Changes in Bacterioplankton Community Structure and Activity with Depth in a Eutrophic Lake

合集下载

Chitin-mediated changes in bacterial communities of the soil,

Chitin-mediated changes in bacterial communities of the soil,rhizosphere and within roots of cotton in relation to nematodecontrolJ.Hallmann 1,R.Rodrõguez-Kabana,J.W.Kloepper *Biological Control Institute,Alabama Agricultural Experiment Station,Department of Plant Pathology,Auburn University,Auburn,AL 36849-5409,USAAccepted 3September 1998AbstractChanges in microbial communities associated with nematode control were studied by comparing population numbers of fungiand bacteria in the soil and in internal root tissues (endorhiza)in non-amended and chitin-amended soils.Addition of chitin to soil at 1%(w/w)eliminated plant-parasitic nematodes in a ®rst planting of cotton cv.`Rowden'and signi®cantly reduced Meloidogyne incognita infestation in a second planting,con®rming long-term nematode suppressiveness induced by this organic amendment.The chitin amendment was associated with an increase in fungal and bacterial populations,especially those with chitinolytic activity.The bacterial communities of soil,rhizosphere and endorhiza were assessed by examining the taxonomic diversity of recoverable bacteria based on identi®cation with fatty acid analysis of sample sizes of 35soil and rhizosphere bacteria and 25endophytic bacteria.All major bacterial species which formed at least 2%of the total population in non-amended soils and rhizospheres also occurred with chitin amendment.In contrast,chitin-amended soils and rhizospheres yielded several species which were not found without chitin amendment,including Aureobacterium testaceum ,Corynebacterium aquaticum and Rathayibacter tritici .Burkholderia cepacia was recovered from both amended and non-amended soils and rhizospheres,but it was most abundant with chitin amendment at the end of the ®rst cotton planting.Soil was probably the major source for bacterial endophytes of cotton roots,since nearly all endophytic bacteria were also found in the soil or rhizosphere.However,two dominant genera in the soil and rhizosphere,Bacillus and Arthrobacter ,were not detected as endophytes.Chitin amendment exhibited a further speci®c in¯uence on the endophytic bacterial community;Phyllobacterium rubiacearum was not a common endophyte following chitin amendment,even though chitin amendment stimulated its populations in non-planted soil.Burkholderia cepacia ,found in similar numbers in the soil of both treatments,was the dominant endophyte in plants grown in chitin-amended soil but rarely colonized cotton roots grown in non-amended soil.These results indicate that application of an organic amendment can lead to modi®cations of the bacterial communities of the soil,rhizosphere and endorhiza.#1999Elsevier Science Ltd.All rights reserved.Keywords:Chitin amendment;Endophytic bacteria;Gossypium hirsutum ;Meloidogyne incognita ;Rhizosphere bacteria1.IntroductionNaturally-occurring nematode suppressiveness has been reported for several agricultural systems (Stirling et al.,1979;Kerry,1982;Kluepfel et al.,1993),but suppressiveness can also be induced by crop rotationwith antagonistic plants such as switchgrass (Panicum virgatum )(Kokalis-Burelle et al.,1995)and velvetbean (Mucuna deeringiana )(Vargas et al.,1994)or organic amendments including pine bark (Kokalis-Burelle et al.,1994),hemicellulose (Culbreath et al.,1985)and chitin (Mankau and Das,1969;Spiegel et al.,1986;Rodrõguez-Kabana and Morgan-Jones,1987).A major component of the suppressiveness of chitin amend-ments is believed to be biotic and several reports con-®rm increased numbers of nematode antagonistic microorganisms associated with chitin-induced sup-Soil Biology and Biochemistry 31(1999)551±5600038-0717/99/$19.00#1999Elsevier Science Ltd.All rights reserved.PII:S 0038-0717(98)00146-1PERGAMON1Present address:Institut fu r P¯anzenkrankheiten,Phytomedizin in Bodeno kosystemen,Nuûallee 9,53115Bonn,Germany.*Corresponding author.Tel.:+1-334-844-5003;fax:+1-334-844-5067;e-mail:jkloeppe@.pressive soils(Godoy et al.,1983;Rodrõguez-Ka bana et al.,1984).Extensive work has been done over the past years on fungi associated with chitin amendments (Godoy et al.,1983;Rodrõguez-Ka bana et al.,1984); however,information on bacterial community struc-ture and the role of bacteria in chitin-induced suppres-siveness is still very limited.We chose chitin amendments as a model system to study the e ect of suppressiveness on bacterial diversity in the soil and endorhiza.Endophytic bacteria were included in this study because they colonize the same root tissues as sedentary plant-parasitic nematodes.This association of endophytic bacteria with nematodes throughout the nematode life cycles makes these bacteria excellent candidates for biocontrol strategies.Chitin is a structural component of some fungi, insects,various crustaceans and nematode eggs.In egg shells of tylenchoid nematodes,chitin is located between the outer vitelline layer and the inner lipid layer and may occur in association with proteins(Bird and Bird,1991).The breakdown of this polymer by chitinases can cause premature hatch,resulting in fewer viable juveniles(Mercer et al.,1992).In the soil, chitinases are produced by some actinomycetes (Mitchell and Alexander,1962),fungi(Mian et al., 1982)and bacteria(Ordentlich et al.,1988;Inbar and Chet,1991),but chitinases are also released by many plants as part of their defense mechanism against var-ious pathogens(Punja and Zhang,1993)and plant-parasitic nematodes(Roberts et al.,1992).Chitinases depolymerize the chitin polymer into N-acetylglucosa-mine and chitobiose.Further microbial activity results in the deamination of the sugar and accumulation of ammonium ions and nitrates(Rodrõguez-Ka bana et al.,1983).Nematicidal concentrations of ammonia in association with a newly formed chitinolytic micro¯ora are believed to cause nematode suppressiveness(Mian et al.,1982;Godoy et al.,1983).Benhamou et al. (1994)have shown that chitosan,the deacetylated de-rivative of chitin,induces systemic plant resistance against Fusarium oxysporum f.sp.radicis-lycopersici in tomato when applied as a seed treatment or soil amendment.This suggests that plant defense mechan-isms might contribute to the overall nematode suppres-sion.Our objective was to determine if the chitin-mediated suppression of plant parasitic nematodes is related to changes in bacterial communities in soils, rhizospheres or within cotton roots.2.Material and methods2.1.Greenhouse studiesThe e ect of chitin on Meloidogyne incognita was studied under greenhouse conditions using soil from the E.V.Smith Research Center,Plant Breeding Unit of the Alabama Agricultural Experiment Station near Tallassee,Alabama,USA.The soil,which was col-lected from a cotton®eld at the end of the season,was a sandy loam with pH=6.5and organic matter con-tent<1%(w/w).The soil was naturally infested with M.incognita,Hoplolaimus spp.and Paratrichodorus spp.at populations of3,7and6nematodes100cmÀ3, respectively,and with free-living species at44nema-todes100cmÀ3.This®eld soil was mixed1:1(w/w) with sand,hereafter referred to as soil.Crustacean chitin¯akes(United States Biochemical Corporation, Cleveland,OH)were ground to pass a2mm sieve and mixed into half of the soil to give a®nal concentration of1%(w/w).Chitin-amended and non-amended soil were transferred into1000cmÀ3cylindrical pots and maintained moist at about60%®eld capacity for3 weeks in a greenhouse at20±308C to allow decompo-sition of the chitin.The total number of pots®lled with either non-amended soil or chitin-amended soil was further divided into two sets of equal numbers with one half of the pots of each treatment seeded with cotton(Gossypium hirsutum L.cv.`Rowden')at3 seeds per pot.The seedlings were thinned to1plant potÀ1after emergence.The experiment consisted of four treatments:non-amended soil without cotton, non-amended soil planted with cotton,chitin-amended soil without cotton,and chitin-amended soil planted with cotton.The experiment was set up as a random-ized complete block design with eight replications.All replications were used for recording plant growth and nematode infestation.To describe soil physical and chemical properties as well as microbial populations, four replications were su cient to demonstrate signi®-cant di erences,so the®rst four replications of each treatment were used.Plants were fertilized in alternate weeks with15ml of Peters'fertilizer(20±20±20)sol-ution(Scotts-Sierra,Marysville,OH).At54d after chitin amendment,the complete cotton plants were harvested.Plant fresh weight was recorded and the roots were processed for isolation of endophy-tic bacteria.The remaining soil was placed back into the pots and replanted with cotton cv.`Rowden'.Due to low numbers of plant-parasitic nematodes in the in-itial soil of the®rst cotton crop,all pots were inocu-lated with approximately3,000eggs of M.incognita8 d after planting using the alginate®lm method described by Rodrõguez-Ka bana et al.(1994).Four ®lms of approximately750eggs®lmÀ1were buried around the cotton root at about1cm depth and1cm distance from the tap root.For the®lms,eggs were extracted from galled cotton roots by the NaOCl-method(Hussey and Barker,1973).Plants were ferti-lized once with15ml Peters'solution3weeks after planting and the experiment was terminated6weeks after the second planting.Plant growth was recordedJ.Hallmann et al./Soil Biology and Biochemistry31(1999)551±560 552and the gall index determined using a0±10scale with 0=no galls and10=maximum gall infestation (Zeck,1971).The experiment lasted for96d from the time of chitin application to the®nal evaluation of the second cotton crop.The experiment comprised four sampling times:d0 (before chitin amendment),d21(before planting),d 54(end of®rst cotton planting)and d98(end of sec-ond cotton planting).Soil and rhizosphere samples of various size were taken to measure pH and to estimate nematode,fungal,bacterial and chitinolytic popu-lations as well as bacterial diversity at each sample time.The term rhizosphere was used for soil thoroughly rooted with cotton.After54d,shoot and root fresh weight,numbers of galls and population sizes of indigenous fungal,bacterial and chitinolytic endophytes as well as endophytic bacterial diversity were determined.At the®nal sampling,only plant growth and gall index were recorded.2.2.Soil pH and nematode populationsFor each sampling,soil was transferred into4-l ca-pacity polyethylene bags,thoroughly mixed and ali-quots were taken to measure the di erent variables. Soil pH was determined in a suspension of10g moist soil and10ml demineralized water after shaking for 30min.For nematode counts,100cmÀ3soil aliquots were incubated for72h using a modi®ed Baerman technique(Rodrõguez-Ka bana and Pope,1981).The numbers of nematodes were determined for the main genera of plant-parasitic nematodes and free living nematodes.2.3.Soil and rhizosphere microbial populationsSoil microbial populations were determined at d0,d 21and d54and rhizosphere populations at d54.Soil (10g)was added to100ml20mM sterile potassium phosphate bu er,pH=7.0.The¯asks were shaken on an orbital shaker at200rpm for30min.A serial dilution was prepared and52m l aliquots of dilutions 10À2,10À4and10À6were plated on three media using a spiral plater(Spiral System,Cincinnati,OH).The media were:5%strength tryptic soy agar(5%TSA) (Difco Laboratories,Detroit,MI)containing150mg cycloheximide lÀ1to support the growth of a broad range of bacteria;Ohio agar(Johnson and Curl,1972) containing150mg streptomycin lÀ1for fungal popu-lations and chitin agar containing0.2%colloidal chitin (Godoy et al.,1982)to estimate total chitinolytic microorganisms.Only microorganisms producing a clearing zone around the colony were counted as chiti-nolytic.2.4.Plant preparation and surface-disinfestation Fresh weights of cotton roots and stems were recorded and the roots were washed with tap water. The roots were then surface-disinfested in a mixture of 1.05%sodium hypochlorite(Clorox1household bleach diluted1:5with tap water)and0.1%Tween20 (Fisher Biotech,Fair Lawn,NJ)for60s followed by three rinses in sterile phosphate bu er.The whole root system was imprinted on full strength TSA to check for root surface contamination.If microbial growth on the TSA check occurred within48h,the surface-disin-festation was considered as incomplete and the samples were rejected.Following the sterility check,roots were triturated in5times the root weight in sterile phos-phate bu er(w/v)with mortar and pestle.The mace-rate was serially diluted in sterile H20and dilutions 100,10À2and10À4were plated on each of the three media indicated above.Agar plates were incubated at 278C for48h(5%TSA,Ohio agar)or96h(chitin agar),respectively.2.5.Bacterial diversitySingle colonies were selected from5%TSA plates by starting at the outside of the plate and transferring each colony along the spiral line onto fresh TSA plates until a total of50colonies was taken per sample.The bacteria were incubated at278C and bacterial purity was checked visually after48h.One loop with ap-proximately50mg bacterial cells was then transferred into1.2ml vials containing0.8ml sterile tryptic soy broth(TSB)(Difco,Detroit,MI)and0.2ml glycerine. The samples were stored in a Nalgene1cryobox at À808C until processed for identi®cation.For bacterial diversity,the®rst35colonies per plate from soil samples were identi®ed,while for endophytes,the®rst 25colonies per plate were identi®ing rarefrac-tion analysis,these numbers were previously reported as su cient for community studies at the genus level (Maha ee and Kloepper,1997).Bacterial identi®cation was based on analysis of fatty acid methyl-esters (FAMEs)of total cellular fatty acids(Sasser,1990). The extraction procedure was as described by McInroy and Kloepper(1995)and samples were processed with the microbial identi®cation system(MIS)of MIDI (Newark,DE).FAME peaks were identi®ed by the MIS software and bacterial isolates were identi®ed using the MIS`Aerobe Library'(Version 3.7). Bacterial strains with a similarity index below0.100 were considered unidenti®ed.Bacterial diversity was characterized at the genus level with two indices:rich-ness(total number of genera)and Hill's diversity num-ber N1which combined richness and evenness (Ludwig and Reynolds,1988).J.Hallmann et al./Soil Biology and Biochemistry31(1999)551±5605532.6.Statistical analysisBacterial and fungal population numbers were trans-formed to log 10cfu g À1soil or root,respectively.Statistical analysis was performed using SAS general linear models and LSD procedures (SAS,Cary,NC).Unless otherwise stated,di erences referred to in the text were signi®cant at P =0.05.3.ResultsThere were no di erences in shoot fresh weights of cotton plants of the ®rst planting grown in soil amended with 1%chitin and those grown in non-amended soil (Table 1).Nevertheless,root fresh weight was signi®cantly reduced for plants grown in chitin-amended soil averaging 320mg plant À1compared to 730mg plant À1for cotton grown in non-amended soil.The shoot to root ratio was signi®cantly higher for plants grown in chitin-amended soil (5.75)than in non-amended soil (2.40).The initial nematode population was 16plant-para-sitic and 44free-living nematodes 100cm À3soil (Table 2).Individual plant-parasitic nematodes were observed throughout the experiment in the non-amended soil but not in chitin-amended soil.Some Meloidogyne spp.galls were formed on plants fromnon-amended soil and no galls were observed in cotton roots from chitin-amended soil (Table 1).Populations of free-living nematodes were signi®cantly higher in chitin-amended soil than in non-amended soil.Soil-pH was initially 7.3and it changed by d 21to 6.9and 6.9for non-amended and chitin-amended soil,respectively.At d 54,pH ranged from 6.6±6.9in non-amended soil and between pH 4.9±5.0for chitin-amended soil.The second cotton planting was set up to evaluate long-term e ects of chitin amendment on nematode suppression.To determine suppressiveness,the soil of each of the four treatments was challenge-inoculated with M.incognita eggs.Cotton grown in chitin-amended soil previously planted with cotton yielded the highest shoot weight (4.99g)(Table 3),whereas shoot weight in non-amended soil was signi®cantly lower for both,previously non-planted (3.75g)and planted (2.41g)soil.Both treatments lacking cotton crops in the ®rst planting resulted in the lowest root weight,with 2.24g for non-amended soil and 2.06g for chitin-amended soil.The shoot to root ratio was lowest for cotton grown in non-amended soil pre-viously planted with cotton (0.83)and highest for cot-ton grown in chitin-amended soil previously without cotton (2.08).Chitin amendment signi®cantly reduced the gall index from 3.71and 3.28,respectively,for cottonTable 1E ect of 1%chitin in soil on plant growth and Meloidogyne incognita infestation of the ®rst cotton planting 54d after amendment Treatment Shoot fresh weight (g)Root fresh weight (mg)Shoot to root ratio Number of galls plant À1Control 1.75a 730a 2.40b 8a Chitin 1.84a 320b 5.75a 0b LSD0.441300.803.3Means with the same letter are not signi®cantly di erent at P =0.05,n =8.Number of replicates =8.Table 2E ects of 1%chitin amendment to soil on cotton cv.`Rowden'on soil nematode populations Nematode taxonNumber of nematodes 100cm À3soil of indicated treatments a over time d 0d 21d 541131234Meloidogyne3000000(Para -)Trichodorus 6100400Hoplolaimus7400000Tylenchorhynchus 0000100Free-living species 4433181443034244338Total6038181443534244338aTreatments were:1=non-amended soil without cotton,2=non-amended soil with cotton,3=chitin-amended soil without cotton,and 4=chitin-amended soil with cotton.J.Hallmann et al./Soil Biology and Biochemistry 31(1999)551±560554grown in non-amended soil previously non-planted and planted with cotton,to0.75and0.86for cotton grown in chitin-amended soil(Table3). Amendment of soil with1%chitin generally resulted in signi®cant increases in soil populations of total cul-turable aerobic bacteria on5%TSA,fungi on Ohio agar,relative to populations in non-amended soil (Table4).In addition,bacterial and fungal popu-lations of the rhizosphere and inside roots,as detected on5%TSA and Ohio agar,were signi®cantly higher, following chitin amendment,at21and54d after planting.Numbers of chitinolytic microorganisms in soil and rhizosphere samples were also signi®cantly enhanced by chitin amendment.The total spectrum of soil bacteria isolated from chitin-amended and non-amended soil is shown in Table5.Gram-positive bacteria accounted for73.8% of the total number of species from the test soil(d0) and remained dominant in non-amended soil at d21 and d54.In contrast,in chitin-amended soil,Gram-positive bacteria were less frequent than Gram-nega-tive bacteria at21and54d.Most species occurred in low numbers throughout the experiment,regardless of treatment or sampling time.Among the predominant taxa,some e ects of chitin-amendment were noted. Arthrobacter and Bacillus were the two dominant Gram-positive bacteria in the test soil(d0)and in the non-amended soil at d21;however,their frequency of recovery was greatly reduced at d21in chitin-amended soil.The frequency of Arthrobacter at d54in soils and rhizospheres was about90%less from chitin-amended than from non-amended treatments.Chitin amend-ment also led to a decreased frequency of recovering Burkholderia spp from soil at d21and54,compared to the non-amended soil;however,rhizosphere popu-lations were higher from chitin-amendments than from the non-amended treatment.Some species which con-tributed at least5%of the total population were only recovered following chitin-amendment,including Aureobacterium testaceum,Corynebacterium aquaticum and Rathayibacter tritici,while all species found in non-amended soil were also recovered from chitin-Table3E ects of1%chitin in soil on plant growth and gall index of Meloidogyne incognita infestation of the second cotton planting96d after amend-mentPrevious treatment a Shoot weight(g)Root weight(g)Shoot to root ratio Gall index b Àchitin,Àcotton 3.75b 2.24bc 1.78ab 3.71aÀchitin,+cotton 2.41c 2.85ab0.83c 3.28a+chitin,Àcotton 4.27ab 2.06c 2.08a0.75b+chitin,+cotton 4.99a 3.22a 1.64b0.86b LSD 1.140.640.44 2.10a In the second cotton planting all treatments were planted with cotton.However,to compare date with the®rst cotton planting,this table lists the treatments as described for the®rst cotton planting.b Gall index on a scale from0±10with0=no galls and10=maximum number of galls (Zeck,1971).Means with the same letter are not signi®cantly di erent at P=0.05,n=8.Table4E ect of1%chitin amendment on microbial populations of soil,rhizosphere and endorhizaSample time(d)Sample(S,R,E)a Chitin amendment(+orÀ)log cfu gÀ15%TSA Ohio agar chitin agar d0SÀ 6.39 3.74 5.29d21SÀ 6.83b 3.67b 5.66b S+8.29a 4.59a7.14ad54SÀ 6.38b 3.80b 5.51b RÀ 6.39b 3.90b 5.51bS+7.11a 6.17a 6.55aR+7.10a 6.17a 6.84aEÀ 5.43b 1.67b 1.41bE+ 5.74a 3.36a 2.87a S=non-planted soil;R=rhizosphere soil;E=endorhiza.b Data were not statistically analyzed,since chitinolytic bacteria were only observed in3out of8roots for the control and5out of8roots grown in chitin-amended soil;presented is the average chitinolytic population for those samples from which chitinolytic microorganisms were recovered.Means with the same letter within the same sampling time are not sig-ni®cantly di erent at P=0.05,n=8.J.Hallmann et al./Soil Biology and Biochemistry31(1999)551±560555amended soil.Analysis of bacterial diversity (Table 6)using measurements of richness and diver-sity,revealed that chitin-amendment resulted in sig-ni®cantly more genera at 54d than non-amendment.Diversity,measured by the N1index,was signi®cantly higher in chitin-amended treatments than non-amended at d 21.The range of endophytic bacteria isolated from sur-face-disinfested cotton roots is presented in Table 7.Except for one strain,all isolated bacteria belonged toTable 5Temporal changes in the isolation frequency a (%)of indigenous soil bacteria a ected by chitin amendment (1%)and planting with cotton cv.`Rowden'Bacterial speciesEBC bSample (S,R)c (Chitin amendment,+/À)day 0day 21day 54S S S S R S R ÀÀ+ÀÀ++Gram-positive A.globiformis 11.5 4.4 3.27.7 6.10.7A.ilicis9.017.87.817.519.6Arthrobacter ,total23.022.913.431.528.50.7 2.1Aureobacterium testaceum 3.512.9Bacillus megaterium 17.29.60.87.7 5.411.39.3B.pumilus 9.08.90.88.4 4.7 2.8 2.1Bacillus ,total36.023.5 1.619.617.615.516.4Corynebacterium aquaticum 1.65.67.1Micrococcus luteusEBC0.80.7 2.7Mycobacterium parafortuitum 0.8Paenibacillus polymyxa 1.6 3.7 5.6 4.10.7 1.4Paenibacillus ,total 5.77.4 2.49.88.2 3.5 2.1Rathayibacter tritici 1.69.222.9Subtotal73.861.528.365.762.140.872.9Gram-negativeAgrobacterium radiobacter EBC 7.9 1.4 2.0 1.4 1.4Burkholderia cepacia EBC 1.5 1.6 2.1 4.17.012.9B.pickettiiEBC 0.8 6.7 2.410.5 2.70.7Burkholderia ,total 0.88.2 4.013.37.57.712.9Cytophaga johnsonae0.79.5Phyllobacterium rubiacearum EBC1.60.7 5.5 4.70.72.1Pseudomonas chlororaphis 1.6 3.0 2.4 2.8 2.7Pseudomonas ,total 3.35.99.5 4.2 3.4Variovorax paradoxus 10.414.22.83.40.7Vibrio cholerae6.3Xanthomonas campestris EBC2.00.7 4.3Subtotal 9.828.158.325.225.750.720.7Unidenti®ed 16.410.413.49.112.28.5 6.4Grand total100100100100100100100Number of species31323132363922aIsolation frequency per treatment is based on 35identi®ed isolates per replication and 4replications per treatment.Predominant species are those isolated at >5%frequency.Less frequently isolated Gram-positive species include Arthrobacter citreus ,A.nicotianae ,A.pascens ,A.proto-phormiae ,A.saperdae ,A.uratoxydans and A.viscosus ;Aureobacterium barkeri ,A.seperdae and A.liquefaciens ;Brevibacillus brevis ;Bacillus cer-eus ,B.chitinosporus ,B.circulans ,B.freudenreichii ,terosporus ,B.longisporus ,B.mycoides ,B.pumilus ,B.sphaericus and B.thuringiensis ;Brevibacterium helvolum ;Cellulomonas biazotae , C.cartae , C.®mi ,and C.gelida ;Clavibacter michiganensis ;Corynebacterium bovis ;Curtobacterium ¯accumfaciens ;Gluconobacter asaii ;Kurthia gibsonii ;Microbacterium imperiale ;Micrococcus kristinae ;Paenibacillus pabuli ;Rhodococcus chubuensis and Staphylococcus aureus .Less frequently isolated Gram-negative species include Acidovorax avenae and A.facilis ;Actinobacillus lignieresii ;Alcaligenes eutophus and A.xykisixydans ;Azospirillum brasilense ;Chryseobacterium balustinum and C.meningosepticum ;Citrobacter freundii ;Comamonas acidovorans and C.testosteroni ;Enterobacter asburiae and E.cancerogenus ;Erwinia amylovora and E.chry-santhemi ;Escherichia coli ;Flavobacterium aquatile and F.yabuuchiae ;Kluyvera cryocrescens ;Methylobacterium mesophilicum and M.zatmanii ,Ochrobactrum anthropi ;Phyllobacterium rubiacearum and P.myrsinacearum ;Pseudomonas cichorii ,P.¯uorescens ,P.marginalis ,P.putida ,P.sac-charophila and P.savastanoi ;Ralstonia solanacearum ;Rhodobacter adriaticus ;Sphingomonas capsulata ;Vibrio ¯uvialis ;and Xanthobacter agilis .b EBC =endorhiza bacterial community.c S =nonplanted soil;R =rhizosphere.J.Hallmann et al./Soil Biology and Biochemistry 31(1999)551±560556Gram-negative species.The endophytic bacterial com-munity of cotton grown in non-amended soil was dominated by Phyllobacterium rubiacearum which accounted for61%of the total population.Other strains with signi®cant occurrence were Burkholderia cepacia(9%),B.pickettii(9%)and Phyllobacterium myrsinacearum(8%).In contrast,the endophytic population isolated from cotton roots grown in chitin-amended soil was dominated by B.cepacia with73% of the recovered population.The number of bacterial species within one plant sample ranged from2to8 species(data not shown);however,all samples were dominated by one strain with a minimum isolation fre-quency of at least44%of the total population. Di erences in the richness and diversity of bacterial endophytes between cotton grown in non-amended and chitin-amended soil were not signi®cantly di erent (Table6).4.DiscussionOur results indicate that amendment of soil with chitin caused soil suppressiveness to plant parasitic nematodes and resulted in changes in the bacterial communities of the soil,rhizosphere and endorhiza.Table6E ect of chitin amendment(1%)on soil bacterial diversity at three sampling times and the endophytic population of cotton roots at the third sampling timeIndex Treatment Day0Day21Day54Endophytes Richness a non-amended7.7510.00a14.50b 4.00a chitin-amended11.75a17.50a 3.75a Diversity N1b non-amended 5.247.02b8.29a 2.48a chitin-amended9.50a12.59a 2.53aa Richness is a measure for number of genera.b Diversity N1(modi®ed by Simpson in Ludwig and Reynolds,1988)is a measure for abundant taxa.Means with the same letter were not signi®cantly di erent at P=0.05,n=4.Table7E ect of chitin amendment on endophytic bacterial communities in cotton rootsBacterial taxon Also found in Mean%of total isolates identi®ed as the indicated taxon asoil rhizosphere non-amended soil chitin-amended soil Gram-positiveMicrococcus luteus++10Subtotal10Gram-negativeAgrobacterium radiobacter++26Bordetella bronchisepticaÀÀ01 Burkholderia cepacia++973B.gladioliÀÀ10B.pickettii++90 Burkholderia,total1973 Comamonas acidovorans+À40 Enterobacter asburiae+À03E.cloacaeÀÀ01E.taylorae+À03Kluyvera cryocrescens+À02 Phyllobacterium myrsinacearum+À80P.rubiacearum+À617 Phyllobacterium,total697 Pseudomonas saccharophila+À01 Salmonella spp.+À03 Xanthomonas campestris++10Subtotal:identi®ed taxa96100Subtotal:unidenti®ed taxa40a Isolations were made from surface-disinfested cotton roots grown in non-amended and chitin-amended(1%)soil54d after chitin amendment, n=4.J.Hallmann et al./Soil Biology and Biochemistry31(1999)551±560557。

galacto-oligosaccharides and other prebiotics

R E V I E W A RT I C L EBacterial metabolism and health-related effects of galacto-oligosaccharides and other prebioticsG.T.Macfarlane,H.Steed and S.MacfarlaneDundee University Gut Group,Ninewells Hospital Medical School,Dundee,UKIntroductionThere is still a widespread belief that while food intake may regulate certain metabolic activities associated with intestinal micro-organisms,changing diet has little effect on the overall composition and structure of microbial communities in the human gut(Macfarlane and Macfar-lane2003).However,the introduction of prebiotics into the diet in recent years has raised a serious challenge to this concept,and it is increasingly being recognized that the species composition of the microbiota,as well as many of its physiological traits,can be modiﬁed by rela-tively small changes in food consumption.The original deﬁnition of a prebiotic was put forward by Gibson and Roberfroid(1995)as‘a nondigestible food ingredient that beneﬁcially affects the host by selectively stimulating the growth and⁄or activity of one or a limited number of bacteria in the colon and thus improves health’.This has recently been amended to‘A prebiotic is a selectively fermented ingredient that allows speciﬁc changes,both in the composition and⁄or activity in the gastrointestinal microbiota that confers beneﬁts upon host well-being and health.’In practice,the beneﬁcial bacteria that serve as targets for prebiotics have been almost exclusively biﬁdobacteria and lactobacilli(Gibson et al.1999;Bouhnik et al.2004).Unlike the situation with probiotics,where allochthonous micro-organisms are being introduced to the gut,and have to compete against established colonic communities,an advantage of using prebiotics to modify gut function is that the target bacteria are already commensal to the large intestine.As a consequence,prebiotics are arguably a more practical and efﬁcient way to manipulate the gut microﬂora than probi-otics.However,the other side of the coin is that if for any reason,such as disease,ageing,antibiotic or drug therapy,the appropriate health-promoting species are notKeywordsbiﬁdobacteria,fermentation,fructo-oligosaccharides,galacto-oligosaccharides,gut bacteria,gut health,immunomodulation, prebiotics.CorrespondenceG.T.Macfarlane,Dundee University Gut Group,Ninewells Hospital Medical School, Dundee DD19SY,UK.E-mail:g.t.macfarlane@2007⁄0649:received23April2007,revised 18June2007and accepted19June2007doi:10.1111/j.1365-2672.2007.03520.x SummaryMost studies involving prebiotic oligosaccharides have been carried out using inulin and its fructo-oligosaccharide(FOS)derivatives,together with various forms of galacto-oligosaccharides(GOS).Although many intestinal bacteria are able to grow on these carbohydrates,most investigations have demonstrated that the growth of biﬁdobacteria,and to a lesser degree lactobacilli,is particu-larly favoured.Because of their safety,stability,organoleptic properties,resis-tance to digestion in the upper bowel and fermentability in the colon,as well as their abilities to promote the growth of beneﬁcial bacteria in the gut,these prebiotics are being increasingly incorporated into the Western diet.Inulin-derived oligosaccharides and GOS are mildly laxative,but can result inﬂatu-lence and osmotic diarrhoea if taken in large amounts.However,their effects on large bowel habit are relatively minor.Although the literature dealing with the health signiﬁcance of prebiotics is not as extensive as that concerning pro-biotics,considerable evidence has accrued showing that consumption of GOS and FOS can have signiﬁcant health beneﬁts,particularly in relation to their putative anti-cancer properties,inﬂuence on mineral absorption,lipid metabo-lism,and anti-inﬂammatory and other immune effects such as atopic disease. In many instances,prebiotics seem to be more effective when used as part of a synbiotic combination.Journal of Applied Microbiology ISSN1364-5072present in the bowel,the prebiotic is unlikely to be effective.Potential prebiotic oligosaccharides can be classiﬁed according to their chemical constituents and degree of polymerization(d.p.),and include manno-oligosaccha-rides(Zentek et al.2002),pectic-oligosaccharides(Olano-Martin et al.2003),soybean-oligosaccharides(Saito et al. 1992),isomalto-oligosaccharides(Morgan et al.1992), xylo-oligosaccharides(Campbell et al.1997)and lactulose (Tuohy et al.2002).However,as reviewed recently(Mac-farlane et al.2006),the vast majority of studies on pre-biotics have focused on inulin,fructo-oligosaccharides (FOS)and galacto-oligosaccharides⁄transgalactosylated-oligosaccharides(GOS⁄TOS).This latter group of carbo-hydrates in particular,has a history of safe commercial use, and are not classiﬁed under the category of novel foods. FOS and GOS are not sensitive to gastric acid and do not serve as substrates for hydrolytic enzymes in the upper digestive tract.Japan was a pioneer in adding short-chain FOS to commercially available foodstuffs,and ‘Neosugar’can be found in more than500food products (Guarner2005).GOS are stable at high temperatures in acidic environments and the caloriﬁc value of these oligo-saccharides is only1Æ7kcal g)1,which makes them of particular interest to the food and drink industry,for both their prebiotic properties,and their use as sweeten-ers,especially in confectionary,acidic beverages and fer-mented milks(Watanuki et al.1996).The caloriﬁc energy value of FOS is similarly low,at1Æ5kcal g)1(Roberfroid 2005).The consumption of8g of oligofructose per day in20healthy subjects has been shown to promote satiety and reduce food intake(Cani et al.2006).Numerous health claims have been made on behalf of prebiotics from investigations undertaken in vivo and in vitro.Much of this work has been carried out with FOS alone,or in combination with inulin,FOS and GOS in combination, and to a lesser extent,with GOS alone(Table1).More-over,a number of studies have been carried out using synbiotics,which are combinations of a probiotic and prebiotic.The rationale here is that the prebiotic stimu-lates growth of the probiotic in the gut,giving the organ-ism a competitive advantage over indigenous species. While most research interest has focused on the ferment-ability and biﬁdogenicity of oligosaccharide prebiotics, in vitro studies have shown that a number of these substances mimic eurkaryotic cell surface receptors that virulent bacteria adhere to as part of the pathogenicity process.For example,GOS was shown to inhibit attach-ment of EPEC(enteropathogenic Escherichia coli)to HEp-2 and Caco-2cells,and to be more effective than either inulin or FOS(Shoaf et al.2006).Moreover,TOS was found to enhance the protective abilities of Biﬁdobacterium breve in mice infected with Salmonella enterica serovar Typhimurium(Asahara et al.2001).In addition to the anti-adherent effects of some oligosaccharides,short-chain chito-oligosaccharides have been reported to have speciﬁc anti-bacterial properties(Sekiguchi et al.1994).If mani-fested in vivo in humans,these qualities could in principle enable prebiotic oligosaccharides to be protective in the small intestine,as well as in the colon;however,it is unclear at the moment whether sufﬁcient amounts could be delivered to have any signiﬁcant therapeutic effects. Galacto-oligosaccharidesGOS in human milkOne of the earliest sources of GOS was human milk, which contains approx.7%carbohydrates,90%of which is lactose,and a variety of oligosaccharides based on lac-tose(ESPGAN Committee on Nutrition.1977;Miller et al.1994).Milk contains a greater proportion of neutral (1%)compared to acidic(0Æ1%)oligosaccharides(Thurl et al.1996;Boehm et al.2005).Oligosaccharides are the third largest component of human milk(Newburg1997), and high levels are found in the colostrum where these substances constitute up to24%of total colostrum carbo-hydrates.Concentrations of these substances in breast milk steadily decrease to between19%and15%in the ﬁrst2months after birth(Miller et al.1994).Oligosac-charides in breast milk can reach concentrations as high as8–12g l)1(Kunz and Rudloff1996;Kunz et al.2000), which is100times greater than in cow’s milk.The princi-pal sugar components of oligosaccharides in human milk are sialic acid,N-acetylglucosamine,l-fucose,d-glucose and d-galactose.This results in a complex mix of over 130different oligosaccharides,because of the great variety of different sugar combinations that are possible(Brand-Miller and McVeagh1999).The type of oligosaccharides produced is inﬂuenced by the mother’s Lewis blood group.Human milk contains a large amount of galactose with the backbone structure based on lactose(galactose–glucose)plus a further external galactose residue.This leads to the formation of three galactosyl-lactoses,3¢,4¢and6¢-galactosyl-lactose(Boehm et al.2005).6¢-Galacto-syllactose is found in amounts ranging between2Æ0–3Æ9mg l)1(Yamashita and Kobata1974),and the total concentration of GOS in human milk is approx.1g l)1 (Angus et al.2005).At the beginning of the last century,it was thought that there were factors in human milk that could promote the growth of certain types of intestinal bacteria(Moro 1900).Biﬁdogenic nucleotides have been detected in human milk(Gil and Rueda2000),and many studies using conventional culture and molecular techniques for bacterial identiﬁcation have shown that breast-fed infantsGalacto-oligosaccharide prebiotics G.T.Macfarlane et al.have an intestinal microbiota that is dominated by biﬁdo-bacteria,which differs from that of infants fed on cow’s milk.These microbiotas are characterized by slightly lower counts of biﬁdobacteria,with greater numbers of more potentially harmful organisms such as clostridia and enterococci(Harmsen et al.2000;Macfarlane et al.2004). Higher levels of ammonia,amines and phenols and other potentially harmful substances have also been found in babies fed powdered milk products(Heavey et al.2003). The preponderance of biﬁdobacteria in breast-fed babies is thought to result from their abilities to utilize oligosac-charides in breast milk,including GOS(Harmsen et al. 2000;Newburg2000),which infants cannot digest in the upper gut(Engfer et al.2000).While it would be difﬁcultTable1Microbiological changes reported in human feeding studies with galacto-oligosaccharides(GOS)Substrate Type of study Delivery Effect on microbiota ReferenceGOS Placebo controlled studyinvolving12subjects 10g prebiotic fed daily for8weeksFaecal excretion ofbiﬁdobacteria andlactobacilli increasedIto et al.(1990)GOS Feeding study with12subjects 2Æ5g prebiotic given per dayfor3weeksIncreased biﬁdobacteria infaeces,reductions innumbers of clostridia andbacteroidesIto et al.(1993)Transgalactosylated-oligosaccharides (TOS)Feeding study with8volunteersSubjects given10g TOS for3weeksSigniﬁcant increases in faecalbiﬁdobacteria,enterobacteria unaffectedBouhnik et al.(1997)TOS Parallel study involving40healthy subjects 7Æ5or15g prebiotic fed perday for3weeksNo effects on biﬁdobacteria.Lactobacilli increased in15g day)1group,noeffects on clostridia,smallreductions in enterobacteriaAlles et al.(1999a)GOS⁄fructo-oligosaccharide (FOS)Placebo-controlled studywith90term infantsInfant formula supplementedwith either4or8g l–1lowmolecular weight GOS andhigh molecular weight FOSfor28daysSigniﬁcantly increasedlactobacilli andbiﬁdobacteria in the twoGOS feeding groups.Biﬁdogenic effects weredose-dependent.Moro et al.(2002)GOS Placebo-controlled studyinvolving30subjects Volunteers given8Æ1g GOSsyrup,8Æ1g GOS plus3·1010Biﬁdobacteriumlactis Bb-12,or3·1010Bif.lactis without GOS for3weeksLittle change in faecalbiﬁdobacteria seen withGOS alone.GOS plus Bif.lactis and ctis on itsown resulted in faecalexcretion of the organism,and reduced numbersof Bif.longumMalinen et al.(2002)GOS⁄FOS (9:1ratio)Double-blinded,randomized-controlled trial(DBRCT)involving20infants aged28–90daysInfant formula supplementedwith0Æ8g100ml–1GOS⁄FOS for6weeksIncreased total biﬁdobacteriacounts in stools.ReducedBif.adolescentis comparedto standard infant formulacontrols.Biﬁdobacteriaspecies composition inprebiotic infants similar tothat found in breast-fedbabies,with Bif.infantis,Bif.breve and Bif.longumpredominatingHaarman and Knol(2005)GOS⁄FOS (9:1ratio)Feeding study involving42preterm infants,15placebos,and a referencegroup given fortiﬁedmothers milkFormula food supplementedwith10g l–1prebioticmixtureBiﬁdobacteria numbersgreatly increased frominitially low levels,comparedto un-supplemented controls.No signiﬁcant effects onbacteroides,clostridia,enterobacteria or yeastsBoehm et al.(2005)G.T.Macfarlane et al.Galacto-oligosaccharide prebioticsto completely replicate all of the constituents of human milk to fortify infant formulas,the incorporation of man-ufactured prebiotics such as GOS can be a useful addition to formula feeds to replicate some of the functional attri-butes associated with breast milk,particularly its biﬁdo-genic effects.However,the ability of GOS to resemble glycoconjugate structures on cell surface receptors used by pathogens for adherence in the gut may also protect babies from infection in early life(Kunz and Rudloff 1996;Newburg1997).Based on the analysis of human milk and the high concentration of galactose,a mixture of10%long-chain FOS(inulin)and90%GOS has been developed to simulate human milk for use in infant for-mulas(Boehm et al.2002;Weaver2003).When this mix-ture was fed to term and preterm infants,it resulted in an increase in intestinal biﬁdobacteria and lactobacilli, with a gut microbiota and faecal fermentation product composition more resembling that of breast-fed infants (Haarman and Knol2005;Moro and Arslanoglu2005). Similar results have been obtained in several other studies involving young children using the same prebiotic combi-nation(see later).Production and structure of GOSGalacto-oligosaccharides is principally formed by enzymic treatment of lactose by b-galactosidase to produce several oligomers of different chain lengths(Prenosil et al.1987). They usually have a d.p.between2and10with a termi-nal glucose.GOS can be produced from lactose in cow’s milk,but the main raw material for its production for commercial products is usually whey-derived lactose (Yanahira et al.1995).This is formed in large amounts as a by-product of the dairy industry,and as cheese produc-tion increases there is a need for more efﬁcient and prac-tical methods of reducing this waste.One way this could be performed is by the production of GOS.GOS is pro-duced by b-galactosidases that have transgalactosylation activities(Fig.1),which results in the formation of4¢-or 6¢-galactosylactose,longer oligosaccharides,transgalac-tosylated disaccharides and nonreducing oligosaccharides (Angus et al.2005).For GOS production,b-galactosidases from various fungi,yeasts and bacteria are usually immo-bilized on microparticle carriers such as ion exchange res-ins,chitosan,cellulose and agarose beads,orﬁbrous supports such as cotton cloths,which leads to the forma-tion of different GOS products(Table2).They usually contain24–55%oligosaccharides,and smaller amounts of lactose,glucose and galactose.The enzymes and condi-tions used determine the various glycosidic linkages in theﬁnal products.b(1,2),b(1,3)and b(1,4)linkages and branched glucose residues occur,while(1,4)and(1,6) linkages are present in the galactan fragment.This vari-ability in glycosidic linkages may be one of the reasons why GOS possess increased resistance to acid digestion (Tomomatsu1994).The amount of GOS produced from lactose has also been shown to depend on the initial con-centrations of lactose present in the reaction mixture,and not on the concentration of b-galactosidase.The resulting products can then be puriﬁed by activated carbon,ﬁltered and concentrated before being added to foodstuffs.One way of increasing the speciﬁcity of GOS prod-ucts may be by using b-GOS synthesized by biﬁdobac-teria in the manufacture of the product,as this could preferentially increase numbers of the speciﬁc organisms used to produce the substrate.Rabiu et al.(2001)dem-onstrated that the oligosaccharide proﬁles ofﬁve biﬁdo-bacterial species(Bif.angulatum,Bif.infantis,Bif. pseudolongum,Bif.angulatum,Bif.biﬁdum)differed from the commercial GOS product oligomate55.With the exception of Bif.adolescentis,all of the organisms displayed increased growth rates on GOS prepared by their own b-galactosidase,compared to the commercial product.Properties of GOSGalacto-oligosaccharides preparations are usually trans-parent and more viscous than high-fructose corn syrups. They show good moisture retention and have a high solu-bility.GOS preparations only have about one third of the sweetness of sucrose(Oku1996)and in vitro studies with oral bacteria have shown these substances to have a lowGalacto-oligosaccharide prebiotics G.T.Macfarlane et al.potential for the development of dental caries(Hartemink et al.1997).Because of their stability,GOS can be incor-porated into a wide variety of foods,where they have a pleasant taste,and can increase the texture and mouth-feel of foods,as well as acting as bulking agents.Because of this,GOS and FOS are currently used in a wide range of commercial commodities,including infant formulas, dairy products,sauces,soups,breakfast cereals,beverages, snack bars,ice creams,bakery products,animal feeds,and as sugar replacements(Yang and Silva1995).Physiological effects of prebioticsTo function most effectively,prebiotics need to be resis-tant to digestive processes in the stomach and small bowel,and to come into contact with bacteria growing in the large intestine.Breath hydrogen excretion generally increases following ingestion of prebiotics,indicating that these substances are fermented by gut micro-organisms, but these measurements do not provide information on how much substrate actually escapes digestion in theTable2Studies using various micro-organisms for the production of galacto-oligosaccharides(GOS)from lactoseMicro-organism System Yield Outcome of study ReferenceAspergillus oryzae Enzyme immobilized oncotton cloth membrane in aplugﬂow reactor GOS production of27%(w⁄w)of initial lactose at50%lactose conversion.70%of GOS producedtrisaccharidesCotton cloth did not affectthe characteristics of theb-galactosidase.Thermalstability of the enzymeincrease25-fold afterimmobilization.Yield severaltimes higher than previouslyreportedAlbayrak andYang(2002)Talaromyces thermophilus b-galactosidase on insolublecarrier Eupergit CMaximum yield34%with80%lactose conversionImmobilization increased thethermostability of theenzymeNakkharat andHaltrich(2006)Lactobacillus reuteri Not stated38%GOS produced atapprox.80%lactoseconversion.Majority ofproducts disaccharidesother than lactoseNo major products with b1–4linkages formedSplechtna et al.(2006)Bacillus stearothermophilus Comparison of enzymeembedded in sodiumalginate,chitosan or gelatinMaximum yield of GOS31Æ2%in stirred reactorwith60%lactoseconcentration.In thepacked bead continuousreactor,the yield was17Æ4–31Æ5%Gelatin proved the bestcarrier for immobilization asdemonstrated by increasedyields.Only20%of the originalyield of the packed bedreactor was lost after140hof reaction timeChenet al.(2001)Escherichia coli GOS produced undercontrollable waterconcentration in reversemicelles Maximum29Æ7%productionof GOS(w⁄w)allolactoseand14%production ofother oligosaccharidesIncreasing the molar ratio ofwater to surfactant.W o,decreased production ofGOSChenet al.(2003)Sterigmatomyces elviae Toluene treated restingbacterial cells used in batchculture or in a newfermentation system withcell growth after theenzymatic reaction todecrease inhibitory glucose37%yield in toluene treatedcells.Increased yield to64%because of newfermentation methodMajor structure found4¢-galactosy-lactose.Yeast high capacity for GOSproduction but lowgalactose production.b-galactosidase had highlevels of transgalactolysationactivity but low levels oflactose-hydrolysis.Highoptimal temperature ofproduction,80°COnishiet al.(1995)Aspergillus candidus Immobilized recombinantb-galactosidase in acontinuous packed-bedreactorMaximum yield32%GOSover20daysRecombinant b-galactosidasehas potential for GOSproductionZhenget al.(2006)G.T.Macfarlane et al.Galacto-oligosaccharide prebioticsupper gut.This is highlighted by the study of Bouhnik et al.(1997),where eight volunteers taking10g TOS per day apparently showed reduced breath H2excretion.In another investigation involving40subjects,feeding up to 15g of TOS per day had no signiﬁcant effects on faecal pH,short-chain fatty acid(SCFA)excretion,faecal bile acid proﬁles,or concentrations of putrefactive metabo-lites;however,faecal nitrogen was increased by8Æ5%,and breath hydrogen by130%(Alles et al.1999a).Together with SCFA,H2and CO2are major products of fermentation in the gut,and excess H2formation,and osmotic diarrhoea caused by high levels of SCFA produc-tion,provide a barrier to the unfettered use of prebiotics. Undesirable symptoms caused by fermentation gases have often been reported in human feeding studies involving prebiotics(Hartemink and Rombouts1997;Pedersen et al.1997).Stone-Dorshow and Levitt(1987)gave12 volunteers15g of FOS per day for12days.Eructation, abdominal pain,bloating andﬂatulence were all signiﬁ-cantly more severe in the FOS group compared to con-trols,while there was no adaptation over the study period.After12days,breath H2measurements following a10-g challenge of prebiotic indicated that there was no difference between the FOS and control groups.Other investigations with FOS in humans,involving doses of5 and20g day–1,have indicated that breath H2increased with substrate challenge,with mildﬂatulence and bor-borygmi occurring generally,although some individuals reported greater discomfort(Kawaguchi et al.1993;Luo et al.1996).Other evidence suggests that feeding10g FOS per day can be well tolerated,but that the consump-tion of14g can lead to the development of gastrointesti-nal symptoms(Gibson et al.1995;Nadeau1999).With respect to GOS,evidence obtained so far from European studies indicates that GOS is safe to add to infant feeds(Moro et al.2006),and a recent report by the EU Sci-entiﬁc Committee on Food has approved the addition of up to0Æ8g100ml)1of a prebiotic mixture comprising 90%GOS and10%FOS in infant formula feeds(Scientiﬁc Committee on Food(SCF)2003).Most of the studies with GOS to date have used a recommended dosage of8to 15g day–1,because,like FOS,the consumption of higher levels can lead to abdominal discomfort,cramping,ﬂatu-lence and diarrhoea(Deguchi1997;Teuri et al.1998; Saavedra and Tschernia2002).The recommended dose to detect a biﬁdogenic effect is thought to be at least10g of GOS per day,but studies in Japan have shown that in healthy adults and the elderly,doses as low as2Æ5g of6¢-GOS can elicit a biﬁdogenic effect(Ito et al.1993; Ishikawa et al.1995;Teuri and Korpela1998).In one recent study,the safety of a commercial GOS syrup(Vivinal;Barculo Domo,Zwolle,Netherlands) containing45%GOS was determined by orally feeding the prebiotic to rats for90days.FOS supplemented with lactose and glucose and reverse osmosis-deionized (RODI)water were used as controls.Although signiﬁcant reductions were found in food consumption(7–13%)in animals fed the GOS syrup,and in the FOS group,com-pared to the RODI controls,no toxological manifestations were observed(Anthony et al.2006).These data indicate that the choice of prebiotic substances to use in food products should be made with the ultimate aim of maxi-mizing potential health beneﬁts,and minimizing unwanted side effects.Fermentation reactions in the large intestine Quantitatively,and in terms of their signiﬁcance in host physiology,SCFA,particularly acetate,propionate and butyrate,are the major end products of microbial fer-mentation processes in the large gut.These metabolites are formed predominantly from polysaccharide,oligosac-charide,protein,peptide and glycoprotein precursors (Macfarlane and Macfarlane2002).Polysaccharides are the most important SCFA progenitors,and many differ-ent types of bacterial hydrolytic enzymes are synthesized by the microbiota to digest these substances and facilitate uptake of their component sugars.In quantitative terms, resistant starches and plant cell wall polysaccharides(cel-luloses and noncellulosic polysaccharides such as arabino-galactans,xylans,pectins,gums and mucilages)are the principal fermentation substrates in the large bowel (Cummings and Englyst1987).Many nutritional,microbiological and host physiologi-cal factors affect fermentation reactions in the large intes-tine,particularly colonic transit time.Other host-related factors that inﬂuence metabolic reactions in the microbi-ota and the types of SCFA that are formed in the gut, include ageing,neuroendocrine system activity,stress, pancreatic and other secretions in the digestive tract, mucus production,disease,drugs and antibiotic therapy. From a microbiological viewpoint,the chemical composi-tion,physical form and amount of substrate available also signiﬁcantly affects the end products of fermentation, which are further dependent on the types and numbers of different bacterial populations in the gut,as well as com-petitive and cooperative interactions between different groups of bacteria in the microbiota.Short-chain fatty acid production is one of the most important physiological processes mediated by colonic micro-organisms.The vast majority of these metabolites are absorbed from the gut,enabling the host to salvage energy from food not digested in the upper bowel.SCFA affect colonic epithelial cell transport processes,energy transduction in colonocytes,growth and cellular differen-tiation,hepatic control of lipid and carbohydrate metabo-Galacto-oligosaccharide prebiotics G.T.Macfarlane et al.lism,and provide energy to muscle,kidney,heart and brain(Cummings1995).Because>95%of SCFA are absorbed from the gut, measurements of these metabolites in stools does not really tell us very much about the fermentability of differ-ent carbohydrates.However,in vitro studies,particularly those involving faecal microbiotas,are useful models for studying fermentation processes.At concentrations of 10g l)1,FOS and GOS were shown to increase acetate and butyrate formation in pH controlled fermenters,with transient accumulation of lactate and succinate(Hopkins and Macfarlane2003).Acetate and lactate formation are consistent with biﬁdobacterial and lactobacillus metabo-lism,but not butyrate production.However,the butyrige-nicity of FOS has been reported by other workers (Topping and Clifton2001),and recent work by Belen-guer et al.(2006)has shown how butyrate-producing spe-cies such as Anaerostipes caccae and Eubacterium halli can cross-feed on lactate produced by Bif.adolescentis growing on FOS,while a nonlactate utilizing,butyrate-forming Roseburia sp.could assimilate carbohydrate fragments formed when the Biﬁdobacterium hydrolysed complex polymeric substrates.Indeed,it is important to realize that while prebiotics are often used to stimulate the growth of beneﬁcial bacteria in the gut,they are not entirely speciﬁc for these target species,and other organ-isms have been shown to be able to ferment these carbo-hydrates.For example clostridia can utilize FOS(Duncan et al.2003),whereas bacteroides and clostridia have been reported to ferment GOS(Ohtsuka et al.1989). Biological signiﬁcance of biﬁdobacteria and lactobacilli in the digestive tractLactobacilli do not occur in great numbers in the human large intestine under normal circumstances;however,sig-niﬁcant biﬁdobacterial communities are usually present in the colon,although their community sizes and species composition can vary greatly in different human popula-tion groups(Finegold et al.1983).Evidence suggests that Bif.adolescentis and Bif.longum predominate in most adults,whereas Bif.breve and Bif.infantis are the main species colonizing the infant gut(Mitsuoka1984;Scard-ovi1986).Biﬁdobacteria,together with some lactobacillus species play an important role in the eco-physiology of the colonic microbiota.These organisms have been linked to increased resistance to infection and diarrhoeal disease (Yamazaki et al.1985;Gorbach et al.1987;Tojo et al. 1987;Saavedra et al.1994),stimulation of immune sys-tem activity(Sekine et al.1985;Kirjavainen et al.2002), as well as protection against cancer(Reddy and Rivenson 1993;Sekine et al.1994).Some biﬁdobacteria and many lactobacillus species manifest strong anti-mutagenic and anti-tumour properties,and in animal models,have pro-phylactic and therapeutic beneﬁts(Hosono et al.1997). This may be related,in part,to immune enhancement effects resulting from the chemical composition and structure of their cell wall components.Biﬁdobacteria are also known to excrete a range of water soluble vitamins,though these processes generally appear to be species and strain dependent(Deguchi et al. 1985).Thus,some strains of Bif.biﬁdum and Bif.infantis produce large quantities of folate,nicotinic acid and thia-mine,while Bif.longum and Bif.breve only form small amounts of these substances.These vitamins do not seem to be made at all by Bif.adolescentis.Pyridoxine and vita-min B12are also produced,but not excreted by Bif.breve, Bif.longum,Bif.biﬁdum,Bif.adolescentis and Bif.infantis. Bacteria growing in the large bowel play an important role in colonization resistance.This can be deﬁned as the mechanisms through which the colonic microbiota pro-tects itself against incursion by invading micro-organisms, thereby playing an important role in the maintenance of paratively little is known in detail about this phenomenon,but it is likely that many different spe-cies,including lactobacilli and biﬁdobacteria(Lievin et al. 2000)are involved.In vitro fermentation studies with mixed cultures of faecal bacteria growing on inulin and GOS(oligomate55)have demonstrated that the oligosac-charides,especially GOS,inhibited growth and toxin pro-duction by Clostridium difﬁcile(Hopkins and Macfarlane 2003).However,while the GOS preparation,in particular, was shown to be biﬁdogenic,stimulating growth of Bif. adolescentis,Bif.angulatum and Bif.biﬁdum,these organ-isms were not responsible for suppressing the pathogen, indicating that other species in the microbiota were pro-tective.In contrast,recent studies have shown that Bif.breve was more effective in protecting mice infected with Esc-herichia coli O157H7,than strains of Bif.biﬁdum and Bif.catenulatum(Asahara et al.2004).Some lactobacillus and biﬁdobacterial species form substances that are antag-onistic to other organisms,such as organic acids,hydro-gen peroxide,diacetyl and bacteriocins.Bacteriocin secretion is growth-associated in some species,and is dependent on carbon availability(Lejeune et al.1998). Some of these small proteins and peptides are highly spe-cies-speciﬁc.In one study,of13biﬁdobacterial isolates tested for bacteriocin formation,only one strain formed a protease-sensitive inhibitory substance,with broad spec-trum activity against other biﬁdobacteria,streptococci, lactobacilli and clostridia,but not Gram-negative organ-isms such as proteus,klebsiella,E.coli or pseudomonas (Meghrous et al.1990).Gibson and Wang(1994) reported that eight different biﬁdobacterial species pro-duced similar antagonistic substances that were inhibitoryG.T.Macfarlane et al.Galacto-oligosaccharide prebiotics。

Bacterial Communities in Bioreactors

Bacterial Communities in Bioreactors Bacterial communities play a crucial role in bioreactors, which are used to treat wastewater and produce biofuels. These communities are responsible for breaking down organic matter and converting it into usable products. However, maintaining a stable and efficient bacterial community can be challenging due to various factors such as environmental conditions and microbial competition.One of the main challenges in maintaining a stable bacterial community is the presence of contaminants in the wastewater. Contaminants such as heavy metals, pesticides, and antibiotics can have a detrimental effect on the bacterial community, leading to a decrease in efficiency and even the death of certain bacterial species. To mitigate this, it is essential to monitor the quality of the wastewater and remove any contaminants before introducing it into the bioreactor.Another challenge is the competition between different bacterial species. In a bioreactor, there are often multiple species present, each with their own metabolic pathways and requirements. This can lead to a situation where certain species dominate the community, while others are suppressed. To address this, it is necessary to carefully select the bacterial species used in the bioreactor and to ensure that they have complementary metabolic pathways.Environmental conditions such as temperature, pH, and oxygen levels also play a critical role in the bacterial community's stability and efficiency. Changes in these conditions can lead to shifts in the community composition and a decrease in efficiency. To maintain a stable community, it is essential to monitor these conditions and adjust them as necessary.In addition to the challenges mentioned above, there are also opportunities for improving the efficiency and stability of bacterial communities in bioreactors. One such opportunity is the use of microbial consortia, which are communities of multiple bacterial species that work together to perform specific functions. By carefully selecting and managing these consortia, it is possible to achieve high levels of efficiency and stability in bioreactors.Another opportunity is the use of genetic engineering to create bacterial strains with specific metabolic pathways. By introducing these strains into the bioreactor, it is possible to enhance the efficiency of the bacterial community and produce specific products such as biofuels or bioplastics.In conclusion, maintaining a stable and efficient bacterial community in bioreactors is crucial for wastewater treatment and biofuel production. While there are numerous challenges to overcome, there are also opportunities for improving the efficiency and stability of these communities. By carefully selecting bacterial species, monitoring environmental conditions, and exploring new technologies such as microbial consortia and genetic engineering, we can continue to improve the performance of bioreactors and advance sustainable bioprocessing.。

1月1日托福阅读真题解析

2013年12月1日托福阅读解析第一篇TOPIC 生物的自我保护有些植物为了防止被食草动物吃掉而发展出许多方法，包括structure method, physical 比method，如叶子很硬，含硅,或者叶子的形状等等。

比起这点更重要的是compound method，它们可以分泌一种化学物质避免被昆虫吃，因为吃了以后昆虫就不再生长了。

比如圆白菜（芥末科植物），分泌一种芥子油，虫子吃了有害健康，所以不吃。

但是有种蝴蝶却吃，因为它们进化了，同时植物也会出现进化这种在进化过程中不同生物的互相影响称为：coevolution。

另外有一种monarch butterfly，吃milk family plant，它能吸收植物里的化学物质，让自己变得有毒并且颜色鲜艳colorful，鸟慢慢记住这个颜色的蝴蝶不能吃，昆虫也常常用color警告捕食者自己有毒。

（有题）然后说色彩鲜艳的动物一般有毒（是动物不是植物），所以捕食者会避免吃它们。

但是后面也发生了进化。

解析：本文属生物学话题，考察生物的保护方式。

生物学一直是托福阅读最稳定的考察话题，也一般都是每次托福考试中相对较简单的文章，因其所谈内容大多会给出很多动植物的举例，是同学在阅读时更容易理解，本文也不例外。

由于生物学的重要性，强烈建议广大考生在托福复习期间找到生物学Biology相关的一些英文教科书或介绍性书籍通读一下，会发现诸多内容都在托福阅读中有闪现。

Plant defense against herbivoryPlant defense against herbivory or host-plant resistance (HPR) describes a range of adaptations evolved by plants which improve their survival and reproduction byreducing the impact of herbivores. Plants can sense being touched, and they can use several strategies to defend against damage caused by herbivores. Many plants produce secondary metabolites, known as allelochemicals, that influence the behavior, growth, or survival of herbivores. These chemical defenses can act as repellents or toxins to herbivores, or reduce plant digestibility.Other defensive strategies used by plants include escaping or avoiding herbivores in time or in place, for example by growing in a location where plants are not easily found or accessed by herbivores, or by changing seasonal growth patterns. Another approach diverts herbivores toward eating non-essential parts, or enhances the ability of a plant to recover from the damage caused by herbivory. Some plants encourage the presence of natural enemies of herbivores, which in turn protect the plant. Each type of defense can be either constitutive (always present in the plant), or induced (produced in reaction to damage or stress caused by herbivores).Historically, insects have been the most significant herbivores, and the evolution of land plants is closely associated with the evolution of insects. While most plant defenses are directed against insects, other defenses have evolved that are aimed at vertebrate herbivores, such as birds and mammals. The study of plant defenses against herbivory is important, not only from an evolutionary view point, but also in the direct impact that these defenses have on agriculture, including human andlivestock food sources; as beneficial 'biological control agents' in biological pest control programs; as well as in the search for plants of medical importance.Co-evolutionHerbivores depend on plants for food, and have evolved mechanisms to obtain this food despite the evolution of a diverse arsenal of plant defenses. Herbivore adaptations to plant defense have been likened to offensive traits and consist of adaptations that allow increased feeding and use of a host plant. Relationships between herbivores and their host plants often results in reciprocal evolutionary change, called co-evolution. When a herbivore eats a plant it selects for plants that can mount a defensive response. In cases where this relationship demonstrates specificity (the evolution of each trait is due to the other), and reciprocity (both traits must evolve), the species are thought to have co-evolved. The "escape and radiation" mechanism for co-evolution presents the idea that adaptations in herbivores and their host plants have been the driving force behind speciation, and have played a role in the radiation of insect species during the age of angiosperms. Some herbivores have evolved ways to hijack plant defenses to their own benefit, by sequestering these chemicals and using them to protect themselves from predators. Plant defenses against herbivores are generally not complete so plants also tend to evolve some tolerance to herbivory.第二篇TOPIC 北美思想启蒙enlightment一开始只有男人才能上学，但后来科学家提出都要上学。

2020年12月13日托福阅读答案解析

2020年12月13日托福阅读答案解析12月13日托福阅读词汇题：Obviously=clearlyWidespread=commonDense=thickThus=consequentlyresultantShallow=smalldepthexerciseProfound=very strongEmergence=riseTactic=strategyAdjacent to=near toParallel=match12月13日托福阅读第一篇题材划分：生物类主要内容：板块运动能够改变生物多样性，提到生物区的划分，少于百分之二十的物种相似度就是不同的区越多说明那里的多样性越高。

比如板块分开的时候，多样性增加，反之亦然。

一个山脉能够把原本的湿润风给挡了，就变成沙漠不适合生长了。

或者一个障碍的形成能够把本来的一个物种分成两个，一南一北，等到在合并的时候，发现北部的能够到南部生活，但南部的很少到北部生活。

相似TPO练习推荐TPO31- Speciation in Geographically Isolated Populations相关背景知识：Speciation is the evolutionary process by which new biological species arise. The biologist Orator F. Cook wasthe first to coin the term 'speciation' for the splitting of lineages or "cladogenesis," as opposed to "anagenesis" or "phyletic evolution" occurring within lineages. Charles Darwin was the first to describe the role of naturalselection in speciation.There is research comparing the intensity of sexual selection in different clades with their number of species.There are four geographic modes of speciation in nature, based on the extent to which speciating populations are isolated from one another： allopatric, peripatric, parapatric, and sympatric. Speciation may also be induced artificially, through animal husbandry, agriculture, or laboratory experiments. Whether genetic drift is a minor or major contributor to speciation is the subject matter of much ongoing discussion.All forms of natural speciation have taken place over the course of evolution; however, debate persists as to the relative importance of each mechanism in driving biodiversity.One example of natural speciation is the diversity of the three-spined stickleback, a marine fish that, after the lastglacial period, has undergone speciation into new freshwater colonies in isolated lakes and streams. Over an estimated 10,000 generations, the sticklebacks show structural differences that are greater than those seen betweendifferent genera of fish including variations in fins, changes in the number or size of their bony plates, variable jaw structure, and color differences.During allopatric speciation, a population splits into two geographically isolated populations (for example, by habitat fragmentation due to geographical change such as mountain formation). The isolated populations then undergo genotypic and/or phenotypic divergence as： (a) they become subjected to dissimilar selective pressures; (b) they independently undergo genetic drift; (c) different mutations arise in the two populations. When the populations come back into contact, they have evolved such that they are reproductively isolated and are no longer capable of exchanging genes. Island genetics is the term associated with the tendency of small, isolated genetic pools to produce unusual traits. Examples include insular dwarfism and the radical changes among certain famous island chains, for example on Komodo. The Galápagos Islands are particularly famous for their influence on Charles Darwin. During his five weeks there he heard that Galáp agos tortoises could be identified by island, and noticed that finches differed from one island to another, but it was only nine months later that he reflected that such facts could show that species were changeable. When he returned to England, his speculation on evolution deepened after experts informed him that these were separate species, not just varieties, and famously that other。

八年级英语询问动物演化单选题50题

八年级英语询问动物演化单选题50题1. The first living things on Earth were single - celled organisms. Which of the following is a characteristic of the evolution from single - celled to multi - celled organisms?A. The ability to live independently without any cooperation among cellsB. The development of specialized cells for different functionsC. A decrease in the overall complexity of the organismD. The loss of the ability to reproduce答案：B。

解析：从单细胞生物到多细胞生物的演化过程中，一个重要的特点就是细胞开始分化出不同的功能，这使得生物可以更好地适应环境，选项B正确。

选项A，多细胞生物的细胞间存在合作，并非独立生存。

选项C，这个过程是生物整体复杂性增加而不是减少。

选项D，多细胞生物的细胞仍然具有繁殖能力，并没有失去繁殖能力。

2. Single - celled organisms like bacteria are very simple in structure. In the evolution to multi - celled organisms, what was an important step?A. Keeping the same simple cell structure for all cellsB. Combining with other single - celled organisms randomlyC. Developing a way to communicate between cellsD. Staying in one place without any movement答案：C。

Microbial Community Composition

Microbial Community Composition Microbial community composition is a fascinating and complex topic that plays a crucial role in various ecosystems and environments. These communities consist of a diverse array of microorganisms, including bacteria, archaea, fungi, and viruses, that interact with each other and their surroundings in intricate ways. The composition of these microbial communities can have significant impacts on the health and functioning of ecosystems, as well as on human health and disease. One perspective to consider when discussing microbial community composition is the importance of diversity within these communities. A diverse microbial community is often more resilient and adaptable to changes in environmental conditions, as different species can perform unique functions and help maintain overall ecosystem stability. For example, in soil ecosystems, a diverse microbial community can contribute to nutrient cycling, soil fertility, and plant health. On the other hand, a loss of microbial diversity can lead to ecosystem imbalances and decreased resilience to disturbances. Another important aspect to consider is the role of keystone species within microbial communities. Keystone species are microbial taxa that have a disproportionate impact on community structure and function, despite their low abundance. These species can play critical roles in driving ecosystem processes, such as nutrient cycling, decomposition, and disease suppression. Identifying and understanding the role of keystone species within microbial communities is essential for predicting and managing ecosystem dynamics. Furthermore, the composition of microbial communities can also have implications for human health and disease. The human microbiome, which consists of trillions of microorganisms living in and on our bodies, plays a crucial role in maintaining our health and well-being. Disruptions to the microbial community composition in the gut, for example, have been linked to various health conditions, including inflammatory bowel disease, obesity, and allergies. Understanding the factors that influence microbial community composition in the human microbiome is essential for developing strategies to promote health and prevent disease. In addition to the ecological and human health implications, microbial community composition is also influenced by a variety of factors, including environmental conditions, host-microbe interactions, and microbial competition. Environmental factors such astemperature, pH, nutrient availability, and oxygen levels can shape the composition of microbial communities in different ecosystems. Host-microbe interactions, such as those between plants and their root-associated microbes, can also influence microbial community composition and function. Moreover, microbial competition for resources and niche space can drive the assembly and structure of microbial communities in complex ways. Overall, microbial community composition is a dynamic and intricate field of study that holds great importance for ecosystem functioning, human health, and disease. By exploring the diversity, keystone species, and factors influencing microbial communities, researchers can gain valuable insights into the complex interactions that drive microbial community dynamics. Understanding and managing microbial community composition is essential for maintaining healthy ecosystems, promoting human health, and addressing global challenges such as climate change and emerging infectious diseases.。

(完整版)托福TPOextra答案解析和原文翻译

TPO 34阅读解析第一篇Population and Climate【P1】地球人口的增长已经对大气和生态环境产生了影响。

化石燃料的燃烧，毁林，城市化，种植大米，养殖家畜，生产作为助推燃料和制冷剂的CFC增加了空气中CO2，甲烷，二氧化氮，二氧化硫灰尘和CFOs 的含量。

约70%的太阳能量穿过大气直射地球表面。

太阳射线提高了土地和海洋表面的温度，随后土地和海洋表面将红外射线反射会太空中。

这能使地球避免温度过高。

但是并不是所有的红外射线被返回会太空中，一些被大气中的气体吸收，然后再次反射回地球表面。

温室气体就是其中吸收了红外射线的一种气体，然后再次反射一些红外线到地球。

二氧化碳，CFC，甲烷和二氧化氮都是温室气体。

大气中温室效应形成和建立的很自然。

事实上，大气中如果没有温室气体，科学家预测地球温度比当前的能够低33度。

【P2】大气中当前二氧化碳浓度是360ppm。

人类活动正在对大气中二氧化碳浓度的增加有着重要的影响，二氧化碳浓度正在快速增长，目前预估在未来50-100年内，浓度将是目前的一倍。

IPCC在1992中做出一份报告，在该份报告中大多数大气科学家中观点一致，预测二氧化碳浓度翻倍可能会将全球气温提高1.4-4.5度。

IPCC在2001年的报告中做出的预测是气温几乎将会提高2倍。

可能发生的气温升高比在冰河时期发生的变化要大很多。

这种温度的升高也不会是一直的，在赤道周围变化最小，而在极点周围的变化则是2-3倍。

这些全球变化的本地化影响很难预测，但是大家一致认为可能会影响洋流的改变，在北半球的一些区域可能增加在冬天发洪水的可能性，在一些区域夏天发生干旱的概率提高，还有海平面的升高也可能会淹没位置较低的国家。

【P3】科学家积极参与地球气候系统中物理，化学和生物成分的调查，为了对温室气体的增加对未来全球气候的影响做出准确预测。

全球环流模型在这个过程中是重要的工具。

这些模型体现包含了当前对大气环流模式，洋流，大陆影响和类似东西所掌握的知识，在变化的环境下预测气候。

托福阅读tpo 3 The Long Term Stability of Ecosystems生态系统的长期稳定原题解析

阅读原文：Plant communities assemble themselves flexibly, and their particular structure depends on the specific history of the area. Ecologists use the term “succession”to refer to the changes that happen in plant communities and ecosystems over time. The first community in a succession is called a pioneer community, while the long-lived community at the end of succession is called a climax community. Pioneer and successional plant communities are said to change over periods from 1 to 500 years. These changes—in plant numbers and the mix of species—are cumulative. Climax communities themselves change but over periods of time greater than about 500 years.植物群体可以自由地聚集，它们特殊的结构取决于聚集区域的具体历史。

生态学家使用“演替”来诠释植物群落和生态系统随着时间推移所发生的变化。

演替中的第一个群落被称作先锋群落，而处于演替最后那个长期生存的群落被称为顶极群落。

先锋群落和紧接着的植物群落的变化周期是从1到500年不等，植物数量和混合种类数量的变化是慢慢积累的。

Gut Microbiota Imbalance

Gut Microbiota ImbalanceThe human large intestine is the main site of microbial colonization where the host is assumed to have co-evolved to allow beneficial interactions with certain members of the intestinal bacteria1. In this ecological niche, bacteria live in close proximity to the epithelial cells that cover the surfaces of the gastrointestinal tract (GIT). Many of these colonic bacteria are able to attach to the mucus layer of the luminal side of the GIT forming colonies. In this way the mucus layer is exposed to commensal bacteria and can tolerate its colonization through a highly developed system that can detect bacterial antigens and that allows the host to distinguish between its resident microbiota and unwelcome allochthonous bacteria such as pathogens2. Remarkably, autochthonous components of the gut microbiota are carrying at the cell surfaces lipoproteins (for Gram positive bacteria) or lipopolysaccharides (for Gram negative bacteria) that act as recognition sites by the host cells.The composition of the colonic microbiota, which consists of autochthonous and allochthonous microorganisms, originates from and depends on a number of factors, including host characteristics such as genotype and immune function, d iet and microbial interactions within the colonic microbiota in the form of cooperation and competition3. As a consequence, the autochthonous human colonic microbiota consists of highly adapted species belonging in only four dominant phyla, but with great diversity between individuals at species/strain level4. On the other hand, the allochthonous colonic microbiota consists of microorganisms originating from ingested food and other environmental factors as well as bacteria from other body parts, resulting in a composition that is less stable and differs greatly over time. Consequently, physiological alterations in the gut and general living conditions such as stress, illness, antibiotic treatment or changes in diet can impair the overall colonic microbiota composition allowing pathogenic or detrimental organisms to become established, hence affecting the health and well being of the host5.The human gut microbiota possesses many metabolic capabilities, which are lacking in the host and thus can be considered as indispensable for human life6. The gut microbiota influences a wide range of physiological features of the host by directing intestinal epithelial cell proliferation and differentiation, pH, and the development of immune system7. Another physiological function of the colonic microbiota is the provision of a barrier against pathogens that contributes to antibacterial defences byproducing antibacterial peptides8. Moreover, the gut microbiota is capable of modulating the intestinal epithelium of the host. In model systems involving human cell lines it has been noted that an appropriate balance of the microbiota induces mucin gene expression, that acts as a barrier and at the same time provides nutrients, like saccharides, for bacterial growth thereby promoting intestinal colonization of the adhering autochthonous bacteria, which helps reduce the chances of host invasion by pathogens9.Due to all the above-mentioned properties of the microbiota, there is a strong incentive for the host to control the shaping of the microbiota through the action of its immune system10. In fact, it has been demonstrated that an imbalance of the intestinal microbiota can lead to local and/or systemic disease state11. The interaction between the gut microbiota and host may therefore allow different kinds of relationships, such as commensalism, mutualism, symbiosis and pathogenicity.ReferencePalmer C, Bik EM, Digiulio DB, Relman DA, Brown PO (2007) Development of the Human Infant Intestinal Microbiota. PLoSBiol5:e177Ley RE, Peterson DA, Gordon JI (2006) Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124:837–848Xu J (2006) Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances. MolEcol 15:1713–1731Ventura M, Canchaya C, Tauch A, Chandra G, Fitzgerald GF, Chater KF, van Sinderen D (2007) Genomics of Actinobacteria: tracing the evolutionary history of an ancient phylum. MicrobiolMolBiol Rev 71:495–548Macpherson AJ, Harris NL. Interactions between commensal intestinal bacteria and the immune system. Nat Rev Immunol 4:478–485, 2004.Savage DC (2001) Microbial biota of the human intestine: a tribute to some pioneering scientists. Curr Issues IntestMicrobiol 2:1–15Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI (2005) Host-bacterial mutualism in the human intestine. Science 307:1915–1920Lievin-Le Moal V, Servin AL (2006) The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota. ClinMicrobiol Rev 19:315–337Destoumieux-Garzon D, Peduzzi J, Rebuffat S (2002) Focus on modified microcins: structural features and mechanisms of action. Biochimie 84:511–519Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI (2005) Obesity alters gut microbial ecology. Proc Natl AcadSci U S A 102:11070–11075Ley RE, Turnbaugh PJ, Klein S, Gordon JI (2006) Microbial ecology: human gut microbes associated with obesity. Nature 444:1022–1023。

【高三二模】上海市浦东新区2023届高三英语二模及答案

上海市浦东新区2023届高三英语二模Listening Comprehension Section ADirections: In Section A, you will hear ten short conversations between two speakers. At the end of each conversation, a question will be asked about what was said. The conversations and the questions will be spoken only once. After you hear a conversation and a question about it, read the four possible answers on your paper, and decide which one is the best answer to the question you have heard.1. A. His suit is too old to wear. B. He doesn’t want to wear a suit.C. He’ll go shopping with the woman.D. He doesn’t want to buy new clothes.2. A. He will look at the timetable first.B.10:30 is a perfect time for the reservation.C.The barber shop is fully booked on Saturday.D.No other customers plan to make appointments at 10:30.3. A. She didn’t buy the ticket. B. The ticket was expensive.C. There are still a few tickets left.D. She doesn’t know how much the ticket cost.4. A. He is quitting the orchestra for academic reasons.B.He is blamed for being a member of the orchestra.C.He doesn’t enjoy being a member of the orchestra.D.He prefers to study rather than travel and perform.5. A. Have a bigger breakfast. B. Make time for lunch in her schedule.C. Take only morning classes next semester.D. Change her schedule after she eats lunch.6. A. She doesn’t know where the calculator is.B.She expects the man to have the calculator repaired.C.She’d like the man to return the calculator by tonight.D.She’s angry for the man forgetting to bring the calculator.7. A. She has lost the credit card. B. They can’t buy meals at a low price.C. None of the restaurants is worth a try.D. The meals are less expensive than expected.8. A. The location of the session has been changed.B.She will definitely go to the session this evening.C.She’ll probably be too tired to walk to the session.D.The session might be canceled because of a heavy snow.9. A. He is usually not bad-tempered. B. He doesn’t like the man.C. He started the semester in a bad mood.D. He has few responsibilities.10. A. The girl may realize her dream with social media.B.The girl can present and record fashion on social media.C.The girl should first learn to make proper use of social media.D.The girl isn’t qualified as an influencer for her lack of taste in fashion.Section BDirections: In Section B, you will hear two short passages and one longer conversation, and you will be asked several questions on each of the passages and the conversation. The passages and the conversation will be read twice, but the questions will be spoken only once. When you hear a question, read the four possible answers on your paper and decide which one would be the best answer to the question you have heard.Questions 11 through 13 are based on the following passage.11. A. A different angle. B. A sharp mind.C. Various ways of workout.D. Exposure to different cultures.12. A. He will accept the new environment easily.B.He is likely to struggle with the travel budget.C.He will have to organize different daily routine.D.He may find road trips more appealing than beach views.13. A. To show travelling may bring health risks.B.To show travelling allows you to meet new people.C.To show travelling can change a person’s outlook on life.D.To show travelling gives you a chance to challenge new things.Questions 14 through 16 are based on the following passage.14. A. To detect potential danger in cold places.B.To generate more heat within their bodies.C.To keep their babies warm in breeding seasons.D.To get rid of extra heat with bigger skin surface.15. A. Wood mice. B. Bats in warm climates.C. Bird species.D. Kangaroos in Australia.16. A. Animals cope with body changes. B. Joel Allen’s rule is out of date.C. Climate change poses threat to species.D. Animals adapt to a warmer world.Questions 17 through 20 are based on the following conversation.17. A. By using cash. B. By entering a password.C. By scanning the code.D. By using a tap-and-go card.18. A. Because it touches the card reader.B.Because it uses the password.C.Because it has a built-in signal receiver.D.Because it receives the flying data.19. A. The bank will cover its clients against the loss.B.Every transaction is completed within half a second.C.People must enter their ID card number for continual use.D.Each payment is restricted to a certain amount of money.20. A. Look for his wallet. B. Apply for a tap-and-go card.C. Borrow cash from the woman.D. Stick to buying things in cash.I.Grammar and Vocabulary Section ADirections: After reading the passage below, fill in the blanks to make the passage coherent and grammatically correct. For the blanks with a given word, fill in each blank with the proper form of the given word; for the other blanks, use one word that best fits each blank.Emily Dobek is a seventh-grader at East Prairie Elementary School. Recently she (21) (win) a national prize by designing a space station for travelling to Mars.Dobek traces her interest in space and the universe back (22) Grade Three when she and her father watched a blood moon — a total lunar eclipse ( 月食 ) — on the roof of their house. She says that night watching the lunar eclipse awakened her passion (23) has yet to run out of fuel.So (24) her teacher, Andrew Smeeton, received information about the national challenge, she immediately had one student in mind.“I knew she would love the challenge and that she would go way beyond in her research,” Smeeton said. “When she started, bone density (骨密度) of astronauts (25) (research) immediately to figure out how to survive on Mars.”According to Dobek’s design, the Mars Storage Station (MSS) will be built (26) (accommodate) the need for sufficient supplies. She explains how her spacecraft —the Adventure —will be joined to a space station before flying to the MSS to load supplies. Her design includesthe Self Growing Farm, and she details (27) it would work with elements on Mars.Then there is physical and leisure activity for the astronauts under Dobek’s design. A simulator( 模拟器) allows astronauts to choose their exercise machine and virtual reality environment. Rooms with circular ceilings allow astronauts to watch (28) (download) shows and even see places on Earth, such as their homes.Chief among her immediate goals, she said, is to inspire (29) with this project. “I want to tell other kids to follow their passions,” Dobek said, “(30)they want to do, they should kind ofjust push for it. They should always try their best.”Section BDirections: Complete the following passage by using the words in the box. Each word can only be used once. Note that there is one word more than you need.Japan saw 799,728 births in 2022, the lowest number on record. That number has nearly halved in the past 40 years; by contrast, Japan recorded more than 1.5 million births in 1982. Japan also reported a(n) 31 high for post-war deaths last year, at more than 1.58 million. Deaths have 32 births in Japan for more than a decade, posing a growing problem for leaders of the world’s third-largest economy. They now face a ballooning elderly population, along with a shrinking workforce to 33 pensions and health care as demand from the aging population increases. Japan’s population has been in 34 decline since its economic boom of the 1980s and stood at 125.5 million in 2021. Its death rate of 1.3 is far below the rate of 2.1 required to maintain a stable population, in the 35 of immigration.The country also has one of the highest life expectancies in the world; in 2020, nearly one in 1,500 people in Japan were aged 100 or older. These concerning trends resulted in a warning from Prime Minister that Japan is “on the edge of not being able to maintain social 36 ” and Japan “simply cannot wait any longer” in solving the problem of its low birth rate. A new government agency will be set up to focus on the issue, with Prime Minister saying that he wants the government to 37 its spending on child-related programs.But money alone might not be able to solve the complex problem, with various social factors contributing to the low birth rate. Japan’s high cost of living, limited space and lack of child care support in cities make it difficult to raise children, meaning fewer couples are having kids. Urban couples are also often far from 38 family in other regions, who could help provide support. In 2022, Japan was ranked one of the world’s most expensive places to raise a child. And yet, thecountry’s economy has slowed down since the early 1990s, meaning frustratingly low wages and little 39 mobility.The average real annual household income declined from $50,600 in 1995 to $43,300 in 2020. Attitudes toward marriage and starting families have also 40 in recent years, with more couples putting off both during the pandemic.II.Reading ComprehensionSection ADirections: For each blank in the following passage there are four words or phrases marked A, B, C and D. Fill in each blank with the word or phrase that best fits the context.A recent series of studies examined the role of talent in the sports world. They focused on three different sports: World Cup soccer, professional basketball, and professional baseball. The results were mixed. For soccer and basketball, the studies revealed that adding talented players to a team is indeed a(n) 41 strategy, but only up to a point. Performance 42 when about 70% of the players were considered top talent. Above that level, the team’s performance began to decline. Interestingly, this trend was not evident in baseball, where additional 43 talent continued to enhance the team’s performance. (Figure 1 and 2)In looking for an explanation for the different results for different sports, the researchers 44 one important factor — the extent to which a good performance by a team requires its members to coordinate (协调) their actions. This task 45 distinguishes baseball from basketball and soccer.In baseball, the performance of individual players is 46 teammates than in soccer and basketball. The researchers concluded that when, during the course of play, task interdependence is high, team performance will47 when there is too much talent in the group. When task interdependence is lower, 48 , individual talent will have a positive effect on team performance.One explanation for this phenomenon is not so far from the pecking order (等级排序) situation among chickens. If a basketball star is pursuing his own personal goals, 49 , trying to gather a high personal point total, he may be less 50 as a team player. He may take a shot himself when it would be better to pass the ball to a teammate, thus making the team’s overall performance suffer. “There is no51 in TEAM,” young children learning to play team sports are often told. Apparently stars 52 follow this basic principle of sportsmanship.Another possibility is that when there is a lot of talent on a team, some players may begin to 53 . This is referred to as the Ringelmann effect. Ringelmann conducted an experiment in which he asked two, three, four, and up to 28 people to participate in a game of tug-of-war. He measured how much force each person used to pull the rope. He found that whenever he added a person to the team, everyone else pulled with less force.54 the ideal team — for sports, business, science, or entertainment — is more complicated than simply hiring the best talent. An A-team may require a 55 — not just A players, but a few generous B players as well.41. A. sensible B. partial C. alarming D. attainable42. A. faded B. peaked C. evolved D. proceeded43. A. team B. creative C. academic D. individual44. A. identified B. overlooked C. considered D. concealed45. A. explanation B. conclusion C. discussion D. interdependence46. A. more suitable for B. more critical to C. less dependent on D. less involved in47. A. swing B. suffer C. endure D. function48. A. in principle B. in a sense C. in other words D. on the other hand49. A. as a rule B. by contrast C. for example D. as a matter of fact50. A. stressed B. genuine C. sensitive D. generous51. A. I B. HE C. THEY D. WE52. A. closely B. rarely C. humbly D. jointly53. A. make less effort B. cause more trouble C. take less advantage D. attach more importance54. A. Inspiring B. Intensifying C. Gathering D. Training55. A. exploration B. balance C. stability D. flexibilitySection BDirections: Read the following three passages. Each passage is followed by several questions or unfinished statements. For each of them there are four choices marked A, B, C and D. Choose the one that fits best according to the information given in the passage you have just read.(A)It was a winter afternoon when, rushing to attend the final show of my art school degree, I caught the heel of my boot on the edge of a pavement. Suddenly, I was flying through the air. Ifthe past two years studying photography had taught me anything, it was an appreciation of how things can change in a thousandth of a second. Light, shadow, colours, all are in a constant state of flux (不断的变动) — as is life. And since crash-landing onto my left shoulder, I have been living through the truth of this wisdom.That day, doctors diagnosed a cracked bone. It was only the next morning when, instinctively, I tried to capture some spectacular sunlight streaming into my kitchen, that I had to face the harsh reality: I could no longer lift my camera, let alone use it. Later that week, a hospital appointment confirmed my worst fear — the arm needed total rest.Soon, I became cantankerous and impatient. I couldn’t travel, I couldn’t go anywhere much. Surfing online, I came across the concept of gratitude interventions and their role in boosting mood and wellbeing. A Californian psychologist, Sonja Lyubomirsky, has pioneered research into using a daily photography practice as a tool for enhanced gratitude. Her instructions are simple, but not necessarily easy. Take photographs throughout the day of things that are central to who you are. Take at least five photos a day. Initially, it felt like a demanding task. But reading how participants assigned to the gratitude interventions had experienced enhanced positive emotions, I decided to persevere.Pain forced me to slow down, because capturing a single iPhone photo was painful. And, yet, the struggle to find anything to feel grateful about, and then to record it, started to dramatically improve both my mood and my images. I began to photograph the most boring details of my days, from my breakfast cup to a red pepper reflected in the window. Despite everything, I found I could find magic in the ordinary. “Life seems repetitive and boring when you don’t notice the uniqueness of each moment and the constant subtle changes that are going on all around you,” writes Andy Karr in a wonderful book o n photography. I agree, but don’t just take my word for it — experience it for yourself.56.What happened to the writer on the winter afternoon?A.She went to her classmate’s degree show in a hurry.B.She tripped over and broke her shoulder bone.C.She was taught an unforgettable lesson on photography.D.She took a photo of a tragic crash-landing to be on show.57.The underlined word “cantankerous” in paragraph 3 is closest in meaning to .A.suspicious of the hospital diagnosisB. desperate to have my injury treatedC. enthusiastic about boosting wellbeingD. bad-tempered and always complaining58.The writer photographed the boring life routine because .A.underlying magic consisted in daily affairsB.gratitude fuelled a struggle against boredomC.it was central to the essence of photographyD.she felt like being assigned demanding tasks59.Which of the following might be the best title for the passage?A.Express Gratitude to WinterB. Become a Master of PhotographyC. Witness Rebirth out of InjuryD. Picture this Beauty in the OrdinaryReviewsFilter byMost commentsRating Newest ENTERTAINMENT | RESTAURANTS | THEATRE | FILM | MUSIC | EXHIBITIONSA notable highlight of the show was the real confidence of the singing. Sam Hall was ajoy to watch, with perfect comic timing, as was Emma Williams, thoroughlybelievable and convincing in her role. These two young talents stole the show , inmy opinion. The only disappointments were the dancing, which showed a lack of originality, and the opening scene, which fell a little flat. Despite the occasional technical flaws, this was a highly enjoyable and greatly impressive production, which the company should be proud of.I was really looking forward to Jonathan Baker’s latest, which is set in an imagined,but realistic, London of the near future. But I found this new effort was let down by theslightly one-dimensional characterization, and the writing is below standard forthe most part: some of the early scenes between Martha and her husband are slightly boring. The superb characterization and ambition that Baker demonstrated in his previous novels seem to be missing here. Baker’s commitment to describing the lives of ordinary people is admirable, but the whole thing is lacking in energy.This is episode number three in the nearly twenty-year-old series, delivering a very attractive andinteresting story and loads of comedy. There is some strong writing and voice acting, but the newepisode chooses to go for a linear (线性的) narrative, with some puzzles included along the way,which is less involving than the theme of exploration and conversation which previous episodes havedepended on. Besides, the puzzles are not particularly engaging, and many of them have been seenbefore in other adventure games. The visuals are extremely impressive, of course, as we have come to expect, this time featuring venues in Catalonia. But overall, a slight disappointment.60. The underlined phrase “stole the show ” most probably means .A. disappointed the producerB. ruined the whole performanceC. attracted the most attentionD. exhibited excessive confidence61. Which of the following statements about Jonathan Baker is TRUE ?A. He excels in one-dimensional characterization.B. His novels center around ordinary people’s lives.C. His novels show consistent super characterization and ambition.D. He is passionate about presenting realistic images of future London.62. The puzzles in episode number three .A. are integrated in the narrativeB. are appealing to the audienceC. deal with the theme of explorationD. are absent in other adventure gamesPlugs across AmericaThe United States has around 150,000 fuel stations to refill fossil-fuel-burning vehicles. Despite the rapid growth of all-electric vehicles in America — 400,000 of them were sold in 2021, up from barely 10,000 in 2012 — the country has only 6,000 fast electric charging stations, the kind that can rapidly charge a battery-powered car.A glance at America’s charging map reveals a lot of charging deserts. This makes sense, as EVs (electric vehicles) still represent less than 3% of new car sales. Large cities have a growing number of fast chargers, but not nearly enough to accommodate so many EVs. Away from cities, these chargers are along interstate highways closely enough to allow electric cars safe passage. Otherwise, they are nearly nonexistent in rural America. And EV stations have a problem that gas stations don’t: “Even the fastest T esla supercharger is still going to take 15 minutes to put a couple hundred miles on the vehicle,” says Jeremy Michalek, a professor at Carnegie Mellon University.Michalek says American charging facilities fall far behind what’s needed for the whole count ry to transition to electric driving. On the bright side, there is time to catch up, because not all Americans will embrace EVs at once. Most early adopters were those with access to a charger at home in their garage or parking space. Those owners can wake up with a full battery and only need to rely on public chargers when they leave town on an extended trip. But as the country gets to higher levels of EV adoption, the current facilities won’t be enough. That is why Michalek says the US needs to prioritize increasing the number of chargers at rest stops along well-traveled highways, especially as more people use electric cars for summer-time road trips.“As we get to higher levels of EV adoption, if we don’t have enough chargers for peak demand, the wait ti mes are going to be unlike what we see with gas stations,” he says.Charging dead zones will be larger as more Americans consider an EV. Renters who do not have the option to install a home charger will be hesitant to go fully electric until they can feel confident a public plug will be there when they need it. And as more households drive only electric vehicles, it will be crucial that people can get to all the places they want to go.In the best case, Michalek envisions public-private cooperation to build a national charging network. The US government has promised to install plugs throughout rural areas, while companies constructing charging stations across America will have a strong motivation to fill in the country’s biggest cities. After all, companies like Electrify America, EVgo, and ChargePoint charge customers of energy they use.63.It can be learned from the 2nd paragraph that .A.there is a shortage of charging stations in the rural areas ofAmericaB.it takes about 15 minutes for an average charger to charge a battery-powered carC.more electric vehicles are sold than fossil-fuel burning cars in large cities in AmericaD.there are enough chargers in America considering the limited sales ofnew electric vehicles64.Which of the following statements would Michalek most probably agree with?panies setting up charging stations are hesitant to go electric.B.Those who already have a home charger don’t have to find a public plug.C.Top priority needs to be given to adding more charging stations across the country.D.There is enough time to establish a charging network, because not many EVs are used.65.What can be inferred about the renters?A.Some renters don’t have the intention to go electric.B.Some renters might not be authorized to install a home charger.C.As more renters are unwilling to use public plugs, the charging dead zones are growing.D.Some renters might not have enough confidence in the public plugs for the safety reasons.66.Who does Michalek expect to work together to establish the charging network nationwide?A.The government and some companies.B. The local government and every household.C. The charging facility providers and every family.D. The federal government and the local rural government.Section CDirections: Read the following passage. Fill in each blank with a proper sentence given in the box. Each sentence can be used only once. Note that there are two more sentences than you need.A.In reality gardens are anything but natural.B.What are the things they have been attracted to?C.They are idealized landscapes with all the mud, pests and dead plants edited out.D.How much better a place the planet would be if gardening was our outlet for this need!E.Much like agriculture, gardening is a universal human desire coded into our cultural DNA.F.The calmness created by owning a tiny green space under my control has a powerful effect on my mind.As a botanist who studies our cultural relationship with plants, I am forever fascinated with what draws people to gardening.Admittedly, connecting with the natural world might seem like an obvious motivation,and undoubtedly it is a key part of the attraction. 67 If they were, we’d abandon anyattempts at design, planting or care and watch how walls of weeds slowly gave way to themass of bushes. But that wouldn’t be gardening, of course, because for all their diversity, theone thing that all gardens have in common is how unnatural they are. 68 Dazzlingplants, water features and glorious blooms is all interconnected well beyond what would naturally occur. Whether it is green lawns created in the driest deserts or a tropical paradise on a stormy North Atlantic island, they are all about shaping the natural world to fit our idea of what it “should” be.As I work on my tiny terrariums ( 玻璃花园) on dark February nights, something magical happens to my brain.69 In a world that has become increasingly uncertain, people are often fuelled by the same psychological desire: the instinctive need to have a bit of control amid chaos.As our world becomes more and more unpredictable and often frightening, gardening seems to be able to appeal to and reach out to a whole new generation, often against all odds.Of course, gardening isn’t the only thing people turn to. The rise of culture conflict s and fixation on body image have also been widely documented as being driven by a psychological need to feel a degree of certainty, control and safety. However, I can’t help but think of these alternatives: 70III.Summary WritingDirections: Read the following passage. Summarize the main idea and the main point(s) of the passage in no more than 60 words. Use your own words as far as possible.71.A plan to restore green spacesThe UK government has revealed a plan to protect and restore England’s wildlife. It focuses on at-risk species by making canals, rivers and streams cleaner and expanding green spaces.The new Environmental Improvement Plan sets goals to create or restore more than 5,000 square kilometres of wildlife habitats across England and restore 400 miles of rivers. It will create or expand 25 national nature reserves. New woodland will also be planted alongside rivers. At the moment, access to green spaces is not equal across the UK. Around 4% of people live more than 10 minutes away from their nearest park. The Environmental Improvement Plan aims to make sure households in England are within a 15-minute walk to a green space.As well as helping more people to get close to nature, the plan should increase England’s biodiversity. A Species Survival Fund will be set up to help some of England’s most endangered animals, such as red squirrels (松鼠) and water rats. The Government has set targets to boost these species by 2030. There are also targets to reduce food waste, glass, metal, paper and plastic by 2028, and to improve the quality of water in rivers.New rules mean that the Government will have to consider the environmental effects of any policy it puts forward. These goals are part of a 25-year plan that was launched in 2018. The aim of the plan is to improve the environment “within a generation”, which is roughly 25 years.Although lots of people have welcomed the plan, not everyone is impressed. Paul de Zylva, from the charity Friends of the Earth, said it wasn’t clear enough how the goals would be met and that many of them were like promises the Government had already made but not yet delivered.IV.TranslationDirections: Translate the following sentences into English, using the words given in the brackets.72.有了无人机，救援人员就可以安全地评估灾区的受灾状况。

广西钦州市浦北县寨圩中学2024学年高考考前提分英语仿真卷(含解析)

广西钦州市浦北县寨圩中学2024学年高考考前提分英语仿真卷注意事项:1．答题前，考生先将自己的姓名、准考证号码填写清楚，将条形码准确粘贴在条形码区域内。

2．答题时请按要求用笔。

3．请按照题号顺序在答题卡各题目的答题区域内作答，超出答题区域书写的答案无效；在草稿纸、试卷上答题无效。

4．作图可先使用铅笔画出，确定后必须用黑色字迹的签字笔描黑。

5．保持卡面清洁，不要折暴、不要弄破、弄皱，不准使用涂改液、修正带、刮纸刀。

第一部分（共20小题，每小题1.5分，满分30分）1．They are smiling. There ______ much trouble solving the problem.A．couldn’t be B．mustn’t beC．can’t have been D．mustn’t have been2．In that remote area, the trees _____ by the volunteers are growing well.A．planted B．planting C．being planted D．to plant3．The high-anxiety focus on reading score may have narrowed student________to knowledge about the world that can improve their reading.A．idea B．aid C．belief D．access4．— Excuse me, do you mind if I open the window?— Well, if you __________. I can put on more clothes.A．can B．mayC．must D．shall5．—Sorry, Mum! I failed the job interview again.—Oh, it's too bad. You________ have made full preparations.A．must B．can C．would D．should6．With economy increasing steadily, people ______ enjoy a life of higher quality.A．should B．can C．may D．must7．I was unaware of the critical points involved, so my choice was quite _______.A．arbitrary B．reasonable C．mechanical D．conventional8．Someone who lacks staying power and perseverance is unlikely to ______ a good researcher.A．make B．turnC．get D．grow9．—The new machines have arrived and are being tested in the workshop.—I’m glad we _____ them in the years ahead.A．will be operating B．have been operatingC．would be operating D．had been operating10．﹣Have you got the results of the final exam？﹣Not yet．It will be a few days ________ we know the full results．（）A．before B．afterC．until D．when11．Y ou should first explore your talents and get to know yourself so as to make a list of _________ you think your real interests lie.A．that B．whatC．where D．how12．Maybe he really should do more exercises from now on. ____, I hope he could get over soon.A．Somehow B．Somewhat C．Anyway D．Anywhere13．—Penny’s baby daughter narrowly escaped drinking the fu rniture polish on the coffee table.—Luckily for her. She ________ sick or even died.A．could have got B．should have gotC．must have got D．will have got14．The 19th Party Congress drew up a blueprint for China’s development in the next three decades and mo re.this blueprint into reality, we must be down-to-earth in our approach, take one step at a time as we move forward and deliver solid outcomes.A．Turning B．TurnedC．Turn D．To turn15．Look, ____ fashionable clothes is she wearing that all the eyes are fixed on her!A．so B．such C．how D．what16．Y ou have to rewrite the monthly report because you _____ many important details.A．turned off B．left outC．dropped by D．put up17．James Smith and his girlfriend went to Chenyi Square to celebrate the New Y ear, never _________.A．returned B．to returnC．returning D．having returned18．The kitchen is often the busiest room in a household, so it’s important to make sure it well.A．smoothes B．functions C．pays D．measures19．The influence of this book might not have been reaching so far, ________for Mr. William, who dared to publish it.A. if had it not been B．is it not being C．had it not been D. its only having been20．— I got that job I wanted at the public library.—___________! That’s good news.A．Go ahead B．Cheers C．Congratulations D．Come on第二部分阅读理解（满分40分）阅读下列短文，从每题所给的A、B、C、D四个选项中，选出最佳选项。

FISH技术的探针要求必须具有较好的特异性

F ISH 技术的探针要求必须具有较好的特异性、灵敏性和良好的组织渗透性。

根据需要合成的DNA或RNA寡核苷酸探针可识别靶序列内一个碱基的变化, 能够用酶学或化学方法进行非放射性标记。

F ISH 技术不仅能提供静止的实验结果, 还可以动态地观察流动水体中的微生物种群变化, 因此FISH 技术被用于监测环境中微生物在时间或空间上的动态变化, 以帮助人们理解环境中微生物种群的组成和生长动力学。

FISH 的首次应用是在1980 年，Bauman [1]等用荧光染料直接标记探针RNA 的3'端，检测到了特异的DNA，1988 年Giovnanoni [2]等将FISH 技术引进到了细菌学，他第一次用放射性同位素标记已知的rRNA 寡核苷酸探针进行细菌的显微镜观察，随后该技术在系统发育微生物学、微生物生态学、微生物的诊断和周围环境的研究中得到了迅速的发展。

目前在单一样品( 如纯培养菌种) 检测中的应用已十分普遍，准确度较高［3，4］，但在复杂样品( 如活性污泥) 检测中的应用仍不成熟，易受到样品中所含杂质的影响，出现假阳性或假阴性信号［5］。

本文采用细菌通用探针EUB338，结合DAPI全细胞染色技术，探讨了针对活性污泥样品的荧光原位杂交实验方法，并对自发荧光和真实信号的辨别要点进行了讨论。

[7] Bauman J G J ，Wiegant J ，Van Duijn P . Cytochemical hybridization with fluorochromelabeled RNA. Ⅲ. Increased sensitivity by the use of anti-fluorescein antibodies［J］. Histochemistry，1981，73（4）: 181-193［8］Giovannoni S J ，DeLong E F ，Olsen G J ，Pace N R . Phylogenetic group-specific oligodeoxynucleotide Probes for identification of single microbial cells［J］. Bacteriol，1988，170（21）: 720-726.［3］万春黎. 同步脱氮脱硫工艺生物强化及种群动态分析初探［M］. 哈尔滨:哈尔滨工业大学，2006.123-127［4］任艳红. 降解五氯酚厌氧生物反应器微生物种群结构的分子特性研究［M］. 浙江: 浙江大学，2004.342-145［5］王明义，袁晓燕，宋雪珍，等. 荧光原位杂交法在检测硫酸盐还原菌中的应用［J］.中国现代医学杂志，2008，18( 3) :302-304.荧光原位杂交技术结合分子生物学的精确性和显微镜的可视性, 可进行微生物的空间分布情况分析和特征性微生物的鉴定与定量分析。

The preterm gut microbiota_ changes associated with NEC

infection together now cause 21% of all deaths in infants born <32 weeks gestation (6): both are likely to be affected by gut microbiota.
Molecular techniques utilize the highly conserved nature of the ribosomal 16S rRNA gene to identify species not easily cultured. The phylogenetic diversity of the gut bacterial community increases over time and community shifts are associated with changes in diet and health (7,8). Modelling gut microbiota in term infants suggests competition between three phyla Bacteroides, Proteobacteria and Firmicutes, which explains most community dynamics (9,10).
Key notes • Both culture and molecular data based on stool from
preterm infants, of median gestational age 27 weeks, show the bacterial community is different in patients diagnosed who develop NEC and LOS compared with healthy infants. • Statistical analysis shows Enterobacteriaceae and Staphylococcaceae were associated with NEC and LOS, respectively. • Our study supports the role of altered bacterial communities in the pathophysiology of these diseases.

专升本英语阅读理解题目

专升本英语阅读理解题目English:"In this paragraph, the author discusses the impact of climate change on biodiversity. They highlight how rising temperatures and changing precipitation patterns are disrupting ecosystems, leadingto shifts in species distributions and migration patterns. The author emphasizes the importance of biodiversity for ecosystem stabilityand resilience, pointing out how loss of species diversity can undermine ecosystem services such as pollination and nutrient cycling. They also discuss the role of human activities, such as deforestation and habitat destruction, in exacerbating these effects. Overall, the paragraph underscores the urgent need for proactive measures to mitigate climate change and protect biodiversity."中文翻译:"在这一段中，作者讨论了气候变化对生物多样性的影响。

他们强调了温度上升和降水模式变化如何扰乱生态系统，导致物种分布和迁移模式发生变化。

托福TPO阅读31原文+答案解析

小编为托福考生们准备了托福阅读TPO31原文,希望各位考生们在TPO真题里能够得到锻炼，祝广大托福考生能够取得理想成绩。

Speciation in Geographically Isolated PopulationsEvolutionary biologists believe that speciation,the formation of a newspecies, often begins whensome kind of physical barrier arises and dividesapopulation of a single species into separatesubpopulations. Physical separationbetween subpopulations promotes the formation of newspecies because once themembers of one subpopulation can no longer mate with members ofanothersubpopulation, they cannot exchange variant genes that arise in one ofthesubpopulations. In the absences of gene flow between the subpopulations,genetic differencesbetween the groups begin to accumulate. Eventually thesubpopulations become sogenetically distinct that they cannot interbreed even ifthe physical barriers between themwere removed. At this point the subpopulationshave evolved into distinct species. This routeto speciation is known asallopatry (“alio-” means “different”，and “patria” means “homeland”).进化生物学家认为物种形成(也就是新物种的产生)经常是某种物理障碍(地理隔离)的出现把一个单一物种群分为隔离的亚种群。

friends and foes and how to alter the balance

WALTHAM International Science Symposium:Nature,Nurture,and the Case for NutritionBacteria in the Gut:Friends and Foes and How to Alter the Balance1 R.A.Rastall2Food and Bioprocessing Sciences Group,School of Food Biosciences,University of Reading,Whiteknights, Reading,RG66AP,UKABSTRACT The activities of the bacteria resident in the colon of companion animals can have an impact upon thehealth of the host.Our understanding of this microbial ecosystem is presently increasing due to the development ofDNA-based microbiological tools that allow identiﬁcation and enumeration of nonculturable microorganisms.Thesetechniques are changing our view of the bacteria that live in the gut,and they are facilitating dietary-interventionapproaches to modulate the colonic ecosystem.This is generally achieved by the feeding of either live bacteria(probiotics)or nondigestible oligosaccharides(prebiotics)that selectively feed the indigenous probiotics.Feedingstudies with a Lactobacillus acidophilus probiotic have shown positive effects on carriage of Clostridium spp.incanines and on recovery from Campylobacter spp.infection in felines.Immune function was improved in bothspecies.Prebiotic feeding studies with lactosucrose and fructo-oligosaccharides in both cats and dogs have shownpositive effects on the microﬂora balance.Recently synbiotic forms(a probiotic together with a prebiotic)targeted atcanines have been developed that show promise as dietary-intervention tools.J.Nutr.134:2022S–2026S,2004.KEY WORDS: bacteria colonic microﬂora probiotics prebiotics Biﬁdobacterium LactobacillusIntroductionThe link between the barrier function of colonic microﬂoraand susceptibility to disease(1)is an area of great interest.This has led to a vibrant,global,functional food industry that isintroducing new products for gut health into markets targeted to humans and companion animals.The gut microﬂoraMost of our knowledge of gut microﬂora comes from studies on humans(2).Microbiologically,the gut can be thought of in terms of three principal regions:the stomach,small intestine, and colon.In terms of microbial population,the stomach has very low bacterial numbers;facultative anaerobes such as lactobacilli,streptococci,and yeast are present at;100colony-forming units(CFU)3per milliliter due to the low environ-mental pH.3The small intestine has a larger bacterial load that consists of facultative anaerobes such as lactobacilli,strepto-cocci,and enterobacteria as well as anaerobes such as Biﬁdobacterium spp.,Bacteroides spp.,and clostridia at levels of;104–108CFU/ml.The most heavily colonized region,however,is the colon,with a total population of1011–1012 CFU/ml of contents(3).The colonic microﬂora is thepredominant target for dietary intervention in the gut ecology, and it is this region that is the subject of this article.Consisting of higher levels of obligate anaerobes and lower levels of facultative aerobes(Fig.1),the colonic microﬂora is very complex.The colonic microﬂora is dominated by strict anaerobes such as Bacteroides spp.,the clostridia and other families within the Clostridium mega-genus(including Ruminococcus spp., Butyrovibrio spp.,Fusobacterium spp.,Eubacterium spp.,and Peptostreptococcus),Biﬁdobacterium spp.,Atopobium spp.,and the peptococci.Facultative anaerobes occur in numbers ;1000-fold lower and include lactobacilli,enterococci,strep-tococci,and Enterobacteriaceae.Yeasts are present only at relatively low numbers of102–104CFU/ml(4).In terms of health,the most signiﬁcant organisms arebelieved to be the biﬁdobacteria(4).Biﬁdobacteria are the major component of the microbial barrier to infection. Biﬁdobacteria produce a range of antimicrobial agents that are active against Gram-positive and-negative organisms(5). Lactobacilli are also health positive and produce a range of antimicrobial agents,but they are present in much lower levels in the human colon.In addition to producing antimicrobial agents,a large population of beneﬁcial bacteria competitively excludes pathogens by occupying receptor sites and competing for space,nutrients,etc.Much of the information presently available regarding colonic microﬂora comes from studies that employed classical microbiological techniques based on agar plates.This poses1Presented as part of the WALTHAM International Science Symposium: Nature,Nurture,and the Case for Nutrition held in Bangkok,Thailand,October28–31,2003.This symposium and the publication of the symposium proceedings were sponsored by the WALTHAM Centre for Pet Nutrition,a division of Mars,Inc. Symposium proceedings were published as a supplement to The Journal of Nutrition.Guest editors for this supplement were D’Ann Finley,James G.Morris,and Quinton R.Rogers,University of California,Davis.2To whom correspondence should be addressed.E-mail:r.a.rastall@reading..3Abbreviations used:CFU,colony-forming unit;FISH,ﬂuorescent in situ hybridization;FOS,fructo-oligosaccharides;GI,gastrointestinal.0022-3166/04$8.00Ó2004American Society for Nutritional Sciences.2022S by guest on April 18, 2015 Downloaded froma problem,however,as colonic microﬂora are thought to contain a high level of biodiversity including many species that cannot be cultured using present techniques.This unculturable ﬂora can only be characterized using DNA-based microbiology methods (6–8).Most of these methods rely on ampliﬁcation,detection,and/or sequencing of diagnostic regions of 16S rRNA genes (8).This culture problem is particularly acute in studies on canines.A recent study (9)illustrates the unreliability of apparently selective agar media for enumeration of canine fecal bacteria:many of the selective media used did not support the growth of the target population (Table 1).Our image of the canine gut ﬂora,then,is based largely on traditional methods of investigation (Fig.2)(10).In one study (11),ﬂuorescent in situ hybridization (FISH)was used to describe the ﬂora on one Labrador dog (Fig.2).The most signiﬁcant aspect of the canine microﬂora is the much lower level of biﬁdobacteria found in canines than in other animals.The feline colonic ﬂora (Fig.3)is even less well characterized (12),and the biﬁdobacteria levels are probably even lower than in canines.In fact,biﬁdobacteria are only intermittently isolated from felines.Dietary tools for changing the balanceProbiotics have been investigated as dietary management tools for many years (13)in human as well as livestock animal studies.The concept is that ingestion of beneﬁcial bacteria leads to colonization of the gut with the added strain,and this then strengthens the gastrointestinal (GI)barrier to disease.Although biﬁdobacteria are the most signiﬁcant health-positive organism in the colon,their obligate anaerobic nature has hindered commercial development.Most commercial pro-biotics are lactobacilli,and several species have been developed for application in humans and livestock animals (13).There are few studies on probiotics in companion animals.One recent study (14)investigated the application of Lactobacillus acidophilus DSM 13241in canines.This strainwas chosen on the basis of its growth characteristics,antimicrobial activity toward pathogens,and survival rate in gut models.Feeding of 23109CFU/d to 15healthy dogs resulted in a signiﬁcant increase in the population of recover-able lactobacilli in the feces with a concomitant decrease in the clostridia population (determined by FISH).The animals displayed no signiﬁcant changes in blood biochemistry,body temperature,or fecal quality.Immune-function studies showed no signiﬁcant changes in haptoglobin level or white bloodcellFIGURE 1Overview of the human colonic microﬂora.Bacterial genera were classiﬁed as health positive,health negative,or health neutral.Bacterial enumeration was by selective media (4).Ps,Pseudomonas .TABLE 1Selectivity of media used in canine colonic microﬂora studies 1Agar Selective for Organism identiﬁed,%Nutrient Total aerobes 98E.coli,2Str.bovis MacConkey Coliforms 98E.coli,2ruminal bacteriaWilkins-ChalgrenTotal anaerobes60Collinsiella intestinalis 27Pectinatus -like sp.11Streptococci bovis 1E.coli1Rothia -like sp.Rogosa Lactobacilli 65Str.bovis 24L.animalis 11L.ruminus BeerensBiﬁdobacteria67Str.bovis 18L.ruminus15Staphylococcus epidermidis Azide Gram-positive cocci93unknown 7Str.bovisReinforced clostridial Clostridia 80Staphylococcus sp.20unknown BacteroidesBacteroides56unknown,33E.coli6Staph.haemolyticus 5L.animalis1Fecal samples from 1healthy adult Labrador dog.Microbial identiﬁcation was performed by sequencing of the 16S rRNA genes(9).FIGURE 2Overview of the canine colonic microﬂora.In a study of healthy adult Shepherd dogs (A ),bacterial groups were enumerated by selective media and conﬁrmed by denaturing gradient gel electrophoresis (10).In a study of one healthy adult Labrador dog (B ),bacterial groups were enumerated by FISH (11).2023SBACTERIA IN THE GUT by guest on April 18, 2015Downloaded fromcount but signiﬁcant increases in serum IgG,monocytes,and neutrophils.Signiﬁcant decreases in plasma nitric oxide levels and the osmotic fragility of red blood cells were observed.The researchers concluded that feeding of the probiotic resulted in positive changes in the gut microbiology and in systemic effects that suggested immune system stimulation as observed in humans after they consumed Lactobacillus spp.(13).An attractive alternative to the feeding of probiotics is the use of prebiotics.A prebiotic is a nondigestible food ingredient that is selectively metabolized by the indigenous probiotic bacteria in the gut (4).Presently all prebiotics are car-bohydrates (15),and a range of carbohydrates exist on the market around the world (Table 2).Using prebiotics is attractive,because we can avoid the drawbacks of using probiotic bacteria such as maintaining viability.Prebiotics can thus be incorporated into a wider range of products and are stable to heat treatment.There is relatively little work published on the use of prebiotics in companion animals (16).Most of the research to date has focused on lactosucrose and fructo-oligosaccharides (FOS).Studies on feeding of lactosucrose have been performed on dogs (17)and cats (12).Feeding of 1.5g of lactosucrose/d to 8healthy dogs for 2wk resulted in statistically signiﬁcant desirable changes to the gut ﬂora as determined by fecal microbiological analysis (based on selective media).A 0.5-log increase in biﬁdobacteria was seen together with a 1.6-log decrease in clostridia levels.Decreases were also seen in toxin levels and fecal ctosucrose was also fed to 8healthy cats at a level of 750mg/d for 2wk (12).This resulted in an increasein the incidence of recovery of biﬁdobacteria and a signiﬁcant 0.9-log increase in lactobacilli numbers.Signiﬁcant decreases of 0.4log were seen with levels of clostridia and Enterobacteriaceae .Toxin levels and fecal odor were also reduced.Several studies are available on FOS consumption in companion animals (16).A representative study (18)fed FOS at 4g/d to 20adult dogs.Fecal bacteriology was investigated by selective media,and bacterial metabolites were measured.Statistically signiﬁcant increases in biﬁdobacteria (0.58log)and lactobacilli (0.86log)numbers were seen together with a small but signiﬁcant decrease in clostridia level of 0.11log.Increases were seen in lactate and butyrate quantities but increases were also observed in ammonia,isovalerate,dimethylsulﬁde,and hydrogen sulﬁde levels.A study in cats (19)was performed by feeding a diet that contained 0.75%FOS for 12wk to 12adult cats.BacteriologyFIGURE 3Overview of the feline colonic microﬂora in a study of 8healthy adult cats.Bacterial enumeration was by selective media (12).TABLE 2Oligosaccharide prebiotics on the world marketOligosaccharideLactulose 1Galacto-oligosaccharide Fructo-oligosaccharide InulinIsomalto-oligosaccharide 1Soybean oligosaccharide 1LactosucroseGentio-oligosaccharide 1Xylo-oligosaccharide 11Found only on the Japanese market (15).TABLE 3Growth rate of canine probiotics on various carbohydrates 1SubstrateGrowth rate,m (310)1L.acidophilus L.mucosae L.reuteri Actilight fructo-oligosaccharides 2 5.5860.24 2.7660.279.9060.28Biotosehigh-maltose syrup 311.7060.678.1060.21 6.5460.30Cellobiose 11.4660.637.6260.15 1.2660.19Gentiobiose 4.7460.45 5.8860.1610.2660.25Glucose 12.0660.498.4060.24 6.9060.29Isomalto-oligosaccharides 4.2060.69 6.1260.19 5.0460.21Lactose 10.2660.558.2860.307.2660.29Laevan 0.0660.030.0660.02 1.6860.17Maltose 10.2060.37 6.9660.12 5.8860.26Melezitose 0.7860.17 3.2460.17 1.8660.20Melibiose 11.3460.427.9860.24 4.7460.25Palatinose 9.4860.509.3660.179.0660.20Panorichhigh-panose syrup 38.3460.348.2860.19 5.8260.11Rafﬁnose 10.8660.467.6860.207.0260.27Stachyose 7.4460.32 6.6660.19 5.7660.33Sucrose 10.3260.48 6.8460.23 5.6460.27Tagatose0.0660.03 1.9260.27 3.8460.23Xylo-oligosaccharides 4.1460.19 3.8460.187.5060.33Xylan0.9660.170.0660.034.4460.251Values are means 6SD of ﬁve replicates (11).2Eridania Beghin-Say,Vilvoorde,Belgium.3Nihon Shokuhin Kako,Tokyo,Japan.2024S SUPPLEMENTby guest on April 18, 2015Downloaded fromwas performed by selective media,and bacterial metabolites were measured.Only one isolation of Biﬁdobacterium sp.was made,but a signiﬁcant increase in lactobacilli number was seen (0.22log).Signiﬁcant decreases in clostridia (1.47log)and Escherichia coli (0.52log)numbers and an increase in bacteroides level (0.56log)were also noted.The combination of a probiotic with a prebiotic to support its viability and activity has been termed a synbiotic (4).An exciting development in the ﬁeld of companion animals is that of synbiotics targeted to particular species.This has been attempted for the ﬁrst time with canine synbiotics.Five candidate lactobacilli,L.acidophilus ,L.murinus ,L.reuteri ,L.mucosae,and L.rhamnosus were isolated from a Labrador dog (11).It is generally held in the context of human and livestock animal nutrition that probiotic strains should originate from the species in which they are to be used (13).Three of these strains,L.mucosae ,L.acidophilus,and L.reuteri ,were then evaluated (11)for their growth on various carbohydrates (Table 3)and antimicrobial activity (Table 4)against Salmonella enterica serotype Typhimurium,enteropathogenic E.coli ,and the toxin-negative mutant of E.coli O157:H7.On the basis of these data,candidate synbiotic forms can be identiﬁed with activity against speciﬁc target pathogens (Table 5).This canine synbiotic concept was taken further in an attempt to manufacture a prebiotic targeted to a particular probiotic organism.Most of the prebiotic oligosaccharides presently on the market are synthesized using enzymatic methods (15).For instance,galacto-oligosaccharides,which are a mixture of b -linked di-to pentasaccharides,are manufactured by the action of b -galactosidase on lactose.The enzyme catalyzes a glycosyl transfer reaction at high lactose levels and transfers galactose (Gal)from lactose,thereby actingTABLE 4Antimicrobial activity of selected probiotics against GI pathogens 1Inhibition zone (diameter,mm )VTECEPECS.entericaserotype typhimurium Growth substrate L.mucosae L.acidophilus L.reuteri L.mucosae L.acidophilus L.reuteri L.mucosae L.acidophilus L.reuteri FOS 2NG NG—NG NG6.560.5NG NG—High maltose syrup 3NG 7.060.3 4.260.5NG 5.360.4 3.060.2NG 6.160.57.160.4Cellobiose ——NG ——NG 3.860.5—N/AGentiobiose 6.160.6——5.060.4——6.060.3 3.260.3 3.260.4Isomalto-oligosaccharides 1.460.5 4.260.5 4.260.6 4.660.4——— 1.760.4 4.560.9Lactose ————— 6.260.5— 4.360.5 5.960.9Maltose 3.860.7— 6.860.8 3.460.5— 1.360.4—— 6.560.7Melibiose ————— 5.660.5———Palatinose —————— 1.360.4 1.060.5—High panose syrup 3 6.260.4—— 5.060.3——9.060.4——Rafﬁnose 6.860.5———— 5.860.7 3.460.5 1.960.5—Stachyose ——NG ——NG ——N/A Sucrose — 1.360.5————— 3.560.4—Xylo-oligosaccharidesNGNG—NGNG3.760.5NGNG—1Inhibition zones around paper disks soaked in cell-free culture supernatants,pH adjusted to pH 7.00.Data are the mean 6SD of ﬁve replicates.NG,no or little growth on this substrate;N/A,not tested;—,no inhibition (11).2Actilight,Eridania Beghin-Say,Vilvoorde,Belgium.3Nihon Shokuhin Kako Co.,Ltd,Tokyo,Japan.TABLE 5Effective synbiotic combinations against GI pathogens 1Probiotic CarbohydratePathogenVeritoxigenic E.coliEnteropathogenic E.coliSalmonella enterica,serotype TyphimuriumL.reuteri Maltose Ö—ÖLactose—ÖÖActilight fructo-oligosaccharides 2—Ö—L.mucosae GentiobioseÖ—ÖPanorich high-panose syrup 3Ö—ÖRafﬁnoseÖ——L.acidophilusBiotose high-maltose syrup 3Ö—Ö1Combinations yielded a zone of inhibition .6.00mm.2Eridania Beghin-Say,Vilvoorde,Belgium.3Nihon Shokuhin Kako,Tokyo,Japan.2025SBACTERIA IN THE GUT by guest on April 18, 2015 Downloaded fromas a glycosyl donor to other lactose molecules (which are acting as glycosyl acceptors)and thus building up higher oligosaccha-ride levels (15).This manufacturing technology can be modiﬁed to utilize enzymes from biﬁdobacteria on the assumption that the resultant products might have enhanced selectivity for the producing organism (20).This approach has been used in an attempt to develop highly targeted synbiotics for canine application.One of the canine probiotics discussed above,L.reuteri ,was selected for further study:a -galactosidase enzyme activity was extracted from cultures of the organism and used to synthesize oligosaccharide mixtures from melibiose (Gal a 1!4glucose)as a glycosyl donor (21).The resultant oligosaccharide mixtures were then evaluated in mixed fecal pH-controlled batch cultures (22).The oligosaccharides were found to be prebiotic in their own right and to act synergistically with added L.reuteri to a greater extent than with added L.acidophilus (Fig.4).Interestingly,the synbiotic of L.reuteri and its synthesized oligosaccharides also stimulated biﬁdobacterial populations,presumably by inhibiting other species that inhibit the biﬁdobacteria.Concluding remarksThe bacterial population within the GI tract of mammals constitutes a metabolically active organ that acts as a signiﬁcant barrier to infection by exogenous pathogenic microorganisms.At present,our picture of human GI-tract ecology is far from complete,even less so for companion animals such as cats and dogs.Rapid development of new DNA-based methods is under way for studying the composition of complex microbial ecosystems such as the colonic microﬂora,and these have not yet been systematically applied to the study of companion animals.Such studies are required if we are to realize the potential of dietary manipulation of this barrier effect.Dietary-management tools already exist in the shape of probiotic microorganisms,prebiotic oligosaccharides,and synbiotic mixtures of the two.There is evidence that these tools do work in dogs and,to a lesser extent,in cats.However,much more research is needed on the effects of the various probiotic strains and prebiotic oligosaccharides in a wide range of breeds.Well-designed feeding studies are required that usemolecular microbiology techniques ideally coupled with more fundamental studies on the mechanisms of action of these agents.Such studies will lead to many new product opportu-nities in the pet-care ﬁeld.LITERATURE CITED1.Rastall,R.A.&Maitin,V.(2002)Prebiotics and synbiotics:towards the next generation.Curr.Opin.Biotechnol.13:490–498.2.Roberfroid,M. B.,Van Loo,J.A. E.&Gibson,G.R.(1998)The biﬁdogenic nature of chicory inulin and its hydrolysis products.J.Nutr.128:11–19.3.Cummings,J.H.,Gibson,G.R.&Macfarlane,G.T.(1989)Quanti-tative estimates of fermentation in the hind gut of man.Acta Vet.Scand.Suppl.86:76–82.4.Gibson,G.R.&Roberfroid,M.B.(1995)Dietary modulation of the human colonic microbiota:introducing the concept of prebiotics.J.Nutr.125:1401–1412.5.Gibson,G.R.&Wang,X.(1994)Regulatory effects of biﬁdobacteria on the growth of other colonic bacteria.J.Appl.Bacteriol.77:412–420.6.McCartney,A.L.,Wenzhi,W.&Tannock,G.W.(1996)Molecular analysis of the composition of the biﬁdobacterial and lactobacillus microﬂora of humans.Appl.Environ.Microbiol.62:4608–4613.ngendijk,P.S.,Schut,F.,Jansen,G.J.,Raangs,G.C.,Kamphuis,G.R.,Wilkinson,M.H.F.&Welling,G.W.(1995)Quantitative ﬂuorescence in situ hybridization of Biﬁdobacterium spp.with genus-speciﬁc 16S rRNA-targeted probes and its application in fecal samples.Appl.Environ.Microbiol.61:3069–3075.8.Harmsen,H.J.M.,Gibson,G.R.,Elfferich,P.,Raangs,G.C.,Wideboer-Veloo,A.C.M.,Argaiz,A.,Roberfroid,M.B.&Welling,G.W.(2000)Com-parison of viable cell counts and ﬂuorescent in situ hybridization using speciﬁc rRNA-based probes for the quantiﬁcation of human fecal bacteria.FEMS Microbiol.Lett.183:125–129.9.Greetham,H.L.,Giffard,C.,Hutson,R.A.,Collins,M.D.&Gibson,G.R.(2002)Bacteriology of the Labrador dog gut:a cultural and genotypic approach.J.Appl.Microbiol.93:640–646.10.Simpson,J.M.,Martineau,B.,Jones,W.E.,Ballam,J.M.&Mackie,R.I.(2002)Characterization of fecal bacterial populations in canines:effects of age,breed and dietary ﬁber.Microb.Ecol.Health Disease 44:186–197.11.Tzortzis,G.,Baillon,M.L.A.,Gibson,G.R.&Rastall,R.A.(2004)Mo-dulation of anti-pathogenic activity in canine-derived Lactobacillus species by carbohydrate growth substrate.J.Appl.Microbiol.96:552–559.12.Terada, A.,Hara,H.,Kato,S.,Kimura,T.,Fujimori,I.,Hara,K.,Maruyama,T.&Mitsuoka,T.(1992)Effect of lactosucrose (4G -b -D -galactosyl-sucrose)on fecal ﬂora and fecal putrefactive products of cats.J.Vet.Med.Sci.55:291–295.13.Tannock,G.W.(2002)Probiotics and Prebiotics:Where Are We Going?Caister Academic Press,Wymondham,UK.14.Baillon,M.L.A.,Marshall-Jones,Z.V.&Butterwick,R.F.(2004)Ef-fects of probiotic Lactobacillus acidophilus strain DSM13241in healthy adult dogs.Am.J.Vet.Res.65:338–343.15.Playne,M.J.&Crittenden,R.(1996)Commercially available oligo-saccharides.Bull.Int.Dairy Found.313:10–22.16.Flickinger, E. A.,Van Loo,J.&Fahey,G. C.(2003)Nutritional responses to the presence of inulin and oligofructose in the diets of domesticated animals:a review.Crit.Rev.Food Sci.Nutr.43:19–60.17.Terada,A.,Hara,H.,Oishi,T.,Matsui,S.,Mitsuoka,T.,Nakajyo,S.,Fujimori,I.&Hara,K.(1992)Effect of dietary lactosucrose on fecal ﬂora and fecal metabolites of dogs.Microb.Ecol.Health Disease 5:87–92.18.Swanson,K.S.,Grieshop,C.M.,Flickinger,E.A.,Bauer,L.L.,Healy,H.P.,Dawson,K.A.,Merchen,N.R.&Fahey,G.C.(2002)Supplemental fructo-oligosaccharides and mannanoligosaccharides inﬂuence immune function,ileal and total tract nutrient digestibilities,microbial populations,and concentrations of protein catabolites in the large bowel of dogs.J.Nutr.132:980–989.19.Sparkes,A.H.,Papasouliotis,K.,Sunvold,G.,Werrett,G.,Gruffydd-Jones,E.A.,Egan,K.,Gruffydd-Jones,T.J.&Reinhart,G.(1998)Effect of supplementation with fructo-oligosaccharides on fecal ﬂora of healthy cats.Am.J.Vet.Res.59:436–440.20.Rabiu,B.A.,Jay,A.J.,Gibson,G.R.&Rastall,R.A.(2001)Synthesis and fermentation properties of novel galacto-oligosaccharides by b -galactosidases from Biﬁdobacterium species.Appl.Environ.Microbiol.67:2526–2530.21.Tzortzis,G.,Jay, A.J.,Baillon,M.L. A.,Gibson,G.R.&Rastall,R.A.(2003)Synthesis of a -galactooligosaccharides with a -galactosidase from Lactobacillus reuteri of canine origin.Appl.Microbiol.Biotechnol.63:286–292.22.Tzortzis,G.,Goulas,A.K.,Baillon,M.L.A.,Gibson,G.R.&Rastall,R.A.(2004)In vitro evaluation of the fermentation properties of galactooligo-saccharides synthesized by a -galactosidase from Lactobacillus reuteri.Appl.Microbiol.Biotechnol.64:106–111.FIGURE 4Fermentation properties of canine-targeted synbiotics.Bars represent percentage changes from the inoculum level in triplicate pH-controlled fecal batch cultures.Bacterial groups were enumerated by FISH (22).2026S SUPPLEMENTby guest on April 18, 2015Downloaded from。

2024年研究生考试英语二考试试题

2024年研究生考试英语二考试试题一、阅读理解（共两篇，每篇5小题，共10小题）阅读下面的短文，然后根据短文内容回答问题。

Passage 1Climate change is a global issue that affects every aspect of our lives. It is important for us to understand the causes and consequences of climate change in order to take effective actions to mitigate its effects. This passage explores the impact of climate change on wildlife and ecosystems.Climate change is causing shifts in the natural habitats of many animal species. As the temperature rises, certain species are being forced to migrate to find suitable conditions. This disrupts the delicate balance of ecosystems and can lead to the extinction of certain species. For example, polar bears, which rely on sea ice for hunting, are facing the threat of habitat loss due to the melting of polar ice caps.Furthermore, climate change is altering the timing of natural events, such as the migration of birds and the flowering of plants. This can have cascading effects on the entire food chain. For instance, if birds arrive too early and the insects they feed on have not yet emerged, the birds may starve. This can disrupt the populations of birds and the insects they regulate, causing imbalances in the ecosystem.In addition to the direct impacts on wildlife, climate change also affects the availability of resources for human populations. Changes in temperature and precipitation patterns can lead to droughts, floods, and the spread of diseases. This, in turn, affects the availability of food and water, and can increase the risk of conflicts over resources.In conclusion, climate change poses a significant threat to wildlife and ecosystems. It is crucial for us to take immediate action to reduce greenhouse gas emissions and protect the environment. By understanding the impacts of climate change, we can work towards creating a sustainable future for both humans and the natural world.1. What is the main topic of the passage?The main topic of the passage is the impact of climate change on wildlife and ecosystems.2. How does climate change affect animal species?Climate change affects animal species by causing shifts in their natural habitats and forcing them to migrate to find suitable conditions.3. What is an example of a species threatened by habitat loss due to climate change?An example of a species threatened by habitat loss due to climate change is the polar bear, which relies on sea ice for hunting.4. How can climate change affect the food chain?Climate change can affect the food chain by altering the timing of natural events, such as the migration of birds and the flowering of plants, which can lead to imbalances in the ecosystem.5. How does climate change impact human populations?Climate change impacts human populations by affecting the availability of resources, such as food and water, and can increase the risk of conflicts over resources.Passage 2The world is experiencing a rapid increase in urbanization, with more people moving to cities in search of better opportunities. This trend poses both challenges and opportunities for urban planning and development. This passage explores the concept of sustainable cities and the benefits they can bring.Sustainable cities are designed to minimize their environmental impact and promote the well-being of their residents. They prioritize the use of renewable energy sources, such as solar and wind power, to reduce greenhouse gas emissions and combat climate change. Sustainable cities also focus on efficient transportation systems, such as publictransit and cycling infrastructure, to reduce reliance on private cars and decrease traffic congestion.In addition to environmental considerations, sustainable cities prioritize the social and economic aspects of urban living. They promote affordable and accessible housing, quality healthcare, and education for all residents. By investing in green spaces, such as parks and gardens, sustainable cities provide opportunities for recreation and improve the overall quality of life.Furthermore, sustainable cities encourage community engagement and participation in decision-making processes. They prioritize citizen involvement in urban planning and development, ensuring that the needs and preferences of the residents are taken into account. This fosters a sense of ownership and belonging among the residents, leading to stronger and more resilient communities.Overall, sustainable cities offer numerous benefits. They help combat climate change, improve the quality of life for residents, and promote social and economic equity. By investing in sustainable urban development, we can create cities that are not only environmentally friendly but also livable and inclusive for all.6. What is the concept of sustainable cities?The concept of sustainable cities is to minimize environmental impact, promote resident well-being, and prioritize renewable energy sources and efficient transportation systems.7. How do sustainable cities reduce greenhouse gas emissions?Sustainable cities reduce greenhouse gas emissions by prioritizing the use of renewable energy sources, such as solar and wind power.8. What social and economic aspects do sustainable cities prioritize?Sustainable cities prioritize affordable and accessible housing, quality healthcare, education, and community engagement.9. What benefits do sustainable cities offer?Sustainable cities offer benefits such as combating climate change, improving the quality of life for residents, and promoting social and economic equity.10. Why is citizen involvement important in sustainable cities?Citizen involvement is important in sustainable cities as it ensures that the needs and preferences of the residents are taken into account, fostering a sense of ownership and belonging in the community. This leads to stronger and more resilient cities.。

Bacterial Communities in Food Systems

Bacterial Communities in Food Systems ChatGPT: Hello there! When it comes to the fascinating world of bacterial communities in food systems, it's a topic that delves into the intricate dance of life in our daily meals. Let's explore this intricate ecosystem from various angles. Firstly, it's crucial to understand that these microorganisms are notjust a byproduct of spoilage, but they play a vital role in food preservation. The good bacteria, known as probiotics, are responsible for maintaining gut health and aiding in digestion. They're found in fermented foods like yogurt, kefir, and sauerkraut, and their presence can be a testament to the food's quality and freshness. The balance between these beneficial bacteria and harmful pathogens is crucial for a safe and nutritious diet. The diversity of bacterial communities in food is truly remarkable. Each type of food, from fruits to meats, harbors its own unique set of bacteria. For instance, the fermentation process in cheese production creates a complex ecosystem with lactic acid bacteria, yeasts, and even molds. This diversity contributes to the unique flavors and textures we enjoy. The sheer variety also makes it challenging for food manufacturers to control and standardize these communities, which can impact the final product's stability and shelf life. Foodborne illnesses, on the other hand, often stem from the presence of pathogenic bacteria. These can be harmful strains that cause diseases like salmonella or listeria in raw or undercooked foods. Food safety regulations and proper handling practices are crucial to minimize the risk of these infections. The role of refrigeration, cooking, and storage in controlling bacterial growth is a critical aspect of food safety in our food systems. The impact of climate change on bacterial communities in food is an emerging concern. Changes in temperature and humidity can alter the growth rates of bacteria and affect the stability of food products. For instance, warmer conditions can lead to faster spoilage and the emergence of new strains that may be resistant to existing treatments. This highlights the need for sustainable food systems that can adapt to these environmental shifts. Lastly, consumer behavior and preferences also shape the bacterial landscape in food. The rise of plant-based diets and interest in organic and minimally processed foods can influence the types of bacteria present in these products. As consumers become more conscious about the healthbenefits of these choices, they are likely to demand more transparency and control over the bacterial composition of their food. In conclusion, bacterial communities in food systems are a fascinating and dynamic aspect of our daily lives. They contribute to food safety, flavor, and health, but also pose challenges in maintaining balance and preventing illness. As we continue to learn and understand these complex ecosystems, it's essential to balance the benefits with the need for food safety and sustainability. So, the next time you enjoy a meal, remember the invisible world of bacteria that make it all possible!。

1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。

A PPLIED AND E NVIRONMENTAL M ICROBIOLOGY,July2002,p.3606–3613Vol.68,No.7 0099-2240/02/$04.00ϩ0DOI:10.1128/AEM.68.7.3606–3613.2002Copyright©2002,American Society for Microbiology.All Rights Reserved.Changes in Bacterioplankton Community Structure and Activity with Depth in a Eutrophic Lake as Revealed by5S rRNA AnalysisKatja Dominik and Manfred G.Ho¨ﬂe*GBF—German Research Center for Biotechnology,Department of Environmental Microbiology,D-38124Braunschweig,GermanyReceived10December2001/Accepted4April2002The community structure of bacterioplankton was studied at different depths(0to25m)of a temperateeutrophic lake(Lake Plußsee in northern Germany)by using comparative5S rRNA analysis.The relativeamounts of taxonomic groups were estimated from5S rRNA bands separated by high-resolution electrophore-parison of partial5S rRNA sequences enabled detection of changes in single taxa over space andduring seasons.Overall,the bacterioplankton community was dominated by3to14abundant(>4%of the total5S rRNA)taxa.In general,the number of5S rRNA bands(i.e.,the number of bacterial taxa)decreased withdepth.In the fall,when thermal stratiﬁcation and chemical stratiﬁcation were much more pronounced thanthey were in the spring,the correlation between the depth layers and the community structure was morepronounced.Therefore,in the fall each layer had its own community structure;i.e.,there were differentcommunity structures in the epilimnion,the metalimnion,and the hypolimnion.Only three5S rRNA bandswere detected in the hypolimnion during the fall,and one band accounted for about70%of the total5S rRNA.The sequences of individual5S rRNA bands from the spring and fall were different for all size classes analyzedexcept two bands,one of which was identiﬁed as Comamonas acidivorans.In the overall analysis of the depthproﬁles,the diversity in the epilimnion contrasted with the reduced diversity of the bacterioplankton commu-nities in the hypolimnion,and large differences occurred in the composition of the epilimnion at differentseasons except for generalists like C.acidivorans.A fundamental concept in limnology is that the water col-umn of a lake is divided into three horizontal layers,the epi-limnion,the metalimnion,and the hypolimnion(22).These strata are shaped by solar radiation and wind.In temperate regions,they are characterized by gradients of temperature and oxygen,and they have different functions for the lake as an ecosystem.The epilimnion is well mixed and acts as a biore-actor for primary production,and the hypolimnion is a sink where biomass accumulates and is remineralized(43).These two parts are connected by the metalimnion or thermocline, often characterized by a steep decrease in temperature.This basic view of the structure of a lake illustrates that depth is a key factor for all limnic life and that depth proﬁles are key features for understanding aquatic ecosystems.The depth dependence of the structure of phyto-and zoo-plankton communities in lakes and oceans has been studied since the beginning of aquatic ecology and has contributed enormously to the understanding of the functioning of fresh-water and marine ecosystems.Despite the recognition of the important role of bacteria in the microbial food web and the contribution of bacteria to the functioning of aquatic ecosys-tems,knowledge about the depth distribution of these organ-isms is still scarce(1,5).Pioneering work has been done on the depth-dependent community structure of bacterioplankton in the oceans by DeLong(6),Giovannoni et al.(15),and Fuhr-man et al.(13).These studies were based on analysis of16S ribosomal DNA(rDNA)clone libraries from deep ocean sam-ples(500to2,000m)and provided insight into the depth distribution of single taxa.Field et al.(12)obtained higher resolution of the variation in bacterial communities with depth by using group-speciﬁc16S rRNA-targeted oligonucleotide probes and environmental bulk DNA-RNA for assessment of the abundance of speciﬁc taxa belonging to the␣subgroup of the Proteobacteria in the Sargasso Sea.However,community structure can be assessed most rapidly by communityﬁnger-print methods that are based on electrophoretic separation of 16S rDNA amplicons or5S rRNA obtained from natural mi-crobial communities(27,30,34,35).Theﬁrst communityﬁn-gerprinting analysis of bacterioplankton with depth was done by using amplicons of the V3region of16S rRNA and archaeal and bacterial primers(29).Øvrea˚s et al.(29)found that the diversity of the bacterial community decreased from the sur-face to the bottom in an estuary with a strong vertical gradient of salinity and oxygen.However,a major problem of PCR-based methods is that they can be biased for some phylogenetic groups,and quantitative assessment of community diversity is difﬁcult(39).Therefore,we have used5S rRNAﬁngerprinting to obtain a direct molecularﬁngerprint from natural commu-nities and have calculated,based on the relative densities of individual5S rRNA bands,the diversity of the bacterioplank-ton during the year(20).The goals of the present study were to compare changes in physical,chemical,and biological parameters with depth at a high spatial resolution(i.e.,every meter)with changes in the structure of the bacterioplankton community and to extend the previous seasonal study in terms of depth(20).The ecosystem chosen was Lake Plußsee,a naturally eutrophic lake in north-ern Germany which has been examined extensively(28).We analyzed the5S rRNA fraction of environmental rRNA ob-*Corresponding author.Mailing address:GBF-German Research Center for Biotechnology,Department of Environmental Microbiol-ogy,Mascheroder Weg1,D-38124Braunschweig,Germany.Phone:49-531-6181-419.Fax:49-531-6181-411.E-mail:mho@gbf.de.3606tained directly(i.e.,without PCR)from the bacterioplankton. The structure of the bacterioplankton community as a function of depth was analyzed based on high-resolution5S rRNA com-munityﬁngerprints and partial or full sequences of individual 5S rRNA.MATERIALS AND METHODSSampling.Water samples were collected from the central part of Lake Plußsee (Schleswig-Holstein,Germany;54°10.0ЈN,10°0.23ЈE)on25April and19Sep-tember1994by using a2.5-liter Ruttner sampler at the following depths:0,1,2, 3,4,5,6,7,8,10,15,20,and25m.The bacterioplankton fraction wasﬁltered onto a sandwich consisting of a glassﬁberﬁlter(type GF/F;diameter,90mm; Whatman Corp.)on top of a polycarbonateﬁlter(pore size,0.2␮m;Nuclepore Corp.)and was stored frozen(Ϫ70°C)for later RNA analysis.The samples had been preﬁltered through a polycarbonateﬁlter(Nuclepore Corp.)with a pore size of3␮m to eliminate larger organisms.Biological background parameters,such as total bacterial counts,total hetero-trophic nanoﬂagellate(HNF)counts,and total particulate chlorophyll a concen-trations,were measured by standard techniques;details are given elsewhere(4). 5S rRNAﬁngerprints of bacterioplankton.5S rRNA analysis of bacterioplank-ton started with extraction of the total RNA directly from the frozenﬁlter sandwiches.This extraction was based on mechanical extraction with glass beads in a bead beater in combination with chemical extraction with phenol and sodium dodecyl sulfate(SDS)(8,16).In short,total environmental RNA was extracted from the sandwiches by using4g of2-and3-mm-diameter glass beads,a high-speed cell disrupter(Microdismembrator;Braun-Diessel Corp.)with a Te-ﬂon extraction cell,and a mixture of5ml of RNA extraction buffer and5ml of buffer-saturated phenol(17).Extraction buffer consisted of50mM sodium acetate,10mM EDTA,and1%SDS(pH4.2).Phenol was saturated with extraction buffer lacking1%SDS until aﬁnal pH of4.2to4.4was reached.A thin slurry(total volume,about12ml)of the twoﬁlters and the extraction mixture was generated by vibration for2min with the cell disrupter.This slurry was then centrifuged for10min at8,500ϫg.After repeated phenol(pH4.2)extraction of the pellet,the supernatants were pooled and treated twice with5ml of chloroform.Total RNA was precipitated from the pooled supernatant by addi-tion of ethanol(2.5times the volume of the supernatant)and storage atϪ20°C overnight.The total RNA obtained by theﬁlter extraction procedure was3Јend labeled with cytidine3Ј,5Ј-[5Ј-32P]bisphosphate(speciﬁc activity,3,000Ci/mmol)by us-ing RNA ligase with an efﬁciency of about62%(7,10).The radioactively labeled RNA was subjected to denaturing high-resolution electrophoresis on a10% polyacrylamide gel(Sequicel;0.4mm by38cm by80cm;Bio-Rad)by using running conditions consisting of a stepwise increase over a5-h period from100 to300W.After electrophoresis,the gel was exposed to X-rayﬁlm or a storage phosphor screen(Molecular Probes Corp.)for30min to several hours.More details concerning RNA extraction and electrophoretic analysis are given else-where(8,16,17,19,40).Comparative analysis of5S rRNAﬁngerprints.The5S rRNA fraction was evaluated quantitatively by scanning the autoradiograms with an optical gel scanner(Hirschmann Corp.)and by using a phosphorimager(Molecular Probes Corp.).In this way the abundance of single5S rRNA bands was quantiﬁed in terms of baseline corrected peak areas and in terms of number of pixels per band, respectively.These arbitrary units were used to calculate the relative abundance of every individual5S rRNA band and its contribution to the total5S rRNA. The diversity of the community was determined by determining the number of 5S rRNA bands per sample and by calculating the Shannon diversity index(H)with the following equation:HϭϪS⌺iϭ1͑n i/N͒log͑n i/N͒,where n i is the relativeabundance of a single taxon(single5S RNA band),N is the total abundance of all taxa,and S is the total number of abundant species.Only bands accounting for more than4%of the total5S rRNA were included in the calculation of the diversity index and the total number of bands.This4%threshold is not based on the detection limit of the5S rRNA analysis,which is less than0.1%.Rather,it is an arbitrary threshold set to make our data analysis comparable with analyses in earlier studies(18).Sequence analysis of single environmental5S rRNA.After high-resolution electrophoresis,32P-labeled5S rRNA bands were excised from the gel,eluted with150␮l of elution buffer(0.5M sodium acetate,0.1%SDS,1mM magnesium chloride,100␮M EDTA;pH7.8),and ethanol precipitated atϪ20°C overnight. Before application to the sequencing gel,5S rRNA was enzymatically digested with base-speciﬁc RNases(9,16).The cleaved5S rRNA of a single excised band was run on a high-resolution sequencing gel(80by40cm;thickness,0.4mm;10%acrylamide LongRanger[FMC Corp.]prepared according to the protocol of the manufacturer)to determine the positions of the bases in comparison to the bases of totally hydrolyzed5S rRNA of Escherichia coli,which was used as the molec-ular size marker.This sequencing gel provided information about the positions of almost all bases of the5S rRNA from the5Јend.Only around2%of the molecule(i.e.,two or three nucleotides at the3Јend)could not be read.These sequences could lead to classiﬁcation or identiﬁcation of the taxon represented by a single band provided that the reference sequence of a known bacterial species is available in the5S rRNA sequence database(33).Also,this sequence information was used to determine the homogeneity of a band(i.e.,if one or more taxa formed the band).Furthermore,it allowed comparison of5S rRNA bands of the same length for samples from different depths and times of the year. Such comparisons were also possible with partial sequences(e.g.,by using only RNase T1to examine the positions of all guanine residues within a5S rRNA band of a speciﬁc size).RESULTSEnvironmental conditions in Lake Plußsee.Physical,chem-ical,and biological background data for spring1994for Lake Plußsee are given in Fig.1.The temperature steadily decreased with depth from1to10m,and thus there was not an extended epilimnion.The oxygen content peaked at2m,corresponding to the top of the chlorophyll a maximum(Fig.1b),indicating that the maximum primary production occurred at this depth. The phosphate concentrations decreased to about50␮g/liter in the upper5m,indicating that the phytoplankton was most active in this layer.Below5m,the phosphate and inorganic nitrogen contents(7;data not shown)increased rapidly to the high hypolimnic concentrations.The chlorophyll a concentra-tions reached65␮g/liter,indicating that there was a well-developed spring phytoplankton bloom and that the lake was eutrophic.The phytoplankton community during this spring bloom was composed of a variety of diatoms and green algae (28).The total bacterial numbers(Fig.1b)in the upper5m were rather constant(more than1.2ϫ107cells/ml)and de-creased steadily with depth.The concentration of HNF peaked at3m at about1ϫ105cells/ml;the concentration then dropped rapidly to less than4ϫ103cells/ml.These back-ground data indicate that there was a fairly normal spring situation for a temperate eutrophic lake with a small,unstable epilimnion(0to1m),an extended metalimnion(1to8m)with maximum primary production close to the surface(2m),and a hypolimnion below8m which was still fully oxic.The fall data(Fig.2)revealed a homogenous epilimnion(0 to5m),a metalimnion with very steep decreases in oxygen concentration and temperature(5to8m),and a fully anoxic hypolimnion at depths below8m.The phosphate concentra-tions were below the detection limit in the epilimnion and increased to rather high levels in the metalimnion.The chlo-rophyll a concentration was low in the epilimnion and peaked at8m(i.e.,in the anoxic hypolimnion).The bacterial concen-tration did not vary signiﬁcantly with depth,but the concen-tration of HNF peaked together with the chlorophyll a con-centration,as it did in the spring.As in the spring,in the fall of 1994Lake Plußsee had the very stable and well-structured depth proﬁle of a naturally eutrophic lake during autumn(22). Changes in the overall structure of the bacterioplankton community with depth.In the spring,the community structure for the depth proﬁle showed three different banding patterns of the5S rRNA that corresponded with the three layers of the lake(Fig.3a).The epilimnion(0to3m)had six or sevenV OL.68,2002DEPTH DISTRIBUTION OF BACTERIOPLANKTON COMMUNITIES3607FIG.1.Physical,chemical,and biological background data for depth pro ﬁles of Lake Plu ßsee during the spring (25April 1994).(a)Temperature (ᮀ),oxygen content ( ),and phosphorus content (Œ);(b)chlorophyll a content (ࡗ),total bacterial number (E ),and number of HNF (‚).FIG.2.Physical,chemical,and biological background data for depth pro ﬁles of Lake Plu ßsee during the fall (19September 1994).(a)Temperature (ᮀ),oxygen content ( ),and phosphorus content (Œ);(b)chlorophyll a content (ࡗ),total bacterial number (E ),and number of HNF (‚).3608DOMINIK AND HO¨FLE A PPL .E NVIRON .M ICROBIOL .abundant bands ranging in size from 116to 123nucleotides (nt),the metalimnion (4to 8m)had the highest number of bands with additional bands in the size range from 109to 115nt,and the hypolimnion (10to 25m)had the lowest number of bands,with one band comprising more than 50%of the total 5S rRNA.At greater depths the community structure changed the most.For example,at 15to 20m the two most abundant bands comprised more than 70%of the total 5S rRNA and the 5S rRNA bands at 122and 123nt had disappeared.The amount of small 5S rRNA (111to 113nt),which comprised about 20%of the total 5S rRNA,increased in the deepest water (25m).During thermal strati ﬁcation in the autumn the number of 5S rRNA bands was high in the epilimnion and was greatly reduced below the thermocline (Fig.3b).Also,in the upper 5m the number of bands,their relative positions,and their relative amounts were very stable.Only at 1m was an addi-tional band at 117nt observed.The bacterioplankton commu-nity of the anoxic hypolimnion (7to 25m)was dominated by a single 5S rRNA band at 121nt (on average,this band ac-counted for 63%of the total 5S rRNA from 10m and below).The results of a detailed quantitative analysis of the amount of 5S rRNA per abundant band in the autoradiograms,ob-tained by using a phosphorimager for quanti ﬁcation,are shown in Fig.4.In both depth pro ﬁles the number of abundant bands was rather limited,ranging from three to seven.Also,these abundant bands (i.e.,bands that accounted for more than 4%of the total 5S rRNA)comprised more than 90%of the total 5S rRNA in most samples (Fig.4B).The spring pro ﬁle was slightly more variable in this respect than the fall pro ﬁle due to the larger number of less abundant bands occurring in the metalimnion.Another general trend for both depth pro ﬁles was that in the epilimnion no single band exceeded a relative abundance of 34%and a small number of bands (three to ﬁve bands),each comprising 10to 30%of the total,represented the majority of the bacterioplankton community.With increas-ing depth one band became dominant in both depth pro ﬁles;i.e.,it had a relative abundance of more than 50%.Further-more,in both pro ﬁles for depths below 15m the dominant band was the same size (121nt)despite the anoxic conditions in the fall.The two depth pro ﬁles did differ,however;when anoxic conditions were reached at depths below 6m,the 123-nt band disappeared and a 113-nt band appeared.In general,changes in the community composition were more gradual in the spring pro ﬁle than in the fall pro ﬁle.To examine overall diversity,the Shannon indices were cal-culated for all samples based on the total number of 5S rRNA bands,not just the number of abundant 5S rRNA bands,and their relative amounts (Fig.5).The total number of bands was determined by counting every band that had a relative abun-dance of more than 0.1%of the total 5S rRNA.This threshold re ﬂects the level of resolution of the 5S rRNA ﬁngerprinting technique when a phosphorimager is used.In contrast,the 4%threshold for the abundant 5S rRNA is an arbitrary threshold that was introduced earlier to make community ﬁngerprints more comprehensible (18).Additionally,the following as-sumptions for calculation of the Shannon diversity index based on the relative amounts of individual 5S rRNA bands were made:(i)RNA was extracted with comparable ef ﬁciencies from all taxa,(ii)5S rRNA was 32P labeled with comparable ef ﬁciencies,and (iii)sequence analysis of individual 5S rRNA shows homogeneity.The validity of these assumptions was demonstrated in calibration experiments with reference cul-tures (7,8,40)or direct sequencing (see below).DuringtheFIG.3.Autoradiograms of 5S rRNA from bacterioplankton obtained at different depths.Samples of the water column were taken at different depths.For detection the 5S rRNA was 3Јend labeled with 32P.(a)Depth pro ﬁle for the spring (25April 1994).Lanes M contained a molecular weight standard consisting of hydrolyzed 5S rRNA from E.coli .The intensity of the bands in the boxed area was electronically reduced for better visibility of the single bands.Each lane is labeled with the depth (in meters)at which the sample examined was obtained.(b)Depth pro ﬁle for the autumn (19September 1994):Lanes M contained a molecular weight standard consisting of hydrolyzed 5S rRNA from E.coli .Each lane is labeled with the depth (in meters)at which the sample examined was obtained.V OL .68,2002DEPTH DISTRIBUTION OF BACTERIOPLANKTON COMMUNITIES 3609spring,the overall diversity increased until 5m and then stayed maximal until 10m.Below 10m,the diversity decreased by a factor of two until 25m (Fig.5a).During the fall,the diversity was highest in the epilimnion and then declined uniformly in the anoxic part of the water column (Fig.5b).In both pro ﬁles the Shannon index generally followed the total number of 5S rRNA bands.The most striking difference was that the maxi-mum diversity occurred in the spring below the maximum chlorophyll a concentration,whereas in the fall the maximum diversity was found above the maximum chlorophyll a concen-tration.Detailed community structure as revealed by 5S rRNA se-quence analysis.A sequence analysis of single 5S rRNA bands was performed by using base-speci ﬁc RNases in order to com-pare 5S rRNA bands from different depths that were the same size and to identify the closest taxonomic relatives by compar-ison with the international sequence database (33).To exam-ine 5S rRNA bands with depth,only partial sequences were determined by using RNase T 1and sometimes RNase U 2di-gests to obtain a G and/or A ladder of the 5S rRNA band for about 80%of the molecule,as described previously (17,20).In the spring,5S rRNA bands at 116,118,120,121,and 123nt were compared.All of the bands analyzed had the same partial sequences from 0to 8m.Below 8m,only a few bands could be sequenced due to the much lower amount of radioactivity per band (Fig.3a).The partial sequence of the 121-nt band from 25m,which dominated the community at this depth,was different from the sequence of the 121-nt band obtained from the upper 8m.In the fall,5S rRNA bands at 116,120,121,122,and 123nt were compared for the upper 5m.In addition,the 121-nt band was sequenced from all samples of the depth pro ﬁle.There were no sequence differences detected in any of the fall 5S rRNA bands with depth;i.e.,all bands sequenced in the upper 5m and the 121-nt band found throughout the water columnwere the same.A comparison of the sequences from the spring with the sequences from the fall showed signi ﬁcant differences for all of the single size classes analyzed except the 116-and 122-nt bands.These bands had identical partial sequences;i.e.,all As and Gs were at the same position in the 5S rRNA for both seasons.The 121-nt band,which was the most abundant 5S rRNA in the hypolimnion in both depth pro ﬁles,had dif-ferent sequences in the two seasons,despite the identical sizes.These sequence data indicate that different taxa dominated the hypolimnion in the spring and in the fall.The only 5S rRNA for which a de ﬁnite phylogenetic relative could be found was the 116-nt band,which had a sequence identical to a Comamonas acidovorans sequence (accession number AJ131594).The presence of C.acidovorans has been documented in samples from Lake Plu ßsee before (11,20).For all other bands except the 123-nt band either the sequence information obtained was not complete enough for alignment with sequences from the international database or no close relative could be found;the closest phylogenetic neighbor of the 123-nt band was in the genera Thiovolum and Campy-lobacter of the ⑀subgroup of the Proteobacteria .DISCUSSIONOverall community structure and activity of bacterioplank-ton at different depths and in different seasons.In contrast to PCR-driven community ﬁngerprints,5S rRNA ﬁngerprints of bacterioplankton re ﬂect directly the amounts of different 5S rRNA from different members of the bacterial community.Therefore,we have de ﬁned a single 5S rRNA band with a homogenous sequence as an operational taxonomic unit (OTU)(20).This OTU de ﬁnition enables us to calculate the Shannon diversity index for each community based on the relative abundance of each 5S rRNA in comparison to the total amount of 5S rRNA for each depth.Two technicalprerequi-FIG.4.(A)Relative amounts of the single abundant 5S rRNA bands for the spring shown in Fig.3a.Different colors indicate 5S rRNA bandsof different sizes that account for more than 4%of the total 5S rRNA calculated as detailed in Materials and Methods.(B)Relative amounts of single abundant 5S rRNA bands for the fall shown in Fig.3b.3610DOMINIK AND HO¨FLE A PPL .E NVIRON .M ICROBIOL .sites for assessment of the relative amounts of 5S rRNA have to be ful ﬁlled;the ef ﬁciencies of extracting RNA from natural bacterioplankton should be similar for all taxa,and the ef ﬁ-ciencies of 32P labeling the 3Јends of the 5S rRNA should be similar.It has been demonstrated that both prerequisites are ful ﬁlled with the methodology used here (7,8,20,40).Another theoretical and technical consideration for calculation of the Shannon index is to set a lower limit for the relative abundance of a single OTU.Such a lower limit has been set at a relative abundance of 4%for clarity (Fig.4)and comparability with past studies (18,20).On the other hand,the detection limit for single 32P-labeled 5S rRNA bands is about 0.1%if adjacent bands are not so large that they completely cover the weak bands next to them in the autoradiogram.This analytical prob-lem can be overcome by using a phosphorimager that enables detection of bands next to each other whose band intensities are more than 4orders of magnitude different.A 0.1%detec-tion limit means that we were able to detect taxa whose con-centrations were as low as 0.5ϫ106to 1ϫ106cells per liter in the 5S rRNA ﬁngerprints of bacterioplankton from Lake Plu ßsee with the described methodology if an average cell density of 5ϫ109to 10ϫ109cells per liter was assumed (Fig.1b and 2b).This detection limit is about 1order of magnitude lower than the 1%limit commonly reported for PCR-based ﬁngerprint methods,such as denaturing gradient gel electro-phoresis or single-strand conformation polymorphism analysis (27,31).In addition,not only does the presence of a 5S rRNA band re ﬂect the abundance of an OTU,but the band also represents an active member of the bacterioplankton commu-nity since the amount of rRNA is correlated with the growth of bacteria (3).Vertical gradients of physical and chemical parameters strongly affect the biology and ecology of all pelagic ecosystems (22).This universal ﬁnding is also re ﬂected in the structure of the bacterioplankton community.The most striking feature of both depth pro ﬁles was the much lower diversity in the hy-polimnion than in the upper layers.The decrease can be seen in the reduction of the Shannon index by about one-half,as well as in the number of bands,which decreased from about 14to 7in both depth pro ﬁles (Fig.5).One exception to this overall trend was the 25-m sample collected in the spring,for which the number of bands increased.Conceivably,the dia-toms which dominate the phytoplankton of Lake Plu ßsee in early spring were still being degraded in the deep hypolimnion after they sank in late March (28,32).When the diversity data for the depth pro ﬁles were com-pared with the data from the seasonal study of bacterioplank-ton in Lake Plu ßsee,it was obvious that the low diversity,asFIG.5.(a)Total number of bands ( )(bands containing more than 0.1%of the total 5S rRNA)and Shannon diversity index (ᮀ)calculated from all 5S rRNA bands at each depth during the spring.The chlorophyll a content (ࡗ)is shown for comparison.(b)Total number of bands ( )and Shannon diversity index (ᮀ)calculated from all 5S rRNA bands at each depth during the fall.The chlorophyll a content (ࡗ)is shown for comparison.V OL .68,2002DEPTH DISTRIBUTION OF BACTERIOPLANKTON COMMUNITIES 3611indicated by a Shannon index of around0.4,in the hypolim-nion was encountered only during the three minima occurring in the epilimnion at special times of the year(20).During most of the year the Shannon index remained in the range found for the epilimnion for both depth proﬁles.Theﬁrst minimum for the diversity index in the seasonal study could be explained by strong grazing by cladocerans,whereas grazing by HNF did not have a signiﬁcant effect on diversity.Grazing by HNF did not directly affect the high diversity in the upper layers of depth proﬁles,where HNF grazing is the major mortality factor in the epilimnion(41).On the other hand,viral lysis was demon-strated to be the key mortality factor in the anoxic hypolimnion of Lake Plußsee,where all grazing was strongly reduced(41). This result corroborates theﬁnding that only very few mem-bers of the bacterioplankton community were active in the hypolimnion because low-diversity bacterial communities can be controlled more easily by viral lysis than high-diversity com-munities(37).The major reason for the low diversity in the hypolimnion for both depth proﬁles might be the decrease in the dissolved organic carbon content,which not only decreased by about50%with depth but also accounted for a far higher proportion of refractory organic compounds in the hypolim-nion than in the epilimnion of Lake Plußsee(26).Depth-dependent and seasonal changes in detailed struc-ture of the bacterioplankton community.The presence and depth distribution of C.acidovorans in Lake Plußsee have been demonstrated previously by immunoﬂuorescence microscopy (11,42).Additionally,a seasonal study in which5S rRNA proﬁling was used showed that C.acidovorans is abundant at a depth of1m during all seasons(20).Both depth proﬁles obtained by using5S rRNA analysis conﬁrmed the presence of C.acidovorans in the whole proﬁle,with an average abundance of about6.0%.(The levels in deeper samples were around3% and therefore are not shown in Fig.4).Thisﬁnding is in contrast to the results obtained by immunoﬂuorescence mi-croscopy that indicated that C.acidovorans cells disappeared at depths below10m in the spring of1993and that there was a signiﬁcant lack of C.acidovorans cells in the metalimnion dur-ing the fall of1996(11,42).The most likely explanation for this ﬁnding is that the monoclonal antibodies used for detection of C.acidovorans cells are speciﬁc for a certain subspecies type, whereas the5S rRNA bands represent the whole taxon.There-fore,C.acidovorans was present in abundance in Lake Plußsee not only during all seasons but also at all depths,including the anoxic hypolimnion.Furthermore,the presence of signiﬁcant amounts of rRNA indicated that this group of bacteria was metabolically active at all depths year round.This supports the hypothesis,put forward earlier(42),that C.acidovorans is a K-strategist well adapted to the pelagic environment of a eu-trophic lake because of its relative resistance to protozoan grazing and its ability to utilize refractory dissolved organic matter.Partial sequencing revealed differences in all bands except the116-and122-nt bands in the different seasons.The strong seasonal differences make sense considering the fact that the phytoplankton communities during the two seasons were com-pletely different and that in the fall the hypolimnion was an-oxic.Detection of members of the⑀subgroup of the Proteobac-teria in the anaerobic water column is unusual but has been observed with clone libraries from coastal marine water sam-ples obtained during an algal bloom and from the chemocline of a meromictic lake in the Swiss Alps(2,21). Comparison of bacterioplankton community structure with other depth distributions.A variety of studies have been per-formed with different molecular methods to understand the depth distribution of the bacterioplankton community(14,25). Most of these studies were based on clone libraries of16S rDNA genes(see reference14for a review),followed by hy-bridization studies and communityﬁngerprinting.While clone library studies provide insight into community composition and diversity,ﬁngerprinting studies reveal the overall structure of the whole community and enable direct comparisons of a va-riety of samples.Fingerprinting studies of the bacterioplankton of a brackish meromictic lake and the central Baltic Sea indi-cated that there was higher diversity in the upper layer than in the anoxic bottom layer,as found for Lake Plußsee(18,29). Recent studies of bacterioplankton in a deep ultraoligotrophic lake and the eastern Mediterranean Sea showed that there were distinct changes with depth but did not reveal substantial decreases in diversity(24,38).Several studies performed with group-speciﬁc rRNA-targeted probes revealed the depth de-pendence of these groups in the water column of the oceans (12,23).The amounts of some of these groups correlated well with microbial processes,such as the intensity of primary pro-duction(36).In general,the following picture appears to emerge for the structure of the bacterioplankton community with depth.Pelagic environments have speciﬁc bacterioplank-ton community structures at speciﬁc depths,and the overall diversity and composition might vary from layer to layer de-pending on the trophic status and the availability of electron acceptors.Conclusions.We demonstrated that there were changes in bacterioplankton community structure with depth in a eutro-phic lake.These changes corresponded well with the three layers,epilimnion,metalimnion,and hypolimnion,of the lake. The more the lake was stratiﬁed,the better the three different communities were separated.Whereas the community compo-sition generally varied with the season,some members of the community,such as C.acidovorans,were abundant at all depths during both seasons.Another common feature was the reduction in the overall diversity of the bacterioplankton with depth.This decrease in diversity means that the diversity of the bacterioplankton community in the hypolimnion was quite low compared to the diversity of the community in the epilimnion, where the diversity is low only two or three times during the year(20).ACKNOWLEDGMENTSWe thank D.Albrecht from the MPI of Limnology in Plo¨n for providing background data.The valuable comments of Ingrid Brettar and Markus G.Weinbauer on an earlier version of the manuscript are greatly acknowledged.Improvement of the English by David Kirch-man is greatly appreciated.This project was supported by funds from Bundesministerium fu¨r Bildung,Wissenschaft,Forschung und Technologie grant BEO-0319433B and Deutsche Forschungsgemeinschaft grant Ho930/2-1.REFERENCES1.Azam,F.,T.Fenchel,J.G.Field,J.S.Gray,L.A.Meyer-Reil,and F.Thingstad.1983.The ecological role of water-column microbes in the sea.Mar.Ecol.Prog.Ser.10:257–263.2.Bossard,P.P.,Y.Santini,D.Gru¨ter,R.Stettler,and R.Bachofen.2000.3612DOMINIK AND HO¨FLE A PPL.E NVIRON.M ICROBIOL.。