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FAIR-CT95-0805
OXEPI: Oxidative enzymes for the pulp and paper industry |
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Contract No: | FAIR-CT95-0805 |
| Date Prepared: | December 1999 | |
| Source: | Final Report Abstract |
The project is divided into three parts covering the three main objectives:
Part A: Development of a lignin depolymerising enzyme system Known, previously characterised, oxidative enzymes were produced on laboratory and pilot scale. Purification methods for laccase were also developed on pilot scale. These enzymes participated in basic mechanistic studies, as controls in the screening for novel, more efficient enzymes and in the development of bleaching and mechanical pulp production based on the best oxidative enzyme.
Lignin model compounds including dimers, oligomers and synthetic lignin (DHP) were synthesised and used for mechanistic studies and to create a new technique for the screening of oxidative enzymes. By scintillation counting of the radioactivity released from kraft pulp previously labelled with 14C-DHP by covalent bonding, it was possible to evaluate the efficiency of new enzymes or enzyme/mediator couples in lignin depolymerisation. The 14C-test system was down-scaled to 40 mg labelled pulp suspended in 1.5 ml buffer, thus allowing for high throughput assays to assist in the screening for novel enzymes and to determine the optimum reaction conditions at minimum amounts of enzymes needed.
Mechanistic studies of enzyme/mediator reactions on lignin model compounds and on pulp lignin revealed that polymerised lignin products with molecular masses higher than 4800 Da were produced when laccase had reacted with pulp, while products lower than 4800 Da indicated depolymerisation when HBT was included as mediator. It can be concluded that the presence of HBT prevents repolymerisation. Laccase/HBT treatment caused significant demethylation of pulp lignin, a decrease in phenolic hydroxyl groups and an increase in carbonyl and carboxyl groups, structural modifications that are important for activating the residual lignin for final bleaching. Phenolic lignin units with biphenyl structure were found to be degraded during laccase/HBT treatment. These structures are prominent in lignin after oxygen delignification and are known to be very stable against most delignification and bleaching chemicals. It was also found with dimeric model compounds that laccase/HBT couples performing well on kraft lignin degraded non-phenolic models at a higher rate.
Kinetic studies into the degradation of linear beta-0-4 models by highly purified MnP and LiP showed that both enzymes more readily oxidise the smaller substances without mediators. These data also suggest that MnP does not directly interact with oligomeric lignin in vivo. A more probable mechanism is that they convey the oxidising potential of its active site by oxidising Mn2+ which in turn acts as a diffusible oxidant. For LiP it was found that it is equally adapted at oxidising small or large aromatic substrates but the result does not preclude that LiP only functions directly with the lignin polymer in nature.
To find the optimal conditions and co-factors involved in bleaching with manganese peroxidase biobleaching of eucalyptus kraft pulp by fungal cultures of Bjerkandera BOS55 was studied in detail using a newly developed in vivo biobleaching assay. It was found that Mn was not essential for biobleaching; organic acids stimulated biobleaching in the presence and absence of Mn; MnP and LiP directly decrease kappa number in vitro. The production of MnP by Bjerkandera BOS55 is stimulated by nitrogen and manganese as well as by organic acids.
Part B: Screening for novel, more efficient enzymes and development of a production system The screening systems were set up co-operatively. It was agreed by all partners that peroxidases (manganese peroxidase) and laccases were the fungal enzymes to be investigated. For novel laccases, the enzyme from Trametes hirsuta was used as a control e.g. the novel enzymes should have improved capabilities. In case of manganese peroxidase the question to be answered was whether this enzyme could fulfil the needs of pulp and paper industry.
A total of 571 fungi and 250 actinomycetes were screened. The enzymes from micro- organisms, selected according to the first steps of the screening scheme, were evaluated for their lignin degrading potential by the 14C-in vitro test system. In order to learn more about the novel enzymes and to evaluate the reliability of the 14C-in vitro test, fungal enzyme preparations were evaluated additionally by an oxygen consumption method, an adopted biobleaching assay and by the usual kappa number determination. Considering all screening results, strain 95290 (KL F10) showed the best performance. Therefore this enzyme was chosen for further evaluation. It was revealed by the 14C-in vitro test and by additional experiments that this enzyme is very stable at 60·C (75% activity after 1 h) and against HBT and that it reacts well up to pH 6. The production level unfortunately was not too high, but sufficient to produce the enzyme for application studies (see below).
The enzyme production by the selected streptomycetes was optimised. The enzyme levels were higher in solid substrate fermentations and the water activity proved to be a key factor. The enzymes produced by streptomycetes showed interesting properties, the production level, however, was not high enough for the evaluation of lignin degradation in the in vitro test system. It is assumed that more knowledge has to be gathered on the reaction conditions of these enzymes.
Medium optimisation for the fungal strain 95290 had to be carried out since soybean- medium used in the screening contained small particles and was not suitable for scale up and enzyme purification. A soluble medium was developed, the optimum conditions concerning oxygen supply and agitation and the inoculum size for scale up were evaluated. Manganese peroxidase production from Bjerkandera BOS55 was optimised in flasks and in stirred tank reactors.
After the stability of the enzyme preparation of strain 95290 was tested and the best conditions for transport were established, crude enzyme preparations from 1,000 litres of fermentations (equal to 4,500,000 nkat) were distributed to the partners. In addition to the tasks of the project, two laccases from strain 95290 were purified and characterised.
MnP production by Bjerkandera BOS55 was investigated. Nutrition and environmental conditions, aeration and agitation rate were optimised and a significant increase in stability and production of the enzyme was achieved. Thus sufficient enzyme for enzyme-aided bleaching trials could be sent to the partners together with a protocol how to perform the assays in order to obtain the best results.
Trametes laccase genes were introduced into Aspergillus awamori under control of glucoamylase promoter and it was tried to produce Trametes laccase in transformants of A. awamori in batch cultures. However, all transformants tested produced only low levels of laccase indicating that the transformation was successful but overexpression could not be achieved.
Part C: Development of oxidative enzyme bleaching and mechanical pulp production. Three different chemical pulps, i.e. pine kraft pulp, pine kraft pulp delignified by a two-stage oxygen treatment and birch peroxyformic acid (MILOX) pulp were used as initial pulps for the delignification studies in the presence of two known mediators (HBT and ABTS) and subsequent TCF bleaching. Some new mediators were also tested. The results show that different pulps behave differently in the laccase/mediator treatments. This may be due to different amounts of residual lignin present in the pulps and partly to the different chemical structure of the residual lignins. The oxygen delignified pulp was most accessible towards the mediated enzyme treatment when delignified with the HBT-aided laccase system followed by alkaline extraction to kappa 3.6 corresponding to a delignification degree of nearly 60% in this stage. Under the same reaction conditions the degree of delignification was 43% for pine kraft pulp and 24-30% for high and low kappa number birch MILLOX pulp, respectively. HBT was slightly more effective on both, a weight and a molar basis, than ABTS. Neither laccase alone nor mediator alone gave any delignification. Results obtained by isolated lignins showed that HBT enhanced the reactivity of laccase even when no pulp matrix was present. Laccase in the presence of HBT is a very selective delignification agent, i.e. a high degree of delignification was obtained, while the pulp carbohydrates remained unaffected. Laccse/HBT did not oxidise the hydroxyl groups of pulp carbohydrates to alkali-labile carbonyl groups. Neither did the hexuronic acid groups of the pulps react.
All the laccase/HBT-treated pulps showed increased reactivity towards alkaline hydrogen peroxide, and as a consequence, oxygen-delignified kraft pulp and MILOX pulp reached full brightness (88-90%) with alkaline peroxide in one and two stages, respectively. Xylanase in the bleaching sequence, either before or together with the laccase/HBT stage, slightly improved the pulp brightness after peroxide bleaching. The brightness was further improved by employing reductive bleaching with sodium dithionite as the final stage. TCF bleaching sequences including a peracetic acid stage or a second mediated laccase stage were also evaluated.
In OQPZ/QP (P: peroxide, Z: ozone) bleaching of pine kraft pulp, the oxygen stage could be replaced by laccase/HBT and subsequent alkaline extraction. Both sequences gave pulps with equal brightness, kappa numbers and viscosities. In the final bleaching of two stage oxygen-delignified pine kraft pulp by a QZP sequence or a longer and gentler QPZ/QP sequence, the Z stage and the PZ stage were successfully replaced by a laccase/HBT stage. Both pulps bleached with the laccase-containing sequence had better papermaking properties than the references. Laccase in the bleaching sequence reduced the energy needed in PFI beating to obtain a certain tensile strength. The most significant improvement by laccase treatment was in the tear strengths, which were 14-17% higher than those of the reference tensile index 70 Nm/g. A further advantage of the laccase/mediator stage in bleaching is that in many cases no significant investments are needed.
Laccases produced from two basidiomycetes, Trametes versicolor and the novel strain 95290 (KL F 10) screened during the OXEPI project were tested in the TMP process at different stages on:
Depending on the enzymatic treatment conditions, the effects on the fibre and pulp properties were different. The laccases seemed to have an impact on the fibre surface chemistry, on the lignin structure, on the mechanical behaviour and on the peroxide bleachability.
If applied on the primary- or secondary-refined pulps, the laccases induced some paper strength and brightness decrease. The modification of the fibre surface chemistry allowed to enhance the improvement of pulp quality during peroxide bleaching. When applied on the screening rejects, the effects on the final pulp quality depended on the nature of the laccases. Strain 95290 from Kaiserslautern induced a better bleachability of the final TMP. Finally, the use of laccases on wood chips was perhaps the most interesting application. Energy savings of up to 25% were observed at pilot plant scale. The pulp quality was slightly decreased but optimisation of this treatment could maintain the pulp strength and brightness. With the Trametes laccase, the pilot plant experiment gave important results and confirmed the results obtained at laboratory scale.
Laboratory bleaching experiments were carried out by the industrial partners on OO-prebleached soft wood kraft pulp (laccase/HBT), on oxygen-prebleached Eucalyptus grandis kraft pulp (Laccase/HBT; MnP), and on oxygen-prebleached Magnefite spruce pulp (laccase/HBT; MnP). On all pulps promising effects were identified when a laccase or MnP step was introduced. The general view of the industrial partners is to continue research and development on pilot scale and to carry out mill trials with a commercial laccase and a cheap and environmentally friendly mediator.
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