Research Article

Received: 8 September 2013

Revised: 1 December 2013

Accepted article published: 6 December 2013

Published online in Wiley Online Library:

( DOI 10.1002/jctb.4286

SO2-catalysed steam pretreatment of quinoa
Cristhian Carrasco,a,b Diego Cuno,b Karin Carlqvist,a Mats Galbea
´ a∗
and Gunnar Liden
BACKGROUND: Quinoa is a pseudo-cereal grown predominantly in South America. The quinoa stalks are lignocellulosic residues,
which have a limited use today. The objective of the current study was to assess the potential of this material as a source of
monosaccharides for fermentation purposes by means of steam pretreatment giving sugars from the hemicellulose part, and
enzymatic hydrolysis of the solid fraction obtained. SO2 catalysed steam pretreatment was carried out with a holding time of
5 min at temperatures between 180 and 220◦ C. The pretreatment was carried out at two different scales, a small reactor of size
0.5 L and a somewhat larger reactor of size 10 L, to allow comparison of scale effects in the pretreatment.
RESULTS: The highest xylose yield in the liquid phase, obtained after pretreatment at 210◦ C, was 80%. In the smaller scale unit,
longer residence times were needed. The enzymatic hydrolysis, at an enzyme loading of 15 FPU g-1 glucan and a WIS loading of
2%, resulted in a glucose yield of 70% based on the original glucan. The overall sugar yield, including the xylan hydrolysed in
the enzymatic treatment, at dilute conditions was 75%.
CONCLUSIONS: SO2 catalysed pretreatment of quinoa straw followed by enzymatic hydrolysis gave a relatively good sugar
yield. However, the yield obtained was somewhat lower than previously reported for similar materials, such as wheat straw and
sugarcane bagasse, steam pretreated with SO2 .
c 2013 Society of Chemical Industry 

Keywords: xylose; quinoa stalks; steam pretreatment; SO2

Quinoa (Chenopodium quinoa Willd.) is a colourful pseudo-cereal,
which can be grown from sea level up to an elevation of 3900 m.
The quinoa is very resistant to drought and large temperature
variation, and it has been grown in the Andean region of South
America for many thousands of years.1,2 The genetic variability
is very large and more than 2500 mutant varieties of more than
170 different species are known.3 The quinoa grain contains more
protein than most other grains (about 15%), and is particularly
rich in the essential amino acid lysine, but also threonine and
methionine.4 The grain does not contain gluten, which makes it
suitable as an alternative to wheat for people sensitive to gluten.
The interest in the crop has increased outside the cultivation
region as the nutritional value has become more widely known,
and 2013 was named ‘The international Year of the Quinoa’ by the
United Nations. All these factors have increased the market value
of quinoa, which is now sold at prices several-fold higher than
soy-bean or wheat.5 The main producers in the world are currently
Bolivia and Peru, with a combined production of 70 000 tons in
2009. Bolivia is the leading exporter of quinoa and the export
value of quinoa for Bolivia exceeded US$40 million in 2009.6 The
increase in production has been quite substantial in recent years,
and cultivation of quinoa is now also made in other areas than the
The quinoa grain contains saponins, which are bitter-tasting
and toxic. Saponins are glycosides of two kinds based on the
aglycone part.8 Most common are the triterpenoid glycosides
J Chem Technol Biotechnol (2014)

but there is also a group of steroid glycosides.9,10 In quinoa, the
saponins are of the triterpenoid type. Before export, the seed coats
(‘mojuelo’), which contain most of the saponins, must be removed
by mechanical and washing processes. The saponins, however,
have a potential value on their own due to their foaming properties
at very low concentrations, which can be used in shampoos, beer
and detergents.11 The biological activity is also of interest, e.g. their
anti-inflammatory and antibacterial/antimicrobial properties.8 In
the context of creating maximum value for the quinoa, not only
the grain is therefore of interest but also the saponins, oils and
colourings could add value. The growing use of quinoa also results
in lignocellulosic waste biomass, seed coat and stalks left in the
field when quinoa grains are harvested. The quinoa stalks are
cylindrical and porous, and their coloration varies according to
genotype. This waste material is commonly used as firewood in
the Bolivian Altiplano.12 Due to the presence of saponins, the
stalks are not well suited as cattle feed, but other uses should

Correspondence to: Gunnar Lid´en, Department of Chemical Engineering, Lund
University, Lund, Sweden. E-mail:

a Department of Chemical Engineering, Lund University, P.O. Box 124, SE-221 00,
Lund, Sweden
b Instituto de Investigaci´on y Desarrollo de Procesos Qu´ımicos, Ingenier´ıa
Qu´ımica, Plaza del Obelisco N◦ 1175, Universidad Mayor de San Andr´es,
La Paz, Bolivia

c 2013 Society of Chemical Industry 

210 and 220◦ C. and the subscript ‘am’ is the peak for the amorphous portion (which includes the amorphous part of cellulose.21 and β-glucosidase activity by the procedure of Berghem and Pettersson. 72 and 96 h for carbohydrate analysis by HPLC. and 15 FPU g−1 glucan.8 and 5.5% w/w. Prior to pretreatment. the slurries were cooled by flashing to atmospheric pressure and subsequently separated into two fractions. e.7 mm) using a portable sieve shaker. Quinoa material was also pretreated in a smaller pretreatment unit located at IIDEPROQ. This procedure was repeated twice for each condition. with an SO2 content of 2. 10.5 L hydrolysis vessels equipped with stirrers operating at 300 rpm.13 The use of quinoa stalks for biogas production using a co-digestion process with aquatic flora from the Titicaca region and manure was studied by Alvarez and ´ 14 The substrate worked well in a co-digestion process.15 Furthermore.17 wheat straw (Carrasco et al. The instrument was calibrated using silicon as standard. The objective of the present work was to assess the use of quinoa stalks as a substrate for lignocellulose-derived sugars. 4. La Paz.. The method chosen was SO2 catalysed steam pretreatment.5% w/w.5 L added corresponded to cellulase activities of 5. for which suitable conditions have previously been developed in our laboratory for several other straw materials. After hydrolysis. hemicellulose and lignin) at 2θ = 18.2% H2 SO4 by soaking (solid:liquid ratio of 1:20) in a 15 L plastic container equipped with a mixing agitator.soci. 74% and then pretreated at 190◦ C for 5 min. but Liden. 48. respectively. 2.3% was loaded into the reactor. unpublished). hydrolysate and fibre residue. The composition of quinoa stalk. the impregnated stalk was placed into a laboratory-scale hydrolysis reactor having a volume of 0. Germany) using an unfiltered Cu Kα radiation source and a rotating sample holder. Both enzyme preparations were kindly provided by Novozymes A/S (Bagsværd. Denmark). 13. A second sample of quinoa stalks was also taken from a second location on the Altiplano near to Batallas and Pucarani.5. The crystallinity index (CrI) was determined from XRD and calculated using the formula:23 Crl = I002 − Iam × 100 I002 where the subscript ‘002’ refers to the intensity for the crystalline portion of biomass (cellulose) at 2θ ≈ 22. XRD patterns were recorded between 5 and 80◦ 2θ with a step width of 0. The SO2 impregnation and pretreatment of the material (300 g dry matter (DM) in each experiment) were carried out as described by Carrasco et al. sugarcane bagasse. In the work at Lund University. and a residence time of 5 min. Milled stalks were further sieved (1–1. Duplicate samples were withdrawn at 0. 190.g.5. Characterization of pretreated material Crystallinity X-ray powder diffraction (XRD) patterns of both unpretretated and pretreated quinoa stalk were performed over a Seifert XRD 3000 TT diffractometer (Seifert. and the amount of Novozyme 188 added corresponded to a β-glucosidase activity of 21 IU g−1 glucan (a total β-glucosidase activity of 25 IU g−1 of glucan). La Paz. The amount of Celluclast 1. The collected stalks were first dried and then size reduced by hammer-milling. The prepared feedstock was stored at 4◦ C until used for acid impregnation and steam pretreatment. A two factorial experiment was made with the temperature levels 190 and 200◦ C. nitrates and oxalates in the stalks.www. Liquid fractions were separated by filtration and subsequently stored at −18 ◦ C before analysis. by filtration. residence time 5 or 10 min and SO2 wileyonlinelibrary. the pretreatment of the material aiming to hydrolyse the hemicellulose sugar fraction was investigated. The filter paper units (FPU) were determined according to Ghose et al. The samples were prepared and impregnated as explained above. Steam pretreatment Pretreatment of quinoa were made both at Lund University. 68◦ W Longitude. 200. The quinoa stalks were pretreated at temperatures of C Carrasco et al.2 g of quinoa with a moisture content of 74. Sweden and at IIDEPROQ.18 The pretreated materials were collected and subsequently separated by filtration. Furthermore. Ahrenburg. a 10 L reactor (with a maximum operating pressure of 28 bar) fitted with a quick-opening ball valve20 was used. Germany) to separate the supernatant and the solid fraction. The total weight was adjusted to 100 g by addition of 0. the milled stalks were extensively washed in a concrete mixer with hot water in order to remove as much as possible of the saponins and other residues. Wehingen. Prior to enzymatic digestion.2◦ (according to most publications). 24. dewatered to a MC of approx.05◦ and 10 s per step at room temperature. Also the use of the substrate for enzyme production has been investigated. The tube voltage and the current were set to 40 kV and 40 mA. c 2013 Society of Chemical Industry  J Chem Technol Biotechnol (2014) .5 L (with an activity of 65 FPU g−1 and β-glucosidase activity of 17 IU g−1 ). novel enzyme producing fungi have been isolated from quinoa. In this case the stalk material was impregnated with 0. HERMLE Labortehnik.22 The enzymatic hydrolysis was made using 2% WIS (10 g of DM pulp) in 0. which in turn can be further used in ethanol production or other fermentative processes. This was made in order to compare the pretreatment performance at different scales. steam pretreatment of quinoa stalks has not been reported yet. the slurries were diluted with MilliQ-water (slurry: water ratio of 1:4) and then centrifuged for 3 min at 2214 × g (Z 513 K. Enzymatic hydrolysis (EH) of the water insoluble solid (WIS) pulps was performed using Celluclast 1. impregnation at 2. paja brava18 and also for spruce. One pretreatment was made with H2 SO4 as catalyst. using a total filling weight of 500 g. at an altitude of 3800 m above mean sea level).org be looked for.16 However. and supplemented with the β-glucosidase preparation Novozyme 188 (activity 376 IU g−1 ). in particular its carbohydrate content was for this reason analysed. The supernatant was then replaced with fresh MilliQ-water and the procedure was repeated four times for each sample. The vessels were kept in a water bath at two temperatures of 37 and 51 ◦ C. The quinoa stalks were then dewatered to a moisture content (MC) of approximately 74% using a hydraulic press.5 L and equipped with a flash collector tank and a steam generator. 6. run at 400 rpm for 1 h. Enzymatic hydrolysis The pretreated fibre material from La Paz was assessed for its digestibility by enzymatic hydrolysis.19 MATERIALS AND METHODS Preparation of quinoa stalks The material used in Lund was 20 kg of quinoa stalks collected from farmers fields near Lake Titicaca (17◦ S Latitude. Bolivia. In these experiments. 4. Following impregnation.1 mol L−1 acetate buffer. the biogas yield using quinoa stalks on its own was relatively low. UMSA.1.0 or 2. The acetate buffer solution was prepared at three pH values of 4. Earlier studies showed the presence of alkaloids.

The HPLC system was an Agilent 1100 Series (Palo Alto.24 The acid-soluble lignin was determined by absorbance at 240 nm. The aluminium cups were pelletized using a hydraulic press to obtain a tablet (40 mm). Germany). formic acid.1 0. USA).6 mm. Hercules.7 0. USA). The measurements were made in duplicate. Samples were washed and dried at 30 ◦ C for 48 h and finally sputter-coated with a thin layer of gold (Balzers SCD004 sputter coater) prior to imaging.4 19.6 mL min−1 eluent of 5 mmol L−1 H2 SO4 using high-performance liquid chromatography (HPLC).01 141.5 µm) protected by a Kromasil C18 guard-column (Scantec Lab.7 15.02 88.4 ± 0.9 17.00 11.2 0. Tokyo.5 4.2 0. The structural carbohydrates.00 5. For total nitrogen the conventional micro N-Kjeldahl was used.3 a The material was collected from near Lake Titicaca (La Paz.00 12. USA).20 µm sterile filters. 3.G1329A (Palo Alto.5 ± 0.6 3. USA). Composition of ash in quinoa stalks (Altiplano) Oxide mg per 100 g DM SiO2 Al2 O3 Fe2 O3 CaO MgO Na2 O K2 O SO3 P2 O5 SrO MoO3 MnO Cr2 O3 CuO Rb2 O ZnO Cl Br 479 ± 0.00 8.6 1.1 0. an auto-injector Agilent ALS .1 0. Karlsruhe.4 2. The measurements were performed at ambient conditions. HMF and furfural.00 90.1% formic acid/water (A) and acetonitrile (B).28 The protein content was estimated using a nitrogen factor of 6. which were quantified using the UV detector at 210 nm. total lignin and acetyl contents were determined according to the method described by Sluiter et al.01 1082 ± 0.4 3. comprising a vacuum degasser Agilent G1322 A (Palo Alto. The material was collected from farms on the Altiplano (La Paz. CA. Sugars were determined quantitatively on an Aminex HPX-87P column (Bio-Rad laboratories. Japan) at 5 kV and an average working distance of 8 mm.26 The WIS content was measured on pretreated stalks following the method proposed by Sluiter et al.2 2. CA. extractives and ash according to NREL standard procedures. CA. MA.0 19.01 187 ± 0. MA.6 5.0 37. Aluminium cups were filled with 1 g of wax and 10 g of the unpretreated milled stalks. USA). The column and guard column was thermostatically controlled at 20 ◦ C.5 ± ± ± ± ± 0. Bolivia).4 1.9 3.00 acetic acid.5 0.24 The sum of Klason lignin and acid-soluble lignin was reported as the total lignin content.1 2.0 0. Analysis of extractives and ash were made as described in Sluiter et al.5 0. Sample spectra were obtained in transmission mode by averaging 64 scans from 4000 to 400 cm−1 at a spectral resolution of 4 cm−1 . USA) equipped with a refractive index (RI) detector Waters 2410 (Milford. USA) and a diodearray detector Agilent G1315B (Palo Alto.00 9.00 47. S¨avedalen. CA. Analysis of inorganic elements (as oxides) in the quinoa stalks was made by X-ray fluorescence (XRF) spectroscopy (S8 Tiger X-Ray Spectrometer.7 2.2 0. The HPLC system used was a Waters 1515 (Milford.03 ± 0. Quantifications were made based on the RI detector signal with the exception for J Chem Technol Biotechnol (2014) www.27 Each assay was made in triplicates. levulinic acid.0 4. Hydrolysates were centrifuged at 16000 × g for 5 min and filtered using 0.5 0.00 2565 ± 0.2 ± ± ± ± ± ± 0.0 ± 0. Bolivia).4 ± 0.37 ± 0.25.8 ± 0. USA) and auto-injector Waters 717 plus (Milford. The solid materials were first carefully dried at 30 ◦ C and then ground with KBr (5 mg of powdered sample and 200 mg of KBr).29 For the quantification of sugars in all the enzymatic hydrolysates.4 0.83 ± 0. a solvent delivery system Agilent QuatPump G1311A (Palo .1 ± ± ± ± ± 0.2 0. Germany). Chemical composition The chemical composition of the solid materials (unpretreated and pretreated stalks) was determined with respect to carbohydrate and lignin content. Morphology Scanning electron microscopy (SEM) images were taken of untreated and pretreated quinoa stalks at magnifications of 100.7 ± 0. furfural and 5-hydroxymethylfurfural (HMF) were analysed using an Aminex HPX-87H column (Bio-Rad laboratories) at 60 ◦ C with 0.soci. CA.0 0.00 9. The mixture was poured into a mold of tableting and then pressed to obtain a transparent tablet. 65% A for 15 min at c 2013 Society of Chemical Industry  wileyonlinelibrary. and the gradient used was 75% A at 0 min.2 ± 0.4 ± ± ± ± ± ± 1.2 ± 0.00 15. MA.25.3 2.1 Table 1. MA.5 ± 0. Acetic acid. USA).00 16. Bruker. Ettlingen. The identification and quantification of saponins in the hydrolysate liquids were made using a Kromasil C18 reversedphase column (150 mm × 4. CA.58 ± 0.25 × N) Ash Titicacaa Altiplanob 35. The composition of the acid hydrolysates was determined using a post-hydrolysis treatment (because of the presence of sugars in oligomeric form) according to the method described by Sluiter et al. The xylooligosaccharides were determined using an Aminex HPX-42A column (Bio-Rad laboratories) under the same conditions. a column oven Agilent ColCom . USA). 5000 and 50 000 times using a JSM-6700 F field emission scanning microscope (JEOL. a direct chromatographic analysis was performed. USA) using MilliQ-water as the eluent at 0.2 0.0 0.5 4.0 0.0 0.6 mL min−1 at 85◦ C. Injection volume was set to 20 µL.02 127.00 709. an ultraviolet (UV) dual absorbance detector Waters 2487 (Milford. The mobile phase was a binary solvent system consisting of 0.G1316A (Palo Alto.6 ± 0.7 4. CA. Composition of quinoa stalks (g per 100 g DM) Component Oligosaccharides Glucan Xylan Arabinan Galactan Mannan Lignin Klason Acid-soluble Extractives Acetyl groups Protein (6.SO2 -catalysed steam pretreatment of quinoa stalks Surface chemistry Fourier transform infrared spectrophotometry (FT-IR) spectra were recorded on a Bruker IFS-66 spectrometer (Bruker Optics GmbH. Sweden). b Table 2.

calcium.3 7.7 1. The acetyl content was also slightly higher than for these materials.64 3.8 1.58 0.1 Figure 1.9 0.07 1.6 11.7 37. with a large fraction of water soluble extractives (about 10%).4 1.7 mL min−1 . but only 25% for quinoa stalks.8 –e a Steam pretreatments using 2.9 2.31 49.8 0.1 5.8 10.7 1.8 3. 0.5 1.17.9 65. respectively (Table 1).7 3.4 59.23 61. and silicon (cf.15 3.3 0.11 0.6 1.30 RESULTS AND DISCUSSION Composition of quinoa stalks The compositions of the two quinoa stalk materials used in the current study were in the expected range for a straw material.2 12.2 0.soci.9 2. but it was clear that the amount of extractives in the quinoa stalks was high.06 0.9 58.0 6.6 2.0 1.0 63.5 0.7 0. stalk.5 0. the fraction of monomers was even higher at lower severities.1 2.9 24.4 1.00 0.35 The H2 SO4 catalyzed pretreatment resulted in a high yield of soluble xylose in the liquid phase after pretreatment (79% of the theoretical).1 2.2 21.3 1. b Steam pretreatments using 0. Sugars and selected other products obtained in the liquid phase and pulp composition after pretreatment of quinoa stalks (Altiplano) (g per 100 g DM) Temperature (◦ C) 180a Time (min) log R0 c CrId(%) Hydrolysate Xylose Monomer Oligomer Total Arabinose Monomer Oligomer Total Glucose Monomer Oligomer Total Mannose Monomer Oligomer Total Galactose Monomer Oligomer Total Formic acid Levulinic acid Acetic acid Furfural HMF Pretreated pulp Glucan Xylan Galactan Klason lignin Acid-soluble lignin Acetyl groups 5 5 5 5 5 3.2% of H SO .3 6. Effect of steam pretreatment By varying the temperatures.8 2.02 1.74 0.8 15. wheat straw (Carrasco et al. analyses in Table 2). the hydrolysis and removal of hemicellulose from the material changed (Table 3).94 4.3 0.0 mL min−1 . and 55% A for 20 min at 1.8 0. 2 4 c Severity factor.3 190a 200a 210a 220a 200b 5 3.7 0.8 0.35 3.47 0.06 0.4 54.79 0.0 1.4 0.1 2.9 1.5 2.16 2.8 1. d Crystallinity index.1 8.6 4.6 2.7 0.06 1. However.04 0.5 0.9 0.2 10.1 0.7 0.6 1.2 3.9 1.9 0.4 1. The fraction of xylose monomers in the soluble xylose fraction was for example higher than 85% at T = 210 ◦ C for wheat straw. corn stover) for which values between 4 and 7% have been reported.64 66.7 11.2 3. Retention times and absorbance spectrum profiles were compared with standards of each identified saponin compound isolated in previous studies. sugarcane bagasse.4 0. i.8 1. The xylan content was somewhat lower than previously reported for similar type materials.7 0.4 4.9 2. To be noted was the high fraction of oligomers in the total ‘xylose’ found in the liquid fraction.6 59. in contrast to these materials also mannan and galactan were found in appreciable amounts in quinoa.33 0.05 0. The analysis was monitored at 210 nm and the absorption spectra of saponins were recorded between 190 and 550 nm.17 corn stover31 or paja brava. whereas the soluble xylose was merely c 2013 Society of Chemical Industry  J Chem Technol Biotechnol (2014) .4 1.98 0. For sugarcane bagasse.7 2.4 0.1 1.6 1.4 0.2 1.8 1. Quinoa stalks are rich in inorganic constituents.1 0.6 6. The fraction of xylo-oligomers was significantly higher than previously found for SO2 catalysed steam pretreatment of wheat straw at similar conditions.5 19.5 1.8 23.2 1.98 0.4 0.12 1. i.05 3.3 0.www.47 0.e.0 1. Similar values apply to paja brava.61 0.8 0. This is similar to other straw materials.3 2.1 1.32. All standard deviations are less than 5%.1 2.7 (80% of the xylan initially present) at the highest.6 7.1 2.4 53.8 0.7 2. also pretreated in the same manner.7 2.03 0.9 11.14 2.6 14.33 The amounts of extractives were analysed only for a few unwashed samples.5 1.4 0.0 0.2 0.7 67.09 0.1 0.9 g per 100 g DM (corresponding to 40% of the xylan initially present) at the lowest and 15. Table 3.18 –e 1.18 The pretreatment of corn stover was studied extensively by Elander et al.4 0.8 3.8 2.3 2.2 1.34 Quinoa stalks also contain rather high amounts of chlorine (Table 2). including also SO2 catalysed pretreatment as well as H2 SO4 catalysed pretreatment in a similar temperature range as the current work. X-ray diffraction patterns measured on quinoa material and solid fraction from pretreated quinoa.7 1.e.5 1.5 2. e Below detectable level.7 0.24 –e 1.9 2.6 64.15 1. The amount of total xylose found in the liquid fraction was 7.5% of SO2 . The ash content was similar to what has been reported for untreated straw or agricultural waste (like wheat straw.7 1.4 C Carrasco et al. submitted).7 1. a xylan content of 18 g and a lignin content of 23 g per 100 g of dry wileyonlinelibrary.5 3.9 23.1 1. in particular potassium.5 8.4 1.5 4.2 0.18 Arabinan was found in amounts similar to wheat straw and paja brava.

e. Yields of sugars in the pretreatment liquid after pretreatment of quinoa stalks (Titicaca) Pretreatment conditions T SO2 (◦ C) (%) 190 190 190 190 200 200 200 200 2.48 3.8 56.5% SO2 C Log R0 = 3.0 1.64.5 83.5% SO2 The arabinose was.6 1.6 Table 4.7 L) at IIDEPROQ (Bolivia). The X-ray measurements of the solid fraction after pretreatment show an increased crystallinity (Table 3 and Fig.9 67. and is somewhat higher than found for wheat straw (Carrasco et al.soci.35 1.7 55.05.23. found predominantly as monomers at a temperature of 200 ◦ C and above.8 6.65 3.8 76. 2.08 2.0 39.90 galactose (g per 100 g) (%) 15. in accordance with what was previously found for wheat straw (Carrasco et al.96 0. The solid fibre fraction remaining after pretreatment holds substantial amounts of xylan . Carrasco et al.64 3.8 61.4 69.8 1. like H2 SO4 .50 2.8 1.98 13.17 0.36 However.0 33. and stays below 1 g per 100 g DM.75 0.94 arabinose (g per 100 g) (%) 0.5 0.96 0. However.35 3.27 3.17 9. B Log R0 = 4.8%) than the H2 SO4 catalyzed case (82.0 19.0 2.66 1.7 3. J Chem Technol Biotechnol (2014) c 2013 Society of Chemical Industry  wileyonlinelibrary.3 51.3 68. This is in contrast to what is found when using a more acidic catalyst. 210 ◦ C. For corn stover.56 0. In accordance with what has previously been found for a sugarcane bagasse.3 glucose (g per 100 g) 0. for which substantially higher amounts of the sugar degradation products are found.2 45.8 3.0 2.36 6.SO2 -catalysed steam pretreatment of quinoa stalks www.32 13.almost 5% xylan even at the most severe pretreatment conditions. and paja brava. (submitted 2013) report for example values above 2 g per 100 g DM for wheat straw at T = 190 ◦ C.44 1.7 3.94 3.5 0.5 t (min) Yields of sugars after steam pretreatment log R0 5 10 5 10 5 10 5 10 xylose (g per 100 g) (%) 0.9 73.8%).64 1. This may be explained by the position of the arabinose units in the hemicellulose.83 1.78 3..9 3.5 2. 0. where they typically branch of a main backbone of xylan.79 2.5 2.0 52. H2 SO4 as catalysts at 200 ◦ C for 5 min (C). This is likely due to the removal of the hemicellulose part. however. 37% for SO2 catalyzed pretreatment. SO2 as catalyst at 180 ◦ C for 5 min (A) and at 220 ◦ C for 5 min (B).8%).5 The pretreatments were made using quinoa stalks from Lake Titicaca and using a steam reactor (0.60 1.45 1.2 24.07 0.51 0.67 10. submitted 2013). a remaining xylan content of 5% was reported after pretreatment at similar conditions (SO2 3%.1 17. FTIR transmittance spectra from quinoa material and solid fraction from pretreated quinoa.1 88. All sugars were measured respect to total sugars (monomers + oligomers). SEM images of pretreated quinoa.10 0. 1).70 2.65 3. A Log R0 = 3.68 1.42 .3 2.2 19.3 37.51 1.6 19.0 2. i.32 Mannose (g per 100 g) (%) 19. wheat straw.8 60. the overall xylose yield after enzymatic hydrolysis (at low WIS loadings) was higher for the SO2 catalyzed pretreatment (91. the amount of pentose degradation products.35 3.49 2. 5 min). 2. This can be compared with the xylan content of 18% in the untreated material.8 18.2% H2SO4 Figure 3.4 81.91 1. the removal was somewhat larger.71 1. primarily furfural (2-furaldehyde) is relatively minor in SO2 pretreatment.5 2. the initial xylan content in the corn stover was higher (22.71 0. 220 °C 1735 210 °C 1510 200 °C 190 °C 180 °C Unpretreated stalk 2400 2200 2000 1800 1600 1400 1200 1000 800 Wavenumber [cm-1] Figure 2.0 2.77 2.64 3.78 (%) 1. unpublished).0 76.41 0.99 3.

7 19 21 31 37 43 66 43 51 49 1. The absorbance at this wavenumber is due to the C–O stretching absorption of the acetyl groups and other carbonyl groups of carboxylic acids.5%.1 4.1 c 2013 Society of Chemical Industry  J Chem Technol Biotechnol (2014) .34 The peak at 1510 cm−1 is a characteristic band for the aromatic skeleton of lignin.5 7. however.1 4.8 13.4 0.7 7.9 28. Reported values after 96 h hydrolysis are given.1 4.5 7.7 15.0 9.5 41.5 1. 33.3 13.8 9.1 4. wileyonlinelibrary.4 0.7 0.7 20. where small droplets appear on the surface (Fig. SO2 = 2.1 14. whereas in La Paz a factorial design was used in the lower temperature range. T = 190◦ C.2 10.3 14. Enzymatic hydrolysis of pretreated quinoa stalks (Titicaca) Hydrolysis conditions T ◦ ( C) Yields of sugars after enzymatic hydrolysis Enzymes −1 (FPU g glucan) (g per 100 g DM) (%) Overall sugars yield (g per 100 g DM) Overall sugar (%) yield pH Glucose Xylose Glucose Xylose Glucose Xylose Glucose Xylose (%) 51 51 51 51 51 51 51 51 51 5 10 15 5 10 15 5 10 15 4. i.0 19.www. low in all hydrolysates (data not shown).7 1.5 0.6 16.2 hydrolysis of xylan was very low when operating at a residence time of 5 min in La Paz.8 3. SO2 = 2.7 0.5 0.9 13. Table 6.5% and T = 200◦ C. when the residence time was prolonged to 10 min. a degree of hydrolysis of the xylan more similar to the results from the larger unit in Lund was obtained.5 17.2 3.8 4.2 14.6 19.2 23 26 35 41 48 71 47 56 54 79 79 79 80 81 83 80 81 81 40 42 49 53 58 74 57 63 62 37 37 37 37 37 37 37 37 37 5 10 15 5 10 15 5 10 15 4.0 0.39.1 4. The material used was the washed fibre fraction after pretreatment at 200 ◦ C. A recent study has shown that aqueous extraction is not particularly efficient for removing saponins from quinoa seeds and a removal of only 32% was reported.1 26.5 14.e.7 2.0 14.5 8.3 18.1 14.3 16.37 The reason was that these compounds could not be extracted successfully – possibly due to the presence of polar ‘sapogenins’ or other ‘unknown’ compounds interfering the extraction.9 3. Table 5.3 0.9 14.5 0.1 14.8 5.6 3.0 26.34.2 24. the amount of saponins were.0 3.6 0.7 22.4 0. the relative heat capacity of the reactor material itself is large in comparison with the steam supply. 3(A)).8 4.0 9. giving a xylose removal of about 80%.1 14.5 2. It is likely that saponins remaining in the material going into the pretreatment reacted further to form sapogenins. Some studies have attributed this to a result of an initial lignin melting and subsequent precipitation on the surface of the pretreated biomass. Composition of quinoa pulp pretreated at 200 ◦ C for 10 min with 2.8 13.1 15. Two treatments were the same.38 In the present work.5% SO2 (Titicaca) Compound g per 100 g DM Glucan Xylan Galactan Lignin Unknown C Carrasco et al.5 17. A wider range of pretreatment temperatures was examined in pretreatment experiments in Lund.8 5.soci. 2.0 14. The decreased transmittance in the pretreated samples agrees with the increase in the amount of lignin due to the removal of hemicellulose.5 5.3 0.5 5.5 12.2 19. in the sense that Surface chemistry and morphology A notable change in the FTIR spectrum occurs around 1740 cm−1 in the transmittance spectra as the pretreatment severity is changed (Fig 2).8 0. time = 5 min.4 3.5 5. 3(C)).6 1.8 13. The H2 SO4 -pretreatment had another effect on the material morphology.9 16.8 18.37 The morphological changes in the surface of pretreated quinoa stalks were observed using SEM (Fig.5 5.8 4.5 0. and the change indicates removal of these groups in agreement with increased acetate in the liquid fraction (Table 3).1 2.0 14.1 4. 3).9 14.6 0.9 4.3 13.9 20.6 0.2 14.6 5. time = 5 min.0 18.0 21. whereas the fibre structures of the harshest pretreated stalk were clearly disrupted (Fig. This allows a comparison to be made of the pretreatment performance at the two different scales.8 4. 3(A). The pretreatment results from La Paz (Table 4) deviated from those in Lund. 10 min.7 2.2 12.40 The cell-walls also appear damaged in relation to Fig.3 14.1 16.2 23 23 36 38 40 67 44 50 53 79 79 81 80 80 82 80 81 81 40 40 49 51 53 71 55 59 61 All experiments were made at a solids loading of 2%. A relatively intact fibre was found at the mildest pretreatment condition (Fig.0 14.3 13.e.1 0.2 4. However.7 18 18 31 34 36 62 40 45 48 2. 3(B)).4 Aromatic compounds dissolved in the liquid phase could not be identified in the current work using methods reported in previous work. which in turn gives problem in the extraction of aromatic compounds from the pretreated liquid.5% SO2 .2 7. Most likely the difference between the pretreatment units indicates a slower heat transfer in the small scale reactor – i.8 4.

The authors are also grateful to Mariano Massa. and Functional Properties. New crops. Penarrieta M. Zacchi G and Liden steam pretreatment and fermentation of enzymatically hydrolyzed sugarcane bagasse. La Paz (200 ◦ C. in Pulses. Enzymatic hydrolysis The material that gave the highest xylose removal in the pretreatment made at IIDEPROQ. The highest yield was obtained at a high enzyme loading (15 FPU g−1 glucan) and at a temperature of 51 ◦ C. ´ E. The composition of the pretreated stalk is shown in Table 5. Nitrates. New York (1993). Phytochem Rev 8:473–490 (2009). Glucose and xylose hydrolysis yield measured during enzymatic hydrolysis of pretreated quinoa. Academic Press. Regional Office for Latin America and the Caribbean. in Advances in Food and Nutrition Research. 4 Stikic R. A cellulolytic Hypocrea strain isolated from South American brave straw produces a modular xylanase. The overall sugar yield (Table 6) is below 75%. Vucelic-Radovic B. 3 Gomez-Pando LR and Eguiluz-de la Barra A. for help with some of the analysis. Biological activities and distribution of plant saponins. although with a slightly higher enzyme loading in the latter. Galbe M and Zacchi G. Coleman C. 147–158 (2007). Jovanovic Z. which can be compared with previously obtained values for wheat straw of 95% at similar conditions. J Chem Technol Biotechnol (2014) 1 Abugoch James LE. Baudel HM. La quinua y la kaniwa: cultivos andinos. 10 Flores Y. for 10 min with 2.-glucosidasas por cultivos Alvarez Aliaga MT. Rev Colombiana Biotecnol 13:66–72 (2011). Carbohydr Res 356:215–223 (2012). Berlin/Heidelberg. Sendelius J. Soto Hern´andez M. 1–31 (2009). and a WIS content of 2%.SO2 -catalysed steam pretreatment of quinoa stalks www. 17 Carrasco C. Instituto Interamericano de Ciencias Agricolas (IICA) (1979). Mendoza Mart´ınez GD. a pH of 4. J Cereal Sci 55:132–138 (2012). Wiley. Biological activities and chemistry of saponins from Chenopodium quinoa Willd. Hahn-H¨agerdal B. Quinoa: an ancient crop to contribute to world food security. ed by Kole C.8. 14 Alvarez R and Lid´en G. The situation for Quinoa and its production in southern Bolivia: from economic success to environmental disaster. sugarcane bagasse and paja brava. Garc´ıa Vel´asquez A and Mendoza Castillo MC. ˜ 12 Tapia M. Pilcher L. galactose and mannose. Department of Chemical Engineering.) with gamma radiation for use in breeding programs.42 Previous studies have shown that these sapogenins inhibit some enzymes. Two-step steam 19 Soderstr om pretreatment of softwood with SO2 impregnation for ethanol production. Galbe M and Liden G. Steam pretreatment and fermentation of the straw material ’Paja Brava’ using simultaneous saccharification and co-fermentation. 9 Sparg SG. 4). Under acidic conditions. Springer. Tejeda L. Solano C. Szengyel Z. in American Society for Agronomy. Sterner O and Almanza GR.) as an ingredient in bread formulations. The glucose yield must be regarded as relatively low for a WIS loading of only 2%. and also the enzymatic hydrolysis gave slightly lower yields than wheat straw.43. Baudel H. Glamoclija D.).10. 16 Cabero K. Enzyme Microbiol Technol 46:64–73 (2010). Colque O. J Biosci Bioeng 111:167–174 (2011). 5 Jacobsen SE. Lund University. Bonifacio A. JEthnopharmacol 94:219–243 (2004). Hydrolysis was made at a temperature of 51◦ C. J Agron Crop Sci 197:390–399 (2011). Waste Manage (Oxford) 28:1933–1940 (2008). Pozzo T. Jellen E. Milojkovic-Opsenica D. ed by Steve LT. ¨ ¨ J. oxalates and alkaloids in two phenological stages of quinoa (Chenopodium quinoa Willd) in irrigated and rainfed conditions. 18 Carrasco C.soci. R´ıos Manr´ıquez N. Demin M. AJPS 4:349–355 (2013). Garay F. Design and operation of a c 2013 Society of Chemical Industry  wileyonlinelibrary. Lid´en G and Karlsson EN. To be noted is the profound effect of pH giving a much higher yield at 4. Produccion ´ de bacterias termofilas ind´ıgenas del altiplano boliviano. SO2-catalyzed M. The worldwide potential for Quinoa (Chenopodium quinoaWilld. An overall glucose yield of up to 70% was obtained at the most favourable conditions (Table 6 and Fig. 6 FAO. CONCLUSIONS Quinoa stalks were found to have a composition similar to other straw materials. 20 Palmqvist E. but with the distinguishing feature that it contains appreciable amounts of many different sugars in the hemicellulose fraction: xylose. Tengborg C and Zacchi G.44 This might explain the somewhat lower enzymatic sugar yields obtained from quinoa stalks in comparison with wheat straw. Hahn-Hagerdal H. ed by Crops AftAoI. Chapter 1 Quinoa (Chenopodium quinoa ACKNOWLEDGEMENTS 80 Glucose Xylose The Swedish International Development Cooperation Agency (SIDA) is gratefully acknowledged for its financial support of this project. Rev Fitotec Mex 27:313–322 (2004).): Composition. Stevens M and Fairbanks D. Light ME and van Staden J. Stenberg K. Lopez Castaneta C. It is thus likely that the use of quinoa stalks as a fermentative feedstock would require somewhat more severe pretreatment conditions and result in slightly lower overall sugar yields than many other agricultural residues.5 L at an enzyme loading of 15 FPU g−1 glucan. Appl Biochem Biotechnol 98–100:5–21 (2002). Roslander C. for example α-glucosidases. There is a wide range of inhibitors which reduce the enzymatic digestions in pretreated feedstocks. The pretreatment gave a somewhat lower xylose extraction than previously found for several other similar materials. 11 Janick J and Simon JE. Centro Internacional de Investigaciones para el Desarrollo (CIID). Diaz C. The hydrolysis yield was found to be affected by all the variables studied.41. Anaerobic co-digestion of aquatic flora and quinoa with manures from Bolivian Altiplano. Agronomical and nutritional evaluation of quinoa seeds (Chenopodium quinoa Willd. Quinoa (Chenopodium quinoa).com/jctb .5 than at 4.8 and 5. Galbe ´ G. Developing genetic variability of quinoa (Chenopodium quinoa Willd. 8 Kuljanabhagavad T and Wink M. ´ ´ ˜ 13 Gutierrez Larrazabal A. Sugar and Tuber Crops. Food Rev Int 19:167–177 (2003). Galbe M. Roslander C. Jacobsen S-E and Milovanovic M. Modig T. Larsson M. saponins are converted to sapogenins. 2 Maughan P. Most of the xylan remaining in the solid fraction was hydrolysed during the enzymatic hydrolysis. Terrazas Siles E and 15 Casablanca Alarcon ´ ´ de &beta. Oleanane-type triterpenes and derivatives from seed coat of Bolivian Chenopodium quinoa genotype ’salar’. arabinose. FAO: UNFaAO. Chemistry.5% SO2 ) was examined for its digestibility in enzymatic hydrolysis. Hydrolysis yield [%] 70 60 50 40 REFERENCES 30 20 10 0 0 2 4 6 24 Time [h] 48 72 96 Figure 4. The enzyme used was Celluclast 1. Rev Boliv Quim 22:71–77 (2005). Nutritional.1. 7 Jacobsen S-E. 56 (2011).

Biomass Bioenergy 32:326–332 (2008). Templeton D and Crocker D. Bioresource Technol 100:3948–3962 (2009). Lemus-Mondaca R. Jakobsson E-L. An empirical method for estimating the degree of crystallinity of native cellulose using the x-ray diffractometer. Paton D. Colorado (2008). Saddler JN and Wyman CE. Eur J Biochem 2:295–305 (1974).) seeds. Determination of protein content in biomass laboratory analytical procedure (LAP) : issue date. Phytochemistry 60:295–299 (2002). Ghose TK. Ticona E. National Renewable Energy Laboratory. Cuantificacion de Saponinas en Residuos de Quinua Real Chenopodium Quinoa Willd. Mastebroek HD. Colorado (2008). Summary of findings from the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI): corn stover 33 34 35 36 37 38 39 40 41 42 43 44 C Carrasco et al. Sluiter J and Templeton D. 12/08/2006. Hames B. Ruiz R. Segal L. Ballantyne K and Aubin AA. Combustion properties of biomass. Sluiter J. National Renewable Energy Laboratory. Golden. Mitchinson C. Galbe M and Zacchi G. Determination of insoluble solids in pretreated biomass material laboratory analytical procedure (LAP) : issue date. Oleanolic acid . Sluiter A. Creely JJ. Sluiter A and Scarlata C. 7/17/2005. Colorado (2008). c 2013 Society of Chemical Industry  J Chem Technol Biotechnol (2014) . and degradation products in liquid fraction process samples laboratory analytical procedure (LAP) : issue date. Jahangir M. Bioresource Technol 58:171–179 (1996). Martin AE Jr and Conrad CM. National Renewable Energy Laboratory. Carrasco C. Mechanism of enzymatic cellulose degradation – isolation and some properties of a beta-glucosidase from Trichoderma viride. US Patent 6355249: 43 (2002). Payne C and Wolfe J. and Saddler J. A kinetic approach to saponin extraction during washing of quinoa (Chenopodium quinoa Willd. Amtul Zehra A. Rev Boliv Quim 29:128–135 (2012). Ruiz R. Text Res J 29:786–794 (1959). Mago G.soci. Thygesen L. JChemTechnolBiotechnol 87:1723–1725 (2012). Galbe M and Lid´en G. Bioresource Technol 98:2503–2510 (2007). Sachin Bharat A. Ruiz R. Golden. Sluiter J and Templeton D. National Renewable Energy Laboratory. and enzyme loadings for bioethanol production. Muir AD. Vega-Galvez A. byproducts. Ladisch MR. 4/25/2008. Ruiz R. Jørgensen H and Elder T. Golden. Ali MS. Felby C. Madhusudana K and Janaswamy Madhusudana R. J Food Process Eng 36:202–210 (2013).org 21 22 23 24 25 26 27 28 29 30 31 32 bench-scale process development unit for the production of ethanol from lignocellulosics. Ashok Kumar T. Linde M. Measurement of cellulase activities. Bura R. Ponnapalli Mangala G.www. Bura R. Penarrieta JM. Hyman D. Hames B. Flores Y and Almanza G. Hussan SSu and Choudhary MI. Baudel HM. Scarlata C. Berghem L and Petterson L. Kristensen J. Quispe-Fuentes I. Hames B. 07/17/2005. Occurrence of sapogenins in leaves and seeds of quinoa (Chenopodium quinoa Willd).an alpha . Steam pretreatment of dilute H2SO4-impregnated wheat straw and SSF with low yeast wileyonlinelibrary. Viswanadh V. Solano C. ¨ Ohgren K. Determination of extractives in biomass laboratory analytical procedure (LAP) : issue date. Cellulose 16:649–659 (2009). Jenkins BM. Chandra R. Lozano M and Ah-Hen K. Limburg H. Miranda M. National Renewable Energy Laboratory. Golden. Radhakrishnan SVS. Golden. Lee YY. Colorado (2008). 03/21/2008. National Renewable Energy Laboratory. Colorado (2008). Pure Appl Chem 59:257–268 (1987). Sluiter A. Biotechnol Biofuels 1:1–9 (2008). Dale BE. Baxter LL. Hames B. Holtzapple M. Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies. Sluiter A. Arabinosylated phenolics obtained from SO2-steam-pretreated sugarcane bagasse. Biotechnol Prog 25:315–322 (2009). Optimization of steam pretreatment of SO2-impregnated corn stover for fuel ethanol production. Saddler J and Zacchi G. ´ Lozano M. Miles Jr TR and Miles TR. Determination of sugars. Process for recovery and purification of saponins and sapogenins from quinoa (Chenopodium quinoa). Appl Biochem Biotechnol 121–124:1055–1067 (2005). Elander RT. Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover. Scarlata C. Determination of ash in biomass laboratory analytical procedure (LAP) : issue date. Determination of structural carbohydrates and lignin in biomass laboratory analytical procedure (LAP) : issue date. Inhibition of α-glucosidase by oleanolic acid and its synthetic derivatives. Influence of Xylan on the enzymatic hydrolysis of steam-pretreated corn stover and hybrid poplar. Cellwall structural changes in wheat straw pretreated for bioethanol production. Gilles T and Marvin HJP. Sluiter J and Templeton D.glucosidase inhibitory and antihyperglycemic active compound from the fruits of Sonneratia caseolaris Open Access J Med Aromat Plants 1:19–23 (2010). J Sci Food Agric 80:152–156 (2000). Galbe M and Zacchi G. Golden. Balan V and Wyman CE. Ohgren K. Fuel Process Technol 54:17–46 (1998). ˜ Carrasco C. Sluiter A. 7/17/2005. Kumar R. Sluiter A. Colorado (2008). Scarlata C. Scarlata C.