senior thesis final product

Acetone, Butanol, and Ethanol Production from

feedstock Using Clostridium beijerinckii NCIMB 8052

Abstract: In this study acetone, butanol and ethanol (ABE) production was

attempted from Corn Stover in small cultures using Clostridium Beijerinckii NCIMB

8052. Prior experiments using C. Beijerinckii have yielded ABE solvents in a range of

14.6g/L to approximately 165.1g/L (conducted in fed batch cultures); With about

60% of solution being butanol. From this study, we successfully produced n-butanol,

and ethanol from corn stover fermentation. We produced approximately 4mL of

butanol and 1mL of ethanol from a 1L (86g) corn stover solution. We had a

productivity of about 3.37% (m/m) n-butanol. Through Gas chromatography and

Infrared Spectroscopy, we were able to identify and confirm the presence of butanol

and ethanol from fermentation. We successfully increased productivity have a

solvent yield in a 4:1 ration- 80% butanol and 20% ethanol. This is a 25% increase

from prior ABE fermentation with a solvent yield ration 3:6:1 with approximately

60% butanol in total solution.

Dylan LaFerriereProfessor BrunetIndependent Research 26 April 2016

Acetone, Butanol, and Ethanol Production 1

Introduction

Acetone-butanol-ethanol (ABE) fermentation is the second most commonly

fermentation process, right behind ethanol production from potatoes (Jones &

Woods, 1986). In the early part of the 20th century, Chemist Chaim Weizmann

discovered that using a particular bacterial strain of Clostridium to digest starch had

produced three organic compounds; acetone, butanol and ethanol. Acetone at this

time was used to make cordite, a low explosive substitute for gunpowder in World

War I (Granstrom, 2010). Acetone was used to reduce the muzzle blast from

automatic weapons used in the war. This demand for acetone allowed for the

Weizmann/ABE process to be economically viable and commercially used. Ethanol

was also used for a good source of fuel, for it had a high energy content close to that

of gasoline.

The ABE process however, does not yield large quantities of acetone-butanol-

ethanol. The fermentation process produces the ABE solvents in a 3:6:1 ratio being

3 parts acetone, 6 parts butanol and 1-part ethanol (V.V Zverlov, 2006). In the mid

1900s, it was discovered that butanol could be produced petrochemically (Qureshi,

2001). Simply, propylene is reacted with an aldehyde-specifically formaldehyde-to

produce butyraldehyde, which is then reduced to n-butanol. However, this process

requires petroleum oil for the production of ABE solvents. In the past 30 years, oil

prices have been increasing dramatically. The increase in oil prices in directly

proportional to the cost of butanol production from a petroleum based process. The

fermentation process for producing butanol eliminates the need for petroleum

because it uses carbohydrates as a medium for alcohol production.


In recent years, extensive research in regards to the ABE process has been

conducted. It is well known that starch is a major component of agricultural crops

(Qureshi, 2001). Starch can be digested by enzymes into sugars such as glucose.

These sugars can then be fermented into acetone, butanol and ethanol by using

strains of clostridia (Formanek, 1997).

Early Experiments

Previous studies regarding butanol production when using clostridia have

focused on yield from starch, starch based materials, agricultural waste and corn. All

of these substrates contain either starch, or glucose. Each of which are capable of

being fermented into ABE solvents.

However, many factors play part in the efficiency of butanol fermentation.

Initial use of the ABE process showed small product yield, low concentration of

products and productivity. Along with this, the cost of substrates (glucose, starch,

agricultural crops) hadn’t been cost effective. In other words, it took more money to

produce the butanol than a company made from distributing it.

In recent years, the interest in butanol fermentation has increased;

experiments have been conducted to find ways to increase ABE production, yield

and productivity all while maintaining a low substrate cost.

Scientist have a strong interest in starch, starch based materials and biomass,

for they all contain fermentable carbohydrates. Along with that, they have also

shown interest in finding other strains of clostridia that may be able to yield greater

values of solvents. Frederic Monot, a French scientist conducted experiments for the

production of acetone and butanol by clostridium acetobutylicum ATCC (American


Type Culture Collection) 824 in different mediums (1982). This experiment tested

the yield of ABE due to its interactions with different mediums, all of which

contained a glucose substrate. His results showed that when in the presence of

magnesium sulfate, cell growth of the clostridium had increased greatly along with

product concentration (approximately 3.23g/L solution). Increased concentrations

of potassium chloride, Iron Sulfate, ammonium acetate and glucose all increased

product concentration (6g/L, 5.5g/L, 4.22g/L, 20.44g/L respectively) and cell

growth.

With the knowledge of the increased product concentration from different

mediums, a new medium containing all tested solutions was established for

maximum product yield. It was suggested that a medium- 50-200mg/L Magnesium

Sulfate, 0.015g/L Potassium chloride, 1-50mg/L Iron Sulfate, 1-2g/L ammonium

acetate, and 20 to 60g/L glucose- be used for optimal solvent production (Monot,

1982). Monot's focus was mainly on the ABE solvent production from glucose.

Recently agricultural waste, starch based materials and other sugars are

more desirable substrates than petroleum in further investigations. Corn fiber

arabinoxylan (CFAX) is a hemicellulose commonly found in cell walls of plants. As it

is composed of sugars- glucose, xylose, galactose, and arabinose (a monosaccharide)

all of which can be fermented into butanol using clostridium. Nashib Qureshi tested

the production of acetone, butanol and acetone from corn fiber xylan using

clostridium acetobutylicum P260 (2006). Corn fiber is a byproduct of corn that is

typically used to feed farm animals. The unique characteristic of corn fiber is that it

contains sixty to seventy percent hydrocarbons, of which thirty is hemicellulose.


Along with that, the xylan fiber-found in corn fiber- molecule contains chains of

sugars such as glucose, xylose, galactose and arabinose suggesting that it is a

suitable substrate for fermentation (Qureshi, 2006). Clostridium acetobutylicum

lacks the enzymes required for hydrolyzing starches.

A hydrolyzing enzyme breaks down starches and carbohydrates into sugars,

which then can be fermented into the ABE solvents by Clostridia (Qureshi, 2006).

Prior to the fermentation of the corn fiber, a control test was conducted on each of

the sugars present in xylan to test if they could be fermented into butanol. Total ABE

solvent production from glucose, xylose, galactose, and arabinose after 72 hours of

fermentation was 21.37g/L, 11.12g/L, 10.09g/L, and 15.8g/L respectively. In

comparison to the study conducted by Monot (production of ABE solvents from

glucose using clostridium acetobutylicum ATCC 824), there is a clear increase in

ABE production. Monot's results showed a maximum ABE production of 20.44g/L

when using glucose as a substrate. However, Qureshi had collected a maximum ABE

production of 21.37g/L with a glucose substrate. Both experiments yielded very

similar solvent concentrations, thus further supporting the fermentation of butanol

from glucose using strains of clostridium acetobutylicum

After fermentation of xylan substrate, there were no results collected, and no

production of ABE solvents. This suggested the xylanase enzyme had not hydrolyzed

CFAX fully, and the bacteria died of starvation. In order to fix this, xylose was added

to the culture so the bacteria would survive until the production of sugars.

Following, a second trial was run in which data was collected. Results show that the

fermentation yielded a total ABE solvent concentration of 24.67g/L, with a product


yield of 0.44, and a productivity of .47g/L*h. In comparison to the control test using

xylan as a substrate, productivity increased by 15%. This is a result of all of the

sugars from xylan being consumed by the Clostridia into ABE solvents (Qureshi,

2006). This evidence further proves increasing levels of production of acetone,

butanol and ethanol solvents through fermentation using clostridium.

Researchers have looked into ABE production using different strains of

clostridium. In previous studies, clostridium acetobutylicum was a common species

of clostridium used in the fermentation of butanol. However, small solvent yields

have discouraged the use of the ABE process with C. acetobutylicum. Recently a new

strain of bacteria has been used, clostridium beijerinckii. Unlike C. acetobutylicum,

this strain of clostridium contains amylase enzymes, this type of enzyme is

responsible for hydrolyzing starches and carbohydrates into sugars and acids,

which then the bacteria can use to ferment into butanol (Formanek, 1997).

Typically, the hydrolysis of starch in the fermentation process is a rate-limiting step

where the fermentation cannot begin until the substrate is hydrolyzed by an

enzyme. In this case, that step is done simultaneously with the fermentation

process. This factor makes clostridium beijerinckii a more ideal culture to use.

Joseph Formanek tested this strain of bacteria in the production of butanol using

6% glucose and maltodextrin as substrates. Clostridium beijerinckii is found as a

wild type referred to as NCIMB 8052. However, research has shown that a mutant

version, clostridium beijerinckii BA101, has a much higher concentration of amylase

enzymes, making it a better culture to use in the fermentation process.


Results from Formanek’s tests show that when using a 6% glucose substrate,

total solvent production from c. beijerinckii was 27.5g/L, of which 18.6g/L was

butanol. In comparison, the wild type strain NCIMB 8052 had yielded a total ABE

solvent concentration of 14.5g/L suggesting the mutant strain BA101 more readily,

and efficiently produces butanol from glucose. When BA101 was tested in a 6%

maltodextrin substrate, total ABE solvent production was measured at 26.1g/L, of

which 18.6g/L was butanol (Formanek, 1997). This evidence helps suggest that

clostridium beijerinckii has a greater solvent production than that of its wild type

strain and in comparison to c. acetobutylicum.

In a similar study conducted in 2001, Nashib Qureshi used the mutant strain

C. beijerinckii to digest starch and produce acetone, butanol and ethanol. His results

had shown that when BA101 was grown on starch in culture, the amylase enzymes

shown an increase in production which further leads to an increase in production

yield. His experiment tested large batch cultures, containing large amounts of sugar

concentrations. When fermentation took place in a batch culture, it produced

51.5g/L of ABE solvents. When fermentation took place in a fed batch culture (for

industrial purposes), it produced a solvent concentration of 165.1g/L. Along with

that, productivity increased from 0.35g/L*h to 0.98g/L*h (Qureshi, 2001). Qureshi's

experiment helped show that clostridium beijerinckii BA101 hydrolyzes starch

more effectively and readily than that of its wild-type strain, and it also

demonstrates an increase in production and productivity. With more knowledge on

the effects of clostridium beijerinckii BA101 in the fermentation of butanol,

investigation on a greater ABE production has taken place.


In 2002 the production of butanol from starch based peanuts and

agricultural waste was conducted. Packaging peanuts were created as an

alternative, biodegradable source for packaging material for transport. With such a

large amount of packaging peanuts produced, there is an abundance of them. As

they are starch based (88.4wt%), they are able to be digested into carbohydrates,

and later fermented into butanol (Jesse, 2002). As a control experiment, starch was

used as a substrate to test for butanol production with BA101. Results from the test

showed that solvent production from starch was 27.4g/L. When starch based

packaging peanuts were used as a substrate, ABE solvent production was recorded

at 21.7g/L, with a productivity of .20g/L*h. Although there was a decrease in solvent

production from packaging peanuts in comparison to starch, the sole fact of butanol

production from starch based peanuts gives hope for future research in this area.

C. Beijerinckii was also tested with agricultural waste to test for ABE solvent

production. Two types of agricultural waste were tested: model waste, and actual

waste. Model waste was composed of packaging peanuts, cracked corn, apples, and

pears diluted to a 1-liter volume with distilled water; whereas the actual waste was

composed of apple drops, packaging peanuts, and cracked corn with 50 milliliters of

distilled water. Model waste was created as a control group to use for comparison in

relation to actual agricultural waste. Acetone, butanol, and ethanol were produced

in both model and actual agricultural waste. However, results showed that ABE

solvent production in model agricultural waste was 20.3g/L in comparison to

14.8g/L from actual agricultural waste (Jesse, 2002) In a similar study conducted by

Nashib Qureshi and Badal C. Saha, they tested the butanol production from wheat


straw hydrolysate using C. beijerinckii P260. Wheat straw is a dry stalk after the

wheat has been removed. It is also another type of agricultural waste, closely related

to the study done by TW Jesse in 2002. Results from the test show that when a

wheat straw substrate is fermented using C. beijerinckii P260, a total ABE solvent

concentration of 25.0g/L, a yield of .42 and productivity of .60g/L*h were collected

(Qureshi, 2007). In a controlled experiment involving glucose and c. beijerinckii

P260, total solvent concentration was at 20.1g/L with a yield of 0.41, and a

productivity of 0.28g/L*h. These results suggest that wheat straw yields larger

production of solvents than does glucose, making it an excellent substrate for

butanol production. These past studies have given great insight to understanding

the production of butanol through fermentation. Several strains of clostridia have

proven to be efficient in converting starch, agricultural wastes and sugars into

useful byproduct such as acetone, butanol, and ethanol.

In moving forward, a focus on the production of ABE solvents from feed

stock/biomass seems to be ideal. Such biomasses are sugar cane, agricultural

residues, corn stover etc. Feedstock has unique characteristics that can be used

effectively in ABE production. The composition of biomass is mainly carbohydrates,

made up almost entirely of sugars: 30-50% cellulose and 20-40% hemicellulose

(Lee, 2007). Cellulose can be broken down commonly into glucose, which is a

common substrate used in the ABE process. If biomass were to be treated with

actively growing clostridium, the sugars present will ferment to produce acetone,

butanol and ethanol solvents. Corn stover is one of few byproducts of biomass that

has not been tested for its production of butanol. Corn stover is a lignocellulosic


biomass-the non-starch, fibrous part of plant material. The biggest desire to use

corn stover is because it is an abundant material, and renewable. In looking at the

chemical composition of corn stover, it is made almost entirely of sugars, both

mono- and polysaccharides. Like most biomass it has a high percentage of cellulose,

and hemicellulose. With such high efficiency of sugars, it seems likely that it is a

good candidate for ABE production. In continuing, corn stover also contains around

20 percent Xylan-a polysaccharide found in plant cell walls. Xylan is also referred to

as a group of hemicellulose chains; this we know can be fermented into ABE

solvents. However, as stated earlier in 2006 Nashib Qureshi measured butanol

production from xylan fiber found in corn stalks using Clostridium acetobutylicum

P260 and found it was a suitable substrate for production; although it was noted

that xylose was added to the medium broth prior to inoculation to prevent

starvation of the bacteria.

To test this hypothesis, corn stover will be blended together into a fine dust

and placed in a dilute solution of a strong acid. This is known as an acid digestion;

the acid will break down sugar and starch bonds in the material, and allow them to

remain in solution. With the starches and sugars broken into the medium, we can

filter the solid waste out. The biomass mixture will then be inoculated in separate

tests with an actively growing culture of Clostridium beijerinckii NCIMB 8052.

Fermentation will be conducted in an anaerobic chamber for 72 hours, and perched

with nitrogen gas to remove any oxygen. ABE solvent production will be tested

through gas chromatography. A calibration curve will be constructed in order to test

for final concentration of products. A calibration curve looks at the area of peaks of


compounds, and that area is analogous to the concentration of that product. The

calibration curve will be a reference for trying to estimate the approximate

concentration of ABE solvents from this study. 1-Butanol is the desired product we

are looking for. If we can find a way to increase the productivity of butanol from the

ABE process, then it would be a more desirable way to create butanol biofuels.

It is beneficial to look at the overall process through a simulation and

understand the chemical reactions that the corn stover substrate will undergo.

Figure 1.1 Procedure Flow Chart

*The above diagram depicts the simulation process of the study. It begins with corn stover to the milling process; followed by acid digestion, fermentation, gas stripping of ABE solvents, condensations and then distillation of ABE solvents

Corn Stover Grind & Mill Sulfuric Acid Digestion Incubate Medium 48Hrs

Inoculate C. Beijernickii NCIMB 8052 Spores in Thioglycolate Medium

Incubate spores 48HrsTransfer Corn Stover

Medium to Fermentation Vessel

Purge Vessel w/ Nitrogen Gas

Transfer C. Beijernickii to fermentation Vessel w/ constant Nitrogen

Gas

Fermentation (72Hrs) Fractional Distillation (up to 100)

Extract Butanol from fermentation

solution/water with Diethyl Ether

Evaporate Diethyl Ether w/ Nitrogen Gas

Gas Chromotography and IR Spectra


Fractional Distillation will remove acetone, butanol, and ethanol separately

in order to measure concentration and product yield. Due to the difference in

colligative properties of each ABE solvent, we can use fractional distillation to

remove each of the three solvents. Acetone being removed first, followed by ethanol

and lastly butanol. The distillation column will be packed with glass beads. We will

stop fractional distillation when the temperature reaches 100℃; this is when water

will be separated, and with the excessive amount of water in solution, this process

would be tedious. To avoid this problem, we will separate n-butanol from the

remaining fermentation mixture with an ether extraction. The ether/butanol layer

will be bubbled with nitrogen gas to evaporate the ether.

Table 1.1 Boiling points of ABE solvents

Compound Boiling Point (°C)

Acetone 56.3

Ethanol 78.3

Water 100

Butanol 117.7

Figure 1.2 Fractional Distillation Diagram


The above image is an apparatus used for fractional distillation. The Bunsen burner will be replaced with a heating mantle to secure the round bottom flask.

Materials and Methods

Bacteria Culture propagation

Clostridium Beijerinckii NCIMB 8052 spores were purchased from

http://www.atcc.org/Products/All/51743.aspx#history, and stored at 4C until

ready for use. In order for proper growth of bacteria, and anaerobic chamber was

required for rehydration, inoculation and incubation of bacteria. To create

anaerobic conditions, we used a sodium Thioglycolate medium to grow the C.

beijerinckii spores. Thioglycolate is an anaerobic medium that reduces oxygen gas in

solution to water. Thioglycolate medium is composed of sodium Thioglycolate,

thioglycolic acid, l-cysteine, methylene blue and 0.05% agar. Sodium Thioglycolate,

thioglycolic acid and L-cysteine are responsible for the reduction of oxygen gas in

solution. Methylene blue is an indicator that turns blue/green when in the presence

of oxygen, and the agar helps slow diffusion of oxygen back into solution.

Thioglycolate is a strange medium, as it diffuses oxygen throughout the medium;

where at the top of the medium there is a large amount of oxygen and at the bottom

of the solution there is zero oxygen present. Due to this effect, Thioglycolate can

grow multiple types of bacteria at the same time. However, in respect to C.

beijerinckii, it will grow spores at the bottom of the solution where there is no

oxygen present.

Figure 3.1 Clostridium beijerinckii and Thioglycolate


http://www.atcc.org/Products/All/51743.aspx#history

*The figure here shows the C. beijerinckii NCIMB 8052 spores growing in Thioglycolate medium. The cloudy sediment at the bottom of the flask is the bacteria growth. The bottom of Thioglycolate medium lacks oxygen, this allows for anaerobic bacteria to propagate.

Corn Stover Pretreatment

Corn stover was a generous donation from May-Val Farms in West Hampton,

Massachusetts. Approximately 86g of corn stover was ground into fine particles

using a blender. The resulting corn stover was suspended in 1L 5M Sulfuric acid. 5M

Sulfuric acid was prepared by diluting 10mL of concentrated Sulfuric acid with

990mL of distilled water. The solution was autoclaved at 121C for 1 hour, and then

cooled to room temperature. The pH of the solution was adjusted to 5.0 with

approximately 60mL of 6M Sodium Hydroxide. The final pH of the system was

recorded at 5.8. The final solution was incubated at 45C for 72 hours.

Approximately 48 hours into incubation, mold became apparent on the surface of


the medium. The flask was removed, autoclaved at 121C and placed back in the

incubator for another 24 hours. The autoclave killed off the mold present in

solution. Following incubation, the flask was removed and the solid corn stover was

decanted from solution using vacuum filtration. The solution was stored at 4C.

Prior to bacteria inoculation and fermentation of corn stover medium, the

media was neutralized to a pH of 7. To do so, 12.49g of Calcium Carbonate were

added to the medium to neutralize the solution. A LabQuest 2 with pH probe was

used to monitor the pH. The resulting pH of the solution was 7.00. The calcium

carbonate reacts with the sulfuric acid present in solution to make an insoluble salt,

calcium sulfate. The salt was removed from the medium through vacuum filtration.

2.5mL of yeast extract (40g/L) was added to the medium. The corn stover medium

was stored at 4C.

Fermentation

Fermentation studies took place in a 5L fermentation vessel with multiple

attachments. There was a bubbler attached to the inside of the system, to which a

line of nitrogen gas was attached. The is a release valve that comes out of the system

for the release of pressure and oxygen purged out by the nitrogen gas. The pressure

release line was attached to a water trap, so that any oxygen that would exit or enter

the trap would get trapped in the water, and would not enter the system. The vessel

was bolted shut to ensure the container is free of oxygen.


Figure 5.1 Fermentation Vessel

*in the photo is the nitrogen gas tank with entrance line on the right hand side of the phot. The pressure release line is coming out the left side of the fermenter, attached to the double trap

A Bioreactor was not suitable for this fermentation, as it could not do

fermentations under anaerobic conditions.

1L of corn stover medium (pH 7) was transferred to the 5L fermentation

vessel. The system was closed, and purged with nitrogen gas to remove any oxygen

present in the system and the medium. The Nitrogen tank was set at 16psi, and the

valve was opened just enough for gas to pas through. There was gentle bubbling

through the solution. 10mL of grown C. beijerinckii was transferred to the vessel

containing the corn stover medium. The system was perched with nitrogen gas for

10 minutes. The system was purged of oxygen once every 24 hours for 3 days. The

fermentation was concluded following 72 consecutive hours. An aliquot of the raw

fermentation mixture was taken, and a Gas chromatograph of the solution was

taken. The spectra showed one large, clear peak with a retention time of 7.01


minutes. The solvents were extracted through fractional distillation and an ether

extraction.

Fractional Distillation

The fermentation solution was placed in a 500mL 3-necked round bottom

flask with an additional funnel and a fractional column attached. The flask was

placed in a sand bath and slowly heated at 100℃. At approximately 78℃, there was

approximately 1mL of solution collected. When the solution was heated to 100℃,

fractional distillation was halted to avoid the removal of water from the

fermentation solution.

Fractional Distillation Module:

*The fractional distillation set-up used for the separation, and purification of ethanol. The set-up included a 500mL 3-necked round bottom flask with an addition funnel and fractional column attached. A thermometer-measured vapor temperature- and a condensation column was attached to the fractional column. A 125mL Erlenmeyer flask was used as the collection glass.


Ether Extraction

The remaining fermentation solution (300m) was set aside for a diethyl ether

extraction. The process was done by placing 50mL of fermentation solution into a

125mL separatory funnel and 25mL of diethyl ether was added. The mixture was

shaken vigorously, and pressure was released from the funnel. The solution was

allowed to settle and the two layers were separated. The bottom aqueous layer was

removed from the funnel and placed in a beaker labeled “waste.” The top layer of

ether and butanol was then extracted and placed in a 400mL beaker. The extraction

was done until all of the fermentation solution has been used. The ether and butanol

solution was then placed in a 100mL test tube, and nitrogen gas was used to flush

the solution. The process caused an evaporation of the ether in solution. The process

was halted when condensation inside the tube no longer occurred. This meant all of

the ether had been evaporated. The remaining butanol was measured at 4mL. The

butanol was placed in a 5mL screw-cap vial. The butanol was characterized through

gas chromatography and infrared spectroscopy.

Analysis

Both products extracted (Ethanol, n-Butanol) were characterized using Gas

Chromatography and Infrared Spectroscopy. The IR spectra allows us to identify

functional groups present in solution, and the GC allows us to confirm the presence

of the two products from fermentation. Infrared spectroscopy sends a beam of

infrared light through the liquid sample. When the frequency of the beam is

equivalent to the absorbance of the vibrational modes for the different bonds

present in the molecule, a peak is electronically plotted on a graph. The different


bonds in the molecule absorb infrared light at different frequencies. The peaks elute

out on the graph. The graph is a relation between wavenumber (1/cm) and

absorbance. When the peak frequencies are collected, they are compared to

tabulated values of standard bond frequencies. This allows us to identify the

functional groups present in the molecule.

Gas chromatography works solely on the boiling point of the product. A

microliter sample of each product is injected into a 10% carbowax column that

contains an inert, polar solid. As the column is heated from 40℃-130℃, as the

product evaporates into the gas phase, a helium carrier gas caries the gaseous

product along the column to a detector, which then identifies the product, and a

peak is eluted out onto a graph at different times-this is also known an retention

time. The peak represents the time it took for the gas to be detected by the GC. Since

each sample evaporates at a specific boiling point, we can compare our

peak/retention times to standard solutions’ retention times to confirm the identity

of the product.

Results

Infrared Spectroscopy

A small sample of each product was placed on the detector of the IR, and the

sample was measured. The IR spectra were clear with minimal contaminants in the

graph.


IR Functional Group wavenumber

Type of Bond Wavenumber (1/cm) Intensity

C = C 1680-1600 Medium

C = N 1650-1550 Medium

Benzene Ring ~1600 and ~1500-1430 Strong to weak

C = O 1780-1650 Strong

C – O 1250-1050 Strong

O – H (alcohol) 3650-3200 Strong, broad

O – H 3300-2500 Strong, Very Broad

C – H 3300-2700 Medium

IR spectra n-Butanol


*The IR spectra of n-butanol produced from fermentation was taken. The large, broad peak at ~3500 1/cm is noted to be an alcohol group. The peak at ~3000 1/cm represents the sp3 carbons present in the butanol molecule.

IR Spectra n-Butanol Standard

*The spectra of standard n-butanol shows a broad peak at ~3400 1/cm and another peak at ~3000 1/cm. The peaks found represent the alcohol and sp3 carbons present in the molecule.

The IR spectra helped us identify the alcohol (O-H) group at ~3450 1/cm. This is the

main group found in the butanol molecule. The peaks at ~3000 1/cm represent the

sp3 carbons found in butanol. The two spectra are identical to each other, and if

superimposed, there would be no discrepancies between the two.

Gas Chromatography

A gas chromatograph spectrum of each product was taken on a Buck

Scientific Model 310 Gas Chromatograph with a 10% carbowax column and helium

carrier gas was used. The initial temperature was set at 40℃, held for 1 minute,


ramped at 10℃/min, and brought to a final temperature of 130℃. A 1μL sample of

each product was injected into the GC. A 5mL aliquot of the fermentation solution

was taken from the vessel directly following the 72 hours of fermentation. A GC

spectrum of the aliquot and each purified product was taken.

GC Retention Times

Solvent PeakRetention time

(min)

Aliquot Retention

Time (min)

Distillation

fractions

Retention Time

(min)

Acetone 1 2.01 - -

Ethanol 2 3.33 3.30 3.26

Water 3 5.50 5.69 5.31

Butanol 4 6.50 7.01 6.36

A GC of standard (acetone, ethanol, water, butanol) samples were run, giving

retention times (min) of 2.01, 3.33, 5.50, 6.50 respectively. The aliquot showed

retention times of 3.30min, 5.69min, and 7.01 minutes. In comparison, the fractional

distillation products showed retention times of 3.26min, 5.31min, and 6.36min.

Productivity and Mass Percent

The productivity of the corn stover fermentation was measured in terms of

total volume butanol produced per grams of corn stover used.

Productivity= Butanol (mL )CornStover (g )

Productivity= 4mL86 (g)

=.0465mL /g


From the data above, we can conclude that for every 86g of corn stover used, we

produced 4mL of butanol yielding a productivity of 0.0465mL/g. To better evaluate

the production of butanol, we calculated the mass percentage of butanol produced.

%Mass= Mass Butanol (g )MassCornStover (g )

×100

%Mass=(4mLButanol )( 0.81g

mL )86 gCornStover

×100

%Mass= 3.24 gbutanol86gCornStover

×100

%Mass=3.77 %

From the mass percent, we have discovered that of the 86g of corn stover, 3.24g of

butanol were produced for a 3.77% productivity.

Conclusion

In conclusion, results show ethanol and butanol have successfully been

produce from agriculture biomass. The fermented corn stover solution was purified

through fractional distillation and each collected solution was observed through gas

chromatography and IR spectra. From the comparison of standard retention times

and standard IR spectra, it can be concluded that the solvents produced are ethanol

and butanol, with a minor product of acetic acid. The overall yield of the

fermentation shows a total of 4mL butanol, having a productivity of 0.04651mL/g or

3.77% (m/m). This fermentation yields a 4:1 ratio of butanol and ethanol; 80%

butanol and 20% ethanol. In comparison to previous research in ABE fermentation

from agricultural waste, the reaction has a 3:6:1 ration acetone, butanol and ethanol.

In previous reactions, only about 60% of total products was butanol. This research


of corn stover as the substrate medium shows a 25% increase of butanol in

comparison to previous research. The United States Department of Agriculture

states that there is approximately 111 million tons of agriculture waste in the US. If

all of the waste was used in fermentation, and knowing a 3.77% production of

butanol, we can conclude that approximately 1.25 billion gallons of butanol could be

produced, or about 29.7 million barrels of butanol. This is a major increase in

production of bio-butanol. However, it is concerning that acetone was not produced

through fermentation. In order for this to be analyzed, a batch fed fermentation

should be conducted to identify all major products of the ABE process.

Since the discovery of the ABE solvents through fermentation of sugars using

Clostridia, extensive research on increased solvent production, yield and

productivity has been done. Ethanol and butanol are two byproducts of the ABE

process, both of which are excellent alternatives for fuel.

In the early 20th century, ethanol was commonly used for fuel production for

its high heating content at 26000MJ/m3 (Granstrom, 2010). However, butanol is a

far more superior fuel substitute to ethanol. Butanol contains approximately 22%

oxygen (Qureshi, 2006) and has a heating content of 29000MJ/m3 (Granstrom,

2010). Butanol is significant for it is nearly identical to gasoline in the way that it

burns. The goal of future research is to have a substantial increase in butanol

production from anaerobic fermentation using inexpensive substrates. It has been

shown, that common fermentation substrates included starch, and sugars. But due

to high economic value, this discouraged the use of fermentation of ABE solvents.

Recent studies have shown that alternative substrates such as agricultural waste,


corn fiber, packaging peanuts, and biomass such as wheat straw have shown

increased production of ABE. Improvements in ABE solvent production, with a 25%

increase of butanol compared to prior research helps support future studies to the

potential commercialization of butanol as a biofuel and safe alternative to

petroleum-derived fuel.


References

"Clostridium Beijerinckii Donker Emend. Keis Et Al. (ATCC® 51743™)." ATCC. ATCC,

2014.

Formanek, Joseph. "Enhanced production by clostridium beijerinckii BA101 grown

in semi defined P2 medium containing 6 percent maltodextrin or glucose."

Applied and Environmental Microbiology 63.6 (1997): 2306-2310.

Granstrom, Tom. "Process for biobutanol." Aalto University: School of Sciences and

Technology (2010):1-22.

Lee, DoKyoung. "Composition of Herbaceous Biomass Feedstock." South Dakota

State University 1.07 (2007): 1-16.

Monot, Frederic. "Acetone and butanol production by clostridium acetobutylicum in

a synthetic medium." Applied and Environmental Microbiology 44.6 (1982):

1318-1324.

Qureshi, Nashib. "Butanol production from wheat straw hydrolysate using

clostridium beijerinckii." Bioprocess Bio-system Engineering 30.1 (2007):

419-427.

Qureshi, Nashib and HP Blascheck. "Recent Advances in ABE fermentation: hyper-

butanol producing clostridium beijerinckii BA101." Journal of Industrial

Microbiology and Biotechnology 27 (2001): 287-291.

Qureshi, Nashib & H.P. Blaschek. “Recovery of butanol from Fermentation Broth by

Gas Stripping.” Renewable Energy 22 (2001): 557-564.


Qureshi, Nashib & Xin-Liang Li. "Butanol Production from Corn Fiber Xylan using

clostridium acetobutylicum." Biotechnology 22 (2006): 673-680.

T.W Jesse. "Production of butanol from starch-based waste packing peanuts and

agricultural waste." Journal of Industrial Microbiology & Biotechnology. 29

(2002): 117-123.

V.V. Zverlov. "Bacterial acetone and butanol production by industrial fermentation

in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery."

Applied Microbiology & Biotechnology 71 (2006): 587-597.


senior thesis final product

Documents