Biodegradation Kinetic Studies of Phenol and P-Cresol in a Batch and Continuous Stirred-Tank Bioreactor with Pseudomonas Putida ATCC 17484 Cells

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Article Biodegradation Kinetic Studies of Phenol and p-Cresol in a Batch and Continuous Stirred-Tank Bioreactor with Pseudomonas putida ATCC 17484 Cells

Yen-Hui Lin * and Yi-Jie Gu

Department of Safety, Health and Environmental Engineering, Central Taiwan University of Science and Technology, 666, Bu-zih Road, Bei-tun District, Taichung 406053, Taiwan; [email protected] * Correspondence: [email protected]; Tel.: +886-4-22391647 (ext. 6861)

Abstract: The biodegradation of phenol, p-cresol, and phenol plus p-cresol mixtures was eval- uated using Pseudomonas putida ATCC 17484 in aerobic batch reactors. Shake-flask experiments were performed separately using growth medium with initial nominal concentrations of phenol (50–600 mg L−1) and p-cresol (50–600 mg L−1) as well as phenol (50–600 mg L−1) plus p-cresol (50–600 mg L−1). The complete degradation of phenol and p-cresol was achieved within 48 h and 48–56 h, respectively, for all initial concentrations of phenol and p-cresol. The maximum cell growth −1 −1 rate using phenol (µmax,P = 0.45 h ) was much faster than that using p-cresol (µmax,C = 0.185 h). −1 The larger Ki value for phenol (310.5 mg L ) revealed that the P. putida cells had a higher resistance to phenol inhibition compared with p-cresol (243.56 mg L−1). A mixture of phenol and p-cresol in batch experiments resulted in the complete removal of phenol within 52–56 h for initial phenol concentrations of 50–500 mg L−1. The time needed to remove p-cresol completely was 48–56 h for initial p-cresol concentrations of 50–500 mg L−1. In the continuous-flow immobilized cells reactor, the degradation efficiency for phenol and p-cresol was 97.6 and 89.1%, respectively, at a stable condition. Keywords: biodegradation; phenol; p-cresol; batch reactor; immobilized cells; continuous stirred- Citation: Lin, Y.-H.; Gu, Y.-J. tank bioreactor Biodegradation Kinetic Studies of Phenol and p-Cresol in a Batch and Continuous Stirred-Tank Bioreactor with Pseudomonas putida ATCC 17484 1. Introduction Processes 2021 9 Cells. , , 133. https:// Phenol and its derivatives are toxic organic components often found in various doi.org/10.3390/pr9010133 petroleum and chemical industries [1]. Phenol has been regarded as a toxic pollutant to aquatic living organisms imparted as low as concentrations of 0.005 mg L−1 [2]. There has Received: 16 December 2020 been serious environmental concern due to the persistent toxicity of phenol and its deriva- Accepted: 6 January 2021 Published: 9 January 2021 tives [3]. Much industrial wastewater contains the major phenol and its derivatives such as phenol and cresols [4,5]. Thus, the removal of these phenolic contaminants to a satisfactorily

Publisher’s Note: MDPI stays neu- low level in wastewater becomes an urgent task. tral with regard to jurisdictional clai- Conventional treatments to phenol and its derivatives including physical and chemical ms in published maps and institutio- methods have major disadvantages such as the cost of operation, production of harm- nal afﬁliations. ful metabolites, incomplete mineralization of the substances, and high cost involved in disposal of chemical waste sludge. In such cases, biological treatment processes seem promising for the complete mineralization of phenol and its derivatives to carbon dioxide and water with innocuous residues [6]. However, the growth of microorganisms suffers Copyright: © 2021 by the authors. Li- from the inhibition of phenol and its derivatives at higher concentration levels. In order to censee MDPI, Basel, Switzerland. overcome the inhibition of phenol and its derivatives, the cell acclimation [7], the appli- This article is an open access article cation of genetically engineered microorganisms [8], and cell immobilization [9,10] have distributed under the terms and con- been recommended. Increasing phenol concentrations successively to cultivate phenol- ditions of the Creative Commons At- degrading bacteria usually requires long lag times. Masque et al. [7] demonstrated that the tribution (CC BY) license (https:// −1 creativecommons.org/licenses/by/ degradation of phenol with 1000 mg L needed to take 20 days. The use of genetically 4.0/). engineered microorganisms gives the potential for an unanticipated ecological inﬂuence

Processes 2021, 9, 133. https://doi.org/10.3390/pr9010133 https://www.mdpi.com/journal/processes Processes 2021, 9, 133 2 of 23

and causes controversy in the application of bioremediation. Thus, cell immobilization gains a better alternative to protect cells from the toxicity of phenol and its derivatives. Biological treatment processes such as activated sludge system, biofilm process, biological contact oxidation, biofilter process, etc. are always environmentally friendly, highly effective, and economic [11]. In those biological systems, Pseudomonas putida as a rod- shaped Gram-negative bacterium has been proved to be effective in removing phenol and its derivatives [12]. A pure culture of P. putida ATCC 17484 with high removal efficiency for phenol and p-cresol biodegradation in a batch system has been demonstrated [13,14]. González et al. [13] used stirred tank and fluidized-bed bioreactors to degrade phenol through immobilized cells of P. putida ATCC 17484. Their experimental results revealed that both bioreactors achieved phenol biodegradation efficiencies higher than 90% even a phenol loading rate in the influent as high as 4 g L−1 d−1. Loh and Ranganath [15] carried out an external-loop fluidized bed airlift bioreactor (EFBAB) by using P. putida ATCC 49451 for the cometabolic biotransformation of 4-chlorophenol (4-CP) in the presence of phenol. Their study found that phenol and 4-CP with feed concentrations of 1600 and 200 mg L−1 had been successfully degraded in EFBAB process. The bacterial strain isolated from the contaminated sites by coke-oven effluent was identified as P. putida that showed a high capacity in degrading phenol concentration up to 1800 mg L−1 and tolerating cyanide up to 340 mg L−1 [16]. The biodegradation of toxic substances using entrapped cells has been utilized since 1975 [9]. The immobilized cells have their potential advantages over free cells for the enhancement of biodegradation efficiency in terms of cell reuse and recovery [17]. The bio- polymeric gel beads used to entrap microbial cells are well-established approaches for cell immobilization [18]. Banerjee et al. [1] successfully presented phenol biodegradation kinetics by immobilized cells in a batch system. However, the kinetic model system based on the simultaneous biodegradation kinetics of phenol and p-cresol in a continuous stirred-tank bioreactor with immobilized cells has never been reported. The knowledge of dual-substrates biodegradation kinetics by immobilized cells is helpful for the design of process facilities for the simultaneous removal of multiple substrates in wastewater. In this study, P. putida ATCC 17484 was entrapped in Ca-alginate gel beads using immobilization methods to evaluate the phenol and p-cresol biodegradation kinetics simultaneously. Moreover, the immobilized kinetic model system to describe the simultaneous biodegradation kinetics of phenol and p-cresol was developed. The goal of this work was to develop the kinetic model system to describe the biodegradation kinetics of phenol and p-cresol simultaneously. The main purposes of this study were to (1) evaluate phenol and p-cresol degradation by free P. putida cells, respectively; (2) estimate the growth yield and maximum specific growth rate of P. putida cells in batch experiments; (3) determine interaction parameters by a sum kinetic equation fitted by experimental data; (4) develop the kinetic model in the immobilized cells system in the continuous stirred bioreactor; (5) conduct continuous-flow experiments to investigate the synchronous biodegradation of phenol and p-cresol by alginate-immobilized P. putida cells; and (6) compare the experimental data and model prediction for the synchronous biodegradation of phenol and p-cresol by alginate-immobilized cells in a continuous stirred-tank bioreactor.

2. Kinetic Model Development 2.1. Growth Kinetics of Free Cells Batch System The speciﬁc growth rate of cells µ (h−1) obtained from the exponential phase in the batch experiment is expressed as [19,20]

ln(X /X ) µ = t 0 (1) t − t0

where Xt and X0 are the cell concentration at time t and t0. The value of µ was determined from the slope of a linear plot of ln(Xt/X0) versus time (t) in the log-growth phase of the curve. Processes 2021, 9, 133 3 of 23

P. putida cells used phenol or p-cresol as a sole carbon source, respectively, in a batch culture system. Phenol or p-cresol as a substrate displayed an inhibition to cell growth at much higher initial concentrations of phenol or p-cresol. Haldane kinetics used to model the cell growth using phenol or p-cresol as a substrate, respectively, was represented by the following equation: µ ·S = max µ 2 (2) Ks + S + S /Ki −1 −1 where µ is the speciﬁc growth rate (h ), µmax is the maximum speciﬁc growth rate (h ), −1 Ks is the half-saturated constant of substrate (mg L ), and Ki is the inhibition constant (mg L−1). The experimental data on the substrate degradation at various combinations of initial concentrations of phenol and p-cresol were utilized for determining the growth yield of P. putida cells according to the following equations [21]:

X − X Y = 0 (3) S0 − S

where Y is the growth yield of cells, X and X0 are the cell concentration and initial cell −1 concentration (mg L ), respectively, and S and S0 are the substrate concentration and initial substrate concentration (mg L−1), respectively. Yoon et al. [22] proposed the sum kinetic model to evaluate the interaction parameters (IC,P and IP,C) based on the individual speciﬁc growth rate.

µ S µ S = max,P P + max,C C µ 2 2 (4) Ks,P + SP + SP/Ki,P + IC,PSC Ks,C + SC + SC/Ki,C + IP,CSP

where P and C indicate phenol and p-cresol, respectively. IC,P indicates the degree to which p-cresol affects the biodegradation of phenol and vice versa. The higher parameter value makes a stronger inhibition on the cells growth [22]. The other bio-kinetic parameters µmax, Ks, and Ki are the same as those obtained from Equation (2) in a single substrate batch system.

2.2. Conceptual Basis of Immobilized Cells in the Continuous Stirred-Tank Bioreactor Figure1 presents the phenol and p-cresol concentration profiles in the bulk liquid, liquid film, and gel bead in the completely-mixed and continuous-flow bioreactors. In the bulk liquid, the phenol and p-cresol concentration profiles are flat lines due to a completely- mixed condition occurred in this phase. Then, the phenol and p-cresol pass through the liquid film and diffuse into the gel bead to constitute the curved profiles of concentration in the gel bead. Processes 2021, 9, x FOR PEER REVIEW 4 of 24

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Sb,P Si,P

Sb,C Ss,P Si,C Immobilized Ss,C cells 0

z R

Gel bead Liquid Film Bulk Liquid

Figure 1. Conceptual basis of concentration proﬁles in an immobilized cells system.

2.3. KineticFigure Model 1. Conceptual of the Entrapped basis of Cell concentration in the Continuous profiles Stirred-Tankin an immobilized Bioreactor cells system. The phenol and p-cresol in the bulk liquid diffused into gel beads through the liquid 2.3. Kinetic Model of the Entrapped Cell in the Continuous Stirred-Tank Bioreactor film and were biodegraded by immobilized cells. Diffusion and biodegradation are two fundamentalThe mechanisms phenol and occurred p-cresol simultaneously in the bulk liquid in this diffused phenomena. into gel According beads through to Fick’s the liquid law andfilm Haldane’s and were kinetics, biodegraded the utilization by immobilized rates of phenol cells. and Diffusionp-cresol and in the biodegradation gel bead with are two an unsteady-statefundamental condition mechanisms were given occurred as [1, 23simultaneo]: usly in this phenomena. According to Fick’s law and Haldane’s kinetics, the utilization rates of phenol and p-cresol in the gel 2 bead∂Ss ,withP an unsteady-state∂ Ss,P 2 ∂S conditions,P were givenµmax, as P[1,23]:SPXs = DeP + − (5) ∂t ∂z2 z ∂z Y K + S + S2 /K + I S ∂S  ∂ 2 S 2 ∂SP s,P P P μ i,P S XC,P C s,P =  s,P + s,P  − max,P P s DeP 2 2 (5) ∂S ∂t∂2S 2 ∂∂Sz z ∂z  Y ()µK +SS X+ S K + I S s,C = s,C + s,C −  P max,s,P C CP s P i,P C,P C DeC 2 2 (6) ∂t ∂z z ∂z YC Ks,C + SC + SC/Ki,C + IP,CSP ∂S  ∂ 2 S 2 ∂S  μ S X s,C = D  s,C + s,C  − max,C C s −1 where Ss,P and Ss,C are∂ the phenoleC  and∂ 2p-cresol concentrations∂  () within+ + the2 gel bead+ (mg L ), (6) t z z z YC K s,C SC SC K i,C I P,C S P DeP and DeC are the effective diffusivity of phenol and p-cresol, respectively, within the porous matrix (cm2 d−1), µ and µ are the maximum specific growth rate of cells s,P s,C max,P maxC −1 where S and S are−1 the phenol and p-cresol concentrations within the gel bead (mg L ), on phenol and p-cresol (h ), respectively, YP and YC are the growth yield of cells on DeP and DeC are the −effective1 diffusivity of phenol and p-cresol, respectively, within the phenol and p-cresol (mg mg2 −1), respectively,μ μ Ks,P and Ks,C are half-saturated constants of porous matrix (cm−1 d ), max,P and maxC are the maximum specific growth rate of cells on phenol and p-cresol (mg L ), respectively,−1 Ki,P and Ki,C are inhibition constants of phenol phenol and− 1p-cresol (h ), respectively, YP and YC are the growth yield of cells on phenol and p-cresol (mg L ), respectively, IC,P and IP,C represent the effect of p-cresol on phenol and p-cresol (mg mg−1), respectively, Ks,P and Ks,C are half-saturated constants of phenol biodegradation and the effect of phenol on p-cresol biodegradation, respectively, and z and p-cresol (mg L−1), respectively, Ki,P and Ki,C are inhibition constants of phenol and p- is the radial distance within the bead. The fluxes of phenol and p-cresol diffused into cresol (mg L−1), respectively, IC,P and IP,C represent the effect of p-cresol on phenol biodeg- the film/bead interface are equivalent to the fluxes diffused out the film/bead interface. radation and the effect of phenol on p-cresol biodegradation, respectively, and z is the The boundary conditions for phenol and p-cresol concentration profiles at the bead center radial distance within the bead. The fluxes of phenol and p-cresol diffused into the and film/bead interface were described as the following equations: film/bead interface are equivalent to the fluxes diffused out the film/bead interface. The boundary conditions for phenol∂ andS p-cresol concentration profiles at the bead center and BC1 : s,P = 0, z = 0 (7) film/bead interface were described∂z as the following equations:

Processes 2021, 9, 133 5 of 23

∂S BC1 : s,C = 0, z = 0 (8) ∂z ∂S BC2 : D s,P = k (S − S ), z = R (9) eP ∂z f P b,P i,P ∂S BC2 : D s,P = k (S − S ), z = R. (10) eC ∂z f C b,C i,C The initial conditions for phenol and p-cresol utilization rates in Equations (6) and (7) were expressed by IC1 : Ss,P = 0, 0 ≤ z ≤ R, t = 0 (11)

IC2 : Ss,C = 0, 0 ≤ z ≤ R, t = 0. (12) The growth of P. putida cells in the bead was written by the following equation:

∂X s = µX . (13) ∂t s The initial condition for the growth of cells in the bead can be represented by

IC : Xs = X0, t = 0 (14)

where X0 is the initial condition of cells in the bead. The mass balance of phenol and p-cresol in the bulk liquid phase and initial conditions for phenol and p-cresol were given by:

dSb,P Q 3Xw = (Sb0,P − Sb,P) − k f P(Sb,P − Ss,P) , z = R (15) dt Vε VερbR

dSb,C Q 3Xw = (Sb0.C − Sb,C) − k f C(Sb,C − Ss,C) , z = R (16) dt Vε VερbR

IC1 : Sb,P = Sb0,P (17)

IC2 : Sb,C = Sb0,C. (18)

In the above equations, Sb,P and Sb,C are the concentration of phenol and p-cresol in −1 the bulk liquid (mg L ), respectively, Sb0,P and Sb0,C are the concentrations of phenol −1 and p-cresol in the feed (mg L ), kfP and kfC are the external mass transfer coefficients of −1 phenol and p-cresol (mg L ), Ss,P and Ss,C are the concentrations of phenol and p-cresol at the liquid/bead interface (mg L−1), Q is the influent flow rate (cm3 d−1), V is the working 3 volume of the reactor (cm ), ε is the porosity of the reactor, Xw is the weight of beads (g), ρb is the density of the gel beads (g/cm3), and R is the radius of gel beads (cm).

3. Materials and Methods 3.1. Chemicals Phenol and p-cresol (>99% purity) purchased from Merck, KGaA, Darmstadt, Ger- many were of analytical grade in this study. One g of phenol and p-cresol, respectively, were dissolved in 1.0 L distilled/deionized water (DIDW) to form the stock solutions. The de- sired concentration containing phenol or p-cresol or phenol plus p-cresol was prepared by using stock solutions. All stock solutions are stored at 4 ◦C prior to use.

3.2. Cell Cultivation for Immobilization Pure culture of P. putida ATCC 17484 with high removal efﬁciency for phenol and p-cresol biodegradation has been demonstrated [14,24,25]. P. putida ATCC 17484 grown in mineral salt medium (MSM) [2,26] with 20 mg L−1 of phenol as the carbon source was incubated and collected at the stationary phase. Then, the cells were centrifuged at 3000 rpm for 10 min. The phosphate buffer saline with pH 7.4 was used for washing cells. Then, the washed cells were used as inoculum for biodegradation. The ﬂasks contained Processes 2021, 9, 133 6 of 23

100 mL MSM with varying initial concentrations of phenol, p-cresol, and phenol plus p- cresol, respectively, from 50 to 600 mg L−1 were prepared to conduct the batch experiments. The MSM was auto-claved at 121 ◦C for 15 min, and phenol as well as p-cresol were sterilized by a membrane ﬁlter.

3.3. Entrapment of Cells in Ca-Alginate Ten mL of centrifuged P. putida cells were mixed with 50 mL of sodium alginate of 2% (w/v) to form the cell–alginate mixture [1]. Then, the cell–alginate mixture was dropped into CaCl2 of 1% (w/v) to form gel beads with a diameter of 3 mm. The phosphate buffer saline (PBS) was used to wash gel beads three times. Then, the gel beads were immersed in ◦ 3 g/L CaCl2 and stored at 4 C for overnight to strengthen the gel formation [11].

3.4. Batch Experiments The varying initial concentration (50–600 mg L−1) of phenol, p-cresol, and phenol plus p-cresol with 200 mL MSM was conducted in batch mode, respectively. The bio-kinetic parameters were estimated from those batch tests. The ﬂasks were placed in the rotary incubator at 30 ◦C and 150 rpm to observe the biodegradation of phenol, p-cresol, and phenol plus p-cresol. Samples were taken from the ﬂasks with a different time interval to measure the concentrations of cells, phenol, and p-cresol.

3.5. Analysis of P. putida Cells, Phenol and p-Cresol The optical density (OD) at 600 nm wavelength was used to represent the cell concentration using a UV/vis spectrophotometer. Based on the calibration curve, the linear rela- −1 tionship between cells concentration (X) and OD600 was X (mg cell L ) = 343.75 × OD600. High-Performance Liquid Chromatograph (HPLC) (Alliance 2695, Waters Corp., Milford, MA, USA) equipped with a UV/vis detector (Waters 2487, Waters Corp., Milford, MA, USA) with an auto-sampler (Waters 2707, Waters Corp., Milford, MA, USA) was setup to analyze the samples. A C18 column with a 150 × 3.9 mm size and packed with 5 µm particle size was applied to analyze the residual phenol and p-cresol concentrations. The wavelength was set at 254 nm in the UV/vis detector. The mobile phase containing potassium phosphate and acetonitrile with a volume ratio of 70/30 was used to elute the samples.

3.6. Continuous-Flow Bioreactor A schematic laboratory-scale completely mixed and continuous-flow bioreactor is illustrated in Figure2. The cylinder shape was composed of glass with an acrylic stand. The shape of the system is cylinder made of glass with an acrylic stand. The bioreactor body was 46.6 cm height and 10 cm diameter. The working volume was 1.568 L as the liquid level was 30 cm. A hydraulic residence time (HRT) was 6 h when the influent flow rate was controlled at 6.272 L d−1 in this study. The volume of gel beads was about 0.96 L, which is approximately 40% of the effective volume. The dissolved oxygen was transported by an air compressor with 1 L/min air flow rate. The circulating water bath was employed to control the bioreactor temperature at 30 ± 0.1 ◦C using the water jacket. The inlet port at the bottom of bioreactor was connected with a digital peristaltic pump using a silicone tubing to provide a flow rate of 11.32 mL min−1. The influent feed contained phenol plus p-cresol as binary substrates with MSM. The pH value was controlled at 7.0 ± 0.1 by adding PBS in the influent feed. Processes 2021, 9, x FOR PEER REVIEW 7 of 24

which is approximately 40% of the effective volume. The dissolved oxygen was transported by an air compressor with 1 L/min air flow rate. The circulating water bath was employed to control the bioreactor temperature at 30 ± 0.1 °C using the water jacket. The inlet port at the bottom of bioreactor was connected with a digital peristaltic pump using −1 Processes 2021, 9, 133 a silicone tubing to provide a flow rate of 11.32 mL min . The influent feed contained7 of 23 phenol plus p-cresol as binary substrates with MSM. The pH value was controlled at 7.0 ± 0.1 by adding PBS in the influent feed.

FigureFigure 2. 2. AA completely completely mixed mixed and and continuous-flow continuous-ﬂow bioreactor. bioreactor.

4.4. Results Results and and Discussion Discussion 4.1.4.1. Biodegradation Biodegradation of of Phenol Phenol or or p-Cresol p-Cresol in in Batch Batch Experiments Experiments Figure3 plots the phenol biodegradation and cell growth with various initial phenol (50–600 mg L−1) and cells concentration (7.68–8.73 mg cell L−1). The required time for phenol complete degradation at all levels was about 24 h (Figure3a). There was no signiﬁcant lag phase that occurred from 50 to 600 mg L of initial phenol concentration. After 24 h, the cell growth reached a constant value ranging from 24.7 to 211 mg cell L−1 under different initial phenol concentration (Figure3b). The time required for complete phenol degradation by P. putida CCRC 14365 was in the range of 6–47 h as the initial phenol concentration increased from 0.27 to 4.25 mM [27]. Kumar et al. [28] conducted a batch Processes 2021, 9, x FOR PEER REVIEW 8 of 24

Figure 3 plots the phenol biodegradation and cell growth with various initial phenol (50–600 mg L−1) and cells concentration (7.68–8.73 mg cell L−1). The required time for phenol complete degradation at all levels was about 24 h (Figure 3a). There was no significant lag phase that occurred from 50 to 600 mg L of initial phenol concentration. After 24 h, the cell growth reached a constant value ranging from 24.7 to 211 mg cell L−1 under different

Processes 2021, 9, 133 initial phenol concentration (Figure 3b). The time required for complete phenol degrada-8 of 23 tion by P. putida CCRC 14365 was in the range of 6–47 h as the initial phenol concentration increased from 0.27 to 4.25 mM [27]. Kumar et al. [28] conducted a batch reactor to evaluate the phenol degradation by P. putida MTCC 1194. They reported that the cells had the abilityreactor to todegrade evaluate an the initial phenol phenol degradation concentration by P. putidaof 1000MTCC mg L 1194.−1 completely They reported in 162 h. that Fig- the −1 urecells 4a hadillustrates the ability the top-cresol degrade degradation an initial phenol varying concentration from 50 to 600 of 1000 mg L mg−1. LIt cancompletely be seen −1 thatin 162the h.needed Figure time4a illustratesfor the complete the p-cresol biodegradation degradation of p varying-cresol ranged from 50 from to 600 24 to mg 52 L h . underIt can varying be seen thatinitial the p-cresol needed concentrations time for the complete of 50–600 biodegradation mg L−1. As illustrated of p-cresol in ranged Figure from4b, −1 the24 lag to 52phase h under of cells varying growth initial wasp -cresolobvious, concentrations and the lag time of 50–600 was about mg L 8 h.. AsThe illustrated time re- quiredin Figure to achieve4b, the a lag steady-state phase of cells cell growth rang wased obvious, from 24 and to 52 the h. lag The time range was of aboutfinal cell 8 h. concentrationThe time required was 21.3 to achieve to 176 mg a steady-state cell L−1. cell growth ranged from 24 to 52 h. The range of ﬁnal cell concentration was 21.3 to 176 mg cell L−1.

700 Initial phenol=50 mg/L, In itial cell = 7.86 m g ce ll/L 600 Initial phenol=100 mg/L In itial cell= 7.68 m g cell/L Initial phenol=200 mg/L 500 In itial cell= 8.48 m g cell/L Initial phenol=300 mg/L In itial cell= 7.75 m g cell/L 400 Initial phenol=400 mg/L In itial cell= 8.69 m g/L Initial phenol=500 mg/L In itial cell= 8.28 m g cell/L 300 Initial phenol=600 mg/L In itial cell= 8.73 m g/L

200 Phenolconcentration (mg/L)

100

Processes 2021, 9, x FOR PEER REVIEW 9 of 24 0 0 102030405060 Time (h)

(a)

In itial cell=7 .86 m g/L In itial cell=7 .68 m g/L In itial cell=8 .48 m g/L In itial cell=7 .75 m g/L In itial cell=8 .69 m g/L In itial cell=8 .28 m g/L In itial cell=8 .73 m g/L 250

200

150

ell concentration (mg/L) ell concentration 100 P. Pudita c 50

0 0 102030405060 Time (h)

(b) Figure 3.3. Time coursecourse ofof thethe changechange inin concentrationconcentration for for various various concentrations concentrations of of initial initial phenol phenol and P.and putida P. putidacells: cells: (a) phenol (a) phenol and (andb) P. ( putidab) P. putidacells. cells.

700 p -cresol=50 m g/L p -cresol=100 mg/L 600 p -cresol=200 mg/L p -cresol=300 mg/L p -cresol=400 mg/L p -cresol=500 mg/L 500 p -cresol=600 mg/L

400

300

-cresol concentration (mg/L) 200 P

100

0 0 102030405060 Time (h)

(a)

Processes 2021, 9, x FOR PEER REVIEW 9 of 24

In itial cell=7 .86 m g/L In itial cell=7 .68 m g/L In itial cell=8 .48 m g/L In itial cell=7 .75 m g/L In itial cell=8 .69 m g/L In itial cell=8 .28 m g/L In itial cell=8 .73 m g/L 250

200

150

ell concentration (mg/L) ell concentration 100 P. Pudita c 50

0 0 102030405060 Time (h)

(b) Processes 2021, 9, 133 9 of 23 Figure 3. Time course of the change in concentration for various concentrations of initial phenol and P. putida cells: (a) phenol and (b) P. putida cells.

700 p -cresol=50 m g/L p -cresol=100 mg/L 600 p -cresol=200 mg/L p -cresol=300 mg/L p -cresol=400 mg/L p -cresol=500 mg/L 500 p -cresol=600 mg/L

400

300

-cresol concentration (mg/L) 200 P

100

0 0 102030405060 Processes 2021, 9, x FOR PEER REVIEW 10 of 24 Time (h)

(a)

200

In itial cell=7 .6 7 m g/L In itial cell=7 .5 3 m g/L In itial cell=8 .2 7 m g/L In itial cell=8 .6 2 m g/L 150 In itial cell=8 .7 1 m g/L In itial cell=7 .9 2 m g/L In itial cell=8 .8 5 m g/L

100 cell concentration (mg/L) concentration cell

50 P. putida

0 0 102030405060 Time (h)

(b) Figure 4.4. Time coursecourse ofof thethe changechange in in concentration concentration for for various various concentrations concentrations of of initial initialp-cresol p-cresol and P.and putida P. putidacells: cells: (a) p-cresol (a) p-cresol and (andb) P. ( putidab) P. putidacells. cells. 4.2. P. putida Cells Growth on Phenol or p-Cresol 4.2. P. putida Cells Growth on Phenol or p-Cresol Figure5a plots ln( X/X0) versus time to determine the specific growth rate of the cell on Figure 5a plots ln(X/X0) versus time to determine the specific growth rate of the cell phenol. The specific growth rate on phenol was ranged from 0.0769 to 0.175 h−1. Figure5b on phenol. The specific growth rate on phenol was ranged from 0.0769 to 0.175 h−1. Figure illustrates the ln(X/X0) plotted with time at varying initial p-cresol concentration and 5b illustrates the ln(X/X0) plotted with time at varying initial p-cresol concentration and cell concentrations. The range of the specific growth rate on p-cresol was 0.0515–0.082 h−1. cell concentrations. The range of the specific growth rate on p-cresol was 0.0515–0.082 h−1. As shown in Figures6 and7, it is noted that the P. putida cells had the maximum specific As shown in Figures 6 and 7, it is noted that the P. putida cells had the maximum specific growth rate as the initial phenol and p-cresol concentrations were approximately 220 and 140growth mg rate L−1 ,as respectively. the initial phenol The cells and growthp-cresol wasconcentrations inhibited when were approximately the initial phenol 220 andand −1 p140-cresol mg L concentrations, respectively. were The greatercells growth than 220was and inhibited 140 mg when L−1, the respectively. initial phenol The and batch p- cresol concentrations were greater than 220 and 140 mg L−1, respectively. The batch exper- experimental data were fitted by Haldane’s kinetics to obtain the bio-kinetic values of µmax imental data were fitted by Haldane’s kinetics to obtain the bio-kinetic values of μmax Ks Ks and Ki on phenol and p-cresol, respectively, by a non-linear least squares regression methodand Ki on using phenol the Excel and softwarep-cresol, [respectively,27]. Haldane’s by equations a non-linear for phenolleast squares and p-cresol regression with themethod best-fit using bio-kinetic the Excel parameters software were[27]. Haldane’s yielded as equations follows: for phenol and p-cresol with the best-fit bio-kinetic parameters were yielded as follows: 0.45SP Phenol: µP = (19) + S + S2 μ = 221.4 0.P45SPP/310.5 Phenol: P + + 2 (19) 221.4 SP SP 310.5

μ = 0.185SC P-cresol: C + + 2 (20) 65.1 SC SC 243.56

where μP and μC are the specific growth rates of P. putida cells on phenol and p-cresol, respectively. The μmax, Ks, and Ki values for phenol were 0.45 h−1, 221.4, and 310.5 mg L−1, respectively. The μmax, Ks, and Ki values for p-cresol were 0.185 h−1, 65.1, and 243.56 mg L−1, respectively. The cells with a higher maximum specific growth rate utilized phenol more faster than p-cresol. In addition, the larger Ks value on phenol resulted in a lower affinity of cells to phenol. The higher Ki value on phenol (310.5 mg L−1) displayed that the cells had a stronger resistance to phenol inhibition than to p-cresol (243.56 mg L−1). A higher inhibition to cells growth by p-cresol than that by phenol was observed, as the initial concentration was over 200 mg L−1. The phenol biodegradation by P. putida shows the μmax value for phenol (0.45 h−1) falling between 0.33 and 0.90 h−1 according to the literature survey [29–31]. The Ki value for phenol obtained here (310.5 mg L−1) also falls within the ranges of 54.1–669.0 mg L−1 [29–31]. The Ks value (221.4 mg L−1) for phenol obtained in this

Processes 2021, 9, 133 10 of 23

0.185S = C P-cresol: µC 2 (20) 65.1 + SC + SC/243.56

where µP and µC are the specific growth rates of P. putida cells on phenol and p-cresol, respec- −1 −1 tively. The µmax, Ks, and Ki values for phenol were 0.45 h , 221.4, and 310.5 mg L , re- −1 −1 spectively. The µmax, Ks, and Ki values for p-cresol were 0.185 h , 65.1, and 243.56 mg L , respectively. The cells with a higher maximum specific growth rate utilized phenol more faster than p-cresol. In addition, the larger Ks value on phenol resulted in a lower affinity −1 of cells to phenol. The higher Ki value on phenol (310.5 mg L ) displayed that the cells had a stronger resistance to phenol inhibition than to p-cresol (243.56 mg L−1). A higher inhibition to cells growth by p-cresol than that by phenol was observed, as the initial con- −1 centration was over 200 mg L . The phenol biodegradation by P. putida shows the µmax − − Processes 2021, 9, x FOR PEER REVIEWvalue for phenol (0.45 h 1) falling between 0.33 and 0.90 h 1 according to the11 of literature 24 −1 survey [29–31]. The Ki value for phenol obtained here (310.5 mg L ) also falls within the −1 −1 ranges of 54.1–669.0 mg L [29–31]. The Ks value (221.4 mg L ) for phenol obtained in thisstudy study was was close close to that to that obtained obtained from fromthe study the studyof Banerjee of Banerjee et al. [32]. et The al. [ 32Ks ].value The ob-Ks value obtainedtained inin their study study was was 190.8 190.8 mg mg L−1.L −1.

3.2 μ =0.0582 h-1 μ =0.0799h-1 2.8 μ =0.0820 h-1 μ =0.0767 h-1 2.4 μ =0.0697 h-1 μ =0.0589 h-1 -1 2 μ =0.0515 h ) 0

1.6 ln(X/X

1.2

0.8

0.4

0 0 102030405060 Time (h ) (a) 3.2 μ =0.0582 h-1 μ =0.0799h-1 2.8 μ =0.0820 h-1 μ =0.0767 h-1 2.4 μ =0.0697 h-1 μ =0.0589 h-1 2 μ =0.0515 h-1 ) 0

1.6 ln(X/X

1.2

0.8

0.4

0 0 102030405060 Time (h)

(b) FigureFigure 5. Batch 5. Batch experiments experiments to evaluate to evaluate the the speciﬁc specific growth growth rate rate under under differentdifferent initial single-substrate single-substrate and and cells cells concen- concentra- tions: (atrations:) phenol (a) ( bphenol) p-cresol. (b) p-cresol.

ProcessesProcessesProcesses 202120212021,, 999,, xx FORFOR PEERPEER REVIEWREVIEW 1212 ofof 2424 , , 133 11 of 23

0.20.2

0.150.15 ) ) -1 -1 (h (h μ μ

0.10.1 Specific growth rate, Specific growth rate, 0.050.05

ExperimentalExperimental ModelModel MSE=3.983x10MSE=3.983x10-5-5

00 00 100 100 200 200 300 300 400 400 500 500 600 600 700 700 InitialInitial phenolphenol concentrationconcentration (mg/L)(mg/L)

FigureFigureFigure 6.6. 6. SpecificSpecificSpecific growthgrowth growth raterate rate ofof of cellscells cells variedvaried varied withwith with variousvarious various initialinitial initial phenolphenol phenol concentrations.concentrations. concentrations. HaldaneHaldane Haldane kineticskineticskinetics waswas was fittedfitted fitted toto to thethe the experimentalexperimental experimental datadata data usingusing using thethe the least-squaresleast-squares least-squares methodology.methodology. methodology. TheThe The maximummaximum maximum μμ -1-1 −1 specificspecificspecific growthgrowth growth raterate rate (( (µmaxmaxmax)) isis) is0.450.45 0.45 hh h,, thethe, thephenolphenol phenol half-saturationhalf-saturation half-saturation constantconstant constant isis 221.4221.4 is 221.4 mg/L,mg/L, mg/L, andand thethe and the phenolphenolphenol inhibitioninhibition inhibition conscons constanttanttant isis is 310.5310.5 310.5 mg/L.mg/L. mg/L.

0.10.1

0.080.08 ) ) -1 -1 (h (h μ μ 0.060.06

0.040.04 Specific growthrate, Specific growthrate,

0.020.02 ExperimentalExperimental ModelModel MM SE=3.954x10 SE=3.954x10-6-6

00 00 100 100 200 200 300 300 400 400 500 500 600 600 700 700 InIn itia itia l l pp -c-c re re s s o o l l cc o o n n c c e e n n tra tra tio tio n n (m(m g g /L /L ) ) FigureFigureFigure 7.7. 7. TheTheThe specificspecific specific growthgrowth growth raterate of of ce cellscellslls variedvaried withwithwith various variousvarious initial initialinitialp pp-cresol-cresol-cresol concentrations. concentrations.concentrations. Haldane Hal-Hal- danedanekinetics kineticskinetics was waswas fitted fittedfitted to the toto experimentalthethe experimentalexperimental data dada usingtata usingusing the thethe least-squares least-squaresleast-squares methodology. methodology.methodology. The TheThe maximum maxi-maxi- μμ −1 −−11 mummumspecific specificspecific growth growthgrowth rate ( raterateµmax (() ismaxmax 0.185)) isis 0.1850.185 h ,hh the,, thethep-cresol pp-cresol-cresol half-saturation half-saturationhalf-saturation constant constantconstant is 65.1isis 65.165.1 mg/L, mg/L,mg/L, and andand the thethephenol phenolphenol inhibition inhibitioninhibition constant constantconstant is243.56 isis 243.56243.56 mg/L. mg/L.mg/L.

AsAsAs plottedplotted plotted inin in FiguresFigures Figures 88 and 9 9,9,, thethethe growthgrowthgrowth yield yieldyield (Y) (Y)(Y) ofofof cellscellscells ononon phenolphenolphenol andandand ppp-cresol-cresol-cresol waswas was estimatedestimatedestimated fromfrom from batchbatch batch teststests tests datadata data usingusing using EquationEquation Equation (3),(3), (3), respectively.respectively. respectively. TheThe The calculatedcalculated calculated valuesvalues values ofof YYof onon Y phenolphenol on phenol andand and pp-cresol-cresolp-cresol areare arelistedlisted listed inin TableTable in Table 1.1. TheThe1. The growthgrowth growth yieldsyields yields onon phenolphenol on phenol werewere were inin thethe in − rangerangethe range ofof 0.337–0.3430.337–0.343 of 0.337–0.343 mgmg mgmg mg−−11 mgunderunder1 under initialinitial initialphenolphenol phenol concentrationsconcentrations concentrations rangingranging ranging fromfrom 5050 from toto 600600 50 −1 −1 mgmgto 600 LL−−11.. mgTheThe L averageaverage. The growthgrowth average yieldyield growth onon singlesingle yield on phenolphenol single (Y(Y phenolPP)) waswas 0.3400.340 (YP) was mgmg mgmg 0.340−−11 andand mg thethe mg stand-stand-and −3 ardardthe deviationdeviation standard value deviationvalue waswas value2.1162.116 × was× 1010−−3 2.1163.. TheThe ×growthgrowth10 . yieldyield The growth onon singlesingle yield pp-cresol-cresol on single (Y(YCC)) variedpvaried-cresol fromfrom (YC ) −1 0.2740.274varied toto from 0.2830.283 0.274 mgmg mgmg to 0.283−−11 toto acquireacquire mg mg aa meanmeanto acquire valuevalue a meanofof 0.2790.279 value asas wellwell of 0.279 asas aa as standardstandard well as adeviationdeviation standard −3 valuevaluedeviation ofof 2.7692.769 value ×× 1010 of−−33 2.769.. AbuhamedAbuhamed× 10 . etet Abuhamed al.al. [33][33] carriedcarried et al. outout [33 ] batchbatch carried teststests out withwith batch variousvarious tests with initialinitial various phe-phe- −1 nolnolinitial concentrationsconcentrations phenol concentrations ofof 10–20010–200 mg ofmg 10–200 LL−−11.. TheirTheir mg Lexperimentexperiment. Their experiment resultsresults revealedrevealed results thatthat revealed thethe YY that valuevalue the −1 waswasY value 0.440.44 wasmgmg mgmg 0.44−−11,, mg whichwhich mg isis greater,greater which thanthan is greater thethe YY than valuevalue the obtaobta Y valueinedined inin obtained thisthis studystudy in due thisdue studytoto thethe lower duelower to initialinitialthe lower phenolphenol initial concentrations.concentrations. phenol concentrations.

Processes 2021, 9, x FOR PEER REVIEW 13 of 24 Processes 2021, 9, 133 12 of 23

20 35 2 y = -0.05884 + 0.34004x , R = 0.9984 y = -0.026101 + 0.33794x R 2 = 0.9994 30

15 25

20 10 15

10 5 Increased cell concentration (mg/L) Increased cell concentration (mg/L) concentration cell Increased

0 0 0 1020304050 0 20406080100 Consumed phenol concentration (mg/L) Consumed phenol concentration (mg/L)

(a) (b)

70 120 2 y = -0.3149 8 + 0.34 307x R = 0.9994 y = -0.35292 + 0.34246x R2 = 0.9993 60 100

50 80

40 60

30 40 20 Increasedconcentration cell (mg/L) Increased cellconcentration (mg/L) 20 10

0 0 0 50100150200250300 0 50 100 150 200 Consum ed phenol concentration (mg/L) Consumed phenol concentration (mg/L)

150 100

80 100

40 50 Increased cell concentrationIncreased (mg/L) cell Increased cell concentration (mg/L) 20

0 0 0 50 100 150 200 250 300 350 400 0 100 200 300 400 500 Consumed phenol concentration (mg/L) Consumed phenol concentration (mg/L)

(e) (f)

Figure 8. Cont.

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250 250 y = 0.28778 + 0.33871x R 2 = 0.9997 y = 0.28778 + 0.33871x R 2 = 0.9997 200 200

150 150

100 100

Increased cell concentration (mg/L) 50

0 0 100200300400500600 0 0Consumed 100200300400500600 phenol concentration (mg/L) Consumed phenol concentration (mg/L) (g) (g) FigureFigure 8. 8. BatchBatch kinetic kinetic tests tests to to determine determine growth growth yield yield on on phenol: phenol: (a (a) )50, 50, (b (b) )100, 100, ( (cc)) 200, 200, ( (dd)) 300, 300, ( (ee)) 400, 400, ( (ff)) 500, 500, and and ( (gg)) Figure 8. Batch kinetic tests to determine growth yield on phenol: (a) 50, (b) 100, (c) 200, (d) 300, (e) 400, (f) 500, and (g) 600600 mg/L. mg/L. 600 mg/L. 14 30 14 y = 0.15305 + 0.27445x R2 = 0.9990 2 30 y = 0.052234 + 0.27788x R = 0.9989 12 y = 0.15305 + 0.27445x R2 = 0.9990 y = 0.052234 + 0.27788x R2 = 0.9989 12 25 10 25

10 20 8 20 8 15 6 15 6 10 4 10

Increased cell concentration (mg/L) concentration Increased cell 4 Increased cell concentration (mg/L) 2 5 Increased cell concentration (mg/L) concentration Increased cell Increased cell concentration (mg/L) 2 5 0 0 0 1020304050 020406080100 0 Consum ed p -c re s o l c o n c e n tra tio n (m g /L ) 0 Consum ed p -cresol concentration (m g/L) 0 1020304050 020406080100 Consum ed p -c re s o l c o n c e n tra tio n (m g /L ) Consum ed p -cresol concentration (m g/L) (a) (b) (a) 100 (b) 60 100 y = -0.20692 + 0.27958x R 2 = 0.9996 60 y = -0.20692 + 0.27958x R 2 = 0.9996 50 80 50 80

40 40 60 60 30 30 40 20 40 20 Increased cell concentration (mg/L)

10 Increased(mg/L)concentration cell 20 Increased cell concentration (mg/L)

10 2 Increased(mg/L)concentration cell 20 y = -0.00062923 + 0.27791x R = 0.9999 2 0 y = -0.00062923 + 0.27791x R = 0.9999 0 50 100 150 200 250 0 0 consumed p -cresol concentration (m g/L) 0 50100150200250300 0 50 100 150 200 250 0 consumed p -cresol concentration (m g/L) 0Consumed 50100150200250300 p -cresol concentration (mg/L)

Figure 9. Cont.

Processes 20212021,, 99,, 133x FOR PEER REVIEW 1514 of of 2324

120 140

100 120

100 80

80 60

40 40 Increased cell concentration (mg/L) concentration cell Increased Increased cell concentrationIncreased cell(mg/L) 20 20 2 y = -1.2385 + 0.28331x R 2 = 0.9989 y = -0 .1 9 5 1 + 0 .2 8 0 3 6 x R = 0.9993 0 0 0 100 200 300 400 500 0 100 200 300 400 500 Consum ed p -cresol concentration (m g/L) Consum ed p -cresol concentration (mg/L)

(e) (f) 200 y = -0.35345 + 0.27989x R 2 = 0.9994

150

100

50 Increased cell concentration (mg/L) concentration cell Increased

0 0 100 200 300 400 500 600 Consum ed p -cresol concentration (mg/L)

(g) Figure 9. Batch kinetic tests to determine growth yield on p-cresol: (a) 50, (b) 100, (c) 200, (d) 300, (e) 400, (f) 500, and (g) Figure 9. Batch kinetic tests to determine growth yield on p-cresol: (a) 50, (b) 100, (c) 200, (d) 300, (e) 400, (f) 500, and (g) 600 mg/L. 600 mg/L.

TableTable 1. Batch Tests to Evaluate Growth Yield (Y)(Y) underunder VariousVarious InitialInitial Concentrations.Concentrations. Bio-Kinetic Parameters Initial Phenol Initial p-Cresol Bio-Kinetic Parameters Run No. InitialConcentration Phenol Concentration (mg L−1) Concentration Initial p-Cresol (mg L−1 )Concentration −1 YP Y−C 1 Run No. YP (mg mg ) YC (mg mg ) (mg L−1) (mg L−1) (mg mg−1) (mg mg−1) 1 50 50 0.340 0.274 12 10050 100 50 0.3380.340 0.2780.274 23 200100 200 100 0.3430.338 0.2780.278 34 300200 300 200 0.3420.343 0.2800.278 45 400300 400 300 0.3400.342 0.2830.280 6 500 500 0.337 0.280 5 400 400 0.340 0.283 7 600 600 0.339 0.280 mean6 -500 - 500 0.3400.337 0.2790.280 standard7 deviation -600 - 600 2.116 × 10−3 0.339 2.769 ×0.28010−3 mean - - 0.340 0.279 standard deviation - - 2.116 × 10−3 2.769 × 10−3

4.3. Phenol Plus p-Cresol in Binary Substrates System The biodegradation of phenol plus p-cresol and the growth of P. putida cells is illustrated in Figure 10. As plotted in Figure 10a, the time required for the complete removal of phenol was 52–56 h for the initial phenol concentration ranging from 50 to 500 mg L−1. However, the phenol removal was about 96.8% as the operating time was 56 h. As shown in Figure 10b, the time needed to remove p-cresol completely was 48–56 h for the initial p-

Processes 2021, 9, 133 15 of 23

4.3. Phenol Plus p-Cresol in Binary Substrates System The biodegradation of phenol plus p-cresol and the growth of P. putida cells is illus- Processes 2021, 9, x FOR PEER REVIEWtrated in Figure 10. As plotted in Figure 10a, the time required for the complete removal16 of 24 of phenol was 52–56 h for the initial phenol concentration ranging from 50 to 500 mg L−1. However, the phenol removal was about 96.8% as the operating time was 56 h. As shown in Figure 10b, the time needed to remove p-cresol completely was 48–56 h for the initial −1 cresolp-cresol concentration concentration between between 50 and 50 and 500 500mg mgL . LAt− 1the. At operating the operating time of time 56 h, of only 56 h, 79.7% only removal79.7% removal efficiency efﬁciency for p-cresol for p-cresol was attained. was attained.

700 Phenol=50 mg/L Phenol=100 mg/L 600 Phenol=200 mg/L Phenol=300 mg/L Phenol=400 mg/L Phenol=500 mg/L 500 Phenol =600 mg/L

400

300

200 Phenol concentration (mg/L)

100

0 0 102030405060 Time (h)

(a)

P -cresol=50 (m g/L) P -cresol=100 m g/L P -cresol=200 m g/L P -cresol=300 m g/L P -cresol=400 m g/L P -cresol=500 m g/L P -cresol=600 m g/L 700

600

500

400

300

-cresol concentration (mg/L) 200 P

100

0 0 8 16 24 32 40 48 56 Time (h)

(b)

Figure 10. Cont.

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ProcessesProcesses2021 2021, ,9 9,, 133 x FOR PEER REVIEW 1716 ofof 2324

350 Phenol=50 mg/L, P -cresol=50 m g/L Phenol=100 mg/L, P -cresol=100 mg/L 300350 Phenol=200 mg/L, P -cresol=200 mg/L Phenol=300 mg/L, P -cresol=300 mg/L Phenol=50 mg/L, P -cresol=50 m g/L Phenol=400 mg/L, P -cresol=400 mg/L Phenol=100 mg/L, P -cresol=100 mg/L Phenol=500 mg/L, P -cresol=500 mg/L 300 Phenol=200 mg/L, P -cresol=200 mg/L 250 Phenol=600 mg/L, P -cresol=600 mg/L Phenol=300 mg/L, P -cresol=300 mg/L Phenol=400 mg/L, P -cresol=400 mg/L Phenol=500 mg/L, P -cresol=500 mg/L 200250 Phenol=600 mg/L, P -cresol=600 mg/L

150200 cell concentration (mg/L)

100150 cell concentration (mg/L) P. putida

10050 P. putida

500 0 102030405060 Time (h)

0 0 102030405060(c) Time (h) Figure 10. Batch experiments in the binary substrates system: (a) phenol biodegradation, (b) p- (c) cresol biodegradation, and (c) P. putida cells growth. FigureFigure 10.10. BatchBatch experimentsexperiments in in the the binary binary substrates substrates system: system: (a ()a phenol) phenol biodegradation, biodegradation, (b ()bp)-cresol p- biodegradation,cresolSeven biodegradation, batch and experiments (c) andP. putida (c) P.cells putidawere growth. cellscarried growth. out at 30 °C to determine the interaction parameters of IC,P and IP,C using the non-linear least-squares regression method [27]. The cell growthSevenSeven in the batch binary experiments system varied were were with carried carried time, out outwhich at at 30 30is °C plotted◦ Cto todetermine determinein Figure the 10c. the interaction The interaction specific pa- −1 growthparametersrameters rates of ofI onC,PI andCbinary,P and IP,C phenolusingIP,C using the plus non-linear thep-cresol non-linear rangedleast-squares least-squaresfrom 0.185 regression to regression0.204 method d under method [27]. different The [ 27cell]. initialThegrowth cell phenol in growth the and binary in p-cresol the system binary contents. varied system Figurewith varied time, 11 presents withwhich time, is the plotted which specific in is Figure growth plotted 10c. rate in The Figure of thespecific 10cellc. −1 −1 onThegrowth phenol specific rates plus growth on p binary-cresol. rates phenol The on binarybio-kine plus p phenol-cresoltic parameters plusrangedp-cresol from of I C,P0.185 ranged and to I P,C from0.204 were 0.185d determinedunder to 0.204 different d by fittingunderinitial differentexperimentalphenol and initial p -cresoldata phenol to contents. sum and thep-cresol model.Figure contents. 11The presents kinetic Figure theparameters 11 specific presents growth(μm the, K specifics, rateand ofKi growth )the in thecell equationrateon phenol of the are cellplus the on psame-cresol. phenol as The plusthose bio-kinep -cresol.presentedtic The parameters in bio-kineticthe Haldane’s of parametersIC,P andequations IP,C ofwere IforC,P determinedphenoland IP, Candwere byp- μ cresoldeterminedfitting biodegradation experimental by fitting data [34]. experimental to The sum best-fitted the datamodel.to sum sumThe kinetic kinetic the model. Equation parameters The (21) kinetic (wasm, K parametersgivens, and as: K i) in (µ them, Kequations, and Ki )are in thethe equationsame as those are the presented same as those in the presented Haldane’s in equations the Haldane’s for phenol equations and for p- phenolcresol biodegradation and0.p45-cresolSP biodegradation [34]. The best-fitted [34]. Thesum0 best-fitted kinetic.185SC Equation sum kinetic (21) was Equation given as: (21) was μ = + given+ as: + 2 + + + 2 + (21) 221.4 SP SP 65.1 4.8SC 65.1 SC SC 243.56 12.7SP 0.45SP 0.185SC . μ = + + + 2 + 0.45SP + + 2 0.185+ SC (21) 221.4TheS valuesPµ =SP of65 IC,P.1 and4 .I8P,CS wereC 65 4.8.1 andS 12.7,C + Srespectively,C 243.56 with12.7 aS correlationP coefficient. (21) 221.4 + S + S2 /65.1 + 4.8S 65.1 + S + S2 /243.56 .+ 12.7S (R2) of 0.989. P P C C C P The values of IC,P and IP,C were 4.8 and 12.7, respectively, with a correlation coefficient (R2) of 0.989. In itial p -cresol concentration (mg/L) 0 100 200 300 400 500 600 700 0.08 In itial p -cresol concentration (mg/L)

0.07 0 100 200 300 400 500 600 700 0.08

0.06 )

-1 0.07 (h μ 0.05 0.06 ) -1

(h 0.04 μ 0.05

0.03 0.04 Specific growth rate, 0.02 0.03 Experimental

Specific growth rate, 0.01 Model 0.02 -7 MSE=9.353x10 0 Experimental 0.010 100200300400500600700Model MSE=9.353x10-7 Initial phenol concentration (m g/L) 0 0 100200300400500600700 Figure 11. Kinetic best-fit of the specific growth rate of P. putida cells on the binary substrates of Initial phenol concentration (m g/L) phenol and p-cresol with a correlation coefficient R2 = 0.9894.

Processes 2021, 9, 133 17 of 23

The values of IC,P and IP,C were 4.8 and 12.7, respectively, with a correlation coefﬁcient (R2) of 0.989.

4.4. Mass Transfer Coefﬁcients Wakao and Smith [35] proposed a random pore model to determine the effective diffusivity in gel beads. Furthermore, Korgel et al. [36] used the random pore model to predict the galactose effective diffusivity in the entrapped cell system. Based on the random pore model, the effective diffusivity (De) is described as 2 De = Ds(1 − βX) (22)

2 −1 −1 where Ds is the diffusion coefficient in gel beads (cm d ); X is cell concentration (g L ); and β is the specific volume of cells (L g−1). The values of X and α are 0.496 g L−1 and 3.842 × 10−3 L g−1 using the proposed measurement methods of Ju and Ho [37]. The diffusion coefficient (Ds) of phenol and p-cresol in gel beads employed in a continuous stirred-tank bioreactor is regarded as the same as that in water. The formula derived from Wilke and Chang [38] was used to calculate the values of Ds for phenol and p-cresol, which 2 −1 was 0.949 and 0.856 cm d , respectively. The effective diffusivity (De) of phenol and p-cresol was 0.945 and 0.853 cm2 d−1, respectively. The following equation was applied to estimate the mass transfer coefficient kf [39] Sh·Ds k f = (23) dp where Sh is Sherwood number = {4 + 1.21(Re)2/3(Sc)2/3}1/2, Re is Reynolds number, and Sc is Schmidt number. The value of Re for both phenol and p-cresol was 38.7. The value of Sc for phenol and p-cresol was 724.7 and 803.4, respectively. The value of Sh for phenol and p-cresol was 33.48 and 34.65, respectively. By substituting these values into Equation (23), −1 the mass transfer coefficient kf for phenol and p-cresol was 105.91 and 98.87 cm d , respectively.

4.5. Biodegradation of Phenol and p-Cresol in Immobilized Cells In order to validate the kinetic model system described above, phenol and p-cresol concentrations in bulk liquid phase estimated by the kinetic model were compared with the experimental results under 125 mg L−1 initial concentrations of phenol and p-cresol, respectively, in the feed. Table2 summarized the bio-kinetic and reactor parameters as well as operating conditions applied in kinetic model simulation as reported earlier in the various batch tests. Figure 12 plots the model-predicted and experimental data of phenol and p-cresol effluent concentrations against time. The effluent curve of phenol concentration consists of three segments. During a half day, the phenol and p-cresol concentrations increased −1 sharply to 81.6 (0.653 Sb0,p) and 89.1 (0.713 Sb0,c) mg L . No significant biodegradation of phenol and p-cresol was carried out during the half day. The phenol and p-cresol concentration curves were considered as the typical dilute-in curves, which is characteristic of a continuous stirred-tank bioreactor, while the bioreactor was filled with only nutrient media at the onset of the tests. The second segment of the phenol and p-cresol curves ran from day 0.5 to day 7, when the curves started to deviate from the peak of the dilute-in curves. The effluent concentrations of phenol and p-cresol leveled off and then decreased. Obviously, the immobilized cell markedly degraded phenol and p-cresol during this period, owing to the active growth of cells. The third segment of the phenol and p-cresol concentration curves ran from day 7 day 29. During this period, the immobilized cells system achieved a steady state, and the effluent concentration phenol and p-cresol was −1 approximately 2.95 (0.0236 Sb0,p) and 13.63 (0.109 Sb0,c) mg L , respectively. The removal efficiency for phenol and p-cresol was 97.6% and 89.1%, respectively, under a steady-state condition. The model simulations are in satisfactory agreement with the test results with a correlation coefficient (R2) of 0.9392 for phenol and 0.9063 for p-cresol. Processes 2021, 9, 133 18 of 23

Table 2. Summary of the Biokinetic and Reactor Parameters, as well as the Operation Conditions for the Model Simulation.

Symbol Parameters Description (Unit) Value Remarks ε reactor porosity (dimensionless) 0.72 measured A total surface area of gel beads (cm2) 3.522 × 104 calculated 2 −1 DeP effective diffusivity of phenol in the gel bead (cm d ) 0.945 calculated 2 −1 DeC effective diffusivity of p-cresol in the gel bead (cm d ) 0.853 calculated −1 kfP mass-transfer coefficient of phenol (cm d ) 105.91 calculated −1 kfC mass-transfer coefficient of p-cresol (cm d ) 98.87 calculated −1 Ki,P inhibition constant of phenol (mg L ) 310.5 measured −1 Ki,C inhibition constant of p-cresol (mg L ) 243.56 measured −1 Ks,P saturation constant of phenol (mg L ) 221.4 measured −1 Ks,C saturation constant of p-cresol (mg L ) 65.1 measured IC,P inhibition of cell growth on phenol due to the presence of p-cresol (dimensionless) 4.8 measured IP,C inhibition of cell growth on p-cresol due to the presence of phenol (dimensionless) 12.7 measured Q influent flow rate (mL d−1) 6.272 × 103 measured −1 Sb0,P concentration of phenol in feed (mg L ) 125.0 measured −1 Sb0,C concentration of p-cresol in feed (mg L ) 125.0 measured V effective working volume (mL) 1.568 × 103 measured −1 YP growth yield of cell on phenol (mg cell [mg phenol] ) 0.340 measured −1 YC growth yield of cell on p-cresol (mg cell [mg p-cresol] ) 0.279 measured Processes 2021, 9, x FOR PEER REVIEW −1 20 of 24 µmax,P maximum specific growth rate of cell on phenol (h ) 0.45 measured −1 µmax,C maximum specific growth rate of cell on p-cresol (h ) 0.185 measured

0.8

P he nol (M odel) 0.7 Phenol (Experimental) R 2 =0.9392 b0

/S 0.6

b P -cresol (Model) P -cre so l (E xp e rim e nta l) 0.5 R 2 =0.9063

0.4

0.3

Substrate concentration, S 0.2

0.1

0 0 5 10 15 20 25 30 Time (days)

FigureFigure 12.12. ExperimentalExperimental resultsresults and and model model prediction prediction in in phenol phenol and andp -cresolp-cresol effluent effluent concentrations. concentrations. The batch bioreactors were performed by Yadzir et al. [40], who found that the encap- sulated4.6. Immobilized cells of Acinetobacter Cells Growth baumannii in the Ca-alginate beads had the ability to remove − phenolFigure up to 13a 2000 presents mg L the1 within immobilized 12 d. In cells their gr study,owth as they a function also found of time that by there model was pre- no lossdiction. of Ca-alginate As can be seen, activity there during is no the five elapsed cycles oftime batch required tests. for Basak immobilized et al. [41 ]cells employed to start sugarcaneto grow. The bagasse model as predicted a low-cost that immobilization the immobilized matrix cells for vigorously cells entrapment grew to in utilized the upflow phe- packednol at a bedtransient-state reactor to assess period phenol from degradation5 to 25 days. under The growth different of influentimmobilized flow rates.cells reached The ex- perimentalup to a maximum results exhibitedvalue of around that the 32 phenol mg L−1 removal. efficiency reached up to 97%, while the feed initial phenol concentration was 2400 mg L−1 and the flow rate was controlled at 4 mL min−1 within the operating times of 54 h. Furthermore, Dong et al. [42] combined a zeolite35 imidazole framework (ZIF-8) with hydrochloric acid-modified SEP (CESEP) to form a nanocomposite CESEP/ZIF-8 for P. putida immobilization, which provided adsorption and biodegradation30 mechanisms for phenol removal. The experimental results exhibited

10 Immobilized cell(mg/L) concentration Immobilized 5

0 0 5 10 15 20 25 30 Time (days) (a)

Processes 2021, 9, x FOR PEER REVIEW 20 of 24

0.8

P he nol (M odel) 0.7 Phenol (Experimental) R 2 =0.9392 b0

/S 0.6

b P -cresol (Model) P -cre so l (E xp e rim e nta l) 0.5 R 2 =0.9063

0.4

0.3

Substrate concentration, S 0.2

0.1

Processes 2021, 9, 133 0 19 of 23 0 5 10 15 20 25 30 Time (days)

Figure 12. Experimental results and model prediction in phenol−1 and p-cresol effluent concentra- thattions. phenol at initial concentrations of 10 and 20 mg L was effectively removed within 13 and 24 h as compared to 21 and 36 h for phenol removal by free P. putida alone. 4.6. Immobilized Cells Growth 4.6. Immobilized Cells Growth Figure 13a presents the immobilized cells growth as a function of time by model pre- Figure 13a presents the immobilized cells growth as a function of time by model diction. As can be seen, there is no the elapsed time required for immobilized cells to start prediction. As can be seen, there is no the elapsed time required for immobilized cells to to grow. The model predicted that the immobilized cells vigorously grew to utilized phe- start to grow. The model predicted that the immobilized cells vigorously grew to utilized nol at a transient-state period from 5 to 25 days. The growth of immobilized cells reached phenol at a transient-state period from 5 to 25 days. The growth of immobilized cells −1 reachedup to a maximum up to a maximum value of valuearound of around32 mg L 32. mg L−1.

10 Immobilized cell(mg/L) concentration Immobilized 5

Processes 2021, 9, x FOR PEER REVIEW 0 21 of 24 0 5 10 15 20 25 30 Time (days) (a)

0.025

0.02 ) -1 d -2

0.015

0.01 Substrate flux (mg cm (mg flux Substrate

0.005 Phenol P -cresol

0 0 5 10 15 20 25 30 Time (days)

(b)

FigureFigure 13.13.Model Model predictionprediction versus versus time time ( a(a)) immobilized immobilized cells cells growth growth (b ()b phenol) phenol and andp -cresolp-cresol ﬂuxes fluxes into bead. into bead.

4.7. Flux into Gel Bead Figure 13b plots the model-predicted fluxes of phenol and p-cresol that diffuses from the bulk liquid into the bead. Flux represents the phenol utilization by immobilized cells. At the beginning of the experiment, the flux started at zero, and the immobilized cell growth was negligible. The flux of the immobilized cells increased abruptly at a logarithmic rate for the first four days. During this period, the immobilized cells vigorously degraded phenol and p-cresol in the bead—thus the difference between the concentrations of phenol and p-cresol in the bulk liquid and that at the bead/liquid interface increased, significantly increasing the fluxes of phenol and p-cresol into the beads due to biological activity. During days 4–29, the phenol and p-cresol concentrations in the effluent reached a constant concentration in a steady-state condition. The fluxes of phenol and p-cresol reached a maximal constant value, respectively, which was approximately 0.0220 and 0.0202 mg cm−2 d−1.

4.8. Phenol Concentration Profiles The concentration variations of phenol and p-cresol along the liquid film and bead phrase attained at 10, 20, and 29 days are illustrated in Figure 14. The concentration profiles for phenol and p-cresol due to the diffusional resistance in the liquid film and bead phases was determined by model prediction. It can be seen that phenol and p-cresol concentrations decreased in the liquid film and bead phases when the operating time increased. The continuous stirred-tank bioreactor achieved the steady state on day 10. The entrapped cells concentration in the bead was about 16 mg L−1, and the cells actively utilized phenol and p-cresol simultaneously for their growth. The concentrations of phenol and p-cresol reduced promptly at 20 days around the center of bead. The values of phenol and p-cresol concentration approached to around zero on day 29. At this operating time, the fluxes of phenol and p-cresol remained a constant value, while a maximal value of the growth of entrapped cells was achieved.

Processes 2021, 9, 133 20 of 23

4.7. Flux into Gel Bead Figure 13b plots the model-predicted fluxes of phenol and p-cresol that diffuses from the bulk liquid into the bead. Flux represents the phenol utilization by immobilized cells. At the beginning of the experiment, the flux started at zero, and the immobilized cell growth was negligible. The flux of the immobilized cells increased abruptly at a logarithmic rate for the first four days. During this period, the immobilized cells vigorously degraded phenol and p-cresol in the bead—thus the difference between the concentrations of phenol and p-cresol in the bulk liquid and that at the bead/liquid interface increased, significantly increasing the fluxes of phenol and p-cresol into the beads due to biological activity. During days 4–29, the phenol and p-cresol concentrations in the effluent reached a constant concentration in a steady-state condition. The fluxes of phenol and p-cresol reached a maximal constant value, respectively, which was approximately 0.0220 and 0.0202 mg cm−2 d−1.

4.8. Phenol Concentration Profiles The concentration variations of phenol and p-cresol along the liquid film and bead phrase attained at 10, 20, and 29 days are illustrated in Figure 14. The concentration profiles for phenol and p-cresol due to the diffusional resistance in the liquid film and bead phases was determined by model prediction. It can be seen that phenol and p-cresol concentrations decreased in the liquid film and bead phases when the operating time increased. The continuous stirred-tank bioreactor achieved the steady state on day 10. The entrapped cells concentration in the bead was about 16 mg L−1, and the cells actively utilized phenol and p-cresol simultaneously for their growth. The concentrations of phenol and p-cresol reduced promptly at 20 days around the center of bead. The values of phenol Processes 2021, 9, x FOR PEER REVIEWand p-cresol concentration approached to around zero on day 29. At this operating22 time,of 24 the fluxes of phenol and p-cresol remained a constant value, while a maximal value of the growth of entrapped cells was achieved.

0.026

0.024

0.022 b0,P /S

s,P 0.02

10 days 0.018 20 days 29 days 0.016

0.014 Phenol concentration, S

0.012

0.01 00.51 0 0.5 1 Bead phase Liquid film

(a) 0.104 Figure 14. Cont.

0.102

bo,C 0.1 /S s,C

0.098 10 days 20 days 0.096 29 days

0.094 -cresol concentration, S concentration, -cresol P

0.092

0.09 00.51 0 0.5 1 Bead phase Liquid film

(b) Figure 14. Model predicted phenol concentration profiles at different operating times: (a) phenol, (b) p-cresol.

5. Conclusions The biodegradation kinetic model for the simultaneous removal of phenol and p-cresol was validated by conducting a continuous stirred-tank bioreactor with immobilized cells in Ca-alginate beads. Diffusion and biodegradation are two major mechanisms considered in the model system. The model agreed with experimental data very well in the continuous-flow reactor. Experimental results demonstrate that the immobilized cells

Processes 2021, 9, x FOR PEER REVIEW 22 of 24

0.026

0.024

0.022 b0,P /S

s,P 0.02

10 days 0.018 20 days 29 days 0.016

0.014 Phenol concentration, S

0.012

0.01 00.51 Processes 2021, 9, 133 0 0.5 1 21 of 23 Bead phase Liquid film

(a) 0.104

0.102

bo,C 0.1 /S s,C

0.098 10 days 20 days 0.096 29 days

0.094 -cresol concentration, S concentration, -cresol P

0.092

0.09 00.51 0 0.5 1 Bead phase Liquid film

(b)

FigureFigure 14. 14. ModelModel predicted predicted phenol phenol concentration concentration profiles proﬁles at different at different operating operating times: times: (a) phenol, (a) phenol, (b)( bp)-cresol.p-cresol.

5. 5.Conclusions Conclusions TheThe biodegradation biodegradation kinetic kinetic model model for for th thee simultaneous simultaneous removal removal of of phenol phenol and p-cresol-cre- solwas was validated validated by by conducting conducting a continuous a continuous stirred-tank stirred-tank bioreactor bioreactor with with immobilized immobilized cells in cellsCa-alginate in Ca-alginate beads. beads. Diffusion Diffusion and biodegradation and biodegradation are two are major two mechanismsmajor mechanisms considered con- in sideredthe model in the system. model Thesystem. model The agreed model with agreed experimental with experimental data very data well very in the well continuous- in the continuous-flowflow reactor. Experimental reactor. Experimental results demonstrate results demonstrate that the immobilized that the immobilized cells process cells yields the high biodegradation of phenol and p-cresol, which was 97.6% and 89.1%, respectively. The fluxes of phenol and p-cresol that diffuse from the bulk liquid into the gel beads increased rapidly, while the entrapped cells in beads grew firmly during the unsteady-state period. The approaches of experiments and kinetic model presented in this study can be applied to layout a pilot-scale or full-scale entrapped cells bioreactor for the simultaneous biodegradation of phenol and p-cresol contaminants from various industrial wastewaters.

Author Contributions: Y.-H.L. conceived and designed the experiments as well as developed the kinetic models and analyzed the experimental data; Y.-J.G. conducted the batch and continuous stirred-tank bioreactors and collected the data. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by a funding from the Ministry of Science and Technology of Taiwan under Contract No. MOST 108-2221-E-166-002. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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