Research Article pubs.acs.org/journal/ascecg
Transformation of Lignin-Derived Aromatics into Nonaromatic Polymeric Substances with Fluorescent Activities (NAPSFA) by Pseudomonas sp. ITH-SA‑1 Noriyuki Iwabuchi,*,† Hajime Takiguchi,† Takashi Hamaguchi,† Hayato Takihara,† Michio Sunairi,† and Hiroshi Matsufuji*,‡ †
Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan ‡ Department of Food Bioscience and Biotechnology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan S Supporting Information *
ABSTRACT: We examined bacteria capable of transforming lignin-derived compounds into industrially or economically valuable substances from the seawater after the Great East Japan Earthquake-caused tsunami. Pseudomonas sp. ITH-SA-1 produced watersoluble fluorescent substances from the lignin-derived aromatic, syringaldehyde (SYAL). They: are polymeric substances derived from 3-O-methyl gallate produced through transformation of SYAL via syringate; are not known compounds reported previously; have excitation/emission peaks at 365/498 nm, respectively; and have an average molecular weight of 7.2 kDa. Despite the fact that aromatic structure generally plays an important role in the planarity and rigidity of organic fluorescent substance, the spectroscopic analyses revealed that aromatic rings were not detected in the molecules. Their activity is particularly rare and the biotransforming capabilities will contribute to the development of new basic techniques for the effective utilization of lignin. KEYWORDS: Nonaromatic Fluorescent substance, Syringaldehyde, Lignin, Pseudomonas sp. ITH-SA-1, Organic fluorescent substances, the Great East Japan Earthquake
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INTRODUCTION Lignin is a highly polymerized aromatic compound composed of phenylpropanoids and is a major component of plant cell walls. Lignin is considered an underutilized natural biomass. Syringaldehyde (SYAL) and vanillin (VNL) are major intermediate metabolites of lignin-derived aromatics,1 and these compounds reportedly have great potential for enhancing the utilization of lignin as a bioresource.2 In addition, Ragauskas et al.3 recently reported that the well-established commercial production of VNL from lignin provides a strong precedent for future innovative advances in lignin valorization. However, techniques for utilizing lignin remain limited to the production of products such as low-value fuels; therefore, to expand the possible uses of lignin as a means of enhancing future sustainability, it will be necessary to develop techniques for transforming lignin and/or lignin-derived aromatic compounds to higher-value products. Efficient and economical techniques are required for the total transformation of lignin into high-value products.4−6 The techniques can be classified as biological, physical, or chemical. The industrial application of “white biotechnology” processes is particularly attractive because it can result in the synthesis of products that require less energy to produce, create less waste during their production, and are easily degradable.7 White © XXXX American Chemical Society
biotechnology uses biological systems to produce chemicals, materials, and energy and is based primarily on biocatalysis and fermentation using molecular genetics, enzyme engineering, and metabolic engineering.8 Several research groups have reported the bioconversion of lignin derivatives by Sphingobium sp. SYK-6 (formerly Sphingomonas paucimobilis SYK-6) into high-value-added materials such as 2-pyrone 4,6-dicarboxylic acid (PDC),9 which can be further converted into high-polymer materials such as polyamide, polyester, or polyurethane or by Oceanimonas doudoroffii into the biodegradable polymer polyhydroxyalkanoate.10 Some marine bacteria are capable of degrading and transforming lignin derivatives, facilitating utilization of the most refractory components in aquatic environments.11 On March 11, 2011, coastal areas in the Touhoku region of Northeast Japan suffered catastrophic damage as a result of the Great East Japan Earthquake and subsequent giant tsunami. As a great deal of onshore soil, dirt, and rubble were carried into the marginal sea by the tsunami, it is presumed that the structures of the native bacterial communities in the affected Received: June 6, 2015 Revised: September 2, 2015
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DOI: 10.1021/acssuschemeng.5b00503 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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In addition, fresh seawater was plated onto separate agar plates, and all plates were incubated at room temperature. Colonies exhibiting pigment-producing activity were picked and purified by single-colony isolation for use in downstream experiments. Culture Conditions for Transformation of SYAL by Pseudomonas sp. ITH-SA-1 and HPLC Analysis of Metabolites in Culture Supernatants. Pseudomonas sp. ITH-SA-1 was precultured in Marine Broth 2216 (MB, Difco) for 4 days at 28 °C with shaking at 110 rpm, after which aliquots of the precultures were transferred into fresh MB supplemented with SYAL (hereafter referred to as MB-SYAL medium) at a final concentration of 1 mg/mL and incubated for 4 days at 28 °C with shaking at 110 rpm. Small aliquots of medium from each culture were withdrawn periodically for analysis of metabolites by reversed-phase HPLC and size-exclusion HPLC after centrifugation and filtration using a 0.45 μm membrane filter. SYAL metabolites were analyzed by reversed-phase HPLC using an Alliance 2690 system (Waters Co.) equipped with photodiode array (996 PDA, Waters Co.) and fluorescence (FS-8020, Tosoh Corp.) detectors. Separation of metabolites was achieved on a Waters Xbridge C18 column (4.6 mm i.d. × 150 mm, 5 μm particle size). Mobile phase A consisted of 0.5% TFA and mobile phase B consisted of methanol/0.5% TFA. Metabolites were eluted using the following gradient: 0−5 min, 5% B; 5−30 min, 5−30% B; 30−40 min, 30−100% B; 40−45 min, 100% B; 45−50 min, 100−5% B. The column was reequilibrated for 15 min after each gradient run. The flow rate was 1.0 mL/min. The detection wavelength was set at 200−450 nm for the photodiode array detector, and elution of the fluorescent substance was detected using the fluorescence detector at excitation and emission wavelengths of 365 and 498 nm, respectively. The column temperature was maintained at 40 °C. Size-exclusion HPLC was carried out using a Tosoh TSK-gel G3000SWXL column (7.8 mm i.d. × 300 mm, 5 μm particle size) and an Agilent HP1100 system equipped with photodiode array (G1315B DAD, Agilent Inc.) and fluorescence (G1321A FLD, Agilent Inc.) detectors. The column was eluted isocratically using 20 mmol/L sodium phosphate buffer (pH 7.0) containing 200 mmol/L sodium chloride at a flow rate of 0.5 mL/min. Detection conditions were as described above. The column was calibrated each time with the following native protein standards; glutamate dehydrogenase (Mr, 290 × 103; Rt, 12.89 min), lactate dehydrogenase (Mr, 142 × 103; Rt, 14.92 min), enolase (Mr, 67 × 103, Rt, 16.46 min), myokinase (Mr, 32 × 103; Rt, 17.84 min), cytochrome c (Mr, 12.4 × 103; Rt, 19.33 min), aprotinin (Mr, 6.51 × 103; Rt, 20.56 min), and Bz-Gly-His-Leu (Mr, 0.43 × 103; Rt, 20.07 min). The number-average molecular weight of fluorescent substances was estimated from the calibration curve (y = 4.51 × 105e−0.545x). The standards were prepared by the addition of aprotinin (0.3 mg) and Bz-Gly-His-Leu (0.3 mg) to the vial of MWmarker proteins (Oriental Yeast Co., Ltd.) and dissolving in 0.2 mL of mobile phase. Standards were analyzed before and after HPLC analysis of the sample. These experiments were done in at least triplicate. The data presented are the means of replicate experiments. Extraction of Fluorescent Substance by Methanol. Pseudomonas sp. ITH-SA-1 was cultured in MB-SYAL (2 L) at 28 °C for 20 days. After centrifugation, the culture supernatant was filtered using a membrane filter, and the filtrate was transferred into a dialysis bag (Spectra/Por Biotech CE, MWCO 3500, Spectrum Laboratory Inc.) and dialyzed for 18 h against Milli-Q water. The dialysis was repeated four times, and the final dialysate was concentrated and freeze-dried (7.8 g). Fluorescent substances were extracted repeatedly with methanol to remove insoluble impurities, particularly water-soluble black pigments, and then filtered. The filtrate was evaporated to dryness, dissolved in a small volume of water, and freeze-dried (2.3 g). The resulting freeze-dried powder (methanol extracts with fluorescent activity; hereafter referred to as MEFA) was sealed tightly in a recovery flask and kept at room temperature until used. UV−vis, ATR-FTIR, and NMR Spectroscopy. UV−vis spectra of MEFA were obtained using a Shimadzu UV-1700 spectrophotometer, and excitation and emission spectra were obtained using a JASCO FP6500 spectrofluorometer. Attenuated total reflectance Fourier-trans-
waters were significantly altered. In our other studies aimed at identifying aromatic compound-degrading bacteria, we examined indigenous bacteria in marine environments of the Pacific nearshore area in the Touhoku region more than 15 years ago, and we reported that Cycloclasticus spp. play a key role in the degradation of various aromatic hydrocarbons in seawater.12,13 During the course of these studies, 16S rDNA-based PCRdenaturing gradient gel electrophoresis (DGGE) analyses of seawater samples taken from Heita Bay in Iwate Prefecture after the earthquake-caused tsunami revealed an increase in the diversity of aromatic hydrocarbon-degrading bacterial consortia (Figure S1, Supporting Information). In addition, the DGGE profiles varied from experiment to experiment, even though seawater samples taken from the same sampling location were used for the enrichment cultures. The same trends were observed in DGGE profiles in experiments using seawater taken from other sampling locations along the Pacific nearshore areas of Iwate and Miyagi after March 11, 2011. This phenomenon might have been due to bacteria derived from onshore materials that were carried away with the floodwaters into the marginal sea. Upon observing these bacterial-ecological changes, we hypothesized that (i) after the tsunami, the seawater harbored various aromatic hydrocarbondegrading bacteria in addition to Cyclcolasticus spp.; and (ii) because the materials carried into the marginal sea of Heita Bay by the tsunami floodwaters included large quantities of woodderived dirt and/or rubble, bacterial consortia that occur naturally in such environments and that are capable of degrading and/or transforming lignin and lignin-derived aromatic compounds were also carried into Heita Bay. Because the development of basic techniques for effective lignin utilization remains a high priority area in both basic research and industry,14,15 we conducted an aggressive screening study focusing on the isolation and identification of bacteria capable of degrading and/or transforming lignin and lignin-derived lowmolecular-weight aromatic compounds. In the present study, we isolated bacteria capable of degrading and/or transforming lignin-derived aromatic compounds into functional, industrially beneficial, and/or economically valuable products using seawater collected from Heita Bay after the March 11, 2011 tsunami. One of the bacteria isolated in this study, Pseudomonas sp. ITH-SA-1, produced fluorescent polymeric substances from lignin-derived aromatic compounds. Subsequent chemical characterization of these polymeric substances revealed that they probably do not contain aromatic rings.
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MATERIALS AND METHODS
Seawater Sampling and Bacterial Screening. Seawater samples were collected from Heita Bay (lat. 39°14′57″ N, long. 141°53′46″ E) in May and June of 2011. For enrichment culture, natural seawater medium (NSW medium)13 was prepared using the sampled seawater, and biphenyl (BPH), phenanthrene (PHN), SYAL, VNL, phydroxybenzaldehyde (PHBA), or water-soluble lignin (LGN) was added to the NSW medium at a final concentration of 1 mg/mL as a carbon source. BPH and PHN were added to NSW medium as described previously.13 Other chemicals were added to NSW medium after dissolving in sterilized Milli-Q water or dimethyl sulfoxide (DMSO). These chemicals were purchased from Wako Pure Chemicals or Nacalai Tesque. Samples were incubated in NSW medium supplemented with the above-mentioned carbon sources for 2 weeks at room temperature with shaking at 110 rpm. Aliquots of the cultures were then plated onto NSW-agar or Marine Agar 2216 (Difco) supplemented with BPH, PHN, SYAL, VNL, PHBA, or LGN. B
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ranging between 4 and 28 °C in MB-SYAL. When SYAL was supplied at a final concentration of 0.01 to 20 mg/mL, the fluorescent substances were produced under aerobic conditions regardless of whether shaking was employed. In addition, fluorescent substances produced by Pseudomonas sp. ITH-SA-1 were observed in the supernatants of some complete media, such as Luria broth (LB), nutrient broth (NB), or Trypto-soy broth (TS) when SYAL was added to the medium (the production of fluorescent substances and growth of ITH-SA-1 are summarized in Table S2, Supporting Information). In contrast, the fluorescent substances were not produced in minimum media amended with SYAL, nor were the fluorescent substances produced by Pseudomonas sp. ITH-SA-1 in MBSYAL under anaerobic conditions, regardless of the cultivation temperature. Pseudomonas sp. ITH-SA-1 could not use SYAL, VNL, or PHBA as a sole carbon and energy source. These data suggest that some other organic compounds are required for the production of fluorescent substances by Pseudomonas sp. ITH-SA-1 (Table S2, Supporting Information). We then examined the relationship between cell growth and changes in the SYAL concentration during culture and found that the concentration of SYAL decreased as the number of cells increased (Figure S2, Supporting Information). These data suggest that the fluorescent substances are derived from SYAL through transformation by Pseudomonas sp. ITH-SA-1. Primary Detection of Major Intermediate Metabolites and Fluorescent Substances Produced by Pseudomonas sp. ITH-SA-1 Cultured in MB-SYAL. To elucidate the metabolic pathway for the production of fluorescent substances by Pseudomonas sp. ITH-SA-1, we analyzed the intermediate metabolites contained in the supernatant of cells cultured in MB-SYAL. Two major peaks (1 and 2) and one broad peak were observed upon reversed-phase HPLC analysis of the culture supernatant with detection at 254 nm (Figure 2a). The area of these peaks increased with increasing incubation time. The broad peak (retention time of approximately 38 min) exhibited fluorescence at excitation and emission wavelengths of 365 and 498 nm, respectively (Figure 2b). These data suggest that Pseudomonas sp. ITH-SA-1 releases fluorescent substances into the supernatant when cultured in MB-SYAL. The structures of peaks 1 and 2 were subsequently identified as 3-O-methyl gallate (3-MGA) and syringate (SYAC), respectively, by MS and NMR analyses. ESI-LC-MS/MS analysis (Waters Quattro Premier XE MS system) of peaks 1 and 2 showed corresponding [M + H]+ protonated molecular ion peaks at m/z 185 (C8H8O5) and m/z 199 (C9H10O5), respectively. The structures were confirmed by 1H- and 13C NMR analyses of the isolated compounds and coinjection HPLC analyses with authentic compounds. When the MB medium was cultured in the absence of SYAL or Pseudomonas sp. ITH-SA-1, the two major intermediate metabolites were not detected in the culture supernatant (data not shown). In addition, gallic acid (retention time of 3.88 min) was detected at trace levels, but expected and/or known compounds such as shikimic acid, 2-pyrone-4,6-dicarboxylate (PDC),1,16 tyrosine, syringaldazine,17 phenylalanine, and fluorescein were not detected in the supernatant of Pseudomonas sp. ITH-SA-1 cultured in MB-SYAL. Pyruvate and oxaloacetate16,18 were also not detected in the supernatant. These results suggest that SYAC and 3-MGA are produced by Pseudomonas sp. ITH-SA-1 through the transformation of SYAL.
form infrared (ATR-FTIR) analysis was performed on a solid sample of MEFA using a JASCO FTIR 4100 spectrometer equipped with a JASCO ATR PRO450-H ATR module. 1H-(500 MHz) and 13C-(150 MHz) nuclear magnetic resonance (NMR) spectra were collected using a JEOL ECA-500 spectrometer in D2O (1H- and 13C NMR spectra of SYAL, SYAC, and 3-MGA were collected in 30% methanold4).
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RESULTS Screening for Bacteria Capable of Degradation and/ or Transformation of Aromatic Compounds. We first examined bacteria capable of degrading and/or transforming various aromatic compounds in seawater. Eighteen strains exhibiting pigment-producing activity were isolated from seawater-based enrichment cultures containing SYAL, VNL or PHBA. No bacteria showing such activities were isolated from the enrichment cultures containing LGN, BPH or PHN in this experiment. Subsequent 16S rRNA gene analysis showed that these isolates belonged to the genera Altererythrobacter, Brevibacterium, Glaciecola, Kocuria, Microbacterium, Novosphingobium, and Pseudomonas (Table S1, Supporting Information). During the course of the study, some isolates produced watersoluble fluorescent substances that were released into the culture supernatant when cells were cultivated in MB-SYAL, -VNL, or -PHBA. To characterize the relationship between the production of fluorescent substances and the substrates added to the medium, the isolates were re-examined by the naked eye after an additional 2 weeks of cultivation. Of the 54 conditions tested (18 strains × 3 substrates), primary strong fluorescence (denoted as +++ in Table S1, Supporting Information) was only observed with Pseudomonas sp. ITH-SA-1 cultured in MBSYAL. Pseudomonas sp. ITH-SA-1 released fluorescent substances together with water-soluble black pigments into the supernatant when SYAL was added to the medium as a substrate (Figure 1a). The fluorescence was not observed inside
Figure 1. Fluorescent substances produced from SYAL by Pseudomonas sp. ITH-SA-1. (a) Fluorescence in the supernatant of Pseudomonas sp. ITH-SA-1 cultured in MB-SYAL; (b) fluorescence of MEFA.
the cells. Because organic fluorescent substances are economically valuable compounds, we chose Pseudomonas sp. ITH-SA1 as the first candidate for further downstream analysis. Production of Fluorescent Substances by Pseudomonas sp. ITH-SA-1 Cultured in MB-SYAL. The fluorescent substances were present in the supernatant when Pseudomonas sp. ITH-SA-1 was cultured in MB-SYAL; thus, we subsequently determined the culture conditions necessary for their production. The fluorescent substances were produced when Pseudomonas sp. ITH-SA-1 was cultured at temperatures C
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added to the MB as a starting substrate decreased substantially during the first 10 days until reaching 0.24 mmol/L, after which the concentration decreased to less than the limit of quantitation (0.03 mmol/L) for the remainder of the experiment. In contrast, the concentration of SYAC in the culture supernatant increased rapidly over the first 7 days, ultimately reaching a maximum concentration of approximately 1.5 mmol/L, which was maintained throughout the remainder of the experiment. 3-MGA reached detectable levels after 3 days, reaching a maximum concentration of approximately 0.5 mmol/L after 10 days, after which the concentration gradually decreased to less than the limit of detection by 35 days. The GA concentration was less than the limit of quantification (0.03 mmol/L) throughout the experiment. The level of fluorescent substances began to increase after 7 days, at which time the level plateaued. After 18 days, the level of fluorescent substances began to increase gradually and continued to increase until the end of the experiment. It appeared that approximately 1.5 mmol/L of SYAL was transformed into fluorescent substances (Figure 3). The SYAL to fluorescent substances transformation frequency was estimated roughly 50%. During the cultivation, a close relationship between decreasing SYAL concentration and increasing SYAC concentration was found. In addition, the concentration of 3-MGA began to increase after SYAC reached its maximum concentration. Other research has shown that SYAL is transformed to 3-MGA via SYAC by Sphingobium sp. SYK61,16 and Pseudomonas putida.18 When considered in relation to previously reported data, our results suggest that the SYAC and 3-MGA released into the culture supernatant by Pseudomonas sp. ITH-SA-1 are derived from SYAL and that this bacterium transforms SYAL to 3-MGA via SYAC. To determine whether SYAL is the only substrate used by Pseudomonas sp. ITH-SA-1 for the production of fluorescent substances, cells were cultured in MB with either SYAC, 3MGA, GA, PDC, pyruvate, or oxaloacetate as the starting substrate, and the supernatants were subsequently examined for the presence of fluorescent substances (Table S2, Supporting Information). Fluorescence was observed in the presence of SYAC or 3-MGA; thus, HPLC analyses were performed to examine the metabolites. When SYAC was added to MB as a
Figure 2. Reversed-phase HPLC of metabolites produced from SYAL by Pseudomonas sp. ITH-SA-1. (a) UV detection at 254 nm; (b) fluorescence detection at excitation and emission wavelengths of 365 and 498 nm, respectively.
Relationship between Bacterial Growth and the Production of Fluorescent Substances via Major Intermediate Metabolites. To better define the relationship between growth of Pseudomonas sp. ITH-SA-1 and the production of fluorescent substances via major intermediate metabolites, 35 day time-course static culture experiments were performed. Bacterial growth and changes in the levels of SYAL, SYAC, and 3-MGA during cultivation were assessed by counting CFUs and by HPLC analysis, respectively. Plots of the estimated concentrations of these compounds in the supernatant versus cultivation time are shown in Figure 3. The cell density reached approximately 108 CFU/mL within 5 days, and this density was maintained throughout the remainder of the experiment. The concentration of SYAL
Figure 3. Relationship between bacterial growth and concentrations of metabolites from SYAL in the supernatant of Pseudomonas sp. ITH-SA-1 cultured in MB-SYAL for 35 days. Black line, bacterial growth (CFU/mL); blue line, concentration of SYAL (mmol/L); orange line, concentration of SYAC (mmol/L); green line, concentration of 3-MGA (mmol/L); red line, sum of peak area of fluorescent substances detected at excitation and emission wavelengths of 365 and 498 nm, respectively. D
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acetone, or DMSO, and MEFA exhibited fluorescence over a wide pH range (pH 2−13) (Figure S3, Supporting Information). The MEFA was also protease- and heat-resistant (95 °C, 60 min), suggesting that the substances are not protein (data not shown). ICP-MS analysis revealed that MEFA contained 2.7% inorganic elements (Na, 6600 ppm; Mg, 6300 ppm; Ca, 5300 ppm; S, 5600 ppm; Fe, 3000 ppm; P, 620 ppm; Zn, 53 ppm; Sr, 130 ppm; Ti, 23 ppm; Mn, 1 ppm; Ba, 8 ppm; B, 17 ppm), which suggests that the fluorescent substances are not inorganic. ATR-FTIR and NMR Analyses. ATR-FTIR spectra of MEFA are shown in Figure 6. The observed signals were broad
starting substrate, only 3-MGA was detected in the supernatant as a major intermediate metabolite. In addition, when 3-MGA was added to MB, no significant intermediate metabolites were detected (data not shown). From these results, we concluded that Pseudomonas sp. ITH-SA-1 probably produces fluorescent substances from downstream intermediate metabolites derived from SYAL, such as 3-MGA. Characterization of Fluorescent Substances Produced by Pseudomonas sp. ITH-SA-1. For chemical characterization, MEFA was prepared from the supernatant of Pseudomonas sp. ITH-SA-1 cultured in MB-SYAL as described in the Materials and Methods. Figure 4 shows UV−vis
Figure 4. (a) UV and (b) excitation and emission spectra of MEFA. The spectra shown in panel B were obtained using 0.1 mg/mL of MEFA.
excitation and emission spectra for MEFA. In the UV−vis spectrum, characteristic absorption was not observed, although weak absorption at 255 and 345 nm was detected (Figure 4a). MEFA had excitation/emission peaks at 365/498 nm, characteristic of a blue-green fluorescent dye (Figure 4b). Size-exclusion HPLC analysis revealed that the average molecular weight of the fluorescent substances was about 7.2 kDa, and no fluorescence was detected from lower-molecularweight compounds (Figure 5). MEFA was soluble in water and methanol but not in ethanol, 1- and 2-propanol, acetonitrile,
Figure 6. ATR-FT/IR spectra of SYAC, SYAL, 3-MGA, and MEFA.
compared to those of SYAL, SYAC, and 3-MGA, suggesting that MEFA is composed of a mixture of various polymeric substances. The peaks at approximately 3400, 1650, and 1100 cm−1 are indicative of absorption due to a hydrogen-bonded hydroxyl group, carbonyl group, and esters, respectively. Surprisingly, clear peaks derived from out-of-plane C−H bending at 900−650 cm−1 or from C=C ring stretching at 1500 and 1650 cm−1 were not observed.19 In addition, no aromatic proton and carbon signals were observed in the 1Hand 13C NMR spectra, respectively, but signals indicative of acyclic hydrocarbons, alcohols, ethers, and carbonyl groups were observed, as shown in Figure 7. These data indicate that the fluorescent polymeric substances do not contain aromatic rings. These findings are particularly surprising in view of the fact that nonaromatic organic compounds had fluorescent activities.
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DISCUSSION In this study involving seawater samples collected from the Pacific Ocean after the Great East Japan Earthquake and subsequent tsunami, we isolated bacteria capable of transforming lignin-derived aromatics into functional, industrially beneficial, or economically valuable compounds. We found that one of the isolates, Pseudomonas sp. ITH-SA-1, produces new fluorescent substances from SYAL. Chemical characterization of the fluorescent substances revealed that the substances are polymeric (Figure 2); the polymeric substances are derived from 3-MGA transformed SYAL via SYAC (Figure 3); and the fluorescent polymeric substances, do not contain aromatic rings (Figures 6 and 7). Because of the absence of aromatic rings, we here designate the compounds “non-aromatic polymeric substances with fluorescent activity” (NAPSFA).
Figure 5. Size-exclusion HPLC analysis of MEFA. (a) UV detection at 210 nm; (b) fluorescence detection at excitation and emission wavelengths of 365 and 498 nm, respectively. The number-average molecular weight of fluorescent substances was estimated from the calibration curve described in Materials and Methods. E
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Figure 7. 1H- and 13C NMR spectra of SYAL, SYAC, 3-MGA, and MEFA. The signals in the 1H- and 13C NMR spectra of SYAL, SYAC, and 3-MGA were assigned on the basis of chemical shifts and the results of pulsed-field gradient heteronuclear multiple quantum coherence and heteronuclear multiple-bond connectivity studies.
Figure 8. Known pathway for the metabolism of SYAL by bacteria, modified as described previously by Masai et al.1 and Kamimura et al.16 Compounds in the box at the top are available as starting materials for the production of NAPSFA by Pseudomonas sp. ITH-SA-1. The compounds within the dashed-line box are unstable intermediate metabolites of SYAL.
and/or SYAC. Masai et al.1 previously reported that the 3-MGA catabolic pathway involves the following steps: (i) conversion of 3-MGA to 4-oxalomesaconate (OMA) via GA; (ii) conversion of 3-MGA to OMA via 4-carboxy-2-hydroxy-6-
Data showing that the NAPSFA do not contain aromatic rings and that the metabolites downstream from 3-MGA contribute to the production of the NAPSFA suggest that their production requires ring-opening polymerization of 3-MGA F
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may assist in the long term recovery and reconstruction of the areas affected by the tsunami following the Great East Japan Earthquake. We believe that the discoveries made in this study will contribute to the recovery effort by enhancing the growth of an industry devoted to the development of novel applications for rare organic fluorescent substances such as NAPSFA.
methoxy-6-oxohexa-2,4-dienoate (CHMOD); and (iii) conversion of 3-MGA to PDC. The authors postulated that the first pathway is the major mechanism of 3-MGA catabolism. In addition, they also reported that OMA is transformed to 4carboxy-4-hydroxy-oxoadipate (CHA) and that the CHA is subsequently degraded to pyruvate and oxaloacetate (Figure 8). The fluorescence was not detected in the culture supernatant following addition of GA, PDC, pyruvate, or oxaloacetate to MB as available starting substrates in the present study (Table S2, Supporting Information). These data suggest that NAPSFA consists of precursors that are chemically unstable intermediate metabolites such as CHMOD and/or unidentified intermediate metabolites. The planarity and rigidity of a fluorescent molecule’s structure generally play an important role in the absorption of light and emission of fluorescence. For this reason, most fluorescent organic compounds are aromatic molecules containing several conjugated double bonds. However, our ATR-FTIR and NMR analyses suggested that NAPSFA does not contain aromatic structures. The mechanism of fluorescence emission by organic fluorescent substances such as NAPSFA is unclear at present. Matsumoto et al.20,21 reported that the topochemical polymerization of conjugated 1,3-diene carbonic acid monomers such as sorbic acid and muconic acid yields a crystalline stereoregular polymer in which some nalkylamines are intercalated to form a layered ammonium polymer crystal. Although the authors did not report any fluorescence associated with the polymer, their findings of conjugated topochemical polymerization and intercalation of nalkylamines provide some clues as to why nonaromatic polymeric substances such as NAPSFA emit fluorescence and what controls the planarity and rigidity of NAPSFA. It is reported that organic-conjugated polymers with lightemitting have attracted considerable interest due to the beneficial properties such as flexibility, low weight, good processability in solution, and are usefully utilized in the optoelectronic and electrochemical fields, for example, multicolor organic electroluminescent diodes,22 organic solar cells,23 chemical sensors24 and so on. Because NAPSFA is one of such polymers, and discovering and/or constructing new materials will contribute to develop such industrial fields; therefore, further analyses of NAPSFA is necessary for investigations of putative synthesis of 1,3-diene carbonic acids such as CHMOD and OMA derived from 3-MGA and intercalation of nalkylamines. To the best of our knowledge, the NAPSFA produced by Pseudomonas sp. ITH-SA-1 are particularly rare organic fluorescent substances. Therefore, it is possible that our discovery may expand the applications of organic fluorescent substances. In this study, we isolated some strains that produce fluorescent substances from lignin-derived aromatics. By the primary characterization of fluorescent substances produced by these strains, each fluorescent substance had different excitation/emission peaks in strain to strain and/or in substrate to substrate (data will appear elsewhere). This suggests that a variety of organic fluorescent substances are produced from lignin-derived aromatics through bioconversion. As organic fluorescent substances are economically valuable compounds, further detailed characterization of the substances isolated in this study and the biotransforming capabilities of our isolates will contribute to the development of new basic techniques for the effective utilization of lignin and lignin-derived aromatics using biotechnology. In addition, the results of our research
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CONCLUSION The Pseudomonas sp. ITH-SA-1, which isolated from the seawater samples after the Great East Japan Earthquake-caused tsunami, released water-soluble fluorescent substances from a lignin-derived aromatic, SYAL, into the culture supernatant. The chemical characterization of NAPSFA revealed that they: (i) are polymeric substances derived from 3-MGA produced through transformation of SYAL via SYAC; (ii) are not known compounds reported previously; (iii) have excitation/emission peaks at 365/498 nm, respectively; and (iv) have an average molecular weight of 7.2 kDa. (v) Despite the fact that aromatic molecule’s structure plays an important role in the planarity and rigidity of organic fluorescent substance, NMR and ATR-FTIR analyses revealed that the fluorescent substances contain a number of carbonyl groups and aliphatic ethers, but not aromatic rings in the molecules. These data indicated that the production of particularly rare organic fluorescent substances derived from lignin-derived aromatics by Pseudomonas sp. ITHSA-1. The biotransforming capabilities will contribute to the development of new basic techniques for the effective utilization of lignin and lignin derivatives using biotechnology.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.5b00503. Details regarding culture conditions, determination of pigment production, fluorescence emission, extraction of total DNA, sequencing of 16S rDNA genes, DGGE profiles for pre- and post-tsunami seawater sampleenrichment cultures supplied with BPH, plotting of cell growth and SYAL concentrations versus cultured times, fluorescence of methanol extracts (MEFA) at pH 2 to pH 13 and in various solvents, pigment-producing activity and fluorescence of the 54 conditions tested (18 isolated strains × 3 substrates, SYAL, VNL, PHBA), and the production of fluorescent substances and growth of Pseudomonas sp. ITH-SA-1 in different media (PDF).
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AUTHOR INFORMATION
Corresponding Authors
*N. Iwabuchi. E-mail:
[email protected]. Tel/Fax: +81-466-84-3354. *H. Matsufuji. E-mail:
[email protected]. Tel/Fax: +81-466-84-3988. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Prof. Yoshihiro Katayama, NUBS, Nihon Univ., for valuable discussions and for kindly providing synthetic PDC. We are grateful to Youichi Ono, NUBIC, Nihon Univ., for illustration assistance in preparing the cover G
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Research Article
ACS Sustainable Chemistry & Engineering
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image. This study was supported by grants-in-aid for Scientific Research (B), Scientific Research (C), and Exploratory Research from the Japan Society for the Promotion of Science (JSPS).
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DOI: 10.1021/acssuschemeng.5b00503 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
