Maintenance requirements for bacteria growing on C1-compounds

The maintenance coefficient, ms (mmol substrate/g cell dry wt hr), of two distinct groups of C1-utilizing bacteria has been determined by growing the organisms in an aerobic continuous culture limited by different C1 growth substrates. For growth on methanol, ms = 2.5 +/- 0.3 for Pseudomonas C; 3.9 +/- 0.7 for Ps. methylotropha (these bacteria utilize methanol via the ribulose monophosphate pathway of formaldehyde fixation); 1.5 +/- 0.2 for Pseudomonas 1, and 2.3 +/- 0.4 for Pseudomonas 135 (the latter bacteria utilize C1-compounds via the serine pathway). For growth on formaldehyde, ms = 1.5 +/- 0.3 for Pseudomonas 1 and 2.7 +/- 0.7 for Pseudomonas 135, whereas on formate the values for ms are 1.0 +/- 0.2 and 4.4 +/- 1.3; respectively. Although the maintenance coefficients did not differ systematically between the two groups of bacteria, the maintenance requirements per generation of the serine pathway bacteria were considerably higher (8.7 vs. 3.9) owing to their slower growth rate. The maximum molar yield values, YMmax (g cell dry wt/mol substrate utilized), corrected for the maintenance energy of bacteria which utilize C1-compounds via the ribulose monophosphate pathway averaged 19.1 when grown on methanol, while the values for bacteria which use the serine pathway averaged 13.5. On formaldehyde an average value of 11.5 is obtained and on formate the average value was 7.4 in the serine pathway bacteria.     

Dimethylsulfone as a growth substrate for novel methylotrophic species of Hyphomicrobium and Arthrobacter

Dimethylsulfone is a major product of the chemical oxidation in the atmosphere of the principal biogenic sulfur gas, dimethylsulfide, but no studies have been reported on the mechanisms for its microbiological degradation. Three novel strains of bacteria have been isolated from enrichment cultures provided with dimethylsulfone as the only carbon and energy substrate. These are novel facultatively methylotrophic species of Hyphonmicrobium and Arthobacter, capable of growth on a range of one-carbon substrates. Cell-free extracts contained activities of enzymes necessary for a reductive/oxidative pathway for dimethylsulfone degradation: membrane-bound-dimethylsulfone and dimethylsulfoxide reductases, dimethylsulfide monooxygenase, and methanethiol oxidase. Enzymatic evidence is also presented for the subsequent oxidation of formaldehyde by formaldehyde and formate dehydrogenases in the Hyphomicrobium strain and by a dissimilatory ribulose monophosphate cycle in the Arthrobacter strains. The strains also grew on dimethylsulfoxide and dimethylsulfide, and dimethylsulfide-grown bacteria oxidized dimethylsulfide and dimethylsulfoxide but not dimethylsulfone. Formaldehyde assimilation was effected in the Hyphomicrobium strain by the serine pathway, but enzymes of the ribulose monophosphate cycle for formaldehyde assimilation were present in the Arthrobacter strains grown on dimethylsulfone. In contrast, one of the Arthrobacter strains was shown to switch to the serine pathway during growth on methanol. Growth yields on dimethylsulfone and formaldehyde were consistent with the occurrence of the serine pathway in Hyphomicrobium strain S1 and the ribulose monophosphate cycle in Arthrobacter strain TGA, and with the proposed reductive pathway for dimethylsulfone degradation in both.     

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.     

Effect of oxygen containing C1 compounds on the growth of methane-assimilating bacteria

Added C1-compounds of the intermediates methanol, formaldehyde, formate and carbon dioxide show a catalytic effect on the growth rate and cell yield of CH4-assimilating bacteria GB 25 with serine pathway. Maximum stimulation is obtained by added amounts of about 20 mg C1-compound/g bacteria dry matter. The influence of C1-compounds decreases as follows: methanol greater than carbon dioxide greater than formate greater than formaldehyde.     

Biochemical properties and phylogeny of hydroxypyruvate reductases from methanotrophic bacteria with different c<Subscript>1</Subscript>-assimilation pathways

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C₁-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C₁-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.     

Assimilation of Formaldehyde and other C1Compounds by Gliocladium deliquescens and Paecilomyces varioti

Two fungi were isolated from soil which grew on 0.1~0.2% formaldehyde as the sole carbon source, and identified as Gliocladium deliquescens and Paecilomyces varioti. Both the strains could grow on 5% methanol and 5% Na-formate, while the former could grow even on 7% methanol. Metabolic pathways were traced through two dimensional paper chromatography and autoradiographic techniques using ¹⁴C-formaldehyde, ¹⁴C-methanol or ¹⁴C-CO₂ as substrates.

The intracellular metabolites were persued and their quantitative variation with time was measured. Along with the fact that serine and malate appeared in the earlier time, then appeared organic acids and amino acids belonging to TCA cycle, and the fact that hydroxy-pyruvate reductase and phosphoenolpyruvate carboxylase activities were much stronger in methanol culture than in ethanol culture, it was concluded that the two fungi followed the serine pathway in assimilating C₁-compounds. Oxidation enzymes of methanol and formaldehyde were also studied, and an oxidizing system was found besides usual NAD linked methanol or formaldehyde dehydrogenases.     

Growth yields of microorganisms on methanol and methane. A theoretical study

An attempt was made to calculate growth yields of microorganisms on methanol and methane on the basis of known biochemical pathways of C₁ metabolism. Since 3-phosphoglycerate is a key intermediate in the assimilation pathways of C₁ compounds, the calculations were based on the assumption that the synthesis of cell material from C₁ substrates can be regarded as a two step process. When Y_ATP on 3-phosphoglycerate was taken as 10.5, a maximal cell yield of organisms of the composition C₄H₈O₂N on methanol was found to be 0.73 g cells/g substrate. For growth on methane a value of 0.91 g cells/g substrate was calculated when a mixed function oxidase was implicated in methane oxidation. These yields were calculated on the basis of the ribulose phosphate pathway of formaldehyde fixation as the major pathway of C₁ assimilation. Yields calculated on the basis of the serine pathway were on an average 20% lower. The calculations disclosed that for growth on methane, at least for Methylococcus capsulatus, a reversed electron transport system is required when methane is oxidized by a mixed function oxidase. The theoretical cell yields on methanol and methane have been compared with experimentally obtained yields and the validity of the estimations of growth yields on the basis of the present calculations is discussed.     

Effect of formaldehyde on growth of obligate methylotroph Methylobacillus flagellatum in a substrate non-limited continuous culture

The ribulose monophosphate cycle methylotroph Methylobacillus flagellatum was grown under oxyturbidostat conditions on mixtures of methanol and formaldehyde. Formaldehyde when added at low concentration (50 mg/l) increased the methanol consumption and the yield of biomass. The presence of 150–300 mg/l of formaldehyde resulted in an increase of the growth rate from 0.74 to about 0.79–0.82 h^-1. The presence of 500 mg/l of formaldehyde in the inflow decreased culture growth characteristics. Activities of methanol dehydrogenase and enzymes participating in formaldehyde oxidation and assimilation were measured. The enzymological profiles obtained are discussed.Abbreviations MDH methanol dehydrogenase - NAD-linked FDDH NAD-linked formaldehyde dehydrogenase - DLFDDH dye-linked formaldehyde dehydrogenase - DLFDH dye-linked formate dehydrogenase - GPDH glucose-6-phosphate dehydrogenase - PGDH 6-phosphogluconate dehydrogenase - RuMP cycle ribulose monophosphate cycle     

Electron microscopy of methanol-utilizing bacteria

Two different groups of methanol-utilizing bacteria were studied by electron microscopy. Bacteria using the serine pathway for the assimilation of methanol were found to have a thin cell envelope (outer membrane, periplasmic area and cytoplasmic membrane). Those using the assimilatory ribulose monophosphate pathway of formaldehyde fixation had a much thicker cell envelope and in the case ofPseudomonas C protrusions of the outer membrane were found.     

Formaldehyde incorporation by a new methylotroph (L3)

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehyde- or methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of C1 compounds. Its C1-oxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle.     

Formaldehyde incorporation by a new methylotroph (L3).

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehyde- or methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of C1 compounds. Its C1-oxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle.     

The first methane-oxidizing bacterium from the upper mixing layer of the deep ocean:Methylomonas pelagica sp. nov.

Methane enrichment of twenty-three 100-ml portions of seawater from three stations in the Sargasso Sea yielded the same obligate type I methanotroph. It is pigmented white, requires NaCl, grows well in seawater with either methane or methanol, but not on other C₁ compounds nor on C–C bonded organic matter, and it uses either ammonia or nitrate but not dinitrogen as a nitrogen source. Formaldehyde is produced in marked amounts from methanol. Growth occurs at 20° and 30°C but not at 10°C and is inhibited in natural sunlight. Representative isolates from each hydrographic station assimilate one-carbon units via the ribulose monophosphate pathway for formaldehyde fixation, and have a DNA base composition of 49 mol% guanine plus cytosine. The type strain, NCMB 2265, has been namedMethylomonas pelagica sp. nov. This upper ocean methanotroph may obtain its C₁ substrates in situ from particles of algal debris that become anoxic, ferment, and accumulate in the thermocline to form a false benthos.     

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

Synthesis of dissimilatory enzymes of serine type methylotrophs under different growth conditions

The synthesis of methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase by pink pigmented facultative methylotrophs (PPFM) has been studied during growth on C₁ and multicarbon substrates. In batch cultures, the methanol dehydrogenase activities were higher during slow growth on non-C₁-compounds than during fast growth on methanol. Derepression of this enzyme also occurred at slow growth in methanol-limited chemostat culture. Formaldehyde dehydrogenase and formate dehydrogenase remained largely repressed during growth on multicarbon substrates.     

Growth of Pseudomonas C on C1 compounds: enzyme activites in extracts of Pseudomonas C cells grown on methanol, formaldehyde, and formate as sole carbon sources.

Pseudomonas C can grow on methanol, formaldehyde, or formate as sole carbon source. It is proposed that the assimilation of carbon by Pseudomonas C grown on different C1 growth substrates proceeds via one of two metabolic pathways, the serine pathway or the allulose pathway (the ribose phosphate cycle of formaldehyde fixation). This contention is based on the distribution of two key enzymes, each of which appears to be specifically involved in one of the assimilation pathways, glycerate dehydrogenase (serine pathway) and hexose phosphate synthetase (allulose pathway). The assimilation of methanol in Pseudomonas C cells appears to occur via the allulose pathway, whereas the utilization of formaldehyde or formate in cells grown on formaldehyde or formate as sole carbon sources appears by the serine pathway. When methanol is present together with formaldehyde or formate in the growth medium, the formaldehyde or formate is utilized by the allulose pathway.     

Methanol metabolism and ammonia assimilation in four methylophilns strains

Methanol assimilation and dissimilation pathways and ammonia assimilation pathway were investigated in four obligate methanol-utilizing bacteria through the detection of key enzymes. Both hexulose phosphate synthetase and hexulose phosphate isomerase, key enzymes of the ribulose monophosphate pathway (RMP) for methanol assimilation were detected whereas four key enzymes (hydroxy pyruvate reductase, isocitrate lyase, malyl-CoA-lyase and glyoxylate aminotransferase) that are characteristic of the serine assimilation pathway were absent. Key enzymes for the two methanol dissimilation pathways, the linear sequence enzymes formaldehyde and formate dehydrogenase and the RMP cyclic sequence enzymes glucose-6-phosphate dehydrogenase and 6-phosphate giuconate dehydrogenase were all detected. Ammonia was assimilated via the glutamate dehydrogenase pathway and not via the glutamine synthetase and glutamate synthase pathway.     

Isolation and Characterization of Methanesulfonic Acid-Degrading Bacteria from the Marine Environment

Two methylotrophic bacterial strains, TR3 and PSCH4, capable of growth on methanesulfonic acid as the sole carbon source were isolated from the marine environment. Methanesulfonic acid metabolism in these strains was initiated by an inducible NADH-dependent monooxygenase, which cleaved methanesulfonic acid into formaldehyde and sulfite. The presence of hydroxypyruvate reductase and the absence of ribulose monophosphate-dependent hexulose monophosphate synthase indicated the presence of the serine pathway for formaldehyde assimilation. Cell suspensions of bacteria grown on methanesulfonic acid completely oxidized methanesulfonic acid to carbon dioxide and sulfite with a methanesulfonic acid/oxygen stoichiometry of 1.0:2.0. Oxygen electrode-substrate studies indicated the dissimilation of formaldehyde to formate and carbon dioxide for energy generation. Carbon dioxide was not fixed by ribulose bisphosphate carboxylase. It was shown that methanol is not an intermediate in methanesulfonic acid metabolism, although these strains grew on methanol and other one-carbon compounds, as well as a variety of heterotrophic carbon sources. These two novel marine facultative methylotrophs have the ability to mineralize methanesulfonic acid and may play a role in the cycling of global organic sulfur.     

Enzymatic preparation of [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol and hydroxypyruvate using the methanol-assimilating system of methylotrophic yeasts

Dihydroxyacetoone synthase (EC 2.2.1.3), which is a key enzyme of the C₁-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO₂. When [¹³C]formaldehyde was used as a substrate with dihydroxyacytone synthase from Candida boidinii 2201, ¹³C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the ¹³C content of each carbon (atoms/100 atoms) was estimated to be 50%. [¹³C]Methanol was also useful for the enrichment of dihydroxyacetone with ¹³C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumalation of ¹³C-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Diyhdroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacytone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1, 3-¹³C]dihydroxyacetone phosphate from [¹³C]methanol; the molar yield of the ester relative to methanol added was 92.5%