INTERNATIONALJOURNAL OF SYSTEMATIC BACTERIOLOGY,Apr. 1983, p. 211-217 Vol. 33, No. 2 OO2O-7713/83/02021l-O7$02.0O/O Copyright 0 1983, International Union of Microbiological Societies Genomic and Physiological Comparisons Between Heterotrophic Thiobacilli and Acidiphilium cryptum, Thiobacillus versutus sp. nov. and Thiobacillus acidophilus nom. rev. ARTHUR P. HARRISON, JR. Division of Biological Sciences, University of Missouri, Columbia, Missouri 6521 1 Acidiphilium cryptum grows in yeast extract media with or without elemental sulfur. The growth rate and the cell yield are not changed by the presence of sulfur, but the pH of the medium drops slightly when sulfur is present, presumably because of gratuitous sulfur oxidation. No growth occurs with sulfur alone. A. cryptum is an obligate chemoorganotroph. Thiobacillus acidophilus grows equally well with either elemental sulfur or glucose as a sole energy source; this organism is a facultative autotroph. Since the name T. acidophilus does not appear on the Approved Lists of Bacterial Names, it is revived here, and strain ATCC 27807 is designated the type strain. Thiobacillus perometabolis is considered a subspecies of Thiobacillus intermedius on the basis of close phenotypic similarity and deoxyribonucleic acid homology. Thiobacillus sp. strain A2 is a distinctive mixotroph that shows little deoxyribonucleic acid homology with other species of Thiobacillus. This organism is named Thiobacillus versutus, and strain ATCC 25364 is designated the type strain. Aerobic, mesophilic, nonsporulating, gram- by Taylor and Hoare (19). These two organisms negative, rod-shaped bacteria which utilize ele- can grow autotrophically with thiosulfate as the mental sulfur or compounds containing oxidiz- energy source, heterotrophically with organic able sulfur as sources of energy were placed by compounds, or mixotrophically by using inor- Vishniac (20) in the genus Thiobacillus Beije- ganic and organic compounds simultaneously. rinck. Some of these organisms are obligate (They have also been referred to as facultative chemolithotrophs, and some are acidophilic autotrophs.) Thiobacillus intermedius also can (i.e., they grow when they are inoculated into grow autotrophically or heterotrophically , but it media at pH 13). grows poorly unless both thiosulfate and an The terms proposed by Rittenberg (15) are organic substance are present (12). Thiobacillus used here to denote nutritional categories. Obli- perometabolis, as originally described by Lon- gate chemolithotrophs acquire their energy from don and Rittenberg (13), cannot grow autotroph- inorganic compounds, and the carbon require- ically, but it does utilize thiosulfate as a source ment of these organisms can be met solely with of energy if a suitable organic substance is C02. Mixotrophs can utilize inorganic and or- present to meet its carbon requirement. Under ganic compounds concurrently. Chemolitho- these circumstances this organism grows as a trophic heterotrophs acquire their energy from chemolithotrophic heterotroph; it can also grow an inorganic compound but require an organic heterotrophically, with organic substances pro- substance to fulfill their carbon requirement. viding both energy and carbon. Obligate chemoorganotrophs acquire their ener- The acidophilic, obligate chemolithotrophs in- gy from organic compounds. clude Thiobacillus thiooxidans and Thiobacillus The non-acidophilic, obligate chemolitho- ferrooxidans. These organisms grow best be- trophs in the genus Thiobacillus include Thioba- tween pH 2 and pH 5; little if any growth occurs cillus thioparus and Thiobacillus neapolitanus. at pH 6. T. thiooxidans and T.ferrooxidans are These species grow best near a neutral pH and mesophiles (like the other species cited above), may grow at pH values above 9; they require growing best at temperatures ranging from 25 to inorganic sulfur as a source of energy. 35°C. T. thiooxidans utilizes inorganic sulfur as a The non-acidophilic mixotrophs include Thio- source of energy, whereas T. ferrooxidans can bacillus novellus (a soil organism named and utilize Fe2+in addition to sulfur as a sole source originally described by Starkey 117, 181 and of energy. If there is a strain of either of these further described by Taylor and Hoare [19]) and two species that can be cultivated in convention- Thiobacillus sp. strain A2, which was described al batch culture with glucose in lieu of sulfur or 21 1 212 HARRISON INT. J. SYST.BACTERIOL. iron, it remains to be described adequately. acidophilus and A. cryptum strains were maintained Strains of T. ferrooxidans that have been on agar slants stored at 5°C for 1 month between claimed to grow with glucose in lieu of Fe2+ transfers. The maintenance medium used was basal have been shown to be contaminated with extra- salts medium 1 supplemented with 1 g of glucose per liter, 0.1 g of Trypticase soy per liter, and 12 g of agar neous microorganisms (1, 3, 7). per liter. (Double-strength agar and sugar were auto- The thermophilic iron-oxidizing bacteria that claved separately and were added to double-strength have been isolated from copper mine leaching basal salts solution [pH 31 after the solutions had dumps (2), which are acidophilic, chemolitho- cooled to 45°C.) T. ferrooxiduns strain m-1 was main- trophic heterotrophs, require an organic supple- tained in liquid ferrous sulfate medium (6) at 5°C for 3 ment in order to grow at 55°C. Ferrous iron weeks between transfers. The other thiobacilli were serves as a source of energy, and the organic maintained on solidified Trypticase soy-glucose (TSG) compound fulfills a particular carbon require- medium slants at pH 6.9 or on solidified basal salts ment (14). medium 1 (pH 6.9) supplemented with 0.5 g of Thioba- Na2S203 5H20 per liter. The acidophilic, facultative autotroph Media. Two mineral salts solutions were used to cillus acidophilus was named and described by prepare the growth media. Basal salts solution 1 Guay and Silver (3). This species was discov- contained (per liter) 2 g of (NHJ2S04, 0.5 g of ered as a contaminant in an allegedly pure K2HP04, and 0.5 g of MgS04.7H20. Basal salts culture of T. ferrooxidans strain TM, Although solution 2 contained (per liter) 0.4 g of NH,Cl, 0.2 g of T. acidophilus grows autotrophically with ele- K2HP04, 0.2 g Of KHZP04, 0.4 g of MgS04 7H20, mental sulfur as a sole source of energy and COz and 0.3 ml of a trace element solution (21). The pH of as the source of carbon, it grows equally well each solution was adjusted with 1 N H2S04 and with glucose as the source of energy in the NaOH. The basal salts solutions were autoclaved at absence of sulfur. This organism is not able to 121°C for 15 min, either as 10-ml volumes in test tubes (17-mm internal diameter by 150 mm) or as 100-ml utilize Fe2+ as a source of energy. volumes in 500-ml Erlenmeyer flasks. Various supple- Acidophilic, obligate chemoorganotrophs ments were added to the basal salts solutions and were have been encountered in the same environ- either autoclaved together with the salts solutions or ments that harbor acidophilic thiobacilli. One sterilized separately (if required) at 10 times the de- species, Acidiphilium cryptum (9,has been iso- sired final concentration and added to the sterile, lated from allegedly pure cultures of T. ferrooxi- cooled basal salts solutions. The following supple- duns (6, 7). Unlike T. acidophilus of Guay and ments were used, individually and in mixtures, as the Silver, another contaminant in cultures of T. experiments required: Trypticase soy broth (dehydrat- ferrooxidans, A. cryptum is not able to utilize ed; BBL Microbiology Systems, Cockeysville, Md.), 0.1 glliter; yeast extract (dehydrated; BBL), 0.1 to 1 inorganic sulfur (or Fe2+)as a source of energy g/liter; Na2S203 5H20, 5 g/liter; glucose, 1 gAiter; in laboratory media, either with or without or- mannitol, 1 g/liter; and powdered sulfur (sublimed ganic supplements. However, in this paper I sulfur; type S-591 Fisher Scientific Co., Pittsburgh, demonstrate that in yeast extract this species Pa.), 10 g/liter. Sulfur was added to the basal salts can oxidize sulfur gratuitously, with neither its solutions and sterilized at 105°C (to prevent melting) growth rate nor its cell yield affected by sulfur for 1 h on 2 successive days. TSG medium contained oxidation. Thus, although it is not a thiobacillus, (per liter) 3 g of Trypticase soy broth (dehydrated; A. cryptum nevertheless may be involved in BBL), 1 g of glucose, and 2 g of K2HP0,; the pH was adjusted to 7. If needed, media were solidified with 12 mineral leaching in acidic mineral environments. g of agar per liter. Numerous gram-negative, acidophilic chemoor- To obtain the 10l2 cells required for DNA extrac- ganotrophs physiologically similar to A, cryptum tion, each culture was grown in 10 liters of appropriate were isolated from acidic mine water and were medium in a 23-liter carboy with forced aeration at examined extensively by Wichlacz and Unz 28°C. Mannitol medium (Table 1) contained 10 g of (22), but these bacteria have not been named. mannitol (reagent grade) in 10 liters of basal salts In this paper I summarize and extend the medium 1. Glucose medium (Table 1)contained 10 g of previous descriptions of mixotrophic species of glucose (reagent grade) in 10 liters of basal salts Thiobacillus. The deoxyribonucleic acid (DNA) medium 2. Thiosulfate medium (Table 1) contained 100 g of Na2S203- 5H20 in 10 liters of basal salts medium analyses described here are the beginning of a 2. Media in carboys were autoclaved at 121°C for 1 h. study of the relationships among these and other Sulfur and ferrous sulfate media (Table 1) contained, bacteria. In addition, species status is accorded respectively, 100 g of powdered sulfur and 400 g of Thiobacillus sp. strain A2T (type strain), and the FeS04.7H20, each in 10 liters of basal salts, and name Thiobacillus acidophilus, which was not were prepared as described previously (6).