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Microbial production of sulphate and sulphide in some Australian soils
Soil Bid. Bmchrm. Vol. 5. pp 773-781.
MICROBIAL
PergamonPress 1973. Printed m GreatBritm
PRODUCTION OF SULPHATE AND SULPHIDE IN SOME AUSTRALIAN SOILS R. J. SWABY C.S.I.R.O.,
Division
and RITA FEDEL
of Soils, Glen Osmond, (Accepted
17 April
South Australia
5064
1973)
Summary-Fifty-six Australian soils were examined for their ability to produce sulphate from sulphur and sulphide from added sulphur, sulphate and cystine. Selective liquid media were used to obtain initial counts and progressive activities of some of the principal organisms involved. Attempts were made to correlate soil properties, initial microbial counts, classes of microorganisms detected and metabolic activities. Almost half of the soils oxidized sulphur very slowly or not at all, most probably due to absence of thiobacilli, particularly 7%. thiooxidans. Even soils which oxidized sulphur rapidly never gave initial counts greater than 10“ cells/g. The majority of soils produced sulphide from sulphur, between one-half and two-thirds of them formed sulphide from cystine but sulphate reduction was very rare. Sulphide escaped more readily from aerobic than from waterlogged soils. Numerous genera were isolated including Drsulfocibrio and Desulfatomaculum capable of reducing sulphur, both aerobically and anaerobically, particularly sporing and non-sporing mesophiles which were sometimes present at 10h cells/g of soil. Sulphate reduction by the genus Desulfovibrio was uncommon, and by the genus DesuIfatomaculum extremely rare. Many genera of sporing and non-sporing mesophiles seemed capable of producing sulphide from cystine when first isolated, but they quickly lost this ability on subculturing. Initial counts of 1Oh cells/g of soil were occasionally found but usually much lower. No significant correlations, only trends, were found suggesting that any soil properties measured affected the occurrence or activities of organisms capable of producing sulphate or sulphide. INTRODUCTION SULPHUR deficiencies are widespread in Australian soils, necessitating the use of elemental sulphur or gypsum, yet little is known of the inorganic sulphur cycle in these soils, or of the microorganisms involved. In a survey of 273 soils from eastern and southern Australia, Swaby and Vitolins (1968) and Vitolins and Swaby (1968) found that 1 per cent of sulphur added to soil oxidized either slowly to sulphate or not at all in almost half of them. However Barrow (1971) in Western Australia reported higher percentages of sulphur oxidized at lower concentrations of sulphur. Autotrophic thiobacilli were more active oxidizers of sulphur than other organisms, particularly Thiobacillus thiooxidans, but the latter was rare in or absent from more than half of these soils. Many Australian soils in the temperate regions differ therefore from others examined elsewhere (cf. Starkey, 1966). Sulphate may be formed also from cysteine via sulphide, but Freney (1960) showed that it may be formed directly in aerated soils. The rate of release of sulphate from plant materials depends on the content of sulphur, the C/S ratio and often the N/S ratio, according to Barrow (1960). Freney (1961) considers that possibly ester sulphates in humus are resistant to enzymic hydrolysis, so are poor suppliers of sulphate. It is usually considered that dissimilatory, anaerobic sulphate reducers of the genera Desuljovibrio and Desulfotomaculum, are responsible for sulphide formation in waterlogged soils; but Bromfield (1953) found Bacillus megaterium capable of producing free sulphide from sulphate in partially sterilized soil. 173
114
R. J. SWABY
AND RI-l-A FEDEL
These few references summarize nearly all that is known of microbial production of sulphate and sulphide in Australian soils. In this paper 56 soils were surveyed for their ability to produce sulphate and sulphide from a number of sulphur compounds added simultaneously. Attempts were also made to estimate the initial numbers or activities of the principal sulphur organisms present to see if they were interdependent. MATERIALS
AND METHODS
Fifty-six soils mainly from eastern and southern Australia were examined including seven Solonized Brown, five Gray Clay, five Solodized Solonetz and Solodic, four Soloth and Prairie, six Red Brown Earth, one Brown Earth, six Red Earth, seven Red Podzolic, 10 Yellow Podzolic and two Podzolic. Soils were sampled from 0 to 10 cm, five cores being combined from sites 10 m apart, then refrigerated at 2°C until examined simultaneously under both friable and waterlogged conditions for their ability to oxidize 1.0 per cent added elemental sulphur to sulphate or to reduce to sulphide (a) 0.1% elemental sulphur, (b) a mixture of 0*30/,ammonium sulphate and 0*2:/dsodium sulphate and (c) O+o/;, cystine hydrochloride, since Frederick, Starkey and Segal(l957) found this organic sulphur compound readily decomposed. At weekly intervals over a period of 10 weeks incubation at 25”C, sulphur oxidation was measured by the drop in pH and the rise of titratable acidity in log aliquots of soil. The activity of Th. thiooxidans was determined approximately by the number of days for soil pH to reach 4.0. Free sulphide was detected by inserting lead acetate paper discs, 5.5 cm dia, in the lids of jars. The number of days was noted to produce a shade of blackening corresponding to 35 gg of sulphide. In addition, suitable dilutions of each soil were added to elective media to estimate initial counts of sulphur organisms by dilution to extinction. Vishniac and Santer’s (1957) liquid inorganic media containing @lx sulphur or O-5’)/;,sodium thiosulphate were used for thiobacilli. Th. t~iuoxida~s was considered present when @005 per cent added brom phenol blue indicated that the initial pH of 5-Ohad dropped to 4-Oand 7%. t~7iopar~swhen 0.005 per cent added brom cresol green indicated a pH change from 6.7 to 4.6. Th. dentitri$cans was detected by Baalsrud and Baalsrud’s (1954) method of trapping nitrogen gas from nitrate medium under a paraffin oil seal. Sulphide producers were detected in modified Postgate’s (1966) liquid medium containing 0.1% sulphur or 0.26% sodium sulphate or @Oloi:,cystine as the only sulphur compounds and no added ferrous salt. Aerobes were grown in tubes, l-5 cm dia, containing 5-Oml of medium whereas anaerobes were cultured in 7.5 ml of medium sealed with 1.5 ml ofparaffin oil. Spores were detected after pasteurizing soil suspensions for 2 min at 90°C. Mesophiles were incubated at 25°C and thermophiles at 55°C. Sulphide was detected by inserting lead acetate paper discs, 1.3 cm dia in the loose screw caps of tubes 1.5 mm x 10 mm containing sulphur and cystine media or by adding iron nails to sulphate media, 3 cm Long and 1.2 mm dia. as proposed by Abd-el-~al~k and Rizk (1960). The rate of sulphide production was determined by the number of days incubation required to give a shade of blackening corresponding to 2 fig of sulphide. RESULTS
AND DISCUSSION
Of the 56 soils examined, Table 1 shows that about l/7 of them rapidly oxidized 13-18 per cent of sulphur to sulphate, 2/5 of soils fairly quickly oxidized 4-13 per cent of sulphur, l/3 slowly oxidized 0~5-4 per cent, while l/7 of soils produced no sulphate at all. Only a few of these soils were known to be deficient in sulphur for plants, the majority of
MICROBIAL
TABLE ~.PERCENTOF
Level of sulphate production High Moderate Low None
PRODUCTION
OF
SULPHATE
SOILS(56) SHOWINGDIFFERENTLEVELS
SULPHIDE
OFSULPHATE
Titratable acidity range (ml 0.1 N NaOH per log soil)
Sulphur oxidized (Z) 12417.6 4.2-12.0 1%4~0 r30.8
Composite samples weeks and titratable samples.
AND
775
PRODUCTION
Soils examined (S;)
7.6-i I.0 267.5 062.5 o-O.5
14.3 41.1 30.4 14.3
(100 g) of 56 soils incubated with 1% sulphur at 25°C for IO acidity back to initial soil pH determined weekly on 10 g sub-
them being sufficient. Shortage of total sulphur could thus not be the reason for their low oxidizing ability, but lack of their only sources of energy, elemental sulphur or sulphide, could be involved. Table 2 shows that the activity of 7%. thiooxidans in these soils was high in only about l/7 of cases, moderate in l/4, low in l/3 and nil in l/4. Soils that oxidized sulphur rapidly usually showed high activity of 2%. thiooxidans, although initial counts never exceeded 100 cells/g of soil, as seen in Table 3. Likewise low oxidation rates were associated with absence of Th. th~ooxiduns. Th. thiopurus and 2%. denitr~~c~ns occurred more often in the soils and in slightly higher initial numbers, particularly when detected on thiosulphate rather than sulphur substrate. Counts obtained by dilution to extinction of thiobacilli, either from pure cultures or from inoculated soils, were lower than predicted, so those initially present in these soils are probably underestimated. Nevertheless, paucity of thiobacilli in many Australian soils could explain their inability to rapidly oxidize sulphur.
TABLE 2. PER CENT OF SOILS(56)SHOWING DIFFERENT ACTIVITIESOF Thiohacih thiooxidms
Activity of 7%. ~~i~~J~. High Moderate Low None
Period to reach pH 4.0 (days)
Soils examined (%I
o-21 22242 43-70 > 70
Composite samples (100 g) of 56 soils incubated with 17: sulphur 10 weeks and pH determjned weekly on IO g subsamples.
14.3 25.0 33.9 26.8 at 25°C for
Sulphide production
Table 4 shows that hydrogen sulphide was detected in the atmosphere above the soil after the addition of elemental sulphur in 88 per cent of aerobic soiis and in 36 per cent of waterlogged ones. Either aerobes capable of reducing sulphur are commoner than anaerobes or probably hydrogen sulphide escapes readily from open soil pores. As expected sulphate reduction to sulphide was not detected under aerobic conditions, although aerobes can supposedly protect some anaerobes from oxygen in soil microcrypts. Even under waterlogged conditions only 7 per cent of soils reduced sulphate, suggesting that this process ii
R. J. SWABY
776
AND
RITA
FEDEL
TAHLL 3. PER CENTOF SOILS(56) CONTAINING VARIOUS NUMBERS OF DIFFERENT THIOBACILLI PROD~JCING SULPHATE FROM TWO SUBSTRATESlN LIQLJIIMEDIA
Substrate Sulphur Positive soils found (‘“,I
Species of Thiohacillus
Thiosulphate Positive soils found Counts/g
Th. thiooxidans Th. thioparus
3.6 1.8
IO2 IO2
Th. denitrijicans
3.6
loz
(‘t,) 3.6 I.2 1.X 9.0 1.8
Counts/g IO2 IO2 IO4 IO’ 104
Composite samples of 56 soils examined for thiobacilli by diluting 1 g lo- ‘, 10-4, 10mh into triplicate tubes of elective liquid media, 5 ml for aerobes and 7.5 ml with paraffin seal for anaerobes, incubated at 25’C for 6 weeks, observed at twice weekly intervals. Th. thiooxidans indicated by change in pH from 5.0 to 3.8, Th. /hioparus by change in pH from 6.7 to 4.6, and T/z. denitrijicans by evolution of nitrogen gas inside Durham tube.
rarer than surmised, possibly because (1) suitable microorganisms were sometimes absent, (2) soil pH was sometimes too acidic for the classical sulphate reducers or, more likely, (3) energy sources were lacking. There was no obvious connection between the presence of plants in the soils collected and sulphate reduction, as found by Dommergues, Jacq and Beck (1969) in saline soils. Although cystine was decomposed to sulphide in 48 per cent of friable soils and in 57 per cent of waterlogged ones, it was by no means universally common. Liberation of 35 pg of sulphide from 100 g of soil containing 1 per cent of sulphur within 3 weeks at 25°C was regarded arbitrarily as rapid. However at this rate over a period of I2 months only 0.06 per cent of added supply would.have been lost. From added sulphur, this rate was achieved in 80 per cent of aerated soils and in 30 per cent of waterlogged ones.
TABLE ~.PER CENTOFSOILS (56) PRODUCINGSULPHIIX
Substrate Sulphur
Sulphate
Cystine
Rate of sulphide production High Moderate Low None High Moderate Low None High Moderate Low None
AT DIFFERENT RATES FROM THREE SUBSTRATES AIXXD TO SOIL
Period to blacken lead acetate (days) 0 21 22m42 43-70 > 70 o-2 I 22 42 43 70 > 70 l&21 22-42 43 70 > 70
Per cent of soils examined Aerobic X0.4 5.4 1.X 12.6 0 0 0
IO0 41.1 5.4 I ,x 51.7
Anaerobic 30.4 3.6 54 60.6 7.1 0 0 Y2.Y 4X.2 7. I 1.X 42.9
Composite samples (100 g) of 56 soils. supplemented with ().I”;, sulphur, or 0.3”,; ammonium sulphate plus 0,2”,, sodium sulphate. or 0.5’:,, I-cystine hydrochloride, incubated at 25’C for IO weeks, under crumbly or waterlogged conditions, and number of days recorded to blacken lead acetate paper with 35 /Lg sulphide.
MICROBIAL
PRODUCTION
OF
SULPHATE
AND
777
SULPHIDE
The rate of escape was maintained throughout the incubation period suggesting that even ferruginous soils cannot entirely prevent it. None of the aerobic soils rapidly reduced added sulphate, but 7 per cent of the anaerobic ones did so. After the addition of cystine, sulphide was liberated quickly from 41 per cent of aerated soils and from 48 per cent of anaerobic ones. Escape of hydrogen sulphide depends on soil pH, texture, contents of iron oxide and of water, thereby making comparisons difficult, but dilute suspensions of soil in aqueous media are free from this problem. Table 5 summarizes the percentage of soil suspensions producing sulphide from three substrates in liquid media and also the initial count of organisms involved. All soil suspensions contained some organisms which evolved sulphide from liquid media containing sulphur under both aerobic and anaerobic conditions at 25°C thus revealing how common this process might be in sulphured soils. From TABLE 5. PER CENT OF SOILS (56) CONTAINING VARIOUS NUMBERS OF DIFFERENT GROUPS OF MICROORGANISMS PRODUCtNGSULPHlDE FROMTHREE SUBSTRATESIN LIQUID MEDIA Substrate Sulphurt Positive soils found (“,,)
Sulphate*
Initial counts/g
Positive soils found (%)
Microbial
group
Aerobic Sporing
mesophiles
35.7 554 8.9
lo2 lo4 IO6
5.4
thermophiles
14.3 I.8
IO2 IO4
0
17.9 31.5 356
IO2 IO4 10h
10.7
23.2 7.1
IO2 lo4
0
mesophiles
50.0 33.9 10.7
lo2 lo4 106
5.4
thermophiles
8.9
10’
3.6 33.9 62.5 3.6
Sporing
Non-sporing and sporing mesophiles
Non-sporing and sporing thermophiles
Anaerobic Sporing
Sporing
Non-sporing and sporing mesophiles
Non-sporing and sporing thermophiles
Cystinet
Initial counts/g
lo2
Positive soils found (%)
Initial counts/g
6.0
IO2
2.0
10’
lo2
26.8 25.0 8.9
102 IO4 lo6
10.0 2.0
10Z IO4
10’
24.0 2.0
10’ IO4
1.8
IO2
0 4.0
IO2 104
10’ IO4 10h
5.4
IO2
33.9 250 5.4
lo* lo4 10h
IO2
0
4.0 2.0
lo* lo4
<102
Composite samples of 56 soils, subsamples of 1 g diluted lo-*, lo-“, 10e6, into triplicate tubes of elective liquid media containing 0.6O/, sodium lactate with 0.26?, sulphur, or 0.26’;/ sodium sulphate or 0.01”; 1-cystine. 5 ml medium for aerobes and 7.5 ml with paraffin oil seal for anaerobes. Metallic iron* or lead acetate paper? indicated production of 2 pg sulphide, sporers detected after pasteurizing tubes for 2 min at 9o”C, incubated at 25°C for mesophiles and 55’C for thermophiles.
77x
R. J. SWABY
AND RI-l-A FEDEL
sulphate media, sulphide was detected from suspensions of 11 per cent of soils incubated aerobically and 5 per cent incubated anaerobically with suitable substrate and pH, thus indicating how rare this process could be in Australian soils. At least 60 per cent of all soil suspensions decomposed cystine to sulphide, both aerobically and anaerobically showing that this reaction could be fairly common. With all substrates, mesophiles occurred more often than thermophiles in the soil suspensions, usually in higher initial counts. Non-sporing organisms of each class commonly occurred in greater numbers than sporers. More detailed examination of the data revealed that in sulphur media under aerobic conditions 9 per cent of the soils gave the highest initial counts of lo6 cells/g for sporing mesophiles, 2 per cent gave counts of 10” cells/g of sporing thermophiles, 36 per cent showed 10h/g for non-sporing plus sporing mesophiles, and 7 per cent contained 104/g non-sporing plus sporing thermophiles. In the same media under anaerobic conditions 11 per cent of the soils revealed the highest initial numbers of lO’/g sporing mesophiles, 9 per cent contained lO’/g sporing thermophiles, 63 per cent reached lO”/g non-sporing plus sporing mesophiles, but only 4 per cent showed lO’/g non-sporing plus sporing thermophiles. Numerous genera were represented in the isolates from both aerobic and anaerobic cultures, capable of reducing elemental sulphur, as found by Starkey (1937), but particularly De.sulfooihrio and Desu~fbtomuculum. In sulphate media, under aerobic conditions, only 5 per cent of soils had initial counts of lO’/g sporing mesophiles; 11 per cent of soils showed lO’/g non-sporing plus sporing mesophiles, but none revealed any thermoplliles. In the same sulphate media, incubated anaerobically, rather similar low counts were obtained. Only Desu@ihrio species were detected amongst the isolates suggesting that Drsu~fotomuc.ulum may not be a common sulphate reducer in soil, although it appears to be able to reduce elemental sulphur. In cystine media, under aerobic conditions, only 6 per cent of soils contained lO’/g sporing mesophiles, 2 per cent gave counts of IO’/g sporing thermophiles, but non-sporing plus sporing mesophiles were often encountered so that 9 per cent of soils showed maximum counts of lob/g, while only 2 per cent of soils revealed 104/g of less common non-sporing plus sporing thermophiles. In the same media, incubated anaerobically, the incidence and counts of the four microbial groups approximated those cultivated aerobically, thus suggesting that probably facultative anaerobes are mainly responsible for the production of hydrogen s~llphide from cystine. Unlike isolates from other media they usually lost the ability to produce sulphide on cystine-ferrous sulphate agar after one subculture but remained viable, suggesting that synergic pairs sometimes may have been necessary. No way wasfound for restoring this reaction to pure isolates including soil passage and cultivation on various media. Although numerous colony types were isolated suggesting that many genera were involved, further classification was not possible. Table 6 summarizes the rate of liberation of sulphide from three substrates in liquid media by the various microbial groups occurring in the soil suspensions. When 2 pg of sulphide were liberated within 6 days it was arbitrarily rated as high, between 6 and 10 days as moderate, between 10 and 14 days as low and > 14 days as nil. Sulphide was produced rapidly from sulphur (I) most frequently by anaerobic, non-sporing plus sporing mesophiles in 80 per cent of soils, (2) less often by aerobic, sporing mesophiles in 70 per cent of cases, (3) by anaerobic sporing mesophiles in 50 per cent of cases, (4) by aerobic, non-sporing plus sporing mesophiles in 32 per cent of soils and finally (5) by the remaining four microbialgroupsin less than 13 per cent of cases, thus emphasizing the relative unimportance of thermophiles. Unlike sulphur, sulphate was rarely reduced rapidly by any of the four mesophilic microbial groups, and never by any thermophiles. From cystine, sulphide was
MICROBIAL
PRODUCTION
OF
SULPHATE
TABLE 6. PER CENT OF SOILS WITH DIFFERENT MICROORGANISMS RATESFROMTHREESUBSTRATESIN
AND
SULPHIDE
PRODUCING HYDROGEN LIQUID MEDIA
119 SULPHIDE AT VARIOUS
Per cent of soils producingsulphide Rate of
Microbial
group
blackening
Aerobic Sporing
mesophiles
High Moderate Low None
69.6 23.2 3.6 3.6
thermophiles
High Moderate Low None
8.9 1.8 89.3
High Moderate Low None
32. I 35.7 14.3 17.9
High Moderate Low None
12.5 1.8 1.8 83.9
mesophiles
High Moderate Low None
50.0 23.2 8.9 17.9
I.8 1.8 0 96.4
4.0 8.0 2.0 86.0
thermophiles
High Moderate Low None
0
0 0 0
2.0 0 2.0 96.0
Sporing
Non-sporing and sporing mesophiles
Non-sporing and sporing thermophiles
Anaerobic Sporing
Sporing
Non-sporing and sporing mesophiles
Non-sporing and sporing thermophiles
Sulphur
3.6 0 96.4
High Moderate Low None
80.3
High Moderate Low None
3.6 0 0 96.4
14.3 0 5.4
Sulphate
Cystine
I.8 1.8 1.8 94.6
2.0 2.0 0 96.0
0 0 0 100
0 0 0 100
3.6 3.6 0 92.8
25.0 8.9 8.9 54.2
0 0 0 100
0 0 0 100
100 1.8 1.8 1.8 94.6 0 0 0 100
17.9 3.6 8.9 69.6
-
0 0 0 100
Composite samples of 56 soils, subsamples of 1 g diluted toe2 into triplicate tubes of elective liquid media containing 0.6% sodium lactate with 0.26:; sulphur, or 0.26’:/, sodium sulphate or 0,01$/i I-cystine, 5 ml medium for aerobes and 7.5 ml with paraffin oil seal for anaerobes, sporers detected after heating for 2 min at 90°C. incubated at 25°C for mesophiles and 55°C for thermophiles. Blackening of metallic iron or lead acetate paper indicated production of 2 pg sulphide. high rate G6 days, moderate 6-10 days, low l&14 days, none > 14 days.
produced at high rates most frequently by aerobic, non-sporing plus sporing mesophiles in 25 per cent of soils, less often by anaerobic, non-sporing plus sporing mesophiles in 18 per cent of cases, next by anaerobic and by aerobic sporing mesophiles in 4 and 2 per cent respectively, and rarely by the remaining four thermophilic groups in O-2 per cent of soils. Indeed thermophiles commonly produced no sulphide at all from cystine, this being the function of mesophiles under both aerobic and anaerobic conditions.
780
R. J. SWABY
AND RITA
FEDEL
Attempts were made by plotting scatter diagrams to correlate soil properties, such as Great Soil Group, texture and pH, with the incidence of metabolic products, such as sulphate from sulphur, sulphide from sulphur, sulphate from cystine in soils, and with the incidence of microorganisms involved. In no cases were correlations sufficiently obvious to warrant calculating correlation coefficients, but a number of tendencies were noted. Thus (1) of the Great Soil Groups, Solonized Brown and Gray Clay, showed no activity of Th. thioo?tidans, (2) heavy textured soils showed lower activity of Th. thiooxidans, (3) clayey soils evolved hydrogen sulphide more slowly from sulphur than sandy ones, (4) sulphate reduction occurred so rarely that correlations could not be tested, (5) light soils evolved hydrogen sulphide aerobically from cystine more slowly than heavy textured soils, and (6) light soils produced hydrogen sulphide anaerobically from cystine more rapidly than heavy soils. Correlations between oxidation and reduction metabolic products and the microorganisms involved were non significant. However, (1) under aerobic conditions, but not anaerobic, there was a tendency for soils with low activity of Th. thiooxidans also to show low reduction of sulphur to sulphide, (2) in general microbial oxidation of sulphur compounds seemed to be independent of reduction. This was surprising since aerobic, autotrophic species of thiobacilli depend on sulphur and sulphide produced by various heterotrophic organisms. If sulphureta conditions exist in soil crumbs as observed by BaasBecking (1925) in muds, with interdependence of sulphur oxidation on the surface and reduction in the interior, then they were not obvious from the data. This might be because dissimilatory reduction of sulphate appears to be rare in Australian soils. It is also likely that hydrogen sulphide produced from sulphur compounds is often chemically rather than microbially oxidized to sulphate. Autotrophic species of Thiohacillus and sulphate-reducing DesuIfouihrio were rare enough to be considered opportunistic bacteria, remaining dormant until stimulated by proper conditions. When comparing sulphur oxidation in soils or in liquid media with the incidence of various thiobacilli again there were no significant correlations. However, soils with low sulphur-oxidizing activity tended to contain no Th. thiooxidans, whereas those with high activity contained this species, but many anomalies occurred. Counting techniques for thiobacilli are inadequate since small numbers are sometimes found when sulphur oxidation is rapid. Yet most associated heterotrophic bacteria when isolated and tested oxidize sulphur only slowly. By comparing sulphide production from three substrates added to soils or to liquid media with the occurrence of various classes of organisms a few tendencies were noticed. Thus, (1) under all conditions sulphide was produced more by sporing and non-sporing mesophiles than by thermophiles, (2) there was a better correlation between evolution of sulphide from soils and evolution from liquid cultures under aerobic than anaerobic conditions, and (3) counts of each class of organisms were better correlated with rates of evolution of hydrogen sulphide from liquid media than from soils where diffusion of gas was restricted. REFERENCES RILK S. G. (1960) ~i1I111rc of Dcslr//,/lol,ih,fo t/csu/pl~ur,i~rr,is. !V’trlm,. Loilt/. 185, 635 636. BAALSKUII K. and BAALSKUII K. S. (1954) Thiobucillus drnirrificuus. Arch. Mikrohiol. 20, 3462. BAAS-BFCKIYG L. G. M. (1925) Studies on the sulphur bacteria. Ann. Bat. Land. 39, 613-650. BAKIWW N. J. (I 960) A comparison of the mineralization of nitrogen and of sulphur from decomposing organic matcriala. ,,t~t.st.J. trqrcc. RPS. 11, 960 969. BAKROW N. J. (1971) Slowly available sulphur fcrtilixrs in bouth-western Australia. I. Elemental sulphur. ,.lusr. J. CJYP.Ayric. und Ani,,,. Hush. 11, 21 I-216.
AIM)-1.1..MAI.1.h and
MICROBIAL
PRODUCTION
OF SULPHATE
AND SULPHIDE
781
BROMFIELD S. M. (1953) Sulphate reduction in partially sterilized soil exposed to air. J. gm. Microhiol. 8, 378-390. DOMMERGUES Y., JACQ V. and BECK GENEVI&. (1969) Influence de l’engorgement sur la sulfato-rtduction rhizosphtrique dans un sol salin. C.R. Acad. Sci. Paris. X8,605-608. FREDERICKL. R., STARKEYR. L. and SEGALW. (1957) Decomposabiiity of some organic sulphur compounds in soil. SoiE Sci. Sue. Am. Proc. 21, 287-292. FRENEYJ. R. (1960) The oxidation of cysteine to sulphate in soil. Atrst. J. hioi. Sci. 13, 387-392. FRENEYJ. R. (1961) Some observations on the nature of organic sulphur compounds in soil. Aust. J. agric. Rrs. 12,424-432. POSTGATE J. R. (1966) Media for sulphur bacteria. Lab. Pracr. 15, 1239-1244. STARKEYR. L. (1937) Formation of sulphide by some sulphur bacteria. J. Butt. 33, 545-57 1. STARKEYR. L. (1966) Oxidation and reduction of sulphur compounds in soils. Soil Sci. 101,297 -306. SWABYR. J. and VIT#LINSMAIJAI. (1968) Sulphur oxidation in Australian soils. ~~u~s~~fj~~s Ninth ~~~~~~ff~~u~z~~ Congrrss qf Soil Scirncr, Adelaide. 4,673~68 1. VISHNIACW. and SANTERM. (1957) The thiobacilli. Butt. Rec. 21, 195-213. VITOL~NS MAIJAI. and SWABYR. J. (1968) Activity of sulphur-oxidizing microorganisms in some Australian soils. Aust. J. Soil Res. 7, 171-183.
MICROBIAL
PergamonPress 1973. Printed m GreatBritm
PRODUCTION OF SULPHATE AND SULPHIDE IN SOME AUSTRALIAN SOILS R. J. SWABY C.S.I.R.O.,
Division
and RITA FEDEL
of Soils, Glen Osmond, (Accepted
17 April
South Australia
5064
1973)
Summary-Fifty-six Australian soils were examined for their ability to produce sulphate from sulphur and sulphide from added sulphur, sulphate and cystine. Selective liquid media were used to obtain initial counts and progressive activities of some of the principal organisms involved. Attempts were made to correlate soil properties, initial microbial counts, classes of microorganisms detected and metabolic activities. Almost half of the soils oxidized sulphur very slowly or not at all, most probably due to absence of thiobacilli, particularly 7%. thiooxidans. Even soils which oxidized sulphur rapidly never gave initial counts greater than 10“ cells/g. The majority of soils produced sulphide from sulphur, between one-half and two-thirds of them formed sulphide from cystine but sulphate reduction was very rare. Sulphide escaped more readily from aerobic than from waterlogged soils. Numerous genera were isolated including Drsulfocibrio and Desulfatomaculum capable of reducing sulphur, both aerobically and anaerobically, particularly sporing and non-sporing mesophiles which were sometimes present at 10h cells/g of soil. Sulphate reduction by the genus Desulfovibrio was uncommon, and by the genus DesuIfatomaculum extremely rare. Many genera of sporing and non-sporing mesophiles seemed capable of producing sulphide from cystine when first isolated, but they quickly lost this ability on subculturing. Initial counts of 1Oh cells/g of soil were occasionally found but usually much lower. No significant correlations, only trends, were found suggesting that any soil properties measured affected the occurrence or activities of organisms capable of producing sulphate or sulphide. INTRODUCTION SULPHUR deficiencies are widespread in Australian soils, necessitating the use of elemental sulphur or gypsum, yet little is known of the inorganic sulphur cycle in these soils, or of the microorganisms involved. In a survey of 273 soils from eastern and southern Australia, Swaby and Vitolins (1968) and Vitolins and Swaby (1968) found that 1 per cent of sulphur added to soil oxidized either slowly to sulphate or not at all in almost half of them. However Barrow (1971) in Western Australia reported higher percentages of sulphur oxidized at lower concentrations of sulphur. Autotrophic thiobacilli were more active oxidizers of sulphur than other organisms, particularly Thiobacillus thiooxidans, but the latter was rare in or absent from more than half of these soils. Many Australian soils in the temperate regions differ therefore from others examined elsewhere (cf. Starkey, 1966). Sulphate may be formed also from cysteine via sulphide, but Freney (1960) showed that it may be formed directly in aerated soils. The rate of release of sulphate from plant materials depends on the content of sulphur, the C/S ratio and often the N/S ratio, according to Barrow (1960). Freney (1961) considers that possibly ester sulphates in humus are resistant to enzymic hydrolysis, so are poor suppliers of sulphate. It is usually considered that dissimilatory, anaerobic sulphate reducers of the genera Desuljovibrio and Desulfotomaculum, are responsible for sulphide formation in waterlogged soils; but Bromfield (1953) found Bacillus megaterium capable of producing free sulphide from sulphate in partially sterilized soil. 173
114
R. J. SWABY
AND RI-l-A FEDEL
These few references summarize nearly all that is known of microbial production of sulphate and sulphide in Australian soils. In this paper 56 soils were surveyed for their ability to produce sulphate and sulphide from a number of sulphur compounds added simultaneously. Attempts were also made to estimate the initial numbers or activities of the principal sulphur organisms present to see if they were interdependent. MATERIALS
AND METHODS
Fifty-six soils mainly from eastern and southern Australia were examined including seven Solonized Brown, five Gray Clay, five Solodized Solonetz and Solodic, four Soloth and Prairie, six Red Brown Earth, one Brown Earth, six Red Earth, seven Red Podzolic, 10 Yellow Podzolic and two Podzolic. Soils were sampled from 0 to 10 cm, five cores being combined from sites 10 m apart, then refrigerated at 2°C until examined simultaneously under both friable and waterlogged conditions for their ability to oxidize 1.0 per cent added elemental sulphur to sulphate or to reduce to sulphide (a) 0.1% elemental sulphur, (b) a mixture of 0*30/,ammonium sulphate and 0*2:/dsodium sulphate and (c) O+o/;, cystine hydrochloride, since Frederick, Starkey and Segal(l957) found this organic sulphur compound readily decomposed. At weekly intervals over a period of 10 weeks incubation at 25”C, sulphur oxidation was measured by the drop in pH and the rise of titratable acidity in log aliquots of soil. The activity of Th. thiooxidans was determined approximately by the number of days for soil pH to reach 4.0. Free sulphide was detected by inserting lead acetate paper discs, 5.5 cm dia, in the lids of jars. The number of days was noted to produce a shade of blackening corresponding to 35 gg of sulphide. In addition, suitable dilutions of each soil were added to elective media to estimate initial counts of sulphur organisms by dilution to extinction. Vishniac and Santer’s (1957) liquid inorganic media containing @lx sulphur or O-5’)/;,sodium thiosulphate were used for thiobacilli. Th. t~iuoxida~s was considered present when @005 per cent added brom phenol blue indicated that the initial pH of 5-Ohad dropped to 4-Oand 7%. t~7iopar~swhen 0.005 per cent added brom cresol green indicated a pH change from 6.7 to 4.6. Th. dentitri$cans was detected by Baalsrud and Baalsrud’s (1954) method of trapping nitrogen gas from nitrate medium under a paraffin oil seal. Sulphide producers were detected in modified Postgate’s (1966) liquid medium containing 0.1% sulphur or 0.26% sodium sulphate or @Oloi:,cystine as the only sulphur compounds and no added ferrous salt. Aerobes were grown in tubes, l-5 cm dia, containing 5-Oml of medium whereas anaerobes were cultured in 7.5 ml of medium sealed with 1.5 ml ofparaffin oil. Spores were detected after pasteurizing soil suspensions for 2 min at 90°C. Mesophiles were incubated at 25°C and thermophiles at 55°C. Sulphide was detected by inserting lead acetate paper discs, 1.3 cm dia in the loose screw caps of tubes 1.5 mm x 10 mm containing sulphur and cystine media or by adding iron nails to sulphate media, 3 cm Long and 1.2 mm dia. as proposed by Abd-el-~al~k and Rizk (1960). The rate of sulphide production was determined by the number of days incubation required to give a shade of blackening corresponding to 2 fig of sulphide. RESULTS
AND DISCUSSION
Of the 56 soils examined, Table 1 shows that about l/7 of them rapidly oxidized 13-18 per cent of sulphur to sulphate, 2/5 of soils fairly quickly oxidized 4-13 per cent of sulphur, l/3 slowly oxidized 0~5-4 per cent, while l/7 of soils produced no sulphate at all. Only a few of these soils were known to be deficient in sulphur for plants, the majority of
MICROBIAL
TABLE ~.PERCENTOF
Level of sulphate production High Moderate Low None
PRODUCTION
OF
SULPHATE
SOILS(56) SHOWINGDIFFERENTLEVELS
SULPHIDE
OFSULPHATE
Titratable acidity range (ml 0.1 N NaOH per log soil)
Sulphur oxidized (Z) 12417.6 4.2-12.0 1%4~0 r30.8
Composite samples weeks and titratable samples.
AND
775
PRODUCTION
Soils examined (S;)
7.6-i I.0 267.5 062.5 o-O.5
14.3 41.1 30.4 14.3
(100 g) of 56 soils incubated with 1% sulphur at 25°C for IO acidity back to initial soil pH determined weekly on 10 g sub-
them being sufficient. Shortage of total sulphur could thus not be the reason for their low oxidizing ability, but lack of their only sources of energy, elemental sulphur or sulphide, could be involved. Table 2 shows that the activity of 7%. thiooxidans in these soils was high in only about l/7 of cases, moderate in l/4, low in l/3 and nil in l/4. Soils that oxidized sulphur rapidly usually showed high activity of 2%. thiooxidans, although initial counts never exceeded 100 cells/g of soil, as seen in Table 3. Likewise low oxidation rates were associated with absence of Th. th~ooxiduns. Th. thiopurus and 2%. denitr~~c~ns occurred more often in the soils and in slightly higher initial numbers, particularly when detected on thiosulphate rather than sulphur substrate. Counts obtained by dilution to extinction of thiobacilli, either from pure cultures or from inoculated soils, were lower than predicted, so those initially present in these soils are probably underestimated. Nevertheless, paucity of thiobacilli in many Australian soils could explain their inability to rapidly oxidize sulphur.
TABLE 2. PER CENT OF SOILS(56)SHOWING DIFFERENT ACTIVITIESOF Thiohacih thiooxidms
Activity of 7%. ~~i~~J~. High Moderate Low None
Period to reach pH 4.0 (days)
Soils examined (%I
o-21 22242 43-70 > 70
Composite samples (100 g) of 56 soils incubated with 17: sulphur 10 weeks and pH determjned weekly on IO g subsamples.
14.3 25.0 33.9 26.8 at 25°C for
Sulphide production
Table 4 shows that hydrogen sulphide was detected in the atmosphere above the soil after the addition of elemental sulphur in 88 per cent of aerobic soiis and in 36 per cent of waterlogged ones. Either aerobes capable of reducing sulphur are commoner than anaerobes or probably hydrogen sulphide escapes readily from open soil pores. As expected sulphate reduction to sulphide was not detected under aerobic conditions, although aerobes can supposedly protect some anaerobes from oxygen in soil microcrypts. Even under waterlogged conditions only 7 per cent of soils reduced sulphate, suggesting that this process ii
R. J. SWABY
776
AND
RITA
FEDEL
TAHLL 3. PER CENTOF SOILS(56) CONTAINING VARIOUS NUMBERS OF DIFFERENT THIOBACILLI PROD~JCING SULPHATE FROM TWO SUBSTRATESlN LIQLJIIMEDIA
Substrate Sulphur Positive soils found (‘“,I
Species of Thiohacillus
Thiosulphate Positive soils found Counts/g
Th. thiooxidans Th. thioparus
3.6 1.8
IO2 IO2
Th. denitrijicans
3.6
loz
(‘t,) 3.6 I.2 1.X 9.0 1.8
Counts/g IO2 IO2 IO4 IO’ 104
Composite samples of 56 soils examined for thiobacilli by diluting 1 g lo- ‘, 10-4, 10mh into triplicate tubes of elective liquid media, 5 ml for aerobes and 7.5 ml with paraffin seal for anaerobes, incubated at 25’C for 6 weeks, observed at twice weekly intervals. Th. thiooxidans indicated by change in pH from 5.0 to 3.8, Th. /hioparus by change in pH from 6.7 to 4.6, and T/z. denitrijicans by evolution of nitrogen gas inside Durham tube.
rarer than surmised, possibly because (1) suitable microorganisms were sometimes absent, (2) soil pH was sometimes too acidic for the classical sulphate reducers or, more likely, (3) energy sources were lacking. There was no obvious connection between the presence of plants in the soils collected and sulphate reduction, as found by Dommergues, Jacq and Beck (1969) in saline soils. Although cystine was decomposed to sulphide in 48 per cent of friable soils and in 57 per cent of waterlogged ones, it was by no means universally common. Liberation of 35 pg of sulphide from 100 g of soil containing 1 per cent of sulphur within 3 weeks at 25°C was regarded arbitrarily as rapid. However at this rate over a period of I2 months only 0.06 per cent of added supply would.have been lost. From added sulphur, this rate was achieved in 80 per cent of aerated soils and in 30 per cent of waterlogged ones.
TABLE ~.PER CENTOFSOILS (56) PRODUCINGSULPHIIX
Substrate Sulphur
Sulphate
Cystine
Rate of sulphide production High Moderate Low None High Moderate Low None High Moderate Low None
AT DIFFERENT RATES FROM THREE SUBSTRATES AIXXD TO SOIL
Period to blacken lead acetate (days) 0 21 22m42 43-70 > 70 o-2 I 22 42 43 70 > 70 l&21 22-42 43 70 > 70
Per cent of soils examined Aerobic X0.4 5.4 1.X 12.6 0 0 0
IO0 41.1 5.4 I ,x 51.7
Anaerobic 30.4 3.6 54 60.6 7.1 0 0 Y2.Y 4X.2 7. I 1.X 42.9
Composite samples (100 g) of 56 soils. supplemented with ().I”;, sulphur, or 0.3”,; ammonium sulphate plus 0,2”,, sodium sulphate. or 0.5’:,, I-cystine hydrochloride, incubated at 25’C for IO weeks, under crumbly or waterlogged conditions, and number of days recorded to blacken lead acetate paper with 35 /Lg sulphide.
MICROBIAL
PRODUCTION
OF
SULPHATE
AND
777
SULPHIDE
The rate of escape was maintained throughout the incubation period suggesting that even ferruginous soils cannot entirely prevent it. None of the aerobic soils rapidly reduced added sulphate, but 7 per cent of the anaerobic ones did so. After the addition of cystine, sulphide was liberated quickly from 41 per cent of aerated soils and from 48 per cent of anaerobic ones. Escape of hydrogen sulphide depends on soil pH, texture, contents of iron oxide and of water, thereby making comparisons difficult, but dilute suspensions of soil in aqueous media are free from this problem. Table 5 summarizes the percentage of soil suspensions producing sulphide from three substrates in liquid media and also the initial count of organisms involved. All soil suspensions contained some organisms which evolved sulphide from liquid media containing sulphur under both aerobic and anaerobic conditions at 25°C thus revealing how common this process might be in sulphured soils. From TABLE 5. PER CENT OF SOILS (56) CONTAINING VARIOUS NUMBERS OF DIFFERENT GROUPS OF MICROORGANISMS PRODUCtNGSULPHlDE FROMTHREE SUBSTRATESIN LIQUID MEDIA Substrate Sulphurt Positive soils found (“,,)
Sulphate*
Initial counts/g
Positive soils found (%)
Microbial
group
Aerobic Sporing
mesophiles
35.7 554 8.9
lo2 lo4 IO6
5.4
thermophiles
14.3 I.8
IO2 IO4
0
17.9 31.5 356
IO2 IO4 10h
10.7
23.2 7.1
IO2 lo4
0
mesophiles
50.0 33.9 10.7
lo2 lo4 106
5.4
thermophiles
8.9
10’
3.6 33.9 62.5 3.6
Sporing
Non-sporing and sporing mesophiles
Non-sporing and sporing thermophiles
Anaerobic Sporing
Sporing
Non-sporing and sporing mesophiles
Non-sporing and sporing thermophiles
Cystinet
Initial counts/g
lo2
Positive soils found (%)
Initial counts/g
6.0
IO2
2.0
10’
lo2
26.8 25.0 8.9
102 IO4 lo6
10.0 2.0
10Z IO4
10’
24.0 2.0
10’ IO4
1.8
IO2
0 4.0
IO2 104
10’ IO4 10h
5.4
IO2
33.9 250 5.4
lo* lo4 10h
IO2
0
4.0 2.0
lo* lo4
<102
Composite samples of 56 soils, subsamples of 1 g diluted lo-*, lo-“, 10e6, into triplicate tubes of elective liquid media containing 0.6O/, sodium lactate with 0.26?, sulphur, or 0.26’;/ sodium sulphate or 0.01”; 1-cystine. 5 ml medium for aerobes and 7.5 ml with paraffin oil seal for anaerobes. Metallic iron* or lead acetate paper? indicated production of 2 pg sulphide, sporers detected after pasteurizing tubes for 2 min at 9o”C, incubated at 25°C for mesophiles and 55’C for thermophiles.
77x
R. J. SWABY
AND RI-l-A FEDEL
sulphate media, sulphide was detected from suspensions of 11 per cent of soils incubated aerobically and 5 per cent incubated anaerobically with suitable substrate and pH, thus indicating how rare this process could be in Australian soils. At least 60 per cent of all soil suspensions decomposed cystine to sulphide, both aerobically and anaerobically showing that this reaction could be fairly common. With all substrates, mesophiles occurred more often than thermophiles in the soil suspensions, usually in higher initial counts. Non-sporing organisms of each class commonly occurred in greater numbers than sporers. More detailed examination of the data revealed that in sulphur media under aerobic conditions 9 per cent of the soils gave the highest initial counts of lo6 cells/g for sporing mesophiles, 2 per cent gave counts of 10” cells/g of sporing thermophiles, 36 per cent showed 10h/g for non-sporing plus sporing mesophiles, and 7 per cent contained 104/g non-sporing plus sporing thermophiles. In the same media under anaerobic conditions 11 per cent of the soils revealed the highest initial numbers of lO’/g sporing mesophiles, 9 per cent contained lO’/g sporing thermophiles, 63 per cent reached lO”/g non-sporing plus sporing mesophiles, but only 4 per cent showed lO’/g non-sporing plus sporing thermophiles. Numerous genera were represented in the isolates from both aerobic and anaerobic cultures, capable of reducing elemental sulphur, as found by Starkey (1937), but particularly De.sulfooihrio and Desu~fbtomuculum. In sulphate media, under aerobic conditions, only 5 per cent of soils had initial counts of lO’/g sporing mesophiles; 11 per cent of soils showed lO’/g non-sporing plus sporing mesophiles, but none revealed any thermoplliles. In the same sulphate media, incubated anaerobically, rather similar low counts were obtained. Only Desu@ihrio species were detected amongst the isolates suggesting that Drsu~fotomuc.ulum may not be a common sulphate reducer in soil, although it appears to be able to reduce elemental sulphur. In cystine media, under aerobic conditions, only 6 per cent of soils contained lO’/g sporing mesophiles, 2 per cent gave counts of IO’/g sporing thermophiles, but non-sporing plus sporing mesophiles were often encountered so that 9 per cent of soils showed maximum counts of lob/g, while only 2 per cent of soils revealed 104/g of less common non-sporing plus sporing thermophiles. In the same media, incubated anaerobically, the incidence and counts of the four microbial groups approximated those cultivated aerobically, thus suggesting that probably facultative anaerobes are mainly responsible for the production of hydrogen s~llphide from cystine. Unlike isolates from other media they usually lost the ability to produce sulphide on cystine-ferrous sulphate agar after one subculture but remained viable, suggesting that synergic pairs sometimes may have been necessary. No way wasfound for restoring this reaction to pure isolates including soil passage and cultivation on various media. Although numerous colony types were isolated suggesting that many genera were involved, further classification was not possible. Table 6 summarizes the rate of liberation of sulphide from three substrates in liquid media by the various microbial groups occurring in the soil suspensions. When 2 pg of sulphide were liberated within 6 days it was arbitrarily rated as high, between 6 and 10 days as moderate, between 10 and 14 days as low and > 14 days as nil. Sulphide was produced rapidly from sulphur (I) most frequently by anaerobic, non-sporing plus sporing mesophiles in 80 per cent of soils, (2) less often by aerobic, sporing mesophiles in 70 per cent of cases, (3) by anaerobic sporing mesophiles in 50 per cent of cases, (4) by aerobic, non-sporing plus sporing mesophiles in 32 per cent of soils and finally (5) by the remaining four microbialgroupsin less than 13 per cent of cases, thus emphasizing the relative unimportance of thermophiles. Unlike sulphur, sulphate was rarely reduced rapidly by any of the four mesophilic microbial groups, and never by any thermophiles. From cystine, sulphide was
MICROBIAL
PRODUCTION
OF
SULPHATE
TABLE 6. PER CENT OF SOILS WITH DIFFERENT MICROORGANISMS RATESFROMTHREESUBSTRATESIN
AND
SULPHIDE
PRODUCING HYDROGEN LIQUID MEDIA
119 SULPHIDE AT VARIOUS
Per cent of soils producingsulphide Rate of
Microbial
group
blackening
Aerobic Sporing
mesophiles
High Moderate Low None
69.6 23.2 3.6 3.6
thermophiles
High Moderate Low None
8.9 1.8 89.3
High Moderate Low None
32. I 35.7 14.3 17.9
High Moderate Low None
12.5 1.8 1.8 83.9
mesophiles
High Moderate Low None
50.0 23.2 8.9 17.9
I.8 1.8 0 96.4
4.0 8.0 2.0 86.0
thermophiles
High Moderate Low None
0
0 0 0
2.0 0 2.0 96.0
Sporing
Non-sporing and sporing mesophiles
Non-sporing and sporing thermophiles
Anaerobic Sporing
Sporing
Non-sporing and sporing mesophiles
Non-sporing and sporing thermophiles
Sulphur
3.6 0 96.4
High Moderate Low None
80.3
High Moderate Low None
3.6 0 0 96.4
14.3 0 5.4
Sulphate
Cystine
I.8 1.8 1.8 94.6
2.0 2.0 0 96.0
0 0 0 100
0 0 0 100
3.6 3.6 0 92.8
25.0 8.9 8.9 54.2
0 0 0 100
0 0 0 100
100 1.8 1.8 1.8 94.6 0 0 0 100
17.9 3.6 8.9 69.6
-
0 0 0 100
Composite samples of 56 soils, subsamples of 1 g diluted toe2 into triplicate tubes of elective liquid media containing 0.6% sodium lactate with 0.26:; sulphur, or 0.26’:/, sodium sulphate or 0,01$/i I-cystine, 5 ml medium for aerobes and 7.5 ml with paraffin oil seal for anaerobes, sporers detected after heating for 2 min at 90°C. incubated at 25°C for mesophiles and 55°C for thermophiles. Blackening of metallic iron or lead acetate paper indicated production of 2 pg sulphide. high rate G6 days, moderate 6-10 days, low l&14 days, none > 14 days.
produced at high rates most frequently by aerobic, non-sporing plus sporing mesophiles in 25 per cent of soils, less often by anaerobic, non-sporing plus sporing mesophiles in 18 per cent of cases, next by anaerobic and by aerobic sporing mesophiles in 4 and 2 per cent respectively, and rarely by the remaining four thermophilic groups in O-2 per cent of soils. Indeed thermophiles commonly produced no sulphide at all from cystine, this being the function of mesophiles under both aerobic and anaerobic conditions.
780
R. J. SWABY
AND RITA
FEDEL
Attempts were made by plotting scatter diagrams to correlate soil properties, such as Great Soil Group, texture and pH, with the incidence of metabolic products, such as sulphate from sulphur, sulphide from sulphur, sulphate from cystine in soils, and with the incidence of microorganisms involved. In no cases were correlations sufficiently obvious to warrant calculating correlation coefficients, but a number of tendencies were noted. Thus (1) of the Great Soil Groups, Solonized Brown and Gray Clay, showed no activity of Th. thioo?tidans, (2) heavy textured soils showed lower activity of Th. thiooxidans, (3) clayey soils evolved hydrogen sulphide more slowly from sulphur than sandy ones, (4) sulphate reduction occurred so rarely that correlations could not be tested, (5) light soils evolved hydrogen sulphide aerobically from cystine more slowly than heavy textured soils, and (6) light soils produced hydrogen sulphide anaerobically from cystine more rapidly than heavy soils. Correlations between oxidation and reduction metabolic products and the microorganisms involved were non significant. However, (1) under aerobic conditions, but not anaerobic, there was a tendency for soils with low activity of Th. thiooxidans also to show low reduction of sulphur to sulphide, (2) in general microbial oxidation of sulphur compounds seemed to be independent of reduction. This was surprising since aerobic, autotrophic species of thiobacilli depend on sulphur and sulphide produced by various heterotrophic organisms. If sulphureta conditions exist in soil crumbs as observed by BaasBecking (1925) in muds, with interdependence of sulphur oxidation on the surface and reduction in the interior, then they were not obvious from the data. This might be because dissimilatory reduction of sulphate appears to be rare in Australian soils. It is also likely that hydrogen sulphide produced from sulphur compounds is often chemically rather than microbially oxidized to sulphate. Autotrophic species of Thiohacillus and sulphate-reducing DesuIfouihrio were rare enough to be considered opportunistic bacteria, remaining dormant until stimulated by proper conditions. When comparing sulphur oxidation in soils or in liquid media with the incidence of various thiobacilli again there were no significant correlations. However, soils with low sulphur-oxidizing activity tended to contain no Th. thiooxidans, whereas those with high activity contained this species, but many anomalies occurred. Counting techniques for thiobacilli are inadequate since small numbers are sometimes found when sulphur oxidation is rapid. Yet most associated heterotrophic bacteria when isolated and tested oxidize sulphur only slowly. By comparing sulphide production from three substrates added to soils or to liquid media with the occurrence of various classes of organisms a few tendencies were noticed. Thus, (1) under all conditions sulphide was produced more by sporing and non-sporing mesophiles than by thermophiles, (2) there was a better correlation between evolution of sulphide from soils and evolution from liquid cultures under aerobic than anaerobic conditions, and (3) counts of each class of organisms were better correlated with rates of evolution of hydrogen sulphide from liquid media than from soils where diffusion of gas was restricted. REFERENCES RILK S. G. (1960) ~i1I111rc of Dcslr//,/lol,ih,fo t/csu/pl~ur,i~rr,is. !V’trlm,. Loilt/. 185, 635 636. BAALSKUII K. and BAALSKUII K. S. (1954) Thiobucillus drnirrificuus. Arch. Mikrohiol. 20, 3462. BAAS-BFCKIYG L. G. M. (1925) Studies on the sulphur bacteria. Ann. Bat. Land. 39, 613-650. BAKIWW N. J. (I 960) A comparison of the mineralization of nitrogen and of sulphur from decomposing organic matcriala. ,,t~t.st.J. trqrcc. RPS. 11, 960 969. BAKROW N. J. (1971) Slowly available sulphur fcrtilixrs in bouth-western Australia. I. Elemental sulphur. ,.lusr. J. CJYP.Ayric. und Ani,,,. Hush. 11, 21 I-216.
AIM)-1.1..MAI.1.h and
MICROBIAL
PRODUCTION
OF SULPHATE
AND SULPHIDE
781
BROMFIELD S. M. (1953) Sulphate reduction in partially sterilized soil exposed to air. J. gm. Microhiol. 8, 378-390. DOMMERGUES Y., JACQ V. and BECK GENEVI&. (1969) Influence de l’engorgement sur la sulfato-rtduction rhizosphtrique dans un sol salin. C.R. Acad. Sci. Paris. X8,605-608. FREDERICKL. R., STARKEYR. L. and SEGALW. (1957) Decomposabiiity of some organic sulphur compounds in soil. SoiE Sci. Sue. Am. Proc. 21, 287-292. FRENEYJ. R. (1960) The oxidation of cysteine to sulphate in soil. Atrst. J. hioi. Sci. 13, 387-392. FRENEYJ. R. (1961) Some observations on the nature of organic sulphur compounds in soil. Aust. J. agric. Rrs. 12,424-432. POSTGATE J. R. (1966) Media for sulphur bacteria. Lab. Pracr. 15, 1239-1244. STARKEYR. L. (1937) Formation of sulphide by some sulphur bacteria. J. Butt. 33, 545-57 1. STARKEYR. L. (1966) Oxidation and reduction of sulphur compounds in soils. Soil Sci. 101,297 -306. SWABYR. J. and VIT#LINSMAIJAI. (1968) Sulphur oxidation in Australian soils. ~~u~s~~fj~~s Ninth ~~~~~~ff~~u~z~~ Congrrss qf Soil Scirncr, Adelaide. 4,673~68 1. VISHNIACW. and SANTERM. (1957) The thiobacilli. Butt. Rec. 21, 195-213. VITOL~NS MAIJAI. and SWABYR. J. (1968) Activity of sulphur-oxidizing microorganisms in some Australian soils. Aust. J. Soil Res. 7, 171-183.