2011 International Conference on Alternative Energy in Developing Countries and Emerging Economies
- 87 -
and amino, and increase the production of unfavorable
furfural compounds, which inhibited bacterial growth
[15,16]
. Research on ethanol production from starch by
open fermentation had been carried out successfully
[17]
.
If the open fermentation could be done on food waste to
produce ethanol, a lot of energy and cost would be saved.
The starch and cellulosic components of the food
waste can be hydrolyzed to monomeric sugars, which
composed mainly of mixture of glucose and xylose. A
complete and efficient conversion of these hexose and
pentose sugars present in the food wastes hydrolysates to
ethanol is a prerequisite for maximizing the profitability
of an industrial process for bioethanol production
[18]
.
Since there is no wild type microorganism that could
efficiently accomplish this process, the utilization of two
microorganisms and the construction of genetically
modified biocatalysts have been two common
approaches. The bacterium
Z. mobilis
is known for better
ethanol productivity and tolerance compared to
S.
cerevisiae
[19]
, it has rarely been employed in such a co-
culture process. A sequential culture of
Z. mobilis
and
Pachysolen tannophilus
has been previously reported
[20]
. Co-culture of
Z. mobilis
and
Pichia stipitis
for
efficient ethanol production on glucose/xylose mixtures
also reported
[21]
.
Bansal and Singh [22]
reported a
comparative study of ethanol production from molasses
using
S. cerevisiae
and
Z. mobilis
. However, no study has
been reported on the co-culture
Z. mobilis
and
C.
shehatae
on ethanol production from glucose/xylose
mixtures substrates.
Recently many statistical experimental design methods
have been employed in bioprocess optimization.
Response surface methodology (RSM) is one such
scientific approach that is useful for developing,
improving and optimizing processes and is used to
analyze the effects of several independent variables on
the system response. This method has been successfully
applied to optimize alcoholic fermentation process
[23,24]
. The present study reports for the first time the
new strain combination of
Z. mobilis
and
C. shehatae
for
ethanol production from food wastes hydrolysates and to
determine the optimum level of fermentation variables
(nitrogen source ((NH
4
)
2
SO
4
), phosphorus source
(KH
2
PO
4
), yeast extract and inoculum size) for ethanol
production under non-sterilized condition using response
surface method.
II. M
ATERIAL AND
M
ETHOD
A. Microorganism
Z. mobilis
TISTR0548
and
C. shehatae
TISTR5843
were obtained from the Thailand Institute of Scientific
and Technological Research (TISTR) culture collection.
Cultures were maintained on agar plates at 4°C with
subculture to fresh media every 2 weeks. Glucose agar
for
Z. mobilis
consisted of 20 g/l glucose, 10 g/l yeast
extract 1 g/l KH
2
PO
4
, 1 g/l MgCl
2
, 1 g/l (NH
4
)
2
SO
4
, 15
g/l agar and pH 6.0. Xylose agar for
C. shehatae
was as
previously described
[25].
Inoculum medium consisted of
10 g/l yeast extract, 1 g/l MgCl
2
, 1 g/l (NH
4
)
2
SO
4
, 1 g/l
KH
2
PO
4
, with 20 g/l glucose for
Z. mobilis
and 20 g/l
xylose for
C. shehatae
. Media were sterilized at 121
q
C
for 15 min and sugars were separately autoclaved from
yeast extract and inorganic salts solution. The strains
were grown at a temperature of 35°C and cultured for 48
h before used in the fermentation.
B. Food Waste Hydrolysate (FWH)
The food waste was collected from Tong-Song
municipal waste management plant, Nakhonsrithammarat
Province, Southern Thailand. Food waste was fermented
by fungi from Look-Pang for 24 hours. It was mixed with
water at ratio 1:1 (v/v) and crushed in to small particles
using liquidizer. It was subsequently incubated at 55
q
C
for 12 h and then hydrolysate was generated with rich in
sugar content (145 g/l,
Table I
).
TABLE
I
C
HARACTERISTICS OF FOOD WASTE HYDROLYSATE
Parameter
Concentration (g/l)
Total carbohydrate
232
Total reducing sugar
164
Total nitrogen
6.5
Ammonium
–
nitrogen
0.45
Total phosphorus
643
Oil
10.6
Total solids
323
Volatile solid
261
Glucose
111
Xylose
23
Sucrose
21
pH
4.2
C. Inoculum Preparation
Inoculum size was expressed as volume of inoculum
medium that cells were from to volume of fermentation
medium that cells were inoculated into. Cells from the
corresponding volume of inoculum medium were firstly
centrifuged to exclude the inoculum medium and
concentrate the cells, and then the cell pellets were re-
suspended with the sterilized yeast extract and inorganic
salts solution to be inoculated into each flask. All inocula
were incubated in 250 ml conical flasks with 25 ml of
inoculum medium at 30
q
C, with a 24 h stationary
incubation for
Z. mobilis
and a 36-48 h shaking
incubation at 150 rpm for
C. shehatae
. Multiple flasks
were simultaneously cultured to get the desirable volume
of inoculum medium and subculture up to three times
was carried out to ensure that all inoculum flasks
contained an identical culture.
D. Ethanol Production
RSM batch fermentation, FWH was neutralized with
1N NaOH adjusting the pH to 5.0, and then FWH without
sterilization was used as a fermentation medium. The
nitrogen source ((NH
4
)
2
SO
4
), phosphorus source
(KH
2
PO
4
) and yeast extract were added into FWH
according to Table 2 with varying between 0.5-2.0, 0.5-
2.0 and 0.3-2.0 g/l, respectively. Batch experiments were
carried out in a series of 250 ml Erlenmeyer flasks,
containing 100 ml of FWH. 5-25% inoculum culture was
dispensed to each flask. The flasks were shaken at 180
