Barley straw, sugarcane bagasse and empty fruit bunches were pretreated under acid- and alkaline conditions. Solid phase was separated from the liquid phase and the concentration of dissolved monomeric sugars, organic acids and furanics was determined. Acid hydrolysis yielded monomeric xylose concentrations (max 20 g/l) whereas for alkaline hydrolysis less than 1 g/l was found. Organic acids and furanics were detected with high concentrations when dried empty fruit bunches were used. Acetic acid was found in all samples (max 3 g/l), concentrations were higher under alkaline conditions

Bakker, R.R.C.

Harmsen, P.F.H.

Lips, S.J.J.

Wageningen Yield 

PRETREATMENT OF VARIOUS FEEDSTOCKS FOR LACTIC ACID 
PRODUCTION; DETECTION OF SUGARS, ORGANIC ACIDS AND 
FURANICS IN LIQUID FRACTIONS 
Paulien Harmsen*, Steef Lips, Robert Bakker
 
Wageningen UR Food and Biobased Research, Bornse Weilanden 9, 6708 WG 
Wageningen, The Netherlands 
*Paulien.Harmsen@wur.nl 
ABSTRACT 
Barley straw, sugarcane bagasse and empty fruit bunches were pretreated under acid- and alkaline 
conditions. Solid phase was separated from the liquid phase and the concentration of dissolved 
monomeric sugars, organic acids and furanics was determined. Acid hydrolysis yielded 
monomeric xylose concentrations (max 20 g/l) whereas for alkaline hydrolysis less than 1 g/l was 
found. Organic acids and furanics were detected with high concentrations when dried empty fruit 
bunches were used. Acetic acid was found in all samples (max 3 g/l), concentrations were higher 
under alkaline conditions. 
I. INTRODUCTION 
The use of lignocellulosic biomass for biofuels has gained much interest the last few decades, but 
also chemical building blocks from renewable resources form a huge potential and in particular 
lactic acid for the production of the biodegradable plastic polylactic acid (PLA). To date, lactic 
acid is mainly produced from starch originating from corn, but a sustainable production process at 
a scale meeting future demands for PLA requires the use of second generation biomass such as 
lignocellulosics. 
Given the large amount of pretreatment methods and lignocellulosic biomass available, a fast 
screening method is necessary to set boundaries and to determine suitable conditions for the 
preparation of enzymatic hydrolysates for fermentation studies at larger scale. In this study three 
model feedstocks were selected, i.e. barley straw, sugarcane bagasse and empty fruit bunch, and 
pretreated by acid- and alkaline hydrolysis. After pretreatment the solid phase was separated from 
the liquid phase, and the composition of the fractions was analyzed. The concentration of 
dissolved monomeric sugars, organic acids and furanics (major inhibitors to enzymatic hydrolysis 
and fermentation to lactic acid) in the liquid phase is described here.  
II. EXPERIMENTAL 
Biomass: barley straw was obtained from the Netherlands, sugarcane bagasse from Thailand and 
empty fruit bunches from Malaysia. The empty fruit bunches were dried at 60 °C before shipment 
to the Netherlands to prevent microbial degradation. Dry matter of all feedstocks was >90%. 
Pretreatment: 0.1L-reactors (not stirred) were filled with biomass and water with a fixed L/S-ratio 
of 10. Acid hydrolysis was performed at 140° C for 30 min with H2SO4-concentrations ranging 
from 0-8 wt% on dry biomass. Alkaline hydrolysis was performed at 120 °C for 60 min with 
NaOH-concentrations ranging from 0-12 wt% on dry biomass. 
Analysis: monomeric sugars were detected on a HPAEC-PAD (Dionex Corporation, USA), 
organic acids and furanics on a HPLC (Waters Pharmaceutical Division, Milford, MA, USA). 
III. RESULTS AND DISCUSSION 
Acid hydrolysis 
Results of the acid hydrolysis of barley straw are presented in figure 1. Xylose concentration was 
maximal 19 g/l while the concentration of glucose and arabinose remained below 3 g/l. Based on 
the amount of xylose dissolved, hydrolysis of hemicellulose has taken place and expected 
degradation products include acetic acid and furfural. These components were indeed detected in 
the hydrolysis liquid as shown in figure 1, but also formic acid and succinic acid were found. 
HMF, a degradation product of glucose, may further degrade to formic acid and levulinic acid, and 
levulinic acid can further oxidize to succinic acid. Given the hydrolysis conditions it is possible 
that glucose degradation (from hemicellulose and/or cellulose) has taken place, but more research 
is necessary to validate the concentration of formic acid and succinic acid in the sample. 
 
Figure 1. Acid hydrolysis of barley straw. Concentration (g/l) of monomeric sugars (left) and 
organic acids/furanics (right) in the liquid phase. Rhamnose, galactose or mannose was < 1 g/l; 
lactic acid and levulinic acid were not detected, 5-HMF was < 0.1 g/l. 
 
For sugarcane bagasse the results are given in figure 2. The amount of dissolved sugars is 
comparable to barley straw with a slightly lower xylose concentration. The amount of acetic acid 
formed is lower (approximately 1 g/l) and the amount of furfural is comparable; no succinic acid 
was detected. 
 
Figure 2. Acid hydrolysis of sugarcane bagasse. Concentration (g/l) of monomeric sugars (left) 
and organic acids/furancis (right) in the liquid phase. Rhamnose, galactose or mannose was < 0.5 
g/l; lactic acid, levulinic acid and succinic acid were not detected, HMF  was < 0.1g/l. 
 
Acid hydrolysis of the dried empty fruit bunches resulted in a liquid with a very low xylose 
content (figure 3); only above 6 wt% H2SO4 the xylose concentration was more than 5 g/l. 
Probably sugar degradation had taken place and this was also confirmed by the amount of organic 
acids detected. High concentrations of formic acid, acetic acid and succinic acid were found; 
levulinic acid was also present in the liquid but the concentration decreased with increasing H2SO4 
content or increasing severity of the pretreatment. It is possible that levulinic acid is further 
oxidized to succinic acid. There is reason to believe that the drying process of the empty fruit 
bunches prior to the pretreatment was too severe and caused (partial) burning or degradation. 
Given the high dry matter content of >90% and the dark brown, black color of the bunches this is 
most likely; fresh bunches normally contain a lot of water (>70%). 
Of the acid pretreatments the combined severity factor R‘0 (Chum, Johnson et al. 1990) was 
calculated. This factor  involves the reaction temperature T (
o
C), reaction time t (min) and pH (to 
include catalytic effects of applied or released acids) and is described by the following formula: 
 
    [ 
 ]   (  
   ) (   
     
     ) 
0
5
10
15
20
0 1 2 3 4 5 6
C
o
n
ce
n
tr
a
ti
o
n
 (
g
/l
)
H2SO4 (wt% on dry fibre)
Xylose Glucose Arabinose
0.0
0.5
1.0
1.5
2.0
2.5
0 1 2 3 4 5 6
H2SO4 (wt% on dry fibre)
Formic acid Acetic acid Succinic acid Furfural
0
5
10
15
20
0.0 1.3 2.7 4.0 5.4 6.8 8.1
C
o
n
ce
n
tr
a
ti
o
n
 (
g
/l
)
H2SO4 (wt% on dry fibre)
Xylose Glucose Arabinose
0.0
0.5
1.0
1.5
2.0
0.0 1.3 2.7 4.0 5.4 6.8 8.1
H2SO4 (wt% on dry fibre)
Formic acid Acetic acid Furfural
 
Figure 3. Acid hydrolysis of empty fruit bunches. Concentration (g/l) of monomeric sugars (left) 
and organic acids/furancis (right) in the liquid phase. Rhamnose, galactose or mannose was < 0.5 
g/l; lactic acid, furfural or HMF  was < 0.2 g/l. 
 
Figure 4 illustrates the relation of the total sugar content in the liquid phase with the severity of the 
acid pretreatment. From this figure can be seen that the amount of sugars from the dried empty 
fruit bunches is lower at the same severity factor compared to barley straw or sugarcane bagasse. 
Furthermore it is clear that the severity factor should be above 0 to for the pretreatment to have 
some impact on the feedstock.  
 
Figure 4. Total sugar concentration (g/l) as function of the combined severity factor R’0 for acid-
hydrolyzed barley straw, sugarcane bagasse and empty fruit bunch. 
 
Alkaline hydrolysis 
For alkaline hydrolysis of barley straw the concentration of dissolved monomeric sugars is almost 
zero (<0.2 g/l), for sugarcane bagasse it is slightly higher (<2 g/l). It is known that for alkaline 
hydrolysis the main mode of action is dissolution of lignin, and to a lesser extent hydrolysis of 
hemicellulose to monomeric sugars. However, it is worthwhile investigating the content of 
oligomeric sugars as previous work showed that released sugars are mainly present as oligomers.  
The concentration of organic acids in alkaline pretreatment liquids for barley straw and sugarcane 
bagasse are presented in figure 5. Typical acid-catalyzed sugar degradation products like 5-HMF 
and furfural were not detected, and also levulinic acid was not found. The formic acid 
concentration was lower compared to acid hydrolysis but the acetic acid concentration was 
comparable or even higher. This illustrates that deacetylation of hemicellulose yielding acetic acid 
is best catalyzed under alkaline conditions. 
Alkaline hydrolysis of dried empty fruit bunches (figure 6) showed different results regarding the 
formation of organic acids, whereas the concentrations of monomeric sugars was comparable to 
barley straw and sugarcane bagasse (<0.1 g/l). For the organic acids high concentrations of 
levulinic acid (maximal 9 g/l) and succinic acid (maximal 3 g/l) were detected, indicating that the 
drying step prior to pretreatment caused degradation of the bunches. 
0
2
4
6
8
10
0.0 1.3 2.7 4.1 5.4 6.8 8.1
C
o
n
c
e
n
tr
a
ti
o
n
 (
g
/l
)
H2SO4 (wt% on dry fibre)
Xylose Glucose Arabinose
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.0 1.3 2.7 4.0 5.4 6.8 8.1
H2SO4 (wt% on dry fibre)
Formic acid Acetic acid Succinic acid Levulinic acid
0
5
10
15
20
25
30
-3 -2 -1 0 1 2
T
o
ta
l 
m
o
n
o
m
e
ri
c 
su
g
a
r 
co
n
ce
n
tr
a
ti
o
n
 (
g
/l
)
log R'0
Barley straw Sugarcane bagasse Empty Fruit Bunch
 
Figure 5. Alkaline hydrolysis of barley straw (left) and sugarcane bagasse (right). Concentration of 
organic acids (g/l). Levulinic acid, 5-HMF and furfural were not detected. 
Figure 6. Alkaline hydrolysis of empty fruit bunch. Concentration of monomeric sugars (left) and 
organic acids (right) in g/l. 5-HMF and furfural were not detected; formic acid was <0.1 g/l. 
IV. CONCLUSIONS 
Results of biomass pretreatment is dependent on a number of parameters, one of them is the type 
of biomass. Differences in amounts of cellulose, hemicellulose, lignin and structural differences of 
those like amount of acetyl groups will influence the results of the pretreatment. Further work will 
focus on combining the results described here with the composition of the raw biomass and 
composition of the solid phases obtained after pretreatment. Furthermore, drying of the biomass 
prior to pretreatment can have a major impact as shown here for the empty fruit bunches. 
Experiments will be repeated with fresh material as empty fruit bunches remain an interesting 
source of fermentable sugars for lactic acid fermentation. 
Type of pretreatment (acid or alkaline hydrolysis) and the applied conditions (time, temperature, 
catalyst concentration) have a major effect on the concentration of dissolved components in the 
liquid phase. Concentrations of xylose, furfural, HMF and hydrolysis yields can also be related to 
the combined severity factor in case of acid pretreatment. This severity factor can be used to 
predict the results of experiments done under various conditions.  
V. ACKNOWLEDGEMENT 
The authors would like to thank the BE-Basic project and Willem Spekking, Hans van der Kolk, 
Alniek van Zeeland, Jacinta van der Putten and Edwin van der Pol for performing the 
pretreatments and the analysis. 
VI. REFERENCES 
Chum, H., D. Johnson, et al. (1990). "Pretreatment-Catalyst effects and the combined severity 
parameter." Applied Biochemistry and Biotechnology 1990, 24-25(1), 1-14 (research article) 
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 2 4 6 8 10 12
C
o
n
ce
n
tr
a
ti
o
n
 (
g
/l
)
NaOH (wt% on dry fibre)
Barley straw
Formic acid Acetic acid Succinic acid
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 2 4 6 8 10 12
NaOH (wt% on dry fibre)
Sugarcane bagasse
Formic acid Acetic acid Lactic acid
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0 2 4 6 8 10 12
C
o
n
ce
n
tr
a
ti
o
n
 (
g
/l
)
NaOH (wt% on dry fibre)
Xylose Glucose Arabinose
0
2
4
6
8
10
0 2 4 6 8 10 12
NaOH (wt% on dry fibre)
Acetic acid Succinic acid Levulinic acid


Pretreatment of various feedstocks for lactic acid production: detection of sugars, organic acids and furanics in liquid fractions

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