Rühle Thilo, Hemschemeier Anja, Melis Anastasios, Happe Thomas
Fakultät für Biologie und Biotechnologie, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
BMC Plant Biol. 2008 Oct 17;8:107. doi: 10.1186/1471-2229-8-107.
Sealed Chlamydomonas reinhardtii cultures evolve significant amounts of hydrogen gas under conditions of sulfur depletion. However, the eukaryotic green alga goes through drastic metabolic changes during this nutritional stress resulting in cell growth inhibition and eventually cell death. This study aimed at isolating C. reinhardtii transformants which produce hydrogen under normal growth conditions to allow a continuous hydrogen metabolism without the stressful impact of nutrient deprivation.
To achieve a steady photobiological hydrogen production, a screening protocol was designed to identify C. reinhardtii DNA insertional mutagenesis transformants with an attenuated photosynthesis to respiration capacity ratio (P/R ratio). The screening protocol entails a new and fast method for mutant strain selection altered in their oxygen production/consumption balance. Out of 9000 transformants, four strains with P/R ratios varying from virtually zero to three were isolated. Strain apr1 was found to have a slightly higher respiration rate and a significantly lower photosynthesis rate than the wild type. Sealed cultures of apr1 became anaerobic in normal growth medium (TAP) under moderate light conditions and induced [FeFe]-hydrogenase activity, yet without significant hydrogen gas evolution. However, Calvin-Benson cycle inactivation of anaerobically adapted apr1 cells in the light led to a 2-3-fold higher in vivo hydrogen production than previously reported for the sulfur-deprived C. reinhardtii wild type.
Attenuated P/R capacity ratio in microalgal mutants constitutes a platform for achieving steady state photobiological hydrogen production. Using this platform, algal hydrogen metabolism can be analyzed without applying nutritional stress. Furthermore, these strains promise to be useful for biotechnological hydrogen generation, since high in vivo hydrogen production rates are achievable under normal growth conditions, when the photosynthesis to respiration capacity ratio is lowered in parallel to down regulated assimilative pathways.
在硫缺乏条件下，密闭培养的莱茵衣藻会释放大量氢气。然而，这种真核绿藻在这种营养胁迫期间会经历剧烈的代谢变化，导致细胞生长受到抑制并最终死亡。本研究旨在分离出在正常生长条件下产生氢气的莱茵衣藻转化体，以实现连续的氢代谢，而不受营养剥夺的压力影响。
为了实现稳定的光生物产氢，设计了一种筛选方案，以鉴定光合作用与呼吸能力比（P/R比）降低的莱茵衣藻DNA插入诱变转化体。该筛选方案需要一种新的快速方法来选择其氧气产生/消耗平衡发生改变的突变菌株。在9000个转化体中，分离出了4个P/R比从几乎为零到3不等的菌株。发现菌株apr1的呼吸速率略高于野生型，而光合作用速率则显著低于野生型。在正常生长培养基（TAP）中，中等光照条件下，apr1的密闭培养物会变成厌氧状态并诱导[FeFe]-氢化酶活性，但没有明显的氢气释放。然而，在光照下厌氧适应的apr1细胞的卡尔文-本森循环失活导致体内产氢量比先前报道的硫缺乏莱茵衣藻野生型高出2至3倍。
微藻突变体中降低的P/R能力比构成了实现稳态光生物产氢的平台。利用这个平台，可以在不施加营养胁迫的情况下分析藻类的氢代谢。此外，这些菌株有望用于生物技术产氢，因为在正常生长条件下，当光合作用与呼吸能力比随着同化途径的下调而降低时，可以实现较高的体内产氢率。
