Enhanced photo-fermentative H₂ production using Rhodobacter sphaeroides by ethanol addition and analysis of soluble microbial products

Background: Biological fermentation routes can provide an environmentally friendly way of producing H₂ since they use renewable biomass as feedstock and proceed under ambient temperature and pressure. In particular, photo-fermentation has superior properties in terms of achieving high H ₂ yield through complete degradation of substrates. However, long-term H₂ production data with stable performance is limited, and this data is essential for practical applications. In the present work, continuous photo-fermentative H₂ production from lactate was attempted using the purple non-sulfur bacterium, Rhodobacter sphaeroides KD131. As a gradual drop in H₂ production was observed, we attempted to add ethanol (0.2% v/v) to the medium. Results: As continuous operation went on, H₂ production was not sustained and showed a negligible H₂ yield (< 0.5 mol H₂/mol lactate_added) within two weeks. Electron balance analysis showed that the reason for the gradual drop in H₂ production was ascribed to the increase in production of soluble microbial products (SMP_s). To see the possible effect of ethanol addition, a batch test was first conducted. The presence of ethanol significantly increased the H₂ yield from 1.15 to 2.20 mol H ₂/mol lactate_added, by suppressing the production of SMPs. The analysis of SMPs by size exclusion chromatography showed that, in the later period of fermentation, more than half of the low molecular weight SMPs (< 1 kDa) were consumed and used for H₂ production when ethanol had been added, while the concentration of SMPs continuously increased in the absence of ethanol. It was found that the addition of ethanol facilitated the utilization of reducing power, resulting in an increase in the cellular levels of NAD ⁺ and NADP⁺. In continuous operation, ethanol addition was effective, such that stable H₂ production was attained with an H₂ yield of 2.5 mol H₂/mol lactate_added. Less than 15% of substrate electrons were used for SMP production, whereas 35% were used in the control. Conclusions: We have found that SMPs are the key factor in photo-fermentative H₂ production, and their production can be suppressed by ethanol addition. However, since external addition of ethanol to the medium represents an extra economic burden, ethanol should be prepared in a cost-effective way.