Improving sustainable hydrogen production from green waste: [FeFe]-hydrogenases quantitative gene expression RT-qPCR analysis in presence of autochthonous consortia

Background: Bio-hydrogen production via dark fermentation of low-value waste is a potent and simple mean of recovering energy, maximising the harvesting of reducing equivalents to produce the cleanest fuel amongst renewables. Following several position papers from companies and public bodies, the hydrogen economy is regaining interest, especially in combination with circular economy and the environmental benefits of short local supply chains, aiming at zero net emission of greenhouse gases (GHG). The biomasses attracting the largest interest are agricultural and urban green wastes (pruning of trees, collected leaves, grass clippings from public parks and boulevards), which are usually employed in compost production, with some concerns over the GHG emission during the process. Here, an alternative application of green wastes, low-value compost and intermediate products (partially composted but unsuitable for completing the process) is studied, pointing at the autochthonous microbial consortium as an already selected source of implementation for biomass degradation and hydrogen production. The biocatalysts investigated as mainly relevant for hydrogen production were the [FeFe]-hydrogenases expressed in Clostridia, given their very high turnover rates. Results: Bio-hydrogen accumulation was related to the modulation of gene expression of multiple [FeFe]-hydrogenases from two strains (Clostridium beijerinckii AM2 and Clostridium tyrobutyricum AM6) isolated from the same waste. Reverse Transcriptase quantitative PCR (RT-qPCR) was applied over a period of 288 h and the RT-qPCR results showed that C. beijerinckii AM2 prevailed over C. tyrobutyricum AM6 and a high expression modulation of the 6 different [FeFe]-hydrogenase genes of C. beijerinckii in the first 23 h was observed, sustaining cumulative hydrogen production of 0.6 to 1.2 ml H2/g VS (volatile solids). These results are promising in terms of hydrogen yields, given that no pre-treatment was applied, and suggested a complex cellular regulation, linking the performance of dark fermentation with key functional genes involved in bio-H2 production in presence of the autochthonous consortium, with different roles, time, and mode of expression of the involved hydrogenases. Conclusions: An applicative outcome of the hydrogenases genes quantitative expression analysis can be foreseen in optimising (on the basis of the acquired functional data) hydrogen production from a nutrient-poor green waste and/or low added value compost, in a perspective of circular bioeconomy.