Modeling of growth, lactate consumption, and volatile fatty acid production by Megasphaera elsdenii cultivated in minimal and complex media

Use of the Pirt and Luedeking-Piret equations permits the determination of the effect of medium composition on the metabolic patterns of Megasphaera elsdenii grown in minimal and complex media with lactate as the major carbon source. To establish the significance of the parameters involved in the Pirt and Luedeking-Piret equations, a quantitative statistical criterion was proposed. In the complex medium, lactate was completely used for growth and product formation, whereas in the minimal medium a fraction of the energy obtained from lactate was used for maintenance purposes. Modeling of VFA production by the Luedeking-Piret equation showed that, independent of the type of medium, acetate and propionate are growth-associated products, while butyrate and valerate are only partially growth-associated. The growth-associated products are related to energy-yielding metabolism and the non-growth-associated products are related to the consumption of reducing equivalents.     

Products, requirements and efficiency of biosynthesis: a quantitative approach

The question of how many grams of an organism can grow heterotrophically from only 1·0 g of glucose and adequate minerals has been put forward many times. Only a few attempts have been made to answer this question theoretically and these attempts were rather rough. In this paper, it is demonstrated that the yield of a growth process may be accurately computed by considering the relevant biochemistry of conversion reactions and the cytological implications of biosynthesis and growth. Oxygen consumption and carbon dioxide production by these processes are also computed. The weight of the biomass synthesized from 1·0 g of substrate and the quantities of gases exchanged are independent of temperature.These results are obtained by adding the individual equations describing the formation of each compound synthesized by the organism from the substrate supplied. The sum represents an equation which accounts for all substrate molecules required for biosynthesis of the carbon skeletons of an end-product, whose chemical composition is given. It is then calculated how much energy is required for the non-synthetic processes which form a part of biosynthesis, such as intra- and intercellular transport of molecules and maintenance of RNA and enzymes. The additional amount of substrate required to provide this energy by combustion is easily calculated. Adding this substrate to the amount used for skeleton synthesis gives an overall equation which quantifies the substrate and oxygen demand as well as carbon dioxide evolution during biosynthesis of 1·0 g biomass. For example, it requires 1·34 g of glucose with adequate ammonia and minerals to synthesize 1·0 g maize plant biomass in darkness; during this process 0·14 g oxygen are consumed and 0·24 g carbon dioxide are produced. It has been described elsewhere that similar results were obtained experimentally with growing plants.Such results depend considerably upon the chemical composition of the biomass being synthesized and upon the state (oxidized or reduced) of the nitrogen source. Other parameters, such as the number of ATP molecules required for protein synthesis, the possibility for utilization of alternative pathways for synthesis or energy production, the presence or absence of compartmentation of synthetic processes and variations in the P/O ratio between two and three, under many conditions affect results of the computation less than 10%.Since maintenance of cellular structures is not considered, the approach concerns the gross yield of biosynthesis. It predicts therefore the dry matter yield of heterotrophic cells from a given quantity of substrate at high relative growth rates.     

Directed modification of Escherichia coli metabolism for the design of threonine-producing strains

The modification of Escherichia coli K-12 metabolism leading to threonine overproduction is the most studied system in synthetic biology that has been used to elaborate the majority of the currently known approaches to constructing microbial producers. They include optimization of biosynthesis through search for rate-limiting stages, modification of substrate and product transport, elimination of side metabolic pathways and degradation systems, reinforcement of the regeneration of coenzymes that are required for product biosynthesis, and exclusion of futile cycles and metabolic pathways with low energy efficiency. Extensive research in functional genomics made it possible to selectively remove the “unnecessary genes,” the functions of which are useless for producing a strain or adversely affect its properties. In total, using various approaches to designing threonine-producing strains, over 150 genome loci that affect more than 30% genes in E. coli were directly modified, thus providing interesting data for researchers in the field of microbial synthesis, as well as in related biological sciences. This review is dedicated to the assessment of genetic engineering modifications in E. coli metabolism (primarily, on the basis of modern patent literature) that ensure threonine overproduction.     

Adaptation of metabolic enzyme activities of Trypanosoma brucei promastigotes to growth rate and carbon regimen.

The insect stage of Trypanosoma brucei adapted the activities of 16 metabolic enzymes to growth rate and carbon source. Cells were grown in chemostats with glucose, rate limiting or in excess, or high concentrations of proline as carbon and energy sources. At each steady state, samples were collected for measurements of substrate and end product concentrations, cellular parameters, and enzyme activities. Correlation coefficients were calculated for all parameters and used to analyze the data set. Rates of substrate consumption and end product formation increased with increasing growth rate. Acetate and succinate were the major nonvolatile end products, but measurable quantities of alanine were also produced. More acetate than succinate was formed during growth on glucose, but growth on proline yielded an equimolar ratio. Growth rate barely affected the relative amounts of end products formed. The end products accounted for the glucose consumed during glucose-limited growth and growth at high rates on excess glucose. A discrepancy, indicating production of CO2, occurred during slow growth on excess glucose and, even more pronounced, in cells growing on proline. The activities of the metabolic enzymes varied by factors of 2 to 40. There was no single enzyme that correlated with consumption of substrate and/or end product formation in all cases. A group of enzymes whose activities rigorously covaried could also not be identified. These findings indicate that T. brucei adapted the activities of each of the metabolic enzymes studied separately. The results of this complex manner of adaptation were more or less constant ratios of the end products and a very efficient energy metabolism.     

Porphyrin Overproduction by Pseudomonas denitrificans: Essentiality of Betaine and Stimulation by Ethionine

Ethionine supplementation of a defined medium for growth of Pseudomonas denitrificans inhibited vitamin B(12) overproduction and led to the elaboration of a red pigment. The pigment was shown to be coproporphyrin III. Inhibition by ethionine of cobalamin synthesis is probably due to interference of methylation of the corrin nucleus by methionine. Accumulation of coproporphyrin III is thought to result from interference by ethionine with the activity of methionine in the coproporphyrinogenase reaction; this would inhibit formation of heme, the feedback inhibitor and corepressor of delta-aminolevulinate synthetase, thus allowing unregulated synthesis of coproporphyrinogen III and its degradation product, coproporphyrin III. Betaine, known to be required for vitamin B12 overproduction, was found to be an essential requirement for porphyrin overproduction in the presence of ethionine. Low-level production of porphyrin, which occurs in the absence of ethionine, also required betaine supplementation. Betaine is thus required for overproduction of both corrins and porphyrins in P. denitrificans.     

Atmospheric constraints on the evolution of metabolism

Earth's early history may have been characterized by coevolution of microbial metabolism and atmospheric composition. Metabolic developments affected the composition of the atmosphere and the resultant changes in the atmosphere stimulated the evolution of new metabolic capabilities.The first organisms were presumably fermenting heterotrophs, exploiting organic molecules abiotically synthesized. These organisms multiplied, developing new biosynthetic capabilities to overcome deficiencies in the abiotic supply of particular compounds, until their growth was limited by the energy source provided by abiotic synthesis of fermentable organic compounds. Further growth required a new energy source, which may have been the chemical energy represented by the mixture of carbon dioxide and hydrogen in the primitive atmosphere. Chemotrophic organisms resembling methane bacteria may have evolved to exploit this source. They would have flourished, along with the heterotrophs that fed on them, until they had decreased the level of atmospheric hydrogen to the point where further extraction of chemical energy from the atmosphere was not possible. Once again, the expansion of life was limited by the availability of energy.The origin of bacterial photosynthesis overcame the second energy crisis. Photosynthetic bacteria could exploit the abundant energy of sunlight while using atmospheric hydrogen and reduced compounds derived from it only as electron donors. Life flourished again, drawing atmospheric hydrogen (replenished only by volcanoes) down to levels so low as to limit even bacterial photosynthesis. Before the full potential of photosynthesis could be exploited the evolution of the metabolic apparatus to process an electron donor of unlimited abundance was necessary. This donor, of course, was water, and the new metabolic process was algal photosynthesis. The oxygen released changed the world from anaerobic to aerobic and made possible the last great advance in energy-yielding metabolism, aerobic respiration.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.     

The auxiliary substrate concept — an approach for overcoming limits of microbial performances

In biotechnological processes, fundamental performances of microorganisms are used. The economy of these processes is essentially determined by the efficiency, velocity (productivity) and quality of the products. Therefore it is a permanent task and challenge for basic and biotechnological research to seek out measures for improving the actually attained parameters. The auxiliary substrate concept supplics an approach. It is based on the fact that chemo-organo-heterotrophic substrates differ in the carbon: energy ratio, thus, growth yield is limited in energy and/or reducing power. It says that, by simultaneous utilization of physiologically similar substrates (mixed substrates), the growth yield increases. The substrates are to combine in such a way that with their simultaneous utilization a minimum of carbon is dissimilated merely for the purpose of the generation of biologically useful energy and/or reducing power. Since all chemo-organo-heterotrophic substrates are more or less energy-deficient, an increase in growth efficiency can be expected if the individual substrates of the mixture are assimilated more efficiently than the respective substrates alone. This may result, for instance, from an immediate assimilation of a substrate (according to the “manner of finished part construction”). An increased growth rate is rather the rule than the exception in mixed substrate utilization. In product syntheses the substrates are, depending on the concrete product and metabolic pathway, either energy-excess or energy-excess or energy-deficient. or, in other words, the processes are energy-generating or energy-consuming, respectively. If this is responsible for discrepancies between the possible yields determined by the carbon metabolism and the experimentally obtained yields, the discrepancies should be able to be decreased and the yields increased by mixing substrates. The substrates are to choose and combine so that, due to simultaneous utilization, the product formation process becomes energy neutral. As a rule, the enhanced efficiency is accompanied by an increased velocity. This does not only apply to syntheses, but also to degradation (and detoxification) reactions. Even supposedly inert compounds or persistent substances can be activated by simultaneous (co-)metabolization of another (an auxiliary substrate, victim substrate or co-substrate) and converted at a considerable rate. It is of interest for syntheses of products but in particular for degradation and decontamination of harmful and waste products in the environment that the residual concentrations of the substrates are smaller than those achieved if the compounds of a mixture are metabolized separately. The auxiliary substrate concept has proven to be fruitful, both for theoretical and practical questions. It was practically already being used before it was formulated (mixed substrate utilization, cometabolism). However, an abundance of regulatory and energetic aspects are waiting to be investigated in more detail.     

Metabolic Influences on Tyrosine Excretion in Bacillus subtilis

The biosynthetic pathway for tyrosine synthesis is regulated both by repression of enzyme synthesis and by feedback inhibition of enzyme activity in Bacillus subtilis. Nevertheless, wild-type cells produce significantly more tyrosine than is required for protein synthesis, and part of this is excreted into the medium. Alteration of nutritional and other environmental conditions of cultivation strongly influenced the amount of tyrosine excretion. The excretion of tyrosine by wild-type cells was compared with that of a regulatory mutant having a feedback-insensitive prephenate dehydrogenase. Tyrosine excretion varied directly with the in vitro activity of prephenate dehydrogenase and inversely with temperature in the two strains. The regulatory mutant excreted five times as much tyrosine as the wild type at all growth temperatures examined. The carbon source used for growth significantly influenced the level of tyrosine excretion. The specific activity of prephenate dehydrogenase was also affected by the carbon source. Incorporation studies with isotopic tyrosine and fluorometric determinations of tyrosine concentrations extractable in hot water were used to measure operationally the tyrosine pools in the mutant and wild-type strains. The effects of various environmental conditions on the synthesis and excretion of tyrosine led to the conclusion that metabolic controls governing end product contrations exist which are completely independent of regulation by feedback inhibition and repression.     

The role of energy-spilling reactions in the growth of Klebsiella aerogenes NCTC 418 in aerobic chemostat culture.

When cell-saturating amounts of glucose and phosphate were added to steady state cultures of Klebsiella aerogenes that were, respectively, glucose- and phosphate-limited, the organisms responded immediately with an increased oxygen consumption rate. This suggested that in neither case was glucose transport the rate-limiting process, and also that organisms must possess effective mechanisms for spilling the excess energy initially generated when a growth-limitation is temporarily relieved. Steady state cultures of mannitol- or glucose-limited organisms also seemingly generated energy at a greater rate than was required for cell synthesis since gluconate-limited cultures consumed oxygen at a lower rate, at each corresponding growth rate, than did mannitol- or glucose-limited cultures, and therefore expressed a higher YO value. Thus, mannitol- and glucose-limitations must be essentially carbon (and not energy) limitations. The excess energy generated by glucose metabolism is one component of "maintenance" and could be used at lower growth rates to maintain an increased solute gradient across the cell membrane, imposed by the addition of 2%, w/v, NaCl to the growth environment. The maintenance rates of oxygen consumption of K. aerogenes also could be caused to increase by adding glucose discontinuously (drop-wise) to a glucose-limited chemostat culture, or by exchanging nitrate for ammonia as the sole utilizable nitrogen source. The significance of these findings to an assessment of the physiological factors circumscribing energy-spilling reactions in aerobic cultures of K. aerogenes is discussed.     

Elemental Composition of Biomass and its Relation to Energy Content, Growth Efficiency, and Growth Yield

Biomass synthesis from primary substrate is a principal featureof growth. A short-cut method for the determination of growthyields and efficiency is presented. Elemental analyses of theproducts (individually or collectively) permit one to calculatedirectly and simply the amount of substrate carbon and electronswhich are conserved, and thus the amount of substrate required,and the respiratory gas exchanges associated with the synthesis.Glucose is taken as a standard substrate with the required amounttermed Glucose Equivalent (GE, mol mol^–1) or Glucose Value(GV, g g^–1). Comparisons of GV with Production Values(PV) calculated from biochemical pathways (Penning de Vries,Brunsting and van Laar, 1974) show that PV = 0.88 ±0.01GV. The glucose requirement also serves as a close predictorof the heat of combustion of the product. Biomass, elemental analysis, energy content, growth efficiency, yield, glucose equivalent     

Analysis of the maximum theoretical yield for the synthesis of erythromycin precursors in Escherichia coli

The heterologous biosynthesis of complex polyketides in Escherichia coli was recently achieved through metabolic engineering. However, it was observed that less than 10% of the propionate carbon source is transformed into the erythromycin precursor, 6-deoxyerythronolide B (6dEB), resulting in a 1.4% molar yield. Therefore, metabolic flux analysis was performed using a model of the Escherichia coli metabolism with the addition of the enzymes required for 6dEB synthesis. The analysis shows that the maximum theoretical yield for 6dEB synthesis in E. coli is 11%. The maintenance energy requirement of E. coli and limitations in the specific oxygen uptake rate can further decrease the yield, suggesting that the observed 6dEB yield of 1.4% can be the result of these two factors. In addition, the results suggest that an increase in the specific carbon and oxygen uptake rates will increase the yield of 6dEB. The use of glucose as an alternative carbon source was also evaluated using metabolic flux analysis and the results suggest that the choice of glucose as the carbon source will allow a small improvement in performance relative to a propionate-based process.     

Comprehensive analysis of metabolic sensitivity of 1,4-butanediol producing Escherichia coli toward substrate and oxygen availability

Nowadays, chemical production of 1,4-butanediol is supplemented by biotechnological processes using a genetically modified Escherichia coli strain, which is an industrial showcase of successful application of metabolic engineering. However, large scale bioprocess performance can be affected by presence of physical and chemical gradients in bioreactors which are a consequence of imperfect mixing and limited oxygen transfer. Hence, upscaling comes along with local and time dependent fluctuations of cultivation conditions. This study emphasizes on scale-up related effects of microbial 1,4-butanediol production by comprehensive bioprocess characterization in lab scale. Due to metabolic network constraints 1,4-butanediol formation takes place under oxygen limited microaerobic conditions, which can be hardly realized in large scale bioreactor. The purpose of this study was to assess the extent to which substrate and oxygen availability influence the productivity. It was found, that the substrate specific product yield and the production rate are higher under substrate excess than under substrate limitation. Furthermore, the level of oxygen supply within microaerobic conditions revealed strong effects on product and by-product formation. Under strong oxygen deprivation nearly 30% of the consumed carbon is converted into 1,4-butanediol, whereas an increase in oxygen supply results in 1,4-butanediol reduction of 77%. Strikingly, increasing oxygen availability leads to strong increase of main by-product acetate as well as doubled carbon dioxide formation. The study provides clear evidence that scale-up of microaerobic bioprocesses constitute a substantial challenge. Although oxygen is strictly required for product formation, the data give clear evidence that terms of anaerobic and especially aerobic conditions strongly interfere with 1,4-butanediol production.     

Constitutive synthesis of enzymes of the protocatechuate pathway and of the beta-ketoadipate uptake system in mutant strains of Pseudomonas putida.

Mutant Pseudomonas putida strains that produce constitutive levels of the beta-ketoadipate uptake system are selected by the sequential transfer of cultures between mineral growth media supplemented with the noninducing growth substrate succinate and growth media containing beta-ketoadipate as the sole carbon and energy source. The mutant strains also produce constitutively three catabolic enzymes that give rise to beta-ketoadipate from the metabolic precursor beta-carboxy-cis, cis-muconate, and thus a single regulatory gene appears to govern the expression of the enzymes as well as the uptake system. The three enzymes that convert beta-carboxy-cis, cis-muconate to beta-ketoadipate are induced to higher levels when the orgainisms are grown with p-hydroxybenzoate (a compound that is catabolized via beta-ketoadipate); the beta-ketoadipate uptake system is partially repressed when the cells are grwon at the expense of p-hydroxybenzoate. The transferase that acts upon beta-ketoadipate remains inducible in the constitutive mutant strains. Thus a minimum of three biosynthetic controls must be exerted over the expression of the five genes. Since the regulatory mutation does not alter the expression of the gene for the transferase, the physiological target of the selection procedure appears to be mutant strains that produce the uptake system constitutively. Levels of the uptake system are higher in uninduced constitutive mutant cultures than in induced cultures of the wild type. Hence procedures analogous to the one we employed may be of general use in obtaining mutant strains that produce high levels of uptake systems.     

Cloning of cellulose synthesis related genes from Acetobacter xylinum ATCC23769 and ATCC53582: comparison of cellulose synthetic ability between strains.

About 14.5 kb of DNA fragments from Acetobacter xylinum ATCC23769 and ATCC53582 were cloned, and their nucleotide sequences were determined. The sequenced DNA regions contained endo-beta-1,4-glucanase, cellulose complementing protein, cellulose synthase subunit AB, C, D and beta-glucosidase genes. The results from a homology search of deduced amino acid sequences between A. xylinum ATCC23769 and ATCC53582 showed that they were highly similar. However, the amount of cellulose production by ATCC53582 was 5 times larger than that of ATCC23769 during a 7-day incubation. In A. xylinum ATCC53582, synthesis of cellulose continued after glucose was consumed, suggesting that a metabolite of glucose, or a component of the medium other than glucose, may be a substrate of cellulose. On the other hand, cell growth of ATCC23769 was twice that of ATCC53582. Glucose is the energy source in A. xylinum as well as the substrate of cellulose synthesis, and the metabolic pathway of glucose in both strains may be different. These results suggest that the synthesis of cellulose and the growth of bacterial cells are contradictory.     

Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry

The substrate-carbon flow can be controlled in continuous bioreactor cultures by the medium composition, for example, by the C/N ratio. The carbon distribution is optimal when a maximum fraction flows into the desired product and the residual is just sufficient to compensate for the dilution of the microbial catalyst. Undershooting of the latter condition is reflected immediately by changes in the Gibbs energy dissipation and cellular states. Two calorimetric measurement principles were applied to optimize the continuous synthesis of polyhydroxybutyrate (PHB) by Variovorax paradoxus DSM4065 during growth with constantly increasing supply rates of fructose or toxic phenol. Firstly, the changed slope of the heat production rate in a complete heat balanced bioreactor (CHB) indicated optimum carbon channeling into PHB. The extent of the alteration depended directly on the toxic properties of the substrate. Secondly, a flow through calorimeter was connected with the bioreactor as a "measurement loop." The optimum substrate carbon distribution was indicated by a sudden change in the heat production rate independent of substrate toxicity. The sudden change was explained mathematically and exploited for the long-term control of phenol conversion into PHB. LASER flow cytometry measurements distinguished between subpopulations with completely different PHB-content. Populations grown on fructose preserved a constant ratio of two subpopulations with double and quadruple sets of DNA. Cells grown on phenol comprised a third subpopulation with a single DNA set. Rising phenol concentrations caused this subpopulation to increase. It may thus be considered as an indicator of chemostress.     

Redox rebalance against genetic perturbations and modulation of central carbon metabolism by the oxidative stress regulation

The micro-aerophilic organisms and aerobes as well as yeast and higher organisms have evolved to gain energy through respiration (via oxidative phosphorylation), thereby enabling them to grow much faster than anaerobes. However, during respiration, reactive oxygen species (ROSs) are inherently (inevitably) generated, and threaten the cell’s survival. Therefore, living organisms (or cells) must furnish the potent defense systems to keep such ROSs at harmless level, where the cofactor balance plays crucial roles. Namely, NADH is the source of energy generation (catabolism) in the respiratory chain reactions, through which ROSs are generated, while NADPH plays important roles not only for the cell synthesis (anabolism) but also for detoxifying ROSs. Therefore, the cell must rebalance the redox ratio by modulating the fluxes of the central carbon metabolism (CCM) by regulating the multi-level regulation machinery upon genetic perturbations and the change in the growth conditions.Here, we discuss about how aerobes accomplish such cofactor homeostasis against redox perturbations. In particular, we consider how single-gene mutants (including pgi, pfk, zwf, gnd and pyk mutants) modulate their metabolisms in relation to cofactor rebalance (and also by adaptive laboratory evolution). We also discuss about how the overproduction of NADPH (by the pathway gene mutation) can be utilized for the efficient production of useful value-added chemicals such as medicinal compounds, polyhydroxyalkanoates, and amino acids, all of which require NADPH in their synthetic pathways. We then discuss about the metabolic responses against oxidative stress, where αketoacids play important roles not only for the coordination between catabolism and anabolism, but also for detoxifying ROSs by non-enzymatic reactions, as well as for reducing the production of ROSs by repressing the activities of the TCA cycle and respiration (via carbon catabolite repression). Thus, we discuss about the mechanisms (basic strategies) that modulate the metabolism from respiration to respiro-fermentative metabolism causing overflow, based on the role of Pyk activity, affecting the NADPH production at the oxidative pentose phosphate (PP) pathway, and the roles of αketoacids for the change in the source of energy generation from the oxidative phosphorylation to the substrate level phosphorylation.     

Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L⁻¹ in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.     

Anaerobic microbial methanol conversion in marine sediments

Methanol is an ubiquitous compound that plays a role in microbial processes as a carbon and energy source, intermediate in metabolic processes or as end product in fermentation. In anoxic environments, methanol can act as the sole carbon and energy source for several guilds of microorganisms: sulfate-reducing microorganisms, nitrate-reducing microorganisms, acetogens and methanogens. In marine sediments, these guilds compete for methanol as their common substrate, employing different biochemical pathways. In this review, we will give an overview of current knowledge of the various ways in which methanol reaches marine sediments, the ecology of microorganisms capable of utilizing methanol and their metabolism. Furthermore, through a metagenomic analysis, we shed light on the unknown diversity of methanol utilizers in marine sediments which is yet to be explored.     

Growth of the photosynthetic bacterium Rhodopseudomonas capsulata chemoautotrophically in darkness with H2 as the energy source.

The phototrophic bacterium Rhodopseudomonas capsulata was found to be capable of growing chemoautotrophically under aerobic conditions in darkness. Growth was strictly dependent on the presence of H2 as the source of energy and reducing power, O2 as the terminal electron acceptor for energy transduction, and CO2 as the sole carbon source; under optimal conditions the generation time was about 6 h. Chemoautotrophically grown cells showed a relatively high content of bacteriochlorophyll a and intracytoplasmic membranes (chromatophores). Experiments with various mutants of R. capsulata, affected in electron transport, indicate that either of the two terminal oxidases of this bacterium can participate in the energy-yielding oxidation of H2. The ability of R. capsulata to multiply in at least five different physiological growth modes suggests that it is one of the most metabolically versatile procaryotes known.