Three chlorotoluene-degrading bacterial strains: Differences in biodegradation potential and cell surface properties

Chemosphere 237 (2019) 124452

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Three chlorotoluene-degrading bacterial strains: Differences in biodegradation potential and cell surface properties Wojciech Smułek a, *, Zefiryn Cybulski b, Urszula Guzik c, Teofil Jesionowski a, Ewa Kaczorek a
, Institute of Chemical Technology and Engineering, Faculty of Chemical Technology, Poznan University of Technology, Berdychowo 4, 60-965 Poznan Poland b
, Poland Department of Microbiology, Greater Poland Cancer Centre, Garbary 15, 61-866 Poznan c
ska 28, 40-032 Katowice, Poland University of Silesia in Katowice, Faculty of Biology and Environmental Protection, Department of Biochemistry, Jagiellon a

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Newly-isolated bacterial strains are efficient chlorotoluenes-degraders
Changes in the cell surface properties indicated the strains adaptation mechanisms
The isolated strains differed significantly in the cellular fatty acids profile
The studied strains have a great potential to be used in the chlorotoluenes removal

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 November 2018 Received in revised form 31 May 2019 Accepted 24 July 2019 Available online 26 July 2019

Pollution of the environment with chlorinated aromatic compounds is a problem of increasing importance, which has stimulated the search for efficient methods for the remediation of contaminated soil and water. Additionally, for better understanding of the significance of bioavailability to biodegradation, investigation of the cell surface properties is necessary. Hence, this study concerns the properties and possible application, in chlorotoluene removal, of three newly isolated environmental bacterial strains from the genera Pseudomonas, Raoultella and Rahnella. The results show the differences in the biochemical profiles of the isolated strains, their cellular fatty acid composition and their hemolytic properties. However, all three strains exhibit high biodegradation potential, degrading not less than 60% of each monochlorotoluene isomer in 21-day experiments. What is more, observations of changes in the cell surface properties indicate the possible adaptation mechanisms of the strains that enable efficient biodegradation of hydrophobic pollutants such as monochlorotoluenes. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: Chang-Ping Yu Keywords: Biodegradation Cell surface properties Cellular fatty acids Chlorotoluene

1. Introduction Among all living organisms, it is the microorganisms, especially bacteria, that demonstrate the greatest ability to adapt to different

* Corresponding author. E-mail address: [email protected] (W. Smułek). https://doi.org/10.1016/j.chemosphere.2019.124452 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

conditions in various ecosystems. Specific bacterial strains inhabit extreme ecological niches, from hot springs to the water of lakes under glaciers (Chen et al., 2016; Poli et al., 2017). The adaptation of microorganisms also concerns the ability to use various organic compounds as carbon and energy sources. Because of their key role in the self-cleaning of the natural environment from toxic contaminants, the strains capable of biodegrading persistent organic

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and Dercova
, pollutants attract a great deal of interest (Murínova 2014). One of the most important groups of organic aromatic compounds of industrial significance are the chloroaromatics. Chlorotoluenes, chloronaphthalenes and chlorobenzene are used as solvents and substrates in the production of pesticides, dyes and disinfectants. It should be noted that global production of chlorotoluene isomers alone reached 130,000 tons in 2000 e of which 65,000 tons constituted a mixture of all three isomers e and that this production is increasing (Dobslaw and Engesser, 2012). The compounds, 2-chlorotoluene and 4-chlorotoluene, are precursors in the synthesis of active ingredients of drugs, pigments and cleaning agents (Rossberg et al., 2006). Contact of living organisms with chlorotoluene isomers leads to weakening of the body, and breathing disorders in mammals (Rossberg et al., 2006). Moreover, chloroaromatic compounds may have toxic effects on microorganisms present in the environment (Lu et al., 2011). Therefore, their removal from the environment is extremely important. The use of biodegradation processes for this purpose not only permits removal of harmful substances from the environment, but also allows them to be transformed into nontoxic compounds or leads to their complete mineralization to inorganic compounds (Bhatt et al., 2007). Regardless of the metabolic pathway used by bacteria, one of the main factors limiting the rate of biodegradation is the bioavailability of contaminants to microorganism cells. In the field of environmental protection, bioavailability is understood as the quantity of a polluting chemical compound that can be collected and biodegraded by microorganisms. Hence, if the bioavailability of a compound is high in given conditions, its biodegradation is limited only by the rate of the biochemical reactions that make up the biodegradation pathway (Maier, 2000). In the biodegradation of aromatic compounds and their chlorinated derivatives, the most important factors include their hydrophobicity and low solubility in water. These parameters significantly limit the bioavailability of these compounds to cells. In addition, halogen derivatives of benzene and toluene undergo significant sorption on soil particles and sediments in water reservoirs, and their low rate of biodegradation leads to their accumulation (Jahan et al., 1999). A relatively small proportion of naturally occurring microorganisms are able to degrade persistent organic pollutants. Bacterial species capable of biodegrading chlorinated aromatic compounds include certain strains from different genera, like Pseudomonas, Burkholderia (Field and Sierra-Alvarez, 2008), Alcaligenes (Field and Sierra-Alvarez, 2004), Achromobacter (Chaudhry and Chapalamadugu, 1991) or Comamonas (Duc, 2017). What is important, in the environment, e.g. soils, wetlands, river sediments, biodegradation is carried out by consortia of bacteria species mainly from described genera (Chen et al., 2015) Nevertheless, in these publications there is practically no information about biodegradation efficiency. Kaczorek et al. (2016) isolated the strain from Raoultella genera, which was able to use toluene and its three monochlorinated derivatives as only carbon and energy source. In turn, Rhodococcus sp. OCT 10 biodegraded only 2-chlorotoluene, but not other isomers, and additionally the biodegradation was observed only at high bacteria concentrations (Dobslaw and Engesser, 2012). The same authors described, that the used strain could be used in three-phase system in biofilters to remove gaseous 2-chlorotoluene (Dobslaw and Engesser, 2018). However, the majority of the above mentioned studies focused on metabolic pathways, but not on the cell surface properties and their impact on biodegradation efficiency. Cell surface properties are considered to have great impact on bioavailability and, consequently on the microbial uptake of hydrophobic compound (Kaczorek et al., 2016).

Hence, the necessity of a deeper insight into the cell surface properties of bacterial strains degrading emerging pollutants, like chlorotoluene. The research work reported in this paper contributes to respond to this necessity. The aim of the study presented was to characterize the properties of bacterial strains demonstrating good ability to biodegrade monochlorotoluene isomers from environmental samples. The first stage of the study included characterization of the isolated strains and assessment of their biodegradation potential. In addition, changes in cell surface properties were identified. These can significantly affect the bioavailability of biodegradable compounds to the cells and, as a consequence, the efficiency of biodegradation. 2. Materials and methods 2.1. Chemicals and culture medium The isomers of chlorotoluene (2-, 3- and 4-chlorotoluene) used in this study were purchased from AlfaAesar (United Kingdom). Other fine chemicals were of per analysis grade and were purchased from Sigma-Aldrich (Germany). Aqueous solutions were prepared with the use of deionized and ultrapurified MiliQ water. The culture medium was prepared according to Dobslaw and Engesser's method (Dobslaw and Engesser, 2012). 2.2. Isolation and identification of bacterial strains 2.2.1. Bacterial strain isolation In order to isolate bacterial strains capable of degrading chlorotoluenes, soil samples contaminated with liquid fuels were taken from different locations in northern Poland. Portions of the soil samples weighing 10 g were suspended in 100 mL of sterile culture medium with the addition of 2 mL of 20% sodium succinate solution. After incubation for 24 h at 30  C, 10 mL of the suspension was transferred to 90 mL of sterile medium, and 1.5 mL of 20% sodium succinate solution and 0.01 mL of a mixture of three chlorotoluene isomers (1:1:1 v/v) was added. Passaging of the culture was repeated every 7 days, successively reducing the amount of succinate added, so that this carbon source was gradually replaced with a mixture of the three chlorotoluene isomers. As a consequence, after one month, the only carbon source was chlorotoluenes at a concentration of 40 mg L1. Refreshing of the culture was continued every 7 days for the next three months, maintaining the aforementioned concentration of chlorotoluene isomers. Furthermore, the strains were tried to isolate them on an agar medium with only chlorotoluene isomers, however, a relatively small amount of biomass was obtained. Of the various microbial solid cultures, Mueller-Hinton agar allowed the most favorable growth of the tested microorganisms, which could effectively biodegrade the chlorotoluene isomers. In the next step, 0.1 mL of the cultures were seeded on MuellereHinton agar medium plates (bioMerieux, Poland) and after 24 h of incubation streaking was used to isolate colonies of individual bacterial strains. The strains were identified using Vitek 2 Compact (bioMerieux, Poland) systems and molecular techniques, according to their 16S rRNA gene sequence (Guzik et al., 2009). 2.2.2. Hemolysis test All three strains were inoculated on plates with Columbia agar containing 5% sheep blood. After incubation for 24 h at 30  C, the color and transparency of the agar medium in the vicinity of the bacterial colonies was observed. These observations enabled assessment of the strains’ ability to cause lysis of red blood cells, which may indirectly indicate the ability of microorganisms to

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produce surface active compounds secreted outside the cell (Youssef et al., 2004; Hassanshahian, 2014).

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according to the method described by Zhang et al. (2013). 2.5. Statistical analysis

2.2.3. Identification of cellular fatty acids Bacterial cells were collected from the tryptone soy agar plates after incubation for 24 h at 30  C and washed twice with 0.85% NaCl to remove residues of the culture medium. Cellular fatty acids were isolated and identified following the MIDI-MIS method, involving analysis of fatty acid methyl esters (FAMEs) using an HP 5890 gas chromatograph (Hewlett Packard, Rolling Meadows, IL, United States) with an HP 25 m  0.2 mm cross-linked methyl-silicone capillary column and helium (1 mL min1) as the carrier gas. The oven temperature program was as follows: initial temperature 170  C, increased at 5  C min1 to 260  C, then at 40  C min1, final temperature 320  C maintained for 1.5 min. Sherlock software (TSBA library, version 3.9, Microbial ID, Newark, NJ, United States) was used for the identification of FAMEs, based on the actual calibration retention times run prior to sample analysis. The unsaturation index (UI) was calculated according to Kaszycki et al. (2013) accordingly to the equation:

UI ¼ ð%16 : 1 þ %18 : 1Þ þ ð%18 : 2x2Þ þ ð%18 : 3x3Þ=100

2.3. Biodegradation test The inoculum for liquid cultures was initiated by adding loops full of cells of each strain from the solid culture into 250 mL sterile glass bottles containing 50 mL of culture medium, 0.1 mL of 2% yeast extract and 1 mL of 20% glucose solution. Then, after 24 h of incubation at 30  C, the cultures were used for inoculation of the final bacterial cultures. In that purpose, the cells were centrifuged (4500 g, 5 min) washed and re-suspended in mineral medium. Thereafter, 18 mL of the medium was placed in 100 mL sterile glass bottles and mixed with 2 mL of the cell suspension (OD600 ¼ 1.0) from the inoculum. Several chlorotoluene isomers were then added to obtain a concentration of 40 mg L1. The cultures were incubated on a rotary shaker at 30  C for 7, 14 and 21 days. After extraction of chlorotoluenes with 20 mL dichloromethane, quantitative analysis of chloroaromatics was performed using a Pegasus 4D GCxGCTOFMS (LECO, USA) gas chromatograph with BPX-5 column (28 m, 250 mm, 0.25 mm). The analysis was performed with helium as a carrier gas (1 mL min1) under a programmed temperature gradient: 50  C for the first 3 min, then increased at 12  C min1 to 140  C, final temperature maintained for 3 min. The quantity of the biodegraded compound in the samples was determined on the basis of a calibration curve, which was plotted using measurements of analogous abiotic samples with known hydrocarbon contents. 2.4. Cell surface properties To evaluate changes in the cell surface properties of the tested strains during chlorotoluene biodegradation, the cell surface hydrophobicity (CSH), zeta potential and inner membrane permeability were measured. Bacterial cultures were prepared analogously as for the biodegradation test. After 7 days of incubation, the cultures were centrifuged (8000 g, 5 min) and washed twice with medium to remove residual chloroaromatics. Cell surface hydrophobicity was evaluated by the MATH method (Kaczorek et al., 2016). The zeta potential of the cells in suspensions was calculated from the Smoluchowski equation (Sze et al., 2003) following electrophoretic mobility measurements made using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., United Kingdom). The inner membrane permeability assay was performed

Each described experiment was repeated three times, and mean values and statistical errors were computed. All results underwent statistical analysis using SigmaPlot 11.0 software. Graphs were prepared using Microsoft Excel 2013. 3. Results and discussion 3.1. Identification and characteristics of strains 3.1.1. Identification and biochemical profile Isolated bacterial strains that were found to grow in the presence of chlorotoluene isomers as the only carbon and energy source were identified as Raoultella planticola SA2, Rahnella aquatilis DA2 and Pseudomonas sp. MChB (Fig. 1). Their nucleotide sequences have been deposited in the GeneBank database of NCBI (Table 1). All these strains are Gram-negative. Environmental strains from the Raoutella, Pseudomonas and Rahnella genera often play an important role in processes of biodegradation of persistent pollutants, including aromatic compounds. Microorganisms belonging to the Pseudomonas genus are among the most common microorganisms found in soils and waters containing hydrocarbon contaminants, such as polychlorinated
sova
et al., 2014), dioxins (Hanano et al., 2014), biphenyls (Duda pesticides, phenol derivatives (Wasi et al., 2013) or hydrocarbons present in crude oil (Tanase et al., 2013). Raoultella bacteria have been used in the biodegradation of drugs such as diclofenac (Domaradzka et al., 2016), and dyes such as 4-nitroaniline (Kausar et al., 2016). Strains of the Rahnella genus also have the ability to degrade aromatic compounds. The Rahnella aquatilis DX2b strain has been shown to be capable of degrading dyes used in the textile industry (Han et al., 2012). Marecik et al. (2008), in a study on the biodegradation of atrazine, have reported that the tested microorganisms capable of degrading that herbicide included three different strains of the species R. aquatilis. In another study, bacterial strains belonging to the same genus were isolated from soil samples taken from the vicinity of an oil refinery, and demonstrated to have the ability to degrade hydrocarbons (Pacwa-Płociniczak et al., 2016). The phenotype of bacterial strains was determined from, among other indications, an analysis of the course of selected biochemical reactions. The Vitek 2 system (bioMerieux, Poland) used to evaluate the biochemical profile of isolated strains is a popular tool used for _ this purpose (Ksia˛ zczyk et al., 2016). In the used method, 46 biochemical reactions were studied. Among them 21 reactions, like neutralization of L-lactic acid or absence of production of hydrogen sulfide, lipase or b-glucuronidase, gave analogous results for all three strains (Table 2). However, the courses of the remaining reactions were different for different strains. That diversity was confirmed by a comparison of biochemical profiles of other chloroaromatic-degrading strains, e.g. Burkholderia phenoliruptrix (Coenye et al., 2004), Defluvibacter lusatiae (Fritsche et al., 1999) or Herbaspirillum chlorophenolicum (Im et al., 2004). This result indicates, that biochemical profile evaluated using standard methods did not allow prediction of the capability of chloroaromatics biodegradation. 3.1.2. Hemolysis The ability of the strains to produce extracellular compounds with surfactant properties was determined on the basis of the hemolysis test (Hassanshahian, 2014). Of the three bacterial strains

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Fig. 1. Neighbor-joining tree showing the phylogenetic position of the chlorotoluene isomer-degrading bacterial strains and related species based on partial 16S rRNA gene sequences. The GenBank accession number for each microorganism used in the analysis is shown in parentheses after the species name. Bootstrap values (expressed as percentage of 100 replicons) are shown at the branch: (a) Rahnella aquatilis DA2, (b) Raoultella planticola SA2, (c) Pseudomonas sp. MChB.

Table 1 List of the investigated strains with accession number in GenBank (NCBI) and strain origin. Genetic identification

GenBank (NCBI) number

Strain origin

Rahnella aquatilis DA2 Raoultella planticola SA2 Pseudomonas sp. MChB

KP096518 KP096517 KU563540

soil from the area around liquid fuel tanks soil from the area around liquid fuel tanks soil from rural areas of ecological cultivation

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Table 2 Positive biochemical reactions of the three investigated strains: Raoultella planticola SA2, Rahnella aquatilis DA2, Pseudomonas sp. MChB. Raoultella planticola SA2

Pseudomonas sp. MChB

Rahnella aquatilis DA2

b-glucosidase production

L-pyrrolydonyl-arylamidase production L-lactate alkalinisation O/129 resistance (comp. Vibrio) succinate alkalinisation D-glucose assimilation mannose assimilation tyrosine-arylamidase production citrate assimilation malonate assimilation

b-glucosidase production L-pyrrolydonyl-arylamidase production saccharose assimilation L-lactate alkalinisation O/129 resistance (comp. Vibrio) maltose assimilation L-pyrrolydonyl-arylamidase production mannitol assimilation D-trehalose assimilation succinate alkalinisation D-glucose assimilation mannose assimilation tyrosine-arylamidase production citrate assimilation D-celobiose assimilation b-xilosidase production malonate assimilation a-galactosidase production b-galactosidase production glucose fermentation D-sorbitol assimilation 5-keto-D-gluconate assimilation phosphatase production

saccharose assimilation L-lactate alkalinisation glicyne arylamidase production O/129 resistance (comp. Vibrio) adonitol assimilation b-N-acetyl-glucosaminidase production maltose assimilation L-pyrrolydonyl-arylamidase production mannitol assimilation D-trehalose assimilation succinate alkalinisation lysine decarboxylation production D-glucose assimilation mannose assimilation tyrosine-arylamidase production citrate assimilation b-N-acetyl-galactosaminidase production Ellman test D-celobiose assimilation b-xilosidase production urease production malonate assimilation a-galactosidase production b-galactosidase production glucose fermentation D-sorbitol assimilation 5-keto-D-gluconate assimilation phosphatase production

investigated in this study, only R. aquatilis DA2 exhibited b-hemolysis (Fig. 2), which may indicate that it produces biosurfactants. The occurrence of b-hemolysis resulting from the action of surfaceactive compounds (Im et al., 2004; Manaargadoo-Catin et al., 2016) may confirm the production of biosurfactants by the microorganisms (Aruko et al., 2002). What is more, the production of biosurfactants by bacterial strains is considered to be one of the adaptive features of the strains that are conducive to the absorption of hydrophobic compounds. The compounds secreted outside the cell can significantly increase the bioavailability of hydrocarbons, for instance by reducing interfacial tension and emulsifying poorly soluble pollutants. An example of a strain exhibiting b-type hemolysis and biodegrading a chloroaromatic compound (2,3,5trichlorophenoxyacetic acid) is Burkholderia phenoliruptrix (Coenye et al., 2004). Literature contains reports on many strains that are capable of effective biodegradation of poorly water soluble hydrocarbons, but do not have the ability to produce biosurfactants

(Hassanshahian, 2014). In the light of the above observations, it should be stated that there is no simple relationship between the production of biosurfactant and the effective biodegradation of hydrophobic impurities. The bioavailability of these compounds can be regulated in a different way, thus surfactants can only perform support functions. 3.1.3. Cellular fatty acids Another parameter characterizing bacterial cells is their cellular fatty acid profile. The content of these acids in the cell membrane of bacteria is affected by a number of environmental factors, such as temperature, pH, osmotic pressure, availability and type of carbon sources, and cell culture age. Modification of the fatty acid profile is also one of the mechanisms by which cells adapt to counteract the negative effects of toxic compounds, including phenol and aliphatic hydrocarbons. Changes in the fluidity of the cell membrane, associated with an increased content of unsaturated, branched, cyclic

Fig. 2. Colonies of tested strains grown for 24 h on Columbia agar with 5% sheep blood: (a) Raoultella planticola SA2, (b) Rahnella aquatilis DA2, (c) Pseudomonas sp. MChB.

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and hydroxy fatty acids, may be a response of cells to the presence of aromatic compounds in their surroundings (Nowak et al., 2016). The isolated bacterial strains differed significantly in the content of individual groups of cellular fatty acids (Fig. 3). The highest content of unbranched saturated fatty acids (above 40%) was found in the cells of the R. planticola SA2 strain. Also in the case of the Pseudomonas sp. MChB strain more than 30% of all fatty acids came from this group. On the other hand, in the cells of the R. aquatilis DA2 strain, the content of saturated unbranched fatty acids was 14%. Among the investigated bacterial strains, the lowest content of branched saturated fatty acids (4%) was found in Pseudomonas sp. MChB cells. Strains with a relatively high content of hydroxy fatty acids include Pseudomonas sp. MChB (15%) and R. planticola SA2 (7%). The content of unsaturated fatty acids was close to 50% in Pseudomonas sp. MChB cells, and only slightly lower (42%) in cells of R. planticola SA2. The difference in the fatty acid composition between R. aquatilis DA2 and the other two strains is also reflected in the unsaturation index (UI). For the first strain this index did not exceed 0.13, while the UI values for R. planticola SA2 and Pseudomonas sp. MChB were three times higher (0.40 and 0.48 respectively). One of the strains described in literature as being capable of degrading chlorine derivatives of aromatic compounds is Herbaspirillum chlorophenolicum. The cells of this bacterial strain, known to biodegrade 4-chlorophenol, contained predominantly saturated 16:0 unbranched fatty acids (34%), 17:0 cyclo (22%) and unsaturated 18:1 u7c (13%) (Im et al., 2004). Oleic acid (18:1 u7c) was also predominant in the cells of Defluvibacter lusatiae, a chlorophenolbiodegrading strain (Fritsche et al., 1999). Moreover, the fatty acids 18:1 u7c and 16:1 u7c dominated in the cells of Rhodopseudomonas palustris, isolated from waste water from a paperproducing plant, and capable of biodegrading 2-chlorophenol (Mutharasaiah et al., 2012). Analysis of the fatty acid profile of Burkholderia phenoliruptrix, which biodegraded 2,4,5-trichlorophenoxyacetic acid, showed the dominance of the fatty acids 16:1 u7c (18%), 16:0 (20%) and 18:1 u7c (38%) (Coenye et al., 2004). A large content (over 20%) of 16:1 u7c and 18:1 u7c fatty acids was also found in the Raoultella and Pseudomonas strains investigated in the present study. One of the features attributed to the cell membranes of Gram-negative bacteria is the relatively low proportion of branched fatty acids. The high content of unsaturated fatty acids allows the cell to modify significantly the fluidity of its membrane by converting cis isomers
and Dercova
, 2014). The literature reto trans isomers (Murínova view and results obtained in our study confirm the diversification

of the fatty acid profile in the cell membrane of strains capable of degrading chlorine derivatives of aromatic compounds. However, a common feature of this group of strains is a large proportion of straight-chain acids, both saturated (including 16:0) and nonsaturated (including 18:1 u7c and 16:1 u7c). At the same time, attention should be paid to the relatively low content of hydroxy fatty acids in the cell membrane. This applies both to the strains described in literature and to those discussed in this paper. This may suggest that these fatty acids may be some kind of strain markers with high resistance to hydrophobic and toxic contaminants, like chlorotoluenes. 3.2. Biodegradation tests A set of biodegradation experiments showed the high potential of all three strains to degrade chlorotoluene isomers. In the case of 2-chlorotoluene (Fig. 4a), the most intensive biodegradation was carried out by Pseudomonas sp. MChB. This strain was able to degrade over 80% of 2-chlorotoluene within 21 days, compared with 51% for R. aquatilis DA2 and 62% for R. planticola SA2 in the same period. However, R. planticola SA2 was the most effective biodegrader of 3-chlorotoluene, reducing its concentration by over 90%. In the first two weeks of the experiment, R. aquatilis DA2 degraded relatively small quantities of 3-chlorotoluene, but after three weeks the degradation yield exceeded 60%. Biological degradation of the last isomer, 4-chlorotoluene, proceeded most effectively in the cultures with R. planticola SA2 and Pseudomonas sp. MChB, with a yield of over 80% in 21 days. As in the experiments with the other two chlorotoluene isomers, the R. aquatilis DA2 strain exhibited lower biodegradation ability than the other two tested strains. It should be emphasized, however, that even this Rahnella strain effectively biodegraded all of the chlorotoluene isomers. The biodegradation of 2-chlorotoluene was the subject of a previous study (Dobslaw and Engesser, 2012). The Rhodococcus sp. OCT 10 strain was able to perform complete dehalogenation of 2chlorotoluene (initial concentration 12.6 mM) within 72 h. However, biodegradation of the other two chlorotoluene isomers by this strain was not observed. Moreover, the biodegradation of toluene occurred only in a co-metabolic system with 2-chlorotoluene as an additional carbon source. This demonstrates the high selectivity of this strain towards aromatic compounds. Haigler and Spain (1989) discussed the biodegradation of chlorine derivatives of benzene by the Pseudomonas sp. JS6 strain. Despite biodegrading chlorobenzene, 1,4-dichlorobenzene and

Fig. 3. Fatty acid content in cells of (a) Raoultella planticola SA2, (b) Rahnella aquatilis DA2, (c) Pseudomonas sp. MChB.

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Fig. 4. Biodegradation of (a) 2-chlorotoluene, (b) 3-chlorotoluene, (c) 4-chlorotoluene by the tested strains.

toluene, these bacteria were not able to degrade 4-chlorotoluene. However, from the culture with 4-chlorotoluene, the Pseudomonas sp. JS21 strain was isolated, formed as a result of a spontaneous mutation from Pseudomonas sp. JS6. It demonstrated the ability to biodegrade a number of aromatic compounds, including 1,4dichlorobenzene, chlorobenzene, benzene, toluene, phenol and

ethylbenzene, as well as all three chlorotoluene isomers. The papers cited here are among only a small number of reports on biodegradable chlorotoluene isomers. Several studies (e.g. Pollmann et al., 2005; Haigler and Spain, 1989; Haro and De Lorenzo, 2001) have been devoted to the detailed analysis of metabolic pathways and genes coding for enzymes involved in the

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biodegradation of chlorotoluene isomers; however, they have not considered the efficiency of biodegradation of these compounds by the described bacterial strains. In contrary, biological degradation of chorinated toluenes was presented by Duc (2017), who found out, that Comamonas testosteroni KT5 utilized more than 90% of toluene as well as 3-chlorotoluene within 60 h at the initial concentration of 2 mM. It indicates several times higher biodegradation ratio, than that those of the strains studied here. Moreover, interesting information bring comparison of biodegradation kinetic of chlorotoluenes-degrading strains. 2-, 3- and 4-chlorotoluene biodegradation rate in cultures with R. planticola SA2 reached 1.5, 2.2 and 2.0 mg L1d1, respectively. In test with R. aquatilis DA2 the biodegradation rate was 1.6 mg L1d1 for all chlorotoluene isomers. Analogically, in case of Pseudomonas sp. MChB strain chlorotoluenes' biodegradation rate did not exceed 2.0 mg L1d1. It should be noticed, that in mentioned publication of Duc (2017) the 3-chlorotoluene biodegradation rate was several times higher than in presented research. However, the strain described by Duc (2017) could biodegrade only one chlorotolene isomer.

cell to limit the penetration of the toxic phenol derivative through the cell membrane. The same mechanism may occur in the Pseudomonas sp. MChB strain. In contrast, Liao et al. (2015) observed that under the influence of pyrene, the permeability of the internal membrane of the Brevibacillus brevis strain increased, leading to the death of some cells. The Stenotrophomonas maltophilia KB2 strain also showed similar effects in the presence of 3-chlorophenol (Nowak et al., 2016). This finding coincides with the results presented in this study for the strain R. aquatilis DA2, demonstrating the increase in permeability of the tested strains in the presence of chlorotoluene isomers. The collected results allow to suppose that the permeability of the cell membrane can be changed by penetration of chlorotoluene isomers between the phospholipids layers. 2- and 4-chlorotoluene, regardless of the initial permeability values, caused that the membrane permeability of all three strains oscillated around 0.2e0.1 mM min1. In turn, 3-chlorotoluene strongly and variously modified the permeability of the cell membrane. This may indicate that the shape of the molecule of this isomer interacts more strongly with the components of the cell membrane than the molecules of the remaining two isomers.

3.3. Cell membrane permeability 3.4. Zeta potential The analysis of inner membrane permeability indicated a variety of cell responses to the presence of different carbon sources (Fig. 5). Both Pseudomonas sp. MChB and R. planticola SA2 strains showed relatively high inner membrane permeability in the cells derived from cultures with glucose (0.47 mM min1 and 0.46 mM min1 respectively). In the presence of chlorotoluene isomers, the cells of these bacterial strains reduced the permeability of their inner membrane by more than 50%. An exception was the biodegradation of 3-chlorotoluene by R. planticola SA2, for which a marked increase in cell membrane permeability was observed. The R. aquatilis DA2 strain reacted differently: its cell membrane exhibited low permeability (0.07 mM min1) in the culture with glucose, and this value increased more than twofold in the presence of chloroaromatic compounds. Changes in cell membrane permeability during the biodegradation of toxic aromatic hydrocarbons were analyzed by Tiwari et al. (2017), who studied the degradation of 2-chloro-4nitrophenol by Cupriavidus strain a3. They observed that in the presence of a biodegradable compound the quantity of porins in the cell membrane decreased. This adaptive mechanism allowed the

Zeta potential measurements show that the nature of changes in the inner cell membrane permeability of R. aquatilis DA2 is different than that in the other two strains (for review see Fig. 6). The cells of R. aquatilis DA2 from glucose cultures, having small zeta potential values, exhibited even lower electrokinetic potential in the process of biodegradation of chlorotoluene isomers. In the strains R. planticola SA2 and Pseudomonas sp. MChB, the presence of chloroaromatic compounds resulted in higher zeta potential values than in the cultures with glucose in spite of the significant difference between the electrokinetic potential values measured for both strains in the glucose cultures (12.7 mV for Pseudomonas sp. MChB and 32 mV for R. planticola SA2). For the majority of bacterial strains, the zeta potential of their cells takes negative values, which is attributed to the functional groups of the chemical molecules that make up the outer cell layers. The zeta potential of Gram-negative cells is determined by phosphoryl and carboxyl groups occurring in the lipopolysaccharides present in the outer cell membrane (Cie
sla et al., 2011). Apart from the influence on the stability of the cell suspension, the zeta

Fig. 5. Inner cell membrane permeability of the tested strains grown for 7 days on different carbon sources.

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Fig. 6. Cell zeta potential of the tested strains grown for 7 days on different carbon sources.

potential may affect the interaction of the cells with molecules present in the solution, such as biodegradable compounds or surfactants. Electrostatic interactions may be one of the most important factors determining the accumulation of molecules of various chemical compounds around cells (Halder et al., 2015). Abbasnezhad et al. (2008) studied changes in the surface properties of Pseudomonas fluorescens LP6a cells and their adhesion to the interface in the presence of different surfactants. The introduction of a cationic surfactant, cetylpyridinium chloride, increased the cell potential from 23 mV to 7 mV, which the authors considered to be the main factor contributing to the increase in cell adhesion to the hydrophobic phase (hexadecane). Bearing in mind the small changes in the potential of zeta, depending on the type of carbon source and the significant influence of type of the chlorotoluene isomer on the membrane permeability, the way in which the test compounds interact with bacterial cells can be concluded on. Because the zeta potential describes the cell's properties resulting from the structure of the outer layers of the cell, and the cell membrane permeability informs about the structure of the cell wall interior, the hypothesis about the penetration of chlorotoluene molecules can be confirmed easily inside the membrane and not their adsorption on the cell surface.

3.5. Cell surface hydrophobicity There were significant differences between the cell surface hydrophobicity (CSH) values of the tested strains (Table 3). Attention should be paid, however, to the similarities in the values for the cultures with 2- and 4-chlorotoluene. For all three strains, CSH values for the cells from 3-chlorotoluene cultures significantly differed from those recorded for the cultures with 2- and 4chlorotoluene, being either lower (R. planticola SA2) or higher

(Pseudomonas sp. MChB and R. aquatilis DA2). Literature chiefly provides reports on changes in the surface properties of cells under the influence of hydrocarbons, and does not supply data on the effect of chlorotoluene isomers. Zhong et al. (2014) have noted a significant increase in the hydrophobicity of Pseudomonas aeruginosa AB93066 during biodegradation of hexadecane. The initial value of cell surface hydrophobicity measured for bacterial cells from a culture with glucose was about 5%. Cells of the same strain taken from the cultures with hexadecane showed much higher hydrophobicity (nearly 65%), independently of the addition of rhamnolipids. The increased biodegradability of pyrene by Achromobacter denitrificans ASU-035 was interpreted by Mawad et al. (2016) as being related to both the increased hydrophobicity of the cells of the strain and the increased activity of dioxygenase enzymes produced by the strain. A reduction in the hydrophobicity of the cell surface (from 78% to about 50%), associated with increasing biodegradability, was observed during the biological degradation of 4-nitrotoluene by Rhodococcus pyridinivorans NT2 (Kundu et al., 2013). Similar observations have been made by Franzetti et al. (2008) in a study of the biodegradation of aliphatic hydrocarbons by three Gordonia species. The authors of both papers linked the reduction in cell adhesion to hydrocarbons to the production of a biosurfactant, belonging to the glycolipid group by the strain. As in the case of measurements of cell membrane permeability, the strongest effect on hydrophobicity had 3-chlorotoluene. The high permeability is accompanied by low hydrophobicity of the cell surface, although no such significant changes in the values of the zeta potential were observed at the same time. This shows that all these three parameters indicate modifications at a different level of cell structure. It can be supposed that high hydrophobicity and low permeability mean that the chlortoluene is retained within the cell wall but not penetrated inside the cell.

Table 3 Cell surface hydrophobicity (CSH) of tested strains grown for 7 days on different carbon sources. Cell surface hydrophobicity (%)

Raoultella planticola SA2 Rahnella aquatilis DA2 Pseudomonas sp. MChB

glucose

2-chlorotoluene

3-chlorotoluene

4-chlorotoluene

18 ± 2 37 ± 3 74 ± 3

21 ± 2 33 ± 2 41 ± 2

5±2 40 ± 2 72 ± 2

17 ± 2 31 ± 1 42 ± 1

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4. Conclusions Three strains capable of degrading chlorotoluene isomers e Raoultella planticola SA2, Rahnella aquatilis DA2 and Pseudomonas sp. MChB e were isolated from soil samples. The strain R. aquatilis DA2 was distinguished from the other two by causing alterations in the surface properties of the cells, which were an increased content of branched fatty acids, and the induced capacity for b-hemolysis. Despite these differences, all three strains demonstrated good ability to biodegrade the chlorotoluenes, achieving a biodegradation yield of over 60% in three weeks. The collected results indicated, that the isolated strains may found application in bioaugmentation processes in biological remediation sites contaminated with chlorotoluenes. Main findings The results significantly increased the present knowledge about adaptation of bacteria strains to degradation of the hydrophobic and hazardous compounds. Acknowledgments This study was supported by the National Science Centre (Poland) under decision number DEC-2015/19/N/NZ9/02423. References Abbasnezhad, H., Gray, M.R., Foght, J.M., 2008. Two different mechanisms for adhesion of Gram-negative bacterium, Pseudomonas fluorescens LP6a, to an oilwater interface. Colloids Surf., B 62 (1), 36e41. Aruko, H., Akeyama, T., Asumi, M., Asa, W., Adashi, T., 2002. Atsunaga, M., Screening of soil bacteria for production of biocleaner. Appl. Biochem. Biotechnol. 98e100, 319e326. Bhatt, P., Kumar, M.S., Mudliar, S., Chakrabarti, T., 2007. Biodegradation of chlorinated compounds e a review. Crit. Rev. Environ. Sci. Technol. 37 (2), 165e198. Chaudhry, G.R., Chapalamadugu, S., 1991. Biodegradation of halogenated organic compounds. Microbiol. Rev. 55 (1), 59e79. Chen, Y., Li, X.-K., Si, J., Wu, G.-J., Tian, L.-D., Xiang, S.-R., 2016. Changes of the bacterial abundance and communities in shallow ice cores from dunde and muztagata glaciers, Western China. Front. Microbiol. 7, 1716. €stner, M., Ko €ser, H., 2015. The dynamics of lowChen, Z., Kuschk, P., Paschke, H., Ka chlorinated benzenes in a pilot-scale constructed wetland and a hydroponic plant root mat treating sulfate-rich groundwater. Environ. Sci. Pollut. Res. Int. 22 (5), 3886e3894. Cie
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