Isotope fractionation (δ13C, δ15N) and microbial community response in degradation of petroleum hydrocarbons by biostimulation in contaminated soil

This study investigated the isotope effects of δ13C and δ15N and microbial response during biodegradation of hydrocarbons by biostimulation with nitrate or compost in the petroleum-contaminated soil. Compost and KNO3 amendments promoted the total petroleum hydrocarbon (TPH) removal accompanied by a significant increase of Actinobacteria and Firmicutes phyla. Soil alpha diversity decreased after 90 days of biostimulation. An inverse significant carbon isotope effect (εc = 16.6 ± 0.8‰) and strong significant nitrogen isotope effect (εN = −24.20 ± 9.54‰) were shown by the KNO3 supplementation. For compost amendment, significant carbon and nitrogen isotope effect were εc = 38.8 ± 1.1‰ and εN = −79.49 ± 16.41‰, respectively. A clear difference of the carbon and nitrogen stable isotope fractionation was evident by KNO3 or compost amendment, which indicated that the mechanisms of petroleum degradation by adding compost or KNO3 may be different.


Introduction
Petroleum is a mixture of various hydrocarbons including aliphatic and aromatic fractions. The exploitation, transportation, and application of crude oil and petroleum products may cause petroleum to seep into the soil which changed physical and chemical properties of soil and led to great damage to the ecological environment around the world (Wang et al. 2016;Shahi et al. 2016a;Cho et al. 2019;Mahmoud and Bagy 2019). Alexander (2000) have confirmed that some small molecular weight hydrocarbons can be biodegraded gradually under natural conditions, while the high molecular weight hydrocarbons may stay in the soil for a long time. Due to the slow degradation of petroleum hydrocarbons in the process of natural attenuation, it is particularly important to stimulate the activity of autochthonous microbial consortia using various amendments, which provides an efficient biostimulation strategy with less disturbance to the soil microecosystem.
Biostimulation, as a promising remediation strategy by enhancing activities of the native microorganisms in soil, has been widely concerned and applied in the past few decades (Meynet et al. 2012;Karthick et al. 2019;Onotasamiderhi et al. 2019;Xu et al. 2019). It is generally believed that the activities of indigenous microorganisms are limited by oxygen, water, nitrogen, phosphorus, and other nutrients (Schjønning et al. 2011;Ali et al. 2016;Liu et al. 2020a). Remediation of soil by supplementing stimulators such as nitrogen, phosphorus, compost, and bulking agent is a common and effective method for contaminant removal. Among all the amendments, inorganic salts containing nitrogen and phosphorus are widely applied in remediation of oil-contaminated soil (Abed et al. 2015;Shahi et al. 2016b;Wu et al. 2019). In addition, compost amendments have shown good effects in stimulating indigenous microorganisms to degrade petroleum hydrocarbons and were considered to have great development potential in the future due to the characteristic of recycling waste (Dadrasnia et al. 2016;Huang et al. 2019 The stable isotope technology, originated from the application in geology and geochemistry, has played an important role in illustrating the carbon and nitrogen isotope fractionation effects in biodegradation of organic pollutants (Fan et al. 2019;Fiorentino et al. 2019). Carbon isotope fractionation occurred when the lighter carbon atom ( 12 C) was involved in the cleavage of C-H chemical bond earlier than those of heavier carbon ( 13 C) during degradation of hydrocarbon compounds. Since petroleum mainly consisted of saturated and aromatic hydrocarbons and asphaltenes, the carbon isotope fractionation effects were inconsistent among different fractions of petroleum hydrocarbons with various molecular weights and structures (Bloom et al. 2000). Previous study has shown that microorganisms drive the 13 C/ 12 C isotope fractionation of hydrocarbons with different molecular weights (Stahl 1980). Li et al. (2018a, b) revealed that the carbon isotope fractionation of polycyclic aromatic hydrocarbons (PAHs) can be used to distinguish the degradation degree of PAHs in crude oil and fuel oil. For nitrogen isotope, more attention is focused on the fractionation effect of nitrogen in nitrates which are widely used as inorganic nitrogen source. Hu et al. (2020) compared the nitrogen isotope fractionation effects of nitrate (NO 3 − ) between the field constructed wetland and the laboratory-simulated denitrification process and concluded that the organisms in the wetland had a greater influence on the 15 N isotope fractionation than that in the laboratory simulation. There were significant differences in carbon isotope fractionation effects for various types of hydrocarbons when environmental conditions such as oxygen, moisture, and nutrition levels changed (Tyler et al. 1994). Previous study illustrated that the 13 C enrichment factor for aromatic hydrocarbons was 1.3 greater in aerobic than in anaerobic conditions, which was caused by the specific enzymes involved in the biodegradation of benzene, toluene, and xylene (Meckenstock et al. 2004). Although the influences of temperature, soil properties, and the contents of soil organic matter on carbon isotope effects of organic pollutants have been well elucidated by many research, few studies have been carried out to investigate the carbon and nitrogen isotope effects during biostimulation remediation of oil-contaminated soil presently.
In this study, three bioremediation methods including enhancing soil humidity, adding inorganic salt compounds and organic compost, were applied to promote petroleum hydrocarbon degradation in an aged pollution soil. The degradation rates of petroleum hydrocarbon, microbial community structures, and isotope effects of 13 C and 15 N during remediation were investigated simultaneously. This work provided the intrinsic relevance between petroleum hydrocarbon removal and the effects of imported nutrition in soil.

Soil and compost
Oil-polluted soil samples were collected from the north of Shaanxi province, one of the main oil exploration areas in China, and the soil had a long pollution history over 5 years. According to previous methods (Wu et al. 2019), the samples obtained from the topsoil of contaminated site were mixed evenly and then packed in sterilization bags for storage at low temperature and transported to the lab. Results of soil property determination are shown in Table 1. The decayed organic fertilizer used in the study was made from pig manure:rice husks with a dry mass ratio of 1:2 and 5.0% charcoal slag heap. The organic compost properties were as follows: N, 20400 mg kg −1 ; P, 6500 mg kg −1 ; K, 36200 mg kg −1 ; Na, 3810 mg kg −1 ; Ca, 21300 mg kg −1 ; Mg, 2420 mg kg −1 ; Al, 1330 mg kg −1 ; S, 1575 mg kg −1 ; Cu, 400 mg kg −1 ; Fe, 1615 mg kg −1 ; and Mn, 284 mg kg −1 .

Biostimulation
Three treatments included the following: (1) CK (control check): addition of 100 mL deionized water to 0.8-kg soil to increase soil moisture content by 15%; (2) MS (mineral salt): amendment of 11.8-g KNO 3 to adjust the ratio of C:N to 100:10 in the 0.8-kg soil; (3) SC (soil compost): addition of 11.8-g compost to 0.8-kg soil. All the biostimulation remediation experiments were carried out in flowerpots and made in triplicate at room temperature for 90 days. In addition, the soil was agitated with sterilized glass rods every 3 days so that the remediation could be performed under aerobic conditions.

Determination of TPH, saturate, and aromatic fractions
Determination of TPH, saturate, and aromatic fractions in soils was based on the methods of Wu et al. (2020). In brief, 1 g of soil was added to 15 mL mixed solvent of n-hexane and Nitrate nitrogen (mg kg −1 ) 20.13 ±0.54 a TPH, total petroleum hydrocarbon methylene chloride with the volume ratio of 1:1 in a weighing bottle of known weight, and the petroleum hydrocarbons in soil were extracted for 10 min by an ultrasonic processor (JY96-IIN, Ningbo Science Biotechnology CO. LTD, China). Then, the mixture was centrifuged at 8000 r min −1 for 10min. The extraction and centrifugation processes were repeated three times and mixed to obtain total petroleum hydrocarbon (TPH) from the soil. Finally, the whole extracts were air-dried and total petroleum hydrocarbon was determined by gravimetric method according to the difference in the weight of the weighing bottle (Mishra et al. 2001). The saturate and aromatic fractions of TPH were obtained according to the description by Wu et al. (2020). Briefly, 5 mL of nhexane was added to the TPH and transferred to the Super Flash Alumina Neutral columns (SF 15-24 g, 20.8 × 112 mm, Agilent Technologies), which was repeated three times and then placed in a fume hood to obtain saturate components. The aromatic components were obtained by adding benzene to the remainders in the Super Flash Alumina Neutral columns, subsequently. The saturate and aromatic fractions were respectively quantified gravimetrically (Gao et al. 2019

Soil total carbon and nitrogen contents, carbon and nitrogen stable isotopes detection
The contents of total carbon and nitrogen, the values of δ 13 C and δ 15 N in soils were determined by elemental analyzer (Vario PYRO cube, Elementar Co., Hanau, Germany) and isotope ratio mass spectrometer (IsoPrime IRMS, Elementar Co., Hanau, Germany), respectively. In detail, 20-mg soil was air-dried, ground, and enclosed in tin foil. Then, they were fully burned at a high temperature of 1120°C and the contents of elements and isotopes were obtained based on the CO 2 and NO X generated. Throughout the measurement process, pure carbon dioxide and nitrogen were used as reference gasses, and helium was used as carrier gas. In addition, isotopic data were calculated according to the following equations: in which R meant ratio of heavier isotope ( 13 C, 15 N) to lighter isotope ( 12 C, 14 N). Besides, Vienna Pee Dee Belemnite and N 2 were defined as the standard with an R value of 0.
The carbon isotope enrichment factors (ε c ) in the CK, MS, and SC-treated samples were respectively calculated by the following equation: where δ 13 C 0 and δ 13 C t are carbon isotope ratios at the beginning and at t days of the experiment, respectively. The letter f represented the ratio of the total carbon content at t days to the initial total carbon content in the soils. Similarly, nitrogen isotope enrichment factors (ε N ) can be obtained using the following equation:

Data analysis
All the data representing the physical and chemical properties of soil appeared in the form of mean ± standard deviation (SD) based on three replicated experiments. Origin software (version 9.0, China) was used to analyze carbon and nitrogen isotope effects in soils during 90 days of remediation. The difference among biostimulation treatments was significant for p values < 0.05.

Results and discussion
Effects of biostimulation on TPH, saturates, and aromatics degradation The degradation tendency of petroleum hydrocarbons during 90 days of biostimulation remediation is shown in Fig. 1. Addition of deionized water, mineral nutrition, and compost reduced the petroleum hydrocarbon contents from 24166 to 21438, 18480, and 18232 mg kg −1 soil, which represented 11.29%, 23.53%, and 24.55% of aged hydrocarbon removal, respectively (Fig. 1a).
Compost or KNO 3 amendment was more effective for stimulating the activities of indigenous microorganisms than that of deionized water. In our previous studies, hydrocarbon removal rates were 16% and 18% after 10 and 8 weeks of remediation by 20% soil moisture contents, respectively (Wu et al. 2019;Wu et al. 2017). Nwankwegu et al. (2016) reported that the organic compost as the source of nutrients and bulking agent promoted 93.31 ± 3.98% of diesel oil degradation, which was more effective than nitrogen and phosphorus fertilizers (71.36 ± 5.60% hydrocarbon degradation rate) amendments in the diesel-contaminated soil for 8 weeks of incubation. In this study, removal rates of petroleum hydrocarbons were lower than those of the previous study. The reason for this may be that the aged hydrocarbons are difficult to degrade by soil microorganisms in the loessal soil.
The saturate hydrocarbon fractions in the untreated oilcontaminated soil were 16394 ± 160 mg kg −1 , and they decreased to 15509 ± 124, 13597 ± 136, and 9076 ± 148 mg kg −1 in the CK, MS, and SC treatments after 90 days of biostimulation, representing removal rates of 3.25%, 17.8%, and 45.1%, respectively (Fig. 1b). The aromatic fractions decreased from 4554 ± 115 mg kg −1 in the untreated soil to 3607 ± 130, 1942 ± 143, and 3507 ± 151 mg kg −1 in the CK, MS, and SC treatments, representing degradation rates of 20.8%, 57.4%, and 23.0%, respectively. Thus, KNO 3 amendment was more effective for removing aromatics while compost addition could degrade more the saturates. Table 2 shows that the number of OTUs based on the 97% similarity threshold was CK (5531) > MS (5317) > SC (5025) in the soil microcosms after 90 days of bioremediation. The ACE index were 10849, 10018, and 9824 and Chao1 index were 8954, 8176, and 7785 in the CK, MS, and SC soil samples, respectively, which indicated that enhancing soil moisture was beneficial to the survival of various soil microorganisms, while KNO 3 and compost supplementation could reduce the richness of soil microorganisms. It can also be concluded from Table 2 that the soil microbial community rehabilitated by deionized water addition had the most evenly distribution because the ranking of the Simpson index was CK (0.0026) < MS (0.0301) < SC (0.0405). The inorganic nitrogen fertilizer and compost had great interference on the uniformity distribution of microbial community. Nwankwegu et al. (2016) confirmed that it was a failure to evaluate the degradation potential of diesel oil by the increase of total heterotrophic bacterial in the soil, because only specific hydrocarbon utilizing microorganisms were related to the removal rate of diesel oil. Our study revealed that although KNO 3 and compost reduced soil microbial species and the community distribution uniformity, the existence of functional hydrocarbon-degrading bacteria may be enhanced which could lead to higher petroleum hydrocarbon degradation in the MS and SC treatments. Fig. 2 reflects the compositions of microbial community in soils in terms of dominant phyla, classes, and genera. Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes phyla were dominant in all three treatments but the relative abundance changed in different treatments (Fig. 2a). The relative abundance of the Proteobacteria,  The dominant classes affiliated to phyla of Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes were Alphaproteobacteria, Actinobacteria, Acidobacteria_Gp7, and Planctomycetia, respectively. Compost and KNO 3 addition had minor effect on the microbial community at the class level, except for the class of Alphaproteobacteria and Deltaproteobacteria (Fig. 2b-e). Firmicutes, Proteobacteria, Acidobacteria, and Actinobacteria phyla have proven to be important in mitigating the long-chain alkane, PAHs, and other types of hydrocarbons pollution in the soil environment (Zhang et al. 2012;Wu et al. 2019;Wu et al. 2020). Wolf et al. (2019) found that soil polluted by pyrene substantially increased the microorganisms belonging to Firmicutes phylum in the clay and sandy loam soils. In addition, phyla of Actinobacteria increased while Acidobacteria and Proteobacteria decreased in the soils remediated by amendments of KNO 3 or sterilized nutrition, indicating that the hydrocarbon degradation may be attributed to the increase of Actinobacteria phylum which could utilize petroleum hydrocarbon as their energy source for cellular metabolic activity. The influences of the biostimulation treatments on the genus level were great (Fig. 2f). Steroidobacter, I n t r a s p o r a n g i u m , B l a s t o p i r e l l u l a , S a c c h a r i b a c t e r i a _ g e n e r a _ i n c e r t a e _ s e d i s , Parcubacteria_genera_incertae_sedis, Aquisphaera, and Rhodococcus were new genera appeared in the MS and SC treatments compared with the CK soil. Specially, Intrasporangium also became a second dominant genus (5.6%) in the SC treatment, which may be related to hydrocarbon degradation. Genera of Solirubrobacter, S p a r t o b a c t e r i a , i n c e r t a e _ s e d i s , a n d W P S -1_genera_incertae_sedis with the relative abundance more than 1% in the CK treatment did not appear in the MS and SC treatments. Gp6 was the most dominant genus in the CK (6.4%), while the relative abundance of Gemmata was the greatest in the MS (3.21%) and SC (5.90%) treatments. Gp6, Rhodococcus, and Gemmata belonged to hydrocarbondegrading microorganisms which were reported in previous study ) Therefore, it can be concluded that the compositions of indigenous flora in oil-contaminated soil was influenced by nutrition amendments (Seklemova et al. 2001), which may be related to the higher hydrocarbon removal rates in the MS and SC treatments.

Carbon and nitrogen depletion
The contents of carbon and nitrogen elements in the contaminated soils were detected by elemental analyzer. The soil total carbon content was 32327 ± 30 mg kg −1 in the initial oilcontaminated soil (Table 3), and TPH content was determined by ultrasonic extraction and gravimetric method with the value of 24166 ± 40.46 mg kg −1 (Table 1). According to the description of Margesin et al. (2007), the proportion of carbon content of TPH was 20541 mg kg −1 which derived from 85% TPH concentration. Thus, soil organic carbon in polluted soil was 11786 mg kg −1 which is calculated by subtracting the carbon proportion of TPH from soil total carbon. Two kinds of method including alkaline potassium persulfate digestion-UV spectrophotometry and elemental analyzer were respectively used to determine soil total nitrogen. The content of soil total nitrogen (STN) was 1005 ± 12.21mg kg −1 (Table 1) by using UV spectrophotometry and 1762 ±8.2 mg kg −1 (Table 3) by elemental analyzer. One possible reason for an obvious difference between them was that there were some special nitrogen forms in the polluted soil which cannot be digested by alkaline potassium persulfate. The contents of soil total carbon (STC) obviously decreased with time in the MS and SC treatments compared with the CK treatment. This result was consistent with the TPH degradation trends in our study (Fig. 1a).
KNO 3 and compost-treated soil samples (MS and SC samples) had lower C:N ratios on the 90th day of bioremediation than earlier days (Table 3), while there was less reduction of the C:N ratio in the deionized water-treated soil. Previous literatures reported that soil microorganisms were suitable for living at a nutritional level with a C:N ratio of 100:10 (Prescott 2002). In this study, the C:N ratios had been maintained at about 10:1 in the CK treatment, and these ratios were close to 10 in the first 30 days during the bioremediation and then decreased to about 8 in the MS treatment, while the C:N ratios were reduced from about 10 to 8 on the 15th day in the SC treatment. Additionally, the TPH content was reduced in all three treatments, which was the main reason for the reduction of soil total carbon and the C:N ratios. The reason for the reduced C:N ratios in the SC and MS treatments may be that the degradation rates of TPH by the amendment of KNO 3 or compost were faster than that in the CK treatment.

Carbon and nitrogen isotope effect
The δ 13 C and δ 15 N of soil samples detected by stable isotope ratio mass spectrometer are shown in Table 3. According to Equations 3 and 4, carbon isotope enrichment factors respectively were ε c = 28.3 ± 0.3‰, ε c = 16.6± 0.8‰, and ε c = 38.8 ± 1.1‰ in the CK, MS, and SC treatments after 90 days of remediation (Fig. 3). Previous studies have proved that microbial degradation of toluene and oxylene caused carbon isotope fractionation. Up to 1 of carbon isotope fractionation factor in the residues of toluene and o-xylene at 99.8% biodegradation was shown (Richnow et al. 2003). Gray et al. (2002) found that both in the biodegradation by a single microorganism and microbial flora, 13 C/ 12 C isotope enrichment in the residual MTBE can reach 8.1‰, while 99.7% of MTBE was degraded. It indicated that the fractionation of carbon isotopes can be used to characterize the biodegradation of organic pollutants. In this paper, the limiting nutrients contained in the biostimulant of KNO 3 or compost were incorporated into oilcontaminated soil, thus promoting the degradation of petroleum hydrocarbons by the native microbial flora, which was the reason for the carbon isotope fractionation in the soil. Furthermore, Fig. 1b shows that KNO 3 supplement was more effective for removing the aromatics while compost addition was beneficial to degrade the saturates. Thus, the significant difference of carbon enrichment factors of ε c = 16.6 ± 0.8‰ in the MS and ε c = 38.8 ± 1.1‰ in the SC treatments may be caused by the degradation of aromatic and saturated hydrocarbons, respectively. Therefore, a significant change in δ 13 C of soil total carbons can be used as an indicator of the petroleum hydrocarbon degradation in the contaminated soil.
The δ 15 N value of soil total nitrogen was variable in three different treatments during 90 days of incubation. And the Fig. 3 Carbon isotope enrichment in soil after adding deionized water (a), KNO 3 (b), and compost (c). Nitrogen isotope enrichment in soil after adding deionized water (d), KNO 3 (e), and compost (f). Errors bars indicate ±SD of triplicate samples nitrogen isotope effects (ε N ) were respectively ε N = −7.23 ± 4.08‰, ε N = −24.20 ± 9.54‰, and ε N = −79.49 ± 16.41‰ in the CK, MS, and SC treatments at the end of the bioremediation. Previous study reported that soil nitrogen isotope fractionation was related to soil moisture and environmental conditions, which affected the nitrogen transformation process led by microorganisms, such as nitrogen assimilation (Nikolenko et al. 2018;Yu et al. 2021). Nikolenko et al. (2018) concluded that the nitrogen isotope enrichment factor varied between −35 and −5% in the different soil types and environmental conditions. Choi and Ro (2003) showed that in the process of N immobilization and assimilation, the soil nitrogen isotope enrichment factor is −10‰. Horel et al. (2015) reported that in the biostimulation remediation of oil-contaminated soil, the petroleum hydrocarbondegrading bacteria could convert nitrogen and hydrocarbons into microbial biomass (N immobilization and assimilation). In this paper, humidity changed the soil microcosmic environment, and KNO 3 and compost addition increased the soil nitrogen content. All the amendments promoted the activity of petroleum hydrocarbon-degrading bacteria. After 90 days of remediation, the nitrogen contents in the soils decreased, which may be due to the assimilation of nitrogen sources into biomass of microorganisms. Therefore, it can be inferred that nitrogen isotope fractionation during the bioremediation was attributed to N immobilization and assimilation by indigenous microorganism after addition of the deionized water, KNO 3 , and compost in the oil-contaminated soil.
To further investigate the enrichment trend of carbon and nitrogen isotopes, carbon and nitrogen dual-isotope relationship in the three different treatments is shown in Fig. 4. The carbon and nitrogen dual stable isotopes showed clear differences due to the different hydrocarbon degradation and nitrogen utilization mechanisms by microbial population among the three treatments (Fig. 4). An inverse significant carbon isotope effect (ε c = 28.3 ± 0.3‰) and a normal significant nitrogen isotope effect (ε N = −7.23 ± 4.08‰) in the CK treatment. On the other hand, an inverse significant carbon isotope effect (ε c = 16.6± 0.8‰) and a strong significant nitrogen isotope effect (ε N = −24.20 ± 9.54‰) in the MS treatment, and a significant carbon isotope effect (ε c = 38.8 ± 1.1‰) as well as a strong significant nitrogen isotope effect (ε N = −79.49 ± 16.41‰) in the SC treatment. Thus, carbon isotope enrichment factors are inverse significant while nitrogen isotope enrichment factors are normal significant in all three treatments. Horel et al. (2015) reported that hydrocarbondegrading bacteria utilize carbon and nitrogen in the form of the average biomass composition (C 5 H 7 O 2 N). Liu et al. (2020b) reflected that microalgae had a strong ability to fix carbon and nitrogen in the fuel oil pollution environment. Base on the difference of carbon and nitrogen isotope fractionation, the effect of carbon isotope fractionation on microalgae was greater than that of nitrogen on it. In this paper, the degradation of hydrocarbons and the decrease of soil δ 13 C made the carbon enrichment factors inverse, while the consumption of nitrogen and the increase of soil δ 15 N make the nitrogen enrichment factors normal. Therefore, it can be concluded that the type of stimulant had a significant effect on the fractionation of soil carbon and nitrogen isotopes.
Additionally, the nitrogen isotope effects based on the δ 15 N of KNO 3 (1.008‰) and compost (5.3‰) made the enrichment factors variable in the MS (ε N = −24.20 ± 9.54‰) and SC (ε N = −79.49 ± 16.41‰) treatments, respectively. The nitrogen enrichment factor in the SC treatment was the largest, followed by the MS treatment, and the CK treatment was the smallest (Fig. 3a), which was consistent with the degradation rate of the TPH in the three treatments, implying that petroleum hydrocarbons were degraded accompanied by the utilization and transformation of nitrogen. The ranking of carbon isotope enrichment factors among the three treatments was the SC > CK > MS, which was inconsistent with the degradation rate of TPH, indicating that the enrichment of carbon isotope in the soils may be affected by the soil microenvironment.
In this study, carbon and nitrogen isotopes showed different enrichment effects after introducing various stimulants to the soil. Microbial diversity and uniformity also changed by different treatments. The isotope enrichment effects are related to the change of the soil microbial community structure or the soil microbial biomass by the amendments . It is necessary to investigate soil microbial biomass for understanding isotope enrichment mechanisms during bioremediation of oil-contaminated soil in future studies.

Conclusions
Biostimulation strategy has proven to be effective for mitigating hydrocarbons pollution in soil, but little is known about the carbon and nitrogen isotope effects during bioremediation. The value of C/N decreased accompanied with the petroleum hydrocarbon biodegradation during remediation by supplement of KNO 3 and compost. Isotope enrichments are one of the important characteristics for biodegradation of petroleum hydrocarbons which are different in polluted soils remediated by various biostimulants. Our results provided a new perspective to investigate the biodegradation mechanisms of petroleum hydrocarbons during bioremediation of petroleumcontaminated soil.