Refermentation and maturation of lambic beer in bottles: a necessary step for gueuze production.

The production of gueuze beers through refermentation and maturation of blends of lambic beer in bottles is a way for lambic brewers to cope with the variability among different lambic beer batches. The resulting gueuze beers are more carbonated than lambic beers and are supposed to possess a unique flavor profile that varies over time. To map this refermentation and maturation process for gueuze production, a blend of lambic beers was made and bottled, whereby one of them was produced with the old wheat landrace Zeeuwse Witte. Through the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high-throughput sequencing of bacterial and fungal amplicons, in combination with metabolite target analysis, new insights into gueuze production were obtained. During the initial stages of refermentation, the conditions in the bottles were similar to those encountered during the maturation phase of lambic beer productions in wooden barrels, which was also reflected microbiologically (presence of Brettanomyces species, Pediococcus damnosus, and Acetobacter lambici) and biochemically (ethanol, higher alcohols, lactic acid, acetic acid, volatile phenolic compounds, and ethyl esters). However, after a few weeks of maturation, a switch from a favorable environment to one with nutrient and dissolved oxygen depletion resulted in several changes. Concerning the microbiology, a sequential prevalence of three lactic acid bacterial species occurred, namely, P. damnosus, Lentilactobacillus buchneri, and Lactobacillus acetotolerans, while the diversity of the yeasts decreased. Concerning the metabolites produced, mainly those of the Brettanomyces yeasts determined the metabolic profiles encountered during later stages of the gueuze production.IMPORTANCEGueuze beers are the result of a refermentation and maturation process of a blend of lambic beers carried out in bottles. These gueuze beers are known to have a long shelf life, and their quality typically varies over time. However, knowledge about gueuze production in bottles is scarce. The present study provided more insights into the varying microbial and metabolite composition of gueuze beers during the first 2 years of this refermentation and maturation process. This will allow gueuze producers to gain more information about the influence of the refermentation and maturation time on their beers. These insights can also be used by gueuze producers to better inform their customers about the quality of young and old gueuze beers.

Microbial characterization of Sichuan Baoning vinegar: lactic acid bacteria, acetic acid bacteria and yeasts.

Sichuan Baoning vinegar, a typical representative of Sichuan bran vinegar, is a famous traditional fermented food made from cereals in China. At present, there are few studies on microbial characterization of culturable microorganisms in solid-state fermentation of Sichuan bran vinegar. To comprehensively understand the diversity of lactic acid bacteria, acetic acid bacteria and yeasts, which play an important role in the fermentation of Sichuan bran vinegar, traditional culture-dependent methods combined with morphological, biochemical, and molecular identification techniques were employed to screen and identify these isolates. A total of 34 lactic acid bacteria isolates, 39 acetic acid bacteria isolates, and 48 yeast isolates were obtained. Lactic acid bacteria were dominated by Enterococcus durans, Leuconostoc citreum, Lactococcus lactis, and Lactiplantibacillus plantarum, respectively. Latilactobacillus sakei was the first discovery in cereal vinegar. Acetic acid bacteria were mainly Acetobacter pomorum and A. pasteurianus. The dominant yeast isolates were Saccharomyces cerevisiae, in addition to four non-Saccharomyces yeasts. DNA fingerprinting revealed that isolates belonging to the same species exhibited intraspecific diversity, and there were differences between phenotypic and genotypic classification results. This study further enriches studies on cereal vinegar and lays a foundation for the development of vinegar starters.

Sorlinia euscelidii gen. nov., sp. nov., a novel acetic acid bacterium isolated from the leafhopper Euscelidius variegatus (Hemiptera: Cicadellidae).

Acetic acid bacteria - belonging to the Acetobacteraceae family - are found in the gut of many sugar-feeding insects. In this study, six strains have been isolated from the hemipteran leafhopper Euscelidius variegatus. While they exhibit high 16S rRNA gene sequence similarities to uncultured members of the Acetobacteraceae family, they could not be unequivocally assigned to any particular type species. Considering the clonality of the six isolates, the EV16PT strain was used as a representative of this group of isolates. The genome sequence of EV16PT is composed of a 2.388 Mbp chromosome, with a DNA G+C content of 57 mol%. Phylogenetic analyses based on the 16S rRNA gene sequence and whole-genome multilocus sequence analysis indicate that EV16PT forms a monophyletic clade with the uncultivated endosymbiont of Diaphorina citri, the Candidatus Kirkpatrickella diaphorinae. Such a phylogenetic clade is positioned between those of Asaia-Swaminathania and Kozakia. The genomic distance metrics based on gene and protein sequences support the proposal that EV16PT is a new species belonging to a yet-undescribed genus. It is a rod-shaped Gram-stain-negative bacterium, strictly aerobic, non-motile, non-spore-forming, showing optimal growth without salt (NaCl) at 30 °C and pH of 6-7. The major quinone is Q10, and the dominant cellular fatty acids (>10%) are C18:l ω7c, C19 : 0 cyclo ω6c, C16 : 0 and C19 : 1 2OH. The polar lipid profile comprises diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine, along with unidentified aminophospholipids, glycophospholipids, aminolipids and lipids. Based on a polyphasic approach, including phylogenetic, phylogenomic, genome relatedness, phenotypic and chemotaxonomic characterisations, EV16PT (= KCTC 8296T, = DSM 117028T) is proposed as a representative of a novel species in a novel genus with the proposed name Sorlinia euscelidii gen. nov., sp. nov., in honour of Prof. Claudia Sorlini, an Italian environmental microbiologist at the University of Milan who inspired the research on microbial diversity, including symbiosis in plants and animals.

As next-generation probiotics: acetic acid bacteria isolated from Kombucha beverages produced with Anatolian hawthorn leaves.

This research examined acetic acid bacteria (AAB) isolated from Kombucha beverages produced with Anatolian hawthorn (Crataegus orientalis) as next-generation probiotics. Eighty-six AAB were isolated from the samples and investigated in terms of biosafety, viability in vitro gastrointestinal conditions, technological and bioactive properties, and also in vitro adhesion abilities. Seventy-six isolates demonstrating γ-hemolysis exhibited resistance to erythromycin and ampicillin. Besides, these isolates survived at low pH and in the presence of bile salts. However, the majority of AAB isolates showed tolerance to phenol, pepsin, and pancreatin. Also, twenty-one isolates showed protease enzyme activity, while eight isolates had amylase enzyme activity. Despite most of the isolates showed viability at 1.5% salt, only 19 isolates survived at 10% salt. Most AAB isolates exhibited inhibition zones ranging from 8 to 26 mm against test bacteria, their antioxidant activities were above 80%. Additionally, some isolates exhibited auto-aggregation ability ranging from 0.66 to 23.62% and co-aggregation ability ranging from 1.18 to 71.32%, while hydrophobicity ranged from 1.32 to 69.87% toward xylene. Finally, the indigenous 76 AAB isolates that had remarkable probiotic properties were characterized based on 16S rRNA gene sequencing, and the isolates belonged to Komagateibacter sp. (64.47%), Komagateibacter saccharivorans (15.79%), K. rhaeticus (11.84%), and Gluconobacter sp. (7.90%). As a result, the isolates identified as Gluconobacter sp. A21, Komagataeibacter sp. A139, Gluconobacter sp. A141, and Komagataeibacter sp. A146, which showed high viability under gastrointestinal conditions, safe and acceptable in terms of technological, bioactive, and adhesion properties and could be evaluated as next-generation probiotics.

Comparative studies on substrate specificity of succinic semialdehyde reductase from Gluconobacter oxydans and glyoxylate reductase from Acetobacter aceti.

Gluconobacter oxydans succinic semialdehyde reductase (GoxSSAR) and Acetobacter aceti glyoxylate reductase (AacGR) represent a novel class in the ß-hydroxyacid dehydrogenases superfamily. Kinetic analyses revealed GoxSSAR's activity with both glyoxylate and succinic semialdehyde, while AacGR is glyoxylate specific. GoxSSAR K167A lost activity with succinic semialdehyde but retained some with glyoxylate, whereas AacGR K175A lost activity. These findings elucidate differences between these homologous enzymes.

Implementation of a Novel Method for Processing Proteins from Acetic Acid Bacteria via Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Acetic acid bacteria (AAB) and other members of the complex microbiotas, whose activity is essential for vinegar production, display biodiversity and richness that is difficult to study in depth due to their highly selective culture conditions. In recent years, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for rapidly identifying thousands of proteins present in microbial communities, offering broader precision and coverage. In this work, a novel method based on LC-MS/MS was established and developed from previous studies. This methodology was tested in three studies, enabling the characterization of three submerged acetification profiles using innovative raw materials (synthetic alcohol medium, fine wine, and craft beer) while working in a semicontinuous mode. The biodiversity of existing microorganisms was clarified, and both the predominant taxa (Komagataeibacter, Acetobacter, Gluconacetobacter, and Gluconobacter) and others never detected in these media (Asaia and Bombella, among others) were identified. The key functions and adaptive metabolic strategies were determined using comparative studies, mainly those related to cellular material biosynthesis, energy-associated pathways, and cellular detoxification processes. This study provides the groundwork for a highly reliable and reproducible method for the characterization of microbial profiles in the vinegar industry.

Chemical Synthesis of Acetobacter pasteurianus Lipid A with a Unique Tetrasaccharide Backbone and Evaluation of Its Immunological Functions.

Lipopolysaccharide (LPS), a cell surface component of Gram-negative bacteria, activates innate immunity. Its active principle is the terminal glycolipid lipid A. Acetobacter pasteurianus is a Gram-negative bacterium used in the fermentation of traditional Japanese black rice vinegar (kurozu). In this study, we focused on A. pasteurianus lipid A, which is a potential immunostimulatory component of kurozu. The active principle structure of A. pasteurianus lipid A has not yet been identified. Herein, we first systematically synthesized three types of A. pasteurianus lipid As containing a common and unique tetrasaccharide backbone. We developed an efficient method for constructing the 2-trehalosamine skeleton utilizing borinic acid-catalyzed glycosylation to afford 1,1'-α,α-glycoside in high yield and stereoselectivity. A common tetrasaccharide intermediate with an orthogonal protecting group pattern was constructed via [2+2] glycosylation. After introducing various fatty acids, all protecting groups were removed to achieve the first chemical synthesis of three distinct types of A. pasteurianus lipid As. After evaluating their immunological function using both human and murine cell lines, we identified the active principles of A. pasteurianus LPS. We also found the unique anomeric structure of A. pasteurianus lipid A contributes to its high chemical stability.

Sourdough production: fermentation strategies, microbial ecology, and use of non-flour ingredients.

Sourdough production is an ancient method to ferment flour from cereals for the manufacturing of baked goods. This review deals with the state-of-the-art of current fermentation strategies for sourdough production and the microbial ecology of mature sourdoughs, with a particular focus on the use of non-flour ingredients. Flour fermentation processes for sourdough production are typically carried out by heterogeneous communities of lactic acid bacteria and yeasts. Acetic acid bacteria may also occur, although their presence and role in sourdough production can be criticized. Based on the inoculum used, sourdough productions can be distinguished in fermentation processes using backslopping procedures, originating from a spontaneously fermented flour-water mixture (Type 1), starter culture-initiated fermentation processes (Type 2), and starter culture-initiated fermentation processes that are followed by backslopping (Type 3). In traditional recipes for the initiation and/or propagation of Type 1 sourdough productions, non-flour ingredients are often added to the flour-water mixture. These ingredients may be the source of an additional microbial inoculum and/or serve as (co-)substrates for fermentation. An example of the former is the addition of yoghurt; an example of the latter is the use of fruit juices. The survival of microorganisms transferred from the ingredients to the fermenting flour-water mixture depends on the competitiveness toward particular strains of the microbial species present under the harsh conditions of the sourdough ecosystem. Their survival and growth is also determined by the presence of the appropriate substrates, whether or not carried over by the ingredients added.

Bombella pluederhausensis sp. nov., Bombella pollinis sp. nov., Bombella saccharophila sp. nov. and Bombella dulcis sp. nov., four Bombella species isolated from the environment of the western honey bee Apis mellifera.

Four strains of members of the genus Bombella were isolated from samples associated with the western honey bee Apis mellifera, which could not be assigned to a species with a validly published name. Strains TMW 2.2543T, TMW 2.2556T, TMW 2.2558T and TMW 2.2559T exhibit in silico DNA-DNA hybridisation (isDDH) and orthologous average nucleotide identity (orthoANI) values below species delineation thresholds compared with all described species of the genus Bombella and with each other. TMW 2.2556T and TMW 2.2558T form their own clade within the genus. The major respiratory quinone of all strains was Q-10. The composition of cellular fatty acids was diverse between strains. All strains stained Gram-negative, were rod-shaped, strictly aerobic, pellicle-forming, catalase-positive, oxidase-negative, mesophilic and grew over a wide pH range; they were halosensitive but glucose-tolerant. Unlike the other studied strains, TMW 2.2558T was non-motile. Phylogenetic, chemotaxonomic and physiological analyses revealed a clear distinction between all the strains and species with validly published names. All the data support the proposition of four novel species within the genus Bombella, namely Bombella pluederhausensis sp. nov., Bombella pollinis sp. nov., Bombella saccharophila sp. nov. and Bombella dulcis sp. nov., with the respective type strains Bombella pluederhausensis sp. nov. TMW 2.2543T (= DSM 114872T, = LMG 32791T), Bombella pollinis sp. nov. TMW 2.2556T (= DSM 114874T, = LMG 32792T), Bombella saccharophila sp. nov. TMW 2.2558T (= DSM 114875T, = LMG 32793T) and Bombella dulcis sp. nov. TMW 2.2559T (= DSM 114877T, = LMG 32794T). Moreover, three genomes available in the NCBI database that have not yet been described as species with validly published names could be assigned to the proposed species. Bombella sp. ESL0378 and Bombella sp. ESL0385 to Bombella pollinis sp. nov. and Bombella sp. AS1 to Bombella saccharophila sp. nov.

Comparative metatranscriptome analysis of Brazilian milk and water kefir beverages.

The present study compared bacterial and fungal diversity of kefir beverages produced using milk (MK) or sugared water (WK) as propagation matrices and grains from the cities of Curitiba (CU) or Salvador (SA), Brazil, by sequencing the complete set of RNA transcripts produced in four products. In Brazil, milk and sugared water are used as matrices to propagate kefir grains. In all beverages, the bacterial community was composed of Lactobacillaceae and Acetobacteraceae. Saccharomycetaceae was the yeast family more abundant in WK, and Dipodascaceae and Pichiaceae in MK. Regarding KEGG mapping of functional orthologs, the four kefir samples shared 70% of KO entries of yeast genes but only 36% of bacterial genes. Concerning main metabolic processes, the relative abundance of transcripts associated with metabolism (energy metabolism) and environmental information processing (membrane transport) had the highest water/milk kefir ratio observed in Firmicutes. In contrast, transcripts associated with genetic information processing (protein translation, folding, sorting, and degradation) oppositely had the lowest water/milk ratios. Concluding, milk and water kefir have quite different communities of microorganisms. Still, the main mapped functional processes are similar, with only quantitative variation in membrane transport and energy acquisition in the water kefir and protein synthesis and turnover in the milk kefir.

Glycoside hydrolase family 32 enzymes from Bombella spp. catalyze the formation of high-molecular weight fructans from sucrose.

AIMS: Acetic acid bacteria of the genus Bombella have not been reported to produce exopolysaccharides (EPS). In this study, the formation of fructans by B. apis TMW 2.1884 and B. mellum TMW 2.1889 was investigated. METHODS AND RESULTS: Out of eight strains from four different Bombella species, only B. apis TMW 2.1884 and B. mellum TMW 2.1889 showed EPS formation with 50 g l-1 sucrose as substrate. Both EPS were identified as high-molecular weight (HMW) polymers (106-107 Da) by asymmetric flow field-flow fractionation coupled to multi angle laser light scattering and UV detecors (AF4-MALLS/UV) and high performance size exclusion chromatography coupled to MALLS and refractive index detectors (HPSEC-MALLS/RI) analyses. Monosaccharide analysis via trifluoroacetic acid hydrolysis showed that both EPS are fructans. Determination of glycosidic linkages by methylation analysis revealed mainly 2,6-linked fructofuranose (Fruf) units with additional 2,1-linked Fruf units (10%) and 2,1,6-Fruf branched units (7%). No glycoside hydrolase (GH) 68 family genes that are typically associated with the formation of HMW fructans in bacteria could be identified in the genomes. Through heterologous expression in Escherichia coli Top10, an enzyme of the GH32 family could be assigned to the catalysis of fructan formation. The identified fructosyltransferases could be clearly differentiated phylogenetically and structurally from other previously described bacterial fructosyltransferases. CONCLUSIONS: The formation of HMW fructans by individual strains of the genus Bombella is catalyzed by enzymes of the GH32 family. Analysis of the fructans revealed an atypical structure consisting of 2,6-linked Fruf units as well as 2,1-linked Fruf units and 2,1,6-Fruf units.

Acetic acid bacteria in agro-wastes: from cheese whey and olive mill wastewater to cellulose.

In this study, cheese whey and olive mill wastewater were investigated as potential feedstocks for producing bacterial cellulose by using acetic acid bacteria strains. Organic acids and phenolic compounds composition were assayed by high-pressure liquid chromatography. Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction were used to investigate modifications in bacterial cellulose chemical and morphological structure. Cheese whey was the most efficient feedstock in terms of bacterial cellulose yield (0.300 g of bacterial cellulose/gram of carbon source consumed). Bacterial cellulose produced in olive mill wastewater presented a more well-defined network compared to pellicles produced in cheese whey, resulting in a smaller fiber diameter in most cases. The analysis of bacterial cellulose chemical structure highlighted the presence of different chemical bonds likely to be caused by the adsorption of olive mill wastewater and cheese whey components. The crystallinity ranged from 45.72 to 80.82%. The acetic acid bacteria strains used in this study were characterized by 16S rRNA gene sequencing, allowing to assign them to Komagataeibacter xylinus and Komagataeibacter rhaeticus species. This study proves the suitability to perform sustainable bioprocesses for producing bacterial cellulose, combining the valorisation of agro-wastes with microbial conversions carried out by acetic acid bacteria. The high versatility in terms of yield, morphology, and fiber diameters obtained in cheese whey and olive mill wastewater contribute to set up fundamental criteria for developing customized bioprocesses depending on the final use of the bacterial cellulose. KEY POINTS: • Cheese whey and olive mill wastewater can be used for bacterial cellulose production. • Bacterial cellulose structure is dependent on the culture medium. • Komagataeibacter strains support the agro-waste conversion in bacterial cellulose.

Analysis of cellulose synthesis in a high-producing acetic acid bacterium Komagataeibacter hansenii.

Bacterial cellulose (BC) represents a renewable biomaterial with unique properties promising for biotechnology and biomedicine. Komagataeibacter hansenii ATCC 53,582 is a well-characterized high-yield producer of BC used in the industry. Its genome encodes three distinct cellulose synthases (CS), bcsAB1, bcsAB2, and bcsAB3, which together with genes for accessory proteins are organized in operons of different complexity. The genetic foundation of its high cellulose-producing phenotype was investigated by constructing chromosomal in-frame deletions of the CSs and of two predicted regulatory diguanylate cyclases (DGC), dgcA and dgcB. Proteomic characterization suggested that BcsAB1 was the decisive CS because of its high expression and its exclusive contribution to the formation of microcrystalline cellulose. BcsAB2 showed a lower expression level but contributes significantly to the tensile strength of BC and alters fiber diameter significantly as judged by scanning electron microscopy. Nevertheless, no distinct extracellular polymeric substance (EPS) from this operon was identified after static cultivation. Although transcription of bcsAB3 was observed, expression of the protein was below the detection limit of proteome analysis. Alike BcsAB2, deletion of BcsAB3 resulted in a visible reduction of the cellulose fiber diameter. The high abundance of BcsD and the accessory proteins CmcAx, CcpAx, and BglxA emphasizes their importance for the proper formation of the cellulosic network. Characterization of deletion mutants lacking the DGC genes dgcA and dgcB suggests a new regulatory mechanism of cellulose synthesis and cell motility in K. hansenii ATCC 53,582. Our findings form the basis for rational tailoring of the characteristics of BC. KEY POINTS: • BcsAB1 induces formation of microcrystalline cellulose fibers. • Modifications by BcsAB2 and BcsAB3 alter diameter of cellulose fibers. • Complex regulatory network of DGCs on cellulose pellicle formation and motility.

Bacterial cellulose: Strategies for its production in the context of bioeconomy.

Bacterial cellulose has advantages over plant-derived cellulose, which make its use for industrial applications easier and more profitable. Its intrinsic properties have been stimulating the global biopolymer market, with strong growth expectations in the coming years. Several bacterial species are capable of producing bacterial cellulose under different culture conditions; in this context, strategies aimed at metabolic engineering and several possibilities of carbon sources have provided opportunities for the bacterial cellulose's biotechnological exploration. In this article, an overview of biosynthesis pathways in different carbon sources for the main producing microorganisms, metabolic flux under different growth conditions, and their influence on the structural and functional characteristics of bacterial cellulose is provided. In addition, the main industrial applications and ways to reduce costs and optimize its production using alternative sources are discussed, contributing to new insights on the exploitation of this biomaterial in the context of the bioeconomy.

The Roles of the Various Cellulose Biosynthesis Operons in Komagataeibacter hansenii ATCC 23769.

Cellulose is the most abundant biopolymer on earth and offers versatile applicability in biotechnology. Bacterial cellulose, especially, is an attractive material because it represents pure microcrystalline cellulose. The cellulose synthase complex of acetic acid bacteria serves as a model for general studies on (bacterial) cellulose synthesis. The genome of Komagataeibacter hansenii ATCC 23769 encodes three cellulose synthase (CS) operons of different sizes and gene compositions. This implies the question of which role each of the three CS-encoding operons, bcsAB1, bcsAB2, and bcsAB3, plays in overall cellulose synthesis. Therefore, we constructed markerless deletions in K. hansenii ATCC 23769, yielding mutant strains that expressed only one of the three CSs. Apparently, BcsAB1 is the only CS that produces fibers of crystalline cellulose. The markerless deletion of bcsAB1 resulted in a nonfiber phenotype in scanning electron microscopy analysis. Expression of the other CSs resulted in a different, nonfibrous extracellular polymeric substance (nfEPS) structure wrapping the cells, which is proposed to contain acetylated cellulose. Transcription analysis revealed that all CSs were expressed continuously and that bcsAB2 showed a higher transcription level than bcsAB1. Moreover, we were able to link the expression of diguanylate cyclase B (dgcB) to cellulose production. IMPORTANCE Acetic acid bacteria form a massive biofilm called "mother of vinegar," which is built of cellulose fibers. Bacterial cellulose is an appealing biomaterial with manifold applications in biomedicine and biotechnology. Because most cellulose-producing acetic acid bacteria express several cellulose synthase operons, a deeper understanding of their contribution to the synthesis of modified forms of cellulose fibers within a natural biofilm is of special interest. For the first time, we were able to identify the contribution of each of the three cellulose synthases to cellulose formation in Komagataeibacter hansenii ATCC 23769 after a chromosomal clean deletion. Moreover, we were able to depict their roles in spatial composition of the biofilm. These findings might be applicable in the future for naturally modified biomaterials with novel properties.

Phylogenomic and comparative analyses support the reclassification of several Komagataeibacter species as novel members of the Novacetimonas gen. nov. and bring new insights into the evolution of cellulose synthase genes.

The genus Komagataeibacter harbours bacteria presenting the ability to produce increased levels of crystalline nanocellulose, as well as strains used in the industrial production of fermented products and beverages. Still, most of the studies of this biotechnologically relevant genus were conducted based on limited phenotypic methodologies and taxonomical classifications. In this work, a detailed analysis of the currently described genus Komagataeibacter was conducted based on phylogenomic analysis, unveiling the phylogenomic relationships within the genus and allowing a detailed phylogenetic analysis of biotechnologically important genes such as those involved in cellulose biosynthesis (bcs genes). Phylogenomic and comparative genomic analysis revealed that several type strains formed an independent genomic group from those of other Komagataeibacter, prompting their reclassification as members of a novel genus, hereby termed Novacetimonas gen. nov. The results support the reclassification of Komagataeibacter hansenii, Komagataeibacter cocois, Komagataeibacter maltaceti and Komagataeibacter pomaceti as novel members of the genus Novacetimonas. The Novacetimonas hansenii species is the proposed representative of the novel genus. Importantly, phylogenetic analysis based on cellulose biosynthesis genes (bcsABCD, bcsAB2XYC2, bcsAB3C3, bcsAB4), showed that the evolutionary history of these genes is closely related to the strain's phylogenomic/taxonomic classification. Hence, the robust taxonomic classification of these bacteria will allow the better characterization and selection of strains for biotechnological applications.

Bacterial cellulose production by Novacetimonas hansenii MSCL 1646 on apple juice.

Biomaterials and biopolymers, such as bacterial cellulose (BC), are becoming increasingly important as sustainable materials with a wide range of potential applications. However, BC industrial production is associated with several difficulties such as low BC production yields and high production costs; therefore, cheap alternative growth media, e.g. apple juice are being studied intensively. The aim of this study is to evaluate BC synthesis under static conditions on apple juice medium (AJM). The optimal concentration of apple juice in unsupplemented AJM for Novacetimonas hansenii MSCL 1646 was shown by its dilution 1:6 with water, which resulted in 0.89 ± 0.01 g/L of dry BC weight after 10 cultivation days. Low BC synthesis can be associated with insufficient N concentration in apple juice; therefore, different organic and inorganic N sources were evaluated in combination with AJM, and beef extract (5 g/L) was found to be the most suitable. Further, AJM optimisation experiment showed the optimal apple juice and beef extract concentrations as 1:2 and 15 g/L respectively, which resulted in 17.27 ± 0.07 g/L of dry BC weight, which is significantly higher than in standard Hestrin-Schramm (HS) medium (4.07 ± 0.02 g/L). Analysis of mechanical and physical properties showed that use of AJM results in changes in BC properties compared with the standard HS medium. Results of the study indicate that apple juice is an effective and cheap C source that in combination with appropriate N source leads to high BC synthesis and makes it suitable for industrial BC production. KEY POINTS: • Low quality apples can be used as raw material for BC production; • Beef extract improves BC synthesis in apple juice medium; • Use of apple juice and beef extract affect mechanical properties of BC.

Comparative pangenomic analyses and biotechnological potential of cocoa-related Acetobacter senegalensis strains.

Acetobacter senegalensis belongs to the group of acetic acid bacteria (AAB) that present potential biotechnological applications, for production of D-gluconate, cellulose and acetic acid. AAB can overcome heat and acid stresses by using strategies involving the overexpression of heat-shock proteins and enzymes from the complex pyrroquinoline-ADH, besides alcohol dehydrogenases (ADH). Nonetheless, the isolation of A. senegalensis and other AAB from food may be challenging due to presence of viable but non-culturable (VBNC) cells and due to uncertainties about nutritional requirements. To contribute for a better understanding of the ecology of AAB, this paper reports on the pangenome analysis of five strains of A. senegalensis recently isolated from a Brazilian spontaneous cocoa fermentation. The results showed biosynthetic clusters exclusively found in some cocoa-related AAB, such as those related to terpene pathways, which are important for flavour development. Genes related to oxidative stress were conserved in all the genomes, with multiple clusters. Moreover, there were genes coding for ADH and putative ABC transporters distributed in core, shell and cloud genomes, while chaperonin-encoding genes were present only in the core and soft-core genomes. Regarding quorum sensing, a response regulator gene was in the shell genome, and the gene encoding for acyl-homoserine lactone efflux protein was in the soft-core genome. There were quorum quenching-related genes, mainly encoding for lactonases, but also for acylases. Moreover, A. senegalensis did not have determinants of virulence or antibiotic resistance, which are good traits for strains intended to be applied in food fermentation.

Membrane-bound D-mannose isomerase of acetic acid bacteria: finding, characterization, and application.

D-Mannose isomerase (EC 5.3.1.7) catalyzing reversible conversion between D-mannose and D-fructose was found in acetic acid bacteria. Cell fractionation confirmed the enzyme to be a typical membrane-bound enzyme, while all sugar isomerases so far reported are cytoplasmic. The optimal enzyme activity was found at pH 5.5, which was clear contrast to the cytoplasmic enzymes having alkaline optimal pH. The enzyme was heat stable and the optimal reaction temperature was observed at around 40 to 60˚C. Purified enzyme after solubilization from membrane fraction showed the total molecular mass of 196 kDa composing of identical four subunits of 48 kDa. Washed cells or immobilized cells were well functional at nearly 80% of conversion ratio from D-mannose to D-fructose and reversely 20-25% of D-fructose to D-mannose. Catalytic properties of the enzyme were discussed with respect to the biotechnological applications to high fructose syrup production from konjac taro.

Periplasmic dehydroshikimate dehydratase combined with quinate oxidation in Gluconobacter oxydans for protocatechuate production.

Protocatechuate (3,4-dihydroxybenzoate) has antioxidant properties and is a raw material for the production of muconic acid, which is a key compound in the synthesis of polymers such as nylon and polyethylene terephthalate. Gluconobacter oxydans strain NBRC3244 has a periplasmic system for oxidation of quinate to produce 3-dehydroquinate. Previously, a periplasmic 3-dehydroshikimate production system was constructed by heterologously expressing Gluconacetobacter diazotrophicus dehydroquinate dehydratase in the periplasm of G. oxydans strain NBRC3244. 3-Dehydroshikimate is converted to protocatechuate by dehydration. In this study, we constructed a G. oxydans strain that expresses the Acinetobacter baylyi quiC gene, which encodes a dehydroshikimate dehydratase of which the subcellular localization is likely the periplasm. We attempted to produce protocatechuate by co-cultivation of two recombinant G. oxydans strains-one expressing the periplasmically targeted dehydroquinate dehydratase and the other expressing A. baylyi dehydroshikimate dehydratase. The co-cultivation system produced protocatechuate from quinate in a nearly quantitative manner.