermentation broth. It was suspected that the strain is under severe metabolic burden due to performing both saccharification and hydrogen production, therefore hindering its performance. Therefore, pre-treatment for saccharification of starch residues to reducing sugar, for easy assimilation by C.G117 was considered.

A systematic evaluation of various pre-treatment strategies was conducted to improve the accessibility of complex carbohydrates in FMW. These included physical methods (autoclaving, milling), chemical methods (acid and water hydrolysis), and enzymatic hydrolysis using both commercial α-amylase and a Bacillus sp. strain with high amylase production potential. A particular focus was placed on identifying both effective and cost-optimized hydrolysis techniques. The introduction of the Bacillus sp. strain significantly improved the saccharification efficiency of FMW, offering a more economical and sustainable solution compared to solely relying on commercial enzymes. Detailed analysis of the pre-treatment methods revealed that a combination of moist heat treatment followed by enzymatic hydrolysis provided the optimal balance of sugar release, cost-effectiveness, and environmental sustainability. We found that Bacillus sp. could produce amylase units up to 7U/ml and effectively hydrolyze starch in the supernatant by nearly 97%. Saccharification of high FMW concentration (60-150g/l) by Bacillus sp. followed by fermentation by C.G117 yielded high hydrogen (3-3.6 l/l) with a shorter lag phase in comparison to direct fermentation improving the hydrogen production rate as well.

Following pre-treatment optimization, a consolidated bioprocess (CBP) approach was implemented to streamline the process. The co-culture system, comprised of the selected C.G117 and Bacillus sp., was further refined using Response Surface Methodology (RSM) with Central Composite Design (CCD). Key parameters such as inoculum ratio, FMW concentration, and pH were systematically adjusted to determine the conditions that maximize biohydrogen yield and substrate conversion efficiency. The optimized CBP achieved remarkable success, yielding over 4 L/L of biohydrogen with high FMW conversion rates (70-90%). This outcome represents a substantial improvement over direct fermentation methods, clearly demonstrating the effectiveness of the co-culture strategy in overcoming the challenges associated with utilizing complex biomass. Detailed analysis revealed that the co-culture system facilitates a synergistic relationship. The Bacillus strain effectively hydrolyzes FMW starch, providing readily accessible sugars for enhanced hydrogen production by the Clostridium strain.

This research firmly establishes FMW as a valuable feedstock for biohydrogen production. The developed co-culture system offers a robust and efficient platform for waste valorization. It generates clean energy while simultaneously addressing sustainable waste management practices. Importantly, this work aligns with the principles of a circular economy, where waste streams are transformed into valuable resources, fostering environmental sustainability and resource efficiency.