Biosurfactant production from Candida lipolytica in bioreactor and evaluation of its toxicity for application as a bioremediation agent

This large-scale production, toxicity, characterization and economic analysis of the biosurfactant from Candida lipolytica UCP 0988 produced in the low-medium formulated with animal fat and corn steep liquor was investigated. The biosurfactant was produced in the stationary phase under 200 rpm in the absence of aeration and reduced the surface tension of the medium from 50 to 28 mN/m after 96 h, yielding 10.0 g/L of isolated biosurfactant in a 2 L bioreactor. The production was maximized in a 50 L bioreactor, reaching 40 g/L biosurfactant and 25 mN/m. The cell biomass was quantified and characterized for use in animal nutrition. Chemical structures of the biosurfactant were identified using FTIR and NMR. The crude biosurfactant was not toxic to the bivalve Anomalocardia brasiliana, to the microcrustacean Artemia salina, or three species of vegetables seeds. The biosurfactant stimulated the degradation of motor oil by the seawater indigenous microorganisms. The results obtained indicate that the biosurfactant produced has great potential to be applied as a bioremediation agent for cleaning oil spills.     

Purification and characterization of an extracellular cholesterol oxidase from a Bordetella species

A soil-isolated bacterium (strain B4) was identified as a species of Bordetella and deposited with the China General Microbiological Culture Collection (code, CGMCC 2229). The bacterium grew in a mineral medium, on cholesterol as a sole source of carbon and energy. Only one metabolite of cholesterol was accumulated in detectable amounts during the strain growth. It was identified as 4-cholesten-3-one. Cholesterol oxidase (COD) (EC 1.1.3.6), which catalyzes cholesterol into this metabolite, was evidenced from the strain. The conditions of the bacterium growth were optimized for extracellular enzyme production, which then reached around 1700 UL⁻¹ within 24 h culturing. The enzyme was purified from the spent medium of the strain to homogeneity on SDS-PAGE, and characterized. Its molecular mass, as estimated by this technique, was 55 kDa. COD showed an optimum activity at pH 7.0. It was completely stable at pH 5.0 and 4 °C for 48 h, and retained 80% at least of its initial activity at pH 4.0 or at a pH of 6.0–10.0. The optimum temperature for its reaction was 37 °C. The thermal stability of COD was appreciable, as 90% or 80% of its initial activity was recovered after 1 h or 2 h incubation at 50 °C. Ag⁺ or Hg⁺ at 1 mM, was inhibitor of COD activity, while Cu²⁺, at the same concentration, was activator. The COD K_m, determined at 37 °C and pH 7.0, was 0.556 mM. The enzyme was stable at pH 7.0 and 37 °C during 24 h mechanical shaking in the presence of 33% (v/v) of either of the solvents, dimethylsulfoxide, ethyl acetate, butanol, chloroform, benzene, xylene or cyclohexane.     

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.     

Production of polyhydroxyalkanoates from whey by Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus HB8 is able to utilize lactose from whey-based media for the biosynthesis of polyhydroxyalkanoates (PHAs) under nitrogen limitation. T. thermophilus can utilize both, glucose and galactose, the products of lactose hydrolysis. When T. thermophilus HB8 was grown in culture media containing 24% (v/v) whey, PHA was accumulated up to 35% (w/w) of its biomass after 24 h of cultivation. The effect of initial phosphate concentration on the PHA production was also investigated. Using an initial phosphate concentration of 50 mM the PHA accumulation was enhanced. Analysis of the produced PHA from T. thermophilous HB8 grown in whey-based media revealed a novel heteropolymer consisting of the short chain length 3-hydroxyvalerate (3HV; 38 mol%) and the medium chain length, 3-hydroxyheptanoate (3HHp; 9.89 mol%), 3-hydroxynanoate (3HN; 16.59 mol%) and 3-hydroxyundecanoate (3HU; 35.42 mol%). Despite the low molecular weight of the produced PHA by T. thermophilus, whey could be an excellent substrate for the production of heteropolymers with unique properties.     

The production of a cold-induced extracellular biopolymer by Pseudomonas fluorescens BM07 under various growth conditions and its role in heavy metals absorption

The psychrotrophic bacterium Pseudomonas fluorescens BM07 was induced to excrete an extracellular biopolymer when cells were grown aerobically at 10 °C and its secretion was inhibited at 30 °C. The biopolymer was easily torn apart from the cells by using a shear force under centrifugation (8700 × g, 30 min) and collected as a well-separated mucoid layer in centrifuge tube. The production of the biopolymer was affected by factors such as the types of carbon and nitrogen sources, temperature, and pH. The best production of 2.5 g/l was obtained when the cells were grown on M1 medium containing 70 mM sucrose and 0.2% (w/v) Casamino Acids. In Kings B enriched medium a maximum biopolymer production of up to 3.4 g/l and growth rate of 2.1 g/l, were achieved using 1:1 ratio of C/N. Addition of NaCl and ethanol to the medium led to a decrease in biopolymer production and growth rate of BM07 strain. FT-IR spectroscopy demonstrated the presence of carboxyl, amine, hydroxyl and methoxyl functional groups in the biopolymer. BM07 biopolymer showed high ion binding capacity with particular preference to uptake cadmium and mercury (∼45 and 70%, respectively). The percentage removal of cobalt, zinc, nickel and copper cations were between 20 and 30%. Overall ion uptake by BM07 biopolymer showed a definite preference for larger over smaller cations (Hg > Cd > Ni > Zn > Cu > Co).     

Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production

Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the production of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chemicals from CO₂: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an organic phase (decane), the strain produced up to 670 mg/L total hydrocarbons containing 435 mg/l of alkanes consisting of 286 mg/l of pentadecane, 131 mg/l of heptadecene, 18 mg/l of heptadecane, and 236 mg/l of hexadecanal. We report here the highest level of alka(e)nes production by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes production by a non-native alkane producer from CO₂ as the sole carbon source.     

Degradation of chitin and production of bioactive materials by bioconversion of squid pens

Serratia marcescens TKU011, a protease- and chitosanase-producing bacterium, the optimized condition for protease and chitosanase production was found after the media were heated at 121 °C for 120 min and the culture was shaken at 25 °C for 5 days in 100 mL of medium containing 1% squid pen powder (SPP) (w/v), 0.1% K₂HPO₄, and 0.05% MgSO₄. An extracellular metalloprotease with novel properties of solvent stable, and alkaline was purified from the culture supernatant of S. marcescens TKU011 with squid pen wastes as the sole carbon/nitrogen source. The enzyme was a monomeric protease with a molecular mass of 48–50 kDa by SDS–PAGE and gel filtration chromatography. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU011 protease were 8, 50 °C, pH 5–11, and <40 °C, respectively. Besides protease and chitosanase, with this method, deproteinization of squid pen for β-chitin, the production of peptide and reducing sugar may be useful for biological applications.     

Glutathione S-transferase pi (GST-pi) inhibition and anti-inflammation activity of the ethyl acetate extract of Streptomyces sp. strain MJM 8637

To investigate the anti-cancer properties of soil-borne actinobacteria, MJM 8637, the glutathione S-transferase pi (GST-pi) assay, anti-tumor necrosis factor (TNF)-α assay, the level of antioxidant potential by DPPH radical scavenging activity, NO scavenging activity, and ABTS radical scavenging activity in ethyl acetate extract were determined. The 16S rDNA sequencing analysis revealed that Streptomyces sp. strain MJM 8637, which was isolated from Hambak Mountain, Korea, has 99.5% similarity to Streptomyces atratus strain NBRC 3897. The physiological and the morphological characteristics of the strain MJM 8637 were also identified. The ethyl acetate extract of MJM 8637 inhibited TNF-α production approximately 61.8% at concentration 100 μg/ml. The IC₅₀ value of the strain MJM 8637 extract on GST-pi was identified to be 120.2 ± 1.6 μg/ml. In DPPH, NO, and ABTS radical scavenging assays, the IC₅₀ values of the strain MJM 8637 extract were found to be 977.2 μg/ml, 1143.7 μg/ml, and 454.4 μg/ml, respectively. The ethyl acetate extract of the strain MJM 8637 showed 97.2 ± 1.3% of cell viability at 100 μg/ml in RAW 264.7 cell viability assay. The results obtained from this study suggest that the ethyl acetate extract of Streptomyces sp. strain MJM 8637 could be considered as a potential source of drug for the cancers that have multidrug resistance with its GST-pi inhibition and anti-inflammation activities, and low cytotoxicity.     

Crude biosurfactant from thermophilic Alcaligenes faecalis: Feasibility in petro-spill bioremediation

The thermophilic bacterium Alcaligenes faecalis isolated from the crude oil contaminated soil of Upper Assam, India. The isolated bacterium was first screened for the ability to produce biosurfactant. The strain growing at 42 °C could produce higher amount of biosurfactant in medium supplemented with 2% (v/v) diesel as sole source of carbon and energy. Biochemical characterizations including FT-IR and MS studies suggested the biosurfactant to be glycolipid. Tensiometric studies revealed that the biosurfactant produced by the bacterial strain could decrease the surface tension (??) at air-water interface from 71.6 to 32.3 mNm⁻¹ after 96 h of growth on hydrocarbon and possessed a low critical micelle concentration (CMC) value of approximately 38 mgl⁻¹, indicating high surface activity. The culture supernatant containing the biosurfactant was found to be functionally stable at varying pH (2-12), temperature (100 and 121 °C) and salinity (1-6% NaCl, w/v) conditions. Both the culture broth and the cell free supernatant exhibited high emulsifying activity against the different hydrocarbons and the crude oil components. The increase in cell surface hydrophobicity and glycolipid production by the strain suggested the existence of biosurfactant enhanced interfacial uptake of the hydrocarbons. Moreover, the partially purified biosurfactant exhibited antimicrobial activity by inhibiting the growth of several bacterial and fungal species. The strain represented a new class of biosurfactant producers and could be a potential candidate for the production of glycolipid biosurfactant which could be useful in a variety of biotechnological and industrial processes, particularly in the oil industry.     

Improved production of lycopene and β-carotene by Blakeslea trispora with oxygen-vectors

Lycopene and β-carotene production were increased when oxygen-vectors, n-hexane and n-dodecane, were added to cultures of Blakeslea trispora because of the enhanced dissolved oxygen concentrations. With 1% (v/v) n-hexane or n-dodecane added in the medium, lycopene production was 51% or 78% higher and β-carotene production was 44% or 65% higher than that of the control, respectively. The highest lycopene and β-carotene production, 533 mg l⁻¹and 596 mg l⁻¹, were obtained when 1% (v/v) n-dodecane and 0.1% (w/v) Span 20 were added together, which were 2.1-fold and 1.8-fold of the control, respectively.     

Characteristics of biosurfactant produced by Pseudomonas aeruginosa S6 isolated from oil-containing wastewater

A biosurfactant-producing strain S6 was isolated from oil-containing wastewater and identified as Pseudomonas aeruginosa based on physiological and biochemical tests together with 16S rDNA sequence analysis. Thin layer chromatography (TLC) and high-performance liquid chromatography electrospray ionization mass spectra (HPLC-ESI-MS) worked together to reveal that the strain S6 produced rhamnolipid biosurfactant. Mass spectrometry confirmed the presence of some major components in the rhamnolipid surfactant showing m/z of 675.8, 529.6, 503.3 and 475.4, which corresponded to RhaRhaC₁₀C_12:1, RhaC_12:1C₁₀, RhaC₁₀C₁₀ and RhaC₈C₁₀, respectively. The biosurfactant produced by strain S6 had the ability to decrease the surface tension of water from 72 to 33.9 mN m^?1, with the critical micelle concentration (CMC) of 50 mg L^?1. Emulsification experiment indicated that this biosurfactant effectively emulsified the crude petroleum and the measurements of surface tension demonstrated that the biosurfactant possessed stable surface activity at variable ranges of pH and salinity. The biosurfactant also exhibited good performance of phenanthrene solubilization with about 23 times higher solubility of phenanthrene in water than the control. Thus, this biosurfactant may have a potential for application in bioremediation of crude oil contamination.     

High-loading oil palm empty fruit bunch saccharification using cellulases from Trichoderma koningii MF6

Strain Trichoderma koningii D-64 was improved for enhanced cellulase production. A potential mutant MF6 was obtained and its enzymes contained filter paper cellulase (FPase), carboxymethylcellulase (CMCase), β-glucosidase and xylanase with respective activities of 2.0, 1.3, 2.0 and 3.0 folds of those for the parental strain. MF6 cellulases showed enhanced hydrolysis performance for the treated lignocellulosic biomass. Hydrolysis of treated oil palm empty fruit bunch (OPEFB), horticulture wastes (HW) and wood chips (WC) resulted in cellulose to glucose conversion of 96.3 ± 2.2%, 98.2 ± 3.0% and 81.9 ± 1.4%, respectively. The corresponding conversions of xylan to xylose were 96.9 ± 1.5%, 95.0 ± 2.2% and 76.1 ± 3.1%. Consistently, high sugar yield of 770–844 mg/g biomass was obtained for high-loading (10–16%, w/v) of OPEFB hydrolysis and sugar titer of 135.1 g/L was obtained for 16% (w/v) OPEFB loading at 96 h. In addition, MF6 enzymes alone performed equally well for high-loading OPEFB hydrolysis compared to the enzyme mixture of β-glucosidase from Aspergillus niger and cellulase from T. reesei Rut C30.     

Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.     

Production,purification, and characterization of two extremely halotolerant,thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101

The halophilic bacterial strain Chromohalobacter sp. TVSP 101 was shown to produce extracellular, halotolerant, alkali-stable and moderately thermophilic α-amylase activity. The culture conditions for higher amylase production were optimized with respect to NaCl, pH, temperature and substrates. Maximum amylase production was achieved in a medium containing 20% NaCl or 15% KCl at pH 9.0 and 37 °C in the presence of 0.5% rice flour and tryptone. Addition of 50 mM CaCl₂ to the medium increased amylase production by 29%. Two kinds of amylase activity, designated amylase I and amylase II, were purified from culture filtrates to homogeneity with molecular masses of 72 and 62 kDa, respectively. Both enzymes had maximal activity at pH 9.0 and 65 °C in the presence of 0–20% (w/v) NaCl but amylase I was much more stable in the absence of NaCl than amylase II. The enzymes efficiently hydrolyzed carbohydrates to yield maltotetraose, maltotriose, maltose, and glucose as the end products.     

Benzimidazole-based antibacterial agents against Francisella tularensis

Francisella tularensis is a highly virulent pathogenic bacterium. In order to identify novel potential antibacterial agents against F. tularensis, libraries of trisubstituted benzimidazoles were screened against F. tularensis LVS strain. In a preliminary screening assay, remarkably, 23 of 2,5,6- and 2,5,7-trisubstituted benzimidazoles showed excellent activity exhibiting greater than 90% growth inhibition at 1 μg/mL. Among those hits, 21 compounds showed MIC₉₀ values in the range of 0.35–48.6 μg/mL after accurate MIC determination. In ex vivo efficacy assays, four of these compounds exhibited 2–3 log reduction in colony forming units (CFU) per mL at concentrations of 10 and 50 μg/mL.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Dye–ligand expanded bed adsorption of G6PDH from highly dense unclarified yeast extract

The application of dye–ligand expanded bed chromatography adsorption (EBA) of glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast extract was undertaken by using a commercially available expanded bed column (20 mm i.d.) and UpFront adsorbent (ρ = 1.5 g/mL) from UpFront Chromatography. The influence of biomass concentration on the adsorption capacity was explored by employing yeast extracts containing various biomass concentrations (5–30%, w/v). It was demonstrated that the biomass concentration had little effect on G6PDH adsorption performance. Feedstock containing 15% (w/v) biomass gave a relatively high recovery yield (>90%) of G6PDH compared to feedstock containing 30% (w/v) biomass, which gave a recovery of 75% G6PDH. Nevertheless, the enzyme specific activity of 7 U mg⁻¹ with a purification factor of 6 was achieved in the feedstock containing biomass concentration of 30% (w/v). The generic applicability of dye–ligand as an affinity tool in expanded bed chromatography is discussed.     

Microbial levan from Brachybacterium phenoliresistens: Characterization and enhancement of production

Levan producing bacteria was isolated from rhizosphere soil. The molecular identification of this isolate was conducted using 16S rRNA, which resulted in a sequenced region of 1298 base pairs. The sequence alignment in the gene bank indicated that this isolate has a high percentage of similarity (99%) to the retrieved consensus sequence of Brachybacterium phenoliresistens strain phenol-A. The produced levan was characterized using TLC, FTIR, 1H NMR and 13C NMR spectroscopy techniques. The effects of nutritional and physical factors on this isolate’s levan production were investigated. The results demonstrated that the optimal sources for carbon and casein during levan production were sucrose and casein, yielding 7.88 g/land 8.12 g/l of levan, respectively. The highest levan yield (7.97 g/l) was obtained at a sucrose concentration of 300 g/l. At an initial pH of 7.8, this bacterium yielded their highest levan production of 7.88 g/l. The optimal incubation period was 72 h with a yield of 8.58 g/l, the optimal temperature was 30 °C and resulted in 7.87 g/l, and the highest levan production yield was obtained at 150 rpm and yielded 8.12 g/l.     

Effect of lignocellulose degradation products on microbial biomass and lipid production by the oleaginous yeast Cryptococcus curvatus

This work investigated effects of lignocellulose degradation products on cell biomass and lipid production by Cryptococcus curvatus. Furfural was found to have the strongest inhibitory effect. For the three phenolic compounds tested, vanillin was the most toxic, while PHB and syringaldehyde showed comparable inhibitions in the concentration range of 0–1.0 g/L. Generally little significant differences on the relative cell biomass and lipid contents at the same concentrations of tested compounds were observed between glucose and xylose as a sole carbon source. At 1.0 g/L of furfural, the cell biomass and lipid content decreased by 78.4% and 61.0% for glucose as well as 72.0% and 59.3% for xylose, respectively. C. curvatus ceased to grow at concentrations of PHB over 1.0 g/L or vanillin over 1.5 g/L. The strain could survive in the presence of syringaldehyde up to 2.0 g/L for glucose or 1.5 g/L for xylose. The compounds’ negative impact was reduced by an increase in inoculum size and a 10% (v/v) seed was detected to be optimal for cell biomass and lipid production. The results demonstrated C. curvatus could effectively utilize most of the dominant monosaccharides and cellobiose existing in lignocellulosic biomass hydrolysate in the presence of toxic compounds.     

Cellulose ethanol production from waste newsprint by simultaneous saccharification and fermentation using Saccharomyces cerevisiae KNU5377

A thermotolerant ethanol-fermenting yeast, Saccharomyces cerevisiae KNU5377, isolated from a sludge of a local industrial complex stream in Korea, was evaluated for its capability for lignocellulosic ethanol production from waste newsprint in high temperature. In this fermentation, most of dry-defibrated waste newspaper was first saccharified at 50 °C for 108 h using a commercial cellulase and, then with the last addition of dry-defibrated newsprints to the pre-saccharified broth, simultaneous saccharification and fermentation (SSF) of 1.0 L of reaction mixture was carried out at 40 °C, slowly being dropped from 50 °C, for further 72 h in a 5 L fermentor by inoculating the overnight culture of KNU5377. The maximum production of 8.4% (v/v) ethanol was obtained when 250 g (w/v)/L of dry-defibrated waste newspaper was used for ethanol production by SSF. These results suggest that S. cerevisiae KNU5377 is very useful for cellulose ethanol production by the SSF system.