Gradual demand of fossils fuels in the world affect the environment. Therefore, to eliminate these effect alternative of fossils fuels that is biofuel : ‘bioethanol’ will be used. These fuels are environmental friendly fuels. As they are obtained from biological biomass of agricultural waste and sugarcane bagasse.

Furthermore, these raw materials first wash with water then grind with mortar. Environmental condition i.e. temperature and pH will be kept at moderate rate.

So that the process is completed in a specific time. To isolate bio-ethanol we use bagasse as a raw material. For the conversion of sugar to ethanol we have bacteria Bacillus cereus and Zymomnas mobilis. They  have the ability to tolerate ethanol up to 16% and also to produce ethanol in 48 hours by adding alpha amylase and cellulase.

alternative fossils fuels biofuel bioethanol

As a natural phenomena when demand increases, ultimately to fulfill the demand; the supply is increased. To overcome the demand of fuels for the survival of life, Bioethanol production increased in most of countries of world. Brazil is the largest producer of bio-ethanol 17.5 billion liter per year. Fossils fuel sources coal, crude oil increase the greenhouse effects on environment. Also affect the ozone layer, depletion of
ozone layer causes chronic diseases i.e. cancer.

Bio-ethanol is  environmental friendly fuel and renewable energy. For the production of this environmental friendly fuel, we use biomass of sugarcane bagasse as a raw materials. Overall, has high level of lignocellulose that can easily converted by bacteria bacilluscereus and zymomonas to ethanol. These bacteria strain has ability to convert lignocellulose into ethanol within 48 hours.

Process of ethanol production completed in four step, pre-treatment, hydrolysis, fermentation and distillation. Each step has ability to increase the efficiency of bio-ethanol. We check the ethanol tolerance concentration with the help of mass chromatography and mass spectrometry. The tolerance of ethanol goes up to 16% with the help of different enzymes alpha amylase.

Literature review

To fulfill the demand of energy and removal of waste, ideally biological process plays a vital role. Bioethanol is one the product of this biological process all over the world. USA and Brazil both are on the top (6.4 billion liter) in the production of bioethanol.

Brazil use sugarcane bagasse for the isolation of bio-ethanol, while in USA it is produced from agriculture waste. For high yield,temperature and pressure of the fermenter will be balanced (51C, 80 bar) throughout the whole process. 

Further, enzyme concentration is also important. For maintenance of enzyme concentration pre-treatment of biomass done by alkali pre-treatment. Enzyme alpha amylase and glycoamylase helps in the breakdown of cellulose bond in sugar.

For the long life of enzyme in fermenter, pH is to be kept according to the bacteria species, each species have their own pH limits for their survival.

Bacteria’s role in ethanol production

Micro-organism is the important part of every biological process, so for ethanol production different bacteria are used like Sacchromyces Cervervesia ,Bacillus cereus and Zymomonas mobilis . Bacillus cereus strain GBPS9 which have high tolerance of ethanol. About 10 m inoculum used in the fermenter that do the process of fermentation within 48 hours. Zymomonas mobilis is the unique for the production of bioethanol, which are genetically modified by recombinant DNA technology.

Moreover have the ethanol tolerance higher than other bacteria. For checking the tolerance of ethanol mass chromatography techniques are used. For large scale production of bio-ethanol, the process consists of five steps grinding,cooking , fermentation , distillation and hydration.In these steps ; in every steps the yield improves.


A. Biomass Sugarcane Bagasse

We collect sugarcane bagasse as biomass from local juice point at Commercial market, Rawalpindi. Enzymes
from National Institute of Health, Islamabad.

B. Microorganisms (Bacteria)

For the ethanol production we need microorganisms, we isolate Bacillus cereus and Zymomonas mobilus from
NIH Islamabad. Which were obtained from cow shed air and soil. Then we culture these bacteria on nutrient agar.
So that we obtained colonies of these bacteria for our process.


There are four steps in process to produce ethanol in our experiment. Each steps has its own importance for ethanol production.


In pre-treatment, we wash bagasse with water so to cleanse soil, sand and dust from it. Then we grind bagasse with mortar and obtained powder form of bagasse. After grinding we add 5% H2O2 in 1000ml conical flask.


We add enzyme Alpha amylase and cellulose that helps in the breakdown of lager molecule of sugar and  lignocellulose into smaller pieces. We keep environmental factor i.e temperature, Humanity at moderate rate.


The innovative area of our research is fermentation. We add the inoculum of Bacillus cereus and Zymomonas mobilus isolated from culture media and inoculate in the fermenter. Where these bacteria ferment the sugar into ethanol. These bacteria tolerate ethanol concentration up to 6%.


The net step after fermentation is distillation. In this step we filter the fermented ethanol. That the residues of
ethanol eliminated and to get pure ethanol. Then these bioethanol is use in market instead of fossils fuels.


We have different parameter Time, temperature, pH and ethanol concentration tolerance. In the above two
experiment, the time in first experiment is 72 hours for the production of ethanol.Tempreture is about 60C and the
pH is 7.0. The tolerance is 12% Which shows that ethanol has 12% tolerance while fermentation. if it is low so we
will wait more than 72 hours while in second when we change their temperature from 60C to 37C, pH from 7.0 to 7.5.

So by changing these parameters ,the result totally changes, tolerance to ethanol concentration goes to 16 % and the production time from 72 hours decreases up to 48 hours. While in discussed paper this process is complete in more than 4 days and the the tolerance of that are up to 6 to 8%.

They used single strand of single bacteria in ethier research. From the above experiment we observe that, each parameter has their own importance, by adding enzymes and Ethanol tolerant Bacteria Zymomonas mobilis and Bacillus cereus we can decrease the production time and the ethanol tolerance.

Additionally ,we have done three more experiments in which we change the same parameter, temperature rises up to 80 C so time goes up to four days. Then we decreases temperature from 37 C so the result we obtained is that the process get stop due to decreases temperature. Same as temperature we change pH from 7.5 to 8 and the 6.0 so result different from each other.


We have done five experiments in which each have different result due to difference in temperature and pH. When we increase the temperature up to 80 and pH to 7.8 so the ethanol tolerance decrease up to 6%. So then we change pH to 7.5 and temperature 75 so then ethanol tolerance concentration increase to 10%. Ethanol production done in four days.

Similarly , then we observe that if we decrease temperature so by that tolerance increases. That are
inversely proportional to each other. Same as temperature pH also inversely proportional to tolerance concentration.
We keep temperature at 70 C and pH at 7.5 on that we obtained results that ethanol tolerance is 12%.

Result obtained in 72 hours. We change temperature to 37 C and pH to 7.0 then we obtained result in 48 hours of
16% tolerance of ethanol.we obtained 3.5 liter of ethanol from 500 gram of bagasse. along with that we obtained
byproduct that are not our need so we discard that. These are the laboratory results if we do this in industry we
increase the quantity of inoculum and enzyme. Additionally, for that we have to use more biomass in large quantity. We check tolerance of ethanol with mass spectrometry.


From this study we concluded that microorganisms strains differ from each other. For the ethanol production
if combine two different bacteria strain (Bacilius and Zymomnas) consequently we can get more yield than use of single bacteria. Also temperature and pH have their own importance for the production of bio-fuel. To obtained
results in short period (48 hours) of time we need to maintained all parameter.

As a result, by production of bio fuels we can prevent environment the disturbance which causes due to fossils fuels. From that disease causing agent like lead, CFC Choloro Fluoro Carbon. That are the main agent of skin cancer and other chronic diseases.

As a result , in  future that very help will full fill the demand of fuel all over the world. While in Pakistan it will be beneficial to overcome the large demand of petrol and diesel.

Furthermore, it will play a vital role in the economy. Which can isolate in cheap process.So that every one can offered for their vehicle. From that our country can do trade with other