AN APPRAISAL OF THE ETHANOL PRODUCTION FROM CASSAVA (MANIHOT ESCULENTA

ABSTRACT

The process of ethanol production generally involves pretreatment, hydrolysis of lignocellulosic biomass to fermentable sugars followed by fermentation of such sugars to ethanol. Waste of the cassava peel (Manihot esculenta) was hydrolysed by using sulphuric acidThis research aimed to produce of bioethanol as an alternative source of fuel using cassava peels as raw materials. Yeast isolated from Durian fruit (Durio zhibetinus) was used in the experiment for fermentation and the concentration of sulphuric acid of hydrolysis process was fermented by yeast for 1; 2; 3; 4; 5; 6; 7; and 8 days. 50 ml of Sodium Hydroxide NaOH was prepared to be added at this step to adjust the pH of the slurry until 5 and the temperature was kept at 25 °C. Nine samples were prepared at different three hydrolysis times at 121°C for 30 minutes, 45 minutes and 60 ninutes. For glucose consumption and ethanol product analysis, 2 ml of the sample were taken out at every 2 days interval until 8 days. During this fermentation process, sugar consumption was measured by DNS method, while quantification of ethanol was analyzed by Gas Chromatography. The result of this study obtained that the best time of hydrolysis process was 45 minute, where the result of concentration of glucose was 11.189 %. By virtue of that, fermentation process was influenced by shaking incubator at 6 days. the optimum concentration of sulphuric acid of the hydrolysis process was 30 minute, and duration time of fermentation process by shaking incubator was 8 days, while the concentration of bioethanol for the highest of hydrolysis and fermentation process was obtained 1.63 % ethanol.Go to:

CHAPTER ONE

1.1 Materials and Methods

The cassava variety TMS 30572 was selected for the study. It is characterized by high yield (44 ton ha⁻¹) and above ground biomass as well as a high content of dry matter (~40%). The leaves and roots of this variety hold high levels of cyanogenic glycosides (HCN-equivalents ~800 mg kg⁻¹, dry weight) compared to other common Ugandan cassava varieties. The different plant parts (stem, leaves, root, and root peels) were collected separately, 5 kg of each, in triplicate. The material was collected uniformly from individual plants to avoid tissue differences, immediately stored at 4°C and transferred to the laboratory.

1.2 Treatment of Feedstocks

Stem and peel samples were chopped, washed, and dried at room temperature for 3 h before manually crushing them with a hammer, allowing the resulting particles to pass through a sieve (size 5 mm). Samples were put into glass bottles prior to hydrolysis. Leaf and root samples were washed, dried for 3 h and chopped into fine particles (<2 cm diameter).

1.3 Compositional Analyses of Feedstocks

The treated samples were analyzed for dry matter, ash, lipid, and protein content as well as for starch. Dry matter content (DMC) was determined by placing the samples in an air-forced oven at 105°C until a constant weight was attained, after which the percentage of dry matter was calculated. The ash content was determined by burning the air-dried samples at 550°C for 8 h in a furnace. The samples were cooled in a desiccator and the percentage ash was calculated. Total organic content of the feedstocks was calculated as the difference in weight between the ash content of the feedstock and the dry matter content. Total protein content of the samples was analyzed using the Bradford method (Bradford 1976) and the sample preparation procedures included analyzing the colored compounds in the stem, peel, and leaf matter, measuring the absorbance of untreated samples prior to the protein test. Starch content of the feedstocks was determined in triplicates (100 g wet weight) after enzymatic hydrolysis, using an enzyme solution comprising amylase (3000 IU ml⁻¹, BDH Laboratories) and amyloglycosidase (60 IU ml⁻¹, SIGMA, Aldrich). The liberated amount of glucose was quantified as described by Dubois et al. (1956). Lipid content of the samples was determined by means of extraction, using a hexane chloroform mixture (1:1, volume ratio), after which the weight loss of the samples was estimated and the percentage lipid content was calculated (Nuwamanya et al. 2010).

1.3 Hydrolyses of Samples

Stem, leaf, root, and peel samples were hydrolyzed using the following hydrolysis methods. Acid and alkaline hydrolyses were carried out on triplicate samples of matter (stem, leaf, peel, and root separately), 200 g each in 200 ml solution, 1 M HCl for the acid hydrolysis, and 1 M NaOH for the alkaline hydrolysis. From each solution, samples for analyses of reducing sugar content were taken on an hourly basis for 8 h and thereafter daily for 5 days. Enzymatic hydrolyses were carried out on triplicate samples of 200 g wet weight, using a combination of different enzymes; amyloglycosidases (60 IU ml⁻¹, SIGMA, Aldrich), amylase (3000 IU ml⁻¹, BDH Laboratories) and cellulases (75 IU ml⁻¹, SIGMA, Aldrich) in a 200-ml solution. The amount of the different sugars liberated by the enzymes was noted along with the environmental conditions under which the liberation occurred. Samples for analyses of the content of reducing sugars were taken from each solution on an hourly basis for 8 h, and thereafter daily for 5 days.

Estimation of Reducing Sugars

The method of Dubois et al. (1956) was used to estimate the total amount of reducing sugars produced during hydrolysis.

1.4 Microorganism Preparation and Sugar Fermentation

The glucose-fermenting microorganism Saccharomyces cerevisiae was used. Cells were grown on 1% (wv⁻¹) yeast extract, 2% (wv⁻¹) peptone, and 2% (wv⁻¹) glucose and mixed with 20% (ww⁻¹) glycerol and subsequently stored in vials at −20°C.

1.5 Sugar Fermentation and Estimation of Fermentation Efficiency

After successive saccharification procedures, the mash was cooled to room temperature (~27°C), keeping the pH at 4.5–5.0. A yeast extract was then added to allow fermentation. Inoculum was produced by inoculating a 1000 ml shake flask (500 ml culture volume constituted from the hydrolysis mixtures above) with 7.5 ml of frozen yeast cells. The contents were subsequently transferred to a 2-l shake flask (1 l culture volume) and consequently used for the fermentation, which was carried on for 6 days with regular mixing of the mash (Sassner et al. 2006). Fermentation efficiency was assessed by measuring the decrease of the amount of reducing sugars in the fermenting solution (beer) and was performed hourly for 8 h and thereafter daily for 6 days.

1.6 Ethanol Recovery and Ethanol Evaluation

Ethanol was separated from the beer (500 ml) using a three-phase distillation procedure in which the first distillation was carried out at a temperature range of 20–94°C to recover the first distillate. The distillate was then redistilled at 90°C twice consecutively, to produce ethanol (60–65%) for further evaluation. The amount of ethanol produced from each 500 ml batch of the beer, was used to calculate the ethanol yield. To ascertain the quality of the ethanol produced, various procedures were utilized as described below.

Visual measurements based on the visible properties of ethanol were used to evaluate the color and aspect of ethanol at different processing stages and concentrations according to National Response Team (NRT) quick reference guide (www.nrt.org/production/NRT/NRTWeb.nsf).

Ethanol clarity was analyzed by transferring samples of the produced ethanol to a cuvette and measuring transmittance of light at 650 nm in a spectrophotometer, using double distilled, deionized water and 95% ethanol as standards. The level of acidity of the processed ethanol was assessed by titration with NaOH (aq.) (Titratable acidity), using phenolphthalein as indicator (Fabro et al. 2006). Titratable acidity was expressed as the total volume of 0.1 M NaOH (aq.) used to neutralize the existing acid. The pH-value of the produced ethanol was appraised using a pH-meter. The conductivity of ethanol was measured with a conductivity meter, with a cell constant equal to 0.1 cm⁻¹. Presence of sulfate and chloride in the produced ethanol was assessed by means of precipitation analysis. A 0.1-M barium chloride solution was added drop wise (up to 0.5 ml) to 0.5 ml ethanol to study possible formation of barium sulfate, a salt insoluble in water solutions. Similarly, presence of chloride ions was studied by drop wise adding up to 0.5 ml of silver nitrate solution to a 0.5-ml ethanol sample and the possible precipitate of solid silver chloride were studied.

To determine lipid content, dried samples were extracted using a hexane chloroform mixture (1:1, volume ratio), after which the weight loss of the samples was estimated and the percentage of lipid content was calculated (Nuwamanya et al. 2010).

The percentage of ethanol in the azeotropic mixture was calculated as below.

i.e. a/v = (m − v_*sgb)/(sga − sgb)/v, where (a/(v) = % ethanol, a is the volume of ethanol, (v − a) = b = volume of water, sga = 0.789 is the specific gravity of the ethanol, sgb = 1 = the specific gravity of water, m = the mass of mixture, v = the volume of mixture.

1.7 Statistical Analysis

The Microsoft Excel software was used for descriptive statistics of the feedstocks used. Composition of untreated feedstocks was defined as the average of at least three independent samples originating from the same lot of material, while in the treated feedstocks, two samples of the test solution were used to estimate the composition of reducing sugars. Further statistics involved the analysis of variance as well as correlation and regression analyses, using the GENSTAT Discovery Edition 3 software. All tests were performed at 5% level of significance. ANOVA was used to test the difference between the means of different parameters for feedstock composition as well as the properties of ethanol produced from different feedstocks. After ANOVA, the least significant differences were used to test the difference in means of quantitative ethanol properties. Correlation analysis highlighted relationships between the different ethanol properties and the composition of feedstocks. Covariance was used to test the effect of other factors on the resulting properties of the ethanol.

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Results

Composition

The amount of dry matter varied among different plant parts with dry matter content of peels (30.5%) and stems (28.77%) comparable to that of roots (38.60%). High levels of protein were observed in above ground parts with leaves having the highest levels (12.3%) and the roots exhibiting the lowest (0.38%). The content of ash was considerably lower in leaves and roots (0.33 and 0.27%, respectively) compared to peels and stems. Lipid contents were comparable in dried peels (0.39%) and leaves (0.36%) and somewhat lower in stems and roots (<0.3%). Composition of plant bio-molecules did not follow a particular pattern in the plants and varied in most instances.

1.8 Production of Reducing Sugars

Hydrolysis of feedstocks, using acid, alkali as well as enzymes, resulted in production of various amounts of reducing sugars (glucose + C_n-sugars, where n = 3, 4, and 5, and other reducing saccharides), as illustrated in Fig. 1. Analysis of variance revealed significant differences in the total reducing sugars produced from the different plant parts (see Table 3). During initial hydrolysis, stems and peels produced the highest amount of reducing sugars compared to roots and leaves, the latter producing the least amount. In roots, the use of cellulase alone resulted in degradation of only cellulosic material, not affecting the starchy material. In leaves, hydrolysis with sodium hydroxide and hydrochloric acid produced larger amounts of reducing sugars than enzymes (Fig. 1). In all plant parts, the enzymes required more preparative stages and time before reducing sugars were produced (Fig. 2). The amount of reducing sugars produced from the leaves did not differ significantly between the different methods of hydrolysis. Hydrolysis continued after 8 h for 5 days, accompanied by a corresponding increase of reducing sugar levels, as illustrated by Fig. 2. After 5 days, the enzyme hydrolysis yielded almost the same amount of reducing sugars as acid and alkaline-hydrolyzed feedstocks (Fig. 2). Percentage relative increase in total reducing sugars ranged between 37% for the peels and 74.8% for the leaves compared to reference material (roots), suggesting that roots are more susceptible to hydrolysis compared to other plant parts.