Alba Martínez I Quer

I went on March 2019 to Trondheim (Norway) to visit SINTEF as part of the BRISK2 funding programme.  

My main objective to make use of BRISK2, is to benefit from a high-throughput and a sequencing platform, that my home university does not have.  

Previously, an enrichment of 3 and 6 month for complex-polymer degrading bacteria was carried, followed with an isolation and screening of the fastest growers. 11 unknown isolates, together with 3 well-known isolates were taken form my home university to SINTEF. 

The main goal is to assemble an artificial consortium with these enriched bacteria and quantify their metabolic activity in comparison with the single strains when degrading lignin. Some reports already tested the improved metabolic activity of consortia in comparison with single strains.  

Due to all the possible combinations wanted to be exploited, a high-throughput approach will be required due to the large work that all the potential combinations will provide. Thanks to BRISK opportunity, I have had access to a high-throughput platform at SINTEF lab. Within this platform, I have used the liquid-handling robot, which allows you to transfer liquid and inoculate bacteria to a high number of microtiter culture plates, producing replicates in a short time-frame. 

Firstly, a test with all the single strains cultured with purified lignin extracted from wheat straw (brought from home university), was performed to assess which method of metabolic activity was better and their variability. BacterTito-Glo was chosen due to its more effectiveness and reliability of the results.  

Once all the conditions were set up, the 91 first combinations (1 strain to 1 strain) of the 14 strains were done manually in a master culture microtiter plate with a conventional substrate as metabolic enhancer (glucose). After 24h of culture, the master microtiter plate was used by a robot to transfer 10% of inoculum to microtiter plates (duplicates, in 100 ul), together with a mineral minimal media. Purified lignin was used as substrate in a granule form, was added carefully in each of the wells manually due to it is a non-soluble substrate.  

After the inoculation and the media transfer by the robot, all the microtiter plates were culture off-line  in aerobic conditions. After 48 hours, all the plates were analysed using a BacterTiter-Glo kit assay and measuring the luminescence of each sample. 14 mixed culture were chosen for the next round and the same procedure was repeated for three times. 

As a result, 273 bacterial combinations were tested. The combinations consisted of the isolates enriched and isolated at AAU. Two different selection steps were performed, where the bacterial consortia with a higher ATP content were selected and combined them till reached up to 8 strains per mixture. 

The combination which involves up to 4 strains was the most successful, and when they were cultured on lignin, the bacterial mixtures were performing better than the single strains, which was a successful result because it confirmed our initial hypothesis of synergistic enzymatic activity performs better in terms of using lignin. 

Also, the bacterial mixtures showed better performance when cultured on lignin than plastic and lignin or plastic, which does not confirm our initial theory about co-substrate cultivation, but it showed specificity for lignin even if both substrates shared the same enzymes to be attacked 

Also, the setting-up of an assay that allows to follow-up a big number of cultures while having a non-soluble substrate and a potential growth of the consortia as a biofilm on the substrate, which makes more difficult to have an accurate measurement, was achieved. 

Genomic sequencing, which would not have been possible without the BRISK2 agreement, of 11 unknown isolates brought from my home university, which a putative enzymatic study will be carried in order to link the putative enzyme found on the genome and the biodegradation lignin activity. Also, a bioinformatics expert that assessed me and assembled the data for me was helping me together with a researcher assistant that helped me with the genomic material processing, that was extremely helpful and gave very important data to my project.