Antonio Bucci

Role of organic matter and clay fraction on migration of Escherichia coli cells through pyroclastic soils, southern Italy

A comparative study on the adsorption of Escherichia coli cells to two different pyroclastic soils collected in southern Italy (carbonate Apennines) was performed in laboratory using surfactant-free solutions and solutions with the surfactants sodium dodecyl sulphate (SDS, anionic) and octylphenoxypolyethoxyethanol (Triton X-100, non-ionic). Both soils are rich in organic matter (up to 35%), but only one contains a clay fraction (2-5%). The experiments demonstrated that E. coli cells are significantly adsorbed to the clay fraction of the soil, while the organic matter content does not play a significant role. The pore size exclusion phenomenon is another factor to consider when analyzing the retention of E. coli cells within such soils. However, despite the existence of different factors that enhance bacterial cells retention, a high percent of E. coli cells is transported through soil media. The not absolute protection of such soils against microbial pollution is supported not only by the results of the column experiments at lab scale, but also by the findings of a field monitoring at site scale.

Winter survival of microbial contaminants in soil：An in situ verification

The aim of the research was to evaluate, at site scale, the influence of freezing and freeze/thaw cycles on the survival of faecal coliforms and faecal enterococci in soil, in a climate change perspective. Before the winter period and during grazing, viable cells of faecal coliforms and faecal enterococci were detected only in the first 10 cm below ground, while, after the winter period and before the new seasonal grazing, a lower number of viable cells of both faecal indicators was detected only in some of the investigated soil profiles, and within the first 5 cm. Taking into consideration the results of specific investigations, we hypothesise that the non-uniform spatial distribution of grass roots within the studied soil can play an important role in influencing this phenomenon, while several abiotic factors do not play any significant role. Taking into account the local trend in the increase of air temperature, a different distribution of microbial pollution over time is expected in spring waters, in future climate scenarios. The progressive increase in air temperature will cause a progressive decrease in freeze/thaw cycles at higher altitudes, minimising cold shocks on microbial cells, and causing spring water pollution also during winter.

Microorganisms as contaminants and natural tracers: a 10-year research in some carbonate aquifers (southern Italy)

Limestone aquifers provide the main drinking water resources of southern Italy. Due to cattle grazing and/or manure spreading, these aquifers are often characterized by microbial contamination of groundwater. The aim of this paper is to summarize the results obtained during a 10-year research carried out in experimental field sites in southern Italy, analyzing (1) the influence of the topsoil of pyroclastic origin on the migration of microbial cells from the ground towards the groundwater, and then on the groundwater vulnerability, (2) the influence of the rainfall regime on the breakthrough at the springs, (3) the reliability of thermotolerant coliforms and fecal enterococci as bacterial indicators of microbial contamination, and (4) the effectiveness of microorganisms as natural tracers for some hydrogeological purposes. The results obtained showed that fecal enterococci are a more reliable indicator than thermotolerant coliforms for detecting contamination and that the entity and distribution over time of microbial contamination of fecal origin are influenced by several factors, such as precipitation regime, thermal regime and existence and thickness of the topsoil of pyroclastic origin. Moreover, the migration of a significant amount of bacterial cells through the topsoil and the underlying carbonate rocks allows the utilization of microorganisms as effective natural tracers, to be coupled with other classic tracers to study the recharge and the flow processes. In a broader perspective, these results can be used to optimize the investigations in other hydrogeological scenarios, with emphasis on those where different water types coexist and interact in same aquifer systems.

Bacterial migration through low-permeability fault zones in compartmentalised aquifer systems: a case study in Southern Italy

*emma.petrella@unimol.it Microbial contamination is frequently detected at different springs discharging within this type of aquifer system (Celico et al., 2004; Naclerio et al., 2008, 2009) and in carbonate environments in general (e.g., Personné et al., 1998; Pronk et al., 2006; Flynn & Sinreich, 2010). Nevertheless, no information is available concerning the role low-permeability fault zones play in the migration of microorganisms between adjacent compartments. Whether a fault zone will act as a conduit, barrier, or combined conduit-barrier system is controlled by the relative percentage of fault core and damage zone structures (e.g., in carbonates, Mollema & Antonellini, 1999; Salvini et al., 1999; Billi et al., 2003; Billi, 2005a; Agosta & Aydin, 2006; Billi et al., 2007; Balsamo et al., 2008; Storti & Balsamo, 2010). A fault zone can impede fluid flow when the fault core is well developed (e.g., Antonellini & Aydin, 1994; Newman & Mitra, 1994; Goddard & Evans, 1995; Caine et al., 1996; Bense et al., 2003; Fairley & Hinds, 2004). Fault cores can include single slip surfaces (Caine et al., 1991), highly indurated, INTRODUCTION

Use of molecular approaches in hydrogeological studies: the case of carbonate aquifers in southern Italy

Waterborne pathogens represent a significant health risk in both developed and developing countries with sensitive sub-populations including children, the elderly, neonates, and immune-compromised people, who are particularly susceptible to enteric infections. Annually, approximately 1.8 billion people utilize a faecally contaminated water source, and waterborne diseases are resulting in up to 2.1 million human mortalities globally. Although groundwater has traditionally been considered less susceptible to contamination by enteric pathogens than surface water due to natural attenuation by overlying strata, the degree of microbial removal attributable to soils and aquifers can vary significantly depending on several factors. Thus, accurate assessment of the variable presence and concentration of microbial contaminants, and the relative importance of potentially causative factors affecting contaminant ingress, is critical in order to develop effective source (well) and resource (aquifer) protection strategies. “Traditional” and molecular microbiological study designs, when coupled with hydrogeological, hydrochemical, isotopic, and geophysical methods, have proven useful for analysis of numerous aspects of subsurface microbial dynamics. Accordingly, this overview paper presents the principal microbial techniques currently being employed (1) to predict and identify sources of faecal contamination in groundwater, (2) to elucidate the dynamics of contaminant migration, and (3) to refine knowledge about the hydrogeological characteristics and behaviours of aquifer systems affected by microbial contamination with an emphasis on carbonate aquifers, which represent an important global water supply. Previous investigations carried out in carbonate aquifers in southern Italy are discussed.RésuméLes agents pathogènes d’origine hydrique représentent un risqué important pour la santé aussi bien dans les pays développés que dans les pays en développement avec des sous-populations sensibles notamment les enfants, les personnes âgées, les nouveaux nés, et les personnes immunodéprimées, qui sont particulièrement sensibles aux infections entériques. Chaque année, environ 1.8 milliard de personnes utilisent une source d’eau contaminée par des déchets fécaux, et les maladies transmises par l’eau entrainent jusqu’à 2.1 millions de décès de personnes dans le monde. Bien que les eaux souterraines aient traditionnellement été considérées comme moins sensibles à la contamination par les pathogènes entériques que les eaux de surface en raison de l’atténuation naturelle lors de l’infiltration au sein des couches géologiques supérieures, le degré d’élimination microbienne attribuable au sol et aux aquifères peut varier de manière significative selon plusieurs facteurs. Ainsi, une évaluation précise de la présence et de la concentration variable des contaminants microbiens, et de l’importance relative des potentiels facteurs de causalité qui influent sur la pénétration des contaminants, est essentielle pour développer des stratégies efficaces de protection des sources (puits) et des ressources (aquifères). Les méthodologies « traditionnelles » et de microbiologie moléculaire, associées aux méthodes hydrogéologiques, hydrochimiques, isotopiques, et géophysiques, se sont révélées utiles pour l’analyse de nombreux aspects de la dynamique microbienne souterraine. Par conséquent, cet article présente les principales techniques microbiennes actuellement utilisées (1) pour prédire et identifier les sources de contamination fécale dans les eaux souterraines, (2) pour élucider la dynamique de migration des contaminants, et (3) pour affiner les connaissances relatives aux caractéristiques hydrogéologiques et aux comportements des systèmes aquifères affecté par une contamination microbienne, en mettant l’accent sur les aquifères carbonatés, qui représentent une importante source d’approvisionnement en eau potable à l’échelle mondiale. Des recherches antérieures réalisées dans les aquifères carbonatés du Sud de l’Italie sont discutées.ResumenLos patógenos transmitidos por el agua representan un riesgo significativo para la salud en países desarrollados y en desarrollo con subpoblaciones sensibles que incluyen niños, ancianos, neonatos y personas inmunocomprometidas, que son particularmente susceptibles a las infecciones entéricas. Anualmente, aproximadamente 1.8 billones de personas utilizan una fuente de agua contaminada por contaminantes fecales, y las enfermedades transmitidas por el agua están causando hasta 2.1 millones de muertes humanas en todo el mundo. Aunque el agua subterránea ha sido tradicionalmente considerada menos susceptible que el agua superficial a la contaminación por patógenos entéricos debido a la atenuación natural por los estratos suprayacentes, el grado de eliminación microbiana atribuible a los suelos y acuíferos puede variar significativamente dependiendo de varios factores. Por lo tanto, una evaluación precisa de la presencia y la concentración variable de contaminantes microbianos, y la importancia relativa de factores potencialmente causales que influyen en la entrada de contaminantes, es crítica para desarrollar estrategias efectivas de protección de fuentes (pozos) y recursos (acuíferos). Los diseños de estudios microbiológicos “tradicionales” y moleculares, junto con métodos hidrogeológicos, hidroquímicos, isotópicos y geofísicos, han demostrado ser útiles para el análisis de numerosos aspectos de la dinámica microbiana subterránea. En este trabajo se presentan las principales técnicas microbianas utilizadas actualmente (1) para predecir e identificar fuentes de contaminación fecal en agua subterránea, (2) para dilucidar la dinámica de la migración de contaminantes, y (3) para refinar el conocimiento sobre las características y comportamientos hidrogeológicos de los sistema acuíferos afectados por la contaminación microbiana con un énfasis en los acuíferos carbonáticos, que representan un importante suministro de agua a nivel mundial. Se discuten investigaciones previas realizadas en acuíferos carbonáticos en el sur de Italia.摘要水生病原体在发达国家和发展中国家对特别容易受到肠道感染的敏感亚群体包括儿童、老人、新生儿以及缺乏免疫的人群具有很大的健康风险。每年大约18亿人在使用粪便污染的水源,每年水传播疾病导致全球210万人死亡。尽管传统上认为,由于上伏地层的自然稀释,地下水与地表水相比不容易受到污染,但土壤和含水层中的微生物消除程度变化非常大,取决于好几个因素。因此,精确评价微生物污染物存在的变化情况及其含量、以及影响污染物进入的潜在因素的相对重要性至关重要,为的就是提出有效的水源(井)和资源(含水层)保护策略。“传统的”和分子微生物研究设计与水文地质、水化学、同位素以及地球物理方法结合一起时,就能证明对于分析地表之下微生物动力学的众多方面非常有用。因此,本篇综述性文章展示了目前采用的主要微生物技术:(1)预测和确认地下水中的粪便污染源;(2)阐明污染物运移的动力学;(3)提炼受到微生物污染的含水层系统水文地质特征和习性方面的知识,重点放在代表全球重要供水水源的碳酸盐岩含水层上。还论述了先前在意大利南部对碳酸盐岩含水层进行的调查。ResumoPatógenos transmitidos pela água representam um risco à saúde significante tanto em países desenvolvidos quanto em desenvolvimento com subpopulações sensíveis incluindo crianças, idosos, neonatos, e pessoas com imunidade comprometida, que são particularmente susceptíveis a infecções entéricas. Anualmente, aproximadamente 1.8 bilhão de pessoas utilizam fontes de água contaminadas por organismos fecais, e doenças de vinculação hídrica tem resultado em mais de 2.1 milhões de mortes globalmente. Apesar das águas subterrâneas terem sido consideradas tradicionalmente menos susceptíveis à contaminação por patógenos entéricos que as águas superficiais devido à atenuação natural dos estratos de cobertura, o grau de remoção microbiana atribuível ao solo e aos aquíferos pode variar significativamente dependendo de diversos fatores. Consequentemente, a avaliação precisa da presença variável e da concentração de contaminantes microbianos, e a importância relativa de fatores potencialmente causais que afetam a entrada de contaminantes, é crítica para desenvolver estratégias eficazes de proteção de fontes (poço) e recursos (aquíferos). Os projetos de estudos microbiológicos “tradicionais” e moleculares, quando combinados com métodos hidrogeológicos, hidroquímicos, isotópicos e geofísicos, têm se mostrado úteis para a análise de vários aspectos da dinâmica microbiana subterrânea. Assim, este artigo de síntese apresenta as principais técnicas microbianas atualmente utilizadas (1) para prever e identificar fontes de contaminação fecal em águas subterrâneas, (2) para elucidar a dinâmica de migração de contaminantes, e (3) para aperfeiçoar o conhecimento sobre as características e comportamentos hidrogeológicos de sistemas aquíferos afetados pela contaminação microbiana, com ênfase nos aquíferos carbonáticos, que representam um importante suprimento de água global. Investigações anteriores realizadas em aquíferos de carbonatos no sul da Itália são discutidas.

Influence of soil on groundwater geochemistry in a carbonate aquifer, southern Italy

The role of soil compositions in influencing groundwater geochemistry in carbonate aquifers is still little known. Nothing is known regarding the influence of pyroclastic soils (andisol) within the carbonate Apennines in central-southern Italy, despite their wide distribution. In this study we analyze some physical and chemical properties of pyroclastic soil at the Acqua dei Faggi experimental site (southern Italy), to assess its influence on groundwater geochemistry. Chemical analyses were carried out on saturated paste extracts and a physical analogue model was developed through two column experiments. Physico-chemical properties of rainwater and spring water, and some microbiological features of the soil medium were also taken into consideration. The studied soil has a great influence in modifying rainwater chemistry during percolation. About the 50% of HCO 3 and Ca 2+ in spring water is due to interaction between percolation water and soil medium, and equilibrium with calcite is reached at this stage. The Na + /K + ratio is buffered by clay minerals in the soil by primary silicates in the pyroclastic cover. Cl and SO 4 2concentrations in spring water are very close to that of soil infiltration water during short-term interaction with soil, but a decline is showed during long-term cause to the anions adsorption effect in the andisol. Chemical and microbiological investigations show the existence of a soil microbial community that allows denitrification and nitrate reduction. Infiltration processes cause anoxic conditions within the soil medium, therefore the absence of NH 4 + in spring water throughout the observation period should be due to anammox processes. These findings suggest that hydrochemistry and spring chemographs may be significantly influenced by several factors, such as relationships between soil and rainwater, vegetation, and microbial communities, which are not necessarily correlated with lithological and structural features of carbonate aquifers.

Hydrodynamics in evaporate-bearing fine-grained successions investigated through an interdisciplinary approach : A test study in southern Italy-hydrogeological behaviour of heterogeneous low-permeability media

Messinian evaporates are widely distributed in the Mediterranean Sea as outcropping sediments in small marginal basins and in marine cores. Progressive filling of subbasins led to the formation of complex aquifer systems in different regions where hypersaline and fresh water coexist and interact in different manner. It also generates a significant diversification of groundwater hydrochemical signature and different microbial communities. In the case study, the hydrogeology and hydrochemistry of the whole system are influenced by good hydraulic connection between the shallower pyroclastic horizon and the underlying evaporate-bearing fine-grained Messinian succession. This is demonstrated by the merge of hydrogeological, chemical, isotopic, and microbiological data. No mixing with deep ascending waters has been observed. As shown by geophysical, hydraulic, and microbiological investigations, the hydraulic heterogeneity of the Messinian bedrock, mainly due to karstified evaporitic interstrata/lenses, causes the hydraulic head to significantly vary with depth. Somewhere, the head increases with the depth’s increase and artesian flow conditions are locally observed. Moreover, the metagenomic investigations demonstrated the existence of a poor hydraulic connection within the evaporate-bearing fine-grained succession at metric and decametric scales, therefore leading to a patchwork of geochemical (and microbiological) subenvironments.

Unraveling the Composition of the Root-Associated Bacterial Microbiota of Phragmites australis and Typha latifolia

Phragmites australis and Typha latifolia are two macrophytes commonly present in natural and artificial wetlands. Roots of these plants engage in interactions with a broad range of microorganisms, collectively referred to as the microbiota. The microbiota contributes to the natural process of phytodepuration, whereby pollutants are removed from contaminated water bodies through plants. The outermost layer of the root corpus, the rhizoplane, is a hot-spot for these interactions where microorganisms establish specialized aggregates designated biofilm. Earlier studies suggest that biofilm-forming members of the microbiota play a crucial role in the process of phytodepuration. However, the composition and recruitment cue of the Phragmites, and Typha microbiota remain poorly understood. We therefore decided to investigate the composition and functional capacities of the bacterial microbiota thriving at the P. australis and T. latifolia root–soil interface. By using 16S rRNA gene Illumina MiSeq sequencing approach we demonstrated that, despite a different composition of the initial basin inoculum, the microbiota associated with the rhizosphere and rhizoplane of P. australis and T. latifolia tends to converge toward a common taxonomic composition dominated by members of the phyla Actinobacteria, Firmicutes, Proteobacteria, and Planctomycetes. This indicates the existence of a selecting process acting at the root–soil interface of these aquatic plants reminiscent of the one observed for land plants. The magnitude of this selection process is maximum at the level of the rhizoplane, where we identified different bacteria enriched in and discriminating between rhizoplane and rhizosphere fractions in a species-dependent and -independent way. This led us to hypothesize that the structural diversification of the rhizoplane community underpins specific metabolic capabilities of the microbiota. We tested this hypothesis by complementing the sequencing survey with a biochemical approach and scanning electron microscopy demonstrating that rhizoplane-enriched bacteria have a bias for biofilm-forming members. Together, our data will be critical to facilitate the rational exploitation of plant–microbiota interactions for phytodepuration.

Beneficial effects of carotenoid-producing cells of Bacillus indicus HU16 in a rat model of diet-induced metabolic syndrome

A well-established rat model of diet-induced metabolic syndrome was used to evaluate the effects of the oral administration of spores or cells of HU16, a carotenoid-producing strain of Bacillus indicus. Symptoms of metabolic syndrome were induced in 90-days old, male Sprague-Dawley rats maintained for eight weeks on a high-fat diet, as previously reported. Parallel groups of animals under the same diet regimen also received a daily dose of 1×1010 cells or spores of B. indicus HU16. Cells of strain HU16 were able to reduce symptoms of metabolic syndrome, plasma markers of inflammation and oxidative markers in plasma and liver to levels similar to those observed in rats under a standard diet. HU16 cells did not affect obesity markers or the accumulation of triglycerides in the liver of treated animals. Denaturing gradient gel electrophoresis analysis showed that the oral administration of HU16 cells did not significantly affect the gut microbiota of high fat-fed rats, suggesting that the observed beneficial effects are not due to a reshaping of the gut microbiota but rather to metabolites produced by HU16 cells.