Faculty of Biology, UNIVERSITY OF BUCHAREST
Marine bacteria for bioplastic production
Under ERA-NET Marine Biotechnology Transnational Cooperation
Contract no. 13/2017 University of Bucharest
Department of Genetics
Duration: 36 months (march 2017-february 2020)
Finance Agency: RO-UEFISCDI
Project responsible from UB
Senior researcher, Prof. dr.
Senior researcher, Prof. dr.
PARTNERS IN THE PROJECT
Name of research organization
UiT – the Arctic University of Norway
Prof. Arne Smalas -project leader
Prof. Knut Irgum
University of Bucharest
Assoc. Prof. Ana-Maria Tanase
The steady increase in microplastic concentration could result in dramatic effects on the vulnerable wildlife of the oceans and marine food supplies. It is therefore of immediate importance to develop novel types of polymeric materials that can be sustainably produced to address these environmental concerns. MARPLAST focuses on polyhydroxyalkanoates (PHAs), a class of biodegradable bioplastics which are considered to be feasible replacements for current petroleum-based plastics. PHAs are polymers occurring in nature, produced among others by bacteria, and with properties similar to oil-derived polypropylene and polyesters, rendering them useful as an attractive biodegradable replacement. However, the naturally occurring PHA production pathways are not sufficiently understood, and currently known technologies for production are too costly to allow for a full-scale replacement. MARPLAST aims to develop and provide tools (bacteria, enzymes, and pathways) to enable efficient production of sustainable and biodegradable bioplastics from low cost unexploited biomass. Focus will be on PHA-producing cold-adapted marine bacteria, which have a range of properties that make them especially suitable for industrial applications. MARPLAST will utilize expertise from the Univ of Tromsø (Norway), Univ of Bucharest (Romania) and Umeå University (Sweden) to make important progress and contributions to the transition to a bio-based European economy.
On this focus of MARPLAST, Romanian Team will isolate new marine bacterial strains from Black Sea, especially those with a high PHA production but will test also all marine bacterial strains previously isolated and deposited to Microbial Collection of Department of Genetics. Bacterial strains will be phenotypically characterized using the Biolog Microbial Identification System and other usual microbiological tests. On molecular level, identification will be based on 16S rDNA sequences. Marine bacteria will be analyzed regarding PHA production by Red Nile fluorescence intensity measurements, in order to establish the best cultivation conditions for the highest production of PHA. Those conditions will be tested also to a larger scale in bioreactor.
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.
The isolation and purification activities of bacterial strains from the Black Sea ecosystem resulted in a number of 82 isolates, out of which 12 were selected as PHA-producing strains. Following molecular analysis of 16S gene sequencing, these strains have been taxonomically identified as belonging to various bacterial species, some of them poorly studied as PHA-producers. For a suitable metabolic and taxonomic characterization, the selected strains were analyzed by Biolog system for their ability to metabolize multiple carbon and nitrogen sources, and in terms of osmotic and ion effects (e.g. tolerance to different salts concentrations). The enzymatic profile, determined by API assays, showed high similarities between the isolates, but BIOLOG tests showed a metabolic diversity of the carbon and nitrogen sources. As expected, high salts tolerance confirmed their provenance from saline and hypersaline marine environments such as those from Norwegian partners. The optimization of growth conditions and PHA production parameters primarily consisted of cultivation on carbon sources of low economic value: molasses, glycerol, starch, fructose, in combination with various nitrogen sources. The results obtained were extremely diverse, each strain analyzed behaved differently in terms of multiplication and PHA production capacity. Of all the strains analyzed, C2/2 Photobacterium seems to have nutritional needs that have led to values of 20-25 DO cultures, which is biotechnologically important. Moreover, the extremely intense fluorescent signal we have obtained confirms PHA production potential of this strain, which belongs to a less studied species with no emphasis on PHA production capacity. Bioreactor tests have shown that C2/2 strain can store up to 70% PHA in the cell and can produce up to 4g PHA / L, results comparable to other studies. Although PHA production for strains N16b Halomonas and C2/10 Leisingera, was slightly lower, they still accumulate up to 40-58% of the dry weight, which are interesting results that can be exploited biotechnologically.
European Conference of Biotechnology, Athena, Greece Aprile 26-28, 2018, Polyhydroxyalkanoate production potential of Black Sea new bacterial isolates, Mereuta I, Chiciudean I, Lascu I, Avramescu SM, Stoica I, Tanase AM, abstract published in Journal of Biotechnology: DOI: 0.1016/j.jbiotec.2018.06.148.
Ioana Mereuta, Iulia Chiciudean, Irina Lascu, Sorin Avramescu, Ana-Maria Tanase, Ileana Stoica, Microbial community screening for bacterial PHA producers Roumanian Biotechnological Letters, 2019 (under review)
Mereuta Ioana, Chiciudean Iulia, Vassu Tatiana, Tanase Ana-Maria, Stoica Ileana Metabolic and molecular profiling of microbial communities following controlled kerosene pollution in Bucharest Botanical Garden pristine soil, Polish Journal of Environmental Sciences, 2019 (accepted)
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.
The last two months of the project were dedicated to industry connection and surveil, in order to understand the needs of the economy. We contact several potential users of the bioplastic material, like Romanian milk factory, or several producers of biodegradable material in order to promote our studies. The industry players were open and interested in our project, even we need to extract the PHA granules in larger amounts. In this matter we wrote a project proposal in the national competition call Experimental demonstration project (PED), PN-III-P2-2.1-PED-2019, Production of biodegradable plastic using marine bacteria strains. With this proposal we intended to obtain PHA granules from larger volum cultures, using 10L bioreacter.
The Norwegian partner has contacted an important PHA producer from USA to promote the project findings and also to show them the potential of using waste fish oil for bioplastic production.
Based on the Marplast project, we updated some of the thematic for the Applied Genetics and Biotechnology Master Program at Faculty of Biology, University of Bucharest, by presenting to the students Microbial bioplastic production, molecular and metabolic mechanisms as new innovative courses, showing them also, in the lab sessions the PHA granules and the up-scale cultivation methods for marine bacteria strains. More than 6 final papers were written by the undergraduate and master students on this subject, and 1 PhD thesis is in progress.