Microb Ecol (1987) 13:47-57
MICROBIAL ECOLOGY 9 Springer-VerlagNew York Inc. 1987
Degradation of Substituted Thiophenes by Bacteria Isolated from Activated Sludge Takahiro Kanagawa* and Don P. Kelly Department of EnvironmentalSciences, Universityof Warwick, CoventryCV4 7AL, England Abstract. Actinomycetes were isolated from activated sludge acclimated to thiophene-2-carboxylic acid (T2C) or 5-methyl-thiophene-2-carboxylic acid (T5M2C). These isolates were apparently identical and were identified as strains of Rhodococcus. The strains could grow on T2C, T5M2C, or thiophene-2-acetic acid as sole sources of carbon and energy, but could not use thiophene, methyl thiophenes, several other substituted thiophenes, dibenzothiophene, dimethyl sulfide, or pyrrole-2-carboxylic acid. T2C was degraded quantitatively to sulfate, and its carbon was converted almost entirely to cell biomass and carbon dioxide. Growth yields indicated about 25% conversion of T2C-carbon to cell-carbon. Growth was not supported by thiosulfate or methionine, nor were these compounds oxidized. Rhodococcus strain TTD-1 grown on T2C oxidized both T2C and T5M2C with an apparent Km of 1.3 x 10 -5 M. Sulfide was also oxidized by T2C-grown organisms. This is the first demonstration of an actinomycete capable of the complete degradation of thiophene derivatives and of their use by it as sole substrates for growth.
Introduction There is considerable current interest in the microbiological degradation of organic sulfur compounds, such as thiophenes and their derivatives, because of their importance as potentially polluting components of coals and oils [ 18]. Most of the published work has been concerned with the breakdown of dibenzothiophene and similar sulfur heterocycles [6, 10, 13, 22]. In some cases the benzothiophenes have been shown to be only partially oxidized by Beijerinckia and Pseudomonas strains, with no breakdown of the thiophene ring by the bacteria [14, 16, 19, 26]. Sulfate was, however, produced by Sulfolobus [10], suggesting that more complete oxidation may be possible. It seems likely that the Pseudomonas strains catalyzed direct oxygenation of dibenzothiophene, resulting in the production of water-soluble hydroxylated derivatives. One Pseudomonas also oxidized methylated thiophenes [22] but the end products * Present address: FermentationResearch Institute,Agencyof Industrial Scienceand Technology, Yatabe, Ibaraki 305, Japan
48
T. Kanagawa and D. P. Kelly
were n o t identified. T h e r e a p p e a r s to h a v e b e e n little s t u d y o f t h e m i c r o b i o l o g y o f the b r e a k d o w n o f c a r b o x y l i c acid o r m e t h y l a t e d d e r i v a t i v e s o f t h i o p h e n e . C r i p p s [4]; ( C r i p p s RE, 1971, P h . D . T h e s i s , U n i v e r s i t y o f W a r w i c k , U K ) isol a t e d a y e l l o w - p i g m e n t e d r o d ( o r g a n i s m R 1) t h a t grew o n t h i o p h e n e - 2 - c a r b o x ylic acid (T2C), t h i o p h e n e - 2 , 5 - c a r b o x y l i c acid, a n d f u r a n - 2 - c a r b o x y l i c acid. B r e a k d o w n o f the T 2 C was a p p a r e n t l y c o m p l e t e , p r o d u c i n g sulfate, C O , f r o m the c a r b o x y l a t e g r o u p , a n d 2 - o x o g l u t a r a t e t h a t c o u l d u n d e r g o t e r m i n a l oxid a t i o n b y the t r i c a r b o x y l i c acid cycle. A n o t h e r o r g a n i s m , p r o b a b l y a Flavobacterium, t h a t was also a b l e to d e g r a d e T 2 C was i s o l a t e d i n d e p e n d e n t l y b y A m p h l e t t a n d C a l l e l y [1]; ( A m p h l e t t M J , 1968, P h . D . T h e s i s , U n i v e r s i t y o f Cardiff, U K ) . A n a e r o b i c b r e a k d o w n o f t h i o p h e n e to H2S b y s e v e r a l b a c t e r i a l isolates has also b e e n r e p o r t e d [15]. This study was u n d e r t a k e n to d e t e r m i n e whether bacteria were present in a c t i v a t e d sludge t h a t were a b l e to use s u b s t i t u t e d t h i o p h e n e s as single s u b s t r a t e s , to i d e n t i f y the t y p e s o f o r g a n i s m i n v o l v e d , a n d to define the r a n g e o f c o m p o u n d s metabolized.
Materials and Methods
Media Basal medium B consisted of 2 g of K2HPO,, 2 g of KH2POa, 0.4 g of NH,CI, 0.4 g of Na2COa, 0.2 g of MgSO4' 7H~O, and 2 ml of trace metal solution [24] in 1 liter of distilled water. Medium BV was prepared by adding 5 ml of vitamin mixture [9] to 1 liter of medium B. Low-sulfate Basal Medium C was the same as Medium BV, except it contained MgC12.6H20 (0.2 g/liter) instead of magnesium sulfate and only 1 ml of trace metal solution per liter. Medium D contained (g/liter) K2HPO4 (6.96), KH2PO, (1.26), NH4C1(0.4), MgC12'6H20 (0.2), and (ml/liter) trace metal solution (1.0), vitamin mixture (5.0). Medium E contained half the phosphate concentration of Medium D. Agar plates were prepared by addition of 1.5% (w/v) agar to Medium BV. Silica gel plates and slants were prepared by sterilizing 90 ml colloidal silica (Snowtex 20, Nissan Chemical Industries, Ltd., Tokyo, Japan) at 120~ for 10 min and, after cooling, adding 6 ml of a separately sterilized solution of 0.2 g K2HPO4, 0.2 g KH2PO,, and 0.04 g Na2CO3. The mixture was then neutralized by the addition of about 2.2 ml 1.7 M HC1 (167 ml cone HCI/liter) and supplemented with the following sterile solutions: 1 ml 4% NH4C1, 0.5 ml 4% MgC12.6H20, 0.1 ml trace metal solution, 0.5 ml vitamin mixture, and sterilized substrate solution. The medium (25 ml portions) was placed in Petri dishes or (10 ml) into universal bottles to make slants. The mixture solidified after 2 or 3 days at room temperature.
Acclimatization of Activated Sludge Activated sludge ("return sludge") from a sewage treatment plant (Minworth, Birmingham, England) was diluted (1 + 2) with tap water, filtered through muslin, washed three times by decantation with tap water, and replicate amounts (100 ml) allowed to settle in 500 ml bottles. The upper phase (30 ml) was removed and replaced by 30 ml of a medium containing (g/liter of tap water) K2HPO4, 0.2; KH2PO4, 0.2; MgSO4"7H20, 0.1; NH4CI, 0.1; yeast extract, 0.1. The unsealed bottles were shaken for 24 hours at 30~ allowed to settle (30 min), 30 ml of the upper phase was removed and replaced with 30 ml of medium containing (0.5 ml or 0.5 g/liter) one of the following compounds: thiophene, 2-methyl thiophene, 3-methyl-thiophene, thiophene-2-carboxylic acid (T2C) and 5-methyl-thiophene-2-carboxylic acid (T5M2C). Bottles were sealed and shaken at 30*(2 for
Bacterial Growth on Substituted Thiophenes
49
about one month with replacement of 30 ml with fresh medium as above at 2-3 day intervals. Decreases of pH were observed in activated sludge acclimated to T2C and T5M2C, but not with the other substrates. After 29 days, 5 ml of the acclimated sludge was inoculated into 50 ml of Medium B containing T2C or T5M2C (0.5 g/liter) in 300 ml flasks. After shaking for 7 days at 300C these cultures were streaked on agar plates o f medium BV containing T2C or T5M2C (0.5 g/liter). The plates were incubated at 30"C for 5 days. Colonies on the plates were subcultured onto other agar plates and silica gel plates. The growth of colonies on the silica gel plates was much better than that on the agar plates. Colonies were purified by three serial subcultures on silica gel plates then inoculated into 50 ml Medium C containing T2C or T5M2C (1 g/liter). After 6 days, the cultures were streaked on silica gel plates. One strain was obtained from the sludge acclimated to T2C and two from that acclimated to T5M2C. These were coded TTD- 1, TTD-2, and TTD-3 respectively and strains TTD- 1 and TTD-3 were partially characterized by the National Collection oflndustrial and Marine Bacteria (Aberdeen, Scotland).
Culture Conditions Isolated strains were maintained on silica gel slants containing T2C or T5M2C (0.5 g/liter). Liquid cultures (50 ml) were grown in 300 ml Erlenmeyer flasks shaken at 30"C, unless otherwise described. A chemostat culture was established at 30~ in an LH modular type Series III fermenter (LH Engineering, Slough, England) using a culture volume of 960 ml, with an impeller speed of 1,000 rpm and aeration at 100 ml/min. The culture was maintained at pH 7.5 by automatic titration with 0.25 M K2CO3. After inoculation (11% v/v), the culture was allowed to establish by batch growth on 5 m M T2C for 24 hours before continuous culture was commenced. The basal medium for chemostat culture contained (g/liter) K2HPO4 (1.74), KH2PO4 (0.34), NH,CI (0.3), MgC12' 6H20 (0.15), trace metal solution (0.75 ml), vitamin mixture (3.75 ml).
Measurement of Substrate Oxidation Oxygen uptake was measured with a teflon-coated Clark oxygen electrode cell (Rank Bros., Cambridge, UK), linked to a chart recorder. A culture (900 ml) of strain TTD-1 was grown on 5 mM T2C at pH 7.5. At the end of exponential growth, a further 5 mM T2C was added. After 20 hours (culture absorbance at 660 nm, 0.37), organisms were harvested by centrifuging at 17,700 x g (5"C) for 10 min, washed once with 0.025 M phosphate, pH 7.5, and resuspended in 9 ml of the same buffer to give about 20 mg dry wt/ml. Oxygen uptake was measured at 30~ in a final volume of 3 ml of 0.025 M phosphate, pH 7.5, containing T2C or T5M2C at concentrations between 0.005 and 2 raM. Reaction was initiated by injecting 0.2 ml cell suspension with a syringe. Oxygen uptake with sulfide was assayed at 30~ using 0.5 mM Na2S and 0.8 ml cell suspension in a final volume of 3 ml as before. Reaction was initiated by injecting the sulfide.
Analytical Methods Growth rate was measured as the increase in culture absorbance at 660 nm (A660) using cuvettes of 1 cm light path. To estimate total organic carbon (TOC) in cultures, samples (4 ml) were centrifuged and the supernatant analyzed for dissolved TOC. The cell-pellet was resuspended in 1.5-4 ml 0.025 M KH2PO 4 and used to determine cell-TOC. TOC was measured with a Beckman model 915-B total organic carbon analyzer. Sulfate was determined by atomic absorption spectrophotometry after precipitation as barium sulfate by measurement o f the residual barium, or turbidimetricaily essentially as described by Kelly and Syrett [11]. Thiosulfate was determined eyanolytically [ 12, 21] and methionine by the nitroprusside method [17].
50
T. Kanagawa and D. P. Kelly
Chemicals Dimethyi phosphorodithioate, dimethyl sulfide, pyrrole-2-carboxylic acid, and thiophene and its derivatives were obtained from Aldrich Chemical Co. The thiophene-carboxylic acids were neutralized with NaOH prior to use in media. All other chemicals were obtained from commercial sources.
Results
Identification of Isolates Strain T T D - 1 was isolated from activated sludge acclimated to T 2 C and strains T T D - 2 and T T D - 3 from T 5 M 2 C - a c c l i m a t e d sludge. All three were characterized initially as very similar n o c a r d i o f o r m actinomycetes. Strains T T D - 1 and T T D - 3 were subjected to identification by the National Collections o f Industrial and Marine Bacteria (Torry Research Station, Aberdeen) and were classified as strains o f the actinomycete genus Rhodococcus [8]. Further work was carried out principally with strain T T D - 1 (Table 1). These isolates are believed to be the first o f an actinomycete capable o f degrading thiophene c o m p o u n d s .
Conditions for Growth of Strains TTD- 1 and TTD- 2 G r o w t h rates (#) o f T T D - 1 on T 2 C (0.2 g/liter) were similar using initial p H values o f 7.1-7.9, with a m a x i m u m at about p H 7.5 (g = 0.06 hour-l). G r o w t h rate was decreased at p H values below p H 6.9 (# = 0.04 h o u r -1) with virtually no growth occurring at p H 6.1 and none at p H 5.7. Strain T T D - 2 showed a similar pattern on T 5 M 2 C with best growth observed at p H 7.3-7.7 and no growth at p H 6.1 or below. Strain T T D - 1 grew well in m e d i a containing 0.025 or 0.05 M phosphate, with slightly faster growth in the former, but grew poorly in 0.1 M phosphate. In m e d i u m D, containing 0.05 M phosphate, initially at p H 7.3-7.4, flasks containing a range o f concentrations o f T 2 C were inoculated (4% v/v) with a culture o f strain T T D - 1 , previously grown on T 2 C at 0.2 g/liter. This showed T 2 C to be a potentially toxic substrate, as growth was initiated earliest at the lowest concentration tested (0.2 g/liter). Using 0.2, 0.5, or 1 g T2C/liter, this relative delay lasted only a few hours and was followed by growth at similar rates (~ = 0.087 hour-~). With 2 g/liter, specific growth rate was lowered to 0.048 h o u r -I, and at 5 g/liter a lag o f about 24 hours was followed by growth at a rate o f about 0.005 h o u r -~. N o growth occurred in 7 days at 10 g T2C/liter.
Range of Substrates Supporting Growth of Strain TTD-1 and TTD-2 Both strains grew heterotrophically on glucose, pyruvate, and acetate but were very restricted in their ability to use organic sulfur c o m p o u n d s as growth substrates (Table 2). Only thiophene-2-carboxylic acid, 5-methyl-thiophene-2-
Bacterial Growth on Substituted Thiophenes
51
Table 1. Characteristics of actinomycete TTD-1" Character Colonial morphology (on Lab M Nutrient agar)
Cell morphology Gram stain Motility Spores Growth at 25 ~ 30 ~ 37 ~ 45 ~ Acid production (Glucose P W S ) Catalase Oxidase, Kovacs
O-F glucose Fatty acids Identification
Observation Round, regular, entire, opaque, white, low convex, slightly wrinkled Diameter < 1 mm after 7 days Diffusible brown pigment produced Branching chains of cells; fragmenting mycelium +/variable None No endospores + + +/-
+ Weakly oxidative Major: straight chain saturated and unsaturated acids; tuberculostearic acid also present These characteristics are consistent with strain TTD-1 (and TTD-3) being strains of the genus Rhodococcus Zopf [I0]
Identification carried out by the NCIMB, Torry Research Station
carboxylic acid, and thiophene-2-acetic acid supported growth or were degraded by the organisms. Thiophene, methyl thiophenes, or thiophene-3-carboxylic acid were not metabolized, and the homologue of thiophene-2-carboxylate, pyrrole-2-carboxylic acid, did not support growth. Dimethyl phosphorodithioate, a substance previously shown to be degraded by mixed cultures of Thiobacillus thioparus and a Pseudomonas [9], could not be used, and autotrophic growth on thiosulfate did not occur. Dimethyl sulfide was also not metabolized. The organisms thus did not exhibit physiological capacities for methylotrophic or chemolithotrophic growth.
Test of Cometabolism of Organic Sulfur Compounds by Strain TTD- 1 Growing on T2C Medium D (50 ml) containing T2C at 0.2 g/liter was supplemented with one of a range of compounds and inoculated (10% v/v) with strain TTD-1 previously grown on T2C alone. Growth was monitored as increase in A66o. The control culture reached maximum turbidity in 2 days and subsequently declined (Table 3). It was apparent that there was no cometabolism of any of the compounds that did not support growth when supplied as sole substrates (Table 3), and that most of the compounds were not significantly inhibitory to growth on T2C. Thiophene-2-acetic acid did cause slower growth when supplied with T2C (Table 3), but when used at 0.2 g/liter, the A66o produced was equivalent
52
T. Kanagawa and D. P. Kelly
Table 2. Substrates supporting the growth of actinomycete strains TTD-1 and TTD-2
Substrate Glucose Pyruvate Acetate DMDTP~ (CH~)2S Na2S203 Thiophene 3-Methyl thiophene 2-Methyl thiophene Thiophene-3-carboxylate Thiophene-2-carboxylate Thiophene-3-ethanol Thiophene-3-acetic acid Thiophene-2-acetic acid Thiophene-2-methylamine 5-Methyl-thiophene-2-carboxylate Pyrrole-2-carboxylic acid Benzothiophene
Concentration (g/liter or ml/liter)
Final pH of cultured Growth ~
TTD- 1
TTD-2
2.0 0.125 2.0 0.5 0. I ml 2.2 0.1 ml 0.1 ml 0.1 ml 0.5 0.5 0.1 ml 0. I 0.05 b 0.1 ml
++ + ++ + + -
6.91 7.41 8.82 7.26 7.28 7.28 7.28 7.28 7.28 7.26 6.77 7.22 7.29 7.17 b 7.28
6.93 7.36 8.74 7.21 7.24 7.25 7.24 7.25 7.24 7.23 6.90 7.18 n.d." n.d. n.d.
0.5 0.5 0.1
+ -
6.77 7.22 7.28
6.99 n.d. 7.25
"Dimethyl phosphorodithioate ~Toxic substrate: sequential additions of 0.05 g/liter made after 9, 13, and 17 days; pH was measured after 20 days c +, positive growth within 5 days; + +, abundant growth within 5 days; --7 negative results recorded after 12 days a Initial pH 7.2-7.3, using Medium C "n.d., not determined to the s u m o f t h a t o f the two s u b s t r a t e s separately, a l t h o u g h its i n i t i a l l y i n h i b itory effect was also a p p a r e n t .
Growth of Strain TTD-1 in Batch and Continuous Culture A c u l t u r e (900 ml) o f T T D - 1 i n a o n e - l i t e r a e r a t e d vessel was g r o w n o n 4.8 m M T 2 C w i t h p H c o n t r o l l e d at p H 7.5. G r o w t h (increase i n A66o), p r o d u c t i o n o f n e w l y a s s i m i l a t e d c e l l - T O C , p r o d u c t i o n o f sulfate, d e c r e a s e i n d i s s o l v e d T O C , a n d decrease i n T 2 C c o n c e n t r a t i o n ( m e a s u r e d b y its U V a b s o r b a n c e i n the c u l t u r e s o l u t i o n ) o c c u r r e d c o n c u r r e n t l y (Fig. 1). E x h a u s t i o n o f T 2 C a n d c e s s a t i o n o f sulfate p r o d u c t i o n o c c u r r e d w h e n g r o w t h (as i n c r e a s e i n A66o a n d c e l I - T O C ) was o n l y a b o u t 60% c o m p l e t e , a l t h o u g h d e c r e a s e i n d i s s o l v e d T O C p a r a l l e l e d g r o w t h u n t i l it ceased (Fig. 1). T h i s i n d i c a t e d t h a t s e c o n d a r y o r g a n i c s u b s t r a t e s were p r o d u c e d f r o m T 2 C d u r i n g its d e g r a d a t i o n . Sulfate p r o d u c t i o n p a r a l l e l e d d i s a p p e a r a n c e o f T 2 C , w i t h the p r o d u c t i o n o f 4.6 (___0.4) m M sulfate f r o m 4.8 m M T 2 C . T h i s s t o i c h i o m e t r y was c o n f i r m e d i n f u r t h e r e x p e r i m e n t s w i t h 2 o r 5 m M T 2 C . T h e rate o f i n c r e a s e i n A660 (Fig. 1) a p p e a r e d to accelerate
Bacterial Growth on Substituted Thiophenes
53
Table 3. Growth of actinomycete strain TTD-1 in media containing thiophene2-carboxylate and a range of other organic sulfur compounds Concentration (g/liter or ml/liter)
Supplementary compound None Thiophene 2-Methyl thiophene 3-Methyl thiophene Thiophene-3-carboxylate Thiophene-3-ethanol Thiophene-3-acetic acid Thiophene-2-acetic acid Thiophene-2-methylamine Benzothiophene Thiosulfate Dimethyl sulfide None Pyrrole-2-carboxylic acid
Culture absorbannce (660 nm) after
-0.1 ml 0.1 ml 0.1 ml 0.2 0.1 ml 0.2 0.2 0.1 ml 0.1 1.25 0.1 ml -0.11
2 days
5 days
9 days
0.122 0.128 0.121 0.125 0.111 0.144 0.014 0.068 0.128 0. I 12 0.110 0.054 0.167 0.028
0.095 0.096 0.096 0.096 0.093 0.096 0.022 0.152 0.108 0.096 0.095 0.045 0.111 0.178
0.088 0.094 0.094 0.091 0.084 0.094 0.061 0.196 0.094 0.094 0.096 0.090 0.071 0.138
Cultures were grown at 30*
MICROBIAL ECOLOGY 9 Springer-VerlagNew York Inc. 1987
Degradation of Substituted Thiophenes by Bacteria Isolated from Activated Sludge Takahiro Kanagawa* and Don P. Kelly Department of EnvironmentalSciences, Universityof Warwick, CoventryCV4 7AL, England Abstract. Actinomycetes were isolated from activated sludge acclimated to thiophene-2-carboxylic acid (T2C) or 5-methyl-thiophene-2-carboxylic acid (T5M2C). These isolates were apparently identical and were identified as strains of Rhodococcus. The strains could grow on T2C, T5M2C, or thiophene-2-acetic acid as sole sources of carbon and energy, but could not use thiophene, methyl thiophenes, several other substituted thiophenes, dibenzothiophene, dimethyl sulfide, or pyrrole-2-carboxylic acid. T2C was degraded quantitatively to sulfate, and its carbon was converted almost entirely to cell biomass and carbon dioxide. Growth yields indicated about 25% conversion of T2C-carbon to cell-carbon. Growth was not supported by thiosulfate or methionine, nor were these compounds oxidized. Rhodococcus strain TTD-1 grown on T2C oxidized both T2C and T5M2C with an apparent Km of 1.3 x 10 -5 M. Sulfide was also oxidized by T2C-grown organisms. This is the first demonstration of an actinomycete capable of the complete degradation of thiophene derivatives and of their use by it as sole substrates for growth.
Introduction There is considerable current interest in the microbiological degradation of organic sulfur compounds, such as thiophenes and their derivatives, because of their importance as potentially polluting components of coals and oils [ 18]. Most of the published work has been concerned with the breakdown of dibenzothiophene and similar sulfur heterocycles [6, 10, 13, 22]. In some cases the benzothiophenes have been shown to be only partially oxidized by Beijerinckia and Pseudomonas strains, with no breakdown of the thiophene ring by the bacteria [14, 16, 19, 26]. Sulfate was, however, produced by Sulfolobus [10], suggesting that more complete oxidation may be possible. It seems likely that the Pseudomonas strains catalyzed direct oxygenation of dibenzothiophene, resulting in the production of water-soluble hydroxylated derivatives. One Pseudomonas also oxidized methylated thiophenes [22] but the end products * Present address: FermentationResearch Institute,Agencyof Industrial Scienceand Technology, Yatabe, Ibaraki 305, Japan
48
T. Kanagawa and D. P. Kelly
were n o t identified. T h e r e a p p e a r s to h a v e b e e n little s t u d y o f t h e m i c r o b i o l o g y o f the b r e a k d o w n o f c a r b o x y l i c acid o r m e t h y l a t e d d e r i v a t i v e s o f t h i o p h e n e . C r i p p s [4]; ( C r i p p s RE, 1971, P h . D . T h e s i s , U n i v e r s i t y o f W a r w i c k , U K ) isol a t e d a y e l l o w - p i g m e n t e d r o d ( o r g a n i s m R 1) t h a t grew o n t h i o p h e n e - 2 - c a r b o x ylic acid (T2C), t h i o p h e n e - 2 , 5 - c a r b o x y l i c acid, a n d f u r a n - 2 - c a r b o x y l i c acid. B r e a k d o w n o f the T 2 C was a p p a r e n t l y c o m p l e t e , p r o d u c i n g sulfate, C O , f r o m the c a r b o x y l a t e g r o u p , a n d 2 - o x o g l u t a r a t e t h a t c o u l d u n d e r g o t e r m i n a l oxid a t i o n b y the t r i c a r b o x y l i c acid cycle. A n o t h e r o r g a n i s m , p r o b a b l y a Flavobacterium, t h a t was also a b l e to d e g r a d e T 2 C was i s o l a t e d i n d e p e n d e n t l y b y A m p h l e t t a n d C a l l e l y [1]; ( A m p h l e t t M J , 1968, P h . D . T h e s i s , U n i v e r s i t y o f Cardiff, U K ) . A n a e r o b i c b r e a k d o w n o f t h i o p h e n e to H2S b y s e v e r a l b a c t e r i a l isolates has also b e e n r e p o r t e d [15]. This study was u n d e r t a k e n to d e t e r m i n e whether bacteria were present in a c t i v a t e d sludge t h a t were a b l e to use s u b s t i t u t e d t h i o p h e n e s as single s u b s t r a t e s , to i d e n t i f y the t y p e s o f o r g a n i s m i n v o l v e d , a n d to define the r a n g e o f c o m p o u n d s metabolized.
Materials and Methods
Media Basal medium B consisted of 2 g of K2HPO,, 2 g of KH2POa, 0.4 g of NH,CI, 0.4 g of Na2COa, 0.2 g of MgSO4' 7H~O, and 2 ml of trace metal solution [24] in 1 liter of distilled water. Medium BV was prepared by adding 5 ml of vitamin mixture [9] to 1 liter of medium B. Low-sulfate Basal Medium C was the same as Medium BV, except it contained MgC12.6H20 (0.2 g/liter) instead of magnesium sulfate and only 1 ml of trace metal solution per liter. Medium D contained (g/liter) K2HPO4 (6.96), KH2PO, (1.26), NH4C1(0.4), MgC12'6H20 (0.2), and (ml/liter) trace metal solution (1.0), vitamin mixture (5.0). Medium E contained half the phosphate concentration of Medium D. Agar plates were prepared by addition of 1.5% (w/v) agar to Medium BV. Silica gel plates and slants were prepared by sterilizing 90 ml colloidal silica (Snowtex 20, Nissan Chemical Industries, Ltd., Tokyo, Japan) at 120~ for 10 min and, after cooling, adding 6 ml of a separately sterilized solution of 0.2 g K2HPO4, 0.2 g KH2PO,, and 0.04 g Na2CO3. The mixture was then neutralized by the addition of about 2.2 ml 1.7 M HC1 (167 ml cone HCI/liter) and supplemented with the following sterile solutions: 1 ml 4% NH4C1, 0.5 ml 4% MgC12.6H20, 0.1 ml trace metal solution, 0.5 ml vitamin mixture, and sterilized substrate solution. The medium (25 ml portions) was placed in Petri dishes or (10 ml) into universal bottles to make slants. The mixture solidified after 2 or 3 days at room temperature.
Acclimatization of Activated Sludge Activated sludge ("return sludge") from a sewage treatment plant (Minworth, Birmingham, England) was diluted (1 + 2) with tap water, filtered through muslin, washed three times by decantation with tap water, and replicate amounts (100 ml) allowed to settle in 500 ml bottles. The upper phase (30 ml) was removed and replaced by 30 ml of a medium containing (g/liter of tap water) K2HPO4, 0.2; KH2PO4, 0.2; MgSO4"7H20, 0.1; NH4CI, 0.1; yeast extract, 0.1. The unsealed bottles were shaken for 24 hours at 30~ allowed to settle (30 min), 30 ml of the upper phase was removed and replaced with 30 ml of medium containing (0.5 ml or 0.5 g/liter) one of the following compounds: thiophene, 2-methyl thiophene, 3-methyl-thiophene, thiophene-2-carboxylic acid (T2C) and 5-methyl-thiophene-2-carboxylic acid (T5M2C). Bottles were sealed and shaken at 30*(2 for
Bacterial Growth on Substituted Thiophenes
49
about one month with replacement of 30 ml with fresh medium as above at 2-3 day intervals. Decreases of pH were observed in activated sludge acclimated to T2C and T5M2C, but not with the other substrates. After 29 days, 5 ml of the acclimated sludge was inoculated into 50 ml of Medium B containing T2C or T5M2C (0.5 g/liter) in 300 ml flasks. After shaking for 7 days at 300C these cultures were streaked on agar plates o f medium BV containing T2C or T5M2C (0.5 g/liter). The plates were incubated at 30"C for 5 days. Colonies on the plates were subcultured onto other agar plates and silica gel plates. The growth of colonies on the silica gel plates was much better than that on the agar plates. Colonies were purified by three serial subcultures on silica gel plates then inoculated into 50 ml Medium C containing T2C or T5M2C (1 g/liter). After 6 days, the cultures were streaked on silica gel plates. One strain was obtained from the sludge acclimated to T2C and two from that acclimated to T5M2C. These were coded TTD- 1, TTD-2, and TTD-3 respectively and strains TTD- 1 and TTD-3 were partially characterized by the National Collection oflndustrial and Marine Bacteria (Aberdeen, Scotland).
Culture Conditions Isolated strains were maintained on silica gel slants containing T2C or T5M2C (0.5 g/liter). Liquid cultures (50 ml) were grown in 300 ml Erlenmeyer flasks shaken at 30"C, unless otherwise described. A chemostat culture was established at 30~ in an LH modular type Series III fermenter (LH Engineering, Slough, England) using a culture volume of 960 ml, with an impeller speed of 1,000 rpm and aeration at 100 ml/min. The culture was maintained at pH 7.5 by automatic titration with 0.25 M K2CO3. After inoculation (11% v/v), the culture was allowed to establish by batch growth on 5 m M T2C for 24 hours before continuous culture was commenced. The basal medium for chemostat culture contained (g/liter) K2HPO4 (1.74), KH2PO4 (0.34), NH,CI (0.3), MgC12' 6H20 (0.15), trace metal solution (0.75 ml), vitamin mixture (3.75 ml).
Measurement of Substrate Oxidation Oxygen uptake was measured with a teflon-coated Clark oxygen electrode cell (Rank Bros., Cambridge, UK), linked to a chart recorder. A culture (900 ml) of strain TTD-1 was grown on 5 mM T2C at pH 7.5. At the end of exponential growth, a further 5 mM T2C was added. After 20 hours (culture absorbance at 660 nm, 0.37), organisms were harvested by centrifuging at 17,700 x g (5"C) for 10 min, washed once with 0.025 M phosphate, pH 7.5, and resuspended in 9 ml of the same buffer to give about 20 mg dry wt/ml. Oxygen uptake was measured at 30~ in a final volume of 3 ml of 0.025 M phosphate, pH 7.5, containing T2C or T5M2C at concentrations between 0.005 and 2 raM. Reaction was initiated by injecting 0.2 ml cell suspension with a syringe. Oxygen uptake with sulfide was assayed at 30~ using 0.5 mM Na2S and 0.8 ml cell suspension in a final volume of 3 ml as before. Reaction was initiated by injecting the sulfide.
Analytical Methods Growth rate was measured as the increase in culture absorbance at 660 nm (A660) using cuvettes of 1 cm light path. To estimate total organic carbon (TOC) in cultures, samples (4 ml) were centrifuged and the supernatant analyzed for dissolved TOC. The cell-pellet was resuspended in 1.5-4 ml 0.025 M KH2PO 4 and used to determine cell-TOC. TOC was measured with a Beckman model 915-B total organic carbon analyzer. Sulfate was determined by atomic absorption spectrophotometry after precipitation as barium sulfate by measurement o f the residual barium, or turbidimetricaily essentially as described by Kelly and Syrett [11]. Thiosulfate was determined eyanolytically [ 12, 21] and methionine by the nitroprusside method [17].
50
T. Kanagawa and D. P. Kelly
Chemicals Dimethyi phosphorodithioate, dimethyl sulfide, pyrrole-2-carboxylic acid, and thiophene and its derivatives were obtained from Aldrich Chemical Co. The thiophene-carboxylic acids were neutralized with NaOH prior to use in media. All other chemicals were obtained from commercial sources.
Results
Identification of Isolates Strain T T D - 1 was isolated from activated sludge acclimated to T 2 C and strains T T D - 2 and T T D - 3 from T 5 M 2 C - a c c l i m a t e d sludge. All three were characterized initially as very similar n o c a r d i o f o r m actinomycetes. Strains T T D - 1 and T T D - 3 were subjected to identification by the National Collections o f Industrial and Marine Bacteria (Torry Research Station, Aberdeen) and were classified as strains o f the actinomycete genus Rhodococcus [8]. Further work was carried out principally with strain T T D - 1 (Table 1). These isolates are believed to be the first o f an actinomycete capable o f degrading thiophene c o m p o u n d s .
Conditions for Growth of Strains TTD- 1 and TTD- 2 G r o w t h rates (#) o f T T D - 1 on T 2 C (0.2 g/liter) were similar using initial p H values o f 7.1-7.9, with a m a x i m u m at about p H 7.5 (g = 0.06 hour-l). G r o w t h rate was decreased at p H values below p H 6.9 (# = 0.04 h o u r -1) with virtually no growth occurring at p H 6.1 and none at p H 5.7. Strain T T D - 2 showed a similar pattern on T 5 M 2 C with best growth observed at p H 7.3-7.7 and no growth at p H 6.1 or below. Strain T T D - 1 grew well in m e d i a containing 0.025 or 0.05 M phosphate, with slightly faster growth in the former, but grew poorly in 0.1 M phosphate. In m e d i u m D, containing 0.05 M phosphate, initially at p H 7.3-7.4, flasks containing a range o f concentrations o f T 2 C were inoculated (4% v/v) with a culture o f strain T T D - 1 , previously grown on T 2 C at 0.2 g/liter. This showed T 2 C to be a potentially toxic substrate, as growth was initiated earliest at the lowest concentration tested (0.2 g/liter). Using 0.2, 0.5, or 1 g T2C/liter, this relative delay lasted only a few hours and was followed by growth at similar rates (~ = 0.087 hour-~). With 2 g/liter, specific growth rate was lowered to 0.048 h o u r -I, and at 5 g/liter a lag o f about 24 hours was followed by growth at a rate o f about 0.005 h o u r -~. N o growth occurred in 7 days at 10 g T2C/liter.
Range of Substrates Supporting Growth of Strain TTD-1 and TTD-2 Both strains grew heterotrophically on glucose, pyruvate, and acetate but were very restricted in their ability to use organic sulfur c o m p o u n d s as growth substrates (Table 2). Only thiophene-2-carboxylic acid, 5-methyl-thiophene-2-
Bacterial Growth on Substituted Thiophenes
51
Table 1. Characteristics of actinomycete TTD-1" Character Colonial morphology (on Lab M Nutrient agar)
Cell morphology Gram stain Motility Spores Growth at 25 ~ 30 ~ 37 ~ 45 ~ Acid production (Glucose P W S ) Catalase Oxidase, Kovacs
O-F glucose Fatty acids Identification
Observation Round, regular, entire, opaque, white, low convex, slightly wrinkled Diameter < 1 mm after 7 days Diffusible brown pigment produced Branching chains of cells; fragmenting mycelium +/variable None No endospores + + +/-
+ Weakly oxidative Major: straight chain saturated and unsaturated acids; tuberculostearic acid also present These characteristics are consistent with strain TTD-1 (and TTD-3) being strains of the genus Rhodococcus Zopf [I0]
Identification carried out by the NCIMB, Torry Research Station
carboxylic acid, and thiophene-2-acetic acid supported growth or were degraded by the organisms. Thiophene, methyl thiophenes, or thiophene-3-carboxylic acid were not metabolized, and the homologue of thiophene-2-carboxylate, pyrrole-2-carboxylic acid, did not support growth. Dimethyl phosphorodithioate, a substance previously shown to be degraded by mixed cultures of Thiobacillus thioparus and a Pseudomonas [9], could not be used, and autotrophic growth on thiosulfate did not occur. Dimethyl sulfide was also not metabolized. The organisms thus did not exhibit physiological capacities for methylotrophic or chemolithotrophic growth.
Test of Cometabolism of Organic Sulfur Compounds by Strain TTD- 1 Growing on T2C Medium D (50 ml) containing T2C at 0.2 g/liter was supplemented with one of a range of compounds and inoculated (10% v/v) with strain TTD-1 previously grown on T2C alone. Growth was monitored as increase in A66o. The control culture reached maximum turbidity in 2 days and subsequently declined (Table 3). It was apparent that there was no cometabolism of any of the compounds that did not support growth when supplied as sole substrates (Table 3), and that most of the compounds were not significantly inhibitory to growth on T2C. Thiophene-2-acetic acid did cause slower growth when supplied with T2C (Table 3), but when used at 0.2 g/liter, the A66o produced was equivalent
52
T. Kanagawa and D. P. Kelly
Table 2. Substrates supporting the growth of actinomycete strains TTD-1 and TTD-2
Substrate Glucose Pyruvate Acetate DMDTP~ (CH~)2S Na2S203 Thiophene 3-Methyl thiophene 2-Methyl thiophene Thiophene-3-carboxylate Thiophene-2-carboxylate Thiophene-3-ethanol Thiophene-3-acetic acid Thiophene-2-acetic acid Thiophene-2-methylamine 5-Methyl-thiophene-2-carboxylate Pyrrole-2-carboxylic acid Benzothiophene
Concentration (g/liter or ml/liter)
Final pH of cultured Growth ~
TTD- 1
TTD-2
2.0 0.125 2.0 0.5 0. I ml 2.2 0.1 ml 0.1 ml 0.1 ml 0.5 0.5 0.1 ml 0. I 0.05 b 0.1 ml
++ + ++ + + -
6.91 7.41 8.82 7.26 7.28 7.28 7.28 7.28 7.28 7.26 6.77 7.22 7.29 7.17 b 7.28
6.93 7.36 8.74 7.21 7.24 7.25 7.24 7.25 7.24 7.23 6.90 7.18 n.d." n.d. n.d.
0.5 0.5 0.1
+ -
6.77 7.22 7.28
6.99 n.d. 7.25
"Dimethyl phosphorodithioate ~Toxic substrate: sequential additions of 0.05 g/liter made after 9, 13, and 17 days; pH was measured after 20 days c +, positive growth within 5 days; + +, abundant growth within 5 days; --7 negative results recorded after 12 days a Initial pH 7.2-7.3, using Medium C "n.d., not determined to the s u m o f t h a t o f the two s u b s t r a t e s separately, a l t h o u g h its i n i t i a l l y i n h i b itory effect was also a p p a r e n t .
Growth of Strain TTD-1 in Batch and Continuous Culture A c u l t u r e (900 ml) o f T T D - 1 i n a o n e - l i t e r a e r a t e d vessel was g r o w n o n 4.8 m M T 2 C w i t h p H c o n t r o l l e d at p H 7.5. G r o w t h (increase i n A66o), p r o d u c t i o n o f n e w l y a s s i m i l a t e d c e l l - T O C , p r o d u c t i o n o f sulfate, d e c r e a s e i n d i s s o l v e d T O C , a n d decrease i n T 2 C c o n c e n t r a t i o n ( m e a s u r e d b y its U V a b s o r b a n c e i n the c u l t u r e s o l u t i o n ) o c c u r r e d c o n c u r r e n t l y (Fig. 1). E x h a u s t i o n o f T 2 C a n d c e s s a t i o n o f sulfate p r o d u c t i o n o c c u r r e d w h e n g r o w t h (as i n c r e a s e i n A66o a n d c e l I - T O C ) was o n l y a b o u t 60% c o m p l e t e , a l t h o u g h d e c r e a s e i n d i s s o l v e d T O C p a r a l l e l e d g r o w t h u n t i l it ceased (Fig. 1). T h i s i n d i c a t e d t h a t s e c o n d a r y o r g a n i c s u b s t r a t e s were p r o d u c e d f r o m T 2 C d u r i n g its d e g r a d a t i o n . Sulfate p r o d u c t i o n p a r a l l e l e d d i s a p p e a r a n c e o f T 2 C , w i t h the p r o d u c t i o n o f 4.6 (___0.4) m M sulfate f r o m 4.8 m M T 2 C . T h i s s t o i c h i o m e t r y was c o n f i r m e d i n f u r t h e r e x p e r i m e n t s w i t h 2 o r 5 m M T 2 C . T h e rate o f i n c r e a s e i n A660 (Fig. 1) a p p e a r e d to accelerate
Bacterial Growth on Substituted Thiophenes
53
Table 3. Growth of actinomycete strain TTD-1 in media containing thiophene2-carboxylate and a range of other organic sulfur compounds Concentration (g/liter or ml/liter)
Supplementary compound None Thiophene 2-Methyl thiophene 3-Methyl thiophene Thiophene-3-carboxylate Thiophene-3-ethanol Thiophene-3-acetic acid Thiophene-2-acetic acid Thiophene-2-methylamine Benzothiophene Thiosulfate Dimethyl sulfide None Pyrrole-2-carboxylic acid
Culture absorbannce (660 nm) after
-0.1 ml 0.1 ml 0.1 ml 0.2 0.1 ml 0.2 0.2 0.1 ml 0.1 1.25 0.1 ml -0.11
2 days
5 days
9 days
0.122 0.128 0.121 0.125 0.111 0.144 0.014 0.068 0.128 0. I 12 0.110 0.054 0.167 0.028
0.095 0.096 0.096 0.096 0.093 0.096 0.022 0.152 0.108 0.096 0.095 0.045 0.111 0.178
0.088 0.094 0.094 0.091 0.084 0.094 0.061 0.196 0.094 0.094 0.096 0.090 0.071 0.138
Cultures were grown at 30*
