Biochemical Engineering Journal 187 (2022) 108665

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Biochemical Engineering Journal

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Effects of oxygen transfer rate on the L(+) lactic acid production by

Rhizopus oryzae NRRL 395 in stirred tank bioreactor
María Rodríguez-Torres a, 1, Juliana Romo-Buchelly a, b, 2, Fernando Orozco-Sánchez a, *, 3
a
Universidad Nacional de Colombia Sede Medellín, Carera 65 59A-110, Medellín, Colombia
b
Sede de Investigaciones Universitarias SIU, Calle 62 52-59, Medellín Colombia

A R T I C L E I N F O A B S T R A C T

Keywords: Lactic acid (LA) is a key product widely used in several industries and can be produced by aerobic microor­
L(+) lactic acid ganisms like the fungus Rhizopus oryzae NRRL 395. In this study, the effect of different oxygen transfer rates on
Rhizopus oryzae the performance of R. oryzae was analyzed in a 3 L stirred tank bioreactor. Inoculum and sucrose initial con­
Oxygen transfer rate
centrations were previously evaluated at the shake flask level in order to choose the initial culture conditions for
Oxygen uptake rate
Damköhler number
the bioreactor trials. In bioreactor, evaluations showed a range of OTRmax between 0.13 and 0.28 kg m− 3 h− 1 and
Effectiveness factor of oxygen consumption DOT of 72–83 %, in which were reached the best values of LA productivity (0.53 g L− 1h− 1), Yps (0.78–0.83 g
g− 1), and LA production (44 g L− 1). Further, a dimensionless analysis determined that LA production is greater in
Damköhler number (Da) ≈ 1 and effectiveness factor (ɳ) ≈ 0.14 conditions. Finally, these results were used in a
comparative dimensionless analysis with other aerobic microbial systems reported in the literature. According to
our knowledge, this is the first study in which the oxygen transfer/oxygen uptake relationships and their effects
on R. oryzae are analyzed in-depth.

in increasing the LA production [6–8].

1. Introduction
Lactic acid, CH3CHOHCOOH, is an organic acid of natural origin
used in the food, textile, chemical, and pharmaceutical industries [1,2].
This acid exists naturally in two isomeric forms: L(+) lactic acid and D(-)
lactic acid, being the last one harmful to humans in high quantities.
Therefore, the L(+) isomer is preferable for medical and food applica­
tions. All over the world, LA is a product in high demand presenting a
market size of USD 2.64 billion in 2018 and a Compound Annual Growth
Rate (CAGR) of 18.7 % [3]. The importance of this product is due to it
being a precursor for the synthesis of numerous value-added chemicals,
including propylene glycol, propylene oxide, acrylates, and biopolymers
[4]. Among these biopolymers is polylactic acid (PLA), a thermoplastic
material that, through the years, has had more presence in the packaging
market because it degrades into harmless products, and due to its
compatibility, it is used for medical applications such as surgical sutures,
orthopedic implants, and drug capsules [5]. The PLA can potentially be
used as a substitute for petroleum-derived plastics such as polyethylene,
polypropylene, and polystyrene; for this reason, there is a great interest
Currently, 90 % of the industrial production of lactic acid comes from
bacterial fermentation [9]. This process presents several obstacles to
being considered an efficient process since its most significant challenge
is to achieve high concentrations of lactic acid with high yield and
productivity using cheap renewable resources [10]. Second-generation
substrates have the potential, but the low processing and absence of
bacterial metabolic pools to degrade them lay out the necessity of pre­
treatment stages to obtain simple sugars that are easy to consume for
microorganisms. Unlike bacterial fermentation, it has been found that
filamentous fungi of the genus Rhizopus can produce lactic acid at lower
costs using agro-industrial residues or by-products, which are able to
transform these complex materials into biomass and high value-added
products [11–13]. The most studied fungus for LA production has
been R. oryzae; in particular, the highest yields have been found with the
strain R. oryzae NRRL 395 [14,15]. This microorganism possesses an
enzymatic battery which enables it to utilize complex residues and
produce only the L(+)-lactic acid isomer efficiently in a single step [13,
16]. It is relevant to highlight that the bioprocess performance, in terms
of yields and productivity, is conditioned by the strain used as well as

* Correspondence to: Universidad Nacional de Colombia, Sede Medellín, Cra. 65 59A-110, Medellín, Colombia.
E-mail addresses: mirodrig@unal.edu.co (M. Rodríguez-Torres), jromobuchelly2@gmail.com (J. Romo-Buchelly), feorozco@unal.edu.co (F. Orozco-Sánchez).
1
ORCID 0000–0002-9637-1070.
2
ORCID 0000–0002-1681-8670.
3
ORCID 0000–0002-4577-0557.

https://doi.org/10.1016/j.bej.2022.108665
Received 14 July 2022; Received in revised form 22 September 2022; Accepted 4 October 2022
Available online 5 October 2022
1369-703X/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M. Rodríguez-Torres et al.
Nomenclature
PLA Polilactic acid.
LA L(+) lactic acid (g L− 1).
ADH Alcohol dehydrogenase.
LAD Lactate dehydrogenase.
C Concentration of dissolved oxygen (kg m− 3).
X Concentration of biomass (kg m− 3).
vvm volume of air per volume of culture per minute.
rpm revolutions per minutes, units of stirring speed.
Yps Product yield coefficient (g g− 1).
Yxs Biomass yield coefficient (g g− 1).
DOT Dissolved oxygen tension (%).
a Specific interfacial area (m− 1).
kL Mass transfer coefficient (m h− 1).
kLa Volumetric oxygen mass transfer coefficient (h− 1).
qO2 specific oxygen uptake rate (kg O2 kg biomass− 1h− 1).
culture conditions, e.g. pH, temperature, substrate, supply oxygen,
among others [17,18]. L(+) lactic acid production by R. oryzae, unlike
bacterial fermentation, is aerobic, so oxygen becomes an essential
nutrient for its primary metabolism using the lactate dehydrogenase
enzyme (LDH). For instance, Thitiprasert et al. [19] reported that the
immobilized culture of R. oryzae in a static bed bioreactor generated
considerable amounts of ethanol under specific culture conditions
associated with the oxygen concentration in the liquid phase. In their
work, there was a change in the metabolic flux of the fungus when the
supply of dissolved oxygen tension (DOT) was increased from 40 % to 80
%, favoring lactate production and limiting ethanol production without
affecting biomass. A DOT of 80 % in the culture process allowed
increasing lactate yield and productivity by 24 % and 43 %, respectively,
and a significant LDH specific activity. In contrast, the enzyme pyruvate
decarboxylase (PDC), which participates in ethanol production, reduced
its activity.
The kLa, OTR, and DOT are some of the most studied parameters to
analyze the oxygen transfer problems in broths containing filamentous
microorganisms [20]. Trials on R. oryzae have revealed the influence of
oxygen on L(+) lactic acid production at the bioreactor level [21–25];
however, there are no known studies quantifying oxygen transfer (OTR)
together its relationship with oxygen demand (OUR) in stirred tank
bioreactors (STR) with this fungus. In bioreactors, the OTR is strongly
influenced by hydrodynamic conditions, which are a function of the
operating conditions, physicochemical culture properties, geometrical
bioreactor configuration and also by the presence of oxygen-consuming
cells [26]. If the agitation speed increases, the mass transfer also in­
creases; hence it could also affect the microbial culture causing oxidative
or hydrodynamic stress in the cells as a consequence of cellular damage.
The hydrodynamic stress has a direct impact on the growth as well as the
generation of different products associated with the metabolism in
different biological systems [27–32].
The use of the modified Damköhler dimensionless numbers (Da) and
oxygen consumption effectiveness factor (η) proposed by Gomez et al.
[27] provide an essential tool to phenomenologically understand the
relationship between oxygen transfer rate (OTR) and oxygen con­
sumption rate (OUR). The modified dimensionless Damköhler number,
Da, determines whether the process is limited by the rate of oxygen
transfer from the gas phase into the culture medium or by the rate of
oxygen utilization by the cells. Thus, Da is defined as the ratio between
the maximum possible rate of oxygen consumption (OURmax) and the
maximum possible mass transfer rate (OTRmax) [26,27]. On the other
hand, the effect of internal mass transfer and oxygen uptake during
fermentation is quantified by considering the effectiveness factor, η
[27]. This dimensionless number relates to the rate of oxygen
2
Biochemical Engineering Journal 187 (2022) 108665
OTR Oxygen transfer rate (kg m− 3 h− 1).
OUR Oxygen uptake rate (kg m− 3 h− 1).
t Time (h).
ɳ Effectiveness factor of oxygen consumption.
Da Modified Damköhler number.
ROS Reactive oxygen species.
STR Stirred Tank Bioreactor.
Subindexes and superindexes
max related to maximum value.
avg related to average value.
O2 related to oxygen.
s related to the substrate.
x related to the biomass.
p related to the product.
* related to equilibrium values.
c related to critical oxygen concentratio.
consumption, taking into account the resistance to oxygen transport into
the microorganism (observed OUR) and the OUR without resistance to
mass transfer (OURmax).
Taking R. oryzae as a model system, this work aimed to evaluate the
effect of oxygen supply, the relationships between OTR and OUR and
their effect on lactic acid generated in cultures of Rhizopus oryzae NRRL
395 at the level of 3 L in STR. To define some culture conditions with
R. oryzae NRRL 395, the effect of different operating variables at the
Erlenmeyer flask level, such as inoculum and sucrose concentration,
were previously evaluated. In addition, the dimensionless numbers Da
and η obtained in the bioreactor were compared with other aerobic
microbial systems in order to establish associations from a biochemical
engineering viewpoint.
2. Materials and methods
2.1. Microorganism
The microorganism used in this study was Rhizopus oryzae NRRL 395,
preserved in PDA agar (Potato Dextrose Agar) at a temperature of 4 ◦ C.
This microorganism was donated by the United States Department of
Agriculture (USDA).
2.2. Activation and obtaining spore suspension
R. oryzae NRRL 395 was grown on potato dextrose agar plate (PDA)
at 30 ◦ C for 5–7 days for its activation and sporulation. After this time,
sterile distilled water was added to each Petri dish to scrape and release
the spores generated in the plates. Measuring of spore concentration was
done using a Neubauer chamber to guarantee an initial concentration of
4 × 106 spores mL− 1 as inoculum in the pre-culture phase [33].
2.3. Pre-culture stage
Initially, the spores were added to a pre-culture medium to allow
their germination and biomass growth. This medium was prepared with
the following composition (g L− 1): sucrose, 50; (NH4)2SO4, 1.35; Mg SO4
* 7 H2O, 0.25; Zn SO4 * 7 H2O, 0.04; KH2PO4 0.30 [34]. During this
phase, the flasks were maintained at 30 ◦ C in orbital shaking at 170 rpm
for 48 h.
2.4. Lactic acid production stage
Once pre-culture was finished, the microorganism was inoculated in
a medium defined for the biomass growth and production of lactic acid
M. Rodríguez-Torres et al. Biochemical Engineering Journal 187 (2022) 108665

[15,35,36]. This medium had the following composition (g L− 1): Table 2

(NH4)2SO4, 1.35; KH2PO4, 0.25; MgSO40.7 H2O, 0.15 y ZnSO4 * 7 H2O, Operating conditions in a STR for L(+) lactic acid production by R. oryzae NRRL
0.04 using sucrose as carbon and energy source. This phase was carried 395.
out both in shake flasks and in a 3 L stirred tank reactor. Run Gas Flow Agitation Gas composition C* x OTRmax
rate (vvm) speed 103 (kg m− 3
(rpm) (kg h− 1)
2.5. Culture conditions for lactic acid production at flask level m− 3)

1 0.40 125 72 % Nitrogen, 1.76 0.01 ±

L(+) lactic acid production was evaluated using 250 mL Erlenmeyer 28% air 0.00
flasks with a medium volume of 100 mL (40 % filling volume) at 30 ◦ C 2 1.00 200 100 % air 6.34 0.13 ±
and 150 rpm for 96 h. It is relevant to point out that under these con­ 0.00
ditions have been reported kLa values among 20 h− 1 by Reynoso- 3 1.00 400 100 % air 6.34 0.28 ±
0.01
Cereceda et al. [37] at flask level. That value corresponds to an inter­
4 1.00 650 100 % air 6.34 0.80 ±
media condition of the kLa evaluated in stirred tank bioreactor at 0.02
OTRmax of 0.13 kg m− 3 h− 1. 5 0.40 350 21 % Oxygen, 79 13.05 1.59 ±
Evaluation of culture conditions in terms of inoculum and sucrose % air 0.03
concentrations were defined taking into account the treatments pro­
posed in Table 1, which included three levels for each variable.
value. In contrast, the C* value corresponding to 13.05 × 10− 3 kg m− 3
Initial and final lactic acid, substrate, and biomass values were tested
was reached with an air-oxygen mixture to increase the DOT to 205.9 %
to determine the lactic acid concentration, product and biomass yield
of the saturation value. The gases mixing air-nitrogen (Run 1) and
coefficients (Yps - Yxs) at the end of the culture. These results allowed us
air-oxygen (Run 5) were maintained at the same airflow rate since the
to choose the best sucrose and inoculum concentrations as culture
beginning of the culture. It is essential to point out that dissolved oxygen
conditions to perform the bioreactor trials.
tension was calibrated bubbling air (100 %), and bubbling nitrogen (0
%) when culture conditions were established (temperature, air flow and
2.6. Evaluation of different levels of oxygen transfer rate (OTR) in a STR stirrer speed) for all trials [31].

The growth and production of lactic acid by R. oryzae NRRL 395 in a 2.7. Evaluation of OUR, kLa, and maximum OTR
3 L glass stirred tank bioreactor STR were evaluated using the best
conditions of inoculum and sucrose concentrations obtained in the flask The measurements of the transfer coefficient (kLa), transfer rate
trials. The bioreactor was operated with one standard Rushton turbine (OTR) and oxygen consumption rate (OUR) were performed using the
and an L-type sparger with 8-holes connected with the Bio-Controller dynamic method [40]. A critical point to consider when using this
ADI-1010 (Applikon Biotechnology B.V.). The online data were moni­ methodology is if electrode response time is less than 1/kLa (tres­
tored and acquired using the Bioexpert Lite software (Applikon
ponse<1/(kLa), the time constant of the measurement probe can be
Biotechnology B.V.). In addition, dissolved oxygen tension (DOT) was neglected, as proposed by Van’t Riet [40] and pointed out Pappenreiter
measured using a polarographic dissolved oxygen tension probe et al. [41]. In our case, the response time of the DOT probe satisfied this
(AppliSens Z010023525), and a pH sensor (AppliSens Z01023551) was statement for all the kLa evaluated. The OUR was calculated considering
used to control the pH culture at 4.70 to favor mainly the lactic acid the variation of oxygen with time, dCo2/dt (without aeration), and the
production [36]. The control of this variable was made by adding a biomass present in the bioreactor (Eq. 1)
CaCO3 concentrated suspension (60 g L− 1) with the help of an ADI 1010
bio-controller throughout the culture process. In addition, the suspen­ OUR = qO2 × X = −
dCo2
(1)
sion of CaCO3 was kept under constant stirring using a Magnetic Stirrer dt
in order to ensure that the CaCO3 addition was homogeneous during all Using a graph of Co2 versus time, the OUR (qO2 x X) was calculated as
culture. Moreover, to prevent the high formation of foam at high OTR the slope of the straight line obtained. The critical oxygen concentration
levels (Run 4 and 5, Table 2), low amounts of a dilute solution of silicone (DOTc) was determined at the point where the variation of %DOT did
foam were added at the beginning of the bioreactor fermentation. not decrease linearly with time [42]. In the case of the OUR measure­
Table 2 shows selected operating conditions (agitation speed and ment in run 1, an air injection was performed for 2 min to raise the DOT
airflow) for the evaluation of different levels of oxygen supply in a to 50 %. Afterwards, the decrease in dissolved oxygen was recorded to
culture medium with R. oryzae NRRL 395. The oxygen solubility in the monitor the metabolic state of the cells under this condition.
liquid phase (C*) was calculated with the models given by Weisenberger On the other hand, the oxygen balance in the fermenter was given by
& Schumpe [38,39], which were considered all components of the cul­ the difference between oxygen supply (OTR) and oxygen demand (OUR)
ture medium (including salts and sucrose composition). The C* value [26,31], as shown in Eqs. (2) and (3).
corresponding to 1.76 × 10− 3 kg m− 3 was obtained with an air-nitrogen
mixture that allowed the DOT decreased to 27.7 % of the saturation dCO2
= OTR − OUR (2)
dt
Table 1 dCO2 ( )
Sucrose and inoculum concentrations for Rhizopus oryzae NRRL 395 cultures in = kL a C∗o2 − CO2 − qO2 × X (3)
dt
Erlenmeyer flask.
In this case, an approximation of the differentials to deltas was made
Treatment Sucrose (gL− 1) Inoculum (gL− 1)
and it was taken into account that Co2 is proportional to DOT to reor­
1 60 0.57 ganize Eq. (4) as follows:
2 60 1.48
( )
3 60 4.69
ΔDOT kL a × C∗o2 − qO2 × X
4 90 0.57
= − kL a × DOT (4)
5 90 1.48 Δt b
6 90 4.69
7 120 0.57 b is the constant of proportionality between Co2 and DOT. b was
8 120 1.48 calculated based on the composition of the culture medium and the
9 120 4.69
conditions of temperature and working pressure giving a value equal to

3
M. Rodríguez-Torres et al. Biochemical Engineering Journal 187 (2022) 108665

6.339 × 10− 5 kg m− 3 (%DOT)− 1 [31,40]. kLa was calculated as the slope Fisher’s Least Significant Difference (LSD). The factorial design was
of the line ΔDOT/Δt vs DOT and OTR through the expression kLa (CO2* - done by ANOVA analysis of variance using a threshold significance level
CO2). of 95 % (p < 0.05).
The calculation of the maximum oxygen transfer rate (OTRmax) was
given when the oxygen concentration in the medium is equal to zero as 3. Results and discussion
shown in Eq. 5.
( ) 3.1. Culture conditions for lactic acid production at the Erlenmeyer flask
OTRmáx = kL a C∗O2 (5) level

kLa, qo2, OTR and OUR values were calculated every 12 h until the end Fig. 1 A shows the lactic acid results obtained by varying the initial
of the cultures. concentrations of substrate and biomass (inoculum) in Erlenmeyer
flasks. The statistical analysis allowed identifying that only the inoculum
2.8. Phenomenological analysis of oxygen transfer concentration has a significant effect on lactic acid production

The modified dimensionless Damköhler number was calculated

taking into account Eq. 6 [26,27]. The maximum qO2 value was deter­
mined from the qO2 values obtained in the bioprocess carried out, giving
a value of 7.26 × 10− 2 kg O2 kg biomass− 1h− 1.
OURmax qO2 max × X
Da = = (6)
OTRmax kL a × C∗
Conversely, the effectiveness factor ɳ (Eq. 7) was determined based
on the ratio of the oxygen consumption rate to the OUR without limi­
tation by mass transfer (OURmax)[27,43].
OUR qO2 × X
ɳ= = (7)
OURmax qO2 max × X
According to results, graphics of Da vs LA and ɳ vs LA were elabo­
rated with the purpose of finding a relationship between oxygen
transfer-uptake process plus lactic acid production. On the other hand,
the results of E. coli [44], Rhodococcus erythropolis [27], Pichia pastoris
[45], Raoultella terrigena [30] and R. oryzae (reported in our paper) were
used to compare the relationship of Damköhler, effectiveness factor and
production of primary metabolites in different microbial systems. The
extraction of data (Da, ɳ, OTRmax, OURmax) were made directly from the
articles of Çalik et al. [44] and Güneş & Çalık [45]. For articles of Gomez
et al. [27] and Rodriguez et al. [30] was used WebPlotdigitizer program
to obtain values of dimensionless numbers from the graphics [46]. In the
case of Raoultella terrigena, were only used data of 3 points of evaluated
culture conditions in the article (400, 700 and 1000 rpm).

2.9. Analytical methods

The biomass concentration was determined by dry weight mea­

surement. The obtained samples were filtered, washed twice with
distilled water, and dried at a temperature of 60 ◦ C until constant weight
[47]. The measure of the final lactic acid concentrations was performed
employing high-performance liquid chromatography (HPLC) using
Shimadzu equipment with Photodiode-Array Detection (PDA) and an
Ultra aqueous C18 Restek column at a constant temperature of 25 ◦ C at a
wavelength of 210 nm. Potassium phosphate 50 mM: acetonitrile (99:1)
with a flow rate of 1 mL min− 1 was used as the mobile phase. Calibration
curve was made using lactic acid solutions diluted in acetonitrile-water
(30:70) and a range of concentration among 0.07–1.00 % v/v (r2
=0.998). In the analytical determination of total sugars, the
phenol-sulfuric colorimetric method was used where the intensity of
orange color was measured at an absorbance of 492 nm and the total
concentration corresponding to a sucrose standard curve [48]. The
calibration curve was: absorbance = 0.0168 * (sucrose concentration)
+ 0.0727 (r2 = 0.999).
Fig. 1. Effect of substrate and initial biomass concentration on growth and
2.10. Design of experiments and statistical analysis lactic acid production by R. oryzae NRRL 395 at the Erlenmeyer flask level. All
analyses were carried out at the end of the culture process. A: lactic acid pro­
All experiments were performed in duplicate and each sample was duction; B: Yps yield coefficient; C: Yxs yield coefficient. Values in each column
analyzed twice (n = 4) and the results were reported with mean values with the same letter did not exhibit statistically significant differences accord­
± the standard error. Differences between means were evaluated by ing to Fisher’s LSD test (p ≤ 0.05).

4
M. Rodríguez-Torres et al.
(p ≤ 0.05). The highest lactic acid concentration at the flask level was
38.02 g L− 1 and was obtained using 0.57 g L− 1 biomass and 60 g L− 1
sucrose (treatment 1). This value is statistically equal to those obtained
by treatments 4 and 5. Based on the results of the analysis, the lowest
initial concentrations of substrate and inoculum were selected to favor
the production of L(+) lactic acid. The study presented by Bulut et al.
[33] evidenced that sucrose concentrations close to 100 g L− 1 had an
inhibitory effect on lactic acid production with values lower than 5 g L− 1
with R. oryzae NRRL 395 strain. Moreover, the highest LA concentration
was 21 g L− 1 employing 50 g L− 1 of sucrose as the initial substrate [33].
The utilization of sucrose for LA production by R. oryzae NRRL 395
was determined by calculating the product yield coefficient (Yps). In
Fig. 1B, the influence of initial sucrose and inoculum concentration can
be seen. Statistical analysis demonstrated that both factors influenced
the product yield coefficient; however, there was no interaction between
the factors, nor their combined effect significantly affected this response
variable (p ≤ 0.05). The highest Yps (0.63 ± 0.05 g g− 1) was obtained
using 60 gL− 1 sucrose and 0.57 g L− 1 inoculum (treatment 1), a different
value from those achieved by the other treatments (p ≤ 0.05). Yin et al.
[15] indicated Yps values over 0.90 g g− 1 utilizing glucose as carbon
source, which suggests that sucrose is metabolized with less preference
by R. oryzae. Nevertheless, their report also highlighted a Yps value of
0.44 g g− 1 for the trial with 120 g L− 1 of sucrose at flask level, which is
related to the obtained value in the present study with the same sub­
strate concentration [15].
On the other side, Fig. 1C shows the results of biomass yield coeffi­
cient (Yxs) obtained by varying the sucrose and inoculum concentration.
It can be observed that treatments with 120 g L− 1 sucrose favor biomass
production, especially in the treatment with higher inoculum concen­
tration (Yxs: 0.12 g g− 1). Statistical analysis for Yxs indicated that inoc­
ulum concentration had no significant effect on biomass-substrate yield,
however, sucrose concentration and the interaction of the evaluated
factors did affect Yxs. In general, it is observed that all the biomass yield
values obtained are very low (Yxs ≤ 0.12), which was expected because
the formation of lactic acid is promoted over biomass production during
this stage.
Given the above, 60 g L− 1 of sucrose and 0.57 g L− 1 of initial
biomass (inoculum) were chosen as culture conditions to carry out in the
STR trials. It is fundamental to point out that the present work aims to
evaluate the effects of oxygen transfer rate on lactic acid production to
find the most adequate conditions to operate a stirred tank bioreactor
using the best culture conditions according to the selected substrate.
3.2. Effect of different oxygen transfer rates (OTR) on the culture process
in a STR
3.2.1. Growth of R. oryzae NRRL 395 in a STR
Table 3 shows the results obtained for final biomass and Yxs for the
oxygen transfer conditions evaluated in the stirred tank bioreactor.
There is no clear trend of the effect of OTRmax on these response vari­
ables; it can only infer that cell growth and Yxs decreased at lower ox­
ygen transfer values. In general, low Yxs coefficients were found in all
experiments, which is in agreement with what was previously concluded
for Yxs results in Erlenmeyer flasks.
Table 3
Results for biomass and biomass-substrate yield (Yxs) under different OTRmax
conditions with R. oryzae NRRL 395 in a stirred tank bioreactor.
Run OTRmax (kg m− 3
h− 1) Final dry biomass (g L− 1) Yxs x 102 (g g− 1)
1 0.01 1.02 ± 0.04a 1.48 ± 0.08a
2 0.13 2.60 ± 0.20b 4.30 ± 0.47b
3 0.28 2.04 ± 0.01c 2.95 ± 0.07c
4 0.80 2.59 ± 0.12b 4.51 ± 0.26b
5 1.59 3.02 ± 0.44d 4.70 ± 0.85b
Values in each column with the same letter did not exhibit statistically signifi­
cant differences according to Fisher’s LSD test (p ≤ 0.05).
5
Biochemical Engineering Journal 187 (2022) 108665
Various authors have reported comparable results to the final
biomass concentration and the Yxs during lactic production by R. oryzae
in the bioreactor. For instance, Thongchul [24] carried out trials with
Rhizopus oryzae NRRL 395 in a rotating fibrous bed bioreactor finding
values of Yxs around 0.05 g g− 1. This Yxs value was similar to those
achieved in the present work (Table 3). Likewise, Yu et al. [36] obtained
a biomass concentration of 4.52 g L− 1 and a product-substrate yield
(Yps) corresponding to 0.87 g g− 1 with the strain R. oryzae ATCC 9363 in
STR [36]. On the other hand, Fu et al. [13] reached higher values for this
variable response. In their study, the lactic acid production was analyzed
in STR by R. oryzae LA-UN-1, obtaining a final biomass concentration of
9.77 g L− 1, equally affecting the Yps yield (0.79 g g− 1) using glucose as
substrate. These values were accomplished at the same point of the best
LA concentration [13]. It is essential to mention that the fungal strains
used for lactic acid production are excellent producers of organic acids.
Therefore, they direct their metabolism to produce acids over biomass
production, so the Yxs yields are low compared to the Yps yield co­
efficients [49].
3.2.2. Dynamics of oxygen transfer during L(+)-lactic acid production
Fig. 2 describes the dynamics of the DOT, kLa, and qO2 variables
under different oxygen transfer conditions. Fig. 2A shows the decrease in
DOT over time in response to cell consumption. The dissolved oxygen
was generally above the critical DOT (DOTc =10.6 ± 0.55 %), except for
the culture with OTRmax of 0.01 kg m− 3 h− 1 which had values of
2.35–7.80 % during almost the entire bioprocess. After 36 h, it is
observed that the DOT stabilized because the cells decreased the specific
oxygen consumption (Fig. 2C). Fig. 2A also depicts that bioprocess
carried out on intermediate conditions of OTRmax (0.13–0.28 kg m− 3
h− 1) maintained a level of DOT around 70–80 % during almost all the
culture. Thitiprasert et al. [50] found that under DOT control of 80 %,
R. oryzae NRRL 395 showed the best performance in lactic acid pro­
duction as well as the lowest ethanol value with respect to the others
DOT evaluated. The results about lactic acid production and produc­
tivity and Yps yield coefficient are explained in items 3.2.3 and 3.2.4;
furthermore, a relationship between these variables and DOT average is
shown in the supplementary material (Fig. SM.1).
The kLa was less variable for the treatments of 0.13, and 0.28 kg m− 3
h− 1(Fig. 2B). Its slight variations could result from the dispersed cells
and the continuous addition of electrolytes such as CaCO3 [26]. As for
culture at 0.80 kg m− 3 h− 1, the decrease in kLa probably was due to the
amount of foam generated by the high stirring speed. The higher vari­
ation in the treatment of 1.59 kg m− 3 h− 1 could be caused by the
required antifoaming addition. Antifoaming type substances cause a
sharp drop in kLa affecting the hydrodynamic parameters of the culture
medium; since the increase in bubble size due to induced coalescence
affects gas retention and hence, the mass transfer area [51]. In addition,
the treatment of 1.59 kg m− 3 h− 1 exhibited more biomass production,
which may also be affected by decreasing kLa. The above is due to the
fact that the presence of cells as solid particles can have a possible effect
on the oxygen transfer rate from bubbles as a physical blockage caused
by the accumulated cells near the interface, as Ju et al. mentioned [52].
Regarding specific oxygen consumption (qO2), Fig. 2C shows that this
variable increases in the first hours of culture and that as the OTRmax
increases, the cells increase oxygen consumption. In all the cases eval­
uated, it can be observed that the highest oxygen consumption activity
occurs during the 20 and 40 h of the culture, and then its consumption
rate decreases. This drop is associated with a reduction in cellular
metabolic activity, as described by Garcia-Ochoa et al. [43]. The
maximum qO2 was 7.26 × 10− 2 kg O2 kg biomass− 1h− 1 and was ob­
tained with an OTR of 0.80 kg m− 3 h− 1.
Fig. 3 shows the maximum qO2 at the different OTR levels. It is
observed that as oxygen supply increases, the rate at which R. oryzae
consumes oxygen increases; however, OTR values higher than
0.80 kg m− 3 h− 1 generate a metabolic decrease during the entire bio­
process. García-Ochoa et al.[43] and Gómez et al. [28] presented in their
M. Rodríguez-Torres et al. Biochemical Engineering Journal 187 (2022) 108665

Fig. 3. Variation of maximum specific rate of oxygen consumption (qO2

maximum) and OTRmax during lactic acid production by R. oryzae NRRL 395.
Values with the same letter did not exhibit statistically significant differences
according to Fisher’s LSD test (p ≤ 0.05).

Table 4
Specific oxygen uptake rate (qo2) in different biological systems.
Microorganism Type of qO2 maximum x102 (kg Reference
bioreactor O2 kg biomass− 1 h− 1)

Azadiractha indica STR 1.60 [32]

Steinernema carpocapsae Erlenmeyer 1.76 [55]
CABA01 flask
Rhizopus oryzae NRRL STR 7.26 This work
395
Rhizopus delemar Erlenmeyer 7.92 [56]
flask
Rhodococcus erythropolis STR 9.80 – 10.37 [28]
IGTS8
Xanthomonas campestris STR 48.38 [43,57]
NRRL B-1459
Pseudomona putida STR 57.60 [43]
Escherichia coli STR 73.50 [44]

is compatible with the results obtained in this study. According to our

Fig. 2. Dynamics of oxygen transfer during lactic acid production by R. oryzae
NRRL 395 in a STR at different OTRmax. A: dissolved oxygen tension (% DOT);
B: kLa and C: specific oxygen uptake rate (qO2).
publications a similar behavior when evaluating the cellular response of
Rhodococcus erythropolis IGTS8 (µ, qO2, X) and the stirring rate, pointing
out that the decrease in these response variables is due to cell damage
derived from hydrodynamic stress [43,53]. In contrast to the above, our
study elucidates how metabolism could also be affected by the high
oxygen transfer rate achieved with an air-oxygen mixture (205.9 % DOT
and OTRmax: 1.59 kg m− 3 h− 1). This treatment was performed under
operating conditions that do not generate cell damage (0.4 vvm and
350 rpm); but the high oxygen concentration and high OTRmax, possibly
caused an oxidative stress environment taking cells to use substrate and
energy for cellular maintenance and resistance for the stress [54].
Authors who have studied the effects of oxygen transfer in cell cul­
tures report the values for qO2 rates in other biological systems. As a
comparison, Table 4 expounds on some of these values inferring that
bacteria present the highest specific oxygen demand with values of 9-
and 26-times superior than fungi and plant cell culture, respectively.
Furthermore, another study done on Rhizopus fungi registered a specific
oxygen consumption rate of 7.92 × 10− 2 kg O2 kg biomass− 1 h− 1, which
knowledge, this study is the first report illustrating that OTR conditions
directly affect the qO2 and the maximum qO2 of a biological system
(Fig. 2C and Fig. 3).
3.2.3. Effect of OTR on the kinetics of sucrose consumption and L(+)-lactic
acid production
Fig. 4 illustrates the kinetics of sucrose consumption and L(+) lactic
acid production under each of the oxygen transfer conditions evaluated.
Fig. 4A shows that there is a greater decrease in sucrose concentration
under the OTR of 1.59 kg m− 3 h− 1, initially; however, after 36 h the
microorganism begins to utilize more sucrose at intermediate oxygen
transfer conditions (OTRmax de 0.13 and 0.28 kg m− 3 h− 1). For the OTR
of 0.80 kg m− 3 h− 1, a a more extended lag phase and a slower rate of
sugar consumption is observed, possibly due to hydrodynamic stress
effects under the operating conditions used (650 rpm, 1 vvm). As is
shown in the literature, high agitation speeds generate higher shear
stress, which leads to a high rate of cell fragmentation and increases the
requirements for cell maintenance [21,58,59]. Bioreactor trials done by
Chotisubha-Anandha et al. [21] indicate a higher concentration of L(+)
lactic acid with increasing agitation speed up to 500 rpm; when the
cultivation was run at 700 rpm, a decrease in LA production was
observed. Although there was an increase in the amount of biomass due
to damage to cell structures, affecting the viability of the microorganism
and reducing the generation of L(+)-lactic acid. Therefore, under these

6
M. Rodríguez-Torres et al.
Fig. 4. Effect of OTRmax on the kinetics of L(+) lactic acid production and sugar
consumption of R. oryzae NRRL 395 in a STR. A: sucrose consumption; B: L(+)
lactic acid production. Points with the same letter in the same figure did not
exhibit statistically significant differences according to Fisher’s LSD test
(p ≤ 0.05). Comparison between means was made at specific times.
conditions, the metabolism is redirected to use the carbon source for
biomass production [21].
Regarding the production of L(+) lactic acid (Fig. 4B), it is observed
that there are no differences during the first 36 h in almost all levels of
OTR followed by a stage where the OTR of 0.13 kg m− 3 h− 1 favors a
higher production of lactic acid over time. At 84 h of culture, both the
OTR of 0.13 kg m− 3 h− 1 and 0.28 kg m− 3 h− 1 reached the same L(+)
lactic acid concentration. It is valid to highlight that the productivity
(considering the time in which the highest product concentration is
reached) of these two oxygen transfer conditions, must be different
since, after 84 h, there is a decrease in the lactic acid concentration
when working with an OTR of 0.28 kg m− 3 h− 1. This phenomenon may
be due to the fact that the microorganism possesses two NAD-dependent
lactate dehydrogenase isoenzymes (LDHA and LDHB) and since sucrose
in the medium has been depleted, possibly there is the activity of the
LDH B isoenzyme whose gene is expressed in the presence of non-
fermentable carbon sources (such as lactate, glycerol, ethanol) [22]. In
this case, R. oryzae NRRL 395 could use the product (lactate) as a sub­
strate for its metabolic activities.
The performance of R. oryzae in terms of substrate consumption
(Fig. 4A), biomass generation (Table 3) and lactic acid production
(Fig. 4B) on OTRmax of 1.59 kg m− 3 h− 1 depicts how microorganism
can drive its metabolism for preservation just as is mentioned by Bai
et al. [54]. In this condition, they obtained slightly more cell biomass, a
reduction of specific uptake rate (although it had the highest oxygen
availability), and low lactic acid production. Perhaps part of this
biomass was not viable for the accumulation of oxidative damage.
7
Biochemical Engineering Journal 187 (2022) 108665
3.2.4. Effect of OTR on Yps, productivity and lactic acid production in a
STR
Fig. 5 describes the relationship between Yps, productivity (calcu­
lated at 84 h of culture) and the production of lactic acid L(+) as a
function of OTRmax. From this figure, a favorable OTR zone for lactic
acid production is established, ranging from 0.13 a 0.28 kg m− 3 h− 1, in
which the highest values are accomplished for each response variable.
Taking into account the energy costs involved in working in this OTR
zone, it would be preferable to select the lowest OTR of the range. It
should be noted that the difference of 0.13 and 0.28 kg m− 3 h− 1 is
generated by agitation speeds corresponding to 200 and 400 rpm, which
implies a higher power expenditure for the operation of the motor. In a
similar way, a favorable zone of DOT for lactic acid production could be
established, ranging from 72 % to 83 % (Fig. 5 and Fig. SM.1). This fact
would suggest that to define a determined value of OTRmax or DOT as
operation condition, both of them could be equivalent to increasing the
lactic acid production.
Although there is no statistical difference in Yps values at OTRmax
between 0.28 and 0.80 kg m− 3 h− 1, the kinetics of sugar consumption
indicate that after 84 h of culture there is a lot of sucrose remaining in
the medium (sucrose concentration = 12.51 g L− 1) when the fungus is
grown at an OTR of 0.80 kg m− 3 h− 1 compared to 3.16 g L− 1 for the
0.28 kg m− 3 h− 1 culture (see Fig. 4A). The obtained results for the cul­
ture at 1.59 kg m− 3 h− 1 reaffirm that exposure of the microorganism to
high oxygen transfer rates could induce an oxidative stress environment.
In this case, these conditions were achieved by increasing the oxygen
concentration in the air flow and intermediate process conditions
(350 rpm and 0.4 vvm) which would allow isolating the effects of oxy­
gen stress from those of hydrodynamic stress. Despite microorganisms
possessing antioxidant enzymes, some metabolic processes could
decrease due to the damage caused by the presence of reactive oxygen
species [29,31,60]. This oxygen effect has also been previously reported
in high values of oxygen concentration or oxygen supply; for aerobic
microorganisms, high oxygen concentrations are related to ROS and cell
damage [54,60]. For instance, Blakeslea trispora, cultured in shake flask
at OTR values higher than 20.50 mmol L− 1h− 1, had high concentrations
of H2O2, causing oxidative stress and cell damage [61].
In addition to oxygen stress, hydrodynamic stress may also
contribute when working with an OTR of 0.80 kg m− 3 h− 1. This is
because the agitation applied during the bioprocess creates shear forces
that may affect the microorganisms in various ways such as: damage to
cell structure, morphological changes, as well as variations in growth
rate and product formation. Exposure to a high shear stress zone may not
cause immediate damage, but may gradually affect the metabolic ac­
tivity of microorganisms due to the hydrodynamic stress to which they
are subjected [28,54,58,62]. In contrast, a decrease in the variables in
Figure at an OTRmax value of 0.01 kg m− 3 h− 1 could be caused by the
low oxygen supply, which conditioned the microorganism to reduce its
metabolic activity.
3.3. Phenomenological analysis of oxygen transfer in R. oryzae NRRL
395 cultures
Fig. 6 presents the relationship between lactic acid production and
the dimensionless numbers ɳ and Da. It is observed at values of Da close
to 1, the highest concentrations of L(+) lactic acid were reached
(Fig. 6A). This suggests that the metabolite production increases at
operating conditions where oxygen supply (OTR) is comparable to ox­
ygen demand; such points were reached at the end of the culture process
of 0.13 and 0.28 kg m− 3 h− 1. The culture with an OTRmax of
0.01 kg m− 3 h− 1 had Da values in the range of 3–4. Thus, it developed
under oxygen-limited conditions (Da >> 1). The oxygen transfer con­
ditions led the microorganism to undergo a DOT below the critical ox­
ygen level and adapt to a very low specific rate of oxygen consumption
compared to maximum qO2. For the case of the culture with OTRmax de
0.80 kg m− 3 h− 1 Da values in the range of 0.089–0.582 were obtained;
M. Rodríguez-Torres et al.
Fig. 5. Relationship between productivity, Yps yield coefficient and L(+) lactic acid production of R. oryzae NRRL 395 in a stirred tank bioreactor obtained at 84 h of
culture process. Points with the same letter into the same line did not exhibit statistically significant differences according to Fisher’s LSD test (p ≤ 0.05).
Fig. 6. Relationship between effectiveness factor and Damköhler number with
L(+) lactic acid production in cultures with R. oryzae NRRL 395 in STR. A.
Damköhler (Da); B. Effectiveness factor (ɳ).
this indicates that OTR > OUR, so there was no limitation during the
whole bioprocess. Nevertheless, higher L(+) lactic acid values were not
achieved possibly due to the effects of oxygen stress and hydrodynamic
8
Biochemical Engineering Journal 187 (2022) 108665
stress. The lowest lactic acid production was given in conditions of high
oxygen transfer (OTR: 1.59 kg m− 3 h− 1) whose Da values were below
0.250. The operating conditions (agitation speed 350 rpm; gas flow 0.40
vvm and air-oxygen mixture to which the microorganism was subjected)
would avoid a possible hydrodynamic stress effect, so the dimensionless
analysis would correctly point out the effect of dissolved oxygen on
lactic acid production.
Fig. 6B describes that the effectiveness factor takes values close to 1
at the beginning of the fungal culture, indicating that the cells are
consuming oxygen at such a high rate when they are approaching the
maximum possible value of oxygen utilization, qO2 max [43]. As culture
time progresses, oxygen consumption decreases, which is reflected in
the decrease in the effectiveness factor. This not only suggests a decrease
in OUR, but also suggests possible oxygen diffusional problems within
the pellets and the mycelial masses formed by the fungus around the
reactor accessories.
Employing the relationship between the dimensionless numbers Da
and ɳ respect to acid production, it is established that the highest lactic
acid concentration is obtained at values at ɳ ≈ 0.14 and Da ≈ 1. This
may be due to diffusional phenomena of oxygen transport through the
mycelium, leading to the fact that the oxygen uptake rate of R. oryzae
NRRL 395 becomes low (works at 14 % of OURmax) and the microor­
ganism works under "normal" conditions, without limitation and minus
excess oxygen (Da ≈ 1).
The relationship between dimensionless numbers ɳ and Da with
respect to L(+) lactic acid (Figs. 6 and 7) is similar to the one reported by
Çalik et al. [44], Gomez et al. [27], Güneş & Çalık, [45] and Rodriguez
et al. [30], for products derived from primary metabolism in E. coli,
Rhodococcus erythropolis, Pichia pastoris and Raoultella terrigena respec­
tively. Fig. 8 shows how the effectiveness factor decreases with
increasing Damköhler number in different biological systems. At the
onset of the culture process or at high OTR levels, values of ɳ close to 1
and Da around 0.1 are present, suggesting that microorganisms are
consuming oxygen at a rate similar to the maximum rate (OURmax) and
that the rate of oxygen transfer is very high compared to oxygen demand
(OTR >> OUR). At this point, the rate of oxygen transfer may be high
enough to generate oxidative stress conditions and possibly the forma­
tion of reactive oxygen species, such as hydrogen peroxide (H2O2), hy­
droxyl radicals (OH. ), and superoxide radical (O.2 − ). High
concentrations of ROS react with cellular components, leading to
M. Rodríguez-Torres et al.
Fig. 7. Relationship between Damköhler number (Da) and effectiveness factor
(ɳ) in cultures with R. oryzae NRRL 395 in a STR.
Fig. 8. Relationship between Damköhler number (Da) and effectiveness factor
(ɳ) in cultures with R. oryzae (our study), E. coli [44], Pichia pastoris [45],
Rhodococcus erythropolis [27], and Raoultella terrigena [30] in stirred
tank bioreactor.
protein oxidation, enzyme inactivation, DNA damage, and ultimately
cell [44,45]. Such ROS can also be produced under conditions of exog­
enous oxidative stress, by exposure of the microorganism to high con­
centrations of dissolved oxygen. Mantzouridou et al. [61] reported the
effect of different levels of ROS on the production of β-carotenoids by the
fungus Blakeslea trispora in flasks. The authors found that the production
of these antioxidant compounds increased from 120.66 to
704.10 mg L− 1, as OTR increased from 2.93 to 20.55 mmol L− 1 h-1;
finally, they point out that there is a linear relationship between
hydrogen peroxide production and β-carotene synthesis [45].
At the end of bioprocesses or under certain operating conditions,
oxygen limitation is usually reached; this would occur because the ox­
ygen demand is so high that the oxygen supplied by the system would be
insufficient to meet the metabolic needs of the cells or microorganisms
(Da >> 1; ɳ < 0.1). Generally, in aerobic cultures with high growth rates
(high biomass formation) or that tend to form cell aggregates, hypoxia
or anoxia conditions may occur. Some cell growth models of filamentous
fungi take into account a critical radius for the size of cell aggregates
since the diffusion of oxygen and nutrients towards the center of the
pellets is limited by the increase in biomass density [54,62].
With the dimensionless analysis performed in Fig. 8, a trend of the
9
Biochemical Engineering Journal 187 (2022) 108665
behavior of Da and ɳ for biological systems can be established. For the
case of Rhodococcus erythropolis, Pichia pastoris and R. oryzae it was
found that the most favorable conditions for growth (R. erythropolis and
Pichia pastoris) and production of the primary metabolite L(+) lactic acid
(R. oryzae) occur at Damköhler values around 1 (OUR≈OTR) [23,34]. At
this point, oxygen transfer may be high enough therefore hypoxic con­
ditions do not occur, but it may also be too low so that an oxygen stress
environment is not generated thus affect the cells [22]. There are few
reports quantifying the possible OTR/OUR relationship and its effect on
primary metabolism. This relationship could be used to understand from
a biochemical engineering view this phenomenon, and to define some
conditions to increase the production of biomass or some metabolites
related to the primary metabolism in aerobic systems.
4. Conclusions
Preliminary evaluations carried out at the Erlenmeyer flask level
allowed determining favorable conditions for inoculum concentration
and substrate concentration (sucrose) for the production of lactic acid by
R. oryzae NRRL 395. At this scale, the treatment with sucrose of 60 g L− 1
and inoculum 0.57 g L− 1 was the most favorable for LA production and
Yps with values of 38.02 g L− 1 and 0.61 g g− 1, respectively. These con­
ditions were subsequently used for the different assays at the bioreactor
level. At the bioreactor, the evaluation of the effects of oxygen transfer
demonstrated that low levels of OTRmax (0.10 kg m− 3 h− 1) decreased
the values of LA concentration, Yps, and productivity as a consequence of
oxygen transfer limitation during the whole bioprocess (Da > 1). For the
case of the OTRmax of 1.59 kg m− 3 h− 1, it is deduced that the decrease in
the results obtained from LA, Yps, and productivity could be a conse­
quence of oxygen stress. Further, it was found that there was a range of
OTRmax (0.13–0.28 kg m− 3 h− 1) where it was possible to operate the
bioreactor without altering productivity, yield, and L(+) lactic acid
production.
The highest lactic acid values were obtained under operating con­
ditions where oxygen transfer was comparable to oxygen demand (Da ≈
1). Under these conditions, oxygen transport through the mycelium may
cause the oxygen consumption rate of R. oryzae to become so low (it used
only 14 % of OURmax) and the microorganism worked under "normal"
conditions, without limitation and without excess oxygen (Da ≈ 1).
Extreme conditions of oxygen transfer generated a decrease in cell
growth, lactic acid production, and productivity. In high OTR values, the
effectiveness factor was closed to 1, indicating that the cells were
consuming O2 at a rate similar to the OURmax. There may be no oxygen
transfer problems (Da around 0.1); however, the OTR can be so high that
an environment of oxygen stress and oxidative stress was possibly
generated in the reactor. Low OTR levels are associated with Da > 1 and
ɳ < 0.1, implying that the cells will have to reduce their oxygen con­
sumption (OUR) because the oxygen transfer rate is not sufficient to
meet the demand of the system. According to our knowledge, this is the
first report which quantifies the OTR/OUR relationship and its effect on
R. oryzae NRRL 395 in depth. These results could be used to define
operation conditions for the production of enzymes or other metabolites
related to the primary aerobic metabolism.
Author contributions
M. Rodriguez-Torres participated in the collection, analysis, inter­
pretation of data, writing and editing the manuscript. J. Romo-
Buchelly participated in the collection of data, analysis, writing and
reviewing the manuscript. F. Orozco-Sánchez participated in the
conception and design of the study, drafted and revised the manuscript,
and approved the final version to be submitted.
Declaration of Competing Interest
The authors declare that they have no known competing financial
M. Rodríguez-Torres et al.
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
M. Rodriguez-Torres is grateful to the Science, Technology and
Innovation Department (MINCIENCIAS) for its financial support and its
internship grant for young researchers. In addition, authors thank to
United States Department of Agriculture (USDA) for its donation of
R. oryzae NRRL 395 strain.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.bej.2022.108665.
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