Organic acid production during methylotrophic growth of Eubacterium limosum B2: displacement towards increased butyric acid yields by supplementing with acetate

Summary During anaerobic growth on methanol/CO₂ the fermentative bacterium Eubacterium limosum B2 produced mixtures of acetic and butyric acids as overflow metabolites. The proportion of each product was shown to vary according to the initial acetate concentration. At low concentrations, acetate provoked a displacement of the organic acid ratio culminating in homobutyric fermentations at 100 mM initial acetate. This metabolic shift was accompanied by a proportionate increase in the methanol dissimilated to CO₂, enabling a constant NAD(P)H₂/NAD(P) metabolite pool to be maintained. Higher initial acetate concentrations could not be balanced by further changes to the substrate stoichiometry and resulted in less rapid growth. The yield of butyric acid was enhanced further by some consumption of acetate. A mathematical model is presented relating initial acetate concentration to butyric acid production.     

Effects of various organic acid supplements on growth rates of Eubacterium limosum B2 on methanol

Summary During anaerobic growth on methanol, Eubacterium limosum B2 produces acetic and butyric acids as overflow metabolites, but can be induced to produce other organic acids. All organic acids (C₂–C₆) tested had a similar effect on growth, although the toxicity of each was different e.g. increasing inhibition by acids of increasing chain length. Inhibition was only observed above a threshold concentration related to the molecular size of the organic acids. At higher concentrations the degree of inhibition was a linear function of concentration. In a mathematical treatment of the data the inhibition constant (K _p) was shown to be proportionate to the threshold value (P _c) of each organic acid and accurately predicted the growth characteristics of Eubacterium limosum on methanol following the addition of organic acid supplements.     

Butyrate production in the acetogen Eubacterium limosum is dependent on the carbon and energy source

Eubacterium limosum KIST612 is one of the few acetogenic bacteria that has the genes encoding for butyrate synthesis from acetyl-CoA, and indeed, E. limosum KIST612 is known to produce butyrate from CO but not from H₂ + CO₂. Butyrate production from CO was only seen in bioreactors with cell recycling or in batch cultures with addition of acetate. Here, we present detailed study on growth of E. limosum KIST612 on different carbon and energy sources with the goal, to find other substrates that lead to butyrate formation. Batch fermentations in serum bottles revealed that acetate was the major product under all conditions investigated. Butyrate formation from the C1 compounds carbon dioxide and hydrogen, carbon monoxide or formate was not observed. However, growth on glucose led to butyrate formation, but only in the stationary growth phase. A maximum of 4.3 mM butyrate was observed, corresponding to a butyrate:glucose ratio of 0.21:1 and a butyrate:acetate ratio of 0.14:1. Interestingly, growth on the C1 substrate methanol also led to butyrate formation in the stationary growth phase with a butyrate:methanol ratio of 0.17:1 and a butyrate:acetate ratio of 0.33:1. Since methanol can be produced chemically from carbon dioxide, this offers the possibility for a combined chemical-biochemical production of butyrate from H₂ + CO₂ using this acetogenic biocatalyst. With the advent of genetic methods in acetogens, butanol production from methanol maybe possible as well.     

Methanol conversion in Eubacterium limosum

The conversion of methanol by cell-free extracts of the acetogenic bacterium Eubacterium limosum was studied. Incubation of mixed cell-free extracts of both E. limosum and Methanobacterium formicicum resulted in methane formation from methanol in the presence of ATP and 2-mercaptoethanesulfonic acid. The separate extracts were not able to perform this reaction. Addition of ferredoxin obtained from Methanosarcina barkeri to the mixed extracts resulted in increased methane formation. The enzyme, responsible for methanol binding in cell-free extract of E. limosum, was inactivated by FAD under N₂ and exhibited maximal activity under an atmosphere of H₂. This enzyme contains a firmly bound cobalamin which was methylated by methanol in the presence of ATP. It was demethylated in the presence of methylcobalamin: coenzyme M methyltransferase obtained from M. barkeri under concomitant formation of methylated coenzyme M. These properties are similar to those of methanol: 5-hydroxybenzimidazolylcobamide methyltransferase from M. barkeri. It was proposed that methylotrophic acetogens and methylotrophic methanogens use similar enzymes in the first step of methanol conversion.Abbreviations HS-CoM 2-mercaptoethanesulfonic acid - CH₃S-CoM 2-(methylthio)ethanesulfonic acid - BrES 2-bromoethanesulfonic acid - TES N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid - MT₁ methanol: 5-hydroxybenzimidazolylcobamide methyltransferase - MT₂ methylcobalamin - HS-CoM methyltransferase - DMBI 5,6-dimethylbenzimidazole and HBI, 5-hydroxybenzimidazole, are -ligands of corrinoids - (S-CoM)₂ 2,2-dithiodiethanesulfonic acid     

High methanol-to-formate ratios induce butanol production in Eubacterium limosum

Unlike gaseous C₁ feedstocks for acetogenic bacteria, there has been less attention on liquid C₁ feedstocks, despite benefits in terms of energy efficiency, mass transfer and integration within existing fermentation infrastructure. Here, we present growth of Eubacterium limosum ATCC8486 using methanol and formate as substrates, finding evidence for the first time of native butanol production. We varied ratios of methanol-to-formate in batch serum bottle fermentations, showing butyrate is the major product (maximum specific rate 220 ± 23 mmol-C gDCW^-1day^-1). Increasing this ratio showed methanol is the key feedstock driving the product spectrum towards more reduced products, such as butanol (maximum titre 2.0 ± 1.1 mM-C). However, both substrates are required for a high growth rate (maximum 0.19 ± 0.011 h^-1) and cell density (maximum 1.2 ± 0.043 gDCW l^-1), with formate being the preferred substrate. In fact, formate and methanol are consumed in two distinct growth phases – growth phase 1, on predominately formate and growth phase 2 on methanol, which must balance. Because the second growth varied according to the first growth on formate, this suggests butanol production is due to overflow metabolism, similar to 2,3-butanediol production in other acetogens. However, further research is required to confirm the butanol production pathway in E. limosum, particularly given, unlike other substrates, methanol likely results in mostly NADH generation, not reduced ferredoxin.     

Anaerobic biodegradation of methyl esters byAcetobacterium woodii andEubacterium limosum

Summary The ability ofAcetobacterium woodii andEubacterium limosum to degrade methyl esters of acetate, propionate, butyrate, and isobutyrate was examined under growing and resting-cell conditions. Both bacteria hydrolyzed the esters to the corresponding carboxylates and methanol under either condition. Methanol was further oxidized to formate under growing but not resting conditions. Unlike the metabolism of phenylmethylethers, no H₂ requirement was evident for ester biotransformation. The hydrolysis of methyl carboxylates is thermodynamically favorable under standard conditions and the mixotrophic metabolism of ester/CO₂ allowed for bacterial growth. These results suggest that the degradation of methyl carboxylates may be a heretofore unrecognized nutritional option for acetogenic bacteria.     

Investigation of the trophic relations between anaerobic microorganisms from an underground gas repository during methanol utilization

Joint cultivation of the dominant strains of acetogenic, sulfate-reducing and methanogenic microorganisms isolated from water samples of the North Stavropol underground gas storage facility (UGSF) was carried out for revealing their probable trophic relationships. It was shown that acetogenic strains Eubacterium limosum AG12 and Sporomusa sphaeroides AG8-2 growing on methanol could form a considerable pool of hydrogen, which may support development of hydrogenotrophic cultures, the methanogen Methanobacterium formicicum MG134, or the sulfate reducer Desulfovibrio desulfuricans SR12. Growth of this sulfate-reducing strain was not stimulated under joint cultivation with Methanosarcina barkeri MGZ3 on methanol, probably due to its inability to take up low hydrogen concentrations observed during methanosarcina development. The results show that acetogens in the UGSF system are the most important consumers of methanol and hydrogen and after exhaustion of the latter and switching over to methanol utilization they can supply hydrogen to other microorganisms, including methanogens and sulfate reducers. The role of methanosarcina in the UGSF increases as the hydrogen and CO₂ reserves are exhausted, and methanogenesis on methanol becomes the main way of its destruction.     

HYDROGEN ION BUFFERING OF CULTURE MEDIA FOR ALGAE FROM MODERATELY ACIDIC,OLIGOTROPHIC WATERS 1

Five hydrogen ion buffers were compared for their usefulness in regulating pH in a model oligotrophic, moderately acidic (pH 6.0) algal growth medium. These were 3,3-dimethylglutaric acid (DMGA), tricarbaliylic acid (TCA), trans-aconitic acid (tAA), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) and 2-(N-morpholino) ethanesulfonic acid (MES). All buffers (2.5 mM) except HEPES limited the reduction of pH in a NH₄⁺-based medium during growth of Chrysochromulina breviturrita Nich. to less than 0.12 units, compared with more than 2 units in an unbuffered medium. Long term growth of C. breviturrita in these media was significantly inhibited (P < 0.05) by TCA and tAA. MES was able to control pH with the minimum amount of NaOH (1.0 mM) added to the medium to adjust to pH 6.0. Four of five bacterial isolates were capable of utilizing tAA as a sole organic-C source, and no isolate could metabolize HEPES or MES. No significant differences (P > 0.05) were found in the maximum growth rates of six algal species (from five classes) in a medium with or without MES buffer, although significantly greater cell yields of Ochromonas danica Prings. were obtained in the buffered medium. MES (pK₄=6.15) was considered to be the most useful buffer in the pH range 5.0–6.5, due to its biological inertness, buffering capacity, the minimal requirement for excess base to adjust pH and its minimal metal complexing ability.     

Production of aflatoxin byAspergillus parasiticus and its control

The aim of the present work was to investigate the production of aflatoxin byAspergillus parasiticus and to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from aflatoxins.Bacillus megatertum andB cereus were suitable for microbiological assay of aflatoxins. Czapek’s-Dox medium was found a suitable medium for isolation of fungi from food samples. The optimal pH for the growth ofA. parasiticus and its productivity of aflatoxin B₁ was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production (NH₄)₂HPO₄ (1.55 gL^-1) and NaNO₂ (1.6 gL^-1) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B₁ production. Zn²⁺ and Co²⁺ supported significantly both fungal growth, as well as, aflatoxin B₁ production at the different tested concentrations. Zn²⁺ was effective when added toA. parasiticus growth medium at the first two days of the culture age. The other tested metal ions expressed variable effects depending on the type of ion and its concentration. Water activity (a_w) was an important factor controlling the growth ofA. parasiticus and toxin production. The minimum a_w for the fungal growth was 0.8 on both coffee beans and rice grains, while a_w of 0.70 caused complete inhibition for the growth and aflatoxin B₁ production. H₂O₂ is a potent inhibitor for growth ofA. parasiticus and its productivity of toxins. NaHCO₃ and C₆H₅COONa converted aflatoxin B₁ to water-soluble form which returned to aflatoxin B₁ by acidity. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B₁ production. Stearic acid supported the fungal growth and decreased the productivity of AFB₁ gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B₁ production. Vitamins C and D₂ were also repressive particularly for aflatoxin production The present study included studying the activities of some enzymes in relation to aflatoxin production during 20-days ofA. parasiticus age in 2-days intervals. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B₁ production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B₁ synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were associated with the increase of aflatoxin B₁ production. All the tested enzymes as well as aflatoxin B₁ production were inhibited by either catechol or phenol.     

Growth yields and the efficiency of oxidative phosphorylation of Paracoccus denitrificans during two- (carbon) substrate-limited growth

Paracoccus denitrificans was grown aerobically during two-(carbon)substrate-limitation on mannitol and methanol in chemostat cultures. Theoretical growth parameters were calculated based on the presence of 2 or 3 sites in the electron-transport chain of Paracoccus denitrificans. Experimental growth parameters determined during two-(carbon)substrate growth were conform to the presence of 3 sites of oxidative phosphorylation, while cells grown only on mannitol possessed 2 sites. The maximum growth yield on adenosine triphosphate (ATP), corrected for maintenance requirements, determined in chemostat experiments in which the methanol concentration is less than 2.11 times the mannitol concentration was 8.6 g of biomass. When the methanol concentration was more than 2.11 times the mannitol concentration the maximum growth yield on adenosine triphosphate decreased due to the more energy consuming process of CO₂-assimilation. Cells use methanol only as energy source to increase the amount of mannitol used for assimilation purposes. When the methanol concentration in chemostat experiments was more than 2.11 times the mannitol concentration, all mannitol was used for assimilation and excess energy derived from methanol was used for CO₂-assimilation via the ribulose-bisphosphate cycle. The synthesis of ribulosebisphosphate carboxylase was repressed when the methanol concentration in chemostat experiments was less than 2.11 times the mannitol concentration or when Paracoccus denitrificans was grown in batch culture on both methanol and mannitol. When in chemostat experiments the methanol concentration was more than 2.11 times the mannitol concentration ribulose-bisphosphate carboxylase activity could be demonstrated and CO₂-assimilation will occur. It is proposed that energy produced in excess activates or derepresses the synthesis of the necessary enzymes of the ribulose-bisphosphate cycle in Paracoccus denitrificans. Consequently growth on any substrate will be carbonas well as energy-limited. When methanol is present in the nutrient cells of Paracoccus denitrificans synthesize a CO-binding type of cytochrome c, which is essential for methanol oxidase activity.The reason for the increase in efficiency of oxidative phosphorylation from 2 to 3 sites is most probably the occurrence of this CO-binding type of cytochrome c in which presence electrons preferentially pass through the a-type cytochrome region of the electron-transport chain.Non Standard Abbreviations X prosthetic group of methanol dehydrogenase - q _substrate specific rate of consumption of substrate (mol/g biomass. h.) - Y _substrate, Y _substrate ^MAX are respectively the growth yield and the maximum growth yield corrected for maintenance requirements (g biomass/mol) - m _substrate maintenance requirement (mol substrate/g biomass) - specific growth rate (h^-1) - M [methanol]/[mannitol] ratio in the nutrient - N part of mannitol that is assimilated when M=o - R _m amount of methanol-equivalents that has the same energy content as 1 mannitol-equivalent - P/O _N , P/O _F , P/O _X is the amount of ATP produced during electron-transport of two electrons from respectively NADH+H⁺, FADH₂ and XH₂ to oxygen     

Physiological and morphological acid resistance ofCandida boidinii

The growth ofCandida boidinii strain 2 in a methanol-limited chemostat at a dilution rate of 0.1/h and a low extracellular pH (2.8–4.0) is characterized by a maximum yield coefficient referred to the methanol consumedY _S of 0.4 g/g and a maximum cell content of nitrogenous compounds of 60%. The cell proteins are rich in essential amino acids. At pH<2.6 or >4.0 the cell concentration decreases due to lower growth rate, accompanied by increased metabolic quotientsQ _S,Q _CO2 andQ _form, and increased activities of dissimilating dehydrogenases. The activity of alcohol oxidase (AO) in intact cells (0.54 IU/mg protein) was unaffected by pH 2.8–3.8 although in a cell-free extract the AO activity decreased at these low pH values after a 10-min incubation. The lower AO activity in cells at pH<2.8 and pH>3.8 brought about increased residual methanol levels in the medium, and also an increased level of riboflavin phosphate, arising probably by the release of FAD from active AO. Catalase activity was completely pH-independent. Cell morphology also showed no changes at pH 2.8–4.2, formation of cell chains being observed only at pH<2.8. However, the ultrastructure of cells grown in the chemostat at pH 2.6, however, did not evince any changes as compared with cells grown, at higher pH apart from a lag in cytokinesis. These findings, which point to acid resistance of strain 2, make it possible to produce biomass from methanol, with a high content of valuable proteins and AO, under nonsterile conditions.     

Relationship of soil hydrogen sulfide level to net carbon assimilation ofPanicum hemitomon andSpartina patens

Panicum hemitomon Schult andSpartina patens (Ait) Muhl. plants from Louisiana Gulf Coast fresh and brackish marshes were subjected to hydrogen sulfide under controlled sediment redox conditions. Net carbon assimilation responses of both species to the combined sediment anaerobiosis and hydrogen sulfide concentrations was measured.Panicum hemitomon was more sensitive to hydrogen sulfide as compared toSpartina patens. Initiation of reduction in net carbon assimilation inP. hemitomon began when H₂S concentrations of soil solution exceeded 0.22 mgl^-1. Reductions in net carbon assimilation inS. patens were also noted at H₂S concentrations exceeding 0.34 mgl^-1. The reduction in net carbon assimilation of both species measured at elevated H₂S concentrations suggests that extreme anaerobiosis and elevated sulfide could contribute to the growth reduction of these species under certain conditions. However based on H₂S concentration in fresh and brackish marsh soil profiles, levels were too low to cause any adverse effects ofPanicum hemitomon. In brackish marsh soils containing hydrogen sulfide of 3.4 mgl^-1 in soil solution, sulfide could be a major factor limiting growth ofS. patens.     

Syntrophic Association by Cocultures of the Methanol- and CO2-H2-Utilizing Species Eubacterium limosum and Pectin-Fermenting Lachnospira multiparus During Growth in a Pectin Medium

Lachnospira multiparus grew very well in an anaerobic 0.2% pectin medium, whereas Eubacterium limosum, which utilizes methanol, H₂-CO₂, and lactate, did not. Cocultures of the two species grew at a somewhat more rapid growth rate than did L. multiparus alone and almost doubled the amount of growth as measured by optical density. In model experiments with cultures transferred once a day with a 2-day retention time, L. multiparus produced mainly acetate, methanol, ethanol, formate, lactate, CO₂, and H₂ from pectin. The coculture produced one-third more acetate, and butyrate and CO₂ were the only other significant end products. The results are discussed in relationship to microbial metabolic interactions and interspecies hydrogen transfer.     

The uptake and incorporation of leucine and cystine by the mycelial and yeastlike phases of Blastomyces dermatitidis

Uptake and incorporation of L-leucine-C¹⁴ and L-cystine-S³⁵ was studied in the mycelial [MP] and yeastlike [YP] phases of the dimorphic fungal pathogen,Blastomyces dermatitidis. Both amino acids entered the cells of the two morphological forms ofB. dermatitidis by a permease-like system at low external concentrations of substrate. At high substrate levels, the amino acids entered the cells by a simple diffusion-like process in addition to the permease-like system. Michaelis-Menten constants [K_m] for L-leucine was found to be 1.1×10^–5 M and 4.4×10^–5 M for the MP and YP phases, respectively. The K_m for L-cystine was found to be 1.0×10^–5 M for the MP and 0.5×10^–5 M for the YP. A requirement for energy supplied by metabolic activity was demonstrated by the inhibition of uptake and incorporation of the amino acids by cells incubated with either 2,4-dinitrophenol or sodium azide. Amino acid uptake was broadly tolerant of hydrogen ion concentration, but definite optima were demonstrated at pH 7.0 to 7.5.     

Mass production of poly-β-hydroxybutyric acid by fully automatic fed-batch culture of methylotroph

Summary Fifty-one methylotrophs were checked with respect to their ability of poly--hydroxybutyric acid (PHB) production from methanol. One of them, Pseudomonas sp. K, was chosen from its good growth on a minimum synthetic medium. Optimal temperature and pH for its growth were 30° C and 7.0, respectively. Concentrations of PO ₄ ^3- and NH ₄ ⁺ in the medium should be kept at low levels. PHB formation was stimulated by deficiency of nutrient such as NH ₄ ⁺ , SO ₄ ^2- , Mg²⁺, Fe²⁺ or Mn²⁺. Among them, nitrogen deficiency was chosen from its effectiveness and easiness for PHB accumulation.The microorganism was cultivated to produce a large amount of poly--hydroxybutyric acid (PHB) from methanol by means of microcomputer-aided fully automatic fed-batch culture technique. During the cultivation, temperature, dissolved oxygen concentration (DO), and methanol concentration in the culture broth were maintained at 30° C 2.5±0.5 ppm and 0.5±0.2 g/l, respectively. Other nutrients, nitrogen source and mineral ions, were also controlled to maintain their initial concentrations in the medium during cell growth phase. When the high cell concentration was achieved (160 g/l), feedings of ammonia and minerals were stopped and only methanol was supplied successively to accumulate PHB. At 175 h, high concentration of PHB (136 g/l) was obtained and total cell concentration became 206 g/l. DO must be maintained above the critical level during the PHB formation phase, too. PHB yield from methanol (g PHB/g methanol) was 0.18 and the maximum PHB content reached 66% of dry weight. Solid PHB produced by the strain had the melting point of 176° C and the average molecular weight of 3.0x10⁵.     

Eubacterium aggreganssp. nov., a New Homoacetogenic Bacterium from Olive Mill Wastewater Treatment Digestor

A strictly anaerobic, homoacetogenic, Gram-positive, non spore-forming bacterium, designated strain SR12^T(T=type strain), was isolated from an anaerobic methanogenic digestor fed with olive mill wastewater. Yeast extract was required for growth but could also be used as sole carbon and energy source. Strain SR12^Tutilized a few carbohydrates (glucose, fructose and sucrose), organic compounds (lactate, crotonate, formate and betaine), alcohols (methanol), the methoxyl group of some methoxylated aromatic compounds, and H₂+CO₂. The end-products of carbohydrate fermentation were acetate, formate, butyrate, H₂and CO₂. End-products from lactate and methoxylated aromatic compounds were acetate and butyrate. Strain SR12^Twas non-motile, formed aggregates, had a G+C content of 55 mol % and grew optimally at 35°C and pH 7.2 on a medium containing glucose. Phylogenetically, strain SR12^Twas related toEubacterium barkeri, E. callanderi, andE. limosumwithE. barkerias the closest relative (similarity of 98%) with which it bears little phenotypic similarity or DNA homology (60%). On the basis of its phenotypic, genotypic, and phylogenetic characteristics, we propose to designate strain SR12^TasEubacterium aggreganssp. nov. The type strain is SR12^T(=DSM 12183).     

Denitrification of nitrate and nitric acid with methanol as carbon source

Summary A methanol/nitrate-medium and anaerobic conditions yielded an enrichment culture which consisted ofHyphomicrobium andParacoccus. This mixed culture proved to be very effective in denitrification of solutions containing high concentrations of nitrate and free nitric acid when grown in a chemostat (D=0.04 h^-1). With 0.1 mol/l nitric acid solution as feed medium the pH in the culture vessel adjusted itself to 5.8. For the reduction of 1 g NO₃–N 2.6 g methanol were consumed and 0.56 g cells were produced.     

Use of an organic buffer for the selection of acid tolerantRhizobium meliloti strains

Summary Nine media used to grow rhizobia were examined for their ability to maintain a stable low pH during the growth ofR. meliloti Large fluctuations in the pH of all media were recorded within 72 h, indicating their unsuitability for use in the selection of acid tolerant rhizobia. Morpholino-ethanesulphonic acid (MES) was assessed for its ability to buffer the pH of the media whilst still permitting rapid growth ofR. meliloti, R. trifolii, andBr. lupini. With 30.7 mM MES, the pH of a defined medium containing galactose, arabinose, and glutamate did not change from the initial value of 5.5 even though rhizobial numbers increased from 10⁴ to 10⁹ cells.ml^–1. Even at a buffer concentration of 15.3 mM, pH only increased from 5.5 to 5.6. There was no effect of the buffer on rhizobial growth.     

Effect of increasing methanol concentrations on physiology and cytology ofCandida boidinii

Concentration of methanol in the medium strongly affected not only the physiology but also the cytology ofCandida boidinii strain 2 cells in a methanol-limited chemostat at a constant dilution rateD 0.1/h and at low pH 3.0. The formation of large cubic peroxisomes with high alcohol oxidase (AO) activity observed at low methanol concentration (S ₀ 3 g/L) disappeared on increasing the methanol concentration in the inflow medium. The AO activity in the cells sharply decreased, followed by accumulation of riboflavin phosphate and residual methanol in the medium. The activity of catalase was relatively stable. At methanol concentrationS ₀>K _I (K ₁ equal to 12 g methanol per L), which included a substantial increase in methanol dissimilation, documented by higher formaldehyde and formate dehydrogenase activities and by lower yield coefficient on methanol, the yeast cells contained large lobe-shaped peroxisomes and a smaller number of larger mitochondria. The cells formed pseudomycelium with a thick septum between the mother and daughter cells.     

Characterization of catalase-negative mutants of methylotrophic yeastHansenula polymorpha

Three recently isolated catalase-negative mutants ofHansenula polymorpha lost the ability to grow on methanol but grew in media containing glucose, ethanol or glycerol. Their incubation in a medium with methanol resulted in an accumulation of hydrogen peroxide and cell death. During growth of a catalase-negative mutant in chemostat on a mixture of methanol and glucose, neither H₂O₂ accumulation nor cell death were observed up to the molar ratio of 10:1 of the two substrates. Cytochrome-c peroxidase and NADH-peroxidase activities were detected in the cells. In methylotrophic yeasts, catalase seems to be an enzyme characteristic of the metabolism of methanol but not needed for the metabolism of multicarbon substrates. The hydrogen peroxide produced during growth of the mutants on mixed substrates is detoxified by cytochrome-c peroxidase and other peroxidases. Translated by Č. Novotny