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Most-probable-number procedure to enumerate So-oxidizing, thiosulfate producing heterotrophs in soil
003%0717188 $3.00 f 0.00 Copyright Q 1988 Pcrgamon Press plc
Soil Bioi. Biochem.Vol. 20, No. 4, pp. 577-578, 1988 Printed in Great Britain. All rights reserved
SHORT COMMUNICATION MOST-PROBABLE-NUMBER PROCEDURE TO ENUMERATE So-OXIDIZING, THIOSULFATE PRODUCING HETEROTROPHS IN SOIL Department
J. R. LAWRENCE and J. J. GERMIDA of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO (Accepted
25 Noaemher
Many heterotrophic microorganisms are capable of oxidizing elemental sulfur (Yagi ef al., 1971; Wainwright and Killham, 1980; Wainwright, 1984; Germida, 1985; Germida et al., 1985). Many of these organisms produce thiosulfate as a major product during elemental sulfur (So) oxidation (Yagi er al., 1971; Germida, 1985; Germida er al., 1985). However, it is unclear whether thiosulfate is produced as an intermediate or a by-product during sulfur oxidation by heterotrophs. Germida (1985) demonstrated synergism on agar media between So-oxidizing heterotrophs that produce thiosulfate and ch~olithoautotrophic thios~fate oxidizers, and suggested that the concerted action of these two groups of organisms was important during So oxidation in soil. Studies on So-oxidizing heterotrophs have been hindered by a lack of suitable media and enumeration procedures. Recent developments include several different solidified media containing various forms of S” which allow for the detection of certain types of heterotrophic sulfur oxidizers, i.e. those that produce large quantities of sulfate and change a pH indicator (Germida, 1985). or those that clear colloidal S” from media (Wainw~ght, 1978; Germida 1985). However, these media do not detect those So-oxidizers which only produce thiosuifate as a product of So oxidation. Here, we describe a simple most-probable-number (MPN) procedure to enumerate populations of S-oxidizing, thiosulfate producing heterotrophs. For MPN determinations, the appropriate medium (2 ml) was added to each well of a microtiter plate (24 wells; 3.5 ml capacity per well; Linbro Plastics Ltd) and then inoculated with the appropriate soil dilution (0.1 ml). Total heterotrophic MPN were enumerated using l/l0 strength trypticase-soy-broth (TSB); turbidity was scored as a positive result. Heterotrophic microorganisms oxidizing So and producing thiosulfate were enumerated using TSB containing 1% (v/v) flowabie sulfur (FS). Flowable sulfur (Stoiier Chemical Co. Inc., Houston) is a creamy, liquid suspension (S&70% w/v) of finely divided (1-2pm dia) So; it was washed, autoclaved and added to microtiter plates as described by Germida (1985). Control microtiter plates contained the TSB medium but no FS. These and uninoculated plates containing TSB plus FS were incubated to detect false positive results (i.e. production of thiosulfate from media components or chemical oxidation). The microtiter plates were incubated in polyethylene bags, on a rotary shaker (100 rev min-I) to ensure aerobic growth at 25 f 2°C. After 7 days, the populations had stabilized and no further increase in MPN was detected by prolonged incubation. Choice of medium could make the assay selective for a group (e.g. fungi) of microorganisms. Ail media components were Difco products. A calorimetric. ferric thiocyanate method (Nor and Tabatabai. 1976) was used to score MPN wells positive
1987)
for thiosuifate production. This involved adding 2 drops of 100 mM KCN and, after 15 min, 4 drops of 330 mM CuCl,.2H,O and 2 drops of a 250mM Fe(NO,),~9H,O: 31OOm~ HNO, solution. Formation of a brown color indicated the presence of both thiosulfate and tetrathionate. Omission of CuCl,.2H,O indicated tetrathionate only, and thus the relative proportion of both oxyanions could be determined. Tetrathionate formation in MPN wells and in broth cultures of soil isolates was however, usually very low. Samples from the Ap horizons of five agricultural soils were used to evaluate the MPN procedure, and as sources of isolates for in airro studies. Samples were collected, maintained at field moisture, stored at 4°C in polyethylene bags, and sieved (2 mm) before analyses. All analyses were performed in duplicate on replicate subsamples. The MPN was determined by reference to the table of Cochran (1950) for use with IO-fold dilutions and 5 tubes per dilution. So-oxidizing heterotrophs that produced thiosulfate as the main product were readily enumerated using the proposed MPN procedure. The addition of chemical reagents to MPN plates and the deveiopment of a distinct brown color allowed visual detection of thiosulfate production. The procedure was very reliable and no false positive wells were detected. The sensitivity of the assay was about 1Opg thiosulfate ml-i; instrumentation could also be used to determine the concentrations of thiosulfate in microtiter wells. Similar calorimetric MPN procedures are routinely used to estimate nitrifying and denitrifying populations in soils (Belser, 1979; Tiedje, 1983). Heterotrophic thiosulfate producers were abundant in the agricultural soils tested with this new MPN procedure (Table 1). The detectable populations ranged from-i.0 x 10’ to 1.0 x 10Bg-‘. i.e. 3-31% of the total heterotroohic population de&ted. in addition, the population ieveis of these organisms were about lO,OOO-foldgreater than populations of V-oxidizing heterotrophs that produced sulfate as the end-product (Table 1). These results indicate the major group of So-oxidizing heterotrophs in soil were those that produce thiosulfate, thus this rapid MPN procedure provided a more complete estimate of heterotrophic Sooxidizers in soil than methods based on the detection of pH changes by acid (SO:-) production. In order to verify the findings of the MPN study and further document the abundance of these So-oxidizers in soil, in vitro f&oxidation studies were conducted on 80 soil isolates. Bacterial and fungal isolates were obtained by spread-plating soil dilutions (sterile tap-water diiuent) on I/l0 strength trypticase soy agar (TSA) and acidified QsH 3.5) Czapek-Dox agar, respectively. Individual colonies were randomly picked and repeatedly streaked for isolation on the same medium. The pr~uction of thiosulfate and
578
Short
communications
Table 1. MPN of total beterotrophs and So-oxidizing heterotrophs in five agricultural soils’
Soil association
pHb
Fox Valley Carrot River Weirdale Elstow Smeaton
6.6 7.4 7.5 6.1 1.5
Total heterotrophs MPN CFU 11061 (lo*) 5.2 4.3 6.0 1.1 4.8
2.5 3.2 7.9 2.0 3.2
S,O;-
Y-oxidizing heterotrophs producers SO:- producers MPN t10? (10’) 4.0 1.0 6.3 1.6 I .o
5.5 6.3 7.8 2.0 3.8
‘Means of duplicate analyses on replicate subsamples. The 95% confidence limits for the MPN values are plus or minus 0.33 of the mean. bpH measured in a I : 1 soil: IO ITIM CaCI, slurry. Table 2. S-oxyanions formed during oxidation of elemental sulfur by isolates from the Elstow soil’
findings of the MPN assays, and demonstrate the apparent ubiquity of heterotrophic sulfur oxidizers in these soils.
figsInIsolates Bacteria B-l B-2 (Bacillus sp.) B-3 B-4 (Bocillrrs sp.) B-5 B-6 B-7 B-8 B-9 B-IO B-11 B-12 B-13 Fungi Frl (Pe~icill~~m sp.) F-2 F-3 (Penicilliumsp.)
F-4
Lo?-
so:-
65 380 68 65 65 61 48 123 14 7 4 76 8
NDb ND ND ND ND ND ND ND ND ND ND ND ND
120 114 49 37
90 100 ND 42
‘Isolates were grown in 5Oml l/IO strength of trypticase-soy-broth containing I % (v/v) Rowable sulfur, on a rotary shaker (150 rev min-‘) for 10 days at 27°C; concentrations of S-ions in excess of controls without FS. Values are means of duplicate determinations on replicate cultures. bNone detected.
Ackno&edgements-This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Sulfur Development Institute of Canada. Contribution No. R519 Saskatchewan Institute of Pedology. REFERENCES Belser L. W. (1979) Population ecology of nitrifying bacteria. Annual Review of Microbiology 33, 309-333. Cochran W. G. (1950) Estimation of bacterial densities by means of the “most probable number”. Biometrics 5, 105-I 16. Germida J. I. (1985) Modified sulfur containing media for studying sulfur oxidizing microorganisms. In Planeray Ecology (D. E. Caldwell, J. A. Brierley and C. L. Brierley, Eds), pp. 333-344. Van Nostrand Reinhold, New York. Germida J. J., Lawrence J. R. and Gupta V. V. S. R. (1985) Microbial oxidation of sulphur in Saskatchewan soils. In Proceedings of Sulfur-84 (J. W. Terry. Ed.), pp. 703-710. The Sulfur Development Institute of Canada, Calgary. Hesse P. R. (1971) A Textbook of Soil Chemical Analysis. Murray, London. Nor Y. M. and Tabatabai M. A. (1976) Extraction and calorimetric determination of thiosulfate and tetrathionate in soils. Soil Science 122, 171-178. Tiedje J. M. (1983) Denitrification. in Methods of Soil An&.&, Part 2. 2nd edn (A. L. Page, R. H. Miller and D. K. Keeney. Eds). pp. IO1I-1026. American Society of Agronomy. Madison. Wainwright M. (3978) A modified sulphur medium for the isolation of sulphur oxidizing fungi. PIonr and Soil 49,
tetrathionate was detected using the method of Nor and Tabatabai (1976) and sulfate was detected turbidimetrically (Hesse, 1971). Over 95% of the isolates oxidized S” with the production of thiosulfate. None of the isolates produced 191-193. detectable quantities of tetrathionate. The bacterial isolates Wainwright M. (1984) Sulfur oxidation in soils. Advances in produced only thiosulfate, whereas three out of four fungal Agronomy 37, 349-396. isolates produced similar quantities of both thiosulfate and Wainwright M. and Killham K. (1980) Sulphur oxidation sulfate (Table 2). Germida (1985) and Germida et al. (1985) by Fusarium solani. Soil Biology & Biochemistry 12, reported similar trends for stock cultures and soil isolates. 5.55-558. i.e. minimal production of tetrathionate, bacterial isolates Yagi S., Katai S. and Kimura T. (1971) Oxidation of produced mainly thiosutfate, and fungal isolates produced elemental sulfur to thiosulfate by Srrepzomvces. ApEed both thiosulfate and sulfate. These results support the ~~cro~iolog~ 22, 157-f 59.
Soil Bioi. Biochem.Vol. 20, No. 4, pp. 577-578, 1988 Printed in Great Britain. All rights reserved
SHORT COMMUNICATION MOST-PROBABLE-NUMBER PROCEDURE TO ENUMERATE So-OXIDIZING, THIOSULFATE PRODUCING HETEROTROPHS IN SOIL Department
J. R. LAWRENCE and J. J. GERMIDA of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO (Accepted
25 Noaemher
Many heterotrophic microorganisms are capable of oxidizing elemental sulfur (Yagi ef al., 1971; Wainwright and Killham, 1980; Wainwright, 1984; Germida, 1985; Germida et al., 1985). Many of these organisms produce thiosulfate as a major product during elemental sulfur (So) oxidation (Yagi er al., 1971; Germida, 1985; Germida er al., 1985). However, it is unclear whether thiosulfate is produced as an intermediate or a by-product during sulfur oxidation by heterotrophs. Germida (1985) demonstrated synergism on agar media between So-oxidizing heterotrophs that produce thiosulfate and ch~olithoautotrophic thios~fate oxidizers, and suggested that the concerted action of these two groups of organisms was important during So oxidation in soil. Studies on So-oxidizing heterotrophs have been hindered by a lack of suitable media and enumeration procedures. Recent developments include several different solidified media containing various forms of S” which allow for the detection of certain types of heterotrophic sulfur oxidizers, i.e. those that produce large quantities of sulfate and change a pH indicator (Germida, 1985). or those that clear colloidal S” from media (Wainw~ght, 1978; Germida 1985). However, these media do not detect those So-oxidizers which only produce thiosuifate as a product of So oxidation. Here, we describe a simple most-probable-number (MPN) procedure to enumerate populations of S-oxidizing, thiosulfate producing heterotrophs. For MPN determinations, the appropriate medium (2 ml) was added to each well of a microtiter plate (24 wells; 3.5 ml capacity per well; Linbro Plastics Ltd) and then inoculated with the appropriate soil dilution (0.1 ml). Total heterotrophic MPN were enumerated using l/l0 strength trypticase-soy-broth (TSB); turbidity was scored as a positive result. Heterotrophic microorganisms oxidizing So and producing thiosulfate were enumerated using TSB containing 1% (v/v) flowabie sulfur (FS). Flowable sulfur (Stoiier Chemical Co. Inc., Houston) is a creamy, liquid suspension (S&70% w/v) of finely divided (1-2pm dia) So; it was washed, autoclaved and added to microtiter plates as described by Germida (1985). Control microtiter plates contained the TSB medium but no FS. These and uninoculated plates containing TSB plus FS were incubated to detect false positive results (i.e. production of thiosulfate from media components or chemical oxidation). The microtiter plates were incubated in polyethylene bags, on a rotary shaker (100 rev min-I) to ensure aerobic growth at 25 f 2°C. After 7 days, the populations had stabilized and no further increase in MPN was detected by prolonged incubation. Choice of medium could make the assay selective for a group (e.g. fungi) of microorganisms. Ail media components were Difco products. A calorimetric. ferric thiocyanate method (Nor and Tabatabai. 1976) was used to score MPN wells positive
1987)
for thiosuifate production. This involved adding 2 drops of 100 mM KCN and, after 15 min, 4 drops of 330 mM CuCl,.2H,O and 2 drops of a 250mM Fe(NO,),~9H,O: 31OOm~ HNO, solution. Formation of a brown color indicated the presence of both thiosulfate and tetrathionate. Omission of CuCl,.2H,O indicated tetrathionate only, and thus the relative proportion of both oxyanions could be determined. Tetrathionate formation in MPN wells and in broth cultures of soil isolates was however, usually very low. Samples from the Ap horizons of five agricultural soils were used to evaluate the MPN procedure, and as sources of isolates for in airro studies. Samples were collected, maintained at field moisture, stored at 4°C in polyethylene bags, and sieved (2 mm) before analyses. All analyses were performed in duplicate on replicate subsamples. The MPN was determined by reference to the table of Cochran (1950) for use with IO-fold dilutions and 5 tubes per dilution. So-oxidizing heterotrophs that produced thiosulfate as the main product were readily enumerated using the proposed MPN procedure. The addition of chemical reagents to MPN plates and the deveiopment of a distinct brown color allowed visual detection of thiosulfate production. The procedure was very reliable and no false positive wells were detected. The sensitivity of the assay was about 1Opg thiosulfate ml-i; instrumentation could also be used to determine the concentrations of thiosulfate in microtiter wells. Similar calorimetric MPN procedures are routinely used to estimate nitrifying and denitrifying populations in soils (Belser, 1979; Tiedje, 1983). Heterotrophic thiosulfate producers were abundant in the agricultural soils tested with this new MPN procedure (Table 1). The detectable populations ranged from-i.0 x 10’ to 1.0 x 10Bg-‘. i.e. 3-31% of the total heterotroohic population de&ted. in addition, the population ieveis of these organisms were about lO,OOO-foldgreater than populations of V-oxidizing heterotrophs that produced sulfate as the end-product (Table 1). These results indicate the major group of So-oxidizing heterotrophs in soil were those that produce thiosulfate, thus this rapid MPN procedure provided a more complete estimate of heterotrophic Sooxidizers in soil than methods based on the detection of pH changes by acid (SO:-) production. In order to verify the findings of the MPN study and further document the abundance of these So-oxidizers in soil, in vitro f&oxidation studies were conducted on 80 soil isolates. Bacterial and fungal isolates were obtained by spread-plating soil dilutions (sterile tap-water diiuent) on I/l0 strength trypticase soy agar (TSA) and acidified QsH 3.5) Czapek-Dox agar, respectively. Individual colonies were randomly picked and repeatedly streaked for isolation on the same medium. The pr~uction of thiosulfate and
578
Short
communications
Table 1. MPN of total beterotrophs and So-oxidizing heterotrophs in five agricultural soils’
Soil association
pHb
Fox Valley Carrot River Weirdale Elstow Smeaton
6.6 7.4 7.5 6.1 1.5
Total heterotrophs MPN CFU 11061 (lo*) 5.2 4.3 6.0 1.1 4.8
2.5 3.2 7.9 2.0 3.2
S,O;-
Y-oxidizing heterotrophs producers SO:- producers MPN t10? (10’) 4.0 1.0 6.3 1.6 I .o
5.5 6.3 7.8 2.0 3.8
‘Means of duplicate analyses on replicate subsamples. The 95% confidence limits for the MPN values are plus or minus 0.33 of the mean. bpH measured in a I : 1 soil: IO ITIM CaCI, slurry. Table 2. S-oxyanions formed during oxidation of elemental sulfur by isolates from the Elstow soil’
findings of the MPN assays, and demonstrate the apparent ubiquity of heterotrophic sulfur oxidizers in these soils.
figsInIsolates Bacteria B-l B-2 (Bacillus sp.) B-3 B-4 (Bocillrrs sp.) B-5 B-6 B-7 B-8 B-9 B-IO B-11 B-12 B-13 Fungi Frl (Pe~icill~~m sp.) F-2 F-3 (Penicilliumsp.)
F-4
Lo?-
so:-
65 380 68 65 65 61 48 123 14 7 4 76 8
NDb ND ND ND ND ND ND ND ND ND ND ND ND
120 114 49 37
90 100 ND 42
‘Isolates were grown in 5Oml l/IO strength of trypticase-soy-broth containing I % (v/v) Rowable sulfur, on a rotary shaker (150 rev min-‘) for 10 days at 27°C; concentrations of S-ions in excess of controls without FS. Values are means of duplicate determinations on replicate cultures. bNone detected.
Ackno&edgements-This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Sulfur Development Institute of Canada. Contribution No. R519 Saskatchewan Institute of Pedology. REFERENCES Belser L. W. (1979) Population ecology of nitrifying bacteria. Annual Review of Microbiology 33, 309-333. Cochran W. G. (1950) Estimation of bacterial densities by means of the “most probable number”. Biometrics 5, 105-I 16. Germida J. I. (1985) Modified sulfur containing media for studying sulfur oxidizing microorganisms. In Planeray Ecology (D. E. Caldwell, J. A. Brierley and C. L. Brierley, Eds), pp. 333-344. Van Nostrand Reinhold, New York. Germida J. J., Lawrence J. R. and Gupta V. V. S. R. (1985) Microbial oxidation of sulphur in Saskatchewan soils. In Proceedings of Sulfur-84 (J. W. Terry. Ed.), pp. 703-710. The Sulfur Development Institute of Canada, Calgary. Hesse P. R. (1971) A Textbook of Soil Chemical Analysis. Murray, London. Nor Y. M. and Tabatabai M. A. (1976) Extraction and calorimetric determination of thiosulfate and tetrathionate in soils. Soil Science 122, 171-178. Tiedje J. M. (1983) Denitrification. in Methods of Soil An&.&, Part 2. 2nd edn (A. L. Page, R. H. Miller and D. K. Keeney. Eds). pp. IO1I-1026. American Society of Agronomy. Madison. Wainwright M. (3978) A modified sulphur medium for the isolation of sulphur oxidizing fungi. PIonr and Soil 49,
tetrathionate was detected using the method of Nor and Tabatabai (1976) and sulfate was detected turbidimetrically (Hesse, 1971). Over 95% of the isolates oxidized S” with the production of thiosulfate. None of the isolates produced 191-193. detectable quantities of tetrathionate. The bacterial isolates Wainwright M. (1984) Sulfur oxidation in soils. Advances in produced only thiosulfate, whereas three out of four fungal Agronomy 37, 349-396. isolates produced similar quantities of both thiosulfate and Wainwright M. and Killham K. (1980) Sulphur oxidation sulfate (Table 2). Germida (1985) and Germida et al. (1985) by Fusarium solani. Soil Biology & Biochemistry 12, reported similar trends for stock cultures and soil isolates. 5.55-558. i.e. minimal production of tetrathionate, bacterial isolates Yagi S., Katai S. and Kimura T. (1971) Oxidation of produced mainly thiosutfate, and fungal isolates produced elemental sulfur to thiosulfate by Srrepzomvces. ApEed both thiosulfate and sulfate. These results support the ~~cro~iolog~ 22, 157-f 59.