Cultivation And Photosynthesis Of Transgenic Synechococcus Sp. PCC7002 With HTNF-α Gene

In recent years, the great progress in the genetic engineering ofcyanobacteria has promised to use transgenic cyanobacteria to producemedicines or solve environmental problems. However, their practicalapplication has been limited by the low density and low growth rate oftransgenic cyanobacteria. One way to achieve the high density cultivationis to exploit a better cultivation condition and mode of the transgeniccyanobacteria. In this thesis, the optimal conditions were investigated to achieve thehigh density cultivation of transgenic Synechococcus sp. PCC7002 withhTNF-α gene. First, the factors affecting the growth of transgenicSynechococcus sp. PCC7002 were studied in the shake flask culture, thenmixotrophic cultivation system in 2.5L air-lift photobioreactor wasexploited for more efficient production of transgenic cyanobacteria , andheterotrophic metabolism , photoautotrophic metabolism and theirinteraction in mixotrophic culture were discussed . (1) Light, temperature, salinity, pH, nitrogen, carbon sources areimportant environmental factors affecting the growth of transgenic VSynechococcus sp. PCC7002. The growth of transgenic Synechococcus sp.PCC7002 was saturated at levels of 100μmol.m-2s-1 light intensity. Theoptimal temperature for cultivation was 35℃. The range of NaClconcentration suiting the cell growth was from 12g/L to 24g/L, and theoptimal concentration was 24g/L. The optimum growth pH was 8.2,toohigh or too low pH resulted in a decline of growth rate. TransgenicSynechococcus sp. PCC7002 could use ammonium or nitrate to fulfill itsnutritional requirement, but nitrate was the preferred nitrogen source andthe optimal concentration was 1g/L NaNO3. Several organic carboncompounds such as Sucrose, Glucose, Glucosamine could acceleratedcell's growth apparently . While, when NaHCO3 1g/L and glucose wereadded simultaneously, the rate of cell growth was improved moresignificantly, suggesting that mixotrophic cultivation system was a bettermode for the maximum productivity of transgenic Synechococcus sp.PCC7002. (2) In the 2.5L photobioreactor, when bubbled with air supplementedwith 3%(v/v)CO2,the organic carbon compounds still accelerated thecell growth, thus the growth rate in mixotrophic culture was higher thanthat in photoautotrophic culture. Glucosamine was proved to be themost suitable organic carbon substance in this condition, and higher gasvelocities could markedly improve cell growth and accelerated theutilizing of the organic carbon source. The light intensity attenuation VImathematical model in the culture system of transgenic Synechococcus sp.PCC7002 with hTNF-α gene was I = I0e?(?0.0263+1.1909?OD750)?d . (3) The physiological characteristics of transgenic Synechococcus sp.PCC7002 with hTNF-α gene were investigated. The optimal temperaturefor photosynthesis was 35℃. The optimal CO2 concentration couldimprove the photosynthetic activity of transgenic Synechococcus sp.PCC7002 . The photosynthetic rate of cells during the beginning andexponential phases of mixotrophic growth were investigated andcompared. It was shown that after inducing or activating sugar uptakesystem, organic carbon sources accelerated cell's growth markedly.Adapted to grow for two days in the presence of sugar, the oxygenevolution rate of mixotrophic culture was double comparing to that ofphotoautotrophic culture. Cells adapted to low CO2 showed a reducedphotosynthetic O2 evolution at zero time after the sugar addition whencompared to photoautotrophic cells,This decline was correlated with adecrease in carbonic anhydrase activity. While, when NaHCO3 1g/Lwas added, the photosynthetic rate of cells was noticeably enhanced inthe presence of organic carbon source, which indicated there was s...