# A Novel Halophilic Bacterium for Sustainable Pollution Control: From Pesticides to Industrial Effluents

Nadia Mihoubi, Samira Ferhat, Mohamed Nedjhioui, Billal Zenati, Sabrina Lekmine, Reguia Boudraa, Mohammad Shamsul Ola, Jie Zhang, Abdeltif Amrane, Hichem Tahraoui

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Nadia Mihoubi, Samira Ferhat, Mohamed Nedjhioui, Billal Zenati, Sabrina Lekmine, et al., A Novel Halophilic Bacterium for Sustainable Pollution Control: From Pesticides to Industrial Effluents. Water, 2025, 17 (6), pp.888. 10.3390/w17060888. [https://doi.org/10.3390/w17060881](https://doi.org/10.3390/w17060881)

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Article

# A Novel Halophilic Bacterium for Sustainable Pollution Control: From Pesticides to Industrial Effluents

Nadia Mihoubi<fnref n="1" />, Samira Ferhat<fnref n="2" />, Mohamed Nedjhioui<fnref n="1" />, Billal Zenati<fnref n="3" />, Sabrina Lekmine<fnref n="4" /> ID, Reguia Boudraa<fnref n="5" /> ID,
Mohammad Shamsul Ola<fnref n="6" /> ID, Jie Zhang<fnref n="7" /> ID, Abdeltif Amrane<fnref n="8" />,* ID and Hichem Tahraoui<fnref n="2" />,<fnref n="8" />,* ID

<figure><img src="image_2.png" /></figure>

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Academic Editors: Baghdad Ouddane
and Sopheak Net

Received: 7 January 2025

Revised: 30 January 2025

Accepted: 2 February 2025

Published: 19 March 2025

**Citation:** Mihoubi, N.; Ferhat, S.; Nedjhioui, M.; Zenati, B.; Lekmine, S.; Boudraa, R.; Ola, M.S.; Zhang, J.; Amrane, A.; Tahraoui, H. A Novel Halophilic Bacterium for Sustainable Pollution Control: From Pesticides to Industrial Effluents. *Water* **2025**, *17*, 888. https://doi.org/10.3390/w17060888

**Copyright:** © 2025 by the authors.
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**Abstract:** This study investigates the bioremediation potential of *Marinobacter-hydrocarbonoclasticus* SDK644, a strain that has been isolated from petroleum-contaminated environments, for the degradation of the herbicide metribuzin and the treatment of slaughterhouse effluent. The strain's bacterial growth and degradation capacity were assessed under varying conditions, including different metribuzin concentrations, pH values, temperatures, and inoculum sizes. The strain demonstrated optimal growth at a metribuzin concentration of 20 mg/L, with an optical density (OD600) of 0.408 after 96 h. At this concentration, 80% of the chemical oxygen demand (COD) was reduced over 144 h. The optimal growth conditions for *M. hydrocarbonoclasticus* SDK644 were identified as a pH of 7 and a temperature of 30 °C, where the enzymatic activity and degradation efficiency were maximized. Additionally, the treatment of slaughterhouse effluent showed significant reductions in organic pollution, with the COD and biochemical oxygen demand (BOD<sub>5</sub>) decreasing by 80% (from 1900 mg/L to 384 mg/L) and 81% (from 1700 mg/L to 320 mg/L), respectively, within seven days. The strain also facilitated ammonium removal and promoted nitrification, indicating its suitability for treating high-organic-load wastewater. Notably, the visual transformation of the effluent, from a dark red color to a clear state, further highlighted the efficiency of the treatment process. This research highlights the adaptability of *M. hydrocarbonoclasticus* SDK644 to a wide range of environmental conditions and its efficiency in biodegrading metribuzin and treating complex wastewater. The findings demonstrate the strain's potential as a sustainable solution for mitigating organic pollution in agricultural runoff, pesticide-contaminated water, and industrial effluents.

**Keywords:** biodegradation; metribuzin; *Marinobacterhydrocarbonoclasticus*; wastewater treatment; COD reduction; environmental microbiology

<footnote n="1">¹ Laboratory of Materials and Environment (LME), Faculty of Science and Technology, University Yahia Fares of Médéa, Médéa 26000, Algeria; nadiamihouona@gmail.com (N.M.); m.nedjhioui@gmail.com (M.N.)</footnote>

<footnote n="2">² Laboratory of Biomaterials and Transport Phenomena (LBMPT), University of MÉDÉA, Nouveau Pôle Urbain, Médéa University, Médéa 26000, Algeria; ferhatsamira@yahoo.fr</footnote>

<footnote n="3">³ National Centre for Research and Development of Fisheries and Aquaculture (CNRDPA), 11 Street Colonel Amirouche, Bou-Ismaïl 42415, Algeria; bilalzenati@gmail.com</footnote>

<footnote n="4">⁴ Biotechnology, Water, Environment and Health Laboratory, Abbes Laghrour University, Khenchela 40000, Algeria; sabrina.lekmin.400@gmail.com</footnote>

<footnote n="5">⁵ Technical Platform for Physico-Chemical Analyzes (PTAPC-Bejaia), Targa Ouzemmour, Bejaia 06000, Algeria; boudraa1414@gmail.com</footnote>

<footnote n="6">⁶ Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia; mola@ksu.edu.sa</footnote>

<footnote n="7">⁷ School of Engineering, Merz Court, Newcastle University, Newcastle upon Tyne NE1 7RU, UK; jie.zhang@newcastle.ac.uk</footnote>

<footnote n="8">⁸ Ecole Nationale Supérieure de Chimie de Rennes, University of Rennes, CNRS, ISCR—UMR6226, 35000 Rennes, France</footnote>

<footnote>* Correspondence: abdeltif.amrane@univ-rennes1.fr (A.A.); hichemm.tahraouii@gmail.com (H.T.)</footnote>
# 1. Introduction

In recent years, the global emphasis on water resources has heightened, driven by a dual commitment to increasing water availability and safeguarding its quality for healthy consumption [1–4]. The alarming state of our rivers has become increasingly apparent, with major water bodies that are downstream from urban areas facing contamination from toxic residues, predominantly stemming from intensive agricultural chemicals (nitrates), industrial discharges, and urban runoff (phosphorus and ammonium) [5–7]. Disturbingly, IFEN's data reveal that 90% of rivers carry pesticides, and 10% do not meet the established drinkability standards [8]. Of particular concern is the impact of discharges from wastewater treatment plants (WWTPs) and urban overflow into rivers, leading to the development of heterotrophic conditions and subsequently causing critically low dissolved oxygen levels, resulting in a spike in fish mortality [9,10].

The gamut of xenobiotic environmental pollutants include pesticides and their chlorination residues, combustion by-products of organic matter, various wastewater types, coal mine discharges (fossil fuels), petroleum processing remnants, dyes, pharmaceuticals, and petrochemical products [11–14].

If not adequately treated, these effluents become a significant source of microcontaminants in the environment, resulting in harmful impacts on aquatic life and human health, such as water toxicity, pathogenic bacteria, and genotoxicity [15,16].

To ensure the preservation and restoration of a hygienically clean environment, there is an imperative need to eliminate these pollutants [17,18]. Consequently, the development of reliable and effective physical and chemical depollution methods becomes indispensable [19,20]. However, many of these reported techniques can be economically burdensome and at times incompatible with maintaining a hygienically sound environment [19,21]. The potential solution lies in a biological approach through bioremediation. This method presents the advantages of being less invasive and more cost-effective through harnessing the efficient purification capabilities of environmental microorganisms [22].

Biological depollution processes, grounded in the microbial biotransformation of pollutants, have become pivotal in addressing organic pollutants and have gained prominence in recent times [23,24]. The microbial degradation of xenobiotics, or bioremediation, emerges as a cost-effective technique for removing pollutants from the environment, involving biological reactions that alter the chemical structure of compounds, consequently reducing their toxicity [25,26]. The past few years have witnessed a surge in experiments aimed at isolating, identifying, characterizing, and even constructing new microorganisms that are equipped with novel metabolic pathways that are capable of biodegrading pesticides and other persistent pollutants [27,28]. Numerous studies highlight the utilization of these substances as the primary source of carbon and energy by complex microbiota or pure strains [29,30].

With the widespread use of pesticides in the agricultural and household sectors, there is an urgent need to consider pesticide remediation technologies for the soil and water environment. Microbial biotechnology, wherein the microorganisms use these chemicals as their carbon, nitrogen, phosphorous, and energy source, has proven effective for bioremediating pesticides from contaminated soil and water environments [31,32]. For example, Pan et al. [33] delved into the degradation of 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) by a newly identified bacterium, *Stenotrophomonas* spin, which was isolated from contaminated soil. Additionally, various other microorganisms, including Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, and Bacillus species, demonstrated their ability to degrade DDT [34]. Furthermore, they exhibited proficiency in degrading cypermethrin, albeit not exceeding 1% [35]. *Pseudomonas putida* and *Pseudomonas mendocina* strains showcased a high capacity to biodegrade cypermethrin and permethrin, achieving
a reduction of up to 90% within 15 days [36]. Bordjiba et al. [37] isolated diverse fungal species from pesticide-contaminated soils, with 53 of them being noted for their capacity to degrade the herbicide metribuzin in a liquid medium.

In a comprehensive study by Vitte et al. [38], the strain *Marinobacterhydrocarbonoclasticus*, which was isolated from a petroleum-contaminated environment, demonstrated remarkable capabilities in degrading cyclic and non-cyclic alkanes. This moderate halophile exhibited efficiency in reducing total nitrogen (TN), nitrate, nitrite, and ammonium [39]. Researchers have reported its ability to thrive in the presence of 20–120 × 10³ mg/L of saline solution, removing 94.2% of nitrate and 80.9% of total nitrogen from synthetic water [40]. Another strain, *Marinobacter* sp. F6, has been acknowledged for its ability to completely remove nitrate and 50% of total nitrogen [41].

This research investigates the bioremediation potential of *Marinobacter hydrocarbonoclasticus* strain SDK644 by evaluating its ability to degrade the pesticide metribuzin and treat organic effluents from slaughterhouses. The strain was cultivated under varying conditions to understand its metabolic responses. This novel study explored the potential of a halophilic bacterium to address both pesticide contamination and industrial wastewater, demonstrating the effectiveness of a bioremediation approach. In contrast to previous studies, which focused on the isolated degradation of hydrocarbons or specific pollutants, this research highlights the versatility of this strain in treating diverse contaminants by optimizing the culture conditions. Moreover, it provides comprehensive information on the adaptability of this bacterial strain under various environmental conditions (salinity, temperature, and pH), offering a novel, ecological, and sustainable approach to mitigate pollution in complex and challenging contexts.

## 2. Materials and Methods

### 2.1. Medium and Reagents

In this study, all chemicals that were used for bacterial cultivation and the subsequent analyses were of analytical grade and sourced from reputable suppliers such as FLUKA (Sigma-Aldrich Co., St. Louis, MO, USA), BIOCHEM (Sigma-Aldrich Co., St. Louis, MO, USA), and SIGMA-ALDRICH (Sigma-Aldrich Co., St. Louis, MO, USA). To cultivate the bacterial strain *Marinobacterhydrocarbonoclasticus* SDK644, a Luria-Bertani (LB) broth was prepared as the base medium. This consisted of 1 × 10⁴ mg/L peptone, 5 × 10³ mg/L yeast extract, and 23 × 10³ mg/L NaCl. The pH of the medium was carefully adjusted to 7.0 ± 0.2 using 1M NaOH. Bacterial cultures were incubated in this LB broth at 30 °C for 24 h to allow for sufficient growth.

After the incubation period, the bacterial biomass was transferred to a mineral salt medium (MSM) to study the bacterial behavior under defined nutritional conditions.

The MSM formulation included 50 mg/L CaCl₂, 23 × 10³ mg/L NaCl, 330 mg/L MgCl₂, 400 mg/L NH₄Cl, 300 mg/L K₂HPO₄, and 300 mg/L KH₂PO₄, with the pH of the medium being maintained at 7.0. A trace metal solution was added to the MSM to ensure that the bacteria had access to essential micronutrients. The trace metal solution was composed of FeCl₂·4H₂O (1500 mg/L), ZnCl₂ (70 mg/L), MnCl₂·6H₂O (100 mg/L), H₃BO₃ (6 m g/L), CoCl₂·6H₂O (190 mg/L), NaMoO₄·2H₂O (36 mg/L), and 6.7 mL/L of concentrated HCl. Sterilization of the medium was achieved by autoclaving at 121 °C for 20 min to ensure that the final culture conditions were free from contamination.

### 2.2. Preparation of Bacterial Strains

The bacterial strain used in this study, *Marinobacter hydrocarbonoclasticus* SDK644, was isolated from Khmisti Fish Harbor, located in Bou-Ismail Bay, Tipaza, Algeria. This harbor is heavily polluted with petroleum products, providing an ideal environment for studying
hydrocarbon-degrading bacteria. *M. hydrocarbonoclasticus* SDK644 is a Gram-negative, non-sporulating, rod-shaped bacterium, as observed under the microscope (Figure 1). It can thrive in highly saline environments, with its tolerance ranging from 0 to 150 10³ mg/L NaCl, and can survive across a broad temperature range of 10 °C to 45 °C. The strain also shows robustness in a pH range of 6.0 to 9.5, making it adaptable to diverse environmental conditions. The strain is capable of utilizing a wide range of hydrocarbons as a carbon source, which was demonstrated through its growth on hydrocarbon-enriched agar media. These characteristics make *M. hydrocarbonoclasticus* SDK644 a promising candidate for bioremediation, particularly in environments that are contaminated by petroleum-based products.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** The shape of the colonies of the SDK644 strain, cultured in LB medium.</figcaption></figure>

A fragment of approximately 1500 bp of the 16S rRNA gene was amplified from the genomic DNA of the isolate, cloned into the pGEM-T Easy vector, and sequenced on both strands to establish additional support for the identification of the SDK644 strain, as shown in Figure 2 [42].

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Phylogenetic tree of strain SDK644 [42].</figcaption></figure>

## 2.3. *Samples*

Two distinct types of samples were used in this study: metribuzin (Sample 1) and slaughterhouse effluent (Sample 2).
Metribuzin (Sample 1): Metribuzin is a triazine herbicide that is commonly used for weed control both before and after crop emergence [43]. The sample that was used in this study is commercially known as Metrixone and was used at a concentration of 70%. Its chemical formula is C<sub>8</sub>H<sub>14</sub>N<sub>4</sub>OS, with a molar mass of 214.3 g/mol. Metribuzin has a solubility of 1050 mg/L in water at 20 °C. The metribuzin that was used for this research was provided by the Agricultural Department of Médéa, Algeria. This herbicide was selected for this study due to its widespread use in agricultural practices and its potential environmental impact [44,45].

Slaughterhouse Effluent (Sample 2): The second sample that was used was effluent collected from a slaughterhouse situated in a known contaminated area. The effluent was highly concentrated and was stored in polyethylene bottles immediately after collection. The samples were transported to the laboratory within 24 h and stored at 4 °C until chemical analyses could be conducted. The physicochemical properties of the effluent, which are summarized in Table 1, indicated that the effluent exceeds the permissible discharge limits for industrial waste, particularly with regard to parameters such as COD, BOD<sub>5</sub>, pH, and temperature.

<table><caption>**Table 1.** Physicochemical characteristics of slaughterhouse discharge.</caption><thead><tr><td>Parameters</td><td>Values</td><td>Limit Values</td></tr></thead><tbody><tr><td>COD (mg/L)</td><td>1700</td><td>800</td></tr><tr><td>BOD<sub>5</sub> (mg/L)</td><td>1600</td><td>250</td></tr><tr><td>pH</td><td>8</td><td>6.5-8.5</td></tr><tr><td>T (°C)</td><td>11.5</td><td>30</td></tr></tbody></table>

## 2.4. Monitoring Bacterial Growth in the Presence of Metribuzin

The ability of *Marinobacterhydrocarbonoclasticus* SDK644 to degrade metribuzin as a sole carbon source was evaluated by monitoring the bacterial growth. OD<sub>600</sub> measurements were taken every 24 h over a period of six days to track the metabolic activity of the strain. The changes in OD<sub>600</sub> indicated bacterial growth and the degradation rate of metribuzin, thus allowing for the assessment of the strain's effectiveness in utilizing the herbicide for its metabolic processes.

## 2.5. Optimization of Growth Conditions and Metribuzin Biodegradation

In 100 mL of mineral salt medium, metribuzin concentrations were adjusted as required. An inoculum was introduced, and the cultures were incubated at 30 °C and 150 rpm in the dark using an LG CAP SHAKER 230V EU USA incubator (LG Electronics Inc., Seoul, Republic of Korea). Optical density was measured every 24 h for a total of 144 h.

Several parameters were tested to optimize the metribuzin biodegradation and determine the bacteria's optimal growth conditions.

* **Metribuzin Concentration:** Various concentrations of metribuzin (2, 20, 50, and 100 mg/L) were added to the MSM, and the bacterial growth was monitored by OD<sub>600</sub> measurements over a period of 144 h. This was used to identify the concentration range that allowed for optimal bacterial growth and metribuzin degradation.

* **pH Levels:** The pH of the medium was adjusted to different values (4.0, 7.0, and 9.0) using HCl and NaOH to assess how the pH affects the growth and degradation efficiency of the strain.

* **Temperature:** The bacterial cultures were incubated at various temperatures (12 °C, 20 °C, 30 °C, and 45 °C) to identify the temperature range in which the strain exhibited the best growth rate and metribuzin degradation capacity.
* Inoculum Volume: Different inoculum volumes (1%, 2%, 4%, and 8% v/v) were tested to determine the optimal inoculum size for efficient growth and degradation.

All these experiments were conducted at 30 °C with shaking at 150 rpm, and OD₆₀₀ was measured every 24 h to assess the bacterial growth.

## 2.6. Degradation of Metribuzin by SDK644

To analyze the extent of metribuzin biodegradation by *Marinobacterhydrocarbonoclasticus* SDK644, the COD was measured. The COD serves as a key parameter for determining the level of organic matter in a sample [46], which decreases as the metribuzin is degraded. The standard method for COD measurement, ISO 15705:2002 [47], was followed to quantify the reduction in COD, thereby indicating the bacterial degradation of metribuzin. The steps of the experiments testing the biodegradation of pesticides and slaughterhouse waste by the strain are summarized in Figure 3.

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Schematic representing the biodegradation of (a) metribuzin by SDK644 and (b) biodegradation of slaughterhouse effluent.</figcaption></figure>

## 2.7. Treatment of Slaughterhouse Effluent

In addition to studying metribuzin degradation, the potential of *M. hydrocarbonoclasticus* SDK644 for treating slaughterhouse effluent was investigated. The bacterial culture, after initial growth in the LB medium, was introduced into the effluent at a concentration of 3% v/v. The cultures were incubated at 30 °C with shaking at 150 rpm, and samples were taken every 24 h for analysis. Parameters such as suspended solids (SSs), COD, and BOD₅ were measured. In addition, nitrate, phosphate, and ammonium levels were quantified using a DR/4000 V spectrophotometer (Hach Company, Loveland, CO, USA) operating in the visible range (300–1000 nm). These analyses provided valuable insights into the effluent's composition and the efficiency of the bacterial strain in treating the polluted water.

# 3. Results

## 3.1. Effects of Different Concentrations of Metribuzin on Cell Growth

This study evaluated the bacterial growth response to various concentrations of metribuzin to determine the optimal conditions for bacterial growth and pesticide degradation. Metribuzin is a commonly used herbicide that can be toxic to multiple organisms,
including bacteria [45,48]. Therefore, it is essential to understand how its concentrations impact bacterial growth, as this will help assess its potential for biodegradation under different environmental conditions [49].

The strain *M. hydrocarbonoclasticus* SDK644 exhibited optimal growth at a metribuzin concentration of 20 mg/L, reaching an OD₆₀₀ of 0.408 after 96 h of incubation. This OD value represented the maximum bacterial proliferation under these conditions (Figure 4), suggesting that 20 mg/L is the most suitable concentration for bacterial growth and subsequent pesticide degradation. The growth at this concentration indicates that the bacteria can effectively utilize metribuzin as a carbon and energy source without being inhibited by its toxic effects, which is crucial for bioremediation applications [50,51].

<figure><img src="image_6.png" /><figcaption>**Figure 4.** Effect of initial concentration of metribuzin on cell growth: (a) histogram; (b) curve.</figcaption></figure>

A slight reduction in growth was observed at a higher concentration of 50 mg/L, with an OD₆₀₀ value of 0.35 after 96 h. While this concentration still allowed for growth, it clearly inhibited bacterial activity to some extent [52]. The reduced growth rate at this concentration may be due to the bacterium experiencing stress due to the increased toxicity of metribuzin, which could limit metabolic and enzymatic activity [53]. High pesticide concentrations can create unfavorable conditions by depleting nutrients or accumulating toxic metabolites, affecting the bacteria's overall growth and degradation capabilities [54,55].

In contrast, at a concentration of 100 mg/L, the growth pattern of *M. hydrocarbonoclasticus* SDK644 was notably different. The bacteria exhibited initial growth within the first 48 h but entered a stationary phase after 96 h, and by 120 h, the growth began to decline sharply. This decline could be due to the inhibitory effects of metribuzin at this high concentration, leading to an accumulation of toxic degradation products or a depletion of energy and nutrient sources in the growth medium [56,57]. The sharp decline in growth after 120 h indicates that although the bacteria could initially tolerate the pesticide, the concentration was ultimately too high for sustained growth, highlighting the toxic threshold of metribuzin for this bacterial strain [58].

Bacterial growth was weak at the lowest concentration of 2 mg/L, and no significant prolife ratio was observed. This suggests that the concentration of metribuzin was insufficient to support bacterial growth, likely due to the lack of a sufficient carbon source relative to the bacterial population size. The weak growth at this concentration underscores the importance of maintaining optimal metribuzin concentrations for effective biodegradation and growth [59].
These results are consistent with previous studies on other bacterial strains, such as the work of Essa et al. [60], which showed reduced growth of Pseudomonas aeruginosa at high concentrations of diazinon, an organophosphate pesticide. Similarly, Zhang et al. [61] reported that 20 mg/L of metribuzin was the optimal concentration for degradation by *Bacillus* sp. N1. The results of this study suggest that *M. hydrocarbonoclasticus* SDK644 is well suited for biodegrading metribuzin at concentrations around 20 mg/L, where the bacteria can thrive and efficiently metabolize the pesticide.

## 3.2. *Effect of pH on Cell Growth*

The pH of the growth medium can significantly influence bacterial growth and metabolism, particularly for bacteria that are involved in the biodegradation of pollutants [62]. In this experiment, the effect of pH on the growth of *M. hydrocarbonoclasticus* SDK644 was tested at three pH values, 4, 7, and 9, representing acidic, neutral, and slightly alkaline environments (Figure 5).

<figure><img src="image_7.png" /><figcaption>**Figure 5.** Effect of pH on bacterial growth: (a) histogram; (b) curve.</figcaption></figure>

The results presented in Figure 5 reveal that the optimal growth of *M. hydrocarbonoclasticus* SDK644 occurred at a pH of 7, the neutral pH. At this pH, the bacterial strain exhibited the highest optical density and the most robust growth, which indicates that a neutral pH supports optimal metabolic and enzymatic activity for the biodegradation of metribuzin. The neutral pH is typically more favorable for bacterial enzymes that are involved in breaking down pollutants, and it allows for more efficient nutrient uptake, both of which are essential for the degradation process.

At a pH of 9, the bacterial growth was slower than at a pH of 7, suggesting that slightly alkaline conditions began to exert inhibitory effects on the strain. An alkaline pH can alter the structure of enzymes and proteins, reducing their effectiveness. Additionally, a high pH can interfere with the overall cellular processes of bacteria, leading to slower growth and a reduced ability to degrade metribuzin [63]. However, despite the reduction in growth, *M. hydrocarbonoclasticus* SDK644 was still able to degrade metribuzin at this pH, although at a slower rate.

At a pH of 4, the bacterial growth was severely inhibited, and there was little to no increase in OD₆₀₀. The acidic environment likely caused disruptions in the integrity of the bacterial cell membrane and the enzymes that are responsible for biodegradation, leading to poor growth and low pesticide degradation. Acidic conditions can cause the denaturation of proteins and enzymes, which impairs the ability of bacteria to metabolize organic compounds such as metribuzin [64].
These results align with the findings of Zhang et al. [61], who observed that the pH had a direct influence on the degradation rate of metribuzin by *Bacillus* sp. N1, with the degradation rate declining significantly at both low and high pH levels. The results of this experiment suggest that *M. hydrocarbonoclasticus* SDK644 is most effective at a neutral pH (7), and any significant deviation from this pH could negatively affect both bacterial growth and metribuzin degradation. This knowledge can be applied to optimize bioremediation strategies by maintaining pH levels near neutral when using this bacterium for pesticide degradation.

### 3.3. *Effect of Temperature on Bacterial Growth*

Temperature is another key environmental factor that impacts bacterial growth and metabolic activities [65]. In this experiment, the effect of temperature on the growth of *M. hydrocarbonoclasticus* SDK644 was assessed at four different temperatures, 12 °C, 20 °C, 30 °C, and 45 °C, which represent a range of low, moderate, and high temperatures.

The results, as shown in Figure 6, indicated that the optimal temperature for bacterial growth and metribuzin degradation was 30 °C. At this temperature, the bacterial strain achieved the highest optical density and demonstrated the most efficient pesticide degradation. This suggests that 30 °C provides the most favorable conditions for the enzymatic processes that are involved in metribuzin degradation. Bacterial enzymes typically function most efficiently at moderate temperatures, where both the protein structure and metabolic activity are not negatively affected by heat stress.

<figure><img src="image_8.png" /><figcaption>**Figure 6.** Effect of temperature on bacterial growth: (a) histogram; (b) curve.</figcaption></figure>

At 20 °C, the bacterial growth was slower than at 30 °C, but growth was still observed, indicating that *M. hydrocarbonoclasticus* SDK644 is capable of surviving and degrading metribuzin at lower temperatures. This resilience at lower temperatures suggests that the strain could be useful in colder environments, where the temperature might not be optimal for other bacteria.

At temperatures of 12 °C and 45 °C, the bacterial growth was notably reduced, with little to no growth being observed at 45 °C. At low temperatures like 12 °C, bacterial enzymatic reactions are generally slow, leading to a reduced degradation rate for metribuzin. On the other hand, high temperatures like 45 °C can cause thermal denaturation of bacterial proteins and enzymes, impairing their function and inhibiting bacterial growth. This temperature range likely exceeds the optimal range for enzymatic activity, leading to a decline in growth and metribuzin degradation.
These results are consistent with findings from previous studies, such as those by Zhang et al. [61], who noted that extreme temperatures could negatively affect the rate of metribuzin degradation. The optimal temperature for *M. hydrocarbonoclasticus* SDK644 was found to be 30 °C, suggesting that for effective bioremediation, this temperature should be maintained. However, the strain's ability to tolerate a range of temperatures suggests that it could still be effective in a variety of environmental conditions, as long as the temperature is not too extreme.

### 3.4. *Effect of Inoculum Concentration on Bacterial Growth*

The inoculum size plays a crucial role in determining the success of a biodegradation process, as it affects the initial bacterial population that is available to degrade the pollutant [62]. In this experiment, the effects of different inoculum concentrations (1%, 2%, 4%, and 8%) on the growth and metribuzin degradation by *M. hydrocarbonoclasticus* SDK644 were assessed over 144 h.

The results, depicted in Figure 7, show that the bacterial growth increased with the inoculum concentration up to 4%. At this inoculum size, the $OD_{600}$ value reached 0.43 after 96 h, indicating optimal bacterial growth. This suggests that a 4% inoculum concentration provides the ideal bacterial population for maximum metribuzin degradation, as it ensures that there are enough bacteria to rapidly degrade the pesticide without causing nutrient depletion or overpopulation.

<figure><img src="image_9.png" /><figcaption>**Figure 7.** Effect of inoculum concentration on bacterial growth: (a) histogram; (b) curve.</figcaption></figure>

At inoculum concentrations of 1% and 2%, the growth was slower and the degradation rate of metribuzin was lower than at other concentrations. This could be due to the initial bacterial population being too small to efficiently degrade the pesticide. Conversely, at an inoculum concentration of 8%, bacterial growth was still observed, but there was a slight decline in the growth rate compared to the 4% inoculum. This could be due to nutrient limitations, as the large initial bacterial population may have exhausted the available sources more quickly, slowing down growth and degradation.

These results highlight the importance of the inoculum size in optimizing bioremediation processes. A 4% inoculum concentration provides the ideal balance between a sufficient bacterial population and adequate nutrient availability for optimal growth and metribuzin degradation.
### 3.5. Degradation of Metribuzin by Strain SDK644

The degradation of metribuzin by *M. hydrocarbonoclasticus* SDK644 was monitored through COD measurements, which indicate the total organic matter that is present in a growth medium. The COD values showed a significant reduction over time, with an 80% reduction in COD after 144 h of incubation (Figure 8). This reduction demonstrated the bacterium's ability to effectively degrade metribuzin, reducing the organic load in the medium.

<figure><img src="image_10.png" /><figcaption>Figure 8. (a) Kinetics of variation in the COD over time ($C_0$ = 20 mg/L) and (b) the pseudo-first-order kinetic model.</figcaption></figure>

COD measurements are a key indicator of biodegradation efficiency, as they reflect the breakdown of organic pollutants into simpler compounds [66,67]. The substantial reduction in COD that was observed in this study confirmed that *hydrocarbonoclasticus* SDK644 is highly effective at degrading metribuzin, further supporting its potential for bioremediation applications. The degradation process could involve the breakdown of metribuzin into nontoxic by-products, ultimately reducing the environmental impact of pesticide contamination [68].

Control tests were conducted using 20 mg/L metribuzin in a mineral salt medium (MSM) to assess its degradation in an aqueous environment. The results indicated no significant change in metribuzin concentration throughout the incubation period. Laboratory studies demonstrated that soil sterilization significantly slowed down metribuzin degradation, with a half-life of 154 days. These findings strongly suggest that metribuzin is biodegraded in soil by a specialized microbial community [69].

The kinetics of metribuzin degradation were investigated using the non-linear first-order model (Equation (1)). This model effectively describes the relationship between the degradation rate and pollutant concentration [70].

$$-\frac{C_t}{t} = k_1 C_t \qquad (1)$$

Integrating Equation (1) yields Equation (2), which describes the kinetics of the first-order model.

$$C_t = C_0 \times e^{-k_1 t} \qquad (2)$$

where $k_1$ (h⁻¹) denotes the rate constant of the first-order reaction, and $C_0$ and $C_t$ are the initial metribuzin concentration and at time t, respectively.

Figure 8b illustrates the evolution of the pollutant concentration over time. The results show a significant decrease in concentration after 144 h of contact with the strain studied. The
degradation rate is estimated as 0.01223 h⁻¹, with a coefficient of determination (R²) of 0.9, indicating a good fit of the model to the experimental data.

Under the same conditions (temperature, pH, and salinity), the biodegradation capacity of *M. hydrocarbonoclasticus* SDK644 on polycyclic aromatic hydrocarbons (PAHs) was evaluated. The results demonstrated that strain SDK644 utilizes PAHs with varying degrees of efficiency. Robust growth was observed with pyrene, with no apparent lag phase required for adaptation to the substrate. The SDK644 strain also utilized anthracene after a short lag phase of 2 days. Growth on naphthalene was stimulated, albeit more slowly, with moderate growth observed after six days of incubation (Table 2). Finally, *M. hydrocarbonoclasticus* SDK644 appeared to be unable to utilize phenanthrene [69].

<table><caption>**Table 2.** Degradation of PAHs by MSDK644.</caption><thead><tr><th>Polluants</th><th>Optic Density (600 nm)</th><th>Operating Condition</th><th>Efficiency (%)</th><th>Reference</th></tr></thead><tbody><tr><td>Pyrène</td><td>1.8</td><td>C₀ = 100 mg/L</td><td>38</td><td></td></tr><tr><td>Anthracène</td><td>1.6</td><td>T = 30 °C</td><td>31</td><td></td></tr><tr><td>Naphtalène</td><td>1.1</td><td>pH = 7</td><td></td><td>[42]</td></tr><tr><td>Phénanthrène</td><td>0.3</td><td>inoculation = 2%<br/>Time = 14 day</td><td>Low effeciency</td><td></td></tr><tr><td>Metribuzine</td><td>0.5</td><td>C₀ = 20 mg/L<br/>T = 30 °C<br/>pH = 7<br/>inoculation = 4%<br/>Time = 6 day</td><td>80</td><td>This work</td></tr></tbody></table>

### 3.6. Treatment of Slaughterhouse Effluent

The treatment of slaughterhouse effluent, a source of organic pollution containing fats, proteins, blood, and other organic compounds, was also evaluated using *M. hydrocarbonoclasticus* SDK644 [69]. The initial COD and BOD₅ values were high, reflecting the significant organic pollution in the raw effluent (Figure 9). Over a 7-day incubation period, significant reductions in both COD and BOD₅ were observed, with efficiencies of 80% and 81%, respectively. These reductions indicated that *hydrocarbonoclasticus* SDK644 is effective in treating organic pollutants in wastewater.

<figure><img src="image_11.png" /><figcaption>**Figure 9.** Degradation of metribuzin compared with a reference sample.</figcaption></figure>
Substantial reductions in COD and BOD₅ were achieved within the first few days, with the COD decreasing from 1900 mg/L to 384 mg/L and BOD₅ from 1700 mg/L to 320 mg/L by the end of the 7-day period (Figure 6). This suggests that the strain's ability to degrade organic matter was not only efficient but also rapid, making it suitable for real-time wastewater treatment applications.

The nitrogen removal mechanism was also assessed, with a decrease in NH₄⁺ concentrations and the formation of nitrates, which is indicative of successful nitrification (Figure 10). However, the formation of nitrates slowed down after day 5, likely due to low dissolved oxygen levels, which can limit the activity of nitrifying bacteria.

<figure><img src="image_12.png" /><figcaption>**Figure 10.** Change in COD, BOD₅, and SS as a function of time (a) and variation in chemical parameters (b).</figcaption></figure>

Overall, these results suggest that *M. hydrocarbonoclasticus* SDK644 has the potential to be used in wastewater treatment applications, including the treatment of slaughter-house effluent, offering a viable solution for reducing organic pollutants and enhancing water quality.

Figure 11 provides a visual representation of the color metamorphosis in the sample, offering a clear comparison between its appearance before and after treatment with the SDK644 strain.

<figure><img src="image_13.png" /><figcaption>**Figure 11.** Original sample (a) and sample after treatment (b).</figcaption></figure>

The solution underwent a notable metamorphosis, shifting from an intense dark red to a state of crystal-clear transparency. This visual transformation serves as a clear indicator of the efficiency and success that were achieved through the treatment process.
### 3.7. *Comparative Analysis*

The results of this study are consistent with previous research on the biodegradation of pesticides and organic pollutants by microbial strains. Studies by Nicoly et al. [71] and Vidal et al. [72] have demonstrated the potential of various bacterial species for removing pollutants from wastewater. This study further confirms the capability of *M. hydrocarbonoclasticus* SDK644 in biodegradation applications, particularly for the removal of pesticides and organic matter from contaminated environments. This research highlights the strain's adaptability to different environmental conditions, such as different pH levels, temperatures, and inoculum sizes. These factors are crucial for optimizing bioremediation processes, ensuring maximum degradation efficiency. The strain's ability to degrade metribuzin at optimal concentrations and its capacity for wastewater treatment further demonstrate its potential as a versatile and effective tool for environmental cleanup. The findings of this study suggest that *M. hydrocarbonoclasticus* SDK644 could be utilized in various bioremediation applications, including the treatment of agricultural runoff, pesticide-contaminated water, and organic wastewater, offering a sustainable and efficient solution for environmental pollution.

## 4. Conclusions

This study highlighted the significant potential of the strain *Marinobacterhydrocarbonoclasticus* SDK644 in the bioremediation of contaminated environments. This strain demonstrated an exceptional ability to degrade the herbicide metribuzin and treat slaughterhouse effluents under optimized conditions. Specifically, it achieved optimal degradation of metribuzin at a concentration of 20 mg/L, with an 80% reduction in COD within 144 h. The ideal condition for its activity was identified as a neutral pH of 7 and a temperature of 30 °C, confirming its efficiency as a bioremediation tool in pesticide-contaminated environments. Regarding the treatment of slaughterhouse effluents, the SDK644 strain achieved a notable 80% reduction in COD and 81% reduction in 5-day BOD<sub>5</sub> after just 7 days of incubation. The ammonium levels also decreased significantly, accompanied by effective nitrification. These results highlight the strain's capacity to treat industrial effluents that are rich in organic matter, thereby reducing their environmental impact. Additionally, the visual transformation of the samples, from an intense dark red to a clear solution, further underscored its effectiveness in the purification process. Finally, the strain exhibited remarkable adaptability to diverse environmental conditions, including saline environments, extreme temperatures (ranging from 12 to 45 °C), and pH levels from slightly acidic (6) to alkaline (9). These characteristics broaden its applicability to various real-world scenarios, including the remediation of industrial effluents and pesticide-contaminated waters. In conclusion, *Marinobacter-hydrocarbonoclasticus* SDK644 offers a sustainable, economical, and eco-friendly solution for managing environmental pollution. Future work should focus on scaling up the process, exploring potential synergies with other microbial strains, and evaluating its long-term stability in field applications.

**Author Contributions:** Conceptualization, N.M., S.F., M.N., B.Z., S.L., R.B., M.S.O., J.Z., A.A. and H.T.; Data curation, N.M., S.F. and A.A.; Formal analysis, N.M., S.F., M.N., B.Z., S.L., M.S.O., J.Z., A.A. and H.T.; Funding acquisition, M.S.O.; Investigation, N.M., S.F., M.N., B.Z., R.B., M.S.O., J.Z., A.A. and H.T.; Methodology, N.M., S.F., M.N., B.Z., S.L., R.B., J.Z., A.A. and H.T.; Project administration, S.F., M.S.O., J.Z., A.A. and H.T.; Resources, N.M., S.F., B.Z., S.L., R.B., M.S.O., J.Z. and H.T.; Software, N.M. and M.S.O.; Supervision, S.F., M.N., J.Z., A.A. and H.T.; Validation, N.M., S.F., M.N., B.Z., S.L., R.B., M.S.O., J.Z., A.A. and H.T.; Visualization, N.M., S.F., M.N., B.Z., R.B., M.S.O., J.Z., A.A. and H.T.; Writing—original draft, N.M.; Writing—review and editing, S.F., M.N., B.Z., S.L., R.B., M.S.O., J.Z., A.A. and H.T. All authors have read and agreed to the published version of the manuscript.
**Funding:** Researchers' supporting project number RSPD2025R710, King Saud University, Riyadh, Saudi Arabia.

**Data Availability Statement:** The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

**Acknowledgments:** The authors would like to thank the funding of Researchers Supporting Project Number (RSPD2025R710), King Saud University, Riyadh, Saudi Arabia.

**Conflicts of Interest:** The authors declare no conflict of interest.

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