Cell wash-out enrichment increases the stability and performance of biohydrogen producing packed-bed reactors and the community transition along the operation time


Heat shock and cell wash-out as strategies to enrich hydrogen producing bacteria.

Enriched communities developed were inoculated in packed bed reactors.

Cell wash-out prompted stability attributed to facultative bacteria.

A transition from hydrogen producing community to a methanogenic one was observed.

Strategies to get a stable hydrogen production in packed bed reactors are proposed.


Most of the fermentative hydrogen production studies based on mixed cultures have shown enrichment of the microbial community by means of a heat treatment. This heat treatment enrichment strategy selects for Clostridum spp., an efficient hydrogen producer; however, other bacteria that may contribute to the systems performance could be excluded. Another enrichment strategy based on high dilution rates selects different taxonomic groups, which may affect hydrogen production and the system stability. In this work, two enrichment strategies were evaluated, heat shock and cell wash-out, for hydrogen production and the system stability in continuous stirred reactors. The enriched communities were then inoculated in packed bed reactors and operated up to 70 days. Both strategies selected hydrogen producing bacteria, mainly Clostridium spp. The highest hydrogen production rate (6.01 L H2/L-d), molar yield (1.29 mol H2/mol glucoseconsumed), and stability were achieved by the wash-out procedure; this high performance was attributed to facultative bacteria like Lactobacillus and Lactococcus. Furthermore, there was a transition within the community (along the operation time in the reactor with cell wash-out inoculum) and a selection for methanogenic activity (due to the long solids retention time).

Graphical abstract


  • Dark fermentation;
  • Facultative bacteria;
  • Heat treatment;
  • Pretreatment

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