ATINER CONFERENCE PAPER SERIES No: LNG2014-1176 Athens Institute for Education and Research ATINER ATINER's Conference Paper Series WAT2018- 2517 Use of Molecular Techniques for Identification of Nitrate Contamination Sources in Groundwater Angelantonio Calabrese Technologist National Research Council, Water Research Institute (IRSA) Italy Vito Felice Uricchio Technologist National Research Council, Water Research Institute (IRSA) Italy Massimo Blonda Researcher National Research Council, Water Research Institute (IRSA) Italy Claudia Campanale Phd Student National Research Council, Water Research Institute (IRSA) Italy 1 ATINER CONFERENCE PAPER SERIES No: WAT2018-2517 An Introduction to ATINER's Conference Paper Series Conference papers are research/policy papers written and presented by academics at one of ATINER’s academic events. ATINER’s association started to publish this conference paper series in 2012. All published conference papers go through an initial peer review aiming at disseminating and improving the ideas expressed in each work. Authors welcome comments. Dr. Gregory T. Papanikos President Athens Institute for Education and Research This paper should be cited as follows: Calabrese, A., Uricchio, V. F., Blonda, M. and Campanele, C. (2018). “Use of Molecular Techniques for Identification of Nitrate Contamination Sources in Groundwater”, Athens: ATINER'S Conference Paper Series, No: WAT2018- 2517. Athens Institute for Education and Research 8 Valaoritou Street, Kolonaki, 10671 Athens, Greece Tel: + 30 210 3634210 Fax: + 30 210 3634209 Email: [email protected] URL: www.atiner.gr URL Conference Papers Series: www.atiner.gr/papers.htm Printed in Athens, Greece by the Athens Institute for Education and Research. All rights reserved. Reproduction is allowed for non-commercial purposes if the source is fully acknowledged. ISSN: 2241-2891 05/09/2018 2 ATINER CONFERENCE PAPER SERIES No: WAT2018-2517 Use of Molecular Techniques for Identification of Nitrate Contamination Sources in Groundwater Angelantonio Calabrese Vito Felice Uricchio Massimo Blonda Claudia Campanale Abstract The groundwater ecosystems are regulated by the limiting environmental factors (absence of light, availability of nourishing, low temperatures, etc.) that select microbial communities well-fitting conditions of “extreme” life. The microbial communities in the groundwater are mainly constituted by well adapted heterotrophy and they are characterized by the state of hydrological chemistry and geologic heterogeneity of the stratum. The microbial communities of the groundwater develop according to the typology of contamination (point or diffused) and the category of contaminants as well. The "classical methods" procedures of microbial community analysis provide for the identification of microorganisms from pure culture isolation. These analyzes are not sufficient to identify most species of microorganisms and are limited to cultivable species which represent a very small percentage of all species present in nature. Research on environmental microbiology has shown that microbial communities play a functional role in controlling ecosystems, which cannot be attributed to individual species. New methodologies have been developed that enable to analyze the structure of microbial communities independently of the isolation phase. These work illustrated a pilot action carried out to identify potentially contaminating sources of nitrate in the ground on the whole Apulian territory (livestock effluents, urban wastewater, sewage sludge), by the correlation between certain sources of contamination and specific bacterial species. Keywords: Bacteria Investigation, Biomolecular Technique, Groundwater, Nitrate Contamination. Acknowledgments: The authors wish to thank the technical staff of the Defense Consortia of each province of the Puglia Region for the considerable technical support provided in the planning and implementation of the inspection and sampling operations envisaged in this pilot action. A special thanks to the Apulia Region for the logistical support and for the financial support given to conduct the activities. 3 ATINER CONFERENCE PAPER SERIES No: WAT2018-2517 Introduction The groundwater ecosystems are limited by the presence of environmental factors (absence of light, limited availability of nourishing, low temperatures, etc.) that condition the existence of peculiar microbial community, sometime in conditions of life so called "extreme." Today it is known that the whole system of the subsoil is colonized by the microorganisms, mainly present in microbial community, constituted by bacteria and Archaea, but also from protozoa and fungi, that develop remarkable roles in the trials biogeochemical. The microbial communities in the groundwater are mainly constituted by well adapted heterotrophy to the underground environment and they are characterizing by hydrological, chemistry and geologic of the stratum. The presence of different biotic and abiotic factors can influence in direct or indirect way the microbial difference in the ecosystems; it is generally characterized by a typical stable bacterial community that is directly influenced by the chemical-physical parameters. It is influenced from the spatial heterogeneity of the geologic unity, from the pressures anthropic on the same one, from the nature of the layer of impending ground the stratum, from the biogeography and from the use of the superficial soil. The stability of the bacterial communities is interrupted if there are spatial variations of the groundwater chemical-physical parameters, caused by a superficial contamination. Such change can determine three different dynamics of transformation of the micro biotic communities present: (i) quantitative variation of already determined present bacterial fetterless in them; (ii) inclusion of new alien bacterial fetterless; (iii) disappearance of some bacterial fetterless - cause the presence of toxic substances - with possible occupation of the ecological site of a new alien microorganism. Besides, in base to the typology of contamination (point or diffused) and the class of contaminants different impacts are had on the microbial communities of the groundwater. The "standard" procedures of analysis of the microbial communities, or "classical methods", they foresee the identification of the microorganisms through the isolation of pure crops, followed by test that they analyze some morph-physiological and biochemical characteristics. These analyses are not enough for the identification of the greatest part of the microorganisms and they are limited to the cultivable species that represent a minimum percentage of all the species present in nature. These tests, introduce besides the serious limit to ask for a notable waste of time, and they economically result often onerous. During the last decades, the searches in the field of the environmental microbiology have shown that microbial communities develop a functional role of control of the ecosystems that is not referable to single species. For these reasons, new methodologies have been developed. One of the most important technic is the biomolecular analysis that is based on the individualization of a specific genomics region of the microorganism DNA. With the development of the DNA analysis it has been reached levels of sensibility ever gotten with the traditional other techniques of survey. These methods are usually rapid and they can be applied for the search both of specific pathogens that of groups of microorganisms. The identification of the present species in the environmental matrix and of the relative 4 ATINER CONFERENCE PAPER SERIES No: WAT2018-2517 DNA allows a direct correlation of the possible contamination with the sources to it connected, also supporting studies of molecular epidemiology and phylogenetic analysis. Molecular diagnosis is generally more sensitive and more specific to traditional culture methods and requires shorter time for identification. The gene sequencing of amplified fragments also allows the identification of the present bacterial species. With specific reference to the definition of the source of nitrate contamination, the identification of the microbiological species present in the groundwater allows to identify the source of contamination. Several studies have shown that certain sources of contamination are related to well-defined bacterial species, in particular anthropogenic contamination can be identified by identification of BIFIDOBACTERIUM (Barrett et al., 2002) and by ENTEROCOCCUS FAECALIS AND FACIUM (Boccia et al., 2002; Eigner et al., 2008), or contamination due to incorrect spillage or manure accumulation can be detected by identifying BACTEROIDES-PREVOTELLA, ENTEROCOCCUS AVIUM, ENTEROCOCCUS CASSELIFLAVUS, ENTEROCOCCUS DURANS, ENTEROCOCCUS GALLINARUM, ENTEROCOCCUS HIRAE, ENTEROCOCCUS SACCHAROLYTICUS (Savichtcheva and Okabe, 2006). For this purpose, a pilot action has been carried out to identify potentially contaminating sources of nitrate in the groundwater on the whole Apulian territory, distinguishing the origin of nitrate from livestock effluents, urban wastewater, sewage sludge. Consequently, the priority objective of this action was to identify with greater certainty the causes of nitrate contamination by distinguishing the agricultural, livestock or civil source. With specific reference to the definition of the source of nitrate contamination, the identification of the species present in the environmental matrix (water) of its nucleotide sequences