ETHANOL PRODUCTION FROM PURE GLYCEROL
USING BACILLUS CEREUS
PROJECT REFERENCE NO.: 38S_B_BE_049
COLLEGE
: NMAM INSTITUTE OF TECHNOLOGY, NITTE, KARKALA
BRANCH
: BIOTECHNOLOGY ENGINEERING
GUIDE
: MR. VENKATESH KAMATH H
STUDENTS : MS. SAMIYABANU
MS. KRUPA M.B.
MR. DEEPAK G.M.
Introduction:
Production of biofuel has seen an enormous amount of upfront along with the discovery of
new raw materials. Along with production of biodiesel, by product, glycerol is also produced.
This glycerol can be converted to another fuel bioethanol using minimal energy
consumption. Fermentation of glycerol can be achieved using suitable microbe that can
assimilate the raw material and can sustain high ethanol concentration. Due to the enormous
cost involved in purification, use of this raw glycerol is limited to only a few applications.
Besides, this raw glycerol can also be used for bioethanol production with minimal
purification steps. In this direction glycerol, can also be considered as a potential carbon
source for bioethanol production. Since glycerol is not a preferential substrate for many
microbes, their metabolic pathway needs to be activated by use of suitable stress factor.
Objectives:
The main goal of this work is to produce ethanol from glycerol feedstock using bacterium
Bacillus cereus isolated from honey. The objectives set to achieve this goal are as follows.
 To develop and standardize simple procedures for analysis of ethanol and glycerol
present in the fermentation sample using single analytical procedure.
 To standardize a protocol for the production of ethanol from glycerol (pure) feedstock
using bacterium Bacillus cereus isolated from honey.
 To study the effect of parameters like time, oxygen requirement (oxygenic and anoxic),
glycerol concentration, temperature on the production of ethanol.
 To use purified glycerol from biodiesel byproduct stream for ethanol production and
optimize the process.
Methodology:
Bacillus cereus (Eq. ATCC 14579T, facultative aerobic bacterium) isolated in the lab from
honey was used as microbial source for fermentation.
Shake Flask studies: Pure glycerol medium was prepared using commercial glycerol and
made to required concentration by diluting with water. Medium pH was adjusted using
phosphoric acid and sodium carbonate. The fermentation samples, after sterilization and
inoculation, were agitated at 100 rpm in shake flasks at room temperature. The glycerol and
ethanol concentration were analyzed using sodium periodate method and potassium
dichromate method respectively. Anaerobic condition was provided by purging CO2 gas
before fermentation.
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Fermentation in Small scale Bioreactor: A small scale bioreactor (40ml working volume)
was constructed as shown in Fig.1. Aeration and agitation conditions were provided with air
pump connected to 40micron membrane filter and stirrer connected to external motor
respectively. Pure glycerol medium as in shake flask studies were used to evaluate
performance of this reactor. Stirrer speed was fixed to 150 rpm.
Use of Raw glycerol & Process Optimization: Raw glycerol of Pongamia was obtained
from Udupi district Biofuel information and demonstration centre, and purified using acid
treatment followed by extraction with isopropanol and charcoal adsorption as per literature
reported method. This glycerol was diluted as per requirement and fermentation was carried
out in mini bioreactor. The process was optimized using response surface methodology. The
ethanol is reported in terms of g/l in fermentation broth, yield is reported as gram ethanol
produced per gram glycerol fed. Ethanol production efficiency is reported as actual ethanol
yield to theoretical maximum yield (0.5 g ethanol/g glycerol).
1: Air pump,
3: Micro filter
5: Agitator
7: Motor
2: Air flow controller
4: Needle Sparger
6: Lid / cap
8: Air vent
Fig. 1. Small scale bioreactor
Results & Discussion:
Shake flask studies with pure glycerol was performed with varying concentration of glycerol
(0.1 to 1M), pH (5, 6, 7 & 8), Aerobic and anaerobic condition for 24 and 48 hours with one
variable approach. It is observed that (Fig.2.) as glycerol concentration increases beyond
0.8M, ethanol production drops. Medium pH of 5 to 6 is best for ethanol production.
Literature reports that in microbial fermentation, at basic pH formate ions are accumulated
which in turn inhibit the enzyme that produces ethanol. Oxygenic stress is essential for
ethanol production by B. cereus as evident from Fig. 3. Since oxygenic stress plays a vital
role, a small reactor was designed to supply controlled air. It was noted that anaerobic
condition favors biomass production and aerobic condition favors ethanol production (Fig. 4).
5
0.020
4
0.015
3
0.010
2
1
0.005
0.04
8
0.03
6
0.02
4
0.01
2
0.00
0
0
0.000
0.1
0.2
0.4
0.5
0.6
0.8
1.0
Glycerol Concentration (M)
Fig. 2. Effect of Glycerol
5.0
7.0
6.0
8.0
Aer - 24 hr
Anaer - 24
Aer - 48
Anaer - 48
10
Biomass concentration,
(dry weight, g/L)
0.025
Yield of ethanol (g Eth/g Gly)
6
12
10
Aerobic
Anaerobic
% Efficiency of ethanol production
0.030
0.05
7
Aerobic
Anaerobic
% Efficiency of ethanol production
Yield of ethanol (g Eth/g Gly)
0.035
8
6
4
2
0
5
6
Fig. 3. Effect of pH
7
8
pH
Medium pH
Fig.4. Biomass concentration
The new bioreactor designed was tested for its performance with aeration condition of
80ml/min (2VVM) and 0.5 & 0.8M pure glycerol. Yield of ethanol was increased by 4-5 times
in comparison with shake flask experiments. The raw glycerol obtained from biodiesel unit
was purified and diluted for optimization experiments using this bioreactor.
Table 1. Variables and constants used for optimization experiment
Code Variables Unit
Coded Levels
Optimal
-1.6818
-1
0
+1
+1.6818
Value
2
Yield of
ethanol
A
B
C
Time
Aeration
Glycerol
pH
Agitation
hr
ml/min
g/L
15
4
8
22.50 33.5 44.50
35.62 82.0 128.38
25.03 50.0 74.97
Constant = 5.0
Constant = 150 rpm
52
160
92
35.9
95.4
68.2
5.0
150
Predicted:
0.4900
Experimental:
0.4789
gE/gG
Response surface methodology with 3 factor – 5 level was used and Minitab v14 was used
for analysis. The parameters varied and optimal conditions obtained experimentally are
given in Table 1. The experiments (15) were conducted with one centre point and the yield of
ethanol was fitted to the reduced quadratic model (Eq.1) using the software. Experiments
were conducted at the estimated optimal conditions (Table 1) to validate the result.
Yield (gE/gG) = -0.764732 + 0.018122*A + 0.007244*B + 0.017118*C -0.000218*A*A -0.000016*B*B
-0.000098*C*C -0.000034*A*B + 0.000012*A*C -0.000043*B*C
2
(R = 0.9349)
(Eq. 1)
Conclusion:
Glycerol, pure as well as raw (purified from biodiesel stream), is a good carbon source for
production of ethanol using a facultative aerobic bacterium Bacillus cereus under aerobic
condition. Further, a small scale bioreactor designed for aerobic fermentation was designed
to provide proper aeration and agitation conditions. The process was optimized (RSM) to
produce 33.5 g/L of ethanol under fermentation condition of 68.2 g/L raw glycerol at a pH of
5.0 with aeration rate of 95.4 ml/min (2.4 VVM) and 35.9 hours of incubation. The Yield and
efficiency of ethanol production was measured to be 0.4789 g ethanol/g glycerol and 95.8%
respectively.
Scope for future work:
This work has shown that ethanol can be produced from raw glycerol in a small scale reactor
set up under aerobic condition. The process can be easily scaled up based on these results.
It is recommended to maintain/control pH between 5 and 6 during fermentation. Mild
agitation condition is sufficient since aeration condition provides good mixing. Effect of other
nutrients along with raw glycerol can be tested for enhancement of ethanol production.
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