Introduction

Environmentally sustainable energy sources are desperately needed because of the quickly expanding industrialization. The majority of the world's wealth is derived from nonrenewable fossil resources including natural gas, coal, and oil. They are employed in the production of fuel, power, and other things (Sarkar et al., 2012). With an increase in population, modes of transportation, and technological development, fossil fuel use rises, resulting in a depletion of natural raw materials. This circumstance causes to hike. In addition, greenhouse gases, which change the climate and worsen air pollution, are produced by fossil fuels. We now need to look for alternative energy sources due to factors such as the daily increase in global energy consumption, global warming, the depletion of fossil fuel resources, and the rising expense of fuels derived from petroleum. These sources need to be affordable, productive, and efficient while emitting fewer pollutants or having no adverse impacts (Nigam & Singh, 2011; Lhawang et al., 2021; Sirigeri et al., 2022). As a biofuel, plant biomass can be regarded as a good alternative fuel source. Due to its advantages of being easily available, affordable, and advantageous for the environment, bioethanol, which may be produced from lignocellulosic biomass, is growing in popularity (Chiaramonti, 2007).

Bioethanol can be used as a clean fuel in cars in the following forms: E85 (85 percent ethanol and 15 percent gasoline), E100 (100 percent ethanol with or without a fuel additive), and oxy-diesel (usually an 80/10 mixture of diesel, ethanol, and additives and blending agents). Additionally, if it is continuously generated in large quantities, ethanol can be utilized to generate energy. In contrast to the United States, where corn is mostly used to make ethanol, sugarcane molasses is used as the main raw material in India (Krishnan et al., 2020).

One can categorize biofuels as first- or second-generation (Agarwal, 2007). Second-generation biofuels are often produced from lignocellulosic biomass, which includes grasses, wood, and stems.

Under research are numerous second-generation biofuels, including biohydrogen, bioethanol, and mixed alcohols (Agarwal, 2007). "First-generation" bioethanol is produced through the fermentation of raw sugars, whereas "second-generation" bioethanol is produced through the use of raw lignocellulosic materials. Preliminary research is being done on the "third generation" of algae-based bioethanol (Rajeswari et al., 2022). In addition, cellulose plant material represents an untapped supply of fermentable sugars for a major application, particularly as non-food agricultural by-products like rice and wheat straw, bagasse, rice husk, etc.

Lignocellulosic Biomass (LB) like wood (soft and hardwood), agricultural by-products (Corn, wheat, and rice straw), grass, and waste, are captivating and durable alternatives. It is also utilized to produce bio-alcohol because it is available in large quantities. In addition, it is less expensive compared to food ingredients (based on sugar and starch). 442 billion liters of bio-alcohol can be produced annually using LB. The production of biofuels from lignocellulosic raw materials has the potential to replace approximately 30% of conventional fuels currently consumed by the United States (Hashim et al., 2022).

 Furthermore, second-generation ethanol is created from lignocellulosic feedstock, In this study, the main goal is to identify the non-edible plant for the synthesis of bioethanol and the objective was the extraction of bioethanol through a fermentation process (Devi et al., 2022). The study examined the plant A.caracasana as well as extracted bioethanol by fermentation using S. cerevisiae.

A.caracasana is a lignocellulosic plant with a high growth rate such plants can generate bioethanol that can be used as fuel for vehicles. The characteristic feature of this plant is rapid growth is essential to fulfilling the demand for daily usage of fuel. Lignin degradation is a crucial process in the extraction of bioethanol. Since A.caracasana is not a source of food for the human population, the extraction of bioethanol from it will not interrupt the production of food. Furthermore, the use of ethanol as an automobile fuel will benefit the environment by cutting emissions.

It is possible to reduce food consumption by producing bioethanol from lignocellulosic biomass such as A.caracasana, which is abundant. A.caracasana sugar concentration enables it to create ethanol. Having a high sugar content in plants means that bioethanol production has a lot of capability. Different pretreatment methods can be utilized in the conversion of sugar to ethanol, such as hydrolysis. A larger sugar extraction can be achieved using these pretreatment procedures, which break down hemicellulose and lignin. physical pretreatment is a typical pretreatment method that is both simple and effective at breaking down lignin. Fermentation follows the pretreatment of the pretreated material. During the fermentation process, sugar is transformed into ethanol (Krishnan et al., 2020).

Bioethanol

Bioethanol fuel contributes significantly to environmental protection since it slows global warming and conserves fossil fuels. It is an alcohol produced from carbohydrates by fermentation. Producing bioethanol from biomass or waste is one method for reducing both crude oil use and environmental pollution. Additionally, lignocellulosic biomasses, such as maize, sugar, molasses, etc., derived from edible sources like trees and grasses are used to produce ethanol feedstocks. Both bioethanol and ethanol have the same physical and chemical properties, but they are made from different materials. In its pure state, bioethanol is indeed a clear, odorless, colorless liquid. It freezes at 112°C and boils at 78°C (Lau et al., 2009) (Figure 1).

MATERIALS AND METHODS

Alternanthera Caracasana commonly known as the khaki weed was chosen as a raw material in this work because it is the most widely grown weed in southern states. Since this weed directly does not compete with the food market, it makes A.caracasana a good choice for bioethanol production. A.caracasana consists of cellulose, hemicellulose, lignin, organic compounds, and some trace compounds; it is a flowering plant species of the family Amaranthaceae it is called a tough weed in some places. A.caracasana weed – Samples were collected in and around the JSS AHER campus in December 2021 (Figure 2).

Pretreatment was carried out by cutting into tiny chunks and thereafter drying for about 7 days, then the dried sample was powdered in a grinder and the sample was preserved at room temperature in a sealed zip lock bag. The pretreatment and biofermentation process are shown in Figure 3. 5g of sample was weighed and to that 100 of 1% HCl (hydrochloric acid) was added, after that the sample was hydrolyzed in an autoclave at 105ºC for 15mins, and the hydrolyzed sample was filtered by filter paper with 110mm (Whatmann cat no 1033.110), under LAF(laminar airflow) to avoid further contamination of the sample. The filtered supernatant was cooled to room temperature and further carried out for fermentation. The yeast S.carevisiae (saccharomyces) or brewer’s yeast was used for fermentation, for 100ml of sample; around 2g of yeast was added and incubated at 37ºC for 76 hours, and pH was adjusted before keeping for fermentation (Saadon et al., 2022).

Distillation is one of the fundamental sample purification techniques used virtually all around laboratories and industries. The liquid product is transferred to a distillation column to distill the alcohol to almost azeotrope levels. Filtered and distilled with the aid of a heat supply, the sample was set in a round bottom flask, condenser, and distillate flask, 100ml ethanol was selected, and it was distilled in the same round bottom flask by heating the ethanol. It is distilled at a boiling point of 78ºC at a temperature range of 20-30ºC, and the resulting distillate is collected. Nevertheless, this purity falls short of the customary minimum purity standard of 99 percent. Thus, the hydrous ethanol must undergo dehydration to become anhydrous ethanol. This was traditionally performed using a molecular sieve. The sample was collected in an airtight container and characterized for chemical analysis.

Analysis of reducing sugars                                                                                                       

The reducing sugars recovered from A.caracasana were analyzed by the DNS method. 3, 5-dinitrosalicylic acid (2-hydroxy-3,5-dinitrobenzoic acid, DNS) combines with some reducing compounds as well as reducing sugars to absorb 540 nm light strongly. Forms as an aromatic compound (in the case of glucose). The free carboxyl (C = O) group presence is identified by this procedure, sometimes known as reducing sugar. This includes the ketone functional groups in fructose and, for example, the oxidation of aldehyde groups in glucose. In alkaline conditions, 3,5-dinitrosalicylic acid (DNS) is changed into 3-amino-5-nitrosalicylic acid at the same time.

rESULTS AND DISCUSSION

Sugar content analysis

The DNS method was used to analyze the sample's sugar content, here the minimum reaction time required for DNS to react was taken about 10-15mins at 100ºC; consequently, we concluded that 20min is the most effective time to stabilize the color development of the standard curve as shown in Figure 4. Now, as the amount of reduced sugar increases, so does the brightness of the orange color. At 540 nm, the optical density of this orange color could be measured. By measuring the absorbance of non-concentration of reducing sugar and amplifying its optical density, a standard curve was made. Based on this standard curve, the concentration of undetermined reducing sugar was obtained.

 This pretreated biomass was hydrolyzed such that the cellulose and hemicellulose polymers within lignocellulosic biomass, which can be fermented to ethanol, are broken down by dilute or concentrated acids. The acid hydrolysis was done by adding 1% Conc.HCL to 5g of pretreated biomass and was autoclaved for about 15 minutes at a temperature of 150ºC which resulted in 0.414mg of reducing sugars from 5g of sample.

Alcohol analysis

The sample supernatant analysis was carried out through a few identification tests for ethanol after autoclave and the results are presented in Table 1.

Table 1. Results compilation for identifying tests of ethanol

FTIR            

Fourier transforms infrared (FTIR) spectroscopy is a technique that employs a mathematical procedure (Fourier transform) to transform raw data (interference program) into a real spectral range. The Fourier transform infrared method is used to find out whether a sample lets or lets go of infrared light. It finds out if the sample has both inorganic and organic materials. Using spectroscopic data in automated spectroscopy software, major chemical groups in the sample will be determined from the range 600-4000cm^-1 based on the infrared absorption frequency.

Two samples were analyzed in FTIR one is a predistillation sample and the other is distilled, the peak shape and its position in the corresponding region determined the functional group by Fourier transform infrared spectroscopy (FTIR) plot for bioethanol in the sample.

FTIR results obtained in this study showed some common peaks. The sample was measured in the frequency range 4000-450 cmˉ ˡ, with a spectral resolution of 50cmˉ ˡ. The spectra would be obtained with an average of 4 scans, analysis was performed on both the samples. The absorption spectrum in Figure 5 and band in Table 2: Shows peaks between the range of 3350.57- 3350.17 cm^-1 which indicates OH stretching vibrations, presence of alcohols, phenols, 1638.05-1637.23cmˉ ˡ indicates alkene group, 604-651 cmˉ ˡ indicates aryl and alkyl halides.

Table 2. FTIR absorption bands.

GC-MS

A PerkinElmer Auto System XL gas chromatograph equipped with a quadrupole mass spectrometer (Model: Turbomass Gold, Waltham, MA, USA), and an RTx-5 Cross bond 5 percent diphenyl/ 95 percent dimethyl polysiloxane capillary column (30m x 0.25mm x 0.25mm film thickness; Restek, USA) was utilized to analyze an ethanolic extract of A. caracassana using gas. Helium gas was used as the carrier, and the gas chromatography and mass spectrometer conditions were 40°C for 2 minutes. For an injection volume of 1 L ramp was adjusted from 5 °C/min to 260 °C/min. The split ratio was 20:1, and the operation lasted 5 minutes. The intake line and source temperatures were 200°C and 180°C, respectively. 10 L of the sample was diluted in 990 L of n-Hexane. As a blank in sample preparation and GC-MS analysis, HPLC-grade n-hexane was utilized. For chemical identification and quantification, the peak area percentage of the chromatograms was employed. Using the NIST 2011 Mass spectral library and their fragmentation pattern, the major constituents of the ethanolic extract were determined.