Bioconversion of Woody 

Biomass to Biofuel and Lignin 

Co-Product Using Sulfite 

Pretreatment to Overcome the 

Recalcitrance of Lignocelluloses 

(SPORL)

J.Y. Zhu

Chao Zhang

Rolland Gleisner

Carl J. Houtman

Xuejun Pan

General Technical Report

FPL–GTR–240

United States Department of Agriculture

Forest 

Service

Forest Products

Laboratory

September

2016

Distillation

Separation

Purication

Ethanol Lignosulfonate

Pretreatment

Steam, SO2

, hydroxide

Neutralization

Whole slurry

Lignin residue

Waste water

No detoxication

No washing

Size reduction

Blow tank to 

wet scrubber

and

fermentation

Enzymatic

saccharication

Pipe to wet 

scrubber

Forest residue roadside pile Grinding and loading
Abstract

Sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) promises to provide efficient bioconversion of woody biomass into bioethanol and lignin 

co-products. Results from several laboratory and pilot-scale 

studies are presented to demonstrate SPORL performance, 

with comparisons to competing technologies. Excellent 

ethanol yields of up to approximately 80% theoretical, 

based on glucan, mannan, and xylan content of wood, were 

achieved at titers over 40 g/L. This high productivity was 

accomplished using low cellulase loadings, 26 mL/kg wood, 

and without detoxification, solid–liquor separation, or 

supplementation of nutrients in fermentation. Lignin sulfonation from SPORL likely contributes to this high efficiency 

by reducing nonproductive binding of cellulase through 

electrostatic repulsion and reduced hydrophobic interactions 

between cellulase and lignin. Finally, a reaction-kineticsbased severity factor—the combined hydrolysis factor 

(CHF)—was developed to facilitate process scale-up and 

for determining optimum operating conditions for balancing 

sugar yield against sugar degradation.

Keywords: Sulfite (SPORL) pretreatment, woody biomass, 

biofuel/bioethanol, enzymatic saccharification/hydrolysis, 

nonproductive cellulase binding, fermentation, reaction 

kinetics/severity, process scale-up, lignosulfonate, lignin 

co-products

September 2016

Zhu, J.Y.; Zhang, Chao; Gleisner, Rolland; Houtman, Carl J.; Pan, Xuejun. 

2016. Bioconversion of woody biomass to biofuel and lignin co-product 

using sulfite pretreatment to overcome the recalcitrance of lignocelluloses 

(SPORL). General Technical Report FPL-GTR-240. Madison, WI: U.S. 

Department of Agriculture, Forest Service, Forest Products Laboratory. 

28 p.

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Contents

1. Introduction .......................................................................................................................................... 1

2. The SPORL Process ............................................................................................................................. 1

2.1 Laboratory Practice of SPORL...................................................................................................... 2

2.2 SPORL Operating Conditions....................................................................................................... 2

3. Reaction Kinetics for Process Optimization and Scale-up................................................................... 3

3.1 Pretreatment Severity—Combined Hydrolysis Factor (CHF) ...................................................... 3

3.2 Using CHF to Predict Hemicellulose Dissolution......................................................................... 4

3.3 Using CHF to Predict Sugar Degradation ..................................................................................... 5

4. Process Optimization and Scale-up Design Using CHF ...................................................................... 5

4.1 Dissolution of Hemicelluloses and Its Relation to Sugar Yield .................................................... 5

4.2 Balance Sugar Yield with Degradation Using Low-Temperature Pretreatment............................ 7

4.3 Using pH-profiling in SPORL to Further Reduce Carbohydrate Degradation ............................. 9

4.4 SPORL Optimization for Hardwoods and Herbaceous Biomass Using CHF............................... 9

4.5 SPORL Process Scale-up Design ................................................................................................ 11

5. Lignin Sulfonation on Nonproductive Cellulase Binding.................................................................. 12

5.1 Lignosulfonate to Enhance Enzymatic Saccharification............................................................. 12

5.2 pH-Induced Electrostatic Repulsion between Sulfonated Lignin and Cellulase......................... 12

6. SPORL Process Performance and Integration for Biofuel and Lignin Co-Product Production......... 15

6.1 Comparison of SPORL with Dilute Acid Pretreatment—Enzymatic Saccharification and 

 Fermentation of Washed Water-Insoluble Solids........................................................................ 15

6.2 Bioethanol Production from Lodgepole Pine—Process Scale-up Demonstration and 

 Comparison with SO2 Steam Explosion...................................................................................... 16

6.3 Bioethanol Production from a Softwood Forest Residue—Scale-up Evaluation........................ 17

6.4 Bioethanol Production from Poplar—Scale-up Evaluation ........................................................ 21

6.5 Properties of Lignosulfonate from SPORL................................................................................. 22

6.6 Production of Lipid from a Softwood Forest Residue ................................................................ 23

6.7 Production of Iso-Butanol and Bio-Jet from a Softwood Forest Residue................................... 24

7. Summary............................................................................................................................................. 24

Acknowledgments................................................................................................................................... 24

Literature Cited ....................................................................................................................................... 25

Bioconversion of Woody Biomass to 

Biofuel and Lignin Co-Product Using 

Sulfite Pretreatment to Overcome the 

Recalcitrance of Lignocelluloses (SPORL)

J.Y. Zhu, Research General Engineer, Forest Products Laboratory, Madison, Wisconsin

Chao Zhang, Visiting Scientist, School of Thermal and Power Engineering, Beihang University, Beijing, China

Rolland Gleisner, Physical Science Technician, Forest Products Laboratory, Madison, Wisconsin

Carl J. Houtman, Chemical Engineer, Forest Products Laboratory, Madison, Wisconsin

Xuejun Pan, Professor, Department of Biological Systems Engineering, University of Wisconsin–Madison, 

Madison, Wisconsin

1. Introduction

In the past two decades, concerns over climate change, energy security, and sustainable economic development renewed 

our nation’s interest in using renewable natural resources for 

producing green energy and biochemical and bioproducts 

using lignocellulosic biomass through the biorefinery concept. The United States has rich forestland with excellent 

productivity. Efficient utilization of forest biomass is critical 

to healthy forest management to protect watersheds and preserve ecosystems. Because of its availability and potential 

of being sustainably produced (Perlack and Stokes 2011) in 

large quantities in many regions of our country, woody biomass is an important biorefinery feedstock. Woody biomass 

also has the advantage over herbaceous biomass because of 

its relatively high density for easing transportation and flexible harvesting schedule for eliminating long-term storage 

(Zhu and Pan 2010).

Efficient and scalable pretreatment and fractionation of a 

variety of lignocellulosic feedstock are key to commercial 

success of bioconversion of lignocellulosic biomass to biofuels, biochemicals, and bioproducts. Unfortunately, when 

dealing with woody biomass such as harvest forest residues, 

especially those from softwood species, few pretreatments 

can effectively overcome its strong recalcitrance to microbial deconstruction for efficient enzymatic saccharification 

and subsequent bioconversion (Zhu and Pan 2010). Sulfite 

pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) (Zhu and others 2009, 2015) has demonstrated 

good performance for efficient bioconversion of woody 

biomass at a pilot scale (Zhu and others 2015; Zhou and 

others 2015a, b). The advantage of SPORL lies in several 

facts: (1) It is commercially scalable because it was developed based on sulfite pulping. (2) The dissolved lignin is a 

lignin co-product (that is, lignosulfonate) and can be directly 

marketed without further processing. (3) Lignin sulfonation 

reduces nonproductive binding of cellulase to lignin, and 

lignosulfonate acts as a surfactant to enhance enzymatic 

saccharification (Wang and others 2013a,b; Zhou and others 

2013a); therefore, washing of pretreated solids is not only 

unnecessary but also not preferred, which can simplify 

process integration and significantly reduce water usage. 

(4) Low sugar degradation into fermentation inhibitors allows direct fermentation of the pretreated whole slurry without detoxification and without compromising sugar yield 

even at lower temperatures (Zhou and others 2014; Gu and 

others 2016). In view of these advantages, the SPORL process may provide a pathway for bioconversion of low-grade 

woody biomass such as short-rotation poplars and forest 

harvesting residues.

2. The SPORL Process

The SPORL process was developed based on sulfite pulping with the understanding that the goal of pretreatment 

is to produce a readily enzymatically digestible solid substrate, different from the goal of chemical pulping, which 

is to achieve maximal delignification while protecting the 

strength of cellulosic fibers. Therefore, the SPORL process 

conditions purposely deviate from those of wood pulping, 

particularly at elevated temperatures and acidic conditions, 

as shown on the T–pH diagram in Figure 1. This is to increase carbohydrate degradation (including dissolution of 

hemicelluloses) because hemicellulose removal is critical to 

increasing cellulose accessibility to cellulase and subsequent 

depolymerization of cellulose (Leu and Zhu 2013). SPORL 

has four characteristics: (1) it dissolves a substantial amount 

of hemicelluloses (Zhu and others 2012; Leu and others 

2013; Zhou and others 2013b; Zhang and others 2015); 

(2) it depolymerizes cellulose; (3) it partially delignifies 

biomass through lignin sulfonation and therefore the dissolved lignin is highly sulfonated (Zhou and others 2013b, 

2014); and (4) the remaining solid lignin is also sulfonated 

(Lou and others 2013). The solid substrate from SPORL 

is not suitable to be used as wood fibers because of its low 

strength due to substantial degradation of polysaccharides 

and insufficient delignification, but it is readily digestible by 

enzymes at low dosages for sugar production (Leu and others 2013; Zhu and others 2011a).
General Technical Report FPL–GTR–240

2

2.1 Laboratory Practice of SPORL

Laboratory study of SPORL can be conducted in wood 

pulping digesters (Fig. 2) using bisulfite such as NaHSO3

and an acid such as H2SO4 to adjust pH to approximately 

2 to prepare pretreatment liquor. When wood chips are 

used instead of fiberized materials, the pretreated materials require a size reduction step that can be accomplished 

by blowing the pretreated materials under pressure, as in 

steam explosion, or disk milling (Fig. 2), or a combination 

of the two. Separation of pretreatment spent liquor from 

pretreated solids is not necessary as SPORL produces a very 

small amount of inhibitors; furthermore, the dissolved lignin–lignosulfonate can enhance enzymatic saccharification 

(Wang and others 2013a,b; Zhou and others 2013a). The 

resultant SPORL whole slurry from size reduction can be 

directly fed to enzymatic hydrolysis and fermentation after 

neutralization without washing (Scheme I in Fig. 2). However, the washed water-insoluble solids (WIS) are often used 

to assess the effectiveness of pretreatment as measured by 

solid substrate enzymatic digestibility (SED) (Scheme II in 

Fig. 2). Overall, SPORL has several unique advantages over 

most competing processes, such as dilute acid (DA), alkaline, aqueous ammonia, ammonia fiber expansion (AFEX), 

and SO2 steam explosion. Specifically, SPORL (1) is very 

effective on softwoods; (2) produces fewer inhibitors than 

DA and acid-catalyzed steam explosion and therefore detoxification is not necessary for high-titer biofuel production 

(Zhou and others 2014); (3) generates lignin sulfonation, 

which reduces nonproductive binding of cellulase to lignin 

through pH mediation (Lou and others 2013); (4) produces 

dissolved lignin–lignosulfonate, which is a surfactant that 

can be used to enhance enzymatic saccharification (Wang 

and others 2013a) and which can also be directly marketed 

as a co-product; and (5) does not require separation of pretreated solids from spent liquor, because of the above-mentioned effects of lignosulfonate and because using unwashed 

solids is preferred, which can substantially simplify process 

integration.

2.2 SPORL Operating Conditions

SPORL should be practiced, as in sulfite wood pulping 

(Bryce 1980), where sulfur dioxide (SO2 ) is adsorbed into 

a hydroxide solution (Zhu and others 2015) to prepare the 

pretreatment solution. For softwoods, typical (minimal) 

chemical loadings of SO2 and a base chemical, such as CaO, 

on wood are at approximately 6.5 and 1.8 wt%, respectively. This results in a pretreatment liquor with an initial 

pH slightly below 2.0 (measured at ambient temperature), 

Figure 1—SPORL (sulfite pretreatment to overcome the 

recalcitrance of lignocelluloses) operating conditions 

compared with sulfite pulping in the temperature–pH 

diagram (from Zhu and others (2015)).

Figure 2—Schematic flow diagram of wood chip pretreatment using SPORL along with two 

schemes of enzymatic saccharification and fermentation at laboratory bench scale: (I) using 

washed water-insoluble solids (WIS) only; (II) using the pretreated whole slurry without solids 

and liquor separation or solids washing.

180

170

160 Pulping

150

140

130

120

0 2 4 6 8

T (°C)

pH

SPORL
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

3

which gives excellent pretreatment (Zhu and others 2015). 

This level of SO2 loading is only approximately 30% of the 

amount used in wood pulping. With SPORL, the typical 

liquor to wood ratio (v/w) is between 3 and 4 and pretreatment temperature can be varied between 140 and 180 °C 

with corresponding pretreatment times of 30–240 min using this minimal SO2 loading. The details for determining 

pretreatment duration using a severity factor at a given temperature are presented Section 4.5. Increased SO2 loading 

can significantly reduce pretreatment duration, as found in 

our recent study (Gu and others 2016).

For easing liquor preparation in laboratory studies, sodium 

(or magnesium, calcium, ammonia, for example) bisulfite 

HSO3

–1 (Zhu and others 2009; Wang and others 2009, 2015) 

or sulfite SO3

–2 (Shuai and others 2010), or a combination of the two, can be used with the addition of an acid to 

adjust pH to approximately 2.0, as discussed in Section 6. 

The term sulfite and bisulfite are used interchangeably in 

SPORL because the amount of the active reagents bisulfite (HSO3

–1), sulfite (SO3–2), and free sulfur dioxide (SO2) 

present in the pretreatment liquor depends on the pH of the 

liquor (Ingruber 1985). Most reported SPORL studies used 

sodium bisulfite (NaHSO3) with sulfuric acid (H2SO4) at 

loadings on wood between 3 and 12 wt% and 1.1 and 2.2 

wt%, respectively, depending on the feedstock (Zhu and 

others 2009, 2012; Leu and others 2013; Zhou and others 

2013b; Zhang and others 2014, 2015; Cheng and others 

2015). The ranges of typical pretreatment conditions are 

listed in Table 1.

3. Reaction Kinetics for Process 

Optimization and Scale-up

Rather than using conventional statistical factorial design 

for SPORL process optimization, a kinetic-based reaction 

model and parameters were developed for SPORL process 

optimization and scale-up design. This is because (1) statistical factorial design assumes only one set of optimal condition in a range studied (that is, monotonic process behavior), 

which is not always true, especially for a process like 

SPORL that involves several process variables and (2) maximal sugar yield does not necessarily translate to maximal 

biofuel yield due to process inhibition in downstream processing such as fermentation. Therefore, process optimization allowing balancing sugar yield with inhibitor formation 

is desirable. This section describes user-friendly reaction 

kinetics models for process optimization and design.

3.1 Pretreatment Severity—Combined 

Hydrolysis Factor (CHF)

The chemical reactions taking place during pretreatment of 

lignocelluloses are very complex, involving hydrolysis of 

polysaccharides, degradation of depolymerized carbohydrates, delignification, and lignin condensation. Developing 

predictive reaction models can help process control and 

optimization in commercial biomass conversion. For wood 

pulping, removing lignin and preserving carbohydrates 

are critically important. Prior reaction kinetic studies were 

therefore primarily focused on delignification (Lourenço 

and others 2011; Santos and others 1997; Miranda and 

Pereira 2002). For bioconversion of lignocelluloses, delignification is relatively less important than dissolution of 

hemicelluloses (Leu and Zhu 2013). Therefore, modeling 

hemicellulose dissolution is the key to predicting substrate 

enzymatic digestibility SED. Effective models have been 

developed to predict acid-catalyzed hydrolysis of xylan 

(Springer 1966; Saeman 1945; Morinelly and others 2009; 

Lu and Mosier 2008; Jacobsen and Wyman 2000; Zhao and 

others 2012). Although these models are useful, it is desirable to develop simple reaction-kinetic-based factors, such 

as reaction severity factor, that combine all reaction conditions into one single parameter, for process scale-up design 

and bioconversion predictions. The H-factor, an integration 

of reaction temperature over time or thermal energy input, 

has been widely used for scaling wood pulping in the pulp 

and paper industry (Brasch and Free 1965; Vroom 1957). 

The combined severity factor (CSF) (Abatzoglou and others 

1992; Chum and others 1990; Larson and others 1999) was 

developed based on H-factor after modification to include 

the effect of a catalyst (Eq. (1)) (that is, acid) on lignocellulose reaction:

p

14.75

100 CSF log exp pH log exp ref  −



 



 



 



 − − = ⋅ 



 



 



 



 − = ⋅ T

t

T T

t

ω

(1)

Predictions of hemicellulose dissolution using CSF were 

good at low severities (that is, hemicellulose dissolution 

below approximately 70%); however, predictions were poor 

at hemicellulose dissolution above 70% under higher severities (Abatzoglou and others 1992) that are of most interest 

for bioconversion of lignocelluloses. The poor prediction 

pH 14.75

100 CSF log exp pH log exp ref  −



 



 



 



 − − = ⋅ 



 



 



 



 − = ⋅ T

t

T T

t

ω

Table 1—Typical pretreatment conditions of SPORL 

for different feedstock

Reaction conditions Softwoods

Hardwoods or

herbaceous

 biomass

Temperature (°C) 140–180 130–170

Time (min) 240–30 240–30

Liquor to wood (L/kg) 2–5 2–6

Catalyst choice I (wt% wood)

A: H2SO4 ~2.0 ~1.0

B: Bisulfite (e.g., NaHSO3) 8–12 3–5

Catalyst choice II (wt% wood)

A: SO2 6.5–20 3.0–10

B: Hydroxide (e.g. CaO) 1.0–2.0 0.5–1.0
General Technical Report FPL–GTR–240

4

at high severities is primarily due to the inhomogeneity of 

hemicelluloses. Biphasic models are often used to improve 

the prediction of hemicellulose dissolution and xylose yield 

(Zhao and others 2012; Zhang and others 2012a).

SPORL has two catalysts, SO2 and a base, and therefore a 

CSF that simply uses pH to account for the effect of acid is 

not applicable. Here we will derive a combined hydrolysis 

factor (CHF) that can accurately predict hemicellulose dissolution by pretreatments using more than one catalyst. The 

hydrolysis of lignocelluloses can be characterized by a phenomenological second-order rate expression, first order in 

catalyst and first order in xylan:

(2)

where XR is the fraction of xylan retained in the solids or 

residual xylan, T is temperature, C is catalyst concentration, 

and k is rate constant as developed by several researchers 

(Abatzoglou and others 1992; Springer and others 1963). 

The following expression was found to fit acid hydrolysis of 

aspen well (Springer and others 1963):

(3)

where Kx is the pseudo first-order rate constant for dissolution of xylan, T is temperature in kelvins, and CH is acid 

molar concentration. Following Eq. (3), we can express the 

rate constant k for a reaction involving two catalysts such as 

SPORL as

(4)

where CA and CB are the initial molar concentrations of the 

two catalysts used in pretreatment, respectively, for example 

acid and bisulfite for SPORL studies conducted in laboratory; α, β, and γ are adjustable parameters; E is the apparent 

activation energy; and R is the universal gas constant of 

8.314J/mole/K. Based on Equation (2), the CHF that combines temperature, chemical concentrations, and pretreatment duration into a single measure as a reaction severity 

can be expressed as (Zhu and others 2012)

(5)

CHF is very similar to CSF (Eq. (1)), but it is more precise 

in defining the effects of temperature, with the introduction 

of activation energy E, reaction chemical loadings, and concentration of each catalyst. CHF was compared with CSF 

for a set of DA pretreatments and two sets of SPORL pretreatments at low acid and pH ranges. As shown in Figure 3, 

excellent linear correlations were obtained.

( ) R

R , ,... dt

k T C CX dX = −

H

H

x 10 0.22219 6171.3 Log 15.083 C

C T

K = − +

( ) E RT CA CB k e α− +β +γ =

( ) ( ) k C C t (C C )t

E RT C C

e A B

A B CHF A B = + + − + +

= α β γ

3.2 Using CHF to Predict Hemicellulose 

Dissolution

To obtain accurate prediction of hemicellulose dissolution 

using CHF, two fractions of hemicelluloses with different 

reaction rates can be assumed to account for the inhomogeneity of hemicelluloses (Kobayashi and Sakai 1956; Maloney and others 1985; Montané and others 2002) similar 

to the biphasic concept (Zhao and others 2012; Zhang and 

others 2012a). Furthermore, the fast and slow hemicelluloses are assumed to have the same fundamental dissolution 

chemistry, and therefore the rate of dissolution can be expressed as

(6a)

(6b)

(6c)

(6d)

where Xf and Xs are fractions of fast and slow residual hemicelluloses such as xylan in hardwood, respectively, with 

XR = Xf + Xs and XR (t = 0) = 1; k in Equation (6a) is the rate 

constant for fast xylan hydrolysis, and f is the ratio of the 

rate constants between the slow and fast xylan hydrolysis 

reactions; θ is the initial fraction of slow reacting xylan. 

Integrating Equations (6a) and (6c) results in the following 

expression for XR, the amount of xylan remaining in the 

solid:

(7)

f

f kCX

dt

dX = −

Xf (t = 0) = (1−θ )XR )

S s fkCX dt

dX = −

Xs(t = 0) = θXR

f

f kCX

dt

dX = −

Xf (t = 0) = (1−θ )XR )

S s fkCX dt

dX = −

Xs(t = 0) = θXR

f

f kCX

dt

dX = −

Xf (t = 0) = (1−θ )XR )

S s fkCX dt

dX = −

Xs(t = 0) = θXR

f

f kCX

dt

dX = −

Xf (t = 0) = (1−θ )XR )

S s fkCX dt

dX = −

Xs(t = 0) = θXR

( ) CHF CHF

R 1 f X e e − − = −θ +θ

Figure 3—Correlations between combined hydrolysis 

factor (CHF) and the conventional combined severity 

factor (CSF) defined in Eq. (1) for a set of dilute acid (DA) 

and two sets of SPORL pretreatments of aspen (from 

Zhu and others (2012)).

0.0 0.8 1.6 2.4 3.2 4.0

CSF

–3.2 –2.4 –1.6 –0.8

4.0

3.2

2.4

1.6

0.8

0.0

–0.8

–1.6

–2.4

–3.2

ln (CHF)

y = –0.35 + 0.95x DA 

y = 0.80 + 0.95x SPORL pH~5

y = 0.20 + 0.95x SPORL pH~2
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

5

Equation (7) is an analytical solution for xylan dissolution 

based on kinetics but expressed in terms of pretreatment 

severity CHF. By fitting the remaining xylan content measured in the pretreated substrates using Equation (7) to CHF, 

the parameters E, α, β, γ, for CHF calculations along with 

the initial fraction of slow reacting xylan, θ, and the ratio 

of the rate constant between slow and fast xylan, f, can be 

obtained.

3.3 Using CHF to Predict Sugar Degradation

The hemicelluloses or cellulose dissolved by acidic pretreatments can be degraded into furan and may be further 

degraded into organic acids. These compounds from sugar 

degradation not only reduce sugar yield but also affect 

downstream processing, such as fermentation, as they can 

inhibit most microorganisms. Hemicellulose dissolution and 

sugar degradation take place simultaneously in pretreatment 

of lignocelluloses. Sequential reactions, however, were assumed here to simplify process modeling. Furthermore, 

furans (furfural and hydroxymethylfurfural (HMF)) were 

assumed as indicator product by neglecting further degradation of furans to acids, and all sugar degradation reactions 

were lumped into one pool. A degradation reaction rate 

constant kd was assumed to follow Arrhenius temperature 

dependence (Saeman 1945; Xiang and others 2004; Qi and 

Lu 2007):

(8)

where αd is an adjustable parameter, Ed is apparent activation energy, R is the universal gas constant of 8.314 J/

mole/K, and T is temperature in kelvins. The rate of sugar 

degradation or furan formation (D) in the pretreatment 

liquor can be expressed as the rate of reduction of dissolved 

carbohydrate:

(9)

where (1 – XR) represents the sum of the amount of carbohydrates dissolved by hydrolysis reactions in a pretreatment 

process. Neglecting cellulose dissolution, which is minimal 

for most pretreatments, Equation (7) can be substituted into 

Equation (9) to result in the following expression for furan 

formation through integration (Zhang and other 2014):

(10)

where t is time in minutes at pretreatment temperature T.

( E RT ) k e d d d − = α

( R ) kd 1 X

dt

dD = −

( ) ( )



 



 − − − − = ⋅ − −CHF − CHF

d 1

CHF

1

CHF

1 1 f e

f

D k t e

θ θ

4. Process Optimization and 

Scale-up Design Using CHF

4.1 Dissolution of Hemicelluloses and Its 

Relation to Sugar Yield

The significance of CHF is that it can not only accurately 

predict hemicellulose dissolution but also correlate enzymatic saccharification and sugar yield. Furthermore, it can 

predict furan formation. This provides the opportunity to 

use CHF for process optimization and scale-up design. As 

shown in Figure 4, the experimentally measured hemicellulose dissolution of aspen (Zhu and othes 2012), poplar 

NE222 (Zhang and others 2015), lodgepole pine (Zhou and 

others 2013b), and Douglas-fir forest residue (Leu and others 2013) under various pretreatment severities using DA 

and SPORL can be accurately predicted by Equation (7). 

Separate fitting of the xylan and mannan dissolution data 

produced better results (Fig. 4d). The fitting parameters of 

these studies are listed in Table 2. In these studies, sulfuric 

acid (H2SO4, catalyst A) and sodium bisulfite (NaHSO3, 

catalyst B) were used. The results in Table 2 suggest that 

E = 100,000 J/mole is a very good number to use as a 

known quantity for softwoods in data fitting, and γ = −10 

can be used for all wood species.

The importance of hemicellulose dissolution in removal of 

recalcitrance of lignocelluloses for enzymatic saccharification cannot be overstated (Leu and Zhu 2013). With the 

excellent prediction of hemicellulose dissolution by CHF, 

good correlations between enzymatic saccharification 

efficiency or sugar yield and CHF are ensured. This can 

be clearly seen from the SED of many DA- and SPORLpretreated aspen substrates (Zhu and others 2012) (Fig. 5a), 

enzymatic hydrolysis glucose yield (EHGY) from SPORLpretreated lodgepole pine WIS (Zhou and others 2013b) 

(Fig. 5b), and EHGY and xylose yield from SPORL-pretreated poplar NE222 (Zhang and others 2015) (Fig. 5c, d). 

The results in Figure 5 clearly indicate that maximal sugar 

yield can be achieved at a minimal CHF of approximately 

20 (CHFopt = 20 for lodgepole pine; CHFopt = 4 for poplar 

NE222). As long as the desired CHFopt is achieved, whether 

through a higher temperature with a short reaction time or 

a lower temperature with a longer reaction time, optimal 

sugar yield is ensured. In other words, instead of using conventional statistical experimental designs to optimize each 

pretreatment condition, such as temperature and time, we 

can simply use the optimal CHF to design desired pretreatment conditions that can meet other constraints, especially 

in commercial-scale productions due to facility limitations. 

Furthermore, in pretreatments such as SPORL with multiple 

process variables, there are often several optimal sets of pretreatment conditions or multiple optimal points. Traditional 

statistical process design may produce only a local optimal, 

depending on the ranges of process variables studied.
General Technical Report FPL–GTR–240

6

Figure 4—Using pretreatment severity CHF for predictions of dissolution of hemicelluloses by DA and SPORL: (a) aspen 

(Zhu and others 2012); (b) lodgepole pine (Zhu and others 2012); (c) poplar NE222 (Zhang and others 2015); (d) Douglas-fir 

forest residue (FS-03) (Leu and others 2013).

Table 2—Fitting parameters in Eqs. (5) and (7) for predicting hemicellulose dissolution 

of different feedstock pretreated by dilute acid and SPORL using catalysts: A = H2SO4, 

B = NaHSO3 (CB = 0 for dilute acid)a

Parameters Aspen NE222 Lodgepole pine Douglas-fir Douglas-fir residue

α 25.6 34.5 28.5 28.5 28.5

β (L/mole) 34.5 18.6 17.0 17.0 17.0

γ (L/mole) −9.9 −9.7 −9.8 −10.0 −10.0

E (J/mole) 90,888 126,200 100,000 100,000 100,000

θ 0.205 0.178 0.320 0.180 0.122

f 0.065 0.078 0.056 0.100 0.021

a

For softwoods, α, β, γ, and E were assumed not to be species dependent. Their values were based on fitting the 

hemicellulose (mannan and xylan) dissolution of lodgepole pine. Data from Zhu and others (2012) and Zhang and 

others (2014, 2015).

(a) (b)

(c) (d)

Hardwoods Softwoods

175

160

150

135

Eq. (7)

Eq. (7), f = 0.021, θ = 0.122

Eq. (7), f = 0.032, θ = 0.177

Eq. (7), f = 0.011, θ = 0.083

T (°C)

Data

Eq. (7)

Eq. (7)

T (°C)

Xylan Mannan

1.0

DA

SPORL pH ~ 5

SPORL pH ~ 2

DA acid variation

Acid and bisulfite variation

0.8

0.6

0.4

0.2

0.0

1.0

0.8

0.6

0.4

0.2

0.0

0 5 10 15 20 25 30

Fraction of xylan retained, 

XR Fraction of xylan retained, 

XR

CHF (min × mole/L)

0 4 8 12 16 20 24 28

CHF (min × mole/L)

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0.5

0.4

0.3

0.2

0.1

0.0

0 20 40 60 80 100 120 140

Fraction of mannan retained, 

XR Fraction of hemicellulose retained, 

XR

CHF (min × mole/L)

0 10 20 30 40 50 60

CHF (min × mole/L)

160 170 180
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

7

4.2 Balance Sugar Yield with Degradation 

Using Low-Temperature Pretreatment

Using CHF to optimize pretreatment also allows balancing 

sugar yield with inhibitor formation without sacrificing enzymatic saccharification efficiency of pretreated substrate. 

Sugar degradation is well known to often have a much higher activation energy than that for hemicellulose dissolution 

for most feedstock (Kamireddy and others 2014). Therefore, 

a lower pretreatment temperature but with a longer pretreatment duration can be used while maintaining the same 

optimal pretreatment severity CHFopt to reduce sugar degradation. Specifically, paired pretreatments at two different 

temperatures, T1 > T2, with identical pretreatment severity 

CHF and chemical loadings can be evaluated. The ratio of 

sugar degradation for any pair pretreatments can be obtained 

based on Equation (10) as

(11)

When Ed > E and T1 > T2, DT1 > DT2. The validity of Equation (10) for predicting sugar degradation was verified using 

59 dilute acid and SPORL pretreatments of lodgepole pine 

in a separate study as shown in Figure 6a (Zhou and others 

2013b).





 



 



 



 − − = ⋅ = 2

1

1

1

exp d

2

1

2

d

1

d

2

1

R T T

E E

t

t

k

k

D

D

T

T

T

T

T

T

Figure 5—Correlating CHF with various sugar recovery measures: (a) aspen substrate enzymatic digestibility (SED) 

(Zhu and others 2012); (b) lodgepole pine enzymatic hydrolysis glucose yield (EHGY) (Zhou and others 2013b); 

(c) poplar NE222 EHGY (Zhang and others 2015); and (d) poplar NE222 xylose yield (Zhang and others 2015).

(a) (b)

(c) (d)

100

90

80

70

50

30

60

40

20

10

480

440

400

360

320

280

240

200

160

120

80

40

0 5 10 15 20 25 30

SED (%) EHGY (g/kg wood)

CHF (min × mole/L)

0.5 1 2 4 8 16 32

CHF (min × mole/L)

80

70

60

50

40

20

30

10

0

35

30

25

20

15

10

5

0

0 20 40 60 80 100

EHGY (% theoretical) Xylose (g/L)

CHF (min × mole/L)

0.5 1 2 4 8 16 32

CHF (min × mole/L)

y = 93 – 72exp(–x/4)

T (°C)

DA

SPORL pH ~ 5

SPORL pH ~ 2

DA acid variation

Acid and bisulfite variation

160 170 180

175

160

150

135

T (°C)

175

160

150

135

T (°C)
General Technical Report FPL–GTR–240

8

Figure 6—Balancing sugar yield with sugar degradation in SPORL: (a) validating sugar degradation model using 

experimentally measured furan data—lodgepole pine (Zhou and others 2013b); (b) predicting sugar degradation 

using pretreatment severity CHF at two temperatures—Douglas-fir (Zhang and others 2014); (c) comparisons of 

substrate enzymatic digestibility (SED) and HMF formation at two SPORL temperatures—Douglas-fir (Zhang and 

others 2014). Numbers in each vertical box are acid concentration (v/v%) in the pretreatment liquor and sodium 

bisulfite charge on wood, respectively.

Paired SPORL pretreatments on Douglas-fir wood chips 

at T1 = 180 °C and T 2 = 165 °C were conducted to verify 

the validity of Equations (10) and (11) (Zhang and others 

2014). Fitting the furans (HMF + furfural) data produced 

Ed = 160, 930 J/mole with E = 100,000 J/mole. As shown 

in Figure 6b, the amount of measured furans from a SPORL 

pretreatment at 180 °C is consistently greater than the 

value from the corresponding pretreatment at 165 °C with 

the same severity CHF. Furthermore, the predicted sugar 

degradations from Equation (10) are in good agreement with 

the measured furan data. The low measured furan value at 

the high severity CHF = 54 is due to the high acid loading that induces substantial degradation of furan to organic 

acids. Moreover, the ratio of the measured furans (DT1=180/

DT 2=165) for each paired SPORL pretreatment was slightly 

below 1.91, as predicted by Equation (11) for most of the 

pairs (not shown), suggesting slightly more furan degradation to organic acids at a higher temperature.

(a) (b)

8

6

4

2

0

Predicted: HMF + furfural (g/L)

7

6

5

4

3

2

1

0

HMF + furfural (g/L)

0 2 4 6 8

Measured: HMF + furfural (g/L) 

0 10 20 30 40 50 60

CHF (min × mole/L)

Data

y = x

T (°C)

165

180

Eq. (10) t (min)

75

30

(c)

T (°C)

165

180

t (min)

75

30

100

80

85

90

95

75

70

SED @ 72 h (%)

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

HMF (g/L)
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

9

Figure 7—A pH–time diagram that schematically shows 

the concept of pH-profiling for pretreating lignocelluloses 

(Cheng and others 2015).

Enzymatic saccharification of these paired SPORL-pretreated WIS suggests that using the same CHF (at the same 

chemical loadings) but at different temperatures (180 °C 

and 165 °C) can indeed produce similar SED. Several runs 

at 165 °C produced SED near or over 90%, although more 

runs at 180 °C achieved the similar SED level, whereas the 

amount of furan formation can be reduced by two to four 

times, as shown in Figure 6c. Furthermore, pretreatment at 

a lower temperature tends to produce slightly higher WIS 

yields to result in a higher EHGY. The approach taken here 

of using low-temperature pretreatment is very different from 

low-severity pretreatment(s) reported in the literature (Chen 

and others 2012), which reduced inhibitor formation at the 

expense of enzymatic saccharification efficiency and EHGY. 

In those cases, additional process steps, such as further size 

reduction or xylanase supplementation, were employed to 

compensate for the reduced SED, which increases capital 

and operating costs.

4.3 Using pH-profiling in SPORL to Further 

Reduce Carbohydrate Degradation

The concept of pH-profiling was developed to further reduce sugar degradation in pretreating feedstock with high 

lignin content, such as forest residues and softwoods, when 

using SPORL (Cheng and others 2015). In this concept, the 

application of acid, such as H2SO4 or SO2, was delayed but 

without changing total acid application (Fig. 7). Theoretically, both temperature and pH profiles can be controlled 

and acid injection can be continuous. The delayed application of acid facilitated delignification in the early stage with 

relatively high pH, whereas reduced acidic reaction time 

resulted in reduced sugar degradation and hemicellulose 

dissolution. The concept is based on two understandings: 

(1) the roles of acid and sulfite on hemicellulose dissolution 

(Zhu and others 2012), delignification through sulfonation 

(Ingruber 1985), sugar degradation (Zhang and others 

2014), and deacetylation (Tune and Van Heiningen 2008); 

and (2) the quantitative effects of delignification and dissolution of hemicelluloses on improving cellulose accessibility and SED (Leu and Zhu 2013; Zhang and others 2014; 

Wang and others 2012a; Yang and Wyman 2004)—that is, 

the negative effect of reduced hemicellulose dissolution 

due to reduced acidic reaction time on substrate cellulose 

accessibility and digestibility can be compensated for by 

improved delignification. Therefore, equivalent enzymatic 

saccharification can be achieved but with lower sugar degradation. SPORL is uniquely suited for adopting the pHprofiling concept because delignification can be achieved 

under acidic conditions with sulfite, which can limit the pH 

range that needs to be controlled. The concept is similar to 

two-stage sulfite pulping (Croon 1965; Lagergren 1964; 

Sanyer and others 1962), but with the goal of reducing sugar 

degradation.

The pH-profiling concept was carried out with Douglas-fir 

forest residue using NaHSO3 and H2SO4 in a laboratory at 

165 °C with a total reaction time of 75 min. Different delay 

times for applying H2SO4 were used. The results were compared with the control run with no delay in H2SO4 application. As shown in Table 3, the three pH-profiling SPORL 

runs produced more delignification but dissolved less hemicellulose than the control run. Furthermore, the amounts of 

furfural, HMF, and acetic acid formation were substantially 

lower than the corresponding value for the control run. SED 

was not significantly affected. EHGY was slightly higher for 

the pH-profiling runs with acid injection delay times of 25 

and 35 min. Enzymatic saccharification and fermentation of 

the pretreated whole slurry of the Douglas-fir forest residue 

at 21% total solids loading produced substantially higher 

ethanol yields from the two SPORL profiling runs than 

from the control run. The ethanol yield for the SPORL pHprofiling runs with 25 min acid delay time was 297 ± 9 L/t 

at a titer of 48.9 g/L compared with 215 ± 42 L/t at 38.6 g/L 

for the control run.

4.4 SPORL Optimization for Hardwoods and 

Herbaceous Biomass Using CHF

Unlike softwood species, hardwoods and herbaceous lignocelluloses contain a substantial amount of acetyl groups 

that can be converted to acetic acid in acidic pretreatments. 

Acetic acid inhibits most organisms such as yeasts and is 

difficult to remove. The discussion in Section 4.2 deals only 

with sugar degradation inhibitors and is not sufficient to address acetic acid inhibition. We developed an optimization 

strategy using CHF to address yeast fermentation inhibition 

by acetic acid at high solids loadings without detoxification 

(Zhang and others 2015). In this strategy, the optimal CHF 

Pretreatment

time

pH Acid or SO2

injection

pH

0 ti tT

T

T (°C)
General Technical Report FPL–GTR–240

10

Table 3—Comparisons of pretreatment performance between normal SPORL (control) and SPORL with pH-profiling at different acid injection timesaRun label

Pretreatment conditions

Pretreated washed solids 

(g/1000g FS-10)

Spent liquor

(g/1000g FS-10) 

(only monomeric sugars were reported)b Enzymatic saccharification

Fermentation

at 21%

solids

Initial

pH

H2SO4

at t = 0

(wt%)

Delay

time ti

(min)

H2SO4

at ti

(wt%)

Final

pH Glucan Xylan

Mannan Klignin

Washed

solid

yield

Glucose Xy- lose Man- nose Fur- fural HMF

Acetic

acid

(g/L)

SED

(%)

EHGY

(% 

theoretical)

Ethanol 

(%

theoretical;

g/L)

Untreated FS-10 410 57 97 293 1,000t0A1B12 (control) 1.79 2.2 0 0 1.45 388.4 13.7 15.8 170.6 612.7 21.8 17.7 54.6 5.0 9.3 5.3 97 83.9 52.8; 38.6t25A4B12 4.06 0 25 2.2 1.72 405.6 22.7 23.7 130.1 603.9 10.0 13.5 32.8 2.2 4.0 3.8 95 85.1 73.1; 48.9t35A4B12 4.06 0 35 2.2 1.40 410.7 20.4 21.8 137.7 616.5 15.7 15.1 38.2 2.0 3.8 4.1 95 86.8t45A4B12 4.06 0 45 2.2 1.66 406.5 21.0 25.7 131.9 622.3 9.1 14.2 30.9 2.0 2.9 3.4 93 80.2 63.7; 45.9aAll pretreatments were conducted using a Douglas-fir forest residue (FS-10) at liquor-to-wood ratio of 3 (L/kg) at 165 oC for total of 75 min and sodium bisulfite (NaHSO3) charge on wood of 12 wt%. Data from Cheng and others 2015. bGlucose, xylose, mannose, furfural and HMF were reported as glucan, xylan, mannan, pentosan, and hexosan, respectively.
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

11

Figure 8—Using combined hydrolysis factor (CHF) for bioconversion of poplar wood NE222 using SPORL: (a) CHF on 

acetic acid formation (Zhang and others 2015); (b) CHF on ethanol yield and productivity (Zhou and others 2015a).

Table 4 lists the calculated pretreatment temperature and 

time scheme for softwoods at the minimal sulfite loading 

and hydroxide loading on wood (SO2 = 6.5 wt%, CaO = 

1.8 wt%) or bisulfite loading between 8 and12 wt% and 

H2SO4 loading approximately 2.2 wt%. We also calculated 

the normalized furan formation at different temperatures 

using Eq. (11). The reduction of furan formation (Table 4) 

is a significant advantage using low-temperature pretreatment. Pretreatment duration greater than 120 min may be 

too long and can negatively affect productivity. Increasing 

SO2 loading over its minimal loading of 6.5 wt% (on wood) 

was not determined purely based on sugar yield, but rather 

based on near complete removal of fast xylan (remaining 

xylan XR = θ). Sugar yield at this pretreatment severity 

CHFXR=θ is near optimal (Fig. 5c, d). CHFXR=θ is used as 

the starting point for optimizing pretreatment of feedstock 

with high acetyl content. Mild pretreatments with severity 

CHF < CHFXR=θ may be necessary to further reduce acetic 

acid formation (Fig. 8a) to achieve maximal biofuel yield 

(Fig. 8b).

4.5 SPORL Process Scale-up Design

Process optimization using conventional statistical experimental design at large scales is always economically 

prohibitive. The low-temperature pretreatment approach 

discussed above can also be applied for process scale-up 

design. Specifically, optimal pretreatment severity measured 

by CHFopt can be obtained from laboratory bench-scale studies as demonstrated in Figure 5. Based on Eq. (5), there are 

many possibilities to design a scale-up pretreatment at a severity of CHFopt by using different combinations of pretreatment temperature and time at fixed chemical loadings. If 

the scale-up facility has constraints in operating temperature 

due to equipment pressure limitation or material corrosion, 

a low-temperature pretreatment can be designed with an 

extended pretreatment duration. On the other hand, if production capacity is the bottleneck, a short pretreatment at an 

elevated temperature can be used. For a given set of chemical loadings, we can calculate this scale-up scheme using 

Eq. (5) (Zhou and others 2015b) as

(12)

up op

up op

1 1

exp

T T

t

R T T

E

t 





























 = −

(a) (b)

28

24

20

16

8

12

4

0

0.5 1 2 4 8 16 32

Acetic acid (g/L)

Ethanol productivity (g/L/h)

CHF (min × mole/L)

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

Ethanol yield (g/g sugar)

CHF (min × mole/L)

Pilot scale

Yield

Productivity

Lab scale

175

160

150

135

T (°C)

Table 4—Process scale-up design model 

Eq. (12) predicted pretreatment temperature 

and time for softwood species using SPORL 

at minimal chemical loadingsa along with 

calculated normalized furan formation using 

simple sugar degradation model Eq. (11)

T (°C) t (min) Normalized furans

180 25–30 1

175 34–40 0.834

170 46–55 0.694

165 62–74 0.575

160 85–102 0.474

155 118–141 0.389

150 164–197 0.317

145 231–277 0.258

140 327–393 0.209

a

SO2 = 6.5 wt% and CaO = 1.8 wt%; or bisulfite loading 

8–12 wt% and H2SO4 loading approximately 2.2 wt%.

where subscript “up” denotes scale-up.
General Technical Report FPL–GTR–240

12

can substantially reduce pretreatment time and thereby 

further reduce furan formation, as we recently found in our 

laboratory.

Table 4 indicates that a pretreatment time of 6 h (240 min) 

or more is required at temperature below 145 °C to obtain 

good pretreatment. Although furan formation can be reduced 

by a factor of 4 at T < 145 °C (Table 4), such a prolonged 

reaction time substantially reduces production capacity 

and increases capital cost for more reactors. One way to 

maintain reaction severity at low temperatures is to increase 

chemical loading without increasing reaction time. Such an 

attempt has been carried in our laboratory with limited experiments (Gu and others 2016). Specifically, we found that 

increasing SO2 loading was very effective in reducing reaction time. Furan formation was very low because of the low 

pretreatment temperature of 140 °C and the leveling of pH 

of the sulfite solution to approximately 1.5 at increased SO2

loadings. One should be able to develop an equation similar 

to Equation (12) to scale reaction time with SO2 loading 

(note that Eq. (12) was developed under fixed chemical 

loading). However, more experiments than those we carried 

out are needed.

5. Lignin Sulfonation on 

Nonproductive Cellulase Binding

Nonproductive binding of cellulase to lignin has been 

recognized as a leading mechanism of lignin inhibition of 

enzymatic saccharification of cellulose (Mansfield and others 1999; Sewalt and others 1997; Berlin and others 2006; 

Palonen and others 2004). To reduce nonproductive cellulase binding, washing pretreated solids to remove dissolved 

lignin is often practiced in most reported studies (Nagle 

and others 2002; Tengborg and others 2001). However, the 

remaining unsolubilized lignin on pretreated solids can still 

bind cellulase to reduce activity. Because of the dissolution 

of hemicelluloses in acidic pretreatments, lignin content in 

pretreated solids is often enriched. Therefore, nonproductive cellulase binding to lignin is unavoidable. Instead of 

using an expensive delignification approach to remove lignin and reduce nonproductive binding, lignin modification 

resulting in a lignin with low affinity to cellulase is a better 

alternative. One of the unique characteristics of SPORL is 

the lignin sulfonation that solubilizes approximately 20% 

to 40% of lignin as lignosulfonate into the pretreatment 

spent liquor. The remaining undissolved lignin on solids is 

also sulfonated. Lignin sulfonation produces a hydrophilic 

lignin that has a low affinity to cellulase because cellulase 

binding is mainly through hydrophobic interactions (Hansen and others 1988; Nakagame and others 2011b; Haynes 

and Norde 1994). Therefore, lignin sulfonation plays a very 

important role in improving sugar yield through reducing 

nonproductive cellulase binding and process simplification 

by eliminating washing of pretreated solids as discussed in 

Section 5.1.

5.1 Lignosulfonate to Enhance Enzymatic 

Saccharification

Non-ionic surfactants have been recognized as being capable of keeping the remaining lignin on a lignocellulosic 

solid substrate from binding cellulase to result in enhanced 

cellulose saccharification (Eriksson and others 2002; Liu 

and others 2010; Borjesson and others 2007; Zheng and 

others 2008). Lignosulfonate is a surfactant and can be used 

to block lignin on solid substrate from interacting with cellulase; however, it is also a lignin that has a hydrophobic 

end, and therefore can bind to cellulase to reduce enzyme 

activity on cellulose (Wang and others 2013a; Liu and others 2010). Different fractions of commercial lignosulfonate 

with different molecular weight and sulfonation were applied to enzymatic saccharification of a hardwood (aspen) 

and a softwood (lodgepole pine) pretreated by SPORL, DA, 

and alkaline processes. The results indicated that application 

of lignosulfonate enhanced saccharification (Zhou and others 2013a). However, the degree of enhancement depended 

on (1) the molecular weight and degree of sulfonation of the 

lignosulfonate applied—the smaller molecular weight fraction that also tends to be more sulfonated performed better 

than the larger molecular weight fraction; (2) the substrate 

lignin—the enhancement for the alkaline-pretreated substrate is greater than for the SPORL-pretreated substrate because the SPORL substrate lignin is sulfonated and already 

has relatively low affinity to cellulase; and (3) the loading of 

lignosulfonate (Fig. 9)—increasing lignosulfonate loading 

resulted in increased saccharification.

Lignosulfonate produced from SPORL tends to have lower 

molecular weight than commercial lignosulfonate but is 

equally sulfonated. When SPORL-pretreated WIS from 

lodgepole pine were mixed with the spent liquor from the 

same SPORL pretreatment and containing lignosulfonate, 

SED of the washed WIS was enhanced by approximately 

60% or more. Furthermore, the enhancement increased with 

an increase in spent liquor application dosage (the effect of 

dissolved sugar in the liquor was accounted for) (Fig. 10). 

The lignosulfonate concentration at 1 g/L in Fig. 10a represents the lignosulfonate level in the pretreated whole slurry. 

This indicated that liquor and solid separation and solid 

washing are not only unnecessary, but also not preferred; 

saccharification and fermentation should use the pretreated 

whole slurry, which can significantly simplify process integration (Fig. 2). This is one of the main advantages of 

SPORL over competing pretreatment technologies.

5.2 pH-Induced Electrostatic Repulsion 

between Sulfonated Lignin and Cellulase

Electrostatic interaction was recently found to play an 

important role in cellulase binding to lignin through pHinduced lignin surface charge (Lou and others 2013) despite 

it being recognized as unimportant for protein binding to 

solid surfaces (Haynes and Norde 1994). Remaining lignin 

on SPORL-pretreated WIS is sulfonated. The surface charge 
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

13

Figure 9—Applications of lignosulfonates of different molecular weight (and sulfonation) on substrate cellulose enzymatic 

digestibility (SCED) of different substrates (Zhou and others 2013a): (a) dilute-acid-pretreated aspen; (b) SPORL-pretreated 

aspen; (c) alkaline-pretreated lodgepole pine; (d) SPORL-pretreated lodgepole pine.

Figure 10—Application of SPORL spent liquor on SPORL water insoluble solids (WIS) on substrate enzymatic digestibility 

(SED) (Wang and others 2013a): (a) effect of spent liquor dosage; (b) effect of different SPORL spent liquor.

(b) 75

70

65

50

55

30

60

40

45

35

0 2 4 6 8 10

SCED @ 72 h (%)

Lignosulfonate concentration (g/L)

AS–SP

SXP1

SXP2

SXP2

(d) 75

70

65

50

55

30

60

40

45

35

0 2 4 6 8 10

SCED @ 72 h (%)

Lignosulfonate concentration (g/L)

LP–SP

SXP1

SXP2

SXP2

(a) 75

70

65

50

55

30

60

40

45

35

0 2 4 6 8 10

SCED @ 72 h (%)

Lignosulfonate concentration (g/L)

AS–DA

SXP1

SXP2

SXP2

(c) 75

70

65

50

55

30

60

40

45

35

0 2 4 6 8 10

SCED @ 72 h (%)

Lignosulfonate concentration (g/L)

LP–KP

SXP1

SXP2

SXP2

(a) (b)

Hydrolysate

No liquor

L-BD4-T85-1

L-BD4-T85-3

L-BD4-T85-5

Klason 

lignin (g/L)

2.7

1.0

0.8

1

3

6

12

24

72

100

90

80

70

60

50

40

30

20

0 20 40 60 80

Enzymatic hydrolysis time (h)

0.0 0.2 0.4 0.6 0.8 1.0

Lignosulfonate as Klason lignin (g/L)

Liquors neutralized by NaOH

SED (%)

90

80

70

60

50

40

30

20

SED (%)

Hydrolysis duration (h)
General Technical Report FPL–GTR–240

14

of this sulfonated lignin can be further increased at elevated 

pH. This was verified by using four enzymatic hydrolysis 

residue lignin substrates produced through extensive enzymatic hydrolysis of one dilute acid and three SPORL-pretreated lodgepole pine WIS samples (Lou and others 2013). 

The remaining protein from the enzymes was thoroughly 

removed using proteinase. The measured zeta-potential (absolute value) was found to increase linearly with pH. At an 

elevated pH > pI (isoelectric point) of the enzyme, the cellulase will also be negatively charged and result in a repulsive 

interaction with the negatively charged lignin. The measured 

cellulase binding to lignin decreased rapidly with increase in 

pH (Fig. 11a). Notice that the amounts of enzyme (CTec2) 

binding to two very different lignin substrates (L-DA with 

no sulfonic acid group and L-SP-B6 highly sulfonated) were 

not much different at pH 4.5 (close to pI of CTec2), suggesting that the lignin surface property does not affect cellulase 

binding at a low pH close to the enzyme pI due to lack of 

electrostatic interactions. However, the difference increased 

when both lignin and enzyme became negatively charged at 

elevated pH (> pI). The amount of CTec2 binding to L-SPB6 was near zero at pH 6.0.

To further illustrate the electrostatic interaction between 

lignin and enzyme, the measured amounts of CTec2 binding 

to the four lignin substrates were plotted against the measured zeta potentials of these lignin substrates at different 

pH (Fig. 11b). CTec2 bindings to lignin were not affected 

by lignin surface charge (also other surface properties, 

such as sulfonic acid group content) at low pH of 4.5 and 

4.8. This is in agreement with discussion above. CTec2 has 

little charge at this pH range because its pI is approximately 

between 4.5 and 4.8 (based on the pI of its major components) (Nakagame and others 2011a; Vinzant and others 

2001; Chirico and Brown 1987; Hui and others 2001) and 

therefore lacks electrostatic repulsion between cellulase and 

lignin. Nonproductive cellulase binding to lignin takes place 

through hydrophobic interactions. At elevated pH > pI, the 

CTec2 binding to lignin decreased linearly with increase in 

lignin surface charge measured by zeta potential (Fig. 11b). 

Furthermore, the lignin substrates (L-SP-B6) with the 

highest sulfonic acid group content resulted in the highest zeta-potential and the lowest cellulase binding to lignin 

(Fig. 11b,c).

This finding of pH-induced electrostatic interaction that 

results in reduction in nonproductive cellulase binding to 

lignin was also verified by a recent study (Rahikainen and 

others 2013). This finding suggested that enzymatic saccharification of lignocelluloses should be conducted at elevated pH > pI of enzyme, rather than at the level suggested 

by enzyme manufacturers (pH 4.8) based on enzymatic 

hydrolysis of pure cellulosic substrates. The benefit can 

be clearly seen from two sets of controlled enzymatic saccharification studies using differently pretreated lodgepole 

pine (SPORL and alkaline) and aspen (SPORL and DA) 

at 2% WIS loading (Lan and others 2013). The pH of the 

Figure 11—Illustrations of pH-induced electrostatic 

interactions between a commercial cellulase (CTec2) and 

lignin on nonproductive binding of cellulase to lignin (Lou 

and others 2013): (a) effect of pH on nonproductive CTec2 

binding; (b) effects of lignin surface charge (zeta-potential) 

on nonproductive CTec2 binding at different pH; (c) effects 

of sulfite charge in SPORL on sulfonic acid group content 

in undissolved solid lignin and nonproductive CTec2 

binding at pH 5.5.

(a)CTec2 binding (mg protein/g lignin)

(b)CTec2 binding to lignin (mg protein/g)

pH

4.5

4.8

5.5

6.0

–26 –24 –22 –20 –18 –16 –14

Zeta potential (mV)

2.5

3.0

2.0

1.5

1.0

0.5

0.0

4.4 4.8 5.2 5.6 6.0

pH

2.5

3.0

2.0

1.5

1.0

0.5

0.0

(c)CTec2 binding (mg protein/g lignin)

Sulfonic acid group content (mg/g lignin)

2.5

0

Sodium bisulfite loading on wood (%)

Cellulase binding at pH 5.5

0.2× sulfonic acid groups 

2 4 6

2.0

1.5

1.0

0.5

0.0

2.5

2.0

1.5

1.0

0.5

0.0

L–DA

y = –1.22x + 8.1, r2 = 0.977

L–SP–B6

y = –1.67x + 10.0, r2 = 0.937
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

15

lignocellulosic substrate suspension was buffered in a range 

of 4–7. Furthermore, measured pH (rather than buffer pH) 

of the suspension was used in data plotting. Results indicate 

that the optimal pH for enzymatic saccharification of all 

lignocellulosic substrates all shifted to a higher pH of approximately of 5.5, whereas optimal pH for Whatman paper 

(pure cellulose) was at approximately 4.8. The gain in enzymatic saccharification efficiency (compared with the efficiency at the enzyme manufacturer recommended pH 4.8) 

varied with pretreatment methods used. The maximum gain 

was from lodgepole pine pretreated by SPORL under acidic 

conditions, whose lignin is highly sulfonated, in agreement 

with the results shown in Fig. 11c. A very high pH 6.0 did 

not produce the highest gain in enzymatic saccharification 

despite nonproductive cellulase binding being the lowest 

(Fig. 11a). This is because enzyme activity on pure cellulose 

is optimal at pH 4.8 and is reduced by approximately 10% 

at pH 6.0, as can be seen from the saccharification of both 

Whatman paper (Fig. 12) and bleached kraft pulp fibers that 

do not contain lignin (Lou and others 2013). Direct correlation between gains in saccharification efficiency and 

reductions in nonproductive binding was observed (Lou and 

others 2013).

6. SPORL Process Performance and 

Integration for Biofuel and Lignin 

Co-Product Production

All the features of SPORL for robust bioconversion of 

lignocellulosic biomass have been discussed so far. This 

section outlines SPORL process performance by first comparing it with other pretreatment methods and then presenting bioethanol and lignin co-product production from 

woody biomass, including underutilized lignocelluloses 

such as forest harvest residue.

6.1 Comparison of SPORL with Dilute Acid 

Pretreatment—Enzymatic Saccharification 

and Fermentation of Washed Water-Insoluble 

Solids

Two studies were specifically designed to compare SPORL 

with DA pretreatment. These two studies simply examined 

the effect of sulfite on the effectiveness of pretreatment of 

a spruce (Shuai and others 2010) and an aspen (Zhu and 

other 2011). All pretreatment conditions such as temperature, time, liquor-to-wood ratio, and H2SO4 loadings were 

identical for DA and SPORL except that NaHSO3 at 3 wt% 

and Na2SO3 at 9 wt% on wood were added in SPORL for 

aspen and spruce, respectively. For the study using spruce 

(Shuai and others 2010), it was obvious that DA could not 

effectively remove the recalcitrance of softwood spruce. The 

advantages of SPORL were very obvious in terms of enzymatic saccharification efficiency of the washed pretreated 

WIS (Zhu and others 2011a). SPORL-pretreated aspen 

WIS was also more digestible than DA with lower inhibitor 

formation. Similar comparative results were also obtained 

when pretreating eucalyptus (Wang and others 2009) and 

poplar woods (Wang and others 2012b).

The second study was carried out according to Scheme II 

shown in Figure 2. Enzymatic saccharification and fermentation runs were conducted at high solid loadings between 

12% and 18% using washed DA- and SPORL-pretreated 

aspen WIS. The DA and SPORL aspen substrates were 

produced under identical conditions except 3 wt% on wood 

NaHSO3 was applied in SPORL (Zhu and others 2011a). 

Under the same cellulase loading of 10 FPU/g glucan, 

less torque and mechanical energy for shear mixing were 

required to liquefy the washed WIS from SPORL than the 

WIS from DA pretreatment. The advantage of SPORL was 

more obvious at a low cellulase loading of 6 FPU/g glucan, 

as shown by the ethanol concentration in the fermentation 

broth in Figure 13a. Because the SPORL WIS had a higher 

glucan content of 66.2% than 61.6% for the DA substrate, a 

comparison of simultaneous saccharification and fermentation efficiency at different cellulase loadings was made. The 

efficiency achieved at 10 FPU/g glucan for the SPORL substrate was equivalent to that achieved at 15 FPU/g glucan 

for the substrate pretreated by DA (Fig. 13b).

Figure 12—Effect of pH on enzymatic saccharification of 

different lignocelluloses measured by substrate enzymatic 

digestibility (SED) (Lan and others 2013): (a) differently 

pretreated lodgepole pine; (b) differently pretreated aspen.

(a)

(b)SED @ 48 h (%) SED @ 48 h (%)

4.0 4.5 5.0 5.5 6.0 6.5 7.0

Measured suspension pH at 0 h

4.0 4.5 5.0 5.5 6.0 6.5 7.0

Measured suspension pH at 0 h

80

70

60

50

40

30

20

0

10

60

70

80

50

40

20

30

10

0

Celluclast 1.5 L:11.3 FPU/g glucan

SP–BD4

SPH–FL

Celluclast 1.5 L (FPU/g glucan)

SP–AS

SPH–AS

DA–AS

7.5 11.3

KF–FL

Whatman 

paper
General Technical Report FPL–GTR–240

16

6.2 Bioethanol Production from Lodgepole 

Pine—Process Scale-up Demonstration and 

Comparison with SO2 Steam Explosion

The concepts of process scale-up design and low-temperature pretreatment to balance sugar yield and degradation 

presented in Section 4 were demonstrated here using a softwood—lodgepole pine. The first study focused on scale-up 

to 2 kg in a 23-L wood pulping digester based on SPORL 

optimization using 150 g wood chips in a 1-L reactor (Zhou 

and others 2013b). The lodgepole pine wood chips BD4 

were produced from a mountain pine beetle killed tree harvested from the Canyon Lakes Ranger District of the Arapaho–Roosevelt National Forest, Colorado (Luo and others 

2010; Zhu and others 2011b). The 2-kg scale-up study was 

carried out at two temperatures so that the advantage of the 

low-temperature pretreatment could be shown (Zhou and 

others 2014). The second study scaled the SPORL process 

up to 50 kg wood chips in a pilot-scale 390-L wood pulping 

digester based on the 2-kg study at the same pretreatment 

temperature of 165 °C. The lodgepole pine wood chips 

BKLP were produced from mountain pine beetle killed 

lodgepole pine trees. These trees were approximately 28 

cm in diameter at breast height (DBH) and harvested from 

Canyon Lakes Ranger District of the Arapaho–Roosevelt 

National Forest, Colorado (GPS location: 13 T 458469 

4492172 (NAD27), elevation 2,620 m).

Enzymatic saccharification results from 59 pretreatment 

runs at a scale of 150 g wood chips were used to design the 

two scale-up studies. As shown in Fig. 5b, the 150-g scale 

study indicated that EHGY was approximately maximized 

Figure 13—Comparison of high titer ethanol productions from washed water insoluble solids (WIS) fraction of aspen 

pretreated by dilute acid (DA) and SPORL (Zhu and others 2011a): (a) time-dependent ethanol concentrations in the 

fermentation broth at two cellulase loadings; (b) terminal ethanol fermentation efficiencies at different cellulase loadings.

(plateaued) at the minimal pretreatment severity CHF of approximately 20. Based on this CHF, pretreatment at 180 °C 

can be designed for 25 min at NaHSO3 and H2SO4 loadings of 8 and 2.2 wt%, respectively, on wood with liquorto-wood ratio 3 (L/kg), which gives an actual CHF = 22.5 

(calculated using Eq. (5) with parameters listed in Table 2) 

for the scale-up pretreatment at 2 kg. Using Eq. (12), a lowtemperature pretreatment at 165 °C can be designed for 

75 min under the same chemical loadings for scale-up pretreatments using 2 kg and 50 kg wood chips.

The 2-kg scale study was carried out according to the experimental flow diagram shown in Figure 14 at both 165 

and 180 °C. The 23-L rotary wood pulping digester was 

heated through a steam jacket. At the end of pretreatment, 

the digester was cooled down by flushing tap water into the 

jacket. The drainable spent liquor was sampled and then 

collected and neutralized. The neutralized liquor was mixed 

and disk-refined with the collected pretreated wet solids that 

contain approximately two-thirds of the spent liquor. The 

refined whole slurry was directly used for quasi-enzymatic 

saccharification and fermentation (q-SSF) using Saccharomyces cerevisiae YRH400 (Hector and others 2011) after 

neutralization to approximately pH 6 without any detoxification, solids and liquor separation, solids washing, or 

supplementing nutrients. Pretreatment at 165 °C resulted in 

a higher solids and carbohydrate recovery than at 180 °C 

(Table 5). For example, glucan loss was only approximately 

2% at 165 °C compared with 15% at 185 °C, whereas 

hemicellulose recovery was approximately 65% at 165 °C 

compared with 50% at 180 °C. Furthermore, total furan 

formation was reduced by approximately 50% from 180 to 

70

50

30

60

40

20

10

0

Ethanol concentration (g/L)

0

90

70

50

80

60

40

30

20

Fermentation efficiency @ 96 h (%)

20 40 60 80 100 120 140 160 180

Fermentation time (h)

6 8 10 12 14 16

Cellulase dosage (FPU/g glucan)

Cellulase (FPU/g glucan)

SPORL

DA

SPORL

DA

15 6

(a) (b)
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

17

Figure 14—Schematic process flow diagram for high titer ethanol and lignosulfonate 

production using SPORL at laboratory bench scale (Zhou and others 2013b).

165 °C, in agreement with the prediction by Eq. (11). These 

advantages produced tangible benefits in q-SSF at high solids of 18% despite the washed WIS from 165 °C being less 

digestible than that from 180 °C (Fig. 15a). Sugar consumption and ethanol production were much faster for the whole 

slurry produced at 165 °C than that at 180 °C (Fig.15b) with 

an equivalent terminal ethanol titer of approximately 47 g/L. 

Because of the higher carbohydrate recovery, pretreatment 

at 165 °C produced a higher ethanol yield of 72% theoretical based on wood glucan and mannan (306 L/t) than 61% 

(260 L/t) at 180 °C (Zhou and others 2014). Ethanol yield 

of 306 L/t is excellent, considering xylose utilization by 

YRH400 was negligible due to low concentration. This suggests that using CHF to scale the SPORL process from 150 

g up to 2 kg and designing low-temperature pretreatment at 

165°C worked well.

Using the CHF design scheme (Eq. (12)) to conduct a 

SPORL pilot-scale study using 50 kg wood chips in a 390-L 

wood pulping digester (Fig. 16) worked equally well. The 

experiment was carried out following the flow diagram 

similar to Fig. 14 except that the digester content was discharged under pressure to a blow tank at the end of pretreatment (Zhou and others 2015b). Noncondensable gases, 

including SO2, were vented to a sodium hydroxide scrubber 

(Fig. 16). The SED of the washed WIS from the pilot scale 

agreed with the results from the 2-kg study (Fig. 15a). Component recoveries are listed in Table 6. Very similar major 

carbohydrate recoveries were achieved when comparing 

the pretreatment at 2-kg (Table 5) with that at 50-kg scale 

(Table 6) at 165 °C, except that a higher mannan recovery 

was obtained at the pilot scale perhaps due to small differences in the wood used. The furans formed were also very 

comparable with higher HMF formed at 2-kg scale, consistent with the higher mannan dissolution observed. Furthermore, q-SSF of the pretreated whole slurry at 20% total 

solids loading without nutrient supplementation produced an 

ethanol yield of 289 L/t, or 72% theoretical based on wood 

glucan, xylan, and mannan content, similar to that achieved 

at 2-kg scale using a different lodgepole pine (BD4). Detailed comparisons between the two scale-up studies at 2 kg 

and 50 kg are shown in Table 7, where ethanol yields were 

based on wood glucan and mannan only.

Few processes demonstrated the capability for high-titer 

ethanol production from softwoods without detoxification. 

The only data available were for spruce using SO2-catalyzed 

steam explosion (Hoyer and other 2013) and were compared 

with the two lodgepole pine scale-up studies discussed 

above. As shown in Table 7, the SO2 steam explosion study 

consumed more energy input in pretreatment at 205 °C, had 

better mixing in enzymatic saccharification with mechanical 

actions, and used 50 times more yeast loading, along with 

supplementation of several nutrients, but produced a lower 

ethanol yield of 72% theoretical (based on wood glucan and 

mannan only) as compared to approximately 80% theoretical obtained from the two SPORL studies.

6.3 Bioethanol Production from a Softwood 

Forest Residue—Scale-up Evaluation

Building upon the several process technologies discussed 

in Sections 4 and 5, process integration was carried out for 

the production of high-titer ethanol and lignosulfonate from 

forest harvest residue using the SPORL technology without 

detoxification and solids washing (Zhu and other 2015). 
General Technical Report FPL–GTR–240

18

Figure 15—Comparison of high titer ethanol production from SPORL-pretreated lodgepole pine at two temperatures: 

(a) comparisons of water insoluble solids (WIS) substrate enzymatic digestibility (SED), including data from a substrate from pilot-scale of 50-kg pretreatment (modified from (Zhou and others (2014)); (b) time-dependent glucose 

utilization and ethanol concentration in the fermentation broth of the pretreated whole slurry (Zhou and others 2014).

Table 5—Chemical composition of untreated lodgepole pine BD4 wood chips and wood component recoveries from 

SPORL at two temperaturesa

Untreated 

wood

Unwashed solidsb Collected spent liquorb Total recovery(%)

165 °C 180 °C 165 °C 180 °C 165 °C 180 °C

Wet weight (kg) 2.281 6.227 5.620 1.726 2.250

Solids content (%) 87.7 31.86 29.98 8.37 9.09

Solids (kg)c 2.0 1.984; 90.0% 1.685; 76.4% 0.144; 6.6% 0.205; 9.3% 96.6 85.7

Klason lignin (%) 28.6 28.31; 99.0% 24.45; 85.5% 0.29; 1.0% 4.15; 14.5% 100.0 100.0

Arabinan (%) 1.7 0.58; 34.2% 0.39; 23.0% 0.06; 3.3% 0.07; 3.9% 37.6 26.9

Galactan (%) 2.9 1.43; 49.3% 1.04; 35.9% 0.40; 13.9% 0.55; 18.9% 63.1 54.8

Glucan (%) 41.9 40.64; 97.0% 34.68; 82.8% 0.60; 1.4% 1.03; 2.5% 98.4 85.2

Mannan (%) 11.7 5.98; 51.1% 4.21; 36.0% 1.70; 14.5% 2.39; 20.4% 65.6 56.4

Xylan (%) 5.5 2.86; 52.0% 1.87; 33.9% 0.53; 9.7% 0.70; 12.7% 61.6 46.6

HMF (%)d 0.43; 3.7% 0.72; 6.1% 0.18 (1.7); 1.5% 0.41 (3.0); 3.5% 5.2 9.6

Furfural (%)d 0.31; 5.7% 0.40; 7.3% 0.13 (1.1); 2.3% 0.23 (1.6); 4.2% 8.0 11.5

Acetic acid (%) 1.87 2.05 0.76 1.17

a

Pretreatments were conducted at sulfuric acid and sodium bisulfite loading on wood of 2.2 and 8%, respectively, for 25 and 75 min at 180 °C and 165 °C, 

respectively. Data from Zhou and others (2014).

b

The numbers after “;” are wt% of theoretical based on untreated wood carbohydrate content.

c

Oven dry (od) weight.

d

Reported as xylan and mannan for furfural and HMF, representing percent of xylan and mannan converted to furfural and HMF, respectively. 

Numbers in parentheses are concentrations measured in the collected spent liquor in g/L.

100

40

80

60

20

0

SED (%)

0

70

50

30

60

40

20

10

0

Concentration (g/L)

20 40 60 80

t (h) t (h)

0 20 40 60 80 100 120 140

(a) (b)

T (°C)

165 (BD4)

180 (BD4)

165 (BKLP pilot scale)

T (°C)

165 

180 

(306 L/tonne)

(260 L/tonne)

Glucose Ethanol (yield)
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

19

Figure 16—Schematic process flow diagram showing high titer ethanol and lignosulfonate production from 

forest residue using SPORL at a pilot scale (Zhou and others 2013b), including grinding and loading of roadside piles of harvest residue and pictures of pilot-scale wood pulping digester and blow tank and caustic wet 

scrubbing of noncondensable gases from SPORL.

Table 6—Chemical composition of untreated BKLP wood chips and wood component recoveries 

from a pilot-scale SPORL run in a 390-L wood pulping digestera

Untreated

wood

Unwashed

solidsb Collectedspent liquorb

Total 

recovery 

(%)

Washed

solidsb

Wet weight (kg) 49.95 109.45 39.95

Solids content (%) 80.1 31.9 15.8

Solids (kg)c 40.0 34.86; 87.2% 6.31; 15.8% 102.9 26.40; 66.0%

Klason lignin (%) 29.85 ± 0.01 22.48 ± 0.39; 75.3% 2.32; 7.8% 83.1 23.01 ± 0.33; 77.1%

Arabinan (%) 1.76 ± 0.02 0.55 ± 0.01; 31.2% 0.00 31.2 0.00

Galactan (%) 3.56 ± 0.04 1.54 ± 0.02; 44.3% 0.74; 20.8% 64.1 0.12 ± 0.11; 7.0%

Glucan (%) 39.00 ± 0.03 36.95 ± 1.53; 94.7% 1.13; 2.9% 97.6 37.46 ± 0.94; 88.4%

Mannan (%) 9.46 ± 0.11 5.95 ± 0.14; 61.4% 2.49; 26.4% 87.8 1.42 ± 0.16; 21.3%

Xylan (%) 7.23 ± 0.01 3.64 ± 0.11; 50.4% 1.16; 16.0% 66.4 1.57 ± 0.12; 37.6%

HMF (%)d 0.30; 3.1% 0.11 (0.8); 1.1% 4.3

Furfural (%)d 0.36; 5.0% 0.13 (1.0); 1.8% 6.8

Acetic acid (%) 1.17 0.43 (4.3)

a

Pretreatment was conducted at sulfuric acid and sodium bisulfite loading on wood of 2.2 and 8%, respectively, for 60 min at 

165 °C with ramping time to 165 °C of 38 min. Data from Zhou and others (2015b).

b

The first numbers of the chemical component data are based 100 kg of untreated wood; the % data are theoretical yields based 

on total solids or component of untreated wood. 

c

Based on oven dry (od) basis of untreated wood 

d

Conversion rates of the compounds indicated. The numbers in parentheses are concentrations measured in the collected spent 

liquor in g/L.

Distillation

Separation

Purication

Ethanol Lignosulfonate

Pretreatment

Steam, SO2

, hydroxide

Neutralization

Whole slurry

Lignin residue

Waste water

No detoxication

No washing

Size reduction

Blow tank to 

wet scrubber

and

fermentation

Enzymatic

saccharication

Pipe to wet 

scrubber

Forest residue roadside pile Grinding and loading
General Technical Report FPL–GTR–240

20

fed to a disk refiner together with the collected wet solids, 

which contains approximately two-thirds of the pretreatment 

spent liquor, resulting in the whole slurry.

The q-SSF was carried out by first liquefying the pretreated 

whole slurry using CTec3 after neutralizing pH to approximately 6.0 and then adding Saccharomyces cerevisiae 

YRH400 for fermentation. The yeast was grown on YPD 

agar plates (Zhou and others 2013b). This q-SSF was conducted without supplementation of nutrients, solids and 

liquor separation, solids washing, or detoxification. At the 

end of fermentation, the residual solids, which contained 

primarily water-insoluble sulfonated lignin, were separated 

using a centrifuge. The supernatant was fed into an in-house 

built membrane filtration system to separate lignosulfonate from smaller molecular weight ethanol and remaining 

soluble sugars. The entire process is relatively simple for 

simultaneous production of bio-ethanol at high titer and 

lignosulfonate.

The overall mass balance of the process is shown in 

Figure 17 for q-SSF at 18% total solids with a cellulase 

(CTec3) loading of 35 mL/kg FS-10. A terminal ethanol 

yield of 284 L/t FS-10, or 70% theoretical based on glucan, 

mannan, and xylan content in FS-10, at a titer of 41.9 g/L 

was achieved. SPORL dissolved approximately 40% of the 

lignin as lignosulfonate, which is readily marketable without 

Roadside piles of Douglas-fir plantation harvest residue 

were ground and then loaded into a truck (Fig. 16). The 

ground materials can be either screened on the conveyer belt 

while loading to the truck or separately screened to remove 

the fine fraction, which has high bark and ash contents 

(Cheng and others 2015; Zhang and others 2012b). The 

screened forest residue (FS-10) had a lower bark and ash 

content than the as-ground material. A pretreatment using 

61.75 kg of FS-10 at a moisture content of 18.6% was conducted in the 390-L rotating wood pulping digester at 145 

°C with a minimal total SO2 loadings of 6.6 wt% on wood 

(oven-dry base), or approximately 23 g/L in the pretreatment 

liquor. Loading of Ca(OH)2 on wood was 2.4 wt%. Final 

liquor-to-wood ratio was 3.55 (L/kg) including the amount 

of moisture used in pre-steaming. The heat-up period to 

145 °C was 37 min. The calculated required pretreatment 

duration was 231–277 min based on the scale-up design of 

Eq. (12) (Table 4) using laboratory bench-scale optimal time 

of 25–30 min at 180 °C (Leu and others 2013). Considering 

the longer heat-up period of 37 min required for the 390-L 

digester relative to that for the 23-L bench-scale digester, a 

pretreatment duration of 240 min was used at 145 °C. At the 

end of pretreatment, the digester contents were discharged 

to a blow tank that was connected to a wet scrubber to eliminate any noncondensable gases, including SO2 (Fig. 16). 

The free drainable liquor was collected, neutralized, and 

Table 7—Comparison of ethanol production of two SPORL studies using lodgepole pine 

and a literature work based on SO2 steam explosion from sprucea

SPORL, 

lodgepole pine

(BKLP)

(pilot-scale 50 kg)

SPORL, 

lodgepole pine

(BD4)

(bench-scale 2 kg)

SO2 steam 

explosion, spruce

Pretreatment 

Temperature (°C) 165 165 205

Duration (min) 60 (38-min ramping) 75 (10-min ramping) 6–7

Liquor to wood ratio (L/kg) 3.0 3.0 ~3.0

Q-SSF

Water-insoluble solids (WIS) (%) 15.1 12.0 13.7

Total wet mass (g) 50 50 1,300

Mixing mode Shaking bed: poor Shaking bed: poor Mechanical mixing: good

Cellulase (FPU/g WIS) 11.5 CTec3 11.1 CTec2 10.0 CTec2 + β-glucosidase

Yeast (g dry cell/L) ~0.1 ~0.1 5

Nutrients for fermentation 

(NH4)2HPO4 (g/L) None None 0.5

MgSO4∙7H2O (g/L) None None 0.025

Yeast extract (g/L) None None 1.0

Liquefaction time (h) 24–26 22 22

Final ethanol production

Ethanol concentration (g/L) 52.2 47.1 47.8

Ethanol yield (% of theoretical)a 82.5 79.2 72.0

a

Data from Zhou and others (2013b) and Hoyer and others (2013).

b

Theoretical yield based on glucan and mannan in the untreated wood.
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

21

further processing—a significant economic advantage over 

competing technologies. Even without mechanical shear 

mixing, an equivalent ethanol yield of 68% theoretical 

was achieved at a lower CTec 3 loading of approximately 

26 mL/kg FS-10 (Zhu and others 2015). It is expected that 

mechanical mixing can further enhance ethanol yield and 

that a low cellulase loading of approximately 25 mL/kg forest residue or 15 FPU/g glucan is sufficient to produce an 

excellent ethanol yield.

To reduce pretreatment duration at low pretreatment temperatures, we increased SO2 loading (as discussed in Section 

4.5). An increase in total SO2 charge on wood by a factor of 

4 to a targeted value of 32 wt% with a combined SO2 (with 

Mg) of 4.4 wt% can reduce pretreatment duration to 60 min 

at 140 °C to produce excellent digestible substrate from 

a Douglas-fir forest residue FS-10 (Gu and others 2016). 

Furan formation was very low, less than 0.5 g/L, using 

liquid-to-wood ratio of 4. Compared with the low SO2 loading study (6.6 wt% on wood) at 145 °C with long pretreatment time of 240 min (Zhu and others 2015), the high SO2

loading study produced a maximal terminal ethanol yield of 

321 L/t FS-10 at a titer of 56.3 g/L, compared with 284 L/t 

FS-10 at 41.9 g/L from the run with low SO2 loading of 

6.6 wt% on wood under the same CTec3 loading of 35 mL/

kg FS-10.

6.4 Bioethanol Production from Poplar—

Scale-up Evaluation

Poplar has been identified as a short-rotation energy crop 

that can grow on marginal land, recycle nutrients, and sequester carbon (Vance and others 2010). Bioconversion of 

poplar wood to biofuels through fermentation of hydrolyzed 

sugars is quite challenging. As a woody biomass, poplar 

species have a relatively higher lignin content than herbaceous biomass and can be highly recalcitrant to enzymatic 

saccharification (Wang and others 2012b; Studer and others 

2011). As discussed in Section 4.4, poplar species also have 

a relatively high content of acetyl group (Gille and Pauly 

2012) that can be easily converted to acetic acid (Tune and 

Van Heiningen 2008; Tian and others 2011), a fermentation 

inhibitor that is difficult to neutralize or distill (Xavier and 

others 2010) and cannot be metabolized by yeast (Wei and 

others 2013). Typical yeast can tolerate only approximately 

15 g/L of acetic acid (Keating and others 2006). De-acetylation using hydroxide requires additional processing (Chen 

and others 2012; Kundu and others 2014). The compounding effect of acetic acid with furan and aromatics makes 

fermentative biofuel production from poplar very difficult 

(Palmqvist and others 1999), with few successes reported.

Here bioethanol production is demonstrated from poplar NE222 using the same 390-L pilot-scale digester for 

Figure 17—Overall mass balance for productions of bioethanol and lignosulfonate from a Douglas-fir 

forest residue (FS-10) using SPORL at a pilot-scale (Zhu and others 2015).
General Technical Report FPL–GTR–240

22

pretreatment as described in the Section 6.3. The experimental design was based on the results from a 1-L scale study 

using CHF. As shown in Figure 8b, the laboratory 1-L scale 

study (Zhang and others 2015) indicated that pretreatment 

severity CHF needed to be reduced from the optimal CHF 

for maximal sugar yield (Fig. 5c,d). A target CHF of 2.7 

was used based on the results in Fig. 8b (Zhou and others 

2015a). Using Eq. (5), the pretreatment time of 45 min was 

determined for pretreatment at 160 °C with a liquor-to-wood 

ratio of 3 L/kg and sulfuric acid and sodium bisulfite charge 

on wood of 1.1 and 4.0 wt%, respectively. The actual CHF 

was 3.3 with a shorter pretreatment time of 40 min (due to 

the longer temperature ramping time of 30 min in the 390-L 

digester) and a sodium bisulfite loading of 3 wt%. The experimental details for pretreatment and q-SSF were similar 

to those described in Section 6.3. Again, the disk-refined 

whole slurry was not detoxified and nutrients were not 

supplemented for direct q-SSF after neutralization to pH 6.0 

using a CTec3 loading of 26 mL/kg untreated wood.

Mass balance for the pretreatment is shown in Table 8 along 

with the furans and acetic acid concentrations of approximately 3 and 13 g/L, respectively, in the collected spent 

liquor. Glucan loss was minimal at approximately 5%. Xylan and mannan dissolutions were approximately 75% and 

55%, respectively. Major sugar consumption and ethanol 

production through q-SSF using YRH400 at three different 

yeast loadings are shown in Figure 18. Glucose was rapidly 

consumed except at very low yeast loading of 0.1 mg/g. 

With the high xylan content of poplar and therefore high xylose concentration, YRH400 was able to consume approximately 25% of the xylose. The low xylose consumption was 

due to the presence of fermentation inhibitors and because 

S. cerevisiae relies on hexose transport, which has low affinity to xylose. Mannose was not completely consumed due to 

low concentration. Overall, the scale-up results agreed with 

laboratory bench-scale experiment (Fig. 8b), suggesting 

that using CHF can properly scale up SPORL pretreatment 

of poplar. A final ethanol yield of 247 L/t wood, or 54.7% 

theoretical based on NE222 glucan, xylan, and mannan content, at titer of 43.6 g/L was obtained using a shaker flask 

for fermentation without shear mixing.

6.5 Properties of Lignosulfonate from SPORL

Very high ethanol yield has been achieved using SPORL 

with limited room for further improvement (as discussed 

in Section 6.4). The fuel market is very large, with a very 

low risk of market saturation. However, fuel is a low-value 

commodity and the return from fuel production alone is 

not economically viable for commercialization. Therefore, 

producing lignin co-products are vital. SPORL has the advantages of being able to partially solubilize lignin through 

sulfonation. The dissolved lignin is a directly marketable 

product as lignosulfonate, which can produce another stream 

of revenue for improving economics.

Table 8—Wood component recoveries from a pilot-scale SPORL run using poplar NE222 along 

with the chemical composition of the untreated NE222a 

Untreated

wood

Unwashed

solidsb Collected spent liquorb

Total 

recovery 

(%)

Washed

solids

Wet weight (kg) 81.90 110.70 32.15

Solids content (%) 49.9 32.9 11.1

Solids (kg)c 40.9 36.45; 89.2% 3.58; 8.8% 97.9 27.52; 67.3%

Klason lignin (%) 23.43 ± 2.03 18.56 ± 0.05; 79.1% 4.87; 20.8% 100.0 16.69 ± 0.03; 71.2%

Arabinan (%) 0.61 ± 0.43 0.21 ± 0.03; 35.1% 0.07; 12.0% 47.1 0.00 ± 0.00; 0.0

Galactan (%) 0.61 ± 0.06 0.32 ± 0.01; 52.6% 0.13; 21.9% 74.6 0.00 ± 0.00; 0.0

Glucan (%) 46.76 ± 3.32 44.36 ± 0.52; 94.9% 0.10; 0.2% 95.1 40.83 ± 0.69; 87.3%

Mannan (%) 2.84 ± 0.03 1.86 ± 0.20; 65.6% 0.18; 6.5% 72.1 1.31 ± 0.03; 46.3%

Xylan (%) 12.74 ± 0.05 8.65 ± 0.06; 67.9% 1.74; 13.6% 81.5 3.24 ± 0.26; 25.4%

HMF (%)d 0.11; 3.9% 0.05 (0.5); 1.7% 5.5

Furfural (%)d 0.59; 4.6% 0.26 (2.4); 2.0% 6.7

Acetic acid (%) 2.43 1.05 (13.4)

a

Pretreatment was conducted in a 390-L rotating wood pulping digester with sulfuric acid and sodium bisulfite loading on wood 

1.1 and 3 wt%, respectively, for 40 min at 160 °C. Data from Zhou and others (2015a).

b

The first numbers of chemical component data are based on 100 kg of untreated wood; the numbers after “;” are wt% of 

theoretical based on total solids or component mass in the amount of untreated wood.

c

Oven dry (od) weight based on untreated wood.

d

Reported as xylan and mannan for furfural and HMF, representing percentage of xylan and mannan converted to furfural and 

HMF, respectively. The numbers in parentheses are concentration measured in the collected spent liquor in g/L.
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

23

Figure 18—Time-dependent ethanol production and sugar 

consumptions during Q-SSF at 20% total solids loading 

without detoxification of the whole slurry of SPORL-pretreated NE222 at a pilot-scale (Zhou and others 2015a): 

(a) ethanol and glucose; (b) xylose; (c) mannose.

Molecular weight and degree of sulfonation are two important properties of lignosulfonate. The spent liquor from the 

pilot-scale pretreatment of BKLP at 165 °C was purified 

using dialysis through a membrane system. The purified 

lignosulfonate was analyzed and compared with a commercial purified softwood lignosulfonate D-748 (Zhou and 

others 2015b). Results indicate that SPORL lignosulfonate 

(LS-SP-BKLP) had similar sulfonation as D-748, with a 

sulfur content of approximately 6 wt%, but it had a lower 

molecular weight of approximately 10,000, compared with 

approximately 40,000 for D-748. Membrane separation of 

lignosulfonate after q-SSF was employed on the substrate 

produced in the pilot-scale studies described in Section 6.3. 

Similar results were also obtained for the lignosulfonate 

from a Douglas-fir forest residue pretreated at 145 °C (Zhu 

and others 2015) after correction for difference in molecular weight calibration. Preliminary testing showed that the 

performance of SPORL lignosulfonate was comparable or 

slightly better as a dispersant for coal water slurry.

6.6 Production of Lipid from a Softwood 

Forest Residue

SPORL-pretreated whole slurries of a Douglas-fir forest residue were used to produce intracellular microbial lipid using 

oleaginous microorganisms. In the first study (Harde and 

others 2016), two SPORL-pretreated Douglas-fir residue 

whole slurries at a high targeted SO2 loading of 32 wt% on 

wood (Gu and others 2016) were fermented using two yeast 

strains, Cryptococcus curvatus NRRL Y-1511 and Rhodosporidium toruloides Y-1091, and two fungi, Mortierella 

isabellina NRRL 1757 and Chaetomium globosum NRRL 

1870, all from the Agricultural Research Service (ARS) 

Culture Collection, Illinois. It was found that high sulfite in 

the whole slurry inhibited fermentation using these organisms. Fortunately overliming using calcium hydroxide at 

50 °C for 30 min with the removal of overliming-produced 

solids through centrifuge was found sufficient to detoxify 

the whole slurry for effective fermentation. Maximal lipid 

yield of 0.15 g/g sugar at lipid concentration of 18.6 g/L was 

achieved from the detoxified whole slurry using Mortierella 

isabellina NRRL 1757.

In the second study (Dien and others 2016), two yeast 

strains, L. tetrasporus NRRL Y-11562 and Y. lipolytica

NRRL YB-437, both from the Agricultural Research Service 

(ARS) Culture Collection, Illinois, were used to ferment 

two SPORL-pretreated Douglas-fir residue whole slurries 

produced using a low sulfite loading with and without pHprofiling (Cheng and others 2015). Using two multiple batch 

culture schemes, lipid yield of 0.174 g/g sugar at titer of 

13.4 g/L and 0.104 g/g at titer 18.1 g/L were achieved without detoxifying the SPORL whole slurry sugars.

0.1

0.4

0.6

Yeast (mg/g)

Xylose (g/L)

30

32

34

26

28

24

22

0 40 80 200

Time (h)

120 160

(b)Mannose (g/L)

0.1

0.4

0.6

200

Yeast (mg/g)

6

8

10

4

2

0

0 40 80

Time (h)

120 160

(c)Glucose (g/L)

60

50

70

80

30

20

40

10

0

Ethanol (g/L)

60

50

70

80

30

20

40

10

0

0 40 80 200

Time (h)

120 160

(a)

0.1

0.4

0.6

Glucose Ethanol Yeast (mg/g)
General Technical Report FPL–GTR–240

24

6.7 Production of Iso-Butanol and Bio-Jet 

from a Softwood Forest Residue

Attempts were made to produce biofuels other than ethanol from SPORL-pretreated softwood forest residue. With 

the financial support of a USDA-NIFA project (No. 2011-

68005-30416) through the Northwest Advanced Renewables 

Alliance (NARA), SPORL-pretreated forest residue has 

been used to produce iso-butanol with subsequent conversion to isoparaffinic kerosene (IPK), or bio-jet. Approximately 60 t of Douglas-fir forest residue were pretreated 

using SPORL at an industrial facility of ZeaChem in Boardman, Oregon. The pretreated materials were shipped to 

an industrial saccharification and fermentation facility of 

ICM in St. Joseph, Missouri, to produce iso-butanol using 

a genetically modified yeast developed by GEVO. The isobutanol will be then shipped to South Hampton Refining in 

Silsbee, Texas, to produce approximately 1,400 gallons of 

IPK. The IPK will be blended with petroleum jet using an 

ASTM-certified process for a commercial flight by Alaska 

Airlines in 2016. Laboratory studies at various scales produced excellent yield of iso-butanol by GEVO. Iso-butanol 

itself is a drop-in biofuel and a valuable building block for 

producing various chemicals.

7. Summary

SPORL as a pretreatment process demonstrated robust performance for bioconversion of very recalcitrant lignocelluloses such as woody biomass and especially softwood forest 

harvest residues. Lignin sulfonation reduces nonproductive 

binding of cellulase to lignin and facilitates the processing 

of the pretreated whole slurry without solid and liquor separation or solids washing, even at high solids loadings. Specifically, the sulfonated lignin on solid substrate is relatively 

hydrophilic and has low affinity to cellulase. Further reduction of nonproductive binding of cellulase can be achieved 

by conducting enzymatic saccharification at elevated pH 

around 5.5 through pH-induced electrostatic repulsion between cellulase and lignin on solid substrate. Moreover, 

the dissolved lignin as lignosulfonate is a surfactant and 

can enhance enzymatic saccharification. Low-temperature 

pretreatments can be designed using a combined hydrolysis 

factor (CHF) as a pretreatment severity indicator that can 

accurately predict hemicellulose dissolution and lead to 

reduced sugar degradation to furans for high-titer biofuel 

production without detoxification. Excellent ethanol yields 

of approximately 72% theoretical of available wood glucan, 

mannan, and xylan were achieved for softwoods through 

fermentation of the SPORL-pretreated whole slurry. The 

q-SSF fermentations generated high titers of over 40 g/L at 

relatively low enzyme dosages of 26 mL/kg wood without 

shear mixing during liquefaction of the pretreated solid substrates. SPORL is also effective for converting hardwood 

to biofuel at high titer without detoxification, despite the 

high acetyl group content in hardwood species. Dissolved 

lignin is a valuable co-product that can be directly marketed 

to improve biofuel production economics. Because SPORL 

was developed based on sulfite pulping, it is scalable for 

commercial production with low risks. Overall, SPORL 

has great commercial potential compared with competing 

technologies.

Acknowledgments

We acknowledge the USDA Forest Service (USFS) for providing initial seed funding and the USFS Woody Biomass, 

Bioenergy, and Bioproducts program (WBBB 2009) for 

initial development of the SPORL process. Major funding 

was also received from a USDA NIFA SBIR Phase I (No. 

2009-33610-19644) and II (No. 2010-33610-21589) project 

through Biopulping International, Inc., Madison, Wisconsin, 

and a USDA NIFA Competitive Grant (No. 2011-68005-

30416) through the Northwest Advanced Renewables Alliance (NARA). We also acknowledge Novozymes North 

America (Franklinton, North Carolina) for providing 

enzymes, Dr. Jerry Gargulak of LignoTech for providing 

commercial lignosulfonate, Drs. Bruce Dien and Ron Hector of USDA Agriculture Research Service, Peoria, Illinois, 

for providing Saccharomyces cerevisiae YRH400 and many 

technical discussions and collaboration. Dr. Zhu acknowledges the contribution by Fred Matt, Diane Dietrich, Kolby 

Hirth, Linda Lorenz, and Tim Scott (all from USFS Forest 

Products Laboratory) for carbohydrates, ethanol, sulfur, and 

molecular weight measurements and other laboratory work. 

Gratitude is also due to Profs. Troy Runge and Xuejun Pan 

of the University of Wisconsin–Madison for allowing us 

access to their laboratory facility, technical discussions, and 

friendship over the years. Dr. Zhu also acknowledges the 

dedication of colleague Rolland Gleisner and many years 

of friendship; Dr. Jose Negron of USFS Rocky Mountain 

Research Station for harvesting mountain pine beetlekilled lodgepole pine trees, collaboration, and excursions 

to National Forests; Dr. Ronald Zalesny of USFS Northern 

Research Station for collaboration on poplar studies; Prof. 

Donald Rockwood of University of Florida on Eucalytus 

study; Prof. John Sessions of Oregon State University 

and the Feedstock team of the NARA Project and Drs. 

Johnway Gao and Dwight Anderson of Weyerhaeuser for 

Douglas-fir forest residue study. Last but not the least, Dr. 

Zhu acknowledges many international visiting students and 

scholars (including funding from their home countries) and 

post-doctorates whose dedication, hard work, and technical 

contributions made the development of the SPORL process 

to the current stage possible, especially Dr. Gaosheng Wang 

of Tianjin University of Science and Technology, Tianjin, 

China, who conducted the initial feasibility study of the 

SPORL process.
Bioconversion of Woody Biomass to Biofuel and Lignin Co-Product Using SPORL

25

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