Patrizia Ricciuti

Short-term effects of municipal solid waste compost amendments on soil carbon and nitrogen content, some enzyme activities and genetic diversity

Abstract. Municipal solid waste (MSW) composts have been frequently used as N and C amendments to improve soil quality and to support plant growth, with the additional benefit of reducing waste disposal costs. However, attention has been paid to the risks of MSW use for the soil environment. The presence of heavy metals in MSW composts can affect some microbiological characteristics of soil such as the structure of the soil microbiota, which are responsible for the transformations making nutrients available to plants. The effects of MSW compost and mineral-N amendments in a 2-year field trial on some physical-chemical properties, some enzyme activities and the genetic diversity of cropped plots (sugar beet-wheat rotation) and uncropped plots were investigated. Variations of pH were not statistically related to MSW compost and mineral-N amendments, or to the presence of the crop. Amendment with MSW compost increased the organic C and total N contents, and dehydrogenase and nitrate reductase activities of soil. In cropped plots amended with MSW compost, dehydrogenase activity was positively correlated with β-glucosidase activity, and both enzyme activities with organic C content. No MSW compost dosage effect was detected. No effects were observed on denaturing gradient gel electrophoresis and amplified rDNA restriction analysis patterns, indicating that no significant change in the bacterial community occurred as a consequence of MSW amendment.

Duodenal and faecal microbiota of celiac children: molecular, phenotype and metabolome characterization

BackgroundEpidemiology of celiac disease (CD) is increasing. CD mainly presents in early childhood with small intestinal villous atrophy and signs of malabsorption. Compared to healthy individuals, CD patients seemed to be characterized by higher numbers of Gram-negative bacteria and lower numbers Gram-positive bacteria.ResultsThis study aimed at investigating the microbiota and metabolome of 19 celiac disease children under gluten-free diet (treated celiac disease, T-CD) and 15 non-celiac children (HC). PCR-denaturing gradient gel electrophoresis (DGGE) analyses by universal and group-specific primers were carried out in duodenal biopsies and faecal samples. Based on the number of PCR-DGGE bands, the diversity of Eubacteria was the higher in duodenal biopsies of T-CD than HC children. Bifidobacteria were only found in faecal samples. With a few exceptions, PCR-DGGE profiles of faecal samples for Lactobacillus and Bifidobacteria differed between T-CD and HC. As shown by culture-dependent methods, the levels of Lactobacillus, Enterococcus and Bifidobacteria were confirmed to be significantly higher (P = 0.028; P = 0.019; and P = 0.023, respectively) in fecal samples of HC than in T-CD children. On the contrary, cell counts (CFU/ml) of presumptive Bacteroides, Staphylococcus, Salmonella, Shighella and Klebsiella were significantly higher (P = 0.014) in T-CD compared to HC children. Enterococcus faecium and Lactobacillus plantarum were the species most diffusely identified. This latter species was also found in all duodenal biopsies of T-CD and HC children. Other bacterial species were identified only in T-CD or HC faecal samples. As shown by Randomly Amplified Polymorphic DNA-PCR analysis, the percentage of strains identified as lactobacilli significantly (P = 0.011) differed between T-CD (ca. 26.5%) and HC (ca. 34.6%) groups. The metabolome of T-CD and HC children was studied using faecal and urine samples which were analyzed by gas-chromatography mass spectrometry-solid-phase microextraction and 1H-Nuclear Magnetic Resonance. As shown by Canonical Discriminant Analysis of Principal Coordinates, the levels of volatile organic compounds and free amino acids in faecal and/or urine samples were markedly affected by CD.ConclusionAs shown by the parallel microbiology and metabolome approach, the gluten-free diet lasting at least two years did not completely restore the microbiota and, consequently, the metabolome of CD children. Some molecules (e.g., ethyl-acetate and octyl-acetate, some short chain fatty acids and free amino acids, and glutamine) seems to be metabolic signatures of CD.

Different Fecal Microbiotas and Volatile Organic Compounds in Treated and Untreated Children with Celiac Disease

ABSTRACT This study aimed at investigating the fecal microbiotas of children with celiac disease (CD) before (U-CD) and after (T-CD) they were fed a gluten-free diet and of healthy children (HC). Brothers or sisters of T-CD were enrolled as HC. Each group consisted of seven children. PCR-denaturing gradient gel electrophoresis (DGGE) analysis with V3 universal primers revealed a unique profile for each fecal sample. PCR-DGGE analysis with group- or genus-specific 16S rRNA gene primers showed that the Lactobacillus community of U-CD changed significantly, while the diversity of the Lactobacillus community of T-CD was quite comparable to that of HC. Compared to HC, the ratio of cultivable lactic acid bacteria and Bifidobacterium to Bacteroides and enterobacteria was lower in T-CD and even lower in U-CD. The percentages of strains identified as lactobacilli differed as follows: HC (ca. 38%) > T-CD (ca. 17%) > U-CD (ca. 10%). Lactobacillus brevis, Lactobacillus rossiae, and Lactobacillus pentosus were identified only in fecal samples from T-CD and HC. Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus gasseri were identified only in several fecal samples from HC. Compared to HC, the composition of Bifidobacterium species of T-CD varied, and it varied even more for U-CD. Forty-seven volatile organic compounds (VOCs) belonging to different chemical classes were identified using gas-chromatography mass spectrometry-solid-phase microextraction analysis. The median concentrations varied markedly for HC, T-CD, and U-CD. Overall, the r2 values for VOC data for brothers and sisters were equal to or lower than those for unrelated HC and T-CD. This study shows the effect of CD pathology on the fecal microbiotas of children.

Taxonomic structure of the yeasts and lactic acid bacteria microbiota of pineapple (Ananas comosus L. Merr.) and use of autochthonous starters for minimally processing

Pichia guilliermondii was the only identified yeast in pineapple fruits. Lactobacillus plantarum and Lactobacillus rossiae were the main identified species of lactic acid bacteria. Typing of lactic acid bacteria differentiated isolates depending on the layers. L. plantarum 1OR12 and L. rossiae 2MR10 were selected within the lactic acid bacteria isolates based on the kinetics of growth and acidification. Five technological options, including minimal processing, were considered for pineapple: heating at 72 degrees C for 15 s (HP); spontaneous fermentation without (FP) or followed by heating (FHP), and fermentation by selected autochthonous L. plantarum 1OR12 and L. rossiae 2MR10 without (SP) or preceded by heating (HSP). After 30 days of storage at 4 degrees C, HSP and SP had a number of lactic acid bacteria 1000 to 1,000,000 times higher than the other processed pineapples. The number of yeasts was the lowest in HSP and SP. The Community Level Catabolic Profiles of processed pineapples indirectly confirmed the capacity of autochthonous starters to dominate during fermentation. HSP and SP also showed the highest antioxidant activity and firmness, the better preservation of the natural colours and were preferred for odour and overall acceptability.

Antioxidant treatment of hindlimb-unloaded mouse counteracts fiber type transition but not atrophy of disused muscles

Oxidative stress was proposed as a trigger of muscle impairment in various muscle diseases. The hindlimb-unloaded (HU) rodent is a model of disuse inducing atrophy and slow-to-fast transition of postural muscles. Here, mice unloaded for 14 days were chronically treated with the selective antioxidant trolox. After HU, atrophy was more pronounced in the slow-twitch soleus muscle (Sol) than in the fast-twitch gastrocnemius and tibialis anterior muscles, and was absent in extensor digitorum longus muscle. In accord with the phenotype transition, HU Sol showed a reduced expression of myosin heavy chain type 2A (MHC-2A) and increase in MHC-2X and MHC-2B isoforms. In parallel, HU Sol displayed an increased sarcolemma chloride conductance related to an increased expression of ClC-1 channels, changes in excitability parameters, a positive shift of the mechanical threshold, and a decrease of the resting cytosolic calcium concentration. Moreover, the level of lipoperoxidation increased proportionally to the degree of atrophy of each muscle type. As expected, trolox treatment fully prevented oxidative stress in HU mice. Atrophy was not prevented but the drug significantly attenuated Sol phenotypic transition and excitability changes. Trolox treatment had no effect on control mice. These results suggest possible benefits of antioxidants in protecting muscle against disuse.

Molecular approaches to investigate herbicide-induced bacterial community changes in soil microcosms

Abstract. Since biochemical and microbiological methods used to study microbial community changes induced by anthropogenic activities can be biased, the impact of two herbicides on soil microorganisms was investigated by culture-independent molecular techniques. The effect of three different amounts (the recommended field dose, tenfold, and 100-fold the dose) of propanil or prometryne on the bacterial community of a clay soil, two modalities of incubation (soil moisture at 70% of the field capacity and a soil-herbicide suspension, 1:10, w:v), and time of incubation were investigated by denaturing gradient gel electrophoresis (DGGE) and amplified rDNA restriction analysis (ARDRA). Two sets of primers for 16S rDNA were used to amplify total soil DNA. Sterile and non-sterile samples were used to determine, by HPLC, the amounts of herbicides adsorbed on soil and transformed by soil microorganisms. Prometryne persisted in soil longer than propanil. Propanil was removed significantly more by non-sterile than by sterile samples, while for prometryne, slight differences were observed. 3,4-Dichloroaniline, a product of propanil hydrolysis, was detected in non-sterile samples and increased with incubation time. Propanil did not affect soil bacteria significantly as indicated by DGGE and ARDRA, with the only exception being the soil-herbicide suspension. Despite a lower utilization of prometryne by soil microorganisms, DGGE analysis showed a more diverse banding than with propanil. Some bands were also detected in the DNA sample extracted from the soil-prometryne suspension, and could be representative of bacterial species utilizing the herbicide as a carbon source, in two very different soil microcosms.

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.

Genetic, Functional, and Metabolic Responses of Soil Microbiota in a Sustainable Olive Orchard

The aim of the present work was to evaluate the effects of two soil management systems so called sustainable treatment (ST) and conventional treatment (CT) on the composition and on genetic, functional, and metabolic diversity of soil microbial communities in a Mediterranean olive orchard. The ST system included no-tillage, integrated chemical fertilization, and organic matter inputs from drip irrigation, spontaneous cover crops, and pruning material. Microbial analyses were carried out by an integrated approach of culture-dependent (microbial cultures and Biolog) and culture-independent methods (denaturing gradient gel electrophoresis [DGGE]). After 7 years of treatment, average olive yield was 8.4 and 3.1 t ha−1 year−1 in ST and CT, respectively. Conventional treatment had a significantly higher number of total culturable bacteria and actinomycetes compared with ST, whereas fungi were significantly lower. In ST, the number of ammonifying bacteria, proteolytic bacteria, and Azotobacter in the wetted areas under the drippers (ST-WET) was significantly higher than along interrows (ST-INTER). The DGGE analysis of microbial 16S/18S rDNA showed differences between ST and CT, whereas 16S/18S rRNA DGGE bands of ST-WET clustered differently from those of CT and ST-INTER. Some Biolog metabolic indexes were significantly different between ST and CT. The results revealed qualitative and quantitative changes of soil microbial communities in response to sustainable agricultural practices that stimulate soil microorganism activity and improve olive yield and fruit quality.

Changes in composition and activity of soil microbial communities in peach and kiwifruit Mediterranean orchards under an innovative management system

The aim of this work was to evaluate the effects of 2 soil management systems, so called ‘innovative’ (INN) and ‘conventional’ (CON), on genetic and metabolic diversity of soil microbial communities of peach and kiwifruit orchards. INN system included minimum tillage, organic matter inputs from compost and cover crops, winter pruning, and adequate irrigation and fertilisation. CON system was characterised by conventional tillage, zero organic input, empirical pruning, strong chemical fertilisation, and excessive irrigation. After 4 years of treatments, soil samples were collected in different orchard sites. In peach and kiwifruit INN orchards, average fruit yields were significantly higher than in CON. INN orchards had a significantly higher total number of bacteria. The patterns of denaturing gradient gel electrophoresis of bacterial 16S rDNA/RNA from peach orchard showed differences between soils under drip emitters and along the inter-rows, whereas those from kiwifruit orchard clearly distinguished between INN and CON for both bacteria (16S rRNA) and fungi (18S rDNA/RNA). Shannon’s substrate diversity index, evaluated by Biolog® metabolic assay, was affected by soil treatment in peach orchard and by soil depth in kiwifruit orchard. Principal component analysis of Biolog® values clearly discriminated INN and CON soils of both orchards. The results revealed qualitative and quantitative changes of soil microbial communities in response to an innovative and sustainable soil management.

Profile of microbial communities on carbonate stones of the medieval church of San Leonardo di Siponto (Italy) by Illumina-based deep sequencing

Comprehensive studies of the biodiversity of the microbial epilithic community on monuments may provide critical insights for clarifying factors involved in the colonization processes. We carried out a high-throughput investigation of the communities colonizing the medieval church of San Leonardo di Siponto (Italy) by Illumina-based deep sequencing. The metagenomic analysis of sequences revealed the presence of Archaea, Bacteria, and Eukarya. Bacteria were Actinobacteria, Proteobacteria, Bacteroidetes, Cyanobacteria, Chloroflexi, Firmicutes and Candidatus Saccharibacteria. The predominant phylum was Actinobacteria, with the orders Actynomycetales and Rubrobacteriales, represented by the genera Pseudokineococcus, Sporichthya, Blastococcus, Arthrobacter, Geodermatophilus, Friedmanniella, Modestobacter, and Rubrobacter, respectively. Cyanobacteria sequences showing strong similarity with an uncultured bacterium sequence were identified. The presence of the green algae Oocystaceae and Trebuxiaceae was revealed. The microbial diversity was explored at qualitative and quantitative levels, evaluating the richness (the number of operational taxonomic units (OTUs)) and the abundance of reads associated with each OTU. The rarefaction curves approached saturation, suggesting that the majority of OTUs were recovered. The results highlighted a structured community, showing low diversity, made up of extremophile organisms adapted to desiccation and UV radiation. Notably, the microbiome appeared to be composed not only of microorganisms possibly involved in biodeterioration but also of carbonatogenic bacteria, such as those belonging to the genus Arthrobacter, which could be useful in bioconservation. Our investigation demonstrated that molecular tools, and in particular the easy-to-run next-generation sequencing, are powerful to perform a microbiological diagnosis in order to plan restoration and protection strategies.