It deals about production of bioethanol from cheese whey which is not sterilized and other characters of the ethanol producing yeasts were also covered

1. Bioethanol production from cheese whey
using yeast, a non-Saccharomyces,
Kluyveromyces marxianus
Asmamaw Tesfaw
1

2. Introduction
Bioethanol
Yeast
Starch
Lignocellulose
s
Wastes
Simple
sugars
Sorting
Hydrolysis
Chemical
Physical
Biological
Agricultural
residues
2

3. natural ecosystems rich in sugar
Yeast
Fermented
Plant
Soil
Compost
3

4. Growth factors
temperature
pH
oxygen
initial sugar concentrations
Optimization of multiple variables: single
factor at a time
Limitations
Which one comes first?
You can’t predict for future?
Solution
Response surface methodology (RSM)
4

5. RSM employ
Statistical and
Mathematical technique
RSM enables to
design experiments
build model
evaluate interactions
look for optimum conditions for responses
reduce the number of experiments
5

6. Cheese whey
Liquid waste
130millions tons word wide
50% water bodies
High BOD and COD
increasing at a rate of 3% per year
Ethiopia
20, 000 tons
Increasing
Objective
to evaluate ethanol production capability of
K. marxianus ETP87 from crude whey.
6

7. MATERIALS AND METHODS
Optimizing growth variables
Temperature: 30, 35, 40
pH: 4, 5, 6
Time:24, 48, 72
Where,
Y = ethanol produced in g/L (dependent output)
0 = intercept
β1, β2, and β3= Linear, quadratic and interaction regression
coefficients for temperature, pH and time respectively
X1, X2, and X3 = independent variable for temperature (degree
centigrade), pH, and time (hours) respectively
ε = random experimental error
7

8. Factor 1
(Temperature, oC)
Factor 2 (pH) Factor 3
(time, hours)
Response (Ethanol
produced, g/L)
35.00 5.00 48.00
30.00 6.00 24.00
30.00 6.00 72.00
26.59 5.00 48.00
35.00 5.00 48.00
35.00 5.00 48.00
40.00 4.00 72.00
35.00 5.00 48.00
35.00 5.00 88.36
30.00 4.00 24.00
43.41 5.00 48.00
40.00 4.00 24.00
35.00 5.00 48.00
40.00 6.00 72.00
35.00 3.32 48.00
40.00 6.00 24.00
30.00 4.00 72.00
35.00 6.68 48.00
35.00 5.00 7.64
35.00 5.00 48.00 8
Experimental design

9. Crude whey
Yeast: K. marxianus ETP87
Experimental condition: non-deproteinized
and deproteinized by heating
Supplementation of whey with molasses
and nitrogen sources
extract (0.55%), peptone (1%), and ammonium
sulfate (0.33%).
Molasses
100% molasses alone
25% whey and 75% molasses
50% whey and 50% molasses
75% whey and 25% molasses
100% whey
9

10. Effect of pH and temperature
pH: crude3.9 and adjusted 5.0
Temperature:25, 30, 35, 40, 45, and 50oC
Lactose determination: reducing sugar
Ethanol determination: GC-MS and
Pycnometer methods
10

11. RESULTS AND DISCUSSION
11

12. Model diagnostics
12
Design-Expert® Software
Ethanol Produced
Color points by value of
Ethanol Produced:
19.25
4
Externally Studentized Residuals
Normal
%
Probability
Normal Plot of Residuals
-4.00 -3.00 -2.00 -1.00 0.00 1.00 2.00 3.00
1
5
10
20
30
50
70
80
90
95
99
Normality test
No need to transform

13. 13
Design-Expert® Software
Ethanol Produced
Color points by value of
Ethanol Produced:
19.25
4
Run Number
DFFITS
DFFITS vs. Run
-6
-4
-2
0
2
4
6
1 4 7 10 13 16 19
2.12132
-2.12132
0
DFFITS

14. Diagnostics Case Statistics Report
Internally Externally
Influence
on
Run Actual Predicted
Studentiz
ed
Studentiz
ed
Cook's
Fitted
Value
Standard
Order Value Value Residual Leverage Residual Residual Distance DFFITS Order
1 5.43 10.43 -5.00 0.607 -2.240 -3.009 0.776 -3.7431 11
2 9.30 8.04 1.26 0.670 0.616 0.596 0.077 0.849 2
3 19.25 14.68 4.57 0.670 2.232 2.990 1.0111 4.2581 6
4 15.52 15.29 0.23 0.166 0.070 0.067 0.000 0.030 16
5 15.02 12.07 2.95 0.670 1.440 1.535 0.421 2.1861 7
6 18.46 15.07 3.39 0.670 1.655 1.842 0.555 2.6231 5
7 9.84 12.91 -3.07 0.607 -1.376 -1.450 0.293 -1.803 9
8 9.47 12.35 -2.88 0.607 -1.291 -1.342 0.258 -1.669 10
9 8.56 7.74 0.82 0.670 0.402 0.384 0.033 0.547 4
10 6.70 7.65 -0.95 0.607 -0.427 -0.409 0.028 -0.509 12
11 14.90 15.29 -0.39 0.166 -0.120 -0.114 0.000 -0.051 20
12 15.78 15.29 0.49 0.166 0.150 0.143 0.000 0.064 15
13 8.13 13.54 -5.41 0.607 -2.424 -3.579 0.908 -4.4511 14
14 15.63 15.29 0.34 0.166 0.104 0.099 0.000 0.044 17
15 7.65 8.01 -0.36 0.670 -0.176 -0.167 0.006 -0.238 3
16 10.22 8.61 1.61 0.670 0.785 0.769 0.125 1.095 8
17 4.00 4.54 -0.54 0.607 -0.243 -0.232 0.009 -0.288 13
18 15.11 15.29 -0.18 0.166 -0.056 -0.053 0.000 -0.024 18
19 7.85 5.25 2.60 0.670 1.272 1.318 0.328 1.876 1
20 15.83 15.29 0.54 0.166 0.166 0.157 0.001 0.070 19
14

15. 15
Central Composite design
Factor 1 Factor 2 Factor 3 Response 1
Std Run
A:Temperatur
e
B:pH C:Time
Ethanol
Produced
11 1 35.00 3.32 48.00 5.43
2 2 40.00 4.00 24.00 9.3
6 3 40.00 4.00 72.00 19.25
16 4 35.00 5.00 48.00 15.52
7 5 30.00 6.00 72.00 15.02
5 6 30.00 4.00 72.00 18.46
9 7 26.59 5.00 48.00 9.84
10 8 43.41 5.00 48.00 9.47
4 9 40.00 6.00 24.00 8.56
12 10 35.00 6.68 48.00 6.7
20 11 35.00 5.00 48.00 14.9
15 12 35.00 5.00 48.00 15.78
14 13 35.00 5.00 88.36 8.13
17 14 35.00 5.00 48.00 15.63
3 15 30.00 6.00 24.00 7.65
8 16 40.00 6.00 72.00 10.22
13 17 35.00 5.00 7.64 4
18 18 35.00 5.00 48.00 15.11
1 19 30.00 4.00 24.00 7.85
19 20 35.00 5.00 48.00 15.83

16. ANOVA: Model validation
Source
Sum of
Squares df Mean Square F Value p-value Prob > F
Model 258.14 9 28.68 12.81 0.0002
A-Temperature 0.49 1 0.49 0.22 0.6493
B-pH 22.63 1 22.63 10.10 0.0098
C-Time 113.49 1 113.49 50.67 < 0.0001
AB 1.13 1 1.13 0.51 0.4933
AC 5.59 1 5.59 2.50 0.1451
BC 8.51 1 8.51 3.80 0.0799
A2 32.89 1 32.89 14.68 0.0033
B2 45.27 1 45.27 20.21 0.0012
C2 48.95 1 48.95 21.85 0.0009
Residual 22.40 10 2.24
Lack of Fit 21.69 5 4.34 30.64 0.0009
Pure Error 0.71 5 0.14
Cor Total 280.53 19
Std. Dev. 1.50 R-Squared 0.9202
Mean 11.88
Adjusted R-
Squared 0.8483
C.V. % 12.60
Predicted R-
Squared 0.4102
16

17. 17
Where
X1 temperature (oC)
X2 pH
X3 incubation time(hours)
Optimum
temperature (34.2oC),
pH (4.43), and
incubation time (71.93 hours)

18. 18
(B)
(A)
(D)
(C)
K. marxianus ETP87 Response surface

19. Whey
sample
s pH
Lactose (g/L) Ethanol (g/L) by
Efficie
ncy
(%)
Biomass
(g/L)
Sampling 2 week
later
microflo
ra on
time of
samplin
g
microflora
2 weeks in
refrigerator
Ethanol by
K.
marxianus
ETP87
Tigist
dairy 4.5
34.4±1.3 27.3±1.1
0 1.2±0.09 11.62±0.7
66.23 7.67±0.5
Amanu
al dairy 3.8
28.6±1.2 21.5±0.9
0.6±0.08 1.7±0.08 10.54±0.8
72.26 6.20±0.4
Househ
old 3.1
18.7±0.8 14.4±0.7
2.3±0.1 1.4±0.07 6.43±0.5
67.42 5.48±0.4
Shola
dairy 4.2
27.8±1.2 26.1±1.1
0 0.8±0.08 12.49±0.9
88.09 7.12±0.7
19
Ethanol production from different whey

20. Ethanol production from non-sterilized and
sterilized whey
Refrigera
tor days
Non Sterilized whey Sterilized whey
Ethanol
produced
% of
reduction
Ethanol
produced
% of
reduction
0 11.71 0 11.68 0
1 11.46 2.13 11.69 +0.09
2 10.34 11.7 11.48 1.71
3 10.97 6.3 10.77 7.79
4 10.01 14.51 11.12 4.79
5 9.22 21.26 10.56 9.59
6 8.61 26.47 10.21 12.59
7 8.18 30.15 9.63 17.55
8 7.54 35.61 9.44 19.18
14 5.67 51.58 8.37 28.34
Average 9.11 10.36
20

21. Effect of whey pH
21
11.74
10.23
11.99
13.29
11.91
13.87
0
2
4
6
8
10
12
14
16
Tigist dairy Amnual
dairy
Shola dairy
Ethanol
produced
(g/L)
Whey samples
Crude (pH 4.5, 3.8,
and 4.2 for Tigist,
Amanual and Shola
dairies, respectively)
Treated (pH 5.00)

22. Molasses supplementation to whey
22
10.02
12.49 12.84
17.28
12.38
0
2
4
6
8
10
12
14
16
18
20
100%
molasses
75%
molasses +
25% whey
50%
molasses +
50% whey
25%
molasses
+75% whey
100% whey
Ethanol
concentration
(g/L)
composition of whey versus cane molasses (percentage)

23. Effects of external nutrient additions to
whey
23
9.37
15.92 15.38
10.24
13.57
10.65
0
2
4
6
8
10
12
14
16
18
Ethanol
concentration
(g/L)
Suppliments

24. Effect of Temperature
24
0
2
4
6
8
10
12
14
25 30 35 40 45 50
Concentration
(g/L)
Temperature (oC)
Ethanol
Biomass

25. CONCLUSIONS AND RECOMMENDATIONS
Putting whey sample in refrigerator doesn’t
guarantee the existence of lactose for more
than 5 days without decreasing in its amount
Ethanol could be produced from crude non-
sterilized whey using K. marxianus
25

26. Thank You
26