II. The Chemical Activity of Bacterial Metabolism.

As with ferment production, so also most other chemical activities of bacteria depend in great measure upon nutrient media. This is most striking when one observes the growth of several varieties of bacteria upon albuminous nutrient media, at one time without sugar and again containing sugar. While in the first case, except for the formation of pigments and some odoriferous substances, there is scarcely any appreciable metabolism, in the second case there often occur striking distinguishing changes characterized by the development of gas and active production. of acid. Thus the organism produces "fermentation" in the medium containing sugar; in the other, none.

Because of the practical (and diagnostic) importance of the ability to produce fermentation, there must first be given a precise definition of the process.

The expression "fermentation" is employed in the literature with the most varying meanings.

1. Many authors call every typical decomposition caused by bacteria "fermentation," and speak, for example, of the putrid fermentation of albuminous bodies.

2. Others limit the word "fermentation to processes which are accompanied by evident gas bubbles. According to this definition, the liberation of nitrogen from saltpeter is as much fermentation as the breaking up of milk-sugar by the Bact. acidi lactici.

3. Still others only speak of fermentation when there. occurs a breaking up of carbohydrates, with or without gas production.

To me the expression "fermentation" is only applicable when it is proved that an organism produces, along with or instead of its other metabolic products, one or more special metabolic product in striking amount; products of metabolism which almost always arise from the merely superficial splitting up of an easily decomposed bacterial nutrient material (splitting fermentation). Oxidation fermentation is more rare (compare below). The essential for fermentation is always the presence of a special nutrient material, which the fungus appropriates very easily, often rejecting materials more

difficult of utilization, which it would reduce in the absence of fermentable objects.

Every fermentation has the object of furnishing a store of energy to the fermenting organism. This is attained. in the splitting fermentation in this way:

In the interior of the bacterial cell the complicated fermentable molecule is decomposed into smaller fragments, and thus energy is set free. I will illustrate this as it occurs in the common fermentations of sugar, where the case is very simple:

Organisms growing with oxygen excluded especially utilize one of these sources of energy, as the source of energy at the command of aerobic varieties, residing in the oxidation of resorbed substances through absorbed oxygen, is cut off. Therefore all anaerobic varieties are endowed with the ability to cause active fermentation of sugar, while many facultative anaerobes only cause fermentation of nutrient media containing sugar when oxygen is excluded.

As already mentioned on page 29, Buchner has discovered that by expression under great weight a ferment (zymase) can be obtained from the bodies of yeast-cells which ferments sugar most intensely. These discoveries have advanced considerably our understanding of the utilization of sugar within the cells of the yeast fungus. We now know that the breaking up of sugar and the resulting gain of power originates simply "through the vital process," but we recognize, in the zymase, the special means which the organism makes use of for this activity. Heretofore it has not been possible to obtain zymase or other intracellular acting ferments from bacteria, yet, for the present, we may suppose that yeasts and bacteria are similar in this respect.

A counterpart to splitting fermentation is the rarer oxidation fermentation, of which the most beautiful example is the formation of acetic acid from alcohol. Here, likewise, there occurs one-sided metabolic activity by the acetic-acid fungus, which obtains a large supply of energy, not through the splitting up of an easily decomposed substance, but through oxidation of resorbed alcohol. The gain of energy is here dependent simply upon a partial development and enhancement of the ordinary phenomena in the nourishment of bacteria. 1

According to this, fermentation products as well as all other products of bacterial cells are metabolic products, and a special separate treatment of fermentation is not demanded. On the contrary, it seems most suitable to arrange the discussion of the single bacterial products acording to whether they originate in nutrient media which contain sugar, or are free from it, and to add something concerning such activities of bacteria as result in the breaking up of salts of fatty acids, alcohols, etc.

1. Pigment Production.

The pigments have been but little studied chemically, yet recently, through several pupils of Migula, at least a provisional survey has become possible. Compare Schneider (A. K. 1, 201) and Thumm (A. K. 1, 291).

The red and yellow pigments which have been thoroughly studied are almost all 2 insoluble in water, but soluble in alcohol, ether, carbon bisulphid, benzol, and chloroform. For the present they may be placed in two groups:

(a) Pigments of the carotin group. Yellow, orange, rose colored, with strong sulphuric acid becoming bluishgreen; with caustic alkalis, orange to red. In some cases the pigments, which often consist of a mixture of

'Certainly splitting fermentation is always a restricted, and only under certain circumstances a highly developed, function.

2 In marked contrast to this are the findings of M. Freund (C. B. XVI, 610), according to which the red and yellow pigments produced by four newly discovered bacteria were always soluble in water and insoluble in alcohol and ether.

several pigments, present great variation. (See Schneider, A. K. 1, 201, regarding spectra and peculiarities.) They are, however, closely related to the widely distributed lipochromes (pigment substances of fats, yolk of egg, etc.) and the carotin of yellow carrots. (Compare Leisenberg and Zopf, C. B. XII, 659.)

(b) Prodigiosin pigments. By prodigiosin I designate the beautiful pigment of the Bact. prodigiosum and its nearest relatives. It is soluble in ether as yellowishbrown and in alcohol as garnet-red. It is turned yellow by alkalis, violet-red by acids, and brownish-red by concentrated sulphuric acid. Zinc and hydrochloric acid reduce the pigment to a colorless leuko-product. The spectroscopic behavior is very characteristic.

Violet pigments. In connection with the bacterium violaceum, and also the Bacterium janthinum, there is produced, according to Schneider (verified by myself), a violet pigment (janthin) which is insoluble in water, readily soluble in alcohol, but insoluble in ether, benzol, and chloroform. If dry, it becomes yellow when treated with concentrated sulphuric acid and emerald-green when treated with caustic potash. In alcoholic solution all strong acids and ammonia produce a green or bluish-green color. With zine and sulphuric acid the color is destroyed (Schneider, l. c.).

The beautiful blue pigment of the Bact. indigonaceum Claessen was very incompletely examined by Claessen and Schneider (l. c.). This pigment is not dissolved by ordinary solvents. Hydrochloric acid gives a transitory blue, turning to a yellowish-brown solution. Also, other acids in dissolving it cause its decomposition. Caustic potash turns the color bluish-green. I am unable to add anything further.

Different from these is the blue pigment produced by the Bacterium syncyaneum (blue milk), which I propose to call syncyanin. It is also entirely independent of the bacterio-fluorescein forms (see below). This pigment was pointed out by Thumm as very unstable; acids turn it steel-blue, in weaker acids it is blue-black, neutral it is black, alkaline it is brownish-black. For details see the special part.

The fluorescent pigments which occur in the cultures of very many bacteria are identical, according to recent investigations by K. Thumm. The pigment which I propose to call bacterio-fluorescein, when dry, is lemonyellow and amorphous. It is soluble in water and dilute alcohol, insoluble in strong alcohol, ether, and carbon bisulphid. The aqueous solution, when concentrated, is orange; when diluted, pale yellow. The solution, when acid in reaction, presents no fluorescence; when neutral, a blue; and when alkaline, a green fluorescence. In the culture the fluorescence is at first blue, and later, because of the ammonia produced by the bacteria, becomes green. The pigment is not sensitive to oxidizing agents. Colorless antecedents (leuko-bodies) are not observed. Phosphoric acid and magnesium appear to be essential for the production of bacterio-fluorescein. (See also E. O. Jordan, Botanical Gazette, XXVII, 19.-ED.)

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We have more exact knowledge concerning the beautiful blue crystalline pigment, pyocyanin (C14H11N2O). It can be easily extracted from cultures of the Bact. pyocyaneum with chloroform, and separated from the bacteriofluorescein. Thumm has entirely overlooked it.

Black-growing varieties of bacteria have been but little studied. According to Marpmann (C. B. L. IV, 21), the black color is usually (always) dependent upon a granular secretion of sulphid of iron. It is thus easily understood why the "pigment-production" stops upon transferring to nutrient media free of iron. The almost black forms of the Bact. coeruleum are, however, certainly not colored by sulphid of iron (Lehmann).

There have been many investigations regarding fluctuation of the chromogenic function. All possible influences which affect the growth of the bacteria unfavorably also lessen the production of pigment. After continued cultivation upon unfavorable media or at unfavorable temperature, etc., the chromogenic function of the descendants may remain permanently reduced. Thus, there occur, for instance, examples of the Bact. syncyaneum which no longer produce a trace of pigment in agar and milk (compare Behr, C. B. vIII, 485), but which color potato darkly in the neighborhood of the culture.