PRODUCTION OF BIO-ETHANOL FROM SUGAR BEET WASTES BY SACCHAROMYCES CEREVISIAE

PRODUCTION OF BIO-ETHANOL FROM SUGAR BEET WASTES BY SACCHAROMYCES CEREVISIAE

Khattab, A.E. ¹ ; S.M. Allam ² ; H.M.Z. El-Khamissi ¹

and K.Y.M. Yousef ²

¹ Agric. Biochem. Dept., Fac. Agric., Al-Azhar Univ., Cairo Egypt.

2 Sugar Crops Research Institute, Agriculture Research Center.

Key Words: acid hydrolysis, sugar beet wastes, bio-ethanol production, Saccharomyces cerevisiae.

ABSTRACT

Bio-ethanol is one of the energy sources that can be produced from renewable sources. Sugar beet wasteswere chosen as a renewable carbon source for ethanol fermentation because it is relatively inexpensive compared with other feedstock considered as food sources. However, saccharification processes are needed to convert cellulose of sugar beet wastes into fermentable or reducing sugars before ethanol fermentation. In this study, hydrolysis of sugar beet wastesand growth parameters of the ethanol fermentation were optimized to obtain maximum ethanol production by S. cerevisiae S288c. The ratio of plant material to acid solution of 1:10 (w/v). Results demonstrated that 0.5% H₂SO₄, 1% H₂SO₄,2% H₂SO₄ and 3% H₂SO₄ at 121ºC for 20 min by autoclave were enough to hydrolyze all cellulose contained in the Sugar beet wastes. The maximum yield of reducing or fermentable sugars was 124.80 mg/g obtained in 1% H₂SO₄. The minimum yield was 70.20 mg/g obtained in 3% H₂SO₄. The yield of bioethanol production by S. cerevisiae S288c was (50.96 g / 100 g) was achieved at pH 5.5, temperature of 30⁰C and inoculums size of 10% (v/v) after 72 hours of fermentation.

INTRODUCTION

Development in industrialization and growth of population have led to a continuous rise in global energy demand. At present, more than 80% of world energy production is from fossil fuel.  However, the depletion of fossil fuel is at an alarming rate and it causes environmental pollution (Láinez, et al., 2019). Therefore, there is a need for sustainable and renewable energy sources that do not affect the environment and ecosystems. Biofuels have emerged recently. Fuel tankers are ideal to meet the energy needs in a sustainable manner (Morais, et al., 2019). More specifically, can be used as an alternative oil sources bioethanol and has become one of the most dominated biofuels industry because the majority of the emissions of carbon dioxide, which contributed to the transport sector. In addition, ethanol has been renovated high energy oxygen content easily stored Zhang, et al., 2019).

Sugar content of sugar beet is about 25% higher than that found in sugarcane, but its production cost is more than twice as the production cost of sugarcane. Nowadays, the revenues of the by-products, that is, molasses, pulp, beet particulate matter, and carbonation lime, significantly offset the production cost of beet sugar. Moreover, mainly due to the financial support provided to sugar industry and its use to produce ethanol fuel, sugar beet cultivation is still increasing in many countries (Erdal, et al., 2007). The root of the beet, called taproot, is composed of 75% water and 25% dry matter. The dry matter comprises about 5% of the pulp and sugars represent 75% of the total dry matter. The pulp is insoluble in water and is composed of cellulose, hemicellulose, lignin, and pectin. The sugar content in sugar beet can vary from 12% to 20% (Marzo, et al., 2019). Bioethanol is primarily produced in the world from sugarcane, sugar beet, corn, and starch, although the preparation process from sugar beets is relatively simple compared to starchy materials (Gumienna, et al., 2016a).

Therefore, this investigation was carried out to study the utilization of sugar beet wastesas a very cheap substrate for the production of Bio-ethanol by Saccharomycescerevisiae S288c.

2. MATERIALS AND METHODS

2.1- Materials:

The sugar beet wastes (Beta vulgaris L.) were collected from Nile Sugar Company, Abu El-Mattamir, El Beheira, Egypt. They were dried at 50^◦C for 48 hours, ground and sieved to get particles with particle size between 500 and 1000 µm. They were stored at room temperature (25 ± 5^◦C) until use.

2.2- Chemicals and Reagents:

Chemicals and reagents of the analytical methods used in present study were sulfuric acid, sodium hydroxide, glucose, benedict's reagent, dinitrosalicylic acid reagent (DNS reagent), sodium sulphite, Rochelle salt (potassium sodium tartrate), phenol, yeast extract, malt extract, sodium chloride, peptone, agar, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, sodium hydroxide and potassium dichromate. They purified and distilled before use. All chemicals were purchased from El- Gamhouria Trading Chemicals and Drugs Company, Egypt.

2.3- Micro-organisms

S. cerevisiae S288c was obtained from Microbial Biotechnology Department, National Research Center (Dokki, Egypt). It was used in this study and maintained on yeast extract-malt extract (YM) agar slant at 4^oC

2.4 - Analytical methods:

2.4.1- Chemical composition of sugar beet wastes:

The chemical composition of potato starch wastes (PSW) was determined according to (Zhao et al., 2005) by Near-Infra Red (NIR) Spectroscopy apparatus, model DA1650, which manufactured by FOSS Corporation. (NIR) spectrometer at wavelength region from (750-2500 nm) of the electromagnetic spectrum, which used to analyze the chemical structure and to take a fingerprint.

2.4.2- Chemical pretreatment:

2.4.2.1- Acid pretreatment:

The pretreatment of sugar beet wastes (25g) was performed in a 250 ml conical flask employing H₂SO₄ 0.5%, 1 %, 2% and 3% (w/v) 1:10 solid-liquid ratio, then put in an autoclave at the temperature at 121°C for 20 min.

After hydrolysis, the resulting solid material (cellulose-lignin) was removed by filtration, washed and dried according (Dussan, et al., 2014).

2.4.2.2- Alkali pretreatment:

            After acid pretreatment (sugar beet wastes ) cellulose-lignin was soaked in a solution of 1.5 % w/v NaOH, 1:20 solid-liquid ratio and temperature at 90°C for 1 h. Afterward, the mixture was filtered to collect the cellulose, washed thoroughly with water and dried according (Dussan, et al., 2014).

2.4.3- Acid Hydrolysis or Saccharification:

            The sugar beet wastes residuals after acid and alkali pretreatment were dried at 80ᵒ C. H₂SO₄ 0.5%, 1 %, 2% and 3% (w/v) 1:10 solid-liquid ratio was added to the samples powder, the mixtures were then autoclaved at 121ᵒC for 15 min. Finally, the samples were cooled down and analyzed for glucose concentration. The pH value of the clear filtrate was adjusted to approximately 6.0-6.5 according (Sheikh, et al., 2016).

2.4.4- Determination of reducing sugars:

2.4.4.1- Qualitative analysis of reducing sugars:

            A biochemical test to detect reducing sugars in solution was devised by the US chemist S. R. Benedict (1884–1936). Benedict's reagent a mixture of copper (II) sulphate and a filtered mixture of hydrated sodium citrate and hydrated sodium carbonate is added to the test solution and boiled. A high concentration of reducing sugars induces the formation of a red precipitate; a lower concentration produces a yellow precipitate. Benedict's test is more sensitive alternative to Fehling's test.

2.4.4.2- Quantitative analysis of reducing sugars:

Total reducing sugars in the hydrolysate of sugar beet wastes were estimated by the dinitrosalicylic acid (DNS) colorimetric method adapted from previous work (Miller, 1961 and Ghose, 1987).

2.4.4.2.1- Preparation of standard solution

The standard glucose stock solution 10 g/L was prepared by dissolving 0.20 g of D-(+)-Glucose anhydrous (C₆H₁₂O₆) in 20 mL of distilled water. Working solutions were prepared daily by appropriate dilution of the stock solution in DI water.

2.4.4.2.2 Preparation of dinitrosalicylic acid reagent

3,5-dinitrosalicylic acid reagent was prepared by dissolving 1 g of 3,5-dinitrosalicylic acid in 20 mL of 2 M NaOH. It was then mixed with potassium sodium tartrate (C₄H₄KNaO₆) solution (30 g of C₄H₄KNaO₆ in 50 mL of distilled water) on a magnetic stirrer hot plate and diluted to 100 mL with DI water.

2.4.4.2.3- Calibration curve:

Calibration curve for estimation of reducing sugar yield was obtained by plotting the absorbance (at 520 nm) vs. concentrations of standard glucose in the range of 0.20-1.00 g/L. The concentrations of glucose were prepared daily by dilution of the stock solution.

2.4.4.2.4- Estimation of reducing sugar yield in the hydrolysate of potato starch wastes.

0.50 mL dinitrosalicylic acid was added to a test tube containing 0.50 mL of standard glucose or the hydrolysate of cellulose. It was then boiled at 100^oC for 10 min and cooled in an ice bath. Afterwards, 5 mL of distilled water was added, shaken and left for 5 min. The absorbance was measured at 520 nm against reagent blank using a UV-Visible spectrophotometer (Nicolet- evolution300- Thermo Electron Corporation). To calculate the quantitative of reducing sugar yield in the form of g/100 g substrate, the following equation was used:

Reducing sugar yield (g/100 g substrate) 14=RCأ— V1 أ—100 g 1000 mLأ— M1">

Where R_C is the reducing sugar concentration (g/L), V₁ is the volume of acid solution (mL), and M₁ is the mass of substrate added (g).

2.5-Fermentation process:

2.5.1- Preparation of inoculums medium:

S. cerevisiaeS288cwas activated on yeast extract-malt extract (YM) agar plates containing (per L): 3 g yeast extract, 3 g malt extract, 5 g peptone and 10g glucose. It was then incubated at 30^oC for 24 h, streaked into YM broth (pH ≈ 5.5) (all ingredients like the Petri dishes of YM agar, except agar powder) and incubated again at 30^oC for 24 h. To prepare inoculums for ethanol production, yeast cell suspensions in the broth were quantified and adjusted to 1×10⁸ cell/mL by using a heamacytometer, according to (Pridham et al., 1957).

2.5.2- Fermentation medium and conditions:

The acid hydrolysate of the cellulose under the appropriate conditions was adjusted to pH ≈ 5.5 using 2 M NaOH, supplemented with following additional nutrients (per L): 1 g yeast extract, 1 g MgSO₄.7H₂O, 2 g (NH₄)₂SO₄ and 5 g KH₂PO₄ (Akaracharanya, et al., 2011) and then used as an ethanol production medium. This medium with a working volume of 100 mL was added to a 250 mL Erlernmeyer flask and sterilized by autoclaving at 121^oC, 15 psi for 30 min. Then, an inoculums suspension of S. cerevisiae S288ccells was loaded into the sterilized medium (10% v/v). The fermentation was operated at 30^oC under static conditions for 72 h. The fermented broth was collected at 6 h time intervals to analyze ethanol concentration.

2.6.-Estimation of bioethanol:

2.6.1-Qualitative estimation

Bioethanol production was examined by Jones reagent (K₂Cr₂O₇+H₂SO₄; Jones 1953). One ml of K₂Cr₂O₇ (2 %), 5 ml of H₂SO₄ (concentrated) and 3 ml of sample were added to Jones reagent. Ethanol was oxidized into acetic acid with potassium dichromate in the presence of sulfuric acid and gave blue-green color. Green color indicates positive test (Caputi, et al., 1968).

2.6.2- Quantitative estimation:

Ethanol production was estimated according to (Sharma, et al., 1991) by detecting the ethanol concentration in each sample after fermentation under all biochemical conditions with High Performance liquid chromatography. The system Thermo (Ultimate 3000) consisted of pump, automatic sample injector, and associated DELL-compatible computer supported with Cromelion interpretation program. A Refracto Max520 refractive index detector was used. The Thermo-hypersil reversed phase C18 column 2.5× 30cm was operated at 30 ºC. The mobile phase was distilled water. After centrifugation of the fermented broth, the supernatant was analyzed for ethanol concentration using a HPLC.            

3- RESULTS AND DISCUSSION

3.1- Chemical composition of sugar beet wastes:

             Data in Table (1) shows the chemical composition of sugar beet wastes. The moisture content recorded 7.61%, the fatcontent 0.53%, the crude protein 10.50%, fiber content 17.55%, ash content 6.04% and the total carbohydrate 57.77%. Results indicate that the sugar beet wastes are very rich of carbohydrates, which represent an important source in the production of bioethanol.

These results conform to Mohdaly, et al., (2009) whomeasured that proximate composition of sugar beet pulp contained 69.0 g /kg⁻¹moisture, 69.2 g / kg⁻¹crude fat, 108.4 g /kg⁻¹crude protein, 66.4 g kg⁻¹ash, 178.2 g/ kg⁻¹crude fiber and 577.8 g/kg⁻¹carbohydrate. In addition to Mohdaly, et al., (2010) who observed that the composition analysis of sugar beet pulp contained moisture, crude fat, crude protein, ash, crude fibre, and carbohydrates being 6.90%, 6.92%, 10.8%, 6.64%, 17.8%, and 57.8%, respectively.

Concerning sugar beet molasses, Razmovski and Vučurović, (2012) measured that the quality parameters of sugar beet molasses were ash 8.36%, total nitrogen 2.78% and total sugar 53.05%. As regards sugar beet pulp, Berłowska, et al., (2016) mentionedthe chemical composition of sugar beet pulp was protein 11.5±0.25 g/kg and carbohydrate 742±42.4 g/kg. Finally, Pińkowska, et al., (2019) recorded that the chemical composition of sugar beet pulp was ash 23.7±1.2 g/kg, total protein 88.4±0.5 g/kg and total carbohydrate 423±5.9%.     

Experimentally, our study is identical with the preceding researches and proved that sugar wastes have a promising future for the production of bioethanol.

Table (1): Chemical composition of sugar beet wastes (Average).

3.2- Determination of reducing sugars:

3.2.1- Qualitative analysis of reducing sugars:

            Table (2) shows the qualitative analysis of reducing sugars after acid hydrolysis or (scarification) of sugar beet wastes by using Benedict's test. The sample A (H₂SO₄ 0.5%), a red precipitate appeared, sample B, (H₂SO₄ 1%) with a red precipitate appeared, sample C, (H₂SO₄ 2%) it showed up an orange precipitate, sample D (H₂SO₄ 2%) an orange precipitate appeared. These results are according to Benedict's test.

Table (2): Qualitative analysis of reducing sugars by Benedict's test

3.2.2- Quantitative analysis of reducing sugars:

Miller (1959) reported that dinitrosalicylic acid (DNS) was used to test for the present of free carbonyl group (C=O) present in reducing sugars. The aldehyde group in reducing sugars was reduced with 3,5-dinitrosalicylic acid to form 3-amino,5-nitrosalicylic acid, which absorbs light strongly at 520 nm, as presented in Figure (1).

Fig. (1). Chemical reaction of glucose with 3,5-dinitrosalicylic acid (Miller, 1959)

It was first introduced as a method to detect reducing substances in urine and has since been widely used, for example, for quantification of carbohydrate levels in blood. It is mainly used in assay of alpha amylase. However, enzymatic methods are usually preferred to DNS due to their specificity.

 The determination of reducing sugars by a UV-Visible spectrophotometer by used DNS method for estimation of total reducing sugar yield is presented in table (3).

Table (3): Estimation of total reducing sugar yield (mg/gm) by a UV-Visible spectrophotometer.

This table observed the dilute H₂SO₄ broke down cellulose and turned it into reducing sugar in the form of glucose. The results revealed that reducing sugar yield increased from 70.20 to 124.80 mg/gm when H₂SO₄ concentrations decreased. The maximum yield of total reducing sugar yield 124.80 mg/g was obtained in 1% H₂SO₄. The minimum yield of total reducing sugar yield 70.20 mg/g was obtained in 3% H₂SO_4, because sulfuric acid in the case of increased concentration removed a molecule of water from glucose and converd it to Hydroxy methyl furfural (HMF) compound. Hydroxymethylfurfural (HMF) is a product of dehydration of hexose sugars such as glucose, fructose and carbohydrates, as presented in Figure (3).

These results are in agreement with Köşebent, (2016) asthey measured that the reducing sugar analysis of sugar beet pulp hydrolysates were 39.38±0.21% (4% H₂SO₄₎ and 35.83±0.18% (2% H₂SO₄). Also, Mushimiyimana, and Tallapragada, (2016) observed that the reducing sugar in sugar beet peels (315.81µg/mL).

            Fig. (2): Formation of HMF from D-glucose (Tao et al., 2011)

3.3- Estimation of bioethanol:

3.3.1- Qualitative estimation

 After fermentation process bioethanol production was examined by Jones reagent (K₂Cr₂O₇+H₂SO₄; Jones 1953). One milliliter of K₂Cr₂O₇ (2 %), 5 ml of H₂SO₄ (concentrated) and 3 ml of sample were added to Jones reagent. Ethanol was oxidized into acetic acid with potassium dichromate in the presence of sulfuric acid and gave a blue-green color. Table (4) showed the results of bioethanol reaction with Jones reagent. It showed yellow color with control and sample before fermentation but it was green color with sample after fermentation. Tiwari, et al., (2015) mentioned that the Bioethanol production was examined by Jones reagent. Ethanol was oxidized into acetic acid with potassium dichromate in the presence of sulfuric acid and gave blue-green color. (Caputi, et al., 1968) reportedthat the ethanol with Jones reagent was green color indicating a positive test.

Table (4): Qualitative estimation of bioethanol by Jones reagent.

3.3.2- Quantitative estimation:

            Ethanol yield by HPLC and productivity obtained by fermentation of the hydrolysate are presented in Table (5). In this study, the hydrolysis of cellulose was performed using dilute H₂SO₄ further fermented to ethanol using S. cerevisiae S288c and it was found that the ethanol yield of 50.96 g/100 g substrate corresponded to a productivity ethanol yield of 0.71(g/ 100g/ h).

These results are in agreement with Magaña, et al., (2011) asthey measured that the yield of bioethanol in sugar beet pulp ranging, was pound to be from 21.9 to 31.0 g L^_1. Also, Vučurović and Razmovski, (2012) reported that the potential of sugar beet pulp (SBP) and dried sugar beet pulp (DSBP) as economically cheap and renewable supports for immobilization of Saccharomyces cerevisiae cells was investigated was observed. A maximum ethanol concentration by 52.26±2.0g/L. On the other hand, Zheng, et al., (2012) observed that the ethanol yield from ensiled sugar beet pulp was nearly 50% higher than raw sugar beet pulp.

Table  (5): Ethanol yield and productivity obtained by fermentation of the cellulose hydrolysate.

Under anaerobic conditions, the pyruvate is further reduced to ethanol with the release of CO2. Theoretically, the yield is 0.511 for ethanol and 0.489 for CO2 on a mass basis of glucose metabolized, as represented by the following Eq. (Zhang, et al., 2010)

C₆H₁₂O₆      14Yeast">          2CH₃CH₂OH   +       CO₂

Glucose 1g                    Ethanol 0.51g         Carbon dioxide 0.49g

CONCLUSION

The hydrolysis of sugar beet wastes by mineral acids was studied. Using 0.5% H₂SO₄, 1% H₂SO₄,2% H₂SO₄ and 3% H₂SO₄ at 121ºC for 20 min by autoclave, the ratio of plant material to acid solution of 1:10 (w/v), the highest fermentable or reducing sugars equivalent of 124.80 mg/g in 1% H₂SO₄. The minimum yield was 70.20 mg/g obtained in 3% H₂SO₄. The yield of bioethanol production by S. cerevisiae S288c was (50.96 g / 100 g) and it was achieved at pH 5.5, temperature of 30⁰C and inoculums size of 10% (v/v) after 72 hours of fermentation.

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إنتاج الإیثانول الحیوی من مخلفات بنجر السکر

 Saccharomyces cerevisiae

أحمد السید خطاب¹ ، صبری محمد علام² ، هیثم احمد زکی الخمیسی¹،

کریم یوسف محمد یوسف²

¹ قسم الکیمیاء الحیویة الزراعیة - کلیة الزراعة- جامعة الأزهر.

2- معهد بحوث المحاصیل السکریة – مرکز البحوث الزراعیة

یعتبر الایثانول الحیوی من أهم مصادر الطاقة الحیویة المتجددة الواعدة فى المستقبل وله فؤائد بیئیة واقتصادیة، و یمکن إنتاجه من مصادر مختلفة. وقد اختیرت مخلفات البطاطس النشویة کمصدر متجدد للکربون لإنتاج الإیثانول لأنها غیر مکلفة نسبیاً مقارنة بالمواد الخام الأخرى التی تعتبر مصادر غذائیة. وتجرى عملیات التحلل المائی بالأحماض المخففة لتحویل مخلفات بنجر السکر إلى سکرات قابلة للتخمیر  (سکرات مختزلة)  قبل  عملیة التخمر لإنتاج

الإیثانول. فی هذه الدراسة تم تحسین التحلل المائی لمخلفات بنجر السکر ومعدلات النمو للخمائر للحصول على أقصى قدر من إنتاج الإیثانول بواسطة S. cerevisiae S288c  وکانت نسبة المواد النباتیة إلى محلول الحمض 1: 10(و/ح) وأظهرت النتائج أن حمض الکبریتیک ترکیز 0,5٪ و1٪ و2٪ و3٪ عند 121ºم  لمدة 20دقیقة بواسطة الأوتوکلاف کانت کافیة لتحلل جمیع النشا الموجود فی مخلفات البطاطس النشویة. وکان الحد الأقصى لکمیة السکریات القابلة للتخمیر أو المختزلة 124,80ملجم/جم تم الحصول علیها عند ترکیز الحمض 1٪. وکان الحد الأدنى من السکریات القابلة للتخمیر أو المختزلة 70,20ملجم/جم  التی تم الحصول علیها عند ترکیز 3 ٪. و تم تحقیق أعلى إنتاج من الإیثانول الحیوی بواسطةS. cerevisiae S288c  وهو ( 50,96 جرام / 100 جم) فی درجة حموضة 5.5، ودرجة حرارة 30ºم وحجم التلقیح 10 ٪ (و / و)  بعد 72 ساعة من التخمیر.