A high performance fermntation process for citric acid producdion by Aspergillus niger

A high performance fermentation process for citric acid production by Aspergillus niger NG GCB -101,using vermiculite as an additive in a stirred bioreactorIkram-ul-Haq 1,Sikander Ali 1,*, ,M.A.Qadeer 2and Javed Iqbal 31Department of Botany,Biotechnology Research Labs,G.C.University,Lahore,Pakistan (E-mail:ikrhaq@)2National Centre of Excellence in Molecular Biology,University of the Punjab,Lahore,Pakistan 3The School of Biological Sciences,University of the Punjab,Lahore,Pakistan*Author for correspondence:Tel.:+92-42-921-1634,Fax:+92-42-724-3198,E-mail:alisbiotech@Present address:Department of Plant Soil Science and General Agriculture,Southern Illinois University atCarbondale,Carbondale,IL 62901,USA.Tel.:+1-618-457-0516,Fax:+1-618-453-7457,E-mail:alis@Received 27January 2003;accepted 11November 2003Keywords:Aspergillus niger ,cane-molasses,citric acid,fermentation,fermentor,vermiculiteSummaryThe present study describes the use of vermiculite for enhanced citric acid productivity by a mutant strain of Aspergillus niger NG GCB -101in a stirred bioreactor of 15.0l capacity.The maximum amount of citric acid (96.10g/l)was obtained with the control 144h after mycelial inoculation.To enhance citric acid production,varying levels of vermiculite were added as an additive into the fermentation medium.The best results were observed when 0.20l g/l vermiculite was added into the medium 24h after inoculation resulting in the production of 146.88g citric acid monohydrate/l.The dry cell mass and residual sugar were 11.75and 55.90g/l,respectively.Mixed mycelial pellets (1.08–1.28mm,dia)were observed in the fermented culture broth.When the culture grown at different vermiculite levels was monitored for Q p ,Q s and q p ,there was a significant enhancement (P £0.05)in these variables over the control (vermiculite-free).Based on these results,it is concluded that vermiculite might affect mycelial morphology and subsequent TCA cycle performance to improve carbon source utilization by the mould,basic parameters for high performance citric acid fermentation.IntroductionThere is a growing trend to enhance microbial citric acid production,due to its wide range of uses in the food and pharmaceutical industries (Haq et al.2001).The raw materials like beet or cane-molasses,starch hydrolysate and glycerol residues are abundantly available for exploitation by microbial fermentations.They contain various levels of available sugars,ranging from 40–60%with additional ionic salts.The production of metabo-lites is strongly dependent on the fungal strain and fermentation medium used (Kubicek &Rohr 1985;Maria &Wladyslaw 1989).A potent strain and a cheap carbon source may also raise the status of the fermen-tation process;either directly or indirectly,and so affect the economics of citric acid fermentation (Majolli &Aguirre 1999).Trace metals present in blackstrap molasses such as iron,zinc,copper and manganese pose problems during submerged citric acid fermentation,therefore concentra-tion of these heavy metals should be maintained for optimal mycelial growth (Neilsen 1992;Pera &Callieri 1997).The addition of vermiculite (0.02–0.10l g/l)intothe medium at the time of inoculation or during the first 40h of fermentation counteracts the deleterious effect of certain trace metals like zinc and copper (Fedoseev et al.1970).The use of soil type vermiculite in fungal fermentations is increasing due to enhancement in proteins and metabolites (Tvuawa &Chandn 1986).The present studies highlight that vermiculite addition may influence pellet formation,facilitate enzyme secre-tion and subsequently increase the citric acid production.Materials and methods OrganismAspergillus niger strain NG GCB -101was obtained from available stock culture of Biotechnology Research Labs ,G.C.University Lahore ,Pakistan and maintained on the potato dextrose agar slants (pH 4.8).Molasses pre-treatmentBlackstrap molasses obtained from Kamalia Sugar Mills,Pakistan was pre-treated according to PandaWorld Journal of Microbiology &Biotechnology 20:463–467,2004.463Ó2004Kluwer Academic Publishers.Printed in theNetherlands.et al.(1984).The sugar content of molasses was typi-cally about45.0%.Thirty-five millilitres of0.05M H2SO4were added to per l molasses medium and placed in a water bath at90±2°C for about1h.After cooling at room temperature,the medium was neutral-ized with lime(CaO)and left overnight for clarification. Two layers were formed,the upper shiny black and the lower yellowish brown,due to the presence of trace metal hydroxides.The clear supernatant was decanted and diluted to15.0%sugar.Inoculum preparationFirst45.0ml Vogel’s medium(0.5%KH2PO4,0.2% NH4NO3,0.4%(NH4)2SO4,0.02%MgSO4,0.1% peptone,0.2%yeast extract at pH5.5)were added to a250ml conicalflask.Chromic acid-washed marble chips(12–15in number)were added to break the mycelial pellets.As a carbon source, 2.0ml glucose (50.0%w/v)was then added into the Vogel’s medium. After autoclaving at121°C for15min and cooling at room temperature,theflask was seeded with a loopful of A.niger conidia aseptically.The inoculum was allowed to grow at30°C in a rotary incubator shaker(Gal-lenkamp,UK,160rev/min)for24h.Next the cells were harvested,centrifuged at8331·g for15min,washed twice with saline water(NaCl0.85%,yeast extract 0.50%)and resuspended in distilled water.Optical density was measured(610nm wavelength)in a double beam UV/VIS scanning spectrophotometer and main-tained at0.5·10)2dilution.Fermentation techniqueA stainless steel stirred bioreactor(GLSC-AF-199-10) of9.0l working volume(total capacity15.0l)was employed for the microbial cultivations.The fermenta-tion medium consisted of clarified blackstrap cane-molasses containing:initial sugar15.0,NH4NO30.025, ash contents0.45,trace metals like iron,zinc,aluminium (0.035%w/v)and K4Fe(CN)6200mg/l(initial pH6.0) was sterilized at121°C(15lb/in.2pressure)for20min. Vermiculite(Analytical grade,Sigma,USA)concentra-tion varied from0.10to0.40l g/l added8–48h after inoculation.Mycelial inoculum was added at a level of 4.0%(v/v).Incubation temperature was kept at30°C for144h.The aeration rate was maintained at1.0l/l/ min with a35.0%dissolved O2level(found optimal). Agitation intensity was200rev/min in all experiments. Sterilized silicone oil(10.0%antifoam AE-II)was used to control foaming during fermentation.Samples were taken every24h.Assay methodsDry cell mass was determined gravimetrically using the dry weight method after Pirt(1975).Citric acid and sugars in the fermented culture broth were determined by the DNS method(Ghose1969).Kinetic parameters and statistical tests for batch fermentations were based on Pirt(1975).Results and discussionThe rates of citric acid production by the mutant strain Aspergillus niger NG GCB-101in a stirred bioreactor with control(vermiculite-free)and experimental batches (0.20l g/l vermiculite added at the time of inoculation) were compared(Figures1and2).The maximum amount of citric acid obtained with the control144h after mycelial inoculation was recorded at96.10g/l.The decreased citric acid production after144h might be due to exhaustion of nutrients in the fermentation medium and the age of the fungus,resulting in the decreased metabolic rates.Citric acid excretion normally decreases at the onset of stationary phase(Panda et al. 1984).The dry cell mass and residual sugar were14.20 and62.08g/l,respectively.Small round fungal pellets (1.25–1.40mm,dia)were found at the optimal citric acid production level.However,experimental batch cultivation gave123.78g/l citric acid productivity only 96h after incubation.Thus reduction in the incubation time period for citric acid production took place.In addition,net citric acid productivity was 1.29-fold higher than the control.To enhance citric acid productivity,the effect of the addition of various vermiculite levels was investigated (Table1).In the present study,vermiculite concentra-tion ranged from0.10to0.40l g/l added at the start of fermentation.However,citric acid biosynthesis was observed to be maximal(124.62g/l)when the concen-tration of vermiculite was maintained at0.20l g/l added 96h after the start of the production phase.The enhancement in citric acid biosynthesis is thus sub-stantial.The dry cell mass and residual sugar were12.00 and54.45g/l,respectively(small round fungal pellets having 1.12mm,dia).However,production of citric acid monohydrate was markedly decreased at higher levels of vermiculite,due to a change of mycelial morphology from pellets to a gelatinous form,substan-tiating thefindings of Gulkhera(1997).The residual sugar and dry cell mass were also reduced.The effect of vermiculite addition time on the citric acid production by Aspergillus niger was also examined (Table2).Samples were taken every24h during the fermentation.As far as citric acid production is con-cerned,the best results(146.88g/l citric acid monohy-drate)were obtained when0.20l g vermiculite/l was added24h after mycelial inoculation.The dry cell mass and residual sugar were slightly lower than the previous experimental batch i.e.,11.75and94.10g/l,respectively (Figure2).Mixed fungal pellets(0.08–1.28mm,dia) overtook small round pellets,which revealed the effect of vermiculite on mycelial cells and subsequent higher product formation(Gulkhera1997).In the present study,the vermiculite might have an effect on the performance of the TCA cycle to enhance its efficiency.464I.Haq et al.The mould capacity to utilize carbon source was also increased,resulting in higher product formation as reported by Fedoseev et al.(1970).A comparison of kinetic parameters for vermiculite addition on citric acid production in the stirred biore-actor is highlighted in Table 3.All values at different concentrations of vermiculite were significantly im-proved for Y x/s ,Y p/s and Y p/x over the control (without vermiculite addition).The maximum growth in terms of volumetric rate for mycelial formation (Q x )96h after incubation was only marginally different during fermen-tation at 0.20and 0.25l g/l,respectively.Similar find-ings have also been reported by Tvuawa &Chandn (1986)and Bruchmann (1961),but our report on citric acid productivity is many fold (<2.8–3.5)higher than these workers.In addition,when the culture was monitored for Q p and q s ,there was a significant enhancement (P £0.05)in these variables at 0.20lg/l0153045607590105120135150024487296120144168192216240Rate of citric acid fermentation with control (h)D r y c e l l m a s s , R e s i d u a l s u g a r , C i t r i c a c i d (g /l )Figure 1.The rate of citric acid fermentation by mutant strain of Aspergillus niger NG GCB -101in stirred bioreactor (Incubation temperature 30°C,initial pH 6.0,aeration rate 1.0l/l/min,initial sugar concentration 150g/l.Control batch i.e.,without vermiculiteaddition).0153045607590105120135150024487296120144168192216240Rate of citric acid fermentation with experimental (h)D r y c e l l m a s s , R e s i d u a l s u g a r , C i t r i c a c i d (g /l )Figure 2.The rate of citric acid fermentation by mutant strain of Aspergillus niger NG GCB -101in stirred bioreactor (Incubation temperature 30°C,initial pH 6.0,aeration rate 1.0l/l/min,initial sugar concentration 150g/l.Experimental batch i.e.,with 0.20l g/l vermiculite addition at the time of inoculation).A high performance fermentation process for citric acid production465vermiculite over other levels 96h after the incubation.In particular,value for q p (i.e.,specific productivity rate)at 0.20l g/l is highly significant 96h after incubation.The maximum Y p/s ,Y p/x ,Q p and q p were several fold improved over those from some other Aspergillus or Cellulomonas spp.(Schweiger 1961;Maria &Wladyslaw 1989;Haq et al.2001).In the present study,the rate coefficient of citric acid production proportional to growth rate was almost constant regardless of oxygen tension (Unpublished data).ConclusionThe improvement in citric acid fermentation technology may be attributed to the impact of additives on mycelial morphology for enhanced yields.In the present inves-tigation,most notable are the reduction in incubation period (144to only 96h)and the commercially accept-able citric acid productivity level (146.88g/l)achieved by adding 0.20l g vermiculite/l into the fermentation medium 24h after mycelial inoculation.We conclude that vermiculite addition somehow enhances the TCA cycle efficiency of mycelial cells for A.niger ,resulting in higher levels of product formation.Table 1.The effect of vermiculite addition on citric acid fermentation of Aspergillus niger NG GCB -101in stirred bioreactor*.Vermiculite concentration (l g/l)Concentration (g/l)Peak hour for maximal citric acid productionMycelial morphologyPellet diameter (mm)Dry cell massResidual sugar Citric acid monohydrate 0.1012.8051.50104.46120Mixed pellets 1.35–1.600.1512.1553.20109.50116Small pellets 1.200.2012.0053.45124.6296Small pellets 1.120.2511.3055.90116.1096Fine pellets 0.650.3011.1056.20102.5592Fluffy mass –0.3510.2061.8589.2684Gelatinous –0.409.5567.6081.8680Gelatinous –*Incubation temperature 30°C,initial pH 6.0,aeration rate 1.0l/l/min,initial sugar concentration 150g/l.Vermiculite was added at the time of inoculation.Samples were taken every 24h of fermentation.Table 2.The effect of time of vermiculite addition on citric acid fermentation by Aspergillus niger NG GCB -101in stirred bioreactor*.Vermiculiteaddition time (h)Concentration (g/l)Mycelial morphologyPellet diameter (mm)Dry cell massResidual sugar Citric acid monohydrate 812.6551.10126.10Small pellets 1.161612.1055.35131.64Small pellets 1.122411.7555.90146.88Mixed pellets 1.08–1.283211.6056.20122.05Mixed pellets1.15–1.554011.5059.40121.16Elongated mycelia –4811.2062.55114.42Dumpy mass–*Incubation temperature 30°C,initial pH 6.0,aeration rate 1.0l/l/min,initial sugar concentration 150g/l,vermiculite concentration 0.20l g/l.Table 3.The comparison of kinetic parameters for vermiculite addition on citric acid fermentation by Aspergillus niger NG-GCB101in stirred bioreactor 96h after the incubation.Kinetic parametersVermiculite conc.(l g/l)0.200.25Specific growth rate l (h )1)0.72540.509Citric acid formation parameters Q p (g/l/h) 1.3250.599Y p/s (g/g)0.9560.662Y p/x (g/g)8.293 6.755q p (g/g cells/h)0.0820.026Substrate consumption parameters Y x/s (g cells/g)0.0100.009Q s (g/l/h)0.2670.180q s (g/g cells/h)0.0480.061Q x (g cells/l/h)0.0240.026Least significant difference 0.5660.312Significance level h P iHSSKinetic parameters:l (h )1)=specific growth rate,Q p =g citric acid produced/l/h,Y p/s =g citric acid produced/g substrate consumed,Y p/x =g citric acid produced/g cells formed,q p =g citric acid produced/g cells/h,Y x/s =g cells/g substrate utilized,Q s =g substrate consumed/l/h,q s =g substrate consumed/g cells/h,Q x =g cells formed/l/h.HS is for the ‘highly significant’while S for ‘significant’values.466I.Haq et al.AcknowledgementsWe are thankful to the officials of PSF,Islamabad, Pakistan who partially funded this project.The com-ments and English proof reading by Dr David A. 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