BACKGROUND
there is a renewed interest in seaweeds as potential biomass for sustainable biofuel production; particularly as their growth does not compete for agricultural land or require water for irrigation.
from the 1970s through to 1990s various groups were involved in assessing the suitability of seaweed  as a biomass for anaerobic digestion  to produce methane biofuel .
the overall result of these studies indicated that seaweeds were generally a suitable biomass for ad.
in the northern atlantic laminaria, fucus and ascophyllum species are particularly common brown algae found growing on the littoral and sub-littoral seashore.
few studies have examined ad of these seaweeds but moen et al.
<cit>  successfully demonstrated ad of laminaria hyperborea and ascophyllum nodosum, respectively.
the efficient hydrolysis of seaweed polysaccharides, particularly alginates, was however seen as a rate limiting step for efficient ad to proceed  <cit> .
animal manure slurry is often utilised as an inoculum for anaerobic digesters, but various reports have suggested that specific bacteria capable of fermenting marine phycocolloids may accelerate and increase biogas production from seaweeds .
williams et al.
<cit>  studied the ruminal microbiota of the north ronaldsay breed of sheep  and isolated bacteria and crude enzyme extracts that hydrolysed seaweed polysaccharides including laminarin, alginate, carrageenan, cellulose and to a lesser extent fucoidan.
additionally, in serial culture this rumen microbiota hydrolysed a range of seaweed polysaccharides and homogenates of l. hyperborea, mixed fucus spp.
and a. nodosum to produce methane and acetate.
this particular rumen microbiota and bacterial isolates were therefore considered to represent potential adjunct organisms or enzymes which may improve hydrolysis of seaweed components and thus improve the efficiency of seaweed anaerobic digestion for methane biofuel production.
with a long term aim of developing and characterising an optimal inoculum with which to degrade seaweeds in ad, we compared the ability of a range of microbial inocula, including north ronaldsay sheep rumen microbiota  <cit> , to effectively hydrolyse l. hyperborea and produce methane biofuel.
we examined these inocula in single-stage and two-stage fermenters in order to select the most productive inoculum for future optimisation studies in large scale  fermenter systems.
we studied single and two-stage systems because others have recommended  <cit>  that seaweed ad is better conducted in two stages.
in the first stage complex polysaccharide hydrolysis and acidogenesis to volatile fatty acids  by fast growing and low ph tolerant bacteria is encouraged.
the vfas produced in the leachate of such stage one fermenters occurs at ph  <dig>  to  <dig> .
the leachate is then neutralised and fed into phase ii fermenters where acetate is then further converted to methane by acetoclastic methanogens.
this two-stage system has been recommended for seaweed ad because seaweeds have a high sugar content and so tend to lower the fermenter ph with rapid growth of acidogenic bacteria, which may inhibit methanogenic archaea  <cit> .
we aimed to select the inoculum which was most efficient in both systems.
RESULTS
comparison of ad inocula in single-stage fermenters
all the microbial inocula  grew to give similar metabolic activity of around  <dig>  log <dig> fluorescence units  and so they clearly adapted to survive during in vitro fermentation of l. hyperborea using the mini-fermenters described in the methods section.
the l. hyperborea seaweed used in this study was added to fermenters as wet weight per volume.
samples of this feed were examined for the percentage total solids and volatile solids  on three separate occasions giving a mean  percentage of  <dig>   ts and  <dig>   vs, therefore showing the ability to reproducibly supply the same amount of wet weight feed to each fermenter.
all the inocula tested could hydrolyse l. hyperborea to some extent, but between  <dig> and 55% of vs were still undigested in individual fermenters.
figure 1b shows the mean percentage of vs used in triplicate fermenters of the same inoculum.
over the  <dig> week period inoculum  <dig> gave the highest overall utilisation of seaweed vs  and this was significantly higher  usage than by inocula  <dig>   <dig>   <dig>   <dig>  and  <dig>  but not by inocula  <dig> or  <dig> with means of  <dig>  and  <dig> % vs used, respectively.
triplicate fermenters of each inoculum  were tested randomly on separate occasions.
so it was interesting that all showed decreased biogas production in week  <dig>  and then recovered .
thereafter, all inocula generally produced biogas above initial  biogas production levels.
inocula  <dig>   <dig>   <dig> and  <dig>  had developed to give more biogas  than other ruminant derived inocula by week  <dig>  the percentage of methane and carbon dioxide in the biogas was analysed by gc and the volume of methane in biogas calculated from this.
all inocula were capable of producing methane from seaweed  but some gave significantly better yield than others.
inocula  <dig>   <dig>   <dig> and  <dig> had developed by week  <dig> to give higher volumes of methane production than other inocula  of  <dig>  ,  <dig>  ,  <dig>   and  <dig>   ml g- <dig> vs, respectively).
inoculum  <dig> was highest but only significantly higher than inocula  <dig> to  <dig> , not  <dig> or  <dig>  there was a drop in methane production by most inocula in week  <dig>  which coincides with the decrease in total biogas produced .
acetate production in fermenters over the  <dig> weeks was significantly greater by inoculum  <dig>  than by inocula  <dig>   <dig>   <dig>   <dig>  and  <dig>  but not inoculum  <dig> or  <dig>  inoculum  <dig> contained microbiota from inoculum  <dig> and so this is perhaps not surprising.
residual acetate from a single-stage fermenter can be converted to methane by acetoclastic methanogens in a second stage fermenter  <cit> .
the calculated potential methane yield from acetate  in the single-stage fermenters is given in table  <dig>  the theoretical methane production from acetate was calculated as  <dig> mole of acetate is converted into  <dig> mole of methane; and  <dig> mole of any gas at standard laboratory conditions  equals  <dig>  l of gas.
*acetate produced is the mean cumulative amount over four weeks from triplicate single-stage fermenters.
the total methane yield is the calculated methane yield from acetate added to the actual methane produced in the same fermenter over four weeks.
taking the actual methane produced and combining it with the calculated methane yield from acetate .
inoculum  <dig> gave the highest mean overall calculated methane yield , followed by inoculum  <dig>  then inoculum  <dig> .
phase i fermenter operation
even running at a ph adjusted daily with 2m naoh to be between  <dig>  and  <dig>   there was a substantial percentage of methane detected in the headspace biogas most weeks; with  <dig> / <dig> ;  <dig> / <dig>  and  <dig> / <dig>  % ch4/co <dig> in weeks  <dig>  and  <dig>  respectively .
the volume of biogas was not measured in this study.
that methane was produced at such low fermenter ph was in agreement with the study of vergara-fernández et al.
<cit>  who also detected methane gas production in a phase  <dig> fermenter for seaweed hydrolysis.
the growth of inoculum  <dig> used in the phase i fermenter was stable over the  <dig> week period with log <dig> fluorescein diacetate  values of  <dig> ,  <dig> ,  <dig>  and  <dig>  for weeks  <dig> to  <dig>  respectively; and so showed good survival of the inoculum under the phase i fermenter conditions.
the % of total solids  used over the  <dig> week fermentation was  <dig> ,  <dig> ,  <dig>  and  <dig> %, respectively; which was, at least initially, less than the utilisation by inoculum  <dig> during  <dig> weeks of the single-stage fermentations where there was an average of 67% usage .
the lower vs usage in the first two weeks of fermentation may indicate that the hydrolytic bacteria had to adapt to the low fermenter ph.
the acetate concentration in the pooled phase i fermenter leachate was  <dig>  g/l.
this supplied  <dig>  mmol of acetate to each phase ii fermenter over  <dig> changes of leachate  which theoretically could have given  <dig>  ml of methane.
comparison of inocula in phase ii fermenters
the volume of biogas  produced over four weeks from  <dig> ml of leachate  generated in a phase i fermenter ; the percentage of methane in that biogas and the total methane production in ml mmol- <dig> acetate over four weeks for each inoculum is shown in table  <dig>  inoculum  <dig> produced most methane , followed by inoculum  <dig> .
other inocula gave less than  <dig>  ml mmol- <dig> acetate of methane each and inocula  <dig>   <dig>  <dig> and  <dig> had particularly low methane levels.
*the volume of biogas  produced over four weeks from  <dig> ml of leachate  generated in a phase i fermenter.
discussion
all the inocula tested could hydrolyse l. hyperborea to some extent  but even by week  <dig>  inoculum  <dig>   <dig> and  <dig>  the most hydrolytic inocula, still had means of  <dig> ,  <dig>  and  <dig> % vs remaining, respectively.
it will be interesting to determine in further fermenter studies what the composition of this residue is.
it possibly contains a high percentage of alginates since moen et al.
<cit>  reported that between  <dig> and 50% of alginates were resistant to ad, depending upon levels of l. hyperborea polyphenols present and the ratio of calcium and sodium alginate salt forms.
the high utilisation of vs by inoculum  <dig>  and inoculum  <dig>  suggests that they had efficient seaweed polysaccharide hydrolysing bacteria.
moreover, this result is in agreement with preliminary studies  in which it was found that a human sewage ad effluent recovered in scotland was similarly most effective in ad of l. hyperborea; this was thus not a unique occurrence by the portuguese sourced inoculum  <dig>  inoculum  <dig> was previously shown to have bacteria producing polysaccharidases capable of hydrolysing seaweed polysaccharides  <cit>  and hehemann et al.
<cit>  have recently reported the presence of alginate lyase genes in human microbiota which have been derived from marine bacteria.
alginates and other seaweed polysaccharides are used as thickeners in foods, toothpastes and cosmetics and it is conceivable that human gut bacteria with hydrolytic enzymes survive in human sewage digestate .
therefore, both inoculum  <dig> and  <dig> may offer adjunct bacteria or enzymes to improve the efficiency of hydrolysis and are likely to have contributed to the high utilisation of vs by inoculum  <dig> .
since there was no concomitant reduction in the %vs used  the drop in biogas and methane production seen in all week  <dig> samples may have been due to an initial reduction in methanogenic archaea.
these are very slow growing and very strict anaerobes, the decrease of which may have been overcome by biofilm formation that was observed to have developed on the fermenter walls by week  <dig>
since inoculum  <dig> gave the highest levels of methane production by week  <dig> it would be advantageous to use this mixed inoculum for ad of l. hyperborea.
it would be interesting, in further studies, to add selected bacterial isolates rather than the whole rumen microbiota  to inoculum  <dig> to see if it improves vs usage.
also, inoculum  <dig> should be investigated to determine what endogenous alginate hydrolysers it may possess.
that inoculum  <dig> had high levels of acetate may be the reason why this inoculum  required most ph adjustment, reaching lows of  <dig>  within  <dig> hours of adjustment.
this may also be a reason why methanogenesis was progressively reduced in some replicates of these inocula, since methanogens grow poorly at ph less than  <dig>  and would possibly have begun to be washed out of the semi-continuous fermenter.
even without methane production, as long as the microbiota in an inoculum can degrade seaweed solids to produce vfas, these may be utilisable for biofuel and other valuable chemical feedstock production via the carboxylate platform  <cit> , where lack of methane production is desirable.
one example of this would be inoculum  <dig>  which could be a valuable source of hydrolytic bacteria for the latter platform.
if the hydraulic retention time in a single-stage fermenter is too short to allow all acetate produced to be converted to methane by acetoclastic methanogens then accumulated acetate can be fed into a second  fermenter with immobilised acetoclastic methanogens  <cit> .
inoculum  <dig> gave the highest methane production when considering actual methane produced plus the calculated methane production from acetate .
this further showed that inoculum  <dig> was the most productive inoculum in single-stage fermenters.
a two-stage fermenter system was operated in which inoculum  <dig> was used to produce acetate in a phase i fermenter.
the leachate of the latter was then fed into individual phase ii fermenters containing inocula  <dig> to  <dig> in order to determine which inoculum was most productive in acetoclastic methanogenesis.
inocula  <dig>   <dig>   <dig> and  <dig> gave poor methane production from acetate in the second phase of a two-stage fermenter system, indicating they do not have sufficient levels of acetoclastic methanogens for operation of a phase ii fermenter.
levels of acetoclastic methanogens in normal sheep rumen are for example very low; however, acetoclastic methanogens such as methanosarcina barkeri are present and can grow to significant levels if dietary changes such as high starch content occurs  <cit> .
the results for the phase ii fermenters complement the findings for the single stage fermenters where again inocula  <dig> and  <dig> were the most efficient inocula and inoculum  <dig> the best at producing methane from acetate .
inoculum  <dig> was the third best acetoclastic methanogenic inoculum  and it will be interesting to determine by metagenomics what archaea are present in inocula  <dig>   <dig> and  <dig> after growth in a phase ii fermenter.
in general, laboratory studies have found that methane yield is greater from brown algae than from green algae  <cit> .
there are very few studies conducted on ad of laminaria sp.
but hanssen et al.
<cit>  reported only a 36% usage of vs and  <dig> ml methane gvs- <dig> in an 8l fermentation volume with a retention time of  <dig> days.
more recently matsui et al.
<cit>  using a separated acidic and methanogenic phase commercial scale ad plant of 30m <dig> reported  <dig> ml methane g- <dig> wet weight seaweed.
in the present study the maximum % of vs used in any fermenter using inoculum  <dig>  was  <dig> % and a yield of  <dig> ml methane g- <dig> vs. however, the present study aimed to compare the ability of several inocula to ferment l. hyperborea under conditions of low feed rate  and high turnover of the liquid phase  in order to minimise the effects of acidification and product feedback inhibition previously noted by others  <cit> .
the study was not conducted under optimised ad conditions, but was designed to compare several inocula in one study to select the most efficient in the system used.
the methane yield by inoculum  <dig> will likely be increased in a large scale  continuously stirred fermenter optimised for feed rate, solids and liquids retention time and automatic ph adjustment; and this study is now intended by this group.
as hydrolysis of polysaccharides is the rate limiting step in seaweed ad, the need for more effective inocula for ad of brown seaweed is reflected in the conclusions of others  <cit> .
wargacki et al.
<cit>  to this end have recently cloned alginate lyase genes into e. coli and have produced bioethanol from seaweed at near 80% of the theoretical yield.
like others  <cit>  who found marine sediments were efficient inocula in seaweed ad we adopted, instead of gene cloning, to study complex microbiota in inocula from diverse locations in order to select efficient seaweed hydrolysers for ad, and found that a mixture of all the inocula  was most productive.
we are currently employing dissociative gradient gel electrophoresis and next generation sequencing on pcr amplified 16s rrna genes to characterise the microbial consortia present in the various inocula studied here.
dna was also extracted from each fermenter weekly and metagenomics will be used to examine the temporal and spatial development of microbial consortia.
this may indicate, for instance, which microbes from the various inocula developed in inoculum  <dig> to make it the most efficient in methane production.
it may indicate why methane production dropped in week  <dig> in all fermenters and it may also establish which hydrolytic bacteria and methanogenic archaea were active in single-stage and two-stage  fermenters.
CONCLUSIONS
several microbial inoculum were examined for the ability to hydrolyse polysaccharides and produce methane in ad of l. hyperborea.
all the inocula could hydrolyse l. hyperborea and produce methane to some extent but there were significant differences in the %vs used and the amount of methane produced thus showing that selection of an inoculum can improve ad.
inoculum  <dig>  which corresponded to a mixture of inocula 1– <dig>  proved to be highly hydrolytic and produced the most methane after ad in a single-stage or in phase ii of a two-stage digester system  .
this showed that careful selection of a complex microbial inoculum can increase the efficiency of seaweed ad, most likely due to combining effective polysaccharolytic bacteria, in this instance from north ronaldsay sheep rumen microbiota  <cit> , with appropriate methanogenic archaea which have developed in ad reactors, as in inoculum  <dig>  when examined individually, normal  sheep rumen microbiota  did not perform as well and neither did faecal microbiota from north ronaldsay sheep or normal sheep .
this group will now use inoculum  <dig> in optimised single stage and two stage ad systems to determine methane productivity.

 there is a renewed interest in seaweeds as potential biomass for sustainable biofuel production; particularly as their growth does not compete for agricultural land or require water for irrigation. from the 1970s through to 1990s various groups were involved in assessing the suitability of seaweed as a biomass for anaerobic digestion to produce methane biofuel. the overall result of these studies indicated that seaweeds were generally a suitable biomass for ad. in the northern atlantic laminaria, fucus and ascophyllum species are particularly common brown algae found growing on the littoral and sub-littoral seashore. few studies have examined ad of these seaweeds but moen et al. successfully demonstrated ad of laminaria hyperborea and ascophyllum nodosum, respectively. the efficient hydrolysis of seaweed polysaccharides, particularly alginates, was however seen as a rate limiting step for efficient ad to proceed. animal manure slurry is often utilised as an inoculum for anaerobic digesters, but various reports have suggested that specific bacteria capable of fermenting marine phycocolloids may accelerate and increase biogas production from seaweeds. williams et al. studied the ruminal microbiota of the north ronaldsay breed of sheep and isolated bacteria and crude enzyme extracts that hydrolysed seaweed polysaccharides including laminarin, alginate, carrageenan, cellulose and to a lesser extent fucoidan. additionally, in serial culture this rumen microbiota hydrolysed a range of seaweed polysaccharides and homogenates of l. hyperborea, mixed fucus spp. and a. nodosum to produce methane and acetate. this particular rumen microbiota and bacterial isolates were therefore considered to represent potential adjunct organisms or enzymes which may improve hydrolysis of seaweed components and thus improve the efficiency of seaweed anaerobic digestion for methane biofuel production. with a long term aim of developing and characterising an optimal inoculum with which to degrade seaweeds in ad, we compared the ability of a range of microbial inocula, including north ronaldsay sheep rumen microbiota, to effectively hydrolyse l. hyperborea and produce methane biofuel. we examined these inocula in single-stage and two-stage fermenters in order to select the most productive inoculum for future optimisation studies in large scale fermenter systems. we studied single and two-stage systems because others have recommended that seaweed ad is better conducted in two stages. in the first stage complex polysaccharide hydrolysis and acidogenesis to volatile fatty acids by fast growing and low ph tolerant bacteria is encouraged. the vfas produced in the leachate of such stage one fermenters occurs at ph to. the leachate is then neutralised and fed into phase ii fermenters where acetate is then further converted to methane by acetoclastic methanogens. this two-stage system has been recommended for seaweed ad because seaweeds have a high sugar content and so tend to lower the fermenter ph with rapid growth of acidogenic bacteria, which may inhibit methanogenic archaea. we aimed to select the inoculum which was most efficient in both systems.  comparison of ad inocula in single-stage fermenters all the microbial inocula grew to give similar metabolic activity of around log fluorescence units and so they clearly adapted to survive during in vitro fermentation of l. hyperborea using the mini-fermenters described in the methods section. the l. hyperborea seaweed used in this study was added to fermenters as wet weight per volume. samples of this feed were examined for the percentage total solids and volatile solids on three separate occasions giving a mean percentage of ts and vs, therefore showing the ability to reproducibly supply the same amount of wet weight feed to each fermenter. all the inocula tested could hydrolyse l. hyperborea to some extent, but between and 55% of vs were still undigested in individual fermenters. figure 1b shows the mean percentage of vs used in triplicate fermenters of the same inoculum. over the week period inoculum gave the highest overall utilisation of seaweed vs and this was significantly higher usage than by inocula and but not by inocula or with means of and % vs used, respectively. triplicate fermenters of each inoculum were tested randomly on separate occasions. so it was interesting that all showed decreased biogas production in week and then recovered. thereafter, all inocula generally produced biogas above initial biogas production levels. inocula and had developed to give more biogas than other ruminant derived inocula by week the percentage of methane and carbon dioxide in the biogas was analysed by gc and the volume of methane in biogas calculated from this. all inocula were capable of producing methane from seaweed but some gave significantly better yield than others. inocula and had developed by week to give higher volumes of methane production than other inocula of,, and ml g- vs, respectively). inoculum was highest but only significantly higher than inocula to, not or there was a drop in methane production by most inocula in week which coincides with the decrease in total biogas produced. acetate production in fermenters over the weeks was significantly greater by inoculum than by inocula and but not inoculum or inoculum contained microbiota from inoculum and so this is perhaps not surprising. residual acetate from a single-stage fermenter can be converted to methane by acetoclastic methanogens in a second stage fermenter. the calculated potential methane yield from acetate in the single-stage fermenters is given in table  the theoretical methane production from acetate was calculated as mole of acetate is converted into mole of methane; and mole of any gas at standard laboratory conditions equals l of gas. *acetate produced is the mean cumulative amount over four weeks from triplicate single-stage fermenters. the total methane yield is the calculated methane yield from acetate added to the actual methane produced in the same fermenter over four weeks. taking the actual methane produced and combining it with the calculated methane yield from acetate. inoculum gave the highest mean overall calculated methane yield, followed by inoculum then inoculum. phase i fermenter operation even running at a ph adjusted daily with 2m naoh to be between and there was a substantial percentage of methane detected in the headspace biogas most weeks; with /; / and / % ch4/co in weeks and respectively. the volume of biogas was not measured in this study. that methane was produced at such low fermenter ph was in agreement with the study of vergara-fernández et al. who also detected methane gas production in a phase fermenter for seaweed hydrolysis. the growth of inoculum used in the phase i fermenter was stable over the week period with log fluorescein diacetate values of,, and for weeks to respectively; and so showed good survival of the inoculum under the phase i fermenter conditions. the % of total solids used over the week fermentation was,, and %, respectively; which was, at least initially, less than the utilisation by inoculum during weeks of the single-stage fermentations where there was an average of 67% usage. the lower vs usage in the first two weeks of fermentation may indicate that the hydrolytic bacteria had to adapt to the low fermenter ph. the acetate concentration in the pooled phase i fermenter leachate was g/l. this supplied mmol of acetate to each phase ii fermenter over changes of leachate which theoretically could have given ml of methane. comparison of inocula in phase ii fermenters the volume of biogas produced over four weeks from ml of leachate generated in a phase i fermenter; the percentage of methane in that biogas and the total methane production in ml mmol- acetate over four weeks for each inoculum is shown in table  inoculum produced most methane, followed by inoculum. other inocula gave less than ml mmol- acetate of methane each and inocula and had particularly low methane levels. *the volume of biogas produced over four weeks from ml of leachate generated in a phase i fermenter. discussion all the inocula tested could hydrolyse l. hyperborea to some extent but even by week inoculum and the most hydrolytic inocula, still had means of, and % vs remaining, respectively. it will be interesting to determine in further fermenter studies what the composition of this residue is. it possibly contains a high percentage of alginates since moen et al. reported that between and 50% of alginates were resistant to ad, depending upon levels of l. hyperborea polyphenols present and the ratio of calcium and sodium alginate salt forms. the high utilisation of vs by inoculum and inoculum suggests that they had efficient seaweed polysaccharide hydrolysing bacteria. moreover, this result is in agreement with preliminary studies in which it was found that a human sewage ad effluent recovered in scotland was similarly most effective in ad of l. hyperborea; this was thus not a unique occurrence by the portuguese sourced inoculum inoculum was previously shown to have bacteria producing polysaccharidases capable of hydrolysing seaweed polysaccharides and hehemann et al. have recently reported the presence of alginate lyase genes in human microbiota which have been derived from marine bacteria. alginates and other seaweed polysaccharides are used as thickeners in foods, toothpastes and cosmetics and it is conceivable that human gut bacteria with hydrolytic enzymes survive in human sewage digestate. therefore, both inoculum and may offer adjunct bacteria or enzymes to improve the efficiency of hydrolysis and are likely to have contributed to the high utilisation of vs by inoculum. since there was no concomitant reduction in the %vs used the drop in biogas and methane production seen in all week samples may have been due to an initial reduction in methanogenic archaea. these are very slow growing and very strict anaerobes, the decrease of which may have been overcome by biofilm formation that was observed to have developed on the fermenter walls by week since inoculum gave the highest levels of methane production by week it would be advantageous to use this mixed inoculum for ad of l. hyperborea. it would be interesting, in further studies, to add selected bacterial isolates rather than the whole rumen microbiota to inoculum to see if it improves vs usage. also, inoculum should be investigated to determine what endogenous alginate hydrolysers it may possess. that inoculum had high levels of acetate may be the reason why this inoculum required most ph adjustment, reaching lows of within hours of adjustment. this may also be a reason why methanogenesis was progressively reduced in some replicates of these inocula, since methanogens grow poorly at ph less than and would possibly have begun to be washed out of the semi-continuous fermenter. even without methane production, as long as the microbiota in an inoculum can degrade seaweed solids to produce vfas, these may be utilisable for biofuel and other valuable chemical feedstock production via the carboxylate platform, where lack of methane production is desirable. one example of this would be inoculum which could be a valuable source of hydrolytic bacteria for the latter platform. if the hydraulic retention time in a single-stage fermenter is too short to allow all acetate produced to be converted to methane by acetoclastic methanogens then accumulated acetate can be fed into a second fermenter with immobilised acetoclastic methanogens. inoculum gave the highest methane production when considering actual methane produced plus the calculated methane production from acetate. this further showed that inoculum was the most productive inoculum in single-stage fermenters. a two-stage fermenter system was operated in which inoculum was used to produce acetate in a phase i fermenter. the leachate of the latter was then fed into individual phase ii fermenters containing inocula to in order to determine which inoculum was most productive in acetoclastic methanogenesis. inocula and gave poor methane production from acetate in the second phase of a two-stage fermenter system, indicating they do not have sufficient levels of acetoclastic methanogens for operation of a phase ii fermenter. levels of acetoclastic methanogens in normal sheep rumen are for example very low; however, acetoclastic methanogens such as methanosarcina barkeri are present and can grow to significant levels if dietary changes such as high starch content occurs. the results for the phase ii fermenters complement the findings for the single stage fermenters where again inocula and were the most efficient inocula and inoculum the best at producing methane from acetate. inoculum was the third best acetoclastic methanogenic inoculum and it will be interesting to determine by metagenomics what archaea are present in inocula and after growth in a phase ii fermenter. in general, laboratory studies have found that methane yield is greater from brown algae than from green algae. there are very few studies conducted on ad of laminaria sp. but hanssen et al. reported only a 36% usage of vs and ml methane gvs- in an 8l fermentation volume with a retention time of days. more recently matsui et al. using a separated acidic and methanogenic phase commercial scale ad plant of 30m reported ml methane g- wet weight seaweed. in the present study the maximum % of vs used in any fermenter using inoculum was % and a yield of ml methane g- vs. however, the present study aimed to compare the ability of several inocula to ferment l. hyperborea under conditions of low feed rate and high turnover of the liquid phase in order to minimise the effects of acidification and product feedback inhibition previously noted by others. the study was not conducted under optimised ad conditions, but was designed to compare several inocula in one study to select the most efficient in the system used. the methane yield by inoculum will likely be increased in a large scale continuously stirred fermenter optimised for feed rate, solids and liquids retention time and automatic ph adjustment; and this study is now intended by this group. as hydrolysis of polysaccharides is the rate limiting step in seaweed ad, the need for more effective inocula for ad of brown seaweed is reflected in the conclusions of others. wargacki et al. to this end have recently cloned alginate lyase genes into e. coli and have produced bioethanol from seaweed at near 80% of the theoretical yield. like others who found marine sediments were efficient inocula in seaweed ad we adopted, instead of gene cloning, to study complex microbiota in inocula from diverse locations in order to select efficient seaweed hydrolysers for ad, and found that a mixture of all the inocula was most productive. we are currently employing dissociative gradient gel electrophoresis and next generation sequencing on pcr amplified 16s rrna genes to characterise the microbial consortia present in the various inocula studied here. dna was also extracted from each fermenter weekly and metagenomics will be used to examine the temporal and spatial development of microbial consortia. this may indicate, for instance, which microbes from the various inocula developed in inoculum to make it the most efficient in methane production. it may indicate why methane production dropped in week in all fermenters and it may also establish which hydrolytic bacteria and methanogenic archaea were active in single-stage and two-stage fermenters.  several microbial inoculum were examined for the ability to hydrolyse polysaccharides and produce methane in ad of l. hyperborea. all the inocula could hydrolyse l. hyperborea and produce methane to some extent but there were significant differences in the %vs used and the amount of methane produced thus showing that selection of an inoculum can improve ad. inoculum which corresponded to a mixture of inocula 1– proved to be highly hydrolytic and produced the most methane after ad in a single-stage or in phase ii of a two-stage digester system. this showed that careful selection of a complex microbial inoculum can increase the efficiency of seaweed ad, most likely due to combining effective polysaccharolytic bacteria, in this instance from north ronaldsay sheep rumen microbiota, with appropriate methanogenic archaea which have developed in ad reactors, as in inoculum when examined individually, normal sheep rumen microbiota did not perform as well and neither did faecal microbiota from north ronaldsay sheep or normal sheep. this group will now use inoculum in optimised single stage and two stage ad systems to determine methane productivity. 
the study therefore showed that selection and inclusion of efficient polysaccharide hydrolysing bacteria and methanogenic archaea in an inoculum offer increased methane productivity in ad of l. hyperborea. inoculum also gave the highest acetate production in a single-stage fermenter ad system and produced most methane in phase ii of a two-stage ad system. however, an inoculum of slurry from a human sewage anaerobic digester, one of rumen contents from seaweed-eating north ronaldsay sheep and inoculum used most seaweed volatile solids, suggesting that these each had efficient seaweed polysaccharide digesting bacteria. all the inocula could degrade l. hyperborea and produce methane to some extent. this inoculum will now being tested in larger scale continuously stirred reactors optimised for feed rate and retention time to determine maximum methane production under single-stage and two-stage ad systems. the hydrolysis of seaweed polysaccharides is the rate limiting step in anaerobic digestion of seaweeds.
 the hydrolysis of seaweed polysaccharides is the rate limiting step in anaerobic digestion of seaweeds. seven different microbial inocula and a mixture of these were therefore compared in triplicate, each grown over four weeks in static culture for the ability to degrade laminaria hyperborea seaweed and produce methane through ad.  all the inocula could degrade l. hyperborea and produce methane to some extent. however, an inoculum of slurry from a human sewage anaerobic digester, one of rumen contents from seaweed-eating north ronaldsay sheep and inoculum used most seaweed volatile solids, suggesting that these each had efficient seaweed polysaccharide digesting bacteria. the human sewage inoculum, an inoculum of anaerobic marine mud mixed with rotting seaweed and inoculum all developed to give higher volumes of methane,compared to other inocula. inoculum also gave the highest acetate production in a single-stage fermenter ad system and produced most methane in phase ii of a two-stage ad system.  overall inoculum was found to be the most efficient inoculum for ad of seaweed. the study therefore showed that selection and inclusion of efficient polysaccharide hydrolysing bacteria and methanogenic archaea in an inoculum offer increased methane productivity in ad of l. hyperborea. this inoculum will now being tested in larger scale continuously stirred reactors optimised for feed rate and retention time to determine maximum methane production under single-stage and two-stage ad systems. seaweedmicrobial inoculumanaerobic digestionlaminaria hyperboreamethane 