In spite of the frequent habitation of compost heaps, nitrate, beds, mud or sewage by nitrifying bacteria and thiobacilli, they are generally incapable of being cultured on a wide range of heterotrophic laboratory media. They may even be inhibited by certain organic nutrients added to the autotrophic medium.
Similarly certain diatoms and green or blue-green algae fail to grow on none or only on a very few of the numerous organic compounds tested, and sometimes even then retain a requirement for light and cannot grow heterotrophically in the dark. Numerous metabolic poisons are, not surprisingly, toxic to autotrophs. The existence of facultatively autotrophic relatives of the obligate autotrophs argues against there being any fundamental structural or chemical difference between the two. This view is supported by analysis which shows the usual sugar and amino acid components in polysaccharide and protein of Nitrosomonas, Thiobacillus, Ferrobacillus and blue-green algae. Similarly, the coenzymes, cytochromes, nucleic acids, vitamins and storage products of obligate autotrophs are similar to those of heterotrophs of various types.
The endogeneous metabolism in the absence of light or of the inorganic substrates is probably similar to that in heterotrophs. For example, Thiobacillus respires endogenously with an R. Q. of 1 and seems to oxidize a stored polysaccharide synthesized from carbon dioxide.
Polyhydroxy-butyric acid is stored by Nitrobacter and by the blue-green alga Chlorogloea10 and presumably provides maintenance energy from its oxidation as it does in Rhodospirillum and Hydrogenomonas. Of course, no growth occurs under such ‘starvation’ conditions, andthere must clearly be some distinction in the obligate autotroph between exogenous organic nutrients and those available within the cell as stored reserve nutrients. Compounds synthesized by autotrophs from carbon dioxide are secreted into the medium to a significant extent. For example, Thiobacillus releases amino acids and phosphatidylinositol into the medium and Ferrobacillus loses pyruvate. These compounds cannot support growth after inorganic substrate exhaustion, but might of course make the environment more favourable for growth: phosphatidylinositol is a wetting agent and may facilitate the attack of elementary sulphur by Thiobacillus. Amino acids in solutionsmight act as chelating agents and provide a bound or ‘buffered’ source of essential trace metals.
Such secretion might thus have some selective advantage to these organisms. It should be noted that these compounds probably escape from intact organisms rather than because of lysis, because both T. thiooxidans and Ferrobacillus seem very resistant to lysis. Clearly, no obvious chemical peculiarity is likely to be the cause of obligate autotrophy.
Many attempts to find stimulation or inhibition of autotrophic growth by organic nutrients have produced relatively few positive results. Reports of inhibition of growth of nitrifiers by some amino acids and peptone could be due to metal chelation or some other secondary effect and have little significance in considering obligate autotrophy. Similarly growth stimulation by hay infusion and yeast extract could be due to supply of essential trace metals. Such a reason could explain the stimulation of ammonia oxidation in Nitrosomonas by corn steep liquor, for inorganic solids obtained from the liquor are just as effective. Also the stimulation of Nitrobacter by yeast extract may be due partly to molybdenum in the extract, although biotin could be stimulatory also as was found by Krulwich & Funk.
Certain organic compounds may stimulate autotrophic metabolism. Algal development in the light may be enhanced by the addition of glucose or acetate to the medium. Glucose was slowly used by T. thiooxidans and produced stimulation of sulphur oxidation and growth.
T. neapolitanus gives faster growth rates and increased yields when supplemented with amino acids. Nitrosomonas was stimulated by pyruvic acid, which also decreased the lag in autotrophic growth. Photosynthetic growth of Chlorobium thiosulfatophilum was enhanced by acetate but, of course, carbon dioxide, light and sulphide were also essential.
These effects could be explained if the stimulatory compounds were used as supplementary carbon sources in a metabolism that was carbon-limited by the rate of carbon-dioxide-fixing reactions or by the synthesis of a particular essential compound such as biotin or a derivative of pyruvate in the examples quoted.
Uptake and metabolism of organic compounds
Evidence for oxidation of externally added organic nutrients by chemoauto- trophs is relatively scanty but the endogenous respiration of Thiobacillus does seem to be increased by organic acids and by ribose, glycerol and glucose, al- though sometimes this stimulation was slight. Nitrobacter, however, is able to oxidize formate quite rapidly and the oxidation seems to involve the same cytochrome system as that involved in nitrite oxidation although the pH optima for the oxidation of the two substrates are different. Formate oxidation is apparently coupled to carbon dioxide fixation and may even allow a very low rate of growth.
Acetate, however, is oxidized only slowly. It is interesting that Ruban claims that Nitrosomonas may be able to attack organically bound ammonia (e. g. in guanine or uric acid) by means of deaminases which liberate ammonia, the normal respiratory substrate.
The obligate photoautotroph, Chlamydomonas moewusii, which could not grow in the dark in a vast range of organic nutrients was, however, capable of oxidizing acetate, pyruvate and succinate. Clearly, a possible explanation is that insufficient energy could be obtained from the oxidations to support growth. Such an explanation has been suggested by Umbreit for obligate chemoautotrophs, which might not be absolutely incapable of using organic materials but require additional energy or carbon supplies from the normal autotrophic systems. Using C-labelled nutrients it has been possible in the last few years to test whether the autotrophs are in fact capable of assimilating organic compounds as well as carbon dioxide. Possibly the earliest experiment was performed by van Niel and Cohen (C.
B. van Niel, personal communication) in 1955, when they found that 14C-valine was incorporated by autotrophically growing Thiobacillus denitrificans, and appeared to a large extent in the cell protein. Here the valine was presumably acting as a supplementary carbon source. Subsequently other Thiobacillus spp.
have been shown to assimilate radioactive amino acids16 and materials like acetate, glucose and glycerol. In one Thio- bacillus at least, the incorporation of such nutrients is dependent on energy from thiosulphate oxidation,17 while it has been found that Nitrobacter can incorporate labelled compounds equally well both in the presence or absence of nitrite oxidation. Work on the stimulation of growth of Nitrosomonas by pyruvate also shows that I4C-pyruvate was incorporated and served as a source of carbon for several amino acids subsequently synthesized by the organisms. Among the photoautotrophs, Chlorobium carries out the light and C02-dependent assimilation of acetate20 and some blue-green algae can incorporate 14C-glucose or acetate by light-dependent mechanisms.
Thus members of all the obligately autotrophic types have been shown to use organic compounds and one must look more closely for an explanation of obligate autotrophy.
Id search of an explanation of obligate autotrophy
In trying to present a reasoned theory to explain the problem one must first accept that there may be no single explanation for all the autotrophs. In fact, there might be several separate factors, any one or combination of which might explain obligate autotrophy in one organism or group of organisms. Some of these possible factors could be: (1) limited permeability to organic nutrients; (2) inability to oxidize them or to obtain energy from their oxidation, hence making chcmo- or photosynthetic energy indispensable; (3) a limited ability to synthesize all the compounds necessary for growth from all or most carbon sources other than carbon dioxide; (4) some biochemical block to growth on excess external organic nutrients, related in some way to the carbon-dioxide- bascd metabolism; (5) self-inhibition by products of the metabolism of organic compounds; or (6) some special dependence either on an intermediate of the inorganic respiratory processes in the chemoautotroph or on light in the photo- autotrophs for some specific reaction in the cells. These possiblities are now examined in the light of available evidence.
1. Permeability and the ‘submarine hypothesis’. Umbrcit expressed the view that an autotroph like Thiobacillus thiooxidans is adapted for life in what he regarded as a ‘toxic environment’, and excluded organic compounds from the cell by selective permeability properties. The autotroph could thus be called a ‘biological submarine’ which excluded a hostile world by being impermeable to all but a few compounds.
No studies seem to have been made of any permease systems in obligate autotrophs, but among the algae,23 Chlorella is unable to use tricarboxylic acid cycle intermediates, most likely indicating that they are unable to enter the cells. A similar explanation might well apply for the diatoms mentioned previously, and similar explanations might apply for the ‘acetate flagellates’: Chlamydomonas spp. which grow on acetate or Krebs’ cycle intermediates but are unable to use sugars. However, Thiobacillus denitrificans is freely permeable to glycerol, on which it cannot grow, and Nitrobacter similarly accumulates materials which do not support growth. It is noteworthy that Thiobacillus neapolitanus assimilates virtually none of any acetate or amino acids supplied to it, unless it simultaneously oxidizes thiosulphate. Similarly acetate assimilation by Anacystis nidulans21 is light-dependent.
This may indicate that permeation by the nutrients occurred only if a source of energy were available. Clearly, however, permeability is not the explanation for all autotrophy. 2. Energy couplingPerhaps the most attractive explanation is to believe that even if the organism is permeable to the nutrient it is unable to grow on it, because of an inability to obtain sufficient energy from its oxidation. Such inability might result from a failure to oxidize the compounds or from a lack of the systems known in heterotrophs for the trapping of energy from such oxidations. Many autotrophs do not oxidize organic compounds to any significant extent and even if they do stimulate respiration they do not support growth.
Lewin showed that even complete oxidation of acetate by Chlamydomonas dysosmos was possible but no growth occurred. The fact that oxidations may occur, shows that there is permeation into the cells, and thus indicates that the oxidations may not yield energy to the organism. Lewin in fact simulated the responses of a strictly autotrophic Chlamydomonas to acetate by treating a wild type strain with the energy-uncoupler 2,4-dinitrophenol. Hempfling ; Vishniac found that although Thiobacillus extracts oxidized NADH its oxidation did not support ATP synthesis. If intact organisms behave in the same way, NAD-linked oxidations would not support growth, unless the cytochrome systems believed to be involved in sulphur oxidations were also involved in NADH oxidation.
The critical factor in Nitrobacter growing with formate may well be that the energy trapping system is the same as with nitrite. The main function of light energy or chemolithotrophic energy in supporting organic assimilations by Chlorobium, blue-green algae and thiobacilli is almost certainly in activating the compounds and providing sufficient energy for synthetic reactions. However, if this were the sole explanation for dependence on light or specific inorganic respiratory substrates, an ‘obligate autotroph might be able to grow on organic compounds in the absence of carbon dioxide provided that the particular energy source is also supplied. However, experiments with Chlamydomonas, Nitrobacter and thiobacilli failed to obtain such growth when even complex mixtures of organic compounds, including extractsof the organisms themselves, were provided.
Thiobacillus neapolitanus, for example, could not develop without C02 but with thiosulphate when supplied with hexoses, pentoses, yeast extract and amino acids, although normal development occurred in the presence of C02. Butler ; Umbreit16 obtained growth of T. thiooxidans on glucose with sulphur in the absence of C02. This result does not mean, however, that total cell synthesis produced from glucose directly, for Suzuki27 showed that this organism could incorporate glucose into aminoacids and other cell components during sulphur oxidation, but that it was also oxidized (possibly by both Embden-Meyerhof-Parnas and pentose-phosphatecycle mechanisms) to liberate carbon dioxide. Consequently much of the cell biosynthesis observed by Butler ; Umbreit could have depended on this liberated C02, which would be providing physiologically ‘normal’ conditions.
With Chlorobium and blue-green algae the light-dependent incorporation of organic compounds also depends on simultaneous carbon dioxide supply. Growth could not thus occur on organic nutrients alone, but in considering the C02 requirement of autotrophs one must recall that it is an essential metabolite to most, if not all, heterotrophs also. The large amounts of glucose and acetate carbon (32% and 18% of the dry weight) assimilated by the Anabaena of Carr ; Pearce28 might similarly have been taken up in part as C02 previously released as oxidation products of, say, an operative tricarboxylic acid cycle.
