Bioreactor Design Guided by Kinetic Modeling: a Sustainable Approach for Catechol-Rich Industrial Wastewater Treatment

HIGHLIGHTS

• B. pseudomycoides efficiently degrades Catechol up to 600 mg.L^-1.

• Growth and degradation follow the integrated Haldane model.

• Kinetic parameters guided design of a 10 L aerobic bioreactor.

• Provides eco-friendly strategy for pharmaceutical wastewater treatment.

Abstract

Aerobic biodegradation of Catechol was investigated using the bacterial strain Bacillus pseudomycoides NBRC 101232, with an initial Catechol concentration of up to 600 mg·L^-1. The primary objective of this study was to evaluate the growth and degradation kinetics of the strain by monitoring biomass production and substrate depletion over time in a shake-flask batch reactor system. Microbial growth behavior was successfully described using the integrated Haldane substrate inhibition model, yielding kinetic parameters of μ_m = 0.056 h^-1, K_S = 28.52 mg·L^-1, and K_i = 126.50 mg·L^-1 at an initial Catechol concentration of 600 mg·L^-1. The average biomass yield coefficient (Y_X/S) was 0.0034 OD₆₀₀ units·L·mg^-1, indicating efficient substrate utilization under inhibitory conditions. The strain exhibited clear substrate inhibition kinetics at elevated Catechol concentrations. Based on the experimentally determined kinetic parameters and previously optimized physicochemical conditions, a preliminary aerobic continuous stirred-tank bioreactor (CSTR) design framework was developed for the treatment of Catechol-containing pharmaceutical wastewater. The integration of microbial kinetics with engineering design enables a rational framework for reactor sizing and operational optimization. This study provides a practical and scalable strategy for the efficient, sustainable, and eco-friendly biodegradation of high-strength Catechol-containing industrial wastewater.

GRAPHICAL ABSTRACT

INTRODUCTION

Water contamination has intensified with rapid industrialization, while readily usable freshwater accounts for only ~0.5% of global water resources, underscoring the urgent need for effective wastewater treatment strategies. Phenolic compounds, including Catechol, are widely discharged from petrochemical, pharmaceutical, and allied industries and are recognized as priority environmental pollutants owing to their persistence and toxicity [¹].

Catechol is considered more inhibitory than phenol and can strongly suppress microbial metabolism even at moderate concentrations, thereby complicating biological treatment processes [²]. Although physicochemical methods are available, bioremediation using microorganisms has emerged as a sustainable and environmentally benign alternative due to its lower energy demand and minimal secondary waste generation [³]. However, treatment efficiency is strongly influenced by substrate concentration, and high-strength phenolic wastewater commonly exhibits substrate inhibition effects that limit process performance [⁴].

To describe inhibitory biodegradation systems, several kinetic models have been proposed, among which the Haldane model is widely accepted for phenolic compounds because of its ability to account for substrate inhibition behaviour [⁵]. Earlier kinetic studies on phenol and Catechol degradation, including the foundational work of Nweke and Okpokwasili [⁶], have demonstrated the importance of kinetic modelling for understanding microbial response under inhibitory conditions and for supporting reactor-oriented process design.

This study is grounded in the hypothesis that Bacillus pseudomycoides NBRC 101232, owing to its metabolic versatility and tolerance to inhibitory conditions, can exhibit predictable growth and Catechol degradation kinetics, and that these kinetics can be successfully translated into a scalable aerobic bioreactor design for the efficient treatment of high-strength industrial effluents. Consistent with this hypothesis, the present work investigates the growth and degradation kinetics of Bacillus pseudomycoides NBRC 101232 and integrates experimentally derived Haldane kinetic parameters into a continuous reactor design framework. By coupling microbial kinetics with reactor engineering, the study aims to establish a replicable framework for eco-friendly, cost-effective, and sustainable treatment of Catechol-rich wastewater.

Urban hospital premises often receive chemically complex effluents containing disinfectants, pharmaceuticals, and aromatic compounds, creating selective pressure for metabolically resilient microorganisms capable of degrading toxic organic pollutants. In this context, Bacillus pseudomycoides NBRC 101232 was isolated from chemically contaminated soil collected within the premises of South Howrah State General Hospital (22.5604° N, 88.304° E), an urban site exposed to mixed anthropogenic pollutants. This environmental origin supports the strain’s relevance for application in real-world wastewater treatment systems and provides a logical basis for its selection in the present study.

By integrating microbial kinetics with engineering-oriented reactor design, this study aims to provide a practical framework for translating laboratory biodegradation data into scalable wastewater treatment systems.

MATERIAL AND METHODS

Bacillus pseudomycoides NBRC 101232 was isolated from contaminated soil collected at South Howrah State General Hospital (22.5604° N, 88.304° E). Detailed procedures for isolation, screening, characterization, identification, and parameter optimization are described in Maity and coauthors (2023) [⁷]. Catechol-contaminated water was synthetically prepared in the laboratory, initially following reported methods [⁸, ⁹], and later refined based on results from preliminary experiments.

Experiments were conducted in 1 L cotton-plugged conical flasks using Mineral Salt Medium (MSM) as culture medium [⁶, ⁹, ¹⁰, ¹¹, ¹², ¹³]. The pH was adjusted with 1N HCl or 1N NaOH and confirmed with a digital pH meter. All chemicals were of analytical or reagent grade (Merck, Germany; Sigma–Aldrich). Catechol was of analytical grade, and MSM powder was reagent grade. Cultures were incubated in a BOD shaker at 140–150 rpm under optimized conditions: temperature 35 °C, pH 5.5, incubation time 72 h, medium volume 600 mL, and inoculum size 6%. These conditions yielded maximum degradation in preliminary studies [⁷]. To evaluate substrate inhibition, the initial Catechol concentration was varied over a range of 50–600 mg·L^-1 (50, 100, 200, 400, 500, and 600 mg·L^-1), while all other operational parameters were kept constant. This concentration range was selected to represent high-strength industrial effluent conditions and to capture inhibitory effects relevant to kinetic modeling and reactor design. Samples were withdrawn at 4 h intervals for analysis. Biomass growth was monitored spectrophotometrically in all flasks, whereas residual Catechol was quantified only in test samples.

A growth control consisting of mineral salt medium inoculated with Bacillus pseudomycoides NBRC 101232 in the absence of Catechol was included to assess baseline biomass development. In addition, an abiotic control containing mineral salt medium and Catechol without inoculum showed no measurable Catechol loss, confirming the absence of non-biological degradation.

Catechol degradation was quantified using a UV–VIS spectrophotometer at 510 nm following the 4-Aminoantipyrine method described by Yang and Humphrey (1975) [¹⁴]. A standard calibration curve was prepared using known Catechol concentrations, and the resulting equation was applied to calculate percentage degradation.

(1) $Y=mX+C$

Where, Y stands for the optical density (OD) of an unknown sample, m stands for slope of the standard curve, X stands for the residual conc. of Catechol in the sample and C represents a constant.

Biomass growth was monitored by aseptically collecting cell samples and measuring optical density (OD) at 600 nm, using Mineral Salt Medium as the blank.

The rates of the Catechol degradation (Q_s) were calculated from the plots of S₀ - S_t (amount of degraded Catechol) vs. t – t₀ (incubation time). Specific growth rates were analyzed from the plots of ln(X/X₀) vs. t – t₀ for each initial concentrations of Catechol. The yield coefficients (Y) were calculated by plotting X – X₀ vs. S₀ – S.

(2) $Qs=\frac{s0-s}{t-t0}$

(3) $ln\frac{x}{x0}=\mu \left(t-t0\right)$

(4) $X-X0=Y\left(S0-S\right)$

Here, S0=initial conc. of Catechol at t0 time (mg.L-1), S=Conc. of Catechol at time t (mg.L^-1), X= Conc. Of biomass at OD₆₀₀ at time t and X₀ = Conc. Of biomass at OD₆₀₀ at time t₀

Microbial growth representation by Haldane equation:

(5) $\mu =\frac{{\mu }_m\text{s}}{K_s+S+\frac{S^2}{K_i}}$

Here, μ=specific growth rate in h^-1, S=Substrate conc. in mg.L^-1, μm=maximum specific growth rate in h^-1, Ks=half saturation coefficient in mg.L^-1, K_i=inhibition coefficient in mg.L^-1

Now, the production of biomass can be represented by:

(6) $\frac{dx}{dt}=\mu X$

Substituting the μ in the equation no 6 from the equation no 5,

(7) $\frac{dx}{dt}=\frac{{\mu }_{m}SX}{K_s+S+\frac{S^2}{K_i}}$

From the Equation no 4, it can be represented that,

(8) $S=S0-\frac{X-X0}{Y}$

Here, X = conc. of biomass, Y = yield coefficient (x/s), S = substrate concentration, t = time, S₀ = initial substrate concentration and X₀ = initial biomass concentration.

Now, in the equation no 7, S can be substituted by the Equation no 8

(9) $\frac{dx}{dt}=\frac{{\mu }_m\left(S_0-\frac{X-X0}{Y}\right)X}{K_s+\left(S_0-\frac{X-X_0}{Y}\right)+\frac{{\left(S_0-\frac{X-X_0}{K_i}\right)}^2}{K_i}}$

Now integrating the both side of the equation no 9, equation no 10 obtained,

(10) $\mu mt=\left[\frac{KsY}{Y{S}_0+X_0}+1+\frac{Y{S}_0+X_0}{K_{i}Y}\right]ln\frac{\text{X}}{\text{X0}}-\frac{KsY}{Y{S}_0+X_0}ln\frac{Y{S}_0+X_0-X}{Y{S}_0}$

The rate of biodegradation can be represented as:

(11) $\frac{ds}{dt}=-\mu \frac{X}{Y}$

Now, substituting the μ from the equation no 5,

(12) $\frac{ds}{dt}=-\frac{{\mu }_{m}SX}{{\left(K_{}+S+\frac{S^2}{K_i}\right)}^Y}$

Applying mass balance,

(13) $\text{X}=\text{Y}\left(\text{S}0-\text{S}\right)+\text{X}0$

Now, in the equation no 12, X can be substituted from the equation no 14

(14) $\frac{ds}{dt}=-\frac{{\mu }_{m}S\left[Y\left(S_0-S\right)+X_0\right]}{{\left(K_S+S+\frac{S^2}{K_i}\right)}^Y}$

Now, integrating the equation no 14, equation no 15 can be achieved,

(15) $\mu \text{mt}=\left[\frac{KsY}{Y{S}_0+X_0}+1+\frac{Y{S}_0+X_0}{K_{i}Y}\right]ln\frac{Y{S}_0+X_0-YS}{X_0}-\frac{KsY}{Y{S}_0+X_0}ln\frac{S}{S_0}$

Equation (10) was fitted to the biomass production data, and kinetic parameters were estimated using non-linear regression implemented in Table Curve 2D (v5.01) employing the Levenberg–Marquardt optimization algorithm. Initial parameter estimates were obtained from yield coefficients derived from X–X₀ versus S₀–S plots. Equation (15) was subsequently applied to model substrate depletion kinetics using the biomass-linked parameters obtained from the fitting procedure. A schematic representation of the catechol degradation pathway and its linkage to kinetic modeling is presented in Figure 6.

All experiments were conducted in triplicate independent runs, and results are presented as mean ± standard deviation (SD). Model adequacy was evaluated using the coefficient of determination (R²) and residual analysis to confirm the randomness and homoscedasticity of errors. Parameter uncertainty was assessed by calculating 95% confidence intervals from the covariance matrix, and standard error propagation was applied to derived kinetic parameters. Replicate variability remained below 5%, indicating effective experimental reproducibility and statistical reliability.

The reactor design framework was developed by integrating experimentally derived batch kinetic parameters into steady-state continuous reactor (CSTR) design equations. Batch studies were initially performed to evaluate the effects of operational variables, including initial Catechol concentration, pH, temperature, inoculum size, and incubation time, on microbial growth and substrate degradation. The optimized operating conditions and kinetic parameters derived from these batch experiments were subsequently used as inputs for reactor design and performance estimation.

The aerobic degradation kinetics of Catechol by Bacillus pseudomycoides NBRC 101232 were described using the Haldane substrate inhibition model. The kinetic constants obtained from model fitting (Table 2) formed the basis for determining hydraulic retention time (HRT), reactor sizing, and key operational parameters.

Thus, the reactor design parameters were not assumed arbitrarily but were systematically derived from experimentally obtained batch kinetic data. The integration of Haldane kinetic constants with HRT-based sizing provides a rational and scalable framework for designing aerobic bioreactors for the treatment of high-strength Catechol-containing wastewater.

Hydraulic retention time (HRT) was selected as the primary design parameter, as it governs substrate–biomass contact time, biomass accumulation, degradation efficiency, and reactor volume requirements. HRT was calculated using:

(16) $HRT=\frac{V}{Q}$

Where V is the reactor volume (L) and Q is the influent flow rate (L·h^-1).

For continuous reactor design, the hydraulic retention time (HRT) was derived from steady-state mass balance principles. In a Continuous Stirred Tank Reactor (CSTR), the dilution rate (D = Q/V) governs reactor performance. At steady state, biomass washout occurs when the dilution rate equals the specific growth rate of the microorganism:

(17) $D=\mu \left(S\right)$

Substituting the Haldane kinetic expression:

(18) $D=\frac{{\mu }_{max}\text{S}}{K_s+S+\frac{S^2}{K_i}}$

Using the experimentally determined kinetic parameters, the allowable dilution rate was estimated to be approximately 0.02–0.025 h^-1, corresponding to a hydraulic retention time (HRT = 1/D) of 40–50 h.

This approach ensures that reactor design is based on continuous-flow kinetics rather than batch-derived scaling relationships.

Proposed Reactor Configuration

An aerated Continuous Stirred Tank Reactor (CSTR) is proposed as the primary configuration for continuous treatment of Catechol-containing wastewater. The reactor operates under completely mixed conditions with controlled aeration and agitation to maintain optimal dissolved oxygen, pH, and temperature for microbial activity (Figure 5).

RESULTS

Degradation of Catechol & biomass production

Bacillus pseudomycoides NBRC 101232 was cultured in six initial Catechol concentrations: 50 mg.L^-1, 100 mg.L^-1, 200 mg.L^-1, 400 mg.L^-1, 500 mg.L^-1, and 600 mg.L^-1, along with a control. The maximum concentration (600 mg. L^-1) was selected based on optimization studies. Degradation time varied with initial concentration: 50 and 100 mg.L^-1 were completely degraded within 4 h, 200 mg.L^-1 in 20 h, 400 mg.L^-1 in 32 h, 500 mg.L^-1 in 60 h, and 600 mg.L^-1 in 72 h.

The effect of Catechol concentration on biomass growth is shown in Figure 1, while biomass yield per unit Catechol consumed (Y_x/s) is presented in Table 1. The maximum yield, 0.0038 OD units·L.mg^-1, was achieved at 500 mg.L^-1.

Figure 1.
Yield coefficients for Bacillus pseudomycoides; growth on Catechol in different initial concentrations. Source: Authors

Table 1
Yield coefficients for the bacterial growth on the Catechol determined from the plots

S0 (mg. L-1)	Yield coefficients (Yx/s) [OD600 units.L.mg-1] for Bacillus pseudomycoides
200	0.0036
400	0.0025
500	0.0038
600	0.0037

Yield coefficients were not determined for 50 and 100 mg.L^-1 Catechol. The time duration of the microbial growth and Catechol degradations at 200 mg.L^-1, 400 mg.L^-1, 500 mg.L^-1 & 600 mg. L^-1 is shown in Figure 2, while growth curves in Catechol medium (600 mg.L^-1) and control are presented in Figure 3. Both maximum OD values and incubation time to reach stationary phase depended on the initial Catechol concentration. At 600 mg.L^-1, Bacillus pseudomycoides achieved the highest OD (2.323), with a corresponding OD of 2.078 under the same conditions. Growth exhibited distinct lag, exponential, and stationary phases; the lag phase lengthened with increasing Catechol concentration, extending the degradation period. The transient stagnation in growth, followed by renewed biomass increase, may be attributed to intermediate metabolites.

Figure 2.
Time duration of the microbial growth and Catechol degradation in case of Bacillus pseudomycoides. Source: Authors

Figure 3.
The growth profile of Bacillus pseudomycoides in the highest Catechol conc. and control medium. Source: Authors

Figure 3 further demonstrates that Catechol served as the sole energy source for the strain. Up to 600 mg. L^-1, growth in Catechol medium consistently exceeded that in the control, with marked differences observed beyond 20 h.

Kinetics of degradation of Catechol and production of biomass

The growth and degradation profiles were well explained by the integrated Haldane substrate inhibition model, with R² > 0.9 from progress curve analysis. Both bacterial growth and Catechol degradation initially increased with rising substrate concentration but declined at higher levels due to inhibition. The Haldane model accurately described the overall growth of Bacillus pseudomycoides across 0 – 600 mg. L^-1 (R²=0.9991) and also fitted the degradation rate effectively (R²=0.9356) (Figure 4). The estimated kinetic parameters for Catechol biodegradation by B. pseudomycoides NBRC 101232 were: Q_s=12.01 mg.L^-1.h^-1, K_S=28.52 mg.L^-1, K_i=126.50 mg.L^-1 and μ_m=0.056 h⁻¹.

Figure 4.
Experimental and predicted specific growth rate and degradation rate of Bacillus pseudomycoides NBRC 101232 during the biodegradation of Catechol. Source: Authors.

Bioreactor Design Based on Continuous Reactor Kinetics

In this context, the kinetic parameters obtained from model fitting are summarized in Table 2. Based on these kinetic parameters, the corresponding continuous reactor design and operating parameters were estimated and are summarized in Table 3.

Table 2
Kinetic Characteristics of Bacillus pseudomycoides NBRC 101232

Parameter	Value	Description
Initial Catechol concentration (S0)	600 mg.L-1	Optimized condition
μm	0.056 h-1	Maximum specific growth rate
Ks	28.52 mg.L-1	Half- saturation constant
Ki	126.50 mg.L-1	Substrate inhibition constant
Qs	12.01 mg.L-1.h-1	Substrate utilization rate
Biomass yield (Yx/s)	0.0034 units. L.mg-1	Conversion efficiency
Growth R2 (Haldane fit)	0.9991	Excellent model fit
Degradation R2 (Haldane fit)	0.9356	Good model fit

Table 3
Design and operating parameters of the continuous stirred tank reactor (CSTR) derived from Haldane kinetic modeling

Parameter	Symbol	Value	Unit	Description / Basis
Influent Catechol concentration	S0	600	mg·L-1	Optimized experimental condition
Maximum specific growth rate	μmax	0.056	h-1	Estimated from Haldane model
Half-saturation constant	KS	28.52	mg·L-1	Haldane kinetic parameter
Inhibition constant	Ki	126.50	mg·L-1	Haldane kinetic parameter
Biomass yield coefficient	YX/S	0.0034	OD units·L·mg-1	Experimental determination
Reactor volume	V	10	L	Laboratory-scale design
Dilution rate	D	0.020–0.028	h-1	Derived from growth kinetics (D = μ)
Hydraulic retention time	HRT	36–50	h	Calculated as HRT = 1/D
Influent flow rate	Q	0.208–0.277	L·h-1	Calculated as Q = V × D
Dissolved oxygen (DO)	–	≥ 2	mg·L-1	Ensures aerobic metabolism
pH	–	5.5 ± 0.2	–	Optimized condition
Temperature	–	35–37	°C	Optimal for microbial growth
Aeration rate	–	1.0–1.5	vvm	Maintains oxygen transfer
Agitation speed	–	140–150	rpm	Ensures mixing and mass transfer
Reactor material	–	Borosilicate glass (lab) / SS316 (pilot)	–	Chemical resistance and scalability
Operation mode	–	Continuous (CSTR)	–	Based on steady-state reactor kinetics

Determination of Optimal HRT

The hydraulic retention time (HRT) was derived from the steady-state dilution rate obtained using Haldane growth kinetics. Based on the estimated dilution rate range (0.020 – 0.028 h^-1), the corresponding HRT was determined to be approximately 36 – 50 h. This range ensures stable reactor operation below the microbial washout threshold while maintaining efficient Catechol degradation under continuous-flow conditions.

Accounting for operational variability, oxygen transfer limitations and maintaining pH/DO stability under continuous operation, a practical design HRT of 36–50 h is recommended.

Figure 5.
Schematic representation of the aerated continuous stirred tank reactor (CSTR) proposed for catechol degradation under steady-state operation. Source: Authors

DISCUSSION

Water pollution by aromatic hydrocarbons is a major concern. Phenolic compounds, due to their high solubility, readily disperse in aquatic systems, disrupting ecosystems and threatening both terrestrial organisms and humans [¹⁵]. Their removal from water, particularly at point sources, is therefore essential. Bioremediation is preferred for its eco-friendly nature.

Efficient bioreactor design requires detailed kinetic analysis of the degrading species. Various models, including the Yano–Koga, Monod [¹²], and the widely used Haldane model [⁶], have been applied. In this study, the integrated Haldane model, incorporating maximum specific growth rate, half-saturation constant, biomass yield, and inhibition constant, was employed to evaluate Catechol degradation by Bacillus pseudomycoides NBRC 101232 at different initial concentrations. Non-linear least-squares curve fitting (Table Curve 2D) was used to estimate kinetic parameters. Biomass yield (Y_x/s) is presented in Table 1, while μ_m, K_s, and K_i are shown in Table 2. The Haldane model fit well with the experimental data (R² > 0.9). In continuous bioreactor systems, reactor performance is governed by the dilution rate and steady-state mass balance rather than batch-based substrate depletion kinetics. By incorporating Haldane growth kinetics into the CSTR design framework, the present study establishes a direct relationship between microbial growth rate and reactor operating conditions. The derived dilution rate (0.020–0.028 h⁻¹) ensures operation below the washout threshold, thereby maintaining stable biomass concentration and efficient catechol degradation under continuous-flow conditions. As shown in Table 4, the obtained parameters are consistent with earlier studies, confirming that biomass production was well described by the model. Results also indicate that Catechol inhibition occurred at higher concentrations.

Table 4
Comparative analysis of Haldane kinetic parameters reported for phenolic compound biodegradation systems

Microorganism	Pollutant	Maximum pollutant concentration (mg.L-1)	μm (h-1)	Ks(mg.L-1)	Ki(mg.L-1)	Reactor condition	Reference
Pseudomonas sp. DAF1	Phenol	0 – 1000	0.025	12.718	1632.086	Batch culture (hydrodynamic conditions not specified)	[6]
Pseudomonas sp. RWW2	Phenol	0 - 1000	0.024	14.628	2986.159	Batch culture (hydrodynamic conditions not specified)	[6]
Candida parapsilopsis	Catechol	36 - 910	0.246	16.95	604.85	Fungal batch culture (pellet-forming system)	[20]
Bacillus simplex	Phenol	0 – 700	1.05	9.14	329.0	Not specified	[21]
Bacillus simplex	Phenol	200 - 1200	1.05	9.14	329	Batch culture	[21]
Bacillus brevis	Phenol	750 – 1750	0.026 – 0.078	2.2 - 29.31	868.0- 2434.7	Batch culture	[22]
Alcaligenes faecalis	Phenol	10 – 1400	0.15	2.22	245.37	Shaken batch culture	[24]
Alcaligenes faecalis	m-cresol	5 – 200	0.0782	1.30	71.77	Shaken batch culture	[24]
Pseudomonas putida LY1	Phenol	0 - 800	0.217	24.4	121.7	Well-mixed batch culture	[25]
Alcaligenes faecalis B6 – 2	Phenol	0- 1128	0.12	469.23	188.16	Batch culture	[26]
Kocuria rhizophila strain 11Y	2, 4- Dicloro Phenol	0 – 400	1.17	568.1	215.63	Batch culture	[27]
Mixed activated sludge	Phenol	250,500,1000	0.117	1869.99	85.80	Suspended-growth batch system	[28]
Glutamicibacter nicotianae MSSRFPD35	Phenol	0 - 1117	0.37	20.29	268.1	Well-agitated batch culture	[29]
Rhodococcus sp. strain SKC	Phenol	1019.0	0.30	36.40	418.79	Batch culture	[30]
Rhodococcus pyridinivorans strain PDB9T NS-1	Phenol	-	0.169	54.21	243.26	Batch culture (hydrodynamic details not reported)	[31]
Serratia odorifera	Phenol	-	0.469	26.6	292	Batch culture	[32]
Bacillus pseudomycoides NBRC 101232	Catechol	0 – 600	0.056	28.52	126.50	Well-mixed aerated suspended-growth system	This study

• Hydrodynamic regimes are described as reported in the respective studies. Detailed mixing intensity, oxygen transfer rates, and mass-transfer coefficients were not consistently specified across publications. Among the systems summarized above, Bacillus pseudomycoides NBRC 101232 demonstrated stable biodegradation of Catechol at concentrations up to 600 mg·L⁻¹ without immobilization, highlighting its potential as a robust suspended-growth biocatalyst for high-strength phenolic wastewater treatment.

Compared with earlier works, this study demonstrates distinct advantages. Pseudomonas putida BCRC 14365 required immobilization and strict pH/temperature control, with reduced efficiency at higher concentrations [¹⁶], whereas our strain degraded Catechol up to 600 mg·L^-1 without immobilization. Aspergillus awamori mineralized Catechol and phenolics but only over several days [¹⁷], while our system achieved complete degradation within 36–50 h. The Nocardia hydrocarbonoxydans pulsed plate bioreactor showed high removal at moderate influent phenol but declined at higher dilution rates [¹⁸]; in contrast, our reactor maintained efficiency at elevated Catechol levels using Haldane-guided design. The hybrid membrane–GAC bioreactor with Pseudomonas putida improved phenol removal but relied on sorption–desorption steps [¹⁹], whereas Bacillus pseudomycoides achieved direct mineralization with minimal sludge.

The kinetic behavior of Bacillus pseudomycoides NBRC 101232 was compared with previously reported microbes such as Candida parapsilopsis, Bacillus simplex, and Bacillus brevis during the degradation of Catechol and phenol. While C. parapsilopsis exhibits a higher maximum growth rate (μ_m ≈ 0.246 h^-1) at elevated Catechol concentrations [²⁰], it is a fungus that requires significantly more time to grow compared to bacterial strains. Bacillus simplex and Bacillus brevis can tolerate higher phenol levels but frequently accumulate toxic intermediates, thereby lowering mineralization efficiency [²¹, ²²]. In contrast, B. pseudomycoides sustained degradation up to 600 mg.L^-1 with complete mineralization and no harmful by-products. Its relatively high inhibition constant (K_i = 126.5 mg.L^-1) further emphasizes its robustness under toxic conditions.

In addition to intrinsic microbial kinetics, reactor hydrodynamics influence apparent biodegradation performance and should be considered when comparing results across studies. Mixing intensity, aeration efficiency, and reactor configuration govern oxygen transfer, substrate distribution, shear conditions, and residence time distribution, which collectively affect observed degradation rates. Variations in reported kinetic parameters (Table 4) may therefore reflect not only microbial physiology but also differences in hydrodynamic regimes under which experiments were conducted. Continuous reactor modeling has been successfully applied in wastewater treatment systems, where kinetic analysis is integrated with reactor design to optimize residence time and degradation efficiency [²³].

In the present study, kinetic parameters were derived under controlled and well-mixed aerated conditions designed to minimize concentration gradients and oxygen limitation. The agitation speed (140–150 rpm) and aeration rate (1.0–1.5 vvm) maintained dissolved oxygen levels ≥2 mg·L^-1, allowing intrinsic Haldane-type inhibition kinetics to predominate. Consequently, comparisons with literature values should be interpreted in light of potential hydrodynamic variability among systems. These considerations emphasize the importance of integrating both kinetic and hydrodynamic aspects when translating batch-derived parameters to scalable reactor design for high-strength phenolic wastewater treatment.

Critical Discussion of Kinetics and Model Limitations

The integrated Haldane model adequately described Catechol biodegradation by Bacillus pseudomycoides NBRC 101232 (R² > 0.9), consistent with reports for Pseudomonas, Rhodococcus, and Candida species (Table 4) [⁶, ²⁰, ³¹]. However, similar to previous investigations, the model assumes single-substrate utilization and does not explicitly account for intermediate metabolite accumulation, oxygen transfer effects, or inducible enzyme regulation, which may influence apparent kinetic parameters at elevated phenolic concentrations [²⁸, ³⁰].

The relatively low biomass yield (Y_X/S = 0.0034 L·mg^-1) observed in this study is comparable to values reported for phenol-degrading Rhodococcus strains and mixed activated sludge systems [²⁸, ³⁰], where a substantial fraction of substrate carbon is diverted toward maintenance energy and detoxification processes rather than cell synthesis. In contrast, higher yields reported for certain Bacillus and Kocuria strains were often obtained at lower substrate concentrations or under less inhibitory conditions [²¹,²⁷], indicating that yield variations are strongly dependent on operational context.

As noted in earlier kinetic analyses [⁶, ²⁹], the classical Haldane framework does not explicitly incorporate hydrodynamic limitations or multi-enzyme regulation mechanisms. Therefore, while suitable for batch-scale kinetic evaluation and reactor sizing, the model may underestimate system complexity under continuous-flow operation or in real industrial effluents. Structured or dynamic models incorporating oxygen transfer and intermediate formation kinetics would provide enhanced predictive capability under such conditions.

Ecological and Industrial Implications

Phenolic compounds and Catechol are recognized priority pollutants due to their toxicity and environmental persistence [¹⁵]. Several biological systems reported in the literature demonstrate effective phenol or Catechol removal; however, performance stability often varies with reactor configuration and substrate loading. For example, Pseudomonas putida–based systems and related bacterial cultures have been shown to achieve high degradation rates under controlled conditions but may require immobilization or strict environmental regulation to maintain stability at elevated concentrations [¹⁶, ¹⁹]. Fungal degraders such as Candida parapsilopsis have demonstrated efficient mineralization capability, although typically with longer treatment durations attributable to slower growth kinetics [¹⁷, ²⁰].

In comparison, Bacillus pseudomycoides NBRC 101232 demonstrated stable biodegradation up to 600 mg·L^-1 Catechol without the need for immobilization or sorptive support, positioning its performance within the upper range reported for suspended bacterial systems (Table 4). Although its maximum specific growth rate (μ_m = 0.056 h^-1) is moderate relative to some fast-growing strains [²¹], its inhibition constant (K_i = 126.5 mg·L^-1) indicates tolerance to inhibitory concentrations.

From an industrial perspective, physicochemical hybrid systems such as membrane-assisted or adsorption-based processes enhance apparent removal efficiency but may rely partially on non-biological mechanisms and generate secondary waste streams [¹⁹]. In contrast, the present system achieved biological degradation under well-controlled aerobic conditions with minimal sludge production, consistent with observations for well-adapted Rhodococcus and Glutamicibacter strains [²⁹, ³⁰].

Collectively, these findings support the translational potential of B. pseudomycoides NBRC 101232 as a robust candidate for sustainable treatment of Catechol -rich industrial effluents.

Figure 6.
Proposed ortho- and meta-cleavage pathways for Catechol degradation by Bacillus pseudomycoides NBRC 101232. Source: Authors

CONCLUSION

This study investigated the biodegradation kinetics of Catechol by Bacillus pseudomycoides NBRC 101232 using the Haldane substrate inhibition model. The strain demonstrated the ability to degrade Catechol concentrations up to 600 mg·L^-1, with kinetic parameters of μ_m = 0.056 h^-1, K_S = 28.52 mg·L^-1, and K_i = 126.50 mg·L^-1, confirming its tolerance to inhibitory substrate levels. Despite moderate inhibition at higher concentrations, the organism maintained stable growth and achieved complete Catechol degradation within 36–50 h under optimized aerobic conditions.

Using these experimentally derived kinetic parameters, a preliminary 10 L aerobic continuous stirred-tank bioreactor (CSTR) design framework was developed based on steady-state reactor kinetics. The proposed reactor configuration provides a conceptual basis for efficient biodegradation with minimal sludge generation and stable operation under high substrate loading.

To the best of our knowledge, this is the first report describing Catechol biodegradation by Bacillus pseudomycoides NBRC 101232 integrated with Haldane-based reactor design. The study therefore contributes both fundamental kinetic insight and practical engineering guidance for the treatment of high-strength phenolic wastewater. Overall, the findings highlight the strong potential of this strain as a robust biological agent for sustainable and scalable bioremediation of Catechol-rich industrial effluents.

• Funding:
  This research received no external funding.
• Institutional Review Board Statement:
  Not applicable.
• Informed Consent Statement:
  Not applicable.

Data Availability Statement:

Research data are only available upon request for corresponding author.

Acknowledgments:

The authors are grateful to the dept. of Food Technology & Biochemical Engineering for their constant support.

• Use of Generative Artificial Intelligence:
  The author declares that did not use the artificial intelligence.

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Edited by

• Editor-in-Chief:
  Bill Jorge Costa

• Associate Editor:
  Ana Cláudia Barana

Publication Dates

• Publication in this collection
  20 July 2026
• Date of issue
  2026

History

• Received
  22 Sept 2025
• Accepted
  08 Apr 2026