Production of ethanol from alkali-pretreated sugarcane bagasse under the influence of different process parameters

Abstract

The present study was designed to produce ethanol from sugarcane bagasse treated with 2.5% NaOH through a simultaneous saccharification and fermentation process using Saccharomyces cerevisiae G1. Various process parameters such as incubation temperature, incubation period, initial pH and nitrogen sources were studied to achieve the maximum yield of ethanol. The results showed that the optimum ethanol yield (5.90%) was achieved at an incubation temperature of 30°C, initial pH 5.5, inoculum size 3% (v/v), after an incubation period of 96 h. Among various nitrogen sources, ammonium sulphate was found to be the most suitable source for ethanol production. These findings indicate that the optimization of process parameters is necessary to make the fermentation process economical, and the medium designed in the present study could be exploited on a commercial scale after suitable processing.

Keywords:

• Ethanol
• Saccharomyces cerevisiae G1
• Simultaneous
• saccharification
• fermentation

Introduction

Pakistan, being an agricultural country, produces a large amount of agricultural waste. About 56.22 million tons of agricultural waste are produced annually, comprising 19.83 million tons from wheat, 12.87 million tons from sugarcane, 12.46 million tons from cotton, 8.16 million tons from rice and 2.90 million tons from maize (ESCAP 2000). Agricultural waste as well as by-products have been extensively used for the production of commercially significant products including fuel-grade bioethanol. Bagasse is generally produced as a by-product during the processing of sugarcane in the sugar industry. About 75% of bagasse is utilized as domestic fuel for the combustion process and the remainder is used as a raw material for making low-value products (Dawson & Boopathy 2008). Sugarcane bagasse is mainly composed of cellulose, hemicellulose and lignin (Dawson 2005); the cellulose and hemicelluloses comprise 40–45% and 25–35%, respectively (Kim & Day 2011; Masarin et al. 2011). Alkaline treatment of bagasse produces 75% of the cellulosic compounds (Peng et al. 2009). After pretreatment, saccharification of alkali-treated bagasse is another important step to produce glucose, which is used for ethanol production in the alcohol industry. Xylose is the other main product of saccharification of lignocellulosic biomass for ethanol fermentation (Sun & Cheng 2002). However, cost-effective alcohol production is still the major issue faced by the alcohol industry for its exploitation as a fuel. Various processes, such as simultaneous saccharification and fermentation (SSF) and separate saccharification and fermentation, have been adopted to reduce the cost and increase the production of alcohol (Zheng et al. 2009; Svetlana et al. 2012).

The present study was designed to use the alkaline-pretreated agroindustrial by-product (bagasse) efficiently for the production of low-cost ethanol by employing SSF. Various process parameters were also optimized to obtain the maximum yield of ethanol.

Materials and methods

Microorganism

The strain of Saccharomyces cerevisiae G1 used in the present work was obtained from the Microbiology Lab, FBRC, PCSIR (Lahore, Pakistan). The culture was revived on a potato dextrose agar (Oxoid) slant after incubation at 30°C for 4 days. The culture was then preserved at 4°C and transferred to new slants every 30 days to keep it viable.

Processing of raw material

The lignocellulosic raw material (sugarcane bagasse) was obtained from Shakarganj Sugar Mills (Faisalabad, Pakistan). Sugarcane bagasse was washed with hot water to remove soil dust and residual sugar, dried in a hot air oven, then sealed in polythene bags.

Pretreatment of bagasse

The processed bagasse was crushed in a hammer beater mill of mesh size 2 mm to convert it into powdered form. Powdered bagasse was then soaked in 2.5% NaOH solution for 1 h and thereafter autoclaved at 126°C for 45 min. The autoclaved sample was filtered and solid residues were washed many times with distilled water up to neutrality (Irfan et al. 2011). After drying in a hot air oven, the solid residues were processed for estimation of cellulose content by the method of Gopal and Ranjhan (1980).

Simultaneous saccharification and fermentation

SSF of pretreated biomass was performed in a 1000 ml conical flask (Pyrex), along with a loop trap as described by Taherzadeh and Karimi (2008). About 20 g of substrate was initially mixed in 300 ml distilled water containing various nutrients, i.e. 0.225 g MgSO₄ and 1.05 g K₂HPO₄, and then autoclaved at 121°C for 15 min at 15 psi. After autoclaving, a saccharifying enzyme was added to the substrate with a ratio of 4 g : 20 g, to make an enzyme–substrate mixture in 300 ml distilled water. The mixture was then inoculated with yeast culture under sterilized conditions and incubated at 30°C at 120 rpm in a water bath shaker (Eyela, Japan). The sample was withdrawn periodically, at 24 h intervals, using sampling pots, for the analysis of sugars and ethanol.

Optimization of process parameters

Various process parameters which influenced on the production of ethanol were studied to increase the yield of alcohol produced by yeast through SSF using the strain of S. cerevisiae G1.

Incubation temperature

SSF was carried out at various incubation temperatures, ranging from 25°C to 45°C, to find the optimum temperature for the maximum yield of alcohol by S. cerevisiae G1.

Incubation period

SSF was conducted for different incubation periods, ranging from 24 to 144 h, at the preoptimized incubation temperature, to maximize the ethanol yield.

Size of inoculum

Different inoculum sizes (1–5%) were studied at the preoptimized conditions to find the best suitable inoculum size of S. cerevisiae G1 for optimum ethanol yield.

Initial pH

Various initial pH values such as 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 were studied to find the optimum pH for the maximum yield of ethanol. Each value was adjusted with 1 N NaOH/HCl prior to sterilization of the growth medium.

Nitrogen sources

Organic sources (yeast extract, peptone, tryptone and urea) and inorganic sources (ammonium sulphate, ammonium nitrate and ammonium chloride), each at 1% concentration, were used to evaluate their effects on ethanol production.

Estimation of sugars

Different sugars, comprising total sugars, reducing sugars and glucose, were estimated. Total sugars were estimated by the method of Dubios et al. (1956). Reducing sugar was estimated according to the method described by Miller (1959), whereas glucose was estimated using an enzyme-based glucose estimation diagnostic kit, developed by PCSIR (Karachi, Pakistan).

Ethanol estimation

The ethanol was determined by the procedure of Captui et al. (1968). The fermented broth (5 ml) was placed in a Pyrex distillation flask containing distilled H₂O (150 ml). The distilled material was collected in a 50 ml measuring cylinder containing 25 ml potassium dichromate solution. About 20 ml of distillate was collected in each sample and the volume reached up to 45 ml. Then, the sample was kept at 60°C for 20 min in a water bath. The sample was cooled down to room temperature and the volume made up to 50 ml. Ethanol was then estimated by measuring the optical density at 600 nm against a blank with a spectrophotometer.

Ethanol yield and efficiency

Ethanol yield and fermentation efficiency were calculated according to the following equations: Theoretically, 1 g of glucose can produce 0.51 g of ethanol.

Statistical analysis

All the experiments were carried out independently (in triplicate) in 250 ml Erlenmeyer flasks. The data represented here are in the form of mean ± SD. Significance is presented as Duncan's multiple range test results in the form of probability (p = 0.05) values, which were obtained using CoStat software.

Results and discussion

Lignocellulosic biomass cannot be saccharified by enzymes to yield high sugar without pretreatment, mainly because the lignin in the plant biomass forms a barrier against enzyme attack (Sewalt et al. 1997). Therefore, pretreatment before saccharification is a necessary step to reduce the lignin content and increase the surface area for better enzyme attack. In the present study, pretreatment gave 74% cellulose and 9% lignin contents, while untreated substrate contained 35% cellulose and 25% lignin contents, as shown in Table 1. Chemical delignification of sugarcane bagasse resulted in decreased lignin content (6.94%) and increased cellulose content (78.184%) (Wahono et al. 2014). Previous studies suggested that sugarcane bagasse is the best substrate for ethanol production among several tested substrates under SSF (Shrivastava et al. 2014). On the basis of high cellulose content, the pretreated substrate was employed in the SSF process to produce ethanol by S. cerevisiae G1.

Table 1. Comparison of the main contents of untreated and treated sugarcane bagasse.

Substrate	Cellulose (%)	Lignin (%)	Total phenol (mg/ml)
Untreated sugarcane bagasse	35 ± 2.0	25 ± 1.5	0.000
Treated sugarcane bagasse	74 ± 3.0	9.0 ± 1.0	0.012 ± 0.002

In SSF, the pretreated substrate was initially loaded with 4 g of commercial enzyme to produce the maximum sugars required for ethanol production, and then inoculated with yeast culture. The inoculated broth was incubated at different temperatures (25–45°C) to find the most suitable incubation temperature for the maximum yield of ethanol. The results showed that maximum ethanol production (5.23%) was obtained at a temperature of 30°C and a further increase in temperature decreased ethanol yield (Table 2). A decline in ethanol production above 30°C has also been reported in another investigation (El-Refai et al. 1992). In another study, an incubation temperature of 39°C was reported for maximum yield of ethanol by S. cerevisiae in SSF (Liu et al. 2014). Elumalai and Thangavelu (2010) optimized incubation temperature by response surface methodology and reported 35°C as an optimum for maximum ethanol production through SSF.

Table 2. Effect of various incubation temperatures on fermentation efficiency and ethanol production by Saccharomyces cerevisiae G1 in the simultaneous saccharification and fermentation process.

Temperature (°C)	Sugar (mg/ml)	Ethanol (%)	Ethanol yield (g/g)	Fermentation efficiency (%)
25	28.321 ± 0.134	2.54	0.0843	16.52
30	20.471 ± 0.069	5.231	0.1377	27
35	20.102 ± 0.099	5.201	0.1356	26.58
40	34.431 ± 0.064	2.231	0.0928	18.19
45	56.912 ± 0.402	0.0421	0.0274	5.37

Note: initial pH = 5; incubation period = 96 h; inoculum size = 2% (v/v).

Table 3. Effect of various incubation periods on fermentation efficiency and ethanol production by Saccharomyces cerevisiae G1 in the simultaneous saccharification and fermentation process.

Incubation period (h)	Sugar (mg/ml)	Ethanol (%)	Ethanol yield (g/g)	Fermentation efficiency (%)
24	38.213 ± 0.125	1.541	0.0761	14.92
48	29.423 ± 0.210	4.951	0.17055	33.44
72	25.912 ± 0.160	5.212	0.1602	31.41
96	21.451 ± 0.152	5.851	0.1581	31.0
120	19.321 ± 0.123	5.751	0.1469	28.80
144	19.212 ± 0.144	5.701	0.1453	28.49

Note: initial pH = 5; incubation temperature = 30°C; inoculum size = 2% (v/v).

Incubation period is another important parameter which directly influences the production cost of any fermentation process. To find the optimum time interval for ethanol production in SSF, fermentation was carried out at various time intervals (24–144 h) and samples were analysed at intervals of 24 h. Maximum ethanol yield was obtained in SSF after 96 h of incubation by S. cerevisiae G1, as shown in Table 3. Higher ethanol production was observed after 72 h incubation in another study (Ballesteros et al. 2004). Variation in incubation period may be due to differences in the behaviour of microorganisms used in the fermentation process as well as the availability of glucose from pretreated biomass in SSF.

To find the optimum inoculum size in ethanol fermentation, the inoculum sizes were varied from 1% to 5% (v/v). The results show that 3% inoculum size resulted in the maximum ethanol production (5.98%), as shown in Table 4. Increase in inoculum concentration beyond this concentration resulted in a decline in ethanol yields. It has been reported previously that the amount of sugar consumed and ethanol produced increased linearly with an increase in initial cell concentration from 2.0% to 10%, while the maximum ethanol yield was obtained with 6% (v/v) inoculum size of S. cerevisiae (Stenberg et al. 2000).

SSF was carried out at different initial pH values (4.0–7.0) at intervals of 0.5 to obtain the maximum output from 2.5% NaOH-treated bagasse. It was found that, of the various pH values, pH 5.5 gave maximum ethanol production (5.992%) from S. cerevisiae G1 (Table 5). At values higher or lower than pH 5.5, a sharp decrease in ethanol production was observed, whereas in another study the best ethanol production rates were observed between pH 4 and 6 (Margaritis et al. 1987). A recent study has shown that the initial pH of 4.8 was optimum for ethanol production in SSF of steam-exploded corn stover by S. cerevisiae (Liu et al. 2014).

Table 4. Effect of various inoculum sizes on fermentation efficiency and ethanol production by Saccharomyces cerevisiae G1 in the simultaneous saccharification and fermentation process.

Inoculum size (%)	Sugar (mg/ml)	Ethanol (%)	Ethanol yield (g/g)	Fermentation efficiency (%)
1	26.401 ± 0.356	4.852	0.1514	29.68
2	20.451 ± 0.344	5.350	0.1407	27.58
3	18.213 ± 0.222	5.989	0.1523	29.86
4	19.145 ± 0.240	5.752	0.1465	28.72
5	19.231 ± 0.218	5.452	0.1391	27.27

Note: initial pH = 5; incubation temperature = 30°C; incubation period = 96 h.

Table 5. Effect of various initial pH values of the growth medium on fermentation efficiency and ethanol production by Saccharomyces cerevisiae G1 in the simultaneous saccharification and fermentation process.

Initial pH	Sugar (mg/ml)	Ethanol (%)	Ethanol yield (g/g)	Fermentation efficiency (%)
4.0	35.351 ± 0.275	3.521	0.1524	29.88
4.5	30.415 ± 0.301	4.452	0.1588	31.14
5.0	24.910 ± 0.255	5.531	0.1649	32.33
5.5	20.122 ± 0.214	5.992	0.1563	30.65
6.0	29.311 ± 0.331	4.982	0.1710	33.53
6.5	36.391 ± 0.291	3.303	0.1497	29.35
7.0	38.912 ± 0.370	2.521	0.1290	25.29

Note: incubation temperature = 30°C; incubation period = 96 h; inoculum size = 3% (v/v).

Nitrogen sources have major effects on the growth and yield of ethanol. Therefore, various organic and inorganic nitrogen sources were studied to select the best nitrogen source for high yield of ethanol in the SSF process. Among various nitrogen sources, ammonium sulphate gave a comparatively better yield of ethanol, as shown in Table 6. These results indicate that yeast cells easily utilized various nitrogen sources to metabolize nitrogenous substances for their growth and other metabolic activities. A similar trend towards an increase in the yield of ethanol by S. cerevisiae 3090 in the presence of nitrogen sources has also been reported in another investigation (Benerji et al. 2010).

Table 6. Effect of various nitrogen sources on fermentation efficiency and ethanol production by Saccharomyces cerevisiae G1 in the simultaneous saccharification and fermentation process.

Nitrogen source		Sugar (mg/ml)	Ethanol (%)	Ethanol yield (g/g)	Fermentation efficiency (%)
Organic	Yeast extract	21.245 ± 0.266	5.821	0.1564	30.67
	Peptone	29.958 ± 0.187	4.921	0.1727	33.86
	Tryptone	30.391 ± 0.127	4.921	0.1754	34.39
	Urea	29.41 ± 0.049	4.281	0.1474	28.90
Inorganic	Ammonium sulphate	20.912 ± 0.188	5.891	0.1569	30.77
	Ammonium nitrate	31.910 ± 0.141	3.981	0.1499	29.39
	Ammonium chloride	30.121 ± 0.473	3.121	0.110	21.57

Note: initial pH = 5.5; incubation temperature = 30°C; incubation period = 96 h; inoculum size = 3% (v/v).

Conclusion

This research was conducted to produce low-cost ethanol from lignocellulose material through the SSF process by employing a locally isolated strain of S. cerevisiae G1. For this purpose, bagasse was treated with 2.5% (w/v) NaOH solution to expose maximum cellulosic contents. This pretreated material was used as a substrate for ethanol production in the SSF process. Various other parameters, including incubation temperature, initial pH, fermentation time and the effect of nitrogen sources on ethanol yield, were analysed in the present study. The results showed that maximum ethanol production (5.90%) was achieved after 96 h of incubation at 30°C in the presence of ammonium nitrate as a nitrogen source. These findings indicate that bagasse, which is a cheap and readily available raw material in Pakistan, has tremendous potential as a substrate for ethanol production. However, there is still a lot of work required before for commercial-scale production can become economically viable.

Acknowledgement

The authors would like to thank the Ministry of Science and Technology (MoST), Islamabad, Pakistan, for providing the financial support to carry out this research work, under the PSDP project entitled ‘Production of bioenergy from plant biomass'.

Disclosure statement

No potential conflict of interest was reported by the authors.