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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.

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