Inoculation concentration modulating the secretion and accumulation pattern of exopolysaccharides.pdf

Inoculation concentration modulating thesecretion and accumulation pattern ofexopolysaccharides in desert cyanobacteriumMicrocoleus vaginatusLong Qian1Li Wu1Lie Yang1Zulin Zhang1,21School of Resources and Environmental Engineering,Wuhan University ofTechnology,Wuhan,Peoples Republic of China2The James Hutton Institute,Craigiebuckler,Aberdeen,UKAbstractCyanobacterial exopolysaccharides(EPS)accumulated duringmicroalgal cultivation have significant application potential inantioxidation,pharmaceutical products,and so on.Inoculationconcentration strongly affects the cultivation cost,biomass,and EPS accumulation.In this study,a high-EPS-excreteddesert cyanobacterium Microcoleus vaginatus was isolated,and the effects of inoculation concentration on biomass,photosynthetic activity,and EPS accumulation were explored.The results showed that the original fluorescence(Fo)provided a good indication to cyanobacterial biomass,whenChl-a concentration was lower than 10 mg L1.Inoculationconcentration significantly affected cyanobacterial biomassand EPS concentration(P 0.05).The two fractions ofEPS,capsular exopolysaccharides(CPS)and releasedexopolysaccharides(RPS)were strongly affect by inoculationconcentration.Other than forming thick sheath(CPS)surrounded the filaments,M.vaginatus excreted higherproportions of RPS to culture medium,and the ratio of RPS toCPS ranged from 1.08 to 1.58 depending on the inoculationconcentration.Additionally,although the biomass and EPSaccumulation increased with inoculation concentration,theincreasing inoculation concentration did not bring to theproportionate increase of the final biomass and EPS yield.Altogether,comprehensively considering the EPS yield andproductivity,inoculation concentration of 0.04 mg Chl-a L1isrecommended for M.vaginatus to produce EPS,with an EPSyield of 94.32 mg L1and EPS productivity of 184.86 mg(mg Chl-a)1L1d1at the end of experiment.2020International Union of Biochemistry and Molecular Biology,Inc.Volume 68,Number 2,Pages 330337,2021Keywords:microalgae,biomass,exopolysaccharides,desert,crust1.IntroductionAs resource and environmental problems became more andmore serious,the healthy development of human society,in-cluding human health,is seriously threatened.Under suchAddress for correspondence:Li Wu,Associate Professor of School ofResources and Environmental Engineering,Wuhan University ofTechnology,Wuhan 430072,Peoples Republic of China.Tel.:[phone];e-mail:;or Lie Yang,AssociateProfessor ofSchool of Resources and Environmental Engineering,WuhanUniversity of Technology,Wuhan,Peoples Republic of China.Tel.:[phone];Fax:[phone];e-mail:;orand Zulin Zhang,Professor of The James Hutton Institute,Craigiebuckler,Aberdeen ABI5 8QH,UK.Tel.:[phone];Fax:[phone];e-mail:Zulin.Zhanghutton.ac.uk.Received 26 December 2019;accepted 21 April 2020DOI:10.1002/bab.1930Published online 17 May 2020 in Wiley Online Library()circumstances,as a sustainable industrial model,microalgalcultivation is considered as an ideal way to deal with both theincreasingly prominent resource and environmental issues.That is because in the process of microalgal cultivation,mi-croalgae not only fix atmospheric CO2to reduce the greenhouseeffect,but also can use some high nutritional waste water orgas as nutrient source for growth,achieving simultaneouslyboth the economic and environmental aims 1,2.When thecultivated microalgae are harvested,microalgal biomass cannot only be used as biofertilizer 3,4 and desertification controlinocula 5;but also can be deeply processed into various foodsand feeds 1,6;as well the lipid and other high added-valueproducts in microalgae can be used for bioenergy and healthindustries 7,8.Especially,during the process of microal-gal cultivation,microalgae would excrete a large amount ofexopolysaccharides(EPS),which have a very broad applica-tion prospect because of their antioxidation,pharmaceuticalactivities,and other chemical and rheological properties9-12.330As the earliest photosynthetic organisms on the Earth,cyanobacteria not only have an important influence on theatmospheric composition,but also provide a basis for theorigin and evolution of the Earth s aerobic organisms13.Cyanobacteria are widely distributed in various habitats onthe Earth,even dry stones and hot spring over 60 C 14,15.That cyanobacteria can adapt to a variety of harshenvironmental conditions,may be attributed to their variousecological strategies and special metabolic mechanisms 16,17.One of them,the excreted EPS play an important role intheir environmental adaptability:(1)as a protective layer outof cyanobacterial cells,the excreted EPS prevent the entranceof external toxic and harmful substances into cells 16,18;(2)the EPS provide a matrix for other extracellular substancessuch as trehalose and sucrose,which can lower the phasetransition temperature(Tm)of plasma membrane and form thecarbohydrate glass to prevent fusion of membrane vesicles 17,19;(3)some specific water stress proteins and UV-absorbingsubstances accumulated in EPS can ensure the stability of cellstructure and alleviate the radiation damage 17,20.Cyanobacterial EPS are a type of polysaccharide sub-stances,which are excreted during the process of cyanobac-terial growth and metabolism 14,19,21,22.Because of theimportant role in environment adaptation and applicationprospects,cyanobacterial EPS have recently received a greatdeal of attention 4,912,21.However,for the time being,allthe prospects can only be stayed at laboratory stage,just be-cause the exopolysaccharide yield is limited.So how to improvecyanobacterial exopolysaccharide yield is an important issueto promote exopolysaccharide application.Many studies haveconfirmed that environmental factors,such as light and tem-perature,have significant effects on the excretion of EPS 16,18,21,23.Especially as the energy source of photosyntheticorganisms,light has a particularly important effect on theexcretion of EPS 22,24,and it was found that cyanobacterialEPS accumulation increased with the increasing light intensityin the range of 2080 E m2Sec124.In the current microalgal cultivation,for most of the con-ditions,it is always provided as the surface light intensity(onthe closest photoreactor surface from the light source)18,25.However,the actual light energy absorbed by microalgalcells may be obviously different from each other in differentcultivations because the self-shading effect caused by differentconcentration will seriously affect the light energy absorption,even if the surface light intensity is consistent and same.There-fore,we speculate that different inoculation concentrationwould not only affect microalgal growth and biomass accumu-lation,but also might have a significant effect on the secretionof EPS.Therefore,in this study,the growth,photosyntheticactivity and EPS secretion were monitored under differentinoculation concentrations of Microcoleus vaginatus,whichhas been wildly found as the dominant species of biological soilcrusts in global arid and semiarid areas 26,27,and is alsoused as the inoculum in desertification control 27.The effectsof inoculation concentration on the secretion and accumulationof EPS were further discussed from the points of microalgalcultivation,biomass,and exopolysaccharide accumulation.Theresults will not only provide guidance for adjusting light inten-sity in the process of microalgal cultivation,but also provideimportant theoretical basis for improving microalgal EPS yieldin practical application.2.Materials and Methods2.1.Microalgal strain and inoculaM.vaginatus isolated from the biological soil crusts of theQubqi Desert(located in the Dalate Banner region of InnerMongolia;4021 N,10951 E)was used in this experiment.Formicroalgal isolation,biological soil crusts were firstly passedthrough a 0.1 mm sieve(pore size),and then 0.1 g crust samplewas diluted with 10 mL sterile distilled water.The mixturewas shaken carefully and placed in darkness overnight.Onthe next day,0.1 mL mixture was inoculated on solid BG11medium(1.3%agar;Eiken,Tokyo,Japan).The inoculationwas placed in a laboratory(25 2C),and illuminated withcool white fluorescent light at 40 E m2Sec1.Fifteen to20 days later,the microalgal colonies were identified andM.vaginatus was isolated.After isolation,M.vaginatus wasinoculated into 500 mL flask of sterilized liquid BG11 mediumto obtain enough microalgal inocula.Microalgal culture wasplaced in the laboratory with an aeration of 0.4 L Min1,andilluminated with cool white fluorescent light at 40 E m2Sec1of surface light intensity(on the closest flask surface from thelight source).2.2.Microalgal inoculation and cultivationTwenty days later,the cultivated inocula of M.vaginatus werethen inoculated into four groups of flasks(500 mL liquid BG11medium)with different inoculation concentrations,whichwere labeled as inoculation concentration(IC)1,2,3,and 4according to the inoculation concentration from low to high(Table 1).The inoculated microalgae were placed in the weaklight intensity of about 5 E m2Sec1overnight,and thencultivated in the laboratory-controlled conditions as mentionedabove(40 E m2Sec1,25 2 C).During the cultivationprocess,the microalgal culture was sampled every seven daysand the chlorophyll-a(Chl-a)concentration,EPS concentrationand chlorophyll fluorescence of the cultivated microalgae weredetermined.2.3.Determination of Chl-a concentrationIn this experiment,Chl-a concentration was taken as a mea-sure of the microalgal biomass 22.For Chl-a concentrationdetermination,4 mL microalgal culture was sampled and cen-trifuged at 8,600 g for 10 Min to remove the supernatant fluids.The Chl-a of sedimentary microalgae was extracted by 4 mL95%ethanol overnight at 4 C.Then Chl-a concentration wasspectrophotometrically measured following the description of28.Biomass productivity(BP,mg Chl-a L1d1)was calculatedBiotechnology and Applied Biochemistry331 14708744,2021,2,Downloaded from https:/ by Xinjiang Branch Of C.A.S,Wiley Online Library on 06/09/2023.See the Terms and Conditions(https:/ Wiley Online Library for rules of use;OA articles are governed by the applicable Creative Commons LicenseBiotechnology andApplied BiochemistryTABLE 1Comparison of microalgal biomass productivity,specific growth rate,EPS(RPS+CPS)yield,productivity,and the ratio of RPSto CPS(RPS/CPS)under the different inoculation concentrations(IC)Inoculationconcentration(mg Chl-a L1)Biomassproductivity(mg Chl-a L1d1)Specificgrowth rate(d1)EPS yield(mg L1)EPSproductivity(mg(mg Chl-a)1L1d1)RPS/CPS0.04 0.01(IC 1)0.65 0.010.39 0.0294.32 11.56184.86 30.381.08 0.110.16 0.09(IC 2)0.82 0.050.31 0.06108.82 28.9976.61 78.931.32 0.501.11 0.30(IC 3)0.85 0.130.18 0.03111.02 1.156.28 0.811.58 0.172.03 0.40(IC 4)0.94 0.180.14 0.00136.54 0.895.29 1.341.28 0.06according to the formula:BP=(CCt CCi)/t,where CCtand CCiare the Chl-a concentration(mg L1)at cultivation time t(d)and right after inoculation,respectively.The specific growthrate(,d1)was calculated according to the formula:=ln(CCt/CCi)/t.2.4.Determination of EPS concentrationIn this study,EPS were operationally divided into two fractions:released exopolysaccharides(RPS)and capsular exopolysac-charides(CPS).For concentration determination,4 mL mi-croalgal culture was sampled and centrifuged at 8,000 rpm for10 Min.After centrifugation,2 mL of the supernatant fluid wasused to determine the RPS concentration,whereas the CPS ofsedimentary microalgae were extracted using 4 mL distilledwater for 6 H at 80 C 24,and 2 mL CPS solution was preparedin the same way as the RPS solution.Then,1 mL 9%phenolsolution and 5 mL concentrated sulfuric acid were added intothe RPS or CPS solution in sequence.The mixture was shakencarefully and placed in the laboratory for 30 Min,and then theabsorbance A485of mixture was spectrometrically measured at485 nm 29.The correlation between the absorbance A485and EPS(RPS+CPS)concentration(EC;mg L1)was calculated accord-ing to the following formula:EC=60.321 A485(R2=0.997;using sucrose as the standard).EPS yield(EY;mg L1)wascalculated according to the formula:EY=ECt ECi,where ECtand ECiare the EPS concentration(mg L1)at cultivation timet and right after inoculation,respectively.The EPS productivity(EP,mg(mg Chl-a)1L1d1)was calculated according to theformula:EP=EY/CCi/V/t,where CCiis the Chl-a concentration(mg L1)right after inoculation,V is the cultivation volume(0.5 L),and t is the cultivation time(d).2.5.Determination of chlorophyll fluorescenceBefore determining chlorophyll fluorescence,2 mL microalgalculture was sampled and adapted in the dark for at least10 Min.Then its fluorescence parameters were measuredusing a plant efficiency analyzer(PEA;Hansatech,Norfolk,UK).For fluorescence determination,a saturating light pulseof 3,000 E m2Sec1was supplied,and fluorescence param-eters Foand Fm(maximum fluorescence)were automaticallyrecorded by the PEA.The fluorescence parameter Fv/Fm(themaximum quantum yield of photosystem II photochemistry)was calculated according to the formula:Fv/Fm=(Fm Fo)/Fm30.2.6.Data analysisAll the experiments were conducted three times as repetitions.The variances of Chl-a,EPS concentration,and Fv/Fmamongdifferent treated groups were analyzed using one-way ANOVA.The effects of inoculation concentration,cultivation time,andtheir interactions on microalgal Chl-a,EPS concentration,and Fv/Fmwere analyzed using two-way ANOVA.All thevariance analysis was performed on SPSS 13.0 software(SPSS Inc.,USA).In addition,curve fitting between microalgalChl-a concentration and Fowas performed to evaluate theirrelationships via Curve Estimation(Hyperbola)using SigmaPlot12.5 software(Systat Software Inc.,San Jose,CA,USA).3.Results and Discussion3.1.Isolation and cultivation of M.vaginatusIn a natural field environment,M.vaginatus does not directlydistribute on crust surface,but dwells in the surface substrate31,where many other microalgae also live together,such assome species of green algae and diatoms 28.Therefore,M.vaginatus can only be artificially cultivated after being isolatedfrom biological soil crusts.In order to separate M.vaginatus,the mixture of crusts and water was inoculated on solid BG11medium in our experiment.After 15 days of cultivation,afew colonies of M.vaginatus could be observed.The coloniesof M.vaginatus were blue-green or dark green;bundlesof microalgal filaments irregularly crept on the mediumsurface.EPS excreted by the microalgal filaments formeda gelatinous and colorless sheath,which surrounded themicroalgal filaments and combined with medium closely.Whena colony of M.vaginatus was picked up and observed under332Inoculation Modulating EPS Accumulation Pattern 14708744,2021,2,Downloaded from https:/ by Xinjiang Branch Of C.A.S,Wiley Online Library on 06/09/2023.See the Terms and Conditions(https:/ Wiley Online Library for rules of use;OA articles are governed by the applicable Creative Commons LicenseFIG 1Morphological characteristics of Mocrocoleusvaginatus.(A)Colon