University of Groningen

Growth of bacteria at low oxygen concentrationsGerritse, Jan

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Chapter 5

Oxic and anoxic growth of a new Citrobacter

species on amino acids

Jan Gerritse and Jan c. Gottschal

In: Archives of Microbiology (1993) 160:51-61

Chapter 5

Oxic and anoxic growth of a new Citrohacter species on amino acids

Jan Gerritse, Jan C. Gottschal

Department of Microbiology; University of Groningen: KerkJaan 30, NL-9751 NN Haren. The Netherlands

Received: 17 November 1992/Accepted: 13 January, 1993

Abstract. A facultatively anaerobic bacterium, strain P-88, was enriched selectively under dual limitation by glutamate and oxygen in a chemostat. The new strain is a gram-negative motile rod. The mol% guanine plus cytosine of the DNA is 51.4 ± 0.6 mol%. The organism grows on citrate as a sole source of carbon and energy, does not form acetoin, does not induce lysine decarboxy­lase and was thus classified as a species of !he genus Citrobacter. A remarkable characteristic of the new isolate is its ability to grow on several amino acids with either a respiratory or a fermentative type of metabolism. Under strictly anoxic conditions glutamate was fer­mented to acetate, H" CO, and ammonia. Asparagine, aspartate and serine could also be fermented . Fur­thermore, all type strains of the genus Citrobacter were shown to have the same fermentative abilities. Based on enzyme activities determined in cell-free extracts a com­bination of the methylaspartate pathway and the mixed acid fermentation of Enterobacteriaceae is proposed to explain the glutamate fermentation pattem observed in cultures of strain P-88. Analysis of the growth of strain P-88 in continuous culture with various degrees of oxygen supply, demonstrated that the bacterium can rapidly switch between oxic and anoxic metabolism. Cultures of strain P-88 grown under oxygen limitation simulta­neously respire and ferment glutamate, suggesting that the organism is particularly well adapted to growth in microoxic environments.

Key words: Citrobacter species - Amino acids - Fa­cultative anaerobic growth - Fermentation of glutamate

Amino acids are quantitatively important sourees of carbon, nitrogen, sulphur and energy for microorganisms in both oxic and anoxic habitats (Barker 1961 ; McIner­ney 1988; Sepers 1979 ; Williams 1975). The metabolism of most amino acids has been studied in detail in several

Correspondence co : J. Gerritse

48

strictly aerobic and anaerobic bacteria. Aerobic bacteria usually convert amino acids di rectly into central metabo­lic intermediates via (oxidative) deamination te the corresponding keto acids (Gottschalk 1986). The de­gradation of amino acids under anoxic conditions has most extensively been studied in clostridia (Andreesen et al. 1989; Barker 1981; Buckel 1991), but several olher strictly anaerobic bacteria, members of the genus Bac­teroides, Fusobacterium, and representatives of the Pep­tostreptococcus group, Eubacterium acidaminophi/um, Se­/enomonas acidaminophi/a, Acidaminococcus fe rmentans, Acidaminobacter hydrogenoformans, Spirochaeta isova/e­rica and Megasphaera e/sdenii are also known to metabo­lize amino acids anoxically (Gharbia and Shah 1991 ; Harwood and Canale-Parola 1983 ; Mclnerney 1988 ; Nanninga et al. 1987; Stams and Hansen 1984; Wallace 1986; Wong et al. 1977 ; Zindel el al. 1988). In addition. some sulphate reducers can grow at lhe expense of amino acids (Widdel 1988) .

In many natural ecosystems (t1uctuating) gradients of molecular oxygen occur. Examples are, the rhizosphere (Patriquin et al. 1983), sediments (Revsbech and J0rgen­sen 1986), stratified aq uatic systems (Rudd and Taylor 1980), and the oral ecosystem (Mettraux et al. 1984) . In these habitats bacteria will be exposed to significant variations in lhe concentration of oxygen. Thus growth conditions may switch over from strictly anoxic to oxygen limited and in some cases to fully oxic. Obviously, lhe natural micro bi al population must be adapted to these changes and it may be envisaged that such environments represent important niches for oxygen toleranl. mi­croaerophilic and facultatively anaerobic bacteria (Bla­kemore et al. 1984; Gottschal and Szewzyk 1985; Krieg and HolTman 1986; Loesche et al. 1983 ; Mundt 1982; Ohta and Gottschal 1988 ; Trolldenier 1977). It is te be expected that facultatively anaerobic amino acids meta­bolizing bacteria are present in these microoxic environ­ments. However, although it has been shown that some [acultative anaerobes like Escherichia co/i, Proteus spp. (Barker 1961) and Citrobacter sp. (Janssen and Harfoot 1990) and the microaerophile Campy/obacter sp. (Laan­broek et al. 1977) may ferment a limited number of amino acids, relatively little is known on the ecology and

a

e

f

physiology of amino acid fennentation by such orga­nisms.

Therefore in the present study the enrichment, isoIa­tion and properties are described of a facultatively anaerobic amino acid fennenting bacterium. This orga­nism, identified as a Citrobacrer sp., was enriched selectiv­ely under oxygen limiting conditions with glutamate as the carbon and energy source. The new isolate is able 10

grow on several amino acids, either oxically with a respiralory metabolism or fermentatively under anoxic conditions. For comparison four known species of the genus Cirrobacrer : Cirrobacrer koseri, Cirrobacrer ama/o­naricus, Cirrobacrer freundii, and Cirrobacrer diversus were studied with respect to their ability to metabolize amino acids fennentatively.

Materials and methods

Orgallisms alld cu/rivarion

Slrain P·88 was isolated from a chemostat enrichment under a dual limltatio" of glUlamate and oxygen at a dilution fale of 0.1 h -I.

The chemostat had been inoculated with a soil sample obtained from the rhizosphere of Corylus sp.

Thc glutamate fennenting strain DKglu21 , obtained from our laboratory collection. had been isolated previously by Nanninga et al. (1986) from an enrichment obtained in an anoxic glutamate­Iimited chemostat inoculate<! with a sample obtained from an anoxic purification plant of a pot3to-starch faclory. This haclerium was tent3tively identified as a slraÎn of Citrobacter freundii by means of the API 20E test system (see below).

Citrobacter freundii DSM 30039T, Citrobacter koser; DSM 4595T• Citrobacler diversus DSM 4570T and Citrobacter ama­fonaTicus DSM 4593T are type strains of lhe genus Cirrobacler and were obtained from the German CollecLion of Microorganisms (DSM), Braunschweig. Gennany. Recently, it has been suggested that. C. koseri and G diversus belong lo lhe same species (Frederik­sen 1990). bUl in this paper the taxonomie classification used by the DSM is accepted .

Bacteria were grown at 30 oe in a phosphate-buffered (25 mM. pH 7.0) minima I medium containing vitamins. trace elements, resazurin and 0.01 % (wjv) yeast extract (GerrilSe et al. 1990). Carbon sources were added from 0.2- 1.0 M stock solutions that were autodaved or filter-sterilized (thermolabile compounds; 0.2 JlITl pore size). When amino acids were added as growth substrates. NH 4 C1 was omitted from the medium.

Anoxic batch-culture media were prepared under a Nratmo­sphere in butyl-stoppered crimp-seal bottles (20- 100 mI) or H ung­ate-tubes (5-10 mi) and reduced with 0.025% (wjv) Na,S . 9 H,O. Anoxic batch-cultures were incubated without shaking.

Growth in oxic batch cultures was perfonned in media without Na 2S. in cotton-plugged Erlenmeyer-nasks in a rotaryincubator at l50rpm.

Growlh in continuo us culture was eondueted as described previously (Gerritse el al. J 990). The culture vessel had a working­volume of 450 mi and eontained probes that allowed continuous measuremenl and regulation of the tempera tu re. pH, redox­potent ia I and dissolved oxygen concentration. The pH was main­tained at 7.0 (± 0.1) by automatic titration with IN NaOH and HCI. The Agj AgCI reference electrode of the pH-electrode was used as the reference cell to obtain redox-readings witb a platinum redox-electrode. A polarographic oxygen probe (Ingold) with a detection limit of approx. 0.1 IlM was used to measurc dissolved oxygen coneentrations. The rate of oxygen consumption by the bacleria in lhe chemostat was quantified by continuously measuring the oxygen concentration aod nOW-rale of gas (N1/air mixtures) entering and leaving the chemostat with a Servomex 1100 oxygen

Oxie and anoxie growth of Citrobaeter sp.

analyzer and gas now meters. respectively. Nitrogen gas was freed of traces of oxygen by passing it over hot copper filings. Media used forchemostat experiments were stored under N 2 aod contained 0.1 % (wjv) KH,PO. instead of the phosphate buffer, resulting in a final pH of 4.7 of the reservoir medium.

Bacteria were maintained on oxic outrient broth (BBL) plates. Purity of cultures was regularly checked by microscopie obser­vations and by plating on agar-plates containing minimal medium amended with 2% (w/v) agar incubated both oxically and under an N 2-atmosphere.

Cel/u/ar and biochemica/ characrerizarion

Detennination of the Gram-eharacter of the eell wal! was done according 10 Gregersen (1978) by staining aod with the KOH method Pseudomonas aeruginosa and Methanosarcina barkeri were used as gram-negative and gram-positive eonlrols, respectively.

The guanine plus cytosine (G + C) content of the DNA was detennined by the DSM on H PLC (Meshbah et al. 1989) af ter isolation of the DNA (Cashio et al. 1977) and hydrolysis with PI nuclease.

The API 20E system for identification of Enlerobacteriaceae and other gram-negative rods was used according te the API System Instruetion Manual , Version A (APT system, Marcy-L'Eloile. France).

Chemica/ ana/ysis

Cells were washed aod suspended in an isotonic 7.5 mM phosphate buffer solution pH 7.0 and stored witb the supernatant at -20 °C for further analysis.

Cell-protein was measured (Lowry et al. 1951) with bovine serum albumin as lhe standard. Cell-earbon, tOlal organic carbon and CO 2 in the supernatant were quantified with a Shimadzu TC-500 carbon analyzer with biphthalate as the standard for dissolved organic carbon. Cell-densities were roulinely measurd as the optical density at 660 nm 00660) in a Vitalab colorimeter (Vital Scientific. Dieren. Tbe Netherlands). Ammonium and formate concentrations were measured colorimetrically with assays according to Richterich (1965) and Lang and Lang (1972), respectively. Organic acids (Nanninga and Gottschal 1985). alcohols (Laan broek et al. 1984), as weil as H2 and CO 2 concentrations in the gas phase of cultures (GerrilSe et al. 1990) were analyzed gas chromatographically. Glutamate was detennined enzymatically (Bemt and Bergmeyer 1970) with glutamate dehydrogenase.

Respiromerry

Maximum specific oxygen consumption rates (QOi") were detenni­ned at 30 oe in a (Yellow Springs-type) biological oxygen monitor equipped with a polarographic electrode. Cells were taken from continuous culture and directly washed and suspended in 5 mi air-saturated buffer. Oxygen tracings were recorded before and after addition of 2 mM substrate. Maximum oxygen consumption rales were determined as the tangent from the concentration versus time plots between 100 and 50% air saturation and were correcled for tbe endogenous oxygen consumption rale. Jt was assumed. that air-saturated buffer contained 250 IlM dissolved O 2 -

Enzyme measurements

Crude extracts were prepared by sonication of cells grown anoxically on 30 mM glulamate. For sonication cells were harvested in tbe late logarithmic growth phase and washed and concenlrated in isotonic 20 mM phosphate buffer pH 7.0 (± 750 mg eell-pro­tein' mi -I) by means of eentrifugation. Crude extracts were kept on ice before determination of enzyme activilies . Enzyme aetivities were measured oxically (unless stated otherwise) in cuvetles (I 'ml; 1 cm) incubated at 30 cC in a Hitachi model 100-60 double-beam spectrophotometer. Assay mixtures contained 0.5-10 ~g cell pro-

49

Chapter 5

tein. 3-Methylaspartase (EC 4.3.1.2) activity was measured by following the production of mesaconate from 3-methylaspartate as indic3ted by lhe increase in absorption at 240 om, caused by the formation of a double bond. Assay condirions were as descri­bed by Hsiang and 6right (1969). Glutamate dehydrogenase (EC 1.4.1.2.) was assayed in the direction of glutamate formation from 2-oxoglutarate, NHt and NADH by foliowing the dis­appearance of NADH at 340 nm (Lerud and Whitely 1971). 2-0xoglutarate reductase (EC 1.1.99.2) was measured as NADH disapP!!arance in a 100 mM phosphate buffer pH 6.5 containing I mM 2-oxoglutarate (Lerud and Whitely 1971). 4-Hydroxybuty­rate dehydrogenase (EC 1.1.1.61) was detennined by foliowing the reduction of NAD + according to the assay described by Gharbia and Shah (1991). Malate dehydrogenase (EC 1.1.1.37) activity was analyzed both in the direction of l-malate formation or, consump­tion by observÎng the change in absorbance at 340 om in assay mixtures containing oxaloacetate and NADH or L-malate and NAD, respectively (Stams et al. 1984). Malie enzyme (EC 1.1.1.39) was assayed by foliowing NADPH formation at 340 om in a 100 mM Tris/HCI bulTerpH 7.4containing 10 mM MnCl" 0.5 mM NADP and 10 mM L-malate. Acetate kinase (EC 2.7.2.1) was measured according 10 the method described by Bergmeyer et al. (1970) for acelale kinase of Escherichia coli. Succinyl-CoA syn­thelase (EC 6.2.1.5) activities were quantified by lhe formation of succinohydroxamate in a 50 mM Tris-HCl bufTer pH 7.2. con­taining MgCl" A TP, CoA, succinate and hydroxylamine (Dijkhui­zen et al. 1980).

Chemicals

Chemicals IlUsed were obtained from commercial companies and were of analytica I grade. Amino acids and organic acids were of "l-" and sugars of "o-configuration".

Results

Enrichment and isolation of a facultatively anaerobic glutamate metabolizing bacterium

A chemostat was inoculated with a soil suspension obtained by mixing approximately 5 g of soil from the rhizosphere of Corylus sp. with anoxic medium and subsequent fittration over a membrane filter (1.2 jlm) to remove protozoa. Af ter an initial period (approx. 10 h) of anoxic batch cultivation at 30 °C, a medium containing 30 mM glutamate as the carbon and energy source was supplied at a dilution rate of 0.1 h - I At the same time the chemostat was carefully aerated in such a way that a redox reading was maintained of - 100 to - 50 mV. After a short (approx. 10 h) initial period of increasing cell-density, oxygen-limited enrichment conditions were established by maintaining a cell-density which was intermediate (00660 = 0.88) to that obtained in an anoxic and an oxic batch-enrichment grown on 30 mM glutamate (00660 = 0.23 and 2.70, respectively). The major products detected in the chemostat were acetate (38-45 mM), ammonium (26-28 mM) and CO2 (27-44 mM).

After enrichment under these conditions over aperiod of approx. 200 h the numerically dominant bacteria were isolated on glutamate-agar plates incubated under anoxic or oxic conditions. All bacteria picked from colonies on the anoxic plates and the majority of those picked from the oxic plates (> 70%) appeared to be capable of

50

growth on glutamate under both anoxic and oxic condi­tions. From the oxic plates a secondary population (=:; 30%) ofbacteria was isolated which could be grown on glutamate under oxic conditions only. No strictly anaerohic glutamate degrading bacteria were obtained. The numerically dominant facultatively anaerobic gluta­mate metabolizing bacterium, provisionally named strain P-88, was isolated in pure culture by several transfers on oxic glutamate-agar plates.

The cells of strain P-88 were rod-shaped, 0.8- 1.0 x 2 -4 jlII1 in size and motile. Electron micrographs

suggested that the flagella were peritrichously attached (Fig. I A) and indicated the presence of pili (Fig. I B). Endospores were never observed. A Gram-negative type of cell-wall was indicated based on Gram-staining and on the basis of the KOH-test. The guanine plus cytosine content of !he DNA was 51.4 ± 0.6 mol%.

Occasionally, virus-like particles were observed inside !he cytoplasm (Fig. IC) and outside of the eells recog­nizable viruses appeared sometimes attached to the outer cell-wall (Fig. 10) or freely in the culture-liquid. In these cultures approximately I virus particIe was observed per 100 bacteria. These viruses remained present in spite of repeated streaking on agar plates, or by serial transfers in liquid media.

Physiological characteristics of strain P-88

Strain P-88 is capable ofboth fermentalive, strictly anoxic growth and of oxic growth on glutamate with a maximum specific growth-rate of 0.24 h - land 0.44 h - I, respecliv­ely. The molar growth yield on glutamate in an anoxic batch culture was 4.1 g cell-carbon per .mol glutamate fermented, corresponding to 2.8 g cell-protein per mol of glutamate. The yield in an oxic batch culture was considerably higher, 31.2 g cell-carbon and 30.2 g cell­protein per mol of glutamate, respectively.

Neither vitamins nor yeast extract were needed for oxic growth on glutamate, but for anoxic growth this strain required some yeast extract (0.1 gfl).

A wide range of carbon sourees supported growth of strain P-88 under both oxic and anoxic conditions (TabIe I). All sugars tested were utilized oxically and anoxically. Most organic acids tested (except forma te) were used oxically, but only a few (citrate, pyruvate and fumarate) were fermented. Strain P-88 grew on glycerol both oxically and anoxically. Under oxic conditions strain P-88 grew on all amino acids tested, except glutamine. Amino acids supporting anoxic growth were asparagine, aspartate, glutamate and serine.

Amino acids fermented and the major fermentation products formed in batch-cultures are listed in Table 2. Ammoni.um, aeetate, H 2 and CO2 were fermentation produets from all amino acids tested, whereas succinate appeared as an important product from aspartate and asparagine. Ouring growth on glutamate and serine only traces « I mM) of succinate, fumarate, lactate, formate and ethanol were formed. Formation of the following acids, propionate, butyrate, pyruvate, malate or the alcohols methanol, propanol or butanediol was never observed.

ii-m In

Iy d. a-in m

.0 IS :d ). ,e ,d Ie

ie ,-, Ie l. IS tI 1I

c n

c e ,i .s i-

f

A

8

Fig. J A-D. Electronmicrographs of log-phase cells of strain P-88 grown on glulamat~ in aD anoxic batch culture. A Peritrichously 3tt3ched flagella , B cell-membrane with pili, C virus-like particles inside the cytoplasm, D idem attached to the eell envelope and freely in the culture liquid. A, Band D Negatively stained with 1%

Table 1. Substrates used for growth of strain Substrate P·88 anaerobic (fermentative) or aerobic

conditions Fonnate Acetate Propionate Pyruvale Lactate Butyrate Succinate Fumarate Malonate Citrate

Alanine Asparagine Aspartate Cysteine Glutamine Glutamate Glycine Hislidine Proline Serine

nd: Not determined

Oxic and anoxic growth of Citrobacter sp.

uranyl-acetate and C ultrathin section fixed with 3% glutaralde­hyde. post-fixed with 1% OsO •. 5% K 2Cr1 0 7 and stained with 1 % uranyl-acetate. Bar represents 0.5 Jlm in A and 0.1 Jlm in 0, C and D

Aerobic Anaerobic Substrate Aerobic Anaerobic

Methanol + Ethanol + n-Propanol + + iso-Propanol + Glycerol + + + + + + Mannitol + + + Sorbitol + + + + Xylose + +

Raffinose + + + Fructose + + + + Glucose + + + + Galactose + + nd Ribose + +

Lactose + + + + Sucrose + + + Cellobiose + + + Maltose + + + + + lnulin

51

Chapter 5

Table 2. Products formed during Cennentative growth of strain P-88 on various amina acids in batch cultures

Substrate Product forma tien (mol/mol substrate)

NH; Acet.te Fonn.te L.ct •• e Succi- CO, nate

Aspa.ragine 1.72 0.43 0.03 < 0.01 0.66 0.45 Aspartate 0.85 0.45 0.03 < 0.01 0.58 0.54 Glutamate 0.90 1.78 0.03 0.Q2 0.03 0.42 Serioe 0.93 0.83 0.15 0.03 0.03 0.43

Using the APr 20E identification system for Entero­bacteriaceae and non-fermenters the following results were obtained: strain P-88 contained beta-galactosidase, arginine dihydrolase, omithine decarboxylase and tryp­tophane desaminase activity. Oxidase, lysine decarboxy­lase, urease and gelatinase activity, the production of indoie, H2S or acetoin could not be demonstrated. Also according to this test-system the following compounds were metabolized: citrate, glucose, mannitol, sorbitol, rhamnose, sucrose, melibiose and arabinose. Inositol and amygdalin were not metabolized. Nitrate was reduced to nitrite, but not to N2. The resulting profile did not yield a positive identification of strain P-88 as a known species, but it indicated that this strain belonged to the genus ei/robae/er.

Because of the indicated relatedness to the genus eitrobae/er, five other species of this genus were tested with respect to their ability to ferment amino acids. ei/robae/er freundii, Citrobae/er koseri, Citrobaeter diver­sus, and Ci/robaeter amalonaticus were obtained directly from the Deutsche Sammlung von Mikroorganismen (Braunschweig). A fifth strain, DKglu21, maintained in our laboratory collection was positively identified (89% degree of certainty) as a strain of G.freundii, using the API 20E system. All five organisms grew anoxically on the same amine acids as strain P-88. Tbe fermentation of glutamate by these Citrobae/er strains yielded the same end products as formed by strain P-88 although, C.freun­dii DSM 30039 and C. amalona/ieus produced more formate (0.3 - 0.5 mol per mol of glutamate) and concom­itantly less H2 and CO2.

The activities of some enzymes detected in cell-free extracts of strain P-88 grown anoxically on glutamate are shown in Table 3. Indeed very high activities of 3-methylaspartase, a key-enzyme of the mesaconate route, were detected in strain P-88. Moreover, all Ci­/robae/er strains used in the present study possessed high activities ofthis enzyme (17- \05 I1mol/min . mg protein). Activities of glutamate dehydrogenase and 2-oxogluta­rate dehydrogenase, enzymes involved in the anoxic degradation of glutamate via the 2-oxoglutarate path­way, and of 4-hydroxybutyrate dehydrogenase, a key-enzyme of the 4-aminobutyrate route, were < 0.01 I1mol/min' mg protein. Enzyme measurements also indicated that mal ic enzyme and acetate kinase were not present in th is bacterium. However, the presence of malate dehydrogenase and succinyl-CoA synthetase in­dicated that this organism may becapable ofsynthesizing

52

Growth-yield Recovery (g/mol substra.e) (%)

H, Cell- C.rbon C.rbon Electrons carbon

< 0.01 0.32 3.84 108 100 < 0.01 0.33 3.96 103 100

0.57 0.34 4.08 91 93 0.53 0.23 2.76 89 95

Table 3. Enzymes detected in cell-free extracts of strain P-88 grown on glutamate in an anaerobic batch<ulture. Activities expressed as: Ilmol/min . mg protein

Enzyme

3-Methylaspartase t-Glutamate dehydrogenase 2-0xoglutarate reduetase 4-Hydroxybutyr.te synthe.ase Mala.edehydrogenase (NAD-form •• ion)

(NADH-formation) Malie enzyme Acetate kinase Succinyl-CoA synthetase

Specific activity

100.0 0.04

< 0.01 < 0.01

6.5 4.3

< 0.01 < 0.01

18.8

A TP via substrate level phosphorylation from succinyl­phosphate.

When strain P-88 was grown in an oxic batch cul­ture, 2-oxoglutarate reductase activity was present (0.066 I1mol/min . mg protein) indicating the induction the tricarboxylic acid cycle in this bacterium under these conditions.

Injluenee of oxygen on the grow/h of strain P-88 in continuous culture

Steady-state cultures

Steady-state chemostat cultures of strain P-88, grown on glutamate as the sole carbon and energy source, were obtained at different dilution rates either anoxically, oxygen limited or oxygen sufficient. In the anoxic steady-state cultures glutamate was fermented to acetate, formate, H2' CO2 and NHt, with liule qualitative and quantitative differences at the various dilution rates (Tabie 4) and in batch culture (Tabie 2). Lactate, succinate, fumarate and ethanol, formed in trace amounts in batch- cultures were not detected (concentrations < 10 I1M) in steady-state cultures. When grown under oxygen limitation, strain P-88 reduced the dissolved oxygen concentration to a value below the detection limit of the probe « 0.1 I1M). Under these conditions less acetate was formed than under strictly anoxic conditions (Tabie 4). H2 nor formate were produced in measurable amounts. At oxygen sufficient conditions (dissolved 02-<:oncentration 100-150 I1M) strain P-88 converted glutamate completely into biomass, CO2 and ammo-

Jns

own

ity

:ul­ent ion ese

in

on ere lIy, xic lte, .nd tes lte, nts lOS jer ,ed nit ess lOS bie 'ed ted 10-

Oxic and anoxic growth of Citrobacter sp.

Table 4. Analysis of glutamate-limited chcmostat cultures of strain P-88 grown at various dilution rales and oxygen supply

Dilution Growth Product fonnation or oxygen consumption Growth-yield Recovery rate (h - ') condition (mol/mol glutamate)

0, NHt Acetate Fonnate CO,

0.05 Anaerobic 0 1.19 2.02 0 0.75 0.09 Anaerobic 0 1.06 1.79 0.01 0.75 0.12 Anaerohic 0 1.12 1.90 0.20 nd 0.19 Anaerobic 0 1.20 1.90 0 0.67 0.09 Ol-limited 0.12 0.96 1.38 0 0.95 0.12 02-Iimited nd nd 0.68 0 0.05 °2-sumdent 0.64 0.48 0 0 0.09 °2-sufficient 0.60 0.56 0 0 0.12 Orsufficient nd 0.70 0 0 0.17 02-sufficient nd 0.58 0 0 0.39 °2-sumcient nd 0.44 0 0

nd : Not determined Not reJiahle due to excessive wall-growth

nium. Residual concentrations of glutamate in the chemoslal, measured in steady-state cultures of strain P-88 at a dilution rate of 0.12 h -', were 0.13-0.16 I1M under anoxic, oxygen limited and oxygen sufficient conditions.

The growth yield on glulamale increased considerably wilh increasing oxygen availability (Tabie 4). For exam­ple, at a dilution rale of 0.09 h -', the percenlages of glutamate-carbon converted inlo bi oma ss were 7.4%, 22.2% and 49.6%, under anoxic, oxygen limited and oxygen sufficient conditions, respectively. Incorporation of (glulamate-) nitrogen into biomass explains the relalively low recovery of ammonium in the supernatant of the oxygen sufficient steady-states.

Maximum specific oxygen consumption rates were measured respiromelrically with resting cell suspensions obtained from glutamate limited steady-state cultures (D = 0.1 h - ') of strain P-88. The specific rates of oxygen consumption measured in situ in steady-state cultures in lhe chemostal were always significantly lower than the potential rates of oxygen consumption measured with various carbon-compounds in the biological oxygen monitor. Furthermore, cells taken from an anoxic culture respired glutamate at a maximum rale approx. 6 times

Table 5. Specific rales of oxygen consumption of strain P-88, and the enrichment from which it was isolated, grown under glutamate Iimitation in continuo us culture (0 = 0.1 h - I) at various oxygen supplies. Specific rales were measured in thc chemostat (qOz), or in a

nd 1.89 2.33 nd nd nd

(g/mol glutamate) ('10)

H, Cell- C.rbon Protein C.rbon Electrons carbon

0.74 0.29 3.41 2.37 102 109 0.71 0.37 4.49 3.11 94 99 nd 0.41 4.94 3.43 0.67 0.27' 3.25' 2.26' 95 105 0 1.11 13.29 9.23 96 91 0 1.46 17.47 12.13 0 3.02 36.20 25.14 98 71 0 2.48 29.78 20.68 96 65 nd 2.58 30.91 21.47 nd 3.09 37.11 25.77 nd 1.91' 22.91' 15.91'

lower than those from an oxygen sufficient culture (Tabie 5). Under oxygen limited conditions the QO~" was intermediate to the rate under anoxic and oxygen sufficient conditions.

Potential intermediates and end-products of gluta­mate fermentation, i.e. methylaspartate, aeetate, formate and succinate, were also respired by strain P-88 (Tab Ie 5) . Generally, the rates obtained with anoxically grown eells were lower than those obtained from cells grown under oxygen limitation or oxygen sufficient conditions. Formate was oxidized at a relatively high rate (0.243 I1mol/min . mg protein) by anoxically grown cells. Oxygen suflïcient and oxygen-limited grown cultures also oxidized formate at the highest rate.

Maximum specific oxygen consumption rates of the (oxygen limited) chemostat-enrichment, from which strain P-88 was isolated (see above), were 1.5 to 4 times higher than those obtained with the oxygen limited pure culture of trus bacterium (Tabie 5).

Transient state cultures

The transition from anoxic growth on glutamate at a dilution rate of 0.09 h -, to oxygen limiled growth was

biological oxygen monitor with cels taken from steady state cultures (QO~1). Substrates were added at 2 mM concentrations. Specific rales are expressed as: ~ol/min . mg prolein

Culture Growth Specific rale of oxygen consumption condition

qO, QOF, with 2 mM of:

Glutamate Methyl- Acetate Formate Succinate aspartatc

Str.in P-88 Anaerobic 0 0.034 0.038 0.018 0.243 0.062 Str.in P-88 02+1imÎted 0.020 0.161 0.110 0.040 0.610 0.408 Str.in P-88 °l+sufficient 0.045 0.221 0.228 0.188 0.633 0.408 Enrichment Oz+limited nd 0.52 nd 0.15 1.38 0.56

53

Chapter S

100

80

60 ,--.. ::::E E 40

C 0 -<)

"-ë 20

Cl> u C 0

U

A

0-" 0/

cf

~-J._._. 1\ ---. , \

\ \ \ \

0.8

0.6

0.4

0.2

10 'r 8\'-

6

4

2

100 6

2

1 10 0.8

0.6

0.4

1

0.2

~~ C

'\ .-----~ '\~ . \ I \ . \

I \ 0-

e \ \

1

0.6

0 . 6

0.4

o o

0.2

\ 10 L-~~~~~_~....J 0.1 i 1 L-...:....~_~_~_..lI 0.1 O. 1 \

0.1 10 15 20 25 30 o 5 10 15 20 25 30 o 10 20 30 40 o 5

Time (h)

Fig. ZA, B. Response of strain P·88 to sudden shirts in oxygen ,upply. A Shifl of an anoxically grown culture (0 = 0.09 h -1: S, (glutamate) = 22 mM) to Qxygen limited conditions. B Changeover from oxygen limiled to oxygen sufficient growlh conditions (C) Response of a sudden change of an oxygen sullicient culture

studied by switching the gas-supply from 100% N2 to a mixture containing 16% air and 84% N2 (v/v). Immediately following the change in gas-supply the production of H 2 stopped and the dissolved oxygen concentration began to increase gradually to a maximum of 6 ~M after 2.4 h, and subsequently decreased again to a level below. the detection limit of the oxygen probe ( < 0.1 ~M) approx . 5 haf ter the introduction of air. Acetate production continued, but to a somewhat lower extent than under strictly anoxic conditions (Fig. 2A). Within two hours following the transition the cell-density began to increase, approaching a new steady-state level of approximately three times the anoxic value.

After the culture had grown under oxygen limited conditions for 9 volume changes (100 hl, the oxygen supply was increased further by sparging air through the culture instead of the air-nitrogen mixture (Fig. 2 B). The dissolved oxygen concentration increased within minutes to measurable concentrations (> 0.1 ~M) and stabilized after several hours at a concentration of 180 to 200 ~M. Following this switch from oxygen limited to oxygen sufficient conditions, the acetate concentration feil dramatically to a very low level at a rate much faster than through dilution with fresh medium. This indicated that acetate was metabolized wh en oxygen was supplied in excess. The biomass of strain P-88 in the culture increased and stabilized, af ter 4 to 5 volume changes under fully oxic conditions, at a value 6.5 times higher then obtained under strictly anoxic conditions.

In order to establish how transition to anoxic conditions takes place an oxic steady state culture of strain P-88 (0 = 0.12 h- 1

) was made anoxic by sub­stituting air by pure N 2-gas. Ouring the first 4-6 h the cells washed out from the culture at a rate corresponding to the dilution rate (Fig. 2C), but suhsequently decreased

S4

(0 = O. I 2 h -1: S, (glulamale) = 15 mM) 10 striclly anoxic condi­tion,. Symbols: Cell-den,ilY (00'6.) (0 ): Acelale (e): Formale (+). The dashed fine indicates the theoretical ra Ie of washout when na growth or metabolism occurred

less rapidly and eventually stahilized at a density much less than during oxic conditions. The production of acetate started within 20 min af ter the switch to anoxic conditions and after 2 h a transient accumulation of formate was observed followed by a gradual decrease to concentrations similar to those obtained in anoxic steady state cultures (not shown).

Discussion

Physiological properties

Strain P-88 is a versatile facultatively anaerobic bacte­rium capable of utilizing a wide range of organic com­pounds as a sole source of carbon and energy. The ability to grow fermentatively with several amino acids is of particular interest since very little is known about anoxic growth on amino acids by facultatively anaerobic bac­teria.

Out of ten different amino acids tested, strain P-88 fermented only four: asparagine, aspartate, glutamate and serine. lt is remarkahle that all other members of the genus Cirrobacter used in this study, Citrobacter freundii. Citrobacter koseri, Citrobacter diversIIs and Ci­trobacter amalonaticus and strain OKglu21 (tentatively/ identified as a strain of C.freundil). fermented the same amino acids as indicated for strain P-88.

The fermentation of glutamate by strain P-88 was studied in some detail. The pattern of product formation during strictly anoxic growth resem bles most c10sely the first of the following three possible stoichiometries for the fermentation of glutamate. The major difference between these fermentations is caused by the different

Idi­late hen

lch of

xic of

: to tdy

:te­m­ity of

xic ac-

-88 ate of Ier 0-Iy/ me

las on the for lee ~nt

electron sink products used to dispose of the reducing equivalents generated:

Glutamate- + 3 H,O -+ 2 acetate- + HeO, + NH; + H+ + H,. (I)

AGo, = - 39.8 kJ/ reaction (Wohlfarth and Buckel 1985).

Glutamate- + 2 H,O -+ 1.67 acetate- + 0.33 propionate- + 0.67 HeO,

+ NH.i + 0.67 H + . (2)

AGo' = -62.9 kJ/ reaction [Nanninga et al. (1987); AGo' calculated from data given by Thauer et al. (1977) and Wohlfarth and Buckel (1985)J.

I Acetate I

Glutamate

3-Methylaspartate

8~0 Mesaconate

H20i0 Citramalate

;--{0 Pyruvate ....... ~

Acetyl-CoA

... ...

• o

Oxic and anoxic growtb of Citrobacter sp_

Glutamate- + 2 H,O -+ aeetate- + 0.5 butyrate- + HeO, + NH;

+O.5H+.

AGo, = - 63.1 kj /reactioD (Wohlfarth and Buckel 1985).

(3)

In the first equation, reducing equivalents are released via the prod uction of H ,. A similar reaction stoichiometry was observed fOr the fermentation of glutamate by pure cultures of Acidaminobacler hydrogenoformans (Stams and Hansen 1984). Although 3-methylaspartase activity was observed in cell-free extracts of A. hydrogenoformans, enzyme measurements indicated that in strain P-88 a different metabolic pathway led to this pattern of product formation. These authors further showed that in co-cultures with a H,-consuming bacterium, a shift in the fermentation pattern of A. hydrogenoformans occur­red and large amounts of propionate were formed. No sucb change was observed in co-cultures of strain P-88 with a H,-utilizing Desulfovibrio sp. as performed in the present study (results not shown) .

A preliminary scheme for the pathway of glutamate fermentation in strain P-88 is proposed on the basis of known fermentation routes and results of the determina­tion of activities of several key enzymes of the proposed route (Fig. 3). The presence of high activities of 3-methylaspartase and the absence of significant activities of glutamate debydrogenase, 2-oxoglutarate reductase and 4-hydroxybutyrate dehydrogenase indicate that the initial steps in the anoxic metabolism of glutamate are those which charactenstically belong to the c1assical mesaconate route present in several members ofthe genus ClosIridium (Buckel and Barker 1974). In this pathway glutamate is first rearranged to 3-methylaspartate by the enzyme glutamate mutase. Also in a threonine auxotro­phic mutant of Escherichia coli, which formed isoleucine from glutamate this enzyme has been demonstrated (Pbillips et al. 1972). The subsequent elimination of ammonium by 3-methylaspartase yields mesaconate, which is then bydrated to yield citramalate. Citramalate is c1eaved by citramalate-Iyase into acetate and pyruvate. Interestingly, this enzyme is very similar to the enzyme

I [~~~~!;;;i ~COA~I ATP

,..------, CD + I Acetate I Succinyl-CoA

'----- CoA 4_---'

Fig. 3. Proposed pathway for the fennenlalion of glutamate by slrain P-88. Compounds in boxes indicate major fennentation products. Compounds in dashed boxes were fonned in minor concentrations. See text for quantilalive details . En:)'mes indicared are : ( J) glutamate rnutase; (2) 3-methylaspartase ; (3) citramalate

ADP +

dehydralase ; (4) citramalale Iyase; (5) pyruvale-formale Iyase; (6) forma Ie hydrogenlyase ; (7) succinyl-CoA synlhelase ; (8) succinale thiokinase. (9; In order to maintain the A TP-generalÏng cycle via succinyl-CoA some anaplerotic ronnation of succinatc is supposedly provided by lhe revcrsed TCA-cycle

55

Chapter S

citrate-Iyase, which oecurs in citrate utilizing Enterobac­teriaceae (Gottschalk 1986).

In c10stridial fermentations pyruvate is subsequently converted into acetate, butyrate and CO 2 (Andreesen et al. 1989; Buckel 1991). However, in Enterobac­teriaceae, grown under anoxic conditions, pyruvate is split into formate and acetyl-CoA by pyruvate-formate Iyase, a characteristic enzyme of the mixed-acid fermen­tation carried out by these baeteria (Gottschalk 1986; Knappe 1987). The formate thus produced is cleaved into H 2 and CO 2 by formate-hydrogen Iyase and ATP is generated by means ofsubstrate level phosphorylation via acetyl-CoA and acetyl-phosphate to acetate. Although strain P-88 formed acetate, formate, H2 and CO 2 , ATP­synthesis in strain P-88 probably did not result directly from aeetyl-phosphate since acetate kinase was not detected in this bacterium. Instead, the presence of succinyl-CoA synthetase and mala te dehydrogenase and the formation oftraee amounts ofsuccinate and fumarate suggested th at A TP may be generated by succinate thiokinase through CoA-transfer from acetyl-CoA to succinate. Such a mechanism of A TP-synthesis has also been reported for the Oagellated protozoan Trichomonas foetus (Lindmark et al. 1989) and recently has been demonstrated in an anaerobic fungus, Neocallimastix L2, (Marvin et al., in preparation). Thus, according to this fermentation pathway one mol of A TP is produced per mol of glutamate femented. When assuming that 40% of the cell dry-weight of strain P-88 consists of carbon a yATP of 10.3 g eell mate rial per mol A TP can be calculated per mol of glutamate fermented in batch culture.

Ecological aspects

When grown in continuous culture under oxygen limitation, both the growth yield of strain P-88 on glutamate, and the CO 2 and acetate production were intermediate to those observed under strictly anoxic and oxygen sufficient conditions. These observations suggest that under these conditions the metabolism of a culture of strain P-88 is fermentative and respiratory at the same time. This phenomenon has been described for several facultative anaerobes grown under a dual limitation of a carbon substrate and oxygen (Harrison and Loveless 1973a; Wimpenny and Necklen 1968 ; Linton et al. 1975 ; Gottsehal and Szewzyk 1985). The fact that under oxygen limitation strain P-88 produced acetate as a fermentation product but neither H 2 nor forma te, may reOect the strong sensitivity of pyruvate-formate Iyase for oxygen (Knappe 1987). Another possibility is that formate (or H 2), produced under these conditions was instanta­neously respired, as such cultures possessed high formate oxidation capacities. Such a situation has been demon­strated indeed for a continuous culture of a marine Vibrio sp. grown under glucose and oxygen limiting conditions. Using radioactively labelled acetate it was shown that acetate was both produced and respired at the same time (Gottschal and Szewzyk 1984).

The latter authors further argued that conditions with a duallimitation of glucose and oxygen strongly favour

S6

the enrichment of facultatively anaerobic bacteria . Indeed, strain P-88, became the dominant organism in an enrichment under dual limitation of glutamate and oxygen. In principle, such growth conditions would also allow the establishment of mixed cultures composed of strictly aerobic and anaerobic bacteria, as such organisms were shown to co-exist in oxygen limited chemostats (Gerritse et al. 1990, 1992). However, under these conditions facultative anaerobes may weil have a selective advantage over both strict aerobes and anaerobes if they are able to simultaneously respire and ferment the main substrate. This situation might be compared with growth under limitation by two distinctly different substrates: a fermentabie and a non-fermentable substrate. Under oxygen limitation this capacity would then enable them to gain more energy and thus produce a higher biomass per mol substate consumed than organisms that can only respire or ferment. Under such conditions especially baeteria with a high yield on either or both of these "two" substrates have a major selective advantage over bacteria with a lower yield (Gottschal 1986).

For several facultative anaerobes such as Vibrio sp. , Beneckea narriegens, E. coti and Klebsiella aerogenes, it was observed that the maximum specific substrate oxidation capacity was not significantly repressed when grown in continuous cultures gassed with N 2 (Gottschal and Szewzyk 1985; Harrison and Loveless 1971a; Linton et al. 1975). Although not all these investigators excluded th at traces of oxygen may have been present in the medium or gas-supply, it is remarkable that these organisms continued to produce all th~ enzymes needed for respiratory metabolism in spi te of the fact that their metabolism was fermentative. Quite the opposite was true for strain P-88. Under anoxic eonditions the specific glutamate oxidation capaeity of this bacterium was 3 to 9 times lower than under oxic or oxygen limited conditions. Strain P-88 adapted its metabolism within 2 to 6 h following a sudden shift in the supply of oxygen. This is comparable to the rate at which C.freundii (Keevil et al. 1979), Vibrio sp. (Gottsehal and Szewzyk 1985), E. co/i and K. aerogenes (Harrison and Loveless 1971 b) were shown to respond to such changes.

Gottsehal and Szewzyk (1984) argued th at this ability to switch rapidly between arespiratory and a fer­mentative mode of metabolism adds considerably to the competiveness of bacteria in an environment with a Ouctuating oxygen supply. As strain P-88 can promptly consurne its own fermentation products as soon as oxygen becomes available and immediately switches over to fermentative growth upon deprivation of oxygen, this bacterium seems weil adapted to such environmental conditidns, for example in the rhizosphere, the environ­ment from which this strain was isolated. The coneentra­tions of oxygen ans carbon substrates, such as sugars, amino acids and othe organic acids, are known to vary considerably due to changes in the consumption of these compounds by rhizosphere-microorganisms and by variations in the excretion by plant roots (Richards 1987). Such changes are induced, for example by varia ti ons in temperature, soil moisture-content and ligbt/dark re­gimes.

n h li (

;a. 10

.nd lso of

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ria

:p .. , it ate len hal :on led the ese led .eir vas ific to

ted 12 en. ,vil 5), . b)

ity 'er­the

a tly ;en to

his tal >n­ra­rs, lry :se by 7). in

re-

The fact that facultatively anaerobic amino acid metabolizing Citrobac/er spp. were enriched from various habitats. selectively on amino acids under oxygen limitation (this study), under strictly anoxic conditions (Nanninga and Gottschal 1986), but also using tech­niques that were not based upon growth on amino acids (Farmer 111 1981; Janssen and Harfoot 1990) indicates th at the capacity to ferment amino acids is weil conserved within the genus of Ci/robac/er and may be more important for the survival of these bacteria in the natural environments than hitherto considered. Moreover, this ability mayalso contribute to the opartunistic pathogenic nature of several members of the genus Ci/robac/er (Farmer 111 1981; Shakazaki 1984).

Taxonomy

Strain P-88 is a gram-negative, facultatively anaerobic, motile rod. The oxidase test was negative. These characteristics place strain P-88 in the family of the Enterobacteriaceae (Brenner 1984). The ability to utilize citrate as a sole carbon and energy source, the absence of lysine decarboxylase activity and the negative Voges­Proskauer test (absence of acetoin production) indicated th at strain P-88 belonged to the genus Ci/robac/er (Shakazaki 1984). The mol% G + C of the DNA of strain P-88 (SlA ± 0.6%) is within the range given for this genus (50- 52%). Over the last decade there have been several changes in the taxonomy of species within the genus Citrobac/er. Shakazaki (1984) distinguished three species: Citrobac/er freundii , Citrobac/er diversus and Citrobacter amalonaticus. More recently, Frederik­sen (1990) proposed Citrobac/er koseri to be the correct name for C. div,ersus. Altough strain P-88 appeared ciosely related to these bacteria, this organism could not be assigned to any one of these species due to the following discriminating characteristics: (i) strain P-88 does not produce indoie. whereas C. koserijC. diversus and C. amalonaticus do ; (i i) strain P-88 does utilize malonate, whereas C.Jreundii and C. amalonaticus do not metabolize this compound (Farmer III 1981).

Cells of Citrobac/er sp. strain P-88 are rod-shaped, 0.8 - 1.0 x 2-4 ~m and motile by means of peritrichously attached nagella. The Gram-reaction is negative and endospores are not formed. The organism was selectively enriched from the rhizosphere of Corylus sp. under dual limitation of glutamate and oxygen. For oxic growth no co-factors are needed, however, for fermentative growth on glutamate the organism requires some yeast extract (0.1 gjl). A wide range of substrates including organic acids, amino acids, sugars and glycerol is utilized as the source of carbon and energy under both oxic as weil as anoxic (fermentative) conditions. Ammonium, acetate, succinate. H 2 and CO 2 (ratio I : I) are the major products during the fermentation of aspartate. asparagine. gluta­mate and serine. Nitrate is reduced to ni tri te. Growth occurs over a pH-range from 4 to 9 and over a temperature-range from 4 to 45 °C (optimum 37 0C).

DNA base ratio: 51A ± 0.6 mol% guanine plus cytosine (HPLC).

Oxie and anoxie growth of Citrobaeter sp.

Acknowledgements. We are grateful to G. BerreIkamp-Lahpor and T. Gomez for technical assistance. and thank F. Marvin-Sikkema and Dr. T. A. Hansen for help with the enzyme measurements. We thank K. A. Sjollema and M. Vecnhuis for the preparation of the electron micrographs. This work was supported by the Netherlands Integrated Soil Research Programme.

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