Microbial degradation of formaldehyde in white mineral dispersions preserved with formaldehyde-releasing biocides

Two different formaldehyde-degrading microorganisms, Pseudomonas putida and Methylobacterium extorquens, were isolated from calcium carbonate slurry containing the formaldehyde-releasing biocide (ethylenedioxy) dimethanol. Their relative formaldehyde biodegradation and formic acid production kinetics were studied in broth and in calcium carbonate slurry for each microorganism individually, as well as in mixed cultures. Furthermore, the minimal inhibition concentration (MIC) was determined. The results indicated that in slurry, M. extorquens is more tolerant of formaldehyde than P. putida. In slurry, microbial-induced oxidation of formaldehyde caused a temporary accumulation of formic acid, which is presumed to be responsible for pH drop and destabilisation of the calcium carbonate slurry suspension systems. In addition, the residual formaldehyde concentration was observed to drive dominance and recovery of individual formaldehyde-resistant microorganisms in the slurry. Overall, this investigation indicated that biodegradation of formaldehyde in calcium carbonate slurry is brought about by alternating dominance of bacterial genera of mixed formaldehyde-resistant microbial populations.     

Conversion of methanol to formic acid through the coupling of the enzyme reactions of alcohol oxidase,catalase and formaldehyde dismutase

Summary A novel process for the production of formic acid from methanol has been developed that involves the coupled reactions of the three enzymes, alcohol oxidase, catalase and formaldehyde dismutase. In this process, methanol is oxidized to formaldehyde by alcohol oxidase and catalase, followed by the formaldehyde dismutase reaction that leads to the formation of methanol and formic acid. Ultimately, the substrate methanol (100 to 200 mM) is completely converted to formic acid, by the recycling of the consecutive enzyme reactions.     

Methanol Inhibition in Continuous Culture of Hansenula polymorpha

Growth inhibition of Hansenula polymorpha DL-1 by methanol, formaldehyde, formate, and formic acid was examined to determine the causes of unstable behavior observed during continuous cultures on methanol. The much greater inhibition of growth by formic acid than by formate and the effect of formic acid excretion and assimilation on pH helped to explain culture dynamics observed after transitory oxygen limitations. Oxygen limitation caused by temporary reduction of agitation in a continuous fermentation caused methanol to accumulate to inhibitory concentrations. Immediately after resumption of agitation, formic acid was produced and caused culture inhibition. To ensure the stability of H. polymorpha in continuous culture, it was therefore necessary to prevent transient methanol accumulation.     

Methanol metabolism in the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae)

Plants produce and release large quantities of methanol, especially when attacked by herbivores. It seems that the herbivores may suffer from methanol intoxication. Here we reported the tolerance to and the metabolism of methanol by Ostrinia furnacalis third-instar larvae. When larvae were exposed to dietary methanol, formaldehyde and formic acid for 72 h, the estimated LC₅₀ value was 28, 40 and 29 mg/g diet, respectively. Toxicity of methanol was enhanced by 4-methylpyrazole, 3-amino-1,2,4-triazole and piperonyl butoxide, and toxicity of formaldehyde was increased by 3-amino-1,2,4-triazole and piperonyl butoxide. However, triphenyl phosphate had little synergistic effects on both methanol and formaldehyde. These data indicate that alcohol dehydrogenase, and probably catalase and cytochrome P450 monooxygenase oxidize methanol to formaldehyde, catalase and cytochrome P450 monooxygenase catalyze formaldehyde to formic acid, water and carbon dioxide, and carboxylesterase may have a minor effect. Several fatty acid methyl esters (FAMEs) were identified from extracts of the frass of larvae which had been exposed to a methanol-contained diet, in contrast to those on a methanol-free artificial diet. In vitro tests revealed that a crude enzyme solution from the larvae could synthesize FAMEs from corresponding fatty acids and methanol. In addition, dietary methanol induced higher esterase activities in the first-, second- and third-instar larvae. These findings demonstrate that both oxidative metabolism and non-oxidative metabolism are partially responsible for methanol elimination in O. furnacalis larvae.     

Biodegradation of high concentrations of formaldehyde using Escherichia coli expressing the formaldehyde dismutase gene of Methylobacterium sp. FD1

In the present study, formaldehyde dismutase from Methylobacterium sp. FD1 was partially purified and analyzed by nanoLC–MS/MS; it was then cloned from the genomic DNA of FD1 by PCR. The open reading frame of the formaldehyde dismutase gene of FD1 was estimated to be 1203 bp in length. The molecular weight and pI of formaldehyde dismutase (401 aa), as deduced from the FD1 gene, were calculated at 42,877.32 and 6.56, respectively. NAD(H)-binding residues and zinc-binding residues were found in the amino acid sequence of the deduced formaldehyde dismutase of FD1 by BLAST search. The resting Escherichia coli cells that were transformed with the FD1 formaldehyde dismutase gene degraded high concentrations of formaldehyde and produced formic acid and methanol that were molar equivalents of one-half of the degraded formaldehyde. The lyophilized cells of the recombinant E. coli also degraded high concentrations of formaldehyde.     

Dismutation and cross-dismutation of aldehydes, and alcohol: aldehyde oxidoreduction by resting-cells of Pseudomonas putida F61-a

Pseudomonas putida F61-a defective in formaldehyde dehydrogenase was derived from the parent strain (F61). The bacterial strain grown on a nutrient broth supplemented with 1% glucose exhibited high formaldehyde dismutase activity. The dismutation and cross-dismutation of aldehydes occurred stoichiometrically in the resting-cells reaction. Many kinds of aliphatic and aromatic aldehydes that are scarcely soluble in water were utilized in these reactions as well as soluble ones. Formaldehyde at an extremely high concentration (0.5 M) was almost completely converted to equimolar amounts of methanol and formic acid by the resting-cells, which could be used three times without a loss of activity. The cross-dismutation between acrolein and formaldehyde occurred efficiently in the resting-cells reaction with 0.1 M each substrate. The alcohol: aldehyde oxidoreduction of the short-chain substrates was also shown by the resting-cells of a mutant (M6) unable to grow on n-propanol.     

Biodegradation of high concentrations of formaldehyde by lyophilized cells of Methylobacterium sp. FD1

In the present study, Methylobacterium sp. FD1 utilizing formaldehyde was isolated from soil. The resting cells of FD1 degraded high concentrations of formaldehyde (~2.7 M) and produced formic acid and methanol that were molar equivalents of one-half of the degraded formaldehyde. This result suggests that formaldehyde degradation by FD1 is caused by formaldehyde dismutase. The optimal temperature and pH for formaldehyde degradation by the resting cells of FD1 were 40 °C and 5–7, respectively. The lyophilized cells of FD1 also degraded high concentrations of formaldehyde. The formaldehyde degradation activity of the lyophilized cells was maintained as the initial activity at 25 °C for 287 days. These results suggest that the lyophilized cells of FD1 are useful as formaldehyde degradation materials.     

Reactions of direct formaldehyde oxidation to CO2 are non-essential for energy supply of yeast methylotrophic growth

Mutants of the methylotrophic yeast Hansenula polymorpha deficient in NAD-dependent formaldehyde or formate dehydrogenases have been isolated. They were more sensitive for exogenous methanol but retained the ability for methylotrophic growth. In the medium with methanol the growth yields of the mutant 356–83 deficient in formaldehyde dehydrogenase and of the wild-type strain were identical (0.34 g cells/g methanol) under chemostat cultivation. These results indicate that enzymes of direct formaldehyde oxidation are not indispensable for methylotrophic growth. At the same time inhibition of tricarboxylic acid cycle has resulted in suppression of growth in the media with multicarbon nonfermentable substrates such as glycerol, succinate, ethanol and dihydroxyacetone as well as with methanol, but not with glucose. In the experiments with the wild-type strain H. polymorpha it has been shown that citrate and dihydroxyacetone inhibit the radioactivity incorporation from ¹⁴C-methanol into CO₂. All obtained data indicate that for the dissimilation of methanol and the supplying of energy for methylotrophic growth, the functioning of tricarboxylic acid cycle reactions as oppossed to those of direct formaldehyde oxidation is essential.     

Oxidation of methanol,formaldehyde and formic acid by methanol-utilizing yeast

Methanol-utilizing yeast,Candida boidini 11 Bh, characterized by high tolerance to methanol during growth, displays even higher tolerance when the oxidation rate by intact cells is tested. Low respiration activity is found even at 22% v/v of methanol. The half-saturation constant was 17–18mM. The half-saturation constants for the two oxidation intermediates, formaldehyde and formic acid were 3.6–4.0 and 30–33mM, respectively. When applied together with standard concentration of methanol, very low concentrations of both intermediates stimulated the oxidation rate. These results are discussed in connection with the relationship between growth and oxidation, the tolerance to high concentrations of inhibitory products and the mechanism of inhibition.     

Formaldehyde and methanol biodegradation with the methylotrophic yeast Hansenula polymorpha. An application to real wastewater treatment

The application of methylotrophic yeast Hansenula polymorpha to the treatment of methanol and formaldehyde-containing wastewater was experimentally verified. Avariety of real wastewater samples originating from chemical industry effluent were examined. The yeast cell culture could grow in the wastewater environment, revealing low trophic requirements and a very high adaptation potential to poor cultivation conditions.The proliferation of cells was accompanied by a concomitant xenobiotic biodegradation. Grown, preadapted cellular suspension at a density of about 1 × 10⁷ cells/ml proved to be able to utilize formaldehyde present in wastewater at concentrations up to1750 mg/l, levels toxic to most microorganisms. The biological waste treatment method presented shows the enhanced potential by means of specific enzymatic activities of monocarbonic compound oxidations through methylotrophic pathway reactions. The need to obtain mutants highly resistant to formaldehyde has also been rationalized.     

The linear oxidation of formaldehyde to CO2 as the proper energy generating sequence for the assimilation of methanol in Acetobacter methanolicus MB58

Acetobacter methanolicus MB58 can grow on methanol. Since this substrate exhibits to be energy deficient there must be a chance to oxidize methanol to CO₂ merely for purpose of energy generation. For the assimilation of methanol the FBP variant of the RuMP pathway is used. Hence methanol can be oxidized cyclically via 6-phosphogluconate. Since Acetobacter methanolicus MB58 possesses all enzymes for a linear oxidation via formate the question arises which of both sequences is responsible for generation of the energy required. In order to clarify this the linear sequence was blocked by inhibiting the formate dehydrogenase with hypophosphite and by mutagenesis inducing mutants defective in formaldehyde or formate dehydrogenase. It has been shown that the linear dissimilatory sequence is indispensable for methylotrophic growth. Although the cyclic oxidation of formaldehyde to CO₂ has not been influenced by hypophosphite and with mutants both the wild type and the formaldehyde dehydrogenase defect mutants cannot grown on methanol. The cyclic oxidation of formaldehyde does not seem to be coupled to a sufficient energy generation, probably it operates only detoxifying and provides reducing equivalents for syntheses. The regulation between assimilation and dissimilation of formaldehyde in Acetobacter methanolicus MB58 is discussed.Abbreviations ATP Adenosine-5-triphosphate - DCPIP 2,6-dichlorphenolindophenol - DW dry weight - ETP electron transport phosphorylation - FBP fructose-1,6-bisphosphate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PMS phenazine methosulfate - RuMP ribulose monophosphate - Ru5P ribulose-5-phosphate - SDS sodiumdodecylsulphate - TCA tricarboxylic acid - TYB toluylene blue Dedicated to Prof. Dr. Dr. S. M. Rapoport on occasion of his 75th birthday     

The synthesis of amino acids and sugars on an inorganic template from constituents of the prebiotic atmosphere

Inelastic Electron Tunnelling Spectroscopy (IETS) has been used to identify the reaction products present on an alumina surface when it is exposed to likely components of the earth's prebiotic atmosphere. The alumina barrier of Al-AlO _x -Pb tunnelling junctions have been exposed to water; aqueous ammonia; wet carbon monoxide gas and to aqueous formaldehyde vapour under normal atmospheric conditions at room temperature. The water spectrum shows strong coincidence with that of a genuine sample of formic acid. It is proposed that atmospheric CO₂ is involved in this surface catalyzed reaction. The aqueous ammonia spectrum is assigned as an amino acid species produced from ammonia, water and atmospheric carbon dioxide. This spectrum compares very closely with the tunnelling spectrum of a genuine sample of glycine. The wet carbon monoxide spectrum and the aqueous formaldehyde spectrum have been produced by an infusion doping process. These spectra of CO and aqueous formaldehyde are assigned as a sugar like polymer or a sugar formed on the alumina surface. A tunnelling spectrum of D(–) fructose has been produced to aid this assignment. The role of an inorganic template such as alumina in the original prebiotic synthesis of amino acids and sugars is considered.     

Mikrobielle Verwertung von Methanol

Summary The oxidation of formaldehyde to carbon dioxide in cell-free extracts of methanol-grown Candida boidinii has been investigated. A specific NAD-dependent formaldehyde dehydrogenase requiring reduced glutathione has been partially purified. Furthermore, a NAD-linked formate dehydrogenase was found in cell-free extracts. The synthesis of these two enzymes is induced by methanol and repressed by glucose. The possible significance of these enzymes in the energy-generating system is discussed.     

Negative chemotaxis inSpirochaeta aurantia

A repellent-gradient tube assay for negative chemotaxis inSpirochaeta aurantia was developed and used to demonstrate that acids, alcohols, and sulfide were effective chemorepellents. The threshold concentrations (the lowest concentration of a repellent that elicited a detectable response) for benzoic acid, salicylic acid, and butyric acid were 3×10^–5 M. For acetic acid, propionic acid,p-aminobenzoic acid, propanol, butanol, and sulfide, threshold concentrations were 10^–3 to 10^–4 M. For formic acid, glyoxylic acid, glycolic acid, lactic acid, malonic acid, succinic acid, fumaric acid, methanol, ethanol, ethanediol, and propanediol, threshold concentrations were 10^–2 to 10^–3 M. Compounds such as methylamine, ethanolamine, formaldehyde, benzene, toluene, phenol, indol, nickel, and various amino acids did not elicit a repellent response. The results of competition experiments suggest that the repellents identified are recognized by three distinct receptors: a weak acid receptor, an alcohol receptor, and a sulfide receptor. The repellent responses to weak acids were maximal at pH 5.5 and decreased with increasing pH, whereas the response to propanol was unaffected by pH over a range of 5.5–8.0. The demonstration of negative chemotaxis inS. aurantia and the identification of distinct classes of repellents will allow further experimentation directed at understanding chemosensory mechanisms in spirochetes.     

C<Subscript>1</Subscript> compounds as auxiliary substrate for engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C₁ compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host.     

Comments on the purported generation of formaldehyde and adduct formation from the sweetener aspartame.

A recent paper by Trocho et al. (1) describes experiments meant to show that formaldehyde adducts are formed when rats are administered the sweetener aspartame. These authors assume that the methanol carbon of aspartame generates formaldehyde which then forms adducts with protein, DNA, and RNA. Doses employed range widely. In this letter, studies which have been published previously and which were not cited by these authors are reviewed in order to put into perspective the disposition of methanol and formaldehyde in monkeys and humans, species relevant to the toxicity of methanol and its toxic metabolite, formic acid.     

Chemoselective Deprotection of Triethylsilyl Ethers

An efficient and selective method was developed for the deprotection of triethylsilyl (TES) ethers using formic acid in methanol (5–10%) or in methylene chloride 2–5%) with excellent yields. TES ethers are selectively deprotected to the corresponding alcohols in high yields using formic acid in methanol under mild reaction conditions. Other hydroxyl protecting groups like t-butyldimethylsilyl (TBDMS) remain unaffected.     

Studies on methanol — oxidizing yeast

Oxidation of methanol, formaldehyde and formic acid was studied in cells and cell-free extract of the yeast Candida boidinii No. 11Bh. Methanol oxidase, an enzyme oxidizing methanol to formaldehyde, was formed inducibly after the addition of methanol to yeast cells. The oxidation of methanol by cell-free extract was dependent on the presence of oxygen and independent of any addition of nicotine-amide nucleotides. Temperature optimum for the oxidation of methanol to formaldehyde was 35 degrees C, pH optimum was 8.5. The Km for methanol was 0.8mM. The cell-free extract exhibited a broad substrate specificity towards primary alcohols (C1--C6). The activity of methanol oxidase was not inhibited by 1mM KCN, EDTA or monoiodoacetic acid. The strongest inhibitory action was exerted by p-chloromercuribenzoate. Both the cells and the cell-free extract contained catalase which participated in the oxidation of methanol to formaldehyde; the enzyme was constitutively formed by the yeast. The pH optimum for the degradation of H2O2 was in the same range as the optimum for methanol oxidation, viz. at 8.5. Catalase was more resistant to high pH than methanol oxidase. The cell-free extract contained also GSH-dependent NAD-formaldehyde dehydrogenase with Km = 0.29mM and NAD-formate dehydrogenase with Km = 55mM.     

Effects of formaldehyde on mitochondrial dysfunction and apoptosis in SK-N-SH neuroblastoma cells

Methanol ingestion is neurotoxic in humans due to its metabolites, formaldehyde and formic acid. Here, we compared the cytotoxicity of methanol and its metabolites on different types of cells. While methanol and formic acid did not affect the viability of the cells, formaldehyde (200–800 μg/mL) was strongly cytotoxic in all cell types tested. We investigated the effects of formaldehyde on oxidative stress, mitochondrial respiratory functions, and apoptosis on the sensitive neuronal SK-N-SH cells. Oxidative stress was induced after 2 h of formaldehyde exposure. Formaldehyde at a concentration of 400 μg/mL for 12 h of treatment greatly reduced cellular adenosine triphosphate (ATP) levels. Confocal microscopy indicated that the mitochondrial membrane potential (MMP) was dose-dependently reduced by formaldehyde. A marked and dose-dependent inhibition of mitochondrial respiratory enzymes, viz., NADH dehydrogenase (complex I), cytochrome c oxidase (complex IV), and oxidative stress-sensitive aconitase was also detected following treatment with formaldehyde. Furthermore, formaldehyde caused a concentration-dependent increase in nuclear fragmentation and in the activities of the apoptosis-initiator caspase-9 and apoptosis-effector caspase-3/-7, indicating apoptosis progression. Our data suggests that formaldehyde exerts strong cytotoxicity, at least in part, by inducing oxidative stress, mitochondrial dysfunction, and eventually apoptosis. Changes in mitochondrial respiratory function and oxidative stress by formaldehyde may therefore be critical in methanol-induced toxicity.