ELSEVIER FEMS Microbiology Ecology I9 (I 996) 227-237

Adaptation of soil bacterial communities to prevailing pH in different soils

Erland B&h *

Received 3 I May 1995: revised 20 January 1996; accepted 6 February 1996

Abstract

The bacterial community response to pH was studied for 16 soils with pH(H,O) ranging between 4 and 8 by measuring thymidine incorporation into bacteria extracted from the soil into a solution using homogenization-centrifugation. The pH of the bacterial solution was altered to six different values with dilute sulfuric acid or different buffers before measuring incorporation. The resulting pH response curve for thymidine incorporation was used to compare bacterial communities from the different soils. There was a correlation between optimum pH for thymidine incorporation and the soil pH(H,O). Even bacterial communities from acid soils had optima corresponding to the soil pH, indicating that they were adapted to these conditions. Thymidine incorporation was also compared with leucine incorporation for some soils. The leucine to thymidine incorporation ratio was constant over the tested pH interval when incorporation values were adjusted for isotope dilution. A good correlation was found between the scores along the first component (explaining 80% of the variation) and soil pH ( r2 = 0.85). if principal component analysis of the pH response curves for thymidine incorporation was used. The pH response curves differed most for the extreme pH values used, and a linear relationship was found between the logarithm of the ratio of thymidine incorporation at pH 4.3 to incorporation at pH 8.2 and the soil pH (r’ = 0.86). Thus, a simplified technique using only two pH values, when measuring the thymidine incorporation. could be used to compare the response to pH of bacterial communities.

Kqvnrds: Soil; pH response; Thymidine incorporation: Bacterial community

1. Introduction

It is well known that bacteria usually are less numerous in relation to fungi in acid forest soils compared to arable soils with a higher pH [1,2]. One explanation for this phenomenon has been that bacte- ria as a group have a higher pH optimum compared

_ Tel: +46 (46) 222 42 64; Fax: +46 (46) 232 41 58; E-mail: erland.baath@mbioekoI.lu.se

to fungi, and thus are less adapted to the more acidic conditions in forest soils (e.g. [ 1,3-61). For example. bacteria that have higher pH optima than the prevail- ing soil pH are frequently isolated from acid soils [7,8], and bacterial counts from such soils can be equal or even higher on media with a neutral pH than on media adjusted to low pH [9,10]. On the other hand, in several studies in forest soils, where the pH has been increased by up to 2 pH units due to liming. ash treatment or alkaline pollution, there was little effect on the bacterial biomass estimated using

016%6496/96/$15.00 0 1996 Federation of European Microbiological Societies. All rights reserved PII SO 168.6496(96)00008-6

acridine orange direct counts [IO- 121. This casts doubt on the conventional explanation that fewer bacteria are found in acid soils due to lack of toler- ance to low pH.

Until now one has been forced to rely on agar plate techniques. when testing the pH response of a bacterial community, either after isolating and testing single isolates, or by performing plate counts on agar medium with different pH. However, due to the selectivity of the agar media, only a minor part of the soil bacterial community can be studied in this way. Furthermore, it is difficult to maintain an exact pH during prolonged incubation of the agar plates. The technique is also time consuming, since an incuba- tion time of several weeks is needed to maximize colony counts [13].

Recently a new technique to measure the response of the soil bacterial community to pH was described [lo]. It used short-term incubations of bacteria ex- tracted directly from soil by homogenization-centri- fugation and measured bacterial community growth rates (estimated using incorporation of labelled thymidine) at different pH values. This technique has been used to detect shifts in the pH response of bacterial communities after treatments such as lim- ing, ash fertilization, prescribed burning and alkaline

Table I pH and organic matter contenr in the 16 soils studied

pollution, which raise soil pH [ 10,12.15]. However, the method has so far only been applied to forest soils with a limited range of pH values.

In the present study the bacterial community re- sponse to pH was studied for different soils ranging between pH 4 and pH 8. One aim was to study whether the bacterial community response to pH was directly related to soil pH over the whole pH inter- val. Special emphasis was placed on acid soils. The second aim was to compare different ways of ex- pressing the effect of soil pH on the bacterial com- munity response to pH. This might enable us to use the pH response as a way of elucidating actual pH values in microhabitats. where pH is difficult to measure using normal procedures.

2. Materials and methods

The soils were chosen in order to cover a wide range of pH. Initially it was tried to obtain soils for which no correlation existed between organic matter content and pH, but this was not possible for the soils with the lowest pH. Forest soils were sampled

Number Soil

I Deciduous forest

2 Coniferous forest

3 Coniferous forest

4 Coniferous forest 5 Arable (gas>)

6 Arable (grass)

7 Garden

8 Arable

9 Deciduous forest

IO Garden

II Garden

I2 Arable (grass)

13 Deciduous forest I4 Garden

I5 Deciduous forest

16 Agricultural

pH(H,O) pH(KCI) PH.,,, Organic matter (57-j

3.90 3.35 4.58 51.1 3.21 3.52 1.55 25.8 4.39 3.67 4.73 3.5 1.58 3.82 1.89 ,’ 8.9 3.90 4.09 5.09 a Il.3 5.10 4.13 5.83 I I.2 6.00 5.28 6.39 13.4 6.23 5.52 6.55 10.4 6.24 5.57 6.36 5.1 6.75 6.12 6.82 4.7 6.88 6.62 6.91 2.9 6.91 7.00 7.02 0.8 7.21 6.61 6.67 rl S.6 7.69 7.31 7.63 8.8 7.83 7.58 7.09 d 9.6 8.10 7.99 7.88 3.3

pH(H,OI was measured in distilled water and pH(KCl) in 0.2 M KCI. pH,,,, was the pH of the bacterial solution extracted by

homogenization-centrifufarion. These values were means of two determinations unless otherwise stated.

‘I Only determined once.

just beneath the litter layer, while O-5 cm was taken for the other soils. The soils were sampled, brought to the laboratory and sieved (mesh size 2 mm) the same day, and then stored at 4°C.

Soil pH was measured in distilled water or KC1 (0.2 M) using 5 g wet weight of soil and 50 ml liquid after shaking on a rotary shaker for 1 h. pH was also measured in all bacterial suspensions (see below) at each measuring occasion. Organic matter content was approximated from loss on ignition measured after 4 h at 600°C. Background data for the soils are presented in Table 1.

2.2. pH respome measurements

The bacterial community response to pH was measured twice for each soil. the first time within a week of sampling and the second time after about two months of storage. The pH response of the

bacterial community was measured on bacteria ex- tracted from soil by homogenization-centrifugation using thymidine incorporation into cold acid-insolu- ble material [ 151. In short, 2.5 to 10 g soil (wet weight), depending on the organic matter content, was homogenized with 200 ml distilled water in a Sorvall Omnimixer, centrifuged at 750 X g for 10 min. and the supernatant collected. To the resulting bacterial suspension [’ H-methyllthymidine was

added (200 nM final concentration, 925 GBq mmol-‘, Amersham. UK), and the incorporation (at 20°C) was stopped after 2 h with formalin. A zero-

time control (formalin added before the labelled thymidine) was always included. Filtration, washing and measurement of incorporated radioactivity were as described by BEith [16].

Before adding thymidine, the pH of the bacterial suspensions was altered to different values using different buffers or dilute H, SO,. On both measur-

4 5

PH

Fig. I, The effect of pH on thymidine incorporation into macromolecules (cold acid insoluble material) of bacteria extracted from a forest

soil (soil 2 in Table I) by homogenization-centrifugation. 0. pH was altered with diluted sulphuric acid. where the sample with the highest

pH was used without any acid (distilled water); 0, pH was altered with citrate-phosphate buffer; 0. pH was altered with phosphate buffer.

Bars denote SE. (n = 3) from separate experiments. Soil pH(H,O) was 4.21 and pH(KCI) was 3.51.

ing occasions. pH was altered to approx. 5.5. 6.2, 7.2 and 8.2 using a potassium phosphate buffer (6.6 mM. final concentration). On the first measuring occasion a citrate-potassium phosphate buffer (I .65 mM citric acid and 3.3 mM K,HPO,. final concen- tration) was used to give pH values of approx. 3.5 and 4.3. This was only done for I2 soils. For the second measurement occasion 0.025 M H,SO, was added to give these low pH values. The actual pH was always measured immediately before the thymi- dine addition. Two replicate measurements were made for each pH level and a distilled water control. The data are expressed as percentages of the thymi- dine incorporation values obtained when only dis- tilled water was used, that is. when the natural pH of the soil prevailed. Values for the zero-time control was always subtracted before these calculations.

The bacteria1 community from soil 2 was also

studied more specifically by altering the pH by small additions of dilute sulfuric acid in order to detect the optimum pH for thymidine incorporation with more precision.

Leucine incorporation was performed simultane- ously with the thymidine incorporation measure- ments by adding L-[U-‘JC]leucine (775 nM final concentration. 1 1.9 GBq mmol-‘, Amersham, UK) together with the radioactive thymidine. This was only made for 4 soils.

The degree of participation (DP) of the added labelled substrates in the incorporation was measured for two soils by the isotope dilution approach [ 171. Different amounts of non-radioactive substrate were added with the labelled substrates. If the reciprocal of disintegrations min-’ are plotted against the amount of added thymidine or leucine. the intercept with the .r-axis will be the amount of exogenous and

PH

Fig. 2. The effect of pH on thymidine incorporation into macromolecules (cold acid insoluble material) of bacteria extracted from soils by

homogenization-centrifugation. Data were expressed as percentages of distilled water controls. pH was altered with dilute sulphuric acid for

the two lowest pH values and with phosphate buffer for the four other values. Soil numbers refer to Table I. 0. soil I: n , soil 7: 0. soil 6;

0. soil 7; a. soil 13: A. hoi1 14: ~1. soil 16.

E. BBBth / FEMS Microbiology Ecolo,~y IY (1996) 227-237 231

de novo synthesised substrate participating in the macromolecular synthesis.

2.3. Statistics

Thymidine incorporation at different pH values was subjected to principal component (PC) analysis, after standardization of the data by dividing with the distilled water controls. In order to separate the soils according to the pH response of their bacterial com- munities, each soil sample was considered one ob- ject. The multivariate calculations were performed using the computer programme SIRIUS [ 181.

3. Results

The pH response of the bacterial community of soil 2 (an acid soil from a coniferous forest) was initially determined by decreasing pH using small increments of added dilute sulfuric acid (Fig. 1). The distilled water control of the extracted bacterial solu- tion had a pH of around 4.8. Decreasing the pH

increased the thymidine incorporation up to an opti- mum slightly above pH 4. Decreasing the pH even more resulted in decreased thymidine incorporation of the bacterial community. The optimum pH of the thymidine incorporation thus corresponded well with the pH(H,O) of the soil (4.21), but was higher than pH(KC1) (3.52).

The addition of the citrate-phosphate buffer to set the two lowest pH values appeared to inhibit thymi- dine incorporation. This can be seen in Fig. 1, where the thymidine incorporation using this buffer is com- pared with the use of dilute sulfuric acid to alter pH. The use of phosphate buffer. however, appeared not to inhibit bacterial incorporation of thymidine. Judg- ing from Fig. 1 the use of this buffer for pH around 5 gave similar values as would have been expected from a pH response curve extended to this pH value. Thus, when not otherwise stated only data using phosphate buffer and dilute sulfuric acid from the second measurement occasion were used to compare the pH response of bacterial communities from dif- ferent soils.

Large differences were found, when the pH re-

50-

; 25-

8 & E s -a O- .a s ‘i: a

3

9

5

6

11

14

16

12

7 'ts

::!__’ -100 -50 0 50 100

Principal component 1

Fig. 3. Principal component analysis of pH response curves for bacterial communities extracted from differents soils using homogenization- centrifugation. pH was altered with dilute sulphuric acid for the two lowest pH values and with phosphate buffer for the four other values.

Numbers refer to soils in Table I.

sponse curves from different soils were compared. This is seen in Fig. 2. where some examples of such curves are shown. The optimum pH for the low pH soils I and 2 was around pH 4. while for the soils with higher pH, the optimum incorporation rate was gradually shifted to a higher pH. Thus, soil 16 with the highest pH (8.10) had a response curve with the highest pH optimum. Similar results. with the optima of the pH response curves correlated with the soil pH(H,O). were found for the other soils tested (data not shown). A significant linear correlation (P < 0.001) was found, when the optimum pH for thymi- dine incorporation was correlated to soil pH(H?O) (Y’ = 0.62. 17 = 16).

It was. however, difficult to exactly determine optimum pH values, since only six different pH values were used for the thymidine incorporation. Instead, in order to compare the different bacterial communities. a principal component (PC) analysis was performed, in which each soil was considered as an object and each pH used for the thymidine incor- poration were the variables (Fig. 3). The first princi-

pal component explained about 80% of the variation in the data. The low pH soils (Nos. I. 2 and 3) were all situated to the left in the PC plot. while the high pH soils (Nos. I3 to 16) were found to the right. Thus. the soils were ordered according to their pH along the first PC axis. This relationship was further underlined by correlating pH(H,0) and the scores for each soil for the first principal component (Fig. 4). A strong linear relationship was found (1.’ = 0.85, 17 = 16, P < 0.001).

Although the citrate-phosphate buffer appeared to inhibit thymidine incorporation (Fig. I), the use of this buffer to set the lower pH values could also be used to differentiate between the pH response of the bacterial communities of the different soils (data not shown). Thus. when a PC analysis was performed on the pH response curves for the 12 soils tested in this way. a picture similar to that in Fig. 3 was seen. The first principal component explained 867~ of the vari- ation in the data, and when the scores were corre- lated against soil pH(H,O), a linear relationship was found (Y’ = 0.85, 12 = 12. P < 0.001).

100 y = 35.1x - 213.9 12 = 0.85

-100 ! I I I I I 3 4 5 6 7 8

Soil pH(H20)

Fig. 4. Correlation between scores for the first principal component from the analysis of the pH response curves and the soil pH(HIO).

Numbers refer to soils in Table I.

E. Biiiith / FEMS Microbiology Ecology 19 (1996) 227-237 333

Inspection of Fig. 2 revealed that the largest differences between bacterial communities were found when thymidine incorporation was measured at extreme pH values. Thus, a good separation be- tween the different bacterial communities should be possible, if the ratio between thymidine incorpora- tion at those extreme pH values are compared. This also appeared to be the case, since the logarithm of the ratio of thymidine incorporation at pH 4.3 to that at pH 8.2 (Fig. 5) or thymidine incorporation at pH 3.5 to that at pH 8.2 (not shown) were negatively and linearly related to soil pH(H,O) (r2 = 0.86 and 0.84, respectively). The negative slope was greater for the latter ratio and would thus be better for separating the different bacterial communities. How- ever, since very low values of thymidine incorpora- tion were found at pH 3.5 for bacterial communities from the high pH soils, and thus the errors in the measurements were large, the variation was higher using the ratio incorporation at pH 3.5 to that at 8.2

Table 2

The effect of pH on degree of participation (DP) of the added

labelled substance in the incorporation into cold acid insoluble

substances of bacteria extracted from soil by homogenization-

centrifugation

pH DP,, DP,,, Leu/TdR Leu/Tdr

(T/c) (c/c) (mol/mol) (mol/mol)

without DP with DP

Soil 7 3.98 50.5 17.6 1.9 14.4

5.90 d 49.1 38.6 Il.6 12.0

8.01 58.7 51.9 14.4 14.0

Soil I6 4.08 100 34.4 15.8 45.9

7.10 100 59.6 19.2 49.0

7.91 a 100 58.1 26.1 45.0

The leucine (Leu) to thymidine (TdR) ratio was either calculated

directly, without considering DP, or after taking account of DP.

’ pH in the bacteria1 solution directly extracted from the soil.

than using the ratio incorporation at pH 4.3 to that at 8.2.

The pH dependent thymidine incorporation was

=-0.37x + 2.25 ra = 0.06

3

Soil pH(H20)

8

Fig. 5. Correlation between the logarithm of the ratio of thymidine incorporation into macromolecules (cold acid insoluble material) at pH

4.3 and 8.2 of bacteria extracted from soils by homogenization-centrifugation and pH(H?O) of the soils. pH was altered with dilute sulphuric acid for the lowest pH value and with phosphate buffer for the highest. Numbers refer to soils in Table I.

233

25-

20-

a E % .s

f

15-

_)

lo-

5-

U Soil 3

Soil 5

Soil 15

Soil 16

1 9

PH

Fig. 6. The effect of pH on the leucine to thymidine incorporation ratio calculated withou compensating for isotope dilution.

compared with leucine incorporation for several soils. If these two methods were affected in a similar way by pH, the ratio leucine to thymidine incorporation should be constant over the whole pH interval tested. However, especially at pH values below 5 the leucine to thymidine incorporation ratio decreased (Fig. 6). This appeared to be due to high isotope dilution and thus low degree of participation of the added leucine in the incorporation at low pH values, while isotope dilution for thymidine incorporation appeared not to be affected by pH (Table 2). The leucine to thymi- dine incorporation ratio was not affected by pH when the values were adjusted for isotope dilution.

4. Discussion

The crucial question, that needs to be answered in order to be able to use thymidine incorporation to determine the pH response of a bacterial community. is if thymidine incorporation at a certain pH only

reflects the activity (growth rate) of the community or if pH per se affects the uptake and incorporation of thymidine irrespective of the origin of the bacte- rial community. The latter could for example be due to pH affecting the chemical form of thymidine, thus affecting the uptake rate. This could not be answered definitely using the present data. However, both thymidine and leucine incorporation gave the same results, when isotope dilution was considered (Table 2). indicating that the result for thymidine incorpora- tion was not due to a direct pH effect. Furthermore, any physical/chemical effects would be the same at one particular pH irrespective of the origin of the bacterial community. Thus, the fact that thymidine incorporation was relatively high at pH 3.5 for bacte- rial communities from acid soils and low for com- munities from soils with high pH (Fig. 2) could only be due to properties of the bacteria.

The bacterial community appeared to be well adapted to the prevailing pH of the soil, irrespective of the soil being acid or having a more neutral pH

E. B&?th / FEMS Micmbiolog~ Ecology 19 C 1996) 227-237 235

(Figs. 1 and 2). This was also found for the denitrify- ing populations of two soils with different pH [19]. where the denitrifying potential of the population from the low pH soil was adapted to the acidic pH conditions prevailing in that soil.

In a study of 15 soils different from the ones used

here, 0.7 X lo’“-7.2 X 10” acridine orange stained

bacteria g- ’ organic matter and a strong negative exponential relationship between bacterial counts and organic matter content of the soil were found ([2], recalculated from their Table I >. A negative correla- tion, but less strong, was also found with the soil pH(H,O). Although it is thus clear that, on an organic matter basis. bacteria are less abundant in low pH soils high in organic matter than in high pH

soils with little organic matter, we cannot deduce which factor is the most important, pH or organic matter, since they are often correlated. However, since the optimum activity for the bacterial commu- nities was found around the actual pH(H,O) of the soils (Figs. 1 and 2). it might indicate that pH per se was not the main factor causing low bacterial counts in acid soils. Thus, earlier explanations (see Intro- duction) of low bacterial numbers in acidic soils being due to the bacterial community not being adapted to the pH conditions might be to simplistic.

One can hypothesize that the bacterial community response curves might indicate under what pH condi- tions the soil organisms are actually growing. It has, for example, been argued that pH in soil will be lowest at particle surfaces. Thus, soil pH measured in a water suspension will overestimate the actual pH for surface inhabiting organisms like bacteria. pH(KCl), which should reflect the approximate ionic strength normally present in the soil solution, should therefore be a more appropriate pH indicating the conditions encountered by soil bacteria. A closer inspection of Fig. 1 indicates, however, that the optimum thymidine incorporation of the bacterial community was slightly below pH(Hz0) of this acid soil, but well above pH(KC1). This might indicate, that pH(H20) may better reflect [H+] encountered by soil bacteria than pH(KC1). A direct technique to measure pH in soil is to extract pore water directly from soil using centrifugation. Extracted pore water had about 0.3 units lower pH than pH(H,O), but about 0.5 units higher than the pH determined after extraction with a salt solution in a forest with

pH(H,O) between 4 and 5.5 [20]. Thus, the fact that the optimum thymidine incorporation of the bacterial community is slightly lower than pH(H,O) in Fig. 1 might indicate that the pH of a directly extracted pore water is the most correct one. at least when it comes to the environment encountered by the bacte- ria.

There are different ways of comparing the pH response curves for the different bacterial communi- ties. The most obvious would be to use the optimum pH directly. However, although the optimum pH was significantly correlated to the soil pH, the degree of explanation was rather low. This was not unex- pected. since the use of only six pH values in the response curve gave low precision in the estimated optimum pH, and also that this approach did not consider the whole pH response curve. One could also fit a mathematical model to the data and com- pare the different cardinal pH values calculated using this model. This has been used to compare pure culture bacterial isolates [21.22]. However, to get a good fit one would need data with a higher resolu- tion than the six different pH values used in the present study, and the models therefore had low precision when tried. Furthermore, the models used for pure cultures do not necessarily hold for commu- nities.

An objective way of comparing different curves without fitting a specific equation but still using all the data points is the use of principal component (PC) analysis. This was, for example, a better way to compare temperature effects on growth of fungal isolates than fitting a model equation [23]. In the present study a very good separation along the first PC axis was found (Fig. 3) and the scores along this axis were well correlated to the soil pH (Fig. 4).

An even easier way of comparing the pH relations of different bacterial communities is to calculate the logarithm of the ratio of thymidine incorporation at two extreme pH values, as illustrated for pH 4.3 and 8.2 in Fig. 5. This will make the measurements very rapid and less expensive than using the full range of pH values. Furthermore, there is no need to have a distilled water control to standardize the incorpora- tion values. Thus, only two replicates for two pH values are needed for each soil sample. Compared to plate counting techniques the use of the thymidine incorporation method in this way will be very cost

efficient when measuring community responses to pH. Furthermore, since usually no more than 100 colonies can be counted on an agar plate, while in the thymidine incorporation technique more than IO’ bacteria are used in the measurements, the former technique will probably also be less reproducible.

Earlier attempts to use the thymidine incorpora- tion technique to study the bacterial community pH response used citrate-phosphate buffer to set the lower pH levels [ 10.141. This buffer inhibits thymi- dine incorporation at the concentrations used (Fig. I), and this explains why the optimum pH for incor- poration was not found at the low pH values ex- pected in the acid soils used in these studies.

On the other hand, B%th et al. [I21 used dilute sulfuric acid to set the lower pH values when mea- suring the impact of different pH increasing mea- sures in two different forest soils on the bacterial community pH response. Their data can therefore be directly compared with the ones presented here, and can be used as a validation data set. Recalculating their data and using Fig. 5 to predict soil pH showed. for example, that the two non-treated control soils with pH(H,O) of 3.93 and 5.2-5.4 had a predicted pH of 3.95 and 5.35. respectively, using the pH response curves for the bacterial communities. By using all 8 treatments studied by B%%th et al. [ 121, a linear regression between predicted soil pH from the bacterial community response and measured soil pH explained a large part of the variation (r’ = 0.79). Thus, a calibration curve, either using a multivariate statistical technique. like PC analysis, as in Fig. 4, or the ratio of two values as in Fig. 5. could be constructed once. Bacterial communities extracted from other soils or environments could then be fitted into this correlation to indicate the pH conditions that had prevailed in their environment.

A similar extraction technique as the one used here for estimating the pH response of a bacterial community have been used to study metal tolerance of soil bacteria [24]. In a later study [25] it was shown that leucine incorporation was as sensitive as thymidine incorporation to measure bacterial toler- ance to metals. This was also the case for pH measurements, when isotope dilution for leucine in- corporation was considered (Table 2). However. the measurements will be both more time consuming and less precise, if isotope dilution has to be esti-

mated for each pH tested. Thus, thymidine incorpo- ration, where isotope dilution was not affected by pH, appeared to be better suited for estimating pH response curves of bacterial communities than leucine incorporation.

Acknowledgements

This study was supported by a grant from the Swedish Natural Science Research Council.

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