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PII: S0009-2509(18)30551-7
DOI: https://doi.org/10.1016/j.ces.2018.07.056
Reference: CES 14410
Please cite this article as: E.N. Tec-Caamal, A. Jiménez-González, S.A. Medina-Moreno, M. Alejandro Lizardi-
Jiménez, Production of an oil-degrading bacterial consortium in an airlift bioreactor: Insights into the mass transfer
of the oil and oxygen, Chemical Engineering Science (2018), doi: https://doi.org/10.1016/j.ces.2018.07.056
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Production of an oil-degrading bacterial consortium in an airlift bioreactor: Insights into the
Alejandro Lizardi-Jiménezb,*
a
Universidad Politécnica de Pachuca, Ex-Hacienda de Santa Bárbara, C.P. 43830, Zempoala
Hidalgo, México.
b
CONACYT - Instituto Tecnológico Superior de Tierra Blanca, Av. Veracruz S/N Esq. Héroes de
Abstract
limited by the mass transport of both the gaseous and oil phases. In our study, the mass
transfer rates of hexadecane and oxygen (HTR/OTR) were evaluated during the production
of oil-degrading bacteria in a 3-L airlift reactor. To improve the HTR/OTR ratios during
kinetics, a high initial concentration of hexadecane (77 g L-1) and constant superficial gas
velocity (UG) in the range of 1.5-3.5 cm s-1 were used. Under these conditions, all initial
HTR/OTR were close to the stoichiometric consumption ratio (SCR, ~0.28), which in turn
decreased at the end of the culture until 0.173 ± 0.013, 0.108 ± 0.006 and 0.123 ± 0.006 (g
HXD) (g O2)-1 for UG of 1.5, 2.5, 3.5 cm s-1, respectively. The evaluation of the Damköhler
numbers suggested an enhancement of HXD mass transport that allowed for obtaining a
1
maximum suspended solids (SS) production of 4.9-6.0 g SS (L h)-1. Since the operation of
the ALB at high superficial gas velocities may lead to high power inputs, the variable UG
technique was used for energy saving, finding similar maximum SS production (6.09 g SS
(L h) -1) to that obtained at a constant UG of 2.5 cm s-1 and an energy saving of 33.4%. The
improvement in the HTR/OTR ratio during ALB performance allowed us to enhance the
Keywords: Mass transfer rate; stoichiometry; hexadecane; oxygen; airlift bioreactor; oil-
degrading consortium.
1. Introduction
phase (Chisti, 1989). The main characteristic of these reactors is a well-defined cyclic flow
pattern caused by a density difference between the riser (up-flow section) and downcomer
(down-flow section) that allows a solid phase can be maintained in recirculation (Klein et
al., 2003). ALBs have been used in several biotechnological applications like biomass and
protein production, wastewater treatment (Guieysse et al., 2011), and alkane biodegradation
by microbial cultures (Denis et al., 2017), due to their low energy consumption, efficient
mixing, homogeneous shear stress, and simple construction (Chisti, 1989; Guieysse et al.,
2011). Power input is an important variable to consider in the operation costs of the ALB.
In agitated and aerated systems, the presence of the gas phase reduces density, which
results in lower power consumption; this represents an advantage over the un-aerated
substrates such as crude oil (Medina-Moreno et al., 2005). Alkanes are important oil
constituents. Hexadecane (HXD) is used as a model alkane molecule due to its easy
used as carbon source in the biodegradation process in an ALB, a mass transfer limitation
could be implied due to the low solubility of HXD in water (5.2 x10-5 mg L-1). However,
microscopic droplets, which enhance the bioavailability of the immiscible substrate (Denis
et al., 2017; Hua and Wang, 2014); this can result in an improvement in biomass
performance of the ALB, since mass transfer of both hydrocarbons and oxygen depends on
hydrodynamics (Denis et al., 2017; García-Ochoa and Gomez, 2009). Although previous
studies only considered the mass transfer of oxygen (kLaO2) as the main parameter of
operation and scale-up of bioreactors (Mehrnia et. Al., 2005), some studies suggest that the
towards the aqueous phase (Medina-Moreno et al., 2013; Lizardi-Jiménez et al., 2012).
This has led to studies on the relationship between the turbulence regime in the reactor and
Jiménez et al., 2012). Hence, it is important to investigate the real behavior of mass
transport phenomena in both the oil and gas phases, considering the stoichiometric
consumption rate (SCR), to propose novel methods of operation and scale-up of ALBs for
dispersed phase fraction) were not able to guarantee a system not limited by HXD mass
transfer. This means that SCR was never reached, suggesting a greater mass transfer of
oxygen than HXD, which could restrict biomass growth. Therefore, novel methods must be
developed to improve hydrocarbon mass transport from the oil to aqueous phase. A suitable
method to improve the HTR/OTR ratio could be an increase in the HXD mass transfer
specific area per unit reactor volume, by increasing the HXD loading (high dispersed phase
fraction, φ). Recent works have emphasized that the specific area for mass transfer is an
important factor that should be considered in assessing ALB performance, because this
parameter is related directly to the hydrocarbon transfer rate (Denis et al., 2017; Lizardi-
This research aimed to investigate the relevant aspects of the mass transfer of the oil
and gas phases during ALB operation. This approach included the use of a high initial
concentration of HXD (77 g L-1) to enhance the HTR/OTR ratios, and the application of the
production of an oil-degrading microbial consortium and for energy saving during a 14-day
batch culture.
2. Experimental
2.1 Materials
4
A microbial consortium composed of four bacterial species (Acinetobacter bouvetii,
Shewanella sp., Defluvibacter lusatiensis, Xanthomonas sp.) was obtained from the
from the rhizosphere of a plant (Cyperus laxus Lam) capable of growing in a hydrocarbon-
contaminated swamp in Mexico. Strains were characterized by their 16s rRNA genes
(Tzintzun-Camacho et al. 2012). The separated bacterial species were grown as a whole in
the reactor. A mineral medium (Medina-Moreno et al., 2005) consisting of (g L-1): 6.75
NaNO3; 2.15 K2HPO4; 1.13 KCl; 1.10 MgSO4·5H2O was used. The mineral medium was
supplemented with 77 g L-1 of hexadecane (reagent grade 99% Sigma Aldrich, MO, USA)
All experiments were performed in a 3 L airlift bioreactor made of Pyrex glass 0.68 m
height, with an internal diameter of 0.08 m and an operating volume of 2.2 L with a
height/diameter ratio (H/D) of 5.5. The concentric tube (0.35 m height and 0.05 m
diameter) was placed 0.035 m from the bottom. Air was supplied into the riser zone
through a perforated L-shaped steel pipe with an internal diameter of 0.005 m and seven
perforations of 0.001 m in diameter with 0.004 m separation (Fig. 1). Three different
constant UG in the range of 1.5-3.5 cm s-1 were used to evaluate all kinetic parameters.
The microbial consortium was cultured and grown in sequential batches for 14 days. After
the end of the batch culture, suspended solids (SS) were harvested at the top outlet of the
5
airlift bioreactor (ALB) and centrifuged (Model Hermle, Z300, Hamburg, Germany) at
4300 g for 5 min. Initial conditions were restored by adding 1 g SS L-1 (wet weight) of the
biomass from the previous culture. Biomass growth was determined using the suspended
solids (SS) technique (Denis et al., 2017). A 7 mL sample of the mixed liquor of the ALB
was collected in the riser at the middle of the concentric tube at days 0, 2, 6, 10 and 14.
Afterwards, 7 mL of hexane was added to the sample to rinse off the HXD attached to the
biomass. The solid phase (including the microbial consortium) was separated and dried in
an oven at 90ºC for 24 h until a constant weight. SS determinations were done in triplicate.
The change in the surface tension of the microbial culture through kinetics was determined
Calibration was performed using pure water (72.8 dynes cm-1) and ethyl alcohol (22.3
dynes cm-1) as the reference. Samples (20 mL) were collocated in a 50 mL glass beaker and
placed onto the tensiometer platform for analysis in duplicate. All experiments were
conducted at 25ºC.
2.2.3 Hydrodynamics
Since microbial cells can produce bioemulsifiers that change the surface tension, viscosity
and density of culture medium, a designed abiotic medium that simulates the
work. The mineral medium (section 2.1) was supplemented with Tween 80 (50 μL L-1) to
reach a final surface tension of 68.9 dynes cm-1 and a viscosity of 1.01 cP. The abiotic
velocity, liquid density and the ratio of the cross-sectional areas of the riser and downcomer
P AT
L g U g (1)
V Ar Ad
where P/V = volumetric power (W m-3), L = liquid density (kg m-3), UG = superficial gas
The overall gas hold-up was calculated according to the volumetric expansion
HG H L
g (2)
HG
where HG is the height of (gas + liquid) and HL are the height of the liquid (unaerated) in
the ALB, respectively. All the hydrodynamic assays were carried out without
microorganisms.
7
Fig. 1. Diagram of the experimental set-up.
A polarographic oxygen sensor (ADI dO2, Applisens, The Netherlands) was used for kLaO2
determinations in triplicate. The dissolved oxygen (DO) sensor was collocated at the
middle of the concentric tube in the downcomer. The kLaO2 was evaluated using the static
gassing-out method. The ALB was first fed with nitrogen gas to remove the dissolved
oxygen from the system. When the dissolved oxygen concentration was close to 0%
saturation, air was delivered from the sparger at three different superficial gas velocities
(UG): 1.5, 2.5, 3.5 cm s-1 (0.75, 1.25 and 1.76 volume of air per volume of reactor per
8
minute, VVM). To assess the probe-response dynamics, the approach suggested by Nielsen
dCL
kL aO 2 (CO* 2 CLO 2 ) (3)
dt
dCe
ke (CLO 2 Ce ) (4)
dt
where CLO 2 is dissolved oxygen concentration in the liquid phase (g L -1), CO* 2 is the
saturation oxygen concentration in the oil-water mixtures (0.04 g L-1) (Ju and Ho, 1989;
Tec-Caamal et al. 2018), and kLaO2 is the volumetric oxygen gas-liquid mass transfer
coefficient (h-1), ke is the electrode time constant (h-1) and Ce is the actual electrode-
Additionally, to determine the effect of the dispersed phase fraction on kLaO2, the
1 yoxygen (t 0)
ln kL a t
1 1 yoxygen (t 0)
yoxygen (t )
1 ( moxygen 1) 1 (moxygen 1) 1 (moxygen 1)
(5)
R is the universal gas constant (J/mol K), moxygen is the solubility ratio (solubility of
oxygen in oil versus solubility in water, dimensionless), VG is volume of the gas phase (m3).
(dimensionless). Afterwards, the slope of the straight line formed by plotting the left
member of Eq. (5) respect to time, gives the kLa for the three-phase system.
9
OTR was calculated as the product of kLaO2 and the mass transfer driving force term
The specific mass transfer area of the HXD macrodroplets (aHXD) was calculated using Eq.
(6):
6
aHXD (6)
d32 HXD
where d32HXD is the Sauter mean diameter of HXD droplets (cm) and φ is the dispersed-
phase fraction (dimensionless), calculated as the volume of HXD divided by the operation
volume of the reactor ( 0.1 ). d32HXD determinations were performed abiotically by the
photographic technique (Ribeiro and Lage, 2004) using a digital camera (Sony SteadyShot,
USA, 21 MP) and image analysis software (ImageJ, v1.49, USA). The oil phase was
distinguish between bubbles and droplets of the moving phases by adding 0.061 g of
taken at each superficial gas velocity and 75 droplets were analyzed. d32HXD was obtained
from two measurements for each droplet (major and minor axis of the droplet), using the
equivalent volume of an ellipse. The number of droplets of HXD was obtained by dividing
the remaining volume of HXD in the reactor by the volume of a single droplet (Table 1).
Determinations of kLaHXD were obtained by the product of the specific area for each UG and
the HXD mass transfer coefficients (kLHXD), collected from previous studies (Lizardi-
Jiménez et al., 2012; Lizardi-Jiménez et al., 2011). More details about the kLHXD
calculations and the acceptability of using these data for our study can be found in studies
10
performed by Lizardi-Jiménez et al. (2013) and Tec-Caamal et al. (2018). HTR was
determined as the product of kLaHXD and the mass transfer driving force term
*
( CHXD CLHXD ), where CLHXD is the HXD concentration in the bulk at time t, and CHXD
*
, is
the HXD saturation concentration in the aqueous phase (which varies from 0.73 and 1.1 g
L-1 through the culture). Considering the change in surface tension of the culture medium,
concentration of HXD in the aqueous phase was measured each day, as follows: an aqueous
sample of 2 mL was added to 150 μL of HXD and vortexed for 2 min. After 30 min of
Since the rate of mass transfer of a hydrocarbon may limit the rate of cell growth and
productivity, the rate-controlling regime of the system was estimated using a modified
HURmax
Da
HTRmax
(7)
where HURmax is the maximum HXD uptake rate and HTRmax is the maximum HXD
transfer rate. HURmax was estimated by fitting experimental data to the Gompertz model
*
(inflection point) and HTRmax was obtained with the driving force term ( CHXD CLHXD ) at
day 0.
11
The presence of HXD emulsifying agents in aqueous phase was determined in triplicate
cell-free supernatant samples (1.5 mL) were vortexed for 2 in with 200 μL of HXD. The
1310, USA) using a flame ionization detector at 300ºC, helium as the carrier gas, and a
column coated with a non-polar phase, 5% phenyl methyl polysiloxane (30 × 0.0025 m;
Thermo Scientific TR-5). Injector and detector temperatures were maintained constant at
250ºC and 300ºC, respectively. Samples were injected in splitless mode. The temperature
program used was: 70ºC as the initial temperature, increased by 45ºC min-1 until 300ºC (6.5
min) and maintained at 300ºC (1.5 min). Each determination was done in triplicate.
Data was evaluated by analysis of variance (one-way ANOVA) with a p ≤ 0.05. Analyses
were carried out using the statistical package SPSS 2011, version 20.0 (IBM Corp.,
Armonk, USA).
Since the physical properties of the culture medium, the aeration rate, agitation related to
local turbulence, and power consumption are relevant variables involved in the
hydrodynamics and the mass transport processes of the reactor (Chisti, 1989), we related
superficial gas velocity (UG) to the mass transfer parameters of oxygen and HXD. Table 1
shows that given a high dispersed phase fraction (φ) and considering a constant Sauter
12
mean diameter (d32HXD) in the whole ALB, a higher number of droplets can be obtained by
increasing UG. Thus, the specific area for mass transfer of HXD (aHXD) could be increased.
This phenomenon may be explained by means of the turbulence regime in the ALB. Fig. 2
displays the behavior of the overall gas hold-up (εg) as a function of power input (P/V),
where εg increases as power input increase. The low surface tension of the oil-water
mixtures could reduce the coalescence between bubbles, which increase their residence
time inside the reactor that may result in higher liquid velocities (Shariati et al., 2007) and
promotes a higher driving force for recirculation (Mehrnia et al., 2005). Recently, Tec-
Caamal et al. (2018) demonstrated that the liquid velocity differences between aqueous and
oil phases, obtained as a result of the differences of hold-up between the two sections of the
systems. Our εg values were slightly lower than those reported by Shariati et al. (2007)
probably due to a higher cross-sectional area (Ad/Ar=1.56) and the different configuration
of the sparger (fewer holes) used in this work. Furthermore, the relationship of d32HXD with
Reynolds differences between the aqueous and oil phase (ReAq-HXD) was demonstrated by
Lizardi-Jiménez et al. (2012), who reported a smaller droplet size in the transition regime
(4000 ≥ ReAq-HXD ≥ 2000). In our system, Reynolds differences between the aqueous and
oil phase (ReAq-HXD) calculated according to Lizardi-Jiménez et al. (2012) were in the
transient regime at UG of 1.5 cm s-1, while for a UG of 2.5 and 3.5 cm s-1 they were in the
turbulent regime for both the riser and downcomer (Table 1). From P/V = 96 to 134 W m-3,
Reaq-HXD changed only 2% for the riser and 1% for the downcomer, which implies that for
UG > 2.5 cm s-1 an increase of UG is not necessary to achieve similar turbulent regimes in
the whole ALB. An extensive discussion of the effect of the dispersed phase fraction on the
turbulence regime of our bioreactor setup was published in a previous study (Tec-Caamal et
13
al., 2018). Those authors shows that because d32HXD depends strongly on turbulence,
however, the cited reference clearly shows that d32HXD decreases linearly with Ug, and Re
stabilizes for Ug greater than 2.5 cm/s. Therefore, d32HXD cannot be inferred by Re when Ug
>2.5cm/s. In other hand, no statistically significant differences were observed between the
Reaq-HXD of both zones of the ALB for each UG tested, it was possible to establish a similar
size of HXD droplets in the whole bulk (riser and downcomer included) (Lizardi-Jiménez
et al., 2012; Tec-Caamal et al., 2018), and the calculated aHXD were used for evaluating the
HXD mass transfer parameters in the ALB. Table 1 shows the relationship of d32HXD with
power input. As expected, the turbulent regime presented a lower d32HXD and therefore a
larger aHXD. This could be because the high UG led to an increase in the bubble rise
diameter. Recently, Angeles et al. (2017) reported similar macrodroplet sizes of diesel,
operating the ALB under a similar range of superficial gas velocities and lower φ.
According to Eq. (6), a highly dispersed phase fraction will result in a larger specific area
per unit reactor volume, due to the greater number of droplets. The increase in the specific
area of HXD for mass transfer resulted in an increase in the volumetric mass transfer
coefficient of HXD (kLaHXD), reaching initial values between 0.353 ± 0.022 and 0.147 ±
0.011 h-1. Additionally, kLaO2 values between 73.89 ± 2.32 and 130.82 ± 0.60 h-1 were
obtained for the increased range of UG. These results suggest that the mass transfer of HXD
to the aqueous phase is still the limiting step in the process (Quijano et al., 2010; Barnett et
al., 1974). As depicted in Fig. 3, the (kLaHXD)/(kLaO2) ratio decreases over time, which
indicates that the mass transfer limitation of both substrates could be accentuated through
kinetics. The kLaO2 data were at least two orders of magnitude higher the kLaHXD results on
each day. Therefore, it is necessary to study new ways of improving hydrocarbon mass
14
transfer parameters, to enhance the bioavailability of immiscible substrates in ALBs for
bioremediation purposes. Due to the importance of considering the HXD and O 2 uptake
during the culture (involved in the SCR), it is necessary to simultaneously evaluate the
mass transfer rates of both substrates (Lizardi-Jiménez et al., 2012), referred to henceforth
Table 1. Initia a tran fer para eter of HXD (da 0). Re ≥ 4000; t r ent re i e.
Turbulence Number of
UG (cm s-1) P/V (W m-3) d32HXD (cm) aHXD (cm-1)
regime droplets
1.5 57.4 Transient 0.099 ± 0.003 464 226 6.13 ± 0.46
2.5 95.7 Turbulent 0.075 ± 0.002 1 055 214 8.08 ± 0.55
3.5 133.9 Turbulent 0.065 ± 0.001 1 603 049 9.30 ± 0.58
15
Fig. 3. The (kLaHXD)/(kLaO2) ratio as a function of time during batch cultures at constant UG.
Due to the low solubility of hydrocarbons in water, oil mass transfer limitations
seem to be more important than oxygen in ALB biodegrading processes. To estimate this
simultaneously evaluate HXD and oxygen mass transfer rates during kinetics, by means of
the calculation of the HTR/OTR ratios at different time points of the culture and compared
with the theoretical stoichiometric consumption ratio (SCR), with a value of ~0.28 (gHXD)
(g O2)-1. HTR/OTR values were representative of the whole ALB and applicable for real
cultivation, since the Reaq-HXD for both riser and downcomer were similar and showed the
same increasing trend (section 3.1). As expected, the highest HTR/OTR ratios were
16
obtained at day 0, where the dispersed phase fraction was maximal, reaching initial
HTR/OTR ratios of 0.237 ± 0.057, 0.261 ± 0.036 and 0.322 ± 0.030 (gHXD) (g O2)-1 for
the increased range of UG. As shown in Fig. 4, these values decreased to reach final values
of 0.173 ± 0.013, 0.108 ± 0.006 and 0.123 ± 0.006 (g HXD) (g O2)-1, which indicate a
decrease of 27, 59 and 62% for low, moderate and high UG, respectively. Nevertheless,
HTR/OTR never reached 0, since HXD was not exhausted through the 14 days of culture.
Due to the direct relationship of the aHXD to HTR, our approach suggests the use of high φ
values on ALB operation to reach HTR/OTR ratios close to the SCR to avoid mass transfer
Lizardi-Jiménez et al. (2012) proposed that the balanced stoichiometric equation for
which indicates that 1 gram of oxygen is required for the oxidation of 0.28 grams of HXD.
HTR/OTR values below of 0.28 g HXD (g O2)-1 could point out HXD mass transfer
limitation, which could impact on both HXD uptake and SS production (Medina-Moreno et
al., 2005; Tec-Caamal et al., 2018). The initial HTR/OTR was close to the SCR values, for
all constant UG assayed. This, in turn, could imply a system not limited by mass transfer of
both substrates. However, these HTR/OTR values only occurred once during cultivation
(Fig. 4). Since mass transport phenomena are related to biodegradation processes (Quijano
in the ALB. On one hand, the HTR/OTR ratios were enhanced compared to the results
obtained by Lizardi-Jiménez et al. (2012), who reported HTR/OTR ratios lower than 0.025
g HXD (g O2)-1 through kinetics. Those authors remark that lower mass transfer ratios may
17
result in lower SS production. On the other hand, our initial HTR/OTR values were
between three and four times higher than those reported by Angeles et al. (2017) in ALB,
using diesel as carbon and energy source. That study indicates that the use of low φ values
(0.016) may lead to HTR/OTR maximum ratios between 0.06-0.08 (g HXD) (g O2-1)-1, in a
similar range of UG tested in our study. An examination of HTR and OTR separately could
explain the improvement of the ratio. First, the kLaO2 was enhanced due to the use of a high
initial dispersed phase fraction, compared with previous studies (Angeles et al., 2017;
Lizardi-Jiménez et al., 2011). This, in turn, would allow us to obtain an increase in the
driving force ( CO* 2 CLO 2 ) (Garcia-Ochoa and Gomez, 2009). Secondly, HTR was
augmented through a double effect: (i) obtaining a high kLaHXD product, and (ii) an increase
*
in the gradient ( CHXD CLHXD ). kLaHXD was not assumed to be constant during the culture
since the dispersed phase fraction diminished as the culture proceeded. Additionally, mass
transfer driving forces between 0.10-0.92 g L-1 were obtained for all UG tested. Lower
*
gradients ( CHXD CLHXD ) were obtained in a previous study (Lizardi-Jiménez et al., 2012).
According to Pirollo et al. (2008), the production of biosurfactants can increase the
saturation concentration of HXD in the aqueous phase. Barnett et al. (1974) suggested that
the production of surface-active compounds can increase the mass transport of hexadecane
from large droplets to microdroplets, through the formation of micelles that act as transport
substrate.
The improvement of the mass transfer of HXD was supported by paying attention to
a modified Damköhler number (Da), which is defined as the ratio of the reaction rate
(maximum uptake rate) to the mass transfer rate (maximum HTR, day 0) (Eq. 7). If Da > 1,
18
the process is mass transfer limited, where a large consumption rate or low diffusivity is
presented; conversely, Da ≤ 1 indi ated a low consumption rate or high diffusivity, and
suggests a process limited by a biochemical reaction rate (Oliver-Salvador et al., 2013). The
obtained Da numbers were between 24 and 39, which suggest a greater consumption rate
compared to the HTRmax; this indicates that our process is still limited by the mass transport
of HXD. Notwithstanding, our Da numbers were lower than those observed in other studies
(110 > Da > 347) that evaluated SS production (Lizardi-Jiménez et al., 2012) and the HXD
uptake mode (Denis et al., 2017; Angeles et al., 2017) in a three-phase ALB at low
dispersed phase fraction (φ < 0.02). Those authors observed a greater mass transfer
limitation of hydrocarbon.
However, HTR/OTR decays at values lower than SCR, which point to a greater mass
transfer limitation over time. This decrease in the mass transfer rates may be explained by a
decrease in kLaHXD, due to the consumption of HXD, which diminishes the number of HXD
droplets and therefore decreases the aHXD. Additionally, kLaO2 decreased because of the
diminished dispersed phase fraction, which acts as an oxygen reservoir; this may reduce the
driving force term ( CO* 2 CLO 2 ) (Tec-Caamal et al., 2018). The effect of the mass transfer
parameters was evaluated on the production of the oil-degrading bacterial consortium and
19
Fig. 4. Quotient of the mass transfer rates of HXD and oxygen as a function of time for
performance, SS production and HXD uptake rate at three different constant UG (1.5, 2.5,
3.5 cm s-1) were evaluated. As shown in Fig. 5, HXD was not depleted at each UG assayed,
reaching final concentrations of: 48.43 ± 6.00, 47.74 ± 2.18 and 30.20 ± 0.39 g HXD L-1 for
low, moderate, and high UG, respectively. As depicted in Fig. 6, the SS concentration at the
end of the culture was higher for moderate and high UG. SS productivity and maximum SS
production (at inflection point) were 0.63 ± 0.01 g SS (L d) -1 and 4.91 ± 0.76 g SS (L d)-1,
respectively, for low UG; 0.80 ± 0.003 g SS (L d)-1 and 6.09 ± 0.003 g SS (L d)-1,
respectively, at moderate UG; and 0.74 ± 0.001 and 4.95 ± 0.006 g SS (L d) -1, respectively,
at higher UG. The observations are in line with previous studies, which have used diesel
that an increase of φ from 0.001 to 0.014, promotes an increase of productivity from 0.17 to
0.71 g SS (L d)-1, but not for maximum SS production. In our study, compared with the
studies, between 4-7 days were needed for degrade 13 grams of hydrocarbon. Our results
show that 6 days were required to biodegrade 27, 31 and 45 grams of HXD at UG of 1.5,
2.5 and 3.5 cm s-1, respectively. Moreover, our results indicate a higher cell yield
coefficient for the middle UG (0.41 g SS g HXD-1) compared with the low and high UG
(0.34 and 0.24 g SS g HXD-1, respectively), which indicates similar SS production with a
marked difference of HXD consumption for all UG. Although the higher biomass yield
coefficient was observed at middle UG, the higher UG present the higher HXD
consumption. This phenomenon may be explained by the increase of the interfacial specific
area of the macro-droplets of HXD under high UG, at which, mass transfer to the emulsified
21
Fig. 5. HXD consumption as a function of time using a constant superficial gas velocity.
reduce the surface tension of the culture medium (Angeles et al., 2017). As depicted in Fig.
7, bioemulsifiers were produced throughout the culture, which could lead to a decrease in
the surface tension with respect to water during the 14-day process, with final values of
69.5, 68.3, 65 dynes cm-1 for the increased range of UG (3-10% variation). These
emulsifiers (possibly emulsan) were probably released by one of the strains of the microbial
demonstrates that emulsifiers were necessary for HXD uptake via the formation of
22
pseudosolubilized oil microdroplets (Pirollo et al., 2008). It is important to remark that high
and moderate UG presented the higher emulsification capacity from day 6 to 14, which
achieve the highest HTR/OTR ratios through the culture, which could allow for obtaining
Recent studies have shown that it is necessary to consider the main mechanisms of
oil consumption (emulsified and direct interfacial uptake) in ALB performance, since mass
transport phenomena of hydrocarbons to the aqueous phase cannot explain the total
consumption of oil (Hua and Wang, 2014; Medina-Moreno et al., 2013). Therefore, studies
The transition regime flows in the ALB obtained at a UG of 1.5 cm s-1 (Table 1)
indicates the possibility of HXD mass transfer limitation (Lizardi-Jiménez et al., 2011).
However, although low emulsification capacity was observed for low UG, similar HXD
emulsified forms have indicated that the specific area for mass transfer is an important
factor to consider in ALB performance, since the rate of hydrocarbon transfer is related to
the amount of HXD that is emulsified from large droplets with a given d32HXD.
With the aim of taking into account the HTR/OTR ratio on the production of the oil-
(2012) was used. The results of the sequential-batch kinetics at constant UG, were used to
establish the variable UG setup, considering the maximum SS production and the
HTR/OTR ratio.
23
Fig. 6. Production of suspended solids as a function of time using constant UG during ALB
operation.
Fig. 7. Surface tension of UG: 1.5 (■), 2.5 (●) and 3.5 (▲) cm s-1 ; and emulsification
capacity (OD600) of UG: 1.5 (□), 2.5 (○) and 3.5 (△) cm s-1 as a function of time.
24
3.4 Biomass production and HXD uptake rate at variable UG
The operation of the ALB at high superficial gas velocities may lead to high power
inputs (Table 1). Therefore, since moderate UG presented higher values of productivity and
maximum SS production, the process with variable UG was started at a UG of 2.5 cm s-1.
Afterwards, the UG was decreased to 1.5 cm s-1. This change was made considering that
comparable maximum SS production was obtained at UG of 1.5 and 3.5 cm s-1, (with a
difference of 76.5 W m-3, Table 1), and similar consumption of HXD was achieved at
moderate and low UG. At Ug = 2.5 cm s-1 SS production is maximum and plateau is
reached with 2 days. However, even in plateau, SS production shows a soft increase in both
Ug = 2.5 and 1.5 cm s-1. Then Ug = 2.5 cm s-1 was used for first 2 days and 1.5 cm s -1 after
day 2 due to less P/V is required. Besides, it is important remark that low UG presented the
highest HTR/OTR ratios. The use of this strategy can reduce the energy consumption
(Lizardi-Jiménez et al., 2012). The variable UG technique was used as follows: a UG of 2.5
cm s-1 was kept until day 2, from this point the UG was decreased to 1.5 cm s-1 and
maintained until the end of the culture. As shown in Fig. 8, the HTR/OTR ratio decreased
to 0.143 ± 0.009 at day 2 and 0.038 ± 0.002 g HXD (g O2)-1 at the end of the culture.
Although our HTR/OTR ratios still displayed an HXD mass transfer limitation (HTR/OTR
< SCR), our values were at least 22 times higher at the beginning of the culture and up to
50 times higher in the following days of the kinetics compared with the values obtained by
Lizardi-Jiménez et al. (2012) using the variable UG technique. Contrary to the conditions
used for ALB operation proposed by those authors, a decrease in UG through the kinetics
was used in our study because the highest HTR/OTR ratios were obtained at low UG.
times higher than hydrocarbon saturation concentration (C* = 0.7 -1.1 g L-1) then system
shows driving force along culture time. In other hand, the fact than surface tension decrease
as culture time increase, even when cells are in plateau phase, should indicate that the cells
try to maintain more HXD in HXD droplets. Emulsification increases as culture time due to
surface tension decrease, leading more HXD in emulsified phase. In both cases here is
technique. Maximum SS production was higher (5.48 ± 0.002 g SS (L d) -1) than that
obtained using constant a UG of 1.5 and 3.5 cm s-1, but lower than that obtained at a UG of
2.5 cm s-1. Although the emulsification capacity was lower (Fig. 7), the HXD consumption
was similar to that obtained at a UG of 2.5 cm s-1. Although low productivity was observed
(0.66 g SS (L d)-1) compared to moderate UG, our study suggests that it is possible to
achieve stable SS production and HXD consumption using the variable UG technique,
which consumed 33.4% less energy. Our results reveal that the quotient of the mass transfer
rates of HXD and oxygen could be used as a criterion to enhance the productivity and
26
Fig. 8. HTR/OTR and OD600 as a function of time using the variable UG technique.
Fig. 9. Production of suspended solids and HXD consumption as a function of time using
27
4. Conclusions
Our work provides relevant insights into the mass transport processes of both the oil and
a high concentration of the organic phase (77 g L-1). These improvements in mass transfer
of HXD and oxygen (SCR) and the actual HTR/OTR ratio of the process. An improved
consortium at constant and variable UG. At the beginning of the culture and under constant
UG, the HTR/OTR ratios were close to the SCR since at time 0 the driving force for mass
*
transfer of HXD ( CHXD CLHXD ) and the specific area for mass transfer (aHXD) was maximal.
The variable UG technique, which enabled the use of less energy, was used successfully to
Acknowledgment
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Highlights
Mass transfer rates of hexadecane and oxygen were close to the stoichiometric
34