AMERICAN JOURNAL OF SCIENTIFIC AND INDUSTRIAL RESEARCH 2011, Science Hu, http://www.scihub.org/AJSIR ISSN: 2153-649X doi:10.5251/ajsir.2011.2.4.694.706

Utilization of sugar refinery waste (molasses) for ethanol production-

using Saccharomyces Cervicae O. A. Osunkoya, N. J. Okwudinka

Department of Chemical Engineering, Covenant University, Ota, Ogun State, Nigeria ABSTRACT

An analysis of the current situation and perspective on biomass-to-ethanol is provided in this study. Various conversion pathways are compared from technical, economic, and environmental points of view. This study also deals mainly with the yield of ethanol from molasses with respect to the dilution ratio and the amount of yeast used for fermentation keeping the temperature and fermentation duration constant. After the study it was observed that with an increase in yeast quantity the ethanol yield decreases in a fluctuating manner until the quantity of yeast is about grams it begins to increase again with the action of the yeast greatly depending on the dilution ratio.

Keywords: Ethanol; Molasses; Sugar Refinery waste; Saccharomyces Cervicae;

INTRODUCTION Liquid bio-fuels are receiving increasing attention worldwide as a result of the growing concerns about oil security of supply and global climate change. In most developing countries, the emerging bio-fuels industry is perceived as an opportunity to enhance economic growth and create or maintain jobs, particularly in rural areas. The liquid bio-fuels market is shared mainly between bio ethanol and biodiesel, with more than 85% market share for the former in 2005. The main advantage of bio ethanol is the possibility to blend it in low proportions with gasoline (5 to 25% bio ethanol by volume) for use, without any significant change, in internal combustion engines. (1)

Aims and objective of study: The aim of this project is to study the dilution rate of molasses and the effect of yeast on the yield of ethanol using batch fermentation.

METHOD AND SCOPE OF STUDY Various methods can be used for the specific purpose of the production of ethanol; this includes Ethylene hydration, Fermentation, Cellulosic ethanol. The above stated methods are pathways to ethanol production but fermentation is applied in this paper.

RELEVANCE OF STUDY This study poses great opportunities for the chemical engineering profession in Covenant University and Nigeria at large.

It also would help in further research areas such as biodegradation of waste products as molasses is a waste product after the refining of sugar. ETHANOL

Fig 2. 1: Chemical Structure of Ethanol

Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colourless liquid. In common usage, it is often referred to simply as alcohol or spirits. Ethanol is a straight-chain alcohol, and its molecular formula is C2H5OH. Its empirical formula is C2H6O. An alternative notation is CH3-CH2-OH, which indicates that the carbon of a methyl group (CH3-) is attached to the carbon of a methylene group (-CH2-), which is attached to the oxygen of a hydroxyl group (-OH). It is a constitutional isomer of di-methyl ether. Ethanol is often abbreviated as EtOH, using the common organic chemistry notation of representing the ethyl group (C2H5) with Et. Ethanol burning with its spectrum depicted. (5) Hydrogen bonding causes pure ethanol to be hygroscopic to the extent that it readily absorbs water from the air. The polar nature of the hydroxyl group

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causes ethanol to dissolve many ionic compounds, notably sodium and potassium hydroxides, magnesium chloride, calcium chloride, ammonium chloride, ammonium bromide, and sodium bromide.(4) Sodium and potassium chlorides are slightly soluble in ethanol.(4) Because the ethanol molecule also has a non polar end, it will also dissolve non-polar substances, including most essential oils (6) and numerous flavoring, coloring, and medicinal agents. The addition of even a few percent of ethanol to water sharply reduces the surface tension of water. This property partially explains the tears of wine phenomenon. Mixtures of ethanol and water that contain more than about 50% ethanol are flammable and easily ignited. Pathways to ethanol production include:

ETHYLENE (ETHENE) HYDRATION

Ethanol is manufactured by reacting ethene with steam. The reaction is reversible, and the formation of the ethanol is exothermic.

Only 5% of the ethylene is converted into ethanol at each pass through the reactor. By removing the ethanol from the equilibrium mixture and recycling the ethene, it is possible to achieve an overall 95% conversion. (7)

THE MECHANISM FOR THE ACID CATALYSED HYDRATION OF ETHENE (7)

Step 1

All of the hydrogen atoms in the phosphoric (V) acid are fairly positively charged because they are attached to a very electronegative oxygen atom.

One of these hydrogens is strongly attracted to the carbon-carbon double bond.

The pi part of the bond breaks and the electrons in it move down to make a new bond with the hydrogen atom. That forces the electrons in the hydrogen-oxygen bond down entirely onto the oxygen.

Step 2

The carbocation (carbonium ion) formed reacts with one of the lone pairs on a water molecule. A carbocation is one which carries a positive charge on a carbon atom. (7)

Step 3

Finally, one of the hydrogens on the oxygen is removed by reaction with the dihydrogenphosphate (V) ion, H2PO4-, formed in the first step. The reversibility of this reaction (7) This reaction is entirely reversible - and so each step is reversible. In the reverse direction, this is a dehydration of ethanol to make ethene. Equations of reaction C2H4 (g) + H2O (g) CH3CH2OH (l).-------------Equation 2.1 C2H4 + H2PO4 CH3CH2PO4H ---------------Equation 2.2

Fig 2 . 2: Ethylene hydration using phosphoric acid catalyst CH3CH2PO4H + H2O CH3CH2OH + H2PO4-----------Equation 2.3

Fig 2.3: Phosphoric acid catalyst regeneration

Fermentation The breakdown of carbohydrates to ethanol, carbon di-oxide and water using micro organisms. (10) The many and varied raw materials used in the manufacture of ethanol via fermentation are conveniently classified under three types of agricultural raw materials (11):

1. Sugar 2. Starches 3. Cellulose materials.

Fermentation from Sugars: Sugars from sugar cane, sugar beets, molasses, and fruits) can be converted to ethanol directly. The most widely used sugar for ethanol fermentation is blackstrap molasses which contains about 35 40 wt% sucrose, 15 20 wt% invert sugars such as glucose and fructose, and 28 35 wt% of non-sugar solids. Blackstrap (syrup) is collected as a by-product of cane sugar manufacture. The molasses is diluted

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to a mash containing 10 20 wt% sugar. After the pH of the mash is adjusted to about 4 5 with mineral acid, it is inoculated with the yeast, and the fermentation is carried out non-aseptically at 20 32C for about 1 3days. The fermented beer, which typically contains 6 10 wt% ethanol, is then set to the product recovery in purification section of the plant. Fermentation from Starches: All potable alcohol and most fermentation industrial alcohol are currently made principally from grains. Fermentation of starch from grain is somewhat more complex than fermentation of sugars because starch must first be converted to sugar and then to ethanol (12). Starch is converted enzymatically to glucose either by diastase presents in sprouting grain or by fungal amylase. The resulting dextrose is fermented to ethanol with the aid of yeast producing CO2 as co-product. A second co-product of unfermented starch, fiber, protein and ash known as distillers grain (a high protein cattle feed) is also produced. Fermentation from Cellulosic Materials: Each step in the process of the conversion of cellulose to ethanol proceeded with 100% yield; almost two-thirds of the mass would disappear during the sequence, most of it as carbon dioxide in the fermentation of glucose to ethanol. This amount of carbon dioxide leads to a disposal problem rather than to a raw material credit. Another problem is that the aqueous acid used to hydrolyze the cellulose in wood to

glucose and other simple sugars destroys much of the sugars in the process. One way of making cellulose wastes more susceptible to hydrolysis is by subjecting them to a short burst of high energy electron beam radiation. An alternative to acid hydrolysis is the use of enzymes. Although they avoid the corrosion problems and loss of fuel product associated with acid hydrolysis, enzymes have their own drawbacks. Enzymatic hydrolysis slows as the glucose product accumulates in a reaction vessel. This end-product inhibition eventually halts the hydrolysis unless some way is found to draw off the glucose as it is formed. (12) Ethanol Fermentation with Bacteria: A great number of bacteria are capable of ethanol formation. Many of these microorganisms, however, generate multiple end products in addition to ethyl alcohol. These include other alcohols (butanol, isopropylalcohol, 2,3-butanediol), organic acid (acetic acid, formic acid, and lactic acids), polyols (arabitol, glycerol and xylitol), ketones (acetone) or various gases (methane, carbon dioxide, hydrogen).(12)

Those microbes that are capable of yielding ethanol as the major product (i.e. a minimum of 1 mol ethanol produced per mol of glucose utilized) are shown in the table below.

Table 2. 1 : Bacteria useful for fermentation Mesophilic Organisms

mmol Ethanol Produced per Mmol Glucose Metabolized

Clostridium sporogenes up to 4.15 a) Clostridium indolis

(pathogenic) 1.96 a)

Clostridium sphenoides 1.8 a) (1.8) b) Clostridium sordelli

(pathogenic) 1.7

Zymomonas mobilis (syn.Anaerobica) (anaerobe)

1.9

Zymomonas mobilis Ssp. Pomaceas

1.7

Spirochaeta aurantia 1.5 (0.8) Spirochaeta stenostrepta 0.84 (1.46)

Spirochaeta litoralis 1.1 (1.4) Erwinia amylovora 1.2

Leuconostoc mesenteroides 1.1 Streptococcus lactis 1.0

Sarcina ventriculi (syn. Zymosarcina)

1.0

Many bacteria (i.e. Enterobacteriaceas, Spirochaeta, Bacteroides, etc.) metabolize glucose by the Embden-Meyerhof pathway. Briefly, this path utilizes 1 mol of glucose to yield 2 mol of pyruvate which are

then de-carbosylated to acetaldehyde and reduced to ethanol. (12) Although many Bacteria metabolize glucose by the Embden-Meyerhof pathway, the

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fermentation can also be analyzed using Michaelis - Menten model for certain bacteria (2) A close look at Zymomonas Mobilis as an ethanol Producing Bacteria Zymomonas mobilis Scientific Classification Kingdom: Bacteria Phylum: Proteobacteria Class: Alpha Proteobacteria Order: Sphingomonadales Family: Sphingomonadaceae Genus: Zymononas Species: Z. mobilis Zymomonas mobilis is a bacterium belonging to the genus Zymomonas. It is notable for its bioethanol-producing capabilities, which surpass yeast in some aspects. It was originally isolated from alcoholic beverages like the African palm wine, the Mexican pulque, and also as a contaminant of cider and beer in European countries. Z. mobilis degrades sugars to pyruvate using the Entner-Doudoroff pathway. The pyruvate is then fermentated to produce ethanol and carbon dioxide as the only products (analogous to yeast). (13) Ethanol Fermentation with Yeast The organisms of primary interest to industrial operations in fermentation of ethanol include Saccharomyces cerevisiae, Saccharomyces uvarum, Schizosaccharomyces pombe, and Kluyveromyces sp. Yeast, under anaerobic conditions; metabolize glucose to ethanol primarily by way of the Embden-Meyerhof pathway. The overall net reaction involves the production of 2 moles each of ethanol, but the yield attained in practical fermentations however does not usually exceed 90 95% of theoretical. This is partly due to the requirement for some nutrient to be utilized in the synthesis of new biomass and other cell maintenance related reactions. A small concentration of oxygen must be provided to the fermenting yeast as it is a necessary component in the biosynthesis of polyunsaturated fats and lipids. Typical amounts of O2 maintained in the broth are 0.05 0.10 mm Hg oxygen tension. The relative requirements for nutrients not utilized in ethanol synthesis are in proportion to the major components of the yeast cell. These include carbon oxygen, nitrogen and hydrogen. To lesser extent quantities of phosphorus, sulfur, potassium, and

magnesium must also be provided for the synthesis of minor components. Minerals (i.e. Mn, Co, Cu, Zn) and organic factors (amino acids, nucleic acids, and vitamins) are required in trace amounts. Yeast are highly susceptible to ethanol inhibition. Concentrations of 1-2% (w/v) are sufficient to retard microbial growth and at 10% (w/v) alcohol, the growth rate of the organism is nearly halted. Saccharomyces cerevisiae Scientific classification Kingdom: Fungi Phylum: Ascomycota Subphylum: Saccharomycotina Class: Saccharomycetes Order: Saccharomycetales Family: Saccharomycetaceae Genus: Saccharomyces Species: S. cerevisiae Binomial name Saccharomyces cerevisiae Comparison of Z. mobilis and S. cerevisiae with

respect to producing bio-ethanol:

ADVANTAGES OF Z. Mobilis over S. cerevisiae

i. higher sugar uptake and ethanol yield, ii. lower biomass production, iii. higher ethanol tolerance, iv. does not require controlled addition of

oxygen during the fermentation, v. Amenability to genetic manipulations.

Disadvantages of Z. Mobilis compared to S. cerevisiae Its utilizable substrate range is restricted to glucose, fructose, and sucrose. Using biotechnological methods, scientists are currently trying to overcome this. A variant of Z. mobilis that is able to use certain pentoses as a carbon source has been developed. An interesting characteristic of Z. mobilis is that its plasma membrane contains hopanoids, pentacyclic compounds similar to eukaryotic sterols. This allows it to have an extraordinary tolerance to ethanol in its environment, around 13%. (13)

PROCEDURE AND METHODOLOGY Materials used in the experimentation

Erlenmeyer flasks Test tubes Rubber pipe or glass tube Retort stand Reflux condenser CaOH

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Stopper/Cork Stop watch Thermometer Molasses Yeast (Saccharomyces Cervisae) 600ml beakers Funnel Filter paper pH meter

PROCESS DESCRIPTION

BASIC EXPERIMENTAL PROCEDURE (3) Pre-treatment of molasses 70 ml of molasses was mixed with 70 ml of water in a 250 ml Erlenmeyer flask. About 0.5 g of yeast was added to the flask and stir gently until everything is well mixed. This reaction was stored in a drawer for one week while the fermentation reaction occurs. A simple distillation column was prepared; the simple distillation was done fairly rapidly (one drop per second) and the alcohol fraction should be collected until just below the boiling point of water. The ethanol was distilled slowly; recording the temperature range for each fraction collected, stopping collection at 97C. The above procedures were repeated for different grams of yeast varying from 0.5 grams of yeast to 7grams of yeast and for each gram of yeast the quantity of water was varied from 35ml to 280ml. The results are recorded and discussed in the succeeding chapter. Precautions taken during the experimentation.

i. The molasses was refrigerated until the experimental date to avoid unwanted action of bacteria.

ii. The yeast was also refrigerated until the experimental date to avoid it being activated.

iii. The flask was stopped with a one-hole rubber stopper containing a bent glass tube. A short rubber hose was attached to the bent glass tube and a short straight section of glass tube was inserted into the other end of the rubber tube. The straight glass tube was dipped into a test tube two-thirds full of limewater (Ca (OH) 2 solution). The test tube of limewater serves as a one-way vapour lock, keeping air from entering the flask while allowing the carbon dioxide to escape.

The results for the experiment are recorded in the succeeding chapter. RESULTS AND DISCUSSION OF RESULTS

Due to the quality of the analysis especially the distillation, a relatively good yield of ethanol is expected, although maybe not the best.

CHARACTERISTICS OF RAW MATERIAL

Table 4.1 shows the average analytical properties of the molasses from BUA sugar refinery. The analysis was carried out as follows:

i. To find the Brix was used at the lab; a portion of the molasses was poured into the sensor of a Brixometer and the reading was taken.

ii. To find the purity 13g of the sample was taken in a beaker and the sample was made up with water to the dilute brix value after which a clarifying reagent called octapol is added and the resulting solution is filtered to get a golden clear liquid then the solution is poured into a saccharimeter and the pol value is taken an the purity (amount of sucrose per sample) is calculated (see appendix B for calculation of purity)

iii. For the Ash and pH values a simple conductivity meter was used to determine the values by dipping the electrodes in the sample.

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Table 4. 1 Characteristic Properties of the Molasses used for the analysis Property Value

Brix Dilute Brix

73.210 36.605

Purity 64.530 Ash 8.238% pH 5.097

Temp 30C Colour Dark consistent brown Density 1.292

TEST EXPERIMENT RESULTS Table 4.2 Test Run Experiment: Premium Yeast Quantity For The Best Yield Of The Ethanol Produced.

The values for the table below were gotten as follows: Ratio of water to molasses = 1:1 (varied for other cases see Appendix II) Amount of yeast = 0.2g Weight of specific gravity bottle (A) = 28.70g

Weight of specific gravity bottle + aqueous ethanol (B) =50.58 g Weight of aqueous ethanol = B-A= 21.88g Volume of ethanol = 50ml Specific gravity of ethanol () = V

m = 0.9338g/ml

Gram ethanol present per 100ml = 42.798g Note: the above calculation was repeated for all the tabulated results henceforth.

Table 4.2 reveals that the increase in the quantity of yeast does not favour the process initially but with the continuous increase the yeast activity increases thereby increasing the yield. New substances might have been formed, but they are still held within the mash while the gases (CO2 and possibly minute quantities of methane) were trapped in CaOH, Observing that 5 grams of yeast gave the maximum ethanol yield the main experimental analysis would be carried out around these ranges (from 1 gram to

7grams with no definitive spacing in the quantities just random weights). ANALYSIS OF THE EFFECT OF DILUTION RATIO The various dilution ratios are as follows 1: 0.5; 1: 1; 1: 2; 1: 3; 1: 4 (being 1 part molasses to 1 part water, 1 part molasses to 2 parts water etc respectively) represented as decimals on the chart, theses ratios would be used for the yeast variations as would be analyzed hitherto. 4.2.2.1 Analysis for samples fermented with 1 gram of yeast

Table 4.3: Premium yeast quantity for the best yield of ethanol analysis (1 gram of yeast)

1gram DENSITY AND WT % OF THE ETHANOLRatio Of Molasses To Water DENSITY WT % 1gram (gram ethanol per 100ml )

0.667 0.888 57.781 52.959 0.500 0.888 57.868 53.028 0.333 0.881 60.991 55.524 0.250 0.863 68.296 61.211 0.200 0.816 86.556 74.492

S/N Weight of Yeast (g) Density of Sample (g/ml)

Wt% of ethanol produced

gram ethanol per 100ml

1 0.200 0.915 46.750 42.798 2 0.500 0.947 31.500 29.853 3 1.200 0.968 20.000 19.373 4 3.000 0.934 40.000 37.380 5 5.000 0.885 62.270 55.108

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Fig 4.1: Chart of Dilution Ratio to yield after 5 days of Fermentation (1 gram of yeast)

Analysis of figure 4.1

Fig 4.2: Chart of Dilution Ratio to yield after 5 days of Fermentation (2 grams of yeast) Analysis of figure 4.2

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According to the chart of the experimental result the yield was best at ratio 1:0.5 (0.2 molasses concentration). From literature this could be caused by the insufficient sugar quantity available for fermentation as the more diluted the culture the less the yield meaning the yeast had very little to ferment thereby producing less ethanol as the quantity of water increased because as the biomass is diluted, the sugar level gradually washes out. Note also that the time taken for the breakdown process might have affected the time available for the yeast to convert most of the converted glucose to ethanol before being worn out (or requiring regeneration) due to the quantity of yeast available.

4.2.2.2 Analysis for 2 grams of yeast. According to the chart of the experimental result the yield was best at ratio 1:2 (0.3333 molasses concentration). From literature this could be caused by the insufficient glucose concentrations at the higher dilution ratios while at lower ratios the glucose quantity might be too high therefore the yeast might not act as well also several strains (e.g. K. marxianus, S.uvarum) have been specially cultured for this purpose (high sugar content mediums).

Table 4. 4 Premium yeast quantity for the best yield of ethanol analysis (2 grams of yeast)

2grams DENSITY AND WT % OF THE ETHANOLRATIO OF MOLASSES TO WATER DENSITY WT % 2grams (gram ethanol per 100ml )

0.667 0.924 40.976 38.851 0.500 0.919 43.630 41.154 0.333 0.855 71.614 63.724 0.250 0.938 33.802 32.485 0.200 0.979 10.433 10.319

4.2.2.3 Analysis for 3 grams of yeast

Table 4.5: Premium yeast quantity for the best yield of ethanol analysis (3 grams of yeast)

RATIO OF MOLASSES TO WATER DENSITY WT % for 3 grams (gram ethanol per 100ml )0.667 0.934 36.208 34.643 0.500 0.938 34.224 32.865 0.333 0.930 37.955 36.195 0.250 0.977 11.706 11.582 0.200 0.974 13.404 13.257

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Fig 4.3: Chart of Dilution Ratio to yield after 5 days of Fermentation (3 grams of yeast) Analysis of figure 4.3

Behaves just like the cultures with 2 grams of yeast only the medium is more unstable since the yeast are not adequate to balance the culture and get a steady graph the yeast are many therefore the sugar

breakdown process is rapid but not as rapid but also as the sugar is being broken down reproduction takes place for the yeast due to this divided attention the yeast are unstable and the yield unstable in return. 4.2.2.4 Analysis for 5 grams of yeast.

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Table 4.5: Premium yeast quantity for the best yield of ethanol analysis (5 grams of yeast)

RATIO OF MOLASSES TO WATER DENSITY WT % 5grams (gram ethanol per 100ml )

0.667 0.928 39.274 37.359 0.500 0.897 53.868 49.775 0.333 0.908 48.718 45.491 0.250 0.933 36.673 35.057 0.200 0.961 21.392 20.986

Fig 4.4: Chart of Dilution Ratio to yield after 5 days of Fermentation (5 grams of yeast)

Analysis of figure 4.4

Due to the increase in yeast quantity the ethanol yield is higher within the less diluted mediums causing the yield to be higher although the yield is higher than the previous two at the lower dilution rate regions the optimum yield still lays at the ratio 1:2.

For every dilution rate the yeast behaved differently although averagely the 1 gram of yeast gave the

purest ethanol quality, it can also be seen that the 7 grams gave the next highest this is to say that the ethanol yield reduces with an increase in yeast concentration until a critical point after which it starts to increase again.

4.2.2.4 Analysis for 7 grams of yeast

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Table 4. 6: Premium yeast quantity for the best yield of ethanol analysis (7 grams of yeast)

RATIO OF MOLASSES TO WATER DENSITY WT % 7grams(gram ethanol per 100ml)

0.667 0.908 48.951 45.687 0.500 0.925 40.478 38.416 0.333 0.912 46.841 43.903 0.250 0.829 81.483 70.936 0.200 0.960 21.802 21.375

Verification Analysis of sample using a UV visible Spectrometer

This verification analysis is based strictly on the comparison of the absorbance of each substance with a pure

stock sample. Figure A.UV Spectroscopy Test result

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1

Fig A.UV Spectroscopy Test result 2

Comparing the two samples based on their wavelengths and absorbance it can be deduced that the samples are relatively close in their electronic transitional structure since they both have an absorbance of close range and this occurs at the same wavelength.

Fig 5. 1: Combined Chart for the Ethanol Yield

CONCLUSION

From the chart above it would be observed that after the 1 gram yeast concentration the 5 gram yeast concentration gave the highest yield, this validates the region chosen for analysis (between 1gram and 5grams of yeast). Although this experimental data

chart shows that yield is highest at more dilution taking 1 gram of yeast into consideration as this seems to have a more systematically defined pattern. It can be concluded that the yield of ethanol is greatly dependent on the quantity of fermentable sugars present in the biomass.

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http://www.chemcases.com/alcohol/alc-03.htm