Bacterial metabolism of ethylene glycol

194

Biochimica etBiophysieaActa, 677 (1981) 194- 199 Elsevier/North-HollandBiomedicalPress

BBA 29713 BACTERIAL METABOLISM OF ETHYLENE GLYCOL ANDREWWILLETTS Department of Biological Sciences, University of Exeter, WashingtonSinger Laboratories, Perry Road, Exeter EX4 4QG, Devon (U.K.)

(Received February 27th, 1981) Key words: Bacterial metabolism; Ethylene glycol

Metabolism of ethylene glycol as the sole source of carbon by a species of Flavobacterium was affected by the dissolved oxygen tension of the growth medium. Under strongly aerobic conditions the diol was exclusively metabolised to glycollate by an initial oxidase, subsequently metabolised to aeetyl-CoA with no net change in ATP, and then oxidised to C02 by the tricarboxylic acid cycle yielding large amounts of reduced nicotinamide nucleotides which were used to generate a net gain in ATP by oxidative phosphorylation. Under microaerophilic conditions, some ethylene glycol after initial metabolism to acetyl-CoA by the oxidase-initiated pathway, was subsequently catabolised to acetyl phosphate and then acetate, yielding a net gain in ATP by substrate-level phosphorylation: additionally some diol was catabolised by an inducible diol dehydratase to acetaldehyde and subsequently reduced to ethanol as a terminal metabolite.

Introduction It has been established that the pathway of propane 1,2-diol metabolism by Flavobacterium sp. NCIB 11 171 is influenced by the degree of aeration of the growth medium [1]. Growth of this bacterium on ethylene glycol (ethane 1,2-diol) has also been shown to be dependent on oxygen availability in the culture medium, and the pathway for metabolism of the diol under strongly aerobic conditions has been characterised [2]. Principally because of the participation of two alcohol oxidase activities in the aerobic metabolism of ethylene glycol by Flavobacterium sp. NCIB 11 171, it was considered possible that low oxygen availability may promote altered metabolism of the diol by this bacterium. Alcohol oxidase-independent metabolism of ethylene glycol to acetaldehyde by a diol dehydratase and subsequent assimilation of the aldehyde via acetate and acetyl-CoA has been detected in aerobic cultures of ethylene glycolgrown Mycobacterium sp. E44 [3]. Diol dehydratasedependent metabolism of ethylene glycol has also been demonstrated in Aerobacter aerogenes grown anaerobically on glycerol [4,5].

The present paper describes some effects of oxygen availability on the metabolism of ethylene glycol by Flavobacterium sp. NCIB 11 171. Materials and Methods Microorganisms, maintenance, growth and preparation o f cell fractions. These were as previously described [1 ]. Batch culture experiments. Growth in ethylene glycol minimal medium [2] was performed in a pH-, temperature-, aeration- and agitation-controUed batch fermenter as described previously [1 ]. Continuous culture experiments. The bacterium was grown in chemostat culture using a 3 1 working volume fermenter linked to a Biotec LP-100 control console. The medium contained (per litre of distilled water) 1 g ethylene glycol plus the mineral salts for batch culture medium described previously [2]. During growth, the pH was maintained at 7.0 by automatic addition of sterile 1 M NaOH or 1 M H2SO4. The temperature was maintained at 27°C. The pO2 in the culture was recorded with a membrane-type galvanic oxygen-electrode which was

0304-4165/0000-0000/$02.50 © 1981 Elsevier/North-HollandBiomedicalPress

195 calibrated in sterile medium at 27°C. The dissolved oxygen tension in the culture medium was calculated from the assumption that pO2 100% of air-saturated medium was 0.209 arm. After the dissolved oxygen tension had been set at the desired value in a growing culture, it was maintained at this value (+-1 mm Hg) by automatic adjustment of the stirrer speed. Under these conditions it was established that a dilution rate of 0.10 h-z varied +-5%. Estimation of bacterial growth in culture. Biomass yield [1 ] and specific growth rate [6] were estimated as previously described. Analytical methods. Protein was determined as previously described [1]. Nitrite was determined by the method of Egami and Taniguchi [7]. Ethanol and glycerol were determined by gas-liquid chromatography using instrumentation and conditions described previously [1 ]. Enzyme assays. The following enzymes were assayed by published methods: acetyl-CoA synthetase, EC 6.2.1.1 [3]; aldehyde dehydrogenase, EC 1.2.1.3 [8]; acetaldehyde dehydrogenase (acetylating), EC 1.2.1.10 [9]; alcohol dehydrogenase, EC 1.1.1.1. [10]; catalase, EC 1.11.1.6 [2]; acetate kinase, EC 2.7.2.1 [11]; diol dehydratase, EC 4.2.1.8 opensystem method [12], closed-system method [3]; ethylene glycol oxidase [2]; glycoUate oxidase, EC 1.1.3.1 [2] ; glycerate kinase, EC 2.7.1.31 [13] ; isocitrate dehydrogenase, EC 1.1.1.41 and EC 1.1.1.42 [14]; isocitrate lyase, EC 4.1.31.1 [15]; pyruvate decarboxylase, EC 4.1.1.1 [16]; phosphate acetyltransferase, EC 2.3.1.8 [ 17 ] ; succinate dehydrogenase, EC 1.3.99.1 [18] ; tartronate semialdehyde reductase, EC 1.1.1.60 [19 ]; tartronate semialdehyde synthetase, EC 4.1.1.47 [20]. Results

Batch culture studies Growth of Flavobacterium sp. NCIB 11 171 on ethylene glycol in batch culture as assessed both by biomass yield and specific growth-rate was dependent on the pertaining dissolved oxygen tension of the medium (Fig. 1). Whereas growth decreased only slightly with decreasing dissolved oxygen tension down to 77.5 nun Hg, progressively poorer growth was observed at dissolved oxygen tension values below 62.0 mm Hg. Post.inoculum samples of ethylene

] 0.10 t-

O5

~: 025

.~o.6o ,~

to,o. to.4o8 to,o

o.1

0.05

lo.lo LO t: / 155 31.0 46", 62£) 77~ 930 1085 1~4.0 139D Dissolved oxygen tension (mmNg)

Fig. 1. Effect of dissolved oxygen tension on the biomass yield, specificgrowth rate and enzyme activity of Flavobae. terium sp. NCIB 11 171 grown on ethylene glycol in batch culture: (o), biomass yield; (s), specific growth rate; (zx), ethyleneglycoloxidaseactivity; (A),diol dehydrataseactivity. Enzyme activities are expressed as ttmol product formed (or substrate utilised) per min per mg protein.

glycol minimal medium maintained at dissolved oxygen tension 124.0 mm Hg contained no detectable ethanol or glycerol after 60 h of bacterial growth, whereas equivalent samples maintained at dissolved oxygen tension 31.0 mm Hg contained ethanol and glycerol up to maxima of 6.4 ~tmol/ml and 3.6 btmol/ml, respectively, after 54 h of bacterial growth. Growth of the bacterium on modified ethylene glycol minimal media containing either 1 g K2SO4 or 5 g KNO3 instead of 1 g KNO3 resulted in similar phenomena. No detectable nitrite accumulated in postinoculum samples of ethylene glycol minimal medium, nitrate-enhanced minimal medium or nitratedeficient minimal medium maintained at either dissolved oxygen tension 124.0 mm Hg or 15.5 mm Hg. Similar experiments with either glycollate or glyoxylate as the sole source of carbon resulted in negligible growth at dissolved oxygen tension values below 62.0 mm Hg. When cell-free extracts of Flavobacterium sp. NCIB 11 171 grown on ethylene glycol at different dissolved oxygen tension values were assayed for ethylene glycol oxidase and diol dehydratase activities (Fig. 1), the oxidase activity decreased with decreasing dissolved oxygen tension values below 77.5 mm Hg whereas the dehydratase activity although not present at dissolved oxygen tension values above 46.5 mm Hg increased with decreasing dissolved oxygen tension values below this level. Due

196

to the limitations of this experimental technique, it was not possible to establish whether the changing specific growth rate resulting from variation in the dissolved oxygen tension of the medium determined the observed differences in enzyme levels or viceversa. Consequently similar experiments were performed with chemostat cultures at a fixed dilution rate thus minimising interference of enzyme synthesis by changing specific growth rate.

~

0.09

E

~, oo8

o. .c 0.07

~

006 0.05

o

004 m

E

Continuous culture studies When Flavobacterium sp. NCIB 11 171 was grown in continuous culture under ethylene glycol limitation at a fixed dilution rate of 0.15 h -1 and the dissolved oxygen tension of the medium was varied from 7.75 mm Hg to 93.0 mm Hg, the resultant changes in the activities of ethylene glycol oxidase and diol dehydratase (Fig. 2) were essentially similar to those observed in batch culture. At dissolved oxygen tension values lower than 16.5 mm Hg the culture

0.10

c

0,03

Q02

uJ 0,01 155 310 4 8 5 62.0 775 g3.0 Dissolved c~ygen tension ( m m H g )

Fig. 2. Effect of dissolved oxygen tension on the enzyme activity in cell fractions of Flavobacterium sp. NCIB 11 171 grown on ethylene glycol in continuous culture at a dilution rate of 0.15 h-t: (o), ethylene glycol oxidase activity (e), diol dehydratase activity. Enzyme activities are expressed as /~mol product formed (or substrate used) per rain per mg protein.

TABLE I EFFECT OF DISSOLVED OXYGEN TENSION ON THE ENZYME ACTIVITY IN CELL FRACTIONS OF Flavobacterium sp. NCIB 11171 GROWN ON ETHYLENE GLYCOL IN CONTINUOUS CULTURE AT A DILUTION RATE OF 0.15 h -1 Cell fractions of strain NCIB 11171 grown at different dissolved oxygen tension values were assayed as described in the Materials and Methods. Diol dehydratase was assayed by the open-system method [12]. All enzymes activities are expressed as #mol product formed (or substrate used) per min per mg protein. Enzyme

Acetate kinase Acetyl-CoA synthetase Aldehyde dehydrogenase Acetaldehyde dehydrogenase (acetylating) Alcohol dehydrogenase Catalase Diol dehydratase Ethylene glycol oxidase Glycollate oxidase Glycerate kinase Isocitrate dehydrogenase Isocitrate lyase Malate synthase Pyruvate decarboxylase Phosphate acetyltransferase Succinate dehydrogenase Tartronic semialdehyde reductase Tartronic semialdehyde synthetase

Dissolved oxygen tension of continuous culture medium (mm Hg) 15.5 mm

23.25 mm

31.0 mm

62.0 mm

93.0 mm

0.362 0 0.676 0 0.396 0.294 0.087 0.025 0.021 0.015 3.960 0 0.549 0 0.196 1.391 0.112 0.306

0.288 0 0.704 0 0.339 0.282 0.055 0.034 0.028 0.019 4.105 0 0.592 0 0.179 1.624 0.129 0.326

0.136 0 0.651 0 0.231 0.319 0.024 0.048 0.036 0.018 4.339 0 0.476 0 0.091 1.809 0.166 0.318

0.008 0 0.692 0 0.116 0.308 0.002 0.060 0.045 0.022 5.367 0 / 0.602 0 0.005 1.746 0.209 0.357

0.006 0 0.645 0 0.091 0.326 0 0.067 0.048 0.024 6.162 0 0.616 0 0.004 1.846 0.246 0.389

197 washed out, probably because oxygen became growthrate limiting to a value below the dilution rate. Identical experiments performed at the lower dilution rate of 0.10 h-1 resulted in similar findings except that the dissolved oxygen tension value at which ethylene oxidase activity began to decrease and diol dehydratase activity began to increase were similarly displaced towards higher values by approximately 15 mm Hg.

Enzymes in cell fractions The specific activities of a number of enzymes either known [2] or suspected to be involved in ethylene glycol metabolism by this bacterium were assayed in cell-free extracts and particulate cellular preparations of strains NCIB 11171 grown in continuous culture at a fixed dilution rate of 0.15 h-1 on ethylene glycol minimal medium at various dissolved oxygen tension values (Table I). No isocitrate lyase, acetyl-CoA synthetase, aldehyde dehydrogenase (acetylating) and pyruvate decarboxyla.~e activities could be detected in any cell fraction using a wide range of assay conditions. The aldehyde dehydrogenase activity present in the bacterium after growth on ethylene glycol at all dissolved oxygen tension values tested exhibited similar activity with acetaldehyde, propionaldehyde, glycolaldehyde, glyoxylate and glycollate as alternative substrates. The diol dehydratase activity present in the bacterium after growth on ethylene glycol at dissolved oxygen tension values below 46.5 mm Hg was active with ethylene glycol, propane 1,2-diol and glycerol as alternative substrates (Km= 4.4, 6.9 and 13.5 mM, respectively) but was inactive with butane 2,3-diol or ethanolamine. The activity of the dehydratase when assayed by the closed-system method [3] was unaffected by the presence of oxygen rather than nitrogen in the atmosphere over the assay mixture, or by storage for 12 h at O°C under oxygen rather than nitrogen prior to assay. Discussion

Oxidase-initiated metabolism of ethylene glycol was induced by growth of Flavobacterium sp. NCIB 11 171 on the diol at all dissolved oxygen tension values tested (7.75 mm-139.5 mm Hg, inclusive),

whereas diol dehydratase-initiated metabolism of the diol by this bacterium was only induced by growth at dissoved oxygen tension values below 46.5 mm Hg (Scheme I). Microaerophilic growth of the bacterium on the diol was poor relative to the equivalent aerobic growth. This may be because the energy-yielding metabolism of the bacterium remained cle-pendent on the oxidase-initiated metabolism of the diol under all pertaining dissolved oxygen tension values. The dehydratase-initiated metabolsirn of the diol apparently served as a terminal fermentation pathway to partially redress the accumulation of reducing power (NADH + H*) under microaerophilic conditions when cytochrome.linked regeneration of NAD by oxygen would be inefficient. Phosphate acetyltransferase and acetate kinase but not acetyl-CoA synthetase or acetaldehyde dehydrogenase (acetylating) were induced during rnicroaerophilic growth of strain NCIB 11 171 on ethylene glycol. This supported the proposal that the bacterium remained dependent on the ethylene glycol oxidase-dependent production of acetyl~2oA rather than the diol dehydratase-dependent production of acetaldehyde as the basis for ATP production by substrate level phosphorylation under conditions precluding efficient oxygen-dependent oxidative phosphorylation. That the assimilation of ethylene glycol by strain NCIB 11 171 during oxidase-dependent microaerophilic growth became oxygen-limited was made evident by the culture wash-out that occurred when the bacterium was grown in continuous culture at dissolved oxygen tension values lower than 15.5 mm Hg. Additionally some of the diol available as a potential growth substrate under microaerphilic conditions was dissipated to ethanol as a terminal metabolite in part explaining the relatively poor biomass yield of the bacterium under these growth conditions. Equivalent phenomena have been recorded previously during microaerophilic metabolism of propane 1,2-diol to n-propanol by this bacterium

[1]. The dissolved oxygen tension-dependent changes in the activities of ethylene glycol oxidase and diol dehydratase were dependent on the specific growthrate of the bacterium during ethylene glycol-dependent growth on continuous culture. The nature of the growth.rate dependence of these phenomena was not charaeterised. A similar uncharacterised growth-rate dependence of denitrification by Klebsiella sp. strain

198

KB~2 when grown in continuous culture has been reported [21]. The results obtained during ethylene glycoldependent growth of the bacterium in nitrateenhanced and nitrate-deficient minimal media suggested that Flavobacterium sp. NCIB 11 171 was unable to operate nitrate-dependent anaerobic respiration. The probable inability of the bacterium to operate cytochrome-dependent catabolism of ethylene glycol under microaerophilic conditions, together with the net zero balance in ATP resulting from the oxidase-initiated catabolism of the diol to pyruvate, suggested that the metabolism of pyruvate to acetylCoA and thus via acetyl phosphate to acetate may

serve an important role in ATP production by substrate level phosphorylation during growth at low dissolved oxygen tension values. The absence of nitrite from post-inocuhim growth medium samples precluded the possibility that nitrite may inhibit oxidative phosphorylation as in Pseudomonas aeruginosa [22]. The ability of strain NCIB 11 171 to grown on glycollate and glyoxylate minimal media under conditions of high effective oxygen tension plus the recorded absence of isocitrate lyase during both aerobic and microaerophilic growth of the bacterium on ethylene glycol suggested that the metabolism of the diol differed from the pathway of ethylene glycol NADH2

NAD ~,

:

ethylene glycol

ethylene glycol . . . . . . . . . . ~. acetaldehyde ...';-..'f.~ ethanol

÷ glycoUate

glycollate

l ...........

glucose-6phosphate ~'

glyoxylate tartronic semialdehyde

glucose-6phosphate ...........

glyoxylate

reversed glycolytic pathway

tartronic semialdehyde l

glycerate

reversed glycolytic pathway

glycerate

ATP" ~-~ ADP ~ - " ~

ATP ~ ADP +--~ J¢

2-phosphoglycerate ....... > 3-phosphoglycerate

2-phosphoglycerate .... ~ 3-phosphoglycerate

phosphoenolpyruvate

phosphoenolpyruvate

ADP ~ ATP

A DP - - - ~ ATP "----~~ pyruvate

pyruvate

$

..........

¥

acetyl-CoA

..........

acetyl-CoA . . . . . . . * acetyl phosphate

..;:.:.

.... +

cetate

," "-.V

ADP ATP

t

oxaloacetate

....

citrate

oxaloacetate

2

citrate

. . . . ..malate

J

\

" ~ 2 x C02 (a)

\

"

/

"-~'2 × CO2

(b)

Scheme I. Pathways of (a) aerobic and (b) microaerophilic metabolism of ethylene glycol by F l a v o b a c t e r i u r n sp. NCIB 11171. Catabolic pathway . . . . . . . . . . *; anabolic pathway oooooooooo÷; amphibolic pathway , ; anaplerotic pathway *

199 metabolism recorded in Mycobacterium E44 [3]. The inability of strain NCIB 11 171 to grow on either glycoUate or glyoxylate minimal media under conditions of low effective oxygen tension may reflect the inability of either substrate to undergo a reaction equivalent to the dehydratase-dependent catabolism of ethylene glycol. The presence of a broad substrate specificity aldehyde dehydrogenase in strain NCIB 11 171 after growth on ethylene glycol at all dissolved oxygen tension values tested may explain the previouslyrecorded but uncharacterised metabolism of glycol aldehyde to glycollate and glyoxylate to oxalate [2] by this bacterium. The same enzyme may in combination with diol dehydratase have metabollsed some ethylene glycol to acetate at low dissolved oxygen tension values. The substrate range of the aldehyde dehydrogenase from ethylene glycol-grown Flavobacterium sp. NCIB 11 171 suggested that the equivalent enzyme present after growth of the bacterium on propane 1,2-diol [1] may represent a single protein entity. The apparent lack of influence of oxygen on the activity of diol dehydratase from strain NCIB 11 171 grown at low dissolved oxygen tension values on ethylene glycol when the enzyme was assayed by the close-system method [12] suggested that the absence of the enzyme during growth of the bacterium at dissolved oxygen tension values above 46.5 mm Hg resulted from the ability of oxygen to repress the synthesis of the enzyme rather than to inhibit preformed enzyme. The activity of the diol dehydratase from ethylene glycol-grown Flavobacterum sp. NCIB 11 171 with propane 1,2-diol as an alternative substrate suggested that the equivalent enzyme present after growth of the bacterium on propane i ,2-diol [1 ] may represent a single protein entity.

References

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