2. FERMENTATION OF CARBOHYDRATES 91 TABLE X I
COMPARISON OF GENERA WHICH DISPLAY A 2,3-BUTANEDIOL FERMENTATION*1
Organism Aerohacter
displayed. For instance, as high as 85 % of the diol-glycerol fermentation has been obtained with B. subtilis, and A. aerogenes carries out a diol-hydrogen fermentation to t h e extent of 90 %.2 1 1 Doubtless these processes are not coupled as implied b y the table b u t occur independently.
Stanier and A d a m s ,1 9 0 in a comparison of the butanediol fermentations have called attention, b y the use of Table X I , to the comparative position of the several organisms from the standpoint of both classification and fer
mentation characteristics. T h e fermentations are similar although morpho
logical characteristics vary widely. Yet there are distinct differences in t h e details of the fermentation. I t is also known t h a t organisms taxonomically related to those in the table display profoundly different fermentative patterns (Pseudomonas Zzndnm'-ethanolic versus Aeromonas (Psevdomonas) hydrophila-but&nediol). Stanier and A d a m s1 9 0 state, " T h u s the mechanism of carbohydrate metabolism cuts sharply across orthodox taxonomic di
visions, a fact t h a t suggests t h a t a particular fermentative mechanism has developed independently in several branches of the bacterial kingdom during t h e course of evolution."
92 W. A. WOOD 7. SYNTHESIS OF ACETOIN AND 2,3-BUTANEDIOL
Pyruvate, formed in glucose fermentation, is the precursor of acetoin and 2,3-butanediol. However, the details of the process are complex and have thus far eluded complete solution. This is due partly to a late recogni
tion of the fact t h a t several variations of the basic mechanism for acetoin synthesis exist individually in different organisms or together in the same organism. Nevertheless, it was recognized t h a t pyruvate a n d / o r acetalde
hyde were precursors of acetoin in yeast and t h a t the process was closely associated with pyruvate decarboxylation. T h e process has therefore been considered to occur in two steps, i.e., cleavage of p y r u v a t e to form enzyme-acetaldehyde, and transfer to free enzyme-acetaldehyde, forming acetoin. T h u s the the roles of carboxylase and carboligase in acetoin synthesis were postulated and long debated.2 1 7"2 2 1 I t has since been shown t h a t highly purified carbox
ylase fractions form acetoin from pyruvate in fixed proportion to the car
boxylase activity.2 2 2 - 2 2 4 Hence, it is likely t h a t these activities cannot be completely separated. Since acetaldehyde alone also yields traces of acetoin,
223-225 ft is c u r re n t l y postulated t h a t an aldehyde-diphosphothiamine com
pound is formed from pyruvate and also slowly from a c e t a l d e h y d e .1 5 , 2 2 β'
2 2 7 Neuberg and associates2 1 7 - 2 2 0 and others 2 2 8 , 2 2 9 observed t h a t the addition
of various aldehydes with pyruvate yielded optically active acyloins. There
fore, aldehydes were considered to be the acceptor in a 2-carbon transfer reaction. Fractions have been obtained with greatly increased acetoin-forming ability relative to carboxylase. For instance, an acetoin-acetoin-forming enzyme system has been separated from the yeast-type carboxylase of P.
lindneri* I t therefore appears t h a t these fractions should be assigned the al
dehyde transfer function of carboligase.
Acetoin is produced from pyruvate b y B. subtilis,2*0 A. aerogenes,231-233 C. acetobutylicum,m and other organisms as follows:
2 pyruvate —* acetoin + 2 CO 2
Since these microorganisms do not contain carboxylase or produce a p preciable acetaldehyde, it is evident t h a t a yeast-type carboxylase and carboligase reactions do not function. Also, enzyme preparations from Aerobacter species which produce acetoin do not utilize added acetalde
h y d e .2 3 2 T h e bacterial process utilizes p y r u v a t e as the aldehyde acceptor with a single enzyme presumably catalyzing the pyruvate decarboxylation and aldehyde transfer functions.2 2 7 I n this case as stable intermediate, ( + ) α-acetolactate, is produced as follows:
2 pyruvate —> ( + ) α -acetolactate + CO2
A stereospecific α-acetolactate decarboxylase then forms acetoin:
( + ) α -acetolactate —• (—) acetoin + CO2
2. FERMENTATION OF CARBOHYDRATES 93 TABLE X I I
OPTICAL ROTATION OF 2,3-BUTANEDIOL PRODUCED BY VARIOUS BACTERIA"
Organism Approximate composition
of mixture Reference Aerobacter aerogenes 5-14% L ( + ) , remainder meso 185, 186 Bacillus polymyxa At least 98% D ( - ) 235, 286, 287 Pseudomonas hydrophila About 8% D(—), remainder meso 191 Bacillus subtilis (Ford) Up to 40% D(—), remainder 198, 218
meso
Serratia marcescens Predominantly meso 111
Serratia plymuthicum Predominantly meso 208
Serratia anolium Predominantly meso 208
a Ledingham and N e i s h .1 8 4
T h e first enzyme resembles carboxylase in t h a t diphosphothiamine and man-ganous ions are required.2 2 9 α-Acetolactate is inert in the yeast, animal, and plant acetoin-forming systems and t h u s does not appear to function in acet
oin synthesis in these cells. T h e identity and characteristics of this p a t h w a y has been further established in S. faecalis23* and in other organisms.2 2 7
2,3-Butanediol is produced b y reduction of acetoin. I n Leuconostoc mes-enteroides, and doubtless in the butanediol-producing organisms, the rever
sible reaction is catalyzed b y butanediol dehydrogenase with the equilib
rium in the direction of reduction.2 1 6
acetoin + D P N H + H+ 2,3-butanediol + DPN+
Hence, the balance between acetoin and 2,3-butanediol is determined b y t h e amount of available hydrogen.
As shown in Table X I I , the three possible stereoisomeric forms of bu
tanediol are produced b y bacteria, presumably b u t not necessarily, from ( —) acetoin. I t is likely t h a t separate stereospecific dehydrogenases exist for all of these forms. Mixtures could arise through the simultaneous action of more t h a n one butanediol dehydrogenase, or through the action of racemizing enzymes. T h e details of the stereospecificity remains to be elucidated, how
ever.
B . H E X O S E MONOPHOSPHATE PATHWAYS
1. V I A PENTOSE PHOSPHATE TO H E X O S E PHOSPHATE
T h e so-called "hexose-monophosphate s h u n t , " originally established as an oxidative mechanism alternate t o glycolysis, is known t o function in several variations in fermentation of hexoses, gluconic and 2-ketogluconic acids, and of pentoses.
T h e point of departure from the Embden-Meyerhof system for the
alter-94 W. A. WOOD
6LUC0SE-6-P04
GLUCOSE
•RIB0SE-5-P04~
RIBUUOIE-5-PC>4 -·PENTOSES XYLUL0SE-5-P04_
'SEDOHEPTULOSE - 7- Ρ Ο 4
•6LYCER
ALDEHYDE-3-ΡΟ4-' 3-2 CLEAVAGE PYRUVATE
FIG. 5. Hexose monophosphate pathways.
nate routes is the oxidation of glucose-6-phosphatebya dehydrogenase origi
nally named "zwischenferment" b y Warburg and Christian.2 3 8 I n the late 1930's this shunt pathway, now known as the Warburg-Dickens-Horecker scheme, was thought to consist of successive oxidations and decarboxyl
ations until the substrate was completely oxidized, or until a smaller inter
mediate enters the tricarboxvlic acid cycle. Although such reactions do form pentose from hexose, the further degradation involves cleavage and trans
fer reactions for pentose phosphate of a type hitherto unknown in metabol
ism (see Fig. 5.) The transferring enzymes involved, transketolase and transaldolase, affect the synthesis of fructose-6-phosphate from pentose phosphate. T h e fructose-6-phosphate can then recycle through the hexose monophosphate pathway under aerobic conditions, or undergo fermenta
tion via the glycolytic system (see Chapter 4).
2. V I A 3-2 CLEAVAGE OF PENTOSE PHOSPHATE
One major fermentation pattern follows the Warburg-Dickens-Horecker route except t h a t pentose phosphate does not undergo transfer and cleav
age reactions to form hexose phosphate esters, b u t is split into 3 and 2 carbon units (Fig. 11), yielding glyceraldehyde-3-phosphate and acetyl phosphate.2 3 9 These are converted in fermentative organisms to lactate and ethanol, respectively.
a. Heterolactic Fermentation. Organisms isolated from a variety of sources, including wines, sauerkraut, silage, and spoiled t o m a t o products, ferment hexoses with the production of ethanol, acetic acid, glycerol, m a n
-2. FERMENTATION OF CARBOHYDRATES 9 5 T A B L E X I I I
GLUCOSE AND FRUCTOSE FERMENTATIONS BY HETEROLACTIC ACID BACTERIA