Electron beam irradiation pretreatment and enzymatic saccharification of used newsprint and paper mill wastes

Radiar. Phys. Chem. Vol. 29, No. 2, pp. 117-120. 1987 Inr. J. Radial. Appl. Instrum.. Parr C Printed in Great Britain. All rights reserved

0146-5724/87 $3.00+ 0.00 Copyright Q 1987Pergamon Journals Ltd

ELECTRON BEAM IRRADIATION PRETREATMENT AND ENZYMATIC SACCHARIFICATION OF USED NEWSPRINT AND- PAPER MILL WASTES A. WAHEED KHAN,’ JEAN-PIERRE LABRIE~ and JOSEPH MCKEOWN~ ‘Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada KIA ()R6 and *Accelerator Physics Branch, Chalk River Nuclear Laboratories, Chalk River, Ontario, Canada KOJ 1JO (Received 18 April 1986; in revisedform

12 June 1986)

Abstract-Electron beam pretreatment of used newsprint, pulp, as well as pulp recovered from clarifier sludge and paper mill sludge, caused the dissociation of cellulose from lignin, and rendered them suitable for enzymatic hydrolysis. A maximum dose of 1 MGy for newsprint and 1.5-2.0 MGy for pulp and paper mill sludge was required to render cellulose present in them in a form whichcould be enzymatically saccharified to 90% of completion. Saccharification approaching the theoretical yield was obtained in 2 days with a cellulolytic enzyme system obtained from Trichoderma reesei. As a result of irradiation, water soluble lignin breakdown products, NaOH- soluble lignin, free cellobiose, glucose, mannose, xylose and their polymers, and acetic acid were produced from these materials.

MATERIALS AND METHODS

INTRODU(JI1ON In the manufacture of paper, both chemical and mechanical processes are used for producing pulp. The chemical processes have a pulp yield of about 45%, and the mechanical processes have a yield of about 80-90%. A pulp and paper mill capable of producing 300,000 tons of pulp annually rejects about 370,000 tons of wood solids with a chemical process, and about 75,000 tons of wood solids with a mechanical process. Although the chemical processes are being phased out to reduce these wastes, there are still many paper mills using these processes. For example, in the province of Quebec in Canada, 28 pulp and paper mills use chemical processes and 31 use mechanical processes. As a result, over 7.9 million tons of waste are produced annually.“’ At present about 2% of the rejected wood solids are recycled, about 47% are incinerated and the remaining 51% are dumped as sludge. From both economical and pollution abatement points of view, reclaiming cellulose from sludge and from used newsprint is important. Earlier work carried out in out laboratories”) and elsewhereo4) has demonstrated the usefulness of electron heam irradiation as a pretreatment for softwood for subsequent enzymatic degradation to glucose. Softwoods are generally used in the manufacture of pulp and paper. This paper describes the effects of irradiation pretreatment on used newsprint and pulp and paper mill waste and their subsequent hydrolysis to sugars by fungal enzymes.

Issued as NRCC No. 216616.

Materials

Newsprint used in these tests was from the Ottawa Citizen Daily; Clarifier sludge, waste sludge and pulp samples were obtained from Canadian International Paper Products Ltd (Gatineau, Quebec), MacMillan Bloedel Ltd (Powell River, B.C.) and Ontario Paper Company (Thorold, Ontario). Clarifier sludge was centrifuged to recover pulp and then washed with water before use. Irradiation treatment Samples were irradiated at the desired doses using a 4 MeV electron beam. During irradiation, samples were kept at room temperature by cooling their containers with water circulation. The irradiation source has been described earlier.(2.5) Saccharljication For saccharification, the irradiated materials and the untreated material used as control were powered using a homogenizer (Omni-Mixer, Sorvall) to obtain a uniform sample. Saccharification was carried out using 5% solids suspended in 50mM citrate+NaOH buffer at pH 4.8, and enzyme preparation containing 30-35 IU of filter paper activity/g of material.‘6’ Controls containing non-irradiated paper or pulp and filter paper (Whatman No. 1) as substrates were also run along with each test. Incubation was carried out under aseptic conditions at 50°C for up to 3 days. A cellulolytic enyzme system was prepared by growing Trichoderma reesei RUT C-30 (NRRL No. 11,400) as described earlier.@) 117

A. WAHEEDKHAN et al.

118

Methods of analyses

drop in their cellulose content, probably due to its Dissociation of lignin and cellulose caused by breakdown into soluble components. On the other hand, the content of NaOH-extractable components irradiation treatment was evaluated from the weight increased as a result of irradiation treatment. In all loss that occurred as a result of washing a known the materials tested over 95% of the dry weight was weight of sample with 0.4% NaOH, followed by accounted for as the combined weight of NaOHwater to remove NaOH. Washing with NaOH extractable components and cellulose. As the ligninremoved lignin, and lignin-, cellukose-, and breakdown products are extractalbe in NaOH, this hemicellulose- breakdown products. Water extracts increase may indicate that the lignocellulosic complex were analysed for the estimation of free sugars, acetic is dissociated as a result of irradiation treatment. acid and lignin-breakdown products. Lignin and The amount of free acetic acid, sugars and U.V. l&&-breakdown products released were estimated absorbing materials present in both water and NaOH by measuring U.V. absorption at 281 nm.1.(7)As the extracts increased with increasing irradiation dose nature of these compounds is not known, their release (Table 2). The HPLC analysis of both the water and is reported as absorptivity in gl-’ cm-’ concenthe NaOH extract showed that the increase in U.V. tration. Furfural was separated from other U.V. ababsorbtion occurred mainly as a result of materials sorbing materials that originate from ligninother than furfural. Furfutal was present only in breakdown, by liquid chromatography (HPLC) using small amounts. The increase in U.V. absorbing matea polypore, PB column (Brownlee Laboratories Inc., rials indicates that the irradiation pretreatment disSanta Clara, California), and a U.V. detector set at 281 nm. The column temperature was kept at 80°C sociated lignin from cellulose by forming both waterproducts and NaOHand deionized water was used as a mobile phase at a soluble lignin-breakdown soluble lignin. Typical U.V. spectra of the lignin flow rate of 0.3 ml min-' . The presence of free volreleased from the newsprint as a result of increasing atile acids, such as acetic acid, was determined by gas chromatography.@’ Sugars released as a result of dose of irradiation are shown in Fig. 1. The release irradiation or enzymatic hydrolysis were estimated by of free acetic acid indicated the breakdown of acetyl a calorimetric method using dinitrosalicylic-acid re- units which are present in the hemicellulose fraction of softwoods.“*’ The HPLC analysis of the water agent,“) and the composition of sugar mixtures was determined by HPLC.‘r”) Cellulose content of the .extract obtained from irradiated samples showed the presence of xylose and mannose along with polymers NaOH-washed samples was determined using the containing these sugars. These sugars and acetate anthrone reagent.(“) appear to originate from the breakdown of a hemicellulose fraction as a result of irradiation. RESULTS AND DISCUSSION

Effect of irradiation on lignocellulosic complex

Enzymatic saccharifcation of irradiated materials

The amount

of water-soluble and NaOH-extractable materials in newsprint, pulp and paper mill wastes increased with increasing radiation dose (Table 1). Irradiation of newsprint at 1 MGy dose and other materials at 2 MGy dose cause a 2-6%

Irradiation pretreatment greatly improved the enzymatic saccharification of used newsprint and pulp and pulp wastes (Fig. 2). An irradiation dose’ of 1 MGy increased the enzymatic saccharification of newsprint from 5.2 to over 90% on the basis of

Table I. Cellulose, water-soluble and sodium hydroxide extractable materials present in some oauer mill oroducts and wastes. before and after irradiation tnatment Dose (MGv)

Water soluble’ (%)

NaOH extractableb f%)

Cellulosec (%j

Newsprint

None 0.5 1.0

IO 20 22

I6 I9 38

60 -

Pulp

None

I3 22 32

14 35 45

56 51

2.0

0 19 27

I7 36 M

50 47

None I .o 2.0

IO 21 30

I9 46 56

47 41

Materials

I .o

2.0 Pulp clarifier’s sludge

None

I .o Paper mill sludge

58

‘Extractable in warm water. bExtractablc in cold IN NaOH. This fraction also contains water-soluble components. CSoluble in 72% H,SO,, expressed as glucose equivalents. -Not determined.

Electron beam irradiation pretreatment Table 2. Formation

119

of lignin-breakdown products, acetic acid, free sugars and furfural increasing dose irradiation Water-soluble

Material

NaOHsxtractable Absorptivity (gl-‘cm-‘)

Acetic acid (mgg-‘)

as a result of

Free sugars’ (m.sg-‘1

Furfural @egg-‘)

Newsprint

None 0.25 0.50 0.75 1.00

0.06 0.18 0.25 0.32 0.38

0.4 0.9 1.1 1.5 1.7

1.8 3.2 4.8 5.8 6.0

0 8.8 15.4 21.7 26.7

0 17 28 38 40

Pulp

None 0.5 1.0 1.5 2.0

0.07 0.23 0.36 0.44 0.61

0.7 1.2 1.6 2.2 3.2

0 2.3 4.9 6.1 6.6

0 0.3 4.9 6.1 6.6

0 22 36 48 65

Pulp from clarifier’s sludge

None 0.5 1.0 1.5 2.0

0.02 0.17 0.29 0.39 0.49

0.6 1.2 2.2 2.9

0 I.1 3.1 4.6 4.9

0 8.7 15.0 22.4 30.5

0 18 26 32 40

Paper mill sludge

None 0.5 1.0 1.5 2.0

0.06 0.18 0.27 0.39 0.44

1.3 2.3 2.7 3.3 3.6

2.8 4.0 5.7 7.4 8.2

0 10.3 18.5 31.4 40.1

0 0 0 T T

‘Expressed

as ghtcose.

1.6

T = trace amounts. cellulose content. A higher irradiation dose than this had little or no further effect on its saccharification. On the other hand, a maximum dose of 1.5-2.0 MGy was required to achieve a similar improvement in the enzymatic saccharification of pulp and pulp waste. This difference in radiation dose requirement for the newsprint and pulp and pulp waste may be due to the processing steps used in the manufacture of newsprint from pulp. This beneficial effect was also obvious from the saccharification of untreated samples. For example, the untreated newsprint gave over 10% higher saccharification than untreated pulp samples. Paper mill sludge had the lowest cellulose content, a dirty black color, and after irradiation treatment it gave the lowest saccharification yield. This may be due to the presence of a number of chemicals and

other substances that are used for bleaching, etc. and are discarded along with unusable pulp, which interfere in the saccharification process. However, after irradiation pretreatment, a little over 60% of the cellulose present in sludge was converted into sugars. The glucose yield and the rate of saccharification increased with increasing irradiation dose (Fig. 3). Near maximum yields were obtained after 2 days of incubation; thereafter, the increase was small. Compared to untreated samples, the saccharification of irradiated samples increased from less than 10% to over 45% on a dry weight basis. In terms of cellulose

l-•

H-0 WASTE

0 230

260

290 WAVELENGTH

320

360

390

(nm)

Fig. 1. Typical U.V.spectra of the NaOH extract from used newsprint, (a) untreated, (b) irradiated at 0.25 MGy, (c) irradiated at 0.5 MGy, (d) irradiated at 0.75 MGy, and (e) irradiated at 1.OMGy.

0.5

IO

1.5

20

IRRADIATION

0.5 DOSE

I.o

SLUDGE I.5

2.0

(M.GY)

Fig. 2. Effect of irradiation pretreatment on the enzymatic saccharification of various paper mill products and wastes. Saccharification is expressed on a dry weight basis (a-a), as well as on the basis of cellulose content of the material (O-O).

A. WAHEEDKHAN

120

l_-•-

(01

.__o

(d)

l-o

Lc)

et

al.

ever, no such inhibition was noted in the case of irradiation pretreated materials. The saccharification of irradiated pulp and untreated filter paper used as control reached a maximum value in about 2 days (Fig. 3). During this time about 75% of the filter paper and 7040% of the cellulose present in the treatment pulp was converted to sugars. It appears that irradiation pretreatment is helpful in dissociating the lignocellulosic complex without the formation of harmful materials that inhibit the activity of cellulolytic enzymes. As a result, it may be possible to eliminate the additional treatment step that requires washing with alkali. Acknowledgements-The

authors with to thank Mrs Erin Meek for excellent technical assistance and Mr R. T. F. Bird for performing the electron beam irradiation of the samples.

INCUBATION

TIME

(DAYS)

Fig. 3. Effect of incubation time on enzymatic saccharification of pulp, (a) untreated, (b) irradiated at O.SMGy, (c) irradiated at l.OMGy, (d) irradiated at 1.5MGy, (e) irradiated at 2.0 MGy, and (f) untreated filter paper used as control. G/G = weight of sugar produced in grams, per gram of dry weight of the substrate.

of the pulp, this saccharification value for samples irradiated at 2 MGy represents over 80% hydrolysis. It is well known that the hydrolysis of cellulosic materials is carried out by synergic action of a number of enzymes and it depends upon the accessibility of these enzymes to cellulose.u3) For maximum accessibility, a complete dissociation between lignin and cellulose, an increase in the surface area and a decrease in the degree of polymerization in cellulose, are essential. Work carried out by Kumakura and Kaetsu(“) has shown that irradiation pretreatment increased the surface area by increasing the crushability of lignocellulosics to a fine powder, and decreased the degree of polymerization in cellulose. Results reported in this paper indicate that irradiation also dissociated the lignocellulosic complex. One of the best known pretreatments for hardwood is steam-explosive decompression.(r5.16) Materials obtained as a result of this treatment require washing with NaOH to remove lignin and ligninbreakdown products which inhibit cellulolytic enzymes and consequently saccharification.“@ Howcontent

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