Cellulosic biofilm formation of Komagataeibacter in kombucha at oil-water interfaces

Abstract

Bacteria forming biofilms at oil-water interfaces have diverse metabolism, they use hydrocarbons as a carbon and energy source. Kombucha is a fermented drink obtained from a complex symbiotic culture of bacteria and yeast, where acetic acid bacteria present in kombucha use sugars as a carbon source to produce cellulosic biofilms. We hypothesize that Komagataeibacteraceae in kombucha can adsorb to and use hydrocarbons as the sole energy source to produce cellulosic biofilms. Hence we characterized a kombucha culture, studied bacterial adsorption and cellulosic biofilm formation of kombucha at the n-decane or mineral oil-kombucha suspension interface. The cellulosic biofilms were imaged using fluorescence microscopy and cryo-scanning electron microscopy, and their time-dependent rheology was measured. Komagataeibacter, the dominant bacterial genus in the kombucha culture, produced cellulosic biofilms with reduced cellulose biomass yield at the oil-kombucha suspension interfaces compared to at the air-kombucha suspension interface. The presence of biosurfactants in the supernatant secreted by the kombucha microbes led to a larger and faster decrease in the interfacial tension on both oil types, leading to the formation of stable and elastic biofilm membranes. The difference in interfacial tension reduction was insignificant already after 2 h of biofilm formation at the mineral oil-kombucha suspension interface compared to kombucha microbes resuspended without biosurfactants but persisted for longer than 24 h in contact with n-decane. We also demonstrate that Komagataeibacter in kombucha can produce elastic cellulosic biofilms using hydrocarbons from the oil interface as the sole source of carbon and energy. Thus Komagataeibacter and kombucha shows the potential of this system for producing valued bacterial cellulose through remediation of hydrocarbon waste.

Keywords: Bacterial cellulose; Biofilm; Interfacial rheology; Interfacial tension; Komagataeibacteraceae; Kombucha; Oil-water interface; Scanning electron microscopy.

Fig. 1

Bacteria adsorbed to and stabilized oil droplets for at least 30 days. A) Representative fluorescence microscope images of Komagataeibacter adhering to oil droplets after 1, 10, 21, and 30 days of incubation at 28 °C. The scale bars denote 50 μm. B) Representative superimposed bright-field and fluorescence images of Komagataeibacter adhering to oil droplet after 30 days of incubation. The scale bars denote 25 μm. The emulsions of n-decane or mineral oil in microbial dispersions obtained through mixing kombucha suspension with n-decane or mineral oil were stained using 3.34 mM SYTO 9 green fluorescent nucleic acid stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Cellulosic biofilm formation of kombucha culture at the kombucha suspension-air/oil interface. A) Representative photographs of cellulosic biofilms formed at the interface after 30 days. The regions within the dashed squares show the membrane pellicle formed at the interfaces. Bacteria produced a thick pellicle layer at the kombucha suspension-air interface compared to the kombucha suspension-oil interfaces. B) Scanning electron micrographs of the pellicle show the surface structure of an interconnected network of cellulose fibres secreted by the Komagataeibacter. The high-pressure-frozen, freeze-fractured pellicles imaged by cryo-scanning electron microscopy reveal the internal structure of the pellicle cellulose matrix embedding Komagataeibacter and yeast. Arrows in red point to Komagataeibacter, black arrows point to yeast, and green arrows point to the cellulose matrix. The scale bars denote 5 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Cellulosic biofilm formation at the air-kombucha suspension interface as a function of time. An air bubble was formed at the tip of an inverted needle and aged in the kombucha suspension maintained at 28 °C for 13 h. The scale bars denote 0.5 mm. Red arrows indicate the formation of cellulose (fibre-like structures) starting to be visible at the interface after 4 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Rheology of bacterial adsorption and cellulosic biofilm formation at the air-kombucha suspension interface. A) Interfacial tension ($\gamma .$) as a function of time measured at the interface. B) Interfacial elasticity measurements (E′- elastic modulus and E'′- viscous modulus) of bacterial adsorption and cellulosic biofilm formation at the interface performed by the oscillating drop method. The E′ and E'′ were measured every 2 h. Error bars represent standard deviations over three replicates. * indicates a statistically significant difference of means with p < 0.05. ** indicates a statistically significant difference of means with p < 0.01. C) The ratio of elastic to viscous modulus (E'/E'′) of the interface as a function of time. Error bars represent standard deviations over three biological replicates. ANOVA tests were performed, and no significant differences were observed between the time points.

Fig. 5

Interfacial tension ($\gamma$) as a function of time measured at the interface between A) n-decane and kombucha suspension, and B) mineral oil and kombucha suspension, in PBS or supernatant and only PBS as a control. C) The ratio of elastic to viscous modulus (E'/E'′) of the interface between oil and kombucha suspension with supernatant at 0 h, 1 h, and 24 h. Error bars represent standard deviations over three replicates. ANOVA tests were performed. * indicates significance (p < 0.05) compared to 0 h and 1 h for their corresponding oil types. No difference in E'/E'′ was observed between oil types.

Fig. 6

Deformation of interfaces of kombucha suspensions (PBS or microbes with supernatant) in contact with A) n-decane or B) mineral oil droplet in response to a large volume reduction. A droplet of oil (5 μL) was formed at the tip of an inverted needle and aged in the kombucha suspension for 24 h. The aged drop is compressed manually by withdrawing 90% of the oil droplet volume and then reinjecting it after observing the compression of the interface. The complete process (withdrawal and reinjection of oil droplet volume) took ∼1 min. Supplementary Videos (Videos S2–S7) were recorded, and snapshots at different stages are displayed in the figure.

Fig. 7

Interfacial rheology of cellulosic biofilms at the oil-water interface. A) Interfacial tension ($\gamma$) as a function of the number of days measured at the interface between i) n-decane (open box) and kombucha suspension, and ii) mineral oil (shaded box) and kombucha suspension. B) The ratio of elastic to viscous modulus (E'/E'′) of the interface between oil (i) n-decane (open box) and ii) mineral oil (shaded box)) and kombucha suspension at every 24 h for 7 days. The kombucha suspension was prepared by centrifugation of cells at 5000 RPM for 5 min and diluted to OD_{600 nm} 0.05 in PBS. * denotes significance (p < 0.05) compared to n-decane and kombucha suspension. C) Representative fluorescence microscope images of cellulosic biofilms formed at n-decane-kombucha suspension interface (a, b), and mineral oil-kombucha suspension interface (c, d) after 7 days. The scale bars denote 20 μm. The oil droplet was pipetted from the tip of the inverted needle onto a clean glass slide, stained using vitality staining solution (3.34 mM SYTO 9 and 20 mM PI) and 25 μM Calcofluor White in PBS for 15 min. Green colour indicates live cells stained with SYTO 9, red colour indicates dead cells stained with PI and blue colour represents cellulose stained with Calcofluor White. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)