R. WElNDLlNG Lederle Laboratories, American Cyanamid Co., Pearl River, N. Y.
Microbial Associations and Antagonisms Frequently, when investigators have attempted to explore complex microbial interrelations of natural environments, by laboratory or field studies, the complexity of microbial relations interlocking in space and time have posed formidable obstacles to comprehensive work. Investigators have concentrated therefore on selected microbial groups of special interest or on microbial associations in specific environments, especially when practical application to these environments was needed. The search for answers to practical problems has led, almost invariably, to the view of the interdependence of adaptive and associative microbial phenomena. And i t appears that this fundamental philosophy has been helpful, in turn, in experimental approaches that have led to success in solving the problems.
A L L living organisms, from man to submicrobe, are adapted to their surroundings-that is, to food and energy supplies, temperature, etc.-and all act and are acted upon by the many factors of this environment. One of these factors comprises other organisms, living or dead. T h e effects of these organisms may be favorable, that is associative, or unfavorable: that is antagonistic. The frequency of microbial interactions may be visualized if one stops for a moment to think of the enormous numbers of microbes. Counts of bacterial cells in a cupful of rich soil or in a tube of culture medium reveal as many organisms as there are people on this planet. These fantastic numbers of microbial cells make u p microscopic life because unlike cells of higher plants and animals, single bacterial cells are capable of living as sdf-sufficient individuals: and these individuals are capable of multiplying to millions and to billions in hours or days. Therefore, in speaking of microbial interactions in natural habitats, such as soil or ivater, we must realize that the microbes live there in microenvironments as populations of millions or billions and that they intermingle on a scale that is impossible for higher plants and animals. At the risk of oversimplifying the complex field of microbial interrelations, we shall attempt a bird‘s-eye view of some principles and topics. T h e folloiving
questions ill be brought u p to provide a somewhat logical order:
1. \Vhat are the techniques used in the study of microbial interrelations? 2. \$‘hat types of interactions can we differen tiate? 3. HOW were complex microbial interactions investigated by laboratory models? 4. \Vhat is the role of antibiotics in nature? Techniques Three approaches have been used. three types of techniques, that are opening lvindows for human eyes to visualize the interactions of the lrorld of microscopic organisms. The first approach is to isolate the numerous microbia! species, define their biochemical functions: and deduce their activity in mixed cultures of natural environments. Isolation can be by special selective media or by nonselective media, such as Lochhead’s technique ( 4 ) which includes subsequent classification of the isolates by nutrient requirements. h-o~v, pure cultures, identification, colony counts? and biochemical tests are basic tools. But by the process of isolating and purifying microbes, we must destroy the very relationships we are discussing here. This is like trying to study social relations of man by considering him apart from society. Moreover, as Beijerinck and Winogradsky have pointed out-in
the laboratory. some of these pure cultures will behave like hothouse plants and \vi11 not carry out the biochemical processes of their natural habitats (5). The second approach seeks to overcome this dilemma by direct observation under the microscope. For instance, the contact slides of Rossi-Cholodny are inserted into soil and taken out at intervals for fixing and staining the adhering microbial populations. Thus: a series of “snapshots” of microbial populations, their types, and interactions. is taken directly from the soil to the microscope. This method can give the rxperienced student picturrs of soil microbial relations, comparable perhaps to the microtome sections by which anatomists visualize the microscopic structure of animal organs. The obvious dra\L.back of this method is the difficulty of identifying the microbial species microscopically. As a third approach. laboratory models of microbial associations have been studied-that is, pure cultures a r r combined in the laboratory under conditions that imitate natural habitats but can be controlled and analyzed. This approach is excellent for the study of interrelations between two organisms under the effcct of varying environments. Choice of conditions predetermines the results of such investigations to some extent. However, they give the important advantage of having analysis and synthesis of interactions going hand in hand. VOL. 48, NO. 9
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The ideal is of course to apply the three approaches to problems and to coordinate them. But obviously the labor required sets severe limitations here.
Types of Microbial Interactions The following is not intended to be a classification of microbial interactions. I t is merely meant to serve as a framework for a rapid review of these interactions to give examples of their concerns or objectives and to lead to a n evaluation of the terminology. Antagonistic effects are most frequently observed in nutrient agar, in the laboratory, and they are most readily demonstrated there by mixed cultures. Examples are :
1. Competition-when fast-growing molds or bacteria spread over nutrient plates with appropriate energy and nutrient supplies, while slower microbes have barely started growing. 2 . Inhibition-when molds that form mats on top of liquid media prevent oxygen from reaching microbes underneath the mat or when organic acids are excreted as waste products, reducing the p H below the tolerance level of bacteria. 3. Toxicity or antibiosis, forms of inhibition by defined substances such as antibiotics-best illustrated by the wellknown clear zones around the penicillium colony that led Fleming to the discovery of penicillin. 4. Parasitism-bacteriophage clearing dense clouds of bacteria in nutrient media within half a n hour by infecting and bursting the cells. 5. Predation-infusoria that ingest bacteria. Associative effects may be thought of as being concerned with the same factors as antagonism but in exactly the opposite way. For example:
1. Balanced growth-when lactic acid bacteria are kept growing in milk because fungi (Oidium lactis) remove the excess of organic acid which would inhibit further growth of the bacteria. 2 . Succession-when yeasts produce alcohol in fruit juices, and acetic acid bacteria take over by using the alcohol as their energy source. 3. Stimulation by nutrilites or vitamins-when microbes that are unable to synthesize biotin thrive near other organisms that produce it in excess (e.g., iVematospora gossypii near Polyporus fungi) ; also, stimulation by minute amounts of antibiotics. 4. Symbiosis-when algal cells live in community with certain fungi to form the lichens, unique forms of life which have been synthesized in the laboratory from their constituents more than 50 years ago, In contrast to antagonistic effects, associative interactions are rarely encountered in the routine of the laboratory. They are more complex and harder to demonstrate definitively. I n nature,
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however, associative effects are widespread. Indeed, as every student of elementary biology knows. they are a t the basis of nature‘s carbon and nitrogen cycles. essential for breaking down organic matter from complex carbohydrates and proteins through a series of steps. Pasteur showed more than 75 years ago how populations of aerobic and anaerobic bacteria derive advantages from one another when the aerobic bacteria make it possible for the anaerobes to grow by reducing the oxygen of a medium to tolerable levels. The intergrading of thr types of associative effects is so pronounced that all the examples mentioned have been considered by some authors as symbiosis, in the broad sense of the term. We have used symbiosis here in the strict sensethat is, of individual microbes living in a mutually dependent fashion, attached to one another, and not of populations living under conditions of some mutual benefit ( I , 2). A similar intergrading occurs in antagonistic effects. Furthermore, there is frequently no sharp line between antagonistic and associative effects. For instance; in certain lichens so much of the advantage of the partnership goes to the fungus that some investigators have considered it as parasitic, not symbiotic. When we evaluate, in this light, the meaning of terms (types of antagonistic and associative effects) surveyed, it becomes evident that these terms are useful primarily to define and analyze individual facets of microbial interrelations. Indeed, the terms may be misleading if they become rigid concepts based on these individual facets, overlooking the essentials of microbial physiology (the interlocking functions of the cells) and the continuous fluctuations of their populations.
Complex Microbial Interactions and Their Study by laboratory Models So far microbial relations have been considered as being either favorable or unfavorable for the organism concerned. Nature, ho\\ever, does not work with black and white only but with all shades of graj’ and with ever-changing colors That is, microbial populations exist in natural media of soils and water bodies as complex dvnamic qsterns in which benefits and damage to members of the system change with time, with the viewpoint of the observer, and from one microenvironment to the other. Fairly stable systems do occur where populations are small-e.g., in the ocean and in other large water bodies (Z), and in soils that contain little organic matter. On the other hand, variable systems of great complexity are common in water or in soil rich in fresh decomposing organic matter (5, 7). Discussion of this topic is limited to two
INDUSTRIAL AND ENGINEERINQ CHEMISTRY
representative investigations of laboratory models. I n order to gain basic information on how microbial interactions affect decomposition of organic matter in soil, Waksman and Hutchings (6) set up a series of laboratory ”models.” They compared the action of pure cultures of certain bacteria, fungi, and actinomycetes. as single cultures, as mixtures, or in succession, on cornstalks, alfalfa, and oat straw. They found that the growth of one group of organisms was greatly modified by the presence of the others. Lignin was decomposed only when acrinomycetes were present. Cellulose decomposition was carried out by several organisms, but it was more effective by certain mixtures of microbcs. None of the individual or mixed populations was biochemically more effective than inoculum from a natural soil. The sequence of succession, the type of organic matter, mineral additions, moisture, and pH, all influenced the microbial populations, their interactions, and their biochemical effects. Entirely different laboratory models have been designed by Gotaas and Oswald ( 7 ) in California to study the decomposition of organic matter in sewage oxidation ponds. They investigated first the biochemical action of pure cultures of a large number of algal and bacterial forms, in several controlled environments, and they determined their reactions to cell numbers, amounts of organic material, light, oxygen, etc. Then, in a second series of models, selected culture of algae and bacteria were mixed under aseptic conditions. Some of these systems gave satisfactory biological balances (“symbioses”). In successful balanced populations, the bacteria get their energy and nutrients by breaking down organic matter and by utilizing the oxygen which is liberated by the algae. The algae draw their energy from photosynthesis and use carbon dioxide and other bacterial breakdown products in the metabolism and growth of their populations. It will be interesting to see whether further development will justify the use of a few selected, individual cultures, or whether adapted mixed populations from soil or water will prove practical, as they have in other investigations of similar purposc.
Role of Antibiotics in Microbial Antagonisms of Natural Habitats In this connection the question is frequently raised : What is the role of antibiotics in nature? The answer is difficult, because few critical studies have been made to determine under natural conditions the relative abundance of populations that produce specific antibiotics, the amounts produced, and their stability. Many hitherto unknown antibiotic substances have been literally unearthed by the search for new and better
WATER PURIFICATION antibiotics. Most of these have proved toxic to animals and plants, but their occurrence has lent support to theories that antibiotics are primarily responsible for some generalized effects in soil and water. Examples cited by the proponents of such theories are : 1. Faster disappearance of typhoid and other pathogenic bacteria from soils and from water rich in organic matter than from clear or sterilized water 2. Toxicity of certain sea water samples to microbes which is, according to ZoBell ( 8 ) ,partly removed by sterile filtration and still more by boiling
In order to substantiate the idea that antibiotic substances produced by microbes are the cause of these effects one would have to show that:
1. Specific substances produced by specific microbes are actually present in detectable amounts 2. They are not readily inactivated in the natural environment by adsorption, etc. 3. Other possible causes such as bacteriophage are excluded A survey of the literature offers no clear-cut evidence that antibiotics act generally, in nature, like magic bullets that destroy human, animal, and plant pathogens wherever antagonistic activities of microbes occur. Probably specific antibiotics act in microenvironments which escape our present methods. But, most antagonistic phenomena in natural microbial populations seem to be far too complex to be attributable to antibiotics only-just as beneficial effects of associations can hardly be attributed to vitamins only.
DISCUSSION..
Courtesy of American Waterways Operators
Tennessee River at Guntersville, Ala.
References (1) Gotaas, H. R., Oswald W.J.,Abstract, Sezeage and Znd. Wastes 26, 930 (1954). (2) Henrici, A. T., Ordal, E. J..“Biology of Bacteria.” 3 r d ed. Wilev. , , New
E’ork, 1948.’ (3) Heukelekian, H . , A n n . Rev. .Microbid. 7, 461 (1953). (4) Lochhead, A. G., Chase, F. E., Soil Sci.5 5 , 185 (1943). ( 5 ) Waksman, S. A , , “Principles of Soil
Microbiology,” 2 n d ed.. Williams and Wilkins, Baltimore, Md., 1932. ( 6 ) Waksman, S. A , Hutchings, I. J., Soil Sci. 43, 77 (1937). ( 7 ) Whipple, G. C., “Microscopy of Drinkinp Water.” Wilev. Xew York, 1927. (8) ZoB& C. E:, “Mahne Microbiology,” Chronica Botanica, Waltham. Mass., 1946.
RECEIVED for review January 12, 1956 ACCEPTEDApril 18, 1956
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Microbiologica I Associations and An tag on isms
A n adequate discussion of the article by Weindling would necessitate a complete understanding of all the biochemical processes of sewage and industrial wastes treatment. There is no current process that uses pure cultures; therefore, microbial associations or antagonisms must occur in all the sanitary engi-
neering biological processes. The broad principles of the biochemical factors in these processes have been intensively studied, but the controlling factors of the biological associations are virtually unknown. I t is extremely difficult to differentiate between the adaptation of one organism to another form of that or-
ganism and the shift in the microbial population because of a change in the conditions present in a waste. In microscopic observations of activated sludge, it is quite readily apparent that there are many organisms a t work in the treatment of the sewage. Zoogloeal organisms are primarily bacteria, VOL. 48, NO. 9
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probably of many different species as indicated by McKinney and Horwood ( I ) , and they are typical associations. Trickling filter films contain zoogloeal organisms in very much the same microbial appearance. In each of these aerobic processes, there are frequently fungi or filamentous strands which may be only a modification of zoogloeal forms or actually different species of microorganisms but which are generally associated with the zoogloeal forms. However, it has been observed that frequently the zoogloeal forms tend to disappear as the food concentration increases, while the filamentous forms tend to increase. Further, even a casual observation of either the trickling filter slime or the activated sludge process Tvill reveal large numbers of free-swimming protozoa. ‘The exact function of protozoa is not known, but there is considerable evidence that they may kill or eat the free-swimming bacteria and thus aid in the production of a mvch clearer effluent: leaving only the clumped or filamentous bacteria which can settle out. I n support of this concept the following observations were made by a new member of my staff. Recently, some filter slime \vas brought into the laboratory and aerated in order to develop activated sludge. I t was observed on the first 2 days to have feiv ciliate protozoa and many free-swimming bacteria. O n the third day it was observed that the ciliates were abundant but that very few free-swimming bacteria remained. In the sludge digestion process, there is a neat illustration of the problem of association of bacterial forms. The first group of organisms to function involves those which can hydrolyze large polymers to break them down into the simple sugars and amino acids. Then these monomers are metabolized to the short chain fatty acids and alcohols \vhich in turn are used by the methane organisms to complete the cycle. The methane organisms cannot use complex foods. When the organic acids are formed, the p H of the sludge drops rapidly to values as low as 5.0. The utilization of the organic acids by the methane organisms rapidly reduces the hydrogen ion concentration. This causes a rise in the p H to values of 7.0 where, with a continuing feed, there is a very neat balance in the rate of organic acid production and methane evolution which provides a proper condition for the functioning of each form of organism present in the sludge digestion process. Another function of the forms responsible for liquefaction of sludge is the production of small amounts of highly reducing substances such as hydrogen sulfide which provide or induce a proper oxidationreduction potential for the activity of the methane organisms. However, this process of producing hydrogen sulfide may
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result in the reversal from a favorable microbial association to an antagonistic situation by the presence of a n excess of sulfate ions which inhibit digestion because of the reduction of large quantities of hydrogen sulfide which is toxic to the methane organisms. In polluted streams, all the various biochemical processes are interrelated. The aerobic processes again use filamentous or colonial bacterial forms on the stones or sticks in the floiving stream and thus reduce the total organic matter. There are large numbers of free-swimming or attached protozoan forms again mixed in with the bacterial or fungal forms but, in the natural stream: higher forms are continually developing. The small crustacea and insect larvae feed rapidly upon the smaller protozoa. The small crustacea and the insect larvae are used as food by the small fish, and these in turn are used by the larger fish. This sequence of biological forms does not fit into microbial associations; but the over-all larger biological associations in the polluted stream can be seen. The sludge on the bottom of the polluted stream may exhibit methane fermentation, and these sludge banks may support large numbers of roundwormsj insect larvae: and earthworms. One of the outstanding studies in recent years concerning microbial associations in stream pollution is that reported by Patrick ( 3 ) rvhere the condition of the stream is assayed on the basis of the microbial population and the variety and the number of species that are present in rhe stream. In a later publication. Patrick (2) showed that the distribution of diatom species in a moderately polluted stream differed greatly from that of a healthy stream. Thus pollution increased the number of individuals in the adaptable species while appearing to eliminate other species. In a severely polluted stream, diatoms disappeared or were prevented from developing upon or adhering to the counting device used. These diatom counts are taken by inserting a glass slide in the stream and then counting the forms that adhere to the slide and that can be identified. (As mentioned by IVeindling, however, this type of stream survey is quite expensive. because it involves wellqualified men to ensure the idenriry of the various forms.) I n the presence of toxic substances which may be present from industrial wastes, it is not known whether the toxic substance causes a change in the nature or identity of the species present or whether different species which are less susceptible to the toxic substance are capable of growing. (It is readily observable, however, that i n the presence of toxic substances marked changes in the appearance of sludges occur. Thus, if the pH of an industrial waste sewage
INDUSTRIAL AND ENGINEERING CHEMISTRY
mixture flowing over a trickling filter increases from a value of approximately 8.5 to something between 9.5 and 10.0: the appearance of the film changrs markedly. This change in the appearance of the film may be only remporar) until more resistant forms of that group originally present develop as in adaptation, or i t may he that there will be a complete new series of microbial associations.) As IYeindling has indicated, there is a possibility of the production of antibiotics by some of the various Corms present in either the sejver or srwage rreatmcnt plant. (Some time ago. the writcr had the opportunity of observing large amounrs of filamentous rorms entering one of the large sewage treatment plants in the City of Atlanta. He was told. upon questioning, that this is a common situation in the spriny of the year in thc Atlanta City seiverage sytem and that many of the srivagc treatment plants disposed of literally tons of the filamentous material at this time. He was at thc plant because of difficulty with thr sludgc digestion at this particular unit. It immediately became a matter of thcoreticai speculation as to whether or not tht. filamentous bodies or debris might carry appreciable amounts of antibiotic material in the sludge into the digesLion tank and that some of the sludge-digestion difficulties might be explained on thc basis of the material added by the organisms in the sludge. It is more likely that it was simply inadequate operation of the sludge-digestion units, but there is also the possibility of a contribution to the sludge-digesrion trouble by an antibiotic action resulting from the growth of these filamentous forms. X comment by the chairman of this symposium added that he had made a similar observation in the City of Rahway, N. J.) The writer agrees with LVeindling and other authors who have indicated that chancr inoculations from the soil 01’ natural flowing waters will, in general, produce the most effective microbial associations or the most favorable adaptation of the particular species which arr available in the close environment of the trickling filter, activated sludge tank, or. polluted stream. Liferafute Cited ( 1 ) McKinney. R. E., Horwood, M. P., Seieagr and Ind. Wastes 24, 117 (1 952 ). ( 2 ) Patrick, R.. Academy of Natural Sciences. Philadelphia, Pa.. Notuiae ,Vaturae No. 259, 1954.
( 3 ) Patrick, R., Sezcage and Ind. Ithsles, 25, 210 (1953); Proc. Acad. Natural Sciences, Philadelphia 101, 277 (1949 j.
ROBERT S. INGOLS Engineering Experiment Station, Georgia Institute of Technology Atlanta, Ga.
