KMITL Sci. J. Vol.8 No.

2 (Section A) July December, 2008

SCREENING FOR PHOSPHATE SOLUBILIZING BACTERIA AND OPTIMUM OF BACTERIAL CULTIVATION BY RESPONSE SURFACE METHODOLOGY
Chalit Nopparat , Marisa Jatupornpipat and Aree Rittiboon Faculty of Science, King Munguts Institute of Technology Ladkrabang, Bangkok 10500, Thailand

ABSTRACT
Phosphorus (P) is one of essential macronutrients for plant growth and reproduction. Plants acquire P from soil solution as phosphate anions. However, phosphate anions are extremely reactive and may be immobilized through precipitation with cations such as Ca2+, Mg2+, Fe3+ and Al3+, depending on the particular properties of a soil. In these forms, phosphate is highly insoluble and unavailable to plants. Application of phosphate solubilizing bacteria (PSB) has been fertilized to increase P uptake and plant growth. So, the aim of this study is to screen for PSB from 24 soil samples and determine the optimal factors in cultivation i.e. pH, temperature and shaking rate. Fifty one bacterial isolates have been screen from 19 Kanchanaburi soils, 2 Chaiyaphum soils, 2 Trang soils and 1 Krabi soil to incubate on Pikovskaya agar supplemented with 0.003% w/v rose bengal for 7 days, and the dimension of clear zone and colony were measured. Twenty two stains of screening appeared to have the highly determinable ratio of clear zone to divide colony. When the ratios of appropriated day have arranged by frequency to make normal distribution at 95% of confidence. A bacterial strain of SD02P3218 has solubilized tricalcium phosphate and given the highest available phosphate 0.82 mg P2O5 ml-1 in liquid medium by CRD and DMRT at 5% of probability from the total of twenty two bacterial strains. This PSB SD02P3218 was used to cultivate in nutrient broth for study of optimal conditions by response surface methodology followed by the central composite design. The optimum conditions of the pH, the temperature and the shaking rate were 8.84, 34.7 C and 174 rpm, respectively. These conditions were used in the bacterial cultivation and a growth curve was determined. It was shown that the bacteria grew less with an average practical OD of 1.792 than with the estimated OD of 1.840 at the 24th hr incubation. KEYWORDS: phosphate solubilizing bacteria, response surface methodology

1. INTRODUCTION
Thailand is known to be one of the developing countries that is also worlds largest net food exporter. The use of biofertilizer can lessen dependence on chemical fertilizer, resulting in cost saving as well as reducing deleterious environmental and human effects [1]. Biofertilizer has been identified as an alternative to chemical fertilizer to increase soil fertility and crop production in sustainable farming [2]. P is one of essential macronutrients for plant growth and reproduction [3]. Plants acquire P from soil solution as phosphate anions. However, phosphate anions are extremely reactive and may be immobilized through precipitation with cations such as Ca2+, Mg2+, Fe3+ and Al3+, depending on the particular properties of a soil. In these forms, phosphate is highly insoluble and unavailable to plants. As the results, the amount available to plant usually is decreased to have hardly a small proportion in the nature [4]. Application of phosphate solubilizing microorganisms (PSMs) has been fertilized to increase P uptake and plant growth [5]. The principle mechanism for mineral phosphate solubilization is the production of organic acids. It is generally accepted that the major mechanism of mineral phosphate solubilization is the action of organic acids synthesized by soil microorganism. Production of organic acids result in acidification of the microbial cell

Corresponding Author. Tel. 66-86672-4827 E-mail. Inoculum18@hotmail.com

KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 and its surroundings [4]. A variety of PSB like is Bacillus, Rhodococcus, Arthrobacter, Serratia, Chryseobacterium, Delftia, Gordonia, Phyllobacterium, etc. [6]. Response surface methodology (RSM) is a combination of statistical and mathematical techniques useful for developing, improving and optimizing processes. Optimization of the response is to determine the conditions that optimize the process. A secondorder model could be used to estimate the response in a narrow region and from the examination of the estimating response surface, the optimum levels or condition for process variables could be chosen [7]. When one has located the region of the optimum response, curvature can be produced. Three factors (k) and three levels factorial experiment are often conducted in order to fit such response surfaces. The total number of test runs (n) in a central composite design (CCD) based on a complete 2k factorial which is n = 2k + 2k + m, where m is the number of replications at a center point of design. This number of treatments is usually less than 3k, so that fewer observations are required than in a 3k factorial. The CCD can be made to be rotated by choosing a = F1/4, where F is the number of factorial points (e.g., F = 2k when a complete factorial is used) [8].

2. MATERIALS AND METHODS

2.1 Soil sample collection and bacterial screening
Bacterial strains have been screen from 19 Kanchanaburi soils, 2 Chaiyaphum soils, 2 Trang soils and 1 Krabi soil of Thailand. Soil samples (~ 2 kg) were collected from the areas where any plants has flourished [9] by digging 0-15 cm in deep and store at 4 C in refrigerator [10]. Each of the samples added to 9 ml of 0.85% w/v physiological saline. Ten fold serial dilution were made to drop 0.1 ml of 104-106 on plates containing the Pikovskaya agar (0.5 g (NH4)2SO4, 0.5 g MgSO4.7H2O, 0.3 g NaCl, 0.3 g KCl, 0.03 g FeSO4.7H2O, 0.02 g MnSO4.H2O, 10.0 g Ca3(PO4)2, 10.0 g Glucose and 15.0 g Agar). The plates were spread and incubated at 37 C for 3-5 days. The different colonies were picked and grown on Pikovskaya agar again. The interest strains were restreaked to purify on same medium [9].

2.2 Bacterial selection on solid cultivation

The fifty one PSB were purified to spot on Pikovskaya agar supplemented with 0.003% w/v rose bengal. The dimention of clear zone and colony were measured after incubation at 37 C for 3, 5 and 7 days [11]. The ratios of clear zone were calculated to divide the dimension of clear zone by the dimention of colony. The fifty one of the ratios were arranged by frequency to make normal distribution. A ratio of cut off was calculated from z score in Equation 1 at 95% confidence. (1) = n

2.3 Tricalcium phosphate solubilization and final pH in liquid cultivation

Twenty two strains have shown 5% of high ratio of clear zone to determine tricalcium phosphate solubilization. The starter was prepared in 25 ml nutrient broth and diluted to 0.500 absorbent of the optical density (OD) at 600 nm. One ml of the starter added to 50 ml Pikrovskaya medium in 250 ml Erlenmeyer flask. The triplicate repetition incubated at 37 C with 200 rpm for 3, 5 and 7 days. Autoclaved and uninoculated medium were served as controls. The cultures were harvested by filtration with Whatman paper filter no. 42. The supernatants were valuated available phosphate (P2O5) with the molybdovanadophosphate method [12] and final pH were determined. The valuation of available phosphates were calculated the highest by complete randomize design (CRD) and Duncans multiple range test (DMRT) at 95% confidence.

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KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008

2.4 Optimization of phosphate solubilizing bacterial cultivation by response surface methodology

PSB SD02P3218 has given the highest available phosphate when cultivated in submerged medium supplemented with tricalcium phosphate. The starter was prepared in 50 ml nutrient broth and diluted to 0.500 absorbent of the OD at 600 nm. One ml of the starter added to 50 ml nutrient broth in a 250 ml Erlenmeyer flask for optimization. The OD at 600 nm was measured when the cultivation was inoculated for 24 hrs. Three factors and three levels of CCD were used to determine the optimal factors of bacterial cultivation with triplicate repetitions. The CCD with a quadratic model was employed [7, 8]. Three independent variables namely pH (x1), temperature (x2) and shaking rate (x3) were chosen. Each independent variable had three levels which were -1, 0 and +1. A total of 15 different combinations were chosen in random order according to a CCD configuration for the three factors. The coded values of independent variables were found from the Equation 2-4 to show in Table 1.

code factor 1 = code factor 2 = code factor 3 =

x1 7.00 3 x 2 37.0 8 x3 50 5
Coded levels

(2)

(3)

(4)

Table 1 Original and coded levels of the independent variables. Independent variables pH temperature shaking rate -1.682 1.95 23.5 116 -1 4.0 29.0 150 0 7.0 37.0 200 +1 10.0 45.0 250 +1.682 12.05 50.4 284

The study was carried out according to the CCD and the experimental treatments used according to the design were shown in Table 2. A second-order polynomial equation was used to express the average OD as a function of independent variables,

y = b0 + b1 x1 + b2 x 2 + b3 x3 + b12 x1 x 2 + b13 x1 x3 + b23 x 2 x3 + b11 x1 + b22 x 2 + b33 x3

where y represents the average OD of the bacterial growth. The coefficients of the response surface equation were determined by using a linear regression on SPSS for WINDOW version 11.5.

(5)

2.5 Quality controls

Potassium dihydrogen phosphate (KH2PO4) was standardization to spike in the supernatants about 0.400 mg ml-1. So, the supernatants were divided into 3 lots included lot 1 (SA05P3258, SA05P3472, SA14P2111, SA24P1304, SA24P2431, SA24P2434, SB03P2205, SD02P2205, SD02P2206, SD02P3216, SD02P3218, SD02P3219, SD02P3220 and SD02P3223); lot 2 (SA24P14102, SA24P2328 and SD02P3324) and lot 3 (SA05P1202, SA05P2226, SA24P1415, SB01P3341 and SD02P1201). Percentage of recovery (%Rec) was calculated recoverable available phosphate from KH2PO4 was spiked. And relative percentage difference (%RPD) was calculated differential replication.

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KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 Table 2 The experimental treatments were shown according to the CCD with three independent variables (code and original levels). Original factor levels 2:Temp.** 3:Shaking*** 1:pH (rpm) (C) Code factor levels 1:pH 2:Temp. 3:Shaking -1 +1 -1 +1 -1 +1 -1 +1 0 0 0 0 -1.682 +1.682 0 0 0 0 0 0

Experimental number*

1 4.00 29.0 150 -1 -1 2 4.00 29.0 250 -1 -1 3 4.00 45.0 150 -1 +1 4 4.00 45.0 250 -1 +1 5 10.00 29.0 150 +1 -1 6 10.00 29.0 250 +1 -1 7 10.00 45.0 150 +1 +1 8 10.00 45.0 250 +1 +1 9 1.95 37.0 200 -1.682 0 10 12.05 37.0 200 +1.682 0 11 7.00 23.5 200 0 -1.682 12 7.00 50.4 200 0 +1.682 13 7.00 37.0 116 0 0 14 7.00 37.0 284 0 0 15 7.00 37.0 200 0 0 16 7.00 37.0 200 0 0 17 7.00 37.0 200 0 0 18 7.00 37.0 200 0 0 19 7.00 37.0 200 0 0 20 7.00 37.0 200 0 0 Remarks: *The experiments in Table 2 were performed in random order. **Temp. = temperature ***Shaking = shaking rate

2.6 Statically analysis

Three replications of data were subjected to analyze of variance (ANOVA) with CRD using SPSS for windows version 11.5 software. DMRT calculated at the 0.05 level of probability to determine difference among the mean. The surface and contour graphs plotted with three dimention using STATISTICA version 7.0 software.

3. RESULTS AND DISCUSSION

The fifty one PSB have been screen from 19 Kanchanaburi soils (SA), 2 Chaiyaphum soils (SB), 2 Trang soils (SD) and 1 Krabi soil (SE) that shown in Table 3. The cultures have been incubation for 3, 5 and 7 days to identify the appropriated day by CRD and DMRT at the 0.05 level of probability to determine difference among the mean. Almost of the cultures have given the highest valuation on the appropriate 7 days for 100.00% of the ratio of clear zone and 81.82% of the available phosphate. Also the appropriated day used to calculate a cut off of selective data and the highest valuation by CRD and DMRT. The twenty two stains of screening appeared the highly determinable ratios of clear zone to show in Table 4 when the ratios of the appropriated day have arranged by frequency to make normal distribution in Figure 1. A ratio of cut off was 2.64 from z score at 95% confidence. The twenty two strains have cultivated in liquid medium supplemented with tricalcium phosphate. The bacteria SD02P3218 gave the highest available phosphate 0.82 mg P2O5 ml-1. KH2PO4 used to spike in the supernatants about 0.400 mg ml-1 that showed %recover 95.34-109.45% and %RPD 1.01-7.13% in Table 5. The supernatant have valuated pH at 25 C to reduce initial pH of medium 6.24 (control) to final pH 4.57-4.64 for PSB SD02P3218 in Table 6.

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KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 Table 3 The number of bacteria have been screen from the 24 soils of Thailand which solubilized tricalcium phosphate on Pikovskaya agar. No. Code of soil samples The number of bacteria 1 SA02 1 2 SA03 -* 3 SA05 5 4 SA07 1 5 SA08 -* 6 SA09 5 7 SA11 -* 8 SA12 -* 9 SA13 -* 10 SA14 1 11 SA16 -* 12 SA17 5 13 SA18 -* 14 SA19 -* 15 SA20 -* 16 SA21 -* 17 SA22 -* 18 SA23 -* 19 SA24 19 20 SB01 2 21 SB03 1 22 SD01 -* 23 SD02 11 24 SE01 -* Total 51 Remark: * The soil samples dont find bacteria to solubilize tricalcium phosphate.

Figure 1 The ratios of clear zone were arranged by frequency to make normal distribution.

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KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 Table 4 The ratios of clear zone of fifty one strains have incubated on Pikrovskaya agar supplemented with 0.003% w/v rose bengal at 37 C for 3, 5 and 7 days. The ratios of clear zone The ratios of clear zone Bacterial Bacterial No. No. code code 3 days 5 days 7 days 3 days 5 days 7 days 1 SA02P2106 1.79a 1.87a 1.95a 29 SA24P2429 1.27a 1.49a 1.69a a a a b ab 2 SA05P1202 2.33 2.74 2.98 30 SA24P2431 2.60 2.73 3.13a b ab a a a 3 SA05P2226 1.49 2.24 3.00 31 SA24P2432 1.20 1.27 1.33a a a a a a 4 SA05P2327 1.33 1.56 1.78 32 SA24P2434 2.98 3.30 3.59a a a a a a 5 SA05P3258 2.80 2.80 2.80 33 SA24P2536 1.62 1.79 1.92a 6 SA05P3472 2.47b 3.11ab 3.53a 34 SA24P2537 1.00b 1.80a 1.93a a a a b ab 7 SA07P3130 2.08 2.33 2.33 35 SA24P3539 1.83 1.89 2.17a a a a a a 8 SA09P1308 1.50 1.69 1.73 36 SA24P3540 1.50 1.75 1.83a a a a a a 9 SA09P1513 1.27 1.29 1.33 37 SA24P3542 2.11 2.15 2.13a a a a b ab 10 SA09P2216 1.74 1.56 1.56 38 SB01P2430 1.07 1.53 1.60a a a a b ab 11 SA09P2428 1.76 1.74 1.77 39 SB01P3341 2.17 2.80 3.33a a a a c b 12 SA09P3334 1.97 2.01 2.15 40 SB03P2205 5.00 6.33 8.15a b ab a c b 13 SA14P2111 2.55 3.10 3.61 41 SD02P1201 1.33 2.17 3.22a a a a b ab 14 SA17P2425 2.13 2.12 2.29 42 SD02P2205 3.17 3.42 3.83a 15 SA17P2432 1.23a 1.29a 1.38a 43 SD02P2206 3.66a 3.42a 3.75a a a a a a 16 SA17P2433 1.67 1.92 1.98 44 SD02P2207 1.58 1.75 2.08a a a a a a 17 SA17P3348 1.29 1.37 1.50 45 SD02P2310 1.62 1.68 1.68a a a a a a 18 SA17P3351 1.83 2.05 2.34 46 SD02P3216 2.63 3.03 3.02a a a a b ab 19 SA24P1201 1.25 1.44 1.42 47 SD02P3218 2.53 2.97 4.03a a a a a a 20 SA24P1303 1.86 1.92 2.02 48 SD02P3219 2.80 2.85 3.44a b a a a a 21 SA24P1304 2.36 3.58 4.25 49 SD02P3220 2.78 3.18 3.42a c b a c b 22 SA24P1305 1.00 1.87 2.07 50 SD02P3323 3.00 3.50 3.83a a a a b a 23 SA24P1408 2.31 2.58 2.58 51 SD02P3324 1.91 2.37 2.68a b a a 24 SA24P14102 1.60 2.60 2.80 Summary 25 SA24P1413 1.96a 2.20a 2.45a a 64.70% 74.51% 100.00% 26 SA24P1415 2.08b 2.90a 3.40a ab 0.00% 17.65% 0.00% 27 SA24P1416 2.07a 2.14a 2.28a b 27.45% 7.84% 0.00% 28 SA24P2328 2.32a 2.67a 3.03a c 7.84% 0.00% 0.00% Remark: Data have gotten the mean of triplicate experiments by CRD. The superscript alphabets have calculated by DMRT at the 0.05 level of probability.

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KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 Table 5 The twenty two strains have given the highly determinable available phosphate to incubate in Pikrovskaya medium supplemented with 1.0% w/v tricalcium phosphate at 37 C for 3, 5 and 7 days. Available phosphate (mg P2O5 ml-1) Bacterial No. code 3 days %Rec %RPD 5 days %Rec %RPD 7 days %Rec %RPD 103.9 101.3 101.3 1 SA05P1202 0.03 1.93 0.02 1.01 0.11k 1.65 6 5 0 103.9 101.3 101.3 2 SA05P2226 0.03 1.93 0.02 1.01 0.03l 1.65 6 5 0 106.7 3 SA05P3258 0.63 4.12 0.40 96.13 4.76 0.38g 95.34 7.13 6 106.7 4 SA05P3472 0.04 4.12 0.04 96.13 4.76 0.04l 95.34 7.13 6 106.7 5 SA14P2111 0.04 4.12 0.05 96.13 4.76 0.04l 95.34 7.13 6 106.7 6 SA24P1304 0.50 4.12 0.22 96.13 4.76 0.50d 95.34 7.13 6 109.4 101.8 7 SA24P14102 0.42 1.94 0.40 96.61 5.02 0.47de 2.66 5 2 103.9 101.3 101.3 8 SA24P1415 0.09 1.93 0.11 1.01 0.22j 1.65 6 5 0 109.4 101.8 9 SA24P2328 0.27 1.94 0.23 96.61 5.02 0.33h 2.66 5 2 106.7 10 SA24P2431 0.48 4.12 0.22 96.13 4.76 0.48de 95.34 7.13 6 106.7 11 SA24P2434 0.46 4.12 0.22 96.13 4.76 0.46e 95.34 7.13 6 103.9 101.3 101.3 12 SB01P3341 0.03 1.93 0.02 1.01 0.03l 1.65 6 5 0 106.7 13 SB03P2205 0.03 4.12 0.04 96.13 4.76 0.03l 95.34 7.13 6 103.9 101.3 101.3 14 SD02P1201 0.01 1.93 0.00 1.01 0.01l 1.65 6 5 0 106.7 15 SD02P2205 0.40 4.12 0.42 96.13 4.76 0.40g 95.34 7.13 6 106.7 16 SD02P2206 0.71 4.12 0.52 96.13 4.76 0.71b 95.34 7.13 6 106.7 17 SD02P3216 0.42 4.12 0.44 96.13 4.76 0.42fg 95.34 7.13 6 106.7 18 SD02P3218 0.82 4.12 0.45 96.13 4.76 0.82a 95.34 7.13 6 106.7 19 SD02P3219 0.68 4.12 0.38 96.13 4.76 0.68c 95.34 7.13 6 106.7 20 SD02P3220 0.63 4.12 0.68 96.13 4.76 0.44ef 95.34 7.13 6 106.7 21 SD02P3323 0.26 4.12 0.28 96.13 4.76 0.26i 95.34 7.13 6 109.4 101.8 22 SD02P3324 0.52 1.94 0.38 96.61 5.02 0.02l 2.66 5 2 Summary 3 days 5 days 7 days a 77.27% 36.36% 81.82% ab 4.54% 0.00% 18.18% b 13.64% 59.09% 0.00% c 4.54% 4.54% 0.00% Remark: Data have gotten the mean of triplicate experiments by CRD. The superscript alphabets have calculated by DMRT at the 0.05 level of probability. After the total 20 experimental treatments have inoculated with the PSB SD02P3218 in nutrient broth for 24 hrs, the OD of growth at 600 nm was measured and shown in Table 7. The average OD and original levels

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KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 were plotted in a response surface and contour graphs by STATISTICA version 7.0 software of Statsoft Inc. Both of the pH and the temperature were determined by Equation 6 which showed a maximum point to consist the pH of 8.84, the temperature of 34.7 C and the average OD of 1.765 in Figure 2. Figure 3 showed the broad peak of 8.84 for the pH, 163 rpm for the shaking rate and 1.498 for the average OD. In the cases of the temperature and the shaking rate were determined by Equation 8 also had broad peaks. Figure 4 showed 34.7 C of the temperature, 174 rpm of the shaking rate and 1.326 of the average OD.

Table 6 The twenty two strains have been the final pH to incubate in Pikrovskaya medium supplemented with 1.0% w/v tricalcium phosphate at 37 C for 3, 5 and 7 days. Final pH Bacterial Initial No. code pH 3 days 5 days 7 days 1 SA05P1202 7.02a 6.35b a 2 SA05P2226 7.02 6.33b a 3 SA05P3258 6.24 5.90b b 4 SA05P3472 6.24 6.75a c 5 SA14P2111 6.24 6.73ab a 6 SA24P1304 6.24 5.95b a 7 SA24P14102 6.24 5.98b a 8 SA24P1415 7.02 5.65b 9 SA24P2328 6.24a 5.87b 10 SA24P2431 6.24a 5.84b a 11 SA24P2434 6.24 5.83bc a 12 SB01P3341 7.02 6.30b b 13 SB03P2205 6.24 6.74a a 14 SD02P1201 6.98 6.41ab a 15 SD02P2205 6.24 5.76c a 16 SD02P2206 6.24 4.46b a 17 SD02P3216 6.24 5.78c 18 SD02P3218 6.24a 4.58b 19 SD02P3219 6.24a 5.57c a 20 SD02P3220 6.24 5.60d a 21 SD02P3323 6.24 6.08b a 22 SD02P3324 6.24 4.74b Remark: Data have gotten the mean of triplicate experiments calculated by DMRT at the 0.05 level of probability. 6.29b 6.19c 5.84b 6.81a 6.89a 5.91b 5.77c 4.88c 5.90b 5.87b 5.81c 6.14c 6.65a 5.90b 5.88b 4.36b 5.88ab 4.57b 5.67bc 5.72c 6.10b 5.81a by CRD. 5.37c 6.20c 5.92b 6.71a 6.62b 5.96b 5.90b 4.77d 5.92b 5.96b 5.95b 6.01d 6.63a 6.23b 5.96b 4.50b 5.92b 4.64b 5.73b 5.86b 6.05b 6.06a The superscript alphabets have (6)

Average OD = -11.0586+1.1335x1+0.4652x2-0.0513x12-0.0071x1x2-0.0059x22

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KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008

Figure 2 The surface and contour graphs plotted between pH and temperature.

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KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 Average OD = -3.0130+0.8773x1-0.0085x3-0.0479x12-0.0003x1x3-(1.8433 10-5)x32 (7)

Figure 3 The surface and contour graphs plotted between pH and shaking rate. Average OD = -5.6539+0.3732x2-0.0056x3-0.0053x22-(1.1458 10-5)x2x3-(1.5049 10-5)x32 (8)

Figure 4 The surface and contour graphs plotted between temperature and shaking rate.

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KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 Table 7 The OD of growth at 600 nm measured when cultivation has inoculated for 24 hrs. OD at 600 nm Experimental Number Replication 1 Replication 2 Replication 3 Average SD 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0.022 0.013 0.008 0.014 1.471 1.484 0.817 0.658 0.022 1.326 1.172 0.468 1.721 1.665 1.806 1.581 1.557 1.630 1.793 1.584 0.029 0.012 0.006 0.015 1.476 1.507 0.863 0.648 0.020 1.271 1.212 0.468 1.765 1.526 1.753 1.658 1.592 1.641 1.834 1.703 0.022 0.012 0.01 0.013 1.601 1.144 0.878 0.675 0.021 0.860 1.146 0.462 1.747 1.658 1.820 1.619 1.534 1.712 1.511 1.543 0.024 0.012 0.008 0.014 1.516 1.378 0.853 0.660 0.021 1.152 1.177 0.466 1.744 1.616 1.793 1.619 1.561 1.661 1.713 1.610 0.0040 0.0006 0.0020 0.0010 0.0737 0.2033 0.0318 0.0137 0.0010 0.2547 0.0332 0.0035 0.0221 0.0783 0.0353 0.0385 0.0292 0.0445 0.1758 0.0831

The second-order polynomial equation was used to express the average OD as a function of independent variables given in Equation 9. This equation was analyzed with a regression at 5% probability by SPSS for WINDOW version 11.5. The ANOVA and coefficient of this equation was also determined with regression analysis to show in Table 8 and 9, respectively. (8)
2 2 2

y = 1.6734 + 0.4577 x1 0.1898 x 2 0.0403 x3 0.1708 x1 x 2 0.0405 x1 x3 0.0046 x 2 x3 0.4706 x1 0.3876 x 2 0.0840 x3
Table 8

ANOVA of bacterial OD of growth at 600 nm analyzed with a regression at 5% probability. Model Sum of squares Degree of freedom Mean square 2.835 0.053 F 53.070 Significant 0.000

Regression 25.519 9 Residual 2.671 50 28.190 Total 59 Remarks: R2 = 0.905 and adjusted R2 = 0.888

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KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 Table 9 Coefficient of the second-order polynomial equation was determined with regression analysis at 5% probability. Factors Constant x1 x2 x3 x1x2 x1x3 x2x3 x12 x22 x32 Coefficients b0 b1 b2 b3 b12 b13 b23 b11 b22 b33 Un standardized coefficients B 1.673 0.458 -0.190 -0.040 -0.171 -0.041 -0.005 -0.471 -0.388 -0.084 Standard error 0.054 0.036 0.036 0.036 0.047 0.047 0.047 0.035 0.035 0.035 t 30.745 12.675 -5.255 -1.117 -3.620 -0.859 -0.098 -13.388 -11.028 -2.390 Significant 0.000 0.000 0.000 0.269 0.001 0.394 0.922 0.000 0.000 0.021

The pH of 8.84, the temperature of 34.7 C and the shaking rates of 174 and 163 rpm were to be specified from Figure 2, 3 and 4. Therefore, the highest average OD 1.840 was chosen for the optimal conditions which showed 8.84 for pH, 34.7 C for temperature and 174 rpm for the shaking rate in Table 10. These conditions were used to cultivate for the bacterial growth in nutrient broth. In Figure 5, PSB SD02P3218 showed 1.792 for the average OD at the 24th hr. Therefore, the practical average OD was less than that of the estimation for 97.39%.

13

2.0

11 10

12 1.5 11

9 8

10

1.0

7 6

9 0.5 8

5 4

0.0 0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96

Time (hr.)

Figure 5

The growth curve, final pH and available cell of the PSB SD02P3218 cultivated in nutrient Time (hr.) vs Average pH Time (hr.) vs temperature of Cell broth with the initial pH of 8.48, the Avaliable average 34.7 C and the shaking rate of 174 rpm. (() average OD of the bacterial growth; () final average pH of the nutrient broth and () decadic logarithm of available average cell)

Time (hr.) vs Average OD

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LOG Avaliable average cell (CFU/ml NB)

Average OD

Average pH

KMITL Sci. J. Vol.8 No.2 (Section A) July December, 2008 Table 10 Average estimated OD was calculated by the second-order polynomial equation. Original factor levels Code factor levels average 1:pH 0.613 0.613 2:Temp. -0.288 -0.288 3:Shaking -0.740 -0.520 estimated OD 1.831 1.840

2:Temp.* 3:Shaking** (rpm) (C) 8.84 34.7 163 8.84 34.7 174 Remarks: *Temp. = temperature **Shaking = shaking rate 1:pH

From the results of this present study, PSB SD02P3218 was able to grow in nutrient broth and it effectively produced the highest average OD 1.797 at the 25th hr. Then, statistically based method using CCD was applied to develop a second-order regression model. The high adequacy of the model was proven by fitting the experimental and predicted values. The response surface methodology is a better choice to optimize biologically process than that of a one-condition-at-a-time because response surface methodology has factor interaction analysis and has less number of treatments than full factorial design [8, 13].

4. CONCLUSIONS
In conclusion, the bacterial strain SD02P3218 has solubilized tricalcium phosphate and showed the highest available phosphate 0.82 mg P2O5 ml-1 in liquid medium. The optimum conditions of the pH, the temperature and the shaking rate were 8.84, 34.7 C and 174 rpm, respectively. These conditions used to cultivate in nutrient broth and a growth curve was determined. It was grown less with an average practical OD of 1.792 than with the estimated OD of 1.840 at the 24th hr incubation.

5. ACKNOWLEDGEMENTS
The authors are grateful to the School of Graduated Studies and Faculty of Science, King Mongkuts Institute of Technology Ladkrabang, Thailand for supporting this work.

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