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Importance of environmental factors on the growth of Thermoactinomyces thalpophilus
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Soil Biol. Biochem. Vol. 30, No. 10/11, pp. 1243±1249, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(98)00059-5 0038-0717/98 $19.00 + 0.00
IMPORTANCE OF ENVIRONMENTAL FACTORS ON THE GROWTH OF THERMOACTINOMYCES THALPOPHILUS A. M. JACKSON and A. S. BALL* Department of Biological Sciences, John Tabor Laboratories, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, U.K. SummaryÐThe germination and growth of endospores from Thermoactinomyces thalpophilus in two soils, a sand silt loam and a silt clay loam was investigated under a range of environmental conditions. A 50-fold increase in the recovery of T. thalpophilus was observed after 28 d growth in silt clay loam soil, when compared to the original inoculum (8.85 104 cfu gÿ1 dry soil), with similar results obtained for this soil over a range of soil moisture contents (5±50% w/w) and at temperatures of 30 and 458C. In contrast, no increase in the recovery of T. thalpophilus was detected when endospores (2.53 105 cfu gÿ1 dry soil) were inoculated into sand silt loam soil. However, when sterile farmyard manure (2% w/w) was added to sand silt loam soil, a 50-fold increase in the numbers of T. thalpophilus isolated was observed over 28 d incubation. Analysis of the sand silt loam soil showed that organic C and N content, pH, and the C-to-N ratio of the soil had all increased signi®cantly upon addition of farmyard manure, resulting in the growth of T. thalpophilus endospores in a soil which previously could not sustain growth. Addition of manure to the silt clay loam led to a 70% increase in recovery of T. thalpophilus. Growth and respiratory measurements of T. thalpophilus released into sterile manure con®rmed the ability of these endospores to show rapid germination, with respiratory activity detected after 7 h, reaching a maximum value (700 mmol CO2 hÿ1 gÿ1 dry manure) 11 h after inoculation, resulting in a 600-fold increase (®nal recovery, 2.38 109 cfu gÿ1 dry manure) in the recovery of T. thalpophilus. We conclude that T. thalpophilus is capable of growth in soils at temperatures as low as 308C and under a range of soil moisture conditions. Further, enhanced growth in soils with additions of manure could be observed. These results support the idea that Thermoactinomyces endospores isolated from ancient soils containing agricultural debris may have remained dormant in soil following a brief period of rapid growth immediately following deposition. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
Thermoactinomyces is a Gram-positive hyphal prokaryote which produces spheroidal heat-resistant endospores (Fergus, 1967) formed singly on substrate and aerial mycelium as a response to growth conditions becoming limited (Lacey, 1989). Although Thermoactinomyces closely resemble actinomycetes in its mode of growth, phylogenetic data show that Thermoactinomyces group with Bacillus (Stackebrandt et al., 1987). Thermoactinomyces is common in agricultural environments, being most numerous in moulding hay (Corbaz et al., 1963), composts (Amner et al., 1988) and manure (Waksman and Corke, 1953; Cross, 1968) that have heated spontaneously to temperatures of 508C or more (Lacey, 1989). This increase in temperature promotes the germination and growth of Thermoactinomyces endospores and often results in the production of >107 endospores gÿ1 dry weight (Cross, 1968). Thermoactinomyces are thought to be dispersed into soil when compost or manure is ploughed into the soil as fertiliser (KuÈster and Locci, 1963; Cross, 1968; Goodfellow and Cross, *Author for correspondence.
1974; Nilsson and Renberg, 1990) or by becoming airborne when decaying plant material is disturbed and then deposited on soil (Cross, 1968; Eggins et al., 1972). The proli®c dispersal of endospores has led to Thermoactinomyces being recovered from most soils, rivers and marine sediments (Cross, 1968; Parduhn and Watterson, 1985; Nilsson and Renberg, 1990; Jackson and Ball, 1994). Once deposited in soil it is thought that these endospores remain dormant. In previous work we have isolated viable Thermoactinomyces endospores from soils stored since 1913 (Jackson et al., 1997), while Unsworth et al. (1977) isolated viable thermophilic Thermoactinomyces from sealed Roman deposits dating from AD 125 and suggested that Thermoactinomyces may be a useful indicator in studies on the agricultural history of soils. However very little is known about the ecology of Thermoactinomyces in soil and whether these organisms are able to germinate and grow in soil. We have examined the eects of a number of environmental factors on the growth of Thermoactinomyces thalpophilus in soil. This species was selected because reports have shown that T. thalpophilus is capable of growth at temperatures as low as 298C
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Table 1. The organic carbon and nitrogen content of sandy loam soil, silt clay soil and farm yard manure used in this study. The eects of farmyard manure (FYM) additions (2% w/w) to soil on soil properties are also included. The results are the means of three replicates with standard errors in brackets Parameter measured Soil description
pH (H2O)
% organic C
% organic N
Sand silt loam Sand silt loam +2% v/v FYM Silt clay loam Silt clay loam +2% v/v FYM FYM
5.7 6.0 7.7 7.8 8.4
0.175 0.410 1.425 1.815 35.59
0.067 0.098 0.124 0.144 2.700
(20.3) (20.3) (20.5) (20.4) (20.3)
(Lacey, 1989). Solar radiation may raise soil temperature above 308C, even in temperate climates (Eggins et al., 1972) and especially in greenhouses where high numbers of Thermoactinomyces have been recovered from the highly organic soils (Fields et al., 1974). The aim of the work was to establish under what conditions, if any could T. thalpophilus endospores germinate and grow in soils.
MATERIALS AND METHODS
Soil analyses Samples were taken during spring 1994 from the top 15-cm of the sample sites. The soils sampled were at Stanway, Essex, a sand silt loam (Wix association; Hodge et al., 1984) and at Great Chesterford, a silt clay loam (Hanslope association; Hodge et al., 1984). The properties of the soil are presented in Table 1. For release studies, sterile soil was sieved (2 mm), air-dried (24 h at 408C) until a steady weight was obtained. Soil (20 g) was then placed in capped containers (25 ml) and sterilised by autoclaving (15 min at 1218C) on three successive days. Soil moisture content was determined by placing triplicate weighed soil samples (5 g) in an oven at 1058C until a steady weight was obtained (24 h). Soil pH was measured by shaking air-dried soil (5 g) in distilled water (25 ml) for 20 min prior to reading pH with a digital meter (Warden Precision Apparatus, Cambridge, U.K.). Soil organic C content was determined using the method described by Walkley and Black (1934). The results were calculated on the basis that 1 ml of NK2Cr2O7 (6 M) is consumed by 3 mg of carbon. The results were multiplied by an arbitrary correction factor (1.33) to calculate the total organic C content (Grimshaw et al., 1989). Soil organic N content was determined using a semi-Kjeldahl digestion (Grimshaw et al., 1989). The amount of ammonium N present in the supernatant was analysed spectrophotometrically (Harwood and Kuhn, 1970). Blanks and standards (ammonium sulphate solution, 0.0±3.0 mg N 0.1 mlÿ1) were prepared and a calibration curve drawn from which the organic N content of the soils was calculated.
(20.01) (20.01) (20.02) (20.03) (20.94)
(20.008) (20.001) (20.003) (20.008) (20.076)
C-to-N ratio 8:1 12:1 19:1 21:1 26:1
The soil C-to-N ratio was calculated from measurements of total soil C and N obtained using a CHNS/O analyser (Perkin Elmer). Strain and growth conditions Thermoactinomyces thalpophilus strain AM1 was originally isolated from a sandy loam and maintained on Czapek Dox yeast extract casamino acid (CYC) agar (Jackson and Ball, 1994). For this study T. thalpophilus was incubated for 48 h at 458C. T. thalpophilus can be distinguished from other Thermoactinomyces by its abundance of white aerial mycelium, resistance to novobiocin (25 mg mlÿ1) and its ability to degrade both starch and tyrosine (Cross and Unsworth, 1977; McCarthy and Cross, 1984). Endospores and aerial mycelium were removed from the agar by agitation in sterile distilled water using a glass spreader. The resulting suspension was ®ltered through glass wool to remove the mycelium before being heated to 908C for 15 min to kill any remaining mycelial fragments (Erikson, 1952), ensuring that any future growth would arise only from endospores. Dilutions of the endospore suspensions were made in sterile distilled water to give approximately 107 viable endospores mlÿ1. Inoculation and reisolation of T. thalpophilus from soil The moisture content of autoclaved soil was adjusted to the required value (5, 15, 25 or 50% w/ w) using sterile distilled water. The soil was inoculated with the endospore suspension to give a viable endospore count of approximately 106 endospores gÿ1 dry soil and the ®nal weight determined. The inoculated soils along with uninoculated controls were then incubated for up to 28 d at the selected temperature (5±458C). Replicates from each soil type inoculated were sampled regularly during the incubation. The moisture content of the soil was maintained by daily measurements of the weight of each sample and the addition of water as required to bring the weight of the sample back to its original weight. T. thalpophilus was recovered from the soil samples by homogenising the whole sample (20 g wet weight soil) in 14 strength Ringers solution containing 10% (v/v) nutrient broth (pH 7.5) using a
Growth of T. thalpophilus
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Fig. 1. Eect of moisture content on the recovery of T. thalpophilus (cfu gÿ1 dry soil), following inoculation of (a) sterile silt clay loam soil and (b) sandy loam soil. Soils were maintained at moisture contents (w/w) of 5% (Q), 15% (R), 25% (T) and 50% (W) and at a temperature of 458C for up to 28 d. Results are the means of three replicates with standard error bars presented.
Waring blender for 15 s (Cross, 1968). Replicate samples (n = 3) of the resulting soil suspensions (0.1 ml) were then spread onto Czapek Dox yeast extract casamino acid agar (Jackson and Ball, 1994) and incubated at 458C for up to 4 d. Results are expressed as the mean number of T. thalpophilus recovered gÿ1 dry soil. Eect of the addition of farmyard manure on proliferation of T. thalpophilus in soil Farmyard manure (2% w/w) was added to airdried soil prior to sterilisation of soils. Following autoclaving, the moisture content was adjusted to 15% (w/w) prior to inoculation of soils as described in Section 2.3. In addition sterile farmyard manure samples (moisture content 83.3%) were also prepared, inoculated and incubated for up to 4 weeks at 308C. Respiratory measurement of growth of T. thalpophilus in farmyard manure Measurements of T. thalpophilus respiration in farmyard manure incubated at 308C were taken using an ADC 225 Mk3 IRGA attached to a six point multichannel selector (ADC WA161). Seven water-jacketed respiration chambers were maintained at 308C and sterile farmyard manure added (80 g). Dierential readings were recorded every 5 min for 30 min to ensure the sterility of the manure. The chambers were then inoculated with T. thalpophilus endospores (106 cfu gÿ1 dry weight manure). Dierential readings were then taken every 5 min over the following 72 h. The rate of carbon dioxide (mg CO2) emission gÿ1 dry manure was then calculated.
RESULTS
Eects of soil type and soil moisture content on growth of T. thalpophilus Sterile silt clay loam soil with moisture contents varying from 5±50% (w/w) was inoculated with an endospore suspension containing 8.17 105 4 ÿ1 (24.51 10 ) endospores ml . The numbers of endospores recovered from soil after inoculation on day 0 was 8.85 104 (29.91 103) cfu gÿ1 dry soil, irrespective of soil moisture content (data not shown). The numbers recovered after 28 d incubation at 458C showed an increase of up to 50-fold in the recovery of cfu in soils at all moisture contents [Fig. 1(a)]. Maximum recovery was obtained at the lowest moisture content (5% w/w) and represents a signi®cant increase in recovery (ANOVA, P < 0.05) compared with recovery in soils with a higher moisture content. A signi®cant decrease (ANOVA, P < 0.05) in the recovery of T. thalpophilus was observed after 28 d in soils held at higher moisture contents (25 and 50% w/w) compared to the numbers recovered in the same soils after 21 d [Fig. 1(a)]. Sterile sandy loam soil with moisture contents ranging from 5±50% (w/w) were inoculated with an endospore suspension containing 2.53 105 4 ÿ1 (21.53 10 ) cfu g dry soil. The recovery of endospores from soil following inoculation on day 0 was found to be similar [9.20 104 (21.30 103 cfu)]. The recovery of T. thalpophilus did not vary signi®cantly from this value throughout the 28 d incubation in soils held at all three moisture contents [Fig. 1(b)].
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Fig. 2. Eect of temperature on the recovery of T. thalpophilus (cfu gÿ1 dry soil), following inoculation of sterile silt clay loam soil. Soils were incubated at either 58C (Q), 158C (R), 258C (T), 308C (w) and 458C (W) and at a moisture content of 15% (w/w) for up to 28 d. Results are the means of three replicates with standard error bars presented.
Eects of temperature on the growth and survival of T. thalpophilus
Fig. 3. Comparison of the growth and recovery of T. thalpophilus in soils (cfu gÿ1 dry soil) (closed symbols) with the growth and recovery in soils amended with 2% (v/w) farmyard manure (open symbols): silt clay loam (triangles), (b) sand silt loam (squares), farmyard manure only (.). All samples (moisture content 15% w/w) were incubated at 308C for up to 28 d. Results are the means of three replicates with standard error bars presented.
Recovery of T. thalpophilus from silt clay soil remained constant at incubation temperatures of 5, 15 and 258C (Fig. 2). However, at an incubation temperature of 308C, an increase in the numbers of T. thalpophilus recovered was observed during the initial 7 d of the experiment, reaching 107 cfu mlÿ1. Recovery remained constant at this level throughout the experiment. Similar numbers of T. thalpophilus were recovered when silt clay soil was inoculated and incubated at 458C (Fig. 2). However this level of recovery was reached after only 3 d incubation. Recovery of T. thalpophilus from sand silt loam soil remained constant at 2.4 106 (21.8 105) cfu gÿ1 dry soil throughout the 28 d incubation irrespective of the incubation temperature (data not shown).
(21.09 107) cfu gÿ1 dry soil over the 28 d; this recovery was 70% higher than the numbers of T. thalpophilus recovered in similar soils without farmyard manure (Fig. 3). The addition of 2% (w/v) farmyard manure to sand loam soil led to a 50-fold increase in the number of T. thalpophilus recovered from 1.36 106 (27.71 105) to 8.59 107 (26.39 105) cfu gÿ1 dry soil. Again, no increase in recovery from the initial inoculum was seen when sand silt soil alone was inoculated with T. thalpophilus (Fig. 3). When T. thalpophilus was inoculated into sterile farmyard manure a 600-fold increase in numbers recovered was observed from a inoculation (27.1 105) to 2.38 109 of 3.71 106 8 ÿ1 (22.96 10 cfu g dry soil) after 28 d incubation (Fig. 3).
Eects of addition of farmyard manure on growth of T. thalpophilus in soil
Respiratory activity of T. thalpophilus released in farmyard manure
Farmyard manure is one of the main sources of T. thalpophilus as it provides suitable conditions for the germination and outgrowth of endospores. The eects of addition of sterile farmyard manure to sterile soil (15% moisture content (w/w)) on the growth of T. thalpophilus was studied by comparing the recovery of T. thalpophilus from sterile soil, with soil containing 2% (w/v) sterile farmyard manure. As a comparison, sterile farmyard manure on its own was also inoculated and T. thalpophilus recovered after 28 d incubation at 308C. The results are presented in Fig. 3. The addition of 2% (w/w) farmyard manure to silt clay soil led to an increase (21.6 105) to 1.26 108 from 1.59 106
Figure 3 shows that the growth of T. thalpophilus in all substrates used appears to be almost complete by the time of the ®rst sample (4 d). To establish the pattern of germination and growth of T. thalpophilus, respiratory activity was followed after release into sterile farmyard manure of endospores [1.08 106 (29.74 104) cfu gÿ1 dry manure] (Fig. 4). Germination and growth of endospores occurred after 7 h of incubation with maximum respiratory activity (700 mmol CO2 hÿ1 gÿ1 dry weight farmyard manure) occurring after 11 h after inoculation. Respiratory activity quickly fell and respiration reached low rates after 19 h. This level of respiration is maintained for a further 72 h after
Growth of T. thalpophilus
Fig. 4. Respiratory activity (mmol CO2 hÿ1 g FYMÿ1) of sterile FYM inoculated with T. thalpophilus endospores (106 cfu gÿ1 FYM) and incubated at 308C for 20 h.
which no respiration could be detected (data not shown). At the end of the experiment (4 d), the number of cfu recovered was 2.00 10923.15 108 cfu gÿ1 dry manure. Analysis of a number of soil properties showed that the addition of farmyard manure to the sandy loam soil increased the organic C signi®cantly (ANOVA, P < 0.05) from 0.175 to 0.410% and the organic N from 0.067 to 0.098% while the soil pH increased from 5.7 to 6.0 (Table 1). Addition of farmyard manure to silt clay loam soil resulted in only a small increase (0.02±0.03%) in organic C and N. Increases in the C-to-N ratio of both soils were increased upon addition of farmyard manure (Table 1). DISCUSSION
A comparison of the number of viable endospores inoculated with the numbers immediately recovered (eciency of extraction) was found to be signi®cantly dierent between the two soils. In sterile silt clay soil, eciency of extraction was only 10%, compared to 36% for sterile sand loam soil. This was presumably due the greater amount of organic C and N material present in the silt clay soil, which hampered extraction of endospores, or to the sedimentation of spores with clay particles. Moisture content did not aect extraction eciencies from either soil. T. thalpophilus grew in the silt clay loam soil under all moisture conditions, with maximum recovery occurring in soils with low moisture content (5% w/w). The recovery of T. thalpophilus was reduced with increasing soil moisture content. This was unlikely to be due to physical dierences in the soil leading to a reduction in extraction eciency as the recovery immediately following inoculation was
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similar irrespective of the soil moisture content. A more likely explanation of this result is that to a decrease in the oxygen availability in soils with higher moisture contents reduce the growth of these highly aerobic Thermoactinomyces. Deploey and Fergus (1975) suggested that although Thermoactinomyces were capable of growth at low oxygen concentrations (>0.1% v/v), sporulation did not occur when the oxygen concentration was <1%. This would also explain the observation made by Erikson (1952) that T. vulgaris was only found growing in the upper 15 cm of compost with a high moisture content (83% v/v). The reduction in the number of cfu isolated at the end of the experiment may indicate some deleterious eect of moisture on endospore viability. No increase in the recovery of T. thalpophilus was observed during incubation in sand silt loam soil at any of the moisture contents examined, suggesting that the endospores failed to germinate. This may be due to the acidity of the pH (pH 5.7) and the low organic C and N content, indicative of nutritional limitation. T. thalpophilus grew in silt clay loam at temperatures of 30 and 458C, with similar levels of recovery detected (approximately 1 107 cfu gÿ1 dry soil). These results con®rm the observations of Lacey (1989) that this organism is capable of growth in silt clay loam soils at 308C. Eggins et al. (1972) had shown that solar radiation even in temperate regions can raise soil temperature over 308C. No growth of T. thalpophilus was observed in sandy loam soils incubated at a range of temperatures from 5 to 458C suggesting that factors other than temperature (e.g. nutritional limitation) prevent T. thalpophilus from growing in this soil. Farmyard manure is one of the main sources of T. thalpophilus. Results from Fig. 3 indicated that T. thalpophilus endospores are capable of rapid germination and outgrowth when inoculated into sterile farmyard manure. To determine whether additions of farmyard manure to soil merely results in the dispersal of endospores from manure to soil or whether the additions allows the germination and growth of T. thalpophilus, sterile farmyard manure was added to soils, and T. thalpophilus endospores released. The amount of added farmyard manure 2% (w/v) was based upon our calculation that this approximates to an addition of 35 t haÿ1, ploughed into the top 15 cm of soil, a value which is equivalent to the additions of farmyard manure at Rothamsted experimental sites (Jackson et al., 1997). The results con®rm that additions of farmyard manure to soil increases the recovery of T. thalpophilus from inoculated soils. The amendment of the sandy loam soil with farmyard manure led to a 50-fold increase in the numbers of T. thalpophilus isolated indicating that this addition enabled endospores to germinate and grow
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in soil. The amendment of silt clay loam soil with farmyard manure led to a 70% increase in recovery of T. thalpophilus after 28 d. The addition of farmyard manure signi®cantly increased the organic N and C content, C-to-N ratio and pH of the sand loam soil resulting in the growth of T. thalpophilus. These results con®rm that addition of farmyard manure not only disperses endospores but also allows the growth of T. thalpophilus in a soil, which was previously unsuitable for growth. They also show that even in soils capable of allowing T. thalpophilus to grow, addition of farmyard manure may result in a larger number of endospores being present. This may, in part explain the high recovery of Thermoactinomyces in soils which have received farmyard manure annually (Jackson et al., 1997). However, these experiments were based on autoclaved soils and composts and it is possible that autoclaving manure may release nutrients and other growth-stimulating substances which may not be available to Thermoactinomyces under natural conditions. The experiments on the growth of endospores from T. thalpophilus suggest that as in composts, endospores grow vegetatively for a short time in soil and then form large numbers of endospores as nutrients become limited (Cross, 1968). Respiratory measurements on the growth of endospores released into sterile compost con®rmed this with a rapid increase in respiration followed within 7 h, by a rapid decline and by 19 h little respiration was observed. During this time the numbers of T. thalpophilus recovered increased 600-fold. Such short periods of Thermoactinomyces growth have been observed previously for T. vulgaris (Erikson, 1952), with white aerial mycelium produced within 18± 24 h incubation, but becoming dormant within 48 h. Thus, it may be possible for Thermoactinomyces endospores in undisturbed soils containing agricultural debris to germinate and grow during a short period, immediately following deposition, under suitable conditions. Therefore viable endospores, isolated in studies such as those described by Unsworth et al. (1977) are indeed likely to be as old as the dates suggested by relic dating. Under suitable environmental conditions T. thalpophilus is capable of germinating in soil. Further, the addition of manure to soils may allow T. thalpophilus to germinate and grow in a soil, which was unsuitable for growth. This may, in part explain why this organisms is consistently isolated in high numbers from a variety of soils.
AcknowledgementsÐWe acknowledge the technical assistance of Dr W. S. Wilson, University of Essex. This work was supported by the Poulter Studentship awarded by the University of Essex to A. M. J.
REFERENCES
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Growth of T. thalpophilus Brierley and L. Brierley, pp. 41±53. Van Nostrand Reinhold, New York. Stackebrandt E., Ludwig W., Weizenegger M., Dorn S., McGill T., Fox G. E., Woese C. R., Schubert W. and Schleier K.-H. (1987) Comparative 16S rRNA oligonucleotide analyses and murein types of round-spore-forming Bacilli and non-spore-forming relatives. Journal of General Microbiology 133, 2523±2529. Unsworth B. A., Cross T., Seaward M. R. D. and Sims R. E. (1977) The longevity of thermoactinomycete endo-
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Soil Biol. Biochem. Vol. 30, No. 10/11, pp. 1243±1249, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(98)00059-5 0038-0717/98 $19.00 + 0.00
IMPORTANCE OF ENVIRONMENTAL FACTORS ON THE GROWTH OF THERMOACTINOMYCES THALPOPHILUS A. M. JACKSON and A. S. BALL* Department of Biological Sciences, John Tabor Laboratories, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, U.K. SummaryÐThe germination and growth of endospores from Thermoactinomyces thalpophilus in two soils, a sand silt loam and a silt clay loam was investigated under a range of environmental conditions. A 50-fold increase in the recovery of T. thalpophilus was observed after 28 d growth in silt clay loam soil, when compared to the original inoculum (8.85 104 cfu gÿ1 dry soil), with similar results obtained for this soil over a range of soil moisture contents (5±50% w/w) and at temperatures of 30 and 458C. In contrast, no increase in the recovery of T. thalpophilus was detected when endospores (2.53 105 cfu gÿ1 dry soil) were inoculated into sand silt loam soil. However, when sterile farmyard manure (2% w/w) was added to sand silt loam soil, a 50-fold increase in the numbers of T. thalpophilus isolated was observed over 28 d incubation. Analysis of the sand silt loam soil showed that organic C and N content, pH, and the C-to-N ratio of the soil had all increased signi®cantly upon addition of farmyard manure, resulting in the growth of T. thalpophilus endospores in a soil which previously could not sustain growth. Addition of manure to the silt clay loam led to a 70% increase in recovery of T. thalpophilus. Growth and respiratory measurements of T. thalpophilus released into sterile manure con®rmed the ability of these endospores to show rapid germination, with respiratory activity detected after 7 h, reaching a maximum value (700 mmol CO2 hÿ1 gÿ1 dry manure) 11 h after inoculation, resulting in a 600-fold increase (®nal recovery, 2.38 109 cfu gÿ1 dry manure) in the recovery of T. thalpophilus. We conclude that T. thalpophilus is capable of growth in soils at temperatures as low as 308C and under a range of soil moisture conditions. Further, enhanced growth in soils with additions of manure could be observed. These results support the idea that Thermoactinomyces endospores isolated from ancient soils containing agricultural debris may have remained dormant in soil following a brief period of rapid growth immediately following deposition. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
Thermoactinomyces is a Gram-positive hyphal prokaryote which produces spheroidal heat-resistant endospores (Fergus, 1967) formed singly on substrate and aerial mycelium as a response to growth conditions becoming limited (Lacey, 1989). Although Thermoactinomyces closely resemble actinomycetes in its mode of growth, phylogenetic data show that Thermoactinomyces group with Bacillus (Stackebrandt et al., 1987). Thermoactinomyces is common in agricultural environments, being most numerous in moulding hay (Corbaz et al., 1963), composts (Amner et al., 1988) and manure (Waksman and Corke, 1953; Cross, 1968) that have heated spontaneously to temperatures of 508C or more (Lacey, 1989). This increase in temperature promotes the germination and growth of Thermoactinomyces endospores and often results in the production of >107 endospores gÿ1 dry weight (Cross, 1968). Thermoactinomyces are thought to be dispersed into soil when compost or manure is ploughed into the soil as fertiliser (KuÈster and Locci, 1963; Cross, 1968; Goodfellow and Cross, *Author for correspondence.
1974; Nilsson and Renberg, 1990) or by becoming airborne when decaying plant material is disturbed and then deposited on soil (Cross, 1968; Eggins et al., 1972). The proli®c dispersal of endospores has led to Thermoactinomyces being recovered from most soils, rivers and marine sediments (Cross, 1968; Parduhn and Watterson, 1985; Nilsson and Renberg, 1990; Jackson and Ball, 1994). Once deposited in soil it is thought that these endospores remain dormant. In previous work we have isolated viable Thermoactinomyces endospores from soils stored since 1913 (Jackson et al., 1997), while Unsworth et al. (1977) isolated viable thermophilic Thermoactinomyces from sealed Roman deposits dating from AD 125 and suggested that Thermoactinomyces may be a useful indicator in studies on the agricultural history of soils. However very little is known about the ecology of Thermoactinomyces in soil and whether these organisms are able to germinate and grow in soil. We have examined the eects of a number of environmental factors on the growth of Thermoactinomyces thalpophilus in soil. This species was selected because reports have shown that T. thalpophilus is capable of growth at temperatures as low as 298C
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Table 1. The organic carbon and nitrogen content of sandy loam soil, silt clay soil and farm yard manure used in this study. The eects of farmyard manure (FYM) additions (2% w/w) to soil on soil properties are also included. The results are the means of three replicates with standard errors in brackets Parameter measured Soil description
pH (H2O)
% organic C
% organic N
Sand silt loam Sand silt loam +2% v/v FYM Silt clay loam Silt clay loam +2% v/v FYM FYM
5.7 6.0 7.7 7.8 8.4
0.175 0.410 1.425 1.815 35.59
0.067 0.098 0.124 0.144 2.700
(20.3) (20.3) (20.5) (20.4) (20.3)
(Lacey, 1989). Solar radiation may raise soil temperature above 308C, even in temperate climates (Eggins et al., 1972) and especially in greenhouses where high numbers of Thermoactinomyces have been recovered from the highly organic soils (Fields et al., 1974). The aim of the work was to establish under what conditions, if any could T. thalpophilus endospores germinate and grow in soils.
MATERIALS AND METHODS
Soil analyses Samples were taken during spring 1994 from the top 15-cm of the sample sites. The soils sampled were at Stanway, Essex, a sand silt loam (Wix association; Hodge et al., 1984) and at Great Chesterford, a silt clay loam (Hanslope association; Hodge et al., 1984). The properties of the soil are presented in Table 1. For release studies, sterile soil was sieved (2 mm), air-dried (24 h at 408C) until a steady weight was obtained. Soil (20 g) was then placed in capped containers (25 ml) and sterilised by autoclaving (15 min at 1218C) on three successive days. Soil moisture content was determined by placing triplicate weighed soil samples (5 g) in an oven at 1058C until a steady weight was obtained (24 h). Soil pH was measured by shaking air-dried soil (5 g) in distilled water (25 ml) for 20 min prior to reading pH with a digital meter (Warden Precision Apparatus, Cambridge, U.K.). Soil organic C content was determined using the method described by Walkley and Black (1934). The results were calculated on the basis that 1 ml of NK2Cr2O7 (6 M) is consumed by 3 mg of carbon. The results were multiplied by an arbitrary correction factor (1.33) to calculate the total organic C content (Grimshaw et al., 1989). Soil organic N content was determined using a semi-Kjeldahl digestion (Grimshaw et al., 1989). The amount of ammonium N present in the supernatant was analysed spectrophotometrically (Harwood and Kuhn, 1970). Blanks and standards (ammonium sulphate solution, 0.0±3.0 mg N 0.1 mlÿ1) were prepared and a calibration curve drawn from which the organic N content of the soils was calculated.
(20.01) (20.01) (20.02) (20.03) (20.94)
(20.008) (20.001) (20.003) (20.008) (20.076)
C-to-N ratio 8:1 12:1 19:1 21:1 26:1
The soil C-to-N ratio was calculated from measurements of total soil C and N obtained using a CHNS/O analyser (Perkin Elmer). Strain and growth conditions Thermoactinomyces thalpophilus strain AM1 was originally isolated from a sandy loam and maintained on Czapek Dox yeast extract casamino acid (CYC) agar (Jackson and Ball, 1994). For this study T. thalpophilus was incubated for 48 h at 458C. T. thalpophilus can be distinguished from other Thermoactinomyces by its abundance of white aerial mycelium, resistance to novobiocin (25 mg mlÿ1) and its ability to degrade both starch and tyrosine (Cross and Unsworth, 1977; McCarthy and Cross, 1984). Endospores and aerial mycelium were removed from the agar by agitation in sterile distilled water using a glass spreader. The resulting suspension was ®ltered through glass wool to remove the mycelium before being heated to 908C for 15 min to kill any remaining mycelial fragments (Erikson, 1952), ensuring that any future growth would arise only from endospores. Dilutions of the endospore suspensions were made in sterile distilled water to give approximately 107 viable endospores mlÿ1. Inoculation and reisolation of T. thalpophilus from soil The moisture content of autoclaved soil was adjusted to the required value (5, 15, 25 or 50% w/ w) using sterile distilled water. The soil was inoculated with the endospore suspension to give a viable endospore count of approximately 106 endospores gÿ1 dry soil and the ®nal weight determined. The inoculated soils along with uninoculated controls were then incubated for up to 28 d at the selected temperature (5±458C). Replicates from each soil type inoculated were sampled regularly during the incubation. The moisture content of the soil was maintained by daily measurements of the weight of each sample and the addition of water as required to bring the weight of the sample back to its original weight. T. thalpophilus was recovered from the soil samples by homogenising the whole sample (20 g wet weight soil) in 14 strength Ringers solution containing 10% (v/v) nutrient broth (pH 7.5) using a
Growth of T. thalpophilus
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Fig. 1. Eect of moisture content on the recovery of T. thalpophilus (cfu gÿ1 dry soil), following inoculation of (a) sterile silt clay loam soil and (b) sandy loam soil. Soils were maintained at moisture contents (w/w) of 5% (Q), 15% (R), 25% (T) and 50% (W) and at a temperature of 458C for up to 28 d. Results are the means of three replicates with standard error bars presented.
Waring blender for 15 s (Cross, 1968). Replicate samples (n = 3) of the resulting soil suspensions (0.1 ml) were then spread onto Czapek Dox yeast extract casamino acid agar (Jackson and Ball, 1994) and incubated at 458C for up to 4 d. Results are expressed as the mean number of T. thalpophilus recovered gÿ1 dry soil. Eect of the addition of farmyard manure on proliferation of T. thalpophilus in soil Farmyard manure (2% w/w) was added to airdried soil prior to sterilisation of soils. Following autoclaving, the moisture content was adjusted to 15% (w/w) prior to inoculation of soils as described in Section 2.3. In addition sterile farmyard manure samples (moisture content 83.3%) were also prepared, inoculated and incubated for up to 4 weeks at 308C. Respiratory measurement of growth of T. thalpophilus in farmyard manure Measurements of T. thalpophilus respiration in farmyard manure incubated at 308C were taken using an ADC 225 Mk3 IRGA attached to a six point multichannel selector (ADC WA161). Seven water-jacketed respiration chambers were maintained at 308C and sterile farmyard manure added (80 g). Dierential readings were recorded every 5 min for 30 min to ensure the sterility of the manure. The chambers were then inoculated with T. thalpophilus endospores (106 cfu gÿ1 dry weight manure). Dierential readings were then taken every 5 min over the following 72 h. The rate of carbon dioxide (mg CO2) emission gÿ1 dry manure was then calculated.
RESULTS
Eects of soil type and soil moisture content on growth of T. thalpophilus Sterile silt clay loam soil with moisture contents varying from 5±50% (w/w) was inoculated with an endospore suspension containing 8.17 105 4 ÿ1 (24.51 10 ) endospores ml . The numbers of endospores recovered from soil after inoculation on day 0 was 8.85 104 (29.91 103) cfu gÿ1 dry soil, irrespective of soil moisture content (data not shown). The numbers recovered after 28 d incubation at 458C showed an increase of up to 50-fold in the recovery of cfu in soils at all moisture contents [Fig. 1(a)]. Maximum recovery was obtained at the lowest moisture content (5% w/w) and represents a signi®cant increase in recovery (ANOVA, P < 0.05) compared with recovery in soils with a higher moisture content. A signi®cant decrease (ANOVA, P < 0.05) in the recovery of T. thalpophilus was observed after 28 d in soils held at higher moisture contents (25 and 50% w/w) compared to the numbers recovered in the same soils after 21 d [Fig. 1(a)]. Sterile sandy loam soil with moisture contents ranging from 5±50% (w/w) were inoculated with an endospore suspension containing 2.53 105 4 ÿ1 (21.53 10 ) cfu g dry soil. The recovery of endospores from soil following inoculation on day 0 was found to be similar [9.20 104 (21.30 103 cfu)]. The recovery of T. thalpophilus did not vary signi®cantly from this value throughout the 28 d incubation in soils held at all three moisture contents [Fig. 1(b)].
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Fig. 2. Eect of temperature on the recovery of T. thalpophilus (cfu gÿ1 dry soil), following inoculation of sterile silt clay loam soil. Soils were incubated at either 58C (Q), 158C (R), 258C (T), 308C (w) and 458C (W) and at a moisture content of 15% (w/w) for up to 28 d. Results are the means of three replicates with standard error bars presented.
Eects of temperature on the growth and survival of T. thalpophilus
Fig. 3. Comparison of the growth and recovery of T. thalpophilus in soils (cfu gÿ1 dry soil) (closed symbols) with the growth and recovery in soils amended with 2% (v/w) farmyard manure (open symbols): silt clay loam (triangles), (b) sand silt loam (squares), farmyard manure only (.). All samples (moisture content 15% w/w) were incubated at 308C for up to 28 d. Results are the means of three replicates with standard error bars presented.
Recovery of T. thalpophilus from silt clay soil remained constant at incubation temperatures of 5, 15 and 258C (Fig. 2). However, at an incubation temperature of 308C, an increase in the numbers of T. thalpophilus recovered was observed during the initial 7 d of the experiment, reaching 107 cfu mlÿ1. Recovery remained constant at this level throughout the experiment. Similar numbers of T. thalpophilus were recovered when silt clay soil was inoculated and incubated at 458C (Fig. 2). However this level of recovery was reached after only 3 d incubation. Recovery of T. thalpophilus from sand silt loam soil remained constant at 2.4 106 (21.8 105) cfu gÿ1 dry soil throughout the 28 d incubation irrespective of the incubation temperature (data not shown).
(21.09 107) cfu gÿ1 dry soil over the 28 d; this recovery was 70% higher than the numbers of T. thalpophilus recovered in similar soils without farmyard manure (Fig. 3). The addition of 2% (w/v) farmyard manure to sand loam soil led to a 50-fold increase in the number of T. thalpophilus recovered from 1.36 106 (27.71 105) to 8.59 107 (26.39 105) cfu gÿ1 dry soil. Again, no increase in recovery from the initial inoculum was seen when sand silt soil alone was inoculated with T. thalpophilus (Fig. 3). When T. thalpophilus was inoculated into sterile farmyard manure a 600-fold increase in numbers recovered was observed from a inoculation (27.1 105) to 2.38 109 of 3.71 106 8 ÿ1 (22.96 10 cfu g dry soil) after 28 d incubation (Fig. 3).
Eects of addition of farmyard manure on growth of T. thalpophilus in soil
Respiratory activity of T. thalpophilus released in farmyard manure
Farmyard manure is one of the main sources of T. thalpophilus as it provides suitable conditions for the germination and outgrowth of endospores. The eects of addition of sterile farmyard manure to sterile soil (15% moisture content (w/w)) on the growth of T. thalpophilus was studied by comparing the recovery of T. thalpophilus from sterile soil, with soil containing 2% (w/v) sterile farmyard manure. As a comparison, sterile farmyard manure on its own was also inoculated and T. thalpophilus recovered after 28 d incubation at 308C. The results are presented in Fig. 3. The addition of 2% (w/w) farmyard manure to silt clay soil led to an increase (21.6 105) to 1.26 108 from 1.59 106
Figure 3 shows that the growth of T. thalpophilus in all substrates used appears to be almost complete by the time of the ®rst sample (4 d). To establish the pattern of germination and growth of T. thalpophilus, respiratory activity was followed after release into sterile farmyard manure of endospores [1.08 106 (29.74 104) cfu gÿ1 dry manure] (Fig. 4). Germination and growth of endospores occurred after 7 h of incubation with maximum respiratory activity (700 mmol CO2 hÿ1 gÿ1 dry weight farmyard manure) occurring after 11 h after inoculation. Respiratory activity quickly fell and respiration reached low rates after 19 h. This level of respiration is maintained for a further 72 h after
Growth of T. thalpophilus
Fig. 4. Respiratory activity (mmol CO2 hÿ1 g FYMÿ1) of sterile FYM inoculated with T. thalpophilus endospores (106 cfu gÿ1 FYM) and incubated at 308C for 20 h.
which no respiration could be detected (data not shown). At the end of the experiment (4 d), the number of cfu recovered was 2.00 10923.15 108 cfu gÿ1 dry manure. Analysis of a number of soil properties showed that the addition of farmyard manure to the sandy loam soil increased the organic C signi®cantly (ANOVA, P < 0.05) from 0.175 to 0.410% and the organic N from 0.067 to 0.098% while the soil pH increased from 5.7 to 6.0 (Table 1). Addition of farmyard manure to silt clay loam soil resulted in only a small increase (0.02±0.03%) in organic C and N. Increases in the C-to-N ratio of both soils were increased upon addition of farmyard manure (Table 1). DISCUSSION
A comparison of the number of viable endospores inoculated with the numbers immediately recovered (eciency of extraction) was found to be signi®cantly dierent between the two soils. In sterile silt clay soil, eciency of extraction was only 10%, compared to 36% for sterile sand loam soil. This was presumably due the greater amount of organic C and N material present in the silt clay soil, which hampered extraction of endospores, or to the sedimentation of spores with clay particles. Moisture content did not aect extraction eciencies from either soil. T. thalpophilus grew in the silt clay loam soil under all moisture conditions, with maximum recovery occurring in soils with low moisture content (5% w/w). The recovery of T. thalpophilus was reduced with increasing soil moisture content. This was unlikely to be due to physical dierences in the soil leading to a reduction in extraction eciency as the recovery immediately following inoculation was
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similar irrespective of the soil moisture content. A more likely explanation of this result is that to a decrease in the oxygen availability in soils with higher moisture contents reduce the growth of these highly aerobic Thermoactinomyces. Deploey and Fergus (1975) suggested that although Thermoactinomyces were capable of growth at low oxygen concentrations (>0.1% v/v), sporulation did not occur when the oxygen concentration was <1%. This would also explain the observation made by Erikson (1952) that T. vulgaris was only found growing in the upper 15 cm of compost with a high moisture content (83% v/v). The reduction in the number of cfu isolated at the end of the experiment may indicate some deleterious eect of moisture on endospore viability. No increase in the recovery of T. thalpophilus was observed during incubation in sand silt loam soil at any of the moisture contents examined, suggesting that the endospores failed to germinate. This may be due to the acidity of the pH (pH 5.7) and the low organic C and N content, indicative of nutritional limitation. T. thalpophilus grew in silt clay loam at temperatures of 30 and 458C, with similar levels of recovery detected (approximately 1 107 cfu gÿ1 dry soil). These results con®rm the observations of Lacey (1989) that this organism is capable of growth in silt clay loam soils at 308C. Eggins et al. (1972) had shown that solar radiation even in temperate regions can raise soil temperature over 308C. No growth of T. thalpophilus was observed in sandy loam soils incubated at a range of temperatures from 5 to 458C suggesting that factors other than temperature (e.g. nutritional limitation) prevent T. thalpophilus from growing in this soil. Farmyard manure is one of the main sources of T. thalpophilus. Results from Fig. 3 indicated that T. thalpophilus endospores are capable of rapid germination and outgrowth when inoculated into sterile farmyard manure. To determine whether additions of farmyard manure to soil merely results in the dispersal of endospores from manure to soil or whether the additions allows the germination and growth of T. thalpophilus, sterile farmyard manure was added to soils, and T. thalpophilus endospores released. The amount of added farmyard manure 2% (w/v) was based upon our calculation that this approximates to an addition of 35 t haÿ1, ploughed into the top 15 cm of soil, a value which is equivalent to the additions of farmyard manure at Rothamsted experimental sites (Jackson et al., 1997). The results con®rm that additions of farmyard manure to soil increases the recovery of T. thalpophilus from inoculated soils. The amendment of the sandy loam soil with farmyard manure led to a 50-fold increase in the numbers of T. thalpophilus isolated indicating that this addition enabled endospores to germinate and grow
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in soil. The amendment of silt clay loam soil with farmyard manure led to a 70% increase in recovery of T. thalpophilus after 28 d. The addition of farmyard manure signi®cantly increased the organic N and C content, C-to-N ratio and pH of the sand loam soil resulting in the growth of T. thalpophilus. These results con®rm that addition of farmyard manure not only disperses endospores but also allows the growth of T. thalpophilus in a soil, which was previously unsuitable for growth. They also show that even in soils capable of allowing T. thalpophilus to grow, addition of farmyard manure may result in a larger number of endospores being present. This may, in part explain the high recovery of Thermoactinomyces in soils which have received farmyard manure annually (Jackson et al., 1997). However, these experiments were based on autoclaved soils and composts and it is possible that autoclaving manure may release nutrients and other growth-stimulating substances which may not be available to Thermoactinomyces under natural conditions. The experiments on the growth of endospores from T. thalpophilus suggest that as in composts, endospores grow vegetatively for a short time in soil and then form large numbers of endospores as nutrients become limited (Cross, 1968). Respiratory measurements on the growth of endospores released into sterile compost con®rmed this with a rapid increase in respiration followed within 7 h, by a rapid decline and by 19 h little respiration was observed. During this time the numbers of T. thalpophilus recovered increased 600-fold. Such short periods of Thermoactinomyces growth have been observed previously for T. vulgaris (Erikson, 1952), with white aerial mycelium produced within 18± 24 h incubation, but becoming dormant within 48 h. Thus, it may be possible for Thermoactinomyces endospores in undisturbed soils containing agricultural debris to germinate and grow during a short period, immediately following deposition, under suitable conditions. Therefore viable endospores, isolated in studies such as those described by Unsworth et al. (1977) are indeed likely to be as old as the dates suggested by relic dating. Under suitable environmental conditions T. thalpophilus is capable of germinating in soil. Further, the addition of manure to soils may allow T. thalpophilus to germinate and grow in a soil, which was unsuitable for growth. This may, in part explain why this organisms is consistently isolated in high numbers from a variety of soils.
AcknowledgementsÐWe acknowledge the technical assistance of Dr W. S. Wilson, University of Essex. This work was supported by the Poulter Studentship awarded by the University of Essex to A. M. J.
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