J. Microbiol. Biotechnol. (2012), 22(11), 1471–1477 http://dx.doi.org/10.4014/jmb.1207.07038 First published online September 6, 2012 pISSN 1017-7825 eISSN 1738-8872

Metabolic Roles of Carotenoid Produced by Non-Photosynthetic Bacterium SKF120101

Jeon, Bo Young1, Bo Young Kim1, Il Lae Jung2, and Doo Hyun Park1*

1Department of Chemical and Biological Engineering, Seokyeong University, Seoul 136-704, Korea 2Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon 305-353, Korea Received: July 23, 2012 / Revised: August 11, 2012 / Accepted: August 16, 2012

Carotenoids produced by non-photosynthetic completely degrade various xenobiotic compounds and protect organisms against lethal photodynamic reactions natural macromolecules [5, 15, 18, 21]. Gordonia alkanivorans and scavenge oxygenic radicals. However, the carotenoid was first isolated from tar-contaminated soil. The species produced by Gordonia alkanivorans SKF120101 is coupled name originated from its alkane-degrading function [3, to reducing power generation. SKF120101 selectively 17]. This catabolic function of G. alkanivorans is useful for produces carotenoid under light conditions. The growth biodegradation of organic contaminants and bioremediation yield of SKF120101 cultivated under light conditions was of contaminated environments. However, the anabolic higher than that under dark condition. In the cyclic function of Gordonia species for carotenoid biosynthesis is voltammetry, both upper and lower voltammograms for not known for its competitiveness and usefulness, because neutral red (NR) immobilized in intact cells of SKF120101 some plants produce much more carotenoid than bacteria. were not shifted in the condition without external redox G. jacobaea MV-1 was reported to be the first Gordonia sources but were commonly shifted downward by glucose strain characterized with regard to carotenoid biosynthesis addition and light. Electric current generation in a biofuel [9]. Carotenoids are best known as auxiliary pigments of cell system (BFCS) catalyzed by harvested cells of the photosynthetic light-harvesting apparatus in photosynthetic SKF120101 was higher under light than dark condition. organisms. Carotenoids synthesized by non-photosynthetic The ratio of electricity generation to glucose consumption bacteria and fungi are also reported to protect against lethal by SKF120101 cultivated in BFCS was higher under light photodynamic reaction, and function as scavengers of than dark condition. The carotenoid produced by SKF120101 oxygenic radicals [24, 33, 36]. The carotenoid produced in catalyzes production of reducing power from light energy, the non-photosynthetic microorganisms was found to be first evaluated by the electrochemical technique used in not coupled to energy metabolism but was an antioxidative this research. compound to scavenge the reactive oxygen species [31]. Keywords: Carotenoid, Gordonia alkanivorans, cyclic The metabolism for light-induced carotenoid production in voltammetry, biofuel cell, neutral red the non-photosynthetic bacterium Myxococcus xanthus was studied in detail [6]. G. alkanivorans SKF120101 also is a light-induced carotenoid producer, based on the Gordonia selective production of reddish color under light condition, The genus is known to be widely distributed in whose growth and glucose consumption increased more natural ecosystems such as soil, water, estuary sand, and under light than dark condition [29]. mangrove rhizosphere, and in artificially operated systems Electrochemistry is a useful tool to study bacterial including oil-producing wells, sewage sludge, activated physiology coupled with the metabolic redox reaction sludge foam, and clinical samples [1, 10]. Some of the Gordonia generated by catabolism and anabolism. Biochemical species belonging to the genus partially or reducing power generated by bacterial catabolism can be *Corresponding author measured by electrochemical techniques of cyclic voltammetry Phone: +82-2-940-7190; Fax: +82-2-919-0345; and biofuel cell system. The BFCS has been employed to E-mail: [email protected] produce electricity from the coupling redox reaction of 1472 Jeon et al. electron mediator and biochemical reducing power. The (Shimadzu UV-1601, Japan). Meanwhile, SKF120101 was cultivated biochemical reducing power is not enough to produce on agar plate medium under dark and light conditions to easily applicable electricity, because the bacterial catabolism compare the color variation induced by the lighting. generated in the BFCS is much slower than the chemical Electrochemical Analysis of SKF120101 reaction generated in chemical fuel cell systems for Cyclic voltammetry was employed in order to analyze electrochemical production of electron-driving force [14, 16]. However, the redox reaction between the working electrode and the neutral red BFCS is useful to electrochemically analyze and compare (NR) immobilized in bacterial cells. It was conducted using a reducing power generated by bacterial catabolism under a voltammetric potentiostat (BAS CV50W, USA) linked to a data specific condition. Biochemical reducing power (NADH) acquisition system. A glassy carbon electrode (BAS MF2013, is converted to electrochemical reducing power via a USA), a platinum wire, and an Ag/AgCl electrode were utilized as a specific electron mediator (neutral red). The electrochemically working electrode, counter electrode, and reference electrode, reduced electron mediator on an anode surface is oxidized respectively. The reaction mixture was composed of 25 mM Tris- in coupling with reduction of oxygen (electron acceptor) HCl buffer (pH 7.5), 5 mM NaCl, and intact cells of SKF120101 on a cathode surface in the BFCS, by which electron- (OD660 = 5.0) modified with NR [27]. Prior to use, the electrodes driving force (voltage) is generated in coupling with were cleaned with an ultrasonic cleaner, and the dissolved oxygen in the reaction mixture was purged by argon (99.999%) gassing. The electron transfer (current) from anode to cathode [23, 28]. -1 scanning rate was 25 mVs over a range of -1200 to 0 mV. During The amount of electrons transferred from the anode to the cyclic voltammetry, variations of the upper and lower voltammograms cathode is proportional to the catabolic activity of bacteria generated by addition of glucose or light were saved in a PC using a for generation of biochemical reducing power [4, 22]. data acquisition system. In this research, we isolated a light-induced carotenoid- producing bacterium, G. alkanivorans SKF120101, and Preparation of Cathode analyzed the metabolic function of the carotenoid using A cathode (diameter 40 mm) was constructed of a cellulose acetate the electrochemical techniques of cyclic voltammetry and film (35 µm thickness; Electron Microscopy Science, USA), a BFCS. Electrochemical reducing power generated by ceramic membrane (2 mm thickness), and a porous carbon plate (10 coupling with catabolism of SKF120101 was selectively mm thickness) as shown in Fig. 1B, which was prepared according increased by light. Electricity generation in the BFCS to the methods used in previous research [19]. The cellulose acetate catalyzed by SKF120101 was significantly increased by film was attached to the ceramic membrane using acetone vapor. The ceramic membrane and porous carbon plate are permeable to light. On the basis of the light-induced metabolic activation water, ions, and a variety of water-soluble compounds; however, a of SKF120101, we suggest that the carotenoid produced cellulose acetate film is semipermeable for selective transfer of gas, by strain SKF120101 may catalyze regeneration of proton, and water molecule. biochemical reducing power with light energy. Immobilization of NR in Graphite Felt Anode One% of polyvinyl alcohol (PVA, MW 89,000~98,000) solution MATERIALS AND METHODS absorbed into graphite felt (40 × 60 × 8 mm; Electrosynthesis GF- S8, USA) was completely dried, by which PVA fiber was directly Isolation and Cultivation of Bacterial Strain combined with graphite fiber and the PVA-graphite felt complex The medium used for isolation of SKF120101 was composed of 10 mM K2HPO4, 10 mM NaH2PO4, 1 g NH4Cl/L, 3 g yeast extract/L,

20 g glucose/L, 0.01 g MgSO4/L, 0.01 g FeSO4/L and 1 L of tap water, and was also used for successive transfer culture and bacterial cultivation in the BFCS. Glucose and yeast extract were excluded from the medium for autotrophic cultivation of SKF120101 under light conditions. Biomass was evaluated by viable cell count, optical density at 660 nm, and dry cell weight. Seventy-two hour-cultivated bacterial cells were harvested and washed twice with distilled water by centrifugation at 5,000 ×g and 4oC for 30 min. The washed cells were dried at 110oC until a constant weight was obtained for determination of dry cell weight, and lyophilized for extraction of reddish compound.

Spectral Analysis of Carotenoid The lyophilized cells of SKF120101 were suspended in acetone and o Fig. 1. Single-cell-type electrochemical bioreactor (100 ml) for incubated in a 150 rpm shaker at 4 C for 60 min. Acetone-extracted measurement of electricity generation from glucose under light reddish compound was obtained by centrifugation at 10,000 ×g and or dark condition. o 4 C for 30 min. The supernatant was spectrally analyzed in the The cathode functions as an electrode and electrolyte by catalyzing range from 800 to 350 nm of wavelength using a spectrophotometer reduction of O2 to H2O. METABOLIC ROLES OF CAROTENOID PRODUCED BY G. ALKANIVORANS 1473 was composed. NR was immobilized in the PVA-graphite felt not by calculation using the voltage measured based on the fixed complex by induction of a covalent bond between the amine of NR value of external resistance. The BFCSs for dark cultivation of and the hydroxyl of PVA using thionyl chloride [13]. Water-soluble bacterial cells were doubly enveloped with aluminum foil and black PVA was converted to water-insoluble PVNR by substitution of the plastic paper to completely block light. The anode and cathode were hydroxyl of PVA by NR. The PVNR-graphite complex is a solid- connected via an ammeter and a voltmeter by a closed circuit with type electron carrier that mediates electron transfer between bacterial 1,000 Ω of external resistance (Fig. 1). cells and the graphite electrode. Analysis of Glucose Consumption Preparation of Biofuel Cell System Bacterial culture was centrifuged at 12,000 ×g and 4oC for 20 min, A single-cell-type BFCS with performance verified in previous followed by filtration through a 0.22 µm membrane filter (Sartorius, research was employed [19]. The total and working volumes of the Germany). Fifty µl of filtrated culture was subsequently injected BFCS were 150 ml and 100 ml. The cathode outside (porous carbon into the HPLC injector. Glucose was analyzed using an HPLC plate) was exposed to the atmosphere, by which electrons originated apparatus (Younglin Instrument, Korea) equipped with an ion from the PVNR-graphite anode and protons originated from exchange column (Bio-Rad Aminex HPX-87H, USA) and a refractive anolyte were effectively reacted with O2. Meanwhile, the cathode index detector (Younglin Instrument Acme9000, Korea). The inside (cellulose acetate film) was brought into contact with anolyte, column and detector were adjusted to a temperature of 35oC. A by which protons dissolved in anolyte were selectively transferred to 0.008N H2SO4 solution was used as the mobile phase of which the the porous carbon cathode, as shown in Fig. 1A. A 15W light bulb flow rate was adjusted to 0.8 ml/min. was used for lighting the BFCS, and the light intensity was adjusted to ~5,000 lux. ESULTS Measurement of Electricity Generated by Harvested Cells R Freshly harvested cells of SKF120101 were used as a catalyst for Isolation of Non-Photosynthetic Bacterium Gordonia the BFCS in order to verify results of cyclic voltammetry obtained alkanivorans SKF120101 using the harvested cells, but NR was immobilized in the graphite Gordonia alkanivorans felt anode instead of bacterial cells. SKF120101 cultivated for 72 h SKF120101 was isolated, using the under light condition (~5,000 lux) was harvested by centrifugation specified medium, from a heterotrophic bacterial community at 4oC and 5,000 ×g for 30 min and then suspended in 25 mM cultivated in organic fertilizer-manufacturing culture and phosphate buffer (pH 7) containing 2 g glucose/L. The bacterial cell identified based on 16S rDNA sequence homology, which suspension (OD660 = 5) prepared in the BFCS was incubated under was registered in the GenBank database system under the light or dark condition at 30oC for 120 min, and the current accession code JQ964108. generation was measured by turning on the switch between anode and cathode (Fig. 1). External resistance and the voltmeter were not Induction of Reddish Compound and Its Spectral employed to selectively compare the current generated under light Analysis and dark conditions. The lighting for colonies of SKF120101 growing on agar Measurement of Electricity Generated by Growing Cells plate induced bacterial cells to be reddish, as shown in Ten ml of SKF120101 culture previously cultivated for 48 h under Fig. 2. The reddish pigment extracted from the reddish cell light condition was inoculated into 90 ml of fresh medium prepared was spectrally analyzed in the range from 800 to 350 nm. in the BFCS. The effect of lighting (~5,000 lux) on electricity The absorption maxima at 452, 478, and 506 nm are (electric power) generation by catalysis of growing cells was characteristic of the typical carotenoid spectrum as shown estimated by actual measurement of both current and voltage, but

Fig. 2. Color variation of growing cells of G. alkanivorans SKF120101 cultivated under light (left) and dark (right) conditions. Fig. 3. Spectrum of the reddish compound extracted from White spots are air bubbles generated inside the agar layer. lyophilized G. alkanivorans SKF120101, using acetone. 1474 Jeon et al.

Table 1. Growth of G. alkanivorans SKF120101 autotrophically cultivated in GA medium without glucose and yeast extract (YE) or heterotrophically cultivated in GA medium under light or dark conditions for 72 h. Growth conditions Analytical factors Light Dark Dry cell weight (g/L) 1.96 1.09 Viable cells (CFU) 3.3 × 109 2.8 × 108 Glucose consumption (mM) 85.5 66.6 22.9 16.4 Growth yield (Y ) glucose (1.96/0.0855) (1.09/0.0666) Autotrophic cultivation Not grown Not grown (w/o glucose and YE) Light intensity was adjusted to 5,000 lux with a 15 watt incandescent light, and the bacterial culture was aerated by 150 rpm of rotary shaking. in Fig. 3, which is very similar to the three absorption maxima at 450, 474, and 504 nm of dehydro-β-carotene [12]. The difference of the absorption maxima between the reddish pigment and dehydro-β-carotene may be caused by a difference of solvent used for extraction [26].

Effect of Light on Growth of Gordonia alkanivorans SKF120101 The selective reddish color of SKF120101 cultivated under light condition (Fig. 2) may be coupled to primary metabolism, because its growth and glucose consumption were greatly activated under the light condition as shown in Table 1. The viable cell number was about 10 times higher and growth yield was about 1.4 times higher under light than dark conditions. SKF120101 was not autotrophically grown in the medium without glucose and yeast extract under light condition. It may be presumed that the growth yield of SKF120101 may be lower under light conditions than the dark, because extra energy may be consumed for biosynthesis of carotenoid coupled to scavenging oxygenic radicals. The growth yield was different from the presumption. The differences of growth yield is a clue that carotenoid produced in SKF120101 may catalyze extra reducing power production from light energy.

Effect of Light on Metabolic Reaction of SKF120101 In cyclic voltammetry for NR immobilized in bacterial cells, upper and lower voltammograms are shifted downward Fig. 4. Cyclic voltammetry for NR immobilized in G. alkanivorans SKF120101. coupled to bacterial catabolism, but are shifted upward (A) Cyclic potential scanning from -1,200 to 0 mV under the dark condition. coupled to bacterial anabolism [27]. The upper and lower Both upper and lower voltammetric peaks for the NR shift gradually voltammetric peaks for NR immobilized in bacterial cells downward (arrow marks) under dark condition with glucose (B) and under were not shifted without external energy source (Fig. 4A). light condition without glucose (C). Meanwhile, both upper and lower voltammetric peaks were shifted downward by addition of glucose (Fig. 4B) oxidized to NRox coupled to current generation (Y-axis and and light (Fig. 4C). These results are a clue that arrow marks in Fig. 4). The higher variation of upper biochemically reduced NR (NRred) coupled to metabolic voltammetric peaks than of lower ones is caused by the oxidation of glucose and by the light is electrochemically higher biochemical reduction of NRox to NRred than METABOLIC ROLES OF CAROTENOID PRODUCED BY G. ALKANIVORANS 1475

Fig. 6. Electricity generation by anaerobically growing cells of Fig. 5. Electricity generation by freshly harvested cells of G. G. alkanivorans alkanivorans SKF120101 in M9 medium supplemented with SKF120101 in 25 mM phosphate buffer containing glucose coupled to glucose consumption under light (solid 10 mM glucose under light (solid symbol) and dark conditions symbol) and dark conditions (open symbol). (open symbol). The anode and cathode were connected via an ampere meter by closed circuit without external resistance. power regeneration. The electricity generation was about 2.5 times higher but glucose consumption was about 1.8 electrochemical oxidation of NRred to NRox. Meanwhile, times higher under the light than dark condition as shown the variation of redox potential (-0.52 V vs. Ag/AgCl) of in Fig. 6. The ratio of electricity generation efficiency (2.5) the voltammetric peaks was not generated in cyclic to glucose consumption activity (1.8) was about 1.4 times, voltammetry performed under three different conditions. It which was similar to the growth yield (Table 1) and current shows that biochemical and electrochemical redox reactions generation by harvested cells (Fig. 5). This result may be were definitely generated coupled to redox reaction of NR. an undiscovered biological phenomenon, that light-induced biosynthesis of carotenoid in G. alkanivorans SKF120101 Electricity Generation by Freshly Harvested Cells of is metabolically coupled not only to additional production SKF120101 of reducing power but also to increase of metabolic Light for intact cells of SKF120101 activated electron activity (Table 1). transfer from the bacterial cells to the electrode in coupling with the redox reaction of NR immobilized in bacterial cells according to the cyclic voltammetry (Fig. 4C). The DISCUSSION current generation in the BFCS (Fig. 1) catalyzed by freshly harvested cells of SKF120101 was an average Carotenoid is an accessory light-harvesting pigment located 1.4~1.6 times higher under the light condition than the in the cell membranes of all photosynthetic organisms and dark condition, as shown in Fig. 5. Time-coursed decrease is essential for the survival of photosynthetic organisms [8, of current is caused by a difference between the biochemical 20]. Carotenoid excited by absorption of blue light energy generation and electrochemical consumption of reducing (Fig. 3) induces excitation of chlorophyll by rearrangement power. The biochemical reducing power is limitedly of electronic levels in the light-harvesting complex, and generated in proportion to metabolic activity, substrate protects chlorophyll from photodynamic reaction by concentration, and metabolite production, but electrochemical transferring its excitation energy to singlet oxygen in plants, reducing power is consumed limitlessly without external algae, and photosynthetic bacteria [2]. The carotenoid of resistance for control of electron flow from the anode to the non-photosynthetic bacteria was seen to be maintained the cathode. From this result, it can be assumed that the by evolutionary conservation of enzymes catalyzing the SKF120101 cultivated under the light condition may earliest biosynthetic reactions [30]. The physiological produce the extra biochemical reducing power from light function of carotenoid synthesized by non-photosynthetic energy by catalysis of carotenoid. bacteria has been known to be an oxygenic radical scavenger and protector against toxic molecules generated Electricity Generation by Growing Cells of SKF120101 by photodynamic reaction [34]. The cellular location of The BFCS is generally operated by growing cells of carotenoid pigments produced by the non-photosynthetic bacteria, because the biochemical reducing power is bacteria was identified to be the cell wall and membrane, generated by primary metabolism coupled to reducing which has structural similarity with those of the 1476 Jeon et al. photosynthetic bacteria, although this is not useful for of auxiliary biochemical reducing power with light energy understanding the metabolic function of carotenoid [25]. under only heterotrophic growth condition. The metabolic function of carotenoid in photosynthetic organisms is to catalyze transfer of the excitation energy to chlorophyll in the light-harvesting complex, by which Acknowledgment electron-driving force is generated in the electron transport system coupled to the photosynthetic reaction center [35]. This work was supported by the New & Renewable The linear system of conjugated C=C bonds of carotenoid Energy of the Korea Institute of Energy Technology provides high reducing potential, making itself potent to be Evaluation and Planning (KETEP) grant funded by the electrochemically excited. The biochemical reducing power Korea governmental Ministry of Knowledge Economy generated in the photosynthetic reaction center can be (2012T1001100334). converted to electrochemical reducing power via a proper electron mediator by using a BFCS [32]. This is a useful technique to evaluate the metabolic function of light- REFERENCES harvesting pigments such as are chlorophylls and carotenoids. 1. 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