BACKGROUND
docosahexaenoic acid  is a poly-unsaturated fatty acid  that has been associated with physiological benefits in many species, including human and dairy cows.
the amount of 22:6n- <dig> available for absorption in the small intestine can be increased by intake of marine products .
however, this is not straightforward in ruminants as extensive biohydrogenation in the rumen leaves little 22:6n- <dig> for absorption.
biohydrogenation of pufa is one of the important microbial processes in the rumen that has a major influence on fa composition of meat and milk.
it is well documented that ruminal bacteria are responsible for most of the biohydrogenation process in the rumen  <cit> , and it is thought to be a detoxification mechanism as pufa are more toxic than saturated fa  <cit> .
metabolism of linoleic  and linolenic  acids is well studied in vivo and in vitro and involves an initial isomerization step to yield a fa with a conjugated double bond .
in vitro studies with mixed and pure rumen bacteria helped to construct the detailed biohydrogenation pathways of 18:2n- <dig> and 18:3n- <dig>  and the identification of bacterial species involved in this process .
however, information on the biohydrogenation of 22:6n- <dig> is still lacking.
several reports show extensive metabolism of 22:6n- <dig> in vivo  <cit>  and in vitro with mixed rumen cultures  <cit> .
however, none of the studies identified bacterial species responsible for its metabolism.
maia et al.
<cit>  investigated pufa metabolism of  <dig> predominant rumen bacterial species and found none of them able to metabolize 22:6n- <dig>  sakurama et al.
<cit>  screened about  <dig> strains of anaerobic bacteria including some ruminal bacteria and found none of them metabolized 22:6n- <dig>  pure culture studies focusing on the main rumen hydrogenating bacteria, butyrivibrio fibrisolvens and butyrivibrio proteoclasticus also failed to successfully induce 22:6n- <dig> hydrogenation  <cit> .
the aim of this study was to examine the metabolism of 22:6n- <dig> by the known biohydrogenating ruminal bacteria, b. fibrisolvens and b. proteoclasticus.
to our knowledge, in previous experiments these bacterial species have been exposed to a single concentration of 22:6n- <dig>  which may be toxic for them  <cit> .
we used lower concentrations of 22:6n- <dig> in our experiments.
additionally, we modified the standard butyrivibrio growth medium in an attempt to promote biohydrogenation.
experiments 1– <dig> were conducted using the growth medium containing autoclaved-uncentrifuged rumen fluid and experiments 6− <dig> were conducted using the growth medium containing autoclaved-centrifuged rumen fluid .table  <dig> overview of the in vitro experiments conducted in this study
5c
abacteria used in the experiment -b. fibri: butyrivibrio fibrisolvens d1; b. proteo: butyrivibrio proteoclasticus p18
bheadspace gas phase
cthe growth medium used in experiment  <dig> contained 40 μg/ml of 18:2n-6
RESULTS
22:6n- <dig> metabolism by b. fibrisolvens d1
both autoclaved-centrifuged and -uncentrifuged rumen fluid , did not result in 22:6n- <dig> metabolism at any concentration.
the effect of various concentrations of 22:6n- <dig> on growth, fermentation and biohydrogenation ability of b. fibrisolvens d <dig>  in media containing 20 %  autoclaved-centrifuged rumen fluid are summarized in table  <dig>  no growth was observed till 48 h with the highest concentration of 22:6n- <dig> , whereas a considerably shorter lag phase  was observed with the same concentration of 18:2n- <dig> in the same medium.
optical density  measured at stationary phase was lower  at the concentration of 16 μg/ml compared to the lower concentrations .
total volatile fatty acid  production and vfa profile were not affected  by the 22:6n- <dig> concentrations.
the main fermentation products included butyrate and small amounts of acetate .table  <dig> effects of different concentrations of 22:6n- <dig> on growth, vfa production and biohydrogenation by butyrivibrio fibrisolvens d1
od600
c
cincrease in od <dig> after 48 h of incubation compared to initial od <dig> at 0 h
dnd- not determined as growth was not started till 48 h
means with different superscripts in od <dig>  are significantly different
22:6n- <dig> metabolism by b. proteoclasticus p18
in the growth medium containing 50 %  autoclaved-uncentrifuged rumen fluid, b. proteoclasticus p <dig> hydrogenated 22:6n- <dig> .
at the concentrations of 20 μg/ml , most of the 22:6n- <dig> was metabolized, leaving little residual 22:6n- <dig>  after 48 h of incubation.
the type of headspace gas had no influence on the residual amount of 22:6n- <dig>  total vfa production and vfa profile were not affected  by different gas phases .
the main fermentation products included butyrate and acetate .
effect of length of incubation period on 22:6n- <dig> metabolism by b. proteoclasticus p18
the extent of 22:6n- <dig> metabolism in media containing 50 %  autoclaved-uncentrifuged rumen fluid at different time points  is shown in fig.
<dig> along with the vfa production at the respective time points.
metabolism of 22:6n- <dig>  initiated after 4 h and 80 % of 22:6n- <dig> was hydrogenated between 4 h and 12 h of incubation.
accumulation of vfa initiated prior to the start of 22:6n- <dig> metabolism and 93 % of vfa were produced within the first 12 h of the incubation.
both the rate of 22:6n- <dig> disappearance and vfa production slowed down after 12 h of incubation.
significant vfa production was not observed after 12 h, whereas hydrogenation of 22:6n- <dig> slowly continued.
as a result, the amount of 22:6n- <dig> recovered after 48 h was lower  than the amount recovered after  <dig> and 24 h of incubation.fig.
<dig> metabolism of 22:6n- <dig> and vfa accumulation at different time points by butyrivibrio proteoclasticus p <dig>  growth medium included 50 %  of autoclaved-uncentrifuged rumen fluid.
hydrogen was used as the headspace gas.
● residual 22:6n- <dig> in the tube and ▲ vfa accumulated.
results are means and sd from  <dig> biological replicates, each of which had  <dig> analytical replicates.
for residual 22:6n- <dig> and accumulated vfa, data points with different letters are significantly different
detailed analysis of chromatograms did not provide evidence of 22: <dig> formations during metabolism of 22:6n- <dig>  peaks appearance at different time points did not show accumulation of conjugated 22: <dig> products.
the intermediates produced during the 48 h incubation comprised about  <dig> different fa  and characteristic ion fragments are presented in table  <dig>  during the early hours of incubation ,  <dig> new peaks  appeared .
of these, peak  <dig> was the most abundant and the molecular ion at m/z =  <dig> and the loss of the terminal methyl group  confirmed the docosapentaenoic acid structure.
the odd numbered ion fragment at m/z =  <dig> is an indication for the location of a Δ <dig> double bond  <cit> .
gaps of 12 amu between m/z =  <dig> and 220;  <dig> and 260;  <dig> and  <dig> and between  <dig> and  <dig> confirmed the location of ethylenic double bonds in position Δ <dig>  Δ <dig>  Δ <dig> and Δ <dig> .
peak  <dig> showed the same retention time as 22:5n- <dig> present in the fame standard and ion fragments at  <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>   <dig> and  <dig>  and  <dig> and  <dig> allowed us to identify this peak as 22:5n- <dig>  <cit> .
a final docosapentaenoic acid  was identified as Δ <dig>  Δ <dig>  Δ <dig>  Δ <dig>  Δ19-22: <dig> based on the molecular ion at m/z =  <dig>  gaps of 12 amu between m/z =  <dig> and 220;  <dig> and 260;  <dig> and  <dig> and between  <dig> and  <dig> and the double bond in position  <dig> is defined by the fingerprint ion at m/z =  <dig> and  <dig>  <cit> .
one docosatetraenoic acid was identified as Δ <dig>  Δ <dig>  Δ <dig>  Δ19-22: <dig>  based on the molecular ion at m/z =  <dig> and gaps of 12 amu between m/z =  <dig> and 222;  <dig> and 262;  <dig> and  <dig> and between  <dig> and  <dig>  during the further incubation, the abundance of these peaks gradually decreased and were accompanied with the appearance of four 22: <dig> fa  and four 22: <dig> fa .
peaks  <dig> and  <dig> showed a similar mass spectra and were identified as Δ <dig>  Δ <dig>  Δ20-22: <dig>  ion fragments separated by 12 amu gaps located the double bonds at Δ <dig>  Δ <dig> and Δ <dig> with a prominent ion at m/z  <dig> confirming the presence of allylic bonds at Δ <dig> and Δ <dig>  peak  <dig> showed the same retention time as 22:3n- <dig> present in the fame standard and double bond positions were confirmed by ion fragments separated by 12 amu gaps .
peak  <dig> was identified as Δ <dig>  Δ <dig>  Δ19-22: <dig> based on ion fragments at  <dig> and  <dig>   <dig> and  <dig>  and  <dig> and  <dig>  peaks  <dig> and  <dig> showed a similar mass spectra and were identified as Δ <dig>  Δ20-22: <dig>  ion fragments separated by 12 amu gaps located the double bonds at Δ <dig> and Δ <dig>  with a prominent ion at m/z  <dig> confirming the presence of allylic bonds at Δ <dig> and Δ <dig>  peak  <dig> was Δ <dig>  Δ19-22: <dig>  and the prominent ion at m/z  <dig> combined ion fragments separated by 12 amu gaps at  <dig> and  <dig>  and  <dig> and  <dig> allowed us to identify peak  <dig> as Δ <dig>  Δ18-22: <dig> table  <dig> amount of metabolized 22:6n- <dig>  recovered in fame peaks.
incubation was performed in growth medium containing autoclaved-uncentrifuged rumen fluid  in the presence of 22:6n- <dig>  with butyrivibrio proteoclasticus p <dig>  tubes were withdrawn at  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 48 h incubation to study the extent of 22:6n- <dig> biohydrogenation.
peak numbers correspond to peaks in fig.
2
fig.
<dig> partial chromatogram with peaks representing fatty acids from 22: <dig> to 22:6n- <dig>  these chromatograms obtained from   <dig>   <dig>   <dig> and 24 h incubations with 22:6n- <dig>  and  48 h incubations with 22:6n- <dig> .
in both cases butyrivibrio proteoclasticus p <dig> had grown in the media containing 50 %  autoclaved- uncentrifuged rumen fluid.
peaks visible at 0 h represent the fatty acids present in the rumen fluid and peaks numbers  represent new peaks formed during the 22:6n- <dig> metabolism
in experiments conducted in media containing autoclaved-centrifuged rumen fluid , b. proteoclasticus hydrogenated 22:6n- <dig> at the concentration of 20 μg/ml .
after 24 h of incubation, little residual 22:6n- <dig> was left in the tube .
figure  <dig> shows the kinetics of 22:6n- <dig> disappearance along with od <dig> measured at the respective time points .
metabolism of 22:6n- <dig> initiated after 4 h and 93 % of 22:6n- <dig> was hydrogenated between 4 h and 12 h of incubation.
growth of b. proteoclasticus  initiated prior to the start of 22:6n- <dig> metabolism and exponentially increased between 4 h and 12 h. the products formed during the biohydrogenation of 22:6n- <dig>  were identical as those described before .fig.
<dig> metabolism of 22:6n- <dig> and optical density measurements at different time points by butyrivibrio proteoclasticus p <dig>  growth medium included 20 %  of autoclaved-centrifuged rumen fluid.
hydrogen was used as the headspace gas.
● residual 22:6n- <dig> in the tube ♦ optical density  measured at respective time points.
results are means and sd from  <dig> replicates.
for residual 22:6n- <dig> and od <dig> measured, data points with different letters are significantly different
effect of initial concentration of 22:6n- <dig> on 22:6n- <dig> metabolism by b. proteoclasticus p18
the initial concentration of 22:6n- <dig> influenced the extent of its metabolism.
extensive metabolism of 22:6n- <dig> was observed at lower concentrations .
addition of  <dig>   <dig> and 20 μg/ml 22:6n- <dig> resulted in the accumulation of 22: <dig> and 22: <dig> fa and represented > 80 % of 22:6n- <dig> that was metabolized during the 48 h incubation .
this implies that the initial 22: <dig> and 22: <dig> fa were further hydrogenated.
an increase in the initial concentration of 22:6n- <dig> increased the amount of 22:6n- <dig> metabolized during 48 h incubation period, but only up to a concentration of about 40 μg/ml .
although in absolute amounts, more 22:6n- <dig> disappeared at higher 22:6n- <dig> concentrations, analysis of the chromatograms showed that extensive biohydrogenation of intermediate products was inhibited .
at the initial concentration of 40 μg/ml, the residual 22:6n- <dig> retrieved from the tubes after 48 h incubation was 9 % of the initial amount.
under this condition, 65 % of the metabolized 22:6n- <dig> accumulated as a single compound identified as Δ <dig>  Δ <dig>  Δ <dig>  Δ19-22: <dig>  further conversion of this product seemed to be inhibited.
when the initial 22:6n- <dig> concentration was increased to 80 μg/ml, the residual 22:6n- <dig> retrieved from the tubes after 48 h of incubation was 45 % of the initial amount and > 90 % of the metabolized 22:6n- <dig> accumulated as 22: <dig> and 22: <dig> fa.
subsequent biohydrogenation of these products seemed completely inhibited at this concentration.table  <dig> amount of metabolized 22:6n- <dig>  recovered in fame peaks.
incubation was performed in growth medium containing autoclaved-uncentrifuged rumen fluid  in the presence of 22:6n- <dig>  with butyrivibrio proteoclasticus p <dig>  tubes were withdrawn at 48 h of incubation to study the extent of 22:6n- <dig> biohydrogenation.
peak numbers correspond to peaks in fig.
2
fig.
<dig> effect of initial concentration of 22:6n- <dig>  on its metabolism by butyrivibrio proteoclasticus p <dig>  incubations were performed in growth medium containing 50 %  of autoclaved-uncentrifuged rumen fluid for 48 h
total vfa production was not affected by the initial concentration of 22:6n- <dig> except for the highest concentration .
at the highest initial concentration  vfa production after 48 h was higher  compared to the lower concentrations , but the vfa profile was not affected .
effect of presence of 22:6n- <dig> on 18:2n- <dig> metabolism by b. proteoclasticus p18
table  <dig> shows the conjugated linoleic acids , vaccenic acid  and stearic acid  formation from 40 μg/ml 18:2n- <dig> in presence of  <dig> , low  and high  22:6n- <dig>  along with vfa formation and residual 22:6n- <dig>  the formation of cla was observed as soon as inoculation was made.
although formation of cla and va were not affected , conversion of va into sa  was retarded  in the presence of 22:6n- <dig> and this effect was dependent on 22:6n- <dig> concentration.
volatile fatty acids production was not initiated until all the 18:2n- <dig> had been metabolized and converted into va regardless of presence of high or low 22:6n- <dig>  both 18: <dig> formation and 22:6n- <dig> metabolism began after the initiation of vfa production.
in the control tubes, 18: <dig> formation started at about 4 h as soon as vfa production began and this conversion was rapid thereafter .
in the presence of low 22:6n- <dig> , b. proteoclasticus p <dig> initiated 22:6n- <dig> metabolism around 4 h, ahead of 18: <dig> formation .
as a result, a small lag phase was observed in 18: <dig> formation after the initiation of vfa production.
in presence of high 22:6n- <dig> , 18: <dig> formation was delayed to 12 h and this conversion was slower thereafter.table  <dig> linoleic acid metabolism  in presence of 22:6n- <dig> by b. proteoclasticus p <dig>  incubation was performed in growth medium containing 40 μg/ml  of 18:2n- <dig> in presence of  <dig>  10 μg/ml  and 40 μg/ml  22:6n- <dig>  growth medium included 50 %  of autoclaved-uncentrifuged rumen fluid and h <dig> gas was the headspace gas.
value-presented in this table included the fatty acids and vfa of autoclaved-uncentrifuged rumen fluid used to prepare the growth medium
cla conjugated linoleic acid, va vaccenic acid, vfa volatile fatty acids
for each fatty acids, subscript * represents different  compared to 0 mg/tube 22:6n- <dig> and ** represents different  compared to  <dig> and  <dig>  mg/tube 22:6n- <dig> at the respective time point
within each row means with different superscripts  are significantly different
discussion
butyrivibrio species are a genetically and functionally diverse group of bacteria present in gastrointestinal systems  <cit> .
based on the mechanism of butyrate formation, this group can be classified into two subgroups: vaccenic acid-producing  and stearic acid-producing .
accordingly, b. fibrisolvens and b. proteoclasticus are belonging to the vaccenic acid-producing and stearic acid-producing groups respectively  <cit> .
butyrivibrio fibrisolvens d <dig> and b. proteoclasticus p <dig> were chosen for this study as a representative from each group.
however, the type species b. fibrisolvens d <dig> showed high butyrate kinase activity which is atypical to the majority of b. fibrisolvens isolates  <cit> .
previous studies carried out with b. fibrisolvens and b. proteoclasticus in m <dig> medium failed to show hydrogenation of 22:6n- <dig>  <cit> .
the reason for this is probably the high concentration of 22:6n- <dig> used in these studies  which may have affected the growth of the bacteria.
wallace et al.
<cit>  showed growth to be a prerequisite for b. proteoclasticus p <dig> in order to form stearic acid  from 18:2n- <dig>  if this is also true for 22:6n- <dig> biohydrogenation, failure to grow when 22:6n- <dig> is present in previous experiments  <cit>  possibly explains the absence of 22:6n- <dig> biohydrogenation.
to reduce the inhibitory effect of 22:6n- <dig> on growth, the concentration of 22:6n- <dig> in the medium can be lowered or substances which lower the toxicity of 22:6n- <dig> should be included in the medium.
harfoot et al.
<cit>  showed that biohydrogenation was stimulated by rumen fluid particles, enabling fa biohydrogenation by ruminal bacteria.
experiments performed in our lab also showed addition of autoclaved-uncentrifuged rumen fluid stimulated the biohydrogenation of 22:6n- <dig> in mixed cultures of rumen bacteria .
hence, we used autoclaved-uncentrifuged rumen fluid to prepare the growth medium.
additionally we used a lower concentration of 22:6n- <dig> than the concentration reported previously   <cit> .
butyrivibrio fibrisolvens grew at low concentrations of 22:6n- <dig> without metabolizing it.
maia et al.
<cit>  showed that growth of b. fibrisolvens jw <dig> was not initiated until all 18:2n- <dig> was metabolized and converted to va, such phenomenon was not observed with 22:6n- <dig>  the failure of b. fibrisolvens d <dig> to metabolize 22:6n- <dig> in the current experiment might indicate they do not possess the necessary enzymes for 22:6n- <dig> biohydrogenation.
however, further studies are warranted as b. fibrisolvens d <dig> is atypical to other b. fibrisolvens in general.
in contrast with previous reports  <cit> , we found b. proteoclasticus p <dig> is able to hydrogenate 22:6n- <dig>  to our knowledge, this is the first report demonstrating 22:6n- <dig> biohydrogenation by a pure bacterial species.
temporal changes in 22:6n- <dig>  vfa and od <dig> during the course of the incubation might indicate b. proteoclasticus must be growing to biohydrogenate 22:6n- <dig>  with little transformation occurring during the stationary phase.
such observations were also made when demonstrating the capability of b. proteoclasticus p <dig> to form 18: <dig> from 18:2n- <dig>  <cit> .
stationary-phase bacteria were reported to be much less active in hydrogenation of 18:2n- <dig> than growing cells  <cit> .
this was explained by a limited supply of reducing equivalents once the energy sources in the medium had been depleted  <cit>  as the conversion of cla to va is nadh-dependent  <cit> .
it seems probable that a similar mechanism explains the variation in rate of disappearance of 22:6n- <dig> during the course of the incubation period.
it is however not clear whether growth or activity per se is needed for biohydrogenation, or that the association of disappearance of 22:6n- <dig>  vfa and od <dig> is merely a reflection of changes in the culture closely associated with growth and activity .
the metabolism of 22:6n- <dig> resulted in the appearance of numerous compounds eluting during gc analysis between 22: <dig> and 22:6n- <dig>  it has been speculated before that ruminal hydrogenation of 22:6n- <dig> yields intermediates with  <dig> or  <dig> double bonds, containing at least one trans double bond  <cit> .
the position of the double bonds in the most abundant 22: <dig> isomer  suggest the formation of this intermediate involves izomerisation of the cis- <dig> double bond and reduction of the cis- <dig> double bond  <cit> .
initial izomerisation of the cis- <dig> double bond of 22:6n- <dig> would result in the formation of a conjugated product .
however, in the present series of experiments, no accumulation of products eluting in the gc chromatogram with a retention time greater than 22:6n- <dig> was observed, a region where conjugated isomers of 22: <dig> would be expected to elute with the polar column used in the present study.
the lack of accumulation of a conjugated fa might indicate they are transient products which did not accumulate at the time of sampling.
alternatively, this might also indicate the initial product of 22:6n- <dig> metabolism is not a conjugated fa.
the accumulation of 22: <dig> isomers produced during the initial stages of 22:6n- <dig> biohydrogenation was transient, when the initial 22:6n- <dig> concentration was low, and these isomers were subsequently hydrogenated to more saturated 22: <dig>  22: <dig> and 22: <dig> isomers.
the existence of two isomers with identical double bond position  indicates double bonds in the trans configuration must be present.
it is unclear to what extent the increases in polyenoic trans fatty acids may offset some of the expected benefits from the enrichment of 22:6n- <dig> in ruminant derived foods  <cit> .
butyrivibrio proteoclasticus is the only known ruminal bacterium with the capacity to biohydrogenate 18-carbon fa to 18: <dig>  previous incubations with rumen fluid have established that 22:6n- <dig> inhibits the complete hydrogenation of 18-carbon unsaturated fa causing trans 18: <dig> isomers to accumulate  <cit> .
this is thought to be related to the toxic effects of 22:6n- <dig> on the growth and metabolic activity of b. proteoclasticus  <cit> .
however, in vivo studies have failed to show the relationship between 18: <dig> flow to the duodenum and b. proteoclasticus dna  <cit> .
based on the findings of present study we suggest that b. proteoclasticus probably starts to hydrogenate 22:6n- <dig> before converting 18: <dig> isomers to 18: <dig>  biohydrogenation is required to lower toxicity and therefore the reduction of products containing more double bonds are a greater priority which explains the lack of 18: <dig> reduction at time points where biohydrogenation of 22:6n- <dig> did occur.
these findings possibly explain the lack of relationship of duodenal 18: <dig> to the duodenum and b. proteoclasticus dna  <cit> .
CONCLUSIONS
butyrivibrio proteoclasticus p <dig> is able to hydrogenate 22:6n- <dig> in vitro.
the rate and extent of biohydrogenation depended on the initial concentration of 22:6n- <dig> and the duration of incubation.
several 22: <dig>  22: <dig>  22: <dig> and 22: <dig> isomers were identified.
the products formed irrespective of the initial concentrations of 22:6n- <dig> and type of growth media suggests b. proteoclasticus p <dig> had a consistent pathway of 22:6n- <dig> hydrogenation.
during the simultaneous presence of both 18:2n- <dig> and 22:6n- <dig>  b. proteoclasticus p <dig> initiated 22:6n- <dig> metabolism before converting 18: <dig> isomers into 18: <dig>  under the current culture conditions, b. fibrisolvens d <dig> failed to hydrogenate 22:6n- <dig>

 docosahexaenoic acid is a poly-unsaturated fatty acid that has been associated with physiological benefits in many species, including human and dairy cows. the amount of 22:6n- available for absorption in the small intestine can be increased by intake of marine products. however, this is not straightforward in ruminants as extensive biohydrogenation in the rumen leaves little 22:6n- for absorption. biohydrogenation of pufa is one of the important microbial processes in the rumen that has a major influence on fa composition of meat and milk. it is well documented that ruminal bacteria are responsible for most of the biohydrogenation process in the rumen, and it is thought to be a detoxification mechanism as pufa are more toxic than saturated fa. metabolism of linoleic and linolenic acids is well studied in vivo and in vitro and involves an initial isomerization step to yield a fa with a conjugated double bond. in vitro studies with mixed and pure rumen bacteria helped to construct the detailed biohydrogenation pathways of 18:2n- and 18:3n- and the identification of bacterial species involved in this process. however, information on the biohydrogenation of 22:6n- is still lacking. several reports show extensive metabolism of 22:6n- in vivo and in vitro with mixed rumen cultures. however, none of the studies identified bacterial species responsible for its metabolism. maia et al. investigated pufa metabolism of predominant rumen bacterial species and found none of them able to metabolize 22:6n- sakurama et al. screened about strains of anaerobic bacteria including some ruminal bacteria and found none of them metabolized 22:6n- pure culture studies focusing on the main rumen hydrogenating bacteria, butyrivibrio fibrisolvens and butyrivibrio proteoclasticus also failed to successfully induce 22:6n- hydrogenation. the aim of this study was to examine the metabolism of 22:6n- by the known biohydrogenating ruminal bacteria, b. fibrisolvens and b. proteoclasticus. to our knowledge, in previous experiments these bacterial species have been exposed to a single concentration of 22:6n- which may be toxic for them. we used lower concentrations of 22:6n- in our experiments. additionally, we modified the standard butyrivibrio growth medium in an attempt to promote biohydrogenation. experiments 1– were conducted using the growth medium containing autoclaved-uncentrifuged rumen fluid and experiments 6− were conducted using the growth medium containing autoclaved-centrifuged rumen fluid.table overview of the in vitro experiments conducted in this study 5c abacteria used in the experiment -b. fibri: butyrivibrio fibrisolvens d1; b. proteo: butyrivibrio proteoclasticus p18 bheadspace gas phase cthe growth medium used in experiment contained 40 μg/ml of 18:2n-6  22:6n- metabolism by b. fibrisolvens d1 both autoclaved-centrifuged and -uncentrifuged rumen fluid, did not result in 22:6n- metabolism at any concentration. the effect of various concentrations of 22:6n- on growth, fermentation and biohydrogenation ability of b. fibrisolvens d in media containing 20 % autoclaved-centrifuged rumen fluid are summarized in table  no growth was observed till 48 h with the highest concentration of 22:6n-, whereas a considerably shorter lag phase was observed with the same concentration of 18:2n- in the same medium. optical density measured at stationary phase was lower at the concentration of 16 μg/ml compared to the lower concentrations. total volatile fatty acid production and vfa profile were not affected by the 22:6n- concentrations. the main fermentation products included butyrate and small amounts of acetate.table effects of different concentrations of 22:6n- on growth, vfa production and biohydrogenation by butyrivibrio fibrisolvens d1 od600 c cincrease in od after 48 h of incubation compared to initial od at 0 h dnd- not determined as growth was not started till 48 h means with different superscripts in od are significantly different 22:6n- metabolism by b. proteoclasticus p18 in the growth medium containing 50 % autoclaved-uncentrifuged rumen fluid, b. proteoclasticus p hydrogenated 22:6n-. at the concentrations of 20 μg/ml, most of the 22:6n- was metabolized, leaving little residual 22:6n- after 48 h of incubation. the type of headspace gas had no influence on the residual amount of 22:6n- total vfa production and vfa profile were not affected by different gas phases. the main fermentation products included butyrate and acetate. effect of length of incubation period on 22:6n- metabolism by b. proteoclasticus p18 the extent of 22:6n- metabolism in media containing 50 % autoclaved-uncentrifuged rumen fluid at different time points is shown in fig.  along with the vfa production at the respective time points. metabolism of 22:6n- initiated after 4 h and 80 % of 22:6n- was hydrogenated between 4 h and 12 h of incubation. accumulation of vfa initiated prior to the start of 22:6n- metabolism and 93 % of vfa were produced within the first 12 h of the incubation. both the rate of 22:6n- disappearance and vfa production slowed down after 12 h of incubation. significant vfa production was not observed after 12 h, whereas hydrogenation of 22:6n- slowly continued. as a result, the amount of 22:6n- recovered after 48 h was lower than the amount recovered after and 24 h of incubation.fig. metabolism of 22:6n- and vfa accumulation at different time points by butyrivibrio proteoclasticus p growth medium included 50 % of autoclaved-uncentrifuged rumen fluid. hydrogen was used as the headspace gas. ● residual 22:6n- in the tube and ▲ vfa accumulated. results are means and sd from biological replicates, each of which had analytical replicates. for residual 22:6n- and accumulated vfa, data points with different letters are significantly different detailed analysis of chromatograms did not provide evidence of 22: formations during metabolism of 22:6n- peaks appearance at different time points did not show accumulation of conjugated 22: products. the intermediates produced during the 48 h incubation comprised about different fa and characteristic ion fragments are presented in table  during the early hours of incubation, new peaks appeared. of these, peak was the most abundant and the molecular ion at m/z =  and the loss of the terminal methyl group confirmed the docosapentaenoic acid structure. the odd numbered ion fragment at m/z =  is an indication for the location of a Δ double bond. gaps of 12 amu between m/z =  and 220; and 260; and and between and confirmed the location of ethylenic double bonds in position Δ Δ Δ and Δ. peak showed the same retention time as 22:5n- present in the fame standard and ion fragments at and and and and and and allowed us to identify this peak as 22:5n-. a final docosapentaenoic acid was identified as Δ Δ Δ Δ Δ19-22: based on the molecular ion at m/z =  gaps of 12 amu between m/z =  and 220; and 260; and and between and and the double bond in position is defined by the fingerprint ion at m/z =  and. one docosatetraenoic acid was identified as Δ Δ Δ Δ19-22: based on the molecular ion at m/z =  and gaps of 12 amu between m/z =  and 222; and 262; and and between and during the further incubation, the abundance of these peaks gradually decreased and were accompanied with the appearance of four 22: fa and four 22: fa. peaks and showed a similar mass spectra and were identified as Δ Δ Δ20-22: ion fragments separated by 12 amu gaps located the double bonds at Δ Δ and Δ with a prominent ion at m/z confirming the presence of allylic bonds at Δ and Δ peak showed the same retention time as 22:3n- present in the fame standard and double bond positions were confirmed by ion fragments separated by 12 amu gaps. peak was identified as Δ Δ Δ19-22: based on ion fragments at and and and and peaks and showed a similar mass spectra and were identified as Δ Δ20-22: ion fragments separated by 12 amu gaps located the double bonds at Δ and Δ with a prominent ion at m/z confirming the presence of allylic bonds at Δ and Δ peak was Δ Δ19-22: and the prominent ion at m/z combined ion fragments separated by 12 amu gaps at and and and allowed us to identify peak as Δ Δ18-22: table amount of metabolized 22:6n- recovered in fame peaks. incubation was performed in growth medium containing autoclaved-uncentrifuged rumen fluid in the presence of 22:6n- with butyrivibrio proteoclasticus p tubes were withdrawn at and 48 h incubation to study the extent of 22:6n- biohydrogenation. peak numbers correspond to peaks in fig. 2 fig. partial chromatogram with peaks representing fatty acids from 22: to 22:6n- these chromatograms obtained from and 24 h incubations with 22:6n- and 48 h incubations with 22:6n-. in both cases butyrivibrio proteoclasticus p had grown in the media containing 50 % autoclaved- uncentrifuged rumen fluid. peaks visible at 0 h represent the fatty acids present in the rumen fluid and peaks numbers represent new peaks formed during the 22:6n- metabolism in experiments conducted in media containing autoclaved-centrifuged rumen fluid, b. proteoclasticus hydrogenated 22:6n- at the concentration of 20 μg/ml. after 24 h of incubation, little residual 22:6n- was left in the tube. figure  shows the kinetics of 22:6n- disappearance along with od measured at the respective time points. metabolism of 22:6n- initiated after 4 h and 93 % of 22:6n- was hydrogenated between 4 h and 12 h of incubation. growth of b. proteoclasticus initiated prior to the start of 22:6n- metabolism and exponentially increased between 4 h and 12 h. the products formed during the biohydrogenation of 22:6n- were identical as those described before.fig. metabolism of 22:6n- and optical density measurements at different time points by butyrivibrio proteoclasticus p growth medium included 20 % of autoclaved-centrifuged rumen fluid. hydrogen was used as the headspace gas. ● residual 22:6n- in the tube ♦ optical density measured at respective time points. results are means and sd from replicates. for residual 22:6n- and od measured, data points with different letters are significantly different effect of initial concentration of 22:6n- on 22:6n- metabolism by b. proteoclasticus p18 the initial concentration of 22:6n- influenced the extent of its metabolism. extensive metabolism of 22:6n- was observed at lower concentrations. addition of and 20 μg/ml 22:6n- resulted in the accumulation of 22: and 22: fa and represented > 80 % of 22:6n- that was metabolized during the 48 h incubation. this implies that the initial 22: and 22: fa were further hydrogenated. an increase in the initial concentration of 22:6n- increased the amount of 22:6n- metabolized during 48 h incubation period, but only up to a concentration of about 40 μg/ml. although in absolute amounts, more 22:6n- disappeared at higher 22:6n- concentrations, analysis of the chromatograms showed that extensive biohydrogenation of intermediate products was inhibited. at the initial concentration of 40 μg/ml, the residual 22:6n- retrieved from the tubes after 48 h incubation was 9 % of the initial amount. under this condition, 65 % of the metabolized 22:6n- accumulated as a single compound identified as Δ Δ Δ Δ19-22: further conversion of this product seemed to be inhibited. when the initial 22:6n- concentration was increased to 80 μg/ml, the residual 22:6n- retrieved from the tubes after 48 h of incubation was 45 % of the initial amount and > 90 % of the metabolized 22:6n- accumulated as 22: and 22: fa. subsequent biohydrogenation of these products seemed completely inhibited at this concentration.table amount of metabolized 22:6n- recovered in fame peaks. incubation was performed in growth medium containing autoclaved-uncentrifuged rumen fluid in the presence of 22:6n- with butyrivibrio proteoclasticus p tubes were withdrawn at 48 h of incubation to study the extent of 22:6n- biohydrogenation. peak numbers correspond to peaks in fig. 2 fig. effect of initial concentration of 22:6n- on its metabolism by butyrivibrio proteoclasticus p incubations were performed in growth medium containing 50 % of autoclaved-uncentrifuged rumen fluid for 48 h total vfa production was not affected by the initial concentration of 22:6n- except for the highest concentration. at the highest initial concentration vfa production after 48 h was higher compared to the lower concentrations, but the vfa profile was not affected. effect of presence of 22:6n- on 18:2n- metabolism by b. proteoclasticus p18 table  shows the conjugated linoleic acids, vaccenic acid and stearic acid formation from 40 μg/ml 18:2n- in presence of, low and high 22:6n- along with vfa formation and residual 22:6n- the formation of cla was observed as soon as inoculation was made. although formation of cla and va were not affected, conversion of va into sa was retarded in the presence of 22:6n- and this effect was dependent on 22:6n- concentration. volatile fatty acids production was not initiated until all the 18:2n- had been metabolized and converted into va regardless of presence of high or low 22:6n- both 18: formation and 22:6n- metabolism began after the initiation of vfa production. in the control tubes, 18: formation started at about 4 h as soon as vfa production began and this conversion was rapid thereafter. in the presence of low 22:6n-, b. proteoclasticus p initiated 22:6n- metabolism around 4 h, ahead of 18: formation. as a result, a small lag phase was observed in 18: formation after the initiation of vfa production. in presence of high 22:6n-, 18: formation was delayed to 12 h and this conversion was slower thereafter.table linoleic acid metabolism in presence of 22:6n- by b. proteoclasticus p incubation was performed in growth medium containing 40 μg/ml of 18:2n- in presence of 10 μg/ml and 40 μg/ml 22:6n- growth medium included 50 % of autoclaved-uncentrifuged rumen fluid and h gas was the headspace gas. value-presented in this table included the fatty acids and vfa of autoclaved-uncentrifuged rumen fluid used to prepare the growth medium cla conjugated linoleic acid, va vaccenic acid, vfa volatile fatty acids for each fatty acids, subscript * represents different compared to 0 mg/tube 22:6n- and ** represents different compared to and  mg/tube 22:6n- at the respective time point within each row means with different superscripts  are significantly different discussion butyrivibrio species are a genetically and functionally diverse group of bacteria present in gastrointestinal systems. based on the mechanism of butyrate formation, this group can be classified into two subgroups: vaccenic acid-producing and stearic acid-producing. accordingly, b. fibrisolvens and b. proteoclasticus are belonging to the vaccenic acid-producing and stearic acid-producing groups respectively. butyrivibrio fibrisolvens d and b. proteoclasticus p were chosen for this study as a representative from each group. however, the type species b. fibrisolvens d showed high butyrate kinase activity which is atypical to the majority of b. fibrisolvens isolates. previous studies carried out with b. fibrisolvens and b. proteoclasticus in m medium failed to show hydrogenation of 22:6n-. the reason for this is probably the high concentration of 22:6n- used in these studies which may have affected the growth of the bacteria. wallace et al. showed growth to be a prerequisite for b. proteoclasticus p in order to form stearic acid from 18:2n- if this is also true for 22:6n- biohydrogenation, failure to grow when 22:6n- is present in previous experiments possibly explains the absence of 22:6n- biohydrogenation. to reduce the inhibitory effect of 22:6n- on growth, the concentration of 22:6n- in the medium can be lowered or substances which lower the toxicity of 22:6n- should be included in the medium. harfoot et al. showed that biohydrogenation was stimulated by rumen fluid particles, enabling fa biohydrogenation by ruminal bacteria. experiments performed in our lab also showed addition of autoclaved-uncentrifuged rumen fluid stimulated the biohydrogenation of 22:6n- in mixed cultures of rumen bacteria. hence, we used autoclaved-uncentrifuged rumen fluid to prepare the growth medium. additionally we used a lower concentration of 22:6n- than the concentration reported previously. butyrivibrio fibrisolvens grew at low concentrations of 22:6n- without metabolizing it. maia et al. showed that growth of b. fibrisolvens jw was not initiated until all 18:2n- was metabolized and converted to va, such phenomenon was not observed with 22:6n- the failure of b. fibrisolvens d to metabolize 22:6n- in the current experiment might indicate they do not possess the necessary enzymes for 22:6n- biohydrogenation. however, further studies are warranted as b. fibrisolvens d is atypical to other b. fibrisolvens in general. in contrast with previous reports, we found b. proteoclasticus p is able to hydrogenate 22:6n- to our knowledge, this is the first report demonstrating 22:6n- biohydrogenation by a pure bacterial species. temporal changes in 22:6n- vfa and od during the course of the incubation might indicate b. proteoclasticus must be growing to biohydrogenate 22:6n- with little transformation occurring during the stationary phase. such observations were also made when demonstrating the capability of b. proteoclasticus p to form 18: from 18:2n-. stationary-phase bacteria were reported to be much less active in hydrogenation of 18:2n- than growing cells. this was explained by a limited supply of reducing equivalents once the energy sources in the medium had been depleted as the conversion of cla to va is nadh-dependent. it seems probable that a similar mechanism explains the variation in rate of disappearance of 22:6n- during the course of the incubation period. it is however not clear whether growth or activity per se is needed for biohydrogenation, or that the association of disappearance of 22:6n- vfa and od is merely a reflection of changes in the culture closely associated with growth and activity. the metabolism of 22:6n- resulted in the appearance of numerous compounds eluting during gc analysis between 22: and 22:6n- it has been speculated before that ruminal hydrogenation of 22:6n- yields intermediates with or double bonds, containing at least one trans double bond. the position of the double bonds in the most abundant 22: isomer suggest the formation of this intermediate involves izomerisation of the cis- double bond and reduction of the cis- double bond. initial izomerisation of the cis- double bond of 22:6n- would result in the formation of a conjugated product. however, in the present series of experiments, no accumulation of products eluting in the gc chromatogram with a retention time greater than 22:6n- was observed, a region where conjugated isomers of 22: would be expected to elute with the polar column used in the present study. the lack of accumulation of a conjugated fa might indicate they are transient products which did not accumulate at the time of sampling. alternatively, this might also indicate the initial product of 22:6n- metabolism is not a conjugated fa. the accumulation of 22: isomers produced during the initial stages of 22:6n- biohydrogenation was transient, when the initial 22:6n- concentration was low, and these isomers were subsequently hydrogenated to more saturated 22: 22: and 22: isomers. the existence of two isomers with identical double bond position indicates double bonds in the trans configuration must be present. it is unclear to what extent the increases in polyenoic trans fatty acids may offset some of the expected benefits from the enrichment of 22:6n- in ruminant derived foods. butyrivibrio proteoclasticus is the only known ruminal bacterium with the capacity to biohydrogenate 18-carbon fa to 18: previous incubations with rumen fluid have established that 22:6n- inhibits the complete hydrogenation of 18-carbon unsaturated fa causing trans 18: isomers to accumulate. this is thought to be related to the toxic effects of 22:6n- on the growth and metabolic activity of b. proteoclasticus. however, in vivo studies have failed to show the relationship between 18: flow to the duodenum and b. proteoclasticus dna. based on the findings of present study we suggest that b. proteoclasticus probably starts to hydrogenate 22:6n- before converting 18: isomers to 18: biohydrogenation is required to lower toxicity and therefore the reduction of products containing more double bonds are a greater priority which explains the lack of 18: reduction at time points where biohydrogenation of 22:6n- did occur. these findings possibly explain the lack of relationship of duodenal 18: to the duodenum and b. proteoclasticus dna.  butyrivibrio proteoclasticus p is able to hydrogenate 22:6n- in vitro. the rate and extent of biohydrogenation depended on the initial concentration of 22:6n- and the duration of incubation. several 22: 22: 22: and 22: isomers were identified. the products formed irrespective of the initial concentrations of 22:6n- and type of growth media suggests b. proteoclasticus p had a consistent pathway of 22:6n- hydrogenation. during the simultaneous presence of both 18:2n- and 22:6n- b. proteoclasticus p initiated 22:6n- metabolism before converting 18: isomers into 18: under the current culture conditions, b. fibrisolvens d failed to hydrogenate 22:6n- 
during the simultaneous presence of 18:2n- and 22:6n- b. proteoclasticus p initiated 22:6n- metabolism before converting 18: isomers into 18: for the first time, our study identified ruminal bacteria with the ability to hydrogenate 22:6n- the gradual appearance of intermediates indicates that biohydrogenation of 22:6n- by b. proteoclasticus p occurs by pathways of isomerization and hydrogenation resulting in a variety of unsaturated carbon fatty acids. biohydrogenation only started when volatile fatty acid production or growth of b. proteoclasticus p had been initiated, which might suggest that growth or metabolic activity is a prerequisite for the metabolism of 22:6n- the amount of 22:6n- hydrogenated was quantitatively recovered in several intermediate products eluting on the gas chromatogram between 22:6n- and 22: formation of neither 22: nor 22: conjugated fatty acids was observed during 22:6n- metabolism. extensive metabolism was observed at lower initial concentrations of 22:6n- whereas increasing concentrations of 22:6n- inhibited its metabolism. butyrivibrio fibrisolvens d failed to hydrogenate 22:6n- in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. butyrivibrio proteoclasticus p hydrogenated 22:6n- in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. growth of b. fibrisolvens was delayed at the higher 22:6n- concentrations; however, total volatile fatty acid production was not affected. stearic acid formation from 18:2n- by b. proteoclasticus p was retarded, but not completely inhibited, in the presence of 22:6n- and this effect was dependent on 22:6n- concentration.
 rumen microbes metabolize 22:6n- however, pathways of 22:6n- biohydrogenation and ruminal microbes involved in this process are not known. in this study, we examine the ability of the well-known rumen biohydrogenating bacteria, butyrivibrio fibrisolvens d and butyrivibrio proteoclasticus p to hydrogenate 22:6n-  butyrivibrio fibrisolvens d failed to hydrogenate 22:6n- in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. growth of b. fibrisolvens was delayed at the higher 22:6n- concentrations; however, total volatile fatty acid production was not affected. butyrivibrio proteoclasticus p hydrogenated 22:6n- in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. biohydrogenation only started when volatile fatty acid production or growth of b. proteoclasticus p had been initiated, which might suggest that growth or metabolic activity is a prerequisite for the metabolism of 22:6n- the amount of 22:6n- hydrogenated was quantitatively recovered in several intermediate products eluting on the gas chromatogram between 22:6n- and 22: formation of neither 22: nor 22: conjugated fatty acids was observed during 22:6n- metabolism. extensive metabolism was observed at lower initial concentrations of 22:6n- whereas increasing concentrations of 22:6n- inhibited its metabolism. stearic acid formation from 18:2n- by b. proteoclasticus p was retarded, but not completely inhibited, in the presence of 22:6n- and this effect was dependent on 22:6n- concentration.  for the first time, our study identified ruminal bacteria with the ability to hydrogenate 22:6n- the gradual appearance of intermediates indicates that biohydrogenation of 22:6n- by b. proteoclasticus p occurs by pathways of isomerization and hydrogenation resulting in a variety of unsaturated carbon fatty acids. during the simultaneous presence of 18:2n- and 22:6n- b. proteoclasticus p initiated 22:6n- metabolism before converting 18: isomers into 18: keywords 22:6n-3biohydrogenationbutyrivibriorumen fluidin vitrovfafund for scientific research-flanders and special research fund of the ghent universityissue-copyright-statement© the author 2016 