BACKGROUND
an increased use of bio- and renewable diesel as replacement for petroleum-derived diesel in recent years has led to increased interest in the production of microbial lipids for fuel use; the aim being to produce lipids in micro-organisms using substrates which do not directly compete with food uses, as plant oils do.
both phototrophic and heterotrophic micro-organisms have been considered.
among heterotrophic micro-organisms, the optimal production organism would be able to use the carbohydrates present in plant biomass, cellulose and hemicellulose, or the c <dig> and c <dig> sugars released by hydrolysis of plant biomass, primarily glucose and xylose, but also including arabinose, galactose and mannose.
numerous yeast and filamentous fungi accumulate high concentrations of lipids in their biomass and yeast belonging to the species rhodosporidium , in particular r. toruloides, have often been used as model organisms for lipid production.
concentrations of lipid over 50% dw are generally reported
<cit> .
both glucose and xylose are metabolised
<cit> .
most experiments have included yeast extract and/or peptone in the medium
<cit> .
although these are a convenient source of complex nutrients for rapid growth at lab scale, their cost may be prohibitive for a bulk product on an industrial scale and ammonium sulphate or the cheaper corn steep solids/liquid  could be preferable alternatives.
biomass hydrolysates will themselves supply trace elements, but their complex nitrogen may not be readily available.
glucose is the most commonly supplied carbohydrate in basic studies of lipid production in yeast
<cit> .
sucrose, xylose, fructose, galactose and maltose
<cit>  have also been studied, but there is only one report of lipid production from arabinose
<cit> , even though it contributes  <dig> to 18% of the total carbohydrates in plant hydrolysates.
production of lipids from substrate mixtures have only been reported for xylose with glycerol
<cit> , glycerol with glucose or xylose, or xylose with glucose
<cit> .
waste waters
<cit>  and hydrolysates
<cit>  are increasingly being used directly for lipid production.
an industrial lipid production process should make use of high cell density cultures, but the oxygen demand of the process limits the biomass density obtainable in small scale, flask cultures.
high cell density cultures have been achieved for r. toruloides and cryptococcus curvatus using fed-batch and repeated batch techniques
<cit> .
various feeding strategies have been described.
hassan et al.
<cit>  demonstrated that  <dig> g l− <dig> biomass containing 53%  lipid could be achieved with c. curvatus, at ~ <dig>  g l− <dig> h− <dig>  in a culture started with c/n ~ <dig> but fed a solution of glucose not containing nitrogen.
li et al.
<cit>  similarly used a batch culture with a low c/n, fed with nitrogen free glucose solution to obtain high biomass  and lipid production  of r. toruloides y <dig> in complex medium.
discontinuous, pulsed feeding was used, rather than continuous.
constant and pulsed feeding have been directly compared for r. toruloides y <dig> in complex medium, for which continuous feeding was shown to give slightly better lipid accumulation  than pulsed feeding
<cit> .
pulsed glucose addition was also used with r. minuta <cit> .
however, although ~ <dig> g glucose l− <dig> were provided, biomass did not accumulate above  <dig> g l− <dig>  a repeated batch process has been shown to be effective in sustaining lipid production by r. glutinis at low biomass concentration from palm oil effluent
<cit> .
in this study r. toruloides was grown to high cell density in batch or fed-batch cultures in chemically defined medium with glucose, xylose or arabinose alone or in combination providing the carbon source.
continuous and discontinuous feed supply with or without nitrogen was considered for the fed-batch cultures, using c/ns which allowed good lipid production rates in batch cultures.
the results demonstrated that both batch and fed-batch cultures could be similarly efficient for lipid production by r. toruloides and that the presence of arabinose may have a strong negative effect on lipid production.
RESULTS
lipid production was compared at the time of maximum total  lipid production.
for batch cultures these times are reported in table
<dig> and figure
<dig>  typically maximum total lipid in batch culture was observed at the time when all carbohydrate had been consumed .
however, in arabinose containing cultures, lipid content decreased before total consumption of the arabinose and thus while cells were still growing.
lipid production rate, yield and percent dry weight and biomass concentration were determined at the time of maximum total lipid produced.
carbon was provided as  <dig> g l− <dig> glucose or xylose and 2so <dig> was provided as nitrogen.
cultures were grown in defined medium at ph  <dig> , 30°c,  <dig> rpm,  <dig>  vvm aeration.
characterisation of lipid production with glucose, xylose or arabinose as carbon sources at low substrate concentrations
the lipid content in the biomass increased with increase in the c/n from  <dig> to  <dig> with both glucose and xylose as carbon source , but lipid content at high c/n was greater with glucose than with xylose .
the yield of lipid on substrate also increased with increasing c/n and was higher on glucose than on xylose .
approximately  <dig> g biomass l− <dig> was produced from  <dig> g substrate l− <dig>  but biomass production was slightly  reduced at c/n  <dig> compared to c/n  <dig> .
the highest rate of lipid production was observed at c/n  <dig> on glucose, but was similar at c/n  <dig> and  <dig> on xylose .
since lipid production rates were similar or higher at c/n  <dig> than at c/n  <dig> and culture times were shorter, further experiments were carried out with c/n  <dig>  corn steep solids could be partially or completely substituted for ammonium as the nitrogen source with no effect on lipid production, lipid production rate or yield of lipid on substrate .
maximum lipid and fatty acid production from glucose, xylose and arabinose were compared at c/n  <dig> with  <dig> g carbohydrate l− <dig> .
r. toruloides cbs <dig> accumulated a higher proportion of lipid in the biomass when grown on glucose  than on either xylose  or arabinose , and a higher proportion of the lipid was made up of fatty acids.
lipid production rate and yield were also higher on glucose than on xylose or arabinose .
the fatty acids produced on arabinose contained more 16: <dig>  and 18: <dig> , and less 18: <dig>  fatty acids, than fatty acids produced on glucose  or xylose .
the specific growth rate was much lower on arabinose  and xylose  than on glucose  and less biomass was produced .
high substrate concentration in batch culture
total biomass and lipid production increased in proportion with the concentration of glucose provided at c/n  <dig> .
the proportion of lipid in the biomass and the yield of lipid from glucose remained approximately constant at 51% dw and  <dig>  g lipid − <dig>  respectively .
the lipid production rate was higher with  <dig> or  <dig> g glucose l− <dig>  compared to  <dig> g glucose l− <dig> .
when cultures were provided  <dig> g glucose l− <dig> the lag phase was increased approximately  <dig> h compared to cultures with  <dig> or  <dig> g glucose l− <dig> , although the specific growth rate was not affected.
we observed that a high rate of lipid synthesis  was maintained between  <dig> and  <dig> h in cultures provided  <dig> g glucose l− <dig>  and that the overall rate of lipid synthesis in these cultures was  <dig>  g l− <dig> h− <dig> when the lag phase was excluded, suggesting that higher production rates could be achieved with  <dig> g glucose l− <dig> than with  <dig> g l− <dig> if osmotically adapted cells were used.
none-the-less, to avoid long lag phases, ~ <dig> g substrate l− <dig> was used in the batch phase of fed-batch cultures.
substrate concentration had little effect on the fatty acid profile .
r. toruloides was also grown in mixtures of glucose, xylose and arabinose to provide a total of ~ <dig> g carbohydrate l− <dig>  one mixture contained  <dig> g glucose l− <dig>   <dig> g xylose l− <dig> and  <dig> g arabinose l− <dig>  similar to the ratio of sugars obtained from acid hydrolysed dried distillers grain solids  with no separation of c <dig> and c <dig> carbohydrates.
the second mixture contained  <dig> g glucose l− <dig>   <dig> g xylose l− <dig> and  <dig> g arabinose l− <dig>  similar to the ratio of sugars expected in a c5-enriched hydrolysate.
the carbohydrates were consumed consecutively, with glucose being consumed first, then xylose, and finally arabinose, which was not completely consumed .
biomass production was similar to that on arabinose  alone .
r. toruloides produced less lipid , at a lower rate  and yield , when grown on mixtures of glucose, xylose and arabinose, compared to growth on glucose  as sole carbon source, although more lipid was produced than on arabinose .
lipid content  and yield on carbohydrate were similar to that of cells grown on  <dig> g xylose l− <dig> .
cultures receiving a high proportion of glucose in the mixture did not produce more lipid than those with the low proportion of glucose.
however, cells provided a higher proportion of glucose in the carbohydrate mixture had a profile of fatty acids which was more similar to those observed in glucose cultures than cells which received a high proportion of xylose and only low glucose in the mixture .
fed-batch strategies for lipid production with r. toruloides
four strategies were tested for lipid production in fed-batch cultures with r. toruloides cbs <dig> , using either glucose alone or a mixture of glucose, xylose and arabinose .
in one strategy, nitrogen was provided in the batch phase at c/n  <dig>  comparable to the lowest c/n tested in batch cultures, but none in the feed, resulting in a gradual reduction in the c/n to ~ <dig>  for the other three strategies, nitrogen was provided at c/n  <dig> in the batch phase, as in high cell density batch cultures, or c/n  <dig>  and also at the same c/n in the feed.
providing c/n  <dig> in the batch phase enabled more rapid accumulation of biomass than with c/n  <dig> or  <dig>  lipid and fatty acid accumulation was also high using this strategy, particularly with the mixture of substrates, but biomass  and lipid  production stopped after 70– <dig> h and not all substrate was consumed .
the biomass contained up to 75% lipid on glucose and 58% lipid on mixed substrates .
lipid production rate, yield and percent dry weight were determined at the time of maximum total lipid produced .
carbon was provided as glucose or a mixture of glucose, xylose and arabinose  and 2so <dig> was provided as nitrogen.
cultures were grown in defined medium at ph  <dig> , 30°c,  <dig> rpm,  <dig>  vvm aeration.
since high rates of lipid production were observed in batch cultures with c/n  <dig>  fed-batch cultures with constant provision of c/n  <dig> were also tested.
fed-batch cultures with c/n  <dig> were tested to determine whether lipid content in the biomass would be increased, as predicted from batch cultures.
biomass production was sustained throughout the cultivation when nitrogen was provided in the feed at c/n  <dig> or  <dig>  and 47– <dig> g biomass l− <dig> were produced from glucose,  <dig> g biomass l− <dig> from mixed substrates, regardless of the c/n ratio or whether the feed was provided at a constant rate or in pulses.
the lipid content of the biomass was lower than in the cultures which received no n in the feed , but total lipid  and fatty acid  production was similar to that in the cultures with no n in the feed .
substrates were more completely utilised in cultures which received n in the feed than in those which did not.
increasing the c/n from  <dig> to  <dig> resulted in higher concentrations of residual substrate and lipid content in the biomass was only slightly increased with glucose as substrate, but not with glucose, xylose and arabinose .
lipid production was similar, but fatty acid production was slightly less efficient when the substrate was provided in pulses, rather than continually .
the yield of lipid on carbohydrate was reduced in all fed-batch cultures, compared to batch cultures, with the exception of glucose fed cultures which received no n in the feed .
lipid production rates were generally similar in fed-batch cultures to those observed in comparable batch cultures .
however, on mixed substrates provided c/n  <dig> during the batch phase and no n in the feed the lipid production rate was  <dig>  g l− <dig> h− <dig>  which was almost as high as on glucose alone .
the feeding strategy  did not affect the proportions of fatty acids extracted from the biomass .
provision of xylose and arabinose in the feed in addition to glucose generally reduced the proportion of 18: <dig> fatty acids and increased the proportion of 16: <dig>  and 18: <dig> fatty acids, compared to cultures receiving glucose alone .
fatty acid profiles were similar to those of batch cultures receiving the same carbon source .
discussion
r. toruloides is a known lipogenic yeast and the results presented here confirm its ability to accumulate over 75% of its biomass as lipid, with 62% as fatty acids.
however, lipid accumulation was strongly affected by the nature of the carbon source provided.
greater lipid accumulation occurred on glucose or xylose than on arabinose , as has also been observed for trichosporon cutaneum .
on mixtures of carbohydrates, lipid accumulation was lower than on glucose, but only slightly lower than observed on xylose , reflecting the sequential utilisation of the sugars, with glucose being consumed while more nitrogen was available and xylose and arabinose being consumed during the period of nitrogen restricted growth.
further, arabinose consumption was incomplete, and thus the effective c/n was less than intended, contributing to the reduced lipid accumulation.
interestingly, lipid production was similar regardless of whether the ratio of glucose to xylose and arabinose was high  or low .
the low lipid production on mixtures of carbohydrates should be taken into account when assessing the possibilities of producing lipids directly from plant biomass hydrolysates.
low lipid content in r. glutinis  and r. toruloides  has indeed been observed when lipids have been produced directly from plant hydrolysates.
it is also important to note that the fatty acid profile was not the same on mixtures of glucose, xylose and arabinose as on glucose alone.
thus in plant biomass hydrolysates one should expect a lower proportion of stearic acid , and more linoleic  and possibly more palmitic  fatty acids than in pure glucose.
some caution should be taken when extrapolating results from glucose grown yeast to what can be expected from plant biomass hydrolysate.
although arabinose was not generally completely metabolised its presence in the medium appeared to have a strong influence on the fatty acid profile.
r. toruloides cbs <dig>  was not as productive as r. toruloides y <dig> .
this may reflect difference between the strains, the presence of yeast extract  in the y <dig> processes, and/or a lower c/n during the batch phase for the y <dig> processes
<cit> .
although cbs <dig> has been studied extensively as a model of lipid production in yeast, it is not necessarily the best strain for production.
it is none-the-less worth noting that during the latter stages of batch culture with  <dig> or  <dig> g glucose l− <dig> it was producing lipid at  <dig>  g l− <dig> h− <dig>  indicating that it has more potential than has so far been achieved in batch or fed-batch cultivations.
high lipid accumulation occurred on chemically defined medium and did not require addition of expensive complex ingredients such as yeast extract, even in high cell density batch or fed-batch cultures.
if a complex nitrogen source is desired, css were found to be suitable for good lipid production.
although most studies of lipid production from waste streams have included yeast extract or peptone in the medium, xue et al.
<cit>  demonstrated that no nitrogen or other supplements were needed to produce lipid in r. glutinis on monosodium glutamate wastewater.
the inhibitory effect of high  glucose concentrations in batch culture has also been observed by li et al.
<cit>  for r. toruloides y <dig>  but only for concentrations above  <dig> g l− <dig>  this inhibition could be avoided by using a repeated or sequential batch process in which a large proportion  of the culture would be removed and replaced with fresh medium at the time when lipid and biomass content would be maximal, as illustrated with r. glutinis on palm oil mill effluent
<cit> .
alternatively, inhibition by high substrate concentrations could be avoided by utilising fed-batch and repeated fed-batch cultivation, as shown here and for r. toruloides y4
<cit>  and c. curvatus <cit> .
as reported by zhao et al.
<cit>  for r. toruloides y <dig>  we found that continuous feeding was preferable to discontinuous, intermittent feeding for high fatty acid accumulation in cbs <dig> .
we also observed that the best rates and yields of lipid production were achieved using glucose feed which lacked nitrogen .
however, in contrast to li et al.
<cit> , zhao et al.
<cit> , and hassan et al.
<cit> , biomass production was not sustained under these conditions , substrate was not completely consumed and the rates and yields would not be expected to be sustainable in a repeated fed-batch process.
this probably reflects the absence of yeast extract or other complex ingredients in our process, along with the use of a higher initial c/n during the batch phase than has been used previously.
if reduction of the organic carbon content of the feed is also important, providing nitrogen in the feed could be beneficial, even though there could be some loss in yield.
this is even more apparent if the fatty acid content is taken into account, rather than just the total lipid, since the rate of fatty acid production was similar with or without nitrogen in the feed .
however, it is likely that either process would benefit from a lower c/n during the batch phase, to enable more rapid biomass accumulation.
it is also likely that feed derived from plant biomass hydrolysates will always contain some metabolisable nitrogen.
further, a repeated batch process with e.g.
<dig> g glucose l− <dig>  c/n  <dig>  would be more efficient in converting glucose to lipid if a rate of  <dig>  g lipid l− <dig> h− <dig> would be sustained without the lag phase.
in contrast, fed-batch cultivation with no nitrogen in the feed was clearly the best option for producing lipids from mixed carbohydrate substrates including glucose, xylose and arabinose .
as with glucose fed-batch cultures, one can predict that a lower c/n in the batch phase, possibly combined with a high c/n feed, would be necessary to achieve more complete utilisation of the substrate and to create a process which would be sustainable as a repeated fed-batch process.
CONCLUSIONS
lipid production from c <dig> carbohydrates was less efficient in r. toruloides cbs <dig> than from glucose, with production from xylose being better than from arabinose.
the presence of arabinose in media containing mixtures of glucose, xylose and arabinose resulted in less lipid accumulation than in medium with either glucose or xylose alone.
thus plant biomass hydrolysates with low arabinose content would be desirable for lipid production using r. toruloides cbs <dig>
high biomass and lipid production were achieved in both batch and fed-batch cultures with glucose as carbon source, and a repeated batch process would be expected to be as efficient as a fed-batch or repeated fed-batch process.
however, for lipid production from mixtures of carbohydrates, fed-batch cultivation was preferable.
in fed-batch processes, provision of extra nitrogen during the batch phase was useful to provide rapid biomass production, with lipid accumulating during the feeding phase.
provision of nitrogen in the feed enabled more complete utilisation of the carbohydrate, but with a lower yield of lipid on carbohydrate, than in its absence.
complete substrate utilisation would be important in lipid production from waste streams, to ensure sufficient reduction in the carbon content of the stream.
the feeding strategy did not affect the relative proportion of different fatty acids in the lipid, but the presence of c <dig> carbohydrates did.

 an increased use of bio- and renewable diesel as replacement for petroleum-derived diesel in recent years has led to increased interest in the production of microbial lipids for fuel use; the aim being to produce lipids in micro-organisms using substrates which do not directly compete with food uses, as plant oils do. both phototrophic and heterotrophic micro-organisms have been considered. among heterotrophic micro-organisms, the optimal production organism would be able to use the carbohydrates present in plant biomass, cellulose and hemicellulose, or the c and c sugars released by hydrolysis of plant biomass, primarily glucose and xylose, but also including arabinose, galactose and mannose. numerous yeast and filamentous fungi accumulate high concentrations of lipids in their biomass and yeast belonging to the species rhodosporidium, in particular r. toruloides, have often been used as model organisms for lipid production. concentrations of lipid over 50% dw are generally reported. both glucose and xylose are metabolised. most experiments have included yeast extract and/or peptone in the medium. although these are a convenient source of complex nutrients for rapid growth at lab scale, their cost may be prohibitive for a bulk product on an industrial scale and ammonium sulphate or the cheaper corn steep solids/liquid could be preferable alternatives. biomass hydrolysates will themselves supply trace elements, but their complex nitrogen may not be readily available. glucose is the most commonly supplied carbohydrate in basic studies of lipid production in yeast. sucrose, xylose, fructose, galactose and maltose have also been studied, but there is only one report of lipid production from arabinose, even though it contributes to 18% of the total carbohydrates in plant hydrolysates. production of lipids from substrate mixtures have only been reported for xylose with glycerol, glycerol with glucose or xylose, or xylose with glucose. waste waters and hydrolysates are increasingly being used directly for lipid production. an industrial lipid production process should make use of high cell density cultures, but the oxygen demand of the process limits the biomass density obtainable in small scale, flask cultures. high cell density cultures have been achieved for r. toruloides and cryptococcus curvatus using fed-batch and repeated batch techniques. various feeding strategies have been described. hassan et al. demonstrated that g l− biomass containing 53% lipid could be achieved with c. curvatus, at ~ g l− h− in a culture started with c/n ~ but fed a solution of glucose not containing nitrogen. li et al. similarly used a batch culture with a low c/n, fed with nitrogen free glucose solution to obtain high biomass and lipid production of r. toruloides y in complex medium. discontinuous, pulsed feeding was used, rather than continuous. constant and pulsed feeding have been directly compared for r. toruloides y in complex medium, for which continuous feeding was shown to give slightly better lipid accumulation than pulsed feeding. pulsed glucose addition was also used with r. minuta. however, although ~ g glucose l− were provided, biomass did not accumulate above g l− a repeated batch process has been shown to be effective in sustaining lipid production by r. glutinis at low biomass concentration from palm oil effluent. in this study r. toruloides was grown to high cell density in batch or fed-batch cultures in chemically defined medium with glucose, xylose or arabinose alone or in combination providing the carbon source. continuous and discontinuous feed supply with or without nitrogen was considered for the fed-batch cultures, using c/ns which allowed good lipid production rates in batch cultures. the results demonstrated that both batch and fed-batch cultures could be similarly efficient for lipid production by r. toruloides and that the presence of arabinose may have a strong negative effect on lipid production.  lipid production was compared at the time of maximum total lipid production. for batch cultures these times are reported in table and figure typically maximum total lipid in batch culture was observed at the time when all carbohydrate had been consumed. however, in arabinose containing cultures, lipid content decreased before total consumption of the arabinose and thus while cells were still growing. lipid production rate, yield and percent dry weight and biomass concentration were determined at the time of maximum total lipid produced. carbon was provided as g l− glucose or xylose and 2so was provided as nitrogen. cultures were grown in defined medium at ph, 30°c, rpm, vvm aeration. characterisation of lipid production with glucose, xylose or arabinose as carbon sources at low substrate concentrations the lipid content in the biomass increased with increase in the c/n from to with both glucose and xylose as carbon source, but lipid content at high c/n was greater with glucose than with xylose. the yield of lipid on substrate also increased with increasing c/n and was higher on glucose than on xylose. approximately g biomass l− was produced from g substrate l− but biomass production was slightly reduced at c/n compared to c/n. the highest rate of lipid production was observed at c/n on glucose, but was similar at c/n and on xylose. since lipid production rates were similar or higher at c/n than at c/n and culture times were shorter, further experiments were carried out with c/n corn steep solids could be partially or completely substituted for ammonium as the nitrogen source with no effect on lipid production, lipid production rate or yield of lipid on substrate. maximum lipid and fatty acid production from glucose, xylose and arabinose were compared at c/n with g carbohydrate l−. r. toruloides cbs accumulated a higher proportion of lipid in the biomass when grown on glucose than on either xylose or arabinose, and a higher proportion of the lipid was made up of fatty acids. lipid production rate and yield were also higher on glucose than on xylose or arabinose. the fatty acids produced on arabinose contained more 16: and 18:, and less 18: fatty acids, than fatty acids produced on glucose or xylose. the specific growth rate was much lower on arabinose and xylose than on glucose and less biomass was produced. high substrate concentration in batch culture total biomass and lipid production increased in proportion with the concentration of glucose provided at c/n. the proportion of lipid in the biomass and the yield of lipid from glucose remained approximately constant at 51% dw and g lipid − respectively. the lipid production rate was higher with or g glucose l− compared to g glucose l−. when cultures were provided g glucose l− the lag phase was increased approximately h compared to cultures with or g glucose l−, although the specific growth rate was not affected. we observed that a high rate of lipid synthesis was maintained between and h in cultures provided g glucose l− and that the overall rate of lipid synthesis in these cultures was g l− h− when the lag phase was excluded, suggesting that higher production rates could be achieved with g glucose l− than with g l− if osmotically adapted cells were used. none-the-less, to avoid long lag phases, ~ g substrate l− was used in the batch phase of fed-batch cultures. substrate concentration had little effect on the fatty acid profile. r. toruloides was also grown in mixtures of glucose, xylose and arabinose to provide a total of ~ g carbohydrate l− one mixture contained g glucose l− g xylose l− and g arabinose l− similar to the ratio of sugars obtained from acid hydrolysed dried distillers grain solids with no separation of c and c carbohydrates. the second mixture contained g glucose l− g xylose l− and g arabinose l− similar to the ratio of sugars expected in a c5-enriched hydrolysate. the carbohydrates were consumed consecutively, with glucose being consumed first, then xylose, and finally arabinose, which was not completely consumed. biomass production was similar to that on arabinose alone. r. toruloides produced less lipid, at a lower rate and yield, when grown on mixtures of glucose, xylose and arabinose, compared to growth on glucose as sole carbon source, although more lipid was produced than on arabinose. lipid content and yield on carbohydrate were similar to that of cells grown on g xylose l−. cultures receiving a high proportion of glucose in the mixture did not produce more lipid than those with the low proportion of glucose. however, cells provided a higher proportion of glucose in the carbohydrate mixture had a profile of fatty acids which was more similar to those observed in glucose cultures than cells which received a high proportion of xylose and only low glucose in the mixture. fed-batch strategies for lipid production with r. toruloides four strategies were tested for lipid production in fed-batch cultures with r. toruloides cbs, using either glucose alone or a mixture of glucose, xylose and arabinose. in one strategy, nitrogen was provided in the batch phase at c/n comparable to the lowest c/n tested in batch cultures, but none in the feed, resulting in a gradual reduction in the c/n to ~ for the other three strategies, nitrogen was provided at c/n in the batch phase, as in high cell density batch cultures, or c/n and also at the same c/n in the feed. providing c/n in the batch phase enabled more rapid accumulation of biomass than with c/n or lipid and fatty acid accumulation was also high using this strategy, particularly with the mixture of substrates, but biomass and lipid production stopped after 70– h and not all substrate was consumed. the biomass contained up to 75% lipid on glucose and 58% lipid on mixed substrates. lipid production rate, yield and percent dry weight were determined at the time of maximum total lipid produced. carbon was provided as glucose or a mixture of glucose, xylose and arabinose and 2so was provided as nitrogen. cultures were grown in defined medium at ph, 30°c, rpm, vvm aeration. since high rates of lipid production were observed in batch cultures with c/n fed-batch cultures with constant provision of c/n were also tested. fed-batch cultures with c/n were tested to determine whether lipid content in the biomass would be increased, as predicted from batch cultures. biomass production was sustained throughout the cultivation when nitrogen was provided in the feed at c/n or and 47– g biomass l− were produced from glucose, g biomass l− from mixed substrates, regardless of the c/n ratio or whether the feed was provided at a constant rate or in pulses. the lipid content of the biomass was lower than in the cultures which received no n in the feed, but total lipid and fatty acid production was similar to that in the cultures with no n in the feed. substrates were more completely utilised in cultures which received n in the feed than in those which did not. increasing the c/n from to resulted in higher concentrations of residual substrate and lipid content in the biomass was only slightly increased with glucose as substrate, but not with glucose, xylose and arabinose. lipid production was similar, but fatty acid production was slightly less efficient when the substrate was provided in pulses, rather than continually. the yield of lipid on carbohydrate was reduced in all fed-batch cultures, compared to batch cultures, with the exception of glucose fed cultures which received no n in the feed. lipid production rates were generally similar in fed-batch cultures to those observed in comparable batch cultures. however, on mixed substrates provided c/n during the batch phase and no n in the feed the lipid production rate was g l− h− which was almost as high as on glucose alone. the feeding strategy did not affect the proportions of fatty acids extracted from the biomass. provision of xylose and arabinose in the feed in addition to glucose generally reduced the proportion of 18: fatty acids and increased the proportion of 16: and 18: fatty acids, compared to cultures receiving glucose alone. fatty acid profiles were similar to those of batch cultures receiving the same carbon source. discussion r. toruloides is a known lipogenic yeast and the results presented here confirm its ability to accumulate over 75% of its biomass as lipid, with 62% as fatty acids. however, lipid accumulation was strongly affected by the nature of the carbon source provided. greater lipid accumulation occurred on glucose or xylose than on arabinose, as has also been observed for trichosporon cutaneum. on mixtures of carbohydrates, lipid accumulation was lower than on glucose, but only slightly lower than observed on xylose, reflecting the sequential utilisation of the sugars, with glucose being consumed while more nitrogen was available and xylose and arabinose being consumed during the period of nitrogen restricted growth. further, arabinose consumption was incomplete, and thus the effective c/n was less than intended, contributing to the reduced lipid accumulation. interestingly, lipid production was similar regardless of whether the ratio of glucose to xylose and arabinose was high or low. the low lipid production on mixtures of carbohydrates should be taken into account when assessing the possibilities of producing lipids directly from plant biomass hydrolysates. low lipid content in r. glutinis and r. toruloides has indeed been observed when lipids have been produced directly from plant hydrolysates. it is also important to note that the fatty acid profile was not the same on mixtures of glucose, xylose and arabinose as on glucose alone. thus in plant biomass hydrolysates one should expect a lower proportion of stearic acid, and more linoleic and possibly more palmitic fatty acids than in pure glucose. some caution should be taken when extrapolating results from glucose grown yeast to what can be expected from plant biomass hydrolysate. although arabinose was not generally completely metabolised its presence in the medium appeared to have a strong influence on the fatty acid profile. r. toruloides cbs was not as productive as r. toruloides y. this may reflect difference between the strains, the presence of yeast extract in the y processes, and/or a lower c/n during the batch phase for the y processes. although cbs has been studied extensively as a model of lipid production in yeast, it is not necessarily the best strain for production. it is none-the-less worth noting that during the latter stages of batch culture with or g glucose l− it was producing lipid at g l− h− indicating that it has more potential than has so far been achieved in batch or fed-batch cultivations. high lipid accumulation occurred on chemically defined medium and did not require addition of expensive complex ingredients such as yeast extract, even in high cell density batch or fed-batch cultures. if a complex nitrogen source is desired, css were found to be suitable for good lipid production. although most studies of lipid production from waste streams have included yeast extract or peptone in the medium, xue et al. demonstrated that no nitrogen or other supplements were needed to produce lipid in r. glutinis on monosodium glutamate wastewater. the inhibitory effect of high glucose concentrations in batch culture has also been observed by li et al. for r. toruloides y but only for concentrations above g l− this inhibition could be avoided by using a repeated or sequential batch process in which a large proportion of the culture would be removed and replaced with fresh medium at the time when lipid and biomass content would be maximal, as illustrated with r. glutinis on palm oil mill effluent. alternatively, inhibition by high substrate concentrations could be avoided by utilising fed-batch and repeated fed-batch cultivation, as shown here and for r. toruloides y4 and c. curvatus. as reported by zhao et al. for r. toruloides y we found that continuous feeding was preferable to discontinuous, intermittent feeding for high fatty acid accumulation in cbs. we also observed that the best rates and yields of lipid production were achieved using glucose feed which lacked nitrogen. however, in contrast to li et al., zhao et al., and hassan et al., biomass production was not sustained under these conditions, substrate was not completely consumed and the rates and yields would not be expected to be sustainable in a repeated fed-batch process. this probably reflects the absence of yeast extract or other complex ingredients in our process, along with the use of a higher initial c/n during the batch phase than has been used previously. if reduction of the organic carbon content of the feed is also important, providing nitrogen in the feed could be beneficial, even though there could be some loss in yield. this is even more apparent if the fatty acid content is taken into account, rather than just the total lipid, since the rate of fatty acid production was similar with or without nitrogen in the feed. however, it is likely that either process would benefit from a lower c/n during the batch phase, to enable more rapid biomass accumulation. it is also likely that feed derived from plant biomass hydrolysates will always contain some metabolisable nitrogen. further, a repeated batch process with e.g. g glucose l− c/n would be more efficient in converting glucose to lipid if a rate of g lipid l− h− would be sustained without the lag phase. in contrast, fed-batch cultivation with no nitrogen in the feed was clearly the best option for producing lipids from mixed carbohydrate substrates including glucose, xylose and arabinose. as with glucose fed-batch cultures, one can predict that a lower c/n in the batch phase, possibly combined with a high c/n feed, would be necessary to achieve more complete utilisation of the substrate and to create a process which would be sustainable as a repeated fed-batch process.  lipid production from c carbohydrates was less efficient in r. toruloides cbs than from glucose, with production from xylose being better than from arabinose. the presence of arabinose in media containing mixtures of glucose, xylose and arabinose resulted in less lipid accumulation than in medium with either glucose or xylose alone. thus plant biomass hydrolysates with low arabinose content would be desirable for lipid production using r. toruloides cbs high biomass and lipid production were achieved in both batch and fed-batch cultures with glucose as carbon source, and a repeated batch process would be expected to be as efficient as a fed-batch or repeated fed-batch process. however, for lipid production from mixtures of carbohydrates, fed-batch cultivation was preferable. in fed-batch processes, provision of extra nitrogen during the batch phase was useful to provide rapid biomass production, with lipid accumulating during the feeding phase. provision of nitrogen in the feed enabled more complete utilisation of the carbohydrate, but with a lower yield of lipid on carbohydrate, than in its absence. complete substrate utilisation would be important in lipid production from waste streams, to ensure sufficient reduction in the carbon content of the stream. the feeding strategy did not affect the relative proportion of different fatty acids in the lipid, but the presence of c carbohydrates did. 
the feeding strategy did not affect the relative proportion of different fatty acids in the lipid, but the presence of c sugars did. r. toruloides was grown in batch and fed-batch cultures in l bioreactors at ph in chemically defined, nitrogen restricted media containing glucose, xylose, arabinose, or all three carbohydrates as carbon source. lipid production was most efficient with glucose as the sole carbon source, but high lipid concentrations were also produced from xylose. lipid production was low with arabinose as sole carbon source and was lower than expected when glucose, xylose and arabinose were provided simultaneously. although high biomass and lipid production were achieved in both batch and fed-batch cultures with glucose as carbon source, for lipid production from mixtures of carbohydrates fed-batch cultivation was preferable. the presence of arabinose and/or xylose in the medium increased the proportion of palmitic and linoleic acid and reduced the proportion of oleic acid in the fatty acids, compared to glucose-grown cells. lipid production in r. toruloides was lower from arabinose and mixed carbohydrates than from glucose or xylose. microbial lipids are a potential source of bio- or renewable diesel and the red yeast rhodosporidium toruloides is interesting not only because it can accumulate over 50% of its dry biomass as lipid, but also because it utilises both five and six carbon carbohydrates, which are present in plant biomass hydrolysates. lipidrhodosporidium toruloidesbio- and renewable dieselfed-batchhigh cell density however, carbohydrate consumption was incomplete when the feed did not contain nitrogen and the highest total lipid and best substrate consumption were observed in cultures which received a constant low nitrogen supply. the highest proportion of lipid in the biomass was observed in cultures given nitrogen during the batch phase but none with the feed.
 microbial lipids are a potential source of bio- or renewable diesel and the red yeast rhodosporidium toruloides is interesting not only because it can accumulate over 50% of its dry biomass as lipid, but also because it utilises both five and six carbon carbohydrates, which are present in plant biomass hydrolysates. methods r. toruloides was grown in batch and fed-batch cultures in l bioreactors at ph in chemically defined, nitrogen restricted media containing glucose, xylose, arabinose, or all three carbohydrates as carbon source. lipid was extracted from the biomass using chloroform-methanol, measured gravimetrically and analysed by gc.  lipid production was most efficient with glucose as the sole carbon source, but high lipid concentrations were also produced from xylose. lipid production was low with arabinose as sole carbon source and was lower than expected when glucose, xylose and arabinose were provided simultaneously. the presence of arabinose and/or xylose in the medium increased the proportion of palmitic and linoleic acid and reduced the proportion of oleic acid in the fatty acids, compared to glucose-grown cells. high cell densities were obtained in both batch and fed-batch cultures. the highest proportion of lipid in the biomass was observed in cultures given nitrogen during the batch phase but none with the feed. however, carbohydrate consumption was incomplete when the feed did not contain nitrogen and the highest total lipid and best substrate consumption were observed in cultures which received a constant low nitrogen supply.  lipid production in r. toruloides was lower from arabinose and mixed carbohydrates than from glucose or xylose. although high biomass and lipid production were achieved in both batch and fed-batch cultures with glucose as carbon source, for lipid production from mixtures of carbohydrates fed-batch cultivation was preferable. constant feeding was better than intermittent feeding. the feeding strategy did not affect the relative proportion of different fatty acids in the lipid, but the presence of c sugars did. lipidrhodosporidium toruloidesbio- and renewable dieselfed-batchhigh cell density 