The history of the ENEA microbial collection

The history of the ENEA microbial collection originates from projects in agricultural, industrial microbiology, and microbial ecology implemented in the 1980s. The collection's evolution mirrors the history of the research center, aligning with its programmatic lines which have been changed and/or renewed over the years. Since the 1980s, ENEA researchers have isolated, characterized, and preserved individual strains and microbial pools (bacteria, fungi, algae, viruses) from diversified environmental matrices with applications initially focused on bioenergy and agri-food fields, and subsequently expanded to environmental, cultural heritage (CH), and health sectors. Over the years, ENEA has developed integrated capabilities and laboratories of excellence, providing advanced services on the topics in question thanks to the availability of specialized laboratories and technologically-equipped halls with pilot-scale bioreaction and downstream processing facilities. In the agricultural and environmental fields, approximately 1500 bacterial isolates have been collected, molecularly, biochemically, and phenotypically identified. These strains have been also characterized for the activities such as promoting plant growth, antagonizing phytopathogenic fungi, nitrogen fixation, phosphorus solubilization, siderophore production, with potential applications in agriculture as biocontrol agents and/or biostimulants as a replacement for chemical fertilizers. In the food sector, bacteria and fungi have been isolated from food productions and characterized as biomarkers for quality and microbiological safety. Others microbial strains from almost extreme (contaminated and degraded) and extreme (Antartide) environments have been identified and characterized for metabolic profile and biotechnological functions in the field of bioremediation, biorestoration and nutraceuticals. The collection also includes strains useful for biotechnological purposes, such as cellulose degradation, and strains isolated from marine environments resistant to low temperatures. Furthermore, strains of freshwater and marine microalgae useful in the bio-based molecules production in the nutraceutical, cosmeceutical, and pharmaceutical sectors have been identified. Microbial strains and consortia, utilized for converting various biomass into liquid and/or gaseous biofuels or products of interest to the chemical industry, and microalgae cultures for the production of energy and/or green chemistry products have been also identified.

The microbial collection, managed by ENEA's Casaccia, Trisaia, and Portici laboratories (in house collection), encompasses numerous microorganisms from environments where the industrial revolution of the last two centuries and modern agricultural management have strongly influenced microbial biodiversity: many species have been negatively affected, while others, in response to biotic and/or abiotic stress, have developed peculiar metabolic characteristics that make them excellent candidates for potential biotechnological exploitation in various sectors. This biodiversity has adapted in a short time, compared with evolutionary time, to compounds that have not co-evolved with microbial communities (xenobiotics). For this reason these microorganisms represent a peculiar experience in evolutionary history. Such microbial diversity deserves to be preserved from its inevitable extinction. In accordance with the Convention on Biodiversity, signed in Rio de Janeiro in June 1992, ex-situ conservation in appropriately managed collections represents the only possibility of preserving this microbial biodiversity, which offers us a mine of still unexplored functions for innovative applications, useful for addressing many of the environmental and health emergencies of the 21st century.

The research activities in agriculture, started in the 1960s at CNEN’s Agricultural Applications Laboratory, led in the 1980s, in the Microbiology laboratory of the Casaccia Research Centre’s Agrobiotechnology Department, to the start of a line of research on soil microbiology, with a particular focus on nitrogen-fixing bacteria. In 1986, work was carried out on the isolation of Rhizobium strains contained in the Italian soils (Rhizobium ciceri and Rhizobium meliloti) capable of nodulating traditionally cultivated legumes (chickpea and alfalfa). The strains were identified with the phenotypic techniques, in use at the time, and characterized for their nitrogen fixation and competition capabilities both in vitro and with tests on plants in the field in view of their possible use as inoculants, leading to the definition of the positive effect of the use of Rhizobium japonicum inoculants. Concurrently, research on Azospirillum spp. priming effects was initiated. Researchers from the ENEA Casaccia Center’s Agronomy laboratory conducted field inoculations with nitrogen-fixing bacteria and plant growth stimulators on Zea mays. In the area relating to the development of the fertility of agricultural soils, a feasibility study was carried out for the construction of a plant in Ferrara (Helizea) for the industrial production of Rhizobium japonicum and other microorganisms of agronomic interest as inoculants for soybean crops and other legumes. This study led to the creation of the first bacterial inoculant production plant in Italy, in view of the further development of soya and other legume crops (https://inis.iaea.org/collection/NCLCollectionStore/_Public/20/013/20013361.pdf). In that period, numerous international collaborations had arisen focused on the study and development of nitrogen-fixing bacteria and plant growth stimulators; among all, the collaboration with prof. Johanna Doebereiner (director of EMBRAPA, Séropedica, Brazil), with prof. Yaacov Okon (Rehovot University, Israel), with prof. Istvan Fendrik of the University of Hannover and with Jos Vanderleyden and Thierry Heulin with whom a close collaboration was established with exchanges of strains and development of European projects.

In the 1980s, the organisation's energy vocation favored activities linked to energy production or the consequences of nuclear applications; following the referendum for the repeal of nuclear power in 1987, the horizons of applications broadened. In recent years, important activities have blossomed which have led to the creation of the current microbial collection. In the field of renewable energy, the first biotechnology patent by ENEA was granted in 1987. It pertained to a biotechnological process involving immobilized yeast cells for continuous bioethanol production from waste in the sugar industry. The process drew interest from Eridania, an industrial player. The fermentation agent used was a yeast strain, Saccharomyces cerevisiae MI810, selected from wines for its high ethanol production capacity and is part of the ENEA collection. During that period, as part of the sub-bituminous coal biolixiviation program with high sulfur content, strains of Thiobacillus ferrooxidans were isolated and an oxidative efficiency mutant was obtained, capable of functioning in extremely acidic environments, as anticipated for the operation of a pre-pilot plant. Research in the field of microbial biotechnology has led to the study of the growth rate of Thiobacillus ferrooxidans in continuous and batch cultures and the determination of parameters necessary to optimize the effectiveness of FeSO₄ oxidation by microorganisms (https://bioagro.sostenibilita.enea.it/pubs/2223). Of particular significance has been the establishment of the experimental plant for bioleaching at the ENEA Casaccia Center.

Starting from the 1990s, with the broadening of the thematic horizons of the organization, research in the field of microbial ecology in agriculture was initiated. This led ENEA researchers to undertake an extensive sampling campaign from the rhizosphere of maize cultivated in the north, central, and southern regions of Italy, as well as in Mexico with the aim of isolation of "Plant Growth Promoting Rhizobacteria" for their use in agriculture as biofertilizers and biocontrol agents against phytopathogens. The campaigns resulted in the collection of over 5000 strains belonging to the Burkholderia cepacia complex (BCC), of which 1500 were molecularly and phenotypically characterized, for their metabolic profile and various functional traits of interest. ENEA's BCC collection represents a valuable international resource and has shed light on the role of microorganisms belonging to the complex in the microbial ecology of agricultural soils, as well as in chronic infections in patients affected by cystic fibrosis (CF). The participation of ENEA researchers in the International Burkholderia cepacia working group (https://ibcwg.org) has led to collaborations with leading experts in taxonomy and molecular typing, including Eshwar Mahenthiralingam (Cardiff University), Peter Vandamme (Ghent University), Cristopher Dowson, and Adam Baldwin (Warwick University). The sequences of BCC strains typed by ENEA researchers using Multi Locus Sequence Typing have become part of the Public databases for molecular typing and microbial genome diversity of the BCC (https://pubmlst.org/organisms/burkholderia-cepacia-complex). ENEA microbiologists, thanks to their expertise in taxonomy and characterization of environmental strains, have led a national-level epidemiological study of clinical strains of the Burkholderia cepacia complex (BCC). They have preserved these strains in their database, originating from various cystic fibrosis regional centers nationwide, and have been the national reference point in the taxonomy of clinical BCC strains.

Starting from the 2000s, other samplings have been carried out within national and international research projects, leading to the isolation of over 1000 microbial strains (of which 200 have been molecularly characterized) from soils subjected to various agricultural management and agronomic practices during isolation campaigns conducted in different seasons, strains were also collected from food matrices and lacustrine environments at various lake depths. Other strains have been collected from food matrices during the monitoring of microbiological shelf-life. In recent years, strains from the ENEA microbial collection equipped with plant growth-promoting activities (phosphorus-solubilizing strains, atmospheric nitrogen fixers, plant growth promoters, hormone and siderophore producers, antagonists of phytopathogens) have been assembled with other beneficial microorganisms from European collections into multifunctional microbial consortia. These consortia have been applied in the field for a more environmentally friendly management of agricultural crops, reducing the need for chemical pesticides and synthetic fertilizers in agriculture, promoting soil fertility and enhancing the health and productivity of plants. ENEA's participation in the Italian Soil Partnership working group and in the national soil research node (Soil-Hub) has further enhanced the microbial collection through collaboration with the national and European soil network with the aim to address and mitigate the impact of climate change on agricultural systems and soil-related ecosystem functions concerning agriculture.

In the same years, the research on strains and microbial consortia with functional characteristics of interest for environmental remediation applications began. Exploration occurred mainly in industrial environments affected by historical contamination. Over time, these conditions shape microbial populations under environmental pressure, fostering the establishment of communities capable of adapting to often prohibitive conditions, such as the presence of high concentrations of organic contaminants, heavy metals, and xenobiotic substances. Microorganisms native to these sites are ideal candidates for bioremediation interventions. Strains were collected from various abandoned mines across Europe and different industrial sites in Italy, as part of national and international projects. More recently, many of these strains, thanks to their unique metabolic characteristics, have found application in the field of Cultural Heritage and are currently employed in bio-restoration interventions, a method of selective bio-cleaning that serves as an alternative to often toxic chemical products. This method allows for the removal of deposits of various origins from works and materials of historical and artistic interest. Since 2013, these applications have involved numerous artworks by renowned artists such as Carracci, Algardi, Balzico, Michelangelo, demonstrating their feasibility in the field. The application in the field of Cultural Heritage has led to the European patent ENEA EP 3046779 B1, of which three strains from the collection, deposited in the DSMZ collection, are an integral part. In recent years, the collection has been enriched with strains from archaeological sites, monuments, and artworks. It currently hosts approximately 900 strains, predominantly bacteria. The strains used for bioremediation and bio-restoration applications belong to risk class 1, as do the strains predominantly found in this in-house collection part.

In 2019, ENEA was invited to join the JRU MIRRI-IT, coordinated by the University of Turin, with which it had signed a Framework Agreement on April 17, 2018, focusing on cooperation, research, and innovation in the fields of environmental sciences, energy, safeguarding cultural heritage, and circular economy. The agreement with the University of Turin enabled researchers not only to address highly complex issues, often from an interdisciplinary perspective but also to share infrastructure and organize seminars, conferences, and dissemination initiatives. In 2019, ENEA became an associated entity of the Joint Research Unit MIRRI-IT (Microbial Resource Research Infrastructure – Italy), established in 2017 with the primary goal of overcoming fragmentation in the availability of resources and services for Microbial Collections. MIRRI-IT (http://www.mirri-it.it/) constitutes the Italian node of the European Research Infrastructure MIRRI (Microbial Resource Research Infrastructure, www.mirri.org), included in the 2016 ESFRI roadmap, representing the largest European research infrastructure in the field of Microbial Collections. The purpose of MIRRI-EU is to create a pan-European infrastructure that ensures the conservation and distribution of microorganisms and their derivatives (appropriately controlled and preserved), along with all associated metadata, to promote knowledge and innovation in both the academic and industrial sectors, with significant implications for biotechnological development.

Following the public notice of the MUR Nr. 0003264 of 12/28/2021 for the presentation of project proposals for "Strengthening and creation of Research Infrastructures" to be financed within the framework of the National Recovery and Resilience Plan - Mission 4 "Education and Research”, Component 2, “From research to business”, ENEA is a partner ( https://www.sus-mirri.it/it/partners/) of the project “Strengthening the Italian MIRRI research infrastructure for bioscience and sustainable bioeconomy” (acronym: SUS-MIRRI.IT), financed by Decree directorial no. 114 of 21 July 2022, within the Investment Line 3.1 "Fund for the creation of an integrated system of research and innovation infrastructures" financed by the European Union - NextGenerationEU" financed by the PNRR which provides support for "the creation of research infrastructures and innovation that connect the industrial sector with the academic one.