Bioresource Technology 107 (2012) 282–286

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Bioresource Technology

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Combination of biological pretreatment with liquid hot water pretreatmentto enhance enzymatic hydrolysis of Populus tomentosa

Wei Wang a, Tongqi Yuan b, Kun Wang b, Baokai Cui a, Yucheng Dai a,⇑a Institute of Microbiology, Beijing Forestry University, Beijing 100083, Chinab Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 November 2011Received in revised form 21 December 2011Accepted 21 December 2011Available online 30 December 2011

Keywords:White rot fungiBiodegradationSaccharificationLignocellulosic biomassEthanol

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.12.116

⇑ Corresponding author. Tel./fax: +86 10 6233 6309E-mail addresses: wish101@yahoo.cn (W. Wang), y

A novel stepwise pretreatment of combination of fungal treatment with liquid hot water (LHW) treat-ment was conducted to enhance the enzymatic hydrolysis of Populus tomentosa. The results showed thatlignin and cellulose increased with the elevating temperature, while significant amount of hemicellulosewas degraded during the LHW pretreatment. A highest hemicellulose removal of 92.33% was observed bycombination of Lenzites betulina C5617 with LHW treatment at 200 �C, which was almost 2 times higherthan that of sole LHW treatment at the same level. Saccharification of poplar co-treated with L. betulinaC5617 and LHW at 200 �C resulted in a 2.66-fold increase of glucose yield than that of sole LHW treat-ment, and an increase (2.25-fold) of glucose yield was obtained by the combination of Trametes ochraceaC6888 with LHW. The combination pretreatment performed well at accelerating the enzymatic hydroly-sis of poplar wood.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Populus tomentosa is a wide distributed tree species in China, asa result of its widespread availability, sustainable production andlow starting value, it has the potential to serve as the feedstockfor production of fuel ethanol.

Conversion of lignocellulosics to ethanol employs three majorsteps including (1) pretreatment to breakdown the lignin and openthe crystalline structure of cellulose, (2) hydrolysis with a combi-nation of enzymes to reduce cellulose to glucose and (3) microbialfermentation of glucose to ethanol (Sun and Cheng, 2002). How-ever, a major barrier in the commercialization of a lignocellulosesbased ethanol process is pretreatment, which constitutes one-thirdof the total production costs (NREL, 2000). Typical physical andchemical pretreatments, such as microwave, ionizing radiation,steam explosion, dilute acid, alkali, and oxidation or varied combi-nation, require special instrument and consume a lot of energy(Mosier et al., 2005b). Moreover, they often generate some inhibi-tors to subsequent enzymatic hydrolysis and microbial fermenta-tion apart from producing acidic or alkaline waste water, whichneeds pre-disposal treatment to ensure environmental safety (Kel-ler et al., 2003).

Microbial pretreatment, as an environmental friendly and lowcost pretreatment approach for enhancing enzymatic saccharifica-tion and fermentation of lignocellulosic biomass to ethanol, is

ll rights reserved.

.uchengd@yahoo.com (Y. Dai).

attracting increasing attention in recent years (Keller et al., 2003;Taniguchi et al., 2005; Hwang et al., 2008; Zhang et al., 2007; Shiet al., 2008, 2009). So far, lots of studies on pretreatments with var-ious white rot fungi have been reported. Four white rot fungi wereapplied to pretreatment of white pine and tulip tree for enzymatichydrolysis, and found that 450 mg glucose/g pretreated wood wasobtained by Trametes versicolor in 30 days (Hwang et al., 2008). Yuet al. (2009a) studied the effect of biological pretreatment with theselective white rot fungus Echinodontium taxodii on enzymatichydrolysis of Chinese willow and China-fir, the results showed thatthe hydrolysis ratios increased 4.7-fold for hardwood and 6.3-foldfor softwood after pretreatment for 120 days. Pretreatment withCeriporiopsis subvermispora for enzymatic hydrolysis of corn stoverwas investigated, and overall glucose yields of 66.61% wereachieved with 35-days microbial pretreatment (Wan and Li,2010). These reports showed a great potential with biological pre-treatment in conversion of lignocellulosic biomass to ethanol.However, relative low efficiency, considerable loss of carbohy-drates and long residence periods are the three major disadvan-tages for the fungal pretreatment (Yu et al., 2009b). Newstrategies should be adopted to overcome these feeble sides.

Recently, liquid water under elevated temperature and pres-sure, namely hot compressed water or hydrothermal processing,has received renewed attention. Liquid hot water (LHW) pretreat-ment has been shown to be effective in pretreatment of lignocellu-losic biomass by partially hydrolyzing the hemicelluloses anddisrupting the lignin and cellulose structures, thus increasing thesurface area (Mosier et al., 2005b). The advantages of LHW

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pretreatment contained: limited corrosion problems, no sludgegeneration, low capital and operational costs and negligible lossof cellulose under normal operating conditions (Liu, 2010).

In order to enhance the efficiency of the fungal pretreatmentand lower the severity requirements of the LHW pretreatment, atwo-step pretreatment was employed. The wood flour of P. tomen-tosa was pretreated with white rot fungi Lenzites betulina and Tra-metes ochracea, respectively, and then dealt with LHW. Thecomponent changes of pretreated wood and sugar yield from enzy-matic hydrolysis were both determined to evaluate the effects ofcombination pretreatments on P. tomentosa.

2. Methods

2.1. Microorganism and inoculums preparation

The two white rot fungi, L. betulina C5617 and T. ochraceaC6888, were isolated from Liaoning and Hebei provinces in china,respectively. The organisms were preserved on 2% (w/v) malt-ex-tract agar (MEA) plates at 4 �C in laboratory. The two fungi wereactivated in 100 mL basic medium (g/L: glucose 20, yeast extract5, KH2PO4 1, MgSO4 0.5, VB1 0.01), and cultured on a rotary shakerat 28 �C with a speed of 150 rpm. Mycelial pellets were harvestedafter 5 days, added 100 mL distilled water and then mixed with alaboratory blender for 30 s at 5000 rpm. This suspension wouldact as inoculums.

2.2. Raw materials

Fresh poplar (P. tomentosa) from countryside of Beijing waschopped into small pieces and air-dried. The samples were ground,and the particles below 0.9 mm were prepared for the subsequentpretreatment with white rot fungi and LHW, respectively.

2.3. Biological pretreatment of Poplar wood

The biological pretreatment was carried out in a 250 mL Erlen-meyer flask with 5 g of air-dried poplar wood and 12.5 mL of dis-tilled water. The samples were sterilized in the autoclave for20 min at 121 �C and inoculated with 5 mL inoculums. The cultureswere incubated statically at 28 �C for 4 weeks. The non-inoculatedsamples were served as the control. All experiments were per-formed in triplicate.

2.4. LHW pretreatment of poplar wood

The experiments were conducted in batch tube reactors fabri-cated from 316 stainless steel tubes, with a length of 4.5 inches,an outside diameter of 1.0 inch, wall thickness of 0.065 inch, anda total volume of 50 mL. The reactor was filled with 2.5 g raw orbio-pretreated poplar wood and 25 mL distilled water to achievea 10% w/v of dry matter mixture. After the slurry was loaded, the316 stainless steel caps were fitted onto each end of the tubes.Then a drying oven was used for heat-up of the tubes. When theoven reached the targeted temperature (140, 160, 180, and200 �C), the residence time began to record. After 30 min, the reac-tion was ended by quenching the tube in room-temperature water,which caused the temperature of the internal tube to drop below100 �C in less than 5 min.

Wet material was vacuum filtered to obtain water-insolubleresidues. The residues after filtration were extensively washed toneutralize with distilled water, and then dried at 35 �C for 24 hfor further analysis (Lu and Zhou, 2011).

2.5. Enzymatic hydrolysis

Commercial cellulase preparation (Celluclast 1.5 L), producedby Tricoderma reesei ATCC 26921, was purchased from Sigma. Atypical hydrolysis mixture consisted of 0.2 g of pretreated sample,10 mL of 50 mM sodium acetate buffer (pH 4.8) supplementedwith 40 lL antibiotics tetracycline and 20 lL cycloheximide, and35 FPU/g substrate of cellulase. The mixture was incubated at50 �C in a rotary shaker at 150 rpm for 96 h. Samples were takenfrom the reaction mixture at certain interval and centrifuged for10 min at 10000 rpm, stored at �20 �C for further assay. Experi-ments were all performed in duplicate.

2.6. Analytical methods

The chemical composition of raw material and pretreated resi-dues was determined according to NREL LAP ‘‘Determination ofStructural Carbohydrates and Lignin in Biomass’’ (Sluiter et al.,2008) using HPAEC. The HPAEC system (Dionex ISC 3000, USA)was equipped with an amperometric detector, AS50 autosampler,a carbopacTM PA-20 column (4 � 250 mm, Dionex), and a guardPA-20 column (3 � 30 mm, Dionex). Cellulose contents were calcu-lated based glucose using anhydro corrections of 0.9, hemicellulosecontents were calculated based the sum of xylose, galactose andarabinose, using 0.88 as anhydro corrections for xylose and arabi-nose, and 0.9 for galactose.

The glucose in the supernatant after enzymatic hydrolysis wasalso analyzed by HPAEC. The glucose yield was calculated asfollows:

Glucose yieldsð%Þ ¼ amount of glucose in enzyme hydrolysate� 0:9amount of cellulouse in pretreated sample

� 100

3. Results and discussion

3.1. Effects of pretreatments on chemical components

After 4 weeks of biological pretreatment with L. betulina C5617and T. ochracea C6888, the bio-pretreated samples and raw mate-rial were further treated by LHW at 140, 160, 180 and 200 �C for30 min, respectively. The composition of different chemical com-ponents in residues after various pretreatments might be seen inFig. 1.

As one of the main components of plant cell wall, lignin limitsthe enzymatic hydrolysis of lignocellulosic biomass by cross-link-ing with cellulose and hemicelluloses (Fan et al., 1987). To exposethe highly ordered crystalline structure of cellulose and facilitatesubstrate access by hydrolytic enzymes, reducing the lignin con-tent of the biomass is expected (Sun and Cheng, 2002). Recently,white rot fungi were thought to be the most promising organismsthat can efficiently metabolize lignin in a variety of lignocellulosicmaterials (Hatakka, 1983; Sawada et al., 1995; Keller et al., 2003).In this work, significant decrease of acid insoluble lignin (AIL) onlyhappened in the step of fungal pretreatment, L. betulina C5617 andT. ochracea C6888 decayed 12.7% and 11.89% of AIL, respectively.When the bio-treated samples and raw materials were subject toLHW, however, an increase of AIL was observed with the increasingtemperature, this is contradictory to previous works concerningLHW pretreatment with flowing hot water through cellulosic bio-mass in a small tubular flowthrough reactor, which performed bet-ter at degrading lignin than that of batch reactor, and high flowrates enhanced lignin removal, especially at elevated temperatures(Liu and Wyman, 2003, 2004a, 2004b). The accumulation of ligninmay result from the condensation and precipitation of the lignin

Fig. 1. Changes in percentage composition of chemical components of P. tomentosaafter various pretreatments. A: sole LHW pretreatment; B: combination of L.betulina C5617 treatment with LHW treatment; C: combination of T. ochraceaC6888 treatment with LHW treatment. Components: Cel, cellulose; HCel, hemicel-lulose; AIL, acid insoluble lignin; ASL, acid soluble lignin; O, other components.

Fig. 2. Time course of enzymatic hydrolysis of P. tomentosa after various pretreat-ments. A: sole LHW pretreatment; B: combination of L. betulina C5617 treatmentwith LHW treatment; C: combination of T. ochracea C6888 treatment with LHWtreatment.

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compounds in the no-flowthrough batch reactor employed in thisstudy. In addition, Interestingly, acid soluble lignin (ASL) demon-strated a decreased trend with the increasing temperature underall LHW conditions, it is likely that the hot water solubilized partof the ASL, since hot water liberates acids and facilitates the break-age of such ether linkages in biomass during biomass hydrolysis(Antal, 1996).

As can be seen in Fig. 1, significant amounts of hemicellulosewere degraded during LHW pretreatment above 180 �C. The high-est decrease of hemicellulose (92.33%) was obtained by combina-tion of L. betulina C5617 treatment with LHW treatment at200 �C, which was almost 2 times larger than that of sole LHWtreatment at the same level, indicating that the initial fungal treat-ment softened the tight structure of lignocellulosic biomass, andpromoted the hemicellulose degradation during LHW treatment.Largely removing hemicelluloses during pretreatment can reduce

Table 1Comparison of liquid hot water pretreatment of different substrates by various process conditions and the obtained glucose yield.

Substrates Process conditions Glucose yield (%) References

Corn stover Batch, 200 �C, 20 min 50.5 Liu and Wyman, 2005Distilled grains Sand bath, pH controled, 160 �C, 24 min 68 Kim et al., 2008Hybrid poplar Sand bath, 200 �C, 10 min, then hot-wash 54 Kim et al., 2009Wheat straw Agitated batch (600 rpm), 200 �C, 40 min 96 Pérez et al., 2007Wheat straw Agitated batch (600 rpm), 214 �C, 2.7 min 90.6 Pérez et al., 2008Eucalyptus grandis 500 rpm agitation, 200 �C, 20 min 41.9 Yu et al., 2010Soybean straw 400 rpm agitation, 210 �C, 10 min 70.76 Wan et al., 2011Populus tomentosa Combination treatment (200 �C, 30 min) 60.26 This study

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the possibility of inhibiting enzymatic hydrolysis and subsequentfermentation by Saccharomyces cerevisiae (Lynd, 1996).

Preservation of the cellulose during pretreatment was expectedto increase the sugar yield of enzymatic hydrolysis. When the un-treated wood flour was pretreated solely by LHW, the celluloseslightly increased from 37.21% to 56.64%, corresponding well tothe rising temperature from 140 �C to 200 �C. Sole fungal treatmentby L. betulina C5617 and T. ochracea C6888 for 4 weeks can onlycause a little increase of the cellulose (7.39% and 10.57%, respec-tively). Regarding to co-treated samples with fungal pretreatmentand LHW pretreatment, the cellulose contents increased signifi-cantly with temperature rising from 140 to 180 �C, and as highas 57.2% of cellulose was obtained by combination of L. betulinaC5617 with LHW treatment at 180 �C, which was even higher thanthat of sole LHW at 200 �C. However, cellulose content graduallydecreased when the treatment temperature rose to 200 �C with re-spect to co-treated samples. This indicated that too high tempera-ture of LHW is not indispensible when the combinationpretreatment was employed.

3.2. Effects of pretreatments on enzymatic hydrolysis

To investigate the efficiency of various pretreatments, the resi-dues of corresponding pretreatments were subsequently submit-ted to enzymatic hydrolysis for 96 h, the time course of whichcan be seen in Fig. 2.

With regard to sole pretreatments, the results showed that 4-week fungal pretreatment significantly enhanced enzymatichydrolysis, though the profile of glucose yield was still low. Theglucose yield of L. betulina C5617 and T. ochracea C6888 increased2.31 and 1.54-fold, respectively, compared to untreated sample(Fig. 2A–C). These increases of glucose yield might be a reflectionof lignin degradation or modification in poplar wood by whiterot fungi L. betulina C5617 and T. ochracea C6888, respectively,which made the accessibility of cellulase to cellulose easier. A sim-ilar result was also obtained when water hyacinth was pretreatedby E. taxodii for 10 days (Ma et al., 2010).

When it went to combination pretreatments, the glucose yieldsof co-treated poplar wood with L. betulina C5617 and LHW (from15.68% to 60.26%) were higher than that of sole LHW treated (from5.89% to 52.24%), which increased 1.15–2.66-fold at the same con-ditions (Fig. 2A and B). Besides, combination of T. ochracea C6888with LHW resulted in an increase (1.12–2.25-fold) of glucose yield(Fig. 2A and C). The maximum glucose yield was 60.26% for thecombination of L. betulina C5617 with LHW at 200 �C. The increaseof enzymatic digestibility of combination pretreatment can beattributed to the synergistic effect of biologic and LHW treatment.Specially, the ratio between glucose yield of combination pretreat-ment and that of sole LHW demonstrated a drop trend (from 2.66to 1.55 and 2.25 to 1.12, respectively) with the rising temperatureof LHW treatment under all conditions, though the glucose yieldsof combination pretreatment increased. The results suggested that

the synergy of combination pretreatment was more promising un-der moderate condition. Taking hemicellulose loss into consider-ation, the reason might be hemicelluloses degradation facilitatethe enzymatic hydrolysis of poplar wood. At elevated temperatureand pressure, LHW pretreatment has been shown to be effective inremoving and solubilizing the hemicelluloses fraction of the recal-citrant cellulosic biomass, and improving the subsequent hydroly-sis efficiency (Ladisch et al., 1998; Mosier et al., 2005a; Zeng et al.,2007). The synergy of combination of fungal pretreatment withLHW pretreatment did play a leading role at initial moderate tem-perature, but this leading role became weak as the temperaturegradually ascended.

As a chemical less and comparative environment-friendly pro-cess, recently, many investigations concerning LHW pretreatmenthave reported (Liu and Wyman, 2005; Pérez et al., 2007, 2008;Kim et al., 2008, 2009; Yu et al., 2010; Wan et al., 2011). Table 1shows a summary of LHW pretreatment of different lignocellulosicmaterials. Liu and Wyman (2005) have reported a glucose yield of50.5% was obtained by LHW treated corn stover at 200 �C for20 min. A similar glucose yield was obtained when hybrid poplarwas co-treated by LHW at 200 �C for 10 min and hot wash (Kimet al., 2009). The effect of LHW pretreatment of Eucalyptus grandisat 200 �C for 20 min was also investigated, 41.9% of glucose yieldwas achieved (Yu et al., 2010). Compared to the previous resultsin Table 1, in general, the combination of fungal pretreatment withLHW pretreatment in this work demonstrate a comparative prom-ising result. Notably, in the LHW-treatment step of this work, therewas no agitation equipment in the hydrothermal reactor and it washeated just in a common drying oven, suggesting this process iseasy to follow. Therefore, the new design of combination pretreat-ment performed well at simplifying the requirements of equip-ments, and can be a suitable candidate to pretreat lignocellulosicbiomass for bioethanol production.

4. Conclusion

A novel design of co-treating P. tomentosa with white rot fungiand LHW was conducted. It showed a better performance on hemi-cellulose removal and cellulose preservation, combination of L.betulina C5617 with LHW treatment at 200 �C removed 92.33% ofhemicellulose. Furthermore, a 2.66-fold increase of glucose yieldthan that of sole LHW treatment was obtained by saccharificationof poplar wood co-treated with L. betulina C5617 and LHW at200 �C. The combination pretreatment presents a promising plat-form on which to develop advanced biomass pretreatmentsystems.

Acknowledgements

This research was supported by the Program for New CenturyExcellent Talents in University and Major State Basic Research Pro-jects of China (973-2010CB732204).

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