The 4-tert-Butylphenol-Utilizing Bacterium Sphingobium fuliginis OMI

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The 4-tert-Butylphenol-Utilizing Bacterium Sphingobium f uliginis OMI Can Degrade Bisphenols via Phenolic Ring Hydroxylation and Meta-Cleavage Pathway Yuka Ogata,† Shohei Goda,† Tadashi Toyama,‡ Kazunari Sei,§ and Michihiko Ike†,* †

Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Research, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan § Department of Health Science, School of Allied Health Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara-Minami, Kanagawa 252-0373, Japan ‡

S Supporting Information *

ABSTRACT: Recently, we showed that Sphingobium f uliginis OMI utilizes 4-tert-butylphenol as a sole carbon and energy source via phenolic ring hydroxylation followed by a metacleavage pathway, and that this strain can degrade various alkylphenols. Here, we showed that strain OMI effectively degrades bisphenol A (BPA) via the pathway in which one or two of the phenolic rings of BPA is initially hydroxylated without any modification of the alkyl group that binds the two phenolic rings, and then the aromatic ring is cleaved via a metacleavage pathway. Strain OMI also degraded other bisphenols, including bis(4-hydroxyphenyl)methane, bis(4hydroxyphenyl)sulfone (BPS), 2,2-bis(4-hydroxyphenyl)butane, bis(4-hydroxyphenyl)ethane, 2,2-bis(4-hydroxy-3methylphenyl)propane, 4,4′-thiodiphenol (TDP), and 4,4′-dihydroxybenzophenone via phenolic ring hydroxylation and metacleavage pathway. To our knowledge, this is the first report to describe the aerobic biodegradation of BPS and TDP. The bisphenols degradation pathway of strain OMI is completely different from the known degradation pathways of BPA or bisphenols, and unique in that it does not appear to be influenced by the chemical structure that binds the two phenolic rings. This newly found pathway may play a certain part in the environmental fate of bisphenols and biotreatment/bioremediation of various bisphenols.

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INTRODUCTION

understand the mechanisms of their biodegradation, including metabolic pathways. Biodegradation of bisphenols has been mostly studied with diphenylalkanes, especially BPA. The currently known bacterial BPA degradation pathways are oxidative skeletal rearrangement and ipso-substitution. The former pathway consists of major and minor pathways, both of which involve the oxidative skeletal rearrangement of an aliphatic methyl group in the BPA molecule. This pathway is used by several BPA-degrading bacteria, the classic example being strain MV1.7−12 In addition, some reports suggest that other diphenylalkanes such as BPP, BPB, and BPE are also degraded via this pathway.7,12 The latter BPA degradation pathway involves hydroxylation at position C4, followed by cleavage at the C−C bond between the phenolic moiety and the isopropyl group of BPA. This pathway is used

Bisphenol A (BPA: 2,2-bis[4-hydroxyphenyl]propane) is an industrially important chemical that is widely and abundantly used in the production of polycarbonates and epoxy resins. Because BPA has been frequently found in the environment1,2 and is known to possess estrogenic activity,3,4 it is recognized as an important hazardous pollutant. Although other bisphenols, such as bisphenol F (BPF: bis[4-hydroxyphenyl]methane), bisphenol S (BPS: bis[4-hydroxyphenyl]sulfone), bisphenol B (BPB: 2,2-bis[4-hydroxyphenyl]butane), bisphenol E (BPE: bis[4-hydroxyphenyl]ethane), and bisphenol P (BPP: 2,2-bis[4hydroxy-3-methylphenyl]propane) have been used in place of BPA in the production of resins and plastics, some reports indicate that these other bisphenols also have estrogenic activity.5,6 Therefore, the environmental fate of bisphenols, including BPA, is of a great concern when attempting to assess their potential risk accurately. In particular, since the biodegradation process has a considerable effect on the environmental fate of bisphenols, we need to completely © 2012 American Chemical Society

Received: Revised: Accepted: Published: 1017

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by the nonylphenol-degrading bacteria Sphingomonas sp. strain TTNP313and Sphingobium xenophagum Bayram14 and by the BPA-utilizing bacterium Cupriavidus basilensis JF1.15 Whether this pathway is also involved in the degradation of other diphenylalkanes has not yet been clarified. Apart from these pathways, only the BPF metabolic pathway used by Sphingobium yanoikuyae strain FM-2 has been well characterized; this degradation pathway proceeds through a Baeyer− Villiger reaction at the methyl moiety of BPF,16 however, this reaction does not occur with other diphenylalkanes. As mentioned above, the known degradation pathways of bisphenols all begin with the breakage of the alkyl groups that bind the two phenolic rings. However, bisphenols composed of two phenolic rings that are bound with sulfur, such as BPS and 4,4′-thiodiphenol (TDP), are known to be recalcitrant to biodegradation, and there are no reports describing their aerobic biodegradation or the microbes that can degrade them.17,18 Recently, we isolated the 4-tert-butylphenol (4-t-BP)-utilizing bacterium Sphingobium f uliginis OMI from the rhizosphere of the giant duckweed Spirodela polyrrhiza.19 This strain can degrade 4-t-BP and various alkylphenols (APs) via a metacleavage pathway. Since 4-t-BP shares structural similarities with BPA, in that both possess phenolic ring(s) and an αquaternary C-atom, we hypothesized that strain OMI may degrade BPA and other diphenylalkanes. Accordingly, we investigated the biodegradation of BPA and other bisphenols by strain OMI and successfully confirmed that this strain can metabolize BPA and other diphenylalkanes via a meta-cleavage pathway after aromatic ring hydroxylation. Furthermore, we demonstrated that strain OMI can degrade BPS and TDP, the aerobic biodegradation of which has not previously been reported, by using this pathway.

experiments were conducted in triplicate. Cell density and BPA concentration were monitored over a 14-day experimental period. In the BPA degradation test, whole cells were added (at OD600 = 1.0) to 3 mL of BPA-MSM (0.25 mM). The whole cell mixture was incubated at 28 °C and 120 rpm in the dark. BPA concentration and metabolic products were periodically monitored. Mineralization of BPA was also monitored as dissolved organic carbon (DOC) in the cell mixture. DOC removal at time t (%) was calculated as follow: {1-[DOC at time t − DOC in control system (without BPA) at time t]/ [initial DOC − initial DOC in control system]} × 100. Inhibition of BPA Degradation in Whole Cells of Strain OMI. To confirm whether a meta-cleavage pathway was involved in BPA degradation by strain OMI, we conducted an inhibition test using 3-fluorocatechol, which is a suicide inactivator of the meta-cleavage pathway.20 Strain OMI was grown to late exponential phase in glucose-MSM (5.0 mM). The culture was then added with 0.4 mM 3-fluorocatechol and incubated for 30 min before the start of the experiment. The cells were then harvested by centrifugation (15 000g, 4 °C, 10 min), washed twice with MSM, and resuspended in BPA-MSM (0.25 mM) with 0.2 mM 3-fluorocatechol at an OD600 = 1.0. The whole cell mixture was incubated at 28 °C and 120 rpm in the dark. BPA concentration was periodically monitored. Growth on and Degradation of Other Bisphenols by Strain OMI. Other bisphenols (BPF, BPS, BPB, BPE, BPP, TDP, and HBP) were also assessed in the utilization and degradation tests using cells of strain OMI grown in 4tBP-MSM (1.0 mM). The utilization and degradation tests were conducted in the same manner as those for BPA. Sterile control experiments without inoculation of strain OMI in utilization and degradation tests were also performed. In addition, degradation inhibition tests for the bisphenols with 3fluorocatehol were performed in the same manner as that for BPA. For the utilization tests, cell density and substrate concentrations were monitored over a 14-day experimental period. For degradation and inhibition tests, substrate concentrations were measured after 24 h of incubation. Analytical Procedures. Cell growth was monitored by the change in the OD600 of the cultures by using a UV1700 spectrophotometer (Shimadzu, Kyoto, Japan). The concentrations of each bisphenol were determined by using highperformance liquid chromatography (HPLC). Bisphenol metabolites were analyzed by means of gas chromatography− mass spectrometry (GC-MS). For HPLC analysis, the samples were acidified with 2 N HCl to pH 2−3, centrifuged (15 000g, 4 °C, 10 min), and the supernatant was analyzed. HPLC analysis was conducted in a Shimadzu HPLC system with a Shimpack VP-ODS column (250 mm × 4.6 mm i.d., particle size 5 μm, Shimadzu). In the HPLC analysis, acetonitrile and water were used at ratios of 2:3 (for BPA and BPS) and 1:1 (for other substrates) as the mobile phase, and detection was done at a wavelength of 280 nm. For the GC-MS analysis of the bisphenols and their metabolites, the culture samples at each sampling point were acidified with 2 N HCl to pH 2−3, shaken for 3 min with a half-volume of 2:1 (v:v) ethyl acetate:n-hexane, and centrifuged (415g, 10 min); the organic layer was then collected. The extract (100 μL) was dried under nitrogen, and then trimethylsilylated (TMS) at 60 °C for 1 h using 100 μL of BSTFA-acetonitrile solution (1:1, v:v). The GC-MS analysis was conducted by using a Varian 450-GC equipped with 220MS (Varian, Santa Clara, CA) and a VF-5 ms capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness, Varian). To

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MATERIALS AND METHODS Chemicals. BPA and other bisphenols (BPF, BPS, BPB, BPE, BPP, TDP, and 4,4′-dihydroxybenzophenone [HBP]) as well as 4-t-BP and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Purity of BPA and BPF were over 99%, while that of BSTFA and other chemicals were over 95 and 98%, respectively. Culture Media. Minimal salts medium (MSM) containing 1.0 g (NH4)2SO4, 1.0 g K2HPO4, 0.2 g NaH2PO4, 0.2 g MgSO4·7H2O, 0.05 g NaCl, 0.05 g CaCl2, 8.3 mg FeCl3·6H2O, 1.4 mg MnCl2·4H2O, 1.17 mg NaMoO4·2H2O, and 1 mg ZnCl2 per liter of water with an appropriate concentration of carbon sources was used for the utilization and degradation tests of BPA and the other bisphenols. MSM containing glucose (glucose-MSM) or 4-t-BP (4tBP-MSM) was used for preculture. Unless otherwise noted, the pH of the media was adjusted to 6.5. Solid agar media were prepared with 1.5% (w/ v) agar. Growth on and Degradation of BPA by Strain OMI. Strain OMI was grown (28 °C, 120 rpm) to the late exponential phase in glucose-MSM (5.0 mM). Cells were harvested by centrifugation (15 000g, 4 °C, 10 min) and rinsed twice with MSM. These cells were then used for BPA utilization and degradation tests. In the BPA utilization test, cells were inoculated at a cell density of 0.02 as determined by the optical density at a wavelength of 600 nm (OD600) in 100 mL of MSM containing BPA (BPA-MSM, 0.25 mM). The fresh culture was incubated at 28 °C and 120 rpm in the dark. The growth 1018

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Time courses of metabolites during the BPA degradation test are also shown in Figure 1 (B) and (C). During the BPA degradation test, seven metabolites with retention times of 22.7 min (metabolite I), 20.3 min (metabolite II), 27.6 min (metabolite III), 24.5 min (metabolite IV), 22.2 min (metabolite V), 9.6 min (metabolite VI), and 12.5 min (metabolite VII) were detected in the culture medium by using GC-MS analysis with TMS-derivatization. The BPA peak decreased rapidly to below the detectable limit within 3 h. The peaks of metabolites I and IV increased rapidly after the BPA peak began to decrease, reached maximum levels at 1 h, and then decreased rapidly to undetectable levels within 3 h. The peaks of metabolites II, V, and VII increased, reached maximum levels at 3 h, and decreased gradually from 3 to 24 h. The peak of metabolite VI increased, reached maximum levels at 6 h, and decreased gradually from 6 to 24 h. The peak of metabolite III increased gradually and accumulated in the culture medium during BPA degradation test. Similar trends in the metabolites formation and removal were observed in repeated experiments (3 times). Identification of Metabolites. We attempted to identify the seven metabolites by using GC-MS with TMS-derivatization. The electron impact mass spectrometry (EI-MS) spectral characteristics of the TMS derivatives of 7 metabolites are shown in Table S1 in the Supporting Information, and their estimated structures are shown in Figure 2. Metabolites I, IV, VI, and VII were tentatively identified, respectively, as 3hydroxy BPA; 2,2-bis(3,4-dihydroxyphenyl)propane; 3-(4hydroxyphenyl)-3-methyl-2-butanone; and 3-(3,4-dihydroxyphenyl)-3-methyl-2-butanone by interpreting their mass spectral patterns. The structures of metabolites II and V were best explained as shown in Figure 2 by interpreting their mass spectral patterns. Metabolites II and V can occur on the metacleavage pathways of metabolites I and IV, respectively, and are located downstream the ring fission. Their ion peaks showed EI-MS spectral characteristics similar to those of other products which are found on the meta-cleavage pathways of certain aromatics, which typically lose CH3, CH3 and CO, and C4H9O2Si.21−23 The meta-cleavage of aromatic compounds can yield two possible products, depending on the ring-cleavage styles, proximal cleavage (2,3-cleavage) and distal cleavage (1,6cleavage). However, we could not identify the ring cleavage style of metabolites II and V by strain OMI here. Metabolite III could not be identified by interpreting its mass spectral data. Removal of Organic Carbon during BPA Degradation by Strain OMI. DOC removals (means ± standard deviation) in the culture medium during the BPA degradation at times 0, 1, 3, and 6 h were 0%, 4.1 ± 2.6%, 10.1 ± 1.2%, and 13.5 ± 0.9%, respectively. The DOC decreased gradually; 13.5% of the carbon in the BPA was removed from the culture medium within 6 h. Inhibition of BPA Degradation in Whole Cells of Strain OMI. The influence of 3-fluorocatechol on degradation of BPA by whole cells of strain OMI was investigated as shown in Figure 3. 3-Fluorocatechol, a meta-cleavage inhibitor, inhibited the degradation of BPA considerably, and further degradation of its catechol products (metabolites I and IV) was also severely inhibited. As shown in Figure 3 (A), the peak of metabolite I increased and accumulated in the culture medium with 3-fluorocatechol at 6 h, though it was effectively degraded below the detectable limit within 3 h in the absence of 3fluorocatechol. Metabolite IV showed similar behavior as

analyze the bisphenols and their metabolites with TMSderivatization, the column temperature was held at 90 °C for 1 min, increased to 150 °C at 15 °C/min, increased to 300 °C at 5 °C/min, and then held at 300 °C for 2 min. The injection temperature was 300 °C. Helium gas (99.998%) was applied as a carrier gas at a flow rate of 1.2 mL/min. DOC was measured with a total organic carbon (TOC) analyzer (TOC-L, Shimadzu). The culture samples at each sampling point were acidified, and twice centrifuged (8000g, 10 min). The supernatant was filtered (0.22 μm) and then analyzed for DOC.

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RESULTS Transformation of BPA by Whole Cells of Strain OMI. The results of the BPA degradation test using whole cells of Sphingobium f uliginis OMI are shown in Figure 1 (A). 0.25 mM BPA was completely removed by cells of strain OMI grown on glucose within 3 h. The BPA utilization test revealed that BPA was not utilized for cell growth by strain OMI (data not shown).

Figure 1. Time courses of BPA (A) and metabolites (B and C) during BPA degradation by whole cells of S. f uliginis OMI. The metabolites with maximum peak levels over 5.0 × 107 are shown in (B) and the metabolites with maximum peak levels up to 1.0 × 107 are shown in (C). The peaks areas of BPA (●), metabolite I (■), metabolite II (▲), metabolite III (⧫), metabolite IV (○), metabolite V (□), metabolite VI (◊), and metabolite VII (Δ) are shown. 1019

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Figure 2. Proposed pathway for biodegradation of BPA by S. f uliginis OMI.

bisphenols is summarized in Table 1. Whole cells of strain OMI grown on 4-t-BP degraded all of the tested bisphenols, BPF, BPE, BPB, BPP, HBP, BPS, and TDP. Hydroxylated products of these bisphenols and metabolites which can found on the meta-cleavage pathways of the corresponding catechols were detected in the culture media. The EI-MS spectral characteristics of the TMS derivatives of these metabolites are shown in Supporting Information Table S2−S8. In contrast, in both degradation inhibition and sterile control tests, the tested bisphenols were not removed over the entire experimental period (Table 1). The results show that removal of bisphenols in degradation tests resulted from degradation by strain OMI rather than abiotic losses. On the other hand, strain OMI could not utilize the tested bisphenols for cell growth.

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Figure 3. Time courses of BPA degradation by whole cells of S. f uliginis OMI without and with 0.2 mM 3-fluorocatechol. (A) Concentrations of BPA without (○) and with (●) 0.2 mM 3fluorocatechol, and peak areas of metabolite I without (□) and with (■) 0.2 mM 3-fluorocatechol are shown. (B) Peak areas of metabolite IV without (Δ) and with (▲) 0.2 mM 3-fluorocatechol are shown.

DISCUSSION The present study revealed that the 4-t-BP-utilizing bacterium S. f uliginis OMI effectively degrades BPA, although BPA does not support its growth. On the basis of the metabolites identified and the behavior of metabolites during BPA degradation test, we proposed the BPA degradation pathway by strain OMI as shown in Figure 2. In this pathway, one of the BPA phenolic rings is initially hydroxylated to 3-hydroxy BPA (metabolite I) and this hydroxylated ring is then cleaved at the meta position, followed by further degradation into downstream

metabolite I (Figure 3 (B)). Similar trends in the metabolites formation were observed in repeated experiments (3 times). Transformation of Various Bisphenols by Strain OMI. The ability of strain OMI to degrade and utilize various 1020

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Table 1. Utilization and Degradation of Various Bisphenols by Whole Cells of S. f uliginis OMI

a Results are shown as means ± standard deviation for three independent experiments. bDegradation ratios (DR) were calculated from each substrate concentration determined by HPLC obtained from 0 and 24-h cultures of strain OMI whole cells as follows: DR (%) = [1-(each substrate concentration at 24 h)/(each substrate concentration at 0 h)] × 100. cHydroxylated one of phenolic rings of each bisphenol was detected. dBoth hydroxylated one of phenolic rings and hydroxylated two phenolic rings of each bisphenol were detected. eHydroxylated two phenolic rings of each bisphenol was detected. fMetabolite produced by further degradation of meta-cleavage product of catecholc was detected. gMetabolites produced by further degradation of meta-cleavage products of catecholsd were detected. hN.D., not detected.

that binds the two phenolic rings of BPA. In the BPA degradation pathway of strain OMI, one or both phenolic rings of BPA is initially hydroxylated without any modification of the alkyl group that binds the two phenolic rings, and this is followed by a meta-cleavage of the aromatic ring(s), which means that BPA degradation by strain OMI is completely different from the known BPA biodegradation pathways. The meta-cleavage pathway is one of the major pathways for the biodegradation of various aromatic compounds; however, this is the first study to demonstrate that a meta-cleavage pathway is involved in the biodegradation of BPA by strain OMI. Strain OMI degraded not only BPA but also various bisphenols, and the hydroxylated products of these bisphenols and/or metabolites which can occur on their meta-cleavage pathways were detected in the culture media (Table 1). The results suggest that phenolic ring hydroxylation and metacleavage were involved in the biodegradation of other bisphenols by strain OMI, like BPA. A particularly noteworthy finding is the ability of strain OMI to degrade BPS and TDP, the phenolic rings of which are bound with sulfur. BPS and TDP are known to be recalcitrant to biodegradation and, to the best of our knowledge, there have been no reports describing their aerobic biodegradation. Therefore, the present study is the first report of the clear biodegradation of BPS and TDP. Because degradation of BPA via oxidative skeletal rearrangement is initiated by hydroxylation of a carbon atom of a methyl group or an α-quaternary carbon in BPA,24 it is theoretically impossible for this degradation pathway to degrade BPF, the two phenolic rings of which are bound with an α-secondary Catom, or to degrade BPS and TDP, the two phenolic rings of

products like metabolite II. It is also possible that both phenolic rings of BPA are hydroxylated (metabolite IV) from metabolite I or simultaneously, and that one of the aromatic rings is then cleaved at the meta position to generate metabolite V by further degradation. Metabolite V may also be formed from metabolite II by the subsequent hydroxylation of the other aromatic ring. Furthermore, metabolites II and V are further transformed into lower degradation products (metabolites VI and VII). This degradation pathway is equivalent to a meta-cleavage pathway of an aromatic ring. This proposed pathway is supported by our finding that degradation of metabolites I and IV by strain OMI was inhibited by 3-fluorocatechol, a suicide inactivator of metacleavage pathways (Figure 3). Analysis of carbon balance during BPA degradation showed that about 13.5% of the carbon in BPA was removed within 6 h. This carbon removal ratio (13.5%) corresponds approximately to the removal of two carbon atoms from BPA containing 15 carbon atoms. The carbon numbers for metabolites II and V correspond to the loss of one carbon atom from BPA, whereas those of metabolites VI and VII correspond to the loss of four carbon atoms. On the other hand, metabolites II, V, VI, and VII accumulated in the culture after 6 h (Figure 1). From these results, two types of products seemed to be present in the culture after 6 h: (i) metabolites II and V and (ii) metabolites VI and VII, produced by further degradation of metabolites II and V. As described in the introduction, the known bacterial BPA degradation pathways to date are oxidative skeletal rearrangement and ipso-substitution. Generally, these degradation pathways are initiated with the breakage of the alkyl group 1021

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which are bound with sulfur. Indeed, Sphingomonas sp. strain BP-7 could degrade BPA, BPB, BPE, and BPP, but not BPF, BPS, and TDP. Also, it has been shown that degradation through ipso-substitution is affected by the chemical structure at the α-position.25−27 The degree of degradation achieved by the two pathways depends on the molecular structure or chemical group that binds the two phenolic rings of the bisphenols. In contrast, the pathway for bisphenols degradation, which involves hydroxylation of the phenolic ring and meta-cleavage, employed by strain OMI seems to enable the degradation of various bisphenols, including BPF, BPS, and TDP, regardless of chemical structure or group that binds the two phenolic rings. Therefore, this newly found degradation pathway will open the door to the development of new technological strategies for the simultaneous biotreatment/bioremediation of various bisphenols. Only two 4-t-BP-utilizing bacteria have been identified to date: Sphingobium f uliginis TIK23 and strain OMI.19 Previous studies have shown that both strains OMI and TIK can degrade various APs via phenol hydroxylation and meta-cleavage pathway. The AP-degrading enzymes with broad substrate specificity in the two strains also seem to contribute to the degradation of a wide range of bisphenols. In fact, like strain OMI, strain TIK can also degrade a variety of bisphenols (unpublished data). Here, we revealed a newly found pathway for the biodegradation of BPA and other bisphenols and showed that strain OMI has practical value as a catalyst for the degradation of various bisphenols, including extremely recalcitrant ones, such as BPS and TDP. Our results provide new insights into the fate and biodegradation of bisphenols in aquatic environments. This degradation pathway may play a part in the environmental fate of bisphenols. In particular, the rhizosphere of aquatic plants seems to be an active spot for this pathway, because both strains OMI and TIK have been isolated from the rhizosphere of aquatic plants. To effectively use the findings obtained in this study for the environmental risk assessment and wastewater treatment or bioremediation technologies for bisphenols, future studies should be conducted to evaluate the toxicity of biodegradation end-products of this degradation pathway, as well as the potential activity and distribution of this degradation pathway in other environments.

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Advanced Low Carbon Technology Research and Development Program of the Japan Science and Technology Agency.

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ASSOCIATED CONTENT

S Supporting Information *

EI-MS spectral data of metabolites formed during BPA degradation (Table S1) and other various bisphenols degradation (Table S2-8) by strain OMI. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-(0)6-6879-7674; fax:+81-(0)6-6879-7675; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by Grants-in-Aid for Encouragement of Young Scientists (A) 21681010, (A) 24681011, and (B) 22710069 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the 1022

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dx.doi.org/10.1021/es303726h | Environ. Sci. Technol. 2013, 47, 1017−1023