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Ecological studies on coccoid bacteria in a pine forest soil—II. growth of bacteria introduced into soil
Soil Bid. Biochern. Vol. 5, pp, 449-462. Pergamon Press 1973. Printed in Great Britain.
ECOLOGICAL STUDIES ON COCCOID BACTERIA IN A PINE FOREST SOIL-II. GROWTH OF BACTERIA INTRODUCED INTO SOIL W. E. LOWE*and T. R. G. GRAY HartIey Botanical Laboratories,
University of LiverpooI, England
(Acce~:ed 12 October 1972) Summary-The growth of five typical but phenetically distinct cocci and Arthrobuc#erstrains, isolated from a pine forest soil, has been investigated. Soil reaction was found to have a marked effect on growth and unless naturally acidic soils were made more alkaline they did not support growth, even in the presence of added nutrients. However, in the presence of fungi which could use the added nutrients, e.g. chitin, mycelial fragments etc., bacterial growth was possible and could be correlated with a decrease in acidity, especially around particles of organic matter. Where the pH rose above 7.9, bacterial growth again decreased. All the bacterial strains tested reacted in the same way, suggesting that they occupied similar microenvironments in both the acidic and alkaline soil horizons examined. Some explanations for the occurrence of nonsporing bacteria in soils in which apparently they cannot grow are suggested. rNTRODWCTION
NATURALselection results in the formation
of groups of microorganisms adapted to their environments and so clusters of bacteria defined by numerical taxonomic techniques ought to be found associated with particular microenvironments within the soil. In a soil with several distinct horizons, the occurrence of bacteria in all horizons may indicate that a microenvironment is common to them while restriction of bacteria to one horizon suggests that there is a microenvironment peculiar to it. Lowe and Gray (1972) have described the isolation of coccoid bacteria from a pine forest soil and their classification by numerical taxonomic procedures. Nine clusters or subclusters of organisms were delimited by these techniques although most soil cocci could be placed in five of these clusters. The most central and typical strains of each of these five major groups were identified and this paper describes experiments designed to obtain information concerning the ability of these apparently typical organisms to grow in the soil from which they had been isolated. By altering the soil environment through the addition of nutrients and the adjustment of pH, some of the factors affecting the growth of the bacteria in two soil horizons were investigated. All the experiments involved the inoculation of sterilized soil with the required organisms, subsequent changes in population size being followed using the dilution plate count technique, together with (in certain cases) measurements of oxygen uptake by respirometry. MATERIALS
AND METHODS
Preparation of soils
Soil from the Al (PI-I 4.5) and C (pH 8.3) horizon of Freshfield soil (Goodfellow, Hill and Gray, 1968) was dried at 45°C for 24 h after removal of large roots and twigs. All the * Present address: Department of Soil Science, University of Saskatchewan, Saskatoon, Canada. 449
450
W. E. LOWE AND T. R. G. GRAY
soil used was from samples taken at one time and stored dry until required. Samples of these soils were mixed in a mortar and 10 g quantities transferred to 20 ml Universal screw top vials. These were sterilized by autoclaving (121 “C for 20 min) on two separate occasions, the vials being incubated at room temperature for 24 h between autoclavings. The soil was dried in a vacuum desiccator after the second autoclaving. Selection of bacterial strains The bacterial strains central to the largest clusters defined by Lowe and Gray (1972) were selected for inoculation into the soil. These were as follows: cluster 1-Micrococcus A34; cluster 2-Staphylococcus A8 1; cluster 3-Arthrobacter type A, A21 ; cluster 4iArthrobacter type B, A49; cluster 4i,-Arthrobacter type B, C39. Organisms in clusters 1, 2, 3 and 4, were isolated mainly from the A, horizon whereas those in cluster 4ii were associated mainly with the C horizon. The principle characteristics of these bacteria are given by Lowe and Gray (1972). Inoculation of soils and adjustment of their moisture content Test organisms, which had been stored under oil at 5°C were streaked onto plates of PYE agar (Goodfellow et al., 1968), checked for purity and transferred to PYE agar slopes in 250 ml medical flat bottles. After incubation for 24 h at 25°C the bacterial growth was suspended in 25 ml amounts of sterile deionized water in 100 ml conical flasks. Cell aggregates were dispersed by agitating suspensions with a magnetic stirrer-bar rotating at approx. 200 rev./min for 15 min. The concentration of cells was estimated using a nephelometer and the number of cells adjusted to approx. 2 x lO’/ml. Sterile soil was inoculated immediately with 0.5 ml of this suspension and the number of viable cells determined using the dilution plate count technique. The moisture content of the soil was adjusted to 15 % (v/w) oven dried soil by the addition of I.0 ml nutrient solution or deionized water. This ensured that the moisture content was equivalent to 60 per cent of the moisture holding capacity of the soils, i.e. pF values of I.6 and 1.4 respectively for the A, and C horizon samples (Shameemullah, Parkinson and Burges, 1971). These levels are typical of the soil under field conditions (Kibble, 1966) although they may not be optimal for bacterial growth. However, preliminary experiments suggested that bacterial growth was not greatly affected by alterations in moisture content from 5 to 20% (v/w). After inoculation and adjustment of the moisture content, each soil sample was mixed thoroughly and maintained at 4°C for 12 h to allow the moisture, nutrients and bacteria to be dispersed throughout the system with little growth of bacteria occurring. Methods for determining growth of bacteria Numbers of bacteria. Bacteria were counted using the dilution plate count technique (Goodfellow et al., 1968). Four replicate plates were prepared at each dilution and the experiments performed in triplicate (unless otherwise stated). The soil remaining in the vials was used for determining pH and moisture content. Oxygen uptake. Oxygen uptake was measured using Warburg constant volume respirometers and the reaction flasks of Parkinson and Coups (1963). The flasks were ethersterilized and the joints greased with sterile Vaseline. The entire contents of the vials inoculated with bacteria were transferred to the bases of the flasks, the flasks attached to the manometers and oxygen uptake measured at 25°C. Control flasks, containing soil inoculated with 0.5 ml sterile deionized water in place of bacterial suspensions, were also set
STUDIES ON COCCOID BACTERIA IN SOIL-H
451
up. Flasks were allowed to equilibrate for 12 h with the manometer taps open and readings were then taken at 12 h intervals. In order to obtain consecutive readings, flasks were prepared in duplicate; the taps of one series were dosed after equilibration and readings taken after 12 h. During the following 12 h period, the taps of the first series of flasks were allowed to remain open to replenish the oxygen supply, and the taps of the second series of flasks closed. After 48 h incubation, the KOH in the reaction vessels were renewed. All experiments were performed in triplicate. Adjustment of soil pH. In certain experiments, the pH of A, horizon soil was made alkaline by adding 0.05% (w/w) CaCO,, followed by thorough mixing in a mortar. The pH of this soil after sterilization was approx. 7 *5. Addition of the same amount of CaCO, to C horizon soil caused no change in pH. In some cases, the pH of the A, horizon soil was also adjusted to 7.5 by the addition of KOH solution.
EXPERIMENTAL
EfSect of pH on bacterial growth in soil The effect of pH on bacterial growth was investigated by inoculating soils at different pH values with various strains. A, horizon soil (pH 4.3) A, horizon soil (CaCO, amended, pH 7*5), A, horizon soil (KOH amended, pH 7*5), C horizon soil (pH 8.1) and C horizon soil (CaCO, amended, pH 8 * I) were prepared. All soils were inoculated with organisms A34, A81, A21, A49 and C39, incubated for 168 h at 25°C and sampled after 24, 48, 72 and 168 h. The results of dilution plate counts as well as oxygen uptake readings, taken at 12 h intervals during the first 5 days of this experiment, are also recorded in Table 1. The numbers of bacteria developing and the oxygen uptake levels in the C horizon soil amended with CaCO, never differed significantly from the numbers developing in unamended C horizon soil and so the former have been omitted. One set of data for A21 is also shown in Fig. 1. The results show clearly that none of the bacteria were able to grow in acid A, soil, even though four of them had been isolated from this soil. No bacteria were isolated from this soil after 24 h, even at dilutions of 10m4and no oxygen uptake was recorded. In alkaline C horizon soil (pH 8. l), all the strains grew and levels of oxygen uptake, although low, were recorded. In A, horizon soil adjusted to an alkaline pH, growth also occurred but always at a higher level than that found in the C horizon. The maximum growth rate was achieved during the first 24-36 h in all cases; after 48 h, numbers of organisms stopped increasing or increased slowly while oxygen uptake levels decreased. A further experiment was performed to determine the pH at which growth in the A, horizon soil was inhibited. Al horizon soil was amended with quantities of CaC03 to give pH values in the range 4 -5-8 a5 at O-5 pH unit intervals after autoclaving; vials of these soils were moistened with 0.5 ml sterile deionized water, sealed and incubated for 14 days. After this time, no further changes in pH were detected. Bacteria were added to these soils and dilution plates prepared from samples taken after 24,48 and 72 h. Because of the large number of dilution plates involved, the treatments were not replicated. However, the results are striking and uniform for the five different bacteria. The results are recorded in Fig. 2 and Table 2 and show that in all cases, while the bacteria survived and grew well at pH 6 -0 and above, at pH 5 *5 and below, populations declined or even disappeared completely. Similar results were obtained by growing bacteria in PYE broths buffered to a similar range of pH values. Virtually no growth occurred at
452
W. E. LOWE AND T. R. G. GRAY
pH values of 5.5 and below, with the exception of organism A21 which grew slowly at pH 5.5 in broth. These results indicate that although the organisms used in this study appear to be phenetitally distinct, their growth patterns following introduction into sterile soil are similar. Isolates grew in a similar manner in sterile A, (pH 7 *5) soil and C horizon soil, irrespective of the horizon from which they were isolated, suggesting that similar microenvironments
TABLE
1. GROWTH
OF BACTERIA IN NUTRITIONALLY UNAMENDED PIf LEVELS
STERILE SOILS AT DIFFERENT
Bacterial nos. per g oven dry soil (x
Organism A21
A81
A49
c39
A34
Al horizon pH 7.5
Time (days)
C horizon pH 8.1
0 1 2 3 71
0.13 0.4 5 0.2 2.1 5 0.4 1.4hO.l 2.2 f 0.2
0.13 1.2 19-S 22.4 17.5
+ f i_ i
0.2 3.6 2.8 3-o
: 3 71
0.14 1.2 3.6 5.4 6.5
+ 0.1 10.3 f 0.3 h 0.3
0.14 5.0 11-o 17.5 30.5
+ * * f
0.8 1.5 2.2 1.3
0 1 2 3 71
0.11 1.4 2.5 1.9 1.3
f + * *
0.2 0.2 0‘2 0.4
0.11 3.4 12.2 9.3 16.0
Jr & & +
o-9 2.2 0.6 1.6
0 1 2 3 71
0.19 0.5 0.6 1.3 1.1
i i_ + f
o-1 0.1 0.1 0.2
0.19 3.1 4.6 4.3 4.8
* i + +
0.1 0.3 0.1 0.2
0.21 2.0 * 2.6 f 2.3 + 2.0 f
0.1 0.1 0.1 0.2
0.21 7.3 9.4 8.9 7.6
5 Zt i i
0,2 0.4 0.3 0.2
0
0 ; 3 71
KaCW
A1 horizon pH 7.5
WW 0.13 2.5 21.0 19.7 19.9 -
lo-‘)
AI horizon pH 4.3 0.13
& i5 rt
0.2 1.2 1.8 3.5
x 0 0 0.14
-
ZI
--
8
-
0.11 0
--
00 0
-
0.19 0
--
8 0
--
0.21 :: 0 0
for bacterial growth exist in both horizons. Growth in the A, horizon (pH 4.3) soil is limited by unfavourable pH and the presence of these same bacteria in natural A, horizon soil in large numbers indicates that either microenvironments of higher pH are present in unsterilized soil or that isolation of microorganisms on neutral media only allows the growth of atypical forms. Eflect of nutrient amendment on bacterial growth in soil
Smith (1967) has shown that alkaline microenvironments can be produced in acid soils through the accumulation of ammonium ions in the region of organic matter. Williams and
STUDIES ON COCCOID BACTERIA
IN SOIL-II
453
Mayfield (1971) demonstrated that acid intolerant streptomycetes could grow in such regions. This was shown by amending the soil with nitrogen containing compounds and a soil fungus which could attack them. Ammonification of the organic matter occurred and regions of high pH were observed around organic particles when these were stained with appropriate indicators. The possibility of such interactions being important for bacteria involved in this investigation was tested. First, the effect of nutrient amendment on the five bacteria was examined and then the effect of adding nutrients and fungi to the soil before bacterial inoculation. The form and concentration of nutrients added to the soil are given in Table 3. TABLE 2. GROWTH OF BACTERIAIN STERILEAl HORIZONSOILAT VARIOUSpH LEVELS Bacterial nos. per g oven dry soil (X lOWe) pH of soil Time (days)
8.5
8.0
I.5
7.0
6.5
6.0
5.5
5.0
4.5
A21
0 1 2 3
1.2 119 214 118
1.2 84 223 113
1.2 121 253 124
1.2 31 98 84
1.2 15 17 23
1.2 14 13 15
1.2 1.3 0.2 0.03
1.2 0.2 0.2 0.02
1.2 0 0 0
A81
0
1.3
: 3
3.2 50 71
1.3 11 51 74
1.3 12 111 98
1.3 9 52 31
1.3 5.3 14 12
1.3 2.1 5.6 4.9
1.3 0.5 0.05 0
1.3 0.1 0 0
1.3 0 0 0
0 1 2 3
1.4 140 180 120
1.4 160 340 280
1.4 51 82 140
1.4 45 76 971
1.4 :; 64
1.4 1.3 8.4 8.3
1.4 0.1 0.1 0
1.4 0.1 0 0
1.4 0 0 0
c39
0 1 2 3
0.9 2.7 14 68
0.9 2.8 35 79
0.9 3.2 34 82
0.9 1.3 21 78
0.9 1.2 9.2 14
0.9 1.0 1.1 3.4
0.9 0.6 0.1 0
0.9 0.1 0.1 0
0.9 0 0 0
A34
0 1 2 3
0.9 39 65 62
0.9 131 116 109
0.9 130 150 160
0.9 83 124 115
0.9 35 62 51
0.9 3,4 2.2 1.2
0.9 1.1 0.2 0.04
0.9 1.0 0.03 0
0.9 0 0 0
Organisms
A49
Arthrobacter type A (A21) and Micrococcus (A34) were the only organisms able to use inorganic nutrients as sole sources of nitrogen but the effects of these nutrients on the other organisms were also investigated; an inability to use inorganic substances in culture media may have been due to lack of other substances, e.g. vitamins, present in soil. As well as adding the nutrients separately to soil, nitrogen, phosphorus plus potassium and magnesium were added together and also together with glucose. The number of dilution plates that had to be prepared was again large so that the experiment was performed only once. As before, there were only minor differences between the results for all five bacteria and only the results for A21 have been plotted (Fig. 3). No growth or survival of bacteria was detected in A, (pH 4.3) soil, even in the presence of added nutrients, while growth did
454
W. E. LOWE AND T. R. G. GRAY
occur in A, (pH 7.5) and C horizon soils in amended and unamended conditions. The growth curves were similar to those described in the previous experiment, more growth and oxygen uptake being associated with the A, horizon soil than the C horizon soil. In the amended soils, the growth yields were higher than in the unamended soils but after 7-10 days, numbers of bacteria had decreased and were very similar to those of unamended soil. Oxygen uptake levels were higher than in unamended soils but the rate of oxygen uptake
I
2
3
Time.
4
5
6
7
days
FIG. 1. Growth of Arthrobacter (type A) A21 in nutritionally unamended soil as judged by changes in population size and oxygen uptake. (0-O) C horizon soil, pH 8.1; (O-O) AI horizon soil, pH adjusted to 7.5 with CaCO,; (X-X) Ai horizon soil, pH adjusted to 7.5 with KOH; (.----+) AI horizon soil, pH 4.3.
was already decreasing after 48 h incubation. In all cases, amendment with casamino acid mixture produced the greatest response. Amendment with inorganic nutrients (N, Mg + S, P + K), produced little if any effect unless added with glucose. The results suggest that Iack of carbon and nitrogen limits bacterial growth in these soils, at suitable pH levels, and that increased metabolism caused by the addition of carbon and nitrogen may in turn be limited by the low concentrations of inorganic salts. The growth curves could be divided into two phases, an initial growth phase during which a rapid increase in numbers and oxygen uptake levels took place and a survival phase
STUDIES ON COCCOID BACTERIA
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455
TABLE 3. FORM AND FINAL CONCENTRATION OF NUTRIENTSADDEDTO STERILESOIL Form and final con~en~ation (%, w/w oven dried soil) of nutrient supplied
Nutrient Carbon Nitrogen Organic Inorganic Fhosphorus~potassium
Glucose* (0.02) Casamino acids? (0.08 N) Ammonium nitrate* (0.08 N) Potassium dihydrogen (0.04 P) (0.06 K) Phosphate* Magnesium sulphate* (0.06 S) (0.04 Mg)
Magnesium/sulphur
* BDH Ltd., Poole, England-Analar grades. _t Difco Labs., Michigan, U.S.A.-Bacto vitamin free casamino acids.
during which numbers remained almost constant or decreased slightly and levels of oxygen consumption were low. For higher plants and animals, the size of a single species population which can develop in a given environment has been termed the carrying capacity for that species (Boughey, 1968) and this remains constant under a given set of environmental conditions. Using the same terminology here, the A, horizon soit when treated with calcium carbonate, has a
>
; \ i \J___._
-_-.-
2
I Time,
3
days
FIG. 2. Growth of Arthrobacter (type A) A21 in sterile A1 horizon soil at various pH levels. (A---& pH 8.5; (a-----a) pH 8% (0-O) pH 7.5; (O-O) pH 7.0; (W---u) pH 6.5; (U-U) pH 6.0; (x-x) pH 5.5; (.----.) pH 5.0; (.-.-.> pH 4.5.
4.56
W. E. LOWE AND T. R, G. GRAY
greater carrying capacity than the C horizon; in both cases, it can be increased by nutrient amendment though it falls again as nutrients become depleted. The occurrence o~artl~c~all}~produced ~lk~~~~em~cr~-e~~l~~ro~rne~fs
Under natural conditions, a variety of fungi are present and able to grow in Freshfield soil. Some of these are capable of breaking down chitin and other nitrogen containing compounds, releasing both carbon dioxide and ammonia, e.g. Trichoderma viride and
Time,
days
FIG. 3. Growth of ~~g~r~~~c#er (type A) AZ in nutritionallyamended so% S~eriltr C horizon soil, pH 8. I : (*---e) no added nutrietnts; (O-e-e-*) plus inorganic N, MgS and PK; (----I plus glucose; (A----& plus glucose plus inorganic N, MgS and PK; (x x ) plus casamino acids. Sterile Al horizon suil, pH adjusted fu 7.5 rvifh CaCu,: (O---O) no added nutrients; (O-+-.-O) plus inorganic, N, MgS and PK; (n-----n, plus glucose; (3-D) plus glucose plus inorganic N, MgS and FK; (o-T7) pfus casamino acids. Sterile A, horizwz soil, pH 4.3: (.----.) ait treatments. Numbers of bacteria expressed per gramme oven dried soil. Oxygen uptake expressed as ~1 02/g oven dry soil/l2 h. Vertical lines represent the 95 per cent confidence limits.
Mortierellu marburgensis (Gray and Baxby, 196s). The effect of the growth of these fungi on
soil pH and bacterial growth was investigated. Chitin was obtained in a relatively impure form containing protein (BDH Ltd., Poole, England) and also in crude form in fungal mycelium and yeast cells, For the preparation of fungal mycebum, T. viride and M. RWb~rge~s~~ were grown in malt extract broth (pH 6-O) in shake culture and harvested after 7 days growth at 25°C. The mycehum was washed in running water for 1 h and rinsed in deionized water. After drying at 105°C for 24 h, mycehum was ground in a mortar and
STUDIES ON COCCOID BACTERIA IN SOIL-II
457
stored dry. Baker’s yeast cells were suspended in water, washed four times and then dried and ground. BDH chitin was used as supplied, except for preliminary grinding in a mortar. Quantities of oven-dried A, horizon soil were amended with varying amounts of chitin, fungal mycelium, yeast cells and casamino acids. Ten gram samples were transferred to vials, sterilized and remoistened with 0.5 ml sterile deionized water and kept sealed for 14 days. pH measured after this time was found to be approximately the same as that of untreated soil. The soils were inoculated with living T. viride or M. marburgensis as follows. Spore suspensions were prepared from fungi grown on malt extract agar for 7 days at 25°C; the spores were harvested and suspended in sterile deionized water. After inoculation,
80
6-5 I a r 6-O ii 5.5
5.0
4.5
4.0 ' 0
I I
I 2
I 3
I 4 Time,
I 5
I 6
I 7
I 6
I 9
I IO
days
4. pH changes in sterilized A, horizon soil amended with varying amounts of sterile dry Trichoderma viride and Mortierella marburgensis mycelium and inoculated with living T. viride mycelium. Soil amendments: (O-O) 12.5% w/w T. viride mycelium; (0-O) 10.0% w/w T. viride mycelium; (A-A) 7.5% w/w T. viride mycelium; (A-A) 5.0% w/w T. viride mycelium; (0-O) 2.5% w/w T. viride mycelium; (W-m) 1.25% w/w T. viride mycelium; (O-.-.-O) 12.5% w/w M. marburgensis mycelium; (A-.-.-A) 7.5% w/w M. marburgensis mycelium ; ( q- .-a - 0) 2.5 ‘A w/w M. marburgensis mycelium. Unamended soils: (s---.) inoculated with T. viride culture; (X-.-.-X) inoculated with M. marburgensis culture.
FIG.
O-5 ml amounts of sterile water were added, bringing the soil moisture content to 15 % (v/w). The soils were aseptically and thoroughly mixed and incubated at 25°C for 10 days. Macro-pH determinations were made on these soils using a glass-calomel electrode pH meter. Micro-pH determinations were made by placing a few drops of bromothymol blue indicator on soil spread on a microscope slide (Williams and Mayfield, 1971). The soil was examined under a low power microscope and the colour of the indicator observed; a blue colour was indicative of a pH value of 6 -0 and above, a yellow colour of pH values below 6 90. The results of the bulk-pH determinations for A, horizon soil amended with various amounts of chitin, fungal mycelium and casamino acids (0.01 per cent) inoculated with T.
458
W. E. LOWE AND T. R. G. GRAY
viride are given in Figs. 4 and 5. The results for M. nzarburgensis were the same and have not been plotted. Amending the soil with dried yeast cells gave the same results as amending soil with chitin and so they have also been omitted. The increases in bulk pH are clearly proportional to the amount of substrate added and hence the amount of ammonia released from the substrate but pH never rose above 8.1. Increases in pH above the level of 6 -0 (at which bacterial growth could commence) were generally evident after 4 days if concentrated amendments were used. pH increases were not observed in soil with growing fungus but no organic amendment or in amended soils with no fungal inoculum. Similar results were obtained using unsterilized A, horizon soil.
6.0
6.5 I 0. x 6.0 '; m 5.5
4.0
f
2
3
4
5 Time,
6
7
8
9
10
days
pH changes in sterilized AI horizon soil amended with varying amounts of chitin and casamino acids and inoculated with living I: viride. Soilamendments: (m-----m) 30% w/w chitin; (&---0) 25 % w/w chitin; (&---A) 20% w/w chitin; (A---& 10% w/w chitin; (e-0) 5% w/w chitin; (O-O) 2.5% w/w chitin; A-_-A 10% w/w chitin; l ---- 0 5% w/w chitin; O---O 2.5% w/w chitin; ( x-.-.-x) 0.01% w/w vitamin-free casamino acids. Control soih (uninoculufed): (a---.) 30% w/w chitin; (X-X) 0.01% w/w vitamin-free casamino acids. FIG. 5.
Examination of soil samples under the microscope showed that the pH changes were not uniform throughout the soil. The observations of Williams and Mayfield (1971) were confirmed, showing that regions of higher pH occurred around organic matter and substrate fragments, as judged by the change in colour of the pH indicator. Table 4 shows the time of appearance of such localized regions with various amendments. It is clear that local changes have occurred before they are evident from the macrodeterminations, but they confirm that substantial amendments must be made before changes can be detected by this rather crude method. Growth of bacteria in artificially produced alkaline micro-environments
Williams and Mayfield (1971) have discussed the basis for localized changes in pH and have shown that acid intolerant streptomycetes can grow and survive in these areas. The growth of the five bacteria used in this study under these conditions was investigated.
STUDIES ON COCCOID BACTERIA
459
IN SOIL-II
Oven-dried A1 horizon soil was amended with 20% (w/w) dried yeast cells, chitin or O-5 ml of a 2 per cent solution of casamino acids. After inoculation with T. viride and incubation for 7 days, all these treatments produced soil with a bulk pH of 7.5. Similar soils amended with 10 % (w/w) dried yeast cells or chitin produced soils with a bulk pH of 6 *5. These soils were inoculated with 0.5 ml of a bacterial suspension (see earlier) and incubated for 10 days at 25°C. Samples were removed after 1, 2, 3 and 10 days and the numbers of bacteria estimated and compared with two controls (soil with no amendment of TABLE 4. TIMETAKENFORTHEAPPEARANCEOFAREASOFSOILWITHA pH ABOVE 6.0 ASJUDGEDBYACHANGE IN COLOUR OF BROMOTHYMOL-BLUE INDICATOR (YELLOW-BLUE). SOIL WAS AMENDED WITH THE SUBSTRATES SHOWNANDINOCULATED WITH A CULTUREOF Trichodermaviride AT ZEROTIME
Amendment
Time (days)
0.1% wlw Casamino acids
0 1
---
2 7 10
----
Time (days)
amenzentt
0
_-
1
--
2
--
7
10
--
2.5% w/w Chitin
5% w/w Chitin
10% w/w Chitin
20% w/w Chitin
25% w/w Chitin
30% w/w Chitin
---
-12.5 %* w/w Myc.
--
__ 1.25% w/w Myc. --
--
--
+-
+-
i- ? --+
T4. + _+
+? ++ -+
:+ -+
2.5% w/w Myc.
5% wlw Myc.
10% wlw Myc.
--
12.5% w/w Myc. --
---
3
_L
+ -
I
+-
+-
++ ? +
++ i-t
-+
-+
Presence or absence of a colour change is given for areas around added substrate and native organic matte for each amendment. Myc. = mycelium. (+) Blue colour definite (areas above pH 6.0 present). (?) Intermediate colour. (-) Blue colour definite (no areas above pH 6.0 present). * No T. viride inoculum. t No amendment but T. viride inoculum added.
T. viride and soil with 20 per cent chitin but no T. viride). Figure 6 shows the results of this experiment for A21. In all cases, bacteria did not survive in the control soils, confirming the pattern observed in earlier experiments. However, bacteria were able to survive in soils at pH 7.5, although there was no evidence of population increase. At pH 6 *5 bacteria survived and grew, population size increasing about tenfold. This is in contrast to the results obtained when soils were amended with CaC03 when growth and population increases were greater at pH 7 *5. It is possible that in this experiment, poor growth at pH 7.5 is due to ammonia toxicity. At pH levels of 7 *O-7*5 and above, the NH3 + NH4+ equilibrium is shifted to the left (Bates and Pinching, 1950). Cell membranes are relatively impermeable to ionized ammonium whereas un-ionized ammonia passes tissue barriers (Warren, 1962) and is toxic to cells even at low concentrations. The inhibition of bacterial cells by ammonia
460
W. E. LOWE AND T. R. G. GRAY
produced during the microbial decomponsition of urea at pH 7-O has been reported by Court et al. (1964). Some ammonia toxicity might even occur in soils at pH 6 - 5 in localized areas of higher pH, possibly accounting for the lower yields of cells in this experiment than in the corresponding one with CaCO,. Lower numbers might also be due to the presence of the fungus, either because of competition for limited nutrients or because of antibiotic production. A further experiment was performed with isolate A21 to investigate the effect of simultaneous addition of bacterium and fungus to the A, horizon soil. Two sets of vials containing 10 g soil were prepared, incorporating 12.5, 7.5 and 5 *O% (w/w) T. viride mycelium as previously described. One set was inoculated with bacteria and a spore suspension of T. viride simultaneously, the other with T. viride spores and then bacteria 18 h later following
Time,
days
FIG. 6. Growth of Arthrobacter
(type A) A21 in sterilized AI horizon soil with the pH adjusted to 6.5 and 7.5 by the fungal decomposition of amino-nitrogen compounds. pH 6.5 soil amendment: (A-A) 10% w/w dried yeast cells; (O-O) 10% w/w chitin. pH 7.5 soilamendment: (A--B 20% w/w dried yeast cells; (0-O) 20% w/w chitin; ( x __ x ) 0 .Ol % w/w casamino acids. pH4.3 soiC(unamended): (‘--.) inoculated with T. viride. pH 4.3 soil (uninoculated) amendment: (.-.) 20% w/w chitin.
incubation at 25°C in a moist incubator and numbers of bacteria determined after 1, 2, 3 and 10 days. Control soils (soil with no amendment but inoculated with both bacteria and T. viride, and similar soil with 12.5 per cent dead T. viride mycelium but no spore inoculum) were also prepared. Bulk-pH values were recorded at each sampling time. No bacteria were isolated from the control soils or from the soils which were amended and simultaneously inoculated with the bacteria and fungal spores. The pH values in these soils either remained at 4.3 or rose slightly to 4.6. Increases in pH occurred in all amended soil inoculated with fungal spores, the values resembling those already recorded (Fig. 7). Numbers of bacteria fell slightly within the first 24 h while the pH rose to values ranging from 4.6 to 5.3 but thereafter rose as the pH increased. However, when pH values of 7-9 and 8.1 were recorded, numbers decreased. These experiments indicate that alkaline microenvironments can allow growth of bacteria to take place in otherwise acid soils but if the accumulation of ammonia occurs as the pH increases, the bacterial growth can decrease. If alkaline niches were not present in the soil
STUDIES ON COCCOID BACTERIA IN SOIL-II
0
I Time
'2 after
3 bacterial
4. inoculation,
461
IO days
FIG. 7. Growth of Arthrobacter (type A) A21 in sterile A1 horizon soil during pH changes following amendment of soil with dried, sterile fungal mycelium and inoculation with living T. viride. Soil amendment: (O-.-.-O) 12.5% w/w M. marburgensis mycelium; (O-0) 12.5% w/w T. viride mycelium; (O-*-.-O) 7.5 % W/W M. nuwburgensis mycelium; (0-O) 7.5 % w/w T. viride mycelium; (A-A) 5.0% w/w T. viride mycelium; (.----.) no amendment. Numbers of bacteria expressed per gramme oven dry soil.
then the bacteria died rapidly and could not be recovered after 24 h. However, suitable environments for survival were able to develop in 18 h, following the addition of a nitrogenous substrate and ammonifying fungus. GENERAL
DISCUSSION
Organisms inoculated into autoclaved soil are able to grow on substances released from soil organic matter and from dead microbes. This supply of readily available substrate is limited and organisms utilizing it soon stop growing. In the present study, this pattern was observed in the alkaline C horizon soil and in the A, horizon soil made alkaline with calcium carbonate. Amendment of the soils led to growth for a longer period of time and increased levels of oxygen uptake. However, if organisms were inoculated into acid soils they failed to grow and disappeared within 24 h unless alkaline microenvironments were present. It has not been possible to demonstrate the existence of alkaline microenvironments in natural A, horizon soil and so one must ask how nonsporing bacteria are able to grow and survive in these soils. There are several possible explanations which may be summarized as follows. (a) Alkaline microenvironments are present in the soil but they are spatially and temporally so restricted that the relatively crude techniques used in this study cannot detect them.
462
W. E. LOWE AND T. R. G. GRAY
(b) Organisms grow in other soil horizons, e.g. on litter, and are continually leached into the A, horizon. (c) Organisms present in the soil can grow in an acidic environment but they do not grow on the neutral isolation media used to detect bacteria. Instead, selection of strains growing in neutral conditions takes place. (d) Cells growing slowly in soil are physiologically different from cells growing rapidly in laboratory media and are better able to survive unfavourable conditions. (e) The disappearance of cells from soil is an artefact caused by a change in soil properties following sterilization, e.g. reIease of toxic substances. It is not possible to determine which of these explanations is the most probable, chiefly because of the lack of techniques and sampling procedures which would enable one to follow events on a restricted scale. It is known that ~rt~r~~~e~er species can exist in two morphological states, either as rods or as cocci, and it is thought that cocci are the predominant forms in the soil. In some strains the transformation of rod to coccus can be brought about by lowering the growth rate (Luscombe and Gray, 1971) and currently we are investigating the possibility that properties other than morphology are changed by alteration of the growth rate. REFERENCES BATESR. G. and PINCHINGG. D. (1950) Dissociation constants of aqueous ammonia at 0 to 50°C from e.m.f. studies of the ammonium salt of a weak acid. J. Am. them. Sot. 72, 1393-1396. BOUGHEYA. S. (1968) Ecology of Populations. Macmillan, New York. COURT M. N., STEPHENSR. C. and WAID J. S. (1964) Toxicity as a cause of the inefficiency of urea as a fertilizer-&. Experimental J. Soii Sci. 15, 49-65. G~DFELLOW M.. HILL I. R. and GRAY T. R. G. (1968) Bacteria in a nine forest soil. In The Ecolo~v of Soil 3acterja (T. ‘R. G. Gray and D. Parkinson, Bds.)‘pp. 500-514. University Press, Liverpool. -. . GRAY T. R. G. and B~VBYP. (1968) Chitin decomposition in soil-II. The ecology of chitinoclastic microorganisms in forest soil. Trans. Br. mycol. Sot. 51,293-309. KIBBLER. A. (1966) Physiological Activity in a Pine-wood Soil. Ph.D thesis, University of Liverpool. LOWE W. E. and GRAY T. R. G. (1972) Ecological studies on coccoid bacteria in a pine forest soil-I. Classification. Soil Biol. Biochem. 4, 459468. LUSCOMBEB. M. and GRAY T. R. G. (1971) Effect of growth rate on the morphology of Arthrobacter cells. J. gen. Microbial.
69, 433-434,
PARKINSOND. and COUPS E. (1963) Microbial activity in a podzol. In Soil Organisms (J. Doeksen and J. van der Drift, Eds) pp. 167-174. North Holland, Amsterdam. SHAMEEMULLAH M., PARKINSOND. and BURGESA. (1971) The influence of soil moisture tension on the fungal populations of a pinewood soil. Can. J. Microbial. 17, 975-986. SMITHJ. H. (1967) Nitrogen gradients and nitrification associated with decomposing corn plants and barley straw in soil. Proc. Soil Sci. Sot. Am. 31, 377-379. WARRENK. S. (1963) Ammonia toxicity and pH. Nature, Land. 195,47-49. WILLIAMSS. T. and MAYFIELDC. I. (1971) Studies on the ecology of a~tinomy~et~ in soil--III. The behaviour of neutrophilic streptomycetes in acid soils. Soil Bioi. Biochetn. 3, 197-208.
ECOLOGICAL STUDIES ON COCCOID BACTERIA IN A PINE FOREST SOIL-II. GROWTH OF BACTERIA INTRODUCED INTO SOIL W. E. LOWE*and T. R. G. GRAY HartIey Botanical Laboratories,
University of LiverpooI, England
(Acce~:ed 12 October 1972) Summary-The growth of five typical but phenetically distinct cocci and Arthrobuc#erstrains, isolated from a pine forest soil, has been investigated. Soil reaction was found to have a marked effect on growth and unless naturally acidic soils were made more alkaline they did not support growth, even in the presence of added nutrients. However, in the presence of fungi which could use the added nutrients, e.g. chitin, mycelial fragments etc., bacterial growth was possible and could be correlated with a decrease in acidity, especially around particles of organic matter. Where the pH rose above 7.9, bacterial growth again decreased. All the bacterial strains tested reacted in the same way, suggesting that they occupied similar microenvironments in both the acidic and alkaline soil horizons examined. Some explanations for the occurrence of nonsporing bacteria in soils in which apparently they cannot grow are suggested. rNTRODWCTION
NATURALselection results in the formation
of groups of microorganisms adapted to their environments and so clusters of bacteria defined by numerical taxonomic techniques ought to be found associated with particular microenvironments within the soil. In a soil with several distinct horizons, the occurrence of bacteria in all horizons may indicate that a microenvironment is common to them while restriction of bacteria to one horizon suggests that there is a microenvironment peculiar to it. Lowe and Gray (1972) have described the isolation of coccoid bacteria from a pine forest soil and their classification by numerical taxonomic procedures. Nine clusters or subclusters of organisms were delimited by these techniques although most soil cocci could be placed in five of these clusters. The most central and typical strains of each of these five major groups were identified and this paper describes experiments designed to obtain information concerning the ability of these apparently typical organisms to grow in the soil from which they had been isolated. By altering the soil environment through the addition of nutrients and the adjustment of pH, some of the factors affecting the growth of the bacteria in two soil horizons were investigated. All the experiments involved the inoculation of sterilized soil with the required organisms, subsequent changes in population size being followed using the dilution plate count technique, together with (in certain cases) measurements of oxygen uptake by respirometry. MATERIALS
AND METHODS
Preparation of soils
Soil from the Al (PI-I 4.5) and C (pH 8.3) horizon of Freshfield soil (Goodfellow, Hill and Gray, 1968) was dried at 45°C for 24 h after removal of large roots and twigs. All the * Present address: Department of Soil Science, University of Saskatchewan, Saskatoon, Canada. 449
450
W. E. LOWE AND T. R. G. GRAY
soil used was from samples taken at one time and stored dry until required. Samples of these soils were mixed in a mortar and 10 g quantities transferred to 20 ml Universal screw top vials. These were sterilized by autoclaving (121 “C for 20 min) on two separate occasions, the vials being incubated at room temperature for 24 h between autoclavings. The soil was dried in a vacuum desiccator after the second autoclaving. Selection of bacterial strains The bacterial strains central to the largest clusters defined by Lowe and Gray (1972) were selected for inoculation into the soil. These were as follows: cluster 1-Micrococcus A34; cluster 2-Staphylococcus A8 1; cluster 3-Arthrobacter type A, A21 ; cluster 4iArthrobacter type B, A49; cluster 4i,-Arthrobacter type B, C39. Organisms in clusters 1, 2, 3 and 4, were isolated mainly from the A, horizon whereas those in cluster 4ii were associated mainly with the C horizon. The principle characteristics of these bacteria are given by Lowe and Gray (1972). Inoculation of soils and adjustment of their moisture content Test organisms, which had been stored under oil at 5°C were streaked onto plates of PYE agar (Goodfellow et al., 1968), checked for purity and transferred to PYE agar slopes in 250 ml medical flat bottles. After incubation for 24 h at 25°C the bacterial growth was suspended in 25 ml amounts of sterile deionized water in 100 ml conical flasks. Cell aggregates were dispersed by agitating suspensions with a magnetic stirrer-bar rotating at approx. 200 rev./min for 15 min. The concentration of cells was estimated using a nephelometer and the number of cells adjusted to approx. 2 x lO’/ml. Sterile soil was inoculated immediately with 0.5 ml of this suspension and the number of viable cells determined using the dilution plate count technique. The moisture content of the soil was adjusted to 15 % (v/w) oven dried soil by the addition of I.0 ml nutrient solution or deionized water. This ensured that the moisture content was equivalent to 60 per cent of the moisture holding capacity of the soils, i.e. pF values of I.6 and 1.4 respectively for the A, and C horizon samples (Shameemullah, Parkinson and Burges, 1971). These levels are typical of the soil under field conditions (Kibble, 1966) although they may not be optimal for bacterial growth. However, preliminary experiments suggested that bacterial growth was not greatly affected by alterations in moisture content from 5 to 20% (v/w). After inoculation and adjustment of the moisture content, each soil sample was mixed thoroughly and maintained at 4°C for 12 h to allow the moisture, nutrients and bacteria to be dispersed throughout the system with little growth of bacteria occurring. Methods for determining growth of bacteria Numbers of bacteria. Bacteria were counted using the dilution plate count technique (Goodfellow et al., 1968). Four replicate plates were prepared at each dilution and the experiments performed in triplicate (unless otherwise stated). The soil remaining in the vials was used for determining pH and moisture content. Oxygen uptake. Oxygen uptake was measured using Warburg constant volume respirometers and the reaction flasks of Parkinson and Coups (1963). The flasks were ethersterilized and the joints greased with sterile Vaseline. The entire contents of the vials inoculated with bacteria were transferred to the bases of the flasks, the flasks attached to the manometers and oxygen uptake measured at 25°C. Control flasks, containing soil inoculated with 0.5 ml sterile deionized water in place of bacterial suspensions, were also set
STUDIES ON COCCOID BACTERIA IN SOIL-H
451
up. Flasks were allowed to equilibrate for 12 h with the manometer taps open and readings were then taken at 12 h intervals. In order to obtain consecutive readings, flasks were prepared in duplicate; the taps of one series were dosed after equilibration and readings taken after 12 h. During the following 12 h period, the taps of the first series of flasks were allowed to remain open to replenish the oxygen supply, and the taps of the second series of flasks closed. After 48 h incubation, the KOH in the reaction vessels were renewed. All experiments were performed in triplicate. Adjustment of soil pH. In certain experiments, the pH of A, horizon soil was made alkaline by adding 0.05% (w/w) CaCO,, followed by thorough mixing in a mortar. The pH of this soil after sterilization was approx. 7 *5. Addition of the same amount of CaCO, to C horizon soil caused no change in pH. In some cases, the pH of the A, horizon soil was also adjusted to 7.5 by the addition of KOH solution.
EXPERIMENTAL
EfSect of pH on bacterial growth in soil The effect of pH on bacterial growth was investigated by inoculating soils at different pH values with various strains. A, horizon soil (pH 4.3) A, horizon soil (CaCO, amended, pH 7*5), A, horizon soil (KOH amended, pH 7*5), C horizon soil (pH 8.1) and C horizon soil (CaCO, amended, pH 8 * I) were prepared. All soils were inoculated with organisms A34, A81, A21, A49 and C39, incubated for 168 h at 25°C and sampled after 24, 48, 72 and 168 h. The results of dilution plate counts as well as oxygen uptake readings, taken at 12 h intervals during the first 5 days of this experiment, are also recorded in Table 1. The numbers of bacteria developing and the oxygen uptake levels in the C horizon soil amended with CaCO, never differed significantly from the numbers developing in unamended C horizon soil and so the former have been omitted. One set of data for A21 is also shown in Fig. 1. The results show clearly that none of the bacteria were able to grow in acid A, soil, even though four of them had been isolated from this soil. No bacteria were isolated from this soil after 24 h, even at dilutions of 10m4and no oxygen uptake was recorded. In alkaline C horizon soil (pH 8. l), all the strains grew and levels of oxygen uptake, although low, were recorded. In A, horizon soil adjusted to an alkaline pH, growth also occurred but always at a higher level than that found in the C horizon. The maximum growth rate was achieved during the first 24-36 h in all cases; after 48 h, numbers of organisms stopped increasing or increased slowly while oxygen uptake levels decreased. A further experiment was performed to determine the pH at which growth in the A, horizon soil was inhibited. Al horizon soil was amended with quantities of CaC03 to give pH values in the range 4 -5-8 a5 at O-5 pH unit intervals after autoclaving; vials of these soils were moistened with 0.5 ml sterile deionized water, sealed and incubated for 14 days. After this time, no further changes in pH were detected. Bacteria were added to these soils and dilution plates prepared from samples taken after 24,48 and 72 h. Because of the large number of dilution plates involved, the treatments were not replicated. However, the results are striking and uniform for the five different bacteria. The results are recorded in Fig. 2 and Table 2 and show that in all cases, while the bacteria survived and grew well at pH 6 -0 and above, at pH 5 *5 and below, populations declined or even disappeared completely. Similar results were obtained by growing bacteria in PYE broths buffered to a similar range of pH values. Virtually no growth occurred at
452
W. E. LOWE AND T. R. G. GRAY
pH values of 5.5 and below, with the exception of organism A21 which grew slowly at pH 5.5 in broth. These results indicate that although the organisms used in this study appear to be phenetitally distinct, their growth patterns following introduction into sterile soil are similar. Isolates grew in a similar manner in sterile A, (pH 7 *5) soil and C horizon soil, irrespective of the horizon from which they were isolated, suggesting that similar microenvironments
TABLE
1. GROWTH
OF BACTERIA IN NUTRITIONALLY UNAMENDED PIf LEVELS
STERILE SOILS AT DIFFERENT
Bacterial nos. per g oven dry soil (x
Organism A21
A81
A49
c39
A34
Al horizon pH 7.5
Time (days)
C horizon pH 8.1
0 1 2 3 71
0.13 0.4 5 0.2 2.1 5 0.4 1.4hO.l 2.2 f 0.2
0.13 1.2 19-S 22.4 17.5
+ f i_ i
0.2 3.6 2.8 3-o
: 3 71
0.14 1.2 3.6 5.4 6.5
+ 0.1 10.3 f 0.3 h 0.3
0.14 5.0 11-o 17.5 30.5
+ * * f
0.8 1.5 2.2 1.3
0 1 2 3 71
0.11 1.4 2.5 1.9 1.3
f + * *
0.2 0.2 0‘2 0.4
0.11 3.4 12.2 9.3 16.0
Jr & & +
o-9 2.2 0.6 1.6
0 1 2 3 71
0.19 0.5 0.6 1.3 1.1
i i_ + f
o-1 0.1 0.1 0.2
0.19 3.1 4.6 4.3 4.8
* i + +
0.1 0.3 0.1 0.2
0.21 2.0 * 2.6 f 2.3 + 2.0 f
0.1 0.1 0.1 0.2
0.21 7.3 9.4 8.9 7.6
5 Zt i i
0,2 0.4 0.3 0.2
0
0 ; 3 71
KaCW
A1 horizon pH 7.5
WW 0.13 2.5 21.0 19.7 19.9 -
lo-‘)
AI horizon pH 4.3 0.13
& i5 rt
0.2 1.2 1.8 3.5
x 0 0 0.14
-
ZI
--
8
-
0.11 0
--
00 0
-
0.19 0
--
8 0
--
0.21 :: 0 0
for bacterial growth exist in both horizons. Growth in the A, horizon (pH 4.3) soil is limited by unfavourable pH and the presence of these same bacteria in natural A, horizon soil in large numbers indicates that either microenvironments of higher pH are present in unsterilized soil or that isolation of microorganisms on neutral media only allows the growth of atypical forms. Eflect of nutrient amendment on bacterial growth in soil
Smith (1967) has shown that alkaline microenvironments can be produced in acid soils through the accumulation of ammonium ions in the region of organic matter. Williams and
STUDIES ON COCCOID BACTERIA
IN SOIL-II
453
Mayfield (1971) demonstrated that acid intolerant streptomycetes could grow in such regions. This was shown by amending the soil with nitrogen containing compounds and a soil fungus which could attack them. Ammonification of the organic matter occurred and regions of high pH were observed around organic particles when these were stained with appropriate indicators. The possibility of such interactions being important for bacteria involved in this investigation was tested. First, the effect of nutrient amendment on the five bacteria was examined and then the effect of adding nutrients and fungi to the soil before bacterial inoculation. The form and concentration of nutrients added to the soil are given in Table 3. TABLE 2. GROWTH OF BACTERIAIN STERILEAl HORIZONSOILAT VARIOUSpH LEVELS Bacterial nos. per g oven dry soil (X lOWe) pH of soil Time (days)
8.5
8.0
I.5
7.0
6.5
6.0
5.5
5.0
4.5
A21
0 1 2 3
1.2 119 214 118
1.2 84 223 113
1.2 121 253 124
1.2 31 98 84
1.2 15 17 23
1.2 14 13 15
1.2 1.3 0.2 0.03
1.2 0.2 0.2 0.02
1.2 0 0 0
A81
0
1.3
: 3
3.2 50 71
1.3 11 51 74
1.3 12 111 98
1.3 9 52 31
1.3 5.3 14 12
1.3 2.1 5.6 4.9
1.3 0.5 0.05 0
1.3 0.1 0 0
1.3 0 0 0
0 1 2 3
1.4 140 180 120
1.4 160 340 280
1.4 51 82 140
1.4 45 76 971
1.4 :; 64
1.4 1.3 8.4 8.3
1.4 0.1 0.1 0
1.4 0.1 0 0
1.4 0 0 0
c39
0 1 2 3
0.9 2.7 14 68
0.9 2.8 35 79
0.9 3.2 34 82
0.9 1.3 21 78
0.9 1.2 9.2 14
0.9 1.0 1.1 3.4
0.9 0.6 0.1 0
0.9 0.1 0.1 0
0.9 0 0 0
A34
0 1 2 3
0.9 39 65 62
0.9 131 116 109
0.9 130 150 160
0.9 83 124 115
0.9 35 62 51
0.9 3,4 2.2 1.2
0.9 1.1 0.2 0.04
0.9 1.0 0.03 0
0.9 0 0 0
Organisms
A49
Arthrobacter type A (A21) and Micrococcus (A34) were the only organisms able to use inorganic nutrients as sole sources of nitrogen but the effects of these nutrients on the other organisms were also investigated; an inability to use inorganic substances in culture media may have been due to lack of other substances, e.g. vitamins, present in soil. As well as adding the nutrients separately to soil, nitrogen, phosphorus plus potassium and magnesium were added together and also together with glucose. The number of dilution plates that had to be prepared was again large so that the experiment was performed only once. As before, there were only minor differences between the results for all five bacteria and only the results for A21 have been plotted (Fig. 3). No growth or survival of bacteria was detected in A, (pH 4.3) soil, even in the presence of added nutrients, while growth did
454
W. E. LOWE AND T. R. G. GRAY
occur in A, (pH 7.5) and C horizon soils in amended and unamended conditions. The growth curves were similar to those described in the previous experiment, more growth and oxygen uptake being associated with the A, horizon soil than the C horizon soil. In the amended soils, the growth yields were higher than in the unamended soils but after 7-10 days, numbers of bacteria had decreased and were very similar to those of unamended soil. Oxygen uptake levels were higher than in unamended soils but the rate of oxygen uptake
I
2
3
Time.
4
5
6
7
days
FIG. 1. Growth of Arthrobacter (type A) A21 in nutritionally unamended soil as judged by changes in population size and oxygen uptake. (0-O) C horizon soil, pH 8.1; (O-O) AI horizon soil, pH adjusted to 7.5 with CaCO,; (X-X) Ai horizon soil, pH adjusted to 7.5 with KOH; (.----+) AI horizon soil, pH 4.3.
was already decreasing after 48 h incubation. In all cases, amendment with casamino acid mixture produced the greatest response. Amendment with inorganic nutrients (N, Mg + S, P + K), produced little if any effect unless added with glucose. The results suggest that Iack of carbon and nitrogen limits bacterial growth in these soils, at suitable pH levels, and that increased metabolism caused by the addition of carbon and nitrogen may in turn be limited by the low concentrations of inorganic salts. The growth curves could be divided into two phases, an initial growth phase during which a rapid increase in numbers and oxygen uptake levels took place and a survival phase
STUDIES ON COCCOID BACTERIA
IN SOIL-II
455
TABLE 3. FORM AND FINAL CONCENTRATION OF NUTRIENTSADDEDTO STERILESOIL Form and final con~en~ation (%, w/w oven dried soil) of nutrient supplied
Nutrient Carbon Nitrogen Organic Inorganic Fhosphorus~potassium
Glucose* (0.02) Casamino acids? (0.08 N) Ammonium nitrate* (0.08 N) Potassium dihydrogen (0.04 P) (0.06 K) Phosphate* Magnesium sulphate* (0.06 S) (0.04 Mg)
Magnesium/sulphur
* BDH Ltd., Poole, England-Analar grades. _t Difco Labs., Michigan, U.S.A.-Bacto vitamin free casamino acids.
during which numbers remained almost constant or decreased slightly and levels of oxygen consumption were low. For higher plants and animals, the size of a single species population which can develop in a given environment has been termed the carrying capacity for that species (Boughey, 1968) and this remains constant under a given set of environmental conditions. Using the same terminology here, the A, horizon soit when treated with calcium carbonate, has a
>
; \ i \J___._
-_-.-
2
I Time,
3
days
FIG. 2. Growth of Arthrobacter (type A) A21 in sterile A1 horizon soil at various pH levels. (A---& pH 8.5; (a-----a) pH 8% (0-O) pH 7.5; (O-O) pH 7.0; (W---u) pH 6.5; (U-U) pH 6.0; (x-x) pH 5.5; (.----.) pH 5.0; (.-.-.> pH 4.5.
4.56
W. E. LOWE AND T. R, G. GRAY
greater carrying capacity than the C horizon; in both cases, it can be increased by nutrient amendment though it falls again as nutrients become depleted. The occurrence o~artl~c~all}~produced ~lk~~~~em~cr~-e~~l~~ro~rne~fs
Under natural conditions, a variety of fungi are present and able to grow in Freshfield soil. Some of these are capable of breaking down chitin and other nitrogen containing compounds, releasing both carbon dioxide and ammonia, e.g. Trichoderma viride and
Time,
days
FIG. 3. Growth of ~~g~r~~~c#er (type A) AZ in nutritionallyamended so% S~eriltr C horizon soil, pH 8. I : (*---e) no added nutrietnts; (O-e-e-*) plus inorganic N, MgS and PK; (----I plus glucose; (A----& plus glucose plus inorganic N, MgS and PK; (x x ) plus casamino acids. Sterile Al horizon suil, pH adjusted fu 7.5 rvifh CaCu,: (O---O) no added nutrients; (O-+-.-O) plus inorganic, N, MgS and PK; (n-----n, plus glucose; (3-D) plus glucose plus inorganic N, MgS and FK; (o-T7) pfus casamino acids. Sterile A, horizwz soil, pH 4.3: (.----.) ait treatments. Numbers of bacteria expressed per gramme oven dried soil. Oxygen uptake expressed as ~1 02/g oven dry soil/l2 h. Vertical lines represent the 95 per cent confidence limits.
Mortierellu marburgensis (Gray and Baxby, 196s). The effect of the growth of these fungi on
soil pH and bacterial growth was investigated. Chitin was obtained in a relatively impure form containing protein (BDH Ltd., Poole, England) and also in crude form in fungal mycelium and yeast cells, For the preparation of fungal mycebum, T. viride and M. RWb~rge~s~~ were grown in malt extract broth (pH 6-O) in shake culture and harvested after 7 days growth at 25°C. The mycehum was washed in running water for 1 h and rinsed in deionized water. After drying at 105°C for 24 h, mycehum was ground in a mortar and
STUDIES ON COCCOID BACTERIA IN SOIL-II
457
stored dry. Baker’s yeast cells were suspended in water, washed four times and then dried and ground. BDH chitin was used as supplied, except for preliminary grinding in a mortar. Quantities of oven-dried A, horizon soil were amended with varying amounts of chitin, fungal mycelium, yeast cells and casamino acids. Ten gram samples were transferred to vials, sterilized and remoistened with 0.5 ml sterile deionized water and kept sealed for 14 days. pH measured after this time was found to be approximately the same as that of untreated soil. The soils were inoculated with living T. viride or M. marburgensis as follows. Spore suspensions were prepared from fungi grown on malt extract agar for 7 days at 25°C; the spores were harvested and suspended in sterile deionized water. After inoculation,
80
6-5 I a r 6-O ii 5.5
5.0
4.5
4.0 ' 0
I I
I 2
I 3
I 4 Time,
I 5
I 6
I 7
I 6
I 9
I IO
days
4. pH changes in sterilized A, horizon soil amended with varying amounts of sterile dry Trichoderma viride and Mortierella marburgensis mycelium and inoculated with living T. viride mycelium. Soil amendments: (O-O) 12.5% w/w T. viride mycelium; (0-O) 10.0% w/w T. viride mycelium; (A-A) 7.5% w/w T. viride mycelium; (A-A) 5.0% w/w T. viride mycelium; (0-O) 2.5% w/w T. viride mycelium; (W-m) 1.25% w/w T. viride mycelium; (O-.-.-O) 12.5% w/w M. marburgensis mycelium; (A-.-.-A) 7.5% w/w M. marburgensis mycelium ; ( q- .-a - 0) 2.5 ‘A w/w M. marburgensis mycelium. Unamended soils: (s---.) inoculated with T. viride culture; (X-.-.-X) inoculated with M. marburgensis culture.
FIG.
O-5 ml amounts of sterile water were added, bringing the soil moisture content to 15 % (v/w). The soils were aseptically and thoroughly mixed and incubated at 25°C for 10 days. Macro-pH determinations were made on these soils using a glass-calomel electrode pH meter. Micro-pH determinations were made by placing a few drops of bromothymol blue indicator on soil spread on a microscope slide (Williams and Mayfield, 1971). The soil was examined under a low power microscope and the colour of the indicator observed; a blue colour was indicative of a pH value of 6 -0 and above, a yellow colour of pH values below 6 90. The results of the bulk-pH determinations for A, horizon soil amended with various amounts of chitin, fungal mycelium and casamino acids (0.01 per cent) inoculated with T.
458
W. E. LOWE AND T. R. G. GRAY
viride are given in Figs. 4 and 5. The results for M. nzarburgensis were the same and have not been plotted. Amending the soil with dried yeast cells gave the same results as amending soil with chitin and so they have also been omitted. The increases in bulk pH are clearly proportional to the amount of substrate added and hence the amount of ammonia released from the substrate but pH never rose above 8.1. Increases in pH above the level of 6 -0 (at which bacterial growth could commence) were generally evident after 4 days if concentrated amendments were used. pH increases were not observed in soil with growing fungus but no organic amendment or in amended soils with no fungal inoculum. Similar results were obtained using unsterilized A, horizon soil.
6.0
6.5 I 0. x 6.0 '; m 5.5
4.0
f
2
3
4
5 Time,
6
7
8
9
10
days
pH changes in sterilized AI horizon soil amended with varying amounts of chitin and casamino acids and inoculated with living I: viride. Soilamendments: (m-----m) 30% w/w chitin; (&---0) 25 % w/w chitin; (&---A) 20% w/w chitin; (A---& 10% w/w chitin; (e-0) 5% w/w chitin; (O-O) 2.5% w/w chitin; A-_-A 10% w/w chitin; l ---- 0 5% w/w chitin; O---O 2.5% w/w chitin; ( x-.-.-x) 0.01% w/w vitamin-free casamino acids. Control soih (uninoculufed): (a---.) 30% w/w chitin; (X-X) 0.01% w/w vitamin-free casamino acids. FIG. 5.
Examination of soil samples under the microscope showed that the pH changes were not uniform throughout the soil. The observations of Williams and Mayfield (1971) were confirmed, showing that regions of higher pH occurred around organic matter and substrate fragments, as judged by the change in colour of the pH indicator. Table 4 shows the time of appearance of such localized regions with various amendments. It is clear that local changes have occurred before they are evident from the macrodeterminations, but they confirm that substantial amendments must be made before changes can be detected by this rather crude method. Growth of bacteria in artificially produced alkaline micro-environments
Williams and Mayfield (1971) have discussed the basis for localized changes in pH and have shown that acid intolerant streptomycetes can grow and survive in these areas. The growth of the five bacteria used in this study under these conditions was investigated.
STUDIES ON COCCOID BACTERIA
459
IN SOIL-II
Oven-dried A1 horizon soil was amended with 20% (w/w) dried yeast cells, chitin or O-5 ml of a 2 per cent solution of casamino acids. After inoculation with T. viride and incubation for 7 days, all these treatments produced soil with a bulk pH of 7.5. Similar soils amended with 10 % (w/w) dried yeast cells or chitin produced soils with a bulk pH of 6 *5. These soils were inoculated with 0.5 ml of a bacterial suspension (see earlier) and incubated for 10 days at 25°C. Samples were removed after 1, 2, 3 and 10 days and the numbers of bacteria estimated and compared with two controls (soil with no amendment of TABLE 4. TIMETAKENFORTHEAPPEARANCEOFAREASOFSOILWITHA pH ABOVE 6.0 ASJUDGEDBYACHANGE IN COLOUR OF BROMOTHYMOL-BLUE INDICATOR (YELLOW-BLUE). SOIL WAS AMENDED WITH THE SUBSTRATES SHOWNANDINOCULATED WITH A CULTUREOF Trichodermaviride AT ZEROTIME
Amendment
Time (days)
0.1% wlw Casamino acids
0 1
---
2 7 10
----
Time (days)
amenzentt
0
_-
1
--
2
--
7
10
--
2.5% w/w Chitin
5% w/w Chitin
10% w/w Chitin
20% w/w Chitin
25% w/w Chitin
30% w/w Chitin
---
-12.5 %* w/w Myc.
--
__ 1.25% w/w Myc. --
--
--
+-
+-
i- ? --+
T4. + _+
+? ++ -+
:+ -+
2.5% w/w Myc.
5% wlw Myc.
10% wlw Myc.
--
12.5% w/w Myc. --
---
3
_L
+ -
I
+-
+-
++ ? +
++ i-t
-+
-+
Presence or absence of a colour change is given for areas around added substrate and native organic matte for each amendment. Myc. = mycelium. (+) Blue colour definite (areas above pH 6.0 present). (?) Intermediate colour. (-) Blue colour definite (no areas above pH 6.0 present). * No T. viride inoculum. t No amendment but T. viride inoculum added.
T. viride and soil with 20 per cent chitin but no T. viride). Figure 6 shows the results of this experiment for A21. In all cases, bacteria did not survive in the control soils, confirming the pattern observed in earlier experiments. However, bacteria were able to survive in soils at pH 7.5, although there was no evidence of population increase. At pH 6 *5 bacteria survived and grew, population size increasing about tenfold. This is in contrast to the results obtained when soils were amended with CaC03 when growth and population increases were greater at pH 7 *5. It is possible that in this experiment, poor growth at pH 7.5 is due to ammonia toxicity. At pH levels of 7 *O-7*5 and above, the NH3 + NH4+ equilibrium is shifted to the left (Bates and Pinching, 1950). Cell membranes are relatively impermeable to ionized ammonium whereas un-ionized ammonia passes tissue barriers (Warren, 1962) and is toxic to cells even at low concentrations. The inhibition of bacterial cells by ammonia
460
W. E. LOWE AND T. R. G. GRAY
produced during the microbial decomponsition of urea at pH 7-O has been reported by Court et al. (1964). Some ammonia toxicity might even occur in soils at pH 6 - 5 in localized areas of higher pH, possibly accounting for the lower yields of cells in this experiment than in the corresponding one with CaCO,. Lower numbers might also be due to the presence of the fungus, either because of competition for limited nutrients or because of antibiotic production. A further experiment was performed with isolate A21 to investigate the effect of simultaneous addition of bacterium and fungus to the A, horizon soil. Two sets of vials containing 10 g soil were prepared, incorporating 12.5, 7.5 and 5 *O% (w/w) T. viride mycelium as previously described. One set was inoculated with bacteria and a spore suspension of T. viride simultaneously, the other with T. viride spores and then bacteria 18 h later following
Time,
days
FIG. 6. Growth of Arthrobacter
(type A) A21 in sterilized AI horizon soil with the pH adjusted to 6.5 and 7.5 by the fungal decomposition of amino-nitrogen compounds. pH 6.5 soil amendment: (A-A) 10% w/w dried yeast cells; (O-O) 10% w/w chitin. pH 7.5 soilamendment: (A--B 20% w/w dried yeast cells; (0-O) 20% w/w chitin; ( x __ x ) 0 .Ol % w/w casamino acids. pH4.3 soiC(unamended): (‘--.) inoculated with T. viride. pH 4.3 soil (uninoculated) amendment: (.-.) 20% w/w chitin.
incubation at 25°C in a moist incubator and numbers of bacteria determined after 1, 2, 3 and 10 days. Control soils (soil with no amendment but inoculated with both bacteria and T. viride, and similar soil with 12.5 per cent dead T. viride mycelium but no spore inoculum) were also prepared. Bulk-pH values were recorded at each sampling time. No bacteria were isolated from the control soils or from the soils which were amended and simultaneously inoculated with the bacteria and fungal spores. The pH values in these soils either remained at 4.3 or rose slightly to 4.6. Increases in pH occurred in all amended soil inoculated with fungal spores, the values resembling those already recorded (Fig. 7). Numbers of bacteria fell slightly within the first 24 h while the pH rose to values ranging from 4.6 to 5.3 but thereafter rose as the pH increased. However, when pH values of 7-9 and 8.1 were recorded, numbers decreased. These experiments indicate that alkaline microenvironments can allow growth of bacteria to take place in otherwise acid soils but if the accumulation of ammonia occurs as the pH increases, the bacterial growth can decrease. If alkaline niches were not present in the soil
STUDIES ON COCCOID BACTERIA IN SOIL-II
0
I Time
'2 after
3 bacterial
4. inoculation,
461
IO days
FIG. 7. Growth of Arthrobacter (type A) A21 in sterile A1 horizon soil during pH changes following amendment of soil with dried, sterile fungal mycelium and inoculation with living T. viride. Soil amendment: (O-.-.-O) 12.5% w/w M. marburgensis mycelium; (O-0) 12.5% w/w T. viride mycelium; (O-*-.-O) 7.5 % W/W M. nuwburgensis mycelium; (0-O) 7.5 % w/w T. viride mycelium; (A-A) 5.0% w/w T. viride mycelium; (.----.) no amendment. Numbers of bacteria expressed per gramme oven dry soil.
then the bacteria died rapidly and could not be recovered after 24 h. However, suitable environments for survival were able to develop in 18 h, following the addition of a nitrogenous substrate and ammonifying fungus. GENERAL
DISCUSSION
Organisms inoculated into autoclaved soil are able to grow on substances released from soil organic matter and from dead microbes. This supply of readily available substrate is limited and organisms utilizing it soon stop growing. In the present study, this pattern was observed in the alkaline C horizon soil and in the A, horizon soil made alkaline with calcium carbonate. Amendment of the soils led to growth for a longer period of time and increased levels of oxygen uptake. However, if organisms were inoculated into acid soils they failed to grow and disappeared within 24 h unless alkaline microenvironments were present. It has not been possible to demonstrate the existence of alkaline microenvironments in natural A, horizon soil and so one must ask how nonsporing bacteria are able to grow and survive in these soils. There are several possible explanations which may be summarized as follows. (a) Alkaline microenvironments are present in the soil but they are spatially and temporally so restricted that the relatively crude techniques used in this study cannot detect them.
462
W. E. LOWE AND T. R. G. GRAY
(b) Organisms grow in other soil horizons, e.g. on litter, and are continually leached into the A, horizon. (c) Organisms present in the soil can grow in an acidic environment but they do not grow on the neutral isolation media used to detect bacteria. Instead, selection of strains growing in neutral conditions takes place. (d) Cells growing slowly in soil are physiologically different from cells growing rapidly in laboratory media and are better able to survive unfavourable conditions. (e) The disappearance of cells from soil is an artefact caused by a change in soil properties following sterilization, e.g. reIease of toxic substances. It is not possible to determine which of these explanations is the most probable, chiefly because of the lack of techniques and sampling procedures which would enable one to follow events on a restricted scale. It is known that ~rt~r~~~e~er species can exist in two morphological states, either as rods or as cocci, and it is thought that cocci are the predominant forms in the soil. In some strains the transformation of rod to coccus can be brought about by lowering the growth rate (Luscombe and Gray, 1971) and currently we are investigating the possibility that properties other than morphology are changed by alteration of the growth rate. REFERENCES BATESR. G. and PINCHINGG. D. (1950) Dissociation constants of aqueous ammonia at 0 to 50°C from e.m.f. studies of the ammonium salt of a weak acid. J. Am. them. Sot. 72, 1393-1396. BOUGHEYA. S. (1968) Ecology of Populations. Macmillan, New York. COURT M. N., STEPHENSR. C. and WAID J. S. (1964) Toxicity as a cause of the inefficiency of urea as a fertilizer-&. Experimental J. Soii Sci. 15, 49-65. G~DFELLOW M.. HILL I. R. and GRAY T. R. G. (1968) Bacteria in a nine forest soil. In The Ecolo~v of Soil 3acterja (T. ‘R. G. Gray and D. Parkinson, Bds.)‘pp. 500-514. University Press, Liverpool. -. . GRAY T. R. G. and B~VBYP. (1968) Chitin decomposition in soil-II. The ecology of chitinoclastic microorganisms in forest soil. Trans. Br. mycol. Sot. 51,293-309. KIBBLER. A. (1966) Physiological Activity in a Pine-wood Soil. Ph.D thesis, University of Liverpool. LOWE W. E. and GRAY T. R. G. (1972) Ecological studies on coccoid bacteria in a pine forest soil-I. Classification. Soil Biol. Biochem. 4, 459468. LUSCOMBEB. M. and GRAY T. R. G. (1971) Effect of growth rate on the morphology of Arthrobacter cells. J. gen. Microbial.
69, 433-434,
PARKINSOND. and COUPS E. (1963) Microbial activity in a podzol. In Soil Organisms (J. Doeksen and J. van der Drift, Eds) pp. 167-174. North Holland, Amsterdam. SHAMEEMULLAH M., PARKINSOND. and BURGESA. (1971) The influence of soil moisture tension on the fungal populations of a pinewood soil. Can. J. Microbial. 17, 975-986. SMITHJ. H. (1967) Nitrogen gradients and nitrification associated with decomposing corn plants and barley straw in soil. Proc. Soil Sci. Sot. Am. 31, 377-379. WARRENK. S. (1963) Ammonia toxicity and pH. Nature, Land. 195,47-49. WILLIAMSS. T. and MAYFIELDC. I. (1971) Studies on the ecology of a~tinomy~et~ in soil--III. The behaviour of neutrophilic streptomycetes in acid soils. Soil Bioi. Biochetn. 3, 197-208.