MARPLAST Summary update 2019

Summary update 2019

Throughout this phase we focused our research on Photobacterium sp. strain C2.2, due to its enormous growth and PHA production potential using inexpensive biomass. Because this strain belongs to a less studied species, Photobacterium ganghwense, we decided to send this strain to sequence its complete genome. In the lab, we continued the optimization of various cultivation parameters, for this strain as well as C2.10 Leisingera sp., N16b Halomonas venusta. We added the PM9 plate reads (which contain various osmolytes) to the existing BIOLOG assays, with different conditions than those used previously for the tested strains. In regards to cultivation parameters, we focused mainly on carbon sources, testing both waste sunflower oil which was used for frying as well as waste fish oil, which highlighted the fact that, out of the two carbon sources, our strains can only successfully grow and accumulate PHA on fish oil. Strains C2.2 and C2.10 were shown to be the most capable of using this carbon source. Strain C2.2 was reanalyzed from the perspective of its capacity to consume carbon and nitrogen through TOC analyses, measuring leftover organic carbon and nitrogen throughout its growth period. PHA accumulated by this strain was quantified through GC-MS on dried cell samples, and the largest amount of PHB produced was obtained using fructose as a sole carbon source, 1.84 g/L, with a PHB content of 51.7% of cell dry weight (CDW). Bioreactor experiments were focused on glycerol and molasses as carbon sources, obtaining cell cultures with optical densities (600nm) ranging from 7 to 96 at a maximum of 48h cultivation time, cell densities much greater than those obtained from shake flask cultures. The largest amount of PHB obtained was from the cultivation of C2.2 with glycerol: 7.375 g PHA/L. Lastly, we analyzed the market growth potential for PHA production at a global scale, for which online press estimates an increase of 25% in the next five years, as well as potential companies which could benefit from the studies done throughout this project. 

Publications:

1. De actualizat : Mereuta I., Tanase A.M., Chiciudean I., Vassu T., Stoica I., 2020. Metabolic and molecular profiling of microbial communities following controlled kerosene pollution in Bucharest Botanical Garden pristine soil, Polish Journal of Environmental Studies,29(1):197–203. IF 1.186

2.       Manuscris: Ioana Mereuta, Irina Lascu, Sorin Avramescu, Hilde Hansen, Iulia Chiciudean, Ileana Stoica, Ana Maria Tanase; Photobacterium ganghwense C2.2: A new promising cell factory for bioplastic production trimis,  Journal of Biotechnology

3.       oral presentation : TănaseA.M., Mereuță I., Lascu I., Avramescu S.M., Chiciudean I., Stoica I., Bioplastic production using Black Sea microorganisms, International Microorganism Day (IMD) – September 17, 2019, Bucharest, Romania,.

4. Poster: Chiciudean I., Mereuță I., Mihalachi E.L., Lascu I., Avramescu S.M., Jablonski P., Christensen M., Hansen H., Stoica I., Tănase A.M., Halotolerant Black Sea bacteria for bioplastics production, Halophiles 2019 Conference, 24 – 28 June, Cluj-Napoca, Romania.
5. Mereuta I., Chiciudean I., Lascu I., Mihalachi E.L., Jablonski P., Avramescu S.M., Stoica I., Tanase A.M., Black Sea newly isolated Photobacterium ganghwense C2.2 a promising candidate for PHA production, Biotechnology Congress 2019, April 11-13, Valencia, Spain.

METAMINE Summary update 2019

Summary update 2019

At this stage of the project we have successfully captured for the first time specimens of Teredo navalis from the Romanian Black Sea area, identified mainly on the basis of posterior pellets. These specimens were much larger than expected after deplition for 6 months in the water. Following dissection, gill tissue was collected and used for enrichment cultures on cellulose or carboxymethylcellulose as carbon source,  In the same time total DNA was isolated and purified, using dedicated extraction kits. The DNA samples obtained were sufficiently concentrated and had an adequate degree of purity for NGS sequencing. Because the majority of DNA in the samples is from the host organism, NGS sequencing was performed in depth and as a result generated a very large volume of sequences. These have been processed and compared using issues available on Github, in command line on Linux operating system. This processing was achieved due to an intense learning and study stage, at the University of Gottingen, using bioinformatics resources and their expertise in metagenomics data processing. Phylogenetic abundance analysis of the microbial communities from the analyzed samples revealed that the strains isolated in pure culture are found within these communities: Labrenzia sp., Swanella sp., Vibrio sp. Some of the isolates obtained presented a clear zone around the  colony when cultivated with cellulose, followed by staining with Congo Red.

Papers

Chiciudean I., Mereuta I.,Lascu I., Borges L.M.S., Filimon A., Stoica I., Tanase A.M., Looking for cellulolytic activity in the Black Sea shipworms gill symbionts, Biotechnology Congress 2019, April 11-13, Valencia, Spain.

Workshops:

1.    International Course in Microbial Ecology “Hands-on training in Prokaryotic and Eukaryotic metagenomics (ICME10)”., University Milano Bicocca 6-10th May 2019, Italy.

2.    7th International Workshop on Prokaryotic Genomics&Bioinformatics, Gottingen, 19-20th September, 2019, Germany.

MARPLAST Summary update 2017

The first phase of the project is focused on bacterial strains selection from marine environments capable of production polyhydroxyalkanoates (PHA) granules. A number of 155 strains from MICROGEN Microbial Collection were tested using Red Nile fluorescence. Thus, we managed to select three PHA producers, subsequently confirmed by GC-MS, also identifying polymer type for the tested conditions. Selected strains were taxonomically identified based on ribosomal RNA 16S as Loktanella sp. P2, Granulosicoccus sp. P4, Sulfitobacter sp. P5. These strains along with ones from the Norway partner, Halomonas sp., Pseudomonas sp., Roseobacter sp., were subjected to metabolic assays comprising API and BIOLOG tests. Resulting data revealed special nutritional demands of tested marine bacteria, oligotrophs for many carbon and nitrogen sources. Thus, we faced several problems when adapting our marine isolates to laboratory conditions, requiring a more detailed analysis of growth media. PHA production was evaluated in a volume of 250 mL, variating the temperature and carbon source. Detection and quantification of PHA granules was conducted based on Red Nile fluorescence, using a 96 well plate reader, also determining cellular density. These assays managed to emphasize the high PHA production potential of MICROGEN Bacterial Collection tested strains. For biotechnological approach, Roseobacter sp., was selected for batch cultures in a 2L bioreactor, recording a lower fluorescence then 250 mL cultures. New isolates from the Black Sea were tested, and from 22 strains only four were identified as PHA producers for selected conditions. Thus, we conclude that all our activities were successfully accomplished.