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Microbial-based products are a promising alternative to agrochemicals in sustainable agriculture. However, little is known about their impact on human health even if some of them, i.e., Bacillus and Paenibacillus species, have been increasingly implicated in different human diseases. In this study, 18 bacteria were isolated from 2 commercial biostimulants, and they were genotypically and phenotypically characterized to highlight specific virulence properties. Some isolated bacteria were identified as belonging to the genus Bacillus by BLAST and RDP analyses, a genus in-depth studied for plant growth-promoting ability. Moreover, 16S rRNA phylogenetic analysis showed that seven isolates grouped with Bacillus species while two and four clustered, respectively, with Neobacillus and Peribacillus. Unusually, bacterial strains belonging to Franconibacter and Stenotrophomonas were isolated from biostimulants. Although Bacillus species are generally considered nonpathogenic, most of the species have shown to swim, swarm, and produced biofilms, that can be related to bacterial virulence. The evaluation of toxins encoding genes revealed that five isolates had the potential ability to produce the enterotoxin T. In conclusion, the pathogenic potential of microorganisms included in commercial products should be deeply verified, in our opinion. The approach proposed in this study could help in this crucial step.

The pressure to increasingly optimize the breeding of livestock monogastric animals resulted in antimicrobials often being misused in an attempt to improve growth performance and counteract diseases in these animals, leading to an increase in the problem of antibiotic resistance. To tackle this problem, the use of probiotics, also known as direct in-feed microbials (DFM), seems to be one of the most promising strategies. Among probiotics, the interest in Bacillus strains has been intensively increased in recent decades in pigs and poultry. The aim of the present review was to evaluate the effectiveness of Bacillus strains as probiotics and as a potential strategy for reducing the misuse of antibiotics in monogastric animals. Thus, the potential modes of action, and the effects on the performance and health of pigs (weaning pigs, lactation and gestation sows) and broilers are discussed. These searches yielded 131 articles (published before January 2021). The present review showed that Bacillus strains could favor growth in terms of the average daily gain (ADG) of post-weaning piglets and broilers, and reduce the incidence of post-weaning diarrhea in pigs by 30% and mortality in broilers by 6–8%. The benefits of Bacillus strains on these parameters showed results comparable to the benefit obtained by the use of antibiotics. Furthermore, the use of Bacillus strains gives promising results in enhancing the local adaptative immune response and in reducing the oxidative stress of broilers. Fewer data were available regarding the effect on sows. Discordant effects have been reported regarding the effect on body weight (BW) and feed intake while a number of studies have supported the hypothesis that feeding probiotics to sows could benefit their reproductive performance, namely the BW and ADG of the litters. Taken all the above-mentioned facts together, this review confirmed the effectiveness of Bacillus strains as probiotics in young pigs and broilers, favoring their health and contributing to a reduction in the misuse of direct in-feed antibiotics. The continuous development and research regarding probiotics will support a decrease in the misuse of antibiotics in livestock production in order to endorse a more sustainable rearing system in the near future.

Surface display systems have been developed to express target molecules on almost all types of biological entities from viruses to mammalian cells and on a variety of synthetic particles. Various approaches have been developed to achieve the display of many different target molecules, aiming at several technological and biomedical applications. Screening of libraries, delivery of drugs or antigens, bio-catalysis, sensing of pollutants and bioremediation are commonly considered as fields of potential application for surface display systems. In this review, the non-recombinant approach to display antigens and enzymes on the surface of bacterial spores is discussed. Examples of molecules displayed on the spore surface and their potential applications are summarized and a mechanism of display is proposed.

Die aseptische Prozesstechnik hat sich zu einer beliebten Technologie entwickelt, um die Haltbarkeit von verpackten Produkten zu verlängern und den Verbrauchern nicht kontaminierte Waren zur Verfügung zu stellen. Im Jahr 2017 wurde der globale Aseptikmarkt auf rund 39,5 Mrd. USD geschätzt. Viele flüssige Lebensmittelprodukte, wie Saft oder Milch, werden täglich mit aseptischen Abfüllmaschinen produziert und an die Kunden geliefert. Moderne Abfüllanlagen können ungefähr 12.000 fertig verpackte Produkte pro Stunde befüllen (z. B. Pure-Pak® Aseptic Filling Line E-PS120A). Sie müssen jedoch routinemäßig validiert werden, um eine kontaminationsfreie Ware zu gewährleisten. Die Verfahren zur Validierung derartiger Maschinen erfolgen häufig mittels mikrobiologischer Analysen, wobei bakterielle Sporen als Testorganismen wegen ihrer hohen Beständigkeit gegenüber verschiedenen Sterilisationsmittels (z.B. gasförmiges Wasserstoffperoxid) verwendet werden. Der Hauptnachteil der vorgenannten Tests ist deren Läufe: Es dauert mindestens 36-48 Stunden, um die Ergebnisse zu erhalten, d.h. die Produkte können nicht ohne das Validierungszertifikat an den Kunden geliefert werden. Nur in diesem Beispiel würden in 36 Stunden ca. 432.000 Produkte für den Versand zurückgestellt. Beim parallelen Betreib mehrerer Maschinen würde diese Zahl linear ansteigen und am Ende wären die Kosten (nur für das Warten auf die Ergebnisse) beträchtlich hoch. Aus diesem Grund ist es sinnvoll, neue Sensortechnologien zu entwickeln, um dieses Problem zu lösen. Daher liegt der Schwerpunkt dieser Arbeit auf der Weiterentwicklung eines sporenbasierten Biosensors; dieser Sensor kann die Lebensfähigkeit von Sporen nach der Sterilisation mit Wasserstoffperoxid bestimmen. Es fehlen jedoch eine reproduzierbare Immobilisierungsstrategie sowie deren Implementierung auf Sensorelementen, sowie eine detailliertere Untersuchung hinsichtlich ihres Funktionsprinzips. In dieser Arbeit wird eine Immobilisierungsstrategie für sporenbasierte Biosensoren entwickelt, um rauen Bedingungen (hohe Temperaturen, oxidierende Umgebung) standzuhalten, wo sie in der aseptischen Verpackung eingesetzt werden. Eine systematische Untersuchung der Wirkung der Oberflächenfunktionalisierung (z.B. Hydroxylierung) auf Sensoren (z.B. Elektrolyt-Isolator-Halbleiter- (EIS-) Chips) wird vorgestellt. Im Anschluss werden Organosilane bezüglich der Immobilisierung von Bakteriensporen auf verschiedenen Sensoroberflächen charakterisiert. Die elektrischen Eigenschaften derImmobilisierungsschicht sowie ihre Beständigkeit gegenüber einem Sterilisationsprozess mit gasförmigem Wasserstoffperoxid werden validiert. Zusätzlich wird ein Sensorarray bestehend aus einem kalorimetrischen Gassensor und einem sporenbasierten Biosensor zur gleichzeitigen Messung der Wasserstoffperoxidkonzentration und der Lebensfähigkeit der Sporen vorgeschlagen, um die Wirksamkeit von Sterilisationsprozessen zu bewerten., Aseptic processing has become a popular technology to increase the shelf-life of packaged products and to provide non-contaminated goods to the consumers. In 2017, the global aseptic market was evaluated to be about 39.5 billion USD. Many liquid food products, like juice or milk, are delivered to customers every day by employing aseptic filling machines. They can operate around 12,000 ready-packaged products per hour (e.g., Pure-Pak® Aseptic Filling Line E-PS120A). However, they need to be routinely validated to guarantee contamination-free goods. The state-of-the-art methods to validate such machines are by means of microbiological analyses, where bacterial spores are used as test organisms because of their high resistance against several sterilants (e.g., gaseous hydrogen peroxide). The main disadvantage of the aforementioned tests is time: it takes at least 36-48 hours to get the results, i.e., the products cannot be delivered to customers without the validation certificate. Just in this example, in 36 hours, 432,000 products would be on hold for dispatchment; if more machines are evaluated, this number would linearly grow and at the end, the costs (only for waiting for the results) would be considerably high. For this reason, it is very valuable to develop new sensor technologies to overcome this issue. Therefore, the main focus of this thesis is on the further development of a spore-based biosensor; this sensor can determine the viability of spores after being sterilized with hydrogen peroxide. However, the immobilization strategy as well as its implementation on sensing elements and a more detailed investigation regarding its operating principle are missing. In this thesis, an immobilization strategy is developed to withstand harsh conditions (high temperatures, oxidizing environment) for spore-based biosensors applied in aseptic processing. A systematic investigation of the surface functionalization’s effect (e.g., hydroxylation) on sensors (e.g., electrolyte-insulator semiconductor (EIS) chips) is presented. Later on, organosilanes are analyzed for the immobilization of bacterial spores on different sensor surfaces. The electrical properties of the immobilization layer are studied as well as its resistance to a sterilization process with gaseous hydrogen peroxide. In addition, a sensor array consisting of a calorimetric gas sensor and a spore-based biosensor to measure hydrogen peroxide concentrations and the spores’ viability at the same time is proposed to evaluate the efficacy of sterilization processes.

Background Spore-forming bacteria of the Bacillus genus are widely used probiotics known to exert their beneficial effects also through the stimulation of the host immune response. The oral delivery of B. toyonensis spores has been shown to improve the immune response to a parenterally administered viral antigen in mice, suggesting that probiotics may increase the efficiency of systemic vaccines. We used the C fragment of the tetanus toxin (TTFC) as a model antigen to evaluate whether a treatment with B. toyonensis spores affected the immune response to a mucosal antigen. Results Purified TTFC was given to mice by the nasal route either as a free protein or adsorbed to B. subtilis spores, a mucosal vaccine delivery system proved effective with several antigens, including TTFC. Spore adsorption was extremely efficient and TTFC was shown to be exposed on the spore surface. Spore-adsorbed TTFC was more efficient than the free antigen in inducing an immune response and the probiotic treatment improved the response, increasing the production of TTFC-specific secretory immunoglobin A (sIgA) and causing a faster production of serum IgG. The analysis of the induced cytokines indicated that also the cellular immune response was increased by the probiotic treatment. A 16S RNA-based analysis of the gut microbial composition did not show dramatic differences due to the probiotic treatment. However, the abundance of members of the Ruminiclostridium 6 genus was found to correlate with the increased immune response of animals immunized with the spore-adsorbed antigen and treated with the probiotic. Conclusion Our results indicate that B. toyonensis spores significantly contribute to the humoral and cellular responses elicited by a mucosal immunization with spore-adsorbed TTFC, pointing to the probiotic treatment as an alternative to the use of adjuvants for mucosal vaccinations.

This study investigated for the first time the decontamination efficacy of a probiotic-based cleaning product containing Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium spores on fresh and reused broiler litters during 3 rearing cycles of 6 wk each. Moreover, the impact of reused litters treated with the cleaning product on the chicken caeca microbiota was assessed at the end of the rearing cycles in comparison to untreated litter. The Bacillus spores provided with the cleaning treatment were able to successfully colonize the reused poultry litters, decreasing the mean counts of total aerobic bacteria, Enterobacteriaceae, and coagulase positive Staphylococci. The decrease of Enterobacteriaceae, mainly represented by the genus Escherichia, was also observed in the caeca of broilers reared on reused litters treated with the cleaning product. Moreover, the treatment retained the caeca content of Ruminococcaceae and Faecalibacterium as well as the level of biodiversity among the bacteria genera colonizing the caeca of animals reared on reused litter. Overall, the results of this study highlight a positive effect of the probiotic-based cleaning strategy on the microbial decontamination of reused litters and on broiler caeca stability, thereby enhancing animal health and prevention of poultry diseases.

The interaction between the enteric microbiota and intestinal cells often involves signal molecules that affect both microbial behaviour and host responses. Examples of such signal molecules are the molecules secreted by bacteria that induce quorum sensing mechanisms in the producing microorganism and signal transduction pathways in the host cells. The pentapeptide competence and sporulation factor (CSF) of Bacillus subtilis is a well characterized quorum sensing factor that controls competence and spore formation in the producing bacterium and induces cytoprotective heat shock proteins in intestinal epithelial cells. We analysed several Bacillus strains isolated from human ileal biopsies of healthy volunteers and observed that some of them were unable to produce CSF but still able to act in a CSF-like fashion on model intestinal epithelial cells. One of those strains belonging to the Bacillus megaterium species secreted at least two factors with effects on intestinal HT29 cells: a peptide smaller than 3 kDa able to induce heat shock protein 27 (hsp27) and p38-MAPK, and a larger molecule able to induce protein kinase B (PKB/Akt) with a pro-proliferative effect.

The use of bacterial strains as agents in bioremediation processes could reduce the harmfulness of potential toxic elements (PTEs) from water and soil with low or even no impact on the natural ecosystems. In this study, two new metal resistant-bacterial strains (Q3 and Q5) of Bacillus sp. were isolated from a sulfurous spring and their potential (as pure cultures or mixed) to remove Pb(II) and Cd(II) from an aqueous matrix was evaluated and optimized using response surface methodology (RSM). The optimal conditions for Cd(II) removal from all tested strains combinations were observed at an initial pH 5, a temperature of 38 &deg, C, and an initial Cd(II) concentration of 50 mg L&minus, 1, while the performance of bacterial strains on Pb(II) removal was strongly correlated to initial pH and temperature conditions. Moreover, the efficiency of bacterial strains in removing both PTEs, Pb(II) and Cd(II), from an aqueous matrix was considerably higher when they were used as a mixed culture rather than pure. According to field emission SEM (FESEM) and EDS analysis, the two bacterial strains showed different mechanisms in removing Cd(II): Bacillus sp. Q5 bio-accumulated Cd(II) in its periplasmic space, whereas Bacillus sp. Q3 bio-accumulated Cd(II) on its cell surface. On the other hand, Pb(II) is removed by chemical precipitation (lead sulfide) induced by both Bacillus sp. Q3 and Q5. This study discloses new aspects of Pb(II) and Cd(II) bioremediation mechanisms in Bacillus species that can be extremely useful for designing and operating novel PTEs bioremediation processes.

The ability to produce an extracellular matrix and form multicellular communities is an adaptive behavior shared by many bacteria. In Bacillus subtilis, the model system for spore-forming bacteria, matrix production is one of the possible differentiation pathways that a cell can follow when vegetative growth is no longer feasible. While in B. subtilis the genetic system controlling matrix production has been studied in detail, it is still unclear whether other spore formers utilize similar mechanisms. We report that SF214, a pigmented strain of Bacillus pumilus isolated from the marine environment, can produce an extracellular matrix relying on orthologs of many of the genes known to be important for matrix synthesis in B. subtilis. We also report a characterization of the carbohydrates forming the extracellular matrix of strain SF214. The isolation and characterization of mutants altered in matrix synthesis, pigmentation, and spore formation suggest that in strain SF214 the three processes are strictly interconnected and regulated by a common molecular mechanism.

Aims To evaluate the antifungal activity of the volatile organic compounds (VOCs) produced by 75 different food-borne Bacillus species against Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, Aspergillus clavatus, Fusarium oxysporum f. sp. lactucae and Moniliophthora perniciosa and to determine the VOCs responsible for the inhibition. Methods and Results Bacillus strains inhibited fungal growth, although with different inhibition grades, with Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus cereus strains as the best antifungal VOCs producers. While M. perniciosa DM4B and F. oxysporum f.sp. lactucae MA28 were the most sensitive fungi, A. parasiticus MG51 showed the greatest resistance to Bacillus VOCs exposure. Thirty-seven compounds were detected by SPME-GC-MS analysis, although similar patterns in volatile compounds were evidenced within the species, interspecific VOCs differences determined different effects on fungal growth. Multiple partial least regression (MPLRS) and antifungal activity of the individual VOCs revealed that only propanone, 1-butanol, 3-methyl-1-butanol, acetic acid, 2-methylpropanoic acid, carbon disulphide, 3-methylbutanoic acid and ethyl acetate were responsible for mycelia inhibition of M. perniciosa DM4B and F. oxysporum f.sp. lactucae MA28. Conclusions The antagonistic activity of the Bacillus VOCs was demonstrated, although it cannot easily be explained through the action of a single molecule, thus a holistic approach could be more appropriate to estimate the fungal growth inhibition. Significance and Impact of the Study VOCs produced by Bacillus from cooked food can be considered as promising antifungal compounds useful in the control of fungal plant pathogens. This study investigates for the first time the correlation between mycelia inhibition of M. perniciosa and F. oxysporum f. sp. lactucae and the VOCs emitted by the Bacillus species.

BACKGROUND Cellulases and xylanases are the key enzymes involved in the conversion of lignocelluloses into fermentable sugars. Western Ghat region (India) has been recognized as an active hot spot for the isolation of new microorganisms. The aim of this work was to isolate new microorganisms producing cellulases and xylanases to be applied in brewer's spent grain saccharification. RESULTS 93 microorganisms were isolated from Western Ghat and screened for the production of cellulase and xylanase activities. Fourteen cellulolytic and seven xylanolytic microorganisms were further screened in liquid culture. Particular attention was focused on the new isolate Bacillus amyloliquefaciens XR44A, producing xylanase activity up to 10.5 U mL−1. A novel endo-1,4-beta xylanase was identified combining zymography and proteomics and recognized as the main enzyme responsible for B. amyloliquefaciens XR44A xylanase activity. The new xylanase activity was partially characterized and its application in saccharification of brewer's spent grain, pretreated by aqueous ammonia soaking, was investigated. CONCLUSION The culture supernatant of B. amyloliquefaciens XR44A with xylanase activity allowed a recovery of around 43% xylose during brewer's spent grain saccharification, similar to the value obtained with a commercial xylanase from Trichoderma viride, and a maximum arabinose yield of 92%, around 2-fold higher than that achieved with the commercial xylanase. © 2014 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

In an effort to prevent the formation of pathogenic biofilms on hydroxyapatite (HA)-based clinical devices and surfaces, we present a study evaluating the antimicrobial efficacy of Spherical biogenic Se-Nanostructures Embedded in Organic material (Bio Se-NEMO-S) produced by Bacillus mycoides SelTE01 in comparison with two different chemical selenium nanoparticle (SeNP) classes. These nanomaterials have been studied as potential antimicrobials for eradication of established HA-grown biofilms, for preventing biofilm formation on HA-coated surfaces and for inhibition of planktonic cell growth of Pseudomonas aeruginosa NCTC 12934 and Staphylococcus aureus ATCC 25923. Bio Se-NEMO resulted more efficacious than those chemically produced in all tested scenarios. Bio Se-NEMO produced by B.mycoides SelTE01 after 6 or 24h of Na 2 SeO 3 exposure show the same effective antibiofilm activity towards both P.aeruginosa and S.aureus strains at 0.078mgml −1 (Bio Se-NEMO 6 ) and 0.3125mgml −1 (Bio Se-NEMO 24 ). Meanwhile, chemically synthesized SeNPs at the highest tested concentration (2.5mgml −1 ) have moderate antimicrobial activity. The confocal laser scanning micrographs demonstrate that the majority of the P.aeruginosa and S.aureus cells exposed to biogenic SeNPs within the biofilm are killed or eradicated. Bio Se-NEMO therefore displayed good antimicrobial activity towards HA-grown biofilms and planktonic cells, becoming possible candidates as new antimicrobials.

Maltose degrading enzyme was immobilized within agar-agar support via entrapment method due to its industrial utilization. The maximum immobilization efficiency (82.77 %) was achieved using 4.0 % agar-agar keeping the diameter of bead up to 3.0 mm. The matrix entrapment showed maximum catalytic activity at pH 7.0 and temperature 65 °C. Substrate saturation kinetics showed that the K m of immobilized enzyme increased from 1.717 to 2.117 mM ml−1 where as Vmax decreased from 8,411 to 7,450 U ml−1 min−1 as compared to free enzyme. The immobilization significantly increased the stability of maltase against various temperatures and immobilized maltase retain 100 % of its original activity after 2 h at 50 °C, whereas the free maltase only showed 60 % residual activity under same condition. The reusability of entrapped maltase showed activity up to 12 cycles and retained 50 % of activity even after 5th cycle. Storage stability of agar entrapped maltase retain 73 % of its initial activity even after 2 months when stored at 30 °C while free enzyme showed only 37 % activity at same storage conditions. .

Contains fulltext : 153742.pdf (Publisher’s version ) (Closed access) BACKGROUND: The impact of prognostic factors in T1G3 non-muscle-invasive bladder cancer (BCa) patients is critical for proper treatment decision making. OBJECTIVE: To assess prognostic factors in patients who received bacillus Calmette-Guerin (BCG) as initial intravesical treatment of T1G3 tumors and to identify a subgroup of high-risk patients who should be considered for more aggressive treatment. DESIGN, SETTING, AND PARTICIPANTS: Individual patient data were collected for 2451 T1G3 patients from 23 centers who received BCG between 1990 and 2011. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Using Cox multivariable regression, the prognostic importance of several clinical variables was assessed for time to recurrence, progression, BCa-specific survival, and overall survival (OS). RESULTS AND LIMITATIONS: With a median follow-up of 5.2 yr, 465 patients (19%) progressed, 509 (21%) underwent cystectomy, and 221 (9%) died because of BCa. In multivariable analyses, the most important prognostic factors for progression were age, tumor size, and concomitant carcinoma in situ (CIS); the most important prognostic factors for BCa-specific survival and OS were age and tumor size. Patients were divided into four risk groups for progression according to the number of adverse factors among age >/= 70 yr, size >/= 3 cm, and presence of CIS. Progression rates at 10 yr ranged from 17% to 52%. BCa-specific death rates at 10 yr were 32% in patients >/= 70 yr with tumor size >/= 3 cm and 13% otherwise. CONCLUSIONS: T1G3 patients >/= 70 yr with tumors >/= 3 cm and concomitant CIS should be treated more aggressively because of the high risk of progression. PATIENT SUMMARY: Although the majority of T1G3 patients can be safely treated with intravesical bacillus Calmette-Guerin, there is a subgroup of T1G3 patients with age >/= 70 yr, tumor size >/= 3 cm, and concomitant CIS who have a high risk of progression and thus require aggressive treatment.

La microflora della rizosfera riveste un ruolo fondamentale sia nella difesa che nello sviluppo e crescita delle piante. Molti studi hanno dimostrato inoltre che i nematodi sono in grado d’influenzarne la composizione. I batteri a loro volta producono una varietà di composti (antibiotici, nutrienti, esoenzimi e altre molecole segnale), in grado d’indurre ulteriori cambiamenti. L’obiettivo di questo lavoro è stato quello di studiare i cambiamenti nella biodiversità della microflora batterica di un terreno proveniente da un’azienda biologica, conseguenti all'introduzione sperimentale di larve di Meloidogyne incognita e al trattamento con un nematocida (fenamifos). Seguendo un approccio metagenomico, l'RNA totale è stato estratto da campioni di 2 g di terreno, sequenziando la regione ipervariabile V4 del 16S rRNA batterico con piattaforma Illumina MiSeq. Le analisi bioinformatiche delle sequenze ottenute sono state eseguite con Pandaseq e Qiime. Sono stati rinvenuti 578 generi afferenti a 359 famiglie e 197 ordini, che definiscono 4878 OTU in 91 classi e 29 phyla, diversamente rappresentate nei campioni analizzati. Non tutte le sequenze hanno permesso un’identificazione di pari livello tassonomico per tutti i batteri presenti, per via della mancanza d’informazioni relative ai livelli tassonomici inferiori nei database di riferimento. Tra i generi comuni a entrambi i trattamenti sono stati osservati: Candidatus Solibacter, Streptomyces, Rubrobacter, Nitrospira e Steroidobacter, con frequenze variabili in ciascun campione. La presenza di M. incognita e il relativo trattamento nematocida hanno comportato l'introduzione o identificazione ex novo di batteri appartenenti a ulteriori taxa rispetto al controllo. L'analisi metagenomica ha anche mostrato una significativa variazione nella diversità batterica in relazione ai trattamenti effettuati sullo stesso terreno, confermando che essa non sia da considerare un dato statico e definitivo, ma sia soggetta a cambiamenti indotti da variabili ambientali presenti anche su scale e volumi molto ridotti. Dall'analisi delle informazioni ottenute dal sequenziamento e nonostante la comune origine dei campioni di terreno, il grado di biodiversità osservato suggerisce una componente iniziale legata all’eterogenea distribuzione di specie all’origine. Su questa base, il trattamento chimico e la presenza di M. incognita hanno indotto ulteriori cambiamenti sulla biodiversità, sia riducendo che incrementando alcuni generi rispetto ad altri. L'analisi della biodiversità osservata in entrambi gli esperimenti indica inoltre che una parte significativa, se non maggioritaria, delle specie batteriche sequenziate è ancora poco conosciuta dal punto di vista tassonomico. Ciò è indicato dalla presenza di OTUs appartenenti a generi come Pseudomonas e Bacillus per le quali non è ancora possibile assegnare una specie. In parallelo, le stesse conclusioni possono essere dedotte dalla presenza di altri taxa, per i quali non è ancora possibile definire una classificazione certa. Le specie di Bacillus associate a nematodi sono risultate numerose, includendo possibilmente potenziali agenti di biocontrollo. L’identificazione di nuovi antagonisti comporta vantaggi non solo economici, ma anche ambientali, grazie alla riduzione dell’impatto dei trattamenti nematocidi, garantendo sicurezza per operatori e consumatori e incrementando la sostenibilità nella gestione della rizosfera. The rhizosphere microflora plays an important role in plants defense, development and growth. Many studies also showed that soil nematodes are capable of influencing its composition. Soil bacteria in turn produce a variety of compounds (antibiotics, nutrients, exoenzymes and other signal molecules), able to induce further changes. The objective of this work was to study the biodiversity changes of the bacterial microflora from an organic farm soil, following the experimental introduction of juveniles of Meloidogyne incognita and a treatment with a nematicide (fenamiphos). Following a metagenomic approach, total RNA was extracted from 2 g soil samples, sequencing the hypervariable V4 region of the bacterial 16S rRNA with an Illumina MiSeq platform. Bioinformatic analyses of the sequences obtained were performed with Pandaseq and Qiime. A totale of 578 genera belonging to 359 families and 197 orders was found, defining 4878 OTUs from 91 classes and 29 phyla, otherwise represented in the samples analyzed. Not all sequences allowed identification at the same taxonomic level for all bacteria, due to the lack of informations on lower taxonomic levels in the reference database. Among the genera common to both treatments we observed: Candidatus Solibacter, Streptomyces, Rubrobacter, Nitrospira and Steroidobacter, with varying frequencies in each sample. The presence of M. incognita and the nematicidal treatment led to the introduction or de novo identification of bacteria belonging to further taxa, when compared to the control. The metagenomic analysis also showed a significant variation in the bacterial diversity in relation to treatments carried out on the same soil, confirming that biodiversity should not be considered as static and determined, but undergoes changes induced by environmental variables also acting on very small scales and volumes. From the analysis of the sequence data obtained and despite the common origin of the soil samples, the degree of biodiversity observed suggests a component linked to the initial, heterogeneous distribution of species. On this basis, the chemical treatment and the presence of M. incognita induced further biodiversity changes, reducing or increasing certain genera more than others. The analysis of biodiversity observed in both experiments also indicates that a significant portion, if not the majority, of the bacterial species is still poorly understood by the taxonomic point of view. This is shown by the presence of OTUs belonging to genera such as Pseudomonas and Bacillus for which it is not yet possible to assign a species. In parallel, the same conclusions can be derived from the presence of other taxa, for which it is not yet possible to define a certain classification. Bacillus species increased in samples with nematodes, possibly including potential biocontrol agents. The identification of novel antagonists involves not only economic but also environmental benefits, thanks to the reduction of chemicals impacts, ensuring safety for operators and consumers and increasing the sustainability of rhizosphere management.

Small peptides show higher antioxidant capacity than native proteins and may be absorbed in the intestine without further digestion. In our study, a protein by-product from rice starch industry was hydrolyzed with commercial proteolytic enzymes (Alcalase, Neutrase, Flavourzyme) and microbial whole cells of Bacillus spp. and the released peptides were tested for antioxidant activity. Among enzymes, Alcalase was the most performing, while microbial proteolytic activity was less efficient. Conversely, the antioxidant activity was higher in the samples obtained by microbial hydrolysis and particularly with Bacillus pumilus AG1. The sequences of low molecular weight antioxidant peptides were determined and analyzed for aminoacidic composition. The results obtained so far suggest that the hydrolytic treatment of this industrial by-product, with selected enzymes and microbial systems, can allow its exploitation for the production of functional additives and supplements rich in antioxidant peptides, to be used in new food formulas for human consumption.

The Bag320 satellite DNA (satDNA) family was studied in seven populations of the stick insects Bacillus atticus (parthenogenetic, unisexual) and Bacillus grandii (bisexual). It was characterized as widespread in all zymoraces of B. atticus and in all subspecies of B. grandii. The copy number of this satellite is higher in the bisexual B. grandii (15%-20% of the genome) than in the parthenogenetic B. atticus (2%- 5% of the genome). The nucleotide sequences of 12 Bag320 clones from B. atticus and 17 from B. grandii differed at 13 characteristic positions by fixed nucleotide substitutions. Thus, nucleotide sequences from both species cluster conspecifically in phylogenetic dendrograms. The nucleotide sequences derived from B. grandii grandii could he clearly discriminated from those of B. grandii benazzii and B. grandii maretimi on the basis of 25 variable sites, although all taxa come from Sicily. In contrast, the Bag320 sequences from B. atticus could not he discriminated accordingly, although they derive from geographically quite distant populations of its three zymoraces (the Italian and Greek B. atticus atticus, the Greek and Turkish B. atticus carius, and the Cyprian B. atticus cyprius). The different rate of evolutionary turnover of the Bag320 satDNA in both species can he related to their different modes of reproduction. This indicates that meiosis and chromosome segregation affect processes in satDNA diversification.

Biodegradable, flexible, and moisture-resistant films were obtained by recycling fennel waste and adding to fennel homogenates the bean protein phaseolin that was modified or not modified by the enzyme transglutaminase. All films were analyzed for their morphology, mechanical properties, water vapor permeability, and susceptibility to biodegradation under soil-like conditions. Our experiments showed that transglutaminase treatment of the phaseolin-containing fennel waste homogenates allowed us to obtain films comparable in their mechanical properties and water vapor permeability to the commercial films Ecoflex and Mater-Bi. Furthermore, biodegradability tests demonstrated that the presence of the enzyme in the film-casting sample significantly influences the integrity of such a product that lasts longer than films obtained either with fennel waste alone or with a mixture of fennel waste and phaseolin. These findings indicate the fennel-phaseolin film prepared in the presence of transglutaminase to be a promising candidate for a new environmentally friendly mulching bioplastic.

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii . At this stage, Staphylococcaceae ( Jeotgalicoccus and Staphylococcus ) and Lactobacillales ( Aerococcus , Lactobacillus , and Weissella ) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli ( Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales ( Corynebacterium and Yania ) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.

A spermine-dependent, polyphenylalanine-synthesizing cell-free system having an optimum activity at 75-85 degrees C, has been developed from the extremely thermoacidophilic archaebacterium Caldariella acidophila. The C. acidophila system is totally insensitive to the EF-Tu targeted antibiotics pulvomycin (at 40 degrees C) and kirromycin (at 47-72 degrees C) contrary to control systems derived from both mesophilic (Escherichia coli) and thermoacidophilic (Bacillus acidocaldarius) eubacteria. The archaebacterial EF-Tu-equivalent factor is also immunologically unrelated to eubacterial EF-Tu and does not cross react with antibodies against Escherichia coli EF-Tu. The pulvomycin and kirromycin reactions thus provide new phyletic markers for archaebacterial ancestry.