Edoardo Puglisi

Soil enzymology: classical and molecular approaches

It is still problematic to use enzyme activities as indicators of soil functions because: (1) enzyme assays determine potential and not real enzyme activities; (2) the meaning of measured enzyme activities is not known; (3) the assumption that a single enzyme activity is an indicator of nutrient dynamics in soil neglects that the many enzyme activities are involved in such dynamic processes; (4) spatio-temporal variations in natural environments are not always considered when measuring enzyme activities; and (5) many direct and indirect effects make difficult the interpretation of the response of the enzyme activity to perturbations, changes in the soil management, changes in the plant cover of soil, etc. This is the first review discussing the links between enzyme-encoding genes and the relative enzyme activity of soil. By combining measurements of enzyme activity in soil with expression (transcriptomics and proteomics) of genes, encoding the relative enzymes may contribute to understanding the mode and timing of microbial communities’ responses to substrate availability and persistence and stabilization of enzymes in the soil.

Bacterial diversity in typical Italian salami at different ripening stages as revealed by high-throughput sequencing of 16S rRNA amplicons.

The bacterial diversity involved in food fermentations is one of the most important factors shaping the final characteristics of traditional foods. Knowledge about this diversity can be greatly improved by the application of high-throughput sequencing technologies (HTS) coupled to the PCR amplification of the 16S rRNA subunit. Here we investigated the bacterial diversity in batches of Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage that is typical of a regional area of Northern Italy. Salami samples from 6 different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw meat at time 0 and casing samples at 21 days of ripening where also analysed, and the effect of starter addition was included in the experimental set-up. Culture-based microbiological analyses and PCR-DGGE were carried out in order to be compared with HTS results. A total of 722,196 high quality sequences were obtained after trimming, paired-reads assembly and quality screening of raw reads obtained by Illumina MiSeq sequencing of the two bacterial 16S hypervariable regions V3 and V4; manual curation of 16S database allowed a correct taxonomical classification at the species for 99.5% of these reads. Results confirmed the presence of main bacterial species involved in the fermentation of salami as assessed by PCR-DGGE, but with a greater extent of resolution and quantitative assessments that are not possible by the mere analyses of gel banding patterns. Thirty-two different Staphylococcus and 33 Lactobacillus species where identified in the salami from different producers, while the whole data set obtained accounted for 13 main families and 98 rare ones, 23 of which were present in at least 10% of the investigated samples, with casings being the major sources of the observed diversity. Multivariate analyses also showed that batches from 6 local producers tend to cluster altogether after 21 days of ripening, thus indicating that HTS has the potential for fine scale differentiation of local fermented foods.

Soil bacterial diversity screening using single 16S rRNA gene V regions coupled with multi-million read generating sequencing technologies.

The novel multi-million read generating sequencing technologies are very promising for resolving the immense soil 16S rRNA gene bacterial diversity. Yet they have a limited maximum sequence length screening ability, restricting studies in screening DNA stretches of single 16S rRNA gene hypervariable (V) regions. The aim of the present study was to assess the effects of properties of four consecutive V regions (V3-6) on commonly applied analytical methodologies in bacterial ecology studies. Using an in silico approach, the performance of each V region was compared with the complete 16S rRNA gene stretch. We assessed related properties of the soil derived bacterial sequence collection of the Ribosomal Database Project (RDP) database and concomitantly performed simulations based on published datasets. Results indicate that overall the most prominent V region for soil bacterial diversity studies was V3, even though it was outperformed in some of the tests. Despite its high performance during most tests, V4 was less conserved along flanking sites, thus reducing its ability for bacterial diversity coverage. V5 performed well in the non-redundant RDP database based analysis. However V5 did not resemble the full-length 16S rRNA gene sequence results as well as V3 and V4 did when the natural sequence frequency and occurrence approximation was considered in the virtual experiment. Although, the highly conserved flanking sequence regions of V6 provide the ability to amplify partial 16S rRNA gene sequences from very diverse owners, it was demonstrated that V6 was the least informative compared to the rest examined V regions. Our results indicate that environment specific database exploration and theoretical assessment of the experimental approach are strongly suggested in 16S rRNA gene based bacterial diversity studies.

Extraction and bioanalysis of the ecotoxicologically relevant fraction of contaminants in sediments

Assessments of the risk connected to the contamination of soils and sediments should rely on a multidisciplinary approach based on both chemical and biological techniques (i.e., the sum of exposure and effects assessment). The dioxin-responsive, chemical-activated luciferase expression (DR-CALUX) bioassay is widely applied for evaluation of the toxicity of sediments after an exhaustive extraction of the contaminants, and results are used for risk assessment purposes. Approaches based on total extraction of contaminants do not take into account the importance of bioavailability and aging processes, thus leading to possible overestimations of risk. In the present work, an approach based on nonexhaustive extraction techniques in combination with an in vitro reporter gene assay was tested on sediment samples contaminated with dioxins, polycyclic aromatic hydrocarbons, polychlorinated biphenyls (PCBs), and other xenobiotics. Tenax and hydroxypropyl-beta-cyclodextrin (HPCD) extractions over time were carried out to determine the bioavailable fractions, whereas the residual fractions were determined by means of a microwave-assisted exhaustive extraction. For both fractions, contaminant concentrations were quantified by gas chromatography-mass spectrophotometry, and the toxic potency was determined by the DR-CALUX assay. Assessments of bioavailable fractions of PCBs by Tenax and HPCD gave comparable results and showed that after several years of aging, a considerable fraction (38-70% of the total content for different PCBs) is still available and ecotoxicologically relevant. Coupling of nonexhaustive extraction and bioanalyses leads to a more realistic and, generally, much lower estimated risk for the toxicity of the extracts as compared to commonly adopted exhaustive techniques.

Effect of air-drying treatment on enzymatic activities of soils affected by anthropogenic activities

The influence of air-drying on dehydrogenase, invertase, β-glucosidase, urease, phosphatase, arylsulphatase and phenoloxidase activities was measured in three soils affected by anthropogenic activities and in control soils sampled from neighbouring areas. The air-drying changed enzyme activity, but the behaviour was neither site-specific nor enzyme-sensitive. Canonical discriminant analysis of enzymatic activities of both moist fresh and air-dried samples was able to discriminate between altered and unaltered soils irrespective of the site. Numerical alteration indices were developed as a linear function of the seven enzyme activities and the one obtained from fresh sample data was more successful. Air-drying apparently alters in unpredictable ways enzyme activities of soils, which could lead to misinterpretation of results.

Bioaccessibility, bioavailability and ecotoxicity of pentachlorophenol in compost amended soils

The influence of compost on the bioaccessibility, bioavailability and ecotoxicity of pentachlorophenol (PCP) as a function of time was studied by means of different chemical and ecotoxicological methods. Experiments were conducted in both sterile and non-sterile microcosms and samplings carried out at 20, 60 and 120d from initial contamination. PCP bioaccessibility, assessed by means of Porapak resin extraction, was around 75% of the applied dose with no aging or compost effects. Two different methods were applied to assess the bioavailability of PCP, respectively, to bacteria and earthworms and linked to ecotoxicological assays (biosensor and earthworm coelomocytes assays). Water extraction was applied to assess the bioavailability to bacteria: results showed that this fraction was always below 50%, with significant decreases as a result of aging processes and compost addition. In non-sterile microcosms, compost amendment increased the amount of PCP biodegraded, while the ecotoxicological assay with the biosensor Pseudomonas fluorescens pUCD607 indicated a higher toxicity in the most degraded samples, thus suggesting the formation of more toxic metabolites. Earthworm body accumulation results were rather in accordance with water extractions data, with decreasing bioavailable amounts as a result of time and compost addition. No compost or aging effects were instead detected by coelomocytes assay. Results indicate how different methods must be applied altogether to assess the bioavailability and ecotoxicity of xenobiotics such as PCP in soil. The addition of compost was also proven as an effective strategy for the remediation of PCP contaminated soils, although issues related to the possible formation of toxic metabolites must be taken into account.

Effects of a humic acid and its size-fractions on the bacterial community of soil rhizosphere under maize (Zea mays L.).

The effects of a humic acid (HA) and its size-fractions on plants carbon deposition and the structure of microbial communities in the rhizosphere soil of maize (Zea mays L.) plants were studied. Experiments were conducted in rhizobox systems that separate an upper soil-plant compartment from a lower compartment, where roots are excluded from the rhizosphere soil by a nylon membrane. The upper rhizobox compartment received the humic additions, whereas, after roots development, the rhizosphere soil in the lower compartment was sampled and sliced into thin layers. The lux-marked biosensor Pseudomonas fluorescens 10586 pUCD607 biosensor showed a significant increase in the deposition of bioavailable sources of carbon in the rhizosphere of soils when treated with bulk HA, but no response was found for treatments with the separated size-fractions. PCR-DGGE molecular fingerprintings revealed that the structure of rhizosphere microbial communities was changed by all humic treatments and that the smaller and more bioavailable size-fractions were more easily degraded by microbial activity than the bulk HA. On the other hand, highly hydrophobic and strongly associated humic molecules in the bulk HA required additional plant rhizodeposition before their bio-transformation could occur. This work highlights the importance of applying advanced biological and biotechnological methods to notice changes occurring in plant rhizodeposition and rhizosphere microbial activity. Moreover, it suggests correlations between the molecular properties of humic matter and their effects on microbial communities in the rhizosphere as mediated by root exudation.

Soil microbial diversity patterns of a lowland spring environment

The Po river plain lowland springs represent unique paradigms of managed environments. Their current locations used to be swamps that were drained 6-7 centuries ago, and they have been in constant use ever since. Our aims were to identify the effects of land use on the microbial communities of these soils, look for associated diversity drivers, and assess the applicability of ecology theories with respect to identified patterns. We screened the microbial diversity across a land use transect via high-throughput sequencing of partial 16S rrRNA gene amplicons. Land use had a major effect on soil properties and microbial community structures. Total organic carbon and pH were major diversity drivers for Bacteria, and pH was important for Archaea. We identified the potential contribution of soil amendments to the indigenous microbial communities, and also gained insights into potential roles of taxa in the organic carbon turnover. Verrucomicrobia coincided with the higher values of the recalcitrant organic carbon. Actinobacteria and Acidobacteria correlated with the more labile organic carbon. Finally, the higher diversity found in the soils less enzymatically active and relatively poorer in nutrients, may be explained to an extent by niche-based theories such as the resource heterogeneity hypothesis and Connells intermediate disturbance hypothesis.

Changes in soil bacterial communities and diversity in response to long-term silver exposure

Silver-induced selective pressure is becoming increasingly important due to the growing use of silver (Ag) as an antimicrobial agent in biomedical and commercial products. With demonstrated links between environmental resistomes and clinical pathogens, it is important to identify microbial profiles related to silver tolerance/resistance. We investigated the effects of ionic Ag stress on soil bacterial communities and identified resistant/persistent bacterial populations. Silver treatments of 50-400 mg Ag kg(-1) soil were established in five soils. Chemical lability measurements using diffusive gradients in thin-film devices confirmed that significant (albeit decreasing) labile Ag concentrations were present throughout the 9-month incubation period. Synchrotron X-ray absorption near edge structure spectroscopy demonstrated that this decreasing lability was due to changes in the Ag speciation to less soluble forms such as Ag(0) and Ag2S. Real-time PCR and Illumina MiSeq screening of 16S rRNA bacterial genes showed β-diversity changes, increasing α-diversity in response to Ag pressure, and immediate and significant reductions in 16S rRNA gene counts with varying degrees of recovery. These effects were more strongly influenced by exposure time than by Ag dose at these rates. Ag-selected dominant OTUs principally resided in known persister taxa (mainly Gram positive), including metal-tolerant bacteria and slow-growing Mycobacteria.

Draft Genome Sequence of Clostridium tyrobutyricum Strain UC7086, Isolated from Grana Padano Cheese with Late-Blowing Defect

ABSTRACT Clostridium tyrobutyricum is considered the main agent of late-blowing defect in the production of hard cheese. Here, we described the draft genome sequences and annotation of C. tyrobutyricum strain UC7086, which was isolated from Grana Padano cheese with blowing defect, and C. tyrobutyricum DSM 2637 type strain in a comparative study.