Archiv fiir Mikrobiologie 46, 329--337 (1963)

Hopkins Marine Station Pacific Grove, California

Thiobaei l lus intermedius nov. sp. A Novel Type of Faeultative Autotroph

By JACK LONDON

With 3 ]?igures in the Text

(Received June 15, 1963)

Faeultative autotrophs belonging to the genus Thiobacillus have been described in the literature. B~IJ~r~K'S observation (1904) that T. de- nitri/icans grows well on organic nutrients is probably the earliest report. Two decades later, TnA~Tw]~r~ (1921, 1924) announced the isolation of another autotrophic denitrifying Thiobacillus which would grow equally well on organic media. The validity of both reports has been disputed. LI~SK~ (1912), who reisolated T. denitri/icans, was unable to obtain growth of his culture in organic media. Similarly, S~A~:~Y (1934), work- ing with subcultures of TRAc~w~r~'s original isolates, was unable to obtain autotrophic growth. Hence, ~hese investigators concluded that the earlier claims resulted from the use of impure cultures. STA~x]sY (1934, 1935), however, isolated a facultative autotroph, T. novellus, whose ability to grow both autotrophieally and heterotrophically is well docu- mented. Subsequently, Lrp~A~ and McL~Es (1940) isolated yet another faeultative autotroph, T. coproliticus, from a piece of Triassic coprolite ; however, this organism has not been studied by others.

During a comprehensive study of the Thiobacillus group, an unusual faeultative autotroph was isolated and characterized. This paper describes the new species, for which the name T. intermedius is proposed.

Methods Cultures were grown in a thiosulfate-mincral medium composed of tapwater

with Na2S2Os. SH20, 1%; NH4CI, 0.1~ KH2PO4, 0.1% ; and MgC12, 0.05% ; adjusted to a p~ of 6.8. When required, the medium was solidified by ~dding 1,5% ~gar. Other organic compounds were added where appropriate. Growth in liquid cultures was measured turbidimctricslly at 540 rote with a photoelectric colorimeter. Manometry was performed by the standard techniques described by U~BR~IT, BVn~IS and STACSS~R (1957), Protein was determined by the biuret technique as modified by LA RIVriSRE (1958).

Results Isolation

Selective cultures designed to enrich for T. thiooxidans were initiated by inoculating a thiosulfate-mineral medium with mud taken from the

Arch, Mikrobiol., Bd. 46 23

330 J~cK LONDON:

bank of a freshwater s tream and incubating at 30 ~ C. I t was expected tha t an initial population of T. thioparus would develop and produce sufficient acid to create optimal conditions for the subsequent growth of T. thiooxidans. The hydrogen ion concentration of the culture increased rapidly during the first week until a p~ of 3.1 was reached, remained practically unaltered for several days, and later increased again to a p~ of 1.9. At this time, aliquots were removed and the dominant organism was isolated from the turbid cultures by repeated streaking on thiosulfate- mineral salts agar.

Seven to ten day 's incubation was required before colonies became visible on thiosuffate agar. Since T. thioparus grows much more rapidly under the same conditions, it was obvious tha t either an aberrant strain of this species or another kind of organism was present. In the hope tha t growth might be enhanced by supplementing the medium with growth factors, a trace of yeast extract (0.0050/0) was added. This caused indeed a marked stimulation of growth, which prompted further investigation of the organism.

E//ect o/ Yeast Extract on Growth

A series of mineral thiosulfate agar plates with increasing concentra- tions of yeast extract was prepared, streaked with a suspension of the pure culture, and examined periodically over a period of ten days. With increasing amounts of yeast extract, there was an increase in the rate of development as well as in the final size of the colonies. At the highest yeast extract concentration tested (0.5~ growth was detectible in three days as compared to six days for the unsupplemented medium, and the final colony diameter was about two to four times as great as on the lat ter (Fig. 1).Finally, with 0.50/0 yeast extract, the colony changed from a round to a lobate form. I t will also be noted tha t sulfur deposition within the colony increases with increasing yeast extract content.

A similar experiment was performed with liquid cultures (100 ml in 500 ml Erlenmeyer flasks), incubated on a ro tary shaker at 300 C. The cultures were periodically neutralized with 10~ Na~CO 3 until the thio- sulfate had been completely oxidized to sulfate. Those in thiosulfate-mine- ral medinm did not become turbid until the eighth day, while in yeast extract supplemented n~dia max imum growth was at tained in less than a week. The t ime required for max imum autotrophie growth varied greatly, depending on the previous history of the culture used as inoculum; lag periods from 4 to 28 days were not uncommon. Final cell yields, expressed as mg protein per ml culture, are shown in Table 1 ; it is clear tha t they de- pend markedly on the amount of yeast extract added.

Other complex materials, e.g. hydrolyzed casein, proteose peptone and yeast autolyzatc, as well as a synthetic mixture of 16 amino acids,

Thiobaciltus intermedius nov.sp. (facultative autotroph) 331

F i g . I a - - e . Effect o f yeas t ex t r ac t on g r o w t h a n d morpho logy o f eolonies o f T. in~ermedius on th iosu l fa te -mineraI med ium. Thiosulfa te- minera l m e d i u m : a W i t h o u t yeas t ex t rac t , 6 0 0 x ; b W i t h 0.05~ yeas t ex t rac t , 4 8 0 x ; e W i t h 0.i~ yeas t ex t rac t , 510 x ; d W i t h 0.5~ yeas t ex t rac t , 3 5 0 • ; e T. thioparus wi thou t

yeas t ex t rac t , 580 x

exert an effect similar ~o that of yeast extract. Stimulation by the amino acid mixture is not as striking as that by the complex materials. The

23*

large cell crops observed in thiosulfate-mineral medium with such additives suggested that the increased cell yield could not be ascribed solely to vitamins or other growth i~etors contributed by the yeast ex-

Table 1. The E//ect o] Yeast Extract o~ the Call Yidd of T. intermedius

Yeast extract Cell protein mg]ml concentration culture

0 0.005~ 0.050/0 0.1~

0.053 0.043 0.13 0.20

7

IQ2 $2 01

After maximum growth, aliquots of cultures were removed and centrifuged. The pellet was washed ~wiee with distilled water and suspended in l ml of water. Follow- ing the addition of Biuret reagent, the suspension was allowed Lo stand for 30 min- utes; the protein conten~ wa~ then determined from spectrophotometrie measure- ments of the absorption at 540 m#.

332 JACK Lo~o~:

"0 I 2 3 ~ 5 6 y 3 9 10 77 12 /3 /4 [5 /5 /7 /S DcVs

Fig. 2. Growth of T. intermedius u~lde~ varied nutritional conditions

tract. This conclusion was supported by the failure of vitamins, purines, pyrimidines and combinations thereof to enhance growth in thiosulfate- mineral medium.

An increase in growth rate and ee]l crop is likewise caused by the addition of glucose, fructose, sucrose, maltose, glutamate and aspartate instead of yeast extract, provided these are supplied in a relatively high concentration (0.2~ Evidence for glucose and glutamate stimulation is presented in Fig.2. Galactose, trehalose, arabinose, gtuconate, ethanol, propanol, glycerol, acetate, propionate, butyrate, suecinate, fumarate and citrate do not enhance growth in thiosulfate-mineral medium.

Thiobacillu8 intermedius nov. sp. (facultative autotroph) 333

In a medium devoid of thiosulfate some growth occurs at the expense of yeast extract, as shown in Fig. 2, but this is very slow and scant. Glucose, fructose, sucrose, maltose, aspartate and glutamate alone do not support growth but ff yeast extract is simultaneously added, growth is abundant though much delayed as compared to tha t in thiosulfate media.

The curves in Fig. 2 indicate that the final cell yields in media with thiosulfate plus organic substrates are practically identical with those in thiosulfate-free media containing both yeast extract and a simple organic compound. This suggests that 0.05~ yeast extract is equivalent to 0.5~ thiosulfate for cell production. However, this conclusion is not justified because these cultures were not periodically neutralized. Consequently, the cultures in thiosulfate media became strongly acid, in contrast to those in thiosulfate-free media, so that growth in the former may be limited by low p~ rather than by the availability of an oxidizable sulfur substratc.

At present, no satisfactory interpretation can be offered for the long lag which occurs before growth is initiated in thiosulfate-free media. The possibility that the lag may be caused by the eventual appearance of mutant cells in such cultures was tested by making transfers from yeast extract-glucose or yeast extract-glutamate cultures in their exponential or early stationary phase into homologous media andinto thiosulfate-glucose or thiosulfate-glutamate solutions without yeast extract. Although the subcultures in thiosulfate-free media developed somewhat faster than before, it still took 6 to 10 days before they were as dense as the parent cultures. In contrast, thiosulfate-containing media developed within 4 days.

Thus a mutation is apparently not responsible for the heterotrophic growth observed with these microorganisms. A similar situation was encountered by P~AFF and PA~'rAr (1956), who found that Saccharo- myces rouxii required 21 to 28 days' incubation in sucrose before a fermentation of that sugar commenced, even when the inoculum was taken from cultures rapidly fermenting sucrose. This "long term adapta- t ion" was likewise unexplained; it was postulated that either (1) auto- lysis of cells released a sucrase into the medium, thereby converting sucrose to monosaccharides or (2) an initial repression existed which was somehow reversed.

The marked stimulation of yeast extract on cell yield in thiosulfate medium can best be explained ffcell yield is proportional to the ATP demand for growth (BAlrcJ~or and ELSDEN 1959). The presence in yeast extract of amino acids, peptides, purines, pyrimidines, etc., which can serve as preformed building blocks for the synthesis of proteins and nucleic acids,

334 JACK Lo~ oo~:

would obviate the need for their total synthesis from CO~, and thus de- crease the ATP requirement for cell production, so that an enhanced growth per unit of available thiosulfate, the source of ATP, becomes understand- able. Such an explanation is in line with the findings of M~CHALIS and g ~ r T ~ B ~ G (1960) tha t Desul/ovibrio desul/uricans can synthesize its cell constituents from yeast extract with the energy gained from the oxida- tion of H 2 during sulfate reduction.

Similarly, the large increase in cell yield in yeast, extract media with- out an oxidizable sulfur compound but supplemented with glucose or glutamate may be explained by assuming that these substrates replace thiosulfate as the source of ATP.

Sulfur Metabolism

Resting cell suspenJsions of T. i n t e r ~ d i u s grown in a thiosulfate- yeast extract-minerM medium oxidize, in addition to thiosulfate, sulfur, sulfide, hexa- and tetrathionate. The mechanism of oxidation of these compounds, which is mediated by a type c eytochrome, appears to be essentially the same as that found in T. thioparus and T. thiooxidans (Lo~])o~ and RJ2TEXB~G 1963).

Table 2 The Main Charac~eristlc8 o/the Facuttativety Autotrophic Thiobacittus Species

Size Motility Growth in nutrient

broth pH range for growth Growth in thiosul-

fate-mineral reed. Growth in suffur-

mineral medium Colonies on thio-

sulfate agar

Growth on yeast extract agar

Growth on nutrient agar

Growth in glucose- mineral medium

Growth in asparagine- mineral medium

T. cop~'oliticus T. novellus T. intermedius

0.1 --0.2 X 6--40 ,a + polar flagellum

+ 10--4.6

+

0.6•

+ 7.8--5.8

§

thin, colorless, de- posited sulfur as frosty film

abundant, ropy, mucoid, cream colored abundant, mucoid, ropy, shiny brown opalescence

+

0.5• + polarflagel~m

7.0--1.9

+

+ tiny watery colo- nies with sulfur deposited occa- sionally not reported

not reported

not reported

not reported +

§ small, sparse de- position of sulfur in center of colony

poor, very thin, spreading, clear

Thiobacillus intermedius nov. sp. (facult~tive autotroph) 335

Classification

The sulfur bacterium here described can develop in strictly mineral media at the expense of inorganic oxidizable sulfur compounds. I t can also grow heterotrophically in media devoid of inorganic energy sources. Hence it must be characterized as a facultative autotroph distinct :from T. thiopar~s and T. thiooxidans.

Although it can grow either autotrophically or heterotrophically, growth is optimal if both an oxidizable sulfur compound and organic substances are provided. This property clearly separates the isolate from T. novellus, whose oxidation of thiosulfate is inhibited by the presence of organic matter (SrAnxEY I934).

In autotrophically grown cultures, the final hydrogen ion concentra- tion reaches a level intermediate between those produced by T. thlo- oxidans and by T. thioparus, T. novellus and T. coproliticus.

The organism clearly represents a new type, for which the name T. intermedius is proposed. A comparison of its properties with those of the other two facultatively autotrophie Thiobacillus species is summarized in Table 2.

Description o/Thiobaciltus intermedius spec. nov.

Cells rod-sh~ped, 0.5 by 1 to 2 #, Grain negative, non-spore forming, motile by means of a single polar flagellum (Fig. 3).

On minerM-thiosulfate agar, colonies are small (less than 1 mm in diameter), yellowish-opaque with raised centers and surrounded by flat, veil-like fringes. The opacity is due to the presence of highly refraetile sulfur droplets in the central portion of the colonies, but sulfur deposition is less pronounced than in colonies of i s. thioparus.The plate cultures pres- ent ~ "dus ty" appearance due to the small size of the colonies and also because of sulfur deposition outside the colonies caused by the marked acid production. Colonies on yeast extract-thiosulfate-mineral agar plates are larger and the bordering fringe becomes lobate.

Cells capable of strictly autotrophic growth; in thiosulfate media the final pl~ falls to between 1.9 and 2.2. Ceils oxidize sulfur, sulfide, and tetrathionate in addition to thiosulfate.

Both the rate and extent of g r o ~ h in thiosulfate media are increased by the addition of yeast extract, glucose, fructose, sucrose, maltose, asparate, or glutamate, bu~ not of galaetose, trehalose, vsrabinose,~ glueo- nate, ethanol, propanol, glycerol, acetate, propionate, bntyrate, sueei- hate, fumarate and citrate.

Slight and delayed growth is obtained in a yeast extract medium devoid of thiosulfa4e; it, is increased greatly by the addition of glucose or glutamate.

336 JACK LOnDOn:

Fig. 3. Thiobacillus intermedius cells stained with 1% nrallyl acetate. MagIiified 6,900 •

Ecological Considerations In retrospect, it is not difficult to envisage the causes tha t led to the

appearance of ~:intermedius as the dominant organism in the elective culture from which it was isolated. The early acid production, resulting in a p~ drop to 3.1, can best be at tr ibuted to the development of T. thio- parus. I t s death and autolysis might well have increased the concentra- tion of complex organic mat te r initially contributed by the mud used as an inoculum, the low PH preventing the rapid decomposition of this mate- rial by ordinary heterotrophic bacteria. At this stage the medium still contained appreciable amounts of oxidizable sulfur compounds, mainly sulfur and tetrathionatc, thus providing opt imum conditions for the subsequent growth of T. intermedius.

I f these inferences are correct, it should be possible to devise specific elective culture methods for the isolation of T. intermedius. The primary

Thiobacillus intermedius nov. sp. (facultative autotroph) 337

cul ture med ium should be a minera l salts solution, supplemented with an

inorganic oxidizable sulfur compound and a small a m o u n t of yeast extract , ad jus ted to a pg of 3 to 4. Inocu la t ion with na t u r a l materials , such as soil or mud, in which T. intermedius is present, should t hen lead to the development and eventua l dominance of this bacter ium.

S u m m a r y

A new Thiobacillus has been isolated which, though capable of s tr ict ly auto t rophic growth, requires the addi t ion of organic substrates to a minerM-thiosulfate med ium for op t imum development . The effects of such supplements on growth have been described and t en t a t i ve ly inter- preted. The organism also grows with yeast ext ract and glucose or gluta- ma te in the absence of sulfur compounds. The new isolate has been named T. intermedius, and a descript ion of the species has been provided.

A c k n o w 1 e d g e m e n t. I very gratefully acknowledge the assistance, advice and encouragement afforded me by Professors C. B. vx>* NIEr~ and S. C. RITTENB~G throughout this study and in the preparation of this manuscript. Also, I wish to thank Dr. I~ArAEL MAI~Tn<EZ for the preparation of the electron mierograph and Dr. Jo~r BENNETT for the assistance in photomicrography.

This work was supported in part by a grant of the National Science Foundation, G-6436, to Dr. C. B. vA~r NI]~L.

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Dr. J. LONDOn-, Department of Bacteriology, University of California, Los Angeles 24, California, USA