Biodegradation of total petroleum hydrocarbons in contaminated soils using indigenous bacterial consortium

Background: Biodegradation of hydrocarbon compounds is a great environmental concern due to their toxic nature and ubiquitous occurrence. In this study, biodegradation potential of oily soils was investigated in an oil field using indigenous bacterial consortium. Methods: The bacterial strains present in the contaminated and non-contaminated soils were identified via DNA extraction using 16S rDNA gene sequencing during six months. Furthermore, total petroleum hydrocarbons (TPH) were removed from oil-contaminated soils. The TPH values were determined using a gas chromatograph equipped with a flame ionization detector (GC-FID). Results: The bacterial consortium identified in oil-contaminated soils (case) belonged to the families Halomonadaceae (91.5%) and Bacillaceae (8.5%), which was significantly different from those identified in non-contaminated soils (control) belonging to the families Enterobacteriaceae (84.6%), Paenibacillaceae (6%), and Bacillaceae (9.4%). It was revealed that the diversity of bacterial strains was less in oil-contaminated soils and varied significantly between case and control samples. Indigenous bacterial consortium was used in oil-contaminated soils without need for amplification of heterogeneous bacteria and the results showed that the identified bacterial strains could be introduced as a sufficient consortium for biodegradation of oil-contaminated soils with similar texture, which is one of the innovative aspects of this research. Conclusion: An oil-contaminated soil sample with TPH concentration of 1640 mg/kg was subjected to bioremediation during 6 months using indigenous bacterial consortium and a TPH removal efficiency of 28.1% was obtained.


Introduction
Hydrocarbon compounds are discharged into the environment, especially to soil, through industrial petroleum-related activities such as drilling, transportation, and storage (1,2). Petroleum contaminants are harmful to the environment because they can remain in the soil pore space and stunt the growth of soil microbes, plants and animals, and can be dissolved in the soil moisture or groundwater and contaminate them, and escape into the atmosphere through volatilization (3). Therefore, environmental pollution with hydrocarbons is a great environmental concern due to their toxic nature and ubiquitous occurrence (4). Bioremediation is an effective and environmentally friendly process that degrades oil contaminants into nontoxic, simple, inorganic compounds using hydrocarbondegrading microorganisms (5). Bioremediation may remove the contaminants to a large extent and has proven successful in many applications to petroleumcontaminated soils and relies on the application of microorganism especially bacteria (6)(7)(8), which can be implemented both in situ (9,10) and ex situ (11,12) bioremediation technologies. Although in situ bioremediation technologies are more environmentally-friendly, cheaper, and easier to perform compared to ex situ alternatives (13), but are usually longer, this drawback could be mitigated by identification and following up the quantitative variations of dominant hydrocarbon-degrading strains in polluted areas (14,15). As a key question, dominant hydrocarbon-degrading strains in a highly contaminated soil were monitored and identified in order to prepare an efficient consortium for application in other polluted soils with similar soil texture. Such process is called "bioaugmentation", the inoculation of exogenous bacteria into contaminated soil (16). When contamination adversely affects the native microorganisms, bioaugmentation gives the opportunity for a successful bioremediation (17). Although bioaugmentation has been reported to be an efficient case-specific process to enhance the biodegradation of hydrocarbons in hydrocarboncontaminated soil. Therefore, if the soil texture and characteristics are known, then, there would be a bigger chance to perform a successful bioaugmentation. Consequently, identification of native hydrocarbondegrading bacteria by molecular methods provide a good understanding of the microbial community composition in polluted soils (18). This research was conducted to find the most suitable method for identifying the whole bacterial consortium. This method was applied using direct DNA extraction by 16S rDNA gene sequencing during six months from oil-contaminated soils by testing a combination of many procedures, and finally, applying physical, chemical, and biochemical processes, which is one of the strength points of this study. Also, indigenous bacterial consortium was used in oil-contaminated soils without need for amplification of heterogeneous bacteria and the results showed that the identified bacterial strains could be introduced as a sufficient consortium for biodegradation of oil-contaminated soils with similar texture, which is one of the innovative aspects of this research. Furthermore, an appropriate total petroleum hydrocarbons (TPH) removal efficiency (28.1%) in oilcontaminated soils was obtained.

Soil sampling
Considering the aims of the present study, variations of TPH content as well as bacterial count in case and control samples were analyzed during six months. The case samples were obtained from pre-determined points due to oil leaks from oil and gas separators. Soil samples were prepared using soil cores from surface layers (0-40 cm depth), which were air-dried. Soil samples were homogenized by shaking (19). Also, the same procedure was carried out in control area without TPH contamination. The conditions of sampling site for control and case samples are presented in Table 1. Sampling was performed in spring season (June) and the biodegradation in the laboratory was done during six months from August 2017 to January 2018. The study was performed in a laboratory scale and in a small pot as container with a capacity of approximately 2 kg. DNA extraction Metagenomic DNA extraction was performed in a harsh manner by combining several lysis methods together. The physical lysis including bead beating with the lysis buffer treatment was done, then, the samples were applied to the enzymatic buffer and incubated with shaking overnight. The next steps for the chemical lysis and purification were continued according to Siddhapura et al (20). Then, the genomic library was amplified by the polymerase chain reaction (PCR) using universal primers. Sequencing was carried out using the Illumina MiSeq platform at Macrogen Company of Korea, in order to examine taxonomic diversity of bacterium. Data were analyzed using QIIME software (21,22).

TPH measurement
The TPH levels in the samples were determined by a gas chromatograph equipped with a flame-ionization detector (GC-FID, Chrompack CP 9001) using an HP-5 capillary column (30 m length, 0.32 mm inner diameter, and 0.2 mm film thickness). Helium was used as the carrier gas with a constant flow rate of 1 mL min -1 . The temperature program was as follows: the column temperature was held at 50°C for 1 minute, and then, ramped to 280°C at 15°C min -1 and held for 5 minutes. The injector and detector temperatures were set at 250 and 320°C, respectively. The injection volume was 1 µL. The detection limit of GC-FID was more than 10 ppb. The obtained data were analyzed using R-Studio or R version 4.0.0 and SPSS version 22.

Results
The contaminated soil was characterized as loamy sand which contained silt (22%), clay (4%) sand (74%), and approximately 30% moisture content. Volatile matter content usually accounts for organic fraction of samples, which was 12% in this case. Variations of TPH content in the oil-contaminated soils are presented in Figure 1. As shown in this figure, there is a significant difference in the removal of TPH during six months. The initial TPH concentration of 1640 mg/kg at the beginning of the experiments decreased to 1179 mg/kg at the end of month six (28.1% removal). Statistical analysis showed that the mean difference in the TPH content between month zero and month six is significant at P≤0.05 ( Table 2). Diversity of bacterial strains in six continuous months and in two categories of oil-contaminated soils (case) and non-contaminated soils (control) are presented in Tables  3 and 4, respectively. The bacterial consortia identified in oil-contaminated soils (case) belonged to the families Halomonadaceae (91.5%) and Bacillaceae (8.5%). The findings are significantly different from those obtained in non-contaminated soils (control) belonging to families Entrobacteriaceae (84.6%), Paenibacillaceae (6%), and Bacillaceae (9.4%). It is very surprising that the diversity of bacterial strains was less in oil-contaminated soils, and generally, the identified strains varied significantly in the case and control samples. Taxonomic diversity and frequency of bacterial community in oil-contaminated soils (case) and noncontaminated (control) soils are presented in Tables 5 and  6, respectively.
Variation of bacterial count in case and control samples during the study period are presented in Figure 2. By comparison of bacterial count changes in case and control samples, it can be concluded that there is no rational relationship between these two conditions. Generally, the total counts of bacterial consortium in control samples is smaller than the case ones.

Discussion
The statistical analysis of TPH concentration showed a significant difference in the TPH concentration in case samples at the beginning and at the end of the experiment (P < 0.05). By comparing the TPH removal rate (28.1%), it can be concluded that the removal rate observed in the oil-contaminated soils can be attributed to the biodegradation activity of native microorganism (23), which is consistent with the results of some studies (24)(25)(26). Feizi et al investigated the TPH bioremediation in polluted soils using bacteria under conditions approximately similar to the present study, and reported the TPH removal efficiency of 17.7%, which is similar to that reported in the present study (28.1%) (27). Safdari et al in a study on the bioremediation of TPH, reported that the initial concentrations of TPH were reduced by 4.5% in the natural attenuation, which is very lower than that reported in the presented study. Also, they found that bioremediation by adding nutrients and bacterial consortia did not significantly enhance TPH biodegradation compared to natural attenuation, which is consistent with the results of the present study that was done without the addition of nutrients and bacterial inoculation (28). A research conducted by Liu et al in an oilfield in northern China showed that after bioremediation for 230 days, the removal efficiency of oil and grease was obtained to be 27%-46%, which is consistent with the results of the present study. Furthermore, they reported an increase in the TPH degrader concentrations in all oily sludge, which is consistent with the results of the present study (29). The high total counts of bacterial consortium in case samples could be attributed to the occurrence of biodegradation and related growth of the bacteria compared to control samples with no pollution as a carbon source (30,31). The bacterial count variations in control samples were less, which can be attributed to almost   constant environmental conditions in terms of pollution, soil texture, and chemical composition of soil (32). For oil-contaminated soil, there is no uniform variation trend. The complicated nature of various hydrocarbons present in the contaminated soils, diversity of biodegrading bacteria with different enzymatic and metabolic functions could be considered as the possible reasons for the observed variations (33).

Conclusion
Variations of microbial count in oil-contaminated soils (case) were monitored and compared with noncontaminated soils (control). Furthermore, the bacterial strains were identified by the extraction of DNA. According to the majority of bacterial consortium in oil-contaminated soils (case) belonged to the families Halomonadaceae (91.5%) and Bacillaceae (8.5%). These findings are significantly different from those identified in non-contaminated soils (control) belonging to the families Entrobacteriaceae (84.6%), Paenibacillaceae (6%), and Bacillaceae (9.4%). It is very surprising that the diversity of bacterial strains was less in oil-contaminated soils, and generally, the identified strains varied significantly in case and control samples. In addition, indigenous bacterial strains were identified as efficient hydrocarbon degraders with a removal efficiency of 28.1% (the initial TPH concentration of 1640 mg/kg) after six months. According to the results, the identified bacterial consortium could be introduced as the efficient consortium for biodegradation of oil-contaminated soils with loamy sand texture.