Elsevier

Bioresource Technology

Volume 123, November 2012, Pages 184-188
Bioresource Technology

Increased hydrogen production in co-culture of Chlamydomonas reinhardtii and Bradyrhizobium japonicum

https://doi.org/10.1016/j.biortech.2012.07.055Get rights and content

Abstract

Co-cultivation of Bradyrhizobium japonicum with Chlamydomonas reinhardtii strain cc849 or the transgenic strain lba, which was hetero-expressed the gene of the soybean leghemoglobin apoprotein Lba in chloroplasts of the strain cc849, in Tris–acetate-phosphate (TAP) or TAP-sulfur free media, improved H2 yield. H2 production was 14 times and growth was 26% higher when strain lba and B. japonicum were co-cultured, as compared with cultivation of the algal strain alone under the same conditions. The increase in respiration rate or fast O2 consumption by about 8 times in the co-cultures was the major reason for the improvement.

Highlights

► Decreasing the oxygen content in the cultures of Chlamydomonas reinhardtii is essential for its hydrogen productivity. ► Co-cultivation of Bradyrhizobium japonicum and C. reinhardtii promoted oxygen consumption and improved hydrogen yield. ► B. japonicum promoted H2 yield and growth of the transgenic alga, lba, by 14 times and 26% respectively.

Introduction

The renewable and environment-friendly generation of H2 in large quantities is a major challenge for the utilization of H2 as an energy resource because, at present, most H2 production depends on effective thermochemical and photoelectrochemical processes that may be highly energy-consuming and damaging to the environment (Sen et al., 2008). Therefore, photobiological H2 production by green microalgae is of interest (Esper et al., 2006).

H2 production in photosynthetic bacteria is mediated primarily by the nitrogenase system that requires high-energy input in the form of ATP (Taygankov et al., 1999). In contrast, H2 production in green algae is mediated primarily by the Fe-hydrogenase system in the absence of ATP-input by accepting electrons from ferredoxin (Fd), a terminal acceptor in the photosynthetic electron transport chain in chloroplast thyllakoids, to reduce protons (Kosourov and Seibert, 2008). Chlamydomonas reinhardtii has been chosen as model species for studying photohydrogen production because of its high Fe-hydrogenase activity, easy cultivation, and clear genetics (Melis, 2007). However, Fe-hydrogenase is highly oxygen sensitive (Forestier et al., 2003), while O2 is also the principal product of algal photosynthesis. Therefore, O2 generation by photosynthesis must be controlled upon illumination. The current control method relies on the use of a sulfur-deficient medium for the inactivation of O2 evolution at algal PSII (Photosystem II) under illumination and creation of anaerobic conditions (Melis et al., 2000) to prolong H2 production. However, the algal H2 productivity in such a system has not yet qualified for commercial viability since the stress of sulfur deprivation eventually leads to reduced ferredoxins oxidization, degradation of photosynthetic complexes and accumulation of toxic metabolites (Melis, 2007, Terauchi et al., 2009, Matthew et al., 2009).

Attempts to enhance H2 productivity in algae have included repeated cycles of light restriction and oxygen depletion with unaffected photosynthesis (Melis et al., 2000), control of photosynthesis by both restricted illumination, and genetic engineering to produce algae that have limited light harvesting mechanisms (Polle et al., 2003). Increasing starch reserves and inhibiting the cyclic electron flow around PSI as well as its respiration metabolism were also investigated (Kruse et al., 2005), as were the introduction of a glucose transporter (Rühle et al., 2008), diminishing sulfur uptake (Chen et al., 2005) and reducing oxygen sensitivity of hydrogenase (King et al., 2006). Co-cultivation of algae and bacteria has also been employed. For instance, Kawaguchi et al. (2001) grew the photosynthetic bacterium Rhodobium marinum along with Lactobacillus amylovorus and algal biomass for metabolizing the algal starch into lactate as an electron donor for hydrogen production by the bacteria. Another example is the co-culture of photosynthetic, hydrogen producing algae (wild type and genetically engineered for reduced sulfate utilization) with the hydrogen-producing bacterium, Rhodospirillium rubrum (Melis and Melnicki, 2006). Kim et al. (2006) also found that cultivation of algal biomass of C. reinhardtii with Clostridium butyricum and Rhodobacter sphaeroides KD131 under anaerobic and photosynthetic fermentation conditions improved H2 production. Similar results have been reported by Miura et al. (1992) and Ohmiya et al. (2003).

Bradyrhizobium japonicum is a symbiotic rhizobium of the soybean, Glycine max. Naturally, it infects the root of host plants and forms root nodules, which contain abundant leghemoglobins (lbs) that have high affinity for O2 and carry O2 to the respiration metabolism, resulting in a low O2 concentration and protection of the oxygen-sensitive nitrogenase activity in the nodules (Appleby, 1984, Kundu et al., 2003). When the lba gene from G. max, encoding the lb apoprotein, was introduced into C. reinhardtii and expressed in its chloroplasts (Wu et al., 2010), rapid O2 consumption and about 1.5-time H2 yield of the wild type, strain cc849 (stain 849) in sulfur-deficient medium were observed. In the present study, B. japonicum was co-cultured with the transgenic alga, lba, (transgenic lba) as well as strain 849 of C. reinhardtii at various inoculation ratios and H2 yields of the co-cultures were monitored.

Section snippets

Cultures and culture conditions

C. reinhardtii strain 849 is a cell wall-deficient mutant (a gift from Professor Dr. Madeline Wu in Hong Kong University of Science and Technology). The transgenic algal strain lba was engineered to express the soybean lba gene in its chloroplasts (Wu et al., 2010). Both algae were grown photoheterotrophically on TAP (Tris–acetate-phosphate, pH 7.0) agar plates or in TAP liquid media under continuous illumination (100 μmol photons m−2 s−1) with cool-white fluorescent light at 25 ± 1 °C. Batch cultures

Co-culture of algae and B. japonicum

Co-cultivation of bacteria and algae with a starting ratio of 1:15 (bacteria to algae) led to algal cell density and chlorophyll content of co-cultures of B. japonicum and the transgenic lba reached values of 3.9 × 107 ml−1 and 50 mg l−1., respectively, nearly 26% and 50% higher than those of single transgenic algal cultures which reached about 3.06 × 107 ml−1 and 33.4 mg l−1, respectively (Fig. 1A and B). Although the maximal chlorophyll content of the co-cultures of the strain 849 and B. japonicum was

Conclusions

Co-cultivating C. reinhardtii, with B. japonicum increased H2 production, perhaps by an increased respiration rate or fast O2 consumption and enhanced growth. Further studies need to be carried out to better define the interactions between the algae and the bacteria.

Acknowledgements

This work was supported by grants from the Key Project of Science and Technology Commission of Shanghai (No. 09160500400), NSCF31140070, the CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, the SOA Key Laboratory for Polar Science, Polar Research Institute of China (KP201107). We also thank the support from the Leading Academic Discipline Project of Shanghai Municipal Education Commission (No. J50401) and the Leading Academic

References (25)

  • C.A. Appleby

    Leghemoglobin and Rhizobium respiration

    Annu. Rev. Plant Physiol.

    (1984)
  • H. Chen et al.

    Role of SulP, a nuclear-encoded chloroplast sulfate permease, in sulfate transport and H2 evolution in Chlamydomonas reinhardtii

    Photosynth. Res.

    (2005)
  • Cited by (0)

    View full text