太空生存太空生存太空生存 (36).pdf

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1、Isolation and characterization of enzyme-producing bacteriaof the silkworm larval gut in bioregenerative lifesupport systemXue Liang1,a,b,c,Yuming Fua,b,c,1,Hong Liua,b,c,naSchool of Biological Science and Medical Engineering,Beihang University,ChinabInstitute of Environmental Biology and Life Suppo

2、rt Technology,Beihang University,ChinacInternational Joint Research Center of Aerospace Biotechnology&Medical Engineering,Beihang University,Chinaa r t i c l e i n f oArticle history:Received 11 June 2015Received in revised form4 July 2015Accepted 6 July 2015Available online 14 July 2015Keywords:Sil

3、kwormBLSSCellulase-producing bacteriaAmylase-producing bacteriaProbioticsa b s t r a c tSilkworm(Bombyx mori L.)larvae were used as an ideal animal protein source forastronauts in bioregenerative life support system(BLSS).Here,we compared thedifferences in bacterial communities of the silkworm larva

4、l gut and cellulase-producingand amylase-producing bacteria between the BLSS rearing way(BRW)and the traditionalrearing way(TRW)through Illumina Miseq sequencing,culture-dependent approach,16SrDNA and ITS sequencing,phylogenetic analysis to find the role of gut bacteria in fooddigestion.The analysis

5、 of Miseq showed that the gut microbiota in the BRW wassignificantly changed than that in the TRW.Results revealed that the isolates can producecellulose-degrading and starch-degrading enzymes of gut bacteria of silkworm in theBRW which decreased compared with that of the TRW,but the number of isola

6、tes bothsecrete cellulase and amylase are equal.The isolates that can produce both enzymes in theTRW were Alternaria sp.Preussia sp and Coprinellus radians.Meanwhile,in the BRW wefound Enterococcus,Erwinia and Pantoea can produce cellulase and amylase.We could usethe dominant populations to make pro

7、biotic products for nutrient absorption and diseaseprevention in BLSS to improve gut microecology,as well as the yield and quality of animalprotein.&2015 IAA.Published by Elsevier Ltd.All rights reserved.1.IntroductionThe insect gut is inhabited by a wide diversity ofmicroorganisms as a result of it

8、s constituting intestinalmicrobial ecosystem.The gut microbiota is involved in thehosts digestion,nutrition,development,resistance topathogens invasion.The composition and structure ofmicrobial are dynamic,which can be varied with changingnutrient availability,physiological environments,and theproxi

9、mity to other organisms 1,2.Loss of microorganismsoften results in abnormal development and reduces survi-val of the insect host 3,4.Based on the theory ofmicroecology,insect relying on gut microbes provides avariety of digestive enzymes,to complete its food diges-tion,nutrient absorption and metabo

10、lism 5.However,few have discussed the possibility that microorganismsmay produce some of the digestive enzymes to provideessential nutrients or assist in important metabolismfunction related to host food ingestion 1.Mulberry silkworm(Bombyx mori L.)is an importanteconomical insect whose importance i

11、s reflected not onlyby its silk production but also by its valuable nutritionalContents lists available at ScienceDirectjournal homepage: Astronauticahttp:/dx.doi.org/10.1016/j.actaastro.2015.07.0100094-5765/&2015 IAA.Published by Elsevier Ltd.All rights reserved.nCorresponding author at:Institute o

12、f Environmental Biology and LifeSupport Technology,School of Biological Science and Medical Engineer-ing,Beihang University,Beijing 100191,China.Tel./fax:86 10 82339837.E-mail address:LH(H.Liu).1These authors contributed equally to this study.Acta Astronautica 116(2015)247253composition.The idea of

13、rearing mulberry silkworm larvaeto provide animal protein for crew in bioregenerative lifesupport system(BLSS)required by long-term missions tothe moon and Mars is widely accepted.This is becausesilkworm has many positive merits such as high proteincontent,reasonable nutrient compositions and amplec

14、ontents,a short lifespan,easy breeding method,smallgrowth room,and little odor and wastewater produced 6.Due to limitation of space and resource,mulberry silk-worm rearing method in BLSS was different from tradi-tional rearing method which only uses mulberry leaves.InBLSS,mulberry silkworms of the f

15、irst three instars(fromthe 1st day to 16th day)were fed with mulberry leaves andfor those of the last two instars(from the 17th day to 25thday)were fed with lettuce leaves 7.In this rearing way,the yield and the growth rate of silkworm larvae rearedusing this method were a little lower than those fe

16、edingon mulberry leaves purely.Prior study has providedevidence that gut microbiota of silkworms fed on lettuceleaves was relatively simple.The appearance of profitlessbacteria in the gut of silkworm under the BLSS rearing waymight break down the balance structure of healthy gutmicrobialcommunity,re

17、sultinginreduceddigestiveenzyme activity 8.It has been reported that many silk-worm intestinal bacteria produce digestive enzymes likeamylase and cellulose 9.Thus,it is potentially possiblethat the change of gut flora due to lettuce leaf feeding maycontribute to the decrease of physiological activit

18、y andcause death of the silkworm.However,how diet composi-tions in BLSS shape gut enzyme-producing bacteria is stillunknown.In this study,the gut bacterial diversity and enzyme-producing bacteria of silkworm larvae reared with thetraditional rearing way(TRW)and BLSS rearing way(BRW)wereinvestigatedu

19、singculturedependent,culture-independent and Illumina Miseq approaches.Thechangesofcellulase-producingbacteria,amylase-producing bacteria in silkworm larvae in response tolettuce leaf feeding were revealed.This study may pro-mote the development of probiotic products of animalprotein under BLSS.2.Ma

20、terials and methods2.1.Silkworm strains and rearing methodsThe silkworm eggs B.mori L.872?871 were boughtfrom Guangtong Silkworm seed Co.Ltd.(Shandong Pro-vince,China).The silkworm eggs were incubated under a12-h light/12-h dark cycle in an artificial cultivation box at25 1C and 80%of relative humid

21、ity.When 20%of eggs hadlittle black dots on the surface,they were shaded withblack gobo for about 48 h to ensure the larvae hatching outat one time.The silkworm larvae were reared withmulberry leaves from the first to third instar and thendivided into two groups:the BLSS breeding group rearedwith st

22、em lettuce leaves and the conventional breedinggroup still reared with mulberry leaves at the beginning ofthe fourth instar 8.2.2.Isolation of gut bacteria and DNA extractionWhen the two groups of silkworms grew to the thirdday of the fifth instar,10 individuals of each group wereselected and subjec

23、ted to starvation overnight.Those silk-worms were surface decontaminated by wiping with 70%ethanol solution and scorched gently in a flame.Thecontent of gut was taken and placed into sterile micro-centrifuge tubes on ice under aseptic condition.Accordingto the screening standard of bacteria,tenfold

24、serial dilu-tion was spread and incubated for inoculation.On eachnutrient agar plate,0.1 mL of intestinal content of twogroups was spread and incubated at 37 1C for 2 days.Allsamples were repeated three times.The media used forthe isolation of bacteria included nutrient agar,potatodextrose agar,Gaus

25、es No.1 agar medium,which wereautoclaved at 121 1C for 15 min,and pH value was adjustedto 9.29.8 10.Colonies of each group were picked out,purified three times by inoculating on the correspondingagar plates,and further transferred to agar slants.Pooled DNA samples of the fifth instar in the BLSS and

26、the conventional breeding groups were composed of DNAextracted from the selected 10 individuals.DNA wasextracted with a Promega DNA Kit(Promega,USA),quan-tified with a BioPhotometer(Eppendorf),and stored at?20 1C until used.2.3.Illumina Miseq sequencing and data analysisHigh-throughput sequencing wa

27、s conducted at MajorbioCo.,Ltd.(Shanghai,China).The bacterial 16S rRNA gene wasamplified with primers 338F(ACTCCTACGGGAGGCAGCA)and806R(GGACTACHVGGGTWTCTAAT)targeting the V3V4region(about 470 bp).The fungal 18S rRNA gene was ampli-fied with primers ITS1-1737F(GGA AGT AAA AGT CGT AACAAGG)and ITS2-2043

28、R(GCT GCG TTC TTC ATC GAT GC)targeting the ITS1ITS2 region(about 246 bp).The PCRamplification was conducted using specific primers withbarcode and high fidelity TrashStart Fastpfu DNA Polymerase(TransGen Biotech,China).PCR amplification was performedin a total volume of 20L containing 4L 5?FastPfu B

29、uffer,2L 2.5 mM dNTPs,0.8L 5M primers,0.4L FastPfuPolymerase and 10 ng DNA template.The bacterial 16S rRNAgene PCR thermal cycle profile was as follows:2 min at 95 1C;28 cycles of 30 s at 95 1C,30 s at 61 1C,45 s at 72 1C;final10 min at 72 1C,and cooling at 10 1C.The fungal 18S rRNAgene PCR profile

30、was similar to the bacterial profile exceptthat it had five more cycles.The Miseq sequencing was collecting the fluorescencesignal to read the sequence of DNA fragment.All sequenceswere divided depending on the similarity level and statisticalanalysis of biological information under 97%similar level

31、 ofOTU.The community structure was analyzed statistically atdifferent classification levels and visual analysis of communitystructure and phylogeny finally.2.4.Screening of cellulase-producing bacteria,amylase-producing bacteriaEach isolate was inoculated on carboxy methyl cellu-lose(CMC)agar plate

32、medium(0.1%CMCnutrient agar,X.Liang et al./Acta Astronautica 116(2015)247253248pH9.29.8)and soluble starch agar plate medium(0.1%starchnutrient agar,pH9.29.8)using a sterilized tooth-pick,and incubated at 30 1C for 48 h 11.2.5.Identification of enzyme-producing bacteriaThe enzyme-producing bacteria

33、were identified by 16SrDNA sequencing.DNA was extracted by boiling bacterialcell suspension in sterile distilled water 12.16S rRNAgenes were amplified with universal primers of 27F(50-GAGTTTGATCCTGGCTCAG-30)and1492R(50-CGGTTACCTTGTTACGACTT-30).PCR amplification was performed in atotal volume of 25L

34、containing 5L of DNA extract,1Lof each primer(10L mL?1),1L of deoxyribonucleotidetriphosphate(dNTP)mixture(10 mmol mL?1),3L of10?Taq PCR buffer(containing Mg2),and 0.5L ofTaq DNA polymerase(TaKaRa,China).Cycling conditionswere as follows:initial denaturation at 95 1C for fiveminutes,30 cycles of 94

35、1C for one minute,55 1C for oneminute,72 1C for one minute,and a final extension at72 1C for five minutes.PCR products were examined byelectrophoresis in a 1%agarose gel,and bands werevisualized by staining with ethidium bromide.PCR pro-ducts were further purified with the QIA quick PCRpurification

36、kit(Quiagen,http:/)andcloned into pMD18-T vector followed by sequencing.SequenceanalysiswasperformedusingtheBLAST(http:/www.ncbi.nlm.nih.gov).For the fungi,the ITSsequencewasamplifiedwithITS1(5-TCCGTAGGT-GAACCTGCGG-3)and ITS4(5-TCCTCCGCTTATTGATATGC-3)(Sangon Biotech).Cycling conditions were as follo

37、ws:94 1C 4 min;30 cycles of 94 1C 30 s;55 1C 30 s;72 1C 45 s,and an end extension of at 72 1C 7 min.Identificationswere based on 16S rRNA gene sequence and ITS sequencesimilarity.Phylogenetic tree of the sequence analysis wasconstructed from a matrix of pairwise genetic distancesby the neighbor-join

38、ing method(MEGA 5.0).The boot-strap analysis of 1000 replicates was performed 13.3.Results3.1.Shifts in microbial community structure andcomposition by MiseqThe total genomic DNA was extracted from the gut ofsilkworm larvae.The average length of sequence was470 bp for bacterial community and 300 bp

39、for fungalcommunity by high-throughput sequencing which wasperformed in the three sample types,namely,5 M,gutcontent sample from the fifth instar larvae of silkworms inTRW;5 L,gut content sample from the fifth instar larvae ofsilkworms in BRW;and 4 L,gut content sample from thefourth instar larvae o

40、f silkworms in BRW.Miseq sequencing generated total of 58,827 and 45,956reads of 1737F2043R(fungi primers)and 338F806R(bacter-ial primers)for the TRW and BRW samples.Low quality readswere filtered using the QIIMEs scripts and a total of 48,420(1737F2043R),38,814(338F806R)effective reads wereobtained

41、 after trimming the adapters,barcodes and primers.After denoising,filtering out chimeras and removing thearchaeal sequences,the fungi libraries of 4 L,5 L and 5 Mcontained 14,555,14,993 and 18,872 effective sequences,respectively.The bacteriallibraries of 4 L,5 L and 5 M contained13,210,14,949 and 1

42、0,655 effective sequences,respectively.Biodiversity of the two group samples was investigatedbased the analyses of OTUs.In terms of OTUs number,fungal communities had the richer diversity(46 OTUs),whereas bacterial one displayed considerably less richness(35 OTUs).Those results were supported by Ven

43、n commu-nities of fungi and bacteria(Fig.1).Diversity and composition of microbial communities inTRW and BRW were diverse at different instars(Fig.2).Each sample profile displayed a unique banding pattern.We have detected that the sample 4 L has more diversitythan 5 M and 5 L in bacterial group(Fig.

44、2A).It had found21 genotypes of bacteria and only 7 genotypes in 5 M and5 L.Meanwhile,sample 5 M has more diversity in fungigroup than 4 L and 5 L,the genotypes of fungi were 28,18and 13,respectively(Fig.2B).This analysis confirmed thatFig.1.Venn of fungal and bacterial communities of BRW and TRW ba

45、sed on Miseq sequencing(A)is bacteria(B)is fungi.X.Liang et al./Acta Astronautica 116(2015)247253249feeding with changed feedstuff at the beginning of thefourth instar resulted in great change in gut microbiota ofsilkworm in the BRW.3.2.Enumeration of the gut microfloraUsing the isolation procedure

46、described above,total of23 isolates were successfully collected from the gut ofsilkworm larvae under the TRW and BRW group.Therewere 14 strains isolated from the TRW(9 strains from NA,4 strains from PDA medium,3 strains from Gauses No.1agar medium).The rest were from the BRW(5 strains fromNA medium,

47、1 strain from PDA medium,3 strains fromGauses No.1 agar medium).Those results showed asimilar phenomenon to those observed by Miseq.3.3.Screening of cellulase-producing and amylase-producing bacteriaAccording to screening the isolates on CMCnutrient agar,after incubating at 30 1C for 48 h,the coloni

48、es with degrada-tion capacity were identified using the Congo red overlaymethod 14.For the Congo red method,plates were floodedwith 0.1%aqueous Congo red for 10 min and then washedwith 5%NaCl solution for 50 min.Cellulose production wasobserved as a clear zone of hydrolysis around bacterialcolony.Si

49、x and three isolates of cellulolytic bacteria wereobtained from TRW and BRW group,respectively.For theisolates of starchnutrient agar,the iodine method starchplates were flooded with iodine solution resulting in darkblue plates with uncolored zones where the starch had beendegraded.The isolates that

50、 could product amylase of theBRW and TRW group were 11 and 6,respectively.As identified above,we found three isolates named M1,M4,M11 were cellulolytic and amylolytic bacteria in theTRW group(Fig.3).Three isolates were all screened fromPDA plate.Meanwhile,L1,L5,L9 were the cellulolytic andamylolytic