Effects of zinc-smelter emissions on forest soil microflora1
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 12/05/14 For personal use only.
MARILYN J. JORDAN^'^ AND MARYP. LECHEVALIER Waksman Institute of Microbiology, R ~ l t g e r sUniversity, The State University of New Jersey, New Brunswick, New Jersey 08903 Accepted July 28, 1975
JORDAN,M. J., and M. P. LECHEVALIER. 1975. Effects of zinc-smelter emissions on forest soil microflora. Can. J. Microbiol. 21: 1855-1865. Within 2 km of a zinc (Zn) smelter in Palmerton, Pennsylvania, near the Lehigh Water Gap, up to 13.5% Zn by weight has been measured in the 0, horizon of the soil, and up t o 8 % Zn in the A, horizon. The total numbers of bacteria, actinomycetes, and fungi (measured by dilution plate counts) were greatly reduced in the most severely Zn-contaminated soils compared with control soils. The reduction of microbial populations may be a partial cause of the decreased rate of litter decomposition at Lehigh Gap. Growth of most bacteria from control sites was reduced by 100 to 200 f l Zn, most actinomycetes by 100 f l Zn, and most fungi by 100 to 1000 phf Zn in thin-Pablum extract agar (TPab). All the tested actinomycetes and non-spore-forming bacteria isolated from Zncontaminated Lehigh Gap soils were Zn-tolerant, growing normally in media containing 600-2000 pM Zn. Most fungi, regardless of source, were capable of at least 50% of normal growth at 700 pM Zn. Zinc-tolerant bacteria, actinomycetes, and fungi were readily isolated from low-Zn soils, suggesting that selection for Zn tolerance may proceed rapidly. Acidophilic Mortierello species have been selectively eliminated near the smelter, apparently spp. because of elevated soil pH. Peyronellaen glomerata (Corda) Goidanich and Coniotl~yri~rm were found only in the high-Zn soils. JORDAN,M. J . , et M. P. LECHEVALIER. 1975. Effects of zinc-smelter emissions on forest soil microflora. Can. J. Microbiol. 21: 1855-1865. La microflore de sols pollues par la deposition de metaux lourds aux environsd'une fonderie d e zinc (Zn) situte Palmerton, Pennsylvania, pres de Lehigh Water Gap a ete comparte avec celle de sols temoins Cloignes de la source de contamination. Les bacteries, les actinomycetes et les champignons Ctaient moins nombreux dans les sols pollues ou les taux de Zn atteignaient 13.5% dans I'horizon 0, et 8% dans I'horizon A, que dans les sols temoins. Cette reduction en rnicroflore peut &Irepartiellement responsable de I'accumulation de litiere observee aux environsde Lehigh Gap. La croissance de la plupart des microorganismes isoles des sols ttmoins est inhibee par I'addition de Zn aux milieux de culture: 100 a 200 pM Zn dans le cas des bacteries, 100 pM Zn pour les actinomycetes et 100 1000 pM Zn dans 'thin-Pablum extract agar' (TPab) dans le c a s des champignons. Par contre tous les actinomycetes et toutes les bacttries non-sporulees isoles des sols richesen Znqui ont eteetudies poussaient normalement en presence de600a2000 pM d e Zn. La plupart des champignons des sols donnaient au moins 50% de leur croissance normale e n presence de 700 pM de Zn. Des bacteries, actinomycetes e t champignons tolerant le Zn ont Cte facilement isoles de sols pauvres en Zn ce qui indique qu'une selection peut facilement se faire quand la teneur en Zn augmente sous la pousste de la pollution. Les especes acidophiles de Mortierella semblent avoir e t t eliminees sel6ctivement pres de la fonderie, apparemment a cause du pH eleve des sols. Peyronellnen glomernta (Corda) Goidanich et Coniotl~yrirrm spp. etaient recupires seulement des sols avec des concentrations de Zn elevees.
Introduction Zinc (Zn) ores have been smelted in Palmerton, Pennsylvania, since 1898. Within 2 km of the 'east plant' smelter up to 89, Zn, 1500 ppm 'Received January 9, 1975. T o r m e r l y M. J. Buchauer. 3Present address: Division of Biology, Section of ~ c o ~ o and g y Systematics, Langmuir Laboratory, Cornell University, Ithaca, New York 14853.
cadmium, 1200 copper, and 1100 ppm lead by weight were found in the A , soil horizon (Buchauer 1973). Washed, oven-dried foliage trees near the smelters contained up to 4500 ppm Zn and 70 ppm Cd by weight. The Lehigh Gap area of Blue Mountain near the smelters was densely forested in the early 1900's ; today it is sparsely vegetated or cornpletely barren over an area of about 485 ha.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 12/05/14 For personal use only.
1856
CAN. J. MICROBIOL. VOL. 21, 1975
Saturation extracts of soils collected at I-km intervals 1 to 5 km from the east plant smelter contained 25-616 pM Zn and 0.2-9 pM Cd (Jordan 1975). Only the soils within 5 km of the smelter appear to have a Zn content high enough to be toxic to native higher plant species (Jordan 1975). The combined stresses of high soil Zn levels and fire appear to be the primary causes of the vegetation damage at Lehigh Gap. Erosion and desiccation are important secondary factors preventing revegetation of the barren areas (Jordan 1975). Elevated levels of metals are not limited t o areas where industrial pollution occurs: recent reports (Kirkham 1974; Page 1974) of the effects on plants of high levels of Zn and Cd and other elements in sludgeamended soils underline the need for e ~ a l u a t i n g the effects of these heavy metal pollutants on a variety of ecosystems. The aim of this research was to study the effects of high soil Zn levels on the soil microflora. The numbers of organisms, species composition, and Zn tolerance of the microflora of metal-contaminated and non-contaminated soils were compared.
metal contents determined by atomic absorption spectrophotometry. A Perkin-Elmer model 303 atomic absorption spectrophotometer, a one-slot burner, and a n acetylene-air gas mixture were used. Soil pH was determined in a 2: 1 water:soil mixture with a Beckman Zeromatic SS-3 p H meter. Total organic matter analyses were carried out by the Rutgers College of ,Agriculture and Environmental Sciences Soil Testing Laboratory. Chromic acid oxidation was used for the A, horizon samples, and loss on ignition for the 0, and A , horizons.
Materials and Methods
Zinc-tolerarrce Tests Two composite samples of the A , horizon were collected at the near and distant lower slope sites in August 1972. Estimates of microbial numbers were obtained by dilution plating on unamended media and media amended with ZnSO, at moderate and high levels previously determined by testing the Zn sensitivity of laboratory strains. Bacterial counts were carried out on YD containing 3 (unamended level), 1000, and 6000 p M Z n ; actinomycete counts on TPab containing 6 (unamended level), 200, and 1000 p M Z n ; and fungal counts on TPab containing 6, 600, and 6000 p M Zn. Representative cultures of organisms isolated on unamended and high-Zn media from both sites were tested for Zn tolerance. These organisms were maintained before testing on the same media as that on which they were originally isolated. The tests were carried out using TPab agar containing 6, 100, 200, 600, 1000, 2000, 10000, and 100 000 V M Zn. The p H was adjusted t o 5.5 for the fungi and 6.5 for the bacteria and actinomycetes. Two plates per Zn level were streaked with fresh aqueous suspensions of three bacterial or actinomycete isolates. The plates were incubated at 23 "C for 4 and 10 days, respectively. Growth was characterized by eye a s normal, less than normal, or no growth. Two plates per Zn level per fungal isolate were inoculated by placinga 3-mm spot of fresh sporesuspension, or a 3 x 3 mm square of mycelium, a t the centerof each plate. Plates were incubated at 23 "C until the maximum colony diameters ranged from 30 t o 80 mm. Growth was evaluated by measuring the diameter of each colony in two directions (DeGroot 1972). The entire experiment
Site Selection and Sar~pling Soil samples were collected in October 1972 and May 1973 on top of Blue Mountain ridge and on the lower north slope, at sites both near and distant from the smelters. These sites will henceforth be referred to as "near ridge top, near lower slope, distant ridge top, and distant lower slope." The near samples were collected at Lehigh Gap, within 2 km of the east plant smelter. The distant ridge top samples were collected at Fox Gap, and the distant lower slope samples at Delaware Water Gap, 40 and 46 km, respectively, east of the east plant smelter. Blue Mountain ridge top soils are Dekalb stony loams, and d o not vary appreciably in physical and chemical properties (Buchauer 1971). At each of the four sites, four soil pits about 0.5 x 0.5 m were excavated to a depth of 25-35 cm. Samples of the 0, (partially decomposed leaf litter), A, (humus), and A3 (mineral soil) horizons were collected from each pit with an alcohol-flamed spatula and placed in sterile petri dishes. For analysis, samples of like horizons from two pits at each site were mixed in the laboratory t o yield two composite samples from each of the three horizons, at each of the four sites. Soil Analyses The samples were air-dried and forced through a 2-mmmesh stainless steel screen. 0, and A , horizon samples were digested with a 4: 1 mixture of concentrated nitric and perchloric acids. A.3 horizon samples were boiled for 2 h in perchloric acid. The resulting solutions were diluted to volume with distilled water, and the heavy
Dilution Plate Co~ints Estimates of the numbers of viable microorganisms were obtained by the dilution plate-count technique (Parkinson et al. -1971). Soil samples were mixed using sterilized glass and stainless steel food choppers and were plated within 24 h of collection. An aliquot was taken for determination of moisture content. Four plates per dilution per sample were spread, using 0.1-ml aliquots of soil suspension per plate. The plates were incubated at 23 "C. Bacterial and fungal colonies were counted after 6 days; the actinomycete colonies after 14 days. Yeast-dextrose (YD) agar (10 g Difco yeast extract, 10 g dextrose, 10 g Difco agar per litre of distilled water) was used for enumeration of bacteria, and thin-Pablum extract agar (TPab) (Lechevalier 1968) prepared with Difco agar and distilled water for enumeration of actinomycetes and fungi. T o the medium used for the enumeration of fungi, 100 pg/ml of neomycin was added to repress bacterial growth.
1857
JORDAN AND LECHEVALIER: Zn-SMELTER EMISSION ON MICROFLORA
TABLE I Typical analyses of the soils used in this study
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 12/05/14 For personal use only.
Total metal content, ppmd Horizon
pHa
% moistureb
0 2
4.0 3.8 3.7
55.4 42.4 27.7
4.1 3.6 3.9
52.3 37.1 21.7
5.3 5.5
67.3 71.7
A1 A3 0 2
A1 AB 0 2
A,
A
% organic matterc
Zn
Distant-ridge top 65.4 19.9 6.6 Distant-lower slope 73.6 26.1 6.0 Near-ridge top 74.8 54.9 6.9 Near-lower slope
Cu
Pb
650 225 74
8.0 3.0 2.7
34 18 11
362 152 22
570 110 44
6.0 3.0 8.5
26 18 13
287 113 22
350 82
2250 300
33 700 22 500 1 310
.Mean of four samples. Ninety-five percent least significant difference (LSD) for O2 horizon
-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 12/05/14 For personal use only.
MARILYN J. JORDAN^'^ AND MARYP. LECHEVALIER Waksman Institute of Microbiology, R ~ l t g e r sUniversity, The State University of New Jersey, New Brunswick, New Jersey 08903 Accepted July 28, 1975
JORDAN,M. J., and M. P. LECHEVALIER. 1975. Effects of zinc-smelter emissions on forest soil microflora. Can. J. Microbiol. 21: 1855-1865. Within 2 km of a zinc (Zn) smelter in Palmerton, Pennsylvania, near the Lehigh Water Gap, up to 13.5% Zn by weight has been measured in the 0, horizon of the soil, and up t o 8 % Zn in the A, horizon. The total numbers of bacteria, actinomycetes, and fungi (measured by dilution plate counts) were greatly reduced in the most severely Zn-contaminated soils compared with control soils. The reduction of microbial populations may be a partial cause of the decreased rate of litter decomposition at Lehigh Gap. Growth of most bacteria from control sites was reduced by 100 to 200 f l Zn, most actinomycetes by 100 f l Zn, and most fungi by 100 to 1000 phf Zn in thin-Pablum extract agar (TPab). All the tested actinomycetes and non-spore-forming bacteria isolated from Zncontaminated Lehigh Gap soils were Zn-tolerant, growing normally in media containing 600-2000 pM Zn. Most fungi, regardless of source, were capable of at least 50% of normal growth at 700 pM Zn. Zinc-tolerant bacteria, actinomycetes, and fungi were readily isolated from low-Zn soils, suggesting that selection for Zn tolerance may proceed rapidly. Acidophilic Mortierello species have been selectively eliminated near the smelter, apparently spp. because of elevated soil pH. Peyronellaen glomerata (Corda) Goidanich and Coniotl~yri~rm were found only in the high-Zn soils. JORDAN,M. J . , et M. P. LECHEVALIER. 1975. Effects of zinc-smelter emissions on forest soil microflora. Can. J. Microbiol. 21: 1855-1865. La microflore de sols pollues par la deposition de metaux lourds aux environsd'une fonderie d e zinc (Zn) situte Palmerton, Pennsylvania, pres de Lehigh Water Gap a ete comparte avec celle de sols temoins Cloignes de la source de contamination. Les bacteries, les actinomycetes et les champignons Ctaient moins nombreux dans les sols pollues ou les taux de Zn atteignaient 13.5% dans I'horizon 0, et 8% dans I'horizon A, que dans les sols temoins. Cette reduction en rnicroflore peut &Irepartiellement responsable de I'accumulation de litiere observee aux environsde Lehigh Gap. La croissance de la plupart des microorganismes isoles des sols ttmoins est inhibee par I'addition de Zn aux milieux de culture: 100 a 200 pM Zn dans le cas des bacteries, 100 pM Zn pour les actinomycetes et 100 1000 pM Zn dans 'thin-Pablum extract agar' (TPab) dans le c a s des champignons. Par contre tous les actinomycetes et toutes les bacttries non-sporulees isoles des sols richesen Znqui ont eteetudies poussaient normalement en presence de600a2000 pM d e Zn. La plupart des champignons des sols donnaient au moins 50% de leur croissance normale e n presence de 700 pM de Zn. Des bacteries, actinomycetes e t champignons tolerant le Zn ont Cte facilement isoles de sols pauvres en Zn ce qui indique qu'une selection peut facilement se faire quand la teneur en Zn augmente sous la pousste de la pollution. Les especes acidophiles de Mortierella semblent avoir e t t eliminees sel6ctivement pres de la fonderie, apparemment a cause du pH eleve des sols. Peyronellnen glomernta (Corda) Goidanich et Coniotl~yrirrm spp. etaient recupires seulement des sols avec des concentrations de Zn elevees.
Introduction Zinc (Zn) ores have been smelted in Palmerton, Pennsylvania, since 1898. Within 2 km of the 'east plant' smelter up to 89, Zn, 1500 ppm 'Received January 9, 1975. T o r m e r l y M. J. Buchauer. 3Present address: Division of Biology, Section of ~ c o ~ o and g y Systematics, Langmuir Laboratory, Cornell University, Ithaca, New York 14853.
cadmium, 1200 copper, and 1100 ppm lead by weight were found in the A , soil horizon (Buchauer 1973). Washed, oven-dried foliage trees near the smelters contained up to 4500 ppm Zn and 70 ppm Cd by weight. The Lehigh Gap area of Blue Mountain near the smelters was densely forested in the early 1900's ; today it is sparsely vegetated or cornpletely barren over an area of about 485 ha.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 12/05/14 For personal use only.
1856
CAN. J. MICROBIOL. VOL. 21, 1975
Saturation extracts of soils collected at I-km intervals 1 to 5 km from the east plant smelter contained 25-616 pM Zn and 0.2-9 pM Cd (Jordan 1975). Only the soils within 5 km of the smelter appear to have a Zn content high enough to be toxic to native higher plant species (Jordan 1975). The combined stresses of high soil Zn levels and fire appear to be the primary causes of the vegetation damage at Lehigh Gap. Erosion and desiccation are important secondary factors preventing revegetation of the barren areas (Jordan 1975). Elevated levels of metals are not limited t o areas where industrial pollution occurs: recent reports (Kirkham 1974; Page 1974) of the effects on plants of high levels of Zn and Cd and other elements in sludgeamended soils underline the need for e ~ a l u a t i n g the effects of these heavy metal pollutants on a variety of ecosystems. The aim of this research was to study the effects of high soil Zn levels on the soil microflora. The numbers of organisms, species composition, and Zn tolerance of the microflora of metal-contaminated and non-contaminated soils were compared.
metal contents determined by atomic absorption spectrophotometry. A Perkin-Elmer model 303 atomic absorption spectrophotometer, a one-slot burner, and a n acetylene-air gas mixture were used. Soil pH was determined in a 2: 1 water:soil mixture with a Beckman Zeromatic SS-3 p H meter. Total organic matter analyses were carried out by the Rutgers College of ,Agriculture and Environmental Sciences Soil Testing Laboratory. Chromic acid oxidation was used for the A, horizon samples, and loss on ignition for the 0, and A , horizons.
Materials and Methods
Zinc-tolerarrce Tests Two composite samples of the A , horizon were collected at the near and distant lower slope sites in August 1972. Estimates of microbial numbers were obtained by dilution plating on unamended media and media amended with ZnSO, at moderate and high levels previously determined by testing the Zn sensitivity of laboratory strains. Bacterial counts were carried out on YD containing 3 (unamended level), 1000, and 6000 p M Z n ; actinomycete counts on TPab containing 6 (unamended level), 200, and 1000 p M Z n ; and fungal counts on TPab containing 6, 600, and 6000 p M Zn. Representative cultures of organisms isolated on unamended and high-Zn media from both sites were tested for Zn tolerance. These organisms were maintained before testing on the same media as that on which they were originally isolated. The tests were carried out using TPab agar containing 6, 100, 200, 600, 1000, 2000, 10000, and 100 000 V M Zn. The p H was adjusted t o 5.5 for the fungi and 6.5 for the bacteria and actinomycetes. Two plates per Zn level were streaked with fresh aqueous suspensions of three bacterial or actinomycete isolates. The plates were incubated at 23 "C for 4 and 10 days, respectively. Growth was characterized by eye a s normal, less than normal, or no growth. Two plates per Zn level per fungal isolate were inoculated by placinga 3-mm spot of fresh sporesuspension, or a 3 x 3 mm square of mycelium, a t the centerof each plate. Plates were incubated at 23 "C until the maximum colony diameters ranged from 30 t o 80 mm. Growth was evaluated by measuring the diameter of each colony in two directions (DeGroot 1972). The entire experiment
Site Selection and Sar~pling Soil samples were collected in October 1972 and May 1973 on top of Blue Mountain ridge and on the lower north slope, at sites both near and distant from the smelters. These sites will henceforth be referred to as "near ridge top, near lower slope, distant ridge top, and distant lower slope." The near samples were collected at Lehigh Gap, within 2 km of the east plant smelter. The distant ridge top samples were collected at Fox Gap, and the distant lower slope samples at Delaware Water Gap, 40 and 46 km, respectively, east of the east plant smelter. Blue Mountain ridge top soils are Dekalb stony loams, and d o not vary appreciably in physical and chemical properties (Buchauer 1971). At each of the four sites, four soil pits about 0.5 x 0.5 m were excavated to a depth of 25-35 cm. Samples of the 0, (partially decomposed leaf litter), A, (humus), and A3 (mineral soil) horizons were collected from each pit with an alcohol-flamed spatula and placed in sterile petri dishes. For analysis, samples of like horizons from two pits at each site were mixed in the laboratory t o yield two composite samples from each of the three horizons, at each of the four sites. Soil Analyses The samples were air-dried and forced through a 2-mmmesh stainless steel screen. 0, and A , horizon samples were digested with a 4: 1 mixture of concentrated nitric and perchloric acids. A.3 horizon samples were boiled for 2 h in perchloric acid. The resulting solutions were diluted to volume with distilled water, and the heavy
Dilution Plate Co~ints Estimates of the numbers of viable microorganisms were obtained by the dilution plate-count technique (Parkinson et al. -1971). Soil samples were mixed using sterilized glass and stainless steel food choppers and were plated within 24 h of collection. An aliquot was taken for determination of moisture content. Four plates per dilution per sample were spread, using 0.1-ml aliquots of soil suspension per plate. The plates were incubated at 23 "C. Bacterial and fungal colonies were counted after 6 days; the actinomycete colonies after 14 days. Yeast-dextrose (YD) agar (10 g Difco yeast extract, 10 g dextrose, 10 g Difco agar per litre of distilled water) was used for enumeration of bacteria, and thin-Pablum extract agar (TPab) (Lechevalier 1968) prepared with Difco agar and distilled water for enumeration of actinomycetes and fungi. T o the medium used for the enumeration of fungi, 100 pg/ml of neomycin was added to repress bacterial growth.
1857
JORDAN AND LECHEVALIER: Zn-SMELTER EMISSION ON MICROFLORA
TABLE I Typical analyses of the soils used in this study
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 12/05/14 For personal use only.
Total metal content, ppmd Horizon
pHa
% moistureb
0 2
4.0 3.8 3.7
55.4 42.4 27.7
4.1 3.6 3.9
52.3 37.1 21.7
5.3 5.5
67.3 71.7
A1 A3 0 2
A1 AB 0 2
A,
A
% organic matterc
Zn
Distant-ridge top 65.4 19.9 6.6 Distant-lower slope 73.6 26.1 6.0 Near-ridge top 74.8 54.9 6.9 Near-lower slope
Cu
Pb
650 225 74
8.0 3.0 2.7
34 18 11
362 152 22
570 110 44
6.0 3.0 8.5
26 18 13
287 113 22
350 82
2250 300
33 700 22 500 1 310
.Mean of four samples. Ninety-five percent least significant difference (LSD) for O2 horizon
-
