Soil Biol. Biochem. Vol. 9, pp. 405 to 410. Pergamon Press 1977. Printed in Great Britain.

SULFUR OXIDATION AND RESPIRATION IN54-YEAR-OLD SOIL SAMPLES

WALTER B. BOLLENUSDA Forest Service, Pacific Northwest Forest and Range Experiment Station,

Forestry Sciences Laboratory, Corvallis, Oregon 97331, U.S.A.

(Accepted 12 March 1977)

Summary—Soil samples in dry storage for 54 yr were shown to retain their ability to respire andto oxidize S. Three of the soils had lower S-oxidizing capacity and three oxidized more S at 1 g kg -than did the samples when originally collected. When the experiment was repeated with all apparatussterilized by autoclaving and S sterilized in flowing steam, a greater proportion of the S was oxidized.This was not due to heat treatment of the S. In all cases, S additions and incubation resulted ina lowering of the soil pH, suggesting that Thiobacillus thiooxidans was responsible and had survivedthe prolonged storage. When the soils, before and after incubation, were added to Thiobacillus media,only Gram-positive bacteria, mostly Bacillus spp., were found.

INTRODUCTION

Spore-forming and other bacteria, remain viable forlong periods in dry soil and other harsh environ-ments. Little is known concerning their potentialability to retain such metabolic activities as nitrifica-tion, sulfur oxidation, and organic matter decomposi-tion during dormancy.

Dry soil samples more than 15-year-old are capableof nitrifying added ammonium sulfate (Fraps andSterges, 1932). Simms and Collins (1960) found thatnitrifiers had strong resistance against drought indesert soils. In a 5-year study of dry zone soils inthe Argentine Patagonia, Garbosky and Giambiagi(1962) observed that where Ca and K were adequate,initially high nitrification was maintained or onlyslightly decreased. In a study of S-oxidizing micro-organisms in some Australian soils, including 23desert sands and 21 desert loams, Vitolins and Swaby(1969) found that S was oxidized not only by autotro-phic Thiobacilli but also by such heterotrophs asArthrobacter aurescens, Bacillus licheniformis, andspecies of Flavobacterium, Alcaligenes and Mycobac-terium. Abbot (1923) showed that Penicillium luteum,another heterotroph, oxidized S to

Desert soils from Antarctica, Chile and Californiahave been shown to have ammonifying, nitrifying andS-oxidizing potential (W. B. Bollen, K. M. Kemper,K. M. Byers and F. Au, unpublished reports).

Although their specific metabolic activities were notdetermined, various bacteria have been cultured fromdesert soils of arid and frigid climates and from driedsoil collections. Representatives of Bacillus, Mycobac-terium, Mycoccus, Nocardia and Streptomyces wereisolated by Bollen and Kemper (unpublished report)from samples of the Chile—Atacama desert, an areaof very low and infrequent rainfall.

Bollen and Byers (unpublished report) isolatedspecies of Bacillus, soil coryneforms and Streptomycesfrom soil samples from the 70-year-old collection ofE. W. Hilgard, late Professor of Soils at the Univer-sity of California, Berkeley. Vela (1974) detectedviable Azotobacter in soils stored in the laboratory

for more than 10 yr. 1 have observed that Azotobacterchroococcum can retain viability for 8-10 yr inshrunken dried agar slope cultures that have becomeso hard that removal from the tube carries adherentflakes of glass. Species of Arthrobacter, related soilcoryneform bacteria, Bacillus, Brevibacterium, Coryne-bacterium, Pseudomonas and Streptomyces constitutethe major groups found in arid lands (Cameron,1969).

Such studies emphasize the remarkable ability ofmany bacteria to survive highly unfavorable environ-ments for long times and to resume active life pro-cesses upon re-establishment of suitable conditions.

In my study, certain stored samples of Oregon soilscollected in 1921 for study of S oxidation (Halversenand Bollen, 1923) were tested again in 1975 and 1976for retention of this property and also for soil respir-ation activity.

MATERIALS AND METHODS

The original 14 samples were collected in 1921 andrepresented four distinct areas of Oregon, but onlysix samples were available for this study: Columbiabasin soils Umatilla medium sand and Stanfield finesand on which alfalfa responded to application offlour sulfur; southern Oregon soils—Salem clay loam,Salem clay loam which had been sulfured and Med-ford silt loam; Willamette Valley soil—Dayton siltloam. These air-dry samples had been stored in946 ml fruit jars with clamped but unsealed lids, inthe attic of a five-story building, subject to tempera-tures as low as —12°C in winter and as high as 40°Cin summer. Descriptions of the various samples aregiven by Halversen and Bollen (1923). Their chemicalcharacteristics are presented in Table 1.

Moisture was determined by drying at 105°C.Water-holding capacity was obtained from the weightof water retained by subsamples after saturation inlarge Gooch crucibles wetted from below and thendrained to constant weight in a saturated atmosphere.For pH, a glass electrode was used on 1:5 w/v

405

Table 1. Chemical characterization of air-dry 54-year-old soil samples*

WaterWater-holding

capacity AshTotal

CKjeldahl

N C: NTotal

(%) (%) pH (%) (/o) (%) ratio parts/106 parts /106

1 Umatilla medium sand 0.97 28.1 6.8 97.86 0.47 0.047 10.00 55 54 Stanfield fine sand, sulfuredt 1.14 29.4 6.8 97.16 0.67 0.083 8.07 110 385 Salem clay loam 3.60 38.7 6.6 95.38 1.86 0.132 14.09 155 86 Salem clay loam, sulfuredt 3.64 40.7 6.8 93.48 1.95 0.151 12.91 135 87 Medford silt loam 1.80 31.1 6.7 93.29 2.03 0.188 10.80 80 6

13 Dayton silt loam 2.05 40.1 5.6 94.30 1.62 0.118 13.61 164 7

* Oven-dry basis.f Flour sulfur applied at 112 kg ha' in March 1919.I Flour sulfur applied at 112 kg ha -1 acre in 1915, and again in 1917.

Sulfur oxidation and respiration in 54-year-old soil samples 407

aqueous soil suspensions while stirring. Soil analyseswere made on subsamples ground to pass a 149 /Im-mesh sieve. Burning in a muffle furnace at 650°Cprovided data on ash and loss on ignition. Total Nwas determined by Kjeldahl procedure and total Cby dry combustion at 950°C (Allison et al., 1965).Total S was determined in 1921 by fusion of 2 g soilwith 14 g sodium peroxide, 0.8 g magnesium powderand 1 g sucrose in an illium sulfur bomb (Parr Instru-ment Co., Moline, Illinois). Subsequent procedure wasas recommended by the Association of Official Agri-cultural Chemists (1920). Sulfates were determinedturbidimetrically (American Public Health Associ-ation, 1955) on water extracts clarified by passagethrough Millipore 0.22 um filters. Analytical data forthese soils are in Tables 1 and 2. Numbers of moldswere determined by plating dilutions with peptone-glucose acid agar; bacteria and streptomyces, withsodium albuminate agar (Fred and Waksman, 1922);and endospores, by plating dilutions of pasteurized1:5 (w/v) soil suspensions with nutrient agar. Forthiobacilli (Vishniac and Santer, 1957), inorganicmedia adjusted to pH 3.5 for Thiobacillus thiooxidansand to pH 6.8 for T. thioparus were used. To detectacid production, 0.25% tri-calcium phosphate wasadded. A clear zone around a colony indicated dis-solving of the insoluble salt. All plates were incubatedat 28°C.

The study comprised two separate experiments.The first was conducted with the usual precautionsand cleanliness employed in soil microbiology, butapparatus and containers were not sterilized. Eachsoil and each treatment was run in duplicate and inrandom order, but the replications were run at differ-ent times over a period of 16 months. For S oxidation

and soil respiration, 100 mg flour sulfur was mixedwith 100 g soil while 100 g untreated soil was usedas a control. The soils were placed in 473-m1 bottlesand distilled water added to adjust moisture contentto 50% of water-holding capacity. The bottles werethen connected to a manifold supplying air washedthrough IN NaOH and saturated Ba(OH) 2 solutionto remove CO 2, through 1N H 2 SO4 and throughwater to maintain soil moisture. Each bottle outletwas connected to a sparger immersed in a test tubecontaining 10 ml 1N NaOH. Carbon evolved as CO2was absorbed by the NaOH. At 7, 14, 21 and 28days after treatment, the tubes of alkali were replacedand CO 2 determined by differential titration with 1Nand 0.08 N H 2 SO4 using a Beckman automatic titra-tor and Cooper's (1941) end points. Analyses for sul-fates and pH values were made on the soil in eachbottle at the end of the 28-day incubation.

To avoid any contamination with S-oxidizingorganisms, the second experiment was conducted withaseptic sampling and all equipment sterilized byautoclaving or flowing steam. Because Waksman(1922) believed that T thiooxidans are usually not ori-ginally present in the soil but are introduced artifi-cially with the S added, the S (precipitated sulfur)used in this experiment was placed in a 250-m1 Erlen-meyer cotton plugged flask and sterilized in flowingsteam for 30 min on three consecutive days. This Sgave no indication of any growth within 30 days at28°C when tested on Waksman's (1922) liquid andsolid media and the Vishniac and Santer (1957)medium. It was found that the unsterilized precipi-tated S, as well as three different brands of agricul-tural flour sulfur tested in a similar manner, did notcarry thiobacilli. The setup was similar to the first

Table 2. Microbial analyses of soils (numbers per gram, oven-dry basis)

SoilNo. Treatment

Molds10 3 g

ThousandsTotal

106 g -1

BacteriaStreptomyces

(%)Endospores

10 3 g - 'Thiobacteria*

at pH 3.5 at pH 6.8

Original samplet > 10$ 0.2 5.0 110 20 1001 Control§ >500 11.4 6.5 4925 1163

1000 parts S/106 16.4 21.6 3.5 1400 2660

Original sample >10 0.2 3.0 120 70 904 Control 3.7 31.3 6.0 6045 5225

1000 parts S/10 6 1.5 39.2 3.5 2125 5280

Original sample > 10 0.4 13.1 710 370 11205 Control 17.0 30.1 23.0 3515 4275

1000 parts S/10 6 1.0 48.0 5.2 6265 4315

Original sample 0.2 1.0 7.9 400 120 7006 Control 4.1 32.4 16.5 4100 6700

1000 parts S/106 5.8 45.5 8.3 6245 5325

Original sample > 10 1.1 3.5 420 90 10007 Control 16.3 38.6 14.5 4800 3225

1000 parts S/106 11.3 43.0 10.9 3005 718

Original sample > 10 0.2 1.8 420 710 71013 Control 22.9 9.3 9.0 4775 333

1000 parts S/106 20.7 2.6 1.5 4135 205

* Isolated on Vishniac and Santer (1957) medium. Not Thiobacillus spp.t Air dry. Not incubated.

> indicates no growth on 1:10 or 1:500 dilutions.§ Incubated 28 days at 28°C with moisture at 50% of water-holding capacity and with continuous aeration; all

sterile apparatus and aseptic procedure.

408 WALTER B. BOLLEN

run except that the brass manifold was steam steri-lized and the CO 2 -free air was passed through ad-ditional I N H 2 SO4 and sterile distilled water, thenover Vishniac-Santer (1957) medium and nutrientagar before entering the manifold. These mediaremained sterile during the course of the experiment.To check for any H 2S or S that could possibly bederived from rubber tubing and stoppers used in theconnections, suitable traps were attached to the mani-fold; no S, sulfide, or sulfate was detected.

In Table 3, the second experiment is referred toas "aseptic" while the first is designated "clean". Atthe end of 28 days incubation in the second experi-ment, dilutions of the soils were plated for molds,bacteria and S oxidizers using the media previouslymentioned. Plate counts were not made in the firstexperiment. All data presented are means of duplicatedeterminations.

RESULTS AND DISCUSSION

Few molds and bacteria were found in the originalair-dry samples and a large proportion of the bacteriawere spore formers (Table 2).

Each of the old soil samples retained capacity tooxidize S with marked lowering of pH (Table 3).

Under the usual testing conditions (apparatus notsterilized), three of the soils oxidized less of the1 g kg -1 added S than in the 1922 study; three oxi-dized more. With sterilized apparatus, however, allthe soils with added S produced more sulfate. Onlyin Dayton silt loam was the difference between runswith clean and sterilized apparatus minor. In twocases, sulfate recovery slightly exceeded 100%, indicat-ing either an analytical error due to the very highdilution required, or perhaps additional sulfate fromoxidation of some soil organic S. Sulfate found inthe controls did not greatly differ between the cleanand sterile apparatus usage. Why more S was con-verted to sulfate with the sterile apparatus is not clear.Possibly more aeration induced by mixing and ad-ditional exposure in taking samples for the secondexperiment could be a factor. Repeating the asepticstudy with the S not sterilized by flowing steam gavesimilar results, showing that the heat treatment didnot render the S more susceptible to oxidation.

Whether or not T. thiooxidans or other species ofbacteria were responsible for the oxidation was notsatisfactorily determined. Microbial analysis weremade on the original soil samples and soils incubatedin aseptic apparatus. In addition to media for heter-trophs, samples were also plated in 1:10 and 1:50dilutions with Thiobacillus medium (Vishniac andSanter, 1957) modified by 0.2% tricalcium phosphatein suspension. One series of plates was poured withthe medium adjusted to pH 3.5 for T. thiooxidans andanother adjusted to pH 6.8 for T. thioporus and loweracid-producing species. All the samples except No.13 gave generally higher counts on the less acidmedium. Incubation of the original samples increasedtheir colony counts (Table 3); with added S, the in-creases in total bacteria were much greater except forDayton silt loam; numbers of Streptomyces, on theother hand, were reduced in all cases.

Colonies on the Thiobacillus media were small,about 2-mm dia, dark cream turning brown in old

cultures, dull and opaque. On the medium adjustedto pH 3.5, clear zones, 1- to 2-mm wide, indicatingsolution of the tricalcium phosphate. were developedwith soils 5, 6 and 7 but not with the others. Bacteriain these soils produced higher acid concentrations.

In no case did the organisms prove to be Thioba-cillus. Gram stains of smears from colonies revealedonly Gram-positive rods, 1.2-1.5 x 3-5 pm, singleand in chains, sometimes in long filaments. Endo-spores appeared in non-swollen sporangia. Transfersto nutrient agar grew well. It seems that Bacillus sp.may be the S-oxidizing agent in the old soil samples.Studies are in progress to characterize the organismand determine its capability for oxidizing sulfur inpure culture. R. J. Swaby (personal communication)finds no correlation between the incidence of T.thiooxidans and the capacity of a soil to oxidize S;but when this species is abundant, then oxidation isusually rapid. It can still be rapid due to other Thio-bacillus species. Certain heterotrophs can oxidize S(Vitolins and Swaby, 1969), but R. J. Swaby (personalcommunication) believes they are relatively unimpor-tant in soils rapidly carrying on the oxidation. I. L.Pepper (personal communication) recently isolatedfrom soil a heterotrophic Micrococcus capable of oxi-dizing elemental S to SO4-.

The very rapid oxidation of S in the Stanfield soilsuggests that T. thiooxidans was responsible, and theapplication of S to the field in 1919 could have builtup the population before the sample was collectedin 1921. This does not hold for the Salem soil, wherethe previous sulfuring gave no advantage over theunsulfured soil. In both cases, the S oxidized in the54-year-old samples was a moderate 5%. Vitolins andSwaby (1969), using 1% S additions and incubationfor 10 weeks at 25°C, considered the amount of oxi-dation to be high at 13 to 60% and moderate at 4to 13%. Based on these criteria and adjusting themto 1 g S kg' additions and incubation for 28 days,two of the soils in Table 2 show a high rate of Soxidation while the others are in the moderate range.Other species could be responsible for retention ofthe capacity for S oxidation, but their persistence for54 yr in the dry soils seems remarkable unless somespore-forming species were the active agents. Vitolinsand Swaby (1969) believe the role of any heterotrophs,either cyst- or spore-formers, is remote with moderateto high S oxidizing rate and low pH values. Our fail-ure, however, to isolate Thiobacillus from any of thesoils, and finding highly acid-tolerant Bacillus in all,points to some member of this spore-forming genusas the S-oxidizer.

Organic matter decomposition, as indicated by CO,production (Table 3), was within the ranges com-monly found for may soils. The extensive decomposi-tion shows the Stanfield soil is unusual, consideringthe low C content. Despite low organic matter con-tents, many sandy and pumice soils have beenobserved to evolve CO 2 more rapidly than finer tex-tured soils much higher in organic matter. This couldbe attributable to better aeration.

Although less remarkable than the persistence ofS oxidizing capacity, the ability of the old, dry soilsto actively evolve CO 2 when remoistened and incu-bated is noteworthy. Colonies of Bacillus species pre-dominated in samples of the dry soils sprinkled di-

Table 3. Sulfur oxidation and CO 2 efflux from 55-year-old samples incubated 28 days*t, clean vs. aseptic procedures

SoilNo.

Apparatusused

pH and S as S0,2, -control 1000 parts S/10 6

pH SO4 - -S pH SO;--S

S oxidizedin 1975-1976

28 days inin 192214 days

C evolved as CO 2control 1000 parts S/10 6

Soil C oxidized to CO2control 1000 parts S/10' cna

W'-'o

parts/106 parts/106 parts/106 % % mg 100 g - ' soil % 'Y.

1 cleanaseptic

6.76.5

3313

4.24.2

85513

52500

5.250.0

28 21.340.6

18.121.6

4.538.63

3.854.60

x','.r.5'

4 clean 7.3 81 4.6 860 789 78.9 38 52.2 50.4 7.79 7.52 aaseptic 6.4 104 4.1 1166 1062 100.0 29.4 21.4 4.39 3.19 Pa

C1

5cleanaseptic

5.65.7

2123

4.64.4

73463

52440

5.244.0

7 63.761.6

53.951.0

3.423.31

2.842.74

co°_P

6 cleanaseptic

5.85.9

2229

5.14.9

771050

551021

5.5100.0

10 63.363.1

51.859.3

3.253.24

2.663.04

6'a'a°

clean 7.0 49 6.3 163 124 12.4 11 85.4 81.8 4.21 4.03 LA7 aseptic 6.9 20 5.2 368 348 34.8 69.2 66.4 3.41 3.27-I,

CD

13clean 5.9 29 4.9 113 84 8.4 2 46.7 40.7 2.88 2.51 .ES'Laseptic 5.9 26 5.3 123 97 9.7 66.2 47.9 4.09 2.96

* At 28°C with moisture adjusted to 50% of water-holding capacity.Data are means of duplicate runs made in random order at different times during 16 months.Halverson and Bollen, 1923.

410 WALTER B. BOLLEN

Nell), onto nutrient agar plates. These bacteria couldbe largely responsible for the CO 2 production.

With each soil, the S addition in the clean experi-ment decreased CO 2 evolution—probably due to theincrease in acidity (Table 3). Organic matter decom-position typically proceeds less rapidly as pH de-creases. On the basis of soil total C, the decrease wasgreatest in the Umatilla soil, which had the lowestC content, and least in the Medford soil, which hadthe most C. In the second experiment, with sterileapparatus and sterile air. CO 2 evolution, with twoexceptions, was greater than in the clean setup. Inonly two cases was more CO 2 evolved from controlsthan from the S-treated soils.

Revival of these activities by bacteria that havesurvived and remained potent for prolonged periodsin dry soils raises questions concerning contributingfactors. Answers will require investigation of thephysico-chemical and biological conditions and pro-cesses involved. The energy relations of water reten-tion and exchange between an air-dry matrix and cellsexisting on hygroscopic films require investigation.

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ALLISON L. E., BoLLE81 W. B. and MOODIE C. D. (1965)Total carbon. Methods of Soil Analysis, Part 2, pp.1356-1360. Am. Soc. Agron. Madison, Wisconsin.

AMERICAN PUBLIC HEALTH ASSOCIATION (1955) Standard

Methods for the Examination of Water, Sewage and In-dustrial Wastes, pp. 197-198. New York.

ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS (1920)Official Methods. Washington, D.C.

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Sims C. M. and COLLINS F. M. (1960) The numbers anddistribution of ammonia oxidizing bacteria in somenorthern territory and South Australian soils. Aust. J.agric. Res. 11, 505-512.

VELA G. R. (1974) Survival of Azotohacter in dry soil. Appl.Microbiol. 28, 77-79.

VISHNIAC W. and SANTER M. (1957) The thiobacilli. Bact.Rey. 21, 195-213.

V ITOLINS M. I. and SWABY R. J. (1969) Activity of sulphur-oxidizing microorganisms in some Australian soils. Aust.J. Soil Res. 7, 171-183.

WAKSMAN S. A. (1922) Microorganisms concerned in theoxidation of sulfur in the soil, IV. A solid medium forthe isolation and cultivation of Thiohacillus thiooxidans.J. Bact. 7, 605-608.