Biological treatment of PAHs using genetically modified local bacterial isolates

Bioremediation technology by microorganisms, which already presents in the contaminated soils, is considered one of the primary mechanisms by which petroleum and other hydrocarbon pollutants can be removed from the environment. Twelve decomposing bacterial isolates were isolated from three polluted sites (Al-Dora oil refinery, Middle Refineries Company from oil wells, and Karbala oil refinery). This study showed high efficacy polycyclic aromatic hydrocarbons (PAHs) of isolated bacteria appropriate to polycyclic aromatic hydrocarbons decomposition. Primary and secondary screening of bacterial isolates has been performed using experiments based on the colour change in the medium resulting from the degradation of hydrocarbons in the nutrient medium. The screening results were three isolates that were characterized according to the basis of morphological and biochemical features and verified by Vitek 2, Pseudomonas aeruginosa, Escherichia coli and Sphingomonas paucimobilis. The mutagenesis process has been carried out by UV irradiation with a wavelength of 254 nm on selected bacterial strains. Experiments have been conducted on PAHs (naphthalene, phenanthrene and acenaphthene. According to the Biodegradation Efficiency result for 28 days, the best phenanthrene degrading bacteria was (Pseudomonas aeruginosa wild type (73.4%), Escherichia coli wild type (71.5%) and Sphingomonas paucimobilis wild type (68.8%). For naphthalin metabolism, the best degradation efficiency was (Pseudomonas aeruginosa wild type (86.2%), Sphingomonas paucimobilis wild type (69%) and Escherichia coli wild type (63%). At the same time, acenaphethene great degradation efficiency was Sphingomonas paucimobilis wild type (72%), Pseudomonas aeruginosa wild type (71%) and Sphingomonas paucimobilis mutant type (66%). We conclude that random mutation’s effect did not increase the degradation ability for most bacterial isolate.


Introduction
One of today's major environmental problems is hydrocarbon pollution from activities related to the petrochemical industry, oil exploration and chemical factories wastes. Hydrocarbon compounds are the most popular waste compounds that have been classified as neurotoxic and carcinogenic organic pollutants. [1]. It is known that polycyclic aromatic hydrocarbons are difficult to degrade because of their low reactivity; The USEnvironmental Protection Agency (US EPA) has listed these substances as priority contaminants in natural resources. However, a wide number of microorganisms slowly degrade these compounds [3]. The biodegradation process of hydrocarbon in contaminated sites by hydrocarbondegrading bacteria was studied [4]. Biodegradation for mixtures of hydrocarbons usually involves the collaboration of more than one type of microorganisms. This process is certainly relevant in contaminants that contain several different compounds, such as crude oil or petroleum, and complete CO2 and H2O mineralization is required in the final process [5]. With the aid of a diverse community of microorganisms, especially the indigenous bacteria found in the soil, a hydrocarbon-contaminated site's microbial bioremediation is completed. These microorganisms will degrade a wide variety of desired constituents present in oily sludge. [13]. A great number of strains of Pseudomonas able to degrade PAHs were isolated from the soil. Other petrochemicals degrading bacteria include Yokenella spp, Alcaligenes spp, Rosemonas  Bacteria can degrade Branched hydrocarbons, aliphatic saturated, saturated hydrocarbons and cyclic hydrocarbons through aerobic and anaerobic pathways [1].
By creating covalent bonds between neighbouring pyrimidine bases, mutation by irradiation or UV exposure harms DNA. In the double helix configuration of DNA, this pyrimidine dimer does not match well and prevents replication and translation. Dimer development normally results in a mutation in deletion. Many types of radiation (based on intensity and wavelength) may have a range of effects, but there are often insertions/deletions. Purine dimers frequently occur [16].
This study aims to isolate, screen, and identify local bacterial isolates isolated from the contaminated site with hydrocarbons. Using UV light irradiation in mutagenesis of bacterial isolates to obtain mutant efficient bacterial strains degrade PAHs by measuring biodegradation efficiency (BE %).

Sample Collection
Oil contaminated soil samples were collected from Al-Doura oil refinery in Baghdad, Al-Wasat Refineries Company/Alkout and Karbala'a oil refinery in the duration ( 5/11/2019 -17/12/2019). Samples were collected and put in the sterile zip-lock polythene bags and transferred to the laboratory for further study.

Isolation of Bacteria
Hydrocarbon degrading bacteria were isolated from soil samples using Bushnell Hass broth medium (BH) (0.2 g of MgSO4, 0.02 g of CaCl2, 1.0 g of KH2PO4, 1.0 g of K2HPO4, 1.0 g of NH4NO3, 0.05 g FeCl3, in 1000 mL of distilled water) containing sterilized crude oil 1% (v/v) as a sole source of carbon [17].
One gram of soil sample was introduced in 500 mL Erlenmeyer flask containing 100 mL of BH medium supplemented with 1 ml (1% v/v) crude oil filtered by Millipore unit using 0.45µm filter paper and incubated at 37ᵒC, 150 rpm for one week.
Then 0.1 mL from 10ˆ¹ and 10ˆ8 serial dilutions spread on BH agar with sterilized crude oil (1% v/v) as the sole source of energy. The plates were incubated at 37ᵒC for 7 days. Pure colonies were obtained, and each of the selected colonies was sub-cultured and purified by grown on a nutrient agar plate.

Screening of oil-degrading Bacteria
The purified is isolates were grown on BH broth in the presence of naphthalene (1% wt/v) as the sole source of carbon and incubated at 37°C, 150 rpm for 7 days. After 7 days, the OD of the bacterial growth culture was measured using a spectrophotometer at 600nm for each isolate.
Then 0.5 ml (from grown isolates only) has been spread on the BH agar plate and added 0.5 ml of crude oil as a drop on the plate to illustrate the growth of degrading bacterial isolates only as a conformation step [4].
The bacteria's ability to degrade hydrocarbons was verified using modified Simmon citrate medium based on Bromothymol blue indicator (also known as bromothymol sulfone phthalein and BTB). 3 experiment was the substitution of Sodium Citrate with naphthalene as the sole source of carbon to ensure the bacteria's ability to degrade the PAHs compound. Bacterial isolate, which grown on crude oil, were inoculated (100 μL) in test tubes containing 4 ml of modified Simmon citrate broth medium supplemented with 0.04 g (1 % w/v) of naphthalene and bromothymol indicator and incubated at 37 °C and green colour disappearance. Yellow colour forming was observed daily [9].

Identification of bacterial isolates
Pure colonies which give positive result (yellow colour) were identified and characterized based on their morphological characteristics and biochemical properties according to the identification scheme of Bergey's Manual of Determinative Bacteriology [19]. Isolates were diagnosed for confirmation by the VITEK 2 system for microbial identification.

Preservation of bacterial strain
The identified bacterial isolates preserved as wild type for other experimental procedure in preservation broth medium by adding 40 ml of glycerol to every 100 ml of brain heart infusion broth for long period preservation, 10 ml dispensed in each well-capped screwed test tube, sterilized by autoclaving, cooled to 37º C and used for preserving bacterial isolates [ 20].

Biodegradation of PAH by bacterial strains.
The three efficient bacterial srtains (S.D1, S.D2 and S.K1) used to degrade PAHs material as the sole source of energy and carbon in BH broth media. 20 ml of BH medium prepared with 0.2 g (1% wt/v) of each (naphthalene, acenaphthene and phenanthrene). Then 20 µm from (S.D1, S.D2 and S.K1) inoculated separately in liquid BH medium and then incubated at 37°C, 150 rpm for 28 days. The optical density (OD) of bacterial culture measured at 600 nm every 5 days to estimate bacterial growth [7].

Mutation of bacterial isolates
The three efficient bacterial strains (S.D1, S.D2 and S.K1) were activated in LB broth at 37°C for 24 hr. Then 500 µm of each bacterial strain spread on three LB agar plates according to the time of mutation (10 min, 30 min and 60 min) by UV light radiation at 254nm wavelength from 40 cm distance. The effect of mutation by UV light was studied on morphological (size, shape and arrangement) and biochemical scale (hemolysis and lactose ferments test).
The clear individual colony of each irradiated plate of bacteria was selected to inoculate in BH media supplemented with 0.2 g of PAH to estimate the biodegradation of PAH.

Biodegradation of PAHs by mutant bacterial strains
Twenty µl of mutated bacteria inoculated in 20 mlBH media [with 0.2 g of (naphthalin, phenathrin and acenphthene) as the sole source of carbon] for 28 days (at 37oC, 150 rpm). The optical density measured every 5 days to estimate and observe the bacterial growth state.

Biodegradation efficiency measurement
The culture media, after 28 days, were dried by the oven (dry heat) in a glass petri-dish. The precipitate was collected and weighed to estimate the Biodegradation Efficiency for both wild and mutated biological process. BE% = Cwt -Swt / Cwt ×100. Were, Cwt: is the control weight. Swt: is the sample weight after 28-day treatment. The powder prepared for FTIR and GC Mass analysis.

Results and Discussion
Bacterial strains were collected and isolated from soil contaminated with oil. Incubation of unknownbacterial isolates in broth Bushnell Hass broth medium (BH) with crude oil as the sole source of carbon provide an appropriate method to reduce the number of isolates depending on the efficiency of HC degradation. This step will reduce the number of bacterial isolates to five unknown isolates.
Another confirmation step uses a modified Simmon citrate medium with another carbon source instead of citrates PAHs in this case (naphthalene) to select the efficient HC degrading bacteria. The addition of bromothymol blue indicator in BH media confirms positive results by changing media from green to yellow due to acidic compound metabolites from oxidation of naphthalene, as shown in Fig (1) below. Bacterial isolates have been identified for validation by VITEK 2, which has been used to confirm the conventional diagnosis. According to the results from the VITEK2 technique, the three isolates (S.D1, S.D2 and S.K1) were identified as Pseudomonas aeruginosa (probability 99%), E.coli (probability 89%) and Sphingomonas paucimobilis (probability 93%), respectively.
The effect of mutation by UV-C light was studied, as shown in Fig (2) decrease in colony size for (E.coli and Sphingomonas) while the colony size increased gradually with irradiation duration Pseudomonas. Lacto-fermenting bacteria (E.coli) show moderate resistance toward UV light for the initial amount. The mutated type of (E.coli) acquired high resistance after irradiation due to their strong and stable gene expression, fig [3]. The optical density measurement of wild and mutant bacteria at 600nm wavelength by spectrophotometer every 5 days for 5 weeks. For each sample indicate bacterial growth with PAH as the sole source of carbon inBH media, wild isolate shows high adaption ability with the increased curve (approximately one week). In contrast, mutated isolates took two weeks to adapt and start the growth stage.
Through the gravimetric method that depends on the sample weight difference before and after biological treatment, approximate biodegradation ratios were obtained, depending on the law: BE% = Cwt -Swt / Cwt ×100. Were, Cwt: is the control weight. Swt: is the sample weight after 28-day treatment.
According to the numbers illustrated in Table (0), initial results can be attained, indicating each bacteria's ability to decompose a specific substance. Concerning naphthalene, the wild Pseudomonas aeruginosa are better decomposer for naphthalene among the rest of the species by up to 88%. In the mutagenic type, they have broken down by up to 75%. It was followed by the wild Sphingomonas paucimobilis, at 75%, and then by 58% for the mutated type. Then E.coli bacteria were considered the least bacteria capable of degrading naphthalene, by 70% for the wild type and 59% for the irradiated type. Before treating naphthalene adopted in the equation, the sample's weight is relatively less than its weight in the medium because naphthalene is a volatile substance, especially during the long study period (28 days).
The tricyclic phenanthrene was degraded with the highest amount through Pseudomonas aeruginosa bacteria by 73% for the wild type and 57% for the irradiated type. That follows it in the second set is E.coli wild type as it degrades the phenanthrene by 71% and the mutated type, which dissolved 65% better than the mutated type. Simultaneously, the Sphingomonas paucimobilis bacteria are less than hydrocarbon decomposition by 68% for the original isolate and 64% for the mutated type. The triple benzene ring acinaphthene is decomposed by the wild Sphingomonas paucimobilis by 72% and mutagenic type by 66%, which is the highest among the original mutagenic isolates of the rest of the species. In comparison, the Pseudomonas aeruginosa bacteria have dissociated the compound relatively less by 71% for the wild type and 33% for the mutated type, which is the lowest percentage among the rest of the mutated isolates. E.coli bacteria degraded the material with a ratio close to both the original and mutagenic isolates, reaching 64% and 60% for the original and mutagenic isolates, respectively.
Through the above table data, we can see the effectiveness of the decomposition of the material for each bacterium. As each material was measured in its pure state and the area of its peak was calculated. The pure naphthalene substance has a peak area of 7179468783 and is considered control. After processing and using it as a source of carbon and energy by the wild type of E. coli bacteria for 28 days, the sample was measured in the GC Mass device. The total area of the peaks decreased to 163557919, meaning that the bacteria degraded the material at a rate of 97.7%. As for the mutated type E. coli, the peaks' total area was 336196087, which is relatively less than the original type. Nevertheless, it degraded naphthalene by up to 95.3%. The result of its treatment is considered good because it was exposed to radiation and was mutated.
Phenanthrine compound with the three rings, the peak area in its pure state was 324904536, and it is considered to control. Then it was slowly decomposed by the wild type Sphingomonas paucimobilis bacteria, as the total areas of the peaks after examining the treated sample were 183193411, meaning that the bacteria consumed the compound by 43.6%. The mutagenic sample gave a peak area of 267739318, meaning that it consumed the compound less. By only 17.5%, which is the lowest among the samples, this may be because the mutagenic bacteria have relatively lost their ability to produce the analytic enzymes.
The tricyclic compound acenaphthene, which contains an ethyl group, was dissolved by E. coli bacteria. The pure peak area of the compound reached 411488724 and was adopted as a control during the equation. The sample with the wild type E. coli had a peak area of 46588045, i.e. a decrease of 88.6%, representing the compound's degradation rate. As for the sample with mutagenic bacteria, the mutated sample area decreased to 79740172, meaning that the mutated type decomposed the compound by 80.6%, close to the original type.

Conclusions
According to the current study and the results given from the experiments, as well as their compatibility with previous studies on the degradation of polycyclic aromatic hydrocarbons by isolating unknown strains of local bacteria in the soil, strains have been screened through some experiments based on the use of crude oil and petrochemical materials as a major source of carbon and energy. Through these laboratory experiments, the number of unknown bacterial isolated from the soil has been reduced. Subsequently, strains with the susceptibility to the decomposition and use of oil and its derivatives from PAHs compounds as the sole source of energy were then used. Some microscopic tests and biochemical diagnostic tests were carried out. The use of VITEK 2 technology to identify the types of isolated strains that the decomposition of oil and PAHs have positively induced compounds through the updated Simmon citrate media. The cultivation of identified bacterial is strains in BH media, with the addition of 0.2 g of naphthalene, phenanthrene and acenaphthene as the sole source of carbon and energy and monitoring the growth rate by measuring the Optical density for 28 days. It was concluded that the bacteria could grow and consume PAHs as an energy source with different growth and degradation levels depending on the type of bacteria. The weight method's rate of degradation of each compound was measured during the 28-day incubation period. The effect of the mutation on the change in bacteria's susceptibility to growth and decomposition has been observed. It was found that the best species of wild and mutant bacteria with the optimum speed of consumption of the substance were mentioned in Table 2 and 3.