| Grow either in autotrophism or heterotrophism, Chlorella species, as the widespreadspherical unicellular microalgae under Chlorella genus of Chlorophyta, might be riched inprotein, lipid and pigment etc. Their growth characteristics and cellular components weresignificantly influenced by the surrounding conditions. Screening and cultivating some ofChlorella species which could utilize the pollutive elements in origanic effluent andmeanwhile accumulate high-value nutrient components, might be beneficial to promoteindustries for implementation of green manufacturing and circulation economy. In this paper,a Chlorella strain was screened and studied in details, for figuring out the relations betweencultivation models and Chlorella cellular components, the effects of NaCl concentration onthe Chlorella metabolism, and also the utilization efficiency of some organic effluent forproduction of Chlorella cell components. The main results were illustrated as follows:A novel rapid method of assaying algal protein content was established, and used toassay Chlorella protein accumulation at taming period and different culture processes. Thenewly exploited assay, so called APPD-UV method, is quite accurate and reproducible fordetermining Chlorella protein content. By this assay method, a tamed strain, named C.vulgaris C9-JN2010was screened out, which grew better in citric acid effluent andaccumulated higher level of protein. Its biomass and specific growth rate were both13.0%higher than the starter strains, and the algal protein content was54.6%, in which the essentialamino acid content was about40.8%of the total. Compared with the starter, the consumptionrates of nitrogen and phosphorus by C. vulgaris C9-JN2010increased by about16.6%and15.3%, respectively. Based on that, biosynthesis of the algal protein and amino acids werecompared under autotrophism, heterotrophism and mixotrophism forms. In the3rdprocess, C.vulgaris C9-JN2010grew better and shew higher protein productivity, with a specific growthrate of0.526d-1and the protein productivity of0.097g·L-1·d-1, respectively. Comparedamong the three processes, Cys, Tyr and Met proportions were significantly different. P-Bdesign, the steepest climbing and CCD experiments used, an algal fermentation medium ofprotein optimized that was applied to cultivate C. vulgaris C9-JN2010, in which the algalbiomass and protein content were1.786g·L-1and47.0%, respectively, with a proteinproductivity of0.210g·L-1·d-1which was116.2%higher than the control. The specific growthrate (0.630d-1), biomass productivity (0.470g·L-1·d-1) and protein productivity (0.220g·L-1·d-1) of C. vulgaris C9-JN2010cultivated in photo-reactor were all higher than those inthe shake flask.Based on nitrogen metabolism regulation, NaCl stress plus high light intensity wassupplemented to accumulate higher level of Chlorella lipid. The autotrophic culture mediumand conditions (inoculation size0.150g·L-1, pH7.0,25℃,4000lux and light cycle16h:8h)which were successively optimized in single factor, P-B design, the steepest climbing and theCCD experiments were used for cultivating C. vulgaris C9-JN2010and biosynthesis of thealgal lipid was regulated. One-step cultivation strategy performed, the algal biomass and itsproductivity were0.755g·L-1and0.126g·L-1·d-1, increased by6.5%and6.8%, respectively. It was found that low nitrogen concentration, NaCl stress, high light intensity and long lightcycle were favorable for lipid synthesis. Moreover, under low nitrogen,2.5%NaCl stress andhigh light intensity （6000lux） together, two-step cultivation strategy was more advantageousto obtain higher biomass (0.744g·L-1) and accumulate lipid, with the higher lipid content andproductivity of26.0%and24.2mg·L-1·d-1that were2.5and1.75times as well as the algaebefore regulation, respectively. Compared with the algae inoculated in the shake flask, thealgal lipid content and productivity in7.5L photo-reactor were higher, reaching25.6%and27.5mg·L-1·d-1, respectively, and the lipid productivity increased by about13.6%.NaCl stress-inducing effects on algal physiological response mechanism and chlorophyllbiosynthesis gene expression of the tamed Chlorella strain were studied. Under free NaCl, thealgal growth was the best with a specific growth rate of0.225d-1, and its chlorophyll andprotein contents were of39.5mg·g-1and50.0%, respectively. However, the algal lipidcontent in mezzo-salinity （2.5%） was higher of around15.5%with a productivity of16.10mg·L-1·d-1which was around3.6times as well as the algae in the free NaCl. NaCl stresswould induce biosynthesis of some antioxidants and enzymes, such as free proline, GSH andMDA etc. It was the first found free proline content （2.3%） in mezzo-salinity was around20.0times as well as the lowest value. Under5.5%NaCl, MDA and GSH contents were70.0and9.4mg·g-1DW which were about2.1and2.4times as well as those in the non-salinity, andthe activities of algal cellular SOD, CAT and POD were greatly increased, with the activitiesof2367.5,50.3and710.4U·g-1DW which were3.0,4.7and2.7times as well as those in thenon-salinity, respectively. For C. vulgaris C9-JN2010individually under free NaCl and3.5%NaCl stress, the algal chlorophyll biosynthesis genes chl B, chl I, chl L and chl N werequantificationally analyzed through RT-PCR, in which the transcriptional levels of these genesfrom the sample in3.5%NaCl were2.1,1.5,4.5, and4.8times as well as those in thefree-NaCl sample, respectively. The results indicated that proper NaCl concentration wouldaccelerate synthesis of numerous antioxidants and enzymes to resist oxidation damageinduced in NaCl stress to the algal cell and promote transcription of its chlorophyllbiosynthesis genes.Nitrogen and phosphorus in the organic effluent were efficiently utilized to biosynthesizealgal protein and lipid by C. vulgaris C9-JN2010. The algae individually inoculated in shakeflask and photo-reactor would remove nutrients in CAE, ME and mixed CAE/ME effluent.First, in the optimal20.0%CAE, the algal biomass and its productivity were1.040g·L-1and0.260g·L-1·d-1in5L photo-reactor, with higher growth rate and removal rate of nutrient,respectively. Eventually, high removal efficiency of CODCr, BOD5and TOC, TN and TP inCAE were all over90.0%. Additionally, ME being in favour of the algal culture in the flask,batch, semi-continuous and continuous cultivation were performed in7.5L photo-reactors,respectively. The algal specific growth rates and biomass productivities in the three processeswere over0.200d-1and0.106g·L-1·d-1, and removals of NH4+-N, TN, TP, CODCrand BOD5were98.3,95.5,90.6,88.3and87.4%, respectively. Moreover, in the optimal （20:80） mixedeffluent, the algal biomass and specific growth rate were of1.187g·L-1and0.237d-1,respectively. Simultaneously, NH4+-N, TN, TP, TOC, BOD5and CODCrwere almost used up （above90.0%）. The algal growth and removal efficiency of pollution factors in thephoto-reactor were higher than those in the shake flask, in which the former were slightlyover those in the CAE and ME separately treated. The above effluents were effectively treatedso that low level residual was close to urban sewage plant wastewater discharge standards(TN≤15.0mg·L-1, TP≤0.5mg·L-1, NH4+-N≤5.0mg·L-1, CODCr≤120.0mg·L-1and BOD5≤60.0mg·L-1). Ultimately, the algal biomass gathered in these effluents was analyzed, and itscrude protein, crude fat, crude fiber, ash and total phosphorus contents were48.0%-55.0%,10.0%-30.0%,3.3%-4.0%,5.0%-5.5%and0.65%-0.80%, respectively, and the essentialamino acid was44.5%of the total protein. Moreover, polyunsaturated fatty acids were about73.4%of the total fatty acids.Above results indicated the promising potencial of utilizing Chlorella to makefermentation waste profitable, especially on supplying the high nutrient feed from Chlorellawith riched essential amino acids and unsaturized fatty acids. |
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