BACKGROUND
itaconic acid is a white crystalline unsaturated c <dig> dicarboxylic acid.
because of its specific favorable properties and the unique structure, itaconic acid is used worldwide as monomer or co-monomer in manufacturing plastics, resins etc  <cit> .
its market price is around  <dig> us$ per kilo  <cit> .
currently, itaconic acid is produced commercially by aspergillus terreus  via submerged fungal fermentations  <cit> .
in a transcriptomics study in a. terreus, several itaconic acid-related genes were identified  <cit> .
among them, cada encoding cis-aconitate decarboxylase  which is the key enzyme for itaconic acid production from citric acid in the tri-carboxylic acid  cycle.
based on its high citric acid producing capability and broad applicability in industry, aspergillus niger  was selected as a novel itaconic acid production host strain in recent work.
expression of cada in a. niger leads to itaconic acid production in a. niger <cit> .
in our attempts to enhance itaconic acid production levels in a. niger cad strains we used genetic modification of the production host and medium optimization.
a first target to enhance itaconic acid production in a. niger cad transformants is glyceraldehyde-3-phosphate dehydrogenase .
gpd, encoded by gpda is a key enzyme in glycolysis which converts glyceraldehyde-3-phosphate into biphosphoglycerate  <cit> .
in our previous transcriptomics analysis, this gene is highly expressed under itaconic acid production conditions  <cit>  and its function in biological pathway may indicate a role in generating an increased flux through glycolysis towards the tca cycle  <cit> .
the relevance of increased glycolytic flux in itaconic acid production is also suggested by the results of tevž and co-workers  <cit> .
they show that overexpression of a modified pfka gene  from a. niger in a. terreus results in enhanced itaconic acid production, probably due to increased glycolytic flux.
therefore, over-expression of gpda in a. niger cad strains might increase itaconic acid producing level via enhanced glycolytic flux.
the second target to enhance itaconic acid production in a. niger cad transformants is favohemoglobin , as heterologous fhb overexpression has been shown to enhance itaconic acid production in a terreus <cit> .
in our study, over-expression of fungal hemoglobin domain  in a. niger is used as a potential enhancer of itaconic acid production.
in addition to genetic modification of a. niger, we also focused on development of an itaconic acid production medium for aspergillus niger.
medium optimization for a. niger and a. terreus has been carried out over the last  <dig> years.
media for citric acid and itaconic acid production were accomplished by various research groups  <cit> .
the required medium components for these two organic acids are quite comparable: high concentration of glucose  and magnesium sulfate, low nitrogen and phosphorus, low but adequate levels of zinc, copper and iron, limited manganese .
since the existing industrial itaconic acid production medium for a. terreus has not yet been adapted to a. niger, we aimed for an optimized itaconic acid production medium for a. niger.
as citric acid is the potential substrate of itaconic acid in a. niger, in our medium design the composition of citric acid production medium was considered as well.
methods
strains /plasmids /fungal transformation
the uridine auxotrophic a. niger strains ab  <dig>  and ab  <dig>  cad  <dig>  pyrg- which was derived from ab  <dig>  via cada insertion  <cit>  were used as parental strain for a. niger transformation.
the plasmid used for overexpressing gpda contained a  <dig>  kb psti a. niger fragment, including the gpda gene, in puc <dig> .
the plasmid for overexpression of hbd <dig>  was previously described  <cit> .
the plasmid pab  <dig>   <cit>  containing the pyrg + gene was used as a selection marker for co-transformation.
to over-express gpda and hbd <dig> in the a. niger cad strain, the plasmids pab 5– <dig> and phbn were co- transformed with pab4- <dig> respectively.
all transformants were further selected for their ability to growth on minimal medium plates  without uridine.
a. niger transformants screening
for screening and selection of ab  <dig>  cad + gpd and hbd transformants, 96-wells micro-titer plates  were filled with a. niger cultivation medium,  <dig> μl medium per well.
the inner  <dig> wells were inoculated with spores from target strains, using sterilized tooth picks.
the outermost wells of the plate were kept empty to prevent the effect of evaporation.
in order to prevent contamination, each cultivation plate was sealed with a sterilized oxygen permeable film directly after spore inoculation.
at the end of cultivation , cultures were harvested and used for hplc analysis.
medium design and culture conditions
based on the reference medium  ,  <dig> new media were generated by differing one component or its amount compared to the reference medium .
to find changes compared to the reference medium, the amount of the differing component was either the maximal or the minimal value of the one used in literature before.
to generate a suitable medium for itaconic acid production in a. niger transformants,  <dig> media and including the reference were analyzed in triplicate.
to prevent cross contamination, each plate was inoculated with only one transformant.
for further media improvement, media which showed a positive effect on itaconic acid production were combined and concentration ranges were extended.
after seeding, all plates were directly sealed with an oxygen permeable film , placed in a plastic air bag and cultivated in a  <dig> °c,  <dig> rpm incubator  for  <dig> hours  or  <dig> hours .
controlled batch fermentations were carried out as follows: media were prepared in demineralized water.
pre-cultures of  <dig> ml  in  <dig> ml baffled erlenmeyer flasks were inoculated for two days.
as described previously  <cit> , fermentations were carried out in  <dig> l benchtop fermentors , at  <dig> degree.
air was used for sparging the bioreactor at a constant flow of  <dig>  vvm , dissolved oxygen tension  was maintained minimal at  <dig> % coupled with stirred speed of 400– <dig> rpm, ph was initiated at  <dig>  adjusted by  <dig>  m h3po <dig>  and kept at  <dig>  by addition of  <dig> m koh .
struktol  was applied as antifoam agent though-out the fermentation.
metabolite analysis
for metabolite analysis,  <dig> μl medium was pipetted from each culture to total recovery vials for high performance liquid chromatography  analysis.
two analysis detection facilities were applied.
the one from waters cooperate an  <dig> h-aminex organic acids column , with  <dig>  m h2so <dig> as eluent and a photodiode array  detector for organic acids and refractive index  detector for sugar like compounds.
the other from thermo fisher , analyzing organic acids through an organic acids column ionpac® ice as <dig>  with  <dig>  mm heptafluorobutyric acid as eluent and a detector of suppressed conductivity cd <dig>  standard compounds  with concentrations of  <dig> mg/l,  <dig> mg/l,  <dig> g/l,  <dig> mg/l,  <dig> mg/l and  <dig> mg/l were used for calibration.
RESULTS
host strain selection and modification
the itaconic acid producing strain ab  <dig>  cad  <dig>  pyrg-  was selected from a limited number of cada transformants  <cit> .
in an attempt to isolate a further improved itaconic acid producing strains, new transformants were generated.
more than  <dig> colonies were screened for their itaconic acid production in a  <dig> well micro-titer plate together with cad  <dig>  as a reference.
as presented in figure 1a, strain cad  <dig>  is shown to be among the best itaconic acid producing strains.
therefore, this strain was used for further genetic modification.
to improve itaconic acid production level, the glyceraldehyde dehydrogenase gene  and the gene encoding a fungal hemoglobin domain  were co-transformed with the pyrg + marker into the selected host ab  <dig>  cad  <dig> .
to select improved itaconic acid-producing co-transformants,  <dig> gpda and  <dig> hbd <dig> transformants were screened in  <dig> well micro-titer plate using reference medium  <cit> .
three strains from each hbd and gpd differing most strongly from cad  <dig>  were selected and analyzed for the presence for extra hbd <dig> or gpda gene copies by southern blot analysis .
two of the three selected hbd <dig> strains  with improved itaconic acid titers contained one  hbd <dig> gene copies compared to cad  <dig> .
unexpectedly, only strain gpd  <dig>  which actually has lower itaconic acid titer is a true gpda transformant , the other two strains are just pyrg + transformants from cad <dig> .
we selected the two hbd <dig> co-transformants , the gpda co-transformant  and two pyrg + transformants of cad  <dig>   for further analysis of different cultivation media.
culture medium design
to optimize the production medium,  <dig> new media were generated based on the reference medium from a. terreus <cit> , by varying several components or their concentrations per medium .
to be able to compare many transformants and media components in one batch, a micro-titer plate screening was used.
after cultivation for about  <dig> hours, cultures from each medium were analyzed for itaconic acid production using hplc.
as indicated in figure 2a, in comparison to reference medium  <dig>  all strains showed increased itaconic acid production in medium  <dig>  in this medium, both pyrg + strains have even higher amount of itaconic acid production than the hbd <dig> and gpda strains .
in contrast, medium  <dig> and  <dig> yield the lowest itaconic acid production.
moreover, transformants grown in medium  <dig> and  <dig> also show improved itaconic acid production compared to the reference, except for transformant gpd  <dig> .
gpd  <dig>  has generally low production level compared to other strains through all  <dig> media.
the itaconic acid inducing components in the best media are phosphate , copper  and potassium .
the production pattern of citric acid is quite similar to itaconic acid in medium  <dig>   <dig>   <dig> and  <dig> .
in contrast, medium  <dig> provided high citric acid but low itaconic acid production.
medium  <dig> and  <dig> showed improved itaconic acid production only to the pyrg + strains.
based on the obtained results, we have retested a subset of the above mentioned media  and a newly designed medium  which combined the potentially itaconic acid inducing attributes  from medium  <dig>   <dig> and  <dig>  results are shown in table  <dig>  this time, strain fhb  <dig>  was tested together with the reference strain pyr +  <dig>  in triplicate.
similar results were obtained as in the initial screen.
medium  <dig> still yields the best itaconic acid production, whereas m20+ did not further improve production.
in total, the standard deviations of the micro-titer plate screening results were less than  <dig> %.
no oxalic acid was produced in the micro-titer plate cultivations.
effect of copper on organic acids production in batch fermentation
the levels of itaconic acid, citric acid and oxalic acid of a. niger transformant ab  <dig>  cad pyr +  <dig>  in medium  <dig> containing three concentrations of copper were analyzed.
as presented in figure 3a, the production rate of itaconic acid is positively correlated with the copper concentration while this correlation is different for citric acid  and oxalic acid .
in the low-copper medium , high levels of oxalic acid are accumulated whereas the highest amount of citric acid is produced in the medium-copper medium.
in addition to organic acids, we determined biomass and glucose consumption.
glucose consumption of a. niger strain ab  <dig>  cad pyr +  <dig>  among all three media is nearly the same .
increase in biomass showed a strong positive correlation with copper levels .
the three main organic acids were quantified and the average concentrations  of duplicate measurements are listed in table  <dig>  as indicated, a two fold increase of the percentage of itaconic acid is obtained in the high-copper medium.
.
the levels are averages of the duplicate measurements  .
discussion
shake flask cultures are commonly used for screening of new a. niger transformants  <cit> .
however, for analysis of large numbers of transformants, this approach is not entirely suitable.
therefore, we used 96-well plates for screening transformants similar to the methods described previously  <cit> .
the screening results are consistent since the standard deviations are less than  <dig> %.
as suggested in our previous study we introduced extra copies of gpda in a. niger strain cad  <dig>  in order to improve itaconic acid production via molecular modification  <cit> .
in addition, based on the research of the hemoglobin domain in a terreus <cit>  and a. oryzae <cit> , extra copies of hbd <dig> were introduced in a. niger strain cad  <dig>  to increase the production level of itaconic acid.
co-transformation resulted in strains with increased itaconic acid levels compared to the parental strain.
however, comparison to pyrg + complemented transformants showed that the increase was the results of complementation of the uridine deficiency alone.
surprisingly, a gpda transformant produced only half amount of itaconic acid in comparison to the parental strain.
however, more detailed analysis of this strain in controlled fermentation did not show significant differences with the control strain.
this may indicate that the observed effect is specific for plate conditions.
although this shows that gpda expression and itaconic acid production were related, forced over-expression in a. niger did not result in increased itaconic acid levels without extending the cultivation period.
based on the various itaconic acid and citric acid production media described in literature , we designed  <dig> media by modifying the amount of various components in reference medium m <dig> .
medium  <dig>   <dig> and  <dig> were shown to have a positive effect on itaconic acid production while medium  <dig> and  <dig> had a negative effect .
nevertheless, all of these five media had high glucose concentration of  <dig> %, which had been identified as an important condition for producing organic acids  <cit> .
the positive effects were observed with extra cu2+  or phosphorus , or replacing na+ and ca2+ with k+ .
lack of cu2+  or increment of fe2+  were shown to result in decreased itaconic acid accumulation.
the effect of phosphorous to organic acids production has not yet been demonstrated, except for a positive trend seen due to its limitation in the used medium  <cit> .
similarly, the effect of k+ has also not been documented previously.
since detailed analysis on trace metal concentration in a. niger media showed that citric acid production could be increased by combining low levels of mn2+ with high levels of fe2+ <cit> , and because fe2+ is a cofactor of hemoglobin to bind oxygen, we investigated this combination using medium  <dig>  this medium contained a high concentration of fe2+ and no mn2+.
however, neither the itaconic acid nor the citric acid level was improved in our 96-well screen.
so, probably under limited oxygen supply in this micro-titer cultivation condition, the oxygen uptake in medium  <dig> had not been improved compared to medium  <dig>
unexpectedly, itaconic acid production was not further improved by combining the maximal value of the positive components of m <dig>   <dig>   <dig> in one medium.
in addition, the media yielding improved itaconic acid production were not the same as the ones for citric acid.
based on the results of our micro-titer plate screenings, we studied the influence of copper in the batch cultivation of aspergillus niger strain ab  <dig>  cad pyr +  <dig> .
itaconic acid production level was proved to be positively related with copper concentration in the medium.
moreover, the best itaconic acid producing condition was medium  <dig> with  <dig>  mm cu2+, which yielded a  <dig>  folds increase in itaconic acid production compared to the reference medium   <cit> .
although the percentage of itaconic acid  among the total organic acids is lower in comparison to our previous study , the production level is two folds higher.
in this respect, it was interesting to note that a. niger was the most copper tolerant among  <dig> fungal species especially under low ph conditions  <cit> , allowing the use of copper for enhancing itaconic acid production in this species.
in our cultivations, copper increment had no toxic effect on biomass growth.
besides, copper ions could increase the production of citric acid as well.
the research group of haq found  <dig>  mm of cu2+ could increase the productivity of citric acid by a. niger <cit> .
similarly in our research with a. niger strain ab  <dig>  cad pyr +  <dig> , highest citric acid production was observed using  <dig>  mm cu2+.
although our current itaconic acid production level  is clearly lower than the levels reached using a. terreus, we improved the level with two folds compared to our previous study.
as illustrated here, medium components influence itaconic acid production levels by a. niger.
the fermentation results also indicate that optimal citric acid production conditions were different from optimal itaconic acid production conditions.
our opinion is that the itaconic acid production using a. niger is very promising, in particular if high yielding citric acid producing strains would be used  <cit> .
as currently this strain produces more citric acid and oxalic acid than itaconic acid, there is opportunity to further improve itaconic acid production by eliminating oxalic acid  <cit>  and a better conversion of citric acid.
moreover, additional process parameters such as temperature, ph, and dissolved oxygen tension could also be explored.
furthermore, to obtain optimal product formation, more elaborate research might be required based on our initial transcriptomics research  <cit> .
CONCLUSIONS
in this study, itaconic acid production level has been significantly improved.
we conclude that micro-titer plate cultivation is suitable for screening large numbers of fungal strains or comparison of various media.
copper is shown to be positively correlated with improved itaconic acid production.
although the role of copper in the metabolic pathway of organic acids is not yet clear, this new insight will allow further medium optimization  <cit> .
the performance of gpda and hbd <dig> transformants in itaconic acid production was not better than the non-transformed control.
the optimal conditions for itaconic acid production are clearly different from those optimal for citric acid or oxalic acid production.
further research is required for itaconic acid production improvement.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
al carried out the strain modification, medium study in the controlled batch cultivation, metabolite analysis and drafted the manuscript.
np participated in the strain modification, medium design and screening.
rz joined the medium study in controlled batch cultivation and related metabolite analysis.
pp supervised the study and approved the final manuscript.
all authors read approved the final manuscript.

 itaconic acid is a white crystalline unsaturated c dicarboxylic acid. because of its specific favorable properties and the unique structure, itaconic acid is used worldwide as monomer or co-monomer in manufacturing plastics, resins etc. its market price is around us$ per kilo. currently, itaconic acid is produced commercially by aspergillus terreus via submerged fungal fermentations. in a transcriptomics study in a. terreus, several itaconic acid-related genes were identified. among them, cada encoding cis-aconitate decarboxylase which is the key enzyme for itaconic acid production from citric acid in the tri-carboxylic acid cycle. based on its high citric acid producing capability and broad applicability in industry, aspergillus niger was selected as a novel itaconic acid production host strain in recent work. expression of cada in a. niger leads to itaconic acid production in a. niger. in our attempts to enhance itaconic acid production levels in a. niger cad strains we used genetic modification of the production host and medium optimization. a first target to enhance itaconic acid production in a. niger cad transformants is glyceraldehyde-3-phosphate dehydrogenase. gpd, encoded by gpda is a key enzyme in glycolysis which converts glyceraldehyde-3-phosphate into biphosphoglycerate. in our previous transcriptomics analysis, this gene is highly expressed under itaconic acid production conditions and its function in biological pathway may indicate a role in generating an increased flux through glycolysis towards the tca cycle. the relevance of increased glycolytic flux in itaconic acid production is also suggested by the results of tevž and co-workers. they show that overexpression of a modified pfka gene from a. niger in a. terreus results in enhanced itaconic acid production, probably due to increased glycolytic flux. therefore, over-expression of gpda in a. niger cad strains might increase itaconic acid producing level via enhanced glycolytic flux. the second target to enhance itaconic acid production in a. niger cad transformants is favohemoglobin, as heterologous fhb overexpression has been shown to enhance itaconic acid production in a terreus. in our study, over-expression of fungal hemoglobin domain in a. niger is used as a potential enhancer of itaconic acid production. in addition to genetic modification of a. niger, we also focused on development of an itaconic acid production medium for aspergillus niger. medium optimization for a. niger and a. terreus has been carried out over the last years. media for citric acid and itaconic acid production were accomplished by various research groups. the required medium components for these two organic acids are quite comparable: high concentration of glucose and magnesium sulfate, low nitrogen and phosphorus, low but adequate levels of zinc, copper and iron, limited manganese. since the existing industrial itaconic acid production medium for a. terreus has not yet been adapted to a. niger, we aimed for an optimized itaconic acid production medium for a. niger. as citric acid is the potential substrate of itaconic acid in a. niger, in our medium design the composition of citric acid production medium was considered as well. methods strains /plasmids /fungal transformation the uridine auxotrophic a. niger strains ab and ab cad pyrg- which was derived from ab via cada insertion were used as parental strain for a. niger transformation. the plasmid used for overexpressing gpda contained a kb psti a. niger fragment, including the gpda gene, in puc. the plasmid for overexpression of hbd was previously described. the plasmid pab containing the pyrg + gene was used as a selection marker for co-transformation. to over-express gpda and hbd in the a. niger cad strain, the plasmids pab 5– and phbn were co- transformed with pab4- respectively. all transformants were further selected for their ability to growth on minimal medium plates without uridine. a. niger transformants screening for screening and selection of ab cad + gpd and hbd transformants, 96-wells micro-titer plates were filled with a. niger cultivation medium, μl medium per well. the inner wells were inoculated with spores from target strains, using sterilized tooth picks. the outermost wells of the plate were kept empty to prevent the effect of evaporation. in order to prevent contamination, each cultivation plate was sealed with a sterilized oxygen permeable film directly after spore inoculation. at the end of cultivation, cultures were harvested and used for hplc analysis. medium design and culture conditions based on the reference medium, new media were generated by differing one component or its amount compared to the reference medium. to find changes compared to the reference medium, the amount of the differing component was either the maximal or the minimal value of the one used in literature before. to generate a suitable medium for itaconic acid production in a. niger transformants, media and including the reference were analyzed in triplicate. to prevent cross contamination, each plate was inoculated with only one transformant. for further media improvement, media which showed a positive effect on itaconic acid production were combined and concentration ranges were extended. after seeding, all plates were directly sealed with an oxygen permeable film, placed in a plastic air bag and cultivated in a °c, rpm incubator for hours or hours. controlled batch fermentations were carried out as follows: media were prepared in demineralized water. pre-cultures of ml in ml baffled erlenmeyer flasks were inoculated for two days. as described previously, fermentations were carried out in l benchtop fermentors, at degree. air was used for sparging the bioreactor at a constant flow of vvm, dissolved oxygen tension was maintained minimal at % coupled with stirred speed of 400– rpm, ph was initiated at adjusted by m h3po and kept at by addition of m koh. struktol was applied as antifoam agent though-out the fermentation. metabolite analysis for metabolite analysis, μl medium was pipetted from each culture to total recovery vials for high performance liquid chromatography analysis. two analysis detection facilities were applied. the one from waters cooperate an h-aminex organic acids column, with m h2so as eluent and a photodiode array detector for organic acids and refractive index detector for sugar like compounds. the other from thermo fisher, analyzing organic acids through an organic acids column ionpac® ice as with mm heptafluorobutyric acid as eluent and a detector of suppressed conductivity cd standard compounds with concentrations of mg/l, mg/l, g/l, mg/l, mg/l and mg/l were used for calibration.  host strain selection and modification the itaconic acid producing strain ab cad pyrg- was selected from a limited number of cada transformants. in an attempt to isolate a further improved itaconic acid producing strains, new transformants were generated. more than colonies were screened for their itaconic acid production in a well micro-titer plate together with cad as a reference. as presented in figure 1a, strain cad is shown to be among the best itaconic acid producing strains. therefore, this strain was used for further genetic modification. to improve itaconic acid production level, the glyceraldehyde dehydrogenase gene and the gene encoding a fungal hemoglobin domain were co-transformed with the pyrg + marker into the selected host ab cad. to select improved itaconic acid-producing co-transformants, gpda and hbd transformants were screened in well micro-titer plate using reference medium. three strains from each hbd and gpd differing most strongly from cad were selected and analyzed for the presence for extra hbd or gpda gene copies by southern blot analysis. two of the three selected hbd strains with improved itaconic acid titers contained one hbd gene copies compared to cad. unexpectedly, only strain gpd which actually has lower itaconic acid titer is a true gpda transformant, the other two strains are just pyrg + transformants from cad. we selected the two hbd co-transformants, the gpda co-transformant and two pyrg + transformants of cad for further analysis of different cultivation media. culture medium design to optimize the production medium, new media were generated based on the reference medium from a. terreus, by varying several components or their concentrations per medium. to be able to compare many transformants and media components in one batch, a micro-titer plate screening was used. after cultivation for about hours, cultures from each medium were analyzed for itaconic acid production using hplc. as indicated in figure 2a, in comparison to reference medium all strains showed increased itaconic acid production in medium in this medium, both pyrg + strains have even higher amount of itaconic acid production than the hbd and gpda strains. in contrast, medium and yield the lowest itaconic acid production. moreover, transformants grown in medium and also show improved itaconic acid production compared to the reference, except for transformant gpd. gpd has generally low production level compared to other strains through all media. the itaconic acid inducing components in the best media are phosphate, copper and potassium. the production pattern of citric acid is quite similar to itaconic acid in medium and. in contrast, medium provided high citric acid but low itaconic acid production. medium and showed improved itaconic acid production only to the pyrg + strains. based on the obtained results, we have retested a subset of the above mentioned media and a newly designed medium which combined the potentially itaconic acid inducing attributes from medium and results are shown in table this time, strain fhb was tested together with the reference strain pyr + in triplicate. similar results were obtained as in the initial screen. medium still yields the best itaconic acid production, whereas m20+ did not further improve production. in total, the standard deviations of the micro-titer plate screening results were less than %. no oxalic acid was produced in the micro-titer plate cultivations. effect of copper on organic acids production in batch fermentation the levels of itaconic acid, citric acid and oxalic acid of a. niger transformant ab cad pyr + in medium containing three concentrations of copper were analyzed. as presented in figure 3a, the production rate of itaconic acid is positively correlated with the copper concentration while this correlation is different for citric acid and oxalic acid. in the low-copper medium, high levels of oxalic acid are accumulated whereas the highest amount of citric acid is produced in the medium-copper medium. in addition to organic acids, we determined biomass and glucose consumption. glucose consumption of a. niger strain ab cad pyr + among all three media is nearly the same. increase in biomass showed a strong positive correlation with copper levels. the three main organic acids were quantified and the average concentrations of duplicate measurements are listed in table as indicated, a two fold increase of the percentage of itaconic acid is obtained in the high-copper medium.. the levels are averages of the duplicate measurements. discussion shake flask cultures are commonly used for screening of new a. niger transformants. however, for analysis of large numbers of transformants, this approach is not entirely suitable. therefore, we used 96-well plates for screening transformants similar to the methods described previously. the screening results are consistent since the standard deviations are less than %. as suggested in our previous study we introduced extra copies of gpda in a. niger strain cad in order to improve itaconic acid production via molecular modification. in addition, based on the research of the hemoglobin domain in a terreus and a. oryzae, extra copies of hbd were introduced in a. niger strain cad to increase the production level of itaconic acid. co-transformation resulted in strains with increased itaconic acid levels compared to the parental strain. however, comparison to pyrg + complemented transformants showed that the increase was the results of complementation of the uridine deficiency alone. surprisingly, a gpda transformant produced only half amount of itaconic acid in comparison to the parental strain. however, more detailed analysis of this strain in controlled fermentation did not show significant differences with the control strain. this may indicate that the observed effect is specific for plate conditions. although this shows that gpda expression and itaconic acid production were related, forced over-expression in a. niger did not result in increased itaconic acid levels without extending the cultivation period. based on the various itaconic acid and citric acid production media described in literature, we designed media by modifying the amount of various components in reference medium m. medium and were shown to have a positive effect on itaconic acid production while medium and had a negative effect. nevertheless, all of these five media had high glucose concentration of %, which had been identified as an important condition for producing organic acids. the positive effects were observed with extra cu2+ or phosphorus, or replacing na+ and ca2+ with k+. lack of cu2+ or increment of fe2+ were shown to result in decreased itaconic acid accumulation. the effect of phosphorous to organic acids production has not yet been demonstrated, except for a positive trend seen due to its limitation in the used medium. similarly, the effect of k+ has also not been documented previously. since detailed analysis on trace metal concentration in a. niger media showed that citric acid production could be increased by combining low levels of mn2+ with high levels of fe2+, and because fe2+ is a cofactor of hemoglobin to bind oxygen, we investigated this combination using medium this medium contained a high concentration of fe2+ and no mn2+. however, neither the itaconic acid nor the citric acid level was improved in our 96-well screen. so, probably under limited oxygen supply in this micro-titer cultivation condition, the oxygen uptake in medium had not been improved compared to medium unexpectedly, itaconic acid production was not further improved by combining the maximal value of the positive components of m in one medium. in addition, the media yielding improved itaconic acid production were not the same as the ones for citric acid. based on the results of our micro-titer plate screenings, we studied the influence of copper in the batch cultivation of aspergillus niger strain ab cad pyr +. itaconic acid production level was proved to be positively related with copper concentration in the medium. moreover, the best itaconic acid producing condition was medium with mm cu2+, which yielded a folds increase in itaconic acid production compared to the reference medium. although the percentage of itaconic acid among the total organic acids is lower in comparison to our previous study, the production level is two folds higher. in this respect, it was interesting to note that a. niger was the most copper tolerant among fungal species especially under low ph conditions, allowing the use of copper for enhancing itaconic acid production in this species. in our cultivations, copper increment had no toxic effect on biomass growth. besides, copper ions could increase the production of citric acid as well. the research group of haq found mm of cu2+ could increase the productivity of citric acid by a. niger. similarly in our research with a. niger strain ab cad pyr +, highest citric acid production was observed using mm cu2+. although our current itaconic acid production level is clearly lower than the levels reached using a. terreus, we improved the level with two folds compared to our previous study. as illustrated here, medium components influence itaconic acid production levels by a. niger. the fermentation results also indicate that optimal citric acid production conditions were different from optimal itaconic acid production conditions. our opinion is that the itaconic acid production using a. niger is very promising, in particular if high yielding citric acid producing strains would be used. as currently this strain produces more citric acid and oxalic acid than itaconic acid, there is opportunity to further improve itaconic acid production by eliminating oxalic acid and a better conversion of citric acid. moreover, additional process parameters such as temperature, ph, and dissolved oxygen tension could also be explored. furthermore, to obtain optimal product formation, more elaborate research might be required based on our initial transcriptomics research.  in this study, itaconic acid production level has been significantly improved. we conclude that micro-titer plate cultivation is suitable for screening large numbers of fungal strains or comparison of various media. copper is shown to be positively correlated with improved itaconic acid production. although the role of copper in the metabolic pathway of organic acids is not yet clear, this new insight will allow further medium optimization. the performance of gpda and hbd transformants in itaconic acid production was not better than the non-transformed control. the optimal conditions for itaconic acid production are clearly different from those optimal for citric acid or oxalic acid production. further research is required for itaconic acid production improvement. competing interests the authors declare that they have no competing interests. authors’ contributions al carried out the strain modification, medium study in the controlled batch cultivation, metabolite analysis and drafted the manuscript. np participated in the strain modification, medium design and screening. rz joined the medium study in controlled batch cultivation and related metabolite analysis. pp supervised the study and approved the final manuscript. all authors read approved the final manuscript. 
expressing the cad gene from aspergillus terreus opened the metabolic pathway towards itaconic acid in a. niger. based on the results of previous transcriptomics studies and research from other groups, two genes: gpda encoding the glyceraldehyde −3-dehydrogenase and hbd encoding a flavohemoglobin domain were overexpressed in a. niger. to analyze large numbers of cultures, we developed an approach for screening both fungal transformants and various media in 96-well micro-titer plates. besides, new media were designed based on a reference medium for a. terreus. aspergillus niger was selected as a host for producing itaconic acid due to its versatile and tolerant character in various growth environments, and its extremely high capacity of accumulating the precursor of itaconic acid: citric acid. the hbd transformants did not improve itaconic acid titer while the gpda transformant decreased the itaconic acid production. using different media, copper was discovered to have a positive influence on itaconic acid production. the performance of gpda and hbd transformants was found not to be beneficial for itaconic acid production using the tested cultivation conditions. medium optimization showed that, copper was positively correlated with improved itaconic acid production. interestingly, the optimal conditions for itaconic acid clearly differ from conditions optimal for citric- and oxalic acid production. effects observed in the micro-titer plate screening were confirmed in controlled batch fermentation. in order to increase the production level, we continued by modifying its genome and optimizing cultivation media.
 aspergillus niger was selected as a host for producing itaconic acid due to its versatile and tolerant character in various growth environments, and its extremely high capacity of accumulating the precursor of itaconic acid: citric acid. expressing the cad gene from aspergillus terreus opened the metabolic pathway towards itaconic acid in a. niger. in order to increase the production level, we continued by modifying its genome and optimizing cultivation media.  based on the results of previous transcriptomics studies and research from other groups, two genes: gpda encoding the glyceraldehyde −3-dehydrogenase and hbd encoding a flavohemoglobin domain were overexpressed in a. niger. besides, new media were designed based on a reference medium for a. terreus. to analyze large numbers of cultures, we developed an approach for screening both fungal transformants and various media in 96-well micro-titer plates. the hbd transformants did not improve itaconic acid titer while the gpda transformant decreased the itaconic acid production. using different media, copper was discovered to have a positive influence on itaconic acid production. effects observed in the micro-titer plate screening were confirmed in controlled batch fermentation.  the performance of gpda and hbd transformants was found not to be beneficial for itaconic acid production using the tested cultivation conditions. medium optimization showed that, copper was positively correlated with improved itaconic acid production. interestingly, the optimal conditions for itaconic acid clearly differ from conditions optimal for citric- and oxalic acid production. 