In this study, the most BC was produced in K. xylinus + Lb. acidophilus and K. xylinus + Lb. helveticus co-cultures. The sudden increase in BC weight between days 12 and 14 in the K. xylinus + Lb. acidophilus and K. xylinus + Lb. helveticus co-cultures may be attributed to increased lactose consumption at this time (Fig. 2A and E). The stabilization of lactose levels in Lb. acidophilus and Lb. helveticus monocultures after 12 days of culture (Fig. 2A and E) suggests that these bacteria no longer consume lactose from the medium and convert it to LA after this time. This is confirmed by the constant pH and LA content in these monocultures after 12 days (Tables 1 and 2). In contrast, in the K. xylinus monoculture, lactose is still consumed between days 12 and 14 (Fig. 2A, C, E). Thus, it can be assumed that K. xylinus is responsible for lactose consumption after 12 days in co-cultures of K. xylinus + Lb. acidophilus and K. xylinus + Lb. helveticus. Based on this, it can be hypothesized that Lb. acidophilus and Lb. helveticus in co-cultures consume lactose until day 12, after which, due to the disappearance of competition for the carbon source, K. xylinus becomes more active and utilizes it for increased BC production.

The positive effect of co-culture on BC biosynthesis was noted in studies by Seto et al. (2006), in which Gluconacetobacter xylinus was cultivated with Lactobacilus mali. Specifically, 4.2 g·L−1 of BC was obtained compared to 1.4 g·L−1 of BC produced in a Ga. xylinus monoculture. As a possible explanation for this phenomenon, the authors suggested an increased activity of enzymes responsible for sucrose metabolism and EPS production by LAB, though this was not proved experimentally. Additionally, attention was drawn to the fact that it is not just EPS, but EPS-producing LAB that promote cellulose production, facilitating physical interaction between cells. Cell coaggregation may support the assembly of BC fibers into matrices and facilitate interactions between cells at the quorum sensing (QS) level (Seto et al. 2006). QS is a phenomenon of chemical communication between bacterial cells, consisting of the synthesis and secretion of signaling molecules into the environment, which participate in regulating various physiological processes (Liu et al. 2018). The effect of QS on increased BC biosynthesis may be associated with the transcriptional regulatory protein LuxR, the increased expression of which has been noted in the co-culture of K. nataicola Q2 and Lb. fermentum SR. QS can also regulate the expression of bcs genes (encoding cellulose synthase) by regulating the level of cyclic diguanylate, which affects the formation of β−1,4glucan chains and the crystallization of microfibrils (Liu et al. 2018; Jiang et al. 2023). The K. xylinus UMCC 2756 strain contains the luxR gene; however, further research is required to confirm this mechanism (Gullo et al. 2019).

Increased BC biosynthesis was also observed in co-cultures of AAB with other microorganisms, e.g., with Lactobacilus fermentum, Bacilus cereus, and Escherichia coli (Liu and Catchmark 2019; Jiang et al. 2023; Li et al. 2023). The mechanisms promoting BC production in different co-cultures may vary. In one scheme, metabolites of one strain increase the activity of enzymes involved in ATP synthesis in AAB, thereby increasing energy levels and promoting cell growth (Li et al. 2023). The second mechanism presumably involves one strain assimilating a compound or metabolite that inhibits a certain metabolic pathway in the second strain. Reducing the content of the inhibitor causes this pathway to be activated. For example, in an E. coli co-culture, it assimilates GA, preventing GA from inhibiting the pathway responsible for BC biosynthesis in acetic bacteria (Liu and Catchmark 2019).

Compared to the monoculture, the consumption of lactose in K. xylinus + Lb. acidophilus and K. xylinus + Lb. helveticus co-cultures was almost twice as high. Nevertheless, when analyzing these two co-cultures, a certain correlation can be observed. In both the K. xylinus + Lb. acidophilus and K. xylinus + Lb. helveticus co-cultures, there was a significant (mild in the case of the co-culture with Lb. helveticus) decrease in the lactose content of the medium after 6–9 days of culture, but this did not reflect a significant increase in BC mass (Fig. 1) during this time. A potential explanation for this phenomenon is that lactose consumption could be related to the dominance of Lb. acidophilus and Lb. helveticus, which assimilated lactose and used it for their own metabolism, e.g., LA or EPS production. This may be confirmed by the observation that in the K. xylinus monoculture, the lactose consumption on days 6–9 is lower than the lactose consumption in the Lb. acidophilus and Lb. helveticus monocultures at this time. Moreover, lactose consumption by Lb. acidophilus and Lb. helveticus was observed until the 12th day of culture, after which time the lactose content of the medium with Lb. acidophilus and Lb. helveticus monocultures did not decrease (Fig. 2A). Assuming that the metabolism of lactose by Lb. acidophilus and Lb. helveticus proceeds similarly in co-cultures with K. xylinus, it can be assumed that after 12 days of culture the remaining lactose in the medium becomes easily available to K. xylinus, since they no longer have to compete with LAB for this sugar. Therefore, K. xylinus may have increased BC production only after 12 days of culture. Low lactose consumption (0.3 g·100 mL−1) in the K. xylinus + Lb. delbrueckii co-culture may suggest that allelopathic interactions occurred between K. xylinus and Lb. delbrueckii, meaning the growth and activity of one strain may have been limited by metabolites produced by the other strain. One possible explanation for this effect is that LAB produce compounds with high antioxidant activity (Xing et al. 2015; Zhang et al. 2017; Antolak et al. 2021). Antioxidants could negatively affect aerobic K. xylinus bacteria, in which the oxidative phosphorylation of sugars is one of the primary mechanisms for obtaining energy (He et al. 2022). Thus, K. xylinus, stimulated to obtain energy through alternative pathways, activated the substrate phosphorylation from non-sugar carbon sources by TCA (Zhong et al. 2013), which would explain the low consumption of lactose, the decreasing content of LA and CA, and the production of AA (Table 2). Another explanation suggests the inhibitory effect of metabolites (aldehydes, ketones, organic acids) of K. xylinus on the growth of Lb. delbrueckii, as evidenced by the lower content of LA, the main metabolite of LAB, compared to the Lb. delbrueckii monoculture. The impact of mutual interactions in the AAB co-culture with LAB was also noted by Xia et al. (2022). In the co-culture of Acetobacter pasteurianus with Lb. helveticus, inhibited growth of Lb. helveticus was observed compared to the monoculture, while the growth of A. pasteurianus remained unchanged. The authors argued that this was related to a higher content of AA in the medium, which could inhibit the growth of Lb. helveticus.

One of our goals in this study was to check the possibility of producing BC in AW where the main carbon source is lactose. According to literature data, one of the challenges associated with obtaining BC from AW is the low ability of AAB to assimilate whey proteins and uptake lactose. This is due to the absence of the lacZ gene in the genomes of most AAB, the expression of which determines the synthesis of β-galactosidase, the enzyme responsible for breaking down lactose into glucose and galactose (Kolesovs and Semjonovs 2020). Brugnoli et al. (2023a) used cheese whey as a medium for BC biosynthesis using, among others, K. xylinus UMCC 2756 and obtained more BC than in Hestrin-Schramm medium. However, before culturing, the whey was treated with β-galactosidase from Aspergillus oryzae, which confirms that K. xylinus UMCC 2756 is not able to degrade lactose into glucose and galactose (Brugnoli et al. 2023a). However, our research has shown that in the K. xylinus UMCC 2756 monoculture, the lactose content decreased, suggesting that probably it is included in some metabolic pathway of K. xylinus UMCC 2756, which enables BC production in AW. It is worth noting that the use of lactose and BC biosynthesis increased in the presence of LA in the medium and at pH 3.5–3.8. This observation is consistent with reports by Li et al. (2023), which showed that glucose consumption increased after medium supplementation with acetoin. Therefore, to increase BC production in pure AW, it is necessary to use other stimulants like LA, as its biosynthesis in AW is not very efficient. Another hypothetical explanation for the lactose consumption by K. xylinus is its oxidation to lactobionic acid (4-O-β-galactopyranosyl-D-gluconic acid, LBA) (Kiryu et al. 2019). LBA is formed as a result of enzymatic oxidation of lactose by the periplasmic membrane–bound quinoprotein glucose dehydrogenase (m-GDH). The presence of m-GDH and its oxidative activity has been proven in 48 AAB strains, with the highest activity found in Komagataeibacter (formerly Gluconacetobacter) medellinensis NBRC3288 (Kiryu et al. 2015). Interestingly, the presence of the gcd gene was found in the genome of K. xylinus UMCC 2756 (GeneBank no. QQBI00000000) (Gullo et al. 2019), which encodes the production of m-GDH. However, these studies did not examine the content of LBA, so further research is needed to confirm the ability of K. xylinus UMCC 2756 to oxidize lactose.

In recent years, the possibility of obtaining BC in media with lactose as the primary carbon source has been studied, but the efficiency was unsatisfactory (Cielecka et al. 2021b). In studies on the cultivation of the Komagataeibacter sp. W1 strain by Wang et al. (2018), only 0.04 g·L−1 of BC was obtained, and during the cultivation of the G. hansenii GH1/2008 strain by Bolgova et al. (2023), only 0.1 g·L−1 was produced. In contrast, Singhsa et al. (2018) examined five strains of K. xylinus and found that the amount of BC produced from lactose may differ not only due to the strain used but also due to the cultivation method (stationary or mixed). Under stationary conditions, the highest BC production (approx. 1.4 g·L−1) from lactose was achieved by the K. xylinus strain, whose efficiency in shaken cultures did not change. In the stationary cultivation of the K. xylinus K1011 and K. xylinus K975 strains, 0.9 g·L−1 and 1.3 g·L−1 of BC was obtained, respectively, while in the mixed cultivation, the efficiency increased to approximately 4.7 g·L−1 and 2.4 g·L−1, respectively (Singhsa et al. 2018). Another example of one of the few studies showing the natural ability of AAB to assimilate lactose (and thus increase BC biosynthesis) is the research by Revin et al. (2018) which demonstrated that the Gluconobacter sucrofermentans B-11267 strain could assimilate lactose from whey and produced 5.45 g·L−1 of BC. A summary of research showing the possibilities of producing BC from AW as a standalone medium or an additive to other media is described in our previous publication (Płoska et al. 2023b).

A characteristic feature of microbial co-cultures is that the metabolites of one microorganism can be substrates entering the metabolic pathways of another one. Metabolism of citrate to acetate is typical of LAB (Oliveira et al. 2012). In homofermentative LAB, the production of acetate and oxaloacetate from citrate is catalyzed by citrate lyase (Fig. 7). The acetate is excreted outside the cell, and the oxaloacetate is decarboxylated to pyruvate by oxaloacetate decarboxylase. In subsequent steps, pyruvate may be further converted to lactate, acetate, formate, ethanol, or four-carbon aromatic compounds such as acetoin and 2,3-butanediol (Quintans, et al. 2008; Pudlik and Lolkema 2011; Bintsis 2018). The latter two metabolites may be relevant in the context of the AAB co-culture. According to Li et al. (2023), acetoin and 2,3-butanediol can positively affect the growth and activity of AAB by increasing the activity of acetoin dehydrogenase, which catalyzes the conversion of acetoin to acetyl-coenzyme A (CoA) and acetaldehyde. Additionally, acetaldehyde may be converted to acetate and then acetyl-CoA by acetaldehyde dehydrogenase and acetyl-CoA synthetase, respectively. An increase in acetyl-CoA concentration leads to the generation of more ATP molecules, which in turn provides the energy necessary for cell growth and BC biosynthesis (Fig. 7). This mechanism was confirmed in the co-culture of K. xylinus with Bacillus cereus, where acetoin and 2,3-butanediol were metabolites of B. cereus. Notably, 4.4 g·L−1 of BC was obtained from the co-culture, compared to 1.2 g·L−1 from the K. xylinus monoculture (Li et al. 2023). A hypothetical scheme illustrating the effect of LAB metabolites on AAB metabolism using citrate and acetoin metabolism as example is shown in Fig. 7.

Hypothetical scheme of metabolite flow in acetic-lactic co-culture using citrate and acetoin metabolism as an example

In AAB, gluconic acid and its derivatives are formed as a result of aerobic glucose metabolism, where they participate in one of two pathways for energy production in the cell. When glucose is the carbon source in the medium, it is phosphorylated by glucokinase to glucose-6-phosphate, which is then converted to 6-phosphogluconolactone. This process is associated with the reduction of nicotinamide adenine dinucleotide phosphate (NADP+) to NADPH, generating energy and transferring electrons to electron acceptors (Liu et al. 2018). In contrast, when glucose is absent from the medium and another carbon source, such as mannitol, lactose, or fructose, is present, GA is produced in very low concentrations or not produced at all (Anguluri et al. 2022). In our study, the primary carbon source in AW was lactose, which justifies the low content of GA. In almost all cultures, the GA content decreased over time, with the exception of the K. xylinus + Lb. helveticus co-culture, where the GA content increased. The decrease in GA content in the medium can be explained by the hypothesis put forward by Liu et al. (2018), who argued that in the absence of glucose in the medium, GA can be converted to glucose-6-phosphate and further used as a carbon source for cell growth and BC production via the pentose phosphate pathway. Interestingly, the increase in GA content in the K. xylinus + Lb. helveticus co-culture may be correlated with the increase in galactose content. This supports the hypothesis about the oxidation of lactose to LBA by K. xylinus. It is possible that Lb. helveticus produces enzymes that enable the hydrolysis of LBA to galactose and GA, thus explaining the observed increase in their content in the medium (Fig. 2F).

The FTIR spectra of BC obtained in the K. xylinus monoculture and acetic-lactic co-cultures were similar (Fig. 4). In all samples, peaks corresponding to bonds and functional groups characteristic of BC were observed, and no new peaks were observed in spectra of BC from co-cultures. In studies by Liu and Catchmark (2019), the spectrum of BC from the Ga. hansenii and E. coli co-culture showed the presence of a peak corresponding to the C–N bonds in glucosamine, which was derived from EPS produced by E. coli. In our study, despite the proven ability of LAB to produce EPS (Table 3), no extra peaks indicating the occurrence of C–N bonds were observed. Nevertheless, from the wavenumber of 1400 to 600 cm−1 (Fig. 4), the peak intensity in the spectra of BC from all co-cultures was higher compared to that in the spectra of BC from the K. xylinus monoculture. Peak intensity is assumed to be directly proportional to the number (per unit volume) of functional groups and bonds occurring in the material (Farhadi and Sohbatzadeh 2023). Therefore, considering the similarity of functional groups and bonds in the chemical structure of BC and EPS, potential interactions between BC and EPS produced by LAB can be assumed. A significant difference in peak intensity in the FTIR spectra was observed in BC from the K. xylinus + Lb. delbrueckii co-culture. In this case, the intensity of the peak corresponding to the hydroxyl group (3336 cm−1) was lower than that seen in the spectra of other BC, which may indicate a lower number of hydroxyl groups and hydrogen bonds. According to Sharma et al. (2023), the intensity of the peak at wavenumber 3336 cm−1 indicates the strength of the intramolecular H3(O)H…O(5) bond, which can be increased in the presence of LA in the medium. This explains the lower intensity of this peak in the spectrum of BC from the K. xylinus + Lb. delbrueckii co-culture, where the lowest LA content was observed (Table 2) compared to BC from other co-cultures and monoculture. It can be assumed that minor variations in the FTIR spectra of BC may have been influenced by different combinations of strains and differences in metabolites in the co-culture system, as mentioned by other researchers (Jiang et al. 2023).

Based on the MRDT values, it was found that the co-cultures K. xylinus + Lb. acidophilus and K. xylinus + Lb. helveticus positively affected the thermal properties of BC, with the highest thermostability exhibited by BC obtained from the co-culture K. xylinus + Lb. acidophilus. The apparent shift in the maximum temperature of BC degradation from the K. xylinus monoculture and the K. xylinus + Lb. helveticus co-culture toward a lower temperature may be related to the low crystallinity of these BC variants and a higher share of the amorphous fraction (unpublished results) compared to BC from the other cultures. According to Cichosz and Masek (2020), among others, the degree of crystallinity and the ratio of crystalline to amorphous areas determine the thermal stability of cellulose. The more amorphous areas in the structure (lower crystallinity), the more easily it degrades. According to Lin et al. (2023), changes in BC crystallinity, and therefore thermostability, can be caused by various interactions in the culture medium during crystallization, such as increasing the viscosity of the medium. Additionally, based on the conducted research, we cannot definitively state that the co-culture product is pure BC. We can suspect that some part of the EPS produced by the co-cultured LAB may have built into the BC structure, but we do not have definite evidence for this. On the basis of our results (including unpublished ones) and based on literature data, we assume that the differences in, among others, the thermal stability of the polymer obtained are due to specific interactions between K. xylinus and the particular strain of LAB.

The mechanical properties of BC depend on many factors such as culture conditions, medium composition, fiber concentration, fiber arrangement, and bacterial strain used to obtain BC (Cielecka et al. 2021a; Brugnoli et al. 2025). Cellulose obtained from the K. xylinus + Lb. acidophilus and K. xylinus + Lb. helveticus co-ultures was characterized by the highest strain at break (Fig. 6A), which could be related to a higher LA content in the media and a lower pH in these culture variants. As already mentioned, the presence of LA in the medium may increase the strength of the intramolecular H3(O)H…O(5) bonds within the glucopyranose ring (Sharma et al. 2023). In turn, the created intra- and intermolecular bonds in cellulose affect not only the physical properties of the polymer, including solubility, hydroxyl reactivity, and crystallinity, but also play an important role in its mechanical properties (Fan et al. 2012). According to Sharma et al. (2023), the addition of LA to the medium significantly increased the strain at break of the produced BC compared to the BC obtained in the medium without LA. These results also confirm the reports by Cielecka et al. (2021b) where LA medium supplementation positively influenced the strain at break. Moreover, it was shown that the strain at break of BC obtained in media with glucose and at different pH varied significantly, which would indicate the influence of the pH of the medium on the mechanical properties of BC. In our research, this hypothesis can be confirmed by the difference between the high strain at break of BC from the K. xylinus + Lb. acidophilus and K. xylinus + Lb. helveticus co-cultures, where the final pH was 3.8 and 3.6, respectively (Table 1), and the low strain at break of BC obtained in the K. xylinus + Lb. delbrueckii co-culture, where the final pH was approx. 6.0. The effect of the pH of the medium can also be observed in the case of Young’s modulus (Fig. 6C); however, in this instance, a reverse trend was noted. The highest Young’s modulus was characterized by BC obtained in the K. xylinus + Lb. delbrueckii co-culture, and it was significantly (p < 0.05) higher than the values for other BC samples. Nevertheless, it is also worth noting that in the case of this co-culture, the high value of Young’s modulus could have been influenced by EPS produced by Lb. delbrueckii (Table 3). Liu and Catchmark (2018) conducted studies which demonstrated that the addition of EPS to the culture medium had a positive effect on the Young’s modulus of BC compared to BC obtained from the Hestrin-Schramm medium without EPS. Moreover, it was observed that depending on the concentration, the addition of EPS may have a different effect on the strain at break and the stress at break. In our study, the highest stress at break value (Fig. 6B) was observed for cellulose obtained in the K. xylinus + Lb. helveticus co-culture (17.18 MPa), and a slightly lower value was recorded for BC produced in the K. xylinus + Lb. delbrueckii co-culture (14.43 MPa). These results can be linked to the arrangement and the largest diameter of BC fibers from these co-culture variants. It is possible that more force was required to tear apart thicker BC fibers, resulting in increased tensile stress (Cielecka et al. 2021a). Similar conclusions were formulated by Liu and Catchmark (2019) who, in research on the Ga. hansenii and E. coli co-culture, showed that the BC obtained from the co-culture was characterized by higher density and thicker fibers compared to BC from the Ga. hansenii monoculture, which affected its mechanical properties. On the other hand, Brugnoli et al. (2025) increased Young’s modulus and stress at break of the BC-hyaluronic acid composite obtained in co-culture of AAB with LAB, explained by high BC content and high crystallinity. Nevertheless, the authors noted the diverse effects of different strains on the mechanical properties of BC, which implies the necessity of proper selection of strains for co-culture.