Production of a new exopolysaccharide (EPS) by Pseudomonas oleovorans NRRL B-14682 grown on glycerol

Pure glycerol and glycerol-rich product (GRP) obtained from the biodiesel industries were used as carbon source for the production of a new extracellular polysaccharide (EPS) by Pseudomonas oleovorans NRRL B-14682. The influence of temperature (20–40 °C) and pH (6.0–8.0) was studied. A temperature of 30 °C and pH control at 6.8 gave the maximum cell growth and EPS production. The culture attained a maximum cell dry weight (CDW) of 9.55 g l⁻¹ and an EPS concentration of 11.82 g l⁻¹ when cultivated with pure glycerol. GRP was a suitable carbon source, as shown by the slightly higher EPS concentration (12.18 g l⁻¹). The EPS productivity obtained with GRP (3.85 g l⁻¹ d⁻¹) was almost twice that obtained with pure glycerol (2.00 g l⁻¹ d⁻¹). Also, the yield on glycerol was higher for the cultivation with GRP (0.36 g g⁻¹) than for pure glycerol (0.28 g g⁻¹). The EPS was a high molecular weight heteropolysaccharide, composed by neutral sugars (37–80 wt% galactose, 2–30 wt% glucose, 0.5–25 wt% mannose and 0.5–20 wt% rhamnose) and containing acyl group substituents (pyruvil, acetyl and succinyl were identified). The EPS forms highly viscous aqueous dispersions with many potential commercial applications.     

Production,characterization and antioxidant activities in vitro of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3

The production, characterization and antioxidant activities in vitro of exopolysaccharides (EPS) from endophytic bacterium Paenibacillus polymyxa EJS-3 were investigated. For EPS production, the preferable culture conditions were 24 °C and pH 8 for 60 h with sucrose and yeast extract as the carbon and nitrogen sources, respectively. Notably, sucrose concentration was the prominent factor, and the maximum yield of EPS (22.82 g/L) was obtained at a sucrose concentration of 160 g/L. The crude EPS was purified by chromatography of DEAE-52 and Sephadex G-100, affording EPS-1 and EPS-2 with molecular weights of 1.22 × 10⁶ and 8.69 × 10⁵ Da, respectively. They were composed of mannose, fructose and glucose in a molar ratio of 2.59:29.83:1 and 4.23:36.59:1, respectively. In addition, both crude and purified EPS showed strong scavenging activities on superoxide and hydroxyl radicals, and their antioxidant activities decreased in the order of crude EPS > EPS-2 > EPS-1.     

Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris

The influence of initial pH value, various nitrogen sources, plant oils, and modes of propagation (shake-flask and static culture) on the production of biomass, exopolysaccharide (EPS), adenosine and, in particular, cordycepin, by Cordyceps militaris CCRC 32219 were investigated. Optimal conditions for mycelial growth, EPS and cordycepin production were observed at relatively low pH. Amongst organic sources, yeast extract (YE) was favorable for EPS and cordycepin production, while corn steep powder (CSP) was favorable for adenosine production. A lower C/N ratio was favorable for adenosine and cordycepin production; however, too low a C/N ratio led to diminished production. All plant oils tested stimulate mycelial growth and EPS production of C. militaris, but they did not show much effect on the adenosine and cordycepin production. A two-stage fermentation process by combining shake-flask fermentation with static culture significantly enhanced cordycepin production. A Box–Behnken experimental design was employed to optimize the production of cordycepin, which showed that the optimum conditions to produce cordycepin by C. militaris CCRC 32219 were at pH 6, YE concentration of 45 g/l and 8.0 day of the shake culture followed by 16 days of the static culture. Under the optimized conditions, the maximum production (2214.5 mg/l) of cordycepin was obtained, which is much higher than those reported up to date.     

Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments

Protease producing halotolerant bacterium was isolated from saltern pond sediment (Tuticorin) and identified as Bacillus licheniformis (TD4) by 16S rRNA gene sequencing. Protease production was enhanced by optimizing the culture conditions. The nutritional factors such as carbon and nitrogen sources, NaCl and also physical parameters like incubation time, pH, agitation, inoculum size were optimized for the maximum yield of protease. Studies on the effect of different carbon and nitrogen sources revealed that xylose and urea enhances the enzyme production. Thus, with selected C–N sources along with 1 M NaCl the maximum protease production (141.46 U/mg) was obtained in the period of 24 h incubation at pH 8 under 250 rpm compared to the initial enzyme production (89.87 U/mg).     

Estimation of genetic parameters for post-weaning traits of Kermani sheep

The objective of the present study was to estimate genetic parameters for post-weaning traits in Kermani sheep. Traits were included 6-month weight (6MW), 9-month weight (9MW), yearling weight (YW), greasy fleece weight at first shearing (GFW) and greasy fleece weights at various shearings (RFW). Data and pedigree information used in this research were collected at Breeding Station of Kermani sheep during 1993–2004. Genetic parameters were estimated with single- and multi-traits analysis using restricted maximum likelihood (REML) procedures, under animal models. Log likelihood ratio test indicated the most appropriate model for 6MW and 9MW should included direct additive genetic effects as well as maternal permanent environmental effects. However the most appropriate model for YW and GFW had only the direct additive genetic effects. The effects of sex, age of dam and year of birth were significant on body weight traits (P < 0.01). GFW was influenced significantly by sex and year of birth (P < 0.01) but was not affected by age of dam (P > 0.05). Type of birth was no significant effect on studied traits (P > 0.05). Also, the age of lamb at weighing time was a significant influence on 6MW, 9MW and YW. Direct heritability estimates for 6MW, 9MW, YW and GFW were 0.32, 0.03, 0.15 and 0.15, respectively. Maternal permanent environmental estimates of 0.09 were obtained for 6MW and 9MW. Genetic correlation estimates between mentioned traits ranged from 0.51 to 0.99. Phenotypic correlations were generally lower than those of genetic correlation and varied from 0.05 to 0.79 for various traits. The environmental correlations estimates between GFW with growth traits were low, but between other traits were positive and high, ranged from 0.54 to 0.72. The value of repeatability estimated for greasy fleece weight was 0.22.     

Biosynthesis of selenium-containing polysaccharides with antioxidant activity in liquid culture of Hericium erinaceum

The effects of inorganic selenium (sodium selenite) and Selol containing selenitetriglycerides synthesized from sunflower oil on mycelial growth and selenium-containing extracellular (EPS) and intracellular (IPS) polysaccharides production were examined in shake flask cultures of Hericium erinaceum.Unlike sodium selenite which inhibited mycelial growth, Selol increased in biomass production in a dose-dependent manner. Selol also dramatically enhanced EPS formation to 2.25 g/L which is 2.5–fold higher than in the control. Selenium content in EPS and IPS obtained from Selol-enriched medium reached a maximum of 4.89 and 4.69 mg/g, respectively.The in vitro antioxidant activities of polysaccharides were evaluated by reducing power, inhibition of lipid peroxidation, and 1,1-diphenyl-dipicrylhydrazyl radicals scavenging assays. The selenium-containing EPS showed an excellent antioxidant activity correlated well with increasing concentrations.The results suggested that selenium-containing EPS from H. erinaceum submerged culture should be explored as a novel selenium source in dietary supplements, with potent antioxidant properties.     

Optimization of biosurfactant production using waste from biodiesel industry in a new membrane assisted bioreactor

The main byproduct of biodiesel production is glycerol. Here, crude glycerol – byproduct of biodiesel industry – was evaluated as sole carbon source in rhamnolipids production by Pseudomonas aeruginosa. The optimal concentration of crude glycerol and sodium nitrate was assessed using response surface methodology, resulting in about 40–50 mg/L.h of rhamnolipids, which was about four times higher than previously reported in the literature. Fermentation parameters were similar to those observed with commercial glycerol as sole carbon source. The optimized medium was suitable for production using simple (22.9 mg/L.h) and fed-batch (32.4 mg/L.h) fermentation in oxygen-controlled bioreactor without foaming formation. Composition and relative abundance of rhamnolipid congeners showed that crude glycerol had little effect on metabolic pathways involved in their production. CMC values were approximately 130 mg/L and 230–260 mg/L for rhamnolipids from crude and commercial glycerol fermentation, respectively, which were about 2–6 times lower than CMC values of synthetic surfactants.     

Statistical media optimization for growth and PHB production from methanol by a methylotrophic bacterium

Media components were optimized by statistical design for cell growth and PHB production of Methylobacterium extorquens DSMZ 1340. Four important components of growth media were optimized by central composite design. The growth increased from an OD = 1.35 for Choi medium as control to an OD = 2.15 for optimal medium. Then media components for PHB production were optimized. Optimization of five important factors was conducted by response surface method. The optimal composition of PHB production medium was found to be at 7.8 (g/L) Na₂HPO₄ · 12H₂O, and surprisingly at zero concentration of (NH₄)₂SO₄, KH₂PO₄, MgSO₄ and MnSO₄. The PHB production was found to be 2.95 (g/L) at this medium. RSM results indicated that a deficiency of nitrogen and magnesium is crucial for PHB accumulation in this microorganism. Also, PHB production was carried out in a 5 L fermentor at the optimum condition which resulted in 9.5 g/L PHB and 15.4 g/L cell dry weight with 62.3% polymer content.     

The P170 expression system enhances hyaluronan molecular weight and production in metabolically-engineered Lactococcus lactis

Recombinant Lactococcus lactis strains based on the P170 expression system were developed for hyaluronan (HA) production, by incorporating genes from the has operon of Streptococcus zooepidemicus and compared with nisin-inducible recombinant L. lactis strains containing the hasABC and hasABD constructs. It was found across all batch and fed-batch experimental studies that HA concentration and molecular weight (MW) were higher for the P170 expression systems than the corresponding NICE-based strains. The highest hyaluronan MW was obtained for all constructs in batch studies at 60 g/L initial glucose concentration, the highest being 2.94 MDa for the P170 strains with hasABC construct (L. lactis APJ3). In fed-batch studies with constant feed rate, the L. lactis APJ3 gave better HA yield (0.03 g/g) than the NICE-based strain. A higher hyaluronan MW was obtained for all strains in pulse fed-batch compared to constant feed experiments, the highest being 2.52 MDa for L. lactis APJ3.     

Optimization and immobilization of amylase obtained from halotolerant bacteria isolated from solar salterns

The objective of the present study was to isolate halotolerant bacteria from the sediment sample collected from Marakanam Solar Salterns, Tamil Nadu, India using NaCl supplemented media and screened for amylase production. Among the 22 isolates recovered, two strains that had immense potential were selected for amylase production and designated as P1 and P2. The phylogenetic analysis revealed that P1 and P2 have highest homology with Pontibacillus chungwhensis (99%) and Bacillus barbaricus (100%). Their amylase activity was optimized to obtain high yield under various temperature, pH and NaCl concentration. P1 and P2 strain showed respective, amylase activity maximum at 35 °C and 40 °C; pH 7.0 and 8.0; 1.5 M and 1.0 M NaCl concentration. Further under optimized conditions, the amylase activity of P1 strain (49.6 U mL^?1) was higher than P2 strain. Therefore, the amylase enzyme isolated from P. chungwhensis P1 was immobilized in sodium alginate beads. Compared to the free enzyme form (49.6 U mL^?1), the immobilized enzyme showed higher amylase activity as 90.3 U mL^?1. The enzyme was further purified partially and the molecular mass was determined as 40 kDa by SDS–PAGE. Thus, high activity of amylase even under increased NaCl concentration would render immense benefits in food processing industries.     

Fast conversion of glycerol to 1,3-propanediol by a new strain of Klebsiella pneumoniae

Five bacterial strains screened from a batch of 39 samples could convert glycerol anaerobically to 1,3-propanediol (1,3-PD). One of the strains, XJ-Li, which could synthesize 1,3-PD with a higher concentration, was identified and characterized. Phylogenetic analysis of the strain XJ-Li included the study of morphology, physiological and biochemical characteristics. In addition, 16SrDNA sequences were created. The results indicated that this strain is a member of Klebsiella pneumoniae. The optimal cultivation parameters for pH and temperature were determined as 8.0 and 40 °C, respectively. The optimized nitrogen source and carbon source were 6.0 g/L of (NH₄)₂SO₄ and 20 g/L of glycerol, respectively. After 8 h in batch fermentation, both the 1,3-PD concentration and glycerol consumption reached the maximum, with 12.2 g/L of 1,3-PD and 1.53 g/L h of productivity, and a molar yield of 1,3-PD to glycerol of 0.75. Fed-batch fermentation also indicated a higher molar yield of 0.70, and the concentration of 1,3-PD reached 38.1 g/L after 66.4 g/L of glycerol consumption. The results of batch and fed-batch fermentations demonstrated that K. pneumoniae XJ-Li would be an excellent 1,3-PD producer.     

Development of a process for the production of nutrient supplements for fermentations based on fungal autolysis

This study verifies the potential of fungal autolysis as an alternative process for the production of nutrient-rich solutions similar to yeast extracts. Autolytic experiments were carried out on fermentation solids derived from either batch or continuous submerged cultivations of Aspergillus awamori on various wheat flour milling streams. The degree of autolysis was not affected by the pH range used (3–6.5), whereas it was severely affected by temperature (30–55 °C), initial solids concentration (10–45 g/L) and incubation time. The enzymatic disruption of the fungal cell wall was identified by image analysis as well as by the reduction in total dry weight and the gradual release of various components, such as free amino nitrogen and phosphorus. The novel method of autolysate recycling enabled the enrichment of the solution with lytic enzymes leading to increased fungal cell degradation rates. In this way, it was made possible to reduce the initial total dry weight by 47% and produce a nutrient-rich solution containing 1.6 g/L free amino nitrogen, 5.3 g/L total nitrogen and 0.5 g/L phosphorus.     

Enhancing production of prodigiosin from Serratia marcescens C3 by statistical experimental design and porous carrier addition strategy

Serratia marcescens C3 produces a natural red-pigment, prodigiosin, which exhibits immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. This work seeks to improve the production of prodigiosin by S. marcescens C3 using various strategies. Starch and peptone were identified as the optimized carbon and nitrogen sources for the production of prodigiosin, yielding a prodigiosin concentration of 2.3 g/L. This value was significantly increased to 6.7 g/L using a carbon/nitrogen ratio of 6/4 (starch/peptone = 16 g/L/10.67 g/L). To enhance prodigiosin production even further, a statistical experimental design methodology was utilized to optimize the composition of the culture medium that is utilized in the production of prodigiosin. Prodigiosin production of 7.07 g/L was achieved when the concentrations of two trace compounds, FeSO₄·4H₂O and MnSO₄·4H₂O, were optimized using the statistical experimental design methodology. Their optimal concentrations were 0.56 mM and 3.25 mM, respectively. Ultimately, the production of prodigiosin was increased from 2.3 g/L to 15.6 g/L, or by a factor of nearly seven by immobilizing microorganisms in 3% calcium alginate beads.     

Bioprocess development of the production of the mutant P-219-L human d-amino acid oxidase for high soluble fraction expression in recombinant Escherichia coli

In order to examine the structure–activity relationship and the substrate specificity of human d-amino acid oxidase (h.DAO), a single amino acid mutation had been established as proline-219-luecine (P-219-L). The gene encoding mutant h.DAO has been cloned and expressed in Escherichia coli BL21 (DE3). It was observed that the host cell was negatively affected by the expressed mutant h.DAO, resulting in a remarkable decrease in the cell growth and consequently the amount of the produced enzyme. To overcome this problem, we investigated several factors that may affect the cell growth rate and the mutant h.DAO production such as optimization of the glucose concentration as a main carbon source and the yeast extract concentration as a main nitrogen source, optimization of dissolved oxygen (DO%) concentration and the addition of benzyl alcohol (BA, which can artificially induce a strong heat shock response at low temperature), to enhance the production of natively folded soluble fraction of the recombinant protein. These parameters were tested on both shake flask level and fed-batch bioreactor level. The Western blot analysis and the enzyme activity assay indicated the higher level of the mutant expression towards enhancement of the conditions by using our designed approach.The specific activity (which was used as an indicator for the level of the desired protein produced = U/mg protein) and the OD_600 nm of the host cells (which was used as an indicator for the cell growth), reached to be 0.061 U/mg protein and 3.44, respectively upon using fed-batch culture system containing the optimized medium composition (15 g/l glucose and 5 g/l yeast extract). While upon using the shake flask level, these values were 0.032 and 1.1, respectively. Enhancement of the cell growth and the enzyme production was noticed after DO% optimization upon using 500 rpm agitation speed and 1.8 v.v.m. (volume volume minute) aeration. The specific activity for the mutant enzyme and the OD_600 nm of the host cells reached to be 0.14 U/mg protein and 7.1, respectively. Finally upon using the optimized culture composition (15 g/l glucose and 5 g/l yeast extract), optimized DO% (using 500 rpm agitation speed and 1.8 v.v.m.) and 0.1 mM BA at the fed-batch bioreactor level, the specific activity and the OD_600 nm of the host cells increased significantly to be 0.21 U/mg protein and 11.3, respectively at 24 h culture. These results indicate the importance of our approaches to overproducing mutant h.DAO in soluble form in E. coli.     

Effect of soybean oil and Tween 80 on the production of botryosphaeran by Botryosphaeria rhodina MAMB-05

The addition of soybean oil and Tween 80 was evaluated with the objective of increasing the production of botryosphaeran, an exopolysaccharide (EPS) of the (1 → 3;1 → 6)-β-d-glucan type produced by the fungus Botryosphaeria rhodina MAMB-05. Factorial design and analysis by response surface methodology was developed to select the main factors that would affect and enhance EPS production. The optimized culture conditions were: 40 g l⁻¹ glucose with 10 ml l⁻¹ soybean oil, and 4.5 ml l⁻¹ Tween 80, during 72 h cultivation at 28 °C (180 rpm) and initial pH 5.7. The predicted result for botryosphaeran production was 8.22 ± 1.36 g l⁻¹, and compared with the experimental value of 7.74 ± 0.13 g l⁻¹. Partial characterization of the botryosphaeran produced under the optimized conditions showed one type of polysaccharide with β-glycosidic linkages containing glucose as monosaccharide.     

d- and l-lactic acid production from fresh sweet potato through simultaneous saccharification and fermentation

The aim of this study was to develop a bioprocess for l- and d-lactic acid production from raw sweet potato through simultaneous saccharification and fermentation by Lactobacillus paracasei and Lactobacillus coryniformis, respectively. The effects of enzyme and nitrogen source concentrations as well as of the ratio of raw material to medium were investigated. At dried material concentrations of 136.36–219.51 g L⁻¹, yields of 90.13–91.17% (w/w) and productivities of 3.41–3.83 g L⁻¹ h⁻¹ were obtained with lactic acid concentrations as high as 198.32 g L⁻¹ for l-lactic acid production. In addition, d-lactic acid was produced with yields of 90.11–84.92% (w/w) and productivities of 2.55–3.11 g L⁻¹ h⁻¹ with a maximum concentration of 186.40 g L⁻¹ at the same concentrations of dried material. The simple and efficient process described in this study will benefit the tuber and root-based lactic acid industries without requiring alterations in plant equipment.     

The degradation of coniferyl alcohol and the complementary production of chlorogenic acids in the growth culture of Streptomyces albogriseolus KF977548 isolated from decaying wood residues

Coniferyl alcohol is one of the major precursors of lignin; the most abundant aromatic compound and a natural resource currently receiving attention because of the value-added metabolites resulting from its degradation. Growth study of Streptomyces albogriseolus KF977548 (strain AOB) isolated from decaying wood residues in a tropical estuarine ecosystem was carried out using coniferyl alcohol as a sole carbon source. Cell growth and metabolite production were monitored at 24 h interval by dry weight measurements and HPLC, LC–MS-DAD analyses. Biochemical and PCR assays were carried out to detect the major catabolic enzymes of interest. Strain AOB utilized coniferyl alcohol completely within 72 h (μ = 0.204 h⁻¹, T_d = 3.4 h). Laccase and peroxidase were released into the growth medium up to 0.099 and 98 μmol/mL respectively. Protocatechuate 3, 4-dioxygenase and demethylase were detected in the genome whilst ortho-adipate pathway was clearly indicated. Growth on coniferyl alcohol or caffeic acid as mono substrates resulted in the production of secondary metabolites identified by HPLC–MS as 1-caffeoylquinic and 3,4,5-tricaffeoylquinic acids, known as chlorogenic acids, in the culture medium. The microbial production of chlorogenic acids from a lignin-related substrate base by strain AOB could arouse a plausible biotechnological process.     

Optimization of culture medium for cordycepin production using Cordyceps militaris mutant obtained by ion beam irradiation

The effect of medium components on cordycepin production by Cordyceps militaris mutant obtained by ion beam irradiation was investigated. According to the response surface analysis using a central composite design for the prospective mutant G81-3, the predicted optimal concentrations of glucose as the carbon source and the yeast extract as the nitrogen source were 86.2 g/l and 93.8 g/l, respectively, and 6.84 g/l cordycepin was obtained. To date, this is the highest value for cordycepin production. The optimal concentrations of glucose and yeast extract for cordycepin production of the mutant was much higher than that of control (wild strain) and the cordycepin production was 2.79 times higher. Therefore, this new mutant will be a promising strain for future higher cordycepin production at industrial levels.     

Emulsifying properties of a marine bacterial exopolysaccharide

Exopolysaccharide produced by a marine Enterobacter cloaceae (designated as EPS 71a) emulsified hexane, benzene, xylene, kerosene, paraffin oil, cottonseed oil, coconut oil, jojoba oil, castor oil, groundnut oil and sunflower oil. However, it could form stable emulsions with groundnut oil and hexane at optimal concentration of 1 mg ml⁻¹. Further increase in concentration of EPS 71a did not show noteworthy increase in emulsification indices. Emulsions with groundnut oil and hexane were stable up to 10 days between pH 2 and 10 and in the presence of sodium chloride in the range of 5–50 mg ml⁻¹ at 35–37 °C. EPS 71a formed stable emulsion with xylene as compared to commercial gums such as arabic, tragacanth, karaya and xanthan.     

Medium optimization for the high yield production of single (+)-terrein by Aspergillus terreus strain PF26 derived from marine sponge Phakellia fusca

Terrein has potential application in the fields of medicine, cosmetology and agriculture, however, the chemical synthesis of terrein with single configuration is a difficult task, and the biosynthesis of terrein always results in low production (ca. 0.33–400 mg/L). In this study, we reported an Aspergillus terreus strain PF26 which could produce (+)-terrein on a high level. After the selection of a suitable basic medium, the component concentrations were optimized using Plackett–Burman design and response surface methodology. Consequently, an optimal medium containing 28.41 g glucose, 23.18 g maltose, 20.00 g mannitol, 8.52 g malt extract, 10.00 g monosodium glutamate 10.00 g NH₄Cl in 1 L ASW was obtained, and a high (+)-terrein production of 3.71 g/L fermentation broth was achieved, which represents the highest fermentation production of (+)-terrein to date. The result highlighted the industry's potential of A. terreus strain PF26 in the production of bioactive (+)-terrein on a large-scale.