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:

Dr Bjørn Altermark

 

UiT - The Arctic University of Norway

Department of Chemistry

Norway

Dr Gustav Vaaje-Kolstad

 

Norwegian University of Life Sciences

Norway

Prof Rolf Daniel

 

Georg-August-Universität Göttingen

Institute of Microbiology and Genetics,

Genomic and Applied Microbiology

Germany

Dr Ana-Maria Tanase

 

University of Bucharest

Department of Genetics

Romania

Dr Raul Bettencourt

 

University of the Azores/OKEANOS

Oceanography and Fisheries

Portugal

 

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.

Summary update 2018

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.

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.