Letter pubs.acs.org/journal/estlcu
Extracellular Electron Transfer Enhances Polyhydroxybutyrate Productivity in Ralstonia eutropha Koichi Nishio,† Yuki Kimoto,† Jieun Song,† Tomohiro Konno,‡ Kazuhiko Ishihara,‡,§ Souichiro Kato,∥,⊥,@ Kazuhito Hashimoto,*,†,∥ and Shuji Nakanishi*,†,∥ †
Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡ Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan § Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ∥ Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan ⊥ Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan @ Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan S Supporting Information *
ABSTRACT: Polyhydroxyalkanoate is a biodegradable and biocompatible polymer that is synthesized in intracellular granules by a wide variety of microorganisms under growthsuppressed conditions. Herein, we report that polyhydroxybutyrate (PHB) productivity in Ralstonia eutropha was accelerated by extracellular electron transfer (EET) using a biocompatible mediator in an electrochemical system. When the electrode potential was poised at 0.6 V (vs the standard hydrogen electrode) where the EET pathway was constructed, the PHB production rate was enhanced by 60% compared to the case without the EET process. The results indicate that the extracellular anode served as an additional electron acceptor for microbial metabolism, resulting in acceleration of glycolysis and hence PHB synthesis.
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INTRODUCTION It is now well-known that polyhydroxyalkanoate is a bioderived polymer that is synthesized in intracellular granules by a wide variety of Gram-positive and -negative organisms under growthsuppressed conditions and has attracted attention because of its biodegradability and biocompatibility.1 Ralstonia eutropha H16 is one of the model organisms used to study the production of polyhydroxybutyrate (PHB) from sugar.2 R. eutropha actively synthesizes PHB during growth-suppressed conditions, such as nitrogen starvation and the stationary phase, whereas the other metabolic activities, including the tricarboxylic acid (TCA) cycle and oxygen respiration, are adversely repressed.3,4 This metabolic repression results in the accumulation of the reduced form of nicotinamide adenine dinucleotide (NAD) in principle, leading to deceleration of the glycolytic pathway. Nitrate is another terminal electron acceptor other than oxygen used by R. eutropha, and nitrate respiration possibly accelerates NAD+/ NADH redox cycling and, in turn, the glycolytic pathway. However, the supply of nitrate relieves nitrogen starvation, resulting in the suppression of PHB synthesis. Therefore, the use of an alternative respiration pathway is desirable for enhancing the metabolic activity for PHB production by R. eutropha under nitrogen starvation conditions. © 2013 American Chemical Society
Extracellular electron transfer (EET) involves the direct or indirect exchange of electrons between intracellular redoxactive species and extracellular solid materials and is recognized as a fundamental process in natural microbial ecosystems.5,6 EET is currently being applied in the electrochemical cultivation of microbes, including the production of electricity from waste biomass in microbial fuel cells,7,8 in which an extracellular electrode serves as an electron acceptor. In the work presented here, we attempt to apply EET to create an additional respiration pathway in R. eutropha. The use of such a pathway would be advantageous for maintaining microbial metabolism in the active state during PHB production without adding electron-accepting nitrate. Herein, we report that the PHB productivity of R. eutropha is enhanced when intracellular electrons are transferred to an extracellular anode via EET in the presence of a biocompatible mediator. Received: Revised: Accepted: Published: 40
September 19, 2013 October 31, 2013 October 31, 2013 October 31, 2013 dx.doi.org/10.1021/ez400085b | Environ. Sci. Technol. Lett. 2014, 1, 40−43
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MATERIALS AND METHODS Culture and Medium. R. eutropha H16 was routinely precultured in Luria-Bertani (LB) medium while being shaken at 30 °C. Nitrogen-deficient defined medium (N-deficient DM) for the electrochemical cultivation of R. eutropha consisted of 4.5 g of K2HPO4, 5.6 g of KH2PO4, 0.8 g of MgSO4·7H2O, 0.082 g of CaCl2·2H2O, and 1 mL of a trace element solution9 (per liter). Electrochemical System. A double-chamber, three-electrode system was used for the electrochemical cultivation of R. eutropha (Figure S1 of the Supporting Information). The two chambers were separated by a Nafion membrane. One chamber was equipped with glassy carbon (7 cm2) on the side as the working electrode, and a Ag/AgCl electrode (saturated KCl) was used as the reference electrode. The other chamber was equipped with a platinum wire as the counter electrode. To perform electrochemical cultivation, 23 mL of defined medium was added to both chambers. Fructose (10 mM) and mediator molecules [1 g/L poly(2-methacryloyloxyethyl phosphorylcholine-co-vinylferrocene) (PMF)10] were added to the chamber with the working electrode. R. eutropha cells precultivated in LB medium were centrifuged and washed twice with N-deficient DM to completely remove LB medium. Subsequently, the cells were suspended in N-deficient DM and injected into the chamber with the working electrode to give an optical density at 600 nm of 1.0. The potential of the working electrode was kept at either 0.6 V [vs the standard hydrogen electrode (SHE)] or an open circuit potential (approximately 0.3 V vs SHE) to keep PMF oxidized or reduced, respectively. The chamber with the working electrode was operated under an air flow (30 mL/ min), and the entire system was operated while being agitated at 30 °C. Quantification of PHB and Fructose. To measure PHB concentration, intracellular PHB was first extracted and monomerized to 3-hydroxybutyrate by the propanolic digestion method11 with a slight modification (details described in the Supporting Information). The concentration of 3-hydroxybutyrate was then measured using an enzymatic assay (F-kit, J. K. International) to estimate the PHB concentration. The level of fructose in the medium was also measured by an enzymatic assay (F-kit, J. K. International). Quantitative Reverse Transcription Polymerase Chain Reactions (qRT-PCR). For the measurement of gene expression, total RNA was extracted from cells using an RNeasy Mini kit and RNase-Free DNase set (Qiagen). qRTPCR was performed using a StepOnePlus Real-Time-PCR System (Applied Biosystems) and the PCR primers listed in Table S1 of the Supporting Information. Standard curves were drawn using dilutions of PCR fragments of target genes (napD, nasD, narK1, and 16S rRNA). The specificity of the quantitative PCR was verified by dissociation curve analyses. In accord with the literature,12 mRNA levels of the target genes (napD, nasD, and narK1) were normalized to that of the 16S rRNA.
in a reduced form under open circuit conditions (approximately 0.3 V vs SHE). Note that the PMF exists in a reduced form even under aerobic conditions under open circuit conditions because ferrocene, the redox-active part of PMF,10 is not reactive to oxygen. Figure 1a shows the time course of PHB
Figure 1. Time courses of (a) PHB concentration, (b) fructose consumption, and (c) microbial current generation during electrochemical cultivation of R. eutropha. Error bars represent standard errors (n = 2): (solid line) open circuit with PMF, (dashed line) 0.6 V with PMF, and (dotted line) 0.6 V without PMF.
production with PMF at 0.6 V (dashed line) and at the open circuit potential (OCP, solid line). The PHB production rate was enhanced by 60% at 0.6 V compared to that produced at the OCP. In the absence of PMF (dotted line), the PHB production rate was lower than that in the presence of PMF regardless of the electrode potential, an effect that may have been due to the ability of PMF to function as a surfactant, which would affect nutrient uptake and cell dispersion, as previously reported.10 We also confirmed that the fructose consumption rate was enhanced when a potential of 0.6 V was applied to the system (Figure 1b). This result indicates that the metabolism from fructose decomposition to PHB synthesis was more active at 0.6 V than at the OCP.
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RESULTS AND DISCUSSION R. eutropha H16 cells were inoculated in a double-chamber, three-electrode reactor for electrochemical cultivation. PMF was added to the reactor as a biocompatible mediator, which does not inhibit microbial metabolic activities.10 As PMF has a midpoint potential of 0.5 V versus SHE,10 it exists in an oxidized form at an electrode potential of 0.6 V versus SHE and 41
dx.doi.org/10.1021/ez400085b | Environ. Sci. Technol. Lett. 2014, 1, 40−43
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are underestimated (or overestimated).] However, the relative expression levels of all three nitrate reductases were highest when the metabolic activity was the most active (at a poised electrode potential of 0.6 V in the presence of PMF). In addition, it was reported that the range of variation in the relative amount of 16S rRNA to the total RNA in R. eutropha H16 was only 6% regardless of PHB productivity,14 which is much lower than the difference shown in Figure 2. Thus, we can conclude that the genes encoding the three nitrate reductases were actively expressed when the EET pathway was constructed. This result suggests that NRP is one of the pathways involved in PMF-mediated EET, and redox cycling of NADH/NAD+ was facilitated by EET via NRP. In summary, this study demonstrates that R. eutropha is capable of using an extracellular electrode as an electron acceptor in addition to oxygen when PMF is provided as an electron mediator, and PHB productivity was accelerated by EET. As glycolysis involves the reduction of NAD+ to NADH, this additional respiration pathway possibly facilitates glycolysis and, in turn, PHB production by accelerating the redox cycling of NAD+/NADH (Figure 3). In addition, extracellular
Chronoamperometry was used to quantify the degree of EET for R. eutropha cultivated at 0.6 V in the presence of PMF (Figure 1c, solid curve). As an anodic current was not observed in the absence of PMF (dotted curve), we concluded that R. eutropha can generate current by EET via PMF. Importantly, the microbial current, PHB production rate, and fructose consumption rate simultaneously increased after approximately 40 h of operation, implying that the construction of the EET pathway affected the metabolism of R. eutropha. The enhanced synthesis of PHB and consumption of fructose associated with the EET should involve acceleration of glycolysis and redox cycling of NADH/NAD+. Considering the redox cycling of NADH/NAD+ in R. eutropha H16 is facilitated by oxygen or nitrate respiration, the participation of the oxygen and/or nitrate respiration pathway in EET needs to be investigated. However, it has been known that the expression of genes involved in nitrate respiration is upregulated, whereas the expression of genes involved in the TCA cycle and oxygen respiration is downregulated during the PHB-producing phase.3,4 Thus, we speculated that the nitrate reduction pathway (NRP) is one possible source of electron donors for PMF during the electrochemical cultivation of R. eutropha. As the redox potential of PMF (0.5 V) is sufficiently more positive than that of NO3−/NO2− (0.42 V13), the transfer of an electron from the nitrate reduction pathway (NRP) to PMF is thermodynamically allowed. On the basis of the consideration described above, we analyzed the expression levels of genes encoding three nitrate reductases, napD (periplasmic nitrate reductase), nasD (assimilatory nitrate reductase), and narK1 (respiratory nitrate reductase) (Figure S2 of the Supporting Information) after electrochemical cultivation of R. eutropha for 72 h (Figure 2) to investigate
Figure 2. Expression levels of napD, nasD, and narK1 genes at 72 h during electrochemical cultivation of R. eutropha. The mRNA levels were normalized to that of the 16S rRNA. Error bars represent standard errors (n = 2).
the relationship between EET and NRP. The analysis revealed that the expression of these genes is upregulated at a poised electrode potential of 0.6 V in the presence of PMF, at which the rate of PHB production was accelerated, even though nitrate and other nitrogen sources were not present in the medium. Note that the data shown in Figure 2 were normalized by the 16S rRNA. The quantitative relationship among the three different experimental conditions is not strictly valid, because the 16S rRNA is not a housekeeping gene and the expression level depends on the metabolic activity. [When the metabolic activity is higher (or lower), the expression level of 16S rRNA is expected to be higher (or lower), and the expression levels of the target genes normalized by 16S rRNA
Figure 3. Schematic illustration for the electrochemical cultivation of R. eutropha: (a) open circuit and (b) 0.6 V.
electrodes can accept electrons from microbes under nitrogen-starved conditions, a feature that is advantageous for not only PHB production but also the accumulation of other valuable cell-storage materials, such as triacylglycerol,15 that are also induced during nitrogen starvation. Thus, we anticipate that EET represents a novel approach for enhancing the microbial production of valuable materials. 42
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Salmonella as a model organism. Appl. Environ. Microbiol. 2004, 70 (6), 3618−3623. (13) Unden, G.; Bongaerts, J. Alternative respiratory pathways of Escherichia coli: Energetics and transcriptional regulation in response to electron acceptors. Biochim. Biophys. Acta 1997, 1320 (3), 217−234. (14) Lawrence, A. G.; Schoenheit, J.; He, A.; Tian, J.; Liu, P.; Stubbe, J.; Sinskey, A. J. Transcriptional analysis of Ralstonia eutropha genes related to poly-(R)-3-hydroxybutyrate homeostasis during batch fermentation. Appl. Microbiol. Biotechnol. 2005, 68 (5), 663−672. (15) Hu, Q.; Sommerfeld, M.; Jarvis, E.; Ghirardi, M.; Posewitz, M.; Seibert, M.; Darzins, A. Microalgal triacylglycerols as feedstocks for biofuel production: Perspectives and advances. Plant J. 2008, 54 (4), 621−639.
ASSOCIATED CONTENT
S Supporting Information *
qRT-PCR and PHB extraction and supplementary figures. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Fax: (+81) 3-58418751. Telephone: (+81) 3-5841-7245. *E-mail:
[email protected]. Fax: (+81) 3-58418751. Telephone: (+81) 3-5841-8389. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Specially Promoted Research (24000010) and partially by a Grant-in-Aid for JSPS Fellows (23·9692).
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REFERENCES
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