Changes in Microbiota Composition during the Anaerobic Digestion of Macroalgae in a Three-Stage Bioreactor.

The use of microalgae as a raw material for biogas production is promising. Macroalgae were mixed with cattle manure, wheat straw, and an inoculant from sewage sludge. Mixing macroalgae with co-substrates increased biogas and methane yield. The research was carried out using a three-stage bioreactor. During biogas production, the dynamics of the composition of the microbiota in the anaerobic chamber of the bioreactor was evaluated. The microbiota composition at different organic load rates (OLRs) of the bioreactor was evaluated. This study also demonstrated that in a three-stage bioreactor, a higher yield of methane in biogas was obtained compared to a single-stage bioreactor. It was found that the most active functional pathway of methane biosynthesis is PWY-6969, which proceeds via the TCA cycle V (2-oxoglutarate synthase). Microbiota composition and methane yield depended on added volatile solids (VSadded). During the research, it was found that after reducing the ORL from 2.44 to 1.09 kg VS/d, the methane yield increased from 175.2 L CH4/kg VSadded to 323.5 L CH4/kg VSadded.

Changes in the Bacterial Communities of Biocomposites with Different Flame Retardants.

In today's world, the use of environmentally friendly materials is strongly encouraged. These materials derive from primary raw materials of plant origin, like fibrous hemp, flax, and bamboo, or recycled materials, such as textiles or residual paper, making them suitable for the growth of microorganisms. Here, we investigate changes in bacterial communities in biocomposites made of hemp shives, corn starch, and either expandable graphite or a Flovan compound as flame retardants. Using Next Generation Sequencing (NGS), we found that after 12 months of incubation at 22 °C with a relative humidity of 65%, Proteobacteria accounted for >99.7% of the microbiome in composites with either flame retardant. By contrast, in the absence of flame retardants, the abundance of Proteobacteria decreased to 32.1%, while Bacteroidetes (36.6%), Actinobacteria (8.4%), and Saccharobacteria (TM7, 14.51%) appeared. Using the increasing concentrations of either expandable graphite or a Flovan compound in an LB medium, we were able to achieve up to a 5-log reduction in the viability of Bacillus subtilis, Pseudomonas aeruginosa, representatives of the Bacillus and Pseudomonas genera, the abundance of which varied in the biocomposites tested. Our results demonstrate that flame retardants act on both Gram-positive and Gram-negative bacteria and suggest that their antimicrobial activities also have to be tested when producing new compounds.

Essential Oils of Mentha arvensis and Cinnamomum cassia Exhibit Distinct Antibacterial Activity at Different Temperatures In Vitro and on Chicken Skin.

The bacterial contamination of meat is a global concern, especially for the risk of Salmonella infection that can lead to health issues. Artificial antibacterial compounds used to preserve fresh meat can have negative health effects. We investigated the potential of natural essential oils (EOs), namely Mentha arvensis (mint) and Cinnamomum cassia (cinnamon) EOs, to prevent contamination of the food pathogen, Salmonella enterica subsp. enterica serotype Typhimurium, in vitro and on chicken skin. The gas chromatography-mass spectrometry (GC-MS) technique was used to determine the compositions of mint EO (MEO) and cinnamon EO (CEO); the most abundant compound in MEO was menthol (68.61%), and the most abundant compound was cinnamaldehyde (83.32%) in CEO. The antibacterial activity of MEO and CEO were examined in vapor and direct contact with S. typhimurium at temperatures of 4 °C, 25 °C, and 37 °C. The minimal inhibitory concentration at 37 °C for MEO and CEO reached 20.83 µL/mL, and the minimal bactericidal concentration of CEO was the same, while for MEO, it was two-fold higher. We report that in most tested conditions in experiments performed in vitro and on chicken skin, CEO exhibits a stronger antibacterial effect than MEO. In the vapor phase, MEO was more effective against S. typhimurium than CEO at 4 °C. In direct contact, the growth of S. typhimurium was inhibited more efficiently by MEO than CEO at small concentrations and a longer exposure time at 37 °C. The exploration of CEO and MEO employment for the inhibition of Salmonella bacteria at different temperatures and conditions expands the possibilities of developing more environment- and consumer-friendly antibacterial protection for raw meat.

Association of ScV-LA Virus with Host Protein Metabolism Determined by Proteomics Analysis and Cognate RNA Sequencing.

Saccharomyces yeasts are highly dispersed in the environment and microbiota of higher organisms. The yeast killing phenotype, encoded by the viral system, was discovered to be a significant property for host survival. Minor alterations in transcription patterns underpin the reciprocal relationship between LA and M viruses and their hosts, suggesting the fine-tuning of the transcriptional landscape. To uncover the principal targets of both viruses, we performed proteomics analysis of virus-enriched subsets of host proteins in virus type-specific manner. The essential pathways of protein metabolism-from biosynthesis and folding to degradation-were found substantially enriched in virus-linked subsets. The fractionation of viruses allowed separation of virus-linked host RNAs, investigated by high-content RNA sequencing. Ribosomal RNA was found to be inherently associated with LA-lus virus, along with other RNAs essential for ribosome biogenesis. This study provides a unique portrayal of yeast virions through the characterization of the associated proteome and cognate RNAs, and offers a background for understanding ScV-LA viral infection persistency.

Adaptive Response of Saccharomyces Hosts to Totiviridae L-A dsRNA Viruses Is Achieved through Intrinsically Balanced Action of Targeted Transcription Factors.

Totiviridae L-A virus is a widespread yeast dsRNA virus. The persistence of the L-A virus alone appears to be symptomless, but the concomitant presence of a satellite M virus provides a killer trait for the host cell. The presence of L-A dsRNA is common in laboratory, industrial, and wild yeasts, but little is known about the impact of the L-A virus on the host's gene expression. In this work, based on high-throughput RNA sequencing data analysis, the impact of the L-A virus on whole-genome expression in three different Saccharomyces paradoxus and S. cerevisiae host strains was analyzed. In the presence of the L-A virus, moderate alterations in gene expression were detected, with the least impact on respiration-deficient cells. Remarkably, the transcriptional adaptation of essential genes was limited to genes involved in ribosome biogenesis. Transcriptional responses to L-A maintenance were, nevertheless, similar to those induced upon stress or nutrient availability. Based on these data, we further dissected yeast transcriptional regulators that, in turn, modulate the cellular L-A dsRNA levels. Our findings point to totivirus-driven fine-tuning of the transcriptional landscape in yeasts and uncover signaling pathways employed by dsRNA viruses to establish the stable, yet allegedly profitless, viral infection of fungi.

The Bacterial Microbiota of Edible Insects Acheta domesticus and Gryllus assimilis Revealed by High Content Analysis.

In the concept of novel food, insects reared under controlled conditions are considered mini livestock. Mass-reared edible insect production is an economically and ecologically beneficial alternative to conventional meat gain. Regarding food safety, insect origin ingredients must comply with food microbial requirements. House crickets (Acheta domesticus) and Jamaican field crickets (Gryllus assimilis) are preferred insect species that are used commercially as food. In this study, we examined cricket-associated bacterial communities using amplicon-based sequencing of the 16S ribosomal RNA gene region (V3-V4). The high taxonomic richness of the bacterial populations inhabiting both tested cricket species was revealed. According to the analysis of alpha and beta diversity, house crickets and Jamaican field crickets displayed significantly different bacterial communities. Investigation of bacterial amplicon sequence variants (ASVs) diversity revealed cricket species as well as surface and entire body-associated bacterial assemblages. The efficiency of crickets processing and microbial safety were evaluated based on viable bacterial counts and identified bacterial species. Among the microorganisms inhabiting both tested cricket species, the potentially pathogenic bacteria are documented. Some bacteria representing identified genera are inhabitants of the gastrointestinal tract of animals and humans, forming a normal intestinal microflora and performing beneficial probiotic functions. The novel information on the edible insect-associated microbiota will contribute to developing strategies for cricket processing to avoid bacteria-caused risks and reap the benefits.

Gut Microbiome of Wild Baltic Salmon (Salmo salar L.) Parr.

Gut microbiota of wild Baltic salmon (a sub-population of Atlantic salmon Salmo salar L.) parr was first analyzed using microbial profiling of the 16S rRNA gene (V3-V4 region) and high taxonomic richness was revealed. At the phylum level, the gut microbiota was dominated by Firmicutes, Actinobacteria, and Proteobacteria, the most numerous of which were Firmicutes. The phylum Tenericutes (mainly assigned to Mycoplasmataceae), which is common both in wild North- and East- Atlantic salmon parr, was not detected in Baltic salmon parr. Across all samples, unique amplicon sequence variants (ASVs) belonging to the unclassified Bacilli, Actinomycetales, and Rhizobiales were identified as the major taxa. Fifteen ASVs at the family level were found in all gut samples of Baltic salmon parr, the majority of which were Mycobacteriaceae, Cryptosporangiaceae, Microbacteriaceae, and Planctomycetaceae. At the genus level, Mycobacterium, Clostridium sensu stricto, and Hyphomicrobium were dominant but at low levels in all gut samples. Our study has revealed that the gut microbial community of wild Baltic salmon parr differs from those of wild North- and East-Atlantic salmon parr. This can be due to biogeographical differences or host-selective pressures, as the Baltic salmon population is believed to have split from the Atlantic salmon population in the Ancylian period.

Mycobiota in the Carposphere of Sour and Sweet Cherries and Antagonistic Features of Potential Biocontrol Yeasts.

Sour cherries (Prunus cerasus L.) and sweet cherries (P. avium L.) are economically important fruits with high potential in the food industry and medicine. In this study, we analyzed fungal communities associated with the carposphere of sour and sweet cherries that were freshly harvested from private plantations and purchased in a food store. Following DNA isolation, a DNA fragment of the ITS2 rRNA gene region of each sample was individually amplified and subjected to high-throughput NGS sequencing. Analysis of 168,933 high-quality reads showed the presence of 690 fungal taxa. Investigation of microbial ASVs diversity revealed plant-dependent and postharvest handling-affected fungal assemblages. Among the microorganisms inhabiting tested berries, potentially beneficial or pathogenic fungi were documented. Numerous cultivable yeasts were isolated from the surface of tested berries and characterized by their antagonistic activity. Some of the isolates, identified as Aureobasidium pullulans, Metschnikowia fructicola, and M. pulcherrima, displayed pronounced activity against potential fungal pathogens and showed attractiveness for disease control.

Inactivation of Bacteria Using Bioactive Nanoparticles and Alternating Magnetic Fields.

Foodborne pathogens are frequently associated with risks and outbreaks of many diseases; therefore, food safety and processing remain a priority to control and minimize these risks. In this work, nisin-loaded magnetic nanoparticles were used and activated by alternating 10 and 125 mT (peak to peak) magnetic fields (AMFs) for biocontrol of bacteria Listeria innocua, a suitable model to study the inactivation of common foodborne pathogen L. monocytogenes. It was shown that L. innocua features high resistance to nisin-based bioactive nanoparticles, however, application of AMFs (15 and 30 min exposure) significantly potentiates the treatment resulting in considerable log reduction of viable cells. The morphological changes and the resulting cellular damage, which was induced by the synergistic treatment, was confirmed using scanning electron microscopy. The thermal effects were also estimated in the study. The results are useful for the development of new methods for treatment of the drug-resistant foodborne pathogens to minimize the risks of invasive infections. The proposed methodology is a contactless alternative to the currently established pulsed-electric field-based treatment in food processing.

Saccharomyces paradoxus Transcriptional Alterations in Cells of Distinct Phenotype and Viral dsRNA Content.

Killer yeasts are attractive antifungal agents with great potential applications in the food industry. Natural Saccharomyces paradoxus isolates provide new dsRNA-based killer systems available for investigation. The presence of viral dsRNA may alter transcriptional profile of S. paradoxus. To test this possibility, a high-throughput RNA sequencing was employed to compare the transcriptomes of S. paradoxus AML 15-66 K66 killer strains after curing them of either M-66 alone or both M-66 and L-A-66 dsRNA viruses. The S. paradoxus cells cured of viral dsRNA(s) showed respiration deficient or altered sporulation patterns. We have identified numerous changes in the transcription profile of genes including those linked to ribosomes and amino acid biosynthesis, as well as mitochondrial function. Our work advance studies of transcriptional adaptations of Saccharomyces spp. induced by changes in phenotype and set of dsRNA viruses, reported for the first time.

Correction: Luksa, J., et al. Fungal Microbiota of Sea Buckthorn Berries at Two Ripening Stages and Volatile Profiling of Potential Biocontrol Yeasts. Microorganisms 2020, 8, 456.

The authors wish to make the following corrections to this paper [...].

Fungal Microbiota of Sea Buckthorn Berries at Two Ripening Stages and Volatile Profiling of Potential Biocontrol Yeasts.

Sea buckthorn, Hippophae rhamnoides L., has considerable potential for landscape reclamation, food, medicinal, and cosmetics industries. In this study, we analyzed fungal microorganism populations associated with carposphere of sea buckthorn harvested in Lithuania. An amplicon metagenomic approach based on the ITS2 region of fungal rDNA was used to reveal the ripening-affected fungal community alterations on sea buckthorn berries. According to alpha and beta diversity analyses, depending on the ripening stage, sea buckthorn displayed significantly different fungal communities. Unripe berries were shown to be prevalent by Aureobasidium, Taphrina, and Cladosporium, while ripe berries were dominated by Aureobasidium and Metschnikowia. The selected yeast strains from unripe and mature berries were applied for volatile organic compounds identification by gas chromatography and mass spectrometry techniques. It was demonstrated that the patterns of volatiles of four yeast species tested were distinct from each other. The current study for the first time revealed the alterations of fungal microorganism communities colonizing the surface of sea buckthorn berries at different ripening stages. The novel information on specific volatile profiles of cultivable sea buckthorn-associated yeasts with a potential role in biocontrol is important for the development of the strategies for plant cultivation and disease management, as well as for the improvement of the quality and preservation of the postharvest berries. Management of the fungal microorganisms present on the surface of berries might be a powerful instrument for control of phytopathogenic and potentially antagonistic microorganisms affecting development and quality of the berries.

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses.

The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the ß-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.

High content analysis of sea buckthorn, black chokeberry, red and white currants microbiota - A pilot study.

The high potential of sea buckthorn, black chokeberry, red and white currants in healthy food industry boosted interest in the plant cultivation. The present study is the first work providing comprehensive information on microbial populations of these berries. Next Generation Sequencing allowed identification of eukaryotic and prokaryotic microorganisms prevalent on specific berries, including uncultivable microorganisms. Our study revealed the broad diversity of berries-associated bacterial and fungal microorganisms. Analysis of representative microbial OTUs showed a clear separation among inhabitants of sea buckthorn, black chokeberry and both currants, indicating plant-defined differences in the composition of the bacterial and fungal microbiota. Among the microorganisms distributed on tested berries, we documented potentially beneficial fungi and bacteria along with potential phytopathogens or those harmful for humans. Thus, plant microbiota appears to be highly relevant for the evaluation of the microbiota impact on food quality and human health.

Distribution of apple and blackcurrant microbiota in Lithuania and the Czech Republic.

The microbial assemblies on the surface of plants correlate with specific climatic features, suggesting a direct link between environmental conditions and microbial inhabitation patterns. At the same time however, microbial communities demonstrate distinct profiles depending on the plant species and region of origin. In this study, we report Next Generation Sequencing-based metagenomic analysis of microbial communities associated with apple and blackcurrant fruits harvested from Lithuania and the Czech Republic. Differences in the taxonomic composition of eukaryotic and prokaryotic microorganisms were observed between plant types. Our results revealed limited geographic differentiation between the bacterial and fungal communities associated with apples. In contrast, blackcurrant berries harvested from different regions demonstrated high diversity in both bacterial and fungal microbiota structures. Among fungal and bacterial microorganisms, we identified both potentially beneficial (Cryptococcus, Hanseniaspora, Massilia, Rhodotorula, Sphingomonas) and phytopathogenic microorganisms (Cladosporium, Pantoea, Phoma, Pseudomonas, Septoria, Taphrina) indicating their important roles in ecological and evolutionary processes.

Different Metabolic Pathways Are Involved in Response of Saccharomyces cerevisiae to L-A and M Viruses.

Competitive and naturally occurring yeast killer phenotype is governed by coinfection with dsRNA viruses. Long-term relationship between the host cell and viruses appear to be beneficial and co-adaptive; however, the impact of viral dsRNA on the host gene expression has barely been investigated. Here, we determined the transcriptomic profiles of the host Saccharomyces cerevisiae upon the loss of the M-2 dsRNA alone and the M-2 along with the L-A-lus dsRNAs. We provide a comprehensive study based on the high-throughput RNA-Seq data, Gene Ontology and the analysis of the interaction networks. We identified 486 genes differentially expressed after curing yeast cells of the M-2 dsRNA and 715 genes affected by the elimination of both M-2 and L-A-lus dsRNAs. We report that most of the transcriptional responses induced by viral dsRNAs are moderate. Differently expressed genes are related to ribosome biogenesis, mitochondrial functions, stress response, biosynthesis of lipids and amino acids. Our study also provided insight into the virus-host and virus-virus interplays.

Impact of pectin esterification on the antimicrobial activity of nisin-loaded pectin particles.

The relationship between pectin structure and the antimicrobial activity of nisin-loaded pectin particles was examined. The antimicrobial activity of five different nisin-loaded pectin particles, i.e., nisin-loaded high methoxyl pectin, low methoxyl pectin, pectic acid, dodecyl pectin with 5.4 and 25% degree of substitution were tested in the pH range of 4.0-7.0 by agar-diffusion assay and agar plate count methods. It was found that the degree of esterification of carboxyl group of galacturonic acid in pectin molecule is important for the antimicrobial activity of nisin-loaded pectin particles. Nisin-loaded particles prepared using pectic acid or the pectin with low degree of esterification exhibit higher antimicrobial activity than nisin-loaded high methoxyl pectin particles. Pectins with free carboxyl groups or of low degree of esterification are the most suitable for particles preparation. Moreover, nisin-loaded pectin particles were active at close to neutral or neutral pH values. Therefore, they could be effectively applied for food preservation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:245-251, 2017.

Correlation between the sorption of dissolved oxygen onto chitosan and its antimicrobial activity against Esherichia coli.

The ability of chitosan to adsorb dissolved oxygen from solution depends on its physical shape and is related to the surface area. Depending on conditions chitosan is capable of adsorbing or releasing oxygen. Chitosan, modificated by the substances possessing antimicrobial activity, such as succinic acid, Pd(II) ions, metallic Pd or Ag, distinctly increases the ability to adsorb the dissolved oxygen. The additional treatment of chitosan with air oxygen or electrochemically produced oxygen also increases the uptake of dissolved oxygen by chitosan. A strong correlation between the amount of oxygen adsorbed onto chitosan and its antimicrobial activity against Esherichia coli has been observed. This finding suggests that one of the sources of antimicrobial activity of chitosan is the ability to sorb dissolved oxygen, along with other well-known factors such as physical state and chemical composition.

Yeast ß-1,6-glucan is a primary target for the Saccharomyces cerevisiae K2 toxin.

Certain Saccharomyces cerevisiae strains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of ß-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-ß-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the ß-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that ß-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.

High-yield expression in Escherichia coli, purification and application of budding yeast K2 killer protein.

Saccharomyces cerevisiae K2 toxin is a highly active extracellular protein, important as a biocontrol agent for biotechnological applications in the wine industry. This protein is produced at negligible levels in yeast, making difficult to isolate it in amounts sufficient for investigation and generation of analysis tools. In this work, we demonstrate the use of a bacterial system for expression of the recombinant K2 protein, suitable for generation of antibodies specific for toxin of the yeast origin. Synthesis of the full-length S. cerevisiae K2 preprotoxin in Escherichia coli was found to be toxic to the host cell, resulting in diminished growth. Such effect was abolished by the introduction of the C-terminal truncation into K2 protein, directing it into non-toxic inclusion body fraction. The obtained protein is of limited solubility thus, facilitating the purification by simple and efficient chromatography-free procedure. The protein aggregates were successfully refolded into a soluble form yielding sufficient amounts of a tag-less truncated K2 protein suitable for polyclonal antibody production. Antibodies were raised in rabbit and found to be specific for detection of both antigen and native S. cerevisiae K2 toxin.