莱茵衣藻与根瘤菌共培养提高产氢及其生理生态学机理
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摘要
生物质能源技术是解决当今世界能源危机、环境危机和粮食危机的重要途径之一。光合微藻制氢技术是未来清洁能源可持续生产的重要组成部分。莱茵衣藻(以下简称衣藻)因为氢化酶活性高、生长速度快、适应力强、培养成本低、遗传背景清晰和分子操作系统成熟,成为研究微藻光合制氢的模式物种。衣藻氢化酶对氧气极其敏感导致其产氢效率极低是制约衣藻产氢技术应用的瓶颈问题。目前国际上提高衣藻产氢效率的策略主要有：(1)抑制PSⅡ光解水放氧活性；(2)改造氢化酶结构提高耐氧性；(3)快速降低培养系统细胞内氧气；(4)基因工程手段构建高效产氢藻株；(5)调控培养条件；(6)以藻类作为有机底物与产氢细菌和光合细菌共培养。其中利用藻菌相互作用产氢的策略是一种环境友好的、且具有良好发展前景的清洁能源生产的新思路。
本课题组的前期工作曾发现将衣藻与环境中易污染细菌共培养可以加快共培养体系内的氧气消耗速度、提高产氢量；进一步将衣藻与日本慢生大豆根瘤菌(以下简称根瘤菌)混合共培养,发现可以显著提高衣藻产氢效率达3.5～17.0倍。为了深入了解根瘤菌促进衣藻产氢代谢的原因,本研究将不同的衣藻藻株与根瘤菌混合共培养,对培养条件进行了优化,对最佳产氢状态下共培养体系中藻菌分布格局、产氢量、耗氧量、氢化酶活性、细胞内外碳水化合物和有机酸等的动态变化进行了检测,取得的主要结果如下：
1.不同衣藻藻株与根瘤菌混合共培养,产氢量都明显提高,最大产氢量提高到约为200μmol·mg-1Chl～278μmol·mg-1Chl;但藻株不同,其最佳产氢条件并不相同：根瘤菌与转基因藻hemHc-lbac最优的产氢条件为30μE·m-2·s-1光照,菌藻体积比为1：80,氢气产量为最大值278μmol·mg-1Chl,是纯藻培养体系80μmol·mg-1Chl的3.5倍；根瘤菌与衣藻cc124共培养的最优条件为200μE·m-2·s-1光照,菌藻体积比为1：80,氢气产量最大值为272μmol·mg-1Chl,是纯藻培养体系16μmol·mg-1Chl的17.0倍；根瘤菌与衣藻cc503共培养的最优条件为200μE·m-2·s-1光照,菌藻体积比为1：20,氢气产量最大值为302μmol·mg-1Chl,是纯藻培养体系69μmol·mg-1Chl的4.4倍。
2.通过设置不同的对照组实验,揭示了根瘤菌与衣藻共培养系统中产氢的是衣藻,而根瘤菌不产生氢气。
3.根瘤菌与衣藻在共培养开始至产氢高峰期,根瘤菌聚集在衣藻细胞周围,有“团聚”现象,而且衣藻的细胞数与叶绿素含量和根瘤菌的细胞数都比未混合培养的对照组明显增加；在共培养的后期,衣藻细胞解体。表明在共培养过程中根瘤菌与衣藻之间产生了阶段性互惠共生。但在产氢高峰期之后,衣藻的生存受到严重胁迫,衣藻开始逐渐解体。
4.藻菌共培养体系中,其呼吸速率和氧气消耗速度都明显比未混合培养的对照组高,这是衣藻产氢量提高的直接原因。转基因藻hemHc-lbac与根瘤菌共培养产氢后的第4天呼吸速率为5.04μmolO2·mgChl-1·h-1,是纯衣藻体系3.0μmolO2·mgChl-1·h-1的1.4倍；转基因藻hemHc-lbac加入根瘤菌产氢后的溶氧值最小为0.57mg/L,而纯藻体系为3.16mg/L,是藻菌体系的5.5倍；衣藻cc124与根瘤菌共培养产氢后第2天呼吸速率为6.05μmolO2·mgChl-1·h-1,是纯衣藻体系5.12μmolO2·mgChl-1·h-1的1.2倍；衣藻cc124加入根瘤菌产氢后的溶氧值最小值为2.18mg/L,而纯藻体系为5.08mg/L,是藻菌体系的2.3倍；衣藻cc503与根瘤菌共培养产氢后第5天的呼吸速率为3.34μmolO2·mgChl-1·h-1,是纯衣藻体系1.26μmolO2·mgChl-1·h-1的2.6倍；衣藻cc503加入根瘤菌产氢后的溶氧值最小为0.47mg/L,而纯藻体系为7.34mg/L,是藻菌体系的15.6倍。
5.在藻菌共培养产氢体系中,衣藻氢化酶活性明显增加,佐证了混合培养体系中氧气消耗速度快是衣藻产氢量提高的重要原因之一。转基因藻hemHc-lbac与根瘤菌共培养产氢后体外氢化酶最高活性约为57.9nmo H2μgChl-1·h-1比未加入根瘤菌的体系提高1.1倍,体内氢化酶最高活性约为25.3nmol H2μgChl-1·h-1,是纯藻培养的1.5倍；衣藻cc124与根瘤菌共培养产氢后,体外氢化酶最高活性约为51.0nmol H2μgChl-1·h-1,比未加入根瘤菌的体系提高1.0倍,体内氢化酶最高活性约为13.0nmol H2μgChl-1·h-1,是纯藻培养体系的3.8倍；衣藻cc503与根瘤菌共培养产氢后,体外氢化酶最高活性约为62.8nmol H2μgChl-1·h-1,比未加入根瘤菌的体系提高2.4倍,体内氢化酶最高活性约为21.9nmol H2μgChl-1·h-1,是纯藻培养体系的2.1倍。
6.在藻菌共培养产氢体系中,衣藻细胞内淀粉含量明显增加,这是导致藻菌共培养后衣藻产氢量提高的另一个原因：转基因藻hemHc-lbac与根瘤菌共培养产氢后,淀粉最高含量为7.23μg/ml,是纯衣藻体系淀粉含量0.87μg/ml的8.3倍；衣藻cc124与根瘤菌共培养产氢后,淀粉最高含量为13.01gg/ml,是纯衣藻体系淀粉含量1.55μg/ml的8.4倍；衣藻cc503与根瘤菌共培养产氢后,淀粉最高含量为13.99μg/ml,是纯衣藻体系淀粉含量3.18μg/ml的4.4倍。
7.加入根瘤菌后,三种衣藻藻株培养体系内的产氢代谢物的变化是不同的,表明不同藻株的产氢代谢有所差别,也是不同藻株与根瘤菌混合培养的最佳产氢条件不同的原因之一。如：三种衣藻藻株培养体系加入根瘤菌后,与未加入根瘤菌的纯衣藻对照体系相比,培养基中的乙酸含量都逐渐降低；但在转基因藻hemHc-lbac和衣藻cc503的体系中加入根瘤菌后,葡萄糖含量随着时间的增加而降低,最后达到最低值,分别为55μg/ml和119μg/ml,而在衣藻cc124的体系内加入根瘤菌后,葡萄糖含量却逐渐增加,最后达到最高值340μg/ml;在加入根瘤菌后,上述三种衣藻藻株的培养体系内的甲酸和乙醇含量都增加,最后甲酸达到最高值依次分别为261.16μg/ml.307.12μg/ml和393.80μg/ml,乙醇的最高值分别为281μg/ml.213μg/ml和150μg/ml。
综上所述：根瘤菌与不同衣藻藻株共培养都能显著促进衣藻产氢,表明衣藻与根瘤菌共培养提高产氢的现象具有普遍性；从共培养的开始至产氢高峰阶段,根瘤菌向衣藻聚集生长、形成“团聚物”,且二者的生长都得到促进,细胞内淀粉含量也增加,表明二者间存在阶段性互惠共生现象；本研究还揭示了在根瘤菌与衣藻共培养系统中,只有衣藻产氢,而根瘤菌不产氢；二者共培养使得体系内耗氧速度加快,是根瘤菌促进衣藻产氢的主要原因；其次,共培养体系内二者细胞数增加和淀粉含量增加是导致衣藻产氢增加的另一个重要原因。本研究首次从生理生态学角度揭示了根瘤菌与衣藻共培养过程中二者的相互作用关系和产氢量提高的原因,为进一步深入开展衣藻产氢的代谢调控、拓展根瘤菌的寄主范围以及利用基因工程手段构建高效产氢藻株等研究奠定了理论和实验基础。
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.
本课题组的前期工作曾发现将衣藻与环境中易污染细菌共培养可以加快共培养体系内的氧气消耗速度、提高产氢量；进一步将衣藻与日本慢生大豆根瘤菌(以下简称根瘤菌)混合共培养,发现可以显著提高衣藻产氢效率达3.5～17.0倍。为了深入了解根瘤菌促进衣藻产氢代谢的原因,本研究将不同的衣藻藻株与根瘤菌混合共培养,对培养条件进行了优化,对最佳产氢状态下共培养体系中藻菌分布格局、产氢量、耗氧量、氢化酶活性、细胞内外碳水化合物和有机酸等的动态变化进行了检测,取得的主要结果如下：
1.不同衣藻藻株与根瘤菌混合共培养,产氢量都明显提高,最大产氢量提高到约为200μmol·mg-1Chl～278μmol·mg-1Chl;但藻株不同,其最佳产氢条件并不相同：根瘤菌与转基因藻hemHc-lbac最优的产氢条件为30μE·m-2·s-1光照,菌藻体积比为1：80,氢气产量为最大值278μmol·mg-1Chl,是纯藻培养体系80μmol·mg-1Chl的3.5倍；根瘤菌与衣藻cc124共培养的最优条件为200μE·m-2·s-1光照,菌藻体积比为1：80,氢气产量最大值为272μmol·mg-1Chl,是纯藻培养体系16μmol·mg-1Chl的17.0倍；根瘤菌与衣藻cc503共培养的最优条件为200μE·m-2·s-1光照,菌藻体积比为1：20,氢气产量最大值为302μmol·mg-1Chl,是纯藻培养体系69μmol·mg-1Chl的4.4倍。
2.通过设置不同的对照组实验,揭示了根瘤菌与衣藻共培养系统中产氢的是衣藻,而根瘤菌不产生氢气。
3.根瘤菌与衣藻在共培养开始至产氢高峰期,根瘤菌聚集在衣藻细胞周围,有“团聚”现象,而且衣藻的细胞数与叶绿素含量和根瘤菌的细胞数都比未混合培养的对照组明显增加；在共培养的后期,衣藻细胞解体。表明在共培养过程中根瘤菌与衣藻之间产生了阶段性互惠共生。但在产氢高峰期之后,衣藻的生存受到严重胁迫,衣藻开始逐渐解体。
4.藻菌共培养体系中,其呼吸速率和氧气消耗速度都明显比未混合培养的对照组高,这是衣藻产氢量提高的直接原因。转基因藻hemHc-lbac与根瘤菌共培养产氢后的第4天呼吸速率为5.04μmolO2·mgChl-1·h-1,是纯衣藻体系3.0μmolO2·mgChl-1·h-1的1.4倍；转基因藻hemHc-lbac加入根瘤菌产氢后的溶氧值最小为0.57mg/L,而纯藻体系为3.16mg/L,是藻菌体系的5.5倍；衣藻cc124与根瘤菌共培养产氢后第2天呼吸速率为6.05μmolO2·mgChl-1·h-1,是纯衣藻体系5.12μmolO2·mgChl-1·h-1的1.2倍；衣藻cc124加入根瘤菌产氢后的溶氧值最小值为2.18mg/L,而纯藻体系为5.08mg/L,是藻菌体系的2.3倍；衣藻cc503与根瘤菌共培养产氢后第5天的呼吸速率为3.34μmolO2·mgChl-1·h-1,是纯衣藻体系1.26μmolO2·mgChl-1·h-1的2.6倍；衣藻cc503加入根瘤菌产氢后的溶氧值最小为0.47mg/L,而纯藻体系为7.34mg/L,是藻菌体系的15.6倍。
5.在藻菌共培养产氢体系中,衣藻氢化酶活性明显增加,佐证了混合培养体系中氧气消耗速度快是衣藻产氢量提高的重要原因之一。转基因藻hemHc-lbac与根瘤菌共培养产氢后体外氢化酶最高活性约为57.9nmo H2μgChl-1·h-1比未加入根瘤菌的体系提高1.1倍,体内氢化酶最高活性约为25.3nmol H2μgChl-1·h-1,是纯藻培养的1.5倍；衣藻cc124与根瘤菌共培养产氢后,体外氢化酶最高活性约为51.0nmol H2μgChl-1·h-1,比未加入根瘤菌的体系提高1.0倍,体内氢化酶最高活性约为13.0nmol H2μgChl-1·h-1,是纯藻培养体系的3.8倍；衣藻cc503与根瘤菌共培养产氢后,体外氢化酶最高活性约为62.8nmol H2μgChl-1·h-1,比未加入根瘤菌的体系提高2.4倍,体内氢化酶最高活性约为21.9nmol H2μgChl-1·h-1,是纯藻培养体系的2.1倍。
6.在藻菌共培养产氢体系中,衣藻细胞内淀粉含量明显增加,这是导致藻菌共培养后衣藻产氢量提高的另一个原因：转基因藻hemHc-lbac与根瘤菌共培养产氢后,淀粉最高含量为7.23μg/ml,是纯衣藻体系淀粉含量0.87μg/ml的8.3倍；衣藻cc124与根瘤菌共培养产氢后,淀粉最高含量为13.01gg/ml,是纯衣藻体系淀粉含量1.55μg/ml的8.4倍；衣藻cc503与根瘤菌共培养产氢后,淀粉最高含量为13.99μg/ml,是纯衣藻体系淀粉含量3.18μg/ml的4.4倍。
7.加入根瘤菌后,三种衣藻藻株培养体系内的产氢代谢物的变化是不同的,表明不同藻株的产氢代谢有所差别,也是不同藻株与根瘤菌混合培养的最佳产氢条件不同的原因之一。如：三种衣藻藻株培养体系加入根瘤菌后,与未加入根瘤菌的纯衣藻对照体系相比,培养基中的乙酸含量都逐渐降低；但在转基因藻hemHc-lbac和衣藻cc503的体系中加入根瘤菌后,葡萄糖含量随着时间的增加而降低,最后达到最低值,分别为55μg/ml和119μg/ml,而在衣藻cc124的体系内加入根瘤菌后,葡萄糖含量却逐渐增加,最后达到最高值340μg/ml;在加入根瘤菌后,上述三种衣藻藻株的培养体系内的甲酸和乙醇含量都增加,最后甲酸达到最高值依次分别为261.16μg/ml.307.12μg/ml和393.80μg/ml,乙醇的最高值分别为281μg/ml.213μg/ml和150μg/ml。
综上所述：根瘤菌与不同衣藻藻株共培养都能显著促进衣藻产氢,表明衣藻与根瘤菌共培养提高产氢的现象具有普遍性；从共培养的开始至产氢高峰阶段,根瘤菌向衣藻聚集生长、形成“团聚物”,且二者的生长都得到促进,细胞内淀粉含量也增加,表明二者间存在阶段性互惠共生现象；本研究还揭示了在根瘤菌与衣藻共培养系统中,只有衣藻产氢,而根瘤菌不产氢；二者共培养使得体系内耗氧速度加快,是根瘤菌促进衣藻产氢的主要原因；其次,共培养体系内二者细胞数增加和淀粉含量增加是导致衣藻产氢增加的另一个重要原因。本研究首次从生理生态学角度揭示了根瘤菌与衣藻共培养过程中二者的相互作用关系和产氢量提高的原因,为进一步深入开展衣藻产氢的代谢调控、拓展根瘤菌的寄主范围以及利用基因工程手段构建高效产氢藻株等研究奠定了理论和实验基础。
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.
引文
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