Effect of molasses and corn steep liquor on phosphate solubilization.

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1 Effect of molasses and corn steep liquor on phosphate solubilization. Molasses is most often used as a raw material in microbial industry. It is a dark, thick, syrup like viscous liquid. It is a byproduct in the production of raw and refined sugars from beet sugar or sugar cane. Among many molasses, blackstrap molasses prepared from sugar-cane and it is the most need sugar for industrial fermentation. The blackstrap molasses is recovered after the separation of crystallized sugar from its mother liquor. This mother liquor contains approximately 52% total sugar of which 30% is sucrose and 22% is invert sugar and is known as blackstrap molasses. Besides sucrose, fresh molasses contains also D-glucose at level of 0.1 or higher per cent. Molasses usually contains 20-25% reducing substances low amount of nitrogen (0.4. to 0.5%) and a low amount of ash (3.5 to 6.0%). White (1948) has pointed out that only about half of the phosphates present in molasses is assimilable by yeast. Molasses supplies carbon to the organism in the medium. Corn steep liquor (CSL) is the water extract byproduct obtained from steeping of corn during commercial production of cornstarch and other corn products. This spent steep water is concentrated to approximately 50% solids and this is known as corn steep liquor, which is used in fermentation industry. It contains considerable quantity of nitrogenous substances, lactic acid, 88

2 reducing substances and ash with many trace elements. Amino aids represent more than 83% of total nitrogen. Although many organisms utilize inorganic nitrogen exclusively, they invariably grow faster if supplemented with organic nitrogen. CSL supplies nitrogen to the organisms in the medium. Molasses has been evaluated for supporting phosphate solubilizing (PS) activity by microorganisms in the compost preparation (Singh et al. 1983). The present in vitro study was carried out to evaluate molasses and CSL for their effect on PS activity. Materials and Methods: Organisms: Geotrichum sp. and /ssatchenkia orienta/is maintained on Pikovskaya's agar slants were used. Culture Media: 1. Pikovskaya's broth 2.Modified Pikovskaya's broth: (i) Glucose in Pikovskaya's broth was replaced with molasses in concentration of 1, 2 and 3% (w/v) (ii) Ammonium sulphate in Pikovskaya's broth as replaced with CSL in concentration of 1, 2 and 3% (w/v) 2. Glucose yeast extract (GYE) broth : The medium was used to prepare yeast inoculum. 89

3 The compositions of Pikovskaya's broth and GYE are mentioned in chapter 2. All media were sterilized by autoclaving for 15 minutes at 15 Ibs p.s.i. (121 C). Molasses: It was procured from the 'Anil Starch', Ahemedabad, Gujarat. The sample contained 63% total sugar (Anthrone's method) and 0.2% P205. Corn-Steep Liquor: It was procured from 'Anil Starch', Ahemedabad, Gujarat. It contained 8.44% total nitrogen, while P205 was absent. Preparation of an inoculum: An inoculum of each culture was prepared as described in chapter 2. Inoculation and growth conditions: Two sets, each of seven fiasks containing 100ml modified Pikovskaya's broth were inoculated aseptically with 1 ml inoculum in each flask. All the inoculated fiasks were incubated at 28 ± 0.2 C for 21 d for phosphate solibilization under static condition and shaken at 12 h intervals. Respective uninoculated fiasks were incubated as control. The experiment was carried out in triplicate. Phosphorus estimation and ph measurement: At periodic intervals, 10 ml medium was withdrawn from each fiask aseptically and centrifuged at 10,000 rpm for 20 minutes. Supernatant was analysed for water soluble P content by chlorostannous reduced molybdophosphoric acid blue method. ( Jackson, 1973) The ph of the medium was determined by Elico ph meter. 90

4 Results and discussion Data in Tables 8.1 and 8.2 indicate that high variation was observed in TCP solubilization on different d at the same concentration and on the same d with different concentratio~,where molasses and corn steep liquor (CSL) were used as carbon and nitrogen source, respectively. In case of Geotrichum sp., maximum TCP solubilization was observed at 2% concentration of both molasses and CSL. For molasses, maximum TCP solubilization was 80.27mg % P 2 0s. The preferential trend for maximum TCP solubilization in presence of different concentrations of molasses was; 2% > 3% > 1% mg % P 2 05 M 1% and 2% concentrations of molasses, maximum TCP solubilization was attained on 21 st d, while for 3% concentration, it was on 15 th d. In presence of CSL, maximum TCP solubilization was mg% P 2 0 S. The preferential order for TCP solubilization with different concentrations of CSL was 2% > 3% > 1% 91

5 1% and 2% concentrations of CSL showed maximum TCP solubilization on 9 th day while at3% CSL, it was attained on 6 th d. In case of I. orientalis maximum TCP solubilization was observed at 2% and 3% concentrations of molasses and CSL, respectively. In presence of molasses, maximum TCP solubilization was mg% P20 S at 2% concentration followed by 3% concentration (67.10 mg% P20 S ) and 1 % concentration (57.99 mg% P20S). For 1% and 2% molasses, maximum TCP solubilization was attained on 21 st d, while for 3% concentration, it was on 1ih d. For CSL, maximum PS activity was 74.74mg% P20 S at 3% concentration followed td" 2% concentration (76.30mg% P20 S) and 1 % concentration (53.18% P20 S ). rd7t:.1 % CSL, maximum TCP solubilization was observed on 9 th d while for 2% and 3% concentrations, it was on 21 st and 18 th d, respectively. PS activity was directly proportional to concentration of CSL. In case of molasses, for both cultures, the ph of the media was tumed more acidic with days of incubation and drop of ph of spent medium was noticed with increase in concentration of molasses from 1 to 3%. Such type of observation also noted by Narsian (1995) for Aspergillus. aculeatus. While in case of CSL, ph of the media moved towards alkaline side for both organisms. The maximum ph of the media was 9.1 for Geotrichum sp. at 3% concentration and 8.9 for I. orientalis at all the three concentrations on 18 th d. A rise in the ph above neutral during phosphate solubilization by microorganisms has been observed by Chhonkar and Subba rao (1967), 92

6 ",..,ol p,,~\ Goswami and Sen (1962), Broomfield (1959) and Narsiarvo(1995). This may be due to phosphate solubilization accompanied by by proton extrusion (NH: 'r; Y'I'f'\eh- I H+ ) mechanism ( Roose and Luckner, 1984; Illmer ",.,~ Sc., '1995) and rise in ph may be ascribed to ammonia production. Considering all above results, we infer that 2% concentration of molasses supported maximum PS activlfy 6It both cultures, Geotrichum sp. and I. orientalis. While 2% and 3% concentrations of CSL enhanced maximum PS activity of Geotrichum sp. and I. orientalis, respectively. Molasses was found inferior to CSL in supporting PS activity Il~ Geotrichum sp. This observation is in corroboration with results noted by Samaha (1998) for Oebaryomyces hansenii and Aspergillus niger and by Dave (1999) for Pseudomonas f1uorescens. Molasses was found superior to CSL for supporting maximum PS activity by I orientalis. This observation is in affirmation with result of A. aculeatus by Narsian (1995). Comparing the readily utilizable carbon and nitrogen sources (glucose and ammonium sulphate, respectively) in the Pikovskaya's medium with molasses and CSL, it was observed that these two industrial raw materials gave comparatively good PS activity, 65.2% for molasses and 69.3% for CSL by Geotrichum sp. while almost 55% for both molasses and CSL, by I. orientalis, close to glucose and ammonium sulphate. It is evident that these two substances can replace glucose and ammonium sulphate, in culture media for the growth of phosphate solubilizing microorganisms. Also if these two 93

7 substances are added to agricultural soil, they can enhance the PS activity of the microorganisms. The solubilization of low grade RP by composing with paddy straw as farm waste was studied by Singh et al. (1983). They found that addition of molasses enhanced the rate of RP solubilization and the highest value (58.2%) was recorded after 120 d of composting.jn earlier study (chap.2), we found that Geotrichum sp and I. orientalis were able to liberate soluble P from ~- RPs. So, molasses and CSL, along with these organisms can be used for production of RP enriched compost. 94

8 Table 8.1 : Effect of molasses and corn steep Liquor(CSL) on TCP solubilization by Geotrichum sp. Molasses and CSL (gm%) Days of Incubation P2O" ph P 2 O. ph P 2 O. ph P 2 O. ph P 2 O. ph P 2 O. ph P 2 O. 21 PH Molasses CSL P solubilized as net mg % P 2 0. after deducting respective control.

9 Table 8.2 : Effect of molasses and corn steep Liquor(CSL) on TCP solubilization by I. orientalis. Molasses and CSL (gm%) Days of Incubation P 2 O S * ph P 2 0 S ph P 2 0 S ph P 2 0 S ph P 2 0 S ph P 2 0 S ph P 2 0 S 21 PH Molasses 1 36_35 5_5 40_04 5_7 31_70 5_ _8 32_82 5_9 34_59 6_0 2 46_ _67 5_3 27_08 5_3 29_78 5_6 49_34 5_ ,8 3 64,37 5_1 52_51 5_3 36,32 4_1 67_10 5,3 55_55 5,6 50,27 5,8 57_99 4_8 76_91 4_ ,7 CSL 1 42_37 6_8 35_87 7_6 53_ _2 34_11 8,3 35,39 8, _ _3 58,99 6_3 45,68 8,1 43,92 7_8 58_02 8,9 3 54_11 6_8 55, ,7 37: _6 76_ _08 8_0 76_30 7_6 52, P solubilized as net mg % P20S after deducting respective control.