Applied Microbiology and Biotechnology (2023) 107:3729–3744

https://doi.org/10.1007/s00253-023-12539-8

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Acetic acid bacteria in agro‑wastes: from cheese whey and olive mill
wastewater to cellulose
Marcello Brugnoli1 · Salvatore La China1 · Federico Lasagni1 · Flora Valeria Romeo2 · Andrea Pulvirenti1 ·
Maria Gullo1,3

Received: 17 January 2023 / Revised: 27 March 2023 / Accepted: 14 April 2023 / Published online: 28 April 2023
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
In this study, cheese whey and olive mill wastewater were investigated as potential feedstocks for producing bacterial
cellulose by using acetic acid bacteria strains. Organic acids and phenolic compounds composition were assayed by
high-pressure liquid chromatography. Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray
diffraction were used to investigate modifications in bacterial cellulose chemical and morphological structure. Cheese
whey was the most efficient feedstock in terms of bacterial cellulose yield (0.300 g of bacterial cellulose/gram of carbon
source consumed). Bacterial cellulose produced in olive mill wastewater presented a more well-defined network compared
to pellicles produced in cheese whey, resulting in a smaller fiber diameter in most cases. The analysis of bacterial cellulose
chemical structure highlighted the presence of different chemical bonds likely to be caused by the adsorption of olive
mill wastewater and cheese whey components. The crystallinity ranged from 45.72 to 80.82%. The acetic acid bacteria
strains used in this study were characterized by 16S rRNA gene sequencing, allowing to assign them to Komagataeibacter
xylinus and Komagataeibacter rhaeticus species. This study proves the suitability to perform sustainable bioprocesses for
producing bacterial cellulose, combining the valorisation of agro-wastes with microbial conversions carried out by acetic
acid bacteria. The high versatility in terms of yield, morphology, and fiber diameters obtained in cheese whey and olive
mill wastewater contribute to set up fundamental criteria for developing customized bioprocesses depending on the final
use of the bacterial cellulose.
Key points
• Cheese whey and olive mill wastewater can be used for bacterial cellulose production.
• Bacterial cellulose structure is dependent on the culture medium.
• Komagataeibacter strains support the agro-waste conversion in bacterial cellulose.

Keywords Biopolymer · Bacterial cellulose · Agro-wastes · Acetic acid bacteria · Komagataeibacter · Biotransformation

Introduction

Nowadays, the processing of foods generates large quantities

* Maria Gullo of by-products rich in organic load, which are increasing
maria.gullo@unimore.it
year by year. The disposal of food by-products poses envi-
1
Unimore Microbial Culture Collection Laboratory, ronmental problems, negatively impacting the ecosystem
Department of Life Sciences, University of Modena due to their severe phytotoxicity and antimicrobial proper-
and Reggio Emilia, Reggio Emilia, Italy ties. Although effective strategies have been studied and
2
Research Centre for Olive, Fruit and Citrus Crops (CREA), applied, the treatment or the utilization of food by-products
Acireale 95024, Italy is still very complex and costly (Maurizzi et al. 2022; Singh
3
National Biodiversity Future Center (NBFC), Palermo 90133, and Krishnaswamy 2022). However, the proper handling
Italy of food agro-wastes may support the mitigation of climate
change and contribute to meet circular economy principles
by producing biobased products.

13
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Among agro-food by-products, two of the most abundant
pollutants are cheese whey (CW), which is the liquid result-
ing from the precipitation and removal of milk casein during
cheese-making, and olive mill wastewater (OMW) that is
primarily composed of vegetation water, and water added
during olive oil production steps. Both by-products are char-
acterized by high chemical and biological oxygen demand,
making the management and disposal critical (Zotta et al.
2020; Foti et al. 2021).
However, CW and OMW are primary sources of highly
nutritional compounds. CW retains 55% of total milk
nutrients, containing lactose (45–50 g/L), soluble proteins
(6–8 g/L), lipids (4–5 g/L), mineral salts (8–10% of dried
extract), and traces of organic acids such as lactic acid and
citric acid (Zotta et al. 2020; Menchik et al. 2019). CW can
be used for animal feedstock, as a substrate for obtaining
lactic beverages, and to produce whey protein concentrate or
whey protein isolate.
OMW is rich in phenolic compounds, such as
hydroxytytosol or tyrosol. Indeed, most of the phenolic
fraction present in olives is found in OMW (Foti et al.
2021). The main reducing sugars are glucose and fructose,
even though the presence of xylose and arabinose has been
also reported (Eroglu et al. 2009). OMW organic acids
are mainly represented by malic and citric acid, whereas
potassium, calcium, and magnesium are the major minerals
(Eroglu et al. 2009; El-Abbassi et al. 2017). Several research
efforts have been done to extract phenolic compounds, fatty
acids, and sterols or to use OMW for producing protein-rich
microbial biomass or biosurfactants (Romeo et al. 2021;
Santi et al. 2008).
The production of biopolymers of microbial origin
is of great relevance in many fields; foods, medical, and
pharmaceutics are the main industrial sectors of interest
(Ullah et al. 2016; Gao et al. 2019; Anguluri et al. 2022b).
However, the production of biopolymers from microbial
conversions is affected by many constraints, such as low
yield and high production costs. To face these limita-
tions, improvements’ strategies include the exploitation
of selected and engineered microbial strains, coupled with
suitable cultivation media and conditions (Gao et al. 2019;
Gilbert et al. 2021; Vadanan et al. 2022). Among biopoly-
mers of microbial origin, bacterial cellulose (BC) produced
by acetic acid bacteria (AAB) is widely studied due to its
characteristics that make it suitable in several applications,
as a native or functionalized biomaterial. The role of AAB
in bioprocesses is mainly investigated for their ability to
perform oxidative fermentations, which lead to the pro-
duction of acetic acid, gluconic acid, and other oxidation
products (Cañete-Rodríguez et al. 2016). However, recently
several studies demonstrated the high versatility of selected
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Applied Microbiology and Biotechnology (2023) 107:3729–3744
AAB strains to produce not only vinegar but also BC, by
fermenting waste products or unconventional raw materi-
als (Cantadori et al. 2022; Di Donna et al. 2020; Luzón-
Quintana et al. 2021; Hussain et al. 2019; Vigentini et al.
2019). Among agro-wastes, both CW and OMW have been
also investigated as suitable feedstocks for producing BC
in different conditions (Carreira et al. 2011; Salari et al.
2019; Sar and Akbas 2022; Kolesovs et al. 2022; Elmekawy
et al. 2014).
Considering the effectiveness of specific AAB strains
in producing BC, previous works highlight that they are
included within Komagataeibacter xylinus and other closely
related species (Ross et al. 1991; Fan et al. 2016, 2016;
Gullo et al. 2017; Semjonovs et al. 2017). Many studies have
focused on the suitability of low-cost and waste substrates,
such as potato peels waste, waste beer yeast, citrus peel,
fermentation wastewater, molasses, or apple juice as
feedstock to produce BC (Lin et al. 2014; Fan et al. 2016;
Zhao et al. 2018; Abdelraof et al. 2019; Guzel and Akpinar
2019; Revin et al. 2021; Kolesovs et al. 2022).
In this study, CW and OMW were evaluated as
substrates to produce BC by AAB, as a contribution to
setting up sustainable routes for converting agro-wastes
into added-value products. Bacterial strains were assigned
to species K. xylinus and K. rhaeticus. A high BC yield
was obtained when CW was used as feedstock, whereas
OMW gave a low yield, but higher crystallinity index (CI).
Moreover, a positive correlation between organic acids and
BC yield was observed both in CW and OMW, whereas,
phenolic compounds negatively affected BC production
from OMW.
Scanning electron microscopy (SEM), X-ray diffractom-
etry (XRD), and Fourier transform-infrared spectroscopy
(FT-IR) were conducted to assess BC characteristics. Results
showed structural and chemical differences depending on
the feedstock used. Overall, the study provides the basis for
the industrial scale-up of tailored BC bioprocesses from
agro-wastes.
Materials and methods
Agri‑food by‑products
CW was supplied from a local cheese factory and OMW
was obtained by a three-phase olive oil extraction system
from the Council for Agricultural Research and Economics
(CREA) located in Acireale, Italy. Samples were stored
at − 20 °C until use. CW and OMW were centrifuged three
times at 6000 rpm at 4 °C for 20 min to separate solid
materials. Prior to centrifugation, CW was treated with
Applied Microbiology and Biotechnology (2023) 107:3729–3744
β-galactosidase from Aspergillus oryzae (Sigma-Aldrich,
Milan, Italy) in shaking condition for 30 min to hydrolyse
lactose into glucose and galactose.
Bacterial strains and culture conditions
AAB strains used in this study (Table 1) have been
previously isolated from black tea and green tea Kombucha
(Mamlouk 2012). Strains are deposited at the UMCC culture
collection (Unimore Microbial Culture Collection, Italy).
AAB strains were first rehydrated from − 80 °C storage
conditions, by cultivation in Hestrin-Schramm medium
(Hestrin and Schramm 1954) (20 g/L glucose anhydrous,
10 g/L yeast extract; 5 g/L polypeptone; 2.7 g/L disodium
phosphate anhydrous and 1.15 g/L citric acid monohydrate)
(HS) in static condition for 4 days. Then, 5% v/v of culture
was transferred in a 100-mL Erlenmeyer flask containing
30 mL of HS broth and cultivated for 4 days as the previous
step of the inoculation in CW and OMW. The cultivation
in food by-products was conducted in 100-mL Erlenmeyer
flasks with a working volume of 30 mL and an inoculum of
5% v/v, both for CW and OMW. Cultures were incubated
for 3, 6, and 9 days at 28 °C under static conditions. OMW
and CW were sterilized by vacuum filtration (0.2 µm, PES
Membrane, Nalgene Rapid-Flow, Thermo Fisher). For each
strain, 3 biological replicates were performed. HS and all
the materials were sterilized by autoclaving at 121 °C, 1 atm
for 15 min.
Harvesting, purification, and quantification
of bacterial cellulose
BC was collected at the end of each time point and washed
following Gullo et al. (2019) procedure with minor
modifications. Briefly, BC pellicles were washed with
deionized water four times and then treated with NaOH
1 M and incubated at 80 °C for 30 min. In the end, BC was
washed with deionized water and dried in an oven at 20 °C
up to complete dehydration. The weighting of the dried BC
films was performed using an analytical balance (Gibertini
E42S, Italy). BC yield was expressed as gram BC produced
per gram of carbon source consumed (g BC/g CCS).
Table 1  Phenotypic traits of Strain
acetic acid bacteria used in this
study and their isolation source. K1A18 (UMCC 3067)
Adapted from Mamlouk, (2012)
K1G23 (UMCC 3068)
and Gullo et al. (2017)
K2A8 (UMCC 3069)
K2G14 (UMCC 3070)
K2G30 (UMCC 2756)
K2G39 (UMCC 2970)
3731
Glucose, fructose, lactose, pH, and total phenolic
compounds assay
D-glucose and D-fructose were determined using the enzy-
matic kit K-SUFRG, whereas lactose using the enzymatic kit
K-LACSU (Megazyme Ltd. Bray, Ireland). Analyzes were
performed following the manufacturer’s instructions. The
concentrations were calculated using MEGA-CALC. Data
were expressed as grams per liter. The pH was measured by
using XSPH 80 PRO STIRRER (Securlab, Italy). Total phe-
nolic compounds (TPC) were determined by Folin-Ciacolteu
(FC) method (Singleton et al. 1999) and expressed as mil-
ligrams of gallic acid equivalent per liter (mg GAE/L).
Analytical determination by high‑pressure liquid
chromatography
Qualitative and quantitative analyzes of organic acids and
phenolic compounds were carried out by injecting 20 µL
into a Jasco LC-Net II/ADC apparatus equipped with a Jasco
pump PU-2080 Plus, a UV detector set at 210 nm for organic
acids determination (Jasco UV-2070 Plus) and PDA detec-
tor for phenolic compounds determination (MD-2010 Plus).
Samples for organic acid determination were diluted with
distilled water, filtered through 0.45 μm PTFE membranes,
and transferred into glass vials for injection. An isocratic
separation of molecules was performed using a Bio-Rad
Aminex HPX-87H column (300 × 7.8 mm) heated at 40 °C
with an Eldex CH-150 oven. The mobile phase was com-
posed of 0.005 N sulfuric acid and 5% of acetonitrile using
a flow of 0.6 mL/min.
To extract phenolic compounds, samples were mixed with
a water/methanol/formic acid (28:70:2, v/v/v) solution in a
ratio of 1:2 (v/v) and incubated for 30 min at 37 °C in a rotat-
ing condition. After incubation, the mixtures were centrifuged
(5000 rpm, 20 min, 4 °C) and the supernatant was filtered
through 0.45 μm PTFE membranes. Molecule separation
occurred through a P ­ urospherR star RP-18 endcapped (5 µm
particle size). Chromatographic conditions were set based on
Aldana-Mejía et al. (2021) with minor modifications. The elu-
ent system was composed of two mobile phases: (A) ­H2O/
formic acid (99.9:0.1, v/v) and (B) acetonitrile/formic acid
Colony morphology Growth on broth Isolation source
Green cellulosic Cellulose layer Black tea Kombucha
Dark cellulose Cellulose layer Black tea Kombucha
Green cellulosic small Cellulose layer Green tea Kombucha
White cellulosic Cellulose layer/deposit Green tea Kombucha
Cellulosic Cellulose layer/deposit Green tea Kombucha
Cellulosic Cellulose Green tea Kombucha
13
3732
(99.9:0.1, v/v). The elution gradient was composed as follows:
the gradient phase started at 1% B for 1 min, then linearly
ramped up to 40% B in 13 min, reached 99% B in the next
13 min, and kept for 5 min to wash the column before return-
ing to 1% B. The analysis was performed in the range of 240,
280, and 360 nm. Quantitative analysis was performed using
calibration curves. Four increasing solutions were prepared
for each standard to identify sample peaks through standard
ID peaks. Calibration standards were determined for each acid
(acetic acid, citric acid, gluconic acid, lactic acid, malic acid,
quinic acid, and succinic acid) and phenolic compound (cat-
echin, chlorogenic acid, cinnamic acid, gallic acid, hydroxy-
tyrosol, p-coumaric acid, and rutin trihydrate) observing a
straight-line relationship between response and concentration.
The regression coefficient was over 0.997 for each standard.
Peak identifications were conducted using the functions pro-
vided by ChromNAV software. HPLC organic acids, catechin,
cinnamic acid, p-coumaric acid, gallic acid, and rutin trihy-
drate HPLC standard were purchased from Sigma-Aldrich
(Milan, Italy). Hydroxytyrosol HPLC standard was purchased
from Extrasynthese (Genay, France).
Scanning electron microscopy
Images of BC samples’ microstructure were obtained using
SEM. Samples were cut (1 × 1 ­cm2), coated with a thin layer
of gold (Au) and mounted on a stainless-steel stub with
double-sided tape. SEM analyzes were carried out using a
field emission Nova NanoSEM 450 (FEI Company-Bruker
corporation, Germany) operating at 10 kV in high vacuum
conditions.
Fourier transform infrared spectroscopy
FFT-IR spectra were recorded using a Vertex 70 (Bruker,
Germany) spectrometer equipped with an attenuated total
reflection (ATR) Golden Gate diamond. All spectra were
recorded in adsorption mode in a wavenumber range of
4000–400 ­cm−1 with an accumulation of 32 scans at 4 ­cm−1.
XRD pattern and crystallinity
To determine the crystalline structure and CI of BC lay-
ers, the XRD patterns were obtained using a Panalytical
X’Pert PRO (Malvern Panalytical, Malvern, UK) diffrac-
tometer with CuKα radiation source (λ = 1.54 Å) at a volt-
age of 40 kV and a filament emission of 40 mA. Samples’
diffracted radiation intensity was measured between 10 and
40° (2θ) with ramping at 1°/min using a zero-background
holder to avoid the detection of any peak not related to the
samples. The CI was calculated using the following formula:
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Applied Microbiology and Biotechnology (2023) 107:3729–3744
CI(%) = sc∕st ∗ 100 (1)
where sc is the sum of diffraction peaks area and it is the
sum of total area.
DNA extraction, 16S rRNA gene sequencing,
and analysis
Genomic DNA extraction was performed on HS medium
liquid cultures previously incubated for 5 days at 28 °C.
Flasks were shaken to free the cells embedded inside the
BC network, then 8 mL of cell cultures were centrifuged
at 10,000 g, 4 °C for 10 min. Genomic DNA from AAB
cultures was extracted by sodium dodecylsulfate (SDS)
proteinase-cethyltrimethyl ammonium bromide (CTAB)
treatment as previously reported (Gullo et al. 2006). gDNA
concentration and quality were evaluated by NanoDrop™
1000 Spectrophotometer (Thermo Fisher Scientific, USA)
and by gel electrophoreses on 1% gel agarose in 1X TBE
buffer. Band sizes were determined using a 100 bp plus DNA
ladder (Invitrogen, Carlsbad, CA, USA). PCR reactions were
performed using primers 27F (AGR​GTT​YGATYMTGG​
CTC​AG) and 1490R (TAC​GGY​TAC​CTT​GTT​ACG​ACTT).
Amplified products were purified using the kit DNA Clean
& Concentrator™-5 (ZymoResearch) and automated
sequenced (Sanger sequencing technique, BIOFAB).
Five Ab1 files were processed using CodonCode aligner
for end-trimming according to primer length and base
quality control. High-quality sequences were aligned against
the 16S rRNA database using the blast algorithm. Identified
bacterial species were used to download the type strains 16S
rRNA sequence from the LPNS database accessed on 12
December 2022 (Meier-Kolthoff et al. 2022). Both reference
and sequenced 16S sequences were aligned all-vs-all using
Muscle v5 (Madeira et al. 2022). The multiple alignments
were imported into MegaX v10.2, and the sequences were
trimmed to the same length. The phylogeny was inferred
by computing a neighbor-joining phylogenetic tree based
on 1000 replicates. The Tamura-nei DNA evolutionary
model was applied using a discrete Gamma distribution to
model evolutionary rate differences among the sequence
site (Tamura and Nei 1993). The obtained newick file was
visualized using the interactive Tree of Life (ITOL) v6.6
(Letunic and Bork 2019). The 16S rRNA sequences were
submitted to GenBank/EMBL/DDBJ and published under
the accession numbers: OQ248684, OQ248685, OQ248686,
OQ248687, and OQ248688.
Data statistical analysis
Experimental data were analyzed using R v 4.2.2 (R Core
Team 2022) at a significance level of p = 0.05 and reported
as the average of the triplicate ± standard deviation. The
Applied Microbiology and Biotechnology (2023) 107:3729–3744
statistical significance was determined using one-way
ANOVA and Tukey post hoc test was used to determine sta-
tistical differences among samples. The Pearson correlation
index was calculated using the Hmisc v4.4–2 R package
(Harrell 2022). The correlation plot was obtained using the
Corrplot v 0.92 package (Wei and Simko 2021).
Results
Bacterial cellulose production in cheese whey
and olive mill wastewater
The production of BC is strictly influenced by several fac-
tors such as the bacterial strain, pH, temperature, the cul-
ture medium and especially, the type and amount of carbon
source provided (Son et al. 2001; Gullo et al. 2017). In this
study, the potential of CW and OMW as feedstocks for BC
production was evaluated by cultivating six AAB strains in
both by-products, for 9 days. Waste liquids were used with-
out any addition of carbon source and HS broth was used as
control. As shown in Fig. 1, the yield in CW was consider-
ably higher than the one reached in OMW, independently
of the bacterial strain. Indeed, BC yield in OMW never
reached 0.100 g/g CCS, ranging between 0.023 (K1A18)
and 0.067 (K2G14) g/g CCS. On the other hand, BC yield
in CW obtained from K2G14, K2G30, and K2G39 exceeded
the yield obtained in the HS medium. The highest BC yields
were achieved culturing K2G14, K2G30, and K2G39 in CW,
reaching 0.200 ± 0.004, 0.240 ± 0.004, and 0.300 ± 0.007 g
BC/g CCS, respectively.
Suitability of cheese whey and olive mill wastewater
to produce bacterial cellulose by acetic acid bacteria
Results of the physico-chemical characterization of CW and
OMW are reported in Tables 2 and 3. CW had an initial
lactose concentration of 40.12 g/L, which was almost com-
pletely hydrolysed after a treatment with β-galactosidase.
Furthermore, no supplementary carbon sources nor nitrogen
sources were added to CW and OMW. Glucose was found
to be the main sugar, with CW having a significant higher
amount compared to OMW. Both by-products had similar
pH values and contained different concentrations of organic
acids, such as lactic acid, citric acid, and succinic acid. In
OMW, a significant amount of total phenolic compounds
(3562 mg GAE/L) was found, which is consistent with the
values reported in the literature (Abbattista et al. 2021; Foti
et al. 2021). Among the analyzed phenolic compounds,
hydroxytyrosol was the major component of phenolic frac-
tion in OMW. Nevertheless, phenolic compounds with high
antimicrobial activity, such as cinnamic acid, were present
at high concentrations, whereas phenolic compounds, with
3733
low antimicrobial activity, were not determined (including
tyrosol), even though is reportedly the second main phe-
nolic compound in OMW (Leouifoudi et al. 2015; Foti et al.
2021). On the other hand, in CW a low concentration of phe-
nolic compounds (82 mg GAE/L) was found, with cinnamic
acid being the only detected and quantified (13.81 mg/L).
However, in the case of CW, the total phenolic value may be
overestimated because of some compounds available in this
kind of treated substrate (free aminoacids, Maillard’s reac-
tion derivative molecules, polymers, etc.) that can react with
FC reagent. Indeed, the HPLC analysis identified a single
phenolic compound.
A general reduction of pH values, sugars, organic acids,
and TPC content was observed both in CW and OMW after
9 days of fermentation by each AAB strain. TPC in OMW
significantly decreased although the concentration of most
of the phenolic compounds detected, such as hydroxytyrosol
or gallic acid, increased. Both in OMW and CW, cinnamic
acid resulted to be the only phenolic compound whose con-
centration decreased. In order to understand the correlation
between the production of BC and the presence of organic
acids and phenolic compounds, Spearman’s correlation
index was calculated. The correlation plot is represented
in Fig. 2. A moderate positive contribution was noted for
the majority of organic acids, except for acetic and succinic
acids (R2 ≥ 0.6; p ≤ 0.05), while cinnamic acid, quinic acid,
and hydroxytyrosol had a strong negative effect on BC pro-
duction (R2 ≤  − 0.6; p ≤ 0.05). Regarding the phenolic com-
pounds, hydroxytyrosol, p-coumaric acid, chlorogenic acid,
and cinnamic acid, they all showed a negative correlation to
BC production (R2 ≥ 0.6; p ≤ 0.05). Nevertheless, rutin trihy-
drate, catechin, and gallic acid, even though moderately, also
had a negative effect on BC production. Likewise, gluconic
acid had a similar correlation to organic acids and phenolic
compounds as BC.
Morphological, chemical, and structural
characterization of bacterial cellulose
SEM images of BC obtained cultivating bacterial strains
in CW and OMW showed the typical individual ribbons
structure. Figure 3 presents the difference in surface mor-
phology among BC pellicles. Samples produced in OMW
showed a more well-defined network compared to that pro-
duced in CW, which resulted in smaller fibers diameter
in most cases (Fig. 3). BC-CW samples were character-
ized by thicker fibers ranging from 39.64 nm in K1A18 to
68.67 nm in K1G23 samples. However, K2G14 produced
the BC with the thinnest fibers (35.00 nm as width aver-
age) in CW. On the other hand, the thickest fiber diameter
was recorded in BC-OMW produced by K2G39 with an
average width of 68.86 nm. The chemical structure of BC
samples was evaluated through ATR-FTIR analysis. FT-IR
13
3734 Applied Microbiology and Biotechnology (2023) 107:3729–3744

Fig. 1  Quantification of bacte-

rial cellulose (BC) yield (g
of BC/g of consumed carbon
source) in cheese-whey (CW),
Hestrin-Schramm medium
(HS), and olive mill wastewater
(OMW) by strains K1A18,
K1G23, K2A8, K2G14, K2G30,
and K2G39. Bar plots indicate
the average BC yield by three
replicates ± standard deviation.
Significant difference among
BC yield is shown by different
letters (p ≤ 0.05)

spectra of BC pellicles produced in HS, CW, and OMW
are shown in Fig. 4. BC characteristic peaks have been
observed in all the pellicles, attributable to O–H stretch-
ing vibrations at around 3340 ­cm−1, to ­CH2 asymmetric
stretching vibration at around 2895 ­cm−1, to O–H deforma-
tion vibration at around 1640 ­cm−1, to ­CH2 stretching and
bending vibration at around 1423 ­cm−1 and 1315 ­cm−1, to
C–O–C stretching vibrations of β-(1–4) glycoside bonds
at around 1154 ­cm−1, and to C–O–H stretching vibration
at around 1040 ­cm−1 or generally to C–O bonds (Castro
et al. 2011; Kiziltas et al. 2015). Results highlighted cel-
lulose I pattern and the purity of BC (Algar et al. 2014;
Gullo et al. 2017; Barbi et al. 2021; Ciecholewska-Juśko
et al. 2021). However, other peaks, attributable to differ-
ent molecules or chemical bonds, were present in most
spectra recorded from BC obtained from CW and OMW.
The peaks at around 2920 ­cm−1 and 2850 ­cm−1 could be
related to the C
­ H2 asymmetric and symmetric stretching

13
Applied Microbiology and Biotechnology (2023) 107:3729–3744 3735

Table 2  Physico-chemical composition of olive mill wastewater (OMW) before and after AAB fermentation
OMW K1A18 K1G23 K2A8 K2G14 K2G30 K2G39

pH 4.85a ± 0.08 4.19bc ± 0.03 4.23bcd ± 0.09 4.26bc ± 0.07 4.05 cd ± 0.09 4.00d ± 0.05 4.30b ± 0.10
a c b bc c d
Glucose (g/L) 15.960 ± 0.369 0.998 ± 0.105 5.638 ± 0.451 1.509 ± 0.343 1.143 ± 0.181 0.067 ± 0.004 2.157b ± 0.187
a bc c bc b bc
Fructose (g/L) 7.873 ± 0.387 4.989 ± 0.510 3.847 ± 0.500 5.102 ± 0.173 5.660 ± 0.424 4.709 ± 0.494 5.044bc ± 0.315
Total phenolic 3562.46a ± 140.47 2405.33c ± 90.05 2061.01d ± 45.99 2302.90c ± 51.91 2061.52d ± 105.66 2980.40a ± 80.08 2542.22b ± 68.33
compounds
(mg GAE/L)
Organic acids (mg/L)
Acetic acid 558.68a ± 31.06 135.51b ± 2.73 93.28c ± 1.71 71.20 cd ± 1.73 59.01d ± 2.69 133.11b ± 1.86 133.03b ± 1.99
Citric acid 5170.66a ± 69.51 934.67 cd ± 95.46 804.01d ± 80.57 962.67bc ± 141.71 889.47d ± 17.25 1324.11b ± 73.17 1130.16bc ± 30.98
b c b c
Gluconic acid - 3523.94 ± 161.93 3086.54 ± 96.29 3593.22 ± 257.73 2829.77 ± 82.55 40,343.64 ± 144.82 3651.20ab ± 116.63
a

a bc bc c bc
Lactic acid 539.46 ± 62.01 110.47 ± 10.98 107.35 ± 3.00 99.06 ± 6.46 113.10 ± 3.77 173.69b ± 7.71 143.32bc ± 4.19
a b c d e b
Malic acid 3683.24 ± 28.59 686.10 ± 20.95 604.59 ± 11.240 551.16 ± 14.92 378.01 ± 7.84 681.65 ± 21.38 522.50d ± 12.53
e d b a d bc
Quinic acid 447.65 ± 20.16 761.38 ± 25.44 937.91 ± 27.23 1185.64 ± 39.56 722.99 ± 37.33 864.53 ± 26.77 783.91 cd ± 34.44
Succinic acid 2266.87a ± 72.73 369.95c ± 35.08 334.78c ± 22.57 368.42c ± 68.63 776.17b ± 18.16 431.17c ± 46.07 448.61c ± 30.13
Phenolic compounds (mg/L)
Catechin 203.03d ± 4.74 416.09a ± 20.52 415.30a ± 14.49 384.41b ± 4.10 383.84b ± 4.43 314.68c ± 3.64 404.50ab ± 7.28
a a a a a c
Chlorogenic acid 75.27 ± 1.53 73.26 ± 1.15 73.19 ± 1.61 73.18 ± 0.94 75.68 ± 0.67 51.75 ± 0.77 67.50b ± 1.30
a c c b b b
Cinnamic acid 293.91 ± 5.50 73.23 ± 2.28 81.01 ± 3.89 94.24 ± 2.52 97.62 ± 1.29 95.35 ± 2.80 72.97c ± 3.02
e a b c ab d
Gallic acid 137.10 ± 1.11 280.91 ± 1.15 269.19 ± 1.27 249.59 ± 7.13 280.33 ± 7.73 222.53 ± 1.99 273.95ab ± 0.82
Hydroxytyrosol 353.80d ± 11.77 432.43b ± 6.72 416.43bc ± 14.93 411.18bc ± 4.52 461.26a ± 9.80 405.11c ± 4.93 411.54bc ± 1.71
p-coumaric acid 36.06bc ± 0.97 39.36a ± 1.08 37.92ab ± 0.81 36.17bcd ± 1.00 36.76bc ± 0.51 34.52d ± 0.73 35.86 cd ± 0.33
Rutin trihydrate 104.798a ± 1.625 213.778b ± 4.927 204.866b ± 4.910 188.557c ± 4.795 204.544b ± 6.562 144.076d ± 3.761 203.757b ± 6.932

Data are expressed as means ± standard deviations. Different letters indicate statistical differences within the same row at p ≤ 0.05

of lipids (Christy et al. 2001; Molina-Ramirez et al. 2017).
The presence of absorbance bands at 1743 ­c m −1 BC-
OMW and 1725 ­cm−1 in BC-CW could be attributable to
carbonyl bonds (C = O) derived from an esterification of
BC’s hydroxyl groups (O–H) with phenolic compounds or
organic acids present in the by-products (Lee and Bismark
2012; Molina-Ramirez et al. 2017; Sar and Akbas 2022).
Bands at around 1575 ­cm−1 and 1540 ­cm−1 correspond to
secondary amide N–H bending and C–N stretching (Rouab-
hia et al. 2014; Molina-Ramirez et al. 2017). XRD patterns
were acquired to evaluate the crystalline structure, and the
crystallinity of BC samples produced in HS medium as
well as any occurred changes once the strains were culti-
vated in OMW and CW. The diffraction diagrams (Fig. 5)
showed three peaks at 2-θ at around 14.4°, 16.8°, and 22.5°
in all the samples corresponding to the crystallographic
planes of (100), (010), and (110) (Guzel and Akpinar 2020;
Revin et al. 2021). These diffraction planes correspond to
the crystalline native cellulose I (Moukamnerd et al. 2020;
Ye et al. 2019).
The use of both by-products reduced the CI on BC com-
pared to the HS medium (Table 4), thus increasing the
amorphous structure, as a result of producing BC in CW
and OMW, confirming the results observed in Fig. 5.
Acetic acid bacteria species assignment by 16S rRNA
gene sequencing
In order to infer the phylogeny of the tested AAB, 9
sequences of Komagataeibacter species were used,
covering most of the species described as BC producers
in the Komagataeibacter genus. The 16S sequence of the
species A. pasteurianus LMG 1262 and G. frateurii IFO
3264 was used as outgroups. After the overall alignment
and sequence trimming, the total length resulted to be
1328 bp. The neighbor-joining phylogenetic tree (Fig. 6)
reports the bootstrap value ≥ 50 to define the accuracy of
branch prediction. A distance matrix was computed (data
not shown), showing an overall dissimilarity percentage of
1.72%. According to the sequence clusterisation, two main
clusters could be identified. The first clade includes the
recently reassigned species Novatecimonas hansenii NCIB
­8746 T (Brandão et al. 2022), the subclade K. rhaeticus
JCM ­17122 T and the K2G14 strain. The subclade was
supported by a bootstrap value of 99%. Between K2G14 and
K. rhaeticus JCM17122, the similarity percentage resulted
to be 100%, attributing K2G14 to the K. rhaeticus species.
The second clade includes the remaining Komagataeibacter
species, highlighting the issue previously described in
the utilization of the 16S rRNA as target sequence for

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3736 Applied Microbiology and Biotechnology (2023) 107:3729–3744

Table 3  Physico-chemical composition of cheese whey (CW) and consequent variation before and after AAB fermentation
CW K1A18 K1G23 K2A8 K2G14 K2G30 K2G39
a bcd bc b cd cd
pH 4.74 ± 0.03 3.69 ± 0.08 3.87 ± 0.06 3.91 ± 0.14 3.61 ± 0.06 3.60 ± 0.18 3.47d ± 0.03
a b b b b b
Glucose (g/L) 35.369 ± 1.058 0.309 ± 0.007 0.514 ± 0.019 0.106 ± 0.012 0.148 ± 0.006 0.351 ± 0.004 0.309b ± 0.007
Fructose (g/L) - - - - - - -
Total phenolic 81.88a ± 4.05 51.36b ± 5.05 55.51b ± 4.81 49.48c ± 2.13 35.48d ± 4.60 40.08d ± 1.46 51.36bc ± 5.05
compounds
(mg GAE/L)
Organic acids (mg/L)
Acetic acid 147.82a ± 7.76 97.96c ± 5.57 114.31b ± 2.38 121.62b ± 8.19 38.13e ± 0.72 76.64d ± 0.55 23.67f ± 2.21
a b c b d b
Citric acid 3844.90 ± 231.16 2338.43 ± 247.11 1346.25 ± 172.35 2450.15 ± 208.22 620.85 ± 8.15 2271.12 ± 19.01 1999.32b ± 159.15
Gluconic acid - 18,501.76 ± 81.56 14,410.02 ± 180.64 17,294.77 ± 168.58 18,770.08 ± 350.78 21,258.34 ± 524.58 20,560.02ab ± 755.23
bc d c bc a

a
Lactic acid 4810.74 ± 72.13 954.69c ± 42.67 399.53e ± 26.23 1086.66b ± 58.12 213.584f ± 10.78 834.07d ± 7.87 177.17f ± 7.51
Malic acid 389.82a ± 22.95 111.97b ± 11.04 84.36b ± 11.03 100.16b ± 0.62 94.26b ± 6.69 102.10b ± 7.31 107.65b ± 19.80
Quinic acid - - - - - - -
Succinic acid 329.80a ± 5.81 249.15b ± 18.50 283.22ab ± 41.11 286.28ab ± 42.41 303.44ab ± 49.831 278.45ab ± 17.46 257.30b ± 17.48
Phenolic compounds
(mg/L)
Catechin - - - - - - -
Chlorogenic acid - - - - - - -
Cinnamic acid 13.81a ± 0.82 4.00b ± 0.11 4.74b ± 0.20 3.86b ± 0.23 5.99b ± 0.19 2.37b ± 0.12 4.00b ± 0.12
Gallic acid - - - - - - -
Hydroxytyrosol - - - - - - -
p-coumaric acid - - - - - - -
Rutin trihydrate - - - - - - -
Data are expressed as means ± standard deviations. Different letters indicate statistical differences within the same row at p ≤ 0.05

Fig. 2  Correlation plot among

bacterial cellulose production,
organic acids and phenolic acids
present in cheese whey and
olive mill wastewater, obtained
using Spearman’s correlation
index. Correlation was con-
sidered positive and negative
based on R2 ≥ 0.6 and ≤  − 0.6,
respectively

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Applied Microbiology and Biotechnology (2023) 107:3729–3744
Fig. 3  Scanning electron microscopic images of the surface of the
bacterial cellulose produced by K1A18 (a–d), K1G23 (b–e), K2A8
(c–f), K2G14 (g–j), K2G30 (h–k), and K2G39 (i–l). The insets show
the fiber diameter distribution in each sample synthesized in cheese
Komagataeibacter phylogeny inferring (Cleenwerck and De
Vos 2008; Papalexandratou et al. 2009). This clade is highly
branched with a dissimilarity among the strain ranging
between 0.3 and 0.9%, highlighting a low divergence among
the Komagataeibacter species. The isolates characterized
in this study were grouped with the type-strain K. xylinus
3737
whey (blue) and olive mill wastewater (green). “μ” indicates the aver-
age diameter (nm) of 100 randomly chosen fibers of bacterial cellu-
lose microfibrils
NCIB ­11664 T and the already described K. xylinus K2G30
(Gullo et al. 2019), allowing to confidently attribute those
strains to K. xylinus species. As previously described (La
China et al. 2021), the strains characterized in this study
were obtained from Kombucha tea, in which member of
Komagataeibacter genus are highly represented.
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3738 Applied Microbiology and Biotechnology (2023) 107:3729–3744

Fig. 4  FT-IR spectra of bacterial cellulose (BC) pellicles produced Shramm (HS) in red, olive mill wastewater (OMW) in green, and
by K1A18 (a), K1G23 (b), K2A8 (c), K2G14 (d), K2G30 (e), and cheese whey (CW) in blue
K2G39 (f). Each color represents a different culture media: Hestrin-

Fig. 5  XRD patterns of bacterial cellulose pellicles produced by Shramm (HS) in red, olive mill wastewater (OMW) in green, and
K1A18 (a), K1G23 (b), K2A8 (c), K2G14 (d), K2G30 (e), and cheese whey (CW) in blue
K2G39 (f). Each color represents a different culture media: Hestrin-

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Applied Microbiology and Biotechnology (2023) 107:3729–3744 3739

Table 4  Crystallinity Index Crystallinity index (%) characteristics of substrates used as culture media (Kolesovs
of bacterial cellulose pellicles and Semjonovs 2020; Brugnoli et al. 2021); however, no
obtained in Hestrin-Schramm Strains HS OMW CW
medium (HS), olive mill
adverse metabolic effects referable to preinoculum condi-
wastewater (OMW) and cheese K1A18 94.77 80.82 61.10 tions were observed. The main differences were related to
whey (CW) K1G23 87.69 72.82 55.12 the yield and the characteristics of BC obtained.
K2A8 78.95 58.48 53.61 OMW showed lower amount of BC compared to
K2G14 94.26 56.75 45.72 that produced in CW. On the other hand, AAB strains
K2G30 84.74 71.32 47.53 highlighted high versatility allowing to grow and produce
K2G39 76.02 58.02 53.55 BC using different carbon sources, as already pointed out
from previous studies on K2G30 strain (Gullo et al. 2019;
Anguluri et al. 2022a). In addition, when strains were
Discussion cultivated in CW, a high BC yield was obtained, overcoming,
in three cases (K2G14, K2G30, and K2G39), the yield
In the present study, the production and the characteristics reached in HS. Tsouko et al. (2015) obtained low BC
of BC obtained from six Komagataeibacter sp. strains yield inoculating K. sucrofermentans DSM ­15973 T strain
cultivated in CW and OMW were evaluated. To the aim in CW using lactose as the sole carbon source. Likewise,
to test the technological robustness of the strains in terms Thompson and Hamilton (2001) and Carreira et al. (2011)
of adaptation to new substrates, liquid cultures from HS reported CW as a feedstock which does not support high
medium were directly transferred in CW and OMW and BC yield. However, given the lack of the gene that encodes
BC production assayed. These strains have been previously β-galactosidase in AAB, through an enzymatic hydrolysis
isolated from kombucha tea (Mamlouk 2012) and studied of CW lactose into glucose and galactose, a high yield of
respecting their phenotypic, molecular, and technological BC could be reached, as reported in the present study and in
aspects relevant for BC production (Gullo et al. 2019; La Salari et al. (2019).
China et al. 2020, 2021). The composition of the substrate is also a key factor since
To date, few publications have investigated BC produc- the presence of nitrogen sources, organic acids or other
tion from OMW (Elmekawy et al. 2014; Sar and Akbas molecules deeply influences BC yield (Bodin et al. 2007).
2022) and on CW (Thompson and Hamilton 2001; Car- Indeed, even though glucose is known to be the key carbon
reira et al. 2011; Tsouko et al. 2015; Lappa et al. 2019). source for cells’ metabolic activity including BC production,
According to our knowledge, this is the first study where previous studies reported that, when organic acids such as
organic acids and phenolic compounds are correlated with lactic or acetic acid are present, bacteria tend to co-utilize
BC produced from CW and OMW, respectively. Cultivating both sources, having a positive effect on BC biosynthesis
strains in CW and OMW gave different outcomes, which (Ross et al. 1991; Revin et al. 2018; Wang et al. 2019). Lac-
could be associated with AAB strains high variability and tic acid, through the oxidation in pyruvic acid, acts as an

Fig. 6  Neighbor-joining phylogenetic tree representing the phy- Tamura model was used to make the tree. Node numbers indicate
logenetic distances of acetic acid bacteria tested and the type strain bootstrap values obtained using 1000 replicates
of nine Komagataeibacter species described as BC producers. The

13
3740
accelerator of tricarboxylic (TCA) cycle, stimulating cells
growth at an early stage of culture development (Son et al.
2001; Liu et al. 2016, 2017b). Acetic acid can be metabo-
lized through the TCA cycle once transformed in acetyl-CoA
or used in the gluconeogenesis pathway increasing glucose
availability (Revin et al. 2018). Organic acids located in
the pathway of the TCA cycle, such as succinic acid, citric
acid, and malic acid, are also beneficial for BC production
(Lu et al. 2015; Revin et al. 2018). Culturing Komagataei-
bacter strains in CW and OMW led to an almost complete
consumption of acetic acid, lactic acid, succinic acid, citric
acid, and malic acid in 9 days of cultivation (Tables 2 and
3), suggesting that they were metabolized, as previously
stated. However, there is also evidence that some organic
acids negatively affect BC production (Lu et al. 2015). The
presence of oxalic and tartaric acids could retard or inhibit
BC production. Gluconic acid, which is produced by AAB
from glucose oxidation, contributes to lower BC yield due to
a decrease in the amount of available glucose and to a reduc-
tion of the pH value (Anguluri et al. 2022a). In this study,
among the tested organic acids, the only significant nega-
tive correlation with BC was found in cinnamic and quinic
acids, associable to the antimicrobial effect that chlorogenic
acid exhibits in synergy with other components (Kabir et al.
2014). However, besides chlorogenic acid, other phenolic
compounds, such as hydroxytyrosol, have a high antimicro-
bial activity against gram + and gram − bacteria, which slow
down bacteria’s growth (Leouifoudi et al. 2014; Foti et al.
2021; Çelik et al. 2021). Along cultivation time, the phenolic
compound’s concentration increased (Tables 2 and 3). The
evolution could be attributable to hydrolysis reactions of
oligomeric phenolic, which are bonded through ester and
glucoside linkages (Feki et al. 2006). The presence of phe-
nolic compounds having strong antimicrobial activity and
high total phenolic content could suppress the BCs produc-
tion, despite the presence of sugars or organic acids (Guzel
and Akpinar 2019). This could explain the reduced BC’s
yield in OMW compared to that obtained in CW. However,
despite the negative effect of phenolic compounds, there is
evidence of the in-situ and ex-situ functionalisation of BC
with such compounds (Le Bourvellec and Renard 2012;
Phan et al. 2015, 2016). Molecule absorption in the BC net-
work is enabled by its unique structure, its porous nature and
the numerous OH groups which potentially interact with a
variety of functional groups, allowing the functionalisation
of BC (Manan et al. 2022). Indeed, authors suggested that
BC spontaneously binds to phenolic compounds through
non-covalent interactions, including hydrogen bonding and
hydrophobic forces (Le Bourvellec and Renard 2012; Phan
et al. 2015; Liu et al. 2017a). BC-phenolic compound asso-
ciation seems to be influenced by various factors such as
pH or phenolic compound molecular weight and chemical
characteristics (e.g., number of aromatic rings). The higher
13
Applied Microbiology and Biotechnology (2023) 107:3729–3744
is the molecular weight the higher seems to be the quantity
of bounds between phenolic compound and BC (Phan et al.
2015).
Li et al. (2021) obtained a BC pellicle bound with phenolic
compounds by cultivating K. rhaeticus K15 in wine pomace
and its hydrolysate. In addition, BC pellicles had outstanding
antioxidant and antibacterial properties, slowly releasing
phenolic compounds. However, the phenolic compound
released was strictly influenced by the BC structural (e.g.,
fiber diameter) and chemical composition (e.g., functional
groups). In this study, in most cases, BC pellicles obtained
from OMW had thinner fiber diameter compared to pellicles
obtained from CW. In addition, the BC from CW presented
several fibrous agglomerates on the surface, mainly
composed of random assembly fibrils (Sheykhnazari et al.
2011). However, the average fiber diameter of BC ribbons
ranged from 35 to 68.86 nm among all the pellicles. These
features are extremely lower than the ribbon size obtained by
Revin et al. (2018). Indeed, they have reported a microfibrils
width between 100 and 180 nm when Gluconacetobacter
(at the present Komagataeibacter) sucrofermentans B-11267
was cultivated in CW. On the other hand, the results of this
study agree with BC fiber diameter obtained from various
foods by-products, such as corinthian currant finishing side-
stream and CW (Bekatorou et al. 2019), citrus peels (Guzel
and Akpinar 2019), agro-industrial wastes (Castro et al.
2011), and OMW (Sar and Akbas 2022).
By using FT-IR analysis, BC characteristic peaks have
been observed in all the pellicles. However, for BC produced
in OMW and CW, other peaks were detected, attributable
to different molecules, highlighting a deep influence of
food by-products on BC functional groups, probably due
to the incorporation of chemicals derived from organic
acids, phenols, lipids or protein fractions. These results are
in accordance with the data published by Molina-Ramírez
et al. (2017) and Sar and Akbas (2022), who produced BC
from CW and OMW.
These changes in the chemical structure drastically
reduced the BC’s CI when produced in OMW and, espe-
cially, in CW. BC is known to have high crystallinity due to
the presence of intra- and inter-molecular hydrogen bonds
generated by the hydroxyl groups (Lee and Bismarck 2012;
Bilgi et al. 2016). Any reaction, which involves a substitu-
tion of a hydroxyl group, causes a reduction of the hydrogen
bonds among molecules, decreasing the degree of polymeri-
zation (Lee and Bismarck 2012; Sar and Akbas 2022). Thus,
the esterification reaction that could have occurred between
BC and organic acids or phenolic compounds caused the
substitution of BC hydroxyl group destroying the crystal-
line structure (Lee and Bismarck 2012). Similar results have
also been reported for BC produced in CW (Molina-Ramirez
et al. 2017; Revin et al. 2018), while CI in OMW was higher
compared to the results reported by Sar and Akbas (2022).
Applied Microbiology and Biotechnology (2023) 107:3729–3744
In this study, the suitability of CW and OMW as feed-
stocks for BC production was demonstrated, obtaining pel-
licles with significant variation in the chemical and mor-
phological structure, compared to standard ones. The six
AAB strains of the Komagataeibacter genus used in this
study are promising candidate for implementing sustainable
bioprocesses for producing BC from agro-wastes. Although
further work is needed to better understand the changes of
the chemical structure observed in the produced BC, these
results confirm the feasibility of the microbial process using
selected AAB for valorising agro-wastes, as CW and OMW.
Acknowledgements We greatly thank Dr. Luciana De Vero for bacte-
rial strain handling at UMCC culture collection and Prof. Fabio Lic-
ciardello for the technical support of HPLC analysis.
Author contribution MB conducted the experiments and wrote the
manuscript. SLC generated the phylogenetic tree. FL reviewed and
edited the manuscript. FVR reviewed and edited the manuscript. AP
reviewed and edited the manuscript. MG conceived, conceptualized,
and provided financial resources.
Funding Part of this work was granted by the European Commission—
NextGenerationEU, Project “Strengthening the MIRRI Italian Research
Infrastructure for Sustainable Bioscience and Bioeconomy”, code n.
IR0000005 and by the Italian Ministry of Agriculture, Food and For-
estry Policies (MIPAAF) (Project INNOLITEC, D.M. 37067/2018)
(Grant numbers [Prot. n. 4739].
Data availability All data generated or analyzed during this study are
included in this published article.
Declarations
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Conflict of interest The authors declare no competing interests.
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