Impact of Storage Time, Rain and Quality of Molasses in the Production of Bioethanol

Due to environmental concerns and the rising prices of petroleum products, usually bio-ethanol is being produced in greater amount from sugar cane molasses in Pakistan. In this work various properties related to molasses are being analyzed by performing experiments at the Distillery Plant, AASML (AlAbbas Sugar Mills Limited), Mirpurkhas, Sindh, Pakistan. Brix, Sucrose, pH, ethyl alcohol, RS (Reducing Sugar), TSI (Total Sugar as an Invert), TS (Total Sugar), purity and presence of micro-organisms are scrutinized. In addition, the effects of quality parameters of pre-fermentation and fermentation for ethyl alcohol production, time impact on stored molasses and consequences of rain on quality of molasses are studied. It was observed from the study that good quality molasses which is the raw material for alcohol production should be used in fermentation process. Molasses must be stored in steel tanks to prevent it from contamination and loss of fermentable sugars. The prefermenters need to steam sterilized, cleaned and washed before inoculation of yeast cells so that the yeast grows healthy to produce maximum ethyl alcohol in fermenters. The water used for hydrolysis of molasses in fermentation should be clean, filtered and good quality nutrients must be used for the growth of yeasts. Finally, efficiency and yield are calculated and t-test has been performed to depict a direct relationship of the fermentable sugar with efficiency.


INTRODUCTION
M olasses, the thick liquid remaining after sucrose has been removed from the mother liquor (of clarified concentrated cane or beet juice), in sugar manufacture. Blackstrap molasses is the syrup from which no more sugar may be removed economically. Therefore, it is called as a by-product of sugar manufacturing process. It has usually been subjected to at least three evaporating and centrifuging cycles to remove the crystalline sucrose. Its analysis varies considerably, depending on many factors, including sugar-mill equipment and operational efficiency, but it may contain approximately 40-45% by weight of

Impact of Storage Time, Rain and Quality of Molasses in the Production of Bioethanol
fermentable sugars and approximately 10% ash (or salts) [1][2]. The composition of black strap molasses varies with the variety of cane and the method of processing. The large amount of inorganic salts in molasses exerting high osmotic pressure and the toxicity to yeast cells are important factors during fermentation. Due to repeated crystallization of sucrose in sugar manufacture, inorganic salts are concentrated which interfere with the enzyme system of the yeast thereby reducing final ethanol yield and typically only 90% of the molasses is utilized [3]. The blackstrap molasses is utilized in numerous industries such as process, polymer, beverages, fuel and farming [4]. Saccharomyces cerevisiae is the species of yeast used for the fermentation of molasses to produce ethyl alcohol with improved efficiency. Nevertheless, molasses is one of the vital components in diverse industrial processes.
The biochemical reaction of yeast with diluted molasses produces enzymes called Zymase that works as a catalyst to convert sugar into ethanol, carbon dioxide and heat [5]. The industrial alcohol production capacity of two units of distillery plant, AASML is 170,000 liters per day and the CO 2 is released around 140 tons per day. CO 2 is captured, liquefied for sale to other industries like beverages companies [6].
The advancement in biomass consumption for power sector is the important factor in many parts of the world due to enhanced concerns on the energy sector and environmental pollution. This is true for the developing countries like Pakistan that is an agricultural country and producing huge amount of biomass. Molasses is an indigenous low-cost feedstock producing ethanol which is used to make E 10 fuel. E 10 fuel is a mixture of 90% petrol and 10% ethanol, i.e. a renewable energy source [7]. Sugarcane is an important crop of Pakistan for energy sector because of the higher proportion of the biomass in the shape of fermentable sugar present in it generating biofuels such as ethanol. Generally, the production system of sugarcane is complex, giving various products and by-products such as, sugar, molasses, baggase and filter cake [8][9][10]. The by-product of sugar milling process baggase is largely used for power generation that supply electricity to the sugar mill, distillery plant, bio-gas plant, due to thepromotion of Independent Power Producers and Small Power Producers [11]. The black strap molasses, a by-product of sugar mill process is currently used as the main feedstock for bioethanol generation. Around 60% of the total bioethanol manufacturing in the year 2013 was from molasses [12][13][14]. Even though ethanol manufacture in countries like Brazil, Thailand and Pakistan has currently developed into an established industry, there are still concerns on the environmental sustainability [15].
Experimental results proved that the quality of molasses collected from the Punjab and KPK (Khyber Pakhtunkhwa) was better than the Sindh and the results were presented in our previous work [16]. Due to availability of good quality fertilizers, chemicals and pesticides, fertile lands, canal and tube well water, better harvesting practices, well defined process of sugar manufacturing and appliance of up-to-date agricultural studies, the quality of molasses collected from these areas is comparatively better. The three main contributions of this work are to investigate the impact of quality of molasses, storage time and rain for the production of ethyl alcohol.
The rest of the paper is organized as follows. Section 2 presents materials and methods, including procedures for determination of Brix, RS, TSI, TS, purity, efficiency and yield. Section 3 elaborated the results and their discussion. Finally, section 4 states the concluding remarks.

MATERIALS AND METHOD
Experimental work was conducted at the laboratory of Distillery plant AASML, Mirwah Gorchani, Mirpurkhas.
The laboratory is ISO-9001 certified and the testing techniques are following the standards. Process diagram of Distillery plant AASML [17] is shown in Fig. 1  (1)

Impact of Storage Time, Rain and Quality of Molasses in the Production of Bioethanol
The mixture is maintained at about 30-32 o C for 40 hours.
During this period the enzymes, invertase and zymase present in yeast bring about the conversion of sugar to ethyl alcohol. During this process; the liquor froths owing to the evolution of CO 2 that is collected and liquefied at CO 2 plant for sale to industries including carbonated beverages, urea production, refrigeration, etc. The

Sampling and Analysis
In this section, analysis procedure for determination of Brix degree, RS, TSI, sucrose, TS, purity and alcohol percentage in pre-fermenters and fermenters are presented [18][19]. The apparatus used during the analysis are:

Process to Determine Brix
Brix is a measure of the density or concentration of sugar solution.
Initially, 200 gm of molasses sample was placed in a beaker and 200 ml of distilled water was mixed in it. Then the sample was shifted in 250 ml glass cylinder and was kept in the icebox at 20 0 C for 20 minutes. Then hydrometer (40-50 range) was placed in the sample for two minutes and the reading was taken and Brix was calculated as:

Process to Determine Reducing Sugar
The term, reducing sugar denotes the reducing substances in cane or its products calculated as invert sugar. 100 ml of diluted sample was taken in 250 ml The reducing sugar [18][19]

Process to Determine TSI
This test determined the TS percentage which illustrated the quality of molasses. In this experiment, 10 gm sample of molasses was taken in a beaker using weighing balance and mixed with small amount of distilled water. The Sucrose plus reducing substances is expressed as TS.

TS = Sucrose + TSI
The percentage ratio of sucrose to the total soluble solids (or Brix) in a sugar product.

Process to Determine Alcohol Percentage in Fermenters and Prefermenters
The alcohol percentage was checked by Ebulliometer apparatus. Hydrometer was filled with 50 ml sample and then heated. When the rise in temperature was stopped, reading was noted and confirmed into reference temperature disk [18][19].

Fermentation Efficiency and Actual Yield
The fermentation Efficiency percentage and actual yield percentage were calculated using the following Equations (9-10).

RESULTS AND DISCUSSION
In this section the effect of prefermentation on fermentation, effect of time on stored molasses, consequences of rain on molasses quality, overall efficiency and yield are discussed.

Effect of Prefermentation on Fermentation
The quality of molasses plays an important role in the fermentation process. Tables  giving higher alcohol percentage than unit-2. Because in unit-1 higher quality molasses was used which demonstrated higher alcohol percent and production than the unit-2 in which low quality molasses was used.
As the result the cell value of unit-1 is more than unit-2.
However, the Brix and pH values of both units are approximately alike.

Time Impact on Stored Molasses
The al. [14] have studied successfully the application weak Saccharomyces cerevisiae strain on sugarcane molasses for the improvement in the industrial alcohol production. Table 3 highlights the changing values of Brix, sucrose

Consequence of Rain on Quality of Molasses
During the rainy season last year the quality of molasses was badly affected which was stored in open ponds. Fig. 5 and Table 4

Relationship of Fermentable Sugar with Efficiency and Yield
In Fig. 6

CONCLUSION
Due to increasing cost of oil, it is common practice to utilize sugarcane and molasses in the production of bioethanol in Pakistan. In this work it has been analyzed that the molasses with enhanced fermentable sugar levels yields more yeast cells and alcohol as compared to molasses having diminutive fermentable sugar. The rain also affects negatively because bacteria's were producing acids at low Brix as the molasses was diluted with rain water. The experimental results also revealed that better quality raw material (molasses) obtained maximum yield Molasses must be stored for a month before use in distillery as fresh molasses is difficult to ferment. Fresh molasses has high foaming tendency, buffering capacity and suspended sludge. It is also suggested that molasses should not be stored more than 6 months as fermentable sugar could be reduced.