1

1 Article

2 Bioremediation properties of a novel DDT-degrading Rhodo-

3 coccus sp. with high cell surface hydrophobicity
4 Jie Qu 1#, Wentao Zheng 2#, Ruya Zhang3, Zhenghua Li 1*

5 1
Shandong Key Laboratory of Biophysics, Shandong Engineering Research Center of Swine Herd Health Big Data and Intelligent Monitoring,
6 Institute of Biophysics, Dezhou University, Dezhou 253023, PR China; qujie@dzu.edu.cn (J.Q.)
7 2
International Laboratory for Applied Biotechnology, Institute of Pharmacy, Chemistry and Biology, Belgorod State University, Belgorod 308015,
8 Russia; zhengwentaoo@126.com (W.Z.)
9 3
Shandong Key Laboratory of Biophysic, Belgorod Institute of Food Sciences, Dezhou University, Dezhou
10 253023, PR China; 18653400720@163.com (R.Z.)
11 * Correspondence: zhenghua0407@163.com
12 #
Co-first author

13 Simple Summary: DDT is a dangerous environmental pollutant that poses a risk to human health due to its bioaccumulation
14 effect. The strain Rhodococcus aetherivorans IcdP1 is able to utilize such aromatic compounds and is therefore promising for
15 environmental biotechnology applications. We investigated the metabolic properties of this strain, analysed changes in cell surface
16 hydrophobicity (CSH) under DDT stress, and determined that the strain regulates its CSH by altering its cell wall composition and
17 structure to adapt to its habitat. We also found that the degradation pathways of DDT in the enhanced remediated contaminated
18 soils include reductive dichlorination and oxidative degradation. Understanding these pathways is essential for monitoring the
19 bioremediation process and assessing the formation of potentially toxic intermediates. The research results will provide guidance
20 for the bioremediation of organic pollutant-contaminated soils.

21 Abstract: Long term residues of organochlorine pesticides (OCPs) in soil are of great concerning because they seriously threaten
22 food security and human health. Rhodococcus aetherivorans IcdP1, isolated from coking plant soil, could degrade 74-88% of 1,1,1-
23 trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) at an initial concentration of 50 mg L -1 in laboratory incubation experiments (30
24 days at 30 °C). Notably, exposure to DDT resulted in the development of even higher cell-surface
Citation: To be added by editorial
25 staff during production.
hydrophobicity (CSH) of strain IcdP1, suggesting that the high hydrophobicity of the strain could
26 enhance the attachment and degradation of DDT. Furthermore, the study of the degradation
27 Academic Editor: Firstname pathways of DDT in contaminated soil revealed that DDT undergoes both reductive
Lastname
28 dichlorination and oxidative degradation. This leads to the formation of various metabolites,
29 Received: date including 1,1-dichloro-2,2-bis (4-chlorophenyl) ethane (DDD), 1,1-dichloro-2,2-bis (4-
30 Revised: date chlorophenyl) ethylene (DDE), 1-chloro-2,2-bis (4-chlorophenyl) ethylene (DDMU), 1-chloro-2,2-
31 Accepted: date bis (4-chlorophenyl) ethane (DDMS), dichlorobenzophenone (DBP), and 1,2-Benzenedicarboxylic
32 Published: date butyl dacyl ester. Therefore, the strain IcdP1 of Rhodococcus aetherivorans presents a valuable
33 biological resource for the bioremediation of DDT-contaminated soils. Its ability to degrade DDT

Copyright: © 2024 by the authors.

Submitted for possible open access
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conditions of the Creative Commons
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3 Biology 2024, 13, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/biology

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34 efficiently, coupled with the increased cell-surface hydrophobicity, makes it a promising candidate
35 for further research and application in environmental restoration projects.

36 Keywords: Rhodococcus aetherivorans; DDT; Cell-surface hydrophobicity; Biodegradation

37

38 1. Introduction

39 Organochlorine pesticides (OCPs) have been widely applied during the past few
40 decades as insecticides for crop protection and for control of vector-borne diseases such
41 as typhus and malaria. 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) is a
42 ubiquitous anthropogenic OCP environmental contaminant that has been banned for
43 over 40 years due to its toxicity, hydrophobicity, and bioaccumulation. However,
44 because of its long half-life (4-35 years), it is still abundant in many developed and
45 developing countries worldwide [1–6]. DDT is an organic compound with low solubility
46 and high chemical stability, composed of chlorinated aliphatic and aromatic structures.
47 It tends to accumulate in food sources and adipose tissues because of its strong
48 lipophilicity [7,8]. All these factors together make it potentially harmful to health and
49 environment. Therefore, it is necessary to develop remediation processes to degrade and
50 eliminate contaminants in the environment.

51 Compared to chemical oxidation, physical adsorption and photooxidation,

52 bioremediation technology has acquired increasing attention and is considered to be a
53 cost-effective and eco-friendly method for the removal of OCPs [9,10]. Certain
54 microorganisms are capable of chemically degrading DDT under both aerobic and
55 anaerobic conditions [9]. Reductive dechlorination appears to be the predominant
56 mechanism for microbial transformation of DDT to such compounds as 1,1-dichloro-2,2-
57 bis (4-chlorophenyl) ethane (DDD), 1,1-dichloro-2,2-bis (4-chlorophenyl) ethylene
58 (DDE), 1-chloro-2,2-bis (4-chlorophenyl) ethylene (DDMU), 1-chloro-2,2-bis (4-
59 chlorophenyl) ethane (DDMS), unsym-bis (4-chlorophenyl) ethylene (DDNU), and
60 dichlorobenzophenone (DBP) [11–13]. In addition, certain bacteria have been found to
61 metabolize DDT by hydroxylation of the aromatic ring via oxidative attack [13–15].

62 The strong hydrophobicity of OCPs in soil results in their low solubility in water,
63 which considerably affects the rate of OCPs degradation by microorganisms. Microbial
64 cell-surface hydrophobicity (CSH) is one of the most important surface properties
65 determining cell adhesion to abiotic and biotic surface [16]. Hydrophobicity of the cell-
66 surface allows microorganisms to congregate with each other and to attach to
67 hydrophobic substrates to survival and nutrient uptake [17]. Thus, the surface properties
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68 of microorganisms are generally considered to be key to survival, as surface components

69 mediate cell-to-cell contact and interactions between microorganisms and their
70 environment [18].

71 Rhodococcus are the dominant aromatic compounds degrading microorganisms

72 which can improve their adaptability to hydrophobic environments by modifying the
73 cell surface composition and structure to support effective biodegradation of aromatic
74 compounds [19,20]. Oligosaccharide, lipid and outer membrane protein composition, as
75 well as mycobacterial acids, have been shown to influence the CSH of Rhodococcus
76 [16,19]. Therefore, the cell structure produced by Rhodococcus plays an important role in
77 the metabolism of aromatic compounds. However, the potential role of CSH in the
78 adaptation of Rhodococcus cells to survive in polluted environments, as well as the
79 underlying mechanisms of CSH, remain unclear.

80 In this study, the strain IcdP1 was able to metabolize DDT by oxidation and
81 dechlorination as its sole carbon and energy source. In addition, studies showed that
82 CSH could affect the degradation ability of the DDT-degrading strain, and there might
83 be a relationship between high CSH and strong degradation ability. Biodegradation
84 assays indicated that it could be a very potential and efficient biocatalyst for
85 biotreatment of industrial effluents and bioremediation of contaminated soils.

86 2. Materials and Methods

87 2.1. Chemicals and culture media

88 Standard samples of DDT (mixture of isomers of o,p’-DDT and p,p’-DDT; purity

89 >99.5%), DDE (purity >98.5%), DDD (purity >99.5%), and DDMU (purity >99.0%) were
90 purchased from Dr. Ehrenstorfer (Augsburg, Germany). Acetone and acetonitrile (HPLC
91 purity grade) were from Fisher Scientific (USA). All other chemicals were analytical
92 grade and were purchased from Beijing Chemical Reagent Co., Beijing, China. Standard
93 samples were dissolved in acetone as stock solutions (10 5 mg L-1), sterilized by
94 membrane filtration, and added to sterile flasks. After acetone was vaporized
95 completely, medium was added to obtain the desired concentrations of samples. Mineral
96 salt medium (MSM) and Luria-Bertani (LB) medium were used to culture the DDT-
97 degrading bacterial strain [21].

98 2.2. Inoculum preparation

99 Strain IcdP1 was precultured in LB medium for 24 hr at 30 °C. Cells were harvested
100 by centrifugation at 6000 ×g for 20 min, washed 3 times with 0.2 mol L -1 Na2HPO4-
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101 NaH2PO4 buffer (pH 7.0) to remove residual nutrients, and resuspended in the same
102 buffer to give ~4×109 CFU mL-1.

103 2.3. Biodegradation DDT by strain IcdP1 in liquid culture

104 Flasks containing 10 mL MSM with 50 mg L-1 DDT as the sole carbon and energy
105 source were inoculated with 100 μL cell suspension prepared as above. A similar flask
106 without bacterial inoculation was used as a control. Each treatment was performed in
107 triplicate. Flasks were incubated at 30 °C with rotary shaking (160 rpm). Growth of the
108 culture and the residual DDT concentration were measured for 30 days. Bacterial growth
109 was determined by monitoring the optical density at 600 nm (OD 600nm) using a
110 spectrophotometer (UV-7200, UNICO, Shanghai).

111 Samples incubated for 2, 4, 6, 14, or 30 days were subjected to gas chromatography
112 (GC) analysis. At each interval, flasks were individually extracted three times with 10
113 mL dichloromethane (CH2Cl2) by ultrasonication for 20 min. Following equilibration for
114 1 h, the lower organic phases of one sample were combined and dried with a Heto-
115 PowerDry LL3000 Freeze Dryer (Thermo Electron Corp., USA) and redissolved in an
116 equal volume of n-hexane. Biodegradation was analyzed by GC-ECD (model 2010,
117 Shimadzu Corp., Japan) equipped with an Rtx-1301 capillary column (30 m × 0.25 mm ×
118 0.25 μm). The operating conditions were: initial column temperature 160 °C for 1 min,
119 then 3 °C min-1 to 200 °C for 2 min, then 4 °C min -1 to 220 °C for 4 min, then 5 °C min -1 to
120 240 °C, and finally held at 240 °C for 10 min; ECD temperature 300 °C; injection volume
121 1.0 μL (at 280 °C); nitrogen as carrier gas at flow rate 8.6 mL min-1.

122 2.4. MATH assay

123 Cell surface hydrophobicity (CSH) of IcdP1 incubated in mineral salt medium with
124 DDT (50 mg L-1) was assessed by the microbial adhesion to the hydrocarbon method
125 (MATH) [22,23] . The hydrocarbon xylene was used to determine the CSH value of
126 IcdP1. The process of MATH was modified as described previously [24]. The CSH value
127 was calculated using the following equation:

128 CSH (%) = 100 %×(Ai - Af) / Ai (1)

129 Ai and Af represent the initial and final optical densities of the aqueous phase at 600 nm,
130 respectively.

131 2.5. Identification of the bacterial cell-surface constituents

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132 Fourier transform infrared analysis was used to analyze cell-surface constituents.
133 Strain IcdP1 grown in 10 mL LB liquid medium at 30 °C for 24 h were harvested by
134 centrifugation at 12,000 rpm for 10 min. The cell pellets were washed thrice in 10mL
135 sonication buffer (50 mM Tris-sulfate (pH 7.5), 1 mM EDTA and 1mM DTT). The
136 washed cells were inoculated into 10 mL MSM with 50 mg L -1 DDT, a similar flask
137 without DDT was used as a control. Strains IcdP1 in MSM medium with and without
138 DDT were collected after 24 hours of cultivation, washed twice, and resuspended in
139 0.9% NaCl solution. Fourier transform infrared (FTIR) spectrophotometry was used to
140 analyze the lyophilized cells with the KBr disk technique, using a Nicolet iS50 FTIR
141 instrument (Thermo Scientific, USA).

142 2.6. Colorimetric assay for dechlorination activity

143 Strain IcdP1 grown in 10 mL LB liquid medium at 30 °C for 24 h were harvested by

144 centrifugation at 12,000 rpm for 10 min. The cell pellets were washed thrice in 10mL
145 sonication buffer (50 mM Tris-sulfate (pH 7.5), 1mM EDTA and 1mM DTT). Washed
146 cells were resuspended in 1 mL sonication buffer and subjected to sonication for 10 min
147 on ice with a Vibra cell ultrasonicator (20% of maximum amplitude, 5 s pulse intervals).
148 The lysate was centrifuged for 10 min at 12,000 rpm to remove cell debris and the cell-
149 free extract was immediately used in the dechlorination assay developed previously
150 [25]. Dechlorination is indicated by the color change of phenol red from red to yellow, in
151 a weakly buffered solution (1 mM HEPES (pH 8.2), 20mM sodium sulfate and 1mM
152 EDTA), as the solution becomes acidic due to HCl formation during dechlorination.

153 2.7. DDT dechlorination rate by strain IcdP1 in liquid culture

154 20 mL chloride ion-free MSM with 50 mg L -1 DDT inoculated with 100 μL cell
155 suspension prepared as mentioned in 2.2 were incubated at 30 °C with rotary shaking
156 (160 rpm). Samples incubated for 2, 4, 6, 14, or 30 days were centrifugated at 12,000 rpm
157 for 10 min and the supernatant was collected for the determination of chloride ion
158 concentration. A itration with silver nitrate (Mohr’s precipitation titration) that detects
159 chloride ion concentration was modified to determind the DDT dechlorination rate by
160 strain IcdP1 in liquid culture. As the silver nitrate solution is added, a colloidal solution
161 of silver chloride forms. The moles of chloride ions reacting can be determined using the
162 following reaction equation, based on the linear relation between spectrum absorption
163 and silver chloride content.

164 Ag+(aq)+ Cl–(aq)→ AgCl(s) (2)

165 2.8. Analysis of DDT metabolites in soil environment

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166 Simulated bioremediation experiments were conducted to evaluate the feasibility of

167 using IcdP1 for removing DDT from soil. Soil samples were collected from an
168 uncontaminated field. Multiple samples were taken from 10 cm below the soil surface
169 and sifted (2 mm mesh size) to remove the stones and blended thoroughly to give one
170 soil sample [13]. Properties of the soil samples were as (g kg -1 of dry weight): total
171 organic carbon, 7.18; total nitrogen, 18.2; total phosphorus, 20.6; pH, 6.5 and has sandy
172 loam texture (sand, 30.53%; silt, 25.12%; clay, 14.35%). A total of 1kg of nonsterile soils
173 were used to investigate the metabolic ability of strain IcdP1. A DDT solution was added
174 to the soil, reaching a final concentration of 50 mg kg-1 in acetone solution. After mixing,
175 two experimental groups were conducted in triplicate: control group and IcdP1-
176 augmented group (1.0×107 CFU g-1 dry soil). To monitor DDT metabolites, three soil
177 samples were taken from each treatment group and mixed for intermediate analysis.

178 Soil samples were extracted by the ultrasonication technique. Each sample (10g dry
179 weight) was placed in a 100-mL Erlenmeyer flask, added with 5 mL deionized water and
180 25 mL acetonitrile, and extracted by ultrasonication for 20 min. The extraction process
181 was repeated three times. All extracts from a particular sample were combined and
182 filtered through a 4-cm Buchner funnel. The filtrates were collected, lyophilized, and
183 dissolved in 2 mL acetone for analysis.

184 Gas Chromatography mass spectrometry (GC-MS) methods were used to analyze
185 DDT and its intermediates in soil environment. A gas chromatograph (model 7890A)
186 coupled with single quadrupole mass spectrometer (model 5975C; Agilent Technologies;
187 Santa Clara, CA, USA) was used. The sample (1 μL) was injected and separated on an
188 HP-5MS capillary GC column (30 m × 0.25 mm i.d., 0.25 μm film; Agilent). The GC-MS
189 operating conditions were set according to Qu et al[13]. The data were acquired using
190 full-scan (m/z 20 - 650) and scan acquisition modes with an 8-minute solvent delay. The
191 Enhanced ChemStation software program (Agilent) was used for analysis.

192 3. Results

193 3.1. Degradation of DDT by IcdP1 in liquid culture

194 The DDT degradation pattern of IcdP1 was determined in liquid MSM (pH 7.0)
195 with DDT (50 mg L-1) as the sole carbon and energy source at 30 °C. The time course of
196 DDT metabolism by IcdP1 was shown in Figure 1. Over a 30-day incubation period,
197 IcdP1 degraded 87.86±5.12% and 74.44±4.80% of o,p’-DDT and p,p’-DDT respectively. At
198 the beginning of incubation, IcdP1 rapidly adapted to the environment without any lag
199 phase. By day 6, it had degraded 39.72±3.65% of o,p'-DDT and 22.37±4.62% of p,p'-DDT,
200 demonstrating its efficiency in degrading these DDT isomers.
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201

202 Figure 1. Biodegradation of DDT by strain IcdP1 during culture in mineral salt medium.

203 3.2. Hydrophobicity of strain IcdP1 during growth on DDT

204 During bacterial growth and DDT degradation, the time curves of CSH were
205 plotted (Figure 1). At the beginning of degradation, the CSH of strain IcdP1 increased
206 rapidly, reaching a maximum hydrophobicity of 0.68 in 4 days, accompanied by rapid
207 DDT degradation. However, in the control group without DDT, the CSH of strain IcdP1
208 decreased significantly. In comparison to the control group, the DDT degradation
209 experimental group consistently exhibited higher CSH during the early stage of the
210 incubation period. As the concentration of DDT decreased, the CSH values also
211 declined. These results suggest a correlation between the concentration of DDT and the
212 CSH of the bacteria, indicating a relationship between high CSH and strong degradation
213 ability.

214 Furthermore, the composition and structure of peptidoglycan, the main component
215 of the cell wall of Gram-positive bacteria, were analyzed under DDT-induced and non-
216 induced conditions because the cell wall plays a key role in the biodegradation and
217 adsorption of DDT. Intact IcdP1 cells underwent FTIR spectral analysis at 4000-400cm -1
218 [26]. The spectra of lipid/acyl chains [3050-2800 cm-1], carbohydrates [1200-800 cm-1] and
219 nucleic acid backbone conformations [1250-800 cm -1] corresponding to DDT-inoculated
220 Icdp1 cells were not significantly different from those of non-inoculated cells, However,
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221 the peak intensities were different (Figure 2A). Additionally, the absorption values of
222 cell wall components of DDT- inoculated cells were significantly lower than those of
223 uninoculated cells. In particular, peptidoglycan contains sugars which are alcohols with
224 polyhydroxyl groups, and 1074 cm-1 belong to C-O stretching vibration of alcohols. Thus,
225 DDT induction had a negative impact on the sugar structure of peptidoglycan, resulting
226 in a decrease in sugar content and lower hydrophilicity compared to other
227 peptidoglycans. The C=O stretching vibration of amide, which is the main site for
228 structural characterization of peptidoglycan in the cell wall, is represented by the 1655
229 cm-1 peak[27]. The study found significant differences in the spectra related to protein
230 absorption in the amide I region (Figure 2B). The FTIR absorption spectra at 1655 cm -1
231 indicated that intact DDT-inoculated cells exhibited lower levels of α-helical secondary
232 structures[26,28], suggesting that DDT-induced hydrophobic strains contain more
233 nonpolar proteins than non-induced strains.

234

235 Figure 2. FTIR spectra of intact cells of DDT-inoculated (red) and non-inoculated (black). (A) FTIR
236 spectra at 4000-400cm-1; (B) FTIR spectra at 1720-1600cm-1.

237 3.3. Colorimetric assay for dechlorination activity

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238 The colorimetric assay for detecting dechlorination activity using a 96-well
239 microtitre plate was applied for this study. The reaction in this assay using 1ul of 12.5
240 mg L-1 stock DDT solution per well is shown in Figure 3. The phenol red indicator
241 turned yellow when cell-free extracts of strain IcdP1 were incubated in the presence of
242 DDT (row B). In contrast, the incubation of sonication buffer blanks or heat-denatured
243 cell-free extracts from strain IcdP1 did not have the color change effect (row A and C).
244 This indicates that cell-free extracts of IcdP1 caused a pH drop when incubated with
245 DDT, due to the formation of HCl during dechlorination. The assay showed that strain
246 IcdP1 expressed the enzyme activity of dechlorinase and this is an effective qualitative
247 test for dechlorination activity.

248

249 Figure 3. Colorimetric assay for DDT dechlorination. Row A1-3: sonication buffer blanks; B1-3:
250 cell-free extract from IcdP1; C1-3: heat-denatured cell-free extract from IcdP1.

251 3.4. DDT dechlorination rate by strain IcdP1 in liquid culture

252 OCPs are highly toxic and anti-degradable due to the existent of chlorine atoms.
253 The number and position of chlorine atoms play an important role in structural stability
254 and biodegradability[27,28]. Therefore, dechlorination of organochlorine is a significant
255 symbol of its biodegradation effect. Aerobic dechlorination usually occurs between the
256 saturated carbon chloride and the saturated carbon chloride, eliminate a molecular of
257 hydrochloric accompanied with the formation of an unsaturated double bond. Silver
258 chloride turbidimetric method was used to examine the release of chlorine ion during
259 DDT degradation.

260 Chloride ion-free MSM with 50 mg L -1 DDT was used in order to avoid interference
261 from other sources of chloride ions. The time course of DDT degradation and the
262 formation rate of chlorine ion by IcdP1 are shown in Figure 4. During the initial phase of
263 incubation, IcdP1 degraded 17.94±3.51% and 21.74±4.38% of DDT by 2 and 4 days,
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264 respectively. The theoretical yields of chlorine ion were 4.59 mg L-1 and 5.49 mg L-1.
265 However, the actual yields of chlorine ion were 0.76±0.09 mg L -1 and 1.16±0.13mg L-1 as
266 shown in Figure 4, which were only about 16.56 and 21.33% of the full theoretical yields
267 of chlorine ion. Six days later, the release amount of chlorine ion began to rise. At this
268 point, the concentration of chloride ion and the releasing rate were 3.74±0.22 mg L -1 and
269 55.40%, respectively. In the later period of culture (30days), the concentration of chloride
270 ion increased to 11.62 mg L-1, this number means that 3 moles of chloride substituent
271 groups can be released from 1 mole of DDT, but at this moment, DDT was not fully
272 degraded, this means that more than 3 chloride ions were released from DDT (with 5
273 chloride substituent groups) by strain IcdP1. From the results obtained, we deduced that
274 at the beginning of the degradation, DDT was transformed into some metabolites which
275 had the same number of chlorine atoms as DDT. In the late stage of culture, DDT was
276 further degraded into low chloride products accompanied by the release of a large
277 number of chloride ions. Therefore, it is further proved that strain IcdP1 has the
278 dechlorination enzyme activity.

279

280 Figure 4. Chloride ion release during degradation of DDT by strain IcdP1.

281 3.5. Products of DDT transformation by IcdP1

282 In order to investigate the feasibility of IcdP1 to remove DDT from soil, the
283 degradation intermediates were detected and identified by GC-MS. The results indicated
284 that there were six intermediates identified when strain IcdP1 grew and degraded DDT
285 in soil samples (Table 1); their structures are shown in Figure 5. These intermediates
286 included A, 1,1-dichloro-2,2-bis (4-chlorophenyl) ethane (DDD); B, 1,1-dichloro-2,2-bis
287 (4-chlorophenyl) ethylene (DDE); F, 1,2-Benzenedicarboxylic butyl dacyl ester, etc. Based
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288 on these detected metabolites, DDT degradation pathways in IcdP1-bioaugmented soils

289 were proposed (Figure 6); DDT could be degraded by reductive dechlorination and
290 oxidative ring-opening, respectively. One was shown to occur via dechlorination and
291 dehydrochlorination at the trichloromethyl group to form DDD (A) and DDE (B),
292 respectively. Subsequent steps were dehydrochlorination or dechlorination to give
293 DDMU (C), hydrogenation to give DDMS (D), dioxygenation to give DDA (not
294 detected), and decarboxylation and hydroxylation to give DBP (E) which was regarded
295 as the terminal metabolite in the DDT biodegradation pathway. The other occurred via
296 hydroxylation at the benzene ring and side chain was removed; in this pathway, DDT
297 was degraded to form a new product, 1,2-Benzenedicarbox5ylic butyl dacyl ester (F),
298 which could eventually enter the central aromatic process and be converted to low
299 molecular weight fatty acids, followed by several transformations into metabolites
300 leading to the TCA cycle.

301

302 Figure 5. Mass spectra and proposed structures of DDT and six metabolites. A: DDD. B: DDE. C:
303 DDMU. D: DDMS. E: DBP. F: 1,2-Benzenedicarboxylic butyl dacyl ester.
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304
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305

306 Figure 6. Proposed pathway for degradation of DDT in soil. The dashed lines denote that these
307 compounds have not been identified in this study.

308

309 4. Discussion
310 DDT is a toxic, hydrophobic and long half-life organochlorine pesticide. Although it
311 has been banned in many countries, DDT and its key metabolites are still found in soil,
312 sediment and groundwater due to its increased bioaccumulation. Rhodococcus
313 aetherivorans IcdP1 could efficiently degrade all DDT isomers (>87.86 of o,p’-DDT and
314 >74.44 of p,p’-DDT within 30 days). Several microorganisms have been shown to
315 degrade DDT as
316 mentioned above, however, none of them belong to Rhodococcus aetherivorans, and
317 none showed the ability to degrade both o,p’-DDT and p,p’-DDT isomers. There was no
318 lag phase in the degradation pattern of DDT isomers by IcdP1. During the initial phase
319 of incubation, IcdP1 rapidly adapted to the environment and degraded 39.72±3.65% and
320 22.37±4.62% of o,p’-DDT and p,p’-DDT, respectively, by day 6. This observation may be
321 due to two reasons, one is that DDT induction alters the CSH of the bacterium and
322 facilitates the uptake of DDT by IcdP1 strain, and the other may be related to the
323 degradation pathway of DDT isomers.
324 Direct attachment to the substrate surface is a key step in the biodegradation of
325 hydrophobic organic compounds. Previous studies have shown that microorganisms
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326 with higher CSH have potential advantages in the bioremediation of persistent organic
327 pollutants [9,29]. To date, the potential role of CSH in the survival of Rhodococcus sp.
328 cells adapted to the polluted environment and the intrinsic mechanisms of CSH remain
329 unclear. The hydrophobicity of specific strains may be influenced by environmental
330 factors and certain cellular properties, and the authors conclude that strain IcdP1
331 regulates its CSH by altering its cell-surface composition to adapt to its habitat. FTIR
332 spectral analysis showed that DDT affected the cell wall composition and structure of
333 IcdP1, which in turn affected its CSH, resulting in the adsorption and binding of DDT on
334 the cell wall surface, and finally crossing the cell membrane and entering the cell for
335 gradual degradation. The changes in the composition and structure of IcdP1 cell well
336 may be induced by the bacterial response to chemical substances. Appropriate
337 concentrations of DDT can induce an increase in CSH without affecting the normal
338 growth of the bacteria, which facilitates the rapid uptake and degradation of DDT.
339 The key reaction during microbial DDT degradation is the removal of the halogen
340 atom. However, there are two distinct microbial degradation pathways for DDT
341 isomers: dechlorination and oxygenation [12]. In previous studies, multiple gene copies
342 encoding ring-hydroxylating oxygenases (RHOs, 50 copies) and cytochrome P450
343 monooxygenases (CYP450, 22 copies) were identified in the genome of strain IcdP1 [29].
344 This might contribute to its efficient degradation ability towards a variety of high-
345 molecular-weight polycyclic aromatic hydrocarbons (HWM-PAHs) with four or more
346 rings, such as pyrene (PYR), benzo[b]fluoranthene (BbF), and indeno[1,2,3-cd]pyrene
347 (IcdP) [21]. Thus, if the metabolism of DDT is initiated by a direct attack on the ring
348 structure via oxygenation, DDT is metabolized to 2,3-dihydrodiol DDT, which
349 undergoes meta-cleavage in a subsequent step [14]. If dechlorination is the first step in
350 the attack, it may occur on the alkyl chain, forming DDD. Our comparison and analysis
351 of the degradation processes of the DDT isomers suggests that IcdP1 possesses both
352 dechlorinating and dioxygenase activities. Based on the degradation intermediates such
353 as DDD (A) and DDE (B), the DDT degradation is initiated by the dechlorination
354 reaction, these metabolites are transformed into DBP (E). Another oxidative ring-
355 opening pathway can be derived from the aromatic ring cleavage product 1,2-
356 benzenedicarboxylic butyl dacyl ester.
357 Most of the earlier studies have demonstrated that DDT is decomposed under
358 anaerobic circumstances. Anaerobic degradation of DDT occurred by reductive
359 dechlorination of the ethane group to DDD or by dehalogenation to DDE, then further
360 degraded by sequential steps involving reductive dechlorination and hydroxylation of
361 the ethane group, resulting in the accumulation of dichlorobenzophenone (DBP) [30–32].
362 However, few research reports the aerobic bacterial degrade DDT by oxidizing phenyl
363 ring at adjacent ortho and meta positions to form hydroxy-DDTs. These compounds
364 would be further metabolized through ring-opening reaction on benzene ring
365 [14,15,33,34]. In IcdP1-bioaugmented soils, one ring-opening metabolite was detected
30 Biology 2024, 13, x FOR PEER REVIEW 15 of 17
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366 and characterized as 1,2-benzenedicarboxylic butyl dacyl ester. This is the first time that
367 this metabolic product was detected in the degradation pathway of DDT. Thus, an
368 alternative pathway exists in which the phenyl ring is initially oxidized by a
369 dioxygenase to form hydroxy-DDT and then undergoes ring cleavage to finally yield
370 1,2-benzenedicarboxylic butyl dacyl ester. 1,2-benzenedicarboxylic butyl dacyl ester may
371 then undergo cleavage of the aromatic rings for further degradation [33,34].

372 5. Conclusions
373 In conclusion, Rhodococcus aetherivorans IcdP1 presents a valuable biological
374 resource for the bioremediation of DDT-contaminated soils. The enhancement of CSH in
375 strain IcdP1 upon exposure to DDT is a significant observation. This increase in
376 hydrophobicity could potentially facilitate better attachment of the bacterial cells to the
377 DDT molecules, thereby improving the efficiency of the degradation process. This
378 adaptation of the bacterial strain to the environmental stressor (DDT) indicates a form of
379 bioaugmentation, where the introduction of specific microorganisms can enhance the
380 natural ability of the environment to degrade pollutants. Furthermore, the study of the
381 degradation pathways of DDT in contaminated soil revealed that DDT undergoes both
382 reductive dichlorination and oxidative degradation. Understanding these pathways is
383 crucial for monitoring the progress of bioremediation efforts and assessing the potential
384 formation of any toxic intermediates. Therefore, the strain IcdP1 will be useful in future
385 OCP biodegradation studies and bioaugmentation of highly OCP-contaminated soils, it
386 is essential to continue studying such microbial strains and their mechanisms of action
387 to develop effective and sustainable strategies for combating the long-term residues of
388 OCPs and safeguarding our environment and health.

389 Author Contributions: Conceptualization, Jie Qu and Wentao Zheng; Data curation, Jie Qu,
390 Wentao Zheng and Ruya Zhang; Formal analysis, Jie Qu; Funding acquisition, Jie Qu and
391 Zhenghua Li; Methodology, Jie Qu and Wentao Zheng; Project administration, Zhenghua Li;
392 Software, Wentao Zheng and Ruya Zhang; Supervision, Zhenghua Li; Validation, Jie Qu and
393 Wentao Zheng; Visualization, Jie Qu and Wentao Zheng; Writing – original draft, Jie Qu and
394 Wentao Zheng; Writing – review & editing, Wentao Zheng and Zhenghua Li. All authors have
395 read and agreed to the published version of the manuscript. J.Q. and W.Z. contributed equally to
396 this work.
397 Funding: This research was funded by the National Natural Science Foundation of China (Grant
398 number 31800106); the Natural Science Foundation of Shandong Province (Grant number
399 ZR2019BC027); and the Talent Incubation Project of Dezhou University (Grant number
400 2019xjrc324).
401 Data Availability Statement: All data needed to evaluate the conclusions in the paper are present
402 in the paper.
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403 Acknowledgments: We thank Chonghui Li for technical assistance with the Fourier transform
404 infrared analysis.
405 Conflicts of Interest: The authors declare no conflicts of interest.

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