Analyst October 1983 VoZ. 108 pp. 1209-1220 1209 Sulphide Ion-selective Electrode Studies Concerning Desdfovibrio Species of Sulphate-reducing Bacteria lsmail K. Al-Hitti Gwilym J. Moody and J. D. R. Thomas Department of Applied Chemistry Redwood Building U WIST Cardifl CF1 3XA The sulphide ion-selective electrode has been used for further studies on the Desulfovibrio species of sulphate-reducing bacteria. Emphasis has been placed on D. vulgaris which thrives in sodium dithionite metabolic medium, which provides an alternative sulphur source to sulphate. The bacterium also grows well in media containing certain organic materials namely, cysteine cystine and glutathione as alternative sulphur sources to sulphate. The bacterium does not grow with methionine as the sulphur source and this is attributed to the relative stability of the C-S-C bonds which are not adjacent to the amino group.The growth of D. vulgaris is retarded with barium sulphate or elemental sulphur as sulphur sources. However under certain circumstances there is less retardation with flowers of sulphur. More general parameters have also been studied including age of starter culture inoculum size poising agents pH and yeast stimulator. The active life of D. gigas was significantly less than for D . desulfuricans and D. vulgaris. Growth was more vigorous as the inoculum size was increased, while the optimum pH was between 7 and 8. Poising agents are less essential for sub-culturing from fresh starter cultures than for older ones while yeast extract accelerates the growth of D.vulgaris. Acetate is confirmed to be inappropriate as an organic carbon nutrient. D. vulgaris is considerably less sensitive to the salinity of the culture media (up to 3% m/V sodium chloride) than D. desulfuricans and D. gigas which were generally inactive even with 1% wz/V sodium chloride. Finally sodium tetraborate(II1) (2% m/ V ) and 2,kdinitrophenol (2% m/V) are confirmed as effective bactericides for D. vulgaris which is the most robust of the three species (D. desulfuricans D. gigas and D. vulgaris) studied in this work. Keywords Sulphate-reducing bacteria Desulfovibrio bacteria ; sulphide ion-selective electrode Various potentiometric methods have been employed for monitoring bacterial growth. Thus, Hussein and Guilbault1.s2 replaced the conventional but tedious turbidimetric monitoring method by nitrate and ammonium ion-selective electrodes for following the decrease in nitrate concentration and increase in ammonium concentration respectively in the presence of E.coZi. The resulting growth curves based on e.m.f. measurements were used to determine the best time for harvesting the bacterial cultures for enzyme extraction. Ion-selective electrodes and other potentiometric probes can provide useful metabolic information concerning pH carbon dioxide and ammonia species for proteus culture^.^ In contrast to an autoanalyser pr~cedure,~ such in siiu methods have the advantage of not con-suming any culture. Sulphate-reducing bacteria derive their energy from the anaerobic reduction of sulphate and the idea of employing potentiometric techniques to monitor their growth was first used by Alico and Liegey.5 This study of the growth pattern of Desd'jovibrio desulfiricarts involved measurements of the redox potentials with a platinum electrode coupled to a saturated calomel electrode alongside turbidimetric measurements of sulphate substrate consumption during growth.The liberation of hydrogen sulphide is the best indication of thriving and multiplication of sulphate-reducing bacteria. This lends itself to monitoring by the sulphide ion-selective electrode and the Orion 94-16A electrode has been used for studying the production of sulphide in cultures of D. desuZ+uricans6 and slurries of estuarine marshland that have been collected with sterilised syringes.' These monitoring modes of sulphide ion-selective electrodes would have been helpful in earlier work e.g.in the failure of growth of sulphate-reducing bacteri 1210 AL-HITTI ef al. SULPHIDE ISE STUDIES CONCERNING Analyst Vol. 108 populations in solid media and where reliable pictures of the activity of the organisms in different samples required comparison of the rates of sulphide formation in liquid media.8 The electrode would also have helped in determining numbers of these bacteria in samples of oil well waters when the rate of hydrogen sulphide formation after inoculation into a culture medium was used.9~10 A detailed study of the role of sulphide ion-selective electrodes for monitoring the growth of various Desulfovibrio species of sulphate-reducing bacteria has recently been reported using both indirect and direct methods.l1,l2 Three species of Desuulfovibrio were studied namely, D.desuZ+uricans D. gigas and D. vulgaris. The sulphide determined by both modes matched the amounts expected from the nutrient sulphate initially present in the culture media and also the sulphide recovered gravimetrically.11 All three species of the bacteria were shown to thrive on the metabolic intermediates sulphite thiosulphate and metabisulphite but they did not survive in the more stable dithionate or in the absence of inorganic sulphur. This paper describes a study of other parameters affecting the growth of sulphate-reducing bacteria namely redox poising agents pH inoculum size stimulators age of starter culture, elemental sulphur insoluble sulphate and inhibitors.Attention has also been given t o their viability in the presence of certain organic sulphur sources other than thioglycollate. Experimental Micro-organisms and Reagents All reagents were of analytical-reagent grade unless otherwise specified. Most of this work was carried out with Desulfovibrio vulgaris Strain No. NClB 8457 1975. Additionally Desulfovibrio desulfiricans Strain No. NClB 8307 1979 and Desulfovibrio gigas, Strain No. NClB 9332 1979 were used for the purposes of comparison. Refrigerated freeze-dried cultures were obtained from the National Collection of Industrial Bacteria (NCIB), Aberdeen. Preparation of Starter Cultures and Sub-cultures of Desulfovibrio Species Cultures of the three Desulfovibrio species were revived13 in a slightly modified version of Postgate's medium for sulphate reducers14J5 as previously described11 in order to provide starter cultures from which the inocula for sub-cultures were taken.Thus the basal medium for growing starter cultures contained K,HPO, 0.25 g; NH,C1 0.50 g; yeast extract (Oxoid Code L21) 0.50 g; MgSO4.7H,O 1.00 g; Na,SO (anhydrous) 0.50 g ; CaC12.2H,0 0.05 g; sodium lactate (70% m/m solution) 2.50 cm3; and doubly de-ionised water 500 cm3. The pH of the medium was adjusted to 7.40 with 5 M sodium hydroxide solution and autoclaved at 125 "C for 30 min. The culture was then cooled and the preparation completed by adding 0.50 g of sodium thioglycollate and 0.50 g of sodium ascorbate as poising agents in order to lower the oxidation - reduction potential of the medium for initiating growth of the sulphate-reducing bacteria.The pH was finally adjusted to 7.80 and the culture was autoclaved at 125 "C for 30 min. The medium was then cooled rapidly and anaerobicity was assured by the vigorous bubbling of white-spot nitrogen for at least 30 min prior to inoculation. The basal medium was then inoculated with the freeze-dried Desulfovibrio species. Any remaining air above the medium was flushed out with nitrogen. The sealed container was incubated at 30 "C and bacterial growth became evident after 2-3 d. This medium called the "starter culture," was used as the source for the sub-cultures. The sub-cultures were set up in basal media as detailed above except that magnesium chloride (0.83 g) was used instead of magnesium sulphate.Appropriate variations in content were made for parameter studies on the sulphur species the organic carbon source pH etc. as described in the text and in Tables I I1 and 111. The fresh basal medium of the sub-culture was aseptically charged with an aliquot of the starter culture (normally 2 cm3). The pH of the sub-cultures was strictly adjusted to the optimum growth range of 7.0-7.5 unless otherwise indicated. Monitoring of Sulphide Produced by Desulfovibrio Bacteria duction namely an indirect method and a direct method. claved prior to inoculating with the starter culture. Two main modes previously described,ll were employed for monitoring the sulphide pro-The sub-culture flasks were auto October 1983 DES ULFOVIBRIO SPECIES OF SULPHATE-REDUCING BACTERIA 121 1 Indiyect method Hydrogen sulphide produced by bacterial action was swept with white-spot nitogen from the culture flask which contained basal medium inoculated with 2 cm3 of starter culture into a monitoring flask fitted with an Orion Model 94-16A sulphide ion-selective electrode and a Corning Model 476002 double-junction reference electrode with an outer 10% potassium nitrate filler solution.Both the culture flask and remote monitoring flask were thermostated at 30 "C. Direct method The culture flask with basal medium after sterilisation and flushing out with white-spot nitrogen was charged with 2 cm3 of starter culture and fitted with an Orion Model 94-16A, sulphide ion-selective electrode a Corning Model 476002 double-junction reference electrode with an outer 10% potassium solution and a combination K E N 4 pH glass electrode.The flask was thermostated at 30 "C and the medium was stirred gently by a magnetic stirrer. During growth the pH was monitored as well as the e.m.f. of the sulphide ion-selective reference electrode pair. Potentiometric Measurements E.m.f. and pH measurements were recorded with a Beckman Model 4500 digital pH-millivoltmeter in conjunction with a Servoscribe Model RE541 potentiometric recorder. Concentrations of sulphide monitored by direct and indirect methods were calculated as previously described.ll Gravimetric Determination of Sulphide Produced by Cultures the monitoring flask of the indirect type of experiments. charged with 0.5 M lead nitrate. weighed.The sulphide produced was determined gravimetrically by precipitation of the sulphide in For these the monitoring flasks were The filtered lead sulphide precipitate was dried at 110 "C and Results and Discussion Studies of Alternative Sulphur Sources Dithionite An earlier study11 showed that the three species of Desulfovibrio in the culture media with lactate as an electron and carbon donor thrived with sulphate as an electron acceptor and also with sulphite thiosulphate and metabisulphite intermediates instead of sulphate. In con-trast all the species failed to grow with the chemically stable dithionate (S,0,2-) in the basal medium and they also failed to grow in the absence of an inorganic sulphur source. Dithionite ions (S,0,2-) are also involved in the metabolic mechanism of inorganic sulphur compounds by sulphate-reducing bacteria (see scheme in reference 11).Experiments with sodium dithionite as the sulphur source indicated that D. vulgaris thrived with a sulphide yield (0.144 g) near to the theoretically expected value (0.139 g). The reaction is stoicheiometric and consistent with the following equation : 3CH,CHOHCOONa + Na,S,O -+ 3CH,COONa + Na,CO + 2C0 + 2H,S + H (1) This supports an earlier conclusion16 that dithionite could effectively replace sulphate for growing D. desulfiricans possibly because dithionite spontaneously decomposed to sulphate.ls Dithionite is a strong reducing agent but it has been shown to be itself reduced rapidly by cells and cell extracts of sulphate-reducing bacteria.17 Also with gaseous hydrogen as a reductant cytochrome C accelerated the reduction of dithionite ions.Thus although dithionite and dithionate differ by just two oxygen atoms only the former is reduced to sulphide by the organisms. Furthermore dithionate is the only 0x0 - sulphur species that is metabolically stable. This is compatible with the view that the dithionate ion is of a different structural type from the higher polythionates.l 1212 Analyst VoZ. 108 Organic szclphur sources Preliminary studies on organic sulphur sources in the culture media of sulphate-reducing bacteria have indicated that such compounds could serve as both organic nutrient and sulphur source.12 However it was emphasised that none of the three species of sulphate-reducing bacteria studied1l,l2 utilise the organic sulphur of thioglycollate (Table I).This confirms Pankhurst’s observation that the organic sulphur of thioglycollate is not ~ti1ised.l~ Several experiments were designed for cystine and cysteine as organic sulphur sources for D. vulgaris species employing the indirect monitoring method (Table I). The first run for each sulphur source (runs 2 and 5 respectively) contained sodium thioglycollate and sodium lactate while for the second runs (3 and 6) sodium thioglycollate was omitted. Both sodium thioglycollate and sodium lactate were omitted from the third runs (4 and 7). AL-HITTI et al. SULPHIDE ISE STUDIES CONCERNING TABLE I TOTAL SULPHIDE PRODUCTION BY D. vulgaris FROM VARIOUS ORGANIC SULPHUR SUBSTRATES INSTEAD OF INORGANIC SOURCES Experiment Organic sulphur source 1 Thioglycollate + lactate 2 3 Cystine without thioglycollate 4 Cystine with thioglycollate and lactate Cystine without thioglycollate and lactate 5 6 Cysteine without thioglycollate 7 8 9 Cysteine with thioglycollate and lactate Cysteine without thioglycollate and lactate Glutathione with thioglycollate and lactate Methionine with thioglycollate and lactate Mass of sulphur sourcelg 0.500 0.479 (0.500)t 0.479 0.479 0.480 (0.500)t 0.480 0.480 1.220 (0.500)t 0.592 (0.500) t Equivalent nutrient sulphur contentlg 0.140 0.127 (0.140) 0.127 0.127 0.127 (0.140) 0.127 0.127 0.127 (0.140) Sulphide content in monitoring flask/g electrode metrically 0.006 None* 0.134 0.151 Sulphide-0.134 0.139 0.120 -0.124 -0.124 0.121 0.116 0.105 0.124 0.128 0.112 -0.122 0.124 Monitoring method Indirect Indirect Indirect Indirect Direct Indirect Indirect Indirect Direct Indirect 0.127 0.004 0.002 Indirect (0.140) Age of starter cultureld 49 52 63 75 208 166 178 190 129 10 16 *Trace amounts only of black precipitate in gravimetry.t Mass of thioglycollate. For both cystine and cysteine the organisms entered their logarithmic phase within 24 h as verified by the rapid production of sulphide detected in the remote monitoring vessel while the growth of D. vdgaris occurred after only 3 d with inorganic sources under comparable circum-stances. The multiplication of D. vulgaris occurred with cystine and cysteine even in the absence of sodium lactate (runs 4 and 7) thus confirming that both cystine and cysteine can function as organic carbon and hydrogen sources.1g The growth of D.vuZgaris during direct monitoring (second runs 4 and 7) in the presence of cystine and cysteine respectively but in the absence of lactate eliminates the possibility that inorganic carbon or trace amounts of carbon dioxide in the nitrogen of the indirect method might have stimulated growth. The metabolic reduction of cystine and cysteine to sulphide was efficient in terms of mass balance and tests for cystine and cysteine in the terminal culture media proved negative. The degradation of cystine and cysteine to sulphide may respectively be represented by20 : HH H H I 1 I I I I I I 4 HOOC-C-C-S-S-C-C-C00H + 2H2O -+ CHSCOOH + HZS + NH3 + C02 + + H2 (2) H,NH HNH, H - - (3) I HS-CH2-C-COOH + 2H2O -+ CH3COOH + H2S + NH3 + C02 + H2 .. October 1983 DES ULFOVIBRIO SPECIES OF SULPHATE-REDUCING BACTERIA 1213 Two additional organic sulphur sources were examined namely glutathione and methionine. Glutathione was degraded as confirmed by the collected sulphide (run 8 Table I) and is consistent with the following equation : I I It I 1 H 0 H CH, I I HOOC-C-CH,CH,C-N-C-H + 6H,O + 4CH3COOH + 3NH + 2C0 + H,S + H (4) I NH2 c=o I HOOC-CH,-NH The low culture pH (7.0-7.3) measured after the experiment had progressed to the stationary stage may be related to the acetic acid which is produced in a relatively greater amount than ammonia [equation (a)].The media containing inorganic sodium salts as sulphur sources tended to become more basic (pH ca. 8.5). No growth of D. vulgaris is observed with methionine as indicated by the very low yield of sulphide obtained (run 9 Table I) The resistance of methionine to microbial degradation may be related to the relative stability of the C-S-C bonds that are remote from the amino group compared with the C-S-S-C bonds in cystine and the C-S-H bonds in cysteine and glutathione, each having an amino group attached to the penultimate carbon away from the sulphur atom. Elemental sulphur as a terminal electron acceptor Elemental sulphur is not included as an intermediate in the sulphur cycle as it has been shown not to be involved in either the reduction or oxidation of sulphide.21 For example, elemental sulphur purified by redistillation was not attacked while colloidal sulphur permitted only slow growth or slow hydrogen absorption when a resting cell suspension was used.16 Confirmatory experiments have been carried out in this study using elemental native sulphur as an alternative sulphur source for D.vulgaris. Direct monitoring experiments with native sulphur showed little growth only as indicated by the low yield of sulphide (0.040 g) compared with the expected value (0.127 g). The native sulphur was autoclaved within the culture flask. However when flowers of sulphur was used under direct and indirect monitoring conditions there was bacterial growth. The flowers of sulphur was either autoclaved within the culture medium or added to the culture medium immediately prior to inoculation with starter culture.In each instance there was bacterial growth and reasonable amounts of sulphide were collected (0.076 g) during indirect monitoring for the growth term of the D. vul-garis with autoclaved flowers of sulphur compared with the expected value (0.127 g). For the non-autoclaved flowers of sulphur experiment the sulphide produced (0.320 g) was also con-siderable in relation to that expected (0.500g) for indirect monitoring. The growth of D. vulgaris in the presence of flowers of sulphur was easier to follow by the indirect method than by the direct method as in the latter instance colloidal sulphur accumulated on the electrodes to give spurious e.m.f. measurements particularly towards the end of experiments.Autoclaved cultures with flowers of sulphur became yellow - blue and the flowers of sulphur powder became solid and settled to the bottom of the culture flask. Utilisation of flowers of sulphur by D. vulgaris may be ascribed to polymerisation of sulphur at the temperature of sterilisation. Polysulphides are more soluble than flowers of sulphur.22 For the non-autoclaved flowers of sulphur the growth of D. vuulgaris is also clearly possible and its utilisation may be due to the partial solubility of this form of sulphur owing to the small particle size.l6 Barium sulphate as a solid sulphur source Experiments were conducted by the indirect monitoring method to determine whether D. vzdgaris can thrive and survive with barium sulphate (0.926 g) as a sulphur source.The bacterium grew slowly and the culture became slightly cloudy and yellow in appearance. The solubility of barium sulphate (0.00246 g dm-3 at 25 "C) corresponds to 1.05 x 1214 AL-HITTI et al. SULPHXDE ISE STUDIES CONCERNING Analyst Vol. 108 concentration and 3.4 x lom4 g dm-3 of sulphur. In relation to this the amount of sulphide monitored by the sulphide ion-selective electrode corresponded to 0.026 g (0.048 g by the gravimetric method). These results indicate that soluble sulphate reduced to sulphide by the bacteria is not replenished sufficiently fast by further dissolution of barium sulphate. The eventual termination of growth may be attributed to insufficient soluble sulphate as an effective energy source. This indicates that barium sulphate is unsuitable as a terminal sulphur source for bacterial growth.General Parameters of Bacterial Growth Inoculum age Growth studies on Desulfovibrio species indicate that the age of starter culture has no effect on sulphide yield for D. vulgaris and D. desuZjuricans.ll However the lag phase lengthens with age. With D. gigas however the age of starter culture influences the activity of the organisms resulting in reduced yields of the sulphide end-product. This has been tested in this work by several experiments using the indirect monitoring method for D. gigas from different ages of starter cultures and using sulphite as inorganic sulphur source. For example, the amount of sulphide produced was 0.128 0.008 and 0.002 g respectively for experiments corresponding to starter cultures of ages 25,45 and 110 d (Fig.1) and confirming the effective lifetime of D. gigas to be shorter than those of D. vulgaris and D. desulfitricans. 10-3 H A 10-5 0 10-7 I O - ~ .c CT -lo-” 0 50 100 150 Time/h 200 250 Fig. 1. Effect of inoculum age on growth of D. gigas expressed as sulphide produced from a sulphite-based nutrient as monitored by the sulphide ion-selective electrode (indirect method). D. gigas is an exceptional species because of its large spirilloid cell and intracellular granule^.^^,^^ These unique characteristics probably enhance accumulation of the micro-organisms and they settle to the bottom of the starter culture. Their cultures always clarified after one month and large colonies were clearly seen in the bottom of the culture flask.In order to compare the robustness of the three species very old starter cultures of 9,5 and 8 months of D. desulfuricans D. gigas and D. vulgaris respectively were regrown using the direct monitoring method. The necessary culture nutrients were autoclaved and added to the old starter cultures of the appropriate bacteria. The pH of the cultures was adjusted to 7.0-7.5 by adding sterilised sodium hydroxide solution. Renewed activity of D. vztlgaris and D. desuZfwicans was observed as can be concluded from the characteristic growth phasell deduced from potential and pH measurements during the experiment (Fig. 2B and C). No growth activity could be detected for the D. gigas culture as indicated by the almost constant sulphide electrode potential and pH measurements during the 12 d of the experiment (Fig.2A). It is noted that the D. gigas system had a high sulphide content (Fig. 2A) which was un-affected by the renewed nutrient culture for reactivating bacterial activity October 1983 DESULFOVIBRI~ SPECIES OF SULPHATE-REDUCING BACTERIA 1215 .-0 50 100 150 200 240 Time/h Fig. 2. Regrowth attempts for starter cultures of three Desulfovibrio species monitored potentio-metrically (direct method). A D. gigas 5 months old (e.m.f. of sulphide ISE system); B D. vulgaris, 8 months old (e.m.f. of sulphide ISE system); C D . desulfuricans 9 months old (e.m.f. of sulphide ISE system) ; and A* B* and C* pH changes for A B and C systems respectively. Efect of inoculum size Indirect monitoring experiments were designed to study the effect of size of the starter culture sample on the growth of D.vzllgaris and on the rate of production of hydrogen sulphide. Three basal media of identical composition were used with inocula of the same age. The basal media were charged with 2 4 and 6 cm3 of starter culture and the growth was followed potentiometrically. As the sample size was increased the rate of production of hydrogen sulphide increased viz. 3.3 x 10-4 7 x 10-4 and 2.7 x mol dm-3 d-l averaged over a 3-d yield. Also the total yields of sulphide were 0.120 0.136 and 0.176 g after 12 d corre-sponding to inocula sizes of 2 4 and 6 cm3 respectively. The increases in the amount of sulphide produced with sample size of the inoculum may be related to (i) an increase in the number of micro-organisms and hence of enzyme mass trans-ferred to the basal medium; (ii) an increase in the sulphide carried over with the starter culture that will lower the reducing potential of the medium; and (iii) although unlikely unreduced sulphate in the starter culture that may increase sulphide concentration as the transferred inoculum was increased in amount.Efect of reducing the amozlnt of redox poising agent The growth of anaerobic bacteria is often stimulated by reducing agents. Thus many bacterial media contain one or more redox poising agents to lower the reducing potential of the media to more negative levels. The role of reducing agents in activating D. vulgaris growth has been studied here in relation to the roles of sodium thioglycollate and sodium ascorbate as well as sulphide brought in with the starter culture.Sulphate was used as the sulphur source and lactate as the carbon and hydrogen or electron donor (see Table I1 for parameters). In experiments 1 and 2 (Table I), sodium glycollate was substituted for sodium thioglycollate but sodium ascorbate redox poising agent was still included. The D. vulgaris grew well with stoicheiometric yields of sulphide and even the pause periodll was absent in experiment 1. The activation and initiation of the organisms in this run were probably due to the presence of ascorbate in addition to sulphide brought in with the very fresh starter culture (age 14 d). Thus sodium thioglycollate can be avoided if the inocula are fresh but it is recommended for inclusion as redox poising agent in sub-culturing if the inocula are from old starter cultures 1216 AL-HITTI et al.SULPHIDE ISE STUDIES CONCERNING TABLE I1 GROWTH OF D. vulgaris UNDER VARIOUS CIRCUMSTANCES OF ADDED POISING Analyst VoZ. 108 AGENTS P H AND LACTATE NUTRIENT Inorganic sulphur Experiment source 1 0.564 g of Na,SO, 2 0.664 g of Na,SO, 3 0.664 g of Na,SO, 0.564 g of Na,SO, 6 NoNa,SO, 6 NoNa,SO, 7 NoNa,SO, 8 0.564 g of Na,SO, 9 0.564 g of Na,SO, 10 0.564 g of Na,SO, Size of pH a t Theoretical Sulphide starter Monitoring the start sulphurlg obtained/g culture/cma method 7.35 7.30 7.35 7.35 7.30 7.20 7.30 9.00 7.50 7.35 0.127 0.127 0.127 0.127 ---0.127 0.127 0.127 0.128 0.123 0.001 0.120 0.006 0.001 0.001 0.006 0.003 0.018 2 2 2 2 2 2 2 2 -2 Indirect Direct Indirect Direct Indirect Direct Direct Direct Direct Indirect Remarks Sub-culture contains 0.5 g of sodium glycollate as alternative to thioglycollate (growth) Sub-culture contains 0.5 g of sodium glycollate as alternative to thioglycollate (growth) Sub-culture lacks thioglycollate and ascorbate (no growth) Sub-culture lacks thioglycollate and ascorbate (growth by sulphide poising) Sub-culture contains normal poising agents (no growth) Sub-culture contains normal poising agents (no growth) Sub-culture lacks thioglycollate (no growth) Sub-culture contains normal poising agents but pH set to 9.0 (no growth) No inoculation of starter culture to the normal sub-culture (no growth) Culture lacks lactate (no growth) For experiments 3 and 4 (Table 11) both sodium thioglycollate and sodium ascorbate were omitted.No meaningful growth could be detected in experiment 3 (sulphide collected was 0.001 g) but a repeat of run 2 showed very slow growth with a long lag phase and more sulphide (0.032 g). With the direct monitoring method (experiment 4) growth was readily observed (Fig. 3) despite the absence of poising agents and the sulphide yield was near stoicheiometric (Table 11). The differences between the indirect (experiment 3 Table 11) and direct (experiment 4, Table 11) monitoring methods can be attributed to the sulphide brought in by the inoculum from the starter culture on the subsequent experimental procedure Thus in the indirect method (experiment 3) this sulphide is swept off by the nitrogen stream while it remains in the culture flask for the direct method (experiment 4).For experiment 4 however the reducing potential capability brought in by the sulphide of the starter culture seems to be sufficient for Time/h Fig. 3. Direct monitoring of growth of D. vulgaris in the absence of poising agents with 0.564 g of Na,SO (0.127 g of S) (sulphide produced = 0.120 g) October 1983 DES ULFOVIBRIO SPECIES OF SULPHATE-REDUCING BACTERIA 1217 the viability of these bacterial populations in the direct monitoring procedure. This is com-patible with the observations of other worker^.^^-^' The need for reducing agents is significant as also shown28 for generation of the enzyme pyrophosphatase involved in the reduction of sulphate to sulphite.The control experiments (5-7 Table 11) confirm that sulphate (or sulphur source) is a prerequisite for growth of the bacteria with the optimum pH adjustment (experiment 8) as well as lactate (or organic carbon) as a major organic source (experiment 10). Efect of fiH Three experiments illustrate the influence of pH on the activity of D. vuzgaris in the indirect monitoring method. Each culture medium was identical except that the initial pH values were set at 6.0 7.0 and 8.0 respectively for the three cultures of D. vulgaris. The respective rates of sulphide production over 3 d were ca. 3 x ca. 7 x and mol dm-3 h-l (Fig. 4). The sulphide yield (0.002 g) was small at pH 6.0 and ceased after 3 d while sulphide continued to be produced at pH 7 and 8 for at least a further 50 h with yields of 0.128 g of sulphide com-pared with the expected 0.127 g (Fig.4). Obviously a pH of 6 is unsuitable for growth while the organisms grow successfully at pH 7 or 8 but growth is faster at a pH of 8 as observed by other w0rkers.~9,30 The highest pH recorded for cultures in an earlier studyl1J2 was ca. 8.6 after 15 d of sulphide monitoring by the indirect method. Precautions are necessary with media at a pH above 8 because calcium magnesium and phosphate ions cause precipitation. For cultures at pH 9 a considerable amount of white precipitate was produced for runs using the direct method of sulphide monitoring and no growth was identified in this medium, thus confirming the pH range of 7.0-8.0 as compatible with the activity of sulphate-reducing bacteria.This range is compatible with the viability of most enzymes that are active in sulphate to sulphide conversion at pH <8 and >6. For example Akagi and CampbelPl discovered that ATP-sulfurylase extracted from D. desulfiricans functioned over a pH range of 6.0-9.0 but was denatured beyond this range. lo-’’ 10-9 ti 1 I I I 50 100 150 Time/h 200 250 Fig. 4. Effect of pH on the growth of D. vulgaris (indirect method). A pH 8; B pH 7; and C, pH 6. Role of yeast extract For this parameter different amounts of yeast extract were added to D. vulgaris cultures and growth was observed by the indirect monitoring method. The inocula (2 cm3) of D. vulgaris were of the same age in each example.Growth of D. vulgaris was extremely poor over 15 d in the absence of yeast extract as shown by the small yield of sulphide (0.002 g) (Fig. 5) but was enhanced by yeast extract and after 6 d the expected 0.128 g of sulphide was produced €or each of the yeast runs. The relevance of yeast extract can be related to its rich content of vitamin B and organic nitrogen and carbon. The incorporation of 0.1% m/V of yeast extract has previously bee 1218 AL-HITTI et al. SULPHIDE ISE STUDIES CONCERNING Analyst Vol. 108 Fig. 5. Influence of yeast extract on the growth of D. vulgaris (indirect method). A 1.0 g of yeast extract; B 0.5 g of yeast extract; and C without yeast extract. shown32 to enhance the growth of bacteria and it is claimed33 that yeast extract is required to complete substrates for example by introducing oxamate and 2-methylpropan-1-01 and where the organic matter of yeast extract is a~similated.~' Acetate as a substrate for D.vulgaris Pure cultures of most sulphate-reducing bacteria dissimilate carbon sources with three or more carbon atoms with acetate and carbon dioxide as the end p r o d u ~ t s ~ ~ ~ ~ ~ and further oxida-tion of acetate coupled to sulphate reduction does not seem to occur. This was checked in this study for D. vuZgaris by using acetate as a carbon source. The indirect monitoring method was used but with calcium hydroxide solution in the monitoring flask instead of sodium hydroxide solution in order to monitor visually the production of carbon dioxide as well as for collecting hydrogen sulphide.The solution became turbid and finally a white precipitate was produced; this was attributed to calcium carbonate according to solubility considerations. The basal medium was also slightly cloudy. The solution in the monitoring flask at the end of the experiment was neutralised with perchloric acid and lead nitrate solution was added. The black lead sulphide precipitate collected corresponded to 0.013 g of sulphide (experiment 1 Table 111). This was consistent with the small amount of sodium sulphate (0.025g) actually reduced (Table 111). TABLE I11 GROWTH OF D. vulgaris IN MEDIA CONTAINING SODIUM ACETATE AS ORGANIC SUBSTRATE INSTEAD OF SODIUM LACTATE Sulphide Reduced expected Sulphide Age of Unreduced Na,SO calculated by sulphide Gravimetric starter Monitoring Experiments Na,SO,/g Na,SO,/g (col.2- col. 3)/g from col. 3/g electrodelg sulphide/g culture/d method Remarks 1 0.564 0.538* 0.026 0.006 - 0.013 127 Indirect No growth 2 0.564 0.538* 0.026 0.006 0.0005 0.004 133 Indirect No growth 3 0.564 0.562* 0.002 0.0004 0.0004 - 183 Direct No growth * Determined gravimetrically by the addition of barium chloride solution to the acidified medium after cessation of the experiment. The experiment was repeated and monitored potentiometrically with a sulphide ion-selective electrode by having 1 M sodium hydroxide solution and 2% ascorbic acid in the monitoring flask (experiment 2 Table 111). An even smaller indication of growth was observed by using the direct monitoring method and where of course nitrogen gas was excluded (experiment 3 Table 111).The purity of the A small yield of sulphide was obtained October 1983 DES ULFOVIBRIO SPECIES OF SULPHATE-REDUCING BACTERIA 1219 white-spot nitrogen was checked by bubbling through calcium hydroxide solution for 2-3 h and only trace amounts of carbon dioxide were found. In conclusion D. vuZgaris does not metabolise acetate thus confirming the view of Postgate.36 Consumption of acetate has however been reported with crude enrichment cultures37 and attributed to the existence of Desulfotomaculum acetoxidans isolated by Widdle and Pfennig.37 These do not utilise the substrates common to Desulfovibrio species such as lactate and pyru-vate but utilise acetate instead in the presence of sulphate as an electron acceptor with the following stoicheiometric reduction of sulphate to sulphide : CH,COONa + Na,SO -+ 2NaHC0 + NaHS .. * - (5) Salt (sodium chloride) tolerance in bacterial growth Sulphate-reducing bacteria are found in natural waters of all salinities from zero to satura-tion point.20 Therefore the sodium chloride concentration of growth media should match that of the habitat from which the organism was obtained for example Desulfovibrio salexigens have an absolute requirement of more than 1% sodium chloride.,O Nevertheless most bacterial samples from very saline environments exhibit more rapid growth in media of some-what less salinity.2* Several runs based on the indirect monitoring method tested sodium chloride tolerance in the growth of Desztlfovibrio species studied in this work.No traces of growth were observed with D. gigas and D. desulfuricans even with just 1% sodium chloride but D. vulgaris thrived well even at 3y0 sodium chloride (15 g per 500 ~ m - ~ ) in the culture. Full yields of sulphide were realised with D. vulgaris (0.124,0.128 and 0.127 g at 1,2 and 3% sodium chloride respectively) compared with very low yields (G0.003 g ) from D. desuljkicans and D. gigas. Thus D. vulgaris is more active and more adaptable environmentally than the other two species, although growth of the species in the presence of 3% sodium chloride was rather slower than at lower levels of the salt. Sodium chloride might be used for inhibiting growth of both D. desulfuricans and D. gigas. Inhibition of Bacterial Growth Minimum inhibitory conditions are influenced by the nature of the test media and by inoculum size.Desulfovibrio species and their resistance to inhibitors can cause conversion problem^.^^^^^ Also the existence of mixed populations shows different patterns of sensitivity to inhibitors to those shown by individual species. Experiments were carried out in this study with D. vulgaris using the indirect monitoring method for testing sodium tetraborate(II1) (2% m/V) and 2,4-dinitrophenol (2% m/V) as inhibitors. For sodium tetraborate(III) the pH of the culture initially around 9.0 was adjusted to 7.4 with sterilised hydrochloric acid after sterilisation. No growth was observed according to the low yield of sulphide (0.007 g) sensed by the sulphide ion-selective electrode. The inhibition of D.vulgaris was attributed to tetraborate(II1) ions while in an earlier study with D. desulfuricans38 similar inhibitors might have been brought about by pH as well as the tetraborate(II1) ions because the pH of this earlier study was not adjusted to the appropriate value for bacterial growth. The pH of one was adjusted to 7.40 with sterilised sodium hydroxide solution after sterilisation of the culture and the other was used at the prepared pH of ca. 4. No growth of D. vzdgaris was identified in either of the cultures as indicated by the low yield of sulphide (0.006 g), thus confirming the pronounced inhibiting effect of DNP. DNP has previously been shown41 to inhibit completely the reduction of sulphate in whole cells but had no effect on the reduction of thiosulphate indicating that DNP prevented the cells from producing the high energy phosphate required for reducing sulphate.Two other cultures for D. vulgaris contained 2% m/V of 2,4-dinitrophenol (DNP). Conclusion The sulphide ion-selective electrode is confirmed as a very useful and convenient means of monitoring the growth patterns of sulphate-reducing bacteria and especially for determinin 1220 AL-HITTI MOODY AND THOMAS the role of various culture media parameters. The study also illustrates a wider role for ion sensors and similar potentiometric devices in the monitoring of fermentation type processes. The authors thank the University of Al-Deen Iraq for paid leave of absence to I. K. A1-H. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. References Hussein W. R. and Guilbault G. G. Anal. Chim. Acta 1974 72 38. Hussein W. R. and Guilbault G. G. Anal. Chim. Acta 1975 76 187. Ladenson J. H. Huebner M. and Marr J. J. Anal. Biochem. 1975 63 56. Mor J. R. Zimmerli A. and Fiechter A. Anal. Biochem. 1973 152 614. Alico R. K. and Liegey F. W. J. Bacteriol. 1966 91 1112. Crombie D. J. Moody G. J. and Thomas J. D. R. Lab. Pract. 1980 29 259. Nedwell D. B. and Barat I. M. Microbiol. Ecol. 1981 7 305. Burker H. J. J . SOC. Chem. Ind. London 1939 58 93. Dostalek M. and Spurny M. Folia Biol. (Prague) 1956 2 338. Spurny M. Dostalek M.and Vlehla S. Folia Biol. (Prague) 1957 3 202. Al-Hitti I. K. Moody G. J. and Thomas J. D. R. Analyst 1983 108 43. Al-Hitti I. K. Moody G. J. and Thomas J. D. R. Anal. Proc. 1983 20 119. Machenzie A. R. “National Collection of Industrial Bacteria,” Aberdeen personal communication, Postgate J. R. in Schlegel H. H. and Kroger E. Editors “Anreichungskulfur and Mutanteri-Pankhurst E. S. in Slapton D. A. and Board R. G. Editors “Isolation Anaerobes,” Academic Postgate J. R. J. Gen. Microbiol. 1951 5 725. Nakatsukasa W. and Akagi J. M. J. Bacteriol. 1969 98 429. Ephraim F. C. “A Textbook of Inorganic Chemistry,” Second Edition Gurney and Jackson, London 1934. Senez J. C. and Leroux-Gilleron J. Bull. SOG. Chim. Biol. 1954 36 553. Postgate J. R. “The Sulphate-reducing Bacteria,” Cambridge University Press Cambridge 1979.Postgate J. R. Annu. Rev. Microbiol. 1959 13 505. Tobolosky A. V. and Eisenberb A. J. J . Am. Chem. Soc. 1959 81 780. Thomas P. J. Microsc. Paris 1972 13 349. Moura J. J . G. Xauier A. V. Bruschi M. LeGall J. Hall D. D. and Cammack R. Biochem. Stephenson M. and Stickland L. H. Biochem. J. 1933 27 1517. Grossman J. P. and Postgate J. R. J. Gen. Microbiol. 1955 12 429. Postgate J. R. J. Bacteriol. 1963 85 1450. Ware D. A. and Postgate J. R. J Gen. Microbiol. 1971 67 145. Postgate J. R. J. Gen. Microbiol. 1951 5 714. Starkey R. L. and Wight K. M. Am. Gas. Assoc. Proc. 1945 25 307. Akagi J. M. and Campbell L. L. J. Bacteriol. 1963 85 1450. Butlin K. R. Adams M. E. and Thomas M. J. Gen. Microbiol. 1949 3 46. Mechalas B. J. and Rittenberg S. C. J. Bacteriol. 1960 80 501. Senez J. C. Bull. SOG. Chim. Biol. 1954 36 541. Grossman J. P. and Postgate J. R. Proc. SOC. Appl. Bacteriol. 1953 16 1. Postgate J. R. personal communication 1980. Widdle F. and Pfennig N. Arch. Microbiol. 1977 112 119. Crombie D. J. PhD Thesis University of Wales 1979. Roberts G. A. H. BY. Corros. J. 1969 4 318. Crombie D. J. Moody G. J. and Thomas J. D. R. Chem. Ind. (London) 1980 500. Peck H. D. J. Biol. Chem. 1960 235 2434. 1976. auslese,” Fischer Stuttgart 1965 p. 190. Press London 1971 p. 223. Biophys. Res. Commun. 1976 72 782. Received April 20th 1983 Accepted May 19th 198