Short communication
Chlorination and biodegradation of lignin
E. Johansson
a, C. Krantz-RuÈlcker
b, B.X. Zhang
c, G. OÈberg
a,*
a
Department of Water and Environmental Studies, LinkoÈping University, SE-583 81 LinkoÈping, Sweden
b
Department of Industrial Environmental Technique, LinkoÈping University, SE-583 81 LinkoÈping, Sweden
c
Institute of Soil Science, Academica Sinica, Nanjing, People's Republic of China
Accepted 16 December 1999
Abstract
Recent research has shown that large amounts of high-molecular weight organic chlorine of unknown origin are present in the terrestrial environment. There are indications that an underlying process may be microorganisms which produce reactive chlorine that chemically degrades organic matter and facilitates degradation of recalcitrant organic matter on one hand, and on the other hand causes a formation of organic chlorine. Our aim was to test one part of this hypothesis by investigating whether reactive chlorine facilitates microbial degradation of lignin. Dierent concentrations of chlorine dioxide were added to the autoclaved lignin suspension. Mycelium of the white-rot fungus P. chrysosporium was used to inoculate ¯asks with the lignin solutions. The evolution of CO2was followed during 8 d of continuous measurement. At the end of the experiment the solutions
were analyzed for organic chlorine. The amount of CO2 evolved was variable, but the results were repeatedable; addition of
chlorine dioxide to the lignin solutions caused an increase in the mineralization by P. chrysosporium that increased with increasing additions of chlorine dioxide. This suggests that exposure of lignin to reactive chlorine enhance its biodegradability. The most likely cause of the observed eect is that the addition of chlorine dioxide initiated a fragmentation and oxidation of the lignin, thus rendering a more easily degraded substrate. However, the results may also be interpreted as if an additional cause to the observed eect is that the chlorination in itself somehow enhanced degradation. The amount of organically-bound chlorine decreased during the incubation, and the decrease was more pronounced with the chlorination of lignin, whereas no change at all was observable in the control batches. This makes it tempting to suggest thatP. chrysosporium rather than having an enzyme system just capable of handling the chlorinated compounds, actually has a system that preferentially degrades such compounds.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Chlorination; Halogenation; Biodegradation; Biohalogenation; Biochlorination; Natural formation; Organochlorine; Organohalogen
Recent research has shown that large amounts of high-molecular weight organic chlorine of unknown origin are present in the terrestrial environment (Asplund and Grimvall, 1991; Grimvall and de Leer, 1995). It is possible that these compounds are formed by soil microorganisms (Hjelm, 1996; OÈberg et al., 1996, 1997; OÈberg, 1998; de Jong and Field, 1997). There are indications that an underlying process may be microorganisms which produce reactive chlorine
that chemically degrades organic matter and facilitates degradation of recalcitrant organic matter on one hand, and on the other hand causes a formation of organic chlorine (OÈberg et al., 1997). Our aim was to test one part of this hypothesis by investigating whether reactive chlorine facilitates microbial degra-dation of lignin.
All chemicals were of analytical grade, unless other-wise stated. Arti®cial alkali lignin (Aldrich) was added to 60 ml of a phosphate buer solution (NaH 2-PO4H2O, pH 3.5, ®nal concentration of lignin 25 mg mlÿ1) in four bottles and autoclaved for 20 min.
Dierent concentrations of chlorine dioxide (0.01, 0.1 or 1 mg Cl lÿ1) were added to the autoclaved lignin
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* Corresponding author. Tel.: +46-1328-2279; fax: +46-1313-3630.
suspension. The bottles with the lignin solution and the chlorine dioxide were placed on a rotary shaker at moderate speed (80 rev minÿ1) for 4 days at room
tem-perature to allow the chlorine dioxide to react with the lignin. The control contained everything except the chlorine dioxide and in all other respects it was treated as the other bottles. The autoclaving of the lignin sol-utions had caused some precipitation and the concen-tration of lignin in the solutions therefore varied. These were determined approximately by decanting the solution and weighing the dry mass of the precipitate. The concentrations of lignin were as follows: 8.7, 12.2, 9.3 and 4.4 mg lÿ1 starting with the control followed
by increasing concentration of chlorine dioxide in the treatments. The suspensions were analyzed for adsorb-able organic halogens (AOX) in six replicates for each bottle. The analyses were performed on an Euroglas 84/85 analyzer (Delft, Holland) according to a stan-dard procedure (DIN, 1985).
Mycelium of the white-rot fungus P. chrysosporium was inoculated on a malt agar plate and allowed to grow for a week at room temperature. A piece of fun-gal-laden agar was then added to a malt solution (100 ml, 2% Bacto malt extract ``Difco'', pH 4.75), placed on a rotary shaker (20 rev minÿ1) and allowed to grow
for 2 weeks at room temperature. Thereafter, the my-celium was harvested by centrifugation (10,000 rev minÿ1 for 15 min), washed three times with sterile
phosphate buer (NaH2PO4H2O, pH 3.5) and hom-ogenized in 100 ml of the same buer. Five ¯asks with lignin solutions had been prepared for each chlorine dioxide treatment (control, 0.01, 0.1 and 1 mg Cl lÿ1,
equalling 20 ¯asks) by adding 9 ml to each autoclaved ¯ask. One ml of the mycelium-buer solution was used to inoculate each of the 20 experimental ¯asks that were placed in an automatic respirometer at room tem-perature (208C; Respirocond III, Nordgren Inventions, UmeaÊ, Sweden). The evolved CO2 was trapped in 10 ml of 0.2 M NaOH during 8 days of continuous measurement. At the end of the experiment, the sol-ution in each of the 20 ¯asks was analyzed for AOX in duplicates.
The experiment was run again with nine dierent concentrations of chlorine dioxide with two or four replicates of each (0.001, 0.01, 0.02, 0.03, 0.06, 0.1, 0.3, 0.6, 1 mg Cl lÿ1). Controls were run in four replicates.
No precipitation of lignin was observed after autoclav-ing and the concentration of lignin was consequently 25 mg mlÿ1in all treatments.
All statistical analyses were 2-tailed at a 5% signi®-cance level. Kruskali-Wallis 1-way ANOVA was used to compare the dierent groups of samples and the correlation analyses were conducted using Kendall's tau b (Helsel and Hirsch, 1992).
The amount of organic chlorine in the lignin was 0.2 mg Cl gÿ1 C in the control (median value). The
amount increased with increasing chlorine concen-tration after the treatment with chlorine dioxide result-ing in median values of 0.3, 4.0 and 10.5 mg Cl gÿ1C,
respectively (Fig. 1). About 25% of the added chlorine was converted to organically-bound chlorine in two of the treatments (0.01 and 0.1 g Cl lÿ1) and about 3% in
the third treatment (1 g Cl lÿ1, data not shown).
The amount of CO2 evolved was variable (0.5±2.2 mg) the ®rst time the experiment was run and no clear pattern was observed. However, the median values showed a clear increasing trend if the values were shown as percentage of respired lignin. Still, no trends were distinguished when analyzing the whole data set statistically (Fig. 2). When comparing the groups, the amount of carbon respired in the group with the high-est amount of organic chlorine was signi®cantly larger than both the control and the group with lowest amounts of chlorine dioxide in the treatment (P = 0.047). No other signi®cant dierences were observed among the groups.
When the experiment was run again with nine con-centrations, the variation among replicates was smal-ler, and the evolved CO2 increased signi®cantly with increasing amounts of chlorine dioxide in the treat-ment (P< 0.001; Fig. 3).
At the end of the incubation, the concentration of organically-bound chlorine in the lignin had decreased signi®cantly in the two groups with highest amounts of
Fig. 1. Organic chlorine-to-carbon ratios in the lignin suspension after addition of chlorine dioxide. Bars represent median, minimum and maximum values of ®ve replicates.
E. Johansson et al. / Soil Biology & Biochemistry 32 (2000) 1029±1032
organic chlorine (P = 0.006; Fig. 4). No signi®cant decrease was observed for the other two groups.
The fraction of the lignin that was mineralized in our study was small, and the dierences between the treatments were equally small. Nevertheless, the results were repeatedable; addition of chlorine dioxide to the lignin solutions caused an increase in the mineraliz-ation by P. chrysosporium. Bergbauer et al. (1991) and Bergbauer and Eggert (1994) found that chlorinated pulp and paper euents were more easily degraded by Trametes versicolor than chlorine-free euent, which is in accord with our results. This suggests that exposure of lignin to reactive chlorine enhances its biodegrad-ability.
The most likely cause of the observed eect is that the addition of chlorine dioxide initiated a fragmenta-tion and oxidafragmenta-tion of the lignin, thus rendering a more easily degraded substrate. It is well known from the pulp and paper industry that addition of chlorine species, such as chlorine dioxide, causes oxidation and fragmentation, thereby reducing the amount of re-sidual lignin in the pulp. It is also well documented that the process also causes organic chlorine to be formed as a by-product (Dence and Annergren, 1979; Ni et al., 1993). This was also observed in our study, and the organic chlorine formed after addition of chlorine dioxide did not appear to hamper the positive eect of the fragmentation and oxidation, such that P. chrysosporium has an enzyme system capable of hand-ling the chlorinated compounds.
Fig. 2. Percent of lignin respired after 7 days of incubation withP. chrysosporium. The ®ve replicates of each chlorine dioxide treatment are shown and the median value is marked.
Fig. 3. Cumulated evolution of CO2after 7 days of incubation with
P. chrysosporium.The two to four replicates of each chlorine dioxide treatment are shown.
Fig. 4. Change in organic chlorine-to-carbon ratios after 7 days of incubation withP. chrysosporium.
Our results may be interpreted as if the chlorine itself enhanced the degradation. The amount of orga-nically-bound chlorine decreased during the incu-bation, and the decrease was more pronounced with the increase in chlorination of lignin, whereas no change at all was observable in the control batches (Fig. 4). This makes it tempting to suggest that P. chrysosporium rather than having an enzyme system just capable of handling the chlorinated compounds, actually has a system that preferentially degrades such compounds.
Our aim was to test a part of a larger hypothesis; i.e. that microorganisms produce reactive chlorine that enables degradation of organic matter with the for-mation of organic chlorine as a by-product. A number of dierent types of enzymes that catalyze chlorination have been identi®ed (e.g. Hager, 1966; Hewson and Hager, 1979; Dunford et al., 1987; KuÈsthardt et al., 1993; van Schijndel et al., 1993; Homann et al., 1998), and the most commonly proposed reaction mechanism is that hypochlorous acid is formed as a reaction intermediate. Studies on chlorine dioxide treatment of lignin have shown that hypochlorous acid is formed in a yield of 50±60% as a reaction inter-mediate (Dence and Annergren, 1979; Ni et al., 1993). Thus, it is likely that the reactions caused by chlorine dioxide in our study resemble, at least partly, the reac-tions caused by microbially-formed hypochlorous acid.
Thus it can be concluded that our study has strengthened the suggested hypothesis, but that it remains to elucidate if microbially-formed reactive chlorine enables degradation of organic matter, and if this process contributes signi®cantly to the organic chlorine found in soil.
Acknowledgements
We thank Dr. Per SandeÂn for advice on statistical issues and fruitful comments on the manuscript. The chlorine dioxide used in the experiments was a kind gift from AssiDomaÈn, SkaÈrblacka, Sweden. The Swed-ish Research Council ®nanced this study.
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