Project METAMINE

Mining the microbiomes from marine wood-digesting bivalves for novel lignocellulose depolymerizing enzymes

Under ERA-NET Marine Biotechnology Transnational Cooperation

Contract no. 1/2018 University of Bucharest

Department of Genetics

Budget 184.000EURO

Duration: 36 months (April 2018- March 2021)

Finance Agency: RO-UEFISCDI

Romanian Team

1. Ana-Maria Tanase, Project responsible for UB
2. Ileana Stoica, Prof. dr.
3. Diana Pelinescu, Assoc. prof. dr.
4. Iulia Chiciudean, PhD student
5. Ioana Mereuta, PhD student

ABSTRACT:

Lignocellulose is a greatly undervalorized biomass and methodologies to convert it to high-value products needs fortification. This plant-derived raw material is the most abundant biomass in Europe and it can be harvested from waste streams found in forest and agricultural industries. A functional bioeconomy depends on the ability to convert lignocellulose to chemicals and fuels, a process called biorefining, which still is in its infancy. A critical step in biorefining is the enzymatic conversion of lignocellulose to soluble sugars and lignin. The cost and the efficiency of enzymes is far from optimal and new enzymes are needed to improve the efficiency and sustainability of lignocellulose depolymerization. Through META-MINE, we will exploit the process strategies of nature’s own micro-biorefinery, the shipworm. Shipworms are voracious animals with respect to their appetite for wood. These marine bivalves are causing severe damage to all wood found in the sea worldwide. Their digestive system is especially intriguing. Wood engulfed by mechanical rasping is digested by enzymes secreted by a community of symbiotic bacteria located in the gill tissue. Current model systems for the study of cellulose degradation are highly complex (e.g. community driven anaerobe systems in ruminants and the intricate secreted enzyme systems of aerobic fungi) and challenging to analyze. The shipworm gill symbionts are specialists in lignocellulose degradation and perform this task by applying a perfected enzyme cocktail in a defined and physiochemically stable environment. Thus, by unravelling the contributions of the individual enzymes in the shipworm cocktail, we have the opportunity to take a leap forward in understanding the fundamental properties of enzymatic lignocellulose degradation. META-MINE will use the shipworms as a model system for a holistic study of marine lignocellulose degradation and mine the metagenomes for novel lignocellulose depolymerizing enzymes.

CONSORTIUM:

Kick-off meeting: From the left: Gustav Vaaje-Kolstad, Seila Pandur, Ioana Mereuta, Luisa Borges, Rolf Daniel, Ana-Maria Tanase, Bjørn Altermark, Iulia Chiciudean and Nelson Simões.

In this first stage of the project, we have constructed a system of wooden panels, which we have anchored in the Black Sea, in order to capture shipworms. For establishing a molecular method to study these organisms capable of using lignocellulosic material for development and growth, such as Teredo navalis, we also captured bivalve specimens belonging to the species Mytilus galloprovincialis from the Black Sea. Following dissection, we took branchial tissue as a biological material for testing DNA isolation kits and analyzing its concentration and quality for use in metagenomic procedures. For the evaluation of bacterial biodiversity in branchial tissue, we quantified 16S rDNA copy number by qRT-PCR, in the same time by cloning the 16S sequences from the bacterial communities of each specimen and determined there individual HRM profile, which allowed us to group the sequences as 51 OTU. We considered it extremely useful to analyze DNA and/or protein sequences of some bacterial enzymes involved in the lignocellulosic material degradation: multifunctional laccases / peroxidase, cellulases / endo β 1-4, glucanases and beta-1,4-xylosidases, respectively. Genebank, Uniprot and CAZy databases were analyzed by multiple alignments with a number of programs or applications such as BLAST, ClustalOmega, Jalview, and constructed phylogenetic trees using MEGA7.

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.

Throughout the past year, we have managed to do a much more detailed analysis of the existing sequencing data. We have generated assemblies from the raw sequencing data of  Black Sea Teredo navalis gill tissue, which were further used to discover the potential that the shipworms’ symbiotic microbial communities have with regard to lignocellulose degradation, as well as their taxonomic diversity and abundance at a read level. Functional annotation based on known amino-acid sequence motifs for lignocellulolytic enzymes (derived from the CAZy database), revealed 9421 such sequences in sample SW01, and 8601 sequences in sample SW02, respectively. These sequences are associated with the catalytically active regions of all major classes of carbohydrate-active enzymes: glycoside hydrolases (GH), glycosyl tranferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE), and auxiliary activities (AA), as well as numerous carbohydrate binding modules (CBM), which allow the CAZymes to bind to their specific substrate.

            Data analysis shows that over 70% of microorganisms identified in the Teredo navalis gill tissue samples belong to the Bacteria domain, regardless of the location the shipworm was collected from. From this domain, approximatively 70% belong to the phylum Proteobacteria, of which a majority is represented by members of the class Gammaproteobacteria (approx. 85%). As was expected, the Cellvibrionaceae represented the dominant family in the class, taking up approx. 80%. Surprisingly, in the case of the studied Black Sea bivalves, only a small percentage of annotated reads in each sample were associated with the Teredinidibacter genus, the major symbiont being Cellvibrionaceae bacterium 1162T, or a similar microorganism. Cellulose enriched cultures presented a relatively large degree of diversity, dominated by members of the Gammaproteobacteria class, and which gradually changed their composition towards a majority of anaerobic bacteria. Isolated strains stood out with their ability to produce carboxymethylcellulose-degrading enzymes capable of producing large amounts of reducing sugars, indicating the ability to cleave numerous glycosidic bonds in a short amount of time. Comparative bioinformatics analyses were not successful in identifying a core set of genes involved exclusively in the endosymbiotic lifestyle.

Posters:

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.

Lascu I., Preda M., Mereuță I., Stoica I., Chiciudean I., Tănase AM., Evaluating the diversity and cellulolytic potential of Black Sea shipworm (Teredo navalis) gill symbionts, Biotechnology Congress 2020, September 24-26,Prague, Czech Republic.

Manuscripts:

Lascu I., Mereuță I., Chiciudean I., Hansen H., Avramescu S.M., Tănase AM., Stoica I., The complete genome sequence of Photobacterium ganghwense C2.2: A new polyhydroxyalkanoate production candidate, submmited to MicrobiologyOpen (under review)

Chiciudean I., Lascu I., (...)Tănase AM., Stoica I., The special case of the shipworms endosymbiont Teredinibacter turnerae and the endosymbiont status,  (under project partners discussions) wil be submmited to Marine Biology Research

The last three months of the project were dedicated to surveying the industrial sector for interest in cellulolytic enzyme technologies, in order to understand the needs of the economy, as well as establishing a line of communication for possible future collaboration. We contacted several potential users of cellulolytic enzymes, such as paper factories, textile fiber producers, and pharmaceutical producers, in order to promote our studies. We are in touch with our project partners and we were engaged in several meetings and discussions regarding the paper contributions of each partner. Based on the computational resources gained by the Metamine project, we managed to reanalyze data from a complete genome and to get a research paper accepted in the MicrobiologyOpen journal. More than 3 final papers will be written by the master students on microbial communities metabarcoding analysis subject and 1 PhD thesis is in progress, Irina Lascu being also employed for 15 months of the project. The knowhow gained in this project is going to be used in another partnership in an already financed national project in collaboration with Faculty of Physics and Faculty of Chemistry.

1. A picture is worth a thousand words: novel photographic evidence on the anatomy of the digestive system of four shipworm species (Bivalvia, Teredinidae), Luisa Borges, Birgit Pheiffer, Seila Pandur, Iulia Chiciudean, Irina Lascu Gustav Vaaje-Kolstad, Ana-Maria Tanase, Nelson Simões, Rolf Daniel, Bjørn Altermark (in progress)
2. First phylogeny of the Teredinidae with species trees, Luisa Borges, Birgit Pheiffer, Seila Pandur, Iulia Chiciudean, Irina Lascu, Gustav Vaaje-Kolstad, Ana-Maria Tanase, Nelson Simões, Rolf Daniel, Bjørn Altermark (in progress).
3. The complete genome sequence of Photobacterium ganghwense C2.2: A new polyhydroxyalkanoate production candidate Ioana Mereuta, Irina Andreea Lascu, Iulia Chiciudean, Hilde Hannsen, Sorin Avramescu, Ana-Maria Tanase, Ileana Stoica. MicrobiologyOpen (accepted in 15.03.2021)