Methylotrophic extremophilic yeast Trichosporon sp.: a soil-derived isolate with potential applications in environmental biotechnology

A yeast isolate revealing unique enzymatic activities and substrate-dependent polymorphism was obtained from autochthonous microflora of soil heavily polluted with oily slurries. By means of standard yeast identification procedures the strain was identified as Trichosporon cutaneum. Further molecular PCR product analyses of ribosomal DNA confirmed the identity of the isolate with the genus Trichosporon. As it grew on methanol as a sole carbon source, the strain appeared to be methylotrophic. Furthermore, it was also able to utilize formaldehyde. A multi-substrate growth potential was shown with several other carbon sources: glucose, glycerol, ethanol as well as petroleum derivatives and phenol. Optimum growth temperature was determined at 25 degrees C, and strong inhibition of growth at 37 degrees C together with the original soil habitat indicated lack of pathogenicity in warm-blooded animals and humans. The unusually high tolerance to xenobiotics such as diesel oil (>30 g/l), methanol (50 g/l), phenol (2 g/l) and formaldehyde (7.5 g/l) proved that the isolate was an extremophilic organism. With high-density cultures, formaldehyde was totally removed at initial concentrations up to 7.5 g/l within 24 h, which is the highest biodegradation capability ever reported. Partial biodegradation of methanol (13 g/l) and diesel fuel (20 g/l) was also observed. Enzymatic studies revealed atypical methylotrophic pathway reactions, lacking alcohol oxidase, as compared with the conventional methylotroph Hansenula polymorpha. However, the activities of glutathione-dependent formaldehyde dehydrogenase, formaldehyde reductase, formate dehydrogenase and unspecific aldehyde dehydrogenase(s) were present. An additional glutathione-dependent aldehyde dehydrogenase activity was also detected. Metabolic and biochemical characteristics of the isolated yeast open up new possibilities for environmental biotechnology. Some potential applications in soil bioremediation and wastewater decontamination are discussed.     

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.     

Bioconversion of methanol to formaldehyde. II. By purified methanol oxidase from modified yeast, Hansenula polymorpha

Modified methylotrophic yeast Hansenula polymorpha (HP A16) that was obtained by repressing leucine oxotrophic yeast; a wild type of Hansenula polymorpha CB4732 was used in this study. The yeast is grown with methanol, which is used as a sole carbon source, using various methanol concentrations and temperatures, and methanol oxidase (MOX) which is a key enzyme of methanol metabolism; production is maximized. Whole yeast cells were cultivated under optimized inoculation conditions; they were separated into two portions. One portion of these cells was directly used in bioconversion of methanol to formaldehyde. The second portion of the free cells was broken into pieces and a crude enzyme extract was obtained. The MOX enzyme in this extract was purified via salt precipitation, dialysis, and chromatographic methods. The purified MOX enzyme of yeast (HP A16) oxidized the methanol to formaldehyde. Optimization of bioconversion conditions was studied to reach maximum activity of enzyme. The optimum temperature and pH were found to be 35 degrees C and pH 8.0 in boric acid/NaOH buffer, and it was stable over the pH range of 6-9, at the 20 degrees C 15 min. A suitable reaction period was found as 50 min. The enzyme indicated low carbon primary alcohols (C2 to C4), as well as methanol. Initially, MOX activity increased with the increase of methanol concentration, but enzyme activity decreased. The apparent Km and Vmax values for methanol substrate of HP A16 MOX were 0.25 mM and 30 U/mg, respectively. The purified MOX enzyme was applied onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis; molecular weight of the enzyme was calculated to be about 670 kDa. Each MOX enzyme is composed of eight identical subunits, each of whose molecular weight is around 82 kDa and which contain eight moles of FAD as the prosthetic group, and the pI of the natural enzyme is found to be 6.4. The purified MOX enzyme was used in the bioconversion of methanol to formaldehyde as a catalyst; this conversion was compared to the conversion percentages of whole cells in our previous article in terms of catalytic performances.     

Isolation and identification of Paracoccus sp. FD3 and evaluation of its formaldehyde degradation kinetics

A formaldehyde-degrading bacterium strain, FD3, was isolated from contaminated soil and identified as Paracoccus sp. based on partial 16S rRNA gene sequence analysis. In batch culture, the bacterium metabolized 5,000 and 8,000 mg/L formaldehyde completely within 16 and 18 h, respectively, at 30°C (pH 7.0) with agitation at 150 rpm. The degradation kinetics was found to follow a first-order model at all initial formaldehyde concentrations with regression values greater than 0.99. Formaldehyde degradation rates increased from 532.37 to 2283.04 mg/L/h as the initial concentration of formaldehyde was increased from 1,000 to 8,000 mg/L. The growth of strain FD3 on formaldehyde as a sole carbon and energy source was well described by the Luong model with a maximal specific growth rate of 0.1754/h, a half-saturation constant of 309.02 mg/L, and a maximum substrate concentration of 3875.53 mg/L. Due to its high tolerance and degradation capacity to formaldehyde, Paracoccus sp., FD3 is considered an excellent candidate for use in degrading formaldehyde in wastewaters.     

汉森酵母表达载体的构建和人血管生成抑制素基因的表达

汉森酵母(H.polymorpha)是一类能以甲醇为唯一碳源和能源的甲基营养酵母,具有高表达外源基因、易于高密度发酵和产业化的特点。应用PCR技术扩增汉森酵母甲醇氧化酶(Methanol oxidase MOX)基因启动子和转录终止序列,并与汉森酵母Leu基因(Hpleu2)和人血管生成抑制素基因一起重组进大肠杆菌质粒pSP72,构建了整合型表达载体pSMA17,采用LiAc法将pSMA17转入汉森酵母A16(leu),筛选出阳性转化子H.polymorpha A16(pSMA17)。转化子在YPGE培养基中培养至对数生长后期,用甲醇进行诱导表达。ELISA和SDSPAGE分析结果证明人血管生成抑制素已获表达,表达产物分泌至培养基中。Western blot结果显示重组的人血管生成抑制素能与抗人纤溶酶原抗血清特异结合,具有免疫原性。     

Studies on the resistance of activated sludge bacteria to high concentrations of methanol, butanol, glycol, cyclohexanone and cyclohexylamine

The resistance of bacterial strains isolated from activated sludge purifying petrochemical wastewaters to high concentrations of methanol, butanol, glycol, cyclohexanone and cyclohexylamine was examined. The strains were found to be resistant to up to 5000 mg/l of methanol, butanol and glycol. Cyclohexylamine in concentration 1500 mg/l completely inhibited the growth of all examined strains whereas cyclohexanone even at concentration 4500 mg/l eliminated only about half of the isolated strains. The highest resistance to cyclohexane derivatives was shown by bacteria belonging to Pseudomonas III. None of the studied strains was, however, able to utilize cyclohexanone and cyclohexylamine as a source of barbon and energy.     

Expression of xylA genes encoding xylose isomerases from Escherichia coli and Streptomyces coelicolor in the methylotrophic yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol at high temperatures. H. polymorpha xylose reductase and xylitol dehydrogenase are involved during the first steps of this fermentation. In this article, expression of bacterial xylA genes coding for xylose isomerases from Escherichia coli or Streptomyces coelicolor in the yeast H. polymorpha was shown. The expression was achieved by integration of the xylA genes driven by the promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene ( HpGAP) into the H. polymorpha genome. Expression of the bacterial xylose isomerase genes restored the ability of the H. polymorpha Deltaxyl1 mutant to grow in a medium with xylose as the sole carbon source. This mutant has a deletion of the XYL1 gene encoding xylose reductase and is not able to grow in the xylose medium. The H. polymorpha Deltaxyl1(xylA) transformants displayed xylose isomerase activities, which were near 20% of that of the bacterial host strain. The transformants did not differ from the yeast wild-type strain with respect to ethanol production in xylose medium.     

Formaldehyde removal in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1

Rhodococcus erythropolis strain UPV-1 is able to grow on phenol as the only carbon and energy source and to remove formaldehyde completely from both synthetic and industrial wastewater. The rate of formaldehyde removal is independent of either initial biomass or formaldehyde concentration. The presence of viable, intact cells is strictly necessary for this removal to take place. Discontinuous and continuous formaldehyde-feed systems were successfully tested with synthetic wastewater in shaken flasks. Once biodegradation was well established in model synthetic wastewater, a real wastewater sample was obtained from a local phenolic and melamine resin-manufacturing company. Incubation of biomass with this wastewater at subtoxic concentrations of formaldehyde resulted in the complete removal of the pollutant. Parameters, such as chemical oxygen demand and toxicity, were assessed as indicators of wastewater cleanup progress.     

Formaldehyde removal in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1

. Rhodococcus erythropolis strain UPV-1 is able to grow on phenol as the only carbon and energy source and to remove formaldehyde completely from both synthetic and industrial wastewater. The rate of formaldehyde removal is independent of either initial biomass or formaldehyde concentration. The presence of viable, intact cells is strictly necessary for this removal to take place. Discontinuous and continuous formaldehyde-feed systems were successfully tested with synthetic wastewater in shaken flasks. Once biodegradation was well established in model synthetic wastewater, a real wastewater sample was obtained from a local phenolic and melamine resin-manufacturing company. Incubation of biomass with this wastewater at subtoxic concentrations of formaldehyde resulted in the complete removal of the pollutant. Parameters, such as chemical oxygen demand and toxicity, were assessed as indicators of wastewater cleanup progress.     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii.

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alcohol oxidases of Kloeckera sp. and Hansenula polymorpha. Catalytic properties and subunit structures.

1. Alcohol oxidase (alcohol: oxygen oxidoreductase) of a thermophilic methanol-utilizing yeast, Hansenula polymorpha DL-1, was isolated in crystalline form. 2. This alcohol oxidase of H. polymorpha was more stable to heat than was the enzyme of Kloeckera sp. This difference in heat stability is compatible with the difference in growth temperatures for both yeasts. 3. The crystalline alcohol oxidases of both yeast oxidized the lower primary alcohols (C-2 to C-4) as well as methanol. The apparent Km values for the methanol of Kloeckera and H. polymorpha enzymes were 0.44 and 0.23 mM, respectively. The enzymes could also oxidize formaldehyde to formate, and were inactivated by relatively low concentrations of hydrogen peroxide. 4. The molecular weight for both enzymes was calculated to be about 670000. Each enzyme is composed of eight identical subunits (molecular weight 83000) and contains eight moles of FAD as the prosthetic group. The NH2-terminal and COOH-terminal amino acids of H. polymorpha enzyme were identified as alanine and phenylalanine, respectively. The octameric subunits model of each enzyme was confirmed by electron micrographs, which showed an octad aggregate, composed of two tetragons face to face.     

Glutathione participation in the regulation of methanol metabolism in yeasts

The activity of enzymes involved in methanol oxidation and assimilation as well as the levels of formaldehyde and glutathione were determined during batch cultivation of Candida boidinii KD1 in a medium with methanol. The distribution of [14C]methanol between oxidative and biosynthetic processes in the yeast was analysed. Changes in the concentrations of formaldehyde and glutathione were found to correlate with the activity of formaldehyde dehydrogenase. The results indicate that an increase in the concentration of reduced glutathione (GSH) at the early logarithmic phase of the yeast growth stimulates formaldehyde oxidation via formate to carbon dioxide whereas a subsequent decrease in the concentration of GSH favours formaldehyde assimilation.     

Isolation of Bacteria Able to Metabolize High Concentrations of Formaldehyde

Summary Nineteen bacterial strains able to degrade and metabolize formaldehyde as a sole carbon source were isolated from soil and wastewater of a formaldehyde production factory. The samples were cultured in complex and mineral salts media containing 370 mg formaldehyde/l. The bacterial strains were identified to be Pseudomonas pseudoalcaligenes, P. aeruginosa, P. testosteroni, P. putida, and Methylobacterium extorquens. After adaptation of these microorganisms to high concentrations of formaldehyde; two isolated strains of M. extorquens (strains ESS and PSS) and four strains of P. pseudoalcaligenes (strains LSW, SSW, NSW and OSS) degraded 1850 mg formaldehyde/l, where as P. pseudoalcaligenes strain OSS completely consumed 3700 mg of formaldehyde/l after 24 h and degraded 70% of 5920 mg of formaldehyde/l after 72 h.     

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.     

Ambient temperature treatment of low strength wastewater using anaerobic sequencing batch reactor

Low strength wastewater having chemical oxygen demands (COD) concentrations of 1000, 800, 600 and 400mg/l were treated at 35, 25, 20 and 15¡C using four anaerobic sequencing batch reactors (ASBRs). Reactor 1 was operated at hydraulic retention time (HRT) of 48h, reactor 2 at 24h HRT, reactor 3 at 16h HRT and reactor 4 at 12h HRT. 80 to 99% soluble COD was removed at the various operational conditions, except during 15¡C treatment of 1000 and 800mg/l COD wastewater at 12h HRT and 1000mg/l COD wastewater at 16h HRT, where excessive loss of biological solids occurred. The ASBR process can be an effective process for the treatment of low concentrated wastewaters which are usually treated aerobically with large amount of sludge production and higher energy expenditures.     

Monitoring the denitrification of wastewater containing high concentrations of nitrate with methanol in a sulfur-packed reactor

Biological denitrification of high nitrate-containing wastewater was examined in a sulfur-packed column using a smaller amount of methanol than required stoichiometrically for heterotrophic denitrification. In the absence of methanol, the observed nitrate removal efficiency was only about 40%, and remained at 400 mg NO(3)(-)-N/l, which was due to an alkalinity deficiency of the pH buffer and of CO(2) as a carbon source. Complete denitrification was achieved by adding approximately 1.4 g methanol/g nitrate-nitrogen (NO(3)(-)-N) to a sulfur-packed reactor. As the methanol concentration increased, the overall nitrate removal efficiency increased. As influent methanol concentrations increased from 285 to 570, 855, and 1,140 mg/l, the value of Delta mg alkalinity as CaCO(3) consumed/Delta mg NO(3)(-)-N removed increased from -1.94 to -0.84, 0.24, and 0.96, and Delta mg SO(4)(2-) produced/Delta mg NO(3)(-)-N removed decreased from 4.42 to 3.57, 2.58, and 1.26, respectively. These results imply the co-occurrence of simultaneous autotrophic and heterotrophic denitrification. Sulfur-utilizing autotrophic denitrification in the presence of a small amount of methanol is very effective at decreasing both sulfate production and alkalinity consumption. Most of methanol added was removed completely in the effluent. A small amount of nitrite accumulated in the mixotrophic column, which was less than 20 mg NO(2)(-) -N/l, while under heterotrophic denitrification conditions, nitrite accumulated steadily and increased to 60 mg NO(2)(-) -N/l with increasing column height.     

Biodegradation of phenol in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1 immobilized in an air-stirred reactor with clarifier

Phenol biodegradation by suspended and immobilized cells of Rhodococcus erythropolis UPV-1 was studied in discontinuous and continuous mode under optimum culture conditions. Phenol-acclimated cells were adsorbed on diatomaceous earth, where they grew actively forming a biofilm of short filaments. Immobilization protected cells against phenol and resulted in a remarkable enhancement of their respiratory activity and a shorter lag phase preceding active phenol degradation. Under optimum operation conditions in a laboratory-scale air-stirred reactor, the immobilized cells were able to completely degrade phenol in synthetic wastewater at a volumetric productivity of 11.5 kg phenol m(-3) day(-1). Phenol biodegradation was also tested in two different industrial wastewaters (WW1 and WW2) obtained from local resin manufacturing companies, which contained both phenols and formaldehyde. In this case, after wastewater conditioning (i.e., dilution, pH, nitrogen and phosphorous sources and micronutrient amendments) the immobilized cells were able to completely remove the formaldehyde present in both waters. Moreover, they biodegraded phenols completely at a rate of 0.5 kg phenol m(-3) day(-1) in the case of WW1 and partially (but at concentrations lower than 50 mg l(-1)) at 0.1 and 1.0 kg phenol m(-3) day(-1) in the cases of WW2 and WW1, respectively.     

Comparative study of semi-specific Aeromonas hydrophila and universal Pseudomonas fluorescens biosensors for BOD measurements in meat industry wastewaters

Aeromonas hydrophila P69.1 (A. hydrophila) was used to construct a semi-specific biosensor to estimate biochemical oxygen demand (BOD) in high fat and grease content wastewaters. A. hydrophila cells were grown in fat containing medium to induce necessary enzymes for transport and degradation of fatty substances. Universal biosensor based on non-specific Pseudomonas fluorescens P75 (P. fluorescens) was used to conduct comparison experiments. Biosensors were calibrated using OECD synthetic wastewater and steady-state method, subsequently several experiments with synthetic and industrial wastewaters were conducted. A linear range up to 45 mg l(-1) BOD(7) was gained using A. hydrophila biosensor, in comparison to 40 mg l(-1) BOD(7) obtained using P. fluorescens biosensors. The lower limit of detection was 5 mg l(-1) BOD(7). Service life of A. hydrophila and P. fluorescens biosensors were 110 and 115 days, respectively. The response time of the biosensors depended on the BOD(7) of measuring solution and was up to 20 min when analyzing different wastewaters. Both biosensors underestimated BOD in meat industry wastewater from 43% up to 71%, but more accurate results could be obtained with A. hydrophila biosensor. Semi-specific A. hydrophila biosensor was able to measure proportion of fat found in wastewater sample, while other refractory compounds remained undetectable to both biosensors.