Zawadi A. Chipeta1, James C. du Preez1 and Lew Christov1,2

Company/Organisation and addresses

1Department of Microbiology, Biochemistry and Food Science/UNESCO MIRCEN, University of the Free State, PO Box 339, 9300 Bloemfontein, South Africa
2Sappi Management Services, PO Box 3252, 1560 Springs, South Africa



spent sulphite liquor, xylanase, Aspergillus oryzae, Aspergillus phoenicis, biobleaching, chlorine dioxide, soda-aq pulp


The utilisation of pulp mill effluents as carbon feedstock for microbial xylanase production is an unexplored field of research. In this study, the use of concentrated spent sulphite liquor (SSLc) as carbon substrate for xylanase production by the filamentous fungi Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157, was investigated. The main sugar in this effluent was D-xylose, with D-glucose and D-galactose each amounting to less than 10% of the total sugar concentration in the effluent.  Shake flask cultivations yielded relatively high xylanase activities of up to 156 and 147 U/ml with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157, respectively, in a medium based on concentrated SSL.  In a medium containing xylan as carbon substrate, the above strains produced xylanase activities of up to only 77 and 74 U/ml, respectively. These results indicated that the SSL concentrate constituted a suitable carbon feedstock for xylanase production by these Aspergillus strains. The xylanase of A. oryzae NRRL 3485 exhibited a maximum activity at an assay pH of 6.5 and at 65oC, whereas the optimum pH and temperature of the xylanase of A. phoenicis ATCC 13157 was 5.0 and 55C, respectively. Pretreatment of soda-aq pulp with crude xylanases obtained from the Aspergillus strains grown on the SSL concentrate, enhanced pulp brightness by up to 1.3 and 1.5 brightness points, respectively, over the control.  The usage of chlorine dioxide could be reduced by 30%, which would impact positively on the environment. The process developed could be advantageous for utilising industrial wastewaters as inexpensive and abundant carbon source for xylanase production and on-site use as a bleaching agent.


Xylanases are produced by a variety of microorganisms and catalyse the hydrolysis of the
$-1,4-glycosidic bonds of xylan.  One of the most important biotechnological applications of xylanolytic enzymes in industry is their use in pulp bleaching.  Xylanase preparations used in pulp bleaching, however, have to be free from cellulolytic enzymes so as to avoid losses in pulp yield and decreased pulp strength and viscosity1,2.

Limited research has been conducted on the production of xylanases using pulp mill effluents as carbon feedstock. Spent sulphite liquor (SSL), generated by the delignification process of wood chips, may contain 11 to 14 % solids with pentose and hexose sugars constituting 20 to 30 % of this fraction, depending on the type of wood used3,4.  The presence of these sugars in relatively high amounts renders SSL an attractive carbon feedstock for microbial cultivation.

We report here on xylanase production by two Aspergillus strains using concentrated spent sulphite liquor as carbon substrates. The crude enzymes were also evaluated in terms of their biobleaching efficacy when applied in pre-treatment of hardwood soda-aq pulp.


3.1 Pulp Mill Effluent
The pulp mill effluent investigated was a spent sulphite liquor concentrate (designated SSLc). The SSLc was used as carbon substrate and was diluted ten-fold with distilled water during medium preparation.

3.2  Sugar Determination
The sugar composition of the above effluent as well as that of the culture supernatants was determined using a Dionex 4000I HPLC (Dionex Corp., Sunnyvale, CA, USA) equipped with a pulse amperometric detector and gold electrode, using a 250 x 4 mm (I.D.) PA1 Carbopac column (Dionex Corp.) with inlet and oven temperatures of 30oC and 1.8 mM NaOH as eluent at a flow rate of 1 ml/min.

3.3  Microorganisms
Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 were evaluated for xylanase production using xylan and the SSL concentrates.  The cultures were maintained on Sabouraud-dextrose agar slants (Biolab, Biolab Diagnostics, Midrand, South Africa) and stored at 4C with subculturing at 12-week intervals.

3.4  Xylanase Production
The two fungal strains were evaluated for xylanase production using SSLc diluted ten-fold with distilled water as the carbon substrate.  Oat spelts xylan (10 g/l, Sigma) was used as carbon substrate in the control medium.  The other medium constituents were (g/l): citric acid, 0.25; yeast extract, 10; (NH4)2SO4, 5; K2HPO4, 5; MgSO4.7H2O, 0.5; CaCl2.2H2O, 0.02 and 1 ml of a trace element solution5.

The media were adjusted to an initial pH of 6.0 prior to autoclaving.  The inoculum was prepared by inoculating 500 ml Erlenmeyer shake flasks containing 100 ml of the appropriate medium with 5 ml of a fungal spore suspension washed from a 5-day old Sabouraud-dextrose agar plate culture with 10 ml of a 0.05 M KH2PO4 solution containing 0.1 % Tween 80. These flasks were incubated on a rotary shaker at 30oC for 48h. Similar shake flasks were subsequently inoculated with 5 ml of this inoculum and incubated on a rotary shaker at 30oC for 5 days.

The xylanase activity in the culture supernatants was determined using the 3,5-dinitrosalycylic acid (DNS) assay6 at pH 6.0 and 50oC with birch wood xylan (Sigma Chemical Co, St Louis, MO, USA) as substrate.  The biomass concentration was gravimetrically determined after centrifugation of 5 ml aliquots of the respective cultures, washing twice with distilled water and drying at 105oC to constant mass.

3.5  Xylanase Temperature and pH Optima
The pH and temperature optima of the crude xylanase preparations (i.e. the culture supernatants) from the SSLc-based cultures were determined.  The xylanase activity in the culture supernatants were assayed at 50 C at different pH values using 0.5 M citrate buffer (pH 4.0, 5.0, 5.5, 6.0), 0.1 M phosphate buffer (pH 6.5, 7.0, 8.0) and 0.1 M carbonate-bicarbonate buffer (pH 9.0, 10.0).  The optimal temperature at pH 6.0 was determined by conducting the enzyme assay in a water bath at temperatures ranging from 40 to 80oC. The reactions were stopped after a 5 min incubation period by the addition of dinitrosalicylic acid and the xylanase activity determined6.

3.6  Biobleaching with Xylanase
Crude xylanase preparations of A. oryzae NRRL 3485 and A. phoenicis ATCC 13157 grown on xylan and SSLc were applied to the unbleached soda-aq pulp.  An enzyme charge of 10 U/g dry pulp equivalent was applied at pH 6.0 and 50C using the xylanase of A. phoenicis and at pH 7.0 and 70oC in the case of A. oryzae.  A pulp consistency of 10% was maintained and the aliquots were incubated for 2 h then chemically bleached using a DED bleaching sequence. Subsequently, the brightness was determined using a Color Touch 2 brightness meter (Technidyne Corp., New Albany, Indiana, USA).


4.1  Sugar Composition of SSL Effluents
The sugar composition of the raw and concentrated SSL is shown in Table 4.1. Overall, concentration of raw SSL increased the sugar content 4.5-5-fold. In both effluents, D-xylose was the predominant sugar constituent, amounting to 85-87 % of the total sugars. The high sugar concentrations present in the SSL rendered these effluents attractive as a carbon feedstock for microbial cultivation.

Table 4.1  Sugar composition of the spent sulphite liquor effluents


Constituents (g/l)





Total sugars













SSL  raw SSL effluent
SSLc concentrated SSL effluent

4.2  Xylanase Production on Xylan and Effluent-based Media
The cultivation profiles for A. oryzae and A. phoenicis using SSLc and xylan as carbon substrates are shown in Fig. 4.1.  Higher xylanase activities were obtained with SSLc than with xylan as carbon source. Both glucose and xylose were readily utilised by these fungi.  After 24 h of cultivation, the concentrations of both glucose and xylose in the effluent -based medium were below 0.2 g/l.  These low sugar levels coincided with the period of rapid xylanase production (Fig. 4.1).  This observation suggested that xylanase production was derepressed when the concentrations of xylose and/or glucose had decreased to below a threshold level. This correlates with findings of others who reported that xylanase production was inhibited by xylose and glucose7,8.  This phenomenon, however, needs further investigation. The acetic acid in the SSLc-based medium was 2.8 g/l and was depleted after 72 and 48 h of cultivation of A. oryzae and A. phoenicis, respectively.

4.4  pH and Temperature Optima of Xylanase
The temperature and pH optimum exhibited by the  A. phoenicis xylanase (55oC and pH 5.0, respectively) was typical of fungal xylanases, whereas the A. oryzae xylanase had an unusually high temperature and pH optimum (65oC and pH 6.5, respectively).

Figure 4.1

Figure 4.1 Profiles of xylanase activity, growth of, sugar and acetic acid utilisation by A. oryzae NRRL 3485 (A) and A. phoenicis ATCC 13157 (B) with xylan and SSL concentrate (SSLc) as the respective carbon substrates in shake flask cultures at 30oC. Open symbols, SSLc; solid symbols, xylan; , total sugars; , acetic acid.

4.5  Application of Xylanases in Biobleaching
Unbleached soda-aq post oxygen pulp was pretreated with crude xylanases from A. oryzae and A. phoenicis grown on xylan and SSLc (Table 4.2).  Using the full chlorine dioxide charge, a biobleaching effect was produced with both xylanases.  The crude xylanase of A. phoenicis, grown on the SSLc, induced the highest gain of 1.5 brightness points over control (Table 4 .2) and overall, the bleaching efficiency of this enzyme was superior to that of the A. oryzae xylanase (Table 4.2).  These results demonstrated that the SSL concentrate had potential to serve as carbon feedstock for the production of fungal xylanases suitable for application in biobleaching in the pulp and paper industry.  Further work, however, is required to determine the optimal enzyme charge for biobleaching.

Table 4.2  Brightness change (points) over control during biobleaching of soda-aq pulp with crude xylanases produced by A. oryzae NRRL 3485a and A. phoenicis ATCC 13157b using xylan and SSLc as carbon substrates

Carbon substrate

Reduction in active chlorine charge over control

0 %






1.0a / 1.5b

0.7 / 0.9

0.4 / 0.7

-0.1 / 0.5

-0.5 / 0.4


1.1 / 1.3

0.9 / 0.7

0.6 / 0.5

0.5 / 0.3

0.3 / 0.1

Brightness of control 87%
SSLc   SSL concentrate


The results indicated the potential of the SSL concentrate to serve as carbon feedstock for microbial enzyme production. Rapid xylanase production coincided with low concentrations of xylose and glucose in the SSL-based medium, suggesting that xylose and/or glucose repressed xylanase production. The effect of xylose and glucose concentration during xylanase production will have to be investigated further.  The pH and temperature optima exhibited by A. oryzae NRRL 3485 was exceptional to fungal xylanases. The application of the crude xylanase preparations in ECF pulp bleaching enhanced brightness by up to 1.5 points over that of control. Alternatively, savings of up to 30% could be attained. The use of the SSLc as inexpensive and abundant carbon feedstock could facilitate the industrial production of enzymes for use in biobleaching. This would result in a decreased consumption of chlorine-based chemicals and also reduce the level of hazardous chlorinated organics in the mill effluent.


The authors wish to acknowledge Sappi Management Services, Sappi Saiccor, the Water Research Commission and the National Research Foundation for funding this project and Sappi Management Services for granting permission to publish this work.


1. Bailey, M. J., and L. Viikari., "Production of xylanases by Aspergillus fumigatus and Aspergillus oryzae on xylan-based media", World J. Microbiol. Biotechnol.  9: 80-84 (1993).

2. Viikari, L., A. Kantelinen, J. Sundquist and M. Linko., "Xylanases in biobleaching: From an idea to the industry", FEMS Microbiol. Rev. 13: 335-350 (1994).

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5. Du Preez, J.C. and van der Walt, J.P. "Fermentation of D-xylose to ethanol by a strain of Candida shehatae", Biotechnol. Lett. 5: 357-362 (1983).

6. Bailey, M.J., Biely, P. and Poutanen, K., "Interlaboratory testing of methods for assay of xylanase activity", J. Biotechnol. 23: 257-270 (1992).

7. Royer, J. C. and Nakas, J. P., "Interrelationship of xylanase induction and cellulase induction of Trichoderma longibrachiatum", Appl. Environ. Microbiol. 56: 2535-2539 (1990).

8. Pardo, A. G., Obertello, M. and Forchiassin, F., "Cellulose and xylan degrading enzymes in Thecotheus pelletieri", Revisita Argentina de Microbiologia. 32: 190-195 (2000).


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