GRAIL e-NEWSLETTER #1 – September 2014

Project Activities Overview

Evaluation of raw material supply, conditioning of the starting materials

A database of biodiesel plants in Europe and worldwide was updated and it will be created maps and questionnaires which will be included with site qualifications, contact details of other biodiesel plants, modification proposal, etc). Another task which is being developed is the analysis of characteristis of available glycerol. At this stage, the coordination of the procurement of relevant glycerol specifications is completed and the procurement of technical and economic requirements of the targeted products are working on it.
Glycerol treatment for further processing is another task which is being carried on with experiments regarding ionic exchange agents and high vacuum distillation.


Optimization of a process developed by Pontificia Universidad Católica de Valparaíso (PUCV) about hydrogen and ethanol production from crude glycerol streams, based on mixed microbial culture biotechnology.
Batch experiments with no other nutrients than glycerol concentration; PUCV: It is been determined the effect of glycerol concentration in the hydrogen and ethanol production, using batch experiments with a synthetic feeding with pure-glycerol (5, 10, 20, 30 or 50 g l-1) containing macro and micronutrients (Table 1). The first results show that higher concentrations of glycerol have a negative effect on the hydrogen yield but not on the ethanol concentration. It was determined the effect of different pretreatments of inoculum on the hydrogen and ethanol production, using a Continuous Stirred Tank Reactors (CSTR). The reactor was inoculated using different sludge (Activated Sludge or Anaerobic Sludge) with and without pretreatment (thermal shock or aeration). The start-up of the operation in the reactor was considered to have a low HRT for biokinetic control. The reactor was fed with glycerol at 10gL-1 and the synthetic feeding medium also contained salts and minerals. The aerobic sludge has an advantage over anaerobic sludge in terms of ethanol production but not with biohydrogen production, where yields were not significantly different and the microbial structure is determined by the biokinetic control with the pretreatments studied.


Table 1. Synthetic effluent composition used in hydrogen production.

The preliminary results show that the higher concentrations of glycerol have a negative effect on the hydrogen yield but not on the ethanol concentration. Although there is a feasibility of using this mixed culture for producing hydrogen and ethanol, simultaneously, it is necessary to find an optimum point because the ethanol concentration would reverse the hydrogen yield. The decrease in the pH during operational days can lead to lower substrate degradation. This must be studied in future batch experiences by adding a buffer to maintain the pH or in continuous culture.

 Development of green chemicals from glycerol

Production of building blocks with focus on 1,3-propanediol

Based on the literature research Clostridium butyricum DSM 10702 was selected for 1,3-propanediol pilot optimization experiments on pure glycerol as a starting substrate. All batch experiments were made in 1 or 2,5 L bioreactors (BioFlow 115, Eppendorf) to obtain reliable and reproducible data. Beside that type strain was used in experiments, the fermentation profile of this strain was quite nonstandard (complete acetic acid utilization, incomplete glycerol utilization, increased butyric acid production etc.).

Butyric acid production and optimisation

AAU has started working trying to adapt/enrich mixed microbial consortia to 2nd generation crude glycerol obtained from DAKA biodiesel in DK. Different adaptation strategies and different growth media are currently being tested. The specific feedstock presents the challenge of severely inhibiting microbial growth as repeated transfers of anaerobic sludge have shown (Figure 3.6.1 a,b). Adaptation/enrichment in fed-batch and continuous mode are alternative strategies to be examined.


Figure 3.6.1 a: Effect of repeated transfers of non pretreated anaerobic sludge on hydrogen production (H2%), taken as an indirect indicator of biomass growth. A clear inhibition is observed after the first transfer (T1) with Minimal Medium, and after 6 transfers (T6) with Basal Medium. MM=Minimal Medium; BA=Basal Medium; KC= kinetic control (transfer every 24 h); EF=end of fermentation (transfer at end of fermentation).


Figure 3.6.1 b: Effect of repeated transfers of heat-shock pretreated anaerobic sludge on hydrogen production (H2%). Also in this case, a clear inhibition can be observed after the first transfer (T1) with Minimal Medium, and after 4 transfers (T4) with Basal Medium. MM=Minimal Medium; BA=Basal Medium; KC= kinetic control (transfer every 24 h); EF=end of fermentation (transfer at end of fermentation).

Development of food supplements from glycerol

The overall aim of this task is to convert the glycerol, by means of biotechnological fermentation processes, into high value compounds for food application purposes as well as their subsequent incorporation into model foods. With this purpose the SMEs involved in this WP, Biozoon (WP Leader) and Centiv, in close collaboration with University of Applied Sciences Bremerhaven have joined forces through the sole food related activities of GRAIL project.


The goal is to obtain i) beta carotene, a vitamin A precursor ii) trehalose, a relatively new disaccharide, iii) carotenoids, widely used as food colorant in a variety of foods, and finally docosahexaenoic acid, an unsaturated fatty acid used in food supplements and for medical purposes. Thereby, key materials and methods have been established from the beginning of the project to create the base of the research. In particular, the biotechnological processes necessary for obtaining the targeted food compounds were proposed. Additionally, the pertinent microorganisms and their growth conditions as well as the development of the most suitable medium composition in combination with glycerol are being defined.


In total 10 preliminary Block Diagrams have been developed using all the available information, mainly from literature, in particular:
• Ethanol & Hydrogen, Butanol and FAGE for the technological line “Glycerol to Biofuels”;
• Butyric Acid/PHA, Polymers & Resins, and 1,3-propanediol for the technological line “Glycerol to Green Chemicals”;
• Trehalose, Vitamin B12, β-carotene and DHA for the technological line “Glycerol to Food Compounds”.
The performed steps to develop this have been at first stage an intensive literature search made by PI, a discussion between rest of the partners in order to agree a template to ask for needed information and a preparation of the initial flowsheet. After that, based on the available information, the preliminary draft of Block Diagrams (figure below) has been sent to the involved partners for comments. At the end, all comments/suggestions received have been implemented on the Block Diagrams and a report was attached to them in order to give a description about the working methodology.


Example of Block Diagram of Glycerol to food compounds Vitamin B12

Environmental credentials and economic analysis

The overall objective of this activity is to control and support the glycerol-based products processing proposed in the project, taking into account environmental, economic and social aspects in order to foster sustainability. Life Cycle Assessment analysis will be performed with the aim to report the potentially positive environmental impact from processing glycerol to the value-added products. Apart from that, through Life Cycle Cost analysis will be determined the costs associated to the processes, in order to optimize and select the best options from an economic perspective; and a Social Impact analysis will be done to maximize the positive social impact and promote the acceptance of the project. Finally, an Environmental Technology Verification will be addressed for those technologies applied from producing glycerol-based products.


Events and news

The partners have participated, during the project, at many events (congress, conference and something like that) and wrote articles on newspaper and a press release indicated in the following table. The future events/articles will be published in each website of them.

Partner Events/Articles
IUCT Articles

  • InKemia-IUCT group desarrollará una biorefinería de glicerina
  • Inkemia liderarà un projecte de R+D de la Unió Europea
  • InKemia-IUCT group consigue un gran proyecto para el desarrollo de una biorefinería de glicerina
  • InKemia-IUCT group consigue un gran proyecto para el desarrollo de una biorefinería de glicerina
  • Comunicación de información relevante InKemia-IUCT group, S.A.
  • InKemia gana un concurso público de la UE y se dispara en el MAB
  • InKemia-IUCT group consigue un gran proyecto europeo de I+D de 6 millones de €
  • InKemia diseña una biorefinería con fondos europeos
  • InKemia de Mollet se hace con un proyecto europeo
  • InKemia patenta en Japón uno de sus productos (S50)
STUBA Biotech 2014 in Prague: Stuba have done a presentation about immobilized system (11 June 2014)

  • Almost 8 Million de € invested in developing the biorefinery approach of glycerol
DBFZ Article

  • DBFZ beteiligt sich an EU-Forschungsvorhaben zu Verwertungsmöglichkeiten von Glycerin
AAU Presentation of a poster about AAU’s work in GRAIL, EU conference of Biomass in Hamburg, 23-26 June 2014
UASB Article

  • Mit Glycerin zum Zucker

  • Attended the Open Doors Day of the European Institutions in Brussels (17 May 2014)
  • Health & Food Days (JAS) organized by the cluster « CRITT Agro-Alimentaire» (18-19 June, La Rochelle)
  • 1st International PLEASURE Conference “SALT-SUGAR-LIPIDS REDUCTION” organized by the EU Pleasure Project coordinated by BZN (partner in GRAIL as well)
  • G. Ruiz Filippi; E. Tapia-Venegas. Inoculum pretreatments selection for hydrogen production from glucose and glycerol.Workshop Latino-Americano de Bio-Hidrogênio, Sao Carlos, Brasil. July 2014


  • Javiera Toledo-Alarcón, Estela Tapia-Venegas, Lea Cabrol, Eric Trably, Gonzalo Ruiz-Filippi. Producción de biohidrógeno y bioetanol utilizando lodo pretrado con aireación y glicerol puro como sustrato.XXXIV Congreso Interamericano AIDIS, Monterrey,México. October 2014


  • Toledo-Alarcón, Javiera ; Tapia-Venegas, Estela ; Cabrol, Lea ; Trably, Eric ; Ruiz-Filippi, Gonzalo Producción de hidrógeno y etanol con lodos mixtos pretratados con aireación y glicerol como sustrato. XI Simposio latinoamericano de digestión anaerobia, La Habana, Cuba, November 2014


  • Carmen G. Contreras, Fernando Silva-Illanes, Javiera Toledo-Alarcón, Estela Tapia-Venegas, Gonzalo Ruiz-Filippi. Efecto de la concentración de sustrato en la producción de hidrógeno en cultivo continuo a partir de glicerol. XI Simposio latinoamericano de digestión anaerobia, La Habana, Cuba, November 2014


  • Fernando Silva-Illanes, Estela Tapia-Venegas, Javiera Toledo-Alarcón, Gonzalo Ruiz-Filippi. Producción de hidrógeno a partir de glicerol mediante una etapa inicial de enriquecimiento aerobio del inóculo. XI Simposio latinoamericano de digestión anaerobia, La Habana, Cuba, November 2014



Please download the e-newsletter#1 here: Newsletter 1 – September 2014