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Improved hydrogen production with expression of hemH and lba genes in chloroplast of Chlamydomonas reinhardtii
Journal of Biotechnology 146 (2010) 120–125
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Improved hydrogen production with expression of hemH and lba genes in chloroplast of Chlamydomonas reinhardtii Shuangxiu Wu ∗ , Rui Huang, Lili Xu, Guangyu Yan, Quanxi Wang Department of Biology, College of Life and Environmental Science, Shanghai Normal University, Guilin Road 100, Shanghai City 200234, China
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Article history: Received 8 November 2009 Received in revised form 22 January 2010 Accepted 29 January 2010
Keywords: Chlamydomonas reinhardtii Leghemoglobin Ferrochelatase Bio-hydrogen production Chloroplast transformation
a b s t r a c t The coding region of both the ferrochelatase gene, hemH, from Bradyrhizobium japonicum, and the leghemoglobin gene, lba, from Glycine max, were transferred into chloroplast of Chlamydomonas reinhardtii. As a result, transgenic C. reinhardtii cultures more rapidly consumed O2 and increased H2 output compared with controls in both sulfur-free and sulfur-containing medium. H2 production of the transgenic algal cultures in sulfur-free medium was 4-fold greater than that of control cultures, ∼3.3 ml bottle−1 . Maximum expression of the hemH-lba fusion protein on day 5 coincided with the lowest levels of O2 content and the highest H2 evolution rate detected over 7 days of anaerobic induction in sulfur-free medium. When the concentration of sulfate in the growth medium was restored to 12.5 or 50 M, O2 consumption and H2 yield decreased more slowly in the transgenic algal cultures than in the control cultures. These results demonstrate that expression of the hemH-lba fusion protein in chloroplast of C. reinhardtii improved their H2 yield by decreasing O2 content in the medium, thereby representing the potential for H2 production in green algae to be improved. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Many photosynthetic green algae, including Chlamydomonas reinhardtii, have been shown to metabolize H2 under anaerobic conditions (Melis, 2007). Under anoxic conditions, expression of [Fe]-hydrogenase (H2 ase) is induced and imported into the chloroplast stroma of green algae to catalyze the production of H+ and e− to form H2 (Happe et al., 1994; Forestier et al., 2003). The H2 ase found in C. reinhardtii has a high specific activity that is between 10- and 100-fold higher than any other H2 ase (Happe and Naber, 1993; Florin et al., 2001), which represents the significant potential for this algae to be developed for a sustainable, clean, solar-powered H2 production system in the future (Ghirardi et al., 2000). However, H2 ase is inactivated by trace amounts of O2 which is concomitantly produced during photosynthesis (Ghirardi et al., 1997). Therefore, light-dependent H2 production by C. reinhardtii is generally only observed up to several minutes after anaerobic induction. This apparent incompatibility of simultaneous O2 and H2 production during the photosynthesis of green algae has inhibited the application of H2 production by C. reinhardtii. Till
Abbreviations: PSII, photosystem II; Spec, spectinomycin; WT, wild type; lb, leghemoglobin; S, sulfur; ORF, open reading frame; ALA, ␦-aminolevulinic acid; Fe, ferro; MM, molecular marker. ∗ Corresponding author. Tel.: +86 21 64322526; fax: +86 21 64322142. E-mail address: [email protected] (S. Wu). 0168-1656/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2010.01.023
2000, Melis and coworkers demonstrated that depletion of sulfur in the culture medium of green algae affected photosystem II (PS II) to dramatically decrease O2 production. As a result, a significant improvement in H2 ase activity and prolonged H2 production over 4–5 days was achieved (Woykoff et al., 1998; Melis et al., 2000). However, H2 productivity by this method was much lower than the predicted capacity for H2 production due to the depletion of electron resources for PS II as a result of sulfur depletion (Melis, 2007; Hemschemeier et al., 2008). Nitrogenase is an enzyme in soybean leguminous nodules that is similar to H2 ase, is very sensitive to O2 but has high nitrogen fixation efficiency under ambient conditions. These characteristics are related to the abundance of leghemoglobins (lbs) that exit in the soybean root nodules to facilitate the diffusion of O2 to respiration bacteroids and buffer the free O2 concentration in the nodules at a very low tension to activate nitrogenase (Appleby, 1984). Therefore, in soybeans, lbs provides the solution to the simultaneous requirement for O2 in respiration processes that provide the large amount of energy needed to support nitrogen fixation and the exclusion of O2 to prevent inactivation of O2 -labile nitrogenase (O’Brain et al., 1987). Active soybean lbs are composed of a global leghemoglobin (lb) apoprotein and heme moiety. The lb apoprotein is encoded by the host plant (Appleby, 1984), while the heme moiety is primarily synthesized by symbiotic bacteria (Nadler and Avissar, 1977) and can also be synthesized in chloroplasts by host plants or green algae (Sangwan and O’Brain, 1991; Santana et al., 1998). Furthermore,
S. Wu et al. / Journal of Biotechnology 146 (2010) 120–125
chloroplasts are the site at which O2 and H2 photo-evolution occur simultaneously. Therefore, we hypothesized that expression of soybean lbs in the chloroplast of C. reinhardtii could affect O2 and H2 evolution. In this work, in order to make the two parts of lb binding easily to form active lb proteins after heteroexpression, the coding regions of both the hemH gene encoding ferrochelatase, the enzyme necessary for catalyzing the last step of heme synthesis from the symbiotic bacterium Bradyrhizobium japonicum (Frustaci and O’Brain, 1992) and the lba gene encoding one of lb apoproteins from host plant Glycine max were cloned as a fusion protein and transformed into the chloroplast of C. reinhardtii. The expression level of the fusion protein in chloroplast of C. reinhardtii and the amount of O2 and H2 in the headspace of the algal culture bottles were subsequently monitored. The data show that O2 content decreased and H2 production increased in these transgenic algal cultures compared with control cultures, thus demonstrating the potential for this method to improve the H2 yield of green algae. 2. Materials and methods 2.1. Materials and growth measurement C. reinhardtii strain, cc849 (a cell wall deficient mutant), was grown photoheterotrophically on TAP (Tris–acetate–phosphate, pH 7.0) agar plates or in TAP liquid medium under continuous illumination (∼100 mol photons m−2 s−1 ) by cool-white fluorescent light at 25 ± 1 ◦ C (Harris, 1989). Algal density was checked using a hemocytometer-type counting chamber or by measuring absorbance at 750 nm (OD750 ). Total algal chlorophyll was extracted using 95% ethanol and its content was assayed spectrophotometrically according to the method of Spreitzer (Harris, 1989). B. japonicum was grown in YEM (yeast extract–mannitol) liquid medium (pH 7.2) in the dark at 28 ± 1 ◦ C (Regensburger et al., 1986). Batch cultures were shaken at 200 rpm until the OD600 reached 0.6–0.8 (logarithmic phase) for 1% inoculation each time. 2.2. Sulfur deprivation For sulfur-free TAP medium (TAP-S), all sulfate compounds were replaced with chloride counterparts. To provide specific concentrations of sulfur in the TAP medium, sulfate was added back to the TAP-S cultures using 100 l aliquots of MgSO4 to achieve final concentrations of 12.5, 25, 50, and 100 M. Parallel additions of 100 l aliquots of distilled water were used for control cultures. 2.3. Long term O2 content and H2 production measurements Algae in the late exponential phase (when chlorophyll concentration was ∼25–28 g/ml) was harvested by centrifugation (3000 × g for 5 min) and washed with TAP-S medium three times to ensure the thorough removal of sulfur. Algae were resuspended in 40 ml of medium with a specific concentration of sulfate and a final concentration of ∼7.5 g Chl ml−1 . Algae suspensions were transferred to cylindrical glass bottles (having a total volume of 55 ml and a 3 cm diameter). The glass bottles were sealed with a rubber gas-tight septum and incubated in the dark for 24 h to induce anaerobic conditions in the liquid medium. Starting on the 2nd day, sealed bottles were placed under continuous illumination of 50 E ml−2 s−1 PAR (on the outer surface of the bottle). Evolved gas was collected from the headspace of the bottle using a gas-tight lockable syringe and injected into a gas chromatograph (AgilentTM 7890) with a thermal conductivity detector in order to monitor concentrations of H2 , O2 , and N2 simultaneously. A 5 Å Molecular Sieve column (2 m × 1/8 mm) was used and argon was the carrier gas.
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2.4. hemH cloning and chloroplast transformation The cloning of hemH from B. japonicum (GenBank accession no. M92427) was previously described (Frustaci and O’Brain, 1992). The hemH gene fragment was cloned into the chloroplast transformation plasmid, cg401-1-lba, which was previously constructed in our laboratory (Yan et al., 2009), using a SacII site to create cg4011-lba-hemH. In addition to the lba (GenBank accession no. V00453) and hemH genes, the plasmid contained the aadA gene for screening and endogenous chloroplast fragments of cpDNA-1 and cpDNA-2 for homologous replacement after transformation (Fig. 1) (Vaistij et al., 2000). 2.5. Chloroplast transformation and transformant screening Biolistic transformation on synchronously dividing algal cells was performed as described by Boynton et al. (1988) using a helium-driven particle gun (Bio-RadTM , Model: PDS1000/He Biolistic particle delivery system). Following transformation, algae were grown on normal TAP plates in the dark for 18 h, washed once with fresh TAP medium, and cultured on TAP plates complemented with 100 g ml−1 Spec under continuous illumination for ∼15 days to screen resistance transformants (Yan et al., 2009). After selection, transformants were subcultured on Spec-TAP plates for at least 6 passages in order to obtain homoplasmicity. 2.6. DNA and RNA extraction, cDNA synthesis, and PCR analysis DNA extraction from C. reinhardtii was performed according to Rochaix and Erickson (1988). Total RNA was extracted from 1 to 2 ml of C. reinhardtii cultures using a QIAGEN Plant Mini Kit. The concentration and purity of extracted RNA was measured using a UV spectrophotometer (EppendorfTM Bio Photometer). First strand cDNAs were synthesized from 2 g of DNA-digested, total RNA according to the reverse-transcription protocol provided by the manufacturer (PromegaTM ). PCR was used to confirm the integration of the hemH and lba genes into the chloroplast DNA of C. reinhardtii in Spec-resistant transformants. Primers used included cpDNA-F (5 -aga cag cca aca ttt tgt ta-3 ) and cpDNA-B (5 -gct tca aaa aca aaa tca aa-3 ). 2.7. Crude protein extraction and Western blot analysis Algal crude protein was extracted according to the method described by Hemschemeier et al. (2008). 100 g of total protein was mixed with loading buffer, heated at 100 ◦ C for 5 min and loaded onto 12% separating gels for SDS polyacrylamidegelelectrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF, Millipore, USA) membrane and the hemH-lba fusion protein was detected using polyclonal anti-hemH and anti-lba antibodies (made by Shanghai Immune Biotech Co., Ltd., China), respectively. Binding of antibodies was visualized using chemiluminescence detected by Amersham ECL Plus Western Blotting Detection System (GE Healthcare, UK) (Sambrook et al., 2001). 3. Results 3.1. Identification of transformants and hemH expression in chloroplasts A total of 3 single algal colonies with Spec-resistance were isolated, representing a transformation efficiency of 0.00075%. After 6 cycles of sub-culturing on Spec-containing TAP plates, total DNA from each transgenic algae was extracted and confirmed to contain both hemH and lba genes in the algal chloroplast DNA using PCR detection. In strain 849 (wild type, WT), a single band of 5400 bp
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Fig. 1. Schematic representation of plasmid, cg401-1-lba-hemH, used for chloroplast transformation of Chlamydomonas reinhardtii.
Fig. 2. Identification of transgenic algae by PCR and reverse-transcription PCR. (A) PCR products amplified from total DNA using primers specific for chloroplast DNA. Lane 1: MM; lane 3: WT; lane 4: transgenic algae. (B) PCR products amplified from algae cDNA using primers specific for the hemH gene and lba gene, respectively. Lane 1: MM; lanes 3 and 5: WT; lane 2: hemH gene from transgenic algae; lane 4: lba gene from transgenic algae.
was amplified, representing the distance between the binding sites of the two primers on the chloroplast DNA of C. reinhardtii (Fig. 2A, lane 3). For the transgenic algae, two fragments were amplified. One fragment was 8229 bp, equal to the distance between the two specific primers when the aadA, hemH, and lba genes were recombined into the chloroplast DNA (Fig. 2A, lane 4), and the second fragment was 5400 bp, the same fragment length as that from WT chloroplast DNA. These results indicated that the hemH and lba genes were correctly integrated into the chloroplast DNA of the transgenic algae. Reverse-transcript PCR products of 1048 and 442 bp were amplified from the total RNA of the transgenic algae using primers specific for hemH (Fig. 2B, lane 2) and lba (Fig. 2B, lane 4), respectively. Sequencing further confirmed that the hemH and lba genes were correctly inserted without mutations. As a control, the same RT-PCR reaction was performed using WT RNA and no fragments were amplified with either set of hemH- and lba-specific primers (Fig. 2B, lanes 3 and 5), respectively. Protein extracts were collected from the transgenic algal cells and WT cells prior to sealing of the cultures (day 0), as well as on days 3, 5, 6, and 7 after cells were anaerobically induced in sulfur-free medium. Western blot analyses using hemH- and lbaspecific antibodies both detected a band of ∼64 kDa, consistent with the predicted size of the hemH-lba fused protein product (Fig. 3). Furthermore, immunodetection analyses demonstrated that the transgenic algae started to accumulate detectable levels of the hemH-lba fusion protein by day 3, with expression reaching its highest level on day 5, and then returning to undetectable levels by day 7 (Fig. 3). Protein extract from WT algae was used as a control and no signal was detected following incubation with hemH-
Fig. 3. Western blot analysis of protein extracts from WT and transgenic algae grown in sulfur-depleted medium. Five mls of cells were harvested at the indicated timepoints after cultures were sealed for anaerobic induction and protein extracts were collected. Each lane contains 100 g total protein and detection was performed using anti-hemH and anti-lba antibodies.
and lba-specific antibodies. 3.2. Growth of transgenic algae The growth and chlorophyll content of the transgenic algae were analyzed using spectrometry (Fig. 4). The growth of the transgenic algae reached its highest levels on 4 and 6 days after inoculation with an OD750 value of 2.8 (corresponding to 6.0–7.0 × 106 cells ml−1 ), which was slightly lower than the 3.2 OD750 value for WT cells (corresponding to 7.0–8.0 × 106 cells ml−1 ). For chlorophyll accumulation, transgenic algae contained 38 mg l−1 , while WT algae contained 33 mg l−1 . These results indicated that the growth of the transgenic algae cultures were slightly inhibited compared with the WT cultures, while the chlorophyll content of the two cultures was largely unaffected. 3.3. Reduced O2 content and increased H2 production are observed for the transgenic algae O2 consumption and H2 production were compared between WT and transgenic algal cultures maintained in TAP medium containing 0–100 M sulfate (Fig. 5). After incubation in the dark for 24 h, O2 content in the headspace of all the cultures began to decrease due to respiration consumption. By day 2, all of the cultures were transferred to incubation conditions including illumination. As a result, small amounts of H2 began to be detectable. Both the O2 consumption rate and the H2 yield for the transgenic algal cultures were higher than that for the WT cultures in all media (Fig. 5A, C, E, and G). Particularly in S-free medium (Fig. 5A), O2 content in the transgenic algal cultures decreased quickly to 4.6% by the 2nd day and then remained constant at 2.9% through day 5. In contrast, O2 content in the WT cultures decreased to 9.5% by the 2nd day and reached a minimum level of 3.7% on day 6. The rate of H2 production increased quickly in transgenic algae to reach a maximum level of 47.0 l mg−1 chl h−1 on day 5, which was 2.8 times higher than that of WT cultures on day 7 (16.7 l mg−1 chl h−1 ) (Fig. 5B). Accordingly, H2 yield in the transgenic algal cultures was 3.3 ml bottle−1 , four times higher than that for WT cultures (0.8 ml bottle−1 ) (Fig. 5A). When sulfate was added back into the sulfur-free medium of transgenic and WT algal cultures, O2 consumption and H2 yield decreased compared to parallel cultures maintained in sulfur-free
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Fig. 4. Growth curve (A) and chlorophyll content kinetics (B) of WT and hemH-lba transgenic C. reinhardtii over 6 days of anaerobic induction culturing. All data are the mean of 3 independent experiments with duplicates or replicates performed for each experiment. The variation in assay values was generally less than 2%.
medium. Furthermore, the decreases detected were directly related to the relative increases in sulfate in the medium (Fig. 5C, E, and G). For example, at sulfate concentrations of 12.5, 25, and 50 M, O2 content on the 2nd day was 5.1, 8.9, and 15.0%, respectively, in the transgenic algal cultures, and 10.1, 11.9, and 18.2%, respectively, in the WT cultures. The lowest levels of O2 detected for the trans-
genic algal cultures were 3.1, 4.1, and 4.3%, respectively, which were much lower than the O2 content detected in the corresponding WT cultures, 10.1, 11.9, and 11.2%, respectively. Furthermore, O2 content in the WT cultures increased rapidly when illumination and sulfur-containing medium were provided. However, in transgenic cultures, this same phenomenon only occurred when the sulfate
Fig. 5. Comparison of H2 production, O2 consumption, and specific H2 evolution rates between WT and hemH-lba transgenic C. reinhardtii in TAP medium containing a final sulfate concentration of 0, 12.5, 25, and 50 M maintained under anaerobic induction conditions for 8 days. The data represent the means of three independent. Variation in assay values was generally less than 10%.
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content of these cultures was >25 M. For H2 yields in the presence of sulfate, transgenic cultures contained 1.2, 0.68, and 0.41 ml H2 per bottle, respectively. These H2 yields were higher than those of the WT cultures, 0.46, 0.47, and 0.22 ml H2 per bottle, respectively. At 100 M sulfate, H2 yield decreased rapidly to less than 0.1 ml per bottle in both transgenic and WT algal cultures due to the high O2 content of the cultures (data not shown). 4. Discussion A fused hemH-lba gene cassette was transferred and expressed in chloroplast of C. reinhardtii. As a result, a more rapid consumption of O2 was detected in the transgenic algal cultures than in the control cultures in the presence and absence of sulfur. Consequently, H2 yields for the transgenic algal cultures were higher than that for the control cultures in both sulfur-free and sulfurcontaining medium (Fig. 5). H2 yields in transgenic algal cultures increased as much as 4-fold in sulfur-free medium, which corresponded with the highest expression levels of the hemH-lba fusion protein (Fig. 3). Furthermore, transgenic cultures had the highest H2 production rate and lowest O2 content (Fig. 5A and B). These results indicate that expression of the hemH-lba fusion protein in chloroplast of C. reinhardtii played a role in reducing O2 content and improving H2 production in the presence or absence of sulfur. The amount of hemH-lba fusion proteins decreased significantly after 6 days of anaerobic incubation (Fig. 3) might be due to the protein degradation under anaerobic stresses for such a so long time. However, H2 ase is rather stable under anaerobic conditions (Forestier et al., 2003). Therefore, the hydrogen production of the algae still continued until 7 days (Fig. 5). Sulfur content in the culture medium of C. reinhardtii has been shown to affect the rate of O2 evolution and the supply of electrons by its effect on PS II activity, thereby influencing photo-O2 evolution and H2 production (Antal et al., 2003; Melis, 2007). Some reports have shown that the addition of sulfate at the beginning of the sulfur deprivation process improved electron transport from PS II to H2 ase thereby increasing the production rate and total yield of H2 (Zhang et al., 2002; Kosourov et al., 2002). However, in the current study, the highest H2 yield was achieved in sulfur-free medium, with H2 yields gradually decreasing with increasing concentrations of sulfate (12.5–100 M) in both transgenic and control cultures (Fig. 5A, C, E, and G). In considering the potential industrial application of this technique in the future, the anaerobic induction procedure was simplified in this work so that air in the headspace of the sealed glass bottles was not replaced with argon gas. Therefore, 15 ml of air was sealed in the headspace of the glass bottles, which allowed O2 to diffuse into the liquid culture gradually and constantly to affect the O2 content in the liquid medium during the anaerobic incubation of both sulfur-free and sulfur-containing cultures. Therefore, even in sulfur-free medium, there was still 4.6% O2 after 24 h of incubation in the absence of light, and 2.9% O2 after several days of anaerobic incubation. This is one possible reason for the lower H2 productivity associated with these experiments compared with other reports. Another reason might be the low expression level of the fusion protein in the transgenic algae (Fig. 3) and partial integration of the hemH-lba fused gene into chloroplast DNA was detected (Fig. 2A). The third possibility might be a limited resource of heme synthesis precursors in algal cells that could inhibit heme formation in the transgenic algae. For example, usually the major tetrapyrrole synthesized in plants and green algae is chlorophyll, which may be up to 2.5 mol g−1 fresh weight. The level of other tetrapyrroles such as siroheme, phytochromobilin, and heme is much lower, with an estimated 2 nmol g−1 fresh weight for mitochondrial heme (Santana et al., 1998). Other considerations include post-transcriptional regulation, differences in
codon usage between soybean and algae, low expression by symbiotic bacterium and alga, or reduced ferrochelatase activity. Due to the low heme levels present in the transgenic algae, ferrochelatase activity and heme quantities could not be measured exactly. However, optimization of the expression of this fusion protein in the chloroplast of C. reinhardtii is ongoing in our laboratory. We also transferred the lba gene, independent of the hemH gene, into chloroplast of C. reinhardtii. In this case, decreased O2 content and improved H2 productivity were also observed (unpublished data), indicating that expression of lb apoprotein could also function as an O2 -transporter in C. reinhardtii cells. Therefore, if the expression level of lb proteins in chloroplast of C. reinhardtii was improved using other biological techniques or culture optimization methods, the potential for improved sustainable and solar-driven bio-H2 production in green algae would be demonstrated. Acknowledgments We thank Professor Mark O’Brain (the State University of New York at Buffalo) for his consultation and advice and Professor Michel Goldschmidt-Clermont (Geneva University) for kindly providing plasmid cg40. This work was supported by grants from the Shanghai Pujiang Intelligent Program (No. 06PJ14075), the Innovation Program of Shanghai Municipal Education Commission (No. 08YE66), the Key Fundamental Project of Shanghai (No. 06JC14091), the Leading Academic Discipline Project of Shanghai Municipal Education Commission (No. J50401) and the Leading Academic Discipline Project of Shanghai Normal University (No. DZL808). References Antal, T.K., Krendeleva, T.E., Laurinavichene, T.V., Makarova, V.V., Ghirardi, M.L., Rubin, A.B., Tsygankov, A.A., Seibert, M., 2003. The dependence of algal H2 production on photosystem II and O2 consumption activities in sulfurdeprived Chlamydomonas reinhardtii cells. Biochim. Biophys. Acta 1607, 153–160. Appleby, C.A., 1984. Leghemoglobin and Rhizobium respiration. Annu. Rev. Plant Physiol. 35, 443–478. Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., Sanford, J.C., 1988. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240, 1534–1538. Florin, L., Tsokoglou, A., Happe, T., 2001. A novel type of iron hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain. J. Biol. Chem. 276, 6125–6132. Forestier, M., King, P., Zhang, L.P., Posewitz, M., Schwarzer, S., Happe, T., Ghirardi, M.L., Seibert, M., 2003. Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions. Eur. J. Biochem. 270, 2750–2758. Frustaci, J.M., O’Brain, M.R., 1992. Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene. J. Bacteriol. 174, 4223–4229. Ghirardi, M.L., Togasaki, R.K., Seibert, M., 1997. Oxygen sensitivity of algal H2 production. Appl. Biochem. Biotechnol. 63, 141–151. Ghirardi, M.L., Zhang, L., Lee, J.W., Flynn, T., Seibert, M., Greenbaum, E., Melis, A., 2000. Microalgea: a green source of renewable H2 . Trends Biotechnol. 18, 506–511. Happe, T., Naber, J.D., 1993. Isolation, characterization, and N-terminal amino acid sequence of hydrogen from the green algae Chlamydomonas reinhardtii. Eur. J. Biochem. 214, 475–481. Happe, T., Mosler, B., Naber, J.D., 1994. Induction, localization and metal content of hydrogenase in the green alga Chlamydomonas reinhardtii. Eur. J. Biochem. 222, 769–774. Harris, E.H., 1989. The Chlamydomonas Sourcebook: a comprehensive guide to biology and laboratory use. Academic Press, Inc., City, New York. Hemschemeier, A., Fouchard, S., Cournac, L., Peltier, G., Happe, T., 2008. Hydrogen production by Chlamydomonas reinhardtii: an elaborate interplay of electron sources and sinks. Planta 227, 397–407. Kosourov, S., Tsygankov, A., Seibert, M., Ghirardi, M.L., 2002. Sustained hydrogen photoproduction by Chlamydomonas reinhardtii: effects of culture parameters. Biotechnol. Bioeng. 78, 731–740. Melis, A., 2007. Photosynthetic H2 metabolism in Chlamydomonas reinhardtii. Planta 226, 1075–1086. Melis, A., Zhang, L., Forestier, M., Ghirardi, M.L., Seibert, M., 2000. Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol. 122, 127–136.
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Improved hydrogen production with expression of hemH and lba genes in chloroplast of Chlamydomonas reinhardtii Shuangxiu Wu ∗ , Rui Huang, Lili Xu, Guangyu Yan, Quanxi Wang Department of Biology, College of Life and Environmental Science, Shanghai Normal University, Guilin Road 100, Shanghai City 200234, China
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Article history: Received 8 November 2009 Received in revised form 22 January 2010 Accepted 29 January 2010
Keywords: Chlamydomonas reinhardtii Leghemoglobin Ferrochelatase Bio-hydrogen production Chloroplast transformation
a b s t r a c t The coding region of both the ferrochelatase gene, hemH, from Bradyrhizobium japonicum, and the leghemoglobin gene, lba, from Glycine max, were transferred into chloroplast of Chlamydomonas reinhardtii. As a result, transgenic C. reinhardtii cultures more rapidly consumed O2 and increased H2 output compared with controls in both sulfur-free and sulfur-containing medium. H2 production of the transgenic algal cultures in sulfur-free medium was 4-fold greater than that of control cultures, ∼3.3 ml bottle−1 . Maximum expression of the hemH-lba fusion protein on day 5 coincided with the lowest levels of O2 content and the highest H2 evolution rate detected over 7 days of anaerobic induction in sulfur-free medium. When the concentration of sulfate in the growth medium was restored to 12.5 or 50 M, O2 consumption and H2 yield decreased more slowly in the transgenic algal cultures than in the control cultures. These results demonstrate that expression of the hemH-lba fusion protein in chloroplast of C. reinhardtii improved their H2 yield by decreasing O2 content in the medium, thereby representing the potential for H2 production in green algae to be improved. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Many photosynthetic green algae, including Chlamydomonas reinhardtii, have been shown to metabolize H2 under anaerobic conditions (Melis, 2007). Under anoxic conditions, expression of [Fe]-hydrogenase (H2 ase) is induced and imported into the chloroplast stroma of green algae to catalyze the production of H+ and e− to form H2 (Happe et al., 1994; Forestier et al., 2003). The H2 ase found in C. reinhardtii has a high specific activity that is between 10- and 100-fold higher than any other H2 ase (Happe and Naber, 1993; Florin et al., 2001), which represents the significant potential for this algae to be developed for a sustainable, clean, solar-powered H2 production system in the future (Ghirardi et al., 2000). However, H2 ase is inactivated by trace amounts of O2 which is concomitantly produced during photosynthesis (Ghirardi et al., 1997). Therefore, light-dependent H2 production by C. reinhardtii is generally only observed up to several minutes after anaerobic induction. This apparent incompatibility of simultaneous O2 and H2 production during the photosynthesis of green algae has inhibited the application of H2 production by C. reinhardtii. Till
Abbreviations: PSII, photosystem II; Spec, spectinomycin; WT, wild type; lb, leghemoglobin; S, sulfur; ORF, open reading frame; ALA, ␦-aminolevulinic acid; Fe, ferro; MM, molecular marker. ∗ Corresponding author. Tel.: +86 21 64322526; fax: +86 21 64322142. E-mail address: [email protected] (S. Wu). 0168-1656/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2010.01.023
2000, Melis and coworkers demonstrated that depletion of sulfur in the culture medium of green algae affected photosystem II (PS II) to dramatically decrease O2 production. As a result, a significant improvement in H2 ase activity and prolonged H2 production over 4–5 days was achieved (Woykoff et al., 1998; Melis et al., 2000). However, H2 productivity by this method was much lower than the predicted capacity for H2 production due to the depletion of electron resources for PS II as a result of sulfur depletion (Melis, 2007; Hemschemeier et al., 2008). Nitrogenase is an enzyme in soybean leguminous nodules that is similar to H2 ase, is very sensitive to O2 but has high nitrogen fixation efficiency under ambient conditions. These characteristics are related to the abundance of leghemoglobins (lbs) that exit in the soybean root nodules to facilitate the diffusion of O2 to respiration bacteroids and buffer the free O2 concentration in the nodules at a very low tension to activate nitrogenase (Appleby, 1984). Therefore, in soybeans, lbs provides the solution to the simultaneous requirement for O2 in respiration processes that provide the large amount of energy needed to support nitrogen fixation and the exclusion of O2 to prevent inactivation of O2 -labile nitrogenase (O’Brain et al., 1987). Active soybean lbs are composed of a global leghemoglobin (lb) apoprotein and heme moiety. The lb apoprotein is encoded by the host plant (Appleby, 1984), while the heme moiety is primarily synthesized by symbiotic bacteria (Nadler and Avissar, 1977) and can also be synthesized in chloroplasts by host plants or green algae (Sangwan and O’Brain, 1991; Santana et al., 1998). Furthermore,
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chloroplasts are the site at which O2 and H2 photo-evolution occur simultaneously. Therefore, we hypothesized that expression of soybean lbs in the chloroplast of C. reinhardtii could affect O2 and H2 evolution. In this work, in order to make the two parts of lb binding easily to form active lb proteins after heteroexpression, the coding regions of both the hemH gene encoding ferrochelatase, the enzyme necessary for catalyzing the last step of heme synthesis from the symbiotic bacterium Bradyrhizobium japonicum (Frustaci and O’Brain, 1992) and the lba gene encoding one of lb apoproteins from host plant Glycine max were cloned as a fusion protein and transformed into the chloroplast of C. reinhardtii. The expression level of the fusion protein in chloroplast of C. reinhardtii and the amount of O2 and H2 in the headspace of the algal culture bottles were subsequently monitored. The data show that O2 content decreased and H2 production increased in these transgenic algal cultures compared with control cultures, thus demonstrating the potential for this method to improve the H2 yield of green algae. 2. Materials and methods 2.1. Materials and growth measurement C. reinhardtii strain, cc849 (a cell wall deficient mutant), was grown photoheterotrophically on TAP (Tris–acetate–phosphate, pH 7.0) agar plates or in TAP liquid medium under continuous illumination (∼100 mol photons m−2 s−1 ) by cool-white fluorescent light at 25 ± 1 ◦ C (Harris, 1989). Algal density was checked using a hemocytometer-type counting chamber or by measuring absorbance at 750 nm (OD750 ). Total algal chlorophyll was extracted using 95% ethanol and its content was assayed spectrophotometrically according to the method of Spreitzer (Harris, 1989). B. japonicum was grown in YEM (yeast extract–mannitol) liquid medium (pH 7.2) in the dark at 28 ± 1 ◦ C (Regensburger et al., 1986). Batch cultures were shaken at 200 rpm until the OD600 reached 0.6–0.8 (logarithmic phase) for 1% inoculation each time. 2.2. Sulfur deprivation For sulfur-free TAP medium (TAP-S), all sulfate compounds were replaced with chloride counterparts. To provide specific concentrations of sulfur in the TAP medium, sulfate was added back to the TAP-S cultures using 100 l aliquots of MgSO4 to achieve final concentrations of 12.5, 25, 50, and 100 M. Parallel additions of 100 l aliquots of distilled water were used for control cultures. 2.3. Long term O2 content and H2 production measurements Algae in the late exponential phase (when chlorophyll concentration was ∼25–28 g/ml) was harvested by centrifugation (3000 × g for 5 min) and washed with TAP-S medium three times to ensure the thorough removal of sulfur. Algae were resuspended in 40 ml of medium with a specific concentration of sulfate and a final concentration of ∼7.5 g Chl ml−1 . Algae suspensions were transferred to cylindrical glass bottles (having a total volume of 55 ml and a 3 cm diameter). The glass bottles were sealed with a rubber gas-tight septum and incubated in the dark for 24 h to induce anaerobic conditions in the liquid medium. Starting on the 2nd day, sealed bottles were placed under continuous illumination of 50 E ml−2 s−1 PAR (on the outer surface of the bottle). Evolved gas was collected from the headspace of the bottle using a gas-tight lockable syringe and injected into a gas chromatograph (AgilentTM 7890) with a thermal conductivity detector in order to monitor concentrations of H2 , O2 , and N2 simultaneously. A 5 Å Molecular Sieve column (2 m × 1/8 mm) was used and argon was the carrier gas.
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2.4. hemH cloning and chloroplast transformation The cloning of hemH from B. japonicum (GenBank accession no. M92427) was previously described (Frustaci and O’Brain, 1992). The hemH gene fragment was cloned into the chloroplast transformation plasmid, cg401-1-lba, which was previously constructed in our laboratory (Yan et al., 2009), using a SacII site to create cg4011-lba-hemH. In addition to the lba (GenBank accession no. V00453) and hemH genes, the plasmid contained the aadA gene for screening and endogenous chloroplast fragments of cpDNA-1 and cpDNA-2 for homologous replacement after transformation (Fig. 1) (Vaistij et al., 2000). 2.5. Chloroplast transformation and transformant screening Biolistic transformation on synchronously dividing algal cells was performed as described by Boynton et al. (1988) using a helium-driven particle gun (Bio-RadTM , Model: PDS1000/He Biolistic particle delivery system). Following transformation, algae were grown on normal TAP plates in the dark for 18 h, washed once with fresh TAP medium, and cultured on TAP plates complemented with 100 g ml−1 Spec under continuous illumination for ∼15 days to screen resistance transformants (Yan et al., 2009). After selection, transformants were subcultured on Spec-TAP plates for at least 6 passages in order to obtain homoplasmicity. 2.6. DNA and RNA extraction, cDNA synthesis, and PCR analysis DNA extraction from C. reinhardtii was performed according to Rochaix and Erickson (1988). Total RNA was extracted from 1 to 2 ml of C. reinhardtii cultures using a QIAGEN Plant Mini Kit. The concentration and purity of extracted RNA was measured using a UV spectrophotometer (EppendorfTM Bio Photometer). First strand cDNAs were synthesized from 2 g of DNA-digested, total RNA according to the reverse-transcription protocol provided by the manufacturer (PromegaTM ). PCR was used to confirm the integration of the hemH and lba genes into the chloroplast DNA of C. reinhardtii in Spec-resistant transformants. Primers used included cpDNA-F (5 -aga cag cca aca ttt tgt ta-3 ) and cpDNA-B (5 -gct tca aaa aca aaa tca aa-3 ). 2.7. Crude protein extraction and Western blot analysis Algal crude protein was extracted according to the method described by Hemschemeier et al. (2008). 100 g of total protein was mixed with loading buffer, heated at 100 ◦ C for 5 min and loaded onto 12% separating gels for SDS polyacrylamidegelelectrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF, Millipore, USA) membrane and the hemH-lba fusion protein was detected using polyclonal anti-hemH and anti-lba antibodies (made by Shanghai Immune Biotech Co., Ltd., China), respectively. Binding of antibodies was visualized using chemiluminescence detected by Amersham ECL Plus Western Blotting Detection System (GE Healthcare, UK) (Sambrook et al., 2001). 3. Results 3.1. Identification of transformants and hemH expression in chloroplasts A total of 3 single algal colonies with Spec-resistance were isolated, representing a transformation efficiency of 0.00075%. After 6 cycles of sub-culturing on Spec-containing TAP plates, total DNA from each transgenic algae was extracted and confirmed to contain both hemH and lba genes in the algal chloroplast DNA using PCR detection. In strain 849 (wild type, WT), a single band of 5400 bp
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Fig. 1. Schematic representation of plasmid, cg401-1-lba-hemH, used for chloroplast transformation of Chlamydomonas reinhardtii.
Fig. 2. Identification of transgenic algae by PCR and reverse-transcription PCR. (A) PCR products amplified from total DNA using primers specific for chloroplast DNA. Lane 1: MM; lane 3: WT; lane 4: transgenic algae. (B) PCR products amplified from algae cDNA using primers specific for the hemH gene and lba gene, respectively. Lane 1: MM; lanes 3 and 5: WT; lane 2: hemH gene from transgenic algae; lane 4: lba gene from transgenic algae.
was amplified, representing the distance between the binding sites of the two primers on the chloroplast DNA of C. reinhardtii (Fig. 2A, lane 3). For the transgenic algae, two fragments were amplified. One fragment was 8229 bp, equal to the distance between the two specific primers when the aadA, hemH, and lba genes were recombined into the chloroplast DNA (Fig. 2A, lane 4), and the second fragment was 5400 bp, the same fragment length as that from WT chloroplast DNA. These results indicated that the hemH and lba genes were correctly integrated into the chloroplast DNA of the transgenic algae. Reverse-transcript PCR products of 1048 and 442 bp were amplified from the total RNA of the transgenic algae using primers specific for hemH (Fig. 2B, lane 2) and lba (Fig. 2B, lane 4), respectively. Sequencing further confirmed that the hemH and lba genes were correctly inserted without mutations. As a control, the same RT-PCR reaction was performed using WT RNA and no fragments were amplified with either set of hemH- and lba-specific primers (Fig. 2B, lanes 3 and 5), respectively. Protein extracts were collected from the transgenic algal cells and WT cells prior to sealing of the cultures (day 0), as well as on days 3, 5, 6, and 7 after cells were anaerobically induced in sulfur-free medium. Western blot analyses using hemH- and lbaspecific antibodies both detected a band of ∼64 kDa, consistent with the predicted size of the hemH-lba fused protein product (Fig. 3). Furthermore, immunodetection analyses demonstrated that the transgenic algae started to accumulate detectable levels of the hemH-lba fusion protein by day 3, with expression reaching its highest level on day 5, and then returning to undetectable levels by day 7 (Fig. 3). Protein extract from WT algae was used as a control and no signal was detected following incubation with hemH-
Fig. 3. Western blot analysis of protein extracts from WT and transgenic algae grown in sulfur-depleted medium. Five mls of cells were harvested at the indicated timepoints after cultures were sealed for anaerobic induction and protein extracts were collected. Each lane contains 100 g total protein and detection was performed using anti-hemH and anti-lba antibodies.
and lba-specific antibodies. 3.2. Growth of transgenic algae The growth and chlorophyll content of the transgenic algae were analyzed using spectrometry (Fig. 4). The growth of the transgenic algae reached its highest levels on 4 and 6 days after inoculation with an OD750 value of 2.8 (corresponding to 6.0–7.0 × 106 cells ml−1 ), which was slightly lower than the 3.2 OD750 value for WT cells (corresponding to 7.0–8.0 × 106 cells ml−1 ). For chlorophyll accumulation, transgenic algae contained 38 mg l−1 , while WT algae contained 33 mg l−1 . These results indicated that the growth of the transgenic algae cultures were slightly inhibited compared with the WT cultures, while the chlorophyll content of the two cultures was largely unaffected. 3.3. Reduced O2 content and increased H2 production are observed for the transgenic algae O2 consumption and H2 production were compared between WT and transgenic algal cultures maintained in TAP medium containing 0–100 M sulfate (Fig. 5). After incubation in the dark for 24 h, O2 content in the headspace of all the cultures began to decrease due to respiration consumption. By day 2, all of the cultures were transferred to incubation conditions including illumination. As a result, small amounts of H2 began to be detectable. Both the O2 consumption rate and the H2 yield for the transgenic algal cultures were higher than that for the WT cultures in all media (Fig. 5A, C, E, and G). Particularly in S-free medium (Fig. 5A), O2 content in the transgenic algal cultures decreased quickly to 4.6% by the 2nd day and then remained constant at 2.9% through day 5. In contrast, O2 content in the WT cultures decreased to 9.5% by the 2nd day and reached a minimum level of 3.7% on day 6. The rate of H2 production increased quickly in transgenic algae to reach a maximum level of 47.0 l mg−1 chl h−1 on day 5, which was 2.8 times higher than that of WT cultures on day 7 (16.7 l mg−1 chl h−1 ) (Fig. 5B). Accordingly, H2 yield in the transgenic algal cultures was 3.3 ml bottle−1 , four times higher than that for WT cultures (0.8 ml bottle−1 ) (Fig. 5A). When sulfate was added back into the sulfur-free medium of transgenic and WT algal cultures, O2 consumption and H2 yield decreased compared to parallel cultures maintained in sulfur-free
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Fig. 4. Growth curve (A) and chlorophyll content kinetics (B) of WT and hemH-lba transgenic C. reinhardtii over 6 days of anaerobic induction culturing. All data are the mean of 3 independent experiments with duplicates or replicates performed for each experiment. The variation in assay values was generally less than 2%.
medium. Furthermore, the decreases detected were directly related to the relative increases in sulfate in the medium (Fig. 5C, E, and G). For example, at sulfate concentrations of 12.5, 25, and 50 M, O2 content on the 2nd day was 5.1, 8.9, and 15.0%, respectively, in the transgenic algal cultures, and 10.1, 11.9, and 18.2%, respectively, in the WT cultures. The lowest levels of O2 detected for the trans-
genic algal cultures were 3.1, 4.1, and 4.3%, respectively, which were much lower than the O2 content detected in the corresponding WT cultures, 10.1, 11.9, and 11.2%, respectively. Furthermore, O2 content in the WT cultures increased rapidly when illumination and sulfur-containing medium were provided. However, in transgenic cultures, this same phenomenon only occurred when the sulfate
Fig. 5. Comparison of H2 production, O2 consumption, and specific H2 evolution rates between WT and hemH-lba transgenic C. reinhardtii in TAP medium containing a final sulfate concentration of 0, 12.5, 25, and 50 M maintained under anaerobic induction conditions for 8 days. The data represent the means of three independent. Variation in assay values was generally less than 10%.
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content of these cultures was >25 M. For H2 yields in the presence of sulfate, transgenic cultures contained 1.2, 0.68, and 0.41 ml H2 per bottle, respectively. These H2 yields were higher than those of the WT cultures, 0.46, 0.47, and 0.22 ml H2 per bottle, respectively. At 100 M sulfate, H2 yield decreased rapidly to less than 0.1 ml per bottle in both transgenic and WT algal cultures due to the high O2 content of the cultures (data not shown). 4. Discussion A fused hemH-lba gene cassette was transferred and expressed in chloroplast of C. reinhardtii. As a result, a more rapid consumption of O2 was detected in the transgenic algal cultures than in the control cultures in the presence and absence of sulfur. Consequently, H2 yields for the transgenic algal cultures were higher than that for the control cultures in both sulfur-free and sulfurcontaining medium (Fig. 5). H2 yields in transgenic algal cultures increased as much as 4-fold in sulfur-free medium, which corresponded with the highest expression levels of the hemH-lba fusion protein (Fig. 3). Furthermore, transgenic cultures had the highest H2 production rate and lowest O2 content (Fig. 5A and B). These results indicate that expression of the hemH-lba fusion protein in chloroplast of C. reinhardtii played a role in reducing O2 content and improving H2 production in the presence or absence of sulfur. The amount of hemH-lba fusion proteins decreased significantly after 6 days of anaerobic incubation (Fig. 3) might be due to the protein degradation under anaerobic stresses for such a so long time. However, H2 ase is rather stable under anaerobic conditions (Forestier et al., 2003). Therefore, the hydrogen production of the algae still continued until 7 days (Fig. 5). Sulfur content in the culture medium of C. reinhardtii has been shown to affect the rate of O2 evolution and the supply of electrons by its effect on PS II activity, thereby influencing photo-O2 evolution and H2 production (Antal et al., 2003; Melis, 2007). Some reports have shown that the addition of sulfate at the beginning of the sulfur deprivation process improved electron transport from PS II to H2 ase thereby increasing the production rate and total yield of H2 (Zhang et al., 2002; Kosourov et al., 2002). However, in the current study, the highest H2 yield was achieved in sulfur-free medium, with H2 yields gradually decreasing with increasing concentrations of sulfate (12.5–100 M) in both transgenic and control cultures (Fig. 5A, C, E, and G). In considering the potential industrial application of this technique in the future, the anaerobic induction procedure was simplified in this work so that air in the headspace of the sealed glass bottles was not replaced with argon gas. Therefore, 15 ml of air was sealed in the headspace of the glass bottles, which allowed O2 to diffuse into the liquid culture gradually and constantly to affect the O2 content in the liquid medium during the anaerobic incubation of both sulfur-free and sulfur-containing cultures. Therefore, even in sulfur-free medium, there was still 4.6% O2 after 24 h of incubation in the absence of light, and 2.9% O2 after several days of anaerobic incubation. This is one possible reason for the lower H2 productivity associated with these experiments compared with other reports. Another reason might be the low expression level of the fusion protein in the transgenic algae (Fig. 3) and partial integration of the hemH-lba fused gene into chloroplast DNA was detected (Fig. 2A). The third possibility might be a limited resource of heme synthesis precursors in algal cells that could inhibit heme formation in the transgenic algae. For example, usually the major tetrapyrrole synthesized in plants and green algae is chlorophyll, which may be up to 2.5 mol g−1 fresh weight. The level of other tetrapyrroles such as siroheme, phytochromobilin, and heme is much lower, with an estimated 2 nmol g−1 fresh weight for mitochondrial heme (Santana et al., 1998). Other considerations include post-transcriptional regulation, differences in
codon usage between soybean and algae, low expression by symbiotic bacterium and alga, or reduced ferrochelatase activity. Due to the low heme levels present in the transgenic algae, ferrochelatase activity and heme quantities could not be measured exactly. However, optimization of the expression of this fusion protein in the chloroplast of C. reinhardtii is ongoing in our laboratory. We also transferred the lba gene, independent of the hemH gene, into chloroplast of C. reinhardtii. In this case, decreased O2 content and improved H2 productivity were also observed (unpublished data), indicating that expression of lb apoprotein could also function as an O2 -transporter in C. reinhardtii cells. Therefore, if the expression level of lb proteins in chloroplast of C. reinhardtii was improved using other biological techniques or culture optimization methods, the potential for improved sustainable and solar-driven bio-H2 production in green algae would be demonstrated. Acknowledgments We thank Professor Mark O’Brain (the State University of New York at Buffalo) for his consultation and advice and Professor Michel Goldschmidt-Clermont (Geneva University) for kindly providing plasmid cg40. This work was supported by grants from the Shanghai Pujiang Intelligent Program (No. 06PJ14075), the Innovation Program of Shanghai Municipal Education Commission (No. 08YE66), the Key Fundamental Project of Shanghai (No. 06JC14091), the Leading Academic Discipline Project of Shanghai Municipal Education Commission (No. J50401) and the Leading Academic Discipline Project of Shanghai Normal University (No. DZL808). References Antal, T.K., Krendeleva, T.E., Laurinavichene, T.V., Makarova, V.V., Ghirardi, M.L., Rubin, A.B., Tsygankov, A.A., Seibert, M., 2003. The dependence of algal H2 production on photosystem II and O2 consumption activities in sulfurdeprived Chlamydomonas reinhardtii cells. Biochim. Biophys. Acta 1607, 153–160. Appleby, C.A., 1984. Leghemoglobin and Rhizobium respiration. Annu. Rev. Plant Physiol. 35, 443–478. Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., Sanford, J.C., 1988. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240, 1534–1538. Florin, L., Tsokoglou, A., Happe, T., 2001. A novel type of iron hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain. J. Biol. Chem. 276, 6125–6132. Forestier, M., King, P., Zhang, L.P., Posewitz, M., Schwarzer, S., Happe, T., Ghirardi, M.L., Seibert, M., 2003. Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions. Eur. J. Biochem. 270, 2750–2758. Frustaci, J.M., O’Brain, M.R., 1992. Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene. J. Bacteriol. 174, 4223–4229. Ghirardi, M.L., Togasaki, R.K., Seibert, M., 1997. Oxygen sensitivity of algal H2 production. Appl. Biochem. Biotechnol. 63, 141–151. Ghirardi, M.L., Zhang, L., Lee, J.W., Flynn, T., Seibert, M., Greenbaum, E., Melis, A., 2000. Microalgea: a green source of renewable H2 . Trends Biotechnol. 18, 506–511. Happe, T., Naber, J.D., 1993. Isolation, characterization, and N-terminal amino acid sequence of hydrogen from the green algae Chlamydomonas reinhardtii. Eur. J. Biochem. 214, 475–481. Happe, T., Mosler, B., Naber, J.D., 1994. Induction, localization and metal content of hydrogenase in the green alga Chlamydomonas reinhardtii. Eur. J. Biochem. 222, 769–774. Harris, E.H., 1989. The Chlamydomonas Sourcebook: a comprehensive guide to biology and laboratory use. Academic Press, Inc., City, New York. Hemschemeier, A., Fouchard, S., Cournac, L., Peltier, G., Happe, T., 2008. Hydrogen production by Chlamydomonas reinhardtii: an elaborate interplay of electron sources and sinks. Planta 227, 397–407. Kosourov, S., Tsygankov, A., Seibert, M., Ghirardi, M.L., 2002. Sustained hydrogen photoproduction by Chlamydomonas reinhardtii: effects of culture parameters. Biotechnol. Bioeng. 78, 731–740. Melis, A., 2007. Photosynthetic H2 metabolism in Chlamydomonas reinhardtii. Planta 226, 1075–1086. Melis, A., Zhang, L., Forestier, M., Ghirardi, M.L., Seibert, M., 2000. Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol. 122, 127–136.
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