Whole genome sequencing analysis of Komagataeibacter nataicola reveals its potential in food waste valorisation for cellulose production.

BACKGROUND: Komagataeibacter nataicola (K. nataicola) is a gram-negative acetic acid bacterium that produces natural bacterial cellulose (BC) as a fermentation product under acidic conditions. The goal of this work was to study the complete genome of K. nataicola and gain insight into the functional genes in K. nataicola that are responsible for BC synthesis in acidic environments. METHODS AND RESULT: The pure culture of K. nataicola was obtained from yeast-glucose-calcium carbonate (YGC) agar, followed by genomic DNA extraction, and subjected to whole genome sequencing on a Nanopore flongle flow cell. The genome of K. nataicola consists of a 3,767,936 bp chromosome with six contigs and 4,557 protein coding sequences. The maximum likelihood phylogenetic tree and average nucleotide identity analysis confirmed that the bacterial isolate was K. nataicola. The gene annotation via RAST server discovered the presence of cellulose synthase, along with three genes associated with lactate utilization and eight genes involved in lactate fermentation that could potentially contribute to the increase in acid concentration during BC synthesis. CONCLUSION: A more comprehensive genome study of K. nataicola may shed light into biological pathway in BC productivity as well as benefit the analysis of metabolites generated and understanding of biological and chemical interactions in BC production later.

Complete genome sequence and transcriptome response to vitamin C supplementation of Novacetimonas hansenii SI1 - producer of highly-stretchable cellulose.

Novacetimonas hansenii SI1, previously known as Komagataeibacter hansenii, produces bacterial nanocellulose (BNC) with unique ability to stretch. The addition of vitamin C in the culture medium increases the porosity of the membranes and their stretchability making them highly moldable. To better understand the genetic background of this strain, we obtained its complete genome sequence using a hybrid sequencing and assembly strategy. We described the functional regions in the genome which are important for the synthesis of BNC and acetan-like II polymer. We next investigated the effect of 1% vitamin C supplementation on the global gene expression profile using RNA sequencing. Our transcriptomic readouts imply that vitamin C functions mainly as a reducing agent. We found that the changes in cellular redox status are balanced by strong repression of the sulfur assimilation pathway. Moreover, in the reduced conditions, glucose oxidation is decreased and alternative pathways for energy generation, such as acetate accumulation, are activated. The presence of vitamin C negatively influences acetan-like II polymer biosynthesis, which may explain the lowered yield and changed mechanical properties of BNC. The results of this study enrich the functional characteristics of the genomes of the efficient producers of the N. hansenii species. Improved understanding of the adaptation to the presence of vitamin C at the molecular level has important guiding significance for influencing the biosynthesis of BNC and its morphology.

Optimization of bacterial cellulose production by Komagataeibacter sucrofermentans in synthetic media and agrifood side streams supplemented with organic acids and vitamins.

The effect of thiamine (TA), ascorbic acid (AA), citric acid, and gallic acid (GA) on bacterial cellulose (BC) production by Komagataeibacter sucrofermentans, in synthetic (Hestrin and Schramm, HS) and natural substrates (industrial raisins finishing side stream extract, FSSE; orange juice, OJ; green tea extract, GTE), was investigated. The Response Surface Methodology was found reliable for BC yield prediction and optimization. Higher yields were achieved in the FSSE substrates, especially those supplemented with AA, TA, and GA (up to 19.4 g BC/L). The yield in the non-fortified substrates was 1.1-5.4 and 11.6-15.7 g/L, in HS and FSSE, respectively. The best yield in the natural non-fortified substrate FSSE-OJ-GTE (50-20-30 %), was 5.9 g/L. The porosity, crystallinity, and antioxidant properties of the produced BC films were affected by both the substrate and the drying method (freeze- or oven-drying). The natural substrates and the process wastewaters can be further exploited towards added value and sustainability. Take Home Message Sentence: Raisin and citrus side-streams can be efficiently combined for bacterial cellulose production, enhanced by other vitamin- and phenolic-rich substrates such as green tea.

Exploring the biotic and abiotic drivers influencing nata de coco production by Komagataeibacter nataicola in pre-fermented coconut water.

In China and Southeast Asia, pre-fermented coconut water is commonly used for the production of nata de coco, a jelly-like fermented food that consists of bacterial cellulose (BC). The inherent natural fermentation process of coconut water introduces uncontrollable variables, which can lead to unstable yields during BC production. This study involved the collection of spontaneously pre-fermented coconut water over a five-month production cycle. The aim was to evaluate the microbiota and metabolite profile, as well as determine its impact on BC synthesis by Komagataeibacter nataicola. Significant variations in the microbial community structure and metabolite profile of pre-fermented coconut water were observed across different production months, these variations had significant effects on BC synthesis by K. nataicola. A total of 52 different bacterial genera and 32 different fungal genera were identified as potential biotic factors that can influence BC production. Additionally, several abiotic factors, including lactate (VIP = 4.92), mannitol (VIP = 4.22), ethanol (VIP = 2.67), and ascorbate (VIP = 1.61), were found to be potential driving forces affecting BC synthesis by K. nataicola. Upon further analysis, the correlation network indicated that 14 biotic factors had a significant contribution to BC production in three strains of K. nataicola. These factors included 8 bacterial genera, such as Limosilactobacillus and Lactiplantibacillus, and 6 fungal genera, such as Meyerozyma and Ogataea. The abiotic factors lactate, mannitol, and ethanol showed a positive correlation with the BC yield. This study provides significant insights into controlling the fermentation processes of pre-fermented coconut water in industrial settings.

Proposal of three novel insect-associated Commensalibacter species: Commensalibacter melissae sp. nov., Commensalibacter communis sp. nov. and Commensalibacter papalotli sp. nov.

A recent modification of the Note to Rule 25a of the International Code for Nomenclature of Bacteria is used a posteriori by the List Editors of the International Journal of Systematic and Evolutionary Microbiology to justify the refusal to validate species protologues published in supplementary material prior to this formal decision. Authors are therefore forced to ask permission to reuse published data for the valid publication of such names. In the present letter we re-publish the species protologues of Commensalibacter melissae sp. nov., Commensalibacter communis sp. nov. and Commensalibacter papalotli sp. nov.

Extracellular membrane vesicles derived from Komagataeibacter oboediens exposed on the International Space Station fuse with artificial eukaryotic membranes in contrast to vesicles of reference bacterium.

Membranous Extracellular Vesicles (EVs) of Gram-negative bacteria are a secretion and delivery system that can disseminate bacterial products and interact with hosts and the environment. EVs of nonpathogenic bacteria deliver their contents by endocytosis into eukaryotic cells, however, no evidence exists for a fusion delivery mechanism. Here, we describe the fusion of exposed to space/Mars-like stressors simulated on the International Space Station vesicles (E-EVs) from Komagataeibacter oboediens to different types of model planar membranes in comparison with the EVs of the ground-based reference strain. The most reliable fusion was achieved with PC:PE:ergosterol or sterol-free PC:PE bilayers. The relative permeability ratio (PK+/PCl-) estimated from the shift of zero current potential according to Goldman-Hodgkin-Katz equation consisted of 4.17 ± 0.48, which coincides with preferential cation selectivity of the EV endogenous channels. The increase in membrane potential from 50 mV to 100 mV induced the fusion of E-EVs with all tested lipid compositions. The fusion of model exosomes with planar bilayer lipid membranes was confirmed by separate step-like increases in its conductance. In contrast, the ground-based reference K. oboediens EVs never induced the fusion event. In our study, we show membrane lipidome perturbations and increased protein aggregation occurred in the exposed samples in the harsh environment when outer membranes of K. oboediens acquired the capability of both homo- and heterotypic fusion possibly by altered membrane fluidity and the pore-forming capability.

Honeybee symbiont Bombella apis could restore larval-to-pupal transition disrupted by antibiotic treatment.

Numerous studies have demonstrated the vital roles of gut microbes in the health, immunity, nutrient metabolism, and behavior of adult worker honeybees. However, a few studies have been conducted on gut microbiota associated with the larval stage of honeybees. In the present study, we explored the role of a gut bacterium in larval development and larval-pupal transition in the Asian honeybee, Apis cerana. First, our examination of gut microbial profiling showed that Bombella apis, a larvae-associated bacterium, was the most dominant bacterium colonized in the fifth instar larvae. Second, we demonstrated that tetracycline, an antibiotic used to treat a honeybee bacterial brood disease, could cause the complete depletion of gut bacteria. This antibiotic-induced gut microbiome depletion in turn, significantly impacted the survivorship, pupation rate and emergence rate of the treated larvae. Furthermore, our analysis of gene expression pattens revealed noteworthy changes in key genes. The expression of genes responsible for encoding storage proteins vitellogenin (vg) and major royal jelly protein 1 (mrjp1) was significantly down-regulated in the tetracycline-treated larvae. Concurrently, the expression of krüppel homolog 1(kr-h1), a pivotal gene in endocrine signaling, increased, whilethe expression of broad-complex (br-c) gene that plays a key role in the ecdysone regulation decreased. These alterations indicated a disruption in the coordination of juvenile hormone and ecdysteroid synthesis. Finally, we cultivated B. apis isolated from the fifth instar worker larval of A. cerana and fed tetracycline-treated larvae with a diet replenished by B. apis. This intervention resulted in a significant improvement in the pupation rate, emergence rate, and overall survival rate of the treated larvae. Our findings demonstrate the positive impact of B. apis on honeybee larvae development, providing new evidence of the functional capacities of gut microbes in honeybee growth and development.

The phylogeny of Acetobacteraceae: photosynthetic traits and deranged respiratory enzymes.

IMPORTANCE: Acetobacteraceae are one of the best known and most extensively studied groups of bacteria, which nowadays encompasses a variety of taxa that are very different from the vinegar-producing species defining the family. Our paper presents the most detailed phylogeny of all current taxa classified as Acetobacteraceae, for which we propose a taxonomic revision. Several of such taxa inhabit some of the most extreme environments on the planet, from the deserts of Antarctica to the Sinai desert, as well as acidic niches in volcanic sites like the one we have been studying in Patagonia. Our work documents the progressive variation of the respiratory chain in early branching Acetobacteraceae into the different respiratory chains of acidophilic taxa such as Acidocella and acetous taxa such as Acetobacter. Remarkably, several genomes retain remnants of ancestral photosynthetic traits and functional bc 1 complexes. Thus, we propose that the common ancestor of Acetobacteraceae was photosynthetic.

Unraveling symbiotic microbial communities, metabolomics and volatilomics profiles of kombucha from diverse regions in China.

Kombucha is a natural fermented beverage (mixed system). This study aimed to unravel the signatures of kombucha in China to achieve tailor-made microbial consortium. Here, biochemical parameters, microbiome, metabolite production and volatile profile were comprehensively compared and characterized across four regions (AH, HN, SD, SX), both commonalities and distinctions were highlighted. The findings revealed that yeast species yeast Starmerella, Zygosaccharomyces, Dekkera, Pichia and bacterium Komagataeibacter, Gluconobacter were the most common microbes. Additionally, the composition, distribution and stability of microbial composition in liquid phase were superior to those in biofilm. The species diversity, differences, marker and association were analyzed across four areas. Metabolite profiles revealed a total of 163 bioactive compounds (23 flavonoids, 13 phenols), and 68 differential metabolites were screened and identified. Moreover, the metabolic pathways of phenylpropanoids biosynthesis were closely linked with the highest number of metabolites, followed by flavonoid biosynthesis. Sixty-five volatile compounds (23 esters) were identified. Finally, the correlation analysis among the microbial composition and volatile and functional metabolites showed that Komagataeibacter, Gluconolactone, Zygosacchaaromycess, Starmerella and Dekkera seemed closely related to bioactive compounds, especially Komagataeibacter displayed positive correlations with 1-hexadecanol, 5-keto-D-gluconate, L-malic acid, 6-aminohexanoate, Starmerella contributed greatly to gluconolactone, thymidine, anabasine, 2-isopropylmalic acid. Additionally, Candida was related to ß-damascenone and α-terpineol, and Arachnomyces and Butyricicoccus showed the consistency of associations with specific esters and alcohols. These findings provided crucial information for creating a stable synthetic microbial community structure, shedding light on fostering stable kombucha and related functional beverages.

Acetan-like heteropolysaccharide production by various Kozakia baliensis strains: Characterization and further insights.

The family of Acetobacteraceae has demonstrated their ability to produce several heteropolysaccharides with a strong structural resemblance to xanthan gum. In this study, we assessed the potential of three isolates of K. baliensis as exopolysaccharide producers, namely K. baliensis SR-745, K. baliensis LMG 27018, and K. baliensis SR-1290. Among these, K. baliensis SR-745 was identified as the most promising candidate, exhibiting a final exopolysaccharide titer of 7.09 (± 0.50) g·L-1 and a productivity of 0.15 (± 0.01) g·L-1·h-1. Subsequent monomer analysis confirmed structural variations for the side chain composition of different strains. A molar subunit ratio of 6:1:1:1 (d-glucose: D-mannose: D-galactose: D-glucuronic acid) for EPS derived from K. baliensis SR-745 and of 3:1:3:1 for K. baliensis LMG 27018 was determined, while the exopolysaccharide produced by K. baliensis SR-1290 consisted of a major share of rhamnose. In-depth rheological polymer characterizations revealed high viscosity rates and predominantly elastic gel character, making polysaccharides of K. baliensis highly interesting for applications in the food and cosmetic industry. Further insights into the fundamental structure-function relationships of biopolymers were obtained by comparing exopolysaccharides derived from K. baliensis to a genetically engineered xanthan variant lacking acetyl and pyruvyl substitutions.

Bioconversion of underutilized brewing by-products into bacterial cellulose by a newly isolated Komagataeibacter rhaeticus strain: A preliminary evaluation of the bioprocess environmental impact.

A novel Komagataeibacter rhaeticus UNIWA AAK2 strain was used to produce bacterial cellulose (BC), valorizing brewers' spent grain (BSG) and brewer's spent yeast (BSY). Under optimal conditions (controlled pH = 6 and 30 g/L sugars), a maximum BC of 4.0 g/L was achieved when BSG aqueous extract (BSGE) was used. The substitution of yeast extract and peptone with BSY autolyzates did not show significant differences on BC concentration and productivity. The FTIR, SEM, and TGA analyses showed that the use of brewing by-products had no effect on the structure and thermal stability of the produced BC, compared to highly-pure and commercial substrates. The LCA of the developed bioprocess revealed that BSGE- and BSY-based media can reduce the carbon footprint of 1 kg dry BC by 76% compared to commercial-based-media. Beer by-products could serve as cost-effective resources to produce value-added and sustainable biopolymers such as BC, while minimizing waste and restructuring the brewing-industry.

Spatio-temporal home range of the dominant rodent species in Mabira central forest reserve, Uganda.

BACKGROUND: Rodents form the largest order among mammals in terms of species diversity, and home range is the area where an individual normally moves during its normal daily activities. Information about rodent home ranges is paramount in the development of effective conservation and management strategies. This is because rodent home range varies within species and different habitats. In Uganda, tropical high altitude forests such as the Mabira Central Forest Reserve are experiencing continuous disturbance. However, information on rodent home range is lacking. Therefore, a two year Capture-Mark-Release (CMR) of rodents was conducted in the intact forest habitat: Wakisi, regenerating forest habitat: Namananga, and the depleted forest habitat: Namawanyi of Mabira Central Forest Reserve in order to determine the dominant rodent species, their home ranges, and factors affecting these home ranges. The home ranges were determined by calculating a minimum convex polygon with an added boundary strip of 5 m. RESULTS: Overall, the most dominant rodent species were: Lophuromys stanleyi, Hylomyscus stella, Praomys jacksoni Mastomys natalensis, Lophuromys ansorgei, and Lemniscomys striatus. H. stella dominated the intact forest habitat, while L. stanleyi was the most dominant both in the regenerating and the depleted forest habitats. L. stanleyi had a larger home range in the depleted forest, and the regenerating forest habitats, respectively. In the regenerating forest habitat, M. natalensis had a larger home range size, followed by L. stanleyi, and L. striatus. While in the intact forest habitat, H. stella had the largest home range followed by P. jacksoni. H. stella, L. striatus, L. stanleyi, M. natalensis, and P. jacksoni were most dominant during the wet season while L. ansorgei was relatively more dominant during the dry season. L. ansorgei, and P. jacksoni had a larger home range in the dry season, and a lower home range in the wet season. H. stella, L. stanleyi, M. natalansis and L.striatus had larger home ranges in the wet season, and lower home ranges in the dry season.   The home ranges of the dominant rodent species varied across the three habitats in Mabira central forest reserve ([Formula: see text], [Formula: see text]). CONCLUSION: The significant variation in home ranges of the dominant rodent species in Mabira Central Forest Reserve depending on the type of habitat presupposes that the rodent management strategies in disturbed forest reserves should focus on the type of habitat.

Production efficiency and properties of bacterial cellulose membranes in a novel grape pomace hydrolysate by Komagataeibacter melomenusus AV436T and Komagataeibacter xylinus LMG 1518.

The microbial production of cellulose using different bacterial species has been extensively examined for various industrial applications. However, the cost-effectiveness of all these biotechnological processes is strongly related to the culture medium for bacterial cellulose (BC) production. Herein, we examined a simple and modified procedure for preparing grape pomace (GP) hydrolysate, without enzymatic treatment, as a sole growth medium for BC production by acetic acid bacteria (AAB). The central composite design (CCD) was used to optimise the GP hydrolysate preparation toward the highest reducing sugar contents (10.4 g/L) and minimal phenolic contents (4.8 g/L). The experimental screening of 4 differently prepared hydrolysates and 20 AAB strains identified the recently described species Komagataeibacter melomenusus AV436T as the most efficient BC producer (up to 1.24 g/L dry BC membrane), followed by Komagataeibacter xylinus LMG 1518 (up to 0.98 g/L dry BC membrane). The membranes were synthesized in only 4 days of bacteria culturing, 1 st day with shaking, followed by 3 days of static incubation. The produced BC membranes in GP-hydrolysates showed, in comparison to the membranes made in a complex RAE medium 34 % reduction of crystallinity index with the presence of diverse cellulose allomorphs, presence of GP-related components within the BC network responsible for the increase of hydrophobicity, the reduction of thermal stability and 48.75 %, 13.6 % and 43 % lower tensile strength, tensile modulus, and elongation, respectively. Here presented study is the first report on utilising a GP-hydrolysate without enzymatic treatment as a sole culture medium for efficient BC production by AAB, with recently described species Komagataeibacter melomenusus AV436T as the most efficient producer in this type of food-waste material. The scale-up protocol of the scheme presented here will be needed for the cost-optimisation of BC production at the industrial levels.

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.

Use of brewer's residual yeast for production of bacterial nanocellulose with Gluconacetobacter hansenii.

Bacterial nanocellulose (BNC) has attained elevated interest due to its versatile structure and high resistance characteristics. Accordingly, efforts have been made in order to reduce its production costs, such as the employment of its by-products as a nutrient broth to yield the microorganism. Residual brewer's yeast is an excellent recourse, due to its high nutritional value and availability. Therefore, research which aimed to contribute to the development of a low cost, efficient and biosustainable technology for BNC production with Gluconacetobacter hansenii was carried out. BNC was obtained from residual brewer's yeast hydrolysate at pH 7.0 and five days of incubation at 30 °C in static culture. The hydrolysate was characterized by the amount of sugars, fatty acids, total proteins and ash content. Subsequently, BNC obtained was characterized in terms of yield, carbon conversion ratio, hydrodynamic size, crystallinity, morphology, Fourier-transform infrared spectroscopy, and surface analysis. Residual brewer's yeast hydrolysate proved to be efficient in BNC production via gluconeogenesis with consumption of alanine, threonine and glycerol, obtaining 1.9 times the yield of the chemically defined broth adopted as standard. Additionally, properties observed in the obtained BNC were equal to those obtained from conventional chemical medium. The research contributed to bacterial nanocellulose production using by-products from the brewing industry.

Changes in the chemical compositions and biological properties of kombucha beverages made from black teas and pineapple peels and cores.

Several raw materials have been used as partial supplements or entire replacements for the main ingredients of kombucha to improve the biological properties of the resulting kombucha beverage. This study used pineapple peels and cores (PPC), byproducts of pineapple processing, as alternative raw materials instead of sugar for kombucha production. Kombuchas were produced from fusions of black tea and PPC at different ratios, and their chemical profiles and biological properties, including antioxidant and antimicrobial activities, were determined and compared with the control kombucha without PPC supplementation. The results showed that PPC contained high amounts of beneficial substances, including sugars, polyphenols, organic acids, vitamins, and minerals. An analysis of the microbial community in a kombucha SCOBY (Symbiotic Cultures of Bacteria and Yeasts) using next-generation sequencing revealed that Acetobacter and Komagataeibacter were the most predominant acetic acid bacteria. Furthermore, Dekkera and Bacillus were also the prominent yeast and bacteria in the kombucha SCOBY. A comparative analysis was performed for kombucha products fermented using black tea and a fusion of black tea and PPC, and the results revealed that the kombucha made from the black tea and PPC infusion exhibited a higher total phenolic content and antioxidant activity than the control kombucha. The antimicrobial properties of the kombucha products made from black tea and the PPC infusion were also greater than those of the control. Several volatile compounds that contributed to the flavor, aroma, and beneficial health properties, such as esters, carboxylic acids, phenols, alcohols, aldehydes, and ketones, were detected in kombucha products made from a fusion of black tea and PPC. This study shows that PPC exhibits high potential as a supplement to the raw material infusion used with black tea for functional kombucha production.

Structural properties of optically clear bacterial cellulose produced by Komagataeibacter hansenii using arabitol.

Bacterial cellulose (BC) exhibits beneficial properties for use in biomedical applications but is limited by its lack of tunable transparency capabilities. To overcome this deficiency, a novel method to synthesize transparent BC materials using an alternative carbon source, namely arabitol, was developed. Characterization of the BC pellicles was performed for yield, transparency, surface morphology, and molecular assembly. Transparent BC was produced using mixtures of glucose and arabitol. Zero percent arabitol pellicles exhibited 25% light transmittance, which increased with increasing arabitol concentration through to 75% light transmittance. While transparency increased, overall BC yield was maintained indicating that the altered transparency may be induced on a micro-scale rather than a macro-scale. Significant differences in fiber diameter and the presence of aromatic signatures were observed. Overall, this research outlines methods for producing BC with tunable optical transparency, while also bringing new insight to insoluble components of exopolymers produced by Komagataeibacter hansenii.

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.

Aristophania vespae gen. nov., sp. nov., isolated from wasps, is related to Bombella and Oecophyllibacter, isolated from bees and ants.

Acetic acid bacteria (family Acetobacteraceae) are found in the gut of most insects. Two clades are currently recognized: Commensalibacter-Entomobacter and Bombella-Oecophyllibacter. The latter group is only found in hymenopteran insects and the described species have been isolated from bees and ants. In this study, two new strains DDB2-T1T (=KACC 21507T=LMG 31759T) and DM15PD (=CCM 9165=DSM 112731=KACC 22353=LMG 32454) were isolated from wasps collected in the Republic of Korea and Germany, respectively. Molecular and phenotypic analysis revealed that the strains are closely related, with 16S rRNA gene sequences showing 100 % identity and genomic average nucleotide identity (ANI) values ≥99 %. The closest related species based on type strain 16S rRNA gene sequences are Swingsia samuiensis, Acetobacter peroxydans, Bombella favorum and Bombella intestini (94.8-94.7% identity), whereas the closest related species based on type strain genome analysis are Saccharibacter floricola and Bombella intestini (ANI values of 68.8 and 68.2 %, respectively). The reconstruction of a phylogenomic tree based on 107 core proteins revealed that the branch leading to DDB2-T1T and DM15PD is localized between Oecophyllibacter and Saccharibacter-Bombella. Further genomic distance metrics such as ANI, percentage of conserved proteins and alignment fraction values were consistent with these strains belonging to a new genus. The key phenotypic characteristics were one MALDI-TOF-MS peak (m/z=4601.9±2.0) and the ability to produce acid from d-arabinose. Based on this polyphasic approach, including phylogenetics, phylogenomics, genome distance calculations, ecology and phenotypic characteristics, we propose to name the novel strains Aristophania vespae gen. nov., sp. nov., with the type strain DDB2-T1T (=KACC 21507T=LMG 31759T).

Bacterial cellulose production by Komagataeibacter hansenii can be improved by successive batch culture.

Bacterial cellulose (BC) is a biopolymer principally synthetized by strains of the genus Komagataeibacter. However, high costs and low production yield make large-scale application difficult. The aim of this work was to evaluate the effects of successive batch culture before fermentation on the ability to increase the capacity of bacterial cellulose biosynthesis by a low-producing strain. The Komagataeibacter hansenii strain ATCC 23,769 was initially cultivated in fermentation broth for two periods of 35 or 56 days under static conditions. At the end of each period of time, they were transferred to new broth to be cultivated again (new batch culture cycle) for 35 or 56 days and carried out in parallel with a 10-day fermentation to determine the quantity of BC produced. As a result, a greater increase was observed after the end of the second and third batch cultures of 56 days (increases of 137% and 187% in relation to the nonbatch cultured strain, respectively). The produced samples presented higher crystallinity and thermal properties but lower water holding capacity. Through this work, it was concluded that the longer the batch culture time was, the greater the increase in the capacity of cellulose biosynthesis, which also depended on the number of successive batch culture cycles carried out.