My transnational access (TA) visit with BRISK2, titled “Biodiesel production from sunflower oil and palm oil with ethanol using Rhizopus oryzae as whole-cell biocatalyst”, was virtually carried out from September 20^th to October 1^st (2 weeks) at the Biorefinery and Bioenergy Center (BIO2C), National Renewable Energy Center (CENER), Spain.

Fatty acid ethyl ester (biodiesel) can be produced from the transesterification reaction between a lipid and ethanol, mediated by catalysts. This research project was aimed at optimizing biodiesel yield using a combination of process variables that impacts transesterification reaction (ethanol-to-oil molar ratio, water content, reaction temperature, and reaction time). Additionally, since the experimental design was developed from the perspective of environmental and commercial sustainability, the materials/methods used contrasted with current commercial biodiesel production materials/methods:

1. Utilizing ethanol which is bio-renewable compared to methanol which is generated from fossil feedstock.
2. Utilizing biocatalysts which require mild process conditions (especially lower temperature and alcohol consumption) and offer efficient and inexpensive product recovery compared to chemical catalysts.

The physical TA visit was originally proposed to be carried out in March 2020. However due to COVID-19 travel restrictions, a virtual TA visit was agreed upon. The BIO2C team conducted the research based on the design and protocol agreed upon between me and them, and maintained correspondence via emails and a video conference appointment at the end of week 1 and 2 to discuss the results and the way forward.

The BIO2C which facilitated the research project is equipped with a pilot plant for biochemical conversion of biomass, and could reproduce all the biochemical conversion steps starting from feedstock pretreatment, fungal fermentation using a 100 L fermenter, lipase activity assay, laboratory scale transesterification using Erlenmeyer flasks, and fatty acid profile/biodiesel yield analysis using a gas chromatography-flame ionization detector (GC-FID).

Prior to the start of TA visit, a pre-incubation of the pure culture of Rhizopus oryzae (ATCC 24563 strain was used due to its well documented lipase activity) was carried out in potato dextrose broth using Erlenmeyer flasks and reciprocal shakers. The following activities were carried out during the actual TA visit:

1. Fatty acid profiling of the vegetable oils: GC-FID analysis to determine the average molecular weight of the oils, and thus the stoichiometric equivalent of ethanol and corresponding weight of oil to be added to the reaction mixture.
2. Fungi preparation: incubation of the fungi using a 60 L basal lamina in a 100 L fermenter for 72 hours; filtration, washing, and oven-drying of the fungi (at 30^oC); lipase assay on the fungi to ascertain its expression of lipase activity.
3. Vegetable oil preparation: autoclaving at over 100°C to eliminate interference by water, native microorganisms, and native enzymes.
4. Transesterification of the vegetable oils: this 72-hour reaction was conducted in Erlenmeyer flasks using reciprocal shakers, with the biocatalyst introduced wholly into the reaction mixture.
5. Biodiesel yield quantification: At specific points in time during the reaction course, 1 ml of the reaction mixture was taken and centrifuged to separate the water and glycerol phase from the biodiesel phase which was then analyzed using GC-FID.

The results obtained showed a biodiesel yield range of 68-76 %, in relation to FAMES yield in the original oil, hereby reaffirming the potentials of biochemical conversion process as an option for producing biodiesel in a manner that ensures long term environmental and commercial sustainability.

Finally, I would like to appreciate the BIO2C team led by Inés del Campo, and supported by Mercedes Munarriz and David Sanchez for their patience and support throughout the period preceding and after the virtual TA visit.