The role of formate for the growth of Rhodococcus opacus UFZ B 408 on pyridine

R. opacus UFZ B 408 is able to use pyridine, a potentially growth-inhibiting substrate, as the sole source of carbon, energy and nitrogen. In a previous publication [1] we reported that with the simultaneous utilization of a second carbon and energy source in carbon-substrate-limited chemostat culture, stable steady states could be achieved at higher dilution rates than with growth on pyridine as the sole substrate. Owing to the higher growth yield during growth on such a substrate mixture, both the specific pyridine consumption rates and the residual pyridine concentrations were lower at similar dilution rates than with growth on pyridine alone. Therefore, the critical growth-inhibitory pyridine concentration was only achieved at a higher dilution rate. With the investigations presented here in carbon-substrate-limited continuous culture, the simultaneous utilization of pyridine and formate by R. opacus UFZ B 408 was studied. The yield coefficient during growth on pyridine as the sole substrate amounted to about 0.55 g dry mass/g pyridine. Theoretically, however, the carbon-metabolism-determined yield coefficient should have been about 0.915 g dry mass/g pyridine. Because of the difference between these two values the conclusion was drawn that pyridine is energetically deficient. That means that during growth on pyridine a part of the substrate was dissimilated to supply the energy required for the incorporation of the pyridine carbon into biomass. Formate cannot be used as a carbon source for growth by R. opacus UFZ B 408. However, with growth on pyridine, formate was oxidized simultaneously. During growth on pyridine/formate mixtures, the yield coefficient could be enhanced up to 0.7 g dry mass/g pyridine. That means that biologically usable energy, generated in the course of the formate oxidation, was used for the assimilation of pyridine carbon. The increase in the yield coefficient was related to the utilization ratio of formate to pyridine in a linear manner. However, the carbon-metabolism-determined yield coefficient of 0.915 g dry mass/g pyridine could not be achieved. That can be put down to the fact that R. opacus UFZ B 408 possesses only a limited capacity to oxidize externally supplied formate. Because of the limited formate oxidation capacity the probability is low that, with simultaneous utilization of formate, stable steady states could be achieved at substantially higher dilution rates than with growth on pyridine alone. Enzymatic studies revealed the induction of both NAD(P)⁺-linked glutaric dialdehyde dehydrogenase and isocitrate lyase during growth on pyridine. Therefore, the conclusion was drawn that pyridine is metabolized by R. opacus UFZ B 408 via the same pathway described for the utilization of pyridine by Nocardia Z1 [2]. This conclusion implies that the ability to oxidize formate represents a metabolic performance which seems not to be directly related to the pyridine metabolism of R. opacus UFZ B 408.     

Increase and limits of growth yields for heterotrophic microorganisms

Substrates for heterotrophic growth of microorganisms are more or less energy-limited. In order to improve the growth yields phenotypic and genetic measures come into consideration. By means of the auxiliary substrate concept the carbon metabolism determined carbon conversion efficiency of about 75% for C₂ compounds (i.e. non-glycolytic, gluconeogenetic substrates) and about 85%, respectively, for C₃ or glycolytic substrates can be attained experimentally. With methylotrophic microorganisms the growth yield might approach to this limit if the methanol oxidizing methanol dehydrogenase (bacteria) and methanol oxidase (yeasts) are replaced by an NAD⁺-linked dehydrogenase. Due to an increase in the overall P/O quotient up to the maximum this limit cannot be reached.     

The effect of mixtures of glucose and formate on the yield and respiration of a chemostat culture of Beneckea natriegens

Beneckea natriegens oxidizes sodium formate constitutively when grown on glucose or glycerol in chemostat culture, but cannot utilize formate as the sole source of carbon and energy for growth. However, when grown on a mixture of glucose and formate (D=0.37 h^-1, pH 7.6) the yield is higher than on glucose alone.The yield, expressed in terms of g bacterial dry weight g^-1 glucose plus formate carbon utilized, gave a linear relationship when plotted against the total heat of combustion of glucose plus formate utilized. Extrapolation of the plot cut the abscissa at a value equivalent to the heat of combustion of formate, which suggests that formate is not utilised as a source of carbon but only energy.In cultures with nitrate as the sole source of nitrogen the yield from glucose was lower than that observed with ammonia but the addition of formate to the culture utilizing nitrate resulted in an increase in the yield from glucose to a value similar to that observed with ammonia.At a culture pH value of 7.65 unused formate (<0.15–227 mM) in the culture supernatant had no effect on respiration spiration or yield, but at a culture pH of 6.7 excess formate caused a marked increase in respiration rate and a large decrease in the yield from glucose; further decrease in the pH value caused washout of the culture. This may be explained by undissociated formic acid causing uncoupling of oxidative phosphorylation.     

Energy and reducing equivalent potential of C2-compounds for microbial growth

Yeasts were chemostatically cultivated on C₂-substrates. Experimental yield figure were 0.77 g/g and 0.57 g/g on ethanol, 0.37 g/g and 0.29 g/g on acetate and ammonium and nitrate, respectively, as the nitrogen sources. Growth on hexadecane/ammonium yielded 0.94 g/g. Simultaneous utilization of formate lead to an increase of growth yield on acetate and hexadecane but not on ethanol. The experimental data were compared to theoretical figures. These results show that growth yield on ethanol/ammonium is carbon-limited, on ethanol/nitrate is NADPH-limited and on acetate with both nitrogen sources is energy-limited. With hexadecane/ammonium growth yield is energy-limited too. According to these results ethanol seems to be the only substrate from which an excess in energy can be obtained.     

Growth rate-dependent expression of phenol-assimilation pathways in Alcaligenes eutrophus JMP 134–the influence of formate as an auxiliary energy source on phenol conversion characteristics

 Alcaligenes eutrophus JMP 134 was continuously (carbon-source-limited) grown on phenol to determine the maximum growth rates (μ_max) as a function of the phenol assimilation pathways expressed. During growth on phenol as the sole source of carbon and energy, an almost exclusive expression of the ortho cleavage pathway (catechol 1,2-dioxygenase) was observed at initially low growth rates. This allowed a μ_max of 0.28 h^-1. The induction of the meta cleavage pathway (catechol 2,3-dioxygenase), which appeared at around 0.25 h^-1, resulted in a further increase in the growth rate to 0.40 h^-1 after the enzyme activities of this pathway had been correspondingly expressed. Hence, two maximum growth rates, one for the ortho and one for the meta cleavage pathway, exist for the growth of A. eutrophus JMP 134 on phenol. Growth on phenol was stimulated by formate, which served as an auxiliary energy source in this strain. The simultaneous utilization of phenol and formate at a molar ratio of 1:5.2 resulted in an increase of the yield coefficient from about 0.75 g dry mass/g phenol to 1.25 g/g. Furthermore, formate exerted a pronounced effect on the growth rate. At a molar ratio of phenol to formate of 1:4.2, the growth rate was increased to 0.42 h^-1, despite the exclusive induction of the ortho cleavage pathway. The meta cleavage pathway was expressed during growth on this substrate mixture at about 0.4 h^-1. However, this did not enable a significant increase of the growth rate beyond 0.4 h^-1. This is attributed to an exhaustion of the capacity for formate oxidation at this rate. The results are discussed with respect to energy production capabilities when phenol is assimilated as an energy-deficient heterotrophic substrate. Received: 13 November 1995/Received revision: 15 April 1996/Accepted: 22 April 1996     

Biochemical limits to microbial growth yields: An analysis of mixed substrate utilization

A theoretical analysis has been made of carbon conversion efficiency during heterotrophic microbial growth. The expectation was that the maximal growth yield occurs when all the substrate is assimilated and the net flow of carbon through dissimilation is zero. This, however, is not identical to a 100% carbon conversion, since assimilatory pathways lead to a net production of CO(2). It can be shown that the amount of CO(2) produced by way of assimilatory processes is dependent upon the nature of the carbon source, but independent of its degree of reduction and varies between 12 and 29% of the substrate carbon. An analysis of published yield data reveals that nearly complete assimilation can occur during growth on substrates with a high energy content. This holds for substrates with a heat of combustion of ca. 550 kJ/mol C, or a degree of reduction higher than 5 (e.g. ethane, ethanol, and methanol). Complete assimilation can also be achieved on substrates with a lower energy content, provided that an auxiliary energy source is present that cannot be used as a carbon source. This is evident from the cell yields reported for Candida utilis grown on glucose plus formate and for Thiobacillus versutus grown on acetate plus thiosulfate. This evaluation of the carbon conversion efficiency during assimilation also made it possible to compare the energy content of the auxiliary energy substrate added with the quantity of the carbon source it had replaced. It will be shown that utilization of the auxiliary energy source may lead to extreme changes in the efficiency of dissimilatory processes.     

Choice between autotrophy and heterotrophy in Pseudomonas oxalaticus. Growth in mixed substrates

1. The type of metabolism adopted by Pseudomonas oxalaticus during growth on a variety of carbon sources was studied. 2. The only substrate upon which autotrophic growth was observed is formate. 3. In mixtures of formate and those substrates upon which the organism can grow faster than on formate, e.g. succinate, lactate or citrate, heterotrophic metabolism results. 4. In mixtures of formate and those substrates upon which the organism can grow at a similar rate to that on formate, e.g. glycollate or glyoxylate, the predominant mode of metabolism adopted is heterotrophic utilization of the C₂ substrate coupled with oxidation of formate as ancillary energy source. 5. P. oxalaticus grows on oxalate 30% slower than on formate. In mixtures of formate and oxalate, the predominant mode of metabolism adopted is autotrophic utilization of formate coupled with oxidation of oxalate as ancillary energy source. 6. In mixtures of formate and those substrates upon which the organism grows at a much lower rate than on formate, e.g. glycerol and malonate, the predominant mode of metabolism adopted is autotrophic utilization of formate. 7. It is concluded that synthesis of the enzymes involved in autotrophic metabolism is controlled by a combination of induction and metabolite repression.     

The influence of carbon catabolism on the auxiliary substrate effect,Paper given at the Reinhardsbrunn Symposium “Phyusiology of Microbial Growth and Differentiation”, May 20–26, 1984

Although the carbon/energy ratios of heterotrophic substrates for microbial growth are different this is not reflected in biomass. Nevertheless the macromolecular composition of cells may vary in dependence on growth conditions this does hardly influence the elementary composition and the growth yield. The energy requirement for synthesis of biomass starting from a central precursor, e.g. phosphoglycerate, can be assumed to be constant, hence any differences in carbon conversion efficiency must be attributed to carbon catabolism up to this precursor. This sequence determines if and to what extent an auxiliary substrate effect is possible. However, one has also to consider changes of the P/O ratio due to simultaneous utilization of substratesd which may account for the increase in growth yield with Hansenula polymorpha growing on a methanol/glucose mixture.     

Regulation of autotrophic and heterotrophic metabolism in Pseudomonas oxalaticus OX1: Growth on mixtures of acetate and formate in continuous culture

Growth of Pseudomonas oxalaticus in carbon- and energy-limited continuous cultures with mixtures of acetate and formate resulted in the simultaneous utilization of both substrates at all dilution rates tested. During growth on these mixtures, acetate repressed the synthesis of ribulosebisphosphate carboxylase. The degree of this repression was dependent on the dilution rate and on the ratio of acetate and formate in the medium reservoir. At fixed acetate and formate concentrations in the inflowing medium of 30 and 100 mM, respectively, and dilution rates above 0.10h^-1, the severe repression of autotrophic enzymes resulted in a marked increase in bacterial dry weight compared to the growth yield of the organisms on the two substrates separately. Also, at these dilution rates a significant increase in isocitrate lyase activity was observed in the cells as compared to growth on acetate alone. This indicated that under these conditions more acetate was assimilated and less dissimilated since acetate was partly replaced by formate as the energy source. When formate was added to the reservoir of an acetate-limited culture (S_R=30 mM), derepression of RuBPCase synthesis was observed at formate concentrations of 50 mM and above. Below this concentration formate only served as an energy source for acetate assimilation; when its concentration was increased above 50 mM a progressively increasing contribution of carbon dioxide fixation to the total carbon assimilation was observed as the activity of RuBPCase in the cells increased. It is concluded that in Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic carbon dioxide fixation via the Calvin cycle is regulated by a repression/derepression mechanism.Abbreviations RuBPCase ribulosebisphosphate carboxylase - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - FDH formate dehydrogenase - S_R concentration of growth-limiting substrate in reservoir     

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

During glucose‐limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β‐lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady‐state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose‐limited chemostat cultures at the dilution rates (D) of both 0.03 h^?1 and 0.05 h^?1 are determined to evaluate the quantitative impact on the physiology of a high‐yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h^?1, while this is not the case at D = 0.05 h^?1. In addition, a higher cytosolic redox status (NADH/NAD⁺), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h^?1, which are virtually absent at D = 0.03 h^?1. In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose‐limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial‐scale scenario.     

Methanol and methylamine utilization result from mutational events in Thiosphaera pantotropha

With choline as carbon source Thiosphaera pantotropha GB17 grew with a doubling time (t_d) of 6 h. The cellular yield was 55.8 g dry cell weight per mol of choline, indicating that its methyl moieties were used for growth. However, T. pantotropha was unable to grow with methanol or with methylamine as carbon source. Mutants were isolated from liquid or from solid media able to grow with methanol (Mox⁺) as carbon or methylamine as nitrogen source (Mam⁺). The Mox⁺ mutant GB17M grew with a mean t_d of 11.7h and a growth yield of 8.9 g dry cell weight per mol of methanol. Diauxic growth of strain GB17M was observed with mixtures of pyruvate and methanol as substrates in batch culture. Methanol led to the formation of methanol dehydrogenase, formate dehydrogenase, ribulosebisphosphate carboxylase and of a soluble cytochrome c-551.5. Tn5-insertional mutants defective in the thiosulfate oxidizing enzyme system or in hydrogenase acquired the Mox⁺ phenotype. However, Tn5-insertional mutants defective in either a c-type cytochrome or the molybdenum cofactor did not mutate to the Mox⁺ phenotype, indicating common functions in thiosulfate and in methanol metabolism.     

Optimization of metabolic pathways for bioconversion of lignocellulose to ethanol through genetic engineering

The optimization of metabolic pathways is of fundamental importance for strategies aimed at improving the economics and yield of the lignocellulose-to-ethanol processes. Although Escherichia coli is capable of metabolizing a wide variety of substrates including hexoses and pentoses, its hexose metabolism is inferior to that of Zymomonas mobilis, an obligate, ethanologenic bacterium. We therefore inserted and expressed Z. mobilis genes encoding essential enzymes involved in the fermentation pathway, alcohol dehydrogenase II (adh II) and pyruvate decarboxylase (pdc), into E. coli, resulting in increased cell growth and ethanol production. Ethanol concentrations of > 30 g/L were obtained on 10% glucose. Additionally, since pyruvate is mainly assimilated through pyruvate formate lyase (pfl) and forms formic acid and acetyl coenzyme A, metabolic redirection was attempted through gene knockout by Red-mediated recombination to decrease the byproducts of pyruvate metabolism. Under microaerobic conditions, pflA- and pflB-mutants produced more ethanol (163% and 207%, respectively) relative to the parent strain, using glucose as a carbon source.     

Circular Dichroism of the Polyoximic Acid Moiety in Polyoxins

Incubation of alkyl aryl sulfides with growing cells of Corynebacterium equi IFO 3730 afforded the corresponding sulfoxides and sulfones. The selectivity for the formation of sulfoxides over sulfones was higher with sulfides which have shorter alkyl chains. When methyl sulfides were used as substrates, formation of the corresponding sulfones was completely suppressed. Sulfoxides were revealed to have 87 ~ 100% optical purities as to the (R) absolute configuration by HPLC analysis. The culture conditions seriously influenced the yield of oxidation products. High conversion of sulfides was attained when the reaction was carried out with growing cells in the logarithmic phase, in medium containing hexadecane as the carbon and energy source.     

Acetogenium kivui,a new thermophilic hydrogen-oxidizing acetogenic bacterium

Hydrogen-oxidizing acetogenic bacteria in pure culture are presently represented by the two mesophilic species, Acetobacterium woodii and Clostridium aceticum. From Lake Kivu we have isolated a Gram negative, chemolithotrophic, thermophilic anaerobe (LKT-1) that oxidizes hydrogen and reduces carbon dioxide to acetic acid. It is a non-motile, non-sporeforming rod, about 0.7m in width and 2–7.5m in length, often occuring in pairs or chains. The cell wall has a banded appearance; the surface layer contains a regular array of particles with six-fold rotational symmetry. No outer membrane is present. The temperature optimum for growth is 66°C, and the pH optimum is 6.4. Organic growth substrates include glucose, mannose, fructose, pyruvate, and formate; acetate is the principal product. The doubling time for growth on hydrogen and carbon dioxide is about 2h. Vitamins are neither required nor stimulatory. Yeast extract and Trypticase enhance the final yield but do not affect the growth rate. Cysteine or sulfide are required and cannot be replaced by thioglycolate or dithiothreitol. LKT-1 was mass cultured on hydrogen and carbon dioxide in a 24.1 fermentor with a yield of 34g (wet weight) of cells. The DNA base composition as determined by buoyant density is 38 mol % guanine plus cytosine. LKT-1 appears only distantly related to physiologically similar bacteria. A new genus Acetogenium is proposed, and the species is Acetogenium kivui.     

Formate as an Auxiliary Substrate for Glucose-Limited Cultivation of Penicillium chrysogenum: Impact on Penicillin G Production and Biomass Yield

Production of β-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol·mol⁻¹, an increasing rate of formate oxidation via a cytosolic NAD⁺-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol·mol⁻¹, the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as β-lactams.     

Respiratory activity of Candida tropicalis during growth on hexadecane and on glucose

Summary Rates of oxygen uptake and the oxygen demand during growth of Candida tropicalis on hexadecane and glucose were determined in batch experiments. Oxygen demand was 2.5 fold higher for the synthesis of one unit of cell mass from hydrocarbon than from glucose. On the other hand specific respiration is of the same order of magnitude for both substrates, e.g. 12 mmoles O₂xh^-1xg^-1 (dry weight) and seems to be a constant of this organism. Higher rates of oxygen supply into the medium had no effect on the specific rates of respiration. Specific growth rates on hexadecane were 2.4 times lower than on glucose. It is concluded, that rates of synthesis of cell components are controlled by the overall capacity of the respiratory pathways.     

硝酸盐对牛粪梭菌甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响

【背景】异于同型产乙酸菌通常利用Wood-Ljungdahl途径将2分子CO₂还原为1分子乙酰辅酶A,Clostridium bovifaecis缺失Wood-Ljungdahl途径甲基支路第1步将CO₂还原为甲酸的甲酸脱氢酶,需甲酸存在时将1分子甲酸和1分子CO₂还原为乙酰辅酶A发生葡萄糖的同型产乙酸型发酵。已有报道显示,硝酸盐也可作为同型产乙酸菌的电子受体,而且对不同同型产乙酸菌的代谢影响有所不同,然而硝酸盐对这种独特的甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响尚不清楚。【目的】探究硝酸盐对C.bovifaecis甲酸脱氢酶缺失型Wood-Ljungdahl途径固碳的影响。【方法】硝酸盐浓度分别为10 mmol/L和30 mmol/L时,以未添加硝酸盐为对照实验,研究C.bovifaecis在葡萄糖+甲酸+CO₂为基质条件下的细菌生长、底物消耗和产物生成情况。【结果】10 mmol/L和30 mmol/L硝酸盐存在时,主要产物乙醇浓度分别为5.80 mmol/L和1.66 mmo...     

Correlation between cell composition and carbon conversion efficiency in microbial growth: a theoretical study

Summary As the macromolecular composition of microorganisms varies during their life cycle it was asked whether, and to what extent such changes exert any influence on substrate consumption, i.e. growth yield and carbon conversion efficiency, respectively. This question was dealt with in a theoretical study by use of the Y _APT ^max -concept. The growth substrates considered were methanol, acetate and glucose; the latter was assumed to be assimilated via both the glycolytic and the oxidative hexosemonophosphate pathway. Five fictitious biomasses were used which were altered in their proportion of polysaccharides, proteins, lipids, RNA and DNA. As a result, only small variations in the individual biomass formulae were obtained. On the basis of the energy balances for the syntheses of all cell constituents it was found that variations in the macromolecular composition of microbial biomass have only a slight effect on carbon conversion efficiency, amounting to maximally 3%. From the material balances it could be calculated that the upper, solely metabolism-determined limit of carbon conversion efficiency is 85% for substrates assimilated glycolytically via phosphoglycerate; for gluconeogenetic substrates, the upper limit was 75%. These limits are essentially determined by carbon loss on the way to amino acid syntheses.Abbreviations Ac acetate - CCE carbon conversion efficiency (%) - EMP Embden-Meyerhof-Parnas (glycolytic) pathway - Gluc glucose - HMP oxidative hexosemonophosphate pathway - m _e maintenance coefficient (mmol g^-1 h^-1) - MeOH methanol - PGA phosphoglycerate, Y, growth yield (g dry weight per g substrate) - Y _ATP growth yield (g dry weight per mole ATP) - specific growth rate (h^-1)     

Growth yield increase linked to caffeate reduction in Acetobacterium woodii

Growth yields were determined with Acetobacterium woodii strain NZva 16 on hydrogen and CO₂, formate, methanol, vanillate, ferulate and fructose in mineral medium in the absence and presence of 0.05% yeast extract. Yeast extract was not essential for growth but enhanced growth yields by 25–100% depending on the substrate fermented. Comparison of yields on formate or methanol allowed calculation of an energy yield in the range of 1.5–2 mol ATP per mol acetate formed during homoacetate fermentation of A. woodii. In the presence of 6 mM caffeate, growth yields were determined with the substrates formate or methanol. Caffeate was reduced to hydrocaffeate and increased growth yields were obtained. An ATP yield of about 1 mol per mol of caffeate reduced was calculated. Cytochromes were not detectable in cell free extracts or membrane preparations.     

Anaerobic energy metabolism of the sulfur-reducing bacterium “Spirillum” 5175 during dissimilatory nitrate reduction to ammonia

In a batch culture experiment the microaerophilic Campylobacter-like bacterium “Spirillum” 5175 derived its energy for growth from the reduction of nitrate to nitrite and nitrite to ammonia. Hereby, formate served as electron donor, acetate as carbon source, and l-cysteine as sulfur source. Nitrite was quantitatively accumulated in the medium during the reduction of nitrate; reduction of nitrite began only after nitrate was exhausted from the medium. The molar growth yield per mol formate consumed, Y_m, was 2.4g/mol for the reduction of nitrate to nitrite and 2.0 g/mol for the conversion of nitrite to ammonia. The gain of ATP per mol of oxidized formate was 20% higher for the reduction of nitrate to nitrite, compared to the reduction of nitrite to ammonia. With succinate as carbon source and nitrite as electron acceptor, Y_m was 3.2g/mol formate, i.e. 60% higher than with acetate as carbon source. No significant amount of nitrous oxide or dinitrogen was produced during growth with nitrate or nitrite both in the presence or absence of acetylene. No growth on nitrous oxide was found. The hexaheme c nitrite reductase of “Spirillum” 5175 was an inducible enzyme. It was present in cells cultivated with nitrate or nitrite as electron acceptor. It was absent in cells grown with fumarate, but appeared in high concentration in “Spirillum” 5175 grown on elemental sulfur. Furthermore, the dissimilatory enzymes nitrate reductase and hexaheme c nitrite reductase were localized in the periplasmic part of the cytoplasmic membrane.