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Journal of Environmental Quality 31:1004-1009 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America

TECHNICAL REPORTS
Waste Management

Kraft Mill Residues Effects on Monterey Pine Growth and Soil Microbial Activity

Miguel Jordan*,a, Miguel Angel Sánchezb, Leandro Padillab, Ricardo Céspedesb, Miguel Ossesc and Bernardo Gonzálezb

a Dep. of Ecology, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Alameda 340, Casilla 114-D, Santiago, Chile
b Dep. of Molecular Genetics and Microbiology, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Alameda 340, Casilla 114-D, Santiago, Chile
c Celulosa Arauco y Constitución, Arauco, Chile

* Corresponding author (mjordan{at}genes.bio.puc.cl)

Received for publication August 20, 2001.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The production of bleached Kraft pulp generates inorganic and organic residues that are usually deposited on the soil surface or land-filled. Studies conducted to address the impact of these wastes on the environment are scarce. In this work, Monterey pine (Pinus radiata D. Don), an important tree for pulping, was evaluated for germination and development under greenhouse conditions in forest soils exposed to solid residues of the cellulose industry using the Kraft process. Soils exposed to 10 to 60% ashes, 10 to 70% fly ashes, or 10 to 30% dregs allowed substantial seed germination and seedling growth. In contrast, soils exposed to low proportions of brown rejects, grits, or a mixture of all these residues were detrimental for germination, plant growth, or both. The strongest negative effect (no germination) was observed with as low as 10% grits. The changes in pH and/or water content caused by solid wastes did not correlate with detrimental effects observed in various soil–residue combinations. No significant changes in the microbial community of soils exposed to these solid residues were observed by determination of culturable counts, or by terminal-restriction fragment length polymorphism analysis of the microbial community DNA. The presence of organic residues did not affect the ability of the soil microbial community to remove typical pulp bleaching chloroaromatics. However, inorganic wastes strongly decreased the removal of such compounds.

Abbreviations: AOX, adsorbable organic halogen • CS, clay soil • GC-MS, gas chromatography–mass spectrometry, SS, sandy soil • T-RFLP, terminal-restriction fragment length polymorphism


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
THE PRODUCTION OF bleached Kraft pulp generates several inorganic residues, including ashes, fly ashes, dregs, and grits as well as organic residues, including primary sludge and brown stock screening rejects (Springer, 1993; Sherman, 1995). Ashes are produced in the power boiler, whereas grits and dregs originate during liquor caustisizing procedures. Primary sludge is cellulose fiber material settled in the clarifier during the treatment of bleaching effluents. Brown rejects, derived from the dewatering press, are mainly composed of semicooked small shives impregnated with some residual black liquor. Aerobic secondary treatment of these effluents also produces a considerable amount of residues as microbial biomass (Graves and Joyce, 1994). These residues are usually burnt and/or deposited on the soil surface or land-filled (Wiegand and Unwin, 1994). However, rigorous studies conducted to assess the impact of these wastes on the environment are scarce (Feagley et al., 1994; Vera and Servelo, 1994; Sherman, 1995). It is likely that some of these residues or their components may be harmful due to their content of chloroorganic compounds and heavy metals. It is also possible that inorganic and organic components of these solid residues may have a positive effect, especially in soils that have low organic matter and nutrient contents. This study focused on the effects that mixing these solid wastes with forest soils had on germination and growth of Monterey pine (Pinus radiata D. Don), an important tree species for pulping. Their effects on the microbial activity of such soils were also addressed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Soil, Seedlings, and Solid Waste Analysis
Two forest soils—a sandy soil (SS) and a clay soil (CS)—from the coast to the inland transect of Southern Chile (SL 37°S), were selected for this study. Chemical properties of these soils are shown in Table 1. The solid residues were collected from the Arauco Kraft pulp mill, located in the same area. The daily residue production corresponds to 150 to 300 wet Mg, about 75000 wet Mg yr-1, consisting of (approx. values): ashes (46%), sludges (23%), fly ashes (11%), dregs (15%), grits (3.0%), and brown rejects (2.0%) (Jordan et al., 2002). The chemical analysis, pH, and water content (by wt.) of the soil, solid residues, and seedlings were performed using standard procedures (Kalra, 1998). Essential elements and heavy metals were determined through atomic absorption spectrometry using a GBC 920 instrument (Victoria, Australia) and a Perkin Elmer, model 2380 (USA), respectively. The organic matter content was measured with a Helios unit (Unicamp, Cambridge, UK), while the adsorbable organic halogen (AOX) content was determined using an ECS 1000 instrument (Euroglas, the Netherlands). Chloroaromatic compounds were determined by gas chromatography–mass spectrometry (GC-MS) (Céspedes et al., 1996). The detection level for this analysis was 10 to 15 µg kg-1. Chloroaromatic compounds used as standards for identification included several mono, di- or trichlorinated derivatives of phenol, guaiacol, veratrol, syringol, and vanillin (Helix Biotech. Vancouver, BC, Canada).


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  		Table 1. Chemical characteristics of Kraft pulp mill solid residues and two forest soils used in this study.{dagger}

 
Growth Responses
One-year-old stratified Monterey pine seeds, obtained from a selected elite tree, were washed in Captan and Benlate (each 0.01%) for 8 h under constant agitation. Seeds were then rinsed in 2% (v/v) sodium hypochlorite, washed in distilled water, and germinated in 250-mL plastic containers under greenhouse conditions. Four seeds were used in each container and each treatment was replicated eight times arranged in a completely random design. Soil and residues were mixed in proportions ranging from 0 to 100% (v/v) of each solid residue plus sandy soil or clay soil. Seedlings were examined for size, after a period of 300 d. Size was determined measuring the height of the aerial part of the plant because is a nondestructive procedure. Shoot-tips and root-tips from selected samples were examined histologically by light microscopy after staining with safranine and fast-green. Growth conditions in the greenhouse were set for a summer ratio of 14 h light to 10 h darkness. Light intensities were 350 to 550 µmol m-1 s-2 from a metal halide light measured at the outer surface of the containers. The temperature range was 16 to 28°C. Seed germination and plant size differences were estimated by comparison of proportions (STATS) and using the Duncan's multiple range test, respectively.

Microbial Soil Responses
Five grams of soil were mixed (1:1, w/w) with each solid residue alone or with a mixture that corresponded to the average solid waste composition produced in the Arauco mill indicated above. Incubations were also performed by adding a mixture of representative pulp bleaching chloroaromatic compounds (Sherman, 1995)—2,4-dichlorophenol, 2,4,6-trichlorophenol, 4,5-dichloroguaiacol, and trichlorosyringol (5 mg kg-1 each)—to the soil–solid residue combination. Before incubation, soils and solid residues were air-dried and sieved (1-mm internal pore diam.). Incubations were performed in open beakers kept in a chamber at 20°C and 65% relative humidity for 0, 4, 30, or 60 d. Every 2 to 3 d, water was added to each beaker to bring the soil to field capacity. Incubation of samples was performed in three replicates. Samples were acetylated after extraction as described by Brezny et al. (1992) and Céspedes et al. (1996). An enrichment procedure to isolate bacteria able to grow in the chloroaromatic compounds indicated above was carried out as described by Matus et al. (1996). Microbial culturable counts were determined as described by Brezny et al. (1993).

Terminal-Restriction Fragment Length Polymorphism Analysis
Terminal-restriction fragment length polymorphism (T-RFLP) tests were performed by the following procedure. Community DNA was obtained using a soil DNA isolation kit (MOBIO, Solana Beach, GA), and used as the template for the polymerase chain reaction. Primer pairs 8F: 5' AGA GTT TGA TCC TGG CTC AG 3', and 1392R: 5' ACG GGC GGT GTG TAC 3', designed for eubacterial rDNA 16S sequences, were used. The primer 8F was labeled with the NED fluorochrome. Each 100-µL reaction (in Taq polymerase buffer) contained: 5 U of Taq polymerase (GIBCO-BRL), 20 pmol of each primer, 0.25 mM of each deoxynucleoside triphosphate, and 1.5 mM MgCl2. Polymerase chain reactions were carried out in a Perkin-Elmer 2400 thermal cycler with the following cycling conditions: 3 min of denaturation at 94°C, 35 cycles of 30 s at 94°C, 45 s at 56°C, and 2 min at 72°C with a final extension step of 3 min at 72°C. The polymerase chain reaction product was digested with the enzyme HhaI and the DNA fragments separated by capillary electrophoresis using a Perkin Elmer ABI Prism 310 sequencer. The T-RFLP profiles were plotted as peak area (abundance) against fragment size. The fragment size was estimated using the internal standard ROX 1000 as reference. Terminal-restriction fragments with a peak height of <80 fluorescence units were not included in the analysis. The area of each peak was standardized with respect to the sum of all peak areas of the sample. The standardized peak areas of the same fragment size in replicate profiles from the same experimental condition were averaged. To evaluate phylotype richness and diversity, the total number of different fragment sizes (between 60 and 600), and the averaged, standardized peak areas were used, respectively. However, it is accepted that each terminal-restriction fragment may represent more than one phylotype and that the peak area does not necessarily correspond to the abundance of each phylotype, because different phylotypes may have a different 16S rDNA dosage. The Shannon-Weiner diversity index (H) was calculated as follows:

where p is the proportion of an individual peak area relative to the sum of all peaks areas (Margalef, 1958). Friedman's nonparametric analysis of variance was carried out to compare experiments using peak areas (Conover, 1980).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Chemical Composition of Solid Residues
The chemical composition of solid waste residues and soils are presented in Table 1. Both soil types had high organic matter contents, but low levels of available N, P, and K, and other extractable elements. In the solid wastes, organic matter varied considerably; primary sludges and brown rejects had higher contents of organic matter, 645 and 946 g kg-1, respectively. The chemical analyses of these residues revealed that several essential elements were present. Calcium, Mg, K, and P, as well as various minor elements were generally found in higher levels compared with the forest soils. With respect to the AOX content, only the primary sludge had a low but significant value. This was expected because chlorinated compounds produced during bleaching become part of the material that settle during primary treatment (Springer, 1993; Sherman, 1995). A search using GC-MS analysis for typical pulp bleaching chlorinated compounds indicated a few, low intensity signals. Only minor signals corresponding to 2,3,5-trichlorophenol and 2,4-dichlorophenol, as well as an unidentified, oligomeric aromatic material, were observed in the primary sludge. The low AOX level and the almost complete absence of chloroaromatic, single-ring compounds is probably related to the fact that the mill is running a bleaching process with a low use of active chlorine species (Céspedes et al., 1996; Vicuña et al., 1997).

Monterey Pine Germination and Plant Growth
The effects on germination and plant growth of Monterey pine cultivated in soils amended with Kraft mill solid residues are summarized in Tables 2 and 3. An improvement in shoot length with respect to the control was observed in the presence of 10 to 60% ashes, 10 to 70% fly ashes, or with 10 to 30% dregs. Increasing proportions of these residues caused a decrease in germination and plant height, but the effect varied according to the type and amount of residue used. In general, the effect of the amount of each residue was not directly related to changes in pH and/or water content (Tables 2 and 3), since similar pH changes produced different effects (compare dregs with grits or brown rejects). In contrast, different changes in water content caused similar effects in germination and growth (compare dregs and primary sludge). Visual examination of selected seedlings showed that higher amounts of each residue or their mixture also resulted in marked reduction of root size. Light microscope observation of shoot and root meristems from these seedlings showed less cell division, while in the shoot and root elongation zone a diametrical cell growth and thickening was frequent (not shown). High proportions of primary sludge caused only a slight decrease in germination, but low proportions were detrimental for plant growth (Table 2). Three of these solid residues: brown rejects, grits, or the solid mixture limited growth and germination, even at low proportions (Table 3). The effect of brown rejects is probably related to poor oxygen supply due to the high water content (1000–5000 g kg-1) of these mixtures. However, the strongest negative effect was observed with grits. This may be explained by the high pH value (Table 3), the formation of a hard sealed crust in the soil surface, the high electrical conductivity (Table 1), and the high soluble Na (approx. 503 mg kg-1) of this residue. Accordingly, the inhibiting effect on germination of this residue, as well as the crust formation, could be reversed by a single application of 0.01% HCl before sowing, which lowered the pH.


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  		Table 2. Germination and growth of Monterey pine seedlings in the presence of clay soil–Kraft mill solid waste mixtures, under greenhouse conditions.{dagger}

 

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  		Table 3. Germination and growth of Monterey pine seedlings in the presence of sandy soil–Kraft mill solid waste mixtures, under greenhouse conditions.{dagger}

 
Seedlings grown for 300 d in contact with 1:1 soil/residue mixtures only showed minor mineral imbalances (Table 4). In addition, the high content of Fe and Mn in ashes, fly ashes, and dregs (Table 1) was not evidenced in the plants exposed to these residues (Table 4). Other studies (Jordan et al., 2002) showed that elements present in residues are not accumulated in leaves of Monterey pine trees growing for long periods close to dumps where these solid residues have been accumulated.


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  		Table 4. Nutrient concentration of shoot tips from Monterey pine seedlings grown for 300 d in the presence of various solid wastes form the Kraft process, under greenhouse conditions.{dagger}

 
Soil Microorganisms
Bacterial or fungal cultivable counts were not significantly affected by the presence of these solid residues in the soils during a 60-d incubation period. In all conditions and times assayed, the bacterial and fungal counts were typically 108 and 104 colony-forming units mL-1, respectively. On the other hand, the presence of primary sludge (the solid residue that contains chloroaromatics) did not allow isolation by enrichment procedures of bacterial strains degrading typical pulp bleaching chlorinated compounds. Negative results were also obtained when determinations of cultivable counts and presence of bacterial decomposers were carried out in soil–solid residue incubations to which a mixture of typical pulp bleaching chloroaromatic compounds was added (see below).

Determination of culturable counts only allows detection of 1 to 5% of soil microorganisms (Amann et al., 1995; Liu et al., 1997). It is possible that the presence of solid residues on soils changes the microbial community and that these variations remain undetected with the determination of viable counts. Therefore, a molecular approach, not based on cultivation, was also used. T-RFLP analysis of community DNA allows detection of 16S rDNA sequences that correspond to the presence of different bacterial species, either culturable or not culturable (Liu et al., 1997; Moeseneder et al., 1999; Osborn et al., 2000). The T-RFLP analysis was carried out with the community DNA obtained from nonincubated SS soil (So) and compared with the SS soil incubated without any amendment (S), or with the solid residue mixture (SR), the chloroorganic (see below) mixture (SC), or both (SRC). The T-RFLP profile (Fig. 1) from the nonincubated soil was significantly different from those of the soil incubated with the residue mixture (SR, p < 0.011), with the chloroorganic mixture (SC, p < 0.026), and with the solid residue plus chloroorganic mixture (SRC, p < 0.007), and was also different from the nonamended, incubated soil (S, p < 0.011). This observation suggests that the primary source of differences was related to the incubation itself rather than the effect of the solid residues and/or the chloroorganic mixture. Correspondingly, richness shown in the Sn T-RFLP profile (37) increased in the profiles of S, SR, SC, and SRC to 55, 58, 55, and 65, respectively. The Shannon-Weiner diversity index (H) also increased from 3.6 in Sn to 4.6, 4.6, 4.4, and 4.8 in S, SR, SC, and SRC, respectively. These results suggest that incubation of soil at 65% humidity and 20°C, conditions that are found in many natural soil habitats, allows bacteria that were at low abundance or metabolically inactive to become more abundant or active and be detectable by the T-RFLP analysis.



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  		Fig. 1. Terminal-restriction fragment length polymorphism profiles from community DNA obtained from soil incubated with Kraft pulp mill solid residues. Each phylotype detected in the soil samples (z axis) is represented as a fragment size (x axis; base pairs, bp) and plotted against its averaged, standardized peak area (y axis). So, nonincubated sandy soil; S, soil incubated without any amendment; or with the solid residue mixture (SR), the chloroorganic mixture (SC), or both (SRC).

 
As is indicated above, only the presence of a few chlorinated aromatic compounds was observed in the primary sludge, which is in contrast with conventionally bleached Kraft mill effluents (Céspedes et al., 1996). However, there is potential for transfer of such compounds from primary sludge to soils. In this context, it is worthwhile to mention that microbial decomposition activity against Kraft bleaching effluent chloroaromatics has been reported in soils (Brezny et al., 1992; González et al., 1995) and in biological, aerobic treatment of Kraft bleaching effluents (Graves and Joyce, 1994; Céspedes et al., 1996). Therefore, we evaluated if the microorganisms from these forest soils were able to degrade typical pulp bleaching compounds, and if the presence of solid wastes decreased or promoted such activity. The results from incubations of each soil–solid residue plus a mixture of typical pulp bleaching chloroaromatics: 2,4-dichlorophenol, 2,4,6-trichlorophenol, 4,5-dichloroguaiacol, and trichlorosyringol (5 mg kg -1 each) are summarized in Table 5. After 30 d of incubation, the microbiota from both soils was able to significantly remove the four compounds of the mixture. In some cases, significant removal was evident after 4 d of incubation (data not shown). Typically, removal at Day 30 represented 80 to 90% of the removal determined after 60 d. No signals for material derived from the degradation of the added compounds were detected by GC-MS (data not shown), suggesting that accumulation of intermediates did not take place. The presence of solid residues affected the extent and rate of chloroaromatic removal. Whereas the organic residue slightly increased the extent of removal, the inorganic residues strongly decreased the removal of all these compounds, suggesting a detrimental effect on soil microbial activity. Among other possibilities, this effect may be related to the changes in pH observed with these inorganic wastes (Table 3). The addition of the solid residue mixture that mainly contained inorganic wastes completely prevented the microbial removal of these chlorinated compounds. Due to the characteristics of the soils and solid residues, an abiotic, sterilized control to account for volatilization, complexation, and/or absorption processes was not run. However, the kinetic profile, and the different extent of removal, suggested that chloroaromatic elimination was mainly a microbial activity.


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  		Table 5. Removal of chloroaromatic compounds in incubations of soils with solid wastes of the cellulose Kraft process.{dagger}

 

   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
The effects of Kraft mill residues on Monterey pine growth and soil microbial activity were studied. Growth responses of Monterey pine seedlings and effect on soil microbial activity depended primarily on type, concentration, and specific chemical characteristics of each residue. These observations suggest that amendment with some of these residues may improve the chemical availability of nutrients by increasing pH and water content. The results of the present report encourage the feasibility of landspreading with the purposes of fertilization and amendment of soils, but other studies are still necessary to address this possibility. Some wastes, like grits, were inhibitory residues to plant growth and soil microbial activity; therefore, they should not be included in a landspreading program without closer examination in controlled research field trials.


   ACKNOWLEDGMENTS
 
This work was supported by grants FONDEF D97/1012 and FONDECYT 8990004. The help of T. Marsh with T-RFLP analysis is acknowledged. We are grateful for the technical assistance of C. Roveraro, C. Jones, J. Beltrán, and M. Yáñez. The advice of R. Vicuña and the support from Bioforest S.A and from Arauco Mill are also acknowledged. M.A. Sánchez was a FUNDACION ANDES undergraduate fellow.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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