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Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli
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Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli Byung Hoon Jo a,b, Jaoon Y.H. Kim b, Jeong Hyun Seo b,c, Hyung Joon Cha a,b,* a
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea b Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea c School of Chemical Engineering, Yeungnam University, Gyeongsan 712-749, Republic of Korea
article info
abstract
Article history:
H2 production under aerobic conditions has been proposed as an alternative method to
Received 16 March 2014
overcome the fundamentally low yield of H2 production by fermentative bacteria by
Received in revised form
maximizing the number of electrons that are available for H2. Here, we engineered Vitre-
26 April 2014
oscilla hemoglobin (VHb) in Escherichia coli to study the effects of this versatile oxygen (O2)-
Accepted 29 April 2014
binding protein on oxic H2 production in a closed batch system that was supplemented
Available online xxx
with glucose. The H2 yields that were obtained with the VHb-expressing E. coli were greatly
Keywords:
tensions. The formate hydrogen lyase (FHL) activity of oxically cultured, VHb-expressing
Biohydrogen
cells was also much higher than that of the negative control cells. Through inhibitor
Vitreoscilla hemoglobin
studies and time-course experiments, VHb was shown to contribute to the improved H2
Escherichia coli
yield primarily by increasing the efficiency of cellular metabolism during the aerobic phase
Aerobic condition
before the onset of H2 production and not by working as an O2-scavenger during H2 pro-
Formate hydrogen lyase
duction. This new approach allowed more substrate to remain to be further utilized for the
enhanced in comparison to the negative control cells in culture that started with high O2
production of more H2 from limited resources. We expect that VHb can be successfully engineered in potential aerobic H2-producing microbial systems to enhance the overall H2 production yield. In addition, the remarkably high FHL activity of oxically grown, VHbexpressing cells may make this engineered strain an attractive whole-cell biocatalyst for converting formate to H2. Copyright ª 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Introduction H2 is a promising alternative fuel and is expected to be a key energy carrier in the future “hydrogen economy” [1]. This
utility arises from several attributes of H2; for example, it has the greatest energy content per mass among known fuels and is environment-friendly because its oxidation by O2 leaves only water as a byproduct. Most H2 that is currently being used is prepared by fossil fuel reforming,
* Corresponding author. Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. Tel.: þ82 54 279 2280; fax: þ82 54 279 2699. E-mail address: [email protected] (H.J. Cha). http://dx.doi.org/10.1016/j.ijhydene.2014.04.209 0360-3199/Copyright ª 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
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which makes the current H2 production process pollute the environment. The use of biological microbial systems is, therefore, an attractive and valuable approach to H2 production because these systems are renewable and carbon neutral [2]. Among the several biological approaches that are possible, dark fermentation is a reasonable choice because it allows for exceedingly high H2 productivity to be achieved, along with high cell density [3]. However, the practical application of this approach is limited by the fundamental obstacle that the efficiency of energy conversion by fermentative H2 metabolism is not sufficiently high. H2 yield should be expanded beyond the upper limit that is found in nature to increase the efficiency of H2 conversion [4]. Several studies have investigated the feasibility of constructing a non-native H2 pathway in the facultative anaerobe Escherichia coli through the utilization of NADH as the electron donor, which is generated through glycolysis [5e7]. However, the total yield of H2 production from these additional pathways cannot exceed the limit that is found in strict anaerobes such as Clostridia [4]. For fermentative H2 production to be commercially viable, the yield should be expanded to become as close as possible to the maximal stoichiometric H2 yield that can be obtained from glucose [8,9]. This expansion could potentially be accomplished by harnessing electrons under more energy-efficient aerobic conditions [4,10e12]. With such an aerobic H2-producing system (which does not exist in nature, nor has it yet been artificially constructed), strategies are required to increase electron flow to the tricarboxylic acid (TCA) cycle (and, in turn, to NADH) to improve H2 production because most electrons flow toward the formation of byproducts such as acetic acid rather than to the TCA cycle, even during aerobic growth [13]. The construction of efficient microbial cell factories also requires that other factors be optimized, including efficient cell growth and high levels of enzyme production. A previous study on the improvement of microalgal H2 production by the expression of leghemoglobin showed that the artificial engineering of cells with an adjuvant molecular component can improve overall H2 metabolism in the presence of O2 [14]. Inspired by the molecular and physiological function of O2 binding and transport by Vitreoscilla hemoglobin (VHb), we attempted to improve fermentative H2 production under oxic conditions by engineering this molecular component in E. coli. VHb is a soluble form of hemoglobin that is synthesized by the obligate aerobic bacterium Vitreoscilla stercoraria to cope with hypoxic environmental conditions [15]. The intracellular expression of this versatile O2-binding protein in various hosts, including E. coli, has been shown to elicit positive effects, such as enhanced protein production, improved cell growth, and elevated metabolite formation [16e18]. In this study, the effect of VHb expression on H2 production was investigated under oxic and anoxic conditions in a closed batch culture with glucose as the substrate for H2 production via the native formate hydrogen lyase (FHL) pathway. The potential biotechnological application of VHb to aerobic H2 production system was also discussed.
Materials and methods Strains, plasmid construction, and general culture conditions All of the DNA manipulations were performed using the E. coli TOP10 strain (Invitrogen, USA), following standard methods. The E. coli strain W3110 (ATCC 27325) was used for VHb expression and H2 production. The vgb gene, which encodes the VHb protein, was amplified by Taq DNA polymerase (Takara, Japan) from a plasmid that contained the gene [17] using the following primers: forward: 50 -CCATGGGTATGTreverse: 50 -GGTACCTTAGACCAGCAAAC-30 , CAGTGGTGGTGGTGGTGGTGCTCGAGTTCAACCGCTTGAGCGTAC-30 (restriction sites are underlined). The forward primer included an NcoI restriction site, and the reverse primer was designed to contain an Acc65I site that was preceded by a Cterminal His6-tag sequence (italicized), which facilitated the detection and purification of the protein. The PCR product was ligated into the plasmid pGEM-T Easy (Promega, USA), and the DNA sequence was confirmed to be correct. The DNA fragment that was generated with the NcoI and Acc65I restriction endonucleases (Fermentas, USA) was inserted into the pTrcHis C plasmid (Invitrogen, USA), which was digested with the same enzymes. This sub-cloning created the plasmid pTrc_VHb_His. Cells were routinely grown and maintained in LuriaeBertani (LB) medium (USB Corp., USA). All cultures were supplemented with appropriate antibiotics (ampicillin, 50 mg/ mL; streptomycin, 10 mg/mL) and cultivated at 37 C in a shaking (220 rpm) incubator (Jeiotech, Korea). All the represented values and error bars in this study indicate the means and standard deviations of at least three independent experiments, respectively.
In vivo H2 production The E. coli W3110 strain was transformed with pTrc_VHb_His (VHbþ) or pTrcHis C (NC; negative control), and the recombinant strain was grown aerobically in 50 mL of M9 minimal medium (6 g/L Na2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, 2 mM MgSO4, and 100 mM CaCl2), 5 g/L of casamino acids (BD Bioscience, USA) and 5 g/L of glucose in 165 mL serum bottles (Wheaton, USA) until the cell density reached an absorbance value of approximately 0.6 at 600 nm (A600). The culture bottles with headspace of w115 mL were then adjusted to an atmosphere of 0%, 10%, or 30% of O2 that was balanced with N2 inside an anaerobic chamber (Coy Laboratory Products, USA). Alternatively, the culture was exposed to ambient air (21% O2) in a clean bench and kept on ice. After ensuring that the insides of the bottles had been equilibrated with the corresponding outside atmosphere using a portable O2 detector (New Cosmos Electric, Japan), the cultures were induced for protein expression with the addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG; Carbosynth, UK), along with 30 mM NiSO4 and FeSO4 and 1 mM Na2MoO4 and Na2O3Se, which are required for FHL maturation. The bottles were sealed tightly with rubber stoppers and aluminum caps and subsequently cultivated until the cultures reached
Please cite this article in press as: Jo BH, et al., Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/ j.ijhydene.2014.04.209
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Fig. 1 e Functional VHb expression in E. coli. (a) Western blot analysis. Cell lysates were loaded onto the gel with the same volumes. Lane: MW, molecular weight marker; VHbD, VHb-expressing cells; NC, negative control cells. (b) Absorption spectra of purified VHb.
maximum H2 production. Samples were collected at a 1 h or 2 h interval for the time-course measurements. For the inhibitor experiments, the cells were treated with suitable inhibitors (200 mM antimycin A dissolved in ethanol or 2 mM NaNO2 in deionized water; Sigma-Aldrich, USA) at the indicated time points. Equivalent amounts of the inhibitor solvents (ethanol or deionized water, respectively) were added to the untreated cultures.
Affinity purification of VHb The recombinant strain was cultured in LB medium in a conical flask, and VHb expression was induced with 1 mM IPTG and 30 mM FeSO4 at mid-log phase. The culture was then continued overnight. During all of the subsequent procedures, the cells and buffers were kept on ice. Cells were harvested by centrifugation at 4000 g for 20 min at 4 C and then resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole; pH 8.0). Cells were disrupted with a sonic dismembrator (Sonics & Materials, USA) for 3 min at a 20% amplitude (3 s pulse on and 20 s pulse off). The disrupted cell suspension was centrifuged at 10,000 g for 20 min at 4 C. The soluble supernatant was loaded onto a NiNTA column (Qiagen, Germany). After washing with buffer (50 mM NaH2PO4, 300 mM NaCl, and 30 mM imidazole; pH 8.0), the reddish VHb fraction was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole; pH 8.0) and finally dialyzed against Tris buffer (50 mM Tris-HCl; pH 8.0). Ammonium persulfate (Bio-Rad, USA) or sodium dithionite was used to oxidize or reduce the purified VHb, respectively.
FHL activity assay All preparations were performed on ice inside of the anaerobic chamber. Cells were harvested, washed, and resuspended in Tris buffer (50 mM; pH 7.0). One milliliter of the cell suspension was treated with 5 mL of 10% (v/v) Triton X-100 solution and incubated for 30 min at room-temperature to permeabilize the cells [19]. FHL activity was assayed with a reaction mixture that contained 1460 mL of Tris buffer (50 mM; pH 7.0), 500 mL of the permeabilized cell suspension, and 40 mL
of 1 M sodium formate (final concentration of 20 mM; SigmaAldrich) in a 9 mL vial with a screw cap and a rubber septum (Supelco, USA). The vial was taken out of the chamber and was briefly treated with N2. After incubation at 37 C, the formation of H2 was analyzed from the gas phase of the vial using gas chromatography.
Measurement of H2 and O2 in the gas phase To make H2 measurements, 20e50 mL of the gas was sampled using a Gas-Tight syringe (Hamilton, USA) from the headspace of the tightly sealed culture bottle and analyzed with a gas chromatography (GC; Younglin Instrument, Korea) that was equipped with a carboxen-1010 PLOT column (Supelco) and a pulsed discharge detector (Valco Instrument, USA). The GC was routinely operated at a flow rate of 10 mL/min with the temperatures of the oven, injector, and detector set at 100 C, 130 C, and 250 C, respectively. For time-course O2 measurements, the flow rate was changed to 2 mL/min, and the temperatures of the oven and the injector were altered to 10 C and 20 C, respectively, to differentiate the retention time of O2 from that of N2. The gas concentrations in the sample aliquots were calculated using the peak areas and standard curves for H2 and O2 that were prepared using a gas mixture (5% H2 and 95% N2 for H2) and ambient air (21% O2 and 79% N2), respectively. The total amount was determined by multiplying the concentrations by the headspace volume of the serum bottle.
Western blotting Samples were separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). After proteins on the gel were transferred onto a nitrocellulose membrane (Whatman, USA), monoclonal anti-His6 IgG (ABM, Canada) and alkaline phosphatase-conjugated antimouse IgG (Sigma-Aldrich) were used as the primary and secondary antibodies, respectively. The His-tagged proteins were chromogenically detected using nitro blue tetrazolium/ 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; SigmaAldrich).
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Analytical methods Cell densities were measured using a UV/Vis spectrophotometer (Mecasys, Korea). Glucose concentrations were determined by measuring the absorbance at 340 nm using a glucose assay kit (Sigma-Aldrich) that was based on glucosedependent NADþ reduction.
Results and discussion Expression of VHb in E. coli VHb exists as a homodimer in which each subunit has a molecular mass of approximately 15.8 kDa [15]. VHb with a Cterminal His-tag (approximately 17 kDa) was expressed in E. coli by IPTG induction under the control of the trc promoter, which allowed us to study the effects of VHb expression at various O2 tensions. This approach is difficult to carry out with the native hypoxia-inducing promoter. Recombinant VHb was expressed under both anoxic and oxic conditions and was verified by both Western blot analysis (Fig. 1(a)) and by examining the apparent reddish color of the cell pellets (data not shown). When chemically oxidized, the purified VHb resulted in the same spectrum as that of the untreated VHb, indicating that it was initially in the oxidized form. Upon reduction, VHb exhibited a shift of the characteristic Soret peak (Fig. 1(b)) [20], which indicated that the protein was functionally mature with a prosthetic heme group.
O2-dependent effect of VHb on H2 accumulation in vivo To investigate O2-related effects of VHb expression on H2 production, H2 accumulations of E. coli cultures were measured after cultivation under anoxic (0%) or oxic (10%, 21%, and 30%) condition. As shown in Fig. 2, VHb was shown to be effective in elevating H2 production in the cultures that started under highly oxic conditions. The VHbþ strain gave 2-
Fig. 2 e Effects of VHb expression on H2 production. The indicated value above each bar represents the time point (in hours) that was required for maximal H2 production after induction. Abbreviations: NC, negative control cells; VHbD, VHb-expressing cells.
fold and 17-fold higher H2 yields than the NC cultures under conditions of 21% and 30% O2, respectively. In contrast, the VHbþ cultures showed reduced H2 production compared with the NC cultures under conditions of anoxia and 10% O2. The maximal H2 productions were attained at different time points and lagged relative to the VHbþ cultures (Fig. 2). FHL is the H2-producing complex on the E. coli cell membrane. It is well known that the expression of FHL is negatively regulated by O2 [21]. The large decline in the H2 production of the NC strain with gradually increasing pO2 was therefore reasonable. In the VHbþ stain, the pattern of H2 production was not intuitively comprehensible; H2 production under conditions of 10% or 21% O2 was similar to that under the anoxic condition, in which highest H2 yield is generally expected. For subsequent experiments, the 21% O2 condition was chosen to be representative of the oxic condition due to the facile preparation (in ambient air) for experimental sets.
Measurement of in vitro FHL activity for H2 production Theoretically, metabolite production can be increased by elevated enzyme activity or by increased substrate availability. Because the intracellular expression of VHb often results in enhanced protein synthesis, the improved synthesis of H2producing enzymes (and therefore H2-producing activity) can be expected to be the factor that is responsible for the enhanced H2 yield in VHbþ cultures under oxic conditions. We therefore measured whole-cell FHL activity (formate / H2) to see whether VHb enhanced cellular enzyme activity for H2 production. As shown in Fig. 3, in vitro whole-cell FHL activity of oxically grown VHbþ cells measured immediately after reaching the maximal H2 production was approximately 2.7fold higher than the NC culture, which appeared to be correlated to some extent with the maximal H2 yield (2-fold) in vivo. Despite this apparent correlation, we were not able to conclude that the improved H2 was due to the higher enzyme activity because the maximal H2 production values were not observed at a fixed time point but were instead obtained at several different time points (Fig. 2). Further examination of the FHL activities in the anoxically grown cells showed that
Fig. 3 e Whole-cell FHL activity according to initial O2 concentrations. Abbreviations: NC, negative control cells; VHbD, VHb-expressing cells.
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Fig. 4 e Effects of inhibitors on H2 production. The asterisk indicates there were significant differences (*p < 0.05, oneway ANOVA). The indicated value above each bar represents the time point (in hours) that was required for maximal H2 production after induction. Abbreviations: NC, negative control cells; VHbD, VHb-expressing cells.
there was no consistent correlation between H2 production in vivo and FHL activity in vitro. For example, the H2 yield by VHbþ (0% O2) was approximately 90% higher than in the NC culture (21% O2) (Fig. 2), even though VHbþ (0% O2) exhibited only an approximate 40% FHL activity relative to the NC culture (21% O2) (Fig. 3).
Inhibitor studies of H2 production under oxic conditions The FHL complex is expressed under anaerobic growth conditions in the presence of formate [21]. Formate production by fermentation and the subsequent H2 production from formate occur only after most of the O2 is consumed by respiration [22]. H2 production in E. coli can therefore be regarded as a ‘physiological marker’ for cells that are experiencing anaerobiosis. The same criterion was applied to our oxic culture system, in which the culture period can be divided into the following two phases: 1) anaerobic phase: the phase after the onset of H2 production; and 2) aerobic phase: the phase where O2 respiration mainly occurs, which is assigned between the initial induction point and the anaerobic phase. Time course measurements of H2 revealed that VHbþ (with an initial O2 of 21%) entered the anaerobic phase approximately 12 h after induction (see Time Course Analyses Section). We therefore performed inhibitor studies to determine how much VHb is directly involved in the process of H2 production. Nitrite, which is an inhibitor of hemoproteins, was previously shown to severely inhibit VHb activity when treated at proper concentrations and to have only slight effects on cellular respiration [23]. Nitrite can also inhibit formate dehydrogenase-H, a formate-oxidizing component of FHL [24]. In our experiment, nitrite was used at a concentration of 2 mM, which did not bring about severe FHL inhibition and allowed H2 production to take place. When nitrite was added to the culture 12 h after induction (anaerobic phase), it did not lead to a critical difference in the H2 yield of the VHbþ strain
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(Fig. 4). On the other hand, when the inhibitor was treated at the induction point (aerobic phase), VHbþ H2 production was significantly lower than in the untreated control. The NC culture maintained approximately the same maximal H2 production regardless of the treatment. A possible explanation for these findings is that the decreased H2 production of the nitrite-treated VHbþ strain may be due to the inhibitory effect of nitrite on respiration. However, this explanation was excluded by performing an additional experiment in which the VHbþ strain was treated with antimycin A, which is an inhibitor that also represses respiration but does not react with VHb [25]. This compound was unable to trigger a negative interference on the final H2 yield. The lagged H2 productions in the inhibitor-treated cells compared to the untreated cells appeared to be because of a slowdown in O2 consumption and/or lowered FHL activity by nitrite inhibition. Collectively, these experimental results suggest that the preparation for the enhanced H2 production was already done by VHb before the onset of H2 production (that is, during aerobic phase). This also implies that VHb did not directly participate in H2 production as a structural component such as O2 scavenger or O2 barrier, which might alleviate O2 inhibition of H2-producing machinery and increase H2 production.
Time course analyses To achieve a more comprehensive understanding of H2 production in our oxic culture system, time course measurements were performed at 2 h intervals for four important factors (H2, cell density, glucose, and O2) (Fig. 5(a)). The maximal H2 production was observed at 6 h or 18 h after induction in the NC or VHbþ cultures, respectively. After reaching the maximal production, the H2 content gradually decreased in the NC culture, presumably due to H2 consumption by endogenous uptake hydrogenase(s) [26]. The VHbþ culture exhibited a slow growth rate compared to the NC culture, but the maximal cell density was approximately 6% higher than in the NC culture. The NC culture consumed glucose much faster than the VHbþ culture and exhausted all of the glucose by 6 h after induction, while the VHbþ culture used up the substrate by 16 h after induction. The O2 uptake rate by NC was also faster than in the VHbþ culture. Overall, VHb expression slowed down cellular processes but was efficient at higher cell densities and led to more H2 production. To identify the factor that was responsible for the enhanced H2 yield by VHb, we compared the four factors in the NC culture at the end of the aerobic phase (4 h after induction) with those in the VHbþ culture (at 12 h after induction) because the preparation for the improved H2 by VHb mainly occurred during the aerobic phase, as previously demonstrated in the inhibitor study. As shown in Fig. 5(b), there were no significant differences in H2 production or cell density between the NC and VHbþ cultures at the beginning of the anaerobic phase. However, the amount of remaining glucose (1.77 g/L) in the VHbþ culture was 1.8-fold higher than in the NC culture (0.98 g/L). In contrast, the VHbþ culture had much lower O2 content (2.3%) than the NC culture (8.5%). The NC culture still possessed a high O2 content even though it showed H2 production that can occur only under anaerobic conditions. This result was likely due to the depletion of O2
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Fig. 5 e (a) Time-course analyses of H2 production, cell growth, glucose consumption, and O2 consumption. The anaerobic phase of the negative control cell (NC) culture is shaded light gray, and the dark gray shaded regions represent the anaerobic phases of both the NC and VHb-expressing cell (VHbD) cultures. (b) Comparisons of H2 production, cell density, remaining glucose, and remaining O2 at the beginning of the anaerobic phase. The data were derived from the data points at 4 h in the NC culture and at 12 h in the VHbD culture as in (a). The asterisk indicates there were significant differences (**p < 0.01, oneway ANOVA).
from the media by vigorous O2 consumption, which the O2 concentration of the headspace did not reflect. It is also possible that the high O2 concentration in the NC culture was responsible for the lower H2 yield than in the VHbþ culture because O2 can inactivate FHL [27] and cease H2 production. However, examination of the time course measurements in the NC cultures revealed that the cells were able to produce H2 until all of the glucose was depleted (Fig. 5(a)), which suggests that FHL inhibition by O2 was not the limiting factor. Assuming that the amount of O2 that was dissolved in the medium was negligible according to Henry’s Law constant, a calculation showed that the NC culture consumed only 0.66 mol-O2/mol-glucose while the VHbþ culture used
1.22 mol-O2/mol-glucose. These comparisons led to the conclusion that the VHbþ culture consumed glucose more efficiently than the NC culture during the aerobic phase. Thus, the increased substrate availability (the 1.8-fold higher amount of remaining glucose) was the responsible factor for the 2-fold higher H2 yield in the VHbþ culture.
Implications for biotechnological applications of H2 production The mechanism of VHb action remains unclear, but the common consensus is that VHb increases the effective intracellular concentration of dissolved O2, which results in
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increased aerobic respiration and more efficient ATP production [16,28]. The increased O2 level may also cause oxidative damage to the cell. VHb can relieve this oxidative stress by indirectly regulating expression of genes such as catalase and superoxide dismutase [29]. However, because O2 is regenerated (that is, not consumed) during the antioxidant reactions and NADH is wasted during the regulation by VHb [29], the protective role of VHb against oxidative stress was not likely to be responsible for the enhanced H2 production under oxic condition. O2 is reduced to H2O by invariably accepting four electrons. In our experiments, the ratio of O2 to electrons (glucose) was higher in the VHbþ culture than in the NC culture during the aerobic phase. This result coincides with the previous explanation and clearly indicates that VHb can reduce the waste of electrons during aerobic conditions [30], providing a chance for the remaining electron pool to be utilized for H2 production. Meanwhile, basic functions can be retained for cell viability and growth, which is the essential requirement for maximizing H2 production [31]. Although this demonstration was performed with E. coli that are capable of producing H2 only under anaerobic conditions (via FHL), and VHbþ cells showed relatively low H2 productivity, we expect that VHb can also be applied to other potential aerobic H2producing systems (Fig. 6) to efficiently enhance H2 yields from limited bioresources. These potential H2 pathways can be constituted by NADH-dependent hydrogenase or by NADH:ferredoxin oxidoreductase and ferredoxin-dependent hydrogenase. If the rate of glucose consumption can be accelerated without affecting relative metabolic fluxes, the H2 productivity of VHb-harboring, aerobic H2-producing cells will also be improved. In addition, the oxically grown VHbþ culture had a total FHL activity that was even higher than in the anoxically grown NC strain (Fig. 3). The reason for this effect is unclear, but we surmise that the increased expression levels of ribosomes and tRNAs by VHb during the aerobic phase [32] may influence and possibly promote the expression of FHL immediately after the phase has shifted to the anaerobic phase. Because it has a high FHL activity and can be easily prepared by cultivation that
Fig. 6 e Suggested explanation for the metabolic shift towards enhanced H2 production by VHb in a designed aerobic H2-producing system. The electron donors (eL) may be NADH or other reducing equivalents.
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starts with ambient air (21% O2) conditions, we suggest that this VHbþ strain can be utilized efficiently and economically as a whole-cell biocatalyst for H2 production from formic acid [33], a material that is considered to be one of the most promising forms of H2 storage in the future of the hydrogen economy [34].
Conclusions VHb was engineered in E. coli to study the effects of this versatile O2-binding protein on oxic H2 production. The recombinant VHb was efficiently expressed under both oxic and anoxic culture conditions. The H2 yields by the VHbþ cells were 2-fold and 17-fold higher than those of the NC cultures under conditions of 21% and 30% O2, respectively. Through in vitro FHL activity assays, inhibitor treatments, and timecourse measurements, the primary factor that was responsible for the increased H2 yield was concluded be the increased substrate availability that resulted from efficient respiration by VHb even though the FHL activity of VHbþ was also higher than in the NC culture. These results may highlight the use of VHb as an attractive tool for enhancing H2 yield in potential aerobic H2-producing systems and for constructing economical whole-cell biocatalysts that produce H2 from formic acid.
Acknowledgment This work was supported the Marine Biotechnology Program (Marine BioMaterials Research Center) and the Manpower Development Program for Marine Energy funded by the Ministry of Oceans and Fisheries, Korea.
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[9] Woodward J, Orr M, Cordray K, Greenbaum E. Enzymatic production of biohydrogen. Nature 2000;405:1014e5. [10] Nath K, Das D. Improvement of fermentative hydrogen production: various approaches. Appl Microbiol Biotechnol 2004;65:520e9. [11] Kim JYH, Jo BH, Cha HJ. Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli. Microb Cell Fact 2010;9. [12] Kim JYH, Jo BH, Cha HJ. Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli. J Biotechnol 2011;155:312e9. [13] van Rijsewijk BRBH, Nanchen A, Nallet S, Kleijn RJ, Sauer U. Large-scale 13C-flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in Escherichia coli. Mol Syst Biol 2011;7. [14] Wu SX, Yan GY, Xu LL, Wang QX, Liu XL. Improvement of hydrogen production with expression of lba gene in chloroplast of Chlamydomonas reinhardtii. Int J Hydrogen Energy 2010;35:13419e26. [15] Ramandeep, Hwang KW, Raje M, Kim KJ, Stark BC, Dikshit KL, et al. Vitreoscilla hemoglobin - intracellular localization and binding to membranes. J Biol Chem 2001;276:24781e9. [16] Zhang L, Li YJ, Wang ZN, Xia Y, Chen WS, Tang KX. Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering. Biotechnol Adv 2007;25:123e36. [17] Kang DG, Kim JYH, Cha HJ. Enhanced detoxification of organophosphates using recombinant Escherichia coli with co-expression of organophosphorus hydrolase and bacterial hemoglobin. Biotechnol Lett 2002;24:879e83. [18] Kim D, Hwang DS, Kang DG, Kim JYH, Cha HJ. Enhancement of mussel adhesive protein production in Escherichia coli by co-expression of bacterial hemoglobin. Biotechnol Prog 2008;24:663e6. [19] Maier RJ, Olson J, Olczak A. Hydrogen-oxidizing capabilities of Helicobacter hepaticus and in vivo availability of the substrate. J Bacteriol 2003;185:2680e2. [20] Liu CY, Webster DA. Spectral characteristics and interconversions of the reduced, oxidized, and oxygenated forms of purified cytochrome o. J Biol Chem 1974;249:4261e6. [21] Sawers RG. Formate and its role in hydrogen production in Escherichia coli. Biochem Soc Trans 2005;33:42e6. [22] Becker S, Vlad D, Schuster S, Pfeiffer P, Unden G. Regulatory O2 tensions for the synthesis of fermentation products in
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Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli Byung Hoon Jo a,b, Jaoon Y.H. Kim b, Jeong Hyun Seo b,c, Hyung Joon Cha a,b,* a
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea b Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea c School of Chemical Engineering, Yeungnam University, Gyeongsan 712-749, Republic of Korea
article info
abstract
Article history:
H2 production under aerobic conditions has been proposed as an alternative method to
Received 16 March 2014
overcome the fundamentally low yield of H2 production by fermentative bacteria by
Received in revised form
maximizing the number of electrons that are available for H2. Here, we engineered Vitre-
26 April 2014
oscilla hemoglobin (VHb) in Escherichia coli to study the effects of this versatile oxygen (O2)-
Accepted 29 April 2014
binding protein on oxic H2 production in a closed batch system that was supplemented
Available online xxx
with glucose. The H2 yields that were obtained with the VHb-expressing E. coli were greatly
Keywords:
tensions. The formate hydrogen lyase (FHL) activity of oxically cultured, VHb-expressing
Biohydrogen
cells was also much higher than that of the negative control cells. Through inhibitor
Vitreoscilla hemoglobin
studies and time-course experiments, VHb was shown to contribute to the improved H2
Escherichia coli
yield primarily by increasing the efficiency of cellular metabolism during the aerobic phase
Aerobic condition
before the onset of H2 production and not by working as an O2-scavenger during H2 pro-
Formate hydrogen lyase
duction. This new approach allowed more substrate to remain to be further utilized for the
enhanced in comparison to the negative control cells in culture that started with high O2
production of more H2 from limited resources. We expect that VHb can be successfully engineered in potential aerobic H2-producing microbial systems to enhance the overall H2 production yield. In addition, the remarkably high FHL activity of oxically grown, VHbexpressing cells may make this engineered strain an attractive whole-cell biocatalyst for converting formate to H2. Copyright ª 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Introduction H2 is a promising alternative fuel and is expected to be a key energy carrier in the future “hydrogen economy” [1]. This
utility arises from several attributes of H2; for example, it has the greatest energy content per mass among known fuels and is environment-friendly because its oxidation by O2 leaves only water as a byproduct. Most H2 that is currently being used is prepared by fossil fuel reforming,
* Corresponding author. Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea. Tel.: þ82 54 279 2280; fax: þ82 54 279 2699. E-mail address: [email protected] (H.J. Cha). http://dx.doi.org/10.1016/j.ijhydene.2014.04.209 0360-3199/Copyright ª 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
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which makes the current H2 production process pollute the environment. The use of biological microbial systems is, therefore, an attractive and valuable approach to H2 production because these systems are renewable and carbon neutral [2]. Among the several biological approaches that are possible, dark fermentation is a reasonable choice because it allows for exceedingly high H2 productivity to be achieved, along with high cell density [3]. However, the practical application of this approach is limited by the fundamental obstacle that the efficiency of energy conversion by fermentative H2 metabolism is not sufficiently high. H2 yield should be expanded beyond the upper limit that is found in nature to increase the efficiency of H2 conversion [4]. Several studies have investigated the feasibility of constructing a non-native H2 pathway in the facultative anaerobe Escherichia coli through the utilization of NADH as the electron donor, which is generated through glycolysis [5e7]. However, the total yield of H2 production from these additional pathways cannot exceed the limit that is found in strict anaerobes such as Clostridia [4]. For fermentative H2 production to be commercially viable, the yield should be expanded to become as close as possible to the maximal stoichiometric H2 yield that can be obtained from glucose [8,9]. This expansion could potentially be accomplished by harnessing electrons under more energy-efficient aerobic conditions [4,10e12]. With such an aerobic H2-producing system (which does not exist in nature, nor has it yet been artificially constructed), strategies are required to increase electron flow to the tricarboxylic acid (TCA) cycle (and, in turn, to NADH) to improve H2 production because most electrons flow toward the formation of byproducts such as acetic acid rather than to the TCA cycle, even during aerobic growth [13]. The construction of efficient microbial cell factories also requires that other factors be optimized, including efficient cell growth and high levels of enzyme production. A previous study on the improvement of microalgal H2 production by the expression of leghemoglobin showed that the artificial engineering of cells with an adjuvant molecular component can improve overall H2 metabolism in the presence of O2 [14]. Inspired by the molecular and physiological function of O2 binding and transport by Vitreoscilla hemoglobin (VHb), we attempted to improve fermentative H2 production under oxic conditions by engineering this molecular component in E. coli. VHb is a soluble form of hemoglobin that is synthesized by the obligate aerobic bacterium Vitreoscilla stercoraria to cope with hypoxic environmental conditions [15]. The intracellular expression of this versatile O2-binding protein in various hosts, including E. coli, has been shown to elicit positive effects, such as enhanced protein production, improved cell growth, and elevated metabolite formation [16e18]. In this study, the effect of VHb expression on H2 production was investigated under oxic and anoxic conditions in a closed batch culture with glucose as the substrate for H2 production via the native formate hydrogen lyase (FHL) pathway. The potential biotechnological application of VHb to aerobic H2 production system was also discussed.
Materials and methods Strains, plasmid construction, and general culture conditions All of the DNA manipulations were performed using the E. coli TOP10 strain (Invitrogen, USA), following standard methods. The E. coli strain W3110 (ATCC 27325) was used for VHb expression and H2 production. The vgb gene, which encodes the VHb protein, was amplified by Taq DNA polymerase (Takara, Japan) from a plasmid that contained the gene [17] using the following primers: forward: 50 -CCATGGGTATGTreverse: 50 -GGTACCTTAGACCAGCAAAC-30 , CAGTGGTGGTGGTGGTGGTGCTCGAGTTCAACCGCTTGAGCGTAC-30 (restriction sites are underlined). The forward primer included an NcoI restriction site, and the reverse primer was designed to contain an Acc65I site that was preceded by a Cterminal His6-tag sequence (italicized), which facilitated the detection and purification of the protein. The PCR product was ligated into the plasmid pGEM-T Easy (Promega, USA), and the DNA sequence was confirmed to be correct. The DNA fragment that was generated with the NcoI and Acc65I restriction endonucleases (Fermentas, USA) was inserted into the pTrcHis C plasmid (Invitrogen, USA), which was digested with the same enzymes. This sub-cloning created the plasmid pTrc_VHb_His. Cells were routinely grown and maintained in LuriaeBertani (LB) medium (USB Corp., USA). All cultures were supplemented with appropriate antibiotics (ampicillin, 50 mg/ mL; streptomycin, 10 mg/mL) and cultivated at 37 C in a shaking (220 rpm) incubator (Jeiotech, Korea). All the represented values and error bars in this study indicate the means and standard deviations of at least three independent experiments, respectively.
In vivo H2 production The E. coli W3110 strain was transformed with pTrc_VHb_His (VHbþ) or pTrcHis C (NC; negative control), and the recombinant strain was grown aerobically in 50 mL of M9 minimal medium (6 g/L Na2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, 2 mM MgSO4, and 100 mM CaCl2), 5 g/L of casamino acids (BD Bioscience, USA) and 5 g/L of glucose in 165 mL serum bottles (Wheaton, USA) until the cell density reached an absorbance value of approximately 0.6 at 600 nm (A600). The culture bottles with headspace of w115 mL were then adjusted to an atmosphere of 0%, 10%, or 30% of O2 that was balanced with N2 inside an anaerobic chamber (Coy Laboratory Products, USA). Alternatively, the culture was exposed to ambient air (21% O2) in a clean bench and kept on ice. After ensuring that the insides of the bottles had been equilibrated with the corresponding outside atmosphere using a portable O2 detector (New Cosmos Electric, Japan), the cultures were induced for protein expression with the addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG; Carbosynth, UK), along with 30 mM NiSO4 and FeSO4 and 1 mM Na2MoO4 and Na2O3Se, which are required for FHL maturation. The bottles were sealed tightly with rubber stoppers and aluminum caps and subsequently cultivated until the cultures reached
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Fig. 1 e Functional VHb expression in E. coli. (a) Western blot analysis. Cell lysates were loaded onto the gel with the same volumes. Lane: MW, molecular weight marker; VHbD, VHb-expressing cells; NC, negative control cells. (b) Absorption spectra of purified VHb.
maximum H2 production. Samples were collected at a 1 h or 2 h interval for the time-course measurements. For the inhibitor experiments, the cells were treated with suitable inhibitors (200 mM antimycin A dissolved in ethanol or 2 mM NaNO2 in deionized water; Sigma-Aldrich, USA) at the indicated time points. Equivalent amounts of the inhibitor solvents (ethanol or deionized water, respectively) were added to the untreated cultures.
Affinity purification of VHb The recombinant strain was cultured in LB medium in a conical flask, and VHb expression was induced with 1 mM IPTG and 30 mM FeSO4 at mid-log phase. The culture was then continued overnight. During all of the subsequent procedures, the cells and buffers were kept on ice. Cells were harvested by centrifugation at 4000 g for 20 min at 4 C and then resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole; pH 8.0). Cells were disrupted with a sonic dismembrator (Sonics & Materials, USA) for 3 min at a 20% amplitude (3 s pulse on and 20 s pulse off). The disrupted cell suspension was centrifuged at 10,000 g for 20 min at 4 C. The soluble supernatant was loaded onto a NiNTA column (Qiagen, Germany). After washing with buffer (50 mM NaH2PO4, 300 mM NaCl, and 30 mM imidazole; pH 8.0), the reddish VHb fraction was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole; pH 8.0) and finally dialyzed against Tris buffer (50 mM Tris-HCl; pH 8.0). Ammonium persulfate (Bio-Rad, USA) or sodium dithionite was used to oxidize or reduce the purified VHb, respectively.
FHL activity assay All preparations were performed on ice inside of the anaerobic chamber. Cells were harvested, washed, and resuspended in Tris buffer (50 mM; pH 7.0). One milliliter of the cell suspension was treated with 5 mL of 10% (v/v) Triton X-100 solution and incubated for 30 min at room-temperature to permeabilize the cells [19]. FHL activity was assayed with a reaction mixture that contained 1460 mL of Tris buffer (50 mM; pH 7.0), 500 mL of the permeabilized cell suspension, and 40 mL
of 1 M sodium formate (final concentration of 20 mM; SigmaAldrich) in a 9 mL vial with a screw cap and a rubber septum (Supelco, USA). The vial was taken out of the chamber and was briefly treated with N2. After incubation at 37 C, the formation of H2 was analyzed from the gas phase of the vial using gas chromatography.
Measurement of H2 and O2 in the gas phase To make H2 measurements, 20e50 mL of the gas was sampled using a Gas-Tight syringe (Hamilton, USA) from the headspace of the tightly sealed culture bottle and analyzed with a gas chromatography (GC; Younglin Instrument, Korea) that was equipped with a carboxen-1010 PLOT column (Supelco) and a pulsed discharge detector (Valco Instrument, USA). The GC was routinely operated at a flow rate of 10 mL/min with the temperatures of the oven, injector, and detector set at 100 C, 130 C, and 250 C, respectively. For time-course O2 measurements, the flow rate was changed to 2 mL/min, and the temperatures of the oven and the injector were altered to 10 C and 20 C, respectively, to differentiate the retention time of O2 from that of N2. The gas concentrations in the sample aliquots were calculated using the peak areas and standard curves for H2 and O2 that were prepared using a gas mixture (5% H2 and 95% N2 for H2) and ambient air (21% O2 and 79% N2), respectively. The total amount was determined by multiplying the concentrations by the headspace volume of the serum bottle.
Western blotting Samples were separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). After proteins on the gel were transferred onto a nitrocellulose membrane (Whatman, USA), monoclonal anti-His6 IgG (ABM, Canada) and alkaline phosphatase-conjugated antimouse IgG (Sigma-Aldrich) were used as the primary and secondary antibodies, respectively. The His-tagged proteins were chromogenically detected using nitro blue tetrazolium/ 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; SigmaAldrich).
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Analytical methods Cell densities were measured using a UV/Vis spectrophotometer (Mecasys, Korea). Glucose concentrations were determined by measuring the absorbance at 340 nm using a glucose assay kit (Sigma-Aldrich) that was based on glucosedependent NADþ reduction.
Results and discussion Expression of VHb in E. coli VHb exists as a homodimer in which each subunit has a molecular mass of approximately 15.8 kDa [15]. VHb with a Cterminal His-tag (approximately 17 kDa) was expressed in E. coli by IPTG induction under the control of the trc promoter, which allowed us to study the effects of VHb expression at various O2 tensions. This approach is difficult to carry out with the native hypoxia-inducing promoter. Recombinant VHb was expressed under both anoxic and oxic conditions and was verified by both Western blot analysis (Fig. 1(a)) and by examining the apparent reddish color of the cell pellets (data not shown). When chemically oxidized, the purified VHb resulted in the same spectrum as that of the untreated VHb, indicating that it was initially in the oxidized form. Upon reduction, VHb exhibited a shift of the characteristic Soret peak (Fig. 1(b)) [20], which indicated that the protein was functionally mature with a prosthetic heme group.
O2-dependent effect of VHb on H2 accumulation in vivo To investigate O2-related effects of VHb expression on H2 production, H2 accumulations of E. coli cultures were measured after cultivation under anoxic (0%) or oxic (10%, 21%, and 30%) condition. As shown in Fig. 2, VHb was shown to be effective in elevating H2 production in the cultures that started under highly oxic conditions. The VHbþ strain gave 2-
Fig. 2 e Effects of VHb expression on H2 production. The indicated value above each bar represents the time point (in hours) that was required for maximal H2 production after induction. Abbreviations: NC, negative control cells; VHbD, VHb-expressing cells.
fold and 17-fold higher H2 yields than the NC cultures under conditions of 21% and 30% O2, respectively. In contrast, the VHbþ cultures showed reduced H2 production compared with the NC cultures under conditions of anoxia and 10% O2. The maximal H2 productions were attained at different time points and lagged relative to the VHbþ cultures (Fig. 2). FHL is the H2-producing complex on the E. coli cell membrane. It is well known that the expression of FHL is negatively regulated by O2 [21]. The large decline in the H2 production of the NC strain with gradually increasing pO2 was therefore reasonable. In the VHbþ stain, the pattern of H2 production was not intuitively comprehensible; H2 production under conditions of 10% or 21% O2 was similar to that under the anoxic condition, in which highest H2 yield is generally expected. For subsequent experiments, the 21% O2 condition was chosen to be representative of the oxic condition due to the facile preparation (in ambient air) for experimental sets.
Measurement of in vitro FHL activity for H2 production Theoretically, metabolite production can be increased by elevated enzyme activity or by increased substrate availability. Because the intracellular expression of VHb often results in enhanced protein synthesis, the improved synthesis of H2producing enzymes (and therefore H2-producing activity) can be expected to be the factor that is responsible for the enhanced H2 yield in VHbþ cultures under oxic conditions. We therefore measured whole-cell FHL activity (formate / H2) to see whether VHb enhanced cellular enzyme activity for H2 production. As shown in Fig. 3, in vitro whole-cell FHL activity of oxically grown VHbþ cells measured immediately after reaching the maximal H2 production was approximately 2.7fold higher than the NC culture, which appeared to be correlated to some extent with the maximal H2 yield (2-fold) in vivo. Despite this apparent correlation, we were not able to conclude that the improved H2 was due to the higher enzyme activity because the maximal H2 production values were not observed at a fixed time point but were instead obtained at several different time points (Fig. 2). Further examination of the FHL activities in the anoxically grown cells showed that
Fig. 3 e Whole-cell FHL activity according to initial O2 concentrations. Abbreviations: NC, negative control cells; VHbD, VHb-expressing cells.
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Fig. 4 e Effects of inhibitors on H2 production. The asterisk indicates there were significant differences (*p < 0.05, oneway ANOVA). The indicated value above each bar represents the time point (in hours) that was required for maximal H2 production after induction. Abbreviations: NC, negative control cells; VHbD, VHb-expressing cells.
there was no consistent correlation between H2 production in vivo and FHL activity in vitro. For example, the H2 yield by VHbþ (0% O2) was approximately 90% higher than in the NC culture (21% O2) (Fig. 2), even though VHbþ (0% O2) exhibited only an approximate 40% FHL activity relative to the NC culture (21% O2) (Fig. 3).
Inhibitor studies of H2 production under oxic conditions The FHL complex is expressed under anaerobic growth conditions in the presence of formate [21]. Formate production by fermentation and the subsequent H2 production from formate occur only after most of the O2 is consumed by respiration [22]. H2 production in E. coli can therefore be regarded as a ‘physiological marker’ for cells that are experiencing anaerobiosis. The same criterion was applied to our oxic culture system, in which the culture period can be divided into the following two phases: 1) anaerobic phase: the phase after the onset of H2 production; and 2) aerobic phase: the phase where O2 respiration mainly occurs, which is assigned between the initial induction point and the anaerobic phase. Time course measurements of H2 revealed that VHbþ (with an initial O2 of 21%) entered the anaerobic phase approximately 12 h after induction (see Time Course Analyses Section). We therefore performed inhibitor studies to determine how much VHb is directly involved in the process of H2 production. Nitrite, which is an inhibitor of hemoproteins, was previously shown to severely inhibit VHb activity when treated at proper concentrations and to have only slight effects on cellular respiration [23]. Nitrite can also inhibit formate dehydrogenase-H, a formate-oxidizing component of FHL [24]. In our experiment, nitrite was used at a concentration of 2 mM, which did not bring about severe FHL inhibition and allowed H2 production to take place. When nitrite was added to the culture 12 h after induction (anaerobic phase), it did not lead to a critical difference in the H2 yield of the VHbþ strain
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(Fig. 4). On the other hand, when the inhibitor was treated at the induction point (aerobic phase), VHbþ H2 production was significantly lower than in the untreated control. The NC culture maintained approximately the same maximal H2 production regardless of the treatment. A possible explanation for these findings is that the decreased H2 production of the nitrite-treated VHbþ strain may be due to the inhibitory effect of nitrite on respiration. However, this explanation was excluded by performing an additional experiment in which the VHbþ strain was treated with antimycin A, which is an inhibitor that also represses respiration but does not react with VHb [25]. This compound was unable to trigger a negative interference on the final H2 yield. The lagged H2 productions in the inhibitor-treated cells compared to the untreated cells appeared to be because of a slowdown in O2 consumption and/or lowered FHL activity by nitrite inhibition. Collectively, these experimental results suggest that the preparation for the enhanced H2 production was already done by VHb before the onset of H2 production (that is, during aerobic phase). This also implies that VHb did not directly participate in H2 production as a structural component such as O2 scavenger or O2 barrier, which might alleviate O2 inhibition of H2-producing machinery and increase H2 production.
Time course analyses To achieve a more comprehensive understanding of H2 production in our oxic culture system, time course measurements were performed at 2 h intervals for four important factors (H2, cell density, glucose, and O2) (Fig. 5(a)). The maximal H2 production was observed at 6 h or 18 h after induction in the NC or VHbþ cultures, respectively. After reaching the maximal production, the H2 content gradually decreased in the NC culture, presumably due to H2 consumption by endogenous uptake hydrogenase(s) [26]. The VHbþ culture exhibited a slow growth rate compared to the NC culture, but the maximal cell density was approximately 6% higher than in the NC culture. The NC culture consumed glucose much faster than the VHbþ culture and exhausted all of the glucose by 6 h after induction, while the VHbþ culture used up the substrate by 16 h after induction. The O2 uptake rate by NC was also faster than in the VHbþ culture. Overall, VHb expression slowed down cellular processes but was efficient at higher cell densities and led to more H2 production. To identify the factor that was responsible for the enhanced H2 yield by VHb, we compared the four factors in the NC culture at the end of the aerobic phase (4 h after induction) with those in the VHbþ culture (at 12 h after induction) because the preparation for the improved H2 by VHb mainly occurred during the aerobic phase, as previously demonstrated in the inhibitor study. As shown in Fig. 5(b), there were no significant differences in H2 production or cell density between the NC and VHbþ cultures at the beginning of the anaerobic phase. However, the amount of remaining glucose (1.77 g/L) in the VHbþ culture was 1.8-fold higher than in the NC culture (0.98 g/L). In contrast, the VHbþ culture had much lower O2 content (2.3%) than the NC culture (8.5%). The NC culture still possessed a high O2 content even though it showed H2 production that can occur only under anaerobic conditions. This result was likely due to the depletion of O2
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Fig. 5 e (a) Time-course analyses of H2 production, cell growth, glucose consumption, and O2 consumption. The anaerobic phase of the negative control cell (NC) culture is shaded light gray, and the dark gray shaded regions represent the anaerobic phases of both the NC and VHb-expressing cell (VHbD) cultures. (b) Comparisons of H2 production, cell density, remaining glucose, and remaining O2 at the beginning of the anaerobic phase. The data were derived from the data points at 4 h in the NC culture and at 12 h in the VHbD culture as in (a). The asterisk indicates there were significant differences (**p < 0.01, oneway ANOVA).
from the media by vigorous O2 consumption, which the O2 concentration of the headspace did not reflect. It is also possible that the high O2 concentration in the NC culture was responsible for the lower H2 yield than in the VHbþ culture because O2 can inactivate FHL [27] and cease H2 production. However, examination of the time course measurements in the NC cultures revealed that the cells were able to produce H2 until all of the glucose was depleted (Fig. 5(a)), which suggests that FHL inhibition by O2 was not the limiting factor. Assuming that the amount of O2 that was dissolved in the medium was negligible according to Henry’s Law constant, a calculation showed that the NC culture consumed only 0.66 mol-O2/mol-glucose while the VHbþ culture used
1.22 mol-O2/mol-glucose. These comparisons led to the conclusion that the VHbþ culture consumed glucose more efficiently than the NC culture during the aerobic phase. Thus, the increased substrate availability (the 1.8-fold higher amount of remaining glucose) was the responsible factor for the 2-fold higher H2 yield in the VHbþ culture.
Implications for biotechnological applications of H2 production The mechanism of VHb action remains unclear, but the common consensus is that VHb increases the effective intracellular concentration of dissolved O2, which results in
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increased aerobic respiration and more efficient ATP production [16,28]. The increased O2 level may also cause oxidative damage to the cell. VHb can relieve this oxidative stress by indirectly regulating expression of genes such as catalase and superoxide dismutase [29]. However, because O2 is regenerated (that is, not consumed) during the antioxidant reactions and NADH is wasted during the regulation by VHb [29], the protective role of VHb against oxidative stress was not likely to be responsible for the enhanced H2 production under oxic condition. O2 is reduced to H2O by invariably accepting four electrons. In our experiments, the ratio of O2 to electrons (glucose) was higher in the VHbþ culture than in the NC culture during the aerobic phase. This result coincides with the previous explanation and clearly indicates that VHb can reduce the waste of electrons during aerobic conditions [30], providing a chance for the remaining electron pool to be utilized for H2 production. Meanwhile, basic functions can be retained for cell viability and growth, which is the essential requirement for maximizing H2 production [31]. Although this demonstration was performed with E. coli that are capable of producing H2 only under anaerobic conditions (via FHL), and VHbþ cells showed relatively low H2 productivity, we expect that VHb can also be applied to other potential aerobic H2producing systems (Fig. 6) to efficiently enhance H2 yields from limited bioresources. These potential H2 pathways can be constituted by NADH-dependent hydrogenase or by NADH:ferredoxin oxidoreductase and ferredoxin-dependent hydrogenase. If the rate of glucose consumption can be accelerated without affecting relative metabolic fluxes, the H2 productivity of VHb-harboring, aerobic H2-producing cells will also be improved. In addition, the oxically grown VHbþ culture had a total FHL activity that was even higher than in the anoxically grown NC strain (Fig. 3). The reason for this effect is unclear, but we surmise that the increased expression levels of ribosomes and tRNAs by VHb during the aerobic phase [32] may influence and possibly promote the expression of FHL immediately after the phase has shifted to the anaerobic phase. Because it has a high FHL activity and can be easily prepared by cultivation that
Fig. 6 e Suggested explanation for the metabolic shift towards enhanced H2 production by VHb in a designed aerobic H2-producing system. The electron donors (eL) may be NADH or other reducing equivalents.
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starts with ambient air (21% O2) conditions, we suggest that this VHbþ strain can be utilized efficiently and economically as a whole-cell biocatalyst for H2 production from formic acid [33], a material that is considered to be one of the most promising forms of H2 storage in the future of the hydrogen economy [34].
Conclusions VHb was engineered in E. coli to study the effects of this versatile O2-binding protein on oxic H2 production. The recombinant VHb was efficiently expressed under both oxic and anoxic culture conditions. The H2 yields by the VHbþ cells were 2-fold and 17-fold higher than those of the NC cultures under conditions of 21% and 30% O2, respectively. Through in vitro FHL activity assays, inhibitor treatments, and timecourse measurements, the primary factor that was responsible for the increased H2 yield was concluded be the increased substrate availability that resulted from efficient respiration by VHb even though the FHL activity of VHbþ was also higher than in the NC culture. These results may highlight the use of VHb as an attractive tool for enhancing H2 yield in potential aerobic H2-producing systems and for constructing economical whole-cell biocatalysts that produce H2 from formic acid.
Acknowledgment This work was supported the Marine Biotechnology Program (Marine BioMaterials Research Center) and the Manpower Development Program for Marine Energy funded by the Ministry of Oceans and Fisheries, Korea.
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Please cite this article in press as: Jo BH, et al., Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli, International Journal of Hydrogen Energy (2014), http://dx.doi.org/10.1016/ j.ijhydene.2014.04.209