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a) Alkaline treatment

Alkaline treatment employs alkaline solutions, such as sodium hydroxide, calcium hydroxide or
ammonia for the biomass treatment, in order to remove lignin and part of hemicellulose and to
efficiently increase the accessibility of cellulose. It will degrade the ester and glycosidic side
chains, resulting in structural alteration of lignin, cellulose swelling, partial decrystallization of
cellulose and partial solvatation of hemicellulose (Karp, 2013). Basically, it is a delignification
process, where a significant amount of hemicellulose is also solubilized.

Figure 1: The structural alteration of bagasse

For our process, we use sodium hydroxide because it can reduce the sugar degradation during the
process and reduces the lignin and hemicellulose content in biomass, increases the surface area,
allowing penetration of water molecules to the inner layers, and also breaks the bonds between
hemicellulose and lignin carbohydrate.
After the organic acid treatment, we will obtained a slurry with 6% to 25% weight solids and the
aqueous slurry is react with alkali of about 0.5% to about 3% by weight and maintaining the
temperature at about 80 C to about 210 C, for a period of about 5 minutes to about 360 minutes
while having pH of about 7 to about 9 will yield 60.8% of cellulose conversion (Zhao, Zhang, &
Liu, 2007). The alkali treatment will completely dissolves the lignin part and cellulose and
hemicelluloses part remain in cake. The slurry is then proceed to the centrifugation to completely
separate the lignin and the cellulose. The dissolved lignin part is separated and washed with
water while cellulose slurry is being brought to the enzymatic hydrolysis for further steps.


Sodium Hydroxide




Sodium Hydroxide



Hemicellulose +

Sodium Hydroxide



b) Enzymatic Hydrolysis
Following pretreatment, enzymatic hydrolysis is needed to further depolymerize the cellulose
component to glucose, which can be used for bioethanol fermentation. In this step, the
remaining cellulose is hydrolyzed to glucose. In this enzymatic hydrolysis reaction, cellulase
enzymes are used to break the chains of sugars that make up the cellulose and releasing
glucose. Cellulose hydrolysis is also called cellulose saccharification because it produces

C6 H 10 O5

)n + n


n C6 H 12 O6

Figure 2: The cellulose hydrolysis to glucose

As the main component of lignocelluloses, cellulose is a biopolymer consisting of many

glucose units connected through -1, 4-glycosidic bonds. D-glucose is the only building block
in many polysaccharides like glycogen, starch and cellulose. Glucose mostly exists in the more
stable hemiacetal form, which has two isomers which are - and -glucose. Glycogen and
starch are branched polymers consisting -glucose, while cellulose molecules are unbranched

chains formed by -glucose (GJ, 2015).

Figure 3: The structure of glucose

As explained, the cellulose structure is rigid and usually required another treatment to break
down cellulose. Thus, in order to break the cellulose we use the enzymatic hydrolysis. The
cellulose containing slurry is being treated with one or more cellulytic enzyme selected from
the group of cellulose which are endoglucanses or endo-1,4-beta-glucanases, exoglucanases or
cellobiohydrolases and beta-glucosidases. This is because there are two types of hydrogen
bonds in cellulose molecules, those that form between the


group and the oxygen in

the pyranose ring within the same molecule and those that form between the



of one molecule and the oxygen of the glycosidic bond of another molecule (Mooney, 2015).
Basically, the beta-1,4 glycosidic bonds are not too difficult to break. However, because of
these hydrogen bonds, cellulose can form very tightly packed crystallites. The temperature
during enzyme hydrolysis is maintain at 30C to 60C for a period of 4 to 24 hours. The pH is
maintained in the range of 4 to 6.

Figure 4: The enzymatic hydrolysis of cellulose

c) Fermentation
After enzymatic hydrolysis, the process is proceed to the last reactive process that are
fermentation and it is in an anaerobic condition. In this process, we are using Sacchoromyces
cerevisiae and it is a type of facultative anaerobe yeast. As we all know, for an aerobic
environment, the yeast will converts sugars into carbon dioxide and water. Meanwhile, in an
anaerobic environment, it converts sugars into carbon dioxide and ethanol.
During the fermentation reaction, the Sacchoromyces cerevisiae is added and heated to the
solution. It will produce zymase, an enzyme that will convert glucose produce from the
enzymatic hydrolysis into bioethanol as shown in Figure 2. The fermentation is supplemented
with proper dosing of nitrogen, micro and macro nutrients at a concentration sufficient to
enhance the growth of microorganism.

C6 H 12 O6


2C 2 H 5 OH +

2CO 2

Figure 5: The ethanol chemical reaction (Klare, 2011)

When Saccharomyces cerevisiae is added, it will ferment the sugars present that is glucose to
produce bioethanol in the absence of oxygen. The glucose is convert into pyruvic acid through
the glycolysis pathways and after that, the pyruvic acid is convert into bioethanol. As the
reaction progresses, CO2 produced needs to be vented off the solution as shown in Figure 5.
d) Seed Fermentation
There are many environmental conditions that affect yeast cell growth and the kinetics of
chemical reactions within living cells. These include the availability of major and minor
nutrients, the temperature, pH, and dissolved oxygen concentration, and the possible presence of
competing organisms. Cell metabolism and growth are maintained with necessary cell nutrients
is being supplied and when one essential nutrient is absent or depleted, the intracellular reactions
are inhibited. The nutrient sources for this experiment are, glucose which is a carbon source,
ammonium chloride which is a nitrogen source and yeast extract which provides all the essential
trace minerals and growth factors. Since glucose is a preferred carbon source for microbial cell
growth, it is often the limiting substrate.
The temperature also affects the growth and productivity of the cells. As S. cerevisiae approach
their optimum growth temperature of 30C their growth rate approximately doubles for every
10C the temperature increases. Past the optimal growth temperature, the growth rate slows and
metabolism may become faster than the diffusion rate within the bioreactor, making the diffusion
of nutrients the rate limiting step (AS, 2015).
The pH inside the bioreactor affects the enzymes inside the cells and changes the rates of
reaction. Yeast cells have evolved so that they can thrive in more acidic environments than many
competing organisms. As yeast cells consume their nitrogen source, hydrogen ions are released
decreasing the pH of the solution (AS, 2015). Buffers are often used to maintain the solution
within the desired pH range.