A host of developments in the world of enzyme research help bring fresh ideas to papermakers keen on using biotechnology to aid mill performance

by Roger Grant

Enzymes come under the microscope

Only a few simple facts are needed to grasp the basics in the field of enzymes. Enzymes are not living matter, they are chemicals which are very specific catalysts. The presence of a catalyst accelerates a reaction, although the enzyme remains unchanged, and being "specific" means that each enzyme catalyzes a very narrow range of (biochemical) reactions. This sort of fine-tuning is not possible with most other chemicals. The downside to this specific nature is that the correct enzymes must be found to catalyze a particular set of reactions and they must be provided with optimum reaction conditions such as temperature and pH.

Enzymes are named, according to international convention, by adding the suffix "ase" to the substance or chemical group whose attack they catalyze. Here "attack" usually means oxidation or hydrolysis. For example, a cellulase catalyzes the attack of cellulose. A few traditional names are still used, however, such as trypsin, which breaks down food in the pancreas. Enzymes are produced by some living organisms, but they are increasingly synthesized to provide the large quantities required. Biotechnological manipulation of their DNA helps to transfer characteristics between enzymes.

Chip pile

Early biopulping visionaries predicted that chemical pulping would take place in the chip pile, but they quickly encountered chip penetration problems. Successes in enzymatically removing pitch from chips has not been forgotten, however. Further work has exploited the dissolution of the fibers' pit membranes that enzymes catalyze, opening up a network of gaps through the chips ⁽¹⁾. This network roughly doubles the longitudinal diffusion rate of caustic soda into sapwood chips (of sycamore and southern pine) and increases the tangential diffusion rate by 15-60%. When applied to kraft pulping, chip pretreatment with enzymes has been shown to help delignification by some 10%. A cellulase/ hemicellulase mixture gave higher pulp brightness, while the inclusion of a pectinase conserved pulp viscosity better.

Dissolving pulp is characterized by its purity, which includes low contents of hemicellulose, lignin and extractives, accompanied by adequate viscosity. A preliminary study indicates that this purity can be achieved using either bleached hardwood kraft market pulp or even high quality wastepaper rich in hardwood fiber as raw materials ⁽²⁾. The processing sequence included two stages of cold alkali extraction, with xylanase treatment in between.

Last year's review (PPI August 1997, p38) reported the recent interest in laccase (a polyphenol oxidase of molecular weight around 70,000), when used in conjunction with a "mediator" (an organic molecule), as a possible second generation bleaching enzyme. HBT (1-hydroxybenzotriazole) is the mediator most widely studied. The Finnish Pulp & Paper Research Institute (KCL) has now provided laboratory evidence that a laccase/HBT stage can replace the oxygen delignification stage (O) in the OQPZ/QP bleaching of pine kraft pulp. In two other TCF (totally chlorine-free) sequences, namely two-stage oxygen delignification of the same pulp followed by either QZP or QPZ/QP, the laccase/HBT stage not only successfully replaced the ozone (Z) stage, but also increased tear strength by 14-17% at a tensile index of 70 Nm/g ⁽³⁾.

These findings hold out promise for Munich's Consortium für Elektrochemische Industrie, which has shown NHA (N-hydroxy-N-phenylacetamide) to give superior delignification than HBT. The consortium call its laccase/NHA approach the Lignozym Process (also LIVIS). Further investigative work has pinpointed the functional group N-OH as an important contributor to the delignification power, and this group is common to both HBT and NHA. The work also showed that laccase/NHA can be applied in a single stage with a xylanase enzyme, whereas laccase/HBT applied similarly deactivates the xylanase ⁽⁴⁾.

Xylanases are well established for pulp bleaching, partly because because of their ability to aid reduction in chlorination chemicals, which translates into lower AOX in the mill effluent. Any accompanying production increase tends to be credited to the debottlenecking of the chlorine dioxide generator. However, Clariant's investigation of brightness development rate with their Cartazyme NST10 shows that enzyme usage not only accelerates the achievement of the first stage target brightness level, but that this effect may also occur in subsequent stages. There may therefore be a production increase available, that can apply in both ECF and TCF bleaching sequences ⁽⁵⁾.

Enzymes tolerant of higher temperature and pH conditions would allow greater flexibility in bleaching sequences and possibly faster delignification. To the commercial xylanases available Iogen Corp's BioBrite HB60C has been added to the list. It has been protein engineered to give optimum activity at about the 60C level. The Krebs Institute, at Sheffield University, has been inspired by the temperature tolerance of enzymes in washing machine powders, and particularly by the Pyroccocus furiosus bacterium that lives around land and sea vents of active volcanoes at temperatures above 100C ⁽⁶⁾. Comparison of its enzymes, with those living at normal temperatures, has shown that the complex cross-linked structure of the bacterium's surface provides the rigidity necessary to withstand high temperatures. Having characterized this structure chemically, they are now investigating the bacterium's genetic blueprint, with a view to inserting the genes responsible into the DNA of commercial enzymes.

KCL is also investigating the effect of enzymes on hexenuronic acids. These acids give kraft pulps their strong affinity for metals (relative to acid-cooked sulfite and mechanical pulps) due to their negatively charged carboxylic groups (-C:OOH), which attract metal cations like an ion exchange resin.

Deinking brightens

The application of enzymes to the deinking process has been comparatively slow, and little in the way of mill results has reached the literature. But now Stora Dalum has described its work with Enzymatic Deinking Technologies (Table 1) ⁽⁷⁾

Table 1 - Results of enzyme usage at Stora Dalum's deinking plant

Dalum's satellite deinking plant produces 220 tons/day of wet lap deinked pulp using mainly mixed office waste. The system includes primary flotation, dispersion, peroxide and FAS (formamidine sulfinic acid) bleaching stages and secondary flotation. Development of the optimum enzyme formulation for Dalum's configuration took more than one year of intensive laboratory research, so it is perhaps not surprising that its nature is not stated. Plant trials included changing the system from conventional high-pH chemical deinking to neutral enzymatic deinking, so as to reduce total cost. The mill was able to increase plant production by 3.8%, which resulted from improved equipment performance - particularly in the dewatering steps.

Rhodia, formerly Rhône-Poulenc, has provided guidelines for optimizing conditions for neutral deinking mixed office waste using its enzyme/surfactant combination (13). A hybrid flotation/washing approach is suggested, with the cellulase enzyme best added in the pulper. Neutral deinking is a departure from both conventional alkaline deinking and the acidic conditions sometimes used for old newspapers.

Deinking by magnetic separation is potentially interesting, except that not all the toner used in copiers and laser printers contains magnetite (typically magnetic iron oxide). For the first time, work at the University of British Columbia (UBC), Vancouver, has compared magnetic deinking with prior enzyme addition against flotation deinking ⁽⁸⁾. The maximum laboratory magnetic deinking efficiency of 94% fell within the commercial flotation range of 92-98.6%. However, the associated yield loss was only 2.8% - as compared with reported commercial flotation losses of up to 15%. Moreover, magnetic deinking involved no chemical additions other than the enzyme. Of the enzyme alternatives tried, a pure commercial endoglucanase/xylanase mixture proved the most effective, but the actual nature of the endoglucanase influenced the outcome.

Out with the trash

Lurking in all paper mill systems is anionic trash or, more scientifically, dissolved and colloidal substances (DCS). They create a variety of practical problems, which a mill tries to prevent by controlling the wet end chemistry. Now laboratory work, also at UBC ⁽⁹⁾, has found that fungal culture filtrates (e.g. from Trametes versicolor) can significantly reduce the DCS content of newsprint mill white water. Similar investigations in Finland ⁽¹⁰⁾ utilized Novo Nordisk's Resinase A lipase enzyme, which is added to stock before the paper machine to counter pitch-producing extractives. When applied to spruce TMP mill white water, this enzyme acted only on the triglycerides, which represented 36% of the lipophilic extractives present. Handsheet tensile strength improved, even though there was no overall change in extractives content.

Slime control is undergoing an evolution analogous to that in medicine when antibiotics were discovered in the 1940s. Prior to that, both medical ailments and slime had mercurial and other compounds thrown at them - not too healthy for humans. In slime biofilm control, these highly toxic compounds were replaced by others, synonymously named microbicides, biocides, fungicides, slimicides, etc. Their virtue lay in their toxicity being more specific to the components of biofilms and less so to human beings.

Enzymes have an even greater specificity, giving them a better chance of attacking the troublesome components. Practical applications have had to wait until research could decipher the nature of biofilms and their mechanisms of formation. Now two new product line announcements mark the clear arrival of enzyme-based antislimes.

The first of these is BetzDearborn's Spectrum family, which was announced in last year's review. It has been developed in response to such pressures as The European Biocides Directive, eco-labelling, ISO 1400 and mill water system closure. It has now become available on the market and among its toolkit of products is a gluconase enzyme, which catalyzes the attack of glucose. Not only does glucose represent a significant proportion of the polysaccharides in the EPS (extracellular polysaccharides) on paper machines (see Figure 1),

but BetzDearborn believes it to be probably the most troublesome. In addition, formation of this troublesome polysaccharide coating is inhibited by new and non-enzymatic materials in Spectrum, which interfere with the slime formation mechanism.

The other company to release enzymes to combat slime is Buckman Laboratories. It has launched its Neoteric family of products - each with its own Busperse and Buzyme number. The enzyme-based members include formulations of amylase enzymes, which catalyze the attack on starches, and protease enzymes, which catalyze the attack on proteins. Together with blended surfactants and (bio)dispersants, they penetrate biofilms, and disperse, remove and prevent their redeposition in the short circulation white water loop during paper machine operation. Other Neoteric biodispersants can be used during neutral pH boilouts, optimally in the pH range 5-8 and temperature range 50-70C, thus avoiding the need to use traditional acidic or alkaline chemicals.

Conventional effluent treatment is one of the oldest applications for enzymes, but renewed interest has arisen from their ability to reduce color. Laboratory work in India ⁽¹¹⁾ and South Africa ⁽¹²⁾ has thrown the spotlight on the fungal strains Rhizopus oryzae and Rhizomucor pusillus respectively. Thapar found the former strain able to reduce the color of first E-stage filtrate by up to 95% in the presence of glucose, as an easily metabolizable cosubstrate, and even by 78% when it was absent. Both strains also significantly reduced the COD and AOX. Typically the first caustic extraction filtrate contains about 80% of pulp mill effluent color.

Dr Roger Grant is editor of the Windows-based encyclopedia Paper Help. He can be contacted by fax on +44.1580.241950 in the UK

References

1. Jacobs, C.J. et al. Effect of enzymatic pretreatment on the diffusion of sodium hydroxide in wood. TAPPI Journal, Jan 1998, p 260. and Effect of enzyme pretreatments on conventional kraft pulping. TAPPI Journal, Feb 1998, p 143.

2. Jackson, L.S. et al. Production of dissolving pulp from recovered paper using enzymes. TAPPI Journal, March 1998, p 171.

3. Poppius-Levlin, K. et al. TCF bleaching of laccase/ mediator-treated kraft pulps. Proc. International Pulp Bleaching Conference 1998, Helsinki, Book 1, p 77.

4. Freudenreich, J. et al. Understanding the Lignozym Process. ibid, p 71.

5. Atkinson, D. et al. Enzymes make pulp bleaching faster. XXIII Jornadas Technicas Papeleras, Madrid, May 1998 (postponed).

6. Designing extremozymes. Chemistry in Britain, Jan 1998, p 18.

7. Knudson, O. et al. Long-term use of enzymatic deinking at Stora Dalum plant. Preprints of 7th International Conference on Biotechnology in the Pulp & Paper Industry, Vancouver, June 1998, p A17.

8. Gübitz, G.M . et al, Effectiveness of two endoglucanases from gloeophyllum sp. in deinking mixed office waste paper. ibid, p C 135.

9. Zhang, X. et al, Influence of accumulated dissolved and colloidal substances on paper properties and the potential of enzyme treatment for component removal. ibid p C 151 and C 155,

10. Buchert, J. et al. Enzymes for the improvement of paper machine runnability. ibid, p A225.

11. Nagarathnamma, R. and Bajpai, P. Decolourization and dechlorination of kraft bleach plant effluent by Rhizopus Oryzae. ibid, p C199.

12. Christov, L.P. et al. Modifying the quality of a bleach plant effluent using Mucoralean and white-rot fungi. ibid, p C203 and p C207.

13. Jobbing, J.M. and Franks, N.E. Enzymatic deinking of mixed office waste: process condition optimization. TAPPI Journal, Sept 1997, p 73.