Paola Grenni

Pharmaceuticals in the environment: Biodegradation and effects on natural microbial communities. A review

Environmental microorganisms play a key role in fundamental ecological processes such as biogeochemical cycling and organic contaminant degradation. Microorganisms comprise a large unexplored reservoir of genetic diversity and metabolic capability providing several ecosystem services, most importantly the maintenance of soil and water quality. Pharmaceutical occurrence in the environment can compromise microbial community structure and activities in different ways. The fate of a pharmaceutical in soil or water depends on numerous factors, including its inherent physic-chemical properties (e.g. water solubility, lipophilicity, vapour pressure), environmental factors and climate conditions (e.g. temperature, incident radiation, pH) and most importantly the presence and activity of microorganisms that possess the ability to biodegrade it. The presence of a natural microbial community is a necessary prerequisite for an effective response to the various chemicals that can contaminate an ecosystem. The recovery from contamination is only possible if toxicity does not hamper microbial activity. This review presents current knowledge on the effects on natural microbial communities of some pharmaceuticals and of some biocides commonly found as environmental microcontaminants.

Effect of urea on degradation of terbuthylazine in soil

Pesticide and nitrate contamination of soil and groundwater from agriculture is an environmental and public health concern worldwide. The herbicide terbuthylazine (CBET) has replaced atrazine in Italy and in many other countries because the use of the latter has been banned because of its adverse environmental impacts. Unlike atrazine, knowledge about the fate of CBET in soil is still not extensive, especially regarding its transformation products, but recent monitoring data show its occurrence and that of its main metabolite, desethyl-terbuthylazine (CBAT), in groundwater above the limit of 0.1 microg/L established by European Union Directive and Italian legislation. The objective of this work was to investigate if the presence of the fertilizer urea affects CBET degradation in the soil. Laboratory CBET degradation experiments in the presence/absence of urea were performed with microbiologically active soil and sterilized soil. Terbuthylazine degradation rates under the different experimental conditions were assessed, and the formation, degradation, and transformation of the metabolite CBAT were also studied. Terbuthylazine degradation was affected by the presence of urea, in terms both of a higher disappearance time of 50% of the initial concentration and of a lower amount of CBAT formed. These findings have practical implications for the real-life assessment of the environmental fate of triazine herbicides in agricultural areas since these herbicides are frequently applied to soils receiving ureic fertilizers.

The role of a groundwater bacterial community in the degradation of the herbicide terbuthylazine

A bacterial community in an aquifer contaminated by s-triazines was studied. Groundwater microcosms were treated with terbuthylazine at a concentration of 100 microg L(-1) and degradation of the herbicide was assessed. The bacterial community structure (abundance and phylogenetic composition) and function (carbon production and cell viability) were analysed. The bacterial community was able to degrade the terbuthylazine; in particular, Betaproteobacteria were involved in the herbicide biotransformation. Identification of some bacterial isolates by PCR amplification of the 16S rRNA gene revealed the presence of two Betaproteobacteria species able to degrade the herbicide: Advenella incenata and Janthinobacterium lividum. PCR detection of the genes encoding s-triazine-degrading enzymes indicated the presence of the atzA and atzB genes in A. incenata and the atzB and atzC genes in J. lividum. The nucleotide sequences of the PCR fragments of the atz genes from these strains were 100% identical to the homologous genes of the Pseudomonas sp. strain ADP. In conclusion, the results show the potential for the use of a natural attenuation strategy in the treatment of aquifers polluted with the terbuthylazine. The two bacteria isolated could facilitate the implementation of effective bioremediation protocols, especially in the case of the significant amounts of herbicide that can be found in groundwater as a result of accidental spills.

Degradation of the antiviral drug oseltamivir carboxylate in surface water samples

Numerous studies have documented that a wide number of pharmaceuticals used in human and veterinary medicine have the potential to enter the aquatic ecosystem. The antiviral prodrug oseltamivir phosphate has received recent attention with regard to its possible use against the highly pathogenic H5N1 virus. This preliminary laboratory study investigated the persistence of the active antiviral drug, oseltamivir carboxylate (OSC), in water samples taken from an irrigation canal. After an initial rapid decrease, OSC concentrations slowly decreased during the remaining incubation period. Approximately 65% of the initial OSC amount remained in water at the end of the 36-day incubation period. A small amount of OSC was lost both from sterilized water and from sterilized water/sediment samples, suggesting a significant role for microbial degradation. Stimulating microbial processes by the addition of sediments resulted in reduced OSC persistence. Presence of OSC (1.5 µg mL−1) did not significantly affect the metabolic potential of the water microbial population, estimated by glyphosate and metolachlor mineralization. In contrast, OSC caused an initial transient decrease in the size of the indigenous microbial population of water samples.

A new fluorescent oligonucleotide probe for in situ detection of s-triazine-degrading Rhodococcus wratislaviensis in contaminated groundwater and soil samples.

A bacterial strain (FPA1) capable of using terbuthylazine, simazine, atrazine, 2-hydroxysimazine, deethylatrazine, isopropylamine or ethylamine as its sole carbon source was isolated from a shallow aquifer chronically contaminated with s-triazine herbicides. Based on its 16S rDNA sequence analysis, the strain FPA1 was identified as Rhodococcus wratislaviensis. The disappearance time of 50% of the initial terbuthylazine concentration in the presence of this strain (DT(50)) was 62days. This strain was also able to mineralise the [U-ring (14)C] triazine-ring, albeit at a slow rate. A 16S rRNA target oligonucleotide probe (RhLu) was designed, and the FISH protocol was optimised, in order to detect R. wratislaviensis in s-triazine-contaminated sites. The RhLu probe gave a positive signal (expressed as % of total DAPI-positive cells) in both the groundwater (2.19+/-0.41%) and soil (2.10+/-0.96%) samples analysed. Using the RhLu probe, R. wratislaviensis can be readily detected, and its population dynamics can be easily monitored, in soil and in water ecosystems contaminated with s-triazine. To the best of our knowledge, this is the first report showing the isolation, from groundwater, of a bacterial strain able to degrade s-triazines.

Reclassification of the members of the genus Tetrathiobacter Ghosh et al. 2005 to the genus Advenella Coenye et al. 2005.

The taxonomic position of the genera Advenella and Tetrathiobacter was examined. 16S rRNA gene sequence analysis revealed that the two genera are closely related, representing a monophyletic cluster with high sequence similarity (98.1-99.7%) within the family Alcaligenaceae. The phenotypic characteristics of the type strains of Advenella incenata, Tetrathiobacter kashmirensis and Tetrathiobacter mimigardefordensis were re-examined using the API 20NE, API ZYM and API 50CH systems. Phylogenetic data together with similarities in phenotypic characteristics, G+C content and cellular acid composition suggest that they should be classified in the same genus. On the basis of the data presented, the two species of the genus Tetrathiobacter should be transferred to the genus Advenella, since this genus has nomenclatural priority. Therefore, Tetrathiobacter kashmirensis and Tetrathiobacter mimigardefordensis should be transferred to the genus Advenella as Advenella kashmirensis comb. nov. (type strain WT001T=LMG 22695T=MTCC7002T) and Advenella mimigardefordensis comb. nov. (type strain DPN7T=DSM 17166T=LMG 22922T). Emended descriptions of Advenella incenata and the genus Advenella are also presented.

Fluorescence in situ hybridization in soil and water ecosystems: a useful method for studying the effect of xenobiotics on bacterial community structure.

The ability of soil and groundwater ecosystems to recover from chemical contamination is primarily dependent on the presence of a microbial community which has the ability to remove it. Nevertheless, there has been a little research into these communities because it is strictly dependent on methods capable of identifying and characterizing their community structure and functioning. The use of molecular methods makes it possible to overcome this kind of identification limitation. In this work, we applied the fluorescence in situ hybridization (FISH) method to different samples, such as soil and groundwater contaminated with s-triazine herbicides (simazine or terbuthylazine) and surface water treated with the pharmaceutical oseltamivir carboxylate (Tamiflu). We compared the bacterial community structure in the presence/absence of these xenobiotics. The use of 16S rRNA-targeted oligonucleotide probes, designed specifically for the main phylogenetic levels (Archaea, Bacteria, α-, β-, γ-, ε-subdivision of Proteobacteria, Planctomycetes, Gram-positive bacteria with a high or low DNA G + C content, Cytophaga-Flavobacter-Bacteroides phylum, and sulfate-reducing bacteria), and a DAPI stain made it possible to assess the structure of the bacterial community and its changes in the presence of these xenobiotics in all the ecosystems studied.

Pharmaceutical waste disposal: assessment of its effects on bacterial communities in soil and groundwater

A preliminary ecological characterisation of an open quarry that had been used for the disposal of pharmaceutical wastes from a factory producing antibiotics was performed. Pharmaceutical wastes and groundwater samples were collected and analysed in order to assess both the bacterial community structure and functioning, and the contamination by organic compounds, including antibiotics. Bacterial abundance measured using the epifluorescence direct count method, cell viability measured by using two fluorescent dyes, species diversity measured by assessing the bacterial community structure using fluorescence in situ hybridisation (FISH) and soil microbial activity based on dehydrogenase activity were used as microbiological indicators to evaluate the ‘quality state’ of the area studied. The overall results show that groundwater has a low-quality state in terms of bacterial viability, activity and diversity, associated with trace contamination by antibiotics and chlorinated volatile organics.

Plant-assisted bioremediation of a historically PCB and heavy metal-contaminated area in Southern Italy

A plant-assisted bioremediation strategy was applied in an area located in Southern Italy, close to the city of Taranto, historically contaminated by polychlorinated biphenyls (PCBs) and heavy metals. A specific poplar clone (Monviso) was selected for its ability to promote organic pollutant degradation in the rhizosphere, as demonstrated elsewhere. Chemical and microbiological analyses were performed at the time of poplar planting in selected plots at different distances from the trunk (0.25-1m) and at different soil depths (0-20 and 20-40cm), at day 420. A significant decrease in PCB congeners and a reduction in all heavy metals was observed where the poplar trees were present. No evidence of PCB and heavy metal reduction was observed in the non poplar-vegetated soil. Microbial analyses (dehydrogenase activity, cell viability, microbial abundance) of the autochthonous microbial community showed an improvement in soil quality. In particular, microbial activity generally increased in the poplar-rhizosphere and a positive effect was observed in some cases at up to 1m distance from the trunk and up to 40cm depth. The Monviso clone was effective in promoting both a general decrease in contaminant occurrence and an increase in microbial activity in the chronically polluted area a little more than one year after planting.

Belowground Microbiota and the Health of Tree Crops

Trees are crucial for sustaining life on our planet. Forests and land devoted to tree crops do not only supply essential edible products to humans and animals, but also additional goods such as paper or wood. They also prevent soil erosion, support microbial, animal, and plant biodiversity, play key roles in nutrient and water cycling processes, and mitigate the effects of climate change acting as carbon dioxide sinks. Hence, the health of forests and tree cropping systems is of particular significance. In particular, soil/rhizosphere/root-associated microbial communities (known as microbiota) are decisive to sustain the fitness, development, and productivity of trees. These benefits rely on processes aiming to enhance nutrient assimilation efficiency (plant growth promotion) and/or to protect against a number of (a)biotic constraints. Moreover, specific members of the microbial communities associated with perennial tree crops interact with soil invertebrate food webs, underpinning many density regulation mechanisms. This review discusses belowground microbiota interactions influencing the growth of tree crops. The study of tree-(micro)organism interactions taking place at the belowground level is crucial to understand how they contribute to processes like carbon sequestration, regulation of ecosystem functioning, and nutrient cycling. A comprehensive understanding of the relationship between roots and their associate microbiota can also facilitate the design of novel sustainable approaches for the benefit of these relevant agro-ecosystems. Here, we summarize the methodological approaches to unravel the composition and function of belowground microbiota, the factors influencing their interaction with tree crops, their benefits and harms, with a focus on representative examples of Biological Control Agents (BCA) used against relevant biotic constraints of tree crops. Finally, we add some concluding remarks and suggest future perspectives concerning the microbiota-assisted management strategies to sustain tree crops.