Globally interest is growing for production of ecological sustainable bio fuels. Ethanol is a significant product of 21st century with its versatile usages and widely consumption across the globe. The U.S. Energy Information Administration's recently released International Energy Outlook 2016 (IEO2016) projects that world energy consumption will grow by 48% between 2012 and 2040. Most of this growth will come from countries that are not in the Organization for Economic Cooperation and Development (OECD), including countries where demand is driven by strong economic growth, particularly in Asia. Non-OECD Asia, including China and India, accounts for more than half of the world's total increase in energy consumption over the projection period.

Pakistan exiting capacity of fuel grade ethanol is 270000 tons per year, while it has a potential of 400000 tons per year¹.

Ethanol is one of the most important renewable liquid biofuels and its use in large scale contributes directly to the reduction of the environmental impact of fossil fuels, particularly in the transportation sector. First generation production technologies are able to convert sucrose or starch hydrolysates into fuel ethanol while second generation technologies are based on the use of lignocellulosic materials for the same purpose. However, this latter production process is much more complicated because it involves different unit operations including raw material preparation, pretreatment, washing to remove inhibitors, enzymatic hydrolysis, hexose and/or pentose fermentation, ethanol recovery and effluent treatment^2,3,4.

The study relates to a simple and economic process to convert Bagasse waste from Sugar mills and albumin waste from Egg processing industries for the cost effective production of ethanol with Saccharomyces cerevisiae.

One important source of great potential, particularly in India is bagasse waste generated from sugar cane. Lignocellulosic biomasses like sugarcane Bagasse is the world renewable resource in the biosphere⁵. The current demand is to make truly economic process for the conversion of sugar cane waste, bagasse for the production of alcohol. Ethanol from bagasse is thrust and more competitive, since global biofuel manufacturers are currently making rapid progress in the development of the cellulose-ethanol technology. There are numerous advantages related to the utilization of ethanol, especially improved quality of the urban air and its association with the reduction of CO₂, heavy metals, nitrogen oxides and hydrocarbon emissions, abundant, renewable and less expensive raw material such as lignocellulosic residues as a source of fermentable sugars for conversion into ethanol⁶.

Sugarcane bagasse is a plentiful byproduct obtained from the sugar industry, a lignocellulosic, residual material derived after the extraction of cane juice which corresponds to about 25% of the total processed sugarcane⁷. It is almost completely burnt by sugar factories themselves as fuel for boilers⁸. Recently, more efforts have been directed toward more efficient utilization of sugarcane bagasse as a raw material for pulp and paper production, boards, animal feed, and products based on fermentation⁹. Like most agricultural residues, bagasse is rich in soluble sugar, cellulose, hemicellulose, and lignin, which promotes research capabilities on bioconversion processes of this material for the production of ethanol¹⁰.

Sugarcane bagasse is an agricultural by-product which has the general composition of 45-50% cellulose, 23-30% hemicellulose, and 20-25 % lignin. The composition varies from geographical location and age of the plant. The pith content is in the range of 10-35%. Sugar cane bagasse is one such readily available raw material, which represents one of the most important Indian agricultural residues, with an estimated annual surplus of 16 million tons. Most of it is currently used to burn directly for steam generation and heat, which not only use inefficient but also, pollute the air⁶.

Lignocellulosic biomasses have only one problem is that enzymatic hydrolysis yield cannot be greater than 20% of the theoretical maximum glucan conversion, even under a high level of enzyme loading or by employing longer reaction time ¹¹. So to enhance this yield lignocellulosic biomasses need some pretreatment methods to alter the structure for greater enzyme accessibility for conversion of cellulose into glucose units. Hence, a suitable pretreatment method must remove structural barriers that limit the conversion of these materials to fuels and chemicals.

Pretreatment is a key step for the successful chemical or biotechnological processing of lignocellulosic materials because it is responsible for the break-down of the chemical association that exist among the main macromolecular components of plant cell wall, cellulose, hemicelluloses and lignin^{2,3,4,12,13,14}. Pretreatment must produce substrates that can be easily converted to fermentable sugars by acid or enzymatic hydrolysis and prevent the release of inhibitors for the subsequent steps of hydrolysis and fermentation². Besides, an ideal pretreatment method must be economically viable and environmentally friendly. Main pretreatment methods are; Milling and grinding, pyrolysis, high-energy radiation, high pressure steaming, alkaline or acid hydrolysis, gas treatment (chlorine dioxide, nitrogen dioxide, sulfur dioxide, ozone), hydrogen peroxide treatment, organic solvent treatment, hydrothermal treatment, steam explosion, wet oxidation and biological treatment¹⁵. The cellulose content present in these substrates is hydrolyzed by mixture of enzymes which converts it into glucose which is important factor in ethanol production from lignocellulosic biomasses¹⁶.

Apparently enzymatic conversion of albumin hydrolysate from egg albumin waste accumulated by egg processing used as inexpensive nitrogen source for the production of ethanol from Saccharomyces cerevisiae. Egg is delicate product that needs to be handled properly, all though the modern facilities are available with technology up gradation but the accumulation of broken eggs during transport from poultry to processing industry, manual or mechanical loading are unavoidable and highly prone to egg breakage. The albumin normally is a monomer with a molecular weight of 66,000 to 69,000; however, it may also form polymers, with proportionally higher molecular weights. Conversion of albumin hydrolysate that serves as nitrogen source enriched in phenylalanine, tyrosine, and methionine.

Ethanol can be produced by variety of microorganisms. Cellulose-to-ethanol bioconversion can be conducted by various anaerobic thermophilic bacteria such as Clostridium thermocellum¹⁷, Zymomonas sp¹⁸., Engineered Escherichia coli¹⁹ as well as by some filamentous fungi including Monilia sp²⁰, Neurosporacrassa²¹, Neurospora sp²²., Zygosaccharomycesrouxii²³, Aspergillus sp²⁴, Trichodermaviride²⁵and Paecilomyces sp²⁶

This study is focused on combination of chemical and enzymes on systematic and stage wise treatment to produce free reducing glucose and free amino acid peptides of the lignocellulosic biomasses (sugarcane Bagasse) and egg albumin waste were mixed in the ratio of 3:1 used for the cost effective production of ethanol from Sacchromyces cervisiae. The unhydrolysed part of Bagasse is further processed for the production insoluble fiber for as dietary supplement.

2. MATERIALS AND METHODS:

The main objective of the present study is to provide an economical and efficient process for the production of ethanol from sugarcane Bagasse waste, which was selected as inexpensive cellulosic feed stock. Apparently enzymatic conversion of albumin hydrolysate from egg albumin waste accumulated by egg processing was investigated as suitable and inexpensive nitrogen source for the production of ethanol from Saccharomyces cerevisiae.

2.1 Sample collection:

Sugarcane bagasse was collected from the local market. It was washed with tap water to remove debris and dried in oven at 65°C. After drying, it was grounded into 200 to 100 micron size of 1000g by using Ultra Centrifugal Mill(ZM 200 - RETSCH)

Egg albumin waste was collected from various egg processing industries.

2.2 Analytical method for Milled Bagasse:

Total Solids (TS), Volatile Solids (VS), Organic matter, Total Organic Carbon (TOC), Total Nitrogen (TN), Total Phosphorus (TP), Total Potassium (TK) were determined according to standard methods²⁷.

2.3 Production of Cellulose using Chemical-Enzyme coupled treatment:

One of the significant problem with enzymatic hydrolysis processes is the large amount of cellulase enzyme required, which increases the cost of the process. We also avoid the soluble inhibitors produced during pretreatment soluble phenolic compounds derived from lignin and disclose the complete removal.

In this study, combination of both chemical and enzyme coupled process for cellulose to glucose conversion. The first process step is a chemical treatment, at alkaline pH 8.5 at 50 deg C temperature break lignin, tannin and to lose the fibrous material.

Understanding the problem of using high concentrated acid hydrolysis the present process demonstrate the combination of chemical and enzymes to produce high levels of ethanol. Enzymatic hydrolysis process works very effectively because of the pretreatment and as the process is couple with acceptable concentrations of alkali and acid and treatment time are chosen to be significantly milder than usual acid hydrolysis process, such that the exposed cellulose surface area is greatly increased to break the fibrous feedstock and coupled with enzyme treatment.

Method: Step wise addition of enzyme to release reducing sugars from Bagasse:

Pretreatment by Chemical method:

Milled Sugarcane bagasse 200 to 100 micron size of 1000g with 5 to 7% pith was soaked with 1:20 (w/w), reverse osmosis water at pH 8.5 with 1 N NaOH and hold at three different temperature 30, 40, 50 deg C for 2 to 4hours, to know the optimum temperature for the release of reducing sugar. Reducing sugar such as cellulose was calculated by DNS assay ²⁸. The soaking enhances the water absorption of the fiber matrix and alkaline pH release the solubility of the lignin and tannin discharges in to aqueous phase²⁹.

Enzymatic treatment:

The left over biomass is washed and adjusted with 1:5 (w/w) reverse osmosis water, neutralized with repeated reverse osmosis water wash and treated with Neutral cellulase (IndiAge® Excel neutral cellulase of Genencor) at 40Deg for different time intervals 60 minutes. Optimum time interval was 60 minutes, releases 20 to 25% reducing sugars. Further the substrate pH was adjusted to 4.5 with 1 N HCl Neutral cellulase was deactivated and further treated with Acid cellulase (DENICELL Acid cellulase (AETL India)) and hold at 40 deg C temperature. The second stage of Acid cellulase addition releases another 15 to 20% of the reducing sugar from bagasse. The stage wise addition of above cited enzyme contributes 40 to 50% conversion of Bagasse in to reducing glucose units. Every stage of the chemical and enzyme treatment the sugar release is calculated with DNS reducing sugar methods²⁸. The performances on enzyme concentration vs. reducing sugar molecule release are calculated for enzyme addition.

DNS assay:²⁸

Add 3 ml of DNS reagent to 3 ml of sample taken after from alkaline treatment, Neutral cellulase (IndiAge® Excel neutral cellulase of Genencor) and acid cellulase (DENICELL Acid cellulase (AETL India)) enzyme treatment in a lightly capped test tube. Heat the mixture at 90ºC for 5-15 minutes to develop the red-brown color. Add 1ml of a 40% potassium sodium tartrate (Rochelle salt) solution to stabilize the color. After cooling to room temperature in a cold water bath, record the absorbance with a spectrophotometer at 575nm.

2.4 Production of albumin hydrolysate using Enzyme treatment:

A process for treatment of Egg effluent broth is cooked at 80°C for 5 minutes added at 50% concentration to RO water and treated with neutral protease and peptidase to convert the albumin in to albumin hydrolysate.

Method: Processing Albumin hydrolysate from egg effluent broth:

Albumin waste processed from egg processing companies was investigates as suitable nitrogen source for the effective production of ethanol on batch wise with the addition of albumin hydrolysate. A method of producing albumin hydrolysate from albumin rich waste liquid discharges from egg process industries involves cooking egg effluent broth at 80 deg C 10 minutes to obtain the 50% of solids with the 90% of protein content, on weight basis which was filter pressed, separated washed with sterile RO water. The solid protein obtained is taken at 50% concentration in RO water and treated with steps of adding Neutral protease (AETL) and endo-proteases (Protamex®) (Novozyme) and exo-peptidases (Flavourzyme®) and neutral peptidase at pH 7 at 40 Deg C for 60 minutes (AETL) to convert the albumin in to solubilized albumin hydrolysate used as potential protein source for the ethanol production from saccharomyces. The reaction was done at 37 Deg C for 60 minutes with subsequent addition of enzymes on stage wise for every 15minutes. The 90% conversion of hydrolyzed albumin hydrolysate is obtained and sterilized at 121°C 15 Psi for 10minutes used for the production of ethanol.

2.5 Production of ethanol from cellulosic and albumin hydrolysate:

The aqueous portion obtained from bagasse waste that contains 50% conversion of bagasse to reducing sugar is mixed with 90% of albumin hydrolysate from egg albumin waste were taken in the different ratio of 1:1, 2:1 and 3:1 of carbohydrate : nitrogen is fermented to produce ethanol by using yeast, the ethanol is recovered and purified by distillation. The process is performed at static fermentation at anaerobic conditions and tested on batch mode.

2.6 Production of dietary cellulose from hydrolyzed Bagasse:

The obtained unhydrolysed bagasse fiber would be free from tannin, lignin, silicates, reducing sugars, dust and anti-nutritional factor. 100 gram solid content of unhydrolysed product from sugarcane bagasse waste was soaked in RO water in 1:7 ratios and treated with 10% w/w food grade hydrogen peroxide in water. The mixture was cooked at 90⁰C for 10 minutes. The mixture was centrifuged, washed, neutralized, pressed and air dried.

3. RESULT AND DISCUSSION:

3.1 Characteristics of Bagasse sample:

General characteristics of milled sugarcane bagasse were shown in the table 3. Milled bagasse sample contains 54.8% of Total Solids (TS), 96.69% of Volatile Solids (VS), 95.92% of Organic matter, 54.05% of Total Organic Carbon (TOC), 1.757% of Total Nitrogen (TN), 0.245% of Total Phosphorus (TP), 2% of Total Potassium (TK) and 28.6 C/N ratio were analyzed in the Table 1.

Table 1: % Yield of Cellulose by Chemical enzyme treatment

S.No	Parameters	Unit	Bagasse
1	Total Solids (TS)	(%)	54.8
2	Volatile Solids (VS)	(%)	96.69
3	Organic matter	(%)	95.92
4	TOC	(%)	54.05
5	Total-N	(%)	1.757
6	Total-P	(%)	0.245
7	Total-K	(%)	2
8	C/N ratio		28.646

3.2 Production of cellulose by Chemical-Enzyme coupled treatment:

Ethanol has been produced from Sugarcane bagasse waste and egg albumin waste.

Chemical treatment:

The main advantages of the alkali pretreatment are removal of lignin and increasing the availability of cellulose for the bacterial metabolism during the anaerobic digestion process³⁰. Also lignin removal is an important part of the pretreatment process, because lignin can effectively inhibit/prevent the cellulase enzymes from hydrolyzing the cellulose. Alkaline pretreatment by adding NaOH solution causes a swelling of the biomass, which increases the internal surface area of the lignocellulose particles, as well as weakening the structural integrity of the lignocellulose and breaking bond linkages between lignin and the other carbohydrates (cellulose and hemicellulose), resulting in greater accessibility and digestibility of the cellulose fraction, and it can be depolymerized into fermentable sugars³¹. In alkaline treatment, 40 deg Celsius is the optimum temperature for the maximum of releasing sugar for 2-4 hrs in soaking was shown in the Table 2 and Graph 1.

Table 2: % yield of Cellulose at different temperature in alkaline treatment

S. No	Treatment	Temperature	Cellulose (%)
1	1N NaOH	30	10
2	1N NaOH	35	13
3	1N NaOH	40	15

Graph 1: % yield of Cellulose at different temperature in alkaline treatment

Enzyme treatment:

Deligninbagasse treated with stage wise addition of neutral cellulase to release 25% of reducing sugars. Further treated with acid cellulase to add up conversion of 10 to 15% of reducing sugars. Chemical-enzyme coupled treatment combined to produce nearly 50 % conversion of bagasse in to reducing sugars was shown in the Table 3 and Graph 2.

3.3 Production of albumin hydrolysate using Enzyme treatment:

Apparently sterilizing the egg effluent broth at 121 deg for 10 minutes ate 15 psi involves the precipitation of solids by 50% on weight basis. Separation of albumin solids by filter press and neutralized with Sterile Ro water and step wise addition of addition of alkaline protease, neutral protease and peptidase to break albumin polymers to release reducing oligopeptides and amino acids at 37 deg C temperature in 4 to 6 hrs time to cover 90 % of albumin into albumin hydrolysate.

3.4 Production of ethanol from cellulosic and albumin hydrolysate:

The product obtained from sugarcane bagasse and egg albumin wastes were mixed in three different ratios 1:1, 2:1 and 3:1and inoculated with yeast (Saccharomyces cerevisiae) for the effective production of ethanol. The process is performed at static fermentation at anaerobic conditions on batch mode. Recovery of ethanol from different ratio of carbohydrate: nitrogen shown in Table 4. It was to conclude that 3:1 ratio of carbohydrate: nitrogen enhances the conversion of ethanol to 79 g/L yield shown in the Graph 3. The process was performed at static fermentation at anaerobic conditions and tested on batch mode.

Table 4: Recovery of ethanol with different C:N

S. No	C:N	Yield (g/L)
1	1:1	34
2	2:1	50
3	3:1	79

Graph 3: Recovery of ethanol with different C:N

3.5 Production of dietary cellulose from unhydrolyzed bagasse:

Unhydrolysed part of Bagasse is further processed for the production insoluble cellulose fiber for as dietary supplement. The process resulted in a 70 % recovery of insoluble cellulose or fibers. The obtained insoluble cellulose fiber was white in color and ground to fine powder with 100 micron particle size. The purity was about 99% and the dietary cellulose has properties of water absorption 1:9 and oil absorption 1:3.

4. CONCLUSION:

To provide an economical and efficient process for the production of ethanol for which sugarcane Bagasse waste was selected as inexpensive cellulosic feed stock. Enzymatic conversion of albumin hydrolysate from egg albumin waste for ethanol production. Carbohydrate produced from bagasse waste by chemical-enzyme coupled treatment and proteins from egg albumin waste by enzyme treatment sources were combined at definite C:N ratio for the fermentation of Saccharomyces cerevisiae, by batch mode of operation to yield a 79 g/L of ethanol. The hydrolyzed bagasse fiber was further processed for dietary cellulose. It was to conclude that Cost effective production of ethanol from bagasse and egg albumin waste using chemical and enzyme treatment overcomes the disadvantages of the prior art in making the economic ethanol from industrial waste.

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Received on 25.11.2022 Modified on 04.01.2023

Asian J. Research Chem. 2023; 16(2):149-154.

DOI: 10.52711/0974-4150.2023.00024

S. No	Treatment	Method	% Yield of Cellulose
1.	Chemical treatment	Alkaline treatment (1N NaOH)	15
2.	Enzyme treatment	Neutral cellulase (IndiAge® Excel neutral cellulase of Genencor)	25
2.	Enzyme treatment	Acid cellulase (DENICELL Acid cellulase (AETL India))	15
	Total Recovery		50