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Accepted Manuscript

Production of an oil-degrading bacterial consortium in an airlift bioreactor: In-


sights into the mass transfer of the oil and oxygen

Edgar N. Tec-Caamal, Angélica Jiménez-González, Sergio A. Medina-Moreno,


Manuel Alejandro Lizardi-Jiménez

PII: S0009-2509(18)30551-7
DOI: https://doi.org/10.1016/j.ces.2018.07.056
Reference: CES 14410

To appear in: Chemical Engineering Science

Received Date: 16 March 2018


Revised Date: 25 June 2018
Accepted Date: 27 July 2018

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

mass transfer of the oil and oxygen.

Edgar N. Tec-Caamala, Angélica Jiménez-Gonzáleza, Sergio A. Medina-Morenoa, Manuel

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

Puebla, Colonia Pemex, C.P. 95180, Tierra Blanca, Veracruz, México

*Corresponding author: chamarripas@yahoo.com.mx

Abstract

The production of oil-degrading bacteria in multiphase airlift reactors (ALBs) could be

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

production of an oil-degrading microbial consortium.

Keywords: Mass transfer rate; stoichiometry; hexadecane; oxygen; airlift bioreactor; oil-

degrading consortium.

1. Introduction

An airlift bioreactor (ALB) is a pneumatic contacting device agitated by a continuous gas

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

systems (García-Ochoa and Gomez, 2009). The production of oil-degrading bacteria


2
(microbial consortium) for environmental purposes often involves the use of insoluble

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

monitoring and well-known biodegradability (Volke-Sepúlveda et al., 2006). When HXD is

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,

microorganisms that use alkanes as an energy source are capable of producing

bioemulsifiers. These molecules can pseudo-solubilize hydrocarbons in the form of

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

production. The production of oil-degrading bacterial consortiums depends on the

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

limiting step in biomass formation and alkane biodegradation is hydrocarbon transfer

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

the mass transfer parameters of hydrocarbons (Jiménez-González et al., 2015; Lizardi-

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

bioremediation purposes (Lizardi-Jiménez et al., 2011). Lizardi-Jiménez et al. (2012),

working with a hydrocarbon-degrading consortium in an ALB, have recently established


3
the relationship between hydrodynamics and simultaneous mass transfer of HXD and

oxygen (evaluated as a ratio, HTR/OTR), considering the stoichiometry to improve the

production of suspended solids (SS). However, the conditions (hydrodynamics and

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-

Jiménez et al., 2012).

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

variable UG technique as suggested by Lizardi-Jiménez et al. (2012) to improve the

production of an oil-degrading microbial consortium and for energy saving during a 14-day

batch culture.

2. Experimental

2.1 Materials

2.1.1 Microbial consortium and culture conditions

4
A microbial consortium composed of four bacterial species (Acinetobacter bouvetii,

Shewanella sp., Defluvibacter lusatiensis, Xanthomonas sp.) was obtained from the

Metropolitan Autonomous University (México). The microbial consortium was isolated

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)

as the sole carbon source. The initial pH was adjusted to 6.5.

2.1.2 Bioreactor set-up

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.

2.2 Analytical methods

2.2.1 Suspended solids

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.

2.2.2 Surface tension

The change in the surface tension of the microbial culture through kinetics was determined

with a bubble pressure tensiometer (SensaDyne, Chem-Dyne Research Corp., USA).

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

physicochemical properties of a hydrocarbon-degrading culture broth was used in this

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

medium was used for hydrodynamic and mass transfer determinations.


6
Volumetric power input (P/V) was determined as a function of the superficial gas

velocity, liquid density and the ratio of the cross-sectional areas of the riser and downcomer

of the ALB, as follows (Chisti and Moo-Young, 1989):

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

velocity (m s-1), g = gravity acceleration (m s-2), Ad = cross-sectional area of the

downcomer (m2), Ar = cross-sectional area of the riser zone (m2).

The overall gas hold-up was calculated according to the volumetric expansion

method proposed by Chisti (1989):

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.

2.2.4 Oxygen mass transfer parameters

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

et al. (2003) was used, by coupling the following Eqs. (3-4):

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-

measured dissolved oxygen concentration (g L-1).

Additionally, to determine the effect of the dispersed phase fraction on kLaO2, the

approximation of Dumont et al. (2006) was used as follows:

   
   
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)

where   VL RT / VG H ; VL is the volume of the emulsion (m3), T is the temperature (K),

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).

φ is the dispersed-phase fraction (dimensionless), yoxygen is the oxygen mole fraction

(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

( CO* 2  CLO 2 ). Oxygen mass transfer parameters were determined abiotically.

2.2.5 HXD transfer parameters and specific area

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

stained with red chili (Capsicum annuum) oleoresin (Bioquimex-Reka, Mexico) to

distinguish between bubbles and droplets of the moving phases by adding 0.061 g of

oleoresin per 1 mL of HXD (Lizardi-Jiménez et al., 2011). A total of 6 photographs were

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,

due to the bioemulsifiers produced by the microbial consortium, the saturation

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

settling, 2 μL samples were analyzed by gas chromatography in triplicate.

2.2.6 Damköhler number

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

Damköhler number, as follows (Merchuk and Gluz, 2002):

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.

2.2.7 Emulsification capacity and HXD degradation

11
The presence of HXD emulsifying agents in aqueous phase was determined in triplicate

a ordin to the ethod e ted o he - a ta i and ande a tee e (2008). Briefly,

cell-free supernatant samples (1.5 mL) were vortexed for 2 in with 200 μL of HXD. The

optical density at 600 nm (OD600) was then registered after 24 h of settling.

Residual HXD was measured by gas chromatography (Thermo Scientific TRACE

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.

2.2.8 Statistical analysis

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).

3. Results and discussion

3.1 Relationship between hydrodynamics and the kLaHXD/kLaO2 ratio

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

ALB, are an important factor that should be considered in three-phase (oil-air-water)

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

velocities, which induced a high degree of turbulence, resulting in a decrease in droplet

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

as HTR and OTR, respectively.

Table 1. Initia a tran fer para eter of HXD (da 0). Re ≥ 4000; t r ent re i e.

4000 ≥ Re ≥ 2000: tran ient f ow. Re ≤ 2000: a inar f ow.

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

Fig. 2. Overall gas hold-up (εg) as a function of volumetric power (P/V).

15
Fig. 3. The (kLaHXD)/(kLaO2) ratio as a function of time during batch cultures at constant UG.

3.2 Stoichiometry and mass transfer rates assessment

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

limitation, a new approach suggested by Lizardi-Jiménez et al. (2012) was used to

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

limitations (Medina-Moreno et al., 2013; Srivastava et al., 2000).

Lizardi-Jiménez et al. (2012) proposed that the balanced stoichiometric equation for

oxidation of HXD by means of a microbial consortium is given by Eq. (8):

CH 2.125  0.06 NO3  1.56O2  0.35CH1.77O0.55 N0.17  0.65CO2  1.81H 2O (8)

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

et al., 2010), an enhancement in HTR/OTR may lead to an improvement in SS production

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

packets of hydrocarbons, which indicates greater bioavailability of the immiscible

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

the biodegradation of HXD.

19
Fig. 4. Quotient of the mass transfer rates of HXD and oxygen as a function of time for

three different constant UG.

3.3 Biomass production and HXD uptake rate at constant UG

To assess the effect of the improvement of the HTR/OTR ratios on ALB

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

(Medina-Moreno et al., 2014) and polycyclic hydrocarbon mixtures (Lizardi-Jiménez et al.,


20
2015) as carbon source in ALB, finding comparable SS productivity. Those studies suggest

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

abovementioned studies, maximum SS productions were enhanced by four-fold. In those

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

form or cell attachment can occur (Denis et al., 2017).

21
Fig. 5. HXD consumption as a function of time using a constant superficial gas velocity.

As proposed in the literature (Barnett et al., 1974), bioemulsifier molecules play an

important role in hydrocarbon biodegradation. The operation of the ALB at a high

dispersed phase fraction could stimulate the production of high concentrations of

emulsifiers (Pirollo et al., 2008). One important property of surface-active compounds is to

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

consortium characterized in a previous study (Tzintzun-Camacho et al., 2012). Fig. 7 also

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

high values of both maximum SS production and HXD uptake.

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

are needed on the hydrodynamics of multiphase bioreactors related to the predominant

mechanism of hydrocarbon uptake by microorganisms in an ALB (Angeles et al., 2017).

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

consumption and maximum SS production compared to UG of 3.5 cm s-1 were observed.

Recent works (Medina-Moreno et al., 2013) studying the consumption of HXD in

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-

degrading bacterial consortium, the variable UG strategy proposed by Lizardi-Jiménez et al.

(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.

HTR/OTR i tate dependent. It depend not on of f id d na i t a o of φ that


25
han e a on t re. However, φ a wa , even at the end of t re, wa aintained h

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

clearly established than not only hydrodynamics affect HTR/OTR.

Fig. 9 shows SS production and HXD biodegradation using the variable UG

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

maximum SS production of an oil-degrading microbial consortium.

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

the variable UG strategy.

27
4. Conclusions

Our work provides relevant insights into the mass transport processes of both the oil and

gaseous phases on the enhanced production of an oil-degrading bacterial consortium, using

a high concentration of the organic phase (77 g L-1). These improvements in mass transfer

were based on a comparison of the suggested theoretical stoichiometric consumption ratio

of HXD and oxygen (SCR) and the actual HTR/OTR ratio of the process. An improved

HTR/OTR ratio was observed during the production of an oil-degrading microbial

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

improve SS production by considering the maximum SS production and productivity, as

well as the HTR/OTR ratio.

Acknowledgment

We acknowledge the Mexican National Council of Science and Technology (CONACyT)

for a scholarship (391507) to E.N. Tec-Caamal.

Declarations of interest: none

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33
Highlights

Mass transfer rates of hexadecane and oxygen were close to the stoichiometric

Damköhler numbers suggested an enhancement of hexadecane mass transport

Production of an oil-degrading consortium was enhanced with mass transfer

34