1. Effect of Fermentation time on Bioethanol production
Figure 2 showed the ethanol production from two microorganisms with treated and untreated solid substrates sugarcane bagasse in 72 hours and 20g of raw bagasse was used in each trial. Ethanol production from the treated substrate was obtained 42g/l with Saccharomyces Cerevisiae while untreated sugarcane bagasse ethanol production was 22g/l with Saccharomyces Cerevisiae , . Ethanol production from treated sugarcane bagasse was obtained 14g/l with Bacillus Subtilis and from untreated sugarcane bagasse 8g/l with Bacillus Subtilis respectively . However, ethanol production from the treated substrate was obtained higher than untreated sugarcane bagasse because of highly concentrated alkaline chemical 35% hydrogen peroxide treatment, with applied pressure during agitation which affects the conversion rate of fermentation by fast reduction of particle size and breaking the chemical bond of sugar – However, in previous work ethanol production was reported 51.5g/l from 100g raw bagasse with three different methods by reusing of liquor residue with 15% hydrogen peroxide and 5% Ca(OH)2 and enzymes loading . Therefore, it’s been observed that the impact of pressure is significant to achieve the increase in ethanol production.
2. Effect of applied pressure on Bioethanol production
Figure (3) represented the relation between applied pressure and the production of bioethanol through batch fermentation. When the pressure was applied during treatment and untreated strongly effected the unconvertable, compacted structure of substrate , . Sugarcane bagasse is challenging to ferment without treatment due to the complex matrix consisting of the three main polymers of lignocellulosic biomass (hemicellulose, lignin, and cellulose) and pretreatment process is principally required to break down –. Simultaneously during the fermentation process microorganisms efficiently utilized the carbon content of the substrate. However, when pressure is applied with inert gas, fused in the pore volume of substrate, reduce the strength of the bond and reduce the particle size of substrate . In previous work, pressure has been applied through steam and CO2, which do not bring any change in pore size of the substrate –. In this work pressure applied with non-reactive gas effecting the conversion rate of substrate indicated by the SEM analysis that amorphous hemicellulose could be easily degraded on applied pressure, reducing the fermentation time and enhancing the bioethanol production, with the pretreated and non-treated substrate. Non-reactive gases are also environment friendly .
3. Fourier-transform infra-red spectroscopy
The Fourier transform infrared (FTIR) was used to observe the structural changes of hemicellulose, lignin, and cellulose during chemical and pressure treatments exposed to sugarcane bagasse. Figure (4) and figure (5) represented the FTIR spectrum of the initial solid substrate (sugarcane bagasse) without treatment referred to as standard. The wavelength range is between (3200-3400cm-1) and its intensity peak is alcohol (O-H). The wavelength range between (2800-3000cm-1 indicates sp3 (C-H) . The region of (1600-1700cm-1) represent (C=C and C=N). Wavelength range between Acyl and phenyl (1100-1350cm-1) (C-O) Trisubstituted alkene (790-840cm-1) , . The region has a significant molecular interaction, making the area highly complicated, involving the superposition of numerous lignin and carbohydrate vibration modes. Due to (C-O) stretching of (C-O-C), the region between 1,100 and 1,200cm-1 has a substantial proportion of hemicellulose and cellulose, with a maximum value around (1,035cm-1). After acid hydrolysis (red line), the region between (1,100 and 1,000cm-1) exhibits two peaks, showing the elimination of hemicellulose . In the region of (1,247cm-1), hemicellulose elimination is also visible . The elimination of lignin appears to have an impact on alkaline hydrolysis. The (C=C) stretching of the aromatic ring in lignin causes the band around (1515cm-1). The FTIR spectrum of native sugarcane bagasse, alkaline hydrolyzed cellulignin, and hydrolyzed substrate in the range (2,700-3,900cm-1). Increased under-curve width asymmetry in the range of (3100-3500 cm-1) is significant indication of the production of alcohols .
4. Gas chromatography mass spectrometry
Gas chromatography-mass spectrometry (GC/MS) was used in the qualitative analysis of ethanol in the fermentation process. Bioethanol obtained from sugarcane bagasse is an integrated system for the analytical equipment by combined alkaline chemical and applied pressure treatment. GC/MS is used to separate analysts and mass spectrometry is used for its identification. The retention time was (4.190 (min) Ret index 759 and quality index SI is 49) of production which is obtained from treated sugarcane bagasse with microorganism B. Subtilis, retention time was (4.380 (min) Ret index 1803 quality index SI 453) of production which is obtained from treated sugarcane bagasse with microorganism S. Cerevisiae however both microorganisms used for bioethanol production .
5. Thermo-gravimetric analysis
The thermal stability and weight loss of the solid substrate sugarcane bagasse were analyzed by TGA at a temperature between 100°C and 600°C as shown in figure (6). The curves that were obtained by the TGA, were divided into different three phases. The different degradation ranges define the removal of cellulose and hemicellulose because sugarcane bagasse mainly consists of lignocellulose, which starts to burn above 250°C. The first phase explained the removal of moisture and in this case, it was noticed at a temperature of 250°C, and during the second phase, a high amount of moisture content was removed at 300°C and 450°C which describes the removal of volatile compounds. The last phase ranges from a temperature of 480°C is shows the significant weight loss was recorded between temperatures 500°C and 600°C is 90% loss of raw sugarcane bagasse, 97% weight loss of treated sugarcane bagasse with microorganism Saccharomyces Cerevisiae, 93% weight loss of untreated sugarcane bagasse with microorganism Saccharomyces Cerevisiae and 92% weight loss of treated sugarcane bagasse with microorganism Bacillus Bubtilis. However the maximum weight loss of 97% showed at this point is the strong evidence of chemical pretreatment and carbon bond degradation at a range between 300°C and 450°C, there was a tremendous amount of volatile matter indicating a combustion reaction , .
6. High-performance liquid chromatography
Figure 7 shows that a significant quantity of cellulosic sugarcane bagasse is consumed during the process of fermentation. In this work, fermented bioethanol was analyzed which was obtained from non-pretreated sugarcane bagasse with microorganism Saccharomyces Cerevisiae and alkaline pretreated sugarcane bagasse with Saccharomyces Cerevisiae as well as alkaline pretreated sugarcane bagasse with Bacillus Subtilis to find the concentration value of bioethanol with the help of (HPLC) analysis. The produced bioethanol was estimated to be a steep peak at a retention time of (2.920, 2.937, and 2.903 min) respectively, related to marketable ethanol ordinary at holding time of 2.892. The variation of retention time and production yield was due to the presence of different microorganisms, alkaline chemical treatment, and applied pressure changes , , .
The structural and morphological changes on the SCB, before and after the chemical and pressure pretreatment was investigated and presented in figure (8). The untreated, raw substrate biomass showed a representative surface of the rigid, orderly, smooth structure. The treated SCB, on the other hand, presented a characteristic vast surface changes, loosened surface structure, due to chemical treatment and applied pressure. The most visible outcome of pretreatment is the loosening of the fibrous structure. The combined Pretreatment of pressure and high concentration of hydrogen peroxide removes the hemicellulose from SCB, weakening the cell wall and pronounced the loose compact structure. The clear presence of tiny pores was observed by the combined pretreatment of SCB. However, before pretreatment structure of native SCB was thick-walled fiber cells compacted with pith. The parallel stripes make up the Fibers which are covered on outside constituted by parallel as well as compacted, as applied pressure during chemical treatment which effects the highly compacted matrix of the substrate to reduce the long chain of carbon and packed fibers with an open structure of carbon loose strong unconvertable structure of hemicellulose, the increased pore size of substrate for faster chemical reaction on SCB. SEM results are strong evidence of pretreatment of SCB with hydrogen peroxide and pressure improved cellulose recovery and effective lignin and hemicellulose removal for enhanced production of fermentable sugars –.