The nature of the Aeromonas fermentation

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Biochemical Journal, 38, 2, pp. 168-178, 1944

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The nature of the Aeromonas fermentation

Stanier, R. Y.; Adams, G. A.

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I944

The

Nature of the Aeromonas Fermentation

BY R. Y. STANIER AND G. A. ADAMS, Divisionof AppliedBiology, National Re8earch Council, Ottawa,Canada

(Received 26 February 1944) The genus Aeromona8 was establishedby Kluyver

& van Niel (1936) for pseudomonads fermenting carbohydrateswith the production of carbon dioxide and hydrogen. Since von Wolzogen Kiihr (1932)

hadpreviously shown thataxnemberofthisgroup, Pseudomonas fermentan8, gave a positive Voges-Proskauer reaction, the existence of a butylene glycol fermentation in the genus was suspected, although the absence ofexactdataontheproducts offermentation left this point unsettled.

During the course ofan intensive investigation

on the bacteriological production of 2:3-butylene

glycol (Adams&Stanier, 1944),it seemed desirable

tomakeasomewhat closer study of this little-known Aeromona8 fermentatior.. The recent discovery of polar flagellation in P.ateu8 hydrophilus (Kulp & Borden, 1942; Guthrie& Hitchner, 1943), taken in

conjunction with its fermentative abilities, mark this organism as an Aeromonas species, and since cultures werereadily available it waschosen as a representative of thegroup.

MATERIALS AND METHODS

Through the kindness ofDr WV. L. Kulp and Dr E. R.

Hitchner we obtained a number ofstrains ofAeromo8as

hydro-phila. Preliminary analyses having shown that all

these organisms were capable of producing substantial

amountsof ethanol and 2:3-butylene glycol fromsugars, a

strain with particularly vigorous fermentative properties

wasselected formoredetailedquantitative investigations.

The basal medium employed consisted ofyeast extract

1-25g.,CaCO,1-0g.,

MgSO4

0-025g., -OM-phosphate buffer

(pH 7.4) 0-65ml., tap water 250ml. The carbohydrates used were sterilized separately in concentrated aqueous

solutionand added tothe basalmedium just before

inocu-lation in amounts givinga final concentration of 1-2 %.

Fermentationswere carriedout at30° and allowedtorun

for3-4days. During this periodaslowstreamof oxygen-freenitrogenwaspassed through the flasks. At the end of

thefermentation, an excess of HCIwasaddedtodissolve

the residual

_CaCO3,

and aeration with nitrogen was

con-tinued for several hoursto sweepoutall theCO..

Carbon dioxidewasdeterminedgravimetrically. Hydro-gen was continuously oxidized by passage over heated

copperoxide anddeterminedaswater. Aftercompletion of

the fermentation, the fermented solution wasneutralized

and made up to500 ml. A measured fraction was made

alkalineand extracted continuously withetherin orderto

removebutyleneglycol and acetoin.The ether extractwas

evaporated, andtheresidue takenupin water. Butylene

glycol wasdeterminedby anunpublished method developed in the Department of Biochemistry at the University of Wisconsin. The butylene glycol is oxidized with periodic

acid, and the excess periodatedeterminediodometrically in neutral solution. Acetoin wasestimated by the method of Langlykke & Peterson (1937). After extraction of the alkaline solution with ether, it was made acid and re-extracted with ether to remove the organic acids, which were taken up in water again afterremoval ofthe ether. The acid extract was steamdistilledandthevolatileportion estimated as acetic acid by titration. The non-volatile fraction contained lactic and succinic acids. The formerwas

determined by the method of Boyland (1928), the latterby the formation of the silver salt. Pyruvicacid,when present, was withheld from going into the ether acid extract by saturating the solution with sodium bisulphite. Ethanol was determined by dichromate oxidation, following frac-tional distillationof a measured fraction ofthe fermentation solution.

Pyruvic acid was determined manometrically by ceric sulphate oxidation(Krebs & Johnson, 1937). Xylome esti-mations were carried out by the copper reduction method ofSchoorl (1929). Glucose was determined by the Hanes (1929) modification of theHagedorn-Jensen procedure.

RESULTS

Carbon balances were obtained for the fermenta-tions of glucose, xylose and pyruvic acid. An attempt was also made to study the fermentation of mannitol but, largely owing to the slow and

incompleteattack on thiscompound,asatisfactory

balance was not obtained.

Fermentaion ofglucose. Theproductsofglucose fermentationbyA.hydroph.ilaare shown in Table1. The mainorganic ones arealways butylene glycol,

ethanol and lacticacid;inaddition,smaller amounts ofacetoin, acetic acid and succinic acid are found. Fluctuations in the quantitative relationships be-tween the various products may occur from one

experiment to the next; thus, C02 production can vary from 140 to 170 mol. per 100 mol. ofglucose fermented,with concomitant differences in the rela-tive amounts of

_C.

andC3 compounds.The ratio of

butylene glycol to ethanol can also fluctuate over

quite a wide range, although it is usually about 1: 1 onamolecular basis.

In order toestablishbeyond doubtthenatureof the glycol produced in this fermentation, a small samplewasisolatedandcharacterized. About151. ofafermented yeast extract-glucose mediumwere

AEROMONAS FERMENTATION

Table 1. Fermentationofglucose by Aeromonas hydrophila Glucose fermented: 5.490 g.

Number of mol./100 mol. of glucose fermented

Products 2:3-Butylene glycol Acetoin Ethanol Acetic acid Lacticacid Succinic acid CO2

Hs

Total Weight (g.) 1-502 0-047 0-730 0-085 0-639 0-130 2-230 0-035 Product 54-7 1-7 52-0 4-6 23-3 3-6 166-2

57.5

Carbon 218-8 6-8 104-0 92 69-9 14-4 166-2 589-3 Calculated

Co2

109-4 3.4 52-0

4-6

-3-6

166-8'

Calculated H2 54.7 3.4 9-2 -3-6 63-7 Oxidation values +_ 164-1 - 3-4 - 104-0 3-6 332-4 336-0

57.7

329-2 Carbonrecovery ... ... ...

CO2

calculated/CO2

observed ...

98-2% 0-997

evaporated to a volume of 41. in an open steam

kettle, saturated with anhydrous

K2CO3,

and

ex-tractedwith twice the volume ofbutanol. Most of the butanol was removedby distillation througha shortVigreux column,theresidualextract was dis-tilled under reduced pressure in a Stedmancolumn

and the pure glycol fraction recovered. Oxidation

of a small sample ofthis glycolwith bromine and

FeCl3

gave a good yield of diacetyl, which was

characterizedbythe

melting-point

of itsbis-phenyl

hydrazone. Theproductionofdiacetylonoxidation

provedconclusivelythat theglycolwas2:3-butylene

glycol. Judgingfrom its opticalrotation (-

0.970),

it consists of a mixture of the 1- and meso-forms with a

prepo'

lerance of the latter. Thus it differs

bothfrom tho 2:3-butylene glycol producedin the

Aerobacter fermentation,whichis a d-mesomixture,

and fromthatproduced in theAerobacillus fermen-tation, which is the pure1-isomer.

Fermentation of xylose. Table 2 presents the

carbon balance for a xylose fermentation by A. hydrophila. In this particular experiment the

butylene glycol-ethanol ratio was somewhat less

H2

calculated/H2

observed ... 1-108

O/R index ... ... ... 1-021

than 1:1, but this is not characteristic; ratios of 1:1 or over have been obtained in other similar

experiments. Thus there are no significant differ-ences, either qualitative or quantitative, between theend-products ofpentose and hexose fermenta-tionbyA.hydrophila. This indicates very strongly that there must be a resynthesis from the xylose (or from the C2 fragments of the xylose) of a

C,

compoundwhich is subsequentlydecomposed in a manner similar to that operative in glucose

dis-similation. Unless one is willing to assume the production ofCO2from a

_C2

compound-r-a reaction unknowninfermentative processes-a simple

calcu-lationshows the necessity for this assumption. Were the end-products ofpentose fermentation derived

directlyfrom the fragmentsofa

_C3-C2

split,the

CO2

production could not exceed 100

mol./100

mol. of pentose fermented, since no CO2 couldbe derived fromthe

_C.

fraction. Furthermore, the production

of

C3

compoundswill reduce thispossiblemaximum of

CO2

productionby an amountequivalenttothe number of mols of

C3

compound formed. In the

experiment shown in Table 2, the observed

C02

Table2. Fermentation ofxylo8ebyAeromonashydrophila

Xylose fermented: 4-010 g.

Numberofmol./100 mol. of xylose fermented

Products 2:3-Butylene glycol Acetoin Ethanol Acetic acid Lacticacid Succinicacid CO2

H2

Total Weight

(g.)

0-939 0-061 0-600 0-150 0-491 0-036 1-584 0-029 Product 39-0 2-6 48-9 9-3 20-4 1-1 134-7 53-9 96-6% 1-041 Carbon 156-0 10-4 97-8 18-6 61-2 4-4 134-7 Calculated CO2 78-0 5-2 48-9 9-3 -1-1 - 483-1 140-3 Calculated H2 39-0 5-2 18-6 -1-1 61-7 Oxidationvalues r 'A +_ 117-0 5-2 - 97-8 1.1 269-4 270-5 Carbonrecovery ...H,c ... ... C02calculated/CO,"observed ... 53-9 273-9 1-126

0-988

Vol.

38

169

H2

calculated,/H2

obsorved ...

O/R

index ... ... ...

R.

Y. STANIER AND G. A. ADAMS

Table 3.

Fermentation

of pyruvic acid by Aeromonas hydrophila

Pyruvicacid fermented:3-530g.

Numberof mol./100 mol. of pyruvic acid fermented

Pyruvicacidfermented Products 2:3-Butyleneglycol Acetoin Ethanol Aceticacid Lacticacid CO2 H2 Total Weight (g.) 3 530 0-125 0-021 0-038 1-800 0-776 1-479 0035 Carbonrecovery ... ...

CO2 calculated/CO2observed

Calculated Calculated Product Carbon CO H2 3-5 0-5 2-1 74-8 21-5 83-8 43-6 14-0 2-0 4-2 149-6 04-5 83-8 318-1 .. . 106-3% .... 1013 7-0 1-0 2-1 74-8 84*9 -3.5 -2-1 74-8 -

21*5

47.7 Oxidation + A 100 167-6 167-6 H2calculated/H2observed ... O/R index ... ... .

production-amountedto135 mol./100 mol. of xylose fermented: i.e. 35 mol. more than the possible maximumfroma

C3-C2

split and58mol.morethan theamounttobeexpected after taking intoaccount the lactic and succinic acid formed. It would be interesting to determine the effect of A.hydrophila on glycol aldehyde, the C2 compound which one mightexpect to result from the primary fission of the pentose molecule.

Fermentation ofpyruvic acid. Aeromona8

hydro-phila causes a vigorous fermentation of pyruvic

acid. The balance sheet for this fermentation is shown in Table 3. Surprisingly enough, the pro-duction of butylene glycol, acetoin and ethanol is largely suppressed, and the chief organic end-productsareacetic and lactic acids. Approximately

three molecules of pyruvic acid are oxidized to acetic acid for every one reduced to lactic acid, andconsequentlyaconsiderable volume of hydrogen

isproduced. The three main reactionsappeartobe:

3CH3.CO.COOH+3H20 3CH COOH+3CO2+6H,

CH3.CO COOH+2H CH,. CHOH COOH,

4H 2H2.

Thus thedismutation of pyruvic acid by A. hy-drophila belongs to the type demonstrated for heterofermentative lactic acid bacteria (Nelson &

Werkman, 1936) and for staphylococci(Krebs,1937),

with thedifferencethatin the latterfermentations there is an equimolecular production of the two

acids and consequentlynohydrogen formation. Thedissimilation of pyruvic acid by A.hydrophila differs notably from that whichoccursin theother two bacterial groups where one finds a butylene glycol fermentation. When growing anaerobically on pyruvic acid, AerobaciUus polymyxa produces principally acetoin and acetic acid(Adams &Stanier, 1944), while under similar conditions Aerobacter

aerogene8 gives rise to butylene glyocl and acetic

aoid(Mickelson, Reynolds & Werkman, 1936).

DISCUSSION

Theseinvestigationshavedefinitely established the existence of a butylene glycol fermentation in a

bacterialgroupwhere itwaspreviouslynot known. Thus there are at present three morphologically

widely separated bacterial families-the Entero-bacteriaceae (Aerobacter andSerratia), the

Bacilla-ceae (Aerobacillue) and the Pseudomonadaceae

(Aeromona8) in whichabutyleneglycolfermentation

occurs. In each family there are also organisms

which carry out entirely different carbohydrate fermentations; in the Pseudomonadaceae, the alco-holic fermentation of Zymomona lindneri: in the Enterobacteriaceae, the mixed acid fermentationof Escherichia coli and other species: and in the

Bacillaceaeawidevariety of dissimilatoryprocesses

(acetone-butanol, acetone-ethanol and other fer-mentations)performed by various organisms.

This absence of correlation between morpho-logical and biochemical characters isof considerable interest from the standpoint of comparative bio-chemistry. Itsuggestsverystronglythat thepresent fermentative mechanisms for the dissimilation of

carbohydrates have developed independently along similar lines in different branches ofthe bacteria.

Thishypothesis is supported by the slightbut signi-ficant differences which characterize a particular fermentationascarriedoutbyorganismsbelonging todifferent morphologicalgroups. Agood example is the differencesinoptical rotationofthebutylene

glycols produced by Aerobacter, Aeromonas and

Aerobacillus.

SUMMARY

1. Aeromona hydrophila,atypicalrepresentative

of the genus Aeromona. Kluyver and van Niel, causes a butylene glycol ferinentation of carbo-hydrates. The end-products from glucose are

2:3-10.5 1-0 4-2 43-6 159-3 1-102 1-052

1944

Vol. 38

AEROMONAS FERMENTATION

171

butylene glycol, ethanol, acetic and lactic acids,

CO2

and H,, with traces of acetoin and succinic acid. The 2:3-butylene glycol is a mixture of the 1- and meso-forms.

2. The products of xylosefermentation arevery similar, both quantitatively and qualitatively, to those of glucose fermentation. This is regarded as

evidenceagainsta simple

C3-C2

splitof the pentose

molecule.

3. The main products of the fermentation of

pyruvic acidare lacticandacetic acids, CO2and

H2.

Approximatelythree molecules ofpyruvicacid are oxidized to acetic acid for every one reduced to lactic acid.

REFERENCES Adams, G. A. & Stanier, R. Y. (1944). Canad. J. Res.

(inthePress).

Boyland, E. (1928). Biochem. J. 22, 236.

Guthrie, R.& Hitchner, E.R.(1943). J.Bact.46,52. Hanes, C. S.(1929). Biochem. J.28,99.

Kluyver,A. J. & van Niel,C. B. (1936). Zbl. Bakt. ia,94,369. Krebs, H. A. (1937). Biochem. J. 31, 661.

-& Johnson,W. A. (1937). Biochem. J. 31, 645. Kulp, W. L. & Borden, D. G.(1942). J.Bact.44, 673.

Langlykke, A. & Peterson, W. H. (1937). Industr. Engng Chem.(Anal. ed.), 9, 163.

Mickelson, M., Reynolds, H. & Werkman, C. H. (1936). Proc.Soc. exp. Biol., N. Y., 34, 748.

Nelson, M. E. & Werkman, C. H. (1936). Iowa St. Coll. J. Sci. 10, 141.

Schoorl,N. (1929). Chem. Weekbl.26,130.

von Wolzogen Kuhr, C. A. H. (1932). Zbl. Bakt. II, 85, 223.

Experiments on the Methylation and Acetylation of

Wool,

Silk

Fibroin,

Collagen

and

Gelatin

BY S. BLACKBURN AND H. PHILLIPS, The Wool

Indu8trie

ResearchA88ociation, Torridon, Leedi (Received 3 March 1944)

In apreviouspaper (Blackburn, Carter& Phillips, 1941) it was shown that woolandsilkfibroincould be O-methylated either by contact with methyl

bromide or iodide at room temperatures, or with methyl sulphate in buffers within the pH range

2-5-8-5. With both proteins the number of

CH3

groups introduced by repeated treatment with

methylsulphatewas greater than couldbe accom-modated on the free carboxyl groups of glutamic

andaspartic acids, andit wassuggestedthatcertain 'activated' peptide linkages methylatedas well as thefreecarboxylgroups of thesalt-linkages. CH3I and

CH3Br

introducedfewerCOH groupsinto wool and silk fibroin thandidmethyl sulphate.

Thepresent paperdescribes the extension of this

work to themethylationof collagen and gelatinby similar methods,and themethylation with methyl sulphate andCH8Brof woolwhich has bieen chemi-cally modified by alkali, acid,

nitrous

acid and

formaldehyde. In addition, the

siaultaneous

ace-tylation and methylation of wool, silk fibroin and collagen by the combined action ofmethanol and

acetic anhydride has been investigated.

METHODS

Methylation of collagenandgelatin with methyl

suphate

andmethyl halides

Thecollagenused in theseinvestigations was a limed hide which had been delimed with acetic acid and then de-hydratedinacetone. The gelatin wasCoignet'sGoldLabel.

The methylation procedure wassimilarto thatdescribed for wool and silk (Blackburn et al. 1941), with certain modifications to suit the propertiesof collagen and gelatin. Both proteins were cut intosmallpiecesand acetate buffer was used for the methylations with methyl sulphate. At theend ofeachperiod ofmethylationabout1 g. ofcollagen was removed, washed in three changes of water and then shaken mechanically with water(25ml.) for 1 hr. It was thendehydrated with acetone,air-driedand conditioned.* The moisture content of the collagen, the physical pro-perties ofwhich appeared to beunaltered,wasdetermined by Barritt & King's method (1926). During methylation, the gelatin absorbed water and swelled greatly. The re-sulting gel and solution was dialyzed incellophan against waterfor 3 days,thewaterbeingchangedfrequently.The resultinggelatinousmasswasdried in vacuobelow 60°.

Before methylation with either CH3Br or _CH3I, the collagen was allowed to reach equilibrium with a borate buffer of pH 8, and then dehydrated with acetone and conditioned. The gelatin was treated dry asbought. After methylation, both proteinswere rinsedinseveralchanges of benzene and then conditioned. When proteins are methylated byeither

_CHsBr

or

CH3I,

onehalogenanion is liberated for each CH3 group introduced. To provide an additional measure of their degree of methylation, the halogen contentof some of themethylated collagens and gelatins was therefore determined by the open Carius method(Peters&VanSlyke, 1932). The methylated gelatin

* _{Allproteins described in this paper as conditioned had}

beenexposed to a controlled atmosphere of 210 and 70% R.H. until they attained a constant weight. Although all the analyses were made on the conditioned proteins, the results are recorded as percentages of the anhydrous material.