<p>Title of the Article Optimization of medium using response surface methodology for maximizing biomass production of plant growth-promoting microbial inoculant Pseudomonas trivialis strain BIHB 745 (MTCC 5336) Name of the Authors 1. Pratibha Vyas1 2. Praveen Rahi1 3. B. S. Chadha2 2 Microbiology Department, Guru Nanak Dev University, Amritsar-143 005, Punjab, India. 4. Arvind Gulati1 Corresponding author Arvind Gulati (Email: [email protected]; [email protected]) Mailing Address 1 Plant Pathology and Microbiology Laboratory, CSIR-IHBT, P.O. Box No. 6, Palampur-176 061, H. P., India. Telephone +91-1894-230411 Fax +91-1894-230433 Journal Name Indian Journal of Microbiology</p><p>Online Resource 1 Range of variables at three different levels with dextrose as the carbon source Levels (g/l) -1 0 +1 Factor Casein enzyme hydrolysate 5.0 17.0 29.0 Soya peptone 0.2 3.0 5.8 NaCl 1.0 5.0 9.0 Dextrose 0.5 2.5 4.5</p><p>KH2PO4 - 2.5 -</p><p>Online Resource 2 Range of variables at three different levels with molasses as the carbon source Levels (g/l) -1 0 +1 Factor Casein enzyme hydrolysate 5.0 17.0 29.0 Soya peptone 0.2 3.0 5.8 NaCl 1.0 5.0 9.0 Molasses 1.0 5.0 9.0</p><p>KH2PO4 - 2.5 - Online Resource 3 l m / 10.5 U F</p><p>C 10</p><p>0 1</p><p> g 9.5 o</p><p>L NB 9 TSB 8.5 KB PMA 8 PM 2 TSB meth 7.5 GYEM 7</p><p>6.5</p><p>6 0 6 9 12 15 18 21 24 30 33 36 42 48 Time (h) Growth of Pseudomonas trivialis BIHB 745 in different media at 28±0.1 °C. NB = nutrient broth, TSB = trypticase soya broth, KB = King’s B medium, PMA = Pseudomonas medium A, PM2 = Pseudomonas medium 2, TSBmeth = TSB with 2 % methanol, GYEM = glucose yeast extract medium. Values are the mean of three replicates. Vertical bars represent standard error of the mean Online Resource 4</p><p>Growth of Pseudomonas trivialis BIHB 745 in different media at 28±0.1 ºC Medium Colony forming Specific growth units/ml (×109) rate Nutrient broth 3.7 0.20 Trypticase soya broth 7.6 0.58 Pseudomonas Medium A 1.3 0.18 Pseudomonas Medium 2 3.8 0.32 Trypticase soya broth with 2% methanol 2.6 0.48 Glucose yeast extract medium 2.3 0.16 King’s B broth 4.2 0.40 Online Resource 5 Optimization of Pseudomonas trivialis BIHB 745 biomass production in growth medium with dextrose as carbon source using Box-Behnken design of RSM Medium Composition (g/l) Biomass (g/l)</p><p>CEH SP NaCl Dextrose KH2PO4 Actual Predicted CFU/ml 17 0.2 1 2.5 2.5 2.02 1.94 6.2 × 109 5 3.0 1 2.5 2.5 1.19 1.23 1.9 × 109 17 5.8 5 0.5 2.5 2.22 2.22 7.7 × 109 17 3.0 1 0.5 2.5 1.63 1.67 3.3 × 109 17 3.0 9 0.5 2.5 1.76 1.82 5.2 × 109 5 3.0 9 2.5 2.5 1.22 1.24 2.2 × 109 5 0.2 5 2.5 2.5 0.87 0.94 8.2 × 108 17 0.2 5 4.5 2.5 2.29 2.25 8.0 × 109 17 5.8 9 2.5 2.5 2.27 2.32 7.7 × 109 17 3.0 1 4.5 2.5 2.33 2.33 8.2 × 109 29 3.0 5 4.5 2.5 2.55 2.63 8.9 × 109 29 3.0 5 0.5 2.5 2.24 2.21 8.9 × 109 17 5.8 1 2.5 2.5 2.29 2.35 8.1 × 109 17 3.0 5 2.5 2.5 2.14 2.21 7.4 × 109 17 0.2 5 0.5 2.5 1.42 1.44 3.9 × 109 29 3.0 1 2.5 2.5 2.59 2.53 9.1 × 109 29 0.2 5 2.5 2.5 2.42 2.5 7.9 × 109 17 3.0 5 2.5 2.5 2.20 2.21 7.5 × 109 29 3.0 9 2.5 2.5 2.49 2.42 8.8 × 109 5 5.8 5 2.5 2.5 1.74 1.70 4.9 × 109 5 3.0 5 4.5 2.5 1.43 1.43 4.1 × 109 17 3.0 5 2.5 2.5 2.22 2.21 7.6 × 109 17 3.0 5 2.5 2.5 2.25 2.21 7.7 × 109 17 3.0 5 2.5 2.5 2.22 2.21 7.6 × 109 17 5.8 5 4.5 2.5 2.39 2.33 8.4 × 109 29 5.8 5 2.5 2.5 2.63 2.61 9.9 × 109 17 3.0 9 4.5 2.5 2.07 2.08 6.8 × 109 5 3.0 5 0.5 2.5 1.02 0.92 9.8 × 108 17 0.2 9 2.5 2.5 1.94 1.86 5.9 × 109 CEH = casein enzyme hydrolysate, SP = soya peptone, CFU = colony forming units Online Resource 6</p><p>Results of regression analysis for maximizing biomass production of Pseudomonas trivialis BIHB 745 in growth medium with dextrose as the carbon source using Box- Behnken design of response surface methodology Term Regression co-efficient CEH 0.1347*** SP 0.2314*** NaCl 0.0862** Dextrose 0.4671*** CEH*CEH -0.0018*** SP*SP -0.0 000 NaCl*NaCl -0 .0054** Dextrose*Dextrose - 0.0359*** CEH*SP - 0.0049*** CEH*NaCl -0. 0007* CEH*Dextrose -0.00 10 SP*NaCl 0.00 13 SP*Dextrose -0. 0312*** NaCl*Dextrose -0.0 122** Coefficient of determination (R2) 0.989 *p ≤ 0.5; ** p ≤ 0.05, ***p ≤ 0.005, CEH = casein enzyme hydrolysate, SP = soya peptone Online Resource 7</p><p>Optimization of Pseudomonas trivialis BIHB 745 biomass production in growth medium with molasses as carbon source using Box-Behnken design of RSM Media Composition (g/l) Biomass (g/l)</p><p>CEH SP NaCl Molasses KH2PO4 Actual Predicted CFU/ml 17 0.2 1 5 2.5 1.84 1.79 5.6 × 109 5 3.0 1 5 2.5 1.05 0.97 9.9 × 108 17 5.8 5 1 2.5 2.00 1.90 6.4 × 109 17 3.0 1 1 2.5 1.24 1.33 1.5 × 109 17 3.0 9 1 2.5 1.30 1.36 2.5 × 109 5 3.0 9 5 2.5 0.95 0.92 9.4 × 108 5 0.2 5 5 2.5 0.65 0.71 6.0 × 108 17 0.2 5 9 2.5 1.95 1.99 6.1 × 109 17 5.8 9 5 2.5 2.20 2.22 7.2 × 109 17 3.0 1 9 2.5 2.04 2.00 6.5 × 109 29 3.0 5 9 2.5 2.22 2.27 7.7 × 109 29 3.0 5 1 2.5 2.00 1.97 8.1 × 109 17 5.8 1 5 2.5 2.10 2.06 7.0 × 109 17 3.0 5 5 2.5 2.05 2.06 6.6 × 109 17 0.2 5 1 2.5 1.00 1.05 9.4 × 108 29 3.0 1 5 2.5 2.25 2.29 7.9 × 109 29 0.2 5 5 2.5 2.30 2.27 8.3× 109 17 3.0 5 5 2.5 1.95 2.06 5.4 × 109 29 3.0 9 5 2.5 2.07 2.15 6.8 × 109 5 5.8 5 5 2.5 1.46 1.51 4.7 × 109 5 3.0 5 9 2.5 1.10 1.17 1.4 × 109 17 3.0 5 5 2.5 2.10 2.14 7.1 × 109 17 3.0 5 5 2.5 2.08 2.06 6.9 × 109 17 3.0 5 5 2.5 2.10 2.06 7.0 × 109 17 5.8 5 9 2.5 2.17 2.12 7.4 × 109 29 5.8 5 5 2.5 2.54 2.50 9.0 × 109 17 3.0 9 9 2.5 1.85 1.78 5.5 × 109 5 3.0 5 1 2.5 0.52 0.45 9.2 × 108 17 0.2 9 5 2.5 1.60 1.53 4.0 × 109 CEH = casein enzyme hydrolysate, SP = soya peptone, CFU = colony forming units</p><p>Online Resource 8 Results of regression analysis for maximizing biomass production of Pseudomonas trivialis BIHB 745 in medium with molasses as the carbon source using Box-Behnken design of response surface methodology Term Regression co-efficient CEH 0.153*** SP 0.203*** NaCl 0.086** Molasses 0.352*** CEH*CEH -0.002*** SP*SP 0.002 NaCl*NaCl -0.009*** Molasses*Molasses -0.018*** CEH*SP -0.004*** CEH*NaCl -0.000 CEH*Molasses -0.002** SP*NaCl 0.008* SP*Molasses -0.017*** NaCl*Molasses -0.004* Coefficient of determination (R2) 0.988 *p ≤ 0.5, **p ≤ 0.05, ***p ≤ 0.005, CEH = casein enzyme hydrolysate, SP = soya peptone Online Resource 9</p><p>Agitation (rpm) 200 6</p><p>) 250 l /</p><p> g 300 (</p><p> s 5 s a m o</p><p> i 4 B</p><p>3</p><p>2</p><p>1</p><p>0</p><p>Time (h) Effect of agitation on Pseudomonas trivialis BIHB 745 biomass production in optimized medium at 14 l working volume in bioreactor</p>