P. Barghini | Università degli Studi della Tuscia (original) (raw)

Papers by P. Barghini

25° Congresso Nazionale della Società Italiana di Microbiologia Generale e Biotecnologie Microbiche (SIMGBM),, 2006

Capsaicin, the major pungent principle in hot pepper fruit, can be hydrolyzed enzymatically to va... more Capsaicin, the major pungent principle in hot pepper fruit, can be hydrolyzed enzymatically to vanillylamine (a natural precursor of vanillin) using a specific acylase from Streptomyces mobaraensis. Production of this enzyme using strain DSM40847 was studied under batch fermentation conditions in stirred tank (STR) and airlift (AR) bioreactors. The process performance in both fermentation devices was different with respect to biomass, enzyme concentration and specific yield (enzyme activity/biomass content); in particular the specific yield was lower in the AR (5.7 mU/g of biomass) than in the STR (6.25 mU/g of biomass). Experiments carried out in STR bioreactors at controlled (DO = 20% of saturation) and uncontrolled dissolved oxygen concentration, and at constant stirrer speeds (300, 450 and 600 rpm) demonstrated that the DO level has no remarkable effect on the production of the capsaicin-hydrolyzing enzyme, which is mainly produced in a cell-associated form.

Food Technology and …, 2004

Yeast extract, Luria-Bertani medium and tryptone were tested as co-substrates for vanillin produc... more Yeast extract, Luria-Bertani medium and tryptone were tested as co-substrates for vanillin production from ferulic acid by resting cells of Escherichia coli JM109/pBB1. Yeast extract proved to be the best component for sustaining such a bioconversion, which is not self-sustained from the bioenergetic point of view. Tests were also performed under variable aeration conditions by simultaneously varying the ratio of medium to vessel volume and the agitation speed. The results of these tests suggest that, under excess aeration, a non-specific oxidase activity was very likely responsible for the oxidation of a significant portion of vanillin to vanillic acid, thus reducing the vanillin yield.

In: La ricerca biotecnologica al servizio del consumatore attraverso l'industria alimentare. ... more In: La ricerca biotecnologica al servizio del consumatore attraverso l'industria alimentare. p. 85-94. Con il patrocinio della Universita' degli Studi= di Bologna. Convegno tenutosi a Bologna il 20-21 novembre 1995 nell'ambito del sottoprogetto 4 del P.F. RAISAConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Ann. …, 2004

The present work deals with a novel bioconversion of ferulate into vanillin using resting cells o... more The present work deals with a novel bioconversion of ferulate into vanillin using resting cells of Escherichia coli strain JM109/pBB1 as a biocatalyst. Biomass recycling from four successive bioconversion steps demonstrated the possibility of employing the proposed resting cell system for the continuous production of vanillin. Among the tested immobilization supports (polyurethane, synthetic sponge and porous glass) the synthetic sponge proved to be the best material in terms of both vanillin formation (C v = 0.080 g l-1) and productivity (Q v = 0.019 g l-1 h-1) at the end of entrapment tests. Thus, it was used in preliminary continuous tests using a fixed-bed column with immobilized E. coli JM109/pBB1 cells. The highest vanillin yield (Y P/S = 0.851 mol mol-1) was obtained at a dilution rate of 0.022 h-1 .

Microbial Cell Factories, 2007

Background Vanillin is one of the most important aromatic flavour compounds used in the food and ... more Background Vanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details. Results Effect of plasmid copy number in metabolic engineering of E. coli for the synthesis of vanillin has been evaluated by the use of genes encoding feruloyl-CoA synthetase and feruloyl hydratase/aldolase from Pseudomonas fluorescens BF13. The higher vanillin production yield was obtained using resting c...

Brazilian Journal of Microbiology, 2003

Vanillin production from ferulate was studied using different recombinant strains of Escherichia ... more Vanillin production from ferulate was studied using different recombinant strains of Escherichia coli. To prevent the occurrence of aerobic conditions and then possible product oxidation, tests were performed in Erlenmeyer flasks under mild mixing (150 rpm). Among other transformants, E. coli JM109(pBB1) appeared to be the best vanillin producer, being able to convert no less than 95% of starting ferulate to the product within 1h. This yield decreased down to 72% after 72h, likely because of a non-specific oxidase activity responsible for vanillin oxidation to vanillate.

Applied Microbiology and Biotechnology, 2008

In Pseudomonas fluorescens BF13, the cluster of genes essential for degradation of ferulic to van... more In Pseudomonas fluorescens BF13, the cluster of genes essential for degradation of ferulic to vanillic acid (ech, vdh and fcs) is expressed in ferulic but not in succinicgrown cells. In the upstream region, we identified a gene, ferR, encoding a protein homologous to transcriptional regulators of the MarR family. A ferR knockout mutant (BF13-89) showed a 3.5-fold increase in expression of an ech-reporter gene fusion compared with the parent strain in succinic-grown cells, indicating that the ferR gene product negatively regulates expression of the ferulic catabolic operon in P. fluorescens BF13. Consistent with the increased expression of the catabolic genes in the ferR mutant, BF13-89 showed a shorter (relative to its FerR + parent) lag phase during carbon source shift from succinic to ferulic acid. However, expression of ech-lacZ fusion did not increase in BF13-89 grown in the presence of ferulic acid, indicating that FerR has a second function as transcriptional activator. Expression of ech-lacZ in a feruloyl-CoA synthetase-deficient strain revealed unambiguously that FerR-mediated activation of the ferulic catabolic operon is dependent on the thioester product of the feruloyl-CoA synthetase reaction.

Applied and Environmental Microbiology, 2000

From a ferulic-acid-degrading Pseudomonas fluorescensstrain (BF13), we have isolated a transposon... more From a ferulic-acid-degrading Pseudomonas fluorescensstrain (BF13), we have isolated a transposon mutant, which retained the ability to bioconvert ferulic acid into vanillic acid but lost the ability to further degrade the latter acid. The mutant, BF13-97, was very stable, and therefore it was suitable to be used as a biocatalyst for the preparative synthesis of vanillic acid from ferulic acid. By use of resting cells we determined the effect on the bioconversion rate of several parameters, such as the addition of nutritional factors, the concentration of the biomass, and the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with cells pregrown on M9 medium containing p-coumaric acid (0.1% [wt/vol]) as a sole carbon source and yeast extract (0.001% [wt/vol]) as a source of nutritional factors. Under these conditions, 1 mg (wet weight) of biomass produced 0.23 mg of vanillic acid per h. The genomic region of BF13-97 flanking the transposon&...

Annals of Microbiology, 2014

ABSTRACT Kandalaksha Bay is an estuarine system located around the North Polar Circle in the Whit... more ABSTRACT Kandalaksha Bay is an estuarine system located around the North Polar Circle in the White Sea (Russia). This peculiar environment, showing big sea level differences during tide cycles causing intense water mixing, is almost unknown concerning its microbial diversity. In this work, seawater bacterial communities, mainly obtained from a coastal area, were studied in order to gather information on their structure and most abundant populations. The study was carried out by cluster analysis of polymerase chain reaction–temperature-gradient gel electrophoresis (PCR-TGGE) fingerprinting of partial 16S-rRNA gene amplicons. Bacterial communities were strongly homogenized by tidal water mixing, especially on surface layers and close to the shore. Samples collected from the intertidal zone and the nearby sea surface grouped together with a high percentage of similarity, while those taken offshore at various depths showed evident differences. Multivariate analysis indicated depth as the most significant environmental parameter causing variations in the community structure. High levels of diversity were revealed by both the Simpson’s index of diversity and the range weighted richness index. The functional organization index suggested that the community was potentially able to preserve its functionality under stressing environmental perturbations. Sequencing of TGGE bands showed that most of the bacteria populations were evolutionarily close to α-proteobacteria. Some γ-proteobacteria and Actinobacteria were revealed too. This work represents the first major contribution to understanding bacterial diversity in Kandalaksha Bay.

Annals of Microbiology, 2015

ABSTRACT Due to huge yearly variations of environmental stressing conditions, Kandalaksha Bay (Ar... more ABSTRACT Due to huge yearly variations of environmental stressing conditions, Kandalaksha Bay (Arctic Circle, White Sea, Russia) could represent a model to study microbial adaptation in extreme environments. This peculiar estuarine system has been scarcely investigated for its microbial diver- sity. In this work, to gather information on their nutritional competencies, seawater planktonic bacteria were studied for their ability to use different carbon sources by the Biolog phenotype microarray assays. Nestedness, a useful statistical tool used in ecology, was employed to underline nutritional differences among microbial groups. In particular, nestedness was used to understand the complex relationship that is established when many nutrients are available for various mi- croorganisms, and to highlight presence of specialists and generalists. Among the studied bacteria, which showed very diverse nutritional abilities, 47% belonged to Pseudomonas, 21% to Serratia and 32% to other Genera. Within Pseudomonas, both highly generalist and highly specialist strains were discovered. However, most of them used organic and/or amino acids as principal carbon sources. In contrast, Serratia strains typically preferred sugars and appeared to be more generalist. On the whole, important differences in specialization levels and nutritional competencies were recorded in strains belonging to the same species. Correlations between phylogenetic and nutritional data were validated by Procrustes analysis.

Ochratoxin A (OTA), a potent nephrotoxic and human carcinogenic agent, is found fre quently in ag... more Ochratoxin A (OTA), a potent nephrotoxic and human carcinogenic agent, is found fre quently in agricultural products such as cereals, coffee, cocoa beans, dried fruits, beers, grape musts and wines. OTA is produced by several molds belonging to species o f Aspergillus and Penicillus. Several studies showed that A. carbonarius is the main OTA-producer in grapes and musts, particu larly during post-harvest. Cell-wall degrading enzymes (CWDE), such as chitinases and glucanases, could reduce fungal contamination on several plant structures and could be used in food technology to reduce occurrence of these contaminants. The Antarctic fungus Lecanicillium muscarium CCFEE 5003 is a strong producer of CWDE, chitinases in particular. In this work, w hite and black grape bunches were inoculated with spore suspension of A. carbonarius to simulate natural contamina tion. Grapes were then treated with crude CWDE solution from L. muscarium in order to study possible inhibition o f the OTA-producer in post-harvest conditions. Presence of contaminants on untreated grapes (control) was very high (> 2000 cfu/ml). On treated bunches residual spores were 95 and 89% less than on the control, in white and red grape, respectively. Light microscopy showed that the enzyme solution caused various spore damages such as cell-wall disruption and protoplast formation and bursting.

A bstract. Hydrogen is a clean energy carrier having great potential as alternative fuel. Despite... more A bstract. Hydrogen is a clean energy carrier having great potential as alternative fuel. Despite its well-established production, by chemical/electrochemical processes, bio-conversion o f organic wastes to hydrogen could be a sustainable alternative since the afore-mentioned methods require a lot o f energy. Anaerobic digestion to bio-methane is considered a winning strategy to transform wastes into energy with reduction o f environmental issues. Chitin is the second most abundant polysaccharide after cellulose. Huge amounts o f chitinous wastes, produced from seafood industry and fungal fermentation plants, represent source o f pollution if improperly disposed. In this paper the feasibility to obtain hydrogen and methane in a two-phase anaerobic bio-process using raw chitin was investigated. After a preliminary aerobic pre-hydrolysis, carried out by the chitinolytic fungus Lecanicillium muscarium CCFEE 5003, H, was obtained by dark fermentation and CH4, subsequently, by further digestion. For best productions, pre-hydrolysis was optimised by response surface methodology. Highest hydrogen (147 ml/1) and methane (7713 ml/1) levels were obtained after 24 days o f dark fermentation and 83 days o f digestion, respectively. However, best productivi ties were obtained at day 14 and 30 for H, and CH4, respectively. This work is the first attempt to use raw chitin to obtain these biofuels by dark fermentation and anaerobic digestion.

Salterns represent peculiar environm ents showing wide gradient o f salinity and high variation o... more Salterns represent peculiar environm ents showing wide gradient o f salinity and high variation o f other chemicophysical parameters. Being subm itted to sudden and repeated fluctua tions o f temperature and water availability, they could be taken as a prototypical example o f global changes. Here, organism s must cope with huge environmental stress. Prokaryotic comm unities are predominant at highest salinity representing a valid model to study these environm ents and possible adaptation strategies. To get a complete picture o f total microbial diversity, cultural and cultureindependent methods must be integrated. In this study, the microbial com m unities o f various ponds within the 'Saline di Tarquinia' saltern, Italy has been studied by cultural and cultural-independent methods (PCR-DGGE). Average bacterial counts ranged from 0.2 to 2.5* 105 cells/ml. Twelve bacte rial strains were isolated in pure culture. The majority (67%) belonged to the y-proteobacteria class. Species affiliated to Bacilli and Actinobacteria were found also. PCR-DGGE fingerprinting showed a m ore com plex situation. In fact, at least 32 bands, theoretically corresponding to single species, w ere detected. Re-amplification o f predominant bands led to define a com posite com m unity mostly constituted by marine and halophilic species. This study represents the first detailed investigation on the bacterial com m unity o f Saline di Tarquinia.

Enzyme and Microbial Technology, 2013

The Antarctic fungus Lecanicillium muscarium CCFEE-5003 was preliminary cultivated in shaken flas... more The Antarctic fungus Lecanicillium muscarium CCFEE-5003 was preliminary cultivated in shaken flasks to check its chitinase production on rough shrimp and crab wastes. Production on shrimp shells was much higher than that on crab shells (104.6±9.3 and 48.6±3.1U/L, respectively). For possible industrial applications, bioprocess optimization was studied on shrimp shells in bioreactor using RSM to state best conditions of pH and substrate concentration. Optimization improved the production by 137% (243.6±17.3). Two chitinolytic enzymes (CHI1 and CHI2) were purified and characterized. CHI1 (MW ca. 61kDa) showed optima at pH 5.5 and 45°C while CHI2 (MW ca. 25kDa) optima were at pH 4.5 and 40°C. Both enzymes maintained high activity levels at 5°C and were inhibited by Fe(++), Hg(++) and Cu(++). CHI2 was strongly allosamidin-sensitive. Both proteins were N-acetyl-hexosaminidases (E.C. 3.2.1.52) but showed different roles in chitin hydrolysis: CHI1 could be defined as "chitobiase" while CHI2 revealed a main "eso-chitinase" activity.

Microbial Cell Factories, 2012

Background: The Antarctic fungus Lecanicillium muscarium CCFEE 5003 is one of the most powerful c... more Background: The Antarctic fungus Lecanicillium muscarium CCFEE 5003 is one of the most powerful chitinolytic organisms. It can produce high level of chitinolytic enzymes in a wide range of temperatures (5-30°C). Chitinolytic enzymes have lot of applications but their industrial production is still rather limited and no cold-active enzymes are produced. In view of massive production of L. muscarium chitinolytic enzymes, its cultivation in bioreactors is mandatory. Microbial cultivation and/or their metabolite production in bioreactors are sometime not possible and must be verified and optimized for possible exploitation. Agitation and aeration are the most important parameters in order to allow process up-scaling to the industrial level. Results: In this study, submerged cultures of L. muscarium CCFEE 5003 were carried out in a 2-L bench-top CSTR bioreactor in order to optimise the production of chitinolytic enzymes. The effect of stirrer speed (range 200-500 rpm) and aeration rate (range 0.5-1.5 vvm) combination was studied, by Response Surface Methodology (RSM), in a medium containing 1.0% yeast nitrogen base and 1% colloidal chitin. Optimization was carried out, within a "quadratic D-optimal" model, using quantitative and quantitative-multilevel factors for aeration and agitation, respectively. The model showed very good correlation parameters (R 2 , 0.931; Q 2 , 0.869) and the maximum of activity (373.0 U/L) was predicted at ca. 327 rpm and 1.1 vvm. However, the experimental data showed that highest activity (383.7 ± 7.8 U/L) was recorded at 1 vvm and 300 rpm. Evident shear effect caused by stirrer speed and, partially, by high aeration rates were observed. Under optimized conditions in bioreactor the fungus was able to produce a higher number of chitinolytic enzymes than those released in shaken flasks. In addition, production was 23% higher. Conclusions: This work demonstrated the attitude of L. muscarium CCFEE 5003 to grow in bench-top bioreactor; outlined the strong influence of aeration and agitation on its growth and enzyme production and identified the optimal conditions for possible production at the industrial level.

Pseudomonas¯uorescens BF13 is especially capable of promoting the formation of vanillic acid duri... more Pseudomonas¯uorescens BF13 is especially capable of promoting the formation of vanillic acid during ferulic acid degradation. We studied the possibility of enhancing the formation of this intermediary metabolite by using suspensions of cells at high density. The bioconversion of ferulic into vanillic acid was affected by several parameters, such as the concentration of the biomass, the amount of ferulic acid that was treated, the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with 6 mg/ml cells pre-grown on p-coumaric acid and 2 mg/ml ferulic acid. Under these conditions the bioconversion rate was 95% in 5 h. Therefore BF13 strain represents a valid biocatalyst for the preparative synthesis of vanillic acid.