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Glycerol Biorefinery Approach for the Production of High Quality Products of Industrial Value

Final Report Summary - GRAIL (Glycerol Biorefinery Approach for the Production of High Quality Products of Industrial Value)

Executive Summary:
The goal of GRAIL is to develop a set of scalable and cost-effective technologies for converting crude glycerol from biodiesel production into a range of biofuels and commercial chemicals.
To accomplish this goal, the project has designed an overall strategy based on three main pillars covering the entire value chain. This includes the evaluation of crude glycerol and purification methods along with the transformation of crude glycerol into high value added products such as biofuels, green chemicals, biopolymers and food supplements. The project is also looking at industrial techno-economical feasibility of glycerol-based products.

Project Context and Objectives:
The global production and consumption of biodiesel is in continually increasing, resulting in a stoichiometric increased generation of crude glycerol, due to its co-production in the transesterification process (Luque et al., 2008, Luque et al., 2010 and Thompson and He, 2006). As a consequence, a vast amount of raw glycerol is generated each year and its value on market is being reduced to the point of becoming a “waste-stream” rather than a valuable “coproduct”. Glycerol prices fell, generating a bankruptcy of companies that produce glycerol chemically, reducing 10 times the price of glycerol in the market (Dharmadi et al, 2006; Hu et al., 2010; Singhabhandhu et al., 2010).

Crude glycerol is available in numerous countries throughout the world, since biodiesel is produced as a response to governments ‘green initiatives’. The total EU27 biodiesel production for 2010 was over 8 million metric tons (UE, 2011), but to date an effective use for the glycerol derived from biodiesel production does not exist on the market. GRAIL project is targeted to integrate and develop existing and novel bio-technologies in order to use glycerol as a competitive biological feedstock in a biorefinery approach. Utilization of today crude glycerol waste stream will also improve the economics and environmental viability of biodiesel (FAME) production.

The overall concept of GRAIL project is the use, exploitation and further development of the state of the art in the field of bio-based products from glycerol and the development research-driven cluster for the use of crude glycerol for the production of high-value platforms, as well as valued end products, harnessing the biotech processes. Therefore GRAIL project has a strong business focus and its ultimate goal is to set up implantation of biorefineries in close relationship with biodiesel.

Main ideas that led us to propose the work

The origin of the GRAIL project lies in the usage of previously achieved knowledge in different already finished projects (e.g.GLYFINERY or PROPANERGY) and integrating that knowledge with new one, herein generated to use waste-glycerol as a carbon source for biotechnological applications. Glycerol is not only cheap and abundant, but its greater degree of reduction, compared with C5 and C6 carbohydrates, offers the opportunity to obtain reduced chemicals at a higher yield. On other hands, glycerol can be readily oxidized, halogenated, etherified, and esterified to obtain alternative commodity chemicals. Glycerol has the advantages of being readily digestible and easily storable over a long period (Ma et al., 2007).

Previous European projects and a growing number of studies focusing on marketable uses for waste-glycerol have proposed several isolated approaches, but their results have failed to integrate a process to resolve the main barriers for the valorisation of this co-product. GRAIL project is born aiming to produce a replicable methodology for using economic and scientific arguments to overcome the main scientific, technological and economical barriers to consider crude glycerol as a suitable feedstock for the production of economically value-added products.

To date, there is no real use for raw glycerol besides from calorific valorisation, which led to an accumulation and as storage or expend as waste cost for the biodiesel corporations, but because of the abundance of glycerol from biodiesel production and the will to continue with renewable energies, there has been major exploration as to uses for glycerol (Leray, 2010). GRAIL, therefore, proposes a "green process” designed for the manufacture of various high value products and biofuels from glycerol side-streams.

The GRAIL consortium is focused on the development of known and new types of applications using glycerol as the starting material. Reactions that already have been extensively applied to convert glycerol into new molecules, such as oxidations, reductions, dehydrations, etherifications, esterifications, etc., will be replaced by biotransformations, at least in some stage of the process and thereby establish a green biorefinery.

This project's aim is to develop a set of technologies for converting waste glycerol from biodiesel production. Our approach includes the transformation of glycerol into
• 1,3 propanediol
• Fatty acid glycerol formal esters
• PolyHydroxyAlkanoates (PHA)
• Hydrogen and Ethanol
• Synthetic coatings, powder coating resins
• Secondary Glycerol Amines
• Biobutanol
• Trehalose
• Cyanocobalamin (Vitamin B12)
• ß-carotene
• Docosahexaenoic acid (DHA)
• Other products
This aim encompasses the following scientific objectives:
1. Review of the state of the art of Commodity Chemicals Derived from Glycerol.
2. Parallel research into completely new options to replace purely chemical transformations by Biotransformations or chemoenzymatic processes.
3. Identification of least 15 Commodity Chemicals and Biofuels to launch the platform.
4. Generation of at least 1 prototype biorefinery for integral use of glycerol as a feedstock for the production of economically value-added products and biofuels.
5. Integral life-cycle analysis and the evaluation of the ecological effects of the glycerol processing to value added products (food additives, green chemicals and biofuels).
6. Integration of Biodiesel and “Bio-Commodity” chemicals production Processes

Specific objectives of GRAIL

Objective 1: Providing enough information on crude glycerol for its introduction in a biorefinery
Target 1.1. Analysis and evaluation of glycerol availability and supply
Target 1.2. Characteristics of available glycerol
Target 1.3. Setting performance specifications for the targeted products
Target 1.4. Glycerol treatment for further processing in order to provide a protocol for the purification of glycerol with the required quality
Target 1.5. Mass and energy balancing for glycerol processing plant concepts in order to provide a good data base

Objective 2: Transformation of glycerol in biofuels
Target 2.1. Biotransformation of Glycerol to hydrogen, ethanol
Target 2.2. Biotransformation of Glycerol to biobutanol
Target 2.3. Biotransformation of Glycerol to FAGE

Objective 3: Transformation of Glycerol in Green Chemicals
Target 3.1. Transformation of Glycerol into propanediol
Target 3.2. Transformation of Glycerol into component of resins and polymers
Target 3.3. Transformation of Glycerol into butyric acid
Target 3.4. Transformation of Glycerol into PHA
Target 3.5. Transformation of glycerol into green reactive diluents and solvents

Objective 4: Conversion of Glycerol into food related compounds
Target 4.1. Conversion of Glycerol into Trehalose
Target 4.2. Conversion of Glycerol into Cyanocobalamin (Vitamin B12)
Target 4.3. Conversion of Glycerol into ß-carotene
Target 4.4. Conversion of Glycerol into Docosahexaenoic acid (DHA)

Project Results:
1. Evaluation of raw material supply and conditioning of the starting materials
GRAIL has investigated current availability of crude glycerine in Europe. This is important in order to rationalize the supply chains involved in the conversion processes proposed in the project. The main conclusion is that the European Union has built a capacity of 22 million tones of biodiesel which translates into a nominal amount of 2.2 million tones per year of crude glycerine. This equals to 44% of the global capacity. At the beginning of the GRAIL project the actual biodiesel production in the European Union was 8 million tones per year. This corresponds to 0.8 million tones of crude glycerine, which represents the most abundant and cheap renewable feedstock in the European Union.
Since biodiesel manufacturing plants use different raw materials, specifically different vegetable oil sources, and use a variety of conversion processes, crude glycerine from a variety of sources representing the most common compositions have been characterized by chemical analysis.
Purification of crude glycerine has been one of the main objectives in GRAIL. The goal has been to develop pre-treatment and purification processes in order to obtain a purified glycerine that satisfies the specifications for the manufacture of resins and food ingredients. Mass and energy balances have been used to simulate two process concepts that integrate both the purification of glycerine and the concomitant production of FAGE, an advanced biofuel. The main result is that the process yields glycerine with sufficient purity to match the very strict specifications of polyurethane based paints and coatings.

2. Glycerol to biofuels
The goal is to develop and demonstrate, both at laboratory and pilot scale, bio-based processes for the production of advanced biofuels from glycerol.
Glycerol to ethanol/hydrogen:
An anaerobic fed-batch process in lab-scale for ethanol production was improved thanks to the isolation of GCLfd20, a microbial community more productive than the reference parental GCL, which allowed a productivity of 36.8±0.3 g/L of ethanol in optimized conditions (80% of increase). The scale-up of this process to 50L was positive. A pervaporation process for the recovery of ethanol from fermentation broths was characterized as a suitable system for the recovery of ethanol at lab-scale.
The ethanologenic process was also improved in microaerophilic conditions at both lab and pilot-scale. Experiments indicated that the yields of ethanol were comparable under microaerophilic conditions and higher under anaerobic conditions than those obtained in lab-scale. For example, a yield of 0.34 geth/gglyc was obtained when 5% of air was used.
The feasibility of a two-step process that converts glycerol into hydrogen with a CSTR reactor and utilizes the effluent in the UASB reactor to produce methane, was successful. Increasing the ORL to 13 kg/m3d-1 and using HRT=13h, 83% of the glycerol energy content was recovered by hydrogen and methane with a 98% of COD removal.
Glycerol to biobutanol:
The conversion of crude glycerol to biobutanol was achieved by means of a fermentation process performed by C. pasteurianum. Cell immobilization by adsorption on PVA particles, and later by entrapment inside, allowed the process intensification. The latter increased butanol productivity up to 3.06±0.30 which represented 6.8 and 1.5 fold increase compared to free cells and adsorbed cells, respectively. At the same time, entrapped cells were able to utilize crude glycerol and form butanol. C. pasteurianum was adapted to grow on crude glycerol in concentrations up to 20-25%, but butanol yield and productivity were both 25 and 13% lower, respectively, than the 100% pure glycerol reference. Volumetric and specific productivities of butanol were both increased of 13% and 16% by the gas stripping integration of a continuous process.
Glycerol to Fatty Acid Glycerol Formal Esters (FAGE):
FAGE was synthesized according to the novel IUCT process using a commercial lipase and it was characterized as a diesel-compatible biofuel. Stability tests showed the biofuel satisfies the required stability in the automotive fuel standards with the usual aditivation protocols that involve the use of commercially available antioxidants.

3. Glycerol to green chemicals
Polyester resins:
The commercial application of purified glycerine obtained with the IUCT process was investigated by Megara. The greek R&D team synthesized glycerine containing polyester resins and formulated different powder coatings. Their performance was subsequently compared with benchmark powder coatings obtained from two different commercial glycerines. The study involved the measurement of key properties of the final products. In most cases the polyester resins prepared with the Grail purified glycerine exhibited similar properties to their countertype commercial grades.
The application of FAGE as emollient in cosmetic applications was investigated in IUCT laboratories. FAGE samples were synthesized from olive, sesame and avocado oils and their physico-chemical properties were correlated with sensorial properties. The study showed that all FAGE emolients displayed excellent performance when compared to equivalent commercial emollients.
FAGE has been investigated as stabilizer of FAME against microbial degradation. Microbial growth in FAME causes the formation of sludge and slimy, slippery and dark coatings in tank walls. As a result of biomass growth in the biofuel, filter clogging in the supply chain infrastructure or vehicles may occur. Metabolites formed during microbial growth contaminate the biofuel leading to out of specifications products. Also acidic metabolites may cause pitting corrosion in aluminum and steel tanks (biocorrosion). An inhibitory test to assess the presevative action of FAGE in FAME. Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergilus niger were used as indicator strains. The experimental results showed a clear inhibitory action when FAGE is blended with FAME at concentrations higher than 20% w/w.
Polyhydroxyalkanoates (PHA) are biobased polymers with enormous interest for their potential commercial applications. PHA can be obtained from crude glycerol through a two step fermentation process. In the first step volatile fatty acids (VFA) and 1,3-propanediol (13PDO) are obtained. In the second step VFA are selectively transformed into PHA. 13PDO can subsequently be recovered by an ammonia assisted method. The laboratory scale process showed that PHA could be recovered with 90% yield and as high as 95% purity and an acceptable molar mass of 200 kg/mol.
Food ingredients:
Fermentation processes have been developed for the production of three important food ingredients, namely DHA, Vitamin B12 and ß-carotene. The work targeted the microbial conversion of glycerol and involved an initial step where a variety of bacteria, microalgae, and fungi were procured and cultivated to obtain the preparatory cultures. Further, fermentation processes were established, followed by optimization and up-scaling at 5 L together with suitable downstream processing and purification procedures for each of the three food ingredients. Finally, the high value compounds were characterized and successfully incorporated into model food products. Specifically, β-carotene was incorporated into a molecular egg containing mango pure, DHA was incorporated to three different fruit juices (banana, peaches and blueberries), and a plum jam was was fortified with vitamin B12.
A sensorial evaluation of all samples was conducted by a panel of 10 judges, and a hedonic test was performed on the products. It was observed that the sensory characteristics were not strongly affected by the DHA concentration. There was no critical factor for the sensory characteristics. Lower acceptances were observed in foods supplemented with higher concentrations of DHA. According to the sensorial analysis, molecular eggs with highest ß-carotene concentration received higher score for colour and smell after taste and touch. Similar scores were registered for taste for all three variants of molecular eggs. In the case of vitamin B12, only slight differences were observed for the taste.
Overall, the fortification of foods was successfully achieved with the ingredients obtained with the fermentation processes developed in the GRAIL project.
Organic Fine Chemicals:
GRAIL has also explored the transformation of crude glycerol into a range of high value added organic fine chemicals. The importance of this chemical conversion is to expand the chemical diversity of commercial specialty chemicals, in particular the generation of novel chemistries with better environmental, health and safety credentials. A second important aspect is that any chemical conversion process should maximize the productivity of chemicals with the desired purity and quality while simultaneously minimizing energy consumption and maximizing the recyclability of catalysts and other auxiliary chemicals.
GRAIL research teams have successfully extracted 1,3-propanediol from fermentation broths using ionic liquids and subsequently synthesized a range of C3 and C6 aldehydes via homogeneous hydrogen transfer initiated dehydration (HTID) of 1,3-propanediol (1,3-PDO) in ionic liquids by means of the iridium catalyst Cp*IrX2(NHC) (Cp* = pentamethylcyclopentadienyl; X = halide; NHC = carbene ligand) complexes as the catalyst precursors. The catalyst precursors were found to be highly recyclable. The successful isolation of highly pure propionaldehyde (yields up to 99 %; selectivity up to 90.8 %) can be easily achieved under reduced pressure, and distillation, with minimal waste. In addition, HTID of 1,3-PDO in ionic liquids is successful also when significant volumes of water are involved in the reaction, and in the presence of air.

4. Engineering evaluation and life cycle assessment of plant concepts and techno-economical feasibility
GRAIL engineering teams have analyzed the laboratory data generated by research teams and performed basic engineering design for a variety of plant concept designs. The engineering data has allowed the iterative optimization and re-design of process conditions. In addition, the engineers have been able to estimate the cost of production of selected chemicals. 1,3-propanediol, FAGE and ethanol are well positioned in terms of cost of production and are sufficiently competitive to start industrial implementation of a commercial plant.
The results have shown that the processes involved in the production of the GRAIL target products range in different levels of optimality and feasibility. The first most important bottleneck is the large amount of process water used in the fermentation of crude glycerol which impact both cost of production and life cycle analysis. Engineering teams have conceived a way around this problem by replacing multiple step water evaporation and distillations by a combination of cascade reverse osmosis and distillation. The second main driver for greenhouse gas emissions is the amount of added yeast. To minimize this process parameter, several optimizations have been performed.
From the environmental point of view, all the systems studied within the project have lower emissions than their reference systems.

5. GRAIL in figures
30 Publications in science journals.
23 General audience publications.
5 Patents (filed or in preparation).
4 Newsletters
54 Oral presentations and posters.
10 Conferences.
32 Seminars and workshops.
53 Woman and 56 men involved in GRAIL.
19 New jobs (16 W, 3 M)

Potential Impact:
GRAIL has been working in parallel to the evolution of the SIRA of the BIobased Industry Consortium (BIC) and the Biobased Industry Joint Undertaking (BBI-JU) and the results in terms of the BIC Key Performance Indicators are as follows that has provided with an specific objectives to ENHANCEMENT OF THE ECONOMIC EFFICIENCY AND ENVIRONMENTAL PERFORMANCE OF EXISTING AND FUTURE BIOREFINERIES to 2020 in terms of the KPIs. Compared to this objectives Grail has reach at least 20% of this objectives already:
KPI 2: Number of new bio-based value chains created/ realised with BBI projects: GRAIL COVER 3 VALUE CHAINS OUT OF THE 10 EXPECTED IN BBI: FOOD, CHEMICALS AND BIOFUEL
KPI 3: Number of grant agreements signed between the project consortia and the EU: AMONG PROJECT PARTNERS WE HAVE ALREADY SUBMITTED 4 PROJECTS IN H2020 AND EXPECT THIS COLLABORATIONS TO BE IN PLACED IN FP9.
KPI 4: Number of new bio-based materials developed (TRL3), validated (TRL 4-5) or demonstrated (TRL 6-7-8) with BBI projects: OUT OF THE 5 BUILDING BLOCKS OF BBI IT IS CLEAR THAT WE HAVE DEMONSTRATED THAT GLYCEROL IS A SUITABLE BUILDING BLOCK.

KPI 6: Number of new bio-based ‘consumer’ products or bio-based applications demonstrated (TRL 6-7-8) with BBI projects: GRAIL CURRENTLY HAVE 1 BIOFUEL, 2 CHEMICALS AND 3 FOOD PRODUCTS IN TRL 6-7

ECONOMIC IMPACTS: most of the products herein develop are economically viable. For example FAGE (WP2) cost calculations of 20%FAGE/FAME compared with FAME represent a reduction of 37 EUR/Tm. This looks that it is not important, however considering that the business plant for this products include the productions of plants of 150.000 Tm, that will mean a manufacturing cost saving of more than 5 million euro.

JOB CREATION IMPACT: currently GRAIL has employed 137 persons from which 16 persons where new employments. Furthermore the industrial implementation of this project will lead to 75 new direct employments in a 5 year period. Moreover, the development of the flag project and the spin-off IABF operations will lead to more than 100 indirect employments in the same 5 year periods. These indirect employmen
ts are related to the building, piping and projecting the flag plant and the dairy operations of this plant

GENDER ISSUES: consortium members has had a policy of setting targets to achieve a gender balance in the workforce and the results of this policy is that out of the 137 persons, 81 researchers were women. Furthermore only 3 new researchers in this period were men. This gender issues will be continue to survive after the project ends and promote gender equality within the consortium members.

ENVIRONMENTAL IMPACT: With these accomplishments, GRAIL will contribute to the 2020 objectives by the reduction of the consumption of biomass for biofuel porpoises and revalorization by revalorization of biowaste in biofuel, chemicals, plastics, coating, and food supplements promoting the use of 10% of the unused resources of the oils from the biodiesel industry.

GRAIL partners where deeply involved in the dissemination of the project. In fact during the project GRAL partners has carried out 54 oral presentations and posters in scientific food, biotech, chemistry and energy fairs providing the published information of grail, has published 23 general articles in popular media, webpages and media briefings announces the latest advances on the project. Furthermore we have organized 10 conferences and fairs 3 of those fully dedicated to GRAIL
1. Open session in Brussels for industrial and policy makers
2. Expoquimia event in Barcelona for industry
3. Show room in the Barcelona Science Museum for industry and academics.
Regarding the last event dedicated to the fair, it was a innovative idea, that involve the presentation of the GRAIL product and demonstration of some of them rather than the usual oral presentation. This event is summarised in two you tube videos already on line.

List of Websites:

Contact details
Dr Carles Estevez
Scientific director of IUCT
Alvarez de castro 63
08100 Mollet del valles