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Enhancement of thermophilic dark fermentative hydrogen production and the use of molecular biology methods for bioprocess monitoring

Research output: Book/ReportDoctoral thesisCollection of Articles


Original languageEnglish
PublisherTampere University
Number of pages172
Publication statusPublished - 12 Dec 2019
Publication typeG5 Doctoral dissertation (article)


Dark fermentation of organic waste materials such as organic fraction of municipal waste and agricultural wastes is a promising technology to produce renewable hydrogen. However, further research and development is required to improve the efficiency and stability of the process. The aim of this thesis was to enhance thermophilic dark fermentative hydrogen production by using
microbial strategies (bioaugmentation and synthetic co-cultures) and by increasing the understanding on the microbial community dynamics especially during stress conditions such as fluctuating temperatures and elevated substrate concentrations.

To study the effects of sudden short-term temperature fluctuations, batch cultures initially incubated at 55°C (control) were subjected to downward (from 55°C to 35°C or 45°C) or upward (from 55°C to 65°C or 75°C) temperature shifts for 48 h after which they were incubated again at 55°C for two consecutive batch cycles. The results showed that sudden, temporal upward and downward temperature fluctuations had a direct impact on the hydrogen yield as well as the microbial community structure. Cultures exposed to downward temperature fluctuation recovered more rapidly enabling almost similar hydrogen yield (92-96%) as the control culture kept at 55 °C. On the contrary, upward temperature shifts from 55 to 65 or 75 °C had more significant negative
effect on dark fermentative hydrogen production as the yield remained significantly lower (54-79%) for the exposed cultures compared to the control culture.

To improve the stability of hydrogen production during temperature fluctuations and to speed up the recovery, mixed microbial consortium undergoing a period of either downward or upward temperature fluctuation was augmented with a synthetic mix culture containing well-known hydrogen producers (Thermotoga, Thermoanaerobacter, Thermoanaerobacterium, Caldicellulosiruptor and Thermocellum spp.) The addition of new species into the native
consortium significantly improved hydrogen production both during and after the fluctuations. However, when the bioaugmentation was applied during the temperature fluctuation, hydrogen production was enhanced.

This study also investigated the dynamics between pure cultures and co-cultures of highly specialized hydrogen producers, Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana. The highest hydrogen yield (2.8 ± 0.1 mol H2 mol-1 glucose) was obtained with a synthetic co-culture which resulted in a 3.3 or 12% increase in hydrogen yield when compared to pure cultures of C. saccharolyticus or T. neapolitana, respectively. Furthermore, quantitative polymerase chain reaction (qPCR) based method for monitoring the growth and contribution of T. neapolitana in synthetic co-cultures was developed. With this method, it was verified that T. neapolitana was an active member of the synthetic co-culture.

The effect of different feed glucose concentrations (from 5.6 to 111.0 mmol L-1) on hydrogen production was investigated with and without augmenting the culture with T. neapolitana. Compared to the control (without T. neapolitana), bioaugmentated culture resulted in higher hydrogen yields in almost all the concentrations studied even though hydrogen yield decreased the feed glucose concentration was increased. The presence of T. neapolitana also had a
significant impact on the metabolite distribution when compared to the control. The number gene copies of T. neapolitana measured with qPCR was higher at the highest initial glucose concentrations. Thus, the results demonstrated that the use of a single strain with the required properties needed in a biological system can be sufficient for improving dark fermentative hydrogen production.

In summary, this study showed that thermophilic dark fermentative hydrogen production can be enhanced by using synthetic co-cultures or bioaugmentation. The highest hydrogen yield in this study was obtained with the synthetic co-culture, although it should be considered that the incubation conditions differed from those used for the mixed cultures in this study. The use of molecular methods such as qPCR and high-throughput sequencing also helped to understand the role of certain species in the microbial consortia and improved the understanding of the microbial community dynamics during stress conditions. The use of molecular methods is thus important as it helps to create a link between the microbial community structure and observed hydrogen production.

Field of science, Statistics Finland