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Optimized standard conditions for determination ofnitrate reduction in propionibacteria

Renée Swart, Karl-Heinz Riedel, Trevor Britz

To cite this version:Renée Swart, Karl-Heinz Riedel, Trevor Britz. Optimized standard conditions for determination ofnitrate reduction in propionibacteria. Le Lait, INRA Editions, 1998, 78 (2), pp.217-226. �hal-00929589�

Lait (1998) 78, 217-226© Inra/Elsevier, Paris

217

Original article

Optimized standard conditions for determinationof nitrate reduction in propionibacteria

Renée Swart", Karl-Heinz Riedela,

Trevor Britz?"

"Department of Microbiology, University of the Orange Free State, Bloemfontein,South Africa

"Department of Food Science, University of Stellenbosch, Private Bag Xl, Matieland 7602,South Africa

(Received 18 February 1997; accepted Il November 1997)

Abstract - Nitrate reduction is currently considered to be a key characteristic for the determi-native grouping of strains of the Propionibacterium but many controversies exist regardingnitrate reduction by members of this genus. The aim was thus to determine the influence of dif-ferent media, nitrate concentrations and pH values under aerobie and anoxie conditions, using theconventional technique and the diazotization coupling method, on the nitrate reducing capabilityof propionibacteria. The low concentrations of nitrite formed during nitrate reduction by thepropionibacteria could not be accurately determined using the conventional technique. Thismethod was found to be reliable only when the initial KN03 concentration exceeded 1.0 mmol/L.ln contrast, with high concentrations of nitrite, the diazotization coupling method was found tobe too sensitive. Nitrate reduction in the yeast extract lactate (Y EL) medium with added glu-cose and KN03, was not effective as only low concentrations of nitrite were produced. Good nitratereduction was obtained on both the YEL and yeast extract (YE) media with or without the addi-tion of KN03 (pH 8.0) to the test media. The presence of nitrite in the YEL culture media of thefive Propionibacterium type strains, un der aerobie conditions (pH 8.0) with 20 mmol/L KN03,was determined using the diazotization coupling method. The type strains of P. acidipropionici,P. acnes and P.freudenreichii subsp.freudenreichii were able to reduce nitrate. In contrast, theP. jensenii and P. thoenii type strains were not able to reduce nitrate under these conditions.Furthermore, P. acidipropionici and P. acnes were both able to further reduce the formed nitriteto gaseous nitrogen. It was found that nitrate reduction was strongly influenced by environmentalfactors such as oxygen, nitrate concentration, pH, media composition, incubation period and thepresence of glucose. Data are presented which permit a more standardized determination ofnitrate reduction of propionibacteria. © Inra/Elsevier, Paris.

Propionibacterium 1nitrate reduction 1environ mental factors

* Correspondence and reprints. E-mail: tjbritz@land.sun.ac.za

218 R. Swart et al.

Résumé - Optimisation des conditions standard pour la détermination de la réductiondu nitrate par les bactéries propioniques. La réduction du nitrate est généralement considéréecomme étant une caractéristique clef pour déterminer des groupes de Propionibacterium mais denombreuses controverses existent concernant la réduction du nitrate par les membres de ce genrede bactéries. Le but était donc de déterminer l'influence de différents milieux, de la concentra-tion en nitrate et des valeurs de pH dans des conditions aérobies et anoxiques sur la capacité deréduction du nitrate par les bactéries propioniques, et ce, en utilisant la technique conventionnelleet la méthode de couplage de diazotisation. Les faibles concentrations en nitrite formé au coursde la réduction du nitrate par les bactéries propioniques ne pouvaient pas être déterminées avecprécision par la technique conventionnelle. Cette méthode s'est avérée fiable uniquement lorsquela concentration initiale de KN03 excédait 1,0 mmol/L. En revanche, avec une concentrationélevée de nitrite, la méthode de couplage de diazotisation s'est avérée trop sensible. La réductiondu nitrate dans le milieu YEL additionné de glucose et de KN03 n'était pas efficace puisqueseulement de faibles concentrations de nitrite étaient produites. Une bonne réduction de nitrateétait obtenue tant sur le milieu YEL que sur celui à l'extrait de levure avec ou sans addition deKN03 (pH 8,0). La présence de nitrite dans le milieu de culture YEL des cinq souches types dePropionibacterium, en aérobiose (pH 8,0) avec 20 mmolfL de KN03, a été déterminée en utili-sant la méthode de couplage de diazotisation. Les souches types de P. acidipropionici, de P. acneset de P.freudenreichii subsp. freudenreichii étaient capables de réduire le nitrate dans ces condi-tions' contrairement aux souches types de P. jensenii et de P. thoenii. De plus, P. aeidipro-pionici et P. acnes étaient capables de réduire le nitrite formé en azote gazeux. La réduction dunitrate s'est révélée fortement influencée par des facteurs environnementaux tels que l'oxygène,la concentration en nitrate, le pH, la composition des milieux, la durée d'incubation et la présencede glucose. Les données présentées permettent une meilleure détermination standardisée de laréduction du nitrate par les bactéries propioniques. © Inra/Elsevier, Paris.

Propionibacterium / réduction du nitrate / facteurs environnementaux

1. INTRODUCTION

The ability of an organism to reducenitrate is influenced by various environ-mental factors, including: medium com-position [2]; pH [3]; dissolved oxygenconcentration [9,25]; light intensity [25];incubation period [2]; nitrate concentra-tion [1]; and other factors [7,10, 18,26].

Many controversies exist [16, 23, 27]regarding nitrate reduction by membersof the Propionibacterium. This is an extre-mely important phenotypic characteristicas it is used as one of the major differentialcharacteristics to separate the four dairyspecies [6]. Kaspar [16], however, repor-ted that ail the dairy Propionibacteriumspecies reduced nitrate to nitrite and fur-ther to nitrous oxide (N20). In contrast,Van Gent-Ruijters et al. [27] reported thatonly one of three strains of 'P. pen tosa-

ceum' was able to reduce nitrate to nitriteand further to N20 or N2. In the currentclassification system, 'P. arabinosum' and'P. pentosaceum' strains were consolida-ted [19] to form the P. acidipropionicispecies, although nitrate reduction wasnegative for the 'P. arabinosum' strains,while certain of the' P. pentosaceum'strains were able to produce nitrite as weilas nitrogen [4]. According to Cumminsand Johnson [6], the other dairy speciescould not reduce nitrate, with the exceptionof P.freudenreichii subsp.freudellreichii.

As nitrate reduction is used as one ofthe major characteristics to separate thespecies of the Propionibacterium [6], theinfluence of environ mental parameters onnitrate reduction needs to be examined.These conditions should thus be optimi-zed so as to validate the application ofnitrate reduction as a differential charac-

Nitrate reduction by Propionibacteria 219

teristic. The aim of this study was to inves-tigate the influence of oxygen, nitrateconcentration, pH, media composition,incubation period, as weIl as the additionof glucose, on the ability of Propionibac-terium strains to reduce nitrate.

2. MATERIALS AND METHODS

2.1. Bacterial strains, culturemaintenance and growth media

Five Propionibacterium type strains (table 1),Escherichia coli ATCC 11775 and Leuconos-toc dextranicum OSM 20484 were included inthis study. The E. coli and the L. dextranicumstrains were included as positive [9, 17] andnegative control s, respectively.

Lyophilized cultures [14] were used to ino-culate yeast extract lactate medium (YEL) toobtain working cultures. The YEL medium(glL) consisted of: yeast extract 6.0; sodiumlactate (70% v/v) 20.0; peptone 2.0; KHzP0410.0; and Tween 80 1.0 mL. The medium wasprepared anaerobically [13] and the pH adjus-ted to 7.2 before sterilization. The dairy pro-pionibacteria were incubated for 4 days at30 oc and the P. acnes strain incubated for6 days at 37 "C in an anaerobie cabinet, using10% Hz: 10% COz: 80% Nz as gas phase.Escherichia coli was incubated aerobically for48 h at 37 "C on nutrient agar (Merck) with1% (rn/v) glucose (pH 7.0) and L. dextrani-CUI1l grown on tomato juice broth (Oifco)

medium (pH 4.8) and incubated aerobically at25 oC for 7 days.

Culture purity was regularly checked andthe production of propionic and acetic acidsas metabolites were monitored gas chromato-graphically for each propionibacterial strain[5].

2.2. Influence of external parameters

The influence of external parameters onnitrate reduction by the P. acidipropionici typestrain was determined using a modified DeMan-Rogosa-Sharpe medium (M-MRS) [8].The M-MRS-medium consisted of20 mmollLsodium phosphate buffer with (g/L): MRS(Merck) 25.0 and added sodium lactate (70%v/v) 20.0. The M-MRS was then used as basisfor the addition of different concentrations ofpotassium nitrate (KN03) (0.0, La, 5.0, 10.0and 15.0 mmollL). The initial pH value of eachassay was also varied (pH 5.0, 6.0, 7.0, 8.0 and9.0).

A pre-inoculum was prepared by growingeach culture under anoxie conditions for fivedays on M-MRS plates. A 10% standard ino-culum was prepared and the M-MRS-mediuminoculated, followed by incubation under aero-bic as weil as anoxie conditions, at 30 oc. AlItests were done in duplicate. Unless otherwisestated, a 10% standard inoculum (00640 = 0.21)of ail strains grown for 5 days was used throu-ghout this study.

Nitrate reduction was determined by mea-suring for the presence of nitrite, using the

Table I.Propionibacterium type strains used in this study.Tableau I.Souches types de Propionibacterium utilisées dans cette étude.

Culture number Species Source

5680419423424

P. acnesP. jenseniiP. thoeniiP. freudenreichii subsp. freudenreichiiP. acidipropionici

ATCC 6919OSM 20535NCFB 568ATCC 6207ATCC 25562

ATCC, American Type and Culture Collection, Rockville, MD, USA; DSM, Deutsche Sammlung vonMikroorganismen und Zellkulturen, Braunschweig, Germany; NCFB, National Collection of Food Bacte-ria, Reading, UK.

220 R. Swart et al.

conventional technique as described byGerhardt et al. [12]. The intensity of the redcolor was scored visually on a scale from 0 to 5.Nitrate reduction was also assayed using thediazotization coupling method described byNicholas and Nason [20], using a BeckmanDUS spectrophotometer.

Nitrate reduction was also determined usingnitrate broth (Difco) as growth medium. Thiswas performed at different pH values (6.0, 7.0,S.O and 9.0) and varying potassium nitrateconcentrations (10 and 20 mmol/L). The pHwas compensated before autoclaving. Theinfluence of various glucose concentrations(0.0,0.2,0.4,0.6, O.Sand 1.0%) (rn/v) in nitratebroth on nitrate reduction by P. acidipropio-nici was also evaluated. Ali tests were perfor-med under aerobic, as weil as anoxie condi-tions. Anoxie conditions were obtained byincubation of the samples in an anaerobie cabi-net. Aerobic conditions were obtained by aero-bic incubation of the static culture.

2.3. Influence of media composition

The optimal pH and KN03 concentration, asdetermined in the previous section, was appliedin this section. The influence of seven diffe-rent media combinations on the nitrate redu-cing capability of P. acidipropionici duringgrowth phase studies was evaluated in this sec-tion. The different media included: YEL brothwith the addition of both 10% glucose and20 mmol/L KN03; YEL broth with or without20 mmollL KN03; yeast extract (6.0 glL) andglucose (10 g/L) (YEG) with or without20 mmollL KN03 and yeast extract (6.0 g/L)(YE) with or without 20 mmol/L KN03. Inspecifie cases KN03 was added to the testmedia to determine if the presence of nitratein the growth media influenced or enhancednitrate reduction.

A 20% standard inoculum (00 1.0at 640 nm)was prepared and the various media combina-tions were incubated at 30 oc. Samples werewithdrawn at four hourly intervals for the ana-lysis of nitrite. Ali tests were performed in tri-plicate and uninoculated tubes served ascontrols. In this section, the presence of nitritewas determined using the diazotization cou-pling method [20].

3. RESUL TS AND DISCUSSION

3.1. Influence of growth mediumand nitrite assay techniques

The data obtained when using nitratebroth (Difco) under aerobic and anoxieconditions at different pH values andnitrate concentrations, showed that whenusing both the conventional (data notshown) and the diazotization couplingmethod, the L. dextranicum strain did notreduce nitrate while the E. coli strain stron-gly reduced the nitrate. This confirmedthe use of these strains as negative andpositive controls [II, 21].

During the study, it was found that theconvention al method was unsuitable fornitrite determination from propionibacte-ria when using the M-MRS-medium, asno nitrate reduction was observed for theP acidipropionici type strain at any pHvalue or at the different KN03 concentra-tions. In contras t, nitrite was formed inlow concentrations by P acidipropioniciin the nitrate broth (table II) under bothaerobic and anoxie conditions at pH 7.0and 8.0 and only under aerobic conditionsat pH 9.0. No nitrite was detected at pH6.0. The data thus indicate that the typeof medium used must be taken into consi-deration as it has an influence on the abi-lit y of an organism to reduce nitrate.Blaszczyk [2] also reported similar results.Nitrate reduction by the P acidipropio-nici strain, using the nitrate broth underthe same conditions, was also determinedusing the diazotisation coupling method(table III). Similar results were obtainedbut this method was more sensitive, sho-wing the highest nitrate reduction at pH8.0 with 20 mmol/L KN03 under aerobicconditions. In contrast, when this methodwas used to determine nitrate reductionfor the E. coli strain, it was found that theresults were not repeatable even when thesamples were diluted. Thus, based on theresults, it was concluded that the con ven-tional technique might not be sensitive

Nitrate reduction by Propionibacteria 221

Table II. Nitrate reduction by P. acidipropionici in nitrate broth after incubation at different pHand KN03 values (mmollL) under aerobic and anoxie conditions using the convention al nitritedetermination technique (data visually scored from 0 to 5).Tableau II. La réduction du nitrate par P. acidipropionici dans le bouillon Nitrate après incubationà différentes valeurs de pH et de KN03 (mmol/L) sous des conditions aérobies et anoxiquesutilisant la technique conventionnelle de détermination du nitrite (données catégorisées visuellementde 0 à 5).

pH

Aerobie

KN03 concentration (mmollL)

10

Anoxie

20 10 20

6.07.08.09.0

o111

o111

o11o

o11o

Table III. Effect of aerobie and anoxie conditions, different KN03 concentrations (mmol/L) andpH values on the ability of P. acidipropionici to reduce nitrate in nitrate broth, using the diazo-tization coupling method (values are in nmol nitrite per mL and are the average of duplicatetests).Tableau III. Effets des conditions aérobies et anoxiques, des différentes concentrations deKN03 (mmollL) et des valeurs de pH sur la capacité du P. acidipropionici à réduire le nitrate dubouillon Nitrate en utilisant la méthode de couplage de déazotisation (les valeurs sont en nmolde nitrite par mL et représentent la moyenne d'un test répété deux fois).

pH KN03 concentration

Aerobic Anoxie

10 mmol/L ± 50 20 mmol/L ± 50 10 mmol/L ± 50 20 mmol/L ± 50

6.07.08.09.0

0.007.08±0.71

12.85 ± 1.064.57 ± 0.71

0.009.09 ± 2.84

18.85 ± 1.063.57±0.71

0.0010.09 ± 2.13

1.31 ± 0.350.00

0.0015.1±4.964.57 ± 3.550.00

enough when low concentrations of nitriteare formed during nitrate reduction by pro-pionibacteria. The data showed that whenthe conventional method was used, it wasonly reliable when the initial KN03concentration in the medium exceeded1.0 mmollL. In contrast, however, whenhigh concentrations of nitrite were for-med, as found for the E. coli strain, the

diazotization coupling method may againbe too sensitive, even if the samples werediluted. Based on these results, it is recom-mended that the conventional method beused to determine nitrate reduction whentesting E. coli and that the diazotizationcoupling method be used when studyingnitrate reduction by Propionibacteriumstrains.

222 R.Swartet al.

3.2. Influence of environmentalfactors

The YEG medium (with and withoutadded nitrate) was not very effective fornitrite determination (figure 1), as onlylow concentrations were produced whenthe P. acidipropionici culture was grownin this medium. Nitrate reduction wasobtained in both the YEL and YE media(both without glucose addition) with orwithout KN03 (pH 8.0) (figure 1). Nonitrite was detected when the P. acidi-propionici culture was grown in the YELmedium with 10% glucose and 20 mmol/LKN03. This was also found when varyingglucose concentrations were used in the

nitrate broth. Care must thus be takenwhen glucose is added to a medium as noor very little nitrate reduction would befound.

The best medium for the assay of nitratereduction, by the P. acidipropionici typestrain, was found to be the YEL medium(figure 1) with the addition of 20 mmol/LKN03 (pH 8.0) to the medium withoutglucose under aerobic conditions. Thismedium was thus used during furtherexperimental studies. Under these condi-tions, enough biomass for the extractionand purification of the enzyme was alsoproduced. In the case of the YE medium,nitrate reduction was found but very Iittlebiomass was produced.

18

16 .:" 014

~~

::J 12E<,0 10 '.E&::....., 8QI.....;: 6

~

:!::z 4

2-.~-./0 '--_-----.L ____ l

0 24 48 72 96 120 144Incubation time (hours)

Figure 1. The influence of media composition on the ability of Propionibacterium acidipro-pionici to reduce nitrate (e YEL plus 20 mmollL KN03; a YEL minus 20 mmollL KN03;• YEG plus 20 mmollL KN03; D YEG minus 20 mmollL KN03; T YE plus 20 mmollL KN03;V YE minus 20 mmollL KN03). Values represent the average of duplicate tests (SD < 3%).Figure 1. L'influence de la composition des milieux sur la capacité de Propionibacterium aci-dipropionici à réduire le nitrate (eYEL plus 20 mmollL de KN03; a YEL moins 20 mmollL deKN03 ; • YEG plus 20 mmollL de KN03 ; D YEG moins 20 mmollL de KN03 ; T YE plus20 mmollL de KN03 ; V YE moins 20 mmollL de KN03). Les valeurs représentent la moyenned'un test répété deux fois (écart type < 3%).

Nitrate reduction by Propionibacteria

The five Propionibaeterium type strains(table 1) were inoculated into YELmedium with 20 mmollL KN03 underaerobic conditions (pH 8.0) and the pre-

---------~ 800A

0.1 .!!!.::200 ~

600 ?""o

400 I

0.01 ........ ,. ••••• - 0

o 70 140 210 280 350

Time (hours)

800

0.1

c ..~ 600

~

400

- 200

•• ,.-~~ 1 ••• 0

o 70 140 210 280 350

0.01

Time (hours)

~--'-~o----r- 800

0.1 .!!!E

200 Z

600 ?'il

400 f

0.01 0

o 70 140 210 280 350

223

sence of nitrite was determined accordingto the diazotisation coupling method atregular intervals (figure 2). The typestrains of P. acidipropionici, P. aenes and

_---------r 800

JoCl

.3

600 ?'"o

400 f

8

.!!!

200 ~0.1

0.01 ........ ,. •• ,..~ 0

o 70 140 210 280 350

Time (hours)

_----------, 800

0.01

600 ?'il

400 f

D ~ ..... - ....•0.1 -

.!!!:5

- 200 Z

0.001 '..,.-~-r ••,... 0

70 140 210 280 350oTime (hours)

Figure 2. Nitrate reduction and growth by the Propionibaeterium type strains in YEL mediumwith 20 mmol/L KN03 at 30 -c and pH 8.0 (A = P. thoenii NCFB 568; B = P.jensenii DSM20535; C = P. aeidipropionici ATCC 25562; D = P. aenes ATCC 6919; E = P.freudenreiehiisubsp.freudenreiehii ATCC 6207) [. = log OD640;. = nitrite - nmol/mL]. Values represent theaverage of triplicate tests (SD < 5%).Figure 2. Réduction du nitrate et croissance des souches types de Propionibaeterium dans lemilieu YEL, avec 20 mmol/L de KN03 à 30 degrés C et à un pH de 8.0 (A = P.thoenii NCFS 568 ;B = P. jensenii DSM 20535 ; C = P. aeidipropionici ATCC 25562 ; D = P. aenes ATCC 6919 ;E = P.freudenreiehii subspfreudenreiehii ATCC 6207) [. = Log D064O;. = Nitrite - nmol/mL].Les valeurs représentent la moyenne d'un test répété à trois reprises (écart type < 5%).

224 R. Swart et al.

P freudenreiehii subsp. freudenreiehiiwere ail able to reduce nitrate (figure 2C,D, E). In contrast, P thoenii and P jen-senii (figure 2A, B) were not able to reducenitrate under these conditions. Further-more, P aeidipropioniei and P aenes(figure 2C, D) were both able to reducethe accumulated nitrite, as can be seenfrom the decrease in the concentration ofnitrite. The reduction of the accumulatednitrite by P freudenreiehii subsp. freu-denreichii progressed at a much lower rate(figure 2E). This can probably be ascri-bed to the presence of a less active nitritereductase enzyme system in this specifiestrain. It was also found in this part of thestudy that nitrate reduction could only bedetected for the three positive type strainsafter 70 h of incubation. This is an impor-tant factor that must be taken into consi-deration when using nitrate reduction asa species differential character.

3.3. Nitrate reduction as speciesseparation criterion

The separation of species within thegenus Propionibaeterium, using the iden-tification system of Cummins and John-son [6], is based on five phenotypic cha-racteristics, including the ability to reducenitrate. Several reports can be found in theliterature relating to problems on the use ofnitrate reduction as differential characte-ristic [22, 23]. The data from this studyconfirm the identification system of Cum-mins and Johnson [6] in that P thoeniiand P jensenii, under the conditions usedin this study, are the only type strains notable to reduce nitrate. The variation of anorganism' s ability to reduce nitrate canpresent a serious problem if this phenoty-pic characteristic is used to differentiatebetween species. This is especially truefor the genus Propionibacterium, as isfound with the species P aeidipropioniei,P jensenii and P thoenii. If a Propioni-baeterium strain is not able to reduce

nitrate, that strain will be classified as amember of the P jensenii or P thoeniispecies, even though the majority of thecharacters would be similar to that ofP acidipropionici [5]. This incorrect iden-tification based on one phenotypic cha-racteristic, will only be apparent if a nurne-rical analysis based on a wider range ofphenotypic data are performed [5].

The results from this study (table Ill)clearly indicate that media composition,aerobic and anoxie conditions, the nitrateconcentration, as weil as pH, have a dra-matie effect on the ability of a strain toreduce nitrate. This study thereforeconfirms the findings of Riedel and Britz[24], that nitrate reduction in the genusPropionibaeterium can be a variable phe-notypic characteristic with external para-meters influencing the ability of an orga-nism to reduce nitrate. The results of Fochtand Verstraete [10] and Blësl and Conrad[3] were also confirmed in this study, inthat pH has a drastic effect on the capa-bility of microorganisms to reduce nitrate.Similarly, Allison and Macfarlane [1]found that culture pH influenced the pro-ducts of dissimilatory nitrate reduction inP aenes. Nitrate was converted to nitrite atalkaline pH (pH 7.5), whereas nitrousoxide was the product of nitrate reductionat a pH of 6.0 [1].

In the genus Propionibaeterium, nitratereductase appears to be constitutive asindicated in the results obtained in the pre-sence and absence of nitrate in the testmedium. Similarly, Kaspar [16] stated thatthe nitrate reductase of the genus Propio-nibaeterium seems to be either constitu-tive or derepressed by anaerobiosis. Accor-ding to Kaspar [16], nitrate appeared tostimulate synthesis of the nitrate reduc-tase in P acidipropionici, while oxygeninhibited nitrous oxide production in bothP acidipropionici and P thoenii. This isin contrast to the results of Kaneko andIshimoto [15], who found that the pre-sence of nitrate in the growth medium

Nitrate reduction by Propionibacteria 225

itself had no effect on the nitrate reduc-tase activity of P. acidipropionici. Thenutritional composition of the culturemedium was also found to have an effecton the ability of an organism to reducenitrate, as found for 'P. pentosaceum' [27]and Paracoccus denitrificans [2].

Blaszczyk [2] also showed that incu-bation period is of great importance. Cul-tures of Paracoccus denitrificans grown innutrient broth required only a 12-h incu-bation period, whereas cultures grown inmineraI media, supplemented with etha-nol, sodium acetate and methanol, requi-red longer periods to reduce the sameamount of nitrate. In this study, P. acnes,P. acidipropionici and P. freudenreichiisubsp. freudenreichii were only able toreduce measurable amounts of nitrate after70 of incubation (figure zc, D, E). Incontrast, it was also reported that specifiegrowth rates of P. acnes were higher inthe presence of nitrate [1] than in mediumwithout nitrate. Thus, the incubation per-iod is also of critical importance whenusing nitrate reduction as a differentialphenotypic characteristic, especially if theincubation period is too short.

The suppression of nitrate reduction byglucose was also observed during thisstudy, confirming the results of Schulpand Stouthamer [26]. Van Gent-Ruijterset al. [27] found that complex media wereunsuitable for use in nitrate reduction stu-dies in 'P. pentosaceum' and that nitratereductase was parti aIly repressed by glu-cose [26].

It is thus recommended that environ-mental factors, such as the pH, nitrateconcentration, aerobic or anaerobie condi-tions and the growth medium, should bestandardized when using nitrate reductionas criterion for differentiation betweenspecies in the genus Propionibacterium.Using the optimum conditions, as foundfor the type strains in this study, morestrains of the genus Propionibacteriumshould be evaluated for the ability to

reduce nitrate. It is also recommended thatthe nitrate reductase gene (NAR) of P. acidi-propionici be further studied and compa-red with that of other genera and spe-cies.

ACKNOWLEDGMENT

This research was funded by the South Afri-can Foundation for Research Development.

REFERENCES

[1] Allison C; Macfarlane G.T., Dissimilatorynitrate reduction by Propionibacterium acnes,Appl. Environ. Microbiol. 55 (1989)2899-2903.

[2] Blaszczyk M., Effect of medium composi-tion on the denitrification of nitrate by Para-coccus denitrificans, Appl. Environ. Micro-biol. 59 (1993) 3951-3953.

[3] Blësl M., Conrad R., Influence of an increa-sed pH on the composition of the nitrate-redu-cing microbial populations in an anaerobi-cally incubated acidic forest soil, Syst. Appl.Microbiol. 15 (1992) 624-627.

[4] Breed R.S., Murray E.G.D., Smith N.R.,Genus 1. Propionibacterium Orla-Jensen1909, in: Breed R.S., Murray E.G.D., SmithN.R., (Eds.) Bergey's Manual of Determina-tive Bacteriology, 7th edn. Williams & Wil-kins Co, Baltimore, MD, USA, 1957.

[5] Britz T.J., Riedel K.H.J., Propionibacteriumspecies diversity in Leerdammer cheese, Int.J. Food Microbiol. 22 (1994) 257-267.

[6] Cummins C.S., Johnson J.L., Genus 1. Pro-pionibacterium Orla-Jensen 1909, in: SneathP.H.A., Mair N.S., Sharpe M.E., Holt J.G.,(Eds.) Bergey's Manual of Systematic Bac-teriology, Vol. 2, Williams & Wilkins Co,Baltimore, MD, USA, 1986.

[7] Dalsgaard T., Bak F., Nitrate reduction in asulfate-reducing bacterium, Desulfovibriodesulfuricans, isolated from rice paddy soil:sulfide inhibition, kinetics, and regulation,Appl. Environ. Microbiol. 60 (1994)291-297.

[8] De Man J.D., Rogosa M., Sharpe M.E., Amedium for the cultivation of Lactobacilli,J. Appl. Bacteriol. 23 (1960) 130-135.

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