# Popular Science Monthly/Volume 62/December 1902/Nitrogen-Fixing Bacteria

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* NITROGEN-FIXING BACTERIA.
By J. G. LIPMAN,
NEW JERSEY AGRICULTURAL EXPERIMENT STATION.
THE soil may truly be regarded as a vast laboratory. The many processes normally taking place in cultivated soils lead to the gradual formation of plant-food, to the solution of the mineral constituents, to the breaking down of the organic molecules into simpler forms, such that are in a condition to furnish the chlorophyl-bearing plants the material for the building up of plant tissue. The cycle of transformation from the simple to the complex and the falling apart of these complex molecules involve the activity of higher plant life, on the one hand, and that of lower organisms, on the other. Primarily it is the energy derived from the sun that, with the cooperation of the living protoplasm, impels the atoms to enter one or another of the innumerable combinations. These atoms are, as Carlyle would put it, 'but the garment of the spirit,' and the atom of carbon or nitrogen, which to-day is in the leaf of the oak or in the brain-cell of man, may on the next day become a structural part of some bacterial spore that is scarcely visible even with magnification of 1,500 or 2,000 diameters. The different kinds of atoms whose presence is essential in order that living tissue may arise, are not many. Among the less than one dozen of these, it is the migration and transmigration of the nitrogen atoms that undoubtedly form the most interesting, as well as the most important, phase of agricultural research. Of all the elements that enter into the composition of vegetable and animal substances, nitrogen is the most expensive, the most evasive, the most difficult to replace. And every person who at all concerns himself with questions as to the origin and the development of the various forms of life, can not be indifferent as to the source of nitrogen in the soil, and the factors that in one way or another affect the store of nitrogen at the disposal of the living world. Whence is the soil nitrogen derived? What conditions are most favorable for the maintenance of an adequate supply of this precious material? What means have we at our disposal for replacing the losses that occur, that in the nature of things must occur?
We should remember of all things, that the great aerial ocean, containing as it does more than 78 per cent, by volume of gaseous nitrogen, does not directly offer that element to the plant world. In order that this nitrogen may become available, it must be combined with other ​elements. Like Coleridge's Mariner, floating on the sea, surrounded by the sea, and yet perishing for lack of water, so plants growing on the bottom of the aerial ocean, with four fifths of its bulk consisting of nitrogen, and that to a depth of 200 miles or more, would yet starve for lack of nitrogen if there were not means in nature's workshop to combine that very inert gas. Now, soils of average fertility contain rather more than .1 per cent of nitrogen; in other words, the soil taken to a depth of one foot contains 3,500 pounds of nitrogen to the acre. The quantity is not constant, because of the various factors that lead either to the increase or decrease of that treasure hoard which it had taken ages to accumulate. In the processes of decay and fermentation, due to the activity of molds and bacteria, much of the combined nitrogen is set free and is returned to the atmosphere; in all processes of burning and explosion great quantities of nitrogen are again liberated. This latter fact led Bunge to say that every shot fired kills whether it hits anything or not, for it takes away that much life-giving substance from living things. Then again, as the insoluble proteid molecules are broken down and changed into simple salts, the nitrogen that thus becomes soluble, if not taken up by the crop, is leached out of the soil, and ultimately finds its way to the ocean. Enormous quantities of nitrates are thus carried by the streams to the sea, there to feed its denizens, to return, perhaps, in very slight measure to the land, though changed into other forms. Thus the great swarms of coarse fish caught for fertilizer purposes return in their bodies the nitrogen that had once traveled to the sea; thus, also, the still extensive nitrate beds of arid South America are a fractional return of what the sea had taken to itself. That the dissolved nitrates poured into the sea year after year by streams great and small do not remain there as such is clearly evidenced by the analysis of sea-water which shows but traces of nitrates. And so it appears that the soluble nitrogen salt, greedily taken up by plants in the field, is also quickly consumed in the sea. Of course, the nitrate deposits of South America were not deposited from the ocean as such, but resulted from the decay of great masses of seaweed.
Then there are opposite tendencies. There are agencies which lead to the increase of the nitrogen stock in the soil. It was Cavendish who first showed that when electric sparks are passed through air confined over a solution of caustic potash, small quantities of oxidized nitrogen are formed. In a similar manner, the electric discharges in the atmosphere cause the formation of small quantities of combined nitrogen, and Berthelot had shown that silent electric discharges also cause the combination of gaseous nitrogen. Similarly it has been claimed that in the burning of gas, coal, wax, etc., slight amounts of nitrogen become combined. All these factors, and others not mentioned, are, however, of ​minor importance as regards the maintaining of the store of combined nitrogen. We should remember that a fair crop of hay will remove from the soil more than 50 pounds of nitrogen, that at times there is removed from the soil 100 to 150 pounds of nitrogen per acre in one season, and remembering that, we can easily appreciate how entirely inadequate the 3 or 4 pounds of nitrogen per acre that are brought down from the atmosphere by dew, rain or snow are for supplying the nitrogen requirements of even a very meager crop.
There must be, then, another factor, or other factors, that are concerned in the supply of the vast quantities of combined nitrogen that are consumed from day to day. The mineral portion of plant food, that portion which constitutes the ash of plants, containing the calcium, magnesium, potassium, sulphur, iron, phosphorus, etc., is derived from the common rocks of the earth's crust. It is otherwise with nitrogen; to be sure, small quantities of it are contained in primitive rocks in iron deposits, in meteoric iron, etc.; yet, speaking generally, nitrogen is not a normal constituent of rocks. It is the atmosphere, and the atmosphere only, which must remain its source for plant and animal life. It is idle to speculate in what condition that nitrogen existed when the earth's crust first began to solidify. It is not likely that it existed as ammonia, for the hydrogen having a greater affinity for oxygen would have combined with the latter. It is not improbable, however, that it existed in combination with oxygen when the temperature of the earth's atmosphere had become sufficiently low. Be it as it may, when the surface rock began to disintegrate and lower plant life first appeared, there was no soil nitrogen. As rock disintegration proceeded, as the rock fragments became finer and offered a more favorable dwelling place for plants and bacteria, the store of nitrogen in the soil began to accumulate. And now we come to those agencies that are of the greatest importance in this gradual increase of the nitrogen store. Small amounts of combined nitrogen formed through electric discharges and brought down to the soil by precipitation would be sufficient in themselves through countless centuries to give rise to vast accumulations, provided that there was a gain only and no loss. But we have already seen that nitrogen is constantly being leached out of the soil. Analytical data at hand show that there is drained away from the land as much as 75 pounds of nitrogen per acre in the form of nitrates, and this certainly is lost to the soil. On some soils the loss is much smaller, on other soils it is even greater, but this, taken together with the amount removed in the crops taken off from year to year, shows clearly that unless there are other means in the economy of nature for drawing upon the great store of atmospheric nitrogen, the present store would soon be exhausted, in fact, it could never have accumulated.
​Untiring research by many men and in many places has taught us that it is the mysterious force in living protoplasm that in its aggressive way reaches out and appropriates the restless molecules of atmospheric nitrogen; that though it destroys it also builds up. Practical experience had taught the ancients that crops of the legume family, crops like clover, beans, lupines, etc., do not exhaust the soil to such an extent as do crops not belonging to the same family. They had learned that after a crop of clover they could raise a larger crop of wheat. Why it was so they did not know, nor did the many generations of farmers who followed them; yet not knowing they availed themselves of the advantages that time had pointed out to them. It was reserved for the men of our generation, for men equipped with the methods of our own day, to illuminate the darkness, to unveil for us still another of nature's mysteries, to show us an intelligent way for replacing the unceasing losses of nitrogen. It was scarcely more than fifteen years ago that Hellriegel and Wilfarth published a series of wonderfully conceived and wonderfully exact experiments that decided for all time a much-debated question, which for a century had taxed the ingenuity of the foremost scientists of Europe. What Boussingault with all his mental penetration and clearness of vision had failed to accomplish, what Lawes and Gilbert with all their painstaking care and admirable equipment had failed to achieve, the German investigators had made clear. They showed conclusively that in the root nodules of leguminous plants there are found certain bacteria that in a way still unknown to us enable the host plant to make use of the gaseous atmospheric nitrogen. We do know that there is a continual struggle between the plant and the invading bacteria; we are justified in believing that the bacteria, compelled by the plant, unlock to it the hitherto inaccessible store of nitrogen. It was in this wise, partly, that the nitrogen accumulation in our soils resulted; it was in this wise that the rich prairie soils, containing at times as much as twenty thousand pounds of combined nitrogen per acre to a depth of one foot, had acquired that nitrogen. This dwelling together of two distinct forms of life with mutual benefit resulting is known as symbiosis, and the symbiotic life of leguminous plants and of the organism known as Bacillus radicicola has made possible to a great extent the terrestrial life of to-day. Yet there is another phase of the question, a phase that but a few years ago had not been recognized. There are bacteria in the soil that can avail themselves of atmospheric nitrogen without the aid of leguminous plants. Recent work in this field of research indicates that such fixation of atmospheric nitrogen is of vast significance, of greater moment, perhaps, than the fixation of nitrogen by legumes. To understand more clearly the relations existing among the many ​forms of bacterial life that are concerned with the transformations of the soil nitrogen it is necessary to consider separately some distinct phases of the nitrogen question. The plant tissues from which life had departed hold in them the nitrogen that had once moved in soluble form through the soil. The nitrogen in the dead plant tissue can not, however, again become a part of the food for other plants; not until it has again been changed into simpler soluble forms. This locking up of the nitrogen in forms slowly decaying, and therefore slowly available, is a wise provision, otherwise the nitrogen would soon be washed out of the soil. Thus we see that the soil nitrogen is contained in an insoluble form in the remains of former plants, and, no matter how much of it the soil contains it is inaccessible to the plant growing upon it until it has been first changed into the simpler forms. Now, as to the agents that produce this transformation. Bacteriology, in general, and soil-bacteriology, in particular, are subjects to which the attention of the scientific world has turned very recently. Of the many hundreds of different species of bacteria living in the soil, but few are known. Nevertheless, even at the present time, enough has been learned to enable us to form a conception, at least, of the changes that take place there. The nitrogen of organic substance, whether plant or animal, usually exists in the form of albuminoids, more frequently termed proteids. These proteid molecules are seized by the soil bacteria and are utilized by them for the formation of their own bodies. Being saprophytic by nature, that is, unable to build up organic substance from the simpler materials, as is done by higher plants, they must derive their energy from the tissues that chlorophyl-bearing plants had fashioned with the aid of sunlight. In availing themselves of this potential energy for their own purposes, they break down the complex molecules; to use a popular expression, they cause decay. Tn order to gain their end, that is, to secure the food contained in the proteid molecules, the bacteria must first change it into a readily diffusible, soluble form. For this purpose the chemical ferments known as enzymes are produced. With the aid of these enzymes, the albuminoid substances are 'peptonized.' In the laboratory such organisms are described as gelatin-liquefying bacteria. A part of the food thus made accessible is appropriated by the microorganisms and in their physiological processes is still further simplified. A part of the carbon is oxidized and escapes into the atmosphere as carbon dioxide, gaseous hydrogen or oxygen is set free, or the two are combined to form water. The nitrogen with which we are here concerned is subject to many changes. In the course of its migration it forms a part of the amid molecules; from these it is split off in the form of ammonia, and this again may be destroyed and gaseous nitrogen set free, or seized by another distinct class of organisms, and oxidized to nitrites ​and nitrates. The last is the form in which the nitrogen is usually taken up by the plants. On the other hand, the nitrates are themselves subject to the opposite forces of deoxidation. There are species of bacteria in the soil which reduce nitrates to nitrites, to ammonia or even to gaseous nitrogen. To recapitulate, then, there take place in the soil processes of nitrification, denitrification and also the fixation of free nitrogen. It was necessary to consider the former two, in order to understand the third. It would be out of place here to speculate upon the manner in which the soil nitrogen is oxidized; it might not be out of place here to consider the possible ways in which nitrogen is set free from its compounds, on the one hand, or is 'fixed,' on the other hand. Quite recently hypotheses have been advanced which would regard the processes of 'fixation' and of 'denitrification' as being very much related phases of the same physiological activities. The investigators who have labored in this field of research, and to whom we owe most of our knowledge on the subject, are Berthelot, Winogradsky, Beyerink and Stoklasa. Berthelot was among the first to observe that soils free from vegetation can increase their store of nitrogen. Winogradsky, after much painstaking search, isolated from the soil an organism, which, in company with two others, can grow in nitrogen-free media and fix considerable quantities of nitrogen in a short time. Beyrink, also, has isolated within the last few months several organisms that possess a similar power, and Stoklasa has done a great deal of careful work to determine just how the fixation of nitrogen is accomplished. Moreover the subject has assumed more than a mere scientific interest within the last three or four years. The firm of Friedrich Bayer and Co., of Elberfeld, Germany, has placed on the market a bacterial culture bearing the fancy name of 'Alinit.' This alinit they claim can under favorable conditions increase the yield of non-leguminous crops 40 per cent. On examination, the alinit proved to be a pure culture of B. ellenbachii, mixed with a starchy material resembling dried and pulverized potatoes. The organism was isolated by a German gentleman-farmer, Caron by name, and named B. ellenbachii after his estate, Ellenbach. This bacillus differs but little from the organism isolated by Du Bary some years earlier, and called by him B. megaterium. This organism, Stoklasa claims, is not only similar to, but identical with B. ellenbachii. The accumulated evidence of several investigators on this point inclines me to the belief that the two are not identical, though very much allied. At any rate, Stoklasa has shown that B. megaterium is capable of fixing atmospheric nitrogen in media containing but traces of fixed nitrogen; it develops and adds to the nitrogen content of the medium by drawing upon the nitrogen of the air. This organism has, as it were, a double nature. In the first place, as just noted, it is capable of fixing atmospheric nitrogen; in the second place, it exerts ​a deoxidizing effect when grown in solutions containing nitrates, with the production of nitrites and ammonia. This double action leads us to inquire into the nature of the physiological processes taking place in either case. His investigations in this field led Stoklasa to assert that there is much in common between the two processes. He believes that the deoxidation of nitrate is due to the action of the bacteria on water, with the liberation of hydrogen and the formation of hydroxyl. Nascent hydrogen is a powerful reducing agent, and would of itself withdraw the oxygen from nitrates to form nitrites, while hydroxyl in contact with ammonia will cause the formation of water and the liberation of nitrogen. Part of this nitrogen is undoubtedly utilized by the bacteria, and the rest is returned to the air. How the inert nitrogen molecule is torn apart and the nascent nitrogen atoms thus formed utilized by the bacteria for their growth is a question that is more difficult of solution. We do know that the amount of nitrogen fixed by B. megaterium is affected by the composition of the nutritive medium. The same is true of denitrification. The molecular structure of some carbohydrates or of organic acids and the arrangements of the atoms in the molecule influence the activity of the bacterial cell and its life processes. I have found, for instance, that the more complex citric acid molecule offers a more favorable source of energy to a denitrifying organism that I have isolated than do either succinic, tartaric or lactic acid. And the laboratory work clearly indicates that the amount of organic substance in the soil, as well as its nature, determines the course of development, and the prevalence of the one or the other of the soil organisms. Certain it is that where the fixation of nitrogen takes place in the soil, it occurs only when its store of nitrogen is very meager. This is analogous to the behavior of the legumes. It has been found that these plants when growing in a soil rich in soluble nitrogen do not to any considerable extent draw upon the atmosphere for that element. It is only when the soil offers little or no nitrogen that the atmospheric treasure house is unlocked for it. All experimental evidence thus far accumulated indicates that there is a struggle between the plant and the bacteria invading its roots, that the latter are so modified by the aggressive activity of their host that they form a fine network of tissue in which the atmospheric nitrogen is captured, as it were. That this assumption is not entirely erroneous is shown by the fact that legumes, inoculated with cultures of B. radicola that are particularly virulent, although they form root tubercles, show nitrogen hunger when there is none in the soil at their disposal, and the microscopic examination reveals bacteria that are not modified as is the case in vigorous plants. In other words, the bacteria resist the encroachment of their host and would not be compelled to furnish it with the nitrogen that it can not get otherwise. In connection with this, the question naturally ​arises whether B. radicola, the bacteria of the legume tubercles, can fix atmospheric nitrogen independently of its life in the legume tubercles. As a matter of fact, Beyerink and Mazè claim to have proved that this organism can fix elementary nitrogen independently of legumes. We should note here the remarkable fact that although this organism is so universally distributed and common in all soils, all attempts to isolate it from the soil directly have not been successful.
There are probably a half a dozen bacteria capable of fixing atmospheric nitrogen known to-day, and there is little doubt that others will be found before long. As it is, we are fully justified in the claim that soil bacteria are a potent, nay, an indispensable, factor in the creation of the world's food. Though they are to most of us an invisible world, though many of us never suspect their existence, they are yet our staunch friends, living their brief life, contributing to a broader life, making it possible for the finite to dream of the infinite.