Development of a Simple Nonbiological Method for Converting Lignin

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Development of a Simple, Non-Biological Method for Converting Lignin-Derived Aromatics into Non-Aromatic Polymeric Substances with Fluorescent Activity (NAPSFA) Noriyuki Iwabuchi, Yuki Sakano, Hajime Takiguchi, Hayato Takihara, Michio Sunairi, and Hiroshi Matsufuji ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01009 • Publication Date (Web): 20 Jun 2016 Downloaded from http://pubs.acs.org on June 22, 2016

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ACS Sustainable Chemistry & Engineering

Development of a Simple, Non-Biological Method for Converting Lignin-Derived Aromatics into NonAromatic Polymeric Substances with Fluorescent Activity (NAPSFA)

Noriyuki Iwabuchi,*,† Yuki Sakano, †Hajime Takiguchi, † 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)

ACS Paragon Plus Environment

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KEYWORDS: non-aromatic polymeric substances with fluorescent activity, Tryptone, 3-Omethyl gallate, high-value-added materials, biomass, lignin

ABSTRACT: We recently reported that Pseudomonas sp. ITH-SA-1 can transform the ligninderived aromatic compound syringaldehyde (SYAL) into non-aromatic polymeric substances with fluorescent activity (NAPSFA). NAPSFA are particularly rare organic substances that fluoresce despite the absence of aromatic rings. In this study, we developed a simple method for producing NAPSFA using a non-biological process. Incubation of the SYAL metabolite, 3-Omethyl gallate (3-MGA) produced by Pseudomonas sp. ITH-SA-1, with Marine broth (MB) or Luria-Bertani (LB) broth produced fluorescence, even in the absence of bacteria, suggesting that fluorescent substances were produced from 3-MGA by a non-biological process. 3-MGA reacted with Tryptone and Peptone, which are the primary nitrogen and phosphate sources in MB and LB, to produce the fluorescent substances. The fluorescent substances produced from the reaction of 3-MGA and Tryptone exhibited excitation/emission peaks at 370/505 nm, respectively, nearly identical to those of the NAPSFA (365/498 nm). The average molecular weight of the fluorescent substances (4.2 kDa) was lower than that of NAPSFA (7.2 kDa). ATRFTIR and NMR analyses revealed that the molecules contained no aromatic rings, similar to NAPSFA. Our results demonstrate that non-aromatic fluorescent substances can be synthesized via a simple chemical reaction.

■INTRODUCTION


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Lignin is considered to be an underutilized natural biomass. Syringaldehyde (SYAL) and vanillin (VNL) are major intermediate metabolites of lignin-derived aromatics1 that reportedly show great potential for enhancing the utilization of lignin as a bioresource.2 Also, future innovative approaches to valorize lignin into value-added fuels, chemicals, and materials have been recently reported and reviewed.

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However, techniques for utilizing lignin remain limited

to the production of materials such as low-value fuels. To expand the possible uses of lignin as a means of enhancing future sustainability, it will therefore be necessary to develop techniques for transforming lignin and/or lignin-derived aromatic compounds to higher-value products. Organic fluorescent substances are economically valuable compounds. Light-emitting organic-conjugated polymers have attracted considerable interest due to their beneficial properties and are utilized in the optoelectronic and electrochemical fields in a variety of products, such as multicolor organic electroluminescent diodes,6 organic solar cells,7 and chemical sensors.8 Because the development of basic techniques that would enable more effective lignin utilization is a high priority area in both basic research and industry, attempts to transform lignin-derived aromatics into organic fluorescent substances is a worthwhile endeavor. We recently found the novel organic fluorescent substances produced by Pseudomonas sp. ITH-SA-1 from SYAL.9 Although aromatic structures play an important role in determining the planarity and rigidity of organic fluorescent substances, our attenuated total reflectance–Fouriertransform infrared (ATR-FTIR) and nuclear magnetic resonance (NMR) analyses revealed that the organic fluorescent substances produced by Pseudomonas sp. ITH-SA-1 do not contain aromatic rings. The finding that these novel non-aromatic organic compounds exhibit fluorescence was surprising, so we designated the compounds “non-aromatic polymeric substances with fluorescent activity” (NAPSFA).


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NAPSFA are derived from 3-O-methyl gallate (3-MGA) generated from SYAL via syringate (SYAC), and their formation requires ring-opening polymerization of 3-MGA and/or SYAC (Figure 1). Because the NAPSFA are high-molecular-weight polymeric substances (7.2 kDa) and are not readily separable even with the aid of modern chromatographic methods, it is difficult to isolate them in pure form for structural analysis. In addition, the predicted ring-opening intermediates in the 3-MGA catabolic pathway (i.e., 4-carboxy-2-hydroxy-6-methoxy-6oxohexa-2,4-dienoate [CHMOD], 4-oxalomesaconate [OMA], and 4-carboxy-4-hydroxyoxoadipate [CHA])1,10 are chemically unstable. Therefore, in this study, we attempted to form NAPSFA from SYAC, 3-MGA, and similar compounds. During the course of the study, we incidentally discovered that fluorescent substances were produced from 3-MGA via a nonbiological process. As this finding could be useful in efforts to determine the chemical structures of the NAPSFA, we intensively investigated the conditions necessary to produce fluorescent substances from 3-MGA. We report here a simplified method for producing non-aromatic fluorescent substances from 3MGA via a non-biological process.

■MATERIALS AND METHODS

Bacteria, Chemicals, and Biological Production of Fluorescent Substances Derived from 3-MGA. Pseudomonas sp. ITH-SA-1 was pre-cultured in Marine Broth 2216 (MB, Difco) for 2 days at 28°C with shaking at 110 rpm, after which aliquots of the pre-cultures were washed. To accelerate short-term production (2-4 days) of fluorescent substances, a 20-100 µL of the washed cell suspension was transferred into 2 mL of fresh complete medium supplemented with various lignin-derived aromatics at a final concentration of 0.5 or 1 mg/mL after dissolving in sterilized


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Milli-Q water or dimethyl sulfoxide (DMSO). In this case, the initial cell density of each sample was approximately 107-108 colony forming units (CFU)/mL. The samples were incubated for 2 days at 28°C with shaking at 110 rpm. In this experiment, MB and Luria-Bertani broth (LB) were used as complete media. When required, minimal medium (MM) was also used to assess the effects of medium components on the production of fluorescent materials. The structures of chemicals used in this study are shown in Figure 1. SYAL (Wako), SYAC (Wako), 3-MGA (EXTRASYNTHESE), gallate (GA, Wako) p-hydroxybenzaldehyde (PHBA, Wako), 3,5-dimethoxy benzoic acid (DMB, Sigma-Aldrich), 3,5-dihydroxy benzoic acid (DHB, Sigma-Aldrich), or vanillin (VNL, Wako) was used as the starting substrate for production of fluorescent materials. The primary fluorescent activity of Pseudomonas sp. ITH-SA-1 in liquid cultures was determined by the naked eye under UV illumination (254 and 365 nm) of the sample. In some experiments, Pseudomonas sp. ITH-SA-1 colonies streaked onto Marine Agar 2216 (MA, Difco) or LB plates were exposed to aromatic compounds as a vapor. The primary fluorescent activity in these cases was also determined by the naked eye. Production of Fluorescent Substances Derived from 3-MGA via a Non-biological Process. Material to be used for production of fluorescent substances was sterilized in an appropriate manner. 3-MGA-DMSO solution (1 g/mL) was added to substrate solution at a final concentration of 0.1% (v/v), and the sample was incubated for 2 to 4 days at 28°C with shaking at 110 rpm. When required, the cultivation time was extended to 1 week. Primary fluorescence of the sample was determined by the naked eye under UV illumination (254 and 365 nm). Purification of Fluorescent Substances Derived from the Reaction of 3-MGA with Tryptone via a Non-biological Process. A 0.3-mL aliquot of 3-MGA-DMSO solution (10


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mg/mL) was added to 5% Tryptone aqueous solution (300 mL), and the solution was incubated for 2 days at 28°C with shaking at 110 rpm. According to our previous study, the reaction solution was then concentrated and freeze-dried. Fluorescent substances were repeatedly extracted with methanol (MeOH) to remove insoluble impurities (particularly water-soluble black pigments and residual unreacted Tryptone) and then filtered. The MeOH extract was subjected to chromatography on a Sephadex LH-20 glass column (25-mm i.d. × 270 mm) to remove unreacted 3-MGA and eluted with MeOH at a flow rate of 1.5 mL/min. The eluate was collected every 5 min into separate tubes, and the fluorescent fractions without unreacted 3MGA were collected after reverse phase–high-performance liquid chromatography (RP-HPLC) analysis. The fractions were evaporated to dryness, dissolved in a small volume of water, and freeze-dried (0.22 g). The resulting freeze-dried powder was sealed tightly in a recovery flask and kept at room temperature until used. RP-HPLC and Size-exclusion HPLC. RP-HPLC analysis of 3-MGA in the reaction solution was performed using a Waters X-bridge C18 column (4.6-mm i.d. × 150 mm, 5-µm particle size) with gradient elution using 0.5% TFA and MeOH, as previously described.9 Size-exclusion HPLC was carried out using a Sepax Zenix-C SEC-80 column (7.8-mm i.d. × 300 mm, 3-µm particle size, MW range 100-50000 Da) and an Agilent HP1100 system equipped with photodiode array and fluorescence detectors (Agilent Inc.). The column was eluted isocratically using 150 mmol/L sodium phosphate buffer (pH 7.0) at a flow rate of 0.8 mL/min. The detection wavelength was set at 200-450 nm for the photodiode array detector, and elution of the fluorescent substances was detected using the fluorescence detector at excitation and emission wavelengths of 365 and 498 nm, respectively. The column was calibrated prior to each analysis using the following native protein standards: myokinase (Mr, 32.0 × 103; Rt, 10.17 min),


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cytochrome c (Mr, 12.4 × 103; Rt, 11.05 min), aprotinin (Mr, 6.51 × 103; Rt, 15.93 min), cyanocobalamine (Mr, 1.355 × 103; Rt, 20.94 min), Bz-Gly-His-Leu (Mr, 0.43 × 103; Rt, 21.90 min), and SYAC (Mr, 0.198 × 103; Rt, 22.50 min). The number-average molecular weight of the fluorescent substances was estimated from the calibration curve (y = 8.21 × 105e−0.325x). Spectral Analysis. Excitation and emission spectra were obtained using a JASCO FP-6500 spectrofluorometer. ATR-FTIR analysis was performed on a solid sample of fluorescent substances using a JASCO FTIR 4100 spectrometer equipped with a JASCO ATR PRO450-H ATR module. 1H-(500 MHz) and

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C-(150 MHz) NMR spectra were collected using a JEOL

ECA-500 spectrometer in D2O (1H- and

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C-NMR spectra of 3-MGA were collected in 30%

methanol-d4).

■RESULTS AND DISCUSSION

Effect of SYAL Analogues on the Production of NAPSFA by Pseudomonas sp. ITH-SA-1. In our static culture experiments analyzing the production of NAPSFA from SYAL by Pseudomonas sp. ITH-SA-1 at 28°C, fluorescence was observed after 7 days. Moreover, the results of additional culture experiments suggested that the initial cell density and the aerobic cultivation greatly affect the rate of fluorescent substance production from SYAL by Pseudomonas sp. ITH-SA-1.9 Therefore, in the present study, we examined whether fluorescence could be observed during the early period of culture. Fluorescence was observed after 2 days when SYAL was added to cultures in MB medium incubated at 28°C with shaking at 110 rpm.


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The presence of molecules exhibiting fluorescent activity was therefore assessed 2 days after starting the cultures in subsequent experiments. To elucidate the importance of functional groups on benzene rings of lignin-derived aromatics with respect to the production of NAPSFA by Pseudomonas sp. ITH-SA-1, we cultured cells with various SYAL analogues (SYAC, 3-MGA, DMB, DHB, GA, PDC, PHB, or VNL) as the starting substrate (Figure 1). In each case, the culture supernatant was assessed for fluorescence by the naked eye after 2 days of cultivation. No fluorescence was observed with aromatic compounds other than SYAL, SYAC, and 3-MGA (Table 1). These results were in agreement with our previous study demonstrating that Pseudomonas sp. ITH-SA-1 produces fluorescent substances via 3-MGA, SYAC, and SYAL. In addition, the absence of fluorescence in samples amended with DHB or DMB suggests that the hydroxyl group at the 4-position of 3-MGA contributes to the fluorescent activity.


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Figure 1. (a) Known pathway for the metabolism of SYAL by bacteria. The pathway was modified as described previously by Masai et al.1 and Kamimura et al.10 These data were compiled from a previous report.9 The compounds within the dashed-line box are unstable intermediate metabolites of SYAL. (b) Chemical structures of lignin-derived aromatics or SYAL analogues used in this study.

Table 1. Production of fluorescent substances by Pseudomonas sp. ITH-SA-1 in different complete media amended with various lignin-derived aromatics.

B.A. and N.B.A. indicate bacterial addition and no bacterial addition, respectively. “2 days” indicates that the samples were cultured for 2 days, and “4 days” indicates that the samples were cultured more than 4 days. +, fluorescent substances were produced in the supernatant; −, fluorescent substances were not produced in the supernatant. The substrates, except PHBA, were added to the medium at a final concentration of 1 mg/mL, and PHBA was added to the medium at a final concentration of 0.5 mg/mL. MB, Marine broth; LB, Luria-Bertani broth; MM, minimal medium.


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The production of fluorescent substances via biological processes was also examined in cultures exposed to the starting material as a vapor. Although no fluorescence was observed in MA exposed to 3-MGA as a vapor, unexpectedly, the 3-MGA placed on the cover was converted into a black liquid during incubation. This phenomenon was observed only with 3-MGA and also occurred with cultures on LB plates. When 3-MGA was added to water and then incubated, the solution also turned black (fluorescence was not observed). RP-HPLC analysis of the black material showed a peak corresponding to 3-MGA and broad peaks derived from polymeric substances, suggesting that 3-MGA polymerizes in the air and in aqueous solution (data not shown). We previously reported that 1) cultured Pseudomonas sp. ITH-SA-1 releases NAPSFA together with water-soluble black pigments into the supernatant when SYAL is added to the medium as a starting material; 2) the NAPSFA and black pigments are polymeric substances; and 3) the production of NAPSFA requires ring-opening polymerization of 3-MGA and/or SYAC. The observations described above in combination with these data suggest that 1) after SYAL is initially metabolized into 3-MGA by Pseudomonas sp. ITH-SA-1, the 3-MGA is released back into the medium; 2) the ring-opening polymerization reaction of the downstream metabolite of 3-MGA occurs in the medium; and 3) black polymeric substances and NAPSFA are produced via a non-biological process. Synthesis of Fluorescent Substances via a Non-biological Process. To confirm that NAPSFA are produced via a non-biological rather than biological process, we examined the production of fluorescent substances in cultures without bacteria. In LB and MB medium containing 3-MGA but without Pseudomonas sp. ITH-SA-1, fluorescence similar to that of a


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blue-green dye was observed upon UV irradiation at 365 nm after 4 days of incubation (Table 1 and Figure 2). However, no fluorescence was observed when 3-MGA was incubated in MM in the absence of bacteria (Figure 2). In addition, no fluorescence was observed in MB or LB without 3-MGA, nor was fluorescence observed when a solution of 3-MGA alone was incubated with constant shaking. From these results, we concluded that only 3-MGA can be transformed into fluorescent substances in MB or LB medium via a non-biological process. We designated the resulting compounds “fluorescent substances derived from 3-MGA by a non-biological process” (FS3NBP). Our results suggest that 3-MGA reacts with one or more organic components contained in MB and LB medium to produce FS3NBP.

Figure 2. Fluorescent substances derived from 3-MGA by a non-biological process. (a) LB with 3-MGA, (b) MB with 3-MGA, and (c) MM with 3-MGA after 4 days of incubation without bacteria. (d) NAPSFA produced from SYAL by Pseudomonas sp. ITH-SA-1. MB, Marine broth; LB, Luria-Bertani broth; MM, minimal medium.


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Effect of Medium Components on the Production of FS3NBP. According to the Becton, Dickinson and Company product catalog (Difco & BBL Manual, 2nd Edition), the approximate formula (per liter) of LB Broth, Lennox is: Tryptone (10.0 g), Yeast Extract (5.0 g), and NaCl (5.0 g), and the approximate formula (per liter) of MB (Marine Broth 2216) is: Peptone (5.0 g), Yeast Extract (1.0 g), and 14 inorganic salts (NaCl, 19.45 g; MgCl2, 5.9 g; MgSO4, 3.24 g; CaCl2, 1.8 g; KCl, 0.55 g, etc.). Media mimicking LB and MB with respect to the organic components Tryptone, Peptone, and Yeast Extract were prepared to examine the role of each component in the production of FS3NBP (Table 2). Fluorescence was detected under all tested conditions, indicating that 3-MGA reacts with these organic compounds in the medium to produce the fluorescent substances. The highest fluorescence intensity (561.1 ± 37.2) was observed when Tryptone was added to 3-MGA solution at a final concentration of 1.0%. Hence, we focused subsequent experiments on Tryptone as the primary organic component contributing to the production of the FS3NBP.

Table 2. Non-biological production of FS3NBP in various media or media components.

“Mimicked LB” and “Mimicked MB” indicated that the organic composition mimicked that of LB and MB medium, respectively. The conditions 1% Tryptone and 0.5% Yeast Extract were derived from LB medium. The conditions 0.5% Peptone and 0.1% Yeast Extract were derived


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from MB medium. Data represent the mean and SD determined from at least 3 replicates from at least 3 independent experiments. MB, Marine broth; LB, Luria-Bertani broth.

We first investigated the effect of varying the Tryptone concentration on the production of FS3NBP. The maximum fluorescence intensity (550.2 ± 40.6) was observed with the addition of 1.0% Tryptone to 3-MGA, with excitation/emission peaks at 370/505 nm. This fluorescence spectrum was almost identical to that of NAPSFA (365/498 nm).9 At higher concentrations of Tryptone, however, the fluorescence intensity declined. The excitation/emission peaks were slightly shifted to 425-450/517-530 nm at final Tryptone concentrations >10%, suggesting that the excitation/emission peaks of the product may differ depending on the Tryptone concentration. From these results, we concluded that the optimal Tryptone concentration for the production of FS3NBP from 3-MGA in solution is 1.0%. The development of this strategy is useful for further chemical characterization of the NAPSFA because it is simpler than the biological process strategy using Pseudomonas sp. ITH-SA-1. This non-biological strategy could facilitate the production of fluorescent substances on a larger scale as well.

Table 3. Effect of Tryptone on the production of FS3NBP.


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Data represent the mean and SD determined from at least 3 replicates from at least 3 independent experiments.

Structural Characteristics of the FS3NBP. For structural characterization, a large amount of the FS3NBP was prepared and purified as described in the Materials and Methods. Figure 3 shows the results of size-exclusion HPLC analyses of FS3NBP and NAPSFA. The chromatogram for FS3NBP showed a broad peak that was attributed to a mixture of fluorescent substances of different molecular weights. The average molecular weight of FS3NBP was about 4.2 kDa, which is lower than that of NAPSFA (7.2 kDa).9 These results and the similar excitation/emission wavelengths of FS3NBP and NAPSFA suggest that in addition to 3-MGA and Tryptone, other as yet unknown compounds contribute to the biological formation of NAPSFA mediated by Pseudomonas sp. ITH-SA-1.


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Figure 3. Size-exclusion HPLC analysis of NAPSFA and FS3NBP. The number-average molecular weight of the fluorescent substances was estimated from the calibration curve, as described in the Materials and Methods.

ATR-FTIR spectra of 3-MGA, NAPSFA, and FN3NBP are shown in Figure 4. The FS3NBP peaks were broad, as were those of the NAPSFA. The peaks at approximately 3400 cm−1, 1650 cm−1, and 1100 cm−1 are indicative of absorption due to a hydrogen-bonded hydroxyl group, carbonyl group, and ester, respectively. The FS3NBP peak at 1100 cm−1 was small compared to that of the NAPSFA. Clear strong 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 in the FS3NBP or NAPSFA spectra. In addition, no aromatic proton or carbon signals were observed in the 1H- and 13

C-NMR spectra, respectively. Signals indicative of acyclic hydrocarbons, alcohols, ethers, and

carbonyl groups were observed, although fewer such signals were observed in FS3NBP spectra than NAPSFA spectra (Figure 5). Tryptone is pancreatic digests of casein (Difco & BBL Manual, 2nd Edition). Therefore, the specific compound(s) in Tryptone may react with 3-MGA to produce the FS3NBP. Although further investigation is needed, these ATR-FTIR and NMR data indicate that similar to the NAPSFA, the FS3NBP do not contain aromatic rings.


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Figure 4. ATR-FTIR spectra of 3-MGA, NAPSFA, and FS3ANBP.

Figure 5. 1H- and 13C-NMR spectra of 3-MGA, Tryptone, FS3NBP, and NAPSFA.

Based upon the results of our present and previous study, we designated the FS3NBP as “NAPFL-syn-Try-3MGA” (non-aromatic-polymeric fluorescent synthetized from Tryptone and 3-MGA). According to this naming convention, the NAPSFA derived from SAYL via Pseudomonas sp. ITH-SA-1 was renamed “NAPFL-bio-SA1-SYAL” (non-aromatic-polymeric fluorescent produced by Pseudomonas sp. ITH-SA-1 from SYAL). Although further studies are needed, our discovery of the non-aromatic fluorescent substances reported here will ultimately benefit efforts to produce other organic fluorescent compounds containing non-aromatic groups. By manipulating the production strategy, a variety of nonaromatic organic fluorescent substances could be produced via biological and/or non-biological


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processes. From this point of view, lignin and its derived aromatics represent very important sources of non-aromatic fluorescent substances.

■CONCLUSIONS

In this study, we examined the formation mechanism of the NAPSFA, and found that they are produced as a result of the reaction of 3-MGA with Tryptone. This finding illustrates a simple method for producing non-aromatic fluorescent substances from lignin-derived biomass. Downstream analyses showed that the resulting fluorescent substances have excitation/emission peaks at 370/505 nm and an average molecular weight of 4.2 kDa. Despite the fact that aromatic structures generally play an important role in determining the planarity and rigidity of organic fluorescent substances, ATR-FTIR and NMR analyses revealed that the molecules we isolated do not contain aromatic rings. These data suggest that non-aromatic fluorescent substances can be easily synthesized via non-biological processes. The strategy used to sythesize these substances could facilitate identification of the non-aromatic group responsible for the fluorescent activity.

■ASSOCIATED CONTENT

■AUTHOR INFORMATION

Corresponding Author


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*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 interests. ■ACKNOWLEDGMENTS

We are grateful to Prof. Yoshihiro Katayama, NUBS, Nihon Univ., for valuable discussions. We are also grateful to Youichi Ono, NUBIC, Nihon Univ., for illustration assistance in preparing the cover image, and Taihei Sasaki, Tomoya Shirai, and Yuka Kadomatsu, Nihon Univ., for technical assistance. This study was supported by grants-in-aid for Scientific Research (B), Scientific Research (C), and Challenging Exploratory Research from the Japan Society for the Promotion of Science (JSPS), the Strategic Research Foundation at Private Universities from Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT), and the NAGASE Science Technology Foundation. ■REFERENCES

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Linger, J. G.; Vardon, D. R.; Guarnieri, M. T.; Karp, E. M.; Hunsinger, G. B.; Franden, M. A.; Johnson, C. W.; Chupka, G.; Strathmann, T. J.; Pienkos, P. T.; Beckham, G. T. Lignin valorization through integrated biological funneling and chemical catalysis. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 12013-12018.

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Lignin and Lignans as Renewable Raw Materials. Chemistry, Technology and Applications. Calvo-Flores, F.; Dobado, J. A.; Isac-García, J.; Martín-Martínez, F. J., Eds.; Wiley, 2015; pp 249-287, pp 289-312, pp 457-463. (ISBN:9781118597866)

(6) Holder, E.; Langeveld, B. M. W.; Schubert, U. S. New trends in the use of transition metalligand complexes for applications in electroluminescent devices. Adv. Mater. 2005, 17, 1109-1121. (7) Günes, S.; Neugebauer, H.; Sariciftci, N. S. Conjugated polymer-based organic solar cells. Chem. Rev. 2007, 107, 1324-1338. (8)

Thomas, S. W.; Joly, G. D.; Swager, T. M. Chemical sensors based on amplifying fluorescent conjugated polymers. Chem. Rev. 2007, 107, 1339-1386.

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Iwabuchi, N.; Takiguchi, H.; Hamaguchi, T.; Takihara, H.; Sunairi, M.; Matsufuji, H. Transformation of lignin-derived aromatics into non-aromatic polymeric substances with


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fluorescent activities (NAPSFA) by Pseudomonas sp. ITH-SA-1. ACS Sustainable Chem. Eng. 2015, 3, 2678-2685. (10) Kamimura, N.; Takamura, K.; Hara, H.; Kasai, D.; Natsume, R.; Senda, T.; Katayama, Y.; Fukuda, M.; Masai, E. Regulatory system of the protocatechuate 4,5-cleavage pathway genes essential for lignin downstream catabolism. J. Bacteriol. 2010, 192, 3394-3405.


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Synopsis A simple, non-biological method for converting lignin-derived aromatics into non-aromatic polymeric substances with fluorescent activity (NAPSFA) from 3-MGA and Tryptone was developed. 254x190mm (96 x 96 DPI)



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Figure 1. (a) Known pathway for the metabolism of SYAL by bacteria. The pathway was modified as described previously by Masai et al.1 and Kamimura et al.7 These data were compiled from a previous report.6 The compounds within the dashed-line box are unstable intermediate metabolites of SYAL. (b) Chemical structures of lignin-derived aromatics or SYAL analogues used in this study. 254x190mm (96 x 96 DPI)


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Figure 2. Fluorescent substances derived from 3-MGA by a non-biological process. (a) LB with 3-MGA, (b) MB with 3-MGA, and (c) MM with 3-MGA after 4 days of incubation without bacteria. (d) NAPSFA produced from SYAL by Pseudomonas sp. ITH-SA-1. MB, Marine broth; LB, Luria-Bertani broth; MM, minimal medium. 254x190mm (96 x 96 DPI)



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Figure 3. Size-exclusion HPLC analysis of NAPSFA and FS3NBP. The number-average molecular weight of the fluorescent substances was estimated from the calibration curve, as described in the Materials and Methods. 254x190mm (96 x 96 DPI)


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Figure 4. ATR-FTIR spectra of 3-MGA, NAPSFA, and FS3ANBP. 254x190mm (96 x 96 DPI)



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Figure 5. 1H- and

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C-NMR spectra of 3-MGA, Tryptone, FS3NBP, and NAPSFA. 254x190mm (96 x 96 DPI)


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Table 1. Production of fluorescent substances by Pseudomonas sp. ITH-SA-1 in different complete media amended with various lignin-derived aromatics. / B.A. and N.B.A. indicate bacterial addition and no bacterial addition, respectively. “2 days” indicates that the samples were cultured for 2 days, and “4 days” indicates that the samples were cultured more than 4 days. +, fluorescent substances were produced in the supernatant; −, fluorescent substances were not produced in the supernatant. The substrates, except PHBA, were added to the medium at a final concentration of 1 mg/mL, and PHBA was added to the medium at a final concentration of 0.5 mg/mL. MB, Marine broth; LB, Luria-Bertani broth; MM, minimal medium. 254x190mm (96 x 96 DPI)



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Table 2. Non-biological production of FS3NBP in various media or media components. / “Mimicked LB” and “Mimicked MB” indicated that the organic composition mimicked that of LB and MB medium, respectively. The conditions 1% Tryptone and 0.5% Yeast Extract were derived from LB medium. The conditions 0.5% Peptone and 0.1% Yeast Extract were derived from MB medium. Data represent the mean and SD determined from at least 3 replicates from at least 3 independent experiments. MB, Marine broth; LB, LuriaBertani broth. 254x190mm (96 x 96 DPI)


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Table 3. Effect of Tryptone on the production of FS3NBP. / Data represent the mean and SD determined from at least 3 replicates from at least 3 independent experiments. 254x190mm (96 x 96 DPI)


Development of a Simple Nonbiological Method for Converting Lignin

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