Optimization of fermentative biohydrogen production by Thermotoga neapolitana

Hydrogen has revealed a great potential as a versatile and non-polluting energy carrier of the future providing a high energy density and an efficiently conversion to usable power. Dark fermentation is one of the most promising biological production processes, but still has to overcome major challenges, most importantly low hydrogen production rates (HPRs) and hydrogen yields (HYs), before its industrial application becomes cost- and energy-efficient.

In this work, we aimed to optimize the hydrogen production via dark fermentation by Thermotoga neapolitana. The main objectives were to enhance the HPR and maintain a high HY using different approaches to counteract process limitations and prevent the most relevant inhibitions. Furthermore, a development of the industrially preferred continuous-flow process was projected.

An increase of the initial biomass concentration from 0.46 to 1.74 g cell dry weight (CDW)/L in batch bioassays resulted in a more than 2-fold enhancement of the HPR up to 654 (±30) mL/L/h (mL of hydrogen produced per L of volume of reactor per hour of reaction or per hour of liquid retention) without negatively affecting the HY. However, while the volumetric productivity increased the specific HPR (per unit of biomass) was negatively correlated with the HPR and the biomass concentration.

Subsequently, we investigated the supersaturation of hydrogen in the liquid phase (H_2aq) in batch bioassays. At 100 rpm agitation H_2aq supersaturated up to 3 times the equilibrium concentration. Increasing the agitation speed diminished the accumulation of H_2aq until an equilibrium between the gas and liquid phase hydrogen was reached with 500 rpm agitation at low cell concentrations. A raise from 200 to 600 rpm gradually reduced H_2aq from 21.9 (± 2.2) to 8.5 (± 0.1) mL/L and approximately doubled the HPR, revealing a direct correlation between the two parameters. Similarly, the addition of K1 carrier and H₂-rich biogas recirculation (GaR) successfully counteracted the accumulation of H_2aq. Accelerating the process by increasing the reactors biomass concentration up to 0.79 g CDW/L, GaR revealed to be more efficient in removing H_2aq than 500 rpm agitation. The application of GaR at 300 and 500 rpm enhanced the HPR by approximately 260% up to 850 (± 71) mL/L/h, compared to a sole 300 rpm agitation, reaching a HY of 3.5 mol H₂/mol glucose. We demonstrated that an insufficient gas-liquid mass transfer leads to the accumulation of H_2aq which inhibits the yield but even more so the rate of dark fermentation.

In the final phase of this project we successfully maintained continuous-flow hydrogen production. Increasing the feed glucose concentration from 11.1 to 41.6 mM diminished the HY from 3.6 (± 0.1) to 1.4 (± 0.1) mol H₂/mol glucose. The HPR increased concomitantly up to approximately 55 mL/L/h at 27.8 mM of glucose, whereas a further increase of feed glucose to 41.6 mM did not enhance the HPR and the acetic acid (AA) concentration. To investigate whether high levels of AA limited the process, the feed AA concentration was gradually increased. However, this revealed no negative effect on continuous dark fermentation up to 240 mM of feed AA and, throughout the 110 days of continuous fermentation, the HY increased by 47%. Decreasing the hydraulic retention time (HRT) from 24 to 7 h also led to a HPR enhancement from 82 (± 1) to 192 (± 4) mL/L/h, while decreasing the HY. Concomitantly, the H_2aq accumulated, directly correlated to the HPR reaching 15.6 mL/L at an HRT of 7 h and 500 rpm agitation. The application of GaR efficiently counteracted the supersaturation of H_2aq and allowed the highest HPR of 277 mL/L/h at a HRT of 5 h.