A Green Chemistry Lab Biosynthesis of Ethanol from Molasses

Introduction

Alcohols are often called green solvents because they have few human health and environmental risks, particularly when compared to solvents like methylene chloride or benzene. Ethanol may be a benign solvent, but what is its source? If the source is petroleum, all we have done is moved the hazard to another part of the process. In fact, until recently ethanol has been synthesized primarily from oil. A growing industrial source of ethanol is blackstrap molasses, a waste product of sugar production.

There are two major sources of sugar: sugar cane in tropical climates and sugar beets in temperate climates, giving most countries a source of sugar. Both sources are processed in a similar way. Sugar is extracted from the beet or cane pulp with hot water, forming the raw juice or mother liquor. Lime, Ca(OH)₂ and carbon dioxide, CO₂, are then added to the mother liquor to precipitate most of the water-soluble non-sugars. The mother liquor is then concentrated by evaporation, increasing the initial 10% to 15% sugar concentration to 60% to 65%. This leaves the solution super-saturated when cooled back to room temperature. Seed crystals are grown until they reach the desired sugar crystal size and then separated from the mother liquor. The initial sugar crystals collected are known as the A-product or white sugar. The evaporation crystallization cycle is repeated forming the light brown B-product or raw sugar. At this point, it is no longer cost effective to produce further sugar product and the mother liquor is now known as blackstrap molasses.

The need to effectively use this waste molasses has grown in the last 30 years as the world-wide demand for sugar has tripled. Global blackstrap molasses production is estimated to be about 60 tons per year. The majority of this waste molasses is combined with the dried beet or cane pulp and used for animal feed. Ethanol production is one growing application of this plentiful material.

In this experiment we will use commercially available molasses. It can be found in three forms: fancy, cooking, and blackstrap. Fancy molasses, also known as Gold Star molasses, is a direct product of sugar cane. Cooking molasses is a blend of fancy and blackstrap molasses. These products may or may not have sulfur added. Sulfur inhibits the breakdown of molasses sugars and will reduce the ethanol yield.

This experiment demonstrates three key green principles: the use of renewable resources, catalysis, and design for degradation.

Cheap petroleum led to its use for the synthesis of most organic compounds. Petroleum is renewable only on a geological timescale and is dwindling rapidly. In comparison, blackstrap molasses comes from a quickly renewable natural product. Sugar beets are harvested annually and sugar cane several times a year. While these crops are renewable, the practices used in growing them can vary from environmentally responsible to environmentally destructive. Even when well intentioned, our use of chemical products can still result in serious environmental harm. Sometimes the petroleum product may actually do the least amount of harm when considering the big picture. Analyzing the choice of resource to used can be a challenging task!

Catalysis is important for reducing the energy consumption and the waste production in a chemical reaction. The reaction in this experiment takes advantage of two enzymes found in yeast. Enzymes are remarkable natural catalysts that are highly chemically selective and significantly reduce the activation energy of a reaction.

Our reaction is an excellent example of design for degradation. Only a small amount of yeast is necessary to initiate the reaction and the yeast reproduces while the reaction is occurring. Once the reaction has reached completion, the yeast dies and the ethanol and all by-products are easily, rapidly, and harmlessly degraded in the environment.

Experimental Theory/Design

Fermentation is one of the oldest chemical arts. The fermentation of molasses is the process used to make rum (although the rum produced from blackstrap molasses is not fit for human consumption). In our experiment, the interest is the synthesis and purification of ethanol by a green reaction pathway.

Molasses is a mixture of monosaccharides and disaccharides and other miscellaneous flavoring agents naturally produced in the sugar cane or sugar beet. Approximately 50% of the molasses mass is sugar. Yeast has two enzymes that convert the saccharides to ethanol. Invertase converts dissacharides (sucrose) to monosaccharides (glucose) by a catalytic hydrolysis (addition of water) reaciton. Glucose is then converted to ethanol and carbon dioxide by the enzyme zymase.

One mole of sucrose will produce four moles of ethanol and four moles of carbon dioxide. When the alcohol level becomes high enough, the yeast will die of alcohol poisoning. The reaction is vapor locked with limewater so that the reaction environment remains oxygen free, keeping the ethanol from further oxidizing.

Boiling occurs when the vapor pressure of a liquid is equal to atmospheric pressure. This is determined using Raoult's law for an ideal gas. Distillations take advantage of this behavior to separate liquid mixtures. Some mixtures of liquids do not behave in this ideal way and instead form azeotropes, solutions that boil at a temperature different from any specific liquid in the solution. Ethanol and water form a binary azetrope, making pure ethanol very difficult to obtain by distillation. At one atmosphere of pressure, pure water boils at 100 ^oC and pure ethanol boils at 78 ^oC. At one atmosphere of pressure, the ethanol-water azeotrope boils at 78.5^oC, producing a vapor that is 95.6% ethanol and 4.4% water.

 

 

 

The Experiment (Biology Class)

 

1. Mix 70 ml of molasses with 70 ml of water in a 250 ml Erlenmeyer flask.
    
    
2. Add about 0.5 g of yeast to the flask and stir gently until well mixed.
    
    
3. Stopper the flask with a one-hole rubber stopper containing a 90^o glass tube.
    
    
4. Attach a short rubber hose to the 90^o glass tube and insert a short straight section of glass rube into the other end of the rubber tube.
    
    
5. Dip the straight glass tube into a test tube two-thirds full of limewater, Ca(OH)₂ solution. The test tube of limewater serves as a one-way vapor lock, keeping air from entering the flask while allowing the carbon dioxide to escape. If air were to enter the flask during the reaction, the ethanol produced would be further oxidized to acetic acid (vinegar).
    
    
6. Store this apparatus in a lab cabinet (in darkness) for one week while the fermentation reaction occurs.

 

 
The Experiment (Chemistry Class)

1. Prepare a simple distillation, decanting the ethanol solution into a 250 ml round bottom (Florance) flask.

   The simple distillation can be done fairly rapidly (one drop per second) and the alcohol fraction should be collected until just below the boiling point of water, 100 ^oC.

2. Prepare a fractional distillation, placing the mixed alcohol product from the simple distillation in another round bottom flask. Distill the ethanol slowly; record the temperature range for each fraction collected - stop collecting at 97 ^oC.

   Fractons are identified by a rapid change in temperature and a concurrent increase/decrease in distillate production.

3. Determine the density of each fraction by massing a 10 ml sample collected with a volumetric pipette. If the collected fraction is less than 10 ml, use the available pipette that is closest in volume to the fraction.
    
4. Use the table below to determine the alcohol content in each fraction. Record your volume of "pure" ethanol collected and add your ethanol to the collection container.

Aqueous Alcohol (EtOH) Content
Density g/ml	% EtOH by wt.	% EtOH by vol.	g EtOH per 100 ml	Density g/ml	% EtOH by wt.	% EtOH by vol.	g EtOH per 100 ml
0.989	5	6.27	4.95	0.856	75	81.30	64.17
0.982	10	12.44	9.82	0.843	80	85.49	67.48
0.975	15	18.54	14.63	0.831	85	89.48	70.63
0.969	20	24.54	19.37	0.828	86	90.25	71.23
0.962	25	30.45	24.04	0.826	87	91.02	71.84
0.954	30	36.25	28.61	0.823	88	91.77	72.43
0.945	35	41.90	33.07	0.821	89	92.53	73.03
0.935	40	47.40	37.41	0.818	90	93.27	73.62
0.925	45	52.72	41.61	0.815	91	93.99	74.19
0.914	50	57.89	45.69	0.813	92	94/72	74.76
0.903	55	62.89	49.64	0.810	93	95.44	75.32
0.891	60	67.74	53.47	0.807	94	96.11	75.86
0.880	65	72.43	57.17	0.804	95	96.79	76.40
0.868	70	76.95	60.74	0.789	100	100.00	78.90

 

Pre-Laboratory Questions

1.	During the fermentation reaction, it is important to prevent air from entering the reaction chamber. Why?
2.	Explain the advantage of fractional distillation over a simple distillation.
3.	What is an azeotrope and why does it limit our ethanol purity even when we are doing a fractional distillation?
4.	Complete the following table of chemical data: Liquids B.P.   (oC) Density   (g/ml) ethanol     water
5.	Consider the chemicals used for this experiment. What realistic hazards are present? What safety procedures are necessary beyond wearing goggles?

Experiment Report   (Chemistry Class)

For this experiment, create a summary report. The report should be a typed, one-page narrative and should include:

• A brief summary of the experiment (one paragraph).
   
• A discussion of your results that should include:
   
  • Volumes and percent ethanol for each fraction collected
     
  • Percent yield of ethanol from molasses
     
  • Percent yield of ethanol from the reactive sugars in the molasses, assuming molasses is approximately half reactive sugars
  
   
• An analysis of error and the quality of your experiment. Did the experiment go well or poorly and can you explain what went wrong? Do you find your results to be reliable?
   
• An evaluation of this experiment in terms of its greenness.