Monitoring of bacterial communities in a phytoremediation plant for the decontamination of polluted marine sediments

This thesis was performed within the “Agriport” European Project (Agricultural Reuse of Polluted Dredged Sediments, Eco-innovation EU Project n. ECO/08/239065). The Agriport project aims at developing a new technology for treating polluted sediments dredged from the seabed of a commercial port through phytoremediation. Thanks to plant activities and microorganism metabolism, it is possible to recover dredged saline sediments by decontaminating and conditioning them until the obtainment of an artificially prepared soil that is reusable in the terrestrial environment. This is an important advantage from the environmental point of view, and allows to partially solve one of the main problems of most commercial ports, that is the accumulation, storage and disposal of polluted dredged sediments. The study was divided into two main parts, involving the study of bacterial community in fresh marine sediments collected in the seabed of an industrial harbor, and the study of bacterial communities involved in the phytoremediation process of marine contaminated sediments. During the first part of the study, an accurate analysis on bacterial communities composition in fresh marine sediments, directly collected by a scuba diver in five sites of Leghorn harbor was performed. Aim of this first part of the study was to assess the impact of pollution on seabed bacterial diversity, structure and activity in the Port of Leghorn. Seabed sediments of commercial ports are often characterized by high levels of pollution, mainly represented by organic matter, heavy metals and hydrocarbons. One of the main characteristic of harbor sites is the removal of organic matter. Consumption capacity of organic matter is mainly due to the activity of microorganisms. The knowledge of bacterial communities dynamics in fresh marine sediments allowed us to compare the data with the results obtained in the second part of the study, involving bacterial community dynamics in the phytoremediation plant, that were monitored for two years. The phytoremediation plant was made up of a sealed 80 m3 basin that was filled with a mixture of dredged sediments (75%) and natural soil (25%). It was planted with three plant species, and has been properly cultivated with optimized fertilization and irrigation for two years. The techniques developed and used in this thesis were Terminal Restriction Fragment Length Polymorphism (T-RFLP) followed by appropriate statistical analysis, and construction of 16S rRNA clone libraries. Results of the first part of the study, focusing on freshly collected marine sediments, showed that the industrial harbor is mainly contaminated by variable levels of petroleum hydrocarbons and heavy metals, which affect the structure and activity of the bacterial population. A stimulatory effect of nutrients on biological activities and bacterial communities was clearly observed. A stimulation of bacterial development driven by petroleum hydrocarbon and heavy metals was also detected, although with less evidence. Library data, phylogenetic analysis, and T-RFLP coupled with in silico digestion of the obtained sequences, evidenced the dominance of Proteobacteria and the high percentage of Bacteroidetes in all sites. The approach highlighted similar bacterial community among samples coming from the five sites, suggesting a modest differentiation among bacterial communities of different harbor seabed sediments, and hence the capacity of bacterial communities to adapt to different levels and types of pollution. The second part of the study,focusing on bacterial community dynamics in the phytoremediation plant, highlighted that, at the beginning of the experimentation (construction of the treatment basin), each component used to assemble the phytoremediation treatment basin was characterized by its own bacterial community which was differentiated from the others. Six months after the construction of the treatment basin the original bacterial communities evolved into a single bacterial community, homogeneously distributed in the whole area. The bacterial community got stabilized after one year from the construction of the treatment basin. The phytoremediation process influenced the development of a specific bacterial community of the treatment basin that is completely different from the bacterial communities harbored by the basin at the beginning of the experimentation

Multidisciplinary integrated characterization of a native Chlorella-like microalgal strain isolated from a municipal landfill leachate

To date, more than one-hundred taxa of green unicellular coccoid organisms have been wrongly included under the genus Chlorella. This is mainly due to the incomplete characterization resulting from studies that have not adopted and integrated the description of cell morphology, metabolic characteristics and genomic features. In this work, a new native Chlorella-like microalgal strain named “SEC_LI_ChL_1”, is described through a multimethod study. This strain was isolated from a pond where municipal landfil leachate flwed before wastewater treatment. The molecular characterization and the phylogenetic reconstructions based on two different DNA marker regions (i.e. the concatenated SSU and ITS rRNA gene and the complete 18S rRNA gene) were combined with light and transmission electron microscope observations, in addition to the analysis of metabolic traits under four different growing conditions (i.e. autotrophy, heterotrophy, mixotrophy and reduced autotrophy). The two separate phylogenetic analyses highlighted that the strain falls within the Chlorella-Micractinium clade, but with two different placements. According to the morphological-ultrastructural and metabolic analyses, “SEC_LI_ChL_1” shares some features with different Chlorella-like microalgae. Interestingly, two peculiar traits rarely or never previously described in Chlorella-like microalgal strains were observed: the association of the cells with endocellular bacteria, and the presence of doublets (i.e. two-celled microalgae aggregates not due to cell division)

Bacterial communities in polluted seabed sediments: a molecular biology assay in Leghorn harbor

Seabed sediments of commercial ports are often characterized by high pollution levels. Differences in number and distribution of bacteria in such areas can be related to distribution of pollutants in the port and to sediment conditions. In this study, the bacterial communities of five sites from Leghorn Harbor seabed were characterized and the main bacterial groups identified. T-RFLP was used for all samples; two 16S rRNA libraries and in silico digestion of clones were used to identify fingerprint profiles. Library data, phylogenetic analysis, and T-RFLP coupled with in silico digestion of the obtained sequences evidenced the dominance of Proteobacteria and the high percentage of Bacteroidetes in all sites. The approach highlighted similar bacterial communities between samples coming from the five sites, suggesting a modest differentiation among bacterial communities of different harbor seabed sediments, and hence the capacity of bacterial communities to adapt to different levels and types of pollution

Free-living ciliates as potential reservoirs for eukaryotic parasites: Occurrence of a trypanosomatid in the macronucleus of Euplotes encysticus

Background: Flagellates of the family Trypanosomatidae are obligate endoparasites, which can be found in various hosts. Several genera infect insects and occur as monoxenous parasites especially in representatives of Diptera and Hemiptera. These trypanosomatid flagellates probably share the worldwide distribution of their hosts, which are often infested by large numbers of endoparasites. Traditionally, their taxonomy was based on morphology, host origin, and life cycle. Here we report the characterization of a trypanosomatid infection detected in a protozoan, a ciliate collected from a polluted freshwater pond in a suburb of New Delhi (India). Methods. Live observations and morphological studies applying light, fluorescence and transmission electron microscopy were conducted. Molecular analyses of host and parasite were performed and used for phylogenetic reconstructions and species (host) or genus level (parasite) identification. Results: Although the morphological characteristics were not revealing, a high similarity of the trypanosomatids 18S rRNA gene sequence to Herpetomonas ztiplika and Herpetomonas trimorpha (Kinetoplastida, Trypanosomatidae), both parasites of biting midges (Culicoides kibunensis and Culicoides truncorum, respectively) allowed the assignment to this genus. The majority of the host population displayed a heavy infection that significantly affected the shape of the host macronucleus, which was the main site of parasite localization. In addition, the growth rate of host cultures, identified as Euplotes encysticus according to cell morphology and 18S rRNA gene sequence, was severely impacted by the infection. Conclusions: The host-parasite system described here represents a recent example of free-living protists acting as environmental reservoirs for parasitic eukaryotic microorganisms

UV-B irradiation effect on microalgae performance in the remediation of effluent derived from the cigarette butt cleaning process

Funding Information: This research was funded by the Fondazione Cassa di Risparmio di Lucca, grant number FOCUS 2019/2021. Publisher Copyright: © 2022 by the authors.Abstract: In this study, the potential of ultraviolet B (UV-B) radiation to alleviate the effects of pollutants in cigarette butt wastewater (CBW) was investigated using different Chlorella sorokiniana strains (F4, R1 and LG1). Microalgae were treated with UV-B (1.7 W m−2 ) for 3 days prior to their exposure to CBW and then incubated for 4 days in the absence or presence of UV-B. UV-B-untreated microalgae were used as the control. Comparative physiological responses, including photosynthetic pigments and non-enzymatic antioxidants, as well as nicotine and nicotyrine removal, were evaluated in 7-day cultures. UV-B treatments did not negatively impact algal chlorophyll or carotenoid production. UV-B acclimation was strain-dependent, correlating with native environment adaptations and genetic constitutions. UV-B as a pretreatment had long-term positive effects on non-enzymatic antioxidant capacity. However, LG1 needed more time to readjust the pro-oxidant/antioxidant balance, as it was the most UV-B-sensitive. Phenolic compounds played an important role in the antioxidant system response to UV-B, while flavonoids did not contribute to the total antioxidant capacity. Although cross-resistance between UV-B and CBW was observed in F4 and R1, only R1 showed nicotine/nicotyrine catabolism induction due to UV-B. Overall, the results suggest that UV-B activates defense pathways associated with resistance or tolerance to nicotine and nicotyrine.In this study, the potential of ultraviolet B (UV-B) radiation to alleviate the effects of pollutants in cigarette butt wastewater (CBW) was investigated using different Chlorella sorokiniana strains (F4, R1 and LG1). Microalgae were treated with UV-B (1.7 W m−2) for 3 days prior to their exposure to CBW and then incubated for 4 days in the absence or presence of UV-B. UV-B-untreated microalgae were used as the control. Comparative physiological responses, including photosynthetic pigments and non-enzymatic antioxidants, as well as nicotine and nicotyrine removal, were evaluated in 7-day cultures. UV-B treatments did not negatively impact algal chlorophyll or carotenoid production. UV-B acclimation was strain-dependent, correlating with native environment adaptations and genetic constitutions. UV-B as a pretreatment had long-term positive effects on non-enzymatic antioxidant capacity. However, LG1 needed more time to readjust the pro-oxidant/antioxidant balance, as it was the most UV-B-sensitive. Phenolic compounds played an important role in the antioxidant system response to UV-B, while flavonoids did not contribute to the total antioxidant capacity. Although cross-resistance between UV-B and CBW was observed in F4 and R1, only R1 showed nicotine/nicotyrine catabolism induction due to UV-B. Overall, the results suggest that UV-B activates defense pathways associated with resistance or tolerance to nicotine and nicotyrine.Peer reviewe

Endophytic and rhizospheric bacterial communities isolated from the medicinal plants Echinacea purpurea and Echinacea angustifolia

In this work we analyzed the composition and structure of cultivable bacterial communities isolated from the stem/leaf and root compartments of two medicinal plants, Echinacea purpurea (L.) Moench and Echinacea angustifolia (DC.) Hell, grown in the same soil, as well as the bacterial community from their rhizospheric soils. Molecular PCR-based techniques were applied to cultivable bacteria isolated from the three compartments of the two plants. The results showed that the two plants and their respective compartments were characterized by different communities, indicating a low degree of strain sharing and a strong selective pressure within plant tissues. Pseudomonas was the most highly represented genus, together with Actinobacteria and Bacillus spp. The presence of distinct bacterial communities in different plant species and among compartments of the same plant species could account for the differences in the medicinal properties of the two plants. [Int Microbiol 2014; 17(3):165-174]Keywords: Echinacea purpurea · Echinacea angustifolia · rhizosphere · medicinal plants · endophyte

Characterization of the endophytic bacterial community of Bituminaria bituminosa plant grown in vitro and its interaction with the plant extract

IntroductionBituminaria bituminosa is a medicinal plant recognized for its phytochemicals, such as furanocoumarins, pterocarpans, and flavonoids. Since the secondary metabolism is influenced by the plant-endophyte interactions, the endophytic bacterial community of B. bituminosa was explored and the possible interactions with the plant were described.Materials and methodsDifferent bacterial strains were isolated from different organs of in vitro plants as shoots, roots, and seeds. The bacterial strains were identified and phenotypically characterized for different traits; strains were also exposed to different concentrations of B. bituminosa plant extract showing different susceptibility, probably determined by different secondary metabolites produced by the plant in the different organs (i.e. aerial parts and roots).Results and discussionBacterial strains showed different phenotypic characteristics; the 6 detected haplotypes were dominated by a single species related to Stenotrophomonas rhizophila. Endophytes isolated from the aerial parts produced a higher indole-3-acetic acid (IAA) amount than those of the roots, while all strains were unable to produce biosurfactants and antagonistic activity toward the other strains. The research opens new perspectives for future analysis addressed to test the susceptibility of the endophytic bacterial community of B. bituminosa toward the pure compounds extracted from the plants, and to investigate the role of these compounds on the distribution of endophytes within the different plant tissues

Remediation capacity of different microalgae in effluents derived from the cigarette butt cleaning process

Funding Information: This research was funded by the Fondazione Cassa di Risparmio di Lucca, grant number FOCUS 2019/2021. Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Microalgal-based remediation is an ecofriendly and cost-effective system for wastewater treatment. This study evaluated the capacity of microalgae in the remediation of wastewater from cleaning process of smoked cigarette butts (CB). At laboratory scale, six strains (one from the family Scenedesmaceae, two Chlamydomonas debaryana and three Chlorella sorokiniana) were exposed to different CB wastewater dilutions to identify toxicity levels reflected in the alteration of microalgal physiological status and to determine the optimal conditions for an effective removal of contaminants. CB wastewater could impact on microalgal chlorophyll and carotenoid production in a concentration-dependent manner. Moreover, the resistance and remediation capacity did not only depend on the microalgal strain, but also on the chemical characteristics of the organic pollutants. In detail, nicotine was the most resistant pollutant to removal by the microalgae tested and its low removal correlated with the inhibition of photosynthetic pigments affecting microalgal growth. Concerning the optimal conditions for an effective bioremediation, this study demonstrated that the Chlamydomonas strain named F2 showed the best removal capacity to organic pollutants at 5% CB wastewater (corresponding to 25 butts L−1 or 5 g CB L−1) maintaining its growth and photosynthetic pigments at control levels.Microalgal-based remediation is an ecofriendly and cost-effective system for wastewater treatment. This study evaluated the capacity of microalgae in the remediation of wastewater from cleaning process of smoked cigarette butts (CB). At laboratory scale, six strains (one from the family Scenedesmaceae, two Chlamydomonas debaryana and three Chlorella sorokiniana) were exposed to different CB wastewater dilutions to identify toxicity levels reflected in the alteration of microalgal physiological status and to determine the optimal conditions for an effective removal of contaminants. CB wastewater could impact on microalgal chlorophyll and carotenoid production in a concentration-dependent manner. Moreover, the resistance and remediation capacity did not only depend on the microalgal strain, but also on the chemical characteristics of the organic pollutants. In detail, nicotine was the most resistant pollutant to removal by the microalgae tested and its low removal correlated with the inhibition of photosynthetic pigments affecting microalgal growth. Concerning the optimal conditions for an effective bioremediation, this study demonstrated that the Chlamydomonas strain named F2 showed the best removal capacity to organic pollutants at 5% CB wastewater (corresponding to 25 butts L−1 or 5 g CB L−1) maintaining its growth and photosynthetic pigments at control levels.Peer reviewe