Artificial symbiosis based bio-process

蓝藻作为水环境中的初级生产者，发挥着重要的作用。由光合作用生长的蓝藻固定的二氧化碳和氮被其他异养生物进一步利用。异养细菌直接利用蓝藻等藻类的排泄物已被普遍接受，此外，降解环境污染物的生物过程--生物修复也受到了极大的关注。为了构建高效、稳定的生物修复体系，常常需要利用微生物群落，这些微生物群落由几株能够降解和/或中介降解污染物的微生物组成。这些微生物群是由异养细菌组成的，因此不适合在低营养条件下应用，如海洋环境。本文重点研究了蓝藻作为异养细菌…营养物质的生产者和供给者的应用更多的细菌，这是有能力的生物修复。我们将这种由自养菌和异养菌组成的系统微生物联合体称为人工共生。本论文旨在构建人工共生体及其在农药生物修复中的应用。首先，我们研究了由固氮蓝藻Anabaena sp.组成的人工共生体模型。PCC7120和大肠埃希氏菌是典型的异养菌。大肠杆菌能在鱼腥藻培养上清液中生长，说明大肠杆菌细胞能代谢鱼腥藻分泌的营养物质。在此基础上，我们进行了人工共生在连续光照和光暗循环下的伪连续维持。这两个系统都能维持200小时以上，两种微生物都没有减少，这表明我们的人工共生思想是可以构建和维持的。然后，我们构建了一个用于杀虫剂生物修复的人工共生系统，对于蓝藻，鱼腥藻。被利用了。异养菌为黄杆菌。它产生一种酶，磷酸三酯酶(PTE)，从而降解有机磷杀虫剂。黄杆菌。能够在Anabeana sp.的培养上清液中生长，并表达足够水平的PTE来降解具有代表性的有机磷杀虫剂苯硫磷。鱼腥藻人工共生体的拟连续培养。和黄杆菌属。是在连续光照和明/暗循环下进行的。通过表达生长黄杆菌的PTE活性，在两种条件下都保持了对苯硫磷的降解能力。通过金属硫蛋白基因的异源表达，成功构建了能在重金属如锌、铜、镉存在下生长的海洋蓝藻重组菌株。这些重组海洋蓝藻菌株将进一步用于构建人工共生生物修复。较少

Cyanobacteria play an important role as primary producers in aquatic environment. Carbon dioxide and nitrogen fixed by photosynthetically growing cyanobacteria are further utilized by other heterotrophic organisms. It has been generally accepted that heterotrophic bacteria directly utilize products excreted from algae, including cyanobacteria.Beside, the biological process for the degradation of environmental pollutants, bioremediation, has been paid significant attention. In order to construct an efficient and stable bioremediation system, microbial consortiums are often utilized, which are composed of several strains of microorganisms capable of the degradation and/or mediating the degradation of pollutants. These microbial consortium is composed of heterotrophic bacteria, therefore, is not suitable for the application in the low-nutrient condition, such as marine environment.Here, we focused on the application of the cyanobacteria as the producer and doner of nutrient for heterotrop … More hic bacteria, which are capable for the bioremediaton. We designated such a systematic microbial consortium composed of autotrophic bacteria and heterotrophic bacteria as an "Artificial Symbiosis". We aimed the construction of an artificial symbiosis and its application for the bioremediation of insecticide.First, we have investigated the model artificial symbiosis, which is composed of a nitrogen fixing cyanobacterium, Anabaena sp. PCC7 120 and Escherichia coli as a representative heterotrophic bacterium. E.coli cells were able to grow in the culture supernatant of Anabaena sp., indicating that E.coli cells can metabolite the nutrients excreted by Anabaena sp.. On the basis of this finding, we carried out the pseudo-continuous maintenance of the artificial symbiosis, under both continuous light illumination and light/dark cycle. Both of the system were revealed to be maintained for more than 200 hours without the decrease in both microorganisms, suggesting that our idea of the artificial symbiosis can be constructed and maintained.Then we have constructed an artificial symbiosis for bioremediation of insecticide, As for the cyanobacterium, Anabaena sp. was used. As for the heterotrophic bacterium, Flavobacterium sp. which produces an enzyme, phosphotriesterase (PTE), thereby degradating organophosphorous insecticides, was used. Flavobacterium sp. Was able to grow in the culture supernatant of Anabeana sp., and also express enough level of PTE for the degradation of phenitrothion, a representative organophosphorous insecticide. Pseudo-continuous cultivation of the artificial symbiosis composed of Anabaena sp. and Flavobacterium sp. was carried out under both continuous light illumination and light/dark cyrcle. The ability of the degradation of phenitrothion has been maintained in the both conditions, by expressing PTE activity of growing Flavobacterium sp. cells.We also succeeded in the construction of a recombinant marine cyanobacterial strain, which can grow in the presence of heavy metals, such as Zn, Cu or Cd, by the heterogenic expression of metallothionein gene. Such recombinant marine cyanobacterial strains will be further utilized for the construction of an artificial symbiosis for bioremediation. Less