Formation and analysis of mannosylerythritol lipids secreted by Pseudozyma aphidis

Pseudozyma aphidis DSM 70725 was found to be a novel producer of mannosylerythritol lipids (MELs). The MELs were quantified by HPLC. Glucose as carbon source for precultivation supported growth well. By contrast, at concentrations >30 g l^–1 in preculture, subsequent MEL formation in the main culture with soybean oil as sole carbon source was reduced. The type of substrate supply considerably influenced MEL formation. High concentrations of soybean oil (80 ml l^–1) at init favored the production process when compared to a stepwise (20 ml l^–1) addition. Mannose or erythritol were suitable second carbon sources that enhanced the MEL yield with soybean oil as preferred primary substrate. After 10 days, a maximum yield of 75 g l^–1 was attained during shake-flask cultivation. Biofuel (rapeseed oil methyl ester) also resulted in high yields of MEL, but glucose reduced the MEL yield. Analysis by GC-MS showed that all fatty acids contained in MEL and derived from soybean oil or related methyl ester were degraded by C2-units to differing extents. The surface (water/air) and interfacial (water/hexadecane) tension of the MELs produced from different carbon sources were reduced to a minimum of 26.2 mN m^–1 and 1 mN m^–1, respectively.     

Efficient production of mannosylerythritol lipids with high hydrophilicity by <Emphasis Type="Italic">Pseudozyma hubeiensis</Emphasis> KM-59

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants known because of their multifunctionality and biocompatibility. A previously isolated yeast strain, Pseudozyma sp. KM-59, mainly produced a hydrophilic MEL, namely MEL-C (4-O-[4′-O-acetyl-2′,3′-di-O-alka(e)noyl-β-d-mannopyranosyl]-d-erythritol). In this study, we taxonomically characterize the strain in detail and investigate the culture conditions. The genetic, morphological, and physiological characteristics of the strain coincided well with those of Pseudozyma hubeiensis. On batch culture for 4 days under optimal conditions, the yield of all MELs was 21.8 g/l; MEL-C comprised approximately 65% of the all MELs. Consequently, on fed-batch culture for 16 days, the yield reached 76.3 g/l; the volumetric productivity was approximately 4.8 g l⁻¹ day⁻¹. We further examined the surface-active and self-assembling properties of the hydrophilic MELs produced by the yeast strain. They showed higher emulsifying activities against soybean oil and a mixture of hydrocarbons (2-methylnaphtarene and hexadecane, 1:1) than the synthetic surfactants tested. On water penetration scans, they efficiently formed lyotropic liquid crystalline phases such as myelines and lamella () in a broad range of their concentrations, indicating higher hydrophilicity than conventional MELs. More interestingly, there was little difference in the liquid crystal formation between the crude product and purified MEL-C. The present glycolipids with high hydrophilicity are thus very likely to have practical potential without further purification and to expand the application of MELs especially their use in washing detergents and oil-in-water-type emulsifiers.     

Improved production of biosurfactant by a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> mutant using vegetable oil refinery wastes

Biosurfactant production by Pseudomonas aeruginosa EBN-8 mutant was studied in shake flasks on separate wastes from canola, soybean and corn oil refineries. Of the substrates tested, canola oil refinery waste (COD=20 g l⁻¹) supplemented with sodium nitrate (at COD/N=20) showed the best microbial growth (4.50 g l⁻¹) and rhamnolipid production (8.50 g l⁻¹), at 10 d of incubation with the specific growth rate of 0.316 h⁻¹ and specific product yield of 0.597 g g⁻¹ h. Its cell-free supernatant showed the critical micelle dilution (CMD) of 150 and surface tension (ST) of 28.5 mN m⁻¹.     

Bioactive hydroxyphenylpyrrole-dicarboxylic acids from a new marine Halomonas sp.: production and structure elucidation

The new marine Halomonas sp. strain GWS-BW-H8hM (DSM 17996) was found to produce 3-(4′-hydroxyphenyl)-4-phenylpyrrole-2,5-dicarboxylic acid (HPPD-1) and 3,4-bis(4′-hydroxy- phenyl)pyrrole-2,5-dicarboxylic acid (HPPD-2). In initial cultivations using marine broth, only low contents of these compounds have been isolated. Improving the conditions and growing the strain on artificial seawater supplemented with tryptone 10 g l⁻¹, yeast extract 5 g l⁻¹, l-tyrosine 0.6 g l⁻¹, glycine 1 g l⁻¹, and glucose 6 g l^-1, the growth-associated HPPD-1 and HPPD-2 production of a 40-l batch cultivation reached the amounts of 47 mg l⁻¹ and 116 mg l⁻¹, respectively, after 65 h. Both compounds showed potent anti-tumor-promoting activities.     

Lactic acid production from sugar-cane juice by a newly isolated Lactobacillus sp.

A newly isolated sucrose-tolerant, lactic acid bacterium, Lactobacillus sp. strain FCP2, was grown on sugar-cane juice (125 g sucrose l⁻¹, 8 g glucose l⁻¹ and 6 g fructose l⁻¹) for 5 days and produced 104 g lactic acid l⁻¹ with 90% yield. A higher yield (96%) and productivity (2.8 g l⁻¹ h⁻¹) were obtained when strain FCP2 was cultured on 3% w/v (25 g sucrose l⁻¹, 2 g glucose l⁻¹ and 1 g fructose l⁻¹) sugar-cane juice for 10 h. Various cheap nitrogen sources such as silk worm larvae, beer yeast autolysate and shrimp wastes were also used as a substitute to yeast extract.     

Construction of the industrial ethanol-producing strain of Saccharomyces cerevisiae able to ferment cellobiose and melibiose

The gene mel1, encoding α-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and α-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanolproducing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular α-galactosidase and β-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC1 co-expressing 2 genes could achieve 0.29 OD600 h⁻¹ and a biomass yield up to 7.8 g l⁻¹ dry cell weight on medium containing 10.0 g l⁻¹ cellobiose and 10.0 g l⁻¹ melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous saccharification and fermentation process. Approximately 36.5 g l⁻¹ ethanol was produced from 100 g of cellulose supplied with 5 g l⁻¹ melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0% of the theoretical yield.     

Discovery of Pseudozyma rugulosa NBRC 10877 as a novel producer of the glycolipid biosurfactants, mannosylerythritol lipids, based on rDNA sequence

The search for a novel producer of glycolipid biosurfactants, mannosylerythritol lipids (MEL) was undertaken based on the analysis of ribosomal DNA sequences on the yeast strains of the genus Pseudozyma. Pseudozyma rugulosa NBRC 10877 was found to produce a large amount of glycolipids from soybean oil. Fluorescence microscopic observation also demonstrated that the strain significantly accumulates polar lipids in the cells. The structure of the glycolipids produced by the strain was analyzed by ¹H and ¹³C nuclear magnetic resonance and gas chromatography–mass spectrometry methods, and was determined to be the same as MEL produced by Pseudozyma antarctica, a well-known MEL producer. The major fatty acids of the present MEL consisted of C8 and C10 acids. Based on high performance liquid chromatography, the composition of the produced MEL was as follows: MEL-A (68%), MEL-B (12%), and MEL-C (20%). To enhance the production of MEL by the novel strain, factors affecting the production, such as carbon and nitrogen sources, were further examined. Soybean oil and sodium nitrate were the best carbon and nitrogen sources, respectively. The supplementation of a MEL precursor, such as erythritol, drastically enhanced the production yield from soybean oil at a rate of 70 to 90%. Under the optimal conditions in a shake culture, a maximum yield, productivity, and yield coefficient (on a weight basis to soybean oil supplied) of 142 g l⁻¹, 5.0 g l⁻¹ day⁻¹, and 0.5 g g⁻¹ were achieved by intermittent feeding of soybean oil and erythritol using the yeast.     

Heterotrophic production of eicosapentaenoid acid by the diatom Nitzschia laevis: effects of silicate and glucose

The effects of silicate and glucose on growth and eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis were studied. By alternately altering the concentrations of silicate (2.7–64 mg l⁻¹) and glucose (1–40 g l⁻¹) in the medium, the highest cell dry weight (ca. 5.5 g l⁻¹) was obtained at 20 g l⁻¹ glucose and 32 mg l⁻¹ silicate, while the highest specific growth rate (ca. 0.65 day⁻¹) was obtained at a relatively low glucose concentration (5 g l⁻¹) and high silicate concentrations (32–64 mg l⁻¹). At glucose levels of 5 and 20 g l⁻¹, EPA content was higher with lower silicate concentrations (2.7 and 16 mg l⁻¹ silicate, respectively), while at a silicate level of 16 mg l⁻¹, higher glucose concentrations (20–40 g l⁻¹) facilitated EPA formation. The highest EPA yield (131 mg l⁻¹) was obtained at 20 g l⁻¹ glucose and 32 mg l⁻¹ silicate, while the highest EPA productivity (15.1 mg l⁻¹ day⁻¹) was obtained at 20 g l⁻¹ glucose and 64 mg l⁻¹ silicate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 218–224. Received 08 May 2000/ Accepted in revised form 21 July 2000     

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.     

Introduction of a stress-responsive gene, yggG, enhances the yield of l-phenylalanine with decreased acetic acid production in a recombinant Escherichia coli

A stress-responsive gene, yggG, was introduced into an l-phenylalanine producer, Escherichia coli AJ12741. In shake-flask culture, the yggG-containing recombinant strain (named AJ12741/pHYGG) produced 6.4 g l-phenylalanine l⁻¹ at the end of culture and its yield on glucose was 0.16 g l-phenylalanine g glucose⁻¹. These values are much higher than those of the original AJ12741 strain (3.7 g l-phenylalanine l⁻¹ and 0.09 g l-phenylalanine g glucose⁻¹, respectively). On the other hand, AJ12741/pHYGG strain produced only 4.5 g acetic acid l⁻¹ and its yield on glucose was about a half of that of the AJ12741 culture. Analysis of gene expression revealed that in late growth phase, the expression levels of genes involved in acetic acid production (pta, ackA, and poxB) were relatively low in AJ12741/pHYGG cells. In particular, the level of poxB expression in AJ12741/pHYGG strains was one-seventh of that of the original strain. These results suggest that the formation of a bottleneck for acetic acid production brings about a metabolic flow favorable to l-phenylalanine synthesis in the recombinant strain over-expressing the yggG gene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microbial production of 1,3-propanediol from glycerol by Klebsiella pneumoniae under micro-aerobic conditions up to a pilot scale

1,3-Propanediol (1,3-PD) can be produced from glycerol by Klebsiella pneumoniae under micro-aerobic conditions. Recently, this fed-batch fermentation process has been successfully scaled up to 1 m³. The final 1,3-PD concentration, molar yield and volumetric productivity of 72 g l⁻¹, 57% and 2.1 g l⁻¹ h⁻¹, respectively, are close to those of 75 g l⁻¹, 61%, and 2.2 g l⁻¹ h⁻¹ under anaerobic conditions. This process would be suitable for the production of 1,3-PD on a large scale.     

Ethrel treatment enhanced isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa, a woody legume

The cell cultures of Pueraria tuberosa, a perennial leguminous lianas, were maintained in modified MS medium (KNO₃ 475 mg l⁻¹, thiamine 1 mg l⁻¹, biotin 1 mg l⁻¹, calcium pantothenate 1 mg l⁻¹) containing 0.1 mg l⁻¹ 2,4,5-trichloroacetic acid and 0.1 mg l⁻¹ kinetin. Isoflavonoids (puerarin, genistin, daidzein, genistein) accumulation in cell suspension cultures was increased by 14-fold to ~12 mg l⁻¹ after 48 h of adding 100 μM ethrel. Ethrel inhibitors (silver nitrate and silver thiosulfate) completely inhibited this effect in the presence of ethrel and isoflavonoids were not detected in the spent medium. The increase was dose dependent and can be explored to trigger high yield of isoflavonoids production.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp. IHI-91 on olive oil

A thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp. strain IHI-91 growing optimally at 65 °C. Lipase activity decreased with increasing dilution rate while lipase productivity showed a maximum of 340 U l⁻¹ h⁻¹ at a dilution rate of 0.4 h⁻¹. Lipase productivity was increased by 50% compared to data from batch fermentations. Up to 70% of the total lipase activity measured was associated to cells and by-products or residual substrate. Kinetic and stoichiometric parameters for the utilisation of olive oil were determined. The maximal biomass output method led to a saturation constant K _S of 0.88 g/l. Both batch growth data and a washout experiment yielded a maximal specific growth rate, μ_max, of 1.0 h⁻¹. Oxygen uptake rates of up to 2.9 g l⁻¹h⁻¹ were calculated and the yield coefficient, Y _X/O, was determined to be 0.29 g dry cell weight/g O₂. From an overall material balance the yield coefficient, Y _X/S, was estimated to be 0.60 g dry cell weight/g olive oil. Received: 8 January 1997 / Received revision: 30 April 1997 / Accepted: 4 May 1997     

Lactic acid production from cellulosic waste by immobilized cells of Lactobacillus delbrueckii

Industrial waste corn cob residue (from xylose manufacturing) without pretreatment was hydrolyzed by cellulase and cellobiase. The cellulosic hydrolysate contained 52.4 g l⁻¹ of glucose and was used as carbon source for lactic acid fermentation by cells of Lactobacillus delbrueckii ZU-S2 immobilized in calcium alginate gel beads. The final concentration of lactic acid and the yield of lactic acid from glucose were 48.7 g l⁻¹ and 95.2%, respectively, which were comparative to the results of pure glucose fermentation. The immobilized cells were quite stable and reusable, and the average yield of lactic acid from glucose in the hydrolysate was 95.0% in 12 repeated batches of fermentation. The suitable dilution rate of continuous fermentation process was 0.13 h⁻¹, and the yield of lactic acid from glucose and the productivity were 92.4% and 5.746 g l⁻¹ h⁻¹, respectively. The production of lactic acid by simultaneous saccharification and fermentation (SSF) process was carried out in a coupling bioreactor, the final concentration of lactic acid was 55.6 g l⁻¹, the conversion efficiency of lactic acid from cellulose was 91.3% and the productivity was 0.927 g l⁻¹ h⁻¹. By using fed-batch technique in the SSF process, the final concentration of lactic acid and the productivity increased to 107.6 g l⁻¹ and 1.345 g l⁻¹ h⁻¹, respectively, while the dosage of cellulase per gram substrate decreased greatly. This research work should advance the bioconversion of renewable cellulosic resources and reduce environmental pollution.     

Efficient production of ectoine using ectoine-excreting strain

Halophilic bacteria strain Halomonas salina DSM 5928 was found to excrete ectoine, suggesting its potential in the development of a new method of ectoine production. We performed HPLC and LC–MS analyses that showed that Halomonas salina DSM 5928 excreted ectoine under constant extracellular osmolarity. Medium adopting monosodium glutamate as a sole source of carbon and nitrogen was beneficial for ectoine synthesis. The total concentration of ectoine was not affected by NaCl concentration in the range 0.5–2 mol l⁻¹. The total concentration of ectoine and productivity in a 10-l fermentor with 0.5 mol l⁻¹ NaCl were 6.9 g l⁻¹ and 7.9 g l⁻¹ d⁻¹, respectively. These findings show that Halomonas salina DSM 5928 efficiently produces ectoine at relatively low NaCl concentration. This research also indicates the potential application of free or immobilized cells for continuous culture to produce ectoine.     

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp.

Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l⁻¹) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l⁻¹ to 180 mg l⁻¹, approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)⁻¹ h⁻¹. A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.     

Carbon source pulsed feeding to attain high yield and high productivity in poly(3-hydroxybutyrate) (PHB) production from soybean oil using <Emphasis Type="Italic">Cupriavidus necator</Emphasis>

Poly(3-hydroxybutyrate) (PHB) biosynthesis from soybean oil by Cupriavidus necator was studied using a bench scale bioreactor. The highest cell concentration (83 g l⁻¹) was achieved using soybean oil at 40 g l⁻¹ and a pulse of the same concentration. The PHB content was 81% (w/w), PHB productivity was 2.5 g l⁻¹ h⁻¹, and the calculated Y_p/s value was 0.85 g g⁻¹. Growth limitation and the onset of PHB biosynthesis took place due to exhaustion of P, and probably also Cu, Ca, and Fe.     

The Green Microalga <Emphasis Type="Italic">Chlorella saccharophila</Emphasis> as a Suitable Source of Oil for Biodiesel Production

The aim of this study was to investigate the potential of the green microalga Chlorella saccharophila as a source of oil for biodiesel production. We evaluated for the first time, the effect of salinity and/or nitrogen depletion (ND) on cell growth, lipid accumulation and lipid profile in this microalga. The fatty acid methyl esters (FAME) identified for C. saccharophila in this study consisted of C-16:0, C-18:0, C-18:1 cis, and C-18:1 trans. Among these, C-18:1 (indicator of biodiesel quality) was the main FAME found, representing approximately 76 and 80% of total FAME under normal and ND growing conditions, respectively. Under a normal growing condition this microalga showed 154.63 mg l⁻¹ d⁻¹, 63.33 mg l⁻¹ d⁻¹, and 103.73 mg l⁻¹ of biomass productivity, lipid productivity, and FAME yield, respectively. The higher biomass productivity (159.58 mg l⁻¹ d⁻¹), lipid productivity (99.33 mg l⁻¹ d⁻¹), and FAME yield (315.53 mg l⁻¹) were obtained under the ND treatment. In comparison to other related studies, our results suggest that C. saccharophila can be considered as a suitable source of oil for biodiesel production.     

Brewer’s spent grain as raw material for lactic acid production by<Emphasis Type="Italic"> Lactobacillus delbrueckii</Emphasis>

Chemically pre-treated brewer’s spent grain was saccharified with cellulase producing a hydrolysate with approx. 50 g glucose l⁻¹. This hydrolysate was used as a fermentation medium without any nutrient supplementation by Lactobacillus delbrueckii, which produced L-lactic acid (5.4 g l⁻¹) at 0.73 g g⁻¹ glucose consumed (73% efficiency). An inoculum of 1 g dry cells l⁻¹ gave the best yield of the process, but the pH decrease affected the microorganism capacity to consume glucose and convert it into lactic acid.