Artificial symbiosis based bio-process

蓝藻在水生环境中扮演着重要的初级生产者的角色。通过光合作用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。人们普遍认为，异养细菌直接利用藻类排泄的产物，包括蓝藻。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系统，通常采用微生物财团，由能够降解和/或介导污染物降解的几株微生物组成。这些微生物联合体是由异养菌组成的，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的营养物的生产者和供给者的应用。我们将这种由自养细菌和异养细菌组成的系统微生物联合体称为“人工共生”。目的是构建一种人工共生体，并将其应用于杀虫剂的生物修复。首先，我们研究了以固氮蓝藻Anabaena sp. PCC7 120和大肠杆菌为代表的异养细菌组成的人工共生模式。大肠杆菌细胞能在水藻培养上清液中生长，说明大肠杆菌细胞能代谢水藻分泌的营养物质。在此基础上，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，而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。在此基础上，构建了一种用于杀虫剂生物修复的人工共生菌体系，其中蓝藻采用了水蓝藻。对于异养细菌，黄杆菌可以产生一种酶，磷酸三酯酶（PTE），从而降解有机磷杀虫剂。Flavobacterium sp.能够在Anabeana sp.的培养上清液中生长，并表达足够水平的PTE来降解苯基硫磷，这是一种代表性的有机磷杀虫剂。在连续光照和明暗循环条件下，对水藻和黄杆菌组成的人工共生进行了伪连续培养。在这两种条件下，通过表达黄杆菌生长细胞的PTE活性，维持了对苯硫磷的降解能力。我们还成功构建了重组海洋蓝藻菌株，该菌株通过金属硫蛋白基因的异质表达，可以在Zn、Cu或Cd等重金属存在下生长。该重组海洋蓝藻菌株将进一步用于构建人工共生进行生物修复。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