Claudia Sorlini

Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities

ABSTRACT ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al., Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the façade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng μl−1, while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri.

A drought resistance-promoting microbiome is selected by root system under desert farming

Background Traditional agro-systems in arid areas are a bulwark for preserving soil stability and fertility, in the sight of “reverse desertification”. Nevertheless, the impact of desert farming practices on the diversity and abundance of the plant associated microbiome is poorly characterized, including its functional role in supporting plant development under drought stress. Methodology/Principal Findings We assessed the structure of the microbiome associated to the drought-sensitive pepper plant (Capsicum annuum L.) cultivated in a traditional Egyptian farm, focusing on microbe contribution to a crucial ecosystem service, i.e. plant growth under water deficit. The root system was dissected by sampling root/soil with a different degree of association to the plant: the endosphere, the rhizosphere and the root surrounding soil that were compared to the uncultivated soil. Bacterial community structure and diversity, determined by using Denaturing Gradient Gel Electrophoresis, differed according to the microhabitat, indicating a selective pressure determined by the plant activity. Similarly, culturable bacteria genera showed different distribution in the three root system fractions. Bacillus spp. (68% of the isolates) were mainly recovered from the endosphere, while rhizosphere and the root surrounding soil fractions were dominated by Klebsiella spp. (61% and 44% respectively). Most of the isolates (95%) presented in vitro multiple plant growth promoting (PGP) activities and stress resistance capabilities, but their distribution was different among the root system fractions analyzed, with enhanced abilities for Bacillus and the rhizobacteria strains. We show that the C. annuum rhizosphere under desert farming enriched populations of PGP bacteria capable of enhancing plant photosynthetic activity and biomass synthesis (up to 40%) under drought stress. Conclusions/Significance Crop cultivation provides critical ecosystem services in arid lands with the plant root system acting as a “resource island” able to attract and select microbial communities endowed with multiple PGP traits that sustain plant development under water limiting conditions.

PCR fingerprinting of whole genomes, the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus licheniformis

Genomic diversity in 21 strains of Bacillus cereus and 10 strains of Bacillus licheniformis was investigated by random amplified polymorphic DNA (RAPD) analysis, which samples the whole genome, and by two PCR fingerprinting techniques sampling the hypervariable spacers between the conserved 16S and 23S rRNA genes of the rRNA gene operon (ITS-PCR) and regions between tRNA genes (tDNA-PCR). RAPD analysis showed a remarkable diversity among strains of B. cereus that was not observed with the rRNA and tRNA intergenic-spacer-targeted PCR, where all the strains showed practically identical fingerprints. A wide variability among the B. cereus strains was also observed in the plasmid profiles, suggesting that the genetic diversity within B. cereus species can arise from plasmid transfer. One contribution to the diversity detected by RAPD analysis was determined by the presence of large extrachromosomal elements that were amplified during RAPD analysis as shown by Southern hybridization experiments. In contrast to the strains of B. cereus, the 10 strains of B. licheniformis were grouped into two clusters which were the same with all the methods employed. The 16S rRNA genes were identical in all 10 strains when examined using single strand conformation polymorphism analysis after digestion with Alul and Rsal. From these data we hypothesize two different evolutionary schemes for the two species.

COMPOSTING OF SOLID AND SLUDGE RESIDUES FROM AGRICULTURAL AND FOOD INDUSTRIES. BIOINDICATORS OF MONITORING AND COMPOST MATURITY

A study to monitor the composting process, to evaluate the effectiveness of bioindicators for the quality and maturity of cured compost obtained by a mixture of winery residues, sludges from dairies and solid residues from food processing (grape-stalks, grape-dregs, rice husks), was conducted. Composting process lasting five months was monitored by chemico-physical, spectroscopic (FTIR, DTG and DSC), microbiological and enzymatic analyses. Biological activities (ATP, DHA contents and several enzymatic activities), impedance variations (DT) of mixed cultures during growth and potential pathogens (E. coli and Salmonella sp.), were determined. The phytotoxicity tests gave a germination index higher than 90% and no significant genotoxic differences between controls and the compost samples were evidenced. Pathogens were not found on the cured compost that can therefore be satisfactorily used as amendment for agricultural crops. However, no single measurement of a composting process factor, biological, chemical or physical, gave a comprehensive view of the quality of a specific composting. We proposed a tool of bioindicators of potential activity and markers in combination for integrated evaluation of monitoring of composting process and compost quality. The responses of several enzymatic activities were positive and indicative of their favorable use capable to reveal even very small changes within microbial population and activity in test and monitoring of compost programmes.

Bacterial communities associated with the rhizosphere of transgenic Bt 176 maize (Zea mays) and its non transgenic counterpart

The effect of transgenic Bt 176 maize on the rhizosphere bacterial community has been studied with a polyphasic approach by comparing the rhizosphere of Bt maize cultivated in greenhouse with that of its non transgenic counterpart grown in the same conditions. In the two plants the bacterial counts of the copiotrophic, oligotrophic and sporeforming bacteria, and the community level catabolic profiling, showed no significant differences; differences between the rhizosphere and bulk soil bacterial communities were evidenced. Automated ribosomal intergenic spacer analysis (ARISA) showed differences also in the rhizosphere communities at different plant ages, as well as between the two plant types. ARISA fingerprinting patterns of soil bacterial communities exposed to root growth solutions, collected from transgenic and non transgenic plants grown in hydroponic conditions, were grouped separately by principal component analysis suggesting that root exudates could determine the selection of different bacterial communities.

Improved plant resistance to drought is promoted by the root‐associated microbiome as a water stress‐dependent trait

Although drought is an increasing problem in agriculture, the contribution of the root-associated bacterial microbiome to plant adaptation to water stress is poorly studied. We investigated if the culturable bacterial microbiome associated with five grapevine rootstocks and the grapevine cultivar Barbera may enhance plant growth under drought stress. Eight isolates, over 510 strains, were tested in vivo for their capacity to support grapevine growth under water stress. The selected strains exhibited a vast array of plant growth promoting (PGP) traits, and confocal microscopy observation of gfp-labelled Acinetobacter and Pseudomonas isolates showed their ability to adhere and colonize both the Arabidopsis and grapevine rhizoplane. Tests on pepper plants fertilized with the selected strains, under both optimal irrigation and drought conditions, showed that PGP activity was a stress-dependent and not a per se feature of the strains. The isolates were capable of increasing shoot and leaf biomass, shoot length, and photosynthetic activity of drought-challenged grapevines, with an enhanced effect in drought-sensitive rootstock. Three isolates were further assayed for PGP capacity under outdoor conditions, exhibiting the ability to increase grapevine root biomass. Overall, the results indicate that PGP bacteria contribute to improve plant adaptation to drought through a water stress-induced promotion ability.

Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes

Aims:  To set up and employ, for the biorestoration of cultural heritage (altered frescoes), an advanced and innovative biotechnology method based on the sequential use of whole viable bacterial cells and specific enzymes.

Advantages of Using Microbial Technology over Traditional Chemical Technology in Removal of Black Crusts from Stone Surfaces of Historical Monuments

ABSTRACT This study compares two cleaning methods, one involving an ammonium carbonate-EDTA mixture and the other involving the sulfate-reducing bacterium Desulfovibrio vulgaris subsp. vulgaris ATCC 29579, for the removal of black crust (containing gypsum) on marble of the Milan Cathedral (Italy). In contrast to the chemical cleaning method, the biological procedure resulted in more homogeneous removal of the surface deposits and preserved the patina noble under the black crust. Whereas both of the treatments converted gypsum to calcite, allowing consolidation, the chemical treatment also formed undesirable sodium sulfate.

Improved Methodology for Bioremoval of Black Crusts on Historical Stone Artworks by Use of Sulfate-Reducing Bacteria

ABSTRACT An improved methodology to remove black crusts from stone by using Desulfovibrio vulgaris subsp. vulgaris ATCC 29579, a sulfate-reducing bacterium, is presented. The strain removed 98% of the sulfates of the crust in a 45-h treatment. Precipitation of black iron sulfide was avoided using filtration of a medium devoid of iron. Among three cell carriers, Carbogel proved to be superior to both sepiolite and Hydrobiogel-97, as it allowed an easy application of the bacteria, kept the system in a state where microbial activity was maintained, and allowed easy removal of the cells after the treatment.

Microorganisms Attack Synthetic Polymers in Items Representing Our Cultural Heritage

With advancements in materials science over the past few decades, there has been a dramatic increase in the use of synthetic polymers by both artists and conservators. Synthetic polymers in items representing our cultural heritage occur either as original constituents of works of art or as materials used for conservation treatment, and these polymers include adhesives, consolidants, and protective coatings. In the 1980s there was a change in the perception of plastics from consumer goods and disposable materials to fashionable, highly collectable pieces with historical and technological significance (27, 34). Now, in their 20th and 21st century collections, most museums and galleries possess objects made from the thousands of different plastics that have been produced. As museums keep acquiring objects that reflect both everyday life and technological and historical events, the proportion of plastics in museums is increasing dramatically. Plastics may be present in objects of everyday life, such as housewares, jewelry, equipment, furniture, information technology, photography, and toys, and more of these objects are entering museum collections and contemporary art (57). In addition, synthetic polymers have been widely employed for treatment of items representing our cultural heritage as adhesives, consolidants, and protective coatings to preserve many artifacts from further deterioration (20, 45). Synthetic polymer conservation has been formally recognized as a research area only since the 1990s, and it was in this period that the worlds most important organization in the field of cultural heritage conservation, the Committee for Conservation of the International Council of Museums, established the Modern Materials and Contemporary Art Working Group. Indeed, owners and curators have begun to notice that objects made of plastics degrade with time, sometimes very rapidly. Importantly, many synthetic polymers appear to deteriorate faster than other materials in museum collections and have a useful lifetime of just decades (57). Synthetic polymeric materials can suffer different forms of deterioration, including chemical (e.g., oxidation), physical (e.g., UV light), and biological. Although many reports in the scientific literature claim that microorganisms are capable of degrading synthetic resins (35, 42, 59, 68), the microbial contamination of synthetic polymers that are used as materials for conservation treatment (29, 32) and in contemporary collections (50) is still underestimated. Indeed, it was only in the 2005-2008 program that the Committee for Conservation of the International Council of Museums Modern Materials and Contemporary Art Working Group embraced “(microbial) biodeterioration” as a research topic (http://icom-cc.icom.museum/Documents/WorkingGroup/ModernMaterials/Modern-materials2005-2008.pdf). Microorganisms can damage the structure and function of synthetic polymers. According to Flemming (22), the main types of damage include (i) biological coating masking surface properties, (ii) increased leaching of additives and monomers that are used as nutrients, (iii) production of metabolites (e.g., acids), (iv) enzymatic attack, (v) physical penetration and disruption, (vi) water accumulation, and (vii) excretion of pigments. Table ​Table11 describes microorganisms and their modes of action for degrading synthetic resins (polyvinyl chloride [PVC], polyurethane, nylon, and acrylics). Barbie dolls, together with many other toys, clothes, and electrical insulation found in museums, are made from PVC (58). The instability of plasticized PVC is frequently manifested as migration of the plasticizers. Colonization of PVCs by fungi, especially black fungi, due to the availability of platicizers on the surface has been assessed several times (30, 51, 67). Webb et al. (67) identified fungal isolates obtained from PVC by PCR amplification and partial sequencing of the internally transcribed spacer regions and the 5.8S rRNA gene or the V3 domain of the 28S rRNA gene. TABLE 1. Microorganisms degrading the synthetic polymers PVC, polyurethane, nylon, and acrylics and their mode of action It has been suggested that biodeterioration of polyurethane polymers, which are products of a polyol based on either a polyester or polyether and a di- or polyisocyanate, occurs through enzymatic action of hydrolases, such as ureases, proteases, and esterases (18, 21, 52). Degradation of polyurethanes by microorganisms in 20th century museum textiles has been reported by many researchers (36, 63). Polyurethanes can also be found in products such as furniture, adhesives, paints, elastomers, coatings, and contemporary art (33, 34, 52). Biodeterioration due to enzymes, presumably including a manganese peroxidase of the basidiomycete Bjerkandera adusta, was also observed for the aliphatic polyamide Nylon-6 fiber (24). Damage to the polymer was assessed by microscopic examination, differential scanning calorimetry, and evaluation of changes in viscosity. One of the reasons for introducing synthetic consolidants and protective compounds in conservation treatments was the expectation that these materials would be more resistant to microbial attack than natural organic products. In 1968 the superintendents at Ostia Antica (Rome, Italy) decided to replace natural organic compounds, which are easily degraded by microorganisms, with acrylic compounds in conservation treatments. Frescoes detached with Paraloid did not show any biodeterioration problem for the first 3 years after application (4). However, as early as the 1950s, some experiments on biodeterioration of polyvinyl acetate resins were reported by the Istituto Centrale del Restauro in Rome, Italy (26). Generally, filamentous fungi were the agents causing deterioration of these materials that were studied the most, especially in early experiments (17, 41, 54, 61), although some bacteria, yeasts, algae, and lichens that are capable of growing on synthetic polymers have been found or isolated (14). Historically, identification of filamentous fungal species has been based on morphological characteristics, both macroscopic and microscopic. These methods may often be time-consuming and inaccurate, which has required the development of identification protocols that are rapid, sensitive, and precise. In the last decade molecular approaches for rapid characterization of fungi on painted items representing our cultural heritage and paint coatings have been developed (46, 53). A protocol for efficient extraction of fungal DNA from micromycetes colonizing painted art objects was developed by Mohlenhoff et al. (46), who claimed to have successfully removed any inhibitors. In particular, melanin can also be present, which is highly resistant to UV light, enzymatic digestion, and chemical breakdown and might be a potent inhibitor of DNA amplification (46). PCR amplification of the 28S rRNA gene and denaturing gradient gel electrophoresis analysis were used to characterize fungal communities. According to Saad et al. (53), fungi are commonly found on paint films as spores other than mycelium; hence, it is necessary to ensure that DNA extraction is effective also for propagules. The method used involves spore lysis by incubation of a specimen with the enzyme Lyticase, followed by bead beating. DNA is then purified from the lysate with a QIAamp DNA mini kit (53). There have also been case studies related to biodeterioration agents other than filamentous fungi. Bacterial biofilms composed of Pseudomonas aeruginosa, Ochrobactrum anthropi, Alcaligenes denitrificans, Xanthomonas maltophila, and Vibrio harveyi formed readily on the surfaces of synthetic materials being considered for use in space applications (28). A yeast isolated from a bronze statue treated with the acrylic-based coating Incralac was found to accelerate the deterioration of the coating itself, as determined by scanning electron microscopy and electrochemical impedance spectroscopy (44). Stones impregnated with Ahydrosil Z, a silicone resin, were recolonized by algae and fungi more quickly than untreated specimens (40). Rapid recolonization by the alga Stichococcus bacillaris was also noticed in the Roman archaeological site at Luni in northern Italy after treatment with an epoxy resin and an acrylic-siliconic resin (19). Finally, lichens were reported to deteriorate a synthetic polyester resin that was used as a consolidant of stucco walls and column capitals in the Roman city Baelo Claudia in Spain (2). The ecological succession of fungi over 10 months on two Brazilian buildings painted with a white acrylic paint was described by Shirakawa et al. (60). Prior to painting, the walls were treated with hypochlorite. In addition to Cladosporium, the main fungal genus identified during the experiment, the other fungal genera detected were Alternaria, Curvularia, Epicoccum, Helminthosporium, Coelomycetes, Monascus, Nigrospora, and Aureobasidium. The yeast population fell to undetectable levels after the third week, and this microbial group was not detected again until 7 months, after which the number of cells increased.