3.3.1 Pre-treatment of cellulosic waste
3.3.2 Saccharification of cellulosic waste
Lignocellulosic biomass contains cellulose, hemicellulose, lignin, and ash combined in a complex structure (Table 3-6). Pre-treatment increases the crystallinity of cellulose, while removing lignin and other inhibitors, thereby enabling its enzymatic degradation. In addition, pretreatment may increase the surface area of the cellulose thereby enhancing its reactivity with the enzyme and thus its transformation.
|
Cellulosic biomass |
Cellulose |
Hemicellulose |
Lignin |
Ash |
|
Bagasse |
41 |
24 |
18 |
2 |
|
Rice straw |
35 |
35 |
6 |
8 |
|
Wood |
40-55 |
20-35 |
25-30 |
0.2-2.0 |
|
(%) | ||||
Bagasse and rice straw were pre-treated with alkali in our studies as shown in Fig. 3-8. This raw material was crudely crushed (to less than 9 mm in diameter), and qualitatively fed into a counter-current extractor in which delignification was effected through counter-current contact of the raw material with sodium hydroxide, followed by washing with warm water. High alkali concentrations in the reaction mixture however caused simultaneous dissolution of pentosan, leading to a reduction in sugar recovery (Fig. 3-9). The optimum biomass to sodium hydroxide ratio for favorable saccharification was determined to be 1.0:0.12. Relationships between alkali concentration, the sugar composition of the enzymatically saccharified solution and the saccharification rate of bagasse are presented in Figs. 3-9 and 3-10. The xylose content of the saccharified solution decreased with increasing alkali concentration, under temperature conditions of 80 to 100°C, over a 1.5 to 2 hour period.
Steam explosion of beech and cedar was conducted, since alkali treatment of these woods after crushing did not result in saccharification.
Figure 3.8 - Basic flow sheet of raw material pre-treatment
Figure 3.9 - Effect of alkali concentration on saccharification
3.3.2.1 Saccharification
3.3.2.2 Recovery and re-use of cellulase
3.3.2.3 Sugar concentration using reverse osmosis
Saccharification of cellulosic waste materials was conducted using mutant strains obtained from Trichoderma reesei QM-9414 by mutation, (Section 3.2.2), as sources of cellulase enzymes (6, 7). A high titer, low cost cellulase was obtained in our laboratory subsequent to extensive studies on optimum culturing of these strains as described in Section 3.2.3.
Changes in the production of typical cellulase enzymes, by Trichoderma reesei KDG-3 over time, are summarized in Fig. 3-11, while the time course of saccharification with bagasse as a substrate is presented in Fig. 3-12. Enzyme requirements for the saccharification process are summarized in Table 3-7.
Saccharification reaction processes were studied using three reactor types: batch, continuous stirred tank reactor (CSTR), and the tubular plug flow process. The plug flow process produced the most favorable results in that large quantities of free enzyme were recoverable from this process at late stages of the reaction. Utilizing this process, a saccharification rate exceeding 80% was achieved at a high saccharification concentration. The adsorption of large quantities of enzyme by saccharification residues, lowered the rate of sugar production. Sugar recovery was however facilitated by stirring.
The saccharification rate and the load on the subsequent ultrafiltration (UF) and reverse osmosis (RO) membrane processes as well as energy requirements of the process, need to be carefully considered in the selection of an appropriate saccharification process. On the basis of currently available data, these processes have both benefits and disadvantages.
Figure 3.10 - Effect of alkali concentration on the saccharification rate and recovery of bagasse
Table 3-7 Cellulase Requirements For The Saccharification of Bagasse
|
Bagasse Concentration [%] |
Saccharification Time [hours] |
||||||
|
1 |
2 |
3 |
4 |
5 |
6 |
24 |
|
|
1 |
30* |
30 |
20 |
20 |
10 |
10 |
10 |
|
3 |
100 |
100 |
40 |
30 |
30 |
30 |
20 |
|
5 |
200 |
200 |
100 |
100 |
100 |
100 |
30 |
|
7 |
- |
- |
200 |
200 |
200 |
100 |
50 |
|
10 |
|
- |
- |
- |
200 |
150 |
50 |
|
15 |
- |
- |
- |
- |
- |
200 |
75 |
|
20 |
- |
- |
- |
- |
- |
200 |
150 |
|
Enzyme: CMCase [U/ml] |
|||||||
Figure 3.11 - Time course of cellulase production by T. reesei KDG-3
Although a fairly efficient cellulase production process providing a high titer cellulase at relatively low cost was developed, cellulase production was still a costly venture. The use of techniques which enable recovery and reuse of cellulase through separation of the sugar solution and cellulase from the saccharification reaction mixture, were therefore investigated using a UF membrane. A basic flow diagram of the UF membrane treatment utilized is presented in Fig. 3-13.
Figure 3.12 - Time course of bagasse saccharification by T. reesei KDG-3
Saccharified solutions of various concentrations were centrifuged, following which unreacted residue was separated, and the filtrate was subjected to UF membrane filtration. The UF membrane filtrate was subsequently concentrated, and the resulting retentate was concentrated and re-used as recovered enzyme. During this procedure, the pressure of the UF membrane was maintained at approximately 3 kg/cm2G, while the temperature of the circulating liquid was maintained between 30 and 40°C. The enzyme solution was concentrated to approximately one-tenth of its original volume. Only small amounts of residue were obtained after centrifugation of saccharified solutions of low-concentration, with sugar recovery exceeding 95%. However, when high-concentration saccharified solutions were employed, large amounts of residue resulted, and sugar recovery subsequent to centrifugation and membrane filtration was somewhat lower, i.e., 83 to 89% after centrifugation and 88% subsequent to UF membrane filtration. The addition of water either to the residue or to the concentrate improved sugar recovery to a level similar to that obtained in the case of low-concentration saccharified solutions. Similar behaviour was observed with respect to enzyme recovery, although the recovery rate for the enzyme was approximately 80%. Recovered enzyme showed normal saccharification activity. UF membranes have now been successfully used for almost four years, without any degradation. These membranes can be safely operated with periodic chemical or ball-washing.
Sugar solutions obtained via saccharification and enzyme recovery using the UF membrane, varied in concentration from 3 to 15%, depending upon the method used in their preparation. When subjected to alcohol fermentation, these sugar solutions produced low alcohol concentrations thus requiring large amounts of energy for alcohol concentration and dehydration. Concentration of sugar solutions to between 25 and 30%, using a RO membrane was studied. A flow diagram of the basic methodology used is shown in Fig. 3-14. RO was conducted by the loose RO method, using a spiral polyamide membrane, at a pressure of 40 to 50 kg/cm G and circulating liquid temperature of 25 to 30°C. In order to prevent contamination of the membrane system, warm-water washing and washing with 0.1% sodium hydroxide were performed every three days. Sugar recovery exceeding 95% was confirmed at a permeation rate of 100 to 300 L/m/d. Permeation of sugar into the filtrate was not detected.
A 25 to 30% sugar concentration was obtained regardless of the sugar concentration of the initial saccharified solution. Table 3-8 shows the results of the RO membrane operation using filtrate derived from UF membrane-treated low-concentration saccharified solutions.
Figure 3.14 - Schematic of sugar concentration by reverse osmosis
Table 3-8 Concentration of Sugar Solutions Using RO Membrane Technology
|
Test run No. |
1 |
2 |
3 |
4 |
|
Volume fed (liter) |
9,180 |
4,840 |
11,460 |
1,900 |
|
Volume of Concentrate (liter) |
600 |
320 |
400 |
630 |
|
Sugar Concentration (%) |
26 |
25 |
28 |
23 |
|
Permeation Flow Velocity (liter/m'/day) |
293 |
130 |
187 |
189 |
