Microbial Ecology of Artisanal Feta and Kefalograviera Cheeses, Part I: Bacterial Community and Its Functional Characteristics with Focus on Lactic Acid Bacteria as Determined by Culture-Dependent Methods and Phenotype Microarrays

Artisanal cheesemaking is still performed using practices and conditions derived from tradition. Feta and Kefalograviera cheeses are very popular in Greece and have met worldwide commercial success. However, there is a lack of knowledge regarding their lactic acid microecosystem composition and species dynamics during ripening. Thus, the aim of the present study was to assess the microecosystem as well as the autochthonous lactic acid microbiota during the ripening of artisanal Feta and Kefalograviera cheeses. For that purpose, raw sheep’s milk intended for cheesemaking, as well as Feta and Kefalograviera cheeses during early and late ripening were analyzed, and the lactic acid microbiota was identified using the classical phenotypic approach, clustering with PCR-RAPD and identification with sequencing of the 16S-rRNA gene, as well as with the Biolog GEN III microplates. In addition, the functional properties of the bacterial community were evaluated using the Biolog EcoPlates, which consists of 31 different carbon sources. In general, concordance between the techniques used was achieved. The most frequently isolated species from raw sheep’s milk were Enteroroccus faecium, Lactiplantibacillus plantarum and Pediococcus pentosaceus. The microecosystem of Feta cheese in the early ripening stage was dominated by Lp. plantarum and E. faecium, whereas, in late ripening, the microecosystem was dominated by Weissella paramesenteroides. The microecosystem of Kefalograviera cheese in the early ripening stage was dominated by Levilactobacillus brevis and E. faecium, and in late ripening by W. paramesenteroides and E. faecium. Finally, Carbohydrates was the main carbon source category that metabolized by all microbial communities, but the extent of their utilization was varied. Kefalograviera samples, especially at early ripening, demonstrated higher metabolic activity compared to Feta cheese. However, dominating species within microbial communities of the cheese samples were not significantly different.

Global Analysis of Saccharomyces cerevisiae Growth in Mucin

Metagenomic profiling of the human gut microbiome has discovered DNA from dietary yeasts like Saccharomyces cerevisiae. However, it is unknown if the S. cerevisiae detected by common metagenomic methods are from dead dietary sources, or from live S. cerevisiae colonizing the gut similar to their close relative Candida albicans. While S. cerevisiae can adapt to minimal oxygen and acidic environments, it has not been explored whether this yeast can metabolize mucin, the large, gel-forming, highly glycosylated proteins representing a major source of carbon in the gut mucosa. We reveal that S. cerevisiae can utilize mucin as their main carbon source, as well as perform both a transcriptome analysis and a chemogenomic screen to identify biological pathways required for this yeast to grow optimally in mucin. In total, 739 genes demonstrate significant differential expression in mucin culture, and deletion of 21 genes impact growth in mucin. Both screens suggest that mitochondrial function is required for proper growth in mucin, and through secondary assays we determine that mucin exposure induces mitogenesis and cellular respiration. We further show that deletion of an uncharacterized ORF, YCR095W-A, led to dysfunction in mitochondrial morphology and oxygen consumption in mucin. Lastly, we demonstrate that Yps7, an aspartyl protease and homologue to mucin-degrading proteins in C. albicans, is important for growth on mucin. Collectively, our work serves as the initial step towards establishing how this common dietary fungus can survive in the mucus environment of the human gut.

Metaproteogenomic Profiling of Chemosynthetic Microbial Biofilms Reveals Metabolic Flexibility During Colonization of a Shallow-Water Gas Vent

Tor Caldara is a shallow-water gas vent located in the Mediterranean Sea, with active venting of CO2 and H2S. At Tor Caldara, filamentous microbial biofilms, mainly composed of Epsilon- and Gammaproteobacteria, grow on substrates exposed to the gas venting. In this study, we took a metaproteogenomic approach to identify the metabolic potential and in situ expression of central metabolic pathways at two stages of biofilm maturation. Our findings indicate that inorganic reduced sulfur species are the main electron donors and CO2 the main carbon source for the filamentous biofilms, which conserve energy by oxygen and nitrate respiration, fix dinitrogen gas and detoxify heavy metals. Three metagenome-assembled genomes (MAGs), representative of key members in the biofilm community, were also recovered. Metaproteomic data show that metabolically active chemoautotrophic sulfide-oxidizing members of the Epsilonproteobacteria dominated the young microbial biofilms, while Gammaproteobacteria become prevalent in the established community. The co-expression of different pathways for sulfide oxidation by these two classes of bacteria suggests exposure to different sulfide concentrations within the biofilms, as well as fine-tuned adaptations of the enzymatic complexes. Taken together, our findings demonstrate a shift in the taxonomic composition and associated metabolic activity of these biofilms in the course of the colonization process.

Rhodotorula Strains Isolated from Seawater That Can Biotransform Raw Glycerol into Docosahexaenoic Acid (DHA) and Carotenoids for Animal Nutrition

Due to the overexploitation of industrial fisheries, as the principal source of fish oil, as well as the increasing replacement of synthetic pigments for animal nutrition, we need to find sustainable sources for these essential nutrient productions. Marine Rhodotorula strains NCYC4007 and NCYC1146 were used to determine the biosynthesis of docosahexaenoic acid (DHA) and carotenoids by biotransforming raw glycerol, a waste product of biodiesel. To evaluate the presence of inhibitory substances in raw glycerol, both strains were also grown in the presence of analytical grade glycerol and glucose as the main carbon source separately. With raw glycerol, NCYC4007 showed a significant correlation between DHA production and intracellular phosphorous concentrations. NCYC1146, a new Rhodotorula strain genetically described in this work, can produce canthaxanthin but only when glycerol is used as a main carbon source. Then, NCYC4007 could synthesize DHA as a phospholipid, and the production of canthaxanthin depends on the kind of carbon source used by NCYC1146. Finally, malate dehydrogenase activity and glucose production can be used as a proxy of the metabolisms in these marine Rhodotorula. This is the first evidence that marine Rhodotorula are capable of synthesizing DHA and canthaxanthin using an alternative and low-cost source of carbon to potentially scale their sustainable production for animal nutrition.

FTIR Analysis of Biodegradation of Polystyrene by Intestinal Bacteria Isolated from Zophobas Morio and Tenebrio Molitor

Since the 1970s, polystyrene (PS) plastic marine pollution has become a global concern. Tenebrio molitor and Zophobas morio beetle larva actively respond to a diet of primarily polystyrene. The tantalizing evidence indicates that the gut bacteria of these beetle larva enables them not only to consume polystyrene, but to successfully biodegrade polystyrene. Heretofore, data collection to verify polystyrene degradation by the gut bacteria of these larva has taken up to six months per test. Our laboratory created a platform by dissolving PS into a liquid mineral culture medium to examine PS degradation by the gut bacteria. Under investigation, PS works as main carbon source supporting the growth of gut bacteria. Fourier-transform Infrared spectroscopy (FTIR) is an effective method which can provide relevant information on chemical changes. This study presents a methodology using FTIR and Visible Light Spectrometry as an efficient methodology to verify the physiological degradation of polystyrene.

Metaproteogenomic profiling of chemosynthetic microbial biofilms reveals metabolic flexibility during colonization of a shallow-water gas vent

Tor Caldara is a shallow-water gas vent located in the Mediterranean Sea, with active venting of CO2, H2S. At Tor Caldara, filamentous microbial biofilms, mainly composed of Epsilon- and Gammaproteobacteria, grow on substrates exposed to the gas venting. In this study, we took a metaproteogenomic approach to identify the metabolic potential and in situ expression of central metabolic pathways at two stages of biofilm maturation. Our findings indicate that inorganic reduced sulfur species are the main electron donors and CO2 the main carbon source for the filamentous biofilms, which conserve energy by oxygen and nitrate respiration, fix dinitrogen gas and detoxify heavy metals. Three metagenome-assembled genomes (MAGs), representative of key members in the biofilm community, were also recovered. Metaproteomic data show that metabolically active chemoautotrophic sulfide-oxidizing members of the Epsilonproteobacteria dominated the young microbial biofilms, while Gammaproteobacteria become prevalent in the established community. The co-expression of different pathways for sulfide oxidation by these two classes of bacteria suggests exposure to different sulfide concentrations within the biofilms, as well as fine-tuned adaptations of the enzymatic complexes. Taken together, our findings demonstrate a shift in the taxonomic composition and associated metabolic activity of these biofilms in the course of the colonization process.

The seawater carbon inventory at the Paleocene–Eocene Thermal Maximum

The Paleocene–Eocene Thermal Maximum (PETM) (55.6 Mya) was a geologically rapid carbon-release event that is considered the closest natural analog to anthropogenic CO2 emissions. Recent work has used boron-based proxies in planktic foraminifera to characterize the extent of surface-ocean acidification that occurred during the event. However, seawater acidity alone provides an incomplete constraint on the nature and source of carbon release. Here, we apply previously undescribed culture calibrations for the B/Ca proxy in planktic foraminifera and use them to calculate relative changes in seawater-dissolved inorganic carbon (DIC) concentration, surmising that Pacific surface-ocean DIC increased by +1,010−646+1,415 µmol/kg during the peak-PETM. Making reasonable assumptions for the pre-PETM oceanic DIC inventory, we provide a fully data-driven estimate of the PETM carbon source. Our reconstruction yields a mean source carbon δ13C of −10‰ and a mean increase in the oceanic C inventory of +14,900 petagrams of carbon (PgC), pointing to volcanic CO2 emissions as the main carbon source responsible for PETM warming.

Enhanced Triacylglycerol Metabolism Contributes to Efficient Oil Utilization and High-Level Production of Salinomycin in Streptomyces albus ZD11

ABSTRACT Streptomyces is well known for biosynthesis of secondary metabolites with diverse bioactivities. Although oils have been employed as carbon sources to produce polyketide antibiotics for several industrial Streptomyces strains, the intrinsic correlation between oil utilization and high production of antibiotics still remains unclear. In this study, we investigated the correlation between oil metabolism and salinomycin biosynthesis in Streptomyces albus ZD11, which employs soybean oil as the main carbon source. Comparative genomic analysis revealed the enrichment of genes related to triacylglycerol (TAG) metabolism in S. albus ZD11. Transcriptomic profiling further confirmed the enhancement of TAG metabolism and acyl coenzyme A biosynthesis in S. albus ZD11. Multiple secreted lipases, which catalyze TAG hydrolysis, were seen to be working in a synergistic and complementary manner in aiding the efficient and stable hydrolyzation of TAGs. Together, our results suggest that enhanced TAG hydrolysis and fatty acid degradation contribute to the high efficiency of oil utilization in S. albus ZD11 in order to provide abundant carbon precursors for cell growth and salinomycin biosynthesis. IMPORTANCE In order to obtain high-level production of antibiotics, oils have been used as the main carbon source for some Streptomyces strains. Based on multiomics analysis, this study provides insight into the relationship between triacylglycerol (TAG) metabolism and antibiotic biosynthesis in S. albus ZD11, an oil-preferring industrial Streptomyces strain. Our investigation into TAG hydrolysis yielded further evidence that this strain utilizes complicated strategies enabling an efficient TAG metabolism. In addition, a novel secreted lipase was identified that exhibited highly hydrolytic activity for medium- and long-chain TAGs. Our findings represent a good start toward clarifying the complicated relationship between TAG catabolism and high-level antibiotic production in the industrial strains.

Siccibacter turicensis from Kangaroo Scats: Possible Implication in Cellulose Digestion

Microbiota in the kangaroo gut degrade cellulose, contributing to the kangaroo’s energy and survival. In this preliminary study, to discover more about the gut microbes that contribute to the survival of kangaroos, cellulose-degrading bacteria were isolated from kangaroo scats by selection on solidified media containing carboxymethyl cellulose as the main carbon source. One frequently occurring aerobic bacterium was Siccibacter turicensis, a microbe previously isolated in fruit powder and from a patient with angular cheilitis. The whole genome sequence of the kangaroo isolate was obtained using the Illumina MiSeq platform. Its sequence shared 97.98% identity of the S. turicensis Type strain, and the ability of the Type strain to degrade cellulose was confirmed. Analysis of the genomic data focused on the cellulose operon. In addition to genes from the operon, we suggest that a gene following the operon may have an important role in regulating cellulose metabolism by signal transduction. This is the first report of S. turicensis found within microbiota of the animal gut. Because of its frequent presence in the kangaroo gut, we suggest that S. turicensis plays a role in cellulose digestion for kangaroos.