Pseudomonas stutzeri Immobilized Sawdust Biochar for Nickel Ion Removal

Abstract

Nickel ions generated from the electroplating industry and stainless steel and battery manufacturing industries contribute to water pollution, harm human health, and pose environmental risks. A long-term, sustainable, and efficient treatment method should be developed to address this issue. Bioremediation in the presence of biochar and microorganisms is a potential approach for metal ion abatement. This study evaluates the feasibility of Pseudomonas stutzeri immobilized sawdust biochar (PSDB) for Ni²⁺ removal. Sawdust biochar was prepared by pyrolyzing in a muffle furnace and was characterized using SEM, FTIR, and BET. The influence of biochar preparation parameters such as pyrolysis temperature, time on biochar yield, and impact on cell immobilization was investigated. The effect of various parameters, such as incubation time, pH, temperature, and biocatalyst dosage, was studied. The total Ni²⁺ in solution was analyzed using inductively coupled plasma optical emission spectrometry. PSDB showed an 83% Ni²⁺ removal efficiency and reusability up to three cycles. FT-IR analysis revealed that the mechanism of Ni²⁺ removal by PSDB was the synergistic effect of adsorption by biochar and bioaccumulation by P. stutzeri. This study presents a novel approach for environmental application by utilizing waste biomass-derived biochar as a carrier support for bacteria and an adsorbent for pollutants.

1. Introduction

The presence of toxic heavy metal ions in the water and wastewater over permissible limits poses a significant threat to the environment and public health. Natural mineralization and anthropogenic activities are the primary cause of excessive heavy metal release into the environment [1]. Heavy metal contamination and its management are challenging due to its persistent nature [2]. Among the heavy metals, Ni²⁺ is commonly found in soil and water systems and can be discharged into the environment from pigment manufacturing processes, nickel alloy industries, tannery industry wastewater, etc. [3]. Ni²⁺ concentrations in fresh water and the oceans are usually less than 0.02 mg L⁻¹ [1]. Excess intake of Ni²⁺ leads to poisoning, and a low intake also causes harm to aquatic organisms [2]. It primarily affects the respiratory tract, cardiovascular system, and immune system, resulting in allergies and asthma, and long-term exposure can lead to cancer [3]. Ni²⁺-containing water needs to be adequately treated because of its toxicity, gradual accumulation in the food chain, and persistence in the ecosystem. Numerous techniques are used to remove Ni²⁺, including adsorption, ion exchange, membrane filtration, and chemical precipitation [4]. Most of these methods are energy intensive and require skilled operators; operational and maintenance costs are high. Membrane filtration technologies can efficiently remove toxic substances, but membrane material production is typically very complicated [5,6]. Further, the generation of toxic sludge is another serious problem [7]. An ion-exchange resin for pollutant treatment is one metal treatment method with a high removal efficiency. However, it is sensitive to other pollutants in the water, and its use is restricted to acidic environments [6]. Hence, sustainable and new techno-economically feasible strategies are necessary for heavy metal treatment.

Bioremediation through metal ion tolerant bacteria offers an alternative, greener approach and has received extensive attention due to its low cost, high efficiency, and environmentally friendly characteristics [8]. Microbial strains can adapt and survive in polluted areas, with subsequent removal or detoxification of the pollutant. The main mechanisms involved in microbial-mediated metal ion removal include biosorption, biotransformation, bioreduction, and bioaccumulation [9]. Adding functional microorganisms to the target wastewater increases the effectiveness of the treatment process and widens its application possibilities. Wu et al. [10] investigated the bioaccumulation potential of a marine Brevibacterium sp. to remove Ni from an aqueous solution and achieved 66.34% removal. Aslam et al. [11] studied the efficiency of three indigenous bacterial strains Stenotrophomonas sp. MB339, Klebsiella pneumoniae MB361, and Staphylococcus sp. MB371 for Cr⁶⁺, Pb²⁺ and Ni²⁺ removal from wastewater and observed 83.51% Cr⁶⁺, 85.30% of Pb²⁺, and 48.78% of Ni²⁺ bioaccumulation from 200 mg L⁻¹ concentration within 24 h at neutral pH. Microbial remediation has a wide range of applications. However, it is limited by microbial death or lack of survival, continuous nutrient supply, interference due to the presence of multiple metal ions, and cell washout [12,13].

Microbial immobilization on a support material can overcome the drawbacks by chemically or physically attaching the free microbial cells to a specific carrier, allowing for easy recovery and keeping them active for longer [14]. Furthermore, cell immobilization technology is critical in wastewater treatment because it can improve microbial stability, mechanical strength, bioremediation capacity, and reusability while providing high biomass and resistance to the toxic environment [15]. The immobilization process relies on the support material. Biochar has gained popularity in environmental applications over the last decade due to its low cost, specific surface area, porosity, and oxygen-containing functional groups and minerals [16,17,18]. Sawdust has been generated in large quantities from woodworking industries, and biomass availability provides a substantial source for biochar production. Biochar can act as an ideal candidate for microbial habitats and support microbial biofilm formation [19], and these microbial-cell-immobilized biochar supports have been used in pollutant removal [20,21]. Sawdust biochar has been previously reported for Ni²⁺ removal [22,23] but was not used as a carrier for microorganism-mediated metal removal, which is the novelty of the current work.

The technology of biochar-immobilized microorganisms combines the benefits of biochar and microorganisms, has a good removal effect, and produces little secondary pollution; thus, it has good prospects in wastewater treatment. Zhang et al. [24] utilized biochar-immobilized microorganism technology to remove antibiotic contamination in aqueous solutions by immobilizing Bacillus subtilis with honeysuckle residue-derived biochar. Zhou et al. [25] investigated salt-tolerant biosurfactant-producing bacteria Vibrio sp. immobilization on corn straw biochar to treat diesel pollution in the ocean. The residual amount of diesel is reduced from 169.2 to 8.91 mg, with a removal rate of up to 94.7%, outperforming biochar (35.2%) and free cells (54.4%). Even though biochar-immobilized microorganisms have been successfully applied in wastewater treatment, very few studies on metal ion removal, particularly Ni²⁺, have been conducted. However, these studies have not focused on the regeneration and stability of immobilized cells, which can be extremely important for their practical application.

The present study used sawdust biochar to immobilize Pseudomonas stutzeri and Ni²⁺ removal from aqueous solutions. The influence of various process parameters such as incubation time, pH, temperature, biocatalyst dosage, and initial metal ion concentration, was studied. The possible mechanisms of metal ion removal by microbial-cell-immobilized biochar were also investigated. Finally, a reusability study of microbial-cell-immobilized biochar was carried out. This study provides a promising approach for the bioremediation of Ni²⁺ contaminated water in practical application and a novel utilization of the waste biomass.

2. Results

2.1. Biochar Yield

Pyrolysis temperature and time are essential factors affecting biochar properties during production. The yield percentage of sawdust biochar (SDB) produced under different conditions is depicted in Figure 1a. These biochar samples are designated SDB–temp (°C)–time (min). The yield of SDB decreased as pyrolysis temperature increased. A maximum yield of 38.17% was observed for SDB–300–30, and the yield subsequently decreased with increasing temperature and time, reaching a minimum of 10.45% for SDB–700–120 °C. A sharp reduction in yield occurred at 700 °C, implying that high molecular weight structures such as lignin and cellulose were completely degraded at higher production temperatures [26]. Thangalazhy-Gopakumar et al. [27] reported that thermal volatiles was further broken down at higher temperatures into low molecular weight liquid and gas instead of biochar.

2.2. Immobilization and Selection of Biochar

The pyrolysis temperature affects the structural changes in biochar and its ability to remove pollutants and immobilize microorganisms [28]. Various combinations of microbial-cell-immobilized biochar were prepared based on production temperature, and the immobilized cell number was calculated in terms of log₁₀ cells g⁻¹ of Pseudomonas stutzeri. Figure 1b depicts the number of cells immobilized on biochar samples. The graph showed that maximum Pseudomonas stutzeri (11.76 log₁₀ cells) was immobilized onto SDB–500–60. A subsequent decrease in the cell number can be observed for the SDB produced at higher temperatures of 700 °C. However, for an extended duration, biochar produced at lower temperatures demonstrated better cell immobilization; for example, SDB–300–120 immobilized 9.7 log₁₀ cells g⁻¹, whereas SDB–300–30 could only immobilize 7 log₁₀ cells g⁻¹.

Immobilized cell number in SDB indicates that biochar provided nutrients for bacterial growth and acted as a support. Leng et al. [29] reported that biochar produced at 500 °C has a larger surface area and pore volume than biochar produced at 700 °C, allowing more bacterial cells to be accommodated and immobilized within the biochar. SDB produced at higher temperatures had fewer immobilized cells because some macropores collapsed at high temperatures, resulting in loss of functional group and reduced biochar nutrients. Huang et al. [28] found that high pyrolysis temperatures clogged the biochar pores, impeding the mass transfer of oxygen and nutrients and inhibiting microorganisms’ ability to remove contaminants.

The selection of biochar was based on the Ni²⁺ removal rate of microbial-cell-immobilized biochar. The removal efficiency of biochar-immobilized microorganisms pyrolyzed at 500 °C was higher than that of biochar-immobilized microorganisms at 300 and 700 °C (Figure 2). It may be due to the pore structure of the biochar providing a relatively stable growth environment for the microorganisms and protecting them from the toxicity of external pollutants, thus enhancing the removal of Ni²⁺ by the immobilized system. Therefore, the higher the immobilization rate, the higher the Ni²⁺ removal rate. As a result, SDB produced at 500 °C–60 min was immobilized; Pseudomonas stutzeri (PSDB) was used as the carrier for further study.

2.3. Characterization of SDB and PSDB

The SEM result of SDB is depicted in Figure 3. The surface morphology of biochar under 500× revealed a porous surface texture, possibly due to the evaporation of volatile matter from sawdust biomass during pyrolysis. Generally, the surface pore volume of the biochar is closely related to its bacterial and metal ion adsorption potential. It was observed that the distribution of honeycomb-like pores on the surface of the biochar could facilitate the adsorption of microorganisms and metal ions. During the metabolic process of microorganisms, biochar pore structure can act as a transport channel for oxygen, micronutrients, and pollutants, making it an excellent carrier for microbial cell immobilization [28].

FESEM analysis of PSDB was carried out after lyophilization (digital camera image of PSDB is represented in Figure S1) showed the presence of rod-shaped Pseudomonas stutzeri, as depicted in Figure 4. The presence of microorganisms on the surface and in the pores of biochar is due to its large specific surface area and a high number of micropores, which have a high adsorption capacity for microorganisms [30]. SEM analysis of SDB showed pore structures greater than 10 µm, and the diameter of Pseudomonas stutzeri is less than 2 µm, which allows the bacteria to enter the pore structure. Digital camera image of PSDB is.

The elemental composition of SDB showed contents of carbon (64.01%) and oxygen (34.21%). This suggests the abundance of oxygen-containing surface functional groups in SDB, which are the main metal adsorption sites [31]. The specific surface area and total pore volume of SDB (

Table 1. BET analysis of SDB and PSDB.

	Surface Area (m2 g−1) SDB	Total Pore Volume (cm3 g−1)	Average Pore Size (nm)
SDB	195.68	0.99	1.01
PSDB	3.19	0.14	0.5

) indicated that the number of micropores formed by pyrolysis and the pore structure developed fairly. This provides space for the growth and reproduction of microorganisms and is more favorable for increasing cell density. Biochar can obtain more effective adsorption sites during metal ion removal due to its loose and porous structure, large total pore volume, and specific surface areas [32]. BET surface area, total pore volume and average pore size of the SDB after immobilization with Pseudomonas stutzeri were much less compared to pristine SDB, confirming the adsorption and colonization of Pseudomonas stutzeri on biochar.

Surface functional groups can provide information about the properties of biochar and the immobilization of microbial cells and their interactions with the pollutant. Figure 5 represents the FTIR analysis of Pseudomonas stutzeri, SDB and PSDB. The main functional groups of SDB involve a broad transmittance band of 3337 cm⁻¹ (–OH); the bands at 1569 and 1409 cm⁻¹ represent the C=C stretching vibration of the biochar and –CH₂ groups [33]. An intense band occurring at 1036 cm⁻¹ is due to C–O stretching and is associated with oxygenated functional groups of cellulose, hemicellulose, and lignin. PSDB displayed O-H stretching vibration of the hydroxyl group, mainly responsible for the peak at 3251 cm⁻¹. Pseudomonas stutzeri spectral peaks at 2850 cm⁻¹ for C–H bending vibrations were introduced in PSDB, which were absent in SDB (Figure S2). Some of the bands in SDB shifted to a lower frequency from 1409 to 1396 cm⁻¹. Increases in peak intensity were more pronounced after the immobilization of Pseudomonas stutzeri for the peak at 1569 cm⁻¹, which represents C–H bending vibrations of fatty acids and lipids present on the bacteria surface. The FT-IR spectrogram indicates that the microorganisms are successfully loaded onto the SDB and that the complex integrates biochar and microbial functional groups, improving the stability and removal capacity of the PSDB.

Table 2. Extractable minerals present in SDB (mg kg⁻¹).

Ba	Fe	Mg	Mn	Zn	K	Cu	Pb
2.21	673.97	61.41	8.69	11.57	12	40	2.7

indicates the extractable nutrients and minerals in SDB. Based on the ICP analysis results, SDB comprises various mineral constituents, such as Fe, Mg, Mn, Zn, K, Cu, Pb, etc., that could support nutrients for the growth of Pseudomonas stutzeri. Metal ion removal can be accompanied by the release of metal cations such as K⁺ and Mg²⁺ present in the biochar as exchangeable cations to increase efficiency [34]. A study by Ye et al. [35] suggested that bacteria could adhere to the mineral-enriched region of biochar.

2.4. Effect of Operating Conditions on Ni²⁺ Removal

2.4.1. Effect of Incubation Time

The influence of incubation time on Ni²⁺ removal efficiency by Pseudomonas stutzeri and PSDB is depicted in Figure 6a. Ni²⁺ removal efficiency increased with incubation time, from 18.6 to 44.6% for Pseudomonas stutzeri and from 38.52 to 63.6% for PSDB from 6 to 42 h. A steep increase in Ni²⁺ removal efficiency was seen until 36 h for PSDB, whereas Pseudomonas stutzeri showed a significant increase until 24 h. Maximum Ni²⁺ removal of 63.6% was observed at 36 h for PSDB, whereas Pseudomonas stutzeri showed only 43% at an initial Ni²⁺ concentration of 50 mg L⁻¹. Maximum removal of Ni²⁺ occurred when most cells had reached the stationary phase, and cell density was the highest (Figure S3). Huang et al. [36], reported that higher cell densities in the stationary phase caused a higher metal ion accumulation. The delay in PSDB reaching maximum capacity compared to Pseudomonas stutzeri was attributed to PSDB’s high complexity of the metal ion removal process. During this process, Ni²⁺ was first attached to the biochar surface and then slowly adsorbed into the internal pores of the biochar and transferred into the bacterial cell wall, causing a delay in equilibrium.

2.4.2. Effect of Initial pH on Ni²⁺ Removal

One of the main factors that influences microbial growth and enzyme activity is pH [37]. The effect of initial pH on Ni²⁺ removal efficiency was evaluated with experiments conducted at Ni²⁺ concentrations of 50 mg L⁻¹, temperature of 37 °C, and contact time of 36 h. Ni²⁺ removal efficiency of PSDB increased from 5.0 to 7.0 and displayed 66.7% at pH 7 (Figure 6b). In the same condition, free cells showed an increase in removal from 26.72% to 44.38%. Ni²⁺ removal capacity of immobilized cells and Pseudomonas stutzeri varied with the change in pH. However, the metal ion removal was more pronounced in the case of the free cell with an increase in pH compared to immobilized cells (Figure S4). That was mainly attributed to the outer surface of PSDB loaded with various functional groups and a certain number of bacteria. This compact outer layer and inner space offered great buffering capacity for Ni²⁺ removal due to the unaffected metabolism in PSDB rather than in free cells. Additionally, microbial cells are prone to modify their protein folding in unfavorable pH conditions, thereby reducing metal removal efficiency. The present result was similar to the findings of Argun [38] and showed the maximum Ni²⁺ removal activity under the optimized conditions of pH ~7.0 using clinoptilolite.

The decrease in Ni²⁺ removal at lower pH is due to the protonation of surface groups by H⁺ ions, which competed with heavy metal ions [11]. The surface groups became deprotonated with the increase in pH, and Ni²⁺ ions were bound to the surface of the biochar. A study carried out by Bogusz et al. [39] indicated that when pH was strongly acidic, the nickel was present in the form of Ni²⁺ in the solution, while in the medium acidic pH, nickel was present in the form of Ni (OH)⁺. The second form of ions was adsorbed easier due to lower electrostatic repulsion between the ion and the surface. Based on these findings, Ni²⁺ removal by PSDB may be followed by precipitation. The removal efficiency in the immobilized system was higher than that of the free cells in the above-tested pH conditions, suggesting that SDB can act as a barrier and carrier for bacteria and ensure the viability of cells, thus improving the total Ni²⁺ removal efficiency.

2.4.3. Effect of Temperature

Temperature affects the stability and configuration of the cell wall by changing the surface functional groups and the binding sites for metal ions [28,29]. PSDB–mediated Ni²⁺ removal was investigated at 25, 30, 35, and 40 °C to study the effect of temperature. PSDB showed a removal efficiency of 39.76% at 25 °C and increased with temperature rise (Figure 7a). The maximum removal efficiency of 64.54% was obtained at 37 °C. Free cells also demonstrated the same trend of increase in Ni²⁺ removal with an increase in temperature from 25 to 37 °C because the temperature is a critical factor that regulates the bacterial metabolism and growth rate by affecting the stability of the cell wall. Pseudomonas stutzeri strains are more active at temperatures between 30 and 40 °C. Thus, the metabolism could be enhanced under optimal temperature, thereby increasing the nickel removal efficiency. Bacterial-mediated metal ion removal mainly occurs through bioaccumulation and appears to be temperature-dependent. Metal solubility and membrane-binding affinity of metal ions to the bacterial cell wall can be increased at an optimum temperature [37], which also accounts for increased removal efficiency. In PSDB, the pore structure enlarges when the temperature increases, creating more surfaces for metal ion adsorption [40]. A decrease in Ni removal at higher temperatures could be due to cell wall disruption or a weak interaction between the active site and metal ions. Finally, the incubation temperature on the removal efficiency of Ni²⁺ showed that both PSDB and free cells possessed the highest activity at 37 °C. Similar results have been reported in the bioaccumulation of Ni²⁺ by Desulfovibrio desulfuricans, where maximum Ni²⁺ removal was obtained at 37 °C [41].

2.4.4. Effect of PSDB Dosage

To enhance Ni²⁺ removal, PSDB with various dosages (0.5, 1 and 2 g L⁻¹) were employed in the Ni²⁺ solution with an initial concentration of 50 mg L⁻¹ at pH 7. As shown in Figure 7b, increasing the dosage of the biocatalyst enhanced the removal efficiency of Ni²⁺. The maximum removal efficiency of 71.6% was achieved with a PSDB dosage of 2 g L⁻¹. The free cell-mediated Ni²⁺ removal efficiency was 54% under the same conditions. An increase in removal efficiency with biocatalyst dosage can be attributed to increased biochar surface area, availability of more adsorption sites, and a functional group for metal removal.

2.4.5. Effect of Initial Ni²⁺ Concentration

The effect of initial metal ion concentration on the removal of Ni²⁺ by PSDB is shown in Figure 8. The removal efficiency decreased from 82.87% to 58.3%, with an increase in Ni²⁺ concentration from 10 to 100 mg L⁻¹. At lower concentrations, all metal ions in the solutions could interact with the cell’s binding sites; thus, the percentage removal of Ni²⁺ was high in the beginning. The decrease in percentage of Ni²⁺ removal with concentration was due to the exhaustion of the adsorption sites available on the biochar for a given dosage. With the increase in Ni²⁺ concentration, cell number decreased from 8 to 4.6 log₁₀ cells, suggesting that total removal was critically linked to cell viability and activity. During metal removal, adsorption sites on the MCB surface were more easily occupied when the initial Ni²⁺ concentration was low, and the bioaccumulation by growing bacteria was inhibited insignificantly, resulting in a significant increase in metal ion removal, while at higher Ni²⁺ concentration, adsorption sites gradually achieved saturation, and bacterial growth was significantly inhibited. Moreover, SDB biochar provided shelter for the free cells due to its pore structures, thereby prolonging the survival of immobilized bacteria in a stressful environment.

2.5. Adsorption Kinetics and Isotherm Study of PSDB

2.5.1. Adsorption Kinetics

The biosorption mechanism of Ni²⁺ by PSDB was studied using pseudo-first-order and pseudo-second-order kinetic models to match the adsorption experiment. The kinetic parameters are presented in

Table 3. Adsorption kinetics and isotherm parameter values for adsorption of Ni²⁺ by PSDB.

Models	Model Parameter	PSDB
Kinetic model
Pseudo-second order	Qe (mg g−1)	32.59
	K2 (g mg−1 h−1)	0.015
	R2	0.99
Isotherm model
Langmuir	Qm (mg g−1)	67.06
	KL (L mg−1)	0.08
	R2	0.99

. The experimental data were confirmed by both models, and the pseudo-second-order model (R² = 0.99) fit the Ni²⁺ adsorption kinetics for PSDB better than that of the pseudo-first-order model (Figure 9). This result was similar to a previous study on Ni adsorption kinetics [42]. It was reported that Ni²⁺ adsorption by Bacillus coagulans immobilized sodium alginate pellets fit the pseudo-second-order model. The pseudo-second-order kinetics implied chemisorption mechanisms by PSDB.

2.5.2. Adsorption Isotherm

The adsorption isotherm experiments were conducted to describe the adsorption behavior of Ni²⁺ by PSDB. Langmuir and Freundlich’s adsorption models were applied to the experimental data (Figure 10). The Ni²⁺ adsorption by PSDB fit the Langmuir model (R² = 0.99) better than the Freundlich and indicated that Ni²⁺ adsorption on PSDB was by monolayer adsorption. All adsorption sites displayed equal Ni²⁺ affinity.

PSDB had a maximum Ni²⁺ adsorption capacity of 67.06 mg g⁻¹ based on the Langmuir isotherm model. In the Langmuir isotherm model, the dimensionless constant parameter K_L is typically used to assess the spontaneity of the adsorption process. A K_L value between 0 and 1 indicates that adsorption is favorable. In this study, the K_L values were between 0 and 1, indicating that the adsorption process was favorable under these conditions. The kinetic and isotherm parameters are presented in Table 3.

According to the literature, the maximum Ni²⁺ adsorption capacities by various microorganism-immobilized material varied based on support material and tested conditions. Changing the biomass may alter the biochar’s pore structure, affecting the immobilization potential and removal efficiency. An et al. [43] conducted experiments using a peanut-shell-immobilized Pseudomonas hibiscicola strain for Ni²⁺ removal and achieved 77.34% removal in 120 h (

Table 4. Comparative data about Ni²⁺ removal by microorganisms immobilized on various support materials.

Support Material	Microorganism	Initial Concentration of Ni2+	Removal Efficiency	Time	Mechanism	Ref.
Loofa sponge	Chlorella sorokiniana	200 mg L−1	60.38 mg g−1	15 min	Biosorption	[44]
Alginate	Bacillus cereus	50 mg L−1	54 mg g−1	-	Adsorption	[45]
Ca-alginate beads	Sargassum sp.	7 mmol L−1	1.69 mmol g−1		Adsorption	[46]
Rice bran	Rhizopus arrhizus	100 mg L−1	6.83 mg g−1	90 min	Biosorption	[47]
Polyacrylamide beads	Enterobacter species	10 mg L−1	42%	30 min	Accumulation	[48]
Polyvinyl alcohol hydrogel	Aspergillus niger, strain B 77	0.9 mg L−1	48.9%	5 min	Biosorption	[49]
Ca-alginate	Aspergillus niger, strain	0.9 mg L−1	54.4%	5 min	Biosorption	[49]
Peanut shell biochar	Pseudomonas hibiscicola strain L1	20 mg L−1	77.34%	120 h	-	[43]
Saw dust biochar	Pseudomonas stutzeri	10 mg L−1	83%	36 h	Adsorption Bioaccumulation	Present study

), whereas the present study reports 83% removal within 36 h.

2.6. Ni²⁺ Removal by SDB, P. stutzeri, and PSDB at Optimized Operating Conditions

Ni²⁺ removal experiments by SDB, Pseudomonas stutzeri, and PSDB were performed at an optimized incubation temperature of 37 °C, initial solution pH of 7, PSDB dosage of 2 g L⁻¹, 36 h of incubation time, and 10 ppm initial metal ion concentration (Figure 11). Results indicated that the free cells removed Ni²⁺ by 67.6% (3.38 mg g⁻¹). Typically, Ni²⁺ removal by free cells could be from bioaccumulation by the bacterial cells [11]. SDB showed 38% (1.9 mg g⁻¹) removal through adsorption of Ni²⁺, whereas cell immobilization on SDB exhibited a significant enhancement of Ni²⁺ removal performance of 82.87% (4.14 mg g⁻¹). Immobilization can cause a higher number of cells attached to biochar. A more significant number of immobilized cells and biochar adsorption capacity could be the reason for the highest Ni²⁺ removal by PSDB. It is suggested that the pore structure of the biochar provides a relatively stable growth environment for the microorganisms and protects them from the toxicity of metal ions.

2.7. Reusability of Immobilized Cell

Regeneration of material is a key factor for their use in water treatment. In order to investigate the continuous application of PSDB, five consecutive experiments were carried out, and the results showed that the initial Ni²⁺ removal efficiency of PSDB was 83%, while the second and third cycles showed 80%, revealing its steady removal efficiency (Figure 12). PSDB maintained a 75% removal rate after the third cycle, indicating the reusability of the immobilized cell. Huang et al. [50] used algae biochar to immobilize Proteus mirabilis PC801 to maintain the removal rate of chromium and observed stability until the third cycle.

Microbial cell count varied from 8 to 4.9 log₁₀ cells after five cycles with 10 mg L⁻¹ Ni²⁺ (Figure 12). This gradually decreases over each cycle due to cell washout or cell death of surface-immobilized cells from direct contact with a Ni²⁺ ion during experiments. A constant number of cells was retained after two cycles of treatment with PSDB. This might be because biochar served as a carrier with multiple porosities and offered enough space and a stable microenvironment for the growth of bacteria [40]. Immobilization facilitated its repeated use, thereby cutting costs and making the use of biocatalysts more viable. In the reusability studies desorption of Ni²⁺ was not performed.

The Ni²⁺ removal mechanisms involved biochar-mediated adsorption and bacterial-mediated bioaccumulation, and Pseudomonas stutzeri played a significant role in removal (Figure 11). In general, desorption of the accumulated metal depends on the physicochemical characteristics of microbial cells and the type and concentration of the desorbent. Conventional desorbing eluents such as HCl, H₂SO₄, HNO₃, sodium carbonate, and EDTA may result in cell damage. Desorptive metal recovery of PSDB is also limited due to the bioaccumulation properties of live cells [51].

2.8. FTIR Analysis of PSDB after Ni Removal

FTIR absorption peaks of PSDB before and after treatment with Ni²⁺ are represented in Figure 13. The changes in peak indicate the attachment of Ni²⁺ with various functional groups existing on the SDB and Pseudomonas stutzeri cell membrane. FTIR spectrum produced by PSDB after Ni²⁺ removal showed significant changes at 3273, 2962, and 1575 cm⁻¹, regions representing the stretching of O–H, C–H, C=C, respectively [52,53]. The peaks representing hydroxyl groups at 3251 and 1020 cm⁻¹ in SDB were changed significantly. After the removal of Ni²⁺, the stretching vibration peak of O−H shifted from 3251 to 3273 cm⁻¹ due to the substitution of some of the hydrogen in O−H, which indicated that the adsorption could occur through the interaction between surface hydroxyl groups and Ni²⁺ [54]. Moreover, the intensity of peaks at 1396 and 1241 cm⁻¹ regions represented the bending of CH₃ and deformation of the C–H group, respectively, reducing to 1384 and 1230 cm⁻¹ after treatment [55,56].

Additionally, a sharp peak obtained at 1081 cm⁻¹ after Ni²⁺ bioaccumulation indicated the attachment of these metals to the C-O groups [57]. In addition, due to the C=C in the aromatic structure’s stretching, the band of heteroaromatic groups shifted from 873 to 868 cm⁻¹. In conclusion, PSDB treatment with Ni²⁺ altered the cell structure by modifying the functional groups on the cell surface, which was important for metal adsorption and bacterial survival in toxic environments. Furthermore, FTIR spectra of -OH, C=H, C=N, and C=C changed significantly, implying that complexation played a vital role in Ni²⁺ removal.

Previous research has shown that biochar can be used as a substrate for colonizing functional microbes and stimulating microbial activity [58,59]. In order to determine the Ni²⁺ removal mechanism of PSDB at the microbial level and confirm the biochar protective nature, the cell total protein concentrations in metal-ion-treated solution were carried out. The supernatant was collected and centrifuged at 13,000× g for 20 min at 4 °C. The total protein concentration was determined using the Bradford method. PSDB showed a protein concentration of 7.42 µg mL⁻¹ for Ni²⁺-treated solution, and free cells showed less protein concentration of 2.16 after treatment. This indicates the collective carrier and protective effect of SDB. Similar to these findings, Huang et al. [50] reported that heavy-metal-resistant bacterium Proteus mirabilis immobilized on cyanobacteria biochar accumulated more protein than a free bacterial system when incubated with Cr⁶⁺.

3. Materials and Methods

3.1. Chemicals

Nickel sulfate heptahydrate (NiSO₄·7H₂O) was procured from CDH Chemicals Ltd., Delhi, India. Luria-Bertani (LB) growth medium was obtained from HIMEDIA Laboratories (Mumbai, India). Bradford reagent for total protein detection was purchased from Sigma Aldrich. Hydrochloric acid and sodium hydroxide pellets were obtained from Loba Chemicals Pvt. Ltd. (Mumbai, India). All the other reagents used for the experiments were of analytical grade, and the solutions were prepared with deionized water.

3.2. Preparation of Biochar

Saw dust biomass was collected from a sawmill in Palakkad, Kerala, India, to prepare biochar. The biomass was rinsed with distilled water and dried in a vacuum oven at 80 °C for 24 h. The dried biomass was placed in a ceramic crucible and pyrolyzed in a muffle furnace with limited oxygen and controlled temperature at a 20 °C min⁻¹ heating rate. Nine biochar were prepared by pyrolyzing at different temperatures (300, 500, and 700 °C) for three different time intervals of 30, 60, and 120 min. The percentage biochar yield for each pyrolyzing temperature and time was calculated using Equation (1) [60].

% Biochar yield = Mass of biochar obtained (final weight)/Mass of biomass loaded (initial weight) × 100

(1)

3.3. Microbial Cell Immobilization

The bacterial strain Pseudomonas stutzeri MTCC 101, used in this study for Ni²⁺ removal, was purchased from Microbial Type Culture Collection (MTCC, Chandigarh, India). For immobilization, Pseudomonas stutzeri was cultivated in LB medium at 37 °C and 160 rpm for 24 h. Then, the biomass was harvested by centrifugation (5000× g, 5 min). The condensed cell suspension was prepared by dissolving the cell pellet in a fresh LB medium. Then, 2% (v/v) inoculum concentration was added into 250 mL conical flask containing sterilized 5 g (dry weight) biochar and 100 mL LB media. Further, it was incubated with constant shaking at 160 rpm for 24 h until the cells were absorbed onto the biochar surface and pore structure [50]. The biochar–bacterial complex was filtered through a Whatman filter paper, washed twice with water to remove free cells, and used for the Ni²⁺ removal experiment.

3.4. Selection of Biochar Based on Immobilization Potential

The number of microorganisms immobilized by the biochar and its removal rate were used as evaluation indicators to select biochar carriers for subsequent studies. The number of free and immobilized Pseudomonas stutzeri was determined using the spread plate count technique on LB agar medium. Briefly, 0.1 g (wet weight) biochar was soaked in 10 mL LB broth for 5 min, sonicated for 10 min, then vigorously mixed twice by vortex mixing for 3 min. The medium with the extracted cells of 0.1 mL was plated; then, the sample was 10-fold serially diluted until 10⁻⁹. Then, 0.1 mL at the dilution was spread on LB agar plates and incubated at 37 °C for 24 h. Bacterial colonies were counted and reported as CFU per gram of SDB, as shown in Equation (2).

CFU g⁻¹ = [(average colony count/0.1 mL)/g of biochar] × dilution factor

(2)

3.5. Characterization of SDB

The microstructural characterization of prepared SDB was carried out using High-Resolution–Field Emission Scanning Electron Microscopy (HR-FESEM) (GEMINI 300, Carl Zeiss). Elemental components were analyzed using Energy Dispersive X-ray analysis or EDAX (EDAX Octane super EDS System-SDD 70 mm). BET-specific surface area, pore volume, and average pore radius of biochar were determined based on nitrogen adsorption at 77.3 K using a surface area analyzer Autosorb-iQ (Quantachrome, Boynton Beach, FL, USA). The specific surface area was calculated according to the Brunauer–Emmett Teller method. The distribution of functional groups on the biochar surface was analyzed by Fourier-transform infrared spectrometer (FTIR) (Nicolet IS50 spectrometer). Extractable nutrient concentrations were determined using inductively coupled plasma—optical emission spectrometry (5100 ICP-OES; Agilent Technologies, Santa Clara, CA, USA) [28]. The elemental analysis was performed using a CHONS analyzer (LECO Truspec Micro Analyser).

3.6. Effect of Operating Conditions on Ni²⁺ Removal

The Ni²⁺ removal experiment was carried out in 250 mL Erlenmeyer flasks of LB medium containing 50 mg L⁻¹ Ni²⁺ solutions. To ensure the consistency of cell count in all treatments, 0.11 g PSDB containing ~10⁹ CFU mL⁻¹ cells and 1.2 mL free cells of Pseudomonas stutzeri (final density ~10⁹ CFU mL⁻¹) were added to 100 mL Ni²⁺ containing LB broth. The effect of culture incubation time on Ni²⁺ was studied for a period of 42 h. Then, 1 g L⁻¹ of biocatalyst (immobilized cell) was added to a different conical flask containing 100 mL of LB broth. Then, the flask was placed in a rotary shaker and agitated at 150 rpm for each of the different contact times chosen (6, 12, 24, 36, and 42 h). The effect of the solution pH 5–8 (adjusted with 0.01 M HNO₃ or 0.01 M NaOH) was studied at the optimal incubation time. The effect of varying temperatures on the Ni²⁺ removal capacity of PSDB was investigated in the temperature range from 25 to 40 °C at optimal pH. The effect of biocatalyst (PSDB) dosage on Ni²⁺ removal was studied in different concentrations ranging from 0.5 to 2 g L⁻¹. The influence of initial metal ion concentration on the removal efficiency was checked in different concentrations of Ni²⁺ ranging from 10 to 100 mg L⁻¹ at optimized biocatalyst dosage.

After incubation, the suspension was filtered using a 0.45 μm pore syringe filter. ICP-OES was used to analyze the different concentrations of Ni²⁺ in the filtrate. The amount of Ni²⁺ removed by the PSDB was evaluated using Equation (3):

% Removal = (C_i − C_f)/C_i × 100

(3)

C_i and C_f (mg L⁻¹) are the initial and final concentrations of the Ni²⁺ present in the aqueous media before and after removal.

Finally, to understand the Ni²⁺ removal mechanism of PSDB at the microbial level, the cell concentration was analyzed at increasing Ni²⁺ concentration, and protein concentrations in the solution were detected at the optimized condition. Cell count was analyzed by the standard plate count method, and protein contents of the supernatant were determined through the Bradford assay [61] using bovine serum albumin as a standard.

3.7. Adsorption Kinetics and Isotherm Study

Ni²⁺ biosorption characteristics of PSDB were evaluated by kinetic models. This study employed pseudo-first-order and pseudo-second-order models to identify the best fit model for the observed data. Experiments were conducted in 250 mL Erlenmeyer flasks containing Ni solution at 100 mL and PSDB 0.1 g. The experiments were conducted under 37 °C, rotational speed of 150 rpm, and sampled at regular intervals at 0, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48 h. Residual metal ions concentration was determined by ICP-OES, and the quantities of adsorbed metal ions (mg g⁻¹) were evaluated by Equation (4).

$$Q_e=\left[\frac{C_i-C_e}{W}\right]\times V$$

(4)

where C_i and C_e are the initial and final concentrations (mg L⁻¹) of Ni, respectively, W is the quantity of biochar (g) used, and V is volume of the solution (L).

Pseudo-first-order and pseudo-second-order models were applied to interpret adsorption kinetics following Equations (5) and (6), while Q_e and Q_t (mg g⁻¹) are the adsorption capacities at equilibrium and time, respectively. K₁ (min⁻¹) and K₂ (g (mg/min) ⁻¹) are the rate constants of the pseudo-first-order and pseudo-second-order models, respectively.

$$\ln \left(Q_e-Q_t\right)=\ln Q_e-K_{1}t$$

(5)

$$\frac{t}{Q_t}=\frac{1}{K_2{Q}_e^2{}^{ }}+\frac{t}{Q_e}$$

(6)

Ni²⁺ solutions ranging from 10 to 50 mg L⁻¹ were prepared in different conical flasks with the same amount of PSDB added (1 g L⁻¹ dry weight) for the adsorption isotherm study. The Langmuir and Freundlich models were applied to estimate Ni²⁺ adsorption isotherms following Equations (7) and (8), respectively. C_e (mg L⁻¹) is Ni²⁺ concentration at the equilibrium period. Q_m (mg g⁻¹), and K_L (L mg⁻¹) are the maximum adsorption capacity and Langmuir sorption constant, respectively. K_F ((mg g⁻¹) (mg L⁻¹)⁻ⁿ) and n are Freundlich sorption constants, which indicate sorption capacity and intensity, respectively.

$$\frac{1}{Q_e}=\frac{1}{Q_m}+\frac{1}{K_L{Q}_m}\frac{1}{C_e}$$

(7)

$$\ln Q_e=\ln K_F+\frac{1}{n}\ln C_e$$

(8)

3.8. Ni²⁺ Removal by SDB, Pseudomonas stutzeri and PSDB at Optimized Operating Conditions

Ni²⁺ removal by SDB, free cells, and PSDB at optimized operating conditions were conducted in triplicate with 10 mg L⁻¹ concentration. Based on the optimized conditions, 2 g L⁻¹ wet weight of PSDB, 2.4 mL of the bacterial cell suspension, and 2 g L⁻¹ wet weight of SDB were considered. All the flasks were incubated in an orbital shaker at 37 °C for 36 h, and the percentage of Ni²⁺ removal was calculated as described previously.

3.9. Experimental Design for the Reusability Study of Immobilized Cell

In order to determine the persistent reusability of immobilized cells, PSDB was repeatedly used in experiments without desorption, and Ni²⁺ removal efficiency was detected [62]. Experiments were carried out in 5 cycles, with PSDB collected by filtration under aseptic conditions. Immobilized cells were washed with sterile distilled water twice to clear the free cells and then reused to test metal removal in optimized culture conditions using an initial metal concentration of 10 mg L⁻¹. All the graphs and data analysis in this study were created and performed with Origin Pro 2022 (OriginLab Corporation, Northampton, MA, USA), and the results are presented as mean ± standard deviation.

4. Conclusions

The biochar-immobilized microorganism technique for pollutant removal is an effective approach in bioremediation. The present study showed that PSDB could effectively remove Ni²⁺ from water. The efficiency of PSDB is maximized when the removal conditions are as follows: incubation temperature of 37 °C, initial solution pH of 7, and PSDB dosage of 2 g L⁻¹. A time of 36 h of incubation resulted in 83% Ni²⁺ removal, which was better than Pseudomonas stutzeri mediated removal. FT-IR analysis showed that functional groups such as O-H, C=H, C=N, and C=C participated in the reaction process. Improved Ni²⁺ removal compared to free cell could be attributed to simultaneous adsorption, precipitation, ion exchange by metal cations (K⁺ and Mg²⁺) and complexation by carboxyl/hydroxyl groups present in the biochar. The adsorption experiment of Ni²⁺ on PSDB deduced that the adsorption isotherms can be described best by the Langmuir model, and the pseudo-second-order model best fits the Ni²⁺ adsorption kinetics, indicating that the process was monolayer and controlled by chemisorption. The results also demonstrated the effective and continuous reuse ability of PSDB. This study indicated that combining functional microbes and biochar could be a promising technology for the green and sustainable treatment of polluted water. Future research should be conducted to determine the efficacy and performance of this particular bacterium-immobilized biochar in soil remediation.