Bioresource Technology 102 (2011) 9308–9312

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Bioresource Technology

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Short Communication

Dilute-sulfuric acid pretreatment of cattails for cellulose conversion

Bo Zhang ⇑, Lijun Wang, Abolghasem Shahbazi, Oumou Diallo, Allante WhitmoreBiological Engineering Program, Department of Natural Resources and Environmental Design, North Carolina A & T State University, 1601 East Market Street, Greensboro,NC 27411, United States

a r t i c l e i n f o

Article history:Received 18 March 2011Received in revised form 3 July 2011Accepted 5 July 2011Available online 18 July 2011

Keywords:BiomassCattailsDilute acid pretreatmentHydrolysisFermentation

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.07.008

⇑ Corresponding author. Tel.: +1 336 334 7787; faxE-mail address: bzhang@ncat.edu (B. Zhang).

a b s t r a c t

The use of aquatic plant cattails to produce biofuel will add value to land and reduce emissions of green-house gases by replacing petroleum products. Dilute-sulfuric acid pretreatment of cattails was studiedusing a Dionex accelerated solvent extractor (ASE) varying acid concentration (0.1–1%), treatment tem-perature (140–180 �C), and residence time (5–10 min). The highest total glucose yield for both the pre-treatment and enzyme hydrolysis stages (97.1% of the cellulose) was reached at a temperature of180 �C, a sulfuric acid concentration of 0.5%, and a time of 5 min. Cattails pretreated with 0.5% sulfuricacid are digestible with similar results at enzyme loadings above 15 FPU/g glucan. Glucose from cattailscellulose can be efficiently fermented to ethanol with an approximately 90% of the theoretical yield. Theresults in this study indicate that cattails are a promising source of feedstock for advanced renewable fuelproduction.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction partially dissolved, increasing cellulose susceptibility to enzymes

For a number of reasons, there has recently been increasinginterest in converting biomass to liquid fuels. Some of those rea-sons include limited availability and increasing demand for fossilfuels, especially in developing countries; increasing price; the needfor national energy independence and safety; and the need forreduction in greenhouse gas (GHG) emissions. To this end, the fed-eral government has been calling for research into ethanol produc-tion from a number of cellulosic sources. The most widelyinvestigated of these sources thus far have been corn stover andcrops grown specifically as energy crops, such as switchgrass andpoplars (Mosier et al., 2005; Kim et al., 2009; Li et al., 2010). How-ever, another viable feedstock could be aquatic plants. The aquaticplants under consideration in this paper are the Typha species,commonly known as cattails. Cattails have been identified as a par-ticularly suitable biomass crop for wetlands because of their supe-riority in productivity (40+ metric ton/ha standing crops), pestresistance, adaptability, and chemical composition (Pratt et al.,1988; Apfelbaum, 1985). The cattails have been used for phyto-remediation in constructed wetlands (Suda et al., 2009). Usingthe cattails as a feedstock for biofuel production will add valueto the land with little environmental impact.

The use of acid hydrolysis for the conversion of cellulose to glu-cose is a process that has been studied for the last 100 years. Diluteacid (0.5–1.0% sulfuric acid) pretreatment at moderate tempera-tures (140–190 �C) can effectively remove and recover most ofthe hemicellulose as dissolved sugars, and lignin is disrupted and

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(Yang and Wyman, 2004; Hsu et al. 2010; Shi et al., 2011a). Underthis method, glucose yields from cellulose increase with hemicellu-lose removal to almost 100% (Knappert et al., 1981).

In our previous study, when a 1 L Parr reactor was used to treatcattails with dilute sulfuric acid, about 42.3% of the cellulose (rawcattails basis) was dissolved into soluble form (Zhang et al., 2009b).The heating rate of the Parr reactor used for the pretreatment wasslow (4 �C/min). It has been reported that heating rate, as governedby the mode of heat transfer, is an important factor affecting thehydrothermal treatment process (Zhang et al., 2008, 2009a). Forthis study, in order to minimize the heat-transfer-related artifactsand optimize the pretreatment processes, cattails underwent a di-lute acid pretreatment process in an accelerated solvent extractor,which provides a heating rate of 25 �C/min. Saccharomyces cerevisi-ae (ATCC 24858) was then used to test the fermentability of glu-cose enzymatically degraded from cattail cellulose.

2. Methods

2.1. Materials

The aerial portions of cattails, Typha latifolia, were chopped withpruning shears, dried at 70 �C for 5 days, and ground in a Wileymill to 1 mm mesh size.

2.2. Biomass analytical procedures

Compositional analysis of biomass was carried out using thelaboratory analytical procedures (LAPs) developed by the National

B. Zhang et al. / Bioresource Technology 102 (2011) 9308–9312 9309

Renewable Energy Laboratory. The moisture content of the bio-mass was determined by the LAP #001 method, and the ash con-tent of the biomass was determined by the LAP #005 method.Structural analyses of the samples were carried out according tothe LAP #002 method. The composition of cattails and pretreatedcattails is listed in Table 1.

2.3. Pretreatment of the feedstock

A Dionex ASE 350 Accelerated Solvent Extractor (Dionex Corpo-ration, Sunnyvale, CA) was used for dilute acid pretreatment of thebiomass below 180 �C. Approximately 2–3 g of ground biomass(composed of cattails) was placed into a tared 66 mL Dionexextraction cell containing a glass fiber filter. Then the appropriatenumber of 150-mL collection vials were weighed and placed ontothe ASE system. The extractor passed 60 mL sulfuric acid into thecell containing biomass. Then the cell was heated to the desiredtemperature (140–180 �C) at a heating rate of 25 �C/min, and thedesired temperature was maintained for 5–15 min. After pretreat-ment, 40 mL of sulfuric acid was passed into the cell to rinse thebiomass and wash out the extractable products. The resultingextractive and the rinsing solution (total about 100 mL) were col-lected in the collection vials. The extraction cell was cooled downto 25 �C by sitting at room temperature for 30 min. The sulfuricacid pretreated biomass was filtered using a 12.5 cm diameterWhatman No. 1 filter paper in a Buchner funnel. Clean deionizedwater was washed through the filter cake until the filtrate pHwas at least 6.

The yield percentage of each fraction from pretreatment is de-fined as:

Table 1Biomass compositiona,b of pretreated cattails.

H2SO4 concentration Treatment temperature (�C) Treatment time

Unpretreated cattails (%)0.1 140 50.1 140 100.1 140 150.1 160 50.1 160 100.1 160 150.1 180 50.1 180 100.1 180 150.5 140 50.5 140 100.5 140 150.5 160 50.5 160 100.5 160 150.5 180 50.5 180 100.5 180 151 140 51 140 101 140 151 160 51 160 101 160 151 180 51 180 101 180 15

–: Not detectable.a Moisture-free basis.b Biomass also contains acid-soluble lignin, extractives, acetyl acid groups, ash, and uc Other sugars represent galactan, arabinan, and mannan.

Pretreated biomass ð%Þ¼ ðweight of pretreated biomass=weight of starting biomassÞ�100

Dissolved solids yield ð%Þ¼ð1�weight of pretreated biomass=weight of starting biomassÞ�100

All experiments and analysis were performed in triplicate.

2.4. Chemical analysis

Liquid samples were filtered through 0.2 lm nylon membranes(Whatman 0.2 lm NYL w/GMF) and analyzed by high-performanceliquid chromatography (HPLC) (Waters, Milford, MA) with a KC-811ion-exclusion column and a Waters 410 refractive index detector todetermine the presence of glucose, arabinose, xylose, galactose,mannose, and ethanol. The mobile phase was 0.1% H3PO4 solutionat a flow rate of 1 mL/min. The temperatures of the detector andcolumn were maintained at 35 and 60 �C, respectively. For eachrun, the external standards of glucose, arabinose, xylose, galactose,mannose, and ethanol were used.

2.5. Digestibility test

Pretreated biomass samples were used in wet form for enzy-matic digestibility tests. A control was prepared with an identicalamount of cattail material that had not been pretreated. The totalamount of glucose released after 48 h of hydrolysis was measuredto calculate the enzymatic digestibility. The conditions of the enzy-matic digestibility tests were 50 �C and pH 4.8 (0.05 M sodium cit-rate buffer). Screw-capped 250-mL Erlenmeyer flasks were used as

(min) Biomass composition (air-dried, % by weight)

Cellulose Xylan Other sugarsc Klason lignin

34.2 11.6 3.2 26.436.1 18.1 3.4 33.334.5 16.5 3.7 33.436.6 14.0 2.9 33.541.1 17.8 3.2 35.039.6 17.6 2.8 37.441.7 15.1 1.9 37.942.8 12.1 1.4 41.741.2 11.3 1.6 41.841.1 14.1 0.9 40.057.1 – – 36.956.6 – – 38.954.6 – – 39.959.7 – – 40.255.2 – – 42.153.3 – – 43.857.0 – – 42.152.5 – –– 46.350.4 – – 48.658.0 – – 37.255.2 – – 38.252.5 – – 38.858.9 – – 39.556.2 – – 40.154.5 – – 42.647.7 – – 48.743.8 – – 51.935.5 – – 62.4

ronic acid groups.

9310 B. Zhang et al. / Bioresource Technology 102 (2011) 9308–9312

reaction vessels and were agitated at 150 rpm in a constant tem-perature incubator shaker. Pretreated cattails were hydrolyzedusing a cellulase loading (Novozyme NS50013) of 7.5, 15, or60 FPU/g glucan. Novozyme b-glucosidase (NS50010) at a loadingof 4.5 CBU/g glucan and hemicellulase (NS22002) at a loading of2.5 FBG/g glucan were also incorporated with the cellulase (Shiet al., 2011b). The pretreated biomass was loaded into the reactorto give an initial glucan concentration in the reactor of 1% (w/v)(i.e., 1 g-glucan/100 mL liquid).

2.6. Fermentation

S. cerevisiae (ATCC 24858) was the yeast organism used to fer-ment the enzymatically released glucose. For ethanol production,8 mL of seed culture were used to inoculate 40 mL YM mediumin a 250-mL Erlenmeyer flask. The cultures were incubated in ashaker at 30 �C and 200 rpm, and grown aerobically overnight.The yeast was harvested at room temperature by centrifugationat 2600 RCF for 15 min. The supernatant was discarded and thecells were transferred to 250-mL screw-capped Erlenmeyer flaskscontaining 100 mL of hydrolysate. The initial cell mass concentra-tion prior to fermentation in each experiment was 8–9 g dryweight/L. The flasks were then tightly capped to allow fermenta-tion to occur under largely anaerobic conditions. The cultures wereplaced in a shaker and incubated at 30 �C. Fermentation sampleswere filtered through 0.2 lm nylon membranes and analyzed byHPLC to determine the presence of ethanol and sugars.

Ext

ract

able

Pro

duct

s (%

of

tota

l bio

mas

s)

0

10

20

30

40

50

140 160

0

20

40

60

80

140 160

0

20

40

60

80

140 160Treatment Tem

Fig. 1. Effect of treatment temperature and time on the yields of extractable products bysulfuric acid.

3. Results and discussion

3.1. Dilute acid pretreatments

Fig. 1 shows how up to 68.4% of the cattails were dissolved dur-ing pretreatment over the dilute-sulfuric acid concentration rangeof 0.1–1%, temperature range of 140–180 �C, and time intervals of5–15 min. The yield of extractable products obtained from the pre-treatment process increased as the final temperature, treatmenttime, acid concentration, and severity factor increased. The sever-ity factor was calculated by the method described in Lloyd and Wy-man (2005). A high-temperature pretreatment (1% dilute-sulfuricacid, 180 �C for 15 min with a severity factor of LogCS 2.3) resultedin the highest total extractables yield.

The compositions of pretreated cattails are summarized in Ta-ble 1. The xylan fraction of cattails can be effectively removed asoligomers or monomers when applying a 0.5% or 1% sulfuric acidsolution. This finding is consistent with our previous study (Zhanget al., 2009b). The highest pretreatment xylose yield of the pre-treatment stage (74.5% of the total xylan) occurred at a tempera-ture of 180 �C, a sulfuric acid concentration of 1%, and a time of15 min (Table 2).

During the pretreatment process of utilizing 0.5% or 1% sulfuricacid, 2–55% of cellulose was also solubilized as oligomers andmonomeric form. The highest glucose yield in the pretreatmentstage (55.4% of the cellulose) was measured at a pretreatment tem-perature of 180 �C, a sulfuric acid concentration of 1%, and a time of

180

5 min

10 min

15 min

180

5 min

10 min

15 min

180

5 min

10 min

15 min

perature (°C)

A

B

C

dilute-sulfuric acid pretreatment. (A) 0.1% Sulfuric acid, (B) 0.5% sulfuric acid, (C) 1%

Table 2The glucose and xylose yields of the dilute-acid pretreated cattails.

H2SO4

concentration(%)

Treatmenttemperature(�C)

Treatmenttime(min)

Severityfactor(LogCS)

Pretreatmentstage xyloseyield (% of thexylan)

Enzymehydrolysis stagexylose yield (% ofthe xylan)

Totalxyloseyield (% ofthe xylan)

Pretreatmentstage glucoseyield (% of thecellulose)

Enzyme hydrolysisstage glucose yield(% of the cellulose)

Totalglucose yield(% of thecellulose)

0.1 140 5 N/A 7.5 ± 0.9 11.5 ± 1.1 19.0 – 21.6 ± 4.8 21.60.1 140 10 N/A 11.3 ± 0.7 11.3 ± 1.3 22.6 – 29.9 ± 1.0 29.90.1 140 15 N/A 15.4 ± 0.8 11.7 ± 3.4 27.1 – 33.2 ± 1.1 33.20.1 160 5 N/A 29.9 ± 3.4 15.0 ± 2.1 44.9 – 41.6 ± 1.0 41.60.1 160 10 N/A 37.2 ± 3.6 18.2 ± 1.9 55.4 – 46.6 ± 2.1 46.60.1 160 15 N/A 39.8 ± 0.5 21.5 ± 4.1 61.3 – 48.8 ± 2.1 48.80.1 180 5 N/A 44.2 ± 1.4 21.0 ± 2.5 65.2 1.1 ± 3.4 57.7 ± 3.6 58.80.1 180 10 N/A 44.8 ± 2.0 27.1 ± 3.1 71.9 1.2 ± 2.5 62.0 ± 2.6 63.20.1 180 15 N/A 50.5 ± 2.1 45.4 ± 5.4 95.9 1.5 ± 2.4 69.9 ± 1.9 71.40.5 140 5 0.2 21.9 ± 1.4 – 21.9 – 38.0 ± 1.3 38.00.5 140 10 0.5 34.6 ± 4.8 – 34.6 – 44.9 ± 2.7 44.90.5 140 15 0.7 42.0 ± 3.6 – 42.0 – 41.4 ± 1.6 41.40.5 160 5 0.8 41.7 ± 2.2 – 41.7 8.2 ± 1.5 63.5 ± 5.3 71.70.5 160 10 1.1 46.9 ± 1.8 – 46.9 9 ± 1.8 65.3 ± 1.2 74.30.5 160 15 1.3 49.8 ± 1.6 – 49.8 10 ± 1.5 71.1 ± 5.0 81.10.5 180 5 1.4 67.1 ± 4.4 – 67.1 14.9 ± 3.5 82.2 ± 2.1 97.10.5 180 10 1.7 68.1 ± 5.4 – 68.1 21 ± 1.7 74.0 ± 1.0 950.5 180 15 1.9 68.5 ± 4.6 – 68.5 26 ± 2.1 70.7 ± 1.5 96.71 140 5 0.6 38.7 ± 1.7 – 38.7 2.2 ± 1.0 41.0 ± 1.8 43.21 140 10 0.9 40.2 ± 4.3 – 40.2 4.6 ± 1.3 45.7 ± 1.7 50.31 140 15 1.1 42.7 ± 4.1 – 42.7 6.6 ± 1.4 47.0 ± 1.4 53.61 160 5 1.2 63.1 ± 3.8 – 63.1 6.1 ± 1.2 60.9 ± 1.5 67.01 160 10 1.5 68.1 ± 4.7 – 68.1 13.1 ± 2.4 63.2 ± 4.7 76.31 160 15 1.7 68.3 ± 1.5 – 68.3 18.7 ± 2.6 69.5 ± 4.3 88.21 180 5 1.8 68.8 ± 1.6 – 68.8 32.3 ± 2.9 64.5 ± 1.8 96.71 180 10 2.1 71.3 ± 3.9 – 71.3 45.9 ± 1.3 49.5 ± 5.1 95.41 180 15 2.3 74.5 ± 1.9 – 74.5 55.4 ± 2.7 40.2 ± 2.6 95.6

–: Not detectable.

B. Zhang et al. / Bioresource Technology 102 (2011) 9308–9312 9311

15 min. While applying a 0.1% sulfuric acid solution, only approx-imately 1% of cellulose was dissolved into soluble form.

3.2. Hydrolysis of cellulose and xylan from cattails following sulfuricacid pretreatments

The pretreatment processes were studied using the followingtreatment variables: acid concentration (0.1%, 0.5%, 1%), reactiontemperatures (140�, 160�, 180 �C at 1500 psi) and residence times(5, 10, 15 min). The pretreated material was then hydrolyzed for48 h with a cellulase loading of 15 FPU/g glucan with b-glucosidaseat a loading of 4.5 CBU/g glucan and hemicellulase at a loading of2.5 FBG/g glucan. The yields of fermentable sugars from the disso-lution of the cattails are listed in Table 2. Either glucose yield or xy-lose yield increased with increasing final treatment temperatureand treatment time. While pretreated with a 0.1% sulfuric acidsolution at 180 �C for 15 min, nearly 70% of the cellulose of cattailswas converted to fermentable glucose within 48-h hydrolysis, andthe xylose yield of the hydrolysis step was 45.4% of the total xylan.The highest glucose yield of the enzyme hydrolysis stage (82.2% ofthe cellulose) was obtained when cattails were pretreated at180 �C for 5 min with 0.5% sulfuric acid. The highest total glucoseyield for both the pretreatment and enzyme hydrolysis stages(97.1% of the cellulose) was reached under the same reaction con-ditions (Table 2).

The extracts (i.e., hydrolytes) were neutralized by adding NaOHsolution until the pH value is 7. Then the pH of the hydrolysateswas adjusted to 5.0 by adding sodium citrate buffer. The extractsafter neutralization were further hydrolyzed using the same en-zymes mentioned above; however, the sugar yields in the extractsonly increased slightly. S. cerevisiae (ATCC 24858) was pre-culturedin 40 mL YM medium as described in Section 2, then the yeast cellswere transferred into the hydrolysates. Yeast cannot utilize theglucose in the extracts, or produce ethanol, indicating the forma-tion of inhibitory compounds of enzymes and fermentation during

the acid pretreatment process. Further detoxification is necessaryfor utilizing the hydrolytes from the acid pretreatment process ofcattails.

3.3. Effect of enzyme loadings on digestibility of pretreated cattails

Cattails pretreated at 180 �C for 5 min with 0.5% sulfuric acidwere used for digestion to compare cellulase loading. An increasein release of sugars was observed as cellulase dosage was in-creased. After 48 h of enzymatic hydrolysis, the glucose yieldswere 67.1, 82.2, and 84.1% of the total cellulose following pretreat-ment with a cellulase loading of 7.5, 15, and 60 FPU/g glucan,respectively. An increase in glucose content of 15.1% of total cellu-lose was seen when the cellulase loading was raised from 7.5 to15 FPU/g glucan, while the glucose production increased by 1.9%of total cellulose when the cellulase loading was increased from15 to 60 FPU/g glucan. These results indicate that cattails pre-treated with 0.5% sulfuric acid are digestible with similar resultsat enzyme loadings above 15 FPU/g glucan, while there is a signif-icant loss of yield at 7.5 FPU/g glucan cellulose loading. The en-zyme dosage requirement is lower than that of using alkaline orhot-water pretreatment (Zhang et al., 2011, 2009b). High sugaryields and low enzyme requirement would make the process moreeconomically sound, and additional research is required to opti-mize the economics of the overall biorefinery process of cattails.

3.4. Fermentation of cellulose from the pretreated cattails

The pretreated biomass of three extraction tubes (solids) werepooled giving a glucan loading of approximately 2 g/100 mL liquid(i.e., 2% (w/v)). The maximum glucose yield and the theoreticalethanol yield from 2 g glucan are 2.22 and 1.14 g/100 mL, respec-tively. The pretreated biomass first was enzymatically hydrolyzedfor 2 days using a cellulase loading of 15 FPU/g glucan, then fol-lowed by a 2-day Simultaneous Saccharificiaton and Fermentation

9312 B. Zhang et al. / Bioresource Technology 102 (2011) 9308–9312

(SSF). The hydrolysate from undiluted pretreated cattails (first 2-day hydrolysis) gave a fermentable glucose yield of 1.7% w/v,which is lower than the maximum glucose yield of 2.2%. Thismay be because of the product inhibition. During the SSF, moreglucan was converted into glucose, and the glucose was rapidlyconverted to ethanol (<8 h). The final ethanol yield is 0.99% w/v,suggesting that glucose produced from cattails cellulose can beefficiently fermented to ethanol. The glucose to ethanol yields wereapproximately 90% of the theoretical yield for this S. cerevisiaestrain. The result is similar to the results of the hot-water pre-treated and NaOH pretreated cattails, which were approximately88.7% and 95% of the theoretical yield.

4. Conclusions

Dilute-sulfuric acid pretreatment of cattails was studied usingan accelerated solvent extractor. The highest total glucose yieldfor the pretreatment/hydrolysis stages was 97.1% of the cellulose.The pretreated biomass required a lower enzyme loading of15 FPU/g glucan. High sugar yields and low enzyme requirementmake the process more economically sound. Glucose from cattailscellulose can be efficiently fermented to ethanol with an approxi-mately 90% of the theoretical yield. The results indicate that cat-tails are a promising source of feedstock for biofuel production,and additional research is required to optimize the economics ofthe overall biorefinery process.

Acknowledgements

This research was supported by a grant from the USDA-CSREESEvans-Allen program and the Agricultural Research Program atNorth Carolina A&T State University.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2011.07.008.

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