Effect of culture media and conditions on polyunsaturated fatty acids production by Mortierella alpina

To improve the polyunsaturated fatty acid (PUFA) production by Mortierella, culture media and conditions were investigated. M. alpina ATCC 32222 had the highest yield of arachidonic acid, gamma-linolenic acid and linoleic acid among 11 test microbes. Soluble starch at 10% and the mixture of KNO3 and yeast extract at 2:1 (w/w) was the best carbon and nitrogen sources for arachidonic acid and total PUFAs production, respectively. The optimal C/N ratio ranged from 5.1 to 9.0. Each gram of carbon produced 17.4 mg of linoleic acid, 17.0 mg of gamma-linolenic acid, 103.0 mg of arachidonic acid and 194.2 mg of total PUFAs at 20 degrees C, while it yielded 21.4 mg of linoleic acid, 25.6 mg of gamma-linolenic acid, 2.6 mg of gamma-linolenic acid, 110.3 mg of arachidonic acid, 4.3 mg of eicosapentaenoic acid and 218.4 mg of total PUFAs at 12 degrees C. A high degree of unsaturation was found at low temperature incubation. Linseed oil supplementation (1%, w/v) increased the PUFAs production and each gram of carbon produced 403.4 mg of alpha-linolenic acid, 123.1 mg of arachidonic acid, 33.6 mg of eicosapentaenoic acid, 1.68 mg of docosahexaenoic acid and 943.2 mg of total PUFAs. From the optimization of culture media and conditions, PUFAs production increased from 30% to 5 times that was optimal for practical use.     

Thermo-tolerant phosphate-solubilizing microbes for multi-functional biofertilizer preparation

In order to prepare the multi-functional biofertilizer, thermo-tolerant phosphate-solubilizing microbes including bacteria, actinomycetes, and fungi were isolated from different compost plants and biofertilizers. Except Streptomycesthermophilus J57 which lacked pectinase, all isolates possessed amylase, CMCase, chitinase, pectinase, protease, lipase, and nitrogenase activities. All isolates could solubilize calcium phosphate and Israel rock phosphate; various isolates could solubilize aluminum phosphate, iron phosphate, and hydroxyapatite. During composting, biofertilizers inoculated with the tested microbes had a significantly higher temperature, ash content, pH, total nitrogen, soluble phosphorus content, and germination rate than non-inoculated biofertilizer; total organic carbon and carbon-to-nitrogen ratio showed the opposite pattern. Adding these microbes can shorten the period of maturity, improve the quality, increase the soluble phosphorus content, and enhance the populations of phosphate-solubilizing and proteolytic microbes in biofertilizers. Therefore, inoculating thermo-tolerant phosphate-solubilizing microbes into agricultural and animal wastes represents a practical strategy for preparing multi-functional biofertilizer.     

Polyunsaturated fatty acids production with a solid-state column reactor

To investigate the potential production of polyunsaturated fatty acids (PUFAs), a solid-state column reactor of rice bran with Mortierella alpina was used. The optimal conditions for PUFAs production were rice bran supplementation with 3.75% (ww(-1)) nitrogen source at initial moisture content 57%, initial pH 6-7, aeration, and incubation at 20 degrees C for 5 days and then at 12 degrees C for 7 days. Each gram of substrate carbon yielded 127 mg of total PUFAs, 12 mg of eicosapentaenoic acid (EPA), 6 mg of arachidonic acid (AA), 5mg of alpha-linolenic acid (ALA), and 117 mg of linoleic acid (LA) after 12 days incubation. Aeration enhanced the productions of AA, EPA, and total PUFAs. Supplementation of the nitrogen source on the fourth day and then a shift to lower temperature on the fifth day increased EPA production.     

Effect of culture media on protease and oxytetracycline production with mycelium and protoplasts of Streptomyces rimosus

For the simultaneous production of protease and oxytetracycline, mycelium and protoplasts of Streptomyces rimosus TM-55 were cultivated in basal medium containing soluble starch, corn steep liquid, ammonium sulphate, calcium carbonate, sodium chloride and soybean oil. Protease and oxytetracycline production increased with decreasing in ratio of culture broth to vessel volume from 1:2 to 1:5. Each ml of broth with 0.286 mg fresh mycelia yielded 168–204 units of protease and 785–972 g of oxytetracycline after replacement of corn steep liquor, sodium chloride and soybean oil with beef extract and sunflower oil, while each ml of broth with 7.5 × 10⁷ protoplasts produced 141–153 units of protease and 504–615 g of oxytetracycline. Protease and oxytetracycline production were low when the pH was 5.1 or 9.0. Soluble starch and ammonium sulphate were the best carbon and nitrogen sources, respectively. Supplementation with calcium carbonate enhanced protease and oxytetracycline production. The productivity of protoplasts decreased sharply when the incubation temperature increased from 28 to 34 °C, while the productivity of mycelium was almost unchanged.     

Cloning and expression of the α-amylase gene and oxytetracycline production in Streptomyces rimosus

An 8.4 kb Sau3AI DNA fragment containing the Streptomyces rimosus TM-55 -amylase gene (amy) was ligated to a vector pIJ702, named pCYL01, and cloned into amylase deficient mutant S. lividans M2 (amy ^–). Subcloning study showed that the amy gene was localized in 3.3 kbKpnI-PstI fragment. The molecular weight of the purified -amylases of S. lividans M2/pCYL01 and S. rimosus TM-55 were estimated to be 65.7 kDa. Different sizes of recombinant plasmids carrying the amy gene had been retransferred into the parental strain of S. rimosus TM-55. Among these S. rimosus transformants, TM-55/pCYL01, TM-55/pCYL12 and TM-55/pCYL36 showed amylase activity 1.36- to 2.05-fold at the seventh day (1.61 to 2.42 units vs 1.18 units), and oxytetracycline (OTC) production 2.00- to 2.50-fold at the ninth day (approximate 140 to 170 g ml^–1 vs 72 g ml^–1), higher than that of S. rimosus TM-55 alone, respectively. These results showed that industrial microorganisms could be improved by genetic and metabolic engineering.     

Cloning, characterization and phylogenetic relationships of stxI, a endoxylanase-encoding gene from Streptomyces thermonitrificans NTU-88

A thermostable xylanase gene (stxI) obtained from Streptomyces thermonitrificans NTU-88 on domain analysis revealed an N-terminal catalytic domain featuring homology to a known xylanase within the glycoside hydrolase family 11. Recombinant STXI retained more than 60% of its activity following its incubation for at 60 degrees C for 24h. These characteristics were close to thermophile and mesophile Streptomyces strains. The main hydrolysis products of xylan degraded by STXI included large xylooligosaccharide fragments. These results indicated that STXI was a typical endoxylanase. As regards the phylogenetic relationships of GH11, STXI and the other xylanase deriving from Streptomyces were included in a subgroup of the aerobic bacterial group. This result implied that the evolutionary relationships between the various xylanases deriving from Streptomyces strains were convergent.     

The identification,purification, and characterization of STXF10 expressed in <Emphasis Type="Italic">Streptomyces thermonitrificans</Emphasis> NTU-88

Multiple xylanolytic enzymes of Streptomyces thermonitrificans NTU-88 were induced by oat-spelt xylan and separated by two-dimensional polyacrylamide and zymogram gels. Nineteen clear spots differed in pI and molecular weight values were found on the zymogram, and only spot one was seen on the corresponding silver-stained gel. These results revealed that multiple xylanases were secreted when S. thermonitrificans NTU-88 was induced and the spot (STXF10), identified as being a glycosyl hydrolase family 10 xylanase, was the predominant one among xylanases. STXF10 showed a tolerance for high temperatures and broad pH ranges and high affinity and hydrolysis efficiency for xylans. Furthermore, it also featured the minor ability to degrade different lignocellulosic substrates. Although S. thermonitrificans NTU-88 possesses multiple xylanases, our results suggest that the major form of xylanase might be selectively and specifically induced depending on the type of substrate to which the microorganism is exposed.     

Microbial diversity of topographical gradient profiles in Fushan forest soils of Taiwan

To evaluate the microbial diversity of Fushan forest soils, the variation of soil properties, microbial populations, and soil DNA with soil depth in three sites of different altitude were analyzed. Microbial population, moisture content, total organic carbon (C_org), and total nitrogen (N_tot) decreased with increasing soil depth. The valley site had the lowest microbial populations among the three tested sites due to the low organic matter content. Bacterial population was the highest among the microbial populations. The ratios of cellulolytic microbes to the total bacteria in organic layers were high, implying their roles in the carbon cycle. The microbial biomass carbon (C_mic) and nitrogen (N_mic) contents ranged from 130.5 to 564.1 μg g⁻¹ and from 16.7 to 95.4 μg g⁻¹, respectively. The valley had the lowest C_mic and N_mic. The organic layer had the highest C_mic and N_mic and decreased with soil depth. Analysis using denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplicons of 16S rDNA showed that the bacterial diversity of the three sites were very similar to each other in the major bands, and the variation was in the minor bands. However, the patterns in PCR-DGGE profile through gradient horizons were different, indicating the prevalence of specific microbes at different horizons. These results suggest that the microbial diversity in the deeper horizons is not simply the diluted analogs of the surface soils and that some microbes dominate only in the deeper horizons. Topography influenced the quantity and diversity of microbial populations.