| Biodiesel has attracted attention in recent years because of the increasing greenhouse effect and the emerging energy crisis.Microalgae has become a research hotspot owing its higher photosynthetic efficiency,quicker growth rate,shorter growth cycle,and greater compatibility with food crops compared with other biodiesel raw materials.The present study used Monoraphidium sp.FXY-10 as the experimental subject.We studied the effects of fulvic acid on the growth and lipid accumulation of Monoraphidium sp.FXY-10 under heterotrophic cultivation condition.And we researched the change on cell growth,oil accumulation,and fatty acid composition of Monoraphidum sp.FXY-10 by combined fulvic acid and heterotrophic-autotrophic two-step cultivation.In addition,we are also studied the influence of fulvic acid on some physiological and biochemical indexes of algae,such as carbohydrate,protein,reactive oxygen species(ROS),enzyme activity of ACCase,PEPCase and ME.The seven genes of lipid biosynthesis-related were cloned.And the expression of these genes were also examined upon exogenous FA application.The correlation analysis was performed to determine the relationship between gene expression and lipid accumulation in Monoraphidium sp.FXY-10 and to obtain further information about physiological and molecular changes triggered by FA.This paper provides an effective strategy to rapid increase the cells and oil content of microalgae,and laids a certain foundation to study the molecular mechanism of lipid biosynthesis and genetic modification of single needle algae.The results are summarized as follows:(1)The biomass was increased to(6.4±0.03)mg L-1,and enhanced about 1.5-fold compared with the control treatment;the biomass yield was up to 422.83 mg L-1 d-1,and enhanced 1.5-fold compared with the control(250.66 mg L-1d-1);the lipid content(120.79 mg L-1d-1)was significantly(p<0.05)enhanced and approximately 2.8-fold compared with the control under the heterotrophic condition with 80 mg L-1 FA-treated.(2)In combined fulvic acid and heterotrophic-autotrophic two step cultivation,the lipid content increased along with the cultivation time;however,the oil production began to decline as time progressed.During the autotrophic period,the lipid content increased from 30.79%to 54.65%in 25 mg L-1 FA supplemented culture after 6 days of treatment;this finding was higher by 4.56%than the control(50.05%)without any FA supplement for DAT-6.In addition,the oil content increased by 23.82%compared with the one-stage heterotrophic culture with 80 mg L-1.Compared with the traditional autotrophic one-stage batch culture,the oil content did not significantly increase;however,the culture time was shortened by two-thirds.In addition,the main fatty acids in the FA-triggered Monoraphidium sp.FXY-10 were C16/C18 fatty acids(97%),which are appropriate for biodiesel production(3)The protein contents increased rapidly from 31.4%to 39.7%under FA treatment.By contrast,carbohydrate content sharply declined from 49.5%to32.5%.Furthermore,the ROS production levels upon exposure to FA treatment on the 6th,8th,and 10th days were 1.46,1.47,and 1.38 times of the control value,respectively.(4)Seven lipid biosynthesis-related genes were cloned,namely,me,dgatl,has ?,accD,pepc,rbcL,and gpat.The changes in gene expression induced by FA for seven lipid biosynthesis-related genes were quantified through real-time PCR.Genes expression analysis showed that FA induced changes in the expression of rbcL,me,accD,kas III,pepc,and gpat was enhanced 2.5,1.3,1.6,1.4,1.7,and 1.5 times,respectively;the expression of dgatl was insignificant change.(5)The Spearman correlation analysis(using SPSS 19.0)was carried out to determine the relevance between key gene expression level and lipid accumulation under FA treatment conditions.The results show that pepc,dgatl,kas III and rbcL and lipid accumulation has no obvious correlation(p>0.05);however,there has significantly associated with accD,gpat and lipid synthesis(p<0.05).(6)The ACC and ME activities were significantly improved in the FA-treated.The ME enzymatic activities of Monoraphidium sp.FXY-10 on days 2,4,and 6 were increased by 4-,2.7-,and 2.1-fold in the FA-treated culture than in the control culture.Howere,PEPCase enzyme activity decreased obviously.In conclusion,our results help enhance our understanding of the underlying molecular changes in response to FA and provide insights into the regulatory mechanism of lipid metabolism in Monoraphidium sp.FXY-10. |
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