Lipase production by diverse phylogenetic clades of Aureobasidium pullulans

Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2–0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains in clade 9, which exhibit a dark olivaceous pigment, produced the highest levels of lipase, with strain NRRL 62034 yielding 0.57 U lipase/ml. By comparison, Candida cylindracea strain NRRL Y-17506 produced 0.05 U lipase/ml under identical conditions. A. pullulans strain NRRL 62034 reached maximal lipase levels in 5 days on lipase induction medium, while A. pullulans strain NRRL Y-2311-1 and strains in clades 4 and 10 were highest after 6 days. A. pullulans strain NRRL Y-2311-1 and strains in clade 9 produced two extracellular proteins in common, at >50 and <37 kDa.     

Olive-mill wastewaters: a promising substrate for microbial lipase production

The present study investigated the valorization of olive-mill wastewater (OMW) by its use as a possible growth medium for the microbial production of extra-cellular lipase. To this end, strains of Geotrichum candidum (NRRL Y-552 and Y-553), Rhizopus arrhizus (NRRL 2286 and ISRIM 383), Rhizopus oryzae (NRRL 6431), Aspergillus oryzae (NRRL 1988 and 495), Aspergillus niger (NRRL 334), Candida cylindracea (NRRL Y-17506) and Penicillium citrinum (NRRL 1841 and 3754, ISRIM 118) were screened. All strains were able to grow on the undiluted OMW, producing extra-cellular lipase activity. C. cylindracea NRRL Y-17506 showed the highest lipase activity on all the typologies of OMW used. Its lipase production on OMW was markedly affected by the type of nitrogen source and was induced by the addition of olive oil. The highest activity (9.23 IU ml(-1)) of the yeast was obtained on OMW supplemented with NH(4)Cl (2.4 g l(-1)) and olive oil (3.0 g l(-1)).     

Production of poly(β-l-malic acid) (PMA) from agricultural biomass substrates by Aureobasidium pullulans

For the first time the production of poly(β-l -malic acid) (PMA) has been achieved using agricultural biomass substrates by the yeast-like fungus Aureobasidium pullulans. Strains NRRL Y-2311-1, NRRL 50382, NRRL 50383, and NRRL 50384, representing diverse isolation sources and phylogenetic clades, produced PMA from alkaline H₂O₂-pretreated corn fiber and wheat straw as sole carbon sources. Pretreated wheat straw was better than pretreated corn fiber, and strain NRRL 50383 gave the highest overall yields of PMA. The addition of CaCO₃ plus supplementary hydrolytic enzymes enhanced PMA production. Four basal media were compared for PMA production, and the best was found to be a N-limited pullulan production medium (PM). In this medium, PMA production took place during growth limitation. Under optimal conditions, strain NRRL 50383 produced more than 20 g PMA/l from 5 % (w/v) pretreated wheat straw in PM with 3 % (w/v) CaCO₃ and supplementary enzymes.     

Purification and characterization of the extracellular beta-glucosidase produced by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 produced a cell-bound beta-glucosidase when grown in complex media containing 50 g of cellobiose per liter. The majority of the enzyme was located on the cell surface and was released into the supernatant upon treatment of intact cells with Zymolyase 60,000. Only about 10% of the total activity was associated with the cytoplasm. The enzyme was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an apparent native molecular mass of about 198,000 Da and appeared to be composed of two subunits with approximate molecular masses of 94,000 Da. The beta-glucosidase contained approximately 30.5% (w/w) carbohydrate. Mannose was the only detected neutral carbohydrate associated with the purified enzyme. The enzyme demonstrated optimal activity at a pH of 4.0 to 5.0. The Km of the purified beta-glucosidase was 6.74 X 10(-2) M for cellobiose and 4.17 X 10(-3) M for p-nitrophenyl-beta-D-glucopyranoside. Glucose did not appear to inhibit the enzyme.     

Isolation, characterization and evaluation of hyper 2-propanol producing bacteria from Singapore environment

Three hyper 2-propanol producing strains were isolated from Singapore environment using an enrichment step and a high through-put screening step. The analysis of the amplified 16S rDNA revealed that the isolates belonged to Clostridium species and they were named as Clostridium sp. BT10-1, Clostridium sp. M10-1 and Clostridium sp. PU31-4. At 1 L scale, the 2-propanol titer of these positive strains was 1.6–2.1 times of that of Clostridium beijerinckii NRRL B593, which is so far the most efficient natural 2-propanol producer. The highest 2-propanol titer was achieved by isolate BT10-6 and it was 5.26 g/L (87.5 mM). These three positive strains BT10-6, M10-1 and PU31-4 consumed glucose almost completely in 40–48 h and gave 2-propanol productivity at 0.132, 0.118 and 0.087 g/L/h, respectively, which is 3.0–4.6 times of 0.029 g/L/h given by C. beijerinckii NRRL B593. Butanol was also produced by these positive strains with a slightly lower butanol titer and higher butanol productivity, compared to butanol control strain C. beijerinckii NCIMB 8052.     

Metschnikowia arizonensis and Metschnikowia dekortorum,two new large-spored yeast species associated with floricolous beetles

Two new haplontic heterothallic species of Metschnikowia were discovered in flowers and associated beetles. Metschnikowia arizonensis was recovered from flowers of cholla cactus (Opuntia echinocarpa) and a specimen of Carpophilus sp. (Coleoptera: Nitidulidae) found in these flowers, in Arizona. Metschnikowia dekortorum was isolated in specimens of the nitidulid beetle Conotelus sp. captured in flowers of two species of Ipomoea in northwestern Guanacaste Province, Costa Rica. The sexual cycle of these yeasts is typical of the large-spored Metschnikowia species, but the asci and spores are intermediate in size between these and other members of the genus. The physiology is consistent with that of most Metschnikowia species except that both species fail to utilize lysine as sole nitrogen source. Also, M. arizonensis utilizes fewer carbon compounds than most species and exhibits considerable variability among strains at this level. Partial ribosomal DNA large-subunit (D1/D2) sequences suggest that M. arizonensis and M. dekortorum are moderately related sister species whose positions are intermediate between the large-spored species Metschnikowia and Metschnikowia hibisci. The type cultures are: M. arizonensis, strains UWO(PS)99-103.3.1=CBS 9064=NRRL Y-27427 (h(+), holotype) and UWO(PS)99-103.4=CBS 9065=NRRL Y-27428 (h(-), isotype); and M. dekortorum, strains UWO(PS)01-142b3=CBS 9063=NRRL Y-27429 (h(+), holotype) and UWO(PS)01-138a3=CBS 9062=NRRL Y-27430 (h(-), isotype).     

Poly(ethylene glycol)-mediated molar mass control of short-chain- and medium-chain-length poly(hydroxyalkanoates) from Pseudomonas oleovorans

Three strains of Pseudomonas oleovorans, a well known poly(hydroxyalkanoate) (PHA) producer, were tested for the ability to control PHA molar mass and end group structure by addition of poly(ethylene glycol) (PEG) to the fermentation medium. Each strain of P. oleovorans - NRRL B-14682 (B-14682), NRRL B-14683 (B-14683), and NRRL B-778 (B-778) - synthesized a different type of PHA from oleic acid when cultured under identical growth conditions. Strain B-14682 produced poly(3-hydroxybutyrate) (PHB), while B-14683 synthesized a medium-chain-length PHA ( mcl-PHA) with a repeat unit composition ranging from C4 to C14 and some mono-unsaturation in the C14 alkyl side chains. Strain B-778 synthesized a mixture of PHB (95 mol%) and mcl-PHA (5 mol%). The addition of 0.5% (v/v) PEG (M(n) =200 g/mol, PEG-200) to the fermentation broth of strains B-14682 and B-778 resulted in chain termination through esterification at the carboxyl terminus of the PHB with PEG chain segments, thus reducing the molar mass by 54% and 23%, respectively. The molar mass of the mcl-PHA produced by strains B-14683 and B-778 also showed a 34% and 47% reduction in the presence of PEG-200, respectively, but no evidence of esterification was present. PEG-400 (M(n) =400 g/mol) had a reduced effect on PHA molar mass. In fact, the molar masses of the mcl-PHA derived from strain B-14683 and both the PHB and mcl-PHA from B-778 were unchanged by PEG-400. In contrast, the PHB produced by B-14682 showed a 35% reduction in molar mass in the presence of PEG-400.     

A comparison of polyssacharides from strains of Aureobasidium pullulans

Abstract Production of pullulan by five strains of Aureobasidium pullulans was compared in three media with three carbohydrate sources. Our goal was to screen strains and media to obtain pullulan in maximal yield, purity, and stability. Pullulan yields and properties were strongly affected by strain specificity, but a single medium performed best with most strains. Sucrose was the preferred carbohydrate for all five strains. A "color variant" strain of Aureobasidium , NRRL Y-12974, possibly representing a distinct species, produced a polysaccharide of intermediate molecular weight which was stable during storage at 4°C, heating to 100°C, and high shearing action. This polysaccharide was 70% pullulanase sensitive and contained the least contaminating melanin pigment.     

Continuous fermentation of feed streams containing D-glucose and D-xylose in a two-stage process utilizing immobilized Saccharomyces cerevisiae and Pachysolen tannophilus

In the U.S., forest and crop residues contain enough glucose and xylose to supply 10 times the country's usage of ethanol and ethylene, but an efficient fermentation scheme is lacking,(1,2,3) To develop a strategy for process design, specific ethanol productivities and yields of Pachysolen tannophilus NRRL Y-2460 and Saccharomyces cerevisiae NRRL Y-2235 were compared. Batch cultures and continuous stirred reactors (CSTR) loaded with immobilized cells were fed glucose and xylose. As expected from previous reports, Y-2235 fermented glucose but not xylose. Y-2460 consumed both sugars but fermented glucose inefficiently relative to Y-2235, and it suffered a diauxic lag lasting 10-20 h when given a sugar mixture. Immobilized Y-2235 exhibited increasing productivity but constant yield with in creasing glucose concentration. In contrast, Y-2460 exhibited an optimum productivity at 30-40 g/L xylose and a declining yield with increasing xylose concentration. Immobilized Y-2235 tolerated more than 100 g/L ethanol while the productivity and yield of Y-2460 fell by 80 and 58%, respectively, as ethanol reached 50 g/L. A 38.8-g/L ethanol stream could be produced as 103 g/L xylose was continuously fed to Y-2460. If it was blended with a 274 g/L glucose stream to give a composite of 23.7 g/L ethanol and 107 g/L glucose, Y-2235 could en rich the ethanol to 75 g/L. Taken together these results suggest use of a two-stage continuous reactor for pro cessing xylose and glucose from lignocellulose. An immobilized Y-2460 CSTR (or cascade) would convert the hemicellulose hydrolyzate. Then downstream, an immobilized Y-2235 plug flow reactor would enrich the hemicellulose-derived ethanol to more than 70 g/L upon addition of cellulose hydrolyzate.     

Blastobotrys serpentis sp. nov., isolated from the intestine of a Trinket snake (Elaphe sp., Colubridae)

Asporogenus yeast strains W113AT and W113B were isolated from the intestine of a dead Trinket snake. The two isolates showed 100% sequence similarity in the D1/D2 domain of the large-subunit (LSU) rRNA gene, internal transcribed spacer (ITS) 1-5.8S rRNA gene-ITS2 region and mitochondrial small-subunit rRNA gene and the cytochrome oxidase II gene sequence and also showed similar phenotypic characteristics. The nearest phylogenetic neighbors of W113AT and W113B based on the sequence of the D1/D2 domain of the LSU rRNA gene were Blastobotrys chiropterorum NRRL Y-17017T and Blastobotrys terrestris NRRL Y-17704T with about 98% similarity. The close affiliation of W113AT and W113B with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T was also evident from the high similarity observed in the nucleotide sequences of the mitochondrial small subunit rRNA (96-97.8%) and the cytochrome oxidase II (95.5-95.6%) genes. In the neighbor-joining phylogenetic trees constructed based on the D1/D2 domain or cytochrome oxidase gene, the isolates clustered with the above-mentioned species. However, the isolates showed a number of differences in their phenotypic properties with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T and hence are regarded as representing a novel member of the genus Blastobotrys, for which the name Blastobotrys serpentis sp. nov. is proposed.     

Metschnikowia santaceciliae,Candida hawaiiana,and Candida kipukae,three new yeast species associated with insects of tropical morning glory

A new haplontic heterothallic species of Metschnikowia and two related asexual yeast species were discovered in morning glory flowers and associated insects. Metschnikowia santaceciliae came from Conotelus (Coleoptera: Nitidulidae) and other insect species associated with flowers of Ipomoea indica (purple morph) in Costa Rica. Candida hawaiiana and Candida kipukae were found in I. indica (syn. I. acuminata) and its insects in Hawai'i, and the former was also isolated in a specimen of Conotelus collected on Merremia tuberosa (Convolvulaceae) in Costa Rica. The three species have nearly identical physiological profiles, typical of the genus Metschnikowia. The sequences of the D1/D2 domains of their large subunit ribosomal DNA confirm that the species belong to the Metschnikowia clade, even though they share a very low degree of inter-relatedness. M. santaceciliae is a sister species to Metschnikowia continentalis. C. kipukae is a basal member of the large-spored Metschnikowia subclade, and C. hawaiiana has a weak affinity to Metschnikowia agaves. Two of the three species appear to be endemic. The type cultures are: Metschnikowia santaceciliae, strains UWO(PS)01-517a1=CBS 9148=NRRL Y-27475 (h(+, holotype) and UWO(PS)01-520a1=CBS 9149=NRRL Y-27476 (h-, isotype); Candida hawaiiana, strain UWO(PS)91-698.3=CBS 9146=NRRL Y-27473; Candida kipukae, strain UWO(PS)00-669.2=CBS 9147=NRRL Y-27474.     

Heavy oils produced by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

From a survey of more than 50 diverse strains of Aureobasidium pullulans, 21 produced extracellular heavy oils. Most oil producers fell into phylogenetic clades 8, 9, and 11. Oil colors ranged from bright yellow to malachite. More than half of the strains produced oil that was fluorescent. In medium containing 5% (w/v) sucrose, oil yields ranged from 0.5 to 6 g oil/l. Strain CU 43 reached stationary growth phase at day 4 while oil yields were maximal at day 6. CU 43 produced bright yellow, highly fluorescent oil that also was visible as intracellular droplets under fluorescent microscopy. Oil was surface active, suggesting that it functions as a biosurfactant. Oil from two strains (CU 43 and NRRL Y-12974) differentially inhibited mammalian cancer cell lines. MALDI-TOF MS spectra suggested that A. pullulans strains produce a family of related oil structures.     

Candida riodocensis and Candida cellae, two new yeast species from the Starmerella clade associated with solitary bees in the Atlantic rain forest of Brazil

Two new ascomycetous yeast species belonging to the Starmerella clade were discovered in nests of two solitary bee species in the Atlantic rain forest of Brazil. Candida riodocensis was isolated from pollen-nectar provisions, larvae and fecal pellets of nests of Megachile sp., and Candida cellae was found in pollen-nectar provisions of Centris tarsata. Analysis of the sequences of the D1/D2 large-subunit ribosomal DNA showed that C. riodocensis is phylogenetically related to C. batistae, and the closest relative of C. cellae is C. etchellsii. The type strains are C. riodocensis UFMG-MG02 (=CBS 10087(T) = NRRL Y-27859(T)) and C. cellae UFMG-PC04 (=CBS 10086(T) = NRRL Y-27860(T)).     

Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations

Summary Three strains ofPichia stipitis and three ofCandida shehatae were compared withPachysolen tannophilus in their abilities to ferment xylose at concentrations as high as 200 g/L when subjected to both aerobic and microaerophilic conditions. Evaluations based on accumulated ethanol concentrations, ethanol productivities, xylose consumption, and ethanol and xylitol yields were determined from batch culture time courses. Of the strains considered,P.stipitis NRRL Y-7124 seemed most promising since it was able to utilize all but 7 g/L of 150 g/L xylose supplied aerobically to produce 52 g/L ethanol at a yield of 0.39 g per gram xylose (76% of theoretical yield) and at a rate comparable to the fastest shown byC.shehatae NRRL Y-12878. For all strains tested, fermentation results from aerobic cultures were more favorable than those from microaerophilic cultures.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Yeasts isolated from plant-associated beetles and other insects: seven novel Candida species near Candida albicans

Yeasts related to Candida albicans were isolated from the digestive tracts of beetles in eight families and various orders of insects such as earwigs, crickets, and roaches, most of which were caught at light traps or in a few cases directly from plant materials. Based on comparisons of DNA sequences and other taxonomic characteristics, a total of 41 isolates were identified as Candida orthopsilosis, Candida pseudorhagii, Candida maltosa, Candida parapsilosis, Candida tropicalis, Candida neerlandica, Lodderomyces elongisporus, and seven new Candida species. The new species and type strains are designated as Candida gigantensis NRRL Y-27736T, Candida bohiensis NRRL Y-27737T, Candida alai NRRL Y-27739T, Candida buenavistaensis NRRL Y-27734T, Candida frijolesensis NRRL Y-48060T, Candida labiduridarum NRRL Y-27940T, and Candida tetrigidarum NRRL Y-48142T. A phylogeny based on SSU and LSU rRNA gene sequences indicated that most of the new species were closely related to members of the C. albicans/L. elongisporus clade, such as C. albicans, Candida dulbliniensis, C. neerlandica, Candida chauliodes, and Candida corydali. Candida alai was placed near this clade, but no closely related sister taxon was identified. The ecology of the insect-associated yeasts is discussed and compared with the results from other studies.     

DWS microrheology of a linear polysaccharide

Diffusing wave spectroscopy has been used to measure the rheological behavior of pullulan (M(w) = 1 x 10(5)) aqueous solutions up to concentration of 40 g/dL. It was found that these solutions were mainly viscous, with the loss modulus G' higher than the elastic modulus G'. The plot of the specific viscosity eta(sp) as a function of pullulan concentration showed two critical concentrations c = 4 g/dL and c = 15 g/dL. For c < c, eta( sp) approximately c(1.25+/-0.05); for c < c < c, eta( sp) approximately c(2+/-0.05); and for c > c, eta( sp) approximately c(4.5+/-0.5). These results are in very good agreement with those reported in the literature.     

Ethanol production by immobilized Saccharomyces cerevisiae, Saccharomyces uvarum, and Zymomonas mobilis

Saccharomyces cerevisiae NRRL Y-2034, S, uvarum NRRL Y-1347, and Zymomonas mobilis NRRL B-806 each were separately immobilized in a Ca-alginate matrix and incubated in the presence of a free-flowing and continuous 1, 3, 5, 10, or 20% (w/w) glucose solution. In general, the yeast cells, converted 100percnt; of the 1, 3, and 5% glucose to alcohol within 48 h and maintained such a conversion rate for at least two weeks. The bacterium converted ca. 90% (w/w) of the 1, 3, and 5% glucose to alcohol continuously for one week. However, both the yeast and bacterium were inhibited in the highest glucose (20% w/w) solution. All of the immobilized cultures produced some alcohol for at least 14 days. Immobilized S. cerevisiae was the best alcohol producer of all of the glucose concentrations; the yeast yielded 4.7 g ethanol/100 g solution within 72 h in the 10% glucose solution. After 7-8 days in the 10% solution, S. cerevisiae produced ethanol at 100% of theoretical yield (5.0 g ethanol/100 g solution), with a gradual decrease in alcohol production by 14 days. Immobillized S. uvarum produced a maximum of 4.0 g ethanol/100 g solution within 2 days and then declined to ca. 1.0 g ethanol/100 g solution after 7 days continuous fermentation in the 10% glucose solution. Zymomonas mobilis reached its maximum ethanol production at 4 days (4.7 g/100 g solution), and then diminished similarly to S. uvarum. The development of a multiple disk shaft eliminated the problem both of uneven distribution of alginate-encapsulated cells and of glucose channeling within the continuous-flow fermentor column. This invention improved alcohol production about threefold for the yeast cells.     

New species and new combinations in the yeast genera Kregervanrija gen. nov., Saturnispora and Candida

Analysis of nucleotide sequences from the domains 1 and 2 of the large-subunit rDNA demonstrated species of the Pichia fluxuum clade to be phylogenetically isolated, and domains 1 and 2 and internal transcribed spacer rDNA sequence analyses of strains phenotypically identified as P. fluxuum resulted in the discovery of three new species. From this work, the following new genus, new species, and new combinations are proposed: Kregervanrija gen. nov.; type species Kregervanrija fluxuum comb. nov. (type strain NRRL YB-4273, CBS 2287); Kregervanrija delftensis comb. nov. (type strain NRRL Y-7119, CBS 2614); Kregervanrija pseudodelftensis sp. nov. (type strain NRRL Y-5494, CBS 10105); Saturnispora besseyi comb. nov. (NRRL YB-4711, CBS 6343); Saturnispora mendoncae sp. nov. (type strain NRRL Y-11515, CBS 5620); and Candida abiesophila sp. nov. (type strain NRRL Y-11514, CBS 5366).     

Energetic analysis of the growth of Methanobrevibacter arboriphilus A2 in hydrogen-limited continuous cultures

Hydrogen-limited chemostat cultures of Methanobrevibacter arboriphilus A2 were carried out. The available electron balance and carbon balance in M. arboriphilus A2 and other methanogenic strains grown on various substrates were well satisfied. This indicates that no extracellular organic products were formed during methanogenic growth. The molar growth yields for methane (Y(X/CH(4) )) were calculated as 1.06-1.42 g cell/mol CH(4) at dilution rate (0.21-0.43 day(-1)). The smaller Y(X/CH(4) ) of M. arboriphilus A2 compared with that of the other methanogenic strains was probably owing to the low growth rate of M. arboriphilus A2. The low value of Y(X/CH(4) ) may be favorable for methane fermentation because less sludge accumulation is expected. The efficiency of free energy transduction to ATP during methane formation from H(2) + CO(2) was 12-17% at the dilution rate (0.21-0.43 day(-1)) assuming that Y(ATP) was 6.5 g/mol and the free energy change of CO(2) reduction to methane with H(2) was -62.8 kJ/mol under physiological conditions.     

Citric acid production by Yarrowia lipolytica: Effect of nitrogen and biomass concentration on yield and productivity

Summary Citric acid yields of Yarrowia lipolytica (NRRL Y-1095) grown on glucose ranged from 0.38–0.77 g/g and were dependent on both biomass and nitrogen concentration (as NH₄Cl and yeast extract). Increasing the biomass concentration by 3% (w/v) increased fermentor productivities from 0.6 to 1.22 g citric acid/L h.