IX. Congress on Sugar and Sugar Cane Derivatives, Havana, Cuba, June 19-22, 2006
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1 IX. Congress on Sugar and Sugar Cane Derivatives, Havana, Cuba, June 19-22, 2006 Production of fuel ethanol from sugarcane bagasse and sugarcane trash. Michael Saska 1, Carlos Martin 2 1 Audubon Sugar Institute, Louisiana State University Agricultural Center, St. Gabriel, LA 70776, USA 2 Department of Chemistry and Chemical Engineering, University of Matanzas, Matanzas, Cuba Abstract Sugarcane bagasse and trash, the left-over residue of leaves and tops, can be converted to ethanol by enzymatic or acid catalyzed hydrolysis and fermentation of the released monosaccharides. Prior to enzymatic hydrolysis, pre-treatment is required. Among the many processes proposed, steam explosion enhanced by first impregnating biomass with dilute acid or alkali promises to be a viable technology in the future. The lower chemical costs of the acid impregnation are to be balanced against less fermentation toxins generated in the alkaline treatment and less corrosive environment in the steam explosion reactor. Introduction Due to increasing concern about the greenhouse effect, crude oil prices and other factors, bioethanol is increasingly considered a viable alternative for gasoline as a vehicle fuel. Lignocellulosic waste materials are abundant and promising feedstocks for industrial production of low-cost bioethanol (Wheals et al., 1999). One of the major lignocellulosic materials to be considered in tropical countries is sugarcane bagasse, the fibrous residue obtained after extracting the juice from sugar cane (Saccharum officinarum) in the sugar production process. Most of the bagasse produced in the sugar industry is used as a fuel for generating the energy required by the sugar mills. However, it is recognized that the energetic demands of sugar factories could be satisfied with 30-50% of the produced bagasse provided that the thermal efficiency of combustion units would be improved. Therefore, a surplus of bagasse would become available for alternative uses, including ethanol production. Bagasse is an interesting raw material for industrial bioconversion processes since it is rich in carbohydrates, including approximately 40% of the dry matter content as cellulose and 30% as hemicelluloses. Furthermore, it is readily available at the sugar mill site, and the cost for harvest and
2 transportation is borne by the sugar production (Pandey and Soccol, 2000). Sugarcane trash, the left-over residue of leaves and tops, which are usually incorporated to the soil or burnt after harvesting, is another potential raw material for ethanol production, and about equal in quantity to bagasse. Figure 1: Prepared sugarcane biomass (with a centimeter scale). Left: CLM (cane leaf matter). Right: bagasse pith. In a lignocellulose-to-ethanol process, hydrolysis is required for the conversion of cellulose and hemicelluloses to fermentable sugars. Enzymatic hydrolysis is a promising method for improving the technology so that bioethanol would become competitive with other fuels on an industrial scale. However, since the access of native cellulose to the enzymes is limited by its close association with hemicelluloses and lignin, a pretreatment step is required to improve the enzymatic convertibility and to lower the cost of the process (Galbe and Zacchi, 2002). The pretreatment is aimed to remove lignin and hemicelluloses, reduce cellulose crystallinity and increase the porosity of the material (Sun and Cheng, 2002), thus enhancing the enzymatic susceptibility of cellulose. An effective pretreatment must (i) produce fibre, in which the cellulose fraction could be hydrolysed by cellulases without significant troubles; (ii) preserve the utility of the hemicelluloses; and (iii) avoid the formation of inhibitory by-products (Laser et al., 2
3 2002). An economical pretreatment should use inexpensive chemicals and require simple equipment and procedures. Several chemical, physical and biological pretreatment methods have been investigated for different lignocellulosic materials (reviewed by Sun and Cheng, 2002). The best pretreatment options are those which combine elements of both physical and chemical methods. In this regard, steam pretreatment, with or without explosion, has been claimed as one of the most efficient and economic options for different lignocellulosic materials (Saddler et al., 1993) and the first commercial biomass ethanol plant at Abener Energia SA in Seville, Spain is reportedly going to be based on the SunOpta Inc. s continuous steam explosion technology. Steam pre-treatment involves heating lignocellulose to high temperatures and pressures, followed by mechanical disruption of the pretreated material either by violent discharge (explosion) into a collecting tank (Saddler et al., 1993) or by mild blending after bleeding the steam pressure down to atmospheric (no explosion) (Ramos, 1992). The high-pressure steam radically modifies the plant cell wall structure, yielding a dark brown material from which partially hydrolysed hemicelluloses are easily recovered by water-washing, leaving a water-insoluble fraction composed of cellulose, residual hemicelluloses and a chemically modified lignin that can be further recovered by different chemical treatments. The eficiency of the pre-treatment can be enhanced if biomass materials are impregnated with acids or alkalis prior to high-pressure steaming. The recent work related to conversion of sugarcane biomass to ethanol has involved pretreatment tests with lime at Audubon Sugar Institute (Saska and Gray, 2006) and Texas A&M University (Chang etal, 1988; Granda, 2004) in the U.S.A., anhydrous ammonia treatment at Michigan Biotechnology Institute, dilute sulfuric acid and sulfur dioxide impregnation at the University of Matanzas and Lund University in Sweden (Martin etal, 2001; Martin etal, 2002), dilute acid hydrolysis at the Institute for Agrobiotechnology in Tulln, Austria (Neureiter etal, 2002) and the Universities in Parana and Lorena in Brazil (Galvao etal, 2001; Cunha and Silva, 2001). In the ammonia (AFEX) process (Holtzapple etal, 1991; Tiedje etal, 2005) at best conditions for sugarcane bagasse, 40% moisture bagasse is contacted at 100 C for 30 minutes under about 25 kg/cm 2 pressure with anhydrous ammonia (2:1 anhydrous ammonia : biomass dry solids). After the reaction, the reactor is discharged, ammonia is vaporized to be recycled, leaving the pre-treated biomass with about 85% cellulose and 60 % hemicellulose digestibility with the standard enzyme loading. Although an efficient pretreatment, it suffers from the high 3
4 cost of ammonia (US$300/tonne) and the yet to be demonstrated ammonia recovery process. The ammonia consumption to neutralize acetic acid from bagasse is unavoidable and combined with physical losses in the recovery process is estimated as at least 5% on biomass dry solids based on the known acetyl content of bagasse. The tests so far have been limited to small (2 to 4L) batch reactor experiments. Another approach based on acid - not enzymatic - hydrolysis of cellulose with dilute sulfuric acid in ethanol is accomplished in the process pursued jointly by Dedini Industrias de Base and Copersucar in Brazil (Rossell etal, 2005). As with all acid pre-treatments, yeast toxins from sugar degradation are a concern but blending the bagasse hydrolysate with conventional syrup or juice and development of more tolerant yeast strains was reported to overcome the difficulties. A 20 kg/hr pilot unit is reported to be in operation, with a yield of 100L ethanol from 1 tonne of raw bagasse. As with the ammonia process, high pressure on the order of 25 to 28 kg/cm 2 is required (150 to 200 C). An altogether different route to ethanol from bagasse has been pursued in the so called MixAlco process at Texas A&M University (Granda etal, 2006). Raw bagasse is blended with 10% lime on dry solids and stored wet in piles similarly as in the Ritter process. Forced aeration of the pile is designed to increase de-lignification rate. After 6 weeks the lignin content is reduced to 10% and after acidification and inoculation bagasse is ready for 1-month long fermentation to a mixture of short-chained aliphatic acids (70% acetic) with anaerobic bacteria. After recovery and purification, the organic acids can either be recovered and marketed, or their ketones hydrogenated to alcohols over Raney nickel. In the present work, the efficiency of the impregnation of sugarcane residual biomass with lime and sulphuric acid prior to steam explosion is evaluated. Steam explosion with acid pre-treatment Effect of impregnation on solubilisation of bagasse main components The yield of fibrous material, glucose, xylose, furan aldehydes and aliphatic acids after pretreatment are shown in Table 1. Glucose and xylose were formed from the hydrolysis of cellulose and xylan, respectively. The furan aldehydes 2-furfural (hereafter referred to as furfural) and 5-hydroxymethyl-2-furfural (hereafter referred to as HMF) were formed as result of 4
5 the degradation of pentoses and hexoses, respectively. The aliphatic acids are acetic acid derived from the deacetylation of hemicelluloses, and formic and levulinic acids resulted from the degradation of the furan aldehydes. A decrease of the yield of fibrous material was observed for all the pretreatments (Table 1). That was due to the solubilisation of hemicelluloses and some of the cellulose, lignin and extractives. The lowest fibre yield was obtained in the H 2 SO 4 - impregnated pretreatment, and that was a result of a major solubilisation of cellulose and hemicelluloses. Under H 2 SO 4 -assisted steam explosion cellulose was hydrolysed to a high extent as can be deduced from the high glucose yield. Roughly 50% of the glucose content in the raw material was released already in the pretreatment step of the H 2 SO 4 -impregnated bagasse. In contrast, only a negligible part of the glucose was released during pretreatment after impregnation with sulphur dioxide or without any impregnation. A high degree of hydrolysis of hemicelluloses was achieved in all the pretreatments. However, the resulting xylose was well preserved in the pretreatments with SO 2 impregnation and without impregnation, whereas in the H 2 SO 4 -impregnated pretreatment a high sugar degradation occurred resulting in high formation of furan aldehydes and aliphatic acids (Table 1). The comparable yield of phenolic compounds observed in all the pretreatments indicate that lignin solubilisation was not affected for any of the impregnation agents. Since furan aldehydes, aliphatic acids and phenolic compounds are inhibitors of ethanolic fermentation, the fermentability of hydrolysates obtained by steam explosion of H 2 SO 4 -impregnated bagasse are porrly fermentable with Saccharomyces cerevisiae. Impregnati ng agent Table 1. Yields after pretreatment 1, g/100 g dry bagasse (DB). Fibre Glucose Xylose Furan aldehydes 2 Aliphatic acids 3 None SO H 2 SO Phenolics 1 Mean values from two replicates are indicated. 2 Sum of furfural and HMF. 3 Sum of acetic, formic and levulinic acids. 4 Total phenolic compounds measured by Folin-Ciocalteu method. 5
6 Effect of impregnation on the enzymatic convertibility of steam-exploded bagasse All steam pretreatment conditions were efficient for enhancing the enzymatic hydrolysis of cellulose and xylan. H 2 SO 4 impregnation was the most effective method for enhancing the enzymatic convertibility of cellulose, but it was outstandingly ineffective for xylan hydrolysis due to the high degradation of xylose that occurred during pretreatment. On the other hand, the pretreatments with SO 2 impregnation and without impregnation yielded materials, whose cellulose and xylan were successfully hydrolysed with the enzyme preparation used. Table 2. Enzymatic convertibility of bagasse polysaccharides, g/100 g dry bagasse (DB). Impregnating agent Cellulose Xylan None SO H 2 SO No pretreatment Steam explosion with alkaline pre-treatment The objective of this work was to build on the previous work with lime and steam explosion pretreatment of sugarcane bagasse (Chang etal, 1998; Granda, 2004; Playne, 1984) but in conceptualizing the process employ only such unit operations that have a reasonable chance of acceptance by the sugarcane industry; by avoiding chemical recovery, need for specialized equipment to handle high pressures or very corrosive acidic media, and reduction of biomass particle size beyond the one that is readily achieved with present-day sugarcane factory equipment. The lime impregnation was done in covered containers, kept without agitation either at ambient temperature or in a temperature-controlled tank set to 50 or 90 C. Batches of typically 0.3 to 3 kg of sugarcane biomass were blended with water, taking into account the residual moisture of 6
7 the biomass, and hydrated lime, mixed thoroughly and allowed to soak either at ambient temperature or in the heated water tank for one day to three weeks. It was found that the enzymatic conversion was improved when prior to the steam treatment, the lime-treated biomass was acidified to ph close to 5 with concentrated hydrochloric acid. Acidification prior to the enzymatic hydrolysis is unavoidable in alkaline pre-treatment in order to satisfy the ph requirements of the cellulolytic enzymes. Therefore, this does not increase the chemical requirements of the overall process but may aid the hemicellulose hydrolysis. This ph is still much higher than in acid or auto-hydrolysis processes so that production of furfural (2-F) and hydroxymethylfurfural (HMF) are minimized. In most tests, the lime-impregnated and acidified (ph 5) biomass was transferred whole without any liquor removed into a 60L reactor that was heated with steam indirectly through a jacket. During reaction the reactor was rotated to induce mixing and improve heat transfer in the reactor during the treatment. After a predetermined period, the reactor was stopped in the upright position and biomass released tangentially into a receiver placed underneath the reactor. The steam-exploded biomass was then collected and kept refrigerated in closed plastic bags until analysis. Enzymatic Saccharification of the steam pre-treated biomass Enzyme hydrolysis was done according to a procedure adapted from NREL s recommended procedures (www1.eere.energy.gov/biomass/analytical_procedures.html#lap-009). To an amount of substrate calculated to contain 0.5 g glucan was added 25mL of 0.1M sodium citrate. Then 0.150mL of 1% cyclohexamide and 0.200mL of 1% tetracycline in 70% ethanol were pipetted into the flask, followed by 0.254mL of 1:1 diluted Spezyme CP cellulase (77 7
8 FPU/mL) from Bio-Cat Inc. and 0.149mL of 1:1 diluted Novozyme 188 cellobiase (282 CBU/mL). The enzyme loading was therefore about 20 FPU and 42 CBU per one gram of glucan. ph was adjusted to 4.8 with a few drops of glacial acetic acid. The flasks were capped and placed in an orbital shaker-incubator set at 50 C. Yield of fermentable sugars from alkali-assisted steam explosion In terms of sugar yield the behavior of CLM and pith appear nearly identical. Increasing the hydrated lime dose or reducing solids loading during impregnation improve the yield while the temperature effect appears to be only secondary by preventing any microbial action at 90 C that at lower temperatures sets in readily at lime doses less than 10%. Low solids load during impregnation appears to lead to higher sugar yield but from the standpoint of industrial design, 10% solids is considered as minimum for a viable process. At this level biomass is fully saturated with little free liquor, yet sufficiently fluid so it can still be handled relatively easily Table 3: Levels or range of experimental variables. BDS = biomass dry solids. AT = ambient temperature (15-25 C). Feedstock CLM, pith Impregnation Biomass solids, kg BDS/100 kg 4, 8,10, 15 Hydrated Lime kg/100 kg BDS 2, 4, 10, 20 Temperature, C AT, 50, 90 Duration, days
9 Steam treatment Steam pressure (jacket), bar 9 Temperature, C Duration, min L5 ph ph Concentration, g/100g L6 ph L5 concentra tion Impregnation time, days L6 concentra tion 9
10 Acetate concentration, mg/l Formate concentration, mg/l L5 Acetate L6 Acetate L5 Formate L6 Formate Impregnation time, days Figure 2: ph and total dissolved solids (TDS) in g/100 g - top graph - and acetate and formate ion concentrations - bottom graph - in the biomass liquor during impregnation in tests two tests (No. 5 and 6) with 10% lime. Note the rapid acetate and formate generation and the corresponding ph drop in test 6 as the bacterial degradation sets in after two days of impregnation. The acetyl content in sugarcane bagasse is known to range from 4 to 5%BDS and that fits well with the lime consumption during bagasse treatment found earlier (Granda, 2004) and is confirmed by our results. The 4% lime dose is just about sufficient to neutralize acetic acid that is quickly released from the biomass under alkaline conditions (Figure 4) with the ph dropping as lime is converted to calcium acetate and, afterwards as infection sets in. Even the 10% lime level ph is not always high enough to maintain ph in the 10+ range long enough and 20% hydrated lime appears needed for safe, infection-free impregnation. Formate levels in solution are negligible as long as the microbial action is kept to minimum at ph > 11 but increase rapidly if the ph drops because of insufficient lime. 10
11 The low severity steam treatment after impregnation is aimed to sterilize the biomass and increase the enzyme saccharification rate. The severity factor (R 0 ), a combination of the effects of time and temperature (Overend, 1987) R 0 = t * e (T-100)/14.75 t = time (min); T = temperature ( C) that was employed here (log R 0 ~ ) is some times lower than was used (log R ) in the acidic pre-treatment. Jollez etal, 1992 reported an 80% glucan conversion at log R 0 = 3.1 with continuous steam explosion alone with no impregnation but with a very high enzyme loading of about 500 IU/g glucan. The low severity requirement for lime-impregnated biomass is expected to have significant benefits for the design and choice of construction materials for the steam treatment reactor. The combined 72-hour glucose xylose yield of 40 g/100gbds for CLM and 38 g/100gbds for pith, respectively was found, corresponding approximately to an 80% glucan, and 50% xylan conversion. The 24-hour conversion and yield are close to 90% of the final 72-hour values. An additional small gain (~ 2%) in yield would come from the minor amounts of arabinose and cellobiose released by the saccharification enzymes that are not reported here. The 40% yield is lower than the 42, 47 and 53% yields after acid impregnation followed by steam explosion at log R 0 = 4.1 (Martin etal, 2002). This is obviously because of the higher initial glucan content in the whole bagasse sample that originated from a Matanzas, Cuba sugar mill (45% BDS) than in our Louisiana samples of CLM and pith (29% BDS). 11
12 90 Conversion, % initial polymer Test 10A-30 Glucan Test 10A-30 Xylan Test 11A-45 Glucan Test 11A-45 Xylan Enzyme hydrolysis time, hours Figure 3: Glucan and xylan conversion (% initial polymer converted to glucose and xylose) in two tests with lime pretreated cane leaves (Test 10A-30) and lime-pretreated bagasse pith (Test 11ª-45). Major difference between acidic and alkaline pre-treatments is the level of furans that are known to inhibit ethanol fermentation. A range of 100-5,000 mg furan/kg total dissolved solids (TDS) is common in acid hydrolysates from even low severity treatments (Galvao, etal, 2001; Martin etal, 2001), 10,000-40,000 mg/kg TDS furan levels were reported in hydrolysates from higher severity auto-hydrolysis or dilute sulfuric acid processes (Martin etal, 2001; Martinez etal, 2000) and still some 5,000 mg/kg TDS after detoxification by overliming (Martinez etal, 2000). In the alkaline tests, no measurable quantities of either furfural (2-F) or hydroxymethyl furfural (HMF) were detected in any of the pre-treatment liquors. Based on comparison with a 50 mg/l HMF/2-F standard (not shown), it is estimated that the furan concentration in the liquor from the alkaline pre-treated biomass is below the detection limit of 10 mg/kg TDS. Whether the absence of furans in alkali-impreganted steam-exploded biomass hydrolysates translates to improved 12
13 fermentability as expected (Martin and Jonsson, 2003) will be tested in future simultaneous saccharification and fermentation (SSF) trials. Besides de-acetylation that is known to be rapid, the exact mechanism of alkaline action on sugarcane biomass is not known. Thermogravimetric analysis (Figure 4) of the three main components of the biomass reveals very different TGA patterns, with xylan decomposing very rapidly within a very narrow range at about 320 C. Cellulose because its higher crystallinity decomposes later, at about 380 C, and lignin breaks down slowly and over a wide range 300 to 500 C. Untreated sugarcane pith (Figure 5) displays both xylan and cellulose peaks, shifted higher by 20 C. After pre-treatment with hydrated lime, pure cellulose behavior remains. Curiously, after treatment with magnesium hydroxide the TGA pattern is shifted lower matching the xylan behavior. Whether this is related to the different effects of the two alkalis on the biomass structure of whether the residual salts catalyze thermal degradation is under investigation. Calcium magnesium acetate is used in the USA as an environmentally friendly chemical for road de-iceing, and the use of magnesium hydroxide or dolomitic lime in pretreatment of biomass could provide a path to commercial co-products in the future biomass ethanol processes. Cell Xyl Lig 1st Deriv :35:53 1/ C C
14 Figure 4: 1st derivative TGA curves of xylan (oat spelts) - narrow-peaked solid line, pure cellulose (Avicel) - dotted line, and lignin (Granit SA) - the wide distribution solid line. Pith Ca Mg 1st Deriv :44:44 1/ C Lab: METTLER e STAR C SW 8.10 Figure 5: 1st derivative TGA curves of bagasse pith (thick solid line), pith after impregnation with hydrated lime (dotted line) and pith after impregnation with magnesium hydroxide (thin solid line). At current prices, it is estimated that the chemical costs (hydrated lime, acid and enzymes) of the present un-optimized process (20% lime dose, 40% sugar yield) are some $0.10 per kg sugar produced, of which $0.04 is for hydrated lime, $0.03 for the neutralization acid and $0.03 for the cost of the industrial enzymes, based on the reported enzyme cost of $0.15 / gallon ethanol (Anon, 2005). The acid requirements can be reduced by pressing the alkali-pretreated fiber prior to acidification. Unlike for bagasse, no equivalent-fuel value or any collection cost need to be assigned to CLM, only the expense of transportation from the field to the mill. At present this averages from $3 to $4 per ton of cane in Louisiana, adding only $0.01/kg fermentable sugar 14
15 produced. Further improvements in the economics of the process are expected from potential coproducts that are under investigation. Conclusions The two-step pretreatment combining low-temperature acid or alkali impregnation followed by low-severity steam explosion treatment has been found effective for pre-treatment of sugarcane biomass. In the alkaline treatment, the sugar yield of 40g/100gBDS was achieved with the standard enzyme loading, at an estimated chemical cost of $0.10 / kg sugar. The process is the reverse of the standard dilute acid - detoxification by overliming process - but avoids the corrosive high-temperature acidic environment and the associated high-cost construction materials, sugar degradation and generation of fermentation toxins. Abbreviations AFEX ammonia freeze explosion CBU cellobiase unit BDS biomass dry solids NREL National Renewable Energy Laboratory TGA thermo-gravometric analysis FPU filter paper unit HMF hydroxymethylfurfural 2-F 2-furfural TDS total dissolved substance 15
16 References Anon (2005) Novozymes and NREL reduce enzyme cost. (accessed April 20, 2005) Chang, V.S., Nagwani, M. and Holtzapple, M.T. (1998) Lime pretreatment of crop residues bagasse and wheat straw. Applied Biochemistry and Biotechnology, 74, Cunha, H.C.M. and Silva, F.T. (2001) Characterization of carbohydrates present in hydrolysate obtained from sugar cane bagasse pretreated by steam explosion. 6th Brazilian Symposium on Chemicals from Wood Components 7, Laser M, Schulman D, Allen SG, Lichwa J, Antal Jr MJ, Lynd LR. A comparison of liquid hot water and steam pretreatments of sugar cane bagasse for bioconversion to ethanol. Bioresource Technol. 2002, 81, Neureiter, M., Danner, H., Thomasser, C. Saidi, B. and Braun, R. (2002) Dilute acid hydrolysis of sugarcane bagasse at varying conditions. App. Biochem.Biotech , Galbe M, Zacchi G. A review of the production of ethanol from softwood. Appl Microbiol Biotechnol 59: (2002). Galvao, M.L., Ramos, L.P., Echerhoff, M.F. and Fontana, J.D. (2001) Autohydrolysis and acid-catalyzed steam explosion of sugarcane bagasse. 6th Brazilian Symposium on Chemicals and Wood Components VII, Granda, C.B. (2004) Sugarcane juice extraction and preservation and long-term lime pretreatment of bagasse. Ph.D. Dissertation, Texas A&M University, College Station, TX. Granda, C.B., Holtzapple, M.T. and Davison, R.R. (2006) The MixAlco process: Ethanol and other higher alcohols production from sugarcane lignocellulosic residues. IX Congress on Sugar and Sugar Cane Derivatives, Havana, Cuba, June Holtzapple, M.T., Jun, J.H., Ashok, G, Patibandla, S.L. and Dale, B.E. (1991) The ammonia freeze explosion (AFEX) process. Applied Biochemistry and Biotechnology 28-9,
17 Jollez, P., Chornet, E. and Overend, R.P. (1992) Steam-aqueous fractionation of sugarcane bagasse. In Advances in thermo-chemical biomass conversion (Ed. by A.V. Bridgewater), , Blackie, London. Martin, C., Galbe, M., Nilvebrant, N.O. and Jonsson, L.J. (2002) Conmparison of the fermentability of enzymatic hydrolysates of sugarcane bagasse pretreated by steam explosion using different impregnating agents. Applied Biochemistry and Biotechnology 98, Martin, C., Wahlbom, F., Galbe, M., Jonsson, L.J. and Hahn-Hagerdal, (2001) Preparation of sugarcane bagasse hydrolysates for alcoholic fermenation by yeast. 6th Brazilian Symposium on Chemicals and Lignins Wood Components Martin, C., Jonsson, LJ. (2003) Comparison of the resistance o findustrial and laboratory inhibitors. Enzyme and Microbial Technology, 32, Martinez, A., Rodriguez, M.E., York, S.W., Preston, J.F. and Ingram, L.O. (2000) Effects of Ca(OH)2 treatment on the toxicity of bagasse hemicellulose hydrolysate. Biotechnology and Bioengineering 69, Overend, R.P. and Chornet, E. (1987) Phil. Trans. Royal Soc. London, A321, Pandey A, Soccol CR. Economic utilization of crop residues for value addition: a futuristic approach. J Sci Ind Research 59:12-22 (2000). Playne, M. J. (1984) Increased digestibility of bagasse by pretreatment with alkalis and steam explosion. Biotechnology and Bioengineering 26, Ramos, L. P. Ph. D. Thesis, University of Ottawa, Canada, Rossell, C., Filho, D.Hilst, A. and leal, M. (2005) saccharification of sugarcane bagasse for ethanol production using the Organosolv process. Intern. Sugar J. 107,
18 Saddler JN, Ramos L and Breul C. Steam pretreatment of lignocellulosic residues. In Bioconversion of forest and agricultural residues, ed by Saddler JN. CAB International, Wallingford, pp (1993 Saska, M. and Gray, M (2006). Pre-treatment of sugarcane leaves and bagasse pith with lime impregnation and steam explosion for enzymatic conversion to fermentable sugars.28 th Symposium on Biotechnology for fuels and chemicals, Nashville, TN, April 30 - May 3. Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technol. 2002, 83, Tiedje, T., Teymouri, F., McCalla, D., Stowers, M., Chung, C.H., Day, D.F. and Rein, P. (2005) Separation of cellulose from hemicellulose and lignin from sugarcane bagasse and cane leaf matter 27th Symposium on Biotechnology for fuels and chemicals, Denver, CO, May 1-4. Wheals AE, Basso LC, Alves DMG and Amorim HV. Fuel ethanol after 25 years. Trends Biotechnol 17: (
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