Indian Journal of Experimental Biology Vol. 39, July 2001. pp. 69 1-696

Gluconic acid production by Aspergillus niger mutant ORS-4.410 in submerged and solid state surface fermentation

Om Vir Singh, Amit Sharma & R P Singh*

Department of Biosciences and Biotechnology, University of Roorkee, Roorkee 247 667, Indi a

Received 14 August 2000; revised 22 Febmary 2001

Aspergillus niger ORS-4.41 0, a mutant of Aspergillus niger ORS-4 was produced by repeated irradiation with UV rays. Treatments wi th chemical mutagnes also resulted into mutant strains. The mutants differed from the parent strain morpho­logically and in gluconic ac id production. The relationship between UV treatment dosage, conidial survival and frequency of mutation showed the maxi mum frequency of positive mutants (25%) was obtained along with a conidial survival of 59% after second stage of UV irradiation. Comparison of gluconic acid production of the parent and mutant ORS-4.41 0 strain showed a significant increase in gluconic acid production that was 87% higher than the wild type st rain . ORS-4.410 strain when transferred every 15 days and monitored for gluconic acid level s for a total period of ten months appeared stable . Mu­tant ORS-4.410 at 12% substrate concentration resulted into significantly higher i.e. 85-87 and 94-97% yields of gluconic acid under submerged and solid state surface conditions respectively. Further increase in substrate concentration appeared inhibitory. Maximum yield o f gluconic acid was obtained after 6 days under submerged condition and decreased on further cultivation. Solid state surface culture condition on the other hand resulted into higher yield after 12 clays of cultivation and similar levels of yie lds continued thereafter.

D-gluconic acid, an oxidative product of D-glucose, is extensively used in food, feed and pharmaceutical industry'. There are several reports on fermentative production of gluconic acid and its salts by various bacterial and mould species. The commonly studied bacterial species are Pesudomonas, Acetobacter2 and Gluconobacter3 while in moulds Penicillium4 and Aspergillus5 have been considered suitable for com­mercial exploitation for the purpose. Attempts have been made for developing improved calcium gluco­nate producing strain viz. Penicillium funiculosum PS26

, A. niger AB 1801 7 by physical and chemical mutagenesis. Further, a gluconic acid and glucose oxidase overproducing mutant Penicillium variable M 80.10 has been developed from Penicillum vriable Pl6 through UV mutagenesis8

. Total worldwide pro­duction of calcium and sodium salts of gluconic acid is estimated to be approximately 50,000-60,000 ton annually and has thus emphasized various groups for extensive investigations towards identifying and de­veloping potent microbial strain and bioprocesses for industrial production of g luconic acid.

Our aim has been to use the sugarcane industry wastes for selecting microbial strains that may signifi­cantly be used for gluconic acid production. We have,

*Correspondent author: Fax: 91-1332-73560; Tel. : 91- 1332-85792; e-mail : rpsbsfbs @rurkiu .ernet.in

therefore, selected the site decomposed with the sug­arcane industry waste and isolated the strain, Asper­gillus niger ORS-4 that has been found to have a re­markable ability for gluconic acid production with a total yield of 75-80% (Ref. 9). Unfortunately the strain is slow growing with moderate sporulation and therefore has a disadvantage for commercial utili za­tion of the strain. We have, therefore, attempted the two step physical and chemical mutagenesis to de­velop a suitable mutant of A. niger ORS-4 and has analyzed the parameters for culture conditions so as to use the strain for commercial production of the glu­conic acid.

Materials and Methods Microorganis~ The wild type strain , Aspergillus

niger ORS-4 was isolated from the site of decom­posed sugarcane industry wastes9 and maintained on potato dextrose agar (PDA) slants by periodical trans­fers.

Mutagenesis and selection-Two step physical mutagenesis was carried out using the spore suspen­sion of A. niger ORS-4 (10 mL, l x l09 spore/mL) in petri dish by UV illumination (2.5 J/m2/s 1

, distance 0.69 m) for different times ranging from 60 sec to 12 min. The conidial suspension (lxl09 spore/mL) of A. niger ORS-4 was treated with N-methyl-n'-nitro­N-nitrosoguanidine (MNNG, 100 J,!g/mL) for 10-60

692 INDIAN J EXP BIOL, JULY 2001

min at 30°C. The hydrated conidial suspension of A. niger ORS-4 were also treated with 0.2 mL of sodium nitrite (5 mg/mL) solution for I 0-60 min at 30°C, which generated the mutagenic agent, nitrous acid, in the sodium acetate buffer (1.8 mL, pH 4.4) 10

. Fol­lowing every treatment, conidial survival after appro­priate dilutions was determined on PDA plates at 30°C and selection of the surviving mutant strains was performed on optimized mineral salt agar medium conta111111g (g/L): NaN03, 2.5; KH2P04, 1.0; Mt;S04.?H20, 0.5 ; KCI , 0.5; FeS04.?H20, 0.01; glyc­erol , 1 5.0; glucose, 20.0; agar, I 5.0; with 0.04% alco­holic solution of bromocresol green used as an indi­cator for rapid screening of highly acidogenic strains on the basis of their ac id unitage value6

. The colonies thus selected were further streaked for purification on identical media.

Growth in liquid medium and culture conditions­Purified spores from 5 days old cultures grown on PDA slants at 30°C were suspended (1010-1012

spores/mL) in 5 mL of sterile 0.05 M phosphate buffer (pH 6.8) containing 0.1 % Tween-80 and 2% of inoculum was used for 250 mL of Erlenmeyer flask containing 50 mL of culture medium containing (g/L): (N H4h HP04, 1.0; KH2P04, 0.5; MgS04.?H20, 0.15 ; CaC03 was added in the medium (pH 6.5 ± 0.1) with continuous shaking ( I 50 rpm) for submerged fermentation process. The solid state surface culture condition was evaluated by usi ng bagasse as the solid support. Before use, the finely powdered bagasse (obtained from sugar mill) was treated overnight with 2N HCI at room temperaure, followed by thorough washing with double di stilled water till the wash gets neutralized. A slurry of medium containing 15% of treated bagasse (70% moisture contents) was inoculated with 5 mL of A. niger spores (as stated above) and incubated for 12 days at 30°C, with occasional shaking to ensure a surface culture; waste culture cultivation gases were removed by connecting the culture flask to the water suction pump with the circulation of fresh air over the fun gal mat on solid surface. After different time intervals the samples were collected by filteration for chemical analysis and growth was followed by mycelium dry weight determination 11 .

Chemical analysis--The unutilized total residual sugar concentration was assayed according to Miller 12. Gluconic ac id formed was determined by assaying the dissolved calcium amount in culture me­dium13. The broth containing gluconic acid was sub­jected to acid hydrolysis and resulting gl uconolactone

was measured by the modified hydroxamate method 14.

Scanning electorn microscopy-Scanning electron microscopy (SEM) of the mutagenized fungal mycelia was performed by fixing with 2% of glutaraldehyde. After dehydrat ion in ethanol series, the samples were air dried, coated with gold and examined by scanning electron microscope (Leo 435VP, England). The my­celia of wild type A. niger ORS-4 were processed and observed in the similar manner.

All the experiments were performed in triplicate and the experimental results represent the mean of three identical cultivations.

Results and Discussion The fungal strain used for the present investigation

was isolated from the sites decomposed with the sug­arcane industry wastes and was characterized as As­pergillus niger ORS-4. This strain was one of the seven isolates and although comparatively had a lower growth rate but produced apparently hi gher level of gluconic acid. Strain ORS-4 was further sub­jected to physical and chemical mutagenesi s for in­creasing the yield and production of gluconic ac id.

Mutagenesis and screening of A. niger ORS-4-Two step UV irradiation and chemical mutagenesis were used for improving the gluconic acid levels produced by A. niger ORS-4. The optimal conditions for muta­genesis were derived by variations in UV and chemi­cal mutagen exposures and by evaluating the conidial survival and the frequency of positive and negative mutants. The distribution of treated colonies accord­ing to the acid unit age value on mineral salt agar acid indicator media and the percentage viability have been shown in Fig. I a-d. Among the range of dosage used for first and second stage UV irradiation , the second stage of UV irradiation (450 J/m2) appeared to ensure the max imum overproducing mutants (25% of the tested colonies) with 59% of cell survival (Fig. lb) . UV irradiation, as expected, of A. niger ORS-4 cells yielded both positve and negati ve mu­tants. The proportion of negative mutants after first stage UV mutagenesis was low compared to the sec­ond stage of UV exposure and thi s may probably be due to the stability of the parental strain used for the initial UV exposure than the mutant strain ORS-4.410 used for the second stage UV exposure. Mutagenic treatments as observed earlier had resulted into gl u­cose oxidase overproducing mutants of P. variabili and A. niger15

• The results observed showed that A. niger ORS-4.410, obtained after second stage of UV

SINGH et a/.: GLUCONIC ACID PRODUCTION BY ASPERGILLUS NIGER MUTA NT

90

80 ~ .:: 60 c 0

- ~ 0

20

0 0

r~ -

- \ - 1'\ -

-

-, 1 r r I 150 300 450 600

(a) f\ r-

1\ r-I- ~ I- \ r-

i\ r-1\ f-

I I r-r I I I

12(() 1 00 8 0 150 300 450 600

u v irr adiation ( J m2 )

(b)

~ 1200 1

1 00

-

- 8 0

-

-6 0 >.

-

f4 -

- 2

-

.D C)

0 > > ...

0

:J lf)

.. 0 K.J

693

Fig. !-Analysis of mutagenesis on conidial survival (%), di stribution (%) of Aspergillus niger ORS-4 colonies on the basis of AU value Treatment given is (a)-1" stage UV irradiat ion; (b)- 11 nd stage UV irradiation;(c)-N-methyl-n'-nitro-N-nit rosoguan idine and (d) -Nitrous acid(-.._, survivabi lity; 0, negative mutants; ~. corresponding mutants; • . posi ti ve mutants).

Table !-Growth and glucon ic acid production after physical and chemical mutagenesis of Aspergillus niger ORS-4 under submerged condition

[Values are mean± SD of 3 replications]

Mutagenic agent Strains of A. niger AU values" Gluconic acid Glucose con- Yield(%) Biomass (g/L) (g/L) sumption (g/L)

None ORS-4 4.1±0.8 49 .00( I OO)b 64.6±7.6 75 .6 9.5±0.8 1st Stage of UV ORS-4. 110 6.0±2.2 68.50( 139) 11 0.2±8.8 62.1 14.3±15 li nd Stage of UV ORS-4.4 10 8.3± 1.8 9 1.79( 187) 105.2±9. 1 87.2 I 1.6± 1.3 MNNG ORSM-4.248 6.1± 1.5 69.50(14 1) 105 .6±9.5 65.7 14.8±2.8 Nitrous acid ORSN-4.340 5.2± 1.2 59.20( 120) 97.3±7.2 60.8 15 .8±2.3

"AU values expressed as diameter of yellow zone/diameter of mycelial zone. h Values given in paranthescs are the %age glucon ic ac id production as compared to wi ld type.

69-+ INDIAN J EXP BIOL, JULY 200 I

treatment ( 450 J/m2) had gluconic acid production,

87% higher than the wild type ORS-4 (Table 1). The gluconic acid overproducing mutant having such higher levels of acid production have not been de­scribed earlier8

· 16

. The treatment with chemical muta­gens i.e. MNNG and nitrous acid led to the mutants with marginally increased ability (20-40%) for glu­conic acid production. The strain ORS-4.410, there­fore, was employed for further investigation in the subsequent experiments. Product stability was moni­tored by subculturing the high ac id producing mutant strain ORS-4.41 0 after every 15 days for a period of 10 months and simultaneously estimating the glucon i.c ac id levels at respective intervals. Barring a minor variation, a stable gluconic acid production was ob­served after the strain ORS-4.41 0 was subcultured subsequently for 20 generations (Fig. 2).

Morphological characterization-Notable changes in the morphological features of the fungal mycelia following physical and chemical mutagenesis were observed as evident in the scanning electron micros­copy of the wild type and mutagenized mycelia. The mutant ORS-4.41 0 after two stage UV mutagenesis had thick, short and globu lar mycelia (Fig. 3b) as compared to the mycelia of the wild type strain ORS-4 that was highly branched, smooth and elongated (Fig. 3a). So far the co-relationship between distinct morphological features and gluconic acid production has not been analyzed and probably the altered bio­chemical architecture and the activated glucose oxi­dase may lead to increased production of gluconic acid.

Fig. 3-Scanning electron micrograph (SEM) of 72 hr old my­celia of (a)- wild type Aspergillus niger ORS-4 and (b)- mutant Aspergillus niger ORS-4.41 0 [Magnification x 1500 (Bar I 0

)..lm)l .

iOOr-----------------------------------------------------,

80 30

...J

:§ 25-60

...J '0 a, () 20 "' "' .~ "' "' c: 40 15 E 0 0 () m ~ Cl 10

20

5

0 ~ •.. 0

0 2 6 7 8 10

Tirne (months)

Fig. 2-Product stability of mutant Aspergillus niger ORS-4. 4 i 0 and the parent strain Aspergillus niger ORS-4. Levels of gluconic acid and biomass was detected after transferring the cultures every 15 days for a tota l period of ten months in submerged culture condition with 12% substrate concentratioin and at an initial pH 6.6 ± 0.1. (ORS-4. 410: E;:l, gluconic acid; • . biomass. ORS-4: 0 , gluconic acid; E:l, biomass)

S INGH era/.: GLUCONIC AC ID PROD UCTIO N BY ASPERGILLUS NIGER MUTANT 695

Analysis of fac tors affecting gluconic acid produc tion by mutant A. niger ORS-4.410-Glucose in vary­ing concentrati ons was used as substrate under sub­merged and sol id state surface cu lture conditi ons for gluconic ac id production and showed that 10-16% of glucose as substrate resu lted into significantly higher production of gluconi c acid by mutant strain A. niger ORS-4.410 (88-90 g/L) than parent strain (45-50 g/L) in submerged cu lture (Fig. 4a), whereas a further higher yield in gluconic ac id production (98%) at 12% substrate concentration was observed in solid state surface cultu re cond ition (Fig. 4b) and was 78% higher than the wild type A. niger ORS-4 strain . The substrate below and above this concentration inhibited the ac id production wi th a marginal increase in cel l

12C,---------------~

( a)

. 30

Substrate (giL)

Fig. 4-Giuconic acid production by the mutant Aspergillus niger ORS-4. 410 and the wild type Aspergillus niger ORS-4 at differ­ent glucose concentrations in (a)- submerged and (b)- solid state surface culture condition. (ORS-4.41 0: A , gluconic acid; • . re­sidual sugar; • . biomass. ORS-4 :L'., gl uconic acid; D , residual sugar; 0, biomass)

mass. It is assumed that sugars by osmosis enter in the fungal cell wal l and the gluconic acid thus diffuses out. The low osmotic rate that may be generated at hi gher concentration of sugar may subsequently lead to a lower rate of gluconic ac id diffusion due to the concentration gradient across the mycelial mat 18

•

Comparatively lower growth for wild type A. niger ORS-4 was observed than mutant strain , A. niger ORS-4.410. The active growth of fungal myceli a of mutant strain ORS-4.41 0 began after 24 and 48 hr of incubation in submerged and surface culture conditi on respectively and was accompanied by the increasing

(a) 35

eo

60

~ 20 '-"

<!)

"' 0 g 0 ~~~~--~--~~~~~~--~~J

6 Fermentation time (hr)

"'0 ' C) 1£011;--------------------------------,

C1)

0 ·;:: 0 gt 6

80

60

20

( b )

2

s 10 12 14 IG

Fem1entation time (d)

30

25

20

15

10

0~ '-' Vl Vl ;s :::: 0

i:i5

35

JO

25

20

\5

10

5

0

Fig. 5-Production of gluconic acid at various intervals under (a)- submerged and (b)- so lid state surface cultivation by mutant Aspergillus niger ORS-4.4 1 0 and wild type As­perg illus niger ORS-4. (ORS-4.41 0 : .6 , gluconic acid; • , residual sugar; e, biomass. ORS-4 : t:,., gl uconic acid; D,

residual sugar; 0, biomass)

- 696 INDIA N J EXP BIOL, JULY 200 1

levels of gluconate production, the gl uconate yield increased exponentially during active growth phase and a maximum acid yield upto 87% after six clays and 95% after 12 clays was obtained in submerged (Fig. Sa) and solid state surface cu lture condition (Fig. Sb) respectively . Beyond this period the gluconic acid concentration was found to drop in submerged cond i­tion whereas the continuous yield of gluconate pro­duction was obtained upto 25 clays under surface cul­tivation process (data not shown) eventhough a very moderate increase in mycelial growth was observed beyond I 0 days of incubation.

We have compared the yields of gluconic acid in solid state surface and in the submerged culture con­clition and former proved to be a better system for achieving higher yields for gluconate production. The addition of bagasse fo r sol id state cultivation had con­siderably increased the surface area and hence aera-. I I . d . h 'd . 17 IX f' tton anc t 1us 111 ucmg t e oxt at ton process · or

increased production of gluconic acid. Surface culti­vation required longer periods for maximum yi eld but alternatively a system well equipped with agitation, aeration , foam and pH control, etc. may be suitably designed to achieve better yields in shorter time peri­ods. The product generated remai n stab le in solid state process and higher with constant yie ld of production can be obtained for longer periods whereas the prod­uct was more susceptib le for decomposition in sub­merged condition. Solid state surface culture concli­tion, was comparatively simpler in operation involv­ing much less power consumption and this simplicity in the operation made solid state culture process an attractive and economically viable proposition for gluconic acid production.

The above observation, therefore, indicated that a gluconic acid overproducing mutant A. niger ORS-4.4 1 0 was obta ined and was stable after periodical transfer for several generations. The stra in ORS-4.410, therefore, can be suitably utilized for large scale production of gluconic acid and in addition to the conventional process of production under sub­merged conditions, the solid state surface process can be economically employed for thi s purpose. Efforts are underway for scaling up the process in order to economize and for industrial exploitation of the de­veloped strain.

Acknowledgement Project ass istantship awarded to AS by UP Council

of Science and Technology, Lucknow is gratefu lly acknow ledged.

References I Roehr M, Kubicek C P & Kiminck J, in Biotechnology,

Product of Primary Metabolites, vol. 6, edited by Rchm H J and Reed G (Verlag Chemic, Weinhcim) 1996, 347.

2 Lock wood L B, in Mircobial Technology, vol. I, edited by Peppler H J and Perlman D (Academi c Press, New York) 1979, 376.

3 Velizarov S & Beschkov V, Biotransformation of glucose to free gluconic acid by Gluconobacter oxydanse. Substrate and product inhibition situat ions. Proc Bioche111. 33 ( 1998) 527.

4 Pctruccioli M, Picc ioni P, Fcnice M & Federici F, Glucose oxidase, catalase and gluconic acid production by immobi­li zed mycelium of Penicillium variable P16, Biotecllllol LetT, 16 ( 1994), 939.

5 Moksia J, Larrochc C & Gros J B, Gluconate product ion by spores of Aspergillus niger, Biotech no/ Lett, 18 ( 1996) 1025.

6 Kundu P & Das A, Utilization of cheap carbohydrate source for product ion of calcium gluconatc by Penicillium.fimicu/o­su/11 mutant MN238, Indian J Exp Bioi . 22 ( 1984) 279.

7 RayS & Banik A K, Development of a mutant strain of As­pergillus niger and optimizat ion of some physical factors for improved calci um gluconatc production, l11dian J Eq> /Jiol, 32 (1994) 865.

R Pctruccioli M, Piccioni P, Federici F & Polsinelli M, Glucose oxidase overproduc ing mutant s of Penicilliu111 \'(/riable (PI6), FEMS Micmbial Leu, 128 ( 1995) 107.

9 Singh 0 V, Pereira B M J & Singh R P, Isolation and cha­ratcrization of a potent fungal strain Aspergillus niger ORS--1 for gluconic acid production, J Sci Indus/ Res, 5R ( 1999) 594.

I 0 Mark we ll J, Frakes L G, Brott E C, Osterman J & Wagner F W, Aspergillus niger mutants with increased glucose oxidase production, Appl Microbial Biotechnol, 30 ( 1989) 166.

II Hat zinikolaou D G & Macris B J, Factors regulating produc­tion of glucose oxidase by Aspergillus niger, En~yme Micob Techno/, 17 ( 1995) 530.

12 Miller G L, Usc of Dinitrosa licy lic acid reagent for determi­nation of reducing sugar, Anal Chem, 31 ( 1959) 426.

13 Lehmann J K, in Biotechnology, vol. 2, edited by Rchm H J and Reed G (Verlag Chemic Wcinheim) 1985, 620.

14 Lien 0 G, Determination of gluconolactonc, ga lactonolac­tonc and their free acid by the hydroxamate method, Anal Chem, 31 ( 1959) 1363.

15 Wittcvcccn C F B, Vandervoort V D, Swart P. Swart K & Visser J, Glucose oxidase overproducing and negative mu ­tants of Aspergillus niger, Appl Micrubiol Bimecllllol. 33 ( 1990) 683.

16 Mandai S K & Chatarjec S P, Improved production of cal­cium gluconatc by mutants of Penici/liwn funiculosum, Curr Sci, 54 (1985) 149.

17 Torres E F, Lopez J C, Rivero M G & Rojes M G, Kinetics of growth of Aspergillus niger during submerged, agar su r­face and solid state fe rmentations, Proc Biochem, 33 ( 1998) 103.

18 Garg K, Bioconvcrsion of Citric acid. Ph.D. Thes is, Univer­si ty of Roorkce. Roorkcc. India, (1990).