Journal of Scientific & Industri al Research

Vol. 62, May 2003, pp 447-452

Bioconversion of Colocasia antiquorum and Aponogetonnatans to Citric Acid by Aspergillus niger - Effect of Metal Ions and Kinetics

A R Angumeenal, P Kamalakannan, H J Prabhu t & D Venkappayya*,

Department of Chemistry, tDepartment o f Chemi cal Engineering, Regional Engineering College, Thiruchirappalli 6200 15

Received: II July 2002, rev. recd: 17 Februrary 2003, accepted: 03 March 2003

Tuber crops belonging to the family A raceae namely Co Locasia antiquarum and Apanoge /olllla/alls are culti vated in large quantities fo r their edible portion. In thi s work, tubers are su itably treated and used as effi cient substrates fo r citric ac id production by fermentation using Aspergillus Il iger. The quantities of citric acid produced using these materials as substrates for bioconversion using Aspergillus Il iger are compared with those produ ced in synthetic medium compris ing g lu cose as substrate. Transition metal ions such as , chromium, molybdenum, cadmium and lead are added at optimum concentration as nu tri tional supplements and their effect on the biosyntheti c route of the citric acid cycle are discussed. Experi mentally observed growth stages are used for mathematical modeling to evaluate the kinetic parameters. The values obtained by calcul at ion agree we ll with the observed ones .

Introduction

Several sugar sources such as molasses 1-8, whey 9 d 10 b I I permeate, ate syrup , anana ex trac t , and root

I " I I b . d b f' . crops -. . were emg use as su strates or CItrIC ac id production. In recent years, a great upsurge has occurred in search of alternati ve materi a ls as substrates for citric ac id production . This made us to se lect Colocasia antiquo rum and Aponogetonnatans (vernacular names - Seppankkizhangu/seppan and Koraikki zhangu/korai) as substrates fo r citri c acid production. E arlie r work 14 described the effect of metal ions on citri c acid producti on by Aspergillus niger with synthetic substrates . In this work , an attempt is made to study the effect of chromium, molybdenum, cadmium and lead ions on the degradation of these natural substrates during the metaboli sm of Aspergillus niger.

Experimental Procedure

Aspergillus niger used in the present study was

procured from NCL, Pune (NRRL 322 - Species fo r C itric Acid Production CMI 3 1276; NCTC 594; WB 322).

" Author fo r correspondence, e-mail : dvenka@ rect.ernet.in

The fermentation medium compmmg (in per cent) glucose ( 14), KN03 (0 .8), K2HP04 (0.1 25) , M gS04.5HzO (0 .02) and ZnS04.7HzO (0 .02) was used as the control. The selected tubers were characte ri sed for the ir c rude fibre l'i ce llulosel 6

hemicellulose l7, starch l8 and min~ral conten~ (determined using AAS). Steaming the pulped tuber with 0.4 N HCl for 1.5 h was found to be the optimum condition for hydrolys ing the maximum soluble sugars . The pH of the hydrolysed substrate was adjusted to be between 2 and 3. Batch fermentation by surface culture was initi ated using the spore suspension of Aspergillus niger. From the third day of inoculation onwards up to the I I th day, the culture broth was filtered and anal ysed for tota l titrable ac idity (volume of 0 .1 N N aOH consumed by I mL of the filtrate), citric l9, succ inic and malic acids (using HPLC), extracellular soluble proteins2°and residual metal ion contents (using AAS). The biomass was analysed for amylase21, lipid22 and

. 20 protem contents .

Results and Discussion

T he morpho logical views of the tuberous species used as substrates for the study are shown in Figure I and F igure 2 . Seppan was found to contain 55.5 per cent starch, 2. 8 per cent c rude fibre and 2 .4

448 J scrlNO RES VOL 62 \ I. \ Y 2003

Figure I - Co locasia antiquofllm

Figure 2 - Aponogetonn atans

per cent cellulose, whereas korai was found to contain 60 per cent starch , 3.8 per cent fibre and 2.8 per cent cellul ose. Hemice llul ose fraction was present in same amounts (O .S per cent) in both the

spec Ies .

Results with Colocasia

With Colocasia as subst rate A.niger was found to exhibit the best amylase activity (68 U / mL / min)

and the substrate sugar concentration was not depleted till the e nd of fermentation . The results of protein and lipid metabolism are g iven in Table I . The metabolic state of A. niger was found to be influenced by adding cadmium (in the form of cadmium chloride, 40 ppm) lead (in the form of lead nitrate, 40 ppm) , molybdenum ( in the form of ammonium molybdate, 10 ppm) and chromium (in the form of potassium dichromate, 20 ppm) to the

ANGUMEENAL et at.: BIOCONV ERS ION OF COLOCASIA ANT/QUORUM & A PONOGETONNA TANS 449

media, as observed earlier with the synthetic substrates 14. The products of the microbial metaboli sm are shown in Table 2. With the control medium 25 giL of citric ac id was produced, whereas it was 18.4 giL with seppan as substrate . The quantity of ci tric acid produced was considerably increased with the metal ion supplemented medi a. The biomass production was also high with metal ions in the medium with increase in extracellular protein content. This could be due to the interaction of these metal ions with the cell wall contents like peptidoglycans and lipoprote ins resulting in the leakage of peptides, nucleotides and other small molecules ac ross the membrane. The highest citric

acid production was in the presence of molybdenum (52 giL) as nutrient.

Results with. Aponogetonnatans

The underground tuber porti on of thi s species was used as the substrate for the product ion of c itric acid .The products of the microbial metaboli sm after fermentation are shown in Table 3. The amount of citric acid produced was higher with this substrate than with the synthetic medium (37.6 giL) . The metal ion supplemented media were found to produce more quantities of citric ac id in a shorter time of 3-5 d . The quantities of ac ids, proteins and biomass produced by A. niger with this substrate are given in Table 4. The

Table 1- Growth characteristics of A.niger with Colocasia as substrate

Incubation peri od , Total titrable Citric ac id , Protein Lipids, Biomass, cl acidi ty, mUmL giL per cent <T

ICP, per cent ECP, gil 00 mL '" 3 3.5 12.5 4.0 0.6660

5 4.4 15 .0 3.4 4.5 4.6 0.7080

7 4.8 15.0 6.6 4.7 14.4 0.9440

9 5.0 18.4 3.0 3.5 23.0 1.200

II 3.4 8.2 2.2 2.5 10.0 0.860

ICP= Intracellul ar proteins, ECP= Extracellul ar proteins

Table 2 - Intluence of nutrient compositio n on the batch fermentation of A.niger with Co locasia as substrate

Contro l Cd (40 ppm) Pb (40 ppm) Mo (10 ppm) Cr (20 ppm)

TTA, mUmL 5.0 8.0 5.6 5.2 5.0

CA, giL 18.4

ICP, per cent 6.6

ECP, gil 00 mL 4.7

Biomass , gil 00 mL 1.200

30.6

5.6

18.0

2. 148

3 1.2

4.4

17.5

2. 130

52.0

2.8

20.0

2.214

TTA = Total titrable ac idity, CA = Citric acid, ICP = Intracellular proteins, ECP = Extracellular proteins

Table 3 - Growth characteri sti cs of A.niger with Aponogetonnatans as subst rate

Incubat ion Total titrable Citric ac id, Protein Lipids, period , d ac idity, giL

ICP, ECP, per cent

mUmL per cent gil 00 mL

3 7.2 34.9 35.3 26.1 15.0

5 10.3 36.3 28.3 26.1 30.0

7 9.9 37.6 20.0 18.0 30.0

9 7.8 20.8 15.5 16.8 32.0

II 7.2 20.3 11.5 11.5 8.0

rcp = Intracellular proteins, ECP = Extracellular proteins

28.0

R.4

30.0

2.0 18

Biomass ,

" '"

2.460

5.023

5.944

7.809

7.482

450 J SCIIND RES VOL 62 MAY 2003

Tahl e 4 - Influence of nu trient compos iti on o n the batch fermentation of A.niger with Aponogetonnatans as substrate

Contro l Cd (40 ppm) Pb (40 ppm) Mo ( 10 ppm) Cr (20 ppm)

ITA, mg/mL 10.3 5. 8 6.8 604 5.8

CA , giL 37.6

ICP, per cent 35.3

ECP, gi l 00 mL 26. 1

Bio mass, gi l 00 mL 7.809

18.0

4.8

34.0

2.022

58.1

lOA

3204

1.6 16

70.8

1804

44 .0

1.206

5.8

4.8

36.0

2. 102

ITA = Total titrabl c acidi ty, CA = Citric acid, ICP = Intrace llular proteins, ECP = Extracellul ar proteins

results revea l that more quantities o f c itric ac id are produced by A.niger in the presence molybdenum and lead. However the total titrable acidity produced was found to be hindered in the metal ion supplemented media ; probabl y due to the e lectron transport c hain being obstructed . Due to this H+ ion re lease is less, and so less total titrable ac idity . With lead and molybdenum as nutrients the biomass weight was less and the c itric ac id produced was hi gh, but it was not so in the presence of chromium and cadmium. Thi s cou ld poss ibly be due to the absence of active cells taking part in citrate production. The cell wal l of A.niger is made up of peptid oglycans and lipoprotein s. The initial stages of g lyco lys is occur in the cytoplasm after which the acety l-CoA formed enters the mitochondria for further metaboli sm. Hence, it was found that the nature of the cell wall as well as the mitochodrial permeability a lters the secretion of citric ac id. The permeability of these are governed by the environment to which they are exposed to . Thus, as A.niger is exposed to the metal ions while using these substrates the permeabi lity is influenced. Meta l ions interact with the cell wall components such as g lucons and mannans , thereby disrupting their arrangement lead ing to the formation of pores. Consequently the permeability through the pores is improved .

Kinetics and Computation

To gain further insight into the corre lation be tween the experimental and experiment based theoretical values mathematical modeling was done. O n the bas is of the kinetic theory of microbi al cell growth and the product concentrati on in fe rmentati on, equations of growth rate and ce ll concentration were deri ved from the general equa ti ons of growth rate23

, a long with equati ons for

product concentration in each phase of microbial ce ll growth .

The rate equations23 used for the modeling studies are give n below:

Microbial Cell Concentration

Exponential growth phase Cx = Cxe exp(kx (8 -8e)).

Declining growth phase Cx = CXM - (CXM -

Cxe) exp[ - kx (CxelCxM - Cxe) (8 - 8e)].

Product Concentration

E xponential growth phase Cp = (-Kpl I Kx)* Cxe{ I-exp (kx(8 - 8e ))} + C pe

Declining growth phase Cp = {[k pi I kx] - [(k p2

Ikx) (CXM I Cxe)]} (CXM - Cxe) { I - exp[-kx (Cxe

I(CXM - Cxe)) ((}-(}e)]} + kp2 CXM (() - (}e) + Cpc.

where,

Cx Microbial concentration at exponential and declining growth phases

Cxe Biomass concentration at criti cal point CXM Maximum biomass concentrati on kx Microbial growth rate 8 T ime in hours

8e Critical point - point with maXlmum microbial growth

Cp Product concentration at exponential and declining growth phases

Cpe Product concentration at the critical point

kp I First production rate constant, and kp2 Second production rate constant.

Determination of variables

A plot of e (incubation time)versus the biomass

weight (weight of A.niger) was obtained fro m which

ANGUMEENAL et al.: BIOCONVERSION OF COLOCAS1A ANT/QUORUM & APONOCETONNATANS 451

the exponential growth phase and declining phases were identified . The area under the exponential growth phase and declining phase are blown up and ana lysed with more accuracy. During the above two phases the amount of citric acid produced by A.niger was also determined, which gave experimental Cx and Cp va lues. The point (on the graph) where the growth of Aspergillus niger is steady is labeled as ec (c ritical point). At this point the corresponding Cxc and Cpe values were noted. Plots of dCx/dt versus eand dCp/dt versus e gave the rate constants kx, kPl, kp2. These values along with ec, Cxc and Cpe were applied in the above equations to get the calcu lated Cx and Cp values

Resulrs of the Modeling Studies

Using the control/synthetic medium the product format ion was found to be associated with growth, which was inferred from the rate constant values obtained (ie., kPI is positive and kP2 is zero). Experimentally determined ex and Cp values were in good agreement with the calculated ones.

With Aponogetonnatans as substrate two production rate constants were obtained viz., kPI and kP2, one being positive and the other negative which indicates the fermentation process to be associated with growth as wel l as with non-growth2

,. The calculated and observed cell and product concentrations are given in Table 5. The growth of Aspergillus niger was observed to increase under experimental conditions in the initial stages of the exponential phase but the production of citric acid was found to decrease in the later stages . This observation indicates that the active cell concentration in the total cell mass of Aspergillus niger is low and hence the total number of microorganism taking part in c itric acid formation is low. Two production rate constants were identified with Colocasia as substrate whose signs are simi lar to the previous case (i e, kPI is positive and kP2 is negat ive) . The quantities of ce ll and product concentration obtained by the modeling studies reveal that citric acid production can be increased if the growth rate can be s lightly improved (kx > 0.0066). With these tuberous sources too the experimentally determined Cx and Cp values were in good agreement with the calculated ones. Statistical ana lysis was done (using computer excel software) to get the correlation between the calculated and

Table 5 - Experimental and calculated Cx and Cp values with Colocasia alld Aponogcton.n.atalls as substrate

Substrate 8, h Experimental Calculated

Cx, CPo Cx, CPo giL giL gIL giL

Colocasia 0 7.5 15.0 6.6 14. 1

12 8.2 16.0 7.2 14.7

24 8.7 16.5 7.9 15.3

36 9.5 16.5 8.7 15 .9

48 10.0 17.0 9.5 16.7

60 10.5 17.0 10.5 17.5

72 11 .0 18.0 11.5 18.3

84 I 1.9 18.0 12.6 19.3

96 12.0 18.5 13.8 20.3

108 11 .9 16.5 I 1. 8 21.4

Apollogetollllatalls 0 25 .0 35 .0 39.7 19. 1

12 30.0 35.0 41.4 21 . I

24 33.0 35.5 44.0 23. I

36 38 .0 36.0 46.3 25.2

48 43.0 37.0 48.8 27.4

60 47.0 37.0 51.3 29 .7

72 51.0 37.5 54. I 32.2

84 56.0 37.5 56.9 34.8

96 60.0 37.5 60.0 37.6

108 65.0 35.0 63. I 40.4

Cx = Biomass and Cp = Citric acid Coefficient of determination (experimental and mode l calculated Cx and Cp values) , r2: foi' Colocasia 0.957 and 0.928 for Cx and Cr, and for Aponogetonnaralls 0.996 and 0.916 for Cx and CPo

observed values. A higher value for r2 (Tab le 5) shows better fitness to the kinetic model used.

Conclusions

The substrates studied III the current investigation were found to be equal in quality to other substrates reported earlier. The metal s, by producing intermediate substances , take part effectively in improving membrane permeability. The amount of citric acid produced with colocasia as substrate was 18.4 giL and with aponogetol1natans 37.6 giL. With colocasia the acid production was

452 1 SCI IND RES VOL 62 MAY 2003

improved considerably in the presence of the metal ions, the highest production be ing in the presence of molybdenum (52 giL) . With aponogetonnatans as substrate, lead at a concentration of 40 ppm was found to produce 58 . 1 giL of citric acid and molybdenum at a concentration of 10 ppm, 70.8 giL. Thus the nutriti onal quality of both the substrates were found to be improved in the presence of metal ions. To arrive at the exact mechanistic path radi oact ive labeling studies are to be done to ide ntify the activated species (either the enzyme or the organ) in A. niger that induces citric acid production.

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