3.5.1 Outline
3.5.2 Pre-treatment of cellulosic biomass
3.5.3 Cellulase production
3.5.4 Saccharification of biomass
3.5.5 Enzyme recovery from biomass
3.5.6 Concentration of sugar solutions
3.5.7 Alcohol fermentation
3.5.8 Alcohol recovery
Various aspects of alcohol fermentation from cellulosic biomass have been discussed thus far. A number of problems still remain to be resolved prior to industrial-scale production of fuel alcohol from cellulosic biomass. As mentioned in the introduction to this chapter, subsequent to our studies on basic and elemental techniques, an integrated pilot plant for the production of alcohol from biomass, was constructed in our laboratory, in order to demonstrate individual processes. Construction of the plant commenced in 1983, and continued in a step-wise manner for 5 years. The final plant was capable of treating 720 kg of raw material per day with the production of 150 to 200 liters of dehydrated fuel alcohol. A process flow diagram of the pilot plant is given in Fig. 3-20, while Fig. 3-21 shows a plan of the plant.
Figure 3.17 - Effect of alcohol concentration on fermentation rates of immobilized continuous fermentation processes
Figure 3.18 - Schematic of an immobilized flash system
Bagasse, rice straw, and beech wood chips, the compositions of which are shown in Table 3-6, were used as cellulosic biomass in the pilot plant. These materials vary in composition, and are thus presumed to differ in three-dimensional structure. Cellulose can either be saccharified by an acid process, or by an enzymatic process using cellulase. For the pilot plant, the enzymatic process was adopted in view of problems associated with the acid process, i.e., the necessity for the use of acid-resistant equipment, technical difficulties relating to the recovery of acid, and the formation of by-products due to excessive decomposition. At the outset, cellulase of a high titer and balanced enzymatic activity was unavailable, which meant that a long period of time was required for saccharification. However, by employing a variety of screening and mutational techniques, a high titer cellulase that could be produced at a low cost was developed. Saccharification from both low- and high-concentration biomass was investigated, and an appraisal of the most suitable types of reactor was made. Owing to the high cost of cellulase, its recovery and re-use were investigated using UF techniques. With the objective of improving the efficiency of alcohol fermentation and thus saving energy, the use of RO as a means of concentrating the sugar solutions was investigated. Alcohol fermentation was conducted by the immobilized flash method using a bioreactor in combination with the flash method for the purpose of accelerating alcohol production while minimizing the influence of alcohol generated on yeast cells. Two methods were used to evaluate the recovery of alcohol: the conventional azeotropic distillation method, and a combination of the super critical fluid extraction (SCFE) and pervaporation methods. In addition, the plant was evaluated as a total system using waste water, which mainly consisted of lignin waste water derived from the pre-treatment step and alcohol fermentation waste water. This waste water was treated by the methane fermentation method using a cell concentration apparatus fitted with a membrane. Low-concentration alcohol leaking from the SCFE-pervaporation, and immobilized flash fermentation steps was recovered using RO. The processing steps employed in the plant are described individually in the following Sections.
Figure 3.19 - Time profile of fermentation using an immobilized flash fermentation system
Figure 3.20 - Flow diagram outlining processes in an integrated bench plant
Figure 3.21 - Schematic of the layout of an integrated bench plant
Pre-treatment of cellulosic biomass was conducted as described in Section 3.3.1. Bagasse and rice straw were crudely crushed and pre-treated with alkali at 90 to 120°C for 1 to 2 hours in a double screw-type counter-current extractor. The optimum ratio by weight of sodium hydroxide to biomass was 0.08 to 0.1. In the case of beech wood chips, since saccharification did not proceed with sodium hydroxide treatment, steam explosion was found to be effective. Steam explosion combined with treatment by alkali and salts produced good results.
Bacteria represented by the genus Cellulomonas, and yeast strains including those of the genus Trichosporon were used for cellulase production. In general, the production of cellulase by fungi was preferred. As discussed in Sections 3.2.2 and 3.2.3, various microorganisms obtained domestically as well as from abroad, were screened, and Trichoderma reesei QM-9414 isolated by the U.S. Army Natick Laboratory was selected as the best strain. Mutants with enhanced enzymatic titer or with specific desirable characteristics were obtained by mutation using QM-9414 as a parent strain.
The semi-batch culture method produced a high-titer cellulase. A culture method incorporating pH shifting was employed to enhance p-glucosidase activity in Trichoderma microorganisms. In addition to scale-up experiments, cheese whey and biomass (bagasse, rice straw, etc.) were investigated as potential inexpensive carbon sources, while soybean meal and residual fermentation cells, were investigated as potential inexpensive nitrogen sources. On the basis of results obtained, a scale-up experiment was carried out in a 1-kL tank using the semi-batch culture method with 10% Avicel (crystalline cellulose) as a carbon source and soybean meal as a nitrogen source, with pH control by the shifting method. The time course of cellulase production by T. reesei CDU-11 under these conditions is shown in Fig. 3-22. High-titer cellulase activities, i.e., 720 U/ml as CMCase, 33 U/ml as FPU, 47 U/ml as Avicelase, and 31 U/ml as p-glucosidase, were obtained. Soluble protein resulting exceeded 5 %.
High-concentration saccharification was conducted by the batch method. An example using bagasse as the biomass source is shown in Fig. 3-12. Low-concentration saccharification was performed by the continuous method, the volume of biomass being adjusted to between 3 and 6%. A saccharification rate of 90 to 95%, was obtained after 5 to 7 hours of reaction, with only a small amount of unreacted biomass residue remaining after the reaction. Beech chips treated by steam explosion (Temp: 220°C2 pressure retention time: 5 min) were also subjected to the saccharification reaction for 24 hours, resulting in a saccharification rate of approximately 75 %. Table 3-7 summarizes cellulase requirements, for the saccharification of biomass, using cellulases prepared in our laboratory.
Figure 3.22 - Time course of high titer cellulase production by T. reesei CDU-11
As explained in Section 3.2.1, cellulase encompasses a group of enzymes having different molecular weights. A UF membrane capable of fractionating molecular weights ranging between 10,000 and 20,000 Dalton, was therefore used to recover cellulase for re-use. While enzyme recovery from low-concentration saccharification solutions was approximately 90%, that from high-concentration saccharification solutions was approximately 75 to 80% owing to cellulase adsorption by saccharification residues. Enzyme recovery in the latter case could be markedly improved by re-slurrying the residue to extract the enzymes. In the pilot plant, tubular-type ultrafiltration equipment with a polystyrene membrane was used.
In order to improve the efficiency of alcohol fermentation, UF-filtered sugar solutions which were also free of enzyme were passed through a polyamide, spiral-type RO membrane, enabling them to be concentrated from between 3 and 15% to a final concentration of approximately 30%. Using this loose RO system, more than 95% of the sugar was recovered over an extended period.
The immobilized flash method was used for alcohol fermentation in the integrated pilot-scale plant. Results are shown in Fig. 3-23. Using this method, it was possible to maintain the alcohol concentration at approximately 5%, while elevating the sugar concentration of the raw material to as high as 30%. Table 3-9 compares fermentation methodologies used in alcohol production. Three immobilized yeast fermentors, each provided with a flash distillation tank, were connected in series in the pilot plant.
In order to efficiently convert xylose contained in the saccharified solutions of biomass, yeast strains were screened and Kluyveromyces cellobiovorus, a strain capable of assimilating cellobiose and producing ethanol by fermentation was newly isolated. This strain was however of poor alcohol resistance, as are other known xylose-assimilating yeast strains. The simultaneous use of the flash method was effective in minimizing the influence of alcohol and efficiently inducing the activity of this strain.
Methods used for recovering alcohol included the distillation method, and the SCFE and osmotic vaporization methods.
In the distillation method, dehydrated ethanol was obtained by ternary azeotropic distillation, with benzene as a solvent. A pressure-type concentrator was employed, with steam at the top of the concentrator as an energy source for the ethanol separation column. Using this methodology, energy savings of 30% over that of the conventional method were achieved.
Figure 3.23 - Time profile of ethanol production, using the immobilized yeast and flash system
In the SCFE, ethanol obtained from the flash distillation step, ranging in concentration from 20 to 30% was brought into contact with supercritical carbon dioxide gas, resulting in 90 % recovery of 85 to 95% concentrated ethanol solutions. By optimizing the extraction pressure and temperature and, at the same time, providing a mist separator at the top of the extractor, ethanol having a composition approaching that of the azeotrope was recoverable. Impurities in the culture broth exhibited the same behavior as that of the ethanol. Studies on osmotic vaporization membrane methods for dehydration, resulted in alcohol concentrations exceeding 99.5 % at a recovery rate of at least 95 %. Analytical values for fuel alcohol obtained by the ternary azeotropic method, and by supercritical carbon dioxide extraction in combination with an osmotic vaporization membrane are shown in Table 3-10. In both methods, the water content of alcohol recovered, was less than 0.5%, indicating that this alcohol may be feasibly used as fuel ethanol. Dehydrated ethanol produced from bagasse was mixed with gasoline and its performance as a fuel for automobiles was evaluated by Cosmo Oil Co., Ltd. Results showed no substantial difference from ordinary reagent ethanol, and the ethanol was deemed suitable for practical use.
Table 3-10 Composition of Dehydrated Ethanol
|
Method of Dehydration |
Composition of Sample (%) |
||||
|
EtOH |
BuOHs |
AmyO Hs |
CH3CHO |
H2O |
|
|
SCFE-Pervaporation |
98.8 |
0.15 |
0.16 |
0.21 |
0.45 |
|
Azeotropic distillation |
99.5 |
0.05 |
0.03 |
0.003 |
0.33 |
|
H2O: wt. % |
Others: v/v% |
||||
