Evaluation of single cell oil from Aureobasidium pullulans var. melanogenum P10 isolated from mangrove ecosystems for biodiesel production

In this study, the yeast strain P10 which was identified to be a member of Aureobasidium pullulans var. melanogenum isolated from the mangrove ecosystems was found to be able to accumulate high content of oil in its cells. After optimization of the medium for lipid production and cell growth by the yeast strain P10, it was found that 8.0 g of glucose per 100 ml, 0.02 g of yeast extract per 100 ml, 0.02 g of ammonium sulfate per 100 ml, pH 6.0 in the medium were the most suitable for lipid production. During 10-l fermentation, a titer was 66.3 g oil per 100 g of cell dry weight, cell mass was 1.3 g per 100 ml, a yield was 0.11 g of oil per g of consumed sugar and a productivity was 0.0009 g of oil per g of consumed sugar per h within 120 h. At the same time, only 0.07 g of reducing sugar per 100 ml was left in the fermented medium. The compositions of the fatty acids produced were C_16:0 (26.7%), C_16:1(1.7%), C_18:0 (6.1%), C_18:1 (44.5%), and C_18:2 (21.0%). The biodiesel produced from the extracted lipid could be burnt well.     

Characterization of marine microalga, Scenedesmus sp. strain JPCC GA0024 toward biofuel production

A marine microalga, strain JPCC GA0024 was selected as high amount of neutral lipid producers from marine microalgal culture collection toward biofuel production. The strain was tentatively identified as Scenedesmus rubescens by 18S rDNA analysis. The growth of strain JPCC GA0024 was influenced by artificial seawater concentrations. The optimum growth of 0.79 g/l was obtained at 100% artificial seawater. The lipid accumulation reached 73.0% of dry cell weight at 100% artificial seawater without additional nutrients for 11 days. Gas chromatography/mass spectrometry analysis indicates that lipid fraction mainly contained hydrocarbons including mainly hexadecane (C₁₆ H₃₄) and 1-docosene (C₂₂ H₄₄). Furthermore, calorimetric analysis revealed that the energy content of strain JPCC GA0024 was 6,160 kcal/kg (25.8 MJ/kg) of calorific value, which was equivalent to the coal engery. The strain JPCC GA0024, S. rubescens, will become a promising resource that can grow as a dominant species in the seawater for the production of both liquid and solid biofuels.     

Single cell oil production from hydrolysates of inulin and extract of tubers of Jerusalem artichoke by Rhodotorula mucilaginosa TJY15a

In this study, we found that Rhodotorula mucilaginosa TJY15a could accumulate 48.8% (w/w) oil from hydrolysate of inulin and its cell dry weight reached 14.8 g/l during the batch cultivation while it could accumulate 48.6% (w/w) oil and 52.2% (w/w) oil from hydrolysate of extract of Jerusalem artichoke tubers and its cell dry weight reached 14.4 g/l and 19.5 g/l during the batch and fed-batch cultivations, respectively. At the end of the fed-batch cultivation, only 0.04% of reducing sugar and 0.08% of total sugar were left in the fermented medium. Over 87.6% of the fatty acids from the yeast strain TJY15a cultivated in the hydrolysate of extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (54.7%). Therefore, the results show that hydrolysates of inulin and extract of Jerusalem artichoke tubers were also the good materials for single cell oil production.     

Production of polyhydroxyalkanoates by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> UWD using swine wastewater: Effect of supplementing glucose,yeast extract,and inorganic salts

This study examined the effect of adding glucose, yeast extract, and inorganic salts to swine wastewater (SWW) in a batch culture on the production of a biodegradable plastic, polyhydroxyalkanoate (PHA). A bacterial strain, Azotobacter vinelandii UWD, was used to produce PHA without limiting the non-carbon nutrients. The addition of glucose (30 g/L) to the SWW medium increased the level of cell growth (4.4∼7.0 times) and PHA production (3.8∼8.5 times) depending upon the dilution of SWW. A 50% dilution of SWW was found to be optimal considering the dry cell weight (9.40 g/L), PHA content (58 wt%), and hydroxyvalerate (HV) mol fraction in the PHA (4.3 mol%). A 75% SWW medium was more advantageous for producing PHA with a higher HV fraction (7.1 mol%) at the expense of losing 22% of PHA production. The undiluted SWW medium produced less than one third of the PHA compared with the 50% SWW medium, but the HV fraction was the highest (10.8 mol%). Regarding the effect of the glucose concentration, at 20 g/L glucose, the dry cell weight and level of PHA production increased to 9.34 g/L (0.63 g PHA/g dry cell weight) and 5.90 g/L, respectively. At 50 g/L glucose, there was no significant increase in PHA production. For the glucose-supplemented (30 g/L) 50% SWW medium, the addition of a nitrogen source (1 g/L of yeast extract) did not increase the level of cell growth or PHA production because the C:N ratio (23:1) was already close to the optimal value (22:1). Better aeration increased the productivity of PHA. External nitrogen supplements (1 g/L of yeast extract) and other essential mineral salts was not necessary for bacterial growth because they were contained in the SWW. These results suggest that SWW is an excellent feedstock for producing larger amounts of the value-added material, PHA, if it is combined with carbohydrate-rich organic waste.     

Phytase production by fermentation of recombinant Pichia pastoris in monosodium glutamate wastewater

A novel method is proposed to produce both phytase and single-cell protein in recombinant Pichia pastoris fermentation using monosodium glutamate wastewater (MSGW) as the basal medium. Recombinant P. pastoris MR33 transformed with a phytase gene (AppA-m) from Escherichia coli was constructed and showed capability to utilize ammonium as the only nitrogen source. The fermentation medium was optimized in shake flasks by single-factor test and response surface methodology. A fed-batch system containing 30% MSGW, 50 g/l glucose, 1.58 g/l CaSO₄, 5.18 g/l MgSO₄ and 6.67 g/l KH₂PO₄ was developed in a 3.7-l bioreactor. The maximum phytase activity in the MSGW medium reached 3,380 U/ml, 84.2% of that in chemically defined medium, and the dry cell weight was 136 g/l. The single-cell protein (SCP; 46.66% dry cell weight) contains a variety of amino acids and is low in fat, which is ideal for utilization in animal feed. Thus, it is feasible to use MSGW medium for the production of enzymes that can be expressed in P. pastoris.     

Cassava as a Cheap Source of Carbon for Rhizobial Inoculant Production using an Amylase-producing Fungus and a Glycerol-producing Yeast

Summary The aim of this research was to develop methods to use low-cost carbon compounds for rhizobial inoculant production. Five raw starch materials; steamed cassava, sticky rice, fresh corn, dry corn and sorghum were tested for sugar production by an amylase-producing fungus. Streamed cassava produced the highest amount of reducing sugar after fermentation. Bradyrhizobium japonicum USDA110, Azorhizobium caulinodans IRBG23, Rhizobium phaseoli TAL1383, Sinorhizobium fredii HH103, and Mesorhizobium ciceri USDA2429 were tested on minimal medium supplemented with reducing sugar obtained from cassava fermentation. All strains, except B. japonicum USDA110, could grow in medium containing cassava sugar derived from 100 g steamed cassava per litre, and the growth rates for these strains were similar to those in medium containing 0.5 (w/v) mannitol. The sugar derived from steamed cassava was further used for production of glycerol using yeast. After 1 day of yeast fermentation, the culture containing glycerol and heat-killed yeast cells, was used to formulate media for culturing bradyrhizobia. A formulation medium, FM4, with a glycerol concentration of 0.6 g/l and yeast cells (OD₆₀₀ = 0.1) supported growth of B. japonicum USDA110 up to 3.61 × 10⁹ c.f.u./ml in 7 days. These results demonstrate that steamed cassava could be used to provide cheap and effective carbon sources for rhizobial inoculant production.     

Heavy oils,principally long-chain n-alkanes secreted by Aureobasidium pullulans var. melanogenum strain P5 isolated from mangrove system

In this study, the yeast strain P5 isolated from a mangrove system was identified to be a strain of Aureobasidium pullulans var. melanogenum and was found to be able to secrete a large amount of heavy oil into medium. After optimization of the medium for heavy oil production and cell growth by the yeast strain P5, it was found that 120.0 g/l of glucose and 0.1 % corn steep liquor were the most suitable for heavy oil production. During 10-l fermentation, the yeast strain P5 produced 32.5 g/l of heavy oil and cell mass was 23.0 g/l within 168 h. The secreted heavy oils contained 66.15 % of the long-chain n-alkanes and 26.4 % of the fatty acids, whereas the compositions of the fatty acids in the yeast cells were only C_16:0 (21.2 %), C_16:1(2.8 %), C_18:0 (2.9 %), C_18:1 (39.8 %), and C_18:2 (33.3 %). We think that the secreted heavy oils may be used as a new source of petroleum in marine environments. This is the first report of yeast cells which can secrete the long-chain n-alkanes.     

Inulinase production by a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the crude inulinase

Marine yeast strain 1, isolated from the surface of a marine alga, was found to secrete a large amount of inulinase into the medium. This marine yeast was identified as a strain of Pichia guilliermondii according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast worked optimally at pH 6.0 and 60°C. The optimal medium for inulinase production was seawater containing 4.0% (w/v) inulin and 0.5% (w/v) yeast extract, while the optimal cultivation conditions for inulinase production were pH 8.0, 28°C and 170 rpm. Under the optimal conditions, over 60 U ml⁻¹ of inulinase activity was produced within 48 h of fermentation in shake flasks. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis, indicating that the crude inulinase had a high exoinulinase activity.     

Metabolic behaviour of aflatoxin producing strain and non-toxigenic strain of Aspergillus flavus to different sources of nitrogen and glucose concentration

The effect of different nitrogen sources and varying glucose concentration on aflatoxin production by a toxigenic and non-toxigenic strain of Aspergillus flavus was studied. Greatest production (3.8 ppm) of aflatoxin B₁ was produced in a synthetic medium when casamino acids were supplied as the nitrogen source. Optimum sugar concentration for aflatoxin B₁ production ranged between 3 and 10 g/100 ml. There was no appreciable difference in the metabolic behaviour between toxigenic and non-toxigenic strains of A. flavus when dry mycelial weight, total proteins, non-protein nitrogen and reducing sugar were the criteria.     

Gene analysis,optimized production and property of marine lipase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> B106 associated with South China Sea sponge <Emphasis Type="Italic">Halichondria rugosa</Emphasis>

Gene cloning, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa were investigated in this paper. A lipase gene with whole ORF encoding 215 amino acids was obtained by PCR, protein domain prediction suggested that the deduced lipase belongs to α/β hydrolases family. Based on single factor Seriatim-Factorial test and Plackett–Burman experimental design, the optimal medium consisted of (per l) 12.5 ml maize oil, 5.0 g beef extract, 2.0 g PO₄ ³⁻ (0.6 g KH₂PO₄, 1.4 g K₂HPO₄), 17.15 g Mg²⁺, 5.0 g yeast extract, 2.282 g CaCl₂ and 5.0 ml Tween80 with artificial sea water. Using this optimum medium, lipase activity and cell concentration were increased by 3.54- and 1.31-fold over that of the basal medium, respectively. This lipase showed tolerance to high salinity, pH and temperature. About 10–20% methanol exhibited a stimulatory effect on the lipase activity, while activity was inhibited by 30–40% methanol, 2-propanol, DMSO, and ethanol. This study provides a valuable resource for marine lipase production and extends our understanding of the possible role of sponge-associated bacteria in the biotransformation of chemical compounds for the sponge host.     

Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells

In this study, it was found that the immobilized inulinase-producing cells of Pichia guilliermondii M-30 could produce 169.3 U/ml of inulinase activity while the free cells of the same yeast strain only produced 124.3 U/ml of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells were co-cultivated with the free cells of Rhodotorula mucilaginosa TJY15a, R. mucilaginosa TJY15a could accumulate 53.2% oil from inulin in its cells and cell dry weight reached 12.2 g/l. Under the similar conditions, R. mucilaginosa TJY15a could accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/l within 48 h. When the co-cultures were grown in 2 l fermentor, R. mucilaginosa TJY15a could accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 19.6 g/l within 48 h. Over 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (50.6%).     

Isolation of a yeast growing on sardine oil and its characteristics

A yeast strain, FO-144Cl, was isolated from a soil sample, using crude sardine oil, which contains a large quantity of poly-unsaturated long-chain fatty acids, as a sole carbon source. This strain was identified as a species of Candida. A medium for its growth was optimized by statistical methods and optimal temperature for the growth was from 28 to 30°C. Among the natural oils and fats tested, the yeast grew best on olive oil and grew better on the crude sardine oil than on a refined one. The yield of dry cells was 17.6 mg/ml after 24 h, using 2% crude sardine oil. The maximum growth rate was 0.36, 0.25, and 0.21 h⁻¹ with crude sardine oil, soybean oil, and olive oil, respectively. The content of crude fat in the yeast cells was 15.1% and half of the total cell lipid was triglyceride. Fatty acid compositions of the lipid and oily fractions left in the medium after cultivation were analyzed. Little unsaturated long-chain fatty acids (>C₁₈) was observed in the cell lipids, but they were left concentrated in the medium.     

Simultaneous production of single cell protein and killer toxin by Wickerhamomyces anomalus HN1-2 isolated from mangrove ecosystem

The yeast Wickerhamomyces anomalus (the previous name was Pichia anomala) HN1-2 isolated from the mangrove ecosystem was found to be able to produce high level of both killer toxin and single cell protein. When the killer yeast cells were grown by batch cultivation in 5-l fermentor, crude protein in the cells, cell mass, reducing sugar, and diameter of the inhibition zone reached 56.0 g per 100 g of cell dry weight, 7.3 g per liter, 9.5 g per liter, and 19.0 mm, respectively within 12 h and this yeast synthesized a large amount of the essential amino acids, such as lysine (7.8%), methionine (1.8%), and leucine (9.0%). The crude killer toxin produced by the killer yeast isolate HN1-2 could kill the cells of Lodderomyces elongisporus, Candida albicans, Metschnikowia bicuspidata, Pichia guilliermondii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Kluyveromyces aestuarii, which were widely distributed in natural marine environments. The results also showed that the undesirable yeast could be avoided during cell growth of the killer yeast.     

Effects of particulate materials and osmoprotectants on very-high-gravity ethanolic fermentation by Saccharomyces cerevisiae.

The effects of osmoprotectants (such as glycine betaine and proline) and particulate materials on the fermentation of very high concentrations of glucose by the brewing strain Saccharomyces cerevisiae (uvarum) NCYC 1324 were studied. The yeast growing at 20 degrees C consumed only 15 g of the sugar per 100 ml from a minimal medium which initially contained 35% (wt/vol) glucose. Supplementing the medium with a mixture of glycine betaine, glycine, and proline increased the amount of sugar fermented to 30.5 g/100 ml. With such supplementation, the viability of the yeast cells was maintained above 80% throughout the fermentation, while it dropped to less than 12% in the unsupplemented controls. Among single additives, glycine was more effective than proline or glycine betaine. On incubating the cultures for 10 days, the viability decreased to only 55% with glycine, while it dropped to 36 and 27%, respectively, with glycine betaine and proline. It is suggested that glycine and proline, known to be poor nitrogen sources for growth, may serve directly or indirectly as osmoprotectants. Nutrients such as tryptone, yeast extract, and a mixture of purine and pyrimidine bases increased the sugar uptake and ethanol production but did not allow the population to maintain the high level of cell viability. While only 43% of the sugar was fermented in unsupplemented medium, the presence of particulate materials such as wheat bran, wheat mash insolubles, alumina, and soy flour increased sugar utilization to 68, 75, 81, and 82%, respectively.     

Overproduction of poly(β-malic acid) (PMA) from glucose by a novel Aureobasidium sp. P6 strain isolated from mangrove system

After over 100 strains of Aureobasidium spp isolated from mangrove system were screened for their ability to produce poly(β-malic acid) (PMA), it was found that Aureobasidium sp. P6 strain among them could produce high level of Ca²⁺-PMA. Fourteen percent glucose and 6.5 % CaCO₃ in the medium were the most suitable for Ca²⁺-PMA production. Then, 100.7 g/l of Ca²⁺-PMA was produced using Aureobasidium sp. P6 strain within 168 h at flask level. During 10-l batch fermentation, when the medium contained 12.0 % glucose, 98.7 g/l of Ca²⁺-PMA in the culture and 14.7 g/l of cell dry weight were obtained within 156 h, leaving 0.34 % reducing sugar in the fermented medium. When glucose concentration in the fermentation medium was 14.0 %, 118.3 g/l of Ca²⁺-PMA in the culture and 16.4 g/l of cell dry weight were obtained within 168 h, leaving 0.4 % reducing sugar in the fermented medium. After purification of Ca²⁺-PMA from the culture and acid hydrolysis of the pure Ca²⁺-PMA, analysis of HPLC showed that Aureobasidium sp. P6 strain only produced two main components of Ca²⁺-PMA and minor amount of calcium malate and that the hydrolysate of PMA was mainly composed of calcium malate. This is the first time to report that the novel yeast strain Aureobasidium sp. P6 strain isolated from the mangrove systems can produce such high amount of Ca²⁺-PMA.     

Availability of substratum enhances ethanol production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Novel additives that act as substratum for attachment of the yeast cells, increased ethanol production in Saccharomyces cerevisiae. The addition of 2 g rice husk, straw, wood shavings, plastic pieces or silica gel to 100 ml medium enhanced ethanol production by 30–40 (v/v). Six distillery strains showed an average enhancement of 34 from 4.1 (v/v) in control to 5.5 (v/v) on addition of rice husk. The cell wall bound glycogen increased by 40–50 mg g ^–1 dry yeast while intracellular glycogen decreased by 10–12 mg g^–1 dry yeast in cells grown in presence of substratum     

Isolation and Characterization of a Thermotolerant Methanol-Utilizing Yeast

A yeast capable of growth on methanol as its sole carbon-energy source was isoalted from soil samples and identified as a strain of Hansenula polymorpha. A continuous enrichment culture at 37 C with a simple mineral salts medium was used to select this organism. The isolate, designated DL-1, has a maximal specific growth rate of 0.22 per h, at pH 4.5 to 5.5 and temperatures of 37 to 42 C, in simple mineral salts medium with methanol (0.5%), biotin, and thiamine. Growth occurred in a chemostat at temperatures up to 50 C, with strong growth at 45 C. The maximal growth yield of the yeast on methanol was 0.36 g of dry cell weight per g of methanol, and the yield on oxygen was 0.37 g of dry cell weight per g of O(2). Protein content of the isolate is 46%, and total nucleic acid content varies from 5.0 to 7.0% with increasing growth rate from 0.08 to 0.20 per h. The amino acid profile of this yeast protein indicates that it could serve as a good source of food protein. Feeding studies with rats show the yeast to have no toxic effects.     

Production of polyesters consisting of medium chain length 3-hydroxyalkanoic acids by Pseudomonas mendocina 0806 from various carbon sources

Pseudomonas mendocina strain 0806 was isolated from oil-contaminated soil and found to produce polyesters consisting of medium chain length 3-hydroxyalkanoates (mclPHAs). The monomers of mclPHAs contained even numbers of carbon atoms, such as 3-hydroxyhexanoate (HHx or C₆), 3-hydroxyoctanoate (HO or C₈), and/or 3-hydroxydecanoate (HD or C₁₀) as major components when grown on many carbon sources unrelated to their monomeric structures, such as glucose, citric acid, and carbon sources related to their monomeric structures, such as myristic acid, octanoate, or oleic acid. On the other hand, PHA containing both even and odd numbers of hydroxyalkanoates (HA) monomers was synthesized when the strain was grown on tridecanoic acid. The molar ratio of carbon to nitrogen (C/N) had a significant effect on PHA composition: the strain produced PHAs containing 97–99% of HD monomer when grown in a glucose ammonium sulfate medium of C/N<20, and 20% HO, and 80% of the HD monomer when growth was conducted in media containing C/N>40. It was demonstrated that the HO/HD ratio in the polymers remained constant in media with a constant C/N ratio, regardless of the glucose concentration. Up to 3.6 g/L cell dry weight containing 45% of PHAs was produced when the strain was grown for 48 h in a medium containing 20 g/L glucose with a C/N ratio of 40.     

Inulinase production by the marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the crude inulinase

The marine yeast strain G7a isolated from sediment of China South Sea was found to secrete a large amount of inulinase into the medium. This marine yeast strain was identified to be a strain of Cryptococcus aureus according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast showed the highest activity at pH 5.0 and 50 °C. The optimal medium for inulinase production was artificial seawater containing inulin 4.0% (w/v), K₂HPO₄ 0.3% (w/v), yeast extract 0.5% (w/v), KCl 0.5% (w/v), CaCl₂ 0.12% (w/v), NaCl 4.0% (w/v) and MgCl₂·6H₂O 0.6% (w/v), while the optimal cultivation conditions for inulinase production were pH 5.0, a temperature of 28 °C and a shaking speed of 170 rpm. Under the optimal conditions, over 85.0 U/ml of inulinase activity was produced within 42 h of fermentation at shake flask level. This is very high level of inulinase activity produced by yeasts. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.     

Torularhodin and torulene are the major contributors to the carotenoid pool of marine Rhodosporidium babjevae (Golubev)

A carotenoid-producing yeast strain, isolated from the sub-arctic, marine copepod Calanus finmarchicus, was identified as Rhodosporidium babjevae (Golubev) according to morphological and biochemical characteristics and phylogenetic inference from the small-subunit ribosomal RNA gene sequence. The total carotenoids content varied with cultivation conditions in the range 66–117 μg per g dry weight. The carotenoid pool, here determined for the first time, was dominated by torularhodin and torulene, which collectively constituted 75–91% of total carotenoids under various regimes of growth. β-Carotene varied in the range 5–23%. A high-peptone/low-yeast extract (weight ratio 38:1) marine growth medium favoured the production of torularhodin, the carotenoid at highest oxidation level, with an average of 63% of total carotenoids. In standard yeast medium (YM; ratio 1.7:1), torularhodin averaged 44%, with increased proportions of the carotenes, torulene and β-carotene. The anticipated metabolic precursor γ-carotene (β,ψ-carotene) constituted a minor fraction (≤8%) under all conditions of growth.