BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Dynamics of bacterial communities during solid-state fermentation using agro-industrial wastes to produce poly-γ-glutamic acid,revealed by real-time PCR and denaturing gradient gel electrophoresis (DGGE)
Xiaoyu Yong &Yaqing Cui &Lihua Chen &Wei Ran &Qirong Shen &Xingming Yang
Received:28March 2011/Revised:6May 2011/Accepted:6May 2011/Published online:14June 2011#Springer-Verlag 2011
Abstract The dynamics of bacterial communities play an important role in solid-state fermentation (SSF).Poly-γ-glutamic acid (γ-PGA)was produced by Bacillus amyloli-quefaciens C1in SSF using dairy manure compost and monosodium glutamate production residuals as basic sub-strates.The production of γ-PGA reached a maximum of 0.6%after 20days fermentation.Real-time polymerase chain reaction showed the amount of total bacteria reached 3.95×10916S rDNA copies/g sample after 30days,which was in good accordance with the 4.80×109CFU/g obtained by plate counting.Denaturing gradient gel electrophoresis profile showed a reduction of microbial diversity during fermentation,while the inoculum,B.amyloliquefaciens C1,was detected as the dominant organism through the whole process.In the mesophilic phase of SSF,Proteobacteria was the dominant microbial,which was replaced by Firmicutes and Actinobacteria in the thermophilic phase.The molecular analysis of the bacterial diversity has significant potential for instructing the maturing process of SSF to produce γ-PGA at a large-scale level,which could be a benefit in the production of high quality and stable SSF products.
Keywords Poly-γ-glutamic acid (γ-PGA).Solid-state fermentation (SSF).Real-time PCR .Denaturing gradient gel electrophoresis (DGGE).Dynamics of bacterial communities
Introduction
A naturally occurring anionic biopolymer,poly-γ-glutamic acid (γ-PGA),which was first discovered as a component in the capsules of Bacillus anthracis (Ivánovics and Erdos 1937),is water-soluble,biodegradable,edible,and non-toxic to human and the environment,making it useful for various applications in food,cosmetics,medicine,agriculture,and the environment (Shih and Van 2001;Ashiuchi 2010).Solid-state fermentation (SSF)has been employed to produce γ-PGA by several groups using soybean curd,sweet potato residues,wheat bran,diary manure,and swine manure alone or in combination as substrate (Chen 2005;Xu et al.2005;Wang et al.2008b ).A large amount of heat is generated during a large-scale SSF,which is the most critical factor for controlling SSF process,since temperature directly affected the growth,spore formation and germination,the metabolic rate,and the population structure of microbials (Pandey 2003;Sasaki et al.2005).Studies have reported that microorganism diversity is different during SSF process because of the initial materials and the fermentation process (Yu et al.2009;Wang et al.2008a ;Zhu et al.2003;Giraffa 2004).Therefore,it is necessary to detect the temperature as well as the microbial diversity during SSF.
Traditional analysis of the microbial diversity in SSF is primarily based on the selective-plating method (Strom 1985a ,b ).However,this culture-dependent technique is known to only detect 0.1%to 1%of the bacterial composition of the total microbial community (Head et al.1998;Amann et al.1995;Daniel 2005).In recent years,culture-independent methods based on molecular biology techniques,such as real-time polymerase chain reaction
X.Yong :Y .Cui :L.Chen :W.Ran :Q.Shen :X.Yang (*)Jiangsu Provincial Key Lab for Organic Solid Waste Utilization,Nanjing Agricultural University,Nanjing 210095,China
e-mail:xingming@https://www.doczj.com/doc/c18568203.html,
Appl Microbiol Biotechnol (2011)92:717–725DOI 10.1007/s00253-011-3375-3
(PCR)and denaturing gradient gel electrophoresis(DGGE), have provide new methods for analyzing the structure and species composition of complex microbial communities in a wide range of samples from soil(Innerebner et al.2006), compost(Danon et al.2008),and SSF products(Yu et al. 2009;Giraffa2004;Haruta et al.2006).
Althoughγ-PGA produced by fermentation(submerged fermentation and SSF)has been previously studied,it has only been performed with pure cultures of isolated strains.In this study,a large-scale SSF was conducted to produceγ-PGA by Bacillus amyloliquefaciens C1 using agro-industrial residues,such as dairy manure compost(DMC)and monosodium glutamate production residuals(MGPR)as basic substrates.Real-time PCR and DGGE were employed to enumerate the total bacteria and detect the microbial communities,respectively,which could lead to a good identification of the different SSF phases and a better determination of the maturing process at the scale-up level.
Materials and methods
The strain used in SSF
The strain B.amyloliquefaciens C1(China General Micro-biological Culture Collection Center(CGMCC)No.3478), isolated by our laboratory with a highγ-PGA production of 18.3g/L in SMF and4.37%in SSF(20g sterile substrate in 250-mL flask),was used in the present study.C1was inoculated into50-mL Luria-Bertani(LB)medium and cultivated on a rotary shaker at170rpm at37°C for14h (mid-log phase of C1)until the cell concentration(OD600) reached2.0.Then,the liquid culture of C1was sub-inoculated to1,000mL LB at a level of2%(v/v)and incubated under the same condition,which was inoculated into a100-L stirred-tank fermentor thereafter.Finally,the liquid seed culture (109–1010CFU/mL)was acquired by submerged fermenta-tion and used as the inoculum adsorbed with rice bran.
Solid-state fermentation(SSF)
The SSF experiment was performed in a private composting facility located in Yixing,Jiangsu Province,China.Seven thousand kilograms DMC supplemented with500kg wheat bran,300kg soybean cake,350kg corn flour,1,350kg MGPR,100kg citric acid,25kg MgSO4,and15kg MnSO4 were used as the substrate(initial moisture was adjusted to approximately50%),which was inoculated with C1at a1% inoculum concentration.
The windrows of SSF were collected and stirred with a shovel car on a concrete floor in a pile2m in diameter, 1.5m in height,and10m in length at the start of the experiment.The temperature in the center zones,the middle zones,and the exterior zones of the piles was measured every day.To increase aeration,the windrows were turned when the temperatures of the exterior zones reached approximately60°C.Samples were collected on days1, 2,4,6,10,14,20,and30and stored at?20°C.
Extraction ofγ-PGA
Fifty milliliters of deionized water was added to20g of fermented matter,and the mixture was agitated at150rpm at room temperature(approximately25°C)for2h.Forty milliliters of the slurry was subjected to centrifugation at 2,000rpm for5min to remove the SSF substrate.The supernatant was subjected to centrifugation at12,000rpm for10min.The resulting supernatant was poured into four volumes of cold ethanol to precipitate theγ-PGA,which was collected by centrifugation at12,000rpm for20min.A 5-mL aliquot of deionized water was added to dissolve the precipitate,and the resulting solution was stored at4°C. Production analysis ofγ-PGA
A2-mL aliquot of the crudeγ-PGA sample solution and 10mL6M HCl were added to a hydrolysis flask and hydrolyzed at110°C for24h under vacuum.The hydrolyzate was evaporated to dryness using a rotary evaporator,followed by the addition of1mL deionized water to dissolve the dry sample.The liquid sample was then filtered through a0.45-μm membrane filter.The final solution was analyzed by a Biochrom30Amino Acid Analyzer(GE,USA).The corresponding unhydrolyzed sample was also analyzed,and the difference in the concentration of free glutamic acid between these two solutions was considered to be theγ-PGA production.γ-PGA production was expressed as a percentage (%,w/w),which indicated the ratio of the weight ofγ-PGA to the sample.
Enumeration of the bacterial populations by plate count
Serial dilutions(10?1to10?8)of the fermented samples at different times were prepared in sterilized water(w/v).A 0.1-mL aliquot of a diluted mixture of10?6to10?8was spread onto LB plates in triplicate.After incubation at37°C for24h,the numbers of colonies on the plates were counted to calculate the number of CFU per gram(CFU=colony-forming unit).
Extraction of the total genomic DNA of SSF samples
The total genomic DNA of the SSF samples were extracted by the method previously described with small modifications (LaMontagne et al.2002)and stored at?20°C.The DNA
extracts were electrophoresed in1%agarose gel and stained with ethidium bromide(EtBr).
Real-time PCR
A pair of primers,P338-358F(5′-AC TCC TAC GGG AGG CAG CAG-3′)and P534-518R(5′-ATT ACC GCG GCT GCT GG-3′),against the V2–V3region of the16S rRNA gene of bacterial groups were used to estimate the total number of16S rRNA gene copies in this study(Ahn et al. 2009).Real-time PCR amplifications were conducted on a 7500Fast Real-Time PCR System(Applied Biosystems) using the SYBR?Premix Ex Taq?(Perfect Real Time)kit (Takara).The standard curves were generated according to Mieszkin et al.(2009).
PCR-DGGE
PCR on the DNA extracts was performed on a S1000?Thermal Cycler(Bio-Rad Laboratories,Richmond,CA, USA)with primers against the V6–V8region of the16S rRNA gene of bacterial groups.The nucleotide sequences of the primers were as follows:U968-GC(5′-GC-Clamp [CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG G]-AAC GCG AAG AAC CTT AC-3′) and L1401(5′-GCG TGT GTA CAA GAC CC-3′)(Zhu et al. 2003;Konstantinov et al.2003).The PCR amplification mixture consisted of5μl10×PCR buffer(Mg2+free),4μl MgCl2(25mM),4μl dNTP mixture(2.5mM each),
0.2μM of each primer,1μL of template DNA,and
1.25units of rTaq polymerase(Takara,Shiga,Japan);the solution was filled up to a volume of50μL with sterile water.The PCR amplification was performed at94°C for 5min,followed by32cycles of94°C for1min,54°C for 1min,and72°C for1min,and final single extension at 72°C for10min.
The DGGE analysis was conducted according to Muyzer et al.(1993)using a DCode system(Bio-Rad Laboratories, Hercules,CA,USA).The PCR products were loaded onto 8%(w/v)polyacrylamide gel(acrylamide:bisacrylamide-mixture37.5:1)with denaturant gradients ranging from 40%to65%[100%denaturant was7M urea plus40%(w/v) formamide].The electrophoresis was initiated by a pre-running step for10min at a voltage of200V;subsequently, the sample was run at a fixed voltage of80V for16h at 60°C.After electrophoresis,the silver staining procedure was performed as described by Bassam et al.(1991).
Sequencing of the excised band from the DGGE gel
Each dominant DGGE band was excised,and the DNA was extracted in50-μl sterile water at4°C overnight.The supernatant(1μl)was used as the template in the re-amplification PCR reaction mixture with the primer set U968and L1401without a GC clamp.The nucleotide sequences sequenced by Genescript Company(Nanjing, China)were aligned in GeneBank(http://blast.ncbi.nlm.nih. gov/)using the BLAST program,and the most homologous sequences were downloaded for multiple-sequence alignments using ClustalX.A phylogenetic tree was constructed using the neighbor-joining method by MEGA software(Version4.1Beta),and1,000bootstraps were employed to assign confidence levels to the nodes in the tree(Asano et al.2010).
Nucleotide sequence accession numbers
The nucleotide sequences of the DGGE bands excised from the DGGE gel reported in this paper have been deposited in the GenBank databases under the following accession numbers:JF706289–JF706308.
Results
Solid-state fermentation
Changes in the temperature of the SSF process are shown in Fig.1,which was identified by an initial short mesophilic period(1–3days),followed by a long thermophilic period (4–29days)and a curing period with a decrease in temperature(30–33days).The temperature of the exterior zones was much higher than that of the middle and the center zones,and a large amount of heat is generated during
Day
T
e
m
p
e
r
a
t
u
r
e
Fig.1Changes in temperature during the solid-state fermentation processes.Temperature were obtained from the exterior zones(filled diamond),the middle zones(filled square),and the center zones(filled triangle),respectively,of the windrows
the SSF process.The highest temperature was recorded as 70.3°C in the exterior zones at 26days.The production of γ-PGA produced in SSF
There was a lag phase up to 4days of the growth of bacteria,after which,the exponential growth phase was observed with a rapid increase in the biomass.The number of viable cells reached a maximum of 4.8×109CFU/g at 30days (Table 1).The increase in γ-PGA production closely followed the growth pattern of the bacteria,which reached a maximum of 0.6%after 20days (Table 1)when the function microbial,strain C1,had established itself in the deeper layers of the substrates of the SSF.Real-time PCR
The standard curves used to quantify the 16S rRNA gene copies of the samples were generated by plotting threshold cycles against the logarithm of the initial number of DNA;these initial values were harbored by the target 16S rRNA gene using serial dilutions of a known concentration of the plasmid DNA as template in real-time PCR analysis beginning with 5.95×107copies/mL and continuing to 5.95×103copies/mL.Each reaction was performed in triplicate,and a high reproducibility between these three independent standard dilutions was obtained.The quantification showed a good linear rela-tionship (R 2=0.999)ranging from 5.95×107gene copies to 5.95×103gene copies of the target gene (Fig.2).In the range of the detection concentration,the real-time PCR efficiency was calculated as 1.06based on the equation Amplification Efficiency ?10à1=slope à1(Bustin et al.
2009).In the standard samples,a melting curve with a single melting peak was detected at 86.1°C,and no primer dimers or other unspecific PCR products were amplified,which indicated that the presence of a single amplicon in the real-time PCR analysis was accurate (date not shown).Table 1shows that 2.203×10916S rDNA gene copies/g sample was detected at the start of the SSF.Once the SSF was conducted,it increased steadily.At the end of the SSF,the 16S rDNA copies of total bacteria were 3.948×109copies/g sample,which was in good accordance with the results obtained by plate counting.PCR-DGGE
The dynamic change of the structure of the microbial community during the SSF process was detected by PCR-DGGE analysis (Fig.3),which showed a clear reduction of microbial diversity during fermentation and remained stable in the late phase of fermentation based on the DGGE banding patterns.Bands 2,8,10,13,and 19,which could be detected throughout the whole period of fermentation,seemed to be the main functional bacteria during the fermentation process.Some of the original bands (5,6,and 17)disappeared after 4to 6days,indicating either the substrates were not suitable as energy sources for these bacteria or their growth was inhibited by the SSF process,such as high temperature.Seven novel bands appeared in the DGGE profiles,and they were detected at different times and temperatures during the process of SSF.Bands 1,3,7,9,and 14emerged at the start of the SSF and throughout the whole process;the bands increased in intensity to become the major bands by day 6.Band 15appeared at day 10and band 16appeared at day 14,which remained weakly visible thereafter.Bands 4and 11were detected at day 2but disappeared at day 10,which indicated that these two bacteria could not survive at high temperatures.Bands 12and 20appeared in the raw substrate at a high denaturing gradient area,which could not be detected at the early stages of the SSF process,appeared again at the middle and late process.
Twenty dominant DGGE bands obtained with primer set U 968–L 1401were isolated and sequenced.Phylogenetic analyses showed that these 20sequences were grouped into 6phyla (Fig.4).Bands 2(JF706290),7(JF706295),and 19(JF706307)were classified as Firmicutes Bacillales .Bands 6(JF706294),10(JF706298),12(JF706300),and 13(JF706301)were detected as Firmicutes Lactobacillales .Bands 1(JF706289),5(JF706293),14(JF706302),15(JF706303),18(JF706306),and 20(JF706308)were relatives of Actinobacteria Actinobacteridae .Bands 16(JF706304)and 9(JF706297);bands 3(JF706291),
4
1518
21
24
27
30
Log10 Copies
C t V a l u e s
Fig.2The standard curve of the real-time PCR generated by plotting the logarithm amount of the initial target copies against the threshold ranging from 5.95×107to 5.95×103gene copies.The quantification showed a good linear relationship (R 2=0.999).The qPCR efficiency was calculates as 1.06based on the equation Amplification Efficiency ?10à1=slope à1,where the slope referred to the log-linear portion of the calibration curve
(JF706292),8(JF706296),and 11(JF706299);and band 17(JF706305)were identified into the Proteobacteria Alphaproteobacteria ,Proteobacteria Deltaproteobacteria ,and Proteobacteria Gammaproteobacteria ,respectively.Band 2(JF706290),which can be detected during the whole SSF process and finally identified as B.amyloliquefa-ciens ,was the dominant organism of the function bacteria C1incubated in the SSF system.
Discussion
Solid organic waste produced by livestock and agro-industries may cause environmental pollution if not properly treated.The utilization of these waste materials can not only assist in eco-friendly treatments but also reduce production cost of microbial product in SSF (Rai et al.2009).So,in the present study,DMC,MGPR,corn flour,and soybean cake were selected as the basic substrates to produce γ-PGA in SSF.The DMC used in the present study contained 33.24%carbon and 1.97%total nitrogen,whereas the free glutamic acid content in MGPR was 9.04%,which indicated that the substrate provided abundant nutrition for the growth and production of γ-PGA of the C1strain.However,in-depth study to enhance γ-PGA production is needed in the following research since only 0.6%γ-PGA was obtained after 20days fermentation in the mixed culture.
Traditional analysis of the microbial diversity in SSF relies on culture-based techniques that are time-consuming and can detect only cultivable cells.In this study,the bacterial populations were enumerated by real-time PCR,which provided one of the most promising uncultured-dependent techniques on research of microbial https://www.doczj.com/doc/c18568203.html,pared with the results obtained by plate counting (Table 1),the results obtained by real-time PCR (3.948×10916S rDNA copies/g sample;Table 1)showed a slightly lower bacterial population.This may be caused by the reduction of nuclear acid in the manipulation of DNA extraction.
PCR-DGGE was employed to give an overall view of the variation of the dominant microbial community during the SSF period.Sequences retrieved from the DGGE profile primarily belonged to the phyla of Firmicute ,Actinobacteria ,and Proteobacteria ,which indicated that these bacterial communities were dominant in the SSF process (Figs.3and 4).To the best of our knowledge,this is the first culture-independent research study that involved dynamic change of the structures of the microbial communities during the SSF process,which produced γ-PGA.The DGGE profile showed that band
2
Fig.3Profiles of PCR-DGGE analyses of the bacterial community structure established with the U968-GC/L1401primer set.A linear denaturant gradients ranging from 40%denaturant at the top of the gel to 65%denaturant at the bottom [100%denaturant was 7M urea plus 40%(w /v )formamide]were https://www.doczj.com/doc/c18568203.html,ne CK means the sample loaded on the DGGE gel was the PCR product using the genome DNA extracted from the pure culture of Bacillus amyloliquefaciens C1as template
Sample (days)
γ-PGA production (%)
Total number of bacteria,CFU/g sample Copy numbers (copies/g sample)
10.02±0.003 2.43E+09±2.75E+08 2.203E+09±3.325E+0820.06±0.007 2.55E+09±1.39E+08 2.526E+09±6.302E+0740.12±0.009 2.74E+09±2.25E+08 2.645E+09±8.854E+0760.25±0.013 2.84E+09±1.87E+08 2.827E+09±6.519E+07100.46±0.011 3.83E+09±7.33E+08 3.002E+09±3.367E+07140.57±0.017 4.33E+09±5.16E+08 3.414E+09±1.421E+08200.60±0.016 4.44E+09±4.68E+08 3.827E+09±1.140E+0830
0.57±0.010
4.80E+09±
5.89E+08
3.948E+09±
4.680E+07
Table 1The γ-PGA production (percent)produced by solid-state fermentation,the total number of bacteria of the SSF samples enumerated by plate counting,and the copy numbers of the V2–V3region of the 16S rDNA gene copies (copies per gram sample)in the SYBR green real-time PCR assay
(JF706290)could be detected during the whole SSF process,which matched the strains of B.amyloliquefa-ciens C1.Thus,the C1strain used as the inoculum became dominant in the community of the SSF,which indicated that C1could be used as a fast adaptation or intrinsic preference for the nutritional and environmental needs
of
Fig.4Unrooted neighbor-joining tree representing the phylogenetic relationship of the 20cloned V6–V8region of 16S rDNA PCR-DGGE bands to the most closely related identified sequences downloaded from NCBI by BLASTn searches.The numbers at the nodes of the tree are bootstrap values for each node with 100bootstrap resampling.The bar represents 0.1substitutions per site
the bacteria during the SSF process.In the present study, some original bands disappeared during the fermentation process(bands5(JF706293),6(JF706294),and17 (JF706305)),suggesting either that they were not suitable for this SSF medium or that their growth was inhibited by the inoculated bacterial strain.
During the SSF process using agro-industrial waste and livestock manure as substrates,a large amount of ammonia-nitrogen(N)generated by the degradation of organic-N was lost in the solution and subsequent NH3emission(Asano et al.2010).Therefore,during the middle and late SSF processes,the fermentation properties could be characterized by the rise of pH(Maeda et al.2010).Bands3(JF706291),11 (JF706299),and17(JF706305),which were characterized as the pseudomonas-like species,were characterized by their connection with the NH3emission(Maeda et al. 2010)and could be detected in stages in which NH3was actively emitted(date not shown).
It is reported that there were two periods in which the structure of the microbial community shifted significantly according to the different temperature-dependent phases (Danon et al.2008);therefore,two different groups of microorganisms were involved in the SSF process:meso-philic and thermophilic microorganisms.The first period occurred during the first12to24h,when the SSF process was initially characterized by a short mesophilic period. During this period,the development of the mesophilic microbial community was very important because it determined whether a smooth transition from mesophilic to thermophilic period was successfully achieved(Nakasaki et al.1985),whereas the activity of mesophilic bacteria may be greatly decreased under a thermophilic temperature. During this period,the Alphaproteobacteria(Band16 (JF706304))in the raw materials was the dominant microbial,which was effective for mass reduction in DMC because of the higher decomposition activity of the microbial community characterized by the predominance of the Proteobacteria(Bands3(JF706291),4(JF706292), 8(JF706296),9(JF706297),11(JF706299),and17 (JF706305))(Tang et al.2007).Another period occurred during the first60to72h,when a sharp increase in temperature was detected(Fig.1)with the active degradation of the organic waste and an increase in the metabolic activity of the thermophilic bacteria;the SSF then made a rapid transition to the thermophilic phase (Fig.1).During this period,the total microbial diversity decreased(Fig.3),while the diversity of thermophilic bacteria increased(Finstein and Morris1975).Most of the species that were identified belonged to the Firmicutes phylum(Bands2(JF706290),6(JF706294),7 (JF706295),10(JF706298),12(JF706300),13 (JF706301),and19(JF706307))and Actinobacteria phylum(Bands1(JF706289),5(JF706293),14(JF706302),15(JF706303),18(JF706306),and20 (JF706308)).Because Firmicute phyla and Actinobacteria phyla typically include spore-forming bacteria(Fracchia et al.2006),they possess a high thermo-tolerance that can be detected during the thermophilic stage in SSF,which showed that they were active or at least survived the high temperatures.Most Firmicutes detected from the DGGE profile in the present study were identified as belonging to the Bacillus group.These results are in accordance with several research studies(Schloss et al.2003;Zhang et al. 2002)that have indicated that the thermophilic aerobic bacilli are important in classical composting processes. The Actinobacteria detected during the latter stages of the 10-day thermophilic composting process became active during the maturing period.Band18(JF706306),which was closely related to members of the Cellulomonas group in this study,revealed that the decomposition of cellulose was performed by cellulolytic bacteria during the SSF process(Tang et al.2006).Bands12(JF706300)and13 (JF706301),which were identified as the Lactobacillus and Lactococcus,respectively,were found at the beginning of the SSF process,when the substrate was relatively wet, and were reported as the typical dominant microorganisms in degrading plant material under conditions with limited oxygen(Peters et al.2000).Thus,they might have been alive in the center zones of the windrows but not able to tolerate the high temperatures.
Some common bacterial pathogens,such as Escherichia or Salmonella species,were not detected in the SSF samples.However,that did not indicated that the SSF bio-solids were Escherichia-and Salmonella-free,but only that they were not the dominant species;the increase in pH could have been elevated enough to discourage the growth of these bacterial species(Filion et al.2009).The DGGE profile and gel-sequencing analysis revealed that there were several species related to uncultured bacterial, such as Actinobacteria Actinobacteridae(Band5 (JF706293))and Proteobacteria Deltaproteobacteria (Band9(JF706297));the role of their relatives in natural environments is unknown.
Real-time PCR and PCR-DGGE have several draw-backs,such as the low purity of the template DNA and primer mismatch,which can result in a biased banding pattern within the DGGE gel.The deployment of next-generation DNA-sequencing technologies such as454 high-throughput sequencing has greatly enhanced capa-bilities to deeply survey the diversity and composition of the bacterial communities within an environmental sample(Petrosino et al.2009).Such method is valuable in that it could provide our best assessments of overall bacterial diversity and community structure and the relative abundances of specific bacterial taxa within SSF substrates(Lauber et al.2009).Therefore,this method
should be employed to analyze the microbial community in future studies.
Acknowledgements The study was financially supported by Chinese Ministry of Science and Technology(2011CB100503)and by Jiangsu Bureau of Science and Technology(BE2010722and BA2008027). References
Ahn J-h,Y-j K,Kim T,H-g S,Kang C,J-o K(2009)Quantitative improvement of16S rDNA DGGE analysis for soil bacterial community using real-time PCR.J Microbiol Methods78:216–222 Amann RI,Ludwig W,Schleifer K(1995)Phylogenetic identification and in situ detection of individual microbial cells without cultivation.Microbiol Rev59:143–169
Asano R,Otawa K,Ozutsumi Y,Yamamoto N(2010)Development and analysis of microbial characteristics of an acidulocomposting system for the treatment of garbage and cattle manure.J Biosci Bioeng110:419–425
Ashiuchi M(2010)Occurrence and biosynthetic mechanism of poly-γ-glutamic acid.In:Hamano Y(ed)Amino-acid homopolymers occurring in nature(microbiology monographs),vol15.Springer, Heidelberg,pp77–94
Bassam BJ,Caetano-Anollés G,Gresshoff PM(1991)Fast and sensitive silver staining of DNA in polyacrylamide gels.Anal Biochem196:80–83
Bustin S,Benes V,Ja G,Hellemans J,Huggett J,Kubista M, Mueller R,Nolan T,Pfaffl MW,Shipley GL,Vandesompele J, Wittwer CT(2009)The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments.Clin Chem55:611–622
Chen X(2005)High yield of poly-γ-glutamic acid from Bacillus subtilis by solid-state fermentation using swine manure as the basis of a solid substrate.Bioresour Technol96:1872–1879 Daniel R(2005)The metagenomics of soil.Nat Rev Microbiol3:470–478
Danon M,Franke-whittle IH,Insam H,Chen Y,Hadar Y(2008) Molecular analysis of bacterial community succession during prolonged compost curing.FEMS Microbiol Ecol65:133–144 Filion M,Novinscak A,Decoste NJ(2009)Characterization of bacterial and fungal communities in composted biosolids over a 2year period using denaturing gradient gel electrophoresis.Can J Microbiol387:375–387
Finstein MS,Morris ML(1975)Microbiology of municipal solid waste composting.Adv Appl Microbiol19:113–151
Fracchia L,Dohrmann AB,Martinotti MG,Tebbe CC(2006) Bacterial diversity in a finished compost and vermicompost: differences revealed by cultivation-independent analyses of PCR-amplified16S rRNA genes.Appl Microbiol Biotechnol71:942–952
Giraffa G(2004)Studying the dynamics of microbial populations during food fermentation.FEMS Microbiol Rev28:251–260 Haruta S,Ueno S,Egawa I,Hashiguchi K,Fujii A,Nagano M,Ishii M,Igarashi Y(2006)Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis.
Int J Food Microbiol109:79–87
Head IM,Saunders JR,Pickup RW(1998)Microbial evolution, diversity,and ecology:a decade of ribosomal RNA analysis of uncultivated microorganisms.Microb Ecol35:1–21 Innerebner G,Knapp B,Vasara T,Romantschuk M,Insam H(2006) Traceability of ammonia-oxidizing bacteria in compost-treated soils.Soil Biol Biochem38:1092–1100Ivánovics G,Erdos L(1937)Ein beitrag zum wesen der kapselsubstanz des milzbrandbazillus.Z Immunitatsforsch 90:5–19
Konstantinov SR,W-y Z,Williams BA,Tamminga S(2003)Effect of fermentable carbohydrates on piglet faecal bacterial com-munities as revealed by denaturing gradient gel electrophoresis analysis of16S ribosomal DNA.FEMS Microbiol Ecol 43:225–235
LaMontagne MG,Michel FC Jr,Holden PA,Reddy CA(2002) Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis.J Microbiol Methods49:255–264
Lauber CL,Hamady M,Knight R,Fierer N(2009)Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale.Appl Environ Microbiol75(15):5111–5120.doi:10.1128/aem.00335-09 Maeda K,Morioka R,Hanajima D(2010)The impact of using mature compost on nitrous oxide emission and the denitrifier community in the cattle manure composting process.Microb Ecol59:25–36 Mieszkin S,Furet J-P,Gr C,Gourmelon Ml(2009)Estimation of pig fecal contamination in a river catchment by real-time PCR using two pig-specific Bacteroidales16S rRNA genetic markers.Appl Environ Microbiol75(10):3045–3054
Muyzer G,Waal ECD,Uitierlinden AG(1993)Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.Appl Environ Microbiol59:695–700
Nakasaki K,Sasaki M,Shoda M,Kubota H(1985)Characteristics of mesophilic bacteria isolated during thermophilic composting of sewage-sludge.Appl Environ Microbiol49:42–45
Pandey A(2003)Solid-state fermentation.Biochem Eng J13:81–84 Peters S,Koschinsky S,Schwieger F,Tebbe CC(2000)Succession of microbial communities during hot composting as detected by PCR–single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes.Appl Environ Microbiol 66:930–936
Petrosino JF,Highlander S,Luna RA,Gibbs RA,Versalovic J(2009) Metagenomic pyrosequencing and microbial identification.Clin Chem55(5):856–866.doi:10.1373/clinchem.2008.107565
Rai SK,Konwarh R,Mukherjee AK(2009)Purification,character-ization and biotechnological application of an alkalineβ-keratinase produced by Bacillus subtilis RM-01in solid-state fermentation using chicken-feather as substrate.Biochem Eng J 45:218–225
Sasaki H,Yano H,Sasaki T,Nakai Y(2005)A survey of ammonia-assimilating micro-organisms in cattle manure composting.J Appl Microbiol99:1356–1363
Schloss PD,Hay AG,Wilson DB,Walker LP(2003)Tracking temporal changes of bacterial community fingerprints during the initial stages of composting.FEMS Microbiol Ecol46:1–9 Shih IL,Van YT(2001)The production of poly-(γ-glutamic acid) from microorganisms and its various applications.Bioresour Technol79:207–225
Strom PF(1985a)Effect of temperature on bacterial species diversity in thermophilic solid-waste composting.Appl Environ Microbiol 50:899–905
Strom PF(1985b)Identification of thermophilic bacteria in solid-waste composting.Appl Environ Microbiol50:906–913
Tang J,Maie N,Tada Y,Katayama A(2006)Characterization of the maturing process of cattle manure compost.Process Biochem 41:380–389
Tang J-c,Shibata A,Zhou Q,Katayama A(2007)Effect of temperature on reaction rate and microbial community in composting of cattle manure with rice straw.J Biosci Bioeng104:321–328
Wang H-Y,Zhang X-J,Zhao L-P,Xu Y(2008a)Analysis and comparison of the bacterial community in fermented grains
during the fermentation for two different styles of Chinese liquor.
J Ind Microbiol Biotechnol35:603–609
Wang Q,Shouwen C,Jibin Z,Ming S,Ziduo L,Ziniu Y(2008b)Co-producing lipopeptides and poly-γ-glutamic acid by solid-state fermentation of Bacillus subtilis using soybean and sweet potato residues and its biocontrol and fertilizer synergistic effects.
Bioresour Technol99:3318–3323
Xu J,Shouwen C,Ziniu Y(2005)Optimization of process parameters for poly-γ-glutamate production under solid state fermentation from Bacillus subtilis CCTCC202048.Process Biochem 40:3075–3081Yu Z,Dong B,Lu W(2009)Dynamics of bacterial community in solid-state fermented feed revealed by16S rRNA.Lett Appl Microbiol49:166–172
Zhang YC,Ronimus RS,Turner N,Zhang Y,Morgan HW(2002) Enumeration of thermophilic species in composts and identification with a random amplification polymorphic DNA(RAPD)protocol.
Syst Appl Microbiol25:618–626
Zhu W-y,Williams BA,Konstantinov SR,Tamminga S(2003) Analysis of16S rDNA reveals bacterial shift during in vitro fermentation of fermentable carbohydrate using piglet faeces as inoculum.Anaerobe9:175–180
操作系统】Windows XP sp3 VOL 微软官方原版XP镜像◆ 相关介绍： 这是微软官方发布的，正版Windows XP sp3系统。 VOL是Volume Licensing for Organizations 的简称，中文即“团体批量许可证”。根据这个许可，当企业或者政府需要大量购买微软操作系统时可以获得优惠。这种产品的光盘卷标带有"VOL"字样，就取 "Volume"前3个字母，以表明是批量。这种版本根据购买数量等又细分为“开放式许可证”(Open License)、“选择式许可证(Select License)”、“企业协议(Enterprise Agreement)”、“学术教育许可证(Academic Volume Licensing)”等5种版本。根据VOL计划规定， VOL产品是不需要激活的。 ◆ 特点： 1. 无须任何破解即可自行激活，100% 通过微软正版验证。 2. 微软官方原版XP镜像，系统更稳定可靠。 ◆ 与Ghost XP的不同： 1. Ghost XP是利用Ghost程序，系统还原安装的XP操作系统。 2. 该正版系统，安装难度比较大。建议对系统安装比较了解的人使用。 3. 因为是官方原版，因此系统无优化、精简和任何第三方软件。 4. 因为是官方原版，因此系统不附带主板芯片主、显卡、声卡等任何硬件驱动程序，需要用户自行安装。 5. 因为是官方原版，因此系统不附带微软后续发布的任何XP系统补丁文件，需要用户自行安装。 6. 安装过程需要有人看守，进行实时操作，无法像Ghost XP一样实现一键安装。 7. 原版系统的“我的文档”是在C盘根目录下。安装前请注意数据备份。 8. 系统安装结束后，相比于Ghost XP系统，开机时间可能稍慢。 9. 安装大约需要20分钟左右的时间。 10. 如果你喜欢Ghost XP系统的安装方式，那么不建议您安装该系统。 11. 请刻盘安装，该镜像用虚拟光驱安装可能出现失败。 12. 安装前，请先记录下安装密钥，以便安装过程中要求输入时措手不及，造成安装中断。 ◆ 系统信息：
物流商零担中转操作教程 Prepared on 22 November 2020
物流商零担中转操作教程物流商-零担中转 注：在客户使用物流商的零担中转模块之前，会出现两种情况：第一种为托运方（发货方）未使用八万物流在线平台；第二种为托运方（发货方）已经使用八万物流在线平台，并且在平台上发了一笔单据给物流商进行零担中转。在以下的流程中会分别对两种情况的操作进行介绍： 1、托运方（发货方）未使用八万物流在线平台。 步骤一：物流商在登录进平台后，点击进入平台的收货管理-零担收货清单模块。 步骤二：点击收货管理-零担收货清单模块的新增承运单按钮。 步骤三：根据托运方留下的物流信息，填写收货凭证，把物流信息录入平台。 1、填写终到站信息，省-市-区/县 2、点击查看按钮，填写托运方信息 3、填写未注册公司信息，并点击保存按钮 4、点击查看按钮，填写提货方信息 5、若提货方未使用平台，填写未注册公司信息，并点击保存按钮；若使用了平台，可通过手机号码搜索到该公司，点击选定按钮 6、填写货物信息 7、填写费用信息，结算方式可供选择 8、填写其他注意事项 步骤四：单据填写完整后，点击保存按钮，保存单据。 步骤五：系统自动跳转至零担收货清单界面，这笔单据已经保存在零担收货系统的清单中，物流状态为：新增单。 步骤六：在零担收货系统的清单中，找到需要中转的单据，点击详单按钮查看收货凭证。 步骤七：在承运收货凭证中，点击增至零担中转按钮。 步骤八：点击进入平台的发货管理-零担中转模块。 步骤九：点击发货管理-零担中转模块的待托运中转按钮，进入托运发货清单。 步骤十：找到需要中转的单据，此时物流状态为待中转，点击详单按钮查看托运发货凭证。 步骤十一：托运发货凭证中核对物流信息，点击查看按钮选择所要中转的物流公司。 步骤十二：中转的物流公司若未使用平台，就填写未注册公司信息并点击白色保存按钮；若使用了平台，可以根据对方手机号码搜索到该物流公司并直接进行选定。 步骤十三：完成中转物流公司的选定后，点击零担中转按钮，进行中转。 步骤十四：点击确定按钮。 步骤十五：单据自动跳转到物流商的发货管理-零担中转模块下的待验货收货中，在托运发货清单里，可以看到需要中转的单据，点击详单按钮可以查看托运发货凭证，此时单据物流状态为待收货 步骤十六：选择的中转物流公司在平台上对中转的单据进行收货操作后，单据会自动跳转至物流商的发货管理-零担中转模块下的发货完成中，在其托运发货清单中有详细记录，此时物流状态为已完成，物流商的零担中转操作完成。 2、托运方（发货方）已经使用八万物流在线平台，并且在平台上发了一笔单据给物流商进行零担中转。 步骤一：物流商在登录进平台后，点击进入平台的收货管理-零担收货清单模块。 步骤二：点击收货管理-零担收货清单模块的待验货收货按钮，进入承运验货收货清单。 步骤三：查询到托运方需要进行零担中转的单据，此时物流状态为待收货，点击收货按钮。 步骤四：进行确认收货。
Windows7 SP1官方原版 以下所有版本都为Windows7 SP1官方原版，请大家放心下载！ 32位与64位操作系统的选择：https://www.doczj.com/doc/c18568203.html,/Win7News/6394.html 最简单的硬盘安装方法：https://www.doczj.com/doc/c18568203.html,/thread-25503-1-1.html 推荐大家下载旗舰版，下载后将sources/ei.cfg删除即可安装所有版本，比如旗舰，专业，家庭版。 ============================================ Windows 7 SP1旗舰版中文版32位： 文件cn_windows_7_ultimate_with_sp1_x86_dvd_u_677486.iso SHA1:B92119F5B732ECE1C0850EDA30134536E18CCCE7 ISO/CRC:76101970 cn_windows_7_ultimate_with_sp1_x86_dvd_u_677486.iso.torrent(99.63 KB, 下载次数: 326189) Windows 7 SP1旗舰版中文版64位： 文件cn_windows_7_ultimate_with_sp1_x64_dvd_u_677408.iso SHA1: 2CE0B2DB34D76ED3F697CE148CB7594432405E23 ISO/CRC: 69F54CA4 cn_windows_7_ultimate_with_sp1_x64_dvd_u_677408.iso.torrent(128.17 KB, 下载次数: 197053)
现代物流管理基础教学 大纲 -CAL-FENGHAI-(2020YEAR-YICAI)_JINGBIAN
教学大纲 课程：现代物流管理基础适用专业：物流管理 系部：工商管理系 2011 年 8 月 19 日 课程教学大纲审核表
《现代物流管理基础》教学大纲 课程代码：030418 学时：52学时 学分：4学分 课程类别：专业课 一、课程的性质与任务 课程的性质：现代物流管理基础是物流管理专业的一门基础专业课。对掌握专业物流知识，了解专业物流公司的经营与管理至关重要，也为学 习其他专业课打下了良好基础。 课程的任务：本课程的任务是掌握有关物流的基本概念, 物流系统所涉及的各种要素,物流信息技术及系统和有关供应链管理知识等。 前导课程：商品学 后续课程：采购与供应管理，运输管理实务，仓储与配送管理，物流管理信息系统，第三方物流，企业物流管理，国际贸易，报关与报检实务等 二、教学基本要求 知识要求：要求学生掌握有关物流的基本概念，物流系统的基本要素，发展现状，将来的趋势等。 能力要求：要求学生能有物流的基本概念，达到一定的物流理论水平，还要具备一定的实践操作能力，尤其是动手能力。 道德要求：具备一定的职业素养，具有爱岗敬业，无私奉献，明礼诚信等素质。 三、教学条件 要求学校要有实习实训基地，同时与企业要有合作和来往，经常可以带学生去企业考察和学习，让学生感受到理论与实践的差距所在。 四、教学内容及学时安排 （一）教学内容 模块一物流管理基本概念 任务一什么是物流、物流管理 任务二什么是物流系统
模块二包装 任务一何谓包装及包装地位 任务二包装材料 任务三常见包装技术 模块三运输 任务一运输的地位 任务二运输方式 任务三运输线路设计 模块四仓储 任务一何谓仓储及仓储地位 任务二仓储作业管理----ABC分类管理法、入库、盘点、出库作业 模块五装卸搬运 任务一装卸搬运概述 任务二装卸搬运合理化 模块六流通加工 任务一流通加工概述 任务二主要的流通加工类型 任务三流通加工合理化 模块七配送 任务一配送概述 任务二配送业务流程 模块八物流信息 任务一物流信息概述 任务二物流信息技术及应用 （二）基本要求 本课程要求学生掌握物流的有关基本概念,物流系统的构成，物流系统各构成要素，供应链物流等。 （三）实验(上机、习题课或讨论课)内容和基本要求 学生在本课程的学习中应在课外进行一定量的阅读、复习和练习, 进一步消化吸收课堂教学内容和掌握分析方法。讨论课中要求学生通过查找有关物流的最新发展动态, 并结合课堂上所学的内容对新发展做出自己的理解。 五、各教学环节学时分配
Microsoft 微软官方原版（正版）系统大全 微软原版Windows 98 Second Edition 简体中文版 https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16446&page=1&extra=#pid125031 微软原版Windows Me 简体中文版 https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16448&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版Windows 2000 Professional 简体中文版 https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16447&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版Windows XP Professional SP3 简体中文版 https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16449&page=1&extra=#pid125073微软原版Windows XP Media Center Edition 2005 简体中文版 https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16451&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版Windows XP Tablet PC Edition 2005 简体中文版 https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16450&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版Windows Server 2003 R2 Enterprise Edition SP2 简体中文版(32位) https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16452&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版Windows Server 2003 R2 Enterprise Edition SP2 简体中文版(64位) https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16453&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版Windows Vista 简体中文版(32位) https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16454&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版Windows Vista 简体中文版(64位) https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16455&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版 Windows7 SP1 各版本下载地址： https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=12387&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版 Windows Server 2008 Datacenter Enterprise and Standard 简体中文版(32位) https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16457&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版 Windows Server 2008 Datacenter Enterprise and Standard 简体中文版(64位) https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16458&highlight=%CE%A2%C8%ED%D4%AD %B0%E6 微软原版 Windows Server 2008 R2 S E D and Web 简体中文版(64位) https://www.doczj.com/doc/c18568203.html,/viewthread.php?tid=16459&highlight=%CE%A2%C8%ED%D4%AD %B0%E6
物流运输公司数据库设 计 Company Document number：WUUT-WUUY-WBBGB-BWYTT-1982GT
内蒙古科技大学 本科生课程设计论文 题目：物流运输公司数据库设计 学生姓名： 学号： 专业：计算机 班级：13级4班 指导教师： 2015 年 12 月 30 日
内蒙古科技大学课程设计任务书
摘要 随着时间的推移，数据库在各个领域都被广泛的运用。数据库技术已经成为现代信息技术的重要组成部分，是现代计算机信息系统和计算机应用系统的基础和核心。 我所做的是物流运输公司物数据库设计，目的是为了让物流运输公司更好的实行自动化管理，减少了投入的人力、精力，能让数据存储在计算机内，能够有组织的，统一管理公司的业务。我利用课本中的知识，通过需求分析，概念设计，逻辑设计，及数据库的实施和运行等阶段，为物流运输公司设计了一个小型数据库。 关键词：SQL SERVER；数据库设计；物流运输公司 目录
前言 这次课程设计就是对本学期所学的《数据库原理及应用》及《SQL server 从零开始学》的实践，这两门课既有较强的理论性，又有较强的实践性的专业基础课程，需要把理论知识和实际应用紧密结合起来。我的课设题目是“物流运输公司数据库设计”，通过对物流公司内部管理来实现数据库的功能。 这学期学习完数据库的理论知识，又学习了SQL语言的运用，最后用自己的电脑实践，用“物流运输公司数据库设计”来举例用理论来联系实践，了解并
掌握了数据库管理系统的基本原理和数据库系统设计的方法，培养了我应用及设计数据库的能力。 通过亲身实践，我了解物流管理的结构，需要了解客户、公司、货物之间的关系，首先，它们被输入到数据库后，能够查询，修改和删除，然后通过建立键的关系，来建立表的联系，然后通过需求分析，了解了需求分析的过程和目的，建立数据字典，概念设计阶段要完成数据抽象与局部视图设计以及视图的集成。逻辑结构设计阶段要把E-R图转化为关系模式。最后是数据库的实施和运行。 第一章：系统分析及设计 主要的需求 物流运输公司A需要建立一个管理数据库存储以下信息： 1.物流运输公司A中主要的实体有员工、订单、运单、车辆、客户。 2.物流运输公司A有多名负责的不同工作的员工：货运员工和维修员工。 3.每一笔订单包含订单编号、货物名称、送货日期、货物数量、客户编号。 4.每一笔运单包含运单编号、订单编号、出车司机、签收日期、出车日期、回车日期。 5.物流运输公司A还会为客户建立客户表，客户表包含客户编号、客户名称、客户性别、客户地址、联系电话。 6.物流运输公司A会建立一个车辆表，包含：车辆编号、车牌号、车辆型号、最大载重、购买金钱。 7.对于维修的车辆，还会建立一个维修记录表，记录维修编号、维修车牌号、开始维修时间、结束维修时间、维修费用、维修地点。 数据库的E-R图 实体E-R模型设计 1.客户E-R模型如图所示。 图客户E-R模型 2.车辆E-R模型如图所示。 图车辆E-R模型 3.订单信息E-R模型如图所示 图订单信息E-R模型 4.运单信息E-R模型如图所示。
蓝桥E R P物流管理软 件操作教程 Standardization of sany group #QS8QHH-HHGX8Q8-GNHHJ8-HHMHGN#
蓝桥物流系统操作方法和岗位职责 一、开单文员岗位职责注意事项： 1、输入单据时要用输入法半角状态输入， 进入系统点营运中心选开单如下图 2、新增，录入相关信息 2、正确输入托运单相关项目（发货人、收货人、收货人电话、发货人电话，运费等）如上图。要签回单,一定在回单要求里面注明份数,然后按F9打印。 3、中转货物一定填经由地，否则到站接收后看到这票货。 二、配载文员岗位职责和注意事项： 1、短途接驳， 在营运中心点短途接如下图 点新增调出库存如下图 双击移出要短驳的单到右边，移完要驳的单，点击填写车辆等信息并完成我，如下图 填写相关车辆信息并点击完成 2、短驳接收 在营运中心点短途接驳，按时间提取短驳的车辆，找到其它公司驳过来的车辆，选定，双击打开，点接收本车就可以了，如下图。 3、分配配载 点开营中心，选分单配载如下图 点击新增并调出库存,并将要发车的移到右边如下图 点击填写车辆等,并填写相关信息(车牌,长途车运费,到站等信息) 提取已配载好的车,然后打印清单
注意,如果发现漏装可以点淌有库存进补装去,如装错了选择单票剔除,如果取消,就点整车做废.完全无娱就点完成本车,这样到站就可以到货确认那里相关车辆,如果不点，到站就在到货确认里面看不到这辆车。 三、货物分流文员岗位职责和注意事项 1、到货接收 在营运中心点到货确认 然后确认到货车辆，也可以查看到货清单。 2、异常登记，在车辆到达仓库在卸货过程发现异常情况（如：少货，多货，破损等情况）都进行登 记。在营运中心点异常登记，点新增就会出现一个填写界面，如下图，注明红圈的为必填项目。 3、到货库存查询，在营运中心点到货库存，按时间，发站提取库存数来掌握仓库现在有多少货未分 流。 4、到货通知 在营运中心点到货通知 按时间提未通知的单据，然后在电话一栏中选已通知，并点通知保存。 5、客户提货 在营运中心中点击客户提货如下图 将客提走的移到右边，并点确认，填提货人相关信息，并战 6、送货上门 在营运中心点送货上门，然后点安排送货，将要送货单，移到右边，并点送货车辆，输入送货信息。如下图： 7、中转货物分流（终端代理） 在营运中心点，终端代理如下图
精心整理正版Windows系统下载+正版密钥 2010-05-3011:49 喜欢正版Windows系统 这是我收集N天后的成果，正版的Windows系统真的很好用，支持正版！大家可以用激活工 具激活！现将本收集的下载地址发布出来，希望大家多多支持！ Windows98第二版（简体中文） 安装序列号：Q99JQ-HVJYX-PGYCY-68GM3-WXT68 安装序列号：Q4G74-6RX2W-MWJVB-HPXHX-HBBXJ 安装序列号：QY7TT-VJ7VG-7QPHY-QXHD3-B838Q WindowsMillenniumEdition(WindowME)（简体中文） 安装序列号：HJPFQ-KXW9C-D7BRJ-JCGB7-Q2DRJ 安装序列号：B6BYC-6T7C3-4PXRW-2XKWB-GYV33 安装序列号：K9KDJ-3XPXY-92WFW-9Q26K-MVRK8 Windows2000PROSP4（简体中文） SerialNumber:XPwithsp3VOL微软原版（简体中文） 文件名:zh-hans_windows_xp_professional_with_service_pack_3_x86_cd_vl_x14-74070.iso 大小:字节 MD5:D142469D0C3953D8E4A6A490A58052EF52837F0F CRC32:FFFFFFFF 邮寄日期(UTC)：5/2/200812:05:18XPprowithsp3VOL微软原版（简体中文）正版密钥： MRX3F-47B9T-2487J-KWKMF-RPWBY(工行版)（强推此号！！！） QC986-27D34-6M3TY-JJXP9-TBGMD(台湾交大学生版) QHYXK-JCJRX-XXY8Y-2KX2X-CCXGD(广州政府版)
（物流管理）第三方物流管理教程案例习题分册
第一章第三方物流概述案例与习题 案例l： 合理搭配客户，实现季节性互补效益 一家上海的民营物流公司在市区配送方面很有优势，一开始他们的客户都是大型的食品企业，这些企业都有一个特点，天气热的季节，食品销售进入淡季，而随着大气转凉，销售量逐渐回升，因此，物流活动也有明显的季节性，考虑到在天热时物流服务能力的闲置，该物流企业意识到应该选择一些在夏天进入销售旺季的产品，在经过市场调研后，他们确定了啤酒和饮料企业作为营销的主攻方向。由于这些啤酒和饮料企业正在为这种季节性波动造成的成本和管理问题发愁，双方一拍即合，很快签定了合同。 经过客户的合理搭配，该物流公司实现了全年物流业务量的相对稳定，取得了明显的经济效益。 （资料来源：郝聚民编著的《第三方物流》16页）思考题：这家物流公司如何进行客户运作整合？这种运作整合有什么价值？ 案例简评：第三方物流整合运作所产生的规模经济效益是递增的，如果运作得好，将导致竞争优势及更大的客户基础这家民营物流公司将大型的食品企业与啤酒和饮料企业的物流业务整合，使得全年物流业务量的相对稳定，取得了明显的经济效益。 案例2: 冠生园集团物流外包 2
冠生园集团是国内惟一一家拥有“冠生园”、“大白兔”两个中国驰名商标的老字号食品集团。集团生产大白兔奶糖、蜂制品系列、酒、冷冻微波食品、面制品等食品，总计达到了2000多个品种，其中糖果销售额近4亿元人民币。近几年市场需求增大了，但运输配送跟不上。集团拥有的货运车辆近100辆，要承担上海市3000多家大小超市和门店的配送，还有北京、大原、深圳等地的货物运输。淡季运力空放，旺季忙不过来，每年维持车队运行的成本费用就达上百万元。 产品规格品种多、市场辐射面大，靠自己配送运输成本高、浪费大。为此，2002年初，冠生国集团下属合资企业达能饼干公司率先做出探索，将公司产品配送运输全部交给第三方物流。物流外包以后，不仅配送准时准点，而且费用要比自己做节省许多。达能公司把节约下来的资金投入到开发新产品与改进包装上，使企业又上了一个新台阶。为此，集团销售部门专门组织各企业到达能公司去学习，决定在集团系统推广它们的做法。经过选择比较，集团委托上海虹鑫物流有限公司作为第三方物流机构，搞“门对门”物流配送。 虹鑫物流与冠生国签约后，通过集约化配送，极大地提高了效率。每天一早，他们在电脑上输入冠生园相关的配送数据，制定出货最佳搭配装车作业图，安排准时、合理的车流路线。货物不管多少，就是两三箱也送。此外按照签约要求，遇到货物损坏，按规定赔偿。一次，整整一车糖果在运往河北途中翻入河中，物流公司掏出5万元，将掉
现代物流管理操作教程 教材内容 Document number【SA80SAB-SAA9SYT-SAATC-SA6UT-SA18】
第一章报关辅助管理 第一节报关单填制要求 本系统将以海关的H2000为标准来填制报关单，所以在操作过程中必须熟练掌握如下内容。 1.备案号：填写《加工贸易登记手册》、《免征税证明》或其他有关备案审批文件的编号。无备案审批文件的报 关单，本栏目免予填报。一份报关单只允许填报一个备案号。 2.进口日期/出口日期：进口日期与运载进口货物的运输工具申报进境的日期一致。出口日期仅在海关打印报关单 证明联时用，其与运载出口货物的运输工具办结出境手续的日期一致。预录入报关单、EDI报关时免于申报。 无实际进出境的报关单填报向海关申报的日期。标准格式为：YYYY/MM/DD。本系统显示格式为：。 3.申报日期：除特殊情况（如预报关或提前报关）外，进口货物申报日期不能早于进口日期，出口货物申报日期 不能晚于出口日期。标准格式为：YYYY/MM/DD。本系统显示格式为：。 4.经营单位：经营单位应符合a.对外签订并执行进出口贸易合同的单位；b.中国境内法人。填写方法是单位名称 +10位编码，缺一不可。 特殊情况下经营单位确定原则： 三资企业委托外贸公司进口投资设备物品的，经营单位填报外商投资企业，并在备注栏注明“委托某某公 司”进口； 援助、赠送、捐赠的货物，填报直接接受货物的单位。 进出口企业之间相互代理进出口，或没有进出口经营权的企业委托有进出口经营权的企业代理进出口的， 填报代理方。 合同的签订者和执行者不是同一企业的，按执行合同的企业填报。 对委托我驻港澳机构成交的货物，国内委托人为经营单位（中国境内法人）。如上海汽车进出口公司委托 香港大兴汽车进出口公司进口汽车，经营单位应填报上海汽车进出口公司。 5.运输工具名称：指载运货物进出境的运输工具的名称或运输工具编号。 海运货物：船名+“/”+航次 空运货物：航班号+进出境日期+“/”+总运单号 6.提运单号：指进出口货物提单或运单的编号。一份报关单只允许填报一个提运单号。进口提前报关本栏为空。 7.征免性质：指海关对进出口货物实施征、减、免税管理的性质类别。一份报关单只允许填报一种征免性质。加 工贸易结转货物本栏为空。 8.许可证号：一份报关单只允许填报一个许可证号，如一份报关单对应多个许可证号的必须拆单申报。 9.批准文号：进口报关单本栏填报《进口付汇核销单》编号。出口报关单本栏填报《出口收汇核销单》编号。进 口免填。 10.成交方式：进口按实际成交方式填报。出口填报FOB，若按CIF或CFR签订合同，在备注栏内注明合同价。无 实际进出境的，进口填报CIF价，出口填报FOB价。 11.装运港/指运港：装运港指进口货物在运抵我国关境前的最后一个境外装运港。指运港指出口货物运往境外的最 终目的港。无实际进出境的，填报“中国境内”。 12.收货单位/发货单位：收货单位即进口货物在境内的最终消费、使用单位。包括自行从境外进口货物的单位及委 托有外贸经营权的企业进口货物的单位。发货单位指出口货物在境内的生产或销售单位。包括自行出口货物的单位及委托有外贸经营权的企业出口的单位。 13.境内目的地/境内货源地：境内目的地指已知的进口货物在国内的消费、使用地或最终运抵地。境内货源地指出 口货物在国内的产地或原始发货地。 14.集装箱号：填报首个集装箱的箱号/规格/自重，其余填在唛头标记栏。非集装箱货物填报为0。如 TEXU360231/20/22000表示箱号为TEXU360231的20’的集装箱，自重为22000KGS。
微软MSDN官方（简体）中文操作系统全下载 这不知是哪位大侠收集的，太全了，从DOS到Windows，从小型系统到大型系统，从桌面系统到专用服务器系统，从最初的Windows3.1到目前的Windows8，以及Windows2008，从16位到32位，再到64位系统，应有尽有。全部提供微软官方的校验文件，这些文件都可以在微软官方MSDN订阅中得到验证，完全正确！ 下载链接电驴下载，可以使用用迅雷下载，建议还是使用电驴下载。你可以根据需要在下载链接那里找到你需要的文件进行下载！太强大了！！！ 产品名称: Windows 3.1 (16-bit) 名称: Windows 3.1 (Simplified Chinese) 文件名: SC_Windows31.exe 文件大小: 8,472,384 SHA1: 65BC761CEFFD6280DA3F7677D6F3DDA2BAEC1E19 邮寄日期(UTC): 2001-03-06 19:19:00 ed2k://|file|SC_Windows31.exe|8472384|84037137FFF3932707F286EC852F2ABC|/ 产品名称: Windows 3.2 (16-bit) 名称: Windows 3.2.12 (Simplified Chinese) 文件名: SC_Windows32_12.exe 文件大小: 12,832,984 SHA1: 1D91AC9EB3CBC1F9C409CF891415BB71E8F594F7 邮寄日期(UTC): 2001-03-06 19:21:00 ed2k://|file|SC_Windows32_12.exe|12832984|A76EB68E35CD62F8B40ECD3E6F5E213F|/ 产品名称: Windows 3.2 (16-bit) 名称: Windows 3.2.144 (Simplified Chinese) 文件名: SC_Windows32_144.exe 文件大小: 12,835,440 SHA1: 363C2A9B8CAA2CC6798DAA80CC9217EF237FDD10 邮寄日期(UTC): 2001-03-06 19:21:00 ed2k://|file|SC_Windows32_144.exe|12835440|782F5AF8A1405D518C181F057FCC4287|/ 产品名称: Windows 98 名称: Windows 98 Second Edition (Simplified Chinese) 文件名: SC_WIN98SE.exe 文件大小: 278,540,368 SHA1: 9014AC7B67FC7697DEA597846F980DB9B3C43CD4 邮寄日期(UTC): 1999-11-04 00:45:00 ed2k://|file|SC_WIN98SE.exe|278540368|939909E688963174901F822123E55F7E|/ 产品名称: Windows Me 名称: Windows? Millennium Edition (Simplified Chinese) 文件名: SC_WINME.exe
物流商零担中转操作教程Newly compiled on November 23, 2020
物流商零担中转操作教程物流商-零担中转 注：在客户使用物流商的零担中转模块之前，会出现两种情况：第一种为托运方（发货方）未使用八万物流在线平台；第二种为托运方（发货方）已经使用八万物流在线平台，并且在平台上发了一笔单据给物流商进行零担中转。在以下的流程中会分别对两种情况的操作进行介绍： 1、托运方（发货方）未使用八万物流在线平台。 步骤一：物流商在登录进平台后，点击进入平台的收货管理-零担收货清单模块。 步骤二：点击收货管理-零担收货清单模块的新增承运单按钮。 步骤三：根据托运方留下的物流信息，填写收货凭证，把物流信息录入平台。 1、填写终到站信息，省-市-区/县 2、点击查看按钮，填写托运方信息 3、填写未注册公司信息，并点击保存按钮 4、点击查看按钮，填写提货方信息 5、若提货方未使用平台，填写未注册公司信息，并点击保存按钮；若使用了平台，可通过手机号码搜索到该公司，点击选定按钮 6、填写货物信息 7、填写费用信息，结算方式可供选择 8、填写其他注意事项 步骤四：单据填写完整后，点击保存按钮，保存单据。 步骤五：系统自动跳转至零担收货清单界面，这笔单据已经保存在零担收货系统的清单中，物流状态为：新增单。 步骤六：在零担收货系统的清单中，找到需要中转的单据，点击详单按钮查看收货凭证。 步骤七：在承运收货凭证中，点击增至零担中转按钮。 步骤八：点击进入平台的发货管理-零担中转模块。 步骤九：点击发货管理-零担中转模块的待托运中转按钮，进入托运发货清单。 步骤十：找到需要中转的单据，此时物流状态为待中转，点击详单按钮查看托运发货凭证。 步骤十一：托运发货凭证中核对物流信息，点击查看按钮选择所要中转的物流公司。 步骤十二：中转的物流公司若未使用平台，就填写未注册公司信息并点击白色保存按钮；若使用了平台，可以根据对方手机号码搜索到该物流公司并直接进行选定。 步骤十三：完成中转物流公司的选定后，点击零担中转按钮，进行中转。 步骤十四：点击确定按钮。 步骤十五：单据自动跳转到物流商的发货管理-零担中转模块下的待验货收货中，在托运发货清单里，可以看到需要中转的单据，点击详单按钮可以查看托运发货凭证，此时单据物流状态为待收货 步骤十六：选择的中转物流公司在平台上对中转的单据进行收货操作后，单据会自动跳转至物流商的发货管理-零担中转模块下的发货完成中，在其托运发货清单中有详细记录，此时物流状态为已完成，物流商的零担中转操作完成。 2、托运方（发货方）已经使用八万物流在线平台，并且在平台上发了一笔单据给物流商进行零担中转。 步骤一：物流商在登录进平台后，点击进入平台的收货管理-零担收货清单模块。 步骤二：点击收货管理-零担收货清单模块的待验货收货按钮，进入承运验货收货清单。 步骤三：查询到托运方需要进行零担中转的单据，此时物流状态为待收货，点击收货按钮。 步骤四：进行确认收货。
现代物流管理教材中习题参考答案 第一章物流发展与物流职场分析 一、单项选择题 1．现代物流产生哪个国家（）？ A.美国 B.日本 C.德国 D.英国 答案：A 2．以下哪一项不是现代物流特征（）。 A.系统化 B.成本最小化 C.一体化 D.网络化 答案：C 3．目前国际标准组织（ISO）制定的物流基础模数尺寸为（）。A.600mm×400mm。 B.300mm×200mm C.500mm×400mm D.600mm×600mm 答案：A 4．客户企业进行物流战略规划和物流体系构建，要求提供（）。A.一体化的物流服务和解决方案 B.稳定性的物流服务和解决方案C.最低成本的物流服务和解决方案 D.高效率的物流服务和解决方案答案：A 5．现代物流业需要的人才是（）。 A.通用性物流人才 B.专业性物流人才 C.效率性物流人才 D.专业性物流人才和通用性物流人才 答案：D
二、多项选择题 1．物流管理的职能包括（）。 A.计划 B.组织 C.领导 D.控制 E.安全 答案：ABCD 2．物流标准化管理包括（）。 A.技术标准化 B.工作标准化 C.服务标准化 D.管理标准化 E.领导标准化 答案：ABD 3．物流服务归纳起来包含了三个要素（）。 A.品质保证 B.技术保证 C.输送保证 D.信息保证 E.备货保证 答案：ACE 4．我国人力资源和社会保障部物流从业人员职业资格证书有（）。 A.物流师 B.高级物流师 C.物流员 D.助理物流员 E.助理物流师 答案：ABCE 5.提升物流职业能力路径有（）。 A.课程学习 B.仿真训练 C.社会实践 D.顶岗实习 E.毕业实习 答案：ABCDE 三、判断题 1．商流与物流合一效率更高。（X）
Windows7官方个版本正版镜像下载地址 简体中文旗舰版： 32位：下载地址：ed2k://|file|cn_windows_7_ultimate_x86_dvd_x15-65907.iso|2604238848|D6F139D7A45E81B 76199DDCCDDC4B509|/ SHA1：B589336602E3B7E134E222ED47FC94938B04354F 64位：下载地址：ed2k://|file|cn_windows_7_ultimate_x64_dvd_x15-66043.iso|3341268992|7DD7FA757CE6D2D B78B6901F81A6907A|/ SHA1：4A98A2F1ED794425674D04A37B70B9763522B0D4 简体中文专业版： 32位：下载地址：ed2k://|file|cn_windows_7_professional_x86_dvd_x15-65790.iso|2604238848|e812fbe758f 05b485c5a858c22060785|h=S5RNBL5JL5NRC3YMLDWIO75YY3UP4ET5|/ SHA1：EBD595C3099CCF57C6FF53810F73339835CFBB9D 64位：下载地址：ed2k://|file|cn_windows_7_professional_x64_dvd_x15-65791.iso|3341268992|3474800521d 169fbf3f5e527cd835156|h=TIYH37L3PBVMNCLT2EX5CSSEGXY6M47W|/ SHA1：5669A51195CD79D73CD18161D51E7E8D43DF53D1 简体中文家庭高级版： 32位：下载地址：ed2k://|file|cn_windows_7_home_premium_x86_dvd_x15-65717.iso|2604238848|98e1eb474f9 2343b06737f227665df1c|h=GZ7FZE7XURI5HNO2L7H45AGWNOLRLRUR|/ SHA1：CBA410DB30FA1561F874E1CC155E575F4A836B37 64位：下载地址：ed2k://|file|cn_windows_7_home_premium_x64_dvd_x15-65718.iso|3341268992|9f976045631 a6a2162abe32fc77c8acc|h=QQZ3UEERJOWWUEXOFTTLWD4JNL4YDLC6|/ SHA1：5566AB6F40B0689702F02DE15804BEB32832D6A6 简体中文企业版： 32位：下载地址：ed2k://|file|cn_windows_7_enterprise_x86_dvd_x15-70737.iso|2465783808|41ABFA74E5735 3B2F35BC33E56BD5202|/ SHA1：50F2900D293C8DF63A9D23125AFEEA7662FF9E54 64位：下载地址：ed2k://|file|cn_windows_7_enterprise_x64_dvd_x15-70741.iso|3203516416|876DCF115C2EE 28D74B178BE1A84AB3B|/ SHA1：EE20DAF2CDEDD71C374E241340DEB651728A69C4
十大物流仿真软件汇总 目录 (一) Flexsim (2) (二) RaLC(乐龙) (2) (三) Witness(SDX) (2) (四) Automod (3) (五) ShowFlow (3) (六) SIMAnimation (4) (七) Arena (4) (八) Supply chain guru (5) (九) Classwarehouse (5) (十) SimLab (5)
(一)Flexsim Flexsim是美国的三维物流仿真软件,能应用于系统建模、仿真以及实现业务流程可视化.Flexsim中的对象参数可以表示基本上所有的存在的实物对象,如机器装备、操作人员、传送带、叉车、仓库、集装箱等,同时数据信息可以用Flexsim丰富的模型库表示出来.Flexsim具有层次结构,可以使用继承来节省开发时间.而且它还是面向对象的开放式软件.对象、视窗、图形用户界面、菜单列表、对象参数等都是非常直观的.由于Flexsim的对象是开放的,所以这些对象可以在不同的用户、库和模型之间进行交换,再结合对象的高度可自定义性,可以大大提高建模的速度.Flexsim的用户性和可移植性扩展了对象和模型的生命周期. (二)RaLC(乐龙) RaLC(乐龙)软件,是上海乐龙人工智能软件有限公司(日本人工智能服务有限公司在华子公司)提供的.它是面向对象的,物流配送中心所使用的基本搬运器械设备即对象事物,包括各种传送带、自动立体仓库、平板车等,以及工作人员的装卸、分拣、叉车搬运等,全部以按钮的形式摆放在工具栏上,而且可以对对象物体的配置来进行设计,用于对各类对象物体的形状和规格建模也十分直观.RaLC 乐龙系列软件在建模速度、建模操作简便性、模拟和仿真精确度等方面都处于世界领先水平. (三)Witness(SDX) Witness(SDX)由英国Lanner集团用数十年系统仿真经验开发出的,它是面
大连电子学校 《现代物流基础》课程标准 一、基本信息 二、课程概述 （一）课程性质 物流一直是电子商务发展的瓶颈问题，《现代物流基础》是电子商务专业的一门职业技术能力课。通过本课程的教学，要求学生熟练地、系统地掌握现代物流管理基础知识、基本理论，掌握现代物流管理相关方法和技能，并能理论联系实际，培养学生的分析问题、判断问题和解决问题的能力，为以后从事电子商务工作打好基础。 （二）设计思路 现代物流基础课程是电子商务专业的基础课程，为学生讲授关于电子商务所涉及的物流相关方面知识，使学生了解什么是物流，并能够解决实际问题的能力，培养学生的独立解决问题的能力，为以后的学习和工作打下基础。 课程设计理念： （1）课程目标既要明确知识点，更要突出能力点。 （2）课程内容主要是“是什么”和“怎么样”。 （3）教学方法采用案例教学、情境教学和实践教学等手段，使学生在学习过程中做到动脑、动手、动口。 （4）在教学方法上，为探究式学习、合作式学习流出充分的时间。 三、课程目标与核心素养 （一）总体目标 通过对本课程的学习，学生将掌握物流管理的基本知识，熟悉这些知识在物流管理中的应用与发展；具备物流管理的基本技能，并能有效解决物流管理中的实际问题；具备搜集和处理信息的能力、获取新知识的能力、分析解决问题的能力以及归纳总结形成经险的能力；具有爱岗敬业、 吃苦耐劳和与他人合作的意志品质，逐步建立创业就业自信；具备探索未知的兴趣和意志力，养成崇尚科学的态度，增强社会责任感，逐步形成科学的世界观和正确的价值观。 （二）具体目标
1.知识目标 （1）掌握运输配送、储存保管、装卸搬运、现代包装、流通加工等物流基本业务 （2）基本掌握物流信息、物流系统、物流网络系统、企业物流 （3）初步会知第三方物流、城市物流、区域物流、国际物流、绿色物流 （4）了解消费者物流、物流政策、现代物流现代物流的基本知识、基本理论、基本技术和组织管理原则 2. 能力目标 （1）专业能力： 掌握现代物流理论与观念、运输配送、储存保管、装卸搬运、现代包装、流通加工等物流基本业务的作业，理解现代物流的基本知识、基本理论、基本技术和组织管理原则 （2）方法能力： 教材与案例分析相结合具有分析和解决的一些实际问题的能力，同时具有一定的学习能力。 （3）社会能力： 具有良好的社会公德和职业道德，有较强的社会主义民主和法制观念；具有良好的人际交流能力，团队合作精神和客户服务意识；取得相应物流专业认证资格，具有一定的就业竞争力。 3. 素养目标 （1）培养学生独立思考、自我学习、勇于表达自己见解的能力。 （2）培养学生从实际出发分析问题、解决问题的能力。 （3）培养学生的团队意识与合作能力。 （4）培养学生良好的职业道德，诚实守信、爱岗敬业、严谨的工作作风、较强的责任心和吃苦耐劳的职业精神。 （5）培养学生较强的适应性与灵活性和自我调控能力。 （6）培养学生较强的适应性与灵活性和自我调控能力。 四、课程内容与参考学时 本课程共安排日本制图基础知识、投影法、图形的表示方法、尺寸的表示方法、装配图的制定方法等共5个教学单元。课程内容及要求的详细情况见表1。 表1 课程内容及要求