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Mutualistic interactions are often subject to exploitation by species that are not directly involved in the mutualism. Understanding which organisms act as such ‘third-party’ species and how they do so is a major challenge in the current study of mutualistic interactions. Here, we show that even species that appear ecologically similar can have contrasting effects as third-party species. We experimentally compared the effects of nectar-inhabiting bacteria and yeasts on the strength of a mutualism between a hummingbird-pollinated shrub, Mimulus aurantiacus, and its pollinators. We found that the common bacterium Gluconobacter sp., but not the common yeast Metschnikowia reukaufii, reduced pollination success, seed set and nectar consumption by pollinators, thereby weakening the plant–pollinator mutualism. We also found that the bacteria reduced nectar pH and total sugar concentration more greatly than the yeasts did and that the bacteria decreased glucose concentration and increased fructose concentration whereas the yeasts affected neither. These distinct changes to nectar chemistry may underlie the microbes' contrasting effects on the mutualism. Our results suggest that it is necessary to understand the determinants of microbial species composition in nectar and their differential modification of floral rewards to explain the mutual benefits that plants and pollinators gain from each other.

A novel bacterial strain of acetic acid bacteria capable of producing riboflavin was isolated from the soil sample collected in Wuhan, China. The isolated strain was identified as Gluconobacter oxydan s FBFS97 based on several phenotype characteristics, biochemicals tests, and 16S rRNA gene sequence conducted. Furthermore, the complete genome sequencing of the isolated strain has showed that it contains a complete operon for the biosynthesis of riboflavin. In order to obtain the maximum concentration of riboflavin production, Gluconobacter oxydans FBFS97 was optimized in shake flask cultures through response surface methodology employing Plackett–Burman design (PBD), and Central composite design (CCD). The results of the pre-experiments displayed that fructose and tryptone were found to be the most suitable sources of carbon and nitrogen for riboflavin production. Then, PBD was conducted for initial screening of eleven minerals (FeSO 4 , FeCl 3 , KH 2 PO 4 , K 2 HPO 4 , MgSO 4 , ZnSO 4 , NaCl, CaCl 2 , KCl, ZnCl 2 , and AlCl 3 .6H 2 O) for their significances on riboflavin production by Gluconobacter oxydans strain FBFS97. The most significant variables affecting on riboflavin production are K 2 HPO 4 and CaCl 2 , the interaction affects and levels of these variables were optimized by CCD. After optimization of the medium compositions for riboflavin production were determined as follows: fructose 25 g/L, tryptone 12.5 g/L, K 2 HPO 4 9 g/L, and CaCl 2 0.06 g/L with maximum riboflavin production 23.24 mg/L.

This paper considers the effect of multiwalled carbon nanotubes (MWCNTs) on the parameters of Gluconobacter oxydans microbial biosensors. MWCNTs were shown not to affect the structural integrity of microbial cells and their respiratory activity. The positive results from using MWCNTs were due to a decrease in the impedance of the electrode. The total impedance of the system decreased significantly, from 9000 kOhm (G. oxydans/chitosan composite) to 600 kOhm (G. oxydans/MWCNTs/chitosan). Modification of the amperometric biosensor with nanotubes led to an increase in the maximal signal from 65 to 869 nA for glucose and from 181 to 1048 nA for ethanol. The biosensor sensitivity also increased 4- and 5-fold, respectively, for each of the substrates. However, the addition of MWCNTs reduced the affinity of respiratory chain enzymes to their substrates (both sugars and alcohols). Moreover, the minimal detection limits were not reduced despite a sensitivity increase. The use of MWCNTs thus improved only some microbial biosensor parameters.

The acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources as a natural part of its metabolism, and this feature has been exploited for many biotechnological applications. The most important enzymes used to harness the biocatalytic oxidative capacity of G. oxydans are the pyrroloquinoline quinone (PQQ)-dependent dehydrogenases. The membrane-bound PQQ-dependent glucose dehydrogenase (mGDH), encoded by gox0265 , was used as model protein for homologous membrane protein production using the previously described Gluconobacter expression vector pBBR1p452. The mgdh gene had ninefold higher expression in the overproduction strain compared to the parental strain. Furthermore, membranes from the overexpression strain had a five- and threefold increase of mGDH activity and oxygen consumption rates, respectively. Oxygen consumption rate of the membrane fraction could not be increased by the addition of a substrate combination of glucose and ethanol in the overproduction strain, indicating that the terminal quinol oxidases of the respiratory chain were rate limiting. In contrast, addition of glucose and ethanol to membranes of the control strain increased oxygen consumption rates approaching the observed rates with G. oxydans overproducing mGDH. The higher glucose oxidation rates of the mGDH overproduction strain corresponded to a 70 % increase of the gluconate production rate compared to the control strain. The high rate of glucose oxidation may be useful in the industrial production of gluconates and ketogluconates, or as whole-cell biosensors. Furthermore, mGDH was purified to homogeneity by one-step strep-tactin affinity chromatography and characterized. To our knowledge, this is the first report of a membrane integral quinoprotein being purified by affinity chromatography and serves as a proof-of-principle for using G. oxydans as a host for membrane protein expression and purification.

Abstract Kombucha, historically an Asian tea-based fermented drink, has recently become trendy in Western countries. Producers claim it bears health-enhancing properties that may come from the tea or metabolites produced by its microbiome. Despite its long history of production, microbial richness and dynamics have not been fully unraveled, especially at an industrial scale. Moreover, the impact of tea type (green or black) on microbial ecology was not studied. Here, we compared microbial communities from industrial-scale black and green tea fermentations, still traditionally carried out by a microbial biofilm, using culture-dependent and metabarcoding approaches. Dominant bacterial species belonged to Acetobacteraceae and to a lesser extent Lactobacteriaceae, while the main identified yeasts corresponded to Dekkera, Hanseniaspora and Zygosaccharomyces during all fermentations. Species richness decreased over the 8-day fermentation. Among acetic acid bacteria, Gluconacetobacter europaeus, Gluconobacter oxydans, G. saccharivorans and Acetobacter peroxydans emerged as dominant species. The main lactic acid bacteria, Oenococcus oeni, was strongly associated with green tea fermentations. Tea type did not influence yeast community, with Dekkera bruxellensis, D. anomala, Zygosaccharomyces bailii and Hanseniaspora valbyensis as most dominant. This study unraveled a distinctive core microbial community which is essential for fermentation control and could lead to Kombucha quality standardization. Microbial ecology of industrial Kombucha fermentations.

Acetic acid bacteria are used in biotechnology due to their ability to incompletely oxidize a great variety of carbohydrates, alcohols, and related compounds in a regio- and stereo-selective manner. These reactions are catalyzed by membrane-bound dehydrogenases (mDHs), often with a broad substrate spectrum. In this study, the promoters of six mDHs of Gluconobacter oxydans 621H were characterized. The constitutive promoter of the alcohol dehydrogenase and the glucose-repressed promoter of the inositol dehydrogenase were used to construct a shuttle vector system for the fully functional expression of mDHs in the multi-deletion strain G. oxydans BP.9 that lacks its mDHs. This system was used to express each mDH of G. oxydans 621H, in order to individually characterize the substrates, they oxidize. From 55 tested compounds, the alcohol dehydrogenase oxidized 30 substrates and the polyol dehydrogenase 25. The substrate spectrum of alcohol dehydrogenase overlapped largely with the aldehyde dehydrogenase and partially with polyol dehydrogenase. Thus, we were able to resolve the overlapping substrate spectra of the main mDHs of G. oxydans 621H. The described approach could also be used for the expression and detailed characterization of substrates used by mDHs from other acetic acid bacteria or a metagenome.

The Gluconobacter phage GC1 is a novel member of the Tectiviridae family isolated from a juice sample collected during dry white wine making. The bacteriophage infects Gluconobacter cerinus, an acetic acid bacterium which represents a spoilage microorganism during wine making, mainly because it is able to produce ethyl alcohol and transform it into acetic acid. Transmission electron microscopy revealed tail-less icosahedral particles with a diameter of ~78 nm. The linear double-stranded DNA genome of GC1 (16,523 base pairs) contains terminal inverted repeats and carries 36 open reading frames, only a handful of which could be functionally annotated. These encode for the key proteins involved in DNA replication (protein-primed family B DNA polymerase) as well as in virion structure and assembly (major capsid protein, genome packaging ATPase (adenosine triphosphatase) and several minor capsid proteins). GC1 is the first tectivirus infecting an alphaproteobacterial host and is thus far the only temperate tectivirus of gram-negative bacteria. Based on distinctive sequence and life-style features, we propose that GC1 represents a new genus within the Tectiviridae, which we tentatively named “Gammatectivirus”. Furthermore, GC1 helps to bridge the gap in the sequence space between alphatectiviruses and betatectiviruses.

Background L-(+)-tartaric acid (L-TA) is an important organic acid, which is produced from the cream of tartar or stereospecific hydrolysis of the cis -epoxysuccinate. The former method is limited by the availability of raw material and the latter is dependent on the petrochemical material. Thus, new processes for the economical preparation of L-TA from carbohydrate or renewable resource would be much more attractive. Production of 5-keto-D-gluconate (5-KGA) from glucose by Gluconobacter oxydans is the first step to produce L-TA. The aim of this work is to enhance 5-KGA accumulation using combinatorial metabolic engineering strategies in G. oxydans . The sldAB gene, encoding sorbitol dehydrogenase, was overexpressed in an industrial strain G. oxydans ZJU2 under a carefully selected promoter, P 0169 . To enhance the efficiency of the oxidation by sldAB , the coenzyme pyrroloquinoline quinone (PQQ) and respiratory chain were engineered. Besides, the role in sldAB overexpression, coenzyme and respiratory chain engineering and their subsequent effects on 5-KGA production were investigated. Results An efficient, stable recombinant strain was constructed, whereas the 5-KGA production could be enhanced. By self-overexpressing the sldAB gene in G. oxydans ZJU2 under the constitutive promoter P 0169 , the resulting strain, G. oxydans ZJU3, produced 122.48 ± 0.41 g/L of 5-KGA. Furthermore, through the coenzyme and respiratory chain engineering, the titer and productivity of 5-KGA reached 144.52 ± 2.94 g/L and 2.26 g/(L · h), respectively, in a 15 L fermenter. It could be further improved the 5-KGA titer by 12.10 % through the fed-batch fermentation under the pH shift and dissolved oxygen tension (DOT) control condition, obtained 162 ± 2.12 g/L with the productivity of 2.53 g/(L · h) within 64 h. Conclusions The 5-KGA production could be significantly enhanced with the combinatorial metabolic engineering strategy in Gluconobacter strain, including sldAB overexpression, coenzyme and respiratory chain engineering. Fed-batch fermentation could further enlarge the positive effect and increase the 5-KGA production. All of these demonstrated that the robust recombinant strain can efficiently produce 5-KGA in larger scale to fulfill the industrial production of L-TA from 5-KGA.

A poorly performing industrial water kefir production process consisting of a first fermentation process, a rest period at low temperature, and a second fermentation process was characterized to elucidate the causes of its low water kefir grain growth and instability. The frozen-stored water kefir grain inoculum was thawed and reactivated during three consecutive prefermentations before the water kefir production process was started. Freezing and thawing damaged the water kefir grains irreversibly, as their structure did not restore during the prefermentations nor the production process. The viable counts of the lactic acid bacteria and yeasts on the water kefir grains and in the liquors were as expected, whereas those of the acetic acid bacteria were high, due to the aerobic fermentation conditions. Nevertheless, the fermentations progressed slowly, which was caused by excessive substrate concentrations resulting in a high osmotic stress. Lactobacillus nagelii , Lactobacillus paracasei , Lactobacillus hilgardii , Leuconostoc mesenteroides , Bifidobacterium aquikefiri , Gluconobacter roseus / oxydans , Gluconobacter cerinus , Saccharomyces cerevisiae , and Zygotorulaspora florentina were the most prevalent microorganisms. Lb. hilgardii , the microorganism thought to be responsible for water kefir grain growth, was not found culture-dependently, which could explain the low water kefir grain growth of this industrial process.

Kombucha is a beverage made by fermenting sugared tea using a symbiotic culture of bacteria belonging to the genus Acetobacter, Gluconobacter , and the yeasts of the genus Saccharomyces along with glucuronic acid, which has health-promoting properties. The paper presents the evaluation of ferments as a potential cosmetic raw material obtained from Yerba Mate after different fermentation times with the addition of Kombucha. Fermented and unfermented extracts were compared in terms of chemical composition and biological activity. The antioxidant potential of obtained ferments was analyzed by evaluating the scavenging of external and intracellular free radicals. Cytotoxicity was determined on keratinocyte and fibroblast cell lines, resulting in significant increase in cell viability for the ferments. The ferments, especially after 14 and 21 days of fermentation showed strong ability to inhibit (about 40% for F21) the activity of lipoxygenase, collagenase and elastase enzymes and long‐lasting hydration after their application on the skin. Moreover, active chemical compounds, including phenolic acids, xanthines and flavonoids were identified by HPLC/ESI–MS. The results showed that both the analyzed Yerba Mate extract and the ferments obtained with Kombucha may be valuable ingredients in cosmetic products.