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culture of penicillium citrinum mtcc1256 was obtained from imtech chandigarh , india , and was maintained on the slants of potato - dextrose agar ( pda ) medium at 4c and subcultured at 30-day intervals .
the culture of p. citrinum was grown on potato glycerol agar ( pga ) slants , and the spore suspension ( 4  10 spores per ml ) was prepared in glycerol water solution ( 15
g / l ) . the inoculum media used in this study were glucose ( 20
g / l ) , glycerol ( 30 g / l ) , peptone ( 8
g / l ) , which were dissolved in a water - soluble extract of soybean meal .
all fermentation experiments were carried out in 250 ml erlenmeyer flasks containing 50 ml of production media , as per the experimental design .
each flask was inoculated at 5% v / v with spore suspension and incubated at 24c and 220 rpm in an orbital shaker for 14 days .
glucose , glycerol , arrowroot , oats , urea , peptone , yeast extract , magnesium sulfate , and calcium chloride were the nine medium constituents selected for the study .
the plackett - burman experimental design , for 11 variables : nine nutritional components ( independent variables ) and two dummy variables [ table 1 ] , were used , to evaluate the relative importance of various nutrients for mevastatin production in the submerged culture . in table 2
high ( + ) and low ( - ) were tested [ table 2 ] .
concentrations of variables at different levels in plackett - burman design for mevastatin production in submerged culture plackett - burman experimental design of 12 trials for 11 variables ( 9 nutrients + 2 dummies ) along with the observed concentration of mevastatin in fermentation broth mevastatin from fermentation broth was extracted according to the procedure given by chakarvarti and sahai , 2002b .
fermentation broth ( 5 ml ) was adjusted to ph 6.5 with either acid ( h3po4 ) or alkali ( aq . naoh ) .
the broth was diluted five - fold with absolute ethanol , filtered through a 0.22 m filter , and analyzed by high performance thin layer chromatography ( hptlc ) ( camag , muttenz , switzerland ) the extracted mevastatin were applied to the plates as 3-mm - width bands with a camag 100 l syringe , using a linomat v ( camag , muttenz , switzerland ) sample applicator on pre - coated silica gel aluminum plates 60f-254 ( 20 cm  10 cm , with 0.2 mm thickness , e. merck , germany ) .
the sample application rate kept at 150 nl / s was employed and the space between two bands was kept at 12.3 mm .
thin layer chromatography was performed using a mixture of toluene , ethyl acetate , and formic acid as the mobile phase , in the ratio of 3:2:1 per volume , in 10  10 cm twin through - glass chambers .
densitometric scanning was performed on a camag tlc scanner iii in absorbance mode , at 238 nm , for mevastatin , after drying in a hot air oven for 10 minutes at 100c .
the effect of each variable was determined according to the procedure given by plackett - burman .
the percentage of contribution of each nutrient parameter was calculated by using design expert 7.1 software of stat ease inc .
culture of penicillium citrinum mtcc1256 was obtained from imtech chandigarh , india , and was maintained on the slants of potato - dextrose agar ( pda ) medium at 4c and subcultured at 30-day intervals .
the culture of p. citrinum was grown on potato glycerol agar ( pga ) slants , and the spore suspension ( 4  10 spores per ml ) was prepared in glycerol water solution ( 15
g / l ) . the inoculum media used in this study were glucose ( 20
g / l ) , which were dissolved in a water - soluble extract of soybean meal .
all fermentation experiments were carried out in 250 ml erlenmeyer flasks containing 50 ml of production media , as per the experimental design .
each flask was inoculated at 5% v / v with spore suspension and incubated at 24c and 220 rpm in an orbital shaker for 14 days .
glucose , glycerol , arrowroot , oats , urea , peptone , yeast extract , magnesium sulfate , and calcium chloride were the nine medium constituents selected for the study .
the plackett - burman experimental design , for 11 variables : nine nutritional components ( independent variables ) and two dummy variables [ table 1 ] , were used , to evaluate the relative importance of various nutrients for mevastatin production in the submerged culture . in table 2 , each row represents a different variable . for each nutrient variable two different concentrations
high ( + ) and low ( - ) were tested [ table 2 ] .
concentrations of variables at different levels in plackett - burman design for mevastatin production in submerged culture plackett - burman experimental design of 12 trials for 11 variables ( 9 nutrients + 2 dummies ) along with the observed concentration of mevastatin in fermentation broth
mevastatin from fermentation broth was extracted according to the procedure given by chakarvarti and sahai , 2002b .
fermentation broth ( 5 ml ) was adjusted to ph 6.5 with either acid ( h3po4 ) or alkali ( aq . naoh ) .
the broth was diluted five - fold with absolute ethanol , filtered through a 0.22 m filter , and analyzed by high performance thin layer chromatography ( hptlc ) ( camag , muttenz , switzerland )
the extracted mevastatin were applied to the plates as 3-mm - width bands with a camag 100 l syringe , using a linomat v ( camag , muttenz , switzerland ) sample applicator on pre - coated silica gel aluminum plates 60f-254 ( 20 cm  10 cm , with 0.2 mm thickness , e. merck , germany ) .
the sample application rate kept at 150 nl / s was employed and the space between two bands was kept at 12.3 mm .
thin layer chromatography was performed using a mixture of toluene , ethyl acetate , and formic acid as the mobile phase , in the ratio of 3:2:1 per volume , in 10  10 cm twin through - glass chambers .
densitometric scanning was performed on a camag tlc scanner iii in absorbance mode , at 238 nm , for mevastatin , after drying in a hot air oven for 10 minutes at 100c .
the effect of each variable was determined according to the procedure given by plackett - burman .
the percentage of contribution of each nutrient parameter was calculated by using design expert 7.1 software of stat ease inc .
screening of the nutrient by the placket - burman experimental design for mevastatin production , using p. citrinum mtcc 1256 , was carried out in this study .
maximum mevastatin production was found in experiment trial 2 ( 589.3 mg / l ) , minimum in 5 ( 38.9 mg / l ) [ table 2 ] , under submerged fermentation .
the effect of the dummy variables 1 and 2 were close to zero [ table 3 ] , which indicates a successful experimental study .
experimental error was calculated and found to be 0.00005 . among the nine nutrient components used in the study , glycerol , peptone , yeast extract , mgso4.7h2o , and
cacl2.2h2o had contributed to a large extent in mevastatin production by p. citrinum mtcc 1256 .
urea and oats had little impact , while glucose and arrowroot contributed moderately [ figure 1 ] .
effect of different medium components on mevastatin production percent contribution and effect of factors used in the plackett - burman design
it has been found that glycerol is useful as a carbon source , it has a high contribution , and is effective in a lesser amount , as glycerol is a significant limiting factor influencing the biosynthesis of statin via growth .
peptone has been found to be a better nitrogen source than yeast extract for mevastatin production by p. citrinum mtcc 11256 .
this is one reason why the growth of p. citrinum ( biomass ) largely depends on the type of nitrogen source .
moreover , a suitable concentration of peptone is important for a higher production of mevastatin ; a lower biomass at lower peptone concentration results in less production of mevastatin . in fungal nutrition , magnesium and calcium are noted as macronutrients and manganese , iron , copper , and zinc as micronutrients , but in the case of mevastatin production by p. citrinum , the contribution by magnesium and calcium is higher than that by iron and manganese .
designing the medium is an open - ended , time - consuming , laborious process , involving a large number of experiments .
the plackett - burman experimental design is a preliminary technique for the rapid illustration of effects of various medium constituents .
it tests each variable at two levels only ; hence , it can not give an exact idea regarding the optimum level of the constituents required in a medium .
therefore , further optimization of selected nutrients , such as , glycerol , peptone , mgso4.7h2o , cacl2.2h2o , and kh2po4 for mevastatin production by penicillium citrinum , is necessary .
objective : mevastain , an hmg - coa reductase inhibitor produced by penicillium citrinum . 
 the placket - burman experimental design was used to identify the important nutrients influencing the production of mevastatin by penicillium citrinum mtcc 1256 under submerged fermentation.materials and methods : nine nutritional parameters , such as , glucose , glycerol , arrowroot , oats , urea , peptone , yeast extract , mgso4.7h2o , and cacl2.2h2o were screened by placket - burman experimental design in 12 experimental run.results:nine nutritional parameters , glucose , glycerol , arrowroot , oats , urea , peptone , yeast extract , mgso4.7h2o , and cacl2.2h2o contributed with 0.8114% , 24.0390% , 2.2786% , 0.1491% , 0.5608% , 47.5029% , 6.8092 % , 4.0980% , 10.5823% respectively towards mevastatin production by penicillium citrinum under submerged fermentation.conclusion:among the nine nutrient components glycerol , peptone , yeast extract , mgso4.7h20 , and cacl2.2h2o contributed to a large extent , urea had little impact , while glucose , oats , and arrowroot contributed moderately on production of mevastatin .