Optimization of microbial consortia and materials composition enhances gluconic acid content in kombucha.

The objective of this study was to optimize the microbial and ingredient composition of kombucha for enhanced production of gluconic acid (GA). Fourteen strains of Komagataeibacter spp. and one yeast strain of Dekkera sp. were isolated from kombucha. Among them, Komagataeibacter swingsii SS1 (SS1) and Komagataeibacter saccharivorans SS11 (SS11) were selected for their high GA production. A rapid reduction of pH, high GA content relative to acetic acid, and high cellulose production were observed in the tea infusion fermented by the microbial consortium (SS1 + SS11 + Dekkera bruxellensis Y24). From the correlation between the materials composition and quality indicators of kombucha, the decrease in pH was the most critical quality indicator of kombucha and the most closely related to GA content. Maximal GA production (11.7 mg/mL) was obtained under the conditions of 1% (w/v) tea extract, 8.5% (w/v) glucose, and 1.5% (v/v) ethanol through the optimization of materials composition by response surface methodology. The GA content of kombucha was enhanced threefold in comparison to general kombucha by fermentation with Komagataeibacter spp. and optimization of the composition of the ingredients. Overall, this study showed that a specific microbial consortium and materials composition could be established by correlation analysis among the ingredients, which results in increased GA levels in kombucha. These findings offer valuable foundational data for both commercial production and quality control of kombucha.

How carbon sources drive cellulose synthesis in two Komagataeibacter xylinus strains.

Bacterial cellulose synthesis from defined media and waste products has attracted increasing interest in the circular economy context for sustainable productions. In this study, a glucose dehydrogenase-deficient Δgdh K2G30 strain of Komagataeibacter xylinus was obtained from the parental wild type through homologous recombination. Both strains were grown in defined substrates and cheese whey as an agri-food waste to assess the effect of gene silencing on bacterial cellulose synthesis and carbon source metabolism. Wild type K2G30 boasted higher bacterial cellulose yields when grown in ethanol-based medium and cheese whey, although showing an overall higher D-gluconic acid synthesis. Conversely, the mutant Δgdh strain preferred D-fructose, D-mannitol, and glycerol to boost bacterial cellulose production, while displaying higher substrate consumption rates and a lower D-gluconic acid synthesis. This study provides an in-depth investigation of two K. xylinus strains, unravelling their suitability for scale-up BC production.

Bacterial cellulose biosynthesis: Optimization strategy using iranian nabat industry waste.

Bacterial cellulose (BC) is a biopolymer has found extensive applications across different fields due to its nanostructure and biomaterial performance. This study focused on optimizing yield of BC produced by Komagataeibacter xylinus CH1, isolated from kombucha SCOBY. The study aimed to use Nabat industry waste (NIW) as a cost-effective alternative carbon source for submerged fermentation. To optimize the fermentation criteria, the central composite design was used with the inoculation amount (1.5-4.5 % VV-1), NIW (0-1%), and fermentation time (3-7 days) as independent variables. The impressive results indicated the yield was enhanced up to 45.543 gL-1 at 3.013 % VV-1 of inoculation, 0.516 % NIW, and 7 days of stirred fermentation. SEM, XRD, FTIR, and TGA were applied to evaluate the characteristics of freeze-dried BC, such as the three-dimensional, porous structure, crystalline peaks, amorphous haloes, and thermal stability. The physicochemical properties of BC including high moisture content (93.022 ± 0.472 %), water absorption rate (569.473 ± 3.739 %), water-holding capacity (1333.016 ± 3.680 %), porosity (166.247 ± 2.055 %), and low water activity (0.296 ± 0.030 %) were achieved. Rheological properties of BC suspensions showed that G' dominated over G″, with tan δ values lower than 1. These characteristics indicate NIW and stirred fermentation conditions are a promising method for producing BC in high yield.

A Review on the Interaction of Acetic Acid Bacteria and Microbes in Food Fermentation: A Microbial Ecology Perspective.

In fermented foods, acetic acid bacteria (AAB), kinds of bacteria with a long history of utilization, contribute to safety, nutritional, and sensory properties primarily through acetic acid fermentation. AAB are commonly found in various fermented foods such as vinegar, sour beer, fermented cocoa and coffee beans, kefir beverages, kombucha, and sourdough. They interact and cooperate with a variety of microorganisms, resulting in the formation of diverse metabolites and the production of fermented foods with distinct flavors. Understanding the interactions between AAB and other microbes is crucial for effectively controlling and utilizing AAB in fermentation processes. However, these microbial interactions are influenced by factors such as strain type, nutritional conditions, ecological niches, and fermentation duration. In this review, we examine the relationships and research methodologies of microbial interactions and interaction studies between AAB and yeasts, lactic acid bacteria (LAB), and bacilli in different food fermentation processes involving these microorganisms. The objective of this review is to identify key interaction models involving AAB and other microorganisms. The insights gained will provide scientific guidance for the effective utilization of AAB as functional microorganisms in food fermentation processes.

Engineering PTS-based glucose metabolism for efficient biosynthesis of bacterial cellulose by Komagataeibacter xylinus.

Bacterial cellulose (BC) is a renewable biomaterial that has attracted significant attention due to its excellent properties and wide applications. Komagataeibacter xylinus CGMCC 2955 is an important BC-producing strain. It primarily produces BC from glucose while simultaneously generating gluconic acid as a by-product, which acidifies the medium and inhibits BC synthesis. To enhance glucose uptake and BC synthesis, we reconstructed the phosphoenolpyruvate-dependent glucose phosphotransferase system (PTSGlc) and strengthened glycolysis by introducing heterologous genes, resulting in a recombinant strain (GX08PTS03; Δgcd::ptsHIcrrE. coli::ptsGE. coli::pfkAE. coli). Strain GX08PTS03 efficiently utilized glucose for BC production without accumulating gluconic acid. Subsequently, the fermentation process was systematically optimized. Under optimal conditions, strain GX08PTS03 produced 7.74 g/L of BC after 6 days of static fermentation, with a BC yield of 0.39 g/g glucose, which were 87.41 % and 77.27 % higher than those of the wild-type strain, respectively. The BC produced by strain GX08PTS03 exhibited a longer fiber diameter along with a lower porosity, significantly higher solid content, crystallinity, tensile strength, and Young's modulus. This study is novel in reporting that the engineered PTSGlc-based glucose metabolism could effectively enhance the production and properties of BC, providing a future outlook for the biopolymer industry.

Antibiotic Resistance in Acetic Acid Bacteria Originating from Vinegar.

Acetic acid bacteria (AAB) are major contributors to the production of fermented vinegar, offering various cultural, culinary, and health benefits. Although the residual unpasteurized AAB after vinegar production are not pathogens, these are necessary and require safety evaluations, including antibiotic resistance, before use as a starter. In this research, we investigated the antibiotic resistance profiles of 26 AAB strains, including various species of Komagataeibacter and Acetobacter, against 10 different antibiotics using the E-test method. All strains exhibited resistance to aztreonam and clindamycin. Komagataeibacter species demonstrated a 50% resistance rate to ciprofloxacin, analogous to Acetobacter species, but showed twice the resistance rates to chloramphenicol and erythromycin. Genomic analysis of K. saccharivorans CV1 identified intrinsic resistance mechanisms, such as multidrug efflux pumps, thereby enhancing our understanding of antibiotic resistance in acetic acid-producing bacteria. These findings enhance understanding of antibiotic resistance in AAB for food safety and new antimicrobial strategies, suggesting the need for standardized testing methods and molecular genetic study.

Enhanced bacterial cellulose production by Komagataeibacter species and Hibiscus sabdariffa herbal tea.

This study proposed Hibiscus sabdariffa as a novel substrate for BC production with Komagataeibacter species and their consortia. K. intermedius is found as the most efficient producer (5.98 g/L BC, 3.56 × 10-2 g-1 h-1 productivity rate) following K. maltaceti (4.44 g/L BC, 2.64 × 10-2 g-1 h-1 productivity rate) and K. nataicola (3.67 g/L BC, 2.18 × 10-2 g-1 h-1 productivity rate). Whereas agitation increased BC production with K. nataicola (1.22-fold, 4.49 g/L BC), K. maltaceti (1.24-fold, 5.52 g/L BC) and K. intermedius (1.27-fold, 7.63 g/L BC), irregular shaped BC was obtained. This could be a novel result as Komagataeibacter consortia increased BC production by 1.17-2.01-fold compared to monocultures resulting as 8.11 g/L BC through the co-cultivation of K. maltaceti-K. intermedius. Maximum increase was found to be 1.75-fold (1.79-fold WHC), occurring with monoculture of K. maltaceti, while 1.94-fold (1.26-fold WHC) with K. maltaceti-K. intermedius consortium when H. sabdariffa-based media compared Hestrin-Schramm media. Based on these results, this could be a novel result as H. sabdariffa-based media may replace the use of HS media in BC production by means of a bioactive content-rich plant and obtaining 3-D ultrafine porous structure with high thermal resistant (∼460-500 °C) BC with mono and co-cultivation of Komagataeibacter species to be used in industrial area.

Fermentation Characteristics of Unripe Citrus unshiu Vinegar Production Using Acetic Acid Bacteria Isolated from Traditional Fermented Vinegars.

Here, we aimed to isolate an acetic acid bacterium that is suitable for the production of unripe Citrus unshiu vinegar from traditional fermented vinegars. We compared the halo sizes of isolates to select a strain with superior acetic acid production capabilities and selected Komagataeibacter kakiaceti P6 (P6) as the final strain. Using Acetobacter pasteurianus CY (CY) and A. pasteurianus KACC 17058 (KACC 17058) as controls, we analyzed the total phenolic compounds, total flavonoid content, antioxidant activities, and organic acids of the selected strain to verify its suitability for acetic acid fermentation. On the 30th day of the fermentation period, P6 showed a total acidity of 4.86%, which was higher than that of control groups (CY, 4.16%; KACC 17058, 4.01%). The total phenolic compounds, total flavonoid content, 1,1-diphenyl-2-picrylhydrazyl scavenging activity, and ferric ion reducing antioxidant power values significantly increased during fermentation with P6 compared with the initial C. unshiu wine, and no significant differences were observed from the vinegars produced by CY and KACC 17058. Moreover, organic acid analysis revealed that the unripe C. unshiu vinegar produced with P6 had an acetic acid content of 26.15 mg/mL, which was significantly higher than those produced with CY and KACC 17058, indicating that the P6 strain effectively produces acetic acid without adversely affecting other quality aspects during fermentation. In conclusion, the novel P6 strain is expected to be used as a starter for fermenting unripe C. unshiu vinegar, and its excellent acetic acid production capabilities suggest potential applications for other vinegars.

Efficiency of freeze- and spray-dried microbial preparation as active dried starter culture in kombucha fermentation.

BACKGROUND: Kombucha is a widely consumed fermented beverage produced by fermenting sweet tea with a symbiotic culture of bacteria and yeast (SCOBY). The dynamic nature of microbial communities in SCOBY may pose challenges to production scale-up due to unpredictable variations in microbial composition. Using identified starter strains is a novel strategy to control microorganism composition, thereby ensuring uniform fermentation quality across diverse batches. However, challenges persist in the cultivation and maintenance of these microbial strains. This study examined the potential of microencapsulated kombucha fermentation starter cultures, specifically Komagataeibacter saccharivorans, Levilactobacillus brevis and Saccharomyces cerevisiae, through spray-drying and freeze-drying. RESULTS: Maltodextrin and gum arabic-maltodextrin were employed as carrier agents. Our results revealed that both spray-dried and freeze-dried samples adhered to physicochemical criteria, with low moisture content (2.18-7.75%) and relatively high solubility (65.75-87.03%) which are appropriate for food application. Freeze-drying demonstrated greater effectiveness in preserving bacterial strain viability (88.30-90.21%) compared to spray drying (74.92-78.66%). Additionally, the freeze-dried starter strains demonstrated similar efficacy in facilitating kombucha fermentation, compared to the SCOBY group. The observations included pH reduction, acetic acid production, α-amylase inhibition and elevated total polyphenol and flavonoid content. Moreover, the biological activity, including antioxidant potential and in vitro tyrosinase inhibition activity, was enhanced in the same pattern. The freeze-dried strains exhibited consistent kombucha fermentation capabilities over a three-month preservation, regardless of storage temperature at 30 or 4 °C. CONCLUSION: These findings highlight the suitability of freeze-dried starter cultures for kombucha production, enable microbial composition control, mitigate contamination risks and ensure consistent product quality. © 2024 Society of Chemical Industry.

[Knockdown of motility-related genes of Komagataeibacter xylinus and its effect on bacterial cellulose synthesis].

Bacterial cellulose (BC) is a biopolymer synthesized by bacteria, which possess excellent characteristics such as high water holding capacity, high crystallinity, and high purity. It is widely used in food, medical, cosmetics, and functional films. Komagataeibacter xylinus is a model strain used in BC synthesis research. In bacteria, motility-related genes are associated with BC synthesis, whereas in Komagataeibacter xylinus CGMCC 2955, the functions of motility-related genes and their effects on BC synthesis are not known. To address this gap, we used the λ Red recombinant system to individually knock out motA, motB, and mot2A respectively, and constructed the knockout strains K. x-ΔmotA, K. x-ΔmotB, and K. x-Δmot2A. Additionally, both motA and motB were disrupted to construct the K. x-ΔmotAB mutant. The results demonstrated that knockout strain K. x-ΔmotAB exhibited the highest BC yield, reaching (5.05±0.26) g/L, which represented an increase of approximately 24% compared to wild-type strains. Furthermore, the BC synthesized by this strain exhibited the lowest porosity, 54.35%, and displayed superior mechanical properties with a Young's modulus of up to 5.21 GPa. As knocking out motA and motB genes in K. xylinus CGMCC 2955 did not reduce BC yield; instead, it promoted BC synthesis. Consequently, this research further deepened our understanding of the relationship between motility and BC synthesis in acetic acid bacteria. The knockouts of motA and motB genes resulted in reduced BC porosity and improved mechanical properties, provides a reference for BC synthesis and membrane structure regulation modification.

Statistical optimization of bioprocess parameters for enhanced production of bacterial cellulose from K. saccharivorans BC-G1.

Bacterial Cellulose (BC) offers a wide range of applications across various industries, including food, biomedical, and textiles, owing to its distinctive properties. Its unique 3D reticulated network of cellulose nanofibers, imparts excellent mechanical qualities, a high water-holding capacity, and thermal stability. Additionally, it possesses remarkable biocompatibility, biodegradability, high crystallinity, and purity. These attributes have offered significant interest in BC within both academic and industrial sectors. However, BC production is associated with high costs due to the use of expensive growth media and low yields. The study reports the potential of our indigenous isolate, Komagataeibacter saccharivorans BC-G1, as BC producer. Statistical optimization of BC production was carried out using Placket-Burman design and Central composite design, by selecting different parameters. Eight significant factors such as temperature, pH, glucose, yeast, peptone, acetic acid, incubation time and % inoculum were studies using ANOVA-based response surface methodology. Results showed that BC yield (8.5 g/L) with 1.8-fold after optimization of parameters. Maximum cellulose production (8.5 ± 1.8 g/L) was obtained using 2% glucose, 0.3% yeast extract, 0.3% peptone, 0.75% (v/v) acetic acid at pH 7.0 for 10 days of incubation with 4% inoculum at 25 °C under static culture. Main effect graph showed incubation time and acetic acid concentration as the most significant parameters affecting BC production in our study. The physicochemical characterization of produced BC was done using FTIR, XRD and SEM techniques.

Production and characterization of bacterial cellulose by Rhizobium sp. isolated from bean root.

Bacterial cellulose (BC) is a natural polymer renowned for its unique physicochemical and mechanical attributes, including notable water-holding capacity, crystallinity, and a pristine fiber network structure. While BC has broad applications spanning agriculture, industry, and medicine, its industrial utilization is hindered by production costs and yield limitations. In this study, Rhizobium sp. was isolated from bean roots and systematically assessed for BC synthesis under optimal conditions, with a comparative analysis against BC produced by Komagataeibacter hansenii. The study revealed that Rhizobium sp. exhibited optimal BC synthesis when supplied with a 1.5% glucose carbon source and a 0.15% yeast extract nitrogen source. Under static conditions at 30 °C and pH 6.5, the most favorable conditions for growth and BC production (2.5 g/L) were identified. Modifications were introduced using nisin to enhance BC properties, and the resulting BC-nisin composites were comprehensively characterized through various techniques, including FE-SEM, FTIR, porosity, swelling, filtration, and antibacterial activity assessments. The results demonstrated that BC produced by Rhizobium sp. displayed properties comparable to K. hansenii-produced BC. Furthermore, the BC-nisin composites exhibited remarkable inhibitory activity against Escherichia coli and Pseudomonas aeruginosa. This study contributes valuable insights into BC's production, modification, and characterization utilizing Rhizobium sp., highlighting the exceptional properties that render it efficacious across diverse applications.

Customized 16S-23S rDNA ITS Amplicon Metagenomics for Acetic Acid Bacteria Species Identification in Vinegars and Kombuchas.

Acetic acid bacteria (AAB) are involved in food and beverage production bioprocesses, like those in vinegar and kombucha. They oxidize sugars and alcohols into various metabolites, resulting in the final products' pleasant taste and aroma. The 16S rDNA amplicon metagenomics using Illumina technology is usually used to follow the microbiological development of these processes. However, the 16S rRNA gene sequences among different species of AAB are very similar, thus not enabling a reliable identification down to the species level but only to the genus. In this study, we have constructed primers for amplifying half of the 16S-23S rRNA gene internal transcribed spacer (ITS) for library construction and further sequencing using Illumina technology. This approach was successfully used to estimate the relative abundance of AAB species in defined consortia. Further application of this method for the analysis of different vinegar and kombucha samples proves it suitable for assessing the relative abundance of AAB species when these bacteria represent a predominant part of a microbial community.

Citrate-buffered Yamanaka medium allows to produce high-yield bacterial nanocellulose in static culture using Komagataeibacter strains isolated from apple cider vinegar.

Bacterial nanocellulose (BNC) is a sustainable, renewable, and eco-friendly nanomaterial, which has gained great attentions in both academic and industrial fields. Two bacterial nanocellulose-producing strains (CVV and CVN) were isolated from apple vinegar sources, presenting high 16S rRNA gene sequence similarities (96%-98%) with Komagataeibacter species. The biofilm was characterized by scanning electron microscopy (SEM), revealing the presence of rod-shaped bacteria intricately embedded in the polymeric matrix composed of nanofibers of bacterial nanocellulose. FTIR spectrum and XRD pattern additionally confirmed the characteristic chemical structure associated with this material. The yields and productivities achieved during 10 days of fermentation were compared with Komagataeibacter xylinus ATCC 53524, resulting in low levels of BNC production. However, a remarkable increase in the BNC yield was achieved for CVV (690% increase) and CVN (750% increase) strains at day 6 of the fermentation upon adding 22 mM citrate buffer into the medium. This effect is mainly attributed to the buffering capacity of the modified Yakamana medium, which allowed to maintain pH close to 4.0 until day 6, though in combination with additional factors including stimulation of the gluconeogenesis pathway and citrate assimilation as a carbon source. In addition, the productivities determined for both isolated strains (0.850 and 0.917 g L-1 d-1) compare favorably to previous works, supporting current efforts to improve fermentation performance in static cultures and the feasibility of scaling-up BNC production in these systems.

Enhancing bacterial cellulose production of Komagataeibacter nataicola through fermented coconut water by Saccharomyces cerevisiae: A metabonomics approach.

Nata de coco, an edible bacterial cellulose (BC) product, is a traditional dessert fermented in coconut water. Production of Nata de coco by Komagataeibacter nataicola is enhanced by pre-fermented coconut water, but its instability is a challenge. Here, BC production by K. nataicola Y19 was significantly improved by Saccharomyces cerevisiae 84-3 through shaping the metabolite profile of the coconut water. Different fermentation time with S. cerevisiae 84-3 resulted in distinct metabolite profiles and different promoting effect on BC yield. Compared to unfermented coconut water, coconut water fermented by S. cerevisiae 84-3 for 1d and 7d enhanced BC yield by 14.1-fold and 5.63-fold, respectively. Analysis between unfermented coconut water and 1d-fermented coconut water showed 129 significantly different metabolites, including organic acids, amino acids, nucleotides, and their derivatives. Prolonged fermentation for 7d changed levels of 155 metabolites belongs to organic acids, amino acids, nucleotides and their derivatives. Spearman correlation analysis further revealed that 17 metabolites were positively correlated with BC yield and 21 metabolites were negatively correlated with BC yield. These metabolites may affect energy metabolism, cell signaling, membrane integrity, and BC production by K. nataicola Y19. The further verification experiment gave the view that BC yield was not only closely related to the types of metabolites but also the concentration of metabolites. This study provides a novel theoretical framework for a highly efficient BC fermentation system utilizing stable fermented coconut water mediums.

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.

Production, Optimization, and Characterization of Bio-cellulose Produced from Komagataeibacter (Acetobacter aceti MTCC 3347) Usage of Food Sources as Media.

INTRODUCTION: Bio-cellulose is a type of cellulose that is produced by some particular group of bacteria, for example, Komagataeibacter (previously known as Acetobacter), due to their natural ability to synthesize exopolysaccharide as a byproduct. Gluconacetobacter xylinus is mostly employed for the production of bio-cellulose throughout the world. Therefore, exploring other commonly available strains, such as Komagataeibacter aceti (Acetobacter aceti), is needed for cellulose production. METHOD: Bio-cellulose is one of the most reliable biomaterials in the limelight because it is highly pure, crystalline, and biocompatible. Hence, it is necessary to enhance the industrial manufacture of bio-cellulose with low costs. Different media such as fruit waste, milk whey, coconut water, sugarcane juice, mannitol broth, and H&S (Hestrin and Schramm's) broth were utilized as a medium for culture growth. Other factors like temperature, pH, and time were also optimized to achieve the highest yield of bio-cellulose. Moreover, after the synthesis of bio-cellulose, its physicochemical and structural properties were evaluated. The results depicted that the highest yield of bio-cellulose (45.735 mg/mL) was found at 30 °C, pH 5, and on the 7th day of incubation. Though every culture media experimented with synthesized bio-cellulose, the maximum production (90.25 mg/mL) was reported in fruit waste media. RESULT: The results also indicated that bio-cellulose has high water-holding capacity and moisture content. XRD results showed that bio-cellulose is highly crystalline in nature (54.825% crystallinity). SEM micrograph demonstrated that bio-cellulose exhibited rod-shaped, highly porous fibers. The FTIR results demonstrated characteristic and broad peaks for O-H at 3336.25 cm-1, which indicated stronger O-H bonding. CONCLUSION: The thermal tests, such as DSC and TGA, indicated that bio-cellulose is a thermally stable material that can withstand temperatures even beyond 500 °C.

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.

Enhanced bacterial cellulose production in Komagataeibacter sucrofermentans: impact of different PQQ-dependent dehydrogenase knockouts and ethanol supplementation.

BACKGROUND: Bacterial cellulose (BC) is a biocompatible material with unique mechanical properties, thus holding a significant industrial potential. Despite many acetic acid bacteria (AAB) being BC overproducers, cost-effective production remains a challenge. The role of pyrroloquinoline quinone (PQQ)-dependent membrane dehydrogenases (mDH) is crucial in the metabolism of AAB since it links substrate incomplete oxidation in the periplasm to energy generation. Specifically, glucose oxidation to gluconic acid substantially lowers environmental pH and hinders BC production. Conversely, ethanol supplementation is known to enhance BC yields in Komagataeibacter spp. by promoting efficient glucose utilization. RESULTS: K. sucrofermentans ATCC 700178 was engineered, knocking out the four PQQ-mDHs, to assess their impact on BC production. The strain KS003, lacking PQQ-dependent glucose dehydrogenase (PQQ-GDH), did not produce gluconic acid and exhibited a 5.77-fold increase in BC production with glucose as the sole carbon source, and a 2.26-fold increase under optimal ethanol supplementation conditions. In contrast, the strain KS004, deficient in the PQQ-dependent alcohol dehydrogenase (PQQ-ADH), showed no significant change in BC yield in the single carbon source experiment but showed a restrained benefit from ethanol supplementation. CONCLUSIONS: The results underscore the critical influence of PQQ-GDH and PQQ-ADH and clarify the effect of ethanol supplementation on BC production in K. sucrofermentans ATCC 700178. This study provides a foundation for further metabolic pathway optimization, emphasizing the importance of diauxic ethanol metabolism for high BC production.

Elevated abundance of Komagataeibacter results in a lower pH in kombucha production; insights from microbiomic and chemical analyses.

Kombucha consumption has grown rapidly worldwide in the last decade, with production at both small- and large scales. The complex fermentation process involves both bacterial and yeast species, but little is known regarding the progression of microbial development during production. We explored the microbial diversity of multiple batches across two kombucha types, i. e commercial scale versus laboratory-made (hereafter "home") kombucha brew using metabarcoding to characterize both fungal and bacterial communities. We found the microbial community of the commercial kombucha brew to be more complex than that of the home brew. Furthermore, PERMANOVA uncovered significant compositional differences between the bacterial (F = 2.68, R2 = 0.23, p = 00.001) and fungal (F = 3.18, R2 = 0.26, p = 00.006) communities between batches. For the home brew, both alpha and beta diversity analyses revealed no significant differences between all batches and replicates. When the microbial diversity of the home and commercial kombucha types were directly compared, the former had higher proportions of Ammoniphilus and Komagataeibacter. The commercial kombucha on the other hand were high in Anoxybacillus, Methylobacterium and Sphingomonas. For the fungal communities, the most dominant fungal genera detected in both kombucha types were similar. Linear model revealed significant correlations between some microorganisms and the sugars and organic acids assayed in this study. For example, rising glucose levels correlated with an increase in the relative abundance of Komagataeibacter (F = 7.115, Adj. R2 = 0.44, p = 00.0003). We believe these results contribute towards achieving a better control of the kombucha fermentation process and may assist in targeted product diversification.