E F F E C T OF S O I L A C I D I F I C A T I O N

O N T H E S O I L M I C R O F L O R A

R. D. BRYANT, E. A. G O R D Y and E. J. L A I S H L E Y

Department of Biology, The University of Calgary, Calgary, Alberta T2N 1N4 Canada

(Received 14 December, 1978; revised 6 April, 1979)

Abstract. The effects of short and long term acidification on a few Alberta soils were studied with respect to bacterial numbers and total soil respiration. Significant reductions in bacterial numbers were observed in both short and long term acidified soils. Total soil activity was severely affected in an acid soil (pH 3.0, long- term) adjacent to a S block. A soil (pH 6.8) 200 m away from this S block when artificially acidified to pH 2.9 significantly reduced soil activity but not as drastically as found in the long term pH 3.0 soil. A garden soil (pH 7.7) which was also acidified to pH 3.2 showed no significant reduction in total soil respiration rate as compared to its unacidified control soil.

These acid soils when amended with organic substrates demonstrated that certain physiological groups of organisms were severely inhibited by this acid condition. The importance of examining more than one parameter when assessing the effect of a potential pollutant on soil activity is discussed.

1. Introduction

A great deal of information has accumulated in the past 10 years on the detrimental effects of acid precipitation from industrial pollutant emissions, including damage to forest vegetation, increased soil acidity and changes in some soil chemical properties (Abrabamsen et al., 1976). With respect to soil acidity, a few reports have indicated that the soil microflora are also adversely affected by increasing acid concentrations (Tamm, 1976; Babich and Stotzky, 1978).

In addition to soil acidification from industrial pollutant emissions, another source is acid mine run-off water which occurs frequently from coal mining and occasionally from ore mining operations caused by microbial oxidation of sulphide minerals found in these mining deposits (Alexander, 1971; Metson, 1977). It has also been found that additions of elemental S to soil results in a rapid reduction in soil pH (Bollen, 1977; Adamczyk-Winiarska et al., 1975). This processs has been attributed to the oxidation of S o to H 2 S O 4 by S oxidizing microorganisms, One effect of these soil acidifications has been to reduce the heterotrophic bacterial populations (Wood, 1975; Adamczyk- Winiarsky et al., 1975), culminating in a reduction of nutrient cycling and organic matter decomposition (Williams and Gray, 1974).

Since the world's production of S has been estimated to exceed the annual amount necessary for traditional uses for the production of H2SO4, fertilizers, etc. for the 1980's (Sundheim and Delegado, 1976), this has generated renewed interest in finding new uses for S. This is especially true in the construction industry with the development and testing of S concretes, asphalts, foams, etc. The properties of these new bonded S materials have been shown in certain applications to be superior and competitive in cost to conventional materials (Gillott et al., 1978); in fact, some products are already being

Water, Air, andSoilPollution 11 (1979). 437-445. 0049-6979/79/0114-0437 $1.35 Copyright © 1979 by D. Reidel Publishing Co., Dordrecht, Holland, and Boston, U.S.A.

438 R.D. BRYANT E T AL.

commercially marketed. In laboratory experiments we have found that Thiobacillus thiooxidans can oxidize the bonded S in these concretesl mortars, and a certain type of asphalt, resulting in the production of HzSO 4 (Laishley and Tyler, 1979). The large scale

use of some of these S construction materials represents a potential new source for localized increased soil acidity due to microbial oxidization of the S in these products.

Data from our field studies in which S and S concrete cylinders were buried indicate that acidification of soils surrounding these cylinders will be a slow process even though large populations of thiobacilli were detected over the past 4 yr (Laishley, 1978; Laishley et al., 1978). The acidification of these soils appears to depend on such factors as soil type, moisture, buffering capacity and the structural integrity of the S construction material itself (Laishley, 1977). However, the fact that the localized acidification will probably be a long term process, should not mean we ignore the potential problem until it occurs.

Since very little data were available on the effects of soil acidification on different groups of soil microorganisms, we initiated this study to obtain information on the short- and long-term effects of soil acidification on the soil microflora. For the purpose of studying long-term soil acidification we used, as our model system, a biological acidified soil which was next to a S block, a by-product of a Sour Gas plant.

2. Study Sites

Two study sites were selected for these experiments. One was located at a S block (approximately 1 x 106 metric tons and already 15 yr in existence) in Southern Alberta, Canada; elemental S in this stockpile was a by-product of the Sour Gas industry recovered by converting the contaminating HzS to S o by the Claus process. Soil samples were collected 1 m and 200 m away from the S block to provide relevant information on the long- and short-term aspects of soil acidification. A garden soil located at the University of Calgary was subsequently sampled to determine if the effects of soil acidification may be a generalized phenomena, at least on the short-term basis.

3. Methods and Materials

To assess the short- and long-term effects of soil acidification on the microbial ecosystem two estimations were made; namely, (1) bacterial populations (quantitative), and (2) soil

respiration via CO2 monitoring.

3.1. SAMPLING TECHNIQUES

3.1.1. Sulphur Stockpile In September 1977, two study sites which were 1 m (S1) and 200m ($200) from the S block were picked. Preliminary experimental sampling had shown that soil around the S pile had pH values of 3 and lower, while soil 200 m away had pH readings around 7.0.

EFFECT OF SOIL ACIDIFICATION ON THE SOIL M1CROFLORA 4 3 9

Unfortunately we do not know when the $1 soil became acidic, however, in studies around a 3 yr old S block in Northern Alberta we encountered similar acidic soil pH measurements. Thus we assumed from the latter observations that the S1 soil has probably been acidified for 12 yr. Soil from each of these locations was randomly core sampled, combined in plastic bags, and stored at 5 °C until analysis commenced.

3.1.2. Garden Soil

Also, in September 1977 the garden site was sampled (pH 7.7). Only one site was chosen and the random core samples were treated as described above. The soil samples from both the S stockpile and garden plot were transported to the University of Calgary for microbial analysis.

3.1.3. Subdivision of Soils

Prior to analysis the $200 soil and the garden soil were each subdivided into two equal portions. One portion of each soil type was set aside for a laboratory acidification treat- ment (pH 3.0). This treatment provided information on the short term effects of acidification (see below). The remaining portion of each soil type was left unamended and served as the controls.

3.1.4. Soil Preparation

All soils were prepared according to the method of Johnen and Drew (1977). To obtain homogeneous soil samples, the soils were slowly air-dried at room temperature for several days. The dried soil was then sieved (2 mm mesh size) and subsequently adjusted to 30% moisture content (by weight) by either adding the appropriate amount of sterile

distilled water or sterile diluted HzSO 4. The amount of acid required to adjust the pH to 3 was predetermined experimentally by adding a range of concentrations of acid water to appropriate 45 g subsamples of sieved, air-dried soil. The pH in the acid treated soils was monitored daily until constant readings were achieved (7 to 10 days). During this 10-day equilibrium period, all soils (acid and non acid) were kept in plastic trays at 28°C and covered with aluminum foil. The foil had several small holes punched through it to allow sufficient gas exchange, while at the same time minimizing moisture loss. Johnen and Drew (1977) showed that the equilibration period was important to overcome any effect on soil activity caused by air drying and/or soil remoisturing (Jenkinson and Powlson, 1976). These equilibrated soils were used for the respiration experiments and heterotrophic bacterial counts.

Soil pH measurements were made on a standard 1:1 mixture of soil to distilled water using a Radiometer pH meter 28.

3.2. HETEROTROPHIC BACTERIAL COUNTS

The total bacterial numbers from the different soils were obtained by standard plate count on peptone-yeast extract agar (Parkinson et al., 1971) after incubation at 28°C

440 R.D. BRYANT E T A L .

for one week. These bacterial counts were done on soils after the equilibration period as described in Section 3.1.4.

3.3. SOIL RESPIRATION

Soil respiration (CO 2 evolution) was used as a general criterion for evaluating biological soil activity. CO2 evolution was measured in a static system by absorbing the evolved gas in standard NaOH solution and determined by precipitation of the formed NaaCO 3 with 2N BaC12, followed by titration of the unreacted NaOH with standard HC1 (Parkinson et al., 1971). Soil samples (15 gm) were sub-sampled from the prepared equilibrated soils (3.1.4.) placed in compartmentalized ink bottles, capped and incubated at 28°C as the unamended control soils (Figures 1 and 2). Soil respiration was then measured at 24 h intervals on five successive days, to ensure that a steady state situation had been reached. An average respiration value was then calculated for each ink bottle, when it gave a consistent CO 2 measurement on at least three consecutive days. CO2 evolution for each soil treatment was performed in triplicate so that statistical interpretation of the result was possible. Also, to gain an estimate of background CO2, 15 ml of CO2-free water was introduced into the ink bottle, in place of the soil. The mean value from three 'blanks' was determined each day and used as a correction factor for the daily COz respired from the soils.

To assess the effect of acidification on specific groups of soil organisms, soil organic substates (starch, cellulose, glucose, casein, and urea) were mixed with the various equilibrated soil (15 gm) in the ink bottles at 80 mg carbon/100 gm of soil (Figures 1 and 2) as described by Johnen and Drew (1977). Respiration rates obtained from these amended soils were measured as described above.

3.4. STATISTICAL ANALYSIS

Data were routinely statistically analyzed using 1-way ANOVA (analysis of variance) test procedures. Before applying the ANOVA, the Bartlett's and F-Max test were applied to the data to test for homogenity of variances. Because the variances of the data must be homogeneous before the ANOVA test can be applied, heteroscedastic data were trans-

formed using the logarithm (x + 1) or the square root transformations.

4. Results

4.1. H E T E R O T R O P H I C B A C T E R I A L C O U N T S

Table I summarizes data on the number of heterotrophic bacteria per gram dry weight of soil. A 1-way ANOVA of these data indicated that bacterial numbers differed significantly with increased acidification. This relationship is apparent in both the short- term ($200, pH 6.8 soil vs. acidified $200 pH 2.9 soil; garden, pH 7.7 vs. garden acidified, pH 3.2) and long-term ($200, pH 6.8 soil vs. S1, pH 3.0 soil) acidifications.

EFFECT OF SOIL ACIDIFICATION ON THE SOIL MICROFLORA

TABLE I

Effect of increased acidification on the heterotrophic bacteria in soils

Soil Number of heterotrophic Soil samples pH bacteria/g dry wt of soil*

1 m from S block (S1) 3.0 4.5 × 104 (+0.7)*** 200 m from S block ($200) 6.8 2.7 × 107 ( +_ 9.34)*** $200 soil + H2SO4"* 2.9 7.8 x 105 ( +_ 0.43)***

Garden soil 7.7 1.9 x 107( + 0.34)*** Garden soil + H2SO4"* 3.2 2.2 × l0 s ( +_ 0.31)***

* Mean of 3 replicate plates. ** Soil pH was adjusted by H2SO 4 as described in test.

*** Statistically significant differences (P = 0.05) from other mean(s) in group.

( + ) Standard deviation.

441

4.2. SOIL RESPIRATION

4.2.1. Sulphur Block Figure 1 summarizes the microbial respiration measurements from soil samples near and away from the S pile. One-way ANOVA's indicated that soil acidification had significantly

different effects on several physiological soil groups. The control soil (minus substrate) as well as the starch, cellulose and urea amended soils all exhibited the same patterns.

There was a progressive decrease in CO 2 evolution from the control soil sampled 200 m

800 [ ] SOIL (pH 6.B) 200 METERS FROM SULPHUR BLOCK

[ ] SOIL (pH 6.8) ACIDIFIED IN LABORATORY TO pH 2.9

[ ~ ] SOIL(pH 3.0) "1 METER FROM SULPHUR BLOCK

- - INDICATES STATISTICALLY SIMILAR MEANS

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600

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.~ 400 ::i;:

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STARCH CELLULOSE GLUCOSE

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",i/A i','x ;~i~i - - I UREA CASEIN CONTROL

Figure 1. The effect of acid conditions on respiration rates of unamended and organically amended soils adjacent to 1 m and 200 m from a S block. The control represents the unamended soils, while the organically amended soils (see methods) are indicated along the abscissa. For each substrate tested under the different soil conditions, those means which showed no significant difference with respect to one another have been

indicated by a horizontal bar.

442 R . D . B R Y A N T E T A L .

from the S block to the control soil which was laboratory acidified ($200, short-term acid treatment) and finally to the acid soil next to the S block (S 1, long-term acidification). This indicated that acidification to pH 3.0 significantly reduced the potential of the soil flora to utilize these growth substrates. Both glucose and casein amendations showed similar trends with respect to long-term acidification, i.e., a significant reduction in CO2 evolution. When considering short-term acid treatments, glucose showed respiratory rates which were significantly higher than the pH 6.8 soil, while casein-treated soil evolved CO= at a similar rate to the pH 6.8 soil.

Additions of the various organic substrates to both acid and non acid soils increased the basal rate of soil respiration when compared to the control soil which did not receive added substrates.

4.2.1. Garden Soil

The efects of short-term acidification on microbial respiration rates of a typical Calgary garden soil are shown in Figure 2. Cellulose, urea and casein additions to acidified and nonacidified soils show that acid soil reduced the soil's potential for degradation of these substrates. No significant differences between respiration rates in the pH 7.7 soil and the

800

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'1 ' 600

" " 400

~ ~00

GARDEN SOIL pH 7.7

[ ~ ] GARDEN SOIL (pH 7.7) ACIDIFIED IN LABORATORY TO pH 3.2

INDICATES STATISTICALLY SIMILAR MEANS

STARCH CELLULOSE GLUCOSE

i / i i / /

i / i i / i

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UREA

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CASEIN CONTROL

Figure 2. The effect of acid conditions on respiration rates on unamended and organically amended garden soil. The control represents the unamended soil while the organically amended soils (see methods) are

indicated along the abscissa. Statically similar means are explained in Figure 1 caption.

E FFE CT OF SOIL ACIDIFICATION ON T HE SOIL M I C R O F L O R A 443

pH 3.2 soil were detected when the substrates starch and glucose were added. Also, the

unamended control (minus substrate) showed no significant response to acidification. Addition of the various substrates to acid and non acid soil also increased the basal

rate of respiration when compared to the unamended control soil.

5. Discussion

In this study it was found that long-term acidification of the soil near the S pile (S1) resulted in a significant reduction of the heterotrophic bacterial population as compared to soil 200 m ($200) from the pile. This effect of acid on the heterotrophic soil counts agrees with the observation of Wood (1975), and Adamczyk-Winiarska et al. (1975). These results though are atypical of those obtained by other drastic soil treatment procedures such as fumigation, heat treatment, irradiation, etc., in which bacterial numbers are immediately reduced, then rapidly increased again after a short period of time (Powlson, 1975). In our case, the bacterial population did not recover in the S1 soil which is consistent with other soil biocide treatments which showed that the recoloniz- ation population could be inhibited by the continual presence of the biocides (Bryant and Parkinson, 1978; Martin, 1963).

In the short term experiments in which the $200 and garden soils were artificially acidified in the laboratory, again we noted a significant reduction in the heterotrophic bacterial counts (see Table I) in the same order of magnitude as occurred in the long-term acidified soil S 1.

Thus, one major effect of soil acidification whether it be short- or long-term is the reduction in heterotrophic bacterial population which will ultimately effect nutrient cycling in the soil.

In the soil respiration experiments, long-term acidification of the S1 soil (unamended control) adversely affected the total soil activity as compared to the $200 (unamended control), while in the short term artificially acidified $200 soil, the soil microflora activity was significantly reduced as compared to the nonacidified $200. Tamm (1976) reported that short-term acidification of a forest humus soil also showed significant reduction in the soil activity. However, no significant difference in soil activity occurred in unamended garden soil whether it was treated with acid or not, which probably reflects the different physical structure and biological composition of this soil as compared with the $200.

These soils were then assessed to see what physiological group of organisms was affected by acidification. This was accomplished by supplementing these soils with a number of common soil organic substrates and comparing their respiration rates to the unamended control soils. The respiration rates for the S1 amended soils containing starch, cellulose or casein were practically identical to the unamended S1 control soil, indicating that the flora responsible for their degradation were essentially nonexistent while glucose and urea metabolizing organisms were still present after long-term exposure to this acid environment. In contrast, the $200 soil showed significant potential for

444 R. D, BRYANT E T A L .

degrading all substrates added as compared to the unamended control. When this soil was acidified in the laboratory, the respiration rates for starch and cellulose were similar to that found in the acidified unamended control $200 soil. This suggests that the starch and cellulose degrading organisms were quite sensitive to a short-term acid exposure, while urea catabolizing organisms were more acid-resistant but their total activity was still significantly reduced as compared to the nonacidified urea supplemented $200 soil. However, casein metabolism was not affected by this acid treatment, while glucose respiration rates were significantly stimulated over the nonacidified glucose amended $200 soil.

It is interesting to note that in the S1, acidified $200 and acidified garden soil, a common trend was observed in the significant reduction in their potential to degrade cellulose and urea suggesting that this might be a generalized soil phenomena caused by severe acidification. However, more soils need to be tested to substantiate this observa- tion. Another fact emerges which is related to the garden soils study which showed no significant difference in soil respiration rate between the unamended acidified and

nonacidified soil. This would indicate that the short-term acidification treatment of the garden soil had no affect on its biological activity. In actual fact, the organisms responsible for utilization of cellulose, urea and casein were adversely affected as compared to the correspondingly amended nonacidified garden soil. This demonstrates the importance of examining more than one parameter when assessing the effect of a potential pollutant on soil activity.

Tamm (1976) reported that acidification inhibited the nitrification process in a forest soil while we have shown that long term acidification of a field soil severely retards its biological potential in degrading protein and complex polysaccharides. The effect of soil acidification could be far-reaching, seriously disturbing other major biological transformation cycles occurring in soils.

Acknowledgments

This investigation was supported by the University of Calgary Interdisciplinary Sulphur Research Group (UNISUL).

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E FFE CT OF SOIL ACIDIFICATION ON T H E SOIL M I C R O F L O R A 445

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