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This study describes the dynamics and complexity of microbial communities producing hydrogen-rich fermentation gas from sugar-beet molasses in five packed-bed reactors (PBRs). The bioreactors constitute a part of a system producing hydrogen from the by-products of the sugar-beet industry that has been operating continuously in one of the Polish sugar factories. PBRs with different working volumes, packing materials, construction and inocula were tested. This study focused on analysis (based on 16S rRNA profiling and shotgun metagenomics sequencing) of the microbial communities selected in the PBRs under the conditions of high (>100 cm 3 /g COD of molasses) and low (<50 cm 3 /g COD of molasses) efficiencies of hydrogen production. The stability and efficiency of the hydrogen production are determined by the composition of dark fermentation microbial communities. The most striking difference between the tested samples is the ratio of hydrogen producers to lactic acid bacteria. The highest efficiency of hydrogen production (130–160 cm 3 /g COD of molasses) was achieved at the ratios of HPB to LAB ≈ 4:2.5 or 2.5:1 as determined by 16S rRNA sequencing or shotgun metagenomics sequencing, respectively. The most abundant  Clostridium  species were  C. pasteurianum  and  C. tyrobutyricum.  A multiple predominance of LAB over HPB (3:1–4:1) or clostridia over LAB (5:1–60:1) results in decreased hydrogen production. Inhibition of hydrogen production was illustrated by overproduction of short chain fatty acids and ethanol. Furthermore, concentration of ethanol might be a relevant marker or factor promoting a metabolic shift in the DF bioreactors processing carbohydrates from hydrogen-yielding toward lactic acid fermentation or solventogenic pathways. The novelty of this study is identifying a community balance between hydrogen producers and lactic acid bacteria for stable hydrogen producing systems. The balance stems from long-term selection of hydrogen-producing microbial community, operating conditions such as bioreactor construction, packing material, hydraulic retention time and substrate concentration. This finding is confirmed by additional analysis of the proportions between HPB and LAB in dark fermentation bioreactors from other studies. The results contribute to the advance of knowledge in the area of relationships and nutritional interactions especially the cross-feeding of lactate between bacteria in dark fermentation microbial communities.

frontiers in microbiology

Dynamics and Complexity of Dark Fermentation Microbial Communities Producing Hydrogen From Sugar Beet Molasses in Continuously Operating Packed Bed Reactors