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Abstract Background The use of industrial Cannabis sativa L. for recreational, cosmeceutical, nutraceutical, and medicinal purposes has gained momentum due to its rich content of valuable phytochemicals, such as cannabidiol (CBD) and cannabigerol (CBG). However, there are concerns regarding the risk of microbial contamination in plants grown outside controlled environments. Microbes associated with hemp can be either epiphytes or endophytes and may pose a risk of infectious illness for humans. Methods Seven Italian hemp genotypes, including Bernabeo, Carmagnola, Carmaleonte, Codimono, CS, Eletta Campana, and Fibranova, were cultivated in two distinct geographic locations, Catania and Rovigo, for three consecutive years from 2019 to 2021. Total aerobic microbes (TAMC), total combined yeasts/moulds (TYMC), the presence of bile-tolerant Gram-negative bacteria, and the absence of Escherichia coli and Salmonella spp. were evaluated and compared. The main phytocannabinoid content was measured and correlated with microbial contamination. Results Most samples analyzed in this study did not meet the European Pharmacopoeia microbiological limits. The detection of potential pathogens, such as E. coli and Salmonella spp., in the samples indicates that the use of inflorescences may represent a possible source of infection. Microbial contamination varied among harvesting seasons and production sites, with agroclimatic conditions influencing microbial load and composition. The presence of potentially pathogenic bacteria was less associated with seasonal climate variability and more likely affected by sporadic contamination from external sources. CBD concentration exhibited a negative correlation with bile-tolerant Gram-negative bacteria and total yeasts/moulds levels. Samples with lower CBD content were more contaminated than those with higher CBD levels, suggesting a potential protective effect of this phytochemical on the plant. Conclusions The threshing residues (inflorescences, floral bracts, and leaves) of industrial hemp varieties represent a valuable product and a source of beneficial phytochemicals that warrants further exploration. While post-harvest sterilization methods may reduce microbiological risks, they may also degrade heat- and light-sensitive bioactive phytochemicals. The most promising strategy involves implementing best agronomic practices to maintain healthy and uncontaminated cultures. Rigorous monitoring and quality certification protocols are essential to mitigate the microbiological risk associated with the consumption of hemp-derived products.

Archaea are an understudied component of the human microbiome. In this study, the gut archaeome and bacteriome of 60 healthy adults from different region were analyzed by whole-genome shotgun sequencing. Archaea were ubiquitously found in a wide range of abundances, reaching up to 7.2 %. The dominant archaeal phylum was Methanobacteriota, specifically the family Methanobacteriaceae, encompassing more than 50 % of Archaea in 50 samples. The previously underestimated Thermoplasmatota, mostly composed of Methanomassiliicoccaceae, dominated in 10 subjects (>50 %) and was present in all others except one. Halobacteriota, the sole other archaeal phylum, occurred in negligible concentration, except for two samples (4.6–4.8 %). This finding confirmed that the human gut archaeome is primarily composed of methanogenic organisms and among the known methanogenic pathway: i) hydrogenotrophic reduction of CO2 is the predominant, being the genus Methanobrevibacter and the species Methanobrevibacter smithii the most abundant in the majority of the samples; ii) the second pathway, that involved Methanomassiliicoccales, was the hydrogenotrophic reduction of methyl-compounds; iii) dismutation of acetate or methyl-compounds seemed to be absent. Co-occurrence analysis allowed to unravel correlations between Archaea and Bacteria that shapes the overall structure of the microbial community, allowing to depict a clearer picture of the human gut archaeome.

Polyalcohols such as arabitol are among the main targets of biorefineries aiming to upcycle wastes and cheap substrates. In previous works Wickerhamomyces anomalus WC 1501 emerged as an excellent arabitol producer utilizing glycerol. Arabitol production by this strain is not growth associated, therefore, in this study, pre-grown cells were entrapped in calcium alginate beads (AB) and utilized for glycerol transformation to arabitol. Flasks experiments aimed to assess the medium composition (i.e., the concentration of inorganic and organic nitrogen sources and phosphates) and to establish the appropriate carrier-to-medium proportion. In flasks, under the best conditions of ammonium limitation and the carrier:medium ratio of 1:3 (w/v), 82.7 g/L glycerol were consumed in 168 h, yielding 31.2 g/L arabitol, with a conversion of 38% and volumetric productivity of 186 mg/mL/h. The process with immobilized cells was transferred to laboratory scale bioreactors with different configurations: stirred tank (STR), packed bed (PBR), fluidized bed (FBR), and airlift (ALR) bioreactors. The STR experienced oxygen limitation due to the need to maintain low stirring to preserve AB integrity and performed worse than flasks. Limitations in diffusion and mass transfer of oxygen and/or nutrients characterized also the PBR and the FBR and were partially relieved only in ALR, where 89.4 g/L glycerol were consumed in 168 h, yielding 38.1 g/L arabitol, with a conversion of 42% and volumetric productivity of 227 mg/mL/h. When the ALR was supplied with successive pulses of concentrated glycerol to replenish the glycerol as it was being consumed, 117 g/L arabitol were generated in 500 h, consuming a total of 285 g/L glycerol, with a 41% and 234 mg/L/h. The study strongly supports the potential of W. anomalus WC 1501 for efficient glycerol-to-arabitol conversion using immobilized cells. While the yeast shows promise by remaining viable and active for extended periods, further optimization is required, especially regarding mixing and oxygenation. Improving the stability of the immobilization process is also crucial for reusing pre-grown cells in multiple cycles, reducing dead times, biomass production costs, and enhancing the economic feasibility of the process.

Abstract Background The production of microbial lipids has attracted considerable interest during the past decade since they can be successfully used to produce biodiesel by catalyzed transesterification with short chain alcohols. Certain yeast species, including several psychrophilic isolates, are oleaginous and accumulate lipids from 20 to 70% of biomass under appropriate cultivation conditions. Among them, Rhodotorula glacialis is a psychrophilic basidiomycetous species capable to accumulate intracellular lipids. Results Rhodotorula glacialis DBVPG 4785 is an oleaginous psychrophilic yeast isolated from a glacial environment. Despite its origin, the strain abundantly grew and accumulated lipids between -3 to 20°C. The temperature did not influence the yield coefficients of both biomass and lipids production, but had positive effect on the growth rate and thus on volumetric productivity of lipid. In glucose-based media, cellular multiplication occurred first, while the lipogenic phase followed whenever the culture was limited by a nutrient other than glucose. The extent of the carbon excess had positive effects on triacylglycerols production, that was maximum with 120 g L-1 glucose, in terms of lipid concentration (19 g L-1), lipid/biomass (68%) and lipid/glucose yields (16%). Both glucose concentration and growth temperature influenced the composition of fatty acids, whose unsaturation degree decreased when the temperature or glucose excess increased. Conclusions This study is the first proposed biotechnological application for Rhodotorula glacialis species, whose oleaginous biomass accumulates high amounts of lipids within a wide range of temperatures through appropriate cultivation C:N ratio. Although R. glacialis DBVPG 4785 is a cold adapted yeast, lipid production occurs over a broad range of temperatures and it can be considered an interesting microorganism for the production of single cell oils.

Leuconostoc encompasses a number of species that frequently appear in foods where they play different roles, ranging from ripening to spoiling. The number of available Leuconostoc genomes has recently increased and enabled the precise taxonomic and phylogenetic delineation of species. Nonetheless, a thorough investigation of the functions and the metabolic potential of Leuconostoc species has never been accomplished. In this study, all the currently available 553 Leuconostoc genomes were downloaded from NCBI GenBank and annotated utilizing specific tools in order to reconstruct the metabolic potential of the genus in terms of carbohydrate hydrolysis and fermentative pathways, transporters, and anabolic potential. The analysis revealed that species cluster based on their metabolic potential, showing unique adaptation and ecological roles. Pentose phosphate and phosphoketolase pathways were highlighted as the main ones of central metabolism. The various identified PTS and ABC transporters showed adaptability to different sugars. The metabolic diversity described in this study not only supports the role of Leuconostoc spp. in natural ecosystems but also highlights their potential in industrial applications, particularly in the fermentation industry where their ability to metabolize a wide range of substrates can be harnessed for the production of various fermented foods and bioproducts.

Mucins are large glycoproteins whose degradation requires the expression of several glycosil hydrolases to catalyze the cleavage of the oligosaccharide chains and release monosaccharides that can be assimilated. In this study, we present a characterization on the strains Clostridium celatum WC0700, Clostridium tertium WC0709, and Paraclostridium bifermentans WC0705. These three strains were previously isolated from enrichment cultures on mucin of fecal samples from healthy subjects and can use mucin as sole carbon and nitrogen source. Genome analysis and in vitro functional analysis of these strains elucidated their physiological and biochemical features. C. celatum WC0700 harbored the highest number of glycosyl hydrolases specific for mucin degradation, while P. bifermentans WC0705 had the least. These predicted differences were confirmed growing the strains on 5 mucin-decorating monosaccharides (L-fucose, N-Acetylneuraminic acid, galactose, N-acetylgalactosamine, and N-acetylglucosamine) as only source of carbon. Fermenting mucin, they all produced formic, acetic, propionic, butyric, isovaleric, and lactic acids, and ethanol; acetic acid was the main primary metabolite. Further catabolic capabilities were investigated, as well as antibiotic susceptibility, biofilm formation, tolerance to oxygen and temperature. The potential pathogenicity of the strains was evaluated through in silico research of virulence factors. The merge between comparative and functional genomics and biochemical/physiological characterization provided a comprehensive view of these mucin degraders, reassuring on the safety of these species and leaving ample scope for deeper investigations on the relationship with the host and for assessing if some relevant health-promoting effect could be ascribed to these SCFA producing species.

Xylitol is a pentose-polyol widely applied in the food and pharmaceutical industry. It can be produced from lignocellulosic biomass, valorizing second-generation feedstocks. Biotechnological production of xylitol requires scalable solutions suitable for industrial scale processes. Immobilized-cells systems offer numerous advantages. Although fungal pellet carriers have gained attention, their application in xylitol production remains unexplored. In this study, the yeast strain P. fermentans WC 1507 was employed for xylitol production. The optimal conditions were observed with free-cell cultures at pH above 3.5, low oxygenation, and medium containing (NH4)2SO4 and yeast extract as nitrogen sources (xylitol titer 79.4 g/L, YP/S 66.3%, and volumetric productivity 1.3 g/L/h). Yeast cells were immobilized using inactive Aspergillus oryzae pellet mycelial carrier (MC) and alginate beads (AB) and were tested in flasks over three consecutive production runs. Additionally, the effect of a 0.2% w/v alginate layer, coating the outer surface of the carriers (cMC and cAB, respectively), was examined. While YP/S values observed with both immobilized and free cells were similar, the immobilized cells exhibited lower final xylitol titer and volumetric productivity, likely due to mass transfer limitations. AB and cAB outperformed MC and cMC. The uncoated AB carriers were tested in a laboratory-scale airlift bioreactor, which demonstrated a progressive increase in xylitol production in a repeated batch process: in the third run, a xylitol titer of 63.0 g/L, YP/S of 61.5%, and volumetric productivity of 0.52 g/L/h were achieved. This study confirmed P. fermentans WC 1507 as a promising strain for xylitol production in both free- and entrapped-cells systems. Considering the performance of the wild strain, a metabolic engineering intervention aiming at further improving the efficiency of xylitol production could be justified. MC and AB proved to be viable supports for cell immobilization, but additional process development is necessary to identify the optimal bioreactor configuration and fermentation conditions.

Abstract Mucins are large glycoproteins consisting of approximately 80% of hetero-oligosaccharides. Gut mucin degraders of healthy subjects were investigated, through a culture dependent and independent approach. The faeces of five healthy adults were subjected to three steps of anaerobic enrichment in a medium with sole mucins as carbon and nitrogen sources. The bacterial community was compared before and after the enrichment by 16S rRNA gene profiling. Bacteria capable of fermenting sugars, such as Anaerotruncus, Holdemania, and Enterococcaceae likely took advantage of the carbohydrate chains. Escherichia coli and Enterobacteriaceae, Peptococcales, the Coriobacteriale Eggerthella, and a variety of Clostridia such as Oscillospiraceae, Anaerotruncus, and Lachnoclostridium, significantly increased and likely participated to the degradation of the protein backbone of mucin. The affinity of E. coli and Enterobacteriaceae for mucin may facilitate the access to the gut mucosa, promoting gut barrier damage and triggering systemic inflammatory responses. Only three species of strict anaerobes able to grow on mucin were isolated from the enrichments of five different microbiota: Clostridium disporicum, Clostridium tertium, and Paraclostridium benzoelyticum. The limited number of species isolated confirms that in the gut the degradation of these glycoproteins results from cooperation and cross-feeding among several species exhibiting different metabolic capabilities.

β-glucuronidases (GUS) of intestinal bacteria remove glucuronic acid from glucoronides, reversing phase II metabolism of the liver and affecting the level of active deconjugated metabolites deriving from drugs or xenobiotics. Two hundred seventy-nine non-redundant GUS sequences are known in the gut microbiota, classified in seven structural categories (NL, L1, L2, mL1, mL2, mL1,2, and NC) with different biocatalytic properties. In the present study, the intestinal metagenome of 60 healthy subjects from five geographically different cohorts was assembled, binned, and mined to determine qualitative and quantitative differences in GUS profile, potentially affecting response to drugs and xenobiotics. Each metagenome harbored 4–70 different GUS, altogether accounting for 218. The amount of intestinal bacteria with at least one GUS gene was highly variable, from 0.7 to 82.2%, 25.7% on average. No significant difference among cohorts could be identified, except for the Ethiopia (ETH) cohort where GUS-encoding bacteria were significantly less abundant. The structural categories were differently distributed among the metagenomes, but without any statistical significance related to the cohorts. GUS profiles were generally dominated by the category NL, followed by mL1, L2, and L1. The GUS categories most involved in the hydrolysis of small molecules, including drugs, are L1 and mL1. Bacteria contributing to these categories belonged to Bacteroides ovatus, Bacteroides dorei, Bacteroides fragilis, Escherichia coli, Eubacterium eligens, Faecalibacterium prausnitzii, Parabacteroides merdae, and Ruminococcus gnavus. Bacteria harboring L1 GUS were generally scarcely abundant (

Sliced cooked ham packaged in a modified atmosphere is a popular ready-to-eat product, subjected to abundant microbial contamination throughout its shelf life that can lead to deterioration of both sensorial properties and safety. In this study, the microbial load and the chemical–physical features of cooked ham of five producers were monitored for a period of 12 days after the opening of the packages (i.e., the secondary shelf life), during which the products were stored in a domestic refrigerator at 5.2 ± 0.6°C. The sensorial properties presented a perceivable decay after 8 days and became unacceptable after 12 days. High-performance liquid chromatography analysis and solid-phase microextraction coupled with gas chromatography profiling of volatile metabolites indicated that lactic acid, ethanol, acetic acid, acetoin, 3-methyl-1-butanol, and 2-3 butanediol were the main metabolites that characterized the evolution of the analyzed cooked ham. The microbiota was monitored by 16S ribosomal RNA gene profiling and culture-dependent techniques. Already at the opening of packages, all the products presented high microbial load, generally dominated by lactic acid bacteria, with evident differences among the products. The increase of lactic acid bacteria somehow protected samples from abundant contamination by other bacteria, concurring with the evolution of more safe products. This role was exerted by numerous Latilactobacillus, Leuconostoc, and Carnobacterium species, among which the most frequently detected were Latilactobacillus sakei, Latilactobacillus sakei carnosum, Leuconostoc mesenteroides, and Carnobacterium divergens. Some products presented more complex communities that encompassed Proteobacteria such as Moellerella wisconsensis, Proteus hauseri, Brochothrix thermosphacta, and less frequently Pseudomonas, Erwinia, and Massilia. Opportunistic pathogenic bacteria such as Escherichia coli and Vibrio sp. were found in small quantities. The yeasts Kazachstania servazzii and Debaryomyces hansenii occurred already at 0 days, whereas various species of Candida (Candida zeylanoides, Candida sake, Candida norvegica, and Candida glaebosa) were abundant only after 12 days. These results indicated that the microbiological contaminants overgrowing during the secondary shelf life did not derive from environmental cross-contamination at the opening of the tray but were already present when the packages were opened, highlighting the phases of production up to the packaging as those crucial in managing the safety risk associated to this product.

Protein catabolism by intestinal bacteria is infamous for releasing many harmful compounds, negatively affecting the health status, both locally and systemically. In a previous study, we enriched in protein degraders the fecal microbiota of five subjects, utilizing a medium containing protein and peptides as sole fermentable substrates and we monitored their evolution by 16S rRNA gene profiling. In the present study, we fused the microbiome data and the data obtained by the analysis of the volatile organic compounds (VOCs) in the headspace of the cultures. Then, we utilized ANOVA simultaneous component analysis (ASCA) to establish a relationship between metabolites and bacteria. In particular, ASCA allowed to separately assess the effect of subject, time, inoculum concentration, and their binary interactions on both microbiome and volatilome data. All the ASCA submodels pointed out a consistent association between indole and Escherichia–Shigella, and the relationship of butyric, 3-methyl butanoic, and benzenepropanoic acids with some bacterial taxa that were major determinants of cultures at 6 h, such as Lachnoclostridiaceae (Lachnoclostridium), Clostridiaceae (Clostridium sensu stricto), and Sutterellaceae (Sutterella and Parasutterella). The metagenome reconstruction with PICRUSt2 and its functional annotation indicated that enrichment in a protein-based medium affected the richness and diversity of functional profiles, in the face of a decrease of richness and evenness of the microbial community. Linear discriminant analysis (LDA) effect size indicated a positive differential abundance (p < 0.05) for the modules of amino acid catabolism that may be at the basis of the changes of VOC profile. In particular, predicted genes encoding functions belonging to the superpathways of ornithine, arginine, and putrescine transformation to GABA and eventually to succinyl-CoA, of methionine degradation, and various routes of breakdown of aromatic compounds yielding succinyl-CoA or acetyl-CoA became significantly more abundant in the metagenome of the bacterial community.

Leuconostoc carnosum is a known colonizer of meat-related food matrices. It reaches remarkably high loads during the shelf life in packaged meat products and plays a role in spoilage, although preservative effects have been proposed for some strains. In this study, the draft genomes of 17 strains of L. carnosum (i.e., all the strains that have been sequenced so far) were compared to decipher their metabolic and functional potential and to determine their role in food transformations. Genome comparison and pathway reconstruction indicated that L. carnosum is a compact group of closely related heterofermentative bacteria sharing most of the metabolic features. Adaptation to a nitrogen-rich environment, such as meat, is evidenced by 23 peptidase genes identified in the core genome and by the autotrophy for nitrogen compounds including several amino acids, vitamins, and cofactors. Genes encoding the decarboxylases yielding biogenic amines were not present. All the strains harbored 1–4 of 32 different plasmids, bearing functions associated to proteins hydrolysis, transport of amino acids and oligopeptides, exopolysaccharides, and various resistances (e.g., to environmental stresses, bacteriophages, and heavy metals). Functions associated to bacteriocin synthesis, secretion, and immunity were also found in plasmids. While genes for lactococcin were found in most plasmids, only three harbored the genes for leucocin B, a class IIa antilisterial bacteriocin. Determinants of antibiotic resistances were absent in both plasmids and chromosomes.

Riboflavin or vitamin B2 is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Despite increased interest in microbial synthesis of this water-soluble vitamin, the metabolic pathway for riboflavin biosynthesis has been characterized in just a handful of bacteria. Here, comparative genome analysis identified the genes involved in the de novo biosynthetic pathway of riboflavin in certain bifidobacterial species, including the human gut commensal Bifidobacterium longum subsp. infantis (B. infantis) ATCC 15697. Using comparative genomics and phylogenomic analysis, we investigated the evolutionary acquisition route of the riboflavin biosynthesis or rib gene cluster in Bifidobacterium and the distribution of riboflavin biosynthesis-associated genes across the genus. Using B. infantis ATCC 15697 as model organism for this pathway, we isolated spontaneous riboflavin overproducers, which had lost transcriptional regulation of the genes required for riboflavin biosynthesis. Among them, one mutant was shown to allow riboflavin release into the medium to a concentration of 60.8 ng mL–1. This mutant increased vitamin B2 concentration in a fecal fermentation system, thus providing promising data for application of this isolate as a functional food ingredient.

Five-carbons polyalcohols, such as xylitol and arabitol, and microbial oils are important targets for biotechnological industries. Polyalcohols can find application as low-calories sweeteners and as building block in the synthesis of valuable compounds, while lipids are interesting for both biofuel and food industry. The osmophilic yeast Wickerhamomyces anomalus WC 1501 was preliminary known to produce arabitol from glycerol. Production kinetics were investigated in this study. Production was not growth-associated and occurred during a nitrogen-limited stationary phase, in presence of an excess of carbon source. Typical bioreactor batch cultures, carried out with 160 g/L glycerol, yielded 16.0 g/L arabitol in 160 h. A fed-batch process was developed, in which growth is carried out batchwise in a balanced medium containing 20 g/L glycerol, and arabitol production is induced at the entrance into the stationary phase with a pulse of concentrated glycerol to provide the remaining 140 g/L carbon source. At the end of the process 18.0 g/L arabitol were generated. Under these conditions, the yeast also accumulated intracellular triacylglycerols, with fatty acids of 16-18 carbons bearing 0 to 2 unsaturations, reaching up the 23% of biomass dry weight. Therefore, W. anomalus WC 1501 is a good candidate for the development of a fermentative process yielding arabitol and has potential also as oleaginous yeast for producing lipids, further improving the interest in this strain for glycerol biorefinery. The utilization of a fed-batch process allows to carry out distinct growth and production phases and thus allows the optimization of both phases separately, in order to achieve the highest concentration of catalytic biomass during growth and the maximum efficiency during production. This strain deserves further investigation to better exploit its biotechnological potential in the valorization of glycerol.

Background.Chlamydia trachomatis (CT) is the agent of the most common bacterial sexually transmitted infection worldwide, with a significant impact on women’s health. Despite the increasing number of studies about the vaginal microbiome in women with CT infections, information about the composition of the anal microbiome is still lacking. Here, we assessed the bacterial community profiles of vaginal and anal ecosystems associated or not with CT infection in a cohort of Caucasian young women. Methods. A total of 26 women, including 10 with a contemporary vaginal and ano-rectal CT infection, were enrolled. Composition of vaginal and anal microbiome was studied by 16S rRNA gene profiling. Co-occurrence networks of bacterial communities and metagenome metabolic functions were determined. Results. In case of CT infection, both vaginal and anal environments were characterized by a degree of dysbiosis. Indeed, the vaginal microbiome of CT-positive women were depleted in lactobacilli, with a significant increase in dysbiosis-associated bacteria (e.g., Sneathia, Parvimonas, Megasphaera), whereas the anal microbiota of CT-infected women was characterized by higher levels of Parvimonas and Pseudomonas and lower levels of Escherichia. Interestingly, the microbiome of anus and vagina had numerous bacterial taxa in common, reflecting a significant microbial ‘sharing’ between the two sites. In the vaginal environment, CT positively correlated with Ezakiella spp. while Gardnerella vaginalis co-occurred with several dysbiosis-related microbes, regardless of CT vaginal infection. The vaginal microbiome of CT-positive females exhibited a higher involvement of chorismate and aromatic amino acid biosynthesis, as well as an increase in mixed acid fermentation. Conclusions. These data could be useful to set up new diagnostic/prognostic tools, offering new perspectives for the control of chlamydial infections.

Unabsorbed proteins reach the colon and are fermented by the microbiota, yielding a variety of harmful metabolites. In the present study, a 16S rRNA gene survey identified the bacterial taxa flourishing in 11 batch fermentations with proteins and peptones as the sole fermentable substrates, inoculated with the feces of six healthy adults. Organic acids, ammonia, and indole resulting from protein breakdown and fermentation accumulated in all of the cultures. Analysis of differential abundances among time-points identified Enterobacteriaceae, Burkholderiaceae, and Desulfovibrionaceae (including Esherichia-Shigella, Sutterella, Parasutterella, and Bilophila) among the bacteria that especially in the cultures with low inoculation load. Lachnospiraceae and Ruminococcaceae also encompassed many taxa that significantly expanded, mainly in cultures inoculated with high inoculation load, and showed the strongest correlation with the production of ammonium, indole, and p-cresol. Anaerotruncus, Dorea, Oscillibacter, Eubacterium oxidoreducens, Lachnoclostridium, Paeniclostridium, and Rombutsia were among them. Other Firmicutes (e.g., Roseburia, Ruminococcus, Lachnospira, Dialister, Erysipelotrichaceae, and Streptococcaceae) and many Bacteroidetes (e.g., Barnesiellaceae, Prevotellaceae, and Rickenelliaceae) decreased. Sequences attributed to Bacteroides, unresolved at the level of species, presented opposite contributions, resulting in no significant changes in the genus. This study sheds light on the multitude of bacterial taxa putatively participating in protein catabolism in the colon. Protein fermentation was confirmed as unfavorable to health, due to both the production of toxic metabolites and the blooming of opportunistic pathogens and pro-inflammatory bacteria.

The potential utilization of black soldier fly (Hermetia illucens) as food or feed is interesting due to the nutritive value and the sustainability of the rearing process. In the present study, larvae and prepupae of H. illucens were reared at 20, 27, and 33 °C, to determine whether temperature affects the whole insect microbiota, described using microbiological risk assessment techniques and 16S rRNA gene survey. The larvae efficiently grew across the tested temperatures. Higher temperatures promoted faster larval development and greater final biomass but also higher mortality. Viable Enterobacteriaceae, Bacillus cereus, Campylobacter, Clostridium perfringens, coagulase-positive staphylococci, Listeriaceae, and Salmonella were detected in prepupae. Campylobacter and Listeriaceae counts got higher with the increasing temperature. Based on 16S rRNA gene analysis, the microbiota of larvae was dominated by Providencia (>60%) and other Proteobateria (mainly Klebsiella) and evolved to a more complex composition in prepupae, with a bloom of Actinobacteria, Bacteroidetes, and Bacilli, while Providencia was still present as the main component. Prepupae largely shared the microbiota with the frass where it was reared, except for few lowly represented taxa. The rearing temperature was negatively associated with the amount of Providencia, and positively associated with a variety of other genera, such as Alcaligenes, Pseudogracilibacillus, Bacillus, Proteus, Enterococcus, Pediococcus, Bordetella, Pseudomonas, and Kerstersia. With respect to the microbiological risk assessment, attention should be paid to abundant genera, such as Bacillus, Myroides, Proteus, Providencia, and Morganella, which encompass species described as opportunistic pathogens, bearing drug resistances or causing severe morbidity.

Arabitol is a sugar alcohol, stereoisomer to xylitol, which is enlisted among the main target for biorefineries. It can serve as low calorie sweetener and as building block in the enantiopure synthesis of immunosuppressive glycolipids, herbicides, and drugs. Several studies described the fermentative production of arabitol by osmophilic yeasts, cultured with high concentrations of D-glucose. The utilization of cheaper carbon sources, such as glycerol or lactose, is of great interest for biorefinery implementation, but information on exploitation to arabitol production is still scarce. In the present study 50 yeasts belonging to 24 ascomycetous species were screened for the ability to grow and produce arabitol in presence of 80 g/L lactose or glycerol. Production from lactose was generally unsuccessful, the best producer being Kluyveromyces lactis WC 1401 with 0.94 g/L in 160 h. Production from glycerol was promising, with Zygosaccharomyces rouxii WC 1206, Pichia guilliermondii CBS 566, Hansenula anomala WC 1501, and Candida freyschussii ATCC 18737 yielding 3 to 4.5 g/L arabitol, with conversion yield (YP/S) ranging from 11 to 21.7%. Batch growth with high initial glycerol amount (160 g/L) resulted in higher production, with H. anomala WC 1501 yielding 10.0 g/L arabitol (YP/S = 12%) in 160 h. Preliminary bioreactor fermentations with H. anomala WC 1501 indicated that production is not growth associated and revealed some major parameters affecting production, such as the pH and the C:N ratio, that will be the target of following studies aiming at process optimization. Cultivation under controlled oxygenation (DOT = 20%) and pH (= 3.0) resulted in improvement in the performance of H. anomala WC 1501, yielding16.1 g/L arabitol. Cultivation in a medium with high C:N ratio, lacking inorganic nitrogen yielded 17.1 g/L arabitol. Therefore, this strain was selected for the development of a fed-batch process, aiming to improve the efficiency of the biomass, generated in the growth phase, and increasing the production in the stationary phase.

Cold-adapted yeasts are increasingly being isolated from glacial environments, including Arctic, Antarctic, and mountain glaciers. Psychrophilic yeast isolates mostly belong to Basidiomycota phylum, such as Cryptococcus, Mrakia, and Rhodotorula, and represent an understudied source of biodiversity for potential biotechnological applications. Since some basidiomycetous yeast genera (e.g. Rhodotorula, Phaffia, etc.) were demonstrated to produce commercially important carotenoids (e.g. ß-carotene, torulene, torularhodin and astaxanthin), the present study aimed to obtain psychrophilic yeast isolates from the surface ice of an Italian glacier to identify new pigment-producers. 23 yeast isolates were obtained. Among them, three isolates giving pigmented colonies were subjected to ITS1/ITS2 sequencing and were attributed to the Basidiomycetous yeasts Dioszegia sp., Rhodotorula mucilaginosa, and Rhodotorula laryngis. The strains were cultured batch-wise in a carbon-rich medium at 15°C until the stationary phase was reached, then the pigments were extracted from freeze-dried biomass using a DMSO:acetone mixture. Visible absorption spectrum and HPLC-DAD analysis revealed the presence of carotenoid pigments. In batch cultures of Dioszegia sp., carotenoid production was growth-associated and yielded up to 3.4 mg/L of a molecule exhibiting an m/z ratio (568) consistent with the molecular weight of xanthophylls bearing 2 OH groups.

A surplus of glycerol is obtained from biodiesel manufacturing and represents a waste product whose applications are lacking. Thus, the use of glycerol as substrate for fermentation processes yielding valuable products is very attractive. In this study, the utilization of glycerol as a growth substrate for the cultivation of oleaginous yeasts was explored with the aim to produce microbial oils. Forty strains of environmental non-conventional yeasts belonging to 19 different species were screened for the ability to grow on glycerol and produce intracellular lipids in a medium containing an excess of this carbon source (C:N = 48:1). Three strains, belonging to the species Candida freyschussii, Pichia farinosa, and Saccharomyces spencerorum, depleted 40 g/L glycerol within 120 h and produced intracellular lipids. freyschussii yielded the highest amounts, lipids accounting for the 33 % of biomass on dry basis. 1H-NMR analysis revealed that the lipid extract did not contain detectable free fatty acids and was composed mostly of triacylglycerols. Lipid composition, determined by GC-MS analysis, was similar to plant oils, and may be optimal feedstock for biodiesel production, being dominated by monounsaturated C16 and C18. As in other oleaginous yeasts, lipid production by C. freyschussii sp. increased with the increase of the C:N ratio of the medium, but growth was inhibited at glycerol concentrations higher than 40 g/L. As a result, lipid production was the highest with 40 g/L glycerol, yielding 4.7 g/L lipids, with a mean volumetric productivity of 0.15 g/L/h. In order to prevent growth inhibition over 40 g/L glycerol and extend the lipogenic phase, different fed-batch strategies were tried. The best performing processes took advantage from the feeding with concentrated media exhibiting the same C:N ratio of the basal medium, leading to very productive high cell density cultures. With the continuous feeding of 20X-concentrated medium, 29 g/L lipids (i.e. the 32 % of biomass) were obtained in 100 h of cultivation, with a mean volumetric productivity of 0.30 g/L/h. The values herein reported are among the highest yield and productivity values ever obtained for fermentative processes exploiting oleaginous fungi to produce lipids from glycerol. Therefore, C. freyschussii could be considered as an interesting microorganism to convert glycerol into microbial oils for biofuel industry.

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Abstract Background d-Arabitol, a five-carbon sugar alcohol, represents a main target of microbial biorefineries aiming to valorize cheap substrates. The yeast Wickerhamomyces anomalus WC 1501 is known to produce arabitol in a glycerol-based nitrogen-limited medium and preliminary fed-batch processes with this yeast were reported to yield 18.0 g/L arabitol. Results Fed-batch fermentations with W. anomalus WC 1501 were optimized using central composite design (CCD). Dissolved oxygen had not a significant effect, while optimum values were found for glycerol concentration (114.5 g/L), pH (5.9), and temperature (32.5 °C), yielding 29 g/L d-arabitol in 160 h, a conversion yield of 0.25 g of arabitol per g of consumed glycerol, and a volumetric productivity of 0.18 g/L/h. CCD optimal conditions were the basis for further improvement, consisting in increasing the cellular density (3✕), applying a constant feeding of glycerol, and increasing temperature during production. The best performing fed-batch fermentations achieved 265 g/L d-arabitol after 325 h, a conversion yield of 0.74 g/g, and a volumetric productivity of 0.82 g/L/h. Conclusion W. anomalus WC 1501 confirmed as an excellent producer of d-arabitol, exhibiting a remarkable capability of transforming pure glycerol. The study reports among the highest values ever reported for microbial transformation of glycerol into d-arabitol, in terms of arabitol titer, conversion yield, and productivity. Graphical Abstract

Hemp hurds are the main byproduct from hemp fibers supply chain and they could represent valuable feedstock of lignocellulosic biomass for biorefineries. The industrial hemp variety "Carmagnola", is characterized by low amounts of ash and high amount of carbohydrates. Alpha-cellulose (44% w/w), hemicellulose (25%), and lignin (23%) were fractionated using an organosolv pretreatment. The enzymatic hydrolysis of the cellulose fraction yielded up to 60% of glucose, that can easily find application as substrate for industrial fermentations. On the other hand, the black liquor originating from hemicellulose contains mainly xylose and minor amounts of other sugars. In the perspective of finding an application of black liquor, 50 yeasts belonging to 24 ascomycetous species were screened both in aerobiosis and anaerobiosis for the production of ethanol and the sugar alcohols xylitol and arabitol from xylose. Pichia fermentans WC 1507, Wickerhamomyces anomalus WC 1501, and Kluyveromyces bacillosporus WC 1404 were found to consume xylose, yielding xylitol in aerobic conditions. In particular, aerobic flask cultures of P. fermentans WC 1507 containing 120 g/L xylose showed the highest xylitol production values, yielding 63.5 g/L xylitol with a YP/S of 71.5%. Black liquor, exploited as a carbon source, has been successfully utilized by the three selected yeast strains at a concentration of 20 g/L in the culture medium, resulting in comparable or higher yields of biomass and xylitol compared to the medium containing pure xylose. A higher concentration of black liquor in the culture medium (to reach 120 g/l of xylose) has determined an inhibition of the growth of yeasts indicating the need for treatments for the removal of inhibitors. These preliminary results can be considered promising for the microbial valorization of lignocellulosic hemp feedstock toward the production of xylitol.

Leuconostoc is a genus of saccharolytic heterofermentative lactic acid bacteria that inhabit plant-derived matrices and a variety of fermented foods (dairy products, dough, milk, vegetables, and meats), contributing to desired fermentation processes or playing a role in food spoilage. At present, the genus encompasses 17 recognized species. In total, 216 deposited genome sequences of Leuconostoc were analyzed, to check the delineation of species and to infer their evolutive genealogy utilizing a minimum evolution tree of Average Nucleotide Identity (ANI) and the core genome alignment. Phylogenomic relationships were compared to those obtained from the analysis of 16S rRNA, pheS, and rpoA genes. All the phylograms were subjected to split decomposition analysis and their topologies were compared to check the ambiguities in the inferred phylogenesis. The minimum evolution ANI tree exhibited the most similar topology with the core genome tree, while single gene trees were less adherent and provided a weaker phylogenetic signal. In particular, the 16S rRNA gene failed to resolve several bifurcations and Leuconostoc species. Based on an ANI threshold of 95%, the organization of the genus Leuconostoc could be amended, redefining the boundaries of the species L. inhae, L. falkenbergense, L. gelidum, L. lactis, L. mesenteroides, and L. pseudomesenteroides. Two strains currently recognized as L. mesenteroides were split into a separate lineage representing a putative species (G16), phylogenetically related to both L. mesenteroides (G18) and L. suionicum (G17). Differences among the four subspecies of L. mesenteroides were not pinpointed by ANI or by the conserved genes. The strains of L. pseudomesenteroides were ascribed to two putative species, G13 and G14, the former including also all the strains presently belonging to L. falkenbergense. L. lactis was split into two phylogenetically related lineages, G9 and G10, putatively corresponding to separate species and both including subgroups that may correspond to subspecies. The species L. gelidum and L. gasicomitatum were closely related but separated into different species, the latter including also L. inhae strains. These results, integrating information of ANI, core genome, and housekeeping genes, complemented the taxonomic delineation with solid information on the phylogenetic lineages evolved within the genus Leuconostoc.

Twelve strains of Leuconostoc carnosum from meat products were investigated in terms of biochemical, physiological, and functional properties. The spectrum of sugars fermented by L. carnosum strains was limited to few mono- and disaccharides, consistently with the natural habitats of the species, including meat and fermented vegetables. The strains were able to grow from 4 to 37°C with an optimum of approximately 32.5°C. The ability to grow at temperatures compatible with refrigeration and in presence of up to 60 g/L NaCl explains the high loads of L. carnosum frequently described in many meat-based products. Six strains produced exopolysaccharides, causing a ropy phenotype of colonies, according to the potential involvement on L. carnosum in the appearance of slime in packed meat products. On the other side, the study provides evidence of a potential protective role of L. carnosum WC0321 and L. carnosum WC0323 against Listeria monocytogenes, consistently with the presence in these strains of the genes encoding leucocin B. Some meat-based products intended to be consumed without cooking may harbor up to 108 CFU/g of L. carnosum; therefore, we investigated the potential impact of this load on health. No strains survived the treatment with simulated gastric juice. Three selected strains were challenged for the capability to colonize a mouse model and their immunomodulatory properties were investigated. The strains did not colonize the intestine of mice during 10 days of daily dietary administration. Intriguingly, despite the loss of viability during the gastrointestinal transit, the strains exhibited different immunomodulatory effect on the maturation of dendritic cells in vivo, the extent of which correlated to the production of exopolysaccharides. The ability to stimulate the mucosal associated immune system in such probiotic-like manner, the general absence of antibiotic resistance genes, and the lack of the biosynthetic pathways for biogenic amines should reassure on the safety of this species, with potential for exploitation of selected starters.

The fungal component of the intestinal microbiota of eight healthy subjects was studied over 12 months using metagenome survey and culture-based approaches. Aspergillus, Candida, Debaryomyces, Malassezia, Penicillium, Pichia, and Saccharomyces were the most recurrent and/or dominant fungal genera, according to metagenomic analysis. The biodiversity of fungal communities was lower and characterized by greater unevenness, when compared to bacterial microbiome. The dissimilarities both among subjects and over the time within the same subject suggested that most of the fungi passed through the gastro-intestinal tract (GIT) without becoming stable colonizers. Certain genera, such as Aspergillus and Penicillium, were isolated in a minority of cases, although they recurred abundantly and frequently in the metagenomics survey, likely being environmental or food-borne fungi that do not inhabit the GIT. Candida genus was recurrently detected. Candida albicans isolates dominated among the cultivable mycobiota and longitudinally persisted, likely as commensals inhabiting the intestine or regularly reaching it from Candida-colonized districts, such as the oral cavity. Other putative colonizers belonged to Candida zeylanoides, Geotrichum candidum, and Rhodotorula mucilaginosa, with persisting biotypes being identified. Phenotyping of fungal isolates indicated that C. albicans adhered to human epithelial cells more efficiently and produced greater amounts of biofilm in vitro than non-albicans Candida (NAC) and non-Candida fungi (NCF). The C. albicans isolates also induced the highest release of HBD-2 by human epithelial cells, further differing from NAC and NCF. Nine representative isolates were administered to mice to evaluate the ability to colonize the intestine. Only two out of three C. albicans strains persisted in stools of animals 2 weeks after the end of the oral administration, whereas NAC and NCF did not. These results confirm the allochthonous nature of most the intestinal fungi, while C. albicans appears to be commonly involved in stable colonization. A combination of specific genetic features in the microbe and in the host likely allow colonization from fungi normally present solely as passengers. It remains to be established if other species identified as potential colonizers, in addition to Candida, are true inhabitants of the GIT or rather reach the intestine spreading from other body districts.

Escherichia coli may innocuously colonize the intestine of healthy subjects or may instigate infections in the gut or in other districts. This study investigated intestinal E. coli isolated from 20 healthy adults. Fifty-one strains were genotyped by molecular fingerprinting and analyzed for genetic and phenotypic traits, encompassing the profile of antibiotic resistance, biofilm production, the presence of surface structures (such as curli and cellulose), and their performance as recipients in conjugation experiments. A phylogroup classification and analysis of 34 virulence determinants, together with genes associated to the pks island (polyketide-peptide genotoxin colibactin) and conjugative elements, was performed. Most of the strains belonged to the phylogroups B1 and B2. The different phylogroups were separated in a principal coordinate space, considering both genetic and functional features, but not considering pulsed-field gel electrophoresis. Within the B2 and F strains, 12 shared the pattern of virulence genes with potential uropathogens. Forty-nine strains were sensitive to all the tested antibiotics. Strains similar to the potential pathogens innocuously inhabited the gut of healthy subjects. However, they may potentially act as etiologic agents of extra-intestinal infections and are susceptible to a wide range of antibiotics. Nevertheless, there is still the possibility to control infections with antibiotic therapy.