Artificial symbiosis based bio-process

蓝藻是水环境中重要的初级生产者。光合生长的蓝藻所固定的二氧化碳和氮被其他异养生物进一步利用。异养细菌直接利用藻类（包括蓝藻）的排泄物已被广泛接受，而降解环境污染物的生物过程--生物修复也受到了广泛关注。为了构建有效和稳定的生物修复系统，通常利用微生物聚生体，其由能够降解和/或介导污染物降解的几种微生物菌株组成。这些微生物群都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。 ...更多信息 具有生物修复能力的hic细菌。我们将这种由自养菌和异养菌组成的系统性微生物群落称为“人工共生体”。本论文以构建人工共生体系及其在农药生物修复中的应用为研究目的，首先，以固氮蓝藻Anabaena sp. PCC 7120和异养细菌大肠杆菌Escherichia coli为代表，构建了人工共生体系。大肠杆菌细胞能够在鱼腥藻的培养上清液中生长，表明大肠杆菌细胞可以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的伪连续维持，在连续光照和光/暗循环。在此基础上，我们构建了一个用于杀虫剂生物修复的人工共生体系，其中蓝藻为鱼腥藻（Anabaena sp.）。至于异养细菌，使用产生酶磷酸三酯酶（PTE）从而降解有机磷杀虫剂的黄杆菌属。黄杆菌能够在Anabeana sp.的培养上清液中生长，并且表达足够水平的PTE以降解具有代表性的有机磷杀虫剂杀螟硫磷。在连续光照和光暗交替条件下，对鱼腥藻和黄杆菌人工共生菌进行了假连续培养。通过表达PTE活性，在两种培养条件下均保持了对杀螟硫磷的降解能力，并通过金属硫蛋白基因的异源表达，成功构建了一株能在重金属（Zn、Cu、Cd）存在下生长的重组海洋蓝藻。这种重组海洋蓝藻菌株将进一步用于构建生物修复的人工共生体。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