Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp.

Background: Pure bacterial strains give better yields when producing H-2 than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H-2 production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research. Results: Four pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20 L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H-2 production (sequence 1); and the H-2-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H-2 yields were obtained for starch fermentations, and the highest yield of 2.91 mol H-2/mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5 L biogas/h was observed for the co-culture (1). In general co-cultures produced H-2 at higher rates than the pure Clostridium cultures, without negatively affecting the H-2 yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H-2. In the second part of the study the co-culture (3) was used to produce H-2 during 13 days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this fermentation. Conclusions: The four pure Clostridium strains and the artificial co-cultures tested in this study were shown to efficiently produce H-2 using glucose and starch as carbon sources. The artificial co-cultures produced H-2 at higher rates than the pure strains, while the H-2 yields were only slightly affected.