Basis of Calculation for Fermentation Medium of Bacillus Subtilis,

Bacillus Licheniformis and Lactobacillus Plantarum

A. Calculating different C/N Ratios

A.1 From Fan medium used for optimum growth of Bacillus Subtilis:
Amount added %Nitrogen
%Carbon
N
(g/L)
(Organic)
(g/L)
Yeast Extract
4
12
0.48
Corn Steep Liquor
15
7.6
48.66
1.14
Ammonium
5
24.13
1.20
Sulfate
Manganese
0.05
Sulfate
Sugar (Glucose)
100
40
pH =6.5
2.82

C
(g/L)
7.30

40
47.3

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
%N
%C
N
C
(grams/Liter)
(g/L)
(g/L)
[2]
Pancreatic digest of
17
14% [1] 52.88%
2.38
8.9896
Casein
[4]
Papaic digest of soyabean
3
7% [1]
42%
0.21
1.26
meal
NaCl
5
Glucose
2.5
40%
1.00
Dibasic potassium
2.5
phosphate
Final pH = 7
7.3
TOTAL
2.38
11.249
6
[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

Water

99.28

99.2

Ash

0.09

0.2

Nitrogen

0.06

0.05

Carbon

0.08

0.27

Calcium

17.12

249

ppm

Iron

0.48

5.3

ppm

Manganes
e
Magnesiu
m
Zinc

0.24

0.02

ppm

0.72

31

ppm

5.12

2.4

ppm

C:N ratio

1.3

5.4

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 Molasses
55.49
Total Sugars
%
C6H12O 29.40
Glucose
%
6
C12H22 11.92
Sucrose
O11
%
C6H12O 12.79
Fructose
%
6
For 100 g of Molasses:
C = 12 g/ mole

H = 1 g/mole
O = 16 g/ mole

Glucose=29.40 g C 6 H 12 O 6 x

Sucrose=11.92 g C12 H 22 O11 x

Fructose=12.79 C6 H 12 O6 x

40 gC
=11.76 grams C
100 g C6 H 12 O6

42.11 g C
=5.02 grams C
100 g C 12 H 22 O11

40 g C
=5.12 grams C
100 g C 6 H 12 O 6

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)

g Carbon 100 g Molasses

g Molasses
x
=36.53
Liter
21.90 g Carbon
Liter

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,

1.48

g N 100 g Fish Protein Hydrolysate 74 g Protein Hydrolysate required

x
=
L
2gN
L

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 Shrimp
Composition
% (dry basis)
Protein
64.73
Lipids
3.57
Ash
28.62
Chitin
3.08
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 Hydrolysate

Composition
%Wet Basis
Moisture
80.54
Protein
13.25
Nitrogen (Protein / 6.25 )
2.12
Lipids
0.698
Ash
5.60
Chitin
0.602

1.48

g N 100 g Shrimp Protein Hydrolysate 69.81 g Protein Hydrolysate

x
=
required
L
2.12 g N
L

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 powder
Table 4. Composition of ISSP (Insoluble Shrimp Powder)
Composition
%
Protein
Protein Nitrogen (P/6.25)
Lipids
Ash
Chitin
Moisture

(dry basis)
29.19
4.67
20.82
30.79
14.20
5.00

For the amount of Shrimp Powder needed:

1.48

g N 100 g Fish Protein Hydrolysate

g ISSP required
x
=31.69
L
4.67 g N
L

Calculating for the mass of Chitin being added,

31.69 g ISSP x

14.20 Chitin
g Chitin
=4.499
100 g ISSP
L

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,

Chitin ,C 8 H 13 O5 N =

(12 x 8)
=47 Carbon
( 12 x 8 )+ (1 x 13 )+ (16 x 5 ) +(14 x 1)

 Amount of carbon present in 31.69 g/L ISSP:

31.69

g ISSP 14.20 g Chitin

47 g Carbon 2.11 grams
x
x
=
Carbon
L
100 g ISSP
100 g Chitin
L

)(

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)

2.11 grams
21.90 g C
8 grams
Carbon+
X=
Carbon
L
100 g molasses
L
X=

26.89 grams
Molasses
L