CHE561CellulisticEthanolPre Treatmenttechnologiesandsimulation

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Ligno-Cellulose Pre-treatment Technologies in Bio-Ethanol Production with a

Related Simulation Study of Economic/Technical Factors using Corn Stover
Feedstock
Research · November 2015

DOI: 10.13140/RG.2.1.5071.4965
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Ligno-Cellulose Pre-treatment Technologies in Bio-Ethanol Production with a Related
Simulation Study of Economic/Technical Factors using Corn Stover Feedstock
Introduction:
There are many factors at work to cause significant increases is gasoline and synthetic chemical precursors derived from
crude oil extract feed stocks. Since crude oil is one of the mainstays of modern civilization, the main worldwide supplies
of oil are under strict control by OPEC and ultimately there is a limited supply of oil, it is necessary for the United States
to invest in renewable energy sources that can sustain the economic and technology base derived from oil based
technologies developed during the course of the 20th century. In addition to economic reasons and environmental
concerns, renewable energy sources are vital to the strategic and security interests of the United States.
Many commercial, government and academic activities around the world are working on developing technology for
renewable Bio-Ethanol energy sources to replace current petroleum energy sources. Ligno-cellulosic material derived
from plant matter and waste stream sources has garnered considerable attention as an untapped source of raw
materials for Bio-Ethanol generation. The use of food crops for the generation of Bio-Ethanol has caused severe
dislocation in worldwide food markets with concomitant large prices increases in basic foodstuffs; an effect which has
led many of the world’s poor to the brink of starvation and cause the generation of severe political instabilities. Ligno-
cellulosic Bio-mass for the generation of Bio-Ethanol and other fuels have a realistic potential to act as a replacement
for the world’s current oil usage given recent upward trends in petroleum fuel prices, the abundance of Ligno-cellulosic
materials and significant improvements in the economics/efficiencies of Bio-Ethanol generation technologies.
Many of the leading technologies for Bio-ethanol production from Ligno-cellulosic materials are based upon high
pressure steam with dilute acid hydrolysis to separate the Lignin and Hemi-cellulose fractions from the cellulose
fraction. Dilute Sulfuric acid (0.5%-1.5%) is most commonly used for acid hydrolysis pre-treatment of wood as a step in
producing Ethanol from shredded/pulverized wood; a process that has been around in commercial scale industrial
processes since the early20th century. Several key modern adaptations of the basic acid hydrolysis process have been
made, such as the “steam explosion” of cellulose pores via sudden decompression of the cellulosic material coming out
of the dilute acid autoclave process. The steam explosion process causes significant increases in exposed surface area
that increases the efficiency of the Cellulase enzyme system (endo /exo-gluconases and β-glucosidase) conversion of
cellulose into glucose. This latter process is termed Saccharification and is also a modern addition to the traditional
process for the digestion of cellulose into fermentable sugars. During the dilute acid autoclaving of Ligno-cellulosic
materials, the hemi-cellulose fraction is subject to acid hydrolysis into C5 (such as Xylans into Xylose) and C6 sugars; the
largest end product being primarily Xylose. The traditional fermentation process has also been given a modern addition
with genetically engineered yeast that is capable of fermenting Xylose as well as Glucose into Ethanol.
In spite of all of the advantages there are downsides to the dilute acid pre-treatment method in that undesirable
impurities are formed, such as acetic acid, phenols, furfurals, and furans resulting from the decomposition of Lignin and
semi-cellulose sugars like Xylose. In current processes the liberation of water soluble Lignins is an undesirable byproduct
of the conversion of hemi-cellulose and cellulose materials into fermentable sugars. Besides the generation of
undesirable contaminates from Xylose sugar breakdown in the dilute acid hydrolysis process, there is a loss of
fermentable Xylose, which can be significant. During the dilute acid hydrolysis step, the free Lignins tend to recombine
with hemi-celluloses and themselves, leading to products that hinder the Saccharification (enzymatic) digestion process
by blocking access to underlying cellulose to the Cellulase enzymes and also by the binding and inactivating of Cellulase
enzymes. In addition to hindering Saccharification process efficiency, water soluble Lignins interfere with the micro-
organisms responsible for the fermentation process, as also do acetic acid, furans and phenols formed during the dilute
acid hydrolysis .
The US Lawrence Berkley and Sandia National Labs have developed a complex simulation of a Ligno-cellulose Bio-
Ethanol production facility reflecting the modern dilute acid hydrolysis pre-treatment, Saccharification and fermentation
processes described above. The simulation was implemented with the Intelligen SuperPro 8 chemical process simulation
software. The goal of working with the simulation is to identify possible process improvements within the simulated
processes that have the potential to significantly affect the economic viability of a real Bio-Ethanol plant processing Corn
Stover as the primary feedstock.
(Aden 2008, BlueFire Ethanol Company 2011, Klein-Marcuschamer 2010, Wooley 2008, zFacts 2008)
Pre-treatment processes
In order to greatly enhance the release of fermentable C5 and C6 sugars during enzymatic saccharification it is necessary
to have a pretreatment process that can dissociate the lignin, hemicellulose and cellulose fractions of Ligno-cellulose
bio-mass used as a feedstock for bio-Ethanol production. A number of pre-treatment regimes have been introduced
such as concentrated acid, dilute acid, hot water, steam, organic "ionic" liquids, inorganic ionic salts, organic non-ionic
surfactants and alkalis like NaOH, lime (Ca[OH]2) and ammonia. All of these methods are basically high temperature,
high pressure hydrolysis digestions. As for acid digestion, sulfuric acid (H2SO4) is generally most popular, but
hydrochloric acid and various carboxylic acids have been used. All of these methods have advantages, but also
disadvantages such as cost, environmental impact and waste products that are toxic to enzymes and microbes used in
downstream processes (saccharification, fermentation). (Achyuthan 2010)
Since all of the above pre-treatment processes are done in high temperature aqueous solution, it is critical to
understand a few things about the properties/role of water in the digestion chemistry. Thermally energetic waters are
very strong nucleophiles and are the primary nucleophiles in hot water/steam and dilute acid pretreatments responsible
for the hydrolysis of ether linkages. Secondly, the role of water as a solvent must not be misunderstood. Water is usually
conceived of as a polar solvent, and so it is, but at high temperatures and pressures (especially above the critical point of
water) the solvent nature of water begins to take on what would be at room temperature chemistry and properties akin
to polar organic solvents at room temperature. This is described as water taking on liquid and gas properties, probably
due to the formation of Chlathrate gas bubbles with steam as the gas. As the nature of the solvent properties of water
evolve towards a quasi-organic solvent nature at high temperatures/pressures, solvation of hydrophobic species such as
poly-phenolic lignin molecules become very useful to the pre-treatment process. (Siskin 1991)
When lignin is not separated from hemicellulose and cellulose by pre-treatment processes applied to Ligno-cellulose
biomass there are a number of difficulties posed for the downstream saccharification process:
1) Lignin's poly-phenolic/hydrophobic character tends to re-bind hemicellulose and cellulose through hydrogen
bonding, polar interactions, Van Der Waals interactions and hydrophobic interactions, thereby physically
blocking access of saccharification enzymes to their carbohydrate substrates.
2) Lignin's will experience covalent condensation reactions with themselves and with hemicellulose if the
pretreatment time/temperature becomes too excessive.
3) Pre-treatment of the Ligno-cellulose produce phenolic and non-phenolic inhibitors of saccharification enzymes
4) Lignin's ultra-molecular structure and hydrophobic character pose a physical barrier to saccharification enzymes
(cellulases and ß-glucosidases).
5) Lignin physically adsorbs saccharification enzymes and thereby inactivating them

6) The strength of lignin inhibition of saccharification enzymes depend strongly the percentage of lignin, its S/G
ratio and its cross-linking density/structure
(Achyuthan 2010)
The Structure of Ligno-cellulose Feedstock’s Affects the Ease of Liberation of Complex Carbohydrates
Lignin in Ligno-cellulosic bio-mass is composed primarily from three phenyl-propanoid monomers: coniferyl alcohol,
sinapyl alcohol and p-coumaryl alcohol. The lignin polymer/large-molecule forms with free radical condensation of the 3
basic monomers. Lignins are soluble in alkaline aqueous media above a pH of 10.5, but precipitate out in neutral and
acidic solution. Lignins are also soluble in acetone, dioxane, dimethyl formamide, and methyl cellusolve. The solubility of
Kraft and other lignins (molecular weight ~2,000-3,000)) in alkaline solution are due to the formation of negatively
charged phenoxy anions. (Lebo 2000, Achyuthan 2010)
Another complication within the structure of Ligno-cellulose feedstock’s is that Hemicellulose effectively "cements"
lignin to the cellulose fraction, so any successful lignin separation process (not to mention a bio-Ethanol production
process) needs to release the hemicellulose fraction from both the cellulose and lignin fractions. To this end, many novel
methods of separating lignin from hemicellulose have been explored such as fungal xylanases used to pretreat wood in
Kraft wood pulping processes. (Angayarkanni 2006)
Production bio-Ethanol is limited by the difficulty to extract complex carbohydrates from Ligno-cellulose feedstock and
completely liberate without degradation the pentose and hexose sugar monomers. The lignin fraction of feedstock’s
represents a formidable barrier to the success of the bio-Ethanol production process saccharification and fermentation
unit operations. It has been shown that the success of pretreatment regimes like dilute (~1%) Sulfuric Acid digestion
(liberates Xylose/Xylans, opens up cellulose chains to enzymatic digestion) depend upon the relative fraction of Lignin in
the feedstock and the relative proportion of the 3 aryl propanoid units making up the highly cross linked, condensed
lignin ultra-molecular structure. In particular, the intent of the Syringyl like (S) lignin structures and the Guaiacyl like (G)
lignin structures are the more important consideration as expressed by the S/G ratio.
The relative lignin content and S/G ratio can non-trivial even within the family and even the same species. For example,
A large sampling of Populus hybrids had lignin fractions range from 22.7-25.8% and S/G ratios from 1.8-2.3. Dilute
H2SO4 hydrolysis of Populus samples with varying lignin content and S/G ratios produced different yields of Xylose. The
Xylose yield from dilute acid digestion of Populus high lignin content and a high S/G ratio (25.8% lignin and S/G=2.3)
produced 30% of the theoretical yield, whereas the Xylose yield from Populus samples undergoing dilute acid digestion
with a low lignin fraction and low S/G ratio (22.7% lignin, S/G=1.8) produced 55% of the theoretical value. These results
indicate that both lignin contents and S/G ratio significantly affected the yield of xylose from dilute acid digestion. These
trends are seen across a number of other tree species and these trends are also seen with the Kraft alkaline digestion
process of wood pulping in the resultant delignification efficiency of the process. (Davison 2006)
A second study expanded upon and reproduced the main features of the Davison 2006 study of lignin content (15.7–
27.9%) and syringyl/guaiacyl (S/G) monomer unit ratios (1.0–3.0) from the undomesticated tree species Populus
trichorcarpa. A sample set of 1,100 core samples were taken giving the best representation to date of the natural
variation of lignin content and S/G ratios in Populus trichorcarpa, including 47 extreme phenotypes. This set of samples
was run through a protocol of saccharification enzyme hydrolysis with and without hot water hydrolysis pre-treatment
to look at total monosaccharide sugar yields to quantitatively assess the resistance of each sample to hydrolysis
digestion. The 47 extreme phenotypes and a sample of 17 "average" cores (lignin ~22.5%, S/G ~2) were evaluated in
significant detail. The study goal was to quantitatively analyze the effect of difficulty of generating free sugars due to
lignin content, S/G ratio, pre-treatment processing parameters, interaction of wood parameters/processing parameters
with anomalous core results and to assess the possibility that other factors within the natural variation of the wood
could play a significant role on sugar release. Both xylan and glucan release were studied along with their respective
monosaccharide monomers xylose (C5) and glucose (C6). Therefore cellulase and xylanase enzymes were jointly utilized.
The release yield of xylan and glucan was found to vary widely amongst the core samples of the 47 different genotypes.
Maximum monosaccharide release was up to 92% of the theoretical maximum. (Studer 2011)
For pre-treated samples there was a strong negative correlation between monosaccharide yields and lignin content for
cores with a S/G ratio less than 2.0. High S/G ratios (≥2.0) correlated with high monosaccharide yields with the effect of
lignin content becoming a minor factor. Interestingly, deeper analysis showed the glucose yield followed the above
pattern, but xylose yield correlated only with the S/G ratio. For cores with no pre-treatment, the monosaccharide yields
were strongly negatively correlated with lignin content for lignin contents below ~20% and no effect attributable to S/G
rations could be apprehended. Certain of the "average" cores had anonymously high/low monosaccharide yields,
strongly suggesting other significant factors were at work besides lignin content and S/G ratio. It is well documented in
the literature that a high S/G ratio enhances the efficiency of Kraft pulping efficiency, but it adversely affects xylose
release in a dilute acid hydrolysis pre-treatment regime. It may be that the actual cell wall structure (e.g. amount and
distribution of lignin) is the other variable in the monosaccharide yield performance of anomalous cores. (Studer 2011)
There was a strong positive dependency of monosaccharide yields attributable to the pre-treatment hot water
temperature, or lack of pre-treatment, as may be reasonably anticipated from known chemical kinetics theory
(Studer 2011):
Table 1
pretreatment monosaccharide yield in Percent of theoretical

temperature grams/g dry feedstock monosaccharide yield
at 140°C 0.17 to 0.58 g/g (23–83%)

at 160°C 0.20 to 0.68 g/g (28–92%)
at 180°C 0.25 to 0.67 g/g (35–91%)
no -
pre-treatment 0.05 to 0.40 g/g (4–56%)
The Guaiacyl (G) like monomers in lignin can covalently crosslink with up to three other monomers, while Syringyl (S) like
monomers can only crosslink with up to two other monomers. The S rich lignin predominantly comprises linear chains
with lower cross linking density than G rich lignin owing to the methoxylated and stearically hindered C-5 position in the
S monomer. The S rich lignin also has a higher fraction of β-β units (resinols), shorter chain lengths and lower average
molecular weights. All of these features of S rich lignin will lead to thermo-mechanical changes such as lower glass
transition temperatures and lower melting temperatures, which can be expected to have an effect on hydrolysis rates in
hot digestion environments. The relative amount of chemically active β-O-4 ether linkages is relatively constant over a
wide range of S/G ratios, depending upon the presence of at least some S monomeric units. The chemically active β-O-4
ether linkages are the primary hydrolysis targets of hot water/steam and dilute acid pre-treatment regimes. The steam
explosion pre-treatment method greatly reduces (by cleavage) the remaining β-O-4 linkages after hydrolysis. The S/G
ratio drops during dilute acid hydrolysis inferring a preferred chemical attack on the syringyl monomer units. This
pattern of enhanced chemical cleavage rates of β-O-4 linkages in S rich lignin also exists under alkaline hydrolysis
conditions. The higher the degree of β-O-4 cleavage that occurs during pre-treatment leads directly to to higher sugar
yields after enzymatic hydrolysis. (Studer 2011, Davison 2006)
Acid Pre-treatment Processes

An "acid soluble" lignin fraction is observed when Ligno-cellulose bio-mass is digested with hot dilute or concentrated
Sulfuric acid (H2SO4). In one study the formation mechanism of acid soluble lignin (ASL) from the lignin portion of beech
wood meals digested with hot 72% sulfuric acid (SA) and elucidated with various time/temperature profiles. The Acid
Soluble Lignin (ASL) fraction in aqueous solution was extractable with Chloroform (CHC13). ASL from beech wood
feedstock was formed by acid hydrolysis and dissolved in 72% H2SO4 for further experimentation. The concentration of
ASL is maximal after the original acid digestion, but solubility of ASL decreases with prolonged heating in 72% H2SO4 till
it reaches a plateau concentration. ASL prepared from hot 72% H2SO4 and boiled in 3% H2SO4 will yield a mixture of
ASL and insoluble Klason lignin, but only half of the ASL left after boiling in 3% H2SO4 is extractable in CHCl3. These data
suggest that the initial acid hydrolysis in 72% H2SO4 (at an appropriate time/temperature) produce relatively low
molecular weight hydrolysis fragments which can undergo condensation to produce higher molecular weight molecules
that undergo internal rearrangements that make these condensed fragments highly resistant to further hot acid attack.
(Yasuda 2001)
Another study further elucidated the formation and chemical structures of water dispersible species of acid soluble
lignin through the use of model lignin aryl analogues. Highly cross-linked lignin can be acted upon by three general
reaction types: phenolation, hydroxymethylation, and sulfonation into water soluble lignosulfonates. Lignin is composed
of three basic aryl propanoid units, so certain aromatic test species that were analogous to lignin aryl units were
selected: compounds of creosol (I) and 5-methoxycreosol (II). These test species were reacted with xylose or xylan in the
presence of apocynol. The goal was to simulate ASL fragment condensation in 72% sulfuric acid. The reaction of test
species I with apocynol yielded condensation products. The reaction of test species II with apocynol did not readily yield
condensation products due to steric hindrance effects from the methoxyl group, but refluxing in 3% H2SO4 did yield
condensation products but did yield C-xyloside. Information on Chloroform extractable (from an acid aqueous phase)
was obtained with other suite of test species consisting of aryl glycerol-ß-aryl ethers combined with guaiacyl (III) and
syringyl (IV) compounds treated using the hot 72% H2SO$ procedure. Test species III gave only insoluble polymerized
products, while test species IV gave insoluble polymerized products and Chloroform soluble low molecular weight
products, which had been dissolved in 3% H2SO4. These results strongly support the view that Klason ASL consist of
water dispersible/soluble syringyl lignin and mono-saccharide units from hemicellulose. The Chloroform soluble fraction
of ASL appear to be relatively low molecular weight, hydrophobic fragments from the hot 72% H2SO4 reaction
environment process. (Yasuda 1998)
After an alkaline digestion pre-treatment process of Ligno-cellulose feedstock it can be shown that not all of the lignin is
removed from the predominantly cellulose solid fraction remaining. This effect can be seen in the common acid dioxane
process used for the extraction of lignin remaining in Kraft process wood pulps. This extraction procedure comprises a
pre-extraction clean up with acetone, then a reflux extraction in a solution of 0.1M HCL in 9:1 dioxane:H20 in an inert
atmosphere. The dioxane:water mixture is an azeotrope boiling point of 88°C. The acid dioxane extraction recovers a
certain amount of high purity lignins (around 40-60%). Enzymatic digestion of Kraft wood pulps (such as in bio-Ethanol
saccharification) would liberate an even higher yield of lignins than typically found in this laboratory method unless
more severe conditions are imposed on the extraction with higher acid content, longer time and higher temperature.
The most likely mechanism responsible for the liberation of lignin is the hydrolysis of covalent linkages between lignin
and cellulose/hemicellulose carbohydrates in the pulp. The acid hydrolysis conditions can be expected to cause lignin to
undergo structural modifications such as the cleavage a-aryl and a-alkyl ethers, and ß-aryl ethers in benzyl alcohol units.
Cleavage of aryl ethers would result in phenolic hydroxyl groups at higher levels than would be found in the natural
wood or Kraft wood pulp. These data suggest that a high temperature, high pressure dilute acid pretreatment step
would still be desirable after an alkaline digestion pretreatment process. (Froass 1996, Lundquist 1973, Gellerstedt 1994,
Jiang 1999, Ragauskas 2011)
Alkaline Pre-treatment Processes

The modern Kraft process for the pulping of wood to recover relatively high purity is an alkaline digestion method that is
the result of almost a century or fundamental research and practical process development work. The extensive
literature and available equipment used in modern wood pulping processes can greatly inform the choice of
pretreatment methods used in Ethanol bio-processes’ using Ligno-cellulose feedstock’s. Within the Kraft wood pulping
process, lignin and hemicellulose fractions freed by high temperature/pressure digestion are removed from the cellulose
fraction in a water soluble matrix known by the nomenclature black liquor. For an Ethanol bio-process the lignin fraction
represents a waste and poison of the saccharification and fermentation sub-processes. The opportunity to remove lignin
as a Ethanol bio-process waste and convert it into a profitable product promises great economic opportunities for the
bio-process production of Ethanol. In addition to the Kraft wood pulping process there are a number of other alkaline
digestion pretreatment methods for the pre-treatment of Ligno-cellulose feedstock’s. (Kumar 2009, Studer 2011)
The chemical reactivity’s of lignin are very important to the success of chemical digestion pretreatment processes used
for bio-Ethanol production, both in separating lignin from the hemicellulose and cellulose fractions of the Ligno-cellulose
feedstock’s and in keeping the lignin separated from these two generic species of interest for the subsequent
saccharification and fermentation processes. The separation of lignin is accomplished by the alkaline or acid hydrolysis
(with adequate time/temperature) of lignin ether bonds to cellulose and hemicellulose. The lignin ultra-molecular
structure is broken down into water soluble/water dispersible super-molecular units through hydrolysis benzyl-diaryl
ether compounds integral to the ultra-molecular structure. It is known that Lignin condensation reactions occur in Kraft
and similar Soda pulping processes if they are too alkaline and/or too much time/temperature is used. In other words,
water dispersed lignin super-molecules can undergo internal alkyl/aromatic structural rearrangements (which
significantly resist further chemical attack) and reform external ether bonds (condensation) with other water dispersed
super-molecular units. A small fraction of the water dispersed/solvable lignin in a alkaline digested Ligno-cellulose
biomass sample remains solubilized after acid precipitation of the acid insoluble fraction. The acid soluble lignin fraction
forms during the hydrolysis process of the acid insoluble lignin method. This lignin fraction can be quantified by
ultraviolet spectroscopy. (Reed 1988)
One study looked at four of the commonly used chemical Ligno-cellulose pre-treatment processes: dilute acid, lime,
aqueous ammonia steeping followed by dilute acid hydrolysis, and sodium hydroxide. There was a direct correlation
between degree lignin removal and saccharification enzymatic digestion efficiency of pre-treated corn stover through
improved accessibility of the enzymes to the cellulose fraction. Pre-treatment of corn stover with 2% NaOH substantially
increased the lignin removal compared with the other pre-treatment regimes. The enzymatic digestion (hydrolysis) of
the NaOH pre-treated corn stover reached an 81.2% yield at 48 hours with 8% substrate concentration and 20 FPU
g/liter of cellulase. Analysis of the sugar liquors showed an increased sugar concentration and lower generation of
process contaminants than with the other three pre-treatments. (Chena 2009)
Ammonium Hydroxide Pretreatment

One study looked at separating lignin liquors produced from Ligno-cellulose feedstock materials using ammonium
hydroxide alkaline digestion pretreatment. In particular this study looked at switch grass as a crop source of Ligno-
cellulose bio-mass. The post pretreatment lignin recovery process consisted of acidification of the pretreatment liquor,
evaporation of the acid treated liquor and recovery of the precipitation lignin with centrifugation, which resulted in the
recovery of a largely pure solid lignin product. The method was capable of recovering 66-95% of the dissolved Lignin in
the switch grass pretreatment liquor. It has been estimated that the break even cost of pilot-scale and industrial-scale
processes are respectively $2,360/MT and $780/MT. (Sherman 2011)
Inorganic salt pre-treatment processes

A number of inorganic salts have been evaluated as pre-treatment agents for corn stover hydrolysis. For example, such
salts as NaCl, KCl, CaCl2, MgCl2, FeCl2, FeSO4, FeCl3, and Fe2(SO4) have been studied. Several advantages accrue from
the use of ionic salts as hydrolyzation inducing agents, such as their low cost, the fact that they can be easily recovered
and to some extent a salting in effect for hydrolysis products can be expected, especially lignin fragments. The choice of
FeCl3 as a hydrolysis agent was found to be particularly fortuitous in one study. A FeCl3 pre-treatment study was
conducted under high pressure, high temperature (140-200°C) hydrolysis digestion of corn stover feedstock. The best
results were obtained with 0.1M FeCL3 at 140 °C for 20 min. Under these conditions with a xylose recovery of 90%-91%
of the theoretical yield could be obtained with less than a 10% loss of the cellulose fraction. Hemicellulose
hydrolysis and removal from the corn stover solids increased 11x with 0.1 M FeCl3 compared to pre-treatment with a
hot water hydrolysis under the same conditions and there was a 6x improvement over a dilute sulfuric acid pre-
treatment at the same pH. (Liua 2009)
Non-Ionic Surfactant Effect on Dilute Acid/Hot Water Pre-treatment and Saccharification

As described elsewhere in this paper, it is well documented that the presence of Lignins during the enzymatic
saccharification process strongly interferes with the action of the cellulase and xylanase enzyme hydrolysis efficiency
through lignin binding of the enzymes or lignin physically blocking the enzyme substrate material. As such, it may be
reasonably projected that the presence of surfactants would enhance the action of dilute acid hydrolysis and the action
of saccharification enzymes by increasing surface interaction with the digestion chemistries and by physically solubilizing
water dispersible lignin molecules.
One study evaluated a suite of surfactants on the efficiencies of dilute acid pre=treatment, hot water pretreatment and
saccharification at temperatures ranging from 140C-220C with corn stover as a feedstock/substrate. The surfactants
studied were: Tween-80, Dodecyl-Benzene Sulfonic Acid (DDBSA), and Polyethylene Glycol 4000 (PEG). It was
experimentally demonstrated that all of these surfactants were salubrious for the solubilizing of lignin during pre-
treatment, the reduction of enzyme/lignin binding and an increase in overall monosaccharide yields. Tween-80 had the
greatest effect on increasing monosaccharide yields. The high cost of purified, active saccharification enzymes is a major
consumables cost factor in the production of bio-Ethanol. Usual cellulase loadings are about 15 FPU/gram of cellulose,
which translates out to about 30 grams of enzyme per liter of Ethyl alcohol produced. With average costs of enzymes
coming in at around $2/Kilogram, it's not hard to project that the cost of enzymes really escalates the total recurring
costs. Therefore, anything that can be done to up saccharification efficiency and thereby reduce enzyme loadings is
going to have a dramatic effect on the profitability of a bio-Ethanol production facility, which is why there has been
some interest generated by the positive effect on saccharification monosaccharide yields by surfactants, including non-
ionic surfactants. Organic solvents have been used on a laboratory scale to remove lignins before saccharification, but
this method is generally too expensive for industrial production of bio-Ethanol. (Qing 2010)
While the exact mechanisms for the actions of surfactants during pre-treatment and saccharification are not known
there are certain effects that can be predicted based upon theoretical considerations and experimental data
(Qing 2010):
1) Alter the substrate surface and pore structure and make the substrate surface physically more accessible to
saccharification enzymes.
2) Alter the substrate surface chemistry and so to make the substrate surface chemically more accessible to
saccharification enzymes (i.e. reduction of hydrophobic effects).
3) Stabilize enzymes and prevent denaturation during saccharification
4) Reduce the binding stability of saccharification enzymes by lignin
5) (5) Partition and sequester lignin in solution and prevent lignin molecules from re-attaching to the substrate
surfaces during saccharification.
The overall experimental protocol called for corn stover soaked in surfactant solution (Tween-80, DDBSA, and PEG)
solution overnight prior to either the dilute acid (140C, 1% sulfuric acid, 40 min) pre-treatment or the hot water-only
(220C, 30 min) pre-treatment. The Lignin removal for corn stover feedstock was about 17.0% for dilute acid pre-
treatment and 7.8% for hot water pre-treatment, where both of these conditions served as the controls (sans
surfactants). The Tween-80 soak increased lignin removal to 25.6% for the dilute acid pre-treatment and 16.7% for the
hot water pre-treatment. The DDBSA soak increased lignin removal to 19.8% for the dilute acid pre-treatment and 13.2%
for the hot water pre-treatment. At best the PEG soak was marginally better than the controls. The data shows that the
dilute acid pre-treatment is superior to the hot water pre-treatment at removing lignin from the feedstock and that
Tween-80 had the greatest effect on pre-treatment efficiency for removing lignin from the bio-mass. (Qing 2010)
The effect of the surfactant soaks prior to pre-treatment on saccharification efficiency were assessed by varying enzyme
loadings from 10-60 FPU/gram glycan and measuring the monosaccharide yields. At the lowest cellulase loading of 10
FPU/gram of glycan, a supplement of 10 CBU/gram of glycan was added. At the highest cellulase loading of 60 FPU/gram
of glycan, a supplement of 120 CBU/gram of glycan was added. After 96 hours of saccharification processing at the
highest enzyme loading the glucose yields were 90-93% of theoretical with all of the surfactant soaks. After 96 hours of
saccharification at the lowest enzyme loading, the Tween-80 soak improved the glucose yield from 78.2% of theoretical
for the control up to 88.1% of theoretical for the surfactant soak. Interestingly, at the lowest enzyme loading, the PEG
soak had a marginal increase in glucose yield while the DDBSA soak actually resulted in a lower glucose yield. Generally
speaking, the Tween-80 soak showed the best overall increase in glycan conversion yields of the 3 surfactants in the
experimental panel. (Qing 2010)
A second round of experiments was conducted on corn stover using a Tween-80 soak and a lowering of the harshness of
the pre-treatment conditions, which used a solids loading of 5%. The new dilute acid regimen was 0.5% sulfuric acid at
160C for 16-24 minutes. The new Hot water regimen was 190C for 10–18 min. These were the time windows where the
highest overall monosaccharide yields were projected to be. A dilute acid pretreatment at 160C with a Tween-80 soak
and saccharification with the lowest enzyme loading, as defined above, achieved better glucose yields than the control
at 20-22 minutes. Xylose yields under these conditions at 18 minutes were better than the control at 18-24 minutes,
after which the control began to have lower xylose yields. At 20 minutes of the dilute acid pre-treatment with the
Tween-80 soak, the glucose yield was 59.8% of theoretical and the xylose yield was 24.3% of theoretical, which
represented about a 10% yield improvement over the controls. Lignin removal from the solids was greater with the
Tween-80 soak than the controls. With the 190C hot water pre-treatment at 16 minutes, the Tween-80 soak had about a
10% improvement in monosaccharide yields over the control and reduced the amount of xylose degradation compared
to the control. The degree of lignin removal from the solids improved with the hot water pre-treatment using the
Tween-80 soak, but it was not as high as that of the dilute acid pre-treatment with the Tween-80 soak. The upshot of the
two experimental panels is that a soak with the right surfactant increases the efficiency of the pre-treatment and
saccharification processes, especially at marginal operating conditions, thereby enhancing the robustness of the overall
process. (Qing 2010)
Two step hybrid acid and alkali pre-treatments

The PureVision process is a good example of a two-step hybrid pre-treatment process possessing some unique features
with an in-line multi-chamber reactor, fractionation of the three primary constituents of Ligno-cellulose and a two stage
hydrolysis pre-treatment regime. The 3 output fractions of the PureVision process are xylose rich liquor, lignin rich liquor
and a 90-97% pure solid cellulose fraction. The multi-stage in-line reactor uses a mechanical transport system to move
solids within and in-between each reactor stage. Shredded Ligno-cellulose feedstock is hopper fed into the first reactor
chamber where it is wetted. Next the mechanical transport system moves the wetted feedstock into the third chamber,
which is the 1st Stage reactor chamber with integral solid-liquid separation capability. A hot Water/dilute acid extraction
takes place in the stage 1 reactor chamber. In later versions of the PureVision process the stage 1 reactor uses hot water
hydrolysis (210C) which pays a penalty in a lower xylose yield (~65%), but with the advantage of avoiding the cost of
Sulfuric acid, avoiding the cost of acid neutralization with a reagent like lime, reducing the material cost of the reactor
chamber and elimination of the generation of Gypsum and attendant disposal costs. However, as discussed elsewhere in
this paper gypsum can be turned into a valuable soil treatment, or could be marketed as a raw material for gypsum
board (drywall). After the dilute acid/hot water hydrolysis in-line batch is processed, the Xylose rich liquor is drained off
and the pre-treated solids are moved into the third chamber that serves as the stage 2 hot alkaline hydrolysis reactor
chamber. The second stage alkaline reactor is run at 220C with NaOH charge of ~0.06g NaOH/g biomass, a value of
about one half of the Kraft process since there is a stage 1 hydrolysis pre-treatment that relieves the alkaline
hydrolysis of having to do all of the work of chemically fractionating the Ligno-cellulose bio-mass. With the separation of
the Xylose rich liquor, the problem of hot alkaline decomposition of Xylose is avoided and most of the lignin moves on
with the cellulose as part of the intermediate solid fraction. After the stage 2 alkaline hydrolysis reactor has processed
the in-line batch, the contents are evacuated into a solid-liquid separation unit operation with the recovery of the Lignin
rich liquor and the output of the solid cellulosic fraction. As each stage of the in-line reactor is complete, the process is
balanced to simultaneously move each chamber's in-line batch onto the next reactor chamber. (Lehrburger 2006,
Kadam 2011)
The xylose rich liquor fraction of the PureVision process can treated for use as raw material for synthetic polymers. The
lignin rich liquor can also be processed and precipitated and used for fuel or as valuable raw material for commercial
and industrial products described elsewhere in this paper. The stage 1 reactor hot dilute acid hydrolysis pretreatment
regime simplifies the purification of the Lignin as the presence of hemicellulose/Xylose complicates the purification and
recovery of the lignin. The cellulosic pulp product can be used for paper products, raw material for polymers or the
production of glucose. The PureVision process can be used as the pre-treatment front end for bio-Ethanol production. A
fractionating process like the PureVision process allows many different business models to be developed for unique
product mixes depending upon market demand and physical access to local/regional markets. PureVision reported an
economic feasibility of the PureVision process for ethanol production with corn stover feedstock superior to that of the
benchmark National Renewable Energy Laboratory (NREL) process. The biggest difference between the two process
economics is the recovery of lignin as a valuable product (~$330/MT) in its own right, The average selling price of lignin
was based upon large fractions of the lignin being diverted to low end uses as a binder in gypsum board, concrete and
feed. (Lehrburger 2006, Kadam 2011)
A study was conducted to evaluate the saccharification enzymatic digestion efficiency of cellulose using brewer's spent
grain as a Ligno-cellulose feedstock. The experimental protocol called for the three regimes: untreated spent grain,
spent grain pre-treated with dilute acid and a two-step pretreatment of dilute acid followed by dilute alkali. The dilute
acid pretreatment yielded hydrolyzed hemicellulose and a cellulose pulp with bound lignin. The two step treatment
resulted in a hydrolyzed hemicellulose liquor, a lignin liquor and cellulose pulp. The saccharification hydrolysis was
conducted with a commercial cellulase (Celluclast 1.5L) with an enzyme/substrate ratio of 45 FPU per gram of 2% (w/v)
substrate concentration at 45°C for 96 hours. The cellulose to glucose yield from the dilute acid pre-treated feedstock
was 3.5x that of the untreated feedstock. The cellulose to glucose conversion yield of the two step dilute acid/alkali pre-
treatment was 4x that of the untreated sample. Therefore, the higher the hemicellulose or lignin fraction in the
cellulose, the lower the enzymatic hydrolysis efficiency will be. The glucose conversion yield was 85.6% of the
theoretical yield. This demonstrated feasibility of using spent brewer's grain as a Ligno-cellulose feedstock for the
production of bio-Ethanol has interesting ramifications for the brewing industry. High quality Bio-Ethanol produced from
brewer's waste material could be used for bio-fuel and some of the product could be diverted, a product mix informed
by economics, into high potency spirits for human consumption. The brewing industry possesses significant knowledge
and process/equipment infrastructure in fermentation and Ethanol distillation. The generation of bio-Ethanol from the
Ligno-cellulosic fraction of grains could have a very salubrious economic effect on regional brewing and also on regional
farming. (Mussatto 2008)
A Comparison of the effectiveness of the most popular Ligno-cellulose pre-treatment regimes:

Over the last several decades thermochemical pre-treatments have emerged as the leading contenders for pre-
treatment of a wide variety of Ligno-cellulose bio-mass feedstock’s and are more likely to be used in commercial
processes because they have shorter processing times, lower reagent usage, higher yields and have lower energy costs.
Generally these pre-treatment regimes may be classified according to pH as acid, near neutral and alkaline pre-
treatments. Steam explosion methods have also become common, though they are usually seen with near neutral
water, dilute acid and SO2 pre-treatment schemes. Dilute acid, SO2, acid flow through and steam explosion (with dilute
acid, SO2) pre-treatments remove most of the hemicellulose fraction and a little of the lignin fraction of Ligno-cellulose
solids. Hot water (near neutral) pre-treatment removes the hemicellulose, but leaves the cellulose and lignin fractions
intact. Highly alkaline pretreatments remove a large fraction of lignin and some hemicelluloses, but AFEX is the
exception in that removes little of any of the 3 main constituents of Ligno-cellulose bio-mass. Of course it should be
noted that time, temperature and pressure play key roles in the action of the several thermochemical pre-treatment
methods, where the highest extremes of these three factors causing the most acceleration of pre-treatment chemical
action. (Kumar 2009a)
In another study a number of physico-chemical and instrumental procedures were used to investigate changes in
feedstock solid chemical and physical features after pretreatment on untreated (UT) corn stover and Poplar by ammonia
fiber expansion (AFEX), ammonia recycled percolation (ARP), hot water/neutral-controlled pH, dilute acid, acid flow
through, lime, and SO2 regimes. Chemical compositions measured were acetyl content (hemicellulose backbone), lignin
content, hemicellulose content and cellulose content. Other attributes measured were biomass crystallinity, degree of
cellulose polymerization, cellulase adsorption capacity of pretreated solids, cellulase adsorption capacity of
enzymatically extracted lignin, copper number, FT-IR spectra, surface morphology characterization with Scanning
Electron Microscopy (SEM) and characterization of the oxygen/carbon ratio with Electron Spectroscopy of Chemical
Analysis (ESCA). (Kumar 2009a)
A number of effects on composition of solids after pre-treatment were characterized for all of the studied popular
regimes. Lime pre-treatment removed the most acetyl groups from corn stover/Poplar feedstock’s and AFEX pre-
treatment removed the least. Low (acid) pH pre-treatments tend to depolymerize cellulose and enhance biomass
crystallinity significantly more than higher pH regimes. Pre-treatments may decrystallize cellulose and change the ratio
of crystalline to amorphous cellulose phases. FT-IR data showed changes in cross-linking density and chemical
compositional changes. It should be noted that many of these effects are dependent on pre-treatment regime and the
particular feedstock source. Some of the pre-treatment regimes were more efficient at opening pores (removal of
xylans) and ripping open cellular cavities (such as steam explosion) within the feedstock solids. (Kumar 2009a)
The Lime pre-treated corn stover feedstock and the acid flow through pre-treated poplar feedstock had the highest
cellulase adsorption capacity. Dilute acid pretreated corn stover solids and hot water pre-treated of Poplar solids had
the lowest cellulase adsorption capacity. Enzymatically liberated lignin from the AFEX pre-treatment had the lowest
cellulase adsorption of any of the other pre-treatments for both corn stover and Poplar feedstock’s. Acetyl side chains
on the hemicellulose backbone may interfere with xylanase activity and the removal of the acetyl side chains may
reduce the amount of enzyme loading needed for effective saccharification of xylan. (Kumar 2009a)
Another important study looked at the comparison of six of the most important/widely studied pre-treatment methods
for Ligno-cellulose feedstock’s and the effect of these pre-treatment methods on the adsorption affinity of cellulase
saccharification enzymes for lignin, hemicellulose and cellulose. The specific pre-treatment methods looked at were:
Ammonia Fiber Expansion (AFEX), Ammonia Recycled Percolation (ARP), controlled (neutral) pH hot water, dilute acid
(DA), Flow Through (FT), lime (Ca[OH]2) and sulfur dioxide (SO2). Solids samples were as processed and supplied by
different institutions specializing in these individual methods. As a control the adsorption and desorption of cellulase
with pure Avicel (hemicellulose/lignin free cellulose) glucan was measured at 4C, with the adsorption and desorption of
cellulase also measure with the prepared pre-treated Polar samples. The adsorption/desorption patters fit a Langmuir
isotherm model. The adsorption/desorption behavior of the cellulase enzymes were also characterized with water
dispersed/dissolved lignin as well as quantitative measurement of the inhibition of cellulase activity by lignin. (Kumar
2009b)
Acid flow through pretreated solids had the maximum cellulase adsorption capacity (St =195 mg/g solid) followed by
dilute acid (S(solids) =170.0 mg/g solid) and lime pretreated solids (St =150.8 mg/g solid), Hot water pre-treated solids
had the lowest maximum cellulase adsorption (St =56 mg/g solid). The maximum adsorption capacity St is from the
Langmuir isotherm equation: [CE] = K*St*[E]/(1+K*[E]). The value of St was characterized for each pre-treatment is
important because it has been shown that the binding capacity of the cellulase enzymes for substrates like glycan from
Avicel or corn stover bio-mass linearly relates to complex carbohydrate hydrolysis rates at 24 hours. A similar effect was
reported for willow wood and has been reported by a number of other researchers. (Kumar 2009b)
AFEX Lignin from AFEX pre-treatment had the lowest cellulase maximum adsorption capacity (St =57 mg/g lignin),
followed by lignin from dilute acid pre-treatment (St =74 mg/g lignin). Lignin from AFEX pre-treatment had the lowest
maximum adsorption capacity for b-glucosidase (St =66.6 mg/g lignin). Lignin from S02 pre-treatment had the highest
maximum cellulase adsorption (St =320 mg/g lignin) followed closely by dilute acid pre-treatment (St =301 mg/g lignin),
illustrating that lignin released from acid pretreatments are particularly good at binding and inactivating cellulases and
thereby severely degrading saccharification efficiency and handing a point to the alkaline pre-treatment regimes.
Curiously, the maximum cellulase effectiveness came from the SO2 pre-treatment followed by dilute acid pre-treatment,
which offsets the increased binding inactivation of lignin from these two pre-treatments. These findings suggest that a
dual, sequential pre-treatment process with the alkaline hydrolysis step conducted first to delignify the feedstock solids,
followed by an acid hydrolysis step would have the potential to really rev up the saccharification process efficiency and
effectiveness. It turns out that lignin from the enzymatic hydrolysis of AFEX pre-treated Poplar pulp and from dilute acid
pre-treated feedstock retarded Avicel cellulose hydrolysis less than lignin from feedstock pre-treated by ARP or acid flow
through. The acid lignin fraction from unpre-treated Poplar solids had the highest inhibition of cellulase enzymes,
confirming that pre-treatments are necessary for efficient saccharification, especially if the pre-treatment process
removes soluble lignins. Cellulase desorption results for lignin released by enzymatic hydrolysis (2 hour incubation @4C)
therefore demonstrated that cellulase binds strongly with lignin for some pretreatments, but for other pre-treatments
cellulase enzymes either partially bind enzyme liberated lignins, or reversibly bind with these lignins and are fully
released upon dilution. (Kumar 2009b)
Table 2
Leading Pretreatment Methods/Conditions, %Glucan/%Xylan Left in Solids, and pre-treated solids composition.
(Kumar 2009b)
Yield of Component Left

in Pre-treated Solids (%)
Pretreatment Pretreatment Conditions Glucan Xylan
AFEX 180C, 700 psi, 2:1 NH3 to 100% 100%
Biomass, 30 min, 23.3% H20, NW
ARP 185C, 400psi, 3.66:1 NH3:dry 93.2% 68.2%
Biomass, 27.5min, 23.49% solid, W
Dilute Acid 190C, 0.02 H2SO4:Dry wt,70 sec, 87.9% 8.3%
30% solids, steam explosion, NW
Flow Through 190C, 0.05% H2SO4,20ml/min,15min 80.2% 5.7%
Hot water 200C,10 min,5 min heat up, NW, 98.0% 42.1%
Neutral pH
Ca(OH)2 65C,0.5:1 Lime: dry Biomass,W1 98.1% 96.2%
SO2 200C, 5 min, 3% SO2, steam 96.9% 9.3%
explosion, NW
(*) Glucan as C6H10O5, Xylan as C5H8O4.

Key: W->washed; NW->non washed; W1->washed/neutralized; ND->not determined.
Table 3
Percent Glucan, Xylan, and Lignin Content of received Poplar Solid Samples
Prepared by Various Pre-treatments and then Chemically Delignified. (Kumar 2009b)
Pretreatment Glucan Xylan Lignin

AFEX 75.8+/-1.6% 23.3+/-0.14% 2.4+/-0.72%
ARP 72.9+/-4.1 20.9+/-0.9 3.0+/-0.8
Dilute Acid 91.7+/-2.4 5.9+/-0.0 3.2+/-1.3
Lime 72.6+/-2.0 23.4+/-1.0 2.2+/-0.6
hot water 88.4+/-0.6 11.5+/-0.1 1.9+/-0.4
SO2 92.7+/-0.8 9.7+/-0.0 1.5+/-0.2
Cellulase effectiveness increased with cellulase/b-glucosidase mass loading for all pretreatments except SO2.
Delignification had a very large impact on cellulase effectiveness for alkaline pre-treatments (AFEX, ARP and lime), but
had a negligible improvement for dilute acid and SO2 pretreatments. In addition, delignification significantly increased
xylose release for AFEX, ARP, lime and hot water pre-treatments. Delignification also increased glucose release for these
same pre-treatments, but to a lesser degree than with xylose release. These results suggest that lignin inhibits enzyme
hydrolysis (access) of xylan more than it does with glycan. However, lignin blocking access of cellulase enzymes to xylan
also retards the action of cellulase on glycan’s owing to the xylans physically blocking cellulase access to glycan’s. Lignin
prepared from the dilute acid pre-treatment had a negligible effect on the enzymatic hydrolysis efficiency of Avicel
cellulose. (Kumar 2009a, Kumar 2009b, Qing 2010)
Separation and Recovery of High Purity Lignin from Alkaline Digestion Liquors
The recovering of lignin from black liquor in a Kraft wood pulping process has been demonstrated with methods such as
the LignoBoost process (below) to provide a valuable species of organic substances for alternative energy and use as a
source of phenols for synthetic chemistry processes. Lignin can serve as raw material for several new products such as
Carbon fibers/materials, Phenols, adhesives/binders, dispersant and metal chelating agents. Phenols and Carbon fibers
are not now currently being produced from renewable raw materials. There are currently two methods in use to remove
lignin from black liquor. One method is the LignoBoost process that precipitates a high purity lignin and the other
method involves the concentration of lignin through ultrafiltration. The filtration properties of lignin filter cakes
produced by precipitation is strongly influenced by the hemicellulose content. It is possible to separate hemicellulose
from lignin in evaporated black liquor prior to processing with the LignoBoost process precipitation and with
ultrafiltration or a combination of ultrafiltration and nano-filtration. A unique combination of membrane filtration and
the LignoBoost concept was used in one study to largely eliminate the hemicellulose fraction from the lignin prior to
precipitation of the lignin with the LignoBoost process. The lignin cake filtration resistance was dramatically lowered
after the removal of the hemicellulose fraction in the black liquor. A hardwood lignin product of high purity could be
produced with a Sodium content of 0.2 w/w% of Total Dry Solids (TDS) and a hemicellulose content of 0.7 w/w% of TDS.
Two concentrated hardwood liquors were used in the study membrane experiments: evaporated (~30% solids) black
liquor and non-evaporated black liquor later concentrated by nano-filtration. Ultrafiltration was done with a 15000
g/mole cut-off ceramic KERASEP membrane (from Novasep, France). The ultrafiltration permeate volume recovered was
66% for evaporated black liquor and 90% for the non-evaporated black liquor. The lignin concentration in the
ultrafiltration permeate of evaporated black liquor was adequate for precipitation, but not so for the ultrafiltration
permeate from non-evaporated black liquor, which had to be concentrated through nano-filtration with an alkali stable,
polymeric NF membrane (Koch Membrane Systems SelRO MPT-36) with a cut off of 1,000g/mole. The nano-filtration
concentrated retentate held the recoverable lignin. (Wallmo 2009, Gellerstedt 2010)
Generally speaking Lignin soluability in aqueous solution (Öhman 2006) may be summarized as:
Relatively high solubility at high pH and/or relatively high ionic strength

Relatively Low solubility at low pH and low ionic strength
(pH=10, Na+ ~5%: solubility ~10%, pH=5, Na+ ~5%: solubility ~5%, pH=2, Na+ ~0.5%: solubility ~0.5%)
Low solubility when a highly alkaline pH (>10.5) has the pH taken down to ~9-10 with CO2 (e.g. Carbonic acid)
Table 4
Lignin solids recovery as a function of pH from black liquor (Lundqvist 2011)
Lignin fractions: 1. 2. 3. 4. 5.
Prec. pH: 10.6 10.0 9.1 5.0 2.0
Isolated solids, g/kg* 110 70 25 65 12
Acid soluble lignin,wt% 3.6 6.6 4.0 9.6 33.5
Klason lignin, wt% 93.6 91.2 95.6 87.7 45.8
Total lignin, wt% 97.2 97.8 99.6 97.3 79.3
Yield, %* 40 63 73 96 100
(*) Cumulative yield, % of total precipitates (282 g/kg black liquor)
Protonation of phenolic hydroxyl groups of lignin at acid to neutral pH result in hydrophobic benzene ring interactions
leading to non-covalent aggregation of dispersed/solubilized lignin super-molecules, which are stabilized by further
polar interactions. These lignin aggregates have been experimentally verified with light scattering and ultrafiltration
methods. The reversible self-assembly/aggregation behavior (poly-dispersivity) of lignins are controlled by
environmental factors such as the "solvent" nature of the solution, pH, ionic strength and temperature and excluded
volume effects from lignin/carbohydrate polymer concentration; all classic signs of a negatively charged bi-layer effect
that has been extensively studied in protein and polymer solubility contexts. This insight therefore, links lignin solubility
to well-studied techniques for solubilization and precipitation which can enhance the ability of a process to separate out
Lignin as a process toxin and turn it into a potential product. The softwood lignins show a greater propensity for
reversible aggregation of lignins than hardwood lignins. (Achyuthan 2010)
The LignoBoost process was developed within the context of recovering water soluble Lignin from the black liquor waste
product of the traditional alkaline hydrolysis, Kraft wood pulping process. Water soluble Lignin from alkaline hydrolysis
of shredded Ligno-cellulosic bio-mass is precipitated with a two-step process consisting of neutralization of the alkaline
Lignin liquor with CO2 causing a precipitate. The precipitate is converted into a dewatered filter cake with a filter press
operation. In order to prevent extremely large filter cake resistances, the filter-cake is dispersed into a slurry with wash
water acidified with H2SO4 at a pH ~1, then re-filtered and washed with wash water acidified with H2SO4 at a pH ~2-2.5.
The LignoBoost process removes Sodium and Potassium with acidified water from the final product filter cake producing
high purity Lignin. Typical uses of Lignin uses are for solid biofuels, a raw material for synthetic chemical industry
(phenols), and the production of activated carbon/carbon fibers and as a dispersant for paints/polymers/Gypsum in dry
wall. As a bio-fuel Lignin is worth about the cost of coal at $110/MT and at $350/MT-$750/MT for other uses as
described above. The filtration resistance of the lignin filter cake is lowered considerably when the hemicelluloses were
removed prior to lignin precipitation. Lignin can be produced with excellent final purity (<0.5 % by weight sodium) and
low yield losses. A conservative estimate of purified lignin energy content using the Lower Heating Value (LHV) method
calculates to an average of 26.7 MJ/kg of dry lignin, as compared to 18-22 MJ/kg for wood or bark. The CAPEX for a
LignoBoost plant is ~$17.2M +/- 20 %, with the assumption that a lignin recovery plant operates 350 days/year with a
utilization rate of 85%, producing 50,000 MT of dry lignin per year. A major operating cost item is CO2, largely from its
transportation. CO2 costs a rate of $128.88/MT, but CO2 produced from flue gasses might reduce the cost by 40 to 50%.
A flue gas CO2 recovery system CAPEX is ~$3.8M. However, a LignoBoost process installed as part of a Ligno-cellulose
bio-ethanol plant would also have a rich source of CO2 as a byproduct of the fermentation process. Other major raw
material costs in a Kraft pulping/LignoBoost process are H2SO4 at $64.44/MT and NaOH at $434.96/MT. However, a
LignoBoost like process within the context of a bio-ethanol conversion process using dilute acid pre-treatment would
considerably mitigate the cost of the H2SO4 by using the acidified water for the hot steam digestion process. (Öhman
2007a, 2007b, 2007c, 2007d, Innventia Company 2011, Tomani 2010, Lundqvist 2011, Wallmo 2009)
Extraction of acetic acid and other process generated contaminants in process streams
There are several industries that deal with unwanted acetic acid that is a waste byproduct of the chemical
manufacturing process and processes to remove waste acetic acid have been well worked out for quite some time. The
pre-treatment and saccharification of xylans release acetic acid from the removal of acetyl groups on the xylan
backbone. The release of the acetic acid detracts from the efficiency of the saccharification process and is actually a
toxin for the fermentation micro-organisms thereby reducing fermentation yields and overall process productivity. The
QVF Acetic acid recovery process is based upon a closed loop liquid-liquid extraction of process streams using Ethyl
Acetate or Methyl Tertiary Butyl Ether - MTBE - as the extractant, followed by a distillation separation of the Acetic acid
from the solvent. The QVF process can take the Acetic acid concentration contamination of process streams down as low
as 0.1%. The optimal location for an acetic acid extraction process is after the Saccharification unit operation was the
simulation process has it at a concentration of 1% by weight. It is likely that furfural/furan and Ethanol could be
extracted as well by the QVF or similar process as well. (QVF 2011)
The Cytec CYANEX 921 extractant (Tri-Octyl-Phosphine Oxide - TOPO) reagent is an interesting alternative solvent to
extract Acetic acid Furfurals and Phenols from effluent streams. The TOPO reagent (MW 396) is a water-immiscible
solvent well adapted to liquid-liquid extraction unit operations. Contaminants (as potential products) and the TOPO
reagent can be separated with a subsequent distillation unit operation. One plant operated successfully by Lenzing AG in
Austria extracts both Acetic acid and Furfural using the TOPO reagent. This plant has been in operation since 1983.
CYANEX 921 has low solubility losses in the aqueous phase, is very stable at elevated distillation temperatures and high
extraction coefficients for acetic acid compared to pure solvating reagents, such as Ethyl Acetate used in the QVF
process described above. (Cytec 2011)
The 1% (w/V%) acetic acid extraction isotherm obtained with a solvent containing 400 g/liter of CYANEX 921 extractant
in DPA is shown in the table below (Cytec 2011). The extraction temperature is 50C with a contact time of 5 minutes. It is
interesting to note that the reference simulation (below) temperature for saccharification is also 50C, suggesting the
placement of a contaminant removal step for the post acid hydrolysis stream feeding the saccharification sub-process.
The aqueous/organic phase partitioning kinetics of acetic acid establish equilibrium pretty much by 30-60 seconds. DPA
is a high boiling (330-379C) biphenyl-alkene solvent and it is available from the Conoco Chemicals Co. (a division of
Conoco).
Table 5
Equilibrium Extraction isotherm Acetic

Acid Concentration (g/liter) (Cytec 2011)
slope Organic Aqueous

O/A phase phase
0.5 8.40 5.80
1 7.50 2.50
2 4.48 1.05
5 1.92 0.40
10 0.92 0.20
Successive McCabe-Thiele interpolations (operating line goes through {0,0} with a slope =1.5) from the extraction
isotherm indicate a very high recovery after three theoretical extraction unit operations with a raffinate/feed
concentration of 10 g/liter (1%) of acetic acid, which is about the nominal concentration of acetic acid from the
hemicellulose fraction of the feedstock after the high temperature/pressure dilute unit operation pretreatment prior to
saccharification in a bio-Ethanol process. The raffinate output stream concentration was found to contain <2 mg/liter of
CYANEX 921, which is the detection limit of the analytical method used.
A buyer's guide for acetic acid extraction equipment can be found at:
http://www.chemicalonline.com/buyersguide.mvc/companylist/4619
A proposal for a Hybrid Two-Step Alkaline/Dilute Acid Pre-treatment Regime, Removal of Process
Toxins and the Production of Ancillary Products
The most widely used and economically viable pre-treatment technologies are the dilute acid and alkaline pre-
treatments. At the same time, these two pre-treatment technologies have the longest track record of academic study
and commercial application. The dilute acid pre-treatment and the alkaline pre-treatment are generally more effective
than hot water pre-treatment. It should be added that sudden decompression from high pressure (explosion) methods
significantly improve surface area and accessibility of the surface to saccharification enzymes. However, each of these
two pre-treatment technologies have their strengths and weaknesses.
The dilute acid pre-treatment generally degrades xylose, sometimes dramatically. Xylose degradation products such as
furfurals/furan act as poisons for the saccharification and fermentation processes. Dilute acid pre-treatment tends to
promote condensation reactions between water dispersible/soluble lignin fragments as well as condensation of lignin
fragments with xylans. An alkaline pre-treatment generally does not degrade xylose unless the pre-treatment conditions
become very severe, therefore they produce significantly less process toxins than dilute acid pre-treatments. Alkaline
pre-treatments have superior lignin removal and recovery compared to dilute acid pre-treatment. However, alkaline
pre-treatment does not remove all of the lignin, but leaves behind a pernicious fraction known as Acid Soluble Lignin
(ASL). Alkaline pre-treatment does a very efficient job of removing the hemicellulose fraction of the Ligno-cellulose
feedstock solids compared to dilute acid pre-treatment. Alkaline pre-treatment of Ligno-cellulose feedstock results in
pre-treated solids with significantly higher cellulase adsorption than with dilute acid pre-treatment. Overall, alkaline pre-
treatments have better monosaccharide yields in the saccharification process than dilute acid pre-treatments.
Perhaps the best known of the dilute acid pre-treatment bio-Ethanol processes and taken herein as the baseline
process is the National Renewable Energy Laboratory (NREL) process (Aden 2008, Brethauer 2010), which has a number
of strengths, including an extensive/detailed process model (by the US Lawrence Berkley and Sandia National Labs)
implemented with the Intelligen Super Pro 8 software.
Based upon the survey of pre-treatment technologies in this paper, there are some important modifications which can
be made to the NREL baseline bio-Ethanol production process. The most important of these modifications would be to
increase saccharification/fermentation efficiency through the removal of process toxins and increase in monosaccharide
yields, as well as the conversion of process wastes into sellable products instead of wastes with a disposal cost. The
changes envisioned are the use of a two-step, hybrid alkaline/dilute acid pre-treatment process and a liquid-liquid
extraction process to remove remaining process wastes/toxins after the over-liming and acidification unit operation. The
hybrid pre-treatment process would involve a lignin recovery process.
The hybrid pre-treatment process with the liquid-liquid toxin extraction is projected to allow for significantly lower
enzyme loadings during saccharification, which could adduce large savings in the most expensive process consumable,
setting aside the cost of the feedstock. The removing of process toxins (acetic acid, furfurals, furans and lignin) to would
cause significant optimization of saccharification efficiency/yields of fermentable monosaccharide’s and an increase in
fermentation process efficiencies through greatly increased fermentation microbe growth rates. The Liquid-Liquid
extraction process can also produce sellable products. It is envisioned that the Liquid-Liquid Extraction placed would be
placed after the over-liming/neutralization processes, which is probably still valuable in the elimination of Acid Soluble
Lignin (ASL), which is a content that makes the Gypsum waste more valuable as a soil treatment or raw material for
Gypsum board/drywall. A large amount of process water can be saved through the elimination of the large scale wash
water evaporation step (to remove acetic acid) that is part of the clarification unit operation that recycles the after
feedstock wash, since the addition of the liquid-liquid toxin extraction would eliminate the need to remove acetic acid
from the wash water by massive evaporation.
Possible sellable products would be: acetic acid, lignin for bioenergy, lignin as a synthetic chemistry raw material, lignin
as a dispersing agent for concrete/paints/Gypsum board, xylitol as a synthetic chemistry raw material, Gypsum as raw
material for Gypsum board, Gypsum as a soil treatment - especially as enriched by other current process wastes such as
lignin/boiler ash/clarifier polymer, CO2 from fermentation, CO2 from fermentation and flue gasses could be used to
generate liquid CO2 for industrial purposes, CO2 could be used for soda water and carbonated beverages. A central CO2
recovery and purification system is envisioned, which can be recovered by bubbling reclaimed/generated gasses through
a Mono-Ethanol Amine (MOA) solution in a closed loop system possessing a heating and partial vacuum to devolve the
purified CO2.
It is proposed to put the alkaline pre-treatment step first to remove the bulk of the lignin and hemicellulose fraction of
the feedstock from the cellulosic solids which are sent to the NREL baseline dilute acid pre-treatment unit operation
with the retention of the steam explosion unit operation after the dilute acid pre-treatment unit operation. The
proposed alkaline agent would be NaCO3 at around a 2% concentration (fresh @pH 11.5-12 spent @pH 10.7-11) as for
2-4 hours at 140C. The post alkaline pre-treatment unit operation would have a solid-liquid separation unit operation
where the liquid would become the raffinate for a modified Ligno-boost process. The first lignin precipitation and acid
neutralization (to pH ~8) would be done with bubbled house CO2 (from the central CO to reclamation system) in a
stirred tank with a post belt filtration unit operation. The filtrate would then be acidified with dilute H2SO4 (to a pH ~2)
in a stirred tank precipitate the remaining non-ASL lignin and devolve all remaining Carbonates to the central CO2
reclamation system and then run through another belt filter unit operation. The recovered lignin precipitates would be
pooled and re-suspended with dilute H2SO4 for a final wash/filter/dry unit operation to produce high purity lignin as a
sellable product. The filtrate from this last step could be re-used within the dilute acid pre-treatment unit operation.
The raffinate with the hemicellulose fraction post lignin removal would be routed to the to over-liming/acid
neutralization unit operation along with the dilute acid pre-treatment liquids to precipitate and remove ASL lignin as
part of the Gypsum waste product. The post over-liming liquids would be heated and routed to the saccharification tank
with the cellulosic solids as in the baseline NREL process. The ASL removed with the Gypsum would serve as a dispersant
fraction for drywall Gypsum or as lignin/soil content for Gypsum waste as a soil treatment. The production of soil
treatment has a significant side benefit with regional distribution in that the productivity of the farming soil would be
replenished and strengthened helping to assure high productivity for food crops and Ligno-cellulose feedstock for the
bio-Ethanol production plant.
The simulation below will explore the impact of some of these considerations on the Super Pro 8 simulation of
operations and economics of a corn stover bio-Ethanol production plant.
Simulation Results and Discussion:

Prior to the simulation runs reported here, many trial runs were undertaken to get a good feel for how the Bio-Ethanol
plant simulation was constructed and how the various processes interacted with each other, as well as conducting
sensitivity analysis to find out where the simulation was vulnerable to non-convergence. Generally speaking, the plant
processes and equipment sizing are very tightly coupled and are generally optimized for the basic process parameters in
the base case simulation. For example, increasing the Corn Stover feed rate by modest amounts either cause the
simulation to not converge or result in simulation errors where the simulation either cannot allocate adequate resources
to a particular unit operation or cannot up size equipment to give enough capacity for a particular unit operation. For
example, any attempt to shorten the 140 hour fermentation time through “simulated” process efficiency improvements
results in a simulation error on the first evaporator (E-501) after the Beer distillation column. At larger material transport
rates, the water treatment loop will run short of water and the simulation will have an error at the belt filter located
after the evaporator E-501. This latter error can be corrected by bringing in more feed water through the input stream
'Water-in' by taking the water input rate from 65 Kg/second to 200 Kg/second.
There are 9 basic simulation runs documented in table 1 below including a simulation run with the base case as
constructed by the authors. One item that becomes clear from the base case study is the very large CAPEX (~$372M)
necessary to build and equip the Bio-Ethanol facility. The very large capitalization costs have a profound effect upon the
economic viability of a real plant, whose output selling price is still severely constrained by the current market prices for
petroleum derived fuels, although this scenario is changing at the time of this writing. The structure of the Bio-Ethanol
generation process and the balancing of the equipment structure in the base case simulation make it unlikely that
adequate productivity gains can be made with the basic equipment to materially affect the plant profitability and the
overall CAPEX payback time of the Bio-Ethanol plant. Therefore, ways to improve the actual process chemistries,
enzymes and yeast must be sought in order to make significant contributions to plant productivity and profitability.
It is appropriate to observe at this point that there are weaknesses/artifacts which may occur from current accounting
practices, which can obscure the truth regarding the economic benefits of particular process changes. More specifically
stated, decisions based upon current cost accounting methods seek to optimize each individual process section/unit
operation, but this practice strictly taken is both naïve and counterproductive when dealing with tightly coupled,
interacting sub-systems and unit operations. Decisions should be based upon optimizing the entire system and not just
the subsystems, as often it can happen that sub-systems can be run under sub-optimal conditions to actually contribute
significantly to optimizing the entire process. Additionally, contemporary cost accounting methods would fully burden
any secondary revenue stream generated from process wastes or process byproducts with the capitalization/labor
overhead of the entire plant; a practice which would make these potential ancillary revenue streams look economically
unattractive, when in fact they are really very beneficial. For example, the base case process design treats 3 output
streams as waste streams with a disposal cost associated with these streams at $27/MT. A little analysis shows that the
Gypsum byproduct from the Over-Liming process could be combined with the other two waste streams - boiler
combustion ash and Stover wash water sediments with clarification polymer - with little cost to produce a desirable soil
treatment that could sell for ~$60/MT.
The base case simulation and simulation runs #1 through #3 look at the effect of the raw feedstock consumption rate.
The yearly gross margin increases from 10.13% for the base case at 83.3 MT/hour to 10.94% at an 86.6 MT/hour feed
rate, which only makes a modest contribution to the plant economics as measured by the CAPEX pay back years. Feed
rates above 88.6 MT/hour actually begin to have significantly negative effects on plant economics, in addition to putting
the simulation in danger of not converging. The latter effect can be seen with the increasing number of iterations to
convergence as the feedstock input rate increases.
In simulation run #4, the possible effect of increasing Ethanol yields by modifying the yeast metabolism though genetic
engineering was tested by changing the fermentation bio-reaction stoichiometry. More specifically, the fermentation
efficiency was upped by changing the microbe ”metabolism” through changing the Glucose/Xylose stoichiometry from
.43 to .47 and taking the Glycerol stoichiometry from 0.06 to 0.02 moles. With nothing else changed, the virtual
modification of the fermentation yeast metabolism resulted in process efficiency gains leading to a gross margin of
14.02%, but this only resulted in a 8-9 month decrease in the CAPEX payback years. Bio-engineering the microbe would
lead to better plant economics, but probably not enough to really make a big difference in the total scheme of things.
Simulation run #5 sought to evaluate the results of improving Saccharification efficiency by doing things to improve
dilute acid hydrolysis pre-treatment effectiveness by removing Lignin to speed up Saccharification. By following
suggestions made in the literature it can be justified to move the Glucose yield from 69% to 80% and Xylose yield from
36% to 65%. The simulation economic calculations show a gross margin decrease from run #4 to a value of 12.99%, but
the way the cash flows work out the CAPEX payback years remains the same at 7.68 years. While significantly improving
the Saccharification efficiency is possible, it only has a modest impact on overall plant economics.
Simulation run #6 sought to reflect co-optimization of both the Saccharification and Fermentation processes, which
resulted in a dramatic effect on over-all plant economics by more than doubling the gross margin (to 25.69%) and taking
the CAPEX pay back years from 8.39 in the base case to 5.79 years. The feedstock input rate in run #6 went from the
83.3 MT/hour in the base case to 88 MT/hour in run #6 to take a bit more advantage of the overall increase in process
efficiency. The increase in fermentation efficiency comes from projecting a 50% increase in the fermentation yeast
growth rate through the removal of Lignin, acetate and furans. The simulation results clearly suggest that upping the
growth rate of the fermentation yeast by changing its chemical environment is more likely to make a significant
difference in process economics than changing fundamental things about the yeast metabolism. Of course one has to
yield the point that some new organism may be found that is a game changer, but with the current state of affairs it
seems to be better to find ways to improve the current performance of organisms currently extant in fermentation
processes.
Simulation run #7 again tests the results of upping the Corn Stover feedstock input rate on plant economics with the
utilizing the “process improvements” implemented for run #6. It can be seen that the gross margin actually declines by
almost a percentage point illustrating the lessons of runs #1 through #3. So the inescapable conclusion here is that the
base case plant design is well balanced amongst the component sub-processes and their unit operations, so that raw
increases in the raw material in feed either has little effect on improving overall productivity or can actually have a
detrimental effect on overall plant economics.
In simulation run #8, the effect of converting process waste into a revenue stream is evaluated. The net effect is that
waste costs experience a yearly reduction of $2,538,308.00 and extra revenue is brought in from waste
conversion/recycling at a yearly sales of $4,319,000.00. The overall yearly impact is a net $6.86M positive impact on
cash flows and plant profitability, as can be seen with the gross margin rising from 10.13% in the base case to 14.27%
when waste is converted to cash. It should also be noted that the magnitude of the economic impact in simulation #8
outshines all other process improvements except for the joint (and interacting) improvement to the Saccharification and
fermentation processes. As suggested above, the temptation to allocate a full overhead burden to this new revenue
stream should be avoided as its true benefits would be masked. Rather it is more appropriate to allocate the full burden
to the primary revenue stream, which is the Bio-Ethanol product.
Table 6
Gross
Stover EtOH Margin IRR
Run Feedstock product CAPEX Net Operating Total Income (% per Payback after
Number Description (MT/hr) (Kg/hr) (US $$) Cost (US$4/yr) (US $$/yr) yr) (Years) taxes
0 Base case 83.3333 11,438.56 $371,764,000.00 $135,552,407.00 $150,834,000.00 10.13% 8.39 11.48%
As supplied by NREL
Up feedstock
1 rate 90 11,049.90 $388,379,000.00 $142,040,472.00 $156,092,000.00 9.00% 8.6 10.55%
Up the feed stock feed rate from base case. ~8 iterations to converge.
Up feedstock
2 rate 86.6 11,733.41 $378,311,000.00 $138,376,841.00 $155,376,000.00 10.94% 8.23 12.27%
Up feedstock
3 rate 88 11,714.16 $377,427,000.00 $144,680,000.00 $154,972,000.00 10.36% 8.33 11.80%
4 Better microbes 83.3333 12,063.10 $375,195,000.00 $136,393,457.00 $158,643,000.00 14.02% 7.68 15.08%
Up fermentation efficiency by bioengineering the microbe for Glucose/Xylose from .43 to .47 moles at the expense of
Glycerol going from 0.06 to 0.02 moles.
Improve
5 Saccharification 83.3333 11,716.56 $381,519,000.00 $142,007,017.00 $163,201,000.00 12.99% 7.68 14.30%
Improve Saccharification efficiency with pre-treatment improvement of removing Lignin to speed up Saccharification.
Glucose yield goes from 69% to 80% and Xylose from 36% to 65%.
Improve pre-
treatment &
6 Fermentation 88 13,803.32 $370,394,000.00 $139,619,645.00 $187,892,000.00 25.69% 5.79 24.92%
Improve Fermentation/Saccharification efficiency with pre-treatment improvement of removing Lignin, acetic acid,
furfurals/furans to speed up Glucose yield goes from 69% to 80% and Xylose from 36% to 65% and fermentation
microbe efficiency (growth) climbs from alpha=1 to alpha=1.5.growth rate of microbes.
Improve pre-
treatment &
7 Fermentation 92 14,018.51 $376,512,000.00 $142,844,134.00 $191,079,000.00 25.24% 5.84 24.61%
Improve Fermentation/Saccharification efficiency with pre-treatment improvement of removing Lignin, acetic acid,
furfurals/furans to speed up Glucose yield goes from 69% to 80% and Xylose from 36% to 65% and fermentation
microbe efficiency (growth) climbs from alpha=1 to alpha=1.5.growth rate of microbes.
Waste to
8 revenue 83.3333 11,438.56 $371,549,000.00 $133,014,099.00 $155,150,000.00 14.27% 7.67 15.08%
The CaSO4 (Gypsum) and boiler ash and trash streams are mixed as a soil treatment for $60/MT. To capture these
waste streams in the simulation the following simulation streams were set to revenue streams: 'To landfill', 'Trash' and
'S-288'. Waste Cost reduction/year: $2,538,308.00. Extra revenue from waste/year: $4,319,000.00.
Conclusions:
Good simulations are invaluable aids in assessing chemical plant and process designs and tradeoffs with their resulting
economics. It is clear that economic considerations are as important as technological considerations when evaluating
new and developing technologies. The cost of developing quality simulations are cheap insurance when projecting the
performance of production facilities having a CAPEX of more than $300M. The best efficiencies and productivity
improvements to be made within the dilute acid hydrolysis/Saccharification Bio-ethanol process are had by co-
optimizing the Saccharification and Fermentations by improving the available fermentable sugars and improving the
growth time of the fermenting yeast by removing pre-treatment toxins that allow more time to produce product within
the base case fermentation cycle time. With higher Saccharification and yeast growth efficiencies the corn Stover
feedstock can go up to 92MT/hour and still achieve serious economic performance increases over the base case.
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Ligno-Cellulose Pre-treatment Technologies in Bio-Ethanol Production with a

Research · November 2015

Pattern Recognition View project

Long range protein-protein attraction View project

The user has requested enhancement of the downloaded file.

5) Lignin physically adsorbs saccharification enzymes and thereby inactivating them

pretreatment monosaccharide yield in Percent of theoretical

at 140°C 0.17 to 0.58 g/g (23–83%)

Acid Pre-treatment Processes

Alkaline Pre-treatment Processes

Ammonium Hydroxide Pretreatment

Inorganic salt pre-treatment processes

Non-Ionic Surfactant Effect on Dilute Acid/Hot Water Pre-treatment and Saccharification

Two step hybrid acid and alkali pre-treatments

A Comparison of the effectiveness of the most popular Ligno-cellulose pre-treatment regimes:

Yield of Component Left

(*) Glucan as C6H10O5, Xylan as C5H8O4.

Pretreatment Glucan Xylan Lignin

Relatively high solubility at high pH and/or relatively high ionic strength

Lignin solids recovery as a function of pH from black liquor (Lundqvist 2011)

(*) Cumulative yield, % of total precipitates (282 g/kg black liquor)

Equilibrium Extraction isotherm Acetic

slope Organic Aqueous

Simulation Results and Discussion:

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