Ethanol production from cellulosic materials of sugarcane scum by bio-fermentation and acid hydrolysis

doi:10.21203/rs.3.rs-1563315/v1

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Research Article

Ethanol production from cellulosic materials of sugarcane scum by bio-fermentation and acid hydrolysis

https://doi.org/10.21203/rs.3.rs-1563315/v1

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posted 20 Apr, 2022

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Bioethanol is a promising candidate and the eco-friendly material for reducing greenhouse gases and it can be a good alternative to fossil fuels. We describe a development of framework for green fuel production. The production of bioethanol as a fuel has been investigated from sugarcane scum (bagasse, which can be hydrolyzed and fermented by high carbon content). In this process, carbohydrates were converted into monomers and then by fermentation of these monomers leads to the production of ethanol. Samples were prepared at different experimental conditions (Saccharomyces and acidic media for hydrolyzing) to carry out the fermentation process. Finally, identification and characterization of ethanol for the best condition was performed by GC-MS (500 gr bagasses, 0.25 gr yeast in acidic media and boiling temperature). The use of cheap yeast is a novelty of this work because it provides commercialization of bioethanol production which is very important in ethanol generation.

Bioethanol

Sugarcane scum

Saccharomyces cerevisiae

Acid hydrolysis

New energy production methods that do not have the disadvantages of classical methods are as common global approaches. These new methods have several major sections such as burning of biomass, production of bioethanol and bio-thermo chemical gases. The main sources for bioethanol production are livestock waste, urban sewage, industrial wastewater, agricultural waste and also it was obtained from the anaerobic fermentation of biomass. In the past few decades, bioethanol has become an important part of renewable fuel sources. The global warming, air pollution and oil futures are the main reasons for interest in ethanol production as a good alternative for oil [1–3]. Nowadays, the raw materials for the production of ethanol are sugar cane juice (sucrose) in Brazil and corn (starch) in America, and there are more attempts to introduce new sources for bioethanol production [4]. Lignocellulosic waste was used as the original source for production of ethanol from 2015 lignocellulosic biomass (LB) and other forms of renewable urban garbage can use for production of bioethanol. According to studies, lignocellulosic waste was used as the original source for production of ethanol from 2015 [5, 6]. The LBs composed of polymeric carbohydrate such as cellulose and hemicellulose. Production of ethanol from LBs is carried out in four steps: pretreatment, hydrolysis, fermentation/recovery and purification, respectively [7–13]. The organic materials receive treatment before beginning the fermentation process [14, 15]. Pretreatment step is consist of an acid pre-treatment, steam explosion, ammonia expansion and alkaline wet oxidation [16]. In pretreatment, LBs structure was deconstructed, following hydrolysis step in which monosaccharides are produced from cellulosic and hemicellulosic hydrolysis. Hydrolysis and fermentation processes can be performed separately or simultaneously. The basic challenge in the production of ethanol from these methods is its production costs, for example; the cost of using the cellulosic enzymes is 30–50% of the total bioethanol production cost. Therefore, the advanced technology is critical to reduce production costs [16–18].

The production of fermentation enzyme using cellulosic substrate can be the solution to reduce the cost of ethanol production. Among the various methods of fermentation, the fermented liquid immersion method has many advantages and widely used. The abilities of this technique can be monitoring and controlling of gas flow rate, pH, temperature and dissolved oxygen [19]. Many strategies have been reported for production of bioethanol. Dias et.al. studied a comprehensive evaluation on 115 types of sugar cane. This assessment evaluated the amount of fiber and efficiency of its fermentation before dilution with acid and enzymatic hydrolysis [20]. There are numerous reports to the evaluation of other parts of the biomass sugar cane (tops, straw) to develop the ethanol production process [21–25]. Many of the remaining lignocellulosic in the sugar cane industry is used to produce ethanol [26]. However, one of the most important factors for production of ethanol from biological waste is low cost in terms of economic. For this purpose, we define the inexpensive method for ethanol production. In this work, different parameters such as pH, temperature, amount of bagasse and water, amount of yeast, Presence and absence of yeast, sample mode (powder and non-powder) were evaluated for determining of ethanol production yield. Also refractive index and Brix value were measured in each step and finally, GC-MS analysis was performed for determination of the produced compounds. Figure 1 is shown the flow chart of this procedure.

Apparatus

The spectra were recorded on an Agilent 5975C gas chromatograph coupled with mass spectroscopy 7890A (Agilent-USA). The column was Agilent J&W DB-5 with a length of 60 meters, diameter of 0.25 mm and film thickness of 0.25 µm (Agilent–USA). The model of refractometer was 2WAGBB (China), andmetrohm 867-pH-meter (Switzerland) was used for adjusting pH.

Reagents

All of the reagents and solvents were of analytical grade and H₂SO₄, MgSO₄, KH₂PO₄, urea and NaOH were purchased from Fluka, Switzerland. Saccharomycescerevisiae was obtained from Razavi Company (Quchan/Iran) and Sugarcane waste (bagasse) was prepared from sugar cane factory in Bahnamir (Mazandaran/northern Iran).

Sample Preparation and Recommended Procedures

The culture medium was prepared as following: 1 gr pepton, 0.6 gr yeast, 0.6 gr bagasse, 1 gr glucose, 0.25 gr NH₄SO₄, 0.1 gr urea, 0.1 gr KH₂PO₄ and 0.2 gr agar that was reminded for 24 h in 28-30 ^oC at incubator. Stock samples were prepared in different experimental conditions. Free bagasse samples were containing only pure molasses. Crushed sugar cane waste samples were boiling for 30 minutes. These samples was remained at pH=2 for 24 hours (for better analysis) and then NaOH (1 N) was added until pH increased to 6. Finally, Saccharomyces was added for carrying out the fermentation process. Each of the samples was in anaerobic conditions at 37 °C in the oven for 48 hours then was filtered with a filter paper. Since the yeast is destroyed in acidic pH, it was added to samples after increasing its pH. After passing sufficient time, each of the samples (50 mL) were again filtered and the solution was collected. After distilling of this solution, refractive index and Brix value was recorded by refractometer. Also, the gas chromatography was used for more investigation and determination of composition of each sample.Then various chemical and physical conditions were investigated to determine the best mod for the production of ethanol. Therefore, 25 droplets from each of the samples, which were prepared in different condition, were filtered and distilled, and then refractive index and Brix value was recorded. The gas chromatography was performed as follow, the carrier gas of helium at 1 mL.min^-1, temperature programing started at 50 ˚C and with ratio of 4 ˚C min^-1 reached to 220˚C and remained in this temperature for 20 min. The synthesized bioethanol (1 µL) was injected to GC and identification of compounds was performed by NIST and WILEY software [26-28].

The effect of different experimental conditions on refractive index, Brix value and ethanol production yield were investigated. The values of refractive index, Brix value and ethanol production yield of samples are presented in table 1 (additional data are given in Online Resource 1). For each sample, 10 times sampling was carried out and each sampling were consisted of 25 drop in different times. Then refractive index, Brix value and ethanol production yield were determined. The Brix value is a measure of the amount of sugar in a solution that depends on the refraction of light. One degree of brix is equal to one gram of sucrose per 100 grams of solution. Brix grade represents the percentage by weight of solids in a solution to the total weight of the solution, or in other words, the weight percentage of solids in solution. So the high or low level of brix is affected by the amount of solid in the products [29-30].

Effect of Temperature

Bagasse is in non-powder form in the samples 1 and 2 and fermentation was carried out without acidic or basic hydrolysis conditions at both room and boiling temperature. The obtained bioethanol in 10th series containing 25 drops of each sample at consecutive times after collecting the distillated samples. As shown in table 1, the most amount of ethanol was in the first 25 drops of both samples 1 and 2. Also, comparison of these results shows that in the boiling temperature due to destruction of cellulosic tissue and better releasing of monomers and increasing ethanol production. It seems that, cellulose structure in bagasse due to boiling condition breaks down well, following more sugar released and more alcohol was produced [25].

Effect of pH

To investigate the effect of pH on the yield of ethanol production, acidic and basic hydrolysis fermentation was performed (samples 3 and 4 in Table 1). Sample 3 shown higher efficiency of fermentation by acid hydrolysis.

Effects of Sample Mode and the Amount of Yeast

In samples 6 to 14 amounts of bagasse and yeast were decreased. The powdered and non-powdered effects of bagasse were examined in ambient and boiling temperature. At the same amounts of water, bagasse and yeast (sample 10 and 13), the powder mode has a greater effect on the fermentation process of ethanol than the non-powdered one. By examining the effect of yeast on samples 3 and 5, it can be seen that the fermentation efficiency is higher in the presence of yeast.

GC-MS Analysis

1 µl of synthesized ethanol in the best condition was injected to the GC-MS device (fig. 2). From table 1 it is noted that this information is corresponding to the tube 3 from sample 8 that has the best conditions for the production of ethanol according to Brix and refractive indexes. In this sample with highest amount of ethanol production, four compounds were identified such as ethanol, carbon dioxide, and acetone and benzyl alcohol. As shown in Table 2, the amount of ethanol was 88.38%.

In summary, we have reported a variety of experimental conditions to access the maximum amount of ethanol production. Cellulose structure in bagasse was broken down well in boiling condition. Using available and inexpensive materials such as yeast is novelty of this work, because it provides the possibility of commercialization of production of bioethanol. Also the obtained GC-MS results of sample confirm the high efficiency of ethanol production. Planning and creating an industrial ethanol production line using widely used and available materials can reduce production costs and create more economical production.

Acknowledge

The authors would like to kindly acknowledge all the supports and funding from Golestan University.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing of interest:

The authors have no relevant financial or non-financial interests to disclose

Authors’ contribution:

Pouyan Asadi: provided the conception and design of the study, acquisition of data, analysis and interpretation of data, Elham Baher: supplied the design of study, analysis and interpretation, Zahra Vaezi: analysis of the results and gave final approval of the version to be submitted, Salma Ehsani Tilami: aided to the analysis of the results.

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Table 1 Results of different experimental condition on Refractive index and Brix value and ethanol production yield

Result			pH			Temperature		Sample mode		Yaest (g)	Water (mL)	bagasses (g)	Test no.
Yield (%)	Brix index	Refractive index	Neutral	basic	acidic	Non-boiling	boiling	Non-Powder	Powder				Test no.
98/09	1/75	1/338	*	-	-	-	*	*	-	0/25	500	500	1
97/78	1	1/335	*	-	-	*	-	*	-	0/25	500	500	2
99/92	3/5	1/345	-	-	*	-	*	*	-	0/25	500	500	3
98/9	1/75	1/338	-	-	*	*	-	*	-	0/25	500	500	4
97/94	1/25	1/336	-	-	*	-	*	*	-	-	500	500	5
97/8	0/75	1/334	-	*	-	-	*	-	*	-	100	20	6
97/87	1	1/335	-	*	-	*	-	-	*	0/06	100	20	7
98/09	1/75	1/338	-	*	-	-	*	-	*	0/06	100	20	8
97/94	1/25	1/336	*	-	-	*	-	-	*	0/06	100	20	9
97/87	1	1/335	*	-	-	-	*	-	*	0/06	100	20	10
98/02	1/5	1/337	-	-	*	-	*	*	-	0/06	100	20	11
97/87	1	1/335	*	-	-	*	-	*	-	0/06	100	20	12
97/8	0/75	1/334	*	-	-	-	*	*	-	0/06	100	20	13
97/8	0/75	1/334	-	-	*	-	*	-	*	0/06	100	20	14

Table 2 Compounds in the sample with the highest efficiency of ethanol production from GC-MS analysis

no.	compound	retention time (min)	extraction yield %

1	CO₂	4.010	0.52
2	Ethanol	4.385	88.38
3	Acetone	4.582	10.71
4	Banzylalcohol	16.297	0.39

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Ethanol production from cellulosic materials of sugarcane scum by bio-fermentation and acid hydrolysis

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Abstract

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Introduction

Experimental

Results And Discussion

Conclusions

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