Inorganic types of fermentation and anaerobic respirations in the evolution of energy-yielding metabolism

We proposed long ago the following sequence as one of the main pathways in the evolution of energy-yielding metabolism: fermentation→nitrate fermentation→nitrate respiration→oxygen respiration. In the present report our concept is presented in a more general form: (1) fermentation→ →(2) fermentation with H₂ release→(3) inorganic types of fermentation→(4) anaerobic respirations →(5) oxygen respiration, based upon recent biological and physical information. The energy-yielding efficiency increased gradually together with the evolution. (2) is characterized by the participation of ferredoxin, (3) by the establishment of electron transfer chain, and (4) by the participation of cytochrome and oxidative phosphorylation. The close relationship between the primary structure of ferredoxins of anaerobic bacteria and that of a cytochrome (cytochromec ₃) was demonstrated. It reveals that the transition from inorganic types of fermentation to anaerobic respirations was direct and accompanied by the transition from ferredoxins to cytochromes, and it further supports our concept that the cytochrome system, and consequently the oxidative phosphorylation, were induced at this evolutionary step. Our concept based upon biological observations is consistent with a physical theory recently proposed by M. Shimizu.     

The biochemical conversions of conjugated dienoic and trienoic fatty acids

The changes in phosphate metabolism induced in yeast by transition from fermentation to respiration have been studied. Orthophosphate added to respiring or fermenting yeast suspensions as Na₂HP³²O₄ is rapidly resorbed and incorporated into adenosine triphosphate (ATP) and other acid-labile fractions. During fermentation, the specific activity of the orthophosphate is higher than that of ATP. This is thought to be mainly due to a heterogeneity in the intracellular orthophosphate. In respiring yeast, pyrophosphate is formed. The specific activity of this pyrophosphate is very high when the cells are maintained from the start of the experiment under aerobic conditions. When respiration follows a prior period of fermentation lasting 30–60 min., an accumulation of lowly labeled pyrophosphate occurs. Concurrently an acidinsoluble phosphate fraction is mobilized. As indicated by labeling relations, this fraction may be an intermediary in the pathway between orthophosphate and pyrophosphate. The possible role of dinucleotides in primary aerobic phosphorylation is reviewed and it is shown that diphosphopyridine nucleotide (DPN) undergoes a temporary resynthesis in yeast during the first 5–6 hr. of respiration. The question whether this phenomenon may be regarded as a secondary consequence of an enzymatic adaptation which involves pyrophosphate accumulation is discussed.     

Ammonia fermentation, a novel anoxic metabolism of nitrate by fungi.

The induction of fungal denitrification by Fusarium oxysporum requires a minimal amount of O(2), although excess O(2) completely represses this process (Zhou, Z., Takaya, N., Sakairi, M. A. C., and Shoun, H. (2001) Arch. Microbiol. 175, 19-25). Here we describe another metabolic mechanism of nitrate in fungal cells, termed ammonia fermentation, that supports growth under conditions more anoxic than those of denitrification. The novel nitrate metabolism of eukaryotes consists of the reduction of nitrate to ammonium coupled with the catabolic oxidation of electron donors to acetate and substrate-level phosphorylation. F. oxysporum thus has two pathways of dissimilatory nitrate reduction that are alternatively expressed in response to environmental O(2) tension. F. oxysporum prefers O(2) respiration when the O(2) supply is sufficient. We discovered that this fungus is the first eukaryotic, facultative anaerobe known to express one of three distinct metabolic energy mechanisms closely depending on environmental O(2) tension. We also showed that ammonia fermentation occurs in many other fungi that are common in soil, suggesting that facultative anaerobes are widely distributed among fungi that have been considered aerobic organisms.     

Effect of inhibitors on acid production by baker’s yeast

Glucose-induced acid extrusion, respiration and anaerobic fermentation in baker’s yeast was studied with the aid of sixteen inhibitors. Uranyl(2+) nitrate affected the acid extrusion more anaerobically than aerobically; the complexing of Mg²⁺ and Ca²⁺ by EDTA at the membrane had no effect. Inhibitors of glycolysis (iodoacetamide, N-ethylmaleimide, fluoride) suppressed acid production markedly, and so did the phosphorylation-blocking arsenate. Fluoroacetate, inhibiting the citric-acid cycle, had no effect. Inhibition by uncouplers depended on their pK_a values: 2,4,6-trinitrophenol (pK_a 0.4) < 2,4-dinitrophenol (4.1) < azide (4.7) < 3-chlorophenylhydrazonomalononitrile (6.0). Inhibition by trinitrophenol was only slightly increased by its acetylation. Cyanide and nonpermeant oligomycin showed practically no effect; inhibition by dicyclohexylcarbodiimide was delayed but potent. The concentration profiles of inhibition of acid production differed from those of respiration and fermentation. Thus, though the acid production is a metabolically dependent process, it does not reflect the intensity of metabolism, except partly in the first half of glycolysis.     

Differential proteomic analysis of Bacillus subtilis nitrate respiration and fermentation in defined medium

A comparative investigation of protein expression by two-dimensional gel electrophoresis was conducted between Bacillus subtilis cultures grown in defined medium under aerobic, anaerobic nitrate respiration, or fermentation conditions. Defined medium specific for either nitrate respiration or fermentation allowed distinction between proteins induced by each individual growth process. Our differential protein profiling analysis between aerobic and anaerobic conditions showed that anaerobic fermentation induced at least 44 proteins and nitrate respiration induced at least 19 proteins compared to aerobic controls. Certain proteins were specifically induced during nitrate respiration or fermentation, while others were induced by both anaerobic processes. Eleven proteins induced by nitrate respiration and/or fermentation were identified by peptide mass matching using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Proteins encoded by feuA, hmp, and ytkD were induced by nitrate respiration. Proteins encoded by pyrR, sucD, trpC, and ywjH were induced by fermentation. Proteins encoded by acuB, pdhC, ydjL, and yvyD were induced by nitrate respiration and fermentation. This proteomic analysis has provided a more complete characterization of B. subtilis anaerobic growth and increased our understanding of its metabolic pathways of nitrate respiration and fermentation.     

Did respiration or photosynthesis come first?

The similarity of the mechanisms in photosynthetic and in oxidative phosphorylation suggests a common origin (conversion hypothesis). It is proposed that an early form of electron flow with oxidative phosphorylation (“prerespiration”), to therminal electron acceptors available in a reducing biosphere, was supplemented by a photocatalyst capable of a redox reaction. In this way, cyclic photophosphorylation arose. Further stages in evolution were reverse electron flow, powered by ATP, to make NADH as a reductant for CO₂, and subsequently noncyclic electron flow. These processes concomitantly provided the oxidants indispensable for full development of oxidative phosphorylation, i.e. for normal respiration: sulphate, O₂, and, with participation of the nitrificants, nitrite and nitrate. Thus prerespiration preceded photosynthesis, and this preceded respiration. It is also suggested that nonredox photoprocesses of the Halobacterium type are not part of the mainstream of bioenergetic evolution. They do not lead to photoprocesses with electron flow.     

Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth.

Bacillus subtilis can grow anaerobically by respiration with nitrate as a terminal electron acceptor. In the absence of external electron acceptors, it grows by fermentation. Identification of fermentation products by using in vivo nuclear magnetic resonance scans of whole cultures indicated that B. subtilis grows by mixed acid-butanediol fermentation but that no formate is produced. An ace mutant that lacks pyruvate dehydrogenase (PDH) activity was unable to grow anaerobically and produced hardly any fermentation product. These results suggest that PDH is involved in most or all acetyl coenzyme A production in B. subtilis under anaerobic conditions, unlike Escherichia coli, which uses pyruvate formate lyase. Nitrate respiration was previously shown to require the ResDE two-component signal transduction system and an anaerobic gene regulator, FNR. Also required are respiratory nitrate reductase, encoded by the narGHJI operon, and moaA, involved in biosynthesis of a molybdopterin cofactor of nitrate reductase. The resD and resDE mutations were shown to moderately affect fermentation, but nitrate reductase activity and fnr are dispensable for fermentative growth. A search for genes involved in fermentation indicated that ftsH is required, and is also needed to a lesser extent for nitrate respiration. These results show that nitrate respiration and fermentation of B. subtilis are governed by divergent regulatory pathways.     

Regulatory O2 tensions for the synthesis of fermentation products in Escherichia coli and relation to aerobic respiration

In an oxystat, the synthesis of the fermentation products formate, acetate, ethanol, lactate, and succinate of Escherichia coli was studied as a function of the O₂ tension (pO₂) in the medium. The pO₂ values that gave rise to half-maximal synthesis of the products (pO_0.5) were 0.2–0.4 mbar for ethanol, acetate, and succinate, and 1 mbar for formate. The pO_0.5 for the expression of the adhE gene encoding alcohol dehydrogenase was approximately 0.8 mbar. Thus, the pO₂ for the onset of fermentation was distinctly lower than that for anaerobic respiration (pO_0.5≤ 5 mbar), which was determined earlier. An essential role for quinol oxidase bd in microaerobic growth was demonstrated. A mutant deficient for quinol oxidase bd produced lactate as a fermentation product during growth at microoxic conditions (approximately 10 mbar O₂), in contrast to the wild-type or a quinol-oxidase-bo-deficient strain. In the presence of nitrate, the amount of lactate was largely decreased. Therefore, under microoxic conditions, the pO₂ appears to be too high for (mixed acid) fermentation to function and too low for aerobic respiration by quinol oxidase bo. Received: 7 February 1997 / Accepted: 2 May 1997     

Denitrification in human dental plaque

Background  

Microbial denitrification is not considered important in human-associated microbial communities. Accordingly, metabolic investigations of the microbial biofilm communities of human dental plaque have focused on aerobic respiration and acid fermentation of carbohydrates, even though it is known that the oral habitat is constantly exposed to nitrate (NO₃ ^-) concentrations in the millimolar range and that dental plaque houses bacteria that can reduce this NO₃ ^- to nitrite (NO₂ ^-).     

Behaviour of nodulatedPisum sativum L. under short-term nitrate stress conditions

Nitrate (20 mM) applied to the root medium of 28-day-old nodulated pea plants (Pisum sativum L., cv. Jupiter) immediately retarded nodule growth and inhibited root nodulation. Acetylene-reducing and H₂-evolving nitrogenase activities were also significantly inhibited. The inhibitory effect of nitrate on nodule respiration was less pronounced while the respiration of roots was increased after the addition of nitrate. The levels of cytosol glutamine synthetase and nitrate reductase in nodule cytosol were permanently decreased from the 4th day after nitrate application. These results indicate that the inhibitory effect of high nitrate concentration on whole nodule metabolism is nonspecific in nature.     

The effect of low concentrations of sodium nitrites and nitrates on respiration and oxidative phosphorylation in rat liver mitochondria]

Nitrite incubation in mitochondria and nitrate intoxication of rats have been studied for their effect on aerobic energetic processes in the liver. Sodium nitrite in concentration of 2 mg/l causes an inhibition of ADP-stimulated respiration and provides uncoupling processes of oxidative phosphorylation and respiration in mitochondria, when adding succinate as a substrate. Low doses of nitrate in vivo promote oxygen economization in mitochondria. Intoxication of rats with nitrate in a dose of 50 mg/l for 30 days induces a decrease of the respiration rate after ADP-phosphorylation and an increase of the coefficient of oxidative phosphorylation efficiency (ADP/O). Intraperitoneal administration of adrenalin in a dose of 25 micrograms/100 g to rats after 30-day nitrate intoxication in a concentration of 10 mg/l induces no typical increase of ADP-stimulated respiration and rate of oxidative phosphorylation and succinate oxidation.     

Anaerobic growth and potential for amino acid production by nitrate respiration in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O₂), while no visible colonies were formed in the absence of O₂. However, in the presence of nitrate (), the organism exhibited limited growth anaerobically with production of nitrite (), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using l-lysine- and l-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.     

Does Dietary Mitigation of Enteric Methane Production Affect Rumen Function and Animal Productivity in Dairy Cows?

It has been suggested that the rumen microbiome and rumen function might be disrupted if methane production in the rumen is decreased. Furthermore concerns have been voiced that geography and management might influence the underlying microbial population and hence the response of the rumen to mitigation strategies. Here we report the effect of the dietary additives: linseed oil and nitrate on methane emissions, rumen fermentation, and the rumen microbiome in two experiments from New Zealand (Dairy 1) and the UK (Dairy 2). Dairy 1 was a randomized block design with 18 multiparous lactating cows. Dairy 2 was a complete replicated 3 x 3 Latin Square using 6 rumen cannulated, lactating dairy cows. Treatments consisted of a control total mixed ration (TMR), supplementation with linseed oil (4% of feed DM) and supplementation with nitrate (2% of feed DM) in both experiments. Methane emissions were measured in open circuit respiration chambers and rumen samples were analyzed for rumen fermentation parameters and microbial population structure using qPCR and next generation sequencing (NGS). Supplementation with nitrate, but not linseed oil, decreased methane yield (g/kg DMI; P<0.02) and increased hydrogen (P<0.03) emissions in both experiments. Furthermore, the effect of nitrate on gaseous emissions was accompanied by an increased rumen acetate to propionate ratio and consistent changes in the rumen microbial populations including a decreased abundance of the main genus Prevotella and a decrease in archaeal mcrA (log₁₀ copies/ g rumen DM content). These results demonstrate that methane emissions can be significantly decreased with nitrate supplementation with only minor, but consistent, effects on the rumen microbial population and its function, with no evidence that the response to dietary additives differed due to geography and different underlying microbial populations.     

Anaerobic C–H Oxyfunctionalization: Coupling of Nitrate Reduction and Quinoline Hydroxylation in Recombinant Pseudomonas putida

Whole‐cell biocatalysis for C–H oxyfunctionalization depends on and is often limited by O₂ mass transfer. In contrast to oxygenases, molybdenum hydroxylases use water instead of O₂ as an oxygen donor and thus have the potential to relieve O₂ mass transfer limitations. Molybdenum hydroxylases may even allow anaerobic oxyfunctionalization when coupled to anaerobic respiration. To evaluate this option, the coupling of quinoline hydroxylation to denitrification is tested under anaerobic conditions employing Pseudomonas putida (P. putida) 86, capable of aerobic growth on quinoline. P. putida 86 reduces both nitrate and nitrite, but at low rates, which does not enable significant growth and quinoline hydroxylation. Introduction of the nitrate reductase from Pseudomonas aeruginosa enables considerable specific quinoline hydroxylation activity (6.9 U g_CDW^?1) under anaerobic conditions with nitrate as an electron acceptor and 2‐hydroxyquinoline as the sole product (further metabolization depends on O₂). Hydroxylation‐derived electrons are efficiently directed to nitrate, accounting for 38% of the respiratory activity. This study shows that molybdenum hydroxylase‐based whole‐cell biocatalysts enable completely anaerobic carbon oxyfunctionalization when coupled to alternative respiration schemes such as nitrate respiration.     

A defect in menadione biosynthesis induces global changes in gene expression in Staphylococcus aureus

Both the high-resolution two-dimensional protein gel electrophoresis technique and full-genome DNA microarrays were used for identification of Staphylococcus aureus genes whose expression was changed by a mutation in menD. Because the electron transport chain is interrupted, the mutant should be unable to use oxygen and nitrate as terminal electron acceptors. Consistent with this, a mutation in menD was found to cause a gene expression pattern typically detected under anaerobic conditions in wild-type cells: proteins involved in glycolytic as well as in fermentation pathways were upregulated, whereas tricarboxylic acid (TCA) cycle enzymes were significantly downregulated. Moreover, the expression of genes encoding enzymes for nitrate respiration and the arginine deiminase pathway was strongly increased in the mutant strain. These results indicate that the menD mutant, just as the site-directed S. aureus hemB mutant, generates ATP from glucose or fructose mainly by substrate phosphorylation and might be defective in utilizing a variety of carbon sources, including TCA cycle intermediates and compounds that generate ATP only via electron transport phosphorylation. Of particular interest is that there are also differences in the gene expression patterns between hemB and menD mutants. While some anaerobically active enzymes were present in equal amounts in both strains (Ldh1, SACOL2535), other classically anaerobic enzymes seem to be present in higher amounts either in the hemB mutant (e.g., PflB, Ald1, IlvA1) or in the menD mutant (arc operon). Only genes involved in nitrate respiration and the ald1 operon seem to be additionally regulated by a depletion of oxygen in the hemB and/or menD mutant.     

Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed-batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO₂ evolution rate, O₂ uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (δ_h) with the fraction of available electrons transferred to oxygen (?_O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ?_O2 than δ_h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.     

Effect of Oxygen and Malate on NO(3) Inhibition of Nitrogenase in Soybean Nodules

Soybean (Glycine max cv Hodgson) nitrogenase activity (C₂H₂ reduction) in the presence or absence of nitrate was studied at various external O₂ tensions. Nitrogenase activity increased with oxygen partial pressure up to 30 kilopascals, which appeared to be the optimum. A parallel increase in ATP/ADP ratios indicated a limitation of respiration rate by low O₂ tensions in the nodule, and the values found for adenine nucleotide ratios suggested that the nitrogenase activity was limited by the rate of ATP regeneration. In the presence of nitrate, the nitrogenase activity was low and less stimulated by increased pO₂, although the nitrite content per gram of nodules decreased from 0.05 to 0.02 micromole when pO₂ increased from 10 to 30 kilopascals. Therefore, the accumulation of nitrite inside the nodule was probably not the major cause of the inhibition. Instead, inhibition by nitrate could be due to competition for reducing power between nitrate reduction and bacteroid or mitochondrial respiration inside the nodule. This is supported by the observation of decrease in ATP/ADP ratios from 1.65, in absence of nitrate, to 0.93 in the presence of this anion at 30 kilopascals O₂. Furthermore, the inhibition was suppressed by the addition, to the plant nutrient solution, of 15 millimolar l-malate, a carbon substrate that is considered to be the major source of reductant for the bacteroids in the symbiosis.     

Long-term effect of linseed plus nitrate fed to dairy cows on enteric methane emission and nitrate and nitrite residuals in milk

A previous study showed the additive methane (CH₄)-mitigating effect of nitrate and linseed fed to non-lactating cows. Before practical application, the use of this new strategy in dairy cows requires further investigation in terms of persistency of methanogenesis reduction and absence of residuals in milk products. The objective of this experiment was to study the long-term effect of linseed plus nitrate on enteric CH₄ emission and performance in dairy cows. We also assessed the effect of this feeding strategy on the presence of nitrate residuals in milk products, total tract digestibility, nitrogen (N) balance and rumen fermentation. A total of 16 lactating Holstein cows were allocated to two groups in a randomised design conducted in parallel for 17 weeks. Diets were on a dry matter (DM) basis: (1) control (54% maize silage, 6% hay and 40% concentrate; CON) or (2) control plus 3.5% added fat from linseed and 1.8% nitrate (LIN+NIT). Diets were equivalent in terms of CP (16%), starch (28%) and NDF (33%), and were offered twice daily. Cows were fed ad libitum, except during weeks 5, 16 and 17 in which feed was restricted to 95% of dry matter intake (DMI) to ensure complete consumption of meals during measurement periods. Milk production and DMI were measured weekly. Nitrate and nitrite concentrations in milk and milk products were determined monthly. Daily CH₄ emission was quantified in open circuit respiration chambers (weeks 5 and 16). Total tract apparent digestibility, N balance and rumen fermentation parameters were determined in week 17. Daily DMI tended to be lower with LIN+NIT from week 4 to 16 (−5.1 kg/day on average). The LIN+NIT diet decreased milk production during 6 non-consecutive weeks (−2.5 kg/day on average). Nitrate or nitrite residuals were not detected in milk and associated products. The LIN+NIT diet reduced CH₄ emission to a similar extent at the beginning and end of the trial (−47%, g/day; −30%, g/kg DMI; −33%, g/kg fat- and protein-corrected milk, on average). Diets did not affect N efficiency and nutrients digestibility. In the rumen, LIN+NIT did not affect protozoa number but reduced total volatile fatty acid (−12%) and propionate (−31%) concentrations. We concluded that linseed plus nitrate may have a long-term CH₄-mitigating effect in dairy cows and that consuming milk products from cows fed nitrate may be safe in terms of nitrate and nitrite residuals. Further work is required to optimise the doses of linseed plus nitrate to avoid reduced cows performance.