The MicroBioDiverSar Project: Exploring the Microbial Biodiversity in Ex Situ Collections of Sardinia

In the last decades, biodiversity preservation has gained growing attention and many strategies, laws and regulations have been enacted by governments with this purpose. The MicroBioDiverSar (MBDS) project, the first one regarding microbiological resources, funded by the Italian Minister of Agricultural, Food and Forestry Policies (Mipaaf) through the Law 194/2015, was aimed at surveying, cataloguing, and managing the microbial resources and the related information of three Sardinian collections (Agris BNSS, Uniss, and Unica). While microorganisms were reordered and inventoried, a federated database, accessible via the web, was designed by the bioinformatician of Ospedale Policlinico San Martino of Genova, according to both international standards and laboratory needs. The resulting MBDS collection boasts a great richness of microbial resources. Indeed, over 21,000 isolates, belonging to over 200 species of bacteria, yeasts, and filamentous fungi isolated from different matrices, mainly food, of animal and vegetable origin, collected in over 50 years, were included in the database. Currently, about 2000 isolates, belonging to 150 species, are available online for both the scientific community and agri-food producers. The huge work done allowed one to know the consistency and the composition of most of the patrimony of the Sardinian microbial collections. Furthermore, the MBDS database has been proposed as a model for other Italian collections that, as the MBDS partners, are part of the Joint Research Unit MIRRI-IT Italian collections network, with the aim of overcoming fragmentation, facing sustainability challenges, and improving the quality of the management of the collections.


Introduction
Agri-food biodiversity includes the variety and variability of animals, plants, and microorganisms that are important for food and agriculture and which are the result of the interactions between the environment, genetic resources, and management systems and practices used by humans [1]. According to what was recently reported by the Food and Agriculture Organization (FAO) in the report 'State of world biodiversity for food and agriculture' [2], the biodiversity at the base of our food systems is declining all over the world; and what is lost-species of plants, animals, and microorganisms-cannot be recovered. In particular, microorganisms play a fundamental functional role in agricultural systems; in fact, they are involved in the production of food (soil fertility, crop nutrition, Research Unit (JRU) MIRRI-IT (http://www.mirri-it.it/ accessed on 24 June 2021) of which Agris, Uniss, and Unica have become associate members, during the development of the MBDS Project. The JRU MIRRI-IT is the Italian node of MIRRI, the pan-European Microbial Resource Research Infrastructure, born with the aim of overcoming the current fragmentation in the availability of resources and services, and enhancing the quality management system of collections, focusing on the needs of stakeholders interested in the biotechnological transfer of the microbial resources. Therefore, the MicroBioDiverSar project, by comparing the partner collections with other Italian ones, characterized by more advanced systems of management and conservation of microorganisms, was a notable growth opportunity for the Sardinian microbial collections.
At the beginning of the Project, within each partner institution, all the information regarding the accessions (e.g., species to which the microorganisms belonged to, their origin, year, and place of collection, and storage methods) were collected. This phase required in-depth documentary research into the paper archives of the host entities, since information on the isolates collected for several decades since the 1960s was not available in digital form, whereas data for the most recent isolates were reported in tabulated spreadsheets, often not homogeneous by structure. In addition, sometimes information on the same isolate was fragmented into different files. Thus, the next step was to create a unique spreadsheet containing information on all the isolates of each collection, following both international rules and the specific internal needs of each laboratory. At the same time, microbial resources have been physically rearranged, codified, and inventoried.
The work carried out highlighted the presence of a great wealth of microbial resources in the Sardinian collections' partners of the project. In fact, about 200 species of bacteria, yeast, and fungi (of which over 150 are already available online) have been included in the database, corresponding to over 21,000 isolates (of which over 2000 are already available online) from different matrices (milk and milk products from sheep, goat and cow; natural starter cultures; rennet paste; lamb digestive tract; table olives and their brines, olive oil; grapes, grape must, wine; beer, wheat, barley, malt; traditional pork sausages; fish intestines, mussels), entered in the collections in over 50 years.
The creation of the federated database is the fundamental tool that, through the web site, has made the list of MBDS collection resources and the related information accessible online, constituting important communication channels for data and microorganisms sharing with the business world that operates in the agri-food sector, and with the scientific community, for study and research purposes.

Database Design and Implementation
In this section, the architectural choices, the software tools, the schema, and the main features of the database mbds_db, implemented in the context of the MicroBioDiverSar project, are presented.

The Database Architecture
Since the involved collections did not yet implement their own database before the project, the first choices were to design one single database schema, to be adopted by all collections, and to implement three distinct databases sharing the schema as well as some reference tables, e.g., for species, geographic locations, growth media. These choices would have allowed all collections to manage their own data autonomously. Since one of the aims of the project was the creation of a common query interface for all catalogues, a centralized and integrated copy of the three databases had to be anyway created for public access.
The overall architecture of the system, then, includes three distinct databases, each implemented and located at the relative collection, and one server, which hosts the centralized integrated database and controls the homogeneities of the reference tables. This simple architecture is viable for the easy and efficient inclusion of further databases, in case more collections would join the project.
The key issue for the success of this common, but distributed, database architecture was then synchronization. Reference tables for common information must be synchronized so that all databases share their values at any time. Moreover, update of information on strains carried out by each collection on its database must be synchronized with the central repository to allow end users to query a constantly up-to-date integrated database.
Regarding the development and production environment, open software was privileged. This led to the adoption of the so-called LAMP environment, which includes Linux as operating system, Apache as web server, MySQL as database management system, and PHP as programming language (hence the acronym LAMP: Linux, Apache, MySQL, PHP). For users willing to implement their database in a Windows environment, we adopted the analogous environment WAMP (where W stays for Windows), and in particular, the Wampserver software (see http://www.wampserver.com/en/ accessed on 24 June 2021). This choice allowed us to have a simple and effective development environment, especially fit for the continuous development that is required in the scientific domain. Moreover, the simplicity of the programming language, joint with the widespread expertise available on both Apache and MySQL, allows users to make simple changes to the applications and the database.

The Database Information and Schema
The information to be included in the database and their format was decided by examining the newly established guidelines for the submission of catalogues to MIRRI-IS, the information system adopted by the Microbial Resource Research Infrastructure (MIRRI), and by comparing it with the needs of the collections participating in the project.
This analysis led to the selection of an original data set which incorporates almost all information defined by MIRRI guidelines, in a proper format for a future submission to the MIRRI-IS, along with many further data that are specific to the needs of the involved collections. It is noteworthy that, due to their different scientific interests (e.g., bacteria against yeasts), not all collections gather the same information for their strains, while the dataset is unique. The full dataset is available from the web site of the MicroBioDiverSar project (http://www.mbds.it/ accessed on 24 June 2021).
This dataset was then analysed from the entity-relationship point of view for its actual implementation in a relational database. The entities of the dataset are summarized in Table 1. The schema of the database is shown in Figure 1. In this figure, section A includes all reference tables, that are shared by the local databases, while section B includes all local tables, whose contents are unique for each collection. Section C includes some additional local tables that are used for managing user access, backup/restore functions, and update of reference tables. The database has been implemented in a relational environment based on the MySQL database management system.

The Features of the Applications: On-Line Integrated Query
Two sets of applications were developed: one for on-line access and query to the integrated database by end users, and one for the actual management of data by the collections.
Access to the integrated database is granted through the website of the MicroBioDi-verSar project. End user authentication is not required. A simple form is available for querying the catalogues by resource type. The main query information is the species of the strain. Moreover, users may query the database by strain number and name, and by substrate and substrate category. An advanced query form is under development.
After the successful execution of a query, the user is faced with the list of retrieved strains that can be printed or exported as an MS Excel file or in PDF. By selecting one of the strain numbers of retrieved strains, the system returns the relative information, grouped in five distinct sections, one each for strain identification, origin, properties, literature, and notes. It is noteworthy that only a subset of strains is included in the query, according to the decision of the involved collection, and that only a subset of available information is included in the output.

The Features of the Applications: Local Database Management
At the collections, access to applications for local database management requires user authentication. Three types of users, having different access rights, have been defined. The administrators can perform all actions, while the curators can manage data, and the other users can only query and see data. Administrators can create as many users as needed. See Table 2 for details on user types and related access rights.
In the following paragraphs, we shortly describe all actions that become available after authentication through the main menu.
Query: Allows all users to query the local database. Two distinct query forms are available at present. The standard query form includes fields for strain number, strain name, project, species, substrate, and substrate category. The advanced query form includes fields for species, substrate, sampling location and date, as well as fields for compound utilization and inhibitors.
Insert: Allows admins and curators to insert new strains in the local database. With this action, only the following information is included in the database: strain number, strain name, project, resource type, species, and strain numbers of the same strain in other collections. Only the resource type and strain name are mandatory. The strain number can be automatically generated by the system, while the species can be specified later, which is useful when the identification has not yet been done. All newly inserted strains are marked as not publicly available.
Update: Allows admins and curators to complete the insertion of data on a strain or update it. The strain must be selected by strain number or strain name. For practical reasons, information is included in distinct sections for data respectively related to strain identity, identification, origin, isolation, properties, inhibitions, utilizations, literature, notes, culture, and storage. An additional section for bacteria properties is also available. Figure 2 reports an example of the application for the management of strain data. It refers to the 'Origin' section of the 'Update' function. Information on strain origin is included in this section and can be inserted or updated according to the needs of the user. Update is supported by autocomplete functions for reference table values, dates, and enumerations.
Duplicate: Allows admins and curators to insert new strains by copying some essential data (e.g., species and origin) from one existing strain. It is useful when many new strains have been collected together, e.g., during a sampling campaign.
Delete: Allows admins and curators to delete strains from the database. Reference lists: Allows admins and curators to see and manage reference lists, while other users may only see and query them. Lists are available for species, habitats, substrates, substrate categories, geographic locations, culture media, chemical compounds, inhibiting compounds, literature, people, and projects (see also Table 1). Figure 3 reports an example of the application for the management of reference tables. It refers to the 'Species' table that includes only those taxons for which at least one strain is present in the databases. The upper list reports existing values. The lower forms allow one to add new taxons from reference databases (namely, Prokaryotic Nomenclature Up-To-Date and Mycobank) and to update or delete one of the existing taxons (e.g., when information on a given species is updated at the reference databases).
Tools: Available to admins only. It allows one to create and restore database backups and to synchronize the local with the federated database by uploading an updated version of the local database to the server. It also allows admin users to check if new versions of the applications are available and to update them.
Options: Allows admins to create and delete users and to define and change their passwords. Each user can autonomously update its password.

The MBDS Microbial Collection
In this section, a description of the three institutions constituting the MBDS Sardinian microbial collection, their history, the main issues, and the microorganisms studied is presented.

BNSS Agris Sardegna Collection
The bacterial collection BNSS was founded in 1967 by Dr. Severino Arrizza and Dr. Antonio Ledda, at the Istituto Zootecnico e Caseario per la Sardegna-Bonassai, Sassari (now merged within Agris Sardegna) [10]. Part of this collection is now included in MBDS, the Sardinian collection established with the project MicroBioDiverSar.
The acquisition of biological material has been done throughout several decades by the isolation of bacteria from different samples, mainly Sardinian PDO and traditional fermented foods. The first group of isolates originated by studies on the use of natural starter cultures (scotta-innesto) in Pecorino Romano cheese production for improving its quality and reducing production losses [11]. In those years, several scotta-innesto cultures were freeze-dried in toto, allowing the preservation of autochthonous strains and their balances within the microbiota before the wide use of selected starters. The collection has been enriched over the years thanks to several projects financed by the Autonomous Region of Sardinia, the Italian Ministry of Agriculture (Mipaaf) and the European Union.
The most common isolation sources are represented by food of animal origin, but in the last years, isolates were taken also from vegetal food; a smaller number of isolates has animal origin (lamb digestive tract, ewe faeces, fish gut). Sheep and goat milk, fermented milk and cheeses, natural starter cultures, traditional Sardinian sausages, natural fermented olives are the main food from which isolates originate. Most of them are PDO or traditional products such as Pecorino Romano PDO cheese [20], Fiore Sardo PDO cheese [21][22][23]], Pecorino Sardo PDO cheese [24,25], other ewe or goat milk products, i.e., Casu axedu, Fresa, Gioddu [26], homemade natural fermented sausage [27], table olives in brine [28,29].
Moreover, the collection contains even lyophilised natural starter cultures not yet included in the online catalogue, although some of them have already been characterized for their genotypic (species and biotypes composition) [30] and phenotypic characteristics [31]. Particularly, their acidifying profile and safety (antibiotic resistance) were investigated, and they were successfully used in some dairy plants for experimental cheese making of Pecorino Romano within an innovation and technology transfer project.
Out of the 5762 identified isolates, 1756 are included in the catalogue available on the MicroBioDiverSar project website http//www.mbds.it/ (accessed on 24 June 2021) for the stakeholders of the productive sector or the scientific community.
Lactiplantibacillus pentosus were collected from natural table olives, both as microbial communities and single isolates, and, after technological characterisation, were used as a starter in experimental and artisanal productions [29]. Latilactobacillus sakei, Latilactobacillus curvatus, and Staphylococcus equorum come from homemade Sardinian sausages, representing the characteristic bacterial microbiota of this kind of products. Isolates coming from fish (Sparus aurata) gut belong to Pseudomonas spp., Sphingomonas paucimobilis, Aeromonas salmonicida, Erwinia persicina, Yersinia bercovieri, Psychrobacter maritimus: some Pseudomonas spp. and Sphingomonas paucimobilis are biosurfatants producers [32] (Table 3).  Some of the isolates of the collection have been tested for technological properties such as acidifying, lipolytic and proteolytic capacity, fermentative profile, production of flavours, and for some safety (antibiotic susceptibility, virulence genetic determinants, bacteriocin production) and probiotic (low pH and bile salt resistance) characteristics [11,21,[33][34][35].

Uniss Collection
The pioneering work of Prof. Augusto Capriotti of the University of Sassari has laid the foundations, since the 1960s, for the creation of a microbial collection consisting of yeasts, bacteria, and fungi isolated from different local food and environmental matrices. This collection has been enriched over the course of 60 years thanks to studies conducted by researchers from the Institute of General and Applied Microbiology of the Department of Agriculture. This collection has been enriched over the course of 60 years thanks to studies conducted by researchers from the Institute of General and Applied Microbiology of the Department of Agriculture. Recently, this historical collection together with that of the Institute of Plant Pathology of the same department was formed in the UNISS collection and became part of the MIRRI-IT (Microbial Resource Research Infrastructure, Italy) collection network. Currently, the microbial isolates are stored at −80 • C and are going to be freeze-dried. The list of genera and species of microorganisms hosted in the UNISS collection is reported in Table 4.  The microbial collection of the Institute of General and Applied Microbiology (MGA-UNISS) is strongly characterized by the presence of wine yeasts which, from 1967 until today, has been implemented thanks to extensive campaigns of isolation and physiological and technological characterization of the isolates. The main technological characteristics taken into consideration have been the fermentative vigour, resistance to SO 2 and pesticides, the ability to adhere to surfaces and form biofilms [36,37], the killer factor [38,39], the ability to produce or reduce malic acid, the synthesis of sulphur products and acetaldehyde. Among wine yeasts, the flor yeasts isolated over 50 years from wines aged under flor are of particular interest and constitute a heritage of microbial biodiversity unique in Sardinia. These yeasts, ascribed to the Saccharomyces cerevisiae species, come largely from musts and wines sampled in the cellars operating in the Vernaccia DOC area of Sardinia (Italy) and are of biotechnological interest in many fields [40,41]. In this aspect, the microbial collection of MGA-UNISS harbours one of the few collections of flor yeasts in the world. The researchers of the yeast group of MBDS are also part of the Italian Oenological Microbiology group which actively works within the International Organization of Vine and Wine, as well as being correspondents of the Academy of Italian Vine and Wine. Eighty-six yeasts and bacteria species of the MGA-UNISS collection, genetically and phenotypically characterized, are included in the MBDS catalogue, starting from the first isolations dating back to 1965 until today. These are mainly yeasts isolated from food and environmental matrices such as olives and olive oil, milk and cheeses, grapes, wine and must, fish, shellfish, meat, soil, and by-products of beer processing such as brewers' spent grain and wastewater [42][43][44]. As for bacteria, since 2012, 91 strains belonging to 36 species have been isolated, genetically characterized, and divided into 50 different biochemical profiles. The bacteria present in the collection are almost entirely of Sardinian origin and derive from five different substrates: olive oil, grape must, wheat, barley, and malt [45][46][47].
The microbial collection of the Institute of Plant Pathology regards specifically filamentous fungi and consists of 86 strains belonging to 15 different species of Fusarium, isolated from uncultivated lands in Sardinia and from plants of agricultural interest (wheat, rice, corn, tomato) between 2001 and 2018. The identification of the different species was carried out starting from monosporic cultures, through the observation of the morphological characteristics, and in many cases, also with the analysis of the Elongation Translation Factor (EF-1-α). Thirty isolates are also deposited in the USDA Northern Regional Research Laboratory (NRRL) culture collection in Peoria, IL, USA.
Many of the microorganisms preserved in the UNISS collection have been selected on the basis their biotechnological potential and can therefore be used as selected starters. Selected starters consist of pure cultures of microorganisms, selected for their technological activities, to be inoculated for the fermentation of matrices that are not always the same as those from which they have been isolated [48]. Microbial isolates of the UNISS collection have been selected for the transformation of food matrices (beer, wine, dairy products, olive, etc.) and industrial by-products (brewers spent grains), as well as for the bioremediation and fertilization of soils. As for the different Fusarium spp. isolates, they can be used for studies on pathogenicity tests on plants of agricultural interest or for the evaluation of the effectiveness of chemical compounds, natural or biological microorganisms, in containing the damage caused by alterative microorganisms (Table 4). An important aspect to consider, strengthening the link between biodiversity preservation and production of local foods with geographical indication, is that microbial collections offer to local producers actions such as isolation, selection, storage, multiplication, production and sale of yeast, and bacterial starters. In the perspective of a circular economy, the UNISS microbial collection will also offer different services for the agri-food and agro-environmental sector, from the production and optimization of microbial starters to waste management and assessment of the quality and safety of fermented products. In addition, the UNISS collection will provide applicative protocols for: (i) bioremediation (bioprotectors, biostimulants, and biofertilizers); (ii) monitoring of microbial processes in the food, environmental, and agricultural fields; (iii) product quality and safety certification. To do that, UNISS will promote the constitution of a spin-off within the framework of a systemic approach to agrifood R&D, as an effective missing link in the technological transfer of food biotechnologies. This is an opportunity for small and medium-sized companies to combine tradition and technologies that can help make fermentation processes manageable without the negative standardization often associated with large-scale industrial production.

Unica Collection
The UNICA microbial collection, started in 1985 by Prof. Francesca Palmas, head of the Hygiene section of the Department of Experimental Biology, at present, belongs to the Department of Medical Sciences and Public Health (DSMSP) of the University of Cagliari. Over the course of the years, several UNICA researchers have isolated and preserved more than 2000 microorganisms with considerable application potential in the agri-food, health, and environmental fields. Among the identified isolates, the most represented genera are the former Lactobacillus and Enterococcus for bacteria, Candida and Debaryomyces for yeasts, and Penicillium and Aspergillus for moulds.
Part of the UNICA-DSMSP microbial collection is now included in MBDS and presently consists of 655 strains comprising bacteria, mainly represented by lactic acid bacteria (belonging to 12 genera and 22 species) and yeasts (15 genera and 31 species), as indicated in Table 5  The strains of the UNICA-DSMSP collection have been isolated from different food matrices, mainly Sardinian dairy products (sheep and goat milk, typical products such as Fiore Sardo, Casu axedu, Pecorino Sardo, Ricotta) but also from honey, mussels, fresh pasta, and fermented meat products [49][50][51][52][53][54][55][56][57][58]. The UNICA-DSMSP microbial collection represents a precious source of resources capable of fostering innovation in the regional agri-food industry and contributing to the qualitative and hygienic-sanitary improvement of the various products, as well as being a tool to protect and enhance microbial biodiversity.
Many strains of autochthonous lactic acid bacteria and yeasts of the UNICA-DSMSP collection have been characterized at a taxonomic, biochemical, molecular, technological, and functional level for their potential use in the food industry.
As summarized in Table 6, the strains have been phenotypically identified based on several taxonomic and biochemical features, and the identification was confirmed by species specific PCR and or by sequencing [59][60][61][62]. The isolates have been also subjected to technological tests relevant for the production of a wide range of fermented foods (production of aromatic substances, acidifying, proteolytic, and lipolytic activity). Several strains were molecularly characterized with the PCR-RAPD (Random Amplified Polymorphic DNA) or PCR-RFLP (Restriction Fragment Length Polymorphism) techniques. It is well known that autochthonous microorganisms, beside conferring typicality and quality to foods, can also be endowed with functional properties of probiotic interest and therefore useful for improving consumer's health [63]. To this end, important traits such as the ability to survive the passage in the digestive tract (with the SSDP: Simulated Stomach-Duodenum Passage test), the stability at extreme pH values and high concentrations of bile salts (essential for the probiotic to reach the intestine in an optimal condition), the ability to adhere to intestinal cells (which represents both the first step in the colonization process and one of the mechanisms by which probiotics exert their action), the ability to antagonize pathogenic and/or spoilage microorganisms through the production of different substances with antimicrobial action (organic acids, hydrogen peroxide, carbon dioxide, bacteriocins) were evaluated [64][65][66][67][68][69][70][71]. The technological potential and biodiversity of the microorganisms present in the UNICA-DSMSP microbial collection can be exploited for different purposes. Over the years, numerous strains have been used in research projects aimed at: (i) their use as fermentation agents (autochthonous starter and/or integrative cultures) with the purpose of obtaining foods with unique qualitative characteristics. The isolated strains were characterized, selected, and used in pilot experiments with subsequent organoleptic, chemical-physical, and microbiological evaluation of the finished products [72][73][74]; (ii) their use as bioprotective culture, based on the ability of some strains to inhibit the development of pathogenic and alterative microorganisms, in order to increase the safety and shelf-life of the products and ensure greater sustainability of the production process [75][76][77]; (iii) the development of innovative/healthy products for new market segments. In recent years, the production of innovative functional foods that represent a driving force for the territory, through the enhancement of typical local products, has emerged among the research priorities in the agri-food field [78]. Our studies have highlighted the presence, in typical artisanal products, of lactic bacteria and yeasts with functional properties of probiotic interest, and some of these have been used successfully in the production of cheeses [65,68,73].

Conclusions
The need to improve the sustainability of crop cultivation and food production is becoming a prominent factor for the next generation industries. Particularly, the use of microbial inoculants with specific metabolic activities represents a feasible and eco-friendly approach to increase crop yields and quality, restore soil fertility, control pathogenic or spoilage organisms, and recycle and dispose of wastes. In this context, microbial collections play a fundamental role as they promote the isolation, characterization, and preservation of such microorganisms, thus enlarging the spectra of potentially useful biotechnological applications for the safeguard of the environment and the sustainability of food production. The work carried in the MicroBioDiverSar Project allowed one to reach some important goals, such as having a fairly precise overview of the consistence of the resources hosted in the ex-situ Sardinian microbial collection (MBDS), create a federated database, based on international standards, for the microorganisms' information storage and management, that is now being used as a template for other Italian collection belonging to the JRU MIRRI-IT, and to build a dedicated website. For the results of the project to continue to have a strategic significance and a concrete value over time, additional implementing decrees of regional and national laws must be issued, and structural funds should be specifically dedicated to microbial resources, thereby ensuring the sustainability of microbial collections and the survival, maintaining, and managing of microorganisms in accordance with internationally recognised quality standards. That is how the collections can aspire to attain biobank status and play a key role for the development of local and global biotechnology, bioeconomy, and social welfare in a sustainable way.

Conflicts of Interest:
The authors declare no conflict of interest.