**1. Introduction**

The attraction of bacterial cellulose (BC) as a natural biopolymer, produced by microorganisms, arises from its main functional properties and subsequent applications, especially in the biomedical, food, and engineering fields [1–3]. Bacteria able to synthesize cellulose can be considered ubiquitous since they have been found to inhabit different ecosystems. They can be isolated from environmental sources (such as soil and plants), from insects, humans, and from food sources [4].

Within the family *Acetobacteraceae*, the genus *Komagataeibacter* includes species, such as *K. xylinus*, *K. europaeus* and *K. hansenii*, that have been described as cellulose producing organisms [5]. The main studied strains belong to the *K. xylinus* species, which is recognized as the model organism for studying the mechanism of BC synthesis [6,7].

One of the most important reservoirs in which it is possible to detect strains belonging to the *Komagataeibacter* genus is kombucha tea, a fermented beverage traditionally produced in Asia, probably originating from the northeast of China [8]. Kombucha tea is characterized by a microbial community in which different microbial groups, mainly yeasts and bacteria, live in a symbiotic lifestyle [9]. Among bacteria groups, acetic acid bacteria are the main functional organisms, although lactic acid bacteria are also found; moreover, among yeasts, strains of the genera *Saccharomyces* and *Zygosaccharomyces* have

**Citation:** La China, S.; De Vero, L.; Anguluri, K.; Brugnoli, M.; Mamlouk, D.; Gullo, M. Kombucha Tea as a Reservoir of Cellulose Producing Bacteria: Assessing Diversity among *Komagataeibacter* Isolates. *Appl. Sci.* **2021**, *11*, 1595. https://doi.org/ 10.3390/app11041595

Academic Editor: Maria Kanellaki Received: 31 December 2020 Accepted: 6 February 2021 Published: 10 February 2021

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been described [10]. Kombucha tea production consists of two fermentation reactions operated by yeasts and acetic acid bacteria [11]. The symbiotic relation between yeast and bacteria is established during the fermentation process, in which the yeasts are capable of hydrolysing sucrose, the main substrate for kombucha tea production, into glucose and fructose, and then ethanol. Acetic acid bacteria use glucose, fructose and ethanol to produce gluconic acid, glucuronic acid, acetic acid, and BC [12,13].

Kombucha tea can be considered as a dynamic environment, rapidly changing its composition, acting as an ecophysiological reservoir of very specialized organisms. Considering the occurrence and function of acetic acid bacteria within kombucha, previous studies have described *K. xylinus* as the main species.

The ability of *K. xylinus* strains to produce BC is highly variable, making the selection of the best ones a crucial step in order to obtain candidate strains for large scale BC production. In fact, the availability of selected organisms in producing BC is a requirement for exploiting them as industrial machinery. The production of BC is mainly influenced by two factors. The first one is represented by the carbon sources used in the medium [14,15]; pure carbon sources could increase the production cost, making the process unsustainable. The second issue is related to the ability of the organism to grow and produce BC in the selected conditions [16]. Some studies have described the abilities of strains belonging to the *Komagataeibacter* genus for producing BC at different yields [5].

In this work, acetic acid bacteria were collected from liquid and pellicle fraction of kombucha tea samples. In total, 46 isolates were screened for their ability to produce BC and for the main phenotypic characteristics. Moreover, they were typed by applying a fingerprint analysis based on (GTG)5 repetitive elements and Enterobacterial Repetitive Intragenic Consensus (ERIC) elements in order to assay the intraspecific genetic diversity.

### **2. Materials and Methods**

#### *2.1. Kombucha Preparation, Sampling and Physico-Chemical Determinations*

Two native local kombucha precultures (P1 and P2) were provided by the National Institute of Applied Sciences and Technology (INSAT, Tunisia), previously cultivated on two different substrates: black tea (P1) and green tea (P2). Each preculture was composed of kombucha cellulose pellicle with approximately 200 mL of starter culture. Duplicates of kombucha, referred to green tea kombucha (GTK) and black tea kombucha (BTK), were prepared and inoculated according to Jayabalan et al. [17]. Briefly, sucrose 10% (*w*/*v*) was added to demineralized water and allowed to boil. Afterwards, 1.2% (*w*/*v*) black or green tea (Les Jardins du thé, Office Tunisien du Commerce, Tunisia) was added and allowed to infuse for about 5 min. After filtration of tea leaves through a sterile sieve, 200 mL of tea was poured into 500 mL glass jars that had been previously sterilized at +121 ◦C for 20 min. Once the sucrose–tea solution had cooled at room temperature, 10% (*v*/*v*) of the fermented tea (of the previous fermentation brew from kombucha with the same origin, in this case black or green tea as substrate, corresponding to the aforementioned starter culture) was added. Then, 3% (*w*/*v*) of representative pellicle fragments cut from the preculture were placed in the culture light side up. The jars were covered with a sterile gauze and fixed with an elastic band. Fermentations were carried out in the dark for 12 days. All experiments were carried out aseptically.

Cell morphology and direct observation of samples were carried out using optical microscopy at 100× of magnification, using C. Zeiss microscope apparatus (Axiolab).

Titratable acidity was determined by neutralizing samples at pH 7.2 with 0.1 N of NaOH (it was assumed that all media acidity was due to acetic acid) and expressed as % (*wt*/*wt*); pH was measured by CRISON, MicropH, 2002 pHmeter. Ethanol, expressed as % (*v*/*v*), was checked by densitometry measure using a hydrostatic balance after distillation.

#### *2.2. Plating and Isolation*

Plating was performed immediately after sampling. Therefore, 10 mL of 0.1% (*w*/*v*) peptone water was added to 1 g of black or green kombucha pellicle in a sterile stomacher

bag that was vigorously shaken for 5 min in a laboratory blender STOMACHER® 400 (Seward, England) to obtain a uniform homogenate. Samples (1 mL each) of the homogenate and liquid phase were 10-fold serially diluted in 0.1% (*w*/*v*) peptone water, from which aliquots (0.1 mL) were plated on GYC medium (50 g/L glucose, 10 g/L yeast extract, 15 g/L CaCO3, 9 g/L bacteriological agar) and ACB agar medium (30 g/L yeast extract, 1 mL of a 22 g/L bromocresol green solution, 20 g/L bacteriological agar). The media were previously autoclaved at 121 ◦C for 15 min and after 20 mL/L of filtered ethanol 95% (*v*/*v*) was added aseptically to the ACB medium. A total of 0.2 mL of cycloheximide hydroalcoholic solution at 25% *m*/*v* was added directly to petri dishes.

Plates were incubated at +28 ◦C for 72–120 h. Colonies were picked up from a suitable dilution of each sample on GYC and ACB agar media, grown for 3–5 days at +28 ◦C and purified through subculturing and plating. Pure kombucha isolates were named and strains were cultivated on the corresponding isolation medium for 3–5 days at +28 ◦C [18]. Long-term preservation methods of the strains were performed according to the standard procedures of the Microbial Resource Research Infrastructure—Italian Joint Research Unit (MIRRI-IT) [19]. Specifically, a seed lot of the strains was stored at −80 ◦C, adding glycerol 50% (*v*/*v*) to the liquid media. The strains with significant cellulose production were deposited in the University of Modena and Reggio Emilia (UNIMORE) Microbial Culture Collection (UMCC, www.umcc.unimore.it, accessed on 3 February 2020).

#### *2.3. Phenotypic Characterization*

Strains were phenotypically characterized by determination of cell morphology, gram staining, catalase activity, potassium hydroxide (KOH) test, consumption of calcium carbonate and oxidation of ethanol and acetic acid on ACB agar medium, as previously reported in Gullo et al. [20].

The BC production test was carried out by collecting the pellicles and boiling them in 4 mL of 5.0% NaOH solution for 2 h. Cellulose was confirmed when the pellicle did not dissolve after boiling [21]. DSMZ (German Collection of Microorganisms and Cell Cultures GmbH) strains *K. xylinus* (DSMZ 2004) and *G. oxydans* (DSMZ 3503) were used as positive and negative controls, respectively. Tests were conducted in triplicate.

The amount of BC was estimated, as reported by Gullo et al. [22]. Briefly, pellicles were washed with distilled water and then treated with 1% NaOH at 90 ◦C for 30 min. Treated BC was washed twice with distilled water, the pH of the residual water was determined; the washing step was repeated until reaching neutral pH. Drying was conducted at 80 ◦C. BC weight was measured by analytical balance (Gibertini E42S) [23]. The yield of BC produced was expressed in grams of dried BC per liter of broth.
