Basis of Calculation for Fermentation Medium of Bacillus Subtilis, Bacillus Licheniformis and Lactobacillus PlantarumA. Calculating different C/N Ratios

A.1 From Fan medium used for optimum growth of Bacillus Subtilis:Amount added(g/L)%Nitrogen%Carbon (Organic)N(g/L)C(g/L)

Yeast Extract4120.48

Corn Steep Liquor157.648.661.147.30

Ammonium Sulfate524.131.20

Manganese Sulfate0.05

Sugar (Glucose)1004040

pH =6.52.8247.3

C/N ratio: 16

A.3 Source: Kumar Et. Al ( 2009) Production of Peptide antifungal antibiotic and biotocontrol activity of Bacillus subtilis, Indian Journal of Experimental Biology Amount (grams/Liter) [2]%N%CN(g/L)C(g/L)

Pancreatic digest of Casein1714% [1]52.88%[4]2.388.9896

Papaic digest of soyabean meal37% [1]42%0.211.26

NaCl5

Glucose2.540%1.00

Dibasic potassium phosphate2.5

Final pH = 77.3

TOTAL2.3811.2496

[1] https://www.ces.ncsu.edu/fletcher/programs/ncorganic/research/soy.html[2] Himediaproduct compositon[3] Acumedia product composition[4] Ramsdell G.A. (1944), Composition of Casein in Milk, Agricultural Research Division, United States)

C/N ratio: 4.7

A.4 Source: Purnawati R. et al (2014), Characterization of Novel Bacillus Thuringiensis isolated from attacus atlas and its growth kinetics in the cultivation media of Tofu whey for Bioinsecticide Production, Journal of Biology, agirculture and Healthcare.Nutrient Broth Tofu Whey

Water99.2899.2%

Ash0.090.2%

Nitrogen0.060.05%

Carbon0.080.27%

Calcium17.12249ppm

Iron0.485.3ppm

Manganese0.240.02ppm

Magnesium0.7231ppm

Zinc5.122.4ppm

C:N ratio1.35.4

In the study, nutrient broth was supplemented with urea to increase production of bacteria and bioinsecticide proteins. Hence, C:N ratio of 5.4 will be used.

B. Calculating for amount of Molasses to be added as C source.Basis: Applicable for all bacteria, calculations will be based on 20 g/L of Glucose Requirement. For Glucose: C6H12O6 = 40% Carbon, 20 g/L Glucose = 8 g/L Carbon Calculating for Carbon in Molasses,Table 1. Composition of MolassesTotal Sugars55.49%

GlucoseC6H12O6 29.40%

SucroseC12H22O1111.92%

FructoseC6H12O612.79%

For 100 g of Molasses:C = 12 g/ moleH = 1 g/moleO = 16 g/ mole

Total % Carbon in 100 g molasses = 11.76 + 5.02 + 5.12 = 21.90 grams/100 grams or 21.90 % Calculating for amount of molasses required in the medium (g/L)

C. Calculating for amount of FISH hydrolysate to be added as Nitrogen Source

With the Basis of C:N ratio of 5.4, 8 g/L Carbon requires 1.48 g/L Nitrogen From Analysis of 2% Nitrogen in Fish Protein Hydrolysate,

D. Calculating for amount of SHRIMP hydrolysate to be added as Nitrogen Source Determining N content of Shrimp hydrolysate:Table 2. Composition of Soluble Fraction of Enzymatic Hydrolysis of ShrimpComposition% (dry basis)

Protein64.73

Lipids3.57

Ash28.62

Chitin3.08

Source: Randriamahatody Z., et al (2011) Proteolysis of shrimp by-products (Peaneus monodon) from Madagascar, Journal of Food Moisture content of Shrimp Hydrolysate (when hydrolysis does not include addition of water) = 80.54 % (Mizani A.M et al, A New Process for Deproteinization of Chitin from Shrimp Head Waste, Proceedings of European Congress of Chemical Engineering)

Table 3. Wet Basis Composition of Shrimp HydrolysateComposition%Wet Basis

Moisture80.54

Protein13.25

Nitrogen (Protein / 6.25 )2.12

Lipids0.698

Ash5.60

Chitin0.602

E. Calculating amount of Shrimp Powder as N source if INSOLUBLE FRACTION OF hydrolysis is used (for higher chitin concentration)

For composition of the shrimp powderTable 4. Composition of ISSP (Insoluble Shrimp Powder)Composition% (dry basis)

Protein29.19

Protein Nitrogen (P/6.25)4.67

Lipids20.82

Ash30.79

Chitin14.20

Moisture5.00

For the amount of Shrimp Powder needed:

Calculating for the mass of Chitin being added,

F. Calculating amount of Molasses as C source if INSOLUBLE FRACTION OF hydrolysis is used (for higher chitin concentration)

In calculating for mass of molasses required, the carbon content present in the Chitin present in ISSP should be considered. For the amount of Carbon present in Chitin,

Amount of carbon present in 31.69 g/L ISSP:

Since the amount of Carbon required is 8 g/L, letting X be the amount of molasses to be added (Molasses = 21.90% C, based on previous calculation)