| Biomass energy technology is one of the most important ways to solve the crises of energy, environment and food of the world. Photosynthetic microalgae hydrogen technology is an important component of sustainable production of clean energy in the future. Chlamydomonas reinhardtii, with high activity of hydrogenase, quick growth, strong adaptability, low cost in cultivation, clear molecular genetic background and mature molecular operation system, has been considered as the most potential and model algal species for development of biological hydrogen production. The low hydrogen production efficiency of C. reinhardtii resulted from the extreme sensitivity of its hydrogenase to oxygen is the bottleneck constraining its application in hydrogen production. Nowadays, there are several methods to improve hydrogen yield of C. reinhardtii:(1) inhibiting the activity of PSII for retarding the process of water photolysis;(2) modifying the structure of hydrogenase to increase its tolerance to oxygen;(3) quickly reducing both intracellular and cultivation medium’s oxygen concentration;(4) constructing high-hydrogen-yield algal strains by genetic engineering technology;(5) optimizing cultivation conditions;(6) co-culturing photosynthetic bacteria and hydrogen-producing bacteria by using algae as organic substrates, among which the strategy of hydrogen production through algae-bacteria interaction is a promising and environment friendly way for clean energy production.In our former work, we find that co-cultivating C. reinhardtii with bacteria which is polluted easily in algal environments can increase oxygen consumption and improve hydrogen production. We further co-cultivated C. reinhardtii and Bradyrhizobium japonicum and resulted in the improvement of hydrogen production by3.5～17.0times. In this study to investigate the mechanism of B. japonicum promoting hydrogen yield of C. reinhardtii, we co-cultivated different C. reinhardtii strains with B. japonicum, optimized cultivation conditions and detected the distribution pattern of algae-bacteria systems, as well as the dynamic changes of hydrogen yield, oxygen consumption, hydrogenase activity, intracellular and medium’s carbohydrates and organic acids in the co-culture systems. The main results were as follows:1. Co-culturing different C. reinhardtii strains with B. japonicum all improved hydrogen yields to200μmol·mg-1Chl～278μmol·mg-1Chl; the optimal conditions of hydrogen production were different among different strains. When co-cultivating the transgenic alga hemHc-lbac with B. japonicum, the optimal hydrogen production condition was under30μE m-2s-1of light intensity and the algae-bacteria volume ratio at1:80, its maximal hydrogen yield was278μmol·mg-1Chl, approximately3.5times of that of the control,80μmolmg·Chl. When co-cultivating C. reinhardtii cc124with B. japonicum, the optimal hydrogen production condition was under200μE m-2s-1of light intensity and the algae-bacteria volume ratio at1:80, the maximal hydrogen yield was272μmol·mg-1Chl, approximately17.0times of that of the control,16μmol·mg-1Chl. When co-cultivating C. reinhardtii cc503with B. japonicum, the optimal hydrogen production condition was under200μE m-2s-1of light intensity and the algae-bacteria volume ratio at1:20, the maximal hydrogen yield was302μmol·mg-1Chl, approximately4.4times of that of the,69μmol·mg-1Chl.2. By setting various control experiments, we revealled that in the co-culture system, C. reinhardtii produced hydrogen but B. japonicum didn’t produce hydrogen.3. From the beginning of the co-cultivation to the stage of maximal hydrogen production, B. japonicum gathered around C. reinhardtii and formed assembly phenomenon. Meanwhile both the cell numbers and chlorophyll contents of C. reinhardtii and the cell numbers of B. japonicum were all obviously more than those of the controls, indicating the mutualistic relationship of C. reinhardtii and B. japonicum happened during this stage. But after that stage, C. reinhardtii disintegrated gradually, showing that C. reinhardtii suffered severe stresses.4. The respiratory rates and oxygen consumption of alage-bacteria co-culture system were higher than those of controls, which should be the direct reason enhancing the hydrogen yields. The respiratory rate of the co-culture system of the transgenic alga hemHc-lbac with B. japonicum was5.04μmolO2·mgChl-1·h-1at the 4th day after co-cultivation, which was1.4times of the control,3.03μmolO2·mgChl-1·h-1. The minimal dissolved oxygen of the co-culture system of transgenic alga hemHc-lbac with B. japonicum after co-cultivation was0.57mg/L, while that of the pure transgenic algal system was3.16mg/L, which was5.5times of that of the co-culture system. The respiratory rate of co-culture system of C. reinhardtii cc124with B. japonicum was6.05μmolO2·mgChl-1·h-1at the2nd day after co-cultivation, which was1.2times of that of the control,5.12umolO2·mgChl-1·h-1. The minimal dissolved oxygen of the pure C. reinhardtii cc124system was5.08mg/L, which was2.3times of that of the co-culture system,2.18mg/L. The respiratory rate of the co-culture system of C. reinhardtii cc503with B. japonicum was3.34μmolO2·mgChl-1·h-1at the5th day after co-cultivation, which was2.6times of that of the control,1.26μmolO2·mgChl-1·h-1. The minimal dissolved oxygen of the pure C. reinhardtii cc503was7.34mg/L, which was15.6times of that of the co-culture system,0.47mg/L.5. The hydrogenase activity greatly improved in the algae-bacteria co-culture system, showing that the quick oxygen consumption was the most important reason for the enhancement of hydrogen production. In vitro and in vivo maximal hydrogenase activity of the transgenic alga hemHc-lbac system and its co-culturing system with B. japonicum were57.9nmol H2μgChl-1·h-1and25.3nmol H2μgChl-1·h-1, which were1.1times and1.5times higher than those of the controls, respectively. In vitro and in vivo maximal hydrogenase activity of C. reinhardtii cc124and its co-culturing system with B. japonicum were51.0nmol H2μgChl-1·h-1and13.0nmol H2μgChl-1·h-1, which were1.0times and3.8times higher than those of the controls, respectively. In vitro and in vivo maximal hydrogenase activity of C. reinhardtii cc503and its co-culturing system with B. japonicum were62.8nmol H2μgChl-1·h-1and21.9nmol H2μgChl-1·h-1, which were2.4times and2.1times higher than those of the controls, respectively.6. In the algae-bacteria co-culture system, the starch contents in algal cells increased greatly, which was another main reason for the improvement of hydrogen production. The maximal starch content of the co-culture system of the transgenic alga hemHc-lbac with B. japonicum was7.23μg/ml, which was8.3times of that of the control,0.87μg/ml. The maximal starch content of the co-culture system of C. reinhardtii cc124with B. japonicum was13.01μg/ml, which was8.4times of that of the control,1.55μg/ml. The maximal starch content of the co-culture system of C. reinhardtii cc124with B. japonicum was13.99μg/ml, which was4.4times of that of the control,3.18μg/ml.7. When B. japonicum being added into the culture systems of the three strains of C. reinhardtii, the metabolites of them were different, which could explain the reasons why the optimal hydrogen production conditions were different among duifferent algal strains. In the co-culture systems of the three strains of C. reinhardtii with B. japonicum, the acetic acid contents in the mediums were all lower than those of the controls. However in the co-culture systems of the transgenic alga hemHc-lbac and C. reinhardtii cc503with B. japonicum, the contents of glucose all decreased along with the extension of co-cultivating time, finally reaching the minimum values at55μg/ml and119μg/ml, respectively. Whereas, in the co-culture systems of C. reinhardtii cc124with B. japonicum, the content of glucose increased, finally reaching the maximal value at340μg/ml. In the co-culture systems of the three strains of C. reinhardtii and B. japonicum in the above orders, the contents of formic acid and ethanol in the mediums increased gradually, finally reached to the maximal vlevels at261.16μg/ml,307.12μg/ml and393.80μg/ml (for formic acid), as well as281μg/ml,213μg/ml and150μg/ml (for ethanol), respectively.In summary, in all the co-culture systems of different strains of C. reinhardtii with B. japonicum, the hydrogen production increased, indicating that the enhancement of hydrogen production is a common phenonmenon when co-culturing C. reinhardtii with B. japonicum. From the beginning of the co-culture to the stage of maximal hydrogen production, B. japonicum gathered around C. reinhardtii and assembled. Meanwhile the algal and bacterial growths increased, as well as the starch contents increased compared with those of the controls, indicating that the staged mutualistic relationship between C. reinhardtii and B. japonicum existed. This study also revealed that when co-culturing C. reinhardtii and B. japonicum, only C. reinhardtii produced hydrogen, but not B. japonicum. In the co-culture systems, the oxygen consumption increased, which was the main reason for B. japonicum to improve the hydrogen yield. Moreover, that the algal cell numbers and starch cntents increased obviously in the co-culture systems was another main reason for B. japonicum to stimulate the hydrogen yield. This study for the first time provided important theoretical and experimental foundations from ecology-physiology angles to reveal the mutual interaction between C. reinhardtii and B. japonicum during the process of cu-cultivation and the reasons of B. japonicum to enhance C. reinhardtii’s hydrogen production. Out study provided important basis for further studies on metabolic regulations of hydrogen production of C. reinhardtii, expanding the host ranges of B. japonicum and constructing high-yield algal strains using genetic engineer technology. |
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