Microbial community structure in anaerobic co-digestion

ORIGINAL PAPER

Microbial community structure in anaerobic co-digestion of grass silage and cow manure in a laboratory continuously stirred tank reactor

Hong Wang ?Katariina Tolvanen ?

Annimari Lehtoma

¨ki ?Jaakko Puhakka ?Jukka Rintala

Received:16December 2008/Accepted:17July 2009/Published online:30July 2009óSpringer Science+Business Media B.V.2009

Abstract The impacts of feeding ratio and loading rate on the microbial community during co-digestion of grass silage with cow manure in an anaerobic laboratory continuously stirred tank reactor were investigated by 16S rRNA gene-based ?ngerprints.The microbial community remained stable when the reactor was fed with cow manure alone and with up to 20%of grass silage in feedstock at an organic loading rate (OLR)of 2kg VS m -3day https://www.sodocs.net/doc/721506855.html,rge changes in the bacterial community were observed when the loading ratio of grass was increased to 40%,while there was little change in the archaeal community.During the increase in OLR from 2to 4kg VS m -3day -1the bacterial community struc-ture showed few differences,whereas Archaea was undetectable.Sequencing of the major DGGE bands indicated that the phylum Bacteriodetes predominated

in the bacterial community.Two unclassi?ed bacteria with high abundance survived throughout the opera-tion of the reactor.

Keywords Microbial community á

Anaerobic digestion áTerminal restriction fragment length polymorphism (T-RFLP)áDenaturing gradient gel electrophoresis (DGGE)áContinuously stirred tank reactor (CSTR)

Introduction

Anaerobic co-digestion of animal manure and organic wastes or crops to methane-rich biogas is increasingly used,as methane can be converted to heat,electricity or replace traf?c biofuel.Besides providing energy,greenhouse gas emissions are also expected to decrease from waste and manure management (Amon et al.2006;Clemens et al.2006).Co-digestion of crops with animal manure has been demonstrated in contin-uously stirred tank reactors (CSTRs)in the laboratory (Fischer et al.1983;Fujita et al.1980;Hashimoto 1983;Hills 1980;Somayaji and Khanna 1994;Wei-land 2003)and on the farm scale (Kaparaju et al.2002).A variety of start-up strategies,feeding com-positions,organic loading rates (OLRs)and hydraulic retention times (HRTs)have been evaluated,but the microbial community structure has only recently been

H.Wang (&)áJ.Rintala

Department of Biological and Environmental Science,

University of Jyva

¨skyla ¨,P.O.Box 35(NSC),40014University of Jyva

¨skyla ¨,Finland e-mail:hong.wang@helsinki.?

K.Tolvanen áJ.Puhakka

Institute of Environmental Engineering and

Biotechnology,Tampere University of Technology,P.O.Box 541,33101Tampere,Finland

Present Address:

A.Lehtoma

¨ki Jyva ¨skyla ¨Innovation Ltd,P.O.Box 27,40101Jyva ¨skyla ¨,Finland

Biodegradation (2010)21:135–146DOI 10.1007/s10532-009-9288-5

linked to operating conditions and digestion perfor-mance.Stable performance of anaerobic digestion systems,as measured by methane yield,can be greatly related to steady microbial community structure (McMahon et al.2001).However,a?exible commu-nity structure can maintain a stable ecosystem function (Fernandez et al.1999,2000).Thus,the link between the performance and microbial community structure remained uncertain.A community-based approach to anaerobic digestion should therefore produce more knowledge about the relations between microbial community structure and operating and functional parameters.

The anaerobic digestion of organic material to methane is a multi-step process mediated by Bacteria and methanogenic Archaea(Chynoweth and Pul-lammanappallil1996).The polymers are hydrolysed into soluble compounds under fermentative condi-tions.Acidogens and acetogenic bacteria convert these intermediates into acetate and one-carbon compounds.These compounds in turn can be con-verted directly by methanogenic Archaea into meth-ane and carbon dioxide.Generally,hydrolysis is considered to be the rate-limiting step in the anaer-obic digestion of particulate organic material(Noike et al.1985;Veeken and Hamelers1999).Therefore, information on the microbial ecology and mecha-nisms of hydrolysis during anaerobic digestion is of importance in seeking to increase the rate of hydro-lysis and the overall ef?ciency of the anaerobic digestion process.A number of bacteria well known for their cellulolytic capabilities,and mostly belong-ing to the order Clostridiales,have been studied (Burrell et al.2004;Chachkhiani et al.2004;Lynd et al.2002;O’Sullivan et al.2005).However,the investigation of microbial community structure tak-ing part in the anaerobic co-digestion of crops and animal manure continue to be lacking.

Comparative analyses of anaerobic microbial com-munities by culture-based methods have limitations because of syntrophic interactions,low growth rates, unknown growth requirements and obligate anaero-biosis.The advent of small-subunit rRNA-based molecular?ngerprinting techniques has made it possible to study complex anaerobic microbial com-munities without the cultivation of microorganisms (Hugenholtz et al.1998).Among these?ngerprinting methods,terminal restriction fragment length poly-morphism(T-RFLP)(Dunbar et al.2001;Kitts2001)and denaturing gradient gel electrophoresis(DGGE) (Muyzer1999)are widely used for the differentiation of communities and for the comparison of the relative phylotype richness in environmental samples.They were also used for identifying speci?c organisms in a community in conjunction with gene sequence infor-mation.Both methods have several advantages over other?ngerprinting methods,since they have high sample throughput,and enable the resolution of a complete target community.Very recently,DGGE and T-RFLP pro?ling have been used in exploring microbial community structure in the anaerobic digestion of industrial wastes(Connaughton et al. 2006)and manure(Mladenovska et al.2006),and co-digestion of food waste and biosolids(McMahon et al. 2001).It is anticipated that knowledge on both the structure of microbial communities and the relations between microbial community structure and digester performance during anaerobic co-digestion of crops and manure would also be obtained by DGGE and T-RFLP pro?ling.

The aim of the present study was to investigate the microbial community structure involved in the co-digestion of grass silage with cow manure,and to assess the impact of the feed component ratio and OLR on the dynamics of the microbial community in a laboratory CSTR.

Materials and methods

Source of biomass

Biomass source have been described in detail by Lehtoma¨ki et al.(2007).In brief,grass silage was initially prepared from grass comprising75%of timothy Phleum pretense and25%of meadow fescue Festuca pratensis grown on a local farm in Central Finland(Kalmari farm,Laukaa).The grasses were harvested at the early?owering stage and chopped after24h of pre-wilting,and were further ensiled in a bunker silo with the addition of a commercial silage additive(lactic acid bacteria inoculant AIV Biopro?t, containing60%Lactobacillus rhamnosus and40% Propionibacterium freudenreichii spp.shermanii, Kemira Growhow Ltd.).The grass silage was further chopped to a particle size of approximately3cm in the laboratory and then stored at-20°C.Before feeding to the reactor,the grass silage was allowed to

thaw overnight at4°C and was then mixed with manure.Cow manure was obtained from a storage tank with a retention time of up to6months on several occasions during the study and stored at4°C. The composition of cow manure batches was also analyzed at each time when they were obtained and showed very little variation.Inoculum was taken from the farm’s mesophilic digester,which treated dairy manure and industrial confectionary by-products.

Reactor set-up and operation

Two5l CSTR reactors were operated at35±1°C and with a continuous stirring rate of300rpm.First the reactors were inoculated with4l of inoculum. Thereafter,the reactors were fed semi-continuously with cow manure at OLR of2kg VS m-3day-1once a day for5days week-1with a HRT of20days for 27days.Before feeding,an equal amount of digestate was withdrawn.The two reactors showed nearly identical speci?c methane yields and VS removals (Lehtoma¨ki et al.2007).Subsequently one reactor was continued similarly for another28days before stopped,while in the other reactor feeding of grass silage along with manure was then initiated by replacing10%of the feedstock volatile solids(VS) with grass silage while maintaining constant OLR and HRT.The proportion of grass silage in the feedstock was then stepwise increased to40%of the feedstock VS.The OLR was then increased?rst to3and then 4kg VS m-3day-1while the HRT was decreased to 18and16days,respectively.Increase in OLR was obtained as feed VS was increased from4.0to5.4and to6.4kg VS/m-3day-1whereas decreased HRT was obtained by slightly increasing the volume of added feed.In total,the318days trial was divided into seven different operational periods(P1–P7)characterized by a change either in the feedstock(grass silage propor-tion)or in the OLR(Table1).

DNA extraction and PCR-ampli?cation of16S rRNA genes

For the microbial community analyses,5ml of sample from the reactor was collected on each sampling day,as shown in Table1,and stored immediately at-80°C.Genomic DNA was extracted from the samples after a physical disruption of the biomass using a FastPrepòInstrument and a Fast DNAòSPIN Kit for Soil(Qbiogene,Inc.,CA,USA) according to the manufacturer’s instructions.

The16S rRNA genes for T-RFLP?ngerprinting were ampli?ed using the PCR primer sets27f-6-carboxy?uorescein(FAM)/1492r(Sait et al.2003) and Ar109f/Ar912rt-FAM(Lueders and Friedrich 2003)speci?c for the bacterial and archaeal16S rRNA genes,respectively.PCR was performed in 100-l l reaction mixture containing approximately 100ng of DNA template,19PCR buffer,200l M of each deoxynucleoside triphosphate,2U of DyNA-zyme TM II DNA polymerase(Finnzymes,Espoo,

Table1Operational conditions and performance of the reactor

Operational periods P1P2P3P4P5P6P7

Run days(day)0–2728–5556–8485–141142–203204–266267–318 Sampling day(day)15;2736;5064;7792;106;134148;162;190218;225;253281;295;309 Grass silage in the feed

(%VS)

0102030404040

OLR(kg VS m-3day-1)2222234

HRT(day)20202020201816

Speci?c methane yield

(m3kg-1VS)a

0.15±0.050.14±0.020.18±0.010.27±0.030.25±0.020.23±0.010.19±0.02 VS removal(%)b26414243465352

pH remained between7.3and7.6and VFA(acetic and propionic acids)concentrations were lower than300mg l-1in the digestates throughout the run

OLR Organic loading rate,HRT Hydraulic retention time

a Calculations as average±SD of the measurements during the last2weeks of each operational period

b Calculated on basis of average values

Finland),and0.5l M of each primer.The PCR conditions consisted of initial denaturation at94°C for3min followed by30cycles of denaturation at 94°C for30s,annealing at55°C for90s and extension at72°C for1min.The?nal extension was carried out at72°C for5min.

The16S rRNA genes for DGGE analysis were ampli?ed by nested PCRs.The primer sets27f/1492r and Ar109f/Ar912rt were used for the initial PCR ampli?cation.Cycle conditions were as follows: initial denaturation at94°C for3min,followed by 30cycles of denaturation at94°C for30s,annealing at60°C for30s,extension at72°C for45s,and?nal extension at72°C for10min.The PCR products were puri?ed with a GFX TM PCR DNA and Gel Band Puri?cation kit(Amersham,NJ,USA)and further used as templates for the second PCR ampli?cation. The primers used for the secondary PCR were universal forward primer533f and reverse primer 907r GC with a40-base pair GC-clamp attached to the 50terminus.The secondary PCR ampli?cation was performed using a touchdown program consisting of initial denaturation at94°C for3min followed by denaturation at94°C for30s,annealing at60°C for 30s and extension at72°C for45s.The annealing temperature was reduced by0.5°C every cycle until it reached50°C.A further10cycles was carried out under these conditions prior to the?nal extension cycle,which was performed at72°C for10min.All PCR products(5l l)were visually veri?ed using1or 1.5%agarose gel electrophoresis and ethidium bro-mide staining(BioRad,CA,USA).

T-RFLP analysis

Prior to digestion,amplicons were puri?ed with a GenElute TM PCR clean-up kit(Sigma)according to the manufacturer’s instructions and were quanti?ed by using NanoDropòND-1000spectrophotometer (NanoDrop Technologies,Inc.,USA).Approxi-mately100ng of puri?ed DNA was digested in a 10-l l reaction volume with10U of MspI and TaqI for Bacteria and Archaea,respectively.The diges-tions were carried out for3h at37°C for MspI and 65°C for TaqI.Fluorescently labeled terminal restriction fragments(T-RFs)were separated on an ABI Prismò3100automated sequencer(Applied Biosystems,CA,USA)using an internal size standard(GeneScan TM1200LIZò;Applied Biosystems,CA,USA).T-RFLP electropherograms were analyzed with GeneMapperòsoftware version 3.1(Applied Biosystems,CA,USA).T-RF sizes between50and1,200bp with a peak area of C50?uorescence units were used in the analysis.The analysis was performed in triplicate for each DNA extraction.The replicate pro?le with a total DNA quantity close to the average DNA quantity of the T-RFLP pro?les in the comparison data set was selected for the analysis.Pro?les from different samples were manually aligned by inspection of the size of peaks in bases according to Sait et al.(2003). T-RF pro?les were then standardized to the pro?le with the smallest total?uorescent units of peak areas following the procedure suggested by Dunbar et al.(2001).After standardization,a percentage threshold was applied to remove all T-RFs that contributed\2%to the total area of all the T-RFs in a T-RFLP pro?le.The relative abundance of a detected T-RF within a given pro?le was then calculated on the basis of the standardized peak area according to Schwarz et al.(2007).

For comparison of the bacterial T-RFLP pro?les,a binary vector representing the presence(1)or absence (0)of T-RFs in the pro?le was constructed for each sample.A Sorensen–Dice similarity coef?cient was used as a measure of the similarity of binary vectors, and a matrix of pairwise comparisons was generated (Jackon et al.1989).Agglomerative hierarchical clustering was performed using the similarity matrix of the Sorensen–Dice coef?cients and the Nearest Neighbor method and was displayed as dendrograms. The analyses were performed by using the SPSS14.0 software for windows(SPSS https://www.sodocs.net/doc/721506855.html,A).

DGGE

DGGE was performed as described by Bodelier et al. (2005)using the INGENYphorU-292system(Ing-eny,The Netherlands).Aliquots of25l l of each of the GC-clamped amplicons were loaded into the6% polyacrylamide gels with denaturing gradient ranging from30to60%where100%denaturant contained 7M urea and40%formamide.The gels were run at 100V and60°C for20h.Following this,the gels were stained for half an hour in Sybr-Gold TM(Invit-rogen,USA)nucleic acid stain(1:10000dilution in 19TAE buffer)and photographed with a Kodak1D

v.3.5.4system(Kodak,USA)equipped with a UV illuminator.

Sequencing of16S rRNA gene fragments

and phylogenetic reconstruction

DGGE bands for sequencing were selected according to the emergence or disappearance of particular bands and the predominant bands for all the samples. Selected bands were excised and suspended in20l l of milli-Q sterilized water for24h at4°C.The purity of selected bands was con?rmed by a second DGGE. The elution from pure bands was used as templates in the PCR reactions,which were conducted with the primer set533f/907r.PCR products were puri?ed for sequencing with Econuclease I-SAP enzyme(Fer-mentas)according to the manufacturer’s instructions. Sequencing was performed on an ABI Prismò3100 sequencer(Applied Biosystems,CA,USA)using an ABI BigDyeòterminator v3.1cycle sequence kit (Applied Biosystems,CA,USA)as speci?ed by the manufacturer.The nucleotide sequence of each band was con?rmed by sequencing two or three parallel bands.

Raw sequence data were assembled and checked with the DNAMAN software package(Lynnon Biosoft,USA).An equal portion(about372bp)of sequences were aligned and uploaded to the MyRDP space maintained at the Ribosomal Databases Project II(Cole et al.2007).A homology search of aligned sequences against the sequences available in dat-abases RDP II(release9.46January2007)was performed with the SeqMatch program at the RDP II. Finally,a phylogenetic tree was constructed by the neighbor-joining method(Saitou and Nei1987)with the PHYLIP3.65software package and the Jukes-Cantor distance model(Felsenstein1985).Bootstrap re-sampling analysis for100replicates was per-formed to estimate the con?dence of the tree topology(Felsenstein1985).

Nucleotide sequence accession numbers

The nucleotide sequences reported in this paper were deposited in the NCBI nucleotide sequence databases under accession numbers EF597504through EF59 7510.Results

The microbial community structure in the reactor was investigated during the seven different periods(P1–P7)from samples taken soon after the feeding or OLR change(early E,day9–15),after ca1HRT operation(middle M,day22–29)and after ca2HRTs operation(late L,day49–50)(Table1).

Bacterial community dynamics

The T-RFLP?ngerprints of bacterial16S rRNA gene fragments revealed a total of32terminal restriction fragments(T-RFs)during the run(Fig.1).In the beginning of the run(P1M)12T-RFs were detected with relatively abundant T-RFs of sizes65,85,157, 536and540bp,which together accounted for73.7% of the pro?le.The inclusion and increase of grass silage in the feedstock led to the reduction or emanation of some of these T-RFs and the appear-ance of some new T-RFs.For instance,T-RFs with sizes of176,186,296,359and536bp disappeared as the proportion of grass in the feedstock increased.On the contrary,there were two T-RFs of87and144bp appeared.Several minor T-RFs of128,133,134and 144bp were detected only during the reactor was fed with30%grass silage in the feedstock.The most richness community(15T-RFs)was detected during this operational period(P4M).

Increase of the OLR also caused the disappearance of the T-RF of85bp and the appearance of a T-RF of 422bp.In addition,T-RFs of83and162bp were undetectable when increasing the OLR(from P5L onwards)whereas a certain amount of T-RF with the size of180bp was present.

The relatively abundant T-RFs of157and540bp occurred throughout the run.The T-RF of65bp was also present in most of the samples,showing at its highest abundance up to36.9%,but it was undetected in several case samples.

The similarity of the bacterial T-RFLP pro?les within the data set was expressed as a Sorensen–Dice pairwise similarity coef?cient.These values were then used to generate distances,and a dendrogram was constructed from these values to depict the similarities between the different pro?les(Fig.2). The dendrogram clearly shows a distinct grouping between the samples before P5L,with a continually increasing proportion of grass in feedstock while

maintaining a constant OLR,and those thereafter.T-RFLP pro?les before P5L were clustered in a group in the dendrogram.T-RFLP pro?les during the increase of the OLR while maintaining the constant feeding ratio were clustered in another group,as shown in Fig.2.Another important ?nding that can be concluded from the dendrogram is that the T-RFLP pro?les during increasing the proportion of grass in the feedstock were more closely related to the previous sampling event but more distinct from the period when the reactor was fed with cow manure alone (P1).In addition,a great difference in T-RFLP pro?les was observed after the proportion of grass was increased to 40%.This suggests that the bacterial community structure was dynamic and in a state of constant change along with the increase in the proportion of grass in the feedstock.Moreover,increasing the grass up to 40%led to a signi?cant change in the bacterial community structure in the reactor.In contrast,there was less difference in the T-RFLP pro?les during the increase in the OLR than during the change in the feed ratio,as shown in Fig.2.

The bacterial DGGE band patterns revealed distinct bands during the runs as shown in Fig.3.The largest number of bands was recorded when 40%of the feedstock was grass and an OLR of 2kg VS m -3day -1was applied (P5L),represented the greatest level of bacterial diversity.Nine bands

were visualized when the reactor was fed with cow manure alone (P1).The DGGE bands also revealed three clearly different band patterns among the analyzed samples.The bacterial community during the reactor was fed with cow manure alone and with up to 20%grass silage in feedstock (P1to P3M)displayed a similar DGGE band pattern,whereas samples taken during feeding with 30–40%

grass

Fig.2Clustering of the bacterial 16S rRNA gene-based T-RFLP pro?les based on Sorensen–Dice similarity coef?cient.P1to P7refer to different operational periods,and E ,M and L refer to the different stages in each period.The dendrogram was constructed by using the Nearest Neighbor method on the basis of the similarity matrix of Sorensen–Dice

coef?cients

Fig.1Relative abundance of bacterial 16S rRNA gene fragments retrieved from the reactor samples

throughout the operational periods P1to P7based on T-RFLP analysis.The operational periods were characterized by a change in either the applied loading ratio of grass silage to manure or the organic loading rate.Each operational period was divided into an early (E )(day 9–15),middle (M )(day 22–29)and late (L )(day 49–50)stage.The length of T-RFs in base pairs (bp)was indicated

silage in feedstock (P4,P5E and P5M)formed another band pattern.The bacterial community after P5L showed a similar band pattern,which exhibited great dissimilarity with the previous band patterns.This was consistent with the results obtained from the T-RFLP pro?les.The bacterial community shift between P5M and P5L might imply that the bacterial structures at P5E and P5M were under transit state while the bacterial structure at P5L was under steady state to feeding condition with 40%grass silage.The DGGE band patterns further indicated during the run the emergence of new bands.For example,eight new bands were detected after P5L,and one of them (B3)appeared as a dominant band.Conversely,evidence of the reduction or elimination of some phylotypes during the run was also observed.Speci?cally,?ve bands were absent from the samples collected during

the increase of the OLR,one of which (B5)appeared as a dominant band in the samples from P1to 3.In addition,DGGE revealed that two phylotypes were abundant in the bacterial community from P1to P3,whereas one phylotype was clearly dominant in the bacterial community after P5L.However,from P4to P5M,it seemed the bacteria were evenly distributed in the community and no individual phylotype could be singled out.Change also occurred in the bacterial community structure in the reactor during the increase in the loading rate,although the changes in community composition were not as signi?cant as those observed in the previous samples taken during the changes in the feed ratio.The observations obtained from the DGGE band patterns were com-parable to those derived from the T-RFLP pro?les.Archaeal community structure

The archaeal 16S rRNA gene could be ampli?ed only from the samples during the increase of grass proportion in the feedstock (before P5L).The archa-eal T-RFLP ?ngerprints indicated a total of four T-RFs as shown in Fig.4,implying lower diversity of the archaeal community than the bacterial population in this reactor.A T-RF of 185bp clearly predomi-nated in the reactor.In addition,the T-RFLP ?ngerprints were undistinguishable until the middle operational period of 30%grass in feedstock (P4M),suggesting the archaeal community remained stable during these operational periods.A T-RF of 281

bp

Fig.3DGGE band patterns of bacterial 16S rRNA gene fragments obtained from the reactor samples throughout the operation.The sequences of the indicated bands (B1to B7)were identi?ed.Each lane represents the bacterial community structure from an individual sampling event.P1to P7refer to different operational periods,and E ,M and L refer to the different stages in each

period

Fig.4Relative abundance of archaeal 16S rRNA gene fragments retrieved from the reactor samples throughout the operational periods P1to P5based on T-RFLP analysis.The operational periods were characterized by a change in the applied loading ratio of grass silage to manure.Each operational period was divided into an early (E )(day 9–15),middle (M )(day 22–29)and late (L )(day 49–50)stage.The length of T-RFs in base pairs (bp)was indicated

with increasing in the relative abundance was detected when increasing the grass proportion from 30to 40%.

The DGGE analysis of archaeal 16S rRNA gene displayed a similar band pattern during increasing the feed ratio of grass in feedstock (data not shown),indicating there was no change in the archaeal community during these operational periods of the reactor.

Sequence analysis of the abundant bacterial groups

A total of seven distinct sequences were identi?https://www.sodocs.net/doc/721506855.html,parative analyses with nucleotide databases and phylogenetic reconstruction revealed that four sequences (B2,B3,B4and B5)were af?liated with the phylum Bacteroidetes while one (B7)belonged to the family Clostridiaceae lineages.In two sequences (B1and B6)phylogenetic identi?cation failed.The phylum Bacteroidetes apparently predominated in the population,indicating that their important role in the co-degradation of cow manure and grass silage within the reactor.The emergence of a Clostridiaceae -like organism during increasing the OLR typi?ed by the sequence illustrated in Fig.5,was noted.In particular,this organism was observed in high abundance at the beginning of increasing the OLR (Fig.3).In addition,the emergence and even the predominance of two Bacteroidetes -like organisms

(B2and 3)were observed after the late operational period at 40%grass in feedstock and the OLR of 2kg VS m -3day -1(Figs.3,5).In parallel,there was apparent reduction in the abundance and even the disappearance of Bacteroidetes -like organism B5.Two unclassi?ed bacterial organisms (B1and B6)more related to the Actinobacteria were detected in all the reactor samples throughout the operation (Figs.3,5).

Discussion

The 16S rRNA gene-based ?ngerprinting techniques have been frequently used to monitor the diversity,structure and dynamics of microbial population in environmental samples.T-RFLP pro?les (Mlade-novska et al.2006)and DGGE band patterns (Bodelier et al.2005;Connaughton et al.2006)were found to re?ect the change in the microbial popula-tion.In the present study,the microbial community dynamics in a CSTR during anaerobic co-digestion of cow manure and grass silage were investigated by 16S rRNA gene-based T-RFLP and DGGE analyses.All the samples were identically prepared and were analysed simultaneously,thus the potential bias should have affected the results equally.Furthermore,two molecular ?ngerprinting techniques were employed to investigate the change in the bacterial community structure during reactor operation,

which

Fig.5Phylogram showing the phylogenetic af?liation of the bacterial partial 16S rRNA gene sequence from re-ampli?ed excised DGGE bands.Bootstrap values from 100replicates are shown for each node .The scale bar represents an estimated 10%difference in nucleotide sequence.The tree is rooted using Escherichia coli as outgroup

eliminated the pitfall of a single technique and supported the interpretation of results derived from one technique.

The microbial community structures revealed by the T-RFLP pro?les were in good agreement with those resolved by DGGE band patterns.However,the number of band detected by the DGGE pattern is relatively low compared to that of T-RFs revealed by the T-RFLP pro?le.A similar observation was made by Moesender et al.(1999),where the T-RFLP approach was more sensitive than DGGE,as indi-cated by the higher number of OTU detected by T-RFLP when investigating marine bacterial com-munities by both methods.Better resolution of complex microbial communities in lake sediment was indicated by T-RFLP than DGGE analysis when the Shannon–Wiener diversity indices obtained by using these two?ngerprinting techniques were com-pared(Koizumi et al.2003;Schwarz et al.2007).

The present16S rRNA gene-based microbial community study revealed that both the bacterial and the archaeal community structure remained stable during operational phases P1to P3M when the reactor was fed with cow manure alone and with up to20%grass silage in feedstock.A bacterial phylotype(B5)within the phylum Bacteriodetes was clearly dominant,and mostly re?ected community composition in the inoculum and cow manure.The phylotype,B5,had94%sequence identity with an uncultured clone,AHU24,which was obtained from a CSTR fed with a synthetic wastewater containing acetate as the sole carbon source(Shigematsu et al. 2003).The Bacteroidetes form a phylogenetically highly diverse group and are known as hydrolytic fermentative degraders of polymers in mainly anaer-obic habitats.Members of the class Bacteroidetes are also abundant in the intestinal tracts and feces of warm-blooded animals,and they are facultative anaerobes.A recent study by Hernon et al.(2006) showed that the most dominant fermentative bacteria belonged to the Bacteroidetes phylum,representing 31%of the cloned library,in a mesophilic anaerobic reactor degrading carbohydrate-rich waste.The mem-bers of the phylum Bacteroidetes capable of fer-menting a range of carbohydrates were further speci?ed by the authors.Bacteria belonging to the phylum Bacteroidetes were also found to be predom-inant in the bacterial community that was able to degrade long-chain fatty acids in a CSTR,suggesting that they play important roles in long-chain fatty acid degradation(Shigematsu et al.2006).The inoculum in the present study was collected from a mesophilic farm digester treating dairy manure and industrial confectionery(sweets and chocolates)by-products.A microbial community capable of ef?cient anaerobic digestion of carbohydrate-rich or fat-rich substrates was therefore present in the inoculum.Feeding with cow manure alone and with a low ratio of grass(10–20%)in the feedstock might have stimulated the growth of microbes able to convert the easily degradable substrates to methane,mainly the VFA present in the manure.The bacteria responsible for cellulose hydrolysis might not have been abundant.

The bacterial population clearly changed when the ratio of grass in the feedstock was increased to40%. Contrary to the bacterial community structure,there was little change in the archaeal population with only a new phylotype appeared at the operational phases P4L,P5E and P5M.The more diverse bacterial population was observed when the reactor was fed with30%grass in the feedstock(P4E to P5E).The study of the reactor performance by Lehtoma¨ki et al. (2007)showed that the highest speci?c methane yield was obtained during this operational period.This result suggests that the versatile substrate resource produced oscillations in the structure of the bacterial community and allowed the coexistence of more species(Saikaly and Oerther2004).However,a large change in bacteria population occurred when the grass ratio was further increased to40%(P5). Compared to the P4,during P5the bacterial popu-lation became less diverse while some individual bacteria became more abundant.Increasing the proportion of grass further to40%decreased the speci?c methane yield by6%as shown by Lehtoma¨ki et al.(2007).Sequence analyses of excised DGGE bands indicated the emergence of two new phylo-types(B2and B3),which belonged to the phylum Bacteroidetes,whereas the dominant phylotype(B5), af?liated also to Bacteroidetes,during P1to P3 declined.The phylotype(B3)predominant in the bacterial community after P5L showed95%sequence identity with the uncultured bacterium clone p-2534-18B5found in the intestinal tracts of Danish pigs (Leser et al.2002).The clone was af?liated with the Rikenella microfusus subgroup(RDP req.no.

2.15.1.2.1).Another abundant phylotype,B2,had 94%sequence identity with the uncultured clone

HsW01-040from gut wall of termites(Nakajima et al.2006).Termites harbor a dense microbial community in their guts,and gut microbes are responsible for the ef?cient decomposition of plant litter.The closest relatives of these phylotypes to the uncultured clones from plant digestive gut microbial communities suggest that these bacteria might play an important role in grass degradation.

A further change in the bacterial community structure was recorded when a number of loading rate increases were applied to the reactor during the ?nal two operational phases P6and P7,although the differences were not as signi?cant as those observed when the grass ratio in the feedstock was increased. The archaeal population could not be detected during the operational phases P5L,P6and P7,which indicated low abundance of the archaeal population in the microbial community.Increasing the loading rate from2to4kg VS m-3day-1caused a reduction in HRT from20to16days.Reduction in HRT may have resulted in enrichment of the microbial species outcompeting for the essential resources in the reactor,whereas species with less competitive advan-tage are removed from the bioreactor because of washout.The relatively low bacteria diversity and high abundance of individual bacteria were observed, with only seven T-RFs being detected at the conclu-sion of P7L.This has also been observed by Dearman et al.(2006),who showed that bacterial diversity during the treatment of organic wastes for hydrogen production increased as HRT was extended.The analyses of the performance of the reactor indicated that the decrease in HRT led to the inef?cient hydrolysis of solid material,mainly cellulose,the decrease of the speci?c methane yield by26%,and the increase of ammonia concentration in the dige-states(Lehtoma¨ki et al.2007).Ammonia was considered inhibitory for aceticlastic methanogens and hydrogenotrophic methanogens under mesophilic conditions(Angelidaki and Ahring1993).A phylo-type,B7,which fell into the family Clostridiaceae was detectable during increasing the OLR(P6and P7).Members of the Clostridiaceae found in a methanogenic land?ll leachate reactor(Burrell et al. 2004;O’Sullivan et al.2005)and in a thermophilic methanogenic bioreactor digesting wastepaper(Shi-ratori et al.2006)are capable of anaerobic cellulose hydrolysis.The Bacteroidetes-like bacteria B3was prevalent in the reactor with the exceptions of P6E,where a Clostridiaceae-like bacterium B7was pre-dominant,and of P7L,where a Bacteroidetes-like bacterium B2was also abundant in the reactor.The evidence indicated that the essential resource and environmental conditions in reactor during these operational phases favoured the proliferation of these bacteria.We assume that the phylotype B2,B3and B7might be important for the degradation of grass silage in the reactor;hence increasing the grass proportion in the feedstock may lead to the enrich-ment of these bacteria.

Signi?cant amounts of the phylogenetic group B1 and B6survived throughout the whole operation,and thus were adaptable to a wide range of conditions. This suggests that the natural population found in the inoculum is at least adequate for the degradation of some components in the feedstock.Unfortunately, conclusions as to the possible functions of these two phylotypes could not be drawn since they failed to fall into any of the known phylogenetic groups, although they were closely related to the phylum Actinobacteria.The Actinobacteria are a group of Gram-positive bacteria,which play an important role in the decomposition of organic materials,such as cellulose and chitin.A few members,such as Actinomyces israelii,can grow under anaerobic conditions(Stackebrandt et al.1997). Conclusions

The microbial community structure remained stable when the reactor was fed with cow manure alone and with up to20%of grass silage in feedstock at an OLR of2kg VS m-3day-1,although the number of the archaeal phylotypes was much smaller than that of the bacterial phylotypes in the reactor.Changes in the bacterial community towards a more diverse bacterial population were observed when the loading ratio of grass was increased to30%.Large changes in bacterial community structure with less observed phylotypes occurred when the grass ratio was further increased to40%.Contrary to the bacterial popula-tion,there was little change in the archaeal popula-tion.The structure of the bacterial community showed fewer differences during the increase in OLR from2to4kg VS m-3day-1than during the changes in the feed ratio.Archaea was undetectable during the increase in OLR,which indicated low

abundance of Archaea in the microbial community. The present community-based studies together with the previous analyses of reactors performance sug-gested a microbial community with most diverse in bacterial population to be related to the highest speci?c methane.Phylotypes which belonged to the phylum Bacteroidetes predominated in the bacterial commu-nity.A Clostridiaceae-like bacterium appeared after the OLR was increased.Two unclassi?ed bacteria with high abundance survived throughout the operation and thus showed adaptability to a wide range of substrate conditions.

Acknowledgments We are grateful to M.Sc.Suvi Huttunen for maintaining the reactor.We thank Leena Siitonen,Eila Korhonen and Elina Virtanen for their dedicated technical assistance.Dr.Anssi Lensu is acknowledged for his statistical expertise.We also thank Dr.Anna Kaksonen for kindly arranging the DGGE experiments.This work was supported by the Finnish Maj and Tor Nessling Foundation. References

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部编版语文一年级上册《10大还是小》教案

10. 大还是小 教学过程 第一课时 【课时目标】 1.会认“时、候”等11个字，会写“自、己、衣”3个字，认识双立人、竖心旁2个偏旁。 2.正确、流利地朗读课文。 【教具准备】 课件、生字卡片 【教学过程】 一、激趣导入，引入课题

1.（课件出示2）出示鸡蛋（一大一小）图片。 同学们，这是什么？（鸡蛋），你发现了什么 呢？（这两个鸡蛋一大一小） 2.师板书“大”和“小”。你认为自己是大还 是小呢？说说原因。 3.有一位小朋友，他自己很矛盾，有时候觉得 自己很大，有时候又觉得自己很小，到底怎么回事 呢？ 今天，我们一起学习《大还是小》一课，一起 去了解、感受这位小朋友的想法。 （板书课题：大还是小）齐读课题。 二、初读课文，检查预习 下面就让我们一起先来看看小作者认为自己是大 的还是小的。 1.自由读课文，注意读准字音，读通句子，做到“三 不”：不错字，不添字，不漏字。 2.要想读好课文，就必须先认识这些生字朋友。 （课件出示3） shí hou jué de zì jǐ hěn kuài chuān yī fu 时候觉得自己很快穿衣服 你认识它们吗？自己试着读一下。 指正：“自”是平舌音，“时、穿”是翘舌音。 “时候”中的“候”，“衣服”中的“服”在这里都 读轻声。 （1）谁能来当小老师带领大家读一读？其他同学 （2）这些字去掉拼音你还认识吗？ （课件出示4） 时候觉得自己很快穿衣 服 我们来开火车读一读。

（3）识记生字： 本课生字以合体字为主，可以运用多种方法帮助学生识记字形、理解字义。 （课件出示5）出示会意字图片：学习会意字“穿”。教师出示老鼠挖掘洞穴的图片，告诉学生：上面是一个“穴”，表示的是野兽居住的洞穴；下面是“牙”，表示野兽用自己的牙齿来挖掘洞穴，是凿通、凿穿的意思。 用熟字组成新词：时间、感觉、得到、很多、大自然、穿过。 小结：识字的时候，我们不仅可以用加一加、换一换的方法，还可以用猜字谜的方法，但要注意编的字谜要合理。 指名认读，齐读。 3.认识了生字朋友，读课文就更容易了。下面我请几位同学接读课文，其他同学边听边想，本文介绍了“我” 什么时候感觉自己很大，什么时候感觉自己很小？ 预设：“我”自己穿衣服和系鞋带的时候感觉自己很大。 预设：4.教师评价学生的朗读。 三、观察生字，指导书写 1.出示生字:自、己、衣（课件出示6） 观察字形，记住它们在田字格中位置。 2.指导写字规律。 自：横平竖直，中间几横之间的间距要均匀。 己：整个字上窄下宽，竖弯钩要圆转。 衣：整个字的重心落在田字格的正中，撇捺舒展，呈三角形；注意笔顺，最后一笔是长捺。

部编版一年级语文上册10大还是小教学反思1

精品教学资料，欢迎老师您参考使用！ 《大还是小》教学反思 孩子们都希望自己快快长大，成为一个独立的人。与此同时，他们也离不开父母的呵护。《大还是小》这篇课文通过3个“有时候”和“更多的时候”把文章紧密地串联起来，形成一个有机整体。课文多处运用对比的方式来展现儿童的世界，儿童的内心是矛盾的，又是充满趣味的。第二自然段的“大”，第四自然段的“小”，就是这种矛盾的具体体现。教学重点为认识“时”“候”等11个生字和双人旁、竖心旁两个偏旁；会写“自”“己”等3个生字；正确、流利地朗读课文，结合插图，体会“我”自相矛盾的内心世界。结合生活体验，说说什么时候觉得自己很大，什么时候觉得自己很小。上完课后，教学效果感觉良好，也有许多的感受、体会。回顾整堂课的教学，总结如下： 一、教学效果 本节课围绕着教学目标，我取得了以下效果： 1.为了实现教学目标，我的教学思路主要还是提示学生读准字音。为了激发学生的识字兴趣用图片的方式来学习会意字“穿”。出示老鼠挖掘洞穴的图片，告诉学生，上面是一个穴表示的是野兽居住的洞穴，下面是“牙”表示野兽用自己的牙齿来挖掘洞穴，是凿通、凿穿的意思。因为学生认知事物的方式不同，鼓励学生用自己喜欢的方式进行识字。组内交流汇报识字方法，效率高。在写字教学中，采取对比学习方式“自”和“白”；“己”“衣”引导观察笔画互相衔接的位置。 2.朗读指导。采取男女生对读、同桌之间对读的形式，引导孩子读出内心成长的感受，体会“大”和“小”的情感变化，当自己觉得很大时，读出自豪之感；当自己觉得自己很小时，读出一种儿童依赖大人的感觉。在熟读的基础上，通过指导读好几个“有时候”和“更多的时候”，读出文章的结构的特点。第一个“有时候”要读出内心的自豪感；第二个“有时候”朗读时语调要有变化，相较于第一个“有时候”在语调上稍微短一点，读出“我觉得自己很小”中的“很小”。学生在朗读中体会到“我”内心世界的自相矛盾。 3.理解运用。从题目入手，学生说说对大和小的理解，能否用到一个人身上，激发学生的学习兴趣。 4.说一说。结合生活实例，将学生带入文本，加深对课文内容的理解。借助句式“有时候，我觉得自己（）。（）的时候，（）的时候，我觉得自己（）”引导学生说感受，把语言学习和内容理解有机结合。 二、成功之处 《语文课程标准》中提出语文课程是一门学习语言文字运用的综合性、实践性课程。读写不分家，学生初步了解课文的基础上，结合生活实例，将学生带入文本，借助句式“有时候，我觉得自己（）。（）的时候，（）的时候，我觉得自己（）”练

拼音13、an en in un ün

13、an en in un ün 第一课时 教学目的： 1．学会前鼻韵母an 、en 2个复韵母，读准字音，认清形，能在四线三格中正确书写。 2．学习声母与an 、en 组成的音节，准确拼读音节，读准三拼音节，复习ü上两点省写规则。 3．学习整体认读音节yuan 。 教学重点： 1．学会韵母an 、en 2个复韵母，读准音，认清形，能正确书写。 2．学会声母与an 、en 组成的音节和整体认读音节。 教学难点： 学会介母是ü的三拼音节，读准音节juan 、quan 、xuan 。 教学过程： 一、谈话导入 我们到目前为止学习了哪些复韵母？能按顺序说说吗？ （ai 、ei 、ui 、ao 、ou 、iu 、ie 、üe、er ）。今天我们一起来学习第13课，再认识几个韵母朋友，请同学们打开书看看。这课书的内容比较多，有信心学好吗？下面我们先来学习前2个韵母及音节。 板书：13 an en 二、看图学习韵母an 、en 1．学习韵母 an （1）出示an 图，问：图上画的是什么？ （2）自己试着发an （安） （3）教师指导发音：把嘴张大，摆好a 的口形，让气 流从前鼻腔里出来，也就是n 的尾音。 （4）学生练习读，体会前鼻韵母的发音方法。 （5）同桌同学互读，纠正发音。 （6）指名读，开火车读。 2．学习韵母 en （1）出示en 图，问：你们看这个人在干什么？ （2）借助“摁”的第四声交成第一声学生练习发en 的音。 （3）en 是由哪两个字母组成的？（e 和n ）发音时，先发e ， 嘴半闭，舌尖抬起抵住上牙床快速读，鼻子出气，一口气读出en 的音。 三、书写韵母an 和en

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10 大还是小 教材解读： 《大还是小》是一篇富有儿童情趣的文章，内容浅显易懂，同时富有教育意义。孩子们都希望自己快快长大，成为一个独立的人。与此同时，他们也离不开父母的呵护。课文通过 3 个“有时候”和“更多的时候”把文章紧密地串联起来，形成一个有机整体。课文多处运用对比的方式来展现儿童的内心世界，儿童的内心世界是矛盾的，又是充满趣味的。第二自然段的“大”，第四自然段的“小”，就是这种矛盾的具体体现。课文配有一幅插图，“大”和“小”的行为都在其上，可以借助课文插图来展开教学。 教学目标： 1.认识“时、候”等 11 个生字和双立人、点横头、竖心旁 3 个偏旁；会写“自、己” 等3个字。 2.正确、流利地朗读课文。结合插图，体会“我”自相矛盾的内心世界。 3.结合生活体验，说说什么时候觉得自己很大，什么时候觉得自己很小。 教学重点、难点： 教学重点：正确、流利地朗读课文。 教学难点：体会“我”自相矛盾的内心世界；会写“己、衣”等 字。 第一课时 一、课时目标： 1.认识“时、候”等 11 个生字，能用不同的识字方法进行识记。学习双立人、点横头、竖心旁 3 个偏旁；会写“自、己”两个生字。 2.正确、流利地朗读课文，初读课文学习质疑并能通过自读自悟解读疑问。 二、教学过程 （一）激趣导入，引出课题，启发质疑 1．出示字卡“大”。

大声读这个字。说说和它意思相反的字是什么吗？ 2．出示字卡“小”。 小声读这个字。(生读：小) 提醒：上课时，回答问题声音不能太小，否则别人就听不到了。老师要看看这节课谁的表现最棒。 3.同时出示字卡“大小”，一起来读一读。 4．质疑：你认为自己是大还是小呢？能说说为什么吗？(指名回答) 5.过渡：有一个小朋友也遇到了这个问题，他是怎么回答的呢？我们这节课就来学习《大还是小》。(板书课文题目) 6．读了课文题目，你有什么疑问？(师生梳理出主要问题) （二）自主探究学习 1.教师出示自读要求 (1)自由朗读课文，遇到不认识的字，借助拼音多读几遍。把词语读正确，句子读通顺。 (2)拼读课前圈画的生字，要读准字音，想办法记住这些生字。 2.根据自探提示先自主学习，然后在小组长的组织下在小组内交流。 （三）初读课文，学习生字 1.检查自主学习情况。 (1)我会读 课件出示词语： 时候觉得穿衣服自己很小快点儿 ①指名开火车朗读，师生正音。 ②齐读。 ③自主选择一个词语说一句话。 ④去掉拼音指名读，齐读。 (2)我会认 ①这些词中有些生字需要我们记住，瞧，它们已经从词中跳出来了，你还能认出它们吗？ 课件出示生字，指名读。

部编人教版一年级语文上册第10课《大还是小》优质教案

10.大还是小 同学们，你们愿意快快长大，还是永远做一个孩子呢？《大还是小》这篇课文的小作者有时候觉得自己很小，有时候觉得自己很大，怎么回事呢，我们一起走进课文看看吧！ 学习目标—要知道 1.能正确流利、有感情地朗读课文。 2.会正确认读“候、穿”等12个生字，学会写“自”等4个字，认识“ㄔ”等个部首。 3.感受小作者要长大心情，能自己的事情自己做。 字词详解—要掌握 2.会认的字

3.多音字 ào （睡觉）de （写得） ? （感觉）d ěi (得学会) 运用：我宁愿睡觉也不看这部电影。我觉得你应该走快点。 他的作文写得很华丽。他们得学会尊重并欣赏这一点。 4.近义词 觉得——感觉 陪——伴 照顾——照看 盼着——希望 5.反义词 大——小 多——少 快——慢 运用：①妈妈买了两个西瓜，一个大一个小。 ②我们班的学生多，二班的学生少。 ③散步时奶奶的脚步总是很慢，而我总是走得飞快。 6.词语听写 自己 妈妈 妹妹 7.一词多义 8.词语拓展 反义词：前——后 左——右 上——下 外——内

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汉语拼音 an en in

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幼儿园大班拼音课《an en in》

活动名称：复韵母an en in 教学目标：1.情感目标：培养幼儿学习拼音的兴趣。 2.态度目标：能够认真的学习拼音。 3.知识目标：学习an en in的认读、四声调及书写格式。 4.能力目标：能掌握声母与an en in拼读。 教学重点：学习an en in的认读、四声调及书写格式。 教学难点：能掌握声母与an en in拼读。 教学准备：课件、拼音卡片。 教学过程： 一、开始部分 1.复习：（课件出示蓝猫形象）小朋友们，你们知道这是谁吗？师：对了，这是小朋友 们最喜欢的蓝猫，蓝猫对小朋友们说：“你们还记得以前学过的拼音吗？它们是谁呢？”出示卡片。 2.小朋友们认得都很棒。今天蓝猫又给你们带来了几位新朋友。请看画面 二、基本部分 （一）认读复韵母an。 （1）出示第一幅图（天安门），“a和n合在一起我们读an，a在前，n在后，我们读，anan an。an在发音时：先发a的音，然后舌尖逐步抬起，顶住上牙床发n的音。 模仿读，个别读，开火车读，齐读。 （2）我们编成了一句识记儿歌便于小朋友记忆“天安门天安门anan an”分组读或请个别幼儿重复读。 （3）an的四声练习。 （4）an的书写格式。（占中格） （二）认读复韵母en 课件出示蓝猫图像：“小朋友们真厉害！认识了一个新朋友，蓝猫送给大家热烈的掌声。现在我们来看看第二个新朋友，出示第二幅图（有关点头的画面）小朋友们看，它们在做什么，仿佛在说什么？ （2）它们仿佛在说：“恩，小朋友们真行！”）那么我们今天所学的新朋友就是复韵母“en” “e和n做朋友时，我们读en，e在前，n在后，en在发音时：先发e的音，然后舌面抬高，气流从鼻腔泄出，发n的音。 我们也编成了一句话识记儿歌“点点头enenen” （3）en的四声练习。 （4）en的书写格式。（占中格） （三）认读复韵母in （1）课件出示蓝猫图像：“小朋友们真厉害！又认识了一个新朋友，蓝猫送给大家热烈的掌声。现在我们来认识最后一个新朋友，出示印章的画面。这个印章里in的发音就是我们今天要学习的。 （2）我们读印章in时，i在前，n在后，我们读in”in在发音时：先发i的音，气流从鼻腔泄出，然后发n的音。 in的前面加一个y就是印章的印的读音完整的样子了，变成了“yin”，它是一个整体认读音节，什么叫整体认读音节呢？添加一个声母后读音仍然和原来的韵母一样的音节（也就是指不用拼读可以直接认读的音节，叫整体认读音节。） 这里我们也有一句识记儿歌是“印章印章in in in” （3）in的四声练习。 （4）in的书写格式。（占中上格） （四）an en in与声母的拼读。

新部编人教版小学一年级语文上册第10课《大还是小》教案及反思

新人教版部编小学一年级语文上册教案及反思 10、大还是小 教学目标： 知识与技能 1.会认“时”“候”“觉”等11个生字，会写“自”“己”“衣”3个生字。掌握3种偏旁“彳”“亠”“忄”。 2.正确、流利地朗读课文，读准字音。 情感态度与价值观 引导学生要学会正视自己，知道什么时候自己很大，什么时候自己又很小。 教学重难点： 1.掌握本课所学生字，能够按笔顺准确、规范地书写生字。 2.正确、流利地朗读课文，读准字音。 教学课时：2课时 第一课时 教学过程： 一、谈话导入 １.师：同学们，每天早上爸爸妈妈送你们上学的时候，当你们看到高年级的哥哥姐姐们自己来上学，有没有很羡慕呢？（生答：有） 2.师：那个时候，你心里是怎么想的呢？（要是我也像哥哥姐姐一样大多好啊！） 3.师：为什么呢？（因为我再大一些，爸爸妈妈就不用每天辛苦送我上学了。）

4.师：有一个小朋友啊，他也和你们一样，有时候能自己系鞋带、穿衣服时，他觉得自己很大；但是有时候呢，够不到按钮、害怕打雷时，他又觉得自己很小。这节课我们一起去认识这位小朋友吧！ 二、看图读文，整体感知 1.让学生自由朗读课文，画出课文生字词，多读几遍。 2.读一读，标出自然段序号。 3.看图，说说图上画了什么。你能根据课文内容说一说吗？（引导学生结合插图说一说。） 4.不明白的地方用横线画下来，并向老师请教。 三、动动脑筋，学习生字 1.看拼音读词语。（课件出示重点词语） 2.课件出示课文生字（去拼音），指名读，开火车读。 3.认读这些生字，并给这些生字找朋友。（口头扩词练习） 4.巧识字形。 （1）师：你们有什么好办法能很快记住这些字的字形吗？ （2）四人小组讨论识记方法。（鼓励学生结合字形和字义巧识巧记。）（3）同桌之间互相说一说你是如何记住的，汇报交流识记方法。 （比一比：己—已自—目） 第二课时 教学过程： 一、创设情境，激发兴趣 1.出示本课生字词卡片，检查学生认读情况。 2.出示课件“图图上小学了”。

部编一上语文10 大还是小【教案】

部编版一年级上册语文10大还是小 1.认识“时、候”等11个生字和双人旁、竖心旁2个偏旁；会写“自、己”等3个生字。 2.正确、流利地朗读课文。结合插图，体会“我”自相矛盾的内心世界。 3.结合生活体验，说说什么时候觉得自己很大，什么时候觉得自己很小。 4.在仿说中迁移运用文中句式。 重点 1.正确、流利地朗读课文。 2.体会“我”自相矛盾的内心世界。 难点 在仿说中迁移运用文中句式，让学生与主人公的心理产生共鸣。 1.字词教学。 生活识字：在教学“穿”时联系学生生活，说说自己还会穿什么，如“穿鞋子、穿裤子”等，在拓展中认读生字，识记生字。 对比识字：“双人旁”是本课新学的偏旁。教学时可引导学生与“单人旁”进行辨析，将“得、很”组合教学，并给“很”组词，通过对“很大、很小、很多、很少”等词语的对读，体会“很”是表示程度的加深，也为后面的朗读指导打好基础。 字义识字：学习“竖心旁”时结合“心”字说说演变过程，从而理解带有“竖心旁”的字大多跟心情有关。 书写生字：“自、己、衣”在写字板块需重点指导。指导“自”时可采用加一加的办法记住字形 “目+ ”；“己”的书写要点是笔顺及书写最后一笔的位置；“衣”要让学生观察笔画的细节，以及几个笔画相互衔接的位置。 2.朗读教学。 本课的语言兼有散文和诗歌的特点，语句有长有短，长句由结构重复的短句组成，如“______的时候，_____的时候，我觉得自己_____”。在指导朗读时，可以采用范读法，让学生能明显判断停顿的位置，进行标注后再反复练习。如：“我自己/穿衣服的时候，我自己/系鞋带的时候，我觉得/自

己很大。”还可采用对比朗读法，一年级的孩子读书时最容易出现拖音拖气的现象，教师通过示范形成对比，让学生正确朗读。评价法也是指导学生朗读的有效途径。如“我自己穿衣服的时候，我自己系鞋带的时候……”教师评价：“你把‘我自己’读得那么响亮，教师听出了你的自豪。” 3.迁移运用。 课文第1、2自然段与第3、4自然段用了以下句式：“有时候，我觉得自己很_____。我自己的时候，我自己______的时候，我觉得自己很_____。”教学时可引导学生联系自身经历进行仿说。不仅做到句式上的迁移运用，还让学生与课文中的主人公产生共鸣。 教学准备 1.借助拼音读课文，认读本课生字。 2.多媒体课件。 教学课时 2课时 第1课时 1.认识“候、得”等生字，注意读准轻声。 2.认识“双人旁”，会写“衣”字。 3.正确、流利地朗读课文。 一、创设情境，引出课题，启发质疑。 1.教师讲故事：在森林王国里住了小白兔一家，一天小白兔欢欢跑到妈妈跟前对妈妈说：“妈妈，妈 妈，你看我长高啦！我是个大孩子了！”同学们，你们同意欢欢的说法吗？说说你的看法。（生自由讨论） 2.质疑：你认为自己是大还是小呢？能说说为什么吗？（生自由回答） 3.有一个小朋友也遇到了这个问题，他是怎么回答的呢？我们这节课就来学习《大还是小》。 4.给课题加上问号，指导学生读出疑问的语气。

部编新人教版一年级语文上册第10课大还是小课堂教学实录

部编新人教版一年级语文上册第10课《大还是小》课堂教学实录 本帖最后由 ljalang 于 XX-10-29 11:31 编辑 10 大还是小 名师教学设计片段 ◆激发识字兴趣，运用多种方法识字(教学难点) 师：看到小朋友们能正确地读出这篇课文，老师心里可高兴了。不仅我高兴，连藏在这篇课文里的十一个生字娃娃也为你们高兴呢。瞧它们出来了！(课件出示：时、候、觉、得、穿、衣、服、自、己、很、快) 师：没有拼音，你们还能叫出它们的名字吗？别急，试着读一读吧。 (学生认读，教师巡视) 师：刚才老师看到有几位小朋友皱起了眉头，看来是碰到了不会认的字，不过没有关系，这很正常。只要我们动动脑筋，想想办法，就一定能认识它们。请你想一想：如果碰到了不会认的字，你会怎么办呢？ 生1：我会看书上的拼音。

生2：我会去查字典。 师：对，字典的确是一位好老师。 生3：我会举手问老师，还可以问旁边的同学。师：你们真聪明，一下子想出了这么多好办法，有了这么多好办法，老师相信你们一定都能跟这些生字娃娃交朋友。好，赶快行动，想办法和这些生字娃娃交朋友吧。 (学生自由认读生字，有的大声认读，有的在看书上的拼音，还有的问起了旁边的同学，老师一会儿看看这个小朋友，一会儿问问那个小朋友。) 师：你们学得这么认真，一定都和生字娃娃交了朋友。老师想来检查一下，请把书合上。看，生字娃娃都跑到老师这里来了。(出示生字卡片)能叫出它们名字的请举手。 师：这些生字娃娃的名字会认了，那这些生字娃娃的模样我们又该怎么记住呢？ (课件出示“很、得”) 师：你有什么发现吗？ 生：它们很像。 师：是呀，这两个生字娃娃长得这么像，怎么把它们区分开呢？

最新人教部编版一年级语文上册《大还是小》说课稿

《大还是小》说课稿 今天我说课的内容是小学语文一年级上册第七单元第10课《大还是小》。下面我将从教材、学情、教学目标、教法学法、教学过程、教学板书六个方面作简单的说明。 一、说教材 《大还是小》是一篇富有儿童情趣的文章，内容浅显易懂，同时富有教育意义。课文用简洁平实的语言写了“我”有时候觉得自己很大，有时候又觉得自己很小，并举了一些具体的事例，让孩子们意识到自己的事情应该自己做。 二、说学情 一年级的小朋友对“长大”非常向往，但是有时候又有一些力不从心的事情，让他们意识到自己还没有长大。这篇课文能让他们对“长大”有更确切的认识，能够让他们意识到自己的事情应该自己做。 三、说教学目标 依据教材和学情，我设定以下教学目标： 1.认识11个生字，会写3个生字。 2.能正确、流利、有感情地朗读课文。 3.理解课文内容，体验长大的快乐。 本课的教学重点：学会本课生字词，能正确、流利、有感情地朗读课文。 教学难点：理解课文内容，体验长大的快乐。 四、说教法学法 语文课程标准提出：“努力建设开放而有活力的语文课程。”所以本节课主要采用媒体演示、自主读书，自主识字、合作学习、合作解疑的方法。学生在教师的引导下动脑、动手、动口。通过自己的劳动获取知识，变被动学习为主动学习。体现“以教师为主导，学生为主体，训练为主线”的原则。 五、说教学过程 （一）谈话导入，激发兴趣 “兴趣是最好的老师。”“兴趣是求知获艺的先导。”因此，上课伊始，我先出示“大”和“小”两个字，让孩子们说说觉得自己是大还是小，为什么？顺势引出课题，激发学生探究课文的兴趣。 （二）初读感知，学习生字

1、老师先范读课文。 2、孩子们借助拼音自由读课文，圈出不认识的字，向小组内其他的同学请教后，多读几遍。 3、学习生字词。首先，课件出示词语，再出示生字进行认读，接着让学生先小组合作交流识记方法，再全班交流，认识双人旁，竖心旁。并利用游戏的方式激发学生的识字兴趣。然后，把生字词放到句中读，再放到文中分段朗读课文。这样，一层层的推进，集中识字与随文识字相结合，字不离词，词不离句，只有将汉字及时纳入词中、句中，并在语言环境中会认、会读，才算真正“会认”，这样的识字也才是有意义。 4、指导写字。出示生字，让学生先自己观察，说说书写时要注意什么，强调笔画顺序。然后老师范写，学生在练习。 新课标提出：要让学生初步感受汉字的形体美。一年级学生是训练写字的关键时期，上课时有选择地渗透一些书法知识可为学生以后的书写奠定良好的基础。 【设计意图：在这一环节中，我从学生实际出发，以“扎实、朴实”为目标，利用课件，认读字词，努力在字词上抓落实，为深入学习课文打下坚实的基础。】（三）合作探究，细读体悟 《语文课程标准》明确指出：阅读是学生的个性化行为，不应以教师的分析代替学生的阅读实践，要十分重视培养学生的自学能力。因此在教学中，要特别注重学法的指导和渗透。 1、自由朗读课文，勾画语句，思考：什么时候觉得自己“很大”，用“____”画出来；“我”什么时候觉得自己“很小”，用“﹏”画出来。在小组内交流，想一想为什么。在汇报交流中，课件相机出示句子引导理解，并进行朗读指导。 2、仿照课文的句式说一说：你什么时候觉得自己很大？什么时候觉得自己很小？ 【设计意图：授人以鱼，不如授人以渔。在这一环节，让学生自己探究并找到答案。，以“画一画”“说一说”“读一读”的方式培养学生的动口、动手、动脑的学习习惯，充分激发了学生的主动意识和进取精神，加深了学生对文本的理解。同时，合作学习的方式，又培养了学生的合作意识和能力。】 （四）联系生活，拓展升华 《语文课程标准》指出：教学要结合课内外资源，多途径的提高学生的语文素养。 1、说一说，你是盼望长大，还是希望一直这样小小的？为什么。在全班交流中对学生进行情感教育，体验长大的快乐。

最新部编人教版一年级语文上册第10课《大还是小》教学设计

10.大还是小

课文10 大还是小 【教学目标】 1. 认识“时、候”等 11 个生字和双立人、点横头、竖心旁 3 个偏旁；会写“自、己”等 3 个字。 2. 正确、流利地朗读课文。结合插图，体会“我”自相矛盾的内心世界。 3. 结合生活体验，说说什么时候觉得自己很大，什么时候觉得自己很小。 【教学重点】 正确、流利地朗读课文。 【教学难点】 体会“我”自相矛盾的内心世界；会写“己、衣”等字。 【课前准备】 1．制作多媒体课件，准备生字词卡片。(教师) 2．借助拼音自主朗读课文，预习课文，标出自然段，圈画生字，拼读生字，记忆生字。(学生) 【课时安排】 2课时 【教学过程】 第一课时 一、激趣导入，引出课题，启发质疑 1．出示字卡“大”。 师：同学们，请大声地读这个字。(生读：大) 师：上课时，回答问题的声音要大。你知道和它意思相反的字是什么吗？(生答：小) 2．出示字卡“小”。 师：请小声地读这个字。(生读：小)上课时，回答问题声音不能太小，否则别人就听不到了。老师要

看看这节课谁的表现最棒。(同时出示字卡“大小”)现在，我们一起来读一读。(生读：大小) 3．质疑：你认为自己是大还是小呢？能说说为什么吗？(指名回答) 师：有一个小朋友也遇到了这个问题，他是怎么回答的呢？我们这节课就来学习《大还是小》。(板书课文题目) 4．读了课文题目，你有什么疑问？ (师生梳理出主要问题) 5．自主探究学习。 教师出示自探提示一。 (1)自由朗读课文，遇到不认识的字，借助拼音多读几遍。把词语读正确，句子读通顺。 (2)拼读课前圈画的生字，要读准字音，想办法记住这些生字。 6．根据自探提示先自主学习，然后在小组长的组织下在小组内交流。 二、初读课文，学习生字 检查自主学习情况。 (1)我会读 时候觉得穿衣服 自己很小快点儿 (2)我会认 ①这些词中有些生字需要我们记住，瞧，它们已经从词中跳出来了，你还能认出它们吗？ 时候觉得自己很穿衣服快 ②识记生字： ③我来考考大家： “我在洞穴里发现了一颗牙。”(穿) 这是我们的识字办法之一——编谜语，猜谜语。接下来要看你们的本领了，说说你们的识字办法吧！(学生自由选择生字说说自己的识字方法。) ④小结：识字的时候，我们不仅可以用加一加、换一换的方法，还可以用猜字谜的方法，但要注意编的字谜要合理。 ⑤指名认读，齐读。 三、写字指导(自、己) 1．交流谈话。 师：你觉得在这十一个生字中哪个字最简单？(己)组一个词好吗？(自己)现在我们就来写好下面这两

最新《an en in un ün》教案

12. an en in un ün 教学目标： 1、学会前鼻韵母an en in un ün，读准音，认清形，能正确书写，学会整体认读音节yuan、yin、yun。 2、掌握关于本节课韵母的简单两拼、三拼音节，能自主拼读 教学重点： 1、对课本导引图片的探索与发现（开发同学们的思维探索能力） 2、掌握本节课韵母 新授课过程： 一、复习回忆 回忆学过的声母、单韵母、复韵母（整体背诵） 二、观察拼音，找到共同点 1.引入：今天，老师请来了五个拼音娃娃，它们是——an、en、in、un、ün，请你们仔细观察它们，你们有什么发现？ 2.学生发现都有n。 3.师：同学们观察的很仔细，这后面都有一个相同的字母n，但是这个小尾巴，在这儿不读n，那么这个音该怎么读呢，请小朋友看老师的手势（这是小朋友上面一排牙齿，这是上齿背、舌头这是舌尖）读的时候要把舌尖轻轻地顶上去，顶到上齿背（做两遍）请小朋友听老师的发音，舌尖顶住，请大家跟老师读一读。发这个音的时候气流要从鼻子里出来，对，轻轻地。谁来试试看。（提问并表扬，再一起来）再次强调（舌尖抵住上齿背，气流从鼻子里出来）设计意图：通过学生自己对拼音的观察，从而发现前鼻韵母的规律，这样能激发学生的学习兴趣，并把韵母记得更牢。 像这样有一个小尾巴n的韵母，我们叫它们前鼻韵母，来跟我说。 这节课我们一起来学这五个前鼻韵母，请大家看图，图上画着什么呢。 三、观察图片，学习韵母和整体认读音节 (一)教学an 1.出示图片（天安门）引导发音 师：图上画着什么呢(生：天安门城楼)对了，这个天安门的安，跟我们这节课要学习的第一个前鼻韵母的发音是一样的。大家仔细看，这个前鼻韵母是由a和n组成的。在读的时候，我们先要做好a的口型，先把嘴巴张大，再把舌尖顶住上齿背，请小朋友们听老师是怎么读的。An an an，跟我来学一学。（生：an an an）谁来试试？ 2.多种方式练读（多次练读）

部编版《an en in un ün》教案

12 an en in un ?n 教案设计 设计说明 《语文课程标准》指出：学生是学习和发展的主体，语文课程必须根据学生身心发展和语文学习的特点，关注学生的个体差异和不同的学习需求，爱护学生的好奇心，充分激发学生的主动意识和进取精神，倡导自主、合作、探究的学习方式。因此，围绕重点，教学设计遵循了趣味性、活动性和开放性三个原则。意在以活动和游戏为主，使儿童在愉快的教学环境中学习拼音，在多种多样的儿童喜闻乐见的活动中提高拼读能力，从而充分发挥汉语拼音帮助识字、学习普通话的作用。 课前准备 1.制作关于an、en、in、un、?n的多媒体课件。(教师) 2.制作关于前鼻韵母an、en、in、un、?n，整体认读音节yin、yun、yuan的音节卡片。(教师) 课时安排 2课时。 教学过程 第一课时 一、观察拼音，尝试发音 1.引入：今天，老师请来了五个拼音娃娃，它们是——an、en、in、un、?n，请你们仔细观察它们，你们有什么发现？ 2.学生发现都有n，尝试发音。 设计意图：通过学生自己对拼音的观察，从而发现前鼻韵母的规律，这样能激发学生的学习兴趣，并把韵母记得更牢。 二、观察图片，学习韵母和整体认读音节 (一)教学an和整体认读音节yuan。 1.出示图片引导发音。 师：请同学们跟随拼音娃娃一起去它们家里看看吧！看，这一家人都在干什么呢？(生：看电视呢)电视上演的是哪儿啊？(生：天安门)天安门的“安”就是旁边这个韵母的发音。谁来试试？发这个音的时候，先做好ɑ的口形，舌头再慢慢往上抬起，感觉音是从鼻子里面发出来的，请看我的口形……谁看清楚了？ 2.多种方式练读。 学生自由练读、指读、齐读、开火车读。 3.探究发音方法。 学生编个顺口溜来记这个韵母的发音，拍着小手一起说说。

前鼻音an en in的拼读

复韵母“an en in”的拼读 ān án ǎn àn bān bán bǎn bàn pān pán pǎn pàn mān mán mǎn màn fān fán fǎn fàn dān dán dǎn dàn tān tán tǎn tàn nān nán nǎn nàn lān lán lǎn làn gān gán gǎn gàn kān kán kǎn kàn hān hán hǎn hàn zhān zhán zhǎn zhàn

chān chán chǎn chàn shān shán shǎn shàn zān zán zǎn zàn cān cán cǎn càn sān sán sǎn sàn yān yán yǎn yàn wān wán wǎn wàn rān rán rǎn ràn ēn ?n ěn an bēn b?n běn ban pēn p?n pěn pan mēn m?n měn man fēn f?n fěn fan

dēn d?n děn dan nēn n?n něn nan gēn g?n gěn gan kēn k?n kěn kan zhēn zh?n zhěn zhan chēn ch?n chěn chan shēn sh?n shěn shan zēn z?n zěn zan cēn c?n cěn can sēn s?n sěn san wēn w?n wěn wan rēn r?n rěn ran

īn ín ǐn ìn bīn bín bǐn bìn pīn pín pǐn pìn mīn mín mǐn mìn nīn nín nǐn nìn līn lín lǐn lìn jīn jín jǐn jìn qīn qín qǐn qìn xīn xín xǐn xìn 整体认读音节： yīn yín yǐn yìn

an en in

an en in an en in 教学目标 1．学会前鼻韵母an、en、in和整体认读音节yin及其四声，读准音，认清形，正确书写。 2．正确认读由声母和an、en、in组成的音节，会读拼音词和拼音句子。教学准备 情境图，歌曲磁带《我爱北京天安门》，。 课时安排：2课时 第一课时 课前游戏 1、做你指我猜的游戏（ai ui iu ye yue ei ao） 2、背儿歌读韵母 一、看图导入 1、小朋友们可真厉害，为了奖励你们，我们一起来看电视。出示情景图 2、谁会开电视机？（请同学开电视） 每个小朋友都跃跃欲试，想来开电视。 3、说说电视里在放什么？ 4、电视里的电视机里在放什么？

基本上能够说完整。 5、听小朋友们在唱什么歌？播放《我爱北京天安门》。 6、学习语境歌：有一首儿歌写的就是这幅图的内容，我们一起来学习。 7、听读儿歌：遥控器，摁一摁，荧屏出现天安门，色彩鲜艳音乐美，小朋友越看越开心。（跟老师读） 8、导出韵母，板贴：an en in 二、指导发音 1．学习韵母an。 (1)教师板书an，过渡叙述：这是天安门中“安”的拼音，去掉声调符号就是我们今天要学习的第一个韵母an。 (2)认清形。(教师指着a。)这个单韵母认识吗?在单韵母a的后面加了个鼻音做尾巴，这样组成的韵母就叫前鼻韵母（跟老师读两遍）。(3)指导读准音。教师告诉学生读这个韵尾要用舌尖抵住上齿龈，不留缝隙，鼻子出气。教师做口形，让学生体会舌尖顶住上齿龈的感觉。教师领读，指名读、学生自由练读，齐读，“开火车”读。 (4)指导读好an的四声。出示ān、án、ǎn、àn，请同学自由读，同桌互读，指名读。 2．学习韵母en。 (1)请学生做一做摁遥控器的动作。 (2)板书en，指导读好音。把“摁”改成第一声会读吗?

an en in教案

《an en in》教学设计 教学要求 1．学会前鼻韵母an、en、in和整体认读音节yin、yuan及其四声，读准音，认清形，正确书写。 2．正确认读由声母和an、en、in组成的音节，会读拼音词。 教学准备 情境图，复韵母卡片、课件 教学过程 第一课时 课前复习： 学过哪些单韵母？( a o e i u ü ) 6个 学过哪些复韵母？( ai ei ui ao ou iu ie ue er ) 8+1个 今天再来学习三个复韵母跟一个整体认读音节。 一、看图导入 1．教师出示情境图，引导学生观察：图画上的小兔正在干什么? 屏幕上出现了什么? 2．学习语境歌。 (1)、同时诵读情境歌。 (遥控器，摁一摁，荧屏映出天安门。色彩鲜艳音乐美，小朋友越看越开心。) (2)、请学生跟着老师念两遍情境歌，引出：天安门、摁、音。 3．教师引导过渡：今天我们就学习an、en、in这几个韵母。 同学们观察这三个复韵母。这些复韵母都是在一个单韵母后面加一个鼻音做尾巴，这样组成的韵母就叫鼻韵母。再说一遍读法。指导读前鼻音。 二、指导发音 1．学习韵母an。 (1)教师领读天安门，过渡叙述：“安”去掉声调要学习的an。领读。 （指导读法：先发a音，舌头迅速抵住上牙龈，鼻子发音） (2)指名读、学生自由练读，齐读，“开火车”读。 (3)老师教an写法。写三个，读三遍。 (4)指导读好an的四声。出示ān、án、ǎn、àn，请同学自由读，同桌互读，指名读。 课件出示简单的拼读。领读，小老师领读，自由读。 2．学习韵母en。 (1)我这里有个遥控器，我请一个同学来摁一下，引出en。 (2)领读：en，指导读好音。（先发e音，舌头迅速抵住上牙龈，鼻子发音） (3) 学生自由练读，齐读，“开火车”读。 (4) 老师教en写法。加声调的方法，加上四个声调。 (5)指导读好en的四声。请同学自由读，同桌互读，指名读。 3．学习韵母in和整体认读音节yin。 (1)同学们看这个韵母（板书in）指导学生自学：你们能运用前面学到的发音方法读这个复韵母吗? (2)检查自读情况，纠正问题。

汉语拼音12 an en in un ün

汉语拼音12 an en in un ?n 教师：龙昌小学张顺英概述： 本课有四部分内容。第一部分是五个前鼻韵母ɑn、en、in、un、?n和三个整体认读音节yuɑn、yin、yun，配有图画。第一幅图是天安门，用“安”提示ɑn的音。第二幅图是圆圈，用“圆”提示yuɑn 的音。第三幅图是一个人在摁门铃，用“摁”提示en的音。第四幅图是小兔在树阴下乘凉，用“阴”提示in和yin的音。第五幅图是蚊子，用“蚊”提示un的音。第六幅图是白云，用“云”提示?n和yun的音。 第二部分是拼音练习。包括两项内容：（1）声母与ɑn、en、in、un、?n的拼音，巩固新学的韵母，复习j、q、x跟?组成音节省写?上两点的规则。（2）看图读音节词语，培养学生认识事物、准确拼音的能力。 第三部分是看图借助汉语拼音认字读韵文。配有一幅山区田园图，图画表现了韵文的意思。韵文中有许多含前鼻韵母的音节，还有要认的五个生字。 第四部分是儿歌，配有图画。儿歌中有多个含前鼻韵母的音节。随儿歌学习“半、云、她”三个字。 学情分析： 针对孩子们的年龄特点，在教学中要力求做到趣味性、富有童趣，让学生在游戏活动中能正确认读五个前鼻韵母和它们组成的音节。另外在学习本课之前，学生已学过9个复韵母，会读声母和韵母组成的音节，能按两相拼的方法拼读音节。对三相拼的拼读方法掌握起来比较困难，老师要加强指导。学生单独拼读一个音节容易，如果连成一句话就困难些，老师可以示范教读，然后叫小朋友们模仿读。 教学目标： 知识与技能： 1.学会前鼻韵母an en in un ?n和整体认读音节yuan yin yun，读准音，记清形，正确书写。 2.学习声母与前鼻韵母组成的音节，准确拼读音节，读准三拼音节，复习?上两点省写规则。 3.能够看图说话，根据音节拼读词语和句子。