الفهرس 
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Abstract
The development of bioethanol production from lignocellulosic residues is one of the greatest challenges nowadays to meet the energy demand sustainably. Lignocellulosic residues as a raw material provide not only low-cost and sustainable products but also a solution to the waste disposal problem. These residues are converted to bioethanol in several sequential steps including pretreatment, hydrolysis, and fermentation. Pretreatment is necessary for breaking the lignin–hemicellulose-cellulose complex, delignification, and disrupting the crystalline structure of cellulose. Hydrolysis of cellulose and hemicellulose was performed to produce fermentable sugars and then sugars were fermented to ethanol.
Rice straw and manure were used in the present study as blending substrates for bioethanol production. Their composition analysis showed that rice straw’s total carbohydrate and lignin contents were 72.3% and 17.1% respectively, and in manure were 63.2% and 25.7%, respectively.
This study aimed to improve the conversion of rice straw and manure into bioethanol using co-culturing of Streptomyces aegyptia and Candida tropicalis and was focused on three trends: process design, optimization of culture conditions, and strain improvement.
Two strategies of process design, separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were performed to compare the efficient bioethanol production. Rice straw and manure were pretreated with Ca(OH)2 and further processed for SHF and SSF. Among both strategies, SSF in form of one-pot hydrolysis and fermentation design gave a maximum ethanol concentration (0.28 g/l) and bioconversion efficiency of 2% after 84 h of culturing at 30˚C.
Substrate adding (batch and fed-batch) in the process design was applied in different modes. In batch mode, all working volume of substrates and medium was applied at the beginning of the culture. In the fed-batch, three substrate-feeding modes were carried out. The first and second modes were one-time feeding at the initial working volume of 75 and 50% of the total working volume designated as (75:25) and (50:50) respectively. The third mode was three-time feeding at the initial working volume of 25% followed by feeding three times with 25% of the total working volume (25:25:25:25). The results showed that the fed-batch system with one-time feeding (75:25) system was higher when compared to other modes with respect to ethanol concentration and bioconversion efficiency of 0.28 g/l and 2%, respectively.
Improving the culture conditions for bioethanol production in submerged mode was investigated using several steps. The first step was the selection of Plackett-Burman (PB) designs and screening of factors that may affect and improve pretreatment, hydrolysis, and fermentation processes. Three parameters were chosen that may improve the pretreatment step namely; Ca(OH)2 loading, time, and temperature of pretreatment. Eleven parameters were chosen that may improve the hydrolysis step namely; the size of rice straw, inoculum size of Streptomyces, pH of hydrolysis medium and medium components of rice straw concentration, manure concentration, KH2PO4, MgSO4.7H2O, ZnSO4.7H2O, FeSO4.7H2O, MnCl2.4H2O and Tween 80. Seven parameters were chosen that may improve the fermentation step namely; inoculum size of Candida, pH of fermentation medium and medium components of manure concentration, rice straw concentration, KH2PO4, MgSO4.7H2O, and FeSO4.7H2O. Statistical procedures were employed to study the effect of process variables on eight responses including, the lignocellulose enzymes activities (Lignin peroxidase, Mn-dependent peroxidase, xylanase, endocellulase, and total cellulase), sugar and ethanol concentration, and the bioconversion efficiency. The results showed optimizing microbial conversion and improving the bioconversion efficiency by 6.6 folds in the first Plackett-Burman (PB) design and 22 folds in the second Plackett-Burman (PB) design compared to the unoptimized process. Maximum ethanol concentration was approximately the same in the two designs (0.76 g/l), but bioconversion efficiency increased in the second Plackett-Burman (PB) design (44.1%) compared to the first design (13.3%). The results indicated that a maximal lignin peroxidase, Mn-dependent peroxidase, and total cellulase activities of 2290 U/min.l, 1400 U/min.l, and 22.3 U/min.l respectively were achieved in the second Plackett-Burman (PB) design. Maximal xylanase and endocellulase activities of 78.1 U/min.l and 57.3 U/min.l respectively were achieved in the first Plackett-Burman (PB) design.
The second step in culture optimization was the selection of five variables (KH2PO4 and tween 80 in the hydrolysis step and KH2PO4, MgSO4, and Yeast inoculum in the fermentation step), which were determined to be significant by Plackett-Burman (PB) designs and further optimized using response surface methodology (RSM) based on Central Composite Design (CCD). The results showed an improvement in bioconversion efficiency (96%) and ethanol concentration (1.59 g/l) under optimum hydrolysis conditions (KH2PO4 6.7 g/l and tween80 5.5 ml/l) and optimum fermentation conditions (KH2PO4 2.5 g/l, MgSO4 3.0 g/l, and 6.5% Yeast inoculum).
Solid-state hydrolysis and fermentation mode were applied to the latter optimal conditions of submerged mode to increase the concentration of ethanol in the medium. Results showed that the ethanol concentration (3.8 g/l) in solid-state mode was greater than in liquid submerged mode (1.6 g/l). However, the bioconversion efficiency in solid-state mode (6.3%) was poorer in comparison with liquid submerged mode (96%).
The strain improvement using UV mutagenesis was applied in this study to Streptomyces aegyptia at five different time exposure levels for improving bioconversion efficiency. An increase of 1.3-fold in ethanol concentration (4.9 g/l) and bioconversion efficiency (8.2%) in the case of mutant exposed to UV radiation for 0.3 min (20 sec) was achieved as compared to ethanol concentration (3.8 g/l) and bioconversion efficiency (6.3%) in the wild-type strain.
Improving the ethanol concentration and bioconversion efficiency in solid-state mode could be performed by optimizing the medium volume applied to the substrates. The medium volumes in hydrolysis and fermentation steps were added in five different levels using response surface methodology (RSM) based on Central Composite Design (CCD). Results showed improving the ethanol concentration (7.4 g/l) with bioconversion efficiency (12.3%) under optimum levels of medium volume in hydrolysis (1.5 ml/g) and fermentation (3.5 ml/g) steps.