Artificial symbiosis based bio-process

蓝藻作为初级生产者在水生环境中发挥着重要作用。光合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。人们普遍认为，异养细菌直接利用藻类（包括蓝藻）的分泌物。此外，降解环境污染物的生物过程——生物修复也受到了广泛的关注。为了构建高效稳定的生物修复系统，通常利用微生物群落，其由多种能够降解和/或介导污染物降解的微生物菌株组成。这些微生物群落由异养细菌组成，因此不适合在低营养条件下应用，例如海洋环境。在这里，我们重点研究了蓝藻作为异养细菌的营养生产者和提供者的应用，这些细菌具有生物修复能力。我们将这种由自养细菌和异养细菌组成的系统微生物群落称为“人工共生”。我们的目标是构建人工共生体及其在杀虫剂生物修复中的应用。首先，我们研究了由固氮蓝藻鱼腥藻组成的模型人工共生体。 PCC7 120和大肠杆菌作为代表性异养细菌。大肠杆菌细胞能够在鱼腥藻培养上清液中生长，表明大肠杆菌细胞可以代谢鱼腥藻分泌的营养物质。基于这一发现，我们在连续光照和光/暗循环下进行了人工共生的伪连续维持。结果表明，这两个系统都维持了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的想法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，至于蓝藻，鱼腥藻属。被使用了。至于异养细菌，黄杆菌属。使用产生磷酸三酯酶（PTE）的酶，从而降解有机磷杀虫剂。黄杆菌属能够在Anabeana sp.的培养物上清液中生长，并且还表达足够水平的PTE以降解代表性有机磷杀虫剂苯螟硫磷。由鱼腥藻组成的人工共生体的伪连续培养。和黄杆菌属。在连续光照和明/暗循环下进行。通过表达生长的黄杆菌属物种的 PTE 活性，在两种条件下都保持了苯螟硫磷的降解能力。我们还通过金属硫蛋白基因的异源表达，成功构建了能够在Zn、Cu、Cd等重金属存在下生长的重组海洋蓝藻菌株。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。较少的

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