FEMS MicrobiologyLetters 96 (1992) 213-218 © 1992 Federation of European MicrobiologicalSocieties0378-1097/92/$05.00 Published by Elsevier

213

FEMSLE 05046

Heterotrophic sulfur reduction by Thermotoga sp. strain FjSS3.B1 P e t e r H. J a n s s e n

~

and H u g h W. M o r g a n

Thermophile and Microbial Biochemistry and Biotechnology Unit, UnirersiO,of Waikato. PricateBag, Hamilton, New Zealand Received 16 June 1992 Accepted 24 June 1992 Key words: Thermotoga; Glucose fermentation; Hydrogen; Sulfur reduction; End product inhibition

1. S U M M A R Y Thermotoga sp. strain FjSS3.B1 was able to reduce sulfur to sulfide when grown on a mineral medium with glucose as the sole carbon and energy source. There was no increase in specific growth yield coupled to sulfur reduction, but the specific growth rate, final growth yield, and tolerance of H 2 were all increased in the presence of sulfur. At dissolved H 2 concentrations, of 550 to 600 p, m o i / I (at 77°C) growth was not possible unless sulfur was added. Glucose was fermented via the Embden-Meyerhof-Parnas pathway to lactate, acetate, H 2 and CO 2 (and other unidentified minor products). The thermodynamic problems associated with the relatively high redox potential electrons from the 1,3-bisphosphoglycerate/glyceraldehyde 3-phosphate couple ( E 0 = - 350 mV) are overcome by reducing sulfur to sulfide (E~ = - 2 7 0 mV) rather than the energeti-

t Correspondence to: P,H. Janssen. Present address: Fakult~it fiir Biologic, Universifiit Konstanz, Postfaeh 5560, W-7750 Konstanz, FRG.

cally unfavourable production of H 2 (E~ = - 414 mV). Under high hydrogen partial pressures there was increased production of lactate as an alternative electron sink. The results indicate that sulfur reduction operates primarily as an electron sink rather than as a detoxification reaction or energy-generating mechanism.

2. I N T R O D U C T I O N The Thermotogales represent a phylogenetically distinct group of thermophilic obligately anaerobic bacteria which apparently separated early in the evolution of the eubacteria [1,2]. Sulfur reduction is a common characteristic of this group, being carried out by Thermotoga maritima [3], Thermotoga neapolitana [4,5], Thermosipho aJ~icanus [ 1], Fercidobacterium nodosum and F. islandicum [6]. In contrast, Thermotoga thermarum [7] and Thermotoga sp. strain FjSS3.B1 [8] are reportedly not able to reduce elemental sulfur. In those strains which do reduce sulfur, the growth yield and stoichiometry of organic end products are not altered [3,4], and in the presence

214 of sulfur growth inhibition by H 2 appears to be overcome [1-4]. Hydrogen is a major end product of glucose fermentation by members of the Thermotogales, and its accumulation inhibits growth [1,3,6,7]. Addition of elemental sulfur did not overcome the inhibitory effect of H 2 in Fervidobacterium spp. [6]. While investigating Thermotoga sp. strain FjSS3.B1 [8], the ability to reduce sulfur and consequent hydrogen tolerance was reinvestigated. The results are presented in this communication.

3. MATERIALS AND METHODS

Thermotoga sp. strain FjSS3.B1 was isolated from geothermal features of an intertidal habitat [8] and stored as a freeze-dried preparation. For use in the present study the culture was maintained by subculture on glucose-containing medium with 2 g yeast extract/l. All incubations were at 77°C. The medium of Rainey et al. [9] was used, with the NaC! and MgCI2.6H20 concentrations increased to 7.0 and 1.2 g / l respectively, and with the addition of 50 mM MOPS. The final pH was 7.0 at 77°C, adjusted with NaOH. Sulfur was added at 5.0 g/! as noted. Media were autoclaved at ll0°C for 30 rain. Yeast extract was not ineluded unless noted otherwise. Glucose, yeast extract, and calcium-sulfide reductant [9] were added after autoclaving from separately sterilized stock solutions. Sulfide was measured by a mieromethod adaptation of the method of Triiper and Schlegel [10]. The growth yield in the presence of sulfur was calculated from the optical density using data obtained from yield experiments in the absence of sulfur. Growth experiments were carried out in 160-ml serum vials containing 100 ml medium. Calculations of hydrogen sulfide concentrations in the headspaee of culture vessels, and of dissolved H 2 concentrations, were made using data from published tables [11,12]. Samples were withdrawn for optical density measurements at 650 nm (10-ram light path) and HPLC analysis using plastic disposable syringes.

Enzyme assays were carried out at 50°C as described elsewhere [13]. All other methods have been previously described [9,13].

4. RESULTS

Thermotoga sp. strain FjSS3.B1 was able to grow on glucose, fermenting it to acetate, lactate, H 2 and CO 2. These products accounted for 7590% of the substrate carbon. The production of other organic products was therefore implied, as found also by other investigators [3]. Continued subculture on glucose was possible in mineral medium in the absence of added growth factors. The addition of yeast extract or peptone increased the growth yield on glucose. All of the added 10 mM glucose was not used. The pH did not change, and factors for culture growth were not limiting (as shown by cultures growing with sulfur, see below). The accumulation of H 2 apparently inhibited the growth of cultures grown on glucose. No growth was observed on glucose in the absence of sulfur once the dissolved H 2 concentration exceeded 200-250 p,mol/! (at 77°C). When elemental sulfur was included in the growth medium there was production of hydrogen sulfide concomitant with growth and glucose utilization (Fig. la). In addition there was an increase in the rate of glucose utilization, and in the growth rate in the presence of sulfur (Fig. la) in comparison to growth without sulfur (Fig. lb). There was no sulfide production from sulfur when uninoculated medium was incubated at 77°C for a week under an H e headspace. Cultures grown under an He headspace (550600/zmol H 2 dissolved per litre medium at 77°C) could not grow, and only 2% of the glucose was metabolized. Addition of sulfur allowed growth with consequent sulfide production, but no production of H 2 was detectable under these conditions, although production of a small amount of H 2 could not be ruled out (due to the high H 2 background). Addition of sulfur to the growth medium allowed production of more cell material (96.0 mg dry weight/I against 42.1 mg/I in the absence of sulfur), but the specific growth yield was not

215 14

Table 2 Enzyme activities detected in crude cell-free extracts of glucose-grown Thermotoga sp. strain FjSS3.BI

~

-12 1 to

o. to

8

~. U.O8 -~ O.Ob ~

Enzyme

-

)

0.04

!

o

20

i

40

1--

tJO 0

t)

l

I

I

20

40

60

Time Ihl Fig. I. Growth curves of Thermotoga sp. strain FjSS3.BI on glucose in the presence {a), and absence (b), of sulfur. Symbols: (e) culture density at 650 nm, (o) glucose, (A) acetate, ( I ) lactate, (E31sulfide.

increased (10.9 g dry w e i g h t / m o l glucose utilized, against 11.3 in the a b s e n c e o f sulfur). T h e production o f H2 was d e c r e a s e d in the p r e s e n c e of sulfur in f a v o u r o f sulfide p r o d u c t i o n (Table 1). T h e final dissolved H 2 c o n c e n t r a t i o n at 77°C was 147/.tmol/1, while in the a b s e n c e o f sulfur it was 205 / . t m o l / I . If the r e d u c i n g equivalents recove r e d as sulfide had b e e n released as H_,, the final c o n c e n t r a t i o n w o u l d have b e e n 326 g m o l / I , h i g h e r t h a n c o n c e n t r a t i o n s r e a c h e d in the absence o f sulfur. T h e r e was also an increase in the a m o u n t of lactate p r o d u c e d u n d e r an H_, h e a d s p a c e . Cult u r e s g r o w n u n d e r a h e a d s p a c e of (initally) N 2 plus C O 2 p r o d u c e d acetate and lactate in a ratio o f 9.0 to 9.2:1 ( a c e t a t e : l a c t a t e ) , regardless of

Phosphofructokinase + ATP ~' + PPi " G-3-P b dehydrogenase +NAD c + MV c Pyruvate synthase + NAD + MV Formate dehydrogenase + NAD + MV MV: NAD oxidoreductase l-tydrogenase +NAD + MV

EC number

Specific activity (/.tmolmin - l mg- t )

2.7.1.1 i 2.7.1.90

0.404 < 0.001

1.2.1.12 -

3.95 < 0.001

1.2.7.1

< 0.001 0.041

1.2.1.2 1.8.1.3

< 0.001 < 0.001 0.368

1.12.1.2 I. 18.99. I

< 0.001 0.384

The specific activities are expressed per mg protein in the crude cell-free extract, assayed at 50°C. " Tested as phosphoryl-donor. " Glyceraldehyde-3-phosphate. c Tested as electron acceptor.

w h e t h e r sulfur was p r e s e n t . U n d e r a h e a d s p a c e c o n t a i n i n g a high initial partial p r e s s u r e o f H E c o r r e s p o n d i n g to a dissolved H 2 c o n c e n t r a t i o n o f 5 5 0 - 6 0 0 p . m o l / I at 77°C ( m o r e t h a n twice that r e a c h e d d u r i n g glucose f e r m e n t a t i o n ) , t h e r e was an increase in lactate p r o d u c t i o n so that the acetate : lactate ration was d e c r e a s e d to 4.8 : 1. G l u c o s e was f e r m e n t e d by the E m b d e n M e y e r h o f - P a r n a s p a t h w a y as indicated by m e a s u r e m e n t of key e n z y m e s o f this p a t h w a y (Table 2). Pyruvate synthase activity w a s m e a s u r e a b l e using M V as the electron a c c e p t o r but not using

Table 1 Substrate utilization and end-pr('ducts of glucose transformation in the presence and absence of sulfur by Thermotoga sp. strain FjSS3.BI Growth conditions

Glucose used (mmol/I)

Products (mmol/I) Lactate

Acetate

H2

- S° + S° - St) + H 2 + S° + H 2

3.73 8.81 0.24 5.32

0.52 1.09 0.00 1.03

4.06 1(I.II3 0.00 4.98

13.31 9.55 0.00 0.00

Sulfide 13.14 17.113

216 NAD, suggesting ferredoxin (or a similar acceptor) is the physiological acceptor for this reaction. NAD was the electron aeeeptor for the other dehydrogenation reactions measured. Good activity of an NAD:MV oxidoreductase activity was also detected, indicating the capacity for the transfer of reducing equivalents from NAD-coupied reactions to ferredoxin and then to proton reduction to be evolved as H2. Hydrogenase activity was also present.

5. DISCUSSION Growth of Thermotoga sp. strain FjSS3.B1 on glucose always resulted in low cell yields and poor substrate utilization. The linear growth kinetics observed suggested either the limitation of a growth factor, or the accumulation of a growth inhibitory product. An extensive study of potential growth factor supplements, including vita* mins, minerals, amino acids, amino sugars, nueleotides and complex growth supplements failed to revea~ ~ growth factor limitation during growth on glucose (Janssen, unpublished). The addition of sulfur to glucose-grown cultures of Thermotoga sp. strain FjSS3.BI resulted in sulfide production from sulfur reduction not linked to a specific growth yield increase. This characteristic was not reported in the original description of the strain [8]. There were however increases in the total amount of growth obtained in a closed system with excess substrate, proportional increases in the amount of substrate used, and increases in the growth rate. Addition of sulfur also overcame the growth inhibition caused by an H z gas headspace above the culture medium, in agreement with investigations on other strains of Thermotoga spp. [1,3,4], and a number of thermophilic Archaea [14-16], The pathway of glucose fermentation suggested the channelling of some electrons derived from the dehydrogenation steps of the EmbdenMeyerhof-Parnas pathway to reduce protons, yielding H2. Increasing H2 concentrations, however, appear to result in a growth inhibition. When the H 2 concentration totally inhibits hydrogenase activity, sulfur reduction and lactate

production remain the only mechanisms for recycling reduced electron carriers. The oxidation step in the transformation of pyruvate to aeetyI-CoA produces electrons with a low enough potential to reduce ferredoxin directly (E6 = - 4 7 0 mV for the pyruvate/acetylCoA couple, E~'~= - 4 0 0 mV for the ferredoxin ox/red couple). However, electrons derived from the 1,3-bisphosphoglycerate/glyceraldehyde-3phosphate couple (E6 = - 3 5 0 mV) have a potential that is too high to reduce ferredoxin except at low H 2 partial pressures. A number of possibilities exist to overcome this energetic problem. The reaction may proceed if the dissolved H z concentration is kept very low, such as has been shown to occur in mixed cultures of thermophiles in which there is interspecies transfer of H2 [15]. Alternatively, the electrons from the 1,3-bisphosphoglycerate/ glyceraidehyde-3-phosphate couple are able to reduce NAD (E~'~= - 3 2 0 mV) which can then reduce pyruvate to lactate (E~ = - 1 9 0 mV) or sulfur to sulfide (Ec~= - 2 7 0 mV). Reduction of pyruvate would result in an almost homolactic fermentation with consequent loss of the acetylP/acetate ATP-forming step. There was a partial shift to increased lactate production under an H z headspace. This therefore represents another mechanism for reoxidizing reduced electron carriers formed during glucose catabolism, but which only operates at high H 2 partial pressures. Sulfide production has been shown in this study to be possible in Thermotoga sp. strain FjSS3.B1. Electrons derived from pyruvate oxidation can thus also be used to reduce sulfur, as must have been the case when the organism was cultured under an H 2 headspace. Sulfur reduction would appear a better strategy than lactate production for the disposal of electrons since it results in an increased flow of substrate carbon to acetate with consequent substrate level ATP formation. There was no specific yield increase during sulfur reduction implying that sulfur reduction is not itself coupled to ATP formation, for example via a chemiosmotic mechanism. It has been suggested that a number of extremely thermophilic bacteria able to reduce sulfur can do so as a detoxification mechanism to

217 o v e r c o m e t h e inhibition o f growth c a u s e d by high partial p r e s s u r e s o f H 2 [14]. H o w e v e r most o f t h e s e o r g a n i s m s can utilize H E as an e l e c t r o n source and t h e r e f o r e are able to couple t h e oxid a t i o n o f H2 to t h e r e d u c t i o n o f S o to conserve m e t a b o l i c energy. Thermotoga spp., in contrast, p r o d u c e H 2 as a metabolic e n d p r o d u c t to disp o s e o f excess e l e c t r o n s (reducing potential) a n d t h u s it w o u l d b e e x p e c t e d that t h e alternative sink in sulfur r e d u c t i o n w o u l d p r o c e e d directly (facilitated f e r m e n t a t i o n [17]) r a t h e r t h a n as a detoxification m e c h a n i s m [14] with initial H 2 evolution t h e n s u b s e q u e n t u p t a k e to reduce S o. T h e p r e s e n c e o f a sulfur r e d u c t a s e accepting e l e c t r o n s f r o m glucose catabolism without H 2 as an interm e d i a t e r e m a i n s to be s h o w n in Thermotoga spp. T h e ability to grow in t h e p r e s e n c e o f S O u n d e r H 2 partial p r e s s u r e s m u c h h i g h e r t h a n t h o s e prod u c e d in t h e a b s e n c e o f sulfur also indicates a direct r e d u c t i o n o f S o r a t h e r t h a n a h y d r o g e n cycling m e c h a n i s m , since t h e s e normally growthinhibitory h y d r o g e n c o n c e n t r a t i o n s would have inhibited t h e initial H E p r o d u c i n g step. Sulfur r e d u c t i o n also b e g a n early in the log p h a s e b e f o r e the h y d r o g e n partial p r e s s u r e r e a c h e d growth inhibitory levels. T h u s sulfur acts as an electron sink to facilitate f e r m e n t a t i o n [17]. Thermotoga sp. strain FjSS3.B1 is t h e r e f o r e able to o v e r c o m e the growth inhibitory effects o f high H 2 partial p r e s s u r e s by routing e l e c t r o n s to sulfur r e d u c t i o n r a t h e r t h a n to t h e energetically unfavourable p r o d u c t i o n o f H 2. In addition, und e r very high H 2 partial pressures, lactate prod u c t i o n a p p e a r s to function as a f u r t h e r e l e c t r o n sink m e c h a n i s m .

ACKNOWLEDGEMENTS This research was f u n d e d by Pacific E n z y m e s Ltd, N e w Z e a l a n d . T h e a u t h o r s t h a n k Bettina R o s n e r for helpful criticism o f t h e manuscript.

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