Transformations of Sulfur
by Microorganisms
Diverse microorganisms participate in transformations of sulfur, many of which command attention as they result in compounds having objectionable odors or causing destruction of industrial equipment. Incompletely oxidized inorganic sulfur compounds and elemental sulfur are oxidized principally b y photosynthetic and chemosynthetic bacteria, but certain heterotrophic microorganisms can also bring about some of the reactions, Two principal sulfur products are formed-under anaerobic conditions, sulfide is the stable product, whereas under aerobic conditions, sulfate is the stable one.
ALL
organisms contain sulfur and are involved in the diverse transformations of sulfur to some degree. SVith some microorganisms these are primarily reactions of assimilation of sulfur. \Vith others. sulfur and sulfur compounds have additional particular significance. They provide energy for the groivth of some microorganisms, and are specific hydrogen donors or acceptors for others. Certain of these are designated sulfur bacteria. Because the term has been used lvith various meanings it would be well to indicate its use in this discourse. Sulfur bacteria are characterized as autotrophic and can utilize sulfur or incompletely oxidized inorganic sulfur compounds as specific reducing agents (direct or indirect hydrogen donors), and assimilate carbon dioxide as their sole source of carbon. With the chemosynthetic bacteria, the hydrogen transfers provide energv for cell development, whereas with the photosynthetic bacteria that utilize radiant energy, they serve to oxidize hydroxyl groups that originate from water in photosynthesis. hficroorganisms concerned in other sulfur transformations are excluded from the category of sulfur organisms. Sulfur is in the same group of the periodic system as oxygen and forms some compounds similar to those of oxygen, but it differs in many respects, such as in the oxidation of the element. Sulfur has six valence electrons, and forms compounds where the valence of the sulfur is 2. 4, and 6-the first as hydrogen sulfide
and the last as sulfate. Sulfur occurs in the free state, and in diverse organic and inorganic compounds, many of which are attacked by microorganisms. It has oxidation levels of from - 2 to +6, the former represented by hydrogen sulfide and the latter by sulfuric acid (70). Some of the organic compounds are indicated in Figures 1 and 2. The most common sulfur-containing amino acids are cysteine, cystine, and methionine. Taurine and cysteic acid may be produced when the amino acids are metabolized by animals. The vitamins thiamine and biotin contain sulfur as do the antibiotics penicillin and gliotoxin. The metabolically important tripeptide, glutathione, contains cysteine. Coenzyme .4 is a mercaptan and the sulfhydryl is the active functional group (70). In the oxidation of pyruvic acid. a-lipoic acid is important and the sulfur groups undergo reduction and other changes. Greenberg (25) lists the following as biochemically important sulfur-containing compounds in the vertebrate: Sulfate, thiosulfate, thiocyanate Cysteine, cystine, methionine, thiolhistidine Betaine, ergothionine Thiamine, biotin 2-Mercoptoethylamine, l i p o i c a c i d , taurine, thetins Microbial transformations of sulfur have counterparts in transformations of nitrogen. Both sulfide and ammonia are
products of decomposition of organic compounds. Both are oxidized by autotrophic bacteria, as are other incompletely oxidized inorganic compounds of sulfur and nitrogen. Sulfate and nitrate are reduced to sulfide and ammonia, respectively, by microorganisms under anaerobic conditions. Elemental sulfur and nitrogen are transformed by microorganisms but there is less specificity in the reactions of sulfur than those of nitrogen. and sulfur undergoes a greater variety of reactions. Furthermore, some microorganisms are sup ported by the energy released from oxidation of sulfur but none has been reported that similarly utilizes nitrogen. The transformations of sulfur are conveniently considered under the following headings : 1. Assimilation of sulfur 2. Decomposition of organic sulfur compounds 3. Oxidation of sulfur compounds 4. Reduction of sulfate and other inorganic sulfur compounds
Assimilation of Sulfur Many microorganisms satisfy their sulfur requirements from sulfate, but some require preformed sulfur-containing amino acids or vitamins. Most of the cell sulfur is in organic compounds. From exhaustive studies of the sulfur metabolism of Escherichia coli, Roberts VOL. 48, NO. 9
SEPTEMBER 1956
1429
Cysteine
Sulfide
HOOC-CH-CH2-SH
I
2"
HOOC-~H-CH?-S-S-CH~-CH-COOH
Cystine
":
hfethionine
*"
Disulfide
I
Thio r t h e r
HOOC-C:H--CH?-CH2-S-CHj I " 1
0
I1
Taurine
Sulfonic acid
CH?-CH?-S--OH I
0
Cvsteic acld
HOOC-C:H--CH:--S-OH
" 2
Lipoic acid
Sulfonic acid
0
HOOC-(CH~)I-CH-CHZ-FH2
s
Disulfide
S
~~
" ?
/
s=c
Thiourea
Carbamide
\
NH?
'3
Penicillin G
/ \
CeH ,-CH?-CO--NH--CH-CH I
1
C=( CH3)2
Thiazole rinq
OC -N-CH-COOH
Thiamine
Thiazole ring
HC'I
CHa S=C--NH!
I
Hac-C
,C=C-CH2-CHrOH T--CH:-F
I1
N--."H
kH-S
I
Cl
0
Biotin
Thiophene ring
I1
C
/ \
HN
NH
I / HC-CH I 1
HE
CH-(CH?)(--C.OOH
s'' Ergothioneine HC=C-CH2-CH-C=0 I 1 I
Figure 1 ,
1430
Hydrosulfide 1
Representative organic sulfur compounds
INDUSTRIAL AND ENGINEERING CHEMISTRY
Abelson, and others (63) found that the cells contained 1.12y0sulfur (dry basis) and that 95% of the sulfur was in the amino acids cysteine, cystine, and methionine. The methionine is entirely in the protein, whereas one half of the cystine and cysteine is in protein and the other half is associated with glutamic acid and glycine in glutathione. There were equal amounts of the sulfur in the amino acids. The culture could meet its sulfur requirements from sulfate and also from other inorganic and organic sulfur compounds, About one third of the sulfur converted to organic compounds was released into the medium. The course of events in conversion of sulfate to amino acids is obscure. Hockenhull (37) postulated the changes in Figure 3 from studies with the fungus AspergilluJ nidulans. The sulfate appeared to be reduced first to sulfite, then to sulfoxylate, dimerized to thiosulfate, and finally became organically bound and transformed to cysteine. With certain microorganisms, as well as with higher organisms, there may be an interconversion of cystine and methionine (22: 25, 32.36, 67). Decomposition of Organic Sulfur Compounds Various sulfur products are formed in the breakdown of organic sulfur compounds. They vary with the compounds attacked, the oganisms concerned, and the environmental conditions. In animal metabolism, taurine, sulfate, and thiosulfate are produced from the sulfur amino acids (22, 25). The most commonly reported product from the bacterial breakdown of the sulfur of cysteine and cystine is sulfide. but other products were also detected (77, 35, 52, 64', 69) 82, 84-87> 701. 703). Commonly the organisms studied were facultative anaerobes. The adaptive enzyme, cysteine desulfhydrase, effects the release of sulfide. Barber and Burrows ( 4 ) repoited the aerobic breakdown of cystine by a bacterium designated Achromobacter cjstznouorum, with production of elemental sulfur as the only sulfur product. Equivalent amounts of sulfur and ammonia were produced indicating equal desulfuration and deamination. Sulfate was reported by hfothes (57) as a product of aerobic decomposition of cystine by various fungi. Garreau (23) recovered as sulfate from 40 to 50% of the sulfur of cysteine, 25 to 30y0 of thtsulfur of taurine, and 167, of the sulfur of methionine after decomposition by Aspergilliis niger for 6 days. Stahl and others (77) reported that in the decomposition of wool by Microsporum gypseum? cystine was produced and its sulfur was transformed to sulfate as follows: Cysteine .-L Cystine -L Sulfenate + Sulfinate -+ Sulfite -P Sulfate
I t is of interest that, with sulfur as strongly reduced as the hydrosulfide in cysteine, sulfate was the sulfur product. Unpublished results (20: 46, 79) indicate that the sulfur of cystine is transformed to various products by different aerobic microorganisms isolated from soil. .4nalyses were made for the following substances: cystine, cysteine, sulfide, elemental sulfur, thiosulfate, tri-, tetra-, and pentathionates, sulfite, dithionate, and sulfate. Sulfide was a n insignificant product. With certain bacteria the principal products were tetrathionate and elemental sulfur. T h e relative amounts of each were affected by changes in the degree of aeration of the substrate. Small amounts of other products, including thiosulfate and dithionate, were formed. The principal sulfur product of other bacteria was sulfate, and the sulfuric acid produced inhibited their grolvth. Certain fungi also produced sulfate as the main sulfur product. A single organic sulfur compound, therefore, may yield different sulfur products on dissimilation by different microorganisms. The aerobic decomposition of cystine by a mixed population such as that in soils results in transformation of practically all of the sulfur to sulfate and there is a n increas: in acidity proportional to the amount of cystine transformed. In poorly buffered substrates the p H may drop sufficiently to inhibit growth of most bacteria. T h e acidity results not only from the sulfuric acid produced, but also from the nitric acid that originates from the ammonia released from the amino acid. Anaerobic decomposition of methionine by Clostridium tetanomorphum resulted in formation of mercaptans (703). Challenger and Charlton (74) identified methyl mercaptan and dimethyl sulfide as products of decomposition of methionine by Scopulariopsis brevicaulis. Stahl, McQue, and others (77) also found that the breakdown of methionine by S. brevicaulis, .M. gypseum, and A . niger yields methyl mercaptan. Challenger observed that dialkyl disulfides were transformed to alkyl sulfide and alkyl methyl sulfide by both S. breoicaulis and Schirophrllum commune (72: 73) : R-S-S-R
+
R-SH
+
R-S-CHz.
This involves fission of the disulfide linkage and methylation of the sulfur of one of the fractions. No methyl mercaptan, methyl sulfide, or hydrogen sulfide were produced from cysteine, cystine, or homocystine by S. brevicuulis (74). Unpublished results obtained by Segal (65) show that the sulfur of methionine is released by certain bacteria as methyl mercaptan, part of which becomes oxidized to dimethyl disulfide. These two products accounted for practically all of the sulfur. These are volatile products
WATER P U R I F I C A T I O N Sulfide (Valence 2 )
H-S-H H-S-S-H
R-S-R R-S-S--K
R-S-H R-S-OH
Sulfenate (Valence 2 ) Sulfinate (Valence 4)
0
/I
R-S-OH 0
Disulfoxide (Valence 4)
I'
o
R-S-S-R
0
0 I
Sulfonate (Valence 6)
R-S-OH I 0
H-S-OH I
0
0
0
Sulfate (Valence 6)
I
1
R-0-'5-OH
HO-S-OH
l
0
0
0
Thiosulfate (Valence 2, 6 )
l~
H-S-S-OH 0
Thiocyanate (Valence 2 )
H-S-CzN
Xanthate (Valence 2 ) Figure 2.
Some types of sulfur compounds
that disappear rapidly from the substrate so that as the amino acid decomposes the sulfur of the medium is depleted until, on completion of methionine decomposition, little or no sulfur remains. T h e volatile products are also produced from methionine added to soil, indicating a possible loss of sulfur proportional to the content of methionine, even with mixed populations of microorganisms. Lluch less acid is produced during decomposition of methionine than of cystine by mixed populations, for little of the sulfur persists in the soil. Furthermore, the mercaptan and disulfide, and possibly other products, inhibit development of associated microorganisms. It was concluded by Quastel and Scholefield (62) that methionine inhibits nitrification in soil. Lees (42) found that methionine \vas not inhibitive to pure cultures. The effect is most likelv due to the products of methionine decomposition by heterotrophic microorganisms and not to methionine itself. Even though there may be a n interconversion of c)-stine and methionine in the metabolism of microorganisms, as lvith higher organisms there is no evidence that this occurs during dissimilation of these compounds, for the products are different. Furthermore, the same culture, adapted to decompose both of the amino acids, produced methyl mercap-
tan and dimethyl disulfide from methionine, and produced sulfate as the principal sulfur product from cystine. Among the other transformations of organic compounds of sulfur is thc liberation of sulfate from ethereal sulfates of phenols, other ring compounds. acids, and carbohydrates. This cleavagc is effected by sulfatases of various bacteria and filamentous fungi (53-5.5, 53). Phenol sulfatase reacts as follo\vs:
Oxidation of Sulfur Compounds Inorganic sulfur compounds including hydrogen sulfide, thiosulfate, polythionates, sulfites: and also elemental sulfur are produced by microorganisms in the aerobic and anaerobic transformation of organic and inorganic compounds of sulfur. or reach waters in waste materials. These can be oxidized by various microorganisms such as photosynthetic sulfur bacteria, colorless sulfur bacteria with relatively large cells sometimes producing filaments, and colorless bacteria of the genus Thiobaczllus. These three groups of bacteria are autotrophic. Various VOL. 48,
NO. 9
SEPTEMBER 1956
1431
HO
\/
0
green sulfur bacteria Chlorohium iiniicola and Chlorohium thiosulfutophilum. Certain of the bacteria oxidize thiosulfate, tetrathionate, and sulfite in addition to sulfide and elemental sulfur. The green bacterium C. thiosulfutophilum, oxidizes sulfide, sulfur, thiosulfate, and tetrathionate to sulfate, whereas C. limicola oxidizes only sulfide and sulfur. The purple sulfur bacteria are also able to substitute various organic compounds, in particular organic acids, for the sulfur materials. All of the purple and green sulfur bacteria can develop in the absence of preformed organic matter, meeting their energy requirements from light, with carbon dioxide as their only source of carbon, and oxidizing reduced sulfur materials. The following is a typical reaction:
Sulfate
/ No
HO
HO
\/
0 Sulfite
/ No
0
H
O
\s/
Sulfoxylate
/ No
H
0
HO
'/
Thiosulfate CO?
-
+ 2HpS light
(CH2O)
+ H e 0 f 2s
I
s I
This can be expressed as a more generalized reaction where H2A may be elemental sulfur, other inorganic sulfur compounds, organic compounds, or even elemental hydrogen with some of the sulfur bacteria :
H
+ serine
i 0
HO
\S/
Po
i S I
COe
Serine thiosulfate
H O
I
H-C-C-C-OH
I/
I I H NH2
H I S H O
I
H-C-C-C-OH I
I1
Cysteine
i
H kH2
Figure 3.
Scheme of sulfate reduction in assimilation b y A. nidulans
heterotrophic microorganisms, including bacteria, actinomycetes, and filamentous fungi also oxidize some of the inorganic sulfur materials. Photosynthetic Sulfur Bacteria. The purple and green sulfur bacteria contain chlorophyll that enables them to develop photosynthetically. They may be light purple, brown, red, or green. They are classified in the families Thiorhodaceae and Chlorohacteriaceae and are single cells. They occur as spheres, rods, vibrios, or spirals, varying in size from less than 1 micron to more than 20 microns. hlany of the organisms are larger than most other bacteria. Since they develop anaerobically with energy provided by light, they are essentially water bacteria. They are most commonly encountered in waters containing sulfide or in bottom material from which sulfide originates. T h e sulfide undergoes oxidation to
1432
sulfate, generally with temporary accumulation of elemental sulfur that occurs as globules within the cells or, Jvith some of the small-celled organisms, outside of the cells (79, 39. 97. 93. 94). The sulfur accumulates outside the cells of the
+ 2H2A
light
(CH20)
+ H20 + 2.4
The (CH20) represents organic compounds in the cell. The role of H2A is more evident from Figure 4 whereby H2A is shown to be the hydrogen donor reacting with the hydroxyl group originating from water, that is attached to the enzyme (39.92). This is similar to green plant photosynthesis except that plants have a mechanism whereby oxygen and water are liberated from the hydroxyl group. Nitrogen fixation by the related purple nonsulfur bacteria was established by Kamen and Gest (33) and was demonstrated for the purple and green sulfur bacteria by Lindstrom, Burris, Tove, and \%Ison (43, 44), and Wall. Ll'agenknecht, and others (98). The purple nonsulfur bacteria (Athiorhoduceue) are similar to the purple sulfur bacteria except that they are unable to develop in substrates lacking preformed organic
Dark Reactions Photochemical
E'
General CHzO H20
+
H2S as electron donor CHPO Hp0
+
E" H2°
Light Pigment Enzymes
Figure 4.
INDUSTRIAL AND ENGINEERING CHEMISTRY
. I __._I_
I
Schematic representation of the mechanism of photosynthesis
WATER P U R I F I C A T I O N compounds and in many cases fail to use incompletely oxidized inorganic sulfur compounds as hydrogen donors.
Colorless Filamentous Sulfur Bacteria. These bacteria, composed of chains of cells, are found in sulfide-containing waters, as are the purple sulfur bacteria. The bacteria are commonly present in swamps, pools, sulfur springs, and also sea water basins where they have access to sulfide, oxygen, and carbon dioxide. They may form a dusty white film or masses of filamentous growth. The white color is due to the sulfur contained in the cells. The fact that Beggiatoa has been reported to occur only in sulfidecontaining waters is evidence that sulfide is important in its development. These sulfur bacteria are presumed to oxidize sulfide to sulfate with temporary accumulation of elemental sulfur as globules in the cells (6, 99-707). Species of Thiothrix are attached to surfaces a t the base, whereas the species Beggiatoa, Thiospirillopsis, and Thioploca are not attached. All are similar to the bluegreen algae of the family Oscillatoriaceae in that they require a solid surface for movement and they progress by a sliding gliding motion. They may be colorless members of this group (67). These bacteria are poorly characterized physiologically because they have been seldom studied in pure culture. Winogradsky reported that they could oxidize two to four times their own cell weight in hydrogen sulfide daily. Ll’inogradsky (707) and Bavendamm (6) concluded that there was little possibility that they use organic compounds. Cataldi ( 7 7 ) failed to obtain groivth of Beggiatoa on a strictly mineral substrate, but she did grow them on organic media. Unlike the purple and green sulfur bacteria, they are aerobic. Other bacteria presumed to be similar physiologically to the colorless filamentous bacteria are colorless bacteria with relatively large spherical, oval, rodshaped, curved, or spiral cells. They have been recovered from sulfide-containing waters and are believed to be sulfur bacteria because they contain globules of sulfur. These are included in the genera Thiospira, Macromonas, Thzouulum, Thiobacterium, and dchromatium and have cells 3 to 5 by 5 to 20 microns or more in size. They have seldom been reported and are poorly characterized, for studies with pure cultures are lacking. Bacteria of the Genus Thiobacillus. Small, colorless, rod-shaped pseudomonads included in the genus T h i o bacillus are commonly encountered in soils and waters. Thev rapidly oxidize sulfur and incompletely oxidized inorganic sulfur compounds chemosynthetically. Most of these bacteria are strictly autotrophic and some have very
unusual physiological characteristics. They are strictly aerobic with one or possibly two exceptions. Thiobacillus thioparus, first described by Nathansohn in 1902 and named by Beijerinck, is a strict autotroph that is primarily a thiosulfate-oxidizing bacterium. I t oxidizes elemental sulfur slowly and also mi-, tetra-, and dithionate, as shown by the folloiving reactions (66, 77, 95) :
+ 4 0 2 + H20 5Na2S01 + H2S04 + 4s Na2S203 + 2 0 2 + H z O Na2S04 + H&O4 2s + 302 + 2H2O 2HzS04 5Na~S203
-+
-
+
The first equation, in Jvhich elemental sulfur is a n important product, represents the transformation brought about in a medium containing 1% thiosulfate. Vishniac (95) concluded that sulfur was produced by the chemical breakdown of pentathionate cataIyzed by thiosulfate and that the bacterial oxidation proceeded to sulfate as indicated bv Figure 5. S2Oa“
I
Figure
5. Oxidation of thiosulfate by T. thioporus
The bacterium grows well near neutrality, and causes the substrate to become somewhat acid (between p H 4.0 and 5.0). The acidity causes the death of the culture. The bacterium is widely distributed in soils and waters and is readily recovered in strictly mineral substrates containing thiosulfate and exposed to air that supplies oxygen and carbon dioxide. Thiobacillus denitrijcans was first described by Beijerinck as a bacterium that oxidizes thiosulfate under anaerobic conditions with reduction of nitrate. Various investigators attempted to cultivate the organism with poor success until recently Baalsrud and Baalsrud (2) established that nitrate, although a n adequate hydrogen acceptor under anaerobic conditions, failed to be assimilated, and ammoniacal nitrogen was required as well. They established that the organism can grow aerobically without nitrate but requires both nitrate and ammonia for anaerobic development. It is a strict autotroph that develops best near neutrality, and oxidizes thiosulfate and elemental sulfur to sulfate under both aerobic and anaerobic conditions. This is shown b>-the following equations:
-
+ 2 0 2 + H20 N a 2 S 0 4+ H P S 0 4 S + 3 0 2 + 2Hz0 2H2SO4
NazS203
-+
5s
+ 6KNOi + 2Hz0 3KzSOa + 2HzS04 f 3Nzt +
Thiosulfate is oxidized more rapidly and some elemental sulfur is produced during this oxidation. A third member of the group, Th io bacillus thiooxidans, was first described by h’aksman and Joffe. It is remarkable in its acid tolerance which is greater than that of any other reported bacterium. I t not only tolerates but produces acidity by sulfur oxidation between p H 0 and 1, and survives for long periods under this acid condition. I t is a strict autotroph, oxidizes elemental sulfur and thiosulfate to sulfate rapidly, and also oxidizes tetrathionate, as shown by the following reactions : 2s
+ 3 0 2 + 2Hz0
-
-*
2H804
+ 2 0 2 + HzO Na2SO4 + H2SOd 2Na2S406+ 7 0 2 + 6H2O 2NanSO4 + 6HzSO4 Na?S203
Its optimum reaction is a t p H 2.0 to 3.0 and it fails to grow appreciably above p H 6.0. I t is commonly encountered in natural environments where very acid reactions have been produced by oxidation of sulfur and incompletely oxidized inorganic sulfur compounds. The culture described as T . thiooxidans assimilates nitrogen from ammonia only. An almost identical culture described by Parker (56-58), as Thiobacillus concretivorus differed principally in that it could use both ammonia and nitrate. An additional organism described as Thiobacillus noLellus is a facultative autotroph that grows slowly in a strictly mineral substrate near neutrality and oxidizes thiosulfate to sulfate (78). The reaction is the same as for this oxidation by T . thiooxidans. I t grows readily on organic substrates. According to Sijderius ( 6 6 ) , it resembles Micrococcus denitrificans in that it is aerobic but able to grow anaerobically, using nitrate as the hydrogen acceptor. I t is not known whether T . nocellus can grow autotrophically under anaerobic conditions as does T . denitrificans. Another member of the genus is remarkable in that it not only oxidizes thiosulfate, but also oxidizes ferrous iron under strongly acid conditions. The reactions are as follows:
-
+ 2 0 2 + H zNa2S04 O + H?SO4 12FeS04 + 3 0 2 + 6 H 2 0 4Fet(SOa)a + 4Fe(OH)3
Na2St03
This bacterium, named Thiobacillus ferrooxidans, was isolated by Colmer, Temple, and Hinkle (76>SS), from acid drainage waters of coal mines where it VOL. 48, NO. 9
SEPTEMBER 1956
1433
causes the aerobic oxidation of ferrous iron resulting in the precipitation of ferric hydrate and the development of red water in streams fed by the mine waters. I t is a strict autotroph that develops under acid conditions a t p H 2.0 0.- lower. I t is the first culture to be well characterized physiologically as a n iron bacterium, for the others grow close to neutrality under conditions where the ferrous iron is oxidized so rapidly by strictly chemical reactions that it is difficult to determine oxidation effected by the bacteria. With this culture it is possible to obtain reproducible oxidation of ferrous iron in an inorganic substrate and growth of the bacterium by this oxidation. Although T. ferrooxidans closely resembles T. thiooxidans as a n oxidizer of thiosulfate, it appears to have a more stable iron-oxidizing than sulfuroxidizing system, for capacity to oxidize thiosulfate is lost by continued cultivation on the iron medium. A similar culture unable to oxidize thiosulfate was reported by Leathen, Kinsd, and Braley (47). Whereas the ability of T. ferrooxidans to oxidize thiosulfate has been questioned ( d o ) , its oxidation of ferrous iron under acid conditions appears to be well established. From sewage that contained thiocyanate from gas wastes, Happold and Key (29) obtained a bacterial culture that decomposes thiocyanate with the formation of ammonia and sulfate, as shown b>-the following reactions:
+
2KSCN 2H20 f 2K2C03 -+ ZKCNO 2K2S 2H20
+
2KCNO 4- 4H20
+
2"s
2K2S
+
+ + 2C02 + 2CO2 + 2KOH
402
+
2K2S04
Summary :
+("4)zSOa 4 0 2 + 4Hp0 + 2 c 0 2 + K2SO4 Na2S203 + 2 0 2 + HQO Na2S04 + H2SOd 2KSCN
+
-+
Further study by Happold, Johnstone, and others (28, 704) indicated that the bacterium is a n aerobic, strict autotroph that oxidizes not only thiocyanate, but also thiosulfate and sulfur, growing close to neutrality (pH 6.8 to 7.6). Thiocyanate can serve as the sole source of energy, nitrogen, carbon, and sulfur for growth of the organism named Thiobacillus thiocyanoxidans. Several of these remarkable bacteria of the genus Thiobacillus are of particular practical importance. .411 oxidize reduced inorganic sulfur materials with sulfate as the principal end product. Not only does this occur under aerobic conditions but it can be effected anaerobically by T . denitrificans in the presence of nitrate. In this case there is loss of nitrogen as N2. T . thiocyanoxidans participates in the purification of wastes from gas plants by oxidizing thiocyanate.
1434
T . thiooxidanr is qenerally responsible for development of stronyly acid conditions resulting from si-lfur oxidation, because it, of all the sulhir bacteria, is particularlv tolerant to acid conditions. Recently drained soils that contained sulfide thus became acid (30) as do soils treated with sulfur for control of potato scab. Large amounts of su!furic acid are formed in the waters of some bituminous coal mines from reducpd sulfur compounds. T . thiooxidans is brlieved to be responsibie for the acid brcausp its cplls are abundant in thrse acid watTrs (76). Large amounts of iron arr dissolved from the rocks by the acid. T . ferro0uidan.r oxidizes the dissolvcd fcrro1.s iron to ferric hydrate that prrcipitat-s ar.d in such large quantit\r as to be an important pollution factor of s t r e a r s and rivers. Destriiction of a Dive jointirg matrrial was ascribed to oxidation of t%e sulfur containrd in the material b s T . fhiooxidans. but failure mav have been due to other C ~ U S C S(78. 27. 3 7 ) . Corrosion of c o n c r t e is caused by the combined action of sulfate-reducipq bacteria and T . thiooridons. The former. produce.: su!fid*,from sulfat-. Hx-drogen sulfide thrs prod:-ced escapes from the anaerobically digesting sewagc or other organic raterial. brcorres dissolved in moisture OP. the concrete \val's or covers of the contzin-rs. and is ovidized to sulfuric acid which camps disinrecration of the concrrte. This \vas reported by Barr and Buchanan in 1912 (,5)%who cited earlier reports of th- S?TC- condition. They Lvere unaware of the possibilitv that the acidity was produced by T. thioouidans. for this bact-riiim \vas unknown. but thry b - l i v 4 that sillfur bacteria \vrre concvn-d. Rlore recently. Parker (.%. 57) reported similar corrosion of a concrete sewer main and recovered from it various sulfur bacteria including bacteria resembling T . thiooxidans. T . thioparus. and T . noveilus (58). In this tvpe of corrosion the initial transformation appears to be chemical oxidation of the sulfide to elemrntal sulfur. Subsequent oxidation involves various microorganisms. the most important of which is T . thiooxidans that producrs sufficient acid to cause disintegration of the concrete. Cast iron and steel may be corroded in the same way. Oxidation by Nonsulfur Microorganisms. Inorganic sulfur compounds are oxidized by various hrterotrophic microorganisms that develop equally \vel1 lvith and without these compounds. Armstrong ( 7 ) reported oxidation of thiosulfate by several fungi. Trautwein ( 9 0 ) described as sulfur bacteria certain cultures that oxidized thiosulfate to tetrathionate. These were found to be heterotrophic bacteria, and this transformation can be brought about by various bacteria, actinomycetes, and filamentous fungi growing on organic substrates (75). The
INDUSTRIAL AND ENGINEERING CHEMISTRY
following is the reaction concerned :
Guittonneau (26) and Guittonneau and Keilling (27) found that elemental sulfur \vas similarly oxidized to thiosulfate and tetrathionate by many heterotrophic bacteria. actinomycetes, and filamentous fungi during development on organic media.
Reduction of Sulfate and Other Inorganic Sulfur Compounds Of particular importance among the reductions of inorganic sulfur compounds is the reduction of sulfate that is brought about principally by a specific group of bacteria. Reduction of incompletely reduced materials such as sulfite. thiosulfate, tetrathionate, and elemental sulfur is much more common and many bacteria and fungi are concrrned in these transformations. hfany bacteria, members of the genus Pseudomonas in particular, were found by Tanner (85) to reduce thiosulfate to sulfide. 4 similar reduction was reported for Proteus vulgaris (50, 86, 87) and yeast (36). Sulfide is produced from thiosulfate and sulfite by thermophilic bacteria (75). Sulfide production from thiosulfate, sulfite. and thiocyanate by various fungi was reported bv Armstrong ( 7 ) . The reduction of tetrathionate to thiosulfate was sufficiently specific for certain intestinal bacteria to serve as a diagnostic test (34. 59). The reaction is as follo\vs: Sa&Os
+ ZH(organic matter) -, Na2SQO. + H&O,
This is essentially the reversal of the oxidation of thiosulfate to tetrathionate common to heterotrophic microorganisms. Elemental sulfur is reduced to sulfide by all microorganisms more or less rapidly. This is due in part to sulfhydryl groups of the cell compounds and to other as yet undefined reactions. The phytochemical reduction due to sulfhydryl is indicated by the following reaction : 2R-SH
+
S
+
R-S-S-R
+
HIS.
In the latter part of the last century: de Rey-Pailhade gave the name "philothion" to the substance believed to be responsible for the hydrogenation of elemental sulfur. It was established subsequently that this reaction is not confined to any one compound but is common to sulfhydryl compounds contained in all cells. Even aerobic sulfur bacteria release some sulfide a t the same time that they oxidize elemental sulfur (76). Furthermore, sulfur is reduced readily in the abscnce of cells a t neutrality by cysteine and glutathione. Miller; XlcCallan, and Weed (49) reported that spores of various fungi produced 0.023 to 6.39 mg. of hydrogen sulfide per gram per hour from sulfur. It
WATER P U R I F I C A T I O N seemed unlikely that the reduction \vas due principally to the sulfhydryl group because the rate of the reaction with spores was higher than with glutathione, and there was little or no reaction with ground spores or spores treated with fungicides. Severtheless, the fact that sodium arsenite, which inactivates the sulfhydryl group, strongly inhibited sulfide production whereas cyanide and azide had little effect suggests action of the sulfhydryl group. If the reaction is due to the sulfhydryl group alone, enzymatic reconstitution of the group and acceleration of the reaction with elemental sulfur would be required. I t was recently reported (45) that sulfur increased production of carbon dioxide by: conidia of fungi and cells of a yeast tested in the absence of carbon in the substrate. Furthermore, the additional carbon dioxide was equal on a molecular basis to the amount of hydrogen sulfide produced, suggesting that the hydrogen was transferred to sulfur instead of oxygen in terminal respiration. Clark and Tanner (75) obtained results suggestive of the reduction of sulfate by thermophilic bacteria. Prevot (60) reported that many anaerobic bacteria reduce sulfite to sulfide and that sulfide is produced even from sulfate by t\vo anaerobes. Ability to reduce sulfate was lost by continued cultivation in laboratory medium. Of particular interest is the observation of Birkinshaw, Findlay: and \Vebb (7) that the fungus Schitophjllum commune produces hydrogen sulfide and methyl mercaptan from sulfate during growth in a glucose medium. It was presumed that the sulfate was reduced to sulfide that became methylated. This appears to have been the first instance of methylation of inorganic sulfur by microorganisms. The most important process of sulfate reduction is that effected by the specific sulfate-reducing bactcria generally referred to as members of the genus Desu~ooibrio. Whether there is more than a single species has not been established satisfactorily. Sevcral species have been described but these differ only in the organic materials they decompose. The culture first described by Beijerinck and the one to which reference is made most frequently is Desulfovibrio desulfuricans. The sulfate-reducing bacteria are strictly anaerobic and use the sulfate as the hydrogen acceptor. Sulfite, thiosulfate, and tetrathionate can be used in place of the sulfate. Various organic materials can be used as hydrogen donors including organic acids, alcohols, amino acids, and carbohydrates. It has even been reported that some cultures oxidize petroleum hydrocarbons. Elemental hydrogen can also be utilized and, when
using it: the culture can develop as an autotroph in a strictly mineral substrate (8, 73, 80). The following are typical reactions:
+
-+
2CH1.CHOH.COONa MgSO4 2CH3.COONa H2S CO? MgC08 H!O
+
4H2
+ CaSOl
-+
H1S
+
Ca(OH),
+
+ 2H20
Sulfate reduction occurs under both moderate and high (55' to 60' C.) temperature, and the cultures appear adaptable to development both as mesophiles and thermophiles (3: 74). The thermophilic cultures produce spores, whereas the mesophilic ones are spore-free. It was noted by '4. J. Kluyver (personal communication) that an anaerobic spore former encountered in spoiled canned food which caused blackening due to iron sulfide showed characteristics identical with those of the bacterium described as Sporovibrio desulfuricans (74). Since this review was prepared, Campbell, Frank, and Hall have identified the bacterium as Clostridium nigrlficans (Bacteriol. Proc., Society American Bacteriologists, Houston, Tex., 1956). This indicates that the asporogenous and sporulating organisms are distinctly different bacteria, contrary to an earlier report (7.1). They develop best near neutrality but can grow over the range of p H 5.5 to 9.0. Sitrogen fixation by sulfate-reducing bacteria has been reported (68). They are widely distributt-d because of their ability to grow on various organic materials and elemental hydrogen, and to tolerate wide ranges of temperature and salt content (47)and high concentrations of sulfide (48). They are readily recovered from soil, mud, fresh and salt water sediments, sewage, and other substrates where organic matter undergoes decomposition. They have been recovered from brines of oil wells and sulfur mines, and are responsible for most of the sulfide occurring as ferrous sulfide in marine sediments and water-logged soils. H>-drogen sulfide, originating from sulfate reduction in impounded waters, or along coastal regions, may be produced
.\erobic corrosion :
2Fe
4 ~ ' 7 H $oyGj ? 2 ~Anodic ~ ~solution of iron 0 2
2Fe
+ 4H
+2 F r . + 4(OH)+ HLO + 3H2O + 1 ] / 2 0 ~
2Fe(OH)'
'/?02
Anaerobic corrosion :
in such quantity as to be obnoxious and darken paint and silverware. Bacterial sulfate reduction concerned in the disintegration of concrete, has also been responsible for the periodic destruction of large numbers of fish in the ocean. I t is probable that deposits of mineral sulfides in sedimentary formations were produced by bacterial sulfate reduction. Furthermore, sulfate-reducing bacteria may have played an important role in the formation of the great deposits of elemental sulfur in the region about the Gulf of Mexico (24, 88). The formations are permeated with brine containing hydrogen sulfide. Sulfate-reducing bacteria have been recovered from the brines issuing a t the ground surface, but it has not been possible to establish with certainty that they were present in the formation. I t is presumed that the bacteria produced sulfide by reduction of sulfate contained in the brines, and prevalent in the formation rocks. The sulfide in turn became oxidized to the elemental sulfur, presumably by some nonbiological reaction. Similar deposition of sulfur occurs in soils periodically flooded by seawater (87). L4ccumulation of elemental sulfur in lakes in North .4fricd in Cyrenaica was reported by Butlin and Postgate (9). I t was presumed to have originated through oxidation of sulfide, produced by bacterial sulfate reduction, by purple and green sulfur bacteria, and by chemical oxidation. The residual cell material of the sulfur bacteria was believed to have served as the source of energy for the sulfate-reducing bacteria. The possibility of using these bacteria in a similar process to produce elemental sulfur from sulfate in sewage and other wastes was suggested. Sulfate-reducing bacteria have been implicated also in anaerobic corrosion of iron and steel. This corrosion is rapid and is not self-stifling. as is rusting, where the adherent oxide film protects the underlying metal. Relationship of the bacteria to anaerobic corrosion was established by von Wolzogen Kuhr (96: 97). This is indicated by the following equations:
-
+
2Fe(OH)?' Corrosion products ZFe(OH)$)
+
2Fe(OH), Summary
+
2H;O
8H20 e 8H' + 80H-' 8H' e 4Fe.. 8H]
Depolarization
nodic solution of iron + + Cas04 + 8H H?S + 2H20 + Ca(OH)2 Depolarization 3Fe.. Fe''++6(OH)H2S -,FeS 3Fe(OH)2) + 2H' Corrosion products Summary 4Fe 4- Cas04 + 4H?O FeS + 3Fe(OH)2 + Ca(0H)n 4Fe
'*
+
-
+
VOL. 48, NO. 9
SEPTEMBER 1956
1435
Under aerobic conditions oxygen is the depolarizing agent that removes hvdrogen from the cathodic areas. Under anaerobic conditions the sulfate-reducing bacteria serve as the depolarizer, oxidizing the hydrogen with sulfate. I n the absence of such a mechanism, one would expect little or no corrosion where oxygen was excluded and, in the absence of galvhnic corrosion, stray current electrolysis or acid corrosion. Unpublished results in the author’s laboratories fail to support the contention that the process described is actually responsible for severe corrosion under anaerobic conditions, although there is no doubt that sulfate-reducing bacteria play a n important role by creating strongly reducing anodes and by producing sulfide that reacts with iron to produce ferrous sulfide that is itself anodic to iron (72). Severe corrosion under the influence of sulfate reduction appears to be influenced by oxygen reaching areas of the iron. These areas serve as cathodes for concentration cells, the anodes of which are areas where ferrous sulfide is present. Iron undergoing anaerobic corrosion has ferrous sulfide and ferrous hydrate in the corrosion products in contact with the metal surface, and these products adhere loosely. Cells of rhe sulfate-reducing bacterium are very abundant in the corrosion products. The corrosion is widespread (SO), but is particularly severe in sea water and marine sediments because of the abundance of sulfate and decomposable organic matter and the high electric conductance of the salt water. I t causes underground corrosion of pipes and other equipment, part of which is in waterlogged soil and part in soil penetrated by oxygen. This corrosion has been encountered as well in gas and gasoline storage tanks, sewerage equipment, and heat exchangers. Control of the sulfate-reducing bacteria is rendered difficult by reason of their common occurrence, their resistance to inhibitors, the inactivation of the inhibitors by the substrate in which the bacteria were active, and the difficulty in penetrating the particulate material in which they are imbedded.
FVebb, R. A , , Biochem. 526-28 11942).
Butlin, K.’R., .4dams, 51. E., Suture 160, 154 (1947). Butlin, K. R., Postgate, J. R., 6th Intern. Microbioi. Congress, Rome 7, 126-43, 1953. Calvin, M., “Glutathione,” pp. 3-26, Academic Press, New York, 1954. Cataldi, M. S..RPL..inst. bacteriol. dep. .Vacl. hig. (Buenos diresj 9, 393-423 (1940). Challenger, F., Adcances in Enzymol. 12, 429-91 (1951). Challenger, F., Chem. Rers. 36, 31561 (1945). Challenger, F., Charlton, P. T., J . Chem. Sac. 1947, 424-9. Clark, F. 34.. Tanner. F. i$’,Zentl. Bakteriol. Parasitenk. 98, 298-31 1 (1 938 1. Colmer. A. R.. Temule. K . L.. Hinkle, hi. E., J . hac>rriol. 59; 317-28 (1950). Desnuelle, P.: Fromageot, C., Enzym010gia 6, 80-7 (1 939). Duecker, W.N‘., Estep, J. I$’., Mayberry, M. G., Schwab, J. I V . , J . A m . H’ater Works Assoc. 40. 715-28 (1 948). Foster, J. W., “Bacterial Physiology,” Academic Press, Yew York, pp. 361-403, 1951. Frederick, L. R., Ph.D. thesis, Rutgers University. 1950. Frederick, L. R., Starkey, R . L., J. -4m. Tt’afer TVorX-s Assoc. 40. 729-36 (1 948). Fromageot, C.. ..iduances in Enzymol. 7, 369-407 (194’7). Garreau, Yvonne, Compt. rend. soc. b i d . 135, 508;lO (1941 j. Ginter, R. L., .‘Problems of Petroleum Geology,” pp. 907-25, A m . Assoc. Petroleum Geol., Tulsa, Okla., 1934. Greenberg, D. hl., “Chemical Pathways of Metabolism,” vol. 2, pp. 148-71. Academic Press, New York: 1954. Guittonneau, G., Compt. rend. 184, 45-6 11927). \ , Guittonneau, G , Keillinq, J., .4nn. dgron. 2, 690-725 (19321. Happold, F. C., Johnstone, K . I., others, J . Gen. .Ificrobiof. 10, 2616 (1954). Happold, F. C.. Key, X., Biochem. J . 31. 1323-9 11937). Harmsen, G. W., Chem. 11Pekblod 35, 495-500 119381. Hockenhull: D.’ J. D., Biochem. Bkpphys. Acta 3 , 326-35 (1949). Horowitz, AT. H., J . Bioi. Chem. 171, 255-64 (1947). Kamen, hi. D., Gest, H., Science 109, 560 (1949). Knox, R.. Gel], P. G. H., Pollock, M. R., J . Hbz. 43, 147-58 11943). Kondo. M., Biochem. Z . 136, 198-202 119231. Korsakova, M. P., .\fzl;robioiogip 2, 251-9 (1933). LaDue, \$. R., J. -4m. FVater Tlrorks ASSOC. 40, 737-50 (1948). Lampen, J. O., Roepke, R . R., Jones, 14. J., drch. Biochem. 13, 55-66 (1947). Larsen. H., “On the hiicrobiology and Biochemistry of the Photosynthetic Green Sulfur Bacteria,” KGL Norske Videnskabers Selskabs Skrifter, Trondheim, 1953. Leathen, W. W., Braley, S. A, Sr., J . Bacteriol. 68, 481 (1955). Leathen, !V. W., Kinsel, N. A , Braley, S. A , , Sr., Bacteriol. Proc. 1955. 46. (42) Lees,H., Biochern. J.52,134-9(1952). \
References ( 1 ) .4rmstrong, G. M.,Ann. ,Missouri Botan. Garden 8, 237-81 (1921). (2) Baalsrud, K., Baalsrud, K . S., Arch. Mikrobiol. 20, 34-62 (1954). (3) Baars, J. K., Dissertation, Delft, Holland, pp. 1-164 (1930). ( 4 ) Barber, H. H., Burrows, R. B., Biochem. J. 30, 599-603 (1936). ( 5 ) Barr, W. M., Buchanan, R. E., Iowa State Coll., Eng.Ex$. Sta. Bull. NO.10, 1-16 (1912). ( 6 ) Bavendamm, W., “Die Farblosen und Roten Schwefelbakterien des Suss-und Salzwassers,” Gustav Fischer, Jena, 1924. ( 7 ) Birkinshaw, J. H., Findlay, W. P. K.,
1436
INDUSTRIAL AND ENGINEERING CHEMISTRY
I
Lindstrom, E. S., Burris, R. H . , Wilson, P. W., J . Bacterial. 58, 31316 (1949). Lindstrom, E. S., Tove, S. R., ‘Wilson, P. W., Science 112, 197-8 (1950). hlccallan, S. E. A , , Miller, L. P., Phytopathology 46, 20 (1956). Manaker, R. A,, Ph.D. thesis, Rutgers University, 1953. hliller, L. P., Contrib. Boyce Thompson Ins!. 15, 437-65 (1949). Ibid., 16, 73-83 (1950). hliller, L. P., McCallan, S. E. A , Weed, R. M.?Ibid., 17, 151-71
J . 6,
1195’31.
3l;tsuh:shi. S , hlatsuo, Y., Japan. J . E xp. Med. 20, 729-36 (1950). Mothes. K., Planta 29, 67-109 (1938). hiyers, J. T., J . Bacterial. 5 , 231-52 (1920). Neuberg, C., Cahill. W.hi.,E n z y mologia 1, 22-38 (1936). Keuberg, C., IVagner. J., Biochem. Z. 161, 492-505 (1925). Ibid., 174, 457-63 (1926). Parker, C. D., Australian J . Exp. Bioi. .\led. 23, 81-90 (1945). Ibid., pp. 91-8. Parker, C. D.: Prisk, J., J . Gen. .Wicrobzd. 8. 344-64 11953). Pollock, M. R . , Knox; R., hiochem. J . 37, 476-81 (1943). Prevot, .A. R.. Ann. Inst. Pasteur 75, 571-4 (1948). Pringsheim, E. G., Bacterial. Reas. 13, 47-98 (1 949). Quastel, J. H.. Scholefield, P. G., Ibid., 15, 1-53 (1951). Roberts. R. B.. Abelson. P. H., others. Carnegze Inst. Khsh. Publ. 607. 318-405 11955 I. V , , Ph D. thesis, Rutgers Univeisity, 1952. Sijderius, R.. Dissertation, Xmsterdam, Holland, 1946. Simmonds, S.,J . Biol. Chem. 174, 717-22 (1948). Sisler. F. D.. ZoBell. C. E.. Science 113, 511-12 (1951). Smvthe, C. V., J . Bioi. Chem. 142, 387-400 (1942). Stadtman, E. R . , “Glutathione,” DD. 191-208. Academic Press, yew York. 1654. Stahl, W. H..McQue, B., others, .4rch. Biochem. 20. 422-32 (1949). Starkey, R. L., Abstract, Pr’oc. 6th Intern. .lficrobioZ. Congress, Rome 7, 347-9 11953). Starkey, R.L.,’ Antonie t’. Leeuicenhoek 12, 193-203 (1947). (74) Starkey, R. L., A4rch. .Mikrobiol. 9, 268-304 (1938). (75) Starkey, R. L., J . Bacieriol. 28, 387400 (1934). (76) Starkev. R. L.. J . Bacteriol. 33, 54571 (i937). (. 7 7.) Starkev. R . L., J . Gen. Physiol. 18, 325-49 (1933 j. (78) Starkey, R. L., Soil Sci. 39, 197-219 (1935). ( 7 9 ) Starkey, R. L., Segal, W., hlanaker, R . A , , .4bstract, Proc. 6th Intern. Alficrobiol. Congress, Rome 1, 256-7 (1953). (80) Starkey, R. L., FYight, K. M., Am. Gas. Assoc. Tech. Rept. Distribution Comm. (1945), 1-108. 181) Subba Rao, XI, S., Dissertation, Indian Institute of Sciences, Bangalore, 1951. ( 8 2 ) Tamiya, iX.,J. Chem. Sac. Japan, Pure Chem. Sect. 72, 118-21, 1214 (1951). \
j
,
I
WATER PURIFICATION ( 8 3 ) Tanko, B., Biochem. 2. 247, 486-90 (1932). ( 8 5 ) Tanner, F. W., J. Am. Chem. SOC. 40, 663-9 (1918). ( 8 4 ) Tanner,\F. W., J . Bacteriol. 2, 585-93 (1917 I. ( 8 6 ) Tarr, H. L A , , Biochem. J . 27, ’596 3 11917) Ibid., pp. 1869-74. Taylor, R. E.: Louisiam Geol. S u m y , Geol. Bull. 11, 1-191 (1938). Temple, K. L., Colmer, .A. R., J . Bacteriol. 62, 605-11 (1951). Trautwein, K., Centr. Bakteriol. Parasitmi. 53; 513-48 (1921 1. (91) van Niel, C. B., Adcances in En:.ymo/. 1. 263-328 119411. (92) van’Niel, C. B:: Am.’Sciel:tist 3 i , 3’183 (1949). ~
(93) van Niel, C. B., .4rch. .Mikrobiol. 3, 1-112 (1931). ( 9 4 ) Ihid., i , 323-358 (1936). (95) Vishniac, \V., J. Bactel-iol. 64, 363-73 (1952). ( 9 6 ) von Wolzogen Kuhr, C. A. H., Tl’ater (Holland) 22, 33-8, 45-8 (1958). ( 9 7 ) von \Volzogen Kuhr. C. .A. H.? van der Vlugt. L. S., Ihid., 16, 147-65 (1934). 198) IL’all, J. S., ivagenknecht, A. C., others, J . Bacteriol. 63, 563-73 (1 952 1. ( 99) \V;nogradsky, S., . 4 m . Inst. Pasteur 3, 50-60 (1889). (100) Ft’inogradsky: S., “Beitrage zur hforphologie und Physiologie der
I--?
DISCUSSION
Bacterien,” vol. I, Arthur Felix, T . p ir--c n y i p (18881. - - -, LVKogradksy, S., Z . Botan. 45, 489-507, 513-23, 529-39, 545-59, 569-76, 585-94, 606-10 (1887). Wohlgemuth, I., Hoppe-Seyler’s Z. Physiol. Chem. 43,469-75 (1905). Woods, D. D., Clifton, (2. C., Biociiem. J . 31, 1774-88 (1937). Youatt. J . B,’, J . Gen. r2ficrohiol. 11, 139-49 (1954). RECEIVED for review January 12, 1956 ACCEPTED April 18, 1956 Journal Series Paper, Xew Jersey Agricultural Experiment Station, Rutgers University, State University of N. J., New Brunswick, Department of .\qicultural Xficrobiology. \
~
...
Transformations of Sulfur D r . Starkey has presented a ver); thorough account of the role of microorganisms in the transformations of sulfur. The numerous valence states of sulfur? the widespread occurrence of this interesting element in diverse organic and inorganic combinations, and the multiplicity of the microorganisms that can be called sulfur bacteria combine to make this a large topic. Of particular inrerest is the recent work on the decomposition of organic sulfur compounds, a hitherto somelvhat neglected field. The practical applications of microbiological transformations of sulfur are of considerable economic importance. One application that should be mentioned concerns the presence of sulfate reducers in the Ivater (usually a brine) that is produced from oil wells. -4 common disposal of this produced brine is reinjection into the oil-bearing formation. 4 s well as being a means of disposal, water injection is used in water flooding of oil formations to increase the amount of oil obtained from the deposit. In the latter case fresh water is usually mixed with the produced water.’ Sulfate-reducing bacteria are commonly found in large numbers in such water systems. Troublesome corrosion in tanks and pipes may be associated with the presence of the bacteria, while a t the point of injection of the water into the sand or limestone oilbearing formation, a n accumulation of iron sulfide and bacterial slime may cause a resistance to flow that makes water injection impractical. I t is not known with certainty to what extent this plugging action is due to the bacteria, but backwashings often show large numbers of sulfate reducers in the injection well. Definitive studies in regard to the contribution of Desulfovibrio desuyuricans to both corrosion and plugging are badly
by Microorganisms
needed. The cost of controlling the bacteria by adding inhibitors to the tvater is a n expense large enough to require the most careful consideration and justifies studies to determine the actual bacterial damage in the different types of water s)-stems used? the different kinds of oil-bearing strata involved, and the expected life and productive capacity of an oil field. An application of even greatereconomic significance is found in the oxidative activities of bacteria associated with the mine waters in contact bvith pyrite and related minerals. Very large numbers of the sulfur-oxidizing bacterium Thiobacillus thiooxidans and the iron-oxidizing bacterium Thiobacillus ferrooxidans exist on the surfaces of damp shales that contain finely divided pyrite. I n the most probable sequence of reactions, iron disulfide is oxidized to ferrous sulfate by a n initial chemical process. The ferrous sulfate is oxidized to ferric sulfate in the acid solution by T.ferrooxidans. As rapidly as the ferric sulfate is formed, it reacts with rhe finely divided iron disulfide present to produce more ferrous sulfate. Elemental sulfur which also results from the reaction between iron disulfide and ferric sulfate is oxidized by T . thiooxiduns to sulfate. The acidity favors the development of these two unusual bacterial species. If a large amount of iron disulfide is present as is the case in the shales and rider coals, associated with some of the major coal seams, enough ferrous sulfate accumulates in the water that passes through a mine to make the effluent highly acid. For example, pH values from 2 to 3 and ferrous iron concentrations of 2000 p.p.m. are known. In highly eroded country where most of the mines are in coal seams that outcrop on a hillside, the volume of water
that passes through a mine is often large. As more mines are opened, the number of surface streams receiving ferrous sulfate increases, and the amount added to the streams increases. Worked out mines continue to add ferrous sulfate to water passing through them long after ownership has ieverted to the state. -4s the polluted water moves downstream the acidity is gradually neutralized by carbonates, and the ferrous sulfate is rapidly oxidized to the ferric state and precipitates on the stream brd. The resulting water is permanently hard because of the high calcium sulfate level that results. Fortunately, not all coal seams produce acid, and many make none. The approach to control is through engineering methods to control water flow and to minimize the exposure of the iron disulfides to air. This can be done best with strip mines. For a discussion of practical methods the publications of S. A. Bralev, obtainable from the Mellon Institute, are recommended. Sulfur oxidation bv the action of bacteria on copper sulfide minerals has been used as a means of working low grade copper ores. Originating with studies on waste rock ore dumps, experimental u.ork showed that bacteria produced soluble sulfates of copper and iron from chalcopyrite, covellite, chalcocite, bornite, tetrahedrite, float concentrate, and pure copper sulfide. Moist aerobic conditions and a nitrogen supply furnished suitable conditions for the oxidative process ( 7 ) . literature Cited (1) Bryner, L. C., Beck, J. V., others, IND.ENG.CHEM.46, 2587 (1954).
KENNETH L. TEMPLE Montana State College, Bozeman, Mont. VOL. 48, NO. 9
SEPTEMBER 1956
1437
