PRACTICAL
ABSTRACT
Fermentation
metabolism are carried by bacteria and fungi but fungi are more widely used in
fermentation process. Most are reliant on oxygen as terminal electron acceptor
during their ATP synthesis while fungi do not grow or grow slowly in
oxygen-poor environments. This practical involve propagation of inoculums was
often carried out before the actual process and monitoring of yeast growth was
often done through microscopy counting chambers at 400magnification. Nutrient
broth was inoculated with bacteria and reading at 660nm as the start. Solution
was put on shaker at 200rpm and reading was taken after one hour followed by
plotting the graph. The reading from spectrophotometer were 0.017, 0.019,
0.026, 0.162, 0.388 for hour one to six respectively. After the yeast obtained
was used in the fermentation of molasses to produce alcohol. Molasses was
diluted to Brix 10 (ph 6) and used to pre-ferment the inoculums followed by
molasses fermentation and sugar. After the initial concentration and
fermentation process (final concentration) was recorded.
INTRODUCTION
Industrial fermentation is the intentional use of fermentation by microorganisms such as bacteria and fungi to make products useful to humans (Donald W et al 2006). Fermented products have applications as food as well as in general industry(Christine L.et al 2010). Some commodity chemicals, such as acetic acid, citric acid, and ethanol are made by fermentation. The rate of fermentation depends on
the concentration of microorganisms, cells, cellular components, and enzymes as
well as temperature, pH and oxygen and carbohydrate concentration (Judith G. et
al 1995). Bacteria and yeast carrying out fermentation metabolism but
the fungi are widely used in fermentation biotechnology. By fermentation, the yeast species Saccharomyces
cerevisiae
converts carbohydrates to carbon dioxide and alcohols (Wang, C. et al 2004).
For thousands of years the carbon dioxide has been used in baking and the alcohol in alcoholic beverages.
Alcoholic beverages are beverages that contain ethanol (C2H5OH). This ethanol is almost
always produced by fermentation (Lubert et al 1975). The metabolism of carbohydrates by certain species of yeasts under
anaerobic or low-oxygen conditions.
Beverages such as mead, wine, beer, or distilled
spirits all use yeast
at some stage of their production (Van Der Drift C. et al 1988). A distilled beverage
is a beverage containing ethanol that has been purified by distillation. Carbohydrate-containing plant
material is fermented by yeast, producing a dilute solution of ethanol in the
process (Justin McCarthy. et al July 2013). Spirits such as whiskey and rum are prepared by distilling these
dilute solutions of ethanol. Components other than ethanol are collected in the
condensate, including water, esters, and other alcohols, which (in
addition to that provided by the oak in which it is aged) account for the flavour of the beverage (Dickinson J. R. et al 1999).
Mixing alcohol with gasoline produces
gasohol. Advantages of fuel blends are that alcohol tends to increase the
octane rating and reduce carbon monoxide (CO) and other tailpipe emissions from
the engine. The octane number of a fuel indicates its resistance to knock
(abnormal combustion in the cylinder). Another advantage is that alcohols can
also be produced from renewable sources (Decker K. et al 1977). .
The primary disadvantage of mixing
methyl and ethyl alcohol with gasoline is that under certain conditions these
alcohols may separate from the gasoline. An engine adjusted to burn gasoline
efficiently will produce less power from alcohol should it separate from the
gasoline William (Kirkwood et al 2004). Separation is caused by the polar nature of the
alcohol molecules and their tendency to absorb water, also a polar substance.
Methyl alcohol is the most likely to separate while butyl alcohol is the least
likely.
Sugar industries produce molasses from
the sugar cane processing. Molasses have 50 -55% concentration of sugar in the
form of sucrose, with chemical formula C12H22O11.
This source of compound is used for preparing ethyl alcohol. Ethanol in the
form of absolute and rectified spirit can be made from molasses (Judith G. et al
1995). . Basis raw
materials for an industry to produce 1 ton of ethyl alcohol requires, molasses up
to 5.6 tons, sulfuric acid 27 kg and ammonium sulphate 2.5 kg.
MATERIAL AND METHODOLOGY
300 ml of Malt Extract Broth (MEB) was
prepared for the whole class by taking 5.3 g (MEB) and dissolve in 300 ml of
water and 300 ml Nutrient Broth (NB) of
was prepared by taking 2.4 g of (NB) dissolved in 300 ml. Both broth media were
autoclaved at 121⁰C for 15minutes.
Nutrient
broth prepared was inoculated with bacteria from last practical session and
reading at 660 nm was taken. The solution was put on shaker at 75 rpm and
reading was taken after every one hour and plotted on graph.
For the yeasts a suspension was made through putting one loop-fully in
the Malt Extract Broth prepared, one drop of the suspension was put on counting
chamber provided, readings were made and submitted in yeast number/ml. Immediately
the inoculated flasks were put on the shaker at 150 rpm (revolution per minute)
for 16 hours, over night. A counting chamber was used again to note the reading
followed by submitting the reading. The
propagated biomasses were kept in fridge for further fermentation process.
PRE-FERMENTATION
500 ml solution of molasses was made for a class; the solution molasses
was diluted to Brix 10⁰ (pH 5 - 6) and used to pre-ferment the inoculum for molasses
fermentation. 200 ml sugar solution of 4% (containing 0.3g/l ammonium sulphate)
was prepared, dispensed into two conical flasks each with 50mL sugar solution. One
flask for commercial yeast and the other two for isolated yeast.
10 ml of the yeast
broth was added in each 50 ml of the solutions prepared followed by putting the
solution on the shaker 150 revolution per minutes (rpm) for 1.5 hours and then
the growth was observed under the microscope. The inoculation was ready for use
in fermentation process.
FERMENTATION
150 ml of molasses
provided was diluted to 23.6⁰ brix and it was dispensed in one conical
flask. 200 ml of sugar solution provided
was diluted to 6%, yeast extract 10g/l and peptone 20 g/l were added, and the
solution dispensed in one conical flask to achieve the highest yields and
compete. pH was adjusted to 6. The diluted media was inoculated and records for
initial sugar concentration were made. The media were incubated at room
temperature without shaking.
Note; addition of
inoculums often was not exceed 5% vol/vol. a starter was always added to make
up a total of 100ml solution. Fermentation medium ws not sterilized as in
industry it is never economical to do so hence the inoculums has to compete
with contaminants. Automatic meter (refractometer) was used to determine the
brix of cane molasses
RESULTS
i)
Different
reading obtained at 660 nm after every one hour by spectrophotometer to determine growth number of microorganism in
different time.
Table 01: Shows absorbance recorded with time.
|
Hour (s)
|
Absorbance (nm)
|
|
1
|
0.017
|
|
2
|
0.019
|
|
3
|
0.026
|
|
4
|
0.162
|
|
5
|
0.388
|
Figure 1: Shows the graph of absorbance against time.
ii) The number of yeast cells obtained by counting chamber under microscope
without dilution are:-
Table 02: Shows the number of yeast cells without dilution in
counting chamber ( i-xvi) under
microscope after one hour of incubation of the sample.
|
Counting chamber
|
Counting chamber
|
Counting chamber
|
Counting chamber
|
|
i)
8
|
v) 8
|
ix) 4
|
xiii) 8
|
|
ii)
5
|
vi) 5
|
x) 8
|
xiv) 2
|
|
iii)
7
|
vii) 6
|
xi) 7
|
xv) 3
|
|
iv)
3
|
viii) 1
|
xii) 10
|
xvi) 6
|
Calculation of number of cell per 1mL
To calculate the
number of cell counted per 1ml.
Total numbers of
cells = 8+5+7+3+8+5+6+1+4+8+7+10+8+2+3+6 = 91
The average of cells = 91/16 = 5.6875 cell/µl
Number of small squares involved in
counting cells = 16
For 1 dilution
Number
of cells/ml = (total of cells x Dilution factor x10000 cell/ml)/number of small
squares involved in counting cells.
Number
of cells/mL = 5.6875 x 1000 cell/ml = 5687.5 cells/ ml
Table 03: Shows the number of yeast cells per ml with dilution of
1 /100 of the sample after 16 hours of incubation of the sample
|
No
of cells per small square
|
No
of cells per small square
|
No
of cells per small square
|
No
of cells per small square
|
|
i.
20
|
v. 16
|
ix.
19
|
xiii.
20
|
|
ii.
21
|
vi. 25
|
x.
26
|
xiv.
21
|
|
iii.
28
|
vii.
14
|
xi.
24
|
xv.
27
|
|
iv.
36
|
viii.
16
|
xii.
19
|
xvi.
24
|
Calculation of number of cell.
Total number of
cells = (20+21+28+36+16+25+14+16+19+26+24+19+20+21+27+24) cell = 356 cells
Number of cell in
each small square = 356/16 = 22.25 cell/µl
Dilution used = 10 -2
Number of cells/ml
= 22.25 x 100 x 1000 cell/ml = 2225000 cell/ml
iii) Table 4: Shows the concentration of the sugar and
molasses after fermentation with yeast isolate and industrial yeast.
Pre-fermentation (initial concentration) and
fermentation process ( final concentration).
|
|
Initial
concentration (brix)
|
Final
concentration ( brix)
|
|
Molasses
inoculated with yeast isolate
|
23.6
|
3. 28
|
|
Sugar inoculated
with industrial yeast
|
6
|
2. 78
|
|
Molasses for
control (industrial yeast)
|
10
|
6.7
|
|
Sugar for
control ( industrial yeast)
|
4
|
3.7
|
DISCUSSION
Figure
one shows that absorbance is directly proportion to time. As the time goes
grows of bacteria in nutrient broth increase with increase in time however at
the initial time growth was very low as bacteria were adapting the new
environment of the media. Bacteria reproduce as microorganisms need access to a
source of energy and the raw materials essential for the construction of
cellular components.
Increase
in cellular constituents leads to a rise in cell number when microorganisms
reproduce. Bacteria consume nutrients in nutrient broth and extract energy
which lead to increase in the reproduction of bacteria as time goes. The number
of bacteria will be reduced as all nutrients in nutrient broth decrease at a
certain time where bacteria fail to reproduce and number of bacteria decrease (Christine L. et al
2010). Also as time goes number of bacteria decrease due to competition.
The
number of microbe in diluted media is greater compare to undiluted one from the
results above as it support growth of bacteria more easily compare to undiluted
one. Scientists use a number of different methods to determine the number of
micro-organisms that are present in a given population. Spectrophotometer
accomplished to measure the optical density of the population, by directly
counting the microorganisms using a haemocytometer, or by serial diluting the
bacteria and plating the diluted bacteria on media that supports the growth of
the micro-organisms (Donald et al 1995). The latter method is somewhat more time
consuming, but provides statistically accurate and repeatable results. This method is also the ideal method for
enumerating microorganisms in a given population because it only identifies the
living organisms in that population.
Microbial
counting is useful in the basic sciences and is used determine the number of
bacteria present for physiological or biochemical studies. For example, if one knows the number of
bacteria present in a culture then one can calculate the amount of protein or
DNA that can be isolated from that population.
Microbial enumeration is also routinely used in the areas of public
health (Berdell R et
al 2010) Food or water
microbiologists test food, milk or water for the numbers of microbial pathogens
to determine if these products are safe for human consumption.
As fermentation
occur concentration of the substrate used decrease as carbohydrate present are
broken down by yeast in to other compound such as alcohol, carbon dioxide and
energy which lead to decrease in concentration of initial substrate used hence
absorbance decrease as concentration of sugar decrease.
CONCLUSSION
As fuel and gas
prices rise and global warming becomes more pervasive, more people are using
biofuels such as alcohol as a way to save money and decrease their consumption
of fossil fuels, petrol and diesel . They release fewer pollutants, such as
carbon dioxide, into the atmosphere, helping decrease heat-trapping gases.
Biofuels are produced from so-called "energy crops" that include
wheat, corn, soybeans and sugarcane, so they are sustainable. And if every
nation can grow its own, there is a high likelihood these biofuels will never
run out.
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