Aerobic Bacterium ‘achromobacter Sp. B10c’ Having Traits to Degrade Phenanthrene and Can Improve Physiology of Alfalfa Plant

Aerobic Bacterium ‘achromobacter Sp. B10c’ Having Traits to Degrade Phenanthrene and Can Improve Physiology of Alfalfa Plant | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Aerobic Bacterium ‘achromobacter Sp. B10c’ Having Traits to Degrade Phenanthrene and Can Improve Physiology of Alfalfa Plant Toseef Majid, Abdul Qadir Ahmad, Mushiada Sandhu, Dan Liu, Etisam Mazhar, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9379295/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Environmental pollution from polycyclic aromatic hydrocarbons (PAHs) poses a significant threat to ecosystems due to their mutagenic and carcinogenic properties. This study focused on the isolation and characterization of a novel bacterial strain, Achromobacter sp. B10c’ from contaminated agricultural soil in Chengdu, China, to evaluate its phenanthrene-degrading potential and its effect on alfalfa ( Medicago Sativa ) plant growth. The 16S rRNA gene sequencing identified the strain with 99.42% identity to other Achromobacter species. Under optimized environmental conditions (pH 7.0 and 30–35°C), Achromobacter sp. B10c’ achieved a maximum phenanthrene degradation rate of 88.33% within 72 hours. Metabolic analysis using GC-MS identified tetrachloroethane and phthalic acid as the primary degradation intermediates. In vivo plant studies revealed that phenanthrene stress (up to 200 mg/L) significantly reduced alfalfa growth parameters, including shoot and root length, biomass, and chlorophyll content. However, inoculation with Achromobacter sp. B10c significantly mitigated these toxic effects, increasing root and shoot lengths by 48% and 35%, respectively, in contaminated treatments. Furthermore, the bacterial strain reduced oxidative stress, evidenced by decreased Malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) levels, while simultaneously enhancing the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). These results suggest that Achromobacter sp. B10c is a highly efficient candidate for the bioremediation of PAH-contaminated sites and can serve as a plant growth-promoting agent under environmental stress. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Anthropogenic activities such as processing of petroleum, industrial treatment and waste combustion leads to promote the increase in pollution mainly Polycyclic aromatic hydrocarbons (PAH). PAHs are chemicals containing two or more than two benzene rings having mutagenic, cytotoxic and carcinogenic capabilities to both plants and animals (Kuppusamy et al., 2017 ). Food chain is the most abundant part for existence of life and these hydrocarbons are directly and indirectly affecting the food chain by accumulation in plants and animals from soil and water bodies (Ghosal et al., 2016 ). Scientists have a lot of concern to eliminate these pollutants from the ecosystem by using chemicals and physicals methods such as lank filling, through incineration, recycling, soil washing, and chemical oxidation but these strategies were not cost effective, not an ecofriendly and overall not suitable. Regarding this issue, bioremediation technique has proven very efficient to remediate PAHs from the environment. By using versatile metabolism of microbes, PAHs become easily to eliminate from the environment (Fahad et al., 2020). Native species of microorganisms are the most important stimulators to degrade PAHs (Kaur et al., 2023 ). Bacteria have potential to disintegrate PAHs aerobically by using ring hydroxylating dioxygenase enzymes encoded by polyaromatic hydrocarbon genes (Song et al. 2015). Rhizobacteria and other soil bacteria play a crucial part in the phytoremediation of hydrocarbons, which is thought to be associated with the stimulation of the rhizodegradation the breakdown of the root microbiome in the root region. This is the manner by which organic contaminants undergo breaking down in the rhizosphere of the plants by microorganisms Abdullah et al. 2020 ). Recently, the detrimental effect of PAHs (Polycyclic Aromatic Hydrocarbons) on plants has been documented by Edwards ( 1983 ), indicating that these compounds reduce photosynthetic enzyme activity, which has antioxidant qualities. PAHs have also been shown to influence lipid peroxidation, cause DNA damage, and limit plant growth (Alkio et al. 2005 ). The present study was conducted to isolate, identify, and characterize a Achromobacter sp. B10. with PAH degrading capability from the PAH contaminated soil, China. We described a unique bacterium with phenanthrene degradation properties without any major pathogenic characteristics that could be used in-vivo for remediation of phenanthrene contaminated sites. Moreover, Achromobacter sp. B10c also had potential in plant growth-promoting traits in the soil contaminated with phenanthrene. 2. Material and methods 2.1. Physico-chemical analysis of agricultural soil This research collected agricultural corn soil from the North of the Southwest Jiaotong University, Xipu Campus, Chengdu, China. The soil was homogenized from the rhizosphere and sieved to < 0.2mm. The soil texture was loamy sand with 6.43pH, grayish-black in color, and 18.49% organic matter (Zhou et al. 2020). 2.2. Isolation of phenanthrene resistance bacterial Strains Dipotassium hydrogen phosphate (K 2 HPO 4 ), ammonium chloride (NH 4 Cl), and other chemicals were all purchased from Chrone Chemicals (Chengdu, China) with purity of > 98%. This experiment was designed in triplicate. One triplicate sample was prepared with contamination of PAH and other contained PAH contaminated concentrations. 120g of soil containing in each replicate. 0.418g of phenanthrene was mixed in 1 mL of acetone, and the mixture was sprayed with phenanthrene. After later, soil was placed in lab conditions for six months for acclimatization of soil microbes. In the 100 mL of sterilized flask of acetone, 1.29mg of phenanthrene was dissolved. The thin layer of acetone was developed, after the acetone was evaporated. In conical flask, mixture of 1g of soil and 50mL of MSM was added at set at rotary shaker 120rpm at 30°C. The solution consisted of 1.25g K2HPO4, 1g Na2HPO4, 1g NH4Cl, 0.5g MgSO4, 0.05g CaCl2, and 0.05g FeSO. Trace elements were 40µg FeSO4, 40µg MnSO4, 20µg ZnSO4, 5µg CuSO4, 4µg CoCl2, 5µg Na2MoO4, 136µg KH2PO4, and 1mg NaCl (Xiao-Hong et al. 2010 ). Following one week of adaptation, 1mL from the flask was transferred into a new sterilized flask, which contained fresh 50mL of MSM and 1.29mg of phenanthrene as per the prior order, and placed in the same circumstances in a rotary shaker. This method was repeated twice. After a month of settling in, 100µL of the resultant MSM was put on the Luria Bertani (LB) agar plate and sprayed with phenanthrene dissolved in acetone. The LB medium comprised (per liter) 10g Tryptophan, 10g NaCl, 14g agar, and 2.5g yeast extract. Following drying, all plates were incubated at 30°C for 48 hours. After 48 hours, eight bacterial strains were isolated from various Petri plates. To further analyze phenanthrene breakdown capacity, each bacterium is placed into a flask containing phenanthrene and MSM, allowing them to grow solely on phenanthrene. For three days, growth was monitored every 24 hours using a UV/VIS spectrophotometer (SQ-4802, USA) with a wavelength of 600 nm. The highest absorption strain was chosen and subsequently evaluated for various phenanthrene concentrations and environmental factors. 2.3. Identification of selected bacteria 16S rRNA method was utilized for identification of chosen strains. The MO BIO Strong Kit for DNA Extraction (MO BIO Laboratories, Carlsbad, CA, USA) was used for the extraction of the genomic DNA from selected strains. Agarose gel electrophoresis was applied to estimate the pureness and concentration of the DNA and delivered for later experiments. The preliminary step of the PCR amplification procedure was initial denaturation, which is about at 94°C for 5min, followed by 34 rounds at 94°C of 30s, 56°C for 30s, and 68°C of 30s, and lastly, at 72°C, a 10min extension was applied, two PCR reactions were conducted for each sample and followed by joining after PCR amplification. For 16S rRNA gene amplification, the specific bacterial primer 27F (5'-AGRGTTYGATYMTGGCTCAG-3') and 1492R (5'RGYTACCTTGTTACGACTT-3') were utilized. The template was purified by magnetic beads, which was verified by gel electrophoresis. PCR amplification was performed after passing the test. Eventually, ABI’s 3730XL sequencer was utilized for detection. In this study the gene sequence identified for 16S rRNA was successfully submitted to the GenBank database at NCBI with the unique identifier KJ676719.1. 2.4. Characterization of phenanthrene degrading bacterial strain To evaluate the growth and ability to breakdown substances of a chosen strain tests were carried out under various environmental settings and levels of phenanthrene. Tests were carried out at varying temperatures such as 25°C, 30°C, 35°C, 40°C and 45°C were given. A range of pH levels was setup for the experiment including 4, 5, 6, 7, 8, and 9. Tests were conducted with varying levels of phenenthrene including 50, 100, 150, 200, 250 milligrams per liter to see if bacteria that can breakdown phenanthrene would work well with different kinds of carbon sources like sucrose, yeast extract, peptone, glycerin, and glucose. 2.5. Identification of metabolite Gas chromatography-mass spectrometer (GC-MS) (Model 7890A, Agilent Tech, and Incorporation. Wilmington, DE, United States of America) was utilized to assess the metabolites of phenanthrene created by Achromobacter sp. B10c . Prior to testing, using a 250mL separatory funnel, 50mL of sample was added, followed by n-hexane (20mL), trichloromethane (5mL), and dichloromethane (10mL); and the organic matter was extracted from the samples. The extraction sequence included polar, weak polar, and non-polar compounds. Then, all these extracts were mixed and additional anhydrous Na 2 SO 4 was added. Then, by evaporation at 30°C in a rotary evaporator, the remaining sample was 3-5mL. After filtration, the sample was assessed using GC-MS spectrometer equipped with a quartz capillary column (30m × 0.25mm × 0.25µm). During the first 15 min, the column temperature was 35°C, and at a rate of 10°C min-1, the temperature was increased to 250°C. The injection volume was 1µL, the temperature of 220°C was maintained at the injection port, the split ratio was 5:1, the volumetric flow rate of the carrier gas (helium) was 3mLmin-1, and the ion source temperature was 220°C, for the testing “SCAN mode,” of the instrument was used and the transmission temperature was 280°C (Ren et al. 2019 ). 2.6. Plant study 2.6.1. Analysis of physical growth parameters of alfalfa plants Seeds were soaked in distilled water for 24h. Later, 10 seeds were sown in each pot. After 60 days of germination, the alfalfa plants were harvested to analyze different physical and physicochemical parameters. The distilled water was used to wash the plants from each treatment to get rid of waste and debris. The harvested alfalfa plants were segregated into shoots, leaves, and roots. The lengths of shoot and root were measured with the help of measuring tape. The fresh weight of the root and shoot was determined using a digital weighing balance. To determine alfalfa plant’s dry shoot and root weights, they were kept for 48 hours in an oven. After completely drying, the root and shoots' dry consequences were measured using a weighing balance. 2.6.2. Analysis of physiological attributes The method of Armon (1949) was followed to determine chlorophyll and carotenoid content. Fresh alfalfa leaves (0.1 g) were ground in 80% acetone using a mortar and pestle in an ice tub and placed overnight at -4°C. The extract was centrifugated at 10,000 rpm for 10 min, and the philological attributes were analyzed for measuring absorbance at 645 nm, 480 nm, and 663 nm wavelengths using a UV-visible spectrophotometer. However, the contents of chlorophyll a and chlorophyll b and carotenoids were determined using the formulas given below: Chlorophyll a= {12.7(OD663-2.69(OD645) ×V/10000×W} …………. (1) Chlorophyll b = {22.9(OD645-4.68(OD663) ×V/10000×W} ……………………. (2) The carotenoids (mg/L) were measured by using the following formula; {A car/ Emx×100} Where Em×100 = 2500 A car = [(OD480) + 0.114(OD663)-0.638(OD 645)] / 2500 ……………………. (3) Where W represents the weights and V represents the extract volume. 2.6.3. Analysis of oxidative parameters The malonaldehyde (MDA) of alfalfa plants was detected by following the procedure of Heath and Packer ( 1968 ). For this purpose, 5 g of fresh leaves were ground using trichloroacetic acid (0.1%). The resultant was centrifuged at 1000rpm at supernatants separated and mixed with TCA and TBA, then heated at 100°C for 30 minutes. After cooling, the solution was again centrifuged at 8000rpm for 5 min. The concentration of MDA was analyzed by measuring absorbance at the wavelength of 532 nm using a UV-visible spectrophotometer. Similarly the concentrations of hydrogen peroxide (H2O2) were determined by adopting the procedure of Jana and Choudhuri ( 1982 ). The fresh leaves of alfalfa plants were ground in the presence of 5 ml of TCA (0.1% w/v) using an ice tub by adding 50 mM potassium phosphate buffer (pH 6.5). The resulting mixture was centrifuged at 6000rpm for 15 min and left at room temperature. The mixture’s absorbance was measured at the wavelength of 390 nm using a UV-visible spectrophotometer after 10 minutes. 2.6.4. Antioxidants The activities of antioxidant enzymes in alfalfa plants exposed to phenanthrene stress were also analyzed in this study. The fresh plant leaves were ground in 5mL of 50Mm sodium phosphate buffer (pH 7) in ice tubes containing 0.5 M ethylenediaminetetraacetic acid and 0.15 M NaCl. The resulting mixture was centrifuged (12000 rpm for 10 min) at 4°C. The obtained supernatants were used for the assay of antioxidant enzymes. APX concentration was determined by adopting the method described by Amako et al. ( 1994 ). A 1000 µL mixture was prepared by taking 700 mL of phosphate buffer, 100 mL of ascorbate solution at a concentration of 0.5 mM, and 100 mL of enzyme extract. The absorbance of the solution was then measured every 20 seconds in a spectrophotometer at a wavelength of 290 nm. The catalase activity (CAT) was measured by adopting the method of Aebi ( 1984 ). The plant's fresh leaves were ground with phosphate buffer (50 mM, pH 7.8). Then, 3 mL of mixture was prepared using 100 µL of enzyme extract, 2.8 mL of phosphate buffer (50 mM, 7.0 pH), and 100 µL of 300 mM hydrogen peroxide with 2 mM Ca. The concentration of CAT was measured by taking the absorbance at 240 nm using a spectrophotometer. The concentration of SOD and POD was determined by following the methodology of Giannopolitis and Ries ( 1977 ) and Chance and Maehly ( 1955 ). A homogenous solution of 0.5 g fresh leaves of alfalfa plants was prepared in 0.05M phosphate buffer at pH 7.8. The resulting mixture was centrifuged at 1200 rpm for 10 minutes. The absorbance of the obtained solution was determined at wavelengths of 560 nm and 470nm using a spectrophotometer. 3. Results 3.1. Isolation of phenanthrene resistance strains Our studies have shown that the agricultural, corn soil of Xipu, Chengdu, have some microbial capacities to degrade the petroleum products, for example, phenanthrene. Phenanthrene-degrading bacterial colonies have been detected surrounded by clear zones of the white spray of phenanthrene. Total of 10 isolates had shown the capability to degrade the phenanthrene. Bacterial strains including B1c, B2c, B3c, B4c, B5c, B60c. B7c, B8c and B9c showed the minimum degradation of < 50% except B10c which exhibited maximum degradation 88.33% of phenanthrene. Almost same pattern of growth OD 600 was observed bacterial strains including B1c, B2c, B3c, B4c, B5c, B60c. B7c, B8c and B9c revealed minimum growth expect B10c which exhibited maximum growth of 1.235 as depicted in Table 1 . Table 1 Potential of 10 selected bacterial isolates for degrading PAH @ 100mgL − 1 and their growth OD 600 . Strain OD (600) % degradation 24 h 48 h 72 h 24 h 48 h 72 h B1c 0.107 0.109 0.115 19.22 27.2 30.88 B2c 0.141 0.148 0.157 13.45 17.99 18.34 B3c 0.108 0.109 0.11 34.2 43.3 45.5 B4c 0.081 0.089 0.094 12.34 14.33 15.99 B5c 0.086 0.093 0.095 11.34 12.22 12.45 B6c 0.107 0.109 0.114 14.44 20.56 20 B7c 0.103 0.19 0.228 20.9 20.65 40.98 B8c 0.095 0.099 0.103 11.88 21.12 25.89 B9c 0.099 0.103 0.106 15.88 17.76 34.98 B10c 0.708 0.943 1.235 34.56 74.09 88.33 3.2. Identification of B10c For more identification, after the isolation of B10c, amplification and sequencing of 16S rRNA gene (1456 bp) was completed Fig. 1 . The database of GenBank was deposited the sequence under KJ676719.1 accession number. Analysis of BlastN showed a high identity (99.42%) with many bacterial strains belonging to species Achromobacter sp. B10c. Phylogenetic tree of B10c was constructed by silico analysis by the method of neighbor joining, utilizing the sequence of 16S rRNA gene and some other strains of bacteria from the database of GenBank. These results reveled that B10c was highly identical with Achromobacter sp. MGT3. 3.3. Characterization of Achromobacter sp. B10c The ability of Achromobacter sp. B10c to degrade phenanthrene at different concentrations is presented in Fig. 2 (A). Achromobacter sp. B10c showed the least growth 0.290 (OD 600 ) and degradation ability (22.22%) in the medium having concentration of PAH 250mgL − 1 followed by 200mgL − 1 with OD 600 (0.483) and degradation (30.02%), the highest elimination of phenanthrene was observed in the mediums containing 50 to 150 mgL − 1 of phenanthrene after 72h of incubation. In Fig. 2 (B) the degradation of phenanthrene at different pH levels are described. In medium containing 100mgL − 1 , the least % degradation of PAH by Achromobacter sp. B10c was observed at pH 9 (58.9%) and at pH 5 (60.9%) and the maximum % degradation at pH 7 (86.8%) followed by pH 8 (78.1%) and pH 6 (77.0%), almost same growth pattern was observed in the case of pH. Generally, 9 pH which is termed as acidic and basic pH is unfavorable for pollutants removal by bacterial strains (Al-Thukair and Malik 2016 ). At different temperatures, B10c showed growth and % degradation variations as depicted in Fig. 2 (C). Results revealed that at moderate temperature 30°C to 35°C maximum growth (1.15–1.26) and % degradation (78–86%) were observed. PAH degradation assay was also observed in different carbon sources as shown in Fig. 2 (D). According to our findings yeast extract has proved itself as a best carbon source with degradation of PAH 90.2% and growth of 1.12 OD 600 . However, PAH degradation in the medium containing sucrose was 79.29 all the tests were phenanthrene-dependent and provided strong evidence of phenanthrene degradation by Achromobacter sp. B10c. 3.4. Identification of metabolites Gas chromatogram of metabolites of phenanthrene is formed during the degradation by Achromobacter sp. B10c shown in Fig. 5 . GC/MS analysis identified two major metabolites of phenanthrene as Tetrachloroethane and Phthalic acid. Retention time (R.T.) of Tetrachloroethane and Phthalic acid were 16.168 and 32.914 respectively. Percentage of Tetrachloroethane and Phthalic acid were 45.51 and 46.01 respectively. Summary of results is shown in Table 2 and peaks are depicted in Fig. 3 . Table 2 Metabolites B10c of Phenanthrene produced by Achromobacter sp. Metabolite GC Rt (min) CAS number Name of compound Percentage 1 16.168 79-34-5 Tetrachloroethane 45.51 2 32.914 1000309-04-4 Phthalic acid 46.01 3.5. Plant Study 3.5.1. Physical growth parameters The results in (Fig. 4 A, B) showed that the shoot and root lengths of alfalfa plants exposed to phenanthrene were significantly reduced compared to control plants. The maximum reduction in the shoot and root lengths of 185% and 141% was observed in the T10 treatment, where 200mg phenanthrene was applied compared to the control treatment. The application bacterial strain Achromobacter sp. B10c significantly increased the shoot and root lengths by degrading the phenanthrene. As compared to treatment T5 (200mg/L phenanthrene), the treatment T10 (200mg/L phenanthrene + Achromobacter ) significantly p > 0.05 increased the root and shoot lengths by 48% and 35%, respectively. Similarly, the shoot and root fresh biomass was significantly decreased in plants exposed to phenanthrene compared to control treatments (Fig. 4 C-D). The root and shoot fresh biomass decreased as the phenanthrene concentration increased from treatment T2 to treatment T5. The maximum reduction in alfalfa plants’ shoot and shoot biomass was observed in the treatment T5 (200mg/L phenanthrene) by 190% and 82% compared to the control treatment plants. The induction of phenanthrene degrading bacterial strain significantly increased the fresh biomass of root and shoot of alfalfa plants. The bacterial strain increased the fresh biomass in T10 (200mg/L phenanthrene + Achromobacter ) by 47% and 17% compared to the treatment T5 (200mg/L phenanthrene). Exposure to phenanthrene decreased alfalfa plants' shoot and root dry biomass with increasing concentration (Fig. 4 , E-F). Results illustrated that the maximum reduction in the dry biomass of alfalfa plants was observed in T5 (200mg/L phenanthrene) by 146% and 111% compared to control treatment T1. The usage of bacterial stain significantly enhanced the dry root and shoot biomass in treatment T10 (200mg/L phenanthrene + Achromobacter ) by 43% and 30% as compared to treatment T5 (200mg/L phenanthrene). 3.5.2. Physiological parameters The chlorophyll content of alfalfa plants in (Fig. 5 ) illustrates that exposure to phenanthrene reduced the concentration of chlorophyll content as compared to the control treatment. The maximum reduction in chlorophyll a and b of alfalfa plants was observed in treatment T5 (200mg/L phenanthrene) by 104% and 45% compared to control treatment T1. However, the induction of the bacterial strain Achromobacter sp. B10c significantly p > 0.05 improved the concentration of chl a and b by 33% and 11% in treatment T10 (200mg/L phenanthrene + Achromobacter ) compared to treatment T5. Similarly, the carotenoids concentration was also improved by the induction of bacterial strain by degrading the concentration of phenanthrene. Figure 5 D showed that the maximum reduction was observed in treatment T5 by 40% compared to control. The application of bacterial strain increased the concentration of carotenoids in treatment T10 by 18% compared to treatment T5 where 200mg of phenanthrene was added. 3.5.3. Physicochemical parameters The exposure of phenanthrene to the plant’s alfalfa generates the oxidative stress of MDA and H 2 O 2, as shown in (Fig. 6 A-B). The maximum activity of MDA and H 2 O 2 was observed in treatment T5 by 54% and 55% compared to the control treatment T1. The oxidative stress was reduced as the bacterial strain was induced. In treatment, T10, the oxidative stress of MDA and H 2 O 2 decreased by 7% and 6% compared to treatment T5. Inoculating bacterial strain to soil contaminated by phenanthrene reduced oxidative stress by improving the antioxidants APX and CAT (Fig. 6 C& F). The exposure of phenanthrene maximum reduces the activity of APX and CAT in treatment T5 by 112% and 79% compared to control treatments. However, adding bacterial stain to the phenanthrene-contaminated soil in treatment (T6-T10) improved the APX and CAT activity significantly p > 0.05 by 24% and 25% compared to treatment T5. Similarly, the phenanthrene decreased the activity SOD and POD in alfalfa plants compared to the control treatment (Fig. 6 D& E). The maximum reduction in the activity of SOD and POD was measured in treatment T5 by 63% and 73% compared to the control treatment. The induction of bacterial stain significantly increased the activity of SOD and POD in treatment T10 by 23% and 24% compared to T5, where 200mg of phenanthrene was added. It concluded that the bacterial strain Achromobacter sp. B10c successfully degraded the phenanthrene concentration in soil by improving the alfalfa physicochemical parameters. 3.5.4. Relationship between physicochemical and physical parameters of alfalfa plants under the stress phenanthrene The Pearson correlation analysis investigated the relationship between phenanthrene stress and various physical and physicochemical parameters of alfalfa plants, as shown in (Fig. 7 ). A positive correlation was observed between MDA and H 2 O 2 contents in alfalfa plants. However, this concentration of MDA and H 2 O 2 showed a strong negative correlation with shoot length, root length, fresh weight of roots and shoot, dry weight of roots, and shoot of alfalfa plants. The MDA and H 2 O 2 contents also negatively correlated with chlorophyll contents, carotenoid content, superoxide dismutase activity, peroxidase activity, catalase activity, and ascorbate peroxidase activity in alfalfa plants. 3.6. Statistical analysis For statistical analysis, complete randomized design (CRD), one-way Analysis of variance (ANOVA) was done by using Statistix (version 8.1) software. The least significant difference test and significant differences for parameters were recorded at P < 0.05. 4. Discussion Anthropogenic activities like use of PHE prevails globally threat to water, soil and human health. Variant species of bacteria have been shown their efficient degradation potential of phenanthrene in the past. In this study phenanthrene degrading bacterial stain was isolated, identified and characterized as Achromobacter sp. B10c. Different genera of Achromobacter sp. having potential to degrade phenanthrene has been reported. Li et al. ( 2021 ) isolated phenanthrene degrading Achromobacter sp. PHED2. Similarly, Achromobacter sp. LH-1, Achromobacter sp. FM6-1 and Achromobacter denitrificans strain PheN1 had shown their potential to degrade phenanthrene (Hou et al. 2018 , Xu et al. 2019 , Zhang et al. 2021 ). Environmental factors have been proven themselves important in degrading phenanthrene. Different pH ranges, temperature variations, carbon source properties and phenanthrene concentrations were used in estimation of phenanthrene degradation by Achromobacter sp. B10c. Figure 3 (A) depicts the degradation of phenanthrene at different concentrations. >80% of degradation was assessed when concentration were 50 to 150mgL − 1 . By increasing concentration up to 250mgL − 1 degradation became drastically down. Almost same pattern was observed by Abdel-Razek et al. ( 2020 ), who isolated the bacterial isolates which were not able to degrade phenanthrene at high concentration. pH is the one of the most important factor in degradation of Phenanthrene. In present study, 5,6,7,8,9 pH were tested Fig. 3 (B). Most acidic and basic solutions couldn’t enhance the degradation potential of Achromobacter sp. B10c. These findings relate to Xu et al. ( 2019 ) who isolated phenanthrene degrading strains FM6-1 and FM8-1 at same pH values. Gordonia sp. SCSIO19801 showed its highest phenanthrene degrading potential at pH 8 (Mai et al. 2021 ). This may be due to unfavorable condition for Achromobacter sp to degrade PHE. Change of temperature may cause change in bacterial degradation of PHE (Xu et al. 2021 ). At temperature of 25°C to 40°C maximum degradation was assessed but by increasing temperature up to 45°C degradation of PHE was significantly reduced. Abdel-Razek et al. ( 2020 ) also observed maximum phenanthrene degradation by M4 and M6 at temperature of 30°C. Phenanthrene, a common environmental pollutant from industrial processes, vehicle emissions, and human activities, adversely affects alfalfa plant physical parameters (Haider et al. 2021 , Gabriele et al. 2021 ). Results showed that the shoot, root lengths, shoots, and root dry and fresh biomass were decreased in those plants where phenanthrene stress was induced (Figure.1F-A). Phenanthrene's toxicity might disrupt plant physiological and biochemical processes, interfering with enzymes and cellular metabolism. It may inhibit essential nutrient uptake in plant roots, leading to nutrient deficiencies that hinder growth (Tarigholizadeh et al. 2021 ). In a study conducted by Li et al. ( 2021 ) similar findings were reported, affirming the significant enhancement of PHE (200 mg/kg) removal from polluted soil through the co-cultivation of these bacteria with maize plants. Interestingly, PHED2 displayed greater efficacy in terms of root dry weight, chlorophyll content, net photosynthesis rate, and transpiration rate in maize plants when compared to PHED1. The study's result illustrated that soil contamination with phenanthrene significantly reduced the physical parameters of alfalfa plants. The reduction in physical growth parameters might be the Root damage that occurs directly in the soil, impairing nutrient and water uptake. Hormonal balance disruption affects plant growth regulation, and altered microbial interactions in the rhizosphere disrupt nutrient uptake and protection against pathogens(Gabriele et al. 2021 ). In a related investigation accompanied by Li et al. ( 2023 ), a comprehensive exploration was undertaken to assess the efficacy of combined microbial-phytoremediation strategies for soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Their research shed light on the considerable potential exhibited by Glomus versiforme and Pseudomonas fluorescens Ps2-6 in significantly augmenting the removal of PAHs. These research outcomes contribute significantly to advancing our comprehension of the underlying mechanisms governing the remediation of PAH-contaminated soils. In the present study, the change in chlorophyll content of the alfalfa plant was studied because chlorophyll serves as a primarily warning signal of plant stress, designating photosynthesis disruptions caused by drought, pollution, and extreme temperatures. Additionally, it provides quantitative data, enabling researchers to assess stress severity and compare different stressors' effects (Krasavina et al. 2014 , Giordano et al. 2021 ). Result of the study showed that the amount of chlorophyll of alfalfa plants exposed to the phenanthrene was decreased as the concentration of phenanthrene increased (Fig. 2 , A-D). When alfalfa plants were grown in phenanthrene contaminated soil, it might enter plant tissues, and it can disrupt the cellular processes, including photosynthesis, due to its toxic nature. This disruption might decrease the production of essential pigments for photosynthesis, like chlorophyll and carotenoids (Tarigholizadeh et al. 2021 ). In a study conducted by Zhu et al. ( 2019 ), the researchers investigated the effects of Phenanthrene (1 mg L − 1 ) nanoparticles of zinc oxide on wheat growth over a 15-day period. Their findings revealed a significant reduction in the chlorophyll concentration within wheat leaves, with a decrease ranging from 0.43 to 0.60-fold as levels of the Phenanthrene was increased. Accumulating of phenanthrene might cause nutrient deficiencies may limit the production of chlorophyll and carotenoids, as these pigments require specific nutrients for their synthesis (Tomar et al. 2019 ). Additionally, phenanthrene exposure might damage plant cell membranes and organelles, including chloroplasts. This damage might disrupt the normal functioning of chloroplasts and reduce the synthesis of pigments (Fonseca et al. 2020 ). This is noteworthy because chlorophyll and carotenoids are crucial pigments for photosynthesis. Chlorophyll captures light energy, while carotenoids protect the plant from excess light while transferring power to chlorophyll. Results were positively corelated by Saeed et al. ( 2022 ) where the isolation of Bacillus marsiflavi strain from oil-contaminated soil in Rawalpindi, Pakistan. Soil experiments conducted under varying oil contamination levels (5%, 10%, and 15%) revealed that Bac 144 substantially increased soil enzymatic activity, resulting in a remarkable 46% hydrocarbon degradation. Furthermore, the application of Bac 144 exhibited notable plant-related benefits, including a remarkable increase in shoot length up to 20% and elevated levels of chlorophyll, carotenoids, and proline content up to 16.85%. However, the result showed that induction of the bacterial strain Achromobacter sp. B10c increased the chlorophyll content (Fig. 2 A-D). The bacterial strain might be metabolized and break down phenanthrene, potentially reducing its concentration in the soil (Khoshru et al. 2020 ). This reduction in phenanthrene levels might alleviate plant stress, enabling them to allocate more resources to growth and physiological processes (Ali et al. 2023 ). Additionally, the decreased toxicity resulting from phenanthrene degradation may enhance plant efficiency, possibly increasing chlorophyll production. Furthermore, phenanthrene degradation might release nutrients previously bound to phenanthrene molecules, making them available for plant uptake. This increased nutrient availability may positively impact chlorophyll synthesis and overall plant health (Sui et al. 2021 ). Similarly, Sheng and Gong ( 2006 ) explored the potential of phenanthrene-degrading bacterium Pseudomonas sp. GF3. Their research investigated its impact on plant growth promotion and phenanthrene removal in induced contaminated soil (0, 100, and 200 mg kg − 1 ). In planted soils with GF3, 84.8% and 70.2% of phenanthrene disappeared at 100 and 200 mg kg − 1 , whereas unplanted soils showed 62.2% and 42.3% removal without GF3. Phenanthrene adversely affected wheat growth under low and high treatments. In this study, the reactive oxygen species (ROS) and antioxidants of alfalfa plants were measured under the stress of phenanthrene at different concentrations along with bacterial strains. ROS measurements help measure oxidative stress started by external factors such as phenanthrene exposure (Spinedi et al. 2021 ). Studying antioxidants elucidates how alfalfa plants combat ROS-induced damage, offering insights into their stress responses, potentially guiding stress tolerance improvements, and aiding in variety selection (Hernandez-Vega 2014 ). Additionally, these parameters are valuable in assessing the impact of stress on plant communities and ecosystems in the context of climate change, pollution, and habitat alterations, providing a broader ecological perspective (Hernández-Vega et al. 2017 ). Phenanthrene, a polycyclic aromatic hydrocarbon (PAH), induces oxidative stress of MDA and H 2 O 2 in alfalfa plants, as shown in Fig. 3 . Results were positively corelated by Saeed et al. ( 2022 ) where the isolation of Bacillus marsiflavi strain from oil-contaminated soil where Bac 144 substantially increased soil enzymatic activity, resulting in a remarkable 46% hydrocarbon degradation. The application of Bac 144 exhibited notable plant-related benefits, including substantial reduction in malondialdehyde content (21%) and a significant boost in antioxidant enzyme activity (24.5%). These findings highlight the promising potential of Bac 144 as an effective candidate for hydrocarbon bioremediation. The oxidative stress might be produced by generating reactive oxygen species (ROS) within plant cells. ROS, like superoxide radicals (O2•-) and hydroxyl radicals (•OH), form via phenanthrene metabolism or auto-oxidation and can harm cellular components. They target cellular structures, including lipids, proteins, and nucleic acids, causing lipid peroxidation and producing malondialdehyde (MDA) as an oxidative stress marker (Tandey et al. 2020 , Barreira 2007 ). Elevated ROS levels might lead to lipid peroxidation in cell membranes, disrupting membrane integrity and function. Exposure to phenanthrene might disrupt the ROS-antioxidant balance in alfalfa plant cells, damaging cell membranes, proteins, and DNA and releasing MDA and H 2 O 2 as oxidative stress markers. To mitigate oxidative stress induced by phenanthrene, the bacterial strain Achromobacter sp. B10c’ was applied to degrade the phenanthrene in the soil around the alfalfa plants (Pandey et al. 2013 , Cui et al. 2013 ). Results in Fig. 3 show that the bacterial stain significantly decreases the activity of oxidants by degrading the phenanthrene in the soil around the alfalfa plants. In a corroborative study conducted by Cui et al. ( 2023 ), the remediation of PAH-contaminated saline-alkali soil was investigated, employing biochar-immobilized Martelella sp. AD-3 in conjunction with Suaeda salsa L ( S. salsa ). Their findings echoed our own, as the combination of biochar-immobilized bacteria and S. salsa (referred to as the MBP group) exhibited a remarkable 91.67% removal efficiency of phenanthrene over a 40-day remediation period. Additionally, the soil's pH and electrical conductivity (EC) recorded reductions of 0.15 and 1.78 ds/m, respectively. Notably, S. salsa displayed substantial enhancements in fresh weight and leaf pigment contents, with increases of 1.30 and 1.35 times, indicating notable improvements in plant growth within PAH-contaminated saline-alkali soil, a trend consistent with our own research outcomes. Results in (Fig. 3 C& F) showed that the phenanthrene stress decreases the activity of CAT and APX. Results of the study were positively corelated with a similar Flocco et al. ( 2002 ) where the interaction between plants and a phenanthrene was investigated using hydroponic cultures of alfalfa as a controlled model system. Over 30 days of monitoring, the presence of plants was found to decrease the half-life of phenanthrene (initial concentration 50 mg L-1) by 2.7 times. However, phenanthrene exposure had detrimental effects on plant growth index, leaf chlorophyll content, and root peroxidase activity. Moreover, damage to plant cell membranes and organelles caused by phenanthrene could impair CAT and APX activity, diminishing their capacity to neutralize ROS (Fig. 3 D& E). Resource allocation within the plant might change in response to stressors like phenanthrene, potentially causing reduced CAT and APX levels as the plant prioritizes other defense mechanisms. The reduction in the amount of CAT and APX levels might also be the phenanthrene interfering with signaling pathways that regulate antioxidant enzyme production, thereby decreasing CAT and APX synthesis (Siddiqi and Husen 2021 , Ranjan et al. 2021 ). In a study reminiscent of our own findings Ye et al. ( 2014 ) conducted a study focusing on the remediation of PAH-contaminated soil, specifically examining the synergistic effects of alfalfa and a microbial consortium comprising Arthrobacter oxydans, Staphylococcus auricularis , and Stenotrophomonas maltophilia on pyrene degradation. Over a 45-day joint treatment period, pyrene concentrations in the rhizosphere soils notably decreased from initial levels of 11.3, 52.5, and 106.0 mg/kg to reduced concentrations of 2.0–3.0, 15.0–18.7, and 41.2–44.8 mg/kg, respectively. This treatment approach resulted in a significant enhancement of both pyrene removal and the overall microbial community diversity in the soil, while also elevating soil dehydrogenase and polyphenol oxidase activities. Conclusion It can be concluded that the bacterial strain Achromobacter sp. B10c, isolated from corn cultivated soil may be the potential microbe having the ability to remediate phenanthrene contaminated soil. 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Increased ZnO nanoparticle toxicity to wheat upon co-exposure to phenanthrene, Environmental Pollution , 247, 108–117, DOI: 10.1016/j.envpol.2019.01.046 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9379295","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":620951129,"identity":"f7e3f80e-ca4f-42a5-a827-3d4889a330af","order_by":0,"name":"Toseef Majid","email":"","orcid":"","institution":"Southwest Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Toseef","middleName":"","lastName":"Majid","suffix":""},{"id":620951130,"identity":"ca987847-623d-4421-b517-0adad0fd2cb1","order_by":1,"name":"Abdul Qadir Ahmad","email":"data:image/png;base64,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","orcid":"","institution":"Bahauddin Zakariya University","correspondingAuthor":true,"prefix":"","firstName":"Abdul","middleName":"Qadir","lastName":"Ahmad","suffix":""},{"id":620951131,"identity":"e70a320b-d5aa-4ea6-ae3f-14fe11b7cd3a","order_by":2,"name":"Mushiada Sandhu","email":"","orcid":"","institution":"Lahore College for Women University","correspondingAuthor":false,"prefix":"","firstName":"Mushiada","middleName":"","lastName":"Sandhu","suffix":""},{"id":620951132,"identity":"abced279-ec1d-4f63-ae23-6f0c2fabc968","order_by":3,"name":"Dan Liu","email":"","orcid":"","institution":"Southwest Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Liu","suffix":""},{"id":620951133,"identity":"475d3a90-1522-411f-9328-4402f95e9bf9","order_by":4,"name":"Etisam Mazhar","email":"","orcid":"","institution":"Government College University","correspondingAuthor":false,"prefix":"","firstName":"Etisam","middleName":"","lastName":"Mazhar","suffix":""},{"id":620951134,"identity":"dde67a2d-1ae9-429a-b93c-6b329dbcf5b6","order_by":5,"name":"Khalid Bilal","email":"","orcid":"","institution":"Bahauddin Zakariya University","correspondingAuthor":false,"prefix":"","firstName":"Khalid","middleName":"","lastName":"Bilal","suffix":""},{"id":620951135,"identity":"7fc53975-412a-4f27-a5ff-4d50dca9f599","order_by":6,"name":"Hina Ghafoor","email":"","orcid":"","institution":"Bahauddin Zakariya University","correspondingAuthor":false,"prefix":"","firstName":"Hina","middleName":"","lastName":"Ghafoor","suffix":""},{"id":620951136,"identity":"b356db34-e333-4902-8a06-a8a41803152a","order_by":7,"name":"Hassaan Ahmed Malik","email":"","orcid":"","institution":"Bahauddin Zakariya University","correspondingAuthor":false,"prefix":"","firstName":"Hassaan","middleName":"Ahmed","lastName":"Malik","suffix":""},{"id":620951137,"identity":"5cf93ae4-3795-4157-ac9d-39d5fcb805c5","order_by":8,"name":"Zubaria Ashraf","email":"","orcid":"","institution":"Government College University","correspondingAuthor":false,"prefix":"","firstName":"Zubaria","middleName":"","lastName":"Ashraf","suffix":""}],"badges":[],"createdAt":"2026-04-10 12:08:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9379295/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9379295/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106755097,"identity":"ea41f948-67fb-41bf-8497-ff7d271c0ec7","added_by":"auto","created_at":"2026-04-13 07:52:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":422360,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining phylogenetic examination ensuing from the numerous sequences of 16S rRNA gene grouping of \u003cem\u003eAchromobacter sp. \u003c/em\u003eB10c with those of other bacterial types detected in the GenBank database. The strain's accession numbers from the database of GenBank, utilized for phylogenetic examination, are given along the names of strains. Values of bootstrap higher than 900‰ are labeled as black circles and a scale bar is shown to express the phylogenetic distance\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9379295/v1/702d68c0c85077c54dcd4922.png"},{"id":106959830,"identity":"ac7667b2-b07c-435b-bc6c-31197e1c919b","added_by":"auto","created_at":"2026-04-15 09:15:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":630725,"visible":true,"origin":"","legend":"\u003cp\u003eCapability of \u003cem\u003eAchromobacter\u003c/em\u003esp. B10c on different environmental factors (A) PHE concentration (B) pH values (C) Temperature (D) Carbon sources\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9379295/v1/436393be28a3dbea7b0e1a56.png"},{"id":106755098,"identity":"68457980-81a7-471d-9668-fed7671a7757","added_by":"auto","created_at":"2026-04-13 07:52:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":606009,"visible":true,"origin":"","legend":"\u003cp\u003eGas chromatographic-mass spectroscopic analysis of intermediates during phenanthrene degradation by \u003cem\u003eAchromobacter sp. \u003c/em\u003eB10c\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9379295/v1/ff9f148915df842182ad5dd3.png"},{"id":106960031,"identity":"282f186a-9c8c-4192-8ea5-140c664c90f3","added_by":"auto","created_at":"2026-04-15 09:18:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2926978,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of phenanthrene along with bacterial strain ‘\u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c’ at different concentrations of T1(Control), T2 (25mg phenanthrene), T3 (50mg phenanthrene), T4 (100mg phenanthrene), T5 (200mg phenanthrene), T6 (\u003cem\u003eAchromobacter\u003c/em\u003e), T7 (25mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e), T8 (50mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e), T9 (100mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e) and T10 (200mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e) on alfalfa shoot length (A), root length (B), Shoot fresh weight (C), Root fresh weight (D), Shoot dry weight (E) and Root dry weight (F) under phenanthrene stress. Data given indicate the average of three replication (n = 3) ± SD. Different letters between the treatments represent the significant difference at p\u0026lt;0.05 under phenanthrene stress conditions\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9379295/v1/a280aecbbbe0e7e4e7ad841e.png"},{"id":106755101,"identity":"c4a07a8e-f054-4e1d-b064-6b9d2e4c236a","added_by":"auto","created_at":"2026-04-13 07:52:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1837961,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of phenanthrene along with bacterial strain ‘\u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c’ at different concentrations of T1(Control), T2 (25mg phenanthrene), T3 (50mg phenanthrene), T4 (100mg phenanthrene), T5 (200mg phenanthrene), T6 (\u003cem\u003eAchromobacter\u003c/em\u003e), T7 (25mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e), T8 (50mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e), T9 (100mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e) and T10 (200mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e) on chlorophyll a (A), chlorophyll b (B), total chlorophyll (C) and carotenoids of the alfalfa plant. Data given indicate the average of three replication (n = 3) ± SD. Different letters between the treatments represent the significant difference at p\u0026lt;0.05 under phenanthrene stress conditions\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9379295/v1/f33cad811cb2242d7ae113c8.png"},{"id":106960088,"identity":"18050d9c-dcc1-4408-8a42-887e64bb6389","added_by":"auto","created_at":"2026-04-15 09:18:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2763095,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of phenanthrene along with bacterial strain ‘\u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c’ at different concentrations of T1(Control), T2 (25mg phenanthrene), T3 (50mg phenanthrene), T4 (100mg phenanthrene), T5 (200mg phenanthrene), T6 (\u003cem\u003eAchromobacter\u003c/em\u003e), T7 (25mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e), T8 (50mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e), T9 (100mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e) and T10 (200mg phenanthrene + \u003cem\u003eAchromobacter\u003c/em\u003e) Monoaldehyde (A), Hydrogen peroxide (B), Ascorbate peroxidase (C), Superoxide dismutase (D), Peroxide dismutase (E) and Catalase (C) of the alfalfa plant. Data given indicate the average of three replication (n = 3) ± SD. Different letters between the treatments represent the significant difference at p\u0026lt;0.05 under phenanthrene stress conditions\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9379295/v1/8fe05f01d3dafd3e722698ba.png"},{"id":106960009,"identity":"67468b78-51bf-4033-8f18-46a1ad0bbbd4","added_by":"auto","created_at":"2026-04-15 09:17:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":374276,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between different physical and physicochemical attributes with phenanthrene stress in alfalfa plants.\u0026nbsp; Abbreviations, SL, shoot length; RL, Root length; SFW, shoot fresh weight; RFW, root fresh weight; SDW, shoot dry weight; RDW, root dry weight; Chla, chlorophyll a; Chlb, chlorophyll b; Tchl, total chlorophyll; Car, carotenoids; APX, ascorbate peroxidase; MDA, malondialdehyde; CAT, Catalase; SOD, superoxide dismutase; POD, peroxide dismutase and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, Hydrogen peroxide\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9379295/v1/cf7d9a3ef53c51dce331f736.png"},{"id":106964291,"identity":"ec8511a9-cb23-4c6e-a991-3bc2d844ff41","added_by":"auto","created_at":"2026-04-15 09:49:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10586988,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9379295/v1/00b10c0f-ecef-4339-9067-0ae99cbb5302.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAerobic Bacterium ‘achromobacter Sp. B10c’ Having Traits to Degrade Phenanthrene and Can Improve Physiology of Alfalfa Plant\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAnthropogenic activities such as processing of petroleum, industrial treatment and waste combustion leads to promote the increase in pollution mainly Polycyclic aromatic hydrocarbons (PAH). PAHs are chemicals containing two or more than two benzene rings having mutagenic, cytotoxic and carcinogenic capabilities to both plants and animals (Kuppusamy et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Food chain is the most abundant part for existence of life and these hydrocarbons are directly and indirectly affecting the food chain by accumulation in plants and animals from soil and water bodies (Ghosal et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Scientists have a lot of concern to eliminate these pollutants from the ecosystem by using chemicals and physicals methods such as lank filling, through incineration, recycling, soil washing, and chemical oxidation but these strategies were not cost effective, not an ecofriendly and overall not suitable. Regarding this issue, bioremediation technique has proven very efficient to remediate PAHs from the environment. By using versatile metabolism of microbes, PAHs become easily to eliminate from the environment (Fahad et al., 2020). Native species of microorganisms are the most important stimulators to degrade PAHs (Kaur et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Bacteria have potential to disintegrate PAHs aerobically by using ring hydroxylating dioxygenase enzymes encoded by polyaromatic hydrocarbon genes (Song et al. 2015). Rhizobacteria and other soil bacteria play a crucial part in the phytoremediation of hydrocarbons, which is thought to be associated with the stimulation of the rhizodegradation the breakdown of the root microbiome in the root region. This is the manner by which organic contaminants undergo breaking down in the rhizosphere of the plants by microorganisms Abdullah et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, the detrimental effect of PAHs (Polycyclic Aromatic Hydrocarbons) on plants has been documented by Edwards (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), indicating that these compounds reduce photosynthetic enzyme activity, which has antioxidant qualities. PAHs have also been shown to influence lipid peroxidation, cause DNA damage, and limit plant growth (Alkio et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study was conducted to isolate, identify, and characterize a Achromobacter sp. B10. with PAH degrading capability from the PAH contaminated soil, China. We described a unique bacterium with phenanthrene degradation properties without any major pathogenic characteristics that could be used in-vivo for remediation of phenanthrene contaminated sites. Moreover, \u003cem\u003eAchromobacter sp.\u003c/em\u003e B10c also had potential in plant growth-promoting traits in the soil contaminated with phenanthrene.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Physico-chemical analysis of agricultural soil\u003c/h2\u003e \u003cp\u003eThis research collected agricultural corn soil from the North of the Southwest Jiaotong University, Xipu Campus, Chengdu, China. The soil was homogenized from the rhizosphere and sieved to \u0026lt;\u0026thinsp;0.2mm. The soil texture was loamy sand with 6.43pH, grayish-black in color, and 18.49% organic matter (Zhou et al. 2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Isolation of phenanthrene resistance bacterial Strains\u003c/h2\u003e \u003cp\u003eDipotassium hydrogen phosphate (K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), ammonium chloride (NH\u003csub\u003e4\u003c/sub\u003eCl), and other chemicals were all purchased from Chrone Chemicals (Chengdu, China) with purity of \u0026gt;\u0026thinsp;98%. This experiment was designed in triplicate. One triplicate sample was prepared with contamination of PAH and other contained PAH contaminated concentrations. 120g of soil containing in each replicate. 0.418g of phenanthrene was mixed in 1 mL of acetone, and the mixture was sprayed with phenanthrene. After later, soil was placed in lab conditions for six months for acclimatization of soil microbes. In the 100 mL of sterilized flask of acetone, 1.29mg of phenanthrene was dissolved. The thin layer of acetone was developed, after the acetone was evaporated. In conical flask, mixture of 1g of soil and 50mL of MSM was added at set at rotary shaker 120rpm at 30\u0026deg;C. The solution consisted of 1.25g K2HPO4, 1g Na2HPO4, 1g NH4Cl, 0.5g MgSO4, 0.05g CaCl2, and 0.05g FeSO. Trace elements were 40\u0026micro;g FeSO4, 40\u0026micro;g MnSO4, 20\u0026micro;g ZnSO4, 5\u0026micro;g CuSO4, 4\u0026micro;g CoCl2, 5\u0026micro;g Na2MoO4, 136\u0026micro;g KH2PO4, and 1mg NaCl (Xiao-Hong et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Following one week of adaptation, 1mL from the flask was transferred into a new sterilized flask, which contained fresh 50mL of MSM and 1.29mg of phenanthrene as per the prior order, and placed in the same circumstances in a rotary shaker. This method was repeated twice. After a month of settling in, 100\u0026micro;L of the resultant MSM was put on the Luria Bertani (LB) agar plate and sprayed with phenanthrene dissolved in acetone. The LB medium comprised (per liter) 10g Tryptophan, 10g NaCl, 14g agar, and 2.5g yeast extract. Following drying, all plates were incubated at 30\u0026deg;C for 48 hours. After 48 hours, eight bacterial strains were isolated from various Petri plates. To further analyze phenanthrene breakdown capacity, each bacterium is placed into a flask containing phenanthrene and MSM, allowing them to grow solely on phenanthrene. For three days, growth was monitored every 24 hours using a UV/VIS spectrophotometer (SQ-4802, USA) with a wavelength of 600 nm. The highest absorption strain was chosen and subsequently evaluated for various phenanthrene concentrations and environmental factors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Identification of selected bacteria\u003c/h2\u003e \u003cp\u003e16S rRNA method was utilized for identification of chosen strains. The MO BIO Strong Kit for DNA Extraction (MO BIO Laboratories, Carlsbad, CA, USA) was used for the extraction of the genomic DNA from selected strains. Agarose gel electrophoresis was applied to estimate the pureness and concentration of the DNA and delivered for later experiments. The preliminary step of the PCR amplification procedure was initial denaturation, which is about at 94\u0026deg;C for 5min, followed by 34 rounds at 94\u0026deg;C of 30s, 56\u0026deg;C for 30s, and 68\u0026deg;C of 30s, and lastly, at 72\u0026deg;C, a 10min extension was applied, two PCR reactions were conducted for each sample and followed by joining after PCR amplification. For 16S rRNA gene amplification, the specific bacterial primer 27F (5'-AGRGTTYGATYMTGGCTCAG-3') and 1492R (5'RGYTACCTTGTTACGACTT-3') were utilized. The template was purified by magnetic beads, which was verified by gel electrophoresis. PCR amplification was performed after passing the test. Eventually, ABI\u0026rsquo;s 3730XL sequencer was utilized for detection. In this study the gene sequence identified for 16S rRNA was successfully submitted to the GenBank database at NCBI with the unique identifier KJ676719.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Characterization of phenanthrene degrading bacterial strain\u003c/h2\u003e \u003cp\u003eTo evaluate the growth and ability to breakdown substances of a chosen strain tests were carried out under various environmental settings and levels of phenanthrene. Tests were carried out at varying temperatures such as 25\u0026deg;C, 30\u0026deg;C, 35\u0026deg;C, 40\u0026deg;C and 45\u0026deg;C were given. A range of pH levels was setup for the experiment including 4, 5, 6, 7, 8, and 9. Tests were conducted with varying levels of phenenthrene including 50, 100, 150, 200, 250 milligrams per liter to see if bacteria that can breakdown phenanthrene would work well with different kinds of carbon sources like sucrose, yeast extract, peptone, glycerin, and glucose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Identification of metabolite\u003c/h2\u003e \u003cp\u003eGas chromatography-mass spectrometer (GC-MS) (Model 7890A, Agilent Tech, and Incorporation. Wilmington, DE, United States of America) was utilized to assess the metabolites of phenanthrene created by \u003cem\u003eAchromobacter sp. B10c\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePrior to testing, using a 250mL separatory funnel, 50mL of sample was added, followed by n-hexane (20mL), trichloromethane (5mL), and dichloromethane (10mL); and the organic matter was extracted from the samples. The extraction sequence included polar, weak polar, and non-polar compounds. Then, all these extracts were mixed and additional anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was added. Then, by evaporation at 30\u0026deg;C in a rotary evaporator, the remaining sample was 3-5mL. After filtration, the sample was assessed using GC-MS spectrometer equipped with a quartz capillary column (30m \u0026times; 0.25mm \u0026times; 0.25\u0026micro;m). During the first 15 min, the column temperature was 35\u0026deg;C, and at a rate of 10\u0026deg;C min-1, the temperature was increased to 250\u0026deg;C. The injection volume was 1\u0026micro;L, the temperature of 220\u0026deg;C was maintained at the injection port, the split ratio was 5:1, the volumetric flow rate of the carrier gas (helium) was 3mLmin-1, and the ion source temperature was 220\u0026deg;C, for the testing \u0026ldquo;SCAN mode,\u0026rdquo; of the instrument was used and the transmission temperature was 280\u0026deg;C (Ren et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Plant study\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Analysis of physical growth parameters of alfalfa plants\u003c/h2\u003e \u003cp\u003eSeeds were soaked in distilled water for 24h. Later, 10 seeds were sown in each pot. After 60 days of germination, the alfalfa plants were harvested to analyze different physical and physicochemical parameters. The distilled water was used to wash the plants from each treatment to get rid of waste and debris. The harvested alfalfa plants were segregated into shoots, leaves, and roots.\u003c/p\u003e \u003cp\u003eThe lengths of shoot and root were measured with the help of measuring tape. The fresh weight of the root and shoot was determined using a digital weighing balance. To determine alfalfa plant\u0026rsquo;s dry shoot and root weights, they were kept for 48 hours in an oven. After completely drying, the root and shoots' dry consequences were measured using a weighing balance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Analysis of physiological attributes\u003c/h2\u003e \u003cp\u003eThe method of Armon (1949) was followed to determine chlorophyll and carotenoid content. Fresh alfalfa leaves (0.1 g) were ground in 80% acetone using a mortar and pestle in an ice tub and placed overnight at -4\u0026deg;C. The extract was centrifugated at 10,000 rpm for 10 min, and the philological attributes were analyzed for measuring absorbance at 645 nm, 480 nm, and 663 nm wavelengths using a UV-visible spectrophotometer. However, the contents of chlorophyll a and chlorophyll b and carotenoids were determined using the formulas given below:\u003c/p\u003e \u003cp\u003eChlorophyll a= {12.7(OD663-2.69(OD645) \u0026times;V/10000\u0026times;W} \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;. (1)\u003c/p\u003e \u003cp\u003eChlorophyll b = {22.9(OD645-4.68(OD663) \u0026times;V/10000\u0026times;W} \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;. (2)\u003c/p\u003e \u003cp\u003eThe carotenoids (mg/L) were measured by using the following formula;\u003c/p\u003e \u003cp\u003e{A car/ Emx\u0026times;100} Where Em\u0026times;100\u0026thinsp;=\u0026thinsp;2500\u003c/p\u003e \u003cp\u003eA car = [(OD480)\u0026thinsp;+\u0026thinsp;0.114(OD663)-0.638(OD 645)] / 2500 \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;. (3)\u003c/p\u003e \u003cp\u003eWhere W represents the weights and V represents the extract volume.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3. Analysis of oxidative parameters\u003c/h2\u003e \u003cp\u003eThe malonaldehyde (MDA) of alfalfa plants was detected by following the procedure of Heath and Packer (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1968\u003c/span\u003e). For this purpose, 5 g of fresh leaves were ground using trichloroacetic acid (0.1%). The resultant was centrifuged at 1000rpm at supernatants separated and mixed with TCA and TBA, then heated at 100\u0026deg;C for 30 minutes. After cooling, the solution was again centrifuged at 8000rpm for 5 min. The concentration of MDA was analyzed by measuring absorbance at the wavelength of 532 nm using a UV-visible spectrophotometer.\u003c/p\u003e \u003cp\u003eSimilarly the concentrations of hydrogen peroxide (H2O2) were determined by adopting the procedure of Jana and Choudhuri (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). The fresh leaves of alfalfa plants were ground in the presence of 5 ml of TCA (0.1% w/v) using an ice tub by adding 50 mM potassium phosphate buffer (pH 6.5). The resulting mixture was centrifuged at 6000rpm for 15 min and left at room temperature. The mixture\u0026rsquo;s absorbance was measured at the wavelength of 390 nm using a UV-visible spectrophotometer after 10 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.6.4. Antioxidants\u003c/h2\u003e \u003cp\u003eThe activities of antioxidant enzymes in alfalfa plants exposed to phenanthrene stress were also analyzed in this study. The fresh plant leaves were ground in 5mL of 50Mm sodium phosphate buffer (pH 7) in ice tubes containing 0.5 M ethylenediaminetetraacetic acid and 0.15 M NaCl. The resulting mixture was centrifuged (12000 rpm for 10 min) at 4\u0026deg;C. The obtained supernatants were used for the assay of antioxidant enzymes. APX concentration was determined by adopting the method described by Amako et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). A 1000 \u0026micro;L mixture was prepared by taking 700 mL of phosphate buffer, 100 mL of ascorbate solution at a concentration of 0.5 mM, and 100 mL of enzyme extract. The absorbance of the solution was then measured every 20 seconds in a spectrophotometer at a wavelength of 290 nm. The catalase activity (CAT) was measured by adopting the method of Aebi (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). The plant's fresh leaves were ground with phosphate buffer (50 mM, pH 7.8). Then, 3 mL of mixture was prepared using 100 \u0026micro;L of enzyme extract, 2.8 mL of phosphate buffer (50 mM, 7.0 pH), and 100 \u0026micro;L of 300 mM hydrogen peroxide with 2 mM Ca. The concentration of CAT was measured by taking the absorbance at 240 nm using a spectrophotometer. The concentration of SOD and POD was determined by following the methodology of Giannopolitis and Ries (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1977\u003c/span\u003e) and Chance and Maehly (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1955\u003c/span\u003e). A homogenous solution of 0.5 g fresh leaves of alfalfa plants was prepared in 0.05M phosphate buffer at pH 7.8. The resulting mixture was centrifuged at 1200 rpm for 10 minutes. The absorbance of the obtained solution was determined at wavelengths of 560 nm and 470nm using a spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Isolation of phenanthrene resistance strains\u003c/h2\u003e \u003cp\u003eOur studies have shown that the agricultural, corn soil of Xipu, Chengdu, have some microbial capacities to degrade the petroleum products, for example, phenanthrene. Phenanthrene-degrading bacterial colonies have been detected surrounded by clear zones of the white spray of phenanthrene. Total of 10 isolates had shown the capability to degrade the phenanthrene. Bacterial strains including B1c, B2c, B3c, B4c, B5c, B60c. B7c, B8c and B9c showed the minimum degradation of \u0026lt;\u0026thinsp;50% except B10c which exhibited maximum degradation 88.33% of phenanthrene. Almost same pattern of growth OD\u003csub\u003e600\u003c/sub\u003e was observed bacterial strains including B1c, B2c, B3c, B4c, B5c, B60c. B7c, B8c and B9c revealed minimum growth expect B10c which exhibited maximum growth of 1.235 as depicted in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePotential of 10 selected bacterial isolates for degrading PAH @ 100mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and their growth OD\u003csub\u003e600\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eOD (600)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e% degradation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e72 h\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB1c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e27.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e30.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB2c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e17.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB3c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e45.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB4c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB5c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB6c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB7c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB8c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e21.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB9c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e17.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eB10c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.708\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.943\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e74.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e88.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Identification of B10c\u003c/h2\u003e \u003cp\u003eFor more identification, after the isolation of B10c, amplification and sequencing of 16S rRNA gene (1456 bp) was completed Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The database of GenBank was deposited the sequence under KJ676719.1 accession number. Analysis of BlastN showed a high identity (99.42%) with many bacterial strains belonging to species \u003cem\u003eAchromobacter sp.\u003c/em\u003e B10c. Phylogenetic tree of B10c was constructed by silico analysis by the method of neighbor joining, utilizing the sequence of 16S rRNA gene and some other strains of bacteria from the database of GenBank. These results reveled that B10c was highly identical with \u003cem\u003eAchromobacter sp.\u003c/em\u003e MGT3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Characterization of \u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c\u003c/h2\u003e \u003cp\u003eThe ability of \u003cem\u003eAchromobacter sp.\u003c/em\u003e B10c to degrade phenanthrene at different concentrations is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(A). \u003cem\u003eAchromobacter sp.\u003c/em\u003e B10c showed the least growth 0.290 (OD\u003csub\u003e600\u003c/sub\u003e) and degradation ability (22.22%) in the medium having concentration of PAH 250mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e followed by 200mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with OD\u003csub\u003e600\u003c/sub\u003e (0.483) and degradation (30.02%), the highest elimination of phenanthrene was observed in the mediums containing 50 to 150 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of phenanthrene after 72h of incubation. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(B) the degradation of phenanthrene at different pH levels are described. In medium containing 100mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the least % degradation of PAH by \u003cem\u003eAchromobacter sp.\u003c/em\u003e B10c was observed at pH 9 (58.9%) and at pH 5 (60.9%) and the maximum % degradation at pH 7 (86.8%) followed by pH 8 (78.1%) and pH 6 (77.0%), almost same growth pattern was observed in the case of pH. Generally, \u0026lt;\u0026thinsp;5 and \u0026gt;\u0026thinsp;9 pH which is termed as acidic and basic pH is unfavorable for pollutants removal by bacterial strains (Al-Thukair and Malik \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). At different temperatures, B10c showed growth and % degradation variations as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(C). Results revealed that at moderate temperature 30\u0026deg;C to 35\u0026deg;C maximum growth (1.15\u0026ndash;1.26) and % degradation (78\u0026ndash;86%) were observed. PAH degradation assay was also observed in different carbon sources as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(D). According to our findings yeast extract has proved itself as a best carbon source with degradation of PAH 90.2% and growth of 1.12 OD\u003csub\u003e600\u003c/sub\u003e. However, PAH degradation in the medium containing sucrose was 79.29 all the tests were phenanthrene-dependent and provided strong evidence of phenanthrene degradation by \u003cem\u003eAchromobacter sp.\u003c/em\u003e B10c.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Identification of metabolites\u003c/h2\u003e \u003cp\u003eGas chromatogram of metabolites of phenanthrene is formed during the degradation by \u003cem\u003eAchromobacter sp.\u003c/em\u003e B10c shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. GC/MS analysis identified two major metabolites of phenanthrene as Tetrachloroethane and Phthalic acid. Retention time (R.T.) of Tetrachloroethane and Phthalic acid were 16.168 and 32.914 respectively. Percentage of Tetrachloroethane and Phthalic acid were 45.51 and 46.01 respectively. Summary of results is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and peaks are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMetabolites B10c of Phenanthrene produced by \u003cem\u003eAchromobacter sp.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetabolite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGC Rt (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAS number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eName of compound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePercentage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16.168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e79-34-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTetrachloroethane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e45.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32.914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e1000309-04-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePhthalic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e46.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Plant Study\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1. Physical growth parameters\u003c/h2\u003e \u003cp\u003eThe results in (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B) showed that the shoot and root lengths of alfalfa plants exposed to phenanthrene were significantly reduced compared to control plants. The maximum reduction in the shoot and root lengths of 185% and 141% was observed in the T10 treatment, where 200mg phenanthrene was applied compared to the control treatment. The application bacterial strain \u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c significantly increased the shoot and root lengths by degrading the phenanthrene. As compared to treatment T5 (200mg/L phenanthrene), the treatment T10 (200mg/L phenanthrene\u0026thinsp;+\u0026thinsp;\u003cem\u003eAchromobacter\u003c/em\u003e) significantly p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 increased the root and shoot lengths by 48% and 35%, respectively.\u003c/p\u003e \u003cp\u003eSimilarly, the shoot and root fresh biomass was significantly decreased in plants exposed to phenanthrene compared to control treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). The root and shoot fresh biomass decreased as the phenanthrene concentration increased from treatment T2 to treatment T5. The maximum reduction in alfalfa plants\u0026rsquo; shoot and shoot biomass was observed in the treatment T5 (200mg/L phenanthrene) by 190% and 82% compared to the control treatment plants. The induction of phenanthrene degrading bacterial strain significantly increased the fresh biomass of root and shoot of alfalfa plants. The bacterial strain increased the fresh biomass in T10 (200mg/L phenanthrene\u0026thinsp;+\u0026thinsp;\u003cem\u003eAchromobacter\u003c/em\u003e) by 47% and 17% compared to the treatment T5 (200mg/L phenanthrene). Exposure to phenanthrene decreased alfalfa plants' shoot and root dry biomass with increasing concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, E-F). Results illustrated that the maximum reduction in the dry biomass of alfalfa plants was observed in T5 (200mg/L phenanthrene) by 146% and 111% compared to control treatment T1. The usage of bacterial stain significantly enhanced the dry root and shoot biomass in treatment T10 (200mg/L phenanthrene\u0026thinsp;+\u0026thinsp;\u003cem\u003eAchromobacter\u003c/em\u003e) by 43% and 30% as compared to treatment T5 (200mg/L phenanthrene).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2. Physiological parameters\u003c/h2\u003e \u003cp\u003eThe chlorophyll content of alfalfa plants in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) illustrates that exposure to phenanthrene reduced the concentration of chlorophyll content as compared to the control treatment. The maximum reduction in chlorophyll a and b of alfalfa plants was observed in treatment T5 (200mg/L phenanthrene) by 104% and 45% compared to control treatment T1. However, the induction of the bacterial strain \u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c significantly p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 improved the concentration of chl a and b by 33% and 11% in treatment T10 (200mg/L phenanthrene\u0026thinsp;+\u0026thinsp;\u003cem\u003eAchromobacter\u003c/em\u003e) compared to treatment T5. Similarly, the carotenoids concentration was also improved by the induction of bacterial strain by degrading the concentration of phenanthrene. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD showed that the maximum reduction was observed in treatment T5 by 40% compared to control. The application of bacterial strain increased the concentration of carotenoids in treatment T10 by 18% compared to treatment T5 where 200mg of phenanthrene was added.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.5.3. Physicochemical parameters\u003c/h2\u003e \u003cp\u003eThe exposure of phenanthrene to the plant\u0026rsquo;s alfalfa generates the oxidative stress of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). The maximum activity of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was observed in treatment T5 by 54% and 55% compared to the control treatment T1. The oxidative stress was reduced as the bacterial strain was induced. In treatment, T10, the oxidative stress of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decreased by 7% and 6% compared to treatment T5. Inoculating bacterial strain to soil contaminated by phenanthrene reduced oxidative stress by improving the antioxidants APX and CAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u0026amp; F). The exposure of phenanthrene maximum reduces the activity of APX and CAT in treatment T5 by 112% and 79% compared to control treatments. However, adding bacterial stain to the phenanthrene-contaminated soil in treatment (T6-T10) improved the APX and CAT activity significantly p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 by 24% and 25% compared to treatment T5. Similarly, the phenanthrene decreased the activity SOD and POD in alfalfa plants compared to the control treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026amp; E). The maximum reduction in the activity of SOD and POD was measured in treatment T5 by 63% and 73% compared to the control treatment. The induction of bacterial stain significantly increased the activity of SOD and POD in treatment T10 by 23% and 24% compared to T5, where 200mg of phenanthrene was added. It concluded that the bacterial strain \u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c successfully degraded the phenanthrene concentration in soil by improving the alfalfa physicochemical parameters.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.5.4. Relationship between physicochemical and physical parameters of alfalfa plants under the stress phenanthrene\u003c/h2\u003e \u003cp\u003eThe Pearson correlation analysis investigated the relationship between phenanthrene stress and various physical and physicochemical parameters of alfalfa plants, as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). A positive correlation was observed between MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents in alfalfa plants. However, this concentration of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e showed a strong negative correlation with shoot length, root length, fresh weight of roots and shoot, dry weight of roots, and shoot of alfalfa plants. The MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents also negatively correlated with chlorophyll contents, carotenoid content, superoxide dismutase activity, peroxidase activity, catalase activity, and ascorbate peroxidase activity in alfalfa plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eFor statistical analysis, complete randomized design (CRD), one-way Analysis of variance (ANOVA) was done by using Statistix (version 8.1) software. The least significant difference test and significant differences for parameters were recorded at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAnthropogenic activities like use of PHE prevails globally threat to water, soil and human health. Variant species of bacteria have been shown their efficient degradation potential of phenanthrene in the past. In this study phenanthrene degrading bacterial stain was isolated, identified and characterized as \u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c. Different genera of \u003cem\u003eAchromobacter\u003c/em\u003e sp. having potential to degrade phenanthrene has been reported. Li et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) isolated phenanthrene degrading \u003cem\u003eAchromobacter\u003c/em\u003e sp. PHED2. Similarly, \u003cem\u003eAchromobacter\u003c/em\u003e sp. LH-1, \u003cem\u003eAchromobacter\u003c/em\u003e sp. FM6-1 and \u003cem\u003eAchromobacter denitrificans\u003c/em\u003e strain PheN1 had shown their potential to degrade phenanthrene (Hou et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Xu et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Zhang et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEnvironmental factors have been proven themselves important in degrading phenanthrene. Different pH ranges, temperature variations, carbon source properties and phenanthrene concentrations were used in estimation of phenanthrene degradation by \u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (A) depicts the degradation of phenanthrene at different concentrations. \u0026gt;80% of degradation was assessed when concentration were 50 to 150mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. By increasing concentration up to 250mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e degradation became drastically down. Almost same pattern was observed by Abdel-Razek et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who isolated the bacterial isolates which were not able to degrade phenanthrene at high concentration. pH is the one of the most important factor in degradation of Phenanthrene. In present study, 5,6,7,8,9 pH were tested Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (B). Most acidic and basic solutions couldn\u0026rsquo;t enhance the degradation potential of \u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c. These findings relate to Xu et al. (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) who isolated phenanthrene degrading strains FM6-1 and FM8-1 at same pH values. \u003cem\u003eGordonia\u003c/em\u003e sp. SCSIO19801 showed its highest phenanthrene degrading potential at pH 8 (Mai et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This may be due to unfavorable condition for \u003cem\u003eAchromobacter\u003c/em\u003e sp to degrade PHE. Change of temperature may cause change in bacterial degradation of PHE (Xu et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). At temperature of 25\u0026deg;C to 40\u0026deg;C maximum degradation was assessed but by increasing temperature up to 45\u0026deg;C degradation of PHE was significantly reduced. Abdel-Razek et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) also observed maximum phenanthrene degradation by M4 and M6 at temperature of 30\u0026deg;C.\u003c/p\u003e \u003cp\u003ePhenanthrene, a common environmental pollutant from industrial processes, vehicle emissions, and human activities, adversely affects alfalfa plant physical parameters (Haider et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Gabriele et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Results showed that the shoot, root lengths, shoots, and root dry and fresh biomass were decreased in those plants where phenanthrene stress was induced (Figure.1F-A). Phenanthrene's toxicity might disrupt plant physiological and biochemical processes, interfering with enzymes and cellular metabolism. It may inhibit essential nutrient uptake in plant roots, leading to nutrient deficiencies that hinder growth (Tarigholizadeh et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In a study conducted by Li et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) similar findings were reported, affirming the significant enhancement of PHE (200 mg/kg) removal from polluted soil through the co-cultivation of these bacteria with maize plants. Interestingly, PHED2 displayed greater efficacy in terms of root dry weight, chlorophyll content, net photosynthesis rate, and transpiration rate in maize plants when compared to PHED1.\u003c/p\u003e \u003cp\u003eThe study's result illustrated that soil contamination with phenanthrene significantly reduced the physical parameters of alfalfa plants. The reduction in physical growth parameters might be the Root damage that occurs directly in the soil, impairing nutrient and water uptake. Hormonal balance disruption affects plant growth regulation, and altered microbial interactions in the rhizosphere disrupt nutrient uptake and protection against pathogens(Gabriele et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In a related investigation accompanied by Li et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), a comprehensive exploration was undertaken to assess the efficacy of combined microbial-phytoremediation strategies for soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Their research shed light on the considerable potential exhibited by \u003cem\u003eGlomus versiforme\u003c/em\u003e and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e Ps2-6 in significantly augmenting the removal of PAHs. These research outcomes contribute significantly to advancing our comprehension of the underlying mechanisms governing the remediation of PAH-contaminated soils.\u003c/p\u003e \u003cp\u003eIn the present study, the change in chlorophyll content of the alfalfa plant was studied because chlorophyll serves as a primarily warning signal of plant stress, designating photosynthesis disruptions caused by drought, pollution, and extreme temperatures. Additionally, it provides quantitative data, enabling researchers to assess stress severity and compare different stressors' effects (Krasavina et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Giordano et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Result of the study showed that the amount of chlorophyll of alfalfa plants exposed to the phenanthrene was decreased as the concentration of phenanthrene increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, A-D). When alfalfa plants were grown in phenanthrene contaminated soil, it might enter plant tissues, and it can disrupt the cellular processes, including photosynthesis, due to its toxic nature. This disruption might decrease the production of essential pigments for photosynthesis, like chlorophyll and carotenoids (Tarigholizadeh et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In a study conducted by Zhu et al. (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the researchers investigated the effects of Phenanthrene (1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) nanoparticles of zinc oxide on wheat growth over a 15-day period. Their findings revealed a significant reduction in the chlorophyll concentration within wheat leaves, with a decrease ranging from 0.43 to 0.60-fold as levels of the Phenanthrene was increased.\u003c/p\u003e \u003cp\u003eAccumulating of phenanthrene might cause nutrient deficiencies may limit the production of chlorophyll and carotenoids, as these pigments require specific nutrients for their synthesis (Tomar et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, phenanthrene exposure might damage plant cell membranes and organelles, including chloroplasts. This damage might disrupt the normal functioning of chloroplasts and reduce the synthesis of pigments (Fonseca et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This is noteworthy because chlorophyll and carotenoids are crucial pigments for photosynthesis. Chlorophyll captures light energy, while carotenoids protect the plant from excess light while transferring power to chlorophyll. Results were positively corelated by Saeed et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) where the isolation of \u003cem\u003eBacillus marsiflavi\u003c/em\u003e strain from oil-contaminated soil in Rawalpindi, Pakistan. Soil experiments conducted under varying oil contamination levels (5%, 10%, and 15%) revealed that Bac 144 substantially increased soil enzymatic activity, resulting in a remarkable 46% hydrocarbon degradation. Furthermore, the application of Bac 144 exhibited notable plant-related benefits, including a remarkable increase in shoot length up to 20% and elevated levels of chlorophyll, carotenoids, and proline content up to 16.85%.\u003c/p\u003e \u003cp\u003eHowever, the result showed that induction of the bacterial strain \u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c increased the chlorophyll content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). The bacterial strain might be metabolized and break down phenanthrene, potentially reducing its concentration in the soil (Khoshru et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This reduction in phenanthrene levels might alleviate plant stress, enabling them to allocate more resources to growth and physiological processes (Ali et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, the decreased toxicity resulting from phenanthrene degradation may enhance plant efficiency, possibly increasing chlorophyll production. Furthermore, phenanthrene degradation might release nutrients previously bound to phenanthrene molecules, making them available for plant uptake. This increased nutrient availability may positively impact chlorophyll synthesis and overall plant health (Sui et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, Sheng and Gong (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) explored the potential of phenanthrene-degrading bacterium \u003cem\u003ePseudomonas\u003c/em\u003e sp. GF3. Their research investigated its impact on plant growth promotion and phenanthrene removal in induced contaminated soil (0, 100, and 200 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). In planted soils with GF3, 84.8% and 70.2% of phenanthrene disappeared at 100 and 200 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, whereas unplanted soils showed 62.2% and 42.3% removal without GF3. Phenanthrene adversely affected wheat growth under low and high treatments.\u003c/p\u003e \u003cp\u003eIn this study, the reactive oxygen species (ROS) and antioxidants of alfalfa plants were measured under the stress of phenanthrene at different concentrations along with bacterial strains. ROS measurements help measure oxidative stress started by external factors such as phenanthrene exposure (Spinedi et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Studying antioxidants elucidates how alfalfa plants combat ROS-induced damage, offering insights into their stress responses, potentially guiding stress tolerance improvements, and aiding in variety selection (Hernandez-Vega \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, these parameters are valuable in assessing the impact of stress on plant communities and ecosystems in the context of climate change, pollution, and habitat alterations, providing a broader ecological perspective (Hern\u0026aacute;ndez-Vega et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Phenanthrene, a polycyclic aromatic hydrocarbon (PAH), induces oxidative stress of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in alfalfa plants, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Results were positively corelated by Saeed et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) where the isolation of \u003cem\u003eBacillus marsiflavi\u003c/em\u003e strain from oil-contaminated soil where Bac 144 substantially increased soil enzymatic activity, resulting in a remarkable 46% hydrocarbon degradation. The application of Bac 144 exhibited notable plant-related benefits, including substantial reduction in malondialdehyde content (21%) and a significant boost in antioxidant enzyme activity (24.5%). These findings highlight the promising potential of Bac 144 as an effective candidate for hydrocarbon bioremediation.\u003c/p\u003e \u003cp\u003eThe oxidative stress might be produced by generating reactive oxygen species (ROS) within plant cells. ROS, like superoxide radicals (O2\u0026bull;-) and hydroxyl radicals (\u0026bull;OH), form via phenanthrene metabolism or auto-oxidation and can harm cellular components. They target cellular structures, including lipids, proteins, and nucleic acids, causing lipid peroxidation and producing malondialdehyde (MDA) as an oxidative stress marker (Tandey et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Barreira \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Elevated ROS levels might lead to lipid peroxidation in cell membranes, disrupting membrane integrity and function. Exposure to phenanthrene might disrupt the ROS-antioxidant balance in alfalfa plant cells, damaging cell membranes, proteins, and DNA and releasing MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as oxidative stress markers. To mitigate oxidative stress induced by phenanthrene, the bacterial strain \u003cem\u003eAchromobacter\u003c/em\u003e sp. B10c\u0026rsquo; was applied to degrade the phenanthrene in the soil around the alfalfa plants (Pandey et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Cui et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Results in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e show that the bacterial stain significantly decreases the activity of oxidants by degrading the phenanthrene in the soil around the alfalfa plants. In a corroborative study conducted by Cui et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the remediation of PAH-contaminated saline-alkali soil was investigated, employing biochar-immobilized \u003cem\u003eMartelella\u003c/em\u003e sp. AD-3 in conjunction with \u003cem\u003eSuaeda salsa\u003c/em\u003e L (\u003cem\u003eS. salsa\u003c/em\u003e). Their findings echoed our own, as the combination of biochar-immobilized bacteria and \u003cem\u003eS. salsa\u003c/em\u003e (referred to as the MBP group) exhibited a remarkable 91.67% removal efficiency of phenanthrene over a 40-day remediation period. Additionally, the soil's pH and electrical conductivity (EC) recorded reductions of 0.15 and 1.78 ds/m, respectively. Notably, S. salsa displayed substantial enhancements in fresh weight and leaf pigment contents, with increases of 1.30 and 1.35 times, indicating notable improvements in plant growth within PAH-contaminated saline-alkali soil, a trend consistent with our own research outcomes.\u003c/p\u003e \u003cp\u003eResults in (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026amp; F) showed that the phenanthrene stress decreases the activity of CAT and APX. Results of the study were positively corelated with a similar Flocco et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) where the interaction between plants and a phenanthrene was investigated using hydroponic cultures of alfalfa as a controlled model system. Over 30 days of monitoring, the presence of plants was found to decrease the half-life of phenanthrene (initial concentration 50 mg L-1) by 2.7 times. However, phenanthrene exposure had detrimental effects on plant growth index, leaf chlorophyll content, and root peroxidase activity. Moreover, damage to plant cell membranes and organelles caused by phenanthrene could impair CAT and APX activity, diminishing their capacity to neutralize ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026amp; E). Resource allocation within the plant might change in response to stressors like phenanthrene, potentially causing reduced CAT and APX levels as the plant prioritizes other defense mechanisms. The reduction in the amount of CAT and APX levels might also be the phenanthrene interfering with signaling pathways that regulate antioxidant enzyme production, thereby decreasing CAT and APX synthesis (Siddiqi and Husen \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Ranjan et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In a study reminiscent of our own findings Ye et al. (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) conducted a study focusing on the remediation of PAH-contaminated soil, specifically examining the synergistic effects of alfalfa and a microbial consortium comprising \u003cem\u003eArthrobacter oxydans, Staphylococcus auricularis\u003c/em\u003e, and \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e on pyrene degradation. Over a 45-day joint treatment period, pyrene concentrations in the rhizosphere soils notably decreased from initial levels of 11.3, 52.5, and 106.0 mg/kg to reduced concentrations of 2.0\u0026ndash;3.0, 15.0\u0026ndash;18.7, and 41.2\u0026ndash;44.8 mg/kg, respectively. This treatment approach resulted in a significant enhancement of both pyrene removal and the overall microbial community diversity in the soil, while also elevating soil dehydrogenase and polyphenol oxidase activities.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIt can be concluded that the bacterial strain \u003cem\u003eAchromobacter sp.\u003c/em\u003e B10c, isolated from corn cultivated soil may be the potential microbe having the ability to remediate phenanthrene contaminated soil. It shows the maximum degradation of phenanthrene among other strains. It can degrade phenanthrene into less toxic metabolites. Moreover, B10c promoted physical and physiological growth of alfalfa plant in PHE contaminated soil.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eToseef Majid, Abdul Qadir Ahmad, Mushiada Sandhu and Liu Dan did research and write this complete article. Etisam Mazhar, Khalid Bilal and Hina Ghafoor prepared characterization data and explain according to litrature. Hassaan Ahmed Malik and Zubaria Ashraf performend activity and prepared results according to experiment.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel-Razek, A., El-Sheikh, H., Suleiman, W., Taha, T. H. \u0026amp; Mohamed, M. (2020). 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Increased ZnO nanoparticle toxicity to wheat upon co-exposure to phenanthrene, \u003cem\u003eEnvironmental Pollution\u003c/em\u003e, 247, 108\u0026ndash;117, DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.envpol.2019.01.046\u003c/span\u003e\u003cspan address=\"10.1016/j.envpol.2019.01.046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9379295/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9379295/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnvironmental pollution from polycyclic aromatic hydrocarbons (PAHs) poses a significant threat to ecosystems due to their mutagenic and carcinogenic properties. This study focused on the isolation and characterization of a novel bacterial strain, Achromobacter sp. B10c\u0026rsquo; from contaminated agricultural soil in Chengdu, China, to evaluate its phenanthrene-degrading potential and its effect on alfalfa (\u003cem\u003eMedicago Sativa\u003c/em\u003e) plant growth. The 16S rRNA gene sequencing identified the strain with 99.42% identity to other Achromobacter species. Under optimized environmental conditions (pH 7.0 and 30\u0026ndash;35\u0026deg;C), Achromobacter sp. B10c\u0026rsquo; achieved a maximum phenanthrene degradation rate of 88.33% within 72 hours. Metabolic analysis using GC-MS identified tetrachloroethane and phthalic acid as the primary degradation intermediates.\u003c/p\u003e \u003cp\u003eIn vivo plant studies revealed that phenanthrene stress (up to 200 mg/L) significantly reduced alfalfa growth parameters, including shoot and root length, biomass, and chlorophyll content. However, inoculation with Achromobacter sp. B10c significantly mitigated these toxic effects, increasing root and shoot lengths by 48% and 35%, respectively, in contaminated treatments. Furthermore, the bacterial strain reduced oxidative stress, evidenced by decreased Malondialdehyde (MDA) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels, while simultaneously enhancing the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). These results suggest that Achromobacter sp. B10c is a highly efficient candidate for the bioremediation of PAH-contaminated sites and can serve as a plant growth-promoting agent under environmental stress.\u003c/p\u003e","manuscriptTitle":"Aerobic Bacterium ‘achromobacter Sp. B10c’ Having Traits to Degrade Phenanthrene and Can Improve Physiology of Alfalfa Plant","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 07:52:23","doi":"10.21203/rs.3.rs-9379295/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dc620463-eeef-4c61-a56e-ec5cc457b4bc","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T07:52:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 07:52:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9379295","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9379295","identity":"rs-9379295","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}