_____________________________________________________________________________________________________ *Corresponding author: E-mail: saad.el-din.hassan@umontreal.ca, saad_botany_22@yahoo.com;

Journal of Advances in Biology & Biotechnology 4(4): 1-12, 2015; Article no.JABB.23613

ISSN: 2394-1081

SCIENCEDOMAIN international www.sciencedomain.org

Biodegradation of Phenanthrene by Klebsiella sp Isolated from Organic Contaminated Sediment

Saad El Din Hassan1*, Said E. Desouky1, Amr Fouda1, Mamdouh S. El-Gamal1

and Ahmed Alemam1

1Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, 11884, Nasr City, Cairo, Egypt.

Authors’ contributions

This work was carried out in collaboration between all authors. Authors SEDH and SED designed the

study and wrote the protocol. Authors SEDH, SED and AF managed the analyses of the study. Authors SED, AF and AA performed the experiments. Author MSEG managed the literature review.

Author SEDH wrote the first draft of the manuscript and performed the statistical analysis. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/JABB/2015/23613

Editor(s): (1) Laura Pastorino, Laboratory of Nanobioscience and Medical Informatic, Dept. Informatics, Bioengineering, Robotics and

Systems Engineering (DIBRIS), University of Genoa, Italy. Reviewers:

(1) Opeyemi Uwangbaoje Lawal, Evangel University Akaeze, Ebonyi State, Nigeria. (2) Volodymyr Chernyshenko, Palladin Institute of Biochemistry, Ukraine.

(3) Shruti Murthy, Bangalore University, India. Complete Peer review History: http://sciencedomain.org/review-history/12806

Received 10 th December 2015 Accepted 24 th December 2015

Published 28 th December 2015

ABSTRACT

Aims: This study report the ability of some bacterial strain to degrade the phenanthrene which is a polycyclic aromatic hydrocarbon and to optimize the condition controlling the phenanthrene biodegradation. Study Design: Collect different petroleum contaminated sediment samples, isolate phenanthrene-degrading bacteria on media supplemented with phenanthrene as a unique carbon source, select and identify the most potent bacterial strain. Finally, some of the most biotic and abiotic parameters were optimized to enhance the bacterial biodegradation of phenanthrene. Place and Duration of Study: The study was performed in Microbial Physiology Lab, Botany & Microbiology Department, Faculty of Science, Al-azhar University, from October 2013 until April 2014. Methodology: Various bacterial strains were isolated on media with phenanthrene (400 ppm) as sole carbon source. The most potent bacterial strain was selected on the basis of their capacity to

Original Research Article

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degrade phenanthrene. Different factors such as pH, temperature, inoculum size, shaking and static condition which affect on the biodegradation of phenanthrene were studied depending on estimation the biodegradation ratio of phenanthrene. Results: The most potent bacterial strain (SB_2.1) which showed the highest biodegradation ratio of phenanthrene was identified as klebsiella sp. on the basis of morphological, physiological, and biochemical characteristics. The results revealed that the optimum parameters for maximum degradation of phenanthrene were observed after 21 days at pH value of 7.0 and temperature of 30°C with shaking speed of 150 rpm and at 2% inoculum concentration. The results showed that, this strain of klebsiella sp degraded approximately 84.9% of phenanthrene at an initial concentration of 400 mg/L under the optimal conditions.

Keywords: Polycyclic aromatic hydrocarbons; phenanthrene; Klebsiella sp.; biodegradation;

bioremediation. ABBREVIATIONS PAHs: Polycyclic Aromatic Hydrocarbons, EPA: Environmental Protection Agency, MHA: Mueller-Hinton Agar, BH: Bushnell-Haas. 1. INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are a group of organic pollutants which occur into the environment from different sources mainly caused by the anthropogenic activities. At present, PAH have become ubiquitous pollutants in soils at oil refineries, gasworks, wood preservation plants, runoff from combustion process [1]. PAHs have attracted particular research attention for decades because of their prolonged persistence, recalcitrance, potential mutagenic and carcinogenic properties [2,3]. The US environmental protection agency (EPA) has listed sixteen PAHs as particularly hazardous agents due to their carcinogenic and mutagenic properties [4]. Among these agents, phenanthrene which is a three ring constituent of coal derivatives and oil fuels and considered as one of the prior pollutants. It has been reported that phenanthrene acts as a human skin photosensitizer, a mild allergen and also a mutagen in bacterial systems [5]. The low water solubility of phenanthrene makes its biodegradation difficult. Phenanthrene has been tested as a model compound to study the potential of microbial transformation, degradation of PAH [6,7]. More researches have been carried out to find the ways and means to remediate PAHs contaminated sites [8,9]. In the contaminated environment, these pollutants undergo transformations involving both biotic and abiotic processes, such as volatilization, adsorption, photolysis, chemical oxidation and microbial degradation [10]. Microbial degradation is a

major process for the successful removal and elimination of PAHs from the environment since it is an efficient, inexpensive, and environmentally safe cleaning method [6]. Bacteria isolated from PAHs polluted environments have shown high biodegradation rates during their application in microbial bioremediation [11]. Hence, the considerable attention has been turned toward the isolation and characterization of PAH degrading bacteria [12]. As a consequence, large number of bacteria with biodegradation ability of PAHs has already been identified [13], phenanthrene degrading bacteria have been reported [6], but the success of bioremediation depends on different biotic and abiotic factors such as bacterial population size, temperature and pH [14]. Although many genera of bacteria have the ability to degrade these recalcitrant compounds and use them as a source of carbon and energy, such phenomena are not commonly encountered in enteric bacteria [15]. The enteric bacteria belong to enterobacteriaceae family are mainly regarded as inhabitants of animal guts [15]. The ability of this group to degrade high molecular weight PAH compounds appears to be an unusual feature, as this phenomenon has been associated with typical soil bacteria. However, very few reports have indicated utilization of aromatic compounds by enterobacteria, particularly those of the genera Klebsiella, Enterobacter, Escherichia, and Hafnia [15-17]. Although there are several reports of bioremediation of high molecular weight of PAHs, research pertaining to biodegradation of these substances by enteric bacteria has been

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relatively rare [15,18]. In addition, there are only few details on the application of these microbes for field bioremediation. Thus, much effort is being made in exploiting this cost effective process by exploring naturally existing diverse bacterial communities ending up with non-hazardous end product in in situ field bioremediation [19]. There are many industrial areas produce large amount of PAHs which expose to the environment and affects human health and aquatic ecosystems and become a severe hazard. Therefore, there is a really requirement to remove PAHs contamination from the environment. One of the most effective and efficient way to remove this contamination is bioremediation by using selected bacteria locally isolated from the contaminated soil that have high capability to degrade PAHs. Although a diverse range of phenanthrene degrading bacterial species and strains have already been isolated, less attention refer toward their capabilities in adjusting different environmental condition through the environmental application of bacterial strain in bioremediation of PAHs. The objectives of the present study were be to isolate and identify phenanthrene-degrading bacteria from organic contaminated sediment, and to evaluate the factors affecting on phenanthrene biodegradation in order to improve the bacterial degradation capacity of phenanthrene. 2. MATERIALS AND METHODS 2.1 Sampling

Three samples were collected from contaminated sediment of Gharbia drain in the Nile Delta, Egypt for isolation of the phenanthrene-degrading bacteria. The samples were collected at summer season where the temperature range was 38-40°C, and the samples were taken from a distance of 20 cm after taking out nearly 5 cm of the soil superficial. The samples were retained in sterile polyethylene bags, closed strongly and stored at 4°C for further study. 2.2 Culture Media and Reagents Phenanthrene (fulka) was obtained from Sigma Aldrich Chemical Co. with a purity > 90% and all other chemicals which employed in this experiments were at analytical grade. The components (g / l) of Bushenll-Haas (BH) medium or mineral media are MgSO4 (0.2), CaCl2 (0.02), KH2PO4 (1), (NH4)3PO4 (1), KNO3

(1) and FeCl3 (0.05), distilled water was added up to 1000 ml/L, and agar 20 g/l was added for media solidification [20]. The pH of the medium was adjusted to 7.0 before sterilization. Nutrient Agar medium consists of (g/l) beef extract (3), peptone (5), NaCl (5) and 20 g/l of agar [21]. pH adjusted to 7.0 before sterilization. 2.3 Isolation and Screening of

Phenanthrene-degrading Bacteria

Bacterial isolation was carried out as described by Zohreh [22] with slight modification as follows: BH broth medium was first cooked and sterilized at 121°C and 1.5 atmospheric pressure for 20 min, this medium was then allowed to cool to 45°C and supplemented with phenanthrene as a unique carbon source. One g of each sediment was inoculated into the 250 mL volume Erlenmeyer flask containing 100 ml BH broth media supplemented with 100 ppm phenanthrene as initial concentration. This medium was incubated at 37°C under shaking (150 rpm) for 7 days. After the incubation period, 1 ml of the previous BH broth media inocubated into new BH agar media supplemented with 100 ppm of phenanthrene to obtain separate colony. Each separate colony was streaked on BH agar media containing 100 ppm phenanthrene for obtaining a pure isolate which preserved in nutrient agar slant for further study. Ten purified isolates were screened on solid media supplemented with different phenanthrene concentrations of 100, 200, 300, 400, 500, and 600 ppm to detect the most potent phenanthrene-degrading bacteria. From the screening profile, the bacterial isolate of SB_2.1 was selected as the most potent bacterial isolate because of its ability to grow on phenanthrene -supplemented media till phenanthrene concentration of 500 ppm. 2.4 Identification of Phenanthrene-

degrading Bacterium The phenanthrene-degrading isolate of SB_2.1 was examined morphologically for colonal characterization such as color and shape on nutrient agar media followed by Gram’s staining. Physiological and biochemical tests were conducted according to Bergey’s Manual of Determinative Bacteriology [23] to identify he phenanthrene-degrading bacterium. Antibiotic susceptibility testing was determined by disc diffusion method on Mueller-Hinton Agar (MHA) plates against the following antibiotics:

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Neomycin, Ampicilin, Cefoperazone/sulbactam, Penicillin G, Ciprofloxacin, Chloramphenicol, Tetracycline, and Refambicin. Briefly, a standard inoculum was prepared by adjusting the bacterial suspension in nutrient broth to final optical density of 0.5 McFarland units (OD620 = 0.08-0.1) and spread evenly onto prepared plates. Plates were kept at room temperature for 10 min. Antibiotic discs were mounted and plates were incubated at 4°C for 2 hr. to allow the diffusion of antibiotics, then all plates incubated overnight at 35°C for 18 hr. After incubation time, the resulted inhibition zone for each tested concentrations of tested antibiotics was observed and the diameter of the zone was measured [24]. 2.5 Preparation the Bacterial Inoculum of

SB_2.1

The most potent bacterial isolate was inoculated in a 250 mL Erlenmeyer flask containing 150 mL nutrient broth, incubated at 37°C under shaking condition (150 rpm) till the growth reached late exponential phase (20 h). Cells were harvested by centrifugation at 5000 rpm for 10 min. The cell pellets were washed with 0.85% normal saline and finally suspended in the same buffer to obtain a cell suspension with an absorbance (A620) of 1.0 and that cell suspention was used as the inoculum as previously mentioned by Zhou et al. [25]. 2.6 Quantitative Assay of Phenanthrene

Degradation Quantitative assay of phenanthrene was carried out as described by Marta et al. [26] by using

hexane as an organic solvent. Briefly, 10 mL of culture was extracted with 10 mL of hexane in a separator funnel, by mixing for two minutes. The resulting organic phase containing phenanthrene was measured at OD275 nm using UV-VISIBLE spectrophotometer (Jenway -6305). The remaining phenanthrene concentrations in the cultures were calculated from the standard curve (r2 = 0.972) as shown in (Fig. 1). Degrading efficiency was calculated using the equation as follows:

Degradation % = (Ci – Cf / Ci) × 100%

Where Ci initial concentration of phenanthrene and Cf final concentration of phenanthrene. 2.7 Effect of Abiotic Factors on

Phenanthrene Degradation 2.7.1 Effect of static and shaking incubation

conditions The experiment was carried out in 250 ml Erlenmeyer flasks containing 100 ml of BH broth medium supplemented with 400 mg/l of phenanthrene as a sole source for carbon and energy. The stock solution (10000 ppm) was prepared by dissolving phenanthrene in ethanol and transferred respective volume into the sterilized flasks and allowed to evaporate ethanol in the laminar hood flow under sterilized condition. Each flask was inoculated by 1 ml of aliquot taken from bacterial inoculum of SB_2.1 as previously mentioned and allowed to grow for one month, at 35°C under static condition. Abiotic controls were prepared without

Fig. 1. Standard curve of phenanthrene from culture media

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inoculation of bacterial isolate, which kept under similar conditions. Inoculated flasks and controls were prepared in triplicate. Above experiment was repeated under shaking condition (150 rpm). Samples were collected at regular intervals of time (3 days) up to 30 days and analyzed for residual phenanthrene as described in section 2.6 to calculate phenanthrene degradation ratio. 2.7.2 Effect of pH and temperature

The effect of pH and temperature on phenanthrene biodegradation by the most potent bacterial isolate was monitored in BH broth media containing 400 mg/l of phenanthrene as a sole carbon source, where 25 mL of sterilized BH broth media was adjusted at different initial pH of 4, 5, 6, 7, 8 and 9, subsequently added into individual flasks. Then each flask was inoculated with (1%, v/v) of aliquot taken from SB_2.1 bacterial inoculum incubated at 35°C on a shaker with the speed of 150 rpm. The same experimental condition was repeated at pH 7.0 and incubated at different temperature of 20ºC, 25ºC, 30ºC, 35ºC, 40ºC and 45ºC. In each experiment and after 21 days of incubation period, the phenanthrene degradation ratio was estimated. 2.7.3 Effect of the inoculum size

The effect of bacterial inoculum size on phenanthrene biodegradation by the most potent strain of SB_2.1 was monitored in BH broth media containing 400 mg/l of phenanthrene as a sole carbon source, 25 mL of sterilized BH broth media was inoculated with different inoculum size of 0.5, 1, 1.5, 2, 2.5, and 3% (v/v), at pH 7 and 30°C, following the same procedure as previously mentioned above. After 21 days of incubation period, the remaining phenanthrene in the medium was quantified.

2.8 Phenanthrene Biodegradation under

the Optimum Conditions Biodegradation experiment was conducted in 250 ml Erlenmeyer flasks containing 100 ml of BH broth media supplemented with 400 mg/l of phenanthrene as a sole source for carbon or energy. The optimum conditions for the highest phenanthrene biodegradation were as the following: pH value of 7.0, temperature at 30°C, inoculation size (2%, v/v) of the bacterial SB_2.1 cell suspension, under shaking speed of 150 rpm, and incubation period of 21 days. The control treatment was prepared at the same

conditions without any bacterial inoculation. Inoculated flasks and controls were prepared in triplicate. After incubation period, 10 mL aliquot from each flask was taken to measure the phenanthrene degradation ratio. 2.9 Statistical Analysis It was necessary to determine the optimization criteria for biodegrading of phenanthrene, therefore, different pH, temperature, inoculum concentration, as well as shaking vs static at different incubation period experiments were performed. Data then analyzed using a two-way model of analysis of variance (ANOVA) to determine the significance between treatments. Also a two-way repeated measure model analysis of the variance (ANOVA) was used to determine the significance between the treated and untreated (control) samples, across the different time points, where the measured were estimated from the same flask. Assumptions of normality were assessed using Shapiro-Wilk’s test while homogeneity of variances was determined using Levene’s test. Data transformation was performed prior to running the ANOVA models. All pairwise-multiple comparisons were performed using Holm-Sidak test. This test is more powerful to detect differences than Tukey’s and Bonferroni’s tests and is recommended as the first line procedure for most multiple comparisons testing (Systat Software, 2011) [27]. SigmaPlot® 12.5 software extended with a statistical package and Graphs were plotted in Microsoft™ Excel® 2013 was used. The graphed values are represented as means and error bars. The error bar represents the standard error means calculated from standard deviations. 3. RESULTS AND DISCUSSION 3.1 Isolation and Identification During this study, ten purified bacterial strains were isolated from three contaminated sediment samples obtained from the Gharbia drain in the Nile Delta, Egypt. These purified isolates had the ability to tolerate high concentration of phenanthrene. In order to select the most potent strain, isolates subjected to different concentrations of phenanthrene on solid media. Data recorded in (Table 1) showed that, seven isolates were Gram negative while three isolates were Gram positive. All ten isolates had the ability to grow on 100 ppm of phenanthrene (the concentration which used for bacterial isolation),

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while seven isolates grew on phenanthrene concentration up to 200 ppm. At 300 ppm of phenanthrene, only four isolates grew on this concentration. The most potent isolate (Code of SB_2.1) had the ability to grow till 500 ppm of phenanthrene; therefore, all experiments were done at 400 ppm (sub lethal dose). The most post potent strain identified by conventional methods. The morphological characters of the most potent bacterial colony on the solid plates was ivory, non-transparent, smooth, and wet with a regular edge, and the margin of the colonies was thin and undulates. After incubation at 30°C for 1 day on solid plates, colony was hemispherical, smooth, semitransparent, and wet with a regular edge. Furthermore, the bacterium was Gram-negative, rod-like, occurring in single and pairs. According to the physiological and biochemical characteristics and instructions from “Bergey’s manual of systematic bacteriology” [23], the most potent strain of SB_2.1 showed high similarity with Klebsiella sp. and thus was identified as Klebsiella sp. Moreover, Klebsiella sp. was tested for antibiotics susceptibility against different antibiotics for approving the identification according to Klebsiella sp. Taxonomy [28]. Many genera and species of enterobacteriace also have typical patterns of resistance and susceptibility to antibiotics, thus the antibiogram patterns of bacterial isolates can also be used as an aid for identification [23]. The antibiotics susceptibility test showed that the zone of inhibition was observed against penicillin, ampicillin, cefoperazone/sulbactam, tetracycline and refambicin antibiotics whereas neomycin, ciprofloxasin and chloramphenicol showed no zone of inhibition (Table 2). Moreover, some strains of the genus Klebsiella sp. which belong to enterobacteriaceae have been reported with the phenanthrene-degradation ability [29,30].

3.2 Effect of Abiotic Factors on Phenanthrene Degradation

The transformation of PAH compounds in the environment is mainly carried out through microbial processes, but there are number of environmental factors affect on the potential degradation of PAH by bacteria [31]. Efforts for degradation of PAHs from environments by bacteria require assessment the concentration of the toxic compounds and the rate at which these compounds will be degraded. Equally important would be considered to understand the environmental factors influencing the degradation process. Therefore, in the present study, the effect of pH, temperature, inoculum size and different incubation conditions such as incubation period, static and shaking were evaluated to obtain the maximum degradation ratio of phenanthrene. The measured remaining phenanthrene and degrading ratio in the medium at regular intervals of time (3 days) up to 30 days from each flask under shaking and static condition are depicted in (Figs. 2A, B). According to the results, it is apparent that the Klebsiella sp. (SB_2.1) showed degradation about 68% of the phenanthrene within 21 day under shaking condition, while under static condition only 62% of phenanthrene degraded at the same time. The maximum degradation (68%) was obtained under shaking condition at 150 rpm because of phenanthrene dissolution and the availability of oxygen increased under shaking condition and thus resulted in increased the rate of degradation. These results differed with Lily et al. [32] which was reported that the ability of Bacillus subtilis BMT4i which started the degrading of Benzo[a]Pyrene (BaP) after 24 hours and continued up to 28 days achieving the maximum degradation of approximately 84.7%.

Table 1. Screening the purified bacterial isolates on phenanthrene biodegradation

Bacterial code

Gram reaction

Phenanthrene concentration (ppm) 100 200 300 400 500 600

SB_1.1 ve+ + + - - - - SB_1.2 ve- + + - - - - SB_1.3 ve- + - - - - - SB_1.4 ve+ + + + - - - SB_2.1 ve- + + + + + - SB_2.2 ve- + + - - - - SB_2.3 ve- + + + - - - SB_3.1 ve+ + + + - - - SB_3.2 ve- + - - - - - SB_3.3 ve- + - - + refer to the ability of bacterial isolate to grow on medium supplemented with phenanthrene as a sole carbon

source. ve+ refer to Gram positive bacteria, ve- to Gram negative bacteria

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Also, Masakorala et al. [33], reported that, Pseudomana sp.USTB-RU have the ability to degrade 86.5% of phenanthrene at an initial concentration of 100 ppm in 8 days. The present study suggested that Klebsiella sp. has the ability to degrade high concentrations of phenanthrene and has potentially useful in bioremediation application of PAHs. Table 2. Effect of different antibiotics against

Klebsiella sp. (strain SB_2.1)

Antibiotics Abbreviation Zone of inhibition (mm)

Neomycin N 16 (S) Ampicilin AM 0 (R) Cefoperazone/ sulbactam

CES 0 (R)

Penicillin G P 0 (R) Ceprofloxasin CIP 18 (S) Chloramphenicol C 15 (S) Tetracycline TE 0 (R) Refambicin RF 0 (R)

S: sensitive, R: resistance The following study showed the effect of pH and temperature on phenanthrene biodegradation by Klebsiella sp. Various of phenanthrene degradation tests were carried out at different pH from 4 to 9 and temperatures from 20 to 45°C. As shown in (Figs. 3A, B), the optimal conditions for Klebsiella sp. (SB_2.1) to give the maximum degradation (66%) was obtained at pH 7.0. The ratio of phenanthrene degradation increased to (76%) at 30°C under the optimum pH of 7.0 and shaking sate of 150 rpm. Previous researchers [1,34] have reported that the optimum conditions for phenanthrene degrading by different bacterial strains at pH 7.0 and temperature of 30°C.

Although, the solubility of phenanthrene increases with the rising temperature in the medium [35], but Klebsiella sp. (SB_2.1) growth reduced at high temperature, highly acidic pH and highly basic pH. The negative impact of these conditions on the enzymatic activity which is vital for growth might be the reason for the observed reduction in growth. In order to find out the optimum inoculum size of Klebsiella sp. (SB_2.1) which needed for faster and higher degradation percentage, the degrading ability was tested at different inoculum concentrations starting from 0.5% to 3% (v/v) (Fig. 4). The rate of degradation increased with increase the inoculum size, reaching maximum value at 2% (v/v). During the Klebsiella sp. growth, lag period decreased by increasing the inoculum size and resulted in higher phenanthrene degradation (78%). As the inoculum of Klebsiella sp. was increased above 2%, it resulted in decreasing degradation and that is in agreement with Abdelhay et al. [36]. Also, Chen et al. [37] reported that, inoculum size was the key factor affecting the speed of phenanthrene biodegradation by Sphingomonas sp. isolated from mangrove sediment. 3.3 The Optimum Condition of

Phenanthrene Biodegradation The rate of biodegradation is influenced by pH, temperature, inocula size, and incubation condition [38,39]. Therefore, biodegradation of phenanthrene by Klebsiella sp. (SB_2.1) was studies by providing those critical factors at the optimum level. The results showed that increasing the biodegradation ratio from 62% at starting experiment to reach 84.9% under the optimum condition within 21 days (Table 3).

Table 3. Phenanthrene degradation by Klebsiella sp. (strain SB_2.1) under the optimum

conditions

Before optimization1 After optimization2 Control3

Klebsiella sp. (strain SB_2.1)

Control

Klebsiella sp. (strain SB_2.1)

Remaining concentration of phenanthrene (mg/l)

387.2±10.34 147.2±2.93b 399.7±3.64 60.3±5.09 a

Degradation ratio (DR) of phenanthrene (%)

0.032 62.0b 0.00075 84.9a*

1Biodegradation of phenanthrene before optimization at pH of 7.5, and temperature of 35°C. 2Biodegradation of phenanthrene after optimization at pH of 7.0, and temperature of 30°C. 3Control (without bacterial inoculation).

Different letters represent the significance between phenanthrene biodegradation by Klebsiella sp. (strain SB_2.1) after and before the optimization. * denote the highest degradation ratio recorded after 21 days of

incubation which significantly different (Holnm-Sidak’s test, DR=84.9%, p= 0.001)

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Fig. 2A. Effect of different incubation times on the phenanthrene biodegradation of by Klebsiella sp. (strain SB_2.1) under shaking (150 rpm) condition

Different letters represent the significance between the presence and absence of the bacterial inoculum at each time of incubation. * denote the highest degradation ratio recorded after 21 days of incubation which significantly

different (Holnm-Sidak’s test, DR=68 %, p= 0.001)

Fig. 2B. Effect of different incubation times on the phenanthrene biodegradation of by Klebsiella sp. (strain SB_2.1) under static condition

Different letters represent the significance between the presence and absence of the bacterial inoculum at each time of incubation. * denote the highest degradation ratio recorded after 21 days of incubation which significantly

different (Holnm-Sidak’s test, DR=62 %, p= 0.001) Analyses of control samples after every 21 days revealed the presence of 95–98% of phenanthrene in the medium, indicating the negligible elimination of phenanthrene during the experiment as previously reported by

Pedetta et al. [6]. Thus, our results implied that the Klebsiella sp. (SB_2.1) has a potential role to efficiently degrade high concentration of phenanthrene under the given conditions.

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Fig. 3A. Effect of different pH value on biodegradation of phenanthrene by Klebsiella sp. (strain SB_2.1)

Different letters represent the significance between the presence and absence of the bacterial inoculum at each time of incubation. * denote the highest degradation ratio recorded at pH 7 which significantly different (Holnm-

Sidak’s test, DR=66 %, p= 0.001)

Fig. 3B. Effect of different temperature on biodegradation of phenanthrene by Klebsiella sp. (strain SB_2.1)

Different letters represent the significance between the presence and absence of the bacterial inoculum at each time of incubation. * denote the highest degradation ratio recorded at 30°C which significantly different (Holnm-

Sidak’s test, DR=76%, p= 0.001)

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Fig. 4. Effect of inoculum size on phenanthrene degradation by Klebsiella sp. (strain SB_2.1) Different letters represent the significance between the presence and absence of the bacterial inoculum at each time of incubation. * denote the highest degradation ratio recorded at inoculum 2% which significantly different

(Holnm-Sidak’s test, DR=78%, p = 0.001) 4. CONCLUSION The data presented here suggested that the biodegradation of phenanthrene by klebsiella sp. (strain SB_2.1) appeared to be feasible to remediate phenanthrene-rich contaminated sites. The specific growth of klebsiella sp. (strain SB_2.1) and the high phenanthrene degradation ratio by this strain throughout the studied phenanthrene concentration (400 ppm) suggested that this strain had efficient capacity in phenantherine degrading. The highest degradation ratio of phenanthrene (400 ppm) by Klebsiella sp. (strain SB_2.1) was 84.9%. The optimal conditions to reach that maximum ratio at pH value of 7.0, temperature of 30°C, and under shaking condition with inoculum size of 2%. This study also shows a new acumen in employing klebsiella sp. (strain SB_2.1) in bioremediation of polycyclic aromatic compound.

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