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Optimizing acid-hydrolysis: a critical step for production of ethanol from mixed wood chips
Biomass and Bioenergy 22 (2002) 401 – 404
Optimizing acid-hydrolysis: a critical step for production of ethanol from mixed wood chips Jamshid Iranmahbooba , Farhad Nadima; ∗ , Sharareh Monemib a The
Environmental Research Institute, University of Connecticut, Route 44, Longley Building, 270 Middle Turnpike U-5210, Storrs, CT 06269-5210, USA b The Department of Biomedical Engineering, University of Connecticut, 260 Glenbrook Rd., U-2157, Storrs, CT USA Received 4 June 2001; received in revised form 10 December 2001; accepted 10 December 2001
Abstract Ethanol can be produced from renewable lignocellulosic materials such as various types of natural woods. The cellulose contents of wood can be converted to ethanol in a two-step process where acid-hydrolysis converts cellulose to glucose sugars by hydrolysis (sacchari0cation) and the resulting sugars can be converted to ethanol by fermentation. The main challenge of producing fuel ethanol from renewable lignocellulosic biomass through acid-hydrolysis and fermentation is overcoming the cost-limiting factors associated with various stages of this technology. In this study, sugar recovery rates from a mixture of wood chips were investigated through three sets of acid-hydrolysis experiments. Wood chips were sorted to include equal ratios (by weight) of softwood and hardwood. Acid concentration and the heating period were the two main factors a4ecting dextrose yields. It was found that with the use of 26% by weight sulfuric acid, highest dextrose yields could be reached within 2 h of heating time. This corresponds to overall conversion e7ciency of mixed wood chip cellulose to dextrose in the range of 78–82% based on theoretical values. ? 2002 Elsevier Science Ltd. All rights reserved. Keywords: Acid-hydrolysis; Biomass; Cellulose; Ethanol; Glucose; Lignin
1. Introduction Advances in technology have allowed the conversion of biomass into fuel ethanol as a potential source of energy. Ethanol is a renewable energy source and can be produced domestically in response to today’s high-energy demand. Due to environmental concerns, the production of sugar from agricultural waste materials, forest residues and municipal solid wastes (MSW) ∗
Corresponding author. Tel.: +1-860-486-6874; fax: +1-860486-5488. E-mail addresses: [email protected] (J. Iranmahboob), [email protected] (F. Nadim), [email protected] (S. Monemi).
and its subsequent conversion to biochemical fuel as an alternative source of energy has gained substantial grounds in the United States. These wastes may be hydrolyzed by acids or enzymes to lower molecular weight carbohydrates and 0nally to monomeric sugars. The United States is the largest producer of wastes in the world [2-kg (4:2 lb) per capita per day] [1]. In terms of energy density, each ton of MSW is equivalent to more than one barrel of oil. It has been estimated that the cost of ethanol, with a feedstock cost of $25=dry US ton will be reduced from its present base of $1.16=gal to about $0.76 by the year 2015 (a reduction of 34%) [2]. Lignocellulosic materials such as agricultural, forest products (hardwood and softwood) and their
0961-9534/02/$ - see front matter ? 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 1 - 9 5 3 4 ( 0 2 ) 0 0 0 1 6 - 8
402
J. Iranmahboob et al. / Biomass and Bioenergy 22 (2002) 401 – 404
residues are renewable resources of energy. Approximately 90% of the dry weight of most plant material is stored in the form of cellulose, hemi-cellulose, pectin, and lignin. Conversion of cellulose and hemi-cellulose from waste materials to sugars provides a feedstock for the production of fuel ethanol and will substantially reduce the amount of wastes that would otherwise exert pressure on municipal land0lls. The main goal of this work was to investigate sugar recovery rates from mixed wood chips originating from a variety of sources with a view to develop an optimal and economically feasible system for production of ethanol as a promising fuel and a base material for other chemical products. The results of this study may bene0t government and private sectors that are interested in the production of fuel and other by-product chemicals from waste materials. 2. Hydrolysis The complex structure of lignocellulose in plants makes a primary protective barrier that prevents cell destruction by bacteria and fungi. In order to break down this structure for conversion of biomass to fuel ethanol, the cellulose and hemi-cellulosic materials must be broken down to corresponding monomers (sugars) so that microorganism can utilize them. There are three major hydrolysis processes for agricultural and wood wastes to produce a variety of sugars capable of making ethanol: dilute acid, concentrated acid, and enzymatic hydrolysis [3,4]. Hemi-cellulose is readily hydrolyzed by dilute acids under moderate conditions, but much more extreme conditions are needed for hydrolysis of cellulose. The main advantage of the low concentration acid in the hydrolysis process is that acid recovery may not be required and there will be no signi0cant losses of acid. However, in a low concentrated acid process, the reaction should be carried out at high temperature and pressure, and due to poor yields of glucose from cellulose in the hydrolysis step the ethanol yield is low. Due to near-quantitative yield of glucose from cellulose, the use of high concentration acid in the hydrolysis process may yield higher quantities of ethanol but di4erent vessels and a good acid recovery process will be required. The dilute acid-hydrolysis process uses high ◦ temperatures (160 C) and pressures (∼10 atm) [5].
The acid concentration in the dilute acid-hydrolysis process is in the range of 2–5% [6,4]. The acid concentration used in concentrated acid-hydrolysis process is in the range of 10 –30% [4]. Lower operating temperatures and pressures are required during the concentrated acid-hydrolysis process. The concentrated acid-hydrolysis involves longer retention times and results in higher ethanol yields than the dilute acid-hydrolysis process [4]. Enzymes produced by a variety of microorganisms are also capable of breaking down lignocellulosic materials to sugars in a longer retention time [7]. Enzymatic hydrolysis requires feedstock pretreatment, enzyme production, and enzyme recovery, which may make this option economically unfeasible. Problems associated with the hydrolysis process need more improvements in order to reduce the operating cost for an ethanol-producing plant that uses wood wastes as feedstock. Development of a feasible process for the acid-hydrolysis of wood chips that could lead high sugar yields and result into less oxidation of the produced dextrose by the concentrated acid were investigated in three sets of bench-scale experiments. 3. Experimental procedure Mixture of 50% hardwood (oak, birch and maple) and 50% softwood (pine and cedar) chips free from ◦ barks were dried at 72 C overnight. The wood chip mixture was rechipped and sieved, and the fraction between 4 and 10 mm was used. Various lignocellulosic raw materials’ composition for one class of hardwood (populus) and one class of softwood (0r) similar to the wood chips used in this study are given in Table 1. Dried, ground wood chips of 200 g were gradually mixed with 500 g of 80% (by weight) H2 SO4 acid to generate a homogeneous paste. The color of the paste turned dark without resulting into severe oxidation of the mixture by acid. Distilled boiling water of 500 g was added to the paste to obtain a 33% (by weight) ◦ acid mixed slurry and was stirred for 30 min at 100 C using a water bath. The mixed slurry was then 0ltrated. The 0ltration time, volume and weight of the 0ltrate, and the weight of 0lter-cake were recorded. The 0ltrate was then heated for 2 h. In order to measure the percent dextrose of the heated 0ltrate using a sugar analyzer (YSI 2700 SELECT) during the
J. Iranmahboob et al. / Biomass and Bioenergy 22 (2002) 401 – 404 Table 1 Composition of various lignocellulosic raw materials in a typical softwood and hardwood
Cellulose (wt%) Hemi-cellulose (wt%) Non-carbohydrate (%) Lignin Ash
Populus tristis (hardwood)
Douglas 7r (softwood)
45.0 30.0
42.0 27.0
40 26% H2SO4
35 30 % Dextrose
Component
403
25 20
33% H2SO4
15 10
20.0 1.0
Carbohydrate (% of sugar equivalent) Glucose 40.0 Mannose 8.0 Galactose NA Xylose 13.0 Arabinose 2.0
28.3 0.2
5
20% H2SO4
0 0
50.0 12.0 1.3 3.4 1.1
Source: Ref. [8].
heating process, samples were taken at 30 min intervals and neutralized (pH: 6 –8) with a 1 N NaOH solution. The same procedure was done for 26% and 20% (by weight) acid mixture slurries.
4. Results and discussion In the hydrolysis of wood wastes, the e4ective acid concentration and the time of heating vary depending on the composition of the wastes such as cellulose, hemicellulose, lignin, ash and pectin. Wood wastes are usually collected and stored as a mixture. Therefore, in this study, acid-hydrolysis was mainly performed on mixed wood chips. The results of hydrolysis of mixed wood chips are shown in Table 2. The 0ltration
0.5
1 1.5 Time (hour)
2
2.5
Fig. 1. The time sequence 0ltrate hydrolysis after treatment of wood chips with sulfuric acid at di4erent concentrations (samples were taken at 30 min intervals). Data points are average values of three experimental runs with error bars indicating the standard error of the means.
time is longer for higher acid concentration because of its high viscosity. The dextrose concentration of the mixed slurry (after 30 min of heating process) was highest for 33% acid slurry. The results of heating process of the 0ltrates for all three acid concentrations, 33%, 26%, and 20% are shown in Fig. 1. The maximum dextrose (sugar) concentration was achieved in 26% (by weight) acid-hydrolysis process batch, in which 0ltrate was heated for 2 h. Lignin is mostly insoluble in mineral acids and only a small fraction of the lignin in a biomass sample is solubilized during the hydrolysis process [9]. With very small quantities of dissolved lignin, the hydrolysis process of cellulose to sugar becomes a simple process and can occur in a less acid concentration mixture. However, at higher acid
Table 2 The results for acid-hydrolysis of mixed wood chips
Filtration time (95% by volume) Filtrate volume Filtrate weight Filter-cake weight Dextrose of the mixed slurry (% by weight) Dextrose of 0ltrate heated for 30 min (% by weight) Dextrose of 0ltrate heated for 60 min (% by weight) Dextrose of 0ltrate heated for 90 min (% by weight) Dextrose of 0ltrate heated for 120 min (% by weight)
33% acid
26% acid
20% acid
60 s 101 ml 130:6 g 65 g 7.89 12.9 17.03 21.13 25.85
40 s 122 ml 147:7 g 52 g 5.51 9.38 14.88 24.21 32.51
30 s 141 ml 164:4 g 30 g 3.83 6.22 8.21 10.24 13.03
404
J. Iranmahboob et al. / Biomass and Bioenergy 22 (2002) 401 – 404
concentrations and heating times, cellulose may be degraded or oxidized during the heating process. Therefore, optimizing acid concentration and the heating time of the 0ltrate are principal factors of the hydrolysis process. 5. Conclusion Cost-e4ective production of sugar from cellulose and hemi-cellulosic material present in waste wood chips through acid-hydrolysis is dependent on the concentration of acid and on the heating time of the 0ltrate. The results of this study indicated that the use of sulfuric acid at moderate concentrations (∼26% by weight) and 2 h of heating time was able to produce the highest yield of dextrose for the production of ethanol. Further research is needed to make this process economically feasible. References [1] Broder JD. A case for biomass utilization. Symposium on Energy Features, Kiawah Island, South Carolina, 1991.
[2] Wooley R, Ruth M, Sheehan J, Ibsen K. Lignocellulosic biomass to ethanol process design and economics utilizing co-current dilute acid prehydrolysis and enzymatic hydrolysis current and future scenarios. National Renewable Energy Laboratory, Biotechnology Center for Fuels and Chemicals, Technical Report, Golden, CO, USA, 1999. p. 72. [3] Broder JD, Barrier JW. Producing ethanol and coproducts from multiple feedstocks. American Society of Agricultural Engineers, St. Joseph, MI, Paper # 88-6007, 1988. [4] Broder JD, Barrier JW, Lee KP, Bulls MM. Biofuels system economics. World Resources Review 1995;7(4):560–9. [5] Barrier JW, Bulls MM. Feedstock availability of biomass and wastes. In: Rowell RM, Schultz TO, Narayan R, editors. Proceedings of the ACS Symposium: Emerging Technologies for Materials and Chemicals from Biomass, American Chemical Society, Washington, DC, 1992. p. 410 –21. [6] Patrick Lee KC, Bulls M, Holmes J, Barrier JW. Hybrid process for the conversion of lignocellulosic materials. Applied Biochemistry and Biotechnology 1997;66:1–23. [7] Ingram LO, Aldrich HC, Borges CC, Causey TB, Martinez A, Morales F, Saleh A, Underwood SA, Yamona LP, York SW, Zaldivar J, Zhou S. Enteric bacterial catalysts for fuel ethanol production. Biotechnological Progress 1999;15(5):855–66. [8] Lee J. Biological conversion of lignocellulosic biomass to ethanol. Journal of Biotechnology 1997;56:1–24. [9] Ehrman T. Determination of acid-soluble lignin in biomass. National Renewable Energy Laboratory, Technical Report NREL-LAP-004, Golden, CO, USA, 1996.
Optimizing acid-hydrolysis: a critical step for production of ethanol from mixed wood chips Jamshid Iranmahbooba , Farhad Nadima; ∗ , Sharareh Monemib a The
Environmental Research Institute, University of Connecticut, Route 44, Longley Building, 270 Middle Turnpike U-5210, Storrs, CT 06269-5210, USA b The Department of Biomedical Engineering, University of Connecticut, 260 Glenbrook Rd., U-2157, Storrs, CT USA Received 4 June 2001; received in revised form 10 December 2001; accepted 10 December 2001
Abstract Ethanol can be produced from renewable lignocellulosic materials such as various types of natural woods. The cellulose contents of wood can be converted to ethanol in a two-step process where acid-hydrolysis converts cellulose to glucose sugars by hydrolysis (sacchari0cation) and the resulting sugars can be converted to ethanol by fermentation. The main challenge of producing fuel ethanol from renewable lignocellulosic biomass through acid-hydrolysis and fermentation is overcoming the cost-limiting factors associated with various stages of this technology. In this study, sugar recovery rates from a mixture of wood chips were investigated through three sets of acid-hydrolysis experiments. Wood chips were sorted to include equal ratios (by weight) of softwood and hardwood. Acid concentration and the heating period were the two main factors a4ecting dextrose yields. It was found that with the use of 26% by weight sulfuric acid, highest dextrose yields could be reached within 2 h of heating time. This corresponds to overall conversion e7ciency of mixed wood chip cellulose to dextrose in the range of 78–82% based on theoretical values. ? 2002 Elsevier Science Ltd. All rights reserved. Keywords: Acid-hydrolysis; Biomass; Cellulose; Ethanol; Glucose; Lignin
1. Introduction Advances in technology have allowed the conversion of biomass into fuel ethanol as a potential source of energy. Ethanol is a renewable energy source and can be produced domestically in response to today’s high-energy demand. Due to environmental concerns, the production of sugar from agricultural waste materials, forest residues and municipal solid wastes (MSW) ∗
Corresponding author. Tel.: +1-860-486-6874; fax: +1-860486-5488. E-mail addresses: [email protected] (J. Iranmahboob), [email protected] (F. Nadim), [email protected] (S. Monemi).
and its subsequent conversion to biochemical fuel as an alternative source of energy has gained substantial grounds in the United States. These wastes may be hydrolyzed by acids or enzymes to lower molecular weight carbohydrates and 0nally to monomeric sugars. The United States is the largest producer of wastes in the world [2-kg (4:2 lb) per capita per day] [1]. In terms of energy density, each ton of MSW is equivalent to more than one barrel of oil. It has been estimated that the cost of ethanol, with a feedstock cost of $25=dry US ton will be reduced from its present base of $1.16=gal to about $0.76 by the year 2015 (a reduction of 34%) [2]. Lignocellulosic materials such as agricultural, forest products (hardwood and softwood) and their
0961-9534/02/$ - see front matter ? 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 1 - 9 5 3 4 ( 0 2 ) 0 0 0 1 6 - 8
402
J. Iranmahboob et al. / Biomass and Bioenergy 22 (2002) 401 – 404
residues are renewable resources of energy. Approximately 90% of the dry weight of most plant material is stored in the form of cellulose, hemi-cellulose, pectin, and lignin. Conversion of cellulose and hemi-cellulose from waste materials to sugars provides a feedstock for the production of fuel ethanol and will substantially reduce the amount of wastes that would otherwise exert pressure on municipal land0lls. The main goal of this work was to investigate sugar recovery rates from mixed wood chips originating from a variety of sources with a view to develop an optimal and economically feasible system for production of ethanol as a promising fuel and a base material for other chemical products. The results of this study may bene0t government and private sectors that are interested in the production of fuel and other by-product chemicals from waste materials. 2. Hydrolysis The complex structure of lignocellulose in plants makes a primary protective barrier that prevents cell destruction by bacteria and fungi. In order to break down this structure for conversion of biomass to fuel ethanol, the cellulose and hemi-cellulosic materials must be broken down to corresponding monomers (sugars) so that microorganism can utilize them. There are three major hydrolysis processes for agricultural and wood wastes to produce a variety of sugars capable of making ethanol: dilute acid, concentrated acid, and enzymatic hydrolysis [3,4]. Hemi-cellulose is readily hydrolyzed by dilute acids under moderate conditions, but much more extreme conditions are needed for hydrolysis of cellulose. The main advantage of the low concentration acid in the hydrolysis process is that acid recovery may not be required and there will be no signi0cant losses of acid. However, in a low concentrated acid process, the reaction should be carried out at high temperature and pressure, and due to poor yields of glucose from cellulose in the hydrolysis step the ethanol yield is low. Due to near-quantitative yield of glucose from cellulose, the use of high concentration acid in the hydrolysis process may yield higher quantities of ethanol but di4erent vessels and a good acid recovery process will be required. The dilute acid-hydrolysis process uses high ◦ temperatures (160 C) and pressures (∼10 atm) [5].
The acid concentration in the dilute acid-hydrolysis process is in the range of 2–5% [6,4]. The acid concentration used in concentrated acid-hydrolysis process is in the range of 10 –30% [4]. Lower operating temperatures and pressures are required during the concentrated acid-hydrolysis process. The concentrated acid-hydrolysis involves longer retention times and results in higher ethanol yields than the dilute acid-hydrolysis process [4]. Enzymes produced by a variety of microorganisms are also capable of breaking down lignocellulosic materials to sugars in a longer retention time [7]. Enzymatic hydrolysis requires feedstock pretreatment, enzyme production, and enzyme recovery, which may make this option economically unfeasible. Problems associated with the hydrolysis process need more improvements in order to reduce the operating cost for an ethanol-producing plant that uses wood wastes as feedstock. Development of a feasible process for the acid-hydrolysis of wood chips that could lead high sugar yields and result into less oxidation of the produced dextrose by the concentrated acid were investigated in three sets of bench-scale experiments. 3. Experimental procedure Mixture of 50% hardwood (oak, birch and maple) and 50% softwood (pine and cedar) chips free from ◦ barks were dried at 72 C overnight. The wood chip mixture was rechipped and sieved, and the fraction between 4 and 10 mm was used. Various lignocellulosic raw materials’ composition for one class of hardwood (populus) and one class of softwood (0r) similar to the wood chips used in this study are given in Table 1. Dried, ground wood chips of 200 g were gradually mixed with 500 g of 80% (by weight) H2 SO4 acid to generate a homogeneous paste. The color of the paste turned dark without resulting into severe oxidation of the mixture by acid. Distilled boiling water of 500 g was added to the paste to obtain a 33% (by weight) ◦ acid mixed slurry and was stirred for 30 min at 100 C using a water bath. The mixed slurry was then 0ltrated. The 0ltration time, volume and weight of the 0ltrate, and the weight of 0lter-cake were recorded. The 0ltrate was then heated for 2 h. In order to measure the percent dextrose of the heated 0ltrate using a sugar analyzer (YSI 2700 SELECT) during the
J. Iranmahboob et al. / Biomass and Bioenergy 22 (2002) 401 – 404 Table 1 Composition of various lignocellulosic raw materials in a typical softwood and hardwood
Cellulose (wt%) Hemi-cellulose (wt%) Non-carbohydrate (%) Lignin Ash
Populus tristis (hardwood)
Douglas 7r (softwood)
45.0 30.0
42.0 27.0
40 26% H2SO4
35 30 % Dextrose
Component
403
25 20
33% H2SO4
15 10
20.0 1.0
Carbohydrate (% of sugar equivalent) Glucose 40.0 Mannose 8.0 Galactose NA Xylose 13.0 Arabinose 2.0
28.3 0.2
5
20% H2SO4
0 0
50.0 12.0 1.3 3.4 1.1
Source: Ref. [8].
heating process, samples were taken at 30 min intervals and neutralized (pH: 6 –8) with a 1 N NaOH solution. The same procedure was done for 26% and 20% (by weight) acid mixture slurries.
4. Results and discussion In the hydrolysis of wood wastes, the e4ective acid concentration and the time of heating vary depending on the composition of the wastes such as cellulose, hemicellulose, lignin, ash and pectin. Wood wastes are usually collected and stored as a mixture. Therefore, in this study, acid-hydrolysis was mainly performed on mixed wood chips. The results of hydrolysis of mixed wood chips are shown in Table 2. The 0ltration
0.5
1 1.5 Time (hour)
2
2.5
Fig. 1. The time sequence 0ltrate hydrolysis after treatment of wood chips with sulfuric acid at di4erent concentrations (samples were taken at 30 min intervals). Data points are average values of three experimental runs with error bars indicating the standard error of the means.
time is longer for higher acid concentration because of its high viscosity. The dextrose concentration of the mixed slurry (after 30 min of heating process) was highest for 33% acid slurry. The results of heating process of the 0ltrates for all three acid concentrations, 33%, 26%, and 20% are shown in Fig. 1. The maximum dextrose (sugar) concentration was achieved in 26% (by weight) acid-hydrolysis process batch, in which 0ltrate was heated for 2 h. Lignin is mostly insoluble in mineral acids and only a small fraction of the lignin in a biomass sample is solubilized during the hydrolysis process [9]. With very small quantities of dissolved lignin, the hydrolysis process of cellulose to sugar becomes a simple process and can occur in a less acid concentration mixture. However, at higher acid
Table 2 The results for acid-hydrolysis of mixed wood chips
Filtration time (95% by volume) Filtrate volume Filtrate weight Filter-cake weight Dextrose of the mixed slurry (% by weight) Dextrose of 0ltrate heated for 30 min (% by weight) Dextrose of 0ltrate heated for 60 min (% by weight) Dextrose of 0ltrate heated for 90 min (% by weight) Dextrose of 0ltrate heated for 120 min (% by weight)
33% acid
26% acid
20% acid
60 s 101 ml 130:6 g 65 g 7.89 12.9 17.03 21.13 25.85
40 s 122 ml 147:7 g 52 g 5.51 9.38 14.88 24.21 32.51
30 s 141 ml 164:4 g 30 g 3.83 6.22 8.21 10.24 13.03
404
J. Iranmahboob et al. / Biomass and Bioenergy 22 (2002) 401 – 404
concentrations and heating times, cellulose may be degraded or oxidized during the heating process. Therefore, optimizing acid concentration and the heating time of the 0ltrate are principal factors of the hydrolysis process. 5. Conclusion Cost-e4ective production of sugar from cellulose and hemi-cellulosic material present in waste wood chips through acid-hydrolysis is dependent on the concentration of acid and on the heating time of the 0ltrate. The results of this study indicated that the use of sulfuric acid at moderate concentrations (∼26% by weight) and 2 h of heating time was able to produce the highest yield of dextrose for the production of ethanol. Further research is needed to make this process economically feasible. References [1] Broder JD. A case for biomass utilization. Symposium on Energy Features, Kiawah Island, South Carolina, 1991.
[2] Wooley R, Ruth M, Sheehan J, Ibsen K. Lignocellulosic biomass to ethanol process design and economics utilizing co-current dilute acid prehydrolysis and enzymatic hydrolysis current and future scenarios. National Renewable Energy Laboratory, Biotechnology Center for Fuels and Chemicals, Technical Report, Golden, CO, USA, 1999. p. 72. [3] Broder JD, Barrier JW. Producing ethanol and coproducts from multiple feedstocks. American Society of Agricultural Engineers, St. Joseph, MI, Paper # 88-6007, 1988. [4] Broder JD, Barrier JW, Lee KP, Bulls MM. Biofuels system economics. World Resources Review 1995;7(4):560–9. [5] Barrier JW, Bulls MM. Feedstock availability of biomass and wastes. In: Rowell RM, Schultz TO, Narayan R, editors. Proceedings of the ACS Symposium: Emerging Technologies for Materials and Chemicals from Biomass, American Chemical Society, Washington, DC, 1992. p. 410 –21. [6] Patrick Lee KC, Bulls M, Holmes J, Barrier JW. Hybrid process for the conversion of lignocellulosic materials. Applied Biochemistry and Biotechnology 1997;66:1–23. [7] Ingram LO, Aldrich HC, Borges CC, Causey TB, Martinez A, Morales F, Saleh A, Underwood SA, Yamona LP, York SW, Zaldivar J, Zhou S. Enteric bacterial catalysts for fuel ethanol production. Biotechnological Progress 1999;15(5):855–66. [8] Lee J. Biological conversion of lignocellulosic biomass to ethanol. Journal of Biotechnology 1997;56:1–24. [9] Ehrman T. Determination of acid-soluble lignin in biomass. National Renewable Energy Laboratory, Technical Report NREL-LAP-004, Golden, CO, USA, 1996.