A Transcriptomic Study Of Hydrogen Production From Chlamydomonas Reinhardtii

Chlamydomonas reinhardtii is a kind of unicellular eukaryotic green algae.It belongs to Chlorophyta and Volvocales.It can produce hydrogen using protons and electons in cells at an anaerobic stress environment,so becomes a model species of biohydrogen study.But in current biohydrogen producing level,it can't meet large-scale industrialized production requirments.So to elucidate the molecular mechanism of biohydrogen production and dig the key functional genes from the point of gene transctription,becomes a research hotspot in the algal biohydrogen production.We choosed the algal strain cc124 and extracted the total RNA at its initialization phase(0 h,24 h),boost phase(72 h,96 h)and declination phase(144 h)during the hydrogen production process,to conduct RNA-Seq using Illumina Hi Seq 3000.We analyzed the differences between RNA-seq samples using bioinformatics tools.At the same time,we also detected contents of starch,glucose,acetic acid,formic acid and alcohol during the experiment.The main results were as follows:(1)We draw the kinetics of algal hydrogen production rate which can be divided into three phases: initialization phase(0 h-24 h),boost phase(24 h-96 h)and declination phase(96 h-144 h).According to the kinetics this process,the highest hydrogen-producing rate was at 96 h and the maximal hydrogen accumulation yield was 3891.07 ?L/mg Chl at 144 h when it was cultured under anaerobic conditions.(2)In our experiment,we built 9 cDNA libraries,including 0 h,24 h,72 h,96 h,144 h and their corresponding control samples,for sequencing.Each library got 6 Gb raw reads.For the 0h sample,we got 61,013,046 raw reads and total 251,403,358 raw reads for other stressed samples,while we got total 255,345,282 raw reads for the control samples.We finally annotated 14849 genes in all the libraries expressed.(3)We analyzed the gene expression model from 0h to 144 h and found out that all the differential genes clustered into four groups.Genes in Cluster 1 were down-regulated gene and their functions were photosynthesis,cellular carbohydrate catabolic process,pigment biosynthesis and metabolic process;Genes in Cluster 3 and Cluster 4 were up-regulated genes and their functions were protein catabolic process,protease endopeptodase activity,cellular amino acid biosynthesis,organic acid biosynthesis process,proton transport and hydrogen transfer.But genes in Cluster 2have no relationship with hydrogen production process.(4)According to the KEGG enchriment results of the differential-expressed genes,we found out that sulfur metabolism-related genes were significantly up-regulated in initialization phase,but down-regulated in declination phase.It was might because of that the algal hydrogen production was at sulfur-deficiency environment,But genes envolved in photosynthesis,glycolysis,TCA cycle,pyruvate metabolism and the pentose phosphate pathway were strongly inhibited,expecially in the boost phase.C.reinhardtii also could produce many amino acids to protect the alagl cell in initialization phase.While genes related to fatty acids of biosynthesis and metabolism were down-regulated in initialization phase,but were up-regulated in boost and declination phases,especially for the synthesis of unsaturated fatty acid.(5)At the beginning of hydrogen production,glycolysis pathway-related genes were up-regulated but down-regulated in the middle phase.This expression pattern was positive correlated to the algal hydrogen-rate kinectic curve.After the further screening and data digging,the up-regulated key genes in this pathway were 6 genes coding HK,PYK,PFK and ADH,and the down-regulated genes were 4 genes coding PYK,PDHB.Up-regulated genes coding PYK were Cre03.144847,Cre05.g234700 sequences,down-regulated genes coding PYK were Cre02.g147900,Cre12.g533550.(6)Pyruvate pathway was strongly supperessed in the process of hydrogen production,especially in the boost phase.The pyruvate is the key intermediate for algae to produce hydrogen.In this pathway,the up-regulated genes were 2 genes coding PDHB and MDH,which can adjust the Krebs cycle.The down-regulated genes were 2 genes coding DLD and DLAT.(7)The TCA cycle is in the central position for the sugar metabolism,protein metabolism and fat metabolism.In TCA cycle pathway,the up-regulated genes were 6genes coding CS,IDH,OGDH,LSC1 and SDHC,and the down-regulated genes were the same as those in the pyruvate pathway.(8)C.reinhardtii can produce hydrogen at the condition of dark or weak light in sulfur deficiency,so most of the transcriptome of photosyhthesis were down-regulated under such conditions.But 4 genes were up-regulated and they code PetJ,ATFD2,LHCBM7 and LHCA3,they can regulate PSII,PSI and cytochrome b6 f.Therefore,even under lack of sulfur conditions,photosynthetic metabolism pathway experienced a complicated process.(9)C.reinhardtii can produce hydrogen at sulfur-deficient environment.In the sulfur metabolic pathway,the up-regulated genes were5 genes coding sat,cysK,cysE,cysU,cysP in the initialization phase while 2 genes coding sat and SIR in the boost phase and one gene sequence coding TST in the declination phase.The down-regulated genes were 3 genes coding cys Q,cysK and metB.(10)At the beginning of the algal producing hydrogen process,the algae accumulated starch and later the starch was degrated.Because glucose derived from starch,they had the same content pattern.Acetic acid is the algal respiration substrates to consume oxygen,so acetic acid was consumed gradually during the hydrogen production process.On the contrary,formic acid and alcohol contents were increased during the hydrogen production process.In summary,during the hydrogen production of C.reinhardtii strain cc124.Sulfur metabolism,carbohydrate metabolism and amino acid metabolism and those regulation actors envolved in these metabolism processes are the main elements effecting algal hydrogen production efficiency.