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. 2022 Jul 20;60(3):1054–1064. doi: 10.1007/s13197-022-05451-y

Improved production of bacterial cellulose by Komagataeibacter europaeus employing fruit extract as carbon source

Yi Sheng Tseng 1,2, Anil Kumar Patel 2,3, Chiu-Wen Chen 1,2,3, Cheng-Di Dong 1,2,3, Reeta Rani Singhania 2,3,✉
PMCID: PMC9998749  PMID: 36908337

Abstract

Bacterial cellulose (BC) has attracted worldwide attention owing to its tremendous properties and versatile applications. BC has huge market demand, however; its production is still limited hence important to explore the economically and technically feasible bioprocess for its improved production. The current study is based on improving the bioprocess for BC production employing Komagataeibacter europeaus 14148. Physico-chemical parameters have been optimized e.g., initial pH, incubation temperature, incubation period, inoculum size, and carbon source for maximum BC production. The study employed crude and/or a defined carbon source in the production medium. Hestrin and Schramm (HS) medium was used for BC production with initial pH 5.5 at 30 °C after 7 days of incubation under static conditions. The yield of BC obtained from fruit juice extracted from orange, papaya, mango and banana were higher than other sugars employed. The maximum BC yield of 3.48 ± 0.16 g/L was obtained with papaya extract having 40 g/L reducing sugar concentration and 3.47 ± 0.05 g/L BC was obtained with orange extract having 40 g/L reducing sugar equivalent in the medium. BC yield was about three-fold higher than standard HS medium. Fruit extracts can be employed as sustainable and economic substrates for BC production to replace glucose and fructose.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-022-05451-y.

Keywords: Bacterial cellulose, Komagataeibacter, Bioprocess, Carbon source, Biorefinery

Introduction

Bacterial cellulose (BC) is a unique nanocrystal material produced by bacteria to protect themselves against harsh chemical environments and ultraviolet radiations and also to get excessive oxygen (Singhania et al. 2022). To be called nanocellulose, the cellulosic material must have at least one dimension at the nanoscale. Chemically it is a polymer of glucose with β-1,4 linkage, however; this biomaterial cannot be considered equivalent to plant cellulose owing to its high purity and excellent physical properties such as crystallinity, thermal stability, mechanical strength and water-absorbing capacity (Wang et al. 2019). The key to cellulose integrity is the presence of bountiful hydroxyl groups existing throughout the polymer chain. It causes the formation of a large number of hydrogen bonds in anhydroglucose units between OH groups and Oxygen atoms (Pogorelova et al. 2020). It finds versatile applications as bioplastics for green packaging, in cosmetics, in medicine and wound healing, the membrane of batteries and specialty paper manufacturing, etc., (Singhania et al. 2022, 2021).

BC has a huge market demand due to various applications but is limited due to its low productivity. It is being produced by aerobic bacteria found in traditional fermented products like acetic acid, vinegar and beverages. Komagataeibacter is the most explored genera of bacteria involved in BC biosynthesis by converting glucose and fructose into glucose-6-phosphate via enzymatic pathway to form β-1,4 glucan chain (Singhania et al. 2022). Several studies on BC production from Komagataeibacter genera such as K. xylinus (Reiniati et al. 2017), K. rhaeticus (Jacek et al. 2021; Thorat and Dastager 2018), K. intermedius (Lin et al. 2016), K. Medellinensis (Molina-Ramírez et al. 2017) and K. europaeus (Dubey et al. 2017) has been undertaken.

Researchers have explored the effects of pH, temperature, inoculum size and a carbon source such as glucose, sucrose, mannitol, fructose, glycerol, xylose, maltose, lactose, mannose and sorbitol and additives (Throat and Dastager 2018; Islam et al. 2017) on BC production. It is necessary before developing any bioprocess; that the physico-chemical parameters must be optimized, as each microbial strain may have different optimal conditions requirement for giving maxiumum products. By employing low-cost agricultural waste instead of pure sugars in the medium, like fruits pulp as carbon source or substrate for BC production the bioprocess can become economically feasible to an extent. Fruit waste like banana, pineapple, strawberry, watermelon, tomato and orange may be employed for BC production (He et al. 2020; Hungund et al. 2013). Fruit juices have been found as a potent carbon source for BC production. K. xylinus produce 0.87 ± 0.02 g/L and 0.8 ± 0.01 g/L BC from sugarcane juice and the coconut juice (Jaroennonthasit et al. 2021).

The objective of the present study was to improve bioprocess for BC production via K. europaeus 14148. This study establishes an improved bioprocess for BC production utilizing residual fruit extract as carbon source. Most of the studies are based on K. xylinus and ours is K. eurapaeus. Also, by characterization of produced cellulose with different carbon source (via FTIR, TGA, SEM and XRD) shows better crystallinity for the one produced with fruit extract when compared to pure glucose or fructose. The current study proves that fruit waste could be considered as a potential carbon source for BC production which could increase the economic feasibility of the bioprocess as well as improves the quality of BC produced.

Materials and methods

Microorganism and chemicals

Bacterial strain Komagataeibacter europaeus BCRC 14148 was purchased from BCRC (HsinChu, Taiwan). It was cultured in a standard HS (Hestrin and Schramm) liquid medium as well as solid medium with agar. For short-term storage, it was cultured in Petri dishes with agar HS medium and kept at 4 °C to be used for 7–10 days and glycerol stock is prepared for long-term storage and kept at  − 80 °C. For inoculum preparation, a loopful of bacteria was inoculated into 50 ml of HS medium in a 250 ml Erlenmeyer flask and incubated at 30 °C for 48 h to be used as inoculum for BC production.

All the carbon sources used in the study except the crude ones were purchased from Sigma (Mo, USA), citric acid was purchased from J.T.Baker. Sodium phosphate dibasic was purchased from Hayashi Pure Chemical (Osaka, Japan). All other chemicals used were analytical grade available commercially.

Fruit extract preparation

Orange, papaya and mango were collected from local market, Kaohsiung, Taiwan. Pulp without peel was mixed with deionized water at ratio 1:2(w/v) and boiled for 1 h then cooled down to room temperature. The extracts of orange (OE), papaya extract (PE) mango extract (ME) and banana extract (BE) were centrifuged at 10,000 rpm for 10 min and the supernatant was filtered by 0.22 μm glass fiber filter (ADVANTEC, Japan) (Dubey et al. 2017).

Media preparation for BC production

HS medium with the following composition as: Glucose, 20 g/L; Peptone, 5 g/L; Yeast extract, 5 g/L; Na2HPO4•12H2O, 3.75 g/L; Citric acid, 1.1 g/L; was prepared for BC production. Initial Medium pH was adjusted to 5.5 by 1 N NaOH and 1 N HCl (Hestrin and Schramm 1954). In case of the carbon source optimization experiment, glucose in the HS medium was replaced by the respective carbon source. Fructose, xylose, sucrose, maltose, glycerol was used as a carbon source in place of glucose in HS media. For fruit extract to be used as a carbon source; OE, PE ME, and BE were added to the HS medium, replacing glucose with 2% reducing sugar. Culture mediums were autoclaved for 15 min at 121 °C at 15 lbs. pressure for sterilization. Production was done in a 3 ml medium taken in culture tubes with a surface area/volume (S/V) ratio of 1.70 and was incubated under static conditions.

Physico-chemical and cultural parameters optimization experiments for BC production

The pre-inoculum was prepared by transferring bacterial colony from agar plate into 3 mL HS medium with initial pH 5.5 under static condition cultivated for 4 days at 30 °C, which was then transferred into 40 mL HS medium and kept under shaking at 140 rpm for 1 day to homogenize. This homogenized culture was used as inoculum and 0.15 mL inoculum was added in the 3 ml production medium.

To study the effect of inoculum size 1–15% v/v inoculum was added on to the production medium. To study the effect of incubation period, 10 sets of the experiment were done in triplicate and incubated for 1–10 days with initial pH 5.5 and incubated at room temperature which was measured as 26 °C ± 1 °C. One set was harvested at each day, i.e. at the interval of 24 h. BC production, final pH and reducing sugar was analyzed for each set. For effect of initial pH on BC production, 5 sets of the experiment were done in triplicates and initial pH was set to 4.0, 5.0, 5.5, 6.0 and 7.0 respectively. All the experiments were incubated for 7 days, unless specified. pH 5.5 was the control.

To study the effect of incubation temperature, production of BC was carried out at 25 °C, 28 °C, 30 °C, 32 °C and 35 °C for 7 days. To screen best carbon source for BC production, defined pure carbon sources such as fructose, xylose, sucrose, maltose, glycerol and glucose as well as crude fruit extract were employed at 2% concentration in HS medium. For different concentrations of carbon source, defined pure carbon source were taken at 2, 4, 6, 8 and 10% w/v concentration, whereas fruit extract was taken at 2 and 4% equivalent reducing sugar concentration.

Monitoring of pH and bacterial growth

The culture sample was taken at 12 h intervals to monitor pH and bacterial cell concentration during cultivation. pH was analyzed by a pH meter. Cell concentration was estimated by measuring the optical density at 600 nm (OD600) and HS medium without bacterial cells was used as blank. Bacterial growth was just indicative as bacterial cellulose may cause interference.

Measurement of sugar concentration

Reducing sugar concentration was estimated by using the dinitrosalicylic acid (DNS) and glucose was used as a standard. DNS reacts with reducing sugar and forms 3-amino-nitrosalicylic acid to give dark orange-red color. 0.5 mL of supernatant was mixed with 0.5 mL DNS solution and kept in a boiling water bath for 5 min incubation. It was cooled using ice flakes and 3 mL deionized water was added and optical density was measured at 540 nm. Glucose and fructose content of fruit extract were analysed by Agilent technological 1260 infinity (Agilent, USA), using 1260 RID (Agilent, USA) and column Coregel 87H3 (300 × 7.8 mm). The column temperature was kept at 55 °C and the mobile phase was 0.005 M H2SO4 with a flow rate of 0.6 mL/min in 20 min.

Bacterial cellulose estimation

After 7 days of incubation, the harvested BC pellicle (the upper floating layer) was treated with 1.0 N NaOH at 60 °C for 2 h to remove the bacteria cell. The BC was washed with deionized water repeatedly until neutral pH and freeze-dried at -53 °C at 7.0 pa for 48 h to reach constant weight. BC was estimated by the gravimetric analysis method. The yield of bacterial cellulose and conversion ratio of utilized reducing sugar to BC was calculated based on the following formula:

Yield of bcaterial celluloseg/L=Freeze-driedweightofbacterialcelluloseVolumeofcultivatedmedium(L) 1
Conversion ratiog/g=Freeze-driedweightofbacterialcellulose(g)Volumeofcultivatedmedium(L) 2

Fourier transfer infrared spectroscopy (FTIR)

Nicolet iN10 spectrophotometer (ThermoFisher Scientific, USA) was used to analyze freeze-dried BC samples by reflection mode. Nitrogen gas is used to maintain inner equilibrium and liquid nitrogen is used to cool detectors. All the spectra were acquired from 4000 to 650 cm-1 with a resolution of 4 cm-1 and 256 scan per sample with 2.5–3.0 reflection energy. All data analysis was done by using OMNICTM software.

X-Ray Diffraction (XRD) analysis

The crystallization of BC was performed by D8 DISCOVER with GADDS (Bruker AXS Gmbh, Karlsruhe, Germany) with Thin Film X-ray Diffractometer at a scanning range of 2θ = 10°–40°. The crystallinity index CrI (%) was calculated by following Eq.

CrI%=Icr-IamIcr 3

where, CrI is the intensity value represented fro the crystalline cellulose 2θ = 22.6 ° and Iam is the intensity value represented for the amorphous cellulose(2θ = 16 o).

Scanning Electron Microscope (SEM) analysis

Scanning electron microscopy (SEM) analysis of BC was done by Hitachi Tabletop TM-3000 Scanning Electron Microscope (FESEM, Hitachi SU8010, Tokyo, Japan) at 10 kV voltage.

Thermogravimetric (TGA) analysis

The thermal stability of BC was analysed by using a thermogravimetric analyzer, TGA (TA Instruments, model TGA SDT Q600, New Castle, DE, USA). The freeze-dried BC sample (10 mg) was heated from room temperature to 600 °C at a heating rate of 10 °C min−1 in N2 atmosphere (40 mL min−1).

Results and discussions

The effect of the incubation period on BC production

BC production was increased continuously with incubation till 7 days at room temperature and then it was decreased further, though the bacterial growth was found to increase till 9 days (Fig. 1a). It was found that there could be an interference of BC during cell density monitoring via spectrophotometer analysis. Maximum BC production was obtained on 7th day having 1.01 ± 0.04 g/L in HS medium and decreased to 0.88 ± 0.06 g/L BC at 10th day of incubation. The lowest pH 3.27 was monitored on 3rd day itself corresponding to 73.77% sugar utilization which is attributed to the accumulation of gluconic acid via consumed glucose (Hwang et al. 1999; Singhania et al. 2022). However, the continuous increase of BC to 7th day being deficient in glucose might attribute to consuming part of gluconic acid to synthesize BC which is reflected with a slight increase in the medium pH (Hwang et al. 1999). Gluconacetobacter xylinus ATCC 10788 produced highest 0.49 g/L BC on fifth day incubated at HS medium. K. xylinus TISTR 975 produced 1.16 ± 0.02 g/L after 10 days of incubation (Jaroennonthasit et al. 2021) which is in accordance with our study. Similarly, K. sucrofermentans DSM15973 in the glucose medium produced 1.7 g L-1 in 10 days (Lee et al. 2021). According to current findings, 7 days was found to be the optimum incubation period and further experiments were carried out for 7 days.

Fig. 1.

Fig. 1

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Effect of a incubation period and b incubation temperature on BC production by K. europaeus 14148

K. europaeus SGP37 cultivated in 50 mL HS medium within 250 mL Erlenmeyer flask (1.06 S/V ratio) produced 9.08 ± 0.24 g/L BC (Dubey et al. 2017) which is almost ninefold higher than the present finding, however, strains are different though the species are similar, also here the surface area/volume (S/V) ratio was 1.70. Acetobacter xylinum 0416 cultivated at low surface area/volume (S/V) ratio produced higher BC compare to high S/V ratio (Azmi et al. 2021) which could also be the reason to a small extent. Kuo et al. (2016) reported higher gluconic acid production, in the high S/V ratio resulting in lower final medium pH and causing inhibition of BC biosynthesis.

The effect of incubation temperature on BC production

The effect of incubation temperature on any bioprocess exerts significant impact as all the microorganisms varies in their temperature requirement owing to its natural habitat as well as its metabolic activities (Singh et al. 2017). Effect of incubation temperature on BC production by K. europaeus 14148 was analyzed at various temperatures (25–35 °C) and at 30 °C maximum BC of 1.32 g/L was obtained (Fig. 1b). It was also corresponding to the maximum sugar utilization. The lowest BC yield and sugar utilization was obtained at 35 °C. This showed incubation temperature above 35 °C was not suitable for BC production by Komagataeibacter (Dubey et al. 2017).

The effect of Inoculum size on BC production

The effect of inoculum size (1.0% to 15%) showed no significant difference (P > 0.05) on BC yield (Fig. 2). In case of bacteria the growth is very fast and hence it can subside the effect on production of metabolites due to short doubling time. Saleh et al. (2020) reported the inoculum size (7% and 9% v/v) showed a negative effect on BC production by K. hannsenii AS. Although the highest BC production was obtained at 7.5% inoculum size (1.04 g/L), the lower inoculum size (1–5%) was found equally good with marginal difference in BC production and hence lower inoculum size may be used for economic feasibility. In this study 1% inoculum was used for economic reasons as there was no significant difference on increasing the inoculum size. The one reason could be the fast growth of bacteria due to which it can easily increase its population subsiding importance of the seed size.

Fig. 2.

Fig. 2

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Effect of inoculum size on BC production by K. europaeus 14148

The effect of initial pH on BC production

Initial pH of the medium shows influence on BC production by K. europaeus 14148. The initial pH of the medium 5.5 obtained maximum BC production of 1.32 ± 0.13 g/L with no significant difference compared to pH 6.0 (P > 0.05) (Fig. 3). This shows initial pH 5.5 to 6 is the optimal range for BC production by K. europaeus 14148. Slight acidic pH (less than pH 7.0) is required for Acetobacter xylinum to produced BC. K. europaeus SGP37 showed improved BC production with increased initial medium pH from 3 to 5 and significantly decreased BC was observed at pH 7 (Dubey et al. 2017). The optimal pH for Komagataeibacter hansenii AS.5 was 5.5 (Saleh et al. 2020). It is in accordance with most of the studies which show that acetic acid bacteria produce BC with initial pH from 5–6. It is in accordance with current findings as well. Based on the results obtained, the optimal culture condition for K. europaeus 14148 to produce BC was 30 °C, pH 5.5, 1% inoculum size and 7 days incubation period under static condition. These optimized conditions were applied for finding the best suitable carbon source and its concentration optimization experiments.

Fig. 3.

Fig. 3

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Effect of initial pH on BC production by K. europaeus 14148

The effect of different carbon source and its concentration on BC production

Komagataeibacter are known to utilize various carbon source and synthesize BC. Among various carbon source fructose showed favorable results on BC production by K. europaeus 14148 and produce maximum of 1.07 ± 0.03 g/L BC but showed no significant difference (P > 0.05) to the control HS medium with glucose (0.97 ± 0.06 g/L) (Fig. 4a). This show fructose and glucose were equally good carbon source for BC production of K. europaeus 14148 followed by glycerol. Dikshit and Kim (2020) reported glycerol was favorable carbon source for BC production by Gluconobacter xylinus, however, in this study medium contained glycerol produced 0.66 g/L BC lower than containing glucose and fructose as carbon source in the production medium. It has been explained that gluconic acid formation may be interrupted by using glycerol due to which the pH might not decrease significantly and thereby not affecting the BC production negatively (Singhania et al. 2022, 2021). During this study, observation made that pH of medium containing glycerol was 5.23 ± 0.07, still it did not support higher BC production which could be due to less growth. K. rhaeticus PG2 showed poor BC obtained from xylose and maltose and sucrose medium (Thorat and Dastager 2018) which is in accordance with current findings too. K. xylinus TISTR 975 and K. europaeus SGP37 show lower BC production on sucrose-based medium, which must be hydrolyzed to glucose and fructose before permeating the cell membrane (Jaroennonthasit et al. 2021; Dubey et al. 2017). Thus, a step of metabolism is increased which involves hydrolysis of sucrose into glucose and fructose, thereby decreasing the BC production. Metabolic pathways for utilization of glucose and fructose and glycerol by Komagataeibacter have been represented which enables us to understand the metabolic pathways that they follow for BC synthesis (Fig. 4b).

Fig. 4.

Fig. 4

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a Effect of carbon sources on BC production by K. europaeus 14148; b Mechanism of utilization of various carbon

source by Komagataeibacter for BC synthesis (Modified from Singhania et al. 2021)

The effect of different glucose concentration on BC yield

The maximum BC production of 1.11 ± 0.12 g/L was obtained from 2% glucose and was reduced in 4% glucose-containing medium (0.97 ± 0.00 g/L) BC. Even with 1% glucose in the medium BC produced was comparatively less. So, 2% and 4% glucose containing HS medium were having significant difference compared to the other glucose concentrations in the medium for BC production (Fig. 5b). There was no increase found further for BC yield by increasing the concentration of glucose in the medium. This indicates that for BC production K. europaeus 14148 can metabolize 2% glucose in the medium. It also reflects that higher glucose content causes more bacterial growth thereby high viscosity which in turn causes deficiency of oxygen not supporting BC production. Though, the maximum conversion ratio was 0.08%, calculated as per Eq. 1 and 2; which was was higher for 1% glucose in the medium when compared to 2% and 4% glucose medium (Fig. 5a). The reason could be that low concentration of glucose controls the growth and there by viscosity causing sufficient oxygen availability, caused less gluconic acid production and there by getting more conversion ratio as the more glucose might be channelizing to form BC, the product. Hence, low glucose medium was suitable for BC production. Semjonovs et al. (2017) reported K. rhaeticus P 1463 and K. hansenii B22 produced lower BC yield, higher gluconic acid with lower final pH and thereby lower conversion ratio at 40 g/L glucose in HS medium compare to 20 g/L glucose medium. Figure 5c showed sugar consumption was increased and final pH was decreased corresponding to the increased glucose concentration of the medium. This indicated high glucose utilization of K. europaeus 14148 and low pH owing to higher acid formation, obtained at 10% glucose medium with 0.54 g/L BC yield.

Fig. 5.

Fig. 5

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The effect of carbon sources and its concentrations on a conversion ratio, b yield of bacterial cellulose, c consumed sugar of Komagataeibacter europeaus

The effect of different fructose concentration on BC yield

Medium with 2% fructose produced the highest BC of 1.38 ± 0.12 g/L. It was observed that irrespective of the concentration of carbon source, fructose supported higher BC production as compared to glucose except at 1% concentration where glucose supported higher BC production as compared to fructose. The BC decreased with increasing fructose concentration in the HS medium (Fig. 5b). K. europaeus SGP37 incubated with fructose as carbon source in HS medium with supplement with ethanol as an energy generated source show improved BC yield (Dubey et al. 2017). Acetobacter xylinum subsp. Sucrofermentuns BPR3OOlA showed reduced BC yield at fructose concentration higher than 45 g/L in production medium and difficulty to maintain a sufficient oxygen supply due to the high viscosity (Naritomi et al. 1998). Semjonovs et al. (2017) reported K. rhaeticus P 1463 and K. hansenii B22 produced higher BC yield at 40 g/L fructose HS medium compared to 20 g/L fructose medium. The final pH of fermented broth was showing decreasing trend with increasing fructose concentration. pH was lowest for highest fructose concentration in the medium as compared to lower concentrations of fructose. Hence higher concentration of fructose must be supporting higher organic acid formation due to metabolism. It also showed higher fructose consumption and the BC yield show decreased trend towards 4% to 10% fructose concentration in the medium (Fig. 5c) and the reason could be similar to the one explained in Sect. 3.5.1.

The effect of various fruit extracts on BC yield

The yield of bacterial cellulose of K. europaeus 14148 was influenced by carbon sources. OE, PE, ME and BE produced 3.34 ± 0.04 g/L, 2.34 ± 0.04 g/L, 1.38 ± 0.09 g/L, 1.31 ± 0.05 g/L respectively (Fig. 4a). These fruits extracts could be the cheaper and sustainable carbon source for BC production leading to the economic feasibility of the bioprocess. There are reports on utilizing fruit juice, pulp and extracts as a carbon source for BC production by various acetic acid bacteria. As these fruits are an excellent source of fructose and it has been reflected from studied carbon source study that fructose supports BC production better than other carbon sources, is the probable reason for higher BC production. It has been reported that orange juice when used as carbon source in HS medium by Acetobacter xylinum NBRC 13,693 showed maximum BC yield compared to pineapple, apple and grape (Kurosumi et al. 2009). Semjonovs et al. (2017) reported K. rhaeticus P 1463 produced higher BC when apple juice was used as carbon source. This showed fruit extract is sustainable economic carbon source for BC production and can be exploited very well as it gave more than threefold higher production of BC. Papaya and orange are rich in many nutrients which could be the reason for supporting more BC production. Thus, the ingredients other than sugars in the fruit juices might exert influence on BC production and substrate inhibition (Kurosumi et al. 2009). Here the fruit juices must not be compared directly with defined sugars in current study as only reducing sugars were estimated and based on that fruit extracts were taken but sucrose presence could not be estimated in this study and the presence of sucrose may increase the total concentration of sugars in the medium. However; from this study, it was found that sucrose did not support BC production. Hence, the effect on BC production could be negligible.

The effect of higher OE and PE concentration on BC yield

The maximum BC yield 3.48 ± 0.16 g/L was obtained with PE at 4 g/100 mL in medium and 3.47 ± 0.05 g/L at OE at 4 g/100 mL in medium (Fig. 5b). The maximum conversion ratio was 0.23% observed at OE at 2 g/100 mL and 0.20% obtained from PE at 1 g/100 mL (Fig. 5a). This shows K. europaeus 14148 had higher sugar utilization efficiency on OE to synthesize BC compared to other fruit extracts employed. Gluconacetobacter persimmonis when incubated in a medium with natural carbon source from muskmelon with ingredients (exclude glucose) of HS medium enhanced BC yield was obtained than orange, watermelon and molasses (Hungund et al. 2013). Acetobacter xylinum NBRC 13,693 incubated in orange extract containing medium produced higher BC than pineapple medium (Kurosumi et al. 2009). This show low-cost fruit extract provided three times higher BC yield than reference HS medium resulting in decreasing the production cost with benefit of containing enhanced ingredients that might be lacking in HS medium. Thus, this result show that fruit extracts are better than defined carbon sources for BC production as it may contain fructose along with several natural ingredients like vitamins etc. The sugar contents of OE and PE containing medium were also analyzed by HPLC in addition to estimating reducing sugar content via DNS method. OE medium contained 6.92 g/L glucose and 11.92 g/L fructose and other sugars were not detected, which comes ~ 1.9% sugars similar to HS medium having 2% glucose; whereas PE contained 9.73 g/L glucose and 10.73 g/L fructose, having 2.46% sugars. OE contains more fructose and even having less total sugars when compared to PE gives similar yield of BC which indicates higher conversion ratio on the basis of sugars utilized. Our study with glucose and fructose proves that better yield is obtained with fructose which is reflected here also.

FTIR measurement of BC

The characteristic absorption bands were estimated by FTIR reflection mode on BC obtained from K. europeaus 14148 (Fig. 6a). The peak observed in the 3500 to 3200 cm-1 regions represent the OH stretching band from O–H hydroxyl group. The typical amorphous cellulose band was showed at 2922 cm-1. The intense peak at 1427 cm-1 is assigned to CH2 symmetric bending which was related to the degree of cellulose crystallinity. The absorption peaks at 1160 cm-1 and 898 cm-1 were assigned to C–O–C anti-symmetric bridge stretching of 1,4 β-D-glucoside (Lotfy et al. 2021) and an amorphous cellulose absorption band with C–O–C stretching at the β (1–4) linkage of glucose polymers (Reiniati et al. 2017) respectively. There was no significant difference of characteristic peaks observed on the spectrum obtained from glucose, fructose, OE and PE. The BC of K. xylinus produced in HS medium, sugarcane, coconut juice appeared to have no difference on FTIR spectrum (Jaroennonthasit et al. 2021). K. hansenii GA2016 obtained BC by citrus peel hydrolysates show the similar FTIR spectra to BC produced in the commercially available nutrients (Güzel and Akpınar 2019). This also supports current study.

Fig. 6.

Fig. 6

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Structure characteristic of BC a FTIR spectra, b X-ray diffraction (XRD) analysis, c Scanning electron microscope (SEM) analysis of BC produced in the medium with glucose, d Scanning electron microscope (SEM) analysis of BC produced in the medium with fructose, e Scanning electron microscope (SEM) analysis of BC produced in the medium with orange extract, f Scanning electron microscope (SEM) analysis of BC produced in the medium with papaya extract, g Thermogravimetric analysis of bacterial cellulose TGA curve and h DTA curve

X-ray diffraction and FTIR are used to estimate the physico-chemical characteristic of cellulose and BC. Through X-ray diffraction provides determined spectrum to identify the physical crystallinity by estimating the peak density of cellulose I and cellulose II. On the other hand, FTIR provide the specific spectrum of the organic function groups and finger print to prove the purity obtained from X-ray (Reiniati et al. 2017). The crystallinity ratio Cr1 and Cr2 converted from FTIR spectrum observed on cellulose was also applicate on BC. The cellulose crystallinity ratio of A1427/A898 (Cr1) and A1372/A2922 (Cr2) were used to present the crystallinity index (Lotfy et al. 2021; Arserim-Uçar et al. 2021). The Crystallinity ratio A1427/A898 reported to reflect the cellulose I fraction of cellulose structure (Oh et al. 2005). The crystallinity ratio converted from peak density showed OE obtained the highest Cr1 2.83 and Cr2 0.73 compared to reference glucose which was 0.66 and 0.28 respectively. Reiniati et al. (2017) reported Cr1 0.56 and Cr2 1.40 of BC obtained from K. xylinus incubated at stirred-tank bioreactor. Kuo et al. (2016) reported Cr1 0.78 and Cr2 0.30 of BC obtained from Gluconacetobacter xylinus incubated at static condition. Utilizing fruit extracts as carbon source in HS medium appeared interesting as it supported improved BC production by K. europaeus 14148 as well as improved BC crystallinity.

The XRD analysis on BC

The characteristic peaks of bacterial cellulose was observed at 2Θ 14.4 °, 16.6 ° and 22.6° which presents the cellulose Iα and amorphous region of BC (Fig. 6b). The crystallinity index of BC from PE and OE was 0.81 and 0.76 both higher than fructose (0.5) and glucose (0.2). These results support crystallinity obtained from FTIR spectrum, glucose and fructose both show lower crystallinity index than OE and PE. K. europaeus SGP37 cultivated at HS and sweet lime pulp waste extract obtained 77.4% and 89.6% XRD crystallinity (Dubey et al. 2018).

The SEM analysis on BC

The average nanofiber diameter of BC produced in glucose, fructose, OE and PE; observed from SEM was 85.21 nm, 53.25 nm, 66.9 nm and 68.00 nm respectively (Fig. 6c-f). 74.29 nm was observed at BC of K. hansenii GA2016 cultivated on HS medium and 47.92 nm ~ 66.32 nm on fruit peel (Güzel and Akpınar 2019). The fiber range was 20–100 nm and average 40-50 nm obtained from K. europaeus SGP37 cultivated on HS medium (Dubey et al. 2017).

The TGA analysis on BC

Figure 6g shows the thermal degradation curves of BC obtained from glucose, fructose, OE and PE containing medium. The TGA curve (Fig. 6g) decrease slowly before 200 °C and PE retained 91% whereas, 93% for fructose. The DTA curve (Fig. 6h) shows the higher weight loss from 310 to 360 °C due to the degradation of cellulose (He et al. 2020). The maximum decomposition temperature of BC which obtained from K. hansenii GA2016 cultivated at lemon, mandarin, orange and grapefruit were from 200 to 360 °C (Güzel and Akpınar 2019). BC produced by A. xylinum showed decomposition temperature as 339.2 °C and 336.4 °C for BC produced by K. rhaeticus TJPU03 (He et al. 2020).

Conclusions

Improved BC production was obtained by K. europaeus 14148 by optimizing physico-chemical and cultural parameters. Maximum BC production was achieved at pH 5.5, 30 °C, 1% inoculum size and 7 days incubation period under static conditions with PE (40 g/L) and OE (40 g/L). This study also indicated fructose as the suitable carbon sources to replace glucose in HS medium. This is the major reason behind better BC production with fruit extract as it contains more fructose than glucose. Therefore, with the use of fruit extracts by K. europaeus for BC production, more feasibility and sustainability is possible. The morphological analysis of BC via SEM, FTIR, XRD and TGA shows the BC produced with fruit extract is superior.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 13 kb) (13.6KB, docx)

Acknowledgements

All the authors are thankful to Taiwan MOST for funding support (Ref. No. 109-2222-E-992-002).

Author Contributions

For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used “Conceptualization, RRS, AKP and YST.; methodology, RRS and YST.; validation, CWC and CDD; formal analysis, YST.; investigation, RRS.; resources, CDD and CWC.; data curation, YST.; writing—original draft preparation, YST and AKP.; writing—review and editing, RRS and AKP.; visualization, RRS.; supervision, CDD.; project administration, CWC.; funding acquisition, CWC and CDD. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Taiwan, MOST, Grant number 109–2222-E-992–002.

Availability of data and material

Not Applicable.

Code availability

Not Applicable.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethics approval

Not Applicable.

Consent to participate

Not Applicable.

Consent for publication

Not Applicable.

Footnotes

Publisher's Note

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Contributor Information

Cheng-Di Dong, Email: cddong@nkust.edu.tw.

Reeta Rani Singhania, Email: reetasinghania@gmail.com.

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Supplementary Materials

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