the human intestinal microbiota is a complex community composed of at least several hundred different species of bacteria with approximately 1010 cells per gram of feces     [ 14 ] .
the intestinal microbiota plays a critical role in human health including colonization resistance , nutrition , metabolism of nondigestible dietary components and xenobiotics , proliferation and differentiation of intestinal mucosal epithelial cells , and homeostasis of the immune system     [ 57 ] .
direct analysis of the intestinal microbiota in the human colon is inherently difficult for routine experiments .
therefore , most studies are conducted with human fecal specimens , animal models , in vitro batch culture , and continuous culture systems that mimic the human gastrointestinal tract .
however , recently , a study using high - throughput anaerobic culture techniques reported that 56% of human fecal microbiota belongs to readily cultured species , over 40% of gut microbiota was uncultured species to date     [ 3 , 8 , 9 ] .
one of the reasons for this limitation generates from the difficulty of providing all of the appropriate nutrients and conditions for growth of the complex intestinal microbiota community . therefore , research to provide more information on in vitro culture conditions as the study by goodman et al
.   would enhance the evaluation of perturbation of the intestinal microbiota by factors that might adversely affect human health .  
molecular techniques that target the 16s rrna gene and other genetic markers have been used to analyze microbial community ecology in the human intestine .
denaturing gradient gel electrophoresis ( dgge ) has been used to monitor differences and changes in the overall microbial community from fecal samples     [ 1012 ] , and quantitative real - time pcr has provided numerical abundance data for fecal microbiota     [ 13 , 14 ] .
recently , application of high - throughput techniques such as pyrosequencing and the human intestinal tract chip ( hitchip ) microarray have been used to obtain deep phylogenetic analysis of intestinal microbiota     [ 12 , 1517 ] . in the present study , dgge , real - time pcr , and pyrosequencing
were used to profile the abundance and diversity of the bacterial community from human fecal inoculum grown under different culture conditions .   the aim of this study was to compare various batch culture conditions for activating and maintaining a complex fecal microbiota community to mimic growth conditions of the gastrointestinal tract .
the culture conditions developed in this investigation can be applied for future research to determine the impact of antimicrobial agents , food contaminants , xenobiotics , probiotics , and dietary supplements on the human intestinal microbiota .
each fecal sample was coded individual a , b , c , and d.   fecal samples were cultured in brain heart infusion ( bhi ) broth , modified high - concentration carbohydrate medium ( hcm ) , or low - concentration carbohydrate medium ( lcm )     [ 18 , 19 ] . the composition of high- and low - carbohydrate media is described in table 1 .
feces were diluted with anaerobic maximum recovery diluent ( mrd ; labm idg , bury , uk ) buffer to a final concentration of 25% ( w / v ) . to compare
the difference of microbiota grown in three media , fecal suspensions were diluted to give an inoculum concentration of 1% ( w / v ) , then inoculated in each medium ( 10  ml of final volume ) , and cultured anaerobically at 37c .
the growth was analyzed by optical density ( od ) and flow cytometry on an accuri c6 fcm ( accuri cytometers , ann arbor , mich , usa ) following the manufacturers instruction with collected samples at each time point . to determine the optimal incubation time and check the metabolic activity of the microbiota , 1% fecal suspension
decolorization of gentian violet indicates the metabolic activity of fecal microbiota     [ 20 , 21 ] .
fecal supernatants were assessed as medium supplements to determine whether unknown growth factors affect in vitro growth of intestinal microbiota .
individual fecal supernatant was prepared from 25% diluted fecal specimens with anaerobic mrd buffer after centrifugation at 11,000  rpm for 30 minutes .
autoclaved fecal supernatant was added to each medium with a final concentration 1% ( v / v ) .
the optimal fecal inoculum concentration was also determined by the inoculating 0.1 to 5% of inoculum to low carbohydrate medium .
the growth of each inoculum was analyzed by optical density at 600  nm and by flow cytometry ( fcm ) .
genomic dna was extracted from 1  ml of samples at each time point using the dna elution accessory kit of the rna power soil total rna isolation kit ( mobio laboratories , carlsbad , calif , usa ) by following the manufacturer 's protocol .
preliminary experiments showed that this kit had the best extraction efficiency ( produce high concentration of dna from same amount of fecal material ) among several kits ( data not shown ) .
to conduct dgge analysis , 16s rrna gene fragments of the v3 region were amplified using primers gc - clamp-340f ( 5-tcc tac ggg agg cag cag-3 ) and 518r ( 5-att acc gcg gct gct gg-3 ) as described     [ 22 , 23 ] . the pcr reaction was performed using a mastercycler gradient instrument ( eppendorf , hauppauge , ny , usa ) , in a final volume of 50  l with 10x taq buffer , dntp mixture ( takara , shiga , japan ) , 10  m of each primer ( mwg - biotech , ebersberg , germany ) , 2  u of taq polymerase ( ex taq ; takara ) , and 1  l of template .
after initial denaturation at 94c for 5 minutes , amplification consisted of 30 cycles of denaturation ( 30 seconds , 94c ) , primer annealing ( 30 seconds , 55c ) , and primer extension ( 30 seconds , 72c ) and a final extension step of 7 minutes at 72c .
the pcr product was checked by using 2% agarose gel electrophoresis and visualized using a gel doc system ( biorad , hercules , calif , usa ) .
pcr products were concentrated and purified with the qiaquick pcr purification kit ( qiagen inc . , valencia , calif , usa ) .   
dgge was conducted using a d - code system ( biorad ) with 8% ( w / v ) polyacrylamide gels contained 40%65% denaturant gradient , 1  mm thick , in 1x tae buffer .
the equal amounts of purified pcr products were loaded on gel , and electrophoresis was performed at 25  v for 15 minutes then at 70  v for 16 hours and 30 minutes at 60c .
the gel was stained in 250  ml of running buffer containing ethidium bromide ( 50  g  ml ) for 15 minutes and then rinsed in 250  ml of running buffer for 20 minutes .
the sequences were identified using blast search on the genbank database and the database of type strains at eztaxon server     .
the profile of dgge gel was analyzed with the bionumerics program , version 6.0 ( applied maths , st .- martens - latem , belgium ) .
cluster analysis of the band pattern was performed using the unweighted pair group method using arithmetic averages ( upgma ) and the similarity between lanes was calculated based on the band position .
the dice coefficient was used to create dendrograms of the dgge profiles obtained from different samples .  
real - time pcr were performed in a final 25  l volume containing 12.5  l of 2x iq sybr green supermix ( biorad ) , 10  m of each primer ( mwg - biotech ) , 1  l of template dna ( tenfold dilution series of standard and samples dna ) or distilled water ( negative control ) .
bact349f ( 5-agg cag cag tdr gga at-3 ) and bact518r ( 5-att acc gcg gct gct gg-3 ) were used to quantify total bacteria , btr275f ( 5-cga tgg ata ggg gtt ctg-3 ) and btr555r ( 5-ccc ttt aaa ccc aat raw tcc gg-3 ) were used for bacteroidetes , firm350f ( 5-ggc agc agt rgg gaa tct tc-3 ) and firm814r ( 5-aca cyt agy act cat cgt tt-3 ) were for firmicutes     [ 2628 ] . the quantifications were performed with three independent real - time pcr runs using the cfx96 real - time pcr detection system ( biorad ) , associated with cfx manager interface software ( version 1.0.1035.131 ; biorad ) .
the amplifications were carried out with the following steps : 50c for 2 minutes , 95c for 10 minutes , and 40 cycles of 95c for 10 seconds and 60c for 30 seconds .
melting curve data were obtained from 60c to 95c at a rate of 0.5c  sec with continuous measurements of the sybr green i signal intensities .
dnas from cultures of escherichia coli atcc25922 , bacteroides eggerthii atcc27754 , and clostridium butyricum atcc19398 were used to construct standard curves for quantification by plotting the ct values obtained from amplification of dilution series .   for pyrosequencing , amplification of genomic dna was performed using barcoded primers , which targeted the v1 to v3 region of the bacterial 16s rrna gene .
the amplification , sequencing , and basic analysis were performed according to the methods described by chun et al .      and completed by chunlab inc .
( seoul , korea ) using a 454 gs flx titanium sequencing system ( roche , branford , conn , usa ) .
briefly , analyzed sequencing reads of each sample were separated by unique barcode and filtered to remove reads , which was shorter than   300  bp or the average quality score of read was less than 25 or containing 2 more ambiguous nucleotides ( ns ) , and then removed chimera products for further analyses     [ 29 , 30 ] .
the extended eztaxon database ( http://www.eztaxon-e.org/ ) , which contains representative sequences of both cultured and uncultured bacteria with hierarchical taxonomic classification , was used for taxonomic assignments .
the pyrosequencing reads were compared with sequences in the eztaxon - e database using blastn search and obtained similarity using pairwise comparison , and then the sequences were assigned a taxonomic classification through using the criteria of   97% sequence identity for species , 94% identity for genus , 90% identity for family , 85% identify for order , 80% identity for class , and 75% identity for phylum .
if the sequence identity was below the cutoff value , the sequence was assigned to the  unclassified  group at each phylogenetic level .
the diversity index and statistical analysis were performed using mothur program with the cutoff value of 97% similarity for assigning phylotypes     .  
bacterial sequences from excised dgge bands were submitted to the genbank database under accession numbers from hq645054 to hq645071 .
the sequence reads from pyrosequencing are available in the embl sra database under the study accession number erp000433 ( http://www.ebi.ac.uk/ena/data/view/erp000433 ) .  
brain heart infusion ( bhi ) , low - concentration carbohydrate ( lcm ) , and high - concentration carbohydrate media ( hcm ) were used for intestinal microbiota growth .
previous studies used hcm in human intestinal continuous culture     [ 18 , 19 ] . however , the digestible carbohydrate concentrations in the large intestine are lower than carbohydrate concentration in high carbohydrate medium     . therefore , we wanted to compare hcm , lcm , and bhi media under the same inoculum and growth conditions
. diluted feces ( 1% ) were inoculated in the different media , and the intestinal microbiota growth was analyzed by spectrophotometer and quantitative real - time pcr ( supplementary figure  1 available at doi : 10.1155/2011/838040 ) .
the growth of intestinal microbiota showed maximum od at 18 hours in lcm and hcm , while the maximum in bhi medium was earlier in the incubation period .  
the 16s rrna genes of cultured bacteria in each medium increased over the incubation period .
the cell number of inoculum was 4.8  10  cells / ml ( mean value of cell numbers in inoculum of three media ) .
the highest cell number was detected at 18 hours in lcm ( 1.85  10  cells / ml ) and hcm ( 1.19  10  cells / ml ) , whereas bhi reached maximum cell numbers ( 1.64  10  cells / ml ) after 18 hours . to determine the metabolic activity of cultured bacteria
, the fecal microbiota cultures were dosed with gentian violet , and the activity was monitored by measuring color disappearance with time .
the microbiota completely decolorized gentian violet after 18 hours of incubation ( supplementary figure  2 ) .
eighteen hours was chosen , because the residence time of readily digestible compounds in intestinal tract is generally within a day .
the growth of intestinal microbiota in different media showed similar maximum od and the total bacterial 16s rrna gene increased over time ( supplementary figure  1 ) .
however , this result did not correlate with the cell numbers determined by flow cytometry .
this difference was most likely caused by the difference of rrna gene copy numbers in each species .  
dgge fingerprinting was used to evaluate the ability of each medium to maintain the initial fecal microbiota .
the dgge banding patterns derived from the initial cultures were compared to those from the 18 hours cultures , and similarity between inoculum and cultured sample was used as the measure of microbiota stability .
overall , the number of bands and the dominant bands were different in each medium ( figure 1(a ) ) .
the band numbers from the in vitro cultures were fewer than the fecal inoculum and formed different profiles from the inoculum on the dgge gel .
the sequences of bands were assigned to the firmicutes , bacteroidetes , and actinobacteria phyla .
firmicutes and bacteroidetes were major phyla in both the fecal inoculum and the in vitro culture .
bands affiliated to bacteroidetes were more dominant in the in vitro culture after 18 hours ( band number 1 , 6 , and 11 ) than at zero time .
the dominant firmicutes bands ( band 2 , 4 , 5 , 10 , and 13 ) at zero time were less dominant in cultured samples .
this result was supported by previous studies that the ratio of bacteroidetes / firmicutes was different between the in vitro intestinal model and the inoculum     [ 12 , 34 ] .
band number 5 contained pairs of dna fragments , because similar sequences had similar denaturant gradient and the length of amplified fragment for dgge ( ranged from 150 to 180  bp ) was insufficient to distinguish similar sequences completely .
bhi and lcm had relatively similar numbers of bands ( 2022 bands ) and hcm had fewer bands ( 14 bands ) .
the lower number of bands on hcm may be due to the high carbohydrate content in the medium .
high carbohydrate promotes the proliferation of bacteroidetes , which possess a larger glycobiome than firmicutes     [ 34 , 35 ] .
cluster analysis showed that the microbiota of lcm was similar to inoculum population ( figure 1(a ) ) .
although the number of bands in bhi medium was similar to that of lcm , their profiles and dominant bands were more different from the inoculum than those in lcm .
moreover , the profile of minor bands in lcm was similar to the profile of inoculum , and the number of minor bands was more abundant than bhi medium .
profiles of lcm after 18 hours displayed the highest similarity ( 78.94% ; mean value of triplicate samples ) with profiles of original inoculum at zero time .
therefore , the lcm medium was used as basal medium for intestinal microbiota growth culture conditions .  
furthermore , they are unique in each individual because of interindividual differences of intestinal microbiota population , dietary habits , and metabolism     .
three different culture conditions ( 1% of fecal inoculum , 1% of fecal inoculum with 1% fecal supernatant , or 2% fecal inoculum ) were compared using dgge profile ( figure 1(b ) ) and revealed relatively similar band patterns . however , the inoculum with 1% fecal supernatant added to lcm was more similar to original inoculum than cultures without fecal supernatant in population cluster analysis ( 80.37% similarity ) .
the inoculum concentration of feces is a significant factor for in vitro culture conditions , because the number and diversity of bacteria can affect growth .
therefore , we determined using dgge and real - time pcr , the optimal inoculum concentration for use in this type of in vitro human fecal culture experiments .
different fecal inoculum suspensions ( 0.1% , 0.5% , 1% , 2% , 3% , 4% , and 5% ) were used to compare the bacterial communities at each concentration .
we did not test concentrations over 5% because of the difficulty with handling the dense and viscous fecal samples .
the dgge profiles of different inoculum concentrations were relatively similar to each other and as expected the interindividual variation of microbial community was found ( figure 2 ) .
although profiles were relatively similar among different inoculum concentrations , small variations of band intensity were observed .
cluster analysis of profiles showed that 1% , 2% , or 3% inoculum cultures were the most similar to original inoculum in individual b and c ( figure 2 ) .
we investigated the change of bacterial communities at each incubation time ( supplementary figure  3 ) .
the communities of original fecal bacteria were similar to those of inoculum and the profiles of bacterial communities were stable after 6 hours of incubation in every inoculum concentration .
in addition , the batch culture was reproducible , as determined by the cluster analysis of triplicate cultures in dgge analysis ( data not shown ) .   the real - time pcr - based quantification
was used to enumerate total bacteria , bacteroidetes and firmicutes in cultures with 1%5% inoculum concentrations of individual - coded a , b , and c samples at 0 and 18 hours of incubation ( table 3 ) .
different numbers of bacteria , bacteroidetes and firmicutes were found in each sample at the varied inoculum concentrations , and their growth was different in the same medium .
the 16s rrna gene copy number of total bacteria increased to 10  copies  ml for all cultured samples , and bacteroidetes and firmicutes reached to 1010  copies  ml .
the 16s rrna gene copies of total bacteria and bacteroidetes in cultured fecal materials from individual c were higher than individual a and b. firmicutes were more abundant in 18 hour cultured samples of individual b than individuals a and c. these results indicated that the different community composition affected the growth of each phylum in fecal microbiota . the increased ratio of bacteroidetes ( 2.29 fold ; mean value of increased copy number ratio ) was more abundant than that of firmicutes ( 1.90 fold ) .
we compared the 16s rrna gene copies of total bacteria in 0.1%3% inoculum cultures of individual d ( supplementary figure  4 ) over time .
the increased numbers of total bacterial 16s rrna gene copies were higher in low concentration of inoculum ( 0.1% and 0.5% ) than the high concentration of inoculum ( 1%3% ) .
the batch culture with higher cell numbers has limited nutrients and there would be more competition for obtaining nutrients .
however , the high concentration of inoculum ( 1%3% ) added to the batch culture could provide more fecal material to facilitate and personalize the cultivation of indigenous microbiota as fecal supernatant     [ 3638 ] .
therefore , 3% inoculum concentration of fecal materials was chosen for in vitro culture conditions since this level would maintain a high cell number of intestinal microbiota and grow a variety of indigenous microbiota . a comparison of the intestinal microbiota of each individual fecal sample before and after culturing was determined by pyrosequencing .
a total of 45,674 reads were obtained from pyrosequencing and 5,843 sequences were removed by filtering process of chimera check , length cutoff , ambiguous base call and average quality check .
therefore , a total of 39,831 sequences were analyzed ( ranging from 3,402 to 8,898 per sample ) after the filtering process ( table 4 ) .
the average length of sequences was 385.39  bp and the observed number of phylotypes ranged from 590 to 1,287 with 92% to 97% good 's coverage .
the richness of samples was investigated by rarefaction curves ( supplementary figure  5 ) .
the changed numbers of observed phylotype and diversity indices of samples from three individuals were different at zero time and after 18 hours of incubation .
samples from individual b had the most similar observed number of phylotypes and shannon indices between the zero time culture and the 18 hour culture among the three individuals .    
the dominant phyla ( firmicutes , bacteroidetes , actinobacteria , proteobacteria , and verrucomicrobia ) from fecal samples of each person were maintained in improved culture condition of this study ( figure 3 ) .
the abundance of firmicutes decreased after 18 hours ( average from 72.39% to 44.95% ) , while bacteroidetes increased from 17.14% to 39.11% in cultured samples .
although the proportion of each phylum was changed in the in vitro cultures , the dominant phyla were maintained after 18 hours of incubation .
these trends are similar to those seen in a previous gut model system analyzed using phylogenetic microarray .
they reported that the abundance of bacteroidetes increased from 52.49% to 75.50% ( ascending model ) , 80.59% ( transverse model ) and 75.60% ( descending model ) , while the firmicutes decreased from 44.57% to 16.81% ( ascending ) , 10.56% ( transverse ) , and 13.23% ( descending ) .
the abundances of actinobacteria ( average 4.91% ) and verrucomicrobia ( 0.21% ) in the present study were higher than observed in the previous model system .   at the genus level ,
a total of 210 genera ( read number 0.01% of total analyzed reads ) were retrieved from the zero time fecal cultures of individual a , b , and c , and 173 genera were obtained from the 18 hour cultured samples ( figure 3 ) .
the dominant genera were bacteroides , subdoligranulum , faecalibacterium , parabacteroides , bifidobacterium , ruminococcus , eubacterium , blautia , roseburia , alistipes , clostridium , escherichia , and dorea ( read number 1% of total analyzed reads ) .
the community profiles of the microbiota from individuals a and c were more similar to each other than to that for individual b , both at zero time and after the 18 hour incubation .
therefore , the bacterial community of each in vitro cultured sample reflected the interindividual uniqueness of the fecal microbiota .
we tested a variety of conditions for the human intestinal microbiota growth in short - term in vitro batch cultures .
the combination of dgge , real - time pcr , and pyrosequencing was sufficient to compare communities of intestinal microbiota in the different cultures .
of the combinations tested , low - concentration carbohydrate medium ( lcm ) supplemented with 1% fecal supernatant and inoculated with a fecal suspension to a final concentration of 3% performed best in maintaining a metabolically active diverse population of bacteria over the 18 hour incubation .
the culture conditions developed in this investigation should be suitable for use in future studies on the impact of xenobiotics on the human intestinal microbiota .
 a stable intestinal microbiota is important in maintaining human physiology and health . 
 although there have been a number of studies using in vitro and in vivo approaches to determine the impact of diet and xenobiotics on intestinal microbiota , there is no consensus for the best in vitro culture conditions for growth of the human gastrointestinal microbiota . to investigate the dynamics and activities of intestinal microbiota , it is important for the culture conditions to support the growth of a wide range of intestinal bacteria and maintain a complex microbial community representative of the human gastrointestinal tract . here , we compared the bacterial community in three culture media : brain heart infusion broth and high- and low - carbohydrate medium with different growth supplements . 
 the bacterial community was analyzed using denaturing gradient gel electrophoresis ( dgge ) , pyrosequencing and real - time pcr . 
 based on the molecular analysis , this study indicated that the 3% fecal inoculum in low - concentration carbohydrate medium with 1% autoclaved fecal supernatant provided enhanced growth conditions to conduct in vitro studies representative of the human intestinal microbiota . 
1. Introduction
2. Material and Methods
3. Results and Discussion
4. Conclusions