The aim of the deliverable 2.1 is to further develop a bioprocess for hydrogen (H2) and ethanol (EtOH) production from crude glycerol increasing the ethanol production up to 40 gl-1. This amount was estimated according to a statistical model based on the production of more than 26 gL-1 of ethanol by preliminary fed-batch fermentation. The bioprocess is based on a selected microbial mixed cultures (called GCL inoculum) which is a functional consortium characterized by Klebsiella spp. (~60%) and Escherichia spp. (~32%)genera, both playing the role of hydrogen and ethanol producers. Working under non sterile conditions using mixed cultures, which are more robust of a pure culture, may lower the operational costs of ethanol production.
Preliminary activities were performed for identifying the substrate suitable for the process. ‘Suitable’ means that the GCL inoculum was able to preserve the target functions (i.e. H2 and EtOH production) when used with the new substrate. The crude glycerol was collected from ItalBiOil srl., a biodiesel manufacturing unit located in Italy, producing biodiesel from vegetable oils.
Two experimental periods characterized the development of D2.1. Firstly, increasing amounts of crude glycerol (from 20 to 80gl-1) were assayed through the fed-batch fermentation using stringent experimental conditions: supplementary substrate was added as the only carbon source once the fermentation reached the stationary phase of production, without removing the microbial culture and/or adding salts into the medium. Afterwards, optimization of the process was carried out by controlling the pH and the hydrogen partial pressure (by nitrogen sparging), as well as by removing the medium and supplementing the salts.
The purpose of increase up to 40 gL-1 the EtOH production was the main effort encountered during the first experimental phase. The average amount of EtOH production was 20.7±0.6 gL-1 (n=6, including the optimized experiments), suggesting that it was the highest amount of EtOH supported by our fermentation processes.
The increase of substrate concentration from 20 to 80 gL-1 did not improve significantly the performance of the fermentations process. The activation of metabolic pathways involved in acetate reduction (as an impurities of the substrate) which consumed H2 and energy (ATP), the metabolic synthesis of toxic bio products (beyond the EtOH) ) were the main factors affecting the EtOH productivity of the GCL inoculum. All these factors explain the lack of reproducibility and replicability with the reference data.
The improvement up to 33.0±3.3 gL-1 (n=3) of EtOH production was later obtained working in optimized experimental conditions. At the same time, the GCL community used more substrate and the glycerol consumption increased from 46.4±3.2 to 78.4±1.0 gL-1. The same metabolic pathways were activated by microorganisms in both cases: the ethanol/oxidative pathway in addition to that involved in the acetic acid metabolism.
These results suggest that the GCL microbial community played a key role in the efficiency of fermentation process since its performance tended to increase with the proceeding of the experimental activities. It is likely that the repeated transfers in the crude glycerol have induced the adaptation (as beneficial mutations) of the microbial community, modifying the diversity at intra- and inter-species level and producing a more ‘specialized’ and ‘resistant’ community.
The ‘new’ behaviour of the mixed microbial culture of GCL is under investigation, but it seems to indicate that the Klebsiella has prevailed over Escherichia community. The experimental activities are still proceeding to the aim of evaluating the stability of the ‘new’ GCL and therefore the replicability of the process.