Isolation, characterization and molecular identification of a halotolerant Bacillus megaterium CTBmeg1 able to grow on halogenated compounds

Abstract Halogenated compounds pose long-term potential risks to the well-being of humans due to their recalcitrance and persistent toxicity. The quest for microorganisms capable of degrading such perilous substances merits urgent consideration. In this study, a new dehalogenase-producing bacterium was isolated from a hypersaline environment (TuzGölü Lake, Turkey), and identified as Bacillus megaterium strain CTBmeg1 (Accession number MK128900). Under culture conditions (pH 8.0, NaCl 20%, 30 °C, 200 rpm, 9 days), the B. megaterium strain CTBmeg1 showed an optimum growth on 10 mmol/L of 2,2-dichloropropionic acid with a doubling time of 26.41 h. Furthermore, the presence of a putative halotolerant dehalogenase gene (dehCTBmeg1) of B. megaterium strain CTBmeg1 was detected and amplified via PCR technique. Bio-prospecting for microorganisms in a highly saline environment capable of utilizing halogenated compounds as the sole carbon may prove to be a practical and safer means for bioremediation of contaminated coastal areas, an increasingly common place predicament faced by many nations.


Introduction
Hypersaline environments, like all other ecosystems, are impacted by industrial activities and frequently contaminated with harmful organic compounds following an escalation in the number of manufacturing activities carried out so close to water bodies [1]. Many naturally occurring hypersaline environments are increasingly threatened by environmental pollution as the result of unscrupulous dumping of industrial effluents into these ecosystems. Moreover, it was estimated that 5% of industrial effluents are saline and hypersaline in nature [2], which could potentially increase the concentration of solutes in affected water bodies. Since food sources, such as fishes and prawns, from marine environments form a substantial proportion of human diet [3], pollution of these ecosystems with antropogenic effluents may lead to higher consumption of contaminated food by humans. This is due to the bioaccumulation of various pollutants in the affected marine life that feeds on contaminated food sources [4,5]. In this context, microbial prospecting may prove as one of the better alternatives for bioremediation of contaminated water bodies [6].
Studies have indicated that hypersaline habitats tend to harbour unique and ancient microbial life capable of surviving or thriving under this harsh environment; such microorganisms are known as halophiles. Halophiles are salt loving and have adapted to thrive in extreme conditions of salinity (35% NaCl), for instance, in specific areas around the world such as the Dead Sea, Jordan [7], Urmia salt lake, Iran [8], Tunisian Solar Saltern [9], Tuzkoy salt mine, Turkey [10], alongside hypersaline environments in south Spain [11] and the Great Salt Lake, USA. [12]. Previous studies have shown that halophilic microorganisms isolated from the hypersaline TuzG€ ol€ u Lake (Turkey) are capable of synthesizing highly efficient halo-stable enzymes which included amylase, cellulase, caseinase, gelatinase, lipase, catalase and oxidase [13,14]. Consequently, halophilic microorganisms may be categorized into five groups: (I) non-halophiles, which grow optimally in less than 0.2 mol/L (< 1% w/v) NaCl, (II) slight halophiles with optimum growth at 0.2-0.5 mol/L (1-3%) NaCl, (III) halotolerant, which are non-halophilic microorganisms but can tolerate high concentrations of salt, in some cases as high as 5.5 mol/L (25% NaCl), (IV) moderate halophiles, growing best between 0.5 and 2.5 mol/L (3-15% NaCl), (V) extreme halophiles with optimum growth between 2.5 and 5.5 mol/L (15-25% NaCl) and ability to grow even at saturated salt concentrations [15].
Pertinently, survival of microorganisms in hypersaline conditions requires specialized cellular adaptations to help preserve the osmotic balance with their external environment. These adaptations involve the existence of organic compatible solutes (osmoprotectants) or the accumulation of variably high concentrations of KCl in their cytoplasms [16,17]. Similar evolution was extended to the enzymes secreted by halophiles, which invariably are metabolically and physiologically stable in a hypersaline environment [18,19]. This is related to the considerably higher proportions of acidic residues in the halophilic protein sequences that confer superior water-binding capacity, thereby better preserving the solubility of their proteins compared to that of the non-halophilic homologues [20].
It has been reported that halogenated compounds, such as 2,2-dichloropropionic acid (2,2-DCP) or commercially known as Dalapon, is the prevalent pollutant found in the environment, typically originating from both natural and human activities. This substance is now widespread in the environment due to our reliance on biocides for weed management in the agricultural industry [21]. The problem is further exacerbated by the fact that 2,2-DCP is highly toxic and resistant to degradation [22], for that reason, this toxic substance could remain in the ecosystem for an extended period of time, long enough to adversely affect marine habitats including commercial and marine fisheries located along the coastal areas [23]. Considering the recalcitrance of such substance, it is as much a local problem as well as a global concern [24][25][26] and the development of safer means to eliminate the seperilous substances from the environment deserves special consideration.
Over the past decade, bacteria isolated from contaminated marine environments have been reported [27][28][29], viz. thermophiles [30] and psychrophiles [31,32]. However, very little is known about the halophiles capable of degrading halogenated compounds [15,20]. These microorganisms are deemed as a good source of biocatalysts and are slowly gaining popularity among the environmental biotechnologists as the cost effective means to clean up polluted environments [33]. The innate stability of halophilic microbes at high salt concentrations justifiably supports their possible use for bioremediation of marine ecosytems polluted with halogenated compounds. Therefore, this present study that aimed at providing insights into the availability of 2,2-DCP degrading bacteria at TuzG€ ol€ u Lake (Turkey) merits special consideration.

Sampling and reagents
The samples were collected from the contaminated hypersaline TuzG€ ol€ u Lake, Turkey (latitude: 39 06 0 30.8.N; longitude: 33 18 0 58.4E) at 1,665 km 2 surface area and it is one of the largest hypersaline lake in the world ( Figure 1) [34]. Using aseptic techniques, the sample was collected and brought back to the laboratory. Chlorinated hydrocarbon, 2,2-DCP was obtained from Sigma Aldrich (USA). Distilled water was used in all media preparation. Analytical grade organic solvents and other chemical reagents were procured from Sigma Aldrich (USA).
Minimal media was prepared in a 250 mL sterile Erlenmeyer flask which contained basal salts (10 mL), trace metals (10 mL) and distilled water to a final volume of 100 mL. The minimal salt media was sterilized by autoclaving at 121 C for 15 min at 15/psi. Then, sterilised 2,2-DCP and NaCl were added to the autoclaved media to the desired concentrations. Using pH meter (Consort C3010, Belgium), pH of the media was adjusted to pH 8 ± 0.2 and incubated with shaking at 200 rpm at 30 C for 9 days. In order to isolate the pure colonies of the halophile, solid medium that consisted of Oxoid bacteriological agar No. 1 (1.5% w/v) was prepared prior to sterilization. The mixed culture of 0.1 mL was spread onto solid minimal media containing 10 mM 2,2-DCP previously filter sterilized through a 0.2 lm nylon-membrane disc and was incubated at 30 C.

Morphological, physiological and biochemical characteristics of the strain
Characterization of the physiological and biochemical properties of the bacterial strain included Gram-staining, motility test, carbohydrate fermentation and gelatin hydrolysis [36]. All results were systematically analyzed according to the Bergey's Manual of Determinative for Bacteriology [37]. Following an overnight incubation in LB medium, tests for oxidase and catalase activity was carried out in 1% (w/v) tetramethylene diamine [38], and consequently observed for evolution of gas and bubble formation in a 2.5% (w/v) hydrogen peroxide solution, respectively [39]. Morphological observation was done under a scanning electron microscope, Hitachi SU5020 FES electron microscope (magnification, of X11.0K, accelerating voltage, 2.0K) equipped with an SE(L) detector at a 16.2 mm working distance. The sample was placed onto an aluminum foil disc and the Leica EM CPD300 critical point dryer (Leica Microsystems GmbH, Germany) was used to dry the sample followed by coating with platinum before viewing. Other biochemical tests: citrate utilization, methyl red, indole production, Voges-Proskauer reactions, urease production and nitrate reduction, were also performed according to a previously described method [39].

PCR amplification of 16S rDNA
Bacterial 16S rDNA universal primers 27 F (5 0 -AGAGA TTGATCCTGGCTCT G-3 0 ) and 1492R (5 0 -GGTTTCCTTG TTACGAC AT-3 0 ) were synthesized by Soygen Biotechnology Laboratories (Istanbul, Turkey). Genomic DNA was extracted using Promega Bacterial DNA Isolation Kit AMBER (Istanbul, Turkey), as described by the manufacturer. PCR conditions were carried out according to the following: initial denaturation The PCR products were purified and sequenced by Soygen Biotechnology Laboratories. The sequence identity was searched by the BLASTn analysis using NCBI database. The 16S rDNA gene sequence data was analyzed on the MEGA7 software package and the phylogenetic tree was constructed using the neighbour-joining method [40].

Investigation of optimum growth conditions
The effects of 2,2-DCP for parameters viz. concentration, pH, temperature, rotary speed and medium salinity on the growth rate of the halophilic bacterial cultures were examined using minimal salts medium supplemented with 2,2-DCP to final concentrations of 5, 10, 20, 30 and 40 mmol/L, respectively. The initial pH in the growth medium was adjusted to pH 6, 7, 8, 9 and 10, using 1 mol/L HCl or 1 mol/L NaOH. The effect of the speed of shaking was investigated for 150, 175, 200, 225 and 250 rpm, respectively. Several concentrations of NaCl in the liquid media were prepared: 50 (5%), 100 (10%), 150 (15%). 200 (20%), 250 (25%) and 300 (30%) g/L. The optimized conditions were then determined by calculating the bacterial growth rate with incubation over a period of 9 days.

Characterization of strain CTBmeg1
Minimal media containing 20% (w/v) of NaCl supplemented with 10 mmol/L of 2,2-DCP was used to screen for new halotolerant microorganisms capable of using this substance as an only source of carbon. The halophilic microorganisms were indeed present in the water samples collected from the hypersaline TuzG€ ol€ u Lake, appearing as faint yellow colonies (2-4 mm in diameter) after 9 days of incubation at 30 C ( Figure  2(a)). Such colonies were not observed on the negative control medium (without 2,2-DCP) (Figure 2(b)). This indicated that the isolate growing on the media supplemented with 2,2-DCP might possess a unique potential in metabolizing 2,2-DCP in the presence of high salt concentration. Development of dark blue colour following Gram staining affirmed that the bacteria were gram-positive (Figure 2(c)).When viewed under the scanning electron microscope (SEM), strain CTBmeg1 appeared to have a bacilli-like shape with width and length between 745 ± 69.5 nm and 1.96 ± 32.7 lm, respectively (Figure 2(d)).The biochemical properties of strain CTBmeg1 were closely affiliated with those of Bacillus megaterium strains YJB3 [41], WS24 [42], G3 [43] and BJC3.1 [44]. The overall properties of strain CTBmeg1 bacterium is tabulated in Table 1.

Identification of strain CTBmeg1
The partial 16S rDNA gene sequence (GenBank accession No. MK128900) of strain CTBmeg1 was obtained as a continuous stretch of 860 bp that was highly similar to that of B. megaterium (97%). The constructed phylogenetic tree from the 16S rDNA sequences revealed that strain CTBmeg1 is closely related to B. megaterium strain WAB2203 (MH169257.1) and other B. megaterium strains (Figure 3). Based on the finding, the bacterial isolate was identified as B. megaterium CTBmeg1.
TuzG€ ol€ u Lake root tissue rhizosphere of wheat root of tobacco sauce Figure 3. Phylogenetic tree of B. megaterium strain CTBmeg1. Note: The accession number for each sequence was obtained from NCBI while an outgroup used the sequence of Pseudomonas putida KMBU4308.

Optimum growth conditions of Bacillus megaterium CTBmeg1
Considering that high multiple significant factors may influence the growth conditions and the consequent bioremediation efficacy of B. megaterium CTBmeg1, such factors were examined in this study. Figure 4(a) illustrates that the bacterial strain CTBmeg1 grew optimally in media containing up to 30 mmol/L of 2,2-DCP. However, concentrations of 2,2-DCP that exceeded 30 mmol/L inhibited the growth of CTBmeg1, presumably due to increased toxicity of the 2,2-DCP [45]. As shown in Figure 4(b), bacterial growth occurred within a medium pH range of pH 7.0-9.0, notably, similar to the natural pH of TuzG€ ol€ u Lake. According to Oren [46] and Litchfield and Gillevet [47], the high concentration of NaCl in thalassohaline brines has resulted in these lakes having slightly alkaline pHs. Still, neutral pH values are generally favourable to support the growth of most halophililic microorganisms [48]. The best growth temperature was observed to occur in a narrower range, between 20 and 35 C (Figure 4(c)), while growth was not observed at 10 C. The halophilic B. megaterium DSM 32T, isolated from hypersaline Lake in Spain, grew over a wider temperature range between 10 and 55 C [49]. The variable growth temperature range seen here between the two bacterial strains might be due to evolutionary adaptation to the different habitats from which they were isolated. Figure 4(d) shows that strain CTBmeg1 was less affected by the change in the rotation speed. Rotation speeds between 150 to 225 rpm are important for sustaining the required amount of dissolved oxygen in the media.  Using the above optimum growth conditions, we aimed the second part of the analysis towards obtaining an efficient bioremediation process by optimizing the NaCl concentration in the growth media. Figure 5 shows the growth curve of B. megaterium CTBmeg1 at different concentrations of NaCl. As seen in the figure, the bacteria showed tolerance towards increasing concentrations of NaCl, reaching as high as 20% (20 g/L or 3.5 mol/L) and exhibited a cell-doubling time of 26.41 h.
It is well known that strains of B. megaterium can tolerate high salt concentrations, for instance, B. megaterium DSM 32 T tolerates up to 25% (250 g/l or 4.5 M) [49]. However, despite strain CTBmeg1 being able to grow in highly saline media, the bacterium species was not highly reliant on salt for growth; thus, in low concentration of salt (5%) the isolate still survived and grew. Halophiles usually necessitate the presence of NaCl for growth, unlike halotolerant organisms, which do not require NaCl but can grow under saline conditions. A possible explanation for this phenomenon is the heterogeneity of the hypersaline habitats, where the salinity can change markedly in space and time. Consequently, highly halophilic microorganisms are periodically eliminated and the euryhaline types are consistently favoured.
Despite the fact that the average salinity level of TuzG€ ol€ u Lake consists of approximately 35% NaCl, this study found that bacterial growth did not occur at concentrations of NaCl higher than 25%. A probable reason for the reduced bacterial growth at 35% NaCl in this study, may be related to the bacteria growing in nutrient poor media that lacked any carbon source other than 2,2-DCP. However, when cultivated in nutrient rich media, notable bacterial growth was observed even at high concentrations of NaCl reaching up to 35%. The outcome seen here corroborates a report by Sorokin et al. [50] that multiplication and survival of microorganisms in saline water is impacted by the availability of nutrients in the environment. Since strain CTBmeg1 can grow in a relatively broad salinity range, this indicated the prospective use of the bacterium as an agent for cleaning up environments of varying salinity, including seawater (3.5% NaCl) polluted by halogenated hydrocarbons.

Dehalogenase gene sequencing and analysis
To further confirm the presence of the dehalogenase gene in B. megaterium CTBmeg1, amplification of the dehalogenase gene was carried out. Figure 6 depicts the produced PCR bands showing sizes similar to that reported by Hill et al. [51]. Therefore, the partial sequence of putative dehalogenase gene (503 bp) was designated as dehCTBmeg1.

Conclusions
A new halogenated hydrocarbon-degrading bacterium CTBmeg1 identified as B. megaterium was isolated from the hypersaline environment of TuzG€ ol€ u Lake, Turkey. The phenotypic and phylogenetic characteristics indicated that the B. megaterium CTBmeg1 showed promising capacity for degradation of halogenated compounds and was adapted to high salinity and pH conditions. Strain CTBmeg1 identified in this study is one of the few haloalkanoic-degrading bacteria belonging to the genus B. megaterium and is the first ever to be isolated from a hypersaline environment. Notably, the findings in this study conveyed the possibility of CTBmeg1 as a cost-effective bioremediation agent for cleaning up marine environments polluted by halogenated compounds.

Disclosure Statement
On behalf of all authors, the corresponding author states that there is no conflict of interest.