Converting biomass into ethanol through fermentation

by
Leng Hong, Grazy
(0630400033)

A Final Year Project Thesis (ENV4071; 3 Credits) submitted in partial fulfillment of the requirements for the degree of

Bachelor of Science (Honours) in Environmental Science

at
BNU-HKBU
UNITED INTERNATIONAL COLLEGE Novembers , 2009

DECLARATION

I hereby declare that all the work done in this Project is of my independent effort. I also certify that I have never submitted the idea and product of this Project for academic or employment credits.

___________________ Leng hong, Grazy (0630400033)

Date: ___________________

Acknowledgements I am grateful to my project supervisor, Dr. C. F. Yu, Assistant Professor of the Environmental Science Program at UIC. He gave me guidance throughout the whole project. Also, I deeply appreciate Prof. Daniel Ruan, the head of the Department of Environmental Science in UIC, for his expert advice, instruction, and technical supports for the whole year.

Thanks are also attributed to Car Wu, Sunshine Chen ,the Laboratory Technician at UIC, who provided useful directions and helpful comments during the project.

Finally, I would like to give credits to all other laboratory technicians, for their valuable advice. And to all my classmates, who provided me with encouragement throughout the whole project.

___________________ Leng Hong, Grazy (0630400033)

Date: _______________

Abstract

Biomass is a common crop waste, especially corn and sugarcane are the main crops planted around the whole world, and large amount of crop waste is discarded or incinerated which cause severely air pollution. Converting biomass into ethanol as a renewable energy is the solution for it. China is the second largest corn producer and mainly produced in the northeast China, the third largest sugarcane producer which mainly distributed in the southern China. Therefore, this experiment mainly focus on these two crops and also compare different efficiency of microzyme, wein makbengj, microzyme on grape skin, liquor makbengj which those can easily perches in market, as well exam the ebullition efficiency of physical treatment. At the same time, using GC-MS to compares with alcoholmeter measurement to determine the accuracy of alcoholmeter. According to the experiment, simulating and ebullition increased ethanol fermatation efficiency, sugarcane performances better in alcohol production. Microzyme efficiency as follow: grape skin microzyme > liquor makbengj > wein makbengj. Alcoholmeter is reliable as the first step measurement.

Key words: biomass; ethanol; fermentation; microzyme;

Content
Declaration …………………………………………………………………………...2
Acknowledgements…………………………………………………………………...3
Abstract …………………………………………………………………………........4
1. Introduction ……………………………………………………………………….6 1.1 Background information of biomass 1.2 Background information of bioethanol 1.3. Chemical of converting biomass to ethanol 1.4. Sources of ethanol 1.5 Environmental impact
2. Objectives ………………………………………………………………………...21
3. Materials and chemical reagents ……………………………………………….22
4. Methodology ……………………………………………………………………..26 4.1 Microzyme activation 4.2 Grape fermentation add with liquor makbengj and wein makbengj 4.3 Niblet, cornstalk, leaf maize add with liquor makbengj
5. Result and discussion …………………………………………………………..31 5.1 Grape fermentation add with liquor makbengj and wein makbengj 5.2 Sugarcane tegument, bagasse, cornstalk, nibel, grape add with cellulose enzyme and wein makbengj. 5.3 GC-MS detection
6. Conclusion ………………………………………………………………………..41
7. Discussion ………………………………………………………………………...42

1. Introduction 1.1 Background information of biomass Biomass, a renewable energy source, is biological material derived from living, or recently living organisms, Manure, garden waste and crop residues are all sources of biomass (L.P. Abrahamson 2000). It is a renewable energy source based on the carbon cycle, which has stored sunlight in the form of chemical energy. As a fuel it may include wood, wood waste, straw, manure, sugar cane, and many other byproducts from a variety of agricultural processes.

There are agricultural products being grown for biofuel production. These include corn, switchgrass, and soybeans, primarily in the United States; rapeseed, wheat and sugar beet primarily in Europe; sugar cane in Brazil; palm oil and miscanthus in Southeast Asia; sorghum and cassava in China; and jatropha in India. Hemp has also been proven to work as a biofuel. Biodegradable outputs from industry, agriculture, forestry and households can be used for biofuel production, using e.g. anaerobic digestion to produce biogas, gasification to produce syngas or by direct combustion. Examples of biodegradable wastes include straw, timber, manure, rice husks, sewage, and food waste. The use of biomass fuels can therefore contribute to waste management as well as fuel security and help to prevent or slow down climate change, although alone they are not a comprehensive solution to these problems.

Biomass is carbon based and is composed of a mixture of organic molecules containing hydrogen, usually including atoms of oxygen, often nitrogen and also small quantities of other atoms, including alkali, alkaline earth and heavy metals. These metals are often found in functional molecules such as the porphyrins which include chlorophyll which contains magnesium.

Biomass energy is derived from three distinct energy sources: wood, waste, and alcohol fuels. Wood energy is derived both from direct use of harvested wood as a fuel and from wood waste streams. The largest source of energy from wood is pulping liquor or “black liquor,” a waste product from processes of the pulp, paper and paperboard industry. Waste energy is the second-largest source of biomass energy. The main contributors of waste energy are municipal solid waste (MSW), manufacturing waste, and landfill gas. Biomass alcohol fuel, or ethanol, is derived almost exclusively from corn. Its principal use is as an oxygenate in gasoline. (EIA 2008)

Biomass can be converted to other usable forms of energy like methane gas or transportation fuels like ethanol and biodiesel. Methane gas is the main ingredient of natural gas. Smelly stuff, like rotting garbage, and agricultural and human waste, release methane gas - also called "landfill gas" or "biogas." Crops like corn and sugar cane can be fermented to produce the transportation fuel, ethanol. Biodiesel, another transportation fuel, can be produced from left-over food products like vegetable oils and animal fats. (EIA 2008)

1.2 Background information of bioethanol Bioethanol is the most common biofuel worldwide,. Alcohol fuels are produced by fermentation of sugars derived from wheat, corn, sugar beets, sugar cane, molasses and any sugar or starch from that alcoholic beverages can be made (like potato and fruit waste, etc.). The ethanol production methods used are enzyme digestion (to release sugars from stored starches), fermentation of the sugars, distillation and drying. The distillation process requires significant energy input for heat (often unsustainable natural gas fossil fuel, but cellulosic biomass such as bagasse, the waste left after sugar cane is pressed to extract its juice, can also be used more sustainably)

Ethanol can be used in petrol engines as a replacement for gasoline; it can be mixed with gasoline to any percentage. Most existing car petrol engines can run on blends of up to 15% bioethanol with petroleum/gasoline. Ethanol has a smaller energy density than gasoline, which means it takes more fuel (volume and mass) to produce the same amount of work. An advantage of ethanol (CH3CH2OH) is that it has a higher octane rating than ethanol-free gasoline available at roadside gas stations which allows an increase of an engine's compression ratio for increased thermal efficiency. In high altitude (thin air) locations, some states mandate a mix of gasoline and ethanol as a winter oxidizer to reduce atmospheric pollution emissions.

Ethanol is also used to fuel bio ethanol fireplaces. As they do not require a chimney and are "flueless", bio ethanol fires (bio-ethanol fireplace 2009) are extremely useful for new build homes and apartments without a flue. The downside to these fireplaces is that the heat output is slightly less than electric and gas fires.

In the current alcohol-from-corn production model in the United States, considering the total energy consumed by farm equipment, cultivation, planting, fertilizers, pesticides, herbicides, and fungicides made from petroleum, irrigation systems, harvesting, transport of feedstock to processing plants, fermentation, distillation, drying, transport to fuel terminals and retail pumps, and lower ethanol fuel energy content, the net energy content value added and delivered to consumers is very small. And, the net benefit (all things considered) does little to reduce un-sustainable imported oil and fossil fuels required to produce the ethanol. (Andrew Bounds 10/09/2007).

Although ethanol-from-corn and other food stocks has implications both in terms of world food prices and limited, yet positive energy yield (in terms of energy delivered to customer/fossil fuels used), the technology has lead to the development of cellulosic ethanol. According to a joint research agenda conducted through the U.S. Department of Energy, the fossil energy ratios (FER) for cellulosic ethanol, corn ethanol, and gasoline are 10.3, 1.36, and 0.81, respectively. (Brinkman, N. et al 2005) (Farrell, A.E. et al. 2006) (Hammerschlag, R. 2006)
1.3. Chemical of converting biomass to ethanol
[pic]
Fig1.3.1. Ethanol 3d stick structure
Glucose (a simple sugar) is created in the plant by photosynthesis. 6 CO2 + 6 H2O + light → C6H12O6 + 6 O2 During ethanol fermentation, glucose is decomposed into ethanol and carbon dioxide. C6H12O6 → 2 C2H5OH+ 2 CO2 + heat During combustion ethanol reacts with oxygen to produce carbon dioxide, water, and heat: C2H5OH + 3 O2 → 2 CO2 + 3 H2O + heat After doubling the combustion reaction because two molecules of ethanol are produced for each glucose molecule, and adding all three reactions together, there are equal numbers of each type of molecule on each side of the equation, and the net reaction for the overall production and consumption of ethanol is just: light → heat The heat of the combustion of ethanol is used to drive the piston in the engine by expanding heated gases. It can be said that sunlight is used to run the engine.

Glucose itself is not the only substance in the plant that is fermented. The simple sugar fructose also undergoes fermentation. Three other compounds in the plant can be fermented after breaking them up by hydrolysis into the glucose or fructose molecules that compose them. Starch and cellulose are molecules that are strings of glucose molecules, and sucrose (ordinary table sugar) is a molecule of glucose bonded to a molecule of fructose. The energy to create fructose in the plant ultimately comes from the metabolism of glucose created by photosynthesis, and so sunlight also provides the energy generated by the fermentation of these other molecules.

1.4. Sources of ethanol Ethanol is a renewable energy source because the energy is generated by using a resource, sunlight, which is naturally replenished. Creation of ethanol starts with photosynthesis causing a feedstock, such as sugar cane or corn, to grow. These feedstocks are processed into ethanol.

About 5% of the ethanol produced in the world in 2003 was actually a petroleum product. It is made by the catalytic hydration of ethylene with sulfuric acid as the catalyst. It can also be obtained via ethylene or acetylene, from calcium carbide, coal, oil gas, and other sources. Two million tons of petroleum-derived ethanol are produced annually. The principal suppliers are plants in the United States, Europe, and South Africa. Petroleum derived ethanol (synthetic ethanol) is chemically identical to bio-ethanol and can be differentiated only by radiocarbon dating. (Murry Tamers 2006)

Bio-ethanol is usually obtained from the conversion of carbon based feedstock. Agricultural feedstocks are considered renewable because they get energy from the sun using photosynthesis, provided that all minerals required for growth (such as nitrogen and phosphorus) are returned to the land. Ethanol can be produced from a variety of feedstocks such as sugar cane, bagasse, miscanthus, sugar beet, sorghum, grain sorghum, switchgrass, barley, hemp, kenaf, potatoes, sweet potatoes, cassava, sunflower, fruit, molasses, corn, stover, grain, wheat, straw, cotton, other biomass, as well as many types of cellulose waste and harvestings, whichever has the best well-to-wheel assessment.

An alternative process to produce bio-ethanol from algae is being developed by the company Algenol. Rather than grow algae and then harvest and ferment it the algae grow in sunlight and produce ethanol directly which is removed without killing the algae. It is claimed the process can produce 6000 gallons per acre per year compared with 400 gallons for corn production. (Martin LaMonica 2008)

Currently, the first generation processes for the production of ethanol from corn use only a small part of the corn plant: the corn kernels are taken from the corn plant and only the starch, which represents about 50% of the dry kernel mass, is transformed into ethanol. Two types of second generation processes are under development. The first type uses enzymes and yeast to convert the plant cellulose into ethanol while the second type uses pyrolysis to convert the whole plant to either a liquid bio-oil or a syngas. Second generation processes can also be used with plants such as grasses, wood or agricultural waste material such as straw.

As ethanol yields improve or different feedstocks are introduced, ethanol production may become more economically feasible in the US. Currently, research on improving ethanol yields from each unit of corn is underway using biotechnology. Also, as long as oil prices remain high, the economical use of other feedstocks, such as cellulose, become viable. By-products such as straw or wood chips can be converted to ethanol. Fast growing species like switchgrass can be grown on land not suitable for other cash crops and yield high levels of ethanol per unit area. (Green Dreams 2007) (Table 1.1 )

Table 1.1 Efficiency of common crops

1.5 Environmental impact All biomass goes through at least some of these steps: it needs to be grown, collected, dried, fermented, and burned. All of these steps require resources and an infrastructure. The total amount of energy input into the process compared to the energy released by burning the resulting ethanol fuel is known as the energy balance (or "Net energy gain"). Table 1.2 compiled in a 2007 by National Geographic Magazine point to modest results for corn ethanol produced in the US: one unit of fossil-fuel energy is required to create 1.3 energy units from the resulting ethanol. The energy balance for sugarcane ethanol produced in Brazil is more favorable, 1:8. Energy balance estimates are not easily produced, thus numerous such reports have been generated that are contradictory. For instance, a separate survey reports that production of ethanol from sugarcane, which requires a tropical climate to grow productively, returns from 8 to 9 units of energy for each unit expended, as compared to corn which only returns about 1.34 units of fuel energy for each unit of energy expended. (Table1.2)

Carbon dioxide, a greenhouse gas, is emitted during fermentation and combustion. However, this is canceled out by the greater uptake of carbon dioxide by the plants as they grow to produce the biomass. When compared to gasoline, depending on the production method, ethanol releases less greenhouse gases.

Compared with conventional unleaded gasoline, ethanol is a particulate-free burning fuel source that combusts with oxygen to form carbon dioxide, water and aldehydes. Gasoline produces 2.44 [[CO2 equivalent]] kg/l and ethanol 1.94 (this is -21% CO2) The Clean Air Act requires the addition of oxygenates to reduce carbon monoxide emissions in the United States. The additive MTBE is currently being phased out due to ground water contamination; hence ethanol becomes an attractive alternative additive. Current production methods include air pollution from the manufacturer of macronutrient fertilizers such as ammonia. (M. Wang 2009)

A study by atmospheric scientists at Stanford University found that E85 fuel would increase the risk of air pollution deaths relative to gasoline by 9% in Los Angeles, USA: a very large, urban, car-based metropolis that is a worst case scenario (San Francisco Chronicle 2009).Ozone levels are significantly increased, thereby increasing photochemical smog and aggravating medical problems such as asthma. (M. Z. Jacobson 2007-03-14).

[pic]
Table 1.2 Energy balance or Net energy gain

2. Objectives In this project, the basic biomass materials were cornstalk and bagasse which were the most common crops from each high and low attitude. The production of ethanol fermentation produced from different biomass (materials) with the addition of different amount enzymes would be studied.

This project was mainly divided into three parts,
1. In the first part, two types of man-made microzyme were activated and added into two fermentation bottles with the same amount of grape then placed the same bottle without adding any mircozyme as the control.
2. In the second part, man-made yeast were added into three bottles of materials, niblet, cornstalk, leaf maize for fermentation.
3. In the final part, the production of ethanol produced from each types of biomass by each mircozymes in studied.

3. Materials and chemical reagents
1. Sugarcane tegument bagasse which separated from the sugarcane lived in Hainan province. (Fig 3.1.1)Cornstalk and nibel came from the local corn field. Jufeng grape came from the local grape garden. (Fig 3.1.2, Fig 3.1.3)

Fig 3.1.1 bagasse and sugarcane tegument

Fig 3.1.2 cornstalk and nibel

Fig 3.1.3 Jufeng grape
2. Fermentation bottle with two different kinds of air locker

Fig 3.2.1 Fermentation bottle as reaction container

Fig 3.2.2 Two kinds of air locker for keeping bacteria in air away from inside bottle
3. Alcoholmeter

Fig 3.3.1 Alcoholmeter for alcohol strength measurement
4. Liquor makbengj, wein makbengj and cellulose enzyme

Fig 3.4.1 Liquor makbengj, wein makbengj and cellulose enzyme for sample treatment
5. Granulated sugar

Fig 3.5.1 granulated sugar as microzyme activator
6. One disintegrator for material comminuting and one experiment setup for condensation

Fig4.6.1 disintegrator for material comminuting &experiment setup for condensation

4. Methodology Arranged six brown fermentation bottles which all made of glass, and each bottle had an air locker at the top from keeping air flowing inside the bottle but the inside-bottle air could come out.

Crumbled each fermentation materials into the smallest pieces, and then filled each material in its corresponding bottle at about 3/4. After putting activated microzymes into fermentation bottle shake bottle so that microzyme could locate equably.

Designed one distillation equipment which made of four condensers, one centigrade palace at the top condenser, one heater and a collection bottle. It was used to separate ethanol from water since ethanol has a lower boiling point (78°C) than water (100°C) so at 78°C ethanol will vaporize and condense first.

4.1 Microzyme activation There were three kinds of microzyme which are used in the experiment: liquor makbengj, wein makbengj and cellulose enzyme. Before adding them into fermentation bottle an activation was needed.

Liquor makbengj activation: prepare a 38°C 2% sugar concentration solution for dilution. Activation will be finished in about 1 hours after solution temperature below 34°C

Wein makbengj activation: addition 1kg into 38°C 5%sugar solution. Keep stirring until all the microzyme dissolved. Activation will be finished in about 17 min and cooled after solution desperation at 30°C.

Cellulose enzyme activation: prepare a solution with a 48°C temperature and a pH at 5 for dissolution. After injection the temperature should maintain at 48°C for at least 40h.

4.2 Grape fermentation with liquor makbengj and wein makbengj added Flushed grape with tap water, be ware of not washing way white yeast colony on the grape skin, crushed every single grape to make a separation between skin and flesh to accelerate the fermentation duration. Then filled all the three fermentation bottles with 3000g grape for each, bottle1 was set up as control without putting any microzyme; 3.0g (0.1%*3000g) activated liquor makbengj and activated wein makbengj were put in to bottle 2 and bottle 3, the concentration of microzyme was 0.1% by weight. The total fermentation duration was 8 days, every 3 days there was a sugar addition, and the addition ratio was 1/20 of total weight so each time 150g sugar was added and the total amount added was 300g.

After fermentation a filtration is needed to extract liquid from solid-liquid mixed solution. Connect filtration bottle with a sucker to accelerate filtration speed.

Final step was to condense, by getting filtrated solution through the distillation equipment.

4.3 Niblet, cornstalk, leaf maize with liquor makbengj added Separated corn into niblet, cornstalk and leaf maize, comminuted each materials by disintegrator in about 0.5cm diameter. Took 500g of each material into a 1L beaker for a 30min boiling, meanwhile maintain pH at 5.0. Put 0.5g(500g*0.1%) activated liquor makbengj in each material and well stirred.

After fermentation a filtration is needed to extract liquid from solid-liquid mixed solution. Connect filtration bottle with a sucker to accelerate filtration speed.

Final step was to condense, by getting filtrated solution through the distillation equipment.

4.4 Sugarcane tegument, bagasse, cornstalk, nibel, grape add with cellulose enzyme and wein makbengj. Decorticated sugarcane into tegument and core, also separated corn in nibel, cornstalk, then comminuted each by disintegrator into smallest chipping. Prepared six fermentation bottles label them as bottle No.1 No.2 No.3 till No.6, the addition of each bottle seen table 4.4.1 After fermentation a filtration is needed to extract liquid from solid-liquid mixed solution. Connect filtration bottle with a sucker to accelerate filtration speed. Final step was to condense, by getting filtrated solution through the distillation equipment.

|Material |Bottle weight |Total weight |Alcohol enzyme |Cellulose enzyme |
|Bagasse |1375g |2950g |500g grape skin |15.75g cellulose |
| | | | |enzyme |
|Bagasse |1375g |2950g |1.5g wein makbeng |15.75g cellulose |
| | | | |enzyme |
|Sugarcane tegument |1375g |2450g |1.07g wein makbeng |10.75g cellulose |
| | | | |enzyme |
|Cornstalk |1375g |3600g |2.25g wein makbeng |22.25g cellulose |
| | | | |enzyme |
|Cornstalk (boiled) |1375g |4000g |2.65g wein makbeng |26.55g cellulose |
| | | | |enzyme |
|nibel |1525g |2775g |/ |/ |

Table 4.4.1 bottle weight, total weight and enzyme weight added
5. Result 5.1 Grape fermentation add with liquor makbengj and wein makbengj
|Materials |Materials weight |Microzyme weight |Volume after |Alcohol strength |
| | | |distillation | |
|Grape(without microzyme)|300g |/ |71ml |71.1 |
|Grape(4.0g liquor |300g |4.0g |17ml |36.7 |
|makbenj addition) | | | | |
|Grape(4.0 wein makbengj |300g |4.0g |72ml |3.1 |
|addition) | | | | |

table-5.1.1ethanoll production after distillation

|Materials |Alcohol strength reading|Alcohol strength |Total ethanol (ml) |Ethanol production per |
| | |calculate | |100g |
|Grape(without microzyme)|73 |71.1 |71 |1.68ml |
|Grape(4.0g liquor |40 |37.6 |17 |0.21ml |
|makbenj addition) | | | | |
|Grape(4.0 wein makbengj |4 |3.1 |72 |0.074ml |
|addition) | | | | |

Table-5.1.2 Grape ethanol production ratio

[pic]
Fig-5.1.3 Grape ethanol production ratio

According to the Table 5.1.1 and Fig 5.1.2, we saw the grape which had no microzyme produced significant much more alcohol than the other two which had microzyme added. Therefore the fig-5.1.2 showed between two microzyme addition grape liquor makbengj appeared better ethanol production ability than wein makbenj.

Therefore, the comparison of ability of ethanol production among 1 natural enzyme and two man-made yeast were following: Natural > liquor makbengj > wein makbengj

5.2 Sugarcane tegument, bagasse, cornstalk, nibel, grape add with cellulose enzyme and wein makbengj.
|Material |Bottle weight |Total weight |Volume after distillation |
|Bagasse |1375g |2950g |700ml |
|Bagasse |1375g |2950g |422ml |
|Sugarcane tegument |1375g |2450g |408ml |
|Cornstalk |1375g |3600g |516.5ml |
|Cornstalk (boiled) |1375g |4000g |231ml |
|nibel |1525g |2775g |92ml |

Table 5.2.1 bottle weight and total weight

|Material |Grape skin |Wein makbengj |Cellulose enzyme |ferv |
|Bagasse |500g |/ |15.75g cellulose enzyme |/ |
|Bagasse |/ |1.5g wein makbengj |15.75g cellulose enzyme |/ |
|Sugarcane tegument |/ |1.5g wein makbengj |10.75g cellulose enzyme |/ |
|Cornstalk |/ |2.25g wein makbengj |22.25g cellulose enzyme |/ |
|Cornstalk (boiled) |/ |2.65g wein makbengj |26.25g cellulose enzyme |yes |
|nibel |/ |/ |/ |/ |

Table 5.2.2 raw materials treatment and addition

|Material |Alcohol strength |Alcohol strength |Total ethanol (ml) |Ethanol production per |
| | |calculated | |100g |
|Bagasse |9 |8.1 |56.7 |3.47ml |
|Bagasse |9 |8.1 |34.18 |1.85ml |
|Sugarcane tegument |2 |1.3 |5.3 |0.49ml |
|Cornstalk |1 |0.1 |0.517 |0.026ml |
|Cornstalk (boiled) |5 |4.2 |9.7 |0.37ml |
|nibel |9 |7.9 |7.27 |0.58ml |

Table 5.2.3 total ethanol volume & ethanol production ratio (every 100g)

Fig 5.2.4 total ethanol volume & ethanol production ratio (every 100g)

The ethanol production ratio was calculated by the formula below:

Ethanol production ratio= (volume of distillation * alcoholstrength calculated) (Total weight-bottle weight)/100g

Table 5.2.2 illustrated four different materials which are classified into six groups by different treatment and enzyme addition. Bottle No.1 and No.2 had the same raw material bagasse while bottle No.1 had a 500g grape addition instated of bottle No.2 wein makbengj addition. Meanwhile bottle No.4 and No.5 had a same enzyme addition but a different raw material treatment, bottle No.4 was not boiled but No.4 was.

There were many comparison set up within those six samples, according to table5.2.3 bottle No1 and No2 had the same raw material, the only different was No.1 added grape instead of wein makbengj added in No.2. Then the result of ethanol production per 100g material bottle No.1 was 3.47ml, however this result should minus the influence of 500g grape addition [(56.7-1.68*5)/15.75=3.07ml] so bottle No.1 was 3.07ml > bottle No.2 1.85ml, bagsse used grape as ethanol enzyme had a better performance.

Compared bottle No.4 and No.5 they both had a same material and enzyme addition only bottle No.5 sample was boiled before fermentation. Therefore the result of each ethanol production ratio had a significant different. Bottle No.4 was 0.026ml < No.5 0.37ml.

Bottle No.2, No.3 and No.4 were a comparison of different materials at the same treatment and enzyme addition. According to table5.2.3 bagasse ethanol production ratio was 1.85ml sugarcane tegument was 0.49ml and cornstalk was 0.026ml.

Bottle No.6 was set up as control without any enzyme addition the only treatment is comminuting by disintegrator. At final the ethanol production ratio is 0.58ml.

Therefore, the ethanol production ability in material was following: bagasse > sugarcane tegument > cornstalk. The ethanol production ability in enzyme addition was following: grape skin > liquor makbengj >wein makbenj . Ebullition and sample size are the main factor that can be economic controlled that affect ethanol production.

5.3 GC-MS detection In order to determine all results were contain ethanol as the main solvent; GC-MS had been used to make a contract with the previous results.

5.3.1 Grape fermentation with addition of liquor makbengj and wein makbengj
Fig 5.3.1 grape (without microzyme)

Fig 5.3.2 grape (liquor makbengj)

Fig 5.3.3 grape (liquor makbengj)

According to Fig 5.3.1, Fig 5.3.2, Fig 5.3.3, each fig had two chart combined, on the left hand side was the turtosis of component in each sample, there were two peaks, first peak time around 1 to 1.1 min was water, the second peak time around 1.1 to 1.2 min represented ethanol (ethyl alcohol), different peak height indicated different ethanol concentration in each sample, the higher peak the higher concentration. The chart on the right hand side was the comparison between sample peak and library control data to determine whether the peak represent ethanol.

Considering those three fig above, according to each comparison chart on the right, every sample second peak on the left chart was all represent ethanol. Therefore, the ethanol peak height by those charts was: grape skin > liquor makbengj >wein makbenj. So it was the same results as alcoholmeter measured.

5.3.2 Sugarcane tegument, bagasse, cornstalk, nibel, grape add with cellulose enzyme and wein makbengj.

Fig 5.2.1 bagasse (grape)

Fig 5.2.2 bagasse (wein makbengj)

Fig 5.2.3 sugarcane tegument

Fig 5.2.4 cornstalk

Fig 5.2.5 cornstalk (boiled)

Fig 5.2.6 nibel Combined all above six figures together, comparison charts on the right all indicated the main solvent of each was ethanol (ethyl alcohol). Meanwhile, ethanol peak height by those charts was: bagasse > sugarcane tegument > cornstalk, ebullition > none ebullition, natural grape skin> wein makbengj. The results were the same as alcoholmeter measured before wards.

6. Conclusion Convert biomass to ethanol is one of the solutions to replace petroleum as gasoline. Choosing the plant that is wildly separated and easily growing is very important. China is the second largest corn producer and mainly produced in the northeast China, China also is the third largest sugarcane producer which mainly distributed in the southern China. So this experiment chose corn and sugarcane as the target materials. In spite of the different raw material has a great impact to ethanol production there are many other effects can affect it. By experiment, the size of material, smaller the size is more ethanol gained, because of microzyme works more efficiently with a small particle, a fiber bonding broken down of material, meanwhile, ebullition also lead to break down fiber bonding so that fermentation can be more sufficiency.

Secondly, different enzyme play a very important role in fermentation efficiency, the result of ethanol production ratio reflected the enzyme on grape skin showing a better performance among wein makbengj and liquor makbengj.

Finally, using sugarcane biomass produces more ethanol than the same weight of corn biomass, in order to increasing ethanol production simulating and ebullition is a very economic treatment, even though enzyme on grape skin perform better than wein makbengj, for mass converting biomass to ethanol production, wein mankbengj has the advantage of being large amount produced and long time store, wein makbengj will be more utilized.

GC-MS results indicated the same results as alcoholmeter measured, although GC-MS is more accuracy and precision method for alcohol determine than alcoholmeter, however alcoholmeter faster, much cheaper and convenience factors make it a very useful as first stage measurement.
7. Discussion At the second part of this experiment which man-made yeast were added into three bottles of materials, niblet, cornstalk, leaf maize for fermentation. However, this part of experiment ends up with no result. There might be three main results: first, in order to maintain pH at 5.0 while ebullition, 10ml nitric acid was added into, this nitric acid may kill the microzyme afterwards. Second, after cornstalk have been comminuted I leave comminuted corntalk and niblet and corn maize for 24 hours in sealed bottle without any treatment, that may lead many other microorganism growing during this period, therefore, these microorganisms are very strong competitor and depress the growth of microzyme. Third, the amount of raw matrial (500g) is too small even ethanol was produced, combine those two factors above, it is very possible too little to be detected.

At the third part, unlike grape in first part experiment, raw materials are solid and hard to be comminuted, so the particle size appears as one of the main factors that affect fermentation. Without disintegrated, hardness for microzyme interacts with raw materials when they are bonded together. Since the equipment limitation, disintegrated particle size can not reach my anticipation, which leads to the entire results are not optimistic; however, this not affects comparison results.

Reference
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