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Conversion of recycled paper sludge to ethanol by SHF and SSF using Pichia stipitis
S. Marques, L. Alves, J.C. Roseiro, F.M. Grio
Departamento de Biotecnologia, INETI, Estrada do Pac o do Lumiar 22, 1649-038 Lisboa, Portugal -

ar t ic l e i n f o
Article history: Received 25 May 2006 Received in revised form 23 October 2007 Accepted 30 October 2007 Available online 11 December 2007 Keywords: Lignocellulosic residues Ethanol Enzymatic hydrolysis Cellulases Xylanases Celluclasts 1.5 L Novozym
s

abs tra ct
The purpose of the present work was to evaluate the possibility of converting recycled paper sludge (RPS), an industrial residue stream with strong environmental impact, into valuable products. The approach used was based on the enzymatic conversion of major sludge components (cellulose and xylan) and the simultaneous (simultaneous saccharication and fermentationSSF) or sequential (separate hydrolysis and fermentationSHF) fermentation of the resulting sugars to ethanol. In the enzymatic hydrolysis step using Celluclasts 1.5 L supplemented with Novozyms 188, a degree of saccharication of 100% was achieved. In relation to ethanol production using the yeast Pichia stipitis CBS 5773, SHF and SSF process efciencies were compared. A slightly higher conversion yield was attained on SHF, corresponding to an ethanol concentration of 19.6 g L1, but 179 h were needed. The SSF process was completed after 48 h of incubation allowing the production of 18.6 g L1 of ethanol from 178.6 g L1 of dried RPS, corresponding to an overall conversion yield of 51% of the available carbohydrates on the initial substrate. These results demonstrate that the biological conversion of sludge to ethanol is efcient even with no pre-treatment or substrate supplementation. & 2007 Elsevier Ltd. All rights reserved.

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Pichia stipitis

1.

Introduction

New difculties emerging from the large increase in paper recycling rates impose urgent solutions. Wastewater sludge from paper recycling mills contains high levels of heavy metals from waste paper inks. Thus it cannot be used for land application as soil amendment and has to be disposed off to landlls, a prohibitively expensive and environmentally harmful end solution [1,2]. Interest has therefore grown in nding novel value-added uses for this residue from the paper industry [35]. Recycled paper sludge (RPS) is basically made up of secondary poor-quality non-recyclable paper bres (bres too short to be retained on bre screens and paper machines). The high lignocellulosic content of this sludge material offers therefore an opportunity as feedstock for bio-products [6].
Corresponding author. Tel.: +351 21 0924721; fax: +351 21 7163636.

Moreover, paper sludge is believed to be one of the most promising feedstock for near-term commercial application of technology for converting cellulosic raw materials into commodity products [7]. In fact, this substrate has some distinctive advantages among cellulosic feedstocks including negative cost at many locations and the potential availability of pre-existing facilities [8,9]. The feasibility of biotechnological recovery of this potentially attractive substrate requires the conversion of all of its major components (cellulose and hemicellulose) to fermentable sugars, which could be further converted to fuels and chemicals, such as ethanol, organic acids or biodegradable plastics [10]. Among these possible products, ethanol possesses a rapidly expanding market, either as an octane enhancer or primary fuel reducing the harmful effects of gasoline consumption [11]. Thereby, the purpose of the

E-mail address: francisco.girio@ineti.pt (F.M. Grio). 0961-9534/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2007.10.011

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present study was the conversion of RPS into ethanol using Pichia stipitis. This yeast has been described to possess the ability to convert both glucose and xylose into ethanol [12], an essential aspect for the economic feasibility of the production process. The proposed biological approach will bring the potential reduction of the residue volume with the consequent reduction on the cost of ultimate disposal of the remaining lignin/inorganic fraction, together with the upgrading of the carbohydrate component of RPS. Hydrolysis of lignocellulosic materials to soluble products can be carried out by chemical (mild acid/alkali) or enzymatic action. Enzymatic saccharication is advantageous when compared to chemical methods, since it is by nature a more specic and cleaner process. It also allows milder operation conditions, leading to reduced formation of biological inhibitory compounds (such as sugar- and lignin-degradation products) and the catalyst is potentially reusable [13]. In fact, because of the higher ethanol yield and lower by-product formation, economic evaluation of a future full-scale plant for production of ethanol from various lignocellulosic raw materials is frequently based on enzymatic hydrolysis instead of conventional acid hydrolysis [14]. Since sludge has already been subject to an extensive mechanical and chemical processing previously imposed on the paper raw material through pulping (during rening, bleaching and drying), polysaccharides in RPS should be much more amenable to enzymatic hydrolysis, as already noted by several authors [8,9,1517]. Therefore, this substrate might require no pretreatment, commonly performed to overcome lignocellulosics recalcitrancy [18]. The two-step process can be run as separate hydrolysis and fermentation (SHF) or as simultaneous saccharication and fermentation (SSF) [19]. SSF has been regarded as the major option because it usually results in higher overall yields and shorter residence times along with the process integration achieved (both steps performed in one reactor) [20]. In the present work, both approaches (SHF and SSF) for RPS conversion to ethanol were implemented and compared for process efciency in terms of product yield and production rate and total residence time.

lignin contents were assayed by means of a quantitative hydrolysis with sulphuric acid according to the method described by Browning [22]. The quantication of the monosaccharides was carried out by high-performance liquid chromatography (Section 2.2). The acid-insoluble residue was considered as Klason lignin, after correction for the acid-insoluble ash. The composition of RPS was determined to be (on a dry weight basis): 34.1% cellulose, 29.3% ash, 20.4% Klason lignin, 7.9% xylan, 4.8% protein and 3.5% fat.

2.2.

Analytical methods

The commercial enzyme preparations applied were previously characterised for catalytic activity at the conditions used for the enzymatic saccharication of sludge. Endo-b-1,4-xylanase (1,4-b-D-xylan xylanhydrolase; EC 3.2.1.8) activity was assayed using 1% (w/v) oat spelts xylan (Sigma, St. Louis, USA) as substrate. Filter paper activity (FPase), describing the cellulolytic activity, was assayed using Whatman number 1 lter paper as substrate. Enzyme activities were expressed in international units (U) as the amount of enzyme required to release 1 mmol per minute of either xylose (endo-b-1,4-xylanase) or glucose (FPase) reducing equivalent under the assay conditions. Reducing sugars were estimated by the dinitrosalycilic acid method [23]. Ethanol, sugars and sugar-degradation products were measured by HPLC using a Waters LC1 module 1 plus (Millford, LA) equipped with a two-serial differential refractive index/ultraviolet detector, the latter being set at a xed wavelength of 280 nm (for hydroxymethylfurfural and furfural). An Aminex HPX-87 H column (Bio-Rad, Hercules, CA, USA) was used, operating at 50 1C with 5 mM H2SO4 as mobile phase at a ow rate of 0.4 mL min1.

2.3.

Enzymatic hydrolysis trials

2.
2.1.

Materials and methods

Substrate

The present study used pressed RPS consisting of the solids resulting from the wastewater treatment facility of a local paper recycling mill (Renova, Torres Novas, Portugal). The asreceived sludge contained calcium carbonate that rendered the resulting suspensions alkaline. Therefore, RPS was neutralised with hydrochloric acid (0.3 g HCl g1 RPS) prior to use and it was chemically characterised. RPS was analysed gravimetrically for water (by oven drying at 105 1C to constant weight) and ash (by igniting at 575 1C for 5 h) contents. Protein content was estimated by the Kjeldahl method [21] using a nitrogen-to-protein conversion factor of 6.25. Fat was determined by extraction with petroleum ether using conventional Soxhlet glassware and gravimetric extract analysis. Hemicellulose, cellulose and

A sample of RPS was suspended in 0.05 M sodium citrate buffer, pH 5.5, for an initial consistency of 3% or 7.5% (w/v), expressed in terms of total carbohydrate mass, and it was steam sterilised by autoclaving (at 121 1C, 1 atm, for 15 min). Sludge on 3% (w/v) suspension consistency was incubated with the lter-sterilised enzyme solution at 50 or 35 1C in an orbital shaker (150 rev min1) for 144 or 72 h, respectively. Sludge on 7.5% (w/v) consistency was incubated at 35 1C during 120 h. Aseptic conditions were maintained throughout the experiments. Enzymatic hydrolysis was performed with a previously selected mixture of two commercial enzyme preparations (cellulolytic and xylanolytic, from Novozymes, Denmark): Celluclasts 1.5 L (exhibiting an FPase activity of 14.7 U mL1 and an endo-b-1,4-xylanase activity of 228.7 U mL1) and Novozyms 188 (exhibiting an FPase activity of 0.6 U mL1 and an endo-b-1,4-xylanase activity of 854.9 U mL1). This mixture was applied at different enzyme loadings containing: Celluclasts 1.5 L on a dosage of 120 (for 3% consistency at 50 1C), 25 (for 3% consistency at 35 1C) or 10 (for 7.5% consistency) U (FPase) g1 carbohydrate plus 0 or 1.0 (for 3% consistency) or 0.4 (for 7.5% consistency) mL of Novozyms 188 g1 carbohydrate on sludge. For each enzyme tested, a

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control enzyme mixture was subjected to the same assay conditions but in the absence of sludge. The hydrolysates obtained, after residual solid removal by ltration (through a membrane lter of 0.45 mm pore size), were analysed for sugar proles by HPLC. Experiments were performed in triplicate.

3.
3.1.

Results and discussion

Enzymatic hydrolysis

2.4. 2.4.1.

Fermentation studies General conditions

The yeast P. stipitis CBS 5773, obtained from Centraalbureau voor Schimmelcultures (CBS, Baarn) culture collection, was used in all fermentation assays. Experiments were performed in 500 mL Erlenmeyer asks containing 100 mL of fermentation medium. Flasks were inoculated with 5% (v/v) of a yeast suspension obtained from growth for 15 h on YMP medium composed of 3 g L1 of yeast extract (Panreac, Barcelona, Spain), 3 g L1 of malt extract (Oxoid, Hampshire, England) and 5 g L1 of peptone (Merck, Darmstadt, Germany) supplemented with 10 g L1 of glucose (Merck, Darmstadt, Germany). Cultivations were carried out in duplicate at 30 1C with 150 rev min1 (orbital shaking). Samples were collected during 72 h, ltered and analysed for ethanol and sugar contents on the cell-free broth obtained. Cell growth (except during SSF) was monitored directly by reading the optical density at 600 nm.

2.4.2.

Separate hydrolysis and fermentation (SHF)

A lter-sterilised hydrolysate produced from RPS (on an initial consistency of 7.5% (w/v)) was used as fermentation medium with no supplement addition. A control fermentation was also run, on YMP medium supplemented with reagent-grade xylose (Merck, Darmstadt, Germany), glucose (Merck, Darmstadt, Germany) and cellobiose (Merck, Darmstadt, Germany) mixed at the same concentrations present in the sludge hydrolysate used for the SHF process.

2.4.3.

Simultaneous saccharication and fermentation (SSF)

A steam-autoclaved (121 1C, 1 atm for 15 min) suspension of sludge in 0.05 M sodium citrate buffer pH 5.5 was used as SSF medium. SSF experiments were started by inoculation with yeast and addition of the lter-sterilised enzyme solution (in the same amounts used to obtain the hydrolysate for SHF and in its subsequent fermentation).

The used RPS material consisted of 42.0% (w/w) of carbohydrates, on a dry matter basis. This value is in agreement with the average carbohydrate content found in a detailed analysis of 15 paper sludges on a previously reported study [24] and it is similar to the value of 39.0% of polysaccharides determined by Nakasaki and Adachi [25] for wastewater sludge from the paper manufacturing industry. Enzymatic saccharication trials, using different conditions (temperature, residence time and substrate and enzyme loadings), were evaluated in terms of sludge conversion through the calculation of the degree of saccharication (DS). DS was based on the total sugar concentration in the nal hydrolysate (corrected for concentration in the control assay) relative to the content of polysaccharides (potential glucose and xylose) in the initial substrate. Firstly, the hydrolysis was run applying Celluclasts 1.5 L at the optimal conditions for activity of the enzyme preparation: 50 1C and pH 5.5. In this trial, applying an extremely high enzyme (FPase) dosage of 120 U g1 carbohydrate, the saccharication occurred in a relatively low extent, giving a DS of 45% after 6 days of incubation (Table 1). The hydrolysate consisted of glucose (73.9%), xylose (14.2%) and cellobiose (11.9%), corresponding to hydrolysis yields of 47% and 34%, respectively, for the xylan and cellulose fractions in the initial substrate. RPS hydrolysis under these conditions was apparently limited by the low enzyme thermostability at 50 1C. This assumption is in agreement with previously reported data showing that the optimum temperature for enzymatic saccharication is not only a function of the raw material and enzyme source, but it is also dependent on the required residence time [19]. The following saccharication experiments were performed at a lower temperature of 35 1C, allowing reasonable enzymatic hydrolysis activities during an extensive period of incubation with, in addition, a lower energy demand. In these trials, with Celluclasts 1.5 L applied on an FPase dosage of 25 U g1 carbohydrate, total solubilisation of RPS carbohydrates was observed, resulting in a DS of 100% after 72 h at 35 1C (Table 1). Monosaccharides (glucose and xylose) were the main products obtained but cellobiose was also present in the hydrolysate as well as minor amounts of xylobiose.

Table 1 Enzymatic hydrolysis yields (expressed as degree of saccharication, DS) and major carbohydrate products obtained from RPS in different conditions Temperature (1C) Sludge consistency % (w/v) Novozyms 188 Incubation time (h) Liberated sugars for hydrolysate (g L1) DS (%)

Glucose
50 35 35 35 3.0 3.0 3.0 7.5 + + 144 72 72 120 9.9 22.6 24.4 51.1

Cellobiose
1.6 1.8 0.0 6.3

Xylose
1.9 4.9 5.6 12.0

Xylobiose
0.0 0.7 0.0 0.0 45 100 100 92

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The achieved complete hydrolysis conrmed that the polysaccharides in RPS are much more susceptible to enzymatic digestion than those in traditional native lignocellulose feedstocks avoiding the need for sludge pre-treatment. The previous hydrolysis mixture was then supplemented with an excess of Novozyms 188, a cellobiase (b-glucosidase), in order to maximise the sludge conversion to monosaccharides (glucose) and to prevent cellobiose accumulation minimising product inhibition [26]. With this enzyme formulation, cellobiose totally disappeared from the hydrolysate maintaining DS at 100%, meaning that all the cellulose present in the substrate was completely hydrolysed to glucose (Table 1). Since in this assay no xylobiose was detected in the hydrolysate, Novozyms 188 apparently also contributed to complete hydrolysis of xylan to xylose. Despite the high extent of hydrolysis of sludge polysaccharides, relatively low sugar concentrations were obtained in the previously described saccharication experiments since low concentrations of substrate were used. Therefore, in order to maximise product concentrations in the hydrolysate to be used for subsequent fermentation, prolonged hydrolysis (for 120 h) of sludge on an initial consistency of 7.5% (w/v) was performed. Above 7.5% (w/v) of initial substrate concentration, it was not possible to have a suspension due to the high amount of solids. With the present conditions, the hydrolysis of sludge polysaccharides was incomplete, with a DS of 92% after a 120-h incubation, but giving rise to higher concentrations of sugars in the hydrolysate, with cellobiose obtained at a signicant concentration (6.3 g L1) (Table 1). From the HPLC analysis of all the hydrolysates, it could be conrmed that biological inhibitory compounds (sugar-degradation products, such as furfural and hydroxymethylfurfural, or lignin-degradation products) were not produced by enzymatic action on this sludge. This is a relevant feature since the overall ethanol yield and the ethanol production rate will depend not only on the sugar yield, but also on the fermentability of the hydrolysate.

reports stating that P. stipitis grown in glucose/xylose mixtures ferments preferentially glucose, since xylose assimilation is competitively inhibited by glucose [12,27,28]. Ethanol was produced along with cell growth in the course of the SHF process (Fig. 1A) as previously observed by other authors working with the same yeast strain [29]. Total glucose, xylose and cellobiose consumption from the enzymatic hydrolysate was observed yielding an ethanol production of 0.25 g ethanol g1 total sugars with a volumetric production rate of 0.33 g ethanol L1 h1 in a 59-h batch fermentation. In this process a maximal ethanol concentration of 19.6 g L1 was obtained. The ethanol yield observed in this study is higher than the value of 0.17 g ethanol g1 total sugars reported by Linden and Hahn-Hagerdal [30] for yeast extract-amended spent sulphite pulping liquor fermented by P. stipitis. In the control fermentation on YMP medium (Fig. 1B), similar behaviour was observed for glucose and xylose assimilation. However, cellobiose consumption was not detected. This difference might arise from the absence of cellobiase activity able to convert cellobiose to glucose. In the SHF process, the commercial enzymes added in the previous hydrolysis step (displaying cellobiase activity) remain active during the fermentation course. Thereby, the total consumption of cellobiose observed for the SHF process might be a result from its hydrolysis to glucose and subsequent fermentation. The maximal ethanol concentration (22.6 g L1) was somewhat higher than in SHF and was obtained after 48 h of cultivation, corresponding to an ethanol volumetric production rate of 0.47 g ethanol g L1 h1 with an ethanol yield of 0.29 g ethanol g1 total sugars. Since small differences were observed in cell growth or alcohol production between the two fermentations (Fig. 1A and B), it may be concluded that the sludge hydrolysate did not produce any signicant inhibitory effect on microbial growth and contained the essential nutrients to support growth and ethanol production, requiring no further supplementation.

3.2. 3.2.1.

Fermentation Separate hydrolysis and fermentation (SHF)

3.2.2.

Simultaneous saccharication and fermentation (SSF)

Hydrolysate obtained from sludge on 7.5% consistency was used as fermentation medium, with no nutritional supplementation, for the SHF process, to produce ethanol with P. stipitis CBS 5773. The fermentation of the sludge hydrolysate (containing 56.2 g L1 glucose, 12.9 g L1 xylose and 6.8 g L1 cellobiose) was compared with that of YMP medium supplemented with reagent-grade glucose, xylose and cellobiose at these same concentrations. The parameters measured during the course of the cultivation were cell growth, sugars consumption and ethanol production. The timecourse proles obtained for both fermentation experiments are represented in Fig. 1A and B. In the SHF process (Fig. 1A), xylose was fermented only after a 15-h period during which the glucose concentration was reduced to 38 g L1, and at a slower rate than that of glucose assimilation. Yeast cells readily consumed glucose to produce ethanol and total consumption occurred within less than 35 h of incubation. This observation is in agreement with previous

Using the same concentrations of sludge and enzyme formulation used to obtain the hydrolysate for SHF and similar fermentation conditions, the enzymatic hydrolysis and the fermentation steps were also simultaneously performed as an SSF process. For SSF, samples were collected only after 6 h of incubation due to the high thickness of the suspension observed during this rst period. The highest ethanol concentration obtained in the SSF process (Fig. 2) was 18.6 g L1 after 48 h of incubation, corresponding to an ethanol volumetric production rate of 0.39 g ethanol L1 h1. This high degree of conversion of polysaccharides into ethanol is in agreement with the signicant decrease observed in polysaccharide content in residual sludge analysis (after extensive solid washing to remove cellular mass). In fact, polysaccharide content in the sludge was reduced to 5.5% (w/w) (on a dry matter basis). The residual sludge collected at the end of the process (after 72 h of cultivation) consisted of (on a dry matter basis): ash, 61.8% (w/w); lignin, 32.8% (w/w); cellulose, 2.4% (w/w) and xylan, 2.9% (w/w).

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Fig. 1 Time course of the fermentation with P. stipitis: (A) SHF process; (B) growth on control YMP medium containing glucose, xylose and cellobiose at the concentrations present in the sludge hydrolysate used for the SHF process. (n) Growth (left axis); () glucose; (m) xylose; (~) cellobiose; and (&) ethanol concentrations (right axis).

Fig. 2 Time course of the SSF process with P. stipitis using RPS: () glucose; (m) xylose; (~) cellobiose; and (&) ethanol concentrations.

In SSF, at least after the rst 6-h cultivation period, no further glucose accumulation was detected and the glucose concentration in the reaction mixture was lower than that obtained for the corresponding time in the hydrolysis stage of

the SHF process. This indicates that yeast cells were metabolically active during the entire course of the fermentation. This also means that enzymatic hydrolysis was the ratelimiting step for ethanol production from RPS for most of the duration of the SSF process as already observed by other authors [31,32]. In order to allow the comparison between SHF and SSF, results were expressed as a percentage of an overall theoretical yield calculated by assuming that all the potential glucose and xylose in the sludge starting material is available for fermentation with a yield of 0.51 g ethanol g1 glucose (or xylose). On this basis, the highest ethanol yield was equivalent to approximately 54% and 51% conversion of the available carbohydrates in the initial sludge, respectively, for SHF and SSF. The obtained ethanol yields may be considered rather low, but they are comparable with data reported in literature. For example, Kadar et al. [3] obtained 58.1% and 59.7% conversion to ethanol for a 72-h SSF of paper sludge using Kluyveromyces marxianus and S. cerevisiae, respectively. The product concentration obtained in the present study for the SSF process (18.6 g L1 after 48 h) is considerably higher than the ethanol concentrations (8.8 and 9.0 g L1) attained in the cited work [3]. However, it has to be noted that these authors used a substantially lower substrate concentration in

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terms of carbohydrate mass (2.7% instead of 7.5% (w/v) in the present study). In the SSF, the extent of carbohydrate conversion into monosaccharides is slightly lower than the one obtained in the enzymatic hydrolysis prior to fermentation in the SHF process. This may appear to be in disagreement with the reduction in end-product inhibition of b-glucosidase that occurs in the course of the SSF process as a result of the rapid fermentation of the released glucose [33]. This observation might be explained by the difference in process temperatures. The SSF process was carried out at the temperature selected for the fermentation step, 30 1C, whereas the hydrolysis of RPS in the SHF was performed at 35 1C, which corresponds to a higher enzyme activity. However, SSF was effective for accelerating ethanol production (as judged by a higher ethanol volumetric production rate) when compared with the SHF process, as shown by other workers [34]. Moreover, the fermentation step on SHF was preceded by a prolonged period of 5 days of enzymatic hydrolysis which must be accounted for in the total residence time, whereas the SSF process was completed after 2 days of incubation. Nowithstanding these results, the SSF process should be improved in order to obtain economically recoverable ethanol concentrations, e.g., greater than 40 g L1 [7,20]. Since a progressive decrease on slurry viscosity was observed during the course of batch conversion, the bioconversion of RPS might be conducted under fed-batch SSF conditions, with periodic addition of fresh substrate, so as to overcome the limit to high substrate concentrations imposed by mixing constraints. This operational procedure will allow the achievement of higher SSF yields at lower enzyme loading, together with higher ethanol concentrations and therefore reducing the product recovery cost, as reported by several authors [79,35,36]. For instance, Fan and Lynd [8], on semicontinuous SSF experiments with paper sludge have achieved a steady state with an average ethanol concentration of 45.2 g L1 corresponding to a cellulose conversion of 95.8%.

plan and the critical reading of the manuscript, Eng. Mario Lopes and Eng. Raquel Pereira of Renova, S.A. (Torres Novas, Portugal) for providing the sludge material and Eng. Claudia Fonseca from Univar Iberia, S.A. for providing the enzymes from Novozymes. The technical assistance of Ceu Penedo and Amelia Marques is also acknowledged.
R E F E R E N C E S

4.

Conclusions

P. stipitis CBS 5773 is able of active fermentation to produce ethanol in a simple and straightforward SSF process using RPS as substrate. This means that there is no sludge component that prevents the use of biological conversion techniques and the process was efcient without the need for any pre-treatment or supplementation of the sludge material. Moreover, RPS has negative cost as a raw material for bioconversion. This process may thereby represent an opportunity for the reduction of an important residue stream generated from the recycling process, having direct benets in the reduction of landlling costs while producing a commercial product. In conclusion, RPS represents a potential resource to be used by other industries to obtain useful value-added products.

Acknowledgements
The authors gratefully acknowledge Prof. Helena Pinheiro (IST) for the valuable discussions concerning the working

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