Continuous cultivation of Debaryomyces hansenii (LAF-3 10 U) on dodecane in synthetic desalter effluent at varying dilution rates on dodecane

Desalter effluent (DE) is typically discharged into a petroleum wastewater treatment plant, but its high salt concentration deteriorates the biological treatment. This study used various dilution rates to investigate the treatment of a synthetic DE containing dodecane under saline conditions using a halotolerant yeast, Debaryomyces hansenii , to determine the optimum substrate concentration for use in continuous stirred-tank reactors (CSTRs). A literature review indicated that this study was the first to examine the biological treatment of DE using D. hansenii in a CSTR system. At a low dode-cane substrate concentration, DE did not inhibit D. hansenii growth, and the experimental data approached the Monod model, with μ max and K s selected as 0.08 h (cid:1) 1 and 1575 mg L (cid:1) 1 , respectively. The optimum removal of chemical oxygen demand (95.7% and 85%) was obtained at dilution rates of 0.007 and 0.026 d (cid:1) 1 . Using D. hansenii in a CSTR system appeared to be a sustainable approach for the biological treatment of DE. Scale-up of these laboratory findings to the industrial scale is required to confirm that petroleum DE can be treated using equalization and filtration tanks as a continuous bioreactor. Adjusting the dilution rate can provide sufficient time for biodegradation and hydrocarbon removal from high salt DE by haloto-lerant yeasts like D. hansenii .

four Arabian crude oils determined salt concentrations of 34.2, 28.5, 14.3 and 5.71 mg L À1 (Aleisa & Akmal, 2015), while another study reported a range of sodium chloride concentrations of up to 250 000 mg L À1 (Bahadori et al., 2012). Another investigation determined that the amount of salt in the form of NaCl in crude oil was 451.4 mg L À1 (Cunha et al., 2008). In general, the salts in crude oil are mainly NaCl and the chlorides and sulfates of calcium and magnesium, such as MgCl 2 , CaCl 2 and MgCl 2 . Chlorides hydrolyze to hydrochloric acid and cause severe corrosion in wastewater treatment components (Pereira et al., 2015). The amount of NaCl in DE at the Imperial Oil facility in Sarnia, ON, was measured at 1000 mg L À1 in a previous study .
Several approaches have been reported for desalter treatment (Al-Otaibi et al., 2005). For example, Pak and Mohammadi (2008) described the use of membrane distillation (MD) and polymeric Teflon microfiltration, which fulfilled the high flow rate required by irrigation water standards (Pak & Mohammadi, 2008). Recently, the installation of an ultrasonicelectric (desalting and dewatering) unit before the desalter gave a salt reduction rate of 94% (Ye et al., 2008) Similarly, the use of reverse osmosis membranes reduced DE salinity from 143 054 to 842.8 mg L À1 and removed up to 99% of the metals (Bijani & Khamehchi, 2019) Other approaches aimed at improving the desalination process and preventing the formation of a rag layer (an undesirable mixture of dispersed oil, water and solids, salt) have included temperature adjustments, fresh water injection, emulsification, application of electric fields and gravity settling (Xu et al., 2006) or withdrawing sample electrically until the result of sampling demonstrates acceptable water-oil separation (Daage et al., 2018).
Many of these approaches, including the use of membranes, incur high maintenance costs, necessitate the replacement of spare parts and require high energy consumption. Furthermore, physicochemical methods are capable of lowering contamination but not eliminating it (Brunori et al., 2005;Cox, 2006;Dabrowski et al., 2004;Mamindy-Pajany et al., 2011). Thus, alternative, cost-effective solutions are needed.
One possible strategy is to treat wastewaters with halotolerant organisms, such as Debaryomyces hansenii, a halotolerant yeast that can effectively metabolize several hydrocarbons. The application of D. hansenii in the treatment of challenging wastewater streams, such as DE, has significant potential, as this yeast can tolerate concentrations of salt (such as NaCl and KCl) as high as 24% (w/v) (Adler et al., 1985) and it is a lipid-accumulating yeast (Breuer & Harms, 2006). D. hansenii is found in salty foods, blue cheese, nd brines, and it has both high respiratory and low fermentative activity (Calahorra et al., 2009;Şahin, 2017). This yeast was previously shown to remove phenols (aromatic compounds)  and dodecane (an n-alkane hydrocarbon) (García-Lugo et al., 2018). Previous studies have shown that high substrate concentrations are inhibitory for yeast cultivation; therefore, continuous cultivation at a steady state in a continuous stirred-tank reactor (CSTR) may be an effective way to control specific growth rates and avoid substrate inhibition. However, only a few studies have investigated the bioremediation of petroleum refinery wastewater in CSTRs by yeast (Gargouri et al., 2015) and bacteria (Gargouri et al., 2011(Gargouri et al., , 2014Jamal & Pugazhendi, 2018;Mizzouri & Shaaban, 2013;Patel & Kumar, 2016;Pugazhendi et al., 2017a;Pugazhendi et al., 2017b;Sponza & Gök, 2010).
In the present study, the cultivation of D. hansenii in a synthetic desalter effluent (SDE) was investigated in a laboratory-scale CSTR using dodecane as a substrate at different dilution rates. The overall aim was to determine the conditions for optimum substrate removal.
The degradation kinetics were modelled, and the kinetic constants were determined.

| Materials
The synthetic desalter components in the mineral medium solution (Van Dijken et al., 1976) are described in Table 1. Dodecane was the only yeast-degradable carbon source used in this study. Tween 20 (a nonionic surfactant) was added to ensure the solubility of the nonpolar hydrocarbon dodecane into the aqueous SDE medium to allow the yeast (D. hansenii) to access this carbon source.
2.1.1 | Chemical composition of the synthetic desalter growth medium All chemicals used in this study were of analytical grade and purchased from Sigma Aldrich (Oakville, Canada). The SDE in 1 L distilled water was prepared as described in Table 1. The amount of NaCl in Imperial Oil DE from Sarnia, ON, was measured by Maxam Laboratories at 1000 mg L À1 ; therefore, the same salt concentration was used in the SDE. The pH of all growth media was adjusted to range between 6 and 6.5. All growth media were autoclaved at 121 C for 20 min before yeast cultivation. Dodecane, as a petroleum hydrocarbon representative, was added to SDE at T A B L E 1 Phase 1 of the synthetic desalter effluent components with dodecane. concentrations of 750 mg L À1 . The theoretical oxygen demand (ThOD) for the oxidation of 1 g of dodecane is 3.43 g (Pitter & Chudoba, 1990). The equivalent of ThOD for dodecane (750 mg L À1 )

Mineral medium component
is 2610 mg L À1 and is within the range of the chemical oxygen demand (COD) in Imperial Oil DE (2500 mg L À1 ). In a typical DE, the total free hydrocarbon content has been reported to be as high as 1000 mg L À1 (Barthe et al., 2015;IPIECA, 2010

| CSTR bioreactor
Hydrocarbon biodegradation was studied in a bioreactor (shown in Figure 1) with a total volume of 1000 mL and a working volume of 500 mL for continuous experiments. The reactor was a Wheaton™ Celstir™ Spinner Flask. Aeration for yeast growth was provided by an impeller and injection from the top of the reactor. The bioreactor was fed continuously with SDE containing dodecane as the sole carbon source.
The reactor was inoculated with 250 mL of precultured (enriched culture) D. hansenii yeast suspension and filled with SDE. The continuous feed was started at dilution rates (D) in the range 0.007-0.045 h À1 . SDE was supplied at the bottom of the reactor using variable flow mini-and ultra-pumps (VWR, 120 mL to 2.2 L min À1 ). Sterile air was passed into the continuous reactor, and the temperature in the storage feed tank was maintained at 25 C. The OD and COD were measured by sampling once a day from the reactor using a sterile syringe. The reactor was monitored by analysing the COD and biomass concentrations. The pH of the culture medium was controlled at pH 6.5 ± 0.3.

| Growth kinetic modelling in the CSTR
In the CSTR setup, a substrate solution (dodecane as the carbon source) is pumped continuously into the reactor, and the culture medium is withdrawn from the bioreactor at the same flow rate.
Therefore, under steady-state conditions, all parameters of the process (volume, reaction rate, concentrations of the substrate, product, biomass, etc.) remain the same and do not change over time. Monod proposed a relationship between μ and S. The Monod equation is shown in Equation (1).
where μ is the specific growth rate, μ max is the maximum specific growth rate in the absence of substrate limitation, S is the concentration of the substrate in the reactor (which is equal to the concentration of the substrate in the liquid leaving the reactor) under steadystate conditions and K s is the substrate concentration that allows the organism to grow at 0.5 μ max .
The CSTR was used for aerobic respiration. The material balance of biomass X can be written as follows: where V is the volume of the reactor (which is constant). Considering that the inlet flow usually contains no microorganisms (X 0 = 0), Equation (2) can be rearranged as Equation (3): Schematic of a continuous stirredtank reactor for dodecane removal from synthetic desalter effluent (SDE).
Therefore, Equation (1) (Monod) under steady-state conditions, when dX/dt = 0, gives the following: The maximum specific growth rate μ max of D. hansenii in SDE with dodecane as the only hydrocarbon was obtained with a batch bioreactor, in which μ max is equal to 0.085 h À1 .
To find the relationship between S versus D, K s must be calculated.
The dilution rate was measured based on adjustment of the peristaltic pump speed and measurement of the time required for accumulation 10 mL of SDE in a graduated cylinder. At each flow rate, the growth rate in the CSTR was measured, and then the dilution rate was calculated. The cell concentration decreases as D (the dilution rate) increases, and it reaches zero when all the microorganisms are washed out; thus, there is no biochemical reaction. Therefore, the concentration of the substrate in the reactor equals that of the feed (S 0 ) (Harvey & Blanch, 1997;Liu, 2012).
The CSTR is a powerful tool for studying microbial processes, and it allows for the control of a specific growth rate by controlling the dilution rate. The microorganisms in the continuous reactor are almost always in the exponential phase of growth. A change in the flow rate of the substrate solution results in a change in D, because D = F/V. If the dilution rate is changed and the substrate concentration in the reactor is measured as a function of the dilution rate, Equation (6)  can be estimated as the ThOD, which, for the oxidation of 1 g of dodecane, was 3.43 g (adapted from Pitter & Chudoba, 1990). A previous study noted that linear correlations exist between total organic carbon (TOC) and COD. (Dubber & Gray, 2010). In this study, because n-dodecane was the only organic carbon source metabolized by the yeast in the SDE, COD was used as a proxy for measuring ndodecane concentrations. The COD measurement can then be directly related to the DE, which is a mixture of Tween 20, SDE and aliphatic compounds. Thus, the total COD in the SDE is a combination of all components and can be determined by Equation (7): where SDE represents the SDE, DD represents dodecane and τ is Tween 20.
Because no organic carbon is present in the SDE, a linear regression relationship can be proposed between COD Total , the TOC of Tween 20 (τ) and n-dodecane ( D ): Our preliminary experiments indicated that Tween 20 alone was not significantly metabolized by the yeast; therefore, Equation (8) can be modified as follows: 2.1.8 | Measurement of n-dodecane removal Dodecane removal was evaluated using Equation (9) and the following formula: where COD i is the influent dodecane concentration (Feed) before adding the yeast and COD e is the final dodecane concentration in the effluent tank.

| Modelling the kinetics of SDE
The Monod equation can be applied to microorganism growth kinetics under steady-state conditions. Because accurate determination of μ max by examining a rectangular hyperbola is difficult, establishing the value of the K s in this way is also difficult. The Lineweaver-Burk plot is the most common linear plot used for growth kinetic analysis by fitting experimental data to the rate equations. When linear data are desired, the reciprocal of the Monod equation plot (Lineweaver-Burk plot) is obtained for illustrative purposes based on kinetic constants (Engelking, 2015;Roskoski, 2011). Linear Equation (11) is obtained by the inverse Monod equation: The Monod equation explains the growth of D. hansenii and the utilization of the substrate in the SDE. When the concentration of the substrate is below the high level that causes inhibition of yeast growth, the maximum specific growth rate and saturated constant can be obtained from the Lineweaver-Burk plot.

| Effect of the dilution rate (D)
The dodecane concentration is one of the important parameters that is monitored to improve the removal efficiency of the treatment system. Table 2 shows the operation parameters, steady-state data, and dodecane removal by COD, as determined throughout the SDE. In batch mode, to determine the effect of Tween 20 on yeast growth, a control experiment was conducted on SDE with Tween 20 but without n-dodecane. The control experiment containing no n-dodecane produced a significantly lower OD 600 value, indicating that yeast could not metabolize Tween 20 considerably and showed an insignificant effect on yeast growth (data not shown). The reactor contains dodecane and SDE/Tween, but no added yeast was carried out in the preliminary study. Preliminary experiments revealed a Dodecane removal efficiency in the range of 85%-95%, when the dilution rate ranged between 0.007 and 0.026 h À1 . However, a dilution rate of 0.007 h À1 was considered best for a CSTR design due to observed fluctuations in the organic load under different conditions. A higher hydrocarbon load could be mineralized and significantly reduced when D = 0.007 h À1 .
The maximum growth rate of D. hansenii was attained during the treatment of SDE in a batch reactor, with a maximum COD of 61% in  The biodegradation rate was gradually increased to 95% at a steady state when the dilution rate was increased to 0.07 h À1 . Table 2 summarizes the change in COD removal and the dilution rate with an initial substrate concentration of 750 mg L À1 . The D was based on the flow rate of the effluent passing through the pump to the bioreactor.  Thus, at a dilution rate of 0.007 h À1 , dodecane was completely utilized, and the maximum cell concentration (OD 600 ) was 0.506 ± 0.03.
Because dodecane was completely utilized at this dilution rate, a maximum COD removal (95%) was observed from 2793 to 136 mg L À1 . Operating the process with decreasing D (0.045, 0.035, F I G U R E 2 Steady-state values of the concentrations of dodecane (□) and chemical oxygen demand (COD) removal () for different dilution rates (D) using Debaryomyces hansenii in a continuous stirred-tank reactor (CSTR). Each data point is the average of triplicate measurements ± standard deviation.

F I G U R E 3
The change in the average cell concentration of Debaryomyces hansenii in synthetic desalter effluent (SDE) at different dilution rates (D) during the continuous aerobic bioreactor operation. Data points are the mean of triplicate readings from a single experiment. The instrument error was 5% of the measured value.
0.031, 0.026 and 0.007 h À1 ) revealed complete utilization of the dodecane, as the COD decreased from 2793 to 1000, 733.3, 618.7 and 136 mg L À1 , respectively (Figure 4). Because dodecane (equivalent to COD) was completely utilized at 0.007 h À1 , subsequent experiments were not conducted at dilution rates lower than 0.007 h À1 .

| Growth kinetic modelling
The substrate concentration used here was not high enough to inhibit the growth of D. hansenii. Therefore, the growth of cells at different dilution rates follows the Monod equation (as shown in Figure 5a). We measured the maximum growth rate and K s , using the experimental data from the Lineweaver-Burk plot. This plot linearizes the hyperbolic curved relationship, and the line produced is easy to extrapolate, allowing for the evaluation of D max = μ max and K s . As we only obtained five data points from the complete curve of substrate versus D, we would have difficulty estimating D max from a direct plot, as shown in Figure 5a. However, as shown in F I G U R E 5 (a) Relationship between the substrate concentration and the rate of growth of Debaryomyces hansenii reaction; (b) Lineweaver-Burk plot of the same kinetic data. Each data point is the average of triplicate measurements ± standard deviation. COD, chemical oxygen demand; D, dilution rate.
Based on the experimental data and the model in Equations (11) and (12), μ max is 0.08 h À1 and K s(Lineweaver-Burk) = 1575.2 mg L À1 (1.575 g L À1 ), and μ max and K s can be applied as the saturated constants of the Monod equation.
Therefore, the kinetic model for SDE, when dodecane is present as an alkane hydrocarbon at a concentration of around 750 mg L À1 or less, is shown in Equation (13): When we observe other kinetics constants in Table 3, it helps us to analyse profoundly and chose the right model. Saudi Arabia under a saline condition by a halophilic bacteria consortium in a CSTR system. They obtained a 94% COD removal after 12 days when the initial concentration of polycyclic aromatic hydrocarbon was 100 mg L À1 and the NaCl concentration was 40 g L À1 (Jamal & Pugazhendi, 2018). In another study, up to 93% of the initial COD of 1025 mg L À1 in petrochemical wastewater was removed within 21 to 25 days using a biosurfactant rhamnolipid in a continuous bioreactor (Sponza & Gök, 2010). In our study, a COD removal of 95% was achieved at an initial COD of 2793 mg L À1 . The petroleum hydrocarbons consisting of C10-C35 n-alkanes were strongly degraded (COD removal of 97%) after 200 days by a microbial consortium in continuous aerobic treatment in a CSTR (Gargouri et al., 2012).

| CONCLUSION
A continuous bioreactor was investigated to determine its capacity for the removal of dodecane in SDE using D. hansenii as the halotolerant organism. A different D for one specific type of wastewater (all components of SDE were kept constant when D was changed) has a profound effect on the efficiency of dodecane removal. The CSTR system showed excellent efficiency for dodecane removal, with a COD removal of 95% (99.9% dodecane removal). The optimum D for the bioreactor was 0.007 h À1 and was below the maximum growth rate at which the washout of the yeast D. hansenii

This study was supported by an Imperial Oil University Research
Grant awarded to Amarjeet Bassi. The funding support is gratefully acknowledged.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.