Promoting Photosynthetic Production of Dammarenediol-II in Chlamydomonas reinhardtii via Gene Loading and Culture Optimization.

Ginsenosides are major bioactive compounds found in Panax ginseng that exhibit various pharmaceutical properties. Dammarenediol-II, the nucleus of dammarane-type ginsenosides, is a promising candidate for pharmacologically active triterpenes. Dammarenediol-II synthase (DDS) cyclizes 2,3-oxidosqualene to produce dammarenediol-II. Based on the native terpenoids synthetic pathway, a dammarane-type ginsenosides synthetic pathway was established in Chlamydomonas reinhardtii by introducing P. ginseng PgDDS, CYP450 enzyme (PgCYP716A47), or/and Arabidopsis thaliana NADPH-cytochrome P450 reductase gene (AtCPR), which is responsible for producing dammarane-type ginsenosides. To enhance productivity, strategies such as "gene loading" and "culture optimizing" were employed. Multiple copies of transgene expression cassettes were introduced into the genome to increase the expression of the key rate-limiting enzyme gene, PgDDS, significantly improving the titer of dammarenediol-II to approximately 0.2 mg/L. Following the culture optimization in an opt2 medium supplemented with 1.5 mM methyl jasmonate under a light:dark regimen, the titer of dammarenediol-II increased more than 13-fold to approximately 2.6 mg/L. The C. reinhardtii strains engineered in this study constitute a good platform for the further production of ginsenosides in microalgae.

[Simultaneously removal of COD, nitrogen and phosphorus from wastewater by coupling treatment system with immobilized algae-bacteria].

A coupling treatment system was developed by employing immobilized Chlamydomonas reinhardti and activated sludge to simultaneously remove COD, nitrogen and phosphorus from wastewater. The amount of wastewater treated by the system was 6 m3 per day, and hydraulic retention time was 12 h. For activated sludge section, as stirring rate of anaerobic tank was 15 r x min(-1) and DO value of aerobic tank was 5 mg x L(-1), COD decreased from about 150 mg x L(-1) to 50 mg x L(-1) and NH4+-N from 20-30 mg x L(-1) to 0.5 mg x L(-1), whereas TP only dropped from 2-3 mg x L(-1) to 1.0 mg x L(-1). For immobilized C. reinhardti section, the suitable conditions were: DO 5 mg x L(-1), illumination intensity 2000 lx, the loading ratio of immobilization pellets 20%, respectively. Under the appropriate conditions of the coupling treatment system, COD, NH4+-N and TP of the effluent were about 15 mg x L(-1), 0.5 mg x L(-1) and 0.5 mg x L(-1), respectively. During 2 months period of continuous treatment, COD, NH4+-N and TP of the effluent kept fairly constant, showing the stability of the coupling wastewater treatment system.

[Study on the removal of nitrogen and phosphorus from wastewater by Chlamydomonas reinhardtii].

Chlamydomonas reinhardtii was employed to remove nitrogen and phosphorus from wastewater. The effects of initial NH4(+) -N and TP concentrations, N/P ratios, Light/Darkness ratios, pH and immobilization on the removal of NH4(+) -N and TP were evaluated. The results showed that C. reinhardtii could almost 100% remove NH4(+) -N and TP as initial concentrations of NH4(+) -N and TP were no more than 55 mg x L(-1) and 7 mg x L(-1), respectively, whereas the removal ratio of NH4(+) -N decreased drastically to 50% with initial NH4(+) -N concentration coming up to 75 mg x L(-1). Under N/P ratios of 5:1 and 10:1, C. reinhardtii could completely remove NH4(+) -N within 3d, while 6 d was needed with N/P ratio being 25: 1. Different from NH4(+) -N removal, the removal ratio of TP could reach almost 100% within 4 d under 3 N/P ratios. With L/D ratios of 24 h: 0 h and 12 h: 12 h, 100% removal of NH4(+) -N and TP could be achieved by C. reinhardtii, but the removal rate under L/D ratio of 24 h: 0 h was relatively faster. The optimal pH range for C. reinhardtii to remove NH4(+) -N and TP was 6-7. After immobilization, the ability of C. reinhardtii to remove NH4(+) -N was significantly enhanced as the removal ratio of NH4(+) -N came up to 100% with initial NH4(+) -N concentration being 75 mg x L(-1). The ability of immobilized C. reinhardtii to remove TP kept stable, whereas the removal rate was slowed slightly.