Introduction

Plasticizers are compounds broadly used to soften and increase the flexibility and elasticity of polymer compounds. Consuming 8 million tons of phthalate annually has made these compounds the most commonly used plasticizer in the world (Mergelsberg et al. 2018; Sheikh and Beg 2019; Ebenau-Jehle et al. 2020). The benefits of using these compounds in manufacturing and increasing the plasticity of PVC, cosmetics, polyethylene, lubricants, adhesives, insecticides, material packaging, plastic compounds, toys, etc. is undeniable. However, due to the lack of chemical and stable bonds between these compounds and the polymer attached to them, they can easily enter the environment (soil, sediments, river, wetlands, landfill, sea, oceans, air) and consequently the human food cycle. As a result of the endocrine-disrupting activity of PAEs, direct and indirect exposure of humans to phthalates can cause numerous diseases including various cancer, reproductive problems, diabetes, obesity, etc. (Kida and Koszelnik et al. 2020; Giuliani et al. 2020). Cleanup and removal of PAEs and their intermediates from the environment are one the foremost strategies for plastic recycling in sustainable plastic waste management that can be preferably carried out using native microorganisms of each ecosystem. Among the various tools used to get rid of phthalate, bioremediation is the most eco-friendly, practical, and ideal way of eliminating this pollutant from the environment (Brandon et al. 2019; Sanz et al. 2020). Furthermore, Orthophthalic acid (PA) and its isomers (isophthalic acid and terephthalic acid) are carcinogenic, mutagenic, and endocrine-disrupting compounds (Bang et al. 2011) that are usually released and accumulated in nature by the breakdown of phthalic acid esters. The stability and half-life of these compounds increase under anaerobic conditions in an aquatic ecosystem such as the sediments of rivers, wetlands, seas, and oceans. The degradation of phthalic acid to CO2 and H2O is the most limiting step in the biodegradation of PAEs. There are many strains whose abilities to degrade this pollutant in aerobic conditions have been proven (Gao and Wen 2016). However, to date, few studies have been conducted on the degradation of this contaminant under anaerobic conditions and very few species enjoying this ability have been presented. More specifically, Pelotomaculum isophthalicum (Qiu et al. 2006), Aromatoleum aromaticum EbN1 (Mergelsberg et al. 2018), Thauera chlorobenzoica 3CB-1, and Azoarcus evansii KB740 (Ebenau-Jehle et al. 2017), Azoarcus sp. PA01 (Junghare et al. 2016), Syntrophorhabdus aromaticivorans (Junghare et al. 2019), and Desulfosarcina cetonica (Geiger et al. 2019) are known to be PA-degrading bacteria under anaerobic conditions. Researchers have also confirmed the biodegradation of PA by denitrifying bacteria in anaerobic conditions (Ebenau-Jehle et al. 2017; Junghare et al. 2019; Boll et al. 2020). Ebenau-Jehle et al. (2020) demonstrated that Thauera chlorobenzoica 3CB-1 and Aromatoleum evansii KB740, enjoying the qualities of facultative anaerobic and denitrifying, use phthalates as a growth substrate under aerobic and nitrate reduction conditions. As the low diversity of bacteria with the competence to degrade PA isomers under anaerobic conditions was discussed above, isolating new strains with this ability from various environments (especially in Iran) is urgent. Anzali international wetland is one of the most important lagoons in Iran and was registered in the Ramsar Convention as an international wetland that supports extremely diverse fauna and flora. Researchers have confirmed that this international wetland is heavily polluted by PAEs (Hassanzade et al. 2014; Shariati et al. 2019). As there were no studies concerning the purification of these pollutants in Anzali international wetland, this study was carried out to draw a range of the objectives including: (1) to evaluate the biological ability of wetland sediments in the breakdown of phthalic acid under anaerobic condition; (2) to obtain an effective consortium in degradation of phthalic acid from sediments of Anzali international wetland; (3) to isolate and identify new native bacteria with the ability to degrade phthalic acid under anaerobic conditions; and (4) to Evaluate of the growth ability of a facultative anaerobic and denitrifying bacterium to grow under anaerobic conditions in the presence of phthalic acid as the only carbon source. Our study was also planned to provide the basic requirements for cleanup and bioremediation of PA or other organic pollutants in natural ecosystems under anaerobic conditions.

Materials and methods

Chemicals

Terephthalic acid, isophthalic acid, and PA were purchased from Sigma Aldrich Chemie GmBH (Hamburg, Germany). Additionally, Acetonitrile and formic acid were of HPLC grade purchased from Carl Roth (Karlsruhe, Germany).

Study area and sampling

Anzali international wetland is a coastal lagoon situated in the North of Iran (Gilan province) and located adjacent to the city of Bandar-e-Anzali, Southwest of the Caspian Sea (the biggest lake in the world) with an area of 193 square kilometers. Anzali wetland was registered in the Ramsar Convention as an international wetland in 1971. Reportedly, The average annual rainfall and evapotranspiration are 1280 and 980 mm, respectively. The catchment area of this wetland is 3610 square kilometers and about 30 rivers from different parts of the province enter this wetland. In fact, the wetland acts as a sediment trap to prevent the entry of pollutants into the Caspian Sea. It is extremely important as spawning and nursery ground for fishes. Anzali international wetland is not only a permanent habitat for various plant and animal species but also a place for wintering and laying birds. There are some parts of the wetland protected including the Siakesheem protected area and Selke wildlife refuge (Hassanzade et al. 2014; Zamani Harghalani et al. 2014; Mousazadeh et al. 2015). Unfortunately, in recent years, due to intense human activities and the entry of a large amount of waste from agricultural to industrial, hospital, and municipal wastewater into Anzali Wetland, this wetland is facing many dangers and threats (Jamshidi and Bastami 2016; Shariati et al. 2019; Alabdeh et al. 2020). In the process of sampling, to prevent the photolysis of PA esters, dark-colored glassware was used. The containers were first washed with distilled water and then with hexane 99% and kept in an oven at 80 °C for 24 h. Samples were taken from a depth of 0–10 cm of the sediments in the Sorkhankol station (Latitude = 363,201, Longitude = 4,144,076) which is heavily contaminated with DEHP and DBP (Hassanzade et al. 2014; Shariati et al. 2019).

Enrichment PA-degrading consortium

In order to enrich the microbial consortium with the ability to degrade PA under anaerobic conditions, 100 ml flasks containing two types of culture media; phosphate medium (K2HPO4 5.6 g/L, NH4CL 0.54 g/L, pH = 7.7–7.8) and carbonate medium (KH2PO4 0.5 g/L, NH4Cl 0.3 g/L, MgSO4, 0.5 g/L, CaCl2 0.1 g/L) were prepared individually. Afterward, the flasks became anaerobic and oxygen was entirely replaced with N2 (3 min cycle, 10 cycles) (FigS. 1), and then, they were sterilized using an autoclave. Following this, trace elements, Selenite, vitamin, and NaHCO3 from anaerobic sterile stocks were added to carbonate media flasks by sterile syringe. Besides, trace elements, vitamins, Selenite, MgSO4, and CaCl2 were added to the flasks containing phosphate culture medium. At this stage, PA isomers and nitrate (anaerobic sterile stocks) with final concentrations of 5 mM and 10 mM, respectively, were added to both media (Rabus and Widdel, 1995; Ebenau-Jehle et al. 2003). Approximately 5 g of the most polluted station sediment was then suspended to the flasks in aseptic and anaerobic conditions and then was incubated at 30 °C. Flask-containing carbonate media + 5 g sediment without any carbon source, were considered as control. Turbidity of the suspension was visually evaluated daily and NO−3/NO−2 indicator (QUANTOFIX Nitrate/Nitrite) was used to determine the amount of nitrite production. Having the best treatment identified, 5 ml of the supernatant was added to the fresh carbonate media + PA isomers + nitrate and incubated at 30 °C. Nitrite production and microbial growth were determined by NO−3 / NO−2 indicator and spectrophotometer (Pharmacia Biotech Ultrospec 3000 PRO UV–VIS Spectrophotometer Haverhill, MA), respectively (Lopez Barragan et al. 2004; Ebenau-Jehle et al. 2017; Sanz et al. 2020).

Biodegradation assay of PA by selected consortium through the photometric method

The fresh carbonate medium was prepared, anaerobized, and sterilized according to the method described above. After adding vitamins, carbonates, trace elements, selenite, orthophthalic acid (5 mM), nitrates (10 mM) under completely anaerobic and sterile conditions (by syringe), 1 mL of the microbial consortium (OD578 = 0.5) was added to the flask (by syringe) and incubated at 30 °C. To measure the growth rate of microbes and the PA biodegradation at each time point, the flask was sampled with a sterile syringe under anaerobic conditions. Microbial growth was measured at 578 nm (OD578) using a spectrophotometer (Pharmacia Biotech Ultrospec 3000 PRO UV–VIS Spectrophotometer Haverhill, MA). To measure the remaining PA in the culture media, after acidifying (pH = 2) with 1 M HCl, the concentration of PA was measured using a spectrophotometer at 276 nm wavelength (ε = 1200 L.mol−1.cm−1) (Ebenau-Jehle et al. 2017).

Biodegradation assay of PA by selected consortium through UPLC

To confirm the degradation of PA by the selected consortium in anaerobic conditions, the remaining PA was measured by UPLC. The carbonate medium with a final concentration of 6 mM (1000 mg/L) of PA, and 10 mM nitrate were prepared according, to what was mentioned above. Then, 1 ml of inoculum (1%) (OD578 = 0.5) was added to each flask and kept at 30 °C. In another test, flasks containing carbonate medium with a concentration of 19 mM (3200 mg/L) PA, 20 mM nitrate, and 5% (v/v) of inoculum were prepared. All flasks were incubated at 30 °C. Also, to ensure complete degradation of PA under anaerobic conditions by this consortium, flasks were fed again with PA after 174 h (22 mM, 3700 mg/L). Each flask was sampled at each time point, and the growth rate and residual PA were measured by Spectrophotometer and UPLC (Waters, MA, USA), respectively (Ebenau-Jehle et al. 2017, 2020; Zhao et al., 2018).

Isolation and identification of cultivable PA-degrading bacteria from the selected consortium

To identify the microbes in the selected consortium, a drop of the fresh suspension was added to the PCR Mix, and following the PCR stage, sequencing and identification were performed. This process was performed with five replications and in all replicates the sequencing result was the same and the desired strain was named SHAn1. For isolation, purification, and identification of cultivable bacteria, dilution series were prepared from the selected consortium and 100 μl of this consortium was cultured on plates containing carbonate media + Agar + PA prepared under anaerobic conditions and incubated in an anaerobic jar (30 °C). After two weeks, two different colonies were grown on the plates. At this point, the selected consortium was examined under a microscope and two different types of colonies were observed with a ratio of about 90:10. On the other hand, preparation of carbonate media + Agar + PA under anaerobic conditions was problematic due to the formation of bubbles in the plates, so phosphate media + PA was used for separation. About 100 μl of the various dilution series of the selected consortium were added to plates containing phosphate media + Agar + PA under anaerobic conditions, and the plates were incubated in an anaerobic jar at N2 pressure of 0.5 bar at 30 °C (Ebenau-Jehle et al. 2017). Besides, the selected consortium was also cultured and incubated on plates containing phosphate media + agar + PA under aerobic conditions. After isolation of strains, the effect of anaerobic conditions on the growth of strains in liquid phosphate media + PA was investigated. Seven colonies were added anaerobically to anaerobic and sterile tubes containing liquid phosphate media + PA (FigS. 2). The results showed that after one week, all strains were able to grow under anaerobic conditions in the presence of orthophthalic acid and NO3−. Then, all seven strains were sequenced and due to the similarity of the sequencing result, the bacterium was named SHAn2. In all sequencing, the 16S rRNA gene was directly amplified without DNA purification (Kai et al. 2019). The pure culture of strains and the fresh culture of the consortium were phylogenetically analyzed using sequencing its 16S rRNA genes, which was concurrently PCR-amplified by the universal primers 27F (5′AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The 50 µL PCR reaction contained 25 µL Red Tag polymerase (GENAXXON bioscience), 2.5 µL forward primer, 2.5 µL reverse primers, 20 µL sterile distilled water, and a small amount of the pure colony or suspension. PCR mixture was applied to the Flex cycler2 Analytik Jena PCR system. The program used for PCR comprises an initial denaturation at 95 °C for 5 min, followed by 34 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s, plus a final step at 72 °C for 5 min. PCR product was qualified by 1% agarose gel electrophoresis and afterward purified by Fast Gene Gel/PCR Extraction Kit (Nippon Genetic Co, Ltd.). It was later quantified by a NanoDrop (PeqLab Biotechnologie GmbH) and then sent out for sequencing (Eurofins Genomics, Germany). The PCR product was sequenced by the Sanger sequencing method with bacterial universal primers: 27F (5′-GAGTTTGATCCTGGCTCAG-3′). Having the gene been sequenced, sequencing, the sequence was edited by using the BioEdit and submitted to GenBank to have it compared against identified sequences by BLAST. Afterward, edited sequences were submitted to NCBI, and accession numbers were received. Finally, the phylogenic trees of each strain were prepared by MEGA 7.0 through the neighbor-joining method (Tang et al. 2016; Zhao et al. 2018).

Biodegradation assay of PA by strain SHAn2

To ensure the ability of aerobic strain SHAn2 to consume PA under anaerobic conditions, flasks containing carbonate media + 12 mM (2000) PA were prepared under completely anaerobic conditions. The inoculum of this strain with OD578 = 0.5 was also prepared under anaerobic conditions. The flasks were inoculated with the 5% (v/v) SHAn2 inoculum size and incubated at 30 °C. At each sampling time, the bacterial growth rate was measured by spectrophotometer (OD578) and residual PA concentration was measured by UPLC according to the methods described above (Benjamin et al. 2016; Ebenau-Jehle et al. 2017).

Analytical method

Extraction of PA

To extract PA from culture media and measure it with UPLC, at each time point, 1 ml of suspension was removed and centrifuged at 20,000 rpm for 10 min and the upper phase was separated. After that, to acidifying (pH = 2), 100 µl of the upper phase was mixed with 900 µl of 1 M HCL. Then, 10 μl of the resulting mixture plus 40 μl of acetonitrile were injected into the UPLC (Ebenau-Jehle et al. 2017; Zhao et al. 2018).

UPLC

Detection and determination of PA were carried out with an Acquity UPLC system coupled to a photodiode array detector. Then, 10 µL of the organic phase and 40 µL acetonitrile were mixed and analyzed on an Acquity UPLC System (Waters) using a BEH-C18 column (Waters, MA, USA) with a column temperature of 30 °C. The chromatographic separations were performed using an acetonitrile + 0.1% FA / water + 0.1% FA gradient at a flow rate of 0.3 mL min−1. The separation was accomplished by increasing the amount of solvent A from 5 to 100% within 6.5 min. PA was identified at 275 nm at a retention time of 5 min. Chromatograms were analyzed using the Empower TM3 (Waters) software. The residual concentrations of PA in the liquid culture were calculated by extrapolating the peak area with standard curves (Ebenau-Jehle et al. 2017; Zhao et al. 2018).

Data analysis

Data related to the biodegradation of PA were collected by three replicates. All figures and chromatograms were produced by Prism 8.0.

Results and discussion

Enrichment process and finding the best consortium

As it can be seen in Table 1, different flasks containing phosphate and carbonate media, sediment supplemented by PA isomers (isophthalic acid, orthophthalic acid, and terephthalic acid), and nitrate were prepared. Carbonate media + sediment without PA and nitrate was also considered as the control. At zero time, no nitrite was detected in the medium, while after 14 days in the carbonate culture medium, nitrite was observed only in orthophthalic acid treatment. Besides, nitrite was found in carbonate media containing isophthalic acid and terephthalic acid after 21 days. When it comes to phosphate media, nitrite was observed in all treatments even in control, so flasks containing carbonate media were selected for further test. Afterward, 5 mL of the last suspension was added to the fresh carbonate media containing PA isomers to prepare the inoculum. As shown in Fig. 1, the microorganisms showed a high ability to break down orthophthalic acid. Bacterial growth (OD578) reached its maximum after 5 days in the presence of orthophthalic acid. Multiple tests revealed that the microbial growth of the consortium reached its maximum after 10 and 20 days in the presence of isophthalic acid and terephthalic acid, respectively. However, the concentration and the peaks of these two isomers could not be detected using UPLC (Figs S3 and S4). Therefore, for further studies, the consortium related to orthophthalic acid was selected and named AnL1.

Table 1 Sediment enrichment with PA isomers in carbonate (C) and phosphate (P) media containing NO3
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Fig. 1
figure 1

a Growth curve of microorganisms in the presence of orthophthalic acid, and b turbidity created in carbonate + consortium AnL1 + orthophthalic acid culture medium

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Biodegradation of PA by consortium AnL1 under anaerobic condition

As shown in Fig. 2, the consortium AnL1 was able to break down 82.5% of 4 mM phthalate (664 mg/L) in 100 h. The results revealed that during the incubation time, PA was degraded by the consortium AnL1, the turbidity of the bacteria increased, and the concentration of PA decreased. Furthermore, the PA biodegradation capability of consortium AnL1 was measured by UPLC. The consortium (1% v/v) could biodegrade 86.86% of 1000 mg/L PA in 150 h under anaerobic conditions without shaking (Fig. 3). In the next step, the degradation of 3200 mg/L PA by the microbial consortium AnL1 (5% v/v inoculum) was investigated under anaerobic conditions (Fig. 4). This consortium could degrade 79.96% 3200 mg/L PA in 166 h. To ensure the ability of this consortium to consume PA under anaerobic conditions, after 174 h, 3700 mg/L of PA was added to the flasks under completely sterile and anaerobic conditions. The results showed that after 16 h, this consortium could degrade 54.90% of the input PA.

Fig. 2
figure 2

Growth curve of AnL1 consortium in the presence of 4 mM (660 mg/L) PA under anaerobic conditions

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Fig. 3
figure 3

a Growth curve of AnL1 consortium in the presence of 6 mM (1000 mg/L) PA under anaerobic conditions measured by UPLC and b PA chromatograms at each time points

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Fig. 4
figure 4

a Growth curve of consortium AnL1 in the presence of 19 mM (3200 mg/L) PA under anaerobic conditions measured by UPLC and b PA chromatograms at each time points

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Cultivable bacteria of the consortium AnL1

Two pure aerobic and anaerobic bacterial strains were isolated from Anzali international wetland after getting enriched in PAEs for one month and designated as SHAn1 and SHAn2, respectively (Fig. 5). SHAn1 is a facultative anaerobic denitrifying, motile, gram-negative, and not spore-producing bacteria. Cells of this strain were rod-shaped (0.6–1.01.2–2.5 µm). According to the BLAST result of its 16S rRNA gene sequence (1398 bp), SHAn belongs to the genus Azoarcus sharing the highest 16S rRNA identity of 99.00 to Azoarcus buckelii strain U120. The results of the ezbiocloud database showed that strain SHAn1 belongs to Aromatoleum aromaticum strain EbN1 with a similarity of 99.93%. Based on Ezbioclude results and phylogenic tree (Fig. 6a), the partial 16S rRNA gene sequence of Aromatoleum aromaticum strain SHAn1 was registered in NCBI GenBank with the accession number MW322985. Azoarcus buckelii was recently reclassified within the Aromatoleum genus (Rabus et al. 2019; Ebenau-Jehle et al. 2017). SHAn2 was a gram-negative bacilli, aerobic, oxidase-positive, non-fermentative, rod-shaped (0.5–0.6 X 1.5–3.0 µm), motile, and not spore-producing bacteria with the colony color spectrum of white to cream. Based on the BLAST result of its 16S rRNA gene sequence (1420 bp), SHAn2 belongs to the Ralstonia pickettii. The partial 16S rRNA gene sequence of Ralstonia pickettii strain SHAn2 was registered in NCBI with the accession number MW290933. In comparison to other studies, which isolated Ralstonia pickettii strains from different places such as agricultural soil (Cheneby et al. 2000) biofilms in plastic water pipes (Ryan et al. 2007), refinery sludge (Al-Zuhair and El-Nass 2012), rhizosphere (Kailasan and Vamanrao, 2015), Mn ore wasteland (Huang et al. 2018), and the sediment of mangrove (Perpetuo et al. 2020), we have isolated and identified this species from sediments of a natural wetland (Anzali international lagoon) which is polluted by DEHP and DBP. Also, The microbial composition of consortium AnL1 (Ralstonia picketti and Aromatoleum aromaticum) is novel between phthalic acid degrading consortia. Furthermore, this is the first report concerning the microbial community and cultivable bacteria of sediments of Anzali international wetland.

Fig. 5
figure 5

Colonies of a Aromatoleum aromaticum strain SHAn1 and b Ralstonia pickettii strain SHAn2 on phosphate media + Agar + PA

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Fig. 6
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a Phylogenic trees of a Aromatoleum aromaticum strain SHAn1 (MW322985) and b Ralstonia pickettii strain SHAn2 (MW290933) isolated from Consortium AnL1

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Biodegradation of PA by Rasltonia pickettii strain SHAn2

Aromatoleum aromaticum is a well-known bacteria in biodegradation of a wide range of organic pollutants especially, PA (Rabus and Widdel 2002; Mechichi et al. 2002; Ebenau-Jehle et al. 2017; Mergelsberg et al. 2018; Boll et al. 2020). However, this study for the first time investigates the biodegradation of PA by one aerobic bacteria Ralstonia pickettii strain SHAn2 under anaerobic conditions. The growth curve (Fig. 7) of strain SHAn2 showed that during the incubation time, while the PA concentration decreased the turbidity increased due to the bacterial growth. Ralstonia pickettii strain SHAn2 was found to be capable of biodegrading 81.57% of 2000 mg/L PA after 80 h. Our study demonstrated that Ralstonia picketti strain SHAn2 has a high ability in the biodegradation of PA under anaerobic conditions when NO3− is used as the electron acceptor. Our results have been confirmed by many researchers that mentioned the high potential of Ralstonia pickettii as the candidate of bioremediation that can survive in harsh conditions with a very low level of nutrients (Ryan et al. 2007). Other studies have so far made it clear that Ralstonia pickettii species can biodegrade Toluene (Kahng et al. 2000), Benzene (Bucheli Witschel et al. 2009), chlorobenzene (Zhang et al. 2011), phenol (Al-Zuhair and El-Nass 2012), chlorophenols (Arora and Bae 2014), Diazinon (Wang and Liu 2016), DDT (Purnomo et al. 2019a, b), biphenyl (Li et al. 2019), PAHs (Li et al. 2020; Sangharak et al. 2020), and Crude oil (Purnomo et al. 2020). To the best of our knowledge, reports of the degradation of phthalates and plastic compounds by this species are very limited. The only research on phthalate biodegradation by Ralstonia pickettii showed that it could degrade 300 mg/L diethyl phthalate (DEP) in aerobic conditions (Perpetuo et al. 2020). As a novel study, we proved that this bacterium (Ralstonia picketti) can also degrade phthalic acid under anaerobic conditions, so another contaminant was added to the list of substances that can be degraded by this bacterium (Ralstonia picketti). The denitrifying capability of Ralstonia pickettii strains and related genes was confirmed by many researchers (Cheneby et al. 2000; Marcus et al. 2006; Takaki et al. 2008, Han et al. 2012; Christianson et al. 2015). Furthermore, R. pickettii are famous for biodegradation of a wide range of organic compounds and consuming them as the sole carbon source due to its ability to survive under both aerobic and oxygen-limited denitrifying conditions (Leahy et al. 1997; Bruins et al. 2000; Baere et al. 2001; Park et al. 2002; Bucheli-Witschel 2009; Purnomo et al. 2018). Udayappan et al. (2017) stated that Ralstonia genus comprises pickettii species that are facultative anaerobic bacteria. Overall, the literature reviews mentioned above have confirmed the degradation of PA by Ralestonia pickettii strain SHAn2 (MW290933) under nitrate-reducing conditions. In comparison with other PA-degrading bacteria in anaerobic conditions, Ralstonia pickettii SHAn2 was more capable and could degrade PA at a faster pace and a higher level of concentration (Table 2).

Fig. 7
figure 7

a Growth curve of Ralstonia pickettii strain SHAn2 in the presence of 12 mM (2000 mg/L) PA under anaerobic conditions measured by UPLC and b PA chromatograms at each time points

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Table 2 Comparison PA biodegradation Ralstonia pickettii in aerobic condition with other well-known strains
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Conclusion

Phthalic acid esters and their derivatives, such as PA, are the main and most widely used plasticizers that enter the human environment. The half-life of these compounds increases in aquatic ecosystems and anaerobic conditions. During the complete degradation of PAEs, the conversion step of PA to H2O and CO2 is the most limiting step. The biodegradation of PAEs and PA from nature through using native microorganisms is the best and most ideal way in eliminating phthalates. In the recent study, consortium AnL1 with the ability to degrade high concentrations of PA (5% inoculum size, 3500 mg/L of phthalic acid) under anaerobic conditions was isolated from sediments of Anzali international wetland that is severely affected by DEHP and DBP contamination. In course of the study, two denitrifying bacteria Aromatoleum aromaticum strain SHAn1 (MW322985) and Ralstonia pickettii strain SHAn2 (MW290933) that are the well-known bacteria in the degradation of aromatic compounds were isolated from consortium AnL1. Surprisingly, results demonstrated that aerobic bacteria Ralstonia pickettii strain SHAn2 could degrade a high concentration of PA under anaerobic conditions. Our findings showed that these bacteria have the potential to be applied in upcoming bioremediation plans of constructed wetlands and then natural wetlands or other ecosystems. However, adding the inoculum of these bacteria to nature requires more comprehensive and supplementary studies in future.