BACKGROUND
the high demand for ethanol in the u.s. has generated large stocks of wet distillers grains  derived as a byproduct from the manufacture of ethanol from corn and sorghum grains.
ethanol production is expected to increase several fold due to the high demand and cost of foreign oil  <cit> .
energy and protein dense dgs are attractive for use as a feed for beef cattle finishing diets; however little is known about the potential influence of dietary dg on fecal microbial community structure.
a better understanding of the microbial population in beef cattle feces could be important in improving nutrient management, increasing animal growth performance, and decreasing odors and/or shedding of pathogens.
a variety of emissions such as ammonia, volatile fatty acids, and hundreds of volatile organic compounds  <cit>  have been tied to beef cattle manure .
volatilization of ammonia has been linked to crude protein content in the diet fed and increased amounts of excreted urinary n  <cit> .
previous studies suggested an association between dried distillers grains  feeding and an increased prevalence and fecal shedding of the foodborne pathogen escherichia coli o157:h <dig> in cattle  <cit> .
a small number of studies have used culture-independent 16s rrna-based  <cit>  and culture-dependent 16s rrna-based methods with dairy cattle feces  <cit> .
clostridium spp were identified as the most dominant taxa across all lactating dairy cows  followed by bacteroides spp  using the culture-independent approach  <cit> .
in this study of holstein dairy cows ,  <dig> different bacterial species were detected corresponding to  <dig> separate genera  <cit> .
several thousand sequences were obtained per sample enabling the detection of populations below  <dig> % abundance.
using culture-dependent methods, a total of  <dig> 16s rrna clones were obtained from three holstein steers and classified at the 98% sequence similarity level  <cit> .
the dominant phyla observed were: firmicutes , bacteroidetes , actinobacteria , and proteobacteria .
a comparison of dairy cattle fed a control diet or fed a diet supplemented with monensin using the culture-dependent 16s rrna method returned  <dig>  16s rrna genes  <cit> .
nearly equivalent abundance levels of firmicutes  and bacteroidetes  were observed across the six lactating holstein cows with proteobacteria comprising the next most abundant group .
culture-dependent and culture-independent 16s rrna methods were also applied with studies involving beef cattle  <cit> .
utilizing classical full length 16s rrna gene sequence analysis a total of  <dig>  otus  were identified from six cattle  <cit> .
a core set of phyla were observed based on  <dig> otus comprised of  <dig>  sequences  with  <dig>  otus found only in individual libraries.
seven phyla were found within six animals with three dominant taxonomic groups; firmicutes, , bacteroidetes  and proteobacteria .
in another small study of beef cattle  the dna pyrosequencing method was applied to the comparison of the effects of three diets on ruminal  and fecal  bacterial assemblages  <cit> .
three diets  in which of  <dig>   <dig>  or 50% of the concentrate portion of the diet was replaced with dried distillers grains  plus solubles were compared.
over  <dig> different bacterial species were detected that belonged to  <dig> separate genera from ruminal samples across all three diets.
in all fecal samples, more than  <dig> different bacterial species were detected corresponding to  <dig> separate genera.
the  <dig> most common genera that accounted for over 85% of the ruminal and fecal bacterial populations were identified.
the firmicutes: bacteroidetes ratio tended to decrease as the proportion of ddgs increased.
in a much larger study involving  <dig> cattle distributed across geographically different locations and six different feeding operations  the dna pyrosequencing method  was used to assess fecal microbial community assemblages  <cit> .
the majority of sequences were distributed across four phyla: firmicutes , bacteroidetes , tenericutes , and proteobacteria .
core taxa were observed across  <dig> different phyla: actinobacteria , bacteroidetes , cyanobacteria , firmicutes , and tenericutes .
using sequence-based clustering and taxonomic analyses, less variability was observed within a particular management practice/location than among different management practices.
animal feeding operations seemed to influence bovine fecal bacterial communities at the phylum and family taxonomic levels much more so than geographic location of the feedlot.
lastly, overall bacterial community composition seemed to be strongly influenced by fecal starch concentrations.
the most responsive phyla to diet were bacteroidetes, firmicutes and proteobacteria.
the relative abundance of bacteroidetes increased with increasing fecal starch concentration, whereas, the abundance of firmicutes decreased with increasing fecal starch concentrations.
in the present study, we used the barcode dna pyrosequencing technique to evaluate the influence of five beef cattle diets on fecal microbial assemblages.
the diets consisted of a traditional diet feed beef cattle in the southern high plains of texas-con , and four diets containing different percentages of dgs in the dietary dry matter;  <dig> c , 5s , 10s , and 15s .
the barcoded dna pyrosequencing method was used to generate 16s otus dataset.
the 16s otus dataset was assigned to various taxonomic classes and each phylogenetic level was analyzed using a variety of statistical tests including unifrac procedures, hierarchal cluster analysis, distance based redundancy analysis , and one-way anova to test the influence of dietary treatments on microbial populations.
we describe significant changes in microbial community structure and diversity that is influenced by these different dgs diets.
RESULTS
general dna sequencing observations
a total of  <dig>  high quality 16s otus were utilized in the analysis .
the total number of high quality 16s otus recovered from each animal is listed in table  <dig>  the average number of otus returned for each diet was: con, 6613;  <dig> c, 6836; 5s, 6042; 10s, 5977; and 15s,  <dig>  rarefaction curves indicated that a high level of microbial diversity was obtained for subsequent analysis of dietary treatments .
in general, no treatment was associated with a loss of sample size for subsequent evaluation of populations across treatments.
the total abundance observed for otus and their associated centroids distributed across treatments are indicated in box plots depicting beta diversity .
the highest abundance was observed in the  <dig> c diet followed closely by the 10s and 15s diets.
the highest animal to animal variation was observed in the 5s diet followed closely by the control diet.
in general, abundance ranges for the diets and their associated centroids were more tightly grouped with the 10s and 15s diets.
the dietary treatment, animal id, and no.
of otus obtained per fecal grab from each animal
the relationship among treatments is indicated in whittaker plots  with each dot representing a species .
the left and top of the graph indicate the presence of the most abundant otus with the bottom and right indicating the occurrence of rare otus.
each dot represents one species and the high steepness of the graph is indicative of unevenly distributed species.
the lengths of the curves also indicate the occurrence of rare otus.
the curves generally overlap one another in this analysis for all dietary treatments; thus, overall microbial diversity were similar.
influence of dgs on fecal microbiota-phyla
four phyla were observed to have a response to dietary treatments .
these are synergistetes , ws <dig> , actinobacteria , and spirochaetes .
a total of  <dig> phyla were observed distributed amongst all beef cattle on all diets .
these are listed in order of average abundance and with their respective ranges : firmicutes , bacteroidetes , spirochaetes , proteobacteria , verrucomicrobia , fibrobacteres , tm <dig> , tenericutes , nitrospirae , actinobacteria , and fusobacteria .
chlamydiae, cyanobacteria, planctomycetes, synergistetes, lentisphaerae, acidobacteria, elusimicrobia, chlorobi, ws <dig>  deinococcus-thermus, chloroflexi, gemmatimonadetes, and deferribacteres were defined as low abundance phyla.
greater than  <dig> % of total bacterial abundance was observed in the first  <dig> phyla, with several remaining phyla represented by  <dig> or less members.
the abundance levels of the top ten phyla averaged based on dietary treatment are presented in figure 3b.
a higher relative abundance of firmicutes was observed when compared to the relative abundance level of bacteroidetes for dgs diets that contain 10% or more dg supplement vs. the con and 5s diets.
however, significant differences were not observed among dietary treatments for abundances of firmicutes  and bacteroidetes  .
a matched pair comparison evaluation of the abundances of firmicutes to bacteroidetes to one another yielded a non-significant response .
a core set of six phyla were observed in all animals regardless of dietary treatment, and they were; firmicutes, bacteroidetes, proteobacteria, tenericutes, nitrospirae, and fusobacteria.
with the exception of one animal  that lacked spirochaetes, seven phyla would have been observed.
a. the distribution of major phyla  based on bacterial counts among  <dig> beef cattle feed five diets.
b. distribution of the most abundant phyla averaged across the dietary treatments.
con = control,  <dig> c = 10% corn, 5s = 5% sorghum, 10s = 10% sorghum, 15s = 15% sorghum.
distribution of bacterial class, order and families by treatment
the response of the most abundant bacteria at the phylogenetic levels of class, order and family is revealed in a series of heat maps  and, for further clarification,  in abundance plots showing both the individual animal response to diet and the averaged response to diet.
for clarity and visualization purposes only the top  <dig> bacterial orders  and the top  <dig> bacterial families  are presented in heat maps.
for corresponding abundance plots, the cutoffs are at the 97-99% abundance levels and orders and families are presented .
with respect to abundance levels of clostridia, bacteroidia, and gammaproteobacteria, animal  <dig> microbial community was the most disparate from all the other animals.
the relative abundance of clostridia was substantially lower and the relative abundance of bacteroidia and gammaproteobacteria were greater .
this effect is expressed at the phylogenetic level of bacterial orders with lower clostridiales and greater bacteroidales and enterobacteriales  down to the level of families with lower abundances of ruminococcaceae and clostridiaceae and greater levels of prevotella .
other animals appeared to be variable with respect to one or two other taxa such as number  <dig>   <dig>  and  <dig> when viewing patterns observed on the heat maps .
influence of dgs on fecal microbiota-genera
a total of  <dig> bacterial species were observed distributed among  <dig> genera across all fecal samples .
a double hierarchal dendrogram was constructed using the upgma clustering method and manhattan distance method with no scaling .
the influence of dg diets on the fecal microbiome was apparent from double hierarchal cluster analysis on the top  <dig> most abundant genera  and clustered by dietary treatment .
with respect to diets, the least apparent phylogenetic distance  observed within the top cluster was with the  <dig> c diet  and the most was with the 5s diets .
prevotella and clostridium occurred together in their own separate cluster, whereas oscillospira, bacteroides, ruminococcus, eubacterium, and oscillibacter resided in the next most distant cluster.
the other  <dig> genera cohabited in another main cluster.
for animal  <dig> the microbial community seemed to be most unlike the other animals and this was apparently a result of a high relative abundance of bacteroidetes and a low relative abundance of firmicutes .
the average abundance by treatment of the top  <dig> genera  and the response of taxa to diet  are presented in additional file 12: table s <dig>  in brief, those taxa that had a treatment response were: clostridium, ruminococcus, oscillibacter, tannerella, parabacteroides, hydrogenoanaerobacterium, pseudoflavonifractor, acetivibrio, ethanoligenens, selenomonas, desulfonispora, and barnesiella.
the top  <dig> species comprised approximately 91% of the total abundance observed  and the following also show a significant response to treatment as detailed above.
these are: clostridium sp., tannerella sp., pseudoflavonifractor capillosus, catabacter sp., hydrogenoanaerobacterium saccharovorans, ruminococcus bromii, and parabacteroides merdae.
a biplot based on dbrda using the unweighted unifrac method identified taxa  that were significantly affected by diets, p =  <dig>  .
taxa most influenced by diet listed alphabetically were: akkermansia, clostridium, escherichia, eubacterium, oscillibacter, oscillospira, prevotella, ruminococcus, tannerella, and treponema.
in figure  <dig> the length and direction of the arrow  with respect to diets indicates their relative positive or negative relationship to that diet.
the ellipses around the animals represent the 95% confidence level, and their distance from one another reflects how closely or distantly the dietary effects are related to one another.
it can be seen that akkermansia, escherichia, and treponema were positively influenced by the 5s and con diets, whereas the  <dig> c is situated to the lower right hand side of the figure indicating a weak response from oscillibacter.
the fecal community associated with the10s dietary treatment was least similar to the other fecal communities.
a moderate influence of the 10s was observed for eubacterium and tannerella, whereas the 15s diet was near the point source eliciting a response from clostridium and oscillospira.
the relative abundance of prevotella seems to be positively influenced by the 5s and con treatments since these diets are located on the lower axis  <dig>  when analyzed using weighted unifrac procedure a significant  but slightly different result was observed regarding the influence of diets on microbial assemblages .
it can be seen that akkermansia and treponema relative abundance were positively influenced by the con diet, whereas, escherichia was orientated at nearly 180° from these two taxa, and was more abundant in the 5s and 15s diets .
eubacterium also had a similar response.
prevotella was oriented to the bottom left hand side of the figure, but it was much more in alignment with escherichia.
discussion
influence of distillers grain diets
deep sequencing of  <dig> individual fecal samples from cattle fed five different diets  provides a detailed view of the beef cattle fecal microbiome.
the barcoded dna pyrosequencing method yielded  <dig>  high quality reads for microbiome comparison.
we detected a core set of six bacterial phyla distributed across all animal fecal samples from all diets.
in addition, we identified a total of  <dig> phyla distributed across a number of the fecal samples associated with the various diets that encompass  <dig> bacterial species distributed across  <dig> genera.
we identified four phyla that were responsive to dietary treatments.
these were synergistetes , ws <dig> , actinobacteria , and spirochaetes .
we also documented  <dig> genera and  <dig> species that responded to dietary treatments.
it can be difficult to make comparisons across these various cattle fecal studies since they have employed a variety of 16s rrna-based sequencing strategies , the number and type of cattle employed in the studies and the types of diets and management practices associated with these diets.
short read lengths and potential biases in evenness  due to primer and template mismatches can result in pyro-sequencing artifacts that potentially affect taxonomic assignment and richness estimates  <cit> .
this is especially so with respect to rare otus.
questions have also been posed and examined regarding the influence of geographical location, climatic conditions, and other localized environmental variables on cattle fecal microbial community structure  <cit> .
animal to animal variation was noted in fecal microbial diversity among beef cattle after controlling for location, climate, animal genetics, and diet  <cit> .
both the number and relative abundance of phyla we observed agree more closely with the distribution of phyla observed in the shanks et al.
<cit>  study than in the callaway et al.
study  <cit> .
this could have been due to the number of cattle in the study  or the size of the 16s otus dataset that was assembled .
both pyrosequencing studies  <cit>  employed different primer locations and different read lengths to generate their datasets.
the v <dig> region was specifically targeted in the shanks study and used short read lengths , whereas that of callaway targeted the v4-v <dig> region .
thus, of the studies described in detail  <cit> , our results generally agree more closely with the findings of shanks and durso, despite using the methodology described by dowd  <cit>  and employed by callaway  <cit> .
one possible explanation is that our choice of primers targeted the v <dig> through v <dig> region of the 16s rrna gene whereas the primer set utilized in the callaway study used the v <dig> to v <dig> region to assess phylogenetic information.
another difference is that all of the cattle in the dowd study  <cit>  were lactating holstein dairy cows and for the callaway study  <cit>  they were jersey dairy cows and angus steers.
a number of taxa appear to fluctuate in response to diets.
two taxa, ruminococcaceae and prevotella spp., had distinct patterns in response to dietary treatments, whereas, the majority of  <dig> taxa identified did not fluctuate across different dietary practices  <cit> .
other taxa identified in this study as being influenced by dietary treatment based on the unifrac procedure were; akkermansia, clostridium, escherichia, eubacterium, oscillibacter, oscillospira, prevotella, ruminococcus, tannerella, and treponema.
two of these, prevotella and ruminococcus, were among those identified by shanks  <cit> .
we noted the presence of phyla in our study that were also present in the massive dna pyrosequencing study of shanks et al.,  <cit>  such as actinobacteria, spirochaetes, verrucomicrobia, cyanobacteria, fibrobacteres, and lentisphaerae.
we also investigated the significance of the response of the dominant of phyla firmicutes and bacteroidetes to dietary treatments because these are highly abundant taxa and are thought to play a key role in energy capture.
we also observed trends in firmicutes and bacteroidetes abundance as have others  <cit> ; however, we could not identify a significant response of these phyla to diet.
the dg diets evaluated in these studies seemed to have a complex effect on fecal microbiota.
several of the procedures used in this study identified a common set of taxa that seem to be responsive to the influence of corn and sorghum dg diets vs. that of the traditional steam-flaked corn diet.
some of these taxa were identified in other studies as responsive to or seemingly influenced by starch content in the diet or the dg diet regardless of the differences in experimental protocols and animals .
the presence of large animal to animal variation is noted in our study using a culture-independent method as well as in a culture dependent approach by durso et al.
<cit> .
however, the importance of a core set of taxa associated with the cattle bovine fecal microbiome is underscored by the fact that this core biome is observable regardless of the scale  of sequencing efforts conducted across studies.
it would appear that at least three phyla, firmicutes, bacteroidetes, and proteobacteria comprise a core set of bacteria across all cattle types.
feeding corn- and sorghum-based dg in steam-flaked corn based diets resulted in significant shifts in the overall fecal microbial community structure ranging from phyla to genera.
ecological and evolutionary theory suggests that more diverse communities can make a greater contribution to ecosystem functioning  <cit> .
if each species uses a slightly different resource and occupies a highly specific niche in the community, a more diverse microbiome should be able to, for example, more efficiently capture energy or be capable of capturing greater amounts of energy or possibly both.
clearly, bacterial communities in the bovine fecal environment are highly adapted and at the same time constrained by selection for existence in this environment.
the presence of core taxa across all these studies implies that these microbes are involved in performing fundamental metabolic functions essential to the collective cattle microbiome.
what the exact metabolic significance of these universal metabolic functions is, and if or how a shift in microbial populations  affects these universal metabolic functions remains to be determined.
daily weight gain and efficiency of weight gain  for the cattle in this experiment decreased linearly  as the dietary concentration of sorghum dg increased; however, these measurements did not differ between corn and sorghum dg fed as 10% of the dietary dm  <cit> .
the relationship between changes in cattle performance and alterations in the microbiome needs further study.
CONCLUSIONS
this is, to our knowledge, the first study using this method to survey the fecal microbiome of beef cattle fed various concentrations of wet dg.
comparison of our results with other cattle dna sequencing studies of beef and dairy cattle from a variety of geographical locations and different management practices identifies a core set of three phyla shared across all cattle.
these three phyla in order of relative abundance are; firmicutes, bacteroidetes, and proteobacteria.
the presence of core taxa across all these studies implies that these microbes are involved in performing fundamental metabolic functions that are essential to the collective cattle microbiome.
the presence of large animal-to-animal variation in cattle microbiome was noted in our study as well as by others.

 the high demand for ethanol in the u.s. has generated large stocks of wet distillers grains derived as a byproduct from the manufacture of ethanol from corn and sorghum grains. ethanol production is expected to increase several fold due to the high demand and cost of foreign oil. energy and protein dense dgs are attractive for use as a feed for beef cattle finishing diets; however little is known about the potential influence of dietary dg on fecal microbial community structure. a better understanding of the microbial population in beef cattle feces could be important in improving nutrient management, increasing animal growth performance, and decreasing odors and/or shedding of pathogens. a variety of emissions such as ammonia, volatile fatty acids, and hundreds of volatile organic compounds have been tied to beef cattle manure. volatilization of ammonia has been linked to crude protein content in the diet fed and increased amounts of excreted urinary n. previous studies suggested an association between dried distillers grains feeding and an increased prevalence and fecal shedding of the foodborne pathogen escherichia coli o157:h in cattle. a small number of studies have used culture-independent 16s rrna-based and culture-dependent 16s rrna-based methods with dairy cattle feces. clostridium spp were identified as the most dominant taxa across all lactating dairy cows followed by bacteroides spp using the culture-independent approach. in this study of holstein dairy cows, different bacterial species were detected corresponding to separate genera. several thousand sequences were obtained per sample enabling the detection of populations below % abundance. using culture-dependent methods, a total of 16s rrna clones were obtained from three holstein steers and classified at the 98% sequence similarity level. the dominant phyla observed were: firmicutes, bacteroidetes, actinobacteria, and proteobacteria. a comparison of dairy cattle fed a control diet or fed a diet supplemented with monensin using the culture-dependent 16s rrna method returned 16s rrna genes. nearly equivalent abundance levels of firmicutes and bacteroidetes were observed across the six lactating holstein cows with proteobacteria comprising the next most abundant group. culture-dependent and culture-independent 16s rrna methods were also applied with studies involving beef cattle. utilizing classical full length 16s rrna gene sequence analysis a total of otus were identified from six cattle. a core set of phyla were observed based on otus comprised of sequences with otus found only in individual libraries. seven phyla were found within six animals with three dominant taxonomic groups; firmicutes,, bacteroidetes and proteobacteria. in another small study of beef cattle the dna pyrosequencing method was applied to the comparison of the effects of three diets on ruminal and fecal bacterial assemblages. three diets in which of or 50% of the concentrate portion of the diet was replaced with dried distillers grains plus solubles were compared. over different bacterial species were detected that belonged to separate genera from ruminal samples across all three diets. in all fecal samples, more than different bacterial species were detected corresponding to separate genera. the most common genera that accounted for over 85% of the ruminal and fecal bacterial populations were identified. the firmicutes: bacteroidetes ratio tended to decrease as the proportion of ddgs increased. in a much larger study involving cattle distributed across geographically different locations and six different feeding operations the dna pyrosequencing method was used to assess fecal microbial community assemblages. the majority of sequences were distributed across four phyla: firmicutes, bacteroidetes, tenericutes, and proteobacteria. core taxa were observed across different phyla: actinobacteria, bacteroidetes, cyanobacteria, firmicutes, and tenericutes. using sequence-based clustering and taxonomic analyses, less variability was observed within a particular management practice/location than among different management practices. animal feeding operations seemed to influence bovine fecal bacterial communities at the phylum and family taxonomic levels much more so than geographic location of the feedlot. lastly, overall bacterial community composition seemed to be strongly influenced by fecal starch concentrations. the most responsive phyla to diet were bacteroidetes, firmicutes and proteobacteria. the relative abundance of bacteroidetes increased with increasing fecal starch concentration, whereas, the abundance of firmicutes decreased with increasing fecal starch concentrations. in the present study, we used the barcode dna pyrosequencing technique to evaluate the influence of five beef cattle diets on fecal microbial assemblages. the diets consisted of a traditional diet feed beef cattle in the southern high plains of texas-con, and four diets containing different percentages of dgs in the dietary dry matter; c, 5s, 10s, and 15s. the barcoded dna pyrosequencing method was used to generate 16s otus dataset. the 16s otus dataset was assigned to various taxonomic classes and each phylogenetic level was analyzed using a variety of statistical tests including unifrac procedures, hierarchal cluster analysis, distance based redundancy analysis, and one-way anova to test the influence of dietary treatments on microbial populations. we describe significant changes in microbial community structure and diversity that is influenced by these different dgs diets.  general dna sequencing observations a total of high quality 16s otus were utilized in the analysis. the total number of high quality 16s otus recovered from each animal is listed in table the average number of otus returned for each diet was: con, 6613; c, 6836; 5s, 6042; 10s, 5977; and 15s, rarefaction curves indicated that a high level of microbial diversity was obtained for subsequent analysis of dietary treatments. in general, no treatment was associated with a loss of sample size for subsequent evaluation of populations across treatments. the total abundance observed for otus and their associated centroids distributed across treatments are indicated in box plots depicting beta diversity. the highest abundance was observed in the c diet followed closely by the 10s and 15s diets. the highest animal to animal variation was observed in the 5s diet followed closely by the control diet. in general, abundance ranges for the diets and their associated centroids were more tightly grouped with the 10s and 15s diets. the dietary treatment, animal id, and no. of otus obtained per fecal grab from each animal the relationship among treatments is indicated in whittaker plots with each dot representing a species. the left and top of the graph indicate the presence of the most abundant otus with the bottom and right indicating the occurrence of rare otus. each dot represents one species and the high steepness of the graph is indicative of unevenly distributed species. the lengths of the curves also indicate the occurrence of rare otus. the curves generally overlap one another in this analysis for all dietary treatments; thus, overall microbial diversity were similar. influence of dgs on fecal microbiota-phyla four phyla were observed to have a response to dietary treatments. these are synergistetes, ws, actinobacteria, and spirochaetes. a total of phyla were observed distributed amongst all beef cattle on all diets. these are listed in order of average abundance and with their respective ranges: firmicutes, bacteroidetes, spirochaetes, proteobacteria, verrucomicrobia, fibrobacteres, tm, tenericutes, nitrospirae, actinobacteria, and fusobacteria. chlamydiae, cyanobacteria, planctomycetes, synergistetes, lentisphaerae, acidobacteria, elusimicrobia, chlorobi, ws deinococcus-thermus, chloroflexi, gemmatimonadetes, and deferribacteres were defined as low abundance phyla. greater than % of total bacterial abundance was observed in the first phyla, with several remaining phyla represented by or less members. the abundance levels of the top ten phyla averaged based on dietary treatment are presented in figure 3b. a higher relative abundance of firmicutes was observed when compared to the relative abundance level of bacteroidetes for dgs diets that contain 10% or more dg supplement vs. the con and 5s diets. however, significant differences were not observed among dietary treatments for abundances of firmicutes and bacteroidetes. a matched pair comparison evaluation of the abundances of firmicutes to bacteroidetes to one another yielded a non-significant response. a core set of six phyla were observed in all animals regardless of dietary treatment, and they were; firmicutes, bacteroidetes, proteobacteria, tenericutes, nitrospirae, and fusobacteria. with the exception of one animal that lacked spirochaetes, seven phyla would have been observed. a. the distribution of major phyla based on bacterial counts among beef cattle feed five diets. b. distribution of the most abundant phyla averaged across the dietary treatments. con = control, c = 10% corn, 5s = 5% sorghum, 10s = 10% sorghum, 15s = 15% sorghum. distribution of bacterial class, order and families by treatment the response of the most abundant bacteria at the phylogenetic levels of class, order and family is revealed in a series of heat maps and, for further clarification, in abundance plots showing both the individual animal response to diet and the averaged response to diet. for clarity and visualization purposes only the top bacterial orders and the top bacterial families are presented in heat maps. for corresponding abundance plots, the cutoffs are at the 97-99% abundance levels and orders and families are presented. with respect to abundance levels of clostridia, bacteroidia, and gammaproteobacteria, animal microbial community was the most disparate from all the other animals. the relative abundance of clostridia was substantially lower and the relative abundance of bacteroidia and gammaproteobacteria were greater. this effect is expressed at the phylogenetic level of bacterial orders with lower clostridiales and greater bacteroidales and enterobacteriales down to the level of families with lower abundances of ruminococcaceae and clostridiaceae and greater levels of prevotella. other animals appeared to be variable with respect to one or two other taxa such as number and when viewing patterns observed on the heat maps. influence of dgs on fecal microbiota-genera a total of bacterial species were observed distributed among genera across all fecal samples. a double hierarchal dendrogram was constructed using the upgma clustering method and manhattan distance method with no scaling. the influence of dg diets on the fecal microbiome was apparent from double hierarchal cluster analysis on the top most abundant genera and clustered by dietary treatment. with respect to diets, the least apparent phylogenetic distance observed within the top cluster was with the c diet and the most was with the 5s diets. prevotella and clostridium occurred together in their own separate cluster, whereas oscillospira, bacteroides, ruminococcus, eubacterium, and oscillibacter resided in the next most distant cluster. the other genera cohabited in another main cluster. for animal the microbial community seemed to be most unlike the other animals and this was apparently a result of a high relative abundance of bacteroidetes and a low relative abundance of firmicutes. the average abundance by treatment of the top genera and the response of taxa to diet are presented in additional file 12: table s in brief, those taxa that had a treatment response were: clostridium, ruminococcus, oscillibacter, tannerella, parabacteroides, hydrogenoanaerobacterium, pseudoflavonifractor, acetivibrio, ethanoligenens, selenomonas, desulfonispora, and barnesiella. the top species comprised approximately 91% of the total abundance observed and the following also show a significant response to treatment as detailed above. these are: clostridium sp., tannerella sp., pseudoflavonifractor capillosus, catabacter sp., hydrogenoanaerobacterium saccharovorans, ruminococcus bromii, and parabacteroides merdae. a biplot based on dbrda using the unweighted unifrac method identified taxa that were significantly affected by diets, p =. taxa most influenced by diet listed alphabetically were: akkermansia, clostridium, escherichia, eubacterium, oscillibacter, oscillospira, prevotella, ruminococcus, tannerella, and treponema. in figure the length and direction of the arrow with respect to diets indicates their relative positive or negative relationship to that diet. the ellipses around the animals represent the 95% confidence level, and their distance from one another reflects how closely or distantly the dietary effects are related to one another. it can be seen that akkermansia, escherichia, and treponema were positively influenced by the 5s and con diets, whereas the c is situated to the lower right hand side of the figure indicating a weak response from oscillibacter. the fecal community associated with the10s dietary treatment was least similar to the other fecal communities. a moderate influence of the 10s was observed for eubacterium and tannerella, whereas the 15s diet was near the point source eliciting a response from clostridium and oscillospira. the relative abundance of prevotella seems to be positively influenced by the 5s and con treatments since these diets are located on the lower axis when analyzed using weighted unifrac procedure a significant but slightly different result was observed regarding the influence of diets on microbial assemblages. it can be seen that akkermansia and treponema relative abundance were positively influenced by the con diet, whereas, escherichia was orientated at nearly 180° from these two taxa, and was more abundant in the 5s and 15s diets. eubacterium also had a similar response. prevotella was oriented to the bottom left hand side of the figure, but it was much more in alignment with escherichia. discussion influence of distillers grain diets deep sequencing of individual fecal samples from cattle fed five different diets provides a detailed view of the beef cattle fecal microbiome. the barcoded dna pyrosequencing method yielded high quality reads for microbiome comparison. we detected a core set of six bacterial phyla distributed across all animal fecal samples from all diets. in addition, we identified a total of phyla distributed across a number of the fecal samples associated with the various diets that encompass bacterial species distributed across genera. we identified four phyla that were responsive to dietary treatments. these were synergistetes, ws, actinobacteria, and spirochaetes. we also documented genera and species that responded to dietary treatments. it can be difficult to make comparisons across these various cattle fecal studies since they have employed a variety of 16s rrna-based sequencing strategies, the number and type of cattle employed in the studies and the types of diets and management practices associated with these diets. short read lengths and potential biases in evenness due to primer and template mismatches can result in pyro-sequencing artifacts that potentially affect taxonomic assignment and richness estimates. this is especially so with respect to rare otus. questions have also been posed and examined regarding the influence of geographical location, climatic conditions, and other localized environmental variables on cattle fecal microbial community structure. animal to animal variation was noted in fecal microbial diversity among beef cattle after controlling for location, climate, animal genetics, and diet. both the number and relative abundance of phyla we observed agree more closely with the distribution of phyla observed in the shanks et al. study than in the callaway et al. study. this could have been due to the number of cattle in the study or the size of the 16s otus dataset that was assembled. both pyrosequencing studies employed different primer locations and different read lengths to generate their datasets. the v region was specifically targeted in the shanks study and used short read lengths, whereas that of callaway targeted the v4-v region. thus, of the studies described in detail, our results generally agree more closely with the findings of shanks and durso, despite using the methodology described by dowd and employed by callaway. one possible explanation is that our choice of primers targeted the v through v region of the 16s rrna gene whereas the primer set utilized in the callaway study used the v to v region to assess phylogenetic information. another difference is that all of the cattle in the dowd study were lactating holstein dairy cows and for the callaway study they were jersey dairy cows and angus steers. a number of taxa appear to fluctuate in response to diets. two taxa, ruminococcaceae and prevotella spp., had distinct patterns in response to dietary treatments, whereas, the majority of taxa identified did not fluctuate across different dietary practices. other taxa identified in this study as being influenced by dietary treatment based on the unifrac procedure were; akkermansia, clostridium, escherichia, eubacterium, oscillibacter, oscillospira, prevotella, ruminococcus, tannerella, and treponema. two of these, prevotella and ruminococcus, were among those identified by shanks. we noted the presence of phyla in our study that were also present in the massive dna pyrosequencing study of shanks et al., such as actinobacteria, spirochaetes, verrucomicrobia, cyanobacteria, fibrobacteres, and lentisphaerae. we also investigated the significance of the response of the dominant of phyla firmicutes and bacteroidetes to dietary treatments because these are highly abundant taxa and are thought to play a key role in energy capture. we also observed trends in firmicutes and bacteroidetes abundance as have others; however, we could not identify a significant response of these phyla to diet. the dg diets evaluated in these studies seemed to have a complex effect on fecal microbiota. several of the procedures used in this study identified a common set of taxa that seem to be responsive to the influence of corn and sorghum dg diets vs. that of the traditional steam-flaked corn diet. some of these taxa were identified in other studies as responsive to or seemingly influenced by starch content in the diet or the dg diet regardless of the differences in experimental protocols and animals. the presence of large animal to animal variation is noted in our study using a culture-independent method as well as in a culture dependent approach by durso et al.. however, the importance of a core set of taxa associated with the cattle bovine fecal microbiome is underscored by the fact that this core biome is observable regardless of the scale of sequencing efforts conducted across studies. it would appear that at least three phyla, firmicutes, bacteroidetes, and proteobacteria comprise a core set of bacteria across all cattle types. feeding corn- and sorghum-based dg in steam-flaked corn based diets resulted in significant shifts in the overall fecal microbial community structure ranging from phyla to genera. ecological and evolutionary theory suggests that more diverse communities can make a greater contribution to ecosystem functioning. if each species uses a slightly different resource and occupies a highly specific niche in the community, a more diverse microbiome should be able to, for example, more efficiently capture energy or be capable of capturing greater amounts of energy or possibly both. clearly, bacterial communities in the bovine fecal environment are highly adapted and at the same time constrained by selection for existence in this environment. the presence of core taxa across all these studies implies that these microbes are involved in performing fundamental metabolic functions essential to the collective cattle microbiome. what the exact metabolic significance of these universal metabolic functions is, and if or how a shift in microbial populations affects these universal metabolic functions remains to be determined. daily weight gain and efficiency of weight gain for the cattle in this experiment decreased linearly as the dietary concentration of sorghum dg increased; however, these measurements did not differ between corn and sorghum dg fed as 10% of the dietary dm. the relationship between changes in cattle performance and alterations in the microbiome needs further study.  this is, to our knowledge, the first study using this method to survey the fecal microbiome of beef cattle fed various concentrations of wet dg. comparison of our results with other cattle dna sequencing studies of beef and dairy cattle from a variety of geographical locations and different management practices identifies a core set of three phyla shared across all cattle. these three phyla in order of relative abundance are; firmicutes, bacteroidetes, and proteobacteria. the presence of core taxa across all these studies implies that these microbes are involved in performing fundamental metabolic functions that are essential to the collective cattle microbiome. the presence of large animal-to-animal variation in cattle microbiome was noted in our study as well as by others. 
comparison of our results with other cattle dna sequencing studies of beef and dairy cattle feces from a variety of geographical locations and different management practices identifies a core set of three phyla shared across all cattle. a total of phyla were observed, distributed amongst all beef cattle on all diets, revealing considerable animal to animal variation, however only six phyla were observed in all animals regardless of dietary treatment. four low abundance phyla significantly responded to dietary treatments: synergistetes, ws, actinobacteria, and spirochaetes. the high demand for ethanol in the u.s. has generated large stocks of wet distillers grains, a byproduct from the manufacture of ethanol from corn and sorghum grains. this is, to our knowledge, the first study using this method to survey the fecal microbiome of beef cattle fed various concentrations of wet dg. these three phyla, in order of relative abundance are; firmicutes, bacteroidetes, and proteobacteria. a better understanding of the microbial population in beef cattle feces could be an important monitoring tool to facilitate goals of improving nutrient management, increasing animal growth performance and decreasing odors and/or shedding of pathogens. five diets consisting of a traditional diet fed to finishing beef cattle in the southern high plains of texas-con, and four concentrations of dg in the dietary dry matter; c, 5s, 10s, and 15s were fed to steers at the texas tech university burnett animal center. the presence of large animal-to-animal variation in cattle microbiome was noted in our study as well as by others. the average abundance and range of abundance, respectively of the core phyla were as follows: firmicutes, bacteroidetes, proteobacteria, tenericutes, nitrospirae, and fusobacteria. feeding dg-based diets resulted in significant shifts in the fecal microbial community structure compared with the traditional con. little is known, however, about the potential influence of dietary dg on fecal microbial community structure.
 the high demand for ethanol in the u.s. has generated large stocks of wet distillers grains, a byproduct from the manufacture of ethanol from corn and sorghum grains. little is known, however, about the potential influence of dietary dg on fecal microbial community structure. a better understanding of the microbial population in beef cattle feces could be an important monitoring tool to facilitate goals of improving nutrient management, increasing animal growth performance and decreasing odors and/or shedding of pathogens. five diets consisting of a traditional diet fed to finishing beef cattle in the southern high plains of texas-con, and four concentrations of dg in the dietary dry matter; c, 5s, 10s, and 15s were fed to steers at the texas tech university burnett animal center. diets were essentially isonitrogenous with a formulated crude protein value of %.  fecal grab samples were obtained from steers and the barcoded dna pyrosequencing method was used to generate 16s operational taxonomic units. a total of phyla were observed, distributed amongst all beef cattle on all diets, revealing considerable animal to animal variation, however only six phyla were observed in all animals regardless of dietary treatment. the average abundance and range of abundance, respectively of the core phyla were as follows: firmicutes, bacteroidetes, proteobacteria, tenericutes, nitrospirae, and fusobacteria. feeding dg-based diets resulted in significant shifts in the fecal microbial community structure compared with the traditional con. four low abundance phyla significantly responded to dietary treatments: synergistetes, ws, actinobacteria, and spirochaetes.  this is, to our knowledge, the first study using this method to survey the fecal microbiome of beef cattle fed various concentrations of wet dg. comparison of our results with other cattle dna sequencing studies of beef and dairy cattle feces from a variety of geographical locations and different management practices identifies a core set of three phyla shared across all cattle. these three phyla, in order of relative abundance are; firmicutes, bacteroidetes, and proteobacteria. the presence of large animal-to-animal variation in cattle microbiome was noted in our study as well as by others. 