Bacteria from Jatropha curcas rhizosphere, degrades aromatic hydrocarbons and promotes growth in Zea mays

Abstract The rhizosphere is one of the most important reservoirs of microorganisms. Because of the microbial metabolic activities, these can be used for various biotechnological, agricultural and environmental purposes. In this study we evaluated five genetically related bacterial strains; Pseudomonas aeruginosa N7B1 (MG457074), Pseudomonas sp. (MG457075), Pseudomonas sp., Bf1 (MG457076) and Pseudomonas aeruginosa F23 (MG457077), isolated from Jatropha curcas rhizosphere, capable of growing and degradating benzene and phenanthrene. The hydrocarbon degradation by these strains was quantified by gas chromatography coupled to mass spectrophotometry. The Pseudomonas aeruginosa N7B1 strain removed 84% of phenanthrene and 45% of benzene in a seven-day period, while the other strains showed a lower hydrocarbon degradation capacity. Another biotechno-logical feature of these strains is maize growth promotion, in a substrate enriched with 0.5% of phenanthrene and 1.0% of benzene. Pseudomonas aeruginosa N7B1 and Pseudomonas aeruginosa F23 showed an increase in root and shoot fresh and dry weight, plant height and root length variables. These results open the possible use of these strains as bioinoculants to promote the growth of maize plants in phenanthrene and benzene polluted soils.


Introduction
Nowadays, great concern has been expressed over the accumulation of Polycyclic Aromatic Hydrocarbons (PAH´s), which are pollutants that include aromatic rings arranged in linear, angular, or clustering forms (Li et al. 2014; Chen et al. 2015). PAH´s have teratogenic, carcinogenic, and mutagenic properties and can pose a huge threat to human health through bioaccumulation in the food chain (Moscoso et al. 2012). This usually occurs in industrial sites and results from the incomplete combustion of organic materials such as coal, oil, and wood. Most PAH´s are recalcitrant in the environment due to their high resistance to nucleophilic attack and low bioavailability (Yuan et al. 2002;Zhang et al. 2006;Cheng et al. 2016).
The elimination of PAH´s using biological candidates, such as microorganisms, is generally preferred because of their ecological nature and profitability. However, a few bacterial genera are capable of degrading polycyclic and heterocyclic aromatic hydrocarbons, particularly anthracene, phenanthrene, and dibenzothiophene (Smalla et al. 2001). It has been suggested that during biodegradation, some microorganisms consume aromatic com-pounds through a series of intrinsic pathways and use them as energy sources, thereby detoxifying contaminants (Gałązka and Gałązka 2015). Muratova  Because of the rhizospheric microorganisms' ecological role and plant-microbe interaction above cited, this study aim was to evaluate the degradation capacity of phenanthrene and benzene by four Pseudomonas strains, to quantify the polyphenol oxidase activity and to compare the growth-promotion in maize under phenanthrene and benzene polluted conditions.

Origin, isolation and 16S rRNA molecular identification of the bacterial strains
Bacterial strains isolated from Jatropha curcas L. rhizosphere, were isolated from plant samples collected in Chiapas, Mexico. Approximately one gram of root was placed into tubes containing 9 ml of 10 mM MgSO 4 .7H 2 O, which were shaken vigorously in order to suspend the rhizosphere microorganisms. From this suspension, 200 μL were inoculated on Baz semisolid medium (0.2% Azelaic acid, 0.02% L-citrulline, 0.04% K 2 HPO 4 , 0.04% KH 2 PO 4 , y 0.02%, MgSO 4 .7H 2 O), and incubated at 28°C for one week, this procedure was repeated twice. After that, bacteria were reseeded and incubated at 28°C for 72 h on solid culture medium supplemented with cycloheximide BAc (100 mg/mL) enriched with phenanthrene 0.5% and benzene 1.0% as only carbon source. The bacterial colonies that were capable of growing on PAH´s and showed different morphology, were purified in BAc medium, the purity of isolated bacterial colony was verified in PY medium. Pure colonies were stored in 70% glycerol at -70°C.
The next procedure with the isolated bacteria was molecular identification by amplification of 16S rRNA gene, using rD1 and fD1 oligonucleotides (Weisburg et al. 1991). PCR conditions were as follows: initial denaturation for 5 min at 94°C; followed by 30 cycles of 30 s of denaturation at 94°C, 45 s of annealing at 60°C, and 1 min of elongation at 72°C; followed by a final 5 min elongation at 72°C. The acquired 970-1000 bp portions of the 16S rRNA gene sequences were deposited in GenBank with MG457074 (Pseudomonas aeruginosa N7B), MG457075 (Pseudomonas sp.), MG457076 (Pseudomonas sp., bf1) and MG457077 (Pseudomonas aeruginosa F23) accession numbers.

Biodegradation kinetics in Bushnell Haas Broth medium (BHB)
Previous to biodegradation kinetics assays, bacterial strains were grown in PY (Peptone Yeast Extract) medium for 24 h at 29°C and shaken at 200 rpm in order to produce inoculum biomass. Then bacterial strains were adjusted to an optical density of 0.2 (10 6 CFU/mL) to 600 nm, and inoculated into flasks containing 200 mL of Bushnell Haas Broth medium (BHB), its composition in g/L is: 0.2 MgSO 4 ·7H 2 O, 0.02 CaCl 2 . 2H 2 O, 1.0 KH 2 PO 4 , 1.0 K 2 HPO 4 , 1.0 NH 4 NO 3 , 0.05 FeCl 3 , and phenanthrene 0.5% and benzene 1.0% as a carbon source. As a positive control, 5.0 g of sodium succinate was used as soluble and biodegradable carbon source. Three independent experiments were carried out with three repetitions each.

Determination of bacterial growth by viable count
Bacterial strains were cultured in BHB media for 168 h at 29°C at 200 rpm, bacterial growth during the aromatic compounds degradation process was determined by plate count method after decimal dilutions, 100 μL of the dilutions was inoculated in Petri dishes with PY culture medium, incubated at 29°C for 48 h; measurements were performed at 0, 24, 48, 72, 96, and 168 h. Three independent experiments were carried out with three repetitions each. The results were analyzed by means comparison with Tukey test, with a significance level of P≤0.05, using "Statistical Analysis System" (SAS) software.

Evaluation of enzymatic activity
The polyphenol oxidase activity was determined using a reaction mixture with 100 mM catechol in 200 mM sodium acetate phosphate buffer solution pH 6.2, and the enzymatic extracts of the aliquots taken every 24 h for 7 days. Oxidation of catechol, was measured spectrophotometrically by an increase of absorbance at 420 nm for 2 min.

Extraction of aromatic hydrocarbons from biodegradation assays in liquid bacterial cultures
The benzene and phenantrene degradation was quantified at 0, 24, 48, 72, 96 and 168 h. From liquid bacterial cultures aliquots of 5 mL were taken, and after that a solid phase extraction technique (SPE) was used in its cartridge mode with C18. The extraction columns were pre-activated with 5 mL methanol, 5 mL hexane and 5 mL mili-Q water, 50 mL of the aqueous sample was passed through the SPE tubes at a pressure of 25 psi and flow rate of 1-2 mL/min. Then 5 mL of mili-Q water was added. Elution of PAHs was performed with 10 mL hexane. The extract was concentrated to dryness under a high argon flow of 99.997% purity (INFRA®) and the extract was resuspended with 1 mL of acetonitrile. The concentrated extract was stored in a 2 mL amber borosilicate vial with screw cap and PTFE septum, cooled to 4°C until chromatographic analysis.

Polycyclic Aromatic hydrocarbons analysis by Gas Chromatography coupled to Mass Spectrometry (GM-MS)
A gas chromatograph (Thermo Trace GC Ultra) coupled to an ion trap mass spectrometer (ITQ 900) (Thermo Fisher Scientific Inc, Austin, TX USA) was used for the determination of AHs. The operating conditions of GC-MS were as follows: 2 μl of sample was used per analysis. The injec-tion was in splitless mode with an autosampler (TriPlus Autosampler). The temperature of the injector was 250°C and the transfer line was 270°C. The temperature of the ion source was 200°C. The components were separated in a capillary column; model TR-5MS, 5% phenylmethylsiloxane (30 m long x 0.25 mm internal diameter) with 0.25 μm thick stationary phase. The oven temperature ramp was at an initial temperature of 70°C held for 1 min, followed by a gradient of 15°C/min to reach 180°C maintained for 1 min, subsequently temperature increments of 5°C/min to 270°C maintained for 15 min (running time: 42 min). The carrier gas was ultra-high purity helium (INFRA) (99.9999%). The mass detector operated in selective ion monitoring mode (SIM).

Growth promoting of Zea mays with Pseudomonas strains PAH´s biodegraders
The Pseudomonas aeruginosa N7B1 (MG457074), Pseudomonas sp. (MG457075), Pseudomonas sp., bf1 (MG457076), and Pseudomonas aeruginosa F23 (MG457077) strains were grown in PY medium for 24 h, 29°C at 200 rpm, then the bacterial cultures were adjusted to an optical density of 0.5 to 600 nm. The corn seeds were disinfected with a 10% hypochlorite solution for 15 min; excess chlorine was removed with washes of sterile distilled water. The seeds were subsequently incubated for 72 h, and maize seedlings were planted in pots containing sterile vermiculite with benzene at 1.0% and phenanthrene 0.5% respectively. Maize plants were inoculated with one milliliter of the bacterial culture, additionally plants were watered with Farheus nutritional solution every third day during four weeks under greenhouse conditions. The following variables were quantified: height, fresh weight and dry weight of roots and aerial part, stem diameter, root length. Experiments were independently performed in triplicate.

Statistical analysis
All data were expressed as a mean of randomized block design experiment under greenhouse conditions. Significant differences in height, root, length, stem diameter, aerial fresh weight, aerial dry weight, root fresh weight, and root dry weight were tested by a one-way analysis of variance (ANOVA) to compare all treatments over the control. The test was followed by Tukey's post-hoc test (where P<0.05). The statistical analysis was carried out using the "Statistical Analysis System" (SAS). Figure 1 shows the kinetic growth with benzene and phenanthrene as the only carbon source. All bacterial strains tested, Pseudomonas aeruginosa N7B1 (MG457074), Pseudomonas sp. (MG457075), Pseudomonas sp., bf1 (MG457076), and Pseudomonas aeruginosa F23 (MG457077), showed typical growth and were capable of growing in both benzene 1.0 % and phenanthrene 0.5%; they showed similar behavior with respect to the control treatment (with sodium succinate).

Bacterial growth in benzene and phenanthrene
After 48 h of culture, Pseudomonas aeruginosa N7B1 and Pseudomonas sp. reached an exponential growth with a magnitude order 10 11 CFU/mL higher than the exponential growth reached by Pseudomonas sp., bf1, and Pseudomonas aeruginosa F23, with a magnitude order of 10 9 CFU/mL. However, between treatments (benzene, phenanthrene, and sodium succinate), there were not statistical differences in the growth of all bacterial strains tested. All bacterial strains finished their growth after 96 h of culture.

Enzymatic activity
Polyphenol oxidase activity was observed at different times intervals during bacterial growth on benzene and phenanthrene. The maximum enzymatic activity was observed in cell-free extracts of Pseudomonas aeruginosa F23 (MG457077) and Pseudomonas aeruginosa N7B1 (MG457074) cultured at 72 h for benzene and 48 h for phenanthrene with (Figure 2). Whereas with Pseudomonas sp. (MG457075), and Pseudomonas sp., bf1 (MG457076) strains, the highest enzymatic activity was observed at 72 h with both substrates (Figure 2).

Pseudomonas strains inoculation and aromatic hydrocarbons effect, on maize growth
After inoculation of all four Pseudomonas strains in maize plants grown in the presence of benzene 1.0% and phenanthrene 0.5%, it was observed that Pseudomonas aeruginosa F23 has the best growth promotion effect (see Table  1). In the presence of benzene and inoculated with Pseudomonas aeruginosa F23, an increase of 16% in height, 26% stem diameter, 6% root length, 40% foliage fresh weight, 108% foliage dry weight, 188% root fresh weight, and 116% root dry weight, was observed compared to maize plants that were only treated with benzene (Table  1).
Also, in maize plants treated with phenanthrene and inoculated with Pseudomonas aeruginosa F23 the best growth promoter effect was observed. According to evaluated variables, it increased 20% highness, 30% stem diameter, 35% root length, 21% foliage fresh weight, 32% foliage dry weight, 94% root fresh weight and 47% root dry weight, with respect to maize plants that were treated with phenanthrene exclusively ( Table 2).

Discussion
Studies on rhizosphere are being focused on exploration of plant-growth promoting bacteria that additionally can degrade organic recalcitrant compounds. Plant-microorganism interactions are happening in the rhizosphere, where there is a liberation of radical exudates, which In this study, four bacterial strains belonging to Pseudomonas genus isolated from Jatropha curcas rhizosphere, were capable of growing and biodegrading PAH´s. They reached a maximum growth rate at 72 h in BHB medium (Figure 1). The microbial growth of Pseudomonas aerug-inosa N7B1 (MG457074), Pseudomonas sp. (MG457075), Pseudomonas sp., bf1 (MG457076), and Pseudomonas aeruginosa F23 (MG457077) strains in benzene 1.0% and phenanthrene 0.5% presence was very similar between them and with respect to the control, which had sodium succinate, a three carbons source easily metabolizable by bacteria in the rhizosphere. These results agree with those  In this study, the Pseudomonas aeruginosa F23 (MG457077) strain isolated from Jatropha curcas rhizosphere had the capacity to remove 84% phenanthrene and 45% benzene ( Figure 3A); Yuan et al. 2002 reported that Pseudomonas fluoresens and Haemophilus spp., degrade 70-100% anthracene, acenaphthene, fluorene, phenanthrene, and pyrene; however, the origin of these strains is a petrochemical residues polluted environment, the reason why they are adapted to the presence of these compounds. de Lima et al. (2016) reported that Pseudomonas veronii 1YdBTEX2 and 1YB2 contain a 2-hydroxy muconic-dehydrogenase component for a single catabolic pathway for benzene degradation.
There are also reports of Pseudomonas sp., JPN2 strain degrading 98.52% phenanthrene at 100 ppm initial concentration after 10 days of incubation (Jin et al. 2016). Although the degradation percentage of these strains with respect to Pseudomonas aeruginosa F23 (MG457077) strain is higher, the degradation rate is 10 times higher and the performance time lapse is shorter. More relevant data from these rhizosphere isolates is that they come from uncontaminated sites, so they are not adapted microorganisms to these compounds presence. Nevertheless, they had the ability to remove up to 84% phenanthrene and 45% benzene ( Figure 3A). Other relevant characteristics of these Pseudomonas strains are the production of biosurfactant, indoleacetic acid, phosphate solubilization, and metal resistance (Wong-Villarreal et al. 2015).
Due to the degradation and plant growth promotion characteristics of Pseudomonas aeruginosa N7B1, Pseudomonas sp., Pseudomonas sp., bf1, and Pseudomonas aeruginosa F23 strains, were inoculated in maize plants, being Pseudomonas aeruginosa N7B1 (MG457074) and Pseudomonas aeruginosa F23 (MG457077) which had an effect on fresh and dry weight increment in roots and shoots, plant height and root length of maize plants increase. Therefore, native strains of Jatropha curcas rhizosphere identified as Pseudomonas aeruginosa N7B1 and Pseudomonas aeruginosa F23 have a clear potential as bio-inoculants to improve the maize plants productivity in the presence of phenanthrene and benzene.

Conclusions
The isolated bacteria from Jatropha curcas plants rhizosphere grew in the presence of hydrocarbons. After seven days, the Pseudomonas aeruginosa N7B1 strain eliminated 84% of phenanthrene and 45% of benzene, and showed the highest capacity of the evaluated strains. The Pseudomonas aeruginosa F23 and Pseudomonas aeruginosa N7B1 strains had a growth promoting effect in maize plants that grew in the presence of 1.0% benzene and 0.5% phenanthrene, this could be observed in the increase of the values of the agronomic variables evaluated. These results open the possibility of using these strains as biofertilizers in soils contaminated with aromatic hydrocarbons.