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.
D2-2 – Operating parameters for batch experiments
To provide information about operational parameters at batch configurations such as, the effect of inoculum, addition of nutrients, pH control and substrate concentration to improve hydrogen and ethanol production
According to the results, the selected inoculum was sludge from an aerobic wastewater treatment plant and an aerobic pretreatment. Also, it was necessary to provide nutrients addition and pH control. The substrate concentration that improves hydrogen and ethanol production is in a range of 40 to 70 g glycerol/L. Finally, the results coming from the analysis of the type of inoculum and pretreatment were also used for a continuous system.
D2.10 Gas stripping removal of ethanol and Cell recycling of cell mass
The main objective is optimization of a process developed by ENEA for production of hydrogen and ethanol from crude glycerol streams based on Mixed Microbial Culture (MMC) biotechnology. The current delivery investigates the possibility of increasing reaction rate by reducing ethanol level in the reactor by gas stripping and increasing cell concentration using recirculation of cell mass.
The delivery has been postponed until end of February 2017.
D 3.3. Screening solvents
The extraction of 1,3-propanediol from its aqueous solutions mimicking fermentation broths, and then from the actual fermentation broths produced by biocatalytic treatment of glycerol, has been targeted.
The capability of different classes of media of extracting 1,3-propanediol out of its water solutions, mimicking the fermentation broths, was tested. Among them, ionic liquids were proven to be the best media for the purpose. A thorough investigation of the capability of a wide range of ionic liquids to extract 1,3-propanediol from its water solution, mimicking the glycerol fermentation broths, and then out of the actual fermentation broths produced and delivered to QUB by STUBA, has been carried out. A range of hydrophobic ionic liquids were synthesised and tested: some of them exhibited promising extraction behaviour. Both high extraction efficiency and stability was achieved. Employing ionic liquids for real fermentation broth extraction, more than 40 % of 1,3-propanediol could be removed after consecutive extractions.
D.3.4 – OPERATING PARAMETERS FOR STABLE MMC OPERATION
The objective was to select and adapt mixed microbial cultures (MMCs), able to ferment crude glycerol generated from 2nd generation biodiesel from animal fat, and produce building-blocks and green chemicals, with a special focus on butyric acid; and 2) to establish stable continuous reactor operations with the selected MMCs.
Different selection and adaptation strategies were tested to obtain MMCs able to convert crude glycerol into preferably butyrate. Different inoculum sources requested different approaches, but showed to be able to acclimatize to the diluted (fat-derived) crude glycerol, without the need of any costly pretreatment (purification) of the feed.
Maximum butyrate production yield was obtained with enriched activated sludge BA_EF, which reached around 0.4 g/g (with 10 g/L substrate).
The inoculum that led to the best butyrate production during steady state in CSTR was obtained through enrichment of anaerobic sludge, grown in MM medium, and was used for subsequent statistical optimization. In general and for all continuous experiments performed with different inocula and media, an HRT of 12h, pH 5.5 and a substrate concentration of 10 g/L (T= 37 °C) allowed to reach a steady state.
D3.10 Sequences of identified butyric acid bacteria
The main objective is development and optimization of a production process for butyric acid from crude glycerol based on Mixed Microbial Culture (MMC) biotechnology. This includes analyses of the composition and development of microbial populations of the MMC during fermentations. The current delivery characterizes the microbial composition of the MMC in general and the butyric acid bacteria in particular. The microbial composition were monitored by Next Generation Sequencing.
Next Generation Sequencing was used as a tool to monitor changes in the microbial composition during fermentations. Changes in the microbial communities at different stages of the fermentations (different transfers) have been monitored by next Generation Sequencing techniques based on DNA sequencing technologies primarily targeting the 16S rDNA region.
For the activated sludge samples, the dominating bacteria in all samples belonged to the phylum Firmicutes in particular the classes Clostridia and Bacilli as well as Gammaproteobacteria. Among the most dominating genera were known glycerol consumers like Clostridium, Klebsiella and Escherichia.
For the anaerobic sludge the dominating genera in the batch test (enriched hexane pretreated crude glycerol) were Clostridium and Klebsiella together with unclassified genera belonging to Proteobacteria while enriched untreated glycerol in fed-batch was dominated by the genus Blautia followed by Clostridia and unclassified genera belonging to Firmicutes.
Both the genera Clostridium and Blautia includes species typical associated with butyric acid production. Species like C. tyrobutyricum, C. butyricum and B. coccoides (reclassified from C. coccoides) are identified in samples from MMC fermentations.
The sulfate reducing bacteria in the inoculum from the anaerobic sludge was diverse, however all genera appeared at very low frequencies all below the cut-off of 1 %. The kinetic control seemed to control the SRBs while the absence of kinetic control allowed for the growth of SRBs leading to the problems relating to the production of H2S.
D3.11. Discovery of new chemical transformations
The conversion of the extracted glycerol fermentation products into value-added chemicals by employing chemocatalysis has been targeted. The glycerol fermentation product 1,3-propanediol has been investigated as the chemical platform.
QUB has shown that the hydrogen transfer initiated deydration (HTID) of 1,3-propanediol in ionic liquids catalysed by recyclable Cp*IrCl2(NHC) (Cp* = pentamethylcyclopentadienyl; NHC = carbene ligand) complexes allows the successful production of a range of C3 and C6 aldehydes as the value-added chemicals. Both propionaldehyde and 2-methyl-pentenal were produced with high conversions, yields and selectivities.In addition, the HTID of 1,3-propanediol in ionic liquids is successful also when significant volumes of water are involved in the reaction. The catalytic system has been proven to be recyclable. The successful synthesis and isolation of value-added chemicals out of the ionic liquid solutions of 1,3-propanediol (mimicking the product of extraction of aqueous glycerol fermentation broth) proves that the combination of Cp*IrX2(NHC) catalysed HTID of 1,3-propanediol in ionic liquids with bio-catalysis has, ultimately, the potential to allow the transformation of waste glycerol into valuable chemicals. This valorisation of waste to chemicals would add significant value and improve the economics of biomass waste utilization. The mono- or multi-step, one-pot, bio- chemo-catalytic process to produce value-added chemicals directly from glycerol will be ultimately tested.
D3.16 – OPERATING PARAMETERS FOR OPTIMAL BUTYRIC ACID PRODUCTIVITY AND YIELD
The objective was to study the distribution of metabolic products during MMC fermentation in continuous mode and statistical optimization of key process parameters for enhanced productivity and yield of butyric acid and 1,3 propanediol.
The enrichment and adaptation of MMCs proved to be a winning strategy to efficiently convert complex substrates, such as crude glycerol derived from animal fat (with no pretreatment). The adaptation allowed the bacteria to grow in CSTR on high feed concentration, performing a high glycerol consumption rate. 1.3 PDO turned out to be the dominant metabolite during steady state, followed by butyrate. The statistical optimization (Inscribed Central Composite Design) allowed to maximize productivity of PDO and butyric acid, with a 5-fold increase compared to steady state results in standard fermentation conditions prior to optimization. The results showed a model with a complex interaction between the key factors (pH, HRT and glycerol concentration), which implied a very careful choice of operating parameters. The model further showed the need for a fine tuning of HRT in combination with the other parameters, in order to maximize productivities and avoid cells wash out. To avoid this problem, preliminary tests and kinetic characterization of the consortium turned out to be fundamental to choose the proper experimental range.
D8.2 – Management and Quality Plan
The main objective of the “Management and Quality Plan” is to define a consistent set of working procedures, processes and best practices in order to ensure Quality standards of the Project outcomes. Moreover, it targets the management of the interaction between the beneficiaries during the work execution as well as the interaction of the consortium with external stakeholders.
The main outcomes of this deliverable is the generation of GRAIL project corporative image through the delivering of a set of standard materials to be used in all communication channels including, internal communication within the consortium, communication with the European Commission, as well as, communication outside the consortium.
Not only a set of rules and quality standards have been established, but also a Quality Assurance Committee has been created. The Quality Assurance Committee is composed by the project coordinator, a scientific expert, a language expert and a dissemination expert, they aim to ensure all the materials created during the project life are in line with those quality standards.
The “Management and Quality Plan” opens the doors to next actions, those actions needed to spread knowledge and information in a standardised manner following the characteristic image of the GRAIL project at the same time it sets the Quality Standards that must be followed to provide uniform and high-quality outcomes to the European Commission and society in general.