INTRODUCTION:

Aspergillus niger has been employed for many years in the research and development related to industrial microbiology¹. It was well known that the growth of Aspergillus niger and subsequently biosorption are strongly affected by medium composition ².Growth of fungus is highly dependent on the availability of carbon, nitrogen, phosphorus, minerals and vitamins^3-5.

Thus in my present study, I was intended to select a suitable synthetic medium by optimizing different nutrients for the optimum growth and subsequently maximum As(III) biosorption by the resistant strain Aspergillus niger X₃₀₀.

MATERIALS AND METHODS:

Microorganism:

An experimentally developed As (III) resistant strain Aspergillus niger X₃₀₀was used throughout the study⁶.

Medium composition used for fungal growth :

The growth medium was composed of: glucose, 10 %; urea, 0.8 %; MgSO₄.7 H₂O, 0.03 %; KH₂PO₄, 0.1 %; K₂HPO₄, 0.1 %; CaCl₂, 0.3 %⁷.

Medium composition for maintenance medium:

Synthetic maintenance medium contains: glucose, 0.1%; urea, 0.6 %; MgSO₄.7 H₂O , 0.01 % ;KH₂PO₄, 0.1 %; K₂HPO₄, 0.1 % ; CaCl₂ , 0.2 % and agar, 4 % as solidifying agent⁷.

Estimation of As(III) :

The concentration of As(III) in the broth was estimated by the method as reported by Cernansky et al., 2007⁸.

Estimation of dry cell / spore weight:

Fungal cells /spores were filtered using Whatmen No.1 filter paper and heated at 70⁰C until it becomes dry and its weight was estimated by electronic weighing machine (ECELON MS- 2690)⁷.

Statistical analysis:

All data were expressed as Mean± SEM. Data were analysed by one way ANOVA followed by Dunett’s post hoc multiple comparison test considering p<0.05 as significant and p<0.01 as highly significant (using Prism 4.0).

Experiments were carried out in six sets using 250 ml Erlenmeyer conical flask ,with pH , 4.5 ; temperature , 30 ⁰C, spore density , 7x 10⁸ spores or cells /ml ; shaker speed, 200 rpm ; volume of medium, 100ml ; age of inoculum, 7 days and incubation period,7 days⁹.

Addition of carbon source in the medium:

Different carbon sources were added one by one (equivalent mole to glucose) by replacing glucose from the growth medium¹⁰.

Addition of nitrogen source in the medium:

Different nitrogen sources were added one by one (equivalent mole to glucose) by replacing urea from the growth medium¹⁰.

Addition of mineral elements in the medium:

Mineral elements were added to the medium directly in the form of their respective salts as mentioned in this paper as proposed by Ganguly and Banik^11,12.

Addition of vitamin B complex in the medium:

Different members of vitamin B complex were added to the medium (0.01-0.03µg/ml) one by one to examine their effect on growth of the fungus¹³.

Addition of amino acids in the medium:

20 amino acids were added to the medium (0.01-0.05mg/ml) one by one to examine their effect on growth of the fungus¹⁴.

Table:1-Selection of Suitable Carbon Source:

Carbon Sources (10%)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry cell/spore weight (gm/L)
⫵Glucose (Control)	1500	301.6±8.616	10.8±0.669
Fructose	1500	**733.4±7.313	**6.9±0.647
Galactose	1500	**631.7±5.642	**6.3±0.613
Lactose	1500	**837.6±4.834	**4.6±0.764
Xylose	1500	**1133.3±6.616	**2.8±0.661
Starch	1500	**1053.7±5.371	**3.2±0.876

(values were expressed as Mean ± SEM , where n=6 ; **p<0.01, ⫵ stands for maximum bio sorption)

Table:2-Optimization of Glucose Concentration

(%)
Glucose concentration (%)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry Cell / spore weight (gm/L)
2.0	1500	**1132.4±4.381	**2.8±0.564
4.0	1500	**722.2±7.413	**6.6±0.316
6.0	1500	**421.3±6.163	**8.7±0.663
8.0	1500	*357.4±3.333	11.0±0.513
10.0(Control)	1500	301.6±8.616	10.8±0.669
⫵12.0	1500	*221.4±6.381	**11.4±0.731
14.0	1500	*254.6±7.314	*11.1±0.661

(values were expressed as Mean ± SEM , where n=6 ; *p<0.05 and **p<0.01, ⫵ stands for maximum bio sorption)

Table:3 Selection of Suitable Nitrogen Source

Nitrogen Sources (0.6%N)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry cell/spore weight (gm/L)
Urea(Control)	1500	221.2±3.683	11.4±0.731
⫵Ammonium sulphate	1500	*200.1±7.313	*11.9±0.661
Di ammonium hydrogen phosphate	1500	224.6±6.166	11.4±0.661
Ammonium Chloride	1500	**304.6±6.183	*10.8±0.753
Ammonium Oxalate	1500	**273.2±4.661	11.1±0.431

(values were expressed as Mean ± SEM , where n=6 ; *p<0.05 and **p<0.01, ⫵ stands for maximum bio sorption)

Table: 4 Optimization of Ammonium Sulphate

% of N	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry Cell / spore weight (gm/L)
0.2	1500	**771.4±6.316	**6.6±0.732
0.4	1500	**454.2±4.631	**8.9±0.777
0.6(Control)	1500	200.1±7.316	11.9±0.661
⫵0.8	1500	*174.4±6.616	*12.4±0.731
1.0	1500	*223.1±4.336	11.6±0.613

(values were expressed as Mean ± SEM , where n=6 ; *p<0.05 and **p<0.01, ⫵ stands for maximum bio sorption)

Table : 5-Effect of K₂HPO₄

K₂HPO₄(gm%)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry Cell / spore weight (gm/L)
0.005	1500	190.4±3.613	12.1±0.636
⫵0.010(control)	1500	176.4±6.661	12.4±0.731
0.015	1500	*192.2±4.136	12.1±0.661

(values were expressed as Mean ± SEM , where n=6 ; *p<0.05, ⫵ stands for maximum bio sorption)

Table : 5 Effect of KH₂PO₄

KH₂PO₄(gm%)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry Cell / spore weight (gm/L)
0.005	1500	182.1±3.913	12.0±0.638
⫵0.010(control)	1500	176.4±6.441	12.4±0.636
0.015	1500	*197.2±6.863	12.3±0.863

(values were expressed as Mean ± SEM , where n=6 ; *p<0.05, ⫵ stands for maximum bio sorption)

Table:6 Effect of CaCl₂,2H₂O

CaCl₂,2H₂O (gm%)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry Cell / spore weight (gm/L)
0.00(control)	1500	176.4±6.613	12.4±0.731
0.01	1500	161.2±4.136	12.6±0.661
0.02	1500	*154.6±3.113	12.6±0.631
⫵0.03	1500	*131.2±7.613	12.8±0.436
0.04	1500	*142.6±6.131	12.8±0.613

(values were expressed as Mean ± SEM , where n=6 ; *p<0.05, ⫵ stands for maximum bio sorption)

Table:6 Effect of NaCl

NaCl (gm%)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry Cell / spore weight (gm/L)
0.00(control)	1500	131.2±4.136	12.8±0.436
0.01	1500	124.2±6.111	13.0±0.661
⫵0.02	1500	*111.1±5.363	*13.3±0.431
0.03	1500	*117.3±6.184	*13.3±0.613

(values were expressed as Mean ± SEM , where n=6 ; *p<0.05,⫵ stands for maximum bio sorption)

Table:7 Effect of MgSO₄.7H₂O

MgSO₄.7H₂O (µg/ml)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry Cell / spore weight (gm/L)
0.01	1500	*127.6±3.613	13.0±0.411
0.02	1500	*121.2±4.331	13.1±0.613
⫵0.03(control)	1500	111.1±5.363	13.3±0.431
0.04	1500	113.3±6.131	13.3±0.461

(values were expressed as Mean ± SEM , where n=6 ; *p<0.05, ⫵ stands for maximum bio sorption)

Table:8 Effect of MnSO₄.4H₂0

MnSO₄.4H₂0(µg/ml)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry Cell / spore weight (gm/L)
0.00(control)	1500	111.1	13.6
0.01	1500	*101.6	13.8
⫵0.02	1500	*96.2	*14.1
0.03	1500	*99.1	13.9

(values were expressed as Mean ± SEM ,*p,0.05 when compared to control, ⫵ stands for maximum bio sorption)

Table:9EFFECT OFFeSO₄.7H₂0

FeSO₄.7H₂0 (µg/ml)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry Cell / spore weight (gm/L)
0.00(control)	1500	96.2±4.331	14.1±0.736
0.01	1500	*96.0±6.316	14.2±0.661
⫵0.02	1500	*94.2±4.361	14.3±0.634
0.03	1500	96.6±4.316	14.3±0.611

(values were expressed as Mean ± SEM ,*p,0.05 when compared to control, ⫵ stands for maximum bio sorption)

Table:10 Effect of Thiamine-HCl

Thiamine-HCl (µg/ml)	Initial concentration of As (III) [mg/L]	Final concentration of As (III) [mg/L]	Dry Cell / spore weight (gm/L)
0.00(control)	1500	94.2±4.361	14.3±0.634
⫵0.01	1500	*89.3±6.136	15.1±0.613
0.02	1500	91.3±7.316	14.8±0.663
0.03	1500	93.6±5.613	14.7±0.436

(values were expressed as Mean ± SEM ,* p,0.05 when compared to control, ⫵ stands for maximum bio sorption)

Zain et al. (2009) used Zapek Dox agar medium (sucrose, 30 gm, sodium nitrate, 2 gm, KH₂PO_4,1 gm, KCl, 0.5gm, MgSO₄.7H₂O, 0.05gm, FeSO₄.7H₂0, 0.002gm) for the growth of Aspergillus terreus².Manjulata S. (2011) used Astha and Howken’s medium (glucose, 5gm; KNO₃, 3.5gm; KH₂PO_4,1.75gm; MgSO₄.7H₂O, 0.75gm and distilled water, 1L) for growth of A. umbrosus.

But in my study, from the above mentioned tables (table 1-10), it can be clearly stated that the fungus Aspergillus niger X₃₀₀grew best with the synthetic medium composed of glucose,12%;

(NH₄)SO₄, 0.8%; K₂HPO₄, 0.01%; KH₂PO₄, 0.01%; CaCl₂. 2H₂O, 0.03%; NaCl, 0.02% ; MgSO₄.7H₂O, 0.03 µg/ml; MnSO₄.4H₂0, 0.02 µg/ml; FeSO₄.7H₂0, 0.02 µg/ml and thiamine-HCl, 0.02µg/ml. But KNO₃, KCl, NH₄VO₃, other members of vitamin B-complex and L-amino acids exhibited non-significant effect whereas CdCl₂.5H₂O and CuSO₄.5H₂O showed adverse effect on cellular growth of the fungus and subsequently on the biosorption. Thus ,the above mentioned synthetic medium is now strongly recommended for further study related to the resistant strain.

ACKNOWLEDGEMENT:

Special gratitude to be given to the department of Chemical Engineering, University of Calcutta, Bose Institute, Kolkata, Indian Institute of Chemical Biology, Kolkata, Department of Food technology and Biochemical Engineering, Jadavpur University for their kind cooperation without which I could not finish the work.

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Received on 24.02.2013 Modified on 10.03.2013

Asian J. Research Chem. 6(4): April 2013; Page 301-304