Table 1 Range of Variables at Three Different Levels with Dextrose As the Carbon Source

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

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

KH2PO4 - 2.5 -

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

KH2PO4 - 2.5 - Online Resource 3 l m / 10.5 U F

g 9.5 o

L NB 9 TSB 8.5 KB PMA 8 PM 2 TSB meth 7.5 GYEM 7

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

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)

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

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

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)

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

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

Agitation (rpm) 200 6

) 250 l /

g 300 (

s 5 s a m o

Time (h) Effect of agitation on Pseudomonas trivialis BIHB 745 biomass production in optimized medium at 14 l working volume in bioreactor